Synthesis and characterization of new phthalhydrazothiazole derivatives: A preliminary investigation on their activity against hepatocellular carcinoma

Article abstract of DOI:10.1002/jhet.138

(Chemical Equation Presented) The synthesis of new 2-(4-substituted thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, 2-(4-oxo-4,5-dihydro-thiazol-2- yl)-2,3-dihydrophthalazine-1,4-diones, and 2-(5-arylidene-4-oxo-4,5- dihydrothiazol-2-yl)-2,3-dihy-drophth

Full text of DOI:10.1002/jhet.138

674
Synthesis and Characterization of New Phthalhydrazothiazole
Derivatives: A Preliminary Investigation on Their Activity
against Hepatocellular Carcinoma
Vol 46
Maria Cristina Cardia,a* Simona Distinto,a Elias Maccioni,a
Antonio Plumitallo,a Laura Sanna,b Maria Luisa Sanna,a and Sara Vigoa
aDipartimento Farmaco Chimico Tecnologico, Via Ospedale, Cagliari 09124, Italy
bDipartimento di Tossicologia, Sezione di Oncologia e Patologia Molecolare,
Via Porcell, Cagliari 09124, Italy
*E-mail: cardiamr@unica.it
Received December 1, 2008
DOI 10.1002/jhet.138
Published online 9 July 2009 in Wiley InterScience (www.interscience.wiley.com).
The synthesis of new 2-(4-substituted thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, 2-(4-oxo-4,5-
dihydro-thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, and 2-(5-arylidene-4-oxo-4,5-dihydrothiazol-2-
yl)-2,3-dihy-drophthalazine-1,4-diones is reported. The introduction of different substituents on the phtha-
lazine, the thiazole and the thiazolinone has been studied. The new compounds have been characterized
and evaluated for their antiproliferative activity against hepatocellular carcinoma, one of the most lethal
tumors. The activity shown by some of these compounds towards liver tumor cells is encouraging.
J. Heterocyclic Chem., 46, 674 (2009).
INTRODUCTION
Hepatocellular carcinoma is one of the five most com-
mon cancers worldwide; it is one of the most common
cancers in males in the world; it has an annual incidence
worldwide of more than 500,000 cases and is very
poorly treated with survival rates of only 23% at one
year and less than 5% at five years [40].
Thiazoles play a prominent role in nature and have
broad applications in agricultural and medicinal chemistry.
As a matter of fact, thiazole derivatives show antitumor
[1,2], anti-hypertensive [3], anti-inflammatory [4,5], anti-
hyperlipidemic [6] and other biological properties [7,8].
Phthalazine derivatives, similarly to other members of
the isomeric diazine series, have found wide application
as therapeutic agents [9–21]. The synthesis and biologi-
cal evaluation of semicarbazide derivatives that exhibit
anticancer activity has been reported [22–24].
According to literature data, the highest diffusion is
observed in China and eastern Asia, middle Africa and
some countries of western Africa, while lower incidence
is evident in Japan, Europe, and America. Nevertheless
a rising trend is observed, due to the increasing of HCV
infection [41]. The prognosis is generally poor, espe-
cially in the African and Chinese population, where sur-
vival time may be as short as eleven weeks from the
onset of symptoms.
Moreover, a number of molecules bearing a tridentate
ligand system, structurally related to thiazolophthala-
zines and thiazolinonephthalazines [25–32], show an in-
hibitory activity towards tumors; it has been shown that
complexation of these compounds results in derivatives
that are potent cytotoxic agents [33].
In this article, we investigate a straightforward and
efficient synthesis of compounds containing thiazole and
phthalazine moieties in the same molecule, as we con-
sider this aspect particularly interesting.
Following our interest in the synthesis and biological
activity of heterocyclic compounds [34–39], we have
synthesized several new 2-(4-substituted thiazol-2-yl)-
2,3-dihydrophthalazine-1,4-diones, 2-(4-oxo-4,5-dihydro-
thiazol-2-yl)-2,3-dihydrophthalazine-1,4-diones, and 2-
(5-arylidene-4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydro-
phthalazine-1,4-diones, in order to assess their capability
of inhibiting tumor cell growth, and in particular their ac-
tivity against hepatocellular carcinoma.
RESULTS AND DISCUSSION
Several 2-(4-substituted thiazol-2-yl)-2,3-dihydroph-
tha-lazine-1,4-diones, 2-(4-oxo-4,5-dihydrothiazol-2-yl)-
2,3-dihydrophthalazine-1,4-diones, and 2-(5-arylidene-4-
oxo - 4 , 5 - dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-
C
V
2009 HeteroCorporation

July 2009
Synthesis and Characterization of New Phthalhydrazothiazole Derivatives:
A Preliminary Investigation on Their Activity against Hepatocellular Carcinoma
675
Scheme 1
diones have been synthesized and their capability of in-
hibiting tumor cell growth investigated.
possible isomers is formed, but the exact structure has not
been investigated at this stage of the study.
The first step in the synthetic pathway (Scheme 1)
consists of the reaction of equimolecular amounts of
phthalic anhydrides 1a–c with thiosemicarbazide 2 in
isopropanol, in the presence of catalytic amounts of ace-
tic acid. By this method [39], 2-carbothioamidophthala-
zines 3a–c can be prepared easily.
Compounds 3a–c are then reacted either with a-halo-
gen ketones or with a-halogen esters to form the substi-
tuted thiazole ring derivatives (5a–c, 7a–c), and the thia-
zolinonic derivatives 9a–c, respectively.
Compounds 11–24 were obtained by reacting com-
pound 9a with different aryl aldehydes in acetic acid and
acetic anhydride. All the obtained compounds were puri-
fied by crystallization from an appropriate solvent, and
1
were fully characterized with the aid of H NMR spec-
troscopy, mass spectrometry, and elemental analysis.
Compounds 5b–c, 7a–c, 9a–c, 11–18 have been eval-
uated for their anti-proliferative activity towards the
FaO Reuber hepatoma cell line, which maintains the
characteristics of hepatocytes, both in vivo and in vitro,
and retains the ability to undergo apoptosis [42].
In the case of compound 3b and its derivatives two pos-
sible regioisomers can be obtained. Apparently, according
to chromatographic and spectral data, only one of the two
To demonstrate the most promising structures and
substitutions for biological activity, we investigated
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

676
M. C. Cardia, S. Distinto, E. Maccioni, A. Plumitallo, L. Sanna, M. L. Sanna, and S. Vigo
Vol 46
Table 1
Percentage of cell vitality with respect to the control (NRU assay).
Concentration (mM)
Cp
1 (mM)
0.5 (mM)
0.25 (mM)
0.1 (mM)
0.05 (mM)
0.01 (mM)
7a
9a
5b
7b
9b
5c
7c
9c
11
12
13
14
15
16
17
18
94.83
60.02
42.68
98.71
100
100
65.20
100
62.17a
40.68
100b
94.83
64.86
94.73
100
96.43
91.35
100
100
100
100
82.03
100
36.53
41.68
90.29
100
98.57
93.87
100
100
100
100
91.70
100
44.41
58.08
94.33
100
100
100
98.01
97.69
100
100
100
100
100
100
100
100
100
100
100
100
69.12
100
42.83a
38.20
88.83
100
99.77
100
94.84
74.94
100
95.12
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
100
67.18
45.86
95.36
100
71.93
94.12
84.30
89.87
98.76
91.11
95.98
89.95
88.55
a Poorly soluble at 1 mM e 0.5 mM.
b Precipitates at 1 mM.
three different scaffolds, namely 2-(4-phenylthiazol-2-
yl)-2,3-dihydrophthalazine-1,4-diones, 2-(4-thiazolinone-
2-yl)-2,3-dihydrophthalazine-1,4-diones, and 2-(4-aryli-
denthiazolinone-2-yl)-2,3-dihydrophthalazine-1,4-diones.
Some of the tested compounds (Table 1) show a fairly
good cytotoxic activity against FaO cells in the NRU
assay.
rine atoms are introduced in the phthalazine moiety,
respectively.
Moreover, the presence of an opportunely functional-
ized aromatic moiety both in the thiazole and in the
thiazolinone rings seems important for biological
properties.
Thus activity is strongly influenced by the nature of
the substituents, and the presence of halogen atoms both
on the phthalazine and on 5-arylidenthiazolinone leads
to an increase in activity.
Compound 11, 2-[5-(4-chlorobenzylidene)-4-oxo-4,5-
dihydrothiazol-2-yl]-2,3-dihydrophthalazine-1,4-dione shows
the best activity with a 44% cell survival at a concentra-
tion of 0.1mM.
These results give an indication towards the design of
new and potentially more active compounds, and sug-
gest that 2-(4-phenylthiazol-2-yl)-2,3-dihydrophthala-
zine-1,4-diones, 2-(4-thiazolinone-2-yl)-2,3-dihydroph-
thalazine-1,4-diones, and 2-(5-arylidenthiazolinone-2-
yl)-2,3-dihydrophthalazine-1,4-diones could be consid-
ered promising scaffolds for cytotoxic compounds.
A similar behavior was observed in compound 12,
2- [5-(2,4-dichlorobenzylidene)-4-oxo-4,5-dihydrothiazol-
2-yl]-2,3-dihydrophthalazine-1,4-dione, indicating that
the presence of one or two chlorine atoms, in the 5-ary-
lidene moiety, increases activity against hepatoma cells.
It should be observed on the other hand that intro-
duction of different substituents in the same scaffold
generally leads to a decrease in biological activity, as
observed for compounds 14, 15, and 18, which have a
3-nitrobenzylidene-, a 3-pyridylene-, and a 4-dimethyl-
aminobenzylidene in position 5 of the thiazolidinone
moiety, respectively.
EXPERIMENTAL
Melting points were uncorrected and were determined on a
Reichert Kofler thermopan apparatus. 1H NMR spectra were
recorded on a Bruker AMX (300 MHz) using tetramethylsilane
(TMS) as internal standard (chemical shifts in d values). Elec-
tron ionization (EI) mass spectra were obtained by a Fisons
QMD 1000 mass spectrometer (70 eV, 200 lA, ion source
temperature 200ꢀC). The samples were introduced directly into
the ion source. Elemental analyses were obtained on a Perkin-
Elmer 240 B microanalyzer.
In the case of compounds 16 and 17, good activity is
observed only at relatively high concentrations.
Compound 9a, which contains an unsubstituted thia-
zolinonic cycle, showed a moderate biological activity.
The substitution of the thiazolinonic ring with a 4-
phenylthiazole or 4-methylthiazole leads to a moderate
increase in biological activity. This is particularly evi-
dent for compounds 5b and 7c, where one or two fluo-
All synthesized compounds were purified by crystallization
from an appropriate solvent (ethanol, ethanol/water or ethanol/
acetic acid).
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2009
Synthesis and Characterization of New Phthalhydrazothiazole Derivatives:
A Preliminary Investigation on Their Activity against Hepatocellular Carcinoma
677
Compounds 3a–c were prepared as reported in the literature
[39].
8.00 (dd, 1H, phth., J ¼ 7.8, 2.7); 10.72 (s, 1H, NH, D2O-
exch.). Anal. Calcd for C12H7F2N3O2S: C, 48.81; H, 2.39; N,
14.23. Found: C, 48.55; H, 2.41; N, 14.17.
General procedure for the synthesis of compounds 5a–c.
The compounds 3a–c (3 mmol) and 2-bromoacetophenone (3.5
mmol) in 100 mL of isopropanol suspension is refluxed, under
vigorous stirring, until complete dissolution of reagents and for
further 4 h. After cooling to room temperature, a solid is
obtained, which is filtered off, washed with isopropyl ether
several times, and dried.
General procedure for the synthesis of compounds 9a-c.
A mixture of compounds 3a–c (0.013 mol) and ethyl bromoa-
cetate (0.016 mol) were suspended in isopropanol (120 mL)
and refluxed for 2 h. A white product was isolated by filtration
and crystallized from acetic acid. The following listed com-
pounds have been synthesized using the same procedure.
2-(4-Oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-
1,4-dione (9a). White solid, m.p. 311–312ꢀC. MS m/z ¼ 261,
yield ¼ 93%. 1H NMR (DMSO-d6): dꢁ 4.10 (s, 2H, CH2-
thiaz.); 7.83–7.91 (m, 4H, phth.); 12.52 (s, 1H, NH, D2O-
exch.). Anal. Calcd for C11H7N3O3S: C, 50.57; H, 2.70; N,
16.08. Found: C, 50.78; H, 2.68; N, 16.11.
5- (or 8-) Fluoro-2-(4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihy-
drophthalazine-1,4-dione (9b). White solid, m.p. 309ꢀC d. MS
m/z ¼ 279, yield ¼ 82%. 1H NMR (DMSO-d6): dꢁ 3.85 (s,
2H, CH2-thiaz.); 7.71–7.76 (m, 2H, phth.); 7.89–7.92 (m, 1H,
phth.); 11.77 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C11H6FN3O3S: C, 47.31; H, 2.17; N, 15.05. Found: C, 47.52;
H, 2.18; N, 14.99.
6,7-Difluoro-2-(4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydroph-
thalazine-1,4-dione (9c). Whitish solid, m.p. 298ꢀC d. MS m/z
¼ 297, yield ¼ 84%. 1H NMR (DMSO-d6): dꢁ 3.97 (s, 2H,
CH2-thiaz.); 8.15–8.26 (m, 2H, phth.); 11.88 (s, 1H, NH, D2O-
exch.). Anal. Calcd for C11H5F2N3O3S: C, 44.45; H, 1.70; N,
14.14. Found: C, 44.21; H, 1.69; N, 14.18.
General procedure for the synthesis of 2-(5-arylidene-4-
oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione
derivatives (11–24). These derivatives were prepared start-
ing from 2-(4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihydrophthala-
zine-1,4-dione (4 mmol) and the appropriate aryl aldehyde
(4 mmol) in acetic acid (20 mL) and acetic anhydride (2 mL).
The reaction mixture was stirred and heated to reflux for
30 min. A colored precipitate was obtained, which was filtered
off and crystallized from the appropriate solvent. Compounds
11–24 were synthesized using this procedure.
2-[5-(4-Chlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-
2,3-dihydrophthalazine-1,4-dione (11). Light yellow solid,
m.p. 346–347ꢀC d. MS m/z ¼ 383–385, yield ¼ 64%. 1H
NMR (DMSO-d6): dꢁ 7.52 (d, 2H, aryl, J ¼ 8.5); 7.58 (d, 2H,
phth., J ¼ 7.3); 7.76 (s, 1H, arylACH¼¼ ); 7.90–7.97 (m, 4H,
aryl þ phth.); 11.80 (1H, s, NH, D2O-exch.). Anal. Calcd for
C18H10ClN3O3S: C, 56.33; H, 2.62; N, 10.95. Found: C,
56.14; H, 2.64; N, 10.90.
2-[5-(2,4-Dichlorobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-
2,3-dihydrophthalazine-1,4-dione (12). Orange solid, m.p. 345–
347ꢀC d. MS m/z ¼ 417–420, yield ¼ 54%. 1H NMR
(DMSO-d6): dꢁ 7.52 (d, 2H, aryl, J ¼ 8.5); 7.59 (d, 2H, phth.,
J ¼ 7.3); 7.76 (s, 1H, arylACH¼¼ ); 7.88–7.97 (m, 3H, aryl þ
phth.); 11.80 (1H, s, NH, D2O-exch.). Anal. Calcd for
C18H9Cl2N3O3S: C, 51.69; H, 2.17; N, 10.05. Found: C,
51.92; H, 2.15; N, 10.10.
2-[5-(3,4-Dimethoxybenzylidene)-4-oxo-4,5-dihydrothiazol-
2-yl]-2,3-dihydrophthalazine-1,4-dione (13). Yellow solid,
m.p. 308–311ꢀC d. MS m/z ¼ 409, yield ¼ 68%. 1H NMR
(DMSO-d6): dꢁ 3.76 (s, 3H, OCH3); 3.78 (s, 3H, OCH3); 6.84
(d, 1H, aryl, J ¼ 8.1); 7.07 (d, 1H, aryl, J ¼ 8.1); 7.24 (s, 1H,
aryl); 7.73 (s, 1H, arylACH¼¼); 7.76–7.94 (m, 4H, phth.);
11.78 (s, 1H, NH, D2O-exch.). Anal. Calcd for C20H15N3O5S:
The following listed compounds were synthesized using the
same procedure.
2-(4-Phenylthiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione
(5a). Whitish solid, m.p. 300ꢀC. MS m/z ¼ 321, yield ¼ 70%.
1H NMR (DMSO-d6): d 7.36 (t, 1H, phth., J ¼ 7.7); 7.47 (t,
2H, phenyl, J ¼ 7.7); 7.90 (s, 1H, C5H-thiaz.); 7.96–8.05 (m,
4H, phth. þ phenyl); 8.10 (d, 1H, phth., J ¼ 7.3); 8.38 (d, 1H,
phth., J ¼ 7.3); 12.42 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C17H11N3O2S: C, 63.54; H, 3.45; N, 13.08. Found: C, 63.71;
H, 3.43; N, 13.13.
5- (or 8-) Fluoro-2-(4-phenylthiazol-2-yl)-2,3-dihydrophtha-
lazine-1,4-dione (5b). Pale yellow solid, m.p. 210–211ꢀC. MS
1
m/z ¼ 339, yield ¼ 79%. H NMR (DMSO-d6): d 7.36 (t, 1H,
phth., J ¼ 7.7); 7.47 (t, 2H, phenyl, J ¼ 7.7); 7.90 (s, 1H,
C5H-thiaz.); 7.96–8.05 (m, 3H, phenyl); 8.10 (d, 1H, phth. J ¼
7.3); 8.38 (d, 1H, phth. J ¼ 7.3); 12.42 (s, 1H, NH, D2O-
exch.). Anal. Calcd for C17H10FN3O2S: C, 60.17; H, 2.97; N,
12.38. Found: C, 59.99; H, 2.96; N, 12.35.
6,7-Difluoro-2-(4-phenylthiazol-2-yl)-2,3-dihydrophthalazine-
1,4-dione (5c). Beige solid, m.p. 229–231ꢀC. MS m/z ¼ 357,
yield ¼ 72%. 1H NMR (DMSO-d6): dꢁ 7.42–7.44 (m, 1H,
phth.); 7.51–7.55 (m, 2H, phenyl); 7.99 (s, 1H, C5H-thiaz.);
8.04–8.07 (m, 2H, phenyl þ phth.); 8.14 (t, 1H, phenyl, J ¼
8.1); 8.42 (t, 1H, phenyl, J ¼ 8.1); 12.82 (s, 1H, NH, D2O-
exch.). Anal. Calcd for C17H9F2N3O2S: C, 57.14; H, 2.54; N,
11.76. Found: C, 56.99; H, 2.55; N, 11.75.
General procedure for the synthesis of compounds 7a–c.
A suspension of compounds 3a–c (2.71 mmol) and chloroace-
tone (3.25 mmol) in 140 mL of isopropanol is refluxed, under
vigorous stirring, until complete dissolution of reagents and then
for further 5 h. The mixture is allowed to cool down to room
temperature thus obtaining a solid, which is filtered off, washed
with isopropyl ether several times, and dried. The following
listed compounds were synthesized using the same procedure.
2-(4-Methylthiazol-2-yl)-2,3-dihydrophthalazine-1,4-dione
(7a). Beige solid, m.p. 249–251ꢀC. MS m/z ¼ 259, yield ¼
1
52%. H NMR (DMSO-d6): dꢁ 2.38 (s, 3H, CH3); 7.08 (s, 1H,
C5H-thiaz.); 7.95–8.08 (m, 3H, phth.); 8.37 (d, 1H, phth. J ¼
7.3); 12.61 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C12H9N3O2S: C, 55.59; H, 3.50; N, 16.21. Found: C, 55.80; H,
3.48; N, 16.15.
5- (or 8-) Fluoro-2-(4-methylthiazol-2-yl)-2,3-dihydrophtha-
lazine-1,4-dione (7b). Beige solid, m.p. 243–245ꢀC. MS m/z ¼
277, yield ¼ 89%. 1H NMR (DMSO-d6): dꢁ 2.13 (s, 3H,
CH3); 6.61 (s, 1H, C5H-thiaz.); 7.76–7.96 (m, 2H, phth.);
7.98–8.03 (m, 1H, phth.); NH not detected. Anal. Calcd for
C12H8FN3O2S: C, 51.98; H, 2.91; N, 15.15. Found: C, 52.19;
H, 2.90; N, 15.11.
6,7-Difluoro-2-(4-methylthiazol-2-yl)-2,3-dihydrophtha-lazine-
1,4-dione (7c). Beige solid, m.p. 254–258ꢀC. MS m/z ¼ 295,
yield ¼ 77%. 1H NMR (DMSO-d6): dꢁ 2.27 (s, 3H, CH3);
6.50 (s, 1H, C5H-thiaz.); 7.65 (dd, 1H, phth., J ¼ 7.8, 2.4);
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M. C. Cardia, S. Distinto, E. Maccioni, A. Plumitallo, L. Sanna, M. L. Sanna, and S. Vigo
Vol 46
C, 58.67; H, 3.69; N, 10.26. Found: C, 58.55; H, 3.70; N,
10.30. Anal. Calcd for C10H10OS: C, 67.38; H, 5.65; S, 17.99.
Found: C, 67.06; H, 5.75; S, 17.69.
d. MS m/z ¼ 394, yield ¼ 37%. 1H NMR (DMSO-d6): dꢁ
7.84–8.09 (m, 9H, phth. þarylACH¼¼ ); 12.27 (s, 1H, NH,
D2O-exch.). Anal. Calcd for C18H10N4O5S: C, 54.82; H, 2.56;
N, 14.21. Found: C, 54.88; H, 2.55; N, 14.18.
2-[5-(3-Nitrobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-2,3-
dihydrophthalazine-1,4-dione (14). Mustard colored solid, m.p.
320–323ꢀC d. MS m/z ¼ 394, yield ¼ 49%. 1H NMR
(DMSO-d6): dꢁ 7.72 (t, 1H, aryl, J ¼ 8.1); 7.88–7.96 (m, 6H,
phth. þarylACH¼¼ ); 8.24 (d, 1H, phth., J ¼ 7.3); 8.45 (s, 1H,
aryl); 11.78 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C18H10N4O5S: C, 54.82; H, 2.56; N, 14.21. Found: C, 55.01;
H, 2.54; N, 14.26.
2-[5-(3-Pyridinylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-2,3-
dihydrophthalazine-1,4-dione (15). Light yellow solid, m.p.
297–299ꢀC d. MS m/z ¼ 350, yield ¼ 67%. 1H NMR
(DMSO-d6): dꢁ 7.48 (t, 1H, pyr. J ¼ 7.8); 7.80 (s, 1H,
ACH¼¼aryl); 7.83–7.94 (m, 5H, phth. þ pyr.); 8.58 (dd, 1H,
pyr, J ¼ 4.6, 1.9); 8.82 (d, 1H, pyr., J ¼ 1.9); 11.78 (s, 1H,
NH, D2O-exch.). Anal. Calcd for C17H10N4O3S: C, 58.28; H,
2.88; N, 15.99. Found: C, 57.98; H, 2.90; N, 16.04.
2-[5-(3,4-Dioxymethylenbenzylidene)-4-oxo-4,5-dihydrothia-
zol-2-yl]-2,3-dihydrophthalazine-1,4-dione (16). Orange solid,
m.p. 333ꢀC d. MS m/z ¼ 393, yield ¼ 75%. 1H NMR
(DMSO-d6): d 6.09 (s, 2H, CH2); 7.04 (d, 1H, aryl, J ¼ 8.1);
7.13 (d, 1H, aryl, J ¼ 8.1); 7.20 (s, 1H, aryl); 7.68 (s, 1H,
ACH¼¼aryl); 7.85–7.93 (m, 4H, phth.); 12.25 (s, 1H, NH,
D2O-exch.). Anal. Calcd for C19H11N3O5S: C, 58.01; H, 2.82;
N, 10.68. Found: C, 57.62; H, 2.80; N, 10.72.
2-[5-(3,4,5-Trimethoxybenzylidene)-4-oxo-4,5-dihydrothia-
zol-2-yl]-2,3-dihydrophthalazine-1,4-dione (17). Yellow solid,
m.p. 320–322ꢀC d. MS m/z ¼ 439, yield ¼ 56%. 1H NMR
(DMSO-d6): dꢁ 3.70 (s, 3H, OCH3); 3.76 (s, 3H, OCH3); 3.78
(s, 3H, OCH3); 6.85 (s, 1H, aryl); 6.97 (s, 1H, aryl); 7.73 (s,
1H, arylACH¼¼); 7.85–7.92 (m, 4H, phth.); 12.22 (s, 1H, NH,
D2O-exch.). Anal. Calcd for C21H17N3O6S: C, 57.40; H, 3.90;
N, 9.56. Found: C, 57.19; H, 3.88; N, 9.53.
2-[5-(4-methoxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-
2,3-dihydrophthalazine-1,4-dione (22). Beige solid, m.p. 301–
1
304ꢀC d. MS m/z ¼ 379, yield ¼ 50%. H NMR (DMSO-d6):
dꢁ 3.86 (s, 3H, OCH3); 7.02 (d, 2H, aryl, J ¼ 8.4); 7.51 (d,
2H, aryl, J ¼ 8.8); 7.70 (s, 1H, arylACH¼¼); 7.86–7.96 (m,
4H, phth.); 11.78 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C19H13N3O4S: C, 60.15; H, 3.45; N, 11.08. Found: C, 59.88;
H, 3.47; N, 11.06.
2-[5-(4-Methylbenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-
2,3-dihydrophthalazine-1,4-dione (23). Yellow solid, m.p.
331–333ꢀC d. MS m/z ¼ 363, yield ¼ 38%. 1H NMR
(DMSO-d6): dꢁ 2.32 (s, 3H, CH3); 7.28 (d, 2H, aryl, J ¼ 8.1);
7.45 (d, 2H, aryl, J ¼ 8.1); 7.72 (s, 1H, arylACH¼¼); 7.76–
7.97 (m, 4H, phth.); 11.79 (s, 1H, NH, D2O-exch.). Anal.
Calcd for C19H13N3O3S: C, 62.80; H, 3.60; N, 11.56. Found:
C, 63.11; H, 3.59; N, 11.60.
2-[5-(3-Hydroxybenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-
2,3-dihydrophthalazine-1,4-dione. (24). Yellow solid, m.p.
1
354ꢀC d. MS m/z ¼ 365, yield ¼ 42%. H NMR (DMSO-d6):
dꢁ 7.22 (d, 1H, aryl, J ¼ 7.7); 7.35 (s, 1H, aryl); 7.44–7.49 (d,
2H, arylþaryl-OH, D2O-exch.); 7.52 (t, 1H, aryl, J ¼ 7.7);
7.75 (s, 1H, ACH¼¼aryl); 7.88–7.94 (m, 4H, phth.); 11.78 (s,
1H, NH, D2O-exch.). Anal. Calcd for C18H11N3O4S: C, 59.17;
H, 3.03; N, 11.50. Found: C, 58.97; H, 3.05; N, 11.45.
Biological assay. The FaO rat hepatoma cell line H-35
Reuber (ICLC ATL99001) was purchased from Interlab Line
Collection (Servizio Biotecnologie, IST, Genoa, Italy). FaO
cells were cultured in flasks (capacity 75 cm2) containing
15 mL Dulbecco’s modified Eagle’s medium (DMEM plus
Glutamax I, Invitrogen S.r.l., Milan, Italy) and then supple-
mented with 100 UI/mL penicillin, 0.1 mg/mL streptomycin
and 10% foetal bovine serum (Mascia Brunelli, Milan, Italy).
Cells were incubated at 37ꢀC in 5% CO2 and 95% O2 atmos-
phere. All experiments were carried out 24 h after seeding.
Before the assay, the cells were treated with a phosphate
buffer (PBS) and the culture medium was substituted with
DMEM without serum.
Cell vitality. The vitality of adhesive cells was determi-
nated with the Neutral Red Uptake assay (NRU) as reported
by Borenfreund and Puerner [43]. As far as the NRU assay is
concerned, the Neutral Red solution (0.4% NR in H2O) was
diluted 1:80 with phosphate buffer (PBS) and incubated at
37ꢀC overnight. Then the solution was centrifuged (2600 rpm)
to remove micro crystals that are insoluble in coloring. Once
treated, the culture medium was vacuumed and substituted
with a NR solution. The cells were incubated in the NR solu-
tion at 37ꢀC for 30 min to allow coloring incorporation of the
lisosomes in the vital cells of the flask. The RN solution was
removed, the cells were rapidly washed with 1% formaldehyde
and a calcium chloride solution was added. To remove incor-
porated NR from vital cells, a mixture (solution) of 1% acetic
acid in ethanol 50% was added. A sample collected from
every well was read using a Perkin-Elmer, 540 nm spectropho-
tometer. The values obtained for the treated cells were
expressed as percentages of the value obtained for the control
cells. All experiments were repeated at lease three times and
were made in triplicate.
2-[5-(4-Dimethylaminobenzylidene)-4-oxo-4,5-di-hydro-thia-
zol-2-yl]-2,3-dihydrophthalazine-1,4-dione (18). Yellow solid,
m.p. 293–294ꢀC d. MS m/z ¼ 392, yield ¼ 70%. 1H NMR
(DMSO-d6): d 3.37 (s, 6H, NCH3); 6.74 (d, 2H, aryl, J ¼ 8.1);
7.36 (d, 2H, aryl, J ¼ 8.1); 7.62 (s, 1H, arylACH¼¼); 7.89–
7.92 (m, 4H, phth.); 11.78 (s, 1H, NH, D2O-exch.). Anal.
Calcd for C20H16N4O3S: C, 61.21; H, 4.11; N, 14.28. Found:
C, 60.97; H, 4.09; N, 14.34.
2-(5-Benzylidene-4-oxo-4,5-dihydrothiazol-2-yl)-2,3-dihy-
drophthalazine-1,4-dione (19). Orange solid, m.p. 331–333ꢀC
1
d.MS m/z ¼ 349, yield ¼ 50%. H NMR (DMSO-d6): d 7.50
(d, 2H, phenyl, J ¼ 7.7); 7.60 (d, 2H, phth., J ¼ 7.3); 7.80 (s,
1H, phenylACH¼¼ ); 7.93–7.98 (m, 5H, phenyl þ phth.);
11.79 (1H, s, NH, D2O-exch.). Anal. Calcd for C18H11N3O3S:
C, 61.88; H, 3.17; N, 12.03. Found: C, 62.12; H, 3.15; N,
11.98.
2-[5-(4-Nitrobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-2,3-
dihydrophthalazine-1,4-dione (20). Dark yellow solid, m.p.
345–348ꢀC d. MS m/z ¼ 394, yield ¼ 60%. 1H NMR
(DMSO-d6): d 7.82 (d, 2H, aryl, J ¼ 8.8); 7.87 (s, 1H,
arylACH¼¼ ); 7.88–7.98 (m, 4H, phth.); 8.26 (d, 2H, aryl, J ¼
8.8); 11.78 (s, 1H, NH, D2O-exch.). Anal. Calcd for
C18H10N4O5S: C, 54.82; H, 2.56; N, 14.21. Found: C, 54.57;
H, 2.55; N, 14.17.
2-[5-(2-Nitrobenzylidene)-4-oxo-4,5-dihydrothiazol-2-yl]-2,3-
dihydrophthalazine-1,4-dione (21). Orange solid, m.p. 327ꢀC
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

July 2009
Synthesis and Characterization of New Phthalhydrazothiazole Derivatives:
A Preliminary Investigation on Their Activity against Hepatocellular Carcinoma
679
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet