Synthesis and structure activity relationship of imidazo[1,2-a]pyridine-8- carboxamides as a novel antimycobacterial lead series

Article abstract of DOI:10.1016/j.bmcl.2013.06.043

Imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead were generated by whole cell screening of a focused library against Mycobacterium tuberculosis. Herein, we describe the synthesis and structure activity relationship evaluation of this class of inhibitors and the optimization of physicochemical properties. These are selective inhibitors of Mycobacterium tuberculosis with no activity on either gram positive or gram negative pathogens.

Full text of DOI:10.1016/j.bmcl.2013.06.043

Bioorganic & Medicinal Chemistry Letters 23 (2013) 4996–5001
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and structure activity relationship
of imidazo[1,2-a]pyridine-8-carboxamides as a novel
antimycobacterial lead series
a
b
a
Sreekanth Ramachandran a, , Manoranjan Panda , Kakoli Mukherjee , Nilanjana Roy Choudhury ,
Subramanyam J. Tantry a, Chaitanya Kumar Kedari a, Vasanthi Ramachandran b, Sreevalli Sharma b,
V. K. Ramya b, Supreeth Guptha b, Vasan K. Sambandamurthy b
⇑
a Department of Medicinal Chemistry, AstraZeneca India Pvt. Ltd, Avishkar, Bellary Road, Hebbal, Bangalore 560024, India
b Department of Biosciences, AstraZeneca India Pvt. Ltd, Avishkar, Bellary Road, Hebbal, Bangalore 560024, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Imidazo[1,2-a]pyridine-8-carboxamides as a novel antimycobacterial lead were generated by whole cell
screening of a focused library against Mycobacterium tuberculosis. Herein, we describe the synthesis and
structure activity relationship evaluation of this class of inhibitors and the optimization of physicochem-
ical properties. These are selective inhibitors of Mycobacterium tuberculosis with no activity on either
gram positive or gram negative pathogens.
Received 15 April 2013
Revised 31 May 2013
Accepted 14 June 2013
Available online 24 June 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
Antimycobacterial activity
Whole cell screening
Structure activity relationship
Physicochemical properties
Tuberculosis (TB) remains the major cause of death from an
infectious disease worldwide.1a,b The emergence of multidrug
resistant (MDR) and extremely drug resistant (XDR) TB has rekin-
dled a significant investment in identifying new drugs to address
this huge unmet medical need.2a,b Hence there is an urgent focus
to bring in new drugs with a novel mechanism of action to combat
both drug-sensitive as well as drug resistant TB.
Various approaches in identifying novel inhibitors of Mycobacte-
rium tuberculosis (Mtb) include both target based as well as whole
cell screening against Mtb or surrogate species such as M. smegma-
tis (Msm) or M. bovis (BCG).3 Despite the availability of the genome
sequence of Mtb,4 numerous efforts to identify novel chemical
starting points against potential targets so far has resulted in lim-
ited success.5 The target based lead generation approaches have of-
ten faced challenges in converting enzymatic activity (IC50) to
cellular potency (MIC) possibly due to the poor bacterial cell wall
permeation. The recent successful identification and development
of agents such as Sirturo™ for MDR-TB,6a,6b PA8246c and other can-
didates in different stages of clinical development can be attributed
to the success of whole cell based screening.
cophores based on structural features derived from known antimy-
cobacterial cell-division inhibitors9
focused library of 500
a
compounds was assembled. Whole cell screening of this library
using M. tuberculosis H37Rv led to the identification of several hits.
One of the actives, was a novel chemical class imidazo[1,2-a]pyri-
dine-8-carboxamides with promising antimycobacterial activity. In
this communication, we present our initial efforts towards the syn-
thesis and structure activity relationship of this class of inhibitors
including optimization of physicochemical properties.
The original hit (1) showed a modest minimum inhibitory con-
centration (MIC) of 16
l
g/mL against M. tuberculosis H37Rv.
F
c
N
B
A
N
1
O
NH2
The synthesis of these class of compounds is shown in the
Scheme 1. Coupling of commercially available 2-aminopyridines
2 with phenacyl bromides 3 in ethanol furnished moderate to good
yields of imidazo[1,2-a]pyridines 4. Hydrolysis of the nitrile using
concentrated sulphuric acid yielded the corresponding amide 5 in
quantitative yield. Suzuki reaction with various boronic acids
In order to compliment our ongoing efforts to discover new TB
drugs including AZD58477 and other compounds,8 we embarked
on a focused library screening against Mtb. Using a set of pharma-
⇑
Corresponding author. Tel.: +91 080 2362 1212; fax: +91 080 2362 1214.
E-mail address: sreekanth.ramachandran@astrazeneca.com (S. Ramachandran).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.06.043

S. Ramachandran et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4996–5001
4997
O
Br
N
R1
Br
(a)
N
+
R1
NH2
Br
N
N
3
4
N
2
(15 - 60 % yield)
R2
Br
N
N
(b)
(C)
R1
R1
N
N
6
5
O
NH2
O
NH2
(quantitative)
(30 - 50 % yield)
R1 = H, 7-OMe
R2 = Aryl, heteroaryl
3-alkoxypyridyl derivatives
N
F
Br
N
N
N
(c)
R1
R1
N
7
O
NH2
O
NH2 5
(~50 % yield)
N
OR3
N
(d)
R1
N
8
O
NH2
(30 - 50 %)
R1 = H, 7-OMe
Scheme 1. Reagents and conditions: (a) EtOH, reflux, 10 h; (b) Con. H2SO4/H2O, 50 °C, 3 h; (c) R2-B(OH)2, PdCl2(dppf)2, CsF, MeOH, 120 °C; (d) R3-OH, NaH, DMF, 6 h.
under standard conditions provided the required imidazo[1,2-
a]pyridines 6 in moderate yields. Similarly, 3-alkoxy pyridine
derivatives 8 were synthesized starting from 5 by Suzuki using
3-fluoropyridine-5-boronic acid followed by SNAr reaction of 7
with various alcohols using sodium hydride as base.
the tertiary amide 10 was inactive. These results led us to hypoth-
esize that at least one H-bond donor at the A-ring is required for
the activity.
The 3-alkoxypyridine derivatives at ring C gave us a diversity
handle to further optimize the MIC. This led to compounds 8c
Systematic medicinal chemistry exploration of 1 resulted in
establishing MIC based SAR for the series with improved Mtb po-
tency and physiochemical properties (Table 1).
and 8d with MICs of 0.25 lg/mL having a tetrahydrofuran and flu-
oroethyl side chains, respectively, but with only marginal improve-
ment in solubility.
Initial SAR exploration was focused on C ring modifications in 1.
A small library synthesis using intermediate 5 allowed the explora-
tion of SAR around the C ring using various aryl and heteroaryl sub-
stituents. This enabled the identification of compound 6a with
eightfold improvement in MIC and thereby fixing the 3-substituted
aryl ring as important for driving MIC. Furthermore, SAR explora-
tion of 6a resulted in 3-methoxy pyridine 8a which improved the
We hypothesized that the series displayed poor aqueous solu-
bility mainly due to high lipophilicity (as measured by LogD) and
the planarity of the scaffold. To improve solubility, we employed
two strategies either using a solubilizing group or breaking the pla-
narity associated with the linking of 3 aromatic rings. The first
strategy that led to compounds such as 3-alkoxypyridine 8e, re-
sulted in less potent compound, while introduction of similar side
chain at secondary amide of imidazopyridine ring 11, retained the
potency with >2000 fold improved solubility compared to 8a.
During the SAR exploration, we identified an alternative ap-
proach to optimize physicochemical properties without compro-
mising the antimycobacterial potency (Fig. 1). This was achieved
MIC to 0.5 lg/ml range. The improvement in MIC with 3-methoxy-
pyridine substituent in the ring C may be due to additional interac-
tions at the active site of the target protein or to an improved cell
permeation across the bacterial cell wall. Converting the primary
amide in to secondary amide 9 retained the MIC of 2 lg/mL, while

4998
S. Ramachandran et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4996–5001
Table 1
Antimycobacterial activity of compounds
R
N
N
R1
N
R2
O
Compound no.
R
R1
R2
Mtb MIC (
l
g/ml)
Aq. solubility (lM)
AZLogDa
X
1
6a
X = F
X = OCH3
N
H
H
H
H
16
2
<1
<1
3.9
3.5
Y
F
8a
8b
Y = OCH3
H
H
H
H
0.5
0.5
<1
<1
3.1
3.7
Y =
F
O
Y =
O
O
8c
8d
8e
H
H
H
H
H
H
0.25
0.25
16
5.3
16
3.4
3.4
2.3
Y =
O
F
Y =
O
320
N
9
10
Y = OCH3
Y = OCH3
Me
Me
H
Me
2
>32
5.5
10
4.1
3.0
11
Y = OCH3
H
4
>2460
2.9
N
a
Predicted LogD.
Figure 1. Matched pairs of 8a with 12 and 13 with improved physico-chemical properties.
by disturbing the intra-molecular hydrogen bonding nature of pri-
mary amide. We hypothesized that a pseudo ring is formed by
intramolecular hydrogen bonding between the primary amide
and the nitrogen of imidazopyridine core (Fig. 2). The placement
of a methoxy group next to the carboxamide resulted in 12 with
improved solubility, reduced intrinsic microsomal clearance and
decreased plasma protein binding. Replacement of an imidaz-
ole[1,2-a]pyridine ring carbon with nitrogen (imidazolopiperazine)
13 led to similar improvements in the molecule. This could be
attributed to the perturbation of the amide group away from the
ring planarity. To explore this further, we performed conforma-
tional analysis using Omega10 followed by geometry optimization
8a
12
13
Torsion angle: 50
360
190
Figure 2. MP2/6-31G(d) optimized geometries of 8a, 12 and 13.

S. Ramachandran et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4996–5001
4999
of the most preferred conformers using quantum mechanical elec-
tronic structure calculation.11 We used the MP2/6-31G(d) method
for the electronic calculations.
imidazolopyridine ring (torsional angle obtained from MP2 geom-
etry optimization is 36°). Similarly, a ring nitrogen at the 7-position
resulted in 13 causing a smaller perturbation. We hypothesized
that the breaking of planarity and hence crystal packing12 plays
an important role in improving solubility and other physicochem-
ical properties for these compounds. Theobserved high metabolic
clearance for compound 8a might be due to the hydroxylation
occurring at 7-position, whereas in compounds 12 and 13 the
7-position is blocked by methoxy and nitrogen, respectively.
Based on these results we have designed and synthesized deriv-
atives 14–16 (Table 2), which further support this hypothesis.
In the most preferred conformer, the amide group in compound
8a is involved in an intramolecular hydrogen bond with the imi-
dazopyridine nitrogen atom. This locks the core ring into a pseu-
do-planar tricyclic system which is rigid. This is reflected in its
poor solubility and other physicochemical properties such as high
plasma protein binding. Introduction of a methoxy group at the
7-position resulted in compound 12, for which the most preferred
conformer has the amide substituent twisted away from the core
Table 2
Improving the aqueous solubility
Compound
Mtb MIC (
lg/mL)
Aq. solubility (lM)
AZLogDa
O
N
O
N
1
120
3.6
N
O
O
NH2
14
O
N
O
4
600
3.3
N
N
O
O
NH2
15
O
N
O
2
40
2.7
N
N
O
N
16
NH2
a
predicted log D.
Table 3
Ring A variations
Compound
Mtb MIC (lg/mL)
Aq. solubility (
lM)
AZLogDa
N
N
O
O
O
N
2
2
3.0
17
O
O
N
N
N
4
2
3
2.8
2.5
N
N
18
N
O
N
N
18
O
NH2
19
a
Predicted LogD.

5000
S. Ramachandran et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4996–5001
Table 4
Compound
Mtb MIC (
lg/mL)
Aq. solubility (lM)
AZLogDa
N
N
O
N
16
22
2.1
N
O
O
O
NH2
20
N
O
N
>32
>32
3.5
2.0
3.0
N
N
NH2
21
N
O
N
<1
F
N
NH2
22
Table 5
Table 7
Invitro DMPK properties of representative compounds
Cytotoxicity index of representative compounds
Compound
Mtb MIC (lM)
A549 IC50 (
lM)
Compound
Hu Clint (
ll/min/mg)
Hu PPB (% free)
LogDa
16
15
8b
13
8d
6.25
12.5
3.12
12.5
0.78
>100
>100
>100
>100
>100
12
8e
8d
56
47
75
2
7.3
1
2.3
ND
3.7
a
Measured LogD at pH 7.4, ND: not determined.
Table 6
Broad-spectrum activity of representative compounds
Broad spectrum panel (lM) Compound
Compound
Compound
8b
8c
8d
Mtb MIC
3.12
0.78
0.78
E. coli MIC
S. aureus
H. influenzae
S. pnuemoniae
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
O
N
N
6a
O
NH2
Replacement of the ring A imidazopyridine with various hetero-
cycles such as benzoxazole, benzimidazole and triazolopyridine re-
sulted in compounds 17–19 (Table 3) which retained good MIC
values thereby suggesting that the series has further potential for
diversification.
A limited SAR exploration around the central phenyl linker (ring
B) showed that unsubstituted phenyl remained the best in terms of
potency and physicochemical properties (Table 4; 20–22)
The series displayed an excellent selectivity index for cytotoxic-
ity (Table 5), where all representative compounds demonstrated
Figure 3. Killing kinetics for compound 6a( g/mL).
l
clearance in human microsomal preparations despite having low
protein binding in the presence of human plasma. In general for
this series, the measured LogD correlates well with predicted LogD
(AZ LogD), which could be helpful in further design and optimiza-
tion of physicochemical properties of this series.
Finally, cidality was measured for compound 6a against repli-
cating Mtb grown in Middlebrook 7H9 broth under aerobic condi-
tions (Fig. 3). This compound displayed a concentration dependent
bacterial kill. A 2Log reduction in bacterial numbers were achieved
no effect on human A549 cell line at 100 lM.
Selected compounds which were profiled for broad spectrum
antibacterial activity (Table 6) showed that these compounds were
highly selective for Mtb with no MIC values observed against gram
negative or gram positive pathogens. The narrow antimycobacteri-
al spectrum of these inhibitors might be attributed to a Mycobacte-
ria specific mode of action or due to selective cell permeability/
efflux in other bacterial pathogens.
at the highest tested concentration of 6a (32 lg/mL).
In conclusion, we have discovered a novel chemotype, the imi-
dazo[1,2-a]pyridine-8-carboxamides, as potent inhibitors of M.
tuberculosis. Initial medicinal chemistry efforts resulted in im-
proved potency and in vitro physicochemical properties with 8d
being the best compound identified as the lead for further progres-
sion. Current efforts are directed towards identifying the mode of
A few representative compounds were also profiled for in-vitro
DMPK properties (Table 7). The series exhibited a medium to high

S. Ramachandran et al. / Bioorg. Med. Chem. Lett. 23 (2013) 4996–5001
5001
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The authors sincerely thanks NM4TB consortium for financial
support, Suresh Rudrapatna, Sandesh Jatheendranath, Sreenivasa-
iah Menasinakai for analytical support, Parvinder Kaur for microbi-
ological assay support and Drs. Tanjore Balganesh, Bheemarao
Ugarkar, Balachandra Bandodkar, Shahul Hameed, and Monalisa
Chatterji for encouragement and helpful scientific discussions.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
06.043.
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