An Overview of the Development of DprE1 Inhibitors for Combating the

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Perspective

An Overview of the Development of DprE1 Inhibitors for Combating the Menace of Tuberculosis Rupesh Vitthalrao Chikhale, Mahesh A. Barmade, Prashant R. Murumkar, and Mange Ram Yadav J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00281 • Publication Date (Web): 31 May 2018 Downloaded from http://pubs.acs.org on May 31, 2018

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Journal of Medicinal Chemistry

An Overview of the Development of DprE1 Inhibitors for Combating the Menace of Tuberculosis

Rupesh V. Chikhale a,b, Mahesh A. Barmade a, Prashant R. Murumkar a, Mange Ram Yadav a*

a. Faculty of Pharmacy, Kalabhavan Campus, The Maharaja Sayajirao University of Baroda, Vadodara, 390 001, India b. School of Health Sciences, Division of Pharmacy and Optometry, University of Manchester, Manchester M13 9PL, UK

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Abstract Decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1), a vital enzyme for cell wall synthesis plays a crucial role in the formation of lipoarabinomannan and arabinogalactan. It was first reported as a druggable target on the basis of inhibitors discovered in high throughput screening of a drug library. Since then, inhibitors with different types of chemical scaffolds have been reported for their activity against this enzyme. Formation of a covalent or noncovalent bond by the interacting ligand with the enzyme causes loss of its catalytic activity which ultimately leads to the death of the mycobacterium. This Perspective describes various DprE1 inhibitors as anti-TB agents reported till date.


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1.

Introduction: History and prevalence

Tuberculosis (TB) is a prehistoric disease that has plagued the mankind since the times known to us. The genus Mycobacterium is reported to have originated some 150 million years ago during the Gondwanaland era.1, 2 Mycobacterial strains prevailing today originated some 15,000-20,000 years ago from a common ancestry, and the currently infecting strains dated some 250-1,000 years back with origin from East Africa.3 This has been evidenced from the fact that the excavated Egyptian mummies had similar skeletal deformities as found in Pott’s disease.4 It spread from the African continent to Europe and then to rest of the world through trade routes. It was found to have spread from humans to the animals, and then the sea creatures spread this disease from the African continent to South America.5,

6

An

extensive impact of TB endemic on health conditions was experienced in Europe during the 16th to 19th centuries.7 This led to extensive efforts for understanding the disease. On 24th March, 1882 Hermann Heinrich Robert Koch announced TB as an infectious disease and Mycobacterium bacillus as the causative microorganism.8, 9 Koch’s discovery, contribution to bacteriology and elucidations on TB etiology won him Noble Prize for physiology in 1905.10 With the invention of anti-TB drugs in 1950s, treatment of TB became possible, TB cases started declining steeply during 1960s and 1970s, and the disease was considered to be completely curable and controllable. However, during 1980s the number of TB cases started rising again due to the emergence of immune suppressive diseases such as HIV, and due to emergence of drug resistant strains in TB bacteria due to mutations. The main reason behind emergence of drug resistance was lack of compliance of the drug regimen by the patients.11 Recently, World Health Organization (WHO) published data from 205 countries comprising of 99 % of the world population, on the prevalence, advances, treatment and diagnosis of TB.12 This report presents an alarming state with rise in cases of multi-drug



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resistant tuberculosis (MDR-TB) in recent years. Despite improvements in the healthcare systems in respect of better diagnosis, treatment and preventive measures, the disease still remains the world’s biggest threat to the human health. In 2014 alone, about 1.5 million people died globally from TB, including 890,000 men, 480,000 women and 140,000 children, and among them 0.4 million were HIV positive. About 6 million new cases were reportedly added to the previous count in 2014 alone, with a frightening estimate of 48,000 cases of MDR-TB. Most of these cases have been reported from South-East Asia and Western Pacific regions accounting for 58 % cases, and the African region accounted for 28 % cases. In the Asian region, countries like India (28 %), Indonesia (10 %) and China (10 %) have been badly affected, making this region highly susceptible to the growing threat of MDR-TB. Development of drug resistance in the bacterium needs to draw more attention because as per one report collected from 105 countries, an estimated 9.7 % of MDR-TB cases are getting converted to Extremely Drug Resistant TB (XDR-TB).12 The occurance of drug resistance to the first line drugs in the TB bacteria caused panic in the healthcare domain and provoked the researchers worldwide to develop novel lead molecules which could be effective against drug resistant mycobacteria. 2.

Mycobacterium tuberculosis infection: Drug targets and therapeutic agents Mycobacterium tuberculosis (Mtb) is the causative organism for tuberculosis and the

infection spreads from an infected subject having active tuberculosis to other persons. The mycobacterium spreads by means of aerosol droplets formed from an infected person on coughing, sneezing, singing and talking. Once the bacilli enter a healthy human being, they target mainly the lungs and start colonizing there. Mtb can cause pulmonary and extrapulmonary types of infections. The pulmonary infection can be an active or a latent infection of the lungs while the extrapulmonary infection can spread to central nervous


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system, lymphatic system, genitourinary system or joints. Activated macrophages of the immune system kill the bacilli by means of phagolysosomes.13, 14 But if the macrophages are not matured then the bacilli entrapped in the phagosomes are left intact allowing them to establish an active infection.15 This phase further develops into two different stages, in one case the Mtb may multiply intracellularly and manifest a replicating state of infection which precipitates as an active TB infection, or it may maintain a dormant or the non-replicating stage called the latent TB. In both the cases the infected person becomes a host for the organism and a potential transmitter for the microbes. The most important factor in Mtb infection is the host immunity; rise in incidents of HIV infections has created conditions conduceive for opportunistic infections causing an endemic situation.16 Rise in MDR-TB has posed a grave situation for its treatment and control of the disease in the human population. MDR-TB is defined as the infection that is resistant to isoniazid 1 and rifampicin 2, a condition attributed mainly to patients’ non-compliance to the prescribed drugs and poor control over self-medication.17 First Line Drugs CH3 CH3 O

NHNH2

O HO H3C O

N

H3CO

CH3 CH3 OH H3C CH3

OH

O

Isoniazid (1)

O

N

NH N OH

CH3 O

HO

O

CH3 O

NH2

N N

Pyrazinamide (3)

N

H3C

H N

N H

CH3

OH Ethambutol (4)

CH3

Rifampicin (2)



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Second Line Drugs NH2 HO H2N

OH

OH

O

HO HO H3C

HO O

NH O OHC OH

OH

NH2

N OH N O NH2 NH2 CH3

OH

O

HO H2N

HO

O

HO

OH O H2N

Streptomycin (5)

O

OH

OH

O

H N

O

NH2

ONH 2

O

OH H2 N

H2N

OH

OH

OH

O

H2N

Kanamycin (6)

OH OH

Amikacin (7)

HO H N

NH2 O H2N

N H O H N

H2N

O NH H N

O

H2N

O

N H3C

Capreomycin (8)

O

OH

N

HN

CH3

H3C

Ofloxacin (9)

N

N O

CH3

Levofloxacin (10)

O

F H

O

OH

N

N

N

O

F

OH

H

N

O

O

F

O

O HN

O

NH2

N H HN

S

S

NH2

NH2 O

N OCH3

H

N

Moxifloxacin (11)

CH3

N

Ethionamide (12)

CH3

Prothionamide (13)

HN O

NH2

Cycloserine (14)

O N O HN

NH O

O OH

N H2N

O Terizidone (15)

OH

p-Aminosalicylic acid (16)


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Drugs for MDR-TB Cl

H3C N

N

NH2

CH3 Cl

O

HO

H N O

N

N H Clofazimine (17)

S N

N

N

O

N O

OH

Amoxicillin (18) + Clavulanic acid (19)

O

O

CH3 NH

F

OH

O

O O

CH3

OH

O

CH3

H N

S H2N

N H

N

CH3 O

Thioacetazone (21)

Linezolid (20)

Figure 1. First line 1-4 and second line 5-16 anti-TB drugs, and the drugs available for the treatment of multi-drug resistant Mtb 17-21. Chemotherapy of TB consists of three lines of therapeutic regimen depending on the response of the subject to the therapy. Initial treatment consists of first line drugs, mostly with a combination of isoniazid 1, rifampicin 2, pyrazinamide 3 and ethambutol 4 (Figure 1). These drugs are generally provided free of cost under national TB missions of various countries in the form of kits consisting of two to three drugs for the convenience of the patient and the physician. Directly Observed Therapy, Short-course (DOTS) is the most successful program adopted or implemented world-wide for treating TB.12 The second line drugs used in the treatment of TB are streptomycin 5, kanamycin 6, amikacin 7, capriomycin 8, ofloxacin 9, levofloxacin 10, moxifloxacin 11,

ethionamide 12, prothionamide 13,

cycloserine 14, terizidone 15 and p-aminosalicylic acid 16.18 In case of MDR-TB, the drugs available for treatment are very limited and consist of clofazimine 17, a combination of amoxicillin 18 and clavulanic acid 19, linezolid 20 and thioacetazone 21.19 A condition where a patient becomes unresponsive to the given drug regimen is termed as XDR-TB which is considered as untreatable at present with the first and second line drugs.20 In the year 2012, 7 ACS Paragon Plus Environment


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the US-Food and Drug Administration (US-FDA) after a span of forty years approved bedaquiline 22, whereas in 2014 European Medicines Agency approved delamanid 23 for the treatment of MDR-TB, but this regimen of treatment spanning for about two years suffers from some cardiovascular risks.21 There are certain other drugs that are used in the treatment of MDR and XDR-TB these are sutezolid 24, rifapentine 25, tedizolid 26, and some of the agents currently in phase three and phase two clinical trials like SQ109 27 and LCB01-0371 28 respectively (Figure 2) may also be tried.22

Figure 2. New drugs 22-26 available for treatment of MDR and XDR TB along with agents 27, 28 in clinical trials for treatment of TB. Scientists worldwide are trying to develop newer and more efficacious drugs for the treatment of TB. Many approaches for the discovery of new anti-TB drugs have been


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adopted. Identification of a drug target is the very first step in the drug discovery process. The post-genomic era with advanced molecular biology techniques, genetic tools and availability of whole genome sequence of Mtb has opened up new avenues for the discovery of new biological targets for chemotherapy. At present, about twenty targets have been identified in the Mtb for the new drug discovery program (Figure 3). Several novel approaches and techniques such as signature-tagged mutagenesis23, recombinant mycobacteriophage technique24 and transposon site hybridization25,

26

, high throughput screening27, next

genenration sequencing28, network based29, 30 and machine learning31 approaches, and threedimensional cell culture32 are successfully adopted for the TB drug discovery paradigm. Some specialised programs like Tuberculosis Animal Research and Gene Evaluation Taskforce (TARGET) program are dedicated for the discovery of anti-TB drugs.33, 34 Highthroughput chemical genomics approach35, proteomics, protein and macromolecular crystallography have significantly contributed to the identification of about 257Mtb protein structures as new potential targets for chemotherapy of TB.36 The progress in discovery of new targets and metabolic pathways of Mtb has remained significant but this could not accelerate the drug discovery process because most of the validated targets were already harnessed, slowing down the pace of the discovery process. Development of a novel anti-TB drug is a challenging task because the Mtb exists in forms like replicating and dormant forms, and a drug should act on the replicating as well as the metabolically inactive form of the bacterium.36 Up till now, anti-TB drug development efforts have remained focused mainly on developing drugs for targeting the actively growing or the replicating forms of Mtb, while it is equally essential to discover newer drugs with efficacy to inhibit and get rid of the dormant form also. Figure 3 and Table 1 describe various targets that have been discovered for inhibiting the active or replicating form and the latent or the dormant form of TB.



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Figure 3. Diagramatic representation of Mtb targets within the cell structure; Coenzyme A Pathway (CoA), cytochrome bc1 complex (QcrB), DNA gyrase, DNA dependent RNA polymerase (RNA pol), CarD, DosR (DevR), isocitrate lyase (Icl), MmpL3, Enoyl-acylcarrier protein-reductase (InhA), β-Ketoacyl ACP synthase (KasA), D-alanyl-D-alanine ligase (Ddl), arabinosyltransferase (EmbAB), maltosyltransferase (GlgE), ATP synthase, L,D-transpeptidase (Ldt), mycolic acid cyclopropanation, Fatty acid synthase (FasI), ribosome, methionine aminopeptidase (Fmt), deformylase (Def), decaprenylphosphoryl-β-Dribose 2’α-epimerase (DprE1/E2), ATP phosphoribosyltransferase (HisG), mycothiol ligase (MshC). (Table 1 contains further details regarding these targets) Drugs currently used in therapy are mentioned outside the cell strucutre with arrows indicating their targets.37 Used with permission from Lamichhane et al. Trends Mol. Med. 2011, 17, 25-33. [Copyright © 2011 Elsevier]


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Table 1. Various targets and their roles in metabolic pathways of Mtb which could be exploited for the discovery of new anti-TB drugs. Entry

Target

Metabolic pathway and role

Reference

1.

MtbGlgE

Maltosyltransferase of Mycobacterium

38, 39

tuberculosis (MtbGlgE) belonging to αamylase family which is essential for metabolism of maltose-1-phosphate

2.

3.

Mycolic acid cyclo-

Mycolic acid synthesis (involved in cell

propane synthetase

wall synthesis)

Cholesterol catabolism

Normal cellular functioning and transport

40, 41

42

mechanism 4.

DprE1/DprE2

Cell wall synthesis

25, 43, 44

5.

MshC

Mycothiol ligase protects Mtb from

45, 46

antibiotic and oxidative stress 6.

HisG

Essential for normal functioning of

47, 48

biosynthetic pathway and growth 7.

AtpE

Growth regulation and ATP synthesis

49, 50

8.

Def

Protein synthesis and normal functioning

51, 52

9.

Methionine

Protein synthesis and normal functioning

53, 54

RelA, a stringent response regulator

55, 56

aminopeptidase 10.

CarD

controls RNA polymerase activity towards production of guanosine tetra/penta phosphate [(p)ppGpp], necessary for maintaining persistent 11 ACS Paragon Plus Environment


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infection 11.

DosR

Acts as dormancy regulator and

57-60

necessary for physiological maintenance 12.

Isocitrate lyase

Essential role in the fatty acid

61, 62

metabolism, virulence and growth regulation in active and dormant phases 13.

L,D-Transpeptidase

Necessary for the generation of the 3-3

63, 64

cross linkage in peptide chain of peptidoglycan layer

14.

Mtb Proteasome complex Essential for protection against reactive

65-73

nitrogen species and protein regulation

15.

Enoyl-acyl carrier

Mycolic acid/Fatty acid biosynthesis

74

Mycolic acid/Fatty acid biosynthesis

75

Arabinosyltransferase

Cell wall synthesis, key building block in

76

(EmbAB)

peptidoglycan biosynthesis

D-Alanyl-D-alanine

Cell wall synthesis, key building block in

ligase (Ddl)

peptidoglycan biosynthesis

protein reductase (InhA) 16.

Beta-ketoacyl-(acylcarrier-protein) synthase III (KasA)

17.

18.

19.

DNA dependent RNA Essential for transcription process

77

78, 79

polymerase (RNApol)

20.

DNA gyrase

Bacterial replication and transcription

80

21.

Ribosomes

Protein synthesis

81

22.

MmpL3

Mycolic acid transport

82 12

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23

QcrB

Mycobacterial energy metabolism

83

24

Coenzyme A pathway

Biosynthesis of lipids

84

25

Acetohydroxy acid

Synthesis of essential amino acids

85, 86

Mycolic acid synthesis

87

synthase (AHAS)

26

3.

FadD32

Decaprenylphosphoryl-β-D-ribose 2′-epimerase 1 (DprE1): A novel target for the discovery of anti-TB drugs Scientists around the world have formed several consortiums and are working together

for developing newer drugs to tackle MDR-TB and XDR-TB.88-91 For drug discovery they are using target-based and the whole cell-based aproaches.92 High throughput screening (HTS) techniques for the discovery of novel molecules were employed initially to screen molecules from the known data bases but these attempts either failed miserably or yeilded little success. However, these methods smoothened the way for anti-TB drug discovery.93 The availability of complete genome for mycobacterium tuberculosis has made it possible to determine the factors responsible for several mutations.94 These mutations play very important role in the development and determination of resistance in Mtb. Identification of candidate genes and their validation has shed light on the mechanism of action of various anti-TB drugs that has helped to identify several new targets. In a breakthrough research Christophe et al.44 and Makarov et al.43 reported decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1) as a novel and highly potential target for the discovery of new anti-TB drugs. This work has paved the way for the development of active drug molecules against Mtb which could specifically target this enzyme.95, 96



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Cell wall is an interface between the intracellular and extracellular environments for a cell. It performs a number of important fundamental processes like defining the structure, protection, transport, virulence and most importantly the pathogenicity. This makes the cell wall an attractive target for drug discovery. A number of processes involved in the formation of cell wall in Mtb are known and have been validated as viable targets for the drug discovery. Drugs like isoniazid 1 and ethambutol 4 are already known to interrupt the biosynthesis of cell wall.97,

98

Along with enoyl-acyl-carrier protein-reductase (InhA), β-

ketoacyl ACP synthase (KasA), arabinosyltransferase (EmbAB), maltosyltransferase (GlgE), ATP synthase, L,D-transpeptidase (Ldt), ATP phosphoribosyltransferase (HisG), mycothiol ligase (MshC), mycolic acid cyclopropanation, FadD32 and decaprenylphosphoryl-β-Dribose 2'-epimerase (DprE1/E2) are some of the novel validated targets in the Mtb cell wall (Table 1, Figure 3) which could be further exploited for the discovery of new drugs for containing the growth of Mtb.37, 87 The most important and unique constituents of Mtb cell wall

are

peptidoglycan-arabinogalactan-mycolic

acid

(PAM)

complex

and

lipoarabinomannan. In the PAM complex the peptidoglycan is covalently bound to arabinogalactan which in turn is bound to mycolic acid through ester linkage.99-101 Lipoarabinomannan is composed of D-arabinofuranose (Araf) and mannopyranosyl residues, which play important roles in many diverse biosynthetic pathways. Decaprenylphosporyl arabinose (DPA) acts as a substsrate for the arabinosyltransferase and plays an important role in the synthesis of mycobacterium cell-wall polysaccherides i.e. arabinogalactan and lipoarabinomannan. DprE1 is a flavoprotein that causes oxidation of decaprenylphosphorylD-ribose (DPR) to decaprenylphosphoryl-2-ketoribose (DPX) which is reduced by the enzyme DprE2 to DPA (Figure 4).95, 99


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O O O P HO O OH HO Site of epimerisation O

FADH2 O HO O

HO

CH3

8

CH3

CH3

Decaprenylphosphoryl-D-ribose (DPR)

H2O2 / menaquinol

FAD

DprE1

CH3

O O O P O

O2 / menaquinone CH3 CH3

CH3 CH3 8 Decaprenylphosphoryl-2-ketoribose (DPX)

DprE2 O HO HO

O O O P O OH

CH3 CH3

CH3 CH3 8 Decaprenylphosphoryl-D-arabinose (DPA)

Figure 4. Epimerisation of 2’-OH group of ribose by DprE1 and DprE2 into arabinose in presence of co-factor FAD. The arrow indicates the site of epimerization. The reaction takes place in presence of free oxygen or menaquinone.102 The crystal structures of DprE1 were first reported in 2012 by Neres et al.103 and Batt et al.104 from Mtb in the native and bound forms. Since then about 23 crystal structures of DprE1 have been made available in the protein data bank (Table 2). DprE1 was isolated by coexpression in Escherichia coli as intense yellow coloured protein with FAD in bound state. The crystals were obtained by the method of ligand co-crystallization through hanging drop technique. The crystal strucutre (PDB ID: 4FDN) of DprE1 possesses various binding sites that are the significant features of this enzyme (Figure 5). It has functional areas like the FAD-binding domain residues 7-196 and 413-461, and the substrate binding domain residues 197-412. The FAD binding domain displays α+β folds with β-sheet strands β1-β4, β5-β9 and α-helices α1-α4 and α11-α13. FAD is deeply seated inside the enzyme but it does not show any covalent interaction with the enzyme as observed in other oxidoreductases. 15 ACS Paragon Plus Environment


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Figure 5. The cartoon representation of crystal strucutre of DprE1 with substrate binding domain and the FAD binding domain.103 Used with permission from Neres et al. Sci. Transl. Med. 2012, 4, 150ra121 [Copyright @ 2012, AAAS].


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Table 2. Crystal strucutres of DprE1 along with their date of release in the Brookhaven Protein Data Bank (PDB) in chronological order, PDB ID, the exact title as mentioned on the PDB website, resolution at which the crystal strucutre was resolved and the species of mycobacterium from which the enzyme was isolated Entry Year (Release

PDB

Co-crystallized Ligand

Resolution Mycobacterium

ID

(Å)

species

4P8H

3.00

M. tuberculosis

Reference

date)

1

2015

105

N F3C

N

N

H3C

S

O

HO

O

NH

29 {(4S)-2-[7-Hydroxyamino-6-methyl-5-trifluoromethyl-1,3benzothiazol-2-yl]-4,5-dihydro-1,3-oxazol-4-yl}(pyrrolidin-1yl)methanone

17


Journal of Medicinal Chemistry 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

2

2015

4PFA

N O N+

F3C

2.56

M. tuberculosis

106

2.30

M. tuberculosis

106

-

HN

S HO

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O

NH

30 7-Hydroxyamino-N-(pyridin-3-ylmethyl)-5-trifluoromethyl1,3-benzothiazole-2-carboxamide 3-oxide 3

2015

4PFD

OCH3 N F3C

N

N

H3C

S

O

NO2

31 [4-(2-Methoxyethyl)piperazin-1-yl][6-methyl-7-nitro-5trifluoromethyl-1,3-benzothiazol- 2-yl]methanone

18


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4


2015

4CVY

M. tuberculosis sulfate ester dioxygenase Rv3406 in complex

2.00

M. tuberculosis

107

1.95

M. tuberculosis

107

2.49

M. tuberculosis

107

with Fe3+ 5

2014

4P8C

O N F3 C

OH

N

NH

CF3

32 6-Trifluoromethyl-3-{[4-(trifluoromethyl)benzyl]amino} quinoxaline-2-carboxylic acid

6

2014

O

4P8K N F3C

N

OH N H OCH3

33 3-(4-Methoxybenzyl)amino-6-trifluoromethylquinoxaline-2carboxylic acid

19



7

2014

4P8L

O N F3C

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2.02

M. tuberculosis

108

2.09

M. tuberculosis

107

OH

N

N H F

34 3-(4-Fluorobenzyl)amino-6-trifluoromethylquinoxaline- 2carboxylic acid 8

2014

O

4P8M N F3C

N

OH N H OC2H5

35 3-(4-Ethoxybenzyl)amino-6-trifluoromethylquinoxaline-2carboxylic acid

20


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9


2014

4P8N

O N F3C

1.79

M. tuberculosis

107

2.20

M. tuberculosis

107

OH

N

F

N H

OCH3

36 3-(3-Fluoro-4-methoxybenzyl)amino-6trifluoromethylquinoxaline-2-carboxylic acid 10

2014

4P8P

O N F3C

OH

N

N H Cl

37 3-[(4-Chlorobenzyl)amino]-6-trifluoromethylquinoxaline-2carboxylic acid

21



11

2014

4P8T

O N F3C

N

Page 22 of 102

2.55

M. tuberculosis

107

2.01

M. tuberculosis

107

OH N H N

38 3-(4-Cyanobenzyl)amino-6-trifluoromethylquinoxaline-2carboxylic acid

12

2014

4P8Y

O N F3C

N

OMe N H OCH3

39 3-(4-Methoxybenzyl)amino-6-trifluoromethylquinoxaline-2carboxylic acid

22


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13


2014

4NCR

N

NO2

1.88

M. tuberculosis

109

2.61

M. tuberculosis

110

N

S N

F3C O

40 2-(4-(Cyclohexylmethyl)piperazin-1-yl)-6-trifluoromethy-8nitro-4H-benzo[e]-1,3-thiazin-4-one 14

2013

4KW5

CH3

O

O

HN S

HN

N

O

O

S

41 Ethyl {2-[(1,3-benzothiazol-2-ylcarbonyl)amino]thiophen-3yl}carbonyl}carbamate 15

2012

4G3T

Nil

2.35

M. smegmatis

111

16

2012

4G3U

Nil

2.69

M. smegmatis

111

17

2012

4AUT

Nil

2.10

M. smegmatis

103 23



18

2012

4F4Q

CH3

Page 24 of 102

2.62

M. smegmatis

103

2.40

M. tuberculosis

104

O NO2

O

N

S N

F3C O

42 8-Nitro-2-[(2S)-2-methyl-1,4-dioxa- 8-azaspiro[4.5]decan-8yl]-6-trifluoromethyl-4H-1,3-benzothiazin-4-one 19

2012

4FDN

NO H N

F3C O

CH3

43 3-Trifluoromethyl-5-nitroso-N-(1-phenylethyl)benzamide

24


Page 25 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47

20


2012

4FDO

NO2

2.40

M. tuberculosis

104

H N

F3C O

CH3

44 3-Trifluoromethyl-5-nitro-N-(1-phenylethyl)benzamide 21

2012

4FDP

Nil

2.23

M. tuberculosis

104

22

2012

4FEH

Nil

2.04

M. tuberculosis

104

23

2012

4FF6

2.60

M. tuberculosis

104

CF3 HO

N H

H N O

CH3

45 3-Trifluoromethyl-5-hydroxyamino-N-(1-phenylethyl)benzamide

25



Page 26 of 102

The co-crystals of DprE1 bound to benzothiazinone (BTZ) and its analogs provided an insight into the mechanism of action of these agents. DprE1 causes an FAD dependent oxidation of DPR to DPX in which the flavin is reduced to FADH2, which gets reoxidised and then participates again in the next cycle.104 It was observed that the co-factor can be reoxidised by menaquinone, a naturally occurring electron acceptor in Mtb, suggesting that this enzyme could be an oxidoreductase instead of purely an oxidase. The crystal structure of DprE1 has helped in understanding the mechanism of action of benzothiazinones; the nitro group of benzothiazinones gets converted to nitroso by the enzyme which makes a covalent interaction with Cys387 residue causing inactivation of the enzyme. During the development of 42, Trefzer et al.108 hypothesised that FADH2 obtained during the conversion of DPR to DPX triggers the non-enzymatic reduction of nitro to nitroso group leading to collapse of the active site and inhibition of the enzymatic activity through formation of covalently bound ligand-DprE1 enzyme complex. However, Tiwari et al. hypothesized that the reported compound inhibited the enzyme activity through an alternate mechanism i.e. cysteine thiol (ate) played a pivotal role in the reduction of nitro group to nitroso intermediate that necessitated the action of FADH2.112 On similar lines, an attempt to determine the efficacy of BTZ derivative 43 revealed that covalent interaction occurred between the nitroso group and Cys387, leading to inhibition of the enzymatic activity.104 4.

Antimicrobial resistance: Covalent modifications of antimicrobial molecules by bacterial strains The graph of antimicrobial resistance (AMR) to the existing marketed drugs is

heading in upward direction continuously and remains a major concern for the future antimicrobial therapy. Hence, unlocking of the exact mechanism of resistance developed in the microbes remains an unmet need of the hour.113 Isoniazid (1), rifampicin (2),


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pyrazinamide (3) and ethambutol (4) are on the WHO list as essential first line anti-TB drugs and resistance (in M. tuberculosis) associated with their use poses a major challenge for the treatment of tuberculosis.114 One of the major causes for the emergence of drug-resistance to the first line anti-TB drugs is the spontaneous gene mutations occuring in M. tuberculosis.115 Isoniazid (1) is one of the most commonly used anti-Tb drug hence it undergoes drugresistance cascade more frequently than the others, and mutations in katG (in multidrugresistant isolate), inhA promoter (in monoresistant isolate), ahpC and kasA genes are mainly responsible for the isoniazid-resistance.116,

117

Mutation in katG gene diminishes the

activation of INH to its active form by reducing the ability of catalase-peroxidase118 whereas mutation in inhA gene results into the overexpression of wild-type enzyme through reduced affinity of the enzyme for NADH.119 Single point mutation in the rpoB gene (which codes for β-subunit of the RNA polymerase) is associated with rifampicin (2) resistance and this region is termed as “rifampicin resistance-determining region”, RRDR.117,

120

In rpoB, mutation

mainly occurs at 81-base pair spanning codons (between 507 and 533), and in most of the studies it was reported to occur particularly at 516, 526 and 531 codons, causing decrease in affinity for the drug which ultimately results into resistance to rifampicin (2). In some instances it was also reported to have double (at 526 and 531 codons) and triple (516, 526 and 531 codons) mutations for eliciting rifampicin drug-resistance.121 Pyrazinamide (3) resistance is mainly associated with the mutations in pncA, rpsA and panD genes and a majority of the reports evidenced the mutations occuring in pncA gene. Although mutations occuring in pncA gene are scattered throughout the gene, but most of them occurred in the open reading frame at 561-base pair region or in its putative promoter 82-base pair region.122-125



Page 28 of 102

Mutations in the embCAB operon genes (embA, B and C) are associated with the resistance to ethambutol (4).126, 127 Based upon the screening results of emb-clinical isolates it was proved that embB gene (576-base pair region) is a major contributor for the development of resistance to ethambutol (4).127, 128 In addition to these embCAB operon genes, mutations in Rv3792 and Rv380c have also been reported for the genesis of ethambutol-resistance.129, 130 Benzothiazinones (BTZs) evolved in 2009 as a newer class of antitubercular agents, having the potential of offering future drug candidates for the treatment of both MDR-TB and XDR-TB.43,

131

In a short period of time, BTZ scaffold has attracted lots of attention of

scientists and researchers throughout the world working in the field of anti-mycobacterials due to the submicromolar MIC values of its derivatives against M. tuberculosis (H37Rv strain). Pharmacological properties of these BTZs have been improved by synthesizing piperazine containing analogs (PBTZ) and one of the molecules, 40 from this class is currently in pre-clinical phase.109 The BTZs have been proved to act as covalent inhibitors of DprE1 enzyme, which plays crucial role in the synthesis of cell wall of the mycobacteria. Keeping in mind the future importance of this class of molecules, Foo et al.132 have characterized DprE1-mediated BTZs’ resistance in M. tuberculosis. The insights obtained from this study revealed that C387 residue of DprE1 acted as a site of mutations which led to the subsequent development of resistance to the BTZs. Overall five mutations on C387 residue [substitution with different amino acids such as glycine (G), alanine (A), serine (S), arginine (N) and threonine (T)] were proved to be responsible for BTZs’ resistance. Among these C387T, C387A and C387S mutations showed higher levels of resistance as compared to the C387N and C387G mutations. These results were corroborated further by in silico studies. In the concluding remarks the authors have stated that mutations at C387 residue were responsible for decreased potency of covalently binding DprE1 inhibitors to a greater


Page 29 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


extent whereas Ty38c mutations led to the development of resistance to the non-covalently binding DprE1 inhibitors. Some of the molecular mechanisms known till date by which bacteria develop resistance towards drugs like hydrolysis, acetylation, adenylation and phosphorylation have been well documented in the literature.133 To find out the exact mechanism of development of AMR, Warrier et al. took previously reported134 bactericidal (to the replicating Mycobacterium tuberculosis) molecule 46 for their study and revealed that N-methylation of the inhibitor molecules could be a prime process for development of antimicrobial resistance. In the initial screening, the authors scrutinized the role of different genes i.e. rv0560c, rv0558 and rv0559c through their overexpression as the possible contributing factors to the resistant phenotypic clones. Among these genes, overexpression of rv0560c contributed for 16-fold more resistance (than rv0558 and rv0559c) by the Mtb against the inhibitor molecule 46. According to the authors’ claim, rv0560c is a gene for S-adenosyl-L-methionine-dependent methyltransferase that methylates the inhibitor 46 at N-5 position. Compound 46 inhibits pure recombinant Mtb DprE1 with an IC50 value of 70 nM whereas in the presence of S-adenosylL-methoinine (SAM) the same compound showed 36-fold increase in IC50 value for DprE1 inhibition that clearly indicated that methylation of compound 46 markedly diminished its activity.135 O

O N

N N H

Me N

46



5.

Page 30 of 102

Development of DprE1 inhibitors High attrition rate in the discovery of newer antitubercular agents led to more

vigorous efforts in this direction. DprE1 has become one of the latest enzyme targets in the discovery for anti-TB drugs. Inhibitors which block the functioning of DprE1 have come in limelight only for the last few years. Researchers are striving tirelessly to develop DprE1 inhibitors as anti-TB agents. In the present review DprE1 inhibitors are broadly classified on the basis of their binding modes with the enzyme as Covalently and Non-covalently binding inhibitors, and are further sub-classified on the basis of the chemical scaffolds they possess such as benzothiazinones, azaindoles, benzothiazoles, benzoquinoxalines, dinitrobenzamides, pyrazolopyridines, and various other aromatic/heteroaromatic moieties that have been explored for the development of newer derivatives as DprE1 inhibitors.110 5.1 Covalently binding DprE1 inhibitors 5.1.1 Benzothiazinone based DprE1 inhibitors First report on the development of nitrobenzothiazinone (BTZ) as an active moiety that can block the arabinan synthesis in Mtb by inhibiting the DprE1 was made through combined efforts of Mollmann lab, Germany; Makarov lab, Moscow and Cole lab at the Global Health Institute, Ecole Polytechnique, Switzerland.43,

136

Synthesis of novel BTZs

with specificity for DprE1 enzyme in Mtb and selectivity for the virulent strain H37Rv over the other seven strains of Mtb was reported. Synthesis of the reported most active compound 42 is depicted in scheme 1. Compound 42 was found to be the most potent compound from the reported series exhibiting antibacterial activity against Mtb (H37Rv) with an MIC of 1 ng/mL compared to isoniazid (INH) MIC of 0.02 to 0.2 mg/mL and ethambutol (EMB) MIC of 1 to 5 mg/mL. Compound 42 caused cell lysis by targeting the cell wall biosynthesis. When tested for in vitro toxicity, compound 42 was found to be safer compared to INH and 30 ACS Paragon Plus Environment

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devoid of anatomical, behavioural and physiological toxicities. Radiolabelled studies have also been performed with the aim to determine its site of action, and it was found that it targeted the synthesis of arabinogalactan that played a crucial role in the formation of a covalent linker between mycolic acid and the peptidoglycan, thus inhibiting the process of cell wall synthesis.

OH HN

O

OH

H3C

O

CH3

HN

H2O

O

p-TsOH 47

S

CS2, NaOH

N O

Na S

48

49

NO2

NO2 Cl

F3C

CH3

O

Cl

SOCl2

COOH

Cl

F3C

50

O

51 Method B

NO2

Method A

NO2

NCS

F3C

NH3

KSCN or NaSCN

Cl

Cl

O 54

F3C

CONH2 52

48 CH3

49

CH3

O NO2 S

N N

F3C

O O

NO2 S

H2O/EtOH

F3C

O 42

N

O

S CONH2 53

Scheme 1. Synthesis of compound 8-nitro-2-(1,4-dioxa-8-azaspiro[4,5]decan-8-yl)-6trifluromethyl-1,3-benzothiazin-4-one 42 (BTZ043)



Page 32 of 102

Neres et al. have reported co-crystal structure of DprE1 obtained from Mycobacterium smegmatis, with inhibitor 42 (PDB 4F4Q).103 Compound 42 was found to be lodged in the substrate binding domain whereas the FAD was found in bound form having a conformation similar to that reported earlier. The compound 42 was observed having covalent bonding with the enzyme. Rest of the other interactions were physical through two water molecules present within the cavity of the active site. Intermolecular interactions of compound 42 were observed with His139, Gln343, Cys394 and Lys425 residues (Figure 6) leading to inactivation of the enzyme and thus inhibition of its activity. Lys425 is an important residue in this interaction as it plays a crucial role in the substrate binding. This finding is consistent with the fact that Lys425 interacts with the 2’-OH group in the substrate leading to oxidation of DPR; thus it is interpreted that mutation in this residue could lead to loss of DprE1 activity. This work also clarified the fact that compound 42 caused a time dependent inactivation of the enzyme unlike other derivatives. This has been verified from the experimental findings wherein it was observed that the nitro group in compound 42 was promptly reduced, leading to an irreversible interaction between Cys394 and the compound 42, causing inactivation of the enzyme. This type of reaction was not observed with other derivatives and this justified the difference in their activities. This study also demonstrated that dinitrobenzamides and nitroquinoxalines also acted as inhibitors of DprE1 but, their crystal structures have not yet been obtained.103, 104


Page 33 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 6. Crystal structure of M. smegmatis DprE1 complexed with FAD and covalently bound compound 42.103 Used with permission from Neres et al. Sci. Transl. Med. 2012, 4, 150ra121 [Copyright @ 2012, AAAS]. Nitro group in the BTZs is known to participate in the formation of covalent bond, but the role of CF3 and spiroketal groups is unclear. Gao et al.137 attempted to study the strucutre activity relationship of the benzothiazinones, for which they designed and synthesized Nalkyl and heterocycle substituted benzothiazinone (BTZ) derivatives with potent inhibitory activity against the Mtb strains H37Ra and H37Rv. BTZ derivatives (55a-55d) were synthesised to obtain compounds with high potency and better pharmacokinetic properties. It was observed that lengthening or branching of an alkyl chain or incorporation of heterocyclic rings as substituents in the BTZ scaffold enhanced the potency of the resulting compounds, and the trifluoro group played an important role in determining the anti-tubercular potency of the piperazine or piperidine analogs. Compounds 55c and 55d having spiropiperidine structures displayed MIC of 0.002 and 0.0001 µM respectively which were lower than or as good as that of compound 42. This rise in activity resulted due to introduction of sulfur in the azaspiro ring system. Compound 55d when subjected to pharmacokinetic evaluation demonstrated good bioavailability.



Page 34 of 102

NO2 R2

S N

R1

O 55a-55d

Compd

55a

55b

R1

MICa (µM)

R2

NO2

N

H37Rv

0.2

NT

0.4

NT

0.002

0.002

0.001

0.0001

CH3

NO2

O N

H37Ra

N

CH3 CH3 CH3

O 55c

CF3

O N S

55d

CF3

S N S

a

42

--

0.004

0.002

INH

--

0.8

0.2

Minimum Inhibitory Concentration, NT= Not tested

Further improvements in the lead compound 42 on the basis of SAR studies led to the synthesis of more lipophilic substituted 2-piperazino-benzothiazinone (PBTZ) derivatives.109 This series provided a variety of compounds with variations in substituents on the N-4 of piperazine ring, but it was found out that introduction of hydrophilic groups such as secondary or tertiary amines, alcohols or carboxylic acids resulted into decreased or loss of activity when compared to compound 42. Other derivatives bearing alkyl substituents on the N-4 position of the piperazine ring were having an added advantage of getting protonated at the amino nitrogen (N-4) of piperidine and possessing an enhanced hydrophobicity due to the alkyl substituents.


Page 35 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


NO2

N S

F3C

N N

O 40, 56-59

R Comp. 40 56 57 58 59

R Cyclohexylmethyl Cyclohexylethyl Heptyl 4-Phenoxybutyl 4-Phenylbutyl

Compound 40 (PBTZ169) was co-crystallized with the enzyme offering the crystal strucutre of the complex DprE1-40 (DprE1-PBTZ169) which provided an insight into the mechanism of inhibition of the enzyme by this benzothiazinone (PDB 4NCR). These compounds (40, 56-59) form adducts with Cys387 residue of the active site causing an irreversible inactivation of the enzymatic activity. Compound 40 is a congener of the previously reported series of BTZs. Compound 40 binds covalently to the enzyme through Cys387, leading to irreversible inactivation of the enzyme (Figure 7).

Figure 7. Crystal structure of M. tuberculosis DprE1 in complexation with compound 40, PBTZ169.109 Used with permission from Makarov et al. EMBO Mol. Med. 2014, 6, 372-383 [Copyright © 2014, John Wiley and Sons]. It was observed that the binding site of compound 40 was the same as that of compound 42. Water molecules were found to bridge hydrogen bond with Leu115 whereas compound 40 interacted with Trp111, Lys134, Phe199, Phe313 and Tyr314 residues. In vivo studies of the compound with zebra fish model were also reported to determine the efficacy



Page 36 of 102

on other mycobacterial infections such as Mycobacterium marinum. Compound 40 was found to be effective at 50 nM in this model proving it to be a highly effective compound in comparison to the standard drug INH. Compounds from PBTZ series were found to be more effective in vivo compared to the BTZs due to the introduction of a piperazine ring in their structures. Compound 40 dissolves faster in the acidic pH and gets absorbed faster compared to the BTZs, and this property is a superior attribute of the newly developed series. In order to improve the activity of PBTZ series Peng et al.138 reported a new series of 1,3benzothiazin-4-one substituted with 4-carbonylpiperazine on similar lines against the Mtb with MIC of 0.008 µM for several compounds. The synthesized compounds were evaluated biologically for the determination of their MIC against Mtb H37Ra. Compound 62 with trifluoromethylphenyl group showed higher solubility compared to compound 42. Compounds containing ortho and meta substituents on the phenyl ring showed somewhat better activity profile than the para substituted derivatives. Efforts were made by the authors to vary the substituents to obtain compounds with different ring sizes of cycloalkyls to admantyl type, 73-79. It was observed that compounds 73 and 74 showed rise in activity from 0.13 to 0.016 µM, but compound 75 having cyclopentyl group showed a dip in the potency with an MIC of 0.031 µM. But increase in the chain length and the ring size caused decreased aqueous solubility of the compounds. To solve the solubility problem, smaller substituents were employed to obtain compounds 80-87. Compound 80 showed improved activity at 0.016 µM with a more hydrophilic nature (clogP = 1.59). In case of compound 83, a rise in solubility to 42.5 µg/mL was obsereved. Compound 87 containing 1-methylpropyl substituent exhibited an MIC of 0.008 µM and a much higher solubility of 104 µg/mL compared to compound 40 (9.07 µg/mL) and 42 (0.94 µg/mL).


Page 37 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


O NO2

N S

R

N N

F3C

O 60-87 Compd

R

MIC (µM)

Compd

R

MIC (µM)

60

4-Nitrophenyl

0.063

74

Cyclobutyl

0.016

61

2-Nitrophenyl

0.13

75

Cyclopentyl

0.031

62

4-(Trifluoromethyl)phenyl

0.0131

76

Cyclohexyl

0.016

63

2-Methoxyphenyl

0.13

77

Cyclohexylmethyl

0.004

64

3-Methoxyphenyl

0.063

78

4-(tert-Butyl)cyclohexyl

0.031

65

4-Methylphenyl

0.031

79

Adamantyl

>0.13

66

4-Ethylphenyl

0.13

80

Methyl

0.016

67

4-tert-Butylphenyl

0.016

81

Ethyl

0.13

68

2-Fluorophenyl

0.008

82

Propyl

0.031

69

3-Fluorophenyl

0.016

83

Butyl

0.031

70

4-Fluorophenyl

0.016

84

(Methylthio)methyl

0.13

71

2-Chlorophenyl

0.13

85

tert-Butyl

0.031

72

4-Chlorophenyl

0.016

86

2-Methylpropyl

0.031

73

Cyclopropyl

0.13

87

1-Methylpropyl

0.008

Benzothiazinones 40 and 42 were extensively studied due to their impressive DprE1 inhibitory activity and selectivity as antitubercular agents. In an attempt to investigate the impact of oxidation of sulfur present in these compounds, the oxidation products 1,3benzothiazinone sulfoxide (BTZ-SO) 88 and 1,3-benzothiazinone sulfone (BTZ-SO2) 89 were prepared and studied in detail for their antitubercular activity and binding to the DprE1 enzyme.139 It is known that the BTZs and PBTZs are activated by the conversion of nitro group to nitroso group mediated by the cofactor FADH2, and the nitroso group subsequently 37 ACS Paragon Plus Environment


Page 38 of 102

reacts with Cys387 residue of the enzyme forming a semimercaptal adduct via covalent interaction. Earlier reports43 have mentioned that BTZs and other nitro derivatives undergo redox activation. Benzothiazinonesulphoxide (BTZ-SO) 88 and benzothiazinonesulphone (BTZ-SO2) 89 were obtained from BTZ on reaction with m-chloroperbenzoic acid (mCPBA) (Scheme 2).

CH3 NO2 S

N N

F3C O 42, BTZ

O

CH3

CH3

O

NO2 O S +

mCPBA 5 days

O

O N N

F3C

NO2 O O S

O

O

N

F3C O

O 88, BTZ-SO

N

89, BTZ-SO2

Scheme 2. Synthesis of BTZ-SO (88) and BTZ-SO2 (89) Compounds 42, 88 and 89 were subjected to evaluation of their antimicrobial and antitubercular activities on various strains, virulent and avirulent as well. It was found out that BTZ 42 and BTZ-SO 88 showed impressive activity against the mycobacterium but not against other bacteria, whereas BTZ-SO2 89 was observed to be poorly active. This study also proved that 42 and 88 were selective towards the mycobacterium. This understanding was further enriched by the work of Trefzer et al.140 demonstrating that DprE1 only reduced BTZ to the nitroso derivative which subsequently got bound to the Cys387 residue. DprE1 enzyme obtained from BTZ resistant strains reduced the nitro group to inert metabolites rendering the molecule inactive for further interaction with Cys387. These experimental results clearly established the mechanism of action of the BTZ derivatives. 5.1.2 Metabolism cascade for the BTZ molecule (42) Benzothiazinones (BTZs) are currently used as the main molecular template for the development of future antitubercular agents. BTZ is a prodrug in which nitro group is 38 ACS Paragon Plus Environment

Page 39 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


converted to nitroso, which on further reaction inhibits DprE1 enzyme. BTZ (42) was found to be a low nanomolar concentration DprE1 inhibitor in the in vitro studies hence it was subjected to in vivo studies. During the in vivo studies plasma samples were stored at room temperature for the purpose of obtaining varying drug concentrations and evaluating the bioactivity of the parent compound at different concentrations. This analysis showed enhancement in drug concentration as well as bioactivity of the parent compound than the original one. Both, the concentrations and the activity got increased proportionately as the storage period was extended. As the obtained results were quite puzzling the authors carried out metabolic studies of BTZ (42) to identify the probable metabolites of BTZ (42). On the basis of chemical reaction, formation of the probable metabolites were confirmed through UV, HPLC, NMR, LC-MS and HRMS analysis. The HRMS for the obtained metabolites showed addition of two hydrogens to the parent compound. It was speculated that the metabolites were formed by enzymatic reduction of BTZ molecule. Characterization of the metabolites (Meisenheimer complexs) through LC-MS analysis confirmed the formation of two isomers i.e. a major 90 and a minor one 91 (Scheme 3). The results obtained from the Me

Me

O NO2 S F3 C

N N

O BTZ (42)

O NaBH4, ACN/H2O rt, fast

H

O O N H

Me

O

N

F3C

O N O

O

N

S

O

H

O

N

S N

F3C

H H OH minor (unstable)

OH major

(m/z = 432.0835)

Me OH O N H H S

N

O O

N

F3C O 90

Me

O

N

H

OH N

S

O

N

F3C HH (m/z = 434.0988)

O

O 91

Scheme 3: Representation for the metabolism of BTZ 42 into 90 and 91. 39 ACS Paragon Plus Environment


Page 40 of 102

chemical reaction got correlated with the in vivo studies, confirming the formation of Meisenheimer complex 91 only. The enzymatic cascade involved in the formation of BTZ metabolite has also been evidenced. A similar metabolite was also obtained in the plasma samples of minipigs and rats which confirmed the presumed reductive enzyme present in the mammals.141 5.1.3 Benzothiazole containing DprE1 inhibitors Most of the benzothiazoles have been discovered on the basis of high throughput screening (HTS). This has been further supplemented by the work reported by Landge et al.106 who discovered some new benzothiazoles possessing antibacterial activity by screening of a database from AstraZeneca Pharmaceuticals drugs library consisting of more than 100,000 compounds. An initial screening criterion was to select compounds having single point activity against M. Bovis and M. Smegmatis at fixed concentrations of 8 µg/mL and 16 µg/mL respectively. Compounds with 80 % growth inhibition were selected in the next step. From this screening process benzothiazole N-oxide was chosen as the lead nucleus. Compound 30 was one of the most potent compounds found from this process with MIC value of 1 µg/mL. The lead compound 30 was further optimised for the development of newer derivatives (Scheme 4). The benzothiazole oxides (BTO) and benzothiazoles (BT) were found to be equipotent, however variations in activity were observed on substituting the second position of the benzothiazole nucleus.


Page 41 of 102 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


R1

NO2

R2

Cl

OCH3

a

O R3

SH

NO2 92a, b

F3C

93

NO2 S

O

N+ O-

OCH3

c F3C

S

O

N+ O-

R4

96-98

94

a: R1 = NO2; R2 = H; R3 = CF3 b: R1 = H; R2 = CH3; R3 = CF3 96: R4 = 1-(2-Methoxyethyl)-4-piperazinyl 97: R4 = Pyridin-2-ylmethylamino 98: R4 = N-Piperidinyl

b NO2 H3C

S

OCH3

F3C

N

O

d

R1

H3C

S

OCH3

F3C

N

O

95

c

R2

S

R4

R3

N

O

99

31, 100, 101 OCH3

N

NO2 S N+ O-

F3C

N

NO2 HN O

30 Mtb MIC = 0.02 µg/mL DprE1 IC50 = 0.026 µM

NO2

NO2

N

H3C

S

N

H3C

S

HN

H3C

S

N

F3C

N

O

F3C

N

O

F3C

N

O

31 Mtb MIC = 2 µg/mL DprE1 IC50 = 1.7 µM

101

100 Mtb MIC = 0.3 µg/mL DprE1 IC50 = 1.20 µM

Mtb MIC = 0.25 µg/mL DprE1 IC50 = 2.20 µM

Scheme 4. Synthesis of 5-trifluoromethyl-7-nitrobenzothiazole derivatives; Reagents and conditions: (a) TEA, EtOH, 0–25 ºC, 75–90%; (b) TEA, EtOH, reflux, 3–4 h, 35–50%; (c) amines, Et3N, CH2Cl2, rt, 12–16 h, 70–80%; (d) KNO3, H2SO4, 0–50 ºC, 90%. Biological activity of these derivatives was determined and they were further explored for their cytotoxicity, genotoxicity and CYP inhibition potential. The BTO (30, 96-98) and the BT analogs were found to be mutagenic. It was also observed that replacement of the nitro group with other substituents would render the molecule inactive. Compound 30 showed Mtb MIC of 0.02 µg/mL and DprE1 IC50 of 0.026 µM. Further, the authors thought of introducing steric hindrance around the nitro group (crowded benzthiazoles, 31, 100 and 101) which could halt the formation of reactive intermediates (responsible for mutagenic property) with the retenetion of DprE1 inhibitory activity. These crowded benzthiazoles (cBT, 31, 100 and 101) showed Mtb MIC values of 0.3, 2.0 and 0.25 µg/ml and significant



Page 42 of 102

DprE1 inhibitory activity with IC50 values of 1.20, 1.7 and 2.20 µM, respectively. Their lipophilicity was determined and it was found that these compounds displayed enhanced lipophilicity than the earlier reported compounds. They were also studied for their killing kinetics and it was reported that these molecules reduced the initial bacterial burden by 3161000 CFU/mL within a week. Compound 30 was co-crystallised with DprE1 enzyme (Figure 8). It was shown that compound 30 formed an adduct with the enzyme and caused prominent interactions with Cys387 residue. This was in agreement with the earlier proposed mechanism of action. The CF3 group occupied the same pocket as the one observed to be occupied in the case of BTZs and PBTZs, and it interacted with Phe369, Lys367 and His132 residues, indicating a similar mechanism of action.

Figure 8. Crystal structure of Mtb DprE1 in complexation with compounds 29, 30 and 40; (A) Active site details of the complex with BTO 30, (green). The residues of the small binding pocket for the CF3 group are shown as a light blue surface for simplicity. (B) Comparison of the binding mode of crowded benzothiazole (cBT) 29 (cyan, PDB 4P8H, unpublished), BTO 30 and PBTZ169 40 (light brown, PDB 4NCR). (C) 2Fo–Fc electron density map contoured at 1.0 sigma showing the refined Cys387–30 covalent adduct. Used with permission from Landge et al., Bioorg. Med. Chem., 2015, 23, 7694-7710 [Copyright © 2015 Elsevier].


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5.1.4 Triazoles as DprE1 inhibitors Triazoles in combination with benzothiazoles have shown their potential for antitubercular activity. The five-membered ring nucleus has been exploited further by researchers for its potential.142 As seen in many earlier cases, the HTS methods have offered many novel biologically active molecules. Stanley et al. reported several novel inhibitors on the basis of cell based HTS assay.27 A collection of already reported 20,000 antibacterial agents were screened against Mtb. A collection of 341,808 additional compounds were also screened against Mtb using 7H12 medium. A nitro substituted triazole 1-(4-(tertbutyl)benzyl)-3-nitro-1H-1,2,4-triazole 102 was found to possess good inhibitory activity against Mtb with an IC90 value of 0.5 µM. The nitro group of the molecule also interacted covalently with the enzyme leading to irreversible inhibition of the enzyme. The report suggested that compounds without a nitro group possessed weak or nil activity indicating that reduction of nitro group to nitroso or some other reactive species is required for binding to the target. To verify this fact, an experiment was conducted on five strains, with one wild type strain and the other four mutants without gene coding for Cys387 in DprE1 enzyme. It was reported that the wild type strain was inhibited by compound 102 but the mutants were not suppressed at all suggesting that irrespective of the type of nucleus, a compound must possess a nitro group for covalent interaction with DprE1 enzyme. CH3 CH3 CH3

N N O2N

N 102

5.1.5 Quinoxaline based DprE1 inhibitors Quinoxalines were discovered to be active against Mtb by Magnet et al. who reported an interesting experiment in which a library of kinase inhibitors containing 12,000 43 ACS Paragon Plus Environment


Page 44 of 102

compounds were screened by cell based assay against Mtb.143 Out of these, three compounds were found to be active at concentrations below 10 µM (Table 3). These compounds were found to be non-mutagenic as well as non-toxic. Compounds 103-105 from this finding were having a common quinoxaline pharmacophore and were found to be specific inhibitors of DprE1. Covalent interactions were obsereved with the enzyme leading to irreversible inhibition of the enzymatic activity which could be attributed to the nitro group present in the discovered molecules. Mechanism of action of these compounds is essentially the same as that of structurally analogous benzothiazinones. Table 3. List of substituents on the quinoxaline pharmacophore with DprE1 inhibitory activity. NO2

R3

N

R2

N

R1

103-105

Compound

R1

R2

R3

MIC (µM)

103

CH3

Phenyl

Br

3.1

104

CH3

Phenyl

CF3

0.75

105

Phenyl

CH3

CF3

6.25

5.1.6 Nitrobenzamide containing DprE1 inhibitors Discovery of nitrobenzamides is another example of sucessful application of HTS technique. Christophe et al.44,

144

carried out screening of a library of 56,984 synthetic

molecules based on the Lipinski’s rule of five for drug likeness and then screened the selected compounds for their antitubercular activity at a single dose concentration. The compounds found to be active (almost 486 compound) were further evaluated using the serial dilution method. Out of these 486 compounds, about 135 compounds were found to have MIC value less than 5 µM, and about 8 % had shown MIC below 1 µM, similar to isoniazid. Cluster analysis of these compounds revealed that 69 compounds had structural similarity to 44 ACS Paragon Plus Environment

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isoniazid and 24 compounds were having a common benzamide scaffold. Till then no reports were available on benzamides as DprE1 inhibitors hence these benzamides were further explored to obtain improved derivatives. Various compounds containing nitrobenzamide phenoxyethyl, alkyl or aryl substituents were synthesised as shown in Scheme 5.

O

O

R1 OH

N OH

O

N

ADDP, PPh3

1. Hydrazine 2.

O

O 106

R2

O Cl

1. R1 N

OH 2. LiAlH4

O R2

R1

H2N

O 2N

N H

O

109

O R1

110

O

O

Cl

O 2N

, TEA

OH

CH2Cl2, 0-rt, 1-2 h R2

111

O

OH O2N

R1

N

R2

OH

108

R2

NO2

HN

O Cl

NO2

107

Br

NO2

R1

112

N

R1 O

CH2Cl2, rt, overnight, ADDP, PPh3

R2

113

Scheme 5. Synthesis of 2-phenoxyethylbenzamides 108 and 3-phenoxypyrrolidin-1-ylphenylmethanones 113 Table 4. Various nitrobenzamides and derivatives with their activities. O O2N

N( ) n R2

Compd

R1

R2

n

O R1

123, 128, 129

MIC

Compd

R1

R2

n

(µM)

MIC (µM)

108a

3-Cl

NO2

0

1.0

108g

Ph

NO2

0

0.2

108b

3-Cl

NH2

0

>20

108h

4-OMe

NO2

0

0.2

108c

3-Cl

NHOH

0

>20

108i

2-F

NO2

0

0.7



Page 46 of 102

108d

3-Cl

H

0

>20

113a

4-OMe

NO2

2

20

108e

2-F

NO2

0

0.08

113b

4-COOMe

NO2

2

>20

108f

3-CF3

NO2

0

0.2

114

4-OMe

NO2

3

20

High content assay was used for the identification of active lead molecules, the synthesised derivatives were evaluated for their DprE1 inhibitory activity, and screened further in the animal model for their ability to reduce the bacterial burden. The compounds obtained from this series offered a range of IC50 values, and accordingly an SAR was framed from this study. It was observed that compounds with nitro group at 3 and 5 positions of the benzene ring possessed high activity, but reduction of the nitro group to hydroxylamine led to loss of activity. When the nitrogen of the amide group was substituted with benzyloxy or phenoxyethyl groups an improvement in activity was observed resulting in MIC values lower than 0.2 µM. Significance of this work lies in the fact that the cyclic benzamides showed MIC values below 80 nM but they lacked the potency in intracellular assay. Compound 108h was assessed for its activity within the primary macrophages. It was found to decrease the conventional CFUs by ten-folds in human and mouse primary cells at a concentration of 5 µM (Table 4). When this compound was screned for its activity on the MDR and XDR strains it reduced the bacterial load but only in the replicating and not in the non-replicating cultures. It was suggested that compound 108h acted by interacting with the Cys387residue of DprE1 thus, preventing cell wall synthesis by inhibiting the formation of DPA. This study offered 108h as the lead molecule with excellent in vitro activity.


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Figure 9. a) Compound 43 (CT325) within DprE1 active site (PDB ID 4FDN) and b) interaction of compound 44 (CT319) with the acive site residues forming H-bonds and van der Waals interactions. Compounds 43 (CT325) and 44 (CT319) were synthesised keeping in mind the structure of benzothiazinone 40 which interacted with the active site residues of DprE1.104 These compounds showed specificity towards Mtb DprE1 and were reported to have good NH2

NO H2O2

H N

F3C O

H N

F3C CH3

O

115

CH3

43 NO2

NO2 1-Phenylethanamine OH

F3C

H N

F3C O

O 116

CH3

44

Scheme 6. Synthesis of N-(1-phenylethyl)-5-trifluoromethylbenzamides 43 (CT325) and 44 (CT319).



Page 48 of 102

inhibitory activity. Compound 43 forms covalent linkage with Cys387 making it an irreversible inhibitor (Figure 9), whereas, compound 44 forms a noncovalent complex with the enzyme. The two compounds 43 and 44 were synthesized in single steps as shown in Scheme 6. 5.2 Non-covalently binding DprE1 Inhibitors 5.2.1 Benzothiazinone based DprE1 inhibitors In their continued efforts for the discovery of novel anti-TB agents Makarov et al.145 replaced the nitro group at 8th position of the benzothiazinone moiety with a pyrrole ring. This replacement resulted in a compound 119 retaining the activity of the BTZ ring system with an MIC of 0.16 µg/mL. Scheme 7 depicts the synthetic routes for the synthesis of pyrrole-BTZ derivatives. In vitro evaluation of these derivatives revealed their DprE1 inhibition at low concentrations similar to the earlier reported BTZ and PBTZ compounds. These non-nitro derivatives had an MIC around 0.16 µg/mL against the Mtb. The IC50 vaules were found to be 10 µM

DprE1 IC50 = 0.1 µM CYP2C9 IC50 = 0.1 µM

N

N H NH

S

O

N

S S

O

Ring 'A'

N

126 (TCA787) DprE1 IC50 = 1.8 µM

O O

Me

N

N H NH

S

O

Ring 'B' N

O

N H NH

Me

N

N H NH

S

O

N

O

N

N

Me

O N

N

N N

N N

127 (TCA582) DprE1 IC50 = 0.06 µM CYP2C9 IC50 = 1.83 µM

128 (TCA711) DprE1 IC50 = 0.03 µM CYP2C9 IC50 = >50 µM

Recently we have reported a series of benzothiazolylpyrimidinecarboxamides as Mtb DprE1 inhibitors.102 Several novel compounds were designed and synthesised as shown in Scheme 8. These compounds were evaluated for their antitubercular activity on the virulent strain H37Rv. It was observed that compounds with para substituents on the phenyl ring showed good inhibitory activity but the unsubstituted derivative showed better activity having an MIC of 0.08 µM and MBC of 7.7 µM.


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H N

NH2

S

NHSCN

N Bromine

NH2

NH2 129

S

130

131 X

Ethyl acetoacetate NaOH

X

H3C O S N

O N H H3C

p-TsOH

O

133a-d

NH

+

NH N 132

H2N

135(a-d)

Compd

O

S

NH2

S

N H

H

S 134

X

MIC (µM)

IC50(µM)

H37Rv

DprE1

135a

H

0.08

7.7±0.8

135b

F

0.09

9.2±1.5

135c

CF3

0.09

11.1±1.8

135d

N(CH3)2

0.08

10.3±2.6

Scheme 8. Synthesis of benzothiazolylpyrimidinecarboxamides 135 (a-d) 5.2.3 Quinoxaline based DprE1 inhibitors A series of quinoxaline derivatives (103-105) reported by Magnet et al.143 further inspired development of newer quinoxaline derivatives by Neres et al.107 Compound 33, a novel quinoxaline derivative was reported as a noncovalently binding non-competitive inhibitor of DprE1 having broad range of bactericidal activity. Phenotype screening of a library consisting of 266 compounds against Mtb was carried out. The lead compound 33 (3((4-methoxybenzyl)amino)-6-trifluoromethylquinoxaline-2-carboxylic acid) was found to be highly effective against the mycobacterium when evaluated using resazurin reduction assay (REMA). Three different compounds from this series were identified having MIC of about 3.1 µM (Scheme 9 and Table 5). SAR of this series revealed that 2-carboxy esters showed 53 ACS Paragon Plus Environment


Page 54 of 102

some activity but not a significant one. Absence of 6-trifluoromethyl moiety caused loss of activity and the presence of this group at para position of the C3 benzylamine group yielded compounds with significant DprE1 inhibition. It was obsereved that presence of 6trifluoromethyl group is necessary for activity and absence of this group caused loss of activity. Compounds with substitutents like methoxy in the phenyl ring of the benzylamino group showed moderate potency such as 33, 36 and 39. Compounds with halogens at the same position showed high to moderate potency like 32, 34 and 37, whereas compounds with nitrile substitutent on this postion showed poor activity as seen in compound 38 (Table 5). These compounds were found to be active against the replicating as well as non-replicating Mtb.

F3C

NH2

O EtO

OEt O

NH2 136

H N

F3C

a

O 137

138

F3C 39 141 142 143 144 145 146

139 5

F3C

6 7 8

4

N

3

N

2

1

F3C

Cl

N

OEt

N

O

140

O

c

H2N

F3C OH

R

O R

H N

O

OEt

N

R 4-OMe 4-CF3 4-F 4-OEt 3-F, 4-OMe 4-Cl 4-CN

b OEt

N

H N

Comp.

O

N

R

H N

d

O

32-38

N

OEt

O 39, 141-146

Scheme 9. Synthetic scheme for quinoxaline derivatives; Reagents and conditions: (a) Ethanol, reflux, 2 h; (b) POCl3, 70 °C, 5 h; (c) Ethanol, reflux overnight; (d) 1N NaOH, Ethanol reflux, 1 h.


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Table 5. Antimycobacterial activity and DprE1 inhibition potential of quinoxaline derivatives (32-39) Comp. R

MIC99 (µM) H37Rv

DprE1

H37Rv

H37Rv

Inhibition

rv3405c

∆rv3406

IC50 (µM)

mutant TRC4

dprE1 L368P

32

4-CF3

6.3

12.5

25

0.12

33

4-OMe

3.1

6.3

12.5

0.041

34

4-F

12.5

25

25

0.072

35

4-OEt

3.1

6.3

12.5

0.088

36

3-F, 4-OMe

6.3-12.5

12.5

25

0.080

37

4-Cl

3.1

12.5

25

0.050

38

4-CN

50

50

100

0.067

39

4-OMe

3.1

6.3

12.5-25

54 % (at 50 µM)

In the co-crystallization studies it was observed that compound 33 occupied the crystal lattice space similar to compound 40 in the DprE1 active site. Complexes of eight of the quinoxalines were studied and all these compounds were seen occupying the same space as occupied by the benzothiazinone 40. This study indicated that the 2-carboxy-6trifluromethyl-quinoxaline core takes the position near to the flavin ring of FAD with trifluoromethyl group housed into a hydrophobic pocket (Figure 11) formed by the residues Val365, Pro316, Cys387, Leu368, His132, Gly133, Lys367, Lys134, Ser22 and Phe369, similar to that shown by compounds 40 and 42.



Page 56 of 102

Figure 11. Crystal structures of DprE1 in complexation with 33 (Ty38c) and analogs. (A) Active site with 34 (Ty36c) bound in a planar conformation (monomer A in the asymmetric unit). (B) Superposed structures of 33 (Ty38c) and six analogs, in the conformations observed in monomer A.107 Used with permission from Neres et al., ACS Chem. Bio., 2015, 10, 705-714. [Copyright © 2015 American Chemical Society] 5.2.4 Thiadiazole as DprE1 inhibitor Batt et al.147 reported a combined use of phenotype screening and target based drug discovery approach leading to the discovery of a series of compounds with DprE1 inhibitory activity. Phenotype screening was carried out on a libraray of 177 compounds known to inhibit the Mtb. The screened compounds were subjected to enzymatic assay for Mtb DprE1 selectivity. Compound 147 ((1-(5-(1H-pyrrol-1-yl)-1,3,4-thiadiazol-2-yl)piperidin-4-yl)(4benzylpiperidin-1-yl)methanone) was found to be highly potent with an IC50 value of 0.054 µM and showed the highest binding affinity (Kd of 0.25 µM) from among the molecules of this series.


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N

N

S N

N N

O 147 IC50= 0.054 µM , Kd= 0.25 µM

5.2.5 Azaindole containing DprE1 inhibitors Several imidazolopyridines were reported to possess antitubercular activity. Compound 148 was reported to have an MIC of 0.017 µM but it lacked the desired potency level (MBC >200 µM) to be an effective anti-TB agent.148,

149

To improve the anti-Tb

activity, the scaffold was morphed in compound 149 leading to improvement in the MBC to 12.5 µM (Scheme 10). The 1,4-azaindoles were further improved by Shirude et al. wherein an MIC based SAR against the Mtb was framed by modulating the substituents on the 1,4azaindole ring 150.150, 151 They reported about twenty three compounds (Scheme 11) with the synthesis of final derivatives as 1H-pyrrolo[3,2-b]pyridine-3-carboxamides 154-157. The most active compounds from this series showed MIC in the range of 0.39 to 0.78 µM. OCH3

H 3C

H3C F N O

NH

morphing

Cl

N

N O

CH3 148 Mtb MIC = 0.017 µM Mtb MBC = >200 µM DprE1 IC50 = >10 µM

N

N

N

Scaffold

N N

N

N

NH

O

150

149 Mtb MIC = 6.25 µM Mtb MBC = 12.5 µM DprE1 IC50 = 0.014 µM

NH F

Mtb MIC = 1.56 - 3.12 µM Mtb MBC = 1.56 - 3.12 µM DprE1 IC50 = 0.010 - 0.017 µM

Scheme 10. Scaffold morphing from imidazolopyridine 148 to 1,4-azaindole 149 as the initial hit and compound 150 as the 1,4-azaindole optimized compound.



Page 58 of 102

These compounds were further studied on various Gram-positive and Gram-negative pathogens but were not found to be effective. On the basis of the obtained results this series was further optimised by synthesizing compound 157 containing dimethylamino group instead of methoxy on the pyrimidine ring. Compound 157 was found to possess IC50 of 0.032 µM against DprE1, Mtb MIC range of 0.5-1.56 µM and Mtb MBC range of 1.56-3.12 µM. Compound 157 was further subjected to in vitro, ex vivo and in vivo studies and was found to be far more superior to the earlier reported compounds. However, these compounds 154-157 suffered from two drawbacks, they inhibited the PDE6 protein complex which is necessary for the normal functioning of the human eyes and possessed suboptimal pharmacokinetic profile.

R1 N

Cl

NH2

R1 + H3C

151

R2

R2

O

NO2

O

N

O O

CH3

R3

152

R1

N

T3P, Et3N

N

Several steps

n

N

OH

O

154-157

153

N N

H3C

OCH3

H3C

NH

N

N

H3C

OCH3

H3C

N

N O

OCH3

H3C N

H3C

N H3C

N

N

N

N

H3C

N

NH

N

N

NH

O

O

Mtb MIC = 0.39 - 1.56 µM Mtb MBC = 0.78 - 1.56 µM DprE1 IC50 = 0.005 - 0.015 µM

N CH3

N O

154

n R3

N H

O

F 155 Mtb MIC =

An Overview of the Development of DprE1 Inhibitors for Combating the

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