Bioorganic & Medicinal Chemistry Letters 24 (2014) 870–879

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Bioorganic & Medicinal Chemistry Letters

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Thiazolopyridone ureas as DNA gyrase B inhibitors: Optimizationof antitubercular activity and efficacy

0960-894X/$ - see front matter � 2014 Elsevier Ltd. All rights reserved.http://dx.doi.org/10.1016/j.bmcl.2013.12.080

⇑ Corresponding author. Tel.: +91 80 23621212x6274; fax: +91 80 23621214.E-mail address: Sandeep.ghorpade@astrazeneca.com (S.R. Ghorpade).

� Current affiliation: Medicines for Malaria Venture (MMV), Switzerland.� Current affiliation: Dr. Reddy’s Laboratories, Hyderabad, India.§ Current affiliation: Quintiles, Bangalore, India.– Current affiliation: Agilent Technologies, Hyderabad, India.k Current affiliation: Vipragen, Mysore, India.€ Epizyme, Inc., Cambridge, MA, USA.

�� Equal contributors.

Ramesh R. Kale a,��, Manoj G. Kale a,��, David Waterson a,�, Anandkumar Raichurkar a, Shahul P. Hameed a,M. R. Manjunatha a, B. K. Kishore Reddy a, Krishnan Malolanarasimhan a,�, Vikas Shinde a,Krishna Koushik a, Lalit Kumar Jena a, Sreenivasaiah Menasinakai a, Vaishali Humnabadkar b,Prashanti Madhavapeddi b, Halesha Basavarajappa b, Sreevalli Sharma b, Radha Nandishaiah b, K. N.Mahesh Kumar c,§, Samit Ganguly c, Vijaykamal Ahuja c,–, Sheshagiri Gaonkar c,k, C. N. Naveen Kumar c,Derek Ogg d, P. Ann Boriack-Sjodin e,€, Vasan K. Sambandamurthy b, Sunita M. de Sousa b,Sandeep R. Ghorpade a,⇑a Department of Medicinal Chemistry, AstraZeneca India Pvt. Ltd, Bellary Road, Hebbal, Bangalore 560024, Indiab Department of Biosciences, AstraZeneca India Pvt. Ltd, Bellary Road, Hebbal, Bangalore 560024, Indiac DMPK and Animal Sciences, AstraZeneca India Pvt. Ltd, Bellary Road, Hebbal, Bangalore 560024, Indiad Discovery Sciences, AstraZeneca, Alderley Park, Macclesfield, UKe Biosciences, Infection iMed, AstraZeneca, Waltham, MA, USA

a r t i c l e i n f o

Article history:Received 7 October 2013Revised 5 December 2013Accepted 19 December 2013Available online 25 December 2013

Keywords:TuberculosisDNA gyraseThiazolopyridone ureasGyrB

a b s t r a c t

Scaffold hopping from the thiazolopyridine ureas led to thiazolopyridone ureas with potent antitubercu-lar activity acting through inhibition of DNA GyrB ATPase activity. Structural diversity was introduced, byextension of substituents from the thiazolopyridone N-4 position, to access hydrophobic interactions inthe ribose pocket of the ATP binding region of GyrB. Further optimization of hydrogen bond interactionswith arginines in site-2 of GyrB active site pocket led to potent inhibition of the enzyme (IC50 2 nM) alongwith potent cellular activity (MIC = 0.1 lM) against Mycobacterium tuberculosis (Mtb). Efficacy wasdemonstrated in an acute mouse model of tuberculosis on oral administration.

� 2014 Elsevier Ltd. All rights reserved.

Tuberculosis (TB) continues to remain one of the major causesof death, claiming close to 1.5 million patients per year.1 The stan-dard treatment is six months long and for drug-resistant TB, thisextends to 18–24 months. There is a high medical need for a short-er, better tolerated regimen for the treatment of drug-sensitive TB,as well as a combination therapy that will be more effective in thetreatment of drug-resistant TB. Cure rates for patients with exten-sively drug resistant (XDR) TB are very low and the advent of

totally drug resistant TB (TDR) leaves very little hope for patientsinfected with such strains.2–4

A new drug must have a novel mechanism of action to beeffective against drug resistant forms of TB. DNA gyrase main-tains the topological state of DNA in the bacterial cell, which isimportant for transcription. A second topoisomerase, toposiomer-ase IV, decatenates DNA and plays a critical role in DNA replica-tion and cell division. M. tuberculosis (Mtb), however, lackstopoisomerase IV and the function of decatenation is performedby DNA gyrase,5 making Mtb DNA gyrase a very attractive drugtarget. DNA gyrase is also clinically validated as a target for thediscovery of new antibacterial agents since the fluoroquinolones,which target this enzyme, are used extensively for treatment ofbacterial infections and are the mainstay of second line treatmentof TB.6 However, the emergence of resistance to the fluoroquino-lones may limit their effectiveness in the long term.7 The fluoro-quinolones inhibit DNA cleavage and reunion, one of the twoactivities present in DNA gyrase. A second mechanism, hitherto

N

N

NH

NNH

O

F

NH

1a 1b

N

N NH

NH2

O

F

O NN

1c

N

S N NH

O

NH

N

NH

Cl Cl

N

S

N

OOH

O

HN

F

N

NHN

NN

ClF

O

OH

1d

FFF

N

NN

N

NNH

ONH

1e

NHNNH

ON

N

1f

F FF

N

NNH

OO

1gN NH

O

NH

SN

FF

F

N

NHN

OO

1i

N

N

N S

NNH

ONH

1h

OO

N

N

NHCl

N S

N

N

A

H

HH2N

N

N

S

NNH

ONH

1j 1k

Figure 1. Known GyrB inhibitors.

1h

N

N S

NNH

ONH

OR

2Access to ribose pocket

1

2345

6

7N

N

N S

NNH

ONH

O

O

1

2

34

5

6 7

Substituents into the ribose pocket

Figure 2. Overlay of ATP (yellow) and thiazolopyridone 2 (cyan) on to the crystal-bound conformation of thiazolopyridine 1h (green) in Spn-ParE (PDB ID: 4mb9).15b

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879 871

underexploited, is the ATPase activity of DNA gyrase whichresides in the GyrB subunit. The ATPase activity can be uncoupledfrom the supercoiling activity, which needs both GyrA and GyrB

subunits, and measured in isolation. Resistance studies to datehave indicated that there is little likelihood of cross resistancebetween inhibitors of the ATPase and the fluoroquinolones8

N

N

SNH

ONH

O

N

N

NH

N

SNH

ONH

O

N

N

3 4

a

Scheme 1. Synthesis of thiazolopyridone urea 4. Reagents and conditions: (a) 35%HBr, 100 �C, 4 h, 20%.

Table 1Comparison of thiazolopyridone 4 with thiazolopyridine 3

Properties 3 4

Msm_GyrB ATPase IC50 (nM) 22 84Mtb_MIC (lM) 2 9

872 R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879

providing an opportunity for drugs effective against fluoroquino-lone-resistant bacteria.

Multiple scaffolds targeting the ATPase activity of DNA gyrasehave recently been reported for example benzimidazole ureas(1a),8 benzthiazole ureas (1b),9 pyrazolethiazoles (1c),10 pyrrola-mides (1d),11 pyridine ureas (1e),12 anilinopyrimidines (1f),13 imi-dazolopyrimidines (1g),14 thiazolopyridine ureas (1h),15 azaindoles(1i),16 pyrrolopyrimidines (1j),17 pyrazinamides (1k).18 Some ofthese, such as the benzimidazole ureas,8c pyrrolamides,11c thiazol-opyridine ureas15 and pyrazinamides18 are reported to have potentactivity against Mtb (Fig. 1).

Here is described the development of a novel series of com-pounds, thiazolopyridone ureas (2 in Fig. 2), that inhibit the ATPaseactivity of Mycobacterium smegmatis (Msm) DNA gyrase with IC50sin the nM range. The compounds inhibit the growth of Mtb both

5 6

10

N

N

SNH2

RO

SNC

NR

O

Het

NR

O

Br NH2

N

NO2

RO

Br

NH

NO2

O

Br

O

Bgf

b

Route B

Route A

a

5

N

NO2

RO

Br

NR

O

Het

3

d

11

Scheme 2. Synthesis of N-4 substituted thiazolopyridone ureas. Reagents and conditioNaHCO3, DME–Water (4:1), 100 �C 4–12 h, or microwave 110–120 �C, 30 min, 60–70%; (50–60%; (e) THF–Toluene (1:1) or DME, ethyl isocynate, Et3N, catalytic dibutyltinoxide, mto 60 �C, 60–90%; (g) (i) Thiocarbonyldiimidazole, Acetonitrile rt, (ii) 7 M ammonia in met20 min, 21%.

in vitro and in vivo demonstrating potential for the compounds tobe progressed towards a candidate drug for the treatment of tuber-culosis. The series was initially explored as a back-up scaffold forthe thiazolopyridine ureas.

Co-crystallization of compounds with Msm or Mtb GyrB wasgenerally unsuccessful. In a couple of cases structures were ob-tained but the crystals took very long to appear, making the longturnaround time impractical from a chemistry perspective. TheGyrB active site is highly conserved across bacterial species andis very similar to that of ParE, the equivalent subunit of topoiso-merase IV.15b As well, the existence, inhouse, of a high throughput,crystallisation system for Spn ParE prompted the use of this systemas a surrogate for Mtu GyrB. Also the data from the thiazolopyri-dine ureas indicated the Spn ParE co-crystal information could beapplied to the optimization of Mtb GryB inhibitors.15b Hence, thecrystal structure of thiazolopyridine urea 1h in complex withStreptococcus pneumoniae (Spn) ParE (PDB ID: 4mb9)15b wasexploited to build a hypothesis for scaffold hopping (Fig. 2). Withthe thiazolopyridine ureas, direct access to the ribose pocket inthe ATP binding site of DNA gyrase is blocked by the N-4 in thethiazolopyridine ring; hence substituents were typically extendedvia the C-5 position to access interactions at the interface of the ri-bose binding and solvent pockets. Modification of thiazolopyridinecore to thiazolopyridone provided an opportunity to extend sub-stituents into the ribose pocket directly from the thiazolopyridone4-N position while keeping other critical interactions similar tothose in the thiazolopyridine core (Fig. 2).

The initial test compound, thiazolopyridone urea 4 with unsub-stituted 4-NH, was synthesized by demethylation of 6-methoxy-thiazolopyridine urea 3 (Scheme 1). Compound 4 retainedmycobacterial GyrB inhibition (IC50s were measured inMycobacterium smegmatis (Msm) DNA GyrB ATPase inhibition

e

7 8

9

12 13

N

SNH

ONH

N

N

SNH

ONH

RO

Br

N

NO2

RO

Het

N

NH2

RO

Het

N

N

SNH2

RO

Het

N

HN S

NH2

R

r

N

N

SNH2

RO

Br

d

c

h

15-32

N

SNH

ONH

3-44

b

14

e

ns: (a) R-Br or R-OMs, DMF, K2CO3, 40–80%; (b) Het-B(OH)2, Tetrakis Palladium,c) Pd/C, ethanol, rt, H2 40 psi 1–2 h, 80–95%; (d) KSCN, Br2, AcOH 10 �C to rt, 3–4 h,icrowave 110 �C, 30 min, 60–70%; (f) SnCl2, Ethanol, or Zn, NH4Cl, Acetone/water, rthanol; 60–70%; (h) Acetic acid, NBS 50 �C, 50–60%; (i) Et3N, DMF, microwave 140 �C,

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879 873

assay.15,18 The Msm enzyme was used instead of the Mtb enzymedue to the much higher specific activity of the Msm protein;manuscript in preparation) and MIC on Mtb although it wasslightly less potent than the thiazolopyridine analogue 3 (Table 1).Encouraged by this finding, N-substituted thiazolopyridone ureaswere explored in order to improve enzyme potency by accessinginteractions in the ribose pocket of GyrB.

Initially the thiazolopyridone ureas, substituted with variousalkyl groups at N-4 position, were synthesized via route A, asdepicted in Scheme 2. The 3-bromo-5-nitropyridone 5 wasalkylated by reacting it with the corresponding alkyl bromide ormesylate in the presence of Na2CO3 with good yields (60%). Thealkylated pyridone intermediate 6 was subjected to Suzuki cou-pling with 3-pyridine boronic acid to yield intermediate 7. Reduc-tion of the nitro group on intermediate 7 was carried out underhydrogenation conditions using Pd/C as a catalyst to prepare the5-aminopyridone derivative 8. This was treated with potassiumthiocynate and Br2 to produce the thiazolopyridone amine 9 in

Figure 3. (a) X-ray co-crystal structure showing key binding interactions of compound 2pose of compound 21 in Msm-GyrB ATP binding site (PDB ID: 4b6c).18

moderate yield. Thiazolopyridone ureas (15–32) were obtainedby heating the corresponding thiazolopyridone amine 9 with ethy-lisocynate at 110 �C in a microwave.

Since the above route was linear, it was unsuitable for rapidanaloging to vary heterocycle substituents at the thiazolopyridoneC-6 position. Also some heterocycles, for example the pyridones,reacted with bromine during the cyclization step. Initial attemptsto react 5-amino-3-bromopyridone 10 with potassium isocyanateto obtain the desired thiazolopyridone amine 13 were unsuccessfuland yielded thiazolopyridone amine 11 as a major product, where-in bromine was replaced with thiocyanate. Hence a two step pro-tocol was followed for the formation of thiazolopyridone amine13, as shown in route B. In this method, the thiourea 12 wasformed by reacting amine 10 with thiocarbonyldiimidazole to formthiocyanate, followed by treatment with ammonia. Thiourea 12was cyclized to thiazolopyridone amine 13 by heating with N-bromosuccinamide in acetic acid. Thiazolopyridone amine 13 wasconverted to urea 14 by heating with ethylisocyanate at 110 �C

1 with selected Spn ParE ATP-binding site residues (PDB code: 4MOT). (b) Docking

874 R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879

in a microwave. Intermediate 14 was then subjected to Suzuki cou-pling with a variety of heterocyclic boronic acids or boronates toyield various thiazolopyridone ureas 33–44.

A variety of substituents extending from the thiazolopyridoneN-4 position, to access the interactions with residues in the ribosepocket of GyrB, were screened for enzyme inhibition and Mtb MIC(Table 1). Even though compound 15, with a methyl group at N-4position, did not improve the IC50 compared to the unsubstitutedanalogue 4, larger alkyl chains like ethyl, isopropyl, cyclopropylm-ethyl and difluoroethyl (Table 1, compounds 16–19) were well tol-erated at this position, leading to marginal improvement inenzyme inhibition and Mtb MIC compared to compound 4. Furtherincreasing the alkyl chain length to isobutyl, isopentyl (compounds20–22) or introduction of bulkier, liphophilic groups like cyclo-hexylmethyl (compound 23) retained potent enzyme inhibitionbut Mtb MICs got weaker, or were lost. But introduction of O inthe cyclohexyl ring resulted in potent Mtb MIC (compound 24).This may be due to the difference in permeation of these com-pounds across the Mtb cell wall, which may be correlated to lipo-philicity of these compounds (refer to Fig. 5b and the related

Table 2Exploration of substituents at thiazolopyridone N-4

NR

O

N

Compounds R Msm_GyrB IC50 (nM) MIC (lM)

Mtb Sauc

4e H 84 9 100

15f * 150 >16 NDh

16 * 30 2 46

17*

40 2 18

18 * 40 1.4 NDh

19 *F

F

40 3 21

20 * 25 8 NDh

21g

*50 10 13

22*

40 7.3 NDh

23*

20 >16 NDh

24*

O14 3 NDh

25 *OH

O2870 >21 NDh

26 *NH2

O107 21 NDh

a Predicted logD using an AstraZeneca prediction tool.b PPB: plasma protein binding.c Sau: Staphylococcus aureus ARC517.d Spn: Streptococcus pneumoniae ARC548, both clinical strains from the AstraZeneca ce 5-Pyrimidine at thiazolopyridone C-6 position.f 6-Fluoro-3-pyridine at thiazolopyridone C-6 position.g Allyl urea derivative.h Not determined.

discussion regarding correlation of predicted logD and MIC). Intro-duction of a carboxylic acid at the N-alkyl region (compound 25)resulted in deterioration of enzyme potency with consequent lossof Mtb MIC. The potency was regained by converting the acid toamide (compound 26), but, even so, the compound had weaker po-tency compared to compounds with hydrophobic side-chains.Overall, substitutions at the N-4 position, by taking advantage ofhydrophobic interactions, were useful in improving the potencyof the compounds.

In the crystal structure of compound 21 in complex with SpnParE (PDB code: 4MOT; binding mode shown in Fig. 3a) it is clearthat the hydrogens of the urea make hydrogen bonds (HB) withthe conserved Asp78. The thiazole ring nitrogen makes awater-mediated HB with Asp78 and Thr172. The pyridine ring atthiazolopyridone C-7 position makes typical p-stacking and HBinteractions with Arg81 and Arg140. The isopentyl alkyl chainattached to N-4 of pyridone binds in the region occupied by theribose/phosphate functional groups of ATP.

Docking studies were performed using Msm GyrB crystal struc-ture (PDB ID: 4b6c)18 and GLIDE and FRED as docking methods

N

SNH

ONH

AZ logD7.4a Hu_PPBb (% free) Solubility pH7.4 (lM)

Spnd

1.6 0.94 NDh NDh

NDh 2.04 NDh NDh

0.8 2.19 11 94

1.6 2.53 21 45

NDh 2.72 5 5

1.3 2.24 21 28

NDh 2.94 7 21

0.4 3.58 3 NDh

NDh 3.17 6 NDh

NDh 3.61 2 7

NDh 2.09 14 25

NDh �0.64 22 7292

NDh 1.36 25 NDh

ollection.13

Table 3Exploration of substituents at thiazolopyridone C-6

N

N

SNH

ONH

O

Het

Compounds Het Msm_GyrB IC50 (nM) MIC (lM) AZ logD7.4a Hu_PPBb (% free) Solubility pH7.4 (lM)

Mtb Sauc Spnd

17

N

*40 2 18 1.6 2.53 21 45

27

N

N*

23 2 22 0.7 1.75 15 7

28

N

*F10 11 NDe NDe 2.65 10 73

29N

*

O23 10 NDe NDe 3.15 5 NDe

30

N

*O6 4 NDe NDe 2.77 8 <5

31

N

*

NC15 21 NDe NDe 2.56 4 NDe

32

N

*NC4 4 NDe NDe 2.2 10 <5

33

*

N

NNN

N <2.5 0.2 1.6 <0.2 2.31 8 2

34N

NNN

N

*

20 1.6 >200 1.6 2.30 7 3

35 N

O

*<2.5 0.2 50 0.4 2.05 42 24

36 N

O

*5 0.5 25 0.4 2.32 7 120

37N

O

*

O5 0.3 50 0.4 1.84 26 42

38 N

O

*

O2.5 0.3 25 0.4 2.17 14 121

39 N

OO

*

175 19 50 >200 1.78 7 577

40

*

N

NN

8 0.6 9 <0.2 2.28 10 17

41

*

N

NN

11 1.3 13 <0.2 2.38 20 12

(continued on next page)

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879 875

Table 3 (continued)

Compounds Het Msm_GyrB IC50 (nM) MIC (lM) AZ logD7.4a Hu_PPBb (% free) Solubility pH7.4 (lM)

Mtb Sauc Spnd

42

*

N

NN

N20 3 >200 6 1.88 18 3

43N

NN

*

16 0.8 3 <0.2 2.32 14 2

44

*

N

NN

217 12.5 >200 3 1.65 26 6

a Predicted logD using an AstraZeneca prediction tool.b PPB: plasma protein binding.c Sau: Staphylococcus aureus ARC517.d Spn: Streptococcus pneumoniae ARC548, both clinical strains from the AstraZeneca collection.13

e Not determined.

876 R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879

(Fig. 3b). HB-interactions and p-stacking similar to those in SpnParE are seen with structures docked into Msm GyrB, Asp79 beingequivalent to Spn Asp78 and Arg82, Arg141 of Msm equivalent toSpn Arg81, Arg140, respectively (Fig. 3b). The Msm active site inthe region surrounding the thiazolopyridone N-4 is hydrophobicand acidic explaining why hydrophobic substituents on the com-pound confer best enzyme inhibition, and suggesting the weakinhibition of compound 25 (vs 16, 17) is due to the acidic carbox-ylic group being repelled out of the protein (Fig. S1 in Supportinginformation). Docking of compounds into the Msm GyrB active sitecould be used to explain the SAR for Msm GyrB ATPase inhibitionand to design new molecules.

Based on the binding mode, HB-interactions of the heterocycleat thiazolopyridone C-6 with Arg141 and Arg82 in the GyrB activesite pocket appear to be critical interactions for GyrB inhibition.Hence it was hypothesised that optimization of these interactionswould lead to improved enzyme inhibition and might improve MtbMICs. Several substituents on the pyridine ring were explored and,in addition, the pyridine itself was replaced with other aromaticheterocycles. Some examples are presented in Table 3. In general,compounds with pyrimidine at C-6 had more potent MICs com-pared to pyridine analogues without a significant change in GyrBIC50 (compare 17 with 27 in Table 2). The reduced liphophilicityof the pyrimidine analogues compared to the pyridine analoguesmay be responsible for improved Mtb MIC, as described in the sec-tion on correlation between predicted logD and MIC and illus-trated graphically in Figure 5. Addition of substitutions like F, CN,and OCH3 on the pyridine ring led to some improvement in GyrBIC50 without any improvement in Mtb MIC (Table 2, compounds28–32). However, addition of 2-methyltetrazole on the C-6 posi-tion of the pyridine ring significantly improved enzyme inhibitionas well as Mtb MIC (Table 2, compound 33). Interestingly, com-pound 34 with 1-methyltetrazole on pyridine C-6 had an inferiorIC50 as well as MIC compared to compound 33 with the 2-tetrazolylisomer. This could be explained by docking into Msm GyrB ATPbinding site (Fig. 4a). The position of the methyl group on the tet-razole was found to influence the orientation of the tetrazolegroup, affecting the distance from Arg141. In compound 33, witha more potent IC50, the N-1 of tetrazole is able to form a HB withArg 141, whereas in compound 34, the 1-methyltetrazole is ori-ented away from Arg141 failing to form a HB with it. Similarly,4-pyridones at the thiazolopyridone C-6 position brought in signif-icant improvement in the potency as seen in compounds 35–38whereas the 3-pyridone analogue 39 was much inferior. In the

matched pair 37 and 39 (Fig. 4b), the carbonyl group of 4-pyridonein 37 makes HB interactions with Arg141, allowing closer interac-tions of urea hydrogens with Asp79. But in the weaker inhibitor,39, the carbonyl of 3-pyridone is exposed to solvent and isdeprived of these interactions. Some N-containing bicyclic hetero-cycles also had good potency (compounds 40–44), but it seemsthat the alignment of nitrogens at the bridge-head was critical. Incompound 43, the 1,2,3-triazolopyridine ring can form HB withArg141 whereas the 1,2,4-triazolopyridine in compound 44 is un-able to form a HB with Arg141 leading to a 40-fold loss in enzymepotency (Fig. 4c).

The SAR and modeling observations suggest that the potency ofinhibition of Msm-GyrB ATPase by the thiazolopyridones is signif-icantly influenced by the strength of cation-Pi stacking and HBinteractions with Arg82 and Arg141, respectively. As well, aliphaticinteractions at the ribose and phosphate binding regions of GyrBand the warhead interactions of the urea hydrogens with Asp79and aliphatic interactions with its surrounding residues are criticalfor the potent inhibition.

The MIC of the series on Mtb correlated well with the IC50 onMsm GyrB (Fig. 5). This is not very surprising, since the Msm andMtb enzyme active sites are well conserved; the IC50 for inhibitionof Mtb DNA gyrase in a supercoiling assay19 was very similar tothat on Msm GyrB ATPase (Table 4). In general, GyrB IC50s below100 nM (pIC50 7) were required to attain Mtb MICs below 1 lM(pMIC 6) and more potent MICs were obtained only below IC50 of10 nM (pIC50 8). However, several compounds with quite potentIC50 had moderate to poor MICs, suggesting access to the enzymewas compromised by poor permeation of the compounds intothe cell or efflux of compounds out of the cell. In general, the com-pounds with predicted log D in the range of 1.5–2.5 (green circles)showed reasonable translation of enzyme inhibition into Mtb MICwhereas compounds exceeding this range either lacked MIC or hadweaker MIC. This correlation was useful in the design of newcompounds.

Some compounds were screened for MIC against Staphylococcusaureus (Sau) and Streptococcus pneumonia (Spn) (Tables 2 and 3).The compounds were mostly equipotent on Spn; but showed onlymoderate MICs against Sau.

In general, these compounds showed good plasma free fractions(human PPB >10% free, Tables 2 and 3). This was mainly influencedby the nature of the substituent at the N-4 position of the thiazol-opyridone ring. The compounds with either smaller hydrophobicsubstituents like ethyl, isopropyl, difluoroethyl (Table 2;

Figure 4. Comparison of docking poses of compounds in the Msm-GyrB ATP binding site. (a) 33 vs 34, (b) 37 vs 39, (c) 43 vs 44.

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879 877

Figure 5. Scatter plot of Mtb pMIC vs Msm GyrB pIC50; pIC50 = �log10(IC50) and pMIC = �log10(MIC) wherein IC50 and MIC are in molar units; colour by calculated logD(AZlogD7.4 was calculated using an AstraZeneca model for logD predictions).

0

1

2

3

4

5

6

7

pre treatment

Untreated/ vehicle

Isoniazid-3 mg/kg

Compound 17-100 mg/kg

Compound 17-300 mg/kg

Compound 17-150 mg/kg bid

Log 1

0 CFU

/Lun

g

Figure 6. Efficacy of compound 17 in an acute mouse model of tuberculosis.22 Theresults of the 300 mg/kg QD dose and the 150 mg/kg bid dose are not statisticallydifferent from each other, resulting in either stasis or a 0.5 log kill vs the number ofbacteria at the start of treatment, respectively. The effect with the 100 mg/kg QDdose was less than with the other two doses and showed retarded growth of Mtb vsthe untreated control group.

Table 4IC50 for inhibition of Mtb DNA gyrase in a supercoiling assay

Compound Msm_GyrB IC50 (nM) Mtb_DNA gyrase_supercoiling IC50 (nM)

17 40 5135 <2.5 30

878 R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879

compounds 16, 17 and 19) or substituents with polar heteroatoms(Table 2; compounds 24–26) at the N-4 position have better plas-ma free fractions (human PPB >10% free) compared to those withlarger liphophilic N-4 substituents like isobutyl, isopentyl, cyclo-hexyl (compounds 20–23, human PPB 2-7% free). But most of thesecompounds exhibited only poor to moderate aqueous solubility(5–121 lM) except for acid derivative 25 presumably due to thehighly crystalline nature of these compounds (melting points>250 �C for most of the compounds). Interestingly, some of thesecompounds showed improved solubility in fed state simulatedintestinal fluid (FeSSIF,20 pH 6.5). For example compound 17 hasFeSSIF solubility of 193 lM compared to 42 lM in phosphate buf-fer at pH 7.4. This could contribute to the plasma exposuresobserved on oral dosing.

Compound 17 showed excellent plasma exposure when dosedorally in mice (AUC 981 lM.h at a dose of 300 mg/kg). It was testedfor in vivo efficacy in an acute infection model21 of tuberculosis inmice (Fig. 6). The lowest dose (100 mg/kg) retarded the growth ofbacteria compared to the untreated mice (vehicle control). Thecompound showed a dose response; a dose of 300 mg/kg QD re-sulted in stasis — that is number of bacteria similar to that of thepre-treatment control, while a dose of 150 mg/kg, bid, resulted ina 0.5 log kill vs the number of bacteria in the pre-treatment con-trol. The results of the 300 mg/kg QD and 150 mg/kg bid dosesare not statistically different from each other (Bonferroni’s Multi-ple Comparison Test). Isoniazid at 3 mg/kg caused a 0.87 log dropin cfu vs the pre-treatment control.

In conclusion the thiazolopyridone ureas, which evolved byscaffold hopping from the thiazolopyridine ureas, show potentantitubercular activity and are active in vivo in a murine modelof tuberculosis. The series has good free fractions in human plasmaprotein binding assays with 42% free as the best value. This is

important, since most antibacterial activity is driven by free com-pound levels. The low solubility observed in the series is a chal-lenge. A robust structure-based SAR is demonstrated in thisstudy, which should help to drive down the MICs. Further optimi-zation of the scaffold is needed to deliver a candidate drug for thetreatment of tuberculosis.

Acknowledgments

The authors thank Suresh Rudrapatna and Sandesh Jatheendr-anath for purification and analytical support, including NMR stud-ies, and Guo Chen and D. Bryan Prince for their contributions to thepreparation of Spn-ParE protein.

R. R. Kale et al. / Bioorg. Med. Chem. Lett. 24 (2014) 870–879 879

Supplementary data

Supplementary data associated with this article can be found,in the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.12.080.

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22. Protocol for the efficacy of compound 17 in an acute infection model oftuberculosis in mouse: Mice were infected with an aerosolized suspension ofMtb. Treatment was started on day 3 post infection and continued for 10 days;in this period there was a 1.7 log increase in the cfu/lung in the vehicle controlgroup. Compounds were administered by oral gavage, either once (QD) ortwice (bid) daily, with isoniazid administered at 3 mg/kg QD serving as acontrol. Compound 17 was administered in three doses-100 mg/kg QD,300 mg/kg QD and 150 mg/kg bid. The dashed line indicates the CFU at thestart of treatment. The number of Mtb in the lungs of each mouse wasmeasured the day following treatment or at the start of treatment for the pre-treatment group. Each bar represents the average CFU per lung of 3 mice; errorbars represent the standard deviation.