Accepted Manuscript Nanomolar inhibitors of Mycobacterium tuberculosis Glutamine synthetase 1: synthesis, biological evaluation and X-ray crystallographic studies Cédric Couturier, Sandra Silve, Renaud Morales, Bernard Pessegue, Sylvie Llopart, Anil Nair, Armin Bauer, Bodo Scheiper, Christoph Pöverlein, Axel Ganzhorn, Sophie Lagrange, Eric Bacqué PII: DOI: Reference:

S0960-894X(15)00142-0 http://dx.doi.org/10.1016/j.bmcl.2015.02.035 BMCL 22447

To appear in:

Bioorganic & Medicinal Chemistry Letters

Received Date: Revised Date: Accepted Date:

5 January 2015 13 February 2015 16 February 2015

Please cite this article as: Couturier, C., Silve, S., Morales, R., Pessegue, B., Llopart, S., Nair, A., Bauer, A., Scheiper, B., Pöverlein, C., Ganzhorn, A., Lagrange, S., Bacqué, E., Nanomolar inhibitors of Mycobacterium tuberculosis Glutamine synthetase 1: synthesis, biological evaluation and X-ray crystallographic studies, Bioorganic & Medicinal Chemistry Letters (2015), doi: http://dx.doi.org/10.1016/j.bmcl.2015.02.035

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Graphical Abstract We have identified by high throughput screening and then optimized a series of imidazo[1,2-a]indeno[1,2-e]pyrazin-4-one that potently inhibit M. tuberculosis glutamine synthetase (GlnA1). Despite possibly nanomolar inhibitions, none of these compounds was active on whole cell Mtb, suggesting that GlnA1 may not be a suitable target to find new anti-tubercular drugs.

N N N H

O

IC50 GlnA1 = 31 nM IC80 Mtb > 30 µM

Bioorganic & Medicinal Chemistry Letters journal homepage: www.elsevi er.com

Nanomolar inhibitors of Mycobacterium tuberculosis Glutamine synthetase 1: synthesis, biological evaluation and X-ray crystallographic studies Cédric Couturiera*, Sandra Silvea, Renaud Moralesb, Bernard Pessegueb; Sylvie Llopartc, Anil Naird, Armin Bauere, Bodo Scheipere, Christoph Pöverleine, Axel Ganzhornf, Sophie Lagrangea and Eric Bacquéa a

Sanofi R&D, TSU Infectious Disease, 195, route d’Espagne BP 13669 31036 Toulouse Cedex 1, France. Sanofi R&D, Lead Generation and Candidate Realization, Structure Design and Informatics, 195, route d’Espagne BP 13669 31036 Toulouse Cedex 1, France c Sanofi R&D SCP Biologics, 195, route d’Espagne BP 13669 31036 Toulouse Cedex 1, France d Sanofi R&D, Lead Generation and Candidate Realization, 2090 East Innovation Park Drive, 85755-1965 OroValley, USA.. e Sanofi Deutschland GmbH, Lead Generation and Candidate Realization, Industriepark Hoechst, 65926 Frankfurt am Main, Germany f Sanofi R&D, DPU early to candidate 16 rue d'Ankara 67080 Strasbourg, France b

ARTICLE INFO

ABSTRACT

Article history: Received Revised Accepted Available online

A series of imidazo[1,2-a]indeno[1,2-e]pyrazin-4-ones that potently inhibit M. tuberculosis glutamine synthetase (GlnA1) has been identified by high throughput screening. Exploration of this series was performed owing to a short chemistry program. Despite possibly nanomolar inhibitions, none of these compounds was active on whole cell Mtb, suggesting that GlnA1 may not be a suitable target to find new anti-tubercular drugs.

Keywords: Tuberculosis High throughput screening X-ray crystallography Glutamine synthetase GlnA1 inhibitors

N N N H

IC50 GlnA1 = 31 nM IC80 Mtb > 30 µM

O

Tuberculosis remains a major global health problem. It causes morbidity among millions of people each year and ranks as the second leading cause of death worldwide among infectious diseases, after the human immunodeficiency virus (HIV). In 2011, the World Health organization (WHO-2012) estimated that 1.4 million people died from TB and that there were 12 million cases of TB worldwide. The TB-burden is highest in Asia and Africa: India/China and Africa respectively account for 40% and 24% of the world’s TB cases. Human Tuberculosis is an airborne infectious disease caused by the bacterium Mycobacterium tuberculosis (Mtb) that mostly affects the lungs. It is a complex disease caused by different bacterial populations that are located within a single host either intracellularly in macrophages or extracellularly in cavities or hypoxic lesions. Some of these microenvironments are conducive to replication, whereas others restrict bacterial growth without necessarily eradicating the infecting micro-organisms.

* Corresponding authors. Tel.: +33 534633128; fax: +33 534632813 email [email protected] (C.C.).

2009 Elsevier Ltd. All rights reserved.

The current duration and complexity of the treatment of drug-sensitive TB infections (6 months associating four drugs) may cause compliance problems, which favour the emergence of multi- and extensive-drug resistant (MDR/XDR) Mtb mutants. Treatments of MDR infections are of particular concern due to their extended duration (up to 2 years), poor safety, high cost and overall poor efficacy. Therefore, there is an urgent need to find new antitubercular drugs with new modes of action that would be efficient on MDR/XDR Mtb mutants and shorten the treatment duration (whatever the type of Mtbinfections) while avoiding relapses and the emergence of resistances, as well as improving compliance. As part of our target-based strategy to attack Mtb, we have investigated nitrogen assimilation as a possible source of novel biochemical targets and in particular, the synthesis of glutamine. In Mtb, four genes (glnA1-4) that are less than 25% homologous to each other, encode glutamine synthetase activities. GlnA2 is involved in the synthesis of D-glutamine, whereas glnA1, glnA3 and glnA4 catalyse the synthesis of L-glutamine1a.

The glnA1 gene encoded protein catalyses the ATP-dependent synthesis of L-glutamine from glutamate and ammonia. Of note, very low levels of glutamine are present in the phagosomes1b, limiting its availability to Mtb. GlnA1 is also involved in the synthesis of poly-Lglutamate/glutamine, a component corresponding to 10% of the cell wall of pathogenic mycobacteria, but absent in non-pathogenic mycobacteria2. The synthesis of this particular cell wall component might be facilitated by the export (about 30%) of the enzyme outside the bacteria in pathogenic, but not in non-pathogenic organisms3. glnA1 has been demonstrated to be essential for Mtb growth in vitro and in vivo and to be involved in Mtb virulence1,24,5. Moreover, only glnA1 is highly expressed, the three other genes glnA2-3-4 being 9 to 15 fold less expressed than GlnA11a, and their encoded activity possibly being 100 fold lower than for GlnA1. If GlnA1 is inhibited, the expression of glnA3, whose basal level is very low, is increased6. However, overexpression is less than 1.5 fold. Therefore, Harth et al, as well as Lee et al, have suggested that glnA2-3-4 might not be crucial for Mtb growth and cannot compensate the lack of glnA1 in vitro and in vivo1a, 5. Glutamine synthetase is also essential in humans for detoxification of ammonia in liver and of glutamate in the brain. However, the primary sequence of the human glnA1 encoded enzyme is poorly homologous to the bacterial protein (less than 20% identity)7. Moreover, the Mtb enzyme is dodecameric, whereas the human protein is decameric. Methionine sulfoximine (MSO) has been described as an inhibitor of Mtb GlnA1 inhibiting Mtb proliferation in vitro (MIC ~ 10-50 µM6,8), inside human mononuclear phagocytes and in vivo in a guinea pig model2,8. MSO is inactive on non-pathogenic mycobacteria2,3a or in the presence of glutamine6. It is mostly active on extracellular glutamine synthase2. In addition, MSO displays high selectivity for the Mtb protein: indeed, this compound is 100 fold less active on the human glutamine synthetase2,8. This data suggests that selective inhibition of Mtb may be possible, with limited risks of on-target in vivo toxicity. Beyond MSO, other ATP-competitive small molecules inhibitors of GlnA19, such as 2 (Figure 1), have been described (IC50 of 0.049 µM and MIC of 2 µg/mL on Mtb 9h).

NH

target involved in both in vitro growth inhibition and Mtb virulence. Therefore a program aimed at finding new, potent inhibitors of GlnA1 was initiated. Here, we describe the discovery, optimisation and structure-activity relationships of a series of GlnA1 inhibitors discovered by HTS. An enzymatic assay based on ADP detection was set up using the purified Mtb GlnA1 (see experimental part). An HTS was run on the GlnA1 activity using Sanofi screening collection (600K, screening performed at 10µM). 1698 actives were selected after dose response including several hit series. Following sorting and prioritization, compound 3 emerged as a promising starting point. This ATP-competitive (data not shown) compound displayed an IC50 of 31 nM against GlnA1 and a lack of cytotoxicity on the human HepG2 cell line. It was also devoid of activity against human glutamine synthetase. Disappointedly enough, compound 3 was devoid of any antibacterial activity against Mtb. In order to better understand the causes of the good affinity of compound 3 and to guide further optimisation efforts, a crystallization platform of the recombinant Mtb-GlnA1 was set up. Mtb-GlnA1 crystallizes in the space group P212121 with 24 monomers in the asymmetric unit to form a dodecamer (Figure. 2). The 24 active sites of the dodecamer are found in long grooves between the subunits. Compound 3 was cocrystallized with Mtb GlnA1. Each site contains clear electron density for the inhibitor whose binding is stabilized by various hydrophobic interactions (Figure 3). The imidazolyl moiety makes a π-stacking with the Trp278 side chain and is also in an edge-face type close contact with the Tyr125 side chain. The planar tetracyclic core lies in a kind of aromatic box formed by Trp278, Tyr125 and Phe228 and complemented by interactions with the hydrophobic part of the Lys357 side chain as well as Ala358. The far edge of the phenyl moiety touches the His274 carbonyl oxygen in a C-HO type interaction and is also surrounded by the Phe228 and Arg360 side chains. The pyrazinone moiety points towards the solvent (Figure 4).

O

O S N OH H2N

N

O MSO 1

N

NH2 N H

N N H O

2

3

Figure 1: Known GlnA1 inhibitors (1 and 2) and hit compound 3

Overall, this analysis suggested that the glnA1 gene product (GlnA1) was an attractive, druggable anti-TB

Fig 2: Scheme of the 24-monomer asymmetric-unit forming a dodecameras observed in the P212121 space group.

comparable (eg 9b, 9c, 9j, 9m and 9n) or even better (see 9i) inhibition of GlnA1 than 3.

R36 H27 K35

Ser 276

F22 W27 Y12 Fig. 3: Close up view of the interaction of compound 3 with GlnA1. Final 2foFc electron density map contoured at 1s is depicted around the inhibitor.

Comparison of this structure with other known X-ray structures (those of 29h and of ADP10) shows that the phenyl moiety of 3 is at essentially the same place as the six-membered pyrimidine ring of ADP. On the other hand, compound 3 doesn’t make any interaction with Ser276 whereas this residue is key in the binding of ADP and of 2 to GlnA1: indeed, N1 of the ADP adenine ring and both nitrogen atoms of the 2-aminopyridine-4-yl moiety of 2 hydrogen bond with Ser276 (Figure 4a and 4b). At this point, a chemistry program was initiated with the objective to explore SARs around 3 and to get antibacterial activities. Synthesis of 3 and its analogues followed the routes described in scheme 1. Starting from 4bromo indan-1-one 4, intermediate 7 was obtained in 2 steps. In the case of variations at R1, the tetracyclic compound 8, obtained by condensation of 7 with ammonium acetate, was cross-coupled with boronic acids to yield compounds 9a-d (pathway 1 in scheme 1). Since this route was flawed by low to moderate yields and harsh reaction conditions, a second pathway was developed. Compound 7 was transformed into the pinacolyl boronic ester 10 via Miyaura borination. The Suzuki cross-coupling with different iodo arenes/heteroarenes was then performed under standard conditions to get access to compound 11. The final compounds 9e-n were obtained by condensation of 11 with various primary amines (pathway 2 of scheme 1). Alternatively, key intermediate 11 was synthesized starting from commercially available benzyl bromides 12 (scheme 2). Substitution with dimethyl malonate followed by saponification and decarboxylation yielded carboxylic acid 14 that was then activated as an acid chloride to undergo an intramolecular Friedel-Crafts acylation to afford 4-arylindan-1-one 15. Bromination of 15 followed by nucleophilic substitution with imidazole 5 completed the synthesis of 11. This approach proved to be superior to the pathway 2 of scheme 1, in terms of yield and convenience for R1= Ph and 3-MeO-phenyl. Prepared compounds 8 and 9a to 9o were then assayed against GlnA1 and Mtb and on HepG2 cell line. As shown in table 1, we were able to identify a few compounds with

Ser

Fig. 4a: Superimposition of GlnA1 bound to 3 (cyan) and to ADP (green) 10 (PDB entry 2BVC) 4b Superimposition of GlnA1 bound to 3 (cyan) and to ligand 2 9h (purple) (PDB entry 3ZXV)

Aryl and heteroaryl substituants at R1 were preferred while the corresponding bromo (8) and NHAc (9n) derivatives were poorly active on the enzyme. Likewise, a quinolin-5-yl was not tolerated at this position. Among the aryl/heteroaryl R1 groups, phenyl (see 3), 3OMe phenyl (see 9b) and 3-pyridyl (see 9c) groups afforded the most potent analogues whereas the 3-Cl phenyl derivative (9a) was much less active. Substitution at R2 was generally detrimental (see the sequence 3, 9f, 9e, 9g and 9d) with the notable exception of 9i, the most active compound of the series, that associates a 3-pyridyl R1 group and an allyl R2 group. We were disappointed to see the lack of improvement provided by the 3-pyridyl group of 9c in comparison with the phenyl group of 3. Based on the examples of ADP and compound 2, we indeed expected the nitrogen atom of this group to be able to hydrogen bond to Ser276 and hence improve affinity. A possible explanation to this observation is that the 3-pyridyl group is not well positioned in 9c to allow hydrogen bonding with Ser276. However, this hydrogen bond may exist in compound 9i because of a slightly different binding mode due to the N1 substituant and this could explain the improved potency of 9i. We were also surprised to see substantial differences in IC50s according to the nature of R2 (see for instance the difference between 3 and 9d or 9c and 9k) whereas x-ray crystallographic model showed that this position points

toward the solvent suggesting that a large variety of substituents was possible at this position. Alternative binding modes for R2 ≠ H cannot be excluded but, in the absence of additional x-ray crystallographic model, we can only speculate. As for 3, analogues of this compound turned out to be highly selective for this Mycobacterium enzyme (data not shown on other enzymes or classical off-target panel). They were generally not or only slightly cytotoxic on HepG2 cell lines (in the 15-30 µM range or > 30 µM). Moreover, all these compounds, including the inhibitors with IC50s in the nanomolar range, were inactive (IC50 > 30µM) or poor inhibitors of Mtb proliferation (IC50 ~ 2030µM). In the later cases (eg 9a and 9d), HepG2 results suggested that these activities might be linked to cytotoxicity rather than to specific antibacterial activities. However, over- or under- expression of GlnA1 were not tested on the inhibitors potentialisation. Br

Br

Br

HN a

N Br

+

b

O

N

5

4

6

7

Br

N

N H

8

N H 9a-c, o-p

O

O

Pathway 2: R2 = Alkyl O

R1

R1 f N N

10

N

11

O

9d-n

O

R2

O

R1

R1 a

O 12

d

e Br

15

O

16

Benzyl CH2CH= CH2 Me (CH2)2OC H3 (CH2)3N( CH3)2 (CH2)2CH =CH2 CH2CH(C H3)2 (CH2)3N( CH3)2

663

29

20

333

> 30

> 30

170

> 30

> 30

219

> 30

23

9i 9j 9k 9l 9m

Pyridin-3yl Pyridin-3yl Pyridin-3yl Pyridin-2yl Pyridin-2yl 3-MeOphenyl -NH-COCH3 quinoline 5-yl

131

> 30

> 30

7

> 30

> 30

66

> 30

> 30

857

> 30

> 30

cPr

456

> 30

> 30

(CH2)3N( CH3)2

84

> 30

15

H

2100

>30

>30

H

7000

>30

>30

Table 1: Structure Activity Relationships of 5,10-dihydro-imidazo[1,2a]indeno[1,2-e]pyrazin-4-one compounds (MIC or IC90 on Mtb gave similare results)

R1

c

9h

9o 14

> 30

Phenyl

9n

O

29

9g

OH

13 R1

O

R1 b

O

Br

31

Phenyl

O

Scheme 1: (a) Br2, DCM, 0 °C to r.t., 16 h; (b) MeCN,NaHCO3, 65 °C; (c) NH4OAc (10 equiv.), AcOH, 110 °C, 4 h; (d) R1B(OH)2, [1,1’-bis(diphenylphosphino)ferrocene]PdCl2 (5 mol%), K3PO4, DME, nBuOH, H2O, 130 °C, microwave, 2 h; (e) bis(pinacolato)diboron, (PPh3)2PdCl2 (5 mol%), KOAc, toluene; (f) R1-I, (PtBu3)2Pd (5 mol%), Cs2CO3, 1,4-dioxane, 60 °C; (g) R2NH2 (10 equiv.), AcOH, 110 °C.

H

9f

N

OO

> 30

Phenyl

N

N OO

> 30

9e

g

N

51

9d

O B

H

H H

N

N e

15

Phenyl Br 3-Clphenyl 3-MeOphenyl Pyridin-3yl Phenyl

O

d

N

22

3 8

9c

c

610

GlnA1 IC50 (nM)

R1

7

H

R2

9b Pathway 1: R2 = H

HepG2 IC50 (µM) > 30 > 30

R1

9a

OO

31 > 30

Mtb IC80 (µM) > 30 > 30

N°

N O

O

O

of poly-L-glutamine/glutamate chains in the cell wall (2). Therefore, at least this essential activity should be accessible to the compounds that should inhibit the bacteria proliferation. In previous similar cases in the field of antibacterial research, compound penetration and target validation have been put forward as possible explanations11. A problem of penetration into the mycobacteria for our compounds seems unlikely: indeed, with experimental logDs typically in the 0.2-3.5 range (eg 3.5 for 3), 3 and its analogues are expected to penetrate easily into the waxy membrane of Mtb although they are not whole-cell inhibitors. Therefore, our results rather led us to doubt of the relevance of GlnA1 as a validated antiTB target.

11

O

Scheme 2: (a) dimethyl malonate, THF, NaH, r. t., 16 h; (b) KOH, EtOH, H2O; HCl, pH < 3 (c) (COCl)2, DCM, 16 h; evavoration; DCM, AlCl3, 0 °C to r. t.; (d) Br2, DCM, 0 °C to r.t., 16 h; (e) 6, MeCN, NaHCO3, 65 °C. Considering the demonstrated essentiality of glnA1 in Mtb, this lack of antibacterial activity despite potent inhibition, was puzzling. GlnA1 is partly located in the cell wall and this later activity is involved in synthesis

During the course of our program, a publication demonstrated that glnA1, though essential, was not a vulnerable target in Mtb11. Indeed, based on the observation that 5% glnA1 mRNAs were still sufficient to allow Mtb proliferation, this paper suggested that the inhibition of GlnA1 should be almost complete to effectively translate into Mtb growth inhibition12. We therefore hypothesized that our compounds, albeit potent in vitro inhibitors of GlnA1, were unable to reach this level of inhibition, perhaps due to the competition with the high

intracellular pool of ATP. The IC50 of these compounds increased linearly with the concentration of ATP present in the enzymatic assay (data not shown). However, it is difficult to extrapolate to the conditions inside bacteria as the intracellular concentrations of the compounds, ATP or glutamine synthase are not known. Noteworthy, compounds are still inactive in anaerobic conditions, whereas the ATP level drops significantly13. In conclusion, following an HTS, a series of MtbGlnA1 inhibitors has been identified and then optimized. Despite potent enzymatic inhibitions, none of our compounds was active on the whole pathogen Mtb. In line with reports on the non-vulnerability of Mtb- glnA1 and the potential for rapid emergence of resistance6, our results support the assumption that Mtb- GlnA1 is not a suitable target for the discovery of new anti-tubercular drugs inhibiting the growth of Mtb though we cannot exclude the possibility that GlnA1 inhibitors may dampen Mtb virulence in vivo. Overall, our results once again illustrate, in the case of Mtb, the challenges associated to targetbased approaches and that other have experienced in their efforts to find new antibiotics11. Literature 1. (a). Harth, G.; Maslesa-Galic, S.; Tullius, M. V.; Horwitz, M. A. Mol. Microbiol 2005, 58, 1157. (b) Tullius, M.V.; Harth, G.; Horwitz, M. A. Infect. Immun. 2003, 71, 3927 2. Harth, G.; Horwitz, M. A. J. Exp. Med. 1999, 189 (9), 1425. 3.(a) Harth, G.; Clemens, D. L.; Horwitz, M. A. Proc Natl Acad Sci U S A. 1994, 91:9342. (b) Chandra, H.; Farhat Basir, S.; Gupta, M.; and Banerjee, N. Microbiology 2010, 156,3669-3677 4. Sassetti, C.M., Boyd, D.H., Rubin, E.J. Molecular Microbiology 2003, 48:77-84. 5. Lee, S.; Jeon, B.-Y.; Bardarov, S.; Chen, M.; Morris, S. L.; Jacobs, W. R. Jr. Infect. Immun. 2006, 74, 6491. 6. Carroll, P.; Waddell, S. J.; Butcher, P. D.; Parish, T. Microb. Drug Resistance, 2011, 17, 351. 7. Krajewski, W. W.; Collins, R.; Holmberg-Schiavone, L.; Jones, T.A.; Karlberg, T.; Mowbray, S.L. J. Mol. Biol. 2008, 375, 217. 8. Harth, G.; Horwitz, M. A. Infect. Immun. 2003, 71, 456. 9. (a).Nordqvist, A., Nilsson, M. T., Lagerlund, O., Muthas, D., Gising, J., Yahiaoui, S., Odell, L. R., Srinivasa, B. R., Larhed, M., Mowbray, S. L. and Karlen, A. Med. Chem. Commun., 2012, 3, 620. (b). Nilsson M. T., Krajewski W. W., Yellagunda S., Prabhumurthy S., Chamarahally G. N., Siddamadappa C., Srinivasa B. R., Yahiaoui S., Larhed M., Karlen A., Jones T. A. and Mowbray S. L., J. Mol. Biol., 2009, 393, 504. (c) Salisu, S., Kenyon, C., and Kaye, P. T. Synthetic Communications 2011, 41, 2216. (d).Gxoyiya S. B, Kaye, P. T., and Kenyon C. Synthetic Communications 2010, 40, 2578. (e) .Odell, L. R., Nilsson, M. T., Gising, J., Lagerlund, O., Muthas, D., Nordqvist, A., Karlén, A., Larhed, M. Bioor. Med. Chem. Let. 2009, 4790. (f). Nordqvist, A., Nilsson, M. T., Rottger, S., Odell, L. R., Krajewski, W. W., Evalena Andersson, C., Larhed, M., Mowbrayc, S. L., and Karlen, A. Bioorg. Med. Chem. 2008, 16, 5501. (g). Marius Mutorwa, M.,

Salisu, S., Blatch, G. L., Kenyon, C., and Kaye, P. T. Synthetic Communications, 2009, 39, 2723. (h) Gising, J., Nilsson, M. T., Odell, L. R., Yahiaoui, S., Lindh, M., Iyer, H., Sinha, A. M., Srinivasa, B. R., Larhed, M., Mowbray, S. L. and Karlén, A. J. Med. Chem. 2012, 55, 2894. (i) Mowbray, S. L., Kathiravan M. K., , Pandey A. A and Odell, L. R. Molecules 2014, 19, 1316. 10. Krajewski W. W, Jones T. A, Mowbray S. L. Proc Natl Acad Sci U S A. 2005, 102, 10499. 11. D. Payne, M.N. Gwynn, D.J. Holmes, D.L. Pompliano Nat. Rev. Drug. Discov. 2007, 6, 29. 12. Carroll, P., Faray-Kele, M. C.; Parish, T. Appl. Environ. Microbiol. 2011, 77, 5040. 13. Koul, A., et al. J. Biol. Chem. 2008, 283(37), 25273 14 Z. Otwinowski, Z., Macromolecular Crystallography, 1997, 276: part A. 307, C.W. Carter, Jr. & R. M. Sweet, Eds., Academic Press. 15. Vagin, A., A., Steiner, R. S., Lebedev, A., A., Potterton, L., McNicholas, S., Long, F. and Murshudov, G., N. Acta Cryst. 2004 D60, 2284. Supplementary data associated with this article can be found in the online version. Coordinates and structure factor data have been deposited at the protein databank with the entry code: 4XYC.