Identification and development of 2-methylimidazo[1,2-a]pyridine-3- carboxamides as Mycobacterium tuberculosis pantothenate synthetase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.05.038

In the present study, we used crystal structure of mycobacterial pantothenate synthetase (PS) bound with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)- 5-methoxy-1H-indol-1-yl) acetic acid inhibitor for virtual screening of antitubercular compound database to identify new scaffolds. One of the identified lead was modified synthetically to obtain thirty novel analogues. These synthesized compounds were evaluated for Mycobacterium tuberculosis (MTB) PS inhibition study, in vitro antimycobacterial activities and cytotoxicity against RAW 264.7 cell line. Among the compounds tested, N′-(1-naphthoyl)- 2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b) was found to be the most active compound with IC₅₀ of 1.90 ± 0.12 μM against MTB PS, MIC of 4.53 μM against MTB with no cytotoxicity at 50 μM. The binding affinity of the most potent inhibitor 5b was further confirmed biophysically through differential scanning fluorimetry.

Full text of DOI:10.1016/j.bmc.2014.05.038

Accepted Manuscript
Identification and development of 2-methylimidazo[1,2-a]pyridine-3-carboxa-
mides as Mycobacterium tuberculosis Pantothenate synthetase inhibitors
Ganesh Samala, Radhika Nallangi, Parthiban Brindha Devi, Shalini Saxena,
Renu Yadav, Jonnalagadda Padma Sridevi, Perumal Yogeeswari, Dharmarajan
Sriram
PII:
S0968-0896(14)00387-3
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.05.038
BMC 11599
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
11 March 2014
7 May 2014
16 May 2014
Please cite this article as: Samala, G., Nallangi, R., Devi, P.B., Saxena, S., Yadav, R., Sridevi, J.P., Yogeeswari, P.,
Sriram, D., Identification and development of 2-methylimidazo[1,2-a]pyridine-3-carboxamides as Mycobacterium
tuberculosis Pantothenate synthetase inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/
10.1016/j.bmc.2014.05.038
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Identification and development of 2-methylimidazo[1,2-a]pyridine-3-
carboxamides as Mycobacterium tuberculosis Pantothenate synthetase
inhibitors
Ganesh Samala, Radhika Nallangi, Parthiban Brindha Devi, Shalini Saxena, Renu Yadav,
Jonnalagadda Padma Sridevi, Perumal Yogeeswari, Dharmarajan Sriram*
Department of Pharmacy, Birla Institute of Technology & Science-Pilani, Hyderabad
Campus, Shameerpet, Hyderabad-500078, India.
ABSTRACT
In the present study, we used crystal structure of mycobacterial pantothenate synthetase (PS)
bound with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid
inhibitor for virtual screening of antitubercular compound database to identify new scaffolds.
One of the identified lead was modified synthetically to obtain thirty novel analogues. These
synthesized compounds were evaluated for Mycobacterium tuberculosis (MTB) PS inhibition
study, in vitro antimycobacterial activities and cytotoxicity against RAW 264.7 cell line.
Among the compounds tested, N'-(1-naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-
carbohydrazide (5b) was found to be the most active compound with IC50 of 1.90±0.12 µM
against MTB PS, MIC of 4.53 µM against MTB with no cytotoxicity at 50 µM. The binding
affinity of the most potent inhibitor 5b was further confirmed biophysically through
differential scanning fluorimetry.
Key words: Tuberculosis, Pantothenate synthetase, Cytotoxicity, 2-Methylimidazo[1,2-
a]pyridine-3-carbohydrazide
*For Correspondence: Email- dsriram@hyderabad.bits-pilani.ac.in
1

1. Introduction
The current treatment for tuberculosis (TB) consists of a multidrug regimen with
rifampin, isoniazid, pyrazinamide, and ethambutol and several major problems are associated
with these drugs. First, the duration and complexity of treatment itself result in non-
compliance leading to suboptimal response, and emergence of resistance1. Secondly, the
adverse events in response to anti-TB drugs also contribute to non-adherence to the regimen
2. Third, increasing incidence of multidrug-resistant (MDR) and extensively drug-resistant
(XDR) TB also seem to be a serious concern3 and fourth, co-infection of TB and HIV is a
problem by itself. Combined treatment of TB and HIV involves high pill count associated
with adherence problems, overlapping toxicity profiles, and drug-drug interactions4. Lastly,
prophylactic therapy of latent TB with isoniazid also revealed problems of non-adherence5.
Thus there is an urgent need to improve treatment by either enhancing the application of
existing agents or introducing new drugs. Pantothenate synthetase (PS; EC 6.3.2.1) enzyme
catalyzes the last step of pantothenate biosynthesis, an ATP-dependent condensation of D-
pantoate, and ß-alanine to form pantothenate6. Pantothenate a major precursor of coenzyme A
and acyl carrier protein, is essential for many intracellular processes including fatty acid
metabolism, cell signalling, and synthesis of polyketides and non-ribosomal peptides7,8.
There are reports that pantothenate biosynthetic pathway could be a potential drug target for
persistent and virulent MTB9. In this work, we discovered novel 2-methylimidazo[1,2-
a]pyridine-3-carboxylic acid derivatives as a novel scaffold as MTB PS inhibitors.
2. Results and discussion
2.1. Designing of the molecules
In the present study, crystal structure of the mycobacterial pantothenate synthetase
(PS) in complex with 2-(2-(benzofuran-2-ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-
yl)acetic acid inhibitor (PDB: 3IVX) having resolution of 1.73A° was used as a framework
2

for virtual screening of known antitubercular compound database to identify lead compounds
against this enzyme. Based on the hit identified, we undertook synthesis of analogues and
evaluated for its biological activity. Analysis of the crystal structure 3IVX revealed two key
hydrogen-bonding interactions by the sulfone oxygen atom with the backbone amide group of
Met40 and the side-chain nitrogen atom of His47. We also found other hydrogen bonding
interactions with Val187, Ser196, Ser197 and His44. The protein crystal structure showed
two hydrophobic pockets- one consisted of Met40, Leu50, Phe157, Thr39 and Pro38 whereas
the other consisted of Lys160, His44 and Gly46. The reference ligand, 2-(2-(benzofuran-2-
ylsulfonylcarbamoyl)-5-methoxy-1H-indol-1-yl) acetic acid was re-docked with the active
site grid of the PS protein to validate the active site cavity. The ligand exhibited Glide score
of -8.83 kcal/mol and was found in the vicinity of amino acids of HIE47, Met40, Val143,
Val139, Pro38, Val142, Leu146, Asp161, Tyr82, Lys160, Ser196, Ser197, HIE44, Thr186,
Val187, Leu50, Ala49, Gly46, and Gly158 residues. Re-docking results showed that the
compound exhibited similar interactions as that of the original crystal structure with a RMSD
of 1.18A°. High-throughput virtual screening of known antitubercular compounds (100
compounds of TAACF and GSK with MIC of less than 6.25 µg/mL)10–13 was performed
using Glide XP (extra precision) docking and we identified 2,6-dimethyl-N-(thiophen-2-
ylmethyl)-2H,3H- imidazo[1,2-a]pyridine-3-carboxamide (Lead 1, GSK358607A) and 6-
acetyl-2-(thiophene-2-carboxamido)-4,5,6,7-tetrahydrothieno[2,3-c]pyridine-3-carboxamide
(Lead 2, SID 92097880) (Fig 1) as potential inhibitors in the PS site interacting with
important amino acid residues like HIE44, Asp160, Ser196, Ser197 and HIE47 with a
docking score of -6.98 and -7.89 respectively. Lead 1 was reported to have good MTB MIC
of 0.19 µM; and we decided to take this lead as a starting point to develop various analogues
to derive structure activity relationship. Docking of the Lead 1 (2,6-dimethyl-N-(thiophen-2-
ylmethyl)-2H,3H-imidazo[1,2-a]pyridine-3-carboxamide) within the active site of the PS
3

protein is illustrated in Fig 2, where the following interactions were observed at the binding
site. One hydrogen bonding interaction was observed between the oxygen atom of
caboxamide and the NH group of the HIE44 amino acid side chain. Apart from hydrogen
bonding; HIE44 was also involved in π-π stacking interaction with the thiophene ring. The N-
(thiophen-2-ylmethyl) was found in the proximity of Met195, Gly46, Thr186, Leu50, Val187,
and Lys106 where it showed hydrophobic interactions with Leu50 and Val187. The nitrogen
atom of amide group was also found to interact with the Asp161. The bicyclic imidazo[1,2-
a]pyridine ring was placed in the proximity of Gly41, Tyr82, Met40, Thr39, Pro38, Phe157
and Gly158 where it showed hydrophobic interaction with Tyr82, Met40 and Pro38. Further
the compound was stabilized by the π-π stacking interaction with one of the important amino
acid residues HIE47 of the active site.
2.2. Chemistry
The target molecules were synthesised by following a three step synthetic protocol
(Scheme), wherein the first step of the reaction was of 2-aminopyridine (1) with 2-
chloroethylacetoacetate in ethanol under reflux conditions to yield the bicyclic compound –
“ethyl 2-methylimidazo[1,2-a]pyridine-3-carboxylate” (2) (Fig 3) in good yield. In the next
step, two types of reactions were carried out on ester group, one was the conversion of ester
group into carboxylic acid (4) using LiOH in Ethanol/Water (1:1), and the other was the
direct conversion of ester into acid hydrazide (3) using 35% aqueous solution of hydrazine
hydrate in ethanol under reflux conditions. Further the 2-methylimidazo[1,2-a]pyridine-3-
carbohydrazide (3) on reaction with various substituted aromatic/aliphatic carboxylic acids in
presence of coupling agents EDCI, and HOBt produced the double amides (5a-j). Reaction of
compound 3 with various substituted aldehydes in ethanol reflux conditions produced the
acid hydrazones (6a-j) in excellent yields. This reaction was faster in presence of catalytic
amount of con. H2SO4, as it formed the final molecules 6b, 6f, and 6h in less than 10 minutes
4

and for others the reaction time ranged from 30 to 60 minutes. During the reaction we
observed the formation of desired product as a solid then reaction mixture was filtered
directly and washed with distilled water, cold ethanol and hexane to obtain pure products
without further purification steps. In the case of simple amides, 2-methylimidazo[1,2-
a]pyridine-3-carboxylic acid (4) was treated with substituted aromatic/aliphatic primary
amines in presence of peptide coupling agent EDCI to produce final compounds (7a-j). The
purity of the synthesized compounds was checked by HPLC and elemental analyses and the
structures were identified by spectral data. In the nuclear magnetic resonance spectra (1H
13
NMR and C NMR), the signals of the respective protons of the prepared derivatives were
verified on the basis of their chemical shifts, multiplicities, and coupling constants. The
elemental analysis results were within ±0.4% of the theoretical values.
2.3. Pantothenate synthetase enzyme inhibition studies
Synthesized compounds were assayed for MTB PS inhibition study that coupled the
AMP produced in the condensation of β-alanine and pantoate with the oxidation of NADH to
NAD+ through myokinase, pyruvate kinase and lactate dehydrogenase. The decrease in
NADH can be monitored spectrophotometrically at 340 nm. In the initial screening at 25µM,
twenty seven compounds showed more than 50% inhibition against MTB PS and were
further studied for IC50 measurements. Compounds showed IC50 in the range of 1.90±0.12
µM to 9.20±0.96 µM. Compound N'-(1-naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-
carbohydrazide (5b) emerged as the most active compound with an IC50 of 1.90±0.12 µM.
Further to support the activity we performed docking for these compounds.
Ccompound 5b showed highest docking score of -8.60 kcal/mol which correlates well with
its potency in the enzyme assay. Closer analysis of this compound in the protein active site
revealed showed three hydrogen bonding interactions with the important amino acid residues
such as HIE44, Ser196, Ser197 and Asp161. In addition to hydrogen bonding interactions,
5

the compound was further stabilized by π-π stacking interaction with Hie44. The carbonyl
oxygen of imidazole ring interacted with the side chain of HIE44. His44 showed a π-π
stacking interaction with the imidazole ring, as well. The nitrogen atom of the amide group of
the imidazole ring interacted with Asp161 and the carbonyl oxygen on the naphthalene
moiety interacted with Ser196. Apart from hydrogen bonding interaction, the compound also
showed hydrophobic interaction with Met40, Thr82, Met195, Pro38, Val139, Leu50 and
Tyr82 and the napthyl ring was stabilized by the positively charged amino acid residues
Arg198 and Arg278 (Fig 4). Compound 5b displayed similar orientations as that of reference
compound. While the compound 5e (2-methyl-N'-(3-nitrobenzoyl)imidazo[1,2-a]pyridine-3-
carbohydrazide) showed less activity IC50=9.20 µM as compared to compound 5b we found
that the 3-nitrobenzohydrazide group was slightly away from the active site (Fig 1,
supplementary information) thus it was not fully placed in the hydrophobic cavity and also
the orientation of the molecule was different. This difference in the activity profile was also
supported by docking score (-7.73 kcal/mol) which was slightly lower than the compound 5b
(-8.60 kcal/mol).
Similarly compounds 6a-j, with substitutions at position 4 of the phenyl ring like
electron withdrawing groups such as fluoro, bromo, trifluoromethyl and substitution with
electron donating groups like hydroxyphenyl, methoxyphenyl, benzyloxyphenyl and
trimethoxyphenyl moieties, showed IC50 range from 3.77 to 8.18 µM. The most active
compound 6c showed good docking score of -8.10 kcal/mol suggesting that this compound
had been well-docked in the active site and showed good PS IC50 of 3.77 µM. It has showed
three hydrogen bondings with Gln72, Gln164 and HIE47. The presence of fluoro group on
the phenyl ring increased its hydrophobicity towards the protein and it was buried in the
hydrophobic cavity surrounded by Val143, Pro38, Phe67, Leu146, val142, and Val139 (Fig
2, supplementary information). As the compound 6e was inactive with IC50 of >25 µM,
6

also showed a low docking score of -4.90 and was found to make only one hydrogen bonding
interaction with the protein (Fig 3, supplementary information). As shown in the figure, 4-
nitrophenyl of compound 6e was not fully occupied in the active site of the protein and apart
from that nitro group of the molecule decreased the hydrophobicity and thus the activity was
less.
Among the compounds 7a-j, compound 7d showed in vitro inhibition of PS enzyme
and also showed a docking score of -6.23. The compound was found to fit very well in the
active site pocket and was surrounded by many hydrophobic amino acid residues such as
Met40, Tyr82, Val139, Val143, Pro38, Met195 and Phe197 (Fig 4, supplementary
information). While the compound 7e and 7j substituted with 2-pyridyl and 4-ethoxyphenyl
respectively were found to be inactive, and was to found to correlate well with their low
docking scores of -5.48 and -3.88 respectively. Docking analysis of these compounds in the
active site revealed that compound 7j showed one hydrogen bonding interaction with the
protein and also was not occupying the hydrophobic cavity of the protein. Even though 4-
ethoxyphenyl belonged to hydrophobic class it was not involved in hydrophobic interaction
which could be due to the bulkiness of the molecule which oriented the molecules away from
the active site (Fig 5, supplementary information). In case of compound 7e, 2-pyridyl
group showed least docking score because of no hydrogen bonding interaction and showed
only π-π stacking interaction (Fig 6, supplementary information). Analysis of docking with
this series of compounds suggested that, interactions with side chains of residues Gln72,
Ser196, Ser197, Asp161, HIE44 and HIE 47 could contribute to the binding strength. All the
compounds substituted with hydrophobic groups showed bioactivity against the panC enzyme
and the compounds having nitro group substitution had shown lesser activity as compared to
hydrophobic substitution. This could be attributed to the fact that the protein has two
7

hydrophobic pockets as described earlier and hence suggest that hydrophobicity of a ligand
could be playing a major role in retaining the activity of compounds.
2.4. In vitro MTB screening
All the synthesized compounds were also screened for their in vitro anti-tubercular
activity against Mycobacterium tuberculosis H37Rv (ATCC27294) using an agar dilution
method with drug concentrations from 50 µg/mL to 0.78µg/mL in duplicates. The minimum
inhibitory concentration (MIC) was determined for each compound which was measured as
the minimum concentration of compound required to completely inhibit the bacterial growth.
Isoniazid, ethambutol, and GSK358607A were used as reference compounds for comparison.
The MIC values of the synthesized compounds along with the standard drug for comparison
are presented in Table 1. All the synthesized compounds showed activity against MTB with
MIC ranging from 4.53 to 98.81 µM. Seven compounds (5b-d, 5f, 5g, 5i and 6d) inhibited
MTB with MIC of <20 µM. Compound 5b was found to be the most active compound in
vitro with MICs of 4.53 ꢀM against log-phase culture of MTB and it was more potent than
ethambutol (MIC 7.64 ꢀM). All the synthesized compounds were less potent than standard
antitubercular compounds like isoniazid and GSK lead compound. When compared to MTB
PS activity; MIC results were found to be different. This might be due to MTB cell wall
penetration problem with these compounds or involvement of MTB efflux pumps. With
respect to structure-MTB activity relationship, the order of activity was double amides (5a-j)
showed better activity followed by acid hydrazones (6a-j) and amides (7a-j). Among double
amides, replacement of phenyl ring (5a) with napthyl ring (5b) enhanced (~10 times) the
potency. Conversion of phenyl to cyclohexyl (5c) and furanyl ring (5d) yielded four times
more potent compounds. Introduction of nitro, chloro, methoxy and benzyloxy groups on
phenyl ring enhanced the activity, whereas 4-methyl group (5h) was found to be detrimental.
In case of acid hydrazones, compound with 4-trifluoromethyl phenyl substituent (6d) showed
8

good potency indicated by its MIC of 9.01 ꢀM. In the case of amides, replacement of phenyl
ring (7a) with benzyl group (7b) enhanced potency up to eight times, but further enlargement
with phenylethyl group (7c) reduced the activity.
2.5. In vitro cytotoxicity studies
Some of the selected compounds were also tested for in vitro cytotoxicity against
RAW 264.7cells at 50 ꢀM concentration using (4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) assay. Percentage inhibition of cells is reported in Table
1. The most promising anti-TB compound 5b showed only 28.42% cytotoxicity at 50ꢀM.
2.6. DSF studies
Furthermore the binding affinity of the most potent analogue was evaluated by
measuring the thermal stability of the protein-ligand complex using the biophysical
differential scanning fluorimetry. Differential scanning flourimetry (DSF) measure
the thermal stability of a target protein and a subsequent increase in protein melting
temperature indicate binding of a ligand to the protein. Protein complexes with ligand were
heated from 25 to 95 °C in steps of 0.1 °C in the presence of a dye called sypro orange. The
flourescence increased when the protein interacted with hydrophobic residues. Positive shift
of Tm corresponding to native protein indicated that stability was increased due to inhibitor
binding. The curves obtained in this are depicted in Fig 5. The protein MTB PS showed a
melting temperature of 49.20 °C, whereas with compound 5b the corresponding Tm was
found to be 51 °C. The difference in the Tm indicated the stability of the native protein when
it was bound with inhibitor.
3. Conclusion
In summary, we identified and synthesized a novel lead 2-methylimidazo[1,2-
a]pyridine-3-carboxylic acid derivatives from a high-throughput virtual screening of known
antitubercular compound database. Many of the compounds showed potent MTB PS
9

inhibition and MTB MIC. Compound 5i showed potency, selectivity, and no cytotoxicity
upto 50 ꢀM and emerged as valid lead for further development. Further structure-activity,
biophysical, pharmacokinetic and metabolism studies should prove fruitful.
4. Experimental Section
4.1. Chemistry
Reagents and solvents obtained from commercial sources were used without further
purification. All the reactions were monitored by thin layer chromatography (TLC) on silica
gel 40 F254 (Merck, Darmstadt, Germany) coated on aluminium plates. All 1H and 13C NMR
spectra were recorded on a Bruker AM-400 and 100 MHz spectrometer, Bruker BioSpin
Corp., Germany. Chemical shifts are reported in parts per million (ppm) using tetramethyl
silane (TMS) as an internal standard. Temperatures are reported in degrees Celsius and are
uncorrected. Compounds were analysed for C, H, N and analytical results obtained were
within ±0.4% of the calculated values for the formula shown. Molecular weights of the
synthesised compounds were checked by (Shimadzu, LCMS-2020) ESI-MS method.
4.1.1. Preparation of ethyl 2-methylimidazo[1,2-a]pyridine-3-carboxylate (2)
2-aminopyridine (4.00 g, 42.50 mmol) and 2-chloroethylacetoacetate (7.08 mL, 51.00 mmol)
were taken in 1,2-Dimethoxyethane (40 mL) and heated at 90 °C for 6 h. The reaction
mixture was concentrated under reduced pressure, diluted with EtOAc (80 mL), washed the
organic layer with H2O (3 × 30 mL). The separated organic layer was dried over anhy
Na2SO4 and concentrated under vacuo to get crude compound. The crude compound was
purified by column chromatography using 15% EtOAc in Hexanes as eluent to get ethyl 2-
methylimidazo[1,2-a]pyridine-3-carboxylate (2) (5.40 g, 62%) as an Off-white solid. ESI-MS
showed 205 [M+H]+ and carried to next step.
4.1.2. Preparation of 2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (3)
10

To the stirred solution of ethyl 2-methylimidazo[1,2-a]pyridine-3-carboxylate (2) (2.70 g) in
Ethanol (30 mL) was added 35% aqueous solution of N2H4.H2O (25 mL) and refluxed for 3
h. The reaction mixture was concentrated to half volume and cooled on ice bath, the solids
formed were filtered and dried in vacuum oven to get 2-methylimidazo[1,2-a]pyridine-3-
carbohydrazide (3) (2.25 g, 89%) as an Off-white solid. ESI-MS showed 191 [M+H]+.
4.1.3. Preparation of 2-methylimidazo[1,2-a]pyridine-3-carboxylic acid (4)
To the stirred solution of ethyl 2-methylimidazo[1,2-a]pyridine-3-carboxylate (2) (2.00 g) in
ethanol/Water (1:1) (30 mL) was added LiOH (4.00 g) and stirred at room temperature for 4
h. The reaction mixture was concentrated to half volume, and added 6N HCl at 0 °C till the
reaction mixture turned to H ~ 6, the solids formed were filtered and dried in vacuum oven
P
to get 2-methylimidazo[1,2-a]pyridine-3-carboxylic acid (4) (1.53 g, 88%) as an Off-white
solid. ESI-MS showed 177 [M+H]+.
4.1.4. General procedure for the synthesis of final molecules (5a-j)
To the stirred solution of carboxylic acid (1.0 equiv), EDCI (1.2 equiv), HOBt (1.2 equiv)
and Et3N (2.5 equiv) in Dichloromethane at 0 °C, was added compound 3 (1.05 equiv) and
allowed stir at rt for 3 h. The reaction mixture was diluted with CH2Cl2 and washed with H2O
and the separated organic layer was concentrated under reduced pressure, purified by column
chromatography using EtOAc/Hexanes as eluent.
4.1.5. N'-Benzoyl-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5a): To the stirred
solution of Benzoic acid (0.4 g, 3.27 mmol), in CH2Cl2 at 0 °C was added EDCI (0.76 g, 3.92
mmol), HOBt (0.53 g, 3.92 mmol), and Et3N (1.02 mL, 7.19 mmol) stirred for few minutes
then was added 2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (0.69 g, 3.60 mmol), and
allowed stir at rt for 3 h, The reaction mixture was diluted with CH2Cl2 and washed with H2O
and the separated organic layer was concentrated under reduced pressure, purified by column
1
chromatography using 25% EtOAc/Hexanes as eluent. MS(ESI) m/z 295 [M+H]+. H NMR
11

(400 MHz, DMSO-d6): δ 10.33 (s, 2H, NH), 8.34 (d, J = 8.4 Hz, 1H, Ar), 8.10 (d, J = 8.0 Hz,
13
2H, Ar), 7.72–7.54 (m, 5H, Ar), 7.34 (t, J = 8.0 Hz, 1H, Ar), 2.61 (s, 3H, CH3); C NMR
(100 MHz, DMSO-d6) δ 172.7, 170.8, 153.6, 149.1, 136.8, 132.5, 126.6, 125.6(2C), 124.5,
121.4(2C), 120.6, 118.4, 116.1, 18.0. Anal. calcd for C16H14N4O2: C, 65.30; H, 4.79; N, 19.04
% Found C, 65.33; H, 4.89; N, 19.11%.
4.1.6. N'-(1-Naphthoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5b):
MS(ESI) m/z 345 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.44 (s, 2H, NH), 8.55 (d, J =
8.8 Hz, 1H, Ar), 8.19 (d, J = 8.4 Hz, 1H, Ar), 7.90–7.72 (m, 3H, Ar), 7.63–7.54 (m, 4H, Ar),
13
7.36 (t, J = 8.4 Hz, 1H, Ar), 7.29 (t, J = 8.4 Hz, 1H, Ar), 2.67 (s, 3H, CH3); C NMR (100
MHz, DMSO-d6) δ 169.9, 167.8, 152.6, 150.1, 136.4, 133.4, 132.4, 130.6, 129.4, 128.4,
127.2, 126.9, 126.0, 125.6, 125.1, 124.2, 120.6, 119.2, 117.9, 19.2. Anal. calcd for
C20H16N4O2: C, 69.76; H, 4.68; N, 16.27 % Found C, 69.83; H, 4.72; N, 16.31%.
4.1.7. N'-(Cyclohexanecarbonyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5c):
1
MS(ESI) m/z 301 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 10.51 (s, 1H, NH), 10.42 (s,
1H, NH), 8.44 (d, J = 8.4 Hz, 1H, Ar), 7.72 (d, J = 8.4 Hz, 1H, Ar), 7.27 (t, J = 8.0 Hz, 1H,
Ar), 7.02 (t, J = 8.4 Hz, 1H, Ar), 2.58 (s, 3H, CH3), 2.22–2.19 (m, 1H, CH), 1.71–1.43 (m,
13
10H, (CH2)5); C NMR (100 MHz, DMSO-d6) δ 170.1, 166.8, 151.6, 149.6, 133.4, 130.4,
127.4, 123.2, 118.8, 48.3, 27.9(2C), 26.3(2C), 26.1, 18.2. Anal. calcd for C16H20N4O2: C,
63.98; H, 6.71; N, 18.65 % Found C, 63.99; H, 6.73; N, 18.71%.
4.1.8.
N'-(Furan-2-carbonyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide
(5d):
MS(ESI) m/z 285 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.53 (s, 2H, NH), 8.39 (d, J =
8.8 Hz, 1H, Ar), 8.22 (d, J = 8.8 Hz, 1H, Ar), 7.83–7.72 (m, 3H, Ar), 7.58–7.40 (m, 2H, Ar),
2.58 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 172.9, 169.2, 157.6, 153.1, 142.4,
139.4, 136.6, 135.2, 133.9, 128.6, 126.0, 123.3, 118.8, 19.1. Anal. calcd for C14H12N4O3: C,
59.15; H, 4.25; N, 19.71 % Found C, 59.23; H, 4.32; N, 19.91%.
12

4.1.9. 2-Methyl-N'-(3-nitrobenzoyl)imidazo[1,2-a]pyridine-3-carbohydrazide (5e):
MS(ESI) m/z 340 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.62 (s, 2H, NH), 8.91 (s, 1H,
Ar), 8.32 (d, J = 8.4 Hz, 1H, Ar), 8.10–7.90 (m, 2H, Ar), 7.81 (t, J = 8.4 Hz, 1H, Ar), 7.72–
7.60 (m, 3H, Ar), 2.58 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 171.2, 170.4, 166.6,
154.1, 145.7, 136.4, 135.6, 134.6, 133.9, 129.4, 127.4, 125.6, 123.3, 120.6, 118.8, 19.8. Anal.
calcd for C16H13N5O4: C, 56.64; H, 3.86; N, 20.64 % Found C, 56.73; H, 3.92; N, 20.71%.
4.1.10. N'-(3,5-Dinitrobenzoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5f):
MS(ESI) m/z 385 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 11.10 (s, 2H, NH), 9.21 (s, 2H,
Ar), 8.91 (s, 1H, Ar), 8.39 (d, J = 8.8 Hz, 1H, Ar), 7.72–7.54 (m, 2H, Ar), 7.30 (d, J = 8.4 Hz,
13
1H, Ar), 2.55 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6) δ 172.6, 171.4, 165.2, 156.2,
148.3(2C), 138.3, 137.1, 132.4(2C), 128.3, 126.6, 124.2, 119.2, 117.6, 20.5. Anal. calcd for
C16H12N6O6: C, 50.01; H, 3.15; N, 21.87 % Found C, 50.03; H, 3.22; N, 21.91%.
4.1.11. N'-(2,4-Dichlorobenzoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5g):
1
MS(ESI) m/z 363 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 10.57 (s, 1H, NH), 10.06 (s,
1H, NH), 8.97 (d, J = 6.8 Hz, 1H, Ar), 7.76 (s, 1H, Ar), 7.71–7.57 (m, 3H, Ar), 7.43 (t, J =
7.2 Hz, 1H, Ar), 7.07 (t, J = 6.8 Hz, 1H, Ar), 2.67 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 164.9, 160.5, 146.7, 145.5, 135.3, 133.3, 131.7, 130.7, 129.5, 127.4, 127.0,
126.9, 116.2, 114.2, 113.3, 15.6. Anal. calcd for C16H12Cl2N4O2: C, 52.91; H, 3.33; N, 15.43
% Found C, 52.94; H, 3.49; N, 15.48%.
4.1.12. 2-Methyl-N'-(4-methylbenzoyl)imidazo[1,2-a]pyridine-3-carbohydrazide (5h):
MS(ESI) m/z 309 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.56 (s, 2H, NH), 8.53 (d, J =
9.2 Hz, 1H, Ar), 8.01–7.74 (m, 3H, Ar), 7.63 (d, J = 8.4 Hz, 2H, Ar), 7.47 (t, J = 8.0 Hz, 1H,
Ar), 7.36 (t, J = 8.0 Hz, 1H, Ar), 2.60 (s, 3H, CH3), 2.41 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 170.8, 169.2, 158.6, 152.1, 137.3, 136.3, 134.6(2C), 132.4, 127.6(2C), 126.2,
13

123.6, 120.4, 118.2, 22.5, 17.9. Anal. calcd for C17H16N4O2: C, 66.22; H, 5.23; N, 18.17 %
Found C, 66.28; H, 5.29; N, 18.29%.
4.1.13. N'-(2-Methoxybenzoyl)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (5i):
MS(ESI) m/z 325 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.72 (s, 2H, NH), 8.39 (d, J =
8.8 Hz, 1H, Ar), 7.99–7.81 (m, 3H, Ar), 7.69–7.54 (m, 3H, Ar), 7.39 (t, J = 8.4 Hz, 1H, Ar),
3.96 (s, 3H, OCH3), 2.61 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 168.8, 167.6,
157.4, 151.9, 144.7, 139.7, 136.2, 134.2, 130.6, 127.4, 125.4, 123.5, 121.6, 119.5, 117.9,
61.2, 18.3. Anal. calcd for C17H16N4O3: C, 62.95; H, 4.97; N, 17.27 % Found C, 62.98; H,
5.02; N, 17.34%.
4.1.14. 2-Methyl-N'-(4-phenoxybenzoyl)imidazo[1,2-a]pyridine-3-carbohydrazide (5j):
MS(ESI) m/z 387 [M+H]+. 1H NMR (300 MHz, DMSO-d6): δ 10.71 (s, 2H, NH), 8.61 (d, J =
8.8 Hz, 1H, Ar), 7.92–7.81 (m, 4H, Ar), 7.69 (d, J = 8.0 Hz, 2H, Ar), 7.54–7.36 (m, 4H, Ar),
13
7.33–7.20 (m, 2H, Ar), 2.66 (s, 3H, CH3); C NMR (75 MHz, DMSO-d6) δ 172.4, 170.3,
168.5, 160.6, 158.1, 155.4, 144.9, 141.4, 136.6, 134.5, 133.2(2C), 132.9, 132.1(2C),
130.2(2C), 128.5, 126.4, 121.6, 119.6, 18.7. Anal. calcd for C22H18N4O3: C, 68.38; H, 4.70;
N, 14.50 % Found C, 68.44; H, 4.79; N, 14.58%.
4.1.15. General procedure for the synthesis of final molecules (6a-j)
2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (3) (1.0 equiv), aldehyde (1.1 equiv),
conc. H2SO4 (cat) were taken in Ethanol and refluxed for 3 min to 1 h. The formed solids
were filtered, dried and triturated with CH2Cl2/Hexanes to get pure products.
4.1.16.
N'-Benzylidene-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide
(6a):
2-
methylimidazo[1,2-a]pyridine-3-carbohydrazide (0.4 g, 2.10 mmol), Benzaldehyde (0.23 mL,
2.31 mmol), conc. H2SO4 (3 drops) were taken in Ethanol (7 mL) and refluxed for 30 min.
The solids in the reaction mixture were filtered, washed with Water, cold Ethanol, Hexanes
and dried in vacuum oven to get (0.49 g, 84%) title compound MS(ESI) m/z 279 [M+H]+. 1H
14

NMR (400 MHz, DMSO-d6): δ 12.33 (s, 1H, NH), 8.91 (s, 1H, Ar), 8.21 (d, J = 8.4 Hz, 1H,
Ar), 7.99 (d, J = 8.4 Hz, 2H, Ar), 7.63–7.44 (m, 5H, Ar), 7.27 (t, J = 8.0 Hz, 1H, Ar), 2.63 (s,
3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 162.6, 158.8, 154.2, 144.9, 134.8, 132.9,
129.6(2C), 127.6, 124.3(2C), 123.2, 119.4, 117.8, 116.1, 17.1. Anal. calcd for C16H14N4O: C,
69.05; H, 5.07; N, 20.13 % Found C, 69.13; H, 5.12; N, 20.19%.
4.1.17. N'-(4-Bromobenzylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (6b):
MS(ESI) m/z 357 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.06 (s, 1H, NH), 9.03 (s, 1H,
Ar), 8.31 (d, J = 9.2 Hz, 1H, Ar), 8.03–7.72 (m, 3H, Ar), 7.63 (d, J = 8.4 Hz, 2H, Ar), 7.47 (t,
13
J = 8.0 Hz, 1H, Ar), 7.29 (t, J = 8.4 Hz, 1H, Ar), 2.56 (s, 3H, CH3); C NMR (100 MHz,
DMSO-d6) δ 166.5, 162.8, 152.4, 146.2, 136.4, 135.8, 133.2(2C), 128.4(2C), 126.9, 125.2,
124.4, 120.6, 118.2, 19.2. Anal. calcd for C16H13BrN4O: C, 53.80; H, 3.67; N, 15.68 % Found
C, 53.93; H, 3.72; N, 15.79%.
4.1.18. N'-(4-Fluorobenzylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (6c):
MS(ESI) m/z 297 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.12 (s, 1H, NH), 8.78 (s, 1H,
Ar), 8.26 (d, J = 8.8 Hz, 1H, Ar), 7.92 (d, J = 8.4 Hz, 2H, Ar), 7.81–7.72 (m, 2H, Ar), 7.54
(d, J = 8.8 Hz, 2H, Ar), 7.39 (t, J = 8.0 Hz, 1H, Ar), 2.62 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 167.2, 163.5, 156.3, 150.3, 144.7, 138.3, 136.3, 135.3(2C), 133.5, 129.7(2C),
125.4, 123.3, 119.6, 18.8. Anal. calcd for C16H13FN4O: C, 64.86; H, 4.42; N, 18.91 % Found
C, 64.93; H, 4.52; N, 18.99%.
4.1.19.
2-Methyl-N'-(4-(trifluoromethyl)benzylidene)imidazo[1,2-a]pyridine-3-
carbohydrazide (6d):
MS(ESI) m/z 347 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 11.91 (s, 1H, NH), 8.68 (s, 1H,
Ar), 8.34 (d, J = 8.4 Hz, 1H, Ar), 7.83 (d, J = 8.4 Hz, 2H, Ar), 7.64 (d, J = 8.8 Hz, 2H, Ar),
7.31–7.22 (m, 2H, Ar), 7.02 (t, J = 8.0 Hz, 1H, Ar), 2.58 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 162.3, 160.5, 153.9, 144.7, 138.2, 136.4, 135.6, 133.6, 132.4(2C), 130.4,
15

128.2(2C), 126.1, 121.6, 118.4, 17.1. Anal. calcd for C17H13F3N4O: C, 58.96; H, 3.78; N,
16.18 % Found C, 58.99; H, 3.82; N, 16.29%.
4.1.20. 2-Methyl-N'-(4-nitrobenzylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (6e):
MS(ESI) m/z 324 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 11.82 (s, 1H, NH), 8.62 (s, 1H,
Ar), 8.55 (d, J = 8.8 Hz, 1H, Ar), 8.23 (d, J = 8.8 Hz, 2H, Ar), 7.94 (d, J = 8.4 Hz, 2H, Ar),
7.54–7.42 (m, 2H, Ar), 7.09 (t, J = 8.4 Hz, 1H, Ar), 2.61 (s, 3H, CH3); 13C NMR (100 MHz,
DMSO-d6) δ 164.3, 162.3, 158.8, 148.2, 144.7, 137.6, 136.3, 134.2, 129.3(2C), 126.6,
123.5(2C), 121.5, 119.1, 16.9. Anal. calcd for C16H13N5O3: C, 59.44; H, 4.05; N, 21.66 %
Found C, 59.49; H, 4.12; N, 21.69%.
4.1.21. N'-(4-Hydroxybenzylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (6f):
MS(ESI) m/z 295 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 12.22 (s, 1H, NH), 9.61 (s, 1H,
Ar), 8.71 (s, 1H, Ar), 8.58 (d, J = 8.4 Hz, 1H, Ar), 7.93 (d, J = 8.8 Hz, 2H, Ar), 7.72–7.54 (m,
13
2H, Ar), 7.04 (d, J = 8.4 Hz, 2H, Ar), 6.99 (t, J = 8.4 Hz, 1H, Ar), 2.56 (s, 3H, CH3); C
NMR (100 MHz, DMSO-d6) δ 170.5, 166.8, 156.3, 144.9, 142.9, 136.4, 133.2, 130.9,
124.2(2C), 123.9, 120.5(2C), 117.5, 116.1, 18.1. Anal. calcd for C16H14N4O2: C, 65.30; H,
4.79; N, 19.04 % Found C, 65.40; H, 4.82; N, 19.09%.
4.1.22. N'-(4-Methoxybenzylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide (6g):
1
MS(ESI) m/z 309 [M+H]+. H NMR (400 MHz, CDCl3): δ 11.92 (s, 1H, NH), 8.49 (s, 1H,
Ar), 8.39 (d, J = 8.4 Hz, 1H, Ar), 7.89 (d, J = 8.4 Hz, 2H, Ar), 7.80 (d, J = 7.6 Hz, 1H, Ar),
7.69–7.32 (m, 4H, Ar), 3.94 (s, 3H, OCH3), 2.56 (s, 3H, CH3); 13C NMR (100 MHz, CDCl3)
δ 170.5, 166.8, 156.3, 144.9, 142.9, 136.4, 133.2, 130.9, 124.2(2C), 123.9, 120.5(2C), 117.5,
116.1, 63.7, 18.1. Anal. calcd for C17H16N4O2: C, 66.22; H, 5.23; N, 18.17 % Found C, 66.26;
H, 5.27; N, 18.29%.
4.1.23.
(6h):
N'-(4-(Benzyloxy)benzylidene)-2-methylimidazo[1,2-a]pyridine-3-carbohydrazide
16

MS(ESI) m/z 385 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 10.72 (s, 1H, NH), 8.61 (s, 1H,
Ar), 8.52 (d, J = 8.8 Hz, 1H, Ar), 8.12–7.82 (m, 6H, Ar), 7.72 (d, J = 7.6 Hz, 2H, Ar), 7.67–
7.45 (m, 3H, Ar), 7.36 (t, J = 8.4 Hz, 1H, Ar), 5.22 (s, 2H, CH2), 2.62 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) δ 169.4, 164.3, 154.1, 148.5, 138.9, 135.2, 134.1, 133.6, 133.0, 130.9,
130.4(2C), 128.5(2C), 127.2, 126.6, 125.1(2C), 123.3(2C), 121.5, 118.7, 16.9. Anal. calcd for
C23H20N4O2: C, 71.86; H, 5.24; N, 14.57 % Found C, 71.96; H, 5.34; N, 14.60%.
4.1.24. 2-Methyl-N'-(3,4,5-trimethoxybenzylidene)imidazo[1,2-a]pyridine-3-carbohydrazide
(6i):
1
MS(ESI) m/z 369 [M+H]+. H NMR (400 MHz, DMSO-d6): δ 10.92 (s, 1H, NH), 8.47–8.35
(m, 2H, Ar), 7.62 (d, J = 8.4 Hz, 1H, Ar), 7.36 (s, 2H, Ar), 7.22–7.15 (m, 2H, Ar), 3.94 (s,
9H, (OCH3)3), 2.55 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 168.3, 166.1, 152.6,
149.2, 146.6, 144.7, 141.5, 137.4, 136.2, 134.3, 133.9, 132.8, 125.1(2C), 119.1, 63.9(2C),
63.0, 17.8. Anal. calcd for C19H20N4O4: C, 61.95; H, 5.47; N, 15.21 % Found C, 62.01; H,
5.53; N, 15.29%.
4.1.25. 2-Methyl-N'-(4-methylbenzylidene)imidazo[1,2-a]pyridine-3-carbohydrazide (6j):
1
MS(ESI) m/z 293 [M+H]+. H NMR (400 MHz, CDCl3): δ 10.88 (s, 1H, NH), 8.54 (s, 1H,
Ar), 8.41 (d, J = 8.0 Hz, 1H, Ar), 7.81 (d, J = 8.4 Hz, 2H, Ar), 7.74 (d, J = 7.2 Hz, 1H, Ar),
7.64–7.48 (m, 3H, Ar), 7.24 (t, J = 7.6 Hz, 1H, Ar), 2.58 (s, 3H, CH3), 2.29 (s, 3H, CH3); 13C
NMR (100 MHz, CDCl3) δ 167.4, 163.8, 152.4, 142.6, 139.5, 135.2, 132.9, 129.5, 125.6,
123.7(2C), 121.5(2C), 119.5, 118.1, 22.5, 17.7. Anal. calcd for C17H16N4O: C, 69.85; H, 5.52;
N, 19.17 % Found C, 69.96; H, 5.67; N, 19.29%.
4.1.26. General procedure for the synthesis of final molecules (7a-j)
To the stirred solution of carboxylic acid (1.0 equiv), EDCI (1.2 equiv), HOBt (1.2 equiv)
and Et3N (2.5 equiv) in Dichloromethane at 0 °C, was added compound 4 (1.05 equiv) and
allowed stir at room temperature for 4 h. The reaction mixture was diluted with CH2Cl2 and
17

washed with H2O and the separated organic layer was concentrated under reduced pressure to
get crude compound. The crude product was purified by column chromatography using
EtOAc/Hexanes as eluent.
4.1.27. 2-Methyl-N-phenylimidazo[1,2-a]pyridine-3-carboxamide (7a): To the stirred
solution of 2-methylimidazo[1,2-a]pyridine-3-carboxylic acid (0.30 g, 1.70 mmol), in CH2Cl2
at 0 °C was added EDCI (0.39 g, 2.04 mmol), HOBt (0.27 g, 2.04 mmol), and Et3N (0.53 mL,
3.74 mmol) stirred for few minutes then was added Aniline (0.17 mL, 1.87 mmol), and
allowed stir at rt for 4 h, The reaction mixture was diluted with CH2Cl2 and washed with H2O
and the separated organic layer was concentrated under reduced pressure, purified by column
chromatography using 20% EtOAc/Hexanes as eluent to get (0.33 g, 78%) title compound.
MS(ESI) m/z 252 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.91 (s, 1H, NH), 8.50 (d, J =
8.4 Hz, 1H, Ar), 7.91–7.72 (m, 3H, Ar), 7.53–7.26 (m, 5H, Ar), 2.67 (s, 3H, CH3); 13C NMR
(100 MHz, DMSO-d6) δ 168.6, 164.9, 156.3, 141.1, 135.1, 133.0, 132.4, 132.0, 127.4, 126.1,
125.6, 123.3, 120.8, 119.1, 18.1. Anal. calcd for C15H13N3O: C, 71.70; H, 5.21; N, 16.72 %
Found C, 71.78; H, 5.38; N, 16.79%.
4.1.28. N-Benzyl-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7b):
MS(ESI) m/z 266 [M+H]+. 1H NMR (400 MHz, CDCl3): δ 9.42 (d, J = 9.6 Hz, 1H), 7.56 (d, J
= 9.6 Hz, 1H), 7.39–7.29 (m, 5H), 6.91–6.82 (m, 3H), 4.72 (d, J = 5.6 Hz, 2H), 2.68 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 167.2, 163.6, 154.7, 144.6, 136.3, 130.9(2C), 128.4(2C),
127.0, 124.4, 121.9, 121.3, 120.4, 50.4, 17.8. Anal. calcd for C16H15N3O: C, 72.43; H, 5.70;
N, 15.84 % Found C, 72.49; H, 5.68; N, 15.92%.
4.1.29. 2-Methyl-N-phenethylimidazo[1,2-a]pyridine-3-carboxamide (7c):
MS(ESI) m/z 280 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.03 (s, 1H, NH), 8.62 (d, J =
8.0 Hz, 1H, Ar), 7.69–7.48 (m, 6H, Ar), 7.38–7.27 (m, 2H, Ar), 3.44 (t, J = 8.4 Hz, 2H, CH2),
13
2.76 (t, J = 8.4 Hz, 2H, CH2), 2.56 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6) δ 166.2,
18

164.8, 156.9, 144.6, 134.6, 132.2(2C), 127.9(2C), 127.3, 125.7, 121.4, 120.5, 119.6, 44.4,
38.4, 18.9. Anal. calcd for C17H17N3O: C, 73.10; H, 6.13; N, 15.04 % Found C, 73.19; H,
6.18; N, 15.12%.
4.1.30. N-Cyclohexyl-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7d):
MS(ESI) m/z 258 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.93 (s, 1H, NH), 8.58 (d, J =
8.0 Hz, 1H, Ar), 7.89–7.74 (m, 2H, Ar), 7.12 (t, J = 8.0 Hz, 1H, Ar), 3.39–3.31 (m, 1H, CH),
13
2.58 (s, 3H, CH3), 1.68–1.11 (m, 10H, (CH2)5); C NMR (100 MHz, DMSO-d6) δ 161.8,
160.8, 155.3, 142.6, 133.9, 129.3, 123.3, 120.4, 49.5, 36.0(2C), 27.4(2C), 26.9, 17.1. Anal.
calcd for C15H19N3O: C, 70.01; H, 7.44; N, 16.33 % Found C, 70.11; H, 7.48; N, 16.42%.
4.1.31. 2-Methyl-N-(pyridin-2-yl)imidazo[1,2-a]pyridine-3-carboxamide (7e):
MS(ESI) m/z 253 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.97 (s, 1H, NH), 8.63 (d, J =
8.0 Hz, 1H, Ar), 8.11 (d, J = 8.0 Hz, 1H, Ar), 7.81–7.64 (m, 3H, Ar), 7.27–7.02 (m, 3H, Ar),
2.61 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 162.8, 161.1, 158.6, 144.7, 139.3,
136.7, 135.0, 129.4, 127.8, 124.3, 121.5, 120.6, 118.9, 17.7. Anal. calcd for C14H12N4O: C,
66.65; H, 4.79; N, 22.21 % Found C, 66.68; H, 4.88; N, 22.29%.
4.1.32. N-(Furan-2-ylmethyl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7f):
MS(ESI) m/z 256 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 8.64 (s, 1H, NH), 8.49 (d, J =
8.4 Hz, 1H, Ar), 7.72–7.58 (m, 3H, Ar), 7.44 (d, J = 8.0 Hz, 1H, Ar), 7.38–7.20 (m, 2H, Ar),
13
5.02 (d, J = 8.0 Hz, 2H, CH2), 2.56 (s, 3H, CH3); C NMR (100 MHz, DMSO-d6) δ 162.9,
161.1, 157.6, 145.4, 139.4, 132.4, 130.5, 128.4, 126.1, 120.3, 119.6, 118.8, 42.1, 18.4. Anal.
calcd for C14H13N3O2: C, 65.87; H, 5.13; N, 16.46 % Found C, 65.93; H, 5.22; N, 16.51%.
4.1.33. N-(4-Bromophenyl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7g):
MS(ESI) m/z 330 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.07 (s, 1H, NH), 8.64 (d, J =
8.4 Hz, 1H, Ar), 7.81 (d, J = 9.2 Hz, 2H, Ar), 7.69–7.54 (m, 4H, Ar), 7.18 (t, J = 8.0 Hz, 1H,
Ar), 2.62 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 162.4, 158.5, 149.9, 139.6, 137.3,
19

133.6(2C), 132.4, 129.6, 127.6, 125.9(2C), 121.5, 119.8, 18.7. Anal. calcd for C15H12BrN3O:
C, 54.56; H, 3.66; N, 12.73 % Found C, 54.63; H, 3.72; N, 12.79%.
4.1.34. N-(4-Chlorophenyl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7h):
MS(ESI) m/z 286 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.27 (s, 1H, NH), 8.67 (d, J =
8.4 Hz, 1H, Ar), 7.90 (d, J = 9.2 Hz, 2H, Ar), 7.68 (d, J = 9.2 Hz, 2H, Ar), 7.63–7.56 (m, 2H,
Ar), 7.21 (t, J = 8.4 Hz, 1H, Ar), 2.64 (s, 3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 163.6,
160.4, 151.2, 139.3, 138.2, 134.4(2C), 133.6, 127.4, 126.2(2C), 124.8, 121.9, 120.4, 18.9.
Anal. calcd for C15H12ClN3O: C, 63.05; H, 4.23; N, 14.71 % Found C, 63.13; H, 4.32; N,
14.78%.
4.1.35. 2-Methyl-N-(3-(trifluoromethyl)phenyl)imidazo[1,2-a]pyridine-3-carboxamide (7i):
MS(ESI) m/z 320 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.21 (s, 1H, NH), 8.54 (d, J =
8.4 Hz, 1H, Ar), 8.17 (s, 1H, Ar), 7.78–7.39 (m, 5H, Ar), 7.18 (t, J = 8.4 Hz, 1H, Ar), 2.66 (s,
3H, CH3); 13C NMR (100 MHz, DMSO-d6) δ 161.9, 160.2, 156.4, 142.5, 138.4, 135.2, 133.9,
130.6, 128.3, 126.4, 125.6, 124.4, 123.9, 121.5, 120.9, 16.9. Anal. calcd for C16H12F3N3O: C,
60.19; H, 3.79; N, 13.16 % Found C, 60.28; H, 3.88; N, 13.29%.
4.1.36. N-(4-Ethoxyphenyl)-2-methylimidazo[1,2-a]pyridine-3-carboxamide (7j):
MS(ESI) m/z 296 [M+H]+. 1H NMR (400 MHz, DMSO-d6): δ 9.47 (s, 1H, NH), 8.58 (d, J =
8.4 Hz, 1H, Ar), 7.72 (d, J = 8.8 Hz, 2H, Ar), 7.58–7.49 (m, 2H, Ar), 7.42–7.26 (m, 3H, Ar),
13
4.19 (q, J = 7.2 Hz, 2H, CH2), 2.68 (s, 3H, CH3), 1.38 (t, J = 7.2 Hz, 3H, CH3); C NMR
(100 MHz, DMSO-d6) δ 162.4, 158.5, 149.9, 139.6, 137.3, 133.6(2C), 132.4, 129.6, 127.6,
125.9(2C), 121.5, 119.8, 69.1, 18.7, 16.2. Anal. calcd for C17H17N3O2: C, 69.14; H, 5.80; N,
14.23 % Found C, 69.23; H, 5.92; N, 14.39%.
4.2. Biological activity
4.2.1. MTB PS screening
20

The MTB panC gene (Rv3602c) encoding the pantothenate synthetase was cloned and
transformed into BL21 (DE3) cells and the expression of the protein was performed as
reported in literature.7 For the assay, in a 96-well plate, 60 mL of PS reagent mix containing
NADH, pantoic acid, β-alanine, ATP, phosphoenol pyruvate, MgCl2, myokinase, pyruvate
kinase, and lactate dehydrogenase in buffer was added. Compounds were then added to plates
in 1-mL volumes. The reaction was initiated with the addition of 39 mL of PS, diluted in
buffer. The test plate was immediately transferred to a micro plate reader, and absorbance
was measured at 340 nm every 12 s for 120 s.7 Percentage inhibition was calculated using
following formula, 100 *1 –compound rate – background rate/full reaction rate – background
rate.
4.2.2. In vitro MTB screening
Two-fold serial dilutions of each test compound/drug were prepared and incorporated
into Middle- brook 7H11 agar medium with oleic acid, albumin, dextrose, and catalase
(OADC) growth supplement to get final concentrations of 50, 25, 12.5, 6.25, 3.13, 1.56, and
0.78 ꢀg/mL. Inoculum of M. tuberculosis H37Rv ATCC 27294 was prepared from fresh
Middlebrook 7H11 agar slants with OADC (Difco) growth supplement adjusted to 1 mg/ mL
(wet weight) in Tween 80 (0.05%) saline diluted to 10−2 to give a concentration of ∼107
cfu/mL14. Five microliters of this bacterial suspension was spotted onto 7H11 agar tubes
containing different concentrations of the drug as discussed above. The tubes were incubated
at 37 °C, and final readings (as MIC in ꢀg/mL) were determined after 28 days. The MIC is
defined as the minimum concentration of compound required to give complete inhibition
ofbacterial growth. This method is similar to that recommended by the National Committee
for Clinical Laboratory Standards for the determination of MIC in triplicate.
4.2.3. In vitro cytotoxicity screening
21

Some compounds were further examined for toxicity in a RAW 264.7 cell line at the
concentration of 50 µM. After 72 h of exposure, viability was assessed on the basis of
cellular conversion of MTT into a formazan product using the Promega Cell Titer 96 non-
radioactive cell proliferation assay.
Acknowledgements
The authors are thankful to Department of Science & Technology (SR/S1/OC-
70/2010), New Delhi, India for their financial assistances. GS (JRF) is thankful to CSIR for
fellowship; DS acknowledges University Grants Commission, New Delhi, India for UGC
Research Award. We also acknowledge Dr David Eisenberg and Dr Shuishu Wang,
Department of Biochemistry & Molecular biology, USUHS, Maryland for providing MTB
PS clone.
References
1. Seita, A. East. Mediterr. Health. J. 2013, 3, 211.
2. Adane, A.A.; Alene, K.A.; Koye, D.N.; Zeleke, B.M PLoS. One. 2013, 11, e78791.
3. Zellweger, J.P. Rev. Mal. Respir. 2011, 8, 1025.
4. Curran, A.; Ribera, E. Expert. Rev. Anti Infect. Ther. 2011, 12, 1115.
5. Parekh, M.J.; Schluger, N.W. Ther. Adv. Respir. Dis. 2013, 6, 351.
6. Zheng, R.; Blanchard, J.S. Biochemistry. 2001, 43, 12904.
7. Samala, G.; Devi, P.B.; Nallangi, R.; Yogeeswari, P.; Sriram, D. Eur. J. Med. Chem.2013,
69, 356.
8. Samala, G.; Devi, P.B.; Nallangi, R.; Sridevi, J.P.; Saxena, S.; Yogeeswari, P.; Sriram, D.
Bioorg. Med. Chem. 2014, 22, 1938.
9. Wang, S.; Eisenberg, D. Protein. Sci. 2003, 5, 1097.
10. Ballell, L.; Bates, R.H.; Young, R.J.; Alvarez-Gomez, D.; Alvarez-Ruiz, E.; Barroso, V.;
22

Blanco, D.; Crespo, B.; Escribano, J.; González, R.; Lozano, S.; Huss, S.; Santos-
Villarejo, A.; Martín-Plaza, J.J.; Mendoza, A.; Rebollo-Lopez, M.J.; Remuiñan-Blanco,
M.; Lavandera, J.L.; Pérez-Herran, E.; Gamo-Benito, F.J.; García-Bustos, J.F.; Barros, D.;
Castro, J.P.; Cammack, N. Chem. Med. Chem. 2013, 2, 313.
11. Reynolds, R.C.; Ananthan, S.; Faaleolea, E.; Hobrath, J.V.; Kwong, C.D.; Maddox, C.;
Rasmussen, L.; Sosa, M.I.; Thammasuvimol, E.; White, E.L.; Zhang, W.; Secrist J.A 3rd,
Tuberculosis. 2012, 1, 72.
12. Ananthan, S.; Faaleolea, E.R.; Goldman, R.C.; Hobrath, J.V.; Kwong, C.D.; Laughon,
B.E.; Maddry, J.A.; Mehta, A.; Rasmussen, L.; Reynolds, R.C.; Secrist, J.A 3rd; Shindo,
N.; Showe, D.N.; Sosa, M.I.; Suling, W.J.; White, E.L. Tuberculosis. 2009, 5, 334.
13. Maddry, J.A.; Ananthan, S.; Goldman, R.C.; Hobrath, J.V.; Kwong, C.D.; Maddox, C;
Rasmussen, L.; Reynolds, R.C.; Secrist, J.A. 3rd; Sosa, M.I.; White, E.L.; Zhang, W.
Tuberculosis. 2009, 5, 354.
14. Leonard, B.; Coronel, J.; Siedner, M. J. Clinical. Microbiology. 2008, 46, 3526.
23

O
CH3
N
CONH2
NH
N
H
S
N
O
N
S
S
O
CH3
H3C
Lead 1, GSK358607A
MIC: 0.19 uM
Lead 2, SID 92097880
MIC: 3.2 ug/mL
Figure 1: Identified lead compounds from high-throughput virtual screening
Figure 2: Binding mode of the (Lead 1) and the interacting pattern in the active site of the PS
protein.
24

N
CH3
N
NH
HN
O
O
c
R
O
O
5a-j
N
N
H3C
OEt
CH3
CH3
OEt
Cl
a
N
b
N
d
N
NH2
NH
NH2
N
O
O
2
CH3
NH
1
3
N
O
e
N
R
6a-j
N
N
CH3
NH
CH3
OH
N
f
N
O
O
R
4
7a-j
Reagents & Conditions: a: 1,2-Dimethoxyethane, 90 °C, 6 h; b: 35% N2H4.H2O, Ethanol. reflux, 3 h;
c: RCOOH, EDCI, HOBt, Et3N, CH2Cl2, 0 °C - rt, 3 h; d: RCHO, Ethanol, reflux, 3 to 60 min; e:
LiOH, Ethanol/H2O (1:1), rt, 6 h; f: RNH2, EDCI, HOBt, Et3N, CH2Cl2, 0 °C - rt, 4 h:
Scheme: Synthetic protocol of compounds
H
N
H
N
H3C
Cl
H
+
N
N
O
N
N
O
O
CH3
OH
CH3
O
- H2O
Cl
Cl
Cl
N
NH2
CH3
1
EtO
EtO
EtO
O
EtO
O
N
N
N
N
H
CH3
CH3
EtO
EtO
O
O
2
Figure 3: Mechanism of conversion of compound 1 to 2
25

Figure 4: Binding mode of the mosst active compound 5b and the interacting patter
n
n
in the
active site of the panC protein.
Figure 5: DSF experiment for commpound 5b (protein-ligand complex, blue) sho
w
w
ing an
increase in the thermal shift of 1.8 °C when compared to the native PS protein (red).

7.60 - 7.54
113.2
H
2.67, s,
15.6
N
7.43, t
J = 7.2 Hz
126.9
CH3
H
N
7.07, t
H
NH
HN
O
J = 6.4 Hz
H
114.2
O
Cl
Cl
8.97, d
J = 6.8 Hz
7.76, s,
H
129.4
129.5
H
7.60 - 7.54
127.4, 127.1
H
Figure 6: 1H and 13C chemical shifts in one of the compounds
27

Table 1: Biological activities of synthesized compounds
N
N
N
CH3
CH3
NH
N
N
CH3
NH
N
NH
HN
O
O
O
N
O
R
R
R
5a-j
6a-j
7a-j
MP
( °C )
Comp
ound
Yiel
d
(%)
PanC IC50
in µM
MTB
MIC in
µM
Cytotoxicity
at 50 µM
(RAW 264.7
cells) %
inhibition
NT
R
Phenyl
81
79
88
69
76
80
87
74
69
89
84
90
88
76
92
87
93
90
91
82
78
82
84
76
69
63
81
83
72
81
180-181
260-261
251-252
186-187
220-221
138-139
251-252
189-190
162-163
142-143
184-186
274-275
250-252
223-224
268-269
277-278
201-202
172-173
206-207
238-240
184-185
>300
5a
5b
5c
5d
5e
5f
5g
5h
5i
3.54±0.18
1.90±0.12
7.70±0.67
8.93±0.53
9.20±0.96
6.48±0.26
5.13±0.24
3.35±0.32
8.21±0.42
7.22±0.29
4.86±0.61
4.43±0.12
3.77±0.08
8.18±0.14
>25
7.49±0.22
6.37±0.12
5.37±0.36
7.05±0.47
7.46±0.45
12.83±0.19
2.74±0.05
5.77±0.03
1.99±0.01
>25
42.37
4.53
Naphthyl
Cyclohexyl
2-Furyl
28.42
31.67
16.76
NT
40.62
20.12
NT
18.96
NT
NT
10.38
10.96
36.76
16.23
17.22
80.9
19.23
32.30
179.2
35.01
21.04
9.01
38.58
21.19
80.91
32.47
67.75
21.33
187.9
23.50
89.29
96.90
98.81
24.41
37.88
87.4
3-Nitrophenyl
3,5-Dinitrophenyl
2,4-Dichlorophenyl
4-Tolyl
2-Methoxyphenyl
4-Phenoxyphenyl
Phenyl
5j
6a
6b
6c
6d
6e
6f
6g
6h
6i
4-Bromophenyl
4-Fluorophenyl
4-Trifluromethylphenyl
4-Nitrophenyl
4-Hydroxyphenyl
4-Methoxyphenyl
4-Benzyloxyphenyl
3,4,5-Trimethoxyphenyl
4-Tolyl
NT
20.94
24.56
NT
19.42
NT
NT
NT
16.66
NT
24.50
NT
NT
NT
20.70
NT
NT
NT
NT
NT
6j
Phenyl
Benzyl
Phenethyl
Cyclohexyl
7a
7b
7c
7d
7e
7f
7g
7h
7i
120-121
155-156
163-164
141-142
155-156
191-192
152-153
160-161
2-Pyridyl
2-Furanylmethyl
4-Bromophenyl
4-Chlorophenyl
3-Trifluromethylphenyl
4-Ethoxyphenyl
Isoniazid
2.81±0.02
6.95±0.34
2.60±0.03
7.01±0.04
>25
78.1
42.23
0.72
7j
>25
7.64
>25
NT
Ethambutol
NT
GSK358607A
8.12±0.03
0.19
28

Graphical Abstract
Identification and development of 2-methylimidazo
[1,2-a]pyridine-3-carboxamides as Mycobacterium
tuberculosis pantothenate synthetase inhibitors
Ganesh Samala, Radhika Nallangi, Parthiban Brindha Devi, Shalini Saxena, Renu Yadav, Jonnalagadda Padma Sridevi,
Perumal Yogeeswari, Dharmarajan Sriram*