Design, Synthesis, and Biological Evaluation of Pyrazolo[1,5-a]pyridine-3-carboxamides as Novel Antitubercular Agents

Article abstract of DOI:10.1021/acsmedchemlett.5b00176

A series of pyrazolo[1,5-a]pyridine-3-carboxamide derivatives were designed and synthesized as new anti-Mycobacterium tuberculosis (Mtb) agents. The compounds exhibit promising in vitro potency with nanomolar MIC values against the drug susceptive H37Rv s

Full text of DOI:10.1021/acsmedchemlett.5b00176

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Letter
Design, Synthesis and Biological Evaluation of Pyrazolo[1,5-
a]pyridine-3-carboxamides as Novel Anti-tubercular Agents
Jian Tang, Bangxing Wang, Tian Wu, Junting Wan, Zheng-Chao Tu, Moses
Njire, Baojie Wan, Scott G. Franzblau, Tianyu Zhang, Xiaoyun Lu, and Ke Ding
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.5b00176 • Publication Date (Web): 11 Jun 2015
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Design, Synthesis and Biological Evaluation of Pyrazolo[1,5-
a]pyridine-3-carboxamides as Novel Anti-tubercular Agents
† § ‖
† ‡ ‖
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Jian Tang, , Bangxing Wang, , , Tian Wu, Junting Wan, Zhengchao Tu, Moses Njire, , § Baojie
,
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Wan,⊥ Scott G. Franzblauc,⊥ Tianyu Zhang,†,* Xiaoyun Lu,†,* and Ke Ding†,*
†State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy
of Science, #190 Kai Yuan Avenue, Guangzhou 510530, China.
‡College of Life Science, Anhui University, 111 Jiulong Road, Hefei 230601, China.
§University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing 100049, China.
⊥Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood
Street, Chicago, IL 60612, USA.
KEYWORDS: Anti-tubercular agent, H37Rv, pyrazolo[1,5-a]pyridine, structure-activity relationship, tuberculosis
ABSTRACT: A series of pyrazolo[1,5-a]pyridine-3-carboxamide derivatives were designed and synthesized as new anti-
Mycobacterium tuberculosis (Mtb) agents. The compounds exhibit promising in vitro potency with nanomolar MIC values
against the drug susceptive H37Rv strain and a panel of clinically isolated multidrug-resistant Mtb (MDR-TB) strains. One
of the representative compounds (5k) significantly reduces the bacterial burden in an autoluminescent H37Ra infected
mouse model, suggesting its promising potential to be a lead compound for future anti-tubercular drug discovery.
Tuberculosis (TB) remains one of the world’s deadliest
pandemic diseases with over 9.0 million new cases and 1.5
million deaths estimated by the World Health Organiza-
tion (WHO) in 2013.1 Despite the forty-year success of the
inexpensive quadruple-drug therapy [a combination of
isoniazid (INH), rifampicin (RIF), pyrazinamide and eth-
ambutol], development and dissemination of multidrug-
resistant (MDR) and extensively drug-resistant (XDR)
Mycobacterium tuberculosis (Mtb) strains, together with
co-infection with Human Immunodeficiency Virus (HIV),
have intensified an urgent need for new anti-TB drug dis-
covery.2,3 Encouragingly, for the first time since 1970s, an
ATP synthase inhibitor bedaquiline4 (1, also known as
TMC207) was approved by the US FDA as a novel active
ingredient of combinational therapies for clinical man-
agement of adult patients with MDR pulmonary TB in
2012.5 However, the drug possesses serious adverse effects
such as cardiac arrhythmias,5 and displayed higher death
rates than that of the placebo group in a clinical investiga-
tion,6 which may limit its wide application in clinical
practice. Several other anti-TB molecules were also identi-
fied with different modes of action.2 For instance, a bicy-
clic nitroimidazofuran pro-drug PA-824 (2) was reported
to kill both replicating and hypoxic nonreplicating Mtb
through a Ddn-mediated activation and has been ad-
vanced to phase II clinical trial.7,8 Imidazo[1,2-a]pyridine
amide (IPA)9,10,11,12,13 analogues [e.g. Q203 (3)9,10 and com-
pound 411] were also discovered to demonstrate strong
inhibitory potencies against a panel of drug-susceptible
and drug-resistant Mtb strains by targeting the QcrB sub-
unit of the menaquinol cytochrome c oxidoreductase (bc1
complex), which is a critical component of mycobacterial
energy metabolism14. Clinical outcomes of the compounds,
particularly their capability against MDR and XDR Mtb
strains in patients, are eagerly awaited. However, given
the fact of only one singular FDA approval in forty years
and the high attrition rate of drug development, it is still
highly valuable to identify new molecules with alternative
scaffolds as effective anti-TB drug candidates.
Figure 1. Representatives of the anti-tubercular agents
and the new designed molecules.
Pyrazolo[1,5-a]pyridine moiety is a drug-like scaffold
which is frequently observed in FDA approved or clinical-
ly investigating drugs including antiallergic agent Ibudi-
last15, platelet aggregation inhibitor KC-76416, and dopa-
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mine D4 antagonist FAUC21317 etc. (Supporting Infor-
Anti-TB activities of compounds 5a-5v were prelimi-
1
2
3
4
5
6
7
8
mation). It shares highly similar 3-dimensional confor-
mation and electronic property to that of imidazo[1,2-
a]pyridine which is the pharmaceutical core of Q203 (3)
and compound 4. Therefore, a series of pyrazolo[1,5-
a]pyridine-3-carboxamide derivatives (5a-5v) were de-
signed as new anti-TB agents by using a scaffold hopping
strategy, in which 2, 5-dimethyl groups were introduced
at first because of the synthetic feasibility and keeping the
geometric similarity to that of compound 4, (Figure 1).
narily screened by using a cost-efficient in vitro assay
against a selectable marker-free avirulent autolumines-
cent H37Ra Mtb strain, in which the bacteria growth was
conveniently monitored by the bioluminescence intensity
without adding any substrates.19,20 The minimum inhibi-
tory concentration (MIC) values of the active compounds
were further determined by a well-established microplate
alamar blue assay (MABA) against H37Rv.21 The MICs are
interpolated values obtained by using an in-house curve-
fitting program. The VERO cell growth inhibition was
also tested using the Cell Counting Kit-8 (CCK-8) assay to
assess the compounds’ potential cytotoxicity. All of the 3
reference compounds (i.e. INH, RIF and Q203) displayed
comparable MIC values to the reported data,10 supporting
the reliability of our screen conditions (Table 1).
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Compounds 5a-5v were readily synthesized using a
straightforward amidation of pyrazolo[1,5-a]pyridine-3-
carboxylic acids 8 or 11 with different primary amines
(Scheme 1). Briefly, an N-amination of substituted pyri-
dines 6 or 9 with O-(2,4-dinitrophenyl)hydroxylamine
(DNPH) or O-mesitylenesulfonylhydroxylamine (MSH),
followed by a 1,3-bipolar cycloaddition with substituted
ethyl propiolate, produced the pivotal intermediate pyra-
zolo[1,5-a]pyridine-3-carboxylates 7 or 10.18 Compounds 7
were hydrolyzed to yield the carboxylic acids 8 which
were coupled with different commercially available or
self-prepared amines (Supporting Information) to pro-
duce the designed compounds 5a-50, 5q and 5r with good
yields (Route A). While due to the difficulty of direct N-
amination of halogen-substituted pyridine, additional
removal of Boc protecting group and diazotization reac-
tion of compounds 10 were needed to convert NHBoc
group to Chlorine substitution for the synthesis of com-
pounds 5p and 5s-5v (Route B).
We were pleased to find the first designed molecule 5a,
which carries an identical p-trifluoromethylbenzyl group
to that in compound 4, exhibited strong antimycobacteri-
al activity against Mtb strain H37Rv and the avirulent Mtb
strain H37Ra with MIC values of 69.1 and 287.9 nM, re-
spectively. Further investigation revealed the 4-CF3 group
could be replaced by a dimethylamino moiety (5b) with-
out obviously affecting the anti-TB potency. When a 1-
piperidinyl (5c) or 4-(trifluoromethyl)piperidin-1-yl (5d)
was introduced at the R3 position, the anti- H37Rv activity
was improved 5-6 folds. The investigation also suggested
that R3 position is well tolerated with large hydrophobic
substituent. For instance, trifluoromethoxyphenyl (5e)
and 4-(4-fluorophenyl)piperazin-1-yl (5f) derivatives dis-
played comparable anti-TB activities to that of 5d. En-
couragingly, compound 5g, which harbors the identical
lipophilic tail to that of Q203, demonstrated the strongest
antimycobacterial activity against both H37Rv and H37Ra
Mtb strains, with MIC values of 7.7 and 5.7 nM, respec-
tively. Moreover, compound 5g did not display obvious
cytotoxity with an IC50 value of > 100 µM in a VERO cell
growth inhibition assay, supporting it as a new promising
starting point for further structure-activity relationship
study.
Scheme 1. Synthesis of Compounds 5a-v a
We first investigated the potential impact of R1 sub-
stituent on the anti-TB activity by altering the substituted
position of a methyl group on the pyridine ring (5g-5j). It
was shown that the 5-position is optimal for substitution
(5g). When the methyl group was merged to 4-, 6-, or 7-
position, the resulting compounds (5h, 5i and 5j) were 2-
149 folds less potent against Mtb H37Rv strain. Further
investigation also revealed that the 5-methyl group (5g)
could be replaced by a methoxyl (5k), ethyl (5m) or chlo-
ride (5p) moiety without obviously affecting the anti-TB
potency. However, a large hydrophobic substituent at this
position is detrimental. For instance, 5-isopropyl (5n) and
5-phenyl (5r) displayed MIC values of 47.4 and 478.9 nM
against Mtb H37Rv strain, which are 6.2 and 62.2 folds
less potent than 5g, respectively. Interestingly, a removal
of the 5-substitutent group (5l) barely affected the
aReagents and conditions: (a) DNPH, MeCN, 40°C, 18h for
all compounds expect 5q, or MSH, DCM, 0°C, 2h for
compound 5q, (b) K2CO3, DMF, rt, 18h, 32-57%; (c) NaOH,
EtOH, H2O, 60°C, 100%; (d) amines, EDCI, HOBt, Et3N,
DMF, 80℃, overnight, 40-87%. (e) (i) TFA, DCM, rt, 2h,
100%. (ii) CuCl, con. HCl, NaNO2 (aq, 0.4M), 0-80°C,
45min, 46-86%.
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Table 1. In Vitro Anti-tubercular Activity of Compounds 5a-v against the Mtb Strain H37Rv and H37Ra, and VERO
Cellular Toxicity
1
2
3
4
5
6
7
8
9
MIC [nM (µg/mL)]
IC50 (µM)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
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25
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Compd.
R1
R2
R3
H37Rva
H37Rab
VEROc
>100
>100
>100
>100
50.74
>100
>100
n.d.e
>100
n.d.
5a
5b
5c
5d
5e
5f
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
5-Me
4-Me
6-Me
7-Me
5-OMe
H
Me
Me
Me
Me
Me
-CF3
69.1 (0.024d)
86.8 (0.028)
16.6 (0.006)
13.9 (0.006)
13.7 (0.006)
13.1 (0.006)
7.7 (0.004)
1148 (0.60)
13.4 (0.007)
114.8 (0.06)
11.1 (0.006)
9.8 (0.005)
11.2 (0.006)
47.2 (0.026)
>1771 (>1.0)
7.4 (0.004)
52.0 (0.03)
478.9 (0.28)
358.7 (0.20)
10.8 (0.006)
17.6 (0.01)
287.9 (0.1)
a
b
c
d
e
f
310.2 (0.1)
27.6-82.8 (0.01-0.03)
2.3 (0.001)
6.8 (0.003)
6.6 (0.003)
5.7 (0.003)
>19136 (>10.0)
574.1 (0.3)
Me
5g
5h
5i
Me
Me
Me
f
f
5j
5k
5l
Me
Me
f
f
5741 (3.0)
5.6 (0.003)
>100
>100
>100
n.d.
Me
f
589.9 (0.3)
5m
5n
5o
5p
5q
5r
5-Et
Me
Me
Me
Me
Me
Me
H
Et
f
f
f
f
f
f
f
f
18.6-55.9 (0.01-0.03)
1816-5448 (1.0-3.0)
>17710 (>10)
18.4 (0.01)
5-i-Pr
5-t-Butyl
5-Cl
5-CF3
5-Phenyl
5-Cl
5-Cl
5-Cl
5-Cl
n.d.
>100
>100
n.d.
1734 (1.0)
>17105 (>10)
18905 (10.0)
53.8 (0.03)
5s
5t
n.d.
>100
>100
n.d.
>100
n.d.
5u
5v
3
INH
RIF
cyclo-Pr
Phenyl
f
f
175.7 (0.1)
1547 (0.94)
10.8 (0.006)
2989 (0.41)
>16528 (>10)
5.4 (0.003)
729 (0.10)
-
-
-
36.4 (0.03)
3.64 (0.003)
n.d.
b
aAnti-TB activity assays against H37Rv were performed using MABA. Anti-TB activity assays against H37Ra were per-
c
formed using the autolumilenscent assay. VERO: African green monkey kidney cell line. The cell growth inhibition was
evaluated using the CCK-8 assay. The activity data in the brackets are reported in µg/mL. eNot determined. Values are
d
means of two or more independent experiments, and the variation is <20%.
suppressing function against H37Rv strain. The impact of
the R2 substituent was also investigated. It was clear that
this position was well tolerated by a small hydrophobic
group such as a methyl (5g), ethyl (5t) or cyclopropyl (5u)
moiety. However, when it was unsubstituted (5s) or phe-
nyl substituted (5v), the resulting compounds were signif-
icantly less potent with MIC values of 358.7 and 1547 nM
against H37Rv strain, respectively.
IC50 and IC90 values of compound 5k are 2.5 and 6.7 nM,
respectively.
Table 2. In Vitro MIC, IC50 and IC90 Values of Com-
pounds 5f, 5g, 5k and 5t against Fluorescent Reporter
Strain of Mtb H37Rv.
Compd.
5f
MIC (nM)
6.3
IC50 (nM)
IC90 (nM)
6.5
2.5
5.1
2.5
31.3
4.1
5g
11.0
12.0
5.7
150
6.9
The anti-mycobacterial activities of compounds 5f, 5g,
5k and 5t were further validated by determining their
MIC, IC50 and IC90 values against the replication of a fluo-
rescent reporter strain of Mtb H37Rv in liquid medium
under aerobic conditions (Table 2).22 It was shown that
compounds 5f and 5k displayed comparable anti-
tubercular effects against H37Rv to that of RIF, whereas
compounds 5g and 5t were moderately less potent. The
5k
5.6
5t
129
RIF
6.5
Encouraged by their strong potencies against the
drug-sensitive Mtb H37Rv strain, compounds 5g, 5k, 5p
and 5t were further evaluated against a panel of clinical
isolated 3495, P71, 9804, 4768, P163 MDR strains23 by us-
ing an autoluminescent assay.19 It was shown that all of
the compounds displayed excellent potencies against the
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resistant Mtb strains with MIC values ranged from 11.1 to as a new promising lead compound for further anti-
1
2
3
4
1914 nM. Particularly, compound 5k exhibited superior
inhibition against five resistant strains with similar MIC
values (11.1-223 nM) to that of the wild-type Mtb H37Rv,
suggesting its promising potential for both drug-sensitive
and resistant Mtb strains.
tubercular drug discovery.
5
6
7
8
9
Table 3. Anti-tubercular Activity of Compounds 5g, 5k,
5p and 5t against Drug-Resistant Clinical Mtb Iso-
lates.
MIC (nM)b
Strai
ns
Re-
sistancea
HRSZ
HRZ
HRZ
EHRSZ
EHSZ
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
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59
60
5g
5k
5p
5t
3495
P71
9804
4768
P163
1914
22.3-223
11.1-223
<11.1
11.1-223
11.1-223
n.d.c
<184
<184
<184
<184
n.d.
71.8-1795
<35.9
35.9-71.8
<35.9
<383
<383
<383
<383
Figure 2. Mean RLU count (± SD) assessed every other
day in live, anesthetized mice and normalized to the base-
line RLU value. Days post initial treatment (x-axis) is plot-
ted against the corresponding RLUdayn/RLUday0 ratio (y-
axis). Blue: vehicle; Purple: 5k 100mg/kg qd; Red: 5k
20mg/kg qd; Green: 5k 4mg/kg qd.
aH, isoniazid; R, rifampicin; S, streptomycin; E, ethambu-
tol; Z, pyrazinamide. Values are means of two independ-
b
c
ent experiments, and the variation is <20%. Not Deter-
mined.
Given its promising anti-mycobacterial activity against
both drug-susceptible and drug-resistant Mtb strains, the
in vivo anti-tubercular efficacy of 5k was evaluated using a
modified real-time monitoring non-invasive mouse model
infected with the selectable marker-free autoluminescent
Mtb strain H37Ra.19,20 The animals were infected with log-
phase autoluminescent Mtb H37Ra via intravenous injec-
tion (2×106 CFU per mouse), and then were repeatedly
administrated with agent 5k once daily via oral gavage for
6 consecutive days. The bacterial burden was measured
by monitoring the bioluminescence intensity (relative
light unit, RLU) from the same batch of live mice every
other day. As shown in Figure 2, compound 5k exhibited
dose-dependent in vivo anti-tubercular activity and was
well tolerated in all of the tested groups with no mortality
(data not shown) observed during treatment. Administra-
tion with CMC-Na (vehicle) permitted a nearly 2.8-fold
increase in RLU over six days; whereas compound 5k with
the three tested doses prevented any increase over base-
line, with RLUdayn/RLUday0 ratios of 0.40, 0.19 and 0.15 in 4,
20 and 100mg/kg/day treated groups, respectively. Espe-
cially, with 100mg/kg/day, compound 5k exhibited a sus-
tained bactericidal activity against Mtb H37Ra, resulting
in a 3.6-fold, 5.0-fold and 6.5-fold decrease in RLU from
baseline on day2, day4 and day6, respectively. These re-
sults strongly suggest the promising potential of com-
pound 5k to serve as a lead compound for further anti-TB
drug discovery.
ASSOCIATED CONTENT
Supporting Information. Experimental procedures for the
synthesis of 5a-5v and self-prepared amines, 1H NMR, and
13C NMR for final compounds, and details of in vitro and in
vivo assays. This material is available free of charge via the
Internet at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
*Tel: +86-20-32015276. Fax: +86-20-32015299. E-mail:
ding_ke@gibh.ac.cn; lu_xiaoyun@gibh.ac.cn;
zhang_tianyu@gibh.ac.cn.
Author Contributions
‖These authors contributed equally.
Funding Sources
We thank financial support from the Key Program of the
Chinese Academy of Sciences (KJZD-EW-L02), Youth Inno-
vation Promotion Association of Chinese Academy of Scienc-
es and Scientific, the Chinese Academy of Sciences 'One
Hundred Talents Program' (Category A, to T.Z.), Technologi-
cal Innovation Program of Foshan City (2013HK100212) and
the Open Project Grant （2014SKLRD-O06） from the State
Key Lab of Respiratory Disease, Guangzhou Medical Univer-
sity. In addition, this work was supported by National Insti-
tutes of Health and the National Institute of Allergy and In-
fectious Diseases, Contract No. HHSN272201100009I.
In summary, a series of pyrazolo[1,5-a]pyridine-3-
carboxamide derivatives were designed as new anti-
tubercular agents by using a scaffold hopping strategy.
The compounds exhibit excellent in vitro inhibitory activ-
ities with low nanomolar MIC values against both drug-
sensitive Mtb strain H37Rv and drug-resistant clinical
isolates. One of the most promising compounds 5k dis-
played significant bacterial burden reduction in the Mtb
H37Ra infected mouse model. This compound may serve
Notes
The authors declare no competing financial interest.
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