Pyrazolo[1,5-a]pyridine-3-carboxamide hybrids: Design, synthesis and evaluation of anti-tubercular activity

Article abstract of DOI:10.1016/j.ejmech.2016.09.030

A series of pyrazolo[1,5-a]pyridine-3-carboxamide hybrids were designed and evaluated as novel anti-tubercular agents. The representative hybrid 7 exhibited promising in?vitro activity against susceptive strain H37Rv and a panel of drug-resistant Mtb strains with MIC values of 0.006?μg/mL and ranged from 0.003 to 0.014?μg/mL, respectively. More importantly, the hybrid 7 also showed very low cytotoxicity, and could significantly reduce the mycobacterial burden in a mouse model infected with autoluminescent H37Ra strain, which may serve as a lead compound for further development of new anti-tubercular agents.

Full text of DOI:10.1016/j.ejmech.2016.09.030

European Journal of Medicinal Chemistry 125 (2017) 41e48
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Research paper
Pyrazolo[1,5-a]pyridine-3-carboxamide hybrids: Design, synthesis
and evaluation of anti-tubercular activity
,
b
,
*
,
,
c
,
,
Xiaoyun Lu a
1, Jian Tang b 1, Shengyang Cui b c, Baojie Wan d, Scott G. Franzblauc d,
Tianyu Zhang b, Xiantao Zhang e, Ke Ding a
,
b
a School of Pharmacy, Jinan University, 601 Huangpu Avenue West, Guangzhou, 510632, China
b State Key Laboratory of Respiratory Diseases, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science, #190, Kai Yuan Avenue,
Guangzhou, 510530, China
c University of Chinese Academy of Sciences, #19 Yuquan Road, Beijing, 100049, China
d Institute for Tuberculosis Research, College of Pharmacy, University of Illinois at Chicago, 833 South Wood Street, Chicago, IL, 60612, United States
e Guangzhou Eggbio Co., Ltd, 3 Ju Quan Road, Science Park, Guangzhou, 510663, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of pyrazolo[1,5-a]pyridine-3-carboxamide hybrids were designed and evaluated as novel anti-
tubercular agents. The representative hybrid 7 exhibited promising in vitro activity against susceptive
Received 27 June 2016
Received in revised form
8 September 2016
Accepted 9 September 2016
Available online 10 September 2016
strain H37Rv and a panel of drug-resistant Mtb strains with MIC values of 0.006
mg/mL and ranged from
0.003 to 0.014 g/mL, respectively. More importantly, the hybrid 7 also showed very low cytotoxicity, and
m
could significantly reduce the mycobacterial burden in a mouse model infected with autoluminescent
H37Ra strain, which may serve as a lead compound for further development of new anti-tubercular
agents.
Keywords:
Anti-tubercular
© 2016 Elsevier Masson SAS. All rights reserved.
Hybrid
Mtb
Pyrazolo[1,5-a]pyridine-3-carboxamide
1. Introduction
effects such as the increased mortality and QT prolongation [5],
which may limit its applications in clinic. Thus, there is an urgent
Mycobacterium tuberculosis (Mtb) is the causative agent of
tuberculosis (TB), which remains one of the world's deadliest
pandemic diseases with over 9.0 million new cases and 1.5 million
deaths every year [1]. WHO recommended a standard regimen for
the treatment of TB by a combination of four first-line drugs
(isoniazid, rifampicin, ethambutol and pyrazinamide) with 6e9
months therapy [2]. However, multidrug-resistant (MDR), exten-
sively drug-resistant (XDR) Mtb strains and comorbidity with HIV
have made TB therapy difficult and ineffective. At present, MDR-TB
is treated with a combination of eight to ten drugs lasting up to
18e24 months [3]. Bedaquiline (TMC207, SIRTURO®) was approved
by FDA in 2012 as part of combination therapy for the treatment of
adults with pulmonary MDR-TB [4]. However, bedaquiline pre-
scription has to include a black-box warning due to serious adverse
need to develop new anti-TB drugs to shorten the treatment
duration and target MDR-TB strains.
Extensive efforts were invested in the identification of novel
anti-TB drugs in academic and pharmaceutical industry. Molecular
hybridization of a lead scaffold and existing drugs has become a
promising strategy to design and discover potent anti-TB drugs
[6,7], with the purpose to enhance activity and modulate physi-
ochemical properties. Pyrazolo[1,5-a]pyridine-3-carboxamide
scaffold is an effective anti-TB pharmacophore published by our
group (Fig. 1) [8]. The study indicated that the incorporation of
lipophilic chain tail into the pyrazolo[1,5-a]pyridine-3-
carboxamide can increase anti-TB activity and improve drugg-
ability. In view of these facts, we envisaged to design new pyrazolo
[1,5-a]pyridine-3-carboxamide hybrids incorporated with frag-
ment of existing clinical anti-TB drugs, which could possibly target
MDR-TB strains. Several clinical representative anti-TB molecules
SQ109 [9], SQ609 [10], PNU-100480 [11], PA-824 [12], TBA-354 [13]
and OPC-67683 [14] (Fig. 1) share similar chain tail to that of pyr-
azolo[1,5-a]pyridine-3-carboxamide. In this study, we designed a
* Corresponding author. School of Pharmacy, Jinan University, 601 Huangpu
Avenue West, Guangzhou, 510632, China.
E-mail address: luxy2016@jnu.edu.cn (X. Lu).
These authors contributed equally.
1
http://dx.doi.org/10.1016/j.ejmech.2016.09.030
0223-5234/© 2016 Elsevier Masson SAS. All rights reserved.

42
X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
Fig. 1. Pyrazolo[1,5-a]pyridine-3-carboxamide core (colored in purple) and representative clinical anti-TB drugs and their tails (colored in blue). (For interpretation of the references
to colour in this figure legend, the reader is referred to the web version of this article.)
new class of pyrazolo[1,5-a]pyridine-3-carboxamide hybrids to
improve the anti-tubercular activity especially for drug resistant
Mtb as outlined in Fig. 1.
tail of SQ109 to 5-chloro-2-ethylpyrazolo[1,5-a]pyridine core,
exhibited strong antimycobacterial activity against H37Ra and
H37Rv comparable to INH, with MIC values of 0.3 and 0.17 mg/mL,
respectively. While incorporation of the tails of SQ609 and PNU-
10048, as in compounds 2 and 3, showed significant loss of po-
tency in both strains. When the tail of TBA-354 was merged to the
5-chloro-2-ethylpyrazolo[1,5-a]pyridine core, the yielded com-
2. Chemistry
As represented in Fig. 2, the synthetic routes of new pyrazolo
[1,5-a]pyridine-3-carboxamide hybrids (1e8) involved amide bond
formation between the corresponding pyrazolo[1,5-a]pyridine-3-
carboxylic acids published by us before [8] and either commer-
cially available or self-prepared amines. The primary amines 1a, 3a,
and 6a for the synthesis of compounds 1, 3 and 6, respectively, were
commercially available. The amine 2a was synthesized by acylation
between piperidine-4-carbonitrile (2b) and commercially available
(3r, 5r, 7r)-adamantane-1-carbonyl chloride (2c) followed by nitrile
reduction with lithium aluminium hydride. Suzuki coupling of 6-
chloronicotinonitrile (5b) or 5-chloropicolinonitrile (7b) and (4-
(trifluoromethoxy)phen-yl)boronic acid using Pd(PPh3)4 and
aqueous Na2CO3, followed by reduction with Raney Nickel, gave the
amines 5a or 7a, respectively. The synthesis of amine 8a was started
by coupling tert-butyl 4-hydroxypiperidine-1-carboxylatec (8b)
and 4-(trifluoromethoxy)phenol (8c), which is followed by nucle-
ophilic and reduction reactions based on our published procedures
[8]. Finally, the title hybrids were readily obtained using a straight
forward amidation of pyrazolo[1,5-a]pyridine-3-carboxylic acids
with amines 1a-8a in the presence of EDCI and HOBt.
pound 4 showed an MIC value of 0.2 mg/mL comparable to that of
compound 1. Further investigations of the substituents on the core
revealed that the replacement of 5-chloro-2-ethylpyrazolo[1,5-a]
pyridine with 2,5-dimethyl- pyrazolo[1,5-a]pyridine (5) could
improve the antimycobacterial activity against H37Ra and H37Rv to
0.03 and 0.06 mg/mL, respectively. However, when the tail of PA-
824 was merged to the 2,5-dimethylpyrazolo[1,5-a]pyridine, the
resulted compound 6 only exhibited moderate activity. Interest-
ingly, shifting the pyridine nitrogen of compound 5 to the ortho
position (7) displayed a remarkable improvement of activity with
MIC values of 0.003 and 0.006 mg/mL against Mtb strain H37Ra and
H37Rv, respectively, which is more potent than INH, and compa-
rable to RIF (Table 1). The 2,5-dimethylpyrazolo[1,5-a]pyridine and
tail of OPC-67683 hybrid 8 was also displayed comparable MIC
activity to that of RIF.
In view of their excellent potencies against Mtb H37Rv strain,
compounds 5 and 7 were further evaluated against five drug-
resistant Mtb isolates under aerobic conditions with INH, RIF and
levofloxacin (LEV) as positive controls (Table 2) [17]. Compounds 5
and 7 also exhibited strong inhibitory activities against all five
different resistant strains with MIC values ranged from 0.003 to
3. Results and discussion
0.079 mg/mL, while INH, RIF and LEV showed significant activity loss
against their corresponding drug-resistant strains. From the results,
it was shown that compound 7 was slightly more active than
compound 5, and exhibited similar potency against both suscepti-
ble strains H37Rv and resistant isolates, suggesting its promising
potential for both drug-sensitive and resistant Mtb strains. More-
over, compound 7 had no cytotoxicity against VERO and HepG2
The minimum inhibitory concentration (MIC) values of all new
compounds were preliminarily screened against avirulent strain
H37Ra using the autoluminescent assay [15], then further deter-
mined against M. tuberculosis H37Rv strain using the microplate
alamar blue assay (MABA) (Table 1) [16]. Isoniazid (INH) and
rifampicin (RIF) were used as the positive drugs.
cells, which displayed the IC50 values of 43
mM and >100 mM,
The first designed hybrid 1, with an incorporation of aliphatic

X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
43
Fig. 2. Synthesis of compounds 1e8. Reagents and conditions: (a) DIPEA, DMF, r.t., 16 h, 71.0%; (b) LAH, anhydrous THF, 0 ꢀC to reflux, overnight, 96.7%; (c) (4-(trifluoromethoxy)
phenyl)boronic acid, Pd(PPh3)4, 2 M Na2CO3 (a.q.), toluene, 110 ꢀC, overnight, 79.9%; (d) Raney Nickel, H2, methanol, r.t., 3.5 h, 58.1%; (e) PPh3, DEAD, THF, r.t., 18 h, 70.4%; (f) i) TFA,
DCM, 0 ꢀC to r.t., 3 h; ii) 4-florobenzonitrile, K2CO3, DMSO, 120 ꢀC, 6 h, 69.2%; (g) LAH, anhydrous THF, 0 ꢀC to r.t., 2 h, 90.6%; (h) EDCI, HOBt, Et3N, DMF, 80 ꢀC, overnight, 50e77%.
respectively (Table 3).
hybrids incorporated with fragment of existing clinical anti-TB
drugs have been designed, synthesized and evaluated for antitu-
bercular activity. Several hybrids exhibited potent anti-
mycobacterial activity against Mtb strain H37Ra and H37Rv with
nanomolar MIC values, which was comparable to that of RIF. The
representative hybrid 7 exhibited promising in vitro activities
against a panel of drug-resistant Mtb strains with MIC values
The antitubercular activity of compound 7 was further evaluated
in vivo using a cost-efficient mouse model infected with the
selectable marker-free autoluminescent Mtb strain H37Ra [8,18]. As
shown in Fig. 3, compound 7 exhibited dose-dependent in vivo
anti-tubercular activity. After 4-day treatment, the RLUdayn/RLUday0
ratios were 0.96 and 0.50 for animals in the 4 and 100 mg/kg/day
treated groups, respectively, while the corresponding number was
ranged from 0.003 to 0.014 mg/mL. Further in vivo studies indicated
about 2.68 in the untreated vehicle group. However, the RLUdayn
/
that compound 7 significantly reduce the mycobacterial burden in
H37Ra infected mouse model, suggesting it as a new lead for
further anti-tubercular drug discovery.
RLUday0 ratios were on the rise on the day 6. The reason needs to be
investigated. Nevertheless, the results suggested the promising
potential of compound 7 as a lead compound for the development
of new anti-TB agents.
5. Experimental section
4. Conclusions
5.1. Chemistry
In this paper, a series of pyrazolo[1,5-a]pyridine-3-carboxamide
General Methods for Chemistry. All reagents and solvents were

44
X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
Table 1
The in vitro antitubercular activity of hybrids 1e8 against the Mtb strains H37Rv and H37Ra.
Compds
Structure
MIC (mg/mL)
H37Raa
H37Rvb
0.17
1
0.30
2
3
3
>1
>10
nd
4
5
0.10
0.03
0.20
0.06
6
7
3.0
0.91
0.003
0.006
8
0.03
0.006
INH
RIF
e
e
0.41
0.03
0.1
0.003
a
Anti-TB activity assays against H37Ra were performed using the autoluminescent assay.
Anti-TB activity assays against H37Rv were performed using MABA.
b
Table 3
Table 2
The cytotoxicity of compound 7 against VERO and HepG2 cells.
Antitubercular activity of compounds 5 and 7 against drug-resistant Mtb strains.
Compd
IC50 (mM)
Compds
MIC (mg/mL)
VEROa
HepG2b
>100
INH-R1a
INH-R2b
RIF-R1c
RIF-R2d
FQ-R1e
7
43
5
7
INH
RIF
LEV
0.079
0.014
>27
0.016
0.44
0.015
0.006
>27
0.007
0.59
0.009
0.003
0.056
0.42
0.016
0.006
0.11
>41
0.63
0.040
0.009
0.088
0.017
10
a
VERO: African green monkey kidney cell line.
HepG2: human liver cells.
b
0.37
a
INH-R1 (isoniazid resistant strain) was derived from H37Rv and is a katG mutant
(Y155* ¼ truncation).
obtained from commercial sources and used without further pu-
rification. Flash chromatography was performed using silica gel
(200e300 mesh). All reactions were monitored by TLC, using silica
gel plates with fluorescence F254 and UV light visualization. 1H NMR
and 13C NMR spectra were recorded either on a 400 MHz (1H,
400 MHz) or 500 MHz (1H, 500 MHz; 13C, 125 MHz) Bruker spec-
trometer. Coupling constants (J) are expressed in hertz (Hz).
b
INH-R2 (isoniazid resistant strain) is a strain ATCC35822.
c
RIF-R1 (rifampicin resistant strain) was derived from H37Rv and is an rpoB
mutant (S522L).
d
RIF-R2 (rifampicin resistant strain) strain ATCC35828.
FQ-R1is a fluoroquinolone-resistant strain derived from H37Rv and is a gyrB
e
mutant (D94 N).
Chemical shifts (
d) of NMR are reported in parts per million (ppm)
units relative to the internal residual solvent peak. The following

X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
45
purified through flash column chromatography to afford the com-
pound 5c (7.62 g, 79.9%) as a white solid. 1H NMR (400 MHz, CDCl3)
d
(ppm) 8.95 (s, 1H), 8.10 (d, J ¼ 8.8 Hz, 2H), 8.03 (dd, J ¼ 8.4, 2.4 Hz,
1H), 7.84 (d, J ¼ 8.4 Hz, 1H), 7.36 (d, J ¼ 8.4 Hz, 1H). MS (ESI) m/z 265
[MþH]þ.
To a stirred solution of 5c (2.0 g, 7.57 mmol) in methanol
(100 mL) was added Raney Nickel (5% e.q.). The mixture was
evacuated and backfilled with hydrogen (three cycles) and stirred
at room temperature for 3.5 h. After the reaction was finished, the
mixture was filtered through celite and concentrated to afford the
compound 5a (1.18 g, 58.1%). 1H NMR (400 MHz, DMSO-d6)
d(ppm)
8.62 (s, 1H), 8.19 (dd, J ¼ 8.8, 2.0 Hz, 2H), 7.94 (d, J ¼ 8.0 Hz, 1H), 7.86
(dd, J ¼ 8.4, 2.4 Hz, 1H), 7.46 (d, J ¼ 8.4 Hz, 2H), 3.78 (s, 2H). MS (ESI)
m/z 269 [MþH]þ.
5.1.3. (5-(4-(trifluoromethoxy)phenyl)pyridin-2-yl) methanamine
(7a)
Fig. 3. Compound 7 dose-dependently inhibits the growth of M.tb H37Ra following 6
consecutive days of administration. Days post initial treatment (x-axis) is plotted
against the corresponding RLUdayn/RLUday0 ratio (y-axis). Blue: vehicle (CMC-Na); Red:
7 100 mg/kg qd; Green: 7 4 mg/kg qd. (For interpretation of the references to colour in
this figure legend, the reader is referred to the web version of this article.)
Compound 7a was prepared by the method similar to that of 5a.
1H NMR (400 MHz, DMSO-d6)
d
(ppm) 8.82 (d, J ¼ 2.0 Hz, 1H), 8.07
(dd, J ¼ 8.4, 2.4 Hz, 2H), 7.85 (d, J ¼ 8.8 Hz, 2H), 7.59 (d, J ¼ 8.0 Hz,
1H), 7.48 (d, J ¼ 8.0 Hz, 2H), 3.92 (s, 2H). MS (ESI) m/z 269 [MþH]þ.
5.1.4. (4-(4-(4-(trifluoromethoxy)phenoxy)piperidin-1-yl)phenyl)
methanamine (8a)
abbreviations were used: br ¼ broad signal, s ¼ singlet, d ¼ doublet,
dd ¼ doublet of doublets, t ¼ triplet, q ¼ quartet, m ¼ multiplet.
Low resolution ESI-MS were recorded on an Agilent 1200 HPLC-MS
mass spectrometer and high resolution ESI-MS on an ABSciex Tri-
pleTOF 5600 plus System ESI-LC-MS/MS mass spectrometer. The
purity of compounds was determined by reverse-phase high per-
formance liquid chromatography (HPLC) analysis to be >95%. HPLC
instrument: Dionex Summit HPLC (column: Diamonsil C18, 5.0 m,
4.6 ꢁ 250 mm (Dikma Technologies); detector: PDA-100 photo-
diode array; pump: p-680A; injector: ASI-100 autoinjector). A flow
rate of 1.0 mL/min was used with a mobile phase of MeOH in H2O
with a 0.1% ammonia.
To a stirred solution of 4-(trifluoromethoxy)phenol (8c, 1.05 g,
5.90 mmol) in THF (50 mL), 4-hydroxy-piperidine-1-carboxylic acid
tert-butyl ester (8b, 1.08 g, 5.37 mmol) was added followed by PPh3
(1.55 g, 5.90 mmol). The resulting reaction mixture was stirred at
r.t. for 15 min. DEAD (1.20 g, 1.17 mmol) was added dropwise at
20 ꢀC and the reaction mixture was stirred at r.t. for 18 h. After
completion of the reaction, the mixture was concentrated under
reduced pressure to obtain the crude material, which was further
diluted with CH2Cl2 (50 mL). The CH2Cl2 layer was washed with
water (3 ꢁ 100 mL) then dried over Na2SO4, filtered and concen-
trated under reduced pressure to obtain the crude material, which
was further purified through silica gel column chromatography to
afford the compound 8d (1.71 g, 70.4%). 1H NMR (400 MHz, CDCl3)
5.1.1. (1-((3r, 5r, 7r)-adamantan-1-ylmethyl)piperidin-4-yl)
methanamine (2a)
d
(ppm) 7.27 (d, J ¼ 8.4 Hz, 2H), 7.07 (d, J ¼ 9.2 Hz, 2H), 4.55e4.58
Piperidine-4-carbonitrile (2b, 1.00 g, 9.1 mmol) was dissolved in
DMF (15 mL) and DIPEA (2, 2.94 g, 22.8 mmol) in an atmosphere of
argon. (3r, 5r, 7r)-adamantane-1-carbonyl chloride (2c, 1.5 g,
7.6 mmol) dissolved in DMF (3.4 mL) was added slowly and the
reaction mixture was stirred at r.t. for 16 h CH2Cl2 and 1 N HCl (aq)
were added and the phases separated. The organic phase was
evaporated to dryness and purified through flash column chro-
matography to afford the compound 2d (1.48 g, 71.0%). MS (ESI) m/z
273 [MþH]þ.
(m, 1H), 3.63e3.67 (m, 2H), 3.15e3.19 (m, 2H), 1.88e1.91 (m, 2H),
1.47e1.55 (m, 2H), 1.40 (s, 9H). MS (ESI) m/z 362 [MþH]þ.
To a solution of 8d (1.36 g, 3.78 mmol) in anhydrous DCM
(15 mL) was added trifluoroacetic acid (4.5 mL) in 0 ꢀC and the
reaction was stirred at room temperature for 3 h. After the reaction
was finished, the mixture was evaporated and the residue was
redissolved with EA. The solution was washed with saturated
NaHCO3 (a.q.) to the aqueous layer pH ¼ 8.0. Organic layer was
separated, washed with brine and evaporated to dryness, and the
crude product was used without further purification.
A solution of 2d (5.49 g, 20.0 mmol) in anhydrous THF (300 mL)
was added LAH (4.6 g,121.0 mmol) at 0 ꢀC. After stirred for 0.5 h, the
mixture was heated to reflux for overnight. The reaction was
quenched with 40 mL 2 M NaOH (a.q.) and the mixture was filtered
through celite and concentrated. The residue was redissolved in
chloroform and washed with water. The organic layer was sepa-
rated and then evaporated to dryness to afford the compound 2a
(5.07 g, 96.7%). MS (ESI) m/z 263 [MþH]þ.
A mixture of the resulting product and anhydrous K2CO3 (1.57 g,
11.34 mmol) in DMSO (23 mL) was added 4-florobenzonitrile
(1.14 g, 9.45 mmol). The reaction was stirred at 120 ꢀC for 6 h. Af-
ter the reaction was finished, the mixture was poured into 100 mL
water with stirring and the precipitated solid was filtrated for
further purification through flash column chromatography to
afford the title compound 8e (0.95 g, 69.23%). 1H NMR (400 MHz,
CDCl3)
d
(ppm) 7.57 (d, J ¼ 8.4 Hz, 2H), 7.15e7.17 (m, 4H), 6.92 (d,
5.1.2. (6-(4-(trifluoromethoxy)phenyl)pyridin-3-yl) methanamine
(4a or 5a)
J ¼ 8.8 Hz, 2H), 4.57 (m, 1H), 3.62e3.67 (m, 2H), 3.34e3.39 (m, 2H),
2.26 (m, 2H), 2.01 (m, 2H). MS (ESI) m/z 363 [MþH]þ.
A
mixture solution of 6-chloronicotinonitrile (5b, 5.0 g,
A solution of 8e (0.95 g, 2.62 mmol) in anhydrous THF (50 mL)
was added LAH (0.31 g, 8.13 mmol) at 0 ꢀC. After stirred for 0.5 h,
the mixture was heated to reflux for additional 2 h. The reaction
was quenched with 3 mL 2 M NaOH (a.q.) and the mixture was
filtered through celite and concentrated. The residue was redis-
solved in EA and washed with brine. The organic layer was sepa-
rated and then evaporated to dryness to afford the title compound
36.1 mmol) and 2 M Na2CO3 (a.q. 36 mL) in toluene (100 mL) was
added (4-(trifluoromethoxy)phenyl)boronic acid (11.12 g,
54.0 mmol) and Pd(PPh3)4 (0.5 g, 0.43 mmol). The mixture was
evacuated and backfilled with argon (three cycles) and stirred at
110 ꢀC for overnight. After the reaction was finished, the mixture
was filtered through celite and concentrated. The residue was

46
X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
8a (0.87 g, 90.6%). MS (ESI) m/z 367 [MþH]þ.
5.1.5.5. 2,5-dimethyl-N-((5-(4-(trifluoromethoxy)phenyl)pyridin-2-
yl)methyl)py-razolo[1,5-a]pyridine-3-carboxamide (5). 1H NMR
5.1.5. General procedure for synthesis of hybrids 1e8
(400 MHz, DMSO-d6):
d
(ppm) 8.86 (s, 1H), 8.55 (d, J ¼ 7.2 Hz, 1H),
To a stirred solution of 5-chloro-2-ethylpyrazolo[1,5-a]pyridine-
3-carboxylic acid or 2,5-dimethylpyrazolo[1,5-a]pyridine-3-
carboxylic acid (1.34 mmol) in DMF (20 mL) was added EDCI
(0.39 g, 2.01 mmol), HOBt (91 mg, 0.67 mmol) and Et3N (0.68 g,
6.7 mmol) at room temperature. After 1 h of stirring, 1ae8a
(1.34 mmol) was added and the reaction was heated to 80 ꢀC for
overnight. The mixture was cooled, and diluted with EA (50 mL),
washed with water and brine, dried with Na2SO4 and concentrated.
The crude product was purified by flash chromatography to afford
the title compounds1e8.
8.09 (m, 2H), 7.86 (d, J ¼ 8.8 Hz, 2H), 7.80 (s, 1H), 7.48 (m, 3H), 6.83
(dd, J ¼ 7.2, 1.6 Hz, 1H), 4.64 (d, J ¼ 6.0 Hz, 2H), 2.59 (s, 3H), 2.39 (s,
3H). 13C NMR (125 MHz, CDCl3):
d(ppm): 164.59, 156.22, 151.26,
149.43, 147.47, 142.19, 137.75, 136.45, 135.36, 134.28, 128.66, 127.60,
122.24, 121.72, 120.62 (q, J ¼ 256 Hz, 1C), 117.57, 115.54, 103.87,
44.31, 21.58, 14.93. HRMS (ESI) calcd for C23H19F3N4O2 [MþH]þ:
441.1533; found 441.1530. HPLC purity ¼ 99.50%, Rt 5.67 min.
5.1.5.6. 2,5-dimethyl-N-(4-(trifluoromethoxy)benzyl) pyrazolo[1,5-a]
pyri-dine-3-carboxamide (6). 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 8.53 (d, J ¼ 7.2 Hz, 1H), 8.04 (t, J ¼ 5.6 Hz, 1H), 7.73 (s, 1H),
5.1.5.1. (E)-5-chloro-N-(3,7-dimethylocta-2,6-dien-1-yl)-2-
7.47 (d, J ¼ 8.8 Hz, 2H), 7.33 (d, J ¼ 8.0 Hz, 2H), 6.81 (dd, J ¼ 7.2,
ethylpyrazolo[1,5-a]py-ridine-3-carboxamide
(1). 1H
NMR
(ppm) 8.71 (d, J ¼ 7.2 Hz, 1H), 7.86 (d,
1.6 Hz, 1H), 4.50, 2.55 (s, 3H), 2.38 (s, 3H). 13C NMR (125 MHz,
(400 MHz, DMSO-d6):
d
CDCl3): d(ppm): 164.54, 150.63, 148.63, 142.24, 137.90, 129.10,
J ¼ 2.0 Hz, 1H), 7.82 (t, J ¼ 5.2 Hz, 1H), 7.00 (dd, J ¼ 7.2, 2.4 Hz, 1H),
5.27 (t, J ¼ 6.4 Hz, 1H), 5.09 (t, J ¼ 6.8 Hz, 1H), 3.87 (t, J ¼ 6.0 Hz, 2H),
2.98 (q, J ¼ 7.6 Hz, 2H), 2.04e2.09 (m, 2H), 1.97e2.00 (m, 2H), 1.68
(s, 3H), 1.62 (s, 3H), 1.56 (s, 3H), 1.24 (t, J ¼ 7.6 Hz, 3H). 13C NMR
127.61, 121.39, 120.61 (q, J ¼ 256 Hz, 1C), 117.459, 115.692, 103.58,
42.82, 21.51, 14.89. HRMS (ESI) calcd for C18H16F3N3O2 [MþH]þ:
364.1267; found 364.1270. HPLC purity ¼ 99.77%, Rt 6.48 min.
(125 MHz, CD3Cl):
d
(ppm): 163.72, 156.73, 141.96, 140.51, 133.14,
5.1.5.7. 2,5-dimethyl-N-((6-(4-(trifluoromethoxy)phenyl) pyridin-3-
yl)methyl)py-razolo[1,5-a]pyridine-3-carboxamide (7). 1H NMR
131.95, 128.95, 123.94, 120.21, 117.21, 114.30, 104.82, 39.65, 37.64,
26.57, 25.81, 22.14, 17.84, 16.51, 13.17. HRMS (ESI) calcd for
C
purity ¼ 99.24%, Rt 15.76 min.
(400 MHz, DMSO-d6):
d
(ppm) 8.68 (s, 1H), 8.53 (d, J ¼ 6.8 Hz, 1H),
20H26ClN3O [MþH]þ: 360.1837; found 360.1836. HPLC
8.20 (d, J ¼ 8.8 Hz, 2H), 8.08 (t, J ¼ 5.8 Hz, 1H), 7.98 (d, J ¼ 8.4 Hz,
2H), 7.88 (dd, J ¼ 8.2, 2.2 Hz, 1H), 7.74 (s, 1H), 7.47 (d, J ¼ 8.0 Hz, 2H),
6.82 (dd, J ¼ 7.2,1.6 Hz,1H), 4.55 (d, J ¼ 5.6 Hz, 2H), 2.56 (s, 3H), 2.39
5.1.5.2. N-((1-((3r,5r,7r)-adamantan-1-ylmethyl)piperidin-4-yl)
methyl)-5-chl- oro-2-ethyl-pyrazolo[1,5-a]pyridine-3-carboxamide
(s, 3H). 13C NMR (125 MHz, CDCl3):
d(ppm): 164.65, 155.40, 150.69,
150.02, 150.01, 149.20, 142.22, 138.09, 137.78, 136.64, 133.49, 128.45,
127.64, 121.19, 120.61 (q, J ¼ 256 Hz, 1C), 117.40, 115.76, 103.44,
40.86, 21.54, 14.92. HRMS (ESI) calcd for C23H19F3N4O2 [MþH]þ:
441.1533; found 441.1530. HPLC purity ¼ 98.41%, Rt 5.24 min.
(2). 1H NMR (400 MHz, DMSO-d6):
d
(ppm) 8.71 (d, J ¼ 7.2 Hz,
1H), 7.84 (d, J ¼ 2.0 Hz, 1H), 7.74 (t, J ¼ 5.6 Hz, 1H), 7.00 (dd, J ¼ 7.2,
2.0 Hz,1H), 3.16 (t, J ¼ 6.0 Hz, 2H), 2.98 (q, J ¼ 7.6 Hz, 2H), 2.68e2.71
(m, 2H), 2.08e2.14 (m, 2H), 1.90 (s, 5H), 1.64e1.67 (m, 3H),
1.56e1.59 (m, 5H), 1.44e1.51 (m, 7H), 1.20e1.28 (m, 5H). 13C NMR
5.1.5.8. 2,5-dimethyl-N-(4-(4-(4-(trifluoromethoxy)phenoxy)piper-
(125 MHz, CD3Cl):
d
(ppm): 163.95, 156.50, 142.08, 133.18, 128.94,
idin-1-
yl)benzyl)pyraz-olo[1,5-a]pyridine-3-carboxamide
(8).
118.05, 114.35, 104.88, 71.22, 56.50, 45.32, 41.11, 37.49, 36.18, 35.19,
30.79, 28.73, 22.27, 13.30. HRMS (ESI) calcd for C27H37ClN4O
[MþH]þ: 469.2729; found 469.2724.
1H NMR (400 MHz, DMSO-d6):
d(ppm) 8.51 (d, J ¼ 8.0 Hz, 1H), 7.90
(t, J ¼ 6.0 Hz, 1H), 7.70 (s, 1H), 7.27 (d, J ¼ 8.4 Hz, 2H), 7.21 (d,
J ¼ 8.4 Hz, 2H), 7.07 (d, J ¼ 9.2 Hz, 2H), 6.93 (d, J ¼ 8.8 Hz, 2H), 6.80
(d, J ¼ 7.2 Hz, 1H), 4.55e4.59 (m, 1H), 4.38 (d, J ¼ 6.0 Hz, 2H),
3.47e3.50 (m, 2H), 2.99e3.04 (m, 2H), 2.53 (s, 3H), 2.37 (s, 3H),
2.02e2.04 (m, 2H), 1.68e1.74 (m, 2H). 13C NMR (125 MHz, CDCl3):
5.1.5.3. 5-chloro-2-ethyl-N-((3-(3-fluoro-4-morpholinophenyl)-2-
oxooxazolidin-5- yl)methyl) pyrazolo [1,5-a]pyridine-3-carboxamide
(3). 1H NMR (400 MHz, DMSO-d6):
d(ppm) 8.71 (d, J ¼ 7.2 Hz,
d(ppm): 164.41, 156.00, 150.80, 150.60, 143.00, 142.99, 142.15,
1H), 8.11 (t, J ¼ 5.6 Hz, 1H), 7.79 (s, 1H), 7.50e7.54 (m, 1H), 7.17e7.19
(m, 1H), 7.00e7.07 (m, 2H), 4.88e4.92 (m, 1H), 4.17 (t, J ¼ 9.2 Hz,
1H), 3.86e3.90 (m, 4H), 3.71e3.73 (m, 2H), 2.92e2.95 (m, 6H), 1.19
137.66, 129.75, 128.93, 127.55, 122.62, 120.72 (q, J ¼ 255 Hz, 1C),
117.51, 117.03, 116.86, 115.56, 103.93, 73.04, 46.80, 43.19, 30.53,
21.53, 14.82. HRMS (ESI) calcd for C29H29F3N4O3 [MþH]þ:
539.2264; found 539.2261. HPLC purity ¼ 97.85%, Rt 7.15 min.
(t, J ¼ 7.2 Hz, 3H). 13C NMR (125 MHz, CD3Cl):
d(ppm): 164.57,
157.26, 156.59, 154.51 (d, J ¼ 30.9 Hz, 1C), 142.03, 136.66 (d,
J ¼ 9.0 Hz, 1C), 133.77, 133.05 (d, J ¼ 10.5 Hz, 1C), 129.15, 118.95 (d,
J ¼ 4.2 Hz, 1C), 117.62, 114.56, 114.02 (d, J ¼ 3.2 Hz, 1C), 107.61 (d,
J ¼ 26.2 Hz, 1C), 103.80, 67.19, 67.07, 51.11 (d, J ¼ 2.8 Hz, 1C), 47.91,
42.10, 22.19, 13.00. HRMS (ESI) calcd for C24H25ClFN5O4 [MþH]þ:
502.1652; found 502.1648. HPLC purity ¼ 99.85%, Rt 5.80 min.
5.2. Biological assay
5.2.1. In vitro anti-tubercular activity using a selectable marker-free
autoluminescent assay against Mtb H37Ra.
UAlRa (Mtb H37Ra:pTYOK) was homogenized with sterile glass
beads in a 50 mL tube containing 5 mL Middlebook 7H9 medium
plus 0.05% Tween 80, 10% v/v oleic acid albumin dextrose catalase
(OADC) supplement (7H9-OADC-Tw). When OD600 reached
0.3e0.5, relative light unit (RLU) count was determined. by putting
5.1.5.4. 5-chloro-2-ethyl-N-((6-(4-(trifluoromethoxy)
pyridin-3- yl)methyl)pyrazolo[1,5-a]pyridine-3-carboxamide (4).
1H NMR (400 MHz, DMSO-d6):
phenyl)
d(ppm) 8.74 (d, J ¼ 7.6 Hz, 1H), 8.68
(s, 1H), 8.32 (t, J ¼ 5.6 Hz, 1H), 8.20 (d, J ¼ 8.8 Hz, 2H), 7.96e8.00 (m,
2H), 7.88 (dd, J ¼ 8.0, 1.6 Hz, 1H), 7.47 (d, J ¼ 8.4 Hz, 2H), 7.04 (dd,
J ¼ 7.2, 2.0 Hz, 1H), 4.55 (d, J ¼ 6.0 Hz, 2H), 3.02 (q, J ¼ 7.2 Hz, 2H),
200
RLU reached 2 million, the effect concentration of compounds 1e8
was assessed over a range of 3-fold increasing from 0.000001 g/
mL to 10 g/mL prepared in 25 L UAlRa broth cuture (RLU diluted
mL culture on the detection hole of the luminometer. When the
m
1.25 (t, J ¼ 7.6 Hz, 3H). 13C NMR (125 MHz, CDCl3):
d
(ppm): 164.03,
m
m
156.84, 155.58, 150.09, 149.25, 142.24, 137.75, 136.70, 133.70, 133.19,
129.11, 128.50, 121.23, 120.65 (q, J ¼ 256 Hz, 1C), 120.52, 117.93,
114.61, 104.14, 41.02, 22.36, 13.12. HRMS (ESI) calcd for
to 2000e4000) grown in 7H9 broth with Tween80. In the treat-
ment group, DMSO was used as negative control and isoniazide
(INH, 10
mL, 1 g/mL and 0.1
counts were determined daily, for 4 days. Analysis of the data, the
m
g/mL, 1
mg/mL and 0.1
mg/mL) and rifampicin (RIF, 10 mg/
C
23H18F3N4O2 [MþH]þ: 475.1143; found 475.1138. HPLC
m
mg/mL) were used as positive control. RLU
purity ¼ 98.03%, Rt 6.11 min.

X. Lu et al. / European Journal of Medicinal Chemistry 125 (2017) 41e48
47
MIClux value is the lowest drug concentration that can achieve the
ratio (RLUdrug/RLUDMSO) less than 10% after treatment.
dissolved to 10 mM with DMSO, and a 3-fold serial dilution of the
compounds from 100 M to 0.003 M was performed. 500 nL of
m
m
compound solution was correspondingly added using Echo520 to
the 384-PP plate and 500 nL DMSO instead of compound solution
was used as the 0% inhibitor control. After coincubation for 72 h,
5.2.2. In vitro anti-tubercular activity using the microplate alamar
blue assay (MABA) against M.tb H37Rv
The test compound MICs against TB were assessed by the MABA
using isoniazid and RIF (supplied by University of Illinois) as posi-
tive controls. The stock solutions of compounds were prepared in
DMSO at a concentration of 5 mg/mL or 2 mg/mL, and the final test
concentrations ranged from 0.05 to 5 mg/mL or from 0.004 to 1 mg/
mL. Two-fold dilutions of compounds were prepared in Mid-
dlebrook 7H12 medium (7H9 broth containing 0.1% w/v casitone,
3 m
l CCK8 (5 mg/mL) was added. 2 h later at 37 ꢀC, the plates were
read in the Envision Multilabel Reader at 450 nm. Cell survival rate
(activity%) was calculated with the formula: activity% ¼ (ODexperi-
ment- ODblank)/(ODcontrol -ODblank) * 100. The resulted data was then
analyzed with corresponding compound concentrations using
GraphPad Prism 5 Demo. The IC50 were fitted using a nonlinear
regression model with a sigmoidal dose response.
5.6
m
g/mL palmitic acid, 5 mg/mL bovine serum albumin, 4 mg/mL
L in 96-well micro-
L inoculum
catalase, filter-sterilized) in a volume of 100
m
5.2.5. HepG2 cytotoxicity assay
plates (black viewplates). M. tuberculosis H37Rv (100
m
The cytotoxicity of compounds was determined by measuring
HepG2 cell viability growth after 3 d in the presence of test com-
pounds. Compounds were prepared as 10-point three-fold serial
dilutions in DMSO. The highest concentration of compound tested
of 2 ꢁ 105 cfu/mL) was added, yielding a final testing volume of
200 ml. The plates were incubated at 37 ꢀC. On the 7th day of in-
cubation 12.5 mL of 20% Tween 80 and 20 mL of Alamar Blue (Trek
Diagnostic, Westlake, OH) were added to the test plate. After in-
cubation at 37 ꢀC for 16e24 h, fluorescence of the wells was
measured (ex 530, em 590 nm). The MICs were interpolated values
obtained by using an in-house curve-fitting program and defined as
the lowest concentration affecting a reduction in fluorescence of
ꢂ90% relative to the mean of replicate bacteria-only controls.
was 100 mM where compounds were soluble in DMSO at 10 mM
HepG2 cells were cultured in complete DMEM, inoculated into 384-
well assay plates containing compounds and incubated for 24 h at
37 ꢀC, 5% CO2. Compounds were added and cells were cultured for a
further 72 h. The final DMSO concentration was 1%. Cell viability
was determined using the CellTiter-Glo® Luminescent Cell Viability
Assay (Promega) and measuring relative luminescent units (RLU).
The dose response curve was fitted using the Lev-
enbergeMarquardt algorithm. The TC50 was defined as the com-
pound concentration that produced 50% loss of cell viability. Each
run included staurosporine as a control.
5.2.3. MIC values of compounds against single drugs resistant
strains
The MIC of compound was determined by measuring bacterial
growth after 5 d in the presence of test compounds. Compounds
were prepared as 10-point two-fold serial dilutions in DMSO and
diluted into 7H9-Tw-OADC medium in 96-well plates with a final
DMSO concentration of 2%. The highest concentration of compound
5.2.6. In vivo anti-tubercular activity of compound 7
UAlRa (M.tb H37Ra:pTYOK) isolated on plates were homoge-
nized with sterile glass beads in a 250 mL flask containing 50 mL
7H9 with Tween80. When RLU reached 2 million/ml, the culture
were used to infect 4-to-6-week-old male BALB/c mice by tail vein
injection. The day after infection (day 0), RLU counts were deter-
mined. The mice were first anesthetized by isoflurane inhalation
and the RLU count was determined by laying the breast of mouse
on the detection hole of the luminometer and measuring light
production for 3 s for twice. Mice with similar RLU readings were
randomly allocated to treatment groups (4 mice/group) and indi-
vidually marked. The treatment groups received: CMC-Na alone as
negative control, 7 (100 mg/kg) and 7 (4 mg/kg). Treatment was
administrated daily, while RLU were detected on day 2, day 4, day 6.
was 200
Each plate included assay controls for background (medium/DMSO
only, no bacterial cells), zero growth (100 M rifampicin) and
mM where compounds were soluble in DMSO at 10 mM.
m
maximum growth (DMSO only), as well as a rifampicin dose
response curve. Plates were inoculated with M. tuberculosis and
incubated for 5 days; growth was measured by OD590. To calculate
the MIC, the 10-point dose response curve was plotted as % growth
and fitted to the Gompertz model using GraphPad Prism 5. The MIC
was defined as the minimum concentration at which growth was
completely inhibited and was calculated from the inflection point
of the fitted curve to the lower asymptote (zero growth). In addition
dose response curves were generated using the Levenberg-
Marquardt algorithm and the concentrations that resulted in 50%
and 90% inhibition of growth were determined (IC50 and IC90
respectively).
Acknowledgments
MIC values were reported when the following quality control
criteria were satisfied:
We thank Guangdong Special Branch Program Technology
Young talents (2014TQ01R341) and Guangdong Natural Science
Funds (2016A030313106) for the financial support. In addition, this
work was supported by National Institutes of Health and the Na-
tional Institute of Allergy and Infectious Diseases, Contract No.
HHSN272201100009I.
ꢃ For each plate
ꢀ No growth in the background (un-inoculated) control wells
ꢀ OD590 > 0.2 in maximum growth wells
ꢀ Rifampicin MIC within 3-fold of the expected value
ꢃ For each compound curve. MIC values were reported if
ꢀ There were 2 points with growth >75%
Appendix A. Supplementary data
ꢀ There were 2 points with growth <75%
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2016.09.030.
ꢃ If no point reached 75% inhibition, the MIC was reported
as > maximum concentration tested.
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