The Design, Synthesis, and Evaluation of Mechanism-Based b - Lactamase Inhibitors

The Design, Synthesis, and Evaluation of Mechanism-

Based b-Lactamase Inhibitors

CWRU 2009

N

S

O

N

HR

OCH3

CH3

CO2Na

Penicillins

NO

N

HR1

O

Cephalosporins

S

R2

CO2Na

NO

CO2Na

Carbapenems

Me (H)OH

R

N

CH3

O

N

HR

O

Monobactams

SO3Na

N

S

OCO2Na

Penems

OH

R

H (OMe)

NO

N

HR1

O

Oxacephems

O

R2

CO2Na

OMe

Major Classes of b-Lactam Antibiotics

Potent, broad-spectrum antibiotics Usually well tolerated Structural similarities include a negatively charged carboxylate, (usually fused bicylic) b-lactam, and C6 appendage

N

S

O

N

HR

OCH3

CH3

CO2Na

Enz-OH

(PBP)HN

S

O

N

HR

OCH3

CH3

CO2NaOEnz

The b-lactam antibiotics interfere with one or more members of a crucial set of bacterial enzymes, known as the penicillin-binding-proteins (PBPs), that are responsible for cross-linking glycan strands through a protruding peptide side chain.

•The b-lactam antibiotics are believed to resemble the D-Ala-D-Ala terminus of the pentapeptide side chain (Strominger Hypothesis)•Bacterial transpeptidases cleave between the two D-Ala residues, to form an intermediate acyl-enzyme, which is then reacted with a free amino moiety (e.g. the w amino group of diaminopimelic acid) to form the cross link.

OH

PeptideChain

Gly

Blocked H2OBlocked

HCR

O

CO2H

NH

O Me

Me

N

S

S

HN

O

O

Me

Me

NH

CO2H

C

O

R H S

HN

O

O

Me

Me

NH

CO2H

C

O

R H

Link

Why are b-lactam antibiotics such good drugs?

• b-Lactam antbiotics still comprise approximately half the commercial antibiotic market.

• Formation of a covalent bond to the target(s) may be an effective strategy for avoiding resistance due to point mutations which lower affinity

• Targeting the bacterial cell wall avoids the necessity to accumulate in cytoplasm, thus avoiding efflux pumps.

• b-lactams do not penetrate most mammalian cell types, resulting in low toxicity (disadvantage when treating atypicals)

• Most commonly observed resistance is due to production of b-lactamase(s)

Resistance to b-Lactam Antibiotics1) Production of one or more enzymes (b-lactamases) that

hydrolytically destroy b-lactam antibiotics 2) Produce PBPs that do not recognize penicillin3) In the case of Gram-negative strains delete outer

membrane porins, which are responsible for the allowing the b-lactams to reach the periplasm and hence the cell wall

4) In the case of Gram-negative strains, upregulate efflux pumps, which are responsible for pumping out foreign substances (including b-lactams).

N

S

O

N

HR

OCH3

CH3

CO2Na

Enz-OH

(b-lactamase) HN

S

O

N

HR

OCH3

CH3

CO2NaOEnz

HN

S

O

N

HR

OCH3

CH3

CO2NaOH

H2O

Enz-OH+

Action of Serine b-Lactamases

The b-Lactamases

• More than 600 different b-lactamases, grouped into four classes A-D• Classes A, C, and D are serine enzymes• Class B are zinc metalloenzymes• Historically, the class A (serine) enzymes were the most prominent• Can be produced in large quantity (hyperexpressed)• Produced in the periplasm of Gram-negative organisms, or extracellularly in Gram-positive strains.

N

S

ON+

CO2-

HN

N O

CO2H

S

NH2N O

NO

HN

O

S

CO2Na

OMe

MeO

Methicillin Ceftazidime

•One early strategy for countering b-lactamase mediated resistance was to design b-lactam antibiotics which would also be poor b-lactamase substrates.•This was achieved by incorporating sterically large substituents at C6 (penicillin) or C7 (cephalosporin).

Bulkygroup

Enzyme

C

R

HN

OCO2H

Me

MeS

N

H H

O

•Unfortunately, this gave rise to new forms of resistance, such as the appearance of a penicillin binding protein with reduced affinity for all b-lactam antibiotics (PBP2a in MRSA) and also the appearance of b-lactamases with enlarged active sites (extended spectrum b-lactamases or ESBLs) that could accommodate the larger antibiotics.

Methicillin-resistant Staphylococcus aureusMRSA

Recent Trends in b-Lactamase-mediated Resistance

• Broad spectrum b-lactamases, known as extended spectrum b-lactamases (ESBLs) capable of hydrolyzing third generation cephalosporins, are disseminated widely (e.g. class A, CTX-M)

• Class C b-lactamases (AmpC) are more widely disseminated, now including many plasmid-mediated AmpCs (e.g. FOX and CMY)

• Classes A and D enzymes have evolved the ability to hydrolyze the carbapenem class of antibiotics. These serine carbapenemases are increasingly widespread (e.g. KPC).

• Class B metallo-b-lactamases are disseminating widely. These enzymes were originally seen in Asia and in Europe, but cases of resistance due to class B b-lactamases are now appearing in the US (e.g. IMP and VIM).

NO

O

CO2Na

OH

NO

O2S

CO2Na

NO

O2S

CO2Na

N NN

Clavulanate Sulbactam Tazobactam

Current Commercial b-Lactamase Inhibitors

•A second approach was to develop inhibitors of b-lactamase•Unfortunately, current commercial inhibitors target only class A enzymes

Since the inhibitors have no independent antibacterial activity (i.e. ability to bind PBPs), they must be coadministered with b-lactam antibiotics

NO

O

CO2Na

OH

Clavulanate

NO

S

CO2Na

Amoxicillin

HN

O

NH2

HO= Augmentin

(GSK)+

NO

O2S

CO2Na

Sulbactam

NO

S

CO2NaAmpicillin

HN

O

NH2

= Unasyn(Pfizer)+

+NO

O2S

CO2Na

N NN

Tazobactam

NO

S

CO2NaPiperacillin

HN

O

HNN

O

NEt

O O

= Zosyn(Wyeth)

How do these commercial inhibitors work?

•Placing sulfur at the sulfone oxidation state predisposes the thiazolidine ring to fragment, producing the iminium ion shown above.•The iminium ion can then tautomerize to the b-aminoacrylate, or be captured by a second active site serine, producing in both cases, a stabilized acyl-enzyme.

N

O2S

OCO2Na

Enz-OHHN

O2S

O

CO2NaO-Ser70

HN+

SO2-

O

CO2NaO-Ser70

HN

SO2-

O

CO2NaOEnz

HN

SO2-

OCO2Na

OEnz

b-Aminoacrylate (Stabilized Acyl-Enzyme)

HN

SO2-

O

CO2NaO-Ser70

OSer130

O

O-Ser70

OSer130

Doubly covalently-boundAcyl-enzyme

How can we build a better mousetrap?

E + I E I E-IMichaelisComplex

InitialAcyl-Enzyme

E-I'StabilizedAcyl-Enzyme

H2OE I'

Complex ofHydrolyzed Inhibitor

E + I'

Irreversible inhibitors offer numerous opportunities for improving the inhibitory efficiency.

enzymatic mechanism

active site dimensions and

binding characteristics

synthetic feasibility

generate a library of prospective inhibitors

Assay againstall relevant enzymes

The Inhibitor Design Process

Initially we focused on designing inhibitors which held the potential to quickly form very stable acyl-enzymes.

N

O2S

OCO2Na

R1

R2

NO

CR1

R2

O2S

OAc

CO2Na

E + I E I E-IMichaelisComplex

InitialAcyl-Enzyme

E-I'StabilizedAcyl-Enzyme

H2OE I'

Complex ofHydrolyzed Inhibitor

E + I'

Focus Here

NO

S

CO2CHPh2

CH2OAc

H2N

NO

S

CO2CHPh2

CH2OAc

O1) Tf2O, Et3N

2) aq HCl

MgBr1)

2) H3O+ NO

S

CO2CHPh2

CH2OAc

OH

1) Tf2O, pyr

2) (t-Bu)2CuCNLi2, -100 oCN

O

S

CO2CHPh2

CH2OAc

CH

t-Bu

1) xs mCPBA

2) TFA, anisole3) NaHCO3

NO

O2S

CO2Na

CH2OAc

CH

t-Bu

N

O

O

OH

CO2Na

N

O2S

CO2NaO

N NN

NO

O2S

CO2Na

CH2OAc

CH

t-Bu

> 2000 M 51.9 M 11.8 M

IC50 Values against the class C b-lactamase derived from Enterobacter cloacae, strain P99

NO

O2S

CO2Na

CH2OAc

CD

t-Bu

HNO

O2S

CO2-

CH2OAc

CD

t-Bu

OEnz

HNO

SO2-

CO2-

CH2OAc

t-Bu

OEnz

EnzOH

Further mechanistic investigations uncovered an isotope effect on the rate of inactivation. A mechanism consistent with this observation is shown below.

StabilizedAcyl-Enzyme

N

S

OCO2CHPh2

H2Ni-PrONO

cat. TFA N

S

OCO2CHPh2

N23 mol % Rh2OAc4

xs propylene oxide N

S

OCO2CHPh2

O

quantitative yield

New chemical methodology facilitated the preparation of new inhibitors.

N

S

OCO2CHPh2

O

RCH=PPh3N

S

OCO2CHPh2

R

xs mCPBAN

O2S

OCO2CHPh2

R

1) TFA, anisole

2) NaHCO3N

O2S

OCO2Na

R

The availability of 6-oxopenicillanate simplifies the synthesis of 6-alkylidene penams, as shown.

N

S

OCO2CHPh2

R1

N

S

OCO2CHPh2

R1O

H

N

SOH

OCO2CHPh2

R1

N

S

OCO2CHPh2

R1 S N

S

1 eq mCPBA

S

NHS

AgOAc

R2CO2H N

S

OCO2CHPh2

R1

O

O

R2

a) R1 = CO2-t-Bu, R2 = CH2O2CCH3

b) R1 = CO2-t-Bu, R2 = CH2O2CCH2Clc) R1 = CO2-t-Bu, R2 = CH2O2CHd) R1 = CO2-t-Bu, R2 = CH2O2CCH2Phe) R1 = CO2Me, R2= CH2O2CCH3

f) R1 = CO2Me, R2 = CH2O2CCH2Cl

N

S

OCO2CHPh2

R1

S S

N

N

S

OCO2CHPh2

R1

O

O

R2

+Ag

N

S+

OCO2CHPh2

R1

-O2CR

A

BN

O

R1

S

CO2CHPh2

CH3

O2CR

A B

+

N

S

OCO2CHPh2

H2N1) alloc-Cl, Et3N

2) 1 eq mCPBA N

S

OCO2CHPh2

allocNHO S

NHS

tol. N

S

O

allocNHSBt

CO2CHPh2

N

S

OCO2CHPh2

allocNHAgOAc

RCO2H

O

O

R a) R = CH3b) R = CH2Ph

c) R = CH2

O-t-Bu

O-t-Bu

N

S

OCO2CHPh2

allocNHO

O

R1(n-Bu)3SnH, AcOH

cat. Pd(PPh3)4N

S

OCO2CHPh2

H2NO

O

R1

N

S

OCO2CHPh2

O

O

R1O1) i-PrONO, cat. TFA

2) cat. Rh2OAc4, propylene oxide

N

Ph3P

R2

N

S

OCO2CHPh2

O

O

R1

N

R2

N

O2S

OCO2Na

O

O

R1

N

R2

1) xs mCPBA2) TFA, anisole

3) NaHCO3

Table 1. Inhibitory activity on Three Representative Serine b-Lactamases IC50 (M)R1 R2 P99 TEM-1 PC1

None (Tazo) CH2C2H2N3 51.9 0.297 2.57CO2Na CH3 4.50 1.8 108CO2Na CH2O2CCH3 0.708 0.180 76.53CO2Na CH2O2CCH2Cl NT 0.196 7.2CO2Na CH2O2CH 0.592 1.84 173CO2Na CH2O2CCH2Ph 0.54 0.0154 579.0CO2Na CH2O2CCH23’,4’-C6H3(OH) 2 0.37 0.105 116CO2Me CH2O2CCH3 9.51 2.72 NTCO2NH2 CH2O2CH3 8.48 0.31 2.21CO2Na CH2Cl 527.0 120.5 2100CO2Me CH2Cl 13.91 44.51 432CO2Na CH=CHCN 6.76 21.67 504CO2Na CH2O2CCH2-S-tet 0.64 0.233 NTCO2Me CH2O2CCH2-S-tet 13.2 2.37 939.7a’-pyr CH2O2CCH3 0.062 0.004 0.66a’-pyr CH2O2CCH2Ph 0.001 0.04 0.39a’-pyr CH2O2CCH2-3’,4’-C6H3 (OH) 2 0.026 0.06

0.7

N

O2S

O

R2

CO2Na

R1

Buynak, J. D. et. al. BMCL 1999, 9, 1997-2002.

Piperacillin PIP:TAZ PIP:JDB/LN-1-255

P. aeruginosa Ps505A1 (AmpC derepressed) >64 16 2

A. sobria ((Asb A, OXA-12, AsbM) 64 64 1

S. marcescens GC 4132 (Amp C, in vivo) 64 32 4

E. coli C600N (no b-lactamase) 2 2 1

E. coli C600N +(TEM-1) >64 4 2

E. coli C600N + (IRT – 2) >64 8 2

E. coli C600N + (SHV – 4) >64 2 2

E. coli C600N + (PSE – 1) 32 1 2

E. coli C600N + (OXA-10) {PSE-2} >64 2 2

E. coli C600N + (MIR-1) 64 8 8

E. coli C600N + (Imi-1) >64 16 8

E. coli 300 + (TEM-1) >64 4 1

E. coli 300 + (ampRampC) 16 4 2

K. Pneumoniae KC 2 (TEM-10) >64 2 4

E. coli GC6265 (TEM-1, in vivo) >64 4 4

N

O2S

OCO2Na

N

O

O

OH

OH

JDB/LN-1-255

N

O2S

OCO2Na

O

NO

NH

N

O2S

OCO2Na

O

NO

OH

OH

NS

NH2 N

O2S

OCO2

-

O

NO

NH

O

HN

NH3+

N

O2S

OCO2Na

O

NO

NH2

O

NH2

N

O2S

OCO2Na

O

NO

NH2

NH3+

N

O2S

OCO2

-

N

NH3+

N

O2S

OCO2

-

O

NO

NH2

HNNH2

NH2+

N

O2S

OCO2

-

O

NO

NH2

NH3+

JDB/SA-3-18JDB/SA-4-11 JDB/SA-4-17 JDB/SA-4-141

JDB/SA-4-157 JDB/SA-4-196 JDB/SA-4-198JDB/LN-1-255

NH2

Inhibition of Representative b-lactamases (IC50, M)Inhibitor TEM-1

E. ColiAmpCP. aeruginosa

AmpCA. baumannii

OXA-40A. baumannii

In serumAmpC P. aeruginosa

JDB/SA-3-18 0.0004 0.008 0.017 0.0060 0.012

JDB/SA-4-11 0.00010 0.185 0.191 0.191 0.028

JDB/SA-4-17 0.00003 0.012 0.020 0.007 0.014

JDB/SA-4-141 0.0002 0.065 0.071 0.583 0.029

JDB/SA-4-157 0.0006 0.201 0.515 0.888 0.080

JDB/SA-4-196 0.0001 0.006 0.015 0.046 0.003

JDB/SA-4-198 0.0001 0.039 0.052 0.079 0.015

JDB/LN-1-255 0.00003 0.006 0.004 0.011 0.082

N

O2S

OCO2Na

O

NO

NH

N

O2S

OCO2Na

O

NO

OH

OH

NS

NH2 N

O2S

OCO2

-

O

NO

NH

O

HN

NH3+

N

O2S

OCO2Na

O

NO

NH2

O

NH2

N

O2S

OCO2Na

O

NO

NH2

NH3+

N

O2S

OCO2

-

N

NH3+

N

O2S

OCO2

-

O

NO

NH2

HNNH2

NH2+

N

O2S

OCO2

-

O

NO

NH2

NH3+

JDB/SA-3-18JDB/SA-4-11 JDB/SA-4-17 JDB/SA-4-141

JDB/SA-4-157 JDB/SA-4-196 JDB/SA-4-198JDB/LN-1-255

NH2

Synergy of Inhibitors with Imipenem Against Resistant P. aeruginosaImipenem

(mg/L)JDB/SA-

3-18(mg/L)

JDB/SA-4-11

(mg/L)

JDB/SA-4-17

(mg/L)

JDB/SA-4-141(mg/L)

JDB/SA-4-157(mg/L)

JDB/SA-4-196(mg/L)

JDB/SA-4-198(mg/L)

JDB/LN-1-255(mg/L)

MIC 20 0 0 0 0 0 0 0 0

0.5 MIC 10 12.5 25 25 12.5 3.125 6.25 12.5 6.25

0.25 MIC 5 100 50 50 25 12.5 12.5 25 25

0.125 MIC

2.5 100 100 100 25 25 12.5 50 50

0.0625 MIC

1.25 >100 >100 >100 50 50 25 100 100

0.0313 MIC

0.625 >100 >100 >100 >100 >100 >100 >100 >100

Inhibition of b-Lactamase (IC50 M)

R Escherichia coli W3310(Class A)

Enterobacter cloacae P99 (Class C)

t-butylmethylidene (allene) >2000 11.8

a-pyridyl 44 1.30

CO2But 0.28 429

tazobactam 1.37 51.9

clavulanic acid 3.3 >2000

NO

NO2S

CO2Na

OAcN

O

CO2S

CO2Na

OAc

H

t-Bu

NO

CO2-t-Bu

O2S

CO2Na

OAc

Initial attempts to improve the cephalosporin series of b-lactamase inhibitors relied on analogy with the cephalosporin antibiotics themselves.

N

S

ON+

CO2-

HN

N O

CO2H

S

NH2N O

Ceftazidime

But these efforts resulted in an abysmal failure!

N

O2S

ON+

CO2-

Ceftazidime-like analog

N

N

O2S

OOAc

CO2-

N

IC50 values against class C P99 b-lactamase

1.3 M >2000 M

HN

O2S

XO

OEnz

N

CO2-

N

O2S

O

OEnz

N

CO2-

Pathway 1 H2ON

O2S

O

OH

N

CO2-

EnzOH+

HN

O2S

XO

OEnz

N

CO2-

N

SO2-

XO

OEnz

N

CO2-

Pathway 2

Stabilized Acyl-enzyme

?

•Since the charge neutral pyridine moiety is a better leaving group than the negatively charged acetate, it is more likely to follow pathway 1 above.•Yet all the inhibitory mechanisms we have proposed follow pathway 2.

Type R1 R2 TEM-1 PC1 P99 GC1

Tazo 0.32 2.8 49.8 3.4

I 2’-py E-CH=CH-CN 0.014 0.72 0.01 0.012

I 2’-py E-CH=CHCO2Me 0.02 0.30 0.20 0.30

I 2’-py E-CH=CHCONH2 0.09 0.10 0.026 0.01

I 2’-py Z-CH=CClCO2Me 0.07 1.4 0.90 0.18

I 2’-py E-CH=CH-CH=CH2 68 75 24 NT

I 2’-py E-CH=CHCO2But NT 240 1.48 NT

I 2’-py E-CH=CHCO2Na 2.5 31 0.31 NT

I 2’-py E-CH=CHNO2 0.07 0.20 0.02 0.10

I 2’-py E-CH=CH-2’-py 0.20 4.3 0.18 NT

I 2’-py E-CH=CH-2”py-N-ox 0.006 8.6 0.60 0.10

I 2’-py CN 2.34 280 0.029 NT

I 2’-thzl E-CH=CHCONH2 0.90 154 0.29 NT

II 2’py E-CH=CHCO2Me 2.9 6.0 0.03 0.06

II 2’-py E-CH=CH-CO2But NT NT 440 150

II 2’-py E-CH=CHCO2Na 2.5 NT 6.60 NT

NO

R1

O2S

CO2Na

R2N

O

O2S

CO2Na

R2

CH2

I II

R1

R TEM-1 InhibitionIC50, M

P99 InhibitionIC50, M

Tazobactam 0.25 101.6CH=CH-CONH2 0.2615 0.022

CH=CH-CONHCH2CF3 0.078 1.18CH=CH-CONHCH2CH2OH 0.0701 0.212CH=CH-CONHCH(CH2) 2 0.240 0.824

CH=CH-CONH-CH2CH2 (CN3H4) 0.0083 0.0055

CH=CH-CONHOH 7.69 0.128CH=CH-CONHC6F5 4.28 0.127

CH=CH-CON(CH2CH2) 2NMe 0.053 6.34CH=CH-CONHCH 2Ph 1.4 0.11

CH=CH-CONHNH 2 0.39 1.1CH=CH-CO-NHC 6H4OH 0.11 0.035

CH=CH-CONHCH 2CO 2Na 4.2 0.31CH=CH-CONH(CH 2) 3NH 2 1.59 4.2

N

O2S

O

N

CO2Na

R

How do my inhibitors work?

N

O2S

OCO2Na

RHN+

SO2-

O

CO2Na

R

O

Ser70

NN

HN

SO2-

O

CO2Na

R

O

Ser70

N+

HN

SO2-

O

CO2Na

R

O

Ser70

N

Stabilized Acyl-Enzyme(several crystal structures now in PDB)

H

- H+

• Intramolecular capture of intermediate imine is more efficient than intermolecular capture (and/or tautomerization)• Inhibitors tend to be more general to all (serine) b-lactamases, since inhibitory mechanism does not depend on enzyme active site groups

Next goal: Prepare penicillin-derived inhibitors of metallo-b-lactamases

Problem: Metallo-b-lactamases are still a small portion of total number of b-lactamase producing strains

Solution: Prepare a single molecule that can function as dual inhibitor of both metallo- and serine-b-lactamases.

Problem: Metallo and serine b-lactamases have profoundly different mechanisms of action.

NO

S

CO2-

R2

R1

Zn1 Zn2

HO

H84

H86

H160

D88

H89

H225OH2

N

O

S

C

R2R1

Zn1 Zn2H84

H86

H160

D88

H89

H225OH2

O

OO

HN

O S

C

R2

R1

Zn1 Zn2H84

H86

H160

D88

H89

H225OH2

O

OH

O

Proposed series of events involved in the hydrolysis of a cephalosporin substrate by the L1 metallo-b-lactamase.

Inhibiting metallo-b-lactamases

Like most metalloenzymes, metallo-b-lactamases are inactivated by good zinc chelators.

Potential problem is that zinc chelating agents would likely be nonspecific, thus resulting in toxicity.

Solution: Generate a zinc chelating moiety that relies on the action of the enzyme itself to achieve optimal inhibitory activity (i.e. generate a mechanism-based metalloenzyme inhibitor).

Proposed Mechanism-based Inhibitors of the Zinc Metallooenzymes

N

O2S

O

HS

CO2Na

H2O

b-lactamase HN

SO2-

O

S

CO2NaO

Zn+2

N

OnS

O

HX

CO2Na

Two epimersTargeted Sulfone and Sulfide oxidation statesBoth alcohol

and thiol

N

S

O

H3N+

CO2-

N

S

O

Br

CO2CHPh2

1) NaNO2, H2SO4, Br2

2) PhC=N2

Br1) t-BuMgBr, -78 oC

2) CH2O, -78 to rtN

S

O

Br

CO2CHPh2

HOCH2

N

S

OCO2CHPh2

HOCH2(n-Bu)3SnH

cat AIBN,

1) MeSO2Cl, DMAP

2) CH3COSCs N

S

OCO2CHPh2

AcSCH2

NaOCH3

-78 to -40 oCN

S

OCO2CHPh2

HSCH2

N

S

OCO2Na

HSCH21) TFA, anisole

2) NaHCO3

N

S

OCO2CHPh2

HSCH2 troc-Cl, DMAP

0 oC to rtN

S

OCO2CHPh2

TrocSCH2

KMnO4/HOAc

CH2Cl2, rt N

O2S

OCO2CHPh2

TrocSCH2

Zn/Cu, HOAc

THF/MeOH, rtN

O2S

OCO2CHPh2

HSCH2 1) m-cresol, 50 oC, 2.5 h

2) NaHCO3N

O2S

OCO2Na

HSCH2

N

S

OCO2CHPh2

HOCH2

2.2 eq mCPBA

CH2Cl2/pH 6.4 BufferN

O2S

OCO2CHPh2

HOCH21) m-cresol, 50 oC

2) NaHCO3

N

O2S

OCO2Na

HOCH2

N

S

OCO2CHPh2

HOCH2 1) m-cresol, 50 oC

2) NaHCO3

N

S

OCO2Na

HOCH2

N

S

OCO2CHPh2

HOCH2

Br Bu3P, anh MeOH

1 h, rtN

S

OCO2CHPh2

HOCH2

H

N

S

OCO2CHPh2

AcSCH2

H1) MsCl, DMAP

2) AcSCs, MeCN

NaOMe, THF/MeOH

-78 oC to -40 oCN

S

OCO2CHPh2

HSCH2

H1) m-cresol, 50 oC, 6h

2) NaHCO3N

S

OCO2Na

HSCH2

H

Compound TEM-1(class A)(Serine)

P99(class C)(Serine)

L1(class B)(Metallo)

BCII(Class B)(Metallo)

Tazobactam 0.122 53.2 >200 >200

752 409 >200 >200

275 96.2 >200 >200

0.65 3.9 72.3 >200

14.6 10.0 >200 >200

Inhibition of Serine and Metallo-b-lactamases IC50 (M)

TEM-1(class A)(Serine)

P99(class C)(Serine)

L1(class B)(Metallo)

BC1(Class B)(Metallo)

Tazobactam 0.122 53.2 >200 >200

601 0.10 32.1 2.9

648 3.75 10.9 1.7

6.8 10.5 0.10 1.4

51.7 7.5 0.30 2.0

Inhibition of Serine and Metallo-b-lactamases IC50 (M)

Van den Akker Strategy: Stabilize the E-b-aminoacrylate intermediate in the active site.

• Designed by analogy with acyl-enzyme of Tazobactam.• This should result in an acyl-enzyme with increased affinity for the site.• May retain occupancy of the site subsequent to hydrolysis of the covalent

ester linkage of the acyl-enzyme.

E + I E I E-IMichaelisComplex

InitialAcyl-Enzyme

E-I'StabilizedAcyl-Enzyme

H2OE I'

Complex ofHydrolyzed Inhibitor

E + I'

Focus Here Focus Here

N

O2S

OCO2Na

Enz-OHHN

O2S

O

CO2NaO-Ser70

HN+

SO2-

O

CO2NaO-Ser70

HN

SO2-

O

CO2NaOEnz

HN

SO2-

OCO2Na

OEnz

b-Aminoacrylate (Stabilized Acyl-Enzyme)

HN

SO2-

O

CO2NaO-Ser70

OSer130

O

O-Ser70

OSer130

Doubly covalently-boundAcyl-enzyme

R

Tightly inBinding Pocket

Design a 2’-substituent that stabilized the E-form of the b-aminoacrylate

N

S

O

BrBr

CO2CHPh2

1) 1 eq mCPBA

2) mercaptobenzothiazole, tol. N

S

O

BrBr

CO2CHPh2

S

N

S

1) AgOAc

2) HO2C(CH2)3CO2CH2CCl3N

S

O

BrBr

CO2CHPh2

O

OOCH2CCl3

O

Zn

CH3CN N

O2S

OCO2CHPh2

O

OOCH2CCl3

O

Zn

HOAc

xs mCPBA

N

O2S

O

BrBr

CO2CHPh2

O

OOCH2CCl3

O

N

O2S

OCO2CHPh2

O

OOH

O

1) TFA, anisole

2) NaHCO3 N

O2S

OCO2Na

O

OONa

O

N

O2S

OCO2Na

O

OONa

O HN

SO2-

O

NaO2C

O

O

-O2CO

Enz

NH3+

K234

EnzOH

Thanks to my collaborators:

Robert BonomoPaul CareyMarion HelfandFocco van den Akker

And my funding sources:

Robert A. Welch FoundationNational Institutes of Health