1,4-azaindole, A potential drug candidate for treatment of tuberculosis

Article abstract of DOI:10.1128/AAC.03233-14

New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant (XDR) Mycobacterium tuberculosis are urgently required to combat the global tuberculosis (TB) threat. Toward this end, we previously reported the identification of 1,4-azaindoles, a promising class of compounds with potent antitubercular activity through noncovalent inhibition of decaprenylphosphoryl-β-D-ribose 2′-epimerase (DprE1). Further, this series was optimized to improve its physicochemical properties and pharmacokinetics in mice. Here, we describe the short-listing of a potential clinical candidate, compound 2, that has potent cellular activity, drug-like properties, efficacy in mouse and rat chronic TB infection models, and minimal in vitro safety risks. We also demonstrate that the compounds, including compound 2, have no antagonistic activity with other anti-TB drugs. Moreover, compound 2 shows synergy with PA824 and TMC207 in vitro, and the synergy effect is translated in vivo with TMC207. The series is predicted to have a low clearance in humans, and the predicted human dose for compound 2 is ≤1 g/day. Altogether, our data suggest that a 1,4-azaindole (compound 2) is a promising candidate for the development of a novel anti-TB drug. Copyright

Full text of DOI:10.1128/AAC.03233-14

AAC Accepts, published online ahead of print on 23 June 2014
Antimicrob. Agents Chemother. doi:10.1128/AAC.03233-14
Copyright © 2014, American Society for Microbiology. All Rights Reserved.
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1,4-azaindole: potential drug candidate for the treatment of tuberculosis
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Monalisa Chatterji,1*# Radha Shandil,2*# M. R. Manjunatha,3 Suresh Solapure,2 Vasanthi
Ramachandran,1 Naveen Kumar,2 Ramanatha Saralaya,2 Vijender Panduga,2 Jitendar
Reddy,2 Prabhakar KR,2 Sreevalli Sharma,1 Claire Sadler,4 Christopher B. Cooper,5 Khisi
Mdluli,5 Pravin S. Iyer,3 Shridhar Narayanan,1 and Pravin S. Shirude3*#
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1Department of Biosciences, IMED Infection, AstraZeneca India, Bellary Road, Hebbal, Bangalore-
560024, India
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2DMPK and Animal Sciences, IMED Infection, AstraZeneca, India
3Department of Medicinal Chemistry, IMED Infection, AstraZeneca, India
4Safety Assessment, IMED, AstraZeneca, Alderly Park, Mereside, UK
5Global Alliance for TB Drug Development, New York, NY, USA
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# Equal contribution
*Corresponding authors
monalisa.chatterji@astrazeneca.com,
pravin.shirude@astrazeneca.com
Tel: +91 80 66213000
radha.shandil@astrazeneca.com
and
Fax: +91 80 23621214
Abstract
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New therapeutic strategies against multidrug-resistant (MDR) and extensively drug-resistant
(XDR) Mycobacterium tuberculosis (Mtb) are urgently required to combat the global
tuberculosis (TB) threat. Towards this end, we have previously reported identification of 1,4-
azaindoles, a promising class of compounds with potent anti-tubercular activity through non-
covalent inhibition of decaprenylphosphoryl-β-D-ribose2’-epimerase (DprE1). Further, the
series was optimized to improve physico-chemical properties and pharmacokinetics in mice.
Here, we describe shortlisting of a potential clinical candidate, compound 2, that has potent
cellular activity, drug-like properties, efficacy in a chronic mouse and rat TB infection models
with minimal in vitro safety risks. We also demonstrate that the compounds including
compound 2, has no antagonistic activity with other anti-TB drugs. Moreover, compound 2
shows synergy with PA-824 and TMC207 in vitro and the synergy effect was translated in
vivo with TMC207. In addition, the series is predicted to have low clearance in human and
the predicted human dose for the compound 2 is ≤1gm/day. Together, our data suggests
that 1,4-azaindole (compound 2) is a promising candidate for development of a novel anti-TB
drug.
INTRODUCTION

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Studies have revealed Mycobacterium tuberculosis (Mtb), an opportunistic intracellular
bacterium, has originated thousands of years ago; genetic analysis of more than 250
contemporary strains, indicate its origin with the emergence of Homo sapiens in Africa more
than 70,000 years ago.1 The spread, evolution and existence of the bacterium to this day, is
an evolutionary success, as well as, a serious threat to humanity.1 Even today, tuberculosis
(TB) is a leading cause of mortality in the world, second only to HIV.1 WHO reported 1.3
million deaths in 2012.2 TB has emerged as a global health emergency as Mtb has acquired
resistance to every drug that has been introduced against it.3 Moreover, multi- (MDR) and
extreme- (XDR) drug resistant strains have made TB therapy ineffective, moving the disease
back to the pre-antibiotic era. The long duration of therapy and associated toxicities, has
challenged patients’ compliance and further assisted development of resistance. A
simplified, relatively safe and shorter duration of therapy is required to combat TB threat.4
Drugs that work by novel mechanisms hence, active on drug resistant strains are urgently
needed to treat patients with drug resistant TB.
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Mtb has a characteristic and complex cell wall architecture involved in multiple functions
related to cellular physiology and pathogenesis.5 Isoniazid, a critical drug in the first line
therapy, inhibits biosynthesis of mycolic acid, a critical component of cell wall.6,7 Ethambutol,
another drug in the first line therapy, also inhibits cell wall synthesis.6,7 Enzymes that are
involved in cell wall biogenesis and/or function have emerged as potential targets for
discovery of antimycobacterials, one such target is decaprenylphosphoryl-β-D-ribose2’-
epimerase (DprE1).6,7 DprE1 along with DprE2 catalyzes the epimerization of
decaprenylphosphoryl-D-ribose to decaprenylphosphoryl-D-arabinose, the sole arabinose
donor for cell wall synthesis.8 A number of DprE1 inhibitors have been reported in the
literature that are bactericidal in vitro and in mouse TB infection models, building confidence
for this target.9-12 Benzothiazinones (e.g. BTZ043), covalent inhibitors of the DprE1 enzyme,
were instrumental in the discovery of the target.9 The nitro-group of this scaffold is converted
to nitroso subsequently forming a covalent link with cysteine at 387 position of the enzyme.9
PBTZ, an improved analogue of BTZ043 has been shown to be effective in an animal model
for TB with a synergistic effect in combination with TMC207.12 PBTZ is in the pre-clinical
phase of development under the sponsorship of iM4TB (Innovative Medicines for
Tuberculosis). TCA1, a non-covalent inhibitor of DprE1 also inhibits MoeW - enzymes
involved in cell wall and molybdenum cofactor biosynthesis.10 TCA1 is currently in a lead
optimization phase as part of TB alliance portfolio.13
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We have previously reported a novel class of DprE1 inhibitors, 1,4-azaindoles.11 This series
emerged from a scaffold morphing effort and demonstrated potent antimycobacterial activity

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and were non-covalent inhibitors of DprE1. In addition, the target site mutations conferring
resistance against azaindoles and BTZ043 were distinct from each other. While C387S/G is
the primary residue involved in BTZ043 resistance, Y314H imparts resistance to azaindoles.
Cross-resistance was not observed between azaindoles and BTZ043, and azaindoles were
found to be equally active on drug sensitive and isoniazid/rifampicin resistant strains. A
robust understanding of the structure activity relationship (SAR) was used to optimize the
series.
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1,4-azaindoles possess small molecular weight, low logD, excellent permeability, no CYP
inhibition, good oral exposures, low in vivo clearance (CL) in rats & dogs and low predicted
human CL, and no major safety liabilities as assessed by a spectrum of in vitro assays.
Moreover, the synthesis of compounds is not complex hence, expected to have low cost of
goods. Hence 1,4-azaindoles is an attractive class for further development towards TB
treatment. During lead optimization, low solubility, high mouse specific clearance and weak
phosphodiesterase6 (PDE6) inhibition were mitigated based on SAR understanding of 1,4-
azaindoles. This has led to the identification of a potential drug candidate for TB treatment.
In this report, the in vivo efficacy of the candidate drug in a chronic mouse and rat model of
tuberculosis has been demonstrated along with its synergy with TMC207. The minimal
safety risk as per the in vitro secondary pharmacology data, no adverse observations during
efficacy study, drug like properties and acceptable predicted human dose, makes 1,4-
azaindole a promising candidate for development.
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Materials and methods:
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Synthesis of selected compounds
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N-(2-hydroxyethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-
pyrrolo[3,2-b]pyridine-3-carboxamide (1):
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1-((6-methoxy-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-pyrrolo[3,2-b]pyridine-3-
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carboxylic acid (75 mg, 0.24 mmol) was added to dichloromethane (10 mL) to give
a
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suspension. Triethyl amine (66.9 mL, 0.48 mmol) was added, followed by 1-
propanephosphonic acid cyclic anhydride (286 mL, 0.48 mmol). The reaction mixture was
stirred at RT for 5 minutes. Ethanol amine (29.0 mL, 0.48 mmol) was added and the mixture
was stirred at RT overnight. After completion of the reaction, the mixture was diluted with
DCM, washed with water and brine. The DCM layer was dried over sodium sulphate and
evaporated to give the crude product. The crude product was was purified by silica gel
column chromatography using MeOH in DCM to afford N-(2-hydroxyethyl)-1-((6-methoxy-5-
methylpyrimidin-4-yl)methyl)-6-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide as a white
solid (1) (Yield 53%).

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ES+ MS m/z: 356.4 (M+1)
1H NMR (300 MHz, DMSO-d6) δ ppm: 2.23 (s, 3 H) 2.39 (s, 3 H) 3.39 - 3.65 (m, 4 H) 3.93 (s,
3 H) 4.84 (t, J=5.09 Hz, 1 H) 5.63 (s, 2 H) 7.74 (s, 1 H) 8.12 (s, 1 H) 8.33 (s, 1 H) 8.41 (s, 1
H) 8.80 (t, J=5.65 Hz, 1 H).
1-((6-(dimethylamino)-5-methylpyrimidin-4-yl)methyl)-N-(2-hydroxyethyl)-6-methyl-1H-
pyrrolo[3,2-b]pyridine-3-carboxamide (2):
To a stirred solution of 1-((6-(dimethylamino)-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-
pyrrolo[3,2-b]pyridine-3-carboxylic acid (7g, 21.51 mmol) in DMF (70 mL), Ethanol amine
(1.97 g, 32.265 mmol), triethylamine (8.99 mL, 64.53 mmoles) and HATU (9.81 g, 25.81
mmoles) were added and the mixture was stirred at room temperature for 2 h. Then the
reaction mixture was poured into water (100 mL) and extracted with ethyl acetate (2 X
200mL). The organic layer was washed with brine and concentrated under reduced
pressure. The crude product was purified by silica gel column chromatography using MeOH
in DCM to afford 1-((6-(dimethylamino)-5-methylpyrimidin-4-yl) methyl)-N-(2-hydroxyethyl)-6-
methyl-1H-pyrrolo [3, 2-b] pyridine-3-carboxamide as a white solid (2) (Yield 60%).
ES+ MS m/z: 369.4 (M+1)
1H NMR (400MHz ,DMSO-d6) δ ppm: 2.27 (s, 3 H), 2.39 (s, 3 H), 2.95 (s, 6 H), 3.45 (q, J=
5.7 Hz, 2 H), 3.54 (q, J= 5.2 Hz, 2 H), 4.81 (t, J= 5.0 Hz, 1 H), 5.52 (s, 2 H), 7.73 (s, 1 H),
8.09 (s, 1 H), 8.22 (s, 1 H), 8.32 (d, J= 1.3 Hz, 1 H), 8.79 (t, J= 5.6 Hz, 1 H).
Microbiology and biochemical assays
Assays to determine cellular activity (MIC, MBC), potency in intracellular THP1 model, and
IC50 fo DprE1 assay was carried out a described before.11 IC50 is compound concentration
that results in 50% inhibition of maximum enzyme activity. MIC referes to the minimal
compound concentratiosn that is able to reduce cell growth by 80% or more compared to the
untreated cells in 7 days, MBC refers to minimal compound concentartion that shows ≥2 log
reduction in colony forming units in 7 days compared to cells at the start of the treatment and
DprE1. For MIC determination 14 drug sensitive clinical isolates, 8 isoniazid (INHR) and one
rifampicin resistant (RIFR) strains were used.
In vitro combination
Activity of 1,4-azaindole in combination with known anti-TB drugs was determined by
checkerboard titration method.14 Briefly, in a 96-well microtiter plate, one drug was diluted
row wise and the second drug was diluted to obtain various combinations of two drugs in a
final volume of 100 µL. Each well of the micro-titre plate was inoculated with 100 µL of Mtb

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H37Rv culture at 5 x 105 CFU/ml. The plates were incubated for 7 days at 37 0C. To
evaluate whether the paired combinations of agents had additive, synergistic, antagonistic or
indifferent effect in inhibiting Mtb, the fractional inhibitory concentration index (ΣFIC) using
the following formula was calculated.
ΣFIC = FA + FB
FA = MIC of drug A in combination/MIC of drug A alone
FB = MIC of drug B in combination / MIC of drug B alone
The synergy was defined as ΣFIC of <0.5, antagonism as a ΣFIC of >4.0, and
additivity/indifference (no interaction) as ΣFIC values between 0.5 and 4.0.
In a combination of two drugs – Indifference equals to the effect of the most active
component, additive equals to the sum of the effects of the individual components, synergy
equals to the effect of the combination that exceeds the additive effects of the individual
components and antagonism equals to the reduced effect of the combination in comparison
with the effect of the more potent individual substance.
All combinations showing a ΣFIC value of <1 were further plated to estimate CFUs. The log10
CFU reduction in 7 days was calculated with respect to untreated control.
In vitro DMPK and safety assays
In vitro DMPK assays to measure aqueous solubility, LogD, plasma protein binding,
metabolic stability (mouse/human microsomal CLint; rat, dog, human hepatocyte CLint), as
well as, safety assays to measure inhibition of hERG channel, Cytochrome P450 enzymes
(CYPs), and a panel of ~65 mammalian targets have been described earlier.11
Pharmacokinetics (PK) and efficacy in animal models (rats and mice)
Methods used for determining the pharmacokinetics in mice and rats after intraveneous
(IVPK) and or oral administration (POPK) of drug, as well as, in vivo efficacy in a mouse and
rat aerosol infection models of TB have been described earlier.11,15 In both the models,
treatment was initiated after four weeks of infection and the total duration of treatment was
for 4 weeks (6 days/week). Animals were sacrificed and viable bacterial counts in the lungs
were enumerated.
Pharmacokinetics (PK) in dog
PK was analysed in healthy Beagle dogs, one male and one female dog per dose group.
For intravenous PK (IVPK), a dose of 5 µmol/kg of the test compound (1) was administered
as solution containing 5% ethanol, 30% PEG-200 & 65% phosphate buffered saline (PBS)
by infusion. For oral PK (POPK), 5 µmol/kg of the compound (1) was administered in a
suspension containing 0.5% hydroxypropyl methycellulose (HPMC) and 0.1% Tween in

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water. Blood samples were collected at 5, 10, 20, 40 mins, 1, 2, 4, 6, 12 h for IVPK and 10,
20, 40 mins, 1, 2, 3, 4, 6, 12 h for POPK. Blood samples were centrifuged to collect plasma
and samples were stored at -200C until analysis using LC-MS/MS.
Prediction of in vivo CL from in vitro CLint
Assuming major route of elimination to be through hepatic metabolism, blood CL in
preclinical species, rat and dog was predicted from in vitro CLint (by in vitro-in vivo correlation
or IVIVC). In vitro CLint was estimated by incubating the test compound with rat or dog
hepatocytes. In vitro CLint was scaled to in vivo CLint as follows.
CLint in vivo = CLint in vitro * scaling factors * fublood/fuinc
The liver weight used for scaling was 40, 32 & 24 g/kg body weight in rat, dog and human
respectively. Hepatocellularity used for scaling was 163, 169 & 120 (X 106) cells/g liver in
rat, dog and human respectively. The fublood or free fraction in blood and fuplasma (unbound
fraction in plasma) were assumed to be similar, while the unbound fraction in the hepatocyte
mix, fuinc was predicted using physicochemical properties.16 Slope and intercept from the
linear regression analysis of IVIVC for a large number of AstraZeneca compounds were
applied for final correction in the scaled in vivo CLint. For prediction of CL, a well stirred
model was used, equation as described below.
Qh × predicted CLintin vivo
Predicted CL =
Qh + predicted CLintin vivo
Where, Qh is the liver blood flow. The values of Qh in rat, dog and human are 72, 55 & 20
mL/min/kg in rat, dog and human, respectively.16
Human dose prediction by allometry
Allometry exponent (b) and coefficient (a) were estimated by linear regression analysis of log
(CL) vs. log (body weight or BW) plot.
CL = a× BW b
Equation for simple allometry:
The steady-state daily AUC required for 1.0 log10CFU reduction in the chronic rat TB
infection model, oral bioavailability (F = ratio of dose normalized AUCs after oral and IV
administration) in rat and human CL predicted by IVIVC were used for human dose
prediction as shown below.
Predicted human dose = (Efficacious AUC in rat*Predicted Hu CL)/F
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Results and discussion
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Selection of potential drug candidate and properties
The 1,4-azaindole series was identified by scaffold morphing approach as described
previously.15 The potency of these compounds against Mtb was mainly improved through

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MIC-based SAR with three points of diversification (amide side chain, a hydrophobic group,
and core ring substitutions). The secondary amide, referred to as the amide side chain is
essential for potency. It could be involved in hydrogen bonding interaction with the target
enzyme, DprE1. Small hydrophobic or hydrophilic amides like methyl cyclopropyl, fluoro-
ethyl or hydroxy ethyl amides were preferred for MIC. The hydrophobic group tolerated
various substituted benzyl and heteroaryl-methyl groups; however, monosubstituted benzyl
groups were less favoured than disubstituted benzyl groups. Substitution of a methyl or
methoxy group at the C-6 position of the 1,4-azaindole improved cellular potency.
Although 1,4-azaindoles showed excellent in vitro and in vivo efficacy against Mtb, these
compounds had poor aqeous solubility, showed high CL in mouse and weakly inhibited the
host PDE6 enzyme activity which could potentially lead to visual acuity. Therefore, the major
focus during lead optimization was to improve these properties while retaining Mtb potency.
The hydroxyethyl amide side chain was crucial to improve aqueous solubility and modulate
physicochemical properties of 1,4-azaindoles. In addition, the hydroxyethyl amide side chain
showed a significant improvement in mouse liver microsome (MLM) stability relative to
hydrophobic amide side chains. The improved MLM stability was attributed to the
compounds with lower lipophillicity as indicated by measured logDs of 2 or less. Structurally
diverse compounds from 1,4-azaindole series were profiled for in vitro PDE6 screening to
understand the PDE6 SAR. The result revealed that 6-methoxy-5-methylpyrimidine-4-yl
group at hydrophobic pocket was the primary cause of PDE6 activity. It was demonstrated
that replacement of 6-methoxy-5-methylpyrimidine-4-yl substituent with 6-(dimethylamino)-5-
methylpyrimidine-4-yl and 6-(difluoromethoxy)-5-methylpyrimidine-4-yl groups helped to
mitigate PDE6 activity.
Thus, overall from an initial hit, 1,4-azaindole series was optimized to have molecules with
small molecular weight, low logD, nanomolar DprE1 IC50s, sub-micromolar MICs, excellent
permeability, no CYP inhibition, good oral exposures, low clearance in mice, rats, dogs &
humans and no major safety liabilities. Relatively straighforward synthesis of 1,4-azaindoles
with no chiral centers ensures low cost of goods, an essential attribute for an anti-TB drug.
Synthesis and characterization of ~250 compounds during lead optimization has led to the
identification of compound 2, as a potential drug candidate for TB (Figure 1).
The specific properties of compound 2 along with an early tool compound 1 are shown in
Table 1. Both the compounds have logD < 2, aqueous solubility >100 µM, similar fuplasma
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nanomolar IC50s against Mtb DprE1 enzyme and potent MICs and MBCs against Mtb. These
compounds were equally active against drug sensitive and drug resistant clinical isolates of
Mtb. They were also active against intracellular Mtb and showed ~1.5 Log10CFU reduction
inside the infected THP1 cells (Table 1, Supplemental Figure 1). On the basis of in vitro

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intrinsic clearance (CLint) using hepatocytes from rat and dog, the predicted in vivo clearance
(CL) for compounds 1 & 2 ranged between 11 and 30% of liver blood flow (% LBF),
indicating low clearance (Table 2). Thus, low CLint in hepatocytes correlated with observed
low CL (< 35% LBF) in rat and dog. Good correlation between the predicted in vivo CL and
observed in vivo CL supported our assumption that hepatic clearance could be the primary
route of elimination of these compounds. IVIVC predicted low CL in human for both the
compounds. High permeability in the Caco2 assay as well as > 85% oral bioavailability in
rat/dog predicts excellent bioavailability in human. Moreover, TB being
a chronic
combination therapy, understanding of safety profile is critical. Cytotoxicity assay in THP-1
(human monocytic cell line) cells showed no inhibition at up to 100 µM. Compounds 1 & 2
did not inhibit the hERG channel at up to 33 µM concentrations, suggesting a low risk of
cardiovascular toxicity. Compounds in this series, including 1 & 2, did not inhibit any of the
cytochrome P450 (CYP450) isoenzymes. A panel of mammalian targets associated with
serious safety liabilities were used to test for inhibition by 1,4-azaindoles. None of the targets
were significantly inhibited except weak inhibition of phosphodiesterase6 (PDE6) enzyme by
compound 1 (PDE6 IC50 4µM). As described above, mitigation of PDE6 inhibition (PDE6 IC50
>100 µM) through systematic medicinal chemistry exploration resulted in the identification of
compound 2.
In vitro combination study with known TB drugs and clinical candidates
Compounds 1 and 2 were tested in combination with various first & second line anti-TB
drugs and clinical/pre-clinical TB candidates in vitro namely, isoniazid, rifampicin,
ethambutol, streptomycin, moxifloxacin, imipenem, meropenem, BTZ043, bedaquiline
(TMC207), and PA824. Antagonism was not observed for any combination tested in this
study. For M. tuberculosis, translation of ΣFIC to bactericidality (reduction in CFU) was used
to rank order combinations. FIC values were used as a criterion to select combinations for
CFU plating. All combinations with ΣFIC <1 were plated for estimation of reduction in CFU
compared to individual drugs treated at MIC. Maximum synergy for compound 2 based on
CFU estimate, was observed with PA824 and TMC207 (Figure 2), which was synergistic
with PA824 (ΣFIC = 0.5 - 0.625) and additive with TMC207 (ΣFIC = 0.625) based on FIC.
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The combination of 2 with TMC207 at sub-MICs showed an additional ~1.5 log10CFU
reduction over maximum individual drug effect (at MIC). Similarly, an additional ≥4 log10CFU
reduction was observed with PA824 (Figure 2). In addition, the combination with
meropenem, rifampicin and streptomycin showed weaker synergy with the compound 2
(Supplemental Figure 2). Similar results were also observed with other compounds from 1,4-
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azaindole series published earlier.11 Interestingly, BTZ043, which also targets DprE1
enzyme, exhibits synergy with TMC207.12,17 The absence of any antagonism with a variety of
tested anti-TB drugs supports the potential of 1,4-azaindoles for combination therapy.
Efficacy and in vivo combination study in a mouse TB infection model
The in vivo efficacy of a representative compound (2) was assessed in BALB/c mice in a
chronic TB infection model, wherein the treatment was started on day 28, post-infection.
After four weeks of treatment with compound 2, the bacterial burden in the lungs was
reduced by ~1 log10CFU/lung at 300 mg/Kg and statistically significant dose dependent
efficacy was observed (Figure 3). The oral exposure of compound 2, assessed from infected
animals showed AUCs of 243 µM.h and total plasma concentrations above the MIC for ~ 15
h at the dose of 300 mg/kg (Table 3). Moreover, the compound 2 (at 100 mg/kg) was also
tested for in vivo combination efficacy with PA824 (50 mg/kg) and TMC207 (25 mg/kg) in the
mouse chronic TB infection model. The bacterial burden in the lungs was reduced by ~0.7
and ~3.5 log10CFU/lung in case of combination with PA824 and TMC207 respectively. Thus,
the synergy observed for 1,4-azaindole (2) with TMC207 (as estimated by CFU reduction,
Figure 2) in vitro translated to in vivo. However, this was not true for the combination with
PA824. The oral exposure of compound 2 assessed from infected animals did not change
significantly when given in combination with PA824 and TMC207 (Table 3). Similarly, the
oral exposures of PA824 and TMC207 remained unchanged when given in combination with
the compound 2 (Supplemental Figure 3 and Supplemental Table 1). Although streptomycin
and meropenem demonstrated synergy in vitro, they were not evaluated in vivo due to being
injectable and observed stability issues respectively. No adverse events were observed
during 4 weeks of repeated dosing during the study.
Correlation of CL across species
Pharmacokinetic profile of compound 1 as a representative of the series in mice, rats and
dogs was determined post IV and oral dosing. The compound 1 showed an oral
bioavailability of 86 and 100 % in rats and dogs respectively. Interestingly, the observed
systemic CL was within 1.5 fold of the predicted value in both rats and dogs, confirming the
hypothesis that hepatic route is indeed the primary route of elimination (Table 2). The
predicted vs observed in vivo CL values were within 1 fold for compound 2 in rats (Table 2).
Hence, 1,4-azaindoles showed good in vitro to in vivo correlation. Further, human CL was
predicted using IVIVC as described in materials and methods. Both the compounds (1 & 2)
were predicted to show low human CL (CLh: < 30% liver blood flow).
Using the observed CL in rat and dog, human CL for compound 1 was also predicted by
simple, two species (rat & dog) allometry (Supplemental Table 2). Predicted mean estimates

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of human CL obtained by allometry (3.3 ml/min/kg) and IVIVC (2.9 ml/min/kg) were very
similar. Steady-state daily AUC required for ≥1.0 log10 CFU reduction in a chronic rat TB
model and predicted human CL, predicted ≤1 gm per day as an efficacious human dose,
assuming complete bioavailability for compound 2 (Table 4). This is a point estimate of the
predicted human dose without considering potential PK variability.
Efficacy study in a rat TB infection model
As described before, a significant challenge associated with the 1,4-azaindole series was
rapid metabolism in the presence of mouse liver microsomes. This made it difficult to explore
efficacy in mouse model of tuberculosis for compounds from 1,4-azaindole series. However,
all compounds in the series had low clearance and significant oral exposures in rats. This
allowed us to assess representative compounds for in vivo efficacy in a rat “chronic” TB
infection model as an alternate model to well established mouse model. After four weeks of
treatment with compounds 1 & 2, the bacterial burden in the lungs was reduced by 0.5 to ≥ 1
log10CFU/ left lobe of the lung and statistically significant dose dependent efficacy was
observed (Figure 4). The oral exposure of compounds 1 & 2, assessed from infected
animals showed AUCs ranging from 166 to 240 µM•h and free plasma concentrations were
maintained above the MIC for 10−24 h (Table 5). No adverse events in the form of body
weight loss, organ weight loss, or gross pathology were observed following 4 weeks of
repeated dosing in the efficacy studies.
In summary, the 1,4-azaindole (2) was identified as a potential clinical candidate for TB
treatment. It also showed synergy with TMC207 and no antagonism with other anti-TB
drugs.

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Tables
TABLE 1. The properties of shortlisted compounds from 1,4 azaindole series.
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aCYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4, bpanel of ~65 high and medium severity
Compound
1
2
DprE1 IC50 (µM)
0.010
0.032
Mtb MIC (µM)
0.78-3.12
1.56
0.5-1.56
1.56-3.12
0.4-3.12
0.78-1.56
200
Mtb MBC (µM)
Drug sensitive clinical isolates MIC (µM)
INHR, RIFR clinical isolates(µM)
DprE1 over-expression strain MIC (µM)
DprE1 C387S/G (BTZ043R) MIC (µM)
Potency in intracellular THP1 model
(log10CFU reduction)
0.4-6.25
0.78-3.12
50-200
0.78-1.56
0.78-1.56
~1.5
~1.5
Cytotoxicity (µM) THP-1
logD
>100
1.8
>100
1.8
Solubility (µM)
170
464
Microsome CLint (µl/min/mg)
Mouse
28
38
CLpred (ml/min/kg) (% LBF)
16 (11)
<4
28 (18)
10
Microsome CLint (µl/min/mg)
Human
CLpred (ml/min/kg) (% LBF)
4 (19)
4
6 (30)
5
Hepatocyte CLint (µl/min/106 cells)
Rat
CLpred (ml/min/kg) (% LBF)
10 (14)
12 (16)
Hepatocyte CLint (µl/min/106 cells)
<1
9 (16)
<1
<1
8 (15)
<1
Dog
CLpred (ml/min/kg) (% LBF)
Hepatocyte CLint (µl/min/106 cells)
Human
CLpred (ml/min/kg) (% LBF)
2 (9)
0.22
11
2 (11)
0.30
5
Human Fuplasma
Caco-2 Papp A to B (1E-6.cm/s)
CYPa inhibition (time dependent inhibition)
IC50 (µM)
>50
>50
hERG IC50 (µM)
>33
4
>33
PDE6 IC50 (µM)
>100
Secondary pharmacology hits IC50 (µM)
No significant hitsb
CL (ml/min/kg) (% LBF)
Rat PK
27 (38)
100
24 (33)
87
F (%)

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targets (binding and functional data) - IC50 >100 µM or >30 µM
TABLE 2. Pharmacokinetic parameters of compound 1 & 2 in rats and dogs
Measured
Predicted
Fold under
Animal Hepatocyte CLint
model (µl/min/106 cells)
Compound
plasma CL in
Blood CLh prediction of
vivo (ml/min/kg) (ml/min/kg)
CL
Rat
Dog
Rat
5.8
1
27
6
20.6
8
1.31
0.75
1.02
1
2
4.8
23.7
23.3
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434
435
436
TABLE 3. Pharmacokinetic parameters (Mean ± SD, n=3) of 2 following multiple oral dose
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administration in Mtb infected male BALB/c mice.
PK Parameter
Dose (mg/kg)
Cmax (µM)
2
2 + PA824
100 + 50
26.3 ± 3.6
53.1 ± 7.4
0.3 ± 0
2 + TMC207
100 + 25
33.7 ± 3
30
100
300
25.8 ± 4.1
13.4 ± 2.1
0.25 ± 0
1.2 ± 0.1
33.6 ± 5.8
65.3 ± 10.7
0.5 ± 0
64.9 ± 8.4
243 ± 59.1
0.3 ± 0.1
2 ± 0.4
AUClast (µM.h)
Tmax (h)
46.3 ± 1.7
0.3 ± 0
T1/2 (h)
2.1 ± 0.2
2.7 ± 0.2
2.8 ± 0.4
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439
440
TABLE 4 . Predicted mean estimates of human CL and dose assuming oral F = 1.0.
Efficacious dose
for ≥1 log CFU
reduction in rat
model (mg/kg)
100
Efficacious
AUC24 in
infected rats
(µM.h)
MW
Predicted
Human CL
(ml/min/kg)
Predicted
Human dose
(mg/70 kg)
Compound
2
368.4
166
3.6
925
441
442
443
444
TABLE 5. Pharmacokinetic parameters (Mean ± SD, n=3) of 1 & 2 following multiple oral
dose (100 mg/kg) administration in Mtb infected male Wistar rats.
Compounds
PK parameter
1
2
Dose (mg/kg)
Cmax (µM)
100
100
46.1 ± 7.7
986.4 ± 274.3
31.5 ± 3.0
166.1 ± 43.9
AUClast (µM.h)

Tmax (h)
T1/2 (h)
5.3 ± 1.2
3.8 ± 0.6
2.0 ± 0.0
4.6 ± 0.0
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448
449
450
451
452
453
Figure Legends
FIG 1: Shortlisted compounds from 1,4 azaindole series.
FIG 2: In vitro combination study of compound 2 with TMC207 and PA824. For PA824 CFU
recovered were <200 per ml (LOQ). Fractional MICs, FA and FB are given in parenthesis for
each drug (*X = fold MIC).
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467
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471
472
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474
FIG 3: A. Summary of efficacy of 2 alone and in combination with PA824 & TMC207 in a
chronic TB infection model in BALB/c mice following 6 days a week oral dosing for four
weeks. The net log10CFU/lung reduction was obtained by subtracting lung bacterial counts
from the vehicle treated control. Compound 2 exhibited statistically significant effect at 300
mg/kg vs untreated controls (*p<0.05). The bacterial counts at the start of treatment (early
control) and at the end of treatment (late control) were 6.0 ± 0.08 log10CFU/lung and 6.24 ±
0.12 log10CFU/lung respectively. B. Time vs. concentration profiles of 2 and in combination
with PA824 and TMC207 following multiple oral administrations at 30, 100 & 300 mg/kg in
chronically Mtb infected mice.
FIG 4: A. Summary of efficacy of 1 & 2 in chronic TB infection model in Wistar Rats following
6 days a week oral dosing for four weeks. The net log10CFU reduction/left lobe of the lung
was obtained by subtracting bacterial counts from the vehicle treated control. Both
compounds exhibited statistically significant effect vs untreated controls (*p<0.05). The
bacterial counts at the start of treatment (early control) and at the end of treatment (late
control) were 6.02 ± 0.11 log10CFU/lung and 6.41± 0.20 log10CFU/lung respectively. B. Time
vs. concentration profiles of 1 & 2 following multiple oral administrations at 100 mg/kg in
chronically Mtb infected rats.

Fig. 1

Fig. 2

Fig. 3A

Fig. 3B
4

Fig. 4A

Fig. 4B
6