Advent of imidazo[1,2-a]pyridine-3-carboxamides with potent multi- and extended drug resistant antituberculosis activity

Article abstract of DOI:10.1021/ml200036r

A set of nine 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides and one 2,6-dimethylimidazo[1,2-a]pyrimidine-3-carboxamide were synthesized. The compounds were evaluated for their in vitro antituberculosis activity versus replicating, nonreplicating, multi- and extensive drug resistant Mtb strains. The MIC₉₀ values of seven of these agents were ≤1 μM against the various tuberculosis strains tested. A representative compound of this class (1) was screened against seven nontubercular strains as well as other nonmycobacteria organisms and demonstrated remarkable microbe selectivity. A transcriptional profiling experiment of Mtb treated with compound 1 was performed to give a preliminary indication of the mode of action. Lastly, the in vivo ADME properties of compounds 1, 3, 4, and 6 were assessed. The 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides are a druglike and synthetically accessible class of anti-TB agents that have excellent selective potency against multi- and extensive drug resistant TB and encouraging pharmacokinetics.

Full text of DOI:10.1021/ml200036r

LETTER
pubs.acs.org/acsmedchemlett
Advent of Imidazo[1,2-a]pyridine-3-carboxamides with Potent
Multi- and Extended Drug Resistant Antituberculosis Activity
Garrett C. Moraski,† Lowell D. Markley,† Philip A. Hipskind,‡ Helena Boshoff,§ Sanghyun Cho,||
Scott G. Franzblau,|| and Marvin J. Miller*,†
†Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, United States
‡Lilly Research Laboratories, Eli Lilly and Company, Indianapolis, Indiana 46285, United States
§Tuberculosis Research Section, Laboratory of Clinical Infectious Diseases, National Institute of Allergy and Infectious Diseases,
National Institutes of Health, Bethesda, Maryland 20892, United States
Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago,
Illinois 60612, United States
S Supporting Information
b
ABSTRACT: A set of nine 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides
and one 2,6-dimethylimidazo[1,2-a]pyrimidine-3-carboxamide were synthesized.
The compounds were evaluated for their in vitro antituberculosis activity versus
replicating, nonreplicating, multi- and extensive drug resistant Mtb strains. The
MIC90 values of seven of these agents were e1 μM against the various tuberculosis
strains tested. A representative compound of this class (1) was screened against seven nontubercular strains as well as other
nonmycobacteria organisms and demonstrated remarkable microbe selectivity. A transcriptional profiling experiment of Mtb
treated with compound 1 was performed to give a preliminary indication of the mode of action. Lastly, the in vivo ADME properties
of compounds 1, 3, 4, and 6 were assessed. The 2,7-dimethylimidazo[1,2-a]pyridine-3-carboxamides are a druglike and synthetically
accessible class of anti-TB agents that have excellent selective potency against multi- and extensive drug resistant TB and
encouraging pharmacokinetics.
KEYWORDS: Antituberculosis, imidazo[1,2-a]pyridine-3-carboxamides, MDR-TB, XDR-TB
uberculosis (TB) is a serious global health risk. More than
one-third of the human population is infected, resulting in an
2-a]pyridine-3-carboxamide scaffold. To our knowledge, the
anti-TB activity of the 2,7-dimethylimidazo[1,2-a]pyridine-3-
carboxamide class is unprecedented. Imidazo[1,2-a]pyridine-3-
nitroso derivatives were reported in 2004 to impart notable anti-
TB activity (MIC = 3.1 μg/mL vs H37Rv-TB) concurrent with
notable toxicity to VERO cells (IC50 = 3.6 μg/mL).6 Also in
2004, rationally designed imidazo[1,2-a]pyridine-3-hydrazones7
were reported but were all inactive against H37Rv-TB at 6.25 μg/
mL. Most recently, in 2009, functionalized 3-amino-imidazo[1,
2-a]pyridines were reported as in vitro Mtb glutamine synthetase
inhibitors but without assessment of the in vitro activity versus
H37Rv-TB.8 While reports on the syntheses of imidazo[1,2-a]-
pyridine-3-carboxamides date to 1965,9 the 2,7-dimethyli-
midazo[1,2-a]pyridine architecture is atypical within the cannon
of medicinal chemistry literature and is unprecedented within the
TB lexicon.
T
estimated 1 700 000 deaths in 2006 (1.5 million in HIV-negative
people and 0.2 million in HIV-positive people).1 Moreover, there
were a staggering 14 400 000 cases estimated worldwide in 2006,
with 83% of the total cases located in the African, South-East
Asia, and Western Pacific regions.1 Mycobacterium tuberculosis,
the causative agent of TB, is an airborne pathogen that can be
spread from one person to another by close contact. Because it
can lie dormant in a latent state for many years, it is a silent killer
among the poor, HIV-infected, immune-compromised, and the
elderly. To make matters worse, multiple drug resistant TB
[MDR-TB, strains that are resistant to first line drugs isoniazid
(INH) and rifampin] and extensively drug resistant TB (XDR-
TB, strains that are resistant to INH and rifampin, as well as any
fluoroquinolone and at least one of three injectable second-line
drugs, such as amikacin, kanamycin, or capreomycin) are on the
rise.2 Most alarming is the emergence of extremely drug resistant
TB “XXDR-TB” (the proposed designation for TB that is
resistant to all first- and second-line TB drugs), which is now
documented.3 In 2008, there were 12 898 cases of TB provision-
ally reported in the United States.4
Since 2007, we have had a collaborative agreement with Dow
AgroScience to screen their compound inventory for inhibitors
of Mtb. This effort, coupled with a program to elaborate novel
heterocylic anti-TB agents from fragment-based studies of myco-
bacterial siderophores,10 led to the identification of an ethyl 2,
7-dimethylimidazo[1,2-a]pyridine-3-carboxylate. This compound
A focus of our laboratories is to facilitate the decline of TB by
the identification of therapeutically effective anti-TB agents to
augment the long dosing regimen of first-line drugs.5 Herein, we
call attention to the in vitro potency of the imidazo[1,
Received: February 8, 2011
Accepted: March 23, 2011
Published: March 23, 2011
r
2011 American Chemical Society
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LETTER
Scheme 1. Synthesis of Imidazo[1,2-a]pyridinesa
a Reagents: (a) Ethyl 2-chloroacetoacetate, DME, reflux, 48 h. (b) (1) LiOH, EtOH; (2) HCl, 56 h. (c) EDC, DMAP, R1, ACN, 16 h.
had weak activity against H37Rv TB (MIC ∼ 65 μM, average) but
was nonetheless an attractive heterocyclic scaffold to optimize as
we did previously with related heterocyclic classes.11
potency) although VERO toxicity (IC50 = 89 μM) was noted.
Compounds 1 and 10 were rescreened in the presence of 4% BSA
(bovine serum albumin) and 10% FBS (fetal equine serum), and
their MICs were found to shift less than 2-fold by ATP and
MABA readouts (see the Supporting Information), indicating
that protein binding is not a problem.
The simplest synthesis of the imidazo[1,2-a]pyridine-3-car-
boxylate ring system12 is the straightforward reaction of 2-amino-
4-picoline with ethyl 2-chloroacetoacetate to give the desired
heterocyclic scaffold in 78% yield (Scheme 1). Saponification
with lithium hydroxide followed by acidic work up gave the free
acid, which was then easily converted to various amide analogues
through classical EDC-mediated couplings in good yields (70%
for 1).
Our initial structureꢀactivity relationship (SAR) strategy eva-
luated a representative panel of nine imidazo[1,2-a]pyridine-3-
carboxamide analogues. The chosen imidazopyridine analogues
included the classical Topliss13 set of benzyl, 4-methoxyphenyl,
4-methylphenyl, 4-chlorophenyl, and 3,4-dichlorophenyl amides.
This set was augmented by ortho- and meta-methoxyphenyl
analogues to probe possible steric effects. Next, because of
potential metabolic issues associated with a benzylic methylene
group, the corresponding aniline was prepared as well as a
fluorine replacement for the chlorine. Finally, we explored the
influence on potency by changing the imidazo[1,2-a]pyridine to
an imidazo[1,2-a]pyrimidine core (as in 10).
Table 1 summarizes the in vitro anti-TB activity of these 10
analogues in three different media (GAS,14 GAST,15 and 7H1214),
their potency against nonreplicating “latent” TB (LORA16), and
an assessment of their toxicity by the VERO17 assay. All
compounds were potent (MIC < 10 μM in the GAS assay
media) with the exception of the aniline derived analogue (9,
MIC > 128 μM), suggesting that in the imidazo[1,2-a]pyridine
series the benzylic position is important for activity. Additionally,
by running the TB assay in three different media (GAS, GAST,
and 7H12), we eliminated concern that the activity of these
compounds might be carbon source dependent, a flaw discovered
in the pyrimidine-imidazoles reported by Pethe and colleagues
at Novartis18 as the GAS and GAST assays use glycerol-alanine
salts as the carbon source, while the 7H12 media use palmitic acid.
SAR analysis based on the whole cell assay readout indicated
that the 3,4-dichloro analogue (7) had diminished activity (MICs
of 9ꢀ14 μM) when compared to the 4-chloro (6, 0.5 μM) and
3-fluoro analogues (8, ∼0.3 μM). There appeared to be a slight
preference for para-substitution in terms of potency (MIC = 2.8
μM for ortho- vs 1.2 μM for meta- vs 0.5 μM for para-methoxy
analogue in the GAS assay media). Comparison of the imidazo-
[1,2-a]pyrimidine analogue (10) to the corresponding imidazo-
[1,2-a]pyridine analogue (3) indicated that the additional nitro-
gen in the heterocyclic core was well tolerated (submicomolar
Encouraged that six of the 10 analogues tested had submicro-
molar MIC values against the H37Rv Mtb strain, we next screened
compounds 1 and 10 against a panel of single drug resistant strains
(Table 2) against controls rifampicin (RIF) and isoniazid (INH)
and then three of the more promising compounds (1, 3, and 8)
against a panel of MDR and XDR clinical strains (Table 2). The
difference in potencies of these compounds against the clinical
strains may be due to the difference in growth media where growth
inhibition for clinical strains was tested in media containing glucose
as well as glycerol as the carbon source, as well as the fact that many
clinical strains exhibit poor growth in vitro since they are not
adapted to laboratory conditions and media, which may affect their
apparent susceptibility to certain inhibitors.
The excellent activity found when these imidazo[1,2-a]-
pyridine agents were tested against the drug resistant strains
compared favorably to the published MIC values of the nitroi-
midazole clinical candidate PA-82419 (MICs against MDR-TB
from 0.03 to 0.25 μg/mL or 0.08 to 0.7 μM, comparatively).
Furthermore, the improved, and indeed outstanding, potency of
these agents against the various drug resistant strains suggests
that they inhibit a novel target. Selectivity screening against
various nontubercular mycobacteria revealed that compounds 1
and 10 are also inhibitors of Mycobacterium avium, Mycobacter-
ium bovis BCG, and Mycobacterium kansasii but not inhibitors of
Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacter-
ium chelonae, and Mycobacterium marinum (Table 3). This
unusual selectivity prompted us to further screen compounds
1, 3, 4, 6, 8, and 10 against a panel of representative nonmyco-
bacterial organisms. Compounds 1, 3, 4, 6, 8, and 10 were all
found to be inactive against the Gram-positive strain of Staphy-
lococcus aureus (MIC > 128 μM), the Gram-negative strain of
Escherichia coli (MIC > 128 μM), and the fungus Candida
albicans (MIC > 128 μM), further suggesting a mycobacterium
specific target of these agents.
The in vivo pharmacokinetics (PK) of compounds 1, 3, 4, and
6 were evaluated in SpragueꢀDawley rats by oral (po) and
intravenous (iv) routes of administration at 10 and 1 mg/kg
dosing levels, respectively (Table 4). Compound 4 had moderate
in vitro rat microsomal stability (69% metabolized, t1/2 = 19 min)
and also displayed promising PK by having the lowest in vivo
clearance (28 mL/min/kg, t1/2 = 0.28 hours by iv). The aqueous
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LETTER
Table 1. In Vitro Evaluation of Compounds 1ꢀ10 against H37Rv-TB in Various Assays and Media (MIC90 in μM), Stability to
Rat Liver (RLM) and Human Liver (HLM) Microsomes and VERO Cellular Toxicity (IC50 in μM)
* Calculated ClogP by ChemDraw version 12.0. GAS, glycerol-alanine-salts media; GAST, iron deficient glycerol-alanine-salts with Tween 80
media; 7H12, 7H9 broth base media with BSA, casein hydrolysate, catalase, palmitic acid; LORA, low oxygen recover assay; VERO, African green
monkey kidney cell line; RLM, rat liver microsomes; and HLM, human liver microsomes. Values reported are the average of three individual
measurements.
solubility for compounds 1, 3, 4, and 6 was measured at 181,
149, 148, and 25 μM, respectively, in phosphate-buffered
saline (PBS) at pH 7.4. Additional in vivo ADME properties
including terminal half-life (t1/2ss), the area under the curve
(AUC), the volume of distribution (Vd) and volume of
distribution at steady state (Vdss) for compounds 1, 3, 4,
and 6 can be found in the Supporting Information. Encour-
aged by the potency, PK, and favorable oral bioavailability of
these imdazo[1,2-a]pyridine agents, we intend to evaluate
various analogues in vivo by the murine gamma knockout
(GKO) infection model, and the results will be reported in due
course.
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LETTER
Finally, curious as to the mechanism of action of these agents,
we performed transcriptional profiling experiments of M. tuber-
culosis treated with compound 1, and comparison to the existing
database of drug-induced transcriptional profiles indicated that
this compound inhibited an aspect of energy generation in the
cell (see the Supporting Information). Thus, compound 1
resulted in up-regulation of the cytochrome bd oxidase, which
is the high oxygen-affinity respiratory enzyme21 observed to be
up-regulated during oxygen restriction as well as inhibition of
respiration by agents such as cyanide, sodium azide, the uncou-
pler carbonyl cyanide m-chlorophenylhydrazone (CCCP), and
the nitric oxide-releasing pro-drug PA-824.22 In addition, this
compound up-regulated the phosphoenolpyruvate carboxyki-
nase, which plays an important role in modulating carbon flow
during cellular energy restriction23 and has previously been
observed to be up-regulated by stresses such as hypoxia, sodium
azide, valinomycin, nigericin, carbonyl cyanide rn-chlorophe-
nylhydrazone, cyanide, PA-824, and the ATP inhibitor dicy-
clohexylcarboxydiimide, that limit energy generation through
respiration.22
All of the data suggest that we have discovered a class of
compounds with promising attributes of synthetic accessibility,
no redox active moieties,19 impressive potency, and selectivity
toward replicating MDR and XDR Mtb strains. This class has
good in vivo ADME properties that potentially can be improved
through further analogue generation. Additionally, compound 1
appears to act by a novel mechanism of action based on transcrip-
tional profiles to known anti-TB agents. With new anti-TB agents
desperately needed, we offer the imidazo[1,2-a]pyridine class as a
potential therapeutic for further development.
Table 2. Potency of Imidazo[1,2-a]pyridines (1, 3, and 8)
and Imidazo[1,2-a]pyrimidine (10) against Single Drug Re-
sistant Strains, MDR-TB and XDR-TB Strains (MIC90 in
μM)a
control/compound ID
strains resistant to drugs RMP
INH
1
3
8
10
RMP
>1
0.01 >8
0.02
0.02
0.23 0.28
1.49
5.84
1.02
5.84
’ ASSOCIATED CONTENT
INH
0.33
0.43 1.07
0.23 1.02
2.24
S
Supporting Information. Full experimental details for
b
kanamycin
compounds synthesized, descriptions of assays, PK data, and
transcriptional profiling as well as copies of relevant NMR
spectra. This material is available free of charge via the Internet
at http://pubs.acs.org.
streptomycin
MDR-HRESP
MDR-HREZSP
MDR-HCPTh
MDR-HREKP
MDR-HRERbb
MDR-HRERbb
MDR-HRERbb
MDR-HREZSKPTh
MDR-HREZRbTh
XDR-HRESPOCTh
XDR-HREPKOTh
XDR-HRESPO
1.01 0.26
0.06 0.06
0.13 0.26
0.13 0.26
1.12
1.12
0.28
’ AUTHOR INFORMATION
0.14 e0.03 0.13
Corresponding Author
*Tel: þ1-574-631-7571. Fax: þ1-574-631-6652. E-mail: mmiller1@
0.14
0.28
0.03 0.34
0.06 0.26
nd.edu.
0.07 e0.03 0.06
0.14
0.07
0.07
0.14
0.06 0.06
0.03 0.07
0.02 0.03
0.02 0.03
Author Contributions
G.C.M. participated in the design, performed the syntheses,
drafted the manuscript, and facilitated all interactions. L.D.M.
participated in the design and coordinated interactions through
Dow AgroSciences. P.A.H. facilitated microsome and PK assess-
ment. H.B. performed MDR and XDR anti-TB assays and the
transcriptional profiling. S.C. and S.G.F. provided anti-TB and
selectivity assays. M.J.M. drafted the manuscript and participated
in the design and direction of the project.
a Abbreviations: H = isoniazid, R = rifampicin, E = ethambutol, Z =
pyrazinamide, S = streptomycin, C = cycloserine, Th = ethionamide, K =
kanamycin, P = p-aminosalicylic acid, Rb = rifabutin, Th = thioaceta-
zone, and O = ofloxacin. b Different clinical strains.20 Values reported are
the average of three individual measurements.
Table 3. Nontubercular Mycobacteria Activity and Selectivity of Imidazo[1,2-a]pyridine (1) and Imidazo[1,2-a]pyrimidine (10)
(MIC90 in μM)a
compound ID
TB-H37Rv
M. abscessus
M. chelonae
M. marinum
M. avium
M. kansasii
M. bovis BCG
M. smegmatis
1
1.07
5.94
0.05
>50
>50
>50
1.32
12.00
<0.78
1.32
12.00
<0.78
0.33
2.78
>50
10
>50
>50
>50
>50
RMP
162.3
150.00
<0.78
<0.78
162.3
a Values reported are the average of three individual measurements.
Table 4. In Vivo PK Evaluation of Imidazo[1,2-a]pyridinesa
compound ID
po Cmax (ng/mL)
po Tmax (h)
iv t1/2 (h)
iv clearance (mL/min/kg)
% F
1
3
4
6
3012
3140
5741
1995
0.25
0.25
0.25
0.31
0.35
0.33
0.28
0.4
91
43
28
51
76
43
50
49
a Values reported are the average of three individual measurements.
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ACS Medicinal Chemistry Letters
LETTER
Funding Sources
(15) De Voss, J. J.; Rutter, K.; Schroeder, B. G.; Su, H.; Zhu, Y.;
Barry, C. E. The salicylate-derived mycobactin siderophores of Myco-
bacterium tuberculosis are essential for growth in macrophages. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 1252–1257.
(16) Cho, S. H.; Warit, S.; Wan, B.; Hwang, C. H.; Pauli, G. F.;
Franzblau, S. G. Low-oxygen-recovery assay for high-throughput screen-
ing of compounds against nonreplicating Mycobacterium tuberculosis.
Antimicrob. Agents Chemother. 2007, 51, 1380–1385.
(17) Falzari, K.; Zhou, Z.; Pan, D.; Liu, H.; Hongmanee, P.;
Franzblau, S. G. In Vitro and In Vivo Activities of Macrolide Derivatives
against Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 2005,
49, 1447–1454.
Funding was provided by NIH AI054193, Dow AgroSciences,
and NSF CHE-0741793. This research was supported in part by
the Intramural Research Program of the NIH, NIAID, and by
Grant 2R01AI054193 from the National Institutes of Health
(NIH) and in part by intermediates provided from Dow Agro-
Sciences. We thank the University of Notre Dame, especially the
Mass Spectrometry and Proteomics Facility (Bill Boggess, Mi-
chelle Joyce, and Nonka Sevova), which is supported by Grant
CHE-0741793 from the NSF.
(18) Pethe, K.; Sequeira, P. C.; Agarwalla, S.; Rhee, K.; Kuhen, K.;
Phong, W. Y.; Patel, V.; Beer, D.; Walker, J. R.; Duraiswamy, J.; Jiricek, J.;
Keller, T. H.; Chatterjee, A.; Tan, M. P.; Ujjini, M.; Roa, S. P. S.;
Camacho, L.; Bifani, P.; Mak, P. A.; Ma, I.; Barnes, S. W. A chemical
genetic screen in Mycobacterium tuberculosis identifies carbon-source-
dependent growth inhibitors devoid of in vivo efficacy. Nature Commun.
2010, 57, 1–8.
(19) Stover, C. K.; Warrener, P.; VanDevanter, D. R.; Sherman,
D. R.; Arain, T. M.; Langhorne, M. H.; Anderson, S. W.; Towell, J. A.;
Yuan, Y.; McMurray, D. N; Kreiswirth, B. N.; Barry, C. E.; Baker, W. R. A
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’ ACKNOWLEDGMENT
We thank Prof. Jennifer DuBois and Dr. Jed Fisher for
profound scientific discussions. The excellent technical assis-
tance of Baojie Wan and Yuehong Wang with anti-TB assays at
UIC is greatly appreciated. Finally, we thank Gail Cassell and the
Lilly Tuberculosis Drug Discovery Initiative for their continued
support of this project.
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