Synthesis of some p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles and their anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2010.08.017

A series of novel p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles derivatives (2a-f, 3a-f and 5-8) were synthesized by initial condensation of p-toluenesulfonylthiosemicarbazide 1 with a series of α-halogenocarbonyls in acetone or dimethy

Full text of DOI:10.1016/j.ejmech.2010.08.017

European Journal of Medicinal Chemistry 45 (2010) 5080e5085
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of some p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-
thiazoles and their anticancer activity
,
a
b
a,
c
,
d
Valentin Zaharia a , Adriana Ignat , Nicolae Palibroda , Bathélémy Ngameni
, Victor Kuete ,
*
**
Charles N. Fokunang c, Marlyse L. Moungang e, Bonaventure T. Ngadjui c
,
e
a Faculty of Pharmacy, “Iuliu Hatieganu” University of Medicine and Pharmacy, Cluj-Napoca, Romania
b National Institute for Isotopic and Molecular Technologies, Cluj-Napoca, Romania
c Faculty of Medicine and Biomedical Science, University of Yaoundé I, Yaoundé, Cameroon
d Faculty of Science, University of Dschang, Dschang, Cameroon
e Faculty of Science, University of Yaoundé I, Yaoundé, Cameroon
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel p-toluenesulfonyl-hydrazinothiazoles and hydrazino-bis-thiazoles derivatives (2aef, 3aef
and 5e8) weresynthesized byinitial condensation of p-toluenesulfonylthiosemicarbazide 1 with a series of
Received 4 June 2010
Received in revised form
3 August 2010
Accepted 6 August 2010
Available online 12 August 2010
a-halogenocarbonyls in acetone or dimethylformamide (DMF)/acetone, mixture. All our synthesized
compounds were submitted for further acylation reaction in the presence of acetic anhydride. The struc-
tures of newly synthesized derivatives 2aef, 3aef and 5e8 were confirmed by IR, 1H-NMR, EIMS spectral
data and elemental analysis. Compounds 2a, 2c, 2d, 2e and 3a showed significant anticancer activities
(IC50 < 10
m
M) on both prostate DU-145 and hepatocarcinoma Hep-G2 cancer cell lines.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
p-Toluenesulfonyl-hydrazinothiazoles
Hydrazino-bis-thiazoles
Cytotoxicity
Prostate cancer
Hepatocarcinoma
1. Introduction
synthetic thiazoles [11,12]. In the previous work, acid hydrazides
were documented as important compounds, due to their high
reactivity usefulness in heterorganic synthesis, as key starting
materials to form various classes of biologically and pharmacologi-
cally active candidates [8,13]. Holla et al. [14] synthesized a series of
aryliden-hydrazinothiazoles and 2-furanyliden-hydrazinothiazoles
with antimicrobial and anti-inflammatory activities. Bhat and Holla
[15] also used a series of acyl-thiosemicarbazide in the synthesis of
thiazolo[2,3-c][1,2,4]triazoles. Such results encouraged us to study
several-hydrazinothiazoles [16e19], for their biological properties.
In view of these reports and in continuation of our previous works in
heterocyclic chemistry, the present study was designed to synthe-
size new hydrazinothiazoles derivatives and investigate their
cytotoxic activities. Compared with the activity of the reference anti-
neoplastic compound (doxorubicin) on both prostate DU-145 and
hepatocarcinoma Hep-G2 cancer cell lines, some of the compounds
can be considered as good anticancer compounds. Compounds 3a,
2e, 2c, 2d, 2a and 1 showed 100% inhibition of DU-145 proliferation
The experimental researchled tothe identification of biologically
active compounds with the sulphonyl-hydrazinic group. The thia-
zolyl group is also of great importance in treating biological systems.
Compounds of this functional group showed antimicrobial [1,2],
antitumoural [2e6], analgesic [1,7], anti-inflammatory and antipy-
retic [1] properties. Also, some synthetic thiazoles exhibited a wide
range of biological activities, such as antitumor, antifilarial, anti-
bacterial, antifungal, and anti-inflammatory [8]. The activities of
hydrazinothiazole compounds, on Monoamine Oxidase Inhibitors
(MAOI) [9], and the anti-thrombotic activities [10] of aryliden-thi-
osemicarbazole compounds were reported. Recent studies demon-
strated significant antimicrobial and anticancer potencies of new
* Corresponding author. Tel.: þ40 264 594148; fax: þ40 264 597257.
** Corresponding author. Faculty of Medicine and Biomedical Science, University
of Yaoundé I, P.O. Box 8664, Yaoundé, Cameroon. Tel.: þ237 76480440; fax: þ237
22221873.
E-mail addresses: vzaharia@umfcluj.ro (V. Zaharia), bath_ngameni@yahoo.fr (B.
Ngameni).
respectively at 34.1; 35.2; 36.2; 38.5; 44.2 and 102
inhibition at 34.1; 35.2; 36.2; 44.2; 51 and 72.5
proliferation was also recorded with 3a, 2e, 2a,1 and 2c respectively
m
M (Fig. 1). Total
m
M of Hep-G2
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.08.017

V. Zaharia et al. / European Journal of Medicinal Chemistry 45 (2010) 5080e5085
5081
160
140
120
100
80
60
40
160
140
120
100
80
compound 1
160
140
120
100
80
160
140
120
100
80
compound 2a
compound 2c
compound 2d
60
60
60
40
40
40
20
20
0
20
20
0
0
0
4.8 9.6 19.2 38.5 76.9
6.38 12.76 25.5
51
102
5.5
11 22.1 44.2 88.3
4.5
9
18.1 36.2 72.5
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
160
140
120
100
80
60
40
compound 2e
160
140
120
100
80
60
40
160
140
120
100
80
compound 3b
compound 3a
60
40
20
20
0
20
0
0
3.9
7.8 15.6 31.2 62.3
4.4 8.8 17.6 35.2 70.4
4.3
8.5
17
34.1 68.1
Concentration (µM)
Concentration (µM)
Concentration (µM)
compound 3c
160
140
120
100
80
160
140
120
100
80
60
40
160
140
120
100
80
60
40
compound 3e
Compound 3d
60
40
20
0
20
20
0
0
3.8
7.6
15.3 30.1 61.1
3.6
7.1 14.2 28.5 56.9
3.6
7.3
14.6 29.1 58.3
Concentration (µM)
Concentration (µM)
Concentration (µM)
compound 3f
160
160
140
120
100
80
60
40
160
140
120
100
80
60
40
160
140
120
100
80
60
40
compound 5
compound 8
Doxorubicin
140
120
100
80
60
40
20
0
20
0
20
0
20
0
4.5
8.9 17.9 35.7 71.4
3.6
7.3 14.5 29.1 58.1
2.9 5.8 11.5 23 46
Concentration (µM)
3.6
7.1
14.2 28.5 56.9
Concentration (µM)
Concentration (µM)
Concentration (µM)
Fig. 1. Effects of the compounds on DU-145.
(Fig. 2). Though compound 1 (the substrate) was active on both DU-
145 and Hep-G2, it can be noted that some of the synthesized
derivatives were more effective, as they presented lower IC50 values.
This includes compound 2a and 2e on the two cancer cell lines.
The derivatives of p-toluenesulfonyl-hydrazinothiazole 2aef were
synthesized with the condensation reaction of p-toluenesulfonylth-
iosemicarbazide 1 [20] and aseriesof
tone; 1,3-dichloroacetone; -bromoacetophenone; 3-chloroacetylace
tone; ethyl -bromoacetoacetate and ethyl -bromoacetoacetate), in
a-halogenocarbonyls (chloroace
a
Though not as active as doxorubicin, compounds 2a (IC50 < 5.65
mM
a
g
on Hep-G2) and2e (IC50 of4.68 Mon DU-145and below4.51 M on
m
m
acetone or DMF/acetone, mixture (Scheme 1). Compounds 3aef were
obtained by the acylation reaction in the presence of acetic anhydride.
A series of compounds were also synthesized that were linked
by two thiazole nucleus separated by the carbonylhydrazine
(eCOeNHeNHe) group. For the synthesis of these organic
compounds, the 4-(2-phenyl-thiazol-4-carbonyl)-thiosemicarba
Hep-G2) showed the best anticancer activities (Table 1). When
regarding the structureeactivity relationship, it can mainly be
deduced from the results obtained with compounds from series 2
(Compounds 2a, 2c, 2d, 2e, 2f) and 3 (Compounds 3a, 3c, 3d, 3e, 3f)
that acetylation significantly reduced the anticancer activity. This
hypothesis is confirmed when analysing the results of compounds 5
and 8 on the two cancer cell lines.
zide 4 was condensed with 1,3-dichloroacetone and ethyl g-bro-
moacetoacetate respectively (Scheme 2).
The thiazolic compounds reacted with acetic anhydride by the
acylation reaction (Scheme 2). In the synthesis of sulfonyl-hydra-
zinothiazole derivatives (Scheme 1), the hydrazinic group takes
part in a double acylation with acetic anhydride, but in the case of
thiazolyl-carbonylhydrazinothiazole, there was instead a mono-
acylation reaction with acetic anhyride.
No condensation and acylation reaction studies on synthesized
p-toluenesulfonyl-hydrazinothiazole 2aef, 3aef and thiazolyl-
carbonylhydrazinothiazole 5e8 derivatives, was previously
2. Results and discussion
For the synthesis of these organic compounds, we used the
Hantzsch type condensation reaction with a p-toluenesulfonylth-
iosemicarbazide 1 as reagents, obtained by acylation of thio-
semicarbazide with the p-toluenesulfochloride. The reaction was
done in the presence of both NaOH and pyridine. The best result
was obtained using the NaOH method.

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V. Zaharia et al. / European Journal of Medicinal Chemistry 45 (2010) 5080e5085
160
140
120
100
80
60
40
160
140
120
100
80
compound 2a
160
140
120
100
80
60
40
160
140
120
100
80
60
40
compound 2c
compound 2d
compound1
60
40
20
20
0
20
0
20
0
0
5.5
11 22.1 44.2 88.3
4.5
9
18.1 36.2 72.5
4.8
9.6 19.2 38.5 76.9
6.38 12.76 25.5 51 102
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
160
140
120
100
80
160
140
120
100
80
160
140
120
100
80
compound 2e
compound 3b
compound 3a
60
60
60
40
40
40
20
20
0
20
0
0
4.4 8.8 17.6 35.2 70.4
3.9 7.8 15.6 31.2 62.3
4.3
8.5
17 34.1 68.1
Concentration (µM)
Concentration (µM)
Concentration (µM)
160
140
120
100
80
compound 3c
160
140
120
100
80
60
40
160
140
120
100
80
compound 3e
compound 3d
60
60
40
40
20
20
0
20
0
0
3.6
7.1 14.2 28.5 56.9
3.6 7.3 14.6 29.1 58.3
3.8
7.6
15.3 30.1 61.1
Concentration (µM)
Concentration (µM)
Concentration (µM)
160
140
120
100
80
160
140
120
100
80
compound 3f
compound 5
160
140
120
100
80
160
140
120
100
80
60
40
compound 8
Doxorubicin
60
60
60
40
40
40
20
20
20
0
20
0
0
0
4.5 8.9 17.9 35.7 71.4
3.6 7.1 14.2 28.5 56.9
3.6
7.3 14.5 29.1 58.1
2.9
5.8
11.5
23
46
Concentration (µM)
Concentration (µM)
Concentration (µM)
Concentration (µM)
Fig. 2. Effects of the compounds on Hep-G2 cells survivals.
reported. The adduct (2aef) and acylated (3aef, 5e8) products
were isolated here for the first time via total synthesis by trans-
formation of synthetics condensate 1 and 4 as a starting material.
The structures of the synthesized compounds were assigned on the
bases of its spectral data, mainly mass, elemental analysis, IR and
1H-NMR (cf. Section 3).
hydrazinothiazoles and hydrazino-bis-thiazoles derivatives as anti-
cancer compounds are currently under investigation in our laboratory.
3. Experimental
3.1. Chemistry
In conclusion, a series of new p-toluenesulfonyl-hydrazinothiazoles
and hydrazino-bis-thiazoles derivatives have been made conveniently
available by direct condensation and acylation of a p-toluenesulfo-
Melting points were determined on open glass capillaries using
an Electrothermal IA 9000 digital melting point apparatus and are
uncorrected. The 1H-NMR spectra were recorded with Bruker WM-
300 and Bruker WM-400 spectrometer in the CDCl3 or acetone-d6.
The mass spectra were recorded using a Varian MAT-311A and the
IR spectra with IR-ART Spectrophotometer. The quantitative
elemental analysis were recorded by the Vario EL analyser.
nylthiosemicarbazide with a series of a-halogenocarbonyls and acetic
anhydride, respectively. Prostate carcinoma appears as the most
common tumor in men [21]. Human liver tumors, particularly hepa-
tocellular carcinoma, are among the most common malignancies
worldwide [22], with an estimated annual incidence of 1 million cases,
mainly in Africa andAsia [23]. Thisexplains the used of both cell lines in
the present work. The cut-off point for good cytotoxic compound has
3.1.1. p-Toluenesulfonylthiosemicarbazide (1)
also been defined as 10
2c, 2d, 2e and 3a showed significant anticancer activities on both
prostate DU-145 and hepatocarcinoma Hep-G2 cancer cell lines with
IC50 values below 10 mM. However, the assays on normal cell lines are
still to be done to confirm the significance of such compounds as
potential anticancer drugs. The studies on the p-toluenesulfonyl-
m
M [24]. Therefore, the target compounds 2a,
Ten millimoles of thiosemicarbazide was dissolved in 5 ml of
pyridine. In the solution obtained, 10 mmol of p-toluenesulfonyl-
chloride (with cooling and shaking), and continuous shaking at
room temperature for 24 h. The same volume of water was added
and then acidified with conc. CH3COOH. The precipitate obtained,
was filtered, purified by re-crystallization in ethanol. Yield 75%;

V. Zaharia et al. / European Journal of Medicinal Chemistry 45 (2010) 5080e5085
5083
Table 1
CH3), 2.40 (s, 3H, CH3), 6.71 (s, 1H, Th-CH), 7.38 (d, 2H, J ¼ 8.4 Hz,
C6H4-meta), 7.86 (d, 2H, J ¼ 8.4 Hz, C6H4-ortho), 9.27 (s, 1H, NH) 9.31
(s, 1H, NH).
Anal. Calcd. for C11H13N3O2S2: C, 46.63%; H, 4.62%; N, 14.83%; S,
22.63%. Found: C, 46.55%; H, 4.56%; N, 14.73%; S, 22.95%.
Activity of the some of the compounds and doxorubicin on DU-145 and Hep-G2
cancer cell lines.
Samples
DU-145
IC50 M)
Hep-G2
IC50 M)
(
m
(m
1
2a
2c
8.98
6.96
9.07
8.98
<5.65
9.16
3.1.2.2. 4-Chloromethyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole
(2b). Yield 66.23%; white-yellow crystals; mp ¼ 188e189 ꢀC; IR
(KBr cmꢁ1): 3269, 3217 (
nNH), 3086, 2955, 2858 (nCH), 1343, 1165
2d
2e
3a
3b
3c
3d
3e
3f
9.84
4.68
7.63
8.77
<4.51
7.36
(n
SO2); EIMS: m/z: 317 (Mþ), 319 (M þ 2), 253, 162, 156, 143, 139,
132, 107, 98, 91 (100%), 77, 71, 65, 59, 45, 39; 1H-NMR (CDCl3)
d
>62.34
>58.27
53.49
>56.95
46.36
49.23
>58.14
<2.94
>62.34
>58.27
57.40
>56.95
10.96
11.23
>58.14
<2.94
(ppm): 2.35 (s, 3H, CH3), 4.39 (s, 2H, CH2Cl), 6.82 (s, 1H, Th-CH), 7.33
(d, 2H, J ¼ 8.42 Hz, C6H4-meta), 7.75 (d, 2H, J ¼ 8.42 Hz, C6H4-ortho),
9.05 (s,1H, NH), 9.37 (s, 1H, NH); Anal. Calcd. for C11H12ClN3O2S2: C,
34.21%; H, 2.87%; N, 9.97%; S, 15.22%. Found: C, 34.20%; H, 2.78%; N,
9.92%; S, 15.18%.
5
8
Doxorubicin
3.1.2.3. 4-Phenyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole
(2c). Yield 66.23%; white-yellow crystal; mp ¼ 179e180 ꢀC; IR (KBr
cmꢁ1): 3261, 3177 (
nNH), 3117, 3078, 2950, 2867 (nCH), 1331, 1164
white crystals; mp ¼ 198e199 ꢀC (mp ¼ 192 ꢀC lit. [25],
mp ¼ 214e215 ꢀC lit. [26]); IR (KBr cmꢁ1): 3453, 3338 (
nNH), 3188,
(n
SO2); EIMS: m/z: 345 (Mþ), 281, 252, 189, 161, 134, 102 (100%), 91,
2993 (nCH), 1339, 1158 (nSO2), 1280 (n
C]S); EIMS: m/z: 245 (Mþ),
77, 65, 63, 45, 39; 1H-NMR (CDCl3)
d(ppm): 2.41 (s, 3H, CH3),
156, 139, 107, 91 (100%), 77, 61, 45, 39; 1H-NMR (CDCl3)
d(ppm):
7.26e7.34 (m, 5H, ArH), 7.72e7.81 (m, 4H, ArH), 7.17 (s, 1H, Th-CH),
9.16 (l.b.,1H, NH), 9.34 (s, 1H, NH); Anal. Calcd. for C16H15N3O2S2: C,
55.63%; H, 4.38%; N,12.16%; S, 18.56%. Found: C, 55.60%; H, 4.33%; N,
12.12%; S, 18.45%.
2.43 (s, 3H, CH3), 7.31 (d, 2H, J ¼ 8.3 Hz, C6H4-meta), 7.82 (d, 2H,
J ¼ 8.3 Hz, C6H4-ortho), 8.48 (l.b., 2H, eNHeNHe), 8.57 (s, 2H, eNH2)
(lit. [27]); Anal. Calcd. for C8H11N3O2S2: C, 41.52%; H, 5.42%; N,
16.14%; S, 24.63%. Found: C, 40.39%; H, 5.03%; N, 15.99%; S, 23.87%.
3.1.2.4. 5-Acetyl-4-methyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thia-
zole (2d). Yield 58.62%; white-yellow crystal; mp ¼ 210e211 ꢀC; IR
3.1.2. General procedure for the synthesis of arylsulfonyl-
hydrazinothiazole (2aef)
Two millimoles of arylsulfonyl-thiosemicarbazide were dissolved
in a mixture of DMF anh. (5 ml) and acetone anh.(5 ml) and then
(KBr cmꢁ1): 3056, 2857 (
nCH), 1598 (nCO), 1325, 1164 (nSO2); EIMS:
m/z: 325 (Mþ), 262, 232, 227, 169, 156, 140, 126, 106, 91, 83, 65, 43
(100%), 39; 1H-NMR (acetone)
d
(ppm): 2.38 (s, 3H, CH3), 2.44 (s, 3H,
2.2 mmol a-halogenocarbonyl-derivative were added. The reaction
CH3), 2.46 (s, 3H, CH3), 7.31 (d, 2H, J ¼ 8.4 Hz, C6H4-meta), 7.76 (d,
2H, J ¼ 8.4 Hz, C6H4-ortho), 9.07 (s, 1H, NH), 9.12 (s, 1H, NH);Anal.
Calcd. for C13H15N3O3S2: C, 47.98%; H, 4,65%; N, 12.91%; S, 19.70%.
Found: C, 47.90%; H, 4.33%; N, 12.62%; S, 19.48%.
mixture was stirred at room temperature for 24 h. The reaction
progress was monitored by TLC. The reaction mixture was cooled
with ice and neutralized with NaHCO3 solution. The precipitate
obtained was filtered and purified by re-crystallization in ethanol.
3.1.2.5. 5-Ethoxycarbonyl-4-methyl-2-[2-(p-toluenesulfonyl)-hydra-
3.1.2.1. 4-Methyl-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole
zino]-thiazole
(2e). Yield
66.43%;
white-yellow
NH), 2937, 2864 (
crystal;
CH),1681
mp ¼ 180e81 ꢀC; IR (KBr cmꢁ1): 3170 (
n
n
(2a). Yield 60.23%; white-yellow crystals; mp ¼ 162e164 ꢀC; IR
(KBr cmꢁ1): 3248, 3206 (
nNH), 3078, 2921, 2857 (nCH), 1314, 1169
(nCO ester), 1333, 1168 (n
SO2); EIMS: m/z: 355 (Mþ), 309, 292, 261,
(n
SO2); EIMS: m/z: 283 (Mþ), 278, 252, 246, 232, 220, 156, 139, 128
200, 171, 155, 138, 126, 107, 91 (100%), 85, 67, 45; 1H-NMR (CDCl3)
d
(100%), 99, 91, 71, 65, 45, 39; 1H-NMR (CDCl3)
d(ppm): 2.07 (s, 3H,
(ppm): 1.35 (t, 3H, J ¼ 6.9 Hz, CH3), 2.34 (s, 3H, CH3), 2.37 (s, 3H,
R
1
R
1
O
O
S
N
S
O
S
NH
S
2
NH
NH
2a-f
+
NH
NH
O
X
R
R
2
2
O
1
Ac O
2
O
R
1
O
N
S
S
N
N
O
R
2
O
3a-f
2a, 3a 2b, 3b 2c, 3c
2d, 3d 2e, 3e
CH3 CH3
COCH3 COOC2H5
2f, 3f
CH2COOC2H5
H
R1
R2
CH3
H
CH2Cl C6H5
H
H
Scheme 1. Synthesis of compounds 3aef.

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V. Zaharia et al. / European Journal of Medicinal Chemistry 45 (2010) 5080e5085
R1
R2
O
N
S
R1
O
O
N
NH
S
NH
NH
2
+
NH
N
NH
H5C6
S
5-6
R2
X
H5C6
S
4
Ac2O
R1
R2
O
N
S
NH
N
N
H5C6
O
S
7-8
5, 7
6, 8
R1
R2
CH2Cl
H
CH2COOC2H5
H
Scheme 2. Synthesis of compound 5e8 deriviatives.
CH3), 4.28 (q, 2H, J ¼ 6.9 Hz, CH2), 7.33 (d, 2H, J ¼ 8.4 Hz, C6H4-meta),
7.75 (d, 2H, J ¼ 8.4 Hz, C6H4-ortho), 9.12 (s, 1H, NH), 9.16 (s, 1H, NH);
Anal. Calcd. for C14H17N3O4S2: C, 47.31%; H, 4.82%; N, 11.82%; S,
18.04%. Found: C, 47.30%; H, 4.80%; N, 11.82%; S, 18.04%.
CH2), 3.99 (q, 2H, J ¼ 7.2 Hz, CH2), 6.82 (s, 1H, Th-CH), 7.43e7.45 (m,
3H, ArH), 7.93e7.96 (m, 2H, ArH), 8.29 (s,1H, Th-CH), 9.24 (s,1H, NH),
10.32(s,1H, NH);Anal. Calcd. forC17H16N4O3S2:C, 52.56%;H, 4.15%;N,
14.42%; S, 16.51%. Found: C, 52.96%; H, 4.12%; N, 14.03%; S, 17.19%.
3.1.2.6. 4-Ethyl acetate-2-[2-(p-toluenesulfonyl)-hydrazino]-thiazole
3.1.4. General procedure of acetylation
(2f). Yield 60.64%; white cyrstals; mp ¼ 192e193 ꢀC; IR (KBr cmꢁ1):
Two millimoles of hydrazinothiazole were treated with 2 ml of
acetic anhydride and two drops (catalytic amounts) of pyridine. The
resulting mixture was heated for 5 min at 139 ꢀC and then stirred at
room temperature for 24 h. Before adding the ethanol solution on
the reaction mixture, acetic anhydride was evaporated under
reduced pressure. The obtained solid was re-crystallized from
ethanol to afford compounds 3aef and 7e8, respectively.
3113, 2982, 2936, 2903 (nCH), 1732, 1707 (nCO ester), 1364, 1168
(
n
SO2); EIMS: m/z: 355 (Mþ), 309, 284, 200, 171, 107, 91 (100%), 65,
43; 1H-NMR (CDCl3)
d
(ppm): 1.07 (t, 3H, J ¼ 7.2 Hz, CH3), 2.35 (s, 3H,
CH3), 3.55 (s, 2H, CH2), 4.01 (q, 2H, J ¼ 7.2 Hz, CH2), 6.83 (s, 1H, Th-
CH), 7.49 (d, 2H, J ¼ 8.4 Hz, C6H4-meta), 7.72 (d, 2H, J ¼ 8.4 Hz, C6H4-
ortho), 8.97 (s, 1H, NH), 9.14 (s, 1H, NH); Anal. Calcd. for
C14H17N3O4S2: C, 47.31%; H, 4.82%; N, 11.82%; O, 18.01%; S, 18.04%.
Found: C, 48.10%; H, 4.48%; N, 11.31%, S, 17.40%.
3.1.4.1. 4-Methyl-2-[2-(p-toluenesulfonyl)-N,N-diacetyl-hydrazino]-
thiazole (3a). Yield 61.64%; white-yellow crystal; mp ¼ 128e130 ꢀC;
3.1.3. General procedure for the synthesis of [2-(2-phenyl-1,3-thiaz
olo-4-carbonyl)-hydrazino]-thiazoles (5,6)
Two millimoles of 4-(2-phenyl-1,3-thiazolo-4-carbonyl)-thio-
semicarbazide [20], were dissolved in a mixture of DMF anh. (3 ml)
IR (KBr cmꢁ1): 3110, 2922 (
nCH), 1695, 1698 (nCO amide), 1367, 1162
(n
SO2); EIMS: m/z: 367 (Mþ), 325, 212,170,128,100, 91, 65, 43 (100%),
39; 1H-NMR (CDCl3)
(ppm): 2.23 (s, 3H, CH3), 2.42 (s, 3H, CH3), 2.46
d
(s, 3H, CH3), 2.48 (s, 3H, CH3), 6.91 (s, 1H, Th-CH), 7.39 (d, 2H,
J ¼ 8.2 Hz, C6H4-meta), 8.04 (d, 2H, J ¼ 8.2 Hz, C6H4-ortho); Anal.
Calcd. for C15H17N3O4S2: C, 49.03%; H, 4.66%; N, 11.44%; S, 17.45%.
Found: C, 49.10%; H, 4.48%; N, 11.31%; S, 17.40%.
and acetone anh.(5 ml) and then 2.2 mmol
a-halogenocarbonyl-
derivative were added. The reaction mixture was stirred at room
temperature for 48 h. The reaction progress was monitored by TLC.
The reaction mixture was cooled with ice and neutralized with
NaHCO3 solution. The precipitate obtained was filtered and purified
by re-crystallization in ethanol.
3.1.4.2. 4-Chloromethyl-2-[2-(p-toluenesulfonyl)-N,N-diacetyl-hydra-
zino]-thiazole
(3b). Yield
65.43%;
white-yellow
crystal;
mp ¼ 152e153 ꢀC; IR (KBr cmꢁ1): 3108, 3074, 3058 (
nCH), 1702 (nCO
3.1.3.1. 4-Chloromethyl-2-[2-(2-phenyl-1,3-thiazolo-4-carbonyl)-hyd
amide), 1358, 1166 (
n
SO2); EIMS: m/z: 401 (Mþ), 403 (M þ 2), 359,
razino]-thiazole (5). Yield 58.63%; white-yellow crystals;
342, 316, 246, 203, 161, 154, 139, 133, 119, 107, 91, 65, 43 (100%), 39;
mp ¼ 220e221 ꢀC; IR (KBr cmꢁ1): 3272, 3170 (
n
NH), 3113, 3091,
1H-NMR (CDCl3)
d(ppm): 2.23 (s, 3H, CH3), 2.41 (s, 3H, CH3), 2.53 (s,
3062, 2950, 2857 (
nCH),1673 (
n
CO amide); EIMS: m/z: 350 (Mþ) 352
3H, CH3), 4.51 (s, 2H, CH2Cl), 6.93 (s, 1H, Th-CH), 7.34 (d, 2H,
J ¼ 8.2 Hz, C6H4-meta), 8.04 (d, 2H, J ¼8.2 Hz, C6H4-ortho); Anal.
Calcd. for C15H16ClN3O4S2: C, 44.83%; H, 4.01%; N, 10.46%; S, 15.95%.
Found: C, 44.83%; H, 4.01%; N, 10.46%; S, 15.95%.
(M þ 2), 204, 187, 160, 121, 101, 94, 77, 71, 57, 36 (100%); 1H-NMR
(CDCl3) d(ppm): 4.5 (s, 2H, CH2Cl), 7.21 (s, 1H, Th-CH), 7.46e7.48 (m,
3H, ArH), 8.02e8.06 (m, 2H, ArH), 8.31 (s,1H, Th-CH), 9.29 (s,1H, NH),
10.42 (s,1H, NH); Anal. Calcd. for C14H11ClN4OS2: C, 47.93%; H, 3.16%;
N, 15.97%; S, 18.28%. Found: C, 47.90%; H, 3.10%; N, 15.65%; S, 18.29%.
3.1.4.3. 4-Phenyl-2-[2-(p-toluenesulfonyl)-N,N-diacetyl-hydrazino]-
thiazole (3c). Yield 56.39%; white crystal; mp ¼ 142e144 ꢀC; IR (KBr
3.1.3.2. 4-Ethyl
acetate-2-[2-(2-phenyl-1,3-thiazolo-4-carbonyl)-
cmꢁ1): 3117, 3048, 2925 (
nCH), 1724, 1704 (nCO amide), 1370, 1169
hydrazino]-thiazole (6). Yield 53.41%; white-yellow crystal;
(n
SO2);EIMS:m/z:429(Mþ), 387, 274, 232 (100%),190,162,134,102, 91,
77, 65, 43; 1H-NMR (CDCl3)
(ppm): 2.23 (s, 3H, CH3), 2.32 (s, 3H, CH3),
mp ¼ 204e205 ꢀC; IR (KBr cmꢁ1): 3270, 3168 (
n
NH), 3113, 3091,
d
3062, 2950, 2857 (
n
CH),1707 (
n
CO ester),1673 (
n
CO amide); EIMS: m/
2.43 (s, 3H, CH3), 7.05 (s,1H, Th-CH), 7.32e7.41 (m, 4H, ArH), 7.81e7.86
(m, 5H, ArH); Anal. Calcd. for C20H19N3O4S2: C, 55.93%; H, 4.46%; N,
9.78%; S, 14.93%. Found: C, 55.93%; H, 4.46%; N, 9.85%; S, 14.90%.
z: 388 (Mþ), 301, 218, 203 (100%), 200,187,160,121,101, 94, 87, 77, 71,
57; 1H-NMR (CDCl3)
(ppm): 1.07 (t, 3H, J ¼ 7.2 Hz, CH3), 3.56 (s, 2H,
d

V. Zaharia et al. / European Journal of Medicinal Chemistry 45 (2010) 5080e5085
5085
3.1.4.4. 5-Acetyl-4-methyl-2-[2-(p-toluenesulfonyl)-N,N-diacetyl-
Briefly, cells were cultured in Dulbecco’s Minimum Essential
Medium (DMEM) supplemented with 5% Foetal Calf Serum (FCS),
gentamicin sulphate (0.004%), glucose (0.57%) and NaHCO3 (0.12%).
Cells were seeded into 96-well flat-bottomed plates at a concen-
tration of 3.0 ꢂ 105 cells per ml. After 24 h, cells were treated with
samples, which were diluted with culture medium to a final
hydrazino]-thiazole
(3d). Yield
65.64%;
white
crystal;
CH), 1731,
mp ¼ 150e151 ꢀC; IR (KBr cmꢁ1): 3078, 3025, 2922 (
n
1719, 1649 (nCO cetone, amide), 1373, 1165 (nSO2); EIMS: m/z: 409
(Mþ), 367, 253, 212, 170, 142, 91, 83, 65, 43 (100%); 1H-NMR (CDCl3)
(ppm): 2.36 (s, 3H, CH3), 2.38 (s, 3H, CH3), 2.41 (s, 3H, CH3), 2.45 (s,
d
3H, CH3), 2.46 (s, 3H, CH3), 7.45 (d, 2H, J ¼ 8.2 Hz, C6H4-meta), 7.79
(d, 2H, J ¼ 8.2 Hz, C6H4-ortho); Anal. Calcd. for C17H19N3O5S2: C,
49.87%; H, 4.68%; N, 10.26%; S, 15.66%. Found: C, 49.70%; H, 4.48%;
N, 10.31%; S, 15.40%.
concentration of 25 mg/ml [29, 30]. XTT labelling reagent (50:1) was
added and the absorbance (560 nm) read after 72 h [29]. Experi-
ments were carried out three times in triplicate. Active samples
(with less than 50% survival) were after an exposure time of 72 h
were serially diluted in a concentration range of 3.12e50
tested. The concentration of the sample that inhibited 50% cell
proliferation (IC50 was determined graphically. Doxorubicin,
a known antitumor agent, was used as positive control. The cells
survival percentage was determined using the formula: % Survival
Cell ¼ (ODT/ODC) ꢂ 100; ODT and ODC being the absorbance of the
test sample-treated group and the control group (0.1% DMSO)
respectively.
mg/ml and
3.1.4.5. 5-Ethoxycarbonyl-4-methyl-2-[2-(p-toluensulfonyl)-N,N-
diacetyl-hydrazino]-thiazole (3e). Yield 61.43%; white-yellow
crystals; mp ¼ 162e163 ꢀC; IR (KBr cmꢁ1): 3106, 3072, 3025, 2981,
)
2905 (nCH), 1730, 1683 (nCO ester, amide), 1370, 1169 (nSO2); EIMS:
m/z: 439 (Mþ), 396, 380, 284, 242, 200, 172, 154, 143, 138, 91, 65, 43
(100%); 1H-NMR (CDCl3)
d
(ppm): 1.38 (t, 3H, J ¼ 7.0 Hz, CH3), 2.24 (s,
3H, CH3), 2.41 (s, 3H, CH3), 2.44 (s, 3H, CH3), 2.46 (s, 3H, CH3), 4.31
(q, 2H, J ¼ 7.0 Hz, CH2), 7.38 (d, 2H, J ¼ 8.2 Hz, C6H4-meta), 7.81 (d,
2H, J ¼ 8.2 Hz, C6H4-ortho); Anal. Calcd. for C18H21N3O6S2: C, 47.31%;
H, 4.82%; N, 11.82%; S, 18.04%. Found: C, 47.30%; H, 4.80%; N, 11.82%;
S, 18.04%.
Acknowledgement
Financial support of this work by the grant CEEX nr.2 CEx-06-11-
55/26.07.2006 of University of Medicine and Pharmacy, Cluj-
Napoca, Romania is gratefully acknowledged.
3.1.4.6. 4-Ethyl acetate-2-[2-(p-toluenesulfonyl)-N,N-diacetyl-hydra-
zino]-thiazole (3f). Yield 60.64%; white crystal; mp ¼ 128e30 ꢀC;
IR (KBr cmꢁ1): 3113, 2982, 2936, 2903 (
nCH), 1732, 1683 (nCO
ester, amide), 1364, 1168 (
366, 324, 284, 242 (100%), 200, 171, 139, 91, 65, 43; 1H-NMR
(CDCl3)
n
SO2); EIMS: m/z: 439 (Mþ), 396, 380,
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3.1.4.7. 4-Chloromethyl-2-[2-(2-phenyl-1,3-thiazolo-4-carbonyl)-N-
acetyl-hydrazino]-thiazole (7). Yield 54.33%; white-yellow crystals;
mp ¼ 181e182 ꢀC; IR (KBr cmꢁ1): 3108, 3082, 3050, 2984, 2961,
ꢀ
2905 (
n
CH), 1700 (
n
CO amide); EIMS: m/z: 392 (Mþ), 341 (M þ 2),
260, 232, 203, 188, 160, 132, 116, 57, 43 (100%); 1H-NMR (CDCl3)
d
(ppm): 2.38 (s, 3H, CH3), 4.52 (s, 2H, CH2Cl), 7.32 (s, 1H, Th-CH),
7.47e7.49 (m, 3H, ArH), 8.04e8.09 (m, 2H, ArH), 8.35 (s, 1H, Th-CH)
9.67 (s, 1H, NH); Anal. Calcd. for C16H13ClN4O2S2: C, 48.91%; H,
3.34%; Cl, 9.02%; N, 14.26%; O, 8.14%; S, 16.32%. Found: C, 48.9%; H,
3.15%; N, 14.34%; S, 16.41%.
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3.1.4.8. 4-Ethylacetate-2-[2-(2-phenyl-1,3-thiazolo-4-carbonyl)-N-
acetyl-hydrazino]-thiazole (8). Yield 58.63%; white-yellow crys-
tals; mp ¼ 160e162 ꢀC; IR (KBr cmꢁ1): 3108, 3082, 3050, 2984,
2961, 2905 (nCH), 1724, 1700 (nCO ester, amide); EIMS: m/z: 430
(Mþ), 387, 356, 342, 314, 200, 188, 160, 116, 57, 43 (100%); 1H-NMR
(CDCl3)
d
(ppm): 1.06 (t, 3H, J ¼ 7.2 Hz, CH3), 2.38 (s, 3H, CH3), 3.58 (s,
2H, CH2), 4.12 (q, 2H, J ¼ 7.2 Hz, CH2), 6.98 (s, 1H, Th-CH), 7.43e7.46
(m, 3H, ArH), 7.93e7.99 (m, 2H, ArH), 8.31 (s, 1H, Th-CH), 9.67 (s, 1H,
eNH); Anal. Calcd. for C19H18N4O4S2: C, 47.93%; H, 3.16%; N, 15.97%;
S, 18.28%. Found: C, 47.90%; H, 3.10%; N, 15.65%; S, 18.29%.
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3.2. Cytotoxicity assay
3.2.1. Antitumor assay on Human DU-145 prostate carcinoma and
Hepatoma Hep-G2 cells
The cytotoxic activity of samples were tested on Human DU-145
(androgen-insensitive prostate cancer cells) and Hepatocarcinoma
Hep-G2, following the XTT (2,3-bis[2-methoxy-4-nitro-5-sulfo-
phenyl]-2H-tetrazolium-5-carboxyanilide inner salt) assay [28e30].