Azaindoles: Noncovalent DprE1 inhibitors from scaffold morphing efforts, kill Mycobacterium tuberculosis and are efficacious in vivo

Article abstract of DOI:10.1021/jm401382v

We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to noncovalently inhibit decaprenylphosphoryl-β-D-ribose2′-epimerase (DprE1). With "drug-like" properties and no expectation of pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-resistant tuberculosis.

Full text of DOI:10.1021/jm401382v

Article
pubs.acs.org/jmc
Azaindoles: Noncovalent DprE1 Inhibitors from Scaffold Morphing
Efforts, Kill Mycobacterium tuberculosis and Are Efficacious in Vivo
Pravin S. Shirude,*,†,# Radha Shandil,§ Claire Sadler,∥ Maruti Naik,† Vinayak Hosagrahara,§
Shahul Hameed,† Vikas Shinde,† Chandramohan Bathula,† Vaishali Humnabadkar,‡ Naveen Kumar,§
Jitendar Reddy,§ Vijender Panduga,§ Sreevalli Sharma,‡ Anisha Ambady,‡ Naina Hegde,‡
James Whiteaker,⊥ Robert E. McLaughlin,⊥ Humphrey Gardner,⊥ Prashanti Madhavapeddi,‡
Vasanthi Ramachandran,‡ Parvinder Kaur,‡ Ashwini Narayan,‡ Supreeth Guptha,‡ Disha Awasthy,‡
Chandan Narayan,‡ Jyothi Mahadevaswamy,‡ KG Vishwas,§ Vijaykamal Ahuja,§ Abhishek Srivastava,§
KR Prabhakar,§ Sowmya Bharath,§ Ramesh Kale,† Manjunatha Ramaiah,† Nilanjana Roy Choudhury,†
Vasan K. Sambandamurthy,‡ Suresh Solapure,§ Pravin S. Iyer,† Shridhar Narayanan,‡
and Monalisa Chatterji*,‡,#
†Department of Medicinal Chemistry, IMED Infection, AstraZeneca India, Bellary Road, Hebbal, Bangalore 560024, India
‡Department of Biosciences, IMED Infection, AstraZeneca, Bangalore 560024, India
§DMPK and Animal Sciences, IMED Infection, AstraZeneca, Bangalore 560024, India
∥Safety Assessment, IMED Infection, AstraZeneca, Alderly Park, Mereside, U.K.
⊥Translational Science, IMED Infection, AstraZeneca, Waltham, Massachusetts, United States
S
* Supporting Information
ABSTRACT: We report 1,4-azaindoles as a new inhibitor class that kills Mycobacterium tuberculosis in vitro and demonstrates
efficacy in mouse tuberculosis models. The series emerged from scaffold morphing efforts and was demonstrated to
noncovalently inhibit decaprenylphosphoryl-β-D-ribose2′-epimerase (DprE1). With “drug-like” properties and no expectation of
pre-existing resistance in the clinic, this chemical class has the potential to be developed as a therapy for drug-sensitive and drug-
resistant tuberculosis.
lives needs to be evaluated in the context of associated safety
risks and the burden of post marketing studies.3
INTRODUCTION
■
Tuberculosis (TB) continues to cause considerable morbidity
and mortality worldwide despite an effective and economical
quadruple drug therapy regimen put in place 40 years ago.1,2
However, long duration of therapy, associated toxicity, and
emergence of multidrug-resistant tuberculosis (MDR-TB) has
created an urgent need for new drugs. The U.S. Food and Drug
Administration’s accelerated approval of Janssen’s Sirturo
(bedaquiline) for MDR-TB ended a four-decade-long drought
of a new TB drug with a novel mechanism of action.3 However,
the impact of Sirturo on the disease landscape and patient’s
Nitro-benzothiazinones (BTZs) are known inhibitors of
decaprenylphosphoryl-β-D-ribose2′-epimerase (DprE1). DprE1
is involved in the conversion of decaprenylphosphoryl-β-D-
ribose (DPR) to decaprenylphosphoryl-β-D-arabinofuranose
(DPA), a precursor of mycobacterial cell wall arabinan.4 This
reaction is catalyzed by a heteromeric enzyme decaprenyl-
phospho-ribose 2′-epimerase (DprE), via a sequential oxida-
Received: September 10, 2013
© XXXX American Chemical Society
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Figure 1. Identification of 1,4-azaindoles. (a) Scaffold morphing: 1, imidazolopyridine; 2 1,4-azaindole, initial hit; 3, 1,4-azaindole-optimized
compound. (b) Structure−activity relationship for 1,4-azaindoles.
tion−reduction mechanism involving decaprenylphosphoryl-2-
keto-β-D-erythro-pentofuranose, (DPX) as an intermediate.
DprE1 is composed of two proteins encoded by the dprE1
and dprE2 genes, and the DprE1 enzyme is an FAD-containing
oxidoreductase while DprE2 is an NADH-dependent reduc-
tase.5,6
compound 1 (Figure 1a) exhibited a potent minimal inhibitory
concentration (MIC, 0.017 μM, Table 1) against Mycobacte-
a
Table 1. Microbiological Properties of Key Compounds
Mtb
Mtb MIC
(μM)
Mtb MBC
(μM)
HBC
DprE1 IC50
(μM)
MMIC
(μM)
The identification of BTZ043 as a covalent inhibitor of
DprE1 with potent antimycobacterial activity confirms the
validity of this target for discovery of novel TB drugs.6
However, it remains to be understood whether noncovalent
inhibition of DprE1 will lead to in vivo efficacy. Moreover, it
also needs be tested if nanomolar-level cellular activity
(observed for BTZ043) is required for DprE1 inhibitors to
be efficacious in vivo. Understanding of the relationship
between mode of inhibition of the target, its translation to
cellular potency, and ultimately efficacy in animal models, will
significantly influence future TB drug discovery efforts directed
at this target. In this paper, we present a scaffold morphing
approach that led to the identification of the “1,4-azaindole”
scaffold as a novel bactericidal inhibitor class. The compounds
in the series have drug like properties, are noncovalent
inhibitors of DprE1, and demonstrate efficacy in mouse
tuberculosis models. These key attributes highlight the
potential of this series to be developed for the treatment of
drug sensitive and drug resistant tuberculosis therapy.
no.
(μM)
1
2
3
4
5
6
0.017
6.25
>200
12.5
>100
>100
50
>10
ND
94
0.014
1.56−3.12
0.39−1.56
<0.39
1.56−3.12
0.78−1.56
<0.39
0.010−0.017
0.005−0.015
0.003−0.015
0.010−0.03
>100
>100
>100
>100
>100
100
1.56−3.12
0.78−1.56
>100
a
ND: not determined.
rium tuberculosis (Mtb) H37Rv but lacked an effective
minimum bactericidal concentration (MBC, >200 μM),
suggesting its static nature. The early scaffold-morphed hit 2
displayed a moderate MIC (6.25 μM) but also, encouragingly,
moderate cidality (MBC, 12.5 μM) (Figure 1a, Table 1). We
have established a MIC-based structure−activity relationship
(SAR) against Mtb. Three points of diversification were
identified for the 1,4-azaindoles, namely the amide side chain,
a hydrophobic group, and core ring substitutions (Figure 1b).
The secondary amide (referred to as the amide side chain in
Figure 1b) is an essential group for maintaining potent MIC
and that could be involved in either intramolecular hydrogen
bonding with nitrogen (N4) of the azaindole ring or hydrogen
bonding with the target enzyme (compound 2−9 vs compound
10, Tables 1 and 2). Small hydrophobic or hydrophilic amides
RESULTS AND DISCUSSION
■
The 1,4-azaindoles described herein were identified from a
scaffold morphing approach starting with a published anti-TB,
non-DprE1 imidazo-pyridine scaffold.7,8 The imidazo-pyridine
B
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a
Table 2. Structure−Activity Relationship of 1,4-Azaindoles
a
ND = not determined.
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a
Table 3. 1,4-Azaindoles: DMPK and Safety Properties
compound
2
3
4
5
6
log D
>4.5
2.1
3.0
2.6
1.8
b
b
b
b
solubility (μM)
3
8
6
7
5
4
124
13
15
24
16
22
human CLpred microsomes (% LBF)
human CLpred hepatocytes (% LBF)
rat CLpred hepatocytes (% LBF)
dog CLpred hepatocytes (% LBF)
human PPB (% free)
10
13
10
ND
35
6
9
20
18
26
ND
<1
25
20
26
9.8
5
5
Caco-2 A−B/B−A (1 × 10−6·cm/s)
ND
ND
28
25/17
>50
>33
38/24
>50
>33
33/17
>50
11/30
>50
c
CYP inhibition (μM)
hERG (μM)
>33
d
>33
secondary pharmacology hits IC50 (μM)
ND
no significant hits
a
b
c
d
ND: not determined. Kinetic solubility in test media >100 μM. CYP1A2, CYP2C9, CYP2C19, CYP2D6, CYP3A4. Panel of 24 high severity
targets (binding and functional data): IC50 >100 μM or >30 μM.
such as methyl cyclopropyl, fluoro-ethyl, and hydroxy ethyl
amides are preferred for cellular potency (compounds 2−5 and
7−8 vs compound 12−14, Tables 1 and 2).
single nucleotide change in DprE1 (Rv3790), resulting in an
amino acid substitution (Tyr → His) at position 314. No
significant secondary targets were observed from the whole
genome sequencing analysis of multiple resistant strains, while
compounds in the 1,4-azaindole series showed resistance to
Y314H mutant strain (Table 4) and cross-resistance was not
observed for series of reference drugs including BTZ043
(Supporting Information Table S4). The cysteine-387 DprE1
mutants (Cys → Ser, Cys → Gly) that impart resistance to
BTZ0436 did not show cross resistance to 1,4-azaindoles
(compounds 3−6, Table 4). Target specificity was further
confirmed by the reduced sensitivity of a DprE1 overexpression
strain (Mtb DprE1 OE) to compounds 3−6 similar to BTZ043
(Table 4). Additionally, the compounds in the series show
potent inhibition of DprE1 enzyme,10 confirming its target
(Table 4). About a thousand-fold shift in IC50 was observed for
purified Msm Y321H (Y314H in Mtb) mutant enzyme
compared to wild-type enzyme, whereas the IC50 for
Cys394G (Cys 387 in Mtb) mutants were comparable to that
of wild-type. The inhibition of mutant enzymes compared to
the wild-type correspond very well with the cellular potency,
thus strengthening the understanding of DprE1 being the target
for this series (Table 4). Mass spectrometric analysis of Msm
DprE1 treated with compound 22 and 23 did not reveal a
covalent adduct, confirming a noncovalent mode of inhibition.
As previously reported,6 BTZ043 showed a covalent adduct
under the same experimental conditions (Supporting Informa-
tion Figure S2). This result confirms a different binding mode
for the 1,4-azaindoles described here in comparison to BTZ043.
Compounds in the series were profiled for in vitro drug
metabolism and pharmacokinetic (DMPK) properties as
depicted in Table 3. The dried DMSO solubility for
compounds 3−5 was lower than compound 6; the improved
solubility can be attributed to hydroxyethyl amide side chain.
The human plasma protein binding (PPB, % free) values for
compounds 3−6 were between 5% and 22%. On the basis of in
vitro intrinsic clearance (Clint) using human microsomes,
human hepatocytes, rat hepatocytes, and dog hepatocytes, the
predicted in vivo clearance (CL) for compounds 3−6 ranged
between 6 and 26% of liver blood flow (% LBF). In contrast,
the in vitro intrinsic clearance was higher from mouse
microsomes (Supporting Information Table S5), suggesting
species specific clearance mechanisms. The permeability as
measured by Caco2 assay suggested that these compounds are
highly permeable with no significant efflux activity. The
compounds in the series did not show inhibition of cytochrome
Additionally, the amide side chain influences physicochemical
properties and in vitro safety parameters (Table 3). The
hydrophobic group tolerates various disubstituted benzyl and
disubstituted heteroaryl-methyl groups; however, monosubsti-
tuted benzyl groups are less favored (compound 2, 9, and 15−
16 vs compound 3−8, Tables 1 and 2). These groups are also
critical to modulate MIC, physicochemical, and in vitro safety
properties (Tables 1−3). Addition of a methyl group at the C-6
position of the 1,4-azaindole improved cellular potency
(compound 3 and 20−22 vs compound 5−6 and 18−19,
Tables 1 and 2). Thus, the scaffold has multiple diversity points
to build in required properties. Overall, the series contains
compounds with submicromolar MICs and MBCs (Table 1,
MBC/MIC ratio 1−2).
The 1,4-azaindole series, although cidal for Mtb and
Mycobacterium smegmatis (Msm), did not show activity against
a broad panel of Gram negative and Gram positive pathogens,
thus suggesting an excellent target pathogen specificity
(Supporting Information Table S1). Compounds in the series
retained their MIC against a panel of drug sensitive and single-
drug resistant clinical isolates of Mtb (Supporting Information
Table S2), establishing a case for their potential use against
drug sensitive and MDR TB infections. Compounds 3 and 4
exhibited time-dependent kill kinetics against replicating Mtb,
with ∼4 log10 reductions in colony forming units (CFU) by day
10 at a concentration of 1−4 fold MIC (Figure 2a,b).
Compounds 3 and 4 were also active against intracellular
Mtb in THP-1 cells, with ∼1−2 log10 reduction in CFU at
concentrations 1−4-fold of MIC (Supporting Information
Figure S1). In addition to potent activity on replicating
bacteria, compound 3 showed weak to moderate activity against
nonreplicating Mtb grown under hypoxic conditions (∼1.5
log10 reduction in CFU at 30 fold of MIC; HBC)9 (Table 1).
Compounds 2−6 were found to be noncytotoxic against a
human lung adenocarcinoma epithelial cell line (A549)
following 72 h of compound exposure (MMIC >100 μM,
Table 1).
Spontaneous resistant mutants to compounds 7 and 8 arose
at a frequency of 2.9 × 10−9 at eight times MIC concentration
(Supporting Information Table S3). Mutant strains showed
about 10−100-fold upward shift in MIC with respect to wild-
type H37Rv strain (Supporting Information Table S3). Whole
genome sequencing of the resistant Mtb mutants revealed a
D
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Figure 2. 1,4-Azaindoles: in vitro cell cidality, ELF PK, and in vivo efficacy. (a) Kinetic of cell cidality for compound 3. (b) Kinetic of cell cidality for
compound 4. (c) ELF PK of compound 3 in healthy mice at 100 mg/kg. (d) ELF PK of compound 4 in healthy mice at 100 mg/kg. (e) Efficacy in
acute TB infection model. (f) Efficacy in chronic TB infection model.
Table 4. Cross-Resistance of 1,4-Azaindoles
Mtb MIC (μM)
DprE1 C387S
Msm DprE1 purified enzyme IC50 (μM)
no.
H37Rv
DprE1 OE
DprE1 C387G
DprE1 Y314H
DprE1 WT
DprE1 C394G
DprE1 Y321H
>10
2.6
3
4
5
6
3.12
0.39
0.39
1.56
50
25
3.12
0.39
0.39
0.39
0.78
0.39
0.39
0.78
>100
>200
>200
>200
0.017
0.006
0.017
0.01
0.015
ND
6.25
100
0.007
0.01
>10
>10
E
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General methods of purification of compounds involved the use of
silica cartridges purchased from Grace Purification systems. The
reactions were monitored by TLC on precoated Merck 60 F254 silica
gel plates and visualized using UV light (254 nm). All compounds
P450 (CYP) enzymes at 50 μM (Table 3), suggesting their low
potential for drug−drug interactions. In vitro safety profiling of
compounds 3−6 against a panel of human targets and cardiac
channels has indicated no major safety liabilities associated with
this series (Table 3).
1
were analyzed for purity by HPLC and characterized by H NMR
using Bruker 300 MHz NMR and/or Bruker 400 MHz NMR
spectrometers. Chemical shifts are reported in ppm (δ) relative to the
residual solvent peak in the corresponding spectra; chloroform δ 7.26,
methanol δ 3.31, DMSO δ 2.5, and coupling constants (J) are reported
in hertz (Hz) (where s = singlet, bs = broad singlet, d = doublet, dd =
double doublet, bd = broad doublet, ddd = double doublet of doublet,
t = triplet, tt = triple triplet, q = quartet, m = multiplet) and analyzed
using ACD NMR data processing software. Mass spectra values are
reported as m/z. All reactions were conducted under nitrogen unless
otherwise noted. Solvents were removed in vacuo on a rotary
evaporator. All final compounds for biological testing were purified by
reverse phase HPLC with >95% purity (Shimadzu HPLC instrument
with a Hamilton reversed phase column (HxSil, C18, 3 μm, 2.1 mm ×
50 mm (H2)). Eluent A, 5% CH3CN in H2O; eluent B, 90% CH3CN
in H2O. A flow rate of 0.2 mL/min was used with UV detection at 254
and 214 nm]
On the basis of in vitro properties, compounds 3−6 were
administered to mice and rats to assess their in vivo PK
parameters. Exposure in mice was measured in the presence of
1-aminobenzotriazole (ABT), a pan-inhibitor of CYP isoforms
used to block mouse specific clearance. Good oral exposure was
observed for compounds 3−6 in both rats and mice
(Supporting Information Figure S3, Supporting Information
Table S6). In rats, good correlation was observed between in
vitro and in vivo clearance (Supporting Information Table S6).
The in vivo efficacy of two representative compounds (3 and 4)
was assessed in BALB/c mice in “acute” and “chronic” TB
infection models.11,12 In the acute infection model, treatment
was initiated 3 days postinfection, whereas treatment was
started on day 28 in the chronic infection model. After four
weeks of treatment with compound 3 and 4, the bacterial
burden in the lungs was reduced by >1.5 log10 CFU and
statistically significant dose dependent efficacy was observed in
both the models (Figure 2e,f). The observed moderate hypoxia
activity for compound 3 does not appear to contribute
significantly to the efficacy results observed in mouse. The
oral exposure of compounds 3 and 4, assessed from infected
animals showed AUCs ranging from 200 to 700 μM·h and free
plasma concentrations were maintained above the MIC for 10−
24 h (%f T > MIC of 25−100), resulting in efficacy in the acute
and chronic TB infection models (Supporting Information
Figure S4, Supporting Information Table S7). Interestingly, the
concentration of compounds 3 and 4 measured in lung
epithelial lining fluid of healthy mice (ELF PK) compared to
the total plasma concentration for both the compounds (Figure
2C, Supporting Information Table S8), demonstrating
significant distribution to the target site. Thus, a good
correlation was observed between plasma and ELF levels and
the pharmacodynamic effect. 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.
Abbreviations: NMP = N-methyl pyrrolidine HCl = hydrochloric
acid DMF = N,N-dimethylformamide NaH = sodium hydride. EI =
electrospray ionization HRMS = high resolution mass spectrometry.
N-(Cyclopropylmethyl)-1-(2-methylbenzyl)-1H-pyrrolo[3,2-
b]pyridine-3-carboxamide (2). 1-(2-Methylbenzyl)-1H-pyrrolo-
[3,2-b]pyridine-3-carboxylic acid (120 mg, 0.45 mmol) was placed in
a 50 mL single-necked flask equipped with a reflux condenser
connected to a nitrogen source. Thionyl chloride (5 mL, 0.45 mmol)
was added, and the mixture was heated at reflux overnight. The excess
thionyl chloride was evaporated to dryness (residue 1). In another
flask, cyclopropanemethylamine (0.195 mL, 2.25 mmol) was dissolved
in dichloromethane (10 mL), and triethylamine (0.312 mL, 2.25
mmol) was added. The mixture was stirred at RT for 10 min, then
added slowly to residue 1 to give a suspension which was stirred at RT
for 1 h. After completion of the reaction, DCM was added and the
mixture was washed with water and brine solution. The DCM layer
was dried over sodium sulfate and evaporated, and the residue was
purified by column using methanol/DCM (yield 53%). ES + MS m/z:
320 (M + 1). 1H NMR (300 MHz, DMSO-d6) δ ppm 0.21−0.34 (m, 2
H) 0.41−0.57 (m, 2 H) 0.98−1.17 (m, 1 H) 2.28 (s, 3 H) 3.28 (t, J =
6.22 Hz, 2 H) 5.55 (s, 2 H) 6.70 (d, J = 7.35 Hz, 1 H) 7.05−7.17 (m, 1
H) 7.17−7.35 (m, 3 H) 7.99 (d, J = 8.29 Hz, 1 H) 8.14 (s, 1 H) 8.53
(d, J = 4.52 Hz, 1 H) 8.82 (t, J = 5.56 Hz, 1 H). 13C NMR (400 MHz,
DMSO-d6) δ ppm 162.6, 143.6, 142.4, 135.8, 135.7, 134.8, 130.4,
129.6, 127.8, 126.9, 126.2, 119.3, 117.3, 110, 48, 42.4, 18.6, 11.2, 3.2.
N-(2-Fluoroethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)-
methyl)-1H-pyrrolo[3,2-b]pyridine-3-carboxamide (3). 1-((6-
Methoxy-5-methylpyrimidin-4-yl)methyl)-1H-pyrrolo[3,2-b]pyridine-
3-carboxylic acid (250 mg, 0.84 mmol) was placed in a 100 mL single-
necked flask equipped with an air condenser connected to a nitrogen
source. DCM (20 mL) was added, followed bytriethyl amine (1.162
mL, 8.38 mmol) to give a clear solution. 1-Propanephosphonic acid
cyclic anhydride (1.497 mL, 2.51 mmol) was added, followed by 2-
fluoroethanamine hydrochloride (83 mg, 0.84 mmol). The reaction
mixture was stirred at RT overnight. After completion of the reaction,
DCM and water was added. The layers were separated, and the DCM
layer was washed with brine, dried over sodium sulfate and evaporated,
and the residue was purified by column using methanol/DCM (yield
52%). ES + MS m/z: 344 (M + 1). 1H NMR (300 MHz, DMSO-d6) δ
ppm 2.25 (s, 20 H) 3.67 (d, J = 5.46 Hz, 7 H) 3.77 (d, J = 5.46 Hz, 7
H) 4.50 (t, J = 4.90 Hz, 7 H) 4.66 (t, J = 4.99 Hz, 7 H) 5.69 (s, 13 H)
7.26 (dd, J = 8.29, 4.71 Hz, 7 H) 7.94 (d, J = 8.48 Hz, 7 H) 8.28 (s, 7
H) 8.41 (s, 6 H) 8.49 (d, J = 4.52 Hz, 7 H) 8.95 (t, J = 5.84 Hz, 7 H).
13C NMR (400 MHz, DMSO-d6) δ ppm 167.2, 163, 161.2, 155, 143.3,
CONCLUSIONS
■
In summary, we have employed a scaffold morphing approach
to discover a novel series of 1,4-azaindoles with antituberculosis
activity. We have established that this novel inhibitor class
exhibits cellular activity via a noncovalent inhibition of DprE1.
This mode of inhibition (MOI) and level of cellular activity
translates and are sufficient to show efficacy in a relevant TB
mouse model. Recent work from Wang et al., identified TCA1,
through cell-based phenotypic screen, which has two cellular
targets, DprE1 and MoeW enzymes, and has been found to be
efficacious in mouse TB models.13 On the basis of in vivo
efficacy, drug like properties and absence of in vitro safety
liabilities, the “1,4-azaindole” series warrants further inves-
tigation for in vivo safety. We believe this series has the
potential to deliver a candidate drug for TB.
EXPERIMENTAL SECTION
■
142.4, 137.1, 130.2, 119.4, 117.2, 114.6, 109.4, 83.8, 82.2, 54.1, 48.1,
38.6, 9.4.
N-(Cyclopropylmethyl)-1-((6-methoxy-5-methylpyrimidin-4-
yl)methyl)-1H-pyrrolo[3,2-b]pyridine-3-carboxamide (4). 1-((6-
Methoxy-5-methylpyrimidin-4-yl)methyl)-1H-pyrrolo[3,2-b]pyridine-
All anhydrous solvents, reagent grade solvents for chromatography,
and starting materials were purchased from either Sigma Aldrich
Chemical Co. or Fisher Scientific. Water was distilled and purified
through a Milli-Q water system (Millipore Corp., Bedford, MA).
F
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3-carboxylic acid (200 mg, 0.67 mmol) was taken in DCM (10 mL). 1-
Propanephosphonic acid cyclic anhydride (427 mg, 1.34 mmol) was
added, followed by the addition of triethyl amine (339 mg, 3.35 mmol)
and cyclopropylmethanamine (95 mg, 1.34 mmol). The reaction mass
was stirred at RT for overnight. After the completion of the reaction,
water was added and extracted with DCM. The organic layer was
washed with water and brine slution. The organic layer was separated
and dried over sodium sulfate. The organic layer was evaporated to get
the residue, which was purified by column chromatography to get the
pure compound (yield 74%). ES + MS m/z: 352.38 (M + 1). 1H NMR
(300 MHz, DMSO-d6) δ ppm 0.02 (q, J = 4.58 Hz, 2 H) 0.18−0.31
(m, 2 H) 0.83 (t, J = 6.88 Hz, 1 H) 2.00 (s, 3 H) 2.98−3.11 (m, 3 H)
3.69 (s, 3 H) 5.43 (s, 2 H) 7.00 (dd, J = 8.29, 4.71 Hz, 1 H) 7.68 (dd, J
= 8.29, 1.13 Hz, 1 H) 7.98 (s, 1 H) 8.17 (s, 1 H) 8.24 (dd, J = 4.71,
1.13 Hz, 1 H) 8.55 (t, J = 5.75 Hz, 1 H). 13C NMR (400 MHz,
DMSO-d6) δ ppm 167.2, 162.7, 161.2, 154.9, 143.3, 142.5, 136.9,
130.2, 119.3, 117.1, 114.6, 109.8, 54.1, 48, 42.4, 11.2, 9.4, 3.1.
Author Contributions
#P.S.S. and M.C. contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the analytical support provided by S. Rudrapatna and
the analytical team. Our sincere thanks to the PD Bangalore,
CMG, Global DMPK, Safety, Biosciences (AZ Boston, USA),
and Discovery Sciences (AZ, Alderly Park, UK) for technical
support in various assays. We thank M. Panda for preliminary
docking studies. We also thank Rajkumar and Subhash for
animal welfare support. We sincerely thank G. Riccardi
(University of Pavia) for providing the DprE1 expression
plasmid. We thank Sudha R. for her contributions towards
DprE1 overexpression strain. We acknowledge N. Dinesh and
S. Ghorpade for scientific discussions. We also thank V.
Balasubramanian, P. Warner, R. Tommasi, and M. Perros for
their constant inspiration, support, and encouragement. This
research was supported by the Global Alliance for Tuberculosis
(GATB).
N-(2-Fluoroethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)-
methyl)-6-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide
(5). 1-((6-Methoxy-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-
pyrrolo[3,2-b]pyridine-3-carboxylic acid (0.190 g, 0.61 mmol), 2-
fluoroethanamine (0.077 g, 1.22 mmol), and TEA (0.254 mL, 1.83
mmol) was added in DCM. After 3 min, 1-propanephosphonic acid
cyclic anhydride (0.484 g, 1.52 mmol) was added. The resulting
reaction mixture was stirred at rt for 50 min. The reaction was diluted
with DCM and water. The DCM layer was extracted and washed with
brine and dried over sodium sulfate and concentrated. Purification was
performed on Waters RP system to get product N-(2-fluoroethyl)-1-
((6-methoxy-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-pyrrolo-
[3,2-b]pyridine-3-carboxamide (0.090 g, 41.4%). ES + MS m/z:
ABBREVIATIONS USED
■
TB, tuberculosis; Mtb, Mycobacterium tuberculosis; DprE1,
decaprenylphosphoryl-β-D-ribose2′-epimerase 1; MDR-TB,
multidrug-resistant tuberculosis; BTZs, nitro-benzothiazinones;
DPR, decaprenylphosphoryl-β-D-ribose; DPA, decaprenylphos-
phoryl-β-D-arabinofuranose; DPX, decaprenylphosphoryl-2-
keto-β-D-erythro-pentofuranose; MBC, minimum bactericidal
concentration; MIC, minimum inhibitory concentration; SAR,
structure−activity relationship; Msm, Mycobacterium smegmatis;
DMPK, drug metabolism and pharmacokinetic; Clint, intrinsic
clearance; PPB, plasma protein binding; CL, clearance; LBF,
liver blood flow; CYP, cytochrome P450 enzymes
1
358.36. H NMR (300 MHz, DMSO-d6) δ ppm 2.17−2.30 (3, 3 H)
2.40 (s, 3 H) 3.59−3.73 (m, 1 H) 3.73−3.83 (m, 1 H) 3.94 (s, 3 H)
4.50 (t, J = 4.99 Hz, 1 H) 4.66 (t, J = 4.99 Hz, 1 H) 5.64 (s, 2 H) 7.76
(s, 1 H) 8.15 (s, 1 H) 8.35 (s, 1 H) 8.42 (s, 1 H) 8.87 (t, J = 5.84 Hz, 1
H). 13C NMR (400 MHz, DMSO-d6) δ ppm 167.1, 163.1, 161.3, 155,
144.4, 140.6, 136.4, 130.3, 126.6, 119, 114.5, 109.3, 83.8, 82.2, 54.1,
47.9, 38.6, 18.3, 9.4.
N-(2-Hydroxyethyl)-1-((6-methoxy-5-methylpyrimidin-4-yl)-
methyl)-6-methyl-1H-pyrrolo[3,2-b]pyridine-3-carboxamide
(6). 1-((6-Methoxy-5-methylpyrimidin-4-yl)methyl)-6-methyl-1H-
pyrrolo[3,2-b]pyridine-3-carboxylic acid (75 mg, 0.24 mmol) was
added to dichloromethane (10 mL) to give a suspension. Triethyl
amine (66.9 mL, 0.48 mmol) was added, followed by 1-propane-
phosphonic acid cyclic anhydride (286 mL, 0.48 mmol). The reaction
mixture was stirred at RT for 5 min. 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 and
washed with water and brine. The DCM layer was dried over sodium
sulfate and evaporated to give the crude product, which was purified by
column chromatography using methanol/dichloromethane as eluent
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1
(yield 52.7%). ES + MS m/z: 356.4 (M + 1). H 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
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D’Haeze, W.; Scherman, M. S.; Brennan, P. J.; McNeil, M. R.; Crick,
D. C. Decaprenylphosphoryl arabinofuranose, the donor of the D-
arabinofuranosyl residues of mycobacterial arabinan, is formed via a
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■
S
* Supporting Information
Details of the synthesis of all compounds and biological assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
̌ ́
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Shandil, R. K.; Balasubramanian, V.; Balganesh, T.; Tyagi, S.; Grosset,
J.; Riccardi, G.; Cole, S. T. Benzothiazinones kill Mycobacterium
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: pravin.shirude@astrazeneca.com. Phone: +91 080
23621212. Fax: +91 080 23621214.
*E-mail: monalisa.chatterji@astrazeneca.com.
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