Synthetic utility of ethylidenethiosemicarbazide: Synthesis and anticancer activity of 1,3-thiazines and thiazoles with imidazole moiety

Article abstract of DOI:10.3987/COM-12-12625

Reactions of ethylidenethiosemicarbazide 3 with DMAD 4 or substituted methylenemalononitriles 8 gave thiazolidin-4-one 6 or 1,3-thiazine derivatives (10, 11), respectively. Also, treatment of 3 with hydrazonoyl halides 12a-i, α-haloketones 15a-d, and chlo

Full text of DOI:10.3987/COM-12-12625

HETEROCYCLES, Vol. 87, No. 2, 2013, pp. -. © 2013 The Japan Institute of Heterocyclic Chemistry
Received, 12th November, 2012, Accepted, 6th December, 2012, Published online, 12th December, 2012
DOI: 10.3987/COM-12-12625
SYNTHETIC UTILITY OF ETHYLIDENETHIOSEMICARBAZIDE:
SYNTHESIS AND ANTI-CANCER ACTIVITY OF 1,3-THIAZINES AND
THIAZOLES WITH IMIDAZOLE MOIETY
Sobhi M. Gomha, Sayed M. Riyadh,* Ikhlass M. Abbas, and Mohammed A.
Bauomi
Department of Chemistry, Faculty of Science, Cairo University, Giza, 12613,
Egypt
*Corresponding author: E-mail: riyadh1993@hotmail.com
Abstract – Reactions of ethylidenethiosemicarbazide 3 with DMAD 4 or
substituted methylenemalononitriles 8 gave thiazolidin-4-one 6 or 1,3-thiazine
derivatives (10, 11), respectively. Also, treatment of 3 with hydrazonoyl halides
12a-i, -haloketones 15a-d, and chloroacetic acid 18 afforded the corresponding
arylazothiazoles 14a-i, thiazoles 17a-d, and thiazolin-4-one derivative 20,
1
respectively. The structures of the synthesized products were confirmed by IR, H
NMR, 13C NMR and mass spectral techniques. The anti-cancer activity of the
selected products against the colon carcinoma cell line (HCT-116) was determined
and the results revealed promising activity of compound 6.
INTRODUCTION
Recent literature is enriched with synthesis and biological activities of alkylidenethiosemicarbazides.
1-(1-Arylethylidene)thiosemicarbazides exhibited potent inhibitory activities against mushroom
tyrosinase1,2 (multifunctional copper-containing enzyme cause dermatological disorders). Also,
1-[1-(heterocyclic)ethylidene]thiosemicarbazides and their metal complexes have been investigated as
potential anticancer agents.3-5 The cytotoxic activity of thiosemicarbazones against human tumor cell
lines has been attributed to their ability to inhibit ribonucleoside diphosphate reductase (RDR), a
rate-limiting enzyme in DNA syntheses that catalyzes the conversion of ribonucleotides into
deoxyribonucleotides.4 On the other hands, alkylidenethiosemicarbazides are reactive building blocks for
construction of bioactive heterocycles such as 1,3,4-thiadiazoles,6 imidazolinones,7 thiazoles,8-11 and
thiazolidin-4-one.9 As a part of our research interest towards developing new routes for the synthesis of a

variety of heterocyclic systems with promising biological and pharmacological activities,12-16 we report in
the present work the synthesis of a new series of 1,3-thiazines and thiazoles bearing imidazole moiety.
RESULTS AND DISCUSSION
1-[1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3) was prepared
via condensation of 5-acetyl-2-mercapto-4-methyl-1-phenyl-1H-imidazole (1)17 with thiosemicarbazide
(2) in absolute ethanol in the presence of a catalytic amount of HCl as depicted in Scheme 1.
Me
Me
H
N
N
N
H2N
NH2
EtOH / HCl
H2N
+
Me
NH
Me
SH
SH
S
S
2
N
N
N
O
Ph
Ph
1
3
Scheme 1
The structure elucidation of the product 3 was substantiated through spectral data. Product 3 can be
1
existed in two geometric structures (E, and Z). In H NMR, when N-H signal is revealed at  =
10.79-10.90 ppm suggesting the E configuration, in which N–H is hydrogen bonded to the solvent.18–20
On the other hand, if N-H signal is revealed at  = 14.69–14.66 ppm indicates the presence of the Z
configuration, in which N–H is hydrogen bonded to the nitrogen of the ring.18–20 in our case, NH signal
was revealed at  = 10.98 ppm which confirms the E-form. Also another signal was observed at  = 10.45
ppm characteristic to mercapto (SH)21 group of imidazole ring.
We commenced our study on the reaction of ethylidenethiosemicarbazide with activated triple bond. It
was reported that, reactions of alkylidenethiosemicarbazides with dimethyl acetylenedicarboxylate
(DMAD) afforded either thiazolidin-4-ones22-24 or 1,3-thiazin-4-ones22,23,25 according to the reaction
conditions. Thus, treatment of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]-
thiosemicarbazide (3) with DMAD in methanol gave the corresponding thiazolidin-4-one derivative 6
or its isomeric structure 1,3-thiazin-4-one derivative 7 (Scheme 2). The structure of the isolated product
1
was inferred from its elemental analysis and spectral data [IR and H NMR]. Its IR spectrum showed
1
absorption bands at v = 3247 (NH), 1704, 1694 (2C=O), and 1606 (C=N) cm-1, its H NMR spectrum
revealed singlet signal at  12.77 ppm (D2O-exchangeable) assignable to (NH) group and another singlet
signal at  6.64 ppm due to vinylic-H.23,26 Revealing of the latter signal of vinylic-H excludes
1,3-thiazin-4-one structure 7. To account for the formation of product 6 we assumed that the reaction
initially proceeds via addition of thiol group in thiosemicarbazone moiety into triple bond to give the
non-isolable intermediate 5. Elimination of methanol molecule from the latter intermediate afforded the

final product 6 (Scheme 2).
Me
N
Me
MeO2C
C
C
CO2Me
+
HS
S
N
N
4
Ph
3
NH
NH
H
MeOH
Me
CO2Me
CO2Me
N
Me
HS
S
N
N
Ph
5
N
H
NH
- MeOH
Me
Me
N
CO2Me
N
CO2Me
Me
Me
S
HS
S
HS
N
N
N
N
Ph
Ph
O
N
N
H
O
N
6
N
H
7
Scheme 2
Next, we investigated the behavior of ethylidenethiosemicarbazide 3 towards substituted
methylenemalononitriles, as activated double bond. Thus, treatment of compound 3 with
benzylidenemalononitrile 8a or ethoxymethylenemalononitrile 8b in refluxing methanol afforded the
respective 1,3-thiazines (10 or 11) (Scheme 3). The reactions proceeded through addition of thiol group in
thiosemicarbazone moiety into activated double bond to give the non-isolable intermediates 9a,b.
Intramolecluar cyclization of intermediate 9a via addition of amino group into nitrile group27 afforded
6H-1,3-thiazine derivative 10. On the other hand, intramolecular addition of intermediate 9b followed by
elimination of ethanol molecule gave the respective 2H-1,3-thiazine derivative 11 (cf. Scheme 3). The
structures of products 10, 11 were established based on analytical and spectral data. The structures of
1,3-thiazines in agreement with literature reports28,29 concerning the reactions of thiosemicarbazides with
substituted methylenemalononitriles.

Me
N
CN
Y
H
Me
C
C
+
HS
S
N
CN
N
8a,b
Ph
3
8a; Y = Ph
8b; Y = EtO
NH
NH
H
MeOH
Me
N
Y
Me
CN
HS
S
N
N
CN
Ph
9a,b
NH
N
H
Y = C6H5
Y = EtO
- EtOH
Me
Me
N
N
Ph
N
CN
CN
Me
N
Me
S
S
HS
HS
N
N
Ph
N
N
Ph
N
NH2
NH2
N
H
11
10
Scheme 3
We extended our study on the reactivity of ethylidenethiosemicarbazide 3 towards halogenated
compounds, such as hydrazonoyl halides, -halocarbonyl compounds and chloroacetic acid. Reaction of
3 with each of the hydrazonoyl halides 12a-i in dioxane under thermal condition in the presence of
triethylamine for 6-8 hrs yielded in each case one isolable product as evidenced by TLC analysis. The
structures of isolated products were evidenced by spectral data together with elemental analyses. For
instance, the IR spectra of products display in each case the absorption bands in the region 3213-3171 and
1
1604-1584 cm-1 due to the (NH) and (C=N) groups, respectively. In H NMR spectra all the products
have characteristic singlet signals in the region  10.83-10.54 ppm (D2O exchangeable) assignable to the
(NH) protons. On the basis of the foregoing results, the isolated products from the reactions of 3 with
12a-i can be assigned 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-
4-substituted-5-arylazothiazole (14a-i) (Scheme 4).

Products 14a-i can be present in three tautomeric forms 14A, 14B, and 14C as shown in Figure 1. To
elucidate the actual tautomeric form of these compounds, their electronic absorption was measured. The
spectra of these compounds (14a, 14g, and 14i) in dioxane showed in each case pair of absorption bands
in the regions 442-447 and 292-296 nm, analogous for the azo chromophore30 and another pair in the
regions 366-373 and 298-306 nm analogous for the hydrazone chromophore31 (Table 1). These finding
excluded tautomeric form 14B. Furthermore, the aromatic stability of thiazole ring excluded tautomeric
form 14C.
Table 1: UV Spectra of compounds 14a, 14g, and 14i
Compound no.

max (log )
max (log )
14a
14g
14i
442 (4.83), 292 (4.94)
444 (4.80), 296 (4.77)
447 (4.61), 295 (4.68)
366 (4.25), 298 (4.82)
373 (4.35), 306 (4.84)
369 (4.50), 298 (5.01)

Finally, ethylidenethiosemicarbazide 3 reacted with -halocarbonyl compounds, namely chloroacetone
15a, phenacyl bromide 15b, 3-chloro-2,4-pentanedione 15c, N-phenyl 2-chloro-3-oxobutanamide 15d,
and chloroacetic acid 18 under thermal conditions in dioxane to afford 2-[2-{1-(2-mercapto-4-methyl-
1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-substituted thiazoles (17a-d), and 2-[2-{1-(2-
mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-oxo-4,5-dihydrothiazole
20,
respectively (Scheme 5). Analytical and spectroscopic data were consistent with the final products 17a-d
and 20.
ANTI-CANCER EVALUATION
The cytotoxicity of synthesized products 6, 10, 11,14a, 14g and 20 was evaluated against human colon
carcinoma cell line (HCT-116) using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) assay and doxorubicin was used as a reference drug (IC50 value of doxorubicin = 0.469 ± 0.03
µg/mL). 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. The results are represented in Tables 2, 3
and Figure 2.
The results revealed that thiazolidin-4-one derivative 6 (IC50 = 1.9 µg/mL) has promising antitumor
activity against colon carcinoma (HCT-116) while 1,3-thiazines (10 and 11) and arylazothiazoles (14a
and 14g) have moderate activity (IC50 = 3.9-4.6 µg/mL). On the other hand, thiazolin-4-one 20 has poor
inhibitory activity against (HCT-116) (IC50 = 33 µg/mL).

Table 2. Viability values of tested compounds against HCT-116
Viability %
Sample conc.
(µg/mL)
50
Doxorubicin
6.82
6
10
11
14a
14g
8.24
20
6.97
8.71
15.94
23.62
32.46
56.28
63.31
100
9.04
10.38
16.53
19.46
29.33
58.72
84.51
100
39.61
54.92
68.19
79.24
88.36
95.80
100
25
8.89
11.28
17.94
26.25
35.12
54.83
100
17.12
29.46
38.84
62.93
75.1
17.08
26.71
35.35
63.87
80.54
100
12.5
6.25
3.125
1.56
0
14.83
16.16
22.28
34.64
100
100

Table 3. IC50 values of tested compounds ± standard deviation against HCT-116
Compound
No.
Compound
No.
IC50
IC50
Doxorubicin
0.469 ± 0.03
14a
4.1± 0.07
6
1.9 ± 0.05
3.9 ± 0.02
4.8. ± 0.08
14g
20
4.6 ± 0.016
33 ±0.18
10
11
Figure 2. Dose-response curves for the cytotoxicity of compounds
6, 10, 11, 14a, 14g, and 20
CONCLUSION
New thiazolidin-4-one, 1,3-thiazines, arylazothiazoles, thiazoles, and thiazolin-4-one have been
synthesized using ethylidenethiosemicarbazide as starting material under thermal conditions.
EXPERIMENTAL
Melting points were measured on an Electrothermal IA 9000 series digital melting point apparatus. 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 300 MHz (1H NMR) or 75 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 analyzes were
measured by using a German made Elementar vario LIII CHNS analyzer. Antitumor activity was
evaluated by the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt.
Hydrazonoyl halides32,33 were prepared as previously reported in the respective literature.
Synthesis of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3).
5-Acetyl-2-mercapto-4-methyl-1-phenyl-1H-imidazole (1) (11.5 g, 50 mmol) was dissolved in 100 mL of
EtOH and stirred with an equimolar quantity of thiosemicarbazide for 24 h at room temperature with
catalytic amounts of HCl. The desired thiosemicarbazone precipitated from reaction mixture was filtered,
washed with EtOH and recrystallized from acetic acid to give pure product of compound 3.
Yellow solid (12.2 g, 80%); mp 229 °C; IR (KBr): v 3439, 3236 (NH2), 3155 (NH), 1587 (C=N) cm-1;
1H-NMR (DMSO-d6): δ 2.35 (s, 3H, CH3), 2.96 (s, 3H, CH3), 4.31 (s, br, 2H, NH2), 6.99-7.66 (m, 5H,
Ar-H), 10.45 (s, 1H, SH), 10.98 (s, 1H, NH); MS m/z (%): 305 (M+, 100), 230 (45), 91 (82), 77 (27). Anal.
Calcd for C13H15N5S2 (305.08): C, 51.12; H, 4.95; N, 22.93; S, 21.00. Found C, 51.31; H, 5.18; N, 23.12;
S, 21.09%.
Synthesis of methyl 2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-
4-oxo-thiazolidin-5-ylidenethanoate (6).
To a solution of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
(0.305 g, 1 mmol) in dry MeOH (20 mL) was added dimethyl acetylenedicarboxylate (0.142 g, 1 mmol).
The solution was refluxed for 2 h. The precipitate was filtered, washed with MeOH, and recrystallized
from EtOH to give product 6.
Canary yellow solid (0.31 g, 75%); mp 352 °C; IR (KBr): v 3247 (NH), 1704, 1694 (2C=O), 1606 (C=N)
1
cm-1; H-NMR (DMSO-d6): δ 2.53 (s, 3H, CH3), 3.35 (s, 3H, CH3), 3.77 (s, 3H, COOCH3), 6.64 (s, 1H,
C=CH), 6.96-7.66 (m, 5H, Ar-H), 10.41 (s, 1H, SH), 12.77 (s, 1H, NH); MS m/z (%): 415 (M+, 68), 229
(68), 118 (68), 77 (100). Anal. Calcd for C18H17N5O3S2 (415.08): C, 52.03; H, 4.12; N, 16.86; S, 15.43.
Found C, 52.21; H, 4.18; N, 16.72; S, 15.29%.
Reactions of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
with benzylidenemalononitrile (8a) and ethoxymethylenemalononitrile (8b).
To a solution of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
(0.305 g, 1 mmol) in dry MeOH (20 mL) was added benzylidenemalononitrile (8a) or
ethoxymethylenemalononitrile (8b) (1 mmol). The solution was refluxed for 2 h. The precipitate was

filtered, washed with methanol, and recrystallized from EtOH to give product 10 or 11, respectively.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-amino-6-phenyl-
6H-1,3-thiazine-5-carbonitrile (10).
Yellow solid (0.32 g, 70%); mp 214 °C; IR (KBr): v 3446-3196 (NH2 + NH), 2264 (C≡N), 1596 (C=N)
1
cm-1; H-NMR (DMSO-d6): δ 2.32 (s, 3H, CH3), 3.35 (s, 3H, CH3), 3.77 (s, 1H, thiazine-H), 4.65 (s, br,
2H, NH2), 7.04-7.75 (m, 10H, Ar-H), 10.41 (s, 1H, SH), 11.37 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 9.31
(CH3), 16.38 (CH3), 52.17 (CH), 108.51 (CN), 117.19, 118.63, 120.62, 120.91, 122.82, 123.45, 123.88,
124.02, 126.18, 128.79, 128.96, 129.52, 139.81, 142.32, 143.45 (Ar-C); MS m/z (%): 459 (M+, 52), 229
(100), 118 (31), 77 (83). Anal. Calcd for C23H21N7S2 (459.13): C, 60.11; H, 4.61; N, 21.33; S, 13.95.
Found C, 60.21; H, 4.48; N, 21.42; S, 13.99%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-amino-1,3-thiazine-
5-carbonitrile (11).
Canary yellow solid (0.27 g, 72%); mp 212 °C; IR (KBr): v 3429, 3238 (NH2), 2258 (C≡N), 1598 (C=N)
1
cm-1; H-NMR (DMSO-d6): δ 2.38 (s, 3H, CH3), 3.05 (s, 3H, CH3), 4.64 (s, br, 2H, NH2), 6.99 (s, 1H,
13
thiazine-H), 7.04-7.68 (m, 5H, Ar-H), 10.63 (s, 1H, SH); C-NMR (DMSO-d6): δ 9.29 (CH3), 16.34
(CH3), 108.41 (CN), 117.09, 118.32, 120.61, 120.66, 122.82, 126.22, 128.89, 128.99, 129.32, 139.84,
142.11, 143.75 (Ar-C); MS m/z (%): 381 (M+, 6), 288 (97), 230 (63), 127 (58), 77 (100). Anal. Calcd for
C17H15N7S2 (381.08): C, 53.52; H, 3.96; N, 25.70; S, 16.81. Found C, 53.41; H, 4.08; N, 25.62; S,
16.91%.
Synthesis
of
2-[2-{1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-
substituted-5-arylazothiazoles (14a-i).
General procedure:
A mixture of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
(0.305 g, 1 mmol) and appropriate hydrazonoyl halides 12a-i (1 mmol) in dioxane (30 mL) containing
triethylamine (0.1 g, 1 mmol) was refluxed for 6-8 h. (monitored by TLC). The formed precipitate was
isolated by filtration, washed with MeOH, dried and recrystallized from appropriate solvent to give
products 14a–i.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-phenyl-
azothiazole (14a).
Red solid (0.36 g, 80%); mp 266 °C; IR (KBr): v 3213 (NH), 1594 (C=N) cm-1; 1H-NMR (DMSO-d6): δ

2.48 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.97-7.63 (m, 10H, Ar-H), 10.50 (s, 1H, SH),
13
10.54 (s, 1H, NH); C-NMR (DMSO-d6):  9.42 (CH3), 13.77 (CH3), 15.01 (CH3), 114.15, 118.45,
120.22, 121.15, 121.85, 123.25, 124.33, 125.75, 127.16, 127.31, 129.73, 131.61, 133.73, 138.61, 143.13
(Ar-C); MS m/z (%): 447 (M+, 41), 229 (61), 77 (100). Anal. Calcd for C22H21N7S2 (447.13): C, 59.04; H,
4.73; N, 21.91; S, 14.33. Found C, 59.21; H, 4.88; N, 21.82; S, 14.19%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-methyl
phenylazo)thiazole (14b).
Red solid (0.38 g, 82%); mp 254 °C; IR (KBr): v 3281 (NH), 1601 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
2.25 (s, 3H, CH3), 2.57 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.56 (s, 3H, CH3), 7.03-7.62 (m, 9H, Ar-H),
10.47 (s, 1H, SH), 10.58 (s, 1H, NH); MS m/z (%): 461 (M+, 12), 229 (77), 91 (100). Anal. Calcd for
C23H23N7S2 (461.15): C, 59.84; H, 5.02; N, 21.24; S, 13.89. Found C, 59.71; H, 4.98; N, 21.32; S,
13.74%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-methoxy-
phenylazo)thiazole (14c).
Red solid (0.37 g, 78%); mp 248 °C; IR (KBr): v 3194 (NH), 1603 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
2.45 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.56 (s, 3H, CH3), 3.87 (s, 3H, OCH3), 7.06-7.72 (m, 9H, Ar-H),
10.48 (s, 1H, SH), 10.62 (s, 1H, NH); MS m/z (%): 477 (M+, 19), 229 (71), 91 (100). Anal. Calcd for
C23H23N7OS2 (477.14): C, 57.84; H, 4.85; N, 20.53; S, 13.43. Found C, 57.72; H, 4.91; N, 20.33; S,
13.24%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-chlorophenylazo)-
thiazole (14d).
Red solid (0.39 g, 75%); mp 262 °C; IR (KBr): v 3191 (NH), 1590 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
2.47 (s, 3H, CH3), 3.30 (s, 3H, CH3), 3.62 (s, 3H, CH3), 7.09-7.83 (m, 9H, Ar-H), 10.51 (s, 1H, SH),
10.80 (s, 1H, NH); MS m/z (%): 483 (M++2, 12), 481 (M+, 30), 229 (100), 111 (50). Anal. Calcd for
C22H20ClN7S2 (481.09): C, 54.82; H, 4.18; N, 20.34; S, 13.30. Found C, 54.78; H, 4.28; N, 20.52; S,
13.19%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-bromo-
phenylazo)thiazole (14e).
Red solid (0.39 g, 75%); mp 270 °C; IR (KBr): v 3171 (NH), 1584 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
2.48 (s, 3H, CH3), 3.31 (s, 3H, CH3), 3.62 (s, 3H, CH3), 7.07-7.84 (m, 9H, Ar-H), 10.50 (s, 1H, SH),

10.83 (s, 1H, NH); MS m/z (%): 527 (M++2, 12), 525 (M+, 13), 229 (65), 77 (100). Anal. Calcd for
C22H20BrN7S2 (525.04): C, 50.19; H, 3.83; N, 18.62; S, 12.18. Found C, 50.28; H, 3.78; N, 18.82; S,
12.29%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(4-nitro-
phenylazo)thiazole (14f).
1
Dark red solid (0.34 g, 70%); mp 224 °C; IR (KBr): v 3190 (NH), 1590 (C=N) cm-1; H-NMR
(DMSO-d6): δ 2.48 (s, 3H, CH3), 3.33 (s, 3H, CH3), 3.71 (s, 3H, CH3), 7.12-7.93 (m, 9H, Ar-H), 10.51 (s,
1H, SH), 10.92 (s, 1H, NH); MS m/z (%): 492 (M+, 35), 229 (97), 118 (76), 77 (100). Anal. Calcd for
C22H20N8O2S2 (492.12): C, 53.64; H, 4.09; N, 22.75; S, 13.02. Found C, 53.78; H, 4.18; N, 22.62; S,
13.15%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenyl-5-phenyl-
azothiazole (14g).
Red solid (0.36 g, 70%); mp 352 °C; IR (KBr): v 3192 (NH), 1602 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
3.24 (s, 3H, CH3), 3.29 (s, 3H, CH3), 6.99-8.29 (m, 15H, Ar-H), 10.53 (s, 1H, SH), 10.65 (s, 1H, NH);
13C-NMR (DMSO-d6):  13.31 (CH3), 15.77 (CH3), 114.17, 115.93, 118.15, 119.33, 120.42, 121.17,
122.15, 123.25, 123.87, 124.36, 125.79, 126.11, 127.26, 127.41, 129.72, 131.57, 133.83, 137.62, 142.13
(Ar-C); MS m/z (%): 509 (M+, 9), 231 (31), 77 (100). Anal. Calcd for C27H23N7S2 (509.15): C, 63.63; H,
4.55; N, 19.24; S, 12.58. Found C, 63.51; H, 4.68; N, 19.12; S, 12.49%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenyl-5-(4-chloro-
phenylazo)thiazole (14h).
Red solid (0.38 g, 70%); mp 316 °C; IR (KBr): v 3193 (NH), 1604 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
3.26 (s, 3H, CH3), 3.32 (s, 3H, CH3), 7.03-8.29 (m, 14H, Ar-H), 10.54 (s, 1H, SH), 10.68 (s, 1H, NH);
MS m/z (%): 545 (M++2, 3), 543 (M+, 9), 231 (54), 111 (50), 77 (100). Anal. Calcd for C27H22ClN7S2
(543.11): C, 59.60; H, 4.08; N, 18.02; S, 11.79. Found C, 59.41; H, 4.18; N, 18.12; S, 11.89%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-(2-thienyl)-5-phenyl-
azothiazole (14i).
Red solid (0.36 g, 70%); mp 268 °C; IR (KBr): v 3198 (NH), 1597 (C=N) cm-1; 1H-NMR (DMSO-d6): δ
3.28 (s, 3H, CH3), 3.31 (s, 3H, CH3), 7.01-8.03 (m, 13H, Ar-H), 10.50 (s, 1H, SH), 10.62 (s, 1H, NH);
13C-NMR (DMSO-d6):  13.33 (CH3), 15.87 (CH3), 113.77, 114.93, 118.15, 119.35, 120.72, 121.13,
122.19, 123.35, 123.82, 124.36, 125.77, 126.18, 127.26, 127.91, 129.79, 132.57, 133.81, 136.61, 143.63

(Ar-C); MS m/z (%): 515 (M+, 9), 231 (31), 77 (100). Anal. Calcd for C25H21N7S3 (515.10): C, 58.23; H,
4.10; N, 19.01; S, 18.65. Found C, 58.41; H, 4.18; N, 19.16; S, 18.49%.
Reactions of 1-[1-(2-mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene]thiosemicarbazide (3)
with -haloketones and -haloacid.
General procedure:
A mixture of 3 (0.305 g, 1 mmol) and -haloketones (15a-d) or chloroacetic acid (18) (1 mmol) in
dioxane (30 mL) was refluxed for 4-6 h. (monitored by TLC). The product started to separate out during
the course of reaction. The solid product was filtered, washed with water, dried and recrystallized from
DMF to give the corresponding compounds 17a-d or 20, respectively.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methylthiazole
(17a).
Yellow solid (0.28 g, 82%); mp 154 °C; IR (KBr): v 3197 (NH), 1606 (C=N) cm-1; 1H-NMR (DMSO-d6):
δ 2.28 (s, 3H, CH3), 3.31 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.18 (s, 1H, thiazole-H), 6.92-7.62 (m, 5H,
13
Ar-H), 10.30 (s, 1H, SH), 10.94 (s, 1H, NH); C-NMR (DMSO-d6):  9.45 (CH3), 13.47 (CH3), 15.33
(CH3), 118.48, 120.53, 121.23, 123.15, 124.33, 127.21, 129.73, 131.61, 133.63, 138.65, 143.43 (Ar-C);
MS m/z (%): 343 (M+, 53), 231 (100), 113 (90), 77 (94). Anal. Calcd for C16H17N5S2 (343.09): C, 55.95;
H, 4.99; N, 20.39; S, 18.67. Found C, 55.81; H, 4.88; N, 20.22; S, 18.59%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-phenylthiazole
(17b).
Yellow solid (0.30 g, 75%); mp 192 °C; IR (KBr): v 3296 (NH), 1616 (C=N) cm-1; 1H-NMR (DMSO-d6):
δ 3.34 (s, 3H, CH3), 3.61 (s, 3H, CH3), 6.22 (s, 1H, thiazole-H), 7.12-7.92 (m, 10H, Ar-H), 10.34 (s, 1H,
13
SH), 10.84 (s, 1H, NH); C-NMR (DMSO-d6):  13.33 (CH3), 15.81 (CH3), 117.15, 119.33, 120.41,
121.17, 122.15, 123.25, 123.87, 124.36, 126.15, 127.24, 127.25, 131.59, 133.73, 137.52, 142.33 (Ar-C);
MS m/z (%): 405 (M+, 52), 231 (78), 176 (91), 134 (100), 113 (55), 77 (96). Anal. Calcd for C21H19N5S2
(405.11): C, 62.19; H, 4.72; N, 17.27; S, 15.81. Found C, 62.11; H, 4.88; N, 17.42; S, 15.69%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-acetyl-
thiazole (17c).
Yellow solid (0.29 g, 75%); mp 112 °C; IR (KBr): v 3214 (NH), 1708 (C=O), 1602 (C=N) cm-1; 1H-NMR
(DMSO-d6): δ 2.31 (s, 3H, CH3), 2.42 (s, 3H, COCH3), 3.31 (s, 3H, CH3), 3.57 (s, 3H, CH3), 6.96-7.64

(m, 5H, Ar-H), 10.30 (s, 1H, SH), 10.90 (s, 1H, NH); MS m/z (%): 385 (M+, 38), 230 (45), 189 (45), 148
(42), 112 (51), 77 (100). Anal. Calcd for C18H19N5OS2 (385.10): C, 56.08; H, 4.97; N, 18.17; S, 16.64.
Found C, 55.91; H, 4.88; N, 18.22; S, 16.59%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-methyl-5-(N-phenyl-
carbamoyl)thiazole (17d).
Yellow solid (0.32 g, 70%); mp 185 °C; IR (KBr): v 3233 (NH), 1671 (C=O), 1600 (C=N) cm-1; 1H-NMR
(DMSO-d6): δ 2.31 (s, 3H, CH3), 3.29 (s, 3H, CH3), 3.49 (s, 3H, CH3), 7.06-7.81 (m, 10H, Ar-H), 10.30
(s, 1H, SH), 10.90 (s, 1H, NH), 11.08 (s, 1H, NH); MS m/z (%): 462 (M+, 13), 343 (26), 252 (98), 141
(100), 77 (69). Anal. Calcd for C23H22N6OS2 (462.13): C, 59.72; H, 4.79; N, 18.17; S, 13.86. Found C,
59.91; H, 4.85; N, 18.12; S, 13.79%.
2-[2-{1-(2-Mercapto-4-methyl-1-phenyl-1H-imidazol-5-yl)ethylidene}hydrazono]-4-oxo-4,5-dihydro-
thiazole (20).
Yellow solid (0.26 g, 75%); mp 278 °C; IR (KBr): v 3246 (NH), 1697 (C=O), 1614 (C=N) cm-1; 1H-NMR
(DMSO-d6): δ 2.35 (s, 3H, CH3), 3.54 (s, 3H, CH3), 3.68 (s, 2H, CH2), 6.93-7.63 (m, 5H, Ar-H), 10.35 (s,
1H, SH), 10.84 (s, 1H, NH); MS m/z (%): 345 (M+, 20), 344 (100), 229 (47), 118 (44), 77 (78). Anal.
Calcd for C15H15N5OS2 (345.07): C, 52.15; H, 4.38; N, 20.27; S, 18.56. Found C, 52.21; H, 4.28; N,
20.15; S, 18.49%.
CYTOTOXIC ACTIVITY
Potential cytotoxicity of the compounds was tested using the method of Skehan et al.,34 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. The relation between surviving fraction and drug
concentration is plotted (Figure 2).
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