Discovery of Imidazo[1,2-a]pyridine Ethers and Squaramides as Selective and Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis

Article abstract of DOI:10.1021/acs.jmedchem.6b01358

The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP) synthase as an attractive target to kill Mycobacterium tuberculosis (Mtb). Herein, we report the discovery of two diverse lead series imidazo[1,2-a]pyridine ethers (IPE) and squaramides (SQA) as inhibitors of mycobacterial ATP synthesis. Through medicinal chemistry exploration, we established a robust structure-activity relationship of these two scaffolds, resulting in nanomolar potencies in an ATP synthesis inhibition assay. A biochemical deconvolution cascade suggested cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be noncytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.

Full text of DOI:10.1021/acs.jmedchem.6b01358

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Article
Discovery of Imidazo[1,2-a]pyridine ethers and Squaramides as Selective and
Potent Inhibitors of Mycobacterial Adenosine Triphosphate (ATP) Synthesis
Subramanyam J Tantry, Shankar D. Markad, Vikas Shinde, Jyothi Bhat, Gayathri Balakrishnan, Amit K
Gupta, Anisha Ambady, Anandkumar V. Raichurkar, Chaitanyakumar Kedari, Sreevalli Sharma, Naina
V Mudugal, Ashwini Narayan, C. N. Naveen Kumar, Robert Nanduri, Sowmya Bharath, Jitendar Reddy,
Vijender Panduga, Prabhakar K R, Karthikeyan Kandaswamy, Ramanatha Saralaya, Parvinder Kaur,
Neela Dinesh, Supreeth Guptha, Kirsty Rich, David Murray, Helen Plant, Marian Preston, Helen Ashton,
Darren Plant, Jarrod Walsh, Peter Alcock, Kathryn Naylor, Matthew Collier, James Whiteaker, Robert
E McLaughlin, Meenakshi Mallya, Manoranjan Panda, Suresh Rudrapatna, Vasanthi Ramachandran,
Radha K Shandil, Vasan K Sambandamurthy, Khisimuzi Mdluli, Christopher B. Cooper, Harvey
Rubin, Takahiro Yano, Pravin S. Iyer, Shridhar Narayanan, Stefan Kavanagh, Kakoli Mukherjee, V.
Balasubramanian, Vinayak P. Hosagrahara, Suresh Solapure, Sudha Ravishankar, and Shahul Hameed P
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b01358 • Publication Date (Web): 11 Jan 2017
Downloaded from http://pubs.acs.org on January 11, 2017
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Journal of Medicinal Chemistry is published by the American Chemical Society. 1155
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Ramachandran, Vasanthi; AstraZeneca,
Shandil, Radha; Astrazeneca India Pvt Ltd
Sambandamurthy, Vasan; AstraZeneca India Pvt Ltd
Mdluli, Khisimuzi; TB Alliance, Research
Cooper, Christopher; Global Alliance for TB Drug Development (TB
Alliance),
Rubin, Harvey; University of Pennsylvania School of Med, Department of
Medicine
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Yano, Takahiro; University of Pennsylvania, Medicine
Iyer, Pravin; Jubilant Innovation
Narayanan, Shridhar; AstraZeneca India Pvt. Ltd., Bioscience
Kavanagh, Stefan; AstraZeneca R&D
Mukherjee, Kakoli; Integral Biosciences,
Balasubramanian , V.; AstraZeneca India Pvt Ltd
Hosagrahara, Vinayak; AstraZeneca India Pvt Ltd
Solapure, Suresh; AstraZeneca India Pvt Ltd
Ravishankar, Sudha; AstraZeneca India Pvt Ltd
Hameed P, Shahul; BUGWORKS, MEDICINAL CHEMISTRY
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Discovery of Imidazo[1,2-a]pyridine ethers and Squaramides
as Selective and Potent Inhibitors of Mycobacterial
Adenosine Triphosphate (ATP) Synthesis
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Subramanyam J. Tantry, Shankar D. Markad, Vikas Shinde, Jyothi Bhat, Gayathri
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Balakrishnan, Amit K. Gupta, Anisha Ambady, Anandkumar Raichurkar, Chaitanyakumar
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Kedari, Sreevalli Sharma, Naina V. Mudugal, Ashwini Narayan, C.N. Naveen Kumar,
†
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Robert Nanduri, Sowmya Bharath, Jitendar Reddy, Vijender Panduga, Prabhakar K.R.,
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Karthikeyan Kandaswamy, Ramanatha Saralaya, Parvinder Kaur, Neela Dinesh, Supreeth
†
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Guptha, Kirsty Rich, David Murray, Helen Plant, Marian Preston, Helen Ashton, Darren
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Plant, Jarrod Walsh, Peter Alcock, Kathryn Naylor, Matthew Collier, James Whiteaker
,
‡
†
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Robert E. McLaughlin, Meenakshi Mallya, Manoranjan Panda, Suresh Rudrapatna, Vasanthi
†
†
†
$
Ramachandran, Radha Shandil, Vasan K. Sambandamurthy, Khisi Mdluli, Christopher B.
$
&
&
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†
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Cooper, Harvey Rubin, Takahiro Yano, Pravin Iyer, Shridhar Narayanan, Stefan Kavanagh,
†
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Kakoli Mukherjee, V. Balasubramanian, Vinayak P. Hosagrahara, Suresh Solapure, Sudha
†
*
†*
Ravishankar Shahul Hameed P,
#
Contributed equally to the work
†
Innovative Medicines, AstraZeneca India Pvt. Ltd., Bellary Road, Hebbal, Bangalore 560024,
India.
‡
AstraZeneca Infection Innovative Medicines, 35 Gatehouse Drive, Waltham, MA 02451, USA.
§
AstraZeneca, Alderley Park, Mereside, Macclesfield, Cheshire, UK. SK10 4TG
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$
th
Global Alliance for TB Drug Development, 40 Wall Street, 24 Floor, New York, NY 10005,
USA.
&
University of Pennsylvania, 111 Clinical Research Building, 415 Curie Boulevard, Philadelphia
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PA 19104, USA
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Corresponding authors
shahulhameed@bugworksresearch.com
mailsuravi01@gmail.com
KEYWORDS:
Oxidative phosphorylation
ATP synthesis
Imidazo[1,2ꢀa]pyridine ethers
Squaramides
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ABSTRACT
The approval of bedaquiline to treat tuberculosis has validated adenosine triphosphate (ATP)
synthase as an attractive target to kill Mycobacterium tuberculosis. Herein, we report the
discovery of two diverse lead series imidazo[1,2ꢀa]pyridine ethers (IPE) and squaramides (SQA)
as inhibitors of mycobacterial ATPꢀsynthesis. Through medicinal chemistry exploration, we
established a robust structure activity relationship of these two scaffolds resulting in nanomolar
potencies in an ATPꢀsynthesis inhibition assay. A biochemical deconvolution cascade suggested
cytochrome c oxidase as the potential target of IPE class of molecules, whereas characterization
of spontaneous resistant mutants of SQAs unambiguously identified ATP synthase as its
molecular target. Absence of cross resistance against bedaquiline resistant mutants suggested a
different binding site for SQAs on ATP synthase. Furthermore, SQAs were found to be nonꢀ
cytotoxic and demonstrated efficacy in a mouse model of tuberculosis infection.
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INTRODUCTION
Tuberculosis (TB) continues to be a major health issue with about 1.5 million deaths reported in
014. One third of the world population is estimated to be exposed to the causative agent M.
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ꢀ3
tuberculosis. Infections with multidrugꢀresistant (MDR) and extensively drugꢀresistant (XDR)
M. tuberculosis strains and coꢀinfection with HIV have further worsened the prospect of
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ꢀ6
controlling this disease. The prolonged treatment regimen for six months with a combination
of four drugs has resulted in poor patient compliance leading to the emergence of MDR and
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XDR strains of M. tuberculosis. The treatment of MDR or XDRꢀTB cases with poorly
efficacious and not so safe second and third line drugs for 12 to 24 months has further
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0
deteriorated the overall success rate of TB treatment. In order to improve the efficacy of TB
treatment and to make an impact on the global war against TB, new antiꢀtubercular agents with a
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potential to reduce the duration of treatment are required. A few existing drugs and several new
chemical entities (NCEs) are being profiled in the clinic to test their effectiveness in treating both
12ꢀ13
drugꢀsensitive and drugꢀresistant TB cases.
Efforts are in progress to test and optimize new
combination of drugs to improve the efficacy of existing drugs and the NCEs emerging out of
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several ongoing research and development programs.
Antiꢀtubercular discovery and development programs select potential drug targets based on a
number of criteria such as, (i) novel target or pathway to avoid chances of cross resistance with
existing drugs, (ii) essentiality of the target or pathway to the survival of M. tuberculosis, (iii)
low propensity for development of resistance and (iv) absence of a human homolog to minimize
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mechanism based toxicity issues. If the chosen target has a human homolog, building in
selectivity during chemistry exploration becomes one of the key requirements as nonꢀselective
inhibitors may result in acute toxicity issues preventing their clinical utility. The recently
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approved drug, bedaquiline to treat drug resistant TB cases is one of the best examples of a
selective antimycobacterial agent. It inhibits mycobacterial ATP synthase with a high selectivity
index eventhough the process of ATP synthesis is highly conserved across both prokaryotes and
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eukaryotes.
The success of bedaquiline in the clinic has resulted in an increased exploration
to identify additional inhibitors of mycobacterial ATP synthase and other novel targets and
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pathways.
ATP synthase is an evolutionarily highly conserved enzyme among prokaryotes and eukaryotes,
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including humans. It supplies cells with bulk of their ATP via the oxidative phosphorylation
(OxꢀPhos) process and hence essential for cell survival. This multiꢀsubunit complex, the last one
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in the oxidative phosphorylation pathway, couples the proton motive force to ATP synthesis.
Hong et al., have provided a comprehensive review of all known ATP synthase inhibitors and
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their utility in scientific research. The wellꢀknown inhibitors of ATP synthase, such as
dicyclohexylꢀcarbodiimide (DCCD), oligomycin and venturicidin, which block ATP synthase
activity through their interaction with subunitꢀc or subunitꢀa/c interface are poorly selective and
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are shown to inhibit both bacterial and mitochondrial ATP synthesis.
The lack of selectivity
and ensuing toxicity has prevented their use in the clinic. Several studies towards understanding
the mechanism of action of ATP synthase inhibitors have led to advances in the methodologies
25ꢀ26
to test for mitochondrial toxicity of drugs in development.
In this regard, the discovery of a
first in class mycobacterial ATP synthase inhibitor, bedaquiline, from a cell based screen, is
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noted as a significant contribution towards tuberculosis drug discovery research. This highly
selective molecule having the ability to reduce the duration of TB treatment because of its potent
activity against both replicating and nonꢀreplicating M. tuberculosis has indicated the possibility
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of identifying selective inhibitors of this mechanism.
Although FDA approved the use of
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bedaquiline for the treatment of tuberculosis, its usage got limited to treat only MDR and XDRꢀ
TB cases because of the drugꢀdrug interaction risk it carried with the front line drug rifampicin.
Additionally, its usage in the clinic carries a blackꢀbox warning due to the QTꢀprolongation risk
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observed, leaving the need to discover safer drugs for TB treatment. However, the discovery of
bedaquiline brought to prominence the criticality of ATP synthase for the survival of M.
tuberculosis and the possibility of exploring the entire oxidative phosphorylation pathway to
identify additional inhibitors of this mechanism. Several attempts made in this direction has
resulted in the identification of a few compounds which inhibit the process of ATP synthesis in
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mycobacteria.
In order to discover and develop a novel, potent and safe antiꢀtubercular agent,
we employed a biochemical screen to identify chemical entities that inhibit the ATP synthesis
pathway. A membrane based biochemical assay (Myc_ATPS), which measured ATP synthesis
through the oxidative phosphorylation (OxꢀPhos) process, was used to screen AstraZeneca’s
(
AZ) corporate compound collection in a high throughput screen (HTS). The high hit rate
observed in the primary screen led to the development and use of counter screens such as delta
pH (ꢀpH) and mammalian mitochondrial ATP synthesis (SMP_ATPS) assays respectively) in
order to shortlist specific and selective inhibitors of mycobacterial ATP synthesis pathway.
Screening of about 900,000 compounds utilizing the cascade presented in Figure 1 yielded about
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200 actives with potent IC (< 10 µM) and good selectivity. Further hit triage that included
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structure activity relationship (SAR) studies to check the potential for chemical diversification
and confirmation of activity through reꢀsynthesis of hits led to the identification of multiple
robust chemical classes. SAR exploration of the potent mycobacterial ATP synthesis inhibitor
2,4ꢀdiaminoquinazolines
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aminopyrazolopyrimidines has been described earlier. Here we describe SAR studies and the
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resulting biological properties of two diverse chemical leads, imidazo[1,2ꢀa]pyridine ethers (IPE)
and squaramides (SQA) (Figure 2), with potent ATP synthesis inhibition and antiꢀtubercular
activity. Utility of a biochemical deconvolution cascade and spontaneous mutant generation
enabled identification of molecular targets of these two scaffolds. A potent and selective
compound from squaramide series was prioritized to demonstrate in vivo proof of principle in a
mouse model of acute tuberculosis infection.
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Figure 1. Schematic of HTS and hit triage
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Figure 2. Prioritized hit series from high throughput screen.
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Published elsewhere.
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CHEMISTRY
Synthesis of imidazo[1,2-a]pyridine ethers
The condensation of 2ꢀaminopyridines (1a-1d) with phenyl glyoxals (2a, 2b) in the presence of
Lewis acid such as boron triflouride etherate afforded imidazo[1,2ꢀa]pyridineꢀ3ꢀols (3a-3e) in
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moderate to excellent yields. The required imidazo[1,2ꢀa]pyridine ethers were obtained in good
yields by alkylation using suitable alkoxyalkyl or aryloxyalkyl halides in the presence of a base
(Scheme 1).
Scheme1. Synthesis of Imidazo[1,2ꢀa]pyridine ethers
In order to introduce a variety of heterocycles at the terminal position of the ether side chain,
imidazo[1,2ꢀa]pyridineꢀ3ꢀols (3a-3e) were treated with 3ꢀbromopropanol in the presence of
K CO as a base to give alcohols (10d, 11e). The terminal hydroxy group was converted into its
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mesyl derivative (12d, 13e) under standard conditions (MsCl/NEt ). Various heteroaryl
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amines/alcohols were introduced by treating the mesyl derivative with corresponding heteroaryl
amines/alcohols in the presence of a base to get imidazo[1,2ꢀa]pyridineꢀ3ꢀethers (Schemes 2 and
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Scheme 2. Synthesis of IPE with heteroaryls
Scheme 3. Synthesis of IPE with heteroarylamines.
The carbon linker of ether side chain was modified to 2 carbon atoms. Imidazo[1,2ꢀa]pyridineꢀ3ꢀ
ols (3e) were treated with 2ꢀbromoethanol in the presence of K CO to give alcohol 25e. The
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terminal hydroxy group was converted to the corresponding amine by a sequence of reactions;
the alcohol was reacted with DPPA to give the azido functionality which in turn was converted to
the corresponding amine 27e under Staudinger reaction conditions. The amine 27e was converted
to the benzylamine derivative 28e by reductive amination using substituted benzaldehyde. The
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amine 27e was also converted to sulfonamide 29e and amide 30e by treatment with the
corresponding sulfonyl chloride and nicotinic acid, respectively (Scheme 4).
10
11
12
13
14
15
16
17
18
19
20
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29
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44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Scheme 4. Synthesis of IPE amide/sulfonamide
Synthesis of Squaramides:
Scheme 5 depicts the synthetic route followed for the synthesis of squaramides. The dichloride,
3,4ꢀdichloroꢀ3ꢀcyclobuteneꢀ1,2ꢀdione was generated in situ by treating squaric acid 31a with
o
thionyl chloride at 0 C and the resulting dichloride was directly subjected to mono arylation
with 4ꢀphenylmorpholine substituted arene in the presence of AlCl as Lewis acid. The
3
o
exothermic reaction was controlled by slow addition of arene at 0 C and the subsequent
purification resulted in mono chloride 31b with moderate yield. The mono chloride 31b could be
treated conveniently with various benzylamines in a parallel mode to yield title compounds 31c-j
in
moderate
yield.
O
O
N
N
N
NH2
H
N
HO
O
OH
O
Cl
Cl
O
Cl
O
SOCl
2
N
Phenylmorpholine
AlCl3, DCM,
o
8
0 C
TEA, 1,4ꢀdioxan,
0ꢀ2 C, 0.5 h
O
O
O
O
o
o
3hr
0 C, 2 h
in situ
3
1a
31b
31c-j
Scheme 5. Synthesis of Squaramides
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6
RESULTS AND DISCUSSION
High throughput screening and hit identification
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
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1
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0
1
2
3
4
5
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9
0
ATP synthesis assays which used inverted membrane vesicles (IMVs) prepared from either
bacterial cytoplasmic membrane or mammalian mitochondrial membrane have been reported
24, 39ꢀ41
previously.
The primary screen used in this study (Myc_ATPS assay), derived from the
4
1
assay reported by Yano et al., measures the ATP synthesized through the process of oxidative
phosphorylation in a luminescence based assay performed with 8 ꢁg/ml of M. smegmatis IMVs
energized with 150 ꢁM NADH. In a single point high throughput screen at 10 µM concentration,
9
00,000 compounds from AstraZeneca’s corporate compound collection were evaluated for the
inhibition of Myc_ATPS assay. This screen yielded inhibitors at a very high hit rate of about
.1% (Figure 1) probably because it also picked up disruptors of proton gradient as ATP
6
synthesis inhibitors in addition to those which inhibited the ATP synthesis pathway. Inhibitors
of proton gradient formation would be nonꢀselective and toxic as they can inhibit proton gradient
across mammalian mitochondrial membrane as well. To weedꢀout such compounds, an assay to
measure the proton gradient across IMVs was designed (ꢁpH assay) by modifiying the
Myc_ATPS assay wherein substrates of ATP synthase (ADP and Pi) were omitted. The assay
was monitored using LysoSensor Green DNDꢀ153, a fluorophore linked to a weak base which
gets partially protonated at neutral pH and fluoresces at acidic pH with excitation maxima at 485
4
2
nm and emission maxima at 523 nm. The output of primary screen, 55K actives, when screened
at 10 ꢁM in a single point ꢁpH assay resulted in the selection of 18K compounds which were
taken for dose response studies to determine IC50 in both the Myc_ATPS and ꢁpH assays
(Figure 1). Mimimum inhibitory concentration (MIC) for M. tuberculosis (minimum
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concentration required to inhibit the growth by 80%) and mammalian cell lines like THP1 and/or
A549 (MMIC – minimum concentration required to inhibit the mammalian cell proliferation by
5
0%) were determined with 1200 compounds having Myc_ATPS IC ≤10 ꢁM and ꢀ pH IC
5
0
50
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
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43
44
45
46
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48
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51
52
53
54
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60
≥
50 ꢁM. In addition, evaluation of identified chemical series for physicochemical properties and
chemical tractability for SAR expansion led to choosing two scaffolds for reꢀsynthesis,
confirmation of activities and follow up SAR (Figure 2).
Structure activity relationship (SAR) studies of imidazo[1,2-a]pyridine ethers
Figure 3. SAR for imidazo[1,2ꢀa]pyridine ethers
All compounds synthesized during SAR studies were tested in the mycobacterial ATP synthesis
assay (Myc_ATPS IC ) and in the antiꢀmycobacterial assay (M. tuberculosis MIC) for assessing
5
0
the potency, as well as in the mammalian cell proliferation assay (MMIC) for assessing
cytotoxicity. Cytotoxicity index, the ratio of MMIC to MIC guided the SAR studies towards the
synthesis of selective inhibitors.
The generic SAR for IPE series is depicted in Figure 3. In general, the position of bridgehead
nitrogen in [1,2ꢀa] format and the presence of a phenyl ring directly attached to the imidazole
portion of IPE core are essential for retaining biological activity (both ATP synthesis inhibition
and M. tuberculosis MIC). Chloro or methyl substitution at R1 position gave best potency,
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
whereas H preferred over F at R3 position for improved potency. The terminal aryl linkage
through –NHꢀ or –Oꢀ at X provided the best potency, whereas the amide (ꢀNHCOꢀ) and
sulphonamide (ꢀNHSO ꢀ) linkages were found to be weakly active. The substituted phenyl is
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
preferred over hetero aromatic rings like pyridine or pyrimidine at the side chain terminal ring
system for maintaining best potency. A detailed SAR for IPE series is presented in tables 1ꢀ 4.
The SAR of IPE series was studied by incorporating various substitutions on imidazo[1,2ꢀ
a]pyridine followed by optimization of the ether side chain. The unsubstituted compound 4a
showed ATP synthesis IC of 0.13 µM and M. tuberculosis MIC of 1.6 µM in the enzymatic and
50
cellular assays, respectively. Introduction of an electron withdrawing and hydrophobic chloro
substitution at 6ꢀposition of the imidazo[1,2ꢀa]pyridine gave compound 4b that showed nearly
eightꢀfold improvement in ATP synthesis inhibition and 3ꢀfold improvement in cellular potency
compared to the unsubstituted compound 4a. In contrast, electron withdrawing and hydrophilic
cyanoꢀsubstituted compound 4c lost activity completely. This data indicated that the hydrophobic
substituents are more favored in the ring for potency improvement. In order to expand the SAR
and confirm the hydrophobic group requirement for activity, we made methyl substituted
compound 4d, which was found to be equipotent to the chloro substituted compound and thus
reinforced the requirement of hydrophobic substituents for potency. There was no significant
drop in potency when the spacer length of ether side chain was shortened from 5 to 4 atoms as
demonstrated by 5a, 5b, 5d and 6d; however, aliphatic and alicyclic groups, compounds 8a and
7b respectively, were not tolerated. Compounds from the first set of design make test and analyze
(DMTA) cycle showed high in vitro metabolic clearance due to the presence of metabolic soft
spots. Introduction of metabolically stable fluoro substitution at para position of the distal phenyl
ring (6d) resulted in a 2ꢀfold improvement in reducing rat in vitro metabolic CLint without
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compromising the potency, e.g., compound 5d (Rat hepatic CL = 394.6 ꢁL/min/10 cells) vs.
int
6
6d (Rat hepatic CL = 186.6 ꢁL/min/10 cells).
int
Table 1. SAR of substituted imidazo[1,2-a]pyridine ethers
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18
19
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22
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60
b
c
d
ATPS IC50
µM)
Mtb MIC
Cꢀ
a
No. R1
R2
R3
LogD
(
(µM)
Index
>62
4
a
H
Cl
H
H
H
H
H
H
H
F
0.13
<0.02
>100
0.03
0.08
0.04
0.05
0.02
1.6
<0.5
>250
<0.5
<0.8
<1
>5
4b
>144
~ 0.4
128
>4.3
>4.2
>4.3
>4.7
>4.1
>5.1
>5.2
4
c
CN
4
d
CH
3
5a
H
74
5
5
b
d
Cl
>18.5
>30.4
6
CH
<1.0
3.1
3
3
6d
CH
7
b
Cl
H
H
36.1
18.9
>200
>15
~0.5
~7
3.3
3.3
8
a
H
a
b
c
d
compound number; Myc_ATPS IC ; M. tuberculosis MIC; Cytotoxicity index.
5
0
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2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Optimization of the ether side chain
The ether side chain was optimized by keeping 2ꢀ(4ꢀfluorophenyl)ꢀ6ꢀmethyl substitution constant
on the imidazo[1,2ꢀa]pyridine ethers. Changing the distal phenyl to pyrimidine (13a) on the
terminal position of ether side chain resulted in reduced LogD and improved solubility (445 ꢁM),
but the compound lost potency significantly. However, replacing phenyl with 3ꢀpyridyl (14e) ring
was well tolerated, but the compound showed low cytotoxicity index. While the introduction of
methyl or fluoro substitution on distal pyridyl ring reduced the cytotoxicity, solubility did not
improve for the pyridyl and phenyl substituted compounds due to high Log D.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Table 2. SAR of ether side chain of imidazo[1,2-a]pyridine ethers
b
c
d
ATPS
Mtb MIC
(ꢁM)
Cꢀ
a
No.
R1
H
R2
R3
LogD
IC (ꢁM)
index
14.5
5
0
1
3a
H
F
5.6
6.9
2.9
3.9
1
4e
CH
3
0.16
<0.9
>8
15e
16e
CH
3
3
F
F
0.06
<0.6
<0.8
>39
>11
4.1
4.1
CH
<0.02
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1
7e
CH
3
F
0.09
6.3
~5
4.2
a
b
c
d
Compound number; Myc_ATPS IC ; M. tuberculosis MIC; Cytotoxicity index.
5
0
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12
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18
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To lower the lipophilicity and demonstrate a solubility handle for the series, the terminal oxygen
atom was modified to basic nitrogen through pKa modulation. The terminal ether of the side
chain modification with 1ꢀmethylpiperazine showed excellent improvement in solubility (674
µM) with diminished ATP synthesis inhibitory activity (compound 4d vs. 18d). However, 1ꢀ
phenylpiperazine substitution was tolerated (IC50 = 0.05 ꢁM) indicating the criticality of the
terminal aryl/heteroaryl substitution for the activity. Further modification of distal phenyl
piperazine into suitably substituted aniline/pyridine amines retained the potency (compounds 20e,
22e and 23e). Replacement of the terminal arylamine group by sulfonamide showed moderate
potency (24e). Compound 19e with 4ꢀFlouroꢀ3ꢀmethylphenyl group was found to be the best
substitution for IPE with IC = 5 nM; MIC = 31 nM and also had a 1000ꢀfold cytotoxicity index.
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1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 3. SAR of ether side chain of imidazo[1,2-a]pyridine ethers
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
b
c
d
ATPS IC50
Mtb MIC
(ꢁM)
Cꢀ
a
No.
R2
R3
LogD
(ꢁM)
index
<0.2
1
8d
H
F
F
6.4
0.005
0.06
>200
0.03
<0.5
2.4
4.1
3.9
1
9e
0e
1050
>113
2
2
2
1e
2e
F
F
0.05
0.08
<0.8
<0.8
>125
>125
4.0
4.2
23e
F
F
0.21
0.8
1.4
3.8
3.9
2
4e
0.7
1.6
ND
a
b
c
d
Compound number; Myc_ATPS IC ; M. tuberculosis MIC; Cytotoxicity index, ND: Not Done
5
0
The ether side chain was further optimized by changing the 3 carbon linker to 2 carbon linker.
The 3ꢀflouroꢀ4ꢀmethylbezyl substitution 28e was tolerated with IC = 0.07 ꢁM; MIC = 0.6 ꢁM
5
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with improved solubility of 22 ꢁM compared to 19e solubility of <1 ꢁM. The sulfonamide 29e
was moderately active, whereas the nicotinamide compound 30e was found to be weakly active
against ATP synthesis inhibition.
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
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
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Table 4. SAR of the chain length of imidazo[1,2-a]pyridine ethers
b
c
ATPS
Mtb MIC
(ꢁM)
a
d
No.
R2
Cꢀindex LogD
IC (ꢁM)
5
0
28e
0.07
0.6
>167
ND
4.0
2
3
9e
0e
0.4
7.2
3.1
4.1
3.4
>200
ND
a
b
c
d
Compound number; Myc_ATPS IC ; M. tuberculosis MIC; Cytotoxicity index, ND: Not Done.
50
In general, as shown in figure 4, the antiꢀtubercular potency (pMIC) correlated well with ATP
synthesis inhibition potency (pIC ) for the IPE series suggesting that the inhibiton of M.
5
0
tuberculosis growth was primarily through inhibition of ATP synthesis.
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Scatter plot of pIC (-log(Myc_ATPS IC ) vs pMIC (-log(Mtb_MIC)).
50
50
Mtb: M. tuberculosis
Structure activity relationship of squaramides series
One of the initial hits, 31c, belonging to squaramide series had demonstrated reasonable potency
both at enzymatic and cellular levels. In an attempt to understand the SAR (Figure 5) and
improve the cellular potency (M. tuberculosis MIC), few analogues were synthesized as
summarized in table 5.
Figure 5. Squaramide SAR
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Table 5: SAR of squaramides
b
c
ATPS IC50
Mtb MIC
(ꢁM)
a
No.
Structure
(
ꢁM)
10
11
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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
3
1c
0.3
6.2
50
3
1d
8.4
3
1e
66.6
0.03
>100
>25
100
0.5
3
1f
31g
>100
>100
3
1h
31i
0.77
9.5
3.12
3
1j
>100
a
b
c
Compound number; Myc_ATPS IC ; M. tuberculosis MIC
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1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Limited SAR exploration around squaramide series suggested that 2ꢀpyridylmethyl substitution
on right hand side (RHS) of the molecule is critical for achieving good enzyme and cellular
potency, whereas on the left hand side (LHS) of the squaramide core, morpholino phenyl
substitution gave the best activity. Replacement of the hydrophobic electron withdrawing CF3
group with hydrophilic cyano substitution on phenyl ring weakened the enzyme potency >200ꢀ
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
fold and cellular potency by 15ꢀfold (compound 31c vs 31e). Further, modification of CF to
3
morpholino ring improved both enzyme and cellular potency >10ꢀfold compared to the initial hit
(31c vs 31f). Improved potency observed with compound 31f may be due to the increased size
and the additional hydrogen bonding potential of morpholino ring. Having observed best potency
with 31f, we fixed morpholino phenyl substitution and started exploring SAR on the amino
group substitution. Replacement of 2ꢀpyridyl methyl with pyrazine ring weakened the enzyme
potency by 25ꢀfold, whereas replacement with benzyl group completely abolished the enzyme
potency (compounds 31f vs 31g and 31i). This modification indicated that the pyridine nitrogen
may be involved in critical hydrogen bonding interaction with the enzyme and thus essential for
maintaining good enzyme potency. An attempt towards restricting the flexibility of amino
methyl linker by alpha methyl substitution 31h and extending the molecule by adding a phenyl
group 31j made the resulting molecule inactive. These results indicated that there may be limited
space available for extending the molecule from the aromatic ring attached to amino portion of
squramide core.
IPE and SQA are specific and selective inhibitors of mycobacterial ATP synthesis
SAR studies established that the compounds in the two novel series inhibited mycobacterial ATP
synthesis. However, it was necessary to ensure that the inhibition observed is not due to
disruption of the proton gradient, an essential event in the process of ATP synthesis or due to
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nonꢀspecific perturbation of the membrane (IMV). During SAR studies, selected sets of
compounds from each of the two series were again profiled in the ꢁpH assay to eliminate the
potential proton gradient disruptors and also in a membrane damage assay to eliminate
compounds which would perturb integrity of the mycobacterial membrane. While the ꢁpH assay
was a modification of the Myc_ATPS assay, membrane damage assay was an adaptation from an
10
11
12
13
14
15
16
17
18
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assay reported earlier for Staphylococcus aureus. Mycobacterium bovis BCG (BCG) was used
as a surrogate in the cell based membrane damage assay due to good translation of MIC between
BCG and M. tuberculosis and the ease of handling BCG. A midꢀlog phase BCG culture was preꢀ
loaded with the fluorophore 3, 3’ꢀ Diethyloxacarbocyanine iodide (DiOC ) before exposure to
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compound. Stacked DioC molecules exhibit a red shift in the emission wavelength at 600 nm
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upon exposure to bacterial cells and an increase in fluorescence is correlated with an increase in
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membrane potential. Compounds like CCCP which dissipate membrane potential served as a
positive control in this assay. We found all the compounds tested had an IC of >100 ꢁM in the
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membrane damage assay with a 100ꢀ1000X window with the IC50 in the Myc_ATPS assay
Table S1). As the process of ATP synthesis is well conserved across both prokaryotes and
(
eukaryotes, to measure the selectivity with which the compounds were inhibiting mycobacterial
ATP synthesis over mammalian mitochondrial ATP synthesis, compounds were routinely tested
in a mammalian mitochondrial ATP synthesis assay (SMP_ATPS) which used subꢀmitochondrial
particles (SMP) from bovine heart mitochondria as a source of mitochondrial OxꢀPhos
components. The ratio of IC in the SMP_ATPS assay to that in Myc_ATPS assay (selectivity
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index) helped in designing molecules during each designꢀmakeꢀtestꢀanalyze (DMTA) cycle. As
seen in Table S1, a good selectivity index for compounds from SQA and IPE series suggested
them to be selective inhibitors of mycobacterial ATP synthesis. Additionally, all compounds
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were also tested for cytotoxicity either in A549 or THP1 cell line (MMIC) to get a measure of
cytotoxicity index (ratio of MMIC to MIC) at the cellular level with respect to M. tuberculosis
MIC. The compounds were also found to selectively inhibit mycobacterial growth as they failed
to inhibit the growth of two Gram positive and two Gram negative organisms tested in a
premilinary MIC assay (Table S2). Together, the data presented in Tables S1 and S2 indicated
that compounds of IPE and SQA series were specific and selective inhibitors of mycobacterial
ATP synthesis.
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Mode of action of SQA and IPE compounds
SQA resistant mutation mapped to subunits-a and c of M. tuberculosis ATP synthase
After the confirmation that the IPE and SQA compounds specifically and selectively inhibited
mycobacterial ATP synthesis pathway, we wanted to further narrow down their mechanism of
action to a particular component in the pathway. In order to identify the exact molecular target
getting inhibited by the SQA and the IPE series compounds, attempts were made to raise
spontaneous resistant mutants against compound 19e from IPE series and 31f from SQA series.
M. tuberculosis cells were plated on 7H10 plates supplemented with 4, 8 and 16ꢀfold MIC of 31f
and upto 100X MIC of 19e. While resistant mutants could not be raised against 19e probably due
to its bacteriostatic property (Table S3) and poor solubility, mutant colonies were obtained at a
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frequency of 10 for 31f. Two colonies from the 4X MIC plate and three from the 8X MIC plate
of 31f were profiled for resistant phenotype against key compounds from the SQA series and a
few reference inhibitors. All the colonies tested were resistant to all the squaramide compounds
(31c, 31i, 31f) with MIC shifting up >8ꢀfold, while the MIC shift for reference inhibitors like
isoniazid and rifampicin were less than or equal to 4ꢀfold (Table 6).
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Table 6: MIC modulation in SQA resistant mutants
Mtb MIC
µM
MIC (µM) in 31f resistant mutants
4.1 4.2 8.1 8.2 8.4
>100 >100 100 100 >100
Series
SQA
Compound
1c
31f
1i
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>100 >100 100 >100 >100
>100 >100 >100 >100 >100
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0.03 0.02 0.03 0.02 0.03
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Rifampicin
Isoniazid
Reference
Mtb: M. tuberculosis H37Rv
Genomic DNA prepared from all these five colonies were analysed by whole genome
sequencing for single nucleotide polymorphism (SNP). A number of observed SNPs were either
in nonꢀessential genes or occurred at very low frequency (data not shown). Only two significant
SNPs occured at high frequency in known essential genes. Table S4 summarizes these SNPs
wherein three of the mutations (K179N) mapped to subunitꢀa (encoded by atpB) of ATP
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synthase and two (D28N) to the subunitꢀc (encoded by atpE) of ATP synthase. SNP at Asp 28
position of subunitꢀc has been observed previously with bedaquiline mutants of M. tuberculosis,
M. fortuitum and M. abscessus, with the M. tuberculosis mutants exhibiting more than or equal
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to 16ꢀfold MIC upꢀshift. SQA compounds were also selective like bedaquiline with respect to
the inhibition of mycobacterial ATP synthesis. Good selectivity index observed with SQA
compounds in the SMP_ATPS assay over that of Myc_ATPS assay could now be explained
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based on the differences in the amino acid present at this particular position (28 amino acid)
between M. tuberculosis and mammalian mitochondrial ATP synthase subunitꢀc, Asp and Ile
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respectively (data not shown). Unlike the case with SQA, no mutations in the atpB gene
encoding subunitꢀa of Fo component of ATP synthase has been reported earlier for
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bedaquiline.
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Proposed binding mode of SQA
Bacterial ATP synthases have a lipophilic intramembrane portion (Fo) with subunit composition
of a b c
and a polar ATPꢀbinding region (F ) consisting of subunits α β γδε that extends into
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the cytoplasm.
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Recent breakthrough in crystallographic efforts revealed that bedaquiline primarily binds at the
interface of two subunitꢀc chains (chain A and chain B) of ATP synthase. Binding of bedaquiline
at this site causes a conformational change of Pheꢀ69 of subunitꢀc providing a hydrophobic space
for its quinoline moiety. Further, carbonyl group of Gluꢀ65 backbone anchors the bedaquiline’s
hydroxyl group through water mediated Hꢀbond. The binding of bedaquiline, thus plugs the
rotation of subunitꢀc chains (Figure 6A). Till date all the bedaquiline resistant M. tuberculosis
strains have the mutations mapped to only subunitꢀc and none to any other subunit of ATP
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synthase,
suggesting that bedaquiline primarily binds at the interface of two subunitꢀc chains
(chain A and chain B) and plugs their rotation.
SNP studies of SQA resistant strain suggested that both subunits ꢀc and ꢀa play a critical tole in
the binding of SQA compounds to ATP synthase. This encouraged us to study the possible
binding modes of SQA to ATP synthase through molecular modeling and generate hypotheses
for the observed SAR.
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Docking studies with SQA class of ATP synthase inhibitors
In order to understand the plausible mode of binding and a rationale for the observed SAR for
SQA compounds, we performed docking studies of SQA molecules over the a/cꢀring interface
construct of M. tuberculosis ATP synthase. This a/cꢀring construct was prepared using crystal
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structure of subunitꢀc ring bound with bedaquiline and the previously reported homology
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model of subunitꢀa, which was developed using E. coli ATP synthase (PDB ID : 1C17) as
template.
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SQA compounds were subjected to the InducedFit docking algorithm of the Schrodinger
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software package where the amino acid residues of proteins were also allowed to move freely
along with docked ligand. The receptor grid (active site of the protein) was generated using the
coꢀcrystallized structure of bedaquiline. The induced fit docking resulted in multiple binding
poses. Post docking analysis was performed to select the best binding pose where the ligands
were first clustered based on their common interactions with residues followed by docking score.
The highest docking score conformation among the largest populated cluster sharing common
interactions was selected as the best binding pose for these ligands.
The binding site interaction of the most active squaramide 31f in the a/cꢀring interface of M.
tuberculosis ATP synthase model is shown in Figure 6B. Pyridine ring and its nitrogen atom of
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1f was found to form πꢀπ interaction and Hꢀbond with Argꢀ186 of subunitꢀa respectively.
Furthermore morpholine oxygen atom was observed to interact with Pheꢀ69 of subunitꢀc through
water mediated Hꢀbond. These Hꢀbond contacts seem to provide firm anchoring to the molecule
at both ends. This diagonal binding of compound 31f at the interface of two subunitꢀc chains
(
chain A and chain B) and subunitꢀa of ATP synthase most likely plugs the rotation process of
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