A Chemocentric approach to anthranilate sulphonamide based therapeutics. Gillian Kiely

1

A chemocentric approach to anthranilate sulfonamide

based therapeutics

Gillian Kiely M B.Sc. (Pharm)

11304851

Based on research carried out under the supervision of John Gilmer

B.A,(Mod),Ph.D.

at

The school of pharmacy and Pharmaceutical sciences ,

Trinity College,

Dublin.

2014

2

Acknowledgements

I would like to express my sincerest gratitude to Dr. John Gilmer for giving me the

opportunity to undertake this project and for his constant support and guidance

throughout.

I would also like to thank Maria Pigott for her patience, assistance and advice from start to

finish.

Finally I would like to thank Gabor, Jason and everyone in the Gilmer group for their advice

and kindness.

3

Table of contents

Abstract …………………………………………………………………………………………………………………………......7

1. Introduction

1.1 What is a chemocentric approach?.........................................................................7

1.2 Sulfonamides……………………………………………………………………………......................…8-9

1.3 Anthranilic acid and anthranilates…………………………………………………………….….10-11

1.4 Anthranilate sulphonamides……………………………………………………………….....…………11

1.5 Anthranilate Sulfonamides as Inhibitors of Methionine Aminopeptidase-

2.............................................................................................................................12-18

1.6 Anthranilate sulfonamides as inhihibitors of PBPs..........................................18-21

1.7 Anthranilate sulfonamides as aldo keto reductase inhibitors…………………......21-22

1.8 Anthranilate sulfonamides as inhibitors of PDE…………………………………………………23

1.9 Anthranilate sulfonamides as antagonists of CCK1 and CCK2

receptors……………………………………………………………………………………………………….…..23-27

1.10 Matrix Metalloproteinases……………………………………………………………………………..27

1.11 Project aims………………………………………………………………………………………………….…28

2. Results and Discussion……………………………………………………………………………………………….29-43

3. Experimental:

3.1Chemistry

3.1.1 General methods……………………………………………………………………………………………44

3.1.2 Synthesis…………………………………………………………………………………………………45-52

3.2 Biological methods

3.2.1MMP-9 Flourogenic assay……………………………………………………………………………….52

4. Conclusion………………………………………………………………………………………………………………………53

References……………………………………………………………………………………………………………………53-54

4

Table of figures

Figure 1.1 Sullfonamide functional group

Figure 1.2 Sulfanilamide structure and its pKa values and para aminobenzoic acid and its

pKa values

Figure 1.3 Anthranilic acid and its pKa values

Figure 1.4 Anthranilate sulfonamide pharmacophore and its pKa values

Figure 1.5 Anthranilate sulfonamide compounds screened for activity against Met AP2






Figure 1.11 Anthranilate sulfonamide compounds screened for activity against PBPs

Figure 1.12 Anthranilate sulfonamide compounds screened for activity against PBPs

Figure 1.13 Anthranilate sulfonamide compounds screened for activity as aldo-keto

reductase inhibitors

Figure 1.14 Anthranilate sulfonamide compounds screened for activity as aldo-keto

reductase inhibitors

Figure 1.15 Anthranilate sulphonamide compound screened for activity as an inhibitor of

phosphodiesterases

Figure 1.16 Anthranilate sulphonamide compound screened for activity as an antagonist of

CCK1 and CCK2 receptors

Figure 1.17 Anthranilate sulfonamide compounds screened for activity as antagonists of

CCK1 and CCK2 receptors

Figure 1.18 The three anthranilate sulfonamide compounds we aimed to synthesise and

investigate in this project

Figure 2.1 Reaction scheme of anthranilic acid and 4-bromophenylsulfonyl chloride

Figure 2.2 HPLC chromatogram of the anthranilic acid starting material

5

Figure 2.3 HPLC chromatogram of the bromobenzenesulfonamide product synthesised

Figure 2.4 Reaction scheme of anthranilic acid and 4-bromophenylsulfonyl chloride with TEA

Figure 2.5 A mechanistic outline of the Suzuki-Miyaura cross-coupling reaction

Figure 2.6 Reaction scheme of the Suzuki reaction

Figure 2.7 HPLC of the phenylboronic acid starting material

Figure 2.8 HPLC of bromobenzenesulfonamide product, the other starting material in this

Suzuki reaction

Figure 2.9 HPLC of the Suzuki reaction mix showing the phenyl phenyl product at RT = 5.543

min

Figure 2.10 Reaction scheme of the Buchwald reaction

Figure 2.11 HPLC of the Buchwald reaction 2 after 1 h at room temperature

Figure 2.12 HPLC of Buchwald reaction 2 after ten minutes in the microwave at 100oC

Figure 2.13 Reaction scheme of the second trial Buchwald reaction

Figure 2.14 HPLC of the second trial Buchwald reaction in toluene after 8h reflux

Figure 2.15 Reaction scheme of 4-nitrobenzenesulfonyl chloride with the

bromobenzenesulfonamide product

Figure 2.16 Graph of Florescence Vs Time for the positive control

Figure 2.17 Graph of fluorescence Vs time for the diaryl ether at 10 μM concentration

Figure 3.1 1H NMR spectrum of 2-(4- bromophenylsulfonamido)benzoic acid

Figure 3.2 13C NMR spectrum of 2-(4- bromophenylsulfonamido)benzoic acid)

Figure 3.3 1H NMR spectrum of 2-(4-nitrophenylulfonylamido)benzoate

Figure 3.4 13C NMR spectrum of 2-(4-nitrophenylulfonylamido)benzoate

Figure 3.5 1H NMR spectrum of 2-([1,1’-biphenyl]-4-ylsulfonamido)benzoic acid

Figure 3.6 13C NMR spectrum of 2-([1,1’-biphenyl]-4-ylsulfonamido)benzoic acid

6

Table of tables

Table 1.1 Results showing the Inhibition of human methionine aminopeptidase type-2


Table 1.3 Results showing the inhibition of penicillin binding proteins

Table 1.4 Results showing the inhibition of CCK1R and CCK2R

Table 2.1 Possible side products of the Suzuki reaction and their retention times

Table 2.2 Palladium source in the Buchwald reaction

Table 2.3 The 6 different ligands used in the Buchwald reaction

Table 2.4 Results for the diaryl ether at a concentration of 10 μM

Table 2.5 Results for all concentrations of the phenyl-o-phenyl product

7

Abstract

A literature review has been completed on the therapeutic value of anthranilate

sulfonamide compounds. In this project, the preparation of three simple anthranilate

sulfonamide compounds is described. Evaluation of the enzymatic activity of each of these

compounds proves that they have some potential as MMP-9 inhibitors, in particular the

phenyl-o-phenyl compound which displayed inhibition in the nano-molar range.

1. Introduction

1.1 What is a chemocentric approach?

There are two principle basic strategies which are commonly used in the rational drug

design process, a chemocentric approach and a target based approach. In a chemo-centric

approach, a compound whose structure has been previously identified and has been shown

to have promising pharmacological ability is studied. This approach takes advantage of the

chemical similarities among compounds related to the candidate drug under the assumption

that structurally similar compounds are likely to exhibit similar physiochemical and

physiological properties. Proteins, for example, can be related through the ligands to which

they bind. An approach such as this can only be utilised when there is prior knowledge of

the ligands which may bind to the target(s) of interest.

Thus, when there is no information about the original compound a chemocentric approach

is futile. Consequently a target based approach is often favoured in modern rational drug

design processes. A target based approach involves identifying a receptor involved in a

disease process, an enzyme, or another biological molecule though to be involved in the

disease pathway and then looking at compounds that could potentially interact with said

target or alter its activityi.

Although it is not necessarily preferred, a chemocentric approach to drug design allows the

full establishment of the target profile of the compounds under development. By

discovering new off-targets, a chemocentric approach may be applied to characterise the

safety profile of a drug or as an essential step of drug reprofilingii.

8

1.2 Sulfonamides Figure1.1

History:

Sulfonamides are a group of drugs with a vast array of medicinal applications which include

antimicrobial, anticancer, anti-inflammatory and antiviral agentsiii. They were the first

effective chemotherapeutic medicinal product to be used for the prevention and treatment

of bacterial infections. The antibacterial properties of sulfonamides were first reported in

1932, when the German scientist Gerhard Domagk observed the effects of the red dye

Prontosil on Streptococcus infections in rodents. The magnitude of this medicinal discovery

and the subsequent widespread use of sulfonamides can be appreciated by the drop in

mortality and morbidity figures for treatable infections that followed.

Due to the discovery of penicillin and as a result, many other antibacterial agents, combined

with the hypersensitivity reactions that were commonly associated with sulfonamides, a

large decline in their use was observed in the 1960siv.

Chemistry:

Sulfonamides are synthetic derivatives of Sulfonilamide that have a similar shape to para-

aminobenzoic acid(PABA). v This is relevant to their antibacterial properties as PABA is a

constituent of folic acid. Thus Sulfanilamide and Sulfonamides are competitive inhibitors of

the enzyme tetrahydrofolate that incorporates PABA into folic acid in bacteria, resulting in

the sulfonamides being bacteriostatic compounds.

9

Figure 1.2

The majority of sulfonamides are insoluble in water, however their sodium salts are readily

soluble in aqueous solution allowing good bioavailability. The key structural requirements

for antimicrobial activity can be seen in Sulfanilamide itself, the aromatic ring attatched to

the sulfonamide functional group is essential. As well as this the para NH2 is crucial and can

only be replaced by a moiety that is capable of being transformed to a free amino in vivo.

However the SO2NH2 is not necessarily needed for antimicrobial activity. Any substitutions

made in the amide group at position 1 show variable effects on the antimicrobial properties

of the resulting sulfonamide. The most potent compounds are produced by substituting the

heterocyclic aromatic nucleivi.

10

1.3 Anthraniliic Acid Figure 1.3

History:

Anthranilic acid (o-amino-benzoic acid, 2-aminobenzoic acid or anthranilate) is an organic

compound with the molecular formula C7H7NO2. The molecule consists of a benzene ring,

hence is classed as aromatic, with two adjacent, or "ortho-" functional groups, a carboxylic

acid and an amine. It was first isolated in 1841 when investigations were being carried out

on the indigo plant (indigofera) and its various componentsvii. Following the discovery of

anthranilic acid, a number of methods were generated to produce the sweet smelling

compund synthetically from different derivitaves of benzoic acid with an alkyl and nitro

group at the ortho position. However the most commonly used method commercially,

which has been proven to have the highest yield, involves treating pthalamide with sodium

hydroxide and then subsequently treating it with sodium hypochlorite. Upon purification a

yield of about 70% anthranilic acid is expected.

Uses:

Anthranilic acid esters have pleasant odours and are thus commonly used in the production

of perfumes, cosmetics and soaps.viii Their use is limited however due to their reactivity with

various aldehydes that can lead to the formation of a Schiff base and subsequent

discolouration of the product.

Anthranilate (the deprotonated form of anthranilic acid) is used by plants and micro-

organisms in the production of tryptophan, an essential amino acid. Anthranilc acid is also

11

employed industrially as an intermediate in the production of azo dyes and saccharin and

also in the pharmaceutical industry in the production of loop diuretics such as furosemide.

Chemistry:

Anthranillic acid is an aromatic acid consisting of a substituted aromatic benzene ring with a

carboxylic acid and an amine in an ortho position to eachother on the ring. As a result of

these functional groups anthanilic acid is amphoteric. In appearance, anthranilic acid is a

white solid when pure with a boiling point of 200o C.ix Anthanilic acid has a pKa of 4.95.

1.4 Anthranilate Sulfonamides:

Overview:

Anthranilate sufonamides are dervitives of anthranlic acid which have a sulfonyl group

attached to the amine directly attached to the benzene ring.

The anthranilate sulfonamide pharmacophore can be seen below in Figure 1.4

Figure 1.4

12

1.5 Anthranilate sulfonamides as Inhibitors of Methionine Aminopeptidase-2

Methionine aminopeptidase 2 (MetAP 2) is an enzyme that is a member of the

dimetallohydrolase family.x It is a cytosolic metalloenzyme that catalyses the hydrolytic

removal of N-terminal methionine residues from proteins before they are fully formed.xi The

biological functions of human methionine aminopeptidases are still not fully understood,

however the siRNA depletion of either MetAP1 or MetAP2 is reported to inhibit the

proliferation of human cells. Thus MetAP2 is a novel target for cancer therapy.

A series of anthranilic acid sulfonamides (compounds 4 and 5) with affinities for human

MetAP2 were identified using affinity selection by mass spectrometry (ASMS) screening in

order to isolate orally active reversible inhibitors of MetAP.

Compound 5 (as seen in Figure 1.5) was seen to have potent inhibition in a MetAP2 enzyme

assay (IC50 10 nM), almost certainly due to the extra ring structure that is not present in

compound 4 (also found in Figure 1.5). However in inhibiting the proliferation of the human

fibrosarcoma-derived cell line HT-1080 (EC50 = 2.4 µM), compound 5 was only reasonably

effective.

The addition of human serum albumin (HSA) decreased the potency considerably in both

assays. This indicates that the two anthranilate sulfonimide compounds may have issues

with protein binding in particularly HSA binding. xii

Figure 1.5

13

In another study carried out by the cancer research centre in Abbott laboratories in the US,

a number of molecules were looked at for inhibition of MetAP2 as a novel approach for

antiangiogenesis and anticancer therapy using affinity selection/mass spectrometry (ASMS).

A series of anthranilic acid sulfonamides with micromolar affinities were isolated and these

affinities were quickly improved upon to produce potent nanomolar inhibitors by chemical

modifications based on information from X-ray crystallography.

The starting point for the study was anthranilic acid sulfonamide 1 (Figure 1.6), with an IC50

of 9 μM for MetAP2, In order to develop a structure–activity relationship from a number of

MetAP2 inhibitors possessing less than 10 μM inhibitory activities against MetAP2.

Sulfonamide is attractive as a starting point due to its synthetic feasibility and good

pharmacokinetics.

Figure 1.6

Based on X ray examination of MetAP2 , It is believed that substituted anthranilic acids with

substituents at 5 and 6 positions could be extremely effective at the active site. Chloro-

substitution at the para-position on the sulfonyl phenyl showed reasonably tight binding

due to a narrow hydrophobic region on the enzyme.xiii

The following 14 anthranilate suldfonamides were screened:

14

Figure 1.7 Taken from M. Kawai et Al. 2006


Compound [E] IC50 (μM) [E] IC50 a (μM) IC50b (μM)

1 9.1 >100 3

2 3.9 >100 2

3 11 0.3

4 1 0.1

5 0.35 >100 0.4

6 0.09 >100 10

7 1.1 60

8 10 100

9 0.019 3.8 0.5

http://www.sciencedirect.com/science/article/pii/S0960894X06003908#tblfn1


15

Compound [E] IC50 (μM) [E] IC50 a (μM) IC50b (μM)

10 0.015 0.5

11 0.009 3.3 0.4

12 0.02 1.9 0.15

13 0.01 2.6 0.18

14 0.027 9.2 0.13

aEnzyme inhibition in the presence of 40 mg/mL of human serum albumin.

b HMVEC cell line growth inhibition.

From the above table it is evident that the modification of anthranilic acid sulfonamide 1

allowed the isolation of inhibitors of human MetAP2 with good to potent activities against

MetAP2 and against human dermal microvascular endothelial cell proliferation. The

compounds were also found to display high affinity to serum albumin.

In another study intent on the optimization of methionine aminopeptidase-2 (MetAP2)

inhibitors containing sulfonamides of 5,6-disubstituted anthranilic acids, a series of aryl

sulfonamides of 5,6-disubstituted anthranilic acids were identified as potent inhibitors of

MetAP2.

It was observed that small alkyl groups and 3-furyl were tolerated at the 5-position of

anthranilic acid, whereas –OCH3, CH3, and Cl were found to be best for the 6-position.

Addition of a 2-aminoethoxy group at the 6-position allowed interaction with the second

Mn2+ however it failed to enhance the overall potency of the molecule.

Introduction of a tertiary amino moiety at the ortho-position of the sulfonyl phenyl ring

resulted in reduced protein binding and improved cellular activity, but led to lower oral

bioavailability.xiv

Initial optimization of 5,6-disubstituted anthranilic acid sulfonamides.



16

Figure 1.8

The most potent compounds, 19g and 19i, displayed potent MetAP2 inhibition with an IC50

of around 20 nM and anti-proliferation activity against HT-1080 cells with an EC50 of around

60 nM.

Figure 1.9

17


R1 R2 Enzyme IC50

(μM)

(No HSA)

Enzyme IC50

(μM) (w/ HSA)

19g

CH3O– CH3CH2– 0.026 0.080

19i

CH3O–

0.015 0.087

The inhibitory effect of anthranilate sufonamides is further demonstrated by the structure

seen below A-800141, which is highly specific for MetAP2. This orally bioavailable inhibitor

exhibits an antiangiogenesis effect and broad anticancer activity in a variety of tumour

xenografts including B cell lymphoma, neuroblastoma, and prostate and colon carcinomas,

either as a single agent or in combination with cytotoxic agents.

A biomarker assay has also been developed to evaluate in vivo MetAP2 inhibition in

circulating mononuclear cells and in tumours. This biomarker assay is based on the N-

terminal methionine status of the MetAP2-specific substrate GAPDH in these cel0ls. In

future it is very possible that these anthranilate sulfonamide MetAP2 inhibitors and GAPDH

biomarker in circulating leukocytes may be used for the development of a cancer treatment.

A-800141 showed potent activity against MetAP2 with an IC50 of 12 nM with a high

selectivity. The only other aminopeptidase examined to date showing inhibition by this

sulfonamide inhibitor at high concentrations is MetAP1.xv

18

Figure 1.10

1.6 Anthranilate sulfonamides as Inhibitors of penicillin binding proteins

Penicillin-binding proteins (PBPs) are well recognised and proven targets for antibacterial

therapy, they are proteins which are categorised by their binding to penicillin. In some

strains of resistant bacteria the resistance is acquired by active-site distortion of PBPs, which

lowers their acylation efficiency for β-lactams. Thus further research into potential inhibitors

of PBPs is crucial.

Anthranilate sulfonamides represent a class of compounds which could potentially be used

as PBP inhibitors. One study tested the following compounds for their effectiveness as

antibacterials.xvi

19

Figure 1.11 taken from Turk, Verlaine et al. 2011

The results of the study can be seen in the table below;

Table 1.3 Results showing the inhibition of penicillin binding proteins

Compound PBP2a RA (%) (IC50) PBP2x5204 RA (%)

(IC50)

PBP5fm RA (%) (IC50)

1 0 38b (391 μM) 100

2 58 123 39 (930 μM )

3 67 80 65

4 83 101 100

5 86 81 73

20

6 0 41 68

7 74 65 72

8 60 103 74

9 0(230 μM) 8b (155 μM) 72

10 17(680 μM) 121 69

11 70 118 61

12 47 (910 μM) 97 34(>1 mM aThe data represents mean values of three separate experiments.

RA=Residual activity of the enzyme at 1 mM inhibitor, unless stated otherwise.

IC50 values were determined in the presence of 0.01% Triton X-100.

bResidual activity of the enzyme at 500 μM inhibitor.

It is clear that these anthranilate sulfonimide compounds have real potential as new

noncovalent inhibitors of PBPs which represent important starting points for development

of more potent inhibitors of PBPs that can target penicillin-resistant bacteria.

Further investigations into the chemical space surrounding the aforementioned compounds

were carried out in order to establish the structure activity relationship for the inhibition of

PBPs. Thus, two more series of naphthalene sulfonamide and anthranilic acid based

compounds were synthesised and examined.

Three different transpeptidases were used to ascertain their inhibitory activity.

The most encouraging result seen between the two series of compounds was compound 52,

with a very promising IC50 of 80 micromolar against PBP21. Two of the molecules in the

series also showed inhibition of PBP1b.

In terms of the structure activity relationship of these series, it was evident from the relative

IC50s for each compound that the carboxyl group on the phenyl ring at the ortho position to

the sulfonamide group is favourable for the inhibition of PBP2a. Furthermore, as seen in

compound 52, the bromine atom which is meta to the carboxyl group is also crucial in

lowering the IC50.

Indeed, the position of the sulfonamide on the naphthalene ring is also seen to be of

importance as the IC50 of compounds with the sulfonamide at position 1 instead of position

2(as in compound 52) is two to three times higher. xvii

21

Figure 1.12

1.7 Anthranilate sulfonamides as aldo keto reductase inhibitors

Aldo-keto reductases are a family of enzymes which catalyse redox transformations involved

in biosynthesis, metabolism, and detoxification. Some aldo-keto reductases have become

targets for the development of new drugs due to the fact that they are involved in the

biosynthesis and inactivation of steroid hormones and prostaglandinsxviii.

In an attempt to identify selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 by

virtual screening of a fragment library, 70 compounds were selected for biochemical

evaluation, some of which being N-aminobenzoic acid derivatives. Of all these new

inhibitors discovered by this process, 15 have known scaffolds including the N-aminobenzoic

acid derivatives seen below.

The anthranilate sulfonamide structure below, compound 14, was found to be a selective

inhibitor of AKR1C3 with a micromolar Ki value of 111 μM. When the activities of the 3-

aminobenzoic acids screened were compared with structurally related derivatives of 5-

aminosalicylic acid, it was observed that the 3-aminobenzoic acids have comparable or

better AKR1C3 inhibitory activities and superior selectivity towards this isoform. The only 4-

aminobenzoic acid derivative identified by this screening, compound 15, 4-(N-

methylphenylsulfonamido)benzoic acid, is a non-selective micromolar AKR1C1-3 inhibitor.xix

22

Figure 1.13

Figure 1.14

23

1.8 Anthranilate sulfonamides as inhibitors of phosphodiesterases

There have been investigative studies carried out into the activity of anthranilic acid-based

compounds as inhibitors of phosphodiesterase. Phosphodiesterases (PDEs) are important in

regulating intracellular concentrations of cAMP thus inhibitors of PDE are currently being

targeted as anti-inflammatory drugs due to their suppressive effects on neutrophil function.

In one particular study a series of anthranilic acid derivatives were synthesized and their

anti-inflammatory effects and underlying mechanisms were examined in human

neutrophils.

In total eight separate compounds based on the pharmacophore below were synthesised

and screened for their anti-inflammatory activity. However both the the neutrophil function

assay data and SAR analysis showed that when a sulfonamide is used as a linker between

ring A and ring B the inhibitory effect of the molecule is decreased immensely.

Figure 1.15

1.9 Anthranilate sulfonamides as antagonists of CCK1 and CCK2 receptors

Cholecystokinin (CCK) is a regulatory hormone mainly located in the gastrointestinal tract as

well as in the central nervous system. There are two G protein coupled receptor subtypes,

24

CCK1 and CCK2, which regulate the main biological functions of CCK in the gastrointestinal

system, including motility, pancreatic enzyme secretion, gastric emptying, and gastric acid

secretion.

CCK1 binds non-sulfated members of the CCK family of peptide hormones and is a key

mediator of pancreatic enzyme secretion and smooth muscle contraction of the gallbladder

and stomach. CCK2 is a type B gastrin receptor, which has a high affinity for both sulfated

and nonsulfated CCK analogues. In the central and peripheral nervous system this receptor

regulates the feeling of fullness and the release of beta-endorphin and dopaminexx.

As a result of the important role of these receptors, a dual antagonist of CCK1R and CCK2R

has been considered as a novel target for the treatment and control of GORD. It has been

put forward that inhibition of CCK1R could help to improve LOS smooth muscle function and

increase the rate of gastric emptying while Inhibition of CCK2R could prove to moderate

gastric acid secretion.

In a study carried out by Johnson & Johnson Pharmaceutical Research in 2009 It was proven

that good CCK1R/CCK2R dual affinity can be applied to certain formerly CCK2R selective

anthranilic amides. As a result of this, several compounds were designed with ∼10×

selectivity for CCK2R/CCK1R that allowed potent in vivo inhibition of gastric acid secretion as

well as inhibition of pancreatic amylase production.

For CCK2R 10 nM affinity was seen with 20–30× selectivity over CCK1R in certain 3,4-dihalo

phenylalanine-derived analogs. The most promising of these compounds, compound 1, can

be seen below in Figure 1.16.xxi

25

Figure 1.16

Most compounds tested were seen to produce a significant amount of inhibition of CCK8s-

stimulated pancreatic amylase secretion around 45–67%. Furthermore, compounds 4a and

4j also potently inhibited pentagastrin-stimulated gastric acid secretion, with oral pED50

values of 5.1 (ED50 = 5.3 mg/kg, 7.9 μmol/kg) for 4a, and 5.8 (ED50 = 1.0 mg/kg, 1.6 μmol/kg)

for 4j.xxii

26

Figure 1.17

Table 1.4 Results showing the inhibition of CCK1R and CCK2R

Compound R1 R2 R3 X CCK1R pKi a CCK2R pKi

a Log ratiob

4a Cl Cl 4-I S 6.7 7.6 0.9

4b Cl Br 4-Br S 6.9 7.9 1.0

4c Cl Br 4-Br CH CH 7.0 8.0 1.0

4d F Br 4-Cl S 6.6 8.0 1.4

4e F Br 4-Cl CH CH 6.4 8.0 1.6

4f F Br 4-Br S 6.6 8.2 1.6

4g F Br 4-Br CH CH 6.5 8.3 1.8

4h F Br 4-I S 6.8 8.3 1.5

4i F Br 4,5-Cl2 CH CH 6.8(6.7)c 8.2(6.6)c 1.4(0.1)c

4j F Br 4,5-Cl2 S 6.8 8.0 1.2

aPercent remaining after 15 min in the presence of pooled human liver microsomes and NADPH.

bApparent compound permeability (10−6 cm/s) from apical to basolateral side of Caco-2 monolayer grown on transwell plates.

cApparent compound permeability (10−6 cm/s) from basolateral to apical side of Caco-2 monolayer grown on transwell plates.

http://www.sciencedirect.com/science/article/pii/S0960894X0901333X#tblfn1






27

It can thus be concluded that the anthranilic sulfonamide compounds designed in this study

have good CCK1R/CCK2R dual affinity. The tenfold selectivity for CCK2R/CCK1R was

displayed by the potent in vivo inhibition of gastric acid secretion as well as inhibition of

pancreatic amylase production. Although the antagonism of the CCK1 receptor was only

moderate, this may actually provide significant benefit for GORD patients as it still allows

periodic gall bladder contraction while also improving lower oesophageal sphincter function

and gastric prokinesis.xxiii

1.10 Matrix Metalloproteinases

Matrix metalloproteinases (MMPs, matrixins) are a family of secreted and membrane-

bound zinc-dependent endopeptidases that have the ability to degrade the components of

the extracellular matrix. These enzymes have a common zinc-binding sequence in their

active site. MMP enzymes are strongly involved in numerous pathological, physiological,

and biological processes including embryogenesis, normal tissue remodeling, wound

healing, and angiogenesis, and in diseases such as atheroma, arthritis, cancer, and tissue

ulceration. As a result of this, inhibitors of MMPs are being developed as potential

therapeutics in order to investigate the involvement of MMPs in various diseasesxxiv.

MMPs are excreted by several host cells such as macrophages, fibroblasts and bone,

epithelial and endothelial cells. Due to the fact that MMPs are excreted all around the body,

it is necessary to make any potential MMP inhibitor highly selective to its target.

The use of hydroxamic acids, carboxylates and thiols as MMP inhibitors has been well

documented- all three of these groups have the ability to bind zinc as they mimic the

peptides that usually bind to the Zn catalytic site in a similar way to that of the

corresponding peptide substances.

28

1.11 Project Aims

Diverse pharmacological effects have been reported for anthranilate sulfonamide

structures. The aim of this project was to review the available literature for applications of

this pharmacophore applying a chemocentric approach to drug design in the synthesis of

sulfonamides from anthranilic acid.

On reviewing the available literature, we decided to focus our synthetic efforts on the

design of MMP inhibitors containing the anthranilate sulfonamide structure. Previous work

in the Gilmer group had demonstrated the ability of this pharmacophore to inhibit MMPs in

the micromolar range. It is widely reported that derivatisation of a carboxylic acid to a

hydroxamate increases MMP inhibitor potency by facilitating bidentate binding to the zinc

atom of the enzyme. However, we decided to focus our attention on binding in the S1’

pocket of the enzyme. The Gilmer group have found that a phenoxyphenyl substituent binds

well in this pocket in work on barbiturate based MMP inhibitors and other groups have

reported good inhibition with a phenyl phenyl sustituent. The sulfonamide group is thought

to direct a substituent towards this pocket as well as increasing affinity by hydrogen

bonding. It is also possible that the group may be involved in binding the zinc atom of the

enzyme together with the carboxylic acid group.

The objectives of this project were to synthesize the compounds below and test them for

MMP 9 inhibitor potency by a fluorogenic assay.

Figure 1.18

29

2. Results and discussion

Sulfonamides can be readily synthesized from an amine and a sulfonyl chloride. In this

project, we used a facile, environmentally friendly synthesis of sulfonamides in water

reported by Deng et al. based on the Hinsberg reaction test.xxv The Hinsberg reaction test

distinguishes between primary, secondary and tertiary amines.

Anthranilic acid was used as a starting material in this reaction. It was necessary to maintain

a pH of eight throughout the reaction due to the fact that as the nucleophilic substitution

reaction proceeds hydrochloric acid is formed. Once the reaction is complete as indicated by

the disappearance of the starting materials on TLC and stabilisation of the pH, acidification

of the reaction mixture precipitates the desired sulfonamide.

Figure 2.1. Reaction scheme of anthranilic acid and 4-bromophenylsulfonyl chloride

HPLC and TLC were used to monitor the progress of the reaction.

30

Figure 2.2 HPLC chromatogram of the anthranilic acid starting material

Figure 2.3 HPLC chromatogram of the bromobenzenesulfonamide product- It can be seen

that all the anthranilic acid starting material at 3.117 in Figure 2.2 has been consumed and

the product is resoluting out at 4.357

Sulfonyl chlorides are sensitive to water hydrolysis and so using water as the solvent for the

reaction may seem surprising. This was not found to be problematic however. It is thought

that eliminating organic solvent from the reaction conditions allows the sulfonyl chloride to

slowly get into the reaction system and so hydrolysis is minimised. Optimal pH is

approximately pH 8. At lower pH, little reaction occurs and at higher pH more hydrolysis will

occur.

The reaction of anthranilic acid with 4-bromophenylsulfonyl chloride was also carried out

under conventional conditions in anhydrous DCM with triethylamine as base. TLC indicated

31

that the desired sulfonamide product was formed but there were also several side-products

and so this method was not pursued further.

A yield of 51.23 percent was achieved by carrying out the reaction in water. The 4-

bromophenylsulfonyl chloride starting material has very low solubility in water and solid

starting material was visible in the reaction mixture even after 24 h of reaction time. This

necessitated the filtration of the reaction mixture before precipitation of the product. In an

effort to improve the yield the reaction was also carried out in aqueous tetrahydrofuran

(1:1) with two equivalents of triethylamine as base. The reaction was stirred at room

temperature for 16 h, followed by the addition of ethyl acetate and 1M HCl. Deionisation of

the acid groups ensures that the product partitions to the organic phase. The organic phase

was separated and the aqueous layer was extracted twice more with ethyl acetate. The

combined organic layers were washed with brine, dried over Na2SO4 and the solvent

removed on a rotary evaporator. This synthetic method had a shorter reaction time and by

TLC appeared to proceed further but the yield achieved was 54% and so similar to that

achieved by the synthesis in water.

Figure 2.4 Reaction scheme of anthranilic acid and 4-bromophenylsulfonyl chloride with TEA

32

Successful synthesis of the bromobenzenesulfonamide provided the starting material for

synthesis of the biphenyl sulfonamide compound using the Suzuki reaction.

The Suzuki reaction is an organic reaction that is classified as a coupling reaction where the

coupling partners are a boronic acid or ester with a vinyl or aryl halide or triflate catalysed

by a palladium(0) complex.

The mechanism of the Suzuki reaction involves oxidative addition of the vinylic or aromatic

halide to the palladium (0) complex which generates a palladium (II) intermediate. The

intermediate undergoes a transmetallation reaction with the alkenyl boronate species. The

product is then expelled from this by reductive elimination resulting in the palladium (0)

catalyst being regenerated.

The base used in this reaction aids the transmetallation step, leading to the borate

directly.xxvi

A mechanistic outline of the Suzuki-Miyaura cross-coupling reaction can be seen below:

Figure 2.5

33

The starting materials used in this reaction were one equivalent of the

bromobenzenesulfonamide product and 1.5 equivalents of phenylboronic acid with K2CO3 as

base and tetraskis(triphenylphosphine)palladium as the source of palladium(0).

Figure 2.6 Reaction scheme of the Suzuki reaction

In the first trial reaction, monitoring by HPLC indicated that a product was formed. Despite

the higher equivalency of phenylboronic acid used it was fully consumed or degraded while

some starting sulfonamide material remained. On working up, it was very difficult to

separate the phenyl phenyl product from this starting material. The reaction was repeated

but in this case an extra equivalent of phenylboronic acid was added during the course of

the reaction to facilitate full consumption of the sulfonamide starting material and simplify

the work-up. HCl (1M) was added to the reaction mixture and the product extracted into

ethyl acetate. The organic layer was dried over Na2SO4 and concentrated under vacuum. It

was purified by flash column chromatography using step gradient elution. The initial mobile

phase used was hexane: ethyl acetate (3:2) with a few drops of acetic acid, then ethyl

acetate with a few drops of acetic acid. Each of the fractions from the column was analysed

by TLC for the phenyl-phenyl product. Once all the TLCs were carried out each of the

fractions found to contain our product were combined in a round bottomed flask and placed

in the rotary evaporator to remove any solvent.

34

Figure 2.7 HPLC of the phenylboronic acid starting material

Figure 2.8 HPLC of bromobenzenesulfonamide product, the other starting material in this

Suzuki reaction

35

Figure 2.9 HPLC of the Suzuki reaction mix showing the phenyl phenyl product at RT = 5.543

min

Possible side products of the Suzuki reaction for which reference materials were available

were excluded by HPLC.

Table 2.1 Possible side products of the Suzuki reaction and their retention times

Product Retention Time (min)

Sulfonamide starting material 4.674

Phenylboronic acid 3.088

Reaction mixture 4.714; 5.564; 8.941

Biphenyl

8.889

3.781

36

Synthesis of the diaryl ether was pursued by Buchwald-Hartwig coupling. The Buchwald–

Hartwig reaction is a chemical reaction used in organic chemistry for the synthesis of

carbon–nitrogen bonds via the palladium-catalyzed cross-coupling of amines with aryl

halides. It can also be used to synthesize carbon-oxygen bonds in the pursuit of diaryl

ethers.

The work of Buchwald and Hartwig showed that palladium can be used to promote

nucleophilic substitution at a vinylic or aromatic centre- a reaction like this is not usually

possible. This reaction proceeds in four distinguishable steps, step 1 involves palladium

insertion into the aryl halide bond in an oxidative addition step. Step two is an oxidative

addition step whereby the ligand allows coordination of the amine or oxygen with

palladium. A similar step is seen in the Suzuki reaction. Addition of the base allows removal

of the hydrogen-bromide and finally reductive elimination forms the product.

The primary byproduct of a Buchwald Hartwig reaction is a beta-hydride elimination product

which yields an aryl species that has had the halide substituted for hydrogen.

Phosphine ligands are used in this reaction due to their bulk- the bulkiness of the ligand

determines the product for the reaction. This reaction is highly dependent on reaction

conditions of starting material, solvent, ligand type and palladium source. Initially we used

Pd2(dba)3 as the palladium source and trialled six different ligands in

dimethoxyethane:water (4:1)

37

Figure 2.10 Reaction scheme of the Buchwald reaction

The starting materials in this reaction were again the bromobenzensulfonamide product and

phenol. The ligands and palladium source were added to each of the six reaction mixtures

under nitrogen and stirred at room temperature for one hour.

Table 2.2 Palladium source (the same for each of the six ligands) in the Buchwald reaction

Mol weight Equivalency Mmol Mass/vol

Pd2(dba)3

dipalladium

915.70 0.2 0.006 0.055g

Table 2.3 The six different ligands used in the Buchwald reaction

Name Mol wgt Equivalency Mmmol Mass/vol

1 Tris(dibenzylidenacetone

(o)(4-(N,N-

Dimethylamino)-phenyl)

di-tert-butylphosphine

265.38 0.1 0.003 0.008g

2 Butyldi-1-

ademantylphosphine

358.54 0.1 0.003 0.0011g

3 2- 476.72 0.1 0.003 0.0014g

38

(Dicylcohexylphosphino)-

2’,4’,6’-tri-i-prpyl-1,1’-

biphenyl

4 2-Di-tert-butyphosphino-

2’,4’,6’-

trisopropylbiphenyl

424.65 0.1 0.003 0.0013g

5 1,1’-

Bis(diphenylphosphino)

ferrocene (DPPF)

554.39 0.1 0.003 0.0017g

6 Tri-o-tolyLphosphine 304.38 0.1 0.003 0.0009g

HPLC of each of the reaction mixtures after 1 h indicated no reaction.

Figure 2.11 HPLC of the Buchwald reaction 2 after 1 h at room temperature

Each of the six reactions were then microwaved for 10 min at 20 W under a control

temperature of 100°C and re-examined by HPLC.

39

Figure 2.12 HPLC of Buchwald reaction 2 after ten minutes in the microwave at 100oC

Reactions 1, 2, 3, 5 and 6 contained identical products and were combined. The combined

mixtures were filtered through 1 cm of celite and rinsed with 2 mL DMA solvent. The solvent

was removed as much as possible from the filtrate and the residue was dispersed in 1M HCl.

This was extracted with ethyl acetate

The components of the reaction mix were separated by flash column chromatography in a

pasteur pipette filled with a plug of cotton wool, 10 mm of sand, 5 cm of silica gel and

another 10 mm of sand (in ascending order). The mobile phase was hexane: ethyl acetate

(3:2) with a few drops of acetic acid. Unfortunately after carrying out the column no ether

was found in any of the fractions collected.

It was decided to carry out another Buchwald reaction using reaction conditions reported by

Burgos et al. for diaryl ether formation. The bulky ligand, tert butylXphos (2-di-tert-

butylphosphino-2′,4′,6′-triisopropylbiphenyl) was used with palladium acetate as the

palladium source and potassium phosphate as base. The reaction was refluxed in toluene at

100°C

40

Figure 2.13 Reaction scheme of the second trial Buchwald reaction

The reaction required prolonged heating but HPLC monitoring after 8 h indicated the

formation of a product with a retention time of 5.6 min.

Figure 2.14 HPLC of the second trial Buchwald reaction in toluene after 8h reflux

After two days of reflux, the product proportion had increased but was still low.

Microwaving the reaction mixture at 20 W with a control temperature of 120°C was found

to speed up the reaction. The reaction mixture was filtered through celite and the filtrate

was concentrated. The residue was dispersed in 1M HCl and the aqueous layer was

extracted with ethyl acetate, dried over Na2SO4 and concentrated. The product was isolated

by column chromatography using hexane: ethyl acetate (1:1)

41

An anthranilate sulfonamide compound containing a nitro group was also synthesized. The

starting materials for this reaction were 4-nitrobenzenesulfonyl chloride and the

bromobenzenesulfonamide product. The method used to carry out this reaction is the same

as the one to make our first compound- the environmentally friendly, facile synthesis of a

sulfonamide in water. We also used this method to produce more of the phenyl-phenyl

derivative using biphenyl-4-sulfonyl chloride as the starting material. This aided in

confirmation of the Suzuki reaction product.

Figure 2.15 Reaction scheme of 4-nitrobenzenesulfonyl chloride with anthranilic acid

Upon successfully synthesizing the diaryl ether, the phenyl-phenyl and the nitro compound

an MMP-9 fluorogenic assay was carried out to quantify the inhibition each of the

compounds would have on the enzyme. The assay measures remaining protease activity

after incubation of activated MMP9 with the candidate inhibitor in TCNB buffer for 30 – 40

min at 37°C. After incubation a suitable substrate is added. Remaining protease activity is

capable of cleaving an amide bond between a fluorescent group and a quencher group. The

resulting increase in fluorescence is linear with time. The positive control used in the assay

contained the buffer, the enzyme and the substrate with no candidate solution present. The

decrease in slope of the test solution compared to the positive control provides a measure

42

of enzyme inhibiton. The candidate solutions were prepared from 500 μM to 50 nM. DMSO

was used where necessary to achieve dissolution but was kept to as low a concentration as

possible as it can interfere with the assay results.

The concentrations tested ranged from 100 μM to 10 nM. As hypothesized, the phenyl-o-

phenyl product had the greatest inhibition, even in the nanomolar range. The phenyl phenyl

product also showed reasonably good inhibition whereas the nitro compound exhibited

poor inhibition of the enzyme.

Representative example of data processing:

Table 1.3 Fluorescence results of the 10 micromolar replicates and the positive control which

did not contain an inhibitor

10 μM

ph-o-ph

3578 3749 4445 4921 5775 7071 7841 9516 10670 11850 13291

10 μM

ph-o-ph

3793 3941 4805 5696 6713 7839 9352 10564 12277 13836 15287

PC 3726 4518 5384 7054 7927 9708 11594 12823 15313 17381 19034

Figure 2.16 Graph of Florescence Vs Time for the positive control

Positive Control

y = 1572.6x + 970.15

R2 = 0.9828

0

5000

10000

15000

20000

0 2 4 6 8 10 12

Time (min)

Flu

ore

scen

ce

43

Figure 2.17 Graph of fluorescence Vs time for the diaryl ether at 10 μM

Upon processing the above data the following results were found.

Table 2.4 Results for the diaryl ether at a 10 μM

Compound Concentratio

n

Slope % Activity % Inhibition Average %

inhibition

Ph-O-Ph 10 μM 804.33 51.15 48.85 48.85

When the nitro compound was tested at the 10 μM level the active % inhibition was not

significant.

Table 2.5 Results for all concentrations of the phenyl-o-phenyl compound

Compound Concentratio

n

Slope % Activity % Inhibition Average %

inhibition

002 1 μM 1058.10 67.28 32.72

002 1 μM 951.91 60.53 39.47 36.09

002 100 nM 1137.20 72.31 27.69

002 100 nM 917.02 58.31 41.69 34.69

002 10 nM 1008.20 64.11 35.89

002 10 nM 1198.60 76.22 23.78 29.84

Ph-O-Ph 10 micromolar

y = 804.33x + 4654.5

R2 = 0.9539

0

2000

4000

6000

8000

10000

12000

14000

16000

0 2 4 6 8 10 12

Time (min)

Flu

ore

scen

ce

44

3. Experimental

3.1 Chemistry

3.1.1 General methods

All chemicals were purchased from Sigma Aldrich (Ireland), except where stated. All the

reactions were monitored using TLC. Uncorrected melting points were measured on a Stuart

Apparatus. Infra-red (IR) spectra were performed on a Perkin Elmer FT-IR Paragon 1000

spectrometer. 1H and 13C nuclear magnetic resonance (NMR) spectra were recorded at 27oC

on a Brucker DPX 400 spectrometer (400.13MHz, 1H; 100.61MHz, 13C) Coupling constants

are reported in Hertz. For 1H-NMR assignments, chemical shifts are reported; shift value

(number of protons, description of absorption, coupling constant(s) where applicable)

Electrospray ionisation mass spectrometry (ESI-MS) was performed in the positive ion mode

on a liquid chromatography time-of-flight mass spectrometer (Micromass LCT, Waters Ltd.,

Manchester UK). The samples were introduced into the ion source via an LC system (Waters

Alliance 2795, Waters Corporation, USA) in acetonitrile:water (70:30% v/v) at 200 μL/min.

The capillary voltage of the mass spectrometer was at 3kV. The sample cone (de-clustering)

voltage was set at 4o V. for exact mass determination, the instrument was externally

calibrated for the mass range m/z 100 to m/z 1000. A lock (reference) mass (m/z 556.2771)

was used. Mass measurement accuracies of < +/- 5ppm were obtained. Compound

purity/homogeneity was confirmed by a combination of NMR, TLC and HPLC.

45

3.1.2 Synthesis

2-(4- bromophenylsulfonamido)benzoic acid

This compound was prepared by two methods.

Method 1

Anthranilic acid (0.5000 g) was weighed out and dispersed in 15 mL water in a round

bottomed flask while being mixed with a stirring bar. The pH of the mixture was adjusted to

pH 8 with 1 M Na2CO3. One equivalent of 4-bromosulfonyl chloride (0.9310 g) was then

added and the pH was continuously monitored and maintained at 8. The reaction was left

overnight to complete. The reaction mixture was filtered and addition of concentrated HCl

to the filtrate to pH 1 caused the product to precipitate out of solution. The precipitate was

collected under vacuum using a buchner flask. This off-white solid was washed with water

and allowed to dry. It did not require further purification.

Percentage yield: 51.23%

Method 2

Antranilic acid (0.5000 g) was dissolved in 10 mL of H2O:THF (1:1). One equivalent of 4-

bromosulfonyl chloride (0.9326 g) and 3 equivalents of triethylamine (1.5 mL) were added

and the reaction mixture was stirred at room temperature. After 16 h, 50 mL ethyl acetate

and 50 mL 1 M HCL were added to the reaction mixture. The organic layer was separated.

The aqueous layer was extracted with ethyl acetate and the combined organic layers were

washed with NaHCO3 solution, dried over Na2SO4 and concentrated to an off-white solid.

This product did not require further purification.


Melting point: 265-269oC

HRMS: calculated for C13H9 BrN04S- = 353.9441, (M-H) = 353.9443 found

1H NMR (DMSO) δ ppm: 7.11-7.23 (m, 1H, Ar-H), 7.46-7.59 (m, 2H, Ar-H), 7.69-7.81 (m, 4H,

Ar-H), 7.91 (dd, j = 8.09, 1.87 Hz, 1H, Ar-H), 11.09-11.21 (br, 1H, NH), 13.5-14.6 (br, 1H,

COOH)

46

13C NMR (DMSO) δ ppm: 117.19, 118.77, 123.69, 127.53, 128.86 (2C), 131.56 (2C), 132.6,

134.52, 137.86, 139.30, 169.62 (COOH)

IR (KBr): v(cm-1) 757.77 (C-H ), 1215.74(C-O) , 2958.81 (N-H)

Figure 3.1 1H NMR spectrum of 2-(4- bromophenylsulfonamido)benzoic acid

Figure 3.2 13C NMR spectrum of 2-(4- bromophenylsulfonamido)benzoic acid

47

2-(4-nitrophenylulfonylamido)benzoate:

Anthranilic acid (0.6188 g) was dissolved in 15 ml water in a round bottomed flask while

being mixed with a stirring bar. The pH of the mixture was adjusted and maintained at a pH

of 8 with 1 M Na2CO3. 4-Nitrobenzene sulfonylchloride 1 g was then added and the pH was

continuously monitored and maintained at 8. Once the pH became constant at 8 and the

reaction was complete 1 M HCl was added to the RBF to acidify the mixture and cause the

product to precipitate out of solution. The contents of the RBF were filtered under pressure

using a buchner flask to yield the pure product as an off white solid which was a powder

when dry. Actual yield = 1.3309 g, theoretical yield: 1.453 g, percentage yield: 84.71% Mp:

231–235oC

MS: calculated for C13H9N206S- = 321.0187. (N-H) = 321.0184 found. 1H NMR (DMSO) δ ppm :

7.14-7.25(t, 1H, CH), 7.45-7.5 ( d,1H, CH), 7.55-7.65 (t,1H,CH), 7.85-7.95 (d,1H,CH), 8.05-8.15

(m, 2H, CH), 8.3-8.4 (m, 2H, CH), 10.8-11.85 (s,1H, broad, NH).

13C NMR (DMSO) δ ppm: 117.92, 119.24 (2C), 124.77, 124.8, 128.55 (2C), 131.59, 134.49,

138.73, 144.08, 150.14, 169.46

IR(KBr): v(cm-1): 756.27(C-H), 1215.12(C-O), 1528.97( N-O), 3019.92(N-H)

Figure 3.3 1H NMR spectrum of 2-(4-nitrophenylulfonylamido)benzoate

48

Figure 3.4 13C NMR spectrum of 2-(4-nitrophenylulfonylamido)benzoate

2-([1,1’-biphenyl]-4-ylsulfonamido)benzoic acid

This compound was synthesized in two ways.

Method 1 – Suzuki coupling of 2-(4-bromophenylsulfonamido)benzoic acid and

phenylboronic acid

2-(4-bromophenylsulfonamido)benzoic acid (0.15 mmol, 0.0550 g) was dissolved in 1 mL of

anhydrous THF. Phenylboronic acid (1.5 eq, 0.225 mmol 0.0274 g), tetrakis

(triphenylphospine)palladium (0) (0.05 eq, 0.0075 mmol, 0.0087 g) and potassium carbonate

(3 eq, 0.45 mmol, 0.0622 g) were added at room temperature. The reaction mixture was

sonicated under N2 to degas and then refluxed under N2 at 75 oC for 12 h over 2 days. A

further equivalent of phenylboronic acid was added during the course of the reaction. After

2 days, the reaction mixture was filtered through celite. THF solvent was removed under

vacuum and the residue was dispersed in 1M HCL. This was extracted with ethyl acetate

three times. The combined organic layers were washed with water, then brine, dried over

Na2SO4 and concentrated. The product was isolated by flash column chromatography using

a step gradient elution of hexane: ethyl acetate (3:2) with a few drops of acetic acid to ethyl

acetate with a few drops of acetic acid.

49

Percentage yield: 63%

Method 2 – Sulphonamide synthesis from anthranilic acid and biphenyl-4-sulfonyl chloride

Anthranilic acid (0.2710 g) was weighed out and dispersed in 15 mL water in a round

bottomed flask while being mixed with a stirring bar. The pH of the mixture was adjusted to

pH 8 with 1 M Na2CO3. One equivalent (0.5000 g) of biphenyl-4-sulfonyl chloride was then

added and the pH was continuously monitored and maintained at 8. Once the pH became

constant at 8 and the reaction was complete 1 M HCl as added to the RBF to acidify the

mixture and cause the product to precipitate out of solution. The contents of the RBF were

filtered using a buchner flask and the collected product was washed with water and allowed

to dry. It was recrystallized from methanol to yield an off white solid.


Melting point: 239-242oC

HR-MS: calculated for C19H14NO4S- = 352.0649 M-H found = 352.0649

1H NMR (DMSO) δ ppm: 7.13 (ddd, J=8.09, 4.35, 4.15 Hz, 1 Ar-H), 7.40 - 7.52 (m, 3 Ar-H),

7.53 - 7.60 (m, 2 Ar-H), 7.65 - 7.77 (m, 2 Ar-H), 7.82 - 7.94 (m, 5 Ar-H), 11.15 - 11.28 (br, 1 N-

H), 13.10-14.70 (br, s, 1 O-H)

13C NMR (DMSO) δ ppm: 116.87, 118.48, 123.55, 127.29 (2C), 127.76 (2C), 127.83 (2C),

128.92, 128.31 (2C) 131.79, 134.79, 137.55, 138.22, 139.96, 145.10, 170.00

IR(KBr) v(cm-1): 757.01(C-H), 1222.36(C-O), 3025.91 (N-H)

50

Figure 3.5 1H NMR spectrum of 2-([1,1’-biphenyl]-4-ylsulfonamido)benzoic acid

Figure 3.6 13C NMR spectrum of 2-([1,1’-biphenyl]-4-ylsulfonamido)benzoic acid

2-(4-phenoxyphenylsulfonamido)benzoic acid

2-(4-bromophenylsulfonamido)benzoic acid (0.1000 g) was weighed out into a round

bottomed flask with 0.0317 g (1.2 eq) of phenol and 0.1788 g (3 eq) of potassium

phosphate. Toluene (3 mL) was added under N2 and the mixture was sonicated to dissolve

and degas. When the aforementioned compounds were dissolved as much as possible

0.0358g (0.3 eq) of tert-butylXphos ligand, 0.0188g (0.3 eq) of palladium acetate were

weighed out and added. The mixture was refluxed at 100oC for 3 days-6 hours each day.

When the reaction was complete the solvent was removed and the residue was dispersed in

51

1M HCl and extracted with ethyl acetate three times. The combined organic layers were

washed with water, then brine, dried over Na2SO4 and concentrated. The product was

isolated by flash column chromatography using hexane: ethyl acetate (3:1) with a few drops

of acetic acid as the mobile phase.


Melting point: 228 - 230oC

1H NMR (DMSO) δ ppm: 7.05 (d, J = 9.12 Hz, 2 Ar-H), 7.08 - 7.15 (m, 3 Ar-H), 7.26

(t, J=7.46 Hz, 1 Ar-H), 7.42 - 7.48 (m, 2 Ar-H), 7.49 - 7.57 (m, 2 Ar-H), 7.81 (d, J=9.12 Hz, 2 Ar-

H), 7.90 (br. s., 1 Ar-H), 10.70 -11.60 (br, 1 N-H), 13.00 – 14.50 (br, 1 O-H)

13C NMR (DMSO) δ ppm: 116.29, 117.51 (2C), 118.41, 120.26 (2C), 123.21, 125.14, 129.51

(2C), 130.40 (2C), 131.59, 132.38, 134.43, 145.14, 154.40, 161.23, 173.62

HR-MS: calculated for C19H14NO5S- = 368.0598, (M-H) found = 368.0594

Figure 3.7 1H NMR spectrum of 2-(4-phenoxyphenylsulfonamido)benzoic acid

52

Figure 3.8 13C NMR data for 2-(4-phenoxyphenylsulfonamido)benzoic acid

3.2 Biological methods

3.2.1 MMP-9 Fluorogenic Assay

Recombinant MMP-9 (R&D Systems, Ireland) was activated by APMA (p-

aminophenylmercuric acetate) at 37oC for 24 h. The synthetic broad-spectrum fluorogenic

substrate (7-methoxycoumarin-4-yl)-acetyl-pro-Leu-Gly-Leu-(3-(2,4-dinitrophenyl)-L-2,3-

diaminopropionyl)-Ala-Arg-NH2 (R & D systems UK) was used to assay MMP-9 activity. The

inhibition of human active MMP-9 was assayed by preincubating MMP-9 (2 nM) and the

inhibitory compounds at varying concentrations in 50 mM Tris-HCl, pH 7.5, containing 150

mM NaCl, 10 mM CaCl2 and Brij 35 at 37oC for 30–45 minutes. An aliquot of substrate (10 μL

of a 50 μM solution) was then added to 90 μL of the preincubated MMP/inhibitor mixture,

and the activity was determined at 37oC by following product release with time. The

fluorescence changes were monitored using a plate reader machine (Fluorstar OPTIMA,

BMG LABTECH) with excitation and emission wavelengths set to 330 and 405nm,

respectively. Reaction rates were measured from the initial 10 min of the reaction profile

where product release was linear with time and plotted as a function of inhibitor dose. From

the resulting inhibition curves, the IC50 value for each inhibitor was calculated by by

nonlinear regression analysis using the Prism 4.0 (SD, CA, USA)

53

4. Conclusion

It can be concluded from the literature review that anthranilate sulfonamides have real

therapeutic potential in a number of different areas. In this project, we have proven that

compounds in this class may be promising inhibitors of MMP-9. The most active compound

synthesised was the di-aryl ether which is consistent with a binding mode in the S1’ pocket

of the enzyme. However the affinity of the pheny-o-phenyl group is well established, it has

good potency in both MMP 2 and MMP 9, thus the next logical step in the improvement of

these compounds should involve substitution on the aromatic ring in order to increase

selectivity for one enzyme over another while maintaining the potency afforded by the

diaryl ether.

i Golan, D. and Tashjian, A. (2012). Principles of pharmacology. Philadelphia: Wolters Kluwer Health/Lippincott Williams & Wilkins. ii Segu, C., Farres, J., Artigas, L. and Mas, J. (n.d.). ASSESSING DRUG TARGET ASSOCIATION FROM BIOLOGICAL

EVIDENCES. iii Investigation on Biological Activities of Anthranilic and Sulfonamide Analogs, S. Dougsoongnuen, A.

Worachartcheean, R. Pingaew, EXCLI Journal 2011;10;155-161 iv Goodman, L., Gilman, A., Hardman, J., Gilman, A. and Limbird, L. (1996). Goodman & Gilman's the

pharmacological basis of therapeutics. New York: McGraw-Hill, Health Professions Division. v Investigation on Biological Activities of Anthranilic and Sulfonamide Analogs, S. Dougsoongnuen, A.

Worachartcheean, R. Pingaew, EXCLI Journal 2011;10;155-161 vi Goodman, L., Gilman, A., Hardman, J., Gilman, A. and Limbird, L. (1996). Goodman & Gilman's the

pharmacological basis of therapeutics. New York: McGraw-Hill, Health Professions Division vii

www.alluredbooks.com/sample_pages/perf_flav_synt_p48_50. viii

pubchem.ncbi.nlm.nih.go ix Pubchem.ncbi.nlm.nih.gov, (2014). anthranilic acid - PubChem Compound Summary. [online] Available at:

http://pubchem.ncbi.nlm.nih.gov//compound/anthranilic%20acid?r=chemical#section=Top [Accessed 21 Oct. 2014]. x Sunil, D., Ranjitha, R., Balaji, S. and Pai, K. (2014). (E)-1-(2, 3-dimethoxyphenyl)-N-(4-methylpyridin-2-yl)

methanimineas a potent anticancer agent against colorectal cancer. International Journal of Pharmaceutical Chemistry, 4(1), pp.11--14. xi Jackson, R. and Hunter, T. (1970). Role of methionine in the initiation of haemoglobin synthesis. Nature, 227,

pp.672--676. xii

Sheppard, G., Wang, J., Kawai, M., Fidanze, S., BaMaung, N., Erickson, S., Barnes, D., Tedrow, J., Kolaczkowski, L., Vasudevan, A. and others, (2006). Discovery and optimization of anthranilic acid sulfonamides as inhibitors of methionine aminopeptidase-2: a structural basis for the reduction of albumin binding. Journal of medicinal chemistry, 49(13), pp.3832--3849. xiii

Kawai, M., BaMaung, N., Fidanze, S., Erickson, S., Tedrow, J., Sanders, W., Vasudevan, A., Park, C., Hutchins, C., Comess, K. and others, (2006). Development of sulfonamide compounds as potent methionine aminopeptidase type II inhibitors with antiproliferative properties. Bioorganic \& medicinal chemistry letters, 16(13), pp.3574--3577. xiv

Wang, G., Mantei, R., Kawai, M., Tedrow, J., Barnes, D., Wang, J., Zhang, Q., Lou, P., Garcia, L., Bouska, J. and others, (2007). Lead optimization of methionine aminopeptidase-2 (MetAP2) inhibitors containing sulfonamides of 5, 6-disubstituted anthranilic acids. Bioorganic \& medicinal chemistry letters, 17(10), pp.2817--2822.

54

xv

Wang, J., Tucker, L., Stavropoulos, J., Zhang, Q., Wang, Y., Bukofzer, G., Niquette, A., Meulbroek, J., Barnes, D., Shen, J. and others, (2008). Correlation of tumor growth suppression and methionine aminopetidase-2 activity blockade using an orally active inhibitor. Proceedings of the National Academy of Sciences, 105(6), pp.1838--1843. xvi

Turk, S., Verlaine, O., Gerards, T., \vZivec, M., Humljan, J., Sosi\vc, I., Amoroso, A., Zervosen, A., Luxen, A., Joris, B. and others, (2011). New noncovalent inhibitors of penicillin-binding proteins from penicillin-resistant bacteria. Plos one, 6(5), p.19418. xvii

Sosivc, I., Turk, S., Sinreih, M., Tro\vst, N., Verlaine, O., Amoroso, A., Zervosen, A., Luxen, A., Joris, B. and Gobec, S. (2012). Exploration of the chemical space of novel naphthalene-sulfonamide and anthranilic Acid-based inhibitors of penicillin-binding proteins. Acta chimica Slovenica, 59(2), pp.280--388. xviii

Barski, O., Tipparaju, S. and Bhatnagar, A. (2008). The aldo-keto reductase superfamily and its role in drug metabolism and detoxification. Drug metabolism reviews, 40(4), pp.553--624. xix

Brozic, P., Turk, S., Adeniji, A., Konc, J., Janezic, D., Penning, T., Lanisnik Rizner, T. and Gobec, S. (2012). Selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 discovered by virtual screening of a fragment library. Journal of medicinal chemistry, 55(17), pp.7417--7424. xx

Varga G, Bálint A, Burghardt B, D'Amato M (2004). "Involvement of endogenous CCK and CCK1 receptors in colonic motor function.". Br. J. Pharmacol. 141 (8): 1275–84 xxi

Liu, J., Deng, X., Fitzgerald, A., Sales, Z., Venkatesan, H. and Mani, N. (2011). Protecting-group-free synthesis of a dual CCK1/CCK2 receptor antagonist. Organic \& biomolecular chemistry, 9(8), pp.2654--2660. xxii

Pippel, M., Boyce, K., Venkatesan, H., Phuong, V., Yan, W., Barrett, T., Lagaud, G., Li, L., Morton, M., Prendergast, C. and others, (2009). Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: Tuning of receptor selectivity and in vivo efficacy. Bioorganic \& medicinal chemistry letters, 19(22), pp.6376--6378. xxiii

Pippel, M., Boyce, K., Venkatesan, H., Phuong, V., Yan, W., Barrett, T., Lagaud, G., Li, L., Morton, M., Prendergast, C. and others, (2009). Anthranilic sulfonamide CCK1/CCK2 dual receptor antagonists II: Tuning of receptor selectivity and in vivo efficacy. Bioorganic \& medicinal chemistry letters, 19(22), pp.6376--6378. xxiv

Visse R, Nagase H. Matrix metalloproteinases and tissue inhibitors of metalloproteinases: structure, function, and biochemistry. Circ Res.2003;92;827–839 xxv

Deng, X. and Mani, N. (2006). A facile, environmentally benign sulfonamide synthesis in water. Green Chem., 8(9), pp.835--838. xxvi

Clayden, J., Greeves, N. and Warren, S. (2012). Organic chemistry. New York (N.Y.): Oxford University Press.

Documents

A Chemocentric approach to anthranilate sulphonamide based therapeutics. Gillian Kiely