Embed Size (px)
Citation preview
Supplementary Material
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 Félix,d,e K. K. Bhasin,a* and Rohit K. Sharmaa*
aDepartment of Chemistry & Centre for Advanced Studies in Chemistry, Panjab University,
Chandigarh, 160014 IndiabDepartment of Microbial Biotechnology, Panjab University, Chandigarh, 160014 IndiacDepartment of Applied Sciences, University Institute of Engineering & Technology (U.I.E.T),
Panjab University, Chandigarh, 160014 IndiadDepartment of Chemistry, CICECO – Aveiro Institute of Materials, University of Aveiro, 3810-
193 Aveiro, PortugaleDepartment of Medical Sciences, iBiMED – Institute of Biomedicine, University of Aveiro,
3810-193 Aveiro, Portugal
S1
Table of Contents
S.No. Contents Page No.
1. Experimental S3-S10
2. 1H-NMR, 13C-NMR of compounds 3a-b, 4a-b S6-S9
3. 1H-NMR, 13C-NMR and Mass spectra of compounds 5a-l S10-S33
4. 77Se NMR of 5a S34
5. Cytotoxicity profile of compounds 5a, 5d and 5e (Figure S1) S35
6. HOMO-LUMO diagram of compounds 5a-l (Figure S2-S3) S36-S37
7. Crystal data and pertinent structure refinement details of 5a, 5d and 5e
(Table S1)
S38
8. Optimized structures of Compounds 5a-l (Table S2) S39
9. Antibacterial results of compounds 5a-l (Table S3) S40
10. Antifungal results of compounds 5a-l (Table S4) S41
11. Cytotoxic selectivity index for compounds 5a, 5e, 5j (Table S5) S42
EXPERIMENTAL
S2
Material and Methods. 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 prepared as per the reported method.1 Column chromatography was
performed using silica gel (99%, 60-120 mesh). 1H and 13C NMR spectra were recorded on a
Bruker Avance II 400 MHz spectrophotometer in CDCl3 or DMSO-d6. 77Se 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.
General procedure for synthesis of 3a-b: To a solution of chloroacetic acid (3.67g, 38.8mmol)
in water (6ml), triethyl amine was added (6.12 ml, 44 mmol) dropwise at room temperature.
After 10 minutes of stirring 2-aminopyridine (4.3 g, 46mmol) was added and was refluxed at
900C for 5h. Then reaction mixture was cooled to room temperature, ethanol was added and
stirred at 50C for 2h to obtain precipitates which were filtered, washed with cold ethanol (4ml),
dried to get 5.3g (89%) of intermediate 2a. This intermediate 2a (5.3g, 34.8 mmol) was dissolved
in toluene (25 ml) and phosphorous oxychloride (9.7 ml, 104 mmol) was added dropwise at
reflux. After refluxing for 16 h reaction mixture was cooled to room temperature and cold water
(100 ml) was added, stirred for 15minutes. Aqueous layer was separated and neutralized with
10% NaOH (aq) and the precipitate was filtered and dissolved in dichloromethane. The aqueous
filtrate was also extracted with dichloromethane (4x20ml). The combined organic layers were
washed with brine, dried over anhydrous sodium sulphate and concentrated under vacuum to
afford a brown powder. This was purified column chromatography on silica gel using hexane:
ethyl acetate (90:10) as eluent to yield 5.2g (98%) of 2-chloroimidazo[1,2-a]pyridine (3a). 2-
chloro-7-methylimidazopyridine (3b) was prepared similarly by using 2-amino-4-picoline
instead of 2-aminopyridine.
2-Chloroimidazo[1,2-a]pyridine (3a): Yield: 74.6% , white crystalline solid, m.p. 76‒770C,1H
NMR (400 MHz, CDCl3) δ (ppm): 7.95 (d, J = 6.8 Hz, 1H), 7.39 (t, J = 4.3 Hz, 2H), 7.09 (ddd,
J= 9.1 Hz, J = 6.9 Hz, J = 1.2 Hz, 1H), 6.71 (td, J = 6.8 Hz, J = 0.9 Hz, 1H). 13C NMR
(100MHz, CDCl3) δ (ppm): 143.7, 135.5, 125.1, 116.8, 112.9, 108.1
S3
2-Chloro-7-methylimidazopyridine (3b): Yield: 72.4%, white crystalline solid, m.p. 85‒870C, 1H NMR (400 MHz, CDCl3) δ (ppm): 7.89 (d, J = 6.9 Hz, 1H), 7.38 (s, 1H), 7.22 (s, 1H), 6.62
(dd, J = 6.9 Hz, J = 1.5 Hz, 1H), 2.36 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm): 144.1,
136.2, 135.0, 124.3, 115.4, 115.2, 107.4, 21.2
General procedure for synthesis of 4a-b: Imidazo[1,2-a]pyridine (3a-b) (40mmol) was added to
sulphuric acid (60ml) under ice bath at 50C. To this solution, nitric acid (6ml) was added
dropwise. After addition, reaction was allowed to stir at room temperature for 3h. The reaction
mixture was poured onto crushed ice and precipitates were filtered by vacuum filtration and
dissolved in dichloromethane. This organic layer was dried on anhydrous sodium sulphate and
concentrated under vacuum to yield pure product.
2-Chloro-3-nitroimidazo[1,2-a]pyridine (4a): Yield: 82%, light brown solid, m.p. 177-1780C, 1H NMR (400 MHz, CDCl3/DMSO-d6) δ (ppm): 9.43 (d, J = 7.0 Hz, 1H), 7.82 (dd, J = 3.4 Hz, J
= 1.2 Hz, 2H), 7.47 (tdd, J = 4.9 Hz, J= 3.9 Hz, J = 1.2 Hz, 1H). 13C NMR (100 MHz,
CDCl3/DMSO) δ (ppm): 142.8, 138.9, 131.8, 127.6, 117.1, 117.0
2-Chloro-7-methyl-3-nitroimidazo[1,2-a]pyridine (4b): Yield: 80%, light brown solid, m.p.
181-1820C, 1H NMR (400 MHz, DMSO-d6) δ (ppm): 9.29 (d, J = 7.1 Hz, 1H), 7.60 (s, 1H), 7.31
(d, J = 7.1 Hz, 1H), 2.56 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ (ppm): 143.9, 143.2, 126.9,
119.3, 115.9, 21.0
Antimicrobial activity. All the bacterial and fungal strains used in the study were obtained from
National Collection of Pathogenic Fungi (NCPF), Post-Graduate Institute of Medical Education
and Research (PGIMER), Chandigarh, India and Microbial Type Culture Collection, Institute of
Microbial Technology (MTCC-IMTECH), Chandigarh, India. The bacterial strains (Escherichia
coli MTCC2961, Staphylococcus aureus MTCC3160, Enterococcus faecalis MTCC439, were
cultured in Muellar Hinton broth (HiMedia, India). The fungal strains Candida albicans
NCPF400034, Candida krusei NCPF440002, Candida parapsilosis NPCPF450002, Candida
kefyr NPCPF410004, Candida tropicalis NPCPF420007, Cryptococcus neoformans (C.
neoformans) NCPF250316, were cultured in yeast extract-peptone-dextrose (YEPD broth,
HiMedia, India) and RPMI-1640 media (HiMedia, India). Aspergillus niger (A. niger)
MTCC282 and Aspergillus fumigatus MTCC2546 were grown on potato dextrose broth. For agar
plates, 2.5% (w/v) bacteriological agar (HiMedia, Mumbai) was added to the medium. The
S4
strains were stored with 15% glycerol at -80°C as frozen stocks. The cells were freshly revived
on respective agar plates from the stock before each experiment.6
.
2-Chloroimidazo[1,2-a]pyridine (3a) 1H, 13C
S5
2-Chloro-7-methylimidazopyridine (3b)1H, 13C
S6
2-Chloro-3-nitroimidazo[1,2-a]pyridine (4a)1H, 13C
S7
2-Chloro-7-methyl-3-nitroimidazo[1,2-a]pyridine (4b)1H, 13C
S8
2-(Phenylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5a) 1H, 13C, Mass spectra
S9
S10
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400
%
0
100SANJEEV SK-2 14 (0.148) Cm (10:33) TOF MS ES+
8.44e3319.98436
317.94040
315.91442
146.01019
79.0713
102.1327
241.9373218.2
285
360.22710
341.92208
321.91270
339.91071337.9405
343.9363
361.3596
S11
2-(o-Tolylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5b)1H, 13C, Mass spectra
S12
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
%
0
100SANJEEV IM-13 24 (0.254) Cm (9:31-42:77) TOF MS ES+
5.36e3334.05362
332.02034
146.0580
330.0550
192.2209
242.0171
356.0970
S13
2-(p-Methoxyphenylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5c)1H, 13C, Mass spectra
S14
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520
%
0
100
SANJEEV IM-3 24 (0.254) Cm (9:34-49:74) TOF MS ES+ 4.83e3350.0
4828
348.01889
346.0521
146.1244
372.0980
352.1459
S15
2-(Mesitylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5d)1H, 13C, Mass spectra
S16
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100SANJEEV IM-4 16 (0.169) Cm (10:31-40:72) TOF MS ES+
1.01e4362.110141
360.14329
358.11221301.2
1063149.0895
163.1361
384.11330364.1
974
382.1557
S17
2-(Pyridin-2-ylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5e)1H, 13C, Mass spectra
S18
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
%
0
100SANJEEV IM-5 9 (0.161) Cm (7:19) TOF MS ES+
4.09e3275.24095
273.21707
272.2486
321.21540
319.2606
277.2428
317.2169
343.21229
345.2127
S19
2-(Phenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5f)1H, 13C, Mass spectra
S20
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100
SANJEEV IM-6 23 (0.243) Cm (9:37-44:66) TOF MS ES+ 1.27e4334.0
12719
332.05238
331.01535301.1
1276
295.0389160.1
389
317.0564
356.03228
354.01315
353.0390
360.3733
S21
2-(4-Methylphenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5g)1H, 13C, Mass
spectra
S22
S23
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950
%
0
100SANJEEV IM-7- 15 (0.248) Cm (1:18) TOF MS ES+
8.47e3370.28473
348.26853
160.14909
346.22611
345.3693
256.1381
161.2268
437.5877399.4
670 457.5416
2-(4-Methoxyphenylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5h)1H, 13C, Mass spectra
S24
S25
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520
%
0
100SANJEEV IM-8 17 (0.179) Cm (11:28-36:48) TOF MS ES+
4.86e3364.04863
362.01928
301.1627
160.1201
360.0490
386.01709
384.0683
366.0478
383.0200
437.2189
388.0185
S26
2-(Mesitylselanyl)-7-methyl-3-nitro-imidazo[1,2-a]pyridine (5i)1H, 13C, Mass spectra
S27
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000
%
0
100
SANJEEV IM-9 9 (0.161) Cm (7:19) TOF MS ES+ 1.75e3376.3
1749
374.3674
160.1493
360.5260
256.197
359.591301.3
87
398.3722
400.3100 475.6
90437.5;61
S28
7-Methyl-3-nitro-2-(pyridin-2-ylselanyl)-imidazo[1,2-a]pyridine (5j)1H, 13C, Mass spectra
S29
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100SANJEEV IM-10 29 (0.305) Cm (9:37-52:92) TOF MS ES+
2.65e4335.026452
333.012710
289.09603
287.04060
286.01130
331.03475
291.0937
357.03407337.0
2798
355.01219
S30
2-(Naphthalen-1-ylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5k)1H, 13C, Mass spectra
S31
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500 520 540
%
0
100
SANJEEV IM-11- 16 (0.169) Cm (9:40-44:71) TOF MS ES+ 3.36e3370.0
3360
368.01337
367.0430202.2
356 366.0340
301.1317
316.2315
392.0735
372.0405
390.0312 437.2
139394.0108
S32
4-(3-Nitro-imidazo[1,2-a]pyridin-2-ylselanyl)-N,N-dimethylbenzenamine(5l)1H, 13C, Mass spectra
S33
WATERS, Q-TOF MICROMASS (LC-MS) SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
m/z80 100 120 140 160 180 200 220 240 260 280 300 320 340 360 380 400 420 440 460 480 500
%
0
100SANJEEV IM-12 21 (0.222) Cm (9:37-48:94) TOF MS ES+
7.64e3363.17641
361.13028
360.1786
149.0583
121.1274
301.2513 335.0
439
365.1699
385.1520
S34
2-(Phenylselanyl)-3-nitro-imidazo[1,2-a]pyridine (5a)77Se NMR
S35
Control 5a 5e 5j0
20
40
60
80
100
120
HeLa HEK- 293
% v
iabi
lity
of c
ells
Figure S1. Mammalian cell cytotoxicity
S36
Figure S2. HOMO-LUMO diagram of compounds 5a-f
S37
Figure S3. HOMO-LUMO diagram of compounds 5g-l
S38
Table S1.Crystal data and pertinent structure refinement details of 5a, 5d and 5eCompound 5a 5d 5eEmpirical formula C13H9N3O2Se C16H15N3O2Se C12H8N4O2Se
Crystal system Monoclinic Triclinic MonoclinicSpace group C2/c C2/ca/Å 19.9416(13) 8.0177(4) 19.9130(15)b/Å 9.2143(6) 8.7865(4) 8.8665(7)c/Å 16.5268(17) 11.8624(6) 16.599(2) /º (90) 104.0020(17) (90)β /º 126.8080(16) 92.9126(17) 126.7922(17)γ /º (90) 110.3394(15) (90)Volume 2431.4(3) 751.88(6) 2346.9(4)Z 8 2 8Dc (Mg m-3) 1.738 1.591 1.807 / (mm-1) 3.089 2.508 3.203F(000) 1264 364 1264range for data collection / º 2.551-30.564 2.696-27.978 2.554-27.197Index ranges -28<h< 28, -13<k<
13, -23<l< 23-10< h< 10, -11<k< 11, -15<l< 15
-25<h< 25, -11<k< 11, -21< l< 21
Reflections collected 21058 15028 14300Independentreflections, [Rint] 3728 [0.0234] 3627 [0.0186] 2596 [0.0265]Data/restraints/ parameters 3728/0/172 3627/0/202 2596/0/172Goodness-of-fit on F2 1.086 1.119 1.091
Final R indicesR1, wR2 [I>2I] 0.0275, 0.0709
[3364]0.0254, 0.0639[3359]
0.0218, 0.0561[2435]
R1, wR2 (all data) 0.0312, 0.0730 0.0282, 0.0655 0.0238, 0.0572Largest diff. peak/hole/e Å-3 1.12/-0.22 0.57/-0.23 0.45/-0.23
S39
TABLE S2: Optimized structures of Compounds with DFT B3LYP 6-31G(d)
Compound Optimized Structure Compound Optimized Structure
5a 5g
5b5h
5c 5i
5d 5j
5e 5k
5f 5l
S40
Table S3: Antibacterial activity of compounds 5a-l
MIC in μM (μg/ml)Bacterial
Strains5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l
P. aeruginosa>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
E. coli7.81
(2.48)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
31.22
(10.41)
>125
(46.03)
>125
(45.25)
K. pneumoniae>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
S. aureus>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
A. baumnnii>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
E. faecalis>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
S. smeltophilia>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
S. dysenteriae>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
S41
Table S4. Antifungal activities of compounds 5a-l
MIC in μM (μg/ml)Fungal strains 5a 5b 5c 5d 5e 5f 5g 5h 5i 5j 5k 5l
C. albicans>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
C. tropicalis>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
C. parapsilosis>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
60
(19.93)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
C. krusei>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
30
(9.96)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
C. neoformans>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
A. niger>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
60
(19.93)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
A. flavus>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
>125
(39.89)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
A. fumigatus>125
(39.77)
>125
(41.52)
>125
(43.52)
>125
(45.03)
30
(9.96)
>125
(41.52)
>125
(43.28)
>125
(45.28)
>125
(46.78)
>125
(41.66)
>125
(46.03)
>125
(45.25)
S42
Table S5 : Cytotoxic selectivity index for compounds 5a, 5e, 5j
5a 5e 5j
HeLa
CC50 46.86 250 187.5
IC5010.5
(E. coli)65.0
(C. krusei)48.4
(E. coli)SI 04.46 03.80 03.87
HEK-293
CC50 58.57 236.20 223.21
IC5010.50
(E. coli)65.00
(C. krusei)48.40
(E. coli)
SI 05.57 03.60 04.60
S43
