Synthesis of novel thiazolone-based compounds containing pyrazoline moiety and evaluation of their anticancer activity

Article abstract of DOI:10.1016/j.ejmech.2008.09.032

To examine the anticancer activity several novel thiazolone-based compounds containing 5-aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl framework were obtained. Reaction of 5-aryl-3-phenyl-4,5-dihydropyrazole with 4-thioxo-2-thiazolidinone or 2-carbethoxymethy

Full text of DOI:10.1016/j.ejmech.2008.09.032

European Journal of Medicinal Chemistry 44 (2009) 1396–1404
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of novel thiazolone-based compounds containing pyrazoline moiety
and evaluation of their anticancer activity
Dmytro Havrylyuk a, Borys Zimenkovsky a, Olexandr Vasylenko c, Lucjusz Zaprutko b, Andrzej Gzella b,
,
Roman Lesyk a
*
a Department of Pharmaceutical, Organic and Bioorganic Chemistry, Danylo Halytsky Lviv National Medical University, Pekarska 69, Lviv 79010, Ukraine
b Department of Organic Chemistry, Poznan University of Medical Sciences, Grunwaldzka 6, Poznan 60-780, Poland
c Institute of Bioorganic Chemistry and Petrochemistry, National Academy of Science of Ukraine, Murmanska 1, Kyiv 02094, Ukraine
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 3 March 2008
Received in revised form 1 September 2008
Accepted 16 September 2008
Available online 7 October 2008
To examine the anticancer activity several novel thiazolone-based compounds containing 5-aryl-3-
phenyl-4,5-dihydro-1H-pyrazol-1-yl framework were obtained. Reaction of 5-aryl-3-phenyl-4,5-dihy-
dropyrazole with 4-thioxo-2-thiazolidinone or 2-carbethoxymethylthio-2-thiazoline-4-one yielded
starting 4- (1 and 2) or 2-substituted (11 and 12) thiazolones which were utilized in Knoevenagel
condensation for obtaining a series of 5-arylidene derivatives 3–10, 13–18. Alternatively 11, 12 and their
5-arylidene derivatives were synthesized by means of 3-phenyl-5-aryl-1-thiocarbamoyl-2-pyrazoline as
S,N-binucleophile via [2 þ 3]-cyclocondensation approach. The structures of compounds were deter-
mined by 1H, 13C NMR, LC–MS, EI-MS and X-ray analysis. The in vitro anticancer activity of synthesized
compounds were tested by the National Cancer Institute and most of them displayed anticancer activity
on leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and breast cancer cell lines. Relations
between structure and activity are discussed, the most efficient anticancer compound 16 was found to be
active with selective influence on colon cancer cell lines, especially on HT 29 (log GI50 ¼ ꢀ6.37).
Ó 2008 Elsevier Masson SAS. All rights reserved.
Keywords:
Thiazolones
Pyrazolines
Knoevenagel reaction
X-ray study
Anticancer activity
1. Introduction
active anticancer compounds [10–12] in this work we try to study
the influence of pyrazoline moiety and thiazolone scaffold combi-
Thiazolidine template is one of the privileged structure frag-
ments in modern medicinal chemistry considering its broad phar-
macological spectrum and affinity for various biotargets of these
class heterocyclic compounds [1]. Some of thiazolidine derivatives,
especially 4-thiazolidinones, are PPAR-receptors agonists showing
hypoglycemic, antineoplastic and anti-inflammatory activities [2],
complex COX-2/5-LOX inhibitors [3] or phospholipase A2 (PLA2) [4]
nation on the anticancer effect. The structural variations were
explored by placing the pyrazoline moiety in positions 2 or 4 and
introduction of different arylidene substituents in position 5 of
thiazolone moiety (Fig. 1). The latter were recently exploited as
bioactive arms on heterocyclic scaffolds useful to pharmacological
effect realization of thiazolidinone based compounds.
possessing anti-inflammatory action, and UDP-MurNAc/L-Ala-
2. Results and discussion
ligase inhibitors with antimicrobial effect [5]. Antineoplastic
properties of 4-thiazolidinones and related heterocycles can most
probably be caused by their affinity to anticancer biotargets, such as
JNK-stimulating phosphatase-1 (JSP-1) [6], tumor necrosis factor
2.1. Chemistry
The general methods for synthesis of target pyrazoline
substituted thiazolones are depicted in Schemes 1 and 2.
TNFa [7], anti-apoptotic biocomplex Bcl-XL-BH3 [8], integrin avb3
receptor [9], etc. It must be emphasized, that combination of
thiazolidine template with other heterocycles is a well-known
approach for drug-like molecules’ build-up, which allows to ach-
ieve new pharmacological profile, action strengthening or toxicity
lowering [1]. As part of our ongoing research in discovery of new
4-(5-Aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-
2(5H)-ones 1 and 2 were obtained by means of 4-thioxo-2-thia-
zolidinone (isorhodanine) reaction with appropriate 5-aryl-3-
phenyl-2-pyrazoline in refluxing ethanol (Scheme 1). Aiming at
the detailed elaboration of structure–activity relationship isomeric
2-(5-aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-4(5H)-
ones 11 and 12 were synthesized (Scheme 2). It should be noted,
that 2-thioxo-4-thiazolidinone (rhodanine) usage for synthesis of
target compounds failed. Therefore with the aim of increasing the
* Corresponding author. Tel.: þ38 0322 75 59 66; fax: þ38 0322 75 77 34.
E-mail address: dr_r_lesyk@org.lviv.net (R. Lesyk).
0223-5234/$ – see front matter Ó 2008 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2008.09.032

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
1397
CH3
O
O
O
N
CH3
OH
N
S
N
O
S
S
O
S
O
N
S
HO
OH
JSP-1 inhibitor
O
O
F
Integrinαvβ3 antagonist
NH
O
IV 563
O
O
N
N
O
O
OH
CH3
CH3
S
N
R1
O
N
Ar'
R2
N
S
O
N
N
Ar
Ar
S
and
N
R1
CH3
N
S
S
S
X
S
N
BH3-1 (Hal = Cl, Br)
Hal
VB 291
N
N
Ar'
O
Inhibitors of antiapoptic proteins
interaction Bcl-XL and BH3
F
S
TNFα-TNFRc-1 inhibitors
F
F
R1
Fig. 1. Structures of anticancer 4-thiazolidinones and rationale for thiazolone-based compounds with pyrazoline moiety synthesis.
rhodanine reactivity, the latter was alkylated via intermediate
triethylammonium salt by ethylchloroacetate in refluxing acetone,
as reported [13]. Following reaction of 2-carbethoxymethylthio-2-
thiazoline-4-one with appropriate 5-aryl-3-phenyl-2-pyrazolines
in refluxing ethanol target 2-(5-aryl-3-phenyl-4,5-dihydro-1H-
pyrazol-1-yl)-1,3-thiazol-4(5H)-ones were obtained. Alternatively
compounds 11 and 12 were synthesized following [2 þ 3]-cyclo-
condensation of 3-phenyl-5-aryl-1-thiocarbamoyl-2-pyrazolines
with chloroacetic acid in the presence of fused sodium acetate in
refluxing acetic acid.
aromatic aldehydes in the presence of fused sodium acetate in
refluxing acetic acid (Scheme 2).
The characterization data of synthesized novel pyrazoline
substituted thiazolones are presented in experimental part.
Analytical and spectral data (1H NMR, 13C NMR, LC–MS, EI-MS)
confirmed the structure of the synthesized compounds.
Protons CH2–CH of pyrazoline fragment in the 1H NMR spectra
of synthesized compounds show characteristic patterns of an AMX
system. The chemical shifts of the protons HA, HM, and Hx have been
assigned to about d w 3.26–3.45, d w 3.96–4.17, and d w 5.68–6.13,
Synthesized compounds 1, 2 and 11, 12 are methylene active
heterocycles. On the other hand, it was previously established
[1,13], that in most cases the presence and nature of moiety in
position 5 of thiazolidinones play the key role in realization and
character of pharmacological effects. The above mentioned thesis
was rationale for synthesis of new 5-arylidene derivatives 3–10,13–
18, using standard Knoevenagel reaction procedure (medium –
respectively, with corresponding coupling constants of JAM ¼ 17.9–
18.6, JAx ¼ 10.4–11.6, and JMx ¼ 2.9–4.5 Hz. The chemical shift for
the methylidene group of 5-arylidene derivatives 3–10 and 13–18 is
insignificantly displaced in weak magnetic field,
d w 9.0 and
d
w 7.5, respectively, and clearly indicated that only Z-isomers were
obtained in Knoevenagel reaction of pyrazoline substituted thia-
zolones with aromatic aldehydes [15].
acetic acid, catalyst
–
fused sodium acetate) [1,13]. Some
Structural features of synthesized 4-(5-aryl-3-phenyl-4,5-dihy-
dro-1H-pyrazol-1-yl)-1,3-thiazol-2(5H)-ones were confirmed by X-
ray crystallographic analysis of exemplified compound 5. As follows
from the X-ray analysis the compound obtained has the structure of
compounds (11, 12, 14, 17 and 18) were prepared alternatively by
one-pot methodology involving reaction of 3-phenyl-5-aryl-1-thi-
ocarbamoyl-2-pyrazolines with chloroacetic acid and appropriate
S
a
b
N
N
NH
O
N
N
S
R
R
N
N
Ar
S
S
O
O
1. R = 2-OH
3. R = 2-OH, Ar = Ph
2. R = 4-OMe
4. R = 2-OH, Ar = 4-Cl-C6H4
5. R = 2-OH, Ar = 4-Br-C6H4
6. R = 2-OH, Ar = 4-OH-C6H4
7. R = 2-OH, Ar = 4-OMe-C6H4
8. R = 4-OMe, Ar = 4-Cl-C6H4
9. R = 4-OMe, Ar = 4-NMe2-C6H4
10. R = 4-OMe, Ar = 4-NO2-C6H4
Scheme 1. Synthesis of novel 4-(5-aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-2(5H)-ones. Reagents, conditions and yields: (a) 5-(2-hydroxy or 4-methoxyphenyl)-3-
phenyl-2-pyrazoline (1.0 equiv), EtOH, reflux 1 h, 62–67%; (b) Ar-CHO (1.1 equiv), AcONa (1.0 equiv), AcOH, reflux 2 h, 52–72%.

1398
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
O
O
H
N
R
S
S
g
c
H2N
S
O
i
N
N
O
N
N
N
NH+
N
S
R = 2-OH, 4-OMe
S
R
Ar
S
R
f
h
d
O
13. R = 2-OH, Ar = 2-Cl-C6H4
14. R = 2-OH, Ar = 4-Cl-C6H4
15. R = 2-OH, Ar = 3-Br-C6H4
16. R = 2-OH, Ar = 3,5-(OMe)2-4-OH-C6H2
17. R = 4-OMe, Ar = 4-NO2-C6H4
18. R = 4-OMe, Ar = 4-NMe2-C6H4
N
O
N
e
N
N
O
S
S
S
O
R
11. R = 2-OH
12. R = 4-OMe
Scheme 2. Synthesis of novel 2-(5-aryl-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-4(5H)-ones. Reagents, conditions and yields: (c) triethylamine (1.0 equiv), i-PrOH, rt 2 h,
88% (Ref. [13]); (d) ClCH2COOEt (1.0 equiv), acetone, reflux 2 h, 62% (Ref. [13]); (e) 5-(2-hydroxy or 4-methoxyphenyl)-3-phenyl-2-pyrazoline (1.0 equiv), EtOH, reflux 1 h, 72–79%;
(f) Ar-CHO (1.2 equiv), AcONa (1.0 equiv), AcOH, reflux 5 h, 59–73%; (g) thiosemicarbazide (1.2 equiv), KOH (2.5 equiv), EtOH, reflux 8 h, 75–80% (Ref. [14]); (i) Ar-CHO (1.2 equiv),
ClCH2COOH (1.0 equiv), AcONa (2.0 equiv), AcOH, reflux 5 h, 65–69%.
5-(4-bromobenzylidene)-4-[5-(2-hydroxyphenyl)-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (5) (Scheme 1,
Fig. 2). The interatomic distances N3–C4 [1.313(4) Å] and C4–N7
[1.341(4) Å], longer than the normal length of the double bond
deviation of the hydroxyphenyl ring from the least squares plane of
the dihydropyrazole ring of 74.88(10)ꢁ is favoured by the inter-
molecular hydrogen bond O24–H24/O33 [O24/O33, 2.656(4) Å],
made by the hydroxyl group of the hydroxyphenolic system and the
carbonyl oxygen atom of the molecule of the crystallization solvent
(dimethylformamide) involved in the formation of the crystal
lattice of 5.
C]N, 1.279(1) Å [16] only by about 8 and 15s, indicate the partly
double character of the two bonds. This suggests that in the solid
state the compound analyzed has a structure intermediate between
that of 5a and that of 5b (Scheme 3).
The bond lengths 1.342(4) Å and 1.289(3) Å confirm the occur-
rence of the double bonds between C5, C25 and N8, C9 atoms,
respectively (Fig. 3).
2.2. Evaluation of anticancer activity in vitro
The five-membered dihydropyrazole ring has an envelope
conformation, with puckering parameters [17] of Q ¼ 0.176(3) Å,
Some new pyrazoline substituted thiazolones (1, 6, 11 and 13–
16) were submitted and evaluated at single concentration of
10ꢀ5 M towards panel of approximately sixty cancer cell lines. The
human tumor cell lines were derived from nine different cancer
types: leukemia, melanoma, lung, colon, CNS, ovarian, renal, pros-
tate and breast cancers. Primary anticancer assays were performed
according to the US NCI protocol, which was described elsewhere
[18–22]. The compounds were added at a single concentration and
the cell culture was incubated for 48 h. End point determinations
were made with a protein binding dye, sulforhodamine B (SRB). The
results for each compound are reported as the percent growth of
treated cells when compared to untreated control cells (Table 1).
4
¼ 319.1(9)ꢁ. The deviation of C11 atom from the almost planar
system of the other four atoms of the heterocyclic ring is 0.286(4) Å.
The conformation of the molecule of compound 5 is stabilized
by the intra- and intermolecular interactions. The intramolecular
hydrogen bond C31–H/S1 [C31/S1, 3.136(4) Å] favours the
arrangement of the p-bromobenzylidene and thiazolidine systems
in the molecule at the angle of 24.44(13)ꢁ, while the hydrogen bond
C25–H/N8 [C25/N8, 2.910(4) Å] stabilizes the almost coplanar
arrangement of the dihydropyrazole and thiazolidine rings. The
dihedral angle made by the rings is only 9.26(13)ꢁ. The significant
Fig. 2. The molecular structure of 5 with displacement ellipsoids drawn at the 30% probability level. H atoms, treated as isotropic, are on an arbitrary scale.

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
1399
cell line: GI50 – molar concentration of the compound that inhibits
50% net cell growth; TGI – molar concentration of the compound
leading to total inhibition; and LC50 – molar concentration of the
compound leading to 50% net cell death. Furthermore a mean graph
midpoints (MG_MID) were calculated for each of the parameters,
giving an average activity parameter over all cell lines for tested
compounds. For the calculation of the MG_MID, insensitive cell
lines are included with the highest concentration tested (Table 2).
The tested compounds showed a broad spectrum of growth
inhibition activity against human tumor cells, as well as some
distinctive patterns of selectivity. Compounds 3, 6 and 16 showed
the highest cytotoxicity and were active against all tested human
tumor cell lines (Table 2). Selectivity pattern analysis of cell lines by
disease origin can definitely affirm selective action of compounds 3
and 6 on leukemia cell lines and compound 16 on colon cancer
(Fig. 4). These compounds appeared to be the most active against
selected individual cell lines with the log GI50 varying from ꢀ6.37
to ꢀ5.70 (Table 3). Compound 3 was found to be a highly active
growth inhibitor of the leukemia cell line RPMI-8226, non-small
cell lung cancer cell line HOR-92, colon cancer line HCT 15 and CNS
cancer cell line U251. Compound 6 showed selectivity on leukemia
cell lines (HL-60(TB) and RPMI-8226), colon cancer line HT-29, CNS
cancer line SNB-75 and melanoma (SK-MEL-28, SK-MEL-5 and
UACC-62). Pyrazoline substituted derivative 16 demonstrates the
most marked effect among all synthesized compounds and
possessed significant activity on non-small cell lung cancer cell line
HOP-62 and colon cancer cell line HT-29 with log GI50 value ꢀ6.12
and ꢀ6.37, respectively.
HO
HO
N
N
N
N+
N
-
N
S
S
O
O
Br
Br
Form a
Form b
Scheme 3. Two mesomeric forms of 5.
Range of growth % shows the lowest and the highest growth %
found among different cancer cell lines.
Finally, compounds 6 and 16 possessed considerable activity and
were selected, whereas compounds 3, 4, 7 and 17 were tested
without preliminary pre-screening stage, in advanced assay against
a panel of approximately sixty tumor cell lines at 10-fold dilutions
of five concentrations (100, 10, 1, 0.1 and 0.01 mM) [18–22]. The
percentage of growth was evaluated spectrophotometrically versus
controls not treated with test agents. A 48-h continuous drug
exposure protocol followed and SRB (sulforodamine B) protein
assay was used to estimate cell viability or growth. Based on the
cytotoxicity assays, three antitumor activity dose–response
parameters were calculated for experimental agents against each
Fig. 3. Molecular packing and hydrogen bonds (dashed lines); H atoms not involved in hydrogen bonds have been omitted for clarity.

1400
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
Table 1
Anticancer screening data in concentration 10ꢀ5 M.
Compound 60 Cell lines assay in 1-dose 10ꢀ5 M conc.
Active (selected for 5-dose 60 cell lines assay)
Mean growth % Range of growth % The most sensitive cell line Growth % of the most sensitive cell line
1
83.59
ꢀ5.39 to 124.97
ꢀ96.31 to 134.28
ꢀ61.48 to 145.90
45.62 to 158.52
ꢀ25.00 to 161.62
ꢀ53.95 to 164.31
ꢀ57.04 to 123.35
CCRF-CEM
(Leukemia)
SK-MEL-5
(Melanoma)
MOLT-4
ꢀ5.39
ꢀ96.31
ꢀ61.48
45.62
Inactive
Active
6
1.13
11
13
14
15
16
101.04
102.39
98.33
88.58
42.59
Inactive
Inactive
Inactive
Inactive
Active
(Leukemia)
SR
(Leukemia)
CCRF-CEM
(Leukemia)
HL-60(TB)
(Leukemia)
DU-145
ꢀ25.00
ꢀ53.95
ꢀ57.04
(Prostate cancer)
NCI web-resources allow to compare selectivity patterns of
clarify the features underlying the antitumor potential of tested
compounds.
tested compounds with standard anticancer agents. Successful
application of COMPARE algorithm could provide the preliminary
information regarding growth inhibition and cell killing mecha-
nism. COMPARE [18,22] analyses were performed for active
compounds at GI50 level, however, obtained correlation coefficients
(r) didn’t allow to distinguish cytotoxicity mechanism of tested
compounds with high probability. Nevertheless moderate correla-
tions compound 4 showed with dichloroallyl lawsone (NSC 126771,
dihydroorotate dehydrogenase inhibitor, r ¼ 0.600 [23]), as well as
compound 6 with tritylcysteine (NSC 83265, aminoacyl-tRNA
synthetases inhibitor with antiproliferative effect against leukemia,
r ¼ 0.537 [24]).
4. Experimental
4.1. Materials and methods
The starting 2-thioxo-4-thiazolidinone [25], 4-thioxo-2-thiazo-
lidinone [26], 5-aryl-3-phenyl-2-pyrazolines [27], 3-phenyl-5-aryl-
1-thiocarbamoyl-2-pyrazolines [14], 2-carbethoxymethylthio-2-
thiazoline-4-one [13] were obtained according to methods
described previously.
Melting points were measured in open capillary tubes on
}
The SAR study revealed that: (1) anticancer activity of
compounds 1, 3, 4, 6, 7, 11 and 13–17 is sensitive to the nature of
substituent in position 5 of thiazolone cycle, (2) introduction of p-
OH group in 5-benzylidene fragment enhanced potency, and (3)
linking position of pyrazoline fragment (2 or 4) with thiazolone
core did not influence antitumor activity.
a BUCHI B-545 melting point apparatus and are uncorrected. The
elemental analyses (C, H, N) were performed using the Perkin–
Elmer 2400 CHN analyzer. Analyses indicated by the symbols of the
elements or functions were within ꢂ0.4% of the theoretical values.
The 1H NMR spectra were recorded on Varian Gemini 300 MHz and
13C NMR spectra on Varian Mercury-400 100 MHz in DMSO-d6 or
DMSO-d6 þ CCl4 mixture using tetramethylsilane (TMS) as an
internal standard. Chemical shifts are reported in ppm units with
3. Conclusions
use of d scale. LC–MS and EI-MS were obtained on Agilent 1100 and
Varian 1200L instruments, respectively.
In the present paper, eighteen new pyrazoline substituted
thiazolones were described. Eleven of synthesized compounds
were tested and most of them displayed antitumor activity on
leukemia, melanoma, lung, colon, CNS, ovarian, renal, prostate and
breast cancers cell lines.
In conclusion, these preliminary results allowed to identify the
most active compounds 3, 6 and 16, especially 5-(4-hydroxy-3,5-
dimethoxybenzylidene)-2-[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihy-
dro-1H-pyrazol-1-yl]thiazol-4(5H)-one (16) could be prospective
antitumor agent (average log GI50 and log TGI values ꢀ5.61 and ꢀ5.14,
respectively) with selective influence on colon cancer cell lines. The
obtained results prove the necessity for further investigations to
4.2. Chemistry
4.2.1. General procedure for synthesis of 4-(5-aryl-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)-1,3-thiazol-2(5H)-ones (1 and 2)
A mixtures of 4-thioxo-2-thiazolidinone (20 mmol) and appro-
priate 5-aryl-3-phenyl-2-pyrazoline (20 mmol) were refluxed for
1 h in 150 ml of ethanol. After cooling to the room temperature
precipitated light brown powder was filtered off, washed with
methanol and recrystallized with n-butanol (1) or DMF:ethanol,1:2
mixture (2).
Table 2
Summary of anticancer screening data at dose-dependent assay.
Compound
Na
log GI50
log TGI
log LC50
N1b
Range
MG_MID
N2b
Range
MG_MID
N3b
Range
MG_MID
3
4
6
7
16
17
54
56
57
57
57
57
54
56
57
54
57
7
ꢀ5.82 to ꢀ4.43
ꢀ5.73 to ꢀ4.22
ꢀ5.78 to ꢀ4.59
ꢀ5.08 to ꢀ4.00
ꢀ6.37 to ꢀ4.74
ꢀ5.28 to ꢀ4.00
ꢀ5.41
ꢀ4.97
ꢀ5.46
ꢀ4.55
ꢀ5.61
ꢀ4.08
52
55
23
34
55
2
ꢀ5.54 to ꢀ4.00
ꢀ5.45 to ꢀ4.00
ꢀ5.48 to ꢀ4.00
ꢀ4.60 to ꢀ4.00
ꢀ5.53 to ꢀ4.37
ꢀ4.36 to ꢀ4.00
ꢀ4.90
ꢀ4.57
ꢀ4.45
ꢀ4.18
ꢀ5.14
ꢀ4.01
37
49
5
11
45
0
ꢀ5.54 to ꢀ4.00
ꢀ5.16 to ꢀ4.00
ꢀ4.31 to ꢀ4.00
ꢀ4.30 to ꢀ4.00
ꢀ5.27 to ꢀ4.00
–
ꢀ4.38
ꢀ4.21
ꢀ4.02
ꢀ4.04
ꢀ4.58
ꢀ4.00
a
N – number of human tumor cell lines tested at the 2nd stage assay.
N1, N2, N3 – number of sensitive cell lines, against which the compound possessed considerable growth inhibition according to mentioned parameter (parameters log GI50
b
,
log TGI and log LC50 ꢃ ꢀ4.00).

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
1401
5.8
5.6
5.4
5.2
5
MGMID
Leukemia
NSCLC
Colon
CNS
Melanoma
Ovarian
Renal
Prostate
Breast
4.8
4.6
4.4
4.2
4
3
4
6
7
16
17
Compounds
Fig. 4. Anticancer selectivity pattern of the most active compounds 3, 4, 6, 7, 16 and 17.
4.2.1.1. 4-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-1,3-thiazol-2(5H)-one (1). Yield 67%, mp 181–183 ꢁC. 1H NMR
(dd, J ¼ 10.6, 3.4 Hz, 1H), 3.94 (dd, J ¼ 18.2, 10.6 Hz, 1H), 3.26 (dd,
J ¼ 18.2, 3.4 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d 176.8 (C]O),
(300 MHz, DMSO-d6 þ CCl4):
d
9.65 (s, 1H), 7.86 (d, J ¼ 8.8 Hz, 2H),
166.6 (C]N, thiaz.), 164.4 (C]N, pyraz.), 154.9 (C–OH), 137.7, 135.8,
132.4, 130.9, 129.9, 129.4, 129.2, 128.3, 127.3, 126.5, 119.7, 116.4, 63.7,
(CHCH2), 40.9 (CHCH2). LC–MS: m/z 426 (95.96%, Mþ þ 1). Anal.
C25H19N3O2S (C, H, N).
7.52–7.43 (m, 3H), 7.10 (t, J ¼ 8.2 Hz, 1H), 6.98 (d, J ¼ 8.0 Hz, 1H),
6.88 (d, J ¼ 8.0 Hz, 1H), 6.75 (t, J ¼ 8.2 Hz, 1H), 5.95 (dd, J ¼ 10.6,
4.0 Hz, 1H), 4.70 (d, J ¼ 18.2 Hz, 1H), 4.58 (d, J ¼ 18.2 Hz, 1H), 4.00
(dd, J ¼ 18.4, 10.6 Hz, 1H), 3.35 (dd, J ¼ 18.4, 4.0 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6):
d
184.2 (C]O), 175.4 (C]N, thiaz.), 161.7
4.2.2.2. 5-(4-Chlorobenzylidene)-4-[5-(2-hydroxyphenyl)-3-phenyl-
(C]N, pyraz.), 154.9 (C–OH), 134.0, 132.0, 130.8, 129.2, 128.9, 127.9,
127.3, 119.6, 116.4, 59.2 (CHCH2), 42.6 (CHCH2), 38.5 (CH2, thiaz.).
Anal. C18H15N3O2S (C, H, N).
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (4). Yield 64%,
mp 225–227 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 9.80 (s, 1H),
9.10 (s, 1H), 7.88 (d, J ¼ 8.8 Hz, 2H), 7.65 (d, J ¼ 9.0 Hz, 2H), 7.48 (d, J
¼ 9.0 Hz, 2H), 7.55–7.45 (m, 3H), 7.07 (t, J ¼ 8.7 Hz, 1H), 6.94 (d,
J ¼ 8.7 Hz, 1H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.74 (t, J ¼ 8.2 Hz, 1H), 6.10
(dd, J ¼ 10.4, 4.0 Hz, 1H), 3.96 (dd, J ¼ 18.4, 10.4 Hz, 1H), 3.26 (dd,
4.2.1.2. 4-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-1,3-thiazol-2(5H)-one (2). Yield 62%, mp 237–238 ꢁC. 1H NMR
(300 MHz, DMSO-d6 þ CCl4):
d
7.85 (d, J ¼ 8.1 Hz, 2H), 7.53–7.46
J ¼ 18.4, 4.0 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d 176.4 (C]O),
(m, 3H), 7.17 (d, J ¼ 8.7 Hz, 2H), 6.87 (d, J ¼ 8.7 Hz, 2H), 5.68 (dd,
J ¼ 11.1, 3.9 Hz, 1H), 4.75 (d, J ¼ 18.1 Hz, 1H), 4.62 (d, J ¼ 18.1 Hz, 1H),
4.07 (dd, J ¼ 18.3, 11.1 Hz, 1H), 3.73 (s, 3H), 3.35 (dd, J ¼ 18.3, 3.9 Hz,
166.4 (C]N, thiaz.), 164.5 (C]N, pyraz.), 154.9 (C–OH), 136.1, 135.4,
134.6, 132.5, 129.8, 129.4, 128.9, 128.4, 126.8, 119.6, 116.4 (3-CH 2-
OH), 63.8 (CHCH2), 41.8 (CHCH2). LC–MS: m/z 460 (94.35%, Mþ þ 1;
3.17%, Mþ þ 2). Anal. C25H18ClN3O2S (C, H, N).
1H). 13C NMR (100 MHz, DMSO-d6):
d 184.2 (C]O), 175.5 (C]N,
thiaz.), 161.3 (C]N, pyraz.), 159.5 (C-OH), 132.9, 132.1, 130.7, 129.6,
128.1, 127.8, 114.8, 63.5 (CHCH2), 55.8 (OCH3), 43.7 (CHCH2), 38.6
(CH2, thiaz.). Anal. C19H17N3O2S (C, H, N).
4.2.2.3. 5-(4-Bromobenzylidene)-4-[5-(2-hydroxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (5). Yield 52%,
mp 212–215 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 9.79 (s, 1H),
4.2.2. General procedure for synthesis of 5-arylidene-4-(5-aryl-3-
phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-2(5H)-ones
(3–10)
A mixture of compounds 1 or 2 (3 mmol), appropriate aldehyde
(3.3 mmol) and anhydrous sodium acetate (3 mmol) were refluxed
for 2 h in glacial acetic acid (10 ml). Obtained powders were filtered
off, washed with methanol and recrystallized with DMF:ethanol or
DMF:acetic acid, 1:2 mixtures.
9.06 (s, 1H), 7.91 (d, J ¼ 8.8 Hz, 2H), 7.68 (d, J ¼ 8.8 Hz, 2H), 7.62
(d, J ¼ 8.8 Hz, 2H), 7.56–7.48 (m, 3H), 7.11 (t, J ¼ 8.7 Hz, 1H), 6.94 (d,
J ¼ 8.7 Hz, 1H), 6.88 (d, J ¼ 8.0 Hz, 1H), 6.72 (t, J ¼ 8.2 Hz, 1H), 6.07
(dd, J ¼ 10.6, 3.2 Hz, 1H), 3.98 (dd, J ¼ 18.2, 10.6 Hz, 1H), 3.26 (dd, J
¼ 18.4, 3.2 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d 176.4 (C]O),
166.5 (C]N, thiaz.), 164.6 (C]N, pyraz.), 154.9 (C–OH), 137.9, 132.9,
132.7, 132.6, 132.4, 130.1, 129.8, 129.4, 128.9, 128.4, 128.1, 126.8,
124.4, 122.4, 119.7, 116.4, 63.8 (CHCH2), 41.8 (CHCH2). Anal.
C25H18BrN3O2S (C, H, N).
4.2.2.1. 5-Benzylidene-4-[5-(2-hydroxyphenyl)-3-phenyl-4,5-dihy-
dro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (3). Yield 63%, mp 204–
4.2.2.4. 5-(4-Hydroxybenzylidene)-4-[5-(2-hydroxyphenyl)-3-phenyl-
206 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d
9.80 (s, 1H), 9.20
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (6). Yield 64%, mp
(s, 1H), 7.86 (d, J ¼ 8.8 Hz, 2H), 7.65 (d, J ¼ 8.8 Hz, 2H), 7.54–7.46
(m, 5H), 7.40 (t, J ¼ 8.8 Hz, 1H), 7.07 (t, J ¼ 8.7 Hz, 1H), 6.90
(d, J ¼ 8.7 Hz, 1H), 6.87 (d, J ¼ 8.0 Hz, 1H), 6.70 (t, J ¼ 8.2 Hz, 1H), 6.13
266–267 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 10.27 (s, 1H),
9.82 (s, 1H), 9.07 (s, 1H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.60 (d, J ¼ 8.8 Hz,
2H), 7.60–7.50 (m, 3H), 6.98 (d, J ¼ 8.8 Hz, 2H), 7.08 (t, J ¼ 8.7 Hz,

1402
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
Table 3
125.9, 119.7 (5-CH, 2-OH), 116.4, 115.5, 63.6 (CHCH2), 56.2 (CH3–O),
40.9 (CHCH2). Anal. C26H21N3O3S (C, H, N).
The influence of compounds 3, 4, 6 and 16 on the growth of individual tumor cell
lines (log GI50 ꢃ ꢀ5.70).
Compound
Disease
Cell line
log GI50
log TGI
4.2.2.6. 5-(4-Chlorobenzylidene)-4-[5-(4-methoxyphenyl)-3-phenyl-
3
Leukemia
Leukemia
CCRF-CEM
RPMI-8226
HOP-62
HOP-92
HCT-15
KM-12
U251
UACC-62
OVCAR-3
786-0
ꢀ5.72
ꢀ5.81
ꢀ5.79
ꢀ5.81
ꢀ5.82
ꢀ5.79
ꢀ5.81
ꢀ5.79
ꢀ5.70
ꢀ5.74
ꢀ5.77
ꢀ5.74
ꢀ5.73
ꢀ5.78
ꢀ5.73
ꢀ5.72
ꢀ5.71
ꢀ5.75
ꢀ5.77
ꢀ5.77
ꢀ5.70
ꢀ5.77
ꢀ5.77
ꢀ6.12
ꢀ5.79
ꢀ5.81
ꢀ5.91
ꢀ6.37
ꢀ5.74
ꢀ5.73
ꢀ5.75
ꢀ5.76
ꢀ5.82
ꢀ5.81
ꢀ5.70
ꢀ5.72
ꢀ5.80
ꢀ5.85
ꢀ5.93
ꢀ5.99
ꢀ5.80
ꢀ5.75
ꢀ5.72
ꢀ5.81
ꢀ5.71
ꢀ5.84
ꢀ5.86
ꢀ5.74
ꢀ5.75
ꢀ5.34
ꢀ5.49
ꢀ5.46
ꢀ5.35
ꢀ5.53
ꢀ5.35
ꢀ5.54
ꢀ5.53
ꢀ5.35
ꢀ5.41
ꢀ5.51
ꢀ5.42
ꢀ5.45
ꢀ5.31
ꢀ5.35
–
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (8). Yield 66%,
mp 231–232 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 9.03
NSC lung cancer
NSC lung cancer
Colon cancer
Colon cancer
CNS cancer
(s, 1H), 7.96 (d, J ¼ 7.2 Hz, 2H), 7.72 (d, J ¼ 8.6 Hz, 2H), 7.60 (d, J
¼ 8.6 Hz, 2H), 7.58–7.50 (m, 3H), 7.20 (d, J ¼ 8.6 Hz, 2H), 6.90
(d, J ¼ 8.6 Hz, 2H), 5.92 (dd, J ¼ 10.8, 3.1 Hz, 1H), 4.01 (dd, J ¼ 18.6,
10.8 Hz, 1H), 3.88 (s, 3H), 3.35 (dd, J ¼ 18.2, 3.1 Hz, 1H). 13C NMR
Melanoma
(100 MHz, DMSO-d6,):
d 176.4 (C]O), 166.6 (C]N, thiaz.), 164.1
Ovarian cancer
Renal cancer
Renal cancer
Breast cancer
Melanoma
Leukemia
Leukemia
Colon cancer
CNS cancer
Melanoma
(C]N, pyraz.), 159.4 (C–OH), 136.2, 135.4, 134.6, 133.1, 132.5, 130.6,
130.0, 129.9, 128.5, 127.8, 114.9, 66.4 (CHCH2), 55.8 (O–CH3), 41.9
(CHCH2). EI-MS (m/z): 473 (Mþ), 475 (Mþ þ 2). Anal. C26H20ClN3O2S
(C, H, N).
SN12C
NCI/ADR-RES
LOX IMVI
HL-60(TB)
RPMI-8226
HT-29
4
6
4.2.2.7. 5-(4-Dimethylaminobenzylidene)-4-[5-(4-methoxyphenyl)-3-
phenyl-4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (9). Yield
SNB-75
ꢀ5.28
57%, mp 233–234 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 9.01
SK-MEL-28
SK-MEL-5
UACC-62
CCRF-CEM
MOLT-4
RPMI-8226
HOP-62
NCI-H460
HCT-116
HCT-15
HT29
KM12
SF-268
SNB-19
SNB-75
U251
LOX IMVI
MALME-3M
SK-MEL-5
IGROV-1
OVCAR-3
ACHN
CAKI-1
SN12C
TK-10
UO-31
DU-145
MCF7
MDA-MB-231/ATCC
HS 578T
MDA-MB-435
T-47D
–
Melanoma
Melanoma
Leukemia
Leukemia
ꢀ5.48
ꢀ5.48
ꢀ5.25
–
(s, 1H), 7.92 (d, J ¼ 7.2 Hz, 2H), 7.60 (d, J ¼ 8.6 Hz, 2H), 7.58–7.50 (m,
3H), 7.46 (d, J ¼ 8.6 Hz, 2H), 7.17 (d, J ¼ 8.6 Hz, 2H), 6.84 (d,
J ¼ 8.6 Hz, 2H), 5.88 (dd, J ¼ 11.0, 2.9 Hz, 1H), 3.96 (dd, J ¼ 18.4,
11.0 Hz, 1H), 3.70 (s, 3H), 3.35 (dd, J ¼ 18.4, 2.9 Hz, 1H), 3.02 (s, 6H).
16
Leukemia
ꢀ5.16
ꢀ4.81
ꢀ5.48
ꢀ5.53
ꢀ5.57
ꢀ5.55
ꢀ5.44
ꢀ5.35
ꢀ5.43
ꢀ5.37
ꢀ5.55
ꢀ5.53
ꢀ5.31
ꢀ4.98
ꢀ5.54
ꢀ5.55
ꢀ4.99
ꢀ5.49
ꢀ5.31
ꢀ5.40
ꢀ5.37
ꢀ5.51
ꢀ5.31
ꢀ5.52
ꢀ5.43
ꢀ5.44
ꢀ5.40
13C NMR (100 MHz, DMSO-d6):
d 177.3 (C]O), 166.7 (C]N, thiaz.),
NSC lung cancer
NSC lung cancer
Colon cancer
Colon cancer
Colon cancer
Colon cancer
CNS cancer
CNS cancer
CNS cancer
CNS cancer
Melanoma
162.3 (C]N, pyraz.), 159.4 (C–OH), 152.1 (4-NMe2), 133.7, 133.6,
133.4, 132.8, 132.2, 130.9, 129.8, 128.1, 127.7, 122.2, 121.1, 114.8, 112.7
(3-CH, 4-NMe2), 66.1 (CHCH2), 55.8 (O-CH3), 41.7 (CHCH2). EI-MS
(m/z): 482 (Mþ). Anal. C28H24N4O2S (C, H, N).
4.2.2.8. 5-(4-Nitrobenzylidene)-4-[5-(4-methoxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (10). Yield 68%,
mp 239–240 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d 9.10 (s, 1H),
Melanoma
Melanoma
8.34 (d, J ¼ 8.8 Hz, 2H), 7.95 (d, J ¼ 8.8 Hz, 2H), 7.93 (d, J ¼ 8.8 Hz,
2H), 7.58–7.54 (m, 3H), 7.23 (d, J ¼ 8.7 Hz, 2H), 6.89 (d, J ¼ 8.7 Hz,
2H), 5.94 (dd, J ¼ 11.0, 3.5 Hz, 1H), 4.03 (dd, J ¼ 18.4, 11.0 Hz, 1H),
3.74 (s, 3H), 3.34 (dd, J ¼ 18.4, 3.5 Hz, 1H). 13C NMR (100 MHz,
Ovarian cancer
Ovarian cancer
Renal cancer
Renal cancer
Renal cancer
Renal cancer
Renal cancer
Prostate cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
Breast cancer
DMSO-d6): d 175.9 (C]O), 166.4 (C]N, thiaz.), 164.7 (C]N, pyraz.),
159.5 (C–O),147.9,142.1,134.9, 133.6,132.9,132.7,131.7,130.4, 129.8,
128.6, 127.9, 124.7, 114.9, 66.6 (CHCH2), 55.8 (O–CH3), 41.9 (CHCH2).
Anal. C26H20N4O4S (C, H, N).
4.2.3. General procedure for synthesis of 2-(5-aryl-3-phenyl-4,5-
dihydro-1H-pyrazol-1-yl)-1,3-thiazol-4(5H)-ones (11 and 12)
Method A. A mixture of 2-carbethoxymethylthio-2-thiazoline-4-
one (20 mmol) and appropriate 5-aryl-3-phenyl-2-pyrazoline
(20 mmol) was refluxed for 1 h in 150 ml of ethanol. After cooling
to the room temperature precipitated light brown powder was
filtered off, washed with methanol and recrystallized with
DMF:ethanol, 1:2 mixture.
Method B. A mixture of 3-phenyl-5-aryl-1-thiocarbamoyl-2-
pyrazoline (10 mmol), chloroacetic acid (10 mmol) and anhydrous
sodium acetate (10 mmol) was refluxed for 3 h in glacial acetic acid
(10 ml). Obtained powder was filtered off, washed with methanol
and recrystallized with DMF:ethanol, 1:2 mixture.
1H), 6.93 (d, J ¼ 8.7 Hz, 1H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.74 (t, J
¼ 8.2 Hz, 1H), 6.00 (dd, J ¼ 11.4, 3.8 Hz, 1H), 3.99 (dd, J ¼ 18.3,
11.4 Hz, 1H), 3.28 (dd, J ¼ 18.3, 3.8 Hz, 1H). 13C NMR (100 MHz,
DMSO-d6): d 177.1 (C]O), 166.8 (C]N, thiaz.), 163.6 (C]N, pyraz.),
160.5 (C–OH-p), 154.9 (C–OH-o), 138.2, 133.4, 132.2, 130.9, 129.8,
129.3, 128.2, 127.0, 126.8, 126.4, 124.6, 119.7, 116.9, 116.4, 63.5
(CHCH2), 36.5 (CHCH2). Anal. C25H19N3O3S (C, H, N).
4.2.3.1. 2-[5-(2-Hydroxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-1,3-thiazol-4(5H)-one (11). Yield 79% (method A), 68%
(method B), mp 255–256 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
4.2.2.5. 5-(4-Methoxybenzylidene)-4-[5-(2-hydroxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-2(5H)-one (7). Yield 72%, mp
256–257 ꢁC.1H NMR (300 MHz, DMSO-d6 þ CCl4):
d
9.60 (s,1H), 9.08
d
9.70 (s, 1H), 7.81 (d, J ¼ 7.4 Hz, 2H), 7.45–7.42 (m, 3H), 7.06
(s, 1H), 7.88 (d, J ¼ 8.8 Hz, 2H), 7.62 (d, J ¼ 8.6 Hz, 2H), 7.53–7.43 (m,
3H), 7.04 (d, J ¼ 8.6 Hz, 2H), 7.08 (t, J ¼ 8.7 Hz,1H), 6.93 (d, J ¼ 8.7 Hz,
1H), 6.88 (d, J ¼ 8.0 Hz, 1H), 6.72 (t, J ¼ 8.2 Hz, 1H), 6.11 (dd, J ¼ 10.6,
3.6 Hz, 1H), 3.92 (dd, J ¼ 18.2, 10.6 Hz, 1H), 3.88 (s, 3H), 3.28 (dd,
(t, J ¼ 7.6 Hz, 1H), 6.87 (d, J ¼ 7.6 Hz, 1H), 6.84 (d, J ¼ 7.6 Hz,1H), 6.71
(t, J ¼ 7.6 Hz, 1H), 5.87 (dd, J ¼ 11.3, 4.2 Hz, 1H), 4.01 (dd, J ¼ 18.2,
11.3 Hz, 1H), 3.79 (s, 2H), 3.30 (dd, J ¼ 18.2, 4.2 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6):
d 187.5 (C]O), 177.8 (C]N, thiaz.), 161.5
J ¼ 18.2, 3.6 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d
176.9 (C]O),
(C]N, pyraz.), 154.9 (C–OH), 132.0, 130.7, 129.6, 129.5, 127.9, 127.5,
126.5, 119.7, 116.4, 61.4 (CHCH2), 42.8 (CHCH2), 39.4 (CH2). Anal.
C18H15N3O2S (C, H, N).
166.7 (C]N, thiaz.), 163.8 (C]N, pyraz.), 161.5 (CH3–O), 154.9 (C–
OH), 137.7, 133.0, 132.3, 130.8, 129.8, 129.4, 128.2, 127.9, 127.1, 126.8,

D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
1403
4.2.3.2. 2-[5-(4-Methoxyphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-
1-yl]-1,3-thiazol-4(5H)-one (12). Yield 72% (method A), 61%
(method B), mp 135–137 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
one (16). Yield 59% (method A), mp 297–298 ꢁC. 1H NMR (300 MHz,
DMSO-d6 þ CCl4): 9.32 (brs, 2H), 7.88 (d, J ¼ 7.6 Hz, 2H), 7.56 (s,1H),
d
7.56–7.42 (m, 3H), 7.12 (t, J ¼ 7.9 Hz,1H), 6.96 (d, J ¼ 8.0 Hz,1H), 6.94
(s, 2H), 6.84 (d, J ¼ 8.0 Hz, 1H), 6.75 (t, J ¼ 7.9 Hz, 1H), 5.90 (dd,
J ¼ 11.6, 4.1 Hz, 1H), 4.09 (dd, J ¼ 17.9, 11.6 Hz, 1H), 3.38 (dd, J ¼ 17.9,
d
7.83 (d, J ¼ 7.3 Hz, 2H), 7.53–7.48 (m, 3H), 7.15 (d, J ¼ 8.7 Hz, 2H),
6.87 (d, J ¼ 8.7 Hz, 2H), 5.72 (dd, J ¼ 11.3, 3.8 Hz, 1H), 4.06 (dd,
J ¼ 18.3, 11.3 Hz, 1H), 3,84 (s, 2H), 3.73 (s, 3H), 3.36 (dd, J ¼ 18.3,
4.1 Hz, 1H), 3.92 (s, 6H). 13C NMR (100 MHz, DMSO-d6):
d 180.1
3.8 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d
187.5 (C]O), 177.9
(C]O),170.5 (C]N, thiaz.),162.1 (C]N, pyraz.),155.1 (C–OH),148.9,
138.8,132.6,132.2,130.5,129.7,128.1,126.2,125.3,124.8,119.7,116.4,
108.4, 61.5, (CHCH2), 56.9 (O–CH3), 42.9 (CHCH2). EI-MS (m/z): 501
(Mþ). Anal. C27H23N3O5S (C, H, N).
(C]N, thiaz.), 161.2 (C]N, pyraz.), 159.4 (C–OH), 133.1, 132.2, 130.5,
129.7, 128.0, 127.8, 114.9, 63.9 (CHCH2), 55.8 (O–CH3), 44.0 (CHCH2),
40.4 (CH2). Anal. C19H17N3O2S (C, H, N).
4.2.4. General procedure for synthesis of 5-arylidene-2-(5-aryl-3-
phenyl-4,5-dihydro-1H-pyrazol-1-yl)-1,3-thiazol-4(5H)-ones
(13–18)
4.2.4.5. 5-(4-Nitrobenzylidene)-2-[5-(4-methoxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-one (17). Yield 73%
(method A), 69% (method B), mp 293–294 ꢁC. 1H NMR (300 MHz,
Method A. A mixture of compounds 11 or 12 (3 mmol), appro-
priate aldehyde (4 mmol) and anhydrous sodium acetate (3 mmol)
was refluxed for 5 h in glacial acetic acid (30 ml). Powder obtained
after cooling was filtered off, washed with methanol and recrys-
tallized with DMF:ethanol or DMF:acetic acid, 1:2 mixtures.
Method B. A mixture of 3-phenyl-5-aryl-1-thiocarbamoyl-2-
pyrazoline (10 mmol), chloroacetic acid (10 mmol), appropriate
aldehyde (12 mmol) and anhydrous sodium acetate (10 mmol) was
refluxed for 5 h in glacial acetic acid (10 ml). Powder obtained after
cooling was filtered off, washed with methanol and recrystallized
with DMF:ethanol or DMF:acetic acid, 1:2 mixtures.
DMSO-d6 þ CCl4):
d
8.35 (d, J ¼ 8.7 Hz, 2H), 7.96 (d, J ¼ 8.7 Hz, 4H),
7.75 (s, 1H), 7.59–7.52 (m, 3H), 7.22 (d, J ¼ 8.6 Hz, 2H), 6.92 (d,
J ¼ 8.6 Hz, 2H), 5.88 (dd, J ¼ 11.1, 3.7 Hz, 1H), 4.17 (dd, J ¼ 18.3,
11.1 Hz, 1H), 3.71 (s, 3H), 3.40 (dd, J ¼ 18.3, 3.7 Hz, 1H). 13C NMR
(100 MHz, DMSO-d6):
d 179.3 (C]O), 170.5 (C]N, thiaz.), 162.9
(C]N, pyraz.), 159.7 (C–OH), 147.8, 140.9, 132.9, 132.6, 132.5, 131.3,
130.1, 129.7, 128.9, 128.2, 128.1, 124.9, 114.9, 64.3 (CHCH2), 55.8 (O–
CH3), 44.2 (CHCH2). EI-MS (m/z): 484 (Mþ). Anal. C26H20N4O4S (C,
H, N).
4.2.4.6. 5-(4-Dimethylaminobenzylidene)-2-[5-(4-methoxyphenyl)-
3-phenyl-4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-one
(18). Yield 59% (method A), 65% (method B), mp 258–259 ꢁC. 1H
4.2.4.1. 5-(2-Chlorobenzylidene)-2-[5-(2-hydroxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-one (13). Yield 59%
(method A), mp 268–269 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
NMR (300 MHz, DMSO-d6 þ CCl4):
d
7.88 (d, J ¼ 7.8 Hz, 2H), 7.56 (s,
1H), 7.55–7.42 (m, 5H), 7.19 (d, J ¼ 8.6 Hz, 2H), 6.91 (d, J ¼ 8.6 Hz, 2H),
6.82 (d, J ¼ 8.8 Hz, 2H), 5.81 (dd, J ¼ 11.2, 4.0 Hz, 1H), 4.12 (dd,
J ¼ 18.4, 11.2 Hz, 1H), 3.71 (s, 3H), 3.45 (dd, J ¼ 18.4, 4.0 Hz, 1H). 13C
d
9.75 (s, 1H), 7.87 (d, J ¼ 7.4 Hz, 2H), 7.85 (s, 1H), 7.70 (d, J ¼ 7.3 Hz,
1H), 7.52–7.42 (m, 5H), 7.39 (t, J ¼ 7.5 Hz, 1H), 7.09 (t, J ¼ 7.1 Hz, 1H),
6.93 (d, J ¼ 7.5 Hz,1H), 6.87 (d, J ¼ 8.0 Hz,1H), 6.73 (t, J ¼ 7.4 Hz,1H),
5.94 (dd, J ¼ 11.4, 4.1 Hz 1H), 4.08 (dd, J ¼ 18.0,11.4 Hz,1H), 3.38 (dd,
NMR (100 MHz, DMSO-d6): d 187.5 (C]O),177.9 (C]N, thiaz.),161.2
(C]N, pyraz.), 159.5 (C–OH), 152.1 (4-NMe2), 133.1, 132.7, 132.2,
130.5, 129.7, 127.9, 127.8, 114.9, 112.7, 63.9 (CHCH2), 55.8 (O–CH3),
44.0 (CHCH2). EI-MS (m/z): 482 (Mþ). Anal. C28H26N4O2S (C, H, N).
J ¼ 18.0, 4.1 Hz, 1H). 13C NMR (100 MHz, DMSO-d6):
d 179.3 (C]O),
170.6 (C]N, thiaz.), 162.9 (C]N, pyraz.), 155.1 (C–OH), 134.9, 132.7,
132.5, 132.4, 131.9, 130.9, 130.4, 129.8, 129.7, 129.6, 128.8, 128.1,
126.3, 125.9, 119.7, 116.4, 61.8 (CHCH2), 42.9 (CHCH2). Anal.
C25H18ClN3O2S (C, H, N).
4.3. Pharmacology
Primary anticancer assay was performed at approximately
sixty human tumor cell lines panel derived from nine neoplastic
diseases, in accordance with the protocol of the Drug Evaluation
Branch, National Cancer Institute, Bethesda [18–22]. Tested
compounds were added to the culture at a single concentration
(10ꢀ5 M) and the cultures were incubated for 48 h. End point
determinations were made with a protein binding dye, sulfo-
rhodamine B (SRB). Results for each tested compound were
reported as the percent of growth of the treated cells when
compared to the untreated control cells. The percentage growth
was evaluated spectrophotometrically versus controls not treated
with test agents. The cytotoxic and/or growth inhibitory effects
of the most active selected compounds were tested in vitro
against the full panel of about 60 human tumor cell lines at 10-
fold dilutions of five concentrations ranging from 10ꢀ4 to
10ꢀ8 M. A 48-h continuous drug exposure protocol was followed
and an SRB protein assay was used to estimate cell viability or
growth.
4.2.4.2. 5-(4-Chlorobenzylidene)-2-[5-(2-hydroxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-one (14). Yield 68%
(method A), 69% (method B), mp 284–286 ꢁC. 1H NMR (300 MHz,
DMSO-d6 þ CCl4):
d
9.74 (s, 1H), 7.88 (d, J ¼ 8.0 Hz, 2H), 7.60 (d,
J ¼ 8.5 Hz, 2H), 7.57 (s, 1H), 7.50–7.42 (m, 5H), 7.08 (t, J ¼ 7.5 Hz, 1H),
6.92 (d, J ¼ 8.0 Hz, 1H), 6.86 (d, J ¼ 8.0 Hz, 1H), 6.73 (t, J ¼ 7.4 Hz,
1H), 5.97 (dd, J ¼ 11.4, 4.5 Hz, 1H), 4.08 (dd, J ¼ 18.1, 11.4 Hz, 1H),
3.38 (dd, J ¼ 18.1, 4.5 Hz,1H). 13C NMR (100 MHz, DMSO-d6):
d 179.7
(C]O), 170.4 (C]N, thiaz.), 162.6 (C]N, pyraz.), 155.1 (C–OH),
135.0, 133.5, 132.3, 131.9, 130.4, 129.9, 129.8, 129.7, 129.6, 128.1,
126.0, 119.7, 116.4, 61.7 (CHCH2), 42.9 (CHCH2). Anal. C25H18ClN3O2S
(C, H, N).
4.2.4.3. 5-(3-Bromobenzylidene)-2-[5-(2-hydroxyphenyl)-3-phenyl-
4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-one (15). Yield 62%
(method A), mp 272–274 ꢁC. 1H NMR (300 MHz, DMSO-d6 þ CCl4):
d
9.84 (s, 1H), 7.89 (d, J ¼ 7.9 Hz, 2H), 7.74 (s, 1H), 7.59 (d, J ¼ 8.6 Hz,
1H), 7.56–7.40 (m, 6H), 7.08 (t, J ¼ 8.2 Hz, 1H), 6.92 (d, J ¼ 8.2 Hz,
1H), 6.86 (d, J ¼ 8.2 Hz, 1H), 6.72 (t, J ¼ 8.2 Hz, 1H), 5.98 (dd, J ¼ 11.3,
4.5 Hz, 1H), 4.08 (dd, J ¼ 18.4, 11.3 Hz, 1H), 3.38 (dd, J ¼ 18.4, 4.5 Hz,
Using the seven absorbance measurements [time zero, (Tz),
control growth in the absence of drug, (C), and test growth in
the presence of drug at the five concentration levels (Ti)],
the percentage growth was calculated at each of the drug
concentrations levels. Percentage growth inhibition was calculated
as:
1H). 13C NMR (100 MHz, DMSO-d6):
d 179.5 (C]O), 170.4 (C]N,
thiaz.), 162.8 (C]N, pyraz.), 155.1 (C–OH), 137.1, 133.1, 132.4, 131.9,
130.5, 130.4, 129.7, 128.6, 128.1, 126.0, 123.1, 119.7, 116.4, 61.8
(CHCH2), 42.9 (CHCH2). Anal. C25H18BrN3O2S (C, H, N).
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢄ ꢅ 100
4.2.4.4. 5-(4-Hydroxy-3,5-dimethoxybenzylidene)-2-[5-(2-hydrox-
yphenyl)-3-phenyl-4,5-dihydro-1H-pyrazol-1-yl]-1,3-thiazol-4(5H)-
for concentrations for which Ti ꢆ Tz,

1404
D. Havrylyuk et al. / European Journal of Medicinal Chemistry 44 (2009) 1396–1404
Acknowledgements
½ðTi ꢀ TzÞ=Tzꢄ ꢅ 100
for concentrations for which Ti < Tz.
We are grateful to Dr. V.L. Narayanan from Drug Synthesis and
Chemistry Branch, National Cancer Institute, Bethesda, MD, USA,
for in vitro evaluation of anticancer activity. This work has been
partially supported by the Polish National Commission for
UNESCO (fellowship for D. Havrylyuk) and by Jozef Mianowski
Fund – Foundation for the Promotion of Science (fellowship for R.
Lesyk).
Three dose–response parameters were calculated for each
compound. Growth inhibition of 50% (GI50) was calculated from
½ðTi ꢀ TzÞ=ðC ꢀ TzÞꢄ ꢅ 100 ¼ 50, which is the drug concentration
resulting in a 50% lower net protein increase in the treated cells
(measured by SRB staining) as compared to the net protein increase
seen in the control cells. The drug concentration resulting in total
growth inhibition (TGI) was calculated from Ti ¼ Tz. The LC50
(concentration of drug resulting in a 50% reduction in the measured
protein at the end of the drug treatment as compared to that at the
beginning) indicating a net loss of cells following treatment was
calculated from ½ðTi ꢀ TzÞ=Tzꢄ ꢅ 100 ¼ ꢀ50. Values were calculated
for each of these three parameters if the level of activity is reached;
however, if the effect was not reached or was exceeded, the value
for that parameter was expressed as more or less than the
maximum or minimum concentration tested. The log GI50, log TGI,
log LC50 were then determined, defined as the mean of the logs of
the individual GI50, TGI, LC50 values. The lowest values are obtained
with the most sensitive cell lines. Compounds having these values
ꢃ4 were declared to be active.
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