Design, one-pot green synthesis and antimicrobial evaluation of novel imidazopyridine bearing pyran bis-heterocycles

Article abstract of DOI:10.1016/j.molstruc.2020.127686

Herein, we report design, one pot synthesis and antibacterial evaluation of novel imidazopyridine bearing pyran bis-heterocycles. The compounds were synthesized in an aqueous solution of gluconic acid under both conventional heating and ultrasound irradiation. The target compounds were obtained in good to moderate yields with yield of 65–88% in 20–60 min under ultrasonic irradiation. The compounds were characterized by spectroscopic methods IR, ¹H NMR, ¹³C NMR, MS and HRMS. X-ray single crystal structure of 7i was also determined. The compounds were evaluated for antibacterial activity by measuring zone of inhibition using disk diffusion method that revealed that some compounds were inhibiting the growth of Gram +ve and Gram -ve bacteria. Result of minimum inhibitory concentration (MIC) showed that 7a, 7h & 7k from a series 7a-7k inhibited the growth of S. aureus. The minimum bactericidal concentration (MBC) value was determined for 7a, 7h & 7k. MBC/MIC ratio of the derivatives 7a, 7k & 7h suggest former two derivatives act as bactericidal agent & later act as bacteriostatic agents against Gram +ve bacteria. Haemolysis results showed that compounds are non-cytotoxic to erythrocytes.

Full text of DOI:10.1016/j.molstruc.2020.127686

Journal Pre-proof
Design, one-pot green synthesis and antimicrobial evaluation of novel
imidazopyridine bearing pyran bis-heterocycles
Ashima Thakur, Gavin Pereira, Chetananda Patel, Vinita Chauhan, Ram Kumar
Dhaked, Abha Sharma
PII:
S0022-2860(20)30009-0
DOI:
https://doi.org/10.1016/j.molstruc.2020.127686
MOLSTR 127686
Reference:
To appear in: Journal of Molecular Structure
Received Date: 11 October 2019
Revised Date: 2 January 2020
Accepted Date: 3 January 2020
Please cite this article as: A. Thakur, G. Pereira, C. Patel, V. Chauhan, R.K. Dhaked, A. Sharma,
Design, one-pot green synthesis and antimicrobial evaluation of novel imidazopyridine bearing
pyran bis-heterocycles, Journal of Molecular Structure (2020), doi: https://doi.org/10.1016/
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Authors
Role
Ashima Thakur
Synthesis and characterization of molecules
under conventional condition
Synthesis and characterization of molecules in
ultrasound irradiation
Gavin Pereira
Chetananda Patel
Vinita Chauhan
Ram Kumar Dhaked
Abha Sharma
Synthesis and characterization of molecules
under conventional condition
Biological activity
Biological activity
Conceptualization

Design, one-pot green synthesis and antimicrobial evaluation of novel
imidazopyridine bearing pyran bis-heterocycles
Ashima Thakur1, Gavin Pereira1, Chetananda Patel1, Vinita Chauhan2, Ram Kumar
Dhaked2, and Abha Sharma1*
1Department of Medicinal Chemistry, National Institute of Pharmaceutical Education
and Research, Raebareli, India
2Biotechnology Division,Defense Research and Development Establishment, Gwalior,
M.P. India
*E-mail: abha.sharma@niperraebareli.edu.in
Abstract
Herein, we report design, one pot synthesis and antibacterial evaluation of novel
imidazopyridine bearing pyran bis-heterocycles. The compounds were synthesized in an
aqueous solution of gluconic acid under both conventional heating and ultrasound irradiation.
The target compounds were obtained in good to moderate yields with yield of 65 to 88% in
20-60 minutes under ultrasonic irradiation. The compounds were characterized by
1
spectroscopic methods IR, H-NMR, 13C-NMR, MS and HRMS. X-ray single crystal
structure of 7i was also determined. The compounds were evaluated for antibacterial activity
by measuring zone of inhibition using disk diffusion method thatrevealed that some
compounds were inhibiting the growth of Gram +ve and Gram -vebacteria. Result of
minimum inhibitory concentration (MIC) showed that 7a, 7h &7k from a series 7a-7k
inhibited the growth of S. aureus. The minimum bactericidal concentration (MBC) value was
determined for 7a, 7h &7k. MBC/MIC ratio of the derivatives 7a, 7k, 7h suggest former two
derivatives act as bactericidal agent & later act as bacteriostatic agents against Gram-positive
bacteria. Haemolysis results showed that compounds are non-cytotoxic to erythrocytes.
1. Introduction
Bis-heterocycles are compounds comprising two or more heterocyclic moieties linked via a
spacer or fused together. There is a growing interest in the synthesis of bis-heterocycles due
to the fact that they exhibit potent biological activities [1-3]. Figure 1 represents some drugs
and bioactive molecules consist of bis-heterocycles[1, 2, 4]. A large number of reports
referring to synthesis of bioactive bis-heterocycles such as sulfone linked pyrrolyl-
oxadiazoles/thiadiazoles/triazoles and pyrazolyl oxadiazoles/thiadiazoles/triazoles as antimicr

obial[5], 2-mercapto benzothiazole linked with 1,2,3-triazoles as anti-inflammatory &anti-
nociceptive activity[6], bis-sulfonamide Schiff bases as carbonic anhydrase inhibitors[7], bis-
isatin derivatives as anti-mycobacterial agent[8] spirochromenes[9] via multistep or one-pot
reaction have been reported. Inspired by literature reports on bis-heterocycles we aim to
synthesize novel bis-heterocyclic scaffold.
Figure 1: Drugs and Bioactive Bis-heterocyclic Molecules
Imidazo[1,2-a]pyridine is an important fused heterocycle possessing various biological
activities such as antibacterial, anticancer, anti-inflammatory, antihypertensive, antiviral,
antiosteoporotic, antiparasitic, and neurodegenerative diseases [10-15]. Some of the
compounds with imidazopyridinescaffold are also used in psychiatry and autoimmune
disorders[16]. Other than this, pyran is an important heterocycle exhibiting a wide variety of
biological activities. For example, pyran demonstrates activities like anticancer, antioxidant,
antimicrobial, antiproliferative, and calcium channel antagonists(Figure 2)[17, 18]. Due to the
importance of these heterocyclic moieties, we planned to combine both pyran and
imidazopyridine moiety in a single structure.

Figure 2: Biologically active imidazopyridine, 2-Amino-3-cyano-4H-pyrans derivatives, and
synthetic targets
Now, we thought about the protocol which can be used to combine imidazo[1,2-a] pyridine
and pyran scaffold. In this direction, gluconic acid aqueous solution (50 wt %, GAAS),
introduced by Gu et al. as a promoting medium and catalyst utilized for many organic
transformations was initially tested for the synthesis of target compound [19]. Gluconic acid
(GA) is a bio-based substance, naturally present in plants, fruits, and other foodstuffs and
commercially available in the market as a 50% aqueous solution. GAAS is a nonvolatile,
noncorrosive, stable, inexpensive, recyclable and biodegradable organic acid. Under normal
condition, GAAS exist in an equilibrium, in which about 5% of glucono- δ-lactone is present.
In particular, when a proton dissociates from GA molecule, it results in the aqueous solution
which makes the solution slightly acidic. This allows the use of GAAS for promoting organic
reactions that need the assistance of a weak acid [20-22]. Thus, such media may constitute an
alternative solvent for the development of organic compounds. Meanwhile, multicomponent
reactions (MCRs) are known to combine more than two components for the construction of
novel and structurally complex molecules in a single pot. It is performed to ensure high atom-
economy, good yields, and to avoid expensive purification processes. It has been established

that MCRs are generally much more environmentally friendly and has emerged as a powerful
synthetic methodology used for the synthesis of drugs and bioactive compounds[23].
The combination of multicomponent reactions, an environmentally benign form of catalyst
and a green solvent has become a promising frontier field of research, which enables
simultaneous growth of both MCRs and green solvents toward ideal organic synthesis[24-
28]. Apart from this, the use of ultrasound irradiation in organic synthesis is now becoming
an emerging powerful technique. The prominent features of the ultrasound-assisted synthesis
are the time required for completion of the reaction is less and products obtained in high yield
[29-31].
A
variety of biologically important scaffolds like pyrazoles[32],
benzoxanthenes[30], dihydroquinazolinones[33], coumarins[34], gem dichloroaziridine[35],
tetrazolopyrimidine[36] have been synthesized with & without catalyst under ultrasound
irradiation. In the view of the above points and as a continuation of our ongoing work
towards the design and development of novel and environmental benign synthetic
methodologies[37-39], herein, we report one-pot synthesis of novel imidazopyridine bearing
pyran moiety via three component condensation reaction of malononitrile, 2-phenylimidazo
[1,2-a]pyridine-3-carbaldehyde and cyclohexanedione using GAAS as an efficient and
reusable promoting medium under conventional and ultrasonication method. In addition, we
have investigated molecules against microbes as microbial infections are causing various
serious diseases such as bacteremia, pneumonia, intra-abdominal & urinary tract infection.
Gram-negative & Gram-positive bacterium’s like Escherichia coli, Klebsiella pneumonia,
Pseudomonas aeruginosa&Staphylococcus aureus are associated with various types of
infections[40]. According to the World Health Organization and Centre for Disease Control
& Prevention data show that death due to bacterial infection is highper year [41, 42]. The
situation becomes dangerous by the emergence of multi, extensive & pan drug resistance
bacterial strains[43, 44]. For example, a nosocomial infection is caused by methicilin
resistance Staphylococcus aureus[45, 46].
2. Results and Discussion
2.1 Chemistry
We started work with the synthesis of precursor 2-phenylimidazo [1,2-a]pyridine-3-
carbaldehyde (4) using reported protocols (Scheme 1). The precursor 2-phenylimidazo[1,2 a]
pyridine carbaldehyde (4) was synthesized in two steps by reacting 2-amino pyridine (1) with

phenacyl bromide (2) first to form 2-phenylimidazo[1,2 a] pyridine (3). Then product (4) was
obtained by Vilsmeier Haack reaction performed on 3.
Scheme 1: Synthesis of 2-phenylimidazo [1,2-a]pyridine-3-carbaldehyde
After the synthesis of precursor (4), we investigated the synthesis of imidazopyridine fused
pyran moiety via MCR in neat at rt using reactants 1(0.2 g,1 mmol), 2 (0.14 g, 1mmol) & 3
(0.063 ml, 1 mmol) as depicted in Table 1 and reaction was monitored via TLC at various
intervals of time for 12 h. Visualization of TLC under UV showed no new spot formation,
indicating no product formation (Table 1, entry 1). Then, the reactions were carried out in
different solvents like water, ethanol, methanol, PEG-400, glycerol, and GAAS at rt for 12 h.
The expected product was not obtained in all tested solvents expect in GAAS, a new spot was
visible on TLC (Table 1, entries 2-7). When the reaction was performed in GAAS at
temperature 60, 80, & 100 ºC, a light spot which was visible on TLC of reaction carried out at
rt (Table 1, entry 7) now appeared as an intense spot on TLC under heating conditions (Table
1, entries 8-10). The maximum yield of product was obtained in GAAS (Table 1, entry 10),
prompted us to check the combination of GAAS with other solvents at 100 ºC. In all the
reaction conditions, a product was formed (Table 1, entries 11-15) but the maximum yield
was obtained in a combination of GAAS with EtOH (Table 1, entry 11). After obtaining
promising results, we increased the amount of GAAS from 1ml to 7ml with 1ml EtOH, with
every time an increment of 1ml GAAS was made (Table 1, entries 16-21). The product yield
was increased till 5ml GAAS and remained the same in the remaining two conditions i.e., 6
and 7ml (Table 1, entries 20 & 21). Therefore, the optimum reaction condition required for
the synthesis of fused pyran moiety with imidazopyridine was GAAS 5ml + EtOH 1ml
(Table 1, Entry 19). Then, the optimum condition was checked for substrate scope. Different
substituted 2-phenylimidazo[1,2-a]pyridine-3-carbaldehyde and cyclohexanedione along with
malononitrile were tested for the synthesis of target compounds. Owing to this we have
synthesized 11 derivatives (7a-7k) in good yield (Table 3). The model reaction was
completed in 2 h. Initially, time was fixed for the reactions but it was found that in some
cases some reactants remained in the reaction mixture so we decided to monitor the reaction
till completion. Thus, different derivatives took different time to give the maximum yield.
Particularly, when dimedone was used for the synthesis of compounds, a solid product was

obtained. The formation of a solid product made the work-up process easier. Different
colored solid products were isolated by filtration and washed with hexane. Then the filtrate
was reduced to ultimately recover GAAS for reuse and finally, the crude products were
crystallized from ethanol. A different observation perceived in the case of 1,3-
cyclohexanedione that no solid product was obtained therefore extracted with ethyl acetate
and washed with water. The product collected in the ethyl acetate layer which on
concentrated under reduced pressure over rotary evaporator gave a solid product. The crude
product was further purified by silica gel column chromatography if needed.
Table 1: Optimization of reaction conditions in conventional methoda
Entry
Solvent
Quantity
Temperature
(οC)
Time (h)
Yieldb (%)
7a
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
No solvent
Water
-
rt
rt
rt
rt
rt
rt
rt
60
80
100
12
12
12
12
12
12
12
12
12
12
-
-
-
-
-
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
1 ml
Ethanol
Methanol
PEG-400
Glycerol
GAAS
GAAS
GAAS
GAAS
-
Trace
Trace
Trace
25
11.
12.
13.
GAAS + EtOH
GAAS+ Water
GAAS+ MeOH
1 ml each
1 ml + 1ml
1 ml + 1ml
1 ml + 1ml
1 ml + 1ml
2 ml + 1ml
3 ml + 1ml
4 ml + 1ml
5 ml + 1ml
6 ml + 1ml
7 ml + 1ml
5 ml + 1ml
5 ml + 1ml
100
100
100
100
100
100
100
100
100
100
100
100
100
4
4
4
4
4
4
4
4
2
2
2
2
2
34
20
28
28
24
43
54
78
88
88
88
83
87
14. GAAS+PEG-400
15. GAAS+ Glycerol
16.
17.
18.
19.
20.
21.
22.
23.
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
cGAAS+ EtOH
dGAAS+ EtOH
aReaction Condition: 1a (1.0 mmol, 2a (1.0 mmol) and 3 (1.0mmol).

bYield: Isolated yield after silica gel chromatography.
C: Recovered GAAS third run
d: 20 mmol scale
GAAS was recovered by concentrating water layer under reduced pressure. The recovered
GAAS was further tested for its catalytic potential for the same reactions. The yield obtained
in the recovered GAAS is given in table 1. Furthermore, on scaling the reactions upto 20
mmol produced a product in almost no significant difference in the yield, indicating an
effective methodology for practical synthesis of target molecules.
Subsequently, this MCR reaction was performed under ultrasonic irradiation using
ultrasonicator. The reactions were performed in optimum solvent conditions (5ml GAAS:
1ml EtOH) as established from table 1. Initially, the reaction was performed at 80oC and
monitored for completion via TLC at various time intervals. Surprisingly, the reaction was
completed in 20 minutes (Table 2, entry 1) which indicates better efficiency of the
ultrasonication method than the conventional method. Furthermore, to determine the effect of
temperature on the reaction time, the reactions were performed at 70, 60, 50 and 40ºC and
were monitored at different intervals for completion via TLC. Notably, the time for
completion of the reaction was found to decrease with increase in temperature (Table 2,
entries 2-5). Hence the optimum conditions were established to be 5ml GAAS: 1ml EtOH at
80ºC for the synthesis of fused pyran moiety with imidazopyridine via ultrasonication method
(Table 2, entry 1). Owing to this the optimized conditions were tested for substrate scope, so
different substituted 2-phenylimidazo[1,2-a]pyridine-3-carbaldehyde and cyclohexandione
along with malononitrile were tested for the synthesis of target compounds. With this, we
have synthesized 11 derivatives (7a-7k) in less time with good yield compared to the
conventional method (Table 3). Initially, 20 minutes time was fixed for the reactions as the
model reaction was completed in this time but it was found that in some cases some reactants
remained in the reaction mixture so we decided to monitor the reaction till completion. Thus,
different derivatives took different time to give the maximum yield.
Table 2: Optimization of reaction in Ultrasonicator
Entry
Solvent
Quantity
Temperature (οC)
Time (h)
Yieldb (%)
7a
88
86
85
84
1.
2.
3.
4.
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
GAAS+ EtOH
5 ml + 1ml
5 ml + 1ml
5 ml + 1ml
5 ml + 1ml
80
70
60
50
0.33
0.66
1.5
3.1

5.
GAAS+ EtOH
5 ml + 1ml
40
3.5
86
Table 3: Synthesis of 7a-7k by conventional and ultrasound method.
Compound
Conventional method
Time (h) Yield (%)
80
Ultrasonication method
Time (h)
0.33
Yield (%)
88
2
5
78
0.66
82
10
62
0.5
65
5
77
0.66
83
9
64
1
66

5
72
76
62
74
0.50
0.50
1
78
78
66
82
11
10
10
0.66
6
80
62
0.66
84
65
11
1

All novel compounds (7a-7k) showed variable colors such as pale yellowish, off-white &
1
13
white and were characterized by spectroscopic techniques(MS, IR, H NMR and C NMR
spectra).Single Crystal X-Ray diffraction studies have been done for the characterization of
Compound 7i which was crystallized by using ethanol under slow evaporation.The
crystallography data indicated that the crystallized compound is C26H24N4O2. The pyran ring
is fused with the imidazopyridine group. TheX-ray crystallographic data of 7i compound
showed monoclinic shape (Figure 3).
Figure 3: a) Crystals of 7i compound b) ORTEP diagram of 7i (CCDC 1865906)
The reaction mechanism of product formation as depicted in scheme 2 involved two
name reactions Knoevenagel condensation and Michael addition followed by cyclization and
tautomerization (37).
Scheme 2: Proposed reaction mechanism of Imidazopyridine fused pyran ring.

2.2 Biological Evaluation
The in vitro antibacterial profile of imidazopyridine fused pyran derivatives 7a-7k was
evaluated by measuring the inhibition zone against Gram-positive (Staphylococcus
aureus,ATCC 25923) and Gram-negative (Escherichia coli, ATCC 25922 and Salmonella
typhi, MTCC 734) bacteria via disk diffusion method.20Among the tested compounds 7a, 7b,
7e, & 7g were found active against S. aureus, 7e against E.coli and 7g and 7h against S.
typhiwith inhibition zone in the range of 8-20mmin different patterns(Table 4). The
compounds 7h & 7k inhibition zone were in the range of 17±1mm & 20±2 mm respectively,
compared to standard vancomycin, 15±1. No substituent is present on imidazopyridine
moiety of 4a derivative showed inhibition zone of 14±1 mm. The other derivatives are
substituted showed different inhibition zone value that indicated that activity is depending on
type & position of the substituent. In 7h derivative fluorine is present on phenyl ring of
imidazopyridine with 7,7-dimethyl-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile,
found less active than 7k but more active than 7a. When only fluorine substituent is attached
to phenyl ring of imidazopyridine ring (7k), the activity was found better than derivatives 7a
and 7h.
Table 4. Antimicrobial activity evaluation of 7a to 7k derivatives against Gram-positive and
Gram-negative bacterial strains using a disc diffusion test
Inhibition zone (mm)
Compound
7a
S. aureus(ATCC 25923)
E.coli(ATCC 25922)
0
S.typhi(MTCC 734)
0
14 ± 1.0#
7b
7c
7d
7e
11 ± 2.0
NA
0
0
NA
NA
0
0
0
11 ± 2.0
11 ± 3 .0
0
7f
0
0
0
0
7g
12 ± 1.0
8.00 ± 2
17 ± 1.0#
0
7h
0
9.00 ± 1
7i
0
0
7j
7k
NA
NA
0
NA
0
20 ± 2.0#
Vancomycin
15 ± 1.0
-
-

Ciprofloxacin
-
22±1
20 ±1
Ciprofloxacin: positive control used for Gram-negative strains. Vancomycin: positive control
used for Gram-positive strains. NA: not active concentrations. Mean diameters of inhibition
zones of the test samples were calculated from three independent experiments. (#) p < 0.05
compared to control.
After measuring the inhibition zone, minimum inhibitory concentrations (MIC) assay was
conducted for the determination of the lowest concentration of compounds require to inhibit
the visible bacterial growth. The compounds 7a, 7h, and 7k inhibited the growth of Gram-
positive (S. aureus) (Table 5). The substituent fluorine is present on the para position of the
phenyl ring of imidazopyridine of 7h & 7k derivatives showed that fluorine was contributing
for activity. 4h was found better than 7k in inhibiting bacterial growth, the former has
dimethyl substituent on cyclohexanone ring which was absent in 7k derivative.
Table 5. Minimum inhibitory concentrations (MICs) for a series of compounds 7a-7k
Compound
7a
S. aureus
125.00
E.coli
NA
S. typhi
NA
7b
7c
7d
7e
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
7f
7g
7h
7i
NA
NA
7.8
NA
NA
NA
NA
NA
NA
-
NA
NA
NA
NA
NA
NA
-
NA
NA
31.25
3.00
-
7j
7k
Vancomycin
Ciprofloxacin
0.80
0.80
Ciprofloxacin: positive control used for Gram-negative strains. Vancomycin: positive control
used for Gram-positive strains. NA: not active at concentrations up to 400 µg/ml
The minimal bactericidal concentration (MBC) values were also determined for 7a, 7h & 7k.
MBC is the lowest concentration of compounds required for microbial death (Table 6). The
ratio of MBC/MIC indicates the type of antimicrobial agent i.e., bactericidal or bacteriostatic.

The ratio of MBC/MIC ≤2 indicate bactericidal effect whereas a ratio of MBC/MIC ≥4 show
bacteriostatic effect [21].
Interestingly, the MBC/MIC ratio of the active derivatives 7a and 7k were ≤2 against Gram-
positive (S. aureus), suggesting that the molecules can be classified as a bactericidal agent
against Gram-positive bacteria. Whereas the MBC/MIC ratio of the active derivative 7h was
≥4 against Gram-positive (S. aureus), suggesting that the compounds can be classified as a
bacteriostatic agent against Gram-positive bacteria.
Table 6.Minimal bactericidal concentration (MBC) of the active compounds 7a, 7h, and 7k
MBC (µg/m1)
Compound
7a
S. aureus
250.00
E.coli
NA
S.typhi
NA
7h
7k
31.25
62.50
NA
NA
NA
NA
Hemolysis tests on humanerythrocytes performed to a series of compounds ‘7a-7k’. No
hemolytic effect was observed for the compounds after 3 hours of incubation. As hemolysis
rate is less than 10% indicate that compounds are non-cytotoxic to erythrocytes according to
literature report (Figure 4)[22].The result of the study showed that there is a need of
modification in derivatives with respect to type & position of the substituent for improving
antibacterial profile them. These antibacterial results show that there is a scope and need for
structural modification of compounds to find more active derivatives against bacterial strains.

Figure 4:Haemolytic profile of compounds (7a to 7k) through haemolysis assay. The
hemolysis obtained from Triton-X 100 was taken as 100% hemolysis and that with respective
concentration of DMSO was taken as negative control. The haemolytic percentage was
observed to be below 10% for all the compounds.
3. Experimental Section
3.1 Materials and methods
All chemicals and reagents were purchased from commercial sources and were used for the
experiments without purification. Reactions were monitored by silica gel coated aluminum
plate containing 60 F254TLC. Visualization was achieved by UV light and 2,4-dinitrophenyl
hydrazine (for aldehyde). Ultrasonication was achieved using Digital Ultrasonic cleaner with
a frequency of 40 kHz & a nominal power of 150 W. Column chromatography was
1
performed with silica gel (60-120 mesh). H were recorded on Bruker DRX 300 and 400
Spectrometers at 300, 400 MHz (1H) and 13C NMR was recorded on Joel 500 NMR
spectrometer at 125 MHz. Experiments were recorded in CDCl3 and DMSO-d6. Chemical
shifts (d) are reported in ppm downfield from an internal TMS standard. Splitting patterns are
described as singlet (s), doublet (d), triplet (t) and multiplet (m). J values are given in Hertz.
ESI mass spectra were recorded on ThermoLcq Advantage Max spectrometer. IR spectra

were recorded on a BRUKER IR spectrophotometer. Melting points were recorded on lab
melting point apparatus and are uncorrected.
3.2 General procedure for synthesis of 2-phenylimidazo[1,2-a]pyridine-3-carbaldehyde
(4):[47]
2-Aminopyridine (0.094 g, 1mmol) was dissolved in ethanol and sodium bicarbonate (0.126
g, 1.5mmol) was added to above solution with stirring at room temperature for 5min. After
5min phenacyl bromide (0.023 g, 1.2mmol) was added. The reaction mixture was heated at
65o for 4-5 h. Completion of the reaction was monitored by TLC. After completion of the
reaction, the reaction mixture was cooled and ethanol was evaporated by rotavapor and then
quenched with water (10ml). The precipitate formed was filtered using Whatman paper. The
precipitate was washed with ice-cold water and recrystallized from ethanol to obtain pure 2-
Phenyl-imidazo[1,2-α]pyridine derivatives.
After this formylation of the 2-Phenyl-imidazo[1,2-α]pyridine was performed using
o
Vilsmeier reagent. It was prepared at 0–4 C by dropping POCl3 (0.37 ml, 4mmol)into a
stirred solution of DMF(0.62 ml, 8mmol). 2-Phenylimidazo [1, 2-α] Pyridine derivatives
(0.194g, 1mmol)in DMF was added to Vilsmeier reagent maintaining stirring and cooling.
The reaction mixture was kept at RT for 2h and then the reaction mixture was refluxed at
90oC for 24 h. After completion of the reaction (monitored by TLC with using DNP), the
reaction mixture was cooled and poured onto ice. The crude product was collected by
filtration and crystallized from EtOH (5 mL) in order to obtain the pure product.
3.3 General procedure for the synthesis of 7a-7k
Conventional method
1,3-Cyclohexanedione/5,5-Dimethyl-1,3-cyclohexanedione (0.14 g, 1mmol) was dissolved
inethanol: GAAS (1ml:5ml).Then, 2-Phenyl–imidazo [1,2-α] pyridine-3-carbaldehyde (0.2
g,1 mmol) and malononitrile (0.063 ml, 1 mmol) was added to the above reaction mixture.
The reaction mixture was stirred at 100 ºC under reflux conditions in a round-bottomed flask
fitted with a reflux condenser. After completion of the reaction (the progress of the reaction
was monitored by TLC using n-hexane: ethylacetate as eluent), the reaction mixture was
cooled and then extracted with water and ethyl acetate. The ethyl acetate layer was dried over
anhydrous sodium sulfate and evaporated on a rotavapor to get the crude product. The crude
product was purified by silica gel (60-120 mesh) column chromatography using n-hexane and
ethyl acetate as eluent to afford the pure product.

Ultrasonication Method
1,3-Cyclohexanedione/5,5-Dimethyl-1,3-cyclohexanedione (0.14 g, 1mmol) was dissolved
inethanol:GAAS (1ml:5ml).Then, 2-Phenyl–imidazo [1,2-α] pyridine-3-carbaldehyde (0.2 g,1
mmol) and malononitrile (0.063 ml, 1 mmol) was added to the above reaction mixture. The
reaction mixture was kept at 80 ºC in a pressure tube under ultrasonication. After completion
of the reaction (the progress of the reaction was monitored by TLC using n-hexane:
ethylacetate as eluent), the reaction mixture was cooled and then extracted with water and
ethyl acetate. The ethyl acetate layer was dried over anhydrous sodium sulfate and evaporated
on a rotavapour to get the crude product. The crude product was purified by silica gel (60-120
mesh) column chromatography using n-hexane and ethyl acetate as eluent to afford the pure
product.
3.4 Antimicrobial Assays
Determination of Diameters of Inhibition Zones
Inhibition zone diameters were determined by the disk diffusion method as described by
Tamokou and co-workers (2011)[48]. Stock solutions of compounds were prepared in 100 %
dimethylsulfoxide (DMSO) solution (Sigma-Aldrich) at a concentration of 10 mg/ml.
Bacterial strains used for the antimicrobial study were Staphylococcus aureus(ATCC 25923)
and Escherichia coli(ATCC 25922) and Salmonella typhi(MTCC 734). The
bacterialsuspension (1.0 x 106CFU m/l)) wasinoculated on a Petri dish containing 1.5 %
Nutrient agar, after drying; two concentrations of (400 and 200µg) of the compound were
applied ontothem. After 24 h at 37 °C, the inhibition zone formed that was measured in
mm.Vancomycin and Ciprofloxacin (Thermo Scientific™) were used as positive controls for
bacteria. Dimethylsulfoxide solution (100%) was used as a negative control. The data were
expressed as the mean ± SEM of three independent experiments. Statistical comparisons
between samples were performed using one-way ANOVA followed student’s t test.
Differences with p <0.05 were taken as significant.
Minimum inhibitory concentration (MIC)
All compounds were subjected to MIC analyses by the microdilution methodfor determining
the minimum concentration of compounds that inhibit bacterial growth. A 100ml volume of
two-fold dilutions (400–1.625mg m/l) was taken in each well of 96-well microplate. No
detectable effect on bacterial growth was observed at 5% DMSO concentration. Then, 100 ml

ofbacterial inoculum containing 106 CFU m/l was added. Negative control was prepared
using the inoculum without compound, whereas vancomycin and ciprofloxacin were used
aspositive controls. The microplates were incubated at 37 °C for 24 h and the result was
determined by visual reading of thelowest concentration with no detectable turbidity
(growth)[49].
Minimal bactericidal concentration (MBC)
To determine MBC value, the culture medium showing no visible growth transfer to agar
plate, incubated for 24h at 37 °C. MBC is defined as theminimum concentration of
compound that showed a 99.9%reduction of the original growth. The MBC was determined
in the well treated with thelowest compound concentration[49].
3.5 Haemolysis assay
Sample erythrocytes were obtained from a healthy human subject, washed 3times with PBS
(pH 7.4) by centrifugation and suspended in thesame buffer according to Sathler and
collaborator (2014). 200µg/ml of the concentration of the compound was incubated with the
erythrocyte suspension for 3 h at 37°C. The optical density of supernatant at 540 nm was
determined to calculate the erythrocytes lysis. 1% Triton X-100 was taken as a positive
control. A value of less than 10% indicates that compounds are nontoxic to erythrocyte
membrane hence, haemocompatible [50]. Assay was performed in triplicates. The percent
haemolysis is calculated using the following formula:
Percent haemolysis =100× [(A540 sample - A540 negative control) ÷ (A540 positive control-
A540 negative control)]
3.6 Spectroscopic data of all compounds
2-amino-7,7-dimethyl-5-oxo-4-(2-phenylimidazo[1,2-a]pyridin-3-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile(7a)
Off white solid (Yield 88%); mp=148-150oC; FT-IR(cm-1) 3617, 2332, 2224, 1668,
1525,1147, 1036; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.85- 0.89 (m, 6 H) 1.23-1.34 (m, 2
H) 2.10-2.18 (m, 1H) 2.19 - 2.20 (m, 1 H) 5.18 (s, 1 H) 6.96–6.99 (m, 2 H) 7.25 - 7.29 (m, 2
13
H)7.38 - 7.47 (m, 2 H) 7.71 (br s, 1H) 7.71-7.72 (br s, 2 H) 7.84 - 8.10 (m, 1 H); C-NMR
(DMSO-d6, 125 MHz): δ= 196.37, 159.77, 158.91, 144.50, 143.76, 135.61, 129.47, 128.70,
128.26, 127.93, 125.50, 124.54, 123.81, 119.38, 118.05, 117.14, 113.16, 112.16, 53.43,

50.48, 31.97, 31.70, 27.72, 27.34, 25.70; MS-ESI calculated for C25H22N4O2 (M+H)+: 411.18,
found 411; HRMS (ESI) m/z [M+H]+calculated for C25H22N4O2; 411.1822 found 411.1812.
2-amino-4-(2-(4-methoxyphenyl)-8-methylimidazo[1,2-a]pyridin-3-yl)-7,7-dimethyl-5-
oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile(7b)
Yellow solid (Yield 80%); mp=156-158oC; FT-IR(cm-1) 3369, 2351, 2189, 1669, 1492,1160,
1
1030; H NMR (400 MHz, DMSO-d6) δ ppm 0.82 - 0.98 (m, 5 H) 0.98 - 1.11 (m, 2 H) 1.99
(br s, 1 H) 2.02 - 2.12 (m, 1 H) 2.13 - 2.36 (m, 1 H) 2.46 - 2.54 (m, 5 H) 2.54 - 2.74 (m, 1 H)
3.35 (s, 7 H) 3.82 (s, 3 H) 5.11 (s, 1 H) 6.85 - 6.89 (m, 1 H) 6.89 – 6.97 (m, 1 H) 7.00 - 7.04
(m, 3 H) 7.06 (br s, 2 H) 7.23 - 7.88 (m, 1 H) 7.88 - 7.90 (m, 1 H); 13C-NMR (DMSO-d6, 125
MHz): δ= 196.47, 168.38, 167.22, 159.79, 158.87, 141.57, 139.50, 131.04, 127.69, 118.83,
118.03, 114.75, 114.33, 111.12, 108.49, 101.97, 55.77, 50.48, 46.56, 33.05, 32.06, 28.23,
22.60, 17.10; MS-ESI calculated for C27H26N4O2 (M+H)+: 455.20, found 455; HRMS (ESI)
m/z [M+H]+calculated for C27H26N4O2; 455.2084 found 455.2074.
2-amino-4-(2-(4-fluorophenyl)-7-methylimidazo[1,2-a]pyridin-3-yl)-7,7-dimethyl-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile(7c)
Yellow solid (Yield 62%); mp=158-160oC; FT-IR(cm-1) 3319, 2950, 2530, 2142, 1675,
1
1432,1116, 1030; H NMR (400 MHz, DMSO-d6) δ ppm 0.98 (s, 6 H) 2.18 (s, 2 H) 2.38 -
2.40 (m, 2H) 5.32 (br s, 2 H) 5.78 (s, 1 H) 6.95 (dd, J=6.91, 1.53 Hz, 1 H) 7.29 - 7.37 (m, 2
13
H) 7.93 - 8.01 (m, 2 H) 8.40 (s, 1 H) 8.51 (d, J=6.97 Hz, 1 H); C-NMR (DMSO-d6, 125
MHz): δ= 196.77, 167.22, 165.86, 165.44, 156.63, 155.41, 144.72, 135.26, 132.06, 131.30,
117.88, 116.25, 115.62, 115.47, 115.26, 115.00, 111.11, 110.90, 50.48, 50.26, 32.28, 32.06,
29.60, 27.48, 27.22, 21.18; MS-ESI calculated C26H23 FN4O2 (M+H)+: 443.18, found 443;
HRMS (ESI) m/z [M+H]+calculated for C26H23 FN4O2; 443.1884 found 443.1852.
2-amino-7,7-dimethyl-4-(8-methyl-2-phenylimidazo[1,2-a]pyridin-3-yl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile(7d)
White solid (Yield 82%); mp=172-174oC; FT-IR(cm-1) 3335, 2945, 2832, 2183, 1681,
1416,1113, 1021; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.82 – 1.02 (m, 6 H) 1.23 - 1.35 (m,
3 H) 2.09 (br s, 1 H) 2.19 - 2.21 (m, 1 H) 2.51 - 2.54 (m, 1 H) ) 2.54 - 2.58 (m, 1 H) 5.14 (s,
1 H) 6.87 - 6.91 (m, 2 H) 6.91 (s, 1H) 7.08-7.10 (m, 1H) 7.25-7.39 (m, 3 H) 7.59 (br s, 1 H)
7.62 - 7.80 (m, 1 H) 7.80 - 7.95 (m, 1 H); 13C-NMR (DMSO-d6, 125 MHz): δ= 196.33,
159.73, 158.66, 144.77, 143.95, 135.77, 129.57, 128.64, 128.23, 127.85, 123.11, 121.58,
120.26, 113.06, 112.16, 108.83, 53.52, 50.50, 31.99, 28.81, 27.62, 27.32, 25.78, 17.17; MS-

ESI calculated C26H24N4O2 (M+H)+: 425.19, found 425; HRMS (ESI) m/z [M+H]+calculated
for C26H24N4O2; 425.1978 found 425.1972.
2-amino-4-(2-(4-fluorophenyl)-8-methylimidazo[1,2-a]pyridin-3-yl)-7,7-dimethyl-5-oxo-
5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile(7e)
Off white solid (Yield 64%); mp=159-161oC; FT-IR(cm-1) 3318, 2943, 2522, 2043, 1659,
1
1449, 1114, 1021; H NMR (400 MHz, METHANOL-d4) δ ppm 1.63-1.87 (m, 7 H) 1.95 -
2.21 (m, 3 H) 2.85 - 3.12 (m, 3 H) 3.24 - 3.35 (m, 5 H) 4.19 (br s, 3 H) 5.92 (s, 1 H) 7.69 -
7.85 (m, 2 H) 7.86 - 8.01 (m, 1H) 8.01 - 8.47 (m, 4 H) 8.47 - 8.52 (m, 1 H) 8.80 (s, 1 H); 13C-
NMR (DMSO-d6, 125 MHz): δ= 196.56, 163.33, 161.51, 151.20, 144.26, 141.77, 131.75,
131.69, 125.66, 124.41, 121.86, 121.86, 120.46, 119.93, 117.83, 117.09, 115.56, 113.87,
101.27, 50.47, 33.02, 31.97, 28.09, 27.39, 25.77, 17.17; MS-ESI calculated C26H23 FN4O2
(M+H)+: 443.18, found 443; HRMS (ESI) m/z [M+H]+calculated for C26H23 FN4O2; 443.1884
found 443.1852.
2-amino-4-(2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile(7f)
Off white solid (Yield 78%); mp=154-156oC; FT-IR(cm-1) 3323, 2948, 2562, 2178, 1678,
1442, 1113, 1021; 1H NMR (400 MHz, DMSO-d6) δ ppm 0.81 - 0.98 (m, 6 H) 0.98 - 1.15 (m,
2 H) 2.04–2.12 (m, 2 H) 5.16 (s, 1 H) 6.96 - 7.04 (m, 4 H) 7.25 - 7.29 (m, 3 H) 7.60 – 7.70
13
(m, 2 H) 7.89 (s, 1H); C-NMR (DMSO-d6, 125 MHz): δ= 196.40, 164.44, 159.75, 159.32,
158.73, 144.26, 143.21, 130.70, 127.86, 125.54, 124.40, 123.71, 119.38, 117.83, 116.85,
114.20, 113.77, 113.07, 55.67, 50.55, 32.00, 31.22, 28.73, 27.33, 25.77, 21.59; MS-ESI
calculated C26H24N4O3(M+H)+: 441.19, found 441.
2-amino-7,7-dimethyl-4-(6-methyl-2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile(7g)
Pale Yellow solid (Yield 76%); mp=152-154oC; FT-IR(cm-1) 3421, 3204, 2960, 2358, 2194,
1
1656, 1499, 1144, 1035; H NMR (400 MHz, DMSO-d6) δ ppm 0.83 – 1.03 (m, 6 H) 1.89 –
2.08 (m, 2 H) 2.27 - 2.50 (m, 2 H) 5.13 (s, 1 H) 6.99 (br s, 1 H) 7.07 - 7.22 (m, 2H) 7.22 -
13
7.37 (m, 3 H) 7.38 - 7.52 (m, 2 H) 7.83 (br s, 1 H); C-NMR (DMSO-d6, 125 MHz): δ=
196.44, 159.85, 158.82, 142.23, 139.94, 138.68, 131.69, 129.52, 129.38, 129.20, 124.10,
118.50, 117.72, 114.20, 112.96, 101.73, 50.28, 46.18, 33.04, 32.17, 28.18, 24.99, 21.43,
18.40; MS-ESI calculated C27H26 N4O2 (M+H)+: 439.21, found 439; HRMS (ESI) m/z
[M+H]+calculated forC27H26 N4O2; 439.2135 found 439.2108.

2-amino-4-(2-(4-fluorophenyl)imidazo[1,2-a]pyridin-3-yl)-7,7-dimethyl-5-oxo-5,6,7,8-
tetrahydro-4H-chromene-3-carbonitrile(7h)
Off white solid (Yield 62%); mp=156-158oC; FT-IR(cm-1) 3313, 2943, 2521, 2223, 2044,
1
1655, 1448, 1114, 1021; H NMR (400 MHz, DMSO-d6) δ ppm 0.86 - 1.02 (m, 6 H) 1.15-
1.35 (m, 2 H) 2.09-2.12 (m, 2 H) 5.15 (s, 1 H) 6.97-7.01 (m, 2 H) 7.27-7.31 (m, 5 H) 7.62-
13
7.71 (m, 2 H) 7.99 (m, 1 H); C-NMR (DMSO-d6, 125 MHz): δ= 196.54, 163.18, 161.29,
159.68, 144.41, 143.48, 131.41, 125.69, 124.69, 123.82, 119.81, 119.34, 118.07, 117.05,
115.50, 113.23, 112.19, 108.64, 53.36, 50.50, 31.98, 28.68, 27.95, 27.40, 25.70; MS-ESI
calculated for C25H21FN4O2(M+H)+: 429.17, found 429; HRMS (ESI) m/z [M+H]+calculated
forC25H21FN4O2; 429.1728 found 429.1725.
2-amino-7,7-dimethyl-5-oxo-4-(2-(p-tolyl)imidazo[1,2-a]pyridin-3-yl)-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile(7i)
Pale Yellow (Yield 74%); mp=150-152oC; FT-IR(cm-1) 3319, 2950, 2530, 2142, 1675, 1432,
1116, 1030; 1H NMR (300 MHz, DMSO-d6) δ ppm 0.62 - 1.08 (m, 6 H) 1.83 - 2.12 (m, 2 H)
2.17 (br s, 1 H) 2.27 - 2.41 (m, 3 H) 2.51 (m, 1 H) 5.15 (s, 1 H) 6.96 - 7.11 (m, 1 H) 7.12 -
7.39 (m, 5 H) 7.48 - 7.61 (m, 1 H) 7.82-7.85 (m, 1H) 8.72 - 8.84 (m, 1 H)); 13C-NMR
(DMSO-d6, 125 MHz): δ= 196.34, 164.42, 159.75, 144.53, 137.15, 132.75, 129.34, 128.77,
125.52, 124.39, 123.74, 119.35, 118.00, 113.08, 112.08, 108.77, 53.43, 50.52, 31.99, 28.78,
28.15, 27.82, 27.31, 21.39; MS-ESI calculated for C26H24N4O2(M+H)+: 425.19, found 425;
HRMS (ESI) m/z [M+H]+calculated forC26H24N4O2; 425.1978 found 425.1975.
2-amino-4-(2-(4-methoxyphenyl)imidazo[1,2-a]pyridin-3-yl)-5-oxo-5,6,7,8-tetrahydro-
4H-chromene-3-carbonitrile(7j)
White solid (Yield 82%); mp=164-166oC; FT-IR(cm-1) 3350, 2946, 2521, 2187, 1631, 1416,
1119, 1030; 1H NMR (400 MHz, DMSO-d6) δ ppm 2.28 (s, 2 H) 2.41 - 2.44 (m, 4 H) 3.86 (s,
3 H) 6.04 (s, 1 H) 7.18 (d, J=8.80 Hz, 2 H) 7.47 (ddd, J=6.63, 5.23, 2.93 Hz, 1 H) 7.85 - 7.90
(m, 5 H) 8.67 (s, 1 H) 8.84 (d, J=6.72 Hz, 1 H); 13C-NMR (DMSO-d6, 125 MHz): δ= 196.37,
157.52, 156.85, 144.18, 137.10, 135.47, 133.18, 129.31, 128.97, 125.42, 124.41, 122.32,
121.46, 119.83, 117.93, 117.36, 113.60, 112.81, 54.15, 50.82, 36.69, 27.08, 21.41, 20.05;
MS-ESI calculated for C24H20N4O3(M+H)+: 413.16, found 413.
2-amino-4-(2-(4-fluorophenyl)imidazo[1,2-a]pyridin-3-yl)-5-oxo-5,6,7,8-tetrahydro-4H-
chromene-3-carbonitrile(7k)
Yellow solid (Yield 65%); mp=165-167oC; FT-IR(cm-1) 3321, 2943, 2529, 2043, 1630, 1449,
1114, 1021; 1H NMR (400 MHz, DMSO-d6) δ ppm 1.24 - 1.40 (m, 1 H) 1.88 - 2.21 (m, 4 H)

2.52 - 2.70 (m, 1 H) 5.12 (s, 1 H) 6.99 (td, J=6.85, 1.10 Hz, 3 H) 7.09 - 7.29 (m, 4 H) 7.30
13
(ddd, J=8.99, 6.72, 1.04 Hz, 2 H) 7.58 -7.89 (m, 1 H); C-NMR (DMSO-d6, 125 MHz): δ=
196.58, 163.18, 161.24, 143.31, 132.38, 131.43, 131.37, 124.66, 119.83, 117.53, 115.41,
115.41, 115.25, 112.78, 60.80, 36.72, 30.14, 27.06, 20.12; MS-ESI calculated for
C23H17FN4O2(M+H)+: 401.14, found 401; HRMS (ESI) m/z [M+H]+calculated
forC23H17FN4O2; 401.1414 found 401.1411.
4. Conclusions
In summary, here we have reported a simple and efficient one-potsyntesis of novel fused
bisheterocyclic compounds (7a-7k) in an aqueous solution of gluconic acid. The protocol
developed is greener, easy to operate, and cost effective. The experiment was performed
using both conventional and ultrasonication methodology in which the later has proven more
efficient for the synthesis. The broad substrate scope, high yield of compounds and solvent
reusability makes this a promising method for synthesis of imidazopyridine bearing pyran
derivatives.Furthermore, the compounds were evaluated in vitro for their antimicrobial
activity. Some compounds of 7a-7kseries were found active in inhibiting the growth of
bacteria. In addition, MIC & MBC values were also determined along with the
hemocompatibility test. Further, the study would be carried out for structural variation of
derivatives in order to enhance antimicrobial activity.
Acknowledgments
The authors are thankful to Dr. S. J. S. Flora, Director for his support in carrying out this
research work. The authors would like to acknowledge the Department of Pharmaceuticals,
Ministry of Chemicals & Fertilizers, Govt. of India for financial support, SAIF, CSIR-CDRI,
Lucknow for the spectral analysis, Mr. Ramesh Singh, Department of Chemistry, Indian
Institute of Technology, Kanpur for single crystal X-ray analysis and Biotechnology
Division, Defense Research and Development Establishment, Gwalior,M.P. India. NIPER-R
communication number is NIPER-R/Communication/065.
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One pot synthesis of novel imidazopyridine bearing pyran bis-heterocycles.
X-ray single crystal structure of 7i.
MIC showed that 7a, 7h & 7k from a series 7a-7k inhibited the growth of S. aureus.
7a, 7k derivatives are bactericidal and 7h is bacteriostatic.
Haemolysis results showed that compounds are non-cytotoxic to erythrocytes.

Declaration of interests
The authors declare that they have no known competing financialinterestsor personal relationships that
could have appeared to influence the work reported in this paper.