A facile green synthesis and anti-cancer activity of bis- arylhydrazononitriles, triazolo[5,1-c][1,2,4]triazine, and 1,3,4-thiadiazolines

Article abstract of DOI:10.3987/COM-13-12696

Coupling of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with diazonium salts of 1,4-benzenediamine (2), 2,6-dichlorobenzene-1,4-diamine (4), and benzidine (6) afforded bis-arylhydrazononitriles 3, 5, and 7, respectively. Also, coupling of 1 with [1,2,4]triazole-3-diazonium sulfate (8) gave the respective [1,2,4]triazolo[5,1-c][1,2,4]triazine derivative 11. On the other hands, treatment of 2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino) acrylonitrile (12) with hydrazonoyl chlorides 13a-h in dioxane, in the presence of chitosan as eco-friendly heterogeneous basic catalyst, under microwave irradiation furnished 1,3,4-thiadiazolines 16a-h, incorporating pyrrole moiety. The anti-cancer activities of the synthesized products were determined against the colon carcinoma (HCT), human laryngeal carcinoma (Hep-2), human medulloblastoma (Daoy), human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma (WiDr) cell lines.

Full text of DOI:10.3987/COM-13-12696

HETEROCYCLES, Vol. 87, No. 5, 2013, pp. 1109 - 1120. © 2013 The Japan Institute of Heterocyclic Chemistry
Received, 8th March, 2013, Accepted, 4th April, 2013, Published online, 8th April, 2013
DOI: 10.3987/COM-13-12696
A FACILE GREEN SYNTHESIS AND ANTI-CANCER ACTIVITY OF
BIS-ARYLHYDRAZONONITRILES, TRIAZOLO[5,1-c][1,2,4]TRIAZINE,
AND 1,3,4-THIADIAZOLINES
Sobhi M. Gomha,1 Khaled D. Khalil,1 Ali M. El-Zanaty,2,3 and Sayed M.
Riyadh*1,2
1) Department of Chemistry, Faculty of Science, Cairo University, Giza, 12613,
Egypt
2) Department of Chemistry, Faculty of Science, King Khalid University, Abha,
9004, KSA
3) Department of Chemistry, Faculty of Science, Beni-Suef University, Egypt
*Corresponding author: E-mail: riyadh1993@hotmail.com
Abstract – Coupling of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with diazonium
salts of 1,4-benzenediamine (2), 2,6-dichlorobenzene-1,4-diamine (4), and
benzidine (6) afforded bis-arylhydrazononitriles 3, 5, and 7, respectively. Also,
coupling of 1 with [1,2,4]triazole-3-diazonium sulfate (8) gave the respective
[1,2,4]triazolo[5,1-c][1,2,4]triazine derivative 11. On the other hands, treatment of
2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino)acrylonitrile
(12) with hydrazonoyl chlorides 13a-h in dioxane, in the presence of chitosan as
eco-friendly heterogeneous basic catalyst, under microwave irradiation furnished
1,3,4-thiadiazolines 16a-h, incorporating pyrrole moiety. The anti-cancer
activities of the synthesized products were determined against the colon
carcinoma (HCT), human laryngeal carcinoma (Hep-2), human medulloblastoma
(Daoy), human breast adenocarcinoma (MCF-7), and human colon
adenocarcinoma (WiDr) cell lines.
Arylazo compounds have multifarious industrial applications such as printing, electronic photography,
liquid crystal displays, laser technology,1 and dye manufacture.2 Also, they are widely used in
chromoionophores because they could exhibit substantial color changes observable by the naked eyes

upon complexation with metal ions3 such as Ca+2, and Pb+2. Moreover, bis-arylazo compounds were
reported to be used as a ratiometric and specific chromogenic sensor for Hg+2 in polar protic solvent.4 The
rich chemistry of azo compounds is also associated with several important biological activities such as
antimicrobial,5 antifungal,6 anticonvulsant,7 antioxidant, and antitumor.8 Recently, the synthesis of
thiadiazoline derivatives9 has attracted considerable attention because these compounds have a wide
range of biological properties. Thiadiazolines could be act as anthelmintics,10 antihypertensive,11
antitumor,12 analgesic,13 and antimicrobial agents.14 In view of the immense importance associated with
arylazo and thiadiazolines and in continuation with our work related to the synthesis and evaluation the
biological activities of aza-heterocycles,15-18 it was envisaged to undertake the synthesis and evaluation of
the anti-cancer activity of a series of novel bis-arylazo and 1,3,4-thiadiazoline compounds. In this context,
we could successfully utilized chitosan as efficient, eco-friendly heterogeneous basic catalyst under
microwave irradiation to afford a novel environmentally benign route for synthesis of 1,3,4-thiadiazoline
compounds.
2-Cyanoacetyl-1-methyl-1H-pyrrole (1),19 prepared via cyanoacetylation of 1-methyl-1H-pyrrole in the
presence of catalytic amount of InCl3, was coupled with diazonium salts of appropriate aromatic amines
[1,4-benzenediamine (2), 2,6-dichlorobenzene-1,4-diamine (4), and benzidine (6)] in molar ratio (2:1) in
ethanol in the presence of sodium acetate to afford the respective bis-hydrazones [3, 5, and 7,
respectively] (Scheme 1). The structures of the products were established on the basis of their elemental
analyses and spectral data (IR, 1H NMR, 13C NMR, MS).
Hydrazones can be formulated in different possible tautomeric structures, namely keto-hydrazone (A),
1
azo-enol (B), and CH-azo tautomer (C) (Figure 1). The H NMR spectra of the studied compounds
showed signal in the region of  = 13.83-13.92 ppm assignable to hydrazone proton (-CH=N-NH-).20
Signal at  = 5.27 ppm,21 which is characteristic for the CH protons of CH-azo form, was not detected.
Also, IR spectra did not reveal broad signal at 3434 cm-1, which is characteristic for OH of azo-enol
form.22 These spectroscopic analyses allow to rule out the azo-enol form (B), CH-azo form (C), and
confirm keto-hydrazone form (A) (Figure 1).
Keto-hydrazone tautomer (A) can be existed in two geometric structures (E and Z). IR spectra of
compounds 3, 5, and 7 showed a shift of the CO bond stretching to lower wave number due to both
conjugation with C=N and formation of a hydrogen bond with the NH group. Furthermore, the downfield
1
shift of hydrazone proton signal ( = 13.83-13.92 ppm) in H NMR is characteristic for the formation of
an intramolecular hydrogen bond of the NH proton for structure (A). These results confirm only the
presence of E- form.23

In continuation of our interest in the synthesis of bridged-head nitrogen heterocyclic systems,24 we have
found that diazotized heterocyclic amines are excellent building blocks for the synthesis of the target
compounds. Thus, coupling of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with [1,2,4]triazole-3-diazonium
o
sulfate (8) in pyridine at 0-5 C afforded 4-amino-3-[(1-methyl-1H-pyrrol-2-yl)carbonyl][1,2,4]triazolo-
[5,1-c][1,2,4]triazine (11)25 (Scheme 2). The structure of the isolated product was elaborated by its
elemental analyses and spectral data. IR spectrum of 11 showed absorption bands at v = 3389, 3221 cm-1
1
assignable to NH2 group besides carbonyl absorption band at v = 1687 cm−1. Also, its H NMR

spectrum revealed a singlet signal at  3.90 ppm (D2O-exchangeable) due to NH2 protons. The
mechanism of formation of product 11 seems to start via initial coupling of diazonium salt on active
methylene group in compound 1 to form the respective non-isolable hydrazone intermediate 9 which then
underwent intramolecular cyclization followed by aromatization to give the isolated product 11 (cf.
Scheme 2).
Next, our study was extended to explore a new synthetic approach to 1,3,4-thiadiazoline derivatives via
green chemical techniques. Thus, stirring of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with phenyl
isothiocyante in dimethylformamide, in the presence of potassium hydroxide, at room temperature gave
the respective 2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino)acrylonitrile (12) as
previously mentioned26 (Scheme 3). Ttreatment of equimolar amounts of 12 with hydrazonoyl chlorides
13a-h in dioxane, in the presence of catalytic amount of chitosan (10% wt), under microwave irradiation
resulted in the formation of 1,3,4-thiadiazolines 16a-h, with reaction times from 3 to 5 min at 300 W of
o
power and temperatures of 150-160 C (Scheme 3). The reaction products were easily obtained in high
yields (90-94%). The spectroscopic data are consistent with structures 16a-h. These compounds showed
the stretching bands of the C≡N group at 2188-2200 cm-1 while the carbonyl bands appeared at
1698-1705 cm-1. 1H NMR revealed a singlet signal at  3.47-3.78 ppm corresponding to (N-CH3) protons
and another multiplet signals at  7.12-8.16 ppm corresponding to aromatic protons. In the 13C NMR
spectrum the presence of a signal at  65.81-65.93 ppm, corresponding to vinylic carbon adjacent to
nitrile group, and another signal at  164.30-165.42 ppm, corresponding to thiadiazoline C-2, confirms
that such structure corresponds to 1,3,4-thiadiazol-2(3H)-ylidene derivatives.27

In Scheme 3, we postulate a plausible mechanism for such reactions. Firstly, nucleophilic displacement of
(SH) group with concurrent elimination of hydrogen chloride afforded intermediates 14a-h. Intramolecular
Michael type addition of (NH) group to vinylic double bond gave non-isolable Michael adducts 15a-h.
Finally, elimination of aniline molecule from intermediates 15a-h gave the isolated products 16a-h.
The anti-cancer activities of the synthesized compounds 3, 5, 7, 11, 16a, 16b, 16f and 16h were
determined against the colon carcinoma (HCT), human laryngeal carcinoma (Hep-2), human
medulloblastoma (Daoy), human breast adenocarcinoma (MCF-7), and human colon adenocarcinoma
(WiDr) cell lines. Data generated were used to plot a dose response curve of which the concentration of
test compounds required to kill 50% of cell population (IC50) was determined. Cytotoxic activity was
expressed as the mean IC50 of three independent experiments (Table 1).

Table 1. Cytotoxic activities of compounds 3, 5, 7, 11, 16a, 16b, 16f, and 16h
against tumor cell lines
Compound
No.
IC50 (g / mL)
Daoy
HCT
18.60
20.41
24.03
> 50
Hep-2
7.03
MCF-7
7.64
WiDr
6.87
3
5
8.14
10.32
14.34
25.31
17.32
16.54
15.68
19.34
11.34
11.74
14.62
26.07
16.98
16.11
15.14
17.62
10.02
13.05
27.48
17.55
17.28
15.78
19.16
7
14.56
11
26.04
16a
16b
16f
16h
36.21
32.87
33.21
38.42
17.05
16.34
15.97
18.91
Cell lines; HCT (colon carcinoma), Hep-2 (human laryngeal carcinoma), Daoy (human medulloblastoma),
MCF-7 (human breast adenocarcinoma), WiDr (human colon adenocarcinoma)
The results revealed that, bis-arylhydrazononitriles 3, 5, and 7 have highest cytotoxicity values compared to
triazolotriazine 11 and 1,3,4-thiadiazolines 16, which could be attributed to formation of azo-hydrazo
isomers.28 Also, 1,3,4-thiadiazolines 16a, 16b, 16f, and 16h have higher cytotoxicity values than
triazolotriazine 11, which could be attributed to size of heterocycles, number of nitrogen atoms in azoles
and azines rings, and the nature of fusion of azoles or azines.28
Novel series of bis-arylhydrazononitriles, triazolotriazine, and 1,3,4-thiadiazolines, bearing pyrrole
moiety were synthesized and evaluated for their anticancer activities. Most of the newly synthesized
products revealed moderate anticancer activity against colon carcinoma (HCT), human laryngeal
carcinoma (Hep-2), human medulloblastoma (Daoy), human breast adenocarcinoma (MCF-7), and human
colon adenocarcinoma (WiDr) cell lines.
EXPERIMENTAL
General
Melting points were measured on a Gallenkamp melting point apparatus (Weiss-Gallenkamp, London,
UK). IR spectra were recorded in potassium bromide discs on Pye Unicam SP 3300 and Shimadzu FTIR
8101 PC infrared spectrophotometers. NMR spectra were recorded on a Varian Mercury VX-300 NMR
spectrometer operating at 400 MHz (1H NMR) or 100 MHz (13C NMR) and run in deuterated

dimethylsulfoxide (DMSO-d6). Chemical shifts were related to that of the solvent. Mass spectra were
recorded on a Shimadzu GCeMS-QP1000 EX mass spectrometer at 70 eV. Elemental analyses were
measured by using a German made Elementar vario LIII CHNS analyzer. Microwave experiments were
carried out using CEM Discover Labmate microwave apparatus (300 W with Chem. Driver Software).
The cytotoxic evaluation of some selected examples was carried out in the Regional Center for Mycology
and Biotechnology of Al-Azhar University, Cairo, Egypt. 2-Cyanoacetyl-1-methyl-1H-pyrrole (1)19 and
hydrazonoyl halides29,30 were prepared as previously reported in the respective literature.
Coupling of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with the appropriate diazonium salt of aromatic
amines
General procedure:
To a cold solution of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) (0.296 g, 2 mmol) in EtOH (20 mL),
containing sodium acetate trihydrate (0.138 g, 1 mmol), was added the appropriate diazonium salt of
aromatic amines [1,4-benzenediamine (2) (0.108 g, 1 mmol), 2,6-dichlorobenzene-1,4-diamine (4) (0.177
g, 1 mmol), and benzidine (6) (0.184 g, 1 mmol)] portion wise over a period of 30 min. After stirring for
o
further 2 h, at 0-5 C, the reaction mixture was diluted with water and extracted with CHCl3. After
evaporation of solvent, the crude product was purified by passing through column chromatography on
silica gel using hexane/EtOAc (5:1) as an eluent.
N',N''-(1,4-Phenylene)bis[2-(1-methyl-1H-pyrrol-2-yl)-2-oxoacetohydrazonoyl cyanide] (3).
Red solid, (0.26 g, 61%), mp 230-232 oC; IR (KBr)  3281, 3154 (2NH), 2249, 2253 (2C≡N), 1691, 1687
1
(2C=O) cm-1; H NMR (DMSO-d6)  = 3.82 (s, 6H, 2 N-CH3), 6.35-7.79 (m, 10H, Ar-H), 13.85 (s, 2H,
D2O-exchangeable, 2NH) ppm; 13C NMR (DMSO-d6)  = 32.62 (2CH3), 116.88 (2C≡N), 110.18, 121.72,
123.54, 129.43, 132.18, 148.22 (Ar-C), 178.12 (2C=O); MS, m/z (%) 426 (M+, 7), 400 (40), 318 (100).
Anal. Calcd for C22H18N8O2 (426.16): C, 61.96; H, 4.25; N, 26.28. Found: C, 62.07; H, 4.38; N, 26.40%.
N',N''-(2,6-Dichloro-1,4-phenylene)bis[2-(1-methyl-1H-pyrrol-2-yl)-2-oxoacetohydrazonoyl cyanide]
(5).
Dark red solid, (0.35 g, 71%), mp 249-251 oC; IR (KBr)  3265, 3158 (2NH), 2248, 2259 (2C≡N), 1698,
1
1685 (2C=O) cm-1; H NMR (DMSO-d6)  = 3.80 (s, 6H, 2 N-CH3), 6.77-7.99 (m, 8H, Ar-H), 13.83 (s,
13
2H, D2O-exchangeable, 2NH) ppm; C NMR (DMSO-d6)  = 32.52 (2CH3), 116.68 (2C≡N), 112.38,
122.21, 123.54, 123.98, 126.87, 129.43, 132.18, 136.37, 148.31 (Ar-C), 179.10 (2C=O); MS, m/z (%) 498
(M++4, 4), 496 (M++2, 12), 494 (M+, 40), 468 (30), 386 (100). Anal. Calcd for C22H16Cl2N8O2 (494.08):

C, 53.35; H, 3.26; N, 22.62. Found: C, 53.27; H, 3.12; N, 22.43%.
N',N''-(Biphenyl-4,4'-diyl)bis[2-(1-methyl-1H-pyrrol-2-yl)-2-oxoacetohydrazonoyl cyanide] (7).
Red solid, (0.35 g, 70%), mp 205-207 oC; IR (KBr)  3251, 3119 (2NH), 2243, 2250 (2C≡N), 1693, 1685
1
(2C=O) cm-1; H NMR (DMSO-d6)  = 3.85 (s, 6H, 2 N-CH3), 6.68-7.82 (m, 14H, Ar-H), 13.92 (s, 2H,
D2O-exchangeable, 2NH) ppm; 13C NMR (DMSO-d6)  = 33.12 (2CH3), 117.28 (2C≡N), 112.38, 120.54,
121.72, 123.54, 128.13, 129.41, 132.18, 142.55, 148.22 (Ar-C), 179.18 (2C=O); MS, m/z (%) 502 (M+,
20), 476 (40), 394 (100). Anal. Calcd for C28H22N8O2 (502.19): C, 66.92; H, 4.41; N, 22.30. Found: C,
67.07; H, 4.34; N, 22.42%.
Coupling of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) with diazonium salt of heterocyclic amine.
To a cold solution of 2-cyanoacetyl-1-methyl-1H-pyrrole (1) (0.148 g, 1 mmol) in pyridine (20 mL), was
added the diazonium salt of 3-amino-[1,2,4]triazole (8) (0.084 g, 1mmol), prepared according to literature
procedures.31 After complete addition of the diazonium salt, the reaction mixture was stirred for a further
2 h in an ice bath, and then poured onto ice/HCl mixture. The solid precipitated was filtered off, washed
with water, dried and crystallized from EtOH to give the respective product 11.
4-Amino-3-[(1-methyl-1H-pyrrol-2-yl)carbonyl][1,2,4]triazolo[5,1-c][1,2,4]triazine (11).
o
1
Yellow solid, (0.16 g, 66%), mp 154-156 C; IR (KBr)  3389, 3221 (NH2), 1687 (C=O) cm-1; H NMR
(DMSO-d6)  3.17 (s, 3H, N-CH3), 3.90 (br, 2H, D2O-exchangeable, NH2), 6.96-7.32 (m, 3H, Ar-H), 8.59
(s, 1H, triazole-H) ppm; MS, m/z (%) 243 (M+, 15), 163 (100), 135 (21). Anal. Calcd for C10H9N7O
(243.09): C, 49.38; H, 3.73; N, 40.31. Found: C, 49.57; H, 3.88; N, 40.57%.
Synthesis of 2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino)acrylonitrile (12).
To a stirred solution of potassium hydroxide (0.56 g, 10 mmol) in DMF (30 mL) was added
2-cyanoacetyl-1-methyl-1H-pyrrole (3) (1.48 g, 10 mmol). After stirring for 30 min, phenyl isothiocyanate
(1.35 g, 10 mmol) was added to the resulting mixture. Stirring was continued overnight. The reaction
mixture was acidified with HCl and the solid product was filtered off, washed with water and dried.
o
Recrystallization from EtOH gave white solid (1.66 g, 70%), mp 156 C; IR (KBr)  3246 (NH), 2201
(C≡N), 1705 (C=O) cm-1; 1H NMR (DMSO-d6)  3.45 (s, 3H, N-CH3), 7.15-7.59 (m, 8H, Ar-H), 10.23 (s,
1H, D2O-exchangeable, NH), 11.58 (s, 1H, SH) ppm; MS, m/z (%) 283 (M+, 7), 266 (17), 108 (100), 93 (85).
Anal. Calcd for C15H13N3OS (283.08): C, 63.58; H, 4.62; N, 14.83; S, 11.32. Found: C, 63.81; H, 4.78; N,
14.99; S, 11.53%.

Reactions of 2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino)acrylonitrile (12) with
hydrazonoyl chlorides under microwave irradiation.
To a solution of 2-[(1-methyl-1H-pyrrol-2-yl)carbonyl]-3-mercapto-3-(phenylamino)acrylonitrile (12)
(0.283 g, 1 mmol) and the appropriate hydrazonoyl chlorides 13a-h (1 mmol of each) in dioxane (10 mL)
was added chitosan (0.1 g) at room temperature. The reaction mixture was irradiated under constant
pressure (11.2 Bar, 150-160 oC) for 3-5 min at a power of 300 W. The hot solution was filtered to remove
chitosan. After cooling, dil. HCl was added till pH became acidic, and the solid product was collected and
recrystallized from EtOH or EtOH/DMF mixture to give products 16a-h. The physical constants together
with the spectral data of 16a-h are listed below.
2-[(3,5-Diphenyl-1,3,4-thiadiazol-2(3H)-ylidene)]-3-(1-methyl-1H-pyrrol-2-yl)-3-oxopropanenitrile
(16a).
Yellow solid (0.35 g, 91%), mp 272 oC; IR (KBr)  2188 (C≡N), 1703 (C=O) cm-1; 1H NMR (DMSO-d6) 
3.47 (s, 3H, N-CH3), 7.12-7.86 (m, 13H, Ar-H) ppm; 13C NMR (DMSO-d6)  36.25, 65.82, 115.98, 119.35,
123.31, 126.12, 126.53, 127.90, 128.23, 129.16, 129.77, 130.04, 131.16, 133.42, 136.02, 156.77, 165.42,
178.13 ppm; MS, m/z (%) 384 (M+, 50), 194 (16), 108 (100), 77 (50), 53 (75). Anal. Calcd for C22H16N4OS
(384.10): C, 68.73; H, 4.19; N, 14.57; S, 8.34. Found: C, 68.46; H, 4.12; N, 14.49; S, 8.19%.
2-[(5-Acetyl-3-phenyl-1,3,4-thiadiazol-2(3H)-ylidene)]-3-(1-methyl-1H-pyrrol-2-yl)-3-oxopropane-
nitrile (16b).
o
1
Yellow solid (0.32 g, 92%), mp 302 C; IR (KBr)  2195 (C≡N), 1704, 1698 (2C=O) cm-1; H NMR
(DMSO-d6)  2.68 (s, 3H, COCH3), 3.68 (s, 3H, N-CH3), 7.16-7.83 (m, 8H, Ar-H) ppm; 13C NMR
(DMSO-d6)  26.73, 36.15, 65.86, 116.08, 119.11, 122.71, 125.82, 126.53, 127.93, 129.33, 133.12, 136.02,
159.78, 165.32, 178.33, 191.77 ppm; MS, m/z (%) 350 (M+, 20), 324 (16), 307 (70), 242 (100), 77 (50).
Anal. Calcd for C18H14N4O2S (350.08): C, 61.70; H, 4.03; N, 15.99; S, 9.15. Found: C, 61.55; H, 3.82; N,
15.69; S, 9.03%.
2-[(5-Acetyl-3-(4-methylphenyl)-1,3,4-thiadiazol-2(3H)-ylidene)]-3-(1-methyl-1H-pyrrol-2-yl)-3-oxo-
propanenitrile (16c).
o
1
Yellow solid (0.33 g, 90%), mp 293 C; IR (KBr)  2192 (C≡N), 1705, 1693 (2C=O) cm-1; H NMR
(DMSO-d6)  2.39 (s, 3H, Ar-CH3), 2.64 (s, 3H, COCH3), 3.75 (s, 3H, N-CH3), 7.13-7.80 (m, 7H, Ar-H)
ppm; MS, m/z (%) 364 (M+, 50), 338 (16), 321 (30), 256 (10), 108 (100). Anal. Calcd for C19H16N4O2S
(364.10): C, 62.62; H, 4.43; N, 15.37; S, 8.80. Found: C, 62.35; H, 4.13; N, 15.60; S, 8.58%.

2-[(5-Acetyl-3-(4-chlorophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)]-3-(1-methyl-1H-pyrrol-2-yl)-3-oxo-
propanenitrile (16d).
o
1
Yellow solid (0.35 g, 90%), mp 237 C; IR (KBr)  2194 (C≡N), 1704, 1695 (2C=O) cm-1; H NMR
(DMSO-d6)  2.67 (s, 3H, COCH3), 3.69 (s, 3H, N-CH3), 7.18-7.89 (m, 7H, Ar-H) ppm; MS, m/z (%) 386
(M++2, 7), 384 (M+, 25), 341 (16), 276 (40), 108 (100), 53 (50). Anal. Calcd for C18H13ClN4O2S (384.04):
C, 56.18; H, 3.40; N, 14.56; S, 8.33. Found: C, 56.37; H, 3.53; N, 14.68; S, 8.51%.
2-[(5-Acetyl-3-(4-bromophenyl)-1,3,4-thiadiazol-2(3H)-ylidene)]-3-(1-methyl-1H-pyrrol-2-yl)-3-oxo-
propanenitrile (16e).
o
1
Yellow solid (0.39 g, 91%), mp 249 C; IR (KBr)  2193 (C≡N), 1705, 1692 (2C=O) cm-1; H NMR
(DMSO-d6)  2.63 (s, 3H, COCH3), 3.66 (s, 3H, N-CH3), 7.15-7.78 (m, 7H, Ar-H) ppm; MS, m/z (%) 430
(M++2, 20), 428 (M+, 22), 385 (40), 319 (50), 108 (100), 53 (50). Anal. Calcd for C18H13BrN4O2S (427.99):
C, 50.36; H, 3.05; N, 13.05; S, 7.47. Found: C, 50.18; H, 3.17; N, 13.18; S, 7.61%.
Ethyl 5-[1-cyano-2-(1-methyl-1H-pyrrol-2-yl)-2-oxoethylidene)]-4-phenyl-4,5-dihydro-1,3,4-
thiadiazole-2-carboxylate (16f).
o
1
Yellow solid (0.35 g, 93%), mp 218 C; IR (KBr)  2200 (C≡N), 1714, 1698 (2C=O) cm-1; H NMR
(DMSO-d6)  1.32 (t, 3H, CH2CH3), 3.74 (s, 3H, N-CH3), 4.53 (q, 2H, CH2CH3), 7.17-7.81 (m, 8H, Ar-H)
ppm; 13C NMR (DMSO-d6)  16.73, 36.11, 58.31, 65.93, 115.98, 119.43, 122.78, 124.82, 126.76, 128.13,
130.13, 132.98, 136.11, 159.88, 164.30, 178.33, 190.77 ppm; MS, m/z (%) 380 (M+, 5), 307 (70), 272 (30),
108 (100), 77 (50). Anal. Calcd for C19H16N4O3S (380.09): C, 59.99; H, 4.24; N, 14.73; S, 8.43. Found: C,
60.15; H, 4.42; N, 14.69; S, 8.63%.
Ethyl 5-[1-cyano-2-(1-methyl-1H-pyrrol-2-yl)-2-oxoethylidene)]-4-(4-methylphenyl)-4,5-dihydro-
1,3,4-thiadiazole-2-carboxylate (16g).
o
1
Yellow solid (0.35 g, 90%), mp 246 C; IR (KBr)  2198 (C≡N), 1718, 1700 (2C=O) cm-1; H NMR
(DMSO-d6)  1.35 (t, 3H, CH2CH3), 2.42 (s, 3H, Ar-CH3), 3.78 (s, 3H, N-CH3), 4.51 (q, 2H, CH2CH3),
7.12-7.87 (m, 7H, Ar-H) ppm; MS, m/z (%) 394 (M+, 15), 321 (40), 286 (30), 108 (100), 91 (20). Anal.
Calcd for C20H18N4O3S (394.11): C, 60.90; H, 4.60; N, 14.20; S, 8.13. Found: C, 60.85; H, 4.40; N, 14.29;
S, 8.23%.
5-[(1-Cyano-2-(1-methyl-1H-pyrrol-2-yl)-2-oxoethylidene)]-N,4-diphenyl-4,5-dihydro-1,3,4-
thiadiazole-2-carboxamide (16h).

o
1
Yellow solid (0.39 g, 91%), mp 226 C; IR (KBr)  2193 (C≡N), 1704, 1665 (2C=O) cm-1; H NMR
(DMSO-d6)  3.49 (s, 3H, N-CH3), 7.14-8.16 (m, 13H, Ar-H), 11.14 (s, 1H, D2O exchangeable, NH) ppm;
13C NMR (DMSO-d6)  36.27, 65.81, 116.04, 119.33, 123.31, 126.17, 126.58, 127.88, 128.28, 129.19,
129.78, 130.54, 131.86, 133.42, 136.11, 156.77, 165.41, 168.19, 178.13 ppm; MS, m/z (%) 427 (M+, 40),
319 (40), 307 (50), 108 (10), 77 (30), 69 (100). Anal. Calcd for C23H17N5O2S (427.11): C, 64.62; H, 4.01; N,
16.38; S, 7.50. Found: C, 64.46; H, 4.12; N, 16.49; S, 7.39%.
Cytotoxic activity
Potential cytotoxicity of the compounds was tested using the method of Skehan et al.32 using
Sulfo-Rhodamine-B stain (SRB). Cells were plated in 96-multiwill plates (104 cells/well) for 24 h before
treatment with the tested compound to allow attachment of cell to the wall of the plate. Different
concentrations of the compound under test (0, 1.56, 3.125, 6.25, 12.5, 25, and 50 µg/mL) were added to the
cell monolayer in triplicate wells individual dose, monolayer cells were incubated with the compounds for
48 h at 37 °C and in atmosphere of 5% CO2. After 48 h, cells were fixed, washed and stained with SRB stain,
excess stain was washed with acetic acid and attached stain was recovered with tris-EDTA buffer, color
intensity was measured in an ELISA reader.
ACKNOWLEDGEMENTS
The support of this work was received from King Khalid University through research grant
(KKU_S112_33).
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