Synthesis of some novel thiazole, thiadiazole and 1,4-phenylene-bis-thiazole derivatives as potent antitumor agents

Article abstract of DOI:10.3987/COM-16-13443

A novel series of 2-ethylidenehydrazono-5-arylazothiazoles 5a-h and 2-ethylidenehydrazono-5-arylazothiazolones 9a-d were prepared by cyclocondensation of hydrazonyl halides 3a-h and 7a-d with ethylidenethiosemicarbazide 2. In addition, reaction of 2 with

Full text of DOI:10.3987/COM-16-13443

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HETEROCYCLES, Vol. 92, No. 5, 2016
HETEROCYCLES, Vol. 92, No. 5, 2016, pp. 954 - 967. © 2016 The Japan Institute of Heterocyclic Chemistry
Received, 23rd February, 2016, Accepted, 15th March, 2016, Published online, 18th March, 2016
DOI: 10.3987/COM-16-13443
SYNTHESIS OF SOME NOVEL THIAZOLE, THIADIAZOLE AND 1,4-
PHENYLENE-BIS-THIAZOLE
ANTITUMOR AGENTS
DERIVATIVES
AS
POTENT
Sobhi M. Gomha,*,1 Fathy M. Abdelrazek,1,2 Aly H. Abdelrahman,1 and
Peter Metz2
1) Chemistry Department, Faculty of Science, Cairo University, Giza 12613,
Egypt, E-mail: s.m.gomha@hotmail.com
2) Institute of Organic Chemistry, TU-Dresden, Dresden 01062, Germany
Abstract – A novel series of 2-ethylidenehydrazono-5-arylazothiazoles 5a-h and
2-ethylidenehydrazono-5-arylazothiazolones
9a-d
were
prepared
by
cyclocondensation of hydrazonyl halides 3a-h and 7a-d
with
ethylidenethiosemicarbazide 2. In addition, reaction of 2 with N-phenyl-
carbohydrazonyl chloride (14), afforded 1,3,4-thiadiazole derivative 17 as the end
product. Moreover, the thiosemicarbazide derivative 2 was reacted with various
bromoacetyl compounds 19a-d and 1,1’-(1,4-phenylene)bis(2-bromoethanone)
(21) furnished the respective thiazole derivatives 20a-d and 1,4-phenylene-bis-
thiazole derivative 22. The structures of the newly synthesized compounds were
established on the basis of spectroscopic evidences and their alternative syntheses.
The newly synthesized compounds were evaluated for their antitumor activities
against hepatocellular carcinoma (HepG2) cell line and the results revealed
promising activities of compounds 5h, 5d, 5g, 5f and 5e with IC50 equal 2.23 ±
0.28, 2.48 ± 0.34, 2.49 ± 0.24, 4.03 ± 0.11, and 5.32 ± 0.27 μM, respectively.
Functionalized thiazole derivatives have received much attention due to their diverse biological activities
such as antimicrobial,1 antiviral,2 cytotoxic and antitumor,3-6 and HIV-protease inhibitory agents.7
Compounds incorporating two thiazole rings either directly connected as in bisthiazoles or through a
linker unit as in bisthiazolyl compounds, show pronounced biological activity as DNA replication
inhibitors in the tumor cells8 and HIV-protease inhibitors.7-9
Pyridine deivatives also play a key role catalyzing both biological and chemical systems. In many
enzymes of living organisms it is the prosthetic pyridine nucleotide (NADP) that is involved in various

HETEROCYCLES, Vol. 92, No. 5, 2016
955
oxidation-reduction processes. Pyridine nucleus is the main constituent in the important vitamins niacin
and pyridoxine (vitamin B6) and also in highly toxic alkaloids such as nicotine. In the pharmaceutical
industry, pyridine forms the nucleus of over 7000 existing drugs. Pyridine ring system is very widely
distributed in nature, especially in plant kingdom. Many important alkaloids such as atropine from Atropa
belladonna, Deadly nightshade, contains saturated pyridine nucleus.10,11
In the last two decades we have been involved in a program aiming at the synthesis of functionally
substituted heterocyclic compounds from cheap laboratory available starting materials with anticipated
biological activities.12-20 Recently, in the frame of our program, some new functionally substituted
thiazoles and / or bisthiazoles were required to be screened for their biological activity as antitumor
agents. It seemed to us that the combination of a thiazole ring with a pyridine ring in one entity may lead
to enhanced biological activity due to the synergistic effect of both rings. 4-Acetylpyridine seemed a
suitable starting material to fulfill this objective.
We commenced our study on the reactions of ethylidenethiosemicarbazone 221 with D-keto hydrazonoyl
halides 3a-h in refluxing dioxane (6-10 h) in the presence of a basic catalyst such as TEA (Scheme 1).
The structures of isolated products 5a-h were evidenced by spectral data together with elemental analyses.
For instance, the IR spectra of products displayed in each case the absorption bands in the region 3431-
1
3397 and 1596-1590 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 G 11.20-10.85 ppm (D2O exchangeable)
assignable to the (NH) protons. On the basis of the foregoing results, the isolated products from the
reactions of
2
with 3a-h can be assigned 4-methyl-5-(aryldiazenyl)-2-(2-(1-(pyridin-4-
yl)ethylidene)hydrazinyl)thiazoles 5a-h (Scheme 1).
Authentic samples of 5a-h could be prepared through alternative synthetic procedures. Thus,
ethylidenethiosemicarbazone 2 was reacted with chloroacetone in EtOH under thermal conditions to give
thiazole derivative 6. Coupling of the latter product 6 with arene diazonium chloride in pyridine afforded
the respective authentic samples 5a-h (Scheme 1).
In a similar manner, when thiosemicarbazone derivative 2 was allowed to react with the ethyl (N-
arylhydrazono)-chloroacetates 7a-d in refluxing dioxane in the presence of triethylamine, it afforded in
each
case
a
single isolable product,
namely,
5-(2-arylhydrazono)-2-(2-(1-(pyridin-4-
yl)ethylidene)hydrazinyl)thiazol-4(5H)-ones 9a-d (Scheme 1). The structures of compounds 9a-d were
confirmed on the bases of spectral data and elemental analyses (see Experimental part).
For example, the IR spectra of the products showed, in each case, one carbonyl band at 1695-1710 cm-1
and two NH bands in the regions 3432–3425 and 3258–3251 cm-1. Their mass spectra of the latter
products revealed in each case, the molecular ion peaks at the expected m/z values and their elemental
analysis data were consistent with their assigned structures.

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HETEROCYCLES, Vol. 92, No. 5, 2016
4-Thiazolidinone compound 10 was obtained by reaction of thiosemicarbazone derivative 2 with ethyl
chloroacetate in EtOH and in the presence of anhydrous sodium acetate. Coupling of the latter product 10
with arenediazonium chloride in pyridine afforded the respective authentic samples 9a-d (Scheme 1).
Scheme 1. Synthesis of the arylazothiazoles 5a-h and 9a-d
Next, the reactivity of thiosemicarbazone derivative 2 towards hydrazonoyl halide, be bereft of a-keto
group, was examined. In the present study, we have established that reaction of
ethylidenethiosemicarbazone 2 with N-phenylcarbohydrazonoyl chloride (14) gave the 1,3,4-thiadiazole
17 as the end product (Scheme 2). The reaction proceeded via S-alkylation,22 with removal of hydrogen
chloride, to give S-alkylated intermediates 15 followed by intramolecular Michael23 type addition under

HETEROCYCLES, Vol. 92, No. 5, 2016
957
the employed reaction conditions, gave cycloadduct 16. Elimination of ammonia from 16 afforded the
same final product 17 (Scheme 2).
Structure 17 was confirmed by elemental analysis, spectral data, and alternative synthesis route. Thus,
methyl 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbodithioate (18) (prepared from refluxing 4-
acetylpyridine (1) with methyl hydrazinecarbodithioate in 2-propanol for 4 h) was reacted with 14 in
ethanol in the presence of TEA at room temperature for 2 h afforded a product identical in all aspects (mp,
mixed mp, and spectra) with 17.
N
NH2
Ph
N
H
N
NH2
+
S
Cl
H
N
dioxane / TEA
N
Me
N
S
Ph
Ph
S
N
N
NHPh
15
Me
14
N
2
Ph
N
N
Ph
H
NH2
N
N
N
- NH3
N
N
EtOH / TEA
-MeSH
N
Me
17
S
Ph
Me
N
Ph
N
16
14
H
H
N
SMe
N
SMe
N
H2N
S
S
1
Me
2-propanol
N
18
Scheme 2. Synthesis of 1,3,4-thiadiazole 17
Moreover, the thiosemicarbazone derivative 2 was reacted with various bromoacetyl compounds 19a-d to
furnish the respective thiazole derivatives 21a-d through the formation of intermediate 20a-d (Scheme 3).
1H NMR spectrum for 21a showed a singlet signal of the methine proton of thiazole ring at δ 7.73 ppm
and the NH proton appeared as another singlet at δ 12.12 ppm. Mass spectrum of compound 21a revealed
the presence of the molecular ion peak at m/z 294. The IR spectrum of compound 21d showed an
absorption peak at 1702 cm-1 due to C=O stretching vibrations.
Finally, compound 2 was reacted with 1,1’-(1,4-phenylene)bis(2-bromoethanone) (21) to give 1,4-
phenylene-bis-thiazole derivative 22 (Scheme 3). Structure 22 was elucidated via elemental analysis and
1
spectral data. H NMR spectrum of 22 showed three singlet signals at ߜ 2.39, 7.33 and 11.73 due to
methyl group, thiazole H5 and NH group, respectively. Its mass spectrum revealed a peak corresponding
to its molecular ion at m/z 510 [cf. Experimental section].

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HETEROCYCLES, Vol. 92, No. 5, 2016
H
N
O
N
NH2
N
N
H
Br
N
Ar
Ar
O
N
NH2
S
S
19a-d
N
2
Me
Ar
Me
S
-H2O
N
EtOH
N
Me
20a-d
21a-d
Ar
N
19-21
O
Br
C6H5-
4-NO2-C6H4-
a
Br
O
b
22
N
c
N
Me
Me
N
Ph
H
N
N
S
N
N
S
N
N
H
d
Me
O
O
23
N
Scheme 3. Synthesis of the thiazoles 20a-d and bis-thiazole 22
ANTICANCER ACTIVITY
The anticancer activity of some newly synthesized compounds was determined against a liver carcinoma
cell line HepG2, using doxorubicin as a reference drug. Data generated were used to plot a dose-response
curve of which the concentration (PM) of test compounds required to kill 50% of cell population (IC50)
was determined. The cytotoxic activity was expressed as the mean IC50 of three independent experiments
(Table 1) and the results revealed that all the tested compounds showed inhibitory activity to the tumor
cell lines in a concentration dependent manner. The small values of IC50 for the selected compounds
indicate that, for more anticancer effect higher concentrations can be used.
The results are represented in Table 1 showed that:
- The in vitro inhibitory activities of tested compounds against the human liver carcinoma (HepG2) have
the descending order as follow: 5h > 5d > 5g > 5f > 5e > 9c > 9d > 5a > 9a > 5b > 9b > 5c.
- The thiazole ring has in vitro inhibitory activity than the thiazolone ring (5a > 9a, 5b > 9b, 5d > 9c,
5g > 9d).
- The introduction of electron-withdrawing group (chlorine atom, bromine atom and nitro group) at the 4-
position of phenyl group at position 5 in the thiazole ring enhances the antitumor activity. In contrast,
introduction of electron-donating group (methyl group and methoxy group) decreases the antitumor
activity.

HETEROCYCLES, Vol. 92, No. 5, 2016
959
Table 1. The in vitro inhibitory activity of tested compounds against a liver carcinoma cell line HepG2
expressed as IC50 values (μM) ± standard deviation from six replicates
Compound No.
X (or Y)
IC50 (PM)
0.72 ± 0.16
19.3 ± 0.25
47.6 ± 0.14
>100
Doxorubicin
-
H
5a
5b
5c
5d
5e
5f
4-Me
4-MeO
4-Cl
2.48 ± 0.34
5.32 ± 0.27
4.03 ± 0.11
2.49 ± 0.24
2.23 ± 0.28
21.8 ± 0.19
49.6 ± 0.16
7.98 ± 0.30
11.3 ± 0.22
3-Cl
4-Br
4-NO2
2,4-(Cl)2
H
5g
5h
9a
9b
9c
9d
p-Me
p-Cl
4-NO2
In our present work,
a
new series of 2-ethylidenehydrazono-5-arylazothiazoles and 2-
ethylidenehydrazono-5-arylazothiazolones were synthesized from reaction of ethylidene-
thiosemicarbazide with various hydrazonoyl halides. The structures of the newly synthesized compounds
were established on the basis of spectroscopic evidences and their synthesis by alternative methods. The
in vitro growth inhibitory activity of the synthesized compounds against hepatocellular carcinoma
(HepG2) cell line was investigated in comparison with doxorubicin as a standard drug using MTT assay
and the results revealed promising activities of compounds 5h, 5d, 5g, 5f and 5e with IC50 equal 2.23 ±
0.28, 2.48 ± 0.34, 2.49 ± 0.24, 4.03 ± 0.11, and 5.32 ± 0.27 μM, respectively.
EXPERIMENTAL
All melting points were determined on an electrothermal Gallenkamp apparatus and are uncorrected.
Solvents were generally distilled and dried by standard literature procedures prior to use. The IR spectra

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HETEROCYCLES, Vol. 92, No. 5, 2016
1
13
were measured on a Pye-Unicam SP300 instrument in potassium bromide discs. The H-NMR and C-
NMR spectra were recorded on a Varian Mercury VXR-300 spectrometer (300 MHz for 1H-NMR and 75
13
MHz for C-NMR) and the chemical shifts were related to that of the solvent DMSO-d6. The mass
spectra were recorded on a GCMSQ1000-EX Shimadzu and GCMS 5988-A HP spectrometers, the
ionizing voltage was 70 eV. Elemental analyses and spectral measurements were carried out by the
microanalytical center at Cairo University and the Analytical Laboratory of the Institute of Organic
Chemistry, Technical University of Dresden, Germany. Antitumor activity was evaluated at the Regional
Center for Mycology and Biotechnology at Al-Azhar University, Cairo, Egypt. Hydrazonoyl halides 3a-h,
7a-d and 14 were prepared as reported in the literature.24
Reactions of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2) with hydrazonoyl chlorides
3a-h
General procedure. A mixture of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2) (0.194 g, 1
mmol) and appropriate hydrazonoyl halides 3a-h (1 mmol) in dioxane (15 mL) containing triethylamine
(0.1 g, 1 mmol) was refluxed until all the starting materials were consumed (3–6 h as monitored by TLC).
Excess of solvent was removed under reduced pressure and the reaction mixture was triturated with
MeOH. The product separated was filtered, washed with MeOH, dried and recrystallized from the proper
solvent to give compounds 5a-h. The products 5a-h together with their physical constants are listed
below.
4-Methyl-5-(phenyldiazenyl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (5a). Red solid
1
(68% yield); mp 170-172 °C (DMF); IR (KBr): v/cm-1 3426 (NH), 3052, 2923 (CH), 1595 (C=N); H-
NMR (DMSO-d6): δ 2.54 (s, 3H, CH3), 2.65 (s, 3H, CH3), 7.05-8.94 (m, 9H, Ar-H), 11.12 (s, 1H, NH);
MS m/z (%): 336 (M+, 13). Anal. Calcd for C17H16N6S (336.12): C, 60.69; H, 4.79; N, 24.98. Found C,
60.53; H, 4.71; N, 24.75%.
4-Methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)-5-(p-tolyldiazenyl)thiazole (5b). Red solid
1
(70% yield); mp 192-194 °C (DMF); IR (KBr): v/cm-1 3415 (NH), 3047, 2934 (CH), 1596 (C=N); H-
NMR (DMSO-d6): δ 2.30 (s, 3H, CH3), 2.52 (s, 3H, CH3), 2.61 (s, 3H, CH3), 6.87-8.94 (m, 8H, Ar-H),
11.03 (s, 1H, NH); MS m/z (%): 350 (M+, 61). Anal. Calcd for C18H18N6S (350.13): C, 61.69; H, 5.18; N,
23.98. Found C, 61.49; H, 5.05; N, 23.77%.
5-((4-Methoxyphenyl)diazenyl)-4-methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (5c).
Red solid (66% yield); mp 210-212 °C (DMF); IR (KBr): v/cm-1 3397 (NH), 3051, 2927 (CH), 1594
1
(C=N); H-NMR (DMSO-d6): δ 2.49 (s, 3H, CH3), 2.58 (s, 3H, CH3), 2.78 (s, 3H, OCH3), 6.98-8.83 (m,
8H, Ar-H), 11.01 (s, 1H, NH); MS m/z (%): 366 (M+, 46). Anal. Calcd for C18H18N6OS (366.13): C,
59.00; H, 4.95; N, 22.93. Found C, 58.86; H, 4.92; N, 22.79%.

HETEROCYCLES, Vol. 92, No. 5, 2016
961
5-((4-Chlorophenyl)diazenyl)-4-methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole
(5d).
Red solid (73% yield); mp 178-180 °C (DMF); IR (KBr): v/cm-1 3404 (NH), 3071, 2971 (CH), 1596
(C=N); 1H-NMR (DMSO-d6): δ 2.54 (s, 3H, CH3), 2.65 (s, 3H, CH3), 7.31-8.95 (m, 8H, Ar-H), 11.06 (s,
1H, NH); MS m/z (%): 372 (M++2, 42), 370 (M+, 13). Anal. Calcd for C17H15ClN6S (370.08): C, 55.06; H,
4.08; N, 22.66. Found C, 54.91; H, 4.01; N, 22.47%.
5-((3-Chlorophenyl)diazenyl)-4-methyl-2-((2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole
(5e).
Red solid (67% yield); mp 187-189 °C (DMF \ EtOH); IR (KBr): v/cm-1 3418 (NH), 3046, 2977 (CH),
1
1592 (C=N); H-NMR (DMSO-d6): δ 2.55 (s, 3H, CH3), 2.63 (s, 3H, CH3), 7.04-8.88 (m, 8H, Ar-H),
10.93 (s, 1H, NH); MS m/z (%): 372 (M++2, 20), 370 (M+, 6). Anal. Calcd for C17H15ClN6S (370.08): C,
55.06; H, 4.08; N, 22.66. Found C, 55.00; H, 4.13; N, 22.57%.
5-((4-Bromophenyl)diazenyl)-4-methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole
(5f).
Red solid (72% yield); mp 172-174 °C (DMF); IR (KBr): v/cm-1 3407 (NH), 3080, 2967 (CH), 1590
(C=N); 1H-NMR (DMSO-d6): δ 2.49 (s, 3H, CH3), 2.60 (s, 3H, CH3), 7.33-8.82 (m, 8H, Ar-H), 10.85 (s,
1H, NH); MS m/z (%): 416 (M++2, 16), 414 (M+, 14). Anal. Calcd for C17H15BrN6S (414.03): C, 49.16; H,
3.64; N, 20.24. Found C, 49.12; H, 3.58; N, 20.17%.
4-Methyl-5-((4-nitrophenyl)diazenyl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole
(5g).
Brown solid (69% yield); mp 219-221 °C (EtOH); IR (KBr): v/cm-1 3425 (NH), 3052, 2914 (CH), 1592
(C=N); 1H-NMR (DMSO-d6): δ 2.49 (s, 3H, CH3), 2.65 (s, 3H, CH3), 7.51-8.75 (m, 8H, Ar-H), 11.09 (s,
1H, NH); MS m/z (%): 381 (M+, 38). Anal. Calcd for C17H15N7O2S (381.10): C, 53.53; H, 3.96; N, 25.71.
Found C, 53.48; H, 3.91; N, 25.66%.
5-((2,4-Dichlorophenyl)diazenyl)-4-methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)-thiazole
(5h). Red solid (73% yield); mp 141-143 °C (DMF); IR (KBr): v/cm-1 3431 (NH), 3054, 2975 (CH), 1590
(C=N); 1H-NMR (DMSO-d6): δ 2.52 (s, 3H, CH3), 2.62 (s, 3H, CH3), 76.94-8.81 (m, 7H, Ar-H), 11.13 (s,
1H, NH); MS m/z (%): 404 (M+, 19). Anal. Calcd for C17H14Cl2N6S (404.04): C, 50.38; H, 3.48; N, 20.74.
Found C, 50.26; H, 3.39; N, 20.64%.
Alternate method for 5a-h
A. Synthesis of 4-methyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (6)
To a solution of thiosemicarbazone 2 (1.98 g, 10 mmol) in EtOH (20 mL), chloroacetone (0.92 g, mmol)
was added. The mixture was refluxed for 4-8 h (monitored by TLC), then left to cool. The solid product
was filtered off, washed with EtOH and recrystalized from dioxane to afford the thiazole derivative 6 as
yellow solid (69% yield); mp 184-186 °C; IR (KBr): v/cm-1 3423 (NH), 3050, 2920 (CH), 1592 (C=N);
1H-NMR (DMSO-d6): δ 2.17 (s, 3H, CH3), 2.27 (s, 3H, CH3), 6.35 (s, 1H, thiazole-H5), 7.66 (d, J = 5.1
Hz, 2H, pyridine-H3,H5), 8.57 (d, J = 5.1 Hz, 2H, pyridine-H2,H6), 11.32 (s, 1H, NH); MS m/z (%): 232

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HETEROCYCLES, Vol. 92, No. 5, 2016
(M+, 100). Anal. Calcd for C11H12N4S (232.08): C, 56.87; H, 5.21; N, 24.12. Found C, 56.75; H, 5.19; N,
24.00%.
B. Coupling of thiazole 6 with arenediazonium chlorides
o
To a solution of 6 (0.464 g, 2 mmol) in pyridine (20 mL), cooled to 0-5 C in an ice bath, was added
portionwise a cold solution of arenediazonium chloride [prepared by diazotizing aniline derivatives (2
mmol) dissolved in hydrochloric acid (6 M, 2 mL) with a solution of sodium nitrite (0.14 g, 2 mmol) in
water (3 mL)]. After complete addition of the diazonium salt, the reaction mixture was stirred for a
further 30 min in an ice bath. The solid that separated was filtered off, washed with water and finally
recrystallized from DMF to give products proved to be identical in all respects (mp, mixed mp and IR
spectra) with compounds 5a-h which obtained from reaction of 2 with 3a-h.
Reactions of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2) with hydrazonoyl chlorides
7a-d. General procedure. A mixture of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2)
(0.194 g, 1 mmol) and appropriate hydrazonoyl halides 7a-d, (1 mmol) in dioxane (15 mL) containing
TEA (0.1 g, 1 mmol) was refluxed until all the starting materials were consumed (3–6 h as monitored by
TLC). Excess of solvent was removed under reduced pressure and the reaction mixture was triturated
with MeOH. The product separated was filtered, washed with MeOH, dried and recrystallized from EtOH
to give compounds 9a-d. The products 9a-d together with their physical constants are listed below.
5-(2-Phenylhydrazono)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4(5H)-one (9a). Yellow
solid (70% yield); mp 175-177 °C; IR (KBr): v/cm-1 3427, 3258 (2NH), 3043, 2974 (CH), 1695 (C=O),
1
1593 (C=N); H-NMR (DMSO-d6): δ 2.33 (s, 3H, CH3), 6.93-8.85 (m, 9H, Ar-H), 10.24, 12.18 (2s, 2H,
2NH); MS m/z (%): 338 (M+, 73). Anal. Calcd for C16H14N6OS (338.09): C, 56.79; H, 4.17; N, 24.84.
Found C, 56.64; H, 4.10; N, 24.79%.
2-(2-(1-(Pyridin-4-yl)ethylidene)hydrazinyl)-5-(2-(p-tolyl)hydrazono)thiazol-4(5H)-one (9b). Yellow
solid (71% yield); mp 160-162 °C; IR (KBr): v/cm-1 3433, 3253 (2NH), 3051, 2922 (CH), 1708 (C=O),
1
1619 (C=N); H-NMR (DMSO-d6): δ 2.21 (s, 3H, CH3), 2.37 (s, 3H, CH3), 7.04-8.70 (m, 8H, Ar-H),
10.42, 12.23 (2s, 2H, 2NH); MS m/z (%): 338 (M+, 73). Anal. Calcd for C17H16N6OS (352.11): C, 57.94;
H, 4.58; N, 23.85. Found C, 57.84; H, 4.47; N, 23.75%.
5-(2-(4-Chlorophenyl)hydrazono)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4(5H)-one
(9c). Yellow solid (72% yield); mp 210-212 °C; IR (KBr): v/cm-1 3425, 3166 (2NH), 3101, 2974 (CH),
1
1708 (C=O), 1597 (C=N); H-NMR (DMSO-d6): δ 2.34 (s, 3H, CH3), 7.10-8.70 (m, 8H, Ar-H), 10.40,
12.15 (2s, 2H, 2NH); MS m/z (%): 372 (M+, 29). Anal. Calcd for C16H13ClN6OS (372.06): C, 51.54; H,
3.51; N, 22.54. Found C, 51.38; H, 3.44; N, 22.46%.
5-(2-(4-Nitrophenyl)hydrazono)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4(5H)-one (9d).
Yellow solid (68% yield); mp 161-163 °C; IR (KBr): v/cm-1 3432, 3251 (2NH), 3054, 2930 (CH), 1710

HETEROCYCLES, Vol. 92, No. 5, 2016
963
1
(C=O), 1594 (C=N); H-NMR (DMSO-d6): δ 2.29 (s, 3H, CH3), 7.37-8.69 (m, 8H, Ar-H), 10.39, 12.09
(2s, 2H, 2NH); MS m/z (%): 383 (M+, 57). Anal. Calcd for C16H13N7O3S (383.08): C, 50.12; H, 3.42; N,
25.57. Found C, 50.03; H, 3.39; N, 25.46%.
Alternate method for 9a-d
A. Synthesis of 2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4(5H)-one (10)
To a mixture of thiosemicarbazone 2 (0.198 g, 1 mmol) and anhydrous sodium acetate (0.33 g, 4 mmol)
in EtOH (20 mL) was added, ethyl chloroacetate (1.22 g, 10 mmol) and then mixture was refluxed for 4-8
h (monitored by TLC), then left to cool. The solid product was filtered off, washed with water and
recrystalized from EtOH to afford the thiazolone derivative 10 as pale brown solid (69% yield); mp 230-
232 °C; IR (KBr): v/cm-1 3431 (NH), 3052, 2975 (CH), 1712 (C=O), 1597 (C=N); 1H-NMR (DMSO-d6):
δ 2.29 (s, 3H, CH3), 3.97 (s, 2H, CH2), 7.73 (d, J = 5.1 Hz, 2H, pyridine-H3,H5), 8.64 (d, J = 5.1 Hz, 2H,
pyridine-H2,H6), 10.39 (s, 1H, NH); 13C-NMR (DMSO-d6): δ 15.91 (CH3), 33.10 (CH2), 123.25, 148.10,
154.73, 163.23, 165.18 (Ar-C), 174.26 (C=O) ppm; MS m/z (%): 234 (M+, 63). Anal. Calcd for
C10H10N4OS (234.06): C, 51.27; H, 4.30; N, 23.91. Found C, 51.19; H, 4.27; N, 23.84%.
B. Coupling of thiazolone derivative 10 with arenediazonium chlorides
o
To a solution of 10 (0.468 g, 2 mmol) in pyridine (20 mL), cooled to 0-5 C in an ice bath, was added
portionwise a cold solution of arenediazonium chloride [prepared by diazotizing aniline derivatives (2
mmol) dissolved in hydrochloric acid (6 M, 2 mL) with a solution of sodium nitrite (0.14 g, 2 mmol) in
water (3 mL)]. After complete addition of the diazonium salt, the reaction mixture was stirred for a
further 30 min in an ice bath. The solid that separated was filtered off, washed with water and finally
recrystallized from DMF to give products proved to be identical in all respects (mp, mixed mp and IR
spectra) with compounds 10a-d which obtained from reaction of 2 with 7a-d.
Reactions of ethylidenethiosemicarbazide 2 with N’-phenylbenzohydrazonoyl chloride (14)
A mixture of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2) (0.194 g, 1 mmol) and N’-
phenylbenzohydrazonoyl chloride (14) (0.230 g, 1 mmol) in dioxane (10 mL) containing triethylamine
(0.1 g, 1 mmol) was refluxed for 5 h. Excess of solvent was removed under reduced pressure and the
reaction mixture was triturated with MeOH. The product separated was filtered, washed with MeOH,
dried and recrystallized from DMF to give 3,5-diphenyl-2-((1-(pyridin-4-yl)ethylidene)hydrazono)-2,3-
dihydro-1,3,4-thiadiazole 17 as white solid (70% yield); mp 147-149 °C; IR (KBr): v/cm-1 3051, 2929
1
(CH), 1593 (C=N); H-NMR (DMSO-d6): δ 2.46 (s, 3H, CH3), 7.24-7.92 (m, 14H, Ar-H); MS m/z (%):
371 (M+, 63). Anal. Calcd for C21H17N5S (371.12): C, 67.90; H, 4.61; N, 18.85. Found C, 67.83; H, 4.44;
N, 18.70%.
Alternate method for 17
A. Synthesis of methyl 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbodithioate (18)

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HETEROCYCLES, Vol. 92, No. 5, 2016
To a solution of 4-acetylpyridine (1) (0.242 g, 2 mmol) in 2-propanol (20 mL) was added methyl
hydrazinecarbodithioate (0.244 g, 10 mmol). The mixture was refluxed for 3 h then left to cool. The solid
product was filtered off and finally recrystalized from EtOH to afford compound 18 as yellow (66%
1
yield); mp 212-214 °C; IR (KBr): v/cm-1 3356 (NH), 3051, 2981 (CH), 1597 (C=N); H-NMR (DMSO-
d6): δ 2.26 (s, 3H, CH3), 2.41 (s, 3H, CH3), 8.11 (d, J = 5.1 Hz, 2H, pyridine-H3,H5), 8.87 (d, J = 5.1 Hz,
2H, pyridine-H2,H6), 11.12 (s, 1H, NH); MS m/z (%): 386 (M+, 21), 273 (55), 92 (26), 77 (100). Anal.
Calcd for C9H11N3S2 (225.04): C, 47.97; H, 4.92; N, 18.65. Found C, 47.88; H, 4.78; N, 18.5%.
B. Reaction of 18 with N’-phenylbenzohydrazonoyl chloride (14)
A mixture of methyl 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbodithioate (18) (0.225 g, 1 mmol) and
N’-phenylbenzohydrazonoyl chloride (14) (0.230 g, 1 mmol) in EtOH (10 mL) containing triethylamine
(0.1 g, 1 mmol) was stirred at room temperature for 2 h. The product separated was filtered, washed with
MeOH, dried and finally recrystallized from dioxane to give product proved to be identical in all respects
(mp, mixed mp, and IR spectra) with compound 17 which obtained from reaction of 2 with 14.
Synthesis of thiazoles 21a-d
General procedure. To a solution of 2-(1-(pyridin-4-yl)ethylidene)hydrazinecarbothioamide (2) (0.194 g,
1 mmol) in EtOH (20 mL), a proper bromoacetyl derivative 19a-d (1 mmol) was added. The mixture was
refluxed for 4-8 h (monitored by TLC), then left to cool. The solid product was filtered off, washed with
EtOH and recrystalized from proper solvent to afford the thiazole derivative 21a-d. The products 21a-d
together with their physical constants are listed below.
4-Phenyl-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (21a). Yellow solid (72% yield); mp
1
226-228 °C (EtOH); IR (KBr): v/cm-1 3429 (NH), 3057, 2922 (CH), 1595 (C=N); H-NMR (DMSO-d6):
δ 2.37 (s, 3H, CH3), 7.73 (s, 1H, thiazole H5), 7.41-7.52 (m, 5H, Ar-H), 7.87 (d, J = 5.1 Hz, 2H, pyridine-
H3,H5), 8.76 (d, J = 5.1 Hz, 2H, pyridine-H2,H6), 12.12 (s, 1H, NH); MS m/z (%): 294 (M+, 51). Anal.
calcd for C16H14N4S (294.09): C, 65.28; H, 4.79; N, 19.03. Found: C, 65.14; H, 4.66; N, 18.93%.
4-(4-Nitrophenyl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (21b). Brown solid (76%
1
yield); mp 281-283 °C (DMF); IR (KBr): v/cm-1 3434 (NH), 3049, 2965 (CH), 1594 (C=N); H-NMR
(DMSO-d6): δ 2.38 (s, 3H, CH3), 7.86 (s, 1H, thiazole H5), 8.06- 8.18 (m, 4H, Ar-H), 8.27 (d, J = 5.1 Hz,
2H, pyridine-H3,H5), 8.83 (d, J = 5.1 Hz, 2H, pyridine-H2,H6), 12.17 (s, 1H, NH); MS m/z (%): 339 (M+,
65). Anal. calcd for C16H13N5O2S (339.08): C, 56.63; H, 3.86; N, 20.64. Found: C, 56.51; H, 3.69; N,
20.53%.
4-(5-Methyl-1-phenyl-1H-pyrazol-4-yl)-2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazole (21c).
Yellow solid, (70% yield); mp 206-208 °C (DMF); IR (KBr): v/cm-1 3344 (NH), 3050, 2982 (CH), 1602
1
(C=N); H-NMR (DMSO-d6): δ 2.37 (s, 3H, CH3), 2.55 (s, 3H, CH3), 7.03-7.98 (m, 6H, Ar-H and
thiazole H5), 7.57 (d, J = 5.1 Hz, 2H, pyridine-H3,H5), 8.41 (s, 1H, pyrazole H3), 8.90 (d, J = 5.1 Hz, 2H,

HETEROCYCLES, Vol. 92, No. 5, 2016
965
pyridine-H2,H6), 11.47 (s, 1H, NH); MS m/z (%): 374 (M+, 39). Anal. calcd for C20H18N6S (374.13): C,
64.15; H, 4.85; N, 22.44. Found: C, 64.04; H, 4.69; N, 22.27%.
3-(2-(2-(1-(Pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4-yl)-2H-chromen-2-one (21d). Yellow solid
(70% yield); mp 263-265 °C (dioxane); IR (KBr): v/cm-1 3423 (NH), 3063, 2959 (CH), 1702 (C=O), 1600
1
(C=N); H-NMR (DMSO-d6): δ 2.36 (s, 3H, CH3), 7.37-7.69 (m, 4H, Ar-H), 7.83 (d, J = 5.1 Hz, 2H,
pyridine-H3,H5), 8.34 (s, 1H, Coumarine H4), 8.58 (s, 1H, thiazole H5), 8.78 (d, J = 5.1 Hz, 2H,
pyridine-H2,H6), 12.07 (s, 1H, NH); MS m/z (%): 362 (M+, 39). Anal. calcd for C19H14N4O2S (362.08): C,
62.97; H, 3.89; N, 15.46. Found: C, 62.83; H, 3.79; N, 15.30%.
Synthesis of 1,4-bis(2-(2-(1-(pyridin-4-yl)ethylidene)hydrazinyl)thiazol-4-yl)benzene (23)
To a solution of thiosemicarbazone 2 (0.198 g, 1 mmol) in EtOH (20 mL), 1,1’-(1,4-phenylene)bis(2-
bromoethanone) (21) (0.634 g, 2 mmol) were added. The mixture was refluxed for 4-8 h (monitored by
TLC), then left to cool. The solid product was filtered off, washed with EtOH and recrystalized from
DMF to afford the bis-thiazole derivative 22 as a white solid (68% yield); mp 327-329 °C; IR (KBr):
1
v/cm-1 3423 (NH), 3063, 2975 (CH), 1593 (C=N); H-NMR (DMSO-d6): δ 2.39 (s, 6H, 2CH3), 7.33 (s,
2H, 2 thiazole H5), 8.02 (d, J = 5.1 Hz, 4H, 2 pyridine-H3,H5), 8.14 (s, 4H, Ar-H), 8.82 (d, J = 5.1 Hz,
4H, 2 pyridine-H2,H6), 11.73 (s, 2H, 2NH); MS m/z (%): 510 (M+, 17). Anal. Calcd for C26H22N8S2
(510.14): C, 61.15; H, 4.34; N, 21.94. Found C, 61.08; H, 4.329; N, 21.76%.
Antitumor Activity
Human liver carcinoma (HepG-2) cell lines were obtained from the American Type Culture Collection
(ATCC, Rockville, MD). The cells were grown on RPMI-1640 medium supplemented with 10%
inactivated fetal calf serum and 50 μg/mL entamycin. The cells were maintained at 37 °C in a humidified
atmosphere with 5% CO2 and were subcultured two to three times aweek.
For antitumor assys, the tumor cell lines were suspended in medium at concentration 5 x 104 cell/well in
corning® 96-well plates (six replicates) to achieve eight concentrations for each compound. Six vehicle
controls with media or 0.5% DMSO were run for each 96 well plate as a control. After incubating for 24
h, the numbers of viable cells were determined by the MTT test. Briefly, the media was removed from the
96 well plate and replaced with 100 μL of fresh culture RPMI 1640 medium without phenol red then 10
μL of the 12 Mm MTT stock solution (5 mg of MTT in 1 mL of PBS) to each well including the
untreated controls. The 96 well plates were then incubated at 37 °C and 5% CO2 for 4 h. An 85 μL aliquot
of the media was removed from the wells, and 50 μL of DMSO was added to each well and mixed
thoroughly with the pipette and incubated at 37 °C for 10 min. then, the optical density was measured at
590 nm with the microplate reader (SunRise, TECAN, Inc, USA) to determine the number of viable cells
and the perecentage of viability was calculated as (1-(ODt/ODc)) x 100% where ODt is the mean optical

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HETEROCYCLES, Vol. 92, No. 5, 2016
density of untreated cells. The relation between surviving cells and drug concentration is plotted to get the
survival curve of each tumor cell line after treatment with the specified compound. The 50% inhibitory
concentration (IC50), the concentration required to cause toxic effects in 50% of intact cells, was
estimated from graphic plots of the dose response curve for each conc. Using Graphad Prism software
(San Diego, CA, USA).25-28
ACKNOWLEDGEMENT
We thank the Alexander von Humboldt-Foundation (Germany) for granting a short research fellowship
(July-September 2015) to F. M. Abdelrazek; during which time a considerable part of the analyses and
the spectra analyses of this work has been carried out.
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