Synthesis, molecular docking and anti-mycobacterial evaluation of new imidazo[1,2-a]pyridine-2-carboxamide derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.10.079

New anti-tubercular agents, imidazo[1,2-a]pyridine-2-carboxamide derivatives (5a-q) have been designed and synthesized. The structural considerations of the designed molecules were further supported by the docking study with a long-chain enoyl-acyl carrier protein reductase (InhA). The chemical structures of the new compounds were characterized by IR, ¹H NMR, ¹³C NMR, HRMS and elemental analysis. In addition, single crystal X-ray diffraction has also been recorded for compound 5f. Compounds were evaluated in vitro against Mycobacterium tuberculosis H37Rv, and cytotoxicity against HEK-293T cell line. Amongst the tested compounds 5j, 5l and 5q were emerged as good anti-tubercular agents with low cytotoxicity. The structure-anti TB activity relationship of these derivatives was explained by molecular docking.

Full text of DOI:10.1016/j.ejmech.2014.10.079

European Journal of Medicinal Chemistry 89 (2015) 616e627
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, molecular docking and anti-mycobacterial evaluation of
new imidazo[1,2-a]pyridine-2-carboxamide derivatives
, e,
Gilish Jose a, T.H. Suresha Kumara a *, Gopalpur Nagendrappa a, H.B.V. Sowmya a,
Dharmarajan Sriram b, Perumal Yogeeswari b, Jonnalagadda Padma Sridevi b,
Tayur N. Guru Row c, Amar A. Hosamani c, P.S. Sujan Ganapathy d, N. Chandrika a,
L.V. Narendra a
a Postgraduate Department of Chemistry, Jain University, 52 Bellary Road, Hebbal, Bangalore, Karnataka 560024, India
b Department of Pharmacy, Birla Institute of Technology and Science-Pilani, Hyderabad 500078, India
c Solid State and Structural Chemistry Unit, Indian Institute of Science, Bangalore, Karnataka 560012, India
d Centre for Advanced Studies in Biosciences, Jain University, 52 Bellary Road, Hebbal, Bangalore, Karnataka 560024, India
e Department of Chemistry, University B.D.T. College of Engineering, Davangere, Karnataka 577004, India
a r t i c l e i n f o
a b s t r a c t
Article history:
New anti-tubercular agents, imidazo[1,2-a]pyridine-2-carboxamide derivatives (5aeq) have been
designed and synthesized. The structural considerations of the designed molecules were further sup-
ported by the docking study with a long-chain enoyl-acyl carrier protein reductase (InhA). The chemical
structures of the new compounds were characterized by IR, 1H NMR, 13C NMR, HRMS and elemental
analysis. In addition, single crystal X-ray diffraction has also been recorded for compound 5f. Compounds
were evaluated in vitro against Mycobacterium tuberculosis H37Rv, and cytotoxicity against HEK-293T cell
line. Amongst the tested compounds 5j, 5l and 5q were emerged as good anti-tubercular agents with low
cytotoxicity. The structure-anti TB activity relationship of these derivatives was explained by molecular
docking.
Received 23 June 2014
Received in revised form
8 October 2014
Accepted 29 October 2014
Available online 30 October 2014
Keywords:
Anti-mycobacterial activity
Imidazopyridine amide
InhA
© 2014 Elsevier Masson SAS. All rights reserved.
Tuberculosis
X-ray crystallography
1. Introduction
accounts for 26% of AIDS related death worldwide [3e7]. In 2012,
an estimated 8.6 million people affected by M. tuberculosis and 1.3
TB is a chronic bacterial infection is often contracted through an
airborne bacterium known as Mycobacterium tuberculosis.
Although most M. tuberculosis infections, known as pulmonary TB,
are in the lungs, 5 to 10 percent of TB patients can develop the
disease in organs such as lymph nodes, bones and joints, eyes, in-
testines, larynx, or the urinary and reproductive systems, skin, and
stomach, known as extrapulmonary TB [1,2]. In recent years, the
emergence of MDR and XDR tuberculosis strains has amplified the
incidences of TB. In addition, TB is a co-infection of HIV-AIDS and
million died from the disease, including 320,000 deaths among
HIV-positive people [8]. To address these issues, design, synthesize
and develop potent anti-mycobacterial agents are necessary.
Imidazopyridine derivatives are very important, versatile motifs
with significant applications in medicinal chemistry [9e14].
Different compounds containing the moiety imidazo[1,2-a]pyridine
have significant biological applications such as anti-mycobacterial
[15e21], anticancer [22,23], antiviral [24e27], antimicrobial
[28e30], anti-HIV [31], antiulcer [32,33], antihelminthic [34] and
anticoccidial activity [35,36]. As a result of our interest in the
synthesis of imidazopyridine-containing compounds and their
antimicrobial activity [37], we initiated a program directed towards
the synthesis of the new imidazopyridine amide derivatives. Imi-
dazopyridine amides (IPA) are a new class of therapeutic agents
against MDR and XDR tuberculosis [18e21]. Recently, Qurient
Therapeutics discovered Q203 (Fig. 1), a highly innovative drug for
MDR and XDR tuberculosis and the first drug in its class with an
original mechanism of action [21]. On the basis of above beneficial
Abbreviations: TB, tuberculosis; MDR, multiple drug-resistant; XDR, extensively
drug-resistant; HIV-AIDS, human immunodeficiency virus-acquired immunodefi-
ciency syndrome; DIPEA, diisopropylethylamine; HATU, 1-[bis(dimethylamino)
methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium-3-oxid hexafluorophosphate; MIC,
minimum inhibitory concentration.
* Corresponding author. Department of Chemistry, University B.D.T. College of
Engineering, Davangere, Karnataka 577004, India.
E-mail addresses: suresha.kumara@gmail.com, suresha.kumara@rediffmail.com
(T.H. Suresha Kumara).
http://dx.doi.org/10.1016/j.ejmech.2014.10.079
0223-5234/© 2014 Elsevier Masson SAS. All rights reserved.

G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
617
information, we earmarked to design new imidazo[1,2-a]pyridine-
2-carboxamide derivatives anticipating enhanced biological activ-
ity to combat this lethal disease. The structural considerations of
the designed molecules were further supported by the docking
study with target enzyme InhA, an enzyme essential for mycolic
acid biosynthesis in M. tuberculosis. The new target compounds
were designed based on the assumptions of Lipinski rule to fulfil
the drug likeness properties of the molecules. We report herein,
synthesis, crystal structure, molecular docking and anti-mycobac-
terial evaluation of new imidazo[1,2-a]pyridine-2-carboxamide
derivatives.
were synthesized by using the same experimental protocol with
different aromatic or aliphatic amines (R1eNH2). The Suzuki re-
action of 4a with 1-methylpyrazole-4-boronic acid pinacol ester,
catalysed by Pd(0) complex in the presence of Na2CO3 gave final
product 5a. IR spectrum of 4a showed that the appearance of the
band at 1650 cmꢀ1 characteristic for amide C]O stretch. 1H NMR
and 13C NMR spectra of 5a were revealed that the presence of the
carboxamide group at position 2, showed a triplet at
d 8.93 ppm
and
d
162.3 ppm, respectively. The band at 1652 cmꢀ1 in the IR
spectrum of 5a characteristic for the amide C]O stretch. The
HRMS spectrum of 5a showed 350.1405 [MþH]þ corresponding
with proposed isotopic mass (349.1338). The obtained elemental
analysis values are in consonance with theoretical data. The
remaining final compounds (5beq) were synthesized using the
same experimental procedure with different aromatic or hetero
aromatic boronic acids (R2). The structures of the final products
were assigned on the basis of their IR, 1H NMR, 13C NMR, HRMS
and elemental analysis. The structure of the compound 5f (Fig. 2)
was unequivocally established by X-ray crystallographic analysis.
The details of the crystal data and structure refinement was
shown in Table 1.
2. Results and discussion
2.1. Chemistry
The synthetic strategy of new polyfunctional imidazo[1,2-a]
pyridine-2-carboxamides were described in Scheme 1. The imi-
dazo[1,2-a]pyridine ring system was accomplished by the
condensation of 2-amino-5-iodopyridine (1) with ethyl bromo-
pyruvate (
ethyl-6-iodo-H-imidazo[1,2-a]pyridine-2-carboxylate (2) [38]. 1H
NMR spectrum of 2 showed a triplet at 1.30 (3H) and a quartet
at 4.30 (2H) ppm corresponds to an ethyl ester group at position
2. 13C NMR spectrum of
showed peaks at 167.3
a-halocarbonyl compound) in refluxing ethanol to gave
d
2.2. Pharmacology
d
2
d
All the synthesized compounds were also screened for their
in vitro anti-tubercular activity against M. tuberculosis H37Rv
(ATCC 27294) using microplate alamar blue assay (MABA) with
(COOeCH2eCH3), 60.3 (OeCH2), 14.8 (CH3) ppm also revealed
that the presence of ethyl ester group. IR spectrum of 2 revealed
that appearance of the band at 1748 cmꢀ1 characteristic for ester
C]O stretch. The ester hydrolysis reaction of 2 with lithium
hydroxide monohydrate gave 6-iodo-H-imidazo[1,2-a]pyridine-
2-carboxylic acid (3). 1H NMR and 13C NMR spectra of 3 exhibited
drug concentrations from 50 mg/mL to 0.78 mg/mL in duplicates.
The MIC was determined for each compound which was
measured as the minimum concentration of compound required
to completely inhibit the bacterial growth. Isoniazid and rifam-
picin were used as reference compounds for comparison. The
in vitro test results for title compounds were tabulated in Table 2
a broad singlet peak at
d 11.50 ppm and a peak at d 167.1 ppm
were assigned for carboxylic acid, while its IR spectrum showed a
strong band at 1755 cmꢀ1 also revealed that the presence of
carboxylic acid. The peptide coupling reaction between 3 and 4-
fluorobenzylamine was accomplished by famous peptide
coupling reagent, HATU and Hunig's base (DIPEA) in DMF gave
scaffold 4a. IR spectrum of 4a showed that the appearance of the
band at 1650 cmꢀ1 characteristic for amide C]O stretch. 1H NMR
as the MIC and the activity ranged from 6.25 to >50
mg/mL.
Various substitutions were attempted at R1 and R2 position of the
imidazo[1,2-a]pyridine core as steps towards the derivation of
structureeactivity relationship and to understand the ideal site
for introducing chemical diversity. Out of the various compounds
tested, compounds 5h, 5j, 5k, 5l, 5o and 5q inhibited mycobac-
terial growth very effectively compared to others in the series
spectrum of 4a showed a triplet at
presence of the carboxamide group at position 2, while its 13C
NMR spectrum showed 162.4 ppm also revealed that the
presence of carboxamide group. The remaining scaffolds (4beg)
d 9.00 ppm revealed that the
with MIC values ranging from 6.25 to 12.5
mg/mL. The highest
d
activity, 6.25
mg/mL was registered for compounds 5j
(R1 ¼ pyridin-4-yl methyl, R2 ¼ 4-chlorophenyl), 5l (R1 ¼ benzo
Fig. 1. Some of the imidazopyridine amide based anti-tubercular agents.

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G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
Scheme 1. Synthesis of imidazo[1,2-a]pyridine-2-carboxamide derivatives; Reagents and conditions: (i) ethyl alcohol, reflux, 4 h; (ii) LiOH, methanol/THF/H2O, 60 ꢂC, 1 h; (iii)
R1eNH2, DIPEA, HATU, DMF, rt, 12 h (iv) R2eB(OH)2, Na2CO3, Pd(PPh3)4, 1,4-dioxane/H2O, 90 ꢂC, 8 h.
[d][1,3]dioxol-5-yl methyl, R2 ¼ 1-methyl-1H-pyrazol-4-yl) and
5q (R1 ¼ tetrahydro-2H-pyran-4-yl, R2 ¼ 1-(2-morpholinoethyl)-
1H-pyrazol-4-yl). It was observed that the presence of the hy-
drophobic substituents on both R1 and R2 position of the imidazo
[1,2-a]pyridine core brought about an enhancement of the anti-
mycobacterial potency as the overall hydrophobic nature might
have enabled these molecules to penetrate the quite complex
mycobacterial cell wall, a major hurdle faced in anti-tubercular
drug discovery. Lipophilicities of the compounds 5aeq and the
standard drugs, which were expressed as logP values, were
determined using the logP method through online http://www.
molinspiration.com/cgi-bin/properties site, as shown in Table 2.
The safety profile of the active leads was also accessed by testing
in vitro cytotoxicity against Human Embryonic Kidney Cell-line
2.3. Molecular docking studies
The molecular docking of imidazo[1,2-a]pyridine carboxamide
motifs (5aeq) with a long-chain enoyl-acyl carrier protein reduc-
tase (InhA) yielded best possible conformations with parameters
including the docking energy, binding energy, inhibition constant
and intermolecular energy (Table 3). The docking results are drawn
based on both binding energy and the number of hydrogen bonds
formed. The intermolecular hydrogen bond interactions play an
important role in adduct binding in InhA, especially the hydrogen
bonds of the pyrophosphate part. The main residues involved in
these interactions are His 265, Tyr 259, Trp 249, Gly 3, Ile 194, and
Arg 173. Compounds (5aeq) showed very good docking energy
ranging from ꢀ12.07 kJ/mol to ꢀ9.42 kJ/mol because target com-
pounds have formed one or more hydrogen bonds with amino acid
residues in the active pocket of InhA (Fig. 3). Compounds contain-
ing the halogen atoms (5aed, 5j, 5n, 5o) also have the ability to
form intermolecular bonds with the active site of InhA in a fashion
that resembles hydrogen bonds. All compounds, except 5c, 5g and
5n have formed two or more hydrogen bonds with amino acid
residues of InhA. The maximum number of hydrogen bonds was
found in the analogue 5o (3 hydrogen bonds) with a binding energy
(ꢀ9.20 kJ molꢀ1), docking energy (ꢀ11.62 kJ molꢀ1) and inhibition
constant (1.79 ꢁ 10ꢀ7 M). In in vitro anti-mycobacterial studies,
compounds 5h, 5j, 5k, 5l, 5o and 5q have emerged as active against
microbe, so it can be predicted as the activity may be due to inhi-
bition of enzyme InhA.
293T (HEK-293T) cells at 50
mg/mL concentration by (4,5-
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT)
assay. Percentage inhibitions of cells are reported in Table 2. The
entire tested compound demonstrated a good safety profile with
very low toxicity towards the HEK cells and showed a good selec-
tivity index (CC50/MIC) indicating the suitability of these com-
pounds for further drug development.
3. Conclusions
In conclusion, new anti-tubercular agents imidazo [1,2-a] pyri-
dine-2-carboxamide derivatives 5aeq was efficiently synthesized.
The activity of these compounds against M. tuberculosis strain
H37Rv was assessed and compounds 5j, 5l and 5q were displayed
good anti-tubercular activity. Their activity depends on both the
substituent's R1 and R2. The in vitro cytotoxicity study against HEK-
293Tcells at 50 mg/mL by MTTassay revealed that the identified hits
are found to be less cytotoxic. The molecular docking of InhA
domain with synthesized compounds revealed that compounds 5h,
5j, 5k, 5l, 5o and 5q having the good inhibitory capability and are
exhibiting the interactions with one or the other amino acids in the
active pocket. With new antiTB agents desperately needed, we
Fig. 2. X-ray crystal structure of 5f.

G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
619
Table 1
reduced pressure. The residue was neutralized with 10% NaHCO3
solution (250 mL). The solid formed was collected by filtration,
washed with water and dried. Off white solid (26.7 g, 94%); mp
135.8e138.6 ꢂC; IR (KBr, nmax, cmꢀ1): 3163 (CeHarom.), 2918
(CeHaliph.), 1748 (C]Oester), 1575 (CeCarom.); 1H NMR (400 MHz,
Crystal data and structure refinement for 5f.
Identification code
Empirical formula
Formula weight
Temperature/K
Crystal system
Space group
a/Å
5f
C18H15N5O2
333.344
296.15
Monoclinic
P21/c
31.097(6)
9.3652(19)
10.816(2)
90.00
90.084(11)
90.00
3149.9(11)
4
1.383
0.090
1368.0
DMSO-D6):
d
8.94 (s, 1H, H-5), 8.43 (s, 1H, H-3), 7.53 (d, J ¼ 9.6 Hz,
1H, H-7), 7.47 (d, J ¼ 9.6 Hz, 1H, H-8), 4.30 (q, J ¼ 7.2 Hz, 2H,
OeCH2eCH3), 1.30 (t, J ¼ 7.2 Hz, 3H, CH2eCH3); 13C NMR (100 MHz,
b/Å
DMSO-D6):
d 167.3 (COOeCH2eCH3), 156.4 (C-5), 149.3 (C-9), 144.5
c/Å
(C-7), 138.8 (C-2), 118.9 (C-3), 118.1 (C-8), 96.2 (C-6), 60.3 (OeCH2),
14.8 (CH3); LC/MS (API-ES) m/z calcd. for C10H9IN2O2: 315.971,
found: 317.0 [MþH]þ; Anal. calcd. for C10H9IN2O2: C, 38.00; H, 2.87;
N, 8.86; found C, 37.98; H, 2.859; N, 8.81.
ꢂ
ꢂ
ꢂ
/
a
/
b
/
g
Volume/Å3
Z
rcalc mg/mm3
m/mmꢀ1
F(000)
4.3. Experimental procedure for the synthesis of 6-iodoH-imidazo
[1,2-a]pyridine-2-carboxylic acid (3)
Crystal size/mm3
Radiation
0.21 ꢁ 0.18 ꢁ 0.26
MoK
a
(
l
¼ 0.71073)
A mixture of compound 2 (26.7 g, 84.46 mmol) and lithium
hydroxide monohydrate (10.63 g, 253.38 mmol) and 270 mL of
methanol/THF/water (7:2:1) were heated at 60 ꢂC for 1 h. The
solvent was concentrated under reduced pressure. The residue was
dissolved in 80 mL of water. The PH of the reaction mixture was
adjusted to 6 by using 10% citric acid solution. The solid formed was
collected by filtration, washed with water and dried. White solid
(22.6 g, 93%); mp 185.9e188.4 ꢂC; IR (KBr, nmax, cmꢀ1): 3166
(CeHarom.), 2520 (OeHacid), 2920 (CeHaliph.), 1755 (C]Oacid), 1576
2
Q
range for data collection
2.62e50ꢂ
Index ranges
ꢀ36 ꢃ h ꢃ 36, ꢀ11 ꢃ k ꢃ 10, ꢀ12 ꢃ l ꢃ 12
Reflections collected
Independent reflections
Data/restraints/parameters
Goodness-of-fit on F2
35,922
5523 [Rint ¼ 0.1491, Rsigma ¼ 0.1080]
5523/0/454
1.016
R1 ¼ 0.0908, wR2 ¼ 0.2394
R1 ¼ 0.1799, wR2 ¼ 0.2817
0.44/ꢀ0.60
Final R indexes [I ꢄ 2
s
(I)]
Final R indexes [all data]
Largest diff. peak/hole/e Åꢀ3
(CeCarom.), 1221 (CeOacid); 1H NMR (400 MHz, DMSO-D6):
d 11.50
(br s, 1H, COOH), 8.94 (s, 1H, H-5), 8.30 (s, 1H, H-3), 7.48 (d,
believe that the new imidazopyridines reported in this work pro-
vide an interesting potential for further optimization. Further
structural modifications of the identified hits are in progress in
order to enhance the efficacy of these compounds active against
M. tuberculosis.
J ¼ 9.6 Hz, 1H, H-7), 7.43 (d, J ¼ 9.6 Hz, 1H, H-8); 13C NMR (100 MHz,
DMSO-D6):
d 167.1 (COOH), 156.4 (C-5), 149.4 (C-9), 144.5 (C-7),
138.9 (C-2), 118.8 (C-3), 118.2 (C-8), 96.1 (C-6); LC/MS (API-ES) m/z
calcd. for C8H5IN2O: 287.940, found: 287.0 [M-H]þ; Anal. calcd. for
C8H5IN2O2: C, 33.36; H, 1.75; N, 9.73; found: C, 33.32; H, 1.758; N,
9.69.
4. Experimental section
4.4. General experimental procedure for the synthesis of 4aeg
4.1. General methods
Synthesis of N-(4-fluorobenzyl)-6-iodo1H-imidazo[1,2-a]pyridine-
2-carboxamide (4a) as an example: To a mixture of compound 3 (1 g,
3.47 mmol), 4-fluorobenzylamine (0.65 g, 5.21 mmol) and DIPEA
(2.4 mL,13.89 mmol) in DMF (10 mL), HATU (1.98 g, 5.21 mmol) was
added portion wise at 0 ꢂC. The resulting reaction mixture was
stirred at rt for 12 h. The solvent was removed under reduced
pressure. The reaction mixture was dissolved in DCM (50 mL) and
the organic layer was washed with water (25 mL) and brine
(25 mL). The organic layer was separated and dried over anhydrous
MgSO4. The organic layer was concentrated in vacuum. The crude
product was purified via flash column chromatography on silica gel
(230e400 mesh) to provide 4a as pale yellow solid (1.27 g, 93%).
All chemicals and solvents were purchased from commercial
suppliers. All reactions were performed under an inert atmosphere
of dry nitrogen using dry solvents. The progress of the reactions
was monitored by using Si gel 60 F254 thin layer chromatography
plates and spots were detected by UV/254 nm. Compounds were
purified by column chromatography on silica gel 230e400 using
petether/ethyl acetate or dichloromethane/methanol solvent mix-
tures as eluent. All the compounds were characterized by IR, 1H
NMR, 13C NMR, HRMS and elemental analysis. Melting points of the
samples were determined in open capillary tubes using Buchi
Melting point B-540 apparatus and are uncorrected. IR spectra were
obtained on a PerkinElmer Frontier™ FT-IR spectrometer, were
recorded in KBr. 1H and 13C NMR spectra were recorded on a Bruker
NMR spectrometer (400 and 100 MHz, respectively) using deuter-
ated DMSO as solvent with TMS as internal standard. The mass
recorded in Agilent LCMS 1100 series instrument using API-ES
positive-ion mode and negative-ion mode. HRMS was recorded in
positive-ion mode by Microflex LT MALDI-ToF mass spectrometer
(Bruker Daltonics, Germany). The CHN analysis was recorded in an
Elementar vario MICRO cube.
4.4.1. N-(4-Fluorobenzyl)-6-iodo1H-imidazo[1,2-a]pyridine-2-
carboxamide (4a)
mp 195.1e197.0 ꢂC; IR (KBr, nmax, cmꢀ1): 3354 (NeHamide), 3163,
3076 (CeHarom.), 2918 (CeHaliph.), 1650 (C]Oamide), 1574
(CeCarom.); 1H NMR (300 MHz, DMSO-D6):
d
9.00 (t, J ¼ 6.0 Hz, 1H,
COeNHeCH2), 8.95 (s, 1H, H-5), 8.26 (s, 1H, H-3), 7.46 (d,
J ¼ 10.4 Hz, 2H, H-20, H-60), 7.36e7.32 (m, 2H, AreH), 7.11 (d,
J
¼
12.0 Hz, 2H, H-30, H-50), 4.41 (d,
J
¼
8.4 Hz, 2H,
162.4
COeNHeCH2eAr); 13C NMR (100 MHz, DMSO-D6):
d
4.2. Experimental procedure for the synthesis of ethyl 6-iodoH-
imidazo[1,2-a]pyridine-2-carboxylate (2)
(COeNH), 162.1 (C-40), 159.7 (C-9), 156.3 (C-5), 144.5 (C-7), 136.0 (C-
10), 135.9 (C-2), 129.4 (C-20), 129.3 (C-60), 118.8 (C-3), 118.2 (C-8),
114.9 (C-30), 114.7 (C-50), 96.2 (C-6), 41.2 (COeNHeCH2eAr); LC/MS
(API-ES) m/z calcd. for C15H11FIN3O: 394.993, found: 396.1 [MþH]þ;
Anal. calcd. for C15H11FIN3O: C, 45.59; H, 2.81; N, 10.63, found: C,
45.56; H, 2.808; N, 10.60.
A mixture of 2-amino-5-iodo pyridine 1 (20 g, 90.90 mmol) and
ethyl bromopyruate (53.17 g, 272.70 mmol) were refluxed in
200 mL of ethanol for 4 h. The solvent was concentrated under

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G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
Table 2
Anti-mycobacterial activity and cytotoxicity of compounds 5aeq.
Compound
R1
R2
MIC (
25
m
g/mL)a
Cytotoxicity at 50
36.12
m
g/mLb
LogP
2.420
5a
5b
5c
25
25
40.8
2.578
4.302
28.12
5d
>50
NT
3.985
5e
5f
>50
NT
1.513
1.672
25
26.12
5g
25
24.8
3.396
5h
5i
12.5
25
34.06
30.12
1.389
1.994
5j
6.25
26.12
31.62
3.268
2.598
5k
12.5

G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
621
Table 2 (continued )
Compound
R1
R2
MIC (
m
g/mL)a
Cytotoxicity at 50
25.56
m
g/mLb
LogP
2.146
5l
6.25
5m
5n
25
29.12
37.80
1.099
5.034
25
5o
5p
12.5
25
22.16
30.7
3.027
1.232
5q
6.25
30.06
3.114
Isoniazid
Rifampicin
e
e
e
e
0.36
0.02
e
e
ꢀ0.70
2.770
a
Anti-mycobacterial activity against M. tuberculosis strain H37Rv.
Percentage of cell inhibition at 50 mg/mL against HEK-293T cells; NT denotes not tested.
b
4.4.2. N-(Furan-2-ylmethyl)-6-iodo1H-imidazo[1,2-a]pyridine-2-
carboxamide (4b)
60), 144.5 (C-7), 139.9 (C-40), 135.1 (C-2), 122.2 (C-30, C-50), 118.6 (C-
3), 118.2 (C-8), 96.3 (C-6), 41.1 (COeNHeCH2eAr); LC/MS (API-ES)
m/z calcd. for C14H11IN4O: 377.998, found: 379.1 [MþH]þ; Anal.
calcd. for C14H11IN4O: C, 44.46; H, 2.93; N, 14.82; found: C, 44.44; H,
2.925; N, 14.83.
Prepared from 3 (1 g, 3.47 mmol), furfurylamine (0.51 g,
5.21 mmol), DIPEA (2.4 mL,13.89 mmol), HATU (1.98 g, 5.21 mmol) in
DMF (10 mL). 4b was formed as a pale brown crystal (1.11 g, 87%); mp
201.2e203.0 ꢂC; 1H NMR (400 MHz, DMSO-D6):
d 8.96 (s, 1H, H-5),
8.78 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2), 8.28 (s, 1H, H-3), 7.55e7.50 (m,
2H, AreH), 7.43 (d, J ¼ 9.6 Hz, 1H, H-50), 6.36 (t, J ¼ 3.2 Hz, 1H, H-40),
6.22 (d, J ¼ 3.1 Hz,1H, H-30), 4.43 (d, J ¼ 6.1 Hz, 2H, COeNHeCH2eAr);
4.4.4. N-(Benzo[d][1,3]dioxol-5-ylmethyl)-6-iodoH-imidazo[1,2-a]
pyridine-2-carboxamide (4d)
Prepared from 3 (1 g, 3.47 mmol), piperonylamine (0.79 g,
5.21 mmol), DIPEA (2.4 mL, 13.89 mmol), HATU (1.98 g, 5.21 mmol)
in DMF (10 mL). 4d was formed as an off white solid (1.34 g, 92%);
13C NMR (100 MHz, DMSO-D6):
d 161.8 (COeNH), 156.3 (C-5), 152.5
(C-9), 144.5 (C-7), 141.8 (C-20), 139.4 (C-50), 135.9 (C-2), 119.0 (C-3),
118.2 (C-8), 110.4 (C-40), 106.6 (C-30), 96.2 (C-6), 35.3
(COeNHeCH2eAr); LC/MS (API-ES) m/z calcd. for [C13H10IN3O2]:
366.982, found: 368.0 [MþH]þ; Anal. calcd. for C13H10IN3O2: C,
42.53; H, 2.75; N, 11.45; found: C, 42.56; H, 2.759; N, 11.43.
mp 211.0e213.2 ꢂC; 1H NMR (300 MHz, DMSO-D6):
d 8.95 (s, 1H, H-
5), 8.88 (t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.26 (s, 1H, H-3), 7.49 (d,
J ¼ 12.0 Hz, 1H, H-7), 7.41 (d, J ¼ 12.0 Hz, 1H, H-8), 6.88 (s, 1H, H-40),
6.83e6.76 (m, 2H, AreH), 5.94 (s, 2H, OeCH2eO), 4.33 (d, J ¼ 5.8 Hz,
2H, COeNHeCH2eAr); 13C NMR (100 MHz, DMSO-D6):
d 161.9
(COeNH), 156.5 (C-5), 147.1 (C-9), 145.9 (H-3a0), 144.5 (C-7), 135.9
(C-2), 133.7 (H-7a0), 120.6 (C-50), 118.8 (C-3), 117.8 (C-60), 118.2 (C-8),
108.1 (C-70), 107.9 (C-40), 100.7 (OeCH2eO), 96.3 (C-6), 41.7
(COeNHeCH2eAr); LC/MS (API-ES) m/z calcd. for C16H12IN3O3:
420.992, found: 422.0 [MþH]þ; Anal. calcd. for C16H12IN3O3: C,
45.63; H, 2.87; N, 9.98; found: C, 45.60; H, 2.871; N, 10.00.
4.4.3. 6-Iodo-N-(pyridin-4-ylmethyl)H-imidazo[1,2-a]pyridine-2-
carboxamide (4c)
Prepared from 3 (1 g, 3.47 mmol), 4-(aminomethyl)pyridine
(0.56 g, 5.21 mmol), DIPEA (2.4 mL, 13.89 mmol), HATU (1.98 g,
5.21 mmol) in DMF (10 mL). 4c was formed as an off white solid
(1.17 g, 89%); mp 165.8e168.3 ꢂC; 1H NMR (400 MHz, DMSO-D6):
d
9.13 (t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.97 (s, 1H, H-5), 8.48 (d,
J ¼ 6.0 Hz, 2H, H-20, H-60), 8.29 (s, 1H, H-3), 7.53 (d, J ¼ 11.1 Hz, 1H,
H-7), 7.45 (d, J ¼ 9.5 Hz, 1H, H-8), 7.29 (d, J ¼ 5.8 Hz, 2H, H-30, H-50),
4.47 (d, J ¼ 6.3 Hz, 1H, COeNHeCH2eAr); 13C NMR (100 MHz,
4.4.5. N-Cyclopropyl-6-iodoH-imidazo[1,2-a]pyridine-2-
carboxamide (4e)
Prepared from 3 (1 g, 3.47 mmol), cyclopropylamine (0.3 g,
5.21 mmol), DIPEA (2.4 mL, 13.89 mmol), HATU (1.98 g, 5.21 mmol)
DMSO-D6): d
162.3 (COeNH), 156.4 (C-5), 149.4 (C-9), 148.7 (C-20, C-

622
G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
Table 3
Molecular docking of imidazo[1,2-a]pyridine-2-carboxamide motifs (5aeq) with InhA.
Compound Binding energy (kJ/mol) Docking energy (kJ/mol) Inhibition constant (M) Intermolecular energy (kJ/mol) H-bonds Bonding
5a
5b
ꢀ8.31
ꢀ9.64
ꢀ9.42
8.12 ꢁ 10ꢀ7
8.60 ꢁ 10ꢀ8
ꢀ9.55
2
2
5a::DRG1:HAZ:INHA:B:HIS265:O
5a::DRG1:NAM:INHA:A:TYR259:HH
5b::DRG1:HBA:INHA:A:GLN32:OE1
5b::DRG1:OAM:INHA:A:TRP249:HE1
5c::DRG1:OAM:INHA:B:ILE194:HN
5d::DRG1:OBE:INHA:A:ILE21:HN
5d::DRG1:OBD:INHA:A:ALA22:HN
5e::DRG1:HAY:INHA:B:GLY3:O
ꢀ11.16
ꢀ10.88
5c
5d
ꢀ10.56
ꢀ9.85
ꢀ12.07
ꢀ11.86
1.81 ꢁ 10ꢀ8
6.02 ꢁ 10ꢀ8
ꢀ11.81
ꢀ12.03
1
2
5e
5f
ꢀ8.75
ꢀ9.46
ꢀ10.16
ꢀ10.95
3.83 ꢁ 10ꢀ7
1.16 ꢁ 10ꢀ7
ꢀ10.00
ꢀ10.71
2
2
5e::DRG1:OAR:INHA:B:TRP249:HE1
5f::DRG1:HAZ:INHA:B:ILE194:O
5f::DRG1:NAQ:INHA:B:ILE194:HN
5g::DRG1:OAM:INHA:B:ILE194:HN
5h::DRG1:OAU:INHA:A:LYS8:HZ2
5h::DRG1:OBC:INHA:B:ASN231:HN
5i::DRG1:NAI:INHA:B:ARG153:HH11
5i::DRG1:OAL:INHA:B:HIS265:HD1
5j::DRG1:NAR:INHA:B:MET98:HN
5j::DRG1:OAM:INHA:B:TYR158:HH
5k::DRG1:NAR:INHA:A:TRP249:HE1
5k::DRG1:NAI:INHA:B:TRP249:HE1
5l::DRG1:HBC:INHA:B:HIS265:O
5l::DRG1:NAM:INHA:B:ARG225:HH11
5m::DRG1:HBE:INHA:A:ILE257:O
5m::DRG1:OAR:INHA:B:ARG173:HH12
5n::DRG1:OAM:INHA:A:TRP249:HE1
5o::DRG1:HAQ:INHA:A:ASP256:OD1
5o::DRG1:NAM:INHA:B:ARG173:HH21
5o::DRG1:OAR:INHA:A:TYR259:HH
5p::DRG1:NAM:INHA:A:THR2:HN1
5p::DRG1:OAR:INHA:B:TRP249:HE1
5q::DRG1:HAL:INHA:B:GLY3:O
5g
5h
ꢀ9.66
ꢀ8.72
ꢀ11.16
ꢀ10.33
8.34 ꢁ 10ꢀ8
4.06 ꢁ 10ꢀ7
ꢀ10.90
ꢀ10.90
1
2
5i
ꢀ8.66
ꢀ9.00
ꢀ8.55
ꢀ8.85
ꢀ9.04
ꢀ10.59
ꢀ10.08
ꢀ10.18
ꢀ9.94
4.51 ꢁ 10ꢀ7
2.53 ꢁ 10ꢀ7
5.38 ꢁ 10ꢀ7
3.25 ꢁ 10ꢀ7
2.37 ꢁ 10ꢀ7
ꢀ10.53
ꢀ10.25
ꢀ10.11
ꢀ10.1
2
2
2
2
2
5j
5k
5l
5m
ꢀ10.28
ꢀ10.91
5n
5o
ꢀ9.53
ꢀ9.20
ꢀ10.95
ꢀ11.62
1.03 ꢁ 10ꢀ7
1.79 ꢁ 10ꢀ7
ꢀ11.09
ꢀ11.69
1
3
5p
5q
ꢀ9.43
ꢀ10.09
ꢀ10.61
1.23 ꢁ 10ꢀ7
4.44 ꢁ 10ꢀ8
ꢀ10.36
ꢀ10.96
2
2
ꢀ10.03
5q::DRG1:OAM:INHA:B:TRP249:HE1
in DMF (10 mL). 4e was formed as an off white solid (0.96 g, 85%);
mp 125.5e128.3 ꢂC; 1H NMR (300 MHz, DMSO-D6):
8.95 (s, 1H, H-
5), 8.38 (d, J ¼ 4.7 Hz, 1H, COeNHeCH), 8.24 (s, 1H, H-3), 7.49 (d,
J ¼ 12.0 Hz, 1H, H-7), 7.39 (d, J ¼ 9.4 Hz, 1H, H-8), 2.86e2.82 (m, 1H,
C-10), 0.65e0.50 (m, 4H, H-20, H-30); 13C NMR (100 MHz, DMSO-D6):
(1.11 g, 86%); mp 111.3e113.6 ꢂC; 1H NMR (300 MHz, DMSO-D6):
d
d
8.95 (s, 1H, H-5), 8.27 (d, J ¼ 8.4 Hz, 1H, COeNHeCH), 8.25 (s, 1H,
H-3), 7.49 (d, J ¼ 9.3 Hz, 1H, H-7), 7.41 (d, J ¼ 9.3 Hz, 1H, H-8),
4.02e3.95 (m, 1H, H-40), 3.84 (d, J ¼ 11.2 Hz, 2H, H-2a0, H-6a0),
3.41e3.35 (dt, J ¼ 11.2, 4.0 Hz, 2H, H-2b0, H-6b0), 1.74e1.66 (m, 4H,
d
163.2 (COeNH), 156.5 (C-5), 144.6 (C-9), 144.4 (C-7), 138.3 (C-2),
H-30, H-50); 13C NMR (100 MHz, DMSO-D6):
d 161.2 (COeNH), 156.4
125.8 (C-3), 118.2 (C-8), 96.3 (C-6), 24.9 (C-10), 5.7 (C-20), 5.5 (C-30);
LC/MS (API-ES) m/z calcd. for C11H10IN3O: 326.987, found: 328.0
[MþH]þ; Anal. calcd. for C11H10IN3O: C, 40.39; H, 3.08; N, 12.85;
found: C, 40.41; H, 3.074; N, 12.85.
(C-5), 147.9 (C-9), 144.5 (C-7), 135.9 (C-2), 118.8 (C-3), 118.2 (C-8),
96.5 (C-6), 66.1 (C-20, C-60), 44.9 (C-40), 32.3 (C-30, C-50); LC/MS (API-
ES) m/z calcd. for C13H14IN3O2: 371.013, found: 372.2 [MþH]þ; Anal.
calcd. for C13H14IN3O2: C, 42.07; H, 3.80; N, 11.32; found C, 42.09; H,
3.809; N, 11.30.
4.4.6. N-(4-(Trifluoromethyl)benzyl)-6-iodoH-imidazo[1,2-a]
pyridine-2-carboxamide (4f)
4.5. General experimental procedure for the synthesis of 5aeq
Prepared from 3 (1 g, 3.47 mmol), 4-(trifluoromethyl)benzyl-
amine (0.91 g, 5.21 mmol), DIPEA (2.4 mL, 13.89 mmol), HATU
(1.98 g, 5.21 mmol) in DMF (10 mL). 4f was formed as a pale yellow
solid (1.44 g, 93%); mp 200.2e202.4 ꢂC; 1H NMR (300 MHz, DMSO-
4.5.1. Synthesis of N-(4-fluorobenzyl)-6-(1-methyl-1H-pyrazol-4-
yl)H-imidazo[1,2-a]pyridine-2-carboxamide (5a) as an example
An oven-dried Schlenk tube was charged with compound 4a
(300 mg, 0.76 mmol), 1-methylpyrazole-4-boronic acid pinacol
ester (236.9 mg, 1.14 mmol), Na2CO3 (241.4 mg, 2.28 mmol) and
9.5:0.5 mixture of 1,4-dioxane/water (10 mL). The Schlenk tube was
capped with a rubber septum, evacuated and backfilled with argon
(this sequence was carried out four times). Tetrakis(-
triphenylphosphine)palladium(0) (43.9 mg, 0.04 mmol) was added
and the Schlenk tube was sealed with a Teflon screw cap. The re-
action mixture was heated to 90 ꢂC for 8 h. The reaction mixture
was filtered through a thin pad of celite (eluted with CH2Cl2) and
the eluent was concentrated under reduced pressure. The crude
product was purified via flash column chromatography on silica gel
(230e400 mesh) to provide 5a as a white solid (225.4 mg, 85%).
D6):
d
9.13 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2), 8.96 (s, 1H, H-5), 8.28 (s,
1H, H-3), 7.66 (d, J ¼ 8.1 Hz, 2H, H-30, H-50), 7.51 (d, J ¼ 8.4 Hz, 2H, H-
20, H-60), 7.49e7.39 (m, 2H, AreH), 4.52 (d, J ¼ 6.2 Hz, 2H,
COeNHeCH2eAr); 13C NMR (100 MHz, DMSO-D6):
d 162.3
(COeNH), 156.5 (C-5), 144.7 (C-9), 144.4 (C-7), 135.4 (C-2), 133.5 (C-
10), 127.8 (C-40), 127.5 (C-20, C-60), 125.1 (C-30, C-50), 125.0 (AreCF3),
118.8 (C-3), 118.2 (C-8), 96.4 (C-6), 41.7 (COeNHeCH2eAr); LC/MS
(API-ES) m/z calcd. for C16H11F3IN3O: 444.99, found 446.0 [MþH]þ;
Anal. calcd. for C16H11F3IN3O: C, 43.17; H, 2.49; N, 9.44; found: C,
43.20; H, 2.504; N, 9.46.
4.4.7. 6-Iodo-N-(tetrahydro-2H-pyran-4-yl)H-imidazo[1,2-a]
pyridine-2-carboxamide (4g)
Prepared from 3 (1 g, 3.47 mmol), 4-aminotetrahydropyran
(0.53 g, 5.21 mmol), DIPEA (2.4 mL, 13.89 mmol), HATU (1.98 g,
5.21 mmol) in DMF (10 mL). 4g was formed as an off white solid
4.5.1.1. N-(4-Fluorobenzyl)-6-(1-methyl-1H-pyrazol-4-yl)H-imidazo
[1,2-a]pyridine-2-carboxamide (5a). mp 220.3e222.6 ꢂC; IR (KBr,
nmax
,
cmꢀ1): 3355 (NeHamide), 3163, 3076 (CeHarom.), 2919

G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
623
Fig. 3. Interaction of compounds 5h, 5j, 5k, 5l, 5o and 5q in the active pocket of InhA.
(CeHaliph.), 1652 (C]Oamide), 1574 (CeCarom.); 1H NMR (400 MHz,
2.28 mmol), Pd(PPh3)4 (43.9 mg, 0.04 mmol) in 1,4-dioxane/water
(10 mL). 5c was formed as a white solid (0.259 g, 86%); mp
199.6e201.9 ꢂC; IR (KBr, nmax, cmꢀ1): 3406 (NeHamide), 3144, 3124,
3091, 3061 (CeHarom.), 2861 (CeHaliph.), 1662 (C]Oamide), 1574
DMSO-D6):
d
8.93 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2), 8.81 (s, 1H, H-5),
8.27 (s, 1H, H-3), 8.15 (s, 1H, H-300), 7.86 (s, 1H, H-500), 7.57 (d,
J ¼ 10.4 Hz, 2H, H-20, H-60), 7.38e7.35 (m, 2H, AreH), 7.12 (t,
J ¼ 8.8 Hz, 2H, H-30, H-50), 4.44 (d, J ¼ 6.4 Hz, 2H, COeNHeCH2eAr),
(CeCarom.); 1H NMR (400 MHz, DMSO-D6):
d
9.03 (t, J ¼ 6.4 Hz, 1H,
3.87 (s, 3H, NeCH3); 13C NMR (100 MHz, DMSO-D6):
d
162.3
COeNHeCH2), 8.97e8.95 (dd, J ¼ 4.0, 1.6 Hz, 1H, H-200), 8.78 (s, 1H,
H-5), 8.42 (s, 1H, H-3), 8.30e8.27 (dd, J ¼ 8.8, 0.8 Hz, 1H, H-800), 8.12
(d, J ¼ 8.4 Hz, 1H, H-400), 7.86 (t, J ¼ 8.4 Hz, 1H, H-700), 7.70 (d,
J ¼ 8.8 Hz, 1H, H-600), 7.55 (d, J ¼ 10.4 Hz, 2H, H-20, H-60), 7.50e7.47
(dd, J ¼ 9.2, 1.6 Hz, 1H, H-300), 7.40e7.36 (m, 2H, AreH), 7.13 (t,
J ¼ 8.8 Hz, 2H, H-30, H-50), 4.46 (d, J ¼ 6.4 Hz, 2H, COeNHeCH2eAr);
(COeNH), 162.1 (C-40), 159.8 (C-9), 142.8 (C-5), 139.7 (C-300), 136.0
(C-10), 135.9 (C-2), 129.4 (C-20), 129.3 (C-60), 128.1 (C-7), 125.8 (C-6),
121.9 (C-500), 118.8 (C-3), 117.3 (C-8), 114.9 (C-30), 114.7 (C-50), 114.6
(C-400), 41.3 (COeNHeCH2eAr), 38.7 (NeCH3); HRMS (MALDI-TOF)
m/z calcd. for C19H16FN5O: 349.1338, found: 350.1405 [MþH]þ
(100%), 372.1235 [MþNa]þ, 388.0978 [MþK]þ; Anal. calcd. for
13C NMR (100 MHz, DMSO-D6): 162.3 (COeNH), 162.0 (C-40), 159.9
d
C
19H16FN5O: C, 65.32; H, 4.62; N, 20.05; found: C, 65.30; H, 4.611; N,
(C-9), 150.6 (C-200), 147.9 (C-8a00), 143.1 (C-5), 140.1 (C-10), 136.0 (C-
2), 135.4 (C-500), 133.5 (C-400), 129.4 (C-20), 129.3 (C-60), 129.1 (C-700),
128.9 (C-7), 127.8 (C-800), 127.3 (C-600), 126.2 (C-6), 124.3 (C-4a00),
121.9 (C-300), 116.8 (C-3), 115.2 (C-8), 115.0 (C-30), 114.7 (C-50), 41.3
(COeNHeCH2eAr); HRMS (MALDI-TOF) m/z calcd. for C24H17FN4O:
396.1386, found: 397.1459 [MþH]þ (100%), 419.1282 [MþNa]þ,
435.1019 [MþK]þ; Anal. calcd. for C24H17FN4O: C, 72.72; H, 4.32; N,
14.13; found: C, 72.74; H, 4.316; N, 14.15.
20.09.
4.5.1.2. N-(4-Fluorobenzyl)-6-(2-aminopyridin-3-yl)H-imidazo[1,2-
a]pyridine-2-carboxamide (5b). Prepared from 4a (300 mg,
0.76 mmol), 2-aminopyridine-3-boronic acid (157.1 mg, 1.14 mmol),
Na2CO3 (241.4 mg, 2.28 mmol), Pd(PPh3)4 (43.9 mg, 0.04 mmol) in
1,4-dioxane/water (10 mL). 5b was formed as an off white solid
(0.23 g, 84%); mp 242.0e244.6 ꢂC; IR (KBr, nmax, cmꢀ1): 3448
(NeHamide), 3284 (NeHamine), 3175, 3138, 3065, 3038 (CeHarom.),
2931 (CeHaliph.), 1651 (C]Oamide), 1606 (NeHamine), 1568
4.5.1.4. 3-(4-(2-((4-Fluorobenzyl)carbamoyl)H-imidazo[1,2-a]pyr-
idin-6-yl)phenyl)propanoic acid (5d). Prepared from 4a (300 mg,
0.76 mmol), 4-(2-carboxyethyl)benzeneboronic acid (220.9 mg,
1.14 mmol), Na2CO3 (241.4 mg, 2.28 mmol), Pd(PPh3)4 (43.9 mg,
0.04 mmol) in 1,4-dioxane/water (10 mL). 5d was formed as a white
solid (0.256 g, 81%); mp 230.1e232.7 ꢂC; IR (KBr, nmax, cmꢀ1): 3298
(NeHamide), 3187, 3088, 3059, 3034 (CeHarom.), 2949, 2921, 2857
(CeHaliph.), 2516 (OeHacid), 1703 (C]Oacid), 1646 (C]Oamide), 1580
(CeCarom.), 1261 (CeNamine); 1H NMR (400 MHz, DMSO-D6):
d 8.96
(t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.64 (s, 1H, H-5), 8.35 (s, 1H, H-3),
7.99e7.97 (dd, J ¼ 4.8,1.6 Hz,1H, H-600), 7.63 (d, J ¼ 9.2 Hz,1H, H-400),
7.42e7.33 (m, 4H, AreH), 7.12 (t, J ¼ 8.8 Hz, 2H, H-30, H-50), 6.65 (t,
J ¼ 4.8 Hz, 1H, H-500), 5.83 (s, 2H, AreNH2), 4.44 (d, J ¼ 6.0 Hz, 2H,
COeNHeCH2eAr); 13C NMR (100 MHz, DMSO-D6):
d 162.3
(COeNH), 162.1 (C-40), 159.8 (C-9), 157.1 (AreC-200eNH2), 147.7 (C-
600), 143.1 (C-5), 139.7 (C-400), 137.8 (C-10), 136.1 (C-2), 129.3 (C-20),
129.2 (C-60), 128.1 (C-7), 126.6 (C-6), 123.5 (C-300), 117.2 (C-3), 116.2
(C-8),115.0 (C-30),114.7 (C-50),112.7 (C-500), 41.3 (COeNHeCH2eAr);
HRMS (MALDI-TOF) m/z calcd. for C20H16FN5O: 361.1338, found:
362.1403 [MþH]þ (100%), 384.1236 [MþNa]þ; Anal. calcd. for
(CeCarom.), 1222 (CeOacid); 1H NMR (400 MHz, DMSO-D6):
d 12.14
(br s,1H, COOH), 8.96 (t, J ¼ 6.4 Hz,1H, COeNHeCH2), 8.91 (s,1H, H-
5), 8.35 (s, 1H, H-3), 7.67e7.61 (m, 4H, AreH), 7.38e7.35 (m, 4H,
AreH), 7.13 (t, J ¼ 8.8 Hz, 2H, H-30, H-50), 4.44 (d, J ¼ 6.4 Hz, 2H,
COeNHeCH2eAr), 2.87 (t, J ¼ 7.2 Hz, 2H, CH2eCH2eAr), 2.56 (t,
J ¼ 7.6 Hz, 2H, HOOCeCH2eCH2); 13C NMR (100 MHz, DMSO-D6):
d
173.7 (COOH), 162.3 (COeNH), 162.0 (C-40), 159.9 (C-9), 143.1 (C-
C
20H16FN5O: C, 66.47; H, 4.46; N,19.38; found: C, 66.50; H, 4.452; N,
5), 140.8 (C-100), 140.0 (C-10), 136.0 (C-2), 133.9 (C-400), 129.4 (C-20),
129.3 (C-60), 129.0 (C-7), 126.4 (C-200, C-600), 126.3 (C-300, C-500), 125.8
(C-6), 124.4 (C-30), 117.1 (C-3), 115.0 (C-8), 114.7 (C-50), 41.3
(COeNHeCH2eAr), 35.1 (CH2eCH2eCOOH), 29.9 (CH2eCH2eAr);
HRMS (MALDI-TOF) m/z calcd. for C24H20FN3O3: 417.1488, found:
19.33.
4.5.1.3. N-(4-Fluorobenzyl)-6-(quinolin-5-yl)H-imidazo[1,2-a]pyri-
dine-2-carboxamide (5c). Prepared from 4a (300 mg, 0.76 mmol),
quinoline-5-boronic acid (197 mg, 1.14 mmol), Na2CO3 (241.4 mg,

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G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
418.1559 [MþH]þ (100%), 440.1385 [MþNa]þ; Anal. calcd. for
391.1172 [MþNa]þ; Anal. calcd. for C22H16N4O2: C, 71.73; H, 4.38; N,
C
24H20FN3O3: C, 69.05; H, 4.83; N, 10.07; found: C, 69.09; H, 4.839;
15.21; found C, 71.76; H, 4.373; N, 15.19.
N, 10.04.
4.5.1.8. N-(Furan-2-ylmethyl)-6-(1-(2-morpholinoethyl)-1H-pyr-
4.5.1.5. N-(Furan-2-ylmethyl)-6-(1-methyl-1H-pyrazol-4-yl)H-imi-
azol-4-yl)H-imidazo[1,2-a]pyridine-2-carboxamide
(5h).
dazo[1,2-a]pyridine-2-carboxamide
(5e). Prepared
from
4b
Prepared from 4b (300 mg, 0.82 mmol),1-(2-morpholinoethyl)-1H-
pyrazole-4-boronic acid pinacol ester (376.5 mg, 1.23 mmol),
Na2CO3 (259.8 mg, 2.45 mmol), Pd(PPh3)4 (47.2 mg, 0.04 mmol) in
1,4-dioxane/water (10 mL). 5h was formed as a pale brown solid
(0.271 g, 79%); mp 222.4e224.7 ꢂC; IR (KBr, nmax, cmꢀ1): 3341
(NeHamide), 3093, 3044, (CeHarom.), 2954, 2856 (CeHaliph.), 1658
(C]Oamide), 1576 (CeCarom.), 1241 (CeO); 1H NMR (400 MHz,
(300 mg, 0.82 mmol), 1-methylpyrazole-4-boronic acid pinacol
ester (255 mg, 1.23 mmol), Na2CO3 (259.8 mg, 2.45 mmol),
Pd(PPh3)4 (47.2 mg, 0.04 mmol) in 1,4-dioxane/water (10 mL). 5e
was formed as a white solid (0.228 g, 87%); mp 188.2e190.6 ꢂC; IR
(KBr, nmax, cmꢀ1): 3286 (NeHamide), 3155, 3087 (CeHarom.), 2929
(CeHaliph.), 1655 (C]Oamide), 1577, 1565 (CeCarom.); 1H NMR
(400 MHz, DMSO-D6):
d
8.82 (s, 1H, H-5), 8.70 (t, J ¼ 6.4 Hz, 1H,
DMSO-D6):
d
8.82 (s, 1H, H-5), 8.69 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2),
COeNHeCH2), 8.28 (s, 1H, H-3), 8.16 (s, 1H, H-300), 7.86 (s, 1H, H-500),
7.61e7.58 (m, 2H, AreH), 7.55 (d, J ¼ 2.8 Hz, 1H, H-50), 6.37 (t,
J ¼ 2.0 Hz, 1H, H-40), 6.24 (d, J ¼ 2.8 Hz, 1H, H-30), 4.45 (d, J ¼ 6.4 Hz,
2H, COeNHeCH2eAr), 3.87 (s, 3H, NeCH3); 13C NMR (100 MHz,
8.28 (s, 1H, H-3), 8.22 (s, 1H, H-300), 7.88 (s, 1H, H-500), 7.61e7.58 (m,
2H, AreH), 7.56e7.54 (dd, J ¼ 6.4,1.2 Hz,1H, H-50), 6.37 (t, J ¼ 2.0 Hz,
1H, H-40), 6.24 (d, J ¼ 2.8 Hz, 1H, H-30), 4.46 (d, J ¼ 6.4 Hz, 2H,
COeNHeCH2eAr), 4.25 (t, J ¼ 6.4Hz, 2H, pyrazole-NeCH2eCH2),
3.55e3.53 (m, 4H, CH2eOeCH2), 2.73 (t, J¼ 6.4 Hz, 2H, morpholine-
NeCH2eCH2), 2.42e2.39 (m, 4H, CH2eNeCH2); 13C NMR (100 MHz,
DMSO-D6):
d
161.8 (COeNH), 152.5 (C-9), 142.7 (C-5), 141.8 (C-20),
139.4 (C-50), 135.9 (C-2), 128.1 (C-7), 126.0 (C-6), 122.0 (C-300), 118.9
(C-3), 117.8 (C-500), 117.3 (C-8), 114.6 (C-400), 110.4 (C-40), 106.6 (C-30),
38.7 (NeCH3), 35.3 (COeNHeCH2eAr); HRMS (MALDI-TOF) m/z
calcd. for C17H15N5O2: 321.1225, found: 322.1301 [MþH]þ (100%),
344.1124 [MþNa]þ, 360.0861 [MþK]þ; Anal. calcd. for C17H15N5O2:
C, 63.54; H, 4.71; N, 21.79; found: C, 63.57; H, 4.703; N, 21.77.
DMSO-D6):
d
161.9 (COeNH), 152.5 (C-9), 142.8 (C-5), 141.8 (C-20),
139.9 (C-50), 139.5 (C-300), 135.8 (C-2), 127.6 (C-7), 125.9 (C-6), 121.9
(C-500), 118.9 (C-3), 117.4 (C-8), 114.6 (C-400), 110.4 (C-40), 106.6 (C-30),
66.1 (CH2eOeCH2), 57.6 (CH2eNeCH2), 53.1 (pyrazole-NeCH2),
48.8 (morpholine-NeCH2), 35.3 (COeNHeCH2eAr); HRMS
(MALDI-TOF) m/z calcd. for C22H24N6O3: 420.1909, found: 421.1985
[MþH]þ (100%), 443.1807 [MþNa]þ; Anal. calcd. for C22H24N6O3: C,
62.84; H, 5.75; N, 19.99; found: C, 62.81; H, 5.742; N, 20.02.
4.5.1.6. 6-(2-Aminopyridin-3-yl)-N-(furan-2-ylmethyl)H-imidazo
[1,2-a]pyridine-2-carboxamide (5f). Prepared from 4b (300 mg,
0.82 mmol), 2-aminopyridine-3-boronic acid (269.8 mg,
1.23 mmol), Na2CO3 (259.8 mg, 2.45 mmol), Pd(PPh3)4 (47.2 mg,
0.04 mmol) in 1,4-dioxane/water (10 mL). 5f was formed as a white
crystal (0.228 g, 84%); mp 250.3e252.9 ꢂC; 1H NMR (400 MHz,
4.5.1.9. N-(Furan-2-ylmethyl)-6-(4-(1-methylpiperazine-4-carbonyl)
phenyl)H-imidazo[1,2-a]pyridine-2-carboxamide
(5i). Prepared
from 4b (300 mg, 0.82 mmol), 4-(4-methylpiperazine-1-carbonyl)
phenyl boronic acid pinacol ester (404.8 mg, 1.23 mmol), Na2CO3
(259.8 mg, 2.45 mmol), Pd(PPh3)4 (47.2 mg, 0.04 mmol) in 1,4-
dioxane/water (10 mL). 5i was formed as a Pale yellow solid
(0.283 g, 78%); mp 233.0e235.3 ꢂC; 1H NMR (400 MHz, DMSO-D6):
DMSO-D6):
d
8.74 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2), 8.64 (s, 1H, H-5),
8.37 (s, 1H, H-3), 7.99e7.97 (dd, J ¼ 4.8, 1.6 Hz, 1H, H-600), 7.63 (d,
J ¼ 9.2 Hz, 1H, H-400), 7.55 (d, J ¼ 2.8 Hz, 1H, H-50), 7.45e7.43 (dd,
J ¼ 7.2, 1.6 Hz, 1H, H-8), 7.36e7.33 (dd, J ¼ 9.6, 2.0 Hz, 1H, H-7), 6.67
(t, J ¼ 4.8 Hz, 1H, H-500), 6.38 (t, J ¼ 1.6 Hz, 1H, H-40), 6.24 (d,
J ¼ 3.2 Hz, 1H, H-30), 5.95 (s, 2H, AreNH2), 4.46 (d, J ¼ 6.0 Hz, 2H,
d
9.02 (s, 1H, H-5), 8.76 (t, J ¼ 5.6 Hz, 1H, COeNHeCH2), 8.39 (s, 1H,
H-3), 7.81e7.67 (m, 4H, AreH), 7.57e7.55 (m, 3H, AreH), 6.37 (t,
J ¼ 2.0 Hz, 1H, H-40), 6.25 (d, J ¼ 2.8 Hz, 1H, H-30), 4.47 (d, J ¼ 5.6 Hz,
2H, COeNHeCH2eAr), 3.63e3.60 (m, 4H, CH2eN(CO)eCH2),
3.01e2.70 (m, 4H, CH2eN(CH3)eCH2), 2.56 (s, 3H, NeCH3); 13C
COeNHeCH2eAr); 13C NMR (100 MHz, DMSO-D6):
d 161.9
(COeNH), 156.8 (AreC-200eNH2), 152.5 (C-9), 147.1 (C-600), 143.1 (C-
5), 141.8 (C-20), 139.6 (C-50), 138.2 (C-2), 128.1 (C-7), 126.7 (C-6),
123.3 (C-400), 117.2 (C-3), 116.5 (C-8), 115.1 (C-300), 112.7 (C-500), 110.4
(C-40), 106.6 (C-30), 35.4 (COeNHeCH2eAr); HRMS (MALDI-TOF) m/
z calcd. for C18H15N5O2: 333.1225, found: 334.1301 [MþH]þ (100%),
356.1121 [MþNa]þ, 372.0860 [MþK]þ; Anal. calcd. for C18H15N5O2:
C, 64.86; H, 4.54; N, 21.01; found C, 64.88; H, 4.549; N, 21.03.
NMR (100 MHz, DMSO-D6):
d 168.6 (piperazine-NeCOeAr), 161.8
(COeNH), 152.5(C-9), 143.2 (C-5), 141.8 (C-20), 139.9 (C-50), 137.6 (C-
100), 134.6 (C-2), 127.9 (C-7), 126.6 (C-400), 126.2 (C-6), 125.2 (C-300,C-
500), 125.1 (C-200,C-600), 117.4 (C-3), 115.3 (C-8), 110.4 (C-40), 106.7 (C-
30), 53.1 (CH2eN(CH3)eCH2), 45.5 (CH2eN(CO)eCH2), 43.7
(NeCH3), 35.3 (COeNHeCH2eAr); HRMS (MALDI-TOF) m/z calcd.
4.5.1.7. N-(Furan-2-ylmethyl)-6-(quinolin-5-yl)H-imidazo[1,2-a]pyr-
idine-2-carboxamide (5g). Prepared from 4b (300 mg, 0.82 mmol),
quinoline-5-boronic acid (212 mg, 1.23 mmol), Na2CO3 (259.8 mg,
2.45 mmol), Pd(PPh3)4 (47.2 mg, 0.04 mmol) in 1,4-dioxane/water
(10 mL). 5g was formed as an off white solid (0.258 g, 86%); mp
201.9e204.1 ꢂC; IR (KBr, nmax, cmꢀ1): 3401 (NeHamide), 3124
(CeHarom.), 2862 (CeHaliph.), 1665 (C]Oamide), 1572 (CeCarom.); 1H
for
C
25H25N5O3: 443.1957, found: 444.2031 [MþH]þ (100%),
466.1853 [MþNa]þ; Anal. calcd. for C25H25N5O3: C, 67.70; H, 5.68; N,
15.79; found: C, 67.68; H, 5.689; N, 15.80.
4.5.1.10. 6-(4-Chlorophenyl)-N-(pyridin-4-ylmethyl)H-imidazo[1,2-
a]pyridine-2-carboxamide (5j). Prepared from 4c (300 mg,
0.79 mmol), 4-chlorophenylboronic acid (186.1 mg, 1.19 mmol),
Na2CO3 (252.2 mg, 2.38 mmol), Pd(PPh3)4 (45.8 mg, 0.04 mmol) in
1,4-dioxane/water (10 mL). 5j was formed as a white solid (0.25 g,
87%); mp 166.8e169.0 ꢂC; IR (KBr, nmax, cmꢀ1): 3409 (NeHamide),
3199, 3136, 3071, 3033 (CeHarom.), 2919 (CeHaliph.),1645 (C]Oamide),
NMR (400 MHz, DMSO-D6):
d
8.97e8.95 (dd, J ¼ 4.0, 1.6 Hz, 1H, H-
200), 8.85 (t, J ¼ 6.0 Hz, 1H, COeNHeCH2), 8.80 (s, 1H, H-5), 8.44 (s,
1H, H-3), 8.29 (d, J ¼ 8.4 Hz, 1H, H-800), 8.12 (d, J ¼ 8.8 Hz, 1H, H-400),
7.86 (t, J ¼ 7.2 Hz, 1H, H-700), 7.74e7.67 (m, 2H, AreH), 7.57e7.47 (m,
3H, AreH), 6.39 (t, J ¼ 2.0 Hz, 1H, H-40), 6.26 (d, J ¼ 2.4 Hz, 1H, H-30),
4.49 (d, J ¼ 6.0 Hz, 2H, COeNHeCH2eAr); 13C NMR (100 MHz,
1602, 1566 (CeCarom.); 1H NMR (400 MHz, DMSO-D6):
d 9.11 (t,
J ¼ 6.4 Hz,1H, COeNHeCH2), 8.97 (s,1H, H-5), 8.49 (d, J ¼ 5.2 Hz, 2H,
H-20, H-60), 8.37 (s, 1H, H-3), 7.74 (d, J ¼ 8.4 Hz, 2H, H-200, H-600), 7.68
(m, 2H, AreH), 7.56 (d, J ¼ 8.4 Hz, 2H, H-300, H-500), 7.30 (d, J ¼ 5.6 Hz,
2H, H-30, H-50), 4.49 (d, J ¼ 6.0 Hz, 2H, COeNHeCH2eAr); 13C NMR
DMSO-D6):
d
161.8 (COeNH), 152.5 (C-9), 150.6 (C-200), 149.7 (C-
8a00), 147.9 (C-500), 143.1 (C-5), 141.8 (C-20), 139.9 (C-50), 136.7 (C-400),
135.4 (C-2), 133.5 (C-700), 130.4 (C-800), 129.4 (C-600), 129.1 (C-4a00),
127.8 (C-7), 126.2 (C-6), 124.3(C-300), 121.9 (C-3), 116.8 (C-8), 110.4
(C-30), 106.6 (C-40), 35.4 (COeNHeCH2eAr); HRMS (MALDI-TOF) m/
z calcd. for C22H16N4O2: 368.1273, found: 369.1349 [MþH]þ (100%),
(100 MHz, DMSO-D6):
d
162.3 (COeNH), 149.4 (C-9), 148.7 (C-20, C-
60), 143.2 (C-5), 139.9 (C-40), 135.1 (C-2), 132.8 (C-100), 129.1 (C-400),
128.3 (C-300, C-500), 126.2 (C-200, C-600), 125.0 (C-7), 124.7 (C-6), 122.2

G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
625
(C-30, C-50), 117.3 (C-3), 115.3 (C-8), 41.1 (COeNHeCH2eAr); HRMS
(MALDI-TOF) m/z calcd. for C20H15ClN4O: 362.0934, found: 363.1010
[MþH]þ (100%), 385.0833 [MþNa]þ; Anal. calcd. for C20H15ClN4O: C,
66.21; H, 4.17; N, 15.44; found: C, 66.19; H, 4.179; N, 15.47.
(CH2eNeCH2), 53.0 (pyrazole-NeCH2), 48.8 (morpholine-NeCH2),
24.9 (C-10), 5.7 (C-20), 5.5 (C-30); HRMS (MALDI-TOF) m/z calcd. for
C
20H24N6O2: 380.1961, found: 381.2035 [MþH]þ (100%), 403.1859
[MþNa]þ; Anal. calcd. for C20H24N6O2: C, 63.14; H, 6.36; N, 22.09;
found: C, 63.19; H, 6.370; N, 22.10.
4.5.1.11. 6-(2-Methoxyphenyl)-N-(pyridin-4-ylmethyl)H-imidazo
[1,2-a]pyridine-2-carboxamide (5k). Prepared from 4c (300 mg,
0.79 mmol), 2-methoxyphenylboronic acid (180.8 mg, 1.19 mmol),
Na2CO3 (252.2 mg, 2.38 mmol), Pd(PPh3)4 (45.8 mg, 0.04 mmol) in
1,4-dioxane/water (10 mL). 5k was formed as a white solid (0.244 g,
86%); mp 144.5e146.9 ꢂC; IR (KBr, nmax, cmꢀ1): 3359 (NeHamide),
3139, 3091, 3029, 3001 (CeHarom.), 2936, 2837 (CeHaliph.),1664 (C]
4.5.1.14. N-(4-(Trifluoromethyl)benzyl)-6-(quinolin-5-yl)H-imidazo
[1,2-a]pyridine-2-carboxamide (5n). Prepared from 4f (300 mg,
0.67 mmol), quinoline-5-boronic acid (175 mg, 1.01 mmol), Na2CO3
(214.3 mg, 2.02 mmol), Pd(PPh3)4 (39 mg, 0.03 mmol) in 1,4-
dioxane/water (10 mL). 5n was formed as a white solid (0.26 g,
86%); mp 177.1e179.6 ꢂC; IR (KBr, nmax, cmꢀ1): 3404 (NeHamide),
3144 (CeHarom.), 1666 (C]Oamide), 1570 (CeCarom.), 1328 (CeF); 1H
O
amide),1600,1570 (CeCarom.); 1H NMR (400 MHz, DMSO-D6):
d 9.10
(t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.71 (s, 1H, H-5), 8.49 (d, J ¼ 6.0 Hz,
2H, H-20, H-60), 8.41 (s, 1H, H-3), 7.61 (d, J ¼ 9.2 Hz, 1H, H-600),
7.51e7.48 (dd, J ¼ 9.6, 1.6 Hz, 1H, H-400), 7.42e7.38 (m, 2H, AreH),
7.30 (d, J ¼ 5.6 Hz, 2H, H-30, H-50), 7.15 (t, J ¼ 4.4 Hz, 1H, H-500), 7.06
(d, J ¼ 6.8 Hz, 1H, H-300), 4.49 (d, J ¼ 6.4 Hz, 2H, COeNHeCH2eAr),
NMR (400 MHz, DMSO-D6):
d
9.16 (t, J ¼ 6.4 Hz, 1H, COeNHeCH2),
8.97e8.95 (dd, J ¼ 4.0,1.6 Hz,1H, H-200), 8.79 (s,1H, H-5), 8.43 (s,1H,
H-3), 8.29 (d, J ¼ 8.4 Hz, 1H, H-800), 8.12 (d, J ¼ 8.4 Hz, 1H, H-400), 7.86
(t, J ¼ 8.4 Hz, 1H, H-700), 7.67 (d, J ¼ 8.0 Hz, 2H, H-30, H-50), 7.53 (d,
J ¼ 8.0 Hz, 2H, H-20, H-60), 7.40e7.36 (m, 2H, AreH), 7.16e7.11 (m,
2H, AreH), 4.56 (d, J ¼ 6.0 Hz, 2H, COeNHeCH2eAr); 13C NMR
3.80 (s, 3H, AreOeCH3); 13C NMR (100 MHz, DMSO-D6):
d 162.5
(COeNH), 156.4 (C-200eOeCH3), 149.4 (C-9), 148.7 (C-20, C-60), 142.9
(C-5), 139.5 (C-40), 135.2 (C-2), 129.6 (C-400), 129.0 (C-600), 126.4 (C-
100), 125.3 (C-7), 123.8 (C-6), 122.2 (C-30, C-50), 120.8 (C-500), 116.0 (C-
3), 115.1 (C-8), 111.8 (C-300), 55.6 (AreOeCH3), 41.2
(COeNHeCH2eAr); HRMS (MALDI-TOF) m/z calcd. for C21H18N4O2:
358.1429, found: 359.1505 [MþH]þ (100%), 381.1326 [MþNa]þ;
Anal. calcd. for C21H18N4O2: C, 70.38; H, 5.06; N, 15.63; found: C,
70.40; H, 5.055; N, 15.61.
(100 MHz, DMSO-D6): d
162.3 (COeNH), 150.7 (C-200), 147.9 (C-8a00),
144.7 (C-9), 143.2 (C-5), 139.9 (C-100), 135.4 (C-2), 133.5 (C-10), 129.4
(C-400), 129.1 (C-700), 129.0 (C-800), 127.9 (C-7), 127.8 (C-40), 127.5 (C-
20, C-60), 127.3 (C-600), 126.2 (C-6), 125.1 (C-30, C-50), 125.0 (AreCF3),
124.3 (C-4a00), 121.9 (C-300), 116.8 (C-3), 115.2 (C-8), 41.7
(COeNHeCH2eAr); HRMS (MALDI-TOF) m/z calcd. for
C
25H17F3N4O: 446.1354, found: 447.1433 [MþH]þ (100%), 469.1251
[MþNa]þ, 485.0989 [MþK]þ; Anal. calcd. for C25H17F3N4O: C, 67.26;
H, 3.84; N, 12.55; found: C, 67.28; H, 3.833; N, 12.52.
4.5.1.12. N-(Benzo[d][1,3]dioxol-5-ylmethyl)-6-(1-methyl-1H-pyr-
azol-4-yl)H-imidazo[1,2-a]pyridine-2-carboxamide
(5l).
4.5.1.15. N-(4-(Trifluoromethyl)benzyl)-6-(1-(2-morpholinoethyl)-
Prepared from 4d (300 mg, 0.71 mmol), 1-methylpyrazole-4-
boronic acid pinacol ester (222.3 mg, 1.07 mmol), Na2CO3
(226.5 mg, 2.14 mmol), Pd(PPh3)4 (41.2 mg, 0.04 mmol) in 1,4-
dioxane/water (10 mL). 5l was formed as a white solid (0.225 g,
84%); mp 251.0e253.4 ꢂC; IR (KBr, nmax, cmꢀ1): 3343 (NeHamide),
3148, 3087 (CeHarom.), 2940, 2921 (CeHaliph.), 1654 (C]Oamide),
1579 (CeCarom.), 1243 (CeOether); 1H NMR (400 MHz, DMSO-D6):
1H-pyrazol-4-yl)H-imidazo[1,2-a]pyridine-2-carboxamide
(5o).
Prepared from 4f (300 mg, 0.67 mmol), 1-(2-morpholinoethyl)-1H-
pyrazole-4-boronic acid pinacol ester (310.5 mg, 1.01 mmol),
Na2CO3 (214.3 mg, 2.02 mmol), Pd(PPh3)4 (39 mg, 0.03 mmol) in
1,4-dioxane/water (10 mL). 5o was formed as a pale yellow solid
(0.265 g, 79%); mp 199.9e202.6 ꢂC; IR (KBr, nmax, cmꢀ1): 3344
(NeHamide), 3140 (CeHarom.), 2994, 2856 (CeHaliph.), 1645 (C]
d
8.83 ((t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.81 (s, 1H, H-5), 8.26 (s, 1H,
O
amide), 1578 (CeCarom.), 1330 (CeF); 1H NMR (400 MHz, DMSO-
D6):
H-3), 8.15 (s, 1H, H-300), 7.86 (s, 1H, H-500), 7.59e7.54 (m, 2H, AreH),
d
9.07 (t, J ¼ 6.4 Hz, 1H, COeNHeCH2), 8.82 (s, 1H, H-5), 8.29 (s,
6.91 (s, 1H, H-40), 6.84e6.78 (m, 2H, AreH), 5.95 (s, 2H, OeCH2eO),
1H, H-3), 8.22 (s, 1H, H-300), 7.88 (s, 1H, H-500), 7.67 (d, J ¼ 8.0 Hz, 2H,
H-30, H-50), 7.60e7.59 (m, 2H, AreH), 7.53 (d, J ¼ 8.0 Hz, 2H, H-20, H-
60), 4.55 (d, J ¼ 6.4 Hz, 2H, COeNHeCH2eAr), 4.25 (t, J ¼ 6.4 Hz, 2H,
pyrazole-NeCH2eCH2), 3.54 (t, J ¼ 4.8 Hz, 4H, CH2eOeCH2), 2.73 (t,
J ¼ 6.4 Hz, 2H, morpholine-NeCH2eCH2), 2.41 (t, J ¼ 4.4 Hz, 4H,
4.36 (d, J ¼ 6.4 Hz, 2H, COeNHeCH2eAr), 3.87 (s, 3H, NeCH3); 13
C
NMR (100 MHz, DMSO-D6): d 161.9 (COeNH), 147.1 (C-9), 145.9 (H-
3a0), 142.8 (C-5), 139.8 (C-300), 135.9 (C-2), 133.7 (H-7a0), 128.1 (C-7),
125.8 (C-6), 121.9 (C-500), 120.6 (C-50), 118.8 (C-3), 117.8 (C-60), 117.3
(C-8), 114.5 (C-400), 108.1 (C-70), 107.9 (C-40), 100.7 (OeCH2eO), 41.7
(COeNHeCH2eAr), 38.7 (NeCH3); HRMS (MALDI-TOF) m/z calcd.
CH2eNeCH2); 13C NMR (100 MHz, DMSO-D6):
d 162.3 (COeNH),
144.7 (C-9), 142.8 (C-5), 139.6 (C-300), 135.8 (C-2), 133.5 (C-10), 127.9
(C-7), 127.6 (C-40), 127.5 (C-20, C-60), 125.9 (C-6), 125.1 (C-30, C-50),
125.0 (AreCF3), 121.9 (C-500), 118.9 (C-3), 117.3 (C-8), 114.7 (C-400),
66.1 (CH2eOeCH2), 57.6 (CH2eNeCH2), 53.1 (pyrazole-NeCH2),
48.8 (morpholine-NeCH2), 41.6 (COeNHeCH2eAr); HRMS (MALDI-
TOF) m/z calcd. for C25H25F3N6O2: 498.1991, found: 499.2065
[MþH]þ (100%), 521.1889 [MþNa]þ, 537.1622 [MþK]þ; Anal. calcd.
for C25H25F3N6O2: C, 60.23; H, 5.05; N, 16.86; found: C, 60.20; H,
5.044; N, 16.89.
for
C
20H17N5O3: 375.1331, found: 376.1402 [MþH]þ (100%),
398.1228 [MþNa]þ; Anal. calcd. for C20H17N5O3: C, 63.99; H, 4.56;
N, 18.66; found: C, 64.01; H, 4.553; N, 18.67.
4.5.1.13. N-Cyclopropyl-6-(1-(2-morpholinoethyl)-1H-pyrazol-4-yl)
H-imidazo[1,2-a]pyridine-2-carboxamide (5m). Prepared from 4e
(300 mg, 0.92 mmol), 1-(2-morpholinoethyl)-1H-pyrazole-4-
boronic acid pinacol ester (422.6 mg,1.38 mmol), Na2CO3 (291.6 mg,
2.75 mmol), Pd(PPh3)4 (53 mg, 0.05 mmol) in 1,4-dioxane/water
(10 mL). 5m was formed as a pale brown solid (0.269 g, 77%); mp
4.5.1.16. 6-(1-Methyl-1H-pyrazol-4-yl)-N-(tetrahydro-2H-pyran-4-
yl)H-imidazo[1,2-a]pyridine-2-carboxamide (5p). Prepared from 4g
(300 mg, 0.81 mmol), 1-methyl-1H-pyrazole-4-boronic acid pina-
col ester (252.3 mg, 1.21 mmol), Na2CO3 (257 mg, 2.42 mmol),
Pd(PPh3)4 (46.7 mg, 0.04 mmol) in 1,4-dioxane/water (10 mL). 5p
was formed as an off white solid (0.216 g, 82%); mp 125.0e127.4 ꢂC;
IR (KBr, nmax, cmꢀ1): 3335 (NeHamide), 3153, 3124, 3043 (CeHarom.),
2952, 2929, 2839 (CeHaliph.), 1640 (C]Oamide), 1558 (CeCarom.); 1H
135.1e137.6 ꢂC; 1H NMR (400 MHz, DMSO-D6):
d 8.81 (s, 1H, H-5),
8.30 (d, J ¼ 4.8 Hz, 1H, COeNHeCH), 8.25 (s, 1H, H-3), 8.21 (s, 1H, H-
300), 7.87 (s,1H, H-500), 7.59e7.54 (m, 2H, AreH), 4.25 (t, J ¼ 6.4 Hz, 2H,
pyrazole-NeCH2eCH2), 3.55e3.51 (m, 4H, CH2eOeCH2), 2.89e2.85
(m, 1H, H-10), 2.73 (t, J ¼ 6.4 Hz, 2H, morpholine-NeCH2eCH2),
2.42e2.37 (m, 4H, CH2eNeCH2), 0.67e0.64 (m, 4H, H-20, H-30); 13C
NMR (100 MHz, DMSO-D6): d 163.2 (COeNH), 144.3 (C-9), 142.7 (C-
5),139.8 (C-300),138.3 (C-2),135.8 (C-7),127.6 (C-6),125.8 (C-3),121.9
NMR (400 MHz, DMSO-D6): d 8.80 (s,1H, H-5), 8.25 (s,1H, H-3), 8.17
(C-500), 117.2 (C-8), 114.3 (C-400), 66.1 (CH2eOeCH2), 57.6
(d, J ¼ 8.4 Hz, 1H, COeNHeCH), 8.15 (s, 1H, H-300), 7.86 (s, 1H, H-500),

626
G. Jose et al. / European Journal of Medicinal Chemistry 89 (2015) 616e627
7.60e7.55 (m, 2H, AreH), 4.04e3.97 (m, 1H, H-40), 3.87 (s, 3H,
NeCH3), 3.85 (d, J ¼ 8.8 Hz, 2H, H-2a0, H-6a0), 3.41e3.34 (dt, J ¼ 11.2,
3.2 Hz, 2H, H-2b0, H-6b0), 1.71e1.65 (m, 4H, H-30, H-50); 13C NMR
control was also prepared on each plate. Sterile water was added to
all perimeter wells to avoid evaporation during the incubation. The
plate was covered, sealed in plastic bags and incubated at 37 ꢂC in
(100 MHz, DMSO-D6):
d
161.2 (COeNH), 147.9 (C-9), 142.7 (C-5),
normal atmosphere. After 7 days incubation, 30 mL of alamar blue
139.8 (C-300), 135.9 (C-2), 128.1 (C-7), 125.9 (C-6), 121.9 (C-500), 118.8
(C-3), 117.2 (C-8), 114.5 (C-400), 66.1 (C-20, C-60), 44.9 (C-40), 38.7
(NeCH3), 32.3 (C-30, C-50); HRMS (MALDI-TOF) m/z calcd. for
solution was added to each well, and the plate was re-incubated
overnight. A change in colour from blue (oxidised state) to pink
(reduced) indicated the growth of bacteria, and the MIC was defined
as the lowest concentration of drug that prevented this change in
colour. Isoniazid and Rifampicin were used as standards.
C
17H19N5O2: 325.1538, found: 326.1611 [MþH]þ (100%), 348.4138
[MþNa]þ; Anal. calcd. for C17H19N5O2: C, 62.75; H, 5.89; N, 21.52;
found: C, 62.74; H, 5.882; N, 21.50.
4.8. Cytotoxicity
4.5.1.17. 6-(Quinolin-5-yl)-N-(tetrahydro-2H-pyran-4-yl)H-imidazo
[1,2-a]pyridine-2-carboxamide (5q). Prepared from 4g (300 mg,
0.81 mmol), quinoline-5-boronic acid (209.7 mg, 1.21 mmol),
Na2CO3 (257 mg, 2.42 mmol), Pd(PPh3)4 (46.7 mg, 0.04 mmol) in
1,4-dioxane/water (10 mL). 5q was formed as an off white solid
(0.253 g, 84%); mp 143.8e146.0 ꢂC; IR (KBr, nmax, cmꢀ1): 3337
(NeHamide), 3099, 3066, 3034 (CeHarom.), 2936, 2848 (CeHaliph.),
1644 (C]Oamide), 1563 (CeCarom.); 1H NMR (400 MHz, DMSO-D6):
All the new compounds synthesized were further examined for
cytotoxicity against Human Embryonic Kidney Cell-line 293T (HEK-
293T) cells at the concentration of 50 mg/mL. After 72 h of expo-
sure, viability was assessed on the basis of cellular conversion of
(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
(MTT) into a formazan product using the Promega Cell Titer 96 non-
radioactive cell proliferation assay.
bromide
d
8.96e8.95 (dd, J ¼ 4.4, 1.6 Hz, 1H, H-200), 8.77 (s, 1H, H-5), 8.41 (s,
1H, H-3), 8.29 (d, J ¼ 8.8 Hz, 1H, H-800), 8.17 (d, J ¼ 8.4 Hz, 1H,
COeNHeCH), 8.12 (d, J ¼ 8.4 Hz, 1H, H-400), 7.86 (t, J ¼ 8.4 Hz, 1H, H-
700), 7.73e7.66 (m, 2H, AreH), 7.55 (d, J ¼ 8.8 Hz, 1H, H-7), 7.49e7.47
(dd, J ¼ 9.2, 1.6 Hz, 1H, H-300), 4.08e3.98 (m, 1H, H-40), 3.87 (d,
J ¼ 11.2 Hz, 2H, H-2a0, H-6a0), 3.41e3.35 (dt, J ¼ 11.2, 4.0 Hz, 2H, H-
2b0, H-6b0), 1.74e1.66 (m, 4H, H-30, H-50); 13C NMR (100 MHz,
4.9. Molecular docking study procedure
The three dimensional structure of target protein InhA, the
enoyl acyl carrier protein reductase (ENR) from M. tuberculosis, is
one of the key enzymes involved in mycobacterial fatty acid elon-
gation cycle, which has been validated as an effective antimicrobial
target, having keyword 2X22 was downloaded from PDB (www.
rcsb.org/pdb) structural database. This file was then opened in
SPDB viewer edited by removing the heteroatoms, adding C ter-
minal oxygen. The active pockets on target protein molecule were
found out using CASTp server [43]. The ligands were drawn using
ChemDraw Ultra 6.0 and assigned with proper 2D orientation
(ChemOffice package). 3D coordinates were prepared using
PRODRG server [44]. Autodock V3.0 was used to perform Auto-
mated Molecular Docking in AMD Athlon (TM)2x2 215 at 2.70 GHz,
with 1.75 GB of RAM. AutoDock 3.0 was compiled and run under
Microsoft Windows XP service pack 3. For docking, grid map re-
quires in AutoDock, the size of the grid box was set at 114, 116 and
100 Å (R, G, and B), and grid center ꢀ12.755, ꢀ22.815, 32.099 for x,
y, and z-coordinates. All torsions were allowed to rotate during
docking. The Lamarckian genetic algorithm and the pseudo-Solis
and Wets methods were applied for minimization, using default
parameters [45]. The newly synthesized compounds were taken as
ligands and docked against target molecule InhA.
DMSO-D6):
d
161.2 (COeNH), 150.6 (C-200), 147.9 (C-9), 143.0 (C-5),
140.3 (C-8a00), 135.4 (C-2), 133.4 (C-500), 129.4 (C-400), 129.0 (C-700),
128.9 (C-800), 127.8 (C-7), 127.2 (C-600), 126.1 (C-6), 124.2 (C-4a00),
121.9 (C-300), 116.7 (C-3), 115.1 (C-8), 66.2 (C-20, C-60), 45.1 (C-40),
32.4 (C-30, C-50); HRMS (MALDI-TOF) m/z calcd. for C22H20N4O2:
372.1586, found: 373.1661 [MþH]þ (100%), 395.1482 [MþNa]þ
395.1484; Anal. calcd. for C22H20N4O2: C, 70.95; H, 5.41; N, 15.04;
found: C, 70.93; H, 5.403; N, 15.03.
4.6. X-ray crystallographic analysis
The X-ray crystallographic analysis of 5f was carried out on a
white crystal, with dimensions 0.21 mm ꢁ 0.18 mm ꢁ 0.26 mm,
grown from the slow evaporation of a dichloromethane and
methanol solvent mixture at room temperature. A suitable crystal
was selected and mounted on a Bruker APEX-II CCD diffractometer.
The crystal was kept at 296.15 K during data collection. Using Olex2
[39], the structure was solved the Superflip [40] structure solution
program using Charge Flipping and refined with the ShelXL [41]
refinement package using Least Squares minimization. CCDC
1007948 contains the supplementary crystallographic data for 5f.
These data can be obtained free of charge via http://www.ccdc.cam.
ac.uk/conts/retrieving.html, or from the Cambridge Crystallo-
graphic Data Centre, 12, Union Road, Cambridge CB2 1EZ, UK; fax:
(þ44) 1223-336-033; or email: deposit@ccdc.cam.ac.uk.
Acknowledgements
The authors are thankful to the authorities of Jain University for
encouragement and financial support for this work. GJ acknowl-
edges the NMR Research Centre, Indian Institute of Science, Ban-
galore, for providing spectral data.
4.7. In vitro Mtb MABA assay procedure
Appendix A. Supplementary data
The anti-mycobacterial activities of compounds 5aeq have been
assessed against M. tuberculosis ATCC 27294 using the microplate
alamar blue assay (MABA) [42]. Briefly, the inoculum was prepared
from fresh LJ medium re-suspended in 7H9-S medium (7H9 broth,
0.1% casitone, 0.5% glycerol, supplemented oleic acid, albumin,
dextrose, and catalase [OADC]), adjusted to a McFarland tube No. 1,
Supplementary data associated with this article can be found in
the online version, at http://dx.doi.org/10.1016/j.ejmech.2014.10.
079. These data include MOL files and InChiKeys of the most
important compounds described in this article.
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