S 1
Pravin Mahajan, 1 Mukesh Nikam, 1 Asha Chate, 1 Urja Nimbalkar, 1 Vrushali Patil, 2 Anil
Bobade, 2 Abhay Chaudhari, 2 Dattatray Deolankar, 3 Balasaheb Javale, 3 Charansingh Gill 1*
1
Department of Chemistry, Dr. Babasaheb Ambedkar Marathwada University, Aurangabad,
Maharashtra 431 004, India
2
Haffkine Institute for Training, Research and Testing, Parel, Mumbai, Maharashtra 400 012,
India
3
National Chemical Laboratory, Pune, Maharashtra 411008, India
Email: chgill16@gmail.com
Supplemental Materials
Pharmacological Evaluation
Antioxidant activity
The reaction was performed in 2 mL of solution containing 0.2 mM freshly prepared DPPH in methanol and different concentration of synthesized compounds (10-100 µg/mL). After incubation at room temperature for 30 min, the absorbance at 517 nm was measured spectrophotometrically
29
. Each experiment was performed in triplicate. The activity was compared with BHT (Butylated Hydroxy Toluene) standard. DPPH radical scavenging capacity was measured by using formula,
Percentage scavenging activity = A control
– A test
/A control
×100
A control
= Absorbance of the control, A test
= Absorbance of the test compounds.
The antioxidant activity was expressed as the concentration of a compound where 50% of its maximal effect is observed (EC
50
) using graph pad prism.

S 2
As shown in Table S 1 , among the tested compounds 3a-3h , compound 3f with EC
50
( 12.21
± 1.89
) showed most promising antioxidant activity compared to standard BHT and compound 3b with EC
50
(
14.98 ± 3.57
) possess good activity. Amongst 5a-h , the compound 5c and 5h are most active for DPPH radical scavenging activity. This result indicated that the sulfur linkage in the compounds 3a-3h may be enhancing the activity than the compounds with no sulfur link 5a-5h .
Table S 1 In vitro antioxidant activity of compounds 3a-3h and 5a-5h
Compounds EC
50
± SD
3a
73.89 ± 1.89
3b
3c
14.98 ± 3.57
70.31 ± 1.90
3d
3e
3f
3g
3h
85.20 ± 1.27
76.11 ± 1.92
12.21 ± 1.89
79.79 ± 1.16
54.66 ± 1.65
BHT
8.25 ± 0.33
Compounds
5a
5b
5c
5d
5e
5f
5g
5h
BHT
EC
50
50.00 ± 2.13
62.31 ± 1.08
34.21 ± 3.54
89.00 ± 1.03
74.23 ± 0.99
64.23 ± 1.00
66.34 ± 0.63
34.03 ± 0.77
8.25 ± 0.33
Values are expressed as mean ± standard deviation (n = 3).
± SD
BHT (Butylated Hydroxy Toluene).
Anti-inflammatory activity
Compounds 3a-h and 5a-h were screened for in vitro anti-inflammatory activity. Fresh whole human blood was collected and it was mixed with equal volumes of sterilized Alsever’s solution (Dextrose 2%, Sodium citrate 0.8%, Citric acid 0.05%, Sodium chloride 0.42% and
Distilled water 100 ml). This blood solution was centrifuged at 3000 rpm for 10 min and was washed three times with equal volume of normal saline. The volume of the blood is measured and reconstituted as 10% v/v suspension with normal saline. The reaction mixture consists of 1.0mL of test sample of different concentrations in normal saline and 0.5mL of 10% HRBC suspension,

S 3
1 ml of 0.2 M phosphate buffer, 1 mL hypo saline were incubated at 37 o
C for 30 min and centrifuged at 3,000 rpm for 30 mins. The hemoglobin content of the supernatant solution was estimated spectrophotometrically at 560 nm. Each experiment was performed in triplicate.
Dichlorofenac sodium was used as standard and distilled water as control in this study. Where the blood control represents 100% lysis or zero percent stability, the percentage of HRBC heamolysis calculated by formula,
% Heamolysis = (O. D. of Control- O. D. of Test sample / O. D. of Control) X 100
The concentration of a compound where 50% of its maximal effect is observed (EC
50
) using graph pad prism was measured.
The anti-inflammatory activity of 1,3,4-oxadiazole derivatives ( Table S 2 ) showed that the compound 3a with EC
50
(15.23 ± 2.18)
was found to possess good anti-inflammatory activity i. e. the compound with S linkage and electron withdrawing group. Among other compounds 3e , 5a ,
5b , 5d showed moderate activity against anti-inflammatory agent.

S 4
Table S 2 In vitro anti-inflammatory activity of compounds 3a-3h and 5a-5h
Compounds
2
3a
3b
3c
3d
3e
3f
3g
3h
DFS
EC
50
± SD
54.00 ± 2.13
15.23 ± 2.18
98.74 ± 1.35
61.02 ± 1.12
111.32 ± 1.10
30.13 ± 0.38
66.19 ± 0.23
68.94 ± 0.54
138.13 ± 0.23
11.70 ± 0.98
Compounds
-
5a
5b
5c
5d
5e
5f
5g
5h
DFS
EC
50
± SD
-
23.20 ± 6.12
27.20 ± 6.12
98.74 ± 1.35
24.01 ± 4.13
61.02 ± 1.12
61.32 ± 1.10
38.13 ± 0.38
66.19 ± 0.23
11.70 ± 0.98
Values are expressed as mean ± standard deviation (n = 3),
DFS- Dichlorofenac sodium.
Antimicrobial activity
Sterile filter paper discs (6mm diameter) were moistened with the test compound solution in Dimethyl sulfoxide of specific concentration 100 µg/disc were carefully placed on the agar cultures plates that had been previously inoculated separately with the microorganisms. The plates were incubated at 37 °C and the results were recorded for antibacterial activity after 14 h and for antifungal activity after 30 h. Ciprofloxacin, streptomycin were used as a standard for the comparison of antibacterial activity and Fluconazole was used as a standard for the comparison of antifungal activity. Each experiment was carried out in triplicate.
The results for antimicrobial studies depicted in Table S 3 revealed that, the tested compounds displayed variable inhibitory effects on the growth of the bacterial and fungal pathogens. In general, compounds were found to exhibit good activity against fungal strains than

S 5 bacterial strains. Amongst all, 3d containing carboxylic acid group was noticeably the most active against bacterial strains E. coli comparable to streptomycin , and P. aeruginosa, S. aureus comparable to ciprofloxacin, which shows the importance of polar acid group for the better activity. Other derivatives mainly 3a , 3g , 3h , 3f exhibited good activity against all bacterial strains.
Antifungal study revealed that the synthesized compounds are more susceptible for fungal strains.
Interestingly, the compound 3c is equally potent against A. niger comparable to Fluconazole with zone of inhibition (16 ± 0.08) . Compound 5c is potentially close against A. niger compared to standard Fluconazole. Compounds 3f , 3g , and 3h of the same series with S linkage, 5d , and 5e of another series are also exhibited good antifungal activity against tested strains. The intermediate 2 was also found to exhibit good activity against E. coli , M. luteus and C. albicans . The above results therefore suggest that introduction of sulfur linkage in the compounds and different substituent noticeably influences the antimicrobial efficiency of the compounds.

S 6
Table S 3 In vitro antimicrobial activities of compounds 3a-3h and 5a-5h
Antibacterial activity a
Antifungal activity a
Compounds
Gram negative Gram positive
E. coli P. aeruginosa S. aureus M. luteus C. albicans A. niger
2
3a
3b
3c
3d
3e
3f
3g
3h
5a
5b
5c
5d
14 ± 0.08
13 ± 0.13
08 ± 0.23
09 ± 0.42
15 ± 0.21
07 ± 0.22
09 ± 0.52
14 ± 0.23
13 ± 0.29
06 ± 0.09
12 ± 0.17
08 ± 0.23
08 ± 0.13
10 ± 0.11
5e
5f
14± 0.11
07 ± 0.13
5g
5h
10 ± 0.11
Ciprofloxacin 20 ± 0.11
Streptomycin 19 ± 0.09
Fluconazole NA
13 ± 0.21 13 ± 0.09 14 ± 0.08 14 ± 0.08 13 ± 0.21
13 ± 0.15 14 ± 0.08 15 ± 0.15 13 ± 0.15 11 ± 0.21
06 ± 0.15 11 ± 0.21 08 ± 0.08 10 ± 0.07 08 ± 0.23
09 ± 0.07 08 ± 0.23 11 ± 0.17 15 ± 0.20 16 ± 0.08
15 ± 0.20 16 ± 0.08 14 ± 0.08 16 ± 0.22 09 ± 0.52
08 ± 0.22 09 ± 0.52 06 ± 0.11 08 ± 0.18 07 ± 0.22
08 ± 0.18 07 ± 0.22 09 ± 0.50 14 ± 0.25 13 ± 0.12
14 ± 0.25 13 ± 0.12 15 ± 0.08 14 ± 0.23 14 ± 0.25
13 ± 0.23 14 ± 0.23 14 ± 0.13 13 ± 0.29 13 ± 0.23
08 ± 0.23 09 ± 0.52 08 ± 0.13 09 ± 0.52 06 ± 0.11
10 ± 0.11 11 ± 0.11 06 ± 0.10 10 ± 0.22 09 ± 0.50
06 ± 0.09 06 ± 0.29 09 ± 0.52 13 ± 0.12 15 ± 0.08
07 ± 0.20 08 ± 0.21 06 ± 0.19 14 ± 0.23 14 ± 0.13
07 ± 0.13 08 ± 0.08 11 ± 0.13 13 ± 0.09 14 ± 0.08
13 ± 0.09 14 ± 0.08 13± 0.13 12 ± 0.52 06 ± 0.11
12 ± 0.19 11 ± 0.11 09 ± 0.52 11 ± 0.13 11 ± 0.19
08 ± 0.20 09 ± 0.53 11 ± 0.15 10 ± 0.11 08 ± 0.20
18 ± 0.12 19 ± 0.10 22 ± 0.13 NA NA
20 ± 0.08 21 ± 0.14 20 ± 0.17 NA NA
NA NA NA 20 ± 0.12 16 ± 0.14 a
Zone of inhibition (Mean three replicate ± standard deviation), NA- Not Applicable.

H
3
C
N
N
S
O
NHNH
2
Figure S 1: LC-MS of compound 2
S 7

S 8
NO
2
85
80
70
60
H
3
C
N
N
S
S
O
N
N
50
1941
40
2848
30
3104.5
1 3 9 5 .7 3 cm -1
20
2921.3
10
1591.72cm-1
1 5 2 4 .3 4 c m -1
-1
4000 3500
Name
Administrator 03
3000 2500 2000 cm-1
Description
Sample 003 By Administrator Date Saturday, April 12 2014
1483
1500
Figure S 2: IR spectrum of 3a
WATERS, Q-TOF MICROMASS (LC-MS)
PRAVIN P-1 9 (0.143) AM (Top,4, Ar,5000.0,556.28,0.70,LS 10); Sm (Mn, 2x3.00); Sb (1,40.00 ); Cm (9:17-(28:60+1:6))
100
450.0667
688
521
691.06cm-1
1163.84cm-1
1343.64cm-1
852.10cm-1
746.89cm-1
710.29cm-1
678.13cm-1
1000 450
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
1: TOF MS ES+
688
NO
2
H
3
C
N
N
S
S
O
N
N
0
445.0869
19
445.6308
64
446.2984
26
448.0261;7
444 446 448 450
472.0469
186
452.0705
146
453.1466
111
452
462.5433
121
454.1099
8
454
459.2544
37
456.4239
19 457.1099
16
456 458
462.2818
69
460.4955
39
460 462
464.4593
32
465.0779
19
464 466
469.0720
32
470.1364
7
473.0513
33
474.0250
15
476.0496
15
468 470 472 474 476 478
478.6315
13
480 m/z
Figure S 3: LC-MS of compound 3a

H
3
C
N
N
S
O
N
N
S
NO
2
S 9
Figure S 4:
1
H NMR spectrum of 3a

H
3
C
N
N
S
O
N
N
S
Figure S 5: LC-MS of compound 3c
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
H
3
C
N
N
S
O
N
N
S
15
7.13
0.93
1.99
2.02
3.00
10 5
Figure S 6:
1
H NMR spectrum of 3c
TMS
0
S 10

N
N S
O
N N
S
S 11
Figure S 7:
1
H NMR spectrum of 3f

S 12
H
3
C
N
N
S
O
N
N
Figure S 8: LC-MS of compound 5a

S 13
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
0.60
0.55
0.50
0.45
0.40
2.03
8.0
5.15
7.5
7.0
H
3
C
N
N
S
O
N
N
6.5
3.00
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure S 9:
1
H NMR spectrum of 5a
2.0
TMS
1.5
1.0
0.5
0.0

S 14
0.55
0.50
0.45
0.40
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
H
3
C
N
N
S
O
N
N
CH
3
TMS
8.0
3.18
2.87
7.5
7.0
6.5
3.17
6.0
5.5
5.0
4.5
4.0
3.5
3.0
2.5
Figure S 10:
1
H NMR spectrum of 5f
2.0
1.5
1.0
0.5
0.0
53
50
45
40
35
30
25
H
3
C
N
N
S
O
N
N
Cl
20
1656.5
15
10 2924.6
1592.12cm-1
3082.6
5
0
-1
4000
1509.20cm -1
3500
Name
Administrator 04
3000 2500 2000 cm-1
Description
Sample 004 By Administrator Date Saturday, April 12 2014
848.79cm-1
1011.02cm-1
1393.33cm-1 755.00cm-1
1277.77cm-1
1444.16cm-1
1500 1000
523.1
677.82cm-1
727.89cm-1
500 400

S 15
Figure S 11: IR spectrum of 5g
WATERS, Q-TOF MICROMASS (LC-MS)
PRAVIN P-2 12 (0.176) AM (Top,4, Ar,5000.0,556.28,0.70,LS 10); Sm (Mn, 2x3.00); Sb (1,40.00 ); Cm (8:15)
100
415.0291
18177
SAIF/CIL,PANJAB UNIVERSITY,CHANDIGARH
1: TOF MS ES+
1.82e4
393.0558
13969
H
3
C
N
N
S
O
N
N
Cl
417.0329
5589
395.0541
4585
0
385.2943
138
386 388
392.0537
69
390 392 394
396.0600
908
398.0563;55
403.0495
23
396 398 400 402
405.0339
43
404 406
409.1551
197
408
411.0657
204
410 412 414 416
Figure S 12: LC-MS of compound 5g
418.0366
1209
418
419.0315
402
420 422
425.1161
76
424 426
427.0838
43
428 m/z