S1
Synthesis of quinoline based [1,2,4]-dithiazolidines through sulfur-sulfur bond
formation and their evaluation as anti-inflammatory and antibacterial agents
Spruha A. Gharad*, Baliram N. Berad & Snehal A. Chavan
P.G. Department of Chemistry, Mahatma Jyotiba Phule Educational Campus,
Rashtrasant Tukadoji Maharaj Nagpur University, Nagpur-440033(India)
Email: spruhagharad@gmail.com
Supplemental Materials
Biological activity
Antibacterial activity
All the newly synthesized compounds were evaluated for their efficacy against the
microorganisms like Escherichia coli, Salmonella typhi, Bacillus subtilis and Staphylococcus
aureus at concentrations 1000, 500,250, 125, 62.5 µg/mL.
Procedure for antibacterial screening:
The media used for antibacterial analysis were Peptone-10 g, NaCl-10g and Yeast extract
5g, in 1000 ml of distilled water. Initially, the stock cultures of bacteria were revived by
inoculating in broth media and grown at 37ºC for 18 hrs. The tubes containing above media
were autoclaved and added with respective concentrations of samples. Each tube was inoculated
with 18 h old cultures (25 μl, 10-4 cfu). A control tube without samples was inoculated and a
tube with only media was maintained as blank. The tubes with Gentamycin were also prepared.
All the tubes were incubated at 37ºC for 24 h on a shaker at 140 rpm, and the growth of bacteria
were noted at 600 nm. The growth in control tubes were measured against media blank where as
the tubes with samples were read against media with compounds at respective concentrations, as
blank. The antibacterial screening results of compounds 5a-g are depicted in Table S1.
S2
Table S 1 Minimum inhibitory concentration of 5a-f in µg/mL against E. coli, S. typhi , B.
subtilis and S. aureus
Minimum inhibitory concentration in µg/mL
Compound
Gram –ve bacteria
Gram +ve bacteria
E. coli
S. typhi
B. subtilis
S. aureus
5a
250
1000
250
250
5b
62.5
500
125
250
5c
125
500
125
250
5d
250
500
250
250
5e
125
500
125
250
5f
500
1000
1000
1000
Gentamycin
62.5
62.5
62.5
62.5
In present protocol, 500 µg/mL- weakly active, 250µg/mL- moderately active, 125µg/mL- good
active, 62.5µg/mL- excellent active
In Vitro Anti-Inflammatory Activity
Procedure for In Vitro Anti-Inflammatory Activity
The assay medium consisting of 3 - 5U hyaluronidase (from Sigma –Aldrich, Bangalore)
in 100µl of 20mM sodium phosphate buffer (pH 7.0) with 77mM sodium chloride, 0.01% BSA
S3
was preincubated with different concentrations (10, 50, 100 μg/mL) of the test compound for 15
min at 37 °C. The assay was commenced by adding 100µl hyaluronic acid (from Sigma –
Aldrich, Bangalore; 0.03% in 300mM sodium phosphate, pH 5.35) to the incubation mixture and
incubated for a further 45 min at 37 °C. The undigested hyaluronic acid was precipitated with
1ml acid albumin solution made up of 0.1% bovine serum albumin in 24mM sodium acetate and
79mM acetic acid (pH 3.75).
After standing at room temperature for 10 min, the absorbance of the reaction mixture
was measured at 600 nm. The absorbance in the absence of enzyme was used as the reference
value for maximum inhibition. The inhibitory activity of test compound was calculated as the
percentage ratio of the absorbance in the presence of test compound vs. absorbance in the
absence of enzyme.
The enzyme activity was checked by control experiment run simultaneously, in which the
enzyme was preincubated with 5µl vehicle instead, and followed by the assay procedures
described above. Compound was tested in a range of 10µg -100µg in the reaction mixture.
Indomethacin was used as reference standard and tested at 50µg concentration. The results of
anti-inflammatory activity are depicted in Table S 2 (Figure S 2).
S4
60
% inhibition
50
40
10µg/mL
30
50µg/mL
100µg/mL
20
10
0
5b
5c
5d
Indomethacin
Figure S 2: Percentage inhibition of hyaluronidase enzyme by the sample
S5
Figure S 3: 1H NMR of 5a
S6
WATERS, Q-TOF MICROMASS (LC-MS)
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
SPRUHA PPDTZ 2 (0.037) Cm (1:2)
TOF MS ES+
3.90e3
474.0
3897
100
N
Cl
N
N
N
%
N
S
476.0
1686
S
5a
477.0
235
0
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
750
Figure S 4: Mass spectrum of 5a
800
850
900
950
1000
S7
RC SAIF PU, Chandigarh
50.3
1746,48
45
1894,45
1864,45
40
2341,42 2101,42
3402,41
2725,40
2551,41
35
30
%T
892,39
2914,36
3050,35
2860,37
1073,34
511,39
596,38 497,38
964,35
918,35
775,34
945,33 852,34
690,32
25
N
1394,25
20
Cl
1455,23
N
755,22
1361,20
1308,18
N
1637,17
N
S
816,26
1049,20
N
15
1193,26
1171,26
1147,25 1022,23
S
1329,15
10
5b
CH3
5
1597,5
1530,5
0.0
4000.0
3600
3200
2800
2400
2000
1800
1600
1400
cm-1
Snehal A-30.sp - 7/17/2014 - Ph-DTZ
Figure S 5: IR spectrum of 5b
1200
1000
800
600
400.0
S8
BRUKER
AVANCE II 400 NMR
Spectrometer
SAIF
Panjab University
Chandigarh
1.2455
2.5468
2.3089
3.3803
3.3306
8.8448
8.5791
8.0964
8.0770
8.0564
7.9661
7.9448
7.8522
7.8330
7.8124
7.6796
7.6605
7.6421
7.6093
7.5891
7.5711
7.5555
7.5373
7.5180
7.5008
7.1468
7.1271
6.8141
6.7939
PHPT-Dtz
Current Data Parameters
NAME
Sep09-2014
EXPNO
310
PROCNO
1
F2 - Acquisition Parameters
Date_
20140909
Time
16.29
INSTRUM
spect
PROBHD
5 mm PABBO BBPULPROG
zg30
TD
65536
SOLVENT
DMSO
NS
8
DS
2
SWH
12019.230 Hz
FIDRES
0.183399 Hz
AQ
2.7263477 sec
RG
1030
DW
41.600 usec
DE
6.00 usec
TE
294.8 K
D1
1.00000000 sec
TD0
1
======== CHANNEL f1 ========
NUC1
1H
P1
10.90 usec
PL1
-3.00 dB
SFO1
400.1324710 MHz
N
Cl
N
N
F2 - Processing parameters
SI
32768
SF
400.1299852 MHz
WDW
EM
SSB
0
LB
0.30 Hz
GB
0
PC
1.00
N
N
S
5b
14
13
12
11
10
9
8
7
3.22
1.07
1.07
1.75
1.11
1.20
1.27
5.31
2.10
CH3
2.00
S
6
5
4
3
2
1
0
ppm
avtar_saifpu@yahoo.co.in
Figure S 6: 1HNMR of 5b
S9
WATERS, Q-TOF MICROMASS (LC-MS)
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
SPREEHA Ph-DTZ 9 (0.167) Cm (2:11)
TOF MS ES+
5.54e3
488.1
5536
%
100
490.1
1726
N
491.1
277
Cl
N
N
N
0
N
100
150
200
S
5b
250
S
300
350
400
450
500
550
600
650
CH3
Figure S 7: Mass spectrum of 5b
700
750
800
850
900
950
m/z
1000
S 10
RC SAIF PU, Chandigarh
65.2
60
55
644,58
1890,57
50
2051,54
596,52
3406,52
45
703,54
2547,51
501,50
568,49
3052,49
3027,48
3001,48
2834,44
2917,42
40
941,44
900,43
863,44
35
528,41
770,39
1107,37
%T 30
1400,34
1330,31
1367,30
25
1634,26
20
1299,26
1174,27 1050,28
1148,25
822,27
1027,23
15
N
1246,15
10
Cl
N
5
N
N
N
0.0
4000.0
3600
3200
H3CO
S
2800
1597,6
1526,3
S
2400
5c
2000
CH3
Snehal A-31.sp - 7/17/2014 - PADTZ
Figure S 8: IR spectrum of 5c
1800
1600
cm-1
1400
1200
1000
800
600
400.0
S 11
Figure S 9: 1H NMR of 5c
S 12
Figure S10: 1H NMR of 5c
S 13
Figure S 11: 13C NMR of 5c
S 14
WATERS, Q-TOF MICROMASS (LC-MS)
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
SPREEHA PADTZ 48 (0.891) Cm (2:75)
TOF MS ES+
1.17e5
518.1
117126
100
N
Cl
N
N
N
N
S
H3CO
S
%
5c
CH3
520.1
46414
521.1
7534
0
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
Figure S 12: Mass spectrum of 5c
750
800
850
900
950
S 15
RC SAIF PU, Chandigarh
70.7
410,69
65
1760,67
1895,65
60
3399,62
55
3089,60
636,65
579,65
2552,62
718,62
596,59
3025,57
2914,58
50
696,58
946,54
905,52
516,51
45
1208,49
40
1284,44 1173,45
1194,44
1142,42 1025,42
1362,39
1086,41
Cl
30
822,38
1326,36
1308,36
N
N
N
25
853,45 751,44
1402,43
N
%T 35
1052,36
1633,33
N
S S
Cl
20
5d
15
1488,19
10
1584,13
1596,12
5
2.0
4000.0
503,56
466,54
775,52
1527,7
3600
3200
2800
2400
2000
1800
1600
1400
cm-1
Snehal A-29.sp - 7/17/2014 - 4CPDTZ
Figure S13: IR spectrum of 5d
1200
1000
800
600
400.0
S 16
S 17
Figure S 14: 1H NMR of 5d
S 18
Figure S 15: 13C NMR of 5d
S 19
WATERS, Q-TOF MICROMASS (LC-MS)
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
SPREEHA 4 CPDTZ 1 (0.019) Cm (1:23)
TOF MS ES+
776
522.1
776
100
N
Cl
524.1
538
N
N
N
N
S S
Cl
%
5d
523.1
218
525.1 526.1
146 140
504.2
55
507.3
28
519.2
513.3
78
73 515.2
511.2
53 517.2
520.2
44
37
36
527.1
39
536.3
139
531.3 533.2
49
42
559.2
134
541.2
122
537.3
80 538.3
58
542.2 543.2
61
66
551.3
29
544.2
25
555.3
557.3
58
50
560.2
53
0
m/z
506
508
510
512
514
516
518
520
522
524
526
528
530
532
534
536
538
540
Figure S 16: Mass spectrum of 5d
542
544
546
548
550
552
554
556
558
560
S 20
N
Cl
N
N
N
N
H3CO
S
S
5e
Figure S 17: IR spectrum of 5e
S 21
WATERS, Q-TOF MICROMASS (LC-MS)
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
SPREEHA PADTZ 18 (0.202) Cm (12:37)
TOF MS ES+
5.04e3
508.2
5040
100
N
Cl
N
N
N
N
454.2
3191
S
H3CO
S
%
5e
510.2
2068
299.2
1597
215.0
1535
455.2
910
96.6
457
143.0
294 170.1 199.0
222 197
233.0
365
274.3
343
605.3
892
506.2
724
345.0 359.3
419 488
359.8
320.2
201
199
511.2
612
606.3
372
476.4 512.2
183
178
604.9
173
840.4
380
841.4
243
701.7
189
0
m/z
100
150
200
250
300
350
400
450
500
550
600
650
700
Figure S 18: Mass spectrum of 5e
750
800
850
900
950
1000
S 22
N
Cl
Cl
N
N
N
N
S
S
5f
Figure S19: IR spectrum of 5f