Fragment Based Identification of Phosphatase Inhibitors ... · Inhibitor library synthesis and evaluation 11 Amide replacement analog synthesis and evaluation 12 ... AEBSF 4-(2-aminoethyl)

Fragment Based Identification of Phosphatase Inhibitors

by

Tyler Daniel Baguley

A dissertation submitted in partial satisfaction of the

requirements for the degree of

Doctor of Philosophy

in

Chemistry

in the

Graduate Division

of the

University of California, Berkeley

Committee in charge:

Professor Jonathan A. Ellman, Co-Chair

Professor Ming C. Hammond, Co-Chair

Professor Matthew B. Francis

Professor Jasper Rine

Fall 2014

© Copyright by


2014

1

Abstract

Fragment Based Identification of Phosphatase Inhibitors

by


Doctor of Philosophy in Chemistry

University of California, Berkeley

Professor Jonathan A. Ellman, Co-Chair

Professor Ming C. Hammond, Co-Chair

Chapter 1. In this chapter, phosphatases are broadly introduced as an enzyme class with

therapeutic implications in a variety of disease areas. Different methods for inhibitor

identification are outlined as well. Finally, a substrate-based fragment approach which has been

developed by the Ellman group for the identification of phosphatase inhibitors is introduced.

Chapter 2. The fragment-based method for the identification of phosphatase inhibitors

introduced in chapter 1 is applied to Mycobacterium tuberculosis protein tyrosine phosphatase

PtpA. Inhibitors incorporating a well established phosphate mimic, the

difluoromethylenephosphonic acid, were explored, resulting in low micromolar inhibitors of

PtpA. The most potent compound was also shown to be selective for PtpA over a variety of

human phosphatases as well as Mycobacterium tuberculosis protein tyrosine phosphatase PtpB.

This inhibitor represents a chemical tool that can be used in conjugation with PtpB selective

inhibitors described previously within the Ellman group to further probe the roles of PtpA and

PtpB in tuberculosis infection.

Chapter 3. The fragment-based approach introduced in chapter 1 is applied to striatal-

enriched protein tyrosine phosphatase (STEP), a brain specific phosphatase that has been

implicated in a number of neuropsychiatric disorders such as Alzheimer’s disease. STEP is a

very promising target for these diseases and was discovered nearly 20 years ago, yet no small

molecule inhibitor existed prior to our work. Through our fragment-based approach, we were

able to identify many low molecular weight (<450 Da), nonpeptidic, single-digit micromolar

mechanism-based STEP inhibitors with greater than 20-fold selectivity across multiple tyrosine

and dual specificity phosphatases. Additionally, significant levels of STEP inhibition in rat

cortical neurons were also observed.

Chapter 4. This chapter discusses the discovery and characterization of benzopentathipins as

redox-reversible inhibitors of STEP, the therapeutically relevant phosphatase introduced in

2

chapter 3. The majority of the chapter focuses on the biochemical characterization of the

benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6 amine hydrochloride (TC-

2153), a unique compound with a cyclic polysulfide that forms a reversible covalent bond with

the catalytic cysteine in STEP. Several analogs of TC-2153 are prepared to scope out not only

what is important for inhibition, but also to identify locations on the molecule that are amenable

to diversification for further compound development. Importantly, TC-2153 is shown to be

active in cell-based secondary assays and in animal behavioral models.

Chapter 5. This chapter outlines the use of seleninic acids as redox-reversible inhibitors of

STEP. The redox-reversible mode of inhibition described in chapter 4 is adapted to seleninic

acids, which have been demonstrated to form stable S–Se bonds with cysteine thiols. This new

PTP pharmacophore is merged with the SAR determined in chapter 3 to attain an inhibitor with

good activity in vitro.

Chapter 6. The development of additions of Knochel-type benzyl zinc reagents to N-tert-

butanesulfinyl aldimines is described. These additions utilize sp3-hybridized reagents that show

good functional group compatability, adding chemoselectively to imines that possess ester and

nitrile functionality. Addition to a glyceraldehyde-derived imine proceeds in high yield and

excellent selectivity and provides entry to hydroxyethylamine-based aspartyl protease inhibitors.

i

Table of Contents

Chapter 1. Protein tyrosine phosphatases as therapeutic targets 1

Protein tyrosine phosphatases 2

Redox-reversible regulation of phosphatases 3

Methods for identifying phosphatase inhibitors 3

High throughput screening 3

X-ray and NMR fragment screens 4

Substrate activity screening 4

Enzymatic assays 5

Substrate screening assay 5

Inhibitor screening assay 6

References 7

Chapter 2. Identification of inhibitors of the Mycobacterium tuberculosis

phosphatase PtpA 9

Introduction 10

Initial scaffold identification 10

Conversion to inhibitors 11

Inhibitor library synthesis and evaluation 11

Amide replacement analog synthesis and evaluation 12

Benzanilide scaffold optimization and evaluation 15

Inhibitor selectivity profile 17

Modeling studies 18

Conclusions 18

Experimental 19

ii

References 31

Chapter 3. Fragment-based identification of inhibitors of striatal-enriched

protein tyrosine phosphatase 35

Introduction 36

Inhibitor scaffold identification 36

Optimization of inhibitor 3.15 39

Optimization of inhibitor 3.16 42

Inhibitor selectivity profile 46

STEP inhibition in neuronal cultures 46

Blood-brain barrier permeability 46

Conclusions 47

Experimental 48

References 77

Chapter 4. Benzopentathiepins as novel redox-reversible inhibitors of STEP 81

Introduction 82

STEP as a therapeutic target 82

Initial high throughput screening results 82

Benzopentathiepins as attractive target molecules 84

Synthesis of TC-2153 84

Mechanism of STEP inhibition by TC-2153 86

Enzymatic characterization of inhibition 86

LC-MS/MS characterization of inhibition 87

Preparation of TC-2153 analogs for STEP inhibition 88

TC-2153 analog synthesis 89

iii

Inhibition of STEP by TC-2153 analogs 90

TC-2153 activity in cell-based secondary assays and in vivo 92

TC-2153 activity in cortical neurons and in vivo 92

TC-2153 specificity in vivo 92

TC-2153 reduces cognitive deficits in 3xTg-AD mice 94

Conclusions 95

Experimental 96

References 108

Chapter 5. Seleninic acids as redox-reversible inhibitors of STEP 111

Introduction 112

Synthesis of seleninic acid inhibitors 113

In vitro evaluation of inhibitors 114

Conclusions 115

Experimental 116

References 122

Chapter 6. Asymmetric additions of Knochel-type benzyl zinc reagents to N-

tert-butanesulfinyl aldimines 123

Introduction 124

Optimization of benzyl zinc additions 125

Evaluation of substrate scope for diastereoselective benzyl zinc addition 126

Stereochemical rationale 126

Preparation of aspartyl protease inhibitor precursors 128

Conclusions 129

Experimental 129

iv

References 141

Appendix 6.1: X-ray crystal data for compound 6.27 145

Appendix 6.2: X-ray crystal data for compound 6.31 157

v

Table of Abbreviations

%R membrane retention (for PAMPA)

[]20

D specific rotation at the sodium D line at 20 °C

[I] inhibitor concentration

[S] substrate concentration

°C degrees Celsius

3xTg-AD triple transgenic Alzheimer’s disease mice

Å angstrom

ABq AB quartet

Abs absorbance

Absmax wavelength of maximum absorbance

Ac acetyl

AcCl acetyl chloride

acetone-d6 deuterated acetone

AcOH acetic acid

AD Alzheimer's disease or 3xTg-AD mouse

AD-TC 3xTg-AD mouse treated with TC-2153

AD-Veh 3xTg-AD mouse treated with vehicle control

AEBSF 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride

ANOVA analysis of variance

app apparent

Ar aryl

Asp aspartic acid

avg average

BBB blood-brain barrier

Bn benzyl

BnBr benzyl bromide

Boc tert-butyloxycarbonyl

Boc2O di-tert-butyl dicarbonate

br broad

br s broad singlet

br t broad triplet

c concentration in grams per deciliter (g/dL)

vi

calcd. calculated

CD3OD dueterated methanol

CN control

cod 1,5-cyclooctadiene

cpd compound

Cys cysteine

NMR chemical shift, in ppm

d doublet

dr diastereomeric ratio

Da dalton

dd double of doublets

dec decomposition

DFMP difluoromethylenephosphonic acid

DIAD diisopropyl azodicarboxylate

DiFMUP 6,8-difluoro-4-methylumbelliferyl phosphate

DM double mutant (3xTg-AD + STEP–/–

)

DMA N,N-dimethylacetamide

DMDO dimethyldioxirane

DME 1,2-Dimethoxyethane

DMF N,N-dimethylformamide

DMSO dimethyl sulfoxide

DMSO-d6 deuterated dimethyl sulfoxide

dppf 1,1′-bis(diphenylphosphino)ferrocene

dt doublet of triplets

dtbpy 4,4-di-tert-butyl bipyridine

DTT dithiothreitol

DUSP dual-specificity protein tyrosine phosphatase

EDTA ethylenediaminetetraacetic acid

ee enantiomeric excess

equiv equivalents

ERK1/2 extracellular signal-regulated kinases 1 and 2

ESI electrospray ionization

Et ethyl

vii

Et2O diethyl ether

Et2SiH2 diethylsilane

Et3SiH triethylsilane

EtOAc ethyl acetate

EtOH ethanol

FAB fast atom bombardment

g gram

GAPDH glyceraldehyde 3-phosphate dehydrogenase

GC gas chromatography

GCMS gas chromatography-mass spectrometry

GluN2B subunit of the NMDAR

GSH reduced glutathione

GSSG oxidized glutathione dimer

GST glutathione S-transferase fusion tag

h hour

HBpin pinacolborane

hept heptet

HPLC high performance liquid chromatography

HRMS high resolution mass spectrometry

HTS high throughput screening

Hz hertz

i.p. intraperitoneal

IC50 half maximal inhibitory concentration

iPr isopropyl

iPr2NH N,N-diisopropylamine

iPrOH isopropanol

IR infrared spectroscopy

J NMR coupling constant

JCF NMR coupling constant between 13

C and 19

F atoms

JPF NMR coupling constant between 31

P and 19

F atoms

KHF2 potassium hydrogen difluoride

Ki the dissociation constant for an enzyme and inhibitor

kinact rate constant of inactivation

viii

Km the Michaelis constant, the dissociation constant for an enzyme and substrate

KO genetic knockout

KOAc potassium acetate

kobs observed rate constant

L liter

wavelength

LCMS liquid chromatography-mass spectrometry

LD50 median lethal dose

LDDN Laboratory for Drug Discovery in Neurodegeneration

LMW-PTP low molecular weight protein tyrosine phosphatase

Ln ligand

micro

M molar

m multiplet

m.p. melting point

m/z mass-to-charge ratio

Me methyl

MeOH methanol

MES 2-(N-morpholino)ethansulfonic acid

MESG 2-amino-6-mercapto-7-methylpurine riboside

mg milligram

mg/mL milligrams per milliliter

MHz megahertz

MIB 3-exo-(morpholino)isoborneol

min minute

mL milliliter

L microliter

M micromolar

m micrometer

mmol millimole

Mnt menthyl

mol mole

Ms mesyl, methanesulfonyl

ix

Mtb Mycobacterium tuberculosis

MTBE methyl tert-butyl ether

MWM Morris water maze

N normal, equivalent concentration

Na3VO4 sodium orthovanadate

NADPH nicotinamide adenine dinucleotide phosphate

NaSH sodium hydrosulfide

NBS N-bromosuccinimide

n-BuLi n-butyllithium

nd not determined

nM nanomolar

nm nanometer

NMDA N-methyl-D-aspartate

NMDAR N-methyl-D-aspartate receptor

NMR nuclear magnetic resonance or nuclear magnetic resonance sprectroscopy

OD600 optical density at 600 nm

p pentet

p probability-value, p-value

PAMPA parallel artificial membrane permeability

Pd(PPh3)4 Tetrakis(triphenylphosphine)palladium(0)

Pe effective permeability (for PAMPA)

Ph phenyl

PK pharmacokinetics

PNP purine nucleoside phosphorylase

pNPP p-nitrophenyl phosphate

PPh3 triphenylphosphine

ppm parts per million

p-protein phosphoprotein

ps picosecond

pSer phosphoserine

pThr phosphothreonine

PTP protein tyrosine phosphatase

pTyr phosphotyrosine

x

Pyk2 proline-rich tyrosine kinase 2

q quartet

RIPA radioimmunoprecipitation assay

ROS reactive oxygen species

rt room/ambient temperature

s singlet or second

S.D. standard deviation

s.e.m. standard error of the mean

S8 or S8 elemental sulfur

SAR structure activity relationship

SAS substrate activity screening

Ser serine

sext sextet

SNAr nucleophilic aromatic substitution

SOD superoxide dismutase

STEP striatal-enriched protein tyrosine phosphatase

STEP–/–

STEP genetic knockout

t triplet

TAT transactivator of transcription fusion tag

TB tuberculosis

TBAF tetra-n-butylammonium fluoride

TBS tert-butyldimethylsilyl

TBSCl tert-butyldimethylsilyl chloride

tBu tert-butyl

TC TC-2153 treated

TC-2153 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride

td triplet of doublets

TEEDA tetraethylethylenediamine

TFA trifluroacetic acid

TFAA trifluroacetic anhydride

THF tetrahydrofuran

Thr threonine

TMS tetramethylsilane

xi

TMSCl chlorotrimethylsilane

TMSI iodotrimethylsilane

tr,major retention time, major component

tr,minor retention time, minor component

tris 2-amino-2-hydroxymethyl-propane-1,3-diol

Trp tryptophan

U units of enzyme activity

U/mL units of enzyme activity per milliliter

UPLC ultra performance liquid chromatography

UV ultraviolet

UV-Vis ultraviolet-visible absorption spectroscopy

Veh vehicle control

Vmax maximum reaction velocity

Woollins' reagent 2,4-Diphenyl-1,3,2,4-diselenadiphosphetan-2,4-diselenide

WT wild-type

WT-TC wild-type mouse treated with TC-2153

WT-Veh wild-type mouse treated with vehicle control

Y tyrosine

xii

Acknowledgements

First and foremost, I need to thank my advisor professor Jonathan Ellman. His guidance,

insight and advice, coupled with his overall knowledge and love of chemistry, have been

invaluable. He has inspired me to be a better scientist and has pushed me at every step along the

way. Working for Jon has been one of the most rewarding aspects of my PhD. He has struck the

perfect balance of openness and willingness to help at every step and the ability to step back and

let the students drive the science which has been very challenging at times, yet rewarding.

I’d also like the thank professor Richmond Sarpong at UC Berkeley. He allowed me as an

incoming graduate student to work in his group prior to enrollment at Berkeley. His group was

the first serious research environment I was part of, and they taught me many of the basics of

how to be a better scientist, as well as the simple things like how to properly run a column. After

the move to Yale, I had the wonderful opportunity to team up with researchers at the Yale School

of Medicine, and I would be remiss not to mention professors Paul Lombroso and Angus Nairn.

Their collaboration through the last few years of my PhD has been invaluable and extremely

rewarding. Paul’s students Jian Xu and Manavi Chatterjee have been the perfect collaborators;

open, helpful, informative, efficient, and overall great scientists.

I am indebted to the Ellman group as a whole, past and present, Berkeley and Yale, graduate

students, postdocs and undergrads alike. You have helped me survive the past 5+ years and

shaped me into the scientist I am today. When I started at Berkeley, my labmates in 908 Latimer

were extremely helpful with the day to day questions, as well as great friends. Denise Colby,

Melissa Herbage, MaryAnn Robak and Melissa Leyva were the ideal labmates to begin my

career. Whether it was annoying Denise or Melissa with trivial science or Leyva about sports

(Go Ducks!) there was never a dull moment in lab.

Rhia Martin, Pete Marsden, Andy Tsai and MaryAnn were always up to go to Yogurt Park or

Chipotle, and to hang out outside of lab. I was sad to leave some of you behind at Berkeley,

because I knew I would miss our weekend meals and overall hanging out (I don’t miss step

aerobics though). I’d also like to thank Katherine Rawls for being my first, and only, true mentor

in the lab. It was great to join in on established projects and you were invaluable to my

development as a scientist. I will always remember Morten Storgaard, Van Yotphan, Jason Ji and

Vivian Lin for their unique laughs and smiles. I’m sad Jason and Vivian didn’t make the trek to

New Haven, but I can’t say I blame them.

Kyle Kimmel, Somenath Chowdhury, Andrew Buesking, Rhia and Andy all made the move

to Yale more bearable, whether it was Rhia’s laugh, skiing with Andy or Somenath’s sense of

humor (I think), I always had a good time and couldn’t have asked for a better group with which

to make the transition across the country. Once at Yale, I had the wonderful opportunity to work

along many talented chemists in 321 CRB. Both Yajing Lian and Kevin Hesp were excellent

(and productive) chemists, and I am glad to have worked alongside both of them. I leave the bay

in capable hands, although I hope Jessica Yuan gets some companions soon.

Although not in my bay, I will remember Tatjana Huber’s smile and Jimmie Weaver’s

breadth of knowledge (not to mention his coffee intake). Eric Phillips had a no nonsense

approach to chemistry which is admirable in its own right, and Simon Duttwyler continued that

tradition once Eric left, essentially training many of the current members of the group on how to

be rigorous with or without a glovebox. Michael Ischay’s ability to be both an optimist and

pessimist at the same time still confuses me, but he knew how to be a scientist, depending on

which day you asked him. Haichao Xu and Joey Stringer were fantastic collaborators, even if I

hardly saw them as they were stuck out at West Campus.

xiii

To the current members of the group, keep on trucking. Hasan KHAAANN (or is it Haroon

Kzing?), Haya (brown Kate) Jamali, Kate (white Haya) Otley and Caroline Tjin should be more

than enough to keep the chemical biology projects rolling. You all are great chemists and

fantastic people (even if you are Canadian, Hasan). Josh, eventually you’ll have a project where

the products are simple to separate, but until then, keep up the good work. Thanks to James

Phelan for, well, everything from hanging out outside of lab to answering my silly questions

which I should know the answer to after five years. Tehetena Mesganaw, it will be a pleasure to

see you back on the best coast shortly. Shuming Chen’s knowledge of computational chemistry

has been an asset to the group and has expanded my understanding of the subject. I have enjoyed

my college football discussions with Apiwat Wangweerawong, and maybe this year the Noles

and the Ducks can meet in the championship game to settle it once and for all (wishful thinking).

To the younger chemists in the group, Jeff Boerth and Scott Kolmar, keep up the good work and

progress will come, I promise. Finally, I’d like to thank Andrew “Wayne” Buesking for being

one of my best friends in the Ellman group over my whole time here. We made the trek from

Berkeley together and now, we’ve made it to the end. I’d also like to thank Andrew’s wife

Melissa for having us over so often, even that time when we invited ourselves when it was

snowing. (If I never live somewhere where snowplows are needed again, it will be too soon).

Finally, I’d like to thank my friends and family who have helped me either directly or

indirectly to pursue this degree in chemistry. First, I’d like to thank my parents, Dan and Tina,

for being supportive every step of the way. I’d like to thank my siblings Jason and Kristen for

both picking holiday weekends to get married, as I love paying double rates for cross-country

flights. I’d like to thank the many math and science teachers that inspired me throughout the

years to pursue knowledge and not grades: Mr. Miller, Mr. Sparkman in high school and Gautam

Bhattacharyya at the University of Oregon to name a few. I’d also like to thank Professor John

Keana at the University of Oregon for giving me a chance as an undergraduate to do chemical

research and really whet my appetite for science.

Last, but certainly not least, I must thank my wonderful wife, friend, partner and (at times)

colleague, Stephanie, without whom graduate school would have been a lot less pleasant. She

has listened to my frustrations in spite of her own, been my first source of editing on all my

manuscripts so I didn’t send something stupid to Jon, and has even helped me with biology

techniques and materials at times. She’s picked up my slack around the house, even though she

has her own job and responsibilities, and made sure I didn’t eat at the carts every day for lunch.

Even though I “dragged her ass to Connecticut,” she’s done it all (mostly) with a smile, and has

only had one psychotic break. I truly have appreciated all her support and understanding.

xiv

1

Chapter 1. Protein tyrosine phosphatases as therapeutic targets

Abstract: This chapter serves as the introduction to my doctoral thesis. Protein tyrosine

phosphatases (PTPs) are introduced as an enzyme class that catalyzes the hydrolysis of

phosphate groups from phosphorylated tyrosine residues in proteins. Additionally, multiple

methods to attain PTP inhibitors which have been utilized with varying degrees of success are

introduced. Finally, substrate activity screening (SAS), a substrate-based fragment identification

approach developed within the Ellman group prior to my arrival, is outlined, including an

introduction to the enzymatic assays performed throughout the thesis.

2

Protein tyrosine phosphatases

Phosphatases are enzymes that catalyze the dephosphorylation of proteins and other

molecules in living organisms. They work in concert with kinases, which catalyze the

phosphorylation of these biomolecules, to control a variety of cellular processes, from cell

growth and differentiation to immune response.1 There are two major classes of protein

phosphatases: protein tyrosine phosphatases (PTPs), which primarily dephosphorylate

phosphotyrosine (pTyr) residues, and the Ser/Thr phosphatases, which target phosphoserine

(pSer) and phosphothreonine (pThr). The PTP superfamily can be further separated into three

subfamilies: classical PTPs, which dephosphorylate only pTyr; dual-specificity PTPs (DUSPs),

which can accommodate pTyr, pSer and pThr; and low molecular weight PTPs (LMW-PTPs),

which share no significant homology with either classical PTPs or DUSPs, but also only

accommodate pTyr residues.2 While Ser/Thr phosphatases are metalloenzymes, all known PTPs

catalyze dephosphorylation through the formation of a covalent phosphocysteine intermediate

followed by subsequent hydrolysis by a solvent water molecule (Figure 1.1).3

Figure 1.1. Catalytic mechanism of protein tyrosine phosphatases (PTPs).

Aberrant PTP activity has been associated with many diseases, including diabetes (PTP1B),

cancer (Cdc25), autoimmune disease (CD45) and neurodegenerative disease (STEP).

Additionally, phosphatases have also been recognized as virulence factors for many infectious

diseases, such as bubonic plague (Yersinia PTP), tuberculosis (Mycobacterium tuberculosis PtpA

and PtpB) and staph infections (Staphylococcus aureus SaPtpA and SaPtpB).4 Generally, this is

accomplished through interrupting signaling cascades associated with host immune response to

the pathogens. As has been extensively reviewed, despite their implication in a variety of disease

areas, phosphatases have proven to be very difficult targets, as demonstrated by the significant

effort by many pharmaceutical companies to develop inhibitors of PTP1B, an enzyme involved

in insulin signaling.5 Although many PTP1B inhibitors have been reported, very few have made

it to clinical trials, and none of those have been approved for use in the clinic.5a,6

The successful

development of phosphatase inhibitors has three major challenges: first, high potency is difficult

to achieve; second, the highly polar character of pharmacophores required for interaction at the

phosphate binding site can limit both cell permeability and oral bioavailability; and third,

because the catalytic domains of PTPs are highly conserved, achieving inhibitor selectivity can

be challenging. For the development of PTP1B, inhibitors with either sufficient oral availability

or selectivity could be achieved, but attributes could not be attained in tandem.

3

Redox-reversible regulation of phosphatases

One important mechanism of PTP regulation involves the generation of endogenous

hydrogen peroxide (H2O2) by the highly controlled activation of NADPH oxidase enzymes in

response to external stimuli such as growth factors, cytokines and hormones.7,8

Inactivation

occurs first by oxidation of the catalytic cysteine residue to the sulfenic acid (Figure 1.2).9 The

half-life of the sulfenic acid is generally quite low in PTPs, because of its high reactivity, and the

sulfenic acid can often react with a neighboring cysteine to form an intramolecular disulfide (as

with the inhibition of PTEN)10

or with the backbone nitrogen to form a cyclic sulfenamide

species (e.g., PTP1B),11

rendering the PTP inactive. Reaction of the oxidized enzymes with low

molecular weight thiols (e.g., reduced glutathione) or protein thiols (e.g., peroxiredoxin/

thioredoxin cycle) regenerates the active enzyme.12

Figure 1.2. Oxidative inactivation of PTPs. (a) Endogenously produced H2O2 inactivates PTPs by

oxidizing the catalytic cysteine thiolate to the sulfenic acid. The sulfenic acid can then undergo further

reactivity with a neighboring cysteine thiol (b) or a backbone nitrogen (c) to form a disulfide or cyclic

sulfenamide respectively.

Within the last decade, there has been extensive research on the biological and chemical roles

of this cysteine oxidation. The Carroll group, among others, has begun to use the innate

reactivity of the sulfenic acid to develop chemical probes to try to detect and understand the role

of cysteine oxidation in PTPs.13

As these questions begin to be answered it allows for other

possibilities. One aspect that has only just begun to be explored is the possibility of redox active

inhibitors for PTPs. In 2013, Barrios and coworkers reported on a pseudo-irreversible redox

inhibitor of the lymphoid tyrosine phosphatase,14

and in the same year the Thompson group

reported on an inhibitor of a LMW-PTP that utilizes a redox based mechanism.15

Methods for identifying phosphatase inhibitors

High throughput screening

High throughput screening (HTS) refers to the screening of thousands of compounds,

sometimes on the order of hundreds of thousands, in an assay in order to identify lead

4

compounds with nanomolar or low micromolar affinity that can be further optimized to provide

potent inhibitors. This method has been used successfully for many clinically relevant enzyme

targets.16

However, using HTS to target phosphatases has been especially challenging resulting

in a high incidence of false positives. This may be due to a variety of factors, including: (1) the

high reactivity of the active site Cys nucleophile,17

(2) the irreversible oxidation of the catalytic

cysteine,17-18

or (3) the tendency of phosphatase inhibitors, which typically have polar head

groups and hydrophobic tails, to form micelles at high concentrations leading to non-specific

inhibition.19

Researchers at Wyeth have commented on this problem, indicating that in a HTS for

PTP1B inhibition, out of over 6,000 of their initial hits, none were real inhibitors upon cross-

validation.18

X-ray and NMR fragment screens

A few fragment-based approaches have also been applied to phosphatases, namely PTP1B. In

a fragment-based screen, smaller libraries (on the order of thousands of compounds) of low

molecular weight (typically <300 Da) are screened to identify lead compounds to be optimized

into potent lead compounds.20

Because the fragment affinities are in the micromolar to

millimolar ranges, these assays are typically done at higher concentrations than traditional HTS,

which results in a higher incidence of false positives. This has led to the need of more

meticulous, and laborious, methods to directly observe protein binding, rather than enzyme

activity inhibition, for phosphatase targets.

Two rigorous fragment-based methods have been used to achieve potent inhibitors of

PTP1B, NMR and X-ray based fragment screening.21

In the NMR based method, changes in the

protein NMR are detected upon fragment binding.22

Because of the detection of direct binding,

the incidence of false positives can be greatly diminished. In the X-ray based screening method,

fragments are co-crystallized into the active site of the enzyme.20b,c

This method also drastically

decreases the number of false positives in the screen and provides direct binding information.

However, crystal structures take time to acquire and structure activity relationship (SAR)

determination by X-ray methods alone, in particular with enzymes that have no previously

published structural data, can be very slow.23

Even though there has been some success with

NMR and X-ray methods in achieving inhibitors of PTP1B, sufficient oral availability and/or

selectivity was not achieved. Additionally, both methods require a large amount of protein and

dedicated use of expensive instrumentation.

Substrate activity screening

To identify phosphatase inhibitors, the Ellman group has developed an alternative, substrate-

based fragment identification method termed substrate activity screening (SAS).24

This approach

addresses the main limitation in fragment-based screening as it pertains to phosphatases: the

identification of weak binding fragment starting points with high fidelity and efficiency. The

SAS method as it applies to phosphatases is outlined in Scheme 1.1.

In the initial screen, a library of O-aryl phosphate substrates is screened to identify phosphate

substrates. The initial substrate library was synthesized by the Ellman group prior to my arrival

and will not be a subject of discussion in this document, except to say that the library consists of

over 150 low molecular weight (<300 Da), diverse and nonpeptidic O-aryl groups. Once the

5

Scheme 1.1. Substrate activity screening (SAS) method for the identification of phosphatase inhibitors

initial substrate fragments are identified, the O-aryl group can be optimized to achieve more

potent substrates. During optimization the substrates are converted to inhibitors by direct

replacement of the phosphate with known non-hydrolyzable phosphate mimetics.

The key feature of the SAS method when compared to other screening methods is this initial

identification step. In this initial fragment identification, the false positives seen in traditional

HTS methods are eliminated, because active site binding and catalysis are needed for signal

production. Additionally, in traditional HTS methods for inhibitor identification, the desired

output is a decrease in signal, which is typically more difficult to quantify than the increase in

signal observed through the SAS method. Due to catalytic substrate turnover, there is also the

added benefit of signal amplification allowing efficient identification of weak binding substrate

fragments. Finally, because there are many non-hydrolyzable phosphate mimetics (Figure 1.3),25

there is flexibility when it comes time to replace the phosphate moiety with a non-hydrolyzable

replacement.

Figure 1.3. Non-hydrolyzable phosphate mimetics for conversion of substrates to inhibitors.

Enzymatic assays

Substrate screening assay

When using SAS, the initial substrate screen uses an established simple, sensitive and high-

throughput sprectrophotometric-based coupled enzyme assay (Scheme 1.2).26

Because inorganic

phosphate is spectrophotometrically silent, the initial substrate screening assay relies upon the

6

action of a secondary enzyme, purine nucleoside phosphorylase (PNP), which phosphorylates the

ribose ring of nucleoside substrate 1.1, releasing ribose-1-phosphate (1.2) and the UV-active

purine base 1.3. Substrate turnover results in a spectrophotometric shift in maximum absorbance

from 330 nm (substrate 1.1) to 360 nm (product 1.3). Importantly, PNP is selective for inorganic

phosphate and the O-aryl phosphate substrates do not interfere with this enzymatic process.

Therefore, this assay can be used continuously to monitor kinetics of inorganic phosphate

released by the phosphatase-catalyzed hydrolysis of the O-aryl phosphate substrates.

Scheme 1.2. Spectrophotometric coupled assay method for detection of inorganic phosphate released by

PTP of interest

Inhibitor screening assay

Inhibitors are assayed in a standard inhibition assay for phosphatases using p-nitrophenyl

phosphate (pNPP) as a chromogenic substrate (Scheme 1.3).27

This is a competitive inhibition

assay in which the absorbance of 1.4 is continuously monitored at 405 nm as pNPP is hydrolyzed

by the phosphatase of interest. The concentration of pNPP is held constant, while the

concentration of the inhibitor is varied, resulting in a dose-response curve from which Ki values

can be determined. Importantly, assays are performed with the addition of Triton X-100

detergent (0.004% to 0.01% total volume) to prevent non-specific aggregation-based inhibition.19

7

Scheme 1.3. Spectrophotometric assay method for PTP inhibitors

References

1. Barford, D.; Das, A. K.; Egloff, M.-P. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 133.

2. Ramponi, G.; Stefani, M. Int. J. Biochem. Cell Biol. 1997, 29, 279.

3. Tonks, N. K. Nat. Rev. Mol. Cell. Bio. 2006, 7, 833.

4. (a) van Huijsduijnen, R. H.; Bombrun, A.; Swinnen, D. Drug Discov. Today 2002, 7, 1013;

(b) Goebel-Goody, S. M.; Baum M,; Paspalas, C. D.; Fernandez, S. M.; Carty, N. C.; Kurup, P.;

Lombroso P. J. Pharmacol. Rev. 2012, 64, 65.

5. (a) Zhang, S.; Zhang, Z.-Y. Drug Discov. Today 2007, 12, 373; (b) Nichols, A. J.; Marshal,

R. D.; Balkan, B. Drug Develop. Res. 2006, 67, 559; (c) van Huijsduijnen, R. H.; Sauer, W. H.

B.; Bombrun, A.; Swinnen, D. J. Med. Chem. 2004, 47, 4142.

6. Combs, A. P. J. Med. Chem. 2009, 53, 2333.

7. General reviews on cysteine redox and its regulation of PTPs: (a) Parsons, Z. D.; Gates, K. S.

Method. Enzymol. 2013, 528, 129; (b) Conte, M. L.; Carroll, K. S. J. Biol. Chem. 2013, 288,

26480.

8. Specific examples: (a) Dickinson, B. C.; Chang, C. J. Nat. Chem. Biol. 2011, 7, 504; (b)

Rhee, S. G. Science 2006, 312, 1882; (c) Tonks, N. K. Nat. Rev. Mol. Cell Biol. 2006, 7, 833; (d)

Truong, T. H.; Carroll, K. S. Biochemistry 2012, 51, 9954.

9. Tanner, J. J.; Parsons, Z. D.; Cummings, A. H.; Zhou, H.; Gates, K. A. Antioxid. Redox

Signal. 2011, 15, 77.

10. Lee, S. R.; Yang, K. S.; Kwon, J.; Lee, C.; Jeong, W.; Rhee, S. G. J. Biol. Chem. 2002, 277,

20336.

11. Salmeen, A.; Anderson, J. N.; Myers, M. P.; Meng, T. C.; Hinks, J. A.; Tonks, N. K.;

Barford, D. Nature 2003, 423, 769.

12. (a) Denu, J. M.; Tanner, K. G. Biochemistry 1998, 37, 5633; (b) Parsons, Z. D.; Gates, K. S.

Biochemistry 2013, 52, 6412; (c) Sivaramakrishnan, S.; Cummings, A. H.; Gates, K. S. Bioorg.

Med. Chem. Lett. 2010, 20, 444; (d) Sivaramakrishnan, S.; Keerthi, K.; Gates, K. S. J. Am.

Chem. Soc. 2005, 127, 10830; (e) Zhou, H.; Singh, H.; Parsons, Z. D.; Lewis, S. M.;

Bhattacharya, S.; Seiner, D. R.; LaButti, J. N.; Reilly, T. J.; Tanner, J. J.; Gates, K. S. J. Am.

Chem. Soc. 2011, 133, 15803; also see reference 9.

13. (a) Leonard, S. E.; Garcia, F. J.; Goodsell, D. S.; Carroll, K. S. Angew. Chem. Int. Ed. 2011,

50, 4423; (b) Gupta, V.; Carroll, K. S. BBA-Gen. Subjects 2014, 1840, 847.

14. Kulkarni, R. A.; Stanford, S. M.; Vellore, N. A.; Krishnamurthy, D.; Bliss, M. R.; Baron, R.;

Bottini, N.; Barrios, A. M. ChemMedChem 2013, 8, 1561.

15. Fuhrmann, J.; Subramanian, V.; Thompson, P. R. ACS Chem. Biol. 2013, 8, 2024.

16. Walters, W.; Namchuk, M. Nat. Rev. Drug Discov. 2003, 2, 259.

8

17. Denu, J.; Dixon, J. Curr. Opin. Chem. Biol. 1998, 2, 633.

18. Moretto, A. F. Kirincich, S. J.; Xu, W. X.; Smith, M. J.; Wan, Z. K.; Wilson, D. P.; Follows,

B. C.; Binnun, E.; Joseph-McCarthy, D.; Foreman, K.; Erbe, D. V.; Zhang, Y. L.; Tam, S. K.;

Tam, S. Y.; Lee, J. Bioorg. Med. Chem. 2006, 14, 2162.

19. (a) Coan, K. E. D.; Maltby, D. A.; Burlingame, A. L.; Shoichet, B. K. J. Med. Chem. 2009,

52, 2067; (b) Shoichet, B. J. Med. Chem. 2006, 49, 7274; (c) Shoichet, B. Drug Discov. Today

2006, 11, 607; (d) Feng, B. Y.; Shoichet, B. K. Nat. Protoc. 2006, 1, 550; (e) Seidler, J.;

McGovern, S. L.; Doman, T. N.; Shoichet, B. K. J. Med. Chem. 2003, 46, 4477; (f) McGovern,

S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. J. Med. Chem. 2003, 46, 4265; (g) McGovern, S.

L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712.

20. (a) Hajduk, P. J.; Greer, J. Nat. Rev. Drug Discov. 2007, 6, 211; (b) Carr, R. A. E; Congreve,

M.; Murray, C. W.; Rees, D. C. Drug Discov. Today 2005, 10, 987; (c) Erlanson, D. A.;

McDowell, R. S.; O’Brien, T. J. Med. Chem., 2004, 47, 3463.

21. (a) Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Hutchins, C. W.; Zhao, H.;

Lubben, T. H.; Ballaron, S. J.; Haasch, D. L.; Kaszubska, W.; Rondinone, C. M.; Trevillyan, J.

M.; Jirousek M. R. J. Med. Chem. 2003, 46, 4232; (b) Szczepankiewicz, B. G.; Liu, G.; Hajduk,

P. J.; Abad-Zapatero, C.; Pei, Z.; Xin, Z.; Lubben, T. H.; Trevillyan, J. M.; Stashko, M. A.;

Ballaron, S. J.; Liang, H.; Huang, F.; Hutchins, C. W.; Fesik, S. W.; Jirousek, M. R. J. Am.

Chem. Soc. 2003, 125, 4087; (c) Shuker, S. B.; Hajduk, P. J.; Meadows, R. P.; Fesik, S. W.

Science, 1996, 274, 1531.

22. (a) Lepre, C. A.; Moore, J. M.; Peng, J. W. Chem. Rev. 2004, 104, 3641; (b) Meyer, B.;

Peters, T. Angew. Chem. Int. Ed. 2003, 42, 864.

23. Hartshorn, M. J.; Murray, C. W.; Cleasby, A.; Fredrickson, M.; Tickle, I. J.; Jhoti, H. J.

Med. Chem. 2005, 48, 403.

24. (a) Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman, J. A. J. Am. Chem. Soc.

2007, 129, 9613; (b) Rawls, K. A.; Lang, T. P.; Takeuchi, J.; Imamura, S.; Baguley, T. D.;

Grundner, C.; Alber, T.; Ellman, J. A. Bioorg. Med. Chem. Lett. 2009, 19, 6851; (c) Baguley, T.

D.; Xu, H.-C.; Chatterjee, M.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. J. Med. Chem. 2013,

56, 7636.

25. (a) Elliott, T. S.; Slowey, A.; Ye, Y.; Conway, S. J. Med. Chem. Commun. 2012, 3, 735; (b)

Burke, T. R.; Kole, H. K.; Roller, P. P. Biochem. Biophys. Res. Commun. 1994, 204, 129; (c)

Combs, A. P.; Yue, E. W.; Bower, M.; Ala, P. J.; Wayland, B.; Douty, B.; Takvorian, A.; Polam,

P.; Wasserman, Z.; Zhu, W.; Crawley, M. L.; Pruitt, J.; Sparks, R.; Glass, B.; Modi, D.;

McLaughlin, E.; Bostrom, L.; Li, M.; Galya, L.; Blom, K.; Hillman, M.; Gonneville, L.; Reid, B.

G.; Wei, M.; Becker-Pasha, M.; Klabe, R.; Huber, R.; Li, Y.; Hollis, G.; Burn, T. C.; Wynn, R.;

Liu, P.; Metcalf, B. J. Med. Chem. 2005, 48, 6544; also see references 12a (J. Med. Chem. 2003,

46, 4232) and 15c (J. Med. Chem. 2013, 56, 7636).

26. Webb, M. R. Proc. Natl. Acad. Sci. U. S. A. 1992, 89, 4884.

27. Montalibet, J.; Skorey, K. I.; Kennedy, B. P. Methods 2005, 35, 2.

9

Chapter 2. Identification of inhibitors of the Mycobacterium tuberculosis phosphatase PtpA

Abstract: This chapter focuses on the identification of phosphatase inhibitors of Mycobacterium

tuberculosis protein tyrosine phosphatase PtpA. Inhibitors incorporating the well established

non-hydrolyzable phosphate mimic, the difluoromethylenephosphonic acid (DFMP), were

explored, resulting in low micromolar inhibitors of PtpA. The basic scaffold of the inhibitors was

identified through the use of the SAS fragment based method by assaying an O-aryl substrate

phosphate library that had previously been generated within the Ellman group. The most potent

inhibitor was also shown to be selective for PtpA over a variety of human phosphatases as well

as Mycobacterium tuberculosis protein tyrosine phosphatase PtpB. This inhibitor represents a

chemical tool that can be used in conjunction with PtpB selective inhibitors described previously

within the Ellman group to further probe the roles of PtpA and PtpB in tuberculosis infection.

The majority of this work has been published (Rawls, K. A.; Lang, P. T.; Takeuchi, J.; Imamura,

S.; Baguley, T. D.; Grundner, C.; Alber, T.; Ellman, J. A., Bioorg. Med. Chem. Lett. 2009, 19,

6851).

10

Authorship

This work was conducted in collaboration with Dr. Katherine Rawls, Dr. Jun Takeuchi, Dr.

Shinichi Imamura, Dr. P. Therese Lang, and Dr. Christoph Grundner. The inhibitor library was

made by Dr. Katherine Rawls, Dr. Jun Takeuchi, Dr. Shinichi Imamura and myself. Dr.

Grundner provided enzyme for the assays, Dr. Lang performed all modeling studies, and Dr.

Rawls and I performed all substrate and inhibitor assays.

Introduction

Tuberculosis (TB) is a chronic, infectious disease caused by Mycobacterium tuberculosis

(Mtb), and is second only to HIV/AIDS as the greatest killer worldwide due to a single infectious

agent. In 2012, 8.6 million people contracted TB, and the disease was responsible for 1.3 million

deaths.1 The current treatment of drug-sensitive strains requires 6–9 months to fully eradicate the

infection. Current frontline treatments2 are hindered by the requirement to penetrate the

unusually thick mycobacterial cell wall3 in order to be effective, and only one drug has been

approved for treatment of TB since 1960.1 Compounding the problem is the development of

drug-resistant strains, caused in large part by a lack of compliance with the lengthy treatment

regimen.1 New Mtb drugs that act on novel targets are needed to shorten treatment time and

address the emergence of antibiotic resistance.

Ever since the discovery of the Yersinia PTP YopH, many host-pathogen interactions have

been found to be dependent on pathogen-secreted phosphatases.4 Mtb encodes three secreted

phosphatases, two of which are protein tyrosine phosphatases, PtpA and PtpB, that are promising

new targets for TB drug development.5 Although genetic deletion of ptpA or ptpB does not affect

Mtb growth in culture,6 it has been shown that these secreted phosphatases not only shut down

critical host cellular processes, but also promote Mtb survival within host macrophages.7 In

particular, PtpA inhibits phagosome acidification and maturation by blocking the recruitment of

the vacuolar H+-ATPase.

7a-b Although not a classical drug target because it is not essential in

vitro, targeting the secreted PtpA in the host macrophage circumvents two central resistance

mechanisms of Mtb; i.e. poor drug permeability due to the Mtb cell wall,3 and pump-mediated

drug efflux.8

Targeting PtpA is an attractive strategy which, in theory, would reduce the ability of Mtb

bacteria to grow and survive in the infected host, thus reducing the lengthy treatment time. Very

few PtpA inhibitors have been reported in the literature thus far, and the novel inhibitors

described in this chapter represent some of the most potent and selective compounds reported to

date.5

Initial scaffold identification

Identification of inhibitors for PtpA began with screening of a previously synthesized 150-

member O-aryl phosphate library.9 On first inspection, it was apparent that ortho and meta

substitution on the substrate fragments was not tolerated as exemplified by substrates 2.01 and

2.02 (Figure 2.1). Importantly, there were several substrate fragment hits with higher affinity

than the simple phenyl phosphate 2.03. Due to its improved solubility over compound 2.07 and it

amenability to modification over 2.08, compound 2.06 was chosen as the initial hit for further

compound optimization.

11

Figure 2.1. Selected results from the initial O-aryl phosphate substrate screening against PtpA.

Conversion to inhibitors

Substrate scaffold 2.06 was converted into inhibitor analogs 2.09 and 2.1010

(Figure 2.2), but

only the non-hydrolyzable difluoromethylenephosphonic acid (DFMP) isostere11

(2.09) was

active against PtpA. Despite the dianionic character of the DFMP isostere, it has been shown to

be both cell permeable and orally bioavailable in animals.12

Compounds containing this isostere

can be easily prepared from advanced synthetic intermediates allowing for rapid analog synthesis

using modified literature procedures.13

Figure 2.2. Conversion of scaffold 2.06 to inhibitors using DFMP and isoxazolecarboxylic acid isosteres

and inhibitory activity against PtpA.

Inhibitor library synthesis and evaluation

Synthesis of benzamide DFMP inhibitors began with a three step sequence to afford

advanced intermediate acid 2.14 (Scheme 2.1).14

Benzyl protection of the commercially available

4-iodobenzoic acid 2.11, followed by a copper mediated organozinc addition with the

commercial diethyl(bromodifluoromethyl)phosphonate affords compound 2.13. Hydrogenolysis

of the benzyl group restores the acid functionality to achieve key intermediate 2.14. With the

acid in hand, a number of commercial amines containing a variety of functionality were

incorporated through amide bond formation, followed by deprotection of the diethyl phosphonate

to afford the desired inhibitors (2.16).

12

Scheme 2.1. Synthesis of DFMP benzamide inhibitors

Upon assaying the initial library of DFMP amide inhibitors against PtpA (Table 2.1),

benzanilide 2.09, the initial inhibitor derived from substrate 2.06 (Figure 2.1), was identified as

the most potent compound (Ki = 24.0 M). Alkyl substituted benzamide compounds 2.17–2.21

showed minimal activity in the assay. The only alkyl derivative that even showed modest activity

was compound 2.23 (Ki = 49.7 M), presumably due to the increased acidity of the NH group

when compared to the other alkyl analogs. Acid analog 2.25 (Figure 2.3) also showed poor

activity, indicating the need of an amide and/or aromatic functionality for activity. However, the

placement of the aromatic group also seems to be important as the N-benzyl substituted 2.24 (Ki

= 43.1 M) was half as potent as 2.09, and the N-phenethyl substituted 2.22, was even less

potent (Ki = 61.6 M).

Amide replacement analog synthesis and evaluation

To investigate the importance of the amide moiety and positioning, we synthesized several

amide replacement analogs (Scheme 2.2). We thought that the position of the amide as well as

the availability of a hydrogen bond donor and acceptor from the amide may play an important

role in binding affinity. We therefore thought to examine the inversion of the amide direction

(2.29), addition of a methylene linker (2.33), removal of the carbonyl (2.36), replacement of

nitrogen with other heteroatoms (2.39), methylation of the amide nitrogen (2.41) and the

replacement of the carbonyl with a sulfonamide group (2.45). Each analog was synthesized in a

fashion similar to the DFMP benzamide inhibitors, but with varying the coupling partners at each

stage of the synthesis. Reverse amide compound 2.29 was synthesized by starting with

commercially available 1-iodo-4-nitrobenzene 2.26 and coupling it with

diethyl(bromodifluoromethyl)phosphonate, followed by hydrogenation to afford amine 2.28,

amide coupling with benzoyl chloride and deprotection to arrive at 2.29. The extended

homoamide 2.33 could be achieved by coupling the commercially available acid chloride 2.30

with aniline followed by the same coupling and deprotection sequence. Amine analog 2.36 was

13

Table 2.1. Initial DFMP amide inhibitor screens against PtpAa

compound R Ki (M) compound R Ki (M)

2.09

24.0 2.21

72.4

2.17 >100 2.23 49.7

2.18

>100 2.24

43.1

2.19

>100 2.22

61.6

2.20

>100

aKi values were determined using at least two independent measurements.

Figure 2.3. Free acid containing DFMP inhibitor screening result against PtpA.

achieved through a reductive amination between 4-iodobenzaldehyde and aniline, followed by

the common coupling and deprotection sequence. Ether 2.39 was synthesized first with a

Mitsunobu reaction between 4-iodobenzyl alcohol and phenol, followed by the same coupling

and deprotection. The N-methylated derivative is synthesized following the previously described

amide coupling of acid 2.14 and N-methylaniline, followed by deprotection of the

diethylphosphonate. Finally, the sulfonamide derivative was synthesized by coupling

commercially available 4-iodobenzenesulfonyl chloride 2.42 with aniline, which is likewise

followed by the coupling and deprotection sequence.

Scheme 2.2. Synthesis of amide replacement analogs 2.29, 2.33, 2.36, 2.39, 2.41 and 2.45

14

15

Each of the amide analogs was tested for activity versus PtpA (Table 2.2), and it was found

that all amide replacements resulted in loss of activity. This could be due to a loss of hydrogen

bonding interactions (2.36, 2.39, 2.41), or to changes associated with the entropic energies of

binding due to an increase in the number of rotatable bonds (2.33, 2.36, 2.39). Interestingly, the

only analog in this series to show any activity was the reverse amide 2.29. It is expected that the

aryl ring would be similarly positioned and only the hydrogen bonding characteristics of the

molecule would be perturbed. These results confirmed that the benzanilide scaffold 2.09 was the

best scaffold to optimize in our further efforts.

Table 2.2. Evaluation of amide replacement analogs for PtpA inhibition

a

compound structure Ki (M) compound Structure Ki (M)

2.29

65.5 2.39

>100

2.33

>100 2.41

>100

2.36

>100 2.45

>100


Benzanilide scaffold optimization and evaluation

Because benzanilide 2.09 was identified as the most potent analog in the inhibition screens

against PtpA, and the amide moiety was shown to be important for potency, a focused

benzanilide library was synthesized in order to determine an SAR and to improve potency of the

compounds (Table 2.3). All focused library members were synthesized in the route discussed for

the benzamide library (Scheme 2.1. Synthesis of DFMP benzamide inhibitors) using

commercially available and diversely substituted anilines. Electron withdrawing groups

generally provided more potent compounds than compounds containing electron donating or

16

neutral substituents (data not shown). Additionally, substitution at the meta and para positions

(2.50–2.51, 2.52–2.55, 2.57–2.58) resulted in compounds with higher potency than substitution

at the ortho position (2.46–2.49). Combining the favorable substituents resulted in compounds

2.56 and 2.59–2.63 which all were more potent than the lead benzanilide 2.09 (24.0 ± 0.9 M).

The most potent of these compounds, 2.63, had a Ki of 1.4 ± 0.3 M.

Table 2.3. Focused benzanilide library screen against PtpA

a

cpd R Ki (M) cpd R Ki (M) cpd R Ki (M)

2.09

24.0 ± 0.9 2.52

22.7 ± 3.2 2.59

6.7 ± 0.4

2.46

>100 2.53

18.5 ± 2.5 2.60

6.0 ± 0.4

2.47

68.0 ± 6.0 2.54

18.2 ± 0.9 2.61

4.9 ± 1.7

2.48

54.8 ± 14.5 2.55

16.8 ± 7.0 2.62

3.3 ± 0.6

2.49

44.6 ± 7.4 2.56

11.4 ± 0.3 2.63

1.4 ± 0.3

2.50

41.8 ± 5.8 2.57

10.7 ± 1.2

2.51

34.9 ± 3.5 2.58

10.3 ± 1.0


In order to rule out non-specific aggregation-based inhibition,15

compound 2.63 was tested

with two different concentrations of enzyme (300 and 600 nM) and two different concentrations

of the detergent Triton X-100 (0.004% and 0.01%, Figure 2.4). The Ki values remained constant

(within experimental error) for each of these conditions, consistent with active site competitive

inhibition. Additionally, the inhibition curves were found to have a Hill coefficient of h = –1.0 ±

0.1, also indicating that the inhibitor was binding into a single enzyme site rather than non-

specific aggregation-based inhibition.

17

Figure 2.4. Ki values of inhibitor 2.63 at two concentrations of PtpA and two concentrations of Triton X-

100 detergent. Ki values were found to be independent of either parameter, indicating real, and not

aggregation-based, inhibition.

Inhibitor selectivity profile

Achieving inhibitor selectivity is a major challenge because of the high structural homology

between PTP active sites.16

Gratifyingly, compound 2.63 was found to be highly selective (>70-

fold) when tested against a panel of PTPs and DUSPs, including TC-Ptp, an essential human

phosphatase important for immune response17

(Table 2.4). This was not unexpected, as low

molecular weight PTPs share little structural homology with classical PTPs and DUSPs.18

However, compound 2.63 was also found to be 11-fold selective over the human low molecular

weight phosphatase, HCPtpA, which shows 38% sequence identity to Mtb PtpA.19

Finally,

compound 2.63 did not inhibit Mtb PtpB; this selectivity should enable the use of this inhibitor as

a tool compound in order to further study the biochemical role of PtpA.

Table 2.4. Selectivity profile of inhibitor 2.63 against a panel of human and Mtb PTPs

a

Mtb PTPs Human PTPs

PtpA PtpB PTP1B TC-Ptp VHR CD45 LAR HCPtpA

Ki (M) 1.4 ± 0.3 >100 >100 >100 >100 >100 >100 14.8 ± 1.9

selectivity -- >70 >70 >70 >70 >70 >70 11 aKi values were determined using at least two independent measurements.

18

Modeling studies

Finally, using AMBER 920

and DOCK 6.4,21

a molecular model of all PtpA inhibitors bound

in the active site of a previously published apo crystal structure of PtpA (PDB ID: 1U2P)19

was

generated (Figure 2.5). The published crystal structure of apo-PtpA was first relaxed using

molecular dynamics in AMBER, followed by docking the compounds into the PtpA active site

with DOCK 6.4. Each of the compounds, including the initial lead benzanilide 2.09 and

optimized inhibitor 2.63, docked such that the DFMP warhead was in direct contact with the

catalytic residues of the protein. Additionally, the scoring function of the docking program

ranked the compounds in the same general order observed experimentally (data not shown),

indicating that the model is reasonably accurate.

Each of the docked compounds exhibited significant hydrogen bonding interactions with

PtpA (Figure 2.5). Nine hydrogen bonds were found between compound 2.09 and PtpA active

site residues, versus seven for compound 2.63. This model of inhibitor-enzyme interactions also

predicted varying degrees of pi-stacking with Trp48, the effectiveness of which depended on the

orientation of the aryl ring and its resulting ability to overlap with the indole ring of Trp48

(Figure 2.5a-b). This binding mode was not completely unexpected, since Trp pi-stacking has

been previously observed in inhibitor-enzyme complexes.22

Further development of SAR around

the benzanilide scaffold could lead to compounds with improved affinity compared to

benzanilide 2.63. In particular, modifications to further improve potency could include

improving pi-stacking efficiency with Trp48, as well as introduction of functionality off of the

pendant anilide ring to extend into an adjacent unfilled enzyme pocket (Figure 2.5c).

Figure 2.5. Model of (a) parent benzanilide 2.09 and (b) optimized benzanilide 2.63 docked in the active

site of PtpA (PDB ID: 1U2P)19

using DOCK 6.4.21

Hydrogen bonds (green lines) between each inhibitor

and active site residues are shown. Trp48 is emphasized to show pi-stacking interactions with each

inhibitor. Also shown is the PtpA binding pocket with inhibitor 2.63-enzyme contact points shown in blue

(c). The arrow indicates the position of an unfilled enzyme pocket adjacent to the docked inhibitor.23

Conclusions

In conclusion, we have identified inhibitors with single-digit micromolar affinity for PtpA

based on the benzanilide scaffold 2.09. SAR optimization resulted in compound 2.63, which,

nearly five years after publication of this work, still represents the most potent and selective

PtpA inhibitor in the literature to date.5

Compound 2.63 was found to be over 70-fold selective

for PtpA over a panel of human PTPs and DUSPs, and 11-fold selective over the highly

19

homologous human HCPtpA, which shows 38% sequence identity to Mtb PtpA. Molecular

modeling highlighted the importance of pi-stacking with Trp48, and hydrogen bonding with

active-site residues. Finally, 2.63, was found to be selective over Mtb PtpB, which allows it to be

a valuable tool compound, along with the PtpB selective inhibitor previously reported by our

group,9 in order to probe the biochemical roles of the Mtb PTPs.

Experimental

General synthetic methods

Unless otherwise noted, all reagents were obtained from commercial suppliers and used

without further purification. Tetrahydrofuran (THF), dichloromethane (CH2Cl2), toluene, and

diethyl ether (Et2O) were dried over alumina under a nitrogen atmosphere. Solvents used for

reactions set up in a nitrogen-filled inert atmosphere box, including THF and toluene, were

additionally degassed with three consecutive freeze pump thaw cycles and stored over 3Å

molecular sieves. Methanol was dried over calcium hydride under a nitrogen atmosphere. All

reactions, unless otherwise stated, were performed under inert atmosphere using syringe,

cannula, and Schlenk techniques, or set up in a nitrogen-filled inert atmosphere box, with flame

or oven-dried glassware. All 1H,

13C,

19F, and

31P NMR spectra were measured with a Bruker

DRX-500, AVB-400, AVQ-400 or AV-300 spectrometer. NMR chemical shifts are reported in

ppm relative to 1,2-difluorobenzene (–138.9) for 19

F NMR and trimethylphosphate (3.0) for 31

P

NMR. Mass spectrometry (HRMS) was carried out by the University of California, Berkeley

Mass Spectrometry Facility.

Synthesis and analytical data for DFMP inhibitors

Synthesis and analytical data for benzamide inhibitor 2.63

Compound 2.13. Compound 2.13 was synthesized according to modified literature

procedures.14

A solution of diethyl(bromodifluoromethyl)phosphonate (13.80 g, 44.0 mmol) in

DMA (20 mL) in a flame-dried 50 mL flask under an N2 atmosphere was slowly added to a

stirred suspension of activated Zn dust (2.88 g, 44.0 mmol) in DMA (20 mL) in a flame-dried

250 mL flask under an N2 atmosphere at 60 °C via cannula addition. After addition was

complete, the resulting mixture was sonicated at ambient temperature for 3 h, followed by

addition of CuBr (6.31 g, 44.0 mmol) in one portion. A solution of benzyl 4-iodobenzoate 2.1214

(5.41 g, 16.0 mmol) in DMA (5 mL) was added dropwise, and the resulting mixture was stirred

for 38 h at ambient temperature. The mixture was diluted with water (50 mL) and Et2O (50 mL),

and was then passed through Celite. The organic layer was separated, washed with brine (1 x 100

mL), dried over anhydrous MgSO4, and filtered. The solvent was removed under reduced

pressure to afford crude product, which was then purified via column chromatography to yield

20

2.13 as a colorless oil (4.68 g, 73% yield). 1H NMR (400 MHz, CDCl3): δ 8.16 (d, J = 8.0 Hz,

2H), 7.70 (d, J = 8.0 Hz, 2H), 7.46–7.36 (m, 5H), 5.39 (s, 2H), 4.26–4.11 (m, 4H), 1.30 (t, J = 7.2 Hz, 6H);

19F NMR (376 MHz, CDCl3): δ –108.59 (d, JPF = 109 Hz);

31P NMR (162 MHz,

CDCl3): δ 5.76 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C19H22F2O5P, 399.1172;

found, 399.1177.

Compound 2.14. Compound 2.14 was synthesized by modified literature procedures.

14 A

solution of 2.13 (2.50 g, 6.28 mmol) in MeOH (5 mL) was added to 10% Pd/C (835 mg, ca. 50%

wet) in MeOH (30 mL). The reaction mixture was stirred for 16 h under H2 atmosphere. The

catalyst was then removed by filtration through Celite, and the solvent was removed under

reduced pressure to give crude 2.14. The crude product was purified by recrystallization from

EtOAc/hexanes to yield 2.14 as a white powder (3.67 g, 86% yield). 1H NMR (400 MHz,

CD3OD): δ 8.15 (d, J = 8.0 Hz, 2H), 7.70 (d, J = 8.0 Hz, 2H), 4.26–4.13 (m, 4H), 1.31 (t, J = 7.2

Hz, 6H); 19

F NMR (376 MHz, CD3OD): δ –111.51 (d, JPF = 109 Hz); 31

P NMR (162 MHz,

CD3OD): δ 5.73 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C12H15F2O5PNa,

331.0523; found, 331.0531.

Compound 2.64. To a solution of 2.14 (98 mg, 0.32 mmol) in dry CH2Cl2 (3 mL) in a flame-

dried 10 mL flask under an N2 atmosphere was added oxalyl chloride (55 L, 0.64 mmol) and a

catalytic amount of DMF. The reaction mixture was stirred for 1 h at ambient temperature,

followed by removal of the solvent under reduced pressure, and drying under high vacuum. The

resulting acid chloride was dissolved in dry CH2Cl2 (2 mL) under an N2 atmosphere and slowly

added to a solution of 4-bromo-3,5-bis(trifluoromethyl)aniline (114 mg, 0.37 mmol) and

triethylamine (67 L, 0.48 mmol) in dry CH2Cl2 (3 mL) in a flame-dried 25 mL flask under an

N2 atmosphere. The reaction mixture was stirred at ambient temperature for 18 h. The solvent

was evaporated under reduced pressure to afford crude 2.64. The crude product was purified via

column chromatography to give 2.64 as a white solid (96 mg, 50% yield). 1H NMR (400 MHz,

CD3OD): δ 9.07 (br s, 1H), 8.40 (s, 2H), 7.92 (d, J = 8.2 Hz, 2H), 7.65 (d, J = 7.8 Hz, 2H), 4.29–

4.16 (m, 4H), 1.33 (t, J = 7.1 Hz, 6H); 19

F NMR (376 MHz, CD3OD): δ –63.47, –108.27 (d, JPF

= 114 Hz); 31

P NMR (162 MHz, CD3OD): δ 4.86 (t, JPF = 114 Hz). MS-ESI (m/z): [M+H]+

calcd. for C20H18BrF8NO4P, 596.99; found, 598.0 and 599.0.

21

Inhibitor 2.63. To a stirred solution of 2.64 (35 mg, 0.06 mmol) in CHCl3 (3 mL) was added

TMSI (33 L, 0.22 mmol). The mixture was stirred for 3 h at ambient temperature. Volatiles

were removed under reduced pressure and the residue was dissolved in MeOH (3 mL) and stirred

at ambient temperature for 18 h. The solvent was removed under reduced pressure to give crude

2.63. The crude product was purified by recrystallization from EtOAc/hexanes to yield 2.63 as a white powder (13 mg, 41% yield).

1H NMR (400 MHz, CD3OD): δ 8.54 (s, 2H), 8.06 (d, J = 8.0

Hz, 2H), 7.77 (d, J = 8.0 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ –64.40, –112.46 (d, JPF =

109 Hz); 31

P NMR (162 MHz, CD3OD): δ 2.55 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+

calcd. for C16H10[79]

BrF8NO4P, 541.9403; found, 541.9406.

General synthesis of other benzamide inhibitors

Benzamide inhibitors 2.09, 2.17–2.24, 2.46–2.62 and 2.41 were synthesized by following the

general procedures described for the synthesis of 2.63, using commercially available amines or

anilines.

Analytical data for benzamide inhibitors

Inhibitor 2.09.

1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.74 (d, J = 8.0 Hz,

2H), 7.69 (d, J = 8.0 Hz, 2H), 7.36 (t, J = 7.6 Hz, 2H), 7.15 (t, J = 7.6 Hz, 1H); 19

F NMR (376 MHz, CD3OD): δ –112.39 (d, JPF = 109 Hz);

31P NMR (162 MHz, CD3OD): δ 2.65 (t, JPF = 109

Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C14H12F2NO4PNa, 350.0370; found, 350.0366.

Inhibitor 2.17.

1H NMR (400 MHz, CD3OD): δ 7.89 (d, 2H, J = 8.1 Hz), 7.68 (d, 2H, J = 8.1

Hz), 3.09 (s, 3H); 19


P NMR (162

MHz, CD3OD): δ 4.34 (br t, JPF = 110 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C9H11O4NF2P,

266.0388; found, 266.0385.

22

Inhibitor 2.18.


Hz), 3.20 (d, 2H, J = 6.9 Hz), 1.93 (nonet, 1H, J = 6.9 Hz), 0.96 (d, 6H, J = 6.9 Hz); 19


31P NMR (162 MHz, CD3OD): δ 4.37 (br t,

JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C12H17O4NF2P, 308.0858; found, 308.0852.

Inhibitor 2.19.


Hz), 3.22 (d, 2H, J = 6.9 Hz), 3.77 (br m, 4H), 3.71–3.62 (br m, 2H), 1.34–1.18 (br m, 3H), 1.02 (br m, 2H);

19F NMR (376 MHz, CD3OD): δ –110.65 (d, JPF = 109 Hz);

31P NMR (162 MHz,

CD3OD): δ 4.63 (br m). HRMS-ESI (m/z): [M+H]+ calcd. for C15H21O4NF2P, 348.1171; found,

348.1166.

Inhibitor 2.20.


Hz), 4.13 (m, 1H), 4.03 (quintet, 2H, J = 7.1 Hz), 3.43 (br m, 2H), 3.12 (br m, 2H), 2.17 (br m, 2H), 3.79 (br m, 2H), 1.24 (t, 3H, J = 7.1 Hz);

19F NMR (376 MHz, CD3OD): δ –108.82 (d, JPF =

99 Hz); 31

P NMR (162 MHz, CD3OD): δ 3.20 (br t, JPF = 99 Hz). HRMS-ESI (m/z): [M+H]+

calcd. for C15H22O4N2F2P, 363.1280; found, 363.1273.

Inhibitor 2.21.


Hz), 3.88–3.84 (m, 1H), 1.95 (br m, 2H), 3.81 (br m, 2H), 3.68 (br m, 1H), 3.45–1.29 (br m, 4H),

1.27–1.19 (br m, 1H); 19


P NMR

(162 MHz, CD3OD): δ 4.72 (br m). HRMS-ESI (m/z): [M+H]+ calcd. for C14H19O4NF2P,

334.1014; found, 334.1020.

23

Inhibitor 2.22.


Hz), 7.34–7.17 (m, 5H), 3.60 (t, 2H, J = 7.4 Hz), 2.91 (t, 2H, J = 7.4 Hz); 19


31P NMR (162 MHz, CD3OD): δ 4.49 (br m). HRMS-ESI

(m/z): [M+H]+ calcd. for C16H17O4NF2P, 356.0858; found, 356.0865.

Inhibitor 2.23.


Hz), 4.10 (q, 2H, JHF = 9.2 Hz); 19

F NMR (376 MHz, CD3OD): δ –72.92 (t, JFH = 9.4 Hz),

–110.78 (d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 4.48 (br m). HRMS-ESI (m/z): [M–

H]– calcd. for C10H8O4NF5P, 332.0117; found, 332.0105.

Inhibitor 2.24.


Hz), 7.36–7.22 (m, 5H), 4.58 (s, 2H); 19

F NMR (376 MHz, CD3OD): δ –110.57 (d, JPF = 109

Hz); 31

P NMR (162 MHz, CD3OD): δ 4.09 (br t, JPF = 109 Hz). HRMS-ESI (m/z): [M+H]+ calcd.

for C15H15O4NF2P, 342.0701; found, 342.0712.

Inhibitor 2.46.

1H NMR (400 MHz, CD3OD): δ 8.03 (d, J = 7.1 Hz, 2H), 7.80–7.71 (m, 3H),

7.29–7.14 (m, 3H); 19

F NMR (376 MHz, CD3OD): δ –110.70 (d, JPF = 111 Hz), –124.20; 31

P

NMR (162 MHz, CD3OD): δ 4.27 (t, JPF = 102 Hz). MS-ESI (m/z): [2M+H]+ calcd. for

C28H23F6N2O8P2, 691.08; found, 691.0.

24

Inhibitor 2.47.

1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.89 (t, J = 2.0 Hz,

1H), 7.75 (d, J = 8.0 Hz, 2H), 7.60 (dd, J = 8.0, 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 8.0, 2.0 Hz, 1H);


31P NMR (162

MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-EI (m/z): [M+H]+ calcd. for

C14H12[35]

ClF2NO4P, 362.0160; found, 362.0169.

Inhibitor 2.48.

1H NMR (400 MHz, CD3OD): δ 8.03 (d, J = 8.0 Hz, 2H), 7.78–7.71 (m, 4H),

7.62–7.60 (m, 1H), 7.54–7.52 (m, 1H); 19


= 109 Hz); 31


calcd. for C15H12F5NO4P, 396.0424; found, 396.0424.

Inhibitor 2.49.


2H), 7.72–7.69 (m, 2H), 7.43 (t, J = 7.6 Hz, 1H), 7.21 (d, J = 7.6 Hz, 1H); 19


31P NMR (162 MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz).

HRMS-FAB (m/z): [M+H]+ calcd. for C14H12

[79]BrF2NO4P, 405.9655; found, 405.9666.

Inhibitor 2.50.


2H), 7.69–7.66 (m, 1H), 7.45 (d, J = 8.0 Hz, 1H), 7.38–7.32 (m, 1H), 6.88 (t, J = 8.0 Hz, 1H); 19

F NMR (376 MHz, CD3OD): δ –112.41 (d, JPF = 109 Hz), –115.00; 31

P NMR (162 MHz,

CD3OD): δ 2.80 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C14H10F3NO4P,

344.0300; found, 344.0292.

25

Inhibitor 2.51.

1H NMR (400 MHz, CD3OD): δ 8.02 (d, J = 8.0 Hz, 2H), 7.89 (t, J = 2.0 Hz,

1H), 7.75 (d, J = 8.0 Hz, 2H), 7.60 (dd, J = 8.0, 2.0 Hz, 1H), 7.34 (d, J = 8.0 Hz, 1H), 7.15 (dd, J = 8.0, 2.0 Hz, 1H);


31P NMR (162

MHz, CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-EI (m/z): [M+H]+ calcd. for

C14H12[35]

ClF2NO4P, 362.0160; found, 362.0169.

Inhibitor 2.52.


2H), 7.70 (dd, J = 8.8, 4.8 Hz, 2H), 7.10 (t, J = 8.8 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ

–112.38 (d, JPF = 109 Hz), –120.72; 31

P NMR (162 MHz, CD3OD): δ 2.71 (t, JPF = 109 Hz).

HRMS-FAB (m/z): [M+Na]+ calcd. for C14H10F3NO4PNa, 390.0095; found, 390.0102.

Inhibitor 2.53.


2H), 7.76 (d, J = 8.0 Hz, 2H), 7.66 (d, J = 8.0 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ –64.41,

–112.37 (d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 2.59 (t, JPF = 109 Hz). HRMS-FAB

(m/z): [M+H]+ calcd. for C15H12F5NO4P, 396.0424; found, 396.0423.

Inhibitor 2.54.

1H NMR (400 MHz, CD3OD): δ 8.03–8.00 (m, 4H), 7.75 (d, J = 8.0 Hz, 1H),

7.67–7.65 (m, 1H), 7.29–7.26 (m, 2H); 19

F NMR (376 MHz, CD3OD): δ –112.27 (d, JPF = 109

Hz); 31


calcd.

for C14H12[79]

BrF2NO4P, 405.9655; found, 405.9650.

26

Inhibitor 2.55.


2H), 7.72 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.0 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ –112.42

(d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 2.60 (t, JPF = 109 Hz). HRMS-ESI (m/z):

[M+2Na–H]+ calcd. for C14H10

[35]ClF2NO4PNa2, 405.9799; found, 405.9815.

Inhibitor 2.56.

1H NMR (400 MHz, CD3OD): δ 8.14 (m, 1H), 8.05–7.92 (m, 3H), 7.82 (s,

1H), 7.75 (m, 1H), 7.31 (m, 1H). MS-ESI (m/z): [M–H]– calcd. for C15H9F6NO4P, 413.0252;

found 413.0.

Inhibitor 2.57.

1H NMR (400 MHz, CD3OD): δ 8.17 (s, 1H), 8.04 (d, J = 8.0 Hz, 2H), 7.95

(d, J = 8.0 Hz, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.56 (t, J = 8.0 Hz, 1H), 7.44 (d, J = 8.0 Hz, 1H); 19

F NMR (376 MHz, CD3OD): δ –65.08, –112.39 (d, JPF = 109 Hz); 31

P NMR (162 MHz,

CD3OD): δ 2.64 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+Na]+ calcd. for C15H11F5NO4PNa,

418.0244; found, 418.0243.

Inhibitor 2.58.


2H), 7.67 (d, J = 8.8 Hz, 2H), 7.50 (d, J = 8.8 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ –112.39

(d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 2.74 (t, JPF = 109 Hz). HRMS-FAB (m/z):

[M+H]+ calcd. for C14H12

[79]BrF2NO4P, 405.9655; found, 405.9650.

27

Inhibitor 2.59.


2H), 7.75 (d, J = 8.0 Hz, 2H), 7.64 (dd, J = 8.8, 2.4 Hz, 1H), 7.50 (d, J = 8.8 Hz, 1H); 19


31P NMR δ (162 MHz, CD3OD): 2.56 (t, JPF =

109 Hz); HRMS-FAB (m/z): [M+H]+ calcd. for C14H10

[35]Cl2F2NO4P, 394.9692; found,

394.9685.

Inhibitor 2.60.


(d, J = 8.8 Hz, 1H), 7.75 (d, J = 8.0 Hz, 2H), 7.59 (d, J = 8.8 Hz, 1H); 19

F NMR (376 MHz, CD3OD): δ –64.96, –112.45 (d, JPF = 109 Hz);

31P NMR (162 MHz, CD3OD): δ 2.62 (t, JPF =

109 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for C15H11

[35]ClF5NO4P, 430.0034; found, 430.0029.

Inhibitor 2.61.


(d, J = 7.2 Hz, 1H), 7.79–7.74 (m, 3H); 19


= 109 Hz); 31


calcd. for C15H11[79]

BrF5NO4P, 473.9529; found, 473.9542.

Inhibitor 2.62.


(d, J = 8.0 Hz, 2H), 7.71 (s, 1H); 19

F NMR (376 MHz, CD3OD): δ –65.38, –112.46 (d, JPF = 109

Hz); 31

P NMR (162 MHz, CD3OD): δ 2.54 (t, JPF = 109 Hz). HRMS-FAB (m/z): [M+H]+ calcd.

for C16H11F8NO4P, 464.0297; found, 464.0301.

28

Compound 2.41.

1H NMR (400 MHz, CD3OD): δ 7.43 (d, J = 8.0 Hz, 2H), 7.39 (d, J = 8.0

Hz, 1H), 7.28 (t, J = 7.6 Hz, 2H), 7.22–7.15 (m, 3H), 3.47 (s, 3H); 19


31P NMR (162 MHz, CD3OD): δ 2.62 (t, JPF = 109 Hz).

HRMS-FAB (m/z): [M+2Na–H]+ calcd. for C15H13F2NO4PNa2, 386.0346; found, 386.0354.

General synthesis of other (non-amide) DFMP inhibitors

Free carboxylic acid containing 2.25 was synthesized by subjecting intermediate 2.14 to the

procedure described for deprotection of 2.64. Inhibitors 2.29, 2.33, 2.36, 2.39 and 2.45 were

synthesized as described in the text (vide supra). All compounds were purified either by

automated reversed-phase C18 column chromatography (linear gradient of 5% to 95%

acetonitrile in water with 0.1% trifluoroacetic acid buffer), or by recrystallization.

Analytical data for other DFMP inhibitors

Inhibitor 2.25.

1H NMR (CD3OD): δ 7.93 (d, 2H, J = 7.4 Hz), 7.72 (d, 2H, J = 7.4 Hz);

19F

NMR (CD3OD): δ –110.78 (d, JPF = 111 Hz); 31

PNMR (CD3OD): δ 3.38 (br m). HRMS-ESI

(m/z): [M–H]– calcd. for C8H6O5F2P, 250.9926; found, 250.9916.

Inhibitor 2.29.


2H), 7.61–7.49 (m, 5H); 19


P NMR

(162 MHz, CD3OD): δ 3.31 (t, JPF = 113 Hz). HRMS-FAB (m/z): [M+H]+ calcd. for

C14H13F2NO4P, 328.0550; found, 328.0545.

Inhibitor 2.33.

1H NMR (400 MHz, CD3OD): δ 3.74 (2H, s), 7.09 (1H, t, J = 7.4 Hz), 7.30

(2H, t, J = 8.0 Hz), 7.47 (2H, d, J = 8.0 Hz), 7.52–7.56 (2H, m), 7.59 (2H, d, J = 8.0 Hz); 19


31P NMR (162 MHz, CD3OD): δ 4.75

29

(t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C15H15F2NO4P, 342.0701; found,

342.0715.

Inhibitor 2.36.

1H NMR (400 MHz, CD3OD): δ 7.48 (ABq, J = 8.4 Hz, 4H), 7.10 (t, J = 7.8

Hz, 2H), 6.68 (d, J = 8.0 Hz, 2H), 6.64 (t, J = 7.4 Hz, 1H), 4.34 (s, 2H); 19

F NMR (376 MHz,

CD3OD): δ –106.70 (d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 2.78 (t, JPF = 108 Hz).

HRMS-ESI (m/z): [M+H]+ calcd. for C14H15F2NO3P, 314.0758; found, 314.0766.

Inhibitor 2.39.


2H), 7.24 (t, J = 7.9 Hz, 2H), 6.93–6.90 (m, 3H), 5.08 (s, 2H); 19

F NMR (376 MHz, CD3OD): δ

–107.45 (d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): δ 7.01 (br t). HRMS-ESI (m/z):

[M+Na]+ calcd. for C14H13F2O4PNa, 337.0412; found, 337.0415.

Inhibitor 2.45.

1H NMR (400 MHz, CD3OD): δ 7.04–7.13 (m, 3H), 7.19–7.25 (m, 2H), 7.70

(d, J = 8.4 Hz, 2H), 7.85 (d, J = 8.4 Hz, 2H); 19

F NMR (376 MHz, CD3OD): δ –110.80 (d, JPF =

106.0 Hz). MS-ESI (m/z): [M+H]+ calcd. for C13H13F2NO5PS, 364.0142; found, 364.0.

Expression and purification of PtpA

The gene for PtpA was amplified from Mtb genomic DNA and cloned into the pET28b

vector (Novagen). Protein was expressed in BL21(DH3) cells (Invitrogen). Transformed bacteria

were grown to an OD600 of 0.8 in terrific broth and protein expression was induced by the

addition of 100 M isopropyl -D-1-thiogalactopyranoside. After 18 h of expression at 20 °C,

cells were harvested and resuspended in buffer A (20 mM Tris pH 7.5, 50 mM NaCl), and

protease inhibitor AEBSF. Cell suspensions were sonicated, the lysates centrifuged for 1 h at

15,000 × g, and the cleared lysate loaded onto a metal affinity column. After elution with an

imidazole gradient, the His6 tag was cleaved by treatment with 1:1,000 (w/w) trypsin for 10 min

at ambient temperature. The protein was further purified by gel filtration on a S75 Superdex

column in buffer A and concentrated to 2.3 mg/mL for phosphatase assays.

30

Assay procedures

Determination of substrate Km

96-well plates were used with reaction volumes of 100 L per well. 30 L of water was

added to each well, followed by 5 L of buffer (stock solution: 1.0 M Tris-HCl, 20 mM MgCl2,

pH 7.5), 40 L of 2-amino-6-mercapto-7-methylpurine riboside (MESG) solution (stock: 1 mM,

400 M in assay), and 10 L of purine nucleotide phosphorylase (PNP) solution (stock: 0.01

U/mL). 5 L of the appropriate substrate dilution, serially diluted 2.5-fold for a total of 8

different concentrations in DMSO, was then added to the wells, and the plate was covered and

incubated at 37 °C for 5 min in a UV-Vis plate reader. The coupled assay was started by addition

of 10 L of a 1 M stock of PtpA (100 nM in assay), and the reaction progress was monitored at

360 nm at 37 °C. The initial rate data collected was used for Michaelis-Menton kinetic analysis,

where the Km could be obtained. Km and Vmax were determined using nonlinear regression

analysis on the substrate-velocity data with the equation v = Vmax*[S]/(Km+[S]).

Determination of inhibitor Ki

96-well plates were used with reaction volumes of 100 L per well. 45 L of water was

added to each well, followed by 20 L of sodium citrate buffer (stock solution: 100 mM sodium

citrate, pH 6.2, 0.02% Triton X-100), 5 L of 20 mM EDTA stock solution (1 mM in assay), 5

L of 20 mM DTT stock solution (1 mM in assay), and 10 L of 1 M PtpA stock solution (100

nM in assay). Then 5 L of the appropriate inhibitor stock solutions, serially diluted 2-fold for a

total of 10 different concentrations in DMSO, was added to the wells, and the plate was covered

and incubated for 5 min at 37 °C in a UV-Vis plate reader. The reaction was started by addition

of 10 L of 2 mM pNPP substrate stock (200 M in assay), and reaction progress was monitored

at 405 nm with continued incubation at 37 °C. The initial rate data collected was used for the

determination of Ki values. The kinetic values were obtained from nonlinear regression of

substrate-velocity curves in the presence of various concentrations of inhibitor using the equation

v = Vmax*[S]/(Km(1+[I]/Ki)+[S]).

Inhibitor-PtpA modeling

Receptor relaxation

The X-ray crystal structure of PtpA (PDB ID 1U2P) was used for all modeling studies.19

To

allow the protein to relax, a short molecular dynamics simulation was run in AMBER 9.0 using

the ff03 force field.20,24

The structure was prepared by removing crystallographic waters and

adding an 8.0 Å octagon of TIP3P water and sodium ions using the LEaP accessory.24

The

system was minimized, slowly melted to 300K, and allowed to equilibrate for 25 ps. The

simulation was continued for an additional 150 ps and selected as the final snapshot for docking.

Active site identification

To identify where inhibitors might bind, water molecules were removed from the structure

produced by the molecular dynamics simulation, and protein surface invaginations were

31

identified using spheres generated by the DOCK accessory SPHGEN.25

The putative binding site

was characterized by selecting all spheres within a 12 Å radius of the chlorine atom bound to the

active-site cysteine nucleophile in the X-ray structure.

Receptor preparation for docking

An octagon of TIP3P waters was built around the receptor using the Chimera AmberTools

module, followed by removal of any water molecules >5 Å from any receptor atom, resulting in

approximately two shells of water molecules.26

All waters <3 Å from the active site spheres

described above were then removed. The Chimera (version 1.3) Dock Prep module was used to

complete the receptor preparation.27

To account for the receptor contribution to the score during

DOCKing, grids were precomputed to store the van der Waals and electrostatic values for the

receptor using the DOCK accessory GRID.27a

Compound preparation for docking

To validate observed structure-activity relationships, structures 2.09 and 2.63 were docked

onto PtpA. Each compound was drawn and converted to SMILE strings using the JME molecular

editor.28

The SMILE strings were used to create rotamer ensembles of three-dimensional

structures in OMEGA.21

All generated conformations were kept for docking, resulting in an

average of 11 conformations per compound. Each conformation was protonated and assigned

AM1-BCC charges using the Chimera (version 1.3) AddH and AddCharge modules.29

Docking procedure

The compound conformations were docked using Grid Score in DOCK 6.4 using default

parameters.21

Each conformation was then rescored and ranked using the PB/SA score. The top

scoring conformation for each compound was used for comparisons. For compounds 2.09 and

2.63, molecular dynamics simulations were also performed on the top-scoring conformation to

explore the validity of the docked poses. The same protocol described in the Receptor Relaxation

section was used. Both simulations were equilibrated after 100 ps, and the simulations were run

for a further 50 ps. The snapshot with the ligand heavy atom RMSD closest to the docked pose

from the final 50 ps of each simulation was selected for analysis.

References

1. World Health Organization, Global Tuberculosis Report 2013:

http://apps.who.int/iris/bitstream/10665/91355/1/9789241564656_eng.pdf

2. Janin, Y. L. Bioorg. Med. Chem. 2007, 15, 2479.

3. Brennan, P.; Nikaido, H. Annu. Rev. Biochem. 1995, 64, 29.

4. (a) Black, D. S.; Baliska, J. B EMBO J. 1997, 16, 2730; (b) Persson, C.; Carballeira, N.;

Wolf‐Watz, H.; Fällman, M. EMBO J. 1997, 16, 2307; (c) Heneberg, P. Curr. Med. Chem. 2012,

19, 1530.

5. Wong, D.; Chao, J. D.; Av-Gay, Y. Trends Microbiol. 2013, 21, 100.

6. Grundner, C.; Cox, J. S.; Alber, T. FEMS Microbiol. Lett. 2008, 287, 181.

32

7. (a) Bach, H.; Papavinasasundaram, K. G.; Wong, D.; Hmama, Z.; Av-Gay Y. Cell Host

Microbe 2008, 3, 316; (b) Wong, D.; Bach, H.; Sun, J.; Hmama, Z.; Av-Gay, Y. Proc. Natl.

Acad. Sci. USA 2011, 108, 19371; (c) Beresford, N. J.; Mulhearn, D.; Szczepankiewicz, B.; Liu,

G.; Johnson, M. E.; Fordham-Skelton, A.; Abad-Zapatero, C.; Cavet, J. S.; Tabernero, L. J.

Antimicrob. Chemother. 2009, 63, 928; (d) Zhou, B.; He, Y.; Zhang, X.; Xu, J.; Luo, Y.; Wang,

Y.; Franzblau, S. G.; Yang, Z.; Chan, R. J.; Liu, Y.; Zheng, J.; Zhang Z.-Y. Proc. Natl. Acad.

Sci. USA 2010, 107, 4573.

8. Louw, G. E.; Warren, R. M.; Gey van Pittius, N. C.; McEvoy, C. R. E.; Van Helden, P. D.;

Victor, T. C. Antimicrob. Agents Chemother. 2009, 53, 3181.

9. Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman, J. A. J. Am. Chem. Soc.

2007, 129, 9613.

10. Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Hutchins, C. W.; Zhao, H.;


M.; Jirousek M. R. J. Med. Chem. 2003, 46, 4232.

11. (a) Sun, J.-P.; Fedorov, A. A.; Lee, S.-Y.; Guo, X.-L.; Shen, K.; Lawrence, D. S.; Almo, S.

C.; Zhang, Z.-Y. J. Biol. Chem. 2003, 278, 12406; (b) Shen, K.; Keng, Y.-F.; Wu, L.; Guo, X.-

L.; Lawrence, D. S.; Zhang, Z.-Y. J. Biol. Chem. 2001, 276, 47311; (c) Yao, Z.-J.; Ye, B.; Wu,

X.-W.; Wang, S.; Wu, L.; Zhang, Z.-Y.; Burke Jr., T. R. Bioorg. Med. Chem. 1998, 6, 1799; (d)

Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.; Zhang Z.-Y. Proc. Natl.

Acad. Sci. USA 1997, 94,

12. Han, Y.; Belley, M.; Bayly, C. I.; Colucci, J.; Dufresne, C.; Giroux, A.; Lau, C. K.; Leblanc,

Y.; McKay, D.; Therien, M.; Wilson, M.-C.; Skorey, K.; Chan, C.-C.; Scapin, G.; Kennedy, B.

P. Bioorg. Med. Chem. Lett. 2008, 18, 3200.

13. (a) Matsui, T.; Takahashi, S.; Matsunaga, N.; Nakamura, K.; Omawari, N.; Sakai, M.;

Kamoshima, W.; Terai, K.; Ohno, H.; Obata, T.; Nakai, H.; Toda, M. Bioorg. Med. Chem. Lett.

2002, 10, 3807; (b) Yokomatsu, T.; Murano, T.; Suemune, K.; Shibuya, S. Tetrahedron 1997,

53, 815.

14. Li, Z.; Yeo, S. L.; Pallen, C. J.; Ganesan, A. Bioorg. Med. Chem. Lett. 1998, 8, 2443.

15. (a) Coan, K. E. D.; Maltby, D. A.; Burlingame, A. L.; Shoichet, B. K. J. Med. Chem. 2009,

52, 2067; (b) Shoichet, B. J. Med. Chem. 2006, 49, 7274; (c) Shoichet, B. Drug Discov. Today

2006, 11, 607; (d) Feng, B. Y.; Shoichet, B. K. Nat. Protoc. 2006, 1, 550; (e) Seidler, J.;

McGovern, S. L.; Doman, T. N.; Shoichet, B. K. J. Med. Chem. 2003, 46, 4477; (f) McGovern,

S. L.; Helfand, B. T.; Feng, B.; Shoichet, B. K. J. Med. Chem. 2003, 46, 4265; (g) McGovern, S.

L.; Caselli, E.; Grigorieff, N.; Shoichet, B. K. J. Med. Chem. 2002, 45, 1712.

16. (a) Lee, H.; Xie, L.; Luo, Y.; Lee, S.-Y.; Lawrence, D. S.; Wang, X. B.; Sotgia, F.; Lisanti,

M. P.; Zhang, Z.-Y. Biochemistry 2006, 45, 234; (b) Moller, N. P. H.; Andersen, H. S.; Jeppesen,

C. B.; Iversen, L. F. Handb. Exp. Pharmacol. 2005, 167, 215; see also reference 5a.

17. Tiganis, T.; Kemp, B. E.; Tonks, N. K. J. Biol. Chem. 1999, 274, 27768.

18. Ramponi, G.; Stefani, M. Int. J. Biochem. Cell Biol. 1997, 29, 279.

19. Madhurantakam, C.; Rajakumara, E.; Mazumdar, P. A.; Saha, B.; Mitra, D.; Wiker, H. G.;

Sankaranarayanan, R.; Das, A. K. J. Bacteriol. 2005, 187, 2175.

20. Case, D. A., et al. AMBER 9, University of California, San Francisco, 2006.

21. Lang, P. T.; Brozell, S. R.; Mukherjee, S.; Pettersen, E. F.; Meng, E. C.; Thomas, V.; Rizzo,

R. C.; Case, D. A.; James, T. L.; Kuntz, I. D. RNA 2009, 15, 1219.

33

22. (a) Zsila, F.; Iwao, Y. BBA-General Subjects 2007, 1770, 797; (b) Zsila, F.; Matsunaga, H.;

Bikádi, Z.; Haginaka, J. BBA-General Subjects 2006, 1760, 1248; (c) Schuettelkopf, A. W.;

Andersen, O. A.; Rao, F. V.; Allwood, M.; Lloyd, C.; Eggleston, I. M.; van Aalten, D. M. F. J.

Biol. Chem. 2006, 281, 27278; (d) Rao, F. V.; Andersen, O. A.; Vora, K. A.; DeMartino, J. A.;

van Aalten, D. M. F. Chem. Biol. 2005, 12, 973; (e) Kryger, G.; Silman, I.; Sussman, J. L.

Structure 1999, 7, 297.

23. Molecular graphics images were produced using the UCSF Chimera package from the

Resource for Biocomputing, Visualization, and Informatics at the University of California, San

Francisco (supported by NIH P41 RR-01081).

24. Duan, Y.; Wu, C.; Chowdhury, S.; Lee, M. C.; Xiong, G.; Zhang, W.; Yang, R.; Cieplak, P.;

Luo, R.; Lee, T.; Caldwell, J.; Wang, J.; Kollman, P. J. Comput. Chem. 2003, 24, 1999.

25. Lee, M. C.; Duan, Y. Proteins 2004, 55, 620.

26. Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R. W.; Klein, M. L. J. Chem.

Phys. 1983, 79, 926.

27. (a) Pettersen, E. F.; Goddard, T. D.; Huang, C. C.; Couch, G. S.; Greenblatt, D. M.; Meng, E.

C.; Ferrin, T. E. J. Comput. Chem. 2004, 25, 1605; (b) Kuntz, I. D.; Blaney, J. M.; Oatley, S. J.;

Langridge, R.; Ferrin, T. E. J. Mol. Biol. 1982, 161, 269.

28. Moustakas, D. T.; Lang, P. T.; Pegg, S.; Pettersen, E.; Kuntz, I. D.; Brooijmans, N.; Rizzo,

R. C. J. Comput.-Aided Mol. Des. 2006, 20, 601.

29. Shoichet, B. K.; Bodian, D. L.; Kuntz, I. D. J. Comput. Chem. 1992, 13, 380.

34

35

Chapter 3. Fragment-based identification of inhibitors of striatal-enriched protein tyrosine

phosphatase

Abstract: In this chapter, the brain-specific phosphatase, STriatal-Enriched protein tyrosine

Phosphatase (STEP) is introduced as a therapeutic target for neurodegenerative diseases. The

substrate-based fragment screening approach from chapters 1 and 2 was used to identify lead

compounds for STEP inhibition. Through this fragment-based approach, we were able to identify

many low molecular weight (<450 Da), nonpeptidic, single-digit micromolar mechanism-based

STEP inhibitors with greater than 20-fold selectivity across multiple human PTPs and DUSPs.

Additionally, significant levels of STEP inhibition in rat cortical neurons were also observed for

our most potent inhibitors. The majority of this work has been published as a full article

(Baguley, T. D.; Xu, H.-C.; Chatterjee, M.; Nairn, A. C.; Lombroso, P. J.; Ellman, J. A. J. Med.

Chem. 2013, 56, 7636).

36

Authorship

This work was conducted in collaboration with Dr. Hai-Chao Xu and Dr. Manavi Chatterjee.

Dr. Xu and I synthesized the inhibitor library. I expressed and purified the enzyme STEP for

assays. The substrate and inhibitor assays were performed by Dr. Xu and myself. Dr. Chatterjee

performed all cell based assays. Blood-brain barrier permeability assays were performed by Pion,

Inc. (Billerica, MA).

Introduction

Synaptic connections provide the physical basis for communication within the brain, and

synaptic plasticity, the ability for synapses to strengthen or weaken between neurons as a result

of molecular signals, is critical to maintaining proper cognitive function. Therefore, disruptions

in synaptic function can lead to impairments in cognition. Synaptic dysregulation has been

implicated in a range of neurodegenerative diseases,1 including Alzheimer’s disease (AD),

2

schizophrenia,3 depression,

4 fragile X syndrome,

5 and drug addiction.

6

One protein that has been implicated in the dysregulation of synaptic plasticity is STriatal-

Enriched protein tyrosine Phosphatase (STEP), which is found in striatum, hippocampus, cortex,

and related regions of the brain. High levels of STEP activity result in the dephosphorylation and

inactivation of several neuronal signaling molecules including extracellular signal-regulated

kinases 1 and 2 (ERK1/2),7 proline-rich tyrosine kinase 2 (Pyk2),

8 mitogen activated protein

kinase p38,9 and the GluN2B subunit of the N-methyl-D-aspartate receptor (NMDAR).

10,11

Dephosphorylation of the kinases inactivates them, while dephosphorylation of GluN2B results

in internalization of NMDA receptors. To test the hypothesis that overexpression of STEP might

contribute to cognitive deficits in AD mouse models, STEP levels were reduced genetically in

AD mice. Progeny null for STEP exhibited significant cognitive improvements and increased

receptor levels on synaptic membranes.11

These results support STEP as a potential target for

drug discovery for the treatment of AD.

As described in chapter 1, the Ellman group has developed a substrate-based fragment-

screening approach to identify small molecule inhibitors of phosphatases termed substrate

activity screening (SAS). In chapter 2, this method was used to identify inhibitors of PtpA from

Mtb. Prior to our work on STEP, there was a single reported ancillary example of a STEP

inhibitor having modest activity and without selectivity or cell data.12

In this chapter, I will

discuss the application of the SAS method to identify low molecular weight (<450 Da)

nonpeptidic STEP inhibitors with single-digit micromolar inhibition, 20-fold selectivity over

multiple human PTPs and DUSPs, and significant activity in rat cortical neurons.

Inhibitor scaffold identification

Identification of inhibitors for STEP began with screening the same previously synthesized

O-aryl phosphate library as was used in chapter 2.13

Initially, there were several promising

fragment substrates (Figure 3.1). Substitution ortho to the phosphate was not well tolerated, but

meta and para substitution seemed to be allowed. Of note, fragment substrates 3.06–3.10 had

much improved Km values relative to the protected phosphotyrosine derivative 3.04, which more

closely resembles the natural phosphotyrosine in PTP substrates.

37

Figure 3.1. Selected initial substrate hits obtained against STEP.

Because of the ease of synthesis of amide containing aryl phosphates (chapter 2, Scheme

2.1), we were first drawn to scaffold 3.09. However, after much effort to optimize the scaffold

through substitution of the aniline ring as was carried out for Mtb PtpA as described in chapter 2,

synthesizing and testing > 50 novel substrates, no substrate gave a more potent lead than the

completely unsubstituted aniline (Figure 3.2). At this point, we decided to identify a non-

hydrolyzable phosphate mimetic that would provide inhibitory activity versus STEP (Figure 3.3).

Upon assaying the DFMP14

(3.12), isothiazolidinone15

(3.13) and isoxazole carboxylic acid16

(3.14) inhibitors, it was apparent that only the DFMP pharmacophore was active for inhibition.

Figure 3.2. Attempted optimization of substrate fragment 3.09 was unsuccessful.

38

Figure 3.3. Conversion of substrate 3.09 into inhibitors by replacement of the phosphate with non-

hydrolyzable mimics demonstrates that DFMP is the only active pharmacophore.

It was at this point that we went back to our original substrate screening results (Figure 3.1).

We then identified 3.06 and 3.08 for further optimization because the biphenyl scaffold has been

regarded as a “privileged scaffold” with druglike properties and because analog preparation is

straightforward using cross-coupling methodology.17

Inhibitors 3.15 and 3.16 (Figure 3.4) were

prepared by replacing the phosphate group with the DFMP isostere, and, gratifyingly, the Ki

values of the inhibitors correlated reasonably well with the Km values of their corresponding

substrates,18

which is desired for substrate analog inhibitors.

Figure 3.4. DFMP inhibitors 3.15 and 3.16 based on privileged substrate scaffolds 3.06 and 3.08.

The biaryl DFMP inhibitors could be conveniently prepared by relying on the Suzuki-

Miyaura cross-coupling reaction (Scheme 3.1). The commercial availability of many diverse

arylboronic acids and boronic esters (ArBLn) enabled rapid access to an initial set of biaryl

inhibitors from the known aryl bromides 3.21 and 3.22.13b,18b

However, for the further

optimization of the inhibitor structures we needed to prepare the requisite more complex

arylboronic acid inputs, which will be the bulk of the synthetic discussion in this chapter.

39

Scheme 3.1. Inhibitor synthesis through a Suzuki-Miyaura cross-coupling strategy

Optimization of inhibitor 3.15

After an initial inhibitor library screen for the inhibitors based on the 3-biphenyl scaffold

(Table 3.1), some trends started to become apparent. Although alkyl substitution at the para

position of the distal ring was beneficial for inhibition (3.39), any substitution larger than a

methyl group (3.38), or inclusion of electron deficient halogens (3.30, 3.36) or electron donating

heteroatoms (3.24–3.25, 3.27–3.28) was met with decreased potency. Likewise, alkyl

substitution at the meta position also led to an increase in potency of the inhibitors, with the -

branched and more bulky isopropyl group (3.43) outperforming the methyl group (3.42).

However, replacement of the alkyl group at the meta position with electron-rich (3.26, 3.29,

3.35, 3.40) or electron-withdrawing (3.32, 3.34) groups was not as beneficial as alkyl

substitution (3.42–3.43). The presence of a hydroxyl group at the ortho position was also

beneficial to the potency of the inhibitors (3.41), but not at the meta (3.29) or para (3.27)

position. This could be due to H-bond donor ability, as the methylated version (3.31) and

halogens (3.33, 3.37) that can accept H-bonds through their lone pairs, all suffered in potency

relative to the free hydroxyl.

At this point, we wanted to explore the additive effects of the ortho hydroxyl (3.41) and meta

alkyl (3.43) groups. Because these more complicated aryl boronic acids were not commercially

available, they had to be synthesized. When the desired alkyl phenols were commercially

available, the 2-hydroxyphenylboronic acids were synthesized by a two-step sequence (Scheme

3.2). First, selective bromination of the phenol (3.44) was accomplished by NBS in the presence

of a catalytic amount of N,N-diisopropylamine.19

The bromo compound was then dilithiated and

treated with trimethyl borate to yield the boronic acids (3.46) after aqueous workup.20

In most

cases, the boronic acid was used crude in the Suzuki-Miyaura cross-coupling reaction, and

although by NMR analysis it appeared that there were multiple species in equilibrium, likely

anhydrides between the hydroxyls on both the aryl rings and the boron centers, by LCMS the

crude products contained a single peak corresponding to the boronic acid mass in all cases.

When the desired alkyl phenols were not commercially available, they had to be synthesized

prior to borylation (Scheme 3.3). This was accomplished through a dilithiation of ortho-

bromophenol and quenching with an aldehyde or ketone. The resulting benzyl alcohols (3.48)

were then treated with triethylsilane (Et3SiH) under acidic conditions to reductively eliminate the

alcohol to yield the requisite 2-alkyl phenols (3.49). While these alkylphenols could be subjected

to the bromination-borylation sequence in Scheme 3.2, in order to increase the efficiency of the

sequence, they were instead subjected to an iridium-catalyzed one-pot silyl-directed ortho-

borylation disclosed by Hartwig and coworkers. Subsequent treatment with potassium hydrogen

difluoride (KHF2) then provided the trifluoroborates 3.50.21

40

Table 3.1. Representative examples from initial 3-biphenyl DFMP inhibitor screening against STEPa

cpd Ar Ki (M) cpd Ar Ki (M) cpd Ar Ki (M)

3.15

337 ± 60 3.30

468 ± 39 3.37

233 ± 23

3.24

>500 3.31

271 ± 17 3.38

222 ± 1

3.25

>500 3.32

267 ± 17 3.39

205 ± 1

3.26

>500 3.33

260 ± 32 3.40

177 ± 8

3.27

>500 3.34

259 ± 53 3.41

168 ± 17

3.28

>500 3.35

240 ± 13 3.42

137 ± 8

3.29

486 ± 2 3.36

238 ± 14 3.43

81 ± 2


Scheme 3.2. Synthesis of arylboronic acids from 2-alkylphenols

Once synthesized, the inhibitors were screened against STEP (Table 3.2). Combining the

meta isopropyl group and the ortho hydroxyl group in a 2,3-orientation (3.51) led to an increase

in potency, while the 2,5-substitution was not advantageous (3.52). We next investigated the

effect of altering the meta alkyl group (3.53–3.60) on inhibitory potency. The ethyl group (3.53)

resulted in 2-fold reduction in inhibitory potency, while the tert-butyl group provided a modest

41

Scheme 3.3. Synthesis of aryltrifluoroborates from 2-bromophenol

enhancement (3.54). Branching at the -carbon was confirmed to be important when comparing

the n-propyl and isobutyl compounds (3.55–3.56). Knowing that -branching was important, we

discovered that a more significant increase in potency was achieved by introducing cycloalkyl

groups (3.57–3.60), with cyclohexyl (3.59) providing the optimal ring size (Ki = 20 ± 3 M).

Finally, introduction of the -hydroxymethylphosphonic acid phosphate mimetic (3.61, Figure

3.5) in place of difluoromethylphosphonic acid resulted in an approximate 2-fold reduction in

potency.

Table 3.2. Screening of focused 3-biphenyl DFMP inhibitors against STEP

a


3.41

168 ± 17 3.53

124 ± 3 3.57

45 ± 2

3.43

81 ± 2 3.54

63 ± 1 3.58

25 ± 4

3.51

69 ± 9 3.55

70 ± 3 3.59

20 ± 3

3.52

95 ± 1 3.56

53 ± 1 3.60

62 ± 1


42

Figure 3.5. Replacement of the DFMP pharmacophore with an -hydroxyphosphonic acid was met with

diminished potency, despite the results from the 4-biphenyl scaffold (vide infra).

Optimization of inhibitor 3.16

A separate inhibitor library based on 4-biphenyl DFMP inhibitor 3.16 was synthesized

utilizing the Suzuki-Miyaura cross-coupling sequence (Scheme 3.1) and was screened for

activity against STEP (Table 3.3). Electron deficient (3.63, 3.65, 3.66), donating (3.67, 3.68) and

even alkyl (3.69, 3.70) groups all proved detrimental to inhibitor potency. Borrowing

information from compound 3.41, we next tried to introduce H-bond donors, and found that they

were detrimental at the ortho (3.62) and para (3.64) positions, but moderately increased inhibitor

potency when placed at the meta position (3.71–3.73), with benzyl alcohol 3.73 providing the

largest increase in potency. However, the greatest potency was observed for benzyl substitution

Table 3.3. Representative examples from initial 4-biphenyl DFMP inhibitor screening against STEP

a


3.16

120 ± 7 3.66

375 ± 30 3.71

113 ± 1

3.62

>500 3.67

231 ± 6 3.72

112 ± 9

3.63

>500 3.68

200 ± 1 3.73

88 ± 3

3.64

450 ± 183 3.69

185 ± 33 3.74

73 ± 3

3.65

400 ± 48 3.70

132 ± 3


43

at the meta position (3.74), which resulted in a near 2-fold enhancement over unsubstituted 3.16.

With this information in hand, we turned toward synthesizing inhibitors with varying

substituents on the aryl ring of the benzyl substituent of inhibitor 3.74. The boronic acid coupling

partners needed for the inhibitor syntheses (Scheme 3.4) were generated first by the addition of

in situ generated 3-bromolithium to a variety of aldehydes (3.75) to yield diarylmethanols, 3.76.

After reductive elimination of the benzyl alcohol, the aryl bromide was converted to the pinacol

boronic ester 3.78 with a Miyaura borylation.22

Scheme 3.4. Synthesis of diarylmethane-based boronic esters for the synthesis of 3.74 analogs

After screening compounds with a variety of substituents on the benzyl ring, it was

discovered that electron deficient halogens were most beneficial for binding (Figure 3.6). Other

substituents were tried (alkyl, alkoxy, etc.; data not shown), but the halogens showed the greatest

increase in potency relative to compound 3.74 (Ki = 73 ± 3 M). The ideal substitution pattern

was the 3,4-dichlorobenzyl inhibitor 3.81 (Ki = 9.6 ± 0.6 M), but the compound had very

limited solubility in aqueous media. The hydroxyl group, which was previously observed to be

well tolerated at the benzylic position (see 3.73, Table 3.3), increased the solubility of the

inhibitor with only a moderate reduction in potency (3.82). Replacing the DFMP pharmacophore

with an -hydroxyphosphonic acid group maintained the desired potency (3.83) as well as

increased the aqueous solubility.

Figure 3.6. Selected screening results versus STEP from the initial focused benzyl inhibitor library.

The -hydroxymethylphosphonic acid inhibitor 3.83 was also prepared by a Suzuki-Miyaura

cross-coupling reaction (Scheme 3.5). Ketone 3.86 was obtained by cross-coupling

ketophosphonic acid 3.8423

with arylboronic acid derivative 3.85, and sodium borohydride

reduction then led to the final -hydroxymethylphosphonic acid compound.

44

Scheme 3.5. Synthesis of -hydroxyphosphonic acid inhibitor 3.83

We next explored the effect of combining the hydroxyl groups in inhibitors 3.82 and 3.83 by

the preparation of the four possible stereoisomers, 3.97–3.100 (Scheme 3.6). The four

stereoisomeric inhibitors were prepared from the enantiomerically pure -hydroxymethyl-

phosphonic acids 3.90 and 3.91 and the enantiomerically enriched boronic esters 3.95 and 3.96

by Suzuki-Miyaura cross-coupling. The synthesis of the enantiomerically pure phosphonic acids

started with the addition of tris-[(1S,2R,5S)-menth-2-yl]phosphite to 4-bromobenzaldehyde to

give a mixture of diastereomers 3.88 and 3.89, which were separated by recrystallization

according to literature procedures for analogous compounds.24

Removal of the menthyl groups

by treatment with TMSCl and NaI afforded phosphonic acids 3.90 and 3.91. The absolute

stereochemistry of the -hydroxyphosphonic acids was confirmed by chemical correlation

through hydrodebromination of 3.91 to the corresponding -hydroxyphenylmethylphosphonic

acid, for which the absolute configuration had previously been determined.25

The catalytic

asymmetric addition of a 3,4-dichlorophenylzinc reagent to 3-bromobenzaldehyde using each

enantiomer of 3-exo-(morpholino)isoborneol (MIB)26

gave the enantiomerically enriched

diarylmethanols 3.93 and 3.94 in 90% ee. Subsequent Miyaura borylation led to the boronic

esters 3.95 and 3.96.22

45

Scheme 3.6. Synthesis of the four stereoisomeric -hydroxymethylphosphonic acid inhibitors 3.97–3.100

With the diastereomerically pure inhibitors in hand, we were able to test their potency against

STEP (Figure 3.7) While the potency is not affected by the stereochemistry of the distal

biarylmethanol (3.97 versus 3.98), the (S)-configuration at the -hydroxyphosphonic acid is

crucial for inhibitory activity. Further evidence of the importance of this particular orientation of

the hydroxyl group was that the (R)-configuration (3.99 and 3.100) provided no better inhibition

than when the hydroxyl was removed completely (3.101).

Figure 3.7. Screening results for diastereomerically pure inhibitors 3.97–3.100 against STEP.

46

Inhibitor selectivity profile

As mentioned in chapter 1, achieving selectivity for PTP inhibition is very challenging due to

the high structural homology among PTP active sites.27

Additionally, knowing the selectivity

needed for therapeutic relevance is very challenging because the therapeutic window is

dependent on a variety of independent factors that are difficult or impossible to predict including

severity of side effects and redundancy in signaling cascades. A drug compound may be

sufficient with 10-fold selectivity, or may require 1000-fold selectivity, but when dealing with

challenging targets such as PTPs, this information is often not known without some first-in-class

inhibitors to use as tool compounds. The best inhibitors from the two biaryl series, compounds

3.59, 3.97, and 3.98, were tested against a panel of PTPs and DUSPs (Table 3.4). Notably, our

most potent inhibitors from the 1,4-biphenyl series 3.97 and 3.98 displayed greater than 20-fold

selectivity against all phosphatases tested, whereas compound 3.59 from the 1,3-biphenyl series

displayed more modest selectivity. These were the first small molecule inhibitors of STEP

reported in the literature with any selectivity data.

Table 3.4. Selectivity profile of inhibitors 3.59, 3.97 and 3.98 against a panel of human PTPs

a

STEP TC-Ptp CD45 LAR MKP5

3.59 Ki, M 20 ± 3 71 ± 2 >500 >900 150 ± 20

selectivity -- 3.6 >25 >45 7.5

3.97 Ki, M 8.9 ± 1.2 164 ± 9 340 ± 40 360 ± 30 340 ± 45

selectivity -- 18 38 40 41

3.98 Ki, M 7.8 ± 0.7 170 ± 20 270 ± 20 390 ± 110 >500

selectivity -- 22 35 50 >65 aKi values were determined using at least two independent measurements.

STEP inhibition in neuronal cultures

The most selective inhibitors, 3.97 and 3.98, were also evaluated for their ability to inhibit

STEP in rat cortical neurons by monitoring the phosphorylation levels of the known STEP

substrates GluN2B, Pyk2 and ERK1/2.7,8,10,11

Clear increases in the phosphorylation levels of

each of the substrates were observed for inhibitor 3.97 with more modest effects observed for

3.98 (Figure 3.8) indicating that the deposphorylation activity of STEP was inhibited. Selectivity

for non-STEP PTP substrate proteins was not assessed in this study.

Blood-brain barrier permeability

Given the promising cell data, the best compound, 3.97, was evaluated for its ability to

passively permeate the blood-brain barrier (BBB) using the well validated parallel artificial

membrane permeability assay (PAMPA) technique.28

In this assay, the compound of interest is

added to a donor well and its ability to passively cross a membrane mimic is assessed through

detection of the compound in an acceptor well which is separated from the donor well only by

the membrane mimic. Although the compound can cross cell membranes, as evidenced in the

above cell data, compound 3.97 did not possess the ability to passively cross the BBB in this

model system (Table 3.5). BBB-PAMPA studies were conducted by Pion, Inc (Billercia, MA) at

47

pH 7.4. The refined effective permeability (Pe) value obtained (average of triplicates) is

summarized in the table along with results for internal highly and low permeable standards,

propranolol and atenolol respectively.

Figure 3.8. Rat cortical neurons were treated with vehicle or 3.97 (a) or 3.98 (b) (concentrations of 0.1, 1 or 10 µM) for 1 h and analyzed by Western blotting. (*p < 0.05; **p < 0.01; ***p < 0.001 one-way

ANOVA, Dunnett’s post hoc). Data represent the phospho-signal normalized to the total substrate protein

signal and GAPDH as a basic expression level control + s.e.m. (n = 3–5 each group).

Table 3.5. BBB-PAMPA assay results for compound 3.97

Compound Avg. Pe

(10–6

cm/s)a Avg. %R

b Avg. logPe Domain, nm

Inhibitor 3.97 <0.1 4 ± 0 260–350

propranolol 68 ± 5 48 ± 3 –4.17 ± 0.03 250–498

atenolol <0.4 1 ± 1 250–498

aeffective permeability measured in assay; results indicated with a “<” sign mean no quantifiable UV

signal was detected in the acceptor compartments. bmembrane retention.

With these results, BBB permeability remains a real challenge for future work. The highly

polar nature of the -hydroxyphosphonic acid motif is likely to be one of the key factors limiting

BBB permeability. Replacement of this motif with less polar, non-hydrolyzable phosphate

mimetics might overcome this problem.29

Alternatively, effective prodrug strategies have also

been developed to mask polar functionality in order to enable BBB permeability.30

Conclusions

Although high STEP activity has been observed in many neuropsychiatric disorders, such as

Alzheimer’s disease, prior to this work there had been no selective and potent inhibitors reported

for potential treatment and study of these diseases. The work in this chapter describes the first

dedicated effort for the identification of selective small-molecule mechanism-based inhibitors of

48

STEP. A library of low molecular weight O-aryl and -heteroaryl phosphate fragments were

screened, which identified both the 4- and the 3-biaryl inhibitors (3.16 and 3.15 respectively) as

promising templates for inhibitor development. SAR of the scaffolds was explored to identify

potent inhibitors from each series, with the most potent inhibitors (3.59, 3.97, and 3.98) all

showing promising selectivity over other human phosphatases tested. Importantly, the most

selective inhibitors 3.97 and 3.98 were able to inhibit STEP in rat cortical neurons as indicated

by the significant increase in phosphorylation levels of STEP substrates.

Experimental



without further purification. Tetrahydrofuran (THF), dioxane, CH2Cl2, and diethyl ether (Et2O)

were passed through a column of activated alumina (type A2, 12 × 32, Purify Co.) under

nitrogen pressure immediately prior to use. All 1H,

19F, and

31P NMR spectra were obtained at

ambient temperature on a Bruker AVB-400 or AVB-500 spectrometer. NMR chemical shifts are

reported in ppm relative to TMS (0.00), CHCl3 (7.26), acetone (2.05) or CH3OH (3.31) for 1H,

trifluoroacetic acid (−76.55) for 19

F, and H3PO4 (0.00) for 31

P. Mass spectrometry (HRMS, ESI)

are reported in m/z. Chromatography was performed either with SiliCycle SiliaFlash P60

230−400 mesh silica gel or by utilizing a Biotage SP1 flash purification system (Biotage model

SP1-B1A) or a Teledyne Isco CombiFlash Rf system. Reversed-phase purifications were

conducted with a Teledyne Isco CombiFlash Rf system equipped with HP C18 gold cartridges.

Product yields are not optimized. Enzymatic assays were carried out on a BioTek Synergy 2

multimode microplate reader. All of the tested substrates and inhibitors displayed ≥ 95% purity

as determined by HPLC or UPLC. BBB PAMPA studies were conducted by Pion, Inc. (Billerica,

MA).

Synthesis of and analytical data for biphenyl inhibitors

Compound 3.21. Iodotrimethylsilane (5.22 mL, 36.6 mmol, 2.2 equiv) was added to a stirred

solution of diethyl compound 3.19 (5.71 g, 16.6 mmol) (synthesized as per a previous report,18b

see Scheme 3.1 for more details) in CH2Cl2 (50 mL, 0.3 M). The reaction solution was stirred at

ambient temperature for 14 h. The volatile components were then removed by a rotary

evaporator under vacuum. Sodium hydroxide (664 mg, 16.6 mmol) in methanol (20 mL) was

added to the resulting residue, and this solution was stirred at ambient temperature for 1 h,

allowing the monosodium salt of the desired compound to precipitate from the solution. The

solvent was removed by filtration. LCMS showed the presence of an undesired byproduct in the

collected crude product; thus, it was purified via reversed-phase gradient column

chromatography (5−100% acetonitrile in water with 0.1% trifluoroacetic acid buffer). Volatile

components were removed, and the resulting water solution was lyophilized to afford the product

49

as an off-white powder (2.75 g, 9.44 mmol, 57%). 1H NMR (400 MHz, CD3OD): δ 7.74 (s, 1H),

7.67 (d, J = 8.0 Hz, 1H), 7.58 (d, J = 7.8 Hz, 1H), 7.41 (t, J = 7.9 Hz, 1H); 19

F NMR (376 MHz, CD3OD): δ −110.13 (d, JPF = 110 Hz);

31P NMR (162 MHz, CD3OD): δ 6.76 (br). HRMS-ESI

(m/z): [M−H]–

calcd. for C7H5[79]

BrF2O3P, 284.9133; found, 284.9129.

Compound 3.22. Compound 3.22 was synthesized similarly to 3.21, and is a known

compound. Analytical data matched the previous report.13b

Synthesis of and analytical data for DMFP inhibitors of the general form 3.23

Synthesis of biaryl inhibitors was accomplished through a Suzuki-Miyaura cross-coupling

from either compound 3.21 or 3.22. Two different procedures were employed.

General synthesis of inhibitors by Suzuki-Miyaura cross-coupling (A). A 1 dram oven-dried

vial was charged with a stir bar, the appropriate aryl bromide (1.0 equiv), organoboron species

(1.5 equiv), potassium carbonate (5 equiv), palladium(II) chloride or palladium(II) acetate (5 mol

%), and (2-biphenyl)dicyclohexylphosphine (10 mol %). After addition of all reagents, solvent

(4:1 dioxane/water) was added to the vial (0.25 M in aryl bromide). The vial was sealed with a

screw top containing a Teflon septum and placed in a preheated heating block at 80 °C, and the

mixture was vigorously stirred for 18 h. The vial was then removed from the heating block and

allowed to cool to ambient temperature, followed by addition of 0.3 volumes of 10 N HCl open

to air. The reaction mixture was diluted with 1 volume of water and 1 volume of methanol,

filtered through a Kimwipe, and purified by reversed-phase gradient column chromatography

(5−100% acetonitrile in water with 0.1% trifluoroacetic acid). Volatile components were

removed, and the resulting water solutions were lyophilized to afford the products.

General synthesis of inhibitors by Suzuki-Miyaura cross coupling (B). A 1 dram oven-dried

vial was charged with a stir bar, the appropriate aryl bromide (1.0 equiv), organoboron species

(1.5 equiv), sodium carbonate (6 equiv), and tetrakis(triphenylphosphine)palladium(0) (10 mol

%). After addition of all reagents, solvent (4:1:1 dimethoxyethane/ethanol/water) was added to

the vial (0.10−0.25 M in aryl bromide). The vial was sealed with a screw top containing a Teflon

septum and placed in a preheated heating block at 80 °C to stir for 4−18 h with vigorous stirring.

The vial was then removed from the heating block and allowed to cool to ambient temperature,

followed by addition of 0.3 volumes of 10 N HCl open to air. The reaction mixture was diluted

50

with 1 volume of water and 1 volume of methanol, filtered through a Kimwipe, and purified by

reversed-phase gradient column chromatography (5−100% acetonitrile in water with 0.1%

trifluoroacetic acid). Volatile components were removed, and the resulting water solutions were

lyophilized to afford the products.

Biphenyl inhibitors 3.15–3.16, 3.24–3.43, and 3.62–3.74 were synthesized following one of

the two general procedures described for the Suzuki-Miyaura cross-coupling (general procedure

A or general procedure B) from aryl bromide 3.21 or 3.22. Analytical data for inhibitors 3.15 and

3.16 match previous literature reports.31,13b

Analytical data for inhibitors 3.24–3.43, and 3.62–3.74

Inhibitor 3.24.

1H NMR (400 MHz, CD3OD): 7.69 (s, 1H), 7.59 (d, J = 6.8 Hz, 1H), 7.49–

7.36 (m, 4H), 6.88 (d, J = 8.7 Hz, 2H), 4.53 (hept, J = 6.0 Hz, 1H), 1.22 (d, J = 6.0 Hz, 6H); 19

F

NMR (376 MHz, CD3OD): –110.79 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.22

(t, JPF = 113 Hz). MS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.08; found, 341.1.

Inhibitor 3.25.

1H NMR (400 MHz, CD3OD): 7.92 (s, 1H), 7.65–7.60 (m, 6H), 7.45 (t, J =

7.8 Hz, 1H), 2.14 (s, 3H);19

F NMR (376 MHz, CD3OD): –108.45 (d, JPF = 98 Hz); 31

P NMR

(162 MHz, CD3OD): 6.02 (t, JPF = 98 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for

C15H15F2NO4P, 342.0701; found, 342.0697.

Inhibitor. 3.26.


7.39 (m, 4H), 7.34–7.24 (m, 2H), 2.05 (s, 3H); 19

F NMR (376 MHz, CD3OD): –110.90 (d, JPF

= 113 Hz); 31

P NMR (162 MHz, CD3OD): 6.95 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+

calcd. for C15H15F2NO4P, 342.0701; found, 342.0694.

51

Inhibitor 3.27.

1H NMR (400 MHz, CD3OD): 7.80 (s, 1H), 7.73–7.66 (m, 1H), 7.56–7.47

(m, 4H), 6.90 (d, J = 8.6 Hz, 2H); 19


P NMR (162 MHz, CD3OD): 7.12 (t, JPF = 114 Hz). MS-ESI (m/z): [M−H]− calcd. for

C13H10F2O4P, 299.03; found, 299.1.

Inhibitor 3.28.

1H NMR (400 MHz, CD3OD): 7.70 (s, 1H), 7.60 (d, J = 6.6 Hz, 1H), 7.48

(d, J = 8.7 Hz, 2H), 7.45–7.38 (m, 2 H), 6.92 (d, J = 8.7 Hz, 2H), 3.74 (s, 3H); 19

F NMR (376

MHz, CD3OD): –110.83 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.12 (t, JPF = 113

Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C14H14F2O4P, 313.0592; found, 313.0573.

Inhibitor 3.29.


7.51 (m, 2H), 7.30 (t, J = 7.9 Hz, 1H), 7.12 (d, J = 7.8 Hz, 1H), 7.08 (t, J = 2.1 Hz, 1H), 6.83 (dd,

J = 8.1, 2.2 Hz, 1H); 19F NMR (376 MHz, CD3OD): –110.90 (d, JPF = 113 Hz); 31

P NMR (162

MHz, CD3OD): 7.03 (t, JPF = 113 Hz). MS-ESI (m/z): [M−H]− calcd. for C13H10F2O4P, 299.03;

found, 299.1.

52

Inhibitor 3.30.


(dd, J = 8.7, 5.3 Hz, 2H), 7.51–7.40 (m, 2H), 7.09 (t, J = 8.8 Hz, 2H); 19

F NMR (376 MHz,

CD3OD): –110.95 (d, JPF = 112 Hz), –117.32; 31

P NMR (162 MHz, CD3OD): 6.97 (t, JPF =

112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11F3O3P, 303.0392; found, 303.0387.

Inhibitor 3.31.


(d, J = 8.0 Hz, 1H), 7.47 (t, J = 7.7 Hz, 1H), 7.35 (d, J = 7.9 Hz, 1H), 7.30 (d, J = 9.1 Hz, 1H),

7.08 (d, J = 8.2 Hz, 1H), 7.02 (t, J = 7.4 Hz, 1H), 3.79 (s, 3H); 19

F NMR (376 MHz, CD3OD):

−109.54 (d, JPF = 114 Hz); 31

P NMR (162 MHz, CD3OD): 7.18 (t, JPF = 114 Hz). HRMS-ESI

(m/z): [M−H]− calcd. for C14H12F2O4P, 313.0447; found, 313.0445.

Inhibitor 3.32.

1H NMR (400 MHz, CD3OD): 7.83–7.75 (m, 3H), 7.70 (d, J = 8.0 Hz, 1H),

7.62–7.48 (m, 4H); 19

F NMR (376 MHz, CD3OD): –64.07, –111.06 (d, JPF = 112 Hz); 31

P

NMR (162 MHz, CD3OD): 6.88 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for

C14H11F5O3P, 353.0360; found, 353.0332.

Inhibitor 3.33.


(d, J = 7.9 Hz, 1H), 7.51–7.35 (m, 2H), 7.34–7.25 (m, 1H), 7.20–7.14 (m, 1H), 7.14–7.07 (m,

53

1H); 19

F NMR (376 MHz, CD3OD): –110.92 (d, JPF = 113 Hz), –120.12; 31

P NMR (162 MHz,

CD3OD): 7.25 (br). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11F3O3P, 303.0392; found,

303.0384.

Inhibitor 3.34.


(s, 1H), 7.82 (d, J = 7.6 Hz, 1H), 7.76 (d, J = 7.8 Hz, 1H), 7.72–7.60 (m, 3H); 19

F NMR (376

MHz, CD3OD): –111.04 (d, JPF = 112 Hz); 31

P NMR (162 MHz, CD3OD): 6.81 (t, JPF = 112

Hz). MS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P, 308.03; found, 308.0.

Inhibitor 3.35.


(d, J = 7.8 Hz, 1H), 7.78 (t, J = 7.7 Hz, 1H), 7.61 (t, J = 7.9 Hz, 1H), 7.44 (d, J = 7.5 Hz, 1H),

7.40 (t, J = 1.9 Hz, 1H), 7.18 (dd, J = 8.3, 2.5 Hz, 1H), 4.08 (s, 3H); 19

F NMR (376 MHz,

CD3OD): –110.82 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.05 (t, JPF = 113 Hz).

HRMS-ESI (m/z): [M+H]+ calcd. for C14H14F2O4P, 313.0592; found, 313.0574.

Inhibitor 3.36.


7.57 (m, 4H), 7.50 (d, J = 8.5 Hz, 2H); 19

F NMR (376 MHz, CD3OD): –110.97 (d, JPF = 112

Hz); 31

P NMR (162 MHz, CD3OD): 6.89 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M+H]+ calcd.

for C13H11[35]

ClF2O3P, 319.0097; found, 319.0110.

54

Inhibitor 3.37.

1H NMR (400 MHz, CD3OD): 7.59–7.51 (m, 2H), 7.51–7.38 (m, 3H),

7.33–7.21 (m, 3H); 19


P NMR (162

MHz, CD3OD): 6.97 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M+H]+ calcd. for C13H11

[35]ClF2O3P,

319.0097; found, 319.0084.

Inhibitor 3.38.

1H NMR (400 MHz, CD3OD): 7.83 (s, 1H), 7.73 (d, J = 7.3 Hz, 1H),

7.60−7.48 (m, 4H), 7.33 (d, J = 8.3 Hz, 2H), 2.95 (hept, J = 6.9 Hz, 1H), 1.28 (d, J = 6.9 Hz,

6H); 19

F NMR (376 MHz, CD3OD): −109.67 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD):

7.11 (br t). HRMS-ESI (m/z): [M–H]– calcd. for C16H16F2O3P, 325.0811; found, 325.0816.

Inhibitor 3.39.

1H NMR (400 MHz, CD3OD): 7.82 (s, 1H), 7.72 (d, J = 7.2 Hz, 1H),

7.60−7.49, (m, 4H), 7.28 (d, J = 8.0 Hz, 2H), 2.38 (s, 3H); 19

F NMR (376 MHz, CD3OD):

−109.69 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.05 (t, JPF = 113 Hz). HRMS-ESI


Inhibitor 3.40.


7.50 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.20 (dd, J = 7.8, 1.3 Hz, 1H), 7.16 (t, J = 2.1 Hz, 1H),

6.94 (dd, J = 8.2, 2.4 Hz, 1H), 4.69 (hept, J = 6.1 Hz, 1H), 1.36 (d, J = 6.0 Hz, 6H); 19

F NMR

55

(376 MHz, CD3OD): –110.87 (d, JPF = 113 Hz); 31


113 Hz). MS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.08; found, 341.1.

Inhibitor 3.41.


(d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.27 (dd, J = 7.5, 1.7 Hz, 1H), 7.17 (td, J = 7.7, 1.6

Hz, 1H), 6.94−6.87 (m, 2H); 19


P

NMR (162 MHz, CD3OD): 7.24 (t, JPF = 114 Hz). HRMS-ESI (m/z): [M−H]−

calcd. for

C13H10F2O4P, 299.0290; found, 299.0285.

Inhibitor 3.42.


(d, J = 8.0 Hz, 1H), 7.53 (t, J = 7.7 Hz, 1H), 7.45 (s, 1H), 7.41 (d, J = 7.7 Hz, 1H), 7.32 (t, J =

7.6 Hz, 1H), 7.18 (d, J = 7.5 Hz, 1H), 2.40 (s, 3H); 19

F NMR (376 MHz, CD3OD): −109.64 (d,

JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.09 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M–

H]– calcd. for C14H12F2O3P, 297.0498; found, 297.0498.

Inhibitor 3.43.


(d, J = 7.8 Hz, 1H), 7.54 (t, J = 7.7 Hz, 1H), 7.48 (s, 1H), 7.43 (d, J = 7.6 Hz, 1H), 7.37 (t, J =

7.6 Hz, 1H), 7.25 (d, J = 7.5 Hz, 1H), 2.97 (hept, J = 6.9 Hz, 1H), 1.29 (d, J = 7.0 Hz, 6H); 19

F

NMR (376 MHz, CD3OD): −109.67 (d, JPF = 113.4 Hz); 31

P NMR (162 MHz, CD3OD): 7.16

(br t). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P, 325.0811; found, 325.0805.

56

Inhibitor 3.62.

1H NMR (400 MHz, DMSO-d6): 9.01 (s, 1H), 7.57, 7.53 (ABq, J = 8.0 Hz,

4H), 7.46−7.29 (m, 4H), 2.74 (s, 3H); 19

F NMR (376 MHz, DMSO-d6): −107.44 (d, JPF = 106

Hz); 31

P NMR (162 MHz, DMSO-d6): 2.83 (t, JPF = 106 Hz). HRMS-ESI (m/z): [M−H]− calcd.

for C14H13F2NO5PS, 376.0226; found, 376.0232.

Inhibitor 3.63.

1H NMR (400 MHz, DMSO-d6): 7.87−7.78 (m, 6H), 7.73 (d, J = 8.0 Hz,

2H); 19

F NMR (376 MHz, DMSO-d6): −111.49 (d, JPF = 112 Hz); 31

P NMR (162 MHz,

DMSO-d6): 5.61 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P,

308.0294; found, 308.0297.

Inhibitor 3.64.

1H NMR (400 MHz, DMSO-d6): 7.70 (d, J = 8.0 Hz, 2H), 7.61 (d, J = 8.0

Hz, 2H), 7.56 (d, J = 8.0 Hz, 2H), 7.41 (d, J = 8.0 Hz, 2H), 4.73 (q, J = 6.4 Hz, 1H), 1.32 (d, J =

6.4 Hz, 3H); 19


P NMR (162 MHz,

DMSO-d6): 2.62 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C15H14F2O4P,

327.0603; found, 327.0608.

57

Inhibitor 3.65.

1H NMR (400 MHz, DMSO-d6): 8.04−7.96 (m, 2H), 7.78−7.71 (m, 5H),

7.67−7.63 (m, 1H); 19


P NMR

(162 MHz, DMSO-d6): 5.81 (t, JPF = 112 Hz). HRMS-ESI (m/z): [M−H]− calcd. for

C14H9F2NO3P, 308.0294; found, 308.0296.

Inhibitor 3.66.


7.67 (d, J = 8.0 Hz, 2H), 7.62−7.60 (m, 1H), 7.59−7.54 (m, 1H); 19

F NMR (376 MHz, DMSO-

d6): −111.15 (d, JPF = 111 Hz); 31

P NMR (162 MHz, DMSO-d6): 5.39 (t, JPF = 111 Hz).

HRMS-ESI (m/z): [M−H]− calcd. for C14H9F2NO3P, 308.0294; found, 308.0298.

Inhibitor 3.67.

1H NMR (400 MHz, DMSO-d6): 7.66, 7.63 (ABq, J = 8.8 Hz, 4H), 7.56 (d,

J = 8.8 Hz, 2H), 6.98 (d, J = 8.8 Hz, 2H), 4.65 (hept, J = 6.0 Hz, 1H), 1.33 (d, J = 6.0 Hz, 6H); 19


P NMR (162 MHz, DMSO-d6):

5.93 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found,

341.0763.

58

Inhibitor 3.68.

1H NMR (400 MHz, DMSO-d6): 7.70−7.65 (m, 4H), 7.34 (t, J = 8.4 Hz,

1H), 7.19−7.13 (m, 2H), 6.92 (ddd, J = 8.4 Hz, 2.4, 0.8 Hz, 1H), 4.67 (hept, J = 6.0 Hz, 1H),

1.33 (d, J = 6.0 Hz, 6H); 19


P NMR

(162 MHz, DMSO-d6): 5.88 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for

C16H16F2O4P, 341.0760; found, 341.0760.

Inhibitor 3.69.


Hz, 2H), 7.58 (d, J = 8.0 Hz, 2H), 7.33 (d, J = 8.0 Hz, 2H), 2.94 (hept, J = 6.4 Hz, 1H), 1.28 (d, J

= 6.4 Hz, 6H); 19


P NMR (162

MHz, DMSO-d6): 5.99 (t, JPF = 114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P,

325.0811; found, 325.0815.

Inhibitor 3.70.


7.27−7.24 (m, 1H), 2.98 (hept, J = 6.4 Hz, 1H), 1.30 (d, J = 6.4 Hz, 6H); 19

F NMR (376 MHz,

DMSO-d6): −111.15 (d, JPF = 113 Hz); 31

P NMR (162 MHz, DMSO-d6): 5.95 (t, JPF = 113

Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O3P, 325.0811; found, 325.0811.

59

Inhibitor 3.71.

1H NMR (400 MHz, DMSO-d6): 9.86 (s, 1H), 7.70 (d, J = 8.0 Hz, 2H),

7.61 (d, J = 8.0 Hz, 2H), 7.47−7.39 (m, 3H), 7.25−7.23 (m, 1H), 3.03 (s, 3H); 19

F NMR (376

MHz, DMSO-d6): −108.08 (d, JPF = 107 Hz); 31

P NMR (162 MHz, DMSO-d6): 2.86 (t, JPF =

107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C14H13F2NO5PS, 376.0226; found, 376.0230.

Inhibitor 3.72.


Hz, 2H), 7.34−7.27 (m, 2H), 7.22−7.18 (m, 2H), 7.12−7.05 (m, 4H), 6.82 (t, J = 7.2 Hz, 1H); 19

F

NMR (376 MHz, DMSO-d6): −108.01 (d, JPF = 107 Hz); 31


2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C19H15F2NO3P, 374.0763; found,

374.0764.

Inhibitor 3.73.

1H NMR (400 MHz, DMSO-d6): 7.73 (d, J = 8.4 Hz, 2H), 7.65−7.56 (m,

3H), 7.53−7.48 (m, 1H), 7.40 (t, J = 7.6 Hz, 1H), 7.37−7.32 (m, 1H), 4.77 (q, J = 6.4 Hz, 1H),

1.34 (d, J = 6.4 Hz, 3H); 19


P

NMR (162 MHz, DMSO-d6): 2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for

C15H14F2O4P, 327.0603; found, 327.0602.

60

Inhibitor 3.74.


3H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.30−7.24 (m, 5H), 7.20−7.15 (m, 1H),

4.01 (s, 2H); 19

F NMR (376 MHz, DMSO-d6) −107.98 (d, JPF = 107 Hz); 31

P NMR (162 MHz,

DMSO-d6): 2.90 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C20H16F2O3P,

373.0811; found, 373.0803.

Synthesis of and analytical data for inhibitors 3.51–3.60

To synthesize inhibitors containing both an ortho hydroxyl and meta alkyl substituent

(general form 3.102), first the boronic acids had to be formed. This process required no

purifications starting from alkylphenols 3.44, so the sequence is written as one procedure

(Scheme 3.7). Compounds 3.51–3.56 and 3.58–3.59 were synthesized with this procedure.

Scheme 3.7. General synthesis of inhibitors 3.102

General synthesis of inhibitors 3.102. Selective ortho-bromination of alkylphenols was

accomplished via a modified literature procedure.19

A solution of N-bromosuccinimide (1.0

equiv) in CH2Cl2 (0.2 M) was added dropwise via cannula to a stirred solution of the phenol in

CH2Cl2 (0.5 M) containing catalytic N,N-diisopropylamine (0.1 equiv), the reaction flask having

been placed in an ambient temperature water bath. The reaction mixture was stirred for 90 min at

which point it was acidified with 1 N HCl to pH < 2.0. The reaction mixture was diluted with 1

volume of water. The layers were separated, and the organic layer was dried over MgSO4. The

volatile material was removed with a rotary evaporator under vacuum to afford the crude

products, generally as yellow to colorless liquids, which were typically used without further

purification.

Borylation of these phenols was accomplished following a literature procedure.20

n-

Butyllitium (2.5 M in hexanes, 2.15 equiv) was added dropwise via syringe to a stirred solution

of the starting o-bromophenol in Et2O (0.2 M) in a dry ice−acetone cold temperature bath. The

cold bath was removed and the reaction solution was allowed to warm to ambient temperature in

61

air for 2.5 h. The reaction flask was resuspended in the dry ice−acetone cold temperature bath.

After the mixture was stirred for 10 min in the cold bath, a solution of trimethyl borate (1.67

equiv, 1.5 M in Et2O) was added. After the mixture was stirred in the cold bath for 30 min, the

reaction mixture was stirred at ambient temperature for 18 h. The reaction mixture was quenched

with 0.5 volumes of 1 N HCl. The layers were separated, and the organic layer was dried over

MgSO4. The volatile material was removed with a rotary evaporator under vacuum to afford the

crude products as orange to brown viscous oils or sticky solids. Note: Although crude products

show multiple sets of peaks in the aromatic region when analyzed by 1H-NMR (potentially the

hydrolytic boroxine species), the crude products show only one major peak on LCMS which

corresponds to the mass of the boronic acids, and the products were used without further

purification.

Final inhibitor conversion was accomplished using general procedure A (vide supra).

Analytical data for each inhibitor is provided.

Inhibitor 3.51.


(d, J = 7.1 Hz, 1H), 7.52 (t, J = 7.6 Hz, 1H), 7.19 (dd, J = 7.8, 1.7 Hz, 1H), 7.04 (dd, J = 7.6, 1.7

Hz, 1H), 6.92 (t, J = 7.6 Hz, 1H), 3.38 (hept, J = 6.9 Hz, 1H), 1.26 (d, J = 6.9 Hz, 6H); 19

F NMR

(376 MHz, CD3OD): −109.81 (d, JPF = 114 Hz); 31


114 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C16H16F2O4P, 341.0760; found, 341.0755.

Inhibitor 3.52.


(d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.7 Hz, 1H), 7.12 (d, J = 2.3 Hz, 1H), 7.06 (dd, J = 8.3, 2.3 Hz,

1H), 6.83 (d, J = 8.3 Hz, 1H), 2.87 (hept, J = 6.9 Hz, 1H), 1.24 (d, J = 6.9 Hz, 6H); 19

F NMR

(376 MHz, CD3OD): −109.48 (d, JPF = 114 Hz); 31



62

Inhibitor 3.53.



Hz, 1H), 6.89 (t, J = 7.6 Hz, 1H), 2.70 (q, J = 7.5 Hz, 2H), 1.23 (t, J = 7.5 Hz, 3H); 19

F NMR

(376 MHz, CD3OD): −109.75 (d, JPF = 114 Hz); 31



Inhibitor 3.54.

1H NMR (400 MHz, CD3OD): 7.68 (s, 1H), 7.63−7.50 (m, 3H), 7.26 (d, J =

7.7 Hz, 1H), 7.02 (d, J = 7.5 Hz, 1H), 6.87 (t, J = 7.7 Hz, 1H), 1.44 (s, 9H); 19

F NMR (376 MHz,

CD3OD): −110.00 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 6.96 (t, JPF = 113 Hz).

HRMS-ESI (m/z): [M−H]− calcd. for C17H18F2O4P, 355.0916; found, 355.0913.

Inhibitor 3.55.


7.37 (m, 2H), 7.00 (d, J = 7.4 Hz, 1H), 6.95 (d, J = 7.6 Hz, 1H), 6.77 (t, J = 7.5 Hz, 1H), 2.59–

2.52 (m, 2H), 1.55 (sext, J = 7.4 Hz, 2H), 0.88 (t, J = 7.4 Hz, 3H); 19

F NMR (376 MHz,

CD3OD): –110.89 (d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.24 (br). HRMS-ESI


63

Inhibitor 3.56.

1H NMR (400 MHz, CD3OD): 7.62 (s, 1H), 7.56–7.37 (m, 3H), 7.05 (d, J =

7.5 Hz, 1H), 6.94 (d, J = 7.5 Hz, 1H), 6.83 (t, J = 7.5 Hz, 1H), 3.12–3.01 (m, 1H), 1.65–1.45 (m,

2H), 1.13 (d, J = 6.9 Hz, 3H), 0.78 (t, J = 7.3 Hz, 3H); 19

F NMR (376 MHz, CD3OD): –110.94

(d, JPF = 113 Hz); 31

P NMR (162 MHz, CD3OD): 7.24 (t, JPF = 113 Hz). HRMS-ESI (m/z):

[M−H]− calcd. for C17H18F2O4P, 355.0916; found, 355.0907.

Inhibitor 3.58.



Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 3.46−3.33 (m, 1H), 2.12−2.01 (m, 2H), 1.92−1.78 (m, 2H),

1.78−1.56 (m, 4H); 19


P NMR (162

MHz, CD3OD) 7.19 (br t). HRMS-ESI (m/z): [M−H]− calcd. for C18H18F2O4P, 367.0916;

found, 367.0912.

Inhibitor 3.59.



Hz, 1H), 6.91 (t, J = 7.6 Hz, 1H), 3.08−2.95 (m, 1H), 1.93−1.82 (m, 4H), 1.82−1.73 (m, 1H),

1.56−1.23 (m, 5H); 19


P NMR (162

MHz, CD3OD): 7.29 (t, JPF = 113 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C19H20F2O4P,

381.1073; found, 381.1068.

64

Inhibitors 3.57 and 3.60 could not be made by the above method, so were made by a different

sequence.

Compound 3.103. Following a previous report, dilithiation of 2-bromophenol was

accomplished.20

2-Bromophenol (3.5 g, 20 mmol, 1.0 equiv) was dissolved in Et2O (55 mL) and

cooled in a dry ice−acetone cold bath. After the mixture was stirred for 10 min, n-butyllithium

(1.6 M in hexanes, 26.5 mL, 42 mmol, 2.1 equiv) was added dropwise by syringe. The cold bath

was removed and the reaction mixture was stirred at ambient temperature for 2 h, resulting in the

dilithiated reagent. Note: LCMS of an acid quenched aliquot confirmed that there was no starting

bromide remaining. An amount of 19 mL (approximately 5 mmol) of the above reagent was

transferred to a reaction flask charged with a stir bar and was suspended in the dry ice−acetone

cold bath. Next, 392 L of cyclobutanone (5.5 mmol, 1.1 equiv) was added neat via syringe. The

reaction mixture was allowed to warm to ambient temperature and was stirred for 14 h. The

reaction was quenched by addition of 10 mL of saturated ammonium chloride. The layers were

separated, and the water layer was washed with 15 mL of Et2O. The combined organic layers

were dried over MgSO4, and the volatile material was removed with a rotary evaporator under

vacuum to afford the crude benzyl alcohol product as a yellow liquid (880 mg). The crude

product (~5 mmol) was dissolved in 5 mL of CH2Cl2 in a 20 mL scintillation vial open to the

atmosphere, and triethylsilane (2.4 mL, 15 mmol, ~3 equiv) was added followed by 2.5 mL of

trifluoroacetic acid (1:0.5 by volume). The reaction mixture was stirred at ambient temperature

for 16 h. The volatile material was removed with a rotary evaporator under vacuum to afford the

crude product. The crude product was purified via gradient column chromatography (2−20%

ethyl acetate in hexanes) to afford the product as a clear oil (550 mg, 67%). 1H NMR (400 MHz,

CDCl3): 7.18 (dt, J = 7.6, 1.3 Hz, 1H), 7.09 (td, J = 7.7, 1.7 Hz, 1H), 6.92 (td, J = 7.4, 1.1 Hz,

1H), 6.76 (dd, J = 7.9, 1.2 Hz, 1H), 4.63 (s, 1H), 3.75−3.58 (m, 1H), 2.46−2.30 (m, 2H),

2.26−2.12 (m, 2H), 2.12−2.00 (m, 1H), 1.95−1.80 (m, 1H). MS-ESI (m/z): [M+H]+

calcd. for

C10H13O, 148.10; found, 149.1.

Compound 3.104. Borylation of 2-cyclobutylphenol (3.103) was accomplished by closely

following a previous report.21

A 1 dram oven-dried vial was charged with a stir bar and brought

into an inert atmosphere glovebox where it was charged with 3.103 (222 mg, 1.5 mmol, 1.0

equiv), diethylsilane (290 L, 2.25 mmol, 1.5 equiv), bis(1,5-cyclooctadiene)diiridium(I)

dichloride (10 mg, 0.015 mmol, 0.01 equiv, 2 mol % Ir), and 3 mL of benzene (0.5 M). The vial

was capped, removed from the inert atmosphere glovebox, and the reaction mixture was stirred

at ambient temperature for 1 h. Solvent was removed with a rotary evaporator under vacuum,

and the reaction vial was brought back into the inert atmosphere glovebox where it was charged

65

with bis(pinacolato)diboron (381 mg, 1.5 mmol, 1.0 equiv), pinacolborane (21.8 L, 0.15 mmol,

10 mol %), bis(1,5-cyclooctadiene)diiridium(I) dichloride (10 mg, 0.015 mmol, 0.01 equiv, 2

mol % Ir), 4,4-di-tert-butylbipyridine (8 mg, 0.03 mmol, 2 mol %), and 3 mL of THF (0.5 M).

The vial was capped, removed from the inert atmosphere glovebox, and placed in a heating block

preheated to 80 °C, and the mixture was stirred vigorously for 2 h. The vial was cooled to

ambient temperature, and saturated potassium hydrogen difluoride (~4.5 M, 3 mL, ~13.5 mmol,

9 equiv) was added. The resulting reaction mixture was stirred vigorously for 18 h. The volatile

material was removed with a rotary evaporator under vacuum to afford the crude product. The

crude product was dissolved in 5 mL of hot acetone, and the insoluble salts were filtered. The

volatile components of the filtrate were removed with a rotary evaporator under vacuum until 1

mL of acetone remained. The solution was transferred to a 15 mL conical centrifuge tube, and

approximately 5 mL of pentane was added to the acetone solution to precipitate the product. The

suspension was centrifuged to pellet the solid product, and the black liquid was removed.

Additional washes were accomplished by adding 5 mL of pentane, resuspending the solid

followed by centrifugation to pellet the solid products for a total of three additional washes.

Removal of residual solvent under vacuum afforded the product as a light gray solid (200 mg,

52%). 1H NMR (400 MHz, acetone-d6): 7.62 (q, J = 11.8 Hz, 1H), 7.15 (d, J = 7.0 Hz, 1H),

6.90 (dd, J = 7.5, 1.8 Hz, 1H), 6.60 (t, J = 7.3 Hz, 1H), 3.74 (p, J = 8.5 Hz, 1H), 2.33−2.18 (m,

2H), 2.15−2.03 (m, 1H), 2.03−1.86 (m, 2H), 1.84−1.71 (m, 1H). MS-ESI (m/z): [M(boronic acid)−H]

– calcd. for C10H12BO3, 191.09; found 191.1.

Inhibitor 3.57. For the Suzuki-Miyaura cross-coupling, general procedure B was followed

with 90 mg (0.35 mmol) of the potassium trifluoroborate 3.104 and 100 mg (0.35 mmol) of

arylbromide 3.21. The procedure yielded 18 mg (15%) of the desired compound as a white

hygroscopic powder. 1H NMR (400 MHz, CD3OD): 7.71 (s, 1H), 7.61−7.54 (m, 2H), 7.49 (t, J

= 7.7 Hz, 1H), 7.20 (dd, J = 7.4, 1.6 Hz, 1H), 7.05 (dd, J = 7.6, 1.7 Hz, 1H), 6.92 (t, J = 7.5 Hz,

1H), 3.82 (p, J = 8.6 Hz, 1H), 2.43−2.32 (m, 2H), 2.23−1.96 (m, 3H), 1.93−1.75 (m, 1H); 19

F

NMR (376 MHz, CD3OD): −109.20 (d, JPF = 109 Hz); 31

P NMR (162 MHz, CD3OD): 6.97

(br t). HRMS-ESI (m/z): [M−H]– calcd. for C17H16F2O4P, 353.0760; found 353.0754.

66

Inhibitor 3.60. Inhibitor 3.60 was synthesized by the same procedures described for 3.57,

yielding the product in a 1:0.4 ratio with residual trifluoroacetic acid (as determined by 19

F

NMR). 1H NMR (400 MHz, CD3OD): 7.72 (s, 1H), 7.68−7.44 (m, 3H), 7.13 (dd, J = 7.6, 1.7

Hz, 1H), 6.99 (dd, J = 7.4, 1.7 Hz, 1H), 6.87 (t, J = 7.6 Hz, 1H), 3.29−3.16 (m, 1H), 2.00−1.49

(m, 12H); 19


P NMR (162 MHz,

CD3OD): 7.01 (br t). HRMS-ESI (m/z): [M−H]– calcd. for C20H22F2O4P, 395.1229; found

395.1225.

Synthesis of and analytical data for inhibitors 3.79–3.83 and 3.61

Compound 3.105. To a solution of 1-bromo-3-iodobenzene 3.17 (2.0 g, 7.1 mmol) in THF

(15 mL) at –78°C under N2 atmosphere was added dropwise n-BuLi (2.5 M in hexanes, 2.8 mL,

7.0 mmol). The reaction mixture was stirred at the same temperature for 0.5 h. 3,4-

Dichlorobenzaldehyde (1.36 g, 7.77 mmol) was added and the reaction mixture was kept at –78

°C for 2 h. Saturated NH4Cl (50 mL) and Et2O (50 mL) were added, and the reaction mixture

was allowed to warm to ambient temperature. The phases were separated and the aqueous phase

was extracted with Et2O (100 mL). The combined organic phase was dried over MgSO4, filtered

and concentrated to yield 3.105 as a viscous oil (1.85 g, 72%). 1H NMR (400 MHz, CDCl3): δ

7.51 (m, 1H), 7.48 (m, 1H), 7.44−7.40 (m, 2H), 7.27−7.16 (m, 3H), 5.74 (d, J = 3.2 Hz, 1H),

2.31 (d, J = 3.2 Hz, 1H).

Compound 3.106. Compound 3.105 (1.80 g, 5.42 mmol) was dissolved in dichloromethane

(6 mL). Et3SiH (3.0 mL, 18.7 mmol) was added at ambient temperature, followed by

trifluoroacetic acid (TFA, 3.0 mL). The resulting solution was stirred at ambient temperature for

15 h. Volatiles were removed under reduced pressure. The residue was purified by automated

silica gel flash column chromatography (80 g flash column, column volume = 125 mL, 60

mL/min, linear gradient of 0−20% Et2O in hexanes over 25 min) to yield 3.106 as a white solid (1.57 g, 92% yield).

1H NMR (400 MHz, CDCl3): δ 7.38−7.35 (m, 2H), 7.31−7.30 (m, 1H), 7.25

67

(d, J = 2.0 Hz, 1H), 7.17 (t, J = 8.0 Hz, 1H), 7.09−7.06 (m, 1H), 7.00 (dd, J = 8.0, 2.0 Hz, 1H),

3.89 (s, 2H).

Compound 3.85. . To a Schlenk flask equipped with a stirring bar was added 3.106 (1.70 g,

5.38 mmol), bis(pinacolato)diboron (B2pin2, 2.73 g, 10.8 mmol), KOAc (2.12 g, 21.6 mmol), and

DMSO (22 mL). Nitrogen was bubbled through the reaction mixture for 0.5 h. Pd(dppf)Cl2 (0.22

g, 0.27 mmol) was added. Nitrogen was bubbled through the reaction mixture for an additional

0.5 h. The reaction mixture was then stirred at 80 °C for 6 h, cooled to ambient temperature, and

poured into water (220 mL). The resulting suspension was extracted with Et2O (2 × 150 mL).

The combined organic extracts was dried over anhydrous MgSO4, concentrated, and the residue

was purified by automated silica gel flash column chromatography (80 g flash column, column

volume = 125 mL, 60 mL/min, linear gradient of 0−20% Et2O in hexanes over 25 min) to yield

3.85 as a white solid (1.60 g, 82% yield). 1H NMR (400 MHz, CDCl3): δ 7.69 (d, J = 8.0 Hz,

1H), 7.64 (s, 1H), 7.32 (t, J = 8.0 Hz, 2H), 7.26−7.22 (m, 2H), 7.0 (d, J = 8.0 Hz, 1H), 3.92 (s,

2H), 1.35 (s, 12H).

Inhibitor 3.81. Compound 3.81 was prepared using general Suzuki-Miyaura cross-coupling

procedure B starting with 80 mg (0.28 mmol) of 3.22 and 152 mg (0.42 mmol) of 3.85. The procedure yielded 34 mg (28%) of 3.81 as a white solid.

1H NMR (400 MHz, DMSO-d6): δ 7.74

(d, J = 8.0 Hz, 2H), 7.62−7.58 (m, 4H), 7.54−7.51 (m, 2H), 7.40 (t, J = 8.0 Hz, 1H), 7.25−7.29 (m, 2H), 4.01 (s, 2H);

19F NMR (376 MHz, DMSO-d6): δ −107.62 (d, JPF = 104 Hz);

31P NMR

(162 MHz, DMSO-d6): δ 2.66 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for

C20H14F2[35]

Cl2O3P, 441.0031; found, 441.0030.

Inhibitors 3.79 and 3.80 were synthesized using the same procedures as 3.81. Analytical data

for each inhibitor is provided.

68

Inhibitor 3.79.


3H), 7.51 (d, J = 8.0 Hz, 1H), 7.39 (t, J = 8.0 Hz, 1H), 7.34−7.29 (m, 2H), 7.24 (d, J = 8.0 Hz,

1H), 7.12−7.06 (m, 2H), 4.00 (s, 2H); 19


Hz), −117.29; 31

P NMR (162 MHz, DMSO-d6): 2.90 (t, JPF = 107 Hz). HRMS-ESI (m/z):

[M−H]− calcd. for C20H15F3O3P, 391.0716; found, 391.0719.

Inhibitor 3.80.


3H), 7.53−7.51 (m, 1H), 7.38 (t, J = 7.6 Hz, 1H), 7.33−7.28 (m, 4H), 7.22 (d, J = 7.6 Hz, 1H),

3.99 (s, 2H); 19


P NMR (162 MHz,

DMSO-d6): 2.59 (t, JPF = 104 Hz). HRMS-ESI (m/z): [M−H]− calcd. for C20H15F2

[35]ClO3P,

407.0421; found, 407.0418.

Compound 3.108. To a solution of known compound 3.107

32 (1.5 g, 5.3 mmol) in THF (45

mL) was added n-BuLi (2.5 M in hexanes, 2.1 mL, 5.3 mmol) dropwise at −78 °C under N2

atmosphere. The resulting solution was stirred at the same temperature for 0.5 h. 3,4-

Dichlorobenzaldehyde (0.93 g, 5.3 mmol in THF (3 mL) was added and the reaction mixture was

stirred at −78 °C for 1 h. 1 N HCl (16.5 mL) and Et2O (100 mL) were added. The reaction

mixture was allowed to warm to ambient temperature. The layers were separated, and the

aqueous layer was extracted with Et2O (2 × 100 mL). The combined organic solution was dried

over anhydrous MgSO4 and concentrated under reduced pressure to give 3.108, which was used

in the following step without purification.

69

Inhibitor 3.82. Inhibitor 3.82 was prepared using general Suzuki-Miyaura cross-coupling

procedure B using 80 mg (0.28 mmol) of 3.22 and 124 mg (0.42 mmol) of 3.108. Inhibitor 3.82

was obtained in 36% yield (46 mg) as a white solid. 1H NMR (400 MHz, DMSO-d6): 7.74 (d, J

= 8.0 Hz, 1H), 7.70 (d, J = 8.0 Hz, 2H), 7.65 (s, 1H), 7.60 (d, J = 8.0 Hz, 2H), 7.56 (d, J = 8.0

Hz, 1H), 7.53 (d, J = 2.0 Hz, 1H), 7.46 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 7.42 (t, J = 8.0 Hz, 1H),

7.31 (d, J = 8.0 Hz, 1H), 6.04 (s, 1H); 19


Hz); 31

P NMR (162 MHz, DMSO-d6): 2.91 (t, JPF = 107 Hz). HRMS-ESI (m/z): [M−H]− calcd.

for C20H14[35]

Cl2F2O4P, 456.9980; found, 456.9979.

Compound 3.86. Compound 3.86 was prepared using general Suzuki-Miyaura cross-

coupling procedure B using 80 mg (0.28 mmol) of compound 3.8423

and 152 mg (0.42 mmol) of

3.85, yielding in 49 mg (42%) as a white solid. 1H NMR (400 MHz, DMSO-d6): 8.26 (d, J =

8.0 Hz, 2H), 7.84 (d, J = 8.0 Hz, 2H), 7.70 (s, 1H), 7.60−7.58 (m, 2H), 7.52 (d, J = 8.0 Hz, 1H),

7.43 (t, J = 8.0 Hz, 1H), 7.32−7.28 (m, 2H), 4.03 (s, 2H); 31


−3.07. HRMS-ESI (m/z): [M−H]− calcd. for C20H14

[35]Cl2O4P, 419.0012; found, 419.0020.

Inhibitor 3.83. To a solution of 3.86 (35 mg, 0.083 mmol) in MeOH (0.2 mL) at 0 °C was

added NaBH4 (16 mg, 0.41 mmol). The reaction mixture was stirred at 0 °C for 20 min. More

NaBH4 (16 mg) was added, and the reaction mixture was stirred for an additional 20 min and

acetic acid (0.1 mL) was then added. The reaction mixture was purified by automated reversed-

phase column chromatography (liquid injection, 50 g C18 column, column volume = 42.6 mL,

40 mL/min, linear gradient of 5−100% acetonitrile/water with 0.1% trifluoroacetic acid over 20

70

min) to yield 3.83 as a white solid (20 mg, 57% yield). 1H NMR (400 MHz, DMSO-d6):

7.58−7.45 (m, 8H), 7.36 (t, J = 7.6 Hz, 1H), 7.28 (dd, J = 7.6, 2.0 Hz, 1H), 7.20 (d, J = 7.6 Hz,

1H), 4.70 (d, J = 14.0 Hz, 1H), 4.00 (s, 2H); 31

P NMR (162 MHz, DMSO-d6): 18.03. HRMS-

ESI (m/z): [M−H]− calcd. for C20H16

[35]Cl2O4P, 421.0169; found, 421.0155.

Inhibitor 3.61 was synthesized using the same procedures as 3.83 beginning from known

monosodium (3-bromobenzoyl)phosphonate.23

Analytical data. 1H NMR (400 MHz, CD3OD):

7.51 (s, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.35−7.26 (m, 2H), 7.04 (dd, J = 7.6, 1.8 Hz, 1H), 6.91

(dd, J = 7.6, 1.7 Hz, 1H), 6.78 (t, J = 7.5 Hz, 1H), 4.84 (d, J = 13.2 Hz, 1H), 3.01−2.86 (m, 1H),

1.86−1.73 (m, 4H), 1.72−1.63 (m, 1H), 1.44−1.55 (m, 5H); 31

P NMR (162 MHz, CD3OD):

22.45. HRMS-ESI (m/z): [M−H]– calcd. for C19H22O5P, 361.1210; found, 361.1215.

Synthesis of and analytical data for diastereomeric inhibitors 3.97–3.100 and inhibitor 3.101

Compounds 3.88 and 3.89. Tris-[(1S,2R,5S)-menth-2-yl]phosphite is prepared according to

literature procedure.24

To a solution of phosphorus trichloride (1.7 mL, 20.0 mmol) in anhydrous

toluene (30 mL) at −20 °C was added slowly a solution of D-menthol (9.4 g, 60.0 mmol) and

triethylamine (10 mL) in toluene (50 mL). Upon complete addition, the cooling bath was

removed and the reaction mixture was stirred at ambient temperature for 5 h. The white

precipitate was filtered off and the filtrate was concentrated under reduced pressure to give tris-

[(1S,2R,5S)-menth-2-yl]phosphite (9.2 g), which was then treated with 4-bromobenzaldehyde

(3.4 g, 18.4 mmol) and chlorotrimethylsilane (5.4 mL, 42.6 mmol) at 0 °C. The suspension was

stirred at 0 °C for 2 h and then at ambient temperature for an additional 1 h. Volatiles were

removed under reduced pressure. The residue was purified by automatic silica gel column

chromatography (220 g flash column, column volume = 334 mL, 150 mL/min, linear gradient of

0−10% hexane/ethyl acetate over 35 min) to give the product as a mixture of diastereoisomers in

a ratio of 1.0:0.7 (8.8 g, 88%). The diastereoisomeric mixture was dissolved in acetonitrile (1

g/100 mL) at 60 °C and cooled to ambient temperature. The solid was collected by filtration and

recrystallized in acetonitrile one more time to give diastereomerically pure 3.89. The filtrate

solution was cooled to 0 °C. The solid was collected and recrystallized in acetonitrile one more

time to give diastereomerically pure 3.88. Compound 3.88. 1H NMR (400 MHz, CDCl3): 7.45

71

(d, J = 8.0 Hz, 2H), 7.37 (dd, J = 8.0, 4.0 Hz, 2H), 4.92 (dd, J = 11.2, 5.2 Hz, 1H), 4.24−4.15 (m,

2H), 3.42 (br, 1H), 2.19−2.14 (m, 1H), 2.06−1.97 (m, 2H), 1.81−1.77 (m, 1H), 1.65−1.59 (m,

4H), 1.41−0.85 (m, 19 H), 0.80 (d, J = 7.2 Hz, 3H), 0.76 (d, J = 6.8 Hz, 3H), 0.69 (d, J = 7.2 Hz,

3H); 31

P NMR (162 MHz, CDCl3): 18.75. Compound 3.89. 1H NMR (400 MHz, CDCl3):

7.45 (d, J = 8.0 Hz, 2H), 7.35 (dd, J = 8.0, 4.0 Hz, 2H), 4.91 (dd, J = 11.2, 5.2 Hz, 1H),

4.26−4.16 (m, 2H), 3.36 (dd, J = 9.2, 4.8 Hz, 1H), 2.16−2.13 (m, 1H), 1.99−1.91 (m, 3H),

1.70−1.59 (m, 4H), 1.42−0.79 (m, 22H), 0.76 (d, J = 7.2 Hz, 3H), 0.72 (d, J = 6.8 Hz, 3H); 31

P

NMR (162 MHz, CDCl3): 19.06.

Compound 3.91. To a suspension of 3.89 (1.8 g, 3.3 mmol) and NaI (2.0 g, 13.3 mmol) in

acetonitrile (20 mL) under nitrogen atmosphere was added chlorotrimethylsilane (1.7 mL, 13.5

mmol). The reaction mixture was refluxed for 10 h and then cooled to ambient temperature. The

precipitates were filtered off. The filtrate was concentrated under reduced pressure. The residue

was purified by automated reversed-phase column chromatography (150 g C18 column, column

volume = 130 mL, 85 mL/min, liquid injection, linear gradient of 5−30% acetonitrile in water

with 0.1% trifluoroacetic acid over 25 min) to give the title compound as a white solid (0.52 g,

59%). 1H NMR (400 MHz, DMSO-d6): 7.48 (d, J = 8.0 Hz, 2H), 7.33 (dd, J = 8.0, 4.0 Hz, 2H),

4.62 (d, J = 12.0 Hz, 1H); 31

P NMR (162 MHz, DMSO-d6): 17.43.

Compound 3.109. A mixture of 3.91 (43 mg, 0.16 mmol) and 10% Pd/C (dry, 16 mg) in

MeOH (4 mL) was stirred under H2 atmosphere (1 atm) for 3 h. The reaction mixture was

filtered through a syringe filter (0.2 m). The filtrate was concentrated to give the title compound

(28 mg, 93%). 1H NMR (400 MHz, DMSO-d6): 7.39−7.17 (m, 5H), 4.64 (d, J = 14.0 Hz, 1H);

31P NMR (162 MHz, DMSO-d6): 18.14.

Compound 3.91 was dissolved in ethanol (0.5 mL), and an excess of cyclohexylamine (100

L, 0.87 mmol) was added. The white precipitate was collected by filtration. []20

D : −10.6 (c

0.77, 50% MeOH/H2O at 20 °C) (lit.25a

−13.8, c 0.77, 50% MeOH/H2O).

72

Compound 3.90. Compound 3.90 (0.22 g) was obtained as a white solid in 45% yield

starting from 3.88 (1.0 g, 1.8 mmol) following the procedure described for the synthesis of

compound 3.91. The 1H NMR and

31P NMR spectra are the same as those of 3.91.

Compound 3.93. Compound 3.93 was prepared by adapting a literature procedure.

26a To a

solution of 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol) in tert-butyl methyl ether (5.50

mL) at −78 °C was added dropwise n-BuLi (2.5 M in hexanes, 1.77, 4.42 mmol). The reaction

mixture was stirred at −78 °C for 1 h. The dry ice−acetone cooling bath was replaced with an

ice−water cooling bath. Anhydrous ZnCl2 (0.63 g, 4.64 mmol) was added. The resulting

suspension was stirred at 0 °C for 1 h. Additional n-BuLi (2.5 M in hexanes, 1.77 mL, 4.42

mmol) was added. The cooling bath was removed, and the reaction mixture was stirred at

ambient temperature for 4.5 h. Toluene (anhydrous, 22.5 mL) and tetraethylethylenediamine

(0.37 mL, 1.77 mmol) were added. The reaction mixture was stirred at ambient temperature for

an additional 1 h. (+)-3-exo-(Morpholino)isoborneol ((+)-MIB,26

27 mg, 0.11 mmol) was added.

and the reaction mixture was cooled to 0 °C and stirred for 0.5 h. 3-Bromobenzaldehyde (0.78 g,

2.21 mmol) was added, and the reaction mixture was stirred at 0 °C for 17 h. Water (30 mL) and

Et2O (30 mL) were added. The phases were separated, and the aqueous layer was extracted with

Et2O (2 × 50 mL). The combined organic phase was dried over anhydrous MgSO4, filtered, and

concentrated. The residue was purified by automated silica gel column chromatography (80 g

flash column, column volume = 125 mL, 60 mL/min, linear gradient of 0−50% ethyl acetate in

hexane over 25 min) to afford 3.93 as a colorless oil (0.51 g, 70%). The enantiomeric excess was

determined by HPLC (AS-H column, isopropanol/hexane = 5:95, flow rate = 0.5 mL/min, tr,major

= 32.3 min, minor enantiomer tr,minor = 35.4 min, 90% ee). 1H NMR (400 MHz, CDCl3): 7.52

(s, 1H), 7.48 (d, J = 2.0 Hz, 1H), 7.44−7.40 (m, 2H), 7.27−7.20 (m, 2H), 7.18 (dd, J = 8.4. 2.0

Hz, 1H), 5.75 (d, J = 3.2 Hz, 1H), 2.30 (d, J = 3.2 Hz, 1H).

Compound 3.94. Compound 3.94 (0.49 g) was obtained as a colorless oil in 67% yield

starting from 3-bromo-1,2-dichlorobenzene (1.00 g, 4.42 mmol) by following the procedure used

for the synthesis of 3.93 but replacing the catalyst (+)-MIB with (−)-MIB. The enantiomeric

excess was determined by HPLC (AS-H column, isopropanol/hexane = 5:95, flow rate = 0.5

73

mL/min, tr,major = 35.4 min, tr,minor = 32.3 min, 90% ee). The 1H NMR spectrum is the same as

that of 3.93.

Compound 3.95. Compound 3.95 (0.28 g) was obtained as a colorless oil in 64% yield

starting from 3.93 (0.38 g, 1.14 mmol) by following the procedure described for the synthesis of

3.85. 1H NMR (400 MHz, CDCl3): 7.80−7.79 (m, 1H), 7.75 (dt, J = 6.8, 1.2 Hz, 1H), 7.51 (dd,

J = 2.4, 0.8 Hz, 1H), 7.44−7.33 (m, 3H), 7.20 (ddd, J = 8.4, 2.4, 0.8 Hz, 1H), 5.80 (d, J = 3.2 Hz,

1H), 2.23 (d, J = 3.2 Hz, 1H), 1.35 (s, 12H).

Compound 3.96. Compound 3.96 was prepared in 58% yield starting from 3.94 (0.50 g, 1.51

mmol) by following the procedure described the synthesis of 3.85. The 1H NMR spectrum of

3.96 is the same as that of 3.95.

Inhibitors 3.97–3.100 were synthesized using the general Suzuki-Miyaura cross-coupling

procedure B beginning with either aryl bromide 3.90 or 3.91 and either aryl boronic ester 3.95 or

3.96. Inhibitor 3.101 was synthesized using the general Suzuki-Miyaura cross-coupling

procedure B beginning with commercially available 4-bromobenzylphosphonic acid and boronic

acid 3.108. Analytical data for each inhibitor is provided.

Inhibitor 3.97.


7.51−7.45 (m, 3H), 7.41−7.36 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.79 (s, 1H), 4.70

(d, J = 14.0 Hz, 1H); 31

P NMR (162 MHz, DMSO-d6): 17.97. HRMS-ESI (m/z): [M−H]− calcd.

for C20H16[35]

Cl2O5P, 437.0118; found, 437.0110.

74

Inhibitor 3.98.


7.51−7.45 (m, 3H), 7.41−7.36 (m, 2H), 7.34 (d, J = 8.0 Hz, 1H), 6.17 (s, 1H), 5.79 (s, 1H), 4.70

(d, J = 14.0 Hz, 1H); 31

P NMR (162 MHz, DMSO-d6): 18.00. HRMS-ESI (m/z): [M−H]− calcd.

for C20H16[35]

Cl2O5P, 437.0118; found, 437.0111.

Inhibitor 3.99. The

1H NMR and

31P NMR spectra are the same as those reported for the

enantiomer, 3.98. HRMS-ESI (m/z): [M−H]− calcd. for C20H16

[35]Cl2O5P, 437.0118; found,

437.0111.

Inhibitor 3.100. The

1H NMR and

31P NMR spectra are the same as those reported for the

enantiomer, 3.97. HRMS-ESI (m/z): [M−H]− calcd. for C20H16

[35]Cl2O5P, 437.0118; found,

437.0112.

75

Inhibitor 3.101.


3H), 7.48 (d, J = 8.0 Hz, 1H), 7.39−7.35 (m, 2H), 7.32−7.30 (m, 3H), 6.15 (br, 1H), 5.78 (s, 1H),

2.97 (d, JHP = 21.2 Hz, 2H); 31

P NMR (162 MHz, DMSO-d6): 20.79. HRMS-ESI (m/z):

[M−H]− calcd. for C20H16

[35]Cl2O4P, 421.0169; found, 421.0162.

Assay procedures

Determination of substrate Km

To facilitate screening in a high-throughput fashion, 96-well plates were used with reaction

volumes of 100 L. An amount of 30 L of water was added to each well, followed by 5 L of

20× buffer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-X 100, pH 7.0), 10 L of 10× DTT (50

mM, 5 mM in assay), 40 L of 2-amino-6-mercapto-7-methylpurine riboside (MESG) solution

(1 mM, 400 M in assay), and 5 L of purine nucleotide phosphorylase (PNP) solution (20

U/mL, 1 U/mL in assay). An amount of 5 L of STEP phosphatase (2 M, 100 nM in assay) was

added, and the 96-well assay plate was incubated at 27 °C for 3 min. The coupled assay was

started by addition of 5 L of the appropriate substrate dilution in DMSO (typically 3.00, 1.20,

0.480, 0.192, 0.077, 0.031, 0.012, 0 mM in assay). The plate was then immediately placed into a

spectrophotometric plate reader, and 20 min of kinetic data was obtained (360 nm, 27 °C). The

initial rate data collected were used for Michaelis−Menten kinetic analysis where the Km was

obtained using the substrate−velocity data with the equation v = (Vmax[S])/(Km+[S]).

Determination of inhibitor Ki

Reaction volumes of 100 L were used in 96-well plates. An amount of 65 L of water was

added to each well, followed by 5 L of 20× buffer (1.0 M imidazole, 1.0 M NaCl, 0.2% Triton-

X 100, pH 7.0), 10 L of 10× DTT (50 mM, 5 mM in assay), 5 L of STEP phosphatase (2 M,

100 nM in assay), and 5 L of the appropriate inhibitor dilution in DMSO (with 2- or 3-fold

serial dilutions). The assay plate was incubated for 5 min at 27 °C, at which point the reaction

was started by addition of 10 L of a 10× pNPP substrate (5 mM, 500 M in assay; Km = 745

). The plate was then immediately placed into a spectrophotometric plate reader, and 20 min

of kinetic data was obtained (405 nm, 27 °C). The initial rate data collected were used for

determination of Ki values. For Ki determination, the kinetic values were obtained directly from

nonlinear regression of substrate−velocity curves in the presence of various concentrations of

inhibitor. Assays were run in at least duplicate using the same inhibitor stock solutions.

For the selectivity assays, the Km of pNPP toward each of the enzymes was determined in the

above assay buffer and used for data analysis. For the assays with the dual-specificity MKP5,

76

due to poor turnover of pNPP, the chromogenic substrate 6,8-difluoro-4-methylumbelliferyl

phosphate (DiFMUP) was used instead. In addition, the assay buffer used was a 10× buffer (0.2

M Tris-HCl, 0.2% Triton-X 100, pH 8.0); 5 mM DTT was used as in the other assays.

Expression and purification of STEP

Protein was expressed from BL21(DE3) cells (Invitrogen) which were pre-transformed with

the pGEX-4T-1 vector (GE Healthcare) containing the full length STEP-GST fusion construct.

This pre-transformed stock was provided by the Lombroso group. Transformed bacteria were

grown to an OD600 of 0.8 in LB broth and protein expression was induced by the addition of

400 M isopropyl -D-1-thiogalactopyranoside. After 18 h of expression at 20 °C, cells were

harvested and resuspended in lysis buffer (1× PBS, pH 7.4 with 0.2% Triton X-100). Cell

suspensions were French pressed (×3), and the lysates centrifuged for 40 min at 21,000 × g, and

the cleared lysate loaded onto a glutathione-sepharose column (GE Healthcare). After elution

with reduced glutathione (10 mM reduced glutathione, 50 mM Tris-HCl, pH 8.0, 2 mM DTT),

the protein was dialyzed into storage buffer (20 mM Tris-HCl, 50 mM NaCl, 1 mM EDTA) and

concentrated to 13.9 mg/mL (193 M) and was aliquoted appropriately for phosphatase assays.

Cell culture and Western blotting

The Yale University Institutional Animal Care and Use Committee approved all procedures.

Primary cortical neurons were isolated from Sprague−Dawley rat embryos (E18) (Charles River

Laboratories, Wilmington, MA) as previously described.33

Briefly, cells were dissociated with

trypsin, resuspended in Hanks’ balanced salt solution, and then plated on poly-D-lysine-coated

plates (1 × 106 cells/well) in neurobasal medium supplemented with 2% B27 (Invitrogen, San

Diego, CA). Neurons were allowed to grow for 18−21 days at 37 °C in a CO2 incubator.

Compounds to be tested were diluted in DMSO and added to the medium at final concentrations

of 0, 0.1, 1, and 10 M and incubated for 1 h at 37 °C in a CO2 incubator. The percentage of

DMSO remained constant (0.1%) in all wells. After incubation, neurons were lysed in

radioimmunoprecipitation assay (RIPA) buffer supplemented with protease inhibitor cocktail

(Roche Applied Science, Indianapolis, IN) and phosphatase inhibitors (NaF and Na3VO4).

Protein concentrations of the samples were estimated using the BCA assay kit (Pierce, Thermo

Scientific).

Samples were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane,

and incubated with phospho-specific antibodies (anti-pY204/187

ERK1/2, anti-pY402

Pyk2, anti-

pY1472

GluN2B) or total protein antibodies (anti-Pyk2, anti-GluN2B, anti-ERK1/2, anti-GAPDH)

overnight at 4 °C. All antibodies used in this study are listed in Table 3.6. Membranes were

washed and incubated in peroxidase-conjugated secondary antibodies (GE Healthcare,

Waukesha, WI). The immunoreactivity was visualized using a chemiluminescent substrate kit

(Pierce Biotechnology, Rockford, IL) and detected using a G:BOX with the image program

GeneSnap (Syngene, Cambridge, U.K.). All densitometric quantifications were performed using

the Image J (NIH) software.

All data are presented as the mean ± SEM. Differences among multiple groups were

evaluated using one-way ANOVA with Dunnett’s post hoc test using Graph Pad Prism 6

software. For all analyses, a p value of < 0.05 indicated a statistically significant difference.

77

Table 3.6. Primary and secondary antibodies used in this study

Antibody Format Immunogen Host Dilution Source

Anti-pTyr1472

GluN2B

Whole IgG,

unconjugated

Synthetic

phosphopeptide Rabbit 1:1000

Cell Signaling

Technology,

Danvers, MA

Anti-GluN2B Whole IgG,

unconjugated

C-terminus of

mouse GluN2B Rabbit 1:1000

Cell Signaling

Technology

Anti-pTyr402

Pyk2

Whole IgG,

unconjugated

Synthetic

phosphopeptide of

human Pyk2

Rabbit 1:1000 Invitrogen

Anti-Pyk2 IgG2a C-terminus of

human Pyk2 Mouse 1:1000

Cell Signaling

Technology

Anti-pTyr204

ERK1/2

Whole IgG,

unconjugated

Synthetic

phosphopeptide Mouse 1:500

Santa Cruz

Biotechnology

Anti-ERK2 Whole IgG,

unconjugated

C-terminus of rat

p44 MAP Kinase Rabbit 1:20,000

Cell Signaling

Technology

Anti-GAPDH,

clone 6c5

IgG1,

unconjugated

Purified protein

from rabbit muscle Mouse 1:20,000 Millipore

Anti-rabbit

Whole IgG

peroxidase-

conjugated

Rabbit Fc Donkey 1:10,000 Amersham

Biosciences

Anti-mouse

Whole IgG

peroxidase-

conjugated

Mouse Fc Sheep 1:5,000 Amersham

Biosciences

References

1. For a general overview of synaptic plasticity in neuropsychiatric disorders, see the following:

(a) Lu scher, C.; Issac, J. T. J. Physiol. 2009, 587, 727; (b) Palop, J. J.; Chin, J.; Mucke, L.

Nature 2006, 443, 768.

2. (a) Selkoe, D. J. Science 2002, 298, 789; (b) Burke, S. N.; Barnes, C. A. Nat. Rev. Neurosci.

2006, 7, 30.

3. (a) Stephan, K. E.; Friston, K. J.; Frith, C. D. Schizophren. Bull. 2009, 35, 509; (b) Stephan,

K. E.; Baldeweg, T.; Friston, K. J. Biol. Psychiatry 2006, 59, 929.

4. Duman, R. S. Eur. Psychiatry 2002, 17, 306.

78

5. (a) Garner, C. C.; Wetmore, D. Z. Adv. Exp. Med. Biol. 2012, 970, 451; (b) Huttenlocher, P.

R. Pediatr. Neurol. 1991, 7, 79; (c) O’Donnell, W. T.; Warren, S. T. Annu. Rev. Neurosci. 2002,

25, 315.

6. Gerdeman, G. L.; Partridge, J. G.; Lupica, C. R.; Lovinger, D. M. Trends Neurosci. 2003, 26,

184.

7. Paul, S.; Nairn, A.; Wang, P.; Lombroso, P. J. Nat. Neurosci. 2003, 6, 34.

8. Xu, J.; Kurup, P.; Bartos, J. A.; Hell, J. W.; Lombroso, P. J. J. Biol. Chem. 2012, 287, 20942.

9. Munoz, J. J.; Tarrega, C.; BlancoAparicio, C.; Pulido, R. Biochem. J. 2003, 372, 193.

10. Snyder, E. M.; Nong, Y.; Almeida, C. G.; Paul, S.; Moran, T.; Choi, E. Y.; Nairn, A. C.;

Salter, M. W.; Lombroso, P. J.; Gouras, G. K.; Greengard, P. Nat. Neurosci. 2005, 8, 1051.

11. (a) Zhang, Y. F.; Kurup, P.; Xu, J.; Carty, N.; Fernandez, S.; Nygaard, H. B.; Pittenger, C.;

Greengard, P.; Strittmatter, S.; Nairn, A. C.; Lombroso, P. J. Proc. Nat. Acad. Sci. U.S.A. 2010,

107, 19014; (b) Zhang, Y. F.; Kurup, P.; Xu, J.; Anderson, G.; Greengard, P.; Nairn, A. C.;

Lombroso, P. J. J. Neurochem. 2011, 119, 664.

12. Sahn, J. J.; Martin, S. F. ACS Comb. Sci. 2012, 14, 496.

13. (a) Soellner, M. B.; Rawls, K. A.; Grundner, C.; Alber, T.; Ellman, J. A. J. Am. Chem. Soc.

2007, 129, 9613; (b) Rawls, K. A.; Lang, P. T.; Takeuchi, J.; Imamura, S.; Baguley, T. D.;

Grundner, C.; Alber, T.; Ellman, J. A. Bioorg. Med. Chem. Lett. 2009, 19, 6851.

14. (a) Sun, J.-P.; Fedorov, A. A.; Lee, S.-Y.; Guo, X.-L.; Shen, K.; Lawrence, D. S.; Almo, S.

C.; Zhang, Z.-Y. J. Biol. Chem. 2003, 278, 12406; (b) Shen, K.; Keng, Y.-F.; Wu, L.; Guo, X.-

L.; Lawrence, D. S.; Zhang, Z.-Y. J. Biol. Chem. 2001, 276, 47311; (c) Yao, Z.-J.; Ye, B.; Wu,

X.-W.; Wang, S.; Wu, L.; Zhang, Z.-Y.; Burke Jr., T. R. Bioorg. Med. Chem. 1998, 6, 1799; (d)

Puius, Y. A.; Zhao, Y.; Sullivan, M.; Lawrence, D. S.; Almo, S. C.; Zhang Z.-Y. Proc. Natl.

Acad. Sci. USA 1997, 94, 13420.

15. Combs, A. P.; Yue, E. W.; Bower, M.; Ala, P. J.; Wayland, B.; Douty, B.; Takvorian, A.;

Polam, P.; Wasserman, Z.; Zhu, W.; Crawley, M. L.; Pruitt, J.; Sparks, R.; Glass, B.; Modi, D.;

McLaughlin, E.; Bostrom, L.; Li, M.; Galya, L.; Blom, K.; Hillman, M.; Gonneville, L.; Reid, B.

G.; Wei, M.; Becker-Pasha, M.; Klabe, R.; Huber, R.; Li, Y.; Hollis, G.; Burn, T. C.; Wynn, R.;

Liu, P.; Metcalf, B. J. Med. Chem. 2005, 48, 6544.

16. Liu, G.; Xin, Z.; Pei, Z.; Hajduk, P. J.; Abad-Zapatero, C.; Hutchins, C. W.; Zhao, H.;


M.; Jirousek M. R. J. Med. Chem. 2003, 46, 4232.

17. Hajduk, P. J.; Bures, M.; Praesstgaard, J.; Fesik, S. W. J. Med. Chem. 2000, 43, 3443; (b)

Horton, D. A.; Bourne, G. T.; Smythe, M. L. Chem. Rev. 2003, 103, 893.

18. (a) Montserat, J.; Chen, L.; Lawrence, D. S.; Zhang, Z.-Y. J. Biol. Chem. 1996, 271, 7868;

(b) Bahta, M.; Lountos, G. T.; Dyas, B.; Kim, S.-E.; Ulrich, R. G.; Waugh, D. S.; Burke, T. R.,

Jr. J. Med. Chem. 2011, 54, 2933; (c) Chen, L.; Montserat, J.; Lawrence, D. S.; Zhang, Z.-Y.

Biochemistry 1996, 35, 9349.

19. Fujisaki, S.; Eguchi, H.; Omura, A.; Okamato, A.; Nishida, A. Bull. Chem. Soc. Jpn. 1993,

66, 1576.

20. Konakahara, T.; Kiran, Y. B.; Okuno, Y.; Ikeda, R.; Sakai, N. Tetrahedron Lett. 2010, 51,

2335.

21. Boebel, T. A.; Hartwig, J. F. J. Am. Chem. Soc. 2008, 130, 7534.

22. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.

79

23. Li, X.; Szardenings, A. K.; Holmes, C. P.; Wang, L.; Bhandari, A.; Shi, L.; Navre, M.; Jang,

L.; Grove, J. R. Tetrahedron Lett. 2006, 47, 19.

24. (a) Kolodiazhnyi, O. I.; Grishkun, E. V.; Sheiko, S.; Demchuk, O.; Thoennessen, H.; Jones,

P.; Schmutzler, R. Russ. Chem. Bull. 1999, 48, 1568; (b) Kolodiazhnyi, O. I.; Sheiko, S.;

Grishkun, E. V. Heteroat. Chem. 2000, 11, 138.

25. (a) Blazis, V. J.; Koeller, K. J.; Spilling, C. D. J. Org. Chem. 1995, 60, 931; (b) Smaardijk,

A. A.; Noorda, S.; Van Bolhuis, F.; Wynberg, H. Tetrahedron Lett. 1985, 26, 493.

26. (a) Salvi, L.; Kim, J. G.; Walsh, P. J. J. Am. Chem. Soc. 2009, 131, 12483; (b) Nugent, W. A.

Chem. Commun. 1999, 1369.

27. (a) Lee, H.; Xie, L.; Luo, Y.; Lee, S.-Y.; Lawrence, D. S.; Wang, X. B.; Sotgia, F.; Lisanti,

M. P.; Zhang, Z.-Y. Biochemistry 2006, 45, 234; (b) Moller, N. P. H.; Andersen, H. S.; Jeppesen,

C. B.; Iversen, L. F. Handb. Exp. Pharmacol. 2005, 167, 215; (c) Zhang, S.; Zhang, Z.-Y. Drug

Discov. Today 2007, 12, 373.

28. (a) Di, L.; Kerns, E. H.; Bezar, I. F.; Petusky, S. L.; Huang, Y. J. Pharm. Sci. 2009, 98, 1980;

(b) Mensch, J.; Melis, A.; Mackie, C.; Verreck, G.; Brewster, M. E.; Augustijns, P. Eur. J.

Pharm. Biopharm. 2010, 74, 495.

29. For recent reviews and leading references on the development of phosphatase inhibitors, see

the following: (a) Vintonyak, V. V.; Waldmann, H.; Rauh, D. Bioorg. Med. Chem. 2011, 19,

2145; (b) Combs, A. P. J. Med. Chem. 2010, 53, 2333; (c) Blaskovich, M. A. T. Curr. Med.

Chem. 2009, 16, 2095.

30. (a) Rautio, J.; Laine, K.; Gynther, M.; Savolainen, J. AAPS J. 2008, 10, 92; (b) Pavan, B.;

Dalpiaz, A.; Ciliberti, N.; Biondi, C.; Manfredini, S.; Vertuani, S. Molecules 2008, 13, 1035; (c)

Prokai, L.; Prokai-Tatrai, K.; Bodor, N. Med. Res. Rev. 2000, 20, 367.

31. Murano, T.; Takechi, H.; Yuasa, Y.; Yokomatsu, T.; Umesue, I.; Soeda, S.; Shimeno, H.;

Shibuya, S. ARKIVOC 2003, 8, 256.

32. Vedso, P.; Olesen, P. H.; Hoeg-Jensen, T. Synlett 2004, 5, 892.

33. Xu, J.; Kurup, P.; Zhang, Y. F.; Goebel-Goody, S.; Wu, P.; Hawasli, A. H.; Baum, M.; Bibb,

J.; Lombroso, P. J. J. Neurosci. 2009, 29, 9330.

80

81

Chapter 4. Benzopentathiepins as novel redox-reversible inhibitors of STEP

Abstract: This chapter discusses the discovery and characterization of benzopentathiepins as

redox-reversible inhibitors of STEP. The majority of the chapter focuses on the biochemical

characterization of the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-

amine hydrochloride (TC-2153). It is established that the cyclic polysulfide pharmacophore

forms a reversible covalent bond with the catalytic cysteine in STEP. Several analogs of TC-

2153 are prepared to define not only what is important for inhibition, but also to identify

locations on the molecule that are amenable to diversification for further compound

development. Finally, TC-2153 is shown to be active in cell-based secondary assays and in

animal behavioral models. The majority of this work has been published as a full article (Xu, J.;

Chatterjee, M.; Baguley, T. D.; Brouillette, J.; Kurup, P.; Ghosh, D.; Kanyo, J.; Zhang, Y.; Seyb,

K.; Ononenyi, C.; Foscue, E.; Anderson, G. M.; Gresack, J.; Cuny, G. D.; Glicksman, M. A.;

Greengard, P.; Lam, T. T.; Tautz, L.; Nairn, A. C.; Ellman, J. A.; Lombroso P. J. PLoS Biol.

2014, 8, e1001923).

82

Authorship

This work was conducted in collaboration with Dr. Jian Xu, Dr. Manavi Chatterjee, Dr.

TuKiet Lam and Jean Kanyo. I synthesized all of the compounds and performed all the in vitro

biochemical characterization of the inhibitors including the IC50 determinations, kinact/Ki

determinations, and dialysis and compound stability experiments. Dr. Jian Xu and Dr. Manavi

Chatterjee, researchers in Dr. Paul Lombroso’s research group (Yale School of Medicine),

conducted the cell based assays and animal behavioral studies presented in this chapter. Jean

Kanyo and Dr. TuKiet Lam (Keck Biotechnology Resource Laboratory, Yale) were responsible

for obtaining the LC-MS/MS data.

Introduction

STEP as a therapeutic target

As mentioned in chapter 3, STEP is a phosphatase discovered by our collaborators at the

Yale School of Medicine that has been implicated in many neurodegenerative diseases, such as

Alzheimer’s disease (AD).1 Genetic deletion of STEP has been shown to improve cognitive

function in 3xTg-AD mice in a variety of behavioral models, including the Morris water maze

(Figure 4.1).1a

In the Morris water maze test, the time it takes a mouse to escape a familiar maze

is recorded. If the mouse has normal cognitive faculties, its escape latency will decrease with

subsequent training sessions. AD model mice are impaired in this task (3xTg-AD), but the

genetic deletion of STEP ameliorated this cognitive deficit (double mutant (DM): 3xTg-AD that

are also STEP–/–

). The Lombroso group has been extremely interested in attaining STEP

inhibitors since its discovery because of these positive biological results.

Figure 4.1. Escape latency of AD mouse models showing the 3xTg-AD disease model mice that are also

genetic knockout STEP–/–

(double mutants, DM) have improved cognitive function over the diseased

model mice.1a

Initial high throughput screening results

Parallel to our efforts in chapter 3, the Lombroso research group conducted a HTS to identify

inhibitors of STEP. After screening a library of around 150,000 compounds from the Laboratory

for Drug Discovery in Neurodegeneration (LDDN) library, eight compounds were selected for

83

further characterization based on chemical structure and IC50 values, which ranged between 1

M and 9.7 M, including compound 4.01 (Figure 4.2). Additionally, studies of these

compounds indicated potent inhibition of STEP activity in neuronal cultures and cortical tissue

after intraperitoneal (i.p.) injections in wild-type mice (data not shown).

Figure 4.2. Compound 4.01 was identified as a lead compound from HTS efforts against STEP.

However, following re-synthesis of several of the lead compounds, including compound

4.01, all exhibited essentially no inhibitory activity towards STEP (Figure 4.3). After preparative

HPLC on compound 4.01 and testing the fractions for inhibitory activity, it was discovered that

the there was a highly active contaminant in the commercial samples of the original library

compounds. After isolating this impurity, it was determined that the active component was

elemental sulfur (S8). Encouragingly, elemental sulfur demonstrated good activity in in vitro

enzyme assays, neuronal cultures and cortical tissue after intraperitoneal (i.p.) injections in wild-

type mice (Figure 4.4).

Figure 4.3. Activity of commercial (a) and resynthesized (b) 4.01 against STEP. Upon resynthesis,

compound 4.01 no longer showed appreciable inhibition.

Figure 4.4. Activity of elemental sulfur (S8) against STEP in (a) in vitro enzyme assays (IC50 = 17.2 ± 0.4

nM), (b) neuronal cultures and (c) cortical tissue after i.p. injections in wild-type mice.

84

Benzopentathiepins as attractive target molecules

As a potential drug or tool compound lead, elemental sulfur is a poor compound for two

primary reasons: first, it is very poorly soluble in aqueous solutions; and second, S8 cannot be

modified to improve physicochemical properties, redox activity, binding affinity, or selectivity.

Therefore, we sought to identify more conventional inhibitor structures that would improve

solubility and enable further refinement through analog preparation and evaluation. We

identified the benzopentathiepin core structure 4.03, which is present in a number of natural

products, as the most promising for further investigation (Figure 4.5). Natural products

incorporating the benzopentathiepin core motif have been reported to have antifungal and

antibacterial activity in cell culture as well as cytotoxicity against human cancer cell lines.2

Additionally, amino substituted derivatives such as varacin (4.04) and 8-(trifluoromethyl)-

1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (TC-2153, 4.05) have reasonable solubility

in aqueous solution.3 TC-2153 was designed as an analog to benzopentathiepin containing

natural products by a research team at a Siberian research institute and reportedly has a low level

of acute toxicity (LD50 > 1,000 mg/kg) and was proposed to cross the blood brain barrier as

evidenced by anxiolytic and anticonvulsant effects in mice.4 Finally, from a medicinal chemistry

point-of-view, we imagined that the scaffold present in TC-2153 would allow for modification to

address any physicochemical properties of interest.

Figure 4.5. Polysulfide containing compounds, including TC-2153 (4.05).

Synthesis of TC-2153

The synthesis of TC-2153 began with the literature synthesis of intermediate 4.08 (Scheme

4.1).5 Commercially available 2-chloro-1,3-dinitro-5-(trifluoromethyl)-benzene (4.06) first

undergoes a double SNAr reaction with sodium N,N-dimethyldithiocarbamate to give

benzodithiolone 4.07 in 40% yield. Further attempts to optimize the yield of this reaction were

unsuccessful because upon hydrolysis of the intermediate iminium ion, either dimethyl amine or

the thiophenol can act as a competent leaving group, with only the former leading to the desired

product. Treatment of intermediate 4.07 with excess sodium hydrosulfide (NaSH) yields the

desired benzopentathiepin intermediate 4.08 in 41% yield. In this reaction, the hydrosulfide acts

as both the terminal reductant to reduce the nitro group to the aniline and as a source of sulfur to

form the pentathiepin ring. Although other ring sizes are possible, the 7-membered pentathiepin

is the thermodynamically most stable species and it adopts a chair-like structure.3b

However, in

addition to the desired product, multiple polysulfidic byproducts are also formed in this reaction,

including nitrotrithiol 4.09, and the dimeric structure 4.10, which greatly complicate the isolation

and purification of compound 4.08. By lowering the concentration in the reaction, less of dimer

4.10 is formed, but the nitro compound 4.09 becomes the major product of the reaction (data not

shown). It was first thought that compound 4.09 is an intermediate product, resulting from partial

85

pentathiepin ring formation prior to nitro reduction. However, resubjecting 4.09 to the reaction

conditions yields no desired product. Based upon this result we hypothesized that the reduction

of the nitro group occurs first, followed by pentathiepin ring formation and that nitro reduction

cannot occur in the presence of the trithiol intermediate.

Scheme 4.1. Original synthesis of intermediate 4.08

In order to test this hypothesis, we elected to selectively reduce the nitro group of 4.07 to

provide aniline 4.11, which would then be subjected to the pentathiepin ring formation step

(Scheme 4.2). Adopting a literature procedure,6 we found that reduction of the nitro group

proceeded cleanly upon treatment with tin (II) chloride and concentrated HCl to yield the desired

aniline 4.11 in 90% yield and high purity with only extractive isolation. Subsequent treatment

with NaSH yields the desired intermediate 4.08 in better yield (55% over two steps) than the

original synthesis, and more importantly eliminates the major byproducts that complicated

purification and resulted in impure product. One other parameter that was crucial to minimizing

formation of dimer 4.10 was the proper workup conditions of the reaction. The reaction must be

quenched with concentrated HCl (1 N, 4 N, and even 10 N HCl led to an increase of dimer

formation, data not shown) and the acidic solution must be neutralized by NaHCO3. Quenching

with NaOH, even at 1 N but especially higher concentrations, led to an increase in dimer

formation (data not shown).

Scheme 4.2. Selective reduction of the nitro group in 4.07 to give 4.11 leads to cleaner conversion to 4.08

Further optimization of this selective nitro reduction strategy led to an optimized synthesis of

TC-2153 (Scheme 4.3). The final reduction conditions of ammonium chloride and zinc dust7

were chosen for ease of scalability with typical yields ranging from 85% to 98% for the

reduction step depending on the scale of the reaction. This synthetic sequence eliminates

contaminating byproducts. Additionally, this sequence has been used to generate > 20 gram

quantities of TC-2153 in high purity, which was needed for ongoing animal studies,

pharmacokinetic (PK) studies and analog synthesis (vide infra).

86

Scheme 4.3. Optimized synthetic route for the preparation of multi-gram quantities of TC-2153

Mechanism of STEP inhibition by TC-2153

Enzymatic characterization of inhibition

With TC-2153 prepared, we could begin to answer the question of if and how it may inhibit

STEP. Initial in vitro assays of the compound indicated irreversible inhibition (Figure 4.6). In the

standard inhibition assay, TC-2153 showed potent inhibition with an IC50 of 24.6 ± 0.8 nM

(Figure 4.6a). To evaluate the mode of inhibition, STEP was incubated with TC-2153, the

sample was subjected to dialysis to remove excess inhibitor, and enzyme activity was determined

(Figure 4.6b). After 24 h of dialysis, STEP remained inhibited, establishing that TC-2153 acts as

an irreversible inhibitor under these conditions. Using the progress curve method,8 inhibition was

also found to be irreversible and the second-order rate of inactivation was determined (Figure

4.6c). A kobs was determined for pNPP in the presence of varying initial inhibitor concentrations

(n ≥ 4). Values were then analyzed with non-linear regression to obtain the kinetic constants:

kinact = 0.0176 ± 0.0007 s–1

; Ki = 115 ± 10 nM; kinact/Ki = 153,000 ± 15,000 M–1

s–1

.

Figure 4.6. TC-2153 irreversibly inhibits STEP under standard assay conditions. (a) The IC50 of TC-2153

is 24.6 ± 0.8 nM. (b) Inhibition is not reversed with dialysis of excess inhibitor. (c) The progress curve

method was used to determine the second-order rate of inactivation: kinact/Ki = 153,000 ± 15,000 M–1

s–1

.

Reduced glutathione (GSH) is a ubiquitous reducing agent in cells,9 and may interact with

TC-2153 because of its polysulfide character. Therefore, STEP inhibition was tested with the

addition of thiols (i.e., GSH and DTT) and inhibition was found to be reversible under these

conditions (Figure 4.7). The addition of GSH (1 mM) decreased the inhibitory activity of TC-

2153 by two orders of magnitude in in vitro assays (Figure 4.7a). Inhibition of STEP by TC-2153

is also reversible through the addition of thiol reducing agents as seen by the recovery of STEP

87

activity after pre-inhibition with TC-2153 (Figure 4.7b). In particular, aliquots of STEP were

incubated with DMSO control or TC-2153 and were then added to assay buffer containing 1 mM

GSH, 1 mM DTT, or water control and allowed to incubate for up to 1 h prior to testing for

enzymatic activity. STEP activity was rapidly recovered by both reductants, with DTT showing a

greater recovery of activity (75% recovery after 1 h, where DMSO control represents 100%

activity) compared to GSH (29% recovery after 1 h).

Figure 4.7. Inhibition of STEP by TC-2153 is reversible through the addition of thiol reducing agents. (a)

The IC50 of TC-2153 is 8.8 ± 0.4 M with addition of 1 mM GSH to the assay. (b) STEP activity is

recovered from pre-inhibited STEP by treatment with thiol reducing agents.

These results suggested an oxidative mechanism for the inhibition of STEP, similar to the

redox-reversible regulation of PTPs discussed in chapter 1. An early hypothesis was that TC-

2153 was decomposing or otherwise generating reactive oxygen species (ROS) under the assay

conditions, but these mechanisms for STEP inhibition were ruled out through a series of

experiments. First, by monitoring the 19

F NMR signal of TC-2153 (details in experimental

section) it was established that TC-2153 was stable and did not degenerate in the assay

conditions. Moreover, upon addition of catalase or superoxide dismutase to the in vitro assay

(Table 4.1), there was no change in inhibition, indicating that TC-2153 is not acting through

generation of ROS.

Table 4.1. In vitro inhibition of STEP by TC-2153

Conditions IC50 (nM)

no additivea 24.6 ± 0.8

+ catalaseb,c

26.2 ± 0.6

+ SODb,d

18.6 ± 0.8

+ catalase + SODb,c,d

24.5 ± 4.2

+ GSHb,e

8,790 ± 430 aMean ± S.D. (n = 4).

bMean ± S.D. (n = 2).

c80 U/mL catalase.

d100 U/mL

superoxide dismutase (SOD). e1 mM reduced glutathione (GSH).

LC-MS/MS characterization of inhibition

In order to further elucidate the inhibition of STEP by TC-2153, LCMS analysis was

performed to determine the intact protein mass of STEP and STEP+TC-2153. The intact protein

analyses suggest a covalent adduct to STEP. Although we were able to obtain the accurate mass

for STEP, we were unable to resolve the heterogeneous mixture of intact STEP+TC-2153 and its

covalent adducts with sufficient accuracy to fully interpret the results.

88

Therefore, we next used high-resolution tandem mass spectrometry to focus upon whether

TC-2153 might modify the active site cysteine of STEP. For these experiments, we used WT

STEP as well as a STEP mutant in which the catalytic cysteine was changed to serine. Greater

than 90% of the primary amino acid sequences were identified by LC-MS/MS for WT STEP or

for the STEP mutant, following in-gel tryptic digestion of STEP from non-denaturing (native)

preparations. We initially analyzed the catalytic cysteine at position 472 of STEP in the absence

of TC-2153 and found a disulfide bridge between Cys465

and Cys472

that presumably forms

following tryptic digestion given that Cys465

and Cys472

are > 20 Å apart from each other in the

3-dimensional X-ray crystal structure of STEP.10

This modification was not observed when the

catalytic site cysteine (Cys472

) was mutated to serine. Incubation of WT STEP with TC-2153

resulted in the presence of a de novo trisulfide within the Cys465

/Cys472

bridge. Consistent with

covalent modification by TC-2153, this modification that was not observed for WT STEP alone

or when the catalytic site cysteine (Cys472

) was mutated to serine (Figure 4.8). The precursor

monoisotopic mass of the trisulfide-containing peptide had a mass error of 4 ppm (~0.011 Da)

based on theoretical mass calculation, which is within the 5 ppm external mass calibration

expected for MS/MS data collected by the linear ion trap instrument used.

Figure 4.8. Detection of trisulfide bridge formation between C

465 and C

472. The peptide sequence in (a)

illustrates the trisulfide bridge along with the b and y-ions assignments detected in the MS/MS

fragmentations spectrum. Part (b) compares the 3D elution profile of the trisulfide peptide (mass =

2746.242 Da). The trisulfide bridge (modified) peptide is only detected in the WT STEP in the presence

of TC-2153. The corresponding disulfide (non-modified) peptide (mass = 2714.254Da) was detected in

WT STEP.

TC-2153 was the only exogenous source of S atoms in these samples. Although the direct

modification, whether whole or partial molecule attachment, cannot be inferred from this data,

these results indicate that the active site cysteine is likely modified by TC-2153 and suggest that

following tryptic digestion a sulfur atom from the benzopentathiepin core is retained giving rise

to the trisulfide which was identified by mass spectrometry.

Preparation of TC-2153 analogs for STEP inhibition

We were next interested in synthesizing derivatives of TC-2153 to be evaluated for inhibition

of STEP. Given the promising biological properties of TC-2153 (vide infra), we were motivated

to define the essential characteristics of the molecule necessary for enzyme inhibition (Figure

4.9). First, we wished to explore the electronic effects of the inhibitor core to assess the

importance of electronics on the redox-reversible inhibition of STEP. Secondly, we wanted to

89

discover sites on the molecule tolerant of substitution to enable modulation of physicochemical

properties such as solubility, for the introduction of reporter groups, or for the introduction of

functionality to facilitate pull down assays and proteomic analysis.

Figure 4.9. Proposed SAR study of TC-2153.

TC-2153 analog synthesis

To prepare the analogs, we first investigated their synthesis using the sequence reported by

Kulikov et. al. (Scheme 4.4),5 which was introduced in Scheme 4.1. Elevated temperatures were

required for the SNAr reactions with sodium dimethyldithiocarbamate when the less activated

substrates 4.12 and 4.13 containing a simple methyl or proton substituent at the R position were

used, with lower temperatures resulting only in a single nucleophilic substitution of the chloride.

Consistent with a previous report,11

treatment of the intermediate nitrodithiolones 4.14 and 4.15

with NaSH yielded little to no desired product and only nitrotrithiol byproducts were formed in

any appreciable yield (e.g., compound 4.09). As was discussed above for the synthesis of TC-

2153, by first reducing the nitro group to the free anilines, the desired synthetic intermediates

could be obtained (Scheme 4.5), and the synthesis of TC-2153 analogs 4.20 and 4.21 could be

completed.

Scheme 4.4. Attempted synthesis of benzopentathiepin compounds 4.16 and 4.17

Scheme 4.5. Completed synthesis of TC-2153 analogs 4.20 and 4.21

Analog 4.24, which does not contain an amine, was synthesized using an analogous

procedure (Scheme 4.6). 2-Chloro-1-nitro-4-(trifluoromethyl)benzene 4.22 was treated with

sodium dimethyldithiocarbamate at elevated temperature to yield the dithialone 4.23. Treatment

with NaSH in DMSO yielded the analog 4.24. The amine provides a convenient handle for

potentially adding solubilizing functionality or chemical labels, which has the potential of being

90

introduced either by alkylation to maintain the basicity of the amine or by acylation. Acylation of

intermediate 4.08 according to literature precedent with either acetyl chloride or trifluoroacetic

anhydride yielded analogs 4.25 and 4.26 respectively (Scheme 4.7).4 Reductive amination of

4.08 with acetaldehyde or benzaldehyde provided N-ethyl and N-benzyl analogs 4.27 and 4.28

(Scheme 4.8).

Scheme 4.6. Synthesis of TC-2153 analog 4.24

Scheme 4.7. Synthesis of TC-2153 analogs 4.25 and 4.26 through acetylation of 4.08

Scheme 4.8. Synthesis of TC-2153 analogs 4.27 and 4.28 through reductive amination of 4.08

Inhibition of STEP by TC-2153 analogs

The inhibitory activity of the analogs was determined against STEP both in the presence and

the absence of 1 mM GSH (Table 4.2). Because of the differential results with TC-2153 (Figure

4.7a), we thought it was prudent to investigate the inhibitor activity under both of these

conditions, as GSH may interact with the inhibitors differentially. In the absence of GSH the

most potent inhibitor in the series was the simple 7-(trifluoromethyl)-benzopentathiepin 4.24 (10

± 1 nM). However, with the absence of the amine substituent, the solubility and thus future

utility of 4.24 is limited. With a benchmark IC50 of 24.6 ± 0.8 nM, TC-2153 (4.05) remains one

of the most potent analogs in this series, along with the other aniline hydrochloride salts 4.20 and

4.20 (25 ± 7 nM) and 4.21 (32 ± 3 nM). Trifluoroacetamide derivative 4.25 also showed good

potency (24 ± 1 nM), while the less electron deficient acetamide derivative 4.26 showed a two-

fold decrease in inhibitory activity (49 ± 2 nM). Alkylated anilines 4.27 and 4.28, which are

slightly less electron deficient than the parent aniline, showed a marked decrease in potency.

91

Interestingly, the byproducts generated in the synthesis of TC-2153 (4.09 and 4.29) also showed

activity against STEP at decreased potency.

When GSH was added to the assay mixture, the same trends generally held, with the acylated

derivatives performing comparably to the free anilines and the alkylated derivatives having

slightly decreased potency. In all cases, there was a decrease in potency by 2–3 orders of

magnitude when GSH was present in the assay.

Table 4.2. Inhibition of STEP in vitro in the presence and absence of GSH

entry compound R1 R

2 IC50 (nM)

a,b IC50 (M)

a,c

1 4.05 (TC-2153) CF3 NH3Cl 24.6 ± 0.8d

8.8 ± 0.4d

2 4.24 CF3 H 10 ± 1 17 ± 2

3 4.20 CH3 NH3Cl 25 ± 7 17 ± 1

4 4.21 H NH3Cl 32 ± 3 33 ± 4

5 4.25 CF3 NHCOCF3 24 ± 1 15 ± 2

6 4.26 CF3 NHAc 49 ± 2 24 ± 1

7 4.27 CF3 NHEt 59 ± 9 > 50

8 4.28 CF3 NHBn 78 ± 3 27 ± 5

9 4.29 -- -- 33 ± 1 20 ± 1

10 4.09 -- -- 145 ± 6 34 ± 4 aAssays were performed in duplicate (mean ± S.D.).

bAssays contained no GSH.

cAssays contained 1 mM

GSH. dAssays were performed in quadruplicate (mean ± S.D.)

Because the inhibition of STEP by TC-2153 was shown to be irreversible in the absence of

GSH (Figure 4.6), we next determined the second order rate of inactivation (kinact/Ki) for all the

inhibitors under these conditions using the progress-curve method (Table 4.3).8 These results

demonstrate that the methyl (4.20) and unsubstituted (4.21) derivatives are about three times less

potent than trifluoromethyl substituted TC-2153 (4.05). Additionally, the alkylation in

derivatives 4.27 and 4.28 was further confirmed to not be beneficial. Even though their Ki values

indicated that binding is not as favored, acylation of the scaffold (4.25 and 4.26) resulted in

inhibitors with higher potency than TC-2153 because of their much higher kinact values relative to

the non-acylated parent structure 4.05.

92

Table 4.3. Second-order rates of inactivation of TC-2153 analogsa

entry compound kinact/Ki (M–1

s–1

) Ki (nM) kinact (s–1

)

1 4.05 (TC-2153) 153,000 ± 15,000 115 ± 10 0.0176 ± 0.0007

2 4.24 135,000 ± 18,000 109 ± 14 0.0147 ± 0.0007

3 4.20 41,000 ± 5,300 116 ± 14 0.0048 ± 0.0002

4 4.21 52,200 ± 8,000 183 ± 25 0.0095 ± 0.0006

5 4.25 161,000 ± 26,000 235 ± 33 0.0378 ± 0.0027

6 4.26 193,000 ± 49,000 123 ± 29 0.0236 ± 0.0022

7 4.27 53,500 ± 8,800 230 ± 34 0.0123 ± 0.0009

8 4.28 33,200 ± 2,400 175 ± 11 0.0058 ± 0.0002

9 4.29 138,000 ± 23,000 124 ± 19 0.0171 ± 0.0011

10 4.09 98,000 ± 20,000 210 ± 38 0.0205 ± 0.0018 aAssays were performed in quadruplicate (mean ± S.D.) and in the absence of GSH.

TC-2153 activity in cell-based secondary assays and in vivo

TC-2153 activity in cortical neurons and in vivo

The activity of TC-2153 was next tested in secondary cell-based assays. Cortical neurons

were treated for 1 h with TC-2153 and Tyr phosphorylation levels of residues that STEP

dephosphorylates on GluN2B (Y1472

), Pyk2 (Y402

), and ERK1/2 (Y204/187

) were determined.

There was a significant increase in the Tyr phosphorylation of all three STEP substrates (Figure

4.10a) (1 M dose: pGluN2B: 2.07 ± 0.15, p < 0.001; pPyk2: 1.81 ± 0.21, p < 0.001; pERK1/2:

2.39 ± 0.18, p < 0.001). The decrease in Tyr phosphorylation in the presence of the highest dose

of TC-2153 (10 M) may be due to off-target effects on positive regulatory PTPs. Similar

inverted-U dose-response curves on Tyr phosphorylation of direct PTP targets has been observed

in previous work with PTP inhibitors.12

It was then determined whether TC-2153 inhibited STEP activity in WT mice in vivo. Six-

month old male mice (C57BL/6) were injected with vehicle or TC-2153 (1, 3, 6, 10 mg/kg, i.p.)

and cortices were removed and processed 3 h post injection. TC-2153 led to a significant

increase in the Tyr phosphorylation of GluN2B, Pyk2, and ERK1/2 (at 10 mg/kg: pGluN2B:

1.66 ± 0.28, p < 0.01; pPyk2: 1.80 ± 0.30, p < 0.05; pERK1/2: 2.52 ± 0.16, p < 0.01) (Figure

4.10b). Together, these results demonstrate that TC-2153 increases the Tyr phosphorylation of

three STEP substrates in intact neurons in culture and in vivo in the cortex of WT mice.

TC-2153 specificity in vivo

To address possible off-target inhibition by TC-2153 in cells, cortical cultures from either

WT or STEP KO mice were treated with TC-2153. Similar to the rat neuronal cultures, there was

an observed increase in the Tyr phosphorylation of STEP substrates in WT mouse cortical

neurons (Figure 4.11, black bars). Consistent with previous findings,13

STEP substrates have

higher basal Tyr phosphorylation levels in STEP KO cultures. TC-2153 failed to increase the

phosphorylation of STEP substrates in the KO cultures (Figure 4.11, grey bar with 0.1 M and 1

M). To exclude a possible ceiling effect, the generic tyrosine phosphatase inhibitor, sodium

orthovanadate (Na3VO4), was added and the Tyr phosphorylation of these substrates was further

93

increased. These results suggest that TC-2153 is relatively specific towards STEP compared to

the generic tyrosine phosphatase inhibitor sodium orthovanadate.

Figure 4.10. (a) Cortical neuronal cultures were treated with TC-2153 and vehicle (0.05, 0.1, 1 and 10

M) for 1 h. Phosphorylation of GluN2B (Y1472

), Pyk2 (Y402

) and ERK1/2 (Y204/187

) were significantly

higher after treatment of cultures with TC-2153 (*p < 0.05, **p < 0.01, ***p < 0.001; one-way ANOVA

with post hoc Bonferroni test). Data represent the phospho-signal normalized to total protein and then to

GAPDH (mean + s.e.m., n = 4). (b) C57BL/6 mice (3–6 months) were injected with TC-2153 (i.p., 1, 3,

6, 10 mg/kg) and were sacrificed 3 h later. Cortices were micro-dissected and lysates spun down to P2

fraction and prepared for western blotting. Tyrosine phosphorylation status was probed with phospho-

specific antibodies to pGluN2B: Tyr1472

, pPyk2: Tyr402

and pERK1/2: Tyr204/187

(*p < 0.05; **p < 0.01;

one-way ANOVA with post hoc Bonferroni test). Data represent the phospho-signal normalized to the

total substrate protein signal and then to GAPDH as a protein expression control (mean + s.e.m., n = 3).

Figure 4.11. TC-2153 failed to increase tyrosine phosphorylation of STEP substrates in STEP KO

cortical neurons. WT and STEP KO cultures were treated with TC-2153 (0.1 and 1 M), vehicle (0.1%

DMSO) or sodium orthovanadate (Na3VO4, 1 mM) for 1 h. Phosphorylation of GluN2B Y1472

, Pyk2 Y402

and ERK1/2 Y204/187

was normalized to total substrate protein level and then to GAPDH as a protein

expression control (*p < 0.05, **p < 0.01 one-way ANOVA with post hoc Bonferroni test, compared with

veh-treated controls, n = 4).

Additionally, there are three highly related PTPs (STEP, HePTP, and PTP-SL) that all

dephosphorylate ERK1/2.14

Only STEP is found in cortex, while HePTP is present in spleen, and

PTP-SL is present in cerebellum, both tissues that lack STEP. In addition, ERK1/2 and Pyk2 are

dephosphorylated by other tyrosine phosphatases outside of the CNS. To further probe this

94

apparent specificity, WT and STEP KO mice were injected with TC-2153 or vehicle, and the Tyr

phosphorylation of ERK1/2 (Y204/187

) and Pyk2 (Y402

) in different tissues was determined (Figure

4.12). There was a significant increase observed in pERK1/2 and pPyk2 in the frontal cortex and

hippocampus, tissues that contain STEP, but not in the cerebellum or in all tissues tested outside

the brain. These results suggest that TC-2153 does not target homologous PTPs known to

dephosphorylate ERK1/2 and Pyk2 when tested in vivo.

Figure 4.12. TC-2153 increased the phosphorylation of ERK1/2 Y

204/187 and Pyk2 Y

402 in frontal cortex

(a) and hippocampus (b), tissues that contain STEP, but not in cerebellum, spleen, kidney or pancreas (c–

f), all tissues that do not have STEP. Mice were injected i.p. with TC-2153 (10 mg/kg; n = 4) or vehicle (n

= 4) and were sacrificed 3 h later. Changes are expressed as the mean ± s.e.m. of pERK1/2 and pPyk2

normalized to total substrate protein level and then to GAPDH as a protein expression control (*p < 0.05,

**p < 0.01; two-way ANOVA followed by Tukey’s H.S.D. test).

TC-2153 reduces cognitive deficits in 3xTg-AD mice

Next, the efficacy of TC-2153 to reverse cognitive deficits in an AD mouse model was tested

in the reference memory version of the Morris water maze (MWM). A three-way ANOVA

analysis revealed a significant genotype × treatment × training day interaction (p < 0.05). Daily

injection of TC-2153 3 h prior to training reversed memory deficits in 3xTg-AD mice on days 5

and 6 of the acquisition phase (p < 0.01) (Figure 4.13a). The longer escape latency of 3xTg-AD

mice injected with vehicle was not attributed to slower swimming speed since no significant

95

differences were found between groups (p > 0.05; two-way ANOVA) (Figure 4.13b). To confirm

memory status, the number of entries in a circular zone located around the previous platform

location (target zone) and in the opposite quadrants was evaluated during the probe trial 24 h

after the last acquisition day. A three-way ANOVA analysis revealed a significant genotype ×

treatment × quadrant interaction (p < 0.004). The 3xTg-AD mice treated with TC-2153 spent as

much time as WT mice in the target zone, while AD mice injected with vehicle showed no

preference for the target zone (Figure 4.13c). All groups had similar escape latencies during the

cued trial when the platform was visible, indicating the absence of sensorimotor or motivational

deficits to escape from water (WTVeh: 15.1 ± 1.7 s; WT-TC: 15.6 ± 1.7 s; AD-Veh: 15.3 ± 3.0 s;

AD-TC: 16.0 ± 2.3 s; mean ± s.e.m.; p < 0.05; two-way ANOVA).

Figure 4.13. WT and 3xTg-AD mice (male, 6-months old) were treated with vehicle or TC-2153 (10

mg/kg, i.p.) and tested MWM tasks. (a) The 3xTg-AD mice injected with vehicle (n = 6) showed longer

escape latency before finding the hidden-platform (3 trials/day; 60s; 30m intertribal interval) when

compared to AD mice treated with TC-2153 (n = 7) or WT mice injected with vehicle (n = 12) or TC-

2153 (n = 13) (three-way ANOVA). *,+ represents a statistical significant variation between AD-Veh

mice and AD-TC or WT-Veh, respectively. (b) Swim speed at each training day was not significantly

different between groups (three-way ANOVA). (c) Number of entries in a circular zone positioned around

the previous platform location and in the opposite quadrants. *represents a statistical significant variation

between AD-TC mice and other groups for the target quadrant. +indicates a difference for the target and

opposite quadrant within each group. Data are mean ± s.e.m. *,+p < 0.05, **,++p < 0.01, ***,+++p <

0.01.

Additionally, TC-2153 treated 6-month old AD 3xTg-AD mice showed significant increases

in performance in the Y-maze (used to evaluate spatial working memory function), open field

and novel object recognition tests (data not shown). Taken together, these results demonstrate

that TC-2153 significantly improved cognitive functioning in mouse models for AD.

Conclusions

After identifying the benzopentathiepin TC-2153 (4.05) as an attractive molecule for

potential STEP inhibition, a reliable and scalable synthesis was developed to access TC-2153 in

high purity and in quantities needed for animal studies. Once synthesized, inhibition of STEP by

TC-2153 was characterized as a redox-reversible mode of inhibition where a sulfur atom from

the pentathiepin ring modified the catalytic Cys. It was also determined that this oxidative

inhibition was not due to generation of ROS.

Additionally, a series of analogs of TC-2153 has been prepared and characterized in in vitro

enzyme assays. Importantly, these results demonstrate that the trifluoromethyl group contributes

96

to the potency of the molecule and that other modifications are tolerated. Most importantly,

acylation of the aniline is accommodated and could provide a site for introducing reporter groups

or functionality for pull down and proteomic applications.

Moreover, TC-2153 was demonstrated to be active in secondary neuronal cortical culture

assays and in vivo. The inhibition in vivo is also selective for tissues that contain STEP and

highly homologous PTPs are not affected in vivo. Importantly, TC-2153 is efficacious in animal

behavioral models, as exemplified by the Morris water maze test where 3xTg-AD mice that were

treated with TC-2153 demonstrated a significant increase in cognitive function. TC-2153 is

currently undergoing PK studies at 7th

Wave Laboratories (Chesterfield, MO). In addition,

animal behavioral studies have progressed to aged Parkinsonian rhesus monkeys (Jay Schneider,

Thomas Jefferson University) and initial results indicate that TC-2153 is able to improve

cognitive impairment in these studies as well.

Experimental

General materials and methods

Synthetic methods

Unless otherwise noted, all reagents and solvents were obtained from commercial suppliers

and used without further purification. Diethyl ether (Et2O) and CH2Cl2 were passed through a

column of activated alumina (type A2, 12 × 32, Purify Co.) under nitrogen pressure immediately

prior to use. All 1H,

19F and

13C NMR spectra were obtained at ambient temperature on a Bruker

AVB-400 or AVB-500 spectrometer. NMR chemical shifts are reported in ppm relative to CHCl3

(7.26), or DMSO (2.50) for 1H, trifluoroacetic acid (−76.55) for

19F, and CDCl3 (77.16), DMSO-

d6 (39.52) or CD3OD (49.00) for 13

C. Mass spectrometry (HRMS, ESI, GCMS) are reported in

m/z. Chromatography was performed with SiliCycle SiliaFlash® P60 230–400 mesh silica gel.

Reversed-phase purifications were conducted with a Teledyne Isco CombiFlash Rf system

equipped with HP C18 gold cartridges. Unless otherwise noted, product yields are not optimized.

Melting points were recorded on an Electrothermal melting Point Apparatus and are uncorrected.

Enzymatic assays were carried out on a BioTek Synergy 2 Multi-Mode Microplate Reader.

Bioassays

p-Nitrophenyl phosphate (pNPP), 2-(N-morpholino)ethanesulfonic acid (MES), sodium

orthovanadate (Na3VO4), ATP, and all buffer components were purchased from Sigma-Aldrich

(St. Louis, MO). Malachite Green reagent kit was purchased from Bioassay system (Hayward,

CA). 6,8-Difluoro-4-methylumbelliferyl phosphate (DiFMUP) and EnzChek phosphatase assay

kit were purchased from Invitrogen (Carlsbad, CA). For some of the biochemical experiments,

WT TAT-STEP46 and TAT-STEP46 (C to S) proteins were produced and purified using standard

procedures.15

97

Ethics statement

The Yale University Institutional Animal Care and Use Committee approved all proposed

use of animals. All animal work was carried out in strict accordance with National Institutes of

Health (NIH) Guidelines for the Care and Use of Laboratory Animals.

Synthesis of TC-2153

Compound 4.07. Modifying a known literature procedure,

5 a 1 L round bottomed flask was

charged with a magnetic stir bar, 135 g (0.500 mol) of 2-chloro-1,3-dinitro-5-(trifluoromethyl)-

benzene 4.06 and 70 mL of DMSO. The reaction flask was suspended in an ambient temperature

water bath. A solution of sodium dimethyldithiocarbamate dihydrate (89.7 g, 0.500 mol) in

DMSO (200 mL) was added dropwise via cannula over 2 h. After addition, the reaction mixtures

was stirred for 1 h in the water bath and was quenched by addition of 400 mL of water. The

reaction mixture was transferred to a larger flask and was diluted to 2 L with water, then was

extracted with CH2Cl2 (3 × 1 L and then 3 × 500 mL). The combined organic layer was

condensed to approximately 2 L, washed with brine (1 × 1 L), and dried by stirring over MgSO4

for 20 min. The remaining solvent was removed on a rotary evaporator. The resulting solid was

purified by SiO2 flash chromatography using 4 L of SiO2 and was dry-loaded onto the column

with approximately 200 mL of SiO2. The product was eluted first with 9:1 and then 4:1

hexanes:EtOAc to yield the product as a light orange solid (61.4 g, 43.6%). Analytical data

correlates with the published report.5

Original synthesis of compound 4.08 with byproducts 4.09 and 4.10. Following a

literature report,5 a 100 mL round bottomed flask was charged with a magnetic stir bar and 4.07

(1.75 g, 6.20 mmol). DMSO (25 mL, 0.25 M) was added to dissolve the starting material and

2.30 g (31.0 mmol, 5 equiv) of NaSH-H2O was added to the reaction as a solid in one portion.

The reaction mixture was stirred for 18 h at ambient temperature. To quench the reaction, 20 mL

of concentrated HCl was added, and the mixture was stirred at ambient temperature for 20 min.

The reaction mixture was neutralized with 10 mL of 10 N NaOH, and solid NaHCO3 to pH 8.0,

and was extracted with EtOAc (2 × 50 mL). The organic layer was washed with NaHCO3 (70

mL) and dried over MgSO4. Solids were removed by filtration and the volatile components were

removed via rotary evaporation. The crude residue was purified via SiO2 flash column

chromatography with an eluent of 6:1 hexanes:MTBE. Compound 4.08 was isolated as an orange

oil (808 mg, 41%). The analytical data correlates with the published report.5 Also isolated from

this reaction were side products 4.09 (658 mg, 37%) and 4.10 (78 mg, 5%). 4.09: red solid, m.p.

113–116 °C (lit.16

110–111 °C). 1H NMR (500 MHz, CDCl3): δ 8.24 (d, J = 1.7 Hz, 1H), 7.82 (d,

J = 1.7 Hz, 1H); 19

F NMR (376 MHz, CDCl3): δ –62.91; 13

C NMR (126 MHz, CDCl3): δ 146.67,

98

146.37, 146.08, 130.80 (q, JCF = 34.8 Hz), 124.18 (q, JCF = 3.4 Hz), 122.62 (q, JCF = 273.1 Hz),

121.32 (q, JCF = 4.0 Hz). GCMS (m/z): 285. 4.10: yellow solid, m.p. 146–152 °C (dec). 1H NMR

(400 MHz, CDCl3): δ 7.31 (d, J = 1.8 Hz, 2H), 6.56 (d, J = 1.8 Hz, 2H), 4.77 (s, 4H). HRMS-ESI

(m/z): [M+H]+ calcd. for C14H9F6N2S6, 510.8989; found, 510.8982.

Compound 4.11. The procedure is based on an existing literature procedure with some

modifications.7 A 2 L round bottomed three-neck Morten flask was charged with 300 mL of

water, 300 mL of EtOH, 214 g of zinc metal (3.27 mol, 15 equiv), 93.3 g (1.74 mol, 8 equiv) of

ammonium chloride and was equipped with a mechanical stirrer. The reaction flask was placed

into an ice water bath. Nitro compound 4.07 (61.3 g, 0.218 mol) was added as a solid portion-

wise with mechanical stirring over 15 min. The ice bath was removed, and the reaction solution

was allowed to warm to ambient temperature with mechanical stirring for 24 h. The reaction

mixture was neutralized with the addition of 100 mL of saturated aqueous NaHCO3 and was

filtered through a fritted funnel to remove solids. The filter cake was washed with water (500

mL) and CH2Cl2 (1 L). The layers were separated and the water layer was extracted with CH2Cl2

(3 × 500 mL). The combined organic layers were condensed to 1 L, washed with brine (1 L), and

dried by stirring over MgSO4 for 20 min. The solvent was removed on a rotary evaporator,

yielding 45.8 g (83.5%) of 4.11 as an off-white solid, m.p. 148–151 °C. 1H NMR (500 MHz,

CDCl3): d 7.19 (d, J = 1.5 Hz, 1H), 6.88 (d, J = 1.5 Hz, 1H), 3.98 (s, 2H); 19

F NMR (376 MHz,

CDCl3): d –62.85; 13

C NMR (126 MHz, CDCl3): d 188.20, 141.63, 134.16, 130.61 (q, JCF = 33.0

Hz), 123.90 (q, JCF = 272.7 Hz), 121.05, 110.28 (q, JCF = 4.1 Hz), 109.84 (q, JCF = 3.7 Hz).

HRMS-ESI (m/z): [M+H]+ calcd for C8H5F3NOS2, 251.9759; found, 251.9163.

Compound 4.08. This reaction was performed on 15 g batches to maintain a reasonable

solvent volume during the extraction steps (vide infra). A 2 L round bottomed three-neck Morten

flask was charged with 22.3 g of NaSH-H2O (300 mmol, 0.5 M final concentration), 500 mL of

DMSO, was equipped with a mechanical stirrer and was placed in an ambient temperature water

bath. Aniline 4.11 (15.1 g, 60.1 mmol) was dissolved in 100 mL of DMSO and was added to the

NaSH suspension dropwise via cannula with positive N2 pressure over 1 h with mechanical

stirring. The reaction mixture was stirred at ambient temperature under N2 for 16 h and was then

placed in an ice water bath. The reaction was quenched with 50 mL of concentrated HCl and the

resulting mixture was stirred for 45 min in the cold water bath. The reaction was neutralized to

pH 8.0 with NaHCO3, first with 50 mL of a saturated aqueous solution, then with solid NaHCO3.

The resulting mixture was diluted to 4 L with water, then was separated into two fractions of 2 L

each. Each fraction was extracted with CH2Cl2 (4 × 1 L). The combined organic layer is

concentrated to 2 L and was washed with saturated NaHCO3 (1 L), then with brine (1 L), then

was dried over Na2SO4 with stirring for 20 min. The solids were removed by filtration, and the

volatile components were removed with a rotary evaporator with an ice water bath. The crude

99

residue was purified via SiO2 flash chromatography with an eluent of 6:1 then 4:1

hexanes:MTBE to yield 4.08 in two fractions. Other solvent systems (hexanes:CH2Cl2,

hexanes:EtOAC, pentane:ether) were unsuccessful in separating the product from the very close

minor dimer impurity 4.10. Pure product was isolated as a yellow oil (8.49 g, 44%), and a second

fraction of product (7.83 g) was isolated as an orange oil, which contains a small amount of

byproduct 4.10 (<10%) and can be purified further. The NMR data of the purified product

correlates with the published data.5

TC-2153 (4.05). Free aniline 4.08 (23.3 g, 72.8 mmol) was dissolved in Et2O (220 mL, 0.33

M) in a 1 L glass beaker with a stir bar. Concentrated HCl (9.5 mL, 110 mmol, 1.5 equiv) was

added dropwise and a yellow precipitate formed. The mixture was stirred at ambient temperature

for 90 min, at which point the solid product 4.05 was isolated via filtration. Drying over vacuum

yielded 18.28 g (70%) of the pure product as a light yellow solid. The filtrate was collected,

concentrated, dissolved in 50 mL of Et2O, acidified with 1 mL of concentrated HCl and filtered

to acquire a second crop of solid. The solid was washed with cold ether to remove any orange

color, providing an additional crop of pure product (2.09 g, 8%), m.p. 142–147 °C (dec). 1H

NMR (400 MHz, DMSO-d6): δ 9.40–7.20 (br s, 3H), 7.17 (d, J = 2.0 Hz, 1H), 7.13 (d, J = 2.0

Hz, 1H); 19

F NMR (376 MHz, DMSO-d6): δ –62.43; 13

C NMR (126 MHz, DMSO-d6): δ 153.40,

146.21, 131.16 (q, JCF = 32.1 Hz), 124.75, 123.02 (q, JCF = 273.3 Hz), 117.29, 113.82 (q, JCF =

4.8 Hz). The analytical data of the purified product correlates with the published data.5

Synthesis of TC-2153 analogs

Analogs 4.25 and 2.26 were prepared from 4.08 following literature methods.4

Compound 4.14. Modifying a literature procedure,

5 commercially available 2-chloro-5-

methyl-1,3-dinitrobenzene 4.12 (1.08 g, 5.00 mmol) and sodium dimethyldithiocarbamate

dihydrate (896 mg, 5.00 mmol) were dissolved in 10 mL of DMSO and sealed in a microwave

vial with a stir bar. The reaction mixture was heated in a microwave reactor at 150 °C for 1 h.

After cooling to ambient temperature, the mixture was diluted to 100 mL with water followed by

extraction with CH2Cl2 (3 × 100 mL). The combined organic layer was washed with water (3 ×

150 mL), and brine (150 mL), and dried over MgSO4. Solids were removed by filtration and the

volatile components were removed via rotary evaporation to yield the crude product 4.14 as a

dark orange solid (644 mg, 57%), which was taken on to the next step without purification.

100

Compound 4.18. Reduction was achieved following the procedure for 4.11 beginning with

190 mg of the crude product 4.14 and 359 mg of NH4Cl and 817 mg of zinc metal. The

procedure yielded 110 mg (67%) of 4.18 as a yellow solid, m.p. 140–141 °C. 1H NMR (400

MHz, CDCl3): δ 6.78–6.76 (m, 1H), 6.50–6.48 (m, 1H), 3.71 (br s, 2H), 2.29 (s, 3H); 13

C NMR

(151 MHz, CDCl3): δ 189.91, 140.91, 138.02, 132.99, 114.50, 114.26, 114.08, 21.45. HRMS-

ESI (m/z): [M+H]+ calcd. for C8H8NOS2, 198.0042; found, 198.0034.

Analog 4.20. Compound 4.20 was generated following the procedures TC-2153 above.

Treatment of 19 mg (0.10 mmol) of 4.18 with 37 mg (0.50 mmol) of NaSH-H2O in DMSO (1

mL) yielded 12 mg of the intermediate free amine 4.16 as an orange residue after purification on

SiO2 column chromatography. This material was immediately converted to the desired HCl salt

via treatment with 1.5 equiv of HCl in Et2O to yield 4.20 (10 mg, 33% over two steps) as an off-white solid, m.p. 164–168 °C (dec).

1H NMR (400 MHz, DMSO-d6): δ 7.16–6.69 (br s, 3H),

6.81 (d, J = 1.9 Hz, 1H), 6.62 (d, J = 1.9 Hz, 1H), 2.16 (s, 3H); 13

C NMR (151 MHz, CD3OD): δ

147.81, 144.76, 140.61, 135.12, 132.57, 125.52, 21.05. HRMS-ESI (m/z): [M–Cl]+ calcd. for

C7H8NS5, 265.9255; found, 265.9248.

Compound 4.15. The procedure for the formation of 4.15 was followed with 1.102 g (5.00

mmol) of 2-chloro-1,3-dinitrobenzene 4.13. Purification by filtration of the intermediate through

a SiO2 plug (3:1 pentane/Et2O) yielded the product as a dark yellow solid (509 mg, 48%), which

was taken onto the next step without further purification.

Compound 4.19. Reduction was achieved through a modified literature procedure.

6 A 100

mL round bottomed flask was charged with 500 mg (2.00 mmol) of the crude product 4.15, 1.14

g (6.00 mmol, 3.0 equiv) of SnCl2 and a magnetic stir bar. Ethanol (15 mL) and concentrated

HCl (15 mL) were added to the reaction flask, which was placed in a preheated 40 °C oil bath.

The reaction mixture was refluxed under N2 for 18 h. The reaction flask was placed into an ice

bath, and the reaction was quenched by the addition of 15 mL of 10 N NaOH via addition funnel.

The still acidic mixture was neutralized with slow addition of saturated NaHCO3 to pH 8.0

(approx. 100 mL total volume). The mixture was extracted with CH2Cl2 (2 × 100 mL), and the

101

combined organic layers were then washed with NaHCO3 (150 mL) and brine (150 mL), then

dried over MgSO4 and filtered. The solvent was removed on a rotary evaporator, and the crude

product was purified via SiO2 flash column chromatography with an eluent of 25% to 35%

diethyl ether in pentane, and yielded 338 mg (77%) of the pure product as an off-white solid,

m.p. 132–134 °C. 1H NMR (400 MHz, CDCl3): δ 7.16 (t, J = 8.0 Hz, 1H), 6.95 (dd, J = 7.9, 1.0

Hz, 1H), 6.67 (dd, J = 8.1, 1.0 Hz, 1H), 3.76 (br s, 2H); 13

C NMR (151 MHz, CDCl3): δ 189.49,

141.17, 133.22, 127.60, 117.46, 113.51, 113.40. HRMS-ESI (m/z): [M+H]+ calcd. for

C7H6NOS2, 183.9885; found, 183.9874.

Analog 4.21. The procedure used for 4.20 was followed with 37 mg (0.20 mmol) of the

amine 4.19. The procedure yielded 14 mg (25% over two steps) of the product as an off-white

solid, m.p. 162–166 °C (dec). 1H NMR (600 MHz, approx. 15% CD3OD in DMSO-d6): δ 7.08–

7.03 (m, 1H), 6.97–6.94 (m, 1H), 6.83–6.79 (m, 1H); 13

C NMR (151 MHz, approx. 15% CD3OD

in DMSO-d6): δ 152.97, 144.97, 131.60, 123.47, 121.69, 118.22. HRMS-ESI (m/z): [M–Cl]+

calcd. for C6H6NS5, 251.9098; found, 251.9097.

Compound 4.23. 2-Chloro-1-nitro-4-(trifluoromethyl)benzene 4.22 (895 mg, 3.50 mmol)

was dissolved in 7 mL of DMSO and was treated with 627 mg (3.50 mmol) of sodium

dimethyldithiocarbamate dihydrate in a sealed vial in a microwave reactor at 200 °C for 20 min.

The mixture was diluted to 70 mL with water and extracted into 70 mL of CH2Cl2. The organic

layer was washed with brine (70 mL) and dried over MgSO4. The solids were removed by

filtration, and the volatile components were removed via rotary evaporation. The crude product

was purified by reversed-phase flash column chromatography with a gradient of 10% to 100%

acetonitrile in water (0.1% TFA). Fractions containing product were combined and the organic

portion of the solvent was evaporated. The aqueous phase was neutralized by addition of

saturated NaHCO3 and was then extracted with ethyl acetate. The organic layer was evaporated

yielding 336 mg (41%) of product 4.23 as a light brown oil, which was taken on directly to the next step.

1H NMR (400 MHz, CDCl3): δ 8.10 (d, J = 2.0 Hz, 1H), 7.98 (d, J = 8.0 Hz, 1H), 7.58

(dd, J = 8.0, 2.0 Hz, 1H).

Analog 4.24. The procedure for pentathiepin formation for 4.20 was followed with 330 mg

of 4.23, without the need for salt formation after the purification. The procedure yielded 63 mg

(15%) of the product as a yellow oil. 1H NMR (400 MHz, CDCl3): δ 8.14 (d, J = 2.0 Hz, 1H),

8.01 (d, J = 8.1 Hz, 1H), 7.61 (dd, J = 8.1, 2.0 Hz, 1H); 19

F NMR (376 MHz, CDCl3): δ –62.97; 13

C NMR (126 MHz, CDCl3): δ 148.13, 145.08, 136.49, 132.84 (q, JCF = 3.6 Hz), 132.37 (q, JCF

= 33.4 Hz), 127.04 (q, JCF = 3.7 Hz), 122.99 (q, JCF = 273.1 Hz). Anal. calcd. for C7H3F3S5: C,

102

27.62; H, 0.99; N, 0.00; S, 52.67; found: C, 27.16; H, 0.96; N, < 0.02; S, 50.80. The 1H NMR

spectrum correlates with the published data.17

Analog 4.27. In a reaction vial charged with a magnetic stir bar, the starting aniline 4.08 (127

mg, 0.400 mmol) was dissolved in 1.8 mL of ethanol, and the vial was placed in a 0 °C ice bath.

Acetaldehyde (2.0 mL of a 0.5 M solution in ethanol, 1.0 mmol, 2.5 equiv) was added via

syringe, followed by 0.2 mL of glacial acetic acid. This solution was stirred 30 min at 0 °C, at

which point, a suspension of 75 mg (1.2 mmol, 3.0 equiv) of NaCNBH3 in EtOH (0.5 mL) was

added to the reaction vial via syringe. The resulting reaction mixture was stirred at 0 °C for 1 h.

The reaction was quenched with the addition of 1.5 mL of concentrated HCl, and then the

mixture was neutralized to pH 8.0 with saturated NaHCO3. The resulting mixture was extracted

into CH2Cl2 (2 × 30 mL), and the combined organic layer was dried over MgSO4. The solids

were removed by filtration, and the volatile components were removed via rotary evaporation.

The resulting residue was purified by SiO2 flash column chromatography with 0% to 10%

EtOAc in hexanes as the eluent. Compound 4.27 was isolated as a yellow solid (15 mg, 11%), m.p. 119–124 °C (dec).

1H NMR (500 MHz, CDCl3): δ 7.24 (d, J = 1.7 Hz, 1H), 6.77 (d, J = 1.7

Hz, 1H), 5.37 (s, 1H), 3.24 (qd, J = 7.2, 5.0 Hz, 2H), 1.34 (t, J = 7.2 Hz, 3H); 19

F NMR (376 MHz, CDCl3): δ –63.64;

13C NMR (126 MHz, CDCl3): δ 151.36, 146.72, 133.34 (q, JCF = 32.3

Hz), 127.53, 123.22 (q, JCF = 273.0 Hz), 118.92 (q, JCF = 3.5 Hz), 108.99 (q, JCF = 4.3 Hz),

38.59, 14.49. HRMS-ESI (m/z): [M+H]+ calcd. for C9H9F3NS5, 347.9285; found, 347.9287.

Analog 4.28. Following the procedure above for 4.27, with a 0.5 M solution of benzaldehyde

in EtOH, the product was obtained as a yellow solid (12 mg, 7%), m.p. 97–104 °C (dec). 1H

NMR (500 MHz, CDCl3): δ 7.43–7.30 (m, 5H), 7.28 (d, J = 1.8 Hz, 1H), 6.80 (d, J = 1.8 Hz,

1H), 5.85 (br t, 1H), 4.43 (d, J = 5.5 Hz, 2H); 19

F NMR (376 MHz, CDCl3): δ –63.66; 13

C NMR

(126 MHz, CDCl3): δ 151.16, 146.77, 137.14, 133.32 (q, JCF = 32.8 Hz), 129.19, 128.10, 127.49,

123.10 (q, JCF = 273.7 Hz), 119.54 (q, JCF = 3.8 Hz), 109.45 (q, JCF = 3.7 Hz), 48.17. HRMS-ESI

(m/z): [M+H]+ calcd. for C14H11F3NS5, 409.9442; found, 409.9437.

Analog 4.29. Diamine 4.10 (5 mg) was dissolved in 0.5 mL of ether and treated with 4 drops

of concentrated HCl to yield 6 mg (quant) of 4.29 as an off-white solid, m.p. 152–155 °C (dec). 1H NMR (400 MHz, DMSO-d6): δ 6.97 (d, J = 1.9 Hz, 1H), 6.65 (d, J = 1.9 Hz, 1H);

19F NMR

103

(376 MHz, DMSO-d6): δ –63.30. HRMS-ESI (m/z): [M–2HCl+H]+ calcd. for C14H9F6N2S6,

510.8989; found, 510.8983.

General procedures for determination of inhibitor IC50

Reaction volumes of 100 μL were used in 96-well plates. 75 L of water was added to each

well, followed by 5 L of 20 × buffer (stock: 1 M imidazole-HCl, pH 7.0, 1 M NaCl, 0.2%

Triton-X 100). Five L of the appropriate inhibitor dilution in DMSO was added, followed by 5

L of STEP (stock: 0.2 M, 10 nM in assay). The assay plate was then incubated at 27 °C for 10

min with shaking. The reaction was started by addition of 10 L of 10 × pNPP substrate (stock: 5

mM, 500 M in assay), and reaction progress was immediately monitored at 405 nm at a

temperature of 27 °C. The initial rate data collected was used for determination of IC50 values.

For IC50 determination, kinetic values were obtained directly from nonlinear regression of

substrate-velocity curves in the presence of various concentrations of inhibitor using one site

competition in GraphPad Prism v5.01 scientific graphing software. The Km value of pNPP for

STEP under these conditions was determined to be 745 M, and was used in the kinetic analysis.

For experiments with catalase or superoxide dismutase (SOD), 10 L of the appropriate

enzyme stocks (catalase: 800 U/mL stock, 80 U/mL in assay; SOD: 1000 U/mL stock, 100 U/mL

in assay) were added prior to addition of the inhibitor and STEP.

For the experiments with glutathione reducing agent, 10 L of glutathione (stock: 10 mM, 1

mM in assay) was added before the inhibitor stocks, and only 65 L of water was added initially

to maintain the 100 L assay volume. Once the inhibitor stocks were added, the assay plate was

allowed to incubate for 10 min at 27 °C with shaking. This was followed by addition of STEP

(stock: 1.0 M, 50 nM in assay) and another 10 min incubation at 27 °C prior to addition of

pNPP substrate.

General procedures for determination of kinact/Ki

The second-order rate constants of inactivation were determined under pseudo-first order

conditions using the progress curve method.8 Assay wells contained a mixture of the inhibitor

(800, 400, 200, 100, 50, 0 nM) and 745 M of pNPP (Km = 745 M) in buffer (50 mM

imidazole-HCl pH 7.0, 50 mM NaCl, 0.01% Triton-X 100). Aliquots of STEP were added to

each well to initiate the assay. The final concentration of STEP was 10 nM. Hydrolysis of pNPP

was monitored spectrophotometrically for 30 min at an absorbance wavelength of 405 nm. To

determine the inhibition parameters, time points for which the control ([I] = 0) was linear were

used. A kobs was calculated for each inhibitor concentration via a nonlinear regression of the data

according to the equation P=(vi/kobs)(1–e^(–kobst)) (where P = product formation, vi = initial rate,

t = time (s)) using Prism v5.01 (GraphPad). Because kobs varied hyperbolically with [I], nonlinear

regression was performed to determine the second-order rate constant, kinact/Ki, using the

equation kobs=kinact[I]/([I]+Ki(1+[S]/Km)). Assays were performed in quadruplicate. The average

and standard deviation of the assays is reported.

104

Dialysis

STEP was diluted into 1 × assay buffer with either inhibitor or DMSO control (final volume:

2.9 mL, final concentration: 1 M STEP, 5 M TC-2153; 50 mM imidazole-HCl, pH 7.0, 50

mM NaCl, 0.001% Triton-X 100, 5% v/v DMSO). The samples were shaken at ambient

temperature for 1 h to inhibit STEP. Each sample was then transferred to a separate Thermo

Scientific Slide-A-Lyzer dialysis cassette with a 10,000 MW cut off and 0.5–3.0 mL sample

volume and was dialyzed into 1 L of 1 × assay buffer over 24 h in a 4 °C cold room. Aliquots of

approximately 100 L were removed from the dialysis cassette at 0, 4, and 24 h time points.

Protein concentration was determined by reading absorbance at 280 nm compared to a standard

curve for STEP. The samples were diluted to 100 nM in 100 L of 1 × assay buffer. The reaction

was started by addition of 10 L of 10 × pNPP substrate (stock: 20 mM, 1.81 mM in assay; total

assay volume: 110 L), and reaction progress was immediately monitored at 405 nm at a

temperature of 27 °C. The initial rate data collected was used to determine enzyme activity

standardized to the DMSO control zero time point.

Determination of TC-2153 stability in imidazole buffer

To monitor the stability of TC-2153 in the imidazole buffer, 20 L of 20 mM TC-2153 stock

in DMSO was added to an Eppendorf tube. The solution was diluted to 400 L (1 mM TC-2153

final concentration, 5% final DMSO) with either water or the pH 7.0 imidazole buffer. The tube

was allowed to incubate at ambient temperature with shaking for 1 h. The mixture was diluted

with 150 L of DMSO-d6 and transferred to an NMR tube containing a capillary of

trifluoroacetic acid as an external standard (–76.55 ppm). The stability of the compound in the

buffer was confirmed by observing no differences in the 19

F NMR spectra (Figure 4.14). As a

control for compound modification, the experiment was repeated with the addition of 1 mM

GSH in the incubation buffer.

Recovery of STEP activity by reducing agents

STEP was diluted to 200 nM in water and aliquots of this stock were mixed with DMSO (5%

by volume) or TC-2153 (5 M final concentration, 5% DMSO by volume) and incubated at

ambient temperature on a shaker for 10 min. Each sample was aliquoted out and 50 L was

transferred to wells of a 96-well microtiter plate containing 40 L of 2 × assay buffer with added

reductant (GSH or DTT, 1 mM final concentration) and shaken for 0, 15, 30, or 60 additional

minutes at ambient temperature. The reaction was started by addition of 10 L of 10 × pNPP

substrate (stock: 20 mM, 2 mM in assay; total assay volume: 100 L), and reaction progress was

immediately monitored at 405 nm at a temperature of 27 °C. The initial rate data collected was

used to determine enzyme activity standardized to the DMSO controls.

105

Figure 4.14. TC-2153 stability in (a) water control, (b) imidazole buffer and (c) 1 mM GSH.

Mass spectrometry

To explore the protein modification(s) of STEP upon TC-2153 inhibition, reduced and

nonreduced gel purified STEP (WT or C472S mutant) proteins were analyzed by high-resolution

tandem mass spectrometry. Briefly, purified STEP WT or C-S mutant proteins (10 g each) were

incubated with vehicle (1% DMSO) or TC-2153 (10 M in 1% DMSO) in assay buffer (50 mM

imidazole-HCl, pH 7.0) at ambient temperature (25 °C) for 30 min. Samples were resolved on

8% SDS-PAGE or non-denaturing PAGE and proteins were visualized by Coomassie Blue

staining. Gel bands were excised and kept at –80 °C until use. Excised gel bands corresponding

to the mutant and WT STEP with and without TC-2153 were in-gel trypsin digested under native

conditions (without reducing agent) overnight. Peptides were extracted from the digested

samples with 80% acetonitrile containing 0.1% TFA, and then dried under SpeedVac. Samples

were then reconstituted in minimum solution containing 0.1% TFA, and loaded onto a RP C18

nanoACQUITY UPLC column (1.7 m BEH130 C18, 75 m×250mm, with a 5 m Symmetry

C18 2G-V/M Trap [180 m×20mm]). Eluted peptides were directly infused into an Orbitrap

Elite LC MS/MS system running data dependent acquisition. Acquired data were processed

utilizing Progenesis LCMS software (Nonlinear Dynamics) and MASCOT Search engine with

user defined possible modification(s) search criteria.

Western blotting

Samples were prepared and resolved by SDS-PAGE, transferred to nitrocellulose membrane

and incubated with phospho-specific antibodies (anti-pY204/187

ERK1/2, anti-pY402

Pyk2, anti-

pY1472

GluN2B) or total protein antibodies (anti-ERK2, anti-Pyk2 and anti-NR2B) overnight at 4

106

°C. All antibodies used are listed in Table 4.4. Immunoreactivity was visualized using a

Chemiluminescent substrate kit (Pierce Biotechnology, Rockford, IL) and detected using a

G:BOX with the image program GeneSnap (Syngene, Cambridge, UK). All densitometric

quantifications were performed using the Genetools program.

Table 4.4. Primary and secondary antibodies used in this study

Antibody Format Immunogen Host Dilution Source

Anti-pTyr1472

GluN2B

Whole IgG,

unconjugated

Synthetic

phosphopeptide Rabbit 1:1000

Cell Signaling

Technology,

Danvers, MA

Anti-GluN2B Whole IgG,

unconjugated

C-terminus of

mouse GluN2B Rabbit 1:1000

Cell Signaling

Technology

Anti-pTyr402

Pyk2

Whole IgG,

unconjugated

Synthetic

phosphopeptide of

human Pyk2

Rabbit 1:1000 Invitrogen

Anti-Pyk2 IgG2a C-terminus of

human Pyk2 Mouse 1:1000

Cell Signaling

Technology

Anti-pTyr204

ERK1/2

Whole IgG,

unconjugated

Synthetic

phosphopeptide Mouse 1:500

Santa Cruz

Biotechnology

Anti-ERK2 Whole IgG,

unconjugated

C-terminus of rat

p44 MAP Kinase Rabbit 1:20,000

Cell Signaling

Technology

Anti-GAPDH,

clone 6c5

IgG1,

unconjugated

Purified protein

from rabbit muscle Mouse 1:20,000 Millipore

Anti-rabbit

Whole IgG

peroxidase-

conjugated

Rabbit Fc Donkey 1:10,000 Amersham

Biosciences

Anti-mouse

Whole IgG

peroxidase-

conjugated

Mouse Fc Sheep 1:5,000 Amersham

Biosciences

In vivo assays

Wild-type male C57BL/6 mice (3–6 months) were used for all studies. An initial dose-

response curve was carried out using TC-2153 (1, 3, 6 and 10 mg/kg, i.p.). Pilot studies were

conducted to optimize the time after i.p. injection when STEP substrates showed maximum Tyr

phosphorylation (1–3 h). Cortical tissues were dissected out 3 h post injection and processed for

107

subcellular fractionation. Brain tissue was homogenized in TEVP buffer containing 10 mM Tris-

HCl, pH 7.6, 320 mM sucrose, 150 mM NaCl, 5 mM EDTA, 5 mM EGTA, 20 mM NaF, 1 mM

Na3VO4, and protease inhibitors. Homogenates were centrifuged at 800 × g to remove nuclei and

large debris (P1). Synaptosomal fractions (P2) were prepared from S1 by centrifugation at 9,200

× g for 15 min. The P2 pellet was washed twice and was resuspended in TEVP buffer. In some

experiments, mice were injected with TC-2153 (3 mg/kg, i.p.) and cortex, cerebellum and spleen

were removed to test for the in vivo inhibition of the highly related PTPs, HePTP and PTP-SL.14

Behavioral analysis

A previous study showed that genetic reduction of STEP significantly reversed cognitive

deficits in 6-month old 3xTg-AD mice.1a

Here we were interested in testing whether

pharmacologic inhibition of STEP with TC-2153 had a similar beneficial effect in this AD

mouse model. We also wanted to test whether TC-2153 had any effects on cognition in WT

mice. The WT cohort was treated as an independent group, and analyses were made

independently for the WT and 3xTg-AD groups. WT or 3xTg-AD mice were randomly allocated

to treatment with either vehicle or TC-2153.

Morris water maze

The reference memory version of the MWM task was performed as described previously.18

A

crossover design was not used in the Morris water maze task, as the mice were randomly

assigned to each treatment condition and can be exposed to the task only once. Briefly, animals

were trained to swim in a 1.4 m diameter pool to find a submerged platform (14 cm in diameter)

located 1 cm below the surface of water (24 °C), rendered opaque by the addition of non-toxic

white paint. Animals were pseudo-randomly started from a different position at each trial and

used distal visual-spatial cues to find the hidden escape platform that remained in the center of

the same quadrant throughout all training days. Training measures included escape latency to

reach the platform, swim speed, and thigmotaxis. When animals failed to find the platform they

were guided to it and remained there for 10 s before removal. 24 h after the acquisition phase, the

platform was removed and a probe trial of 90 s was given to evaluate the number of entries in a

circular zone (three times the platform diameter) positioned around the previous platform

location (target zone) and in the opposite quadrants. To assess visual deficits and motivation to

escape from water, the probe test was followed by a cued task (60 s; three trials per animal)

during which the platform was visible. The visible platform was moved to different locations

between each trial. After each trial, animals were immediately placed under a warming lamp to

dry to prevent hypothermia. The experimenter was blind to mouse genotype when administering

TC-2153 or vehicle to AD mice (AD-TC, n =7; AD-Veh, n = 6) or WT mice (WT-TC, n = 13;

WT-Veh, n= 12). Behavioral data from training, probe, and cued trials were acquired and

analyzed using the ANY-maze automated tracking system (Stoelting, IL, USA).

Data analysis

For the Morris water maze training and probe sessions, a 3-way repeated measures analysis

of variance (ANOVA) with 2 between-subject (Genotype, Treatment) and 1 within-subject

(Training day or Quadrant) factor was used. Escape latency (training) and number of entries

108

(probe) were the dependent measures (StatView, Cary, NC). Swim speed and escape latency

during the probe and cued trials, respectively were analyzed using a 2-way ANOVA with

Genotype and Treatment as the between subject factors. Post-hoc analyses were conducted on

significant results. For cell-based assays, one-way ANOVA with post hoc Bonferroni test was

used to determine statistical significance. All data were expressed as mean ± s.e.m.

References

1. (a) Zhang, Y. F.; Kurup, P.; Xu, J.; Carty, N.; Fernandez, S.; Nygaard, H. B.; Pittenger, C.;

Greengard, P.; Strittmatter, S.; Nairn, A. C.; Lombroso, P. J. Proc. Nat. Acad. Sci. U.S.A. 2010,

107, 19014; (b) Zhang, Y. F.; Kurup, P.; Xu, J.; Anderson, G.; Greengard, P.; Nairn, A. C.;

Lombroso, P. J. J. Neurochem. 2011, 119, 664; (c) Paul, S.; Nairn, A.; Wang, P.; Lombroso, P. J.

Nat. Neurosci. 2003, 6, 34; (d) Xu, J.; Kurup, P.; Bartos, J. A.; Hell, J. W.; Lombroso, P. J. J.

Biol. Chem. 2012, 287, 20942; (e) Munoz, J. J.; Tarrega, C.; BlancoAparicio, C.; Pulido, R.

Biochem. J. 2003, 372, 193; (f) Snyder, E. M.; Nong, Y.; Almeida, C. G.; Paul, S.; Moran, T.;

Choi, E. Y.; Nairn, A. C.; Salter, M. W.; Lombroso, P. J.; Gouras, G. K.; Greengard, P. Nat.

Neurosci. 2005, 8, 1051; also see the discussion in the introduction to chapter 3.

2. (a) Davidson, B. S.; Molinski, T. F.; Barrows, L. R.; Ireland, C. M. J. Am. Chem. Soc. 1991

113, 4709; (b) Kulikov, A. V.; Tikhonova, M. A.; Kulikova, E. A.; Volcho, K. P.; Khomenko, T.

M.; Salakhutdinov, N. F.; Popova, N. K. Psychopharmacology 2012, 221, 469.

3. (a) Kulikov, A. V.; Tikhonova, M. A.; Kulikova, E. A.; Khomenko, T. M.; Korchagina, D.

V.; Volcho, K. P.; Salachutdinov, H. F.; Popova, N. K. Molec. Biol. 2011, 45, 282; (b)

Konstantinova, L.S.; Rakitin, O. A.; Rees, C. W. Chem. Rev. 2004, 104, 2617.

4. Khomenko, T. M.; Tolstikova, T. G.; Bolkunov, A. V.; Dolgikh, M. P.; Pavlova, A. V.;

Korchagina, D. V.; Volcho, K. P.; Salakhutdinov, N. F. Lett. Drug Des. Discov. 2009, 6, 464.

5. Kulikov, A. V.; Tikhonova, M. A.; Kulikova, E. A.; Khomenko, T. M.; Korchagina, D. V.;

Volcho, K. P.; Salakhutdinov, N. F.; Popova, N. K. Molec. Biol. 2011, 45, 251.

6. Nihei, M.; Kurihara, M.; Mizutani, J.; Nishihara, H. J. Am. Chem. Soc. 2003, 125, 2964.

7. Gao, Y.; Ren, Q.; Wu, H.; Li, M.; Wang, J. Chem. Commun. 2010, 46, 9232.

8. Bieth, J. G. Methods Enzymol. 1995, 248, 59.

9. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711.

10. Eswaran, J.; von Kries, J. P.; Marsden, B.; Longman, E.; Debreczeni, J. E.; Ugochukwu, E.;

Turnbull, A.; Lee, W. H.; Knapp, S.; Barr, A. J. Biochem. J. 2006, 395, 483.

11. Khomenko, T. M.; Korchagina, D. V.; Komarova, N. I.; Volcho, K. P.; Salakhutdinov, N. F.

Lett. Org. Chem. 2011, 8, 193.

12. (a) Sergienko, E.; Xu, J.; Liu, W. H.; Dahl, R.; Critton, D. A.; Su, Y.; Brown, B. T.; Chan,

X.; Yang, L.; Bobkova, E. V.; Vasile, S.; Yuan, H.; Rascon, J.; Colayco, S.; Sidique, S.; Cosford,

N. D. P.; Chung, T. D. Y.; Mustelin, T.; Page, R.; Lombroso, P. J.; Tautz, L. ACS Chem. Biol.

2012, 367; (b) Vang, T.; Liu, W. H.; Delacroix, L.; Wu, S.; Vasile, S.; Dahl, R.; Yang, L.;

Musumeci, L.; Francis, D.; Landskron, J.; Tasken, K.; Tremblay, M. L.; Lie, B. A.; Page, R.;

Mustelin, T.; Rahmouni, S.; Rickert, R. C.; Tautz, L. Nat. Chem. Biol. 2012, 8, 437.

13. (a) Zhang, Y.; Venkitaramani, D. V.; Gladding, C. M.; Zhang, Y.; Kurup, P.; Molnar, E.;

Collingridge, G. L.; Lombroso, P. J. J. Neurosci. 2008, 28, 10561; (b) Venkitaramani, D. V.;

Paul, S.; Zhang, Y.; Kurup, P.; Ding, L.; Tressler, L.; Allen, M.; Sacca, R.; Picciotto, M. R.; L.,

Paul J. Synapse 2008, 63, 69; see also references 1a and 1c.

109

14. (a) Zanke, B.; Squire, J.; Griesser, H.; Henry, M.; Suzuki, H.; Patterson, B.; Minden, M.;

Mak, T. W. Leukemia 1994, 8, 236; (b) Hendriks, W.; Schepens, J.; Brugman, C.; Zeeuwen, P.;

Wieringa, B. Biochem. J. 1995, 305, 499; (c) Ogata, M.; Sawada, M.; Fujino, Y.; Hamaoka, T. J.

Biol. Chem. 1995, 270, 2337; (d) Sharma, E.; Lombroso, P. J. J. Biol. Chem. 1995, 270, 49.

15. Xu, J.; Kurup, P3.; Zhang, Y.; Goebel-Goody, S. M.; Wu, P. H.; Hawasli, A. H.; Baum, M.

L.; Bibb, J. A.; Lombroso, P. J. J. Neurosci. 2009, 29, 9330.

16. Rasheed, K.; Warkentin, J. D. J. Org. Chem. 1980, 45, 4806.

17. Chenard, B. L. J. Org. Chem. 1984, 49, 1224.

18. Brouillette, J.; Quirion, R. Neurobiol. Aging 2008, 29, 1721.

110

111

Chapter 5. Seleninic acids as redox-reversible inhibitors of STEP

Abstract: This chapter outlines the use of seleninic acids as redox-reversible inhibitors of STEP.

The redox-reversible mode of inhibition described in chapter 4 is adapted to seleninic acids,

which have been demonstrated to form stable S–Se bonds with cysteine thiols. This new PTP

pharmacophore is merged with the SAR determined in chapter 3 to attain an inhibitor with good

activity in vitro. This work is unpublished.

112

Authorship

I carried out all of the work described in this chapter. I synthesized all of the compounds and

determined their in vitro inhibition against STEP.

Introduction

Selenium is an essential micronutrient of many living species, including humans, because the

selenocysteine amino acid is necessary for the activity of a few important antioxidant enzymes.

In high doses, however, selenium can be toxic. In 2007, Plateau and coworkers were able to

account for some of this toxicity by studying its effects in Saccharomyces cerevisiae.1 Selenite

(SeO32–

, 5.01) reacts with readily with excess reduced glutathione (GSH) to generate hydrogen

selenide (HSe–, 5.04), which is toxic in S. cerevisiae (Scheme 5.1). Other reduction products,

such as selenodiglutathione 5.02, reactive oxygen species (ROS) or elemental Se0, were found to

not be toxic. These results demonstrate that selenium can undergo multiple redox reactions with

bio-relevant thiols.

Scheme 5.1. Selenium redox in vivo with GSH

In 2008, Zhang and coworkers reported the first use of a seleninic acid as a phosphatase

inhibitor.2 They synthesized tyrosine derived seleninic acid 5.06 and characterized its irreversible

inhibition consistent with forming a selenium–sulfur bond at the active site cysteine residue of

both YopH and PTP1B. Seleninic acid 5.06 had a kinact/Ki of 91 ± 20 M–1

s–1

against YopH and

only 14 ± 10 M–1

s–1

against PTP1B. Additionally, they demonstrated that seleninic acid 5.06

forms a covalent bond with cysteine derivative 5.07 and were able to characterize selenosulfide

adduct 5.08 (Scheme 5.2). Finally, to directly demonstrate the irreversible adduct, they obtained

a crystal structure showing a covalent bond between compound 5.06 and the active site of

PTP1B.

Scheme 5.2. Seleninic acid 5.6 forms a covalent adduct with cysteine derivative 5.7

Another important example of selenium redox in vivo is the catalytic mechanism of

thioredoxin reductase (TrxR) (Figure 5.1).3 As the only known enzyme to catalyze the reduction

of thioredoxin (Trx),4 it is a central component of the Trx system which exists in all living cells

and is important for maintaining the cellular redox environment.5 In one of the active sites of

TrxR is a selenocysteine residue which forms a selenosulfide with a neighboring cysteine upon

113

oxidation by Trx. The selenosulfide is reduced to the free selenol and thiol through a series of

reactions that utilize NADPH as the terminal reductant.

Figure 5.1. Selenosulfide redox cycling in Trx/TrxR system.

Combining the facts that selenosulfides have been formed from seleninic acids with active

site cysteines in PTP active sites and that they possess reversible redox in vivo as exemplified by

the TrxR cycle, we hoped that we could obtain redox-reversible inhibitors by introducing the

seleninic acid functional group as a pharmacophore. This pharmacophore should react in in vitro

assays similarly to the pentathiepins in chapter 4, and because it can be incorporated into

druglike scaffolds, it should enable more straightforward modulation of physicochemical

properties.

Synthesis of seleninic acid inhibitors

Utilizing the procedures of Zhang, et. al., we first synthesized seleninic acid 5.13 (Scheme

5.3).2 First, (2-phenyl)-selenoacetic acid 5.10 is generated by treatment of phenylacetic acid with

Woollins’ reagent (the Se analog of Lawesson's reagent). The selenoacetic acid then functions as

the nucleophile in a Mitsunobu reaction yielding the selenoester 5.12. Treatment with

dimethyldioxirane (DMDO)6 yields the desired seleninic acid 5.13.

Scheme 5.3. Synthesis of seleninic acid inhibitor 5.13

With the simple biphenyl inhibitor synthesized, we next sought to incorporate the SAR

optimized scaffold of 3.97 and 3.98 from chapter 3 (Scheme 5.4). Lithiation of 1-bromo-3-

iodobenzene followed by addition into 3,4-dichlorobenzaldehyde yielded aryl bromide 5.15.

114

Subsequent Miyaura borylation7 gave boronate ester 5.16. Protection of the benzyl alcohol as the

TBS ether followed by Suzuki-Miyaura cross-coupling reaction with 4-bromobenzyl alcohol

afforded benzyl alcohol 5.18. The Mitsunobu reaction and DMDO oxidation afforded seleninic

acid 5.20, which was deprotected with TBAF to afford the final seleninic acid inhibitor, 5.21.

Scheme 5.4. Synthesis of seleninic inhibitor 5.21

In chapter 3 we found that the -hydroxyphosphonic acid was more potent than the simple

methylene phosphonic acid. However, primarily out of concern for compound stability, the

literature precedented methylene seleninic acids were synthesized to test the viability of the

seleninic acid as an inhibitor. In this study, we desired to test the ability of the seleninic acid to

function as a redox-reversible pharmacophore for PTP inhibition without the added complication

of potential compound instability.

In vitro evaluation of inhibitors

As was the case in chapter 4 with the pentathiepins, we thought it was important to test the

activity of seleninic acids 5.13 and 5.21 both with and without the ubiquitous biological reducing

agent GSH (Table 5.1).8 Gratifyingly, the compound with the SAR optimized scaffold, 5.21, was

more potent than the simple biphenyl derivative, 5.13. Additionally, compound 5.21 was as

potent as TC-2153 in the absence of GSH. Interestingly, when GSH was added to the enzyme

assay, the potency of 5.21 was not affected nearly as much as that of TC-2153, maintaining sub-

M potency. This suggests that either the seleninic acid pharmacophore in 5.21 is not as

115

susceptible to the non-productive reaction with GSH, or that it forms a single stable adduct that

is still able to inhibit STEP at a reduced potency.

Table 5.1. IC50 values of seleninic acids against STEP compared to TC-2153

compound IC50 (nM)a,b

IC50 (M) a,c

seleninic acid 5.13 70 ± 10 > 50

seleninic acid 5.21 27 ± 7 0.53 ± 0.02

pentathiepin TC-2153 24.6 ± 0.8d

8.8 ± 0.4d

aAssays were performed in duplicate (mean ± S.D.).

bAssays contained no

GSH. cAssays contained 1 mM GSH.

dAssays were performed in

quadruplicate (mean ± S.D.)

The second-order rate of inactivation for each of the seleninic acid inhibitors was then

determined using the progress curve method (Figure 5.2).9 A kobs was determined for pNPP in

the presence of varying initial inhibitor concentrations (n = 4). Values were then analyzed with

non-linear regression to obtain the kinetic constants: 5.13: kinact = 0.0170 ± 0.0029 s–1

; Ki = 1,011

± 300 nM; kinact/Ki = 16,800 ± 5,700 M–1

s–1

; 5.21: kinact = 0.0141 ± 0.0009 s–1

; Ki = 367 ± 63 nM;

kinact/Ki = 38,400 ± 7,000 M–1

s–1

. Consistent with the results in the IC50 assays and with the

elaborated scaffold providing an increase in binding, the Ki of 5.21 was approximately three

times more potent than the simple biphenyl scaffold, while their kinact values were the same

(within experimental error). For comparison, the only other seleninic acid PTP inhibitor reported

to date, 5.06, had a kinact/Ki of 91 ± 18 M–1

s–1

against YopH, considerably worse than the activity

of inhibitor 5.21 against STEP.

Figure 5.2. The progress curve method was used to determine the second-order rates of inactivation for

(a) inhibitor 5.13 (kinact/Ki = 16,800 ± 5,700 M–1

s–1

) and (b) inhibitor 5.21 (kinact/Ki = 38,400 ± 7,000

M–1

s–1

).

Conclusions

Seleninic acids have been shown to be redox-reversible inhibitors of STEP. Like the

pentathiepins in chapter 4, inhibition is dependent on the presence of GSH in the assays.

Moreover, the seleninic acid does not appear to be as affected by GSH, indicating that it perhaps

is not as reactive as the pentathiepin, or that it forms a stable adduct with GSH that can also

function as an inhibitor. Currently, inhibitors 5.13 and 5.21 have been submitted to our

collaborators in Dr. Paul Lombroso’s group to test for activity in neuronal cultures. Additionally,

116

researchers at AstraZeneca are working on obtaining crystal structures of the inhibitors in

complex with STEP. Through personal correspondence, they have been interested in obtaining

STEP inhibitors for the last two years but have not been able to generate anything as potent as

the inhibitors that we have disclosed in chapters 3, 4 and 5.

Experimental



without further purification. Tetrahydrofuran (THF), dioxane, CH2Cl2, and diethyl ether (Et2O)

were passed through a column of activated alumina (type A2, 12 × 32, Purify Co.) under

nitrogen pressure immediately prior to use. All 1H,

13C, and

77Se NMR spectra were obtained at

ambient temperature on a Bruker AVB-400, AVB-500 or AVB-600 spectrometer. NMR

chemical shifts are reported in ppm relative to TMS (0.00), CHCl3 (7.26), or CH3OH (3.31) for 1H, CDCl3 (77.16), or CD3OD (49.00) for

13C, and PhSeSePh (460.0) for

77Se. Mass

spectrometry (HRMS, ESI) are reported in m/z. Chromatography was performed either with

SiliCycle SiliaFlash P60 230−400 mesh silica gel or by utilizing a Biotage SP1 flash purification

system (Biotage model SP1-B1A). Reversed-phase purifications were conducted with a

Teledyne Isco CombiFlash Rf system equipped with HP C18 gold cartridges. Syringe filtrations

were performed with Millex-HN 0.45 µm Nylon 33 mm syringe filters. Product yields are not

optimized. Enzymatic assays were carried out on a BioTek Synergy 2 multimode microplate

reader.

Synthesis of inhibitors 5.13 and 5.21

Compound 5.12. Selenoester 5.12 was prepared by modifying a literature procedure for a

related compound.2 A solution of 1.10 g (4.20 mmol) of triphenylphosphine in 10 mL of THF

was stirred at 0 °C. Diisopropyl azodicarboxylate (94%, 904 mg, 4.20 mmol) was added

dropwise, and the reaction mixture was maintained at 0 °C for 5 min until the white

phsophonium intermediate was formed. A solution of 4-phenylbenzyl alcohol 5.11 (387 mg, 2.10

mmol) in 10 mL of THF was added dropwise by syringe. After 5 min of stirring, 20 mL of a

toluene solution of (2-phenyl)-selenoacetic acid 5.10 was added dropwise by syringe through a

syringe filter [5.10 was prepared by heating at 105 °C a 20 mL of toluene solution of 863 mg

(6.33 mmol) of phenylacetic acid and 1.00 g (1.90 mmol) of Woollins’ reagent for 1 h]. The

reaction mixture was allowed to warm to ambient temperature and then was stirred for 3 h. The

solution was concentrated under reduced pressure. Purification by flash chromatography on SiO2

with 95:5 hexanes/ethyl acetate yielded 230 mg (30%) of pure 5.12 as a pink solid, m.p. 83–86 °C.

1H NMR (500 MHz, CDCl3): δ 7.57 (d, J = 7.5 Hz, 2H), 7.50 (d, J = 8.1 Hz, 2H), 7.44 (app t,

117

J = 7.7 Hz, 2H), 7.40–7.28 (m, 8H), 4.18 (s, 2H), 3.89 (s, 2H); 13

C NMR (151 MHz, CDCl3): δ

200.36, 140.82, 139.98, 138.09, 132.87, 130.19, 129.44, 128.86, 128.84, 127.91, 127.43, 127.37,

127.12, 54.02, 29.20; 77

Se NMR (95 MHz, CDCl3): δ 635.17. MS-ESI (m/z): [M+H]+ calcd. for

C21H19O[80]

Se, 367.06; found, 367.2.

Inhibitor 5.13. Seleninic acid 5.13 was prepared following a literature procedure for a

related compound.2 Dimethyldioxirane (DMDO) was added to a stirred solution of 5.12 (170 mg,

0.47 mmol) in 5 mL of acetone until all of 5.12 was consumed according to LCMS analysis

(total ~10 mL of a 0.16 M solution of DMDO in acetone, 3.5 equiv). The reaction mixture was

concentrated, and the crude residue was purified via reversed-phase gradient column

chromatography (5−100% acetonitrile in water with 0.1% trifluoroacetic acid buffer) to yield

seleninic acid 5.13 as a white crystalline solid, m.p. 118–119 °C. 1H NMR (500 MHz, CD3OD):

δ 7.63 (m, 4H), 7.51–7.39 (m, 4H), 7.34 (t, J = 7.4 Hz, 1H), 4.29, 4.25 (ABq, J = 12.0 Hz, 2H); 13

C NMR (126 MHz, CD3OD): δ 142.58, 141.71, 132.17, 129.93, 129.42, 128.62, 128.33,

127.95, 62.14; 77

Se NMR (96 MHz, CD3OD): δ 1291.68. HRMS-ESI (m/z): [M–H]– calcd. for

C13H11O2[80]

Se, 278.9930; found, 279.0068 (Figure 5.3).

Figure 5.3. HRMS of 5.13. Major contributors to the main peaks are labeled with stable Se isotopes.

Natural abundances of Se isotopes: 74

Se, 0.87%; 76

Se, 9.36%; 77

Se, 7.63%; 78

Se, 23.78%; 80

Se, 49.61%; 82

Se, 8.73%.

118

Compound 5.15. The synthesis of compound 5.15 is described in chapter 3. It is identified in

chapter 3 as compound 3.105. 1H NMR provided here for reference.

1H NMR (400 MHz,

CDCl3): δ 7.51 (m, 1H), 7.48 (m, 1H), 7.44−7.40 (m, 2H), 7.27−7.16 (m, 3H), 5.74 (d, J = 3.2

Hz, 1H), 2.31 (d, J = 3.2 Hz, 1H).

Compound 5.16. Following a literature procedure,

10 a 250 mL flame-dried round bottom

flask was charged with a magnetic stir bar, crude 5.15 (6.65 g, 20.0 mmol),

bis(pinacolato)diboron (10.16 g, 40.0 mmol), potassium acetate (5.89 g, 60.0 mmol) and DMSO

(80 mL). The solution was sparged with N2 at ambient temperature with stirring for 30 min.

Pd(dppf)Cl2 (816 mg, 1.00 mmol, 5 mol %) was added to the solution, which was sparged with

N2 for an additional 30 min. The reaction flask was fitted with a rubber septum and placed in a

pre-heated 80 °C oil bath, and the reaction mixture was stirred under N2 for 6 h. The reaction

flask was removed from the oil bath, and the mixture was allowed to cool to ambient

temperature. The reaction mixture was diluted with 700 mL of water and extracted into Et2O (3 ×

700 mL). The combined organic layer was washed with brine (1 L) and the volatiles were

removed under reduced pressure. The crude residue was purified by silica gel chromatography

with an eluent of 4:1 hexanes:ethyl acetate, yielding 5.16 as a colorless oil (3.34 g, 44% over 2 steps).

1H NMR (400 MHz, CDCl3): δ 7.80–7.79 (m, 1H), 7.75 (d, J = 7.2 Hz, 1H), 7.51 (d, J =

2.0 Hz, 1H), 7.44–7.31 (m, 3H), 7.20 (dd, J = 8.4, 2.1 Hz, 1H), 5.79 (s, 1H), 2.04 (s, 1H), 1.34 (s, 12H);

13C NMR (151 MHz, CDCl3): δ 144.03, 142.40, 134.82, 132.93, 132.61, 131.41, 130.49,

129.53, 128.54, 128.45, 126.00, 84.13, 75.32, 25.02, 25.00.NMR spectral data of racemic 5.16

was identical to literature spectra of each of the enantioenriched isomers.10

Compound 5.17. A 250 mL flame-dried round bottom flask was charged with a magnetic

stir bar, 5.16 (4.55 g, 12.0 mmol), imidazole (6.54 g, 96.0 mmol), and TBSCl (7.23 g, 48.0

mmol), and the flask was flushed with N2. DMF (15 mL) was added, and the reaction mixture

was stirred at ambient temperature with monitoring by TLC. After 1 h, the reaction was stopped

by addition of 20 mL of saturated aqueous NaHCO3, and the mixture was diluted with 200 mL of

water. The resulting suspension was extracted with CH2Cl2 (4 × 150 mL), and the combined

organic layer was washed with water (400 mL) and brine (400 mL), dried over MgSO4, filtered

and the volatiles were removed under reduced pressure. The crude residue was purified by silica

gel chromatography with an eluent of 9:1 hexanes:ethyl acetate, yielding 5.17 as a white solid

119

(4.18 g, 71%); m.p. 102–105 °C. 1H NMR (500 MHz, CDCl3): δ 7.75–7.67 (m, 2H), 7.50–7.47

(m, 2H), 7.37–7.31 (m, 2H), 7.21 (dd, J = 8.3, 2.0 Hz, 1H), 5.72 (s, 1H), 1.35 (s, 12H), 0.92 (s, 9H), 0.02 (s, 3H), –0.05 (s, 3H);

13C NMR (126 MHz, CDCl3): δ 145.80, 143.48, 134.21, 132.62,

132.30, 130.80, 130.31, 129.16, 128.19, 128.17, 125.70, 83.96, 75.79, 25.94, 25.06, 24.98, 18.41,

-4.63, -4.74.

Compound 5.18. A 250 mL round bottom flask was charged with a magnetic stir bar, 5.17

(4.08 g, 8.27 mmol), 4-bromobenzyl alcohol (1.55 g, 8.27 mmol), sodium carbonate (5.26 g, 49.6

mmol), Pd(PPh3)4 (485 mg, 0.420 mmol) and 42 mL of a solvent mixture of 4:1:1

DME:EtOH:H2O. The reaction mixture was sparged with N2 at ambient temperature with stirring

for 45 min. The reaction flask was fitted with a rubber septum and placed in a pre-heated 85 °C

oil bath, and the reaction mixture was stirred under N2 for 20 h. The reaction mixture was

allowed to cool to ambient temperature then was filtered through a pad of Celite; the filter cake

was washed with EtOAc. Volatiles were removed under reduced pressure, and the resulting

crude residue was purified by silica gel chromatography with an eluent of 4:1 hexanes:ethyl

acetate, yielding 5.18 as a yellow oil (3.15 g, 77 %). 1H NMR (500 MHz, CDCl3): δ 7.61–7.55

(m, 3H), 7.53 (s, 1H), 7.51–7.43 (m, 3H), 7.42–7.36 (m, 2H), 7.32 (d, J = 7.7 Hz, 1H), 7.29–7.20

(m, 1H), 5.78 (s, 1H), 4.75 (s, 2H), 1.98 (s, 1H), 0.96 (s, 9H), 0.06 (s, 3H), 0.03 (s, 3H); 13

C

NMR (126 MHz, CDCl3): 145.60, 144.79, 141.10, 140.50, 140.14, 132.40, 131.00, 130.39,

129.03, 128.21, 127.58, 127.41, 126.37, 125.66, 125.33, 124.95, 75.77, 65.13, 25.93, 18.41,

–4.62, –4.69. HRMS-ESI (m/z): [M+H–H2O]+ calcd. for C26H29

[35]Cl2OSi, 455.1359; found,

455.1356.

Compound 5.19. Compound 5.19 was prepared following the procedure for 5.12 beginning

with 2.00 g (4.20 mmol) of 5.18. The procedure yielded 650 mg (24%) of 5.19 as an orange oil. 1H NMR (400 MHz, CDCl3): δ 7.51 (br t, 1H), 7.49 (d, J = 1.9 Hz, 1H), 7.48–7.39 (m, 3H),

7.40–7.23 (m, 10H), 7.21 (dd, J = 8.4, 2.0 Hz, 1H), 5.73 (s, 1H), 4.15 (s, 2H), 3.88 (s, 2H), 0.94 (s, 9H), 0.03 (s, 3H), –0.00 (s, 3H);

13C NMR (151 MHz, CDCl3): δ 200.36, 145.61, 144.77,

141.01, 139.77, 138.32, 132.87, 132.41, 130.99, 130.40, 130.21, 129.49, 129.01, 128.85, 128.20,

127.93, 127.43, 126.30, 125.65, 125.29, 124.91, 75.79, 54.04, 29.18, 25.95, 18.43, –4.60, –4.69;

120

77Se NMR (95 MHz, CDCl3): 635.94. MS-ESI (m/z): [M+H–C6H5COSeH]

+ calcd. for

C26H29[35]

Cl2OSi, 455.14; found, 455.1.

Compound 5.20. Compound 5.20 was prepared following the procedure for 5.13 beginning

with 630 mg (0.96 mmol) of 5.19. The reaction required a total of ~24 mL of a 0.16 M solution

of DMDO in acetone (4.0 equiv) for all of 5.19 to be consumed (monitored by LCMS). The

crude residue was purified first by reversed-phase gradient column chromatography (20−100%

acetonitrile in water with 0.1% trifluoroacetic acid buffer), then by flash chromatography on

SiO2 with an eluent of 94:5:1 CH2Cl2/MeOH/formic acid to yield 5.20 (180 mg, 33%) as a red

viscous oil. The product was contaminated with ~5–10% of an unknown impurity that was co-

purified after both regular and reversed-phase purification. 1H NMR is provided for the desired

product. 1H NMR (400 MHz, CDCl3): δ 7.59–7.27 (m, 10H), 7.20 (dd, J = 8.3, 2.0 Hz, 1H), 5.73

(s, 1H), 4.43 (s, 2H), 0.92 (s, 9H), 0.02 (s, 3H), –0.01 (s, 3H). HRMS-ESI (m/z): [M+H–

Se(OH)2]+ calcd. for C26H29

[35]Cl2OSi, 455.1359; found, 455.1374.

Inhibitor 5.21. To a stirred solution of 5.20 (169 mg, 0.288 mmol) in THF (3 mL) at ambient

temperature was added tetra-n-butylammonium fluoride (1.0 M solution in THF, 0.29 mL, 0.29

mmol, 1 equiv) dropwise via syringe. After 30 min all of 5.20 was consumed (monitoring by

LCMS), and the reaction was concentrated. The crude residue was purified by reversed-phase

gradient column chromatography (10−100% acetonitrile in water with 0.1% formic acid buffer)

to yield 5.21 (40 mg, 31%) as a white solid, m.p. 95–99 °C. 1H NMR (500 MHz, CD3OD): δ

7.64–7.55 (m, 4H), 7.51 (d, J = 7.7 Hz, 1H), 7.47–7.37 (m, 4H), 7.32 (d, J = 7.7 Hz, 1H), 7.29

(d, J = 8.5 Hz, 1H), 5.81 (s, 1H), 4.27, 4.23 (ABq, J = 12.0 Hz, 2H); 13

C NMR (151 MHz, CD3OD): δ 146.97, 145.88, 142.31, 141.97, 133.16, 132.20, 131.82, 131.43, 130.16, 129.54,

128.35, 127.52, 127.23, 126.96, 126.19, 75.51, 62.12; 77

Se NMR (95 MHz, CD3OD): δ 1291.16.

HRMS-ESI (m/z): [M–H]– calcd. for C20H15

[35]Cl2O3

[80]Se, 452.9569; found, 452.9601 (Figure

5.4).

121

Figure 5.4. HRMS of 5.21. Major contributors to the main peaks are labeled with stable Se and Cl

isotopes. Natural abundances of Se isotopes: 74

Se, 0.87%; 76

Se, 9.36%; 77

Se, 7.63%; 78

Se, 23.78%; 80

Se,

49.61%; 82

Se, 8.73%. Natural abundance of Cl isotopes: 35

Cl, 75.77%; 37

Cl, 24.23%.

General procedures for determination of inhibitor IC50

Reaction volumes of 100 μL were used in 96-well plates. 75 L of water was added to each

well, followed by 5 L of 20 × buffer (stock: 1 M imidazole-HCl, pH 7.0, 1 M NaCl, 0.2%

Triton-X 100). Five L of the appropriate inhibitor dilution in DMSO was added, followed by 5

L of STEP (stock: 0.2 M, 10 nM in assay). The assay plate was then incubated at 27 °C for 10

min with shaking. The reaction was started by addition of 10 L of 10 × pNPP substrate (stock: 5

mM, 500 M in assay), and reaction progress was immediately monitored at 405 nm at a

temperature of 27 °C. The initial rate data collected was used for determination of IC50 values.

For IC50 determination, kinetic values were obtained directly from nonlinear regression of

substrate-velocity curves in the presence of various concentrations of inhibitor using one site

competition in GraphPad Prism v5.01 scientific graphing software. The Km value of pNPP for

STEP under these conditions was determined to be 745 M, and this value was used in the

kinetic analysis.

For the experiments with glutathione reducing agent, 10 L of glutathione (stock: 10 mM, 1

mM in assay) was added before the inhibitor stocks, and only 65 L of water was added initially

to maintain the 100 L assay volume. Once the inhibitor stocks were added, the assay plate was

allowed to incubate for 10 min at 27 °C with shaking. This was followed by addition of STEP

(stock: 1.0 M, 50 nM in assay) and another 10 min incubation at 27 °C prior to addition of

pNPP substrate.

General procedures for determination of kinact/Ki

The second-order rate constants of inactivation were determined under pseudo-first order

conditions using the progress curve method.9 Assay wells contained a mixture of the inhibitor

(2000, 666.7, 222.2, 74.1, 24.7, 0 nM) and 745 M of pNPP (Km = 745 M) in buffer (50 mM

imidazole-HCl pH 7.0, 50 mM NaCl, 0.01% Triton-X 100). Aliquots of STEP were added to

each well to initiate the assay. The final concentration of STEP was 10 nM. Hydrolysis of pNPP

122

was monitored spectrophotometrically for 30 min at an absorbance wavelength of 405 nm. To

determine the inhibition parameters, time points for which the control ([I] = 0) was linear were

used. A kobs was calculated for each inhibitor concentration via a nonlinear regression of the data

according to the equation P=(vi/kobs)(1–e^(–kobst)) (where P = product formation, vi = initial rate,

t = time (s)) using Prism v5.01 (GraphPad). Because kobs varied hyperbolically with [I], nonlinear

regression was performed to determine the second-order rate constant, kinact/Ki, using the

equation kobs=kinact[I]/([I]+Ki(1+[S]/Km)). Assays were performed in quadruplicate. The average

and standard deviation of the assays is reported.

References

1. Tarze, A.; Dauplais, M.; Grigoras, I.; Lazard, M.; Ha-Duong, N.-T.; Barbier, F.; Blanquet, S.;

Plateau, P. J. Biol. Chem. 2007, 282, 8759.

2. Abdo, M.; Liu, S.; Zhou, B.; Walls, C. D.; Wu, L.; Knapp, S.; Zhang, Z.-Y. J. Am. Chem.

Soc. 2008, 130, 13196.

3. Holmgren, A.; Lu, J. Biochem. Biophys. Res. Commun. 2010, 396, 120.

4. Mustacich, D.; Powis, G. Biochem. J. 2000, 346, 1.

5. (a) Meyer, Y.; Buchanan, B. B.; Vignols, F.; Reichheld, J.-P. Annu. Rev.Genet. 2009, 43,

335; (b) Lillig, C. H.; Holmgren, A. Antioxid. Redox Signaling 2007, 9, 25.

6. Crandall, J. K.; Curci, R.; D'Accolti, L; Fusco, C. e-EROS Encyclopedia of Reagents for

Organic Synthesis 2005.

7. Ishiyama, T.; Murata, M.; Miyaura, N. J. Org. Chem. 1995, 60, 7508.

8. Meister, A.; Anderson, M. E. Annu. Rev. Biochem. 1983, 52, 711.

9. Bieth, J. G. Methods Enzymol. 1995, 248, 59.

10. Baguley, T. D.; Xu, H.-C.; Chatterjee, M.; Nairn, A. C.; Lombroso, P. J.; Ellman J. A. J.

Med. Chem., 2013, 56, 7636; also see chapter 3.

123

Chapter 6. Asymmetric additions of Knochel-type benzyl zinc reagents to N-tert-

butanesulfinyl aldimines

Abstract: Early in my graduate career, I wanted to be exposed to more of the chemistry in the

Ellman group than just the chemical biology aspects. There was a need in a project area that I

thought would provide me with valuable experience in organic synthesis. This chapter details the

development of a reaction for the addition of Knochel-type benzyl zinc reagents to N-tert-

butanesulfinyl aldimines. Notably, additions of these sp3-hybridized reagents demonstrate good

functional-group compatibility, adding chemoselectively to imines in the presence of esters and

nitriles under convenient ambient temperature conditions. Moreover, addition to a

glyceraldehyde-derived N-tert-butanesulfinyl imine proceeds in high yield and with exceptional

selectivity to provide rapid entry to hydroxyethylamine-based aspartyl protease inhibitors. The

majority of this work has been published (Buesking, A. W.; Baguley, T. D.; Ellman, J. A. Org.

Lett. 2011, 13, 964).

124

Authorship

The majority of the work described in this chapter was completed in collaboration with Dr.

Andrew Buesking, a fellow graduate student. Dr. Buesking and I both were involved in the

synthesis of the starting materials and the reaction optimization. Dr. Chris Incarvito (Yale

Chemical and Biophysical Instrumentation Center) solved the crystal structures and provided the

crystal data reports for products 6.27 and 6.31.

Introduction

The addition of nucleophiles to N-tert-butanesulfinyl imines is one of the most extensively

used approaches for the asymmetric synthesis of amines (Scheme 6.1).1 The popularity of these

methods results from several key features of the N-tert-butanesulfinyl imine substrates and their

corresponding sulfinyl-protected amine products. First, N-tert-butanesulfinyl imines (6.02) are

readily prepared in a single step, typically in high yield, from aldehydes and ketones with diverse

steric and electronic properties. Second, the imines are activated to nucleophilic addition, which

often proceeds with high diastereoselectivity. Third, imines 6.02 show good stability to

hydrolysis and tautomerization. Finally, after reaction with a nucleophile the N-tert-

butanesulfinyl amines 6.03 can undergo a mild deprotection to the final enantiomerically

enriched amine products (6.04).

Scheme 6.1. Asymmetric synthesis of amines via N-tert-butanesulfinamide chemistry

Grignard and organolithium reagents were the first nucleophiles to be used in this synthetic

process and proceed with high yields and diastereoselectivities for a broad range of coupling

partners.2 Still, these methods suffer from poor functional group compatibility, the need for low

reaction temperatures and limited commercial availability of the organometallic reagents, a

liability for analog synthesis. Rhodium-catalyzed additions of boron reagents to N-tert-

butanesulfinyl aldimines greatly expanded the breadth of functionality that may be present

during the addition step.3 However, these methods are currently limited to the coupling of aryl

and vinyl boron reagents, which are sp2-hybridized. Therefore, additions of sp

3-hybridized

organometallic reagents that also proceed with broad functional group compatibility would

represent a significant advance, but prior to our work, only functional group tolerant additions of

allylzinc4 and allylindium reagents

5 had primarily been reported.

Due to their high functional group compatibility, organozinc reagents have become

increasingly popular, particularly in Negishi cross coupling.6 While typical organozinc halides

(i.e., RZnX) do not react directly with aldehydes, ketones, or imines, Knochel and coworkers

have reported that benzyl zinc reagents prepared using a mixture of Mg0, LiCl, and ZnCl2 add

efficiently to aldehydes and ketones in high yields at ambient temperature (Scheme 6.1).7

Additionally, these sp3-hybridized organozinc reagents (6.06 or 6.07) demonstrate good

functional-group compatibility, tolerating both ester and nitrile groups. Because of these features,

125

we desired to apply these Knochel-type organozinc reagents in diastereoselective additions to N-

tert-butanesulfinyl aldimines.89

Scheme 6.2. Preparation of benzyl zinc reagents 6.06 and 6.07 and addition to aldehydes and ketones

Optimization of benzyl zinc additions

We began by investigating the addition of the unsubstituted benzyl zinc reagent 6.09 to the p-

methoxy- and p-methylcarboxy-substituted aromatic imine substrates 6.10 and 6.11, respectively

(Table 6.1). For benzyl zinc reagent 6.09, a 30 minute reagent preparation time was found to

produce the optimal results (entries 1 and 3). Extended reagent preparation time was found to

result in lower diastereoselectivity (entry 2) as well as overaddition into the ester functional

group (entry 4). Because appropriate reagent preparation time was found to vary with the quality

Table 6.1. Optimization of benzyl zinc reagent additions

entry preparation time imine product yield,a % dr

b

1 30 min 6.10 6.12 70 92:8

2 3 h 6.10 6.12 76 81:19

3 30 min 6.11 6.13 77 93:7

4 3 h 6.11 6.13 overadditionc nd

d

5e 30 min 6.11 6.13 83 90:10

6f 30 min 6.11 6.13 88 93:7

aDetermined by

1H NMR relative to 1,3,5-trimethoxybenzene as an external standard.

bDetermined by

1H

NMR. c57% triple addition product, 37% double addition product.

dNot determined.

eReagent stored in a

Schlenk flask under static N2 for 20 days. f4.0 equiv of 6.09.

126

of the magnesium used, reagent preparations were monitored by GC analysis, following

disappearance of benzyl chloride over time after quenching an aliquot of the reaction mixture

with water.

These negative effects were not observed when the reagent was filtered away from excess

Mg0 immediately after consumption of the benzyl chloride starting material (as determined by

GC analysis). Reagent stored in a Schlenk flask for 20 days reacted similarly to freshly prepared

reagent although a decrease in concentration from 0.33 M to 0.19 M10

was observed (entry 5).

Furthermore, overaddition was not observed with a large excess of benzyl zinc reagent prepared

under the standard conditions (entry 6). Together, these results suggest that the presence of

excess Mg0 after formation of the initial desired benzyl zinc reagent leads to the formation of a

second more reactive and less selective organometallic reagent.

Evaluation of substrate scope for diastereoselective benzyl zinc addition

We next evaluated a range of N-tert-butanesulfinyl aldimine substrates and organozinc

coupling partners under these optimal conditions (Table 6.2). Reactions with both electron-rich

(entry 1) and electron-poor aromatic imines (entries 2–5) proceeded with excellent yields and

good diastereoselectivity. Para, meta and ortho substitution were all well tolerated (entries 3–5).

As expected, ortho substitution (entry 5) resulted in a slightly diminished yield but proceeded

with very high diastereoselectivity. Additions to ester and nitrile substituted imines (entries 2, 7,

9, and 10) demonstrated the functional group compatibility of this method. Furthermore, the

benzyl reagent added to the 3-pyridyl imine substrate in high yield and with good

diastereoselectivity (entries 6 and 8), indicating that nitrogen heterocycles that often interfere

with transition metal catalyzed additions, such as Rh-catalyzed arylboronic acid additions,3 do

not interfere with these MgCl2-enhanced benzyl zinc reagent additions. Additions to alkyl imine

substrates (entries 11 and 12) also proceeded in good yields although with only moderate

selectivity. Finally, electron-neutral, electron-rich, and electron-poor organozinc coupling

partners reacted smoothly and provided good stereoselectivity and functional group tolerance

(entries 2, 7 and 9).

Further highlighting the functional group compatibility of this method, organozinc reagent

6.33, which contains an ester substituent, was added to both unsubstituted and nitrile-substituted

aromatic imines 6.34 and 6.20 with good yields and moderate to good selectivity (Scheme 6.3).

However, these reactions required the more reactive dibenzyl zinc reagent 6.07, prepared with

0.55 equiv of ZnCl2 (Scheme 6.2. Preparation of benzyl zinc reagents 6.06 and 6.07 and addition

to aldehydes and ketones).

Stereochemical rationale

The sense of induction for these addition reactions was determined by rigorously establishing

the absolute configurations of addition product 6.12 by chemical correlation and products 6.27

and 6.31 by X-ray structural analysis (Figure 6.1). The sense of induction is consistent with an

open transition state as proposed for a number of N-tert-butanesulfinyl imine addition reactions

(Scheme 6.4).1 Presumably, the excess of coordinating ions in conjunction with the use of a

coordinating solvent favor this transition state over a chelating transition state.

127

Table 6.2. Benzyl zinc additions to N-tert-butanesulfinyl aldimines

entry benzyl zinc X imine R product yield,a % dr

b

1 6.09 H 6.10 4-OMeC6H4 6.12 85 90:10c

2 6.09 H 6.11 4-CO2MeC6H4 6.13 86 92:8

3 6.09 H 6.16 4-ClMeC6H4 6.23 87 92:8

4 6.09 H 6.17 3-ClMeC6H4 6.24 86 92:8

5 6.09 H 6.18 2-ClMeC6H4 6.25 79 >99:1

6 6.09 H 6.19 3-Py 6.26 98 96:4

7 6.14 OMe 6.11 4-CO2MeC6H4 6.27 69 94:6d

8 6.14 OMe 6.19 3-Py 6.28 42 98:2

9 6.15 F 6.11 4-CO2MeC6H4 6.29 86 94:6

10 6.15 F 6.20 4-CNMeC6H4 6.30 83 98:2

11 6.15 F 6.21 tBu 6.31 77 76:24d,e

12 6.15 F 6.22 PhCH2CH2 6.32 76 77:23f

aIsolated yield of mixture of diastereomers after purification by chromatography.

bDetermined by HPLC

comparison to authentic diastereomers. cAbsolute configuration determined by comparison of the optical

rotation of the amine obtained upon sulfinyl deprotection to literature values (see experimental). dAbsolute configuration was determined by X-ray crystallography.

eDetermined by mass balance of

separately isolated diastereomers. fDetermined by

1H and

19F NMR.

Scheme 6.3. Ester-substituted dibenzyl zinc additions

Scheme 6.4. Stereochemical rationale for the reaction

128

Figure 6.1. Crystal structures of inhibitor 6.27 (a) and 6.31 (b).

Preparation of aspartyl protease inhibitor precursors

We also believed that this methodology offered an efficient new route to aspartyl protease

inhibitors including inhibitors of β-secretase (BACE 1) and HIV protease, which are under

development as potential treatments for Alzheimer’s disease and HIV, respectively.11

Two key

features made this class of molecules an attractive target. First, aspartyl protease inhibitors

commonly feature a hydroxyethylamine isostere of phenylalanine, which could be derived from

a glyceraldehyde-derived imine and a benzyl zinc reagent (Scheme 6.5). Second, the introduction

of functionalized benzyl rings has been utlilized to drive potency and to improve

pharmacokinetic properties.12

Scheme 6.5. Retrosynthetic approach to aspartyl protease inhibitors

To evaluate benzyl zinc additions for aspartyl protease inhibitor synthesis, we focused on the

synthesis of anti-3-amino-4-arylbutane-1,2-diol derivatives (Scheme 6.6). Benzyl zinc reagent

6.09 added to imine 6.37, prepared from isopropylidene-protected glyceraldehyde, in high yield

and with exceptionally high selectivity. Importantly, the stereochemistry obtained is that most

commonly found in hydroxyethylamine-based protease inhibitors. Addition to imine 6.40 also

proceeded in high yield but with modest selectivity for the syn diastereomer. For imine 6.40 the

face selectivity provided by the sulfinyl group presumably opposes and modestly overrides the

stereochemical bias provided by the -stereocenter, whereas for imine 6.37 both the sulfinyl

group and the -stereocenter favor the same product. Diastereomer 6.38 was readily converted to

N-Boc-3-amino-1,2-diol 6.39 by simultaneous deprotection of the sulfinyl and isopropylidene

protecting groups followed by Boc protection of the amine functionality. N-Boc-3-amino-1,2-

diols with the stereochemistry present in 6.39 provide direct access to hydroxyethylamine

inhibitors.13

Oxidative conversion of 6.39 to N-Boc-(S)-phenylalanine also enabled rigorous

assignment of the configuration at the amine stereocenter.

129

Scheme 6.6. Addition to glyceraldehyde-derived imines for the synthesis of 3-amino-1,2-diols

Conclusions

The MgCl2-enhanced addition of benzyl zinc reagents to N-tert-butanesulfinyl imines occurs

readily at ambient temperature. Good yields, selectivity, and functional group tolerance are

observed for a variety of aromatic imines. Moreover, ester-substituted dibenzyl zinc reagents add

to nitrile-substituted imines without cross-reactivity, further highlighting the functional-group

compatible nature of the transformation. Although benzyl additions to aliphatic imines generally

showed only moderate selectivity, addition to sulfinyl imine 6.37, prepared from isopropylidene-

protected glyceraldehyde, proceeded in high yield and with very high selectivity, indicating that

this method should allow the rapid introduction of a variety of functionalized benzyl substituents

into hydroxyethylamine-based aspartyl protease inhibitors.

Experimental



without further purification. Benzyl chlorides were filtered through neutral Al2O3 (Brockman I)

immediately prior to use. Magnesium turnings were purchased new from Riedel-de Haën

(Sigma-Aldrich). Zinc chloride (H2O, oxide, OH < 100 ppm; 99.99% Zn) was purchased from

Strem, and lithium chloride, from Fluka (Sigma-Aldrich). Tetrahydrofuran (THF) was passed

through a column of activated alumina under nitrogen, and methanol was distilled from CaH2.

Salt solutions were prepared as previously described by Knochel and coworkers.7a

All reactions

involving air-sensitive and moisture-sensitive reagents were carried out using syringes, tripled

flushed with nitrogen before use. Syringe filtrations were performed with Millex-HN 0.45 µm

Nylon 33 mm syringe filters. Glassware was dried overnight at 140 ˚C or flame-dried under

vacuum prior to use. Chromatography was performed with Merck 60 230–240 mesh silica gel, or

utilizing a Biotage SP Flash Purification System (Biotage No. SP1-B1A). NMR spectra were

obtained at ambient temperature on a Bruker AVB-400 or AVB-500 spectrometer. NMR

130

chemical shifts are reported in ppm relative to CHCl3 (7.26), or TMS (0.00) for 1H,

trifluoroacetic acid (−76.55) for 19

F, and CDCl3 (77.16) or TMS (0.00) for 13

C. Determinations

of diastereomeric ratios were performed using an Agilent 1100 series HPLC equipped with a

normal-phase silica column (Microsorb Si 100 Å packing) and a multi-wavelength detector;

samples were dissolved in 3:1 hexanes:iPrOH. HPLC methods were developed using the authentic diastereomers of products 6.12–6.13, 6.23–6.30, 6.35–6.36 and 6.38 prepared from the

compound of interest14

or via unselective Grignard addition to imine 6.37. IR spectra were

recorded on a Nicolet 6700 FTIR spectrometer, and only partial data are provided. Melting

points were acquired using a Mel-Temp apparatus, and they are reported uncorrected. Specific

rotations were determined using a Perkin-Elmer 341 polarimeter with a sodium lamp, and

concentrations are reported in g/dL. Mass spectra (HRMS) analysis was performed by the Yale

Protein Expression Database facility on a 9.4T Bruker Qe FT-ICR MS.

Preparation of benzyl zinc reagents

General procedure. To an appropriately sized Schlenk flask was added magnesium turnings

(2.5 equiv). The flask was flushed with N2, and then a THF solution (1.0 mL/mmol of the benzyl

chloride) of ZnCl2 (1.1 M) and LiCl (1.5 M) was added via syringe. The appropriate benzyl

chloride (1.0 equiv) and THF (0.5 mL/mmol of the benzyl chloride) were combined in a separate

round bottom flask. The original Schlenk flask was placed in an ambient temperature water bath,

and the benzyl chloride solution was added dropwise via syringe. The water bath was removed

upon addition, and the heterogeneous reaction mixture was stirred vigorously for the specified

time. Reaction times were determined by GC analysis, using complete consumption of the

benzyl chloride as the endpoint. The reaction mixture was removed from the magnesium into a

syringe and then passed through a syringe filter into a second Schlenk flask for storage. Reagent

concentrations were determined via iodometric titration.10

Benzyl zinc reagent 6.09. The general procedure was followed with 669 mg (27.5 mmol) of

magnesium turnings, 11.0 mL of ZnCl2/LiCl solution, and 1.3 mL (11 mmol) of benzyl chloride

in 5.5 mL of THF. The heterogeneous reaction mixture was stirred 30 min after addition of the

benzyl chloride solution. After transfer, iodometric titration determined a concentration of 0.43

M.

4-Methoxybenzyl zinc reagent 6.14. The general procedure was followed with 243 mg

(10.0 mmol) of magnesium turnings, 4.0 mL of ZnCl2/LiCl solution, and 0.54 mL (4.0 mmol) of

4-methoxybenzyl chloride in 2.0 mL of THF. The heterogeneous reaction mixture was stirred for

30 min after addition of the benzyl chloride solution. After transfer, iodometric titration

determined a concentration of 0.39 M.

131

4-Fluorobenzyl zinc reagent 6.15. The general procedure was followed with 608 mg (25.0

mmol) of magnesium turnings, 10.0 mL of ZnCl2/LiCl solution, and 1.20 mL (10.0 mmol) of 4-

fluorobenzyl chloride in 5.0 mL of THF. The heterogeneous reaction mixture was stirred for 35

min after addition of the benzyl chloride solution. After transfer, iodometric titration determined

a concentration of 0.36 M.

Di(3-ethylcarboxybenzyl) zinc reagent 6.33. To a 100 mL Schlenk flask was added 1.65 g

(68.0 mmol, 2.50 equiv) of magnesium turnings. The flask was flushed with N2, and then a THF

solution (13.6 mL) of ZnCl2 (1.1 M, 0.55 equiv) and LiCl (1.5 M, 0.75 equiv) was added. Ethyl

3-(chloromethyl)benzoate (4.60 mL, 27.2 mmol, 1.0 equiv) and THF (24.0 mL) were combined

in a separate round bottom flask. The original Schlenk flask was placed in an ambient

temperature water bath. The benzyl chloride solution was added dropwise via cannula over 8

min. Due to a strong exotherm, the reaction flask remained in the water bath while the reaction

mixture was stirred vigorously for 35 min, the endpoint determined from GC analysis. The

reaction mixture was removed from the magnesium via cannula filtration into a second Schlenk

flask. A 1.0 mL aliquot of reagent was stirred with 1.0 mL of 1.1 M ZnCl2 / 1.5 M LiCl solution

of THF, at which point, iodometric titration determined a concentration of 0.22 M of the

monobenzyl zinc species, and thus 0.11 M of the dibenzyl reagent.

Synthesis of N-tert-butanesulfinyl imine starting materials

Sulfinyl imines 6.10, 6.16, 6.19, 6.34,15

6.2116

and 6.2017

were synthesized according to

literature procedures.

General Procedure. Cs2CO3 (1.2 equiv) and tert-butanesulfinamide (1.2 equiv) were added

to an appropriately sized round bottom flask. The flask was flushed with N2 followed by the

addition of CH2Cl2 and the aldehyde (1.0 equiv, 0.2 M). The flask was equipped with a reflux

condenser, and the reaction mixture was refluxed in a 46 °C oil bath for 14–18 h. The mixture

was filtered through a pad of Celite and concentrated under reduced pressure. The desired

product was isolated via silica gel chromatography, visualizing by UV.

Sulfinyl imine 6.11. The general procedure was followed with 2.38 g (7.31 mmol) of

Cs2CO3, 0.886 g (7.31 mmol) of R-(–)-tert-butanesulfinamide and 1.00 g (6.09 mmol) of 4-

carbomethoxybenzaldehyde to afford the imine product 6.11 (1.53 g, 94%) as a white solid, m.p.

132

92.7–93.2 °C. IR (in CHCl3): 2955, 1724, 1280, 1109, 1084 cm–1

. []20

D –71.1 (c 0.78, CHCl3).

1H NMR (400 MHz, CDCl3): 8.63 (s, 1H), 8.13 (d, J = 8.4 Hz, 2H), 7.91 (d, J = 8.4 Hz, 2H),

3.94 (s, 3H), 1.27 (s, 9H); 13

C NMR (101 MHz, CDCl3): 166.47, 162.07, 137.70, 133.43,

130.32, 129.38, 58.33, 52.65, 22.85. HRMS (m/z): [M+H]+ calcd. for C13H18NO3S, 268.1002;

found, 268.0999.


Cs2CO3, 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide and 1.13 mL (10.0 mmol) of 3-

chlorobenzaldehyde to afford the imine product 6.17 (2.40 g, 98%) as an off-white solid, m.p.

34.5–35.3 °C. IR (in CHCl3): 2981, 1601, 1566, 1212, 1082 cm–1

. []20

D –77.3 (c 1.27, CHCl3).

1H NMR (400 MHz, CDCl3): 8.48 (s, 1H), 7.80 (s, 1H), 7.63 (d, J = 7.5 Hz, 1H), 7.41 (d, J =

8.7 Hz, 1H), 7.35 (apparent t, J = 7.8 Hz, 1H), 1.21 (s, 9H); 13

C NMR (101 MHz, CDCl3): δ

161.48, 135.68, 135.18, 132.34, 130.31, 128.63, 127.96, 58.02, 22.66. HRMS (m/z): [M+H]+

calcd. for C11H15NOS[35]

Cl, 244.0557; found, 244.0553.


Cs2CO3, 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide and 1.13 mL (10.0 mmol) of 2-

chlorobenzaldehyde to afford the imine product 6.18 (2.36 g, 97%) as a white solid. Analytical

data were consistent with previous literature reports.18

Sulfinyl imine 6.22. To a 100 mL round bottom flask was added 1.92 g (12.0 mmol) CuSO4

and 1.45 g (12.0 mmol) of R-(–)-tert-butanesulfinamide. The flask was flushed with N2, then

CH2Cl2 (50 mL) was added, followed by 3-phenylpropanal (90% purity, 1.51 mL, 10.0 mmol).

The suspension was stirred vigorously for 22h then filtered through Celite and concentrated

under vacuum. Purification by flash chromatography afforded imine product 6.22 (0.961 g, 40%)

as a clear oil. Analytical data were consistent with previous literature reports.19

133

Addition of benzyl zinc reagents to N-tert-butanesulfinyl imines

General Procedure. A 25-mL round bottom flask was charged with imine (0.500 mmol) and

flushed with N2. The imine was then dissolved in THF (0.50 mL). The benzyl zinc reagent was

added dropwise via syringe, and the reaction mixture was stirred 24 h at ambient temperature.

The reaction mixture was then cooled to 0 °C, diluted with EtOAc, and the reaction was

quenched with 15 mL of sat. NH4Cl (aq). The aqueous layer was extracted with EtOAc (3 × 20

mL), and the combined organic layers were dried over NaSO4, filtered, and concentrated under

reduced pressure. The desired product was isolated using silica gel chromatography, visualizing

by UV and PMA staining. All products were isolated as a mixture of diastereomers, and NMR

data corresponds to the major diastereomer unless otherwise noted.

Product 6.12. The general procedure was followed with 120 mg (0.500 mmol) of imine 6.10

and 2.8 mL (1.0 mmol) of freshly prepared benzyl zinc reagent 6.09 (0.36 M). Purification by

flash chromatography afforded product 6.12 (142 mg, 85%, 90:10 dr) as a white solid, m.p. 92.9–94.4 °C. HPLC (silica column, hexanes:EtOH, 99:1 to 98:2 over 55 min, 0.5 mL/min, λ =

210 nm): tr,minor = 14.7 min, tr,major = 11.7 min. IR (in CHCl3): 3330, 2960, 1425, 1051, 1023

cm–1

. 1H NMR (400 MHz, CDCl3): δ 7.23–7.12 (m, 5H), 7.03–6.93 (m, 2H), 6.82 (d, J = 8.7 Hz,

2H), 4.70–4.35 (m, 1H), 3.78 (s, 3H), 3.46 (d, J = 3.6 Hz, 1H), 3.28 (dd, J = 13.4, 6.5 Hz, 1H), 2.98 (dd, J = 13.4, 7.8 Hz, 1H), 1.15 (s, 9H);

13C NMR (126 MHz, CDCl3): δ 159.27, 137.76,

133.90, 129.81, 128.63, 128.33, 126.52, 114.00, 60.37, 55.99, 55.37, 43.70, 22.68. HRMS (m/z):

[M+H]+ calcd. for C19H26NO2S, 332.1679; found, 332.1675.

Free amine 6.42. (Note: compound 6.42 was synthesized to determine sense of induction of

the benzyl zinc addition through chemical correlation). A flame-dried scintillation vial was

charged sulfinyl amine 6.12 (20.7 mg, 0.0624 mmol, 93:7 dr). The product was dissolved in dry

MeOH (4.0 mL), and 4.0 M HCl in dioxane (80 L, 0.32 mmol) was added dropwise via syringe.

The reaction mixture was stirred 3 h at ambient temperature. The reaction mixture was then

concentrated under reduced pressure, diluted with EtOAc, and washed with sat. NaHCO3 (aq).

The aqueous layer was extracted twice more with EtOAc. The combined organic layers were

dried over MgSO4, filtered, and concentrated under reduced pressure. The product was isolated

as a clear, yellow oil (11.9 mg, 87%) and used without further purification. Analytical data were

consistent with those previously reported. []20

D –97.0 (c 0.70, 1:1 MeOH:0.1N HCl) [lit. []20

D

–91.9 (c 1.00, 85% ee, 1:1 MeOH:0.1N HCl)].20

134



flash chromatography afforded product 6.13 (154 mg, 86%, 92:8 dr) as a clear, amorphous solid. HPLC (silica column, 98.5:1.5 hexanes:EtOH, 1.0 mL/min, λ = 254 nm): tr,minor = 20.8 min,

tr,major = 23.3 min. IR (in CHCl3): 3240, 2953, 1718, 1275, 1046 cm–1

. 1H NMR (400 MHz,

CDCl3): δ 7.97 (d, J = 8.3 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.24–7.14 (m, 3H), 7.00 (dd, J =

7.6, 1.4 Hz, 2H), 4.78–4.53 (m, 1H), 3.90 (s, 3H), 3.75 (d, J = 4.8 Hz, 1H), 3.30 (dd, J = 13.4, 6.7 Hz, 1H), 3.02 (dd, J = 13.4, 7.5 Hz, 1H), 1.16 (s, 9H);

13C NMR (101 MHz, CDCl3): δ

166.67, 146.89, 136.83, 129.74, 129.51, 129.47, 128.25, 127.26, 126.59, 60.61, 56.09, 52.03,

43.53, 22.41. HRMS (m/z): [M+Na]+ calcd. for C20H25NO3SNa, 382.1447; found, 382.1429.



flash chromatography afforded product 6.23 (146 mg, 87%, 92:8 dr) as a white solid, m.p. 129.7–130.6 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =

14.7 min, tr,major = 11.7 min. IR (in CHCl3): 3201, 2957, 1493, 1043, 1015 cm–1

. 1H NMR (500

MHz, CDCl3): δ 7.26 (t, J = 4.2 Hz, 3H), 7.18 (m, 4H), 7.05–6.90 (m, 2H), 4.57 (m, 1H), 3.50 (d,

J = 3.9 Hz, 1H), 3.27 (dd, J = 13.4, 6.5 Hz, 1H), 2.97 (dd, J = 13.4, 7.7 Hz, 1H), 1.15 (s, 9H); 13

C

NMR (126 MHz, CDCl3): δ 140.22, 136.99, 133.59, 129.60, 128.70, 128.33, 126.64, 60.24,

56.05, 43.55, 22.49. HRMS (m/z): [M+H]+ calcd. for C18H23NOS

[35]Cl, 336.1183; found,

336.1184.



flash chromatography afforded product 6.24 (144 mg, 86%, 92:8 dr) as a white solid, m.p.

116.7–117.4 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =


. 1H NMR (400

MHz, CDCl3): δ 7.28–7.15 (m, 6H), 7.12–7.06 (m, 1H), 7.04–6.96 (m, 2H), 4.62–4.50 (m, 1H),

135

3.52 (br s, 1H), 3.26 (dd, J = 13.5, 6.7 Hz, 1H), 2.99 (dd, J = 13.4, 7.5 Hz, 1H), 1.15 (s, 9H); 13

C NMR (126 MHz, CDCl3): δ 143.95, 137.06, 134.55, 129.94, 129.74, 128.49, 128.21, 127.44,

126.83, 125.85, 60.57, 56.26, 43.68, 22.63. HRMS (m/z): [M+H]+ calcd. for C18H23NOS

[35]Cl,

336.1183; found, 336.1187.



flash chromatography afforded product 6.25 (133 mg, 79%, >99:1 dr) as a white solid, m.p. 109.4–110.2 °C. HPLC (silica column, 95:5 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =


. 1H NMR (500

MHz, CDCl3): δ 7.44–7.31 (m, 2H), 7.31–7.18 (m, 5H), 7.12 (d, J = 7.0 Hz, 2H), 5.15–4.92 (m,

1H), 3.76 (d, J = 6.3 Hz, 1H), 3.19 (dd, J = 13.7, 5.5 Hz, 1H), 3.05 (dd, J = 13.7, 8.2 Hz, 1H), 1.06 (s, 9H).

13C NMR (126 MHz, CDCl3): δ 139.25, 137.26, 132.82, 129.83, 129.65, 128.80,

128.66, 128.39, 127.05, 126.76, 58.29, 56.37, 42.65, 22.33. HRMS (m/z): [M+Na]+ calcd. for

C18H22NOS[35]

ClNa, 358.1003; found, 358.1000.



flash chromatography afforded product 6.26 (149 mg, 98%, 96:4 dr), which became a waxy solid

upon storage at 0 °C. HPLC (silica column, 85:15 hexanes (0.1% v/v Et2NH):iPrOH, 0.5

mL/min, λ = 254 nm): tr,minor = 18.8 min, tr,major = 25.6 min. IR (in CHCl3): 3204, 2957, 1476,

1428, 1052 cm–1

. 1H NMR (400 MHz, CDCl3): δ 8.60–8.40 (m, 2H), 7.59–7.46 (m, 1H), 7.25–

7.13 (m, 4H), 7.07–6.89 (m, 2H), 4.72–4.52 (m, 1H), 3.63 (d, J = 4.3 Hz, 1H), 3.32 (dd, J = 13.4, 6.7 Hz, 1H), 3.00 (dd, J = 13.4, 7.7 Hz, 1H), 1.14 (s, 9H);


149.35, 149.04, 137.23, 136.69, 135.12, 129.69, 128.54, 126.91, 123.45, 58.82, 56.28, 43.48,

22.57. HRMS (m/z): [M+H]+ calcd. for C17H23N2OS, 303.1526; found, 303.1526.

136



flash chromatography afforded product 6.27 (134 mg, 69%, 96:4 dr) as a white solid, m.p. 108.6–111.8 °C. HPLC (silica column, 98.5:1.5 hexanes:EtOH, 0.5 mL/min, λ = 210 nm): tr,minor

= 39.2 min, tr,major = 42.0 min. IR (neat): 2960, 1716, 1514, 1276, 1246, 1055 cm–1

. 1H NMR

(400 MHz, CDCl3): δ 7.88 (d, J = 8.0 Hz, 2H), 7.21 (d, J = 8.2 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H),

6.65 (d, J = 8.4 Hz, 2H), 4.60–4.50 (m, 1H), 3.83 (s, 3H), 3.68 (s, 3H), 3.52 (d, J = 3.9 Hz, 1H),

3.18 (dd, J = 13.5, 6.2 Hz, 1H), 2.87 (dd, J = 13.5, 7.8 Hz, 1H), 1.10 (s, 9H); 13

C NMR (101 MHz, CDCl3): δ 166.98, 158.49, 147.07, 130.76, 129.97, 129.75, 128.85, 127.58, 113.87, 60.68,


found, 390.1732.



flash chromatography afforded product 6.28 (69.6 mg, 42%, 98:2 dr) which became a waxy,

yellow solid upon storage at 0 °C. HPLC (silica column, 85:15 hexanes (0.1% v/v EtN2H):

iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 13.9 min, tr,major = 15.2 min. IR (in CHCl3): 2961,

2925, 1513, 1249, 1040 cm–1

. 1H NMR (400 MHz, CDCl3): δ 8.54–8.42 (m, 2H), 7.63–7.39 (m,

1H), 7.26–7.14 (m, 1H), 6.92–6.75 (m, 2H), 6.75–6.64 (m, 2H), 4.62–4.42 (m, 1H), 3.69 (s, 3H),

3.66 (d, J = 4.9 Hz, 1H), 3.19 (dd, J = 13.6, 6.6 Hz, 1H), 2.89 (dd, J = 13.6, 7.6 Hz, 1H), 1.09 (s, 9H);

13C NMR (126 MHz, CDCl3): δ 158.62, 148.98, 148.83, 138.11, 136.18, 130.72, 128.44,

123.88, 114.03, 58.94, 56.40, 55.34, 42.63, 22.63. HRMS (m/z): [M+H]+ calcd. for

C18H25N2O2S, 333.1631; found, 333.1621.



flash chromatography afforded product 6.29 (163 mg, 86%, 94:6 dr) as an amorphous solid.

137

HPLC (silica column, 99:1 hexanes:EtOH, 1.0 mL/min, λ = 210 nm): tr,minor = 24.1 min, tr,major =

27.8 min. IR (neat): 3238, 2954, 1718, 1509, 1276, 1046 cm–1

. 1H NMR (400 MHz, CDCl3): δ

7.95 (d, J = 8.3 Hz, 2H), 7.26 (d, J = 8.3 Hz, 2H), 6.93–6.83 (m, 4H), 4.65–4.58 (m, 1H), 3.90 (s,

3H), 3.55 (d, J = 4.1 Hz, 1H), 3.30 (dd, J = 13.5, 6.2 Hz, 1H), 2.96 (dd, J = 13.5, 7.9 Hz, 1H), 1.18 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 166.90, 146.66, 132.65 (d, JCF = 3.1 Hz), 131.24

(d, JCF = 7.8 Hz), 130.08, 129.97, 127.54, 115.44, 115.23, 60.56, 56.32, 52.37, 42.72, 22.71; 19

F NMR (376 MHz, CDCl3) δ –116.44. HRMS (m/z): [M+Na]

+ calcd. for C20H24NO3FSNa,

400.1353; found, 400.1345.



flash chromatography afforded product 6.30 (143 mg, 83%, 97:3 dr) as a white solid, m.p. 118.2–119.5 °C. HPLC (silica column, 90:10 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor =

14.2 min, tr,major = 12.8 min. IR (neat): 3236, 2954, 2225, 1509, 1044 cm–1

. 1H NMR (400 MHz,

CDCl3): δ 7.60 (d, J = 8.1 Hz, 2H), 7.32 (d, J = 8.2 Hz, 2H), 6.93–6.89 (m, 4H), 4.65–4.58 (m,

1H), 3.53 (d, J = 4.6 Hz, 1H), 3.28 (dd, J = 13.7, 6.4 Hz, 1H), 2.96 (dd, J = 13.6, 7.7 Hz, 1H), 1.17 (s, 9H);

13C NMR (126 MHz, CDCl3): δ 146.98, 132.63, 132.26 (d, JCF = 3.2 Hz), 131.21

(d, JCF = 8.1 Hz), 128.30, 118.71, 115.65, 115.49, 112.15, 60.72, 56.52, 42.74, 22.69; 19

F NMR (376 MHz, CDCl3): δ –115.92. HRMS (m/z): [M+H]

+ calcd. for C19H22N2OFS, 345.1431; found,

345.1429.



flash chromatography afforded separable diastereomeric products 6.31a (major) and 6.31b

(minor) in a 76:24 dr. 6.31a (RS,R): 88 mg, 59%, white solid, m.p. 117.4–118.3 °C. IR (neat):

2956, 1510, 1459, 1214, 1043 cm–1

. 1H NMR (400 MHz, CDCl3): δ 7.14–7.09 (m, 2H), 6.98–

6.91 (m, 2H), 3.26 (m, 1H), 3.11 (d, J = 7.2 Hz, 1H), 3.03 (dd, J = 14.2, 2.4 Hz, 1H), 2.43 (dd, J = 14.2, 10.6 Hz, 1H), 1.06 (s, 9H), 0.92 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 135.67 (d, JCF =

3.2 Hz), 131.12 (d, JCF = 7.9 Hz), 115.34, 115.13, 67.38, 56.25, 37.95, 35.18, 27.35, 22.63; 19

F NMR (376 MHz, CDCl3): δ –117.63. HRMS (m/z): [M+H]

+ calcd. for C16H27NOFS, 300.1792;

found, 300.1788. 6.31b (RS,S): 28 mg, 18%, white solid, m.p. 90.8–91.5 °C. IR (in CHCl3):

2960, 1510, 1476, 1223, 1062 cm–1

. 1H NMR (400 MHz, CDCl3): δ 7.25–7.19 (m, 2H), 7.02–

6.96 (m, 2H), 3.24 (m, 1H), 3.15–3.02 (m, 2H), 2.63 (dd, J = 14.6, 8.4 Hz, 1H), 1.14 (s, 9H),

138

0.96 (s, 9H); 13

C NMR (101 MHz, CDCl3): δ 134.50 (d, JCF = 3.3 Hz), 131.22 (d, JCF = 7.7 Hz),

115.55, 115.34, 66.04, 56.55, 37.58, 36.21, 27.12, 22.96; 19

F NMR (376 MHz, CDCl3): δ

–116.75. HRMS (m/z): [M+H]+ calcd. for C16H27NOFS, 300.1792; found, 300.1787.



flash chromatography afforded product 6.32 (131 mg, 76%) as a clear oil. IR (in CHCl3): 3224, 2926, 1510, 1221, 1052 cm

–1.

1H NMR (400 MHz, CDCl3): δ 7.27–7.16 (m, 2H), 7.16–7.09 (m,

3H), 7.09–7.02 (m, 2H), 6.96–6.85 (m, 2H), 3.43–3.33 (m, 1H), 3.05 (d, J = 6.2 Hz, 1H), 2.79–2.63 (m, 4H), 1.97–1.80 (m, 2H), 1.03 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 141.45, 134.18

(d, JCF = 3.1 Hz), 131.22 (d, JCF = 7.8 Hz), 128.68, 128.67, 126.22, 115.45, 115.24, 57.98, 56.03,

42.02, 37.10, 32.22, 22.72; 19

F NMR (376 MHz, CDCl3): –117.04. HRMS (m/z): [M+H]+ calcd.

for C20H27NOFS, 348.1792; found, 348.1774.


and 4.8 mL (1.1 mmol) of freshly prepared dibenzyl reagent 6.33 (0.22 M). Purification by flash

chromatography afforded product 6.35 (145 mg, 73%, 80:20 dr) as an oil. HPLC (silica column, 93:7 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 16.7 min, tr,major = 14.8 min. IR (in

CHCl3): 3018, 2983, 1713, 1281, 1214 cm–1

. 1H NMR (400 MHz, CDCl3): δ 7.78 (d, J = 7.8 Hz,

1H), 7.65 (s, 1H), 7.28–7.10 (m, 6H), 7.03 (d, J = 7.6 Hz, 1H), 4.59–4.51 (m, 1H), 4.28 (q, J =

7.1 Hz, 2H), 3.48 (d, J = 3.3 Hz, 1H), 3.27 (dd, J = 13.5, 6.4 Hz, 1H), 3.00 (dd, J = 13.4, 7.7 Hz, 1H), 1.31 (t, J = 7.1 Hz, 3H), 1.10 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 166.70, 141.49,

137.89, 134.42, 130.91, 130.53, 128.82, 128.34, 128.22, 127.93, 127.45, 61.13, 60.69, 56.18,

43.36, 22.75, 14.53. HRMS (m/z): [M+H]+ calcd. for C21H28NO3S, 374.1784; found, 374.1776.


and 4.6 mL (1.0 mmol) of freshly prepared dibenzyl reagent 6.33 (0.22 M). Purification by flash

139

chromatography afforded product 6.36 (147 mg, 74%, 97:3) as an amorphous solid. HPLC (silica column, 88:12 hexanes:iPrOH, 0.5 mL/min, λ = 210 nm): tr,minor = 17.2 min, tr,major = 20.8 min.

IR (in CHCl3): 3246, 3018, 2982, 1715, 1281, 1214, 1055 cm–1

. 1H NMR (400 MHz, CDCl3): δ

7.87 (d, J = 7.8 Hz, 1H), 7.70 (s, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 8.3 Hz, 2H), 7.28 (t,

J = 7.6 Hz, 1H), 7.10 (d, J = 7.7 Hz, 1H), 4.70–4.64 (m, 1H), 4.35 (q, J = 7.1 Hz, 2H), 3.59 (d, J

= 5.2 Hz, 1H), 3.32 (dd, J = 13.6, 6.8 Hz, 1H), 3.06 (dd, J = 13.6, 7.4 Hz, 1H), 1.38 (t, J = 7.1 Hz, 3H), 1.15 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 166.47, 146.89, 136.90, 134.20, 132.63,

130.83, 130.72, 128.66, 128.34, 128.23, 118.70, 112.08, 61.29, 60.67, 56.55, 43.22, 22.65, 14.52.

HRMS (m/z): [M+H]+ calcd. for C22H27N2O3S, 399.1737; found, 399.1731.

Synthesis of aspartyl protease inhibitor precursors

Sulfinyl imine 6.37. To a 250 mL round bottom flask was added 505 mg (3.88 mmol) of

(2R)-2,3-O-isopropylideneglyceraldehyde21

and 699 mg (5.77 mmol) of S-(+)-tert-

butanesulfinamide. The flask was flushed very briefly with N2, then CH2Cl2 (96 mL) and TiOEt4

(4.0 mL, 19.2 mmol) were added. The reaction mixture was stirred for 6 h. The flask was then

cooled to 0 °C, and 4 mL of cold water was added. The resulting mixture was filtered through

Celite, washing with EtOAc. The filtrate was washed with 20 mL of brine, and the aqueous layer

was extracted twice more with EtOAc. The combined organic layers were then concentrated

under vacuum. Purification by flash chromatography afforded imine product 6.37 (584 mg, 65%)

as a light yellow oil. IR (neat): 2984, 1625, 1372, 1219, 1061 cm–1

. 1H NMR (400 MHz, CDCl3):

δ 8.05 (d, J = 4.1 Hz, 1H), 4.98–4.66 (m, 1H), 4.42–4.12 (m, 1H), 4.03 (dd, J = 8.6, 5.1 Hz, 1H),

1.44 (s, 3H), 1.41 (s, 3H), 1.19 (s, 9H); 13

C NMR (101 MHz, CDCl3): δ 168.15, 110.97, 77.07,


found, 234.11580.

Product 6.38. The general procedure for additions of benzyl zinc reagents to N-tert-

butanesulfinyl imines was followed with 120 mg (0.516 mmol) of imine 6.37 and 3.1 mL (1.0

mmol) of freshly prepared benzyl zinc reagent 6.09 (0.33 M). Purification by flash

chromatography afforded product 6.38 (146 mg, 87%, >99:1 dr) as an off-white solid, m.p. 113.1–114.4 °C. HPLC (silica column, hexanes:iPrOH, 95:5, 0.5 mL/min, λ = 254 nm): tr,minor =

27.0 min (only observed in authentic diastereomer mixture prepared by Grignard reagent

addition), tr,major = 24.7 min. IR (in CHCl3) 3221, 1455, 1370, 1062 cm–1

. 1H NMR (400 MHz,

CDCl3): δ 7.42–7.07 (m, 5H), 4.20–4.12 (m, 1H), 4.12–4.04 (m, 1H), 3.96 (dd, J = 8.4, 6.1 Hz,

1H), 3.74–3.61 (m, 1H), 3.48 (d, J = 6.1 Hz, 1H), 3.01 (dd, J = 14.0, 7.4 Hz, 1H), 2.77 (dd, J = 14.0, 6.2 Hz, 1H), 1.48 (s, 3H), 1.33 (s, 3H), 1.11 (s, 9H);


140

137.50, 129.67, 128.64, 126.73, 109.50, 65.99, 58.64, 56.23, 37.76, 26.64, 24.95, 22.61. HRMS

(m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found, 326.1774.

Compound 6.39. A 25 mL round bottom flask was charged with product 6.38 (97 mg, 0.30

mmol). The flask was placed in an ambient temperature water bath, and a 4.0 M solution of HCl

in dioxane (6.0 mL, 24 mmol) was added. The bath was removed, and the reaction mixture was

stirred. After 10 min, 21.6 L of water (1.20 mmol) was added, and the reaction mixture was

stirred for 20 h. Et2O (10 mL) was then added slowly to the solution. The resulting white

precipitate was collected by vacuum filtration and washed with Et2O. The precipitation and

collection steps were repeated twice more on the filtrate with 5 mL of Et2O, concentrating the

sample at atmospheric pressure before the final precipitation. The precipitate was dissolved in

MeOH, transferred to a 25 mL round bottom flask, and concentration under vacuum to afford the

amine hydrochloride product (62.8 mg). Dioxane (3.75 mL), water (0.188 mL, 5% v/v) and

Boc2O (73.1 L, 0.328 mmol) were added to the flask. The flask was placed in a water bath, and

NEt3 (48.4 L, 0.347 mmol) was added. The flask was removed from the water bath, and the

reaction mixture was stirred 3 h at ambient temperature. The reaction mixture was then

concentrated under vacuum. Purification by flash chromatography afforded product 6.39 (74 mg,

86%) as a white solid. Analytical data were consistent with previous literature reports.22

1H NMR

(400 MHz, CDCl3): δ 7.45–7.11 (m, 5H), 4.55 (d, J = 8.6 Hz, 1H), 3.87–3.76 (m, 1H), 3.65 (dd,

J = 10.7, 7.9 Hz, 2H), 3.50–3.27 (m, 2H), 3.10 (dd, J = 14.2, 4.1 Hz, 1H), 2.91 (dd, J = 14.2, 7.7 Hz, 1H), 2.77 (d, J = 9.0 Hz, 1H), 1.38 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 157.21, 137.43,

129.59, 128.78, 126.79, 80.57, 73.19, 63.02, 52.38, 36.60, 28.38.

N-(tert-Butoxycarbonyl)-L-phenylalanine (6.43). (Note: compound 6.43 was synthesized to

determine sense of induction of the benzyl zinc addition through chemical correlation). To a 10

mL round bottom flask was added 25 mg (0.089 mmol) of 6.39. Dioxane (0.40 mL) and water

(0.160 mL) were added, followed by Na2CO3 (5.8 mg, 0.055 mmol), KMnO4 (3.0 mg, 0.019

mmol), and NaIO4 (79 mg, 0.37 mmol) were added. The reaction mixture was stirred for 17 h.

The reaction mixture was then diluted with 5 mL of EtOAc, and the resulting solution was

washed with sat. NaHCO3 (aq.). The aqueous layer was washed with EtOAc and then acidified

with NaHSO4 (aq.) to pH 1, and was extracted with EtOAc (3 × 20 mL). The combined organic

layers were then concentrated under vacuum. Due to the presence of impurities, a second acid-

base extraction was required to afford amino acid product 6.43 (10 mg, 42%) as a clear oil.

Analytical data were consistent with previous literature reports.23

[]20

D +21.3 (c 0.667, EtOH)

[lit. []20

D +23.85 (c 2, EtOH)

24 and []20

D +25.2 (c 1, EtOH)

25].

141

Sulfinyl imine 6.40. Imine 6.40 was prepared analogously to imine 6.38 utilizing R-(–)-tert-

butanesulfinamide. The reaction afforded imine product 6.40 (439 mg, 48%) as a light yellow oil. IR (neat): 2984, 1626, 1372, 1218, 1059 cm

–1.

1H NMR (400 MHz, CDCl3): δ 8.02 (d, J =

4.6 Hz, 1H), 4.94–4.74 (m, 1H), 4.43–4.17 (m, 1H), 4.01 (dd, J = 8.7, 5.5 Hz, 1H), 1.46 (s, 3H), 1.41 (s, 3H), 1.20 (s, 9H);

13C NMR (101 MHz, CDCl3): δ 167.40, 111.04, 76.85, 67.18, 57.22,

26.43, 25.46, 22.40. HRMS (m/z): [M+H]+ calcd. for C10H20NO3S, 234.11584; found,

234.11580.

Product 6.41. The general procedure for additions of benzyl zinc reagents to N-tert-

butanesulfinyl imines was followed with 85.3 mg (0.366 mmol) of imine 6.40 and 2.0 mL (0.73

mmol) of freshly prepared benzyl zinc reagent 6.09 (0.37 M). Purification by flash

chromatography afforded separable diastereomeric products 6.41a (major) and 6.41b (minor) in

73:27 dr. 6.41a (RS,R,S): 70.0 mg, 59%, clear oil which became a white solid upon

concentration from CH2Cl2/CDCl3, m.p. 66.9–69.0 °C. IR (in CHCl3): 2986, 1372, 1216, 1057,

750 cm–1

. 1H NMR (400 MHz, CDCl3): δ 7.21 (apparent t, J = 7.2 Hz, 2H), 7.13 (apparent t, J =

8.3 Hz, 3H), 4.05 (td, J = 6.4, 3.3 Hz, 1H), 3.94–3.86 (m, 2H), 3.82 (dd, J = 8.6, 6.7 Hz, 1H),

3.51–3.42 (m, 1H), 2.82 (d, J = 7.4 Hz, 2H), 1.39 (s, 3H), 1.27 (s, 3H), 1.05 (s, 9H); 13

C NMR (126 MHz, CDCl3): δ 137.94, 129.72, 128.59, 126.69, 109.30, 76.39, 66.38, 57.95, 55.88, 40.86,

26.64, 25.21, 22.77. HRMS (m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found, 326.1778.

6.41b (RS,S,S): 26.4 mg, 22%, white solid, m.p. 87.6–90.0 °C. IR (in CHCl3): 2983, 2926, 1370, 1214, 1065, 754 cm

–1.

1H NMR (400 MHz, CDCl3): δ 7.32–7.23 (m, 3H), 7.21–7.13 (m, 2H),

3.97–3.82 (m, 1H), 3.79–3.63 (m, 2H), 3.53–3.41 (m, 1H), 3.22 (d, J = 9.2 Hz, 1H), 3.14 (dd, J =

13.6, 4.1 Hz, 1H), 3.04 (dd, J = 13.6, 4.9 Hz, 1H), 1.40 (s, 3H), 1.30 (s, 3H), 1.07 (s, 9H); 13

C NMR (126 MHz, CDCl3): δ 135.63, 131.22, 128.67, 126.89, 109.84, 75.78, 67.96, 59.30, 56.29,

37.97, 27.06, 25.60, 22.72. HRMS (m/z): [M+H]+ calcd. for C17H28NO3S, 326.1784; found,

326.1778.

References

1. For reviews on tert-butanesulfinyl imine chemistry, see: (a) Robak, M. T.; Herbage, M. A.;

Ellman, J. A. Chem. Rev. 2010, 110, 3600; (b) Ferreira, F.; Botuha, C.; Chemla, F.; Perez-Luna,

A. Chem. Soc. Rev. 2009, 38, 1162.

2. For initial reports, see: (a) Cogan, D. A.; Liu, G.; Ellman, J. Tetrahedron 1999, 55, 8883; (b)

Liu, G.; Cogan, D. A.; Ellman, J. A. J. Am. Chem. Soc. 1997, 119, 9913; (c) Pflum, D. A.;

142

Krishnamurthy, D.; Han, Z.; Wald, S. A.; Senanayake, C. H. Tetrahedron Lett. 2002, 43, 923; (d)

Plobeck, N.; Powell, D. Tetrahedron: Asymmetry 2002, 13, 303.

3. For rhodium-catalyzed additions to N-tert-butanesulfinyl imines, see: (a) Weix, D. J.; Shi, Y.;

Ellman, J. A. J. Am. Chem. Soc. 2005, 127, 1092; (b) Bolshan, Y.; Batey, R. A. Org. Lett. 2005,

7, 1481; (c) Beenen, M. A.; Weix, D. J.; Ellman, J. A. J. Am. Chem. Soc. 2006, 128, 6304; (d)

Truong, V. L.; Pfeiffer, J. Y. Tetrahedron Lett. 2009, 50, 1633; (e) Brak, K.; Ellman, J. A. J. Am.

Chem. Soc. 2009, 131, 3850; (f) Brak, K.; Ellman, J. A. J. Org. Chem. 2010, 75, 3147.

4. (a) Sun, X.-W.; Xu, M.-H.; Lin, G.-Q. Org. Lett. 2006, 8, 4979; (b) Kolodney, G.; Sklute, G.;

Perrone, S.; Knochel, P.; Marek, I. Angew. Chem., Int. Ed. 2007, 46, 9291; (c) Sun, X.-W.; Liu,

M.; Xu, M.-H. Lin, G.-Q. Org. Lett. 2008, 10, 1259; (d) Reddy, L. R.; Hu, B.; Prashad, M. Org.

Lett. 2008, 10, 3109; (e) Shen, A.; Liu, M.; Jia, Z.-S.; Xu, M.-H.; Lin, G.-Q. Org. Lett. 2010, 12,

5154.

5. (a) Cooper, I. R.; Grigg, R.; MacLachlan, W. S.; Thornton-Pett, M.; Sridharan, V. Chem.

Commun. 2002, 1372; (b) Foubelo, F.; Yus, M. Tetrahedron: Asymmetry 2004, 15, 3823; (c)

Gonzalez-Gomez, J. C.; Medjahdi, M.; Foubelo, F.; Yus, M. J. Org. Chem. 2010, 75, 6308.

6. (a) Knochel, P.; Schade, M. A.; Bernhardt, S.; Manolikakes, G.; Metzger, A.; Piller, F. M.;

Rohbogner, C. J.; Mosrin, M. Beilstein. J. Org. Chem. 2011, 7, 1261; (b) Jana, R.; Pathak, T. P.;

Sigman, M. S. Chem. Rev. 2011, 111, 1417.

7. (a) Metzger, A.; Bernhardt, S.; Manolikakes, G.; Knochel, P. Angew. Chem. Int. Ed. 2010,

49, 4665; (b) Piller, F. M.; Metzger, A.; Schade, M. A.; Haag, B. A.; Gavryushin, A.; Knochel,

P. Chem. Eur. J. 2009, 15, 7192.

8. For studies on the addition of organozincates, prepared from Grignard and dialkylzinc

reagents, to N-tert-butanesulfinyl imines, see: (a) Almansa, R.; Guijarro, D.; Yus, M.

Tetrahedron: Asymmetry 2008, 19, 603; (b) Almansa, R.; Guijarro, D.; Yus, M. Tetrahedron:

Asymmetry 2008, 19, 2484; (c) Almansa, R.; Guijarro, D.; Yus, M. Tetrahedron. Lett. 2009, 50,

3198.

9. Subsequent work has applied this approach to the synthesis of phenylalanine derivatives: (a)

Yang, J.; Min, Q.-Q.; He, Y.; Zhang, X. Tetrahedron: Asymmetry 2011, 52, 4675; (b) Lin, L.;

Fu, X.; Ma, X.; Zhang, J.; Wang, R. Synlett 2012, 2559.

10. For detailed description of the iodometric titration method used to determine benzyl zinc

reagent concentration, see Krasovskiy, A.; Knochel, P. Synthesis 2006, 5, 890.

11. (a) Harried, S. S.; Croghan,M. D.; Kaller, M. R.; Lopez, P.; Zhong, W.; Hungate, R.; Reider,

P. J. J. Org. Chem. 2009, 74, 5975; (b) Ghosh, A. K. J. Med. Chem. 2009, 52, 2163; (c) Izawa,

K.; Onishi, T. Chem. Rev. 2006, 106, 2811; (d) Huang, W.-H.; Sheng, R.; Hu, Y.-Z. Curr. Med.

Chem. 2009, 16, 1806.

12. (a) Kortum, S. W.; Benson, T. E.; Bienkowski, M. J.; Emmons, T. L.; Prince, D. B; Paddock,

D. J.; Tomasselli, A. G.; Moon, J. B.; LaBorde, A.; TenBrink, R. E. Bioorg. Med. Chem. Lett.

2007, 17, 3378; (b) He, G.-X.; Yang, Z.-Y.; Williams, M.; Callebaut, C.; Cihlar, T.; Murray, B.

P.; Yang, C.; Mitchell, M. L.; Liu, H.; Wang, J.; Arimilli, M.; Eisenberg, E.; Stray, K. M.; Tsai,

L. K.; Hatada, M.; Chen, X.; Chen, J. M.; Wang, Y.; Lee, M. S.; Strickley, R. G.; Iwata, Q.;

Zheng, X.; Kim, C. U.; Swaminathan, S.; Desai, M. C.; Lee, W. A.; Xu, L. Med. Chem.

Commun. 2011, 2, 1093; (c) Dineen, T. A.; Weiss, M. M; Williamson, T.; Acton, P.; Babu-Khan,

S.; Bartberger, M. D.; Brown, J.; Chen, K.; Cheng, Y.; Citron, M.; Croghan, M. D.; Dunn II, R.

T.; Esmay, J.; Graceffa, R. F.; Harried, S. S.; Hickman, D.; Hitchcock, S. A.; Horne, D. B.;

143

Huang, H.; Imbeah-Ampiah, R.; Judd, T.; Kaller, M. R.; Kreiman, C. R.; La, D. S.; Li, V.;

Lopez, P.; Louie, S.; Monenschein, H.; Nguyen, T. T.; Pennington, L. D.; San Miguel, T.;

Sickmier, E. A.; Vargas, H. M.; Wahl, R. C.; Wen, P. H.; Whittington, D. A.; Wood, S.; Xue, Q.;

Yang, B. H.; Patel, V. F.; Zhong, W. J. Med. Chem. 2012, 55, 9025.

13. Branalt, J.; Kvarnstrom, I.; Classon, B.; Samuelsson, B.; Nillroth, U.; Danielson, U. H.;

Karlen, A.; Hallberg, A. Tetrahedron Lett. 1997, 38, 3483.

14. Brak, K.; Barrett, K. T.; Ellman, J. A. J. Org. Chem. 2009, 74, 3606.

15. Higashibayashi, S.; Tohmiya, H.; Mori, T.; Hashimoto, K.; Nakata, M. Synlett 2004, 457.

16. Huang, Z.; Zhang, M.; Wang, Y.; Qin, Y. Synlett 2005, 1334.

17. Nielsen, L.; Lindsay, K. B.; Faber, J.; Nielsen, N. C.; Skrydstrup, T. J. Org. Chem. 2007, 72,

10035.

18. Cheng, L.; Liu, L.; Sui, Y.; Wang, D.; Chen, Y.-J. Tetrahedron: Asymmetry 2007, 18, 1833.

19. Schlenk, L. B.; Ellman, J. A. Org. Lett. 2004, 6, 3621.

20. Moreau, P.; Essiz, M.; Mérour, J.-Y.; Bouzard, D. Tetrahedron: Asymmetry 1997, 8, 591.

21. Kadirvel, M.; Stimpson, W. T.; Moumene-Afifi, S.; Arsic, B.; Glynn, N.; Halliday, N.;

Williams, P.; Gilbert, P.; McBain, A. J.; Freeman, S.; Gardiner, J. M. Bioorg. Med. Chem. Lett.

2010, 20, 2625.

22. (a) Badorry, R.; Diaz-de-Villegas, M. D.; Galvez, J. Tetrahedron: Asymmetry. 2009, 20,

2226; (b) Ghosh, A. K.; Fidanze, S. J. Org. Chem. 1998, 63, 6146.

23. Rele, S. M.; Iyer, S. S.; Chaikof, E. L. Tetrahedron Lett. 2007, 48, 5055.

24. Wunsch, E.; Heidrich, H.-G.; Grassman, W. Chemische Berichte. 1964, 97, 1818.

25. (a) Giacomelli, G.; Porcheddu, A.;Salaris, M. Org. Lett. 2003, 5, 2715; (b) De Luca, L.;

Giacomelli, G.; Nieddu, G. J. Org. Chem. 2007, 72, 3955.

144

145

Appendix 6.1: X-ray crystal data for compound 6.27

146

Figure A6.1.1. X-ray crystal structure of 6.27 with thermal ellipsoids drawn at the 50% probability level.

Data collection parameters

A colorless needle crystal of SO4NC21H27 having approximate dimensions of 0.10 x 0.05 x 0.05

mm was mounted in a loop. All measurements were made on a Rigaku Saturn724 CCD

diffractometer using graphite monochromated Cu-K radiation.

The crystal-to-detector distance was 50.00 mm.

Cell constants and an orientation matrix for data collection corresponded to a primitive

orthorhombic cell with dimensions:

a = 5.57254(10) Å

b = 11.2226(2) Å

c = 32.524(2) Å

V = 2034.02(15) Å3

For Z = 4 and F.W. = 389.51, the calculated density is 1.272 g/cm3. The reflection conditions of:

h00: h = 2n

0k0: k = 2n

00l: l = 2n

uniquely determine the space group to be:

P212121 (#19)

147

The data were collected at a temperature of –180 ± 1 °C to a maximum 2 value of 131.8°. A

total of 321 oscillation images were collected. A sweep of data was done using scans from –

48.0 to 132.0° in 2.0° step, at = 45.0° and = 135.0°. The exposure rate was 15.0 [s/°]. The

detector swing angle was 42.00°. A second sweep was performed using scans from 0.0 to

180.0° in 2.0° step, at = 45.0° and = 135.0°. The exposure rate was 30.0 [s/°]. The detector

swing angle was 90.00°. Another sweep was performed using scans from 1.0 to 171.0° in 2.0°

step, at = 45.0° and = 0.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was

90.00°. Another sweep was performed using scans from 98.0 to 150.0° in 2.0° step, at =

30.0° and = 0.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was 90.00°.

Another sweep was performed using scans from 70.0 to 130.0° in 2.0° step, at = 30.0° and

= 180.0°. The exposure rate was 30.0 [s/°]. The detector swing angle was 90.00°. The crystal-to-

detector distance was 50.00 mm. Readout was performed in the 0.090 mm pixel mode.

Data reduction parameters

Of the 8763 reflections that were collected, 3362 were unique (Rint = 0.0689). Data were

collected and processed using CrystalClear (Rigaku).1

The linear absorption coefficient, , for Cu-K radiation is 16.260 cm–1

. An empirical

absorption correction was applied which resulted in transmission factors ranging from 0.513 to

0.922. The data were corrected for Lorentz and polarization effects.

Structure solution and refinement

The structure was solved by direct methods2 and expanded using Fourier techniques. The

non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding

model. The final cycle of full-matrix least-squares refinement3 on F

2 was based on 3351

observed reflections and 248 variable parameters and converged (largest parameter shift was

0.00 times its esd) with unweighted and weighted agreement factors of:

R1 = ||Fo| – |Fc|| / |Fo| = 0.0691

wR2 = [ ( w (Fo2 – Fc

2)2 )/ w (Fo

2)2]1/2

= 0.2016

The standard deviation of an observation of unit weight4 was 1.12. Unit weights were used.

The maximum and minimum peaks on the final difference Fourier map corresponded to 0.79 and

–0.37 e–/Å

3, respectively. The absolute structure was deduced based on Flack parameter, –

0.00(4), using 1309 Friedel pairs.5

Neutral atom scattering factors were taken from Cromer and Waber.6 Anomalous dispersion

effects were included in Fcalc;7 the values for f' and f" were those of Creagh and McAuley.

8

The values for the mass attenuation coefficients are those of Creagh and Hubbell.9 All

148

calculations were performed using the CrystalStructure10

crystallographic software package

except for refinement, which was performed using SHELXL-97.11

References

1. CrystalClear: Rigaku Corporation, 1999. CrystalClear Software User's Guide, Molecular

Structure Corporation, (c) 2000.J.W.Pflugrath (1999) Acta Cryst. D55, 1718–1725.

2. SIR2004: M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro,

C. Giacovazzo, G. Polidori, R. Spagna (2005)

3. Least Squares function minimized: (SHELXL97)

w(Fo2–Fc

2)2 where w = Least Squares weights.

4. Standard deviation of an observation of unit weight:

[w(Fo2–Fc

2)2/(No–Nv)]

1/2 where No = number of observations, Nv = number of variables

5. Flack, H. D. (1983), Acta Cryst. A39, 876–881.

6. Cromer, D. T. & Waber, J. T.; "International Tables for X-ray Crystallography", Vol. IV, The

Kynoch Press, Birmingham, England, Table 2.2 A (1974).

7. Ibers, J. A. & Hamilton, W. C.; Acta Crystallogr., 17, 781 (1964).

8. Creagh, D. C. & McAuley, W.J .; "International Tables for Crystallography", Vol C, (A.J.C.

Wilson, ed.), Kluwer Academic Publishers, Boston, Table 4.2.6.8, pages 219–222 (1992).

9. Creagh, D. C. & Hubbell, J.H..; "International Tables for Crystallography", Vol C, (A.J.C.


10. CrystalStructure 4.0: Crystal Structure Analysis Package, Rigaku and Rigaku Americas

(2000–2010). 9009 New Trails Dr. The Woodlands TX 77381 USA.

11. SHELX97: Sheldrick, G.M. (1997).

149

Table A6.1.1. Crystal data and structure refinement

Crystal data

Chemical formula SO4NC21H27

Mr 389.51

Crystal system, space group Orthorhombic, P212121

Temperature (K) 93

a, b, c (Å) 5.57254 (10), 11.2226 (2), 32.524 (2)

V (Å3) 2034.02 (15)

Z 4

Radiation type Cu Kα

µ (mm−1

) 1.63

Crystal size (mm) 0.10 × 0.05 × 0.05

Data collection

Diffractometer Rigaku Saturn724 CCD

diffractometer

Absorption correction Multi-scan

Jacobson, R. (1998) Private communication

Tmin, Tmax 0.513, 0.922

No. of measured, independent and

observed [F2 > 2.0σ(F

2)] reflections

8763, 3351, 2672

Rint 0.069

(sin θ/λ)max (Å−1

) 0.592

Refinementa

R[F2 > 2σ(F

2)], wR(F

2), S 0.069, 0.202, 1.12

No. of reflections 3351

No. of parameters 248

H-atom treatment H atoms treated by a mixture of independent and

constrained refinement

Δρmax, Δρmin (e Å−3

) 0.79, −0.37

Absolute structure Flack, H. D. (1983), Acta Cryst. A39, 876–881.

1309 Friedel Pairs

Absolute structure parameter −0.00 (4) aRefinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are

based on F2. R-factor (gt) are based on F. The threshold expression of F

2 > 2.0 σ(F

2) is used only for

calculating R-factor (gt).

150

Table A6.1.2. Fractional atomic coordinates and equivalent isotropic displacement parameters (Å2)

atom x y z Beqa

S(1) 0.6429 (2) 0.96741 (13) 0.83005 (3) 4.73(3)

O(1) 0.4970 (9) 0.8597 (5) 0.82691 (12) 8.54(14)

O(2) 0.9719 (6) 0.4497 (3) 1.00033 (10) 4.77(7)

O(3) 1.3115 (5) 0.4525 (3) 0.96300 (9) 4.13(6)

O(4) 0.9617 (7) 1.5076 (3) 0.84357 (10) 5.25(8)

N(1) 0.8947 (7) 0.9472 (4) 0.85383 (11) 4.02(7)

C(1) 0.7607 (9) 0.9940 (5) 0.77831 (15) 4.87(10)

C(2) 0.9182 (14) 1.1049 (5) 0.7800 (2) 6.80(15)

C(3) 0.9074 (14) 0.8860 (6) 0.76333 (16) 6.26(14)

C(4) 0.5441 (12) 1.0116 (9) 0.75071 (19) 8.17(20)

C(5) 0.9023 (8) 0.9548 (4) 0.89899 (12) 3.58(7)

C(6) 0.9498 (8) 0.8356 (4) 0.91884 (13) 3.60(8)

C(7) 0.7955 (8) 0.7865 (4) 0.94808 (14) 3.89(8)

C(8) 0.8438 (8) 0.6776 (4) 0.96646 (12) 3.64(8)

C(9) 1.0483 (8) 0.6135 (4) 0.95581 (13) 3.58(8)

C(10) 1.2055 (8) 0.6616 (4) 0.92652 (13) 3.80(8)

C(11) 1.1569 (9) 0.7699 (4) 0.90809 (13) 3.90(8)

C(12) 1.1001 (8) 0.4983 (4) 0.97557 (13) 3.91(8)

C(13) 1.3799 (10) 0.3396 (4) 0.97945 (14) 4.25(8)

C(14) 1.0902 (8) 1.0464 (4) 0.91261 (14) 4.02(8)

C(15) 1.0521 (8) 1.1696 (4) 0.89535 (14) 3.94(8)

C(16) 1.2199 (9) 1.2224 (5) 0.86990 (15) 4.42(9)

C(17) 1.1876 (10) 1.3330 (5) 0.85314 (15) 4.54(10)

C(18) 0.9813 (10) 1.3971 (4) 0.86197 (14) 4.16(9)

C(19) 0.8101 (9) 1.3488 (4) 0.88779 (14) 4.17(9)

C(20) 0.8469 (9) 1.2356 (4) 0.90403 (14) 4.07(8)

C(21) 0.7524 (12) 1.5758 (5) 0.85211 (18) 5.67(12) a

Beq = 8/3 2(U11(aa*)2 + U22(bb*)

2 + U33(cc*)

2 + 2U12(aa*bb*)cos + 2U13(aa*cc*)cos + 2U23(bb*cc*)cos )

151

Table A6.1.3. Fractional atomic coordinates and isotropic displacement parameters for hydrogens (Å2)

atom x y z Biso

H(2A) 0.8254 1.1716 0.7912 8.16

H(2B) 0.9729 1.1248 0.7521 8.16

H(2C) 1.0574 1.0896 0.7976 8.16

H(3A) 0.9591 0.8993 0.7349 7.52

H(3B) 0.8078 0.8141 0.7646 7.52

H(3C) 1.0486 0.8757 0.7810 7.52

H(4A) 0.5978 1.0309 0.7228 9.80

H(4B) 0.4457 1.0770 0.7614 9.80

H(4C) 0.4488 0.9382 0.7501 9.80

H(5) 0.7419 0.9833 0.9086 4.30

H(7) 0.6542 0.8287 0.9556 4.66

H(8) 0.7365 0.6465 0.9865 4.37

H(10) 1.3469 0.6192 0.9193 4.56

H(11) 1.2640 0.8006 0.8880 4.69

H(13A) 1.3566 0.3399 1.0093 5.10

H(13B) 1.5492 0.3245 0.9732 5.10

H(13C) 1.2808 0.2769 0.9671 5.10

H(14A) 1.0889 1.0513 0.9430 4.82

H(14B) 1.2509 1.0179 0.9041 4.82

H(16) 1.3633 1.1802 0.8638 5.31

H(17) 1.3063 1.3658 0.8355 5.45

H(19) 0.6691 1.3924 0.8944 5.00

H(20) 0.7282 1.2024 0.9216 4.88

H(21A) 0.7431 1.5917 0.8817 6.81

H(21B) 0.6100 1.5312 0.8434 6.81

H(21C) 0.7600 1.6514 0.8371 6.81

H(1N) 1.028(11) 0.905(5) 0.8438(19) 5.8(14)

152

Table A6.1.4. Atomic displacement parameters (Å2)

a

atom U11

U22

U33

U12

U13

U23

S(1) 0.0537 (6) 0.0866 (8) 0.0395 (6) −0.0059 (6) −0.0022 (5) 0.0067 (6)

O(1) 0.120 (3) 0.154 (4) 0.051 (2) −0.084 (3) −0.017 (2) 0.015 (3)

O(2) 0.0638 (18) 0.0606 (18) 0.057 (2) −0.0011 (17) 0.0060 (17) 0.0111 (16)

O(3) 0.0551 (17) 0.0538 (16) 0.0478 (17) 0.0069 (14) 0.0050 (14) 0.0117 (14)

O(4) 0.099 (3) 0.0523 (19) 0.0477 (18) −0.0058 (18) −0.0026 (19) 0.0014 (14)

N(1) 0.054 (2) 0.065 (2) 0.0338 (18) 0.017 (2) −0.0050 (17) −0.0023 (16)

C(1) 0.058 (3) 0.083 (3) 0.043 (3) 0.000 (3) −0.007 (2) 0.005 (2)

C(2) 0.114 (5) 0.082 (4) 0.062 (3) −0.007 (4) 0.007 (4) 0.017 (3)

C(3) 0.106 (5) 0.089 (4) 0.043 (3) 0.001 (4) 0.008 (3) −0.015 (3)

C(4) 0.080 (4) 0.178 (8) 0.053 (3) 0.008 (5) −0.010 (3) 0.017 (4)

C(5) 0.050 (2) 0.054 (2) 0.032 (2) 0.005 (2) 0.0029 (19) −0.0027 (17)

C(6) 0.048 (2) 0.054 (2) 0.034 (2) 0.0011 (19) 0.000 (2) −0.0032 (18)

C(7) 0.048 (2) 0.059 (3) 0.040 (2) 0.0043 (19) 0.000 (2) −0.0014 (19)

C(8) 0.048 (2) 0.061 (2) 0.029 (2) −0.002 (2) 0.002 (2) 0.0007 (17)

C(9) 0.046 (2) 0.053 (2) 0.037 (2) −0.0030 (18) −0.0009 (19) 0.0045 (18)

C(10) 0.046 (2) 0.058 (3) 0.041 (2) −0.001 (2) 0.0080 (19) 0.0031 (19)

C(11) 0.051 (2) 0.057 (2) 0.040 (2) 0.001 (2) 0.005 (2) 0.0072 (19)

C(12) 0.050 (2) 0.059 (3) 0.040 (2) −0.004 (2) 0.002 (2) 0.0042 (19)

C(13) 0.065 (3) 0.051 (2) 0.045 (2) 0.005 (2) −0.004 (2) 0.0052 (19)

C(14) 0.053 (2) 0.055 (2) 0.044 (2) −0.001 (2) −0.003 (2) 0.003 (2)

C(15) 0.053 (2) 0.052 (2) 0.044 (2) 0.002 (2) −0.002 (2) 0.003 (2)

C(16) 0.057 (3) 0.063 (3) 0.048 (3) −0.001 (2) 0.001 (2) −0.002 (2)

C(17) 0.067 (3) 0.063 (3) 0.043 (3) −0.013 (2) 0.003 (2) −0.002 (2)

C(18) 0.069 (3) 0.048 (2) 0.041 (2) −0.007 (2) −0.005 (2) 0.0001 (19)

C(19) 0.057 (3) 0.056 (2) 0.045 (2) 0.006 (2) −0.009 (2) 0.002 (2)

C(20) 0.055 (2) 0.061 (3) 0.039 (2) 0.004 (2) −0.005 (2) 0.0044 (19)

C(21) 0.101 (4) 0.055 (3) 0.060 (3) 0.003 (3) −0.022 (3) 0.002 (2) a The general temperature factor expression:

exp(–22(a*2U11h

2 + b*

2U22k

2 + c*

2U33l

2 + 2a*b*U12hk + 2a*c*U13hl + 2b*c*U23kl))

153

Table A6.1.5. Bond lengths (Å)

bond length bond length

S(1)—O(1) 1.461 (6) N(1)—H(1N) 0.94 (6)

S(1)—N(1) 1.618 (4) C(2)—H(2A) 0.980

S(1)—C(1) 1.831 (5) C(2)—H(2B) 0.980

O(2)—C(12) 1.207 (5) C(2)—H(2C) 0.980

O(3)—C(12) 1.349 (5) C(3)—H(3A) 0.980

O(3)—C(13) 1.427 (5) C(3)—H(3B) 0.980

O(4)—C(18) 1.381 (6) C(3)—H(3C) 0.980

O(4)—C(21) 1.422 (7) C(4)—H(4A) 0.980

N(1)—C(5) 1.472 (5) C(4)—H(4B) 0.980

C(1)—C(2) 1.524 (9) C(4)—H(4C) 0.980

C(1)—C(3) 1.541 (8) C(5)—H(5) 1.000

C(1)—C(4) 1.517 (8) C(7)—H(7) 0.950

C(5)—C(6) 1.509 (6) C(8)—H(8) 0.950

C(5)—C(14) 1.533 (6) C(10)—H(10) 0.950

C(6)—C(7) 1.395 (6) C(11)—H(11) 0.950

C(6)—C(11) 1.414 (6) C(13)—H(13A) 0.980

C(7)—C(8) 1.387 (6) C(13)—H(13B) 0.980

C(8)—C(9) 1.391 (6) C(13)—H(13C) 0.980

C(9)—C(10) 1.402 (6) C(14)—H(14A) 0.990

C(9)—C(12) 1.473 (6) C(14)—H(14B) 0.990

C(10)—C(11) 1.382 (6) C(16)—H(16) 0.950

C(14)—C(15) 1.507 (6) C(17)—H(17) 0.950

C(15)—C(16) 1.382 (7) C(19)—H(19) 0.950

C(15)—C(20) 1.392 (7) C(20)—H(20) 0.950

C(16)—C(17) 1.367 (7) C(21)—H(21A) 0.980

C(17)—C(18) 1.386 (7) C(21)—H(21B) 0.980

C(18)—C(19) 1.382 (7) C(21)—H(21C) 0.980

C(19)—C(20) 1.391 (7)

154

Table A6.1.6. Bond angles (°)

bonds angle bonds angle

O(1)—S(1)—N(1) 113.6 (3) C(8)—C(9)—C(10) 118.8 (4)

O(1)—S(1)—C(1) 105.7 (2) C(8)—C(9)—C(12) 120.4 (4)

N(1)—S(1)—C(1) 98.7 (2) C(10)—C(9)—C(12) 120.8 (4)

C(12)—O(3)—C(13) 117.3 (4) C(9)—C(10)—C(11) 120.7 (4)

C(18)—O(4)—C(21) 117.6 (4) C(6)—C(11)—C(10) 120.7 (4)

S(1)—N(1)—C(5) 119.6 (3) O(2)—C(12)—O(3) 123.2 (4)

S(1)—C(1)—C(2) 107.9 (4) O(2)—C(12)—C(9) 124.9 (4)

S(1)—C(1)—C(3) 110.6 (4) O(3)—C(12)—C(9) 111.9 (4)

S(1)—C(1)—C(4) 106.2 (4) C(5)—C(14)—C(15) 114.3 (4)

C(2)—C(1)—C(3) 110.4 (5) C(14)—C(15)—C(16) 121.4 (4)

C(2)—C(1)—C(4) 111.9 (6) C(14)—C(15)—C(20) 121.9 (4)

C(3)—C(1)—C(4) 109.7 (5) C(16)—C(15)—C(20) 116.7 (4)

N(1)—C(5)—C(6) 112.4 (4) C(15)—C(16)—C(17) 122.6 (5)

N(1)—C(5)—C(14) 110.3 (3) C(16)—C(17)—C(18) 119.8 (5)

C(6)—C(5)—C(14) 110.6 (3) O(4)—C(18)—C(17) 116.2 (4)

C(5)—C(6)—C(7) 122.2 (4) O(4)—C(18)—C(19) 124.1 (5)

C(5)—C(6)—C(11) 120.0 (4) C(17)—C(18)—C(19) 119.7 (4)

C(7)—C(6)—C(11) 117.8 (4) C(18)—C(19)—C(20) 119.1 (4)

C(6)—C(7)—C(8) 121.5 (4) C(15)—C(20)—C(19) 122.0 (4)

C(7)—C(8)—C(9) 120.5 (4)

155

Table A6.1.7. Bond angles involving hydrogens (°)


S(1)—N(1)—H(1N) 126 (4) C(11)—C(10)—H(10) 119.661

C(5)—N(1)—H(1N) 111 (4) C(6)—C(11)—H(11) 119.628

C(1)—C(2)—H(2A) 109.468 C(10)—C(11)—H(11) 119.626

C(1)—C(2)—H(2B) 109.47 O(3)—C(13)—H(13A) 109.471

C(1)—C(2)—H(2C) 109.47 O(3)—C(13)—H(13B) 109.466

H(2A)—C(2)—H(2B) 109.473 O(3)—C(13)—H(13C) 109.468

H(2A)—C(2)—H(2C) 109.475 H(13A)—C(13)—H(13B) 109.475

H(2B)—C(2)—H(2C) 109.471 H(13A)—C(13)—H(13C) 109.483

C(1)—C(3)—H(3A) 109.477 H(13B)—C(13)—H(13C) 109.464

C(1)—C(3)—H(3B) 109.466 C(5)—C(14)—H(14A) 108.688

C(1)—C(3)—H(3C) 109.457 C(5)—C(14)—H(14B) 108.685

H(3A)—C(3)—H(3B) 109.487 C(15)—C(14)—H(14A) 108.691

H(3A)—C(3)—H(3C) 109.483 C(15)—C(14)—H(14B) 108.686

H(3B)—C(3)—H(3C) 109.458 H(14A)—C(14)—H(14B) 107.615

C(1)—C(4)—H(4A) 109.473 C(15)—C(16)—H(16) 118.681

C(1)—C(4)—H(4B) 109.465 C(17)—C(16)—H(16) 118.675

C(1)—C(4)—H(4C) 109.475 C(16)—C(17)—H(17) 120.093

H(4A)—C(4)—H(4B) 109.48 C(18)—C(17)—H(17) 120.099

H(4A)—C(4)—H(4C) 109.467 C(18)—C(19)—H(19) 120.419

H(4B)—C(4)—H(4C) 109.467 C(20)—C(19)—H(19) 120.446

N(1)—C(5)—H(5) 107.801 C(15)—C(20)—H(20) 118.977

C(6)—C(5)—H(5) 107.804 C(19)—C(20)—H(20) 118.986

C(14)—C(5)—H(5) 107.801 O(4)—C(21)—H(21A) 109.47

C(6)—C(7)—H(7) 119.26 O(4)—C(21)—H(21B) 109.466

C(8)—C(7)—H(7) 119.287 O(4)—C(21)—H(21C) 109.474

C(7)—C(8)—H(8) 119.766 H(21A)—C(21)—H(21B) 109.474

C(9)—C(8)—H(8) 119.759 H(21A)—C(21)—H(21C) 109.475

C(9)—C(10)—H(10) 119.642 H(21B)—C(21)—H(21C) 109.469

156

Table A6.1.8. Torsion angles (°)a


O(1)—S(1)—N(1)—C(5) −85.4 (3) C(11)—C(6)—C(7)—C(8) 0.7 (6)

O(1)—S(1)—C(1)—C(3) −58.3 (4) C(6)—C(7)—C(8)—C(9) −0.6 (6)

O(1)—S(1)—C(1)—C(4) 60.7 (4) C(7)—C(8)—C(9)—C(10) 0.6 (6)

N(1)—S(1)—C(1)—C(2) −61.5 (3) C(8)—C(9)—C(10)—C(11) −0.9 (6)

N(1)—S(1)—C(1)—C(3) 59.3 (3) C(8)—C(9)—C(12)—O(2) 2.8 (7)

C(13)—O(3)—C(12)—O(2) 1.6 (6) C(10)—C(9)—C(12)—O(3) 2.2 (6)

C(21)—O(4)—C(18)—C(19) 0.4 (6) C(9)—C(10)—C(11)—C(6) 1.0 (6)

S(1)—N(1)—C(5)—C(6) 111.3 (3) C(5)—C(14)—C(15)—C(16) −117.8 (4)

S(1)—N(1)—C(5)—C(14) −124.8 (3) C(5)—C(14)—C(15)—C(20) 62.0 (5)

N(1)—C(5)—C(6)—C(7) −123.9 (4) C(16)—C(15)—C(20)—C(19) 0.5 (7)

N(1)—C(5)—C(6)—C(11) 56.4 (5) C(20)—C(15)—C(16)—C(17) −1.2 (7)

N(1)—C(5)—C(14)—C(15) 57.0 (4) C(15)—C(16)—C(17)—C(18) 0.8 (7)

C(14)—C(5)—C(6)—C(7) 112.3 (4) C(16)—C(17)—C(18)—C(19) 0.2 (7)

C(14)—C(5)—C(6)—C(11) −67.4 (5) C(17)—C(18)—C(19)—C(20) −0.9 (7)

C(7)—C(6)—C(11)—C(10) −0.9 (6) C(18)—C(19)—C(20)—C(15) 0.5 (7) aThose having bond angles > 160 ° are excluded.

157

Appendix 6.2: X-ray crystal data for compound 6.31

158

Figure A6.2.1. X-ray crystal structure of 6.31 with thermal ellipsoids drawn at the 50% probability level.

Data collection parameters

A colorless plate crystal of SFONC16H26 having approximate dimensions of 0.18 x 0.10 x 0.08

mm was mounted in a loop. All measurements were made on a Rigaku R-AXIS RAPID imaging

plate diffractometer using graphite monochromated Cu-K radiation.

The crystal-to-detector distance was 127.40 mm.

Cell constants and an orientation matrix for data collection corresponded to a primitive

orthorhombic cell with dimensions:

a = 8.7454(2) Å

b = 10.5113(3) Å

c = 18.0667(13) Å

V = 1660.79(13) Å3

For Z = 4 and F.W. = 299.45, the calculated density is 1.198 g/cm3. The reflection conditions of:

h00: h = 2n

0k0: k = 2n

00l: l = 2n

uniquely determine the space group to be:

P212121 (#19)

159

The data were collected at a temperature of –180 + 1 °C to a maximum 2 value of 136.4°. A

total of 63 oscillation images were collected. A sweep of data was done using scans from 20.0

to 200.0° in 5.0° step, at = 54.0° and = 270.0°. The exposure rate was 48.0 [s/°]. A second

sweep was performed using scans from 20.0 to 125.0° in 5.0° step, at = 0.0° and = 90.0°.

The exposure rate was 48.0 [s/°]. Another sweep was performed using scans from 70.0 to

100.0° in 5.0° step, at = 54.0° and = 90.0°. The exposure rate was 48.0 [s/°]. The crystal-to-

detector distance was 127.40 mm. Readout was performed in the 0.100 mm pixel mode.

Data reduction parameters

Of the 6921 reflections that were collected, 2705 were unique (Rint = 0.0702).

The linear absorption coefficient, , for Cu-K radiation is 17.822 cm–1

. An empirical

absorption correction was applied which resulted in transmission factors ranging from 0.621 to

0.867. The data were corrected for Lorentz and polarization effects. A correction for secondary

extinction1 was applied (coefficient = 0.002850).

Structure solution and refinement

The structure was solved by direct methods2 and expanded using Fourier techniques. The

non-hydrogen atoms were refined anisotropically. Hydrogen atoms were refined using the riding

model. The final cycle of full-matrix least-squares refinement3 on F

2 was based on 2687

observed reflections and 186 variable parameters and converged (largest parameter shift was

0.00 times its esd) with unweighted and weighted agreement factors of:

R1 = ||Fo| – |Fc|| / |Fo| = 0.0769

wR2 = [ ( w (Fo2 – Fc

2)2 )/ w (Fo

2)2]1/2

= 0.2081

The standard deviation of an observation of unit weight4 was 1.12. Unit weights were used.

The maximum and minimum peaks on the final difference Fourier map corresponded to 0.77 and

–0.57 e–/Å

3, respectively. The absolute structure was deduced based on Flack parameter, 0.06(5),

using 959 Friedel pairs.5

Neutral atom scattering factors were taken from Cromer and Waber.6 Anomalous dispersion

effects were included in Fcalc;7 the values for f' and f" were those of Creagh and McAuley.

8

The values for the mass attenuation coefficients are those of Creagh and Hubbell.9 All

calculations were performed using the CrystalStructure10

crystallographic software package

except for refinement, which was performed using SHELXL-97.11

160

References

1. Larson, A.C. (1970), Crystallographic Computing, 291–294. F.R. Ahmed, ed. Munksgaard,

Copenhagen (equation 22, with V replaced by the cell volume).

2. SIR2004: M.C. Burla, R. Caliandro, M. Camalli, B. Carrozzini, G.L. Cascarano, L. De Caro,

C. Giacovazzo, G. Polidori, R. Spagna (2005)

3. Least Squares function minimized: (SHELXL97)

w(Fo2–Fc

2)2 where w = Least Squares weights.

4. Standard deviation of an observation of unit weight:

[w(Fo2–Fc

2)2/(No–Nv)]

1/2 where No = number of observations, Nv = number of variables

5. Flack, H. D. (1983), Acta Cryst. A39, 876–881.

6. Cromer, D. T. & Waber, J. T.; "International Tables for X-ray Crystallography", Vol. IV, The

Kynoch Press, Birmingham, England, Table 2.2 A (1974).

7. Ibers, J. A. & Hamilton, W. C.; Acta Crystallogr., 17, 781 (1964).

8. Creagh, D. C. & McAuley, W.J .; "International Tables for Crystallography", Vol C, (A.J.C.


9. Creagh, D. C. & Hubbell, J.H..; "International Tables for Crystallography", Vol C, (A.J.C.


10. CrystalStructure 4.0: Crystal Structure Analysis Package, Rigaku and Rigaku Americas

(2000–2010). 9009 New Trails Dr. The Woodlands TX 77381 USA.

11. SHELX97: Sheldrick, G.M. (1997).

161

Table A6.2.1. Crystal data and structure refinement

Crystal data

Chemical formula SFONC16H26

Mr 299.45

Crystal system, space group Orthorhombic, P212121

Temperature (K) 93

a, b, c (Å) 8.7454 (2), 10.5113 (3), 18.0667 (13)

V (Å3) 1660.79 (13)

Z 4

Radiation type Cu Kα

µ (mm−1

) 1.78

Crystal size (mm) 0.18 × 0.10 × 0.08

Data collection

Diffractometer Rigaku R-AXIS RAPID imaging plate

diffractometer

Absorption correction Multi-scan

Higashi, T. (1995). Program for Absorption

Correction. Rigaku Corporation, Tokyo, Japan.

Tmin, Tmax 0.621, 0.867

No. of measured, independent and

observed [F2 > 2.0σ(F

2)] reflections

6921, 2687, 1620

Rint 0.070

(sin θ/λ)max (Å−1

) 0.602

Refinementa

R[F2 > 2σ(F

2)], wR(F

2), S 0.077, 0.208, 1.12

No. of reflections 2687

No. of parameters 186

H-atom treatment H atoms treated by a mixture of independent and

constrained refinement

Δρmax, Δρmin (e Å−3

) 0.77, −0.57

Absolute structure Flack, H. D. (1983), Acta Cryst. A39, 876–881.

959 Friedel Pairs

Absolute structure parameter 0.06 (5) aRefinement was performed using all reflections. The weighted R-factor (wR) and goodness of fit (S) are

based on F2. R-factor (gt) are based on F. The threshold expression of F

2 > 2.0 σ(F

2) is used only for

calculating R-factor (gt).

162

Table A6.2.2. Fractional atomic coordinates and equivalent isotropic displacement parameters (Å2)

atom x y z Beqa

S(1) 0.47767 (18) 0.78195 (15) 0.38887 (9) 2.83(4)

F(1) 1.2008 (4) 0.8138 (3) 0.2114 (2) 3.70(9)

O(1) 0.3777 (4) 0.7220 (4) 0.4473 (2) 2.93(9)

N(1) 0.6157 (6) 0.8618 (5) 0.4287 (3) 2.59(11)

C(1) 0.5818 (7) 0.6484 (5) 0.3468 (3) 2.23(12)

C(2) 0.4592 (6) 0.5600 (6) 0.3162 (3) 3.33(14)

C(3) 0.6790 (7) 0.5800 (6) 0.4046 (3) 3.19(14)

C(4) 0.6763 (7) 0.7046 (6) 0.2831 (3) 3.06(13)

C(5) 0.6415 (7) 0.9996 (6) 0.4140 (3) 2.48(12)

C(6) 0.5310 (7) 1.0894 (6) 0.4567 (4) 2.80(13)

C(7) 0.3666 (7) 1.0567 (6) 0.4380 (4) 2.94(14)

C(8) 0.5575 (7) 1.0797 (6) 0.5409 (3) 3.37(14)

C(9) 0.5609 (7) 1.2271 (6) 0.4313 (4) 3.42(14)

C(10) 0.8133 (6) 1.0246 (6) 0.4267 (4) 2.81(13)

C(11) 0.9156 (7) 0.9652 (6) 0.3700 (4) 2.42(12)

C(12) 0.9228 (7) 1.0179 (6) 0.2987 (4) 2.84(13)

C(13) 1.0177 (8) 0.9661 (6) 0.2453 (4) 3.06(13)

C(14) 1.1021 (7) 0.8619 (6) 0.2639 (4) 2.80(13)

C(15) 1.1000 (7) 0.8069 (6) 0.3324 (4) 2.96(13)

C(16) 1.0032 (6) 0.8592 (5) 0.3860 (3) 2.60(12) a

Beq = 8/3 2(U11(aa*)2 + U22(bb*)

2 + U33(cc*)

2 + 2U12(aa*bb*)cos + 2U13(aa*cc*)cos + 2U23(bb*cc*)cos )

163

Table A6.2.3. Fractional atomic coordinates and isotropic displacement parameters for hydrogens (Å2)

atom x y z Biso

H(2A) 0.5077 0.4914 0.2879 3.99

H(2B) 0.3905 0.6082 0.2838 3.99

H(2C) 0.4004 0.5236 0.3572 3.99

H(3A) 0.7676 0.6328 0.4172 3.83

H(3B) 0.7141 0.4985 0.3845 3.83

H(3C) 0.6177 0.5649 0.4491 3.83

H(4A) 0.7325 0.6362 0.2581 3.67

H(4B) 0.7489 0.7670 0.3029 3.67

H(4C) 0.6079 0.7463 0.2477 3.67

H(5) 0.6218 1.0133 0.3601 2.98

H(7A) 0.2979 1.1169 0.4628 3.53

H(7B) 0.3439 0.9701 0.4549 3.53

H(7C) 0.3518 1.0621 0.3844 3.53

H(8A) 0.5347 0.9930 0.5575 4.05

H(8B) 0.4903 1.1399 0.5665 4.05

H(8C) 0.6643 1.1000 0.5522 4.05

H(9A) 0.6661 1.2512 0.4437 4.11

H(9B) 0.4895 1.2846 0.4563 4.11

H(9C) 0.5462 1.2331 0.3776 4.11

H(10A) 0.8310 1.1176 0.4269 3.37

H(10B) 0.8420 0.9917 0.4762 3.37

H(12) 0.8619 1.0900 0.2870 3.41

H(13) 1.0238 1.0021 0.1971 3.68

H(15) 1.1624 0.7353 0.3433 3.55

H(16) 0.9976 0.8216 0.4337 3.12

H(1N) 0.692 (6) 0.821 (5) 0.462 (3) 2.9(14)

164

Table A6.2.4. Atomic displacement parameters (Å2)

a

atom U11

U22

U33

U12

U13

U23

S(1) 0.0288 (8) 0.0319 (9) 0.0469 (10) 0.0007 (8) −0.0011 (8) 0.0002 (9)

F(1) 0.041 (2) 0.039 (2) 0.060 (3) 0.0005 (19) 0.021 (2) −0.003 (2)

O(1) 0.025 (2) 0.037 (3) 0.050 (3) −0.001 (2) 0.010 (2) 0.008 (2)

N(1) 0.026 (3) 0.029 (3) 0.043 (4) −0.005 (2) −0.010 (3) −0.002 (3)

C(1) 0.019 (3) 0.023 (3) 0.042 (4) −0.002 (3) 0.002 (3) −0.005 (3)

C(2) 0.029 (4) 0.036 (4) 0.062 (5) −0.002 (3) −0.007 (4) −0.004 (3)

C(3) 0.034 (4) 0.033 (4) 0.054 (5) 0.009 (3) −0.005 (4) 0.007 (4)

C(4) 0.035 (4) 0.038 (4) 0.043 (4) 0.004 (3) 0.005 (3) −0.002 (4)

C(5) 0.029 (3) 0.023 (3) 0.043 (4) −0.004 (3) 0.000 (3) 0.005 (3)

C(6) 0.033 (4) 0.031 (4) 0.042 (4) 0.003 (3) 0.001 (3) −0.001 (3)

C(7) 0.035 (4) 0.035 (4) 0.041 (4) 0.000 (3) 0.004 (3) −0.003 (3)

C(8) 0.042 (4) 0.047 (4) 0.040 (4) 0.000 (3) 0.001 (3) −0.001 (4)

C(9) 0.041 (4) 0.031 (4) 0.059 (5) 0.000 (3) 0.013 (3) 0.002 (4)

C(10) 0.027 (3) 0.026 (4) 0.054 (4) −0.009 (3) 0.000 (3) −0.003 (3)

C(11) 0.019 (3) 0.026 (3) 0.047 (4) 0.000 (3) −0.004 (3) −0.001 (3)

C(12) 0.028 (3) 0.032 (4) 0.048 (4) 0.000 (3) −0.002 (3) 0.011 (3)

C(13) 0.030 (4) 0.038 (4) 0.049 (4) 0.010 (3) 0.008 (3) 0.009 (3)

C(14) 0.026 (3) 0.035 (4) 0.045 (4) 0.002 (3) 0.011 (3) −0.003 (3)

C(15) 0.031 (3) 0.028 (4) 0.054 (4) −0.005 (3) −0.009 (3) 0.003 (3)

C(16) 0.027 (3) 0.030 (3) 0.042 (4) 0.004 (3) 0.001 (3) 0.006 (3) a The general temperature factor expression:

exp(–22(a*2U11h

2 + b*

2U22k

2 + c*

2U33l

2 + 2a*b*U12hk + 2a*c*U13hl + 2b*c*U23kl))

165

Table A6.2.5. Bond lengths (Å)

bond length bond length

S(1)—O(1) 1.508 (4) C(2)—H(2C) 0.980

S(1)—N(1) 1.637 (5) C(3)—H(3A) 0.980

S(1)—C(1) 1.838 (6) C(3)—H(3B) 0.980

F(1)—C(14) 1.378 (7) C(3)—H(3C) 0.980

N(1)—C(5) 1.489 (8) C(4)—H(4A) 0.980

C(1)—C(2) 1.523 (8) C(4)—H(4B) 0.980

C(1)—C(3) 1.526 (8) C(4)—H(4C) 0.980

C(1)—C(4) 1.534 (8) C(5)—H(5) 1.000

C(5)—C(6) 1.557 (9) C(7)—H(7A) 0.980

C(5)—C(10) 1.542 (8) C(7)—H(7B) 0.980

C(6)—C(7) 1.516 (9) C(7)—H(7C) 0.980

C(6)—C(8) 1.541 (8) C(8)—H(8A) 0.980

C(6)—C(9) 1.541 (9) C(8)—H(8B) 0.980

C(10)—C(11) 1.497 (8) C(8)—H(8C) 0.980

C(11)—C(12) 1.404 (9) C(9)—H(9A) 0.980

C(11)—C(16) 1.383 (8) C(9)—H(9B) 0.980

C(12)—C(13) 1.384 (9) C(9)—H(9C) 0.980

C(13)—C(14) 1.364 (9) C(10)—H(10A) 0.990

C(14)—C(15) 1.366 (9) C(10)—H(10B) 0.990

C(15)—C(16) 1.398 (9) C(12)—H(12) 0.950

N(1)—H(1N) 1.00 (5) C(13)—H(13) 0.950

C(2)—H(2A) 0.980 C(15)—H(15) 0.950

C(2)—H(2B) 0.980 C(16)—H(16) 0.950

166

Table A6.2.6. Bond angles (°)


O(1)—S(1)—N(1) 109.5 (3) C(5)—C(6)—C(9) 108.5 (5)

O(1)—S(1)—C(1) 104.9 (3) C(7)—C(6)—C(8) 110.3 (5)

N(1)—S(1)—C(1) 102.0 (3) C(7)—C(6)—C(9) 107.9 (5)

S(1)—N(1)—C(5) 122.2 (4) C(8)—C(6)—C(9) 109.4 (5)

S(1)—C(1)—C(2) 105.5 (4) C(5)—C(10)—C(11) 114.1 (5)

S(1)—C(1)—C(3) 110.6 (4) C(10)—C(11)—C(12) 119.4 (5)

S(1)—C(1)—C(4) 106.4 (4) C(10)—C(11)—C(16) 121.6 (6)

C(2)—C(1)—C(3) 110.7 (5) C(12)—C(11)—C(16) 119.0 (6)

C(2)—C(1)—C(4) 110.0 (5) C(11)—C(12)—C(13) 120.8 (6)

C(3)—C(1)—C(4) 113.2 (5) C(12)—C(13)—C(14) 117.9 (6)

N(1)—C(5)—C(6) 114.0 (5) F(1)—C(14)—C(13) 117.6 (6)

N(1)—C(5)—C(10) 106.7 (5) F(1)—C(14)—C(15) 118.5 (5)

C(6)—C(5)—C(10) 115.3 (5) C(13)—C(14)—C(15) 123.9 (6)

C(5)—C(6)—C(7) 109.9 (5) C(14)—C(15)—C(16) 117.9 (6)

C(5)—C(6)—C(8) 110.8 (5) C(11)—C(16)—C(15) 120.5 (6)

167

Table A6.2.7. Bond angles involving hydrogens (°)


S(1)—N(1)—H(1N) 122 (3) H(7A)—C(7)—H(7C) 109.473

C(5)—N(1)—H(1N) 115 (3) H(7B)—C(7)—H(7C) 109.477

C(1)—C(2)—H(2A) 109.468 C(6)—C(8)—H(8A) 109.464

C(1)—C(2)—H(2B) 109.473 C(6)—C(8)—H(8B) 109.473

C(1)—C(2)—H(2C) 109.478 C(6)—C(8)—H(8C) 109.469

H(2A)—C(2)—H(2B) 109.469 H(8A)—C(8)—H(8B) 109.475

H(2A)—C(2)—H(2C) 109.467 H(8A)—C(8)—H(8C) 109.475

H(2B)—C(2)—H(2C) 109.472 H(8B)—C(8)—H(8C) 109.472

C(1)—C(3)—H(3A) 109.471 C(6)—C(9)—H(9A) 109.472

C(1)—C(3)—H(3B) 109.468 C(6)—C(9)—H(9B) 109.475

C(1)—C(3)—H(3C) 109.475 C(6)—C(9)—H(9C) 109.471

H(3A)—C(3)—H(3B) 109.469 H(9A)—C(9)—H(9B) 109.469

H(3A)—C(3)—H(3C) 109.47 H(9A)—C(9)—H(9C) 109.464

H(3B)—C(3)—H(3C) 109.474 H(9B)—C(9)—H(9C) 109.475

C(1)—C(4)—H(4A) 109.472 C(5)—C(10)—H(10A) 108.726

C(1)—C(4)—H(4B) 109.479 C(5)—C(10)—H(10B) 108.727

C(1)—C(4)—H(4C) 109.475 C(11)—C(10)—H(10A) 108.718

H(4A)—C(4)—H(4B) 109.465 C(11)—C(10)—H(10B) 108.726

H(4A)—C(4)—H(4C) 109.467 H(10A)—C(10)—H(10B) 107.642

H(4B)—C(4)—H(4C) 109.469 C(11)—C(12)—H(12) 119.62

N(1)—C(5)—H(5) 106.749 C(13)—C(12)—H(12) 119.628

C(6)—C(5)—H(5) 106.751 C(12)—C(13)—H(13) 121.035

C(10)—C(5)—H(5) 106.753 C(14)—C(13)—H(13) 121.045

C(6)—C(7)—H(7A) 109.47 C(14)—C(15)—H(15) 121.037

C(6)—C(7)—H(7B) 109.47 C(16)—C(15)—H(15) 121.041

C(6)—C(7)—H(7C) 109.468 C(11)—C(16)—H(16) 119.743

H(7A)—C(7)—H(7B) 109.47 C(15)—C(16)—H(16) 119.733

168

Table A6.2.8. Torsion angles (°)a


O(1)—S(1)—N(1)—C(5) −123.6 (4) C(10)—C(5)—C(6)—C(8) −59.1 (6)

O(1)—S(1)—C(1)—C(2) 59.1 (4) C(10)—C(5)—C(6)—C(9) 61.0 (6)

O(1)—S(1)—C(1)—C(3) −60.6 (4) C(5)—C(10)—C(11)—C(12) 73.5 (6)

N(1)—S(1)—C(1)—C(3) 53.6 (4) C(5)—C(10)—C(11)—C(16) −106.4 (6)

N(1)—S(1)—C(1)—C(4) −69.8 (4) C(12)—C(11)—C(16)—C(15) 1.3 (8)

C(1)—S(1)—N(1)—C(5) 125.7 (4) C(16)—C(11)—C(12)—C(13) −0.9 (8)

S(1)—N(1)—C(5)—C(6) 79.2 (5) C(11)—C(12)—C(13)—C(14) 0.7 (9)

S(1)—N(1)—C(5)—C(10) −152.3 (3) C(12)—C(13)—C(14)—C(15) −1.0 (9)

N(1)—C(5)—C(6)—C(7) −57.3 (6) C(13)—C(14)—C(15)—C(16) 1.4 (9)

N(1)—C(5)—C(6)—C(8) 64.9 (6) C(14)—C(15)—C(16)—C(11) −1.5 (8)

N(1)—C(5)—C(10)—C(11) 69.2 (6) aThose having bond angles > 160 ° are excluded.

Documents

Fragment Based Identification of Phosphatase Inhibitors ... · Inhibitor library synthesis and evaluation 11 Amide replacement analog synthesis and evaluation 12 ... AEBSF 4-(2-aminoethyl)