Synthesis, antioxidant, and antitumor evaluation of certain new N-substituted-2-amino-1,3,4-thiadiazoles

Article abstract of DOI:10.1007/s00044-012-0336-z

Reaction of 2-amino-1,3,4-thiadiazole (1) with chloroacetyl chloride afforded the chloroacetamide 2 which used as starting compound for the synthesis of 2-thiocyanatoacetamide 3 and N-(1,3,4-thiadiazol-2-yl)acetamides 5-9 via reaction of 1 with various re

Full text of DOI:10.1007/s00044-012-0336-z

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2013) 22:3556–3565
DOI 10.1007/s00044-012-0336-z
ORIGINAL RESEARCH
Synthesis, antioxidant, and antitumor evaluation of certain new
N-substituted-2-amino-1,3,4-thiadiazoles
•
Wafaa S. Hamama Moustafa A. Gouda
•
•
Marwa H. Badr Hanafi H. Zoorob
Received: 24 April 2011 / Accepted: 7 November 2012 / Published online: 27 November 2012
Ó Springer Science+Business Media New York 2012
Abstract Reaction of 2-amino-1,3,4-thiadiazole (1) with
chloroacetyl chloride afforded the chloroacetamide 2
which used as starting compound for the synthesis of
Introduction
1,3,4-Thiadiazoles are a valuable group of bioactive com-
pounds showing antituberculosis, anticancer, antibacterial,
antifungal, and anticonvulsant activities (Clerici et al., 2001;
Prasad et al., 1986; Madapa et al., 2009; Sharma and Bahel,
1982; Jatav et al., 2008; Gagnon et al., 2009). Moreover,
N-substituted 2-amino-1,3,4-thiadiazoles bearing acetyl and
halo nucleus show broad range of biological activities
(Molyneux, 2004; Re et al., 1999). Furthermore, thiadiazole
substituted by amide groups possesses antifungal activities
(Liu et al., 2009), antioxidant properties, and radioprotective
effects (Cressier et al., 2009). 2-Amino-1,3,4-thiadiazole
acts as a building block for the synthesis of 1,3,4-thiadiazole
derivatives and various intermediates (Matysiak and Niew-
iadomy, 2006; Matysiak and Opolski, 2006; Terzioglu and
Gu¨rsoy, 2003; Bhati and Kumar, 2008), possessing biolog-
ical activities. Therefore, some new amide derivatives were
synthesized, starting from 2-amino-1,3,4-thiadiazole (Lauer
and Zenchoff, 1976), in order to investigate their antioxidant
and antitumor activities.
2-thiocyanatoacetamide
3
and N-(1,3,4-thiadiazol-2-yl)
acetamides 5–9 via reaction of 1 with various reagents.
Treatment of 9 with 4-(piperidin-1-yl)benzaldehyde or
DMF-DMA afforded the arylidenes 10 and 11, respec-
tively. Cyclization of the later compound with hydrazine
hydrate gave the pyrazole derivative 12. Furthermore,
coupling of 9 with 4,6-dimethyl-1H-pyrazolo[3,4-b]pyri-
din-3-diazonium chloride afforded hydrazone derivative
13, which cyclized in acetic acid to afford 1,2,4-triazine
derivative 14. Moreover, 1,3,4-thiadiazoles 15, 19, 22, and
23 were achieved via reaction of 1 with different nucleo-
philes. Finally, 1-phenyl-1H-pyrazol-5(4H)-one when
subjected to react either with 15 or with diazonium salt of 1
afforded pyrazole derivative 16 or bis-1,3,4-thiadiazole
derivative 18, respectively. Some of these compounds were
screened for their cytotoxicity and antioxidant activities
which showed promising results.
Keywords Thiadiazoles ꢀ Synthesis ꢀ
Antitumor and antioxidant activities
Results and discussion
Chemistry
The target compounds were synthesized as outlined in
Schemes 1, 2, 3, 4. Reaction of 2-amino-1,3,4-thiadiazole
(1) with chloroacetyl chloride afforded chloroacetamide
derivative 2 which gave the corresponding thiocyanate 3
upon treatment with ammonium thiocyanate upon refluxing
in ethanol. Attempts for the preparation of thiazoline 4
through cyclization of 3 under the influence of pyridine
were failed. Treatment of 2 with piperazine, N-phenyl
W. S. Hamama (&) ꢀ M. A. Gouda ꢀ M. H. Badr ꢀ H. H. Zoorob
Chemistry Department, Faculty of Science, Mansoura
University, El-Gomhoria Street, Mansoura 35516, Egypt
e-mail: wshamama@yahoo.com
Present Address:
M. A. Gouda
Department of Chemistry, Faculty of Science and Arts, Ulla,
Taibah University, Medina, Kingdom of Saudi Arabia
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3557
Scheme 1 Synthesis of 2-chloro-N-(1,3,4-thiadiazol-2-yl)acetamide (2) and its reactions with different nucleophiles
piperazine, 2-amino-1,3,4-thiadiazole, and 2-amino-1,3,4-
thiadiazol-5-thiol afforded the corresponding thiadiazole
derivatives 5–8, respectively. Moreover, heating of 2 with
potassium cyanate in dimethylformamide (DMF) afforded
cyanoacetamide 9. In another route, compound 9 was
obtained in good yield via cyanoethylation of 1 with 3,5-
dimethyl-1-cyanoacetyl pyrazole (Ried and Schleimer,
1958), in dioxane (Scheme 1).
exhibit significant bacteriostatic, bactericidal, and fungi-
cidal activities (Potts, 1986). These biological data
prompted us to synthesize some new pyrazole derivatives
incorporating 2-amino-1,3,4-thiadiazole moiety, thus the
aminopyrazole 12 was obtained via treatment of 9 with
dimethylformamide dimethyl acetal (DMF-DMA) in dry
dioxane followed by cyclization of the formed 2-cyano-
3-dimethylamino-N-(1,3,4-thiadiazol-yl)-acrylamide (11) with
hydrazine hydrate. In continuation of our interest in the
synthesis of nitrogen heterocyclic systems (Hamama et al.,
2012a, c), we have found that diazotized heterocyclic
amine is an excellent building block for the synthesis of the
target compounds. Thus, coupling of compound 9 with 4,6-
dimethyl-1H-pyrazolo[3,4-b]pyridin-3-diazonium chloride
(El-Dean et al., 1991), in pyridine at 0–5 °C afforded the
Condensation of compound 9 with 4-(piperidin-1-
yl)benzaldehyde in pyridine afforded the corresponding
arylidene derivative 10. Pyrazole nucleus has pronounced
pharmacological applications as antianxiety and antipyretic
(Wustrow et al., 1998), analgesic, and anti-inflammatory
drugs (Eid et al., 1978; Menozzi et al., 1997; Penning
et al., 1997) and certain alkyl pyrazoles were reported to
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Scheme 2 Reactions of 2-cyano-N-(1,3,4-thiadiazol-2-yl)acetamide (9) with aldehyde, DMF-DMA and diazonium salt
Scheme 3 Synthesis of pyrazol-5(4H)-one 16 and bis(1,3,4-thiadiazole) 18
corresponding hydrazono compound 13. When compound
13 is refluxed in acetic acid, it can be cyclized to pyraz-
olo[5,1-c][1,2,4]triazine derivative 14 (Scheme 2).
Thus, treatment of 1 with 4-flourobenzaldehyde in DMF/
K2CO3 afforded 4-(1,3,4-thiadiazol-2-ylamino)benzalde-
hyde 15, which condensed with 1-phenyl-1H-pyrazol-
5(4H)-one to afford the corresponding arylidene derivative
16. Attempts for the investigation of the reactivity of dia-
zonium salt of 1 toward 1-phenyl-1H-pyrazol-5(4H)-one
Furthermore, we also investigated the reactivity of
1 toward nucleophilic substitution reaction with the aim
of preparing 4-(1,3,4-thiadiazol-2-ylamino)benzaldehyde.
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Scheme 4 Reactions of 2-amino-1,3,4-thiadiazole (1) with different halogenated compounds
(Vogel et al., 1994), in pyridine to give (4Z)-4-(2-(1,3,4-
thiadiazol-2-yl)hydrazono)-1-phenyl-1H-pyrazol-5-(4H-pyrazol-
5(4H)-one (17) were failed, but we obtained (E)-(1,3,
4-thiadiazol-2-ylimino)-2-(1,3,4-thiadiazol-2-yl)hydrazine
(18) (Scheme 3).
Table 1 ABTS antioxidant activity assay N-substituted-2-amino-
1,3,4-thiadiazoles derivatives
Compound no.
ABTS
Absorbance of samples
% Inhibition
Control of ABTS
0.498
0.057
0.356
0.363
0.370
0.370
0.395
0.145
0.355
0.380
0.404
0.240
0.366
0.261
0.376
0.366
28.91
0.309
0.0
Moreover, compound 1 reacted with 4-chlorobenzoyl-
chloride in dioxane/K2CO3 to give the corresponding
N-aroyl derivative 19. Subsequently, reaction of compound
19 with phosphorus oxychloride in dioxane followed by
in situ addition of sodium azide did not furnish the corre-
sponding tetrazole derivative 21. Finally, treatment of
compound 1 with one equivalent amount of 4-chlorobenzyl
chloride afforded the N-(4-chlorobenzyl) derivative 22
(bioisostere of 19). Moreover, compound 23 was achieved
when two equivalent of 4-chlorobenzyl chloride were used
(Scheme 4).
Ascorbic acid
88.55
28.51
27.10
25.70
25.70
20.68
70.88
28.71
23.69
18.87
51.80
26.50
41.56
24.49
26.50
0.354
37.95
1
2
3
6
8
9
10
11
12
13
15
16
18
19
22
23
Biological activity
ABTS antioxidant assay
The antioxidant activity of the synthesized compounds was
evaluated by Lissi et al., 1999. Some of the N-substituted-
2-amino-1,3,4-thiadiazoles exhibited an antioxidant effect
as shown in Table 1. Compared with the control
(L-ascorbic acid), the antioxidant potency of compounds 9
was found to be high, while compounds 13 and 16 showed
a moderate antioxidant activity and the rest tested com-
pounds showed a weak activity. On the other hand, com-
pounds 9, 10, 13, 16, 22, and 23 exhibited a high
antioxidant activity compared to the starting material 1.
From the structure activity relationship (SAR) we found
that, the presence of 4-chlorobenzyl, cyanoacetyl or pyra-
zole moiety in the 2-amino-1,3,4-thiadiazoles enhanced the
antioxidant activity of the 2-amino-1,3,4-thiadiazoles.
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Table 2 Assay for bleomycin-dependent DNA damage (DNA)
Table 3 In vitro cytotoxicity of N-substituted-2-amino-1,3,4-thi-
adiazoles derivatives (Ehrlich ascites cells dead %)
Compound no.
Bleomycin-dependent DNA damage
Absorbance of samples
Compound no. % Dead
ED100 (lg/cm3) ED50 (lg/cm3) ED25 (lg/cm3)
Ascorbic acid
0.098
0.090
0.102
0.099
0.096
0.101
0.080
0.114
0.127
0.102
0.109
0.100
0.105
0.103
0.100
0.108
0.121
1
5-FU
1
97.0
92.9
28.0
37.0
46.1
39.1
42.5
26.0
19.0
80.7
38.3
31.0
42.1
61.5
52.0
6.9
63.0
56.0
16.0
18.0
24.0
18.9
27.0
14.1
10.2
48.2
17.9
17.0
21.0
33.0
24.0
13.0
23.0
16.0
40.0
37.0
7.8
2
3
2
6
5
8.7
8
6
13.0
9.4
9
7
10
11
12
13
15
16
18
19
22
23
8
14.4
7.8
11
13
14
15
16
17
18
19
20
21
23
5.0
25.0
8.7
8.8
10.0
19.3
11.0
6.9
41.0
29.0
12.0
7.4
Bleomycin-dependent DNA damage
Where, ED100, ED50, and ED25 are the effective doses at 25, 50, and
100 ll, respectively, of the compounds used. The dead % refers to the
% of the dead tumor cells and 5-fluorouracil (5-FU) is 5-fluorouracil
as a well-known cytotoxic agent
Damage to DNA in the presence of a Bleomycin-Fe
complex has been adopted as a sensitive and specific
method to examine potential pro-oxidant agents (Gutter-
idge et al., 1981). If the samples to be tested are able to
reduce the bleomycin-Fe3? to Bleomycin-Fe2?, DNA
degradation in this system will be stimulated, resulting in a
positive test for pro-oxidant activity. DNA degradation is
accompanied by the formation of a product similar to
Malondialdehyde (MDA). L-ascorbic acid, a reducing
agent, can reduce Fe3? to Fe2?. Table 2 showed that
compounds 1, 6, and 9 showed a high pro-antioxidant
action compared to the L-ascorbic acid, whereas com-
pounds 23, 10, and 11 have weak effects. On the other hand
the rest of compounds showed good pro-oxidant action in
this system.
compounds 1 and 9 showed high activity, whereas com-
pounds 8, 17, 18, 19, and 21 showed moderate activity on
the other hand the rest of compounds have weak activities.
From the structure activity relationship’s (SAR’s) we found
that N-substitution of 2-amino-1,2,3-thiadiazol decrease the
antitumor activity of 2-amino-1,3,4-thiadiazole.
Conclusion
The prepared new ring systems seem to be interesting for
biological studies. Furthermore, the present investigation
offers rapid and effective new procedures for the synthesis
of a new class of N-substituted-2-amino-1,3,4-thiadiazoles.
The new compounds were investigated for antioxidant
activity. Compounds 1 and 9 exhibited a high antioxidant
activity when compared to the ascorbic acid; these com-
pounds manifested the best protective effect against DNA
damage induced by Bleomycin. Furthermore, N-substi-
tuted-2-amino-1,3,4-thiadiazoles were tested for cytotox-
icity against well-known established model EAC in vitro.
The data showed clearly that N-substitution of 2-amino-
1,2,3-thiadiazol decrease the antitumor activity of 2-amino-
1,3,4-thiadiazole.
Antitumor
Effect of drugs on the viability of Ehrlich ascites cells
(EAC) in vitro (Hamama et al., 2012b): To examine
whether these substances have a direct cytotoxic effect on
Ehrlich ascites cells (EAC) viability, the percentage of
viable cells was estimated by the trypan blue (Sheeja et al.,
1997), exclusion test. Seventeen N-substituted-2-amino-
1,3,4-thiadiazoles were tested for cytotoxicity against well-
known established model EAC in vitro. Results for the
ED100, ED50, and ED25 values of the active compounds are
summarized in Table 3. The data showed clearly that
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Med Chem Res (2013) 22:3556–3565
Experimental section
General
3561
of 2; HRMS(micrOTOF):m/z for C4H4ClN3OS, Calcd.,
177.61 Found, 175.9788(M?-H).
N-(1,3,4-Thiadiazol-2-yl)-2-thiocyanatoacetamide (3)
All melting points are in degree centigrade (uncorrected)
and were determined on Gallenkamp electric melting point
apparatus. Elemental analyses were carried out at Micro
analytical Center, Faculty of Science, Cairo University. IR
spectra were recorded (KBr), (t9 cm-1) on a Mattson 5000
FTIR Spectrophotometer at Micro analytical Center Fac-
ulty of Science, Mansoura University. The 1H NMR
spectra of 3, 6, 7, 8, 10, 12, 13, 16, and 18 were measured
on a Varian Spectrophotometer at 300 MHz, using TMS as
an internal reference and DMSO-d6 or CDCl3 as solvent at
Chemistry Department, Faculty of Science, Cairo Univer-
sity. The 1H NMR and 13C NMR spectra of 2, 9, 11, 19, 22,
and 23 were acquainted on a JEOL ECX-400 spectrometer
Chemistry department, School of Engineering and Science
University of Jacobs, Bremen, Germany, operating at
400 MHz for the 1H NMR and 100 MHz for the 13C NMR
at room temperature in CDCl3 or DMSO-d6, using a 5 mm
probe. High Resolution Mass Spectra (HRMS) were
recorded using both a Bruker HCT ultra and a high reso-
lution (Bruker Daltonics micrOTOF) instruments from
methanol or dichloromethane solutions using the positive
Electrospray Ionization Mode (ESI). Mass spectra were
recorded on (Kratos, 70 eV) MS equipment and/or a Var-
ian MAT 311 A Spectrometer, at Microanalytical Center,
Faculty of Science, Cairo University. Reaction mixtures
were monitored by thin layer chromatography (TLC) using
EM science silica gel coated plates with visualization by
irradiation with ultraviolet lamp. Biological activities were
screened for the tested compounds at Pharmacognosy
Department, Faculty of Pharmacy, Mansoura University,
Mansoura, Egypt.
A mixture of 2 (0.89 g, 5 mmol), ammonium thiocyanate
(0.38 g, 5 mmol) in ethanol (20 mL) was refluxed for 2 h.
The reaction mixture was left to cool and the formed pre-
cipitate was filtered off, dried, and recrystallized from
DMF to furnish 3. Yield 80 % (yellow crystals); m.p.
270 °C (DMF); Rf = 0.25 [pet. ether (60–80)/ethyl acetate
(1:4)]; IR (KBr) mmax (cm-1) 2918 (CH, str.), 2260 (CN),
1
1715 (C=O); H NMR (DMSO-d6) d (ppm) 4.09 (d, 2H,
CH2), 9.29 (s, 1H, CH, thiadiazole), 12.7 (s, 1H, NH).
Anal. Calcd for C5H4N4OS2 (200.24): C, 29.99; H, 2.01; N,
27.98 %. Found: C, 30.03; H, 2.08; N, 28.07 %.
Reaction of chloroacetamide derivative 2 with different
1ory and 2ory amines
General procedure: A mixture of 2 (0.89 g, 5 mmol),
appropriate amines namely; piperazine (0.43 g, 5 mmol),
N-phenyl piperazine (0.81 g, 5 mmol), 2-amino-1,3,4-
thiadiazole (0.51 g, 5 mmol) or 2-amino-1,2,3-thiadiazole-
5-thiol (0.67 g, 5 mmol) and TEA (0.7 mL, 5 mmol) in
dioxane was refluxed for 2 h. The reaction mixture was left
to cool and the formed precipitate was filtered off, dried,
and recrystallized from DMF to furnish the corresponding
thiadiazoles 5–8, respectively.
2-(4-(2-(5-Amino-1,3,4-thiadiazol-2-ylthio)acetyl)
piperazin-1-yl)-N-(1,3,4-thiadiazol-2-yl)acetamide (5)
Yield 60 % (pale green crystals); m.p. 210 °C (ethanol);
Rf = 0.6[pet. ether (80–100)/ethyl acetate (1:1)]; IR (KBr)
mmax (cm-1) 3442 (br, 2NH), 2829, 2874 (CH, aliphatic),
1713 (br, 2CO); MS: m/z (%) = 369 (M??1, 1.4), 283
(0.8), 245 (0.8), 202 (2.9), 176 (2.3), 139 (100), 112 (25.4).
Anal. Calcd for C12H16N8O2S2 (368.44): C, 39.12; H, 4.38;
N, 30.41 %. Found: C, 39.17; H, 4.43; N, 30.46 %.
Chemistry
2-Chloro-N-(1,3,4-thiadiazol-2-yl)acetamide (2)
A mixture of 1 (0.51 g, 5 mmol), chloroacetyl chloride
(0.4 mL, 5 mmol) in dioxane (20 mL) was refluxed for
5 h. The reaction mixture was left to cool, poured into ice
cold water and the precipitated solid was filtered off, dried,
and crystallized from ethanol to give 2. Yield 80 % (white
crystals); m.p. 200 °C; Rf = 0.38 [pet. ether 60–80)/ethyl
acetate (2:3)]; IR (KBr) mmax (cm-1) 3171 (NH), 2900 (CH,
str.), 1709 (C=O); 1H NMR (DMSO-d6) d (ppm) 4.4 (s, 2H,
CH2), 9.1 (s, 1H, CH, thiadiazole); 13C NMR (DMSO-d6) d
(ppm) 165.9 (C=O), 159.1 (C2, thiadiazole), 149.6 (C5,
thiadiazole), 42.8 (CH2). MS (ESI, -98.7 v) (?) ESI
showed two quasi-molecular ion peaks at 199.7 (M??Na)
and 376.8(2M??Na) pointing 177.5 as the molecular mass
2-(4-Phenyl piperazine-1-yl)-N-(1,3,4-thiadiazol-2-
yl)acetamide (6)
Yield 70 % (white crystals); m.p. 191 °C (ethanol);
Rf = 0.34 [pet. ether (60–80)/ethyl acetate (3:7)]; IR (KBr)
mmax (cm-1) 3451 (NH), 2938 (CH, str.), 1696, 1598
(C=C); 1H NMR (CDCl3) d (ppm) 2.82–2.95 (m, 2H,
piperazine), 3.22–3.41 (m, 2H, piperazine), 3.69 (s, 2H,
CH2), 6.85–7.66 (m, 5H, Ar–H), 9.19 (s, 1H, CH, thiadi-
azole), 11.80 (s, 1H, NH); MS: m/z (%) = 303 (M?, 2.62),
273 (3.3), 220 (6.7), 195 (100), 178 (12.6), 152 (11.3), 133
(6.4), 122 (7.1), 85 (76.2), 77 (35.2), 63 (51.3). Anal. Calcd
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(E)-2-Cyano-3-(4-(piperidin-1-yl)phenyl-N-(1,3,4-
for C14H17N5OS (303.38): C, 55.42; H, 5.65; N, 23.08 %.
Found: C, 55.47; H, 5.71; N, 23.13 %.
thiadiazol-2-yl)acrylamide (10)
A mixture of 9 (0.84 g, 5 mmol), 4-(piperidin-1-yl)benz-
aldehyde (0.95 g, 5 mmol) in pyridine (7 mL) was refluxed
for 6 h then the reaction mixture was left to cool and
poured into ice cold water. The formed precipitate filtered
off, dried, and crystallized from ethanol to give 10. Yield
70 % (reddish brown crystals); m.p. 268 °C (ethanol);
Rf = 0.4[pet. ether (60–80)/ethyl acetate (1:1.5)]; IR (KBr)
mmax (cm-1) 3394 (NH), 2937 (CH, str.), 2201 (CN), 1678
2-(1,3,4-Thiadiazol-2-ylamino)-N-(1,3,4-thiadiazol-2-yl)
acetamide (7)
Yield 50 % (yellow crystals); m.p. 226 °C (ethanol);
Rf = 0.24 [pet. ether (60–80)/ethyl acetate (3:7)]; IR (KBr)
m
max (cm-1) 3183 (NH), 2980 (CH, str.), 1702 (C=O), 1616
1
(C=N); H NMR (DMSO-d6) d (ppm) 4.45 (s, 2H, CH2),
8.94 (s, 1H, CH, thiadiazole), 9.22 (s, 1H, CH, thiadiazole),
10.42 (s, 1H, NH), 13.16 (s, 1H, NHCO). Anal. Calcd for
C6H6N6OS2 (242.28): C, 29.74; H, 2.50; N, 34.69 %.
Found: C, 29.71; H, 2.46; N, 34.64 %.
1
(C=O), 1608 (C=N); H NMR (CDCl3) d (ppm) 1.60–1.78
(m, 6H, 3CH2), 3.44–3.52 (m, 4H, CH2N), 7.96 (d, 2H,
Ar–H, J = 9 Hz), 6.85 (d, 2H, Ar–H, J = 9 Hz), 8.27 (s,
1H, methine), 8.87 (s, 1H, C-5H, thiadiazole), 12.15 (s, 1H,
NHCO). Anal. Calcd for C17H17N5OS (339.41): C, 60.16;
H, 5.05; N, 20.63 %. Found: C, 60.27; H, 5.14; N,
20.72 %.
2-(5-Amino-1,3,4-thiadiazol-2-ylthio)-N-(1,3,4-thiadiazol-
2-yl) acetamide (8)
Yield 80 % (white crystals); m.p above 300 °C (DMF);
Rf = 0.17[pet. Ether (60–80)/ethyl acetate (3:7)]; IR (KBr)
mmax (cm-1) 3315 (NH2), 3132 (NH), 1678 (C=O), 1592
2-Cyano-3-(dimethylamino)-N-(1,3,4-thiadiazol-2-yl)
acrylamide (11)
1
(C=C); H NMR (DMSO-d6) d (ppm) 4.13 (s, 2H, CH2),
A mixture of 9 (0.84 g, 5 mmol) and DMF-DMA
(0.66 mL, 5 mmol) in DMF (5 mL) was heated at 60 °C.
The reaction mixture was left to cool and the precipitated
solid was collected by filtration and recrystallized from
ethanol to give 11. Yield 60 % (reddish brown crystals);
m.p. 224 °C (ethanol); Rf = 0.25 [pet. ether (60–80)/ethyl
acetate (2:5)]; IR (KBr) mmax (cm-1) 3217 (NH), 2935 (CH,
str.), 2199 (CN), 1674(C=O), 1611 (C=N); 1H NMR
(DMSO-d6) d (ppm) 3.20 (s, 3H, CH3), 3.27 (s, 3H, CH3),
8.02 (s, 1H, CH, methine), 9.11 (s, 1H, C-5H, thiadiazole),
9.83 (s, 1H, NH); 13C NMR (DMSO-d6) d (ppm)
160(C=O), 157.9 (C2, thiadiazole), 157.7 (C3-acrylamide),
148.7 (C5, thiadiazole), 71.4 (C2, acrylamide), 118.7 (CN),
47.7 (2 CH3); HRMS(micrOTOF):m/z for C8H9N5OS,
Calcd., 223.25. Found, 246.039(M??Na), 469.0920
(2M??Na).
7.30 (s, 1H, NH2), 9.18 (s, 1H, CH, thiadiazole), 12.81
(s, 1H, NH). Anal. Calcd for C6H6N6OS3 (274.35): C,
26.27; H, 2.20; N, 30.63 %. Found: C, 26.34; H, 2.28; N,
30.67 %.
Synthesis of 2-cyano-N-(1,3,4-thiadiazol-2-yl)
acetamide (9)
Method A: A mixture of 2 (0.89 g, 5 mmol) and potassium
cyanide (0.39 g, 6 mmol) in DMF (15 mL) was heated at
60 °C for 8 h. The reaction mixture was left to cool then
poured into ice cold water and the formed precipitate was
filtered off, dried, and crystallized from ethanol to give 9.
Method B: A mixture of 1 (1.42 g, 14 mmol) and 3-(3,
4-dimethyl-1H-pyrazol-1-yl)-3-oxopropanenitrile (2.28 g,
14 mmol) in dioxane (20 mL) was refluxed for 5 h. The
solvent was evaporated under vacuum and the residue was
crystallized form ethanol to give 9. Yield 90 % (white
crystals); m.p. 241 °C (ethanol); Rf = 0.11 [pet. ether
(60–80)/ethyl acetate (2:3)]; IR (KBr) mmax (cm-1) 3423
3-Amino-N-(1,3,4-thiadiazol-2-yl)-1H-pyrazole-4-
carboxamide (12)
1
A mixture of 11 (1.12 g, 5 mmol), hydrazine hydrate
(0.24 mL, 5 mmol) in ethanol (10 mL) was heated under
reflux for 2 h. The formed precipitate was filtered off,
washed with ethanol, and recrystallization from DMF to
give 12. Yield 70 % (white crystals); m.p. 286 °C (DMF);
Rf = 0.3[pet. ether (60–80)/ethyl acetate (2:5)]; IR (KBr)
mmax (cm-1) 3472, 3362, 3204 (NH2, NH), 1670 (C=O),
(br, NH), 2859 (CH, str.), 2181 (CN), 1610 (br, CO); H
NMR (DMSO-d6) d (ppm) 4.05 (s, 2H, CH2), 9.17 (s, 1H,
C-5H, thiadiazole), 12.11 (s, 1H, NHCO); 13C NMR
(DMSO-d6) d (ppm) 162.9 (C=O), 158.9 (C2), 149.6 (C5),
115.5(CN), 49.1 (CH2); MS (ESI, -98.7v) (?)ESI showed
one quasi-molecular ion peak at 190.8 (M??Na), the
(-)-ESI mass spectrum showed one quasi-molecular ion
peak at 166.7 ([M-2H]) pointing 168 as the molecular mass
of 9; HRMS(micrOTOF):m/z for C5H4N4OS, Calcd.,
168.18. Found, 167.0040 (M?-H).
1
1622 (C=N); H NMR (DMSO-d6) d (ppm) 6.2 (s, 2H,
NH2), 8.09 (s, 1H, C5-H, pyrazole), 9.09 (s, 1H, C5-H,
thiadiazole), 11.88 (s, 1H, NH, pyrazole), 11.21 (s, 1H,
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Med Chem Res (2013) 22:3556–3565
3563
(4E)-4-(4-(1,3,4-Thiadiazol-2-ylamino)benzylidene)-3-
NHCO). Anal. Calcd for C6H6N6OS (210.22): C, 34.28; H,
2.88; N, 39.98 %. Found: C, 34.35; H, 2.94; N, 39.94 %.
methyl-1-phenyl-1H-pyrazol-5(4H)-one (16)
A mixture of 15 (1.03 g, 5 mmol), 3-methyl-1-phenyl-1H-
pyrazol-5(4H)-one (0.87 g, 5 mmol) and piperidine (0.5 mL,
5 mmol) in ethanol (20 mL) was refluxed for 2 h. The
reaction mixture was left to cool and the formed precipitate
was filtered off, dried, and recrystallized from DMF to give
16. Yield 65 % (red crystals); m.p. 210 °C (DMF);
Rf = 0.48[pet. ether (60–80)/ethyl acetate (4:3)]; IR (KBr)
mmax (cm-1) 3421 (NH), 2918 (CH, str.), 1668 (C=O), 1572
2-(2-(4-Methyl-1H-pyrazolo[3,4-b]pyridine-3-yl)diazenyl)-
2-cyano-N-(1,3,4-thia-diazol-2-yl)acetamide (13)
To a well-stirred cooled solution of 4,6-dimethyl-1H-pyr-
azolo[3,4-b]pyridin-3-amine (0.81 g, 5 mmol) in conc.
HCl (3 mL), a solution of NaNO2 (0.35 g, 5.1 mmol in
5 mL H2O) was added dropwise. The above cooled dia-
zonium solution was added slowly to a well-stirred solution
of 9 (0.84 g, 5 mmol) in pyridine (10 mL). The reaction
mixture was stirred for 2 h. The crude product was filtered
off, dried well, and recrystallized from the EtOH-benzene
to afford 13. Yield 80 % (reddish brown crystals); m.p.
above 300 °C (DMF); Rf = 0.4 [ethyl acetate/methanol
20:3]; IR (KBr) mmax (cm-1) 3386, 3268, (2NH), 2925 (CH,
str.), 2194 (CN), 1681 (C=O); 1H NMR (DMSO-d6) d
(ppm) 2.84 (s, 3H, CH3), 2.95 (s, 3H, CH3), 6.96 (s, 1H,
C5-H, pyridine), 9.11 (s, 1H, C5-H, thiadiazole), 11.92
(s, 1H, NH, pyrazole), 12.91 (s, 1H, NHCO), 14.12 (s, 1H,
NH–N=C). Anal. Calcd for C13H11N9OS (341.35): C,
45.74; H, 3.25; N, 36.93 %. Found: C, 45.85; H, 3.32; N,
37.02 %.
1
(C=C); H NMR (CDCl3) d (ppm) 2.32 (s, 3H, CH3), 6.72-
8.03 (m, 6H, Ar–H, NH), 8.55 (s, 1H, methine), 8.58 (s, C5-H,
thiadiazole). MS: m/z (%) = 361 (M?, 0.23), 360 (M?-1,
0.23), 277 (1.3), 261 (1.3), 218 (1.4), 185 (8.2), 172 (100),
156 (12.9), 128 (17.8), 91 (7.1), 78 (32.8), 63 (42.4), 43
(26.8). Anal. Calcd for C19H15N5OS (361.42): C, 63.14; H,
4.18; N, 19.38 %. Found: C, 63.22; H, 4.19; N, 19.46 %.
(E)-1-(1,2,3-Thiadiazol-2-ylimino)-2-(1,2,3-thiadiazol-2-
yl)hydrazine (18)
Method A: To a well-stirred cooled solution of 1 (0.51 g,
5 mmol) in conc. HCl (3 mL), a solution of NaNO2
(0.35 g, 5.1 mmol in 5 mL H2O) was added dropwise. The
above cooled diazonium solution was added slowly to a
well-stirred solution of 1-phenyl-1H-pyrazol-5(4H)-one
(0.87 g, 5 mmol) in ethanol (20 mL) and sodium acetate
(1.64 g, 20 mmol). The reaction mixture was stirred for
2 h. The crude product was filtered off, dried well, and
recrystallized from the EtOH to give 18.
8-Amino-2,4-dimethyl-1,5,6,8a,9-pentaazafluorene-7-
carboxylic acid (1,3,4-thiadiazol-2-yl)-amide (14)
A suspension of 13 (1.71 g, 5 mmol) in acetic acid
(10 mL) was refluxed for 7 h. The reaction mixture was
poured into ice cold water. The formed precipitate was
filtered off, dried and recrystallized from DMF to give 14.
Yield 50 % (yellow crystals); m.p. above 300 °C (DMF);
Rf = 0.4 [ethyl acetate containing drops of methanol]; IR
(KBr) mmax (cm-1) 3442, 3372, 3262 (NH2, NH), 1677
(C=O), 1600 (C=C). Anal. Calcd for C13H11N9OS
(341.35): C, 45.74; H, 3.25; N, 36.93 %. Found: C, 45.83;
H, 3.31; N, 36.97 %.
Method B: Hydrochloric acid (0.9 mL) in water
(3.4 mL) was added to 1 (0.61 g, 6 mmol) in ice cold water
then add sodium nitrite (0.2 g in 0.5 mL water) drop wise
for 10 min. The reaction was stirred for 30 min., then raise
pH by adding sodium acetate (0.9 g) in water (1.8 mL),
stirring the mixture for 30 min. The formed precipitate was
filtered off, washed with water, and recrystallized from
ethanol to afford 18. Yield 85 % (yellow crystals); m.p.
228 °C (ethanol); Rf = 0.6[ethyl acetate containing drops
of methanol]; IR (KBr) mmax (cm-1) 3454 (NH), 1570
4-(1,3,4-Thiadiazol-2-ylamino)benzaldehyde (15)
1
(–N=N–); H NMR (DMSO-d6) d (ppm) 9.26 (br., s, 2H,
A mixture of 1 (0.51 g, 5 mmol), p-fluorobenzaldehyde
(0.62 g, 5 mmol) and potassium carbonate (0.69 g,
5 mmol) in DMF (10 mL) was refluxed on water bath for
10 h, poured into ice-water and the resultant solid was
filtered and recrystallized from ethanol to give 15. Yield
90 % (yellow crystals); m.p. 73 °C (ethanol); Rf = 0.48
[pet.ether(80–100)/ethyl acetate (4:1)]; IR (KBr) mmax
(cm-1) 3417 (NH), 2908 (CH, str.), 1662 (CHO), 1598
(C=C); MS: m/z (%) = 203 (M?-2H, 0.98), 176 (4.9), 148
(8.4), 121 (5.2), 106 (3.5), 95 (3.7), 78 (44.0), 63 (81.6), 45
(100). Anal. Calcd for C9H7N3OS (205.24): C, 52.67; H,
3.44; N, 20.47 %. Found: C, 52.61; H, 3.47; N, 20.43 %.
C5-H, thiadiazole), 14.13 (br, s, 1H, NH); MS (EI, 76 eV)
m/z (%) = 213 (M?, 4.27), 113 (100), 114 (5.32), 84
(19.25), 45 (84.9). Anal. Calcd for C4H3N7S2 (213.24): C,
22.53; H, 1.42; N, 45.98 %. Found: C, 22.61; H, 1.47; N,
46.04 %.
Reaction of 2-amino-1,3,4-thiadiazole (1) with 4-
chlorobenzoyl chloride and 4-chlorobenzyl chloride
Generalprocedure: A mixture of 1 (0.51 g, 5 mmol), appro-
priate aromatic chloro derivatives namely; 4-chlorobenzoyl
123

3564
Med Chem Res (2013) 22:3556–3565
chloride (0.88 g, 5 mmol), 4-chlorobenzyl chloride
(0.81 g, 5 mmol) or 4-chlorobenzyl chloride (1.61 g,
10 mmol) in dioxane (20 mL) was refluxed for 2 h, The
reaction mixture was left to cool and the formed precipitate
was filtered off, dried, and recrystallized from ethanol to
furnish 19, 22, and 23, respectively.
cation derived from ABTS [2,20-azino-bis (3-ethyl ben-
zothiazoline-6-sulfonic acid)] was prepared by reaction of
ABTS (60 ll) with MnO2 (3 mL, 25 mg/mL) in (5 mL)
aqueous buffer solution (pH 7). After shaking the solution
for a few minutes, it was centrifuged and filtered. The
Absorbance (A control) of the resulting green–blue solu-
tion (ABTS radical solution) was recorded at kmax 734 nm.
The absorbance (A test) was measured upon the addition of
(20 ll of 1 mg/mL) solution of the tested sample in spec-
troscopic grade MeOH/buffer (1:1 v/v) to the ABTS
solution. The inhibition ratio (%) was calculated using the
following formula: % Inhibition = [A (control) - A (test)/
A (control)] 9 100
4-Chloro-N-(1,3,4-thiadiazol-2-yl)benzamide (19)
Yield 65 % (white crystals); m.p. 270 °C (ethanol); Rf = 0.6
[pet. ether 60–80)/ethyl acetate (3:7)]; IR (KBr) mmax (cm-1
)
3437 (NH), 1672 (C=O); 13C NMR (DMSO-d6) d (ppm)
165.1 (C=O), 160.3 (C2, thiadiazole), 149.5 (C5, thiadia-
zole), 138.3, 131.2, 130.8 (2C), 129 (2C); MS (ESI, -98.7 v)
(?)-ESI mass spectrum showed one quasi-molecular ion
peak at 261.8 (M??Na), (-)-ESI mass spectrum showed
one quasi-molecular ion peak at 237.7 (M?-H) pointing 239
as the molecular mass of 23; HRMS(micrOTOF): m/z for
C9H6N3OSCl, Calcd., 239.5. Found, 237.9866 (M?-H).
Ascorbic acid (20 ll, 2 mM) solution was used as a
standard antioxidant (positive control). Blank sample was
run using solvent without ABTS (Table 2).
Bleomycin-dependent DNA damage
The assay was done according to Aeschbach et al., 1994
and Chan and Tang, 1996, with minor modifications. The
reaction mixture (0.5 mL) contained DNA (0.5 mg/mL),
Bleomycin sulfate (0.05 mg/mL), MgCl2 (5 mM), FeCl3
(50 mM) and the samples were dissolved in DMSO to be
tested at concentration (20 ll of 1 mg/mL). L-Ascorbic
acid was used as a positive control. The mixture was
incubated at 37 °C for 1 h. The reaction was terminated by
the addition of 0.05 mL EDTA (0.1 M). The color was
developed by adding thiobarbituric acid (TBA) (0.5 mL)
(1 %, w/v) and HCl (0.5 mL) (25 %, v/v) followed by
heating at 80 °C for 10 min. After centrifugation, the
extent of DNA damage was measured by the increase in
absorbance at 532 nm.
N-(4-Chlorobenzyl)-1,3,4-thiadiazol-2-amine (22)
Yield 30 % (yellow crystals); m.p 143 °C (ethanol);
Rf = 0.34 [pet. ether (80–100)/ethyl acetate (4:2.5)]; IR
(KBr) mmax (cm-1) 3417 (NH), 2946(CH, str.), 1596
1
(C=C), H NMR (DMSO-d6) d (ppm) 5.5 (s, 2H, CH2),
7.3–7.4 (m, 4H, Ar–H), 8.8 (s, 1H, CH, thiadiazole), 10.6
(s, 1H, NH); 13C NMR (DMSO-d6) d (ppm) 167.4 (C2,
thiadiazole), 146 (C5, thiadiazole), 133.7, 133.4, 130.7,
129.3 (2C), 39.4 (CH2); HRMS(micrOTOF):m/z for
C9H8ClN3S, Calcd., 225.7. Found, 226.0218(M ? H).
(5E)-N-(3-(4-Chlorobenzyl)-1,3,4-thiadiazol-2(3H)-
ylidene)(4-chlorophenyl) methan-amin (23)
Yield 40 % (yellow crystals); m.p. 227 °C (ethanol);
Rf = 0.73 [pet. ether (60–80)/ethyl acetate (2:3)]; IR (KBr)
mmax (cm-1) 2901 (CH, str.), 1631 (C=N), 1H NMR
(DMSO-d6) d (ppm): 4.2 (s, 2H, CH2–N=C), 5.3 (s, 2H,
CH2–N–N), 7.2–7.4 (m, 8H, Ar–H), 8.6 (s, 1H, CH thia-
diazole); 13C NMR (DMSO-d6) d (ppm) 159.5 (C2, thia-
diazole), 146.2 (C5, thiadiazole), 131.1 (2C‘), 130.4 (2C),
129.1 (2C), 128.7 (2C), 39.8 (CH2), 39.6 (CH2). Anal.
Calcd for C16H13Cl2N3S (350.27): C, 54.86; H, 3.74; N,
12.00 %. Found: C, 54.96; H, 3.82; N, 12.08 %.
Antitumor activity
Different concentrations of the tested compounds were
prepared (ED100, ED50, and ED25 1 g mL-1 DMSO). The
amount of DMSO was adjusted to give a final concentra-
tion of 0.1 %. Ascites fluid obtained was aseptically aspi-
rated from the peritoneal cavity of the donor animal
(National Cancer Institute, Cairo, Egypt), which contains
Ehrlich cell. The cells were grown partially floating and
attach in AQ4 a suspension culture (RPMI 1660 medium,
Sigma Chemical, St. Louis), supplemented with 10 % fetal
bovine serum (GIBCO, UK). They were maintained at
37 °C in humidified atmosphere with 5 % CO2 for 2 h. The
viability of the cell used in control experiments (DMSO
only without drug) exceeded 95 % as determined by
microscopic examination using a hemocytometer and try-
pan blue stain (stain only the dead cells).
Materials and methods
Antioxidant activity screening assay; ABTS method (Lissi
et al., 1999)
Antioxidant activity determinations were evaluated from
the bleaching of ABTS derived radical cations. The radical
123

Med Chem Res (2013) 22:3556–3565
3565
3D-QSAR study of N-(5-substituted-1,3,4-thiadiazol-2-yl)cyclo-
propanecarboxamides. Eur J Med Chem 44:2782–2786
Madapa S, Tusi Z, Sridhar D, Kumar A, Siddiqi MI, Srivastava K,
Rizvi A, Tripathi R, Puri SK, Keshava GBS, Shukla PK, Batra S
(2009) Search for new pharmacophores for antimalarial activity.
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Matysiak J, Niewiadomy A (2006) Application of sulfinylbis(2,4-
dihydroxy-thiobenzoyl) in the synthesis of N-substituted 2-amino-
5-(2,4-dihydroxyphenyl)-1,3,4-thiadiazoles. Synth Commun 36:
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