Facile synthesis, structural evaluation, antimicrobial activity and synergistic effects of novel imidazo[1,2-a]pyridine based organoselenium compounds

Article abstract of DOI:10.1016/j.ejmech.2016.07.076

A simple and efficient method has been described to synthesize the hitherto unknown imidazo[1,2-a]pyridine selenides (5a-l) by reaction of 2-chloroimidazo [1,2-a]pyridines with aryl/heteroaryl selenols, generated in situ by reduction of various diselenide

Full text of DOI:10.1016/j.ejmech.2016.07.076

Accepted Manuscript
Facile synthesis, structural evaluation, antimicrobial activity and synergistic effects of
novel imidazo[1,2-a]pyridine based organoselenium compounds
Sanjeev Kumar, Nidhi Sharma, Indresh K. Maurya, Aman K. Bhasin, Nishima
Wangoo, Paula Brandão, Vítor Félix, K.K. Bhasin, Rohit K. Sharma
PII:
S0223-5234(16)30641-9
10.1016/j.ejmech.2016.07.076
EJMECH 8794
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 June 2016
Revised Date: 28 July 2016
Accepted Date: 31 July 2016
Please cite this article as: S. Kumar, N. Sharma, I.K. Maurya, A.K. Bhasin, N. Wangoo, P. Brandão,
V. Félix, K.K. Bhasin, R.K. Sharma, Facile synthesis, structural evaluation, antimicrobial activity and
synergistic effects of novel imidazo[1,2-a]pyridine based organoselenium compounds, European Journal
of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.076.
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Graphical Abstract

ACCEPTED MANUSCRIPT
Facile synthesis, structural evaluation, antimicrobial activity and synergistic effects of
novel imidazo[1,2-a]pyridine based organoselenium compounds
Sanjeev Kumar a, Nidhi Sharma a, Indresh K. Maurya b, Aman K. Bhasin a, Nishima Wangoo c,
Paula Brandão d, Vítor Félixd,e, K. K. Bhasin a,*, and Rohit K. Sharmaa,*
aDepartment of Chemistry & Centre for Advanced Studies in Chemistry, Panjab University,
Chandigarh, 160014 India
bDepartment of Microbial Biotechnology, Panjab University, Chandigarh, 160014 India
cDepartment of Applied Sciences, University Institute of Engineering & Technology (U.I.E.T),
Panjab University, Chandigarh, 160014 India
dDepartment of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, 3810-
193 Aveiro, Portugal
eDepartment of Medical Sciences, iBiMED – Institute of Biomedicine, University of Aveiro,
3810-193 Aveiro, Portugal
*Corresponding authors
(R.K.S.) Tel: +91 172 2534409. E-mail: rohitksg@pu.ac.in
(K.K.B) Tel: +91 172 2534407. E-mail: kkbhasin@pu.ac.in
ABSTRACT: A simple and efficient method has been described to synthesize the hitherto
unknown imidazo[1,2-a]pyridine selenides (5a-l) by reaction of 2-chloroimidazo[1,2-a]pyridines
with aryl/heteroaryl selenols, generated in situ by reduction of various diselenides with
hypophosphorous acid. The crystal structures of 3-nitro-2-(phenylselanyl)-imidazo[1,2-
a]pyridine (5a), 2-(mesitylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5d) and 3-nitro-2-(pyridin-2-
ylselanyl)-imidazo[1,2-a]pyridine (5e) were confirmed by X-ray crystallography and the DFT
calculations were performed to determine various structural parameters which were correlated
with the X-ray crystal structures. The synthesized compounds were subjected to antimicrobial
evaluation and it was found that compounds 5a and 5j were active against gram negative
bacterium E. coli whereas compound 5e was active against different fungal strains. Time kill
assay was performed to understand the microbial activity of synthesized organoselenium
compounds and the toxicity of these compounds was evaluated against human cell lines.
Synergistic effects of active compounds 5a and 5e were tested with existing antibiotic drugs
which exhibited that the antibiotic combination with synthesized organoselenium compounds
efficiently enhanced the antimicrobial activity.
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1. Introduction
Organoselenium chemistry has been an area of continuous research since selenium and its
compounds were identified as micronutrient for several bacteria, mammals and birds [1]. The
relevance of organoselenium compounds has seen an upward thrust particularly after 1970’s with
discovery of many new attractive compounds with various important synthetic and biological
applicability [2]. Several synthetic methods have been developed to synthesize organoselenium
compounds till date, which generally include the use of expensive catalysts and various transition
metals routinely employing harsh reaction conditions and long reaction times [3]. This has led to
continuous search for finding better, inexpensive and environment friendly approaches for
synthesis of organoselenium compounds. The recent efforts in this regard have yielded an
efficient and eco-friendly method in absence of solvent under microwave irradiation to obtain
selenoesters [4]. Similarly, the use of hypophosphorous acid in glycerol as an efficient
environment friendly system for reducing diselenides in the preparation of monoselenides has
also been described [5]. In another example, a metal free approach was followed to achieve
synthesis of unsymmetrical selenides in different ionic liquids [6]. Moreover, unsymmetrical
arylselanyl anilines have been reported to be synthesized under metal and base free conditions
[7].
In context of the biological applications of organoselenium compounds, these have been
reported to mimic the function of antioxidant selenoenzyme Glutathione peroxidase (Gpx) [8]. In
a recent report, novel ebselenol compounds have been identified as multifunctional antioxidants
with regenerable radical-trapping and enhanced hydroperoxide-decomposing capabilities [9].
Selenium has been found to be present in number of selenoproteins and enzymes as
selenocysteine residue at the active site [10]. In fact, selenocysteine is found to be 21st
proteinogenic amino acid which has active selenol group present in active site of Gpx and similar
enzymes [10-11]. Despite the perceived toxicity aspects related to selenium based compounds, a
number of selenium containing molecules are being developed as novel pharmacological agents
[12-13] including anticancer [14,15], anti-inflammatory [16], antitumour [17] and membrane
permeabilization [18] agents. The new age drugs such as Ethaselen (anticancer) and Ebselen
(anti-inflammatory) contain selenium and are termed among the important discoveries in
organoselenium and medicinal chemistry [19].
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The application of selenium enriched compounds containing one or two heteroaromatic
moieties have been observed to be more in biological systems compared to their non-heteroaryl
analogues [12]. This observation is based on the experimental evidences which suggest that
many heteroaryl moieties such as pyrimidine, pyridine, quinoline, imidazole, ferrocene, indole
and pyrazole in association with selenium exhibit moderate to good biological results [20].
However, imidazo[1,2-a]pyridine, which is an aromatic heterocycle with bridgehead nitrogen
atom [21], has not been sufficiently explored for applications resulting from conjugation with
selenium, although chemistry of unconjugated imidazo[1,2-a]pyridine has been intensively
investigated in last few decades [22]. Recently, solvent- and metal-free chalcogenation of
imidazo[1,2-a]pyridines with regioselectivity at C-3 position has been effectively reported [23].
The imidazo[1,2-a]pyridine based compounds are of immense interest as these have been
reported to be exhibiting wide range of biological effects, notably in form of antiviral, antifungal,
antimalarial, antiparasitic, herbicide, anticancer, anti-inflammatory and antibacterial activities
[24]. Keeping the focus on greener strategies for synthesis of organoselenium derivatives, herein
we report the synthesis and applications of novel imidazo[1,2-a]pyridine based organoselenium
compounds. To best of our knowledge, this is the first report describing the chemical synthesis,
structural determination, computational analysis, antimicrobial evaluation and synergistic effects
of novel nitro substituted imidazo[1,2-a]pyridine derived organoselenium compounds.
2. Results and discussions
2.1 Chemistry
The synthetic route adopted for preparation of organoselenium compounds based on
imidazo[1,2-a]pyridine herein is depicted in Scheme 1. Initially, 2-chloroimidazo[1,2-a]pyridine
was synthesized in a two-step procedure from 2-aminopyridine which included the first step
involving the condensation reaction of 2-aminopyridine with chloroacetic acid in the presence of
triethylamine in water as a solvent to afford 2-(2-iminopyridin-1(2H)-yl)acetic acid (2a) [21].
Compound 2a was further refluxed with phosphorous oxychloride to facilitate the intramolecular
cyclisation for preparation of 2-chloroimidazo[1,2-a]pyridine (3a) in good yield. It was further
observed that nucleophilic substitution of chlorine atom at C-2 position in 3a is not entirely a
feasible process as this position is not adequately nucleophilic in nature [25]. Moreover, the
attack of nucleophile may not allowed either by SN1 (as there is no driving force for chloride to
leave by itself) or SN2 mechanism (as the incoming nucleophile is unlikely to attack from inner
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side of the five membered ring).Thus, the introduction an electron withdrawing group at C-3
position of substituted imidazo[1,2-a]pyridine (3a-b) seemed to be essential for facilitation of
nucleophilic aromatic substitution of chlorine atom. Thus, nitration of compounds (3a-b) was
carried out using nitric acid and sulphuric acid to afford nitro substituted imidazo[1,2-a]pyridine
(4a-b) in excellent yields [26].
Scheme 1. Synthesis of organoselenium derivatives of imidazo[1,2-a]pyridines
A modified procedure of already reported method [5] was further used to cleave the Se-Se
bond of diselenides to generate selenide anion which is sufficiently nucleophilic in nature to
substitute chlorine atom of imidazo[1,2-a]pyridine (4a-b). In this modified procedure, ethanol
was used as a solvent instead of glycerol to improve the yield in our case and also generating
selenol at lower temperature (600C) as compared to earlier reported temperature conditions
(900C). The versatility of the present multi-step synthetic approach was further evaluated by
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using different diselenides [27] such as 2,2’-ditolyl diselenide, 4,4’-ditolyl diselenide, 4,4’-
dianisyl diselenide, 2,2’-dimesityl diselenide, 2,2’-dipyridyl diselenide, 4,4’(N,N’-
dimethylaniline) diselenide and dinaphthyl diselenide to yield desired compounds (5a-l). In
addition, the reaction was also investigated with methyl substituted imidazo[1,2-a]pyridine
which showed negligible effect on yield.
2.2 Crystallographic studies
The structures of compounds 5a, 5d and 5e were determined by single crystal X-ray
crystallography. Suitable single crystal of compounds 5a, 5d and 5e were grown at room
temperature in dichloromethane:hexane (30:70) by slow evaporation for X-ray diffraction. The
crystal lattices of selenium derivatives 5a and 5e are built up from asymmetric unit composed of
a single molecule. Views of asymmetric units of 5a, 5d and 5e are shown in Fig. 1 (i, ii, iii
respectively) with the relevant atomic labeling scheme adopted. The three molecular structures
are overlapping apart the phenyl moieties, as depicted in Fig. 1(iv), the carbon atoms are shown
in green (5a), cyan (5d) and magenta (5e), while the hydrogen, nitrogen and oxygen atoms are
shown in white, blue and red, respectively In fact, the overlay between the nitro-substituted
imidazo[1,2-a]pyridine moieties of 5a with 5d and 5e leads to the root mean square deviations
(ii)
(i)
(iv)
(iii)
Fig. 1. Molecular diagrams of 5a (i) 5d (ii) and 5e (iii) with thermal ellipsoids drawn at the 50%
probability level, Overlap between 5a, 5d and 5e(iv)
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(RMSD) of only 0.049 and 0.067 Å, respectively whereas the RMSD between 5d and 5e is 0.058
Å. Furthermore, the dihedral angle between the phenyl ring and imidazo[1,2-a]pyridine moiety is
similar in compounds 5a (62.6o) and 5e (62.7o).
In contrast, these two moieties adopt an almost orthogonal disposition in 5a with a dihedral
angle of 89.5o, which is probably imposed by the minimization of steric interactions between the
methyl phenyl substituents and the imidazole ring. In addition, packing effects, as depicted in
Fig.1 (iv), can also affect the spatial disposition of the substituents around the selenium center.
The bond lengths and bond angles involving the selenium center (from X-ray diffraction data)
(i)
(ii)
(iv)
(iii)
Fig. 2.Crystal packing features with the C–H···O bonds drawn as green dashed lines of 5a (i) and 5d (iii);
and C–H···Se short contacts as yellow dashed lines of 5a (ii) and 5d (iv) respectively
(i)
(ii)
Fig. 3. Crystal packing features of 5e, with the C–H···O bonds drawn as green dashed lines (i) and C–
H··Se short contacts as yellow dashed lines (ii).
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are listed in Table 1. In all the three crystals, it is consistently observed that Se-C6 bond length to
the phenyl group is slightly longer than Se-C7 bond length to the imidazo[1,2-a]pyridine moiety.
This may be explained on basis of electron withdrawing ability of nitro group which in turn is
attached at C15 position of imidazo[1,2-a]pyridine rendering the Se-C7 bond shorter than the
usual length. The most relevant intermolecular interactions observed in the crystal structures of
compounds 5a, 5d and 5e are C–H···O hydrogen bonds followed by C–H···Se short contacts in
compounds 5a and 5e, and C···Se in 5d. In the crystal structure of compound 5a, the molecules
Table 1. Comparison between theoretical and experimental bond parameters around Se atom in
5a, 5d and 5e
X-ray crystallography
Bond length (Å)
5e* 5a
Theoretical calculation
5a
5d
5d
5e*
Se-C6
Se-C7
1.916 1.932 1.930 1.932 1.934 1.939
1.883 1.880 1.885 1.886 1.882 1.885
Bond Angle (°)
C6-Se-C7
97.0
97.8
96.56
97.8
98.7 105.3
Se-C6-C1 120.4 120.4 115.9 119.8 119.2 107.4
Se-C6-C5 119.4 118.6 120.2 119.8 119.0 128.7
Se-C7-N8 124.2 124.4 123.9 124.4 124.1 126.8
C1=N1 for 5e
*
are self-assembled in dimers (Fig. 2 (i)), through two C–H···O hydrogen with O···C distance of
2.885(2) Å and a C–H···O angle of 114°.In compound 5d, 1-D network of C–H···O hydrogen
bonding interactions are constructed from a centro-symmetric motif composed of four molecules
linked by six hydrogen bonds with O···C distances of 3.172(2) Å and 3.229(2) Å and C–H···O
angles of 134° and 126°, respectively (Fig. 2 (iii)). The crystal structure of 5e also exhibits a 1-D
network of C–H···O hydrogen bonding interactions with C···O distances of 3.322(3) and
3.466(2) Å. In addition, adjacent molecules are disposed in an anti-parallel disposition (Fig. 3
(i)). The crystal structures of 5a and 5e display C–H···Se short contacts with H···Se distances of
3.053 and 3.067 Å, respectively, leading to the formation of a C–H···Se 1-D network of contacts
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(Fig. 2 (ii) and Fig. 3 (ii)). On the other hand, neighboring molecules of compound 5d, adopting
an antiparallel disposition are separated by short C···Se contacts of 3.369 Å (Fig. 2 (iv)).
2.3 Theoretical study
The gas phase structures of synthesized compounds 5a-l were optimized (Supporting
Information, Table S2) using density functional theory (DFT) and employing Becke’s three
parameter exchange functional (B3) in conjunction with the correlation functional proposed by
Lee, Yang and Parr (LYP) and 6-31 G(d) basis set (B3LYP/6-31G(d) [28]. The calculated Se-C
and Se-C7 distances in compounds 5a, 5d and 5e are in good agreement with those obtained
from their crystal structures (Table 1). Also, the calculated C-Se-C bond angle in compounds 5a
and 5d is in close proximity to that obtained from corresponding crystal structure while in the
compound 5e; this angle was observed to be slightly lower in crystal structure. The probable
reason for this slight deviation may be crystal packing effects.
Table 2. C-Se bond lengths (Å) and C-Se-C angle (º) together with HOMO and LUMO energies
of 5a-l calculated using B3LYP/6-31G(d)
Se-C6 Se-C7 C6-Se- HOMO
(Å) (Å) C7 (°) (a. u.)*
LUMO
(a. u.)
Energy
Gap (eV)
1.932 1.886 97.81
1.934 1.884 98.50
1.929 1.887 98.07
1.934 1.882 98.73
-0.22404 -0.08134 3.883
-0.22274 -0.08175 3.836
-0.22386 -0.08103 3.886
-0.21961 -0.08013 3.795
5a
5b
5c
5d
5e
5f
1.939 1.885 105.25 -0.21813 -0.08595 3.596
1.932 1.886 97.95
1.929 1.887 98.00
1.929 1.887 97.84
1.935 1.883 98.60
-0.22152 -0.07756 3.917
-0.22000 -0.07646 3.906
-0.22138 -0.07724 3.922
-0.21703 -0.07687 3.814
5g
5h
5i
1.938 1.885 105.20 -0.21590 -0.08219 3.638
1.933 1.899 100.55 -0.20603 -0.08460 3.304
5j
5k
5l
1.920 1.888 98.59
-0.19478 -0.07545 3.247
*1 a. u. = 27.212eV
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The calculated Se-C bond of all the synthesized compounds (5a-l) have been reported in
Table 2 together with energy values of highest occupied molecular orbital (HOMO) and lowest
unoccupied molecular orbital (LUMO). The C6-Se bond length was found to be in the range of
1.929-1.939 Å and C7-Se bond length between 1.882-1.899 Å, with both in agreement with the
experimental values observed for compounds 5a, 5d and 5e in crystal state. The phenyl, tolyl and
anisyl substituted imidazo[1,2-a]pyridine selenides (5a-c, 5f-h) had higher HOMO-LUMO gap
as compared to pyridine, mesityl, naphthyl substituted selenides (5d-e, 5i-l). A general probable
reason for such an observation is that electron withdrawing groups on phenyl ring tend to
stabilize HOMO thereby lowering its energy while keeping the LUMO energy almost unchanged
whereas electron donating groups tend to destabilize the HOMO state and augment its energy.
The HOMO of compound 5d with three methyl (electron donating) groups on phenyl ring is
higher in energy than compounds 5a, 5b and 5c (with lesser or no electron donating groups). For
similar reasons, the energy gap of derivative 5i (R= CH3, R’= mesityl) is less than corresponding
compounds 5f, 5g and 5h (R’= phenyl, tolyl, anisyl). Also, the presence of methyl group on
imidazo[1,2-a]pyridine ring system slightly increases the energy gap compared to unsubstituted
system because of more destabilization of LUMO than HOMO (Table 2). In case of pyridyl
substituted imidazo[1,2-a]pyridine selenides (5e, 5j)large stabilization of LUMO was observed
which makes HOMO and LUMO to come closer and thereby decreasing the energy gap
considerably. For compounds 5a-j, HOMO is mainly composed of imidazo[1,2-a]pyridine ring
system (Supporting Information, Fig. S2), however, compounds 5k and 5l have naphthyl and N,
N’-dimethylphenyl moieties as the major contributors towards HOMO, respectively (Supporting
Information, Fig. S3). This is one of the main reasons for the relatively large destabilization of
HOMO and also stabilization of LUMO subsequently decreasing the energy gap.
2.4 Antimicrobial Activity
The synthesized compounds (5a-l) were evaluated for antimicrobial activities against
different bacterial and fungal strains (Supporting Information, Table S3-S4). In this context, the
synthesized derivatives 5a (R = H; R’ = phenyl) and 5j (R = CH3; R’ = pyridyl) were observed to
exhibit antibacterial activity with MIC value 2.48 µg/ml and 10.41 µg/ml respectively against
Gram-negative bacteria E. coli (Table 3). These observed activities, in particular the one
observed for compound 5a against E. coli, have been found to be more potent as compared to the
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few reports concerning antimicrobial activity of selenide based compounds in literature so far
[29] and quite similar to the Rifampicin (standard).However, in case of activity against tested
fungal strains, only compound 5e was found to be exhibiting multi-spectrum activity against A.
fumigates, C. krusei, A. niger, and C. parapsilosis with observed MIC values of 9.96, 9.96,19.93
and 19.93 µg/ml respectively (Table 3).
Table 3. Antibacterial and antifungal activities of compounds 5a, 5e and 5j [in MIC[b] (µg/ml)]
Compound
E.
A.
C.
A.
C.
coli[a] fumigatus[a] krusei[a] niger[a] parapsilosis[a]
2.48
>39.77
>39.77 >39.77
9.96 19.93
>41.66 >41.66
>39.77
19.93
5a
5e
5j
>39.89 9.96
10.41 >41.66
>41.66
[a] Standard used: Rifampicin (MIC = 1.02 µg/ml for E. coli); Amphotericin B (MIC = 0.18 µg/ml for A.
fumigatus, MIC = 0.36 µg/ml for C. krusei, MIC = 0.18 µg/ml for A. niger, MIC = 0.18 µg/ml for C.
parapsilosis). [b] MIC: Minimum Inhibitory Concentration
2.4.1 Cytotoxicity
The toxicity effect of the three active relatively active compounds (5a, 5e, 5j) was
evaluated to determine their safety profile against HEK-293 and HeLa cells at their MIC values.
The compounds 5a, 5e, and 5j were found to exhibit less than 25% toxicity against both HeLa
and HEK-293 cells (Fig. S1). Considering the toxicity issues concerning some of the earlier
reported organoselenium analogues, the presently compounds have demonstrated acceptable
mammalian cytotoxicity that is further authenticated by calculating selectivity index (SI) of the
active compounds, which is described as theratio of cytotoxicity to biological activity (SI =
CC50 of HEK-293 or HeLa/IC50 of microorganisms) of compounds against microorganisms
were calculated. Notably all the three compounds (5a, 5e and 5j) with antimicrobial activity
(MIC) against E. coli [2.48 µg/ml (5a), 10.41 µg/ml (5j)] and C. krusei [9.96 µg/ml (5e)], A.
fumigates [9.96 µg/ml (5e)], possess good selectivity index against HeLa cells [4.46 (5a), 3.80
(5e) and 3.87 (5j)] and HEK-293 cells [5.57 (5a), 3.60 (5e) and 4.60 (5j)]. (Supporting
Information, Table S5).
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2.4.2 Time kill assay
Further, to account for the efficacy of active compounds, the time kill kinetic studies were
performed in which colony forming units (CFUs) of the E. coli and C. krusei were rapidly
reduced after treatment with compounds 5a (at MIC = 2.48 µg/ml) and 5e (at MIC = 9.96
µg/ml), respectively (Fig. 4). It was observed that the maximum killing of E. coli and C. krusei
cells with compounds 5a and 5e respectively, were observed after about 20 h in comparison to
the used control. Therefore, it can be safely interpreted that the compound 5a is successfully
inhibiting the growth of the Gram negative bacterium E. coli and the same effect is being
pronounced upon by compound 5e against the fungal strain C. krusei.
18
16
B
16
A
Control (C. krusei)
Control (E. coli)
14
12
10
8
14
12
10
8
6
6
Treated (E. coli + 5a)
Treated(C. krusei + 5e)
4
4
2
2
0
0
0
4
8
12
16
20
24
0
4
8
12
16
20
24
Time (hours)
Time (hours)
Fig. 4.Time Kill assay of compound 5a against E. coli (A) and compound 5e against C. krusei (B)
2.4.3 FE-SEM analysis
The morphological effects of active compounds 5a and 5e on the E. coli and C. krusei
respectively were investigated using Field Emission Scanning Electron Microscopy (FE-SEM).
A change in both E. coli and C. krusei cell morphology was observed when treated with 5a and
5e at their MIC value. Increased roughness and damage of the cell wall was observed after 2h of
incubation which confirmed the antimicrobial effect of the tested active compounds (Fig. 5).
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II
I
IV
III
Fig. 5. FE SEM images of (I) Control cells (E. coli, 15000X, 8.1mm); (II) 5a treated E. coli cells
(15000X, 8.1mm); (III) Control (C. krusei, 7500X, 20mm); (IV) 5e treated C. krusei cells (7500X,
20mm)
2.4.4 Synergy
The evaluation of combinations of new lead compounds and existing antibiotics is an
effective way of enhancing theantimicrobial potency. Keeping this in mind, in vitro efficacies of
compounds 5a and 5e were evaluated by checker board dilution assay with well-known
antibacterial drugs suchas kanamycin (KAN), Rifampicin (RIF) and antifungal drugs
Amphotericin B (Amp B) and Fluconazole (FLC) against E. coli and C. krusei respectively
(Table 4). Interestingly, the synergistic results with known drugs showed much better
antimicrobial efficacies at less dosage. The fractional inhibitory concentration index [FICI =
(MIC of drug in combination/MIC of drug alone) + (MIC of the tested compound in
combination/MIC of the tested compound alone) and considered to be synergistic if value of
FICI ≤ 0.5], was determined for combinations of concentrations.
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Table 4. Synergistic studies of compounds 5a and 5e with known antibiotics against E. coli and
C. krusei respectively
MIC(ꢀg/ml) MIC(ꢀg/ml) MIC(ꢀg/ml) FICI
Effect
Synergy
KAN
0.58
5a
2.48
5a
KAN + 5a
0.07 + 0.62
RIF + 5a
0.370
RIF
0.175 Synergy
0.372 Synergy
1.02
2.48
5e
0.052 + 0.31
Amp B + 5e
0.022 + 2.49
FLC + 5e
Amp B
0.18
9.96
5e
FLC
0.03
No
0.623
Synergy
9.96
0.0037+4.98
The corresponding results for compound 5a showed effective synergy with antibacterial
drugs (KAN, RIF) WITH FICI = 0.37 for KAN and FICI = 0.175 for RIF in inhibiting E. coli
growth. Further, the combination of non-cidal concentration of 5a resulted in potentiating of
KAN by 8 fold (MIC of 0.58 µg/ml to 0.07 µg/ml) and RIF by ~20 fold (MIC of 1.02 µg/ml to
0.052 µg/ml) against E. coli (Table 4). Similarly, the compound 5e also demonstrated synergistic
effect with antifungal drug Amp B with FICI = 0.372 in inhibiting C. krusei growth. The MIC
for Amp B was lowered by ~8 fold with 5e (MIC of 0.18 to 0.022 µg/ml) against C. krusei.
Instead, compound 5e does not exhibit same kind of synergistic effect with FLC with the
observed FICI value of 0.623 although the MIC for FLC was lowered by 8 fold with 5e (MIC of
0.03 µg/ml to 0.0037 µg/ml) against C. krusei (Table 4).
3. Conclusions
An efficient route for the synthesis of organoselenium compounds based on heterocyclic
moiety imidazo[1,2-a]pyridine has been developed and reported herein. The selenylation of
imidazo[1,2-a]pyridine at its C-2 position has been carried out leading to the formation of
compounds 5a-l. The use of hypophosphorous acid in selenol generating step provides advantage
over conventional methods in terms of greener and metal free conditions. Out of all synthesized
compounds, three derivatives 5a, 5d and 5e have been thoroughly characterized using X-ray
crystallography. The theoretical optimization of synthesized compounds (5a-l) was performed to
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understand their three dimensional structure and effect of different structural substitutions on the
HOMO-LUMO energy levels. The single crystal X-ray structure parameters were found to be in
acceptable close agreement with theoretically generated values. Further, the synthesized
compounds were biologically evaluated against a host of bacterial and fungal strains leading to
demonstration of broad spectrum of activities against few of them. Specifically, compounds 5a
(2.48 µg/ml against E. coli.), 5j (10.41 µg/ml against E. coli.) and 5e (9.96 µg/ml against both A.
fumigates and C. krusei) were found be exhibiting potent MIC values against listed strains. As
the selenium based derivatives generally generate lot of curiosity regarding their toxicity, the
safety profile of synthesized compounds exhibiting antimicrobial activity was evaluated by
performing cytotoxicity studies against HEK-293 and HeLa cells which revealed that the tested
compounds exhibited acceptable cytotoxicity. This was further confirmed by the good selectivity
index values obtained against E. coli (with 5a, 5j) and C. krusei (with 5e) in comparison to the
tested HEK-293 and HeLa cells clearly indicating the selective action of these compounds
against the micro-organisms versus mammalian cells. The microbial action of compounds 5a and
5e was further explored through time kill assay studies giving more meaningful measurement of
antimicrobial activity. FE-SEM analysis also confirmed the morphological dissociation of
bacterial and fungal cell membrane on treatment with tested compounds 5a and 5e on the E. coli
and C. krusei respectively. Synergistic investigations of combinations of the most potent
compounds 5a and 5e with existing antibiotics were performed to understand the effect on
antimicrobial potency. In this respect, the FICI values obtained for these studies clearly
suggested that compound 5a exhibited enhanced synergy with Kanamycin and Rifampicin, and
similar effect was noted for compound 5e with Amphotericin B. Thus, considering the
antimicrobial activity along with enhanced synergistic effects and relatively low toxicity, the
reported organoselenium compounds serve as good candidates for usage as lead compounds for
novel antimicrobials alone as well as in combination with known antibiotics.
4. Experimental Section
4.1 Chemistry
All the experimental procedure involving selenium was carried out in dry and oxygen free
nitrogen atmosphere. All the solvents used were dried and purified prior to use. Selenium (Sigma
Aldrich, purity >99.0%) was purchased and stored in dessicator prior to use. Hypophosphorous
acid, (SDFCL, 50% wt % in H2O) was used as received and all the diselenide used were
14

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prepared as per the reported method.55 Column chromatography was performed using silica gel
1
13
(99%, 60-120 mesh). H and C NMR spectra were recorded on a Bruker Avance II 400 MHz
77
spectrophotometer in CDCl3 or DMSO-d6. Se NMR was recorded on Bruker BioSpin GmbH
operating at 76.31MHz. Infrared spectra were obtained on a Thermo Scientific Fisher
spectrometer and mass spectrometry was carried out using Waters Q-TOFF micromass whereas
Field Emission Scanning Electron Microscopy (FE-SEM) was performed on a Hitachi, SU8010
electron microscope, operating at 10-15kV
4.1.1 General procedure for synthesis of selenides (5a-l):
Hypophosphorous acid, 50 wt% in water (0.2 ml) was added to a solution of diaryl
diselenide (1.0 mmol) in ethanol (25ml) under inert atmosphere, the resulting solution was
refluxed for 30min changing color from yellow to colorless. After this reaction was cooled to
room temperature followed by addition of corresponding imidazo[1,2-a]pyridine (4a-b) (2.0
mmol), stirred at 600C till completion of reaction as monitored by thin layer chromatography
(TLC). After that reaction mixture was poured into water and extracted with ethyl acetate
(3x15ml), dried over Na2SO4 and concentrated in vacuo. The residue was purified by column
chromatography (hexane/ethyl acetate, 5:1 (v/v)) to give product 5a (89%).
4.1.2 2-(Phenylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5a): Yield: 89%, yellow solid, m.p.:
1770C, δ (ppm): 9.40 (d, J = 6.9 Hz, 1H), 7.77 (dd, J = 7.4 Hz, J = 1.5 Hz, 2H), 7.60 – 7.54 (m,
13
2H), 7.47 – 7.42 (m, 3H), 7.20 – 7.16 (m, 1H); C NMR (100MHz, CDCl3) δ (ppm): 151.6,
147.3, 136.3, 131.3, 129.4, 127.6, 125.7, 117.1, 115.6; ); 77Se NMR (76.31MHz, CDCl3) δ
(ppm): 453.9; IR (neat, ν cm-1) 1629, 1452, 1379, 1327, 1123, 921, 736, 430. ES-MS: m/z 319.9
[M+H]+
4.1.3 2-(o-Tolylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5b): Yield: 91%, yellow solid, m.p.:
1
2000C, H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.44 (d, J = 6.9 Hz, 1H), 7.79 (d, J =
7.4 Hz, 1H), 7.60 – 7.53 (m, 2H), 7.42 (dd, J = 4.9, 2.1 Hz, 2H), 7.24 (dt, J = 5.8 Hz, J = 2.3 Hz,
1H), 7.18 (td, J = 6.7, 1.8 Hz, 1H), 2.49 (s, 3H); 13C NMR (100 MHz, CDCl3/DMSO-d6) δ
(ppm): 151.5, 147.4, 143.3, 137.7, 131.2, 130.5, 130.3, 127.6, 126.7, 117.1, 115.4, 23.0; IR (neat,
ν cm-1) 3099, 1627, 1458, 1375, 1315, 1120, 921, 736, 433ES-MS: m/z 334.0 [M+H]+
4.1.4 2-(p-Methoxyphenylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5c): Yield: 85%, yellow
solid, m.p.: 1520C, 1H NMR (400 MHz, CDCl3) δ (ppm): 9.42 (d, J = 6.9 Hz, 1H), 7.69 – 7.65
15

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(m, 2H), 7.62 – 7.56 (m, 2H), 7.18 (td, J = 6.8 Hz, J = 1.5 Hz, 1H), 6.97 (d, J = 8.8 Hz, 2H), 3.86
13
(s, 3H). C NMR (100 MHz, CDCl3) δ(ppm): 160.6, 152.4, 147.3, 137.9, 131.6, 131.2, 127.6,
117.8, 117.2, 117.1, 116.0, 115.5, 115.1, 55.3; IR (neat, ν cm-1) 2961, 1628, 1449, 1375, 1320,
1127, 935, 747,437 ES-MS: m/z 350.0 [M+H]+
4.1.5 2-(Mesitylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5d): Yield: 82%, yellow solid, m.p.:
1780C, 1H NMR (400 MHz, CDCl3) δ (ppm): 9.35 (d, J = 6.9 Hz, 1H), 7.47 (q, J = 8.4 Hz, 2H),
7.08 (td, J = 6.8 Hz, J = 1.5 Hz, 1H), 6.96 (s, 2H), 2.38 (s, 6H), 2.26 (s, 3H); 13C NMR(100MHz,
CDCl3) δ(ppm): 151.8, 147.4, 143.7, 139.9, 131.1, 128.9, 127.7, 124.4, 117.0, 115.3, 24.1, 21.2;
IR (neat, ν cm-1) 2912, 1626, 1453, 1369, 1128, 935, 747, 437 ES-MS: m/z 362.0 [M+H]+
4.1.6 2-(Pyridin-2-ylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5e): Yield: 76%, orange red solid,
1
m.p.: 1620C, H NMR (400 MHz, DMSO-d6) δ (ppm): 9.41 (d, J = 6.9 Hz, 1H), 8.57 (dd, J =
4.8 Hz, J = 1.1 Hz, 1H), 8.20 (d, J = 7.9 Hz, 1H), 7.82 (td, J = 7.7 Hz, J = 1.9 Hz, 1H), 7.77–7.76
(m, 2H), 7.43–7.36 (m, 2H); 13C NMR(100MHz, CDCl3/DMSO-d6) δ(ppm): 151.8, 149.7,
148.8, 146.8, 136.8, 132.2, 130.5, 127.6, 123.1, 116.5, 116.2; IR (neat, ν cm-1) 1629, 1569, 1466,
1444, 1380, 1208, 1129, 922, 577, 430 ES-MS: m/z 321.2 [M+H]+
4.1.7 2-(Phenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5f): Yield: 79%, light yellow
1
solid, m.p.: 1410C, H NMR (400 MHz, DMSO-d6) δ (ppm): 9.20 (d, J = 7.0 Hz, 1H), 7.73 (d,
13
J= 6.5 Hz, 2H), 7.49 (s, 1H), 7.47 – 7.42 (m, 3H), 7.18 (d, J = 6.8 Hz, 1H), 2.45 (s, 3H); C
NMR(100MHz, CDCl3/DMSO-d6) δ(ppm): 150.4, 147.1, 144.0, 135.8, 129.0, 128.9, 128.7,
126.7, 125.6, 118.0, 115.4, 21.0; IR (neat, ν cm-1) 2916, 1635, 1469, 1374, 1323, 1144, 891, 735,
465 ES-MS: m/z 334.0 [M+H]+
4.1.8 2-(4-Methylphenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5g): Yield: 81%,
yellow solid, m.p.: 1650C, 1H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.19 (d, J = 7.0 Hz,
1H), 7.59 (d, J = 8.0 Hz, 2H), 7.52 (s, 1H), 7.25 (d, J = 7.9 Hz, 2H), 7.21 (dd, J = 7.0 Hz, J = 1.5
13
Hz, 1H), 2.44 (s, 3H), 2.39 (s, 3H); C NMR (100MHz, CDCl3/DMSO) δ(ppm): 150.8, 147.1,
144.2, 138.8, 135.9, 129.8, 126.9, 122.0, 119.5, 118.1, 115.9, 115.3 20.9, 20.8; IR (neat, ν cm-1)
2961, 1636, 1457, 1322, 1145, 851, 737, 486, 405 ES-MS: m/z 348.2 [M+H]+
4.1.9 2-(4-Methoxyphenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5h): Yield: 78%,
yellow solid, m.p.: 1840C, 1H NMR (400 MHz, CDCl3) δ (ppm): 9.27 (d, J = 7.0 Hz, 1H), 7.66
13
(d, J = 8.8 Hz, 2H), 7.38 (s, 1H), 7.08 – 6.78 (m, 3H), 3.86 (s, 3H), 2.47 (s, 3H); C NMR
(100MHz, CDCl3) δ(ppm): 160.6, 152.8, 147.8, 143.5, 137.9, 126.8, 117.7, 116.1, 115.1, 55.3,
16

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21.6; IR(neat, ν cm-1) 3042, 1636, 1448, 1374, 1323, 1202, 842, 520, 439 ES-MS: m/z 364.0
[M+H]+
4.1.10 2-(Mesitylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5i):Yield: 83%, yellow solid,
m.p.: 1770C, 1H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.17 (d, J = 7.0 Hz, 1H), 7.42 (s,
1H), 7.10 (dd, J = 7.0 Hz, 1.4 Hz, 1H), 6.94 (s, 2H), 2.38 (s, 3H), 2.33 (s, 6H), 2.24 (s, 3H); 13C
NMR(100MHz, CDCl3/DMSO-d6) δ (ppm): 150.7, 147.2, 143.9, 143.0, 139.0, 128.4, 126.8,
124.3, 117.8, 115.3, 23.6, 21.0, 20.6; IR(neat, ν cm-1) 2917, 1636, 1463, 1373, 1323, 1204, 1145,
840, 747, 430 ES-MS: m/z 376.2 [M+H]+
4.1.11 7-Methyl-3-nitro-2-(pyridin-2-ylselanyl)-imidazo[1,2-a]pyridine (5j): Yield:76%, yellow
1
solid, m.p.: 1590C, H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.18 (d, J = 7.0 Hz, 1H),
8.48 (d, J = 4.1 Hz, 1H), 8.12 (d, J = 7.9 Hz, 1H), 7.70 (t, J = 7.3 Hz, 1H), 7.42 (s, 1H), 7.30–
13
7.27 (m, 1H), 7.10 (d, J = 6.9 Hz, 1H), 2.43 (s, 3H); C NMR (100MHz, CDCl3/DMSO-d6)
δ(ppm): 151.9, 149.5, 149.2, 147.1, 144.0, 136.7, 130.4, 126.5, 122.9, 118.1, 115.4, 21.1;
IR(neat, ν cm-1) 2951, 1640, 1466, 1443, 1325, 1142, 985, 734, 431 ES-MS: m/z 335.0 [M+H]+
4.1.12 2-(Naphthalen-1-ylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5k): Yield 78%, light yellow
1
solid, m.p.: 2460C, H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.40 (d, J = 6.9 Hz, 1H),
8.24 (d, J = 8.3 Hz, 1H), 8.08 (t, J = 8.3 Hz, 2H), 7.98 (d, J = 7.5 Hz, 1H), 7.67–7.63 (m, 1H),
7.59–7.57 (m, 2H), 7.55 – 7.54 (m, 1H), 7.51 (ddd, J = 8.2, 6.9, 1.4 Hz, 1H), 7.34 (td, J = 7.0,
1.2 Hz, 1H); 13C NMR(100MHz, CDCl3/DMSO-d6) δ (ppm): 150.2, 146.8, 136.6, 134.8, 133.7,
132.0, 130.7, 128.3, 127.8, 127.6, 126.2, 125.8, 124.8, 116.5, 116.0; IR (neat, ν cm-1) 1628,
1448, 1329, 1118, 922, 742, 577 ES-MS: m/z 370.0 [M+H]+
4.1.13 4-(3-Nitro-imidazo[1,2-a]pyridin-2-ylselanyl)-N,N-dimethylbenzenamine (5l): Yield 74%,
red solid, m.p.: 2300C, 1H NMR (400 MHz, DMSO) δ 9.35 (d, J = 6.9 Hz, 1H), 7.77 – 7.75 (m,
13
2H), 7.50–7.48 (m, 2H), 7.39 (td, J = 6.7, 1.9 Hz, 1H), 6.80– 6.76 (m, 2H), 2.98 (s, 6H)); C
NMR (75MHz, CDCl3/CCl4) δ (ppm):181.0, 137.9, 128.4, 117.2, 114.9, 113.2, 40.4; IR (neat, ν
cm-1) 3092, 2876, 1624, 1438, 1319, 1188, 1060, 916, 742, 576 ES-MS: m/z 363.1 [M+H]+
4.2 X-ray crystallographic studies
The X-ray single crystal data of 5a, 5d and 5e were collected with monochromated Mo-
Kα radiation (λ = 0.71073 Å) on a Bruker SMART Apex II diffractometer equipped with a CCD
area detector at 180(2) K. The crystals were positioned at 40 mm from the CCD and the spots
were measured using a counting time of 5s for 5a and 5e, and 10 s for 5d. Data reduction of each
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compound was carried out using the SAINT-NT software package. Multi-scan absorption
correction was applied to all intensity data using the SADABS programme. The structures were
solved by a combination of direct methods and subsequent difference Fourier syntheses followed
by successive refinements by full matrix least squares on F2 using the SHELX-2013 suite. The
hydrogen atoms were inserted at ideal geometric positions and refined with Uiso =1.2Ueq of the
parent carbon atom. Molecular and the crystal packing diagrams were drawn with the Mercury
software package. The crystal data together with pertinent refinement details are summarized in
Supporting Information, Table S1. The crystal structures were deposited with the Cambridge
Crystallographic Database Centre (CCDC) and given the numbers CCDC 1447675-1447677.
4.3 Computational studies
The ground state geometries of all the synthesized compounds were optimized at the
[B3LYP/6-31G(d)] level of theory without any symmetry restriction. All calculations were
performed with Gaussian 03 software package [28].
4.4 Antimicrobial studies
The bacterial strains were grown overnight and were diluted in Mueller–Hinton broth to a
cell density of 105 CFU (colony forming unit)/ml. 100µl of this culture and compounds 5a-l (80
µg/ml to 0.035µg/ml) were added into the 96-well flat bottomed microtiter plate (HiMedia,
India). The plate was incubated at 37ºC without shaking for 25h. The visual and optical density
at 600nm was measured using microplate reader (BioRed, Model 680). The antifungal activities
against fungal species were performed according to the Clinical and Laboratory Standards
Institute (CLSI, formerly NCCL) M-27-A3 and M-38-A2 in RPMI-1640 medium by broth
microdilution methods. The concentrations of compounds 5a-l ranged between 0.035µg/ml and
80µg/ml. The 96 well flat bottom microtiter plates were incubated without shaking at 30°C for
48 h. The visual and optical density was determination at 492 nm using a microplate reader
(BioRed, Model 680) were used for growth inhibition.
4.4.1 Time Kill assay analysis
E. coli cells (~1 x 105 CFU/ml) were inoculated in MHB medium containing 5a (2.48
µg/ml). The tube were incubated (37°C; 200rpm) and 100 µl aliquots were removed at pre-
determined time points (0, 4, 8, 12, 16, 20 and 24 h). The aliquots were serially diluted (10 fold)
in saline and plated on the MHA agar plates. The numbers of colonies were counted after
incubating the plates at 30°C for 25h. Similarly C. krusei cells (~1 x 104 CFU/ml) were
18

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inoculated in RPMI-1640 medium containing 5e. The tubes were incubated (30°C; 200 rpm), and
100 µl aliquots were removed at pre-determined time points (4, 8, 12, 16, and 24 h).
4.4.2 Synergy studies
The interaction of compound 5a with well-known antibacterial drugs Kanamycin and
Rifampicin; compound 5e with antifungal drugs Amphotericin B and Fluconazole (at sub MIC)
were evaluated by the checkerboard method and expressed as the fractional inhibitory
concentration index (FICI). The FICI was interpreted as synergistic when it was ≤ 0.5, as
antagonistic when > 4.0, and any value in between as indifferent.
4.4.3 FE-SEM analysis
E. coli and C. krusei cell suspensions from overnight grown cultures were prepared in
MHB and RPMI-1640 medium (pH 7) respectively. Compound 5a (at 2.48µg/ml) was added to
the E. coli cells (~1 x 105 CFU/ml), whereas 5e (at 9.96 µg/ml) was added to the C. krusei (~1 x
104 CFU/ml) and incubated at 30°C for 2h.
4.4.5 Cytotoxicity analysis
Cytotoxicity analysis of compounds 5a, 5e and 5j against HEK-293 (normal human
embryonic kidney cells) and HeLa (cervical cancer cells) by done using MTT (3-(4,5)-
dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) protocol. Briefly, the cells (5x104
/well) were cultured in RPMI-1640 medium supplemented with 10% fetal bovine serum in 96-
well microtiter plate at 37ºC for overnight. The next day, compounds 5a, 5e and 5j at MIC were
added to the cells in separate wells and incubated at 37ºC for 18 hours. The cells were further
incubated at 37ºC for 3 to 5h in 20µl of MTT solution (5 mg/ml) in PBS. The supernatant
(120µl) was removed, 100µl DMSO was added, and the resulting suspension was mixed to
dissolve the formazan crystals. The percent viability of cells was calculated by the ratio of
O.D570 of treated cells to the O.D570 of untreated cells. Untreated cells and 10% dimethyl
sulfoxide (DMSO) were taken as negative and positive control respectively.
Acknowledgements
This work is supported by Science and Engineering Research Board (SERB), India (grant no.
SB/SO/BB/0040/2013), Department of Science and Technology (DST-PURSE), India, BiMED-
Institute of Biomedicine (FCT Ref. UID/BIM/04501/2013) and CICECO-Aveiro Institute of
Materials (FCT Ref. UID/CTM/50011/2013). SK and NS thank CSIR, New Delhi, India for their
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research fellowships (grant no. 09/135/(0674)/2012/EMR-I and 09/135(0675)/2012-EMR-I).
KKB is grateful to CSIR, India for Emeritus Scientist Scheme India 21(0967)/13/EMR-II.
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Agarwal, S. K. Guchhait, J. Med. Chem. 54 (2011) 5013‒5030; i) R. B. Lacerda, D. Lima,
D. Silva, Bioorg. Med. Chem. 17 (2009) 74‒84.
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Highlights
• Synthesis of novel imidazo[1,2-a]pyridine based organoselenium compounds is
achieved.
• Structural characterization is performed using X-ray and computational analysis.
• The antimicrobial evaluation using time kill assay and FE-SEM analysis gave
good results.
• The synergistic studies with antibiotics exhibit enhanced antimicrobial activity.