Synthesis of new pyrazole derivatives and their anticancer evaluation

Article abstract of DOI:10.1016/j.ejmech.2010.07.064

A series of functionally substituted pyrazole compounds have been synthesized and evaluated in vitro for their antiproliferative effects on a panel of 60 cellular lines, according to the National Cancer Institute screening protocol. Three of the 12 tested compounds showed moderate antitumor activity, one of them being chosen for the 5-dose assay and presented logGI₅₀ values up to -5.75.

Full text of DOI:10.1016/j.ejmech.2010.07.064

European Journal of Medicinal Chemistry 45 (2010) 4914e4919
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European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis of new pyrazole derivatives and their anticancer evaluation
,
b
a
George Mihai Nitulescu a , Constantin Draghici , Alexandru Vasile Missir
*
a Department of Pharmaceutical Chemistry, Faculty of Pharmacy, “Carol Davila” University of Medicine and Pharmacy, Traian Vuia 6, Bucharest 020956, Romania
b The Organic Chemistry Centre of Romanian Academy “Costin D. Nenitescu”, Splaiul Independentei, 202B, Bucharest 060023, Romania
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of functionally substituted pyrazole compounds have been synthesized and evaluated in vitro for
their antiproliferative effects on a panel of 60 cellular lines, according to the National Cancer Institute
screening protocol. Three of the 12 tested compounds showed moderate antitumor activity, one of them
being chosen for the 5-dose assay and presented logGI50 values up to ꢀ5.75.
Received 23 June 2010
Received in revised form
25 July 2010
Accepted 29 July 2010
Available online 10 August 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
1-Methyl-1H-pyrazole-4-carboxylic acid
Acyl thiourea
Thiazolylidenes
Anticancer activity
Pyrazole amines
1. Introduction
effects of some pyrazole amide derivatives are mediated by inhibition
of the RaseNet pathway and microtubule depolymerization [15].
Pyrazole showed promising anticancer effects and in the 1960s,
it was evaluated in Phase I studies as an antitumor agent in man,
but, even in doses of 0.15 mmol/kg/day it proved too toxic for
human use because of development of signs of hepato-toxicity [1].
Trying to overcome this toxicity, 1-carboxamidopyrazole and
1-thiocarbamoylpyrazole were synthesized and they showed
significant anticancer effects on animal experiments, but failed to
pass the clinical evaluation [2,3]. In search for better antitumor
treatment, a large series of pyrazole derivatives were synthesized
and tested over the years, the use of this powerful pharmacophore
being very popular and modern [4e6].
In the last decade several pyrazole derivatives proved to have
potent anticancer action by the inhibition of the cyclin-dependent
kinases. (CDKs). CDKs are members of the large family of protein
kinases and are responsabile for the eukariotic cell cycle regulation;
they are intesively studied for their cancer implication [7e11]. Based
on the model of 1-carboxamidopyrazole, a series of pyrazole deriva-
tives containing an urea scaffold proved to have antiproliferative
effects by inhibitingAurora kinaseactivity [12,13]. Some other protein
kinases are targets of pyrazole based derivatives, some aryl- and
heteroaryl-substituted pyrazole act as inhibitors of the transforming
growth factor-beta type I receptor kinase domain [14]. The anticancer
Based on the model of these aforementioned pyrazole urea
derivatives, 1-thyocarbamoylpyrazole and some antiproliferative
acyl thioureas [16e18], we designed and synthesized some
chimeric thiourea-pyrazole derivatives. Fig. 1 shows relevant anti-
tumor pyrazole derivatives, a pattern for our compounds design.
2. Results and discussion
2.1. Chemistry
The new compounds were synthesized by the general methods
outlined in Schemes 1 and 2. The precursor 1-methyl-1H-pyrazole-
4-carboxylic acid (1) was converted into the 1-methyl-1H-pyr-
azole-4-carbonyl chloride (2) using thionyl chloride as chlorination
reagent and was treated with ammonium isothiocyanate to afford
1-methyl-1H-pyrazole-4-carbonyl isothiocyanate (3). This was
converted into the corresponding N-(1-methyl-1H-pyrazole-4-
carbonyl)-N0-(aryl)-thioureas (4aej) by adding various substituted
anilines and refluxing in dry acetone. The 5-(1-methyl-1H-pyrazol-
4-yl)-1-phenyl-1,2-dihydro-1,2,4-triazole-3-thione
(5)
was
obtained in one step reaction from 1-methyl-1H-pyrazole-4-
carbonyl isothiocyanate and phenylhydrazine. The solvent was
changed to acetonitrile to prevent the hydrozone formation.
The structures of the synthesized compounds were confirmed
by spectral data. For the compounds 4aej the 1H NMR spectra
* Corresponding author. Tel.: þ40 213180739.
E-mail address: nitulescu_mihai@yahoo.com (G.M. Nitulescu).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.064

G.M. Nitulescu et al. / European Journal of Medicinal Chemistry 45 (2010) 4914e4919
4915
Fig. 1. Structures of antitumor pyrazoles as rational compounds design template.
showed two broad singlets for the thiourea moiety,
12.50e11.99 ppm (eNHeC¼S) and 11.60e11.20 ppm (eNHeC¼O).
The hydrogens of the pyrazole ring appeared as two singlets
w8.59 ppm and w8.21 ppm, values indifferent to the aryl substi-
tution. These values are lower in the compound 5, 8.32 ppm and
8.00 ppm. In the 13C NMR spectra the acyl thiourea carbons
appeared at 181.1e178.3 ppm (C¼S) and 163.8e162.9 ppm (C¼O).
The pyrazole ring appeared as 141.1e140.3 ppm (C-5),
136.3e134.5 ppm (C-3) and 116.1e115.4 ppm (C-4). The NeCH3
presented a signal in the range of 39.7e39.0 ppm.
treating the new acyl thioureas with bromoacetone under basic
conditions [19]. The pyrazole hydrogens appeared with lower
values in the 1H NMR spectra, w7.78 ppm and w7.48 ppm, because
of the internal H-bond disappearance. Distinctive is the thiazoly-
lidene hydrogen singlet appearing at 6.81e6.75 ppm.
By the structural rearrangement of the compounds 4 skeleton
we designed the compound 7a and the structural analogues 7bec.
The compounds were synthesized transforming benzoyl chloride in
benzoyl isothiocyanate which was treated with the corresponding
pyrazole amine.
In order to better evaluate the structureeactivity relationship
we synthesized some thiazol-2(3H)-ylidene derivatives (6aeb) by
The compounds 7aec 1H NMR spectra present two broad singlets
for the thiourea group, 13.17e12.46 ppm and 11.80e11.56 ppm and
Scheme 1. Reagents: (a) SOCl2, C2H4Cl2, reflux; (b) NH4SCN, (CH3)2CO, reflux; (c) ReC6H4eNH2, (CH3)2CO, reflux; (d) (C2H5)3N, BrCH2COCH3; (e) C6H5NHNH2, CH3CN, reflux.

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G.M. Nitulescu et al. / European Journal of Medicinal Chemistry 45 (2010) 4914e4919
Scheme 2. Reagents: (a) NH4SCN, (CH3)2CO, reflux; (b) (CH3)2CO, reflux.
onebroadsinglet13.37e13.10ppmforthepyrazoleNH, incompounds
7a and 7c, the pyrazole CH as a singlet and the aromatic hydrogens in
the range of 7.99e7.38 ppm. In the 13C NMR spectra the acyl thiourea
carbons appeared at 180.1e170.0 ppm (C¼S) and 168.8e168.0 ppm
(C¼O). The pyrazole moiety appeared as 147.8e140.8 ppm (C-5),
147.8e138.3 ppm (C-3) and 102.9e96.1 ppm (C-4).
similar to the chemical classification, emphasizing on the struc-
ture’s importance for the antitumor action.
The tested N-(1-methyl-1H-pyrazole-4-carbonyl)-N0-(aryl)-
thioureas show little effect on the cancer cells, excepting some few
cell lines, which show a growth inhibition over 50%. By including
the thiourea moiety in
a 1,2,4-triazole-3-thione ring, as in
The new acyl thioureas are characterized by IR absorptions in
the ranges of 3350e3300, 3250e3200 for the free and associated
NH, 1650e1675 for carbonyl and 1230e1250 cmꢀ1 for thiocarbonyl
group. In the thiazoliden-2-imines (6aeb) the carbonyl vibration
band shifts to lower values because of the conjugation with the
imine bond.
compound 5, the inhibitory effect dissappears.
However, the transformation of the thiourea group in a thiazol-2
(3H)-ylidene ring significantly improves the anticancer effects. The
compounds6a and 6bshare similaranticancer profiles, the best action
being noticed againstACHN renal cells, DU-145 prostate cancer, on SK-
MEL-2 melanocytes and the colon cells HCT-15 and HT29.
Using the retro-inversion of thiourea substituents of compound
4a, the resulting 7a has insignifiant effects on the cellular growth of
most cell lines tested, except for four lines that are strongly stim-
ulated, with growth precentages in the range of 161.3 and 209.2.
The anticancer effect is greatly increased by adding a phenyl group
on the position 1 of the pyrazole ring (7b), the stimulatory effect
being significaly reduced, and the best inhibitory effects are noticed
on the leukemia cells. This inversion of acyl and alkyl function of
the pyrazole ring on thiourea moiety is important for the antitumor
action.
The compound’s purity was certified by elemental analyses, the
results being within ꢁ0.4 of the theoretical values.
2.2. Anticancer activity
A number of 12 compounds were selected by the National Cancer
Institute (NCI) for in vitro anticancer evaluation on 60 different
human tumor cell lines, representing leukemia, melanoma and
cancers of the lung, colon, brain, ovary, breast, prostate, and kidney.
The screening was performed according to the NCI protocol. The
results for each tested compounds are reported as growth percent-
ages compared with the untreated control cells after 48 h of drug
exposure. The compounds were tested initially at a single high dose
of 10ꢀ5 M and one compound was chosen and tested on 5-dose assay
using 1 log serial dilutions from 10ꢀ8 to 10ꢀ4 M. The results are
reported as percent growth of the tested cells comparing with the
control cells values. The screening results are presented in Table 1.
The positive values under 100 show a growth inhibitory effect and
the negative values indicate a cytotoxic effect.
Statistically examining the growth inhibition percent results (GI)
for the 12 tested compounds, using the hierarchical cluster analysis
based on the Euclidean distances between variables (Dx,y) and
Ward’s method we obtained the dendogram presented in Fig. 2. The
Euclidean distances are also used as dissimilarity measure to
compare each compound’s results with the control. This method
permits a better classification based on the anticancer effect and the
identification of some structureeactivity relationship.
Table 1
Antitumor activity of compounds at 10ꢀ5 M against cancer cells.
Test compound Range of
growth %
Mean growth % Most sensitive cells
min
max
7c
ꢀ19.9 116.3
70.1
ꢀ19.9(TKꢀ10/R)
1.0 (HCT-116/C)
8.9 (NCI-H522/L)
ꢀ11.8(ACHN/R)
0.3 (DU-145/P)
ꢀ15.7(ACHN/R)
1.6 (DU-145/P)
22.6 (MDA-MB-435/M)
47.2 (NCI-H522/L)
62.5 (NCI-H522/L)
79.7 (UACC-62/M)
68.7 (HOP-92/L)
40.5 (MOLT-4/Lk)
69.7 (CAKI-1/R)
e
6b
6a
ꢀ11.8 119.8
ꢀ15.7 128.1
80.5
82.5
7b
4g
4a
4h
4e
4b
4j
22.6 125.7
47.2 128.9
84.6
91.1
62.5 120.1 101.0
79.7 190.5 101.0
68.7 118.9 102.2
40.5 138.7 105.9
69.7 148.9 107.4
Based on this cluster classification, we can divide the
compounds in two major groups, one (A) containing the N-(1-
methyl-1H-pyrazole-4-carbonyl)-N0-(aryl)-thioureas
and
the
7a
5
89
95
209.2 109.9
143.5 110.5
structurally similar compound 5, with low Euclidean distances to
the control values, and the other (B) containing the thiazolylidene
derivatives and the pyrazole amine thioureas, presenting high
Euclidean distances to the control. This effect-based classification is
e
R e renal cancer, C e colon cancer, L e non-small cell lung cancer, P e prostate
cancer, M e melanoma, Lk e leukemia, B e breast cancer, N e CNS cancer, O e
ovarian cancer.

G.M. Nitulescu et al. / European Journal of Medicinal Chemistry 45 (2010) 4914e4919
4917
effect of 7c we think the changing of the methyl group in 7a with
a 4-bromophenyl in 7c offered the lipophilic and bulky area nec-
cesary for the CDKs inhibition. Following the model of 6aeb, the
future transformation of the compounds 7bec in thiazolylidene
derivatives may be very useful.
3. Conclusion
A set of pyrazole based small molecules were designed and
synthesized as potential antitumor agents. They were tested in an
in vitro assay against 60 types of human cancer cells to evaluate
their anticancer effects. Three compounds presented significant
growth inhibitory effects on the tested cancer cells. The preliminary
SAR reveals that the substituted pyrazolyl thiourea derivatives are
more active then the related N-[(1-methyl-1H-pyrazole-4-yl)
carbonyl]-thiourea derivatives. However, the transformation of the
latter in thiazolylidene derivatives enhance the anticancer effects.
The promising results obtained with the compound N-benzoyl-N0-
(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thiourea (7c) and the SAR
results encourage us to develop new antitumor substances of the
pyrazole derivatives class.
Fig. 2. Hierarchical cluster classification of the compounds.
The best antitumor action is achieved for the compound 7c,
a close analog of 7b, having an additional bromine atom and
a pyrazole eNHe group free to form hydrogen bonds. The
compound has promising antitumor effects, with growth inhibition
percents over 50% on 12 cell lines and was chosen for advance tests
on a 5 fold range of concentrations. The results are presented in
Table 2 and are expressed as logGI50, logTGI and logLC50. These are
logarithmized molar concentration producing a 50% growth inhi-
bition (GI50), a total growth inhibition (TGI) and a 50% cellular death
(LC50), respectively. The values were calculated if the target level of
activity was reached, if not the value is expressed as more than the
highest tested concentration.
The compound 7c presented logGI50 values under ꢀ4 in 43 of the
60 cancer lines, values ranging from ꢀ4.12 to ꢀ5.75, with an average
of ꢀ5.02. The best results were recorded on the ovarian cancer cells
with values ranging from ꢀ4.66 to ꢀ5.37, with an average of ꢀ5.14,
and the renal cancer cells, with values between ꢀ4.40 and ꢀ5.49. A
number of 24 tested cancer cell lines presented logTGI values under
ꢀ4, the best value being noted on the colon cancer cells HCT-116.
Only in 11 cell lines, compound 7c registred logLC50 values under ꢀ4.
Considering the design premises of these compounds, we
expect the antitumor effect to be created by CDKs inhibition.
Comparing the stimulatory effect of compound 7a to the inhibitory
4. Experimental
4.1. Chemistry
All starting materials and solvents were purchased from common
commercial suppliers and used without purification, unless other-
wise noted. The acetone was dried over potassium carbonate and
distillated, and the ammonium thiocyanate by heating at 100 ꢂC. The
melting points were measured in open capillary tubes on an Elec-
trothermal 9100 apparatus and are uncorrected. The elemental
analyses were performed on a Perkin Elmer CHNS/O Analyser Series
II 2400 apparatus. The NMR spectra were recorded on a Varian
Gemini 300BB instrument at room temperature, operating at
300 MHz for 1H and 75.075 MHz for 13C. The chemical shifts were
recorded as
d values in ppm units downfield to tetramethylsilane,
used as internal standard. The coupling constants values are repor-
ted in hertz and the splitting patterns are abbreviated as follows: s,
singlet; d, doublet; t, triplet; m, multiplet; b, broad. The IR spectra
were recorded on a JASCO FT/IR-4200 spectrometer with a diamond
crystal ATR PRO450-S accessory.
Table 2
The most sensitive cancer cells to 7c.
Cancer type
Leukemia
Most sensitive cells logGI50 logTGI
logLC50
4.1.1. General synthesis procedure for compounds 4aej
SR
ꢀ4.93
ꢀ4.39 >ꢀ4
A solution of 1-methyl-1H-pyrazole-4-carboxylic acid (0.1 mol)
in anhydrous 1,2-dichlorethane is refluxed for 3 h with thionyl
chloride (14.5 mL, 0.2 mol). The solvent and the excess thionyl
chloride are removed by reduced pressure distillation. The raw
obtained 1-methyl-1H-pyrazole-4-carbonyl chloride (10 mmol) is
dissolved in anhydrous acetone (30 mL), added to a solution of
ammonium thiocyanate (10 mmol) in dry acetone and refluxed for
1 h. The ammonium chloride is removed by filtration and the
suitable substituted aniline (10 mmol) dissolved in anhydrous
acetone is added while stirring. The mixture is heated under reflux
for 1 h and then poured into ten times its volume of cold water
when the N-(1-methyl-1H-pyrazole-4-carbonyl)-N0-(aryl)-thio-
ureas (4aej) precipitated as solids, or slowly crystallizing oils. The
compounds were recrystallized from isopropanol. The character-
izations of 4aeb and 4g are presented in previous papers.
Non-Small cell lung cancer A549/ATCC
ꢀ5.63
ꢀ5.35
ꢀ5.03
ꢀ4.85
ꢀ4.1
NCI-H322M
NCI-H23
ꢀ4.35 >ꢀ4
ꢀ4.30 >ꢀ4
Colon cancer
CNS cancer
HCT-116
ꢀ5.75
ꢀ5.32
ꢀ4.61
>ꢀ4
SF-539
SNB-75
SF-295
ꢀ5.47
ꢀ5.21
ꢀ5.01
>ꢀ4
ꢀ4.57
ꢀ4.05
ꢀ4.44 >ꢀ4
Melanoma
LOX IMVI
ꢀ5.26
ꢀ5.18
>ꢀ4
>ꢀ4
MALME-3M
ꢀ4.04 >ꢀ4
Ovarian cancer
OVCAR-4
OVCAR-8
NCI/ADR-RES
OVCAR-3
SK-OV-3
ꢀ5.37
ꢀ5.33
ꢀ5.31
ꢀ5.30
ꢀ5.14
ꢀ4.62 >ꢀ4
ꢀ4.68
ꢀ4.16
ꢀ4.20 >ꢀ4
ꢀ4.69
ꢀ4.66
ꢀ4.15
ꢀ4.28
Renal cancer
RXF 393
786e0
UO-31
ꢀ5.50
ꢀ5.49
ꢀ5.39
ꢀ4.82
ꢀ4.93
ꢀ4.67
ꢀ4.32
ꢀ4.32
ꢀ4.07
4.1.1.1. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(2-methoxy-
phenyl)-thiourea (4c). Yield 65%, mp 180e181 ꢂC. IR (cmꢀ1): 1659
(C¼O). 1H NMR (DMSO-d6, ppm): 12.30 (s, 1H, eNHeCSe), 11.28 (s,
1H, NH), 8.60 (s, 1H, eNeN¼CHe), 8.53 (dd, J ¼ 8.1 Hz, J ¼ 1.5 Hz,
1H, ArH), 8.21 (s, 1H, ¼ NeNeCHe), 7.22 (td, J ¼ 8.1 Hz, J ¼ 1.5 Hz,
Prostate cancer
Breast cancer
DU-145
ꢀ5.03
ꢀ5.56
>ꢀ4
>ꢀ4
HS 578T
ꢀ4.47 >ꢀ4
ꢀ4.65 >ꢀ4
MDA-MB-231/ATCC ꢀ5.42

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G.M. Nitulescu et al. / European Journal of Medicinal Chemistry 45 (2010) 4914e4919
1H, ArH), 7.12 (dd, J ¼ 8.1 Hz, J ¼ 1.5 Hz,1H, ArH), 6.98 (td, J ¼ 8.1 Hz,
J ¼ 1.5 Hz, 1H, ArH), 3.89 (s, 3H, CH3), 3.86 (s, 3H, OCH3). 13C NMR
(DMSO-d6, ppm): 178.32, 163.07, 150.73, 140.47, 134.67, 126.96,
126.65, 123.43, 119.80, 115.52, 111.36, 56.06, 39.11. Calcd. for
C13H14N4O2S: C, 53.78; H, 4.86; N,19.30; S,11.04. Found: C, 53.44; H,
4.99; N, 19.72; S, 11.06%.
1H, ¼ NeNeCHe), 7.56 (d, J ¼ 8.1 Hz, 2H, ArH), 7.39 (t, J ¼ 8.1 Hz,1H,
ArH), 3.90 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 180.88, 163.47,
141.05, 135.33, 134.83, 134.39, 130.40, 129.13, 115.99, 39.70. Calcd.
for C12H10Cl2N4OS: C, 43.78; H, 3.06; N, 17.02; S, 9.74; Found: C,
43.84; H, 2.97; N, 16.92; S, 9.88%.
4.1.2. Synthesis procedure for compound 5
4.1.1.2. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(3-methoxy-
phenyl)-thiourea (4d). Yield 67%, mp 164e165 ꢂC. IR (cmꢀ1): 1665
(C¼O). 1H NMR (DMSO-d6, ppm): 12.50 (s, 1H, eNHeCSe), 11.37 (s,
1H, NH), 8.60 (s,1H, eNeN¼CHe), 8.21 (s,1H, ¼ NeNeCHe), 7.43 (t,
J ¼ 2.1 Hz, 1H, ArH), 7.31 (t, J ¼ 8.1 Hz, 1H, ArH), 7.18 (dd, J ¼ 8.1 Hz,
J ¼ 2.1 Hz, 1H, ArH), 6.83 (ddd, J ¼ 8.1 Hz, J ¼ 2.1 Hz, J ¼ 0.8 Hz, 1H,
ArH), 3.89 (s, 3H, CH3), 3.76 (s, 3H, OCH3).13C NMR (DMSO-d6, ppm):
179.69, 163.74, 159.94, 141.01, 139.66, 135.21, 130.10, 116.88, 116.13,
112.41,110.32, 55.85, 39.68. Calcd. for C13H14N4O2S: C, 53.78; H, 4.86;
N, 19.30; S, 11.04. Found: C, 53.52; H, 5.04; N, 19.65; S, 10.94%.
The 1-methyl-1H-pyrazole-4-carbonyl chloride (10 mmol) is dis-
solved in anhydrous acetonitrile (30 mL), added to a solution of
ammonium thiocyanate (0.76 g, 10 mmol) and refluxed for 1 h. The
ammonium chloride is removed by filtration and an acetonitrile
solution of phenylhydrazine (10 mmol) is added. The mixture is
heated under reflux for 1 h and then poured into ten times its volume
of cold water. The compound was recrystallized from isopropanol.
4.1.2.1. 5-(1-Methyl-1H-pyrazol-4-yl)-1-phenyl-1,2-dihydro-1,2,4-
triazole-3-thione (5). Yield 65%, mp 228e230 ꢂC 1H NMR (DMSO-
d6, ppm): 8.32 (s, 1H, eNeN¼CHe), 8.00 (s, 1H, ¼ NeNeCHe), 7.99
(bd, 2H, ArH), 7.53 (tt, J ¼ 7.4 Hz, J ¼ 1.5 Hz, 2H, ArH), 7.41 (tt,
J ¼ 7.4 Hz, J ¼ 1.5 Hz, 1H, ArH), 3.93 (s, 3H, CH3). 13C NMR (DMSO-d6,
ppm): 165.08, 144.42, 137.75, 137.32, 130.42, 128.70, 127.71, 123.79,
107.24, 39.01. Calcd. for C12H11N5S: C, 56.01; H, 4.31; N, 27.22; S,
12.46; Found: C, 55.88; H, 4.36; N, 27.03; S, 12.55%.
4.1.1.3. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(4-methoxy-
phenyl)-thiourea (4e). Yield 73%, mp 192e193 ꢂC. IR (cmꢀ1): 1666
(C¼O). 1H NMR (DMSO-d6, ppm): 12.50 (s, 1H, eNHeCSe), 11.27 (s,
1H, eNHeCOe), 8.58 (s, 1H, -NeN¼CH-), 8.20 (s, 1H, ¼ NeNeCHe),
7.52 (d, J ¼ 9.1 Hz, 2H, ArH), 6.95 (d, J ¼ 9.1 Hz, 2H, ArH), 3.89 (s, 3H,
CH3), 3.76 (s, 3H, OCH3). 13C NMR (DMSO-d6, ppm): 180.06, 163.68,
158.02, 140.96, 135.15, 131.46, 126.61, 115.48, 114.40, 55.91, 39.66.
Calcd. for C13H14N4O2S: C, 53.78; H, 4.86; N, 19.30; S, 11.04; Found:
C, 53.44; H, 4.87; N, 19.58; S, 10.96%.
4.1.3. General synthesis procedure for compounds 6aeb
To
a
solution of N-(1-methyl-1H-pyrazole-4-carbonyl)-N0-
(aryl)-thioureas (5 mmol) and triethylamine (15 mmol) in acetone
we added, while stirring, a solution of bromoacetone, prepared in
situ, in the dropping funnel, from bromine (5 mmol) and acetone.
The reaction mixture is stirred for 2 h, the solvent is removed by
distillation and the solid is washed with water. The compounds
were recrystallized from ethyl acetate.
4.1.1.4. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(3-chloro-
phenyl)-thiourea (4f). Yield 72%, mp 168e169 ꢂC. IR (cmꢀ1): 1670
(C¼O).1H NMR (DMSO-d6, ppm): 12.50 (bs,1H, eNHeCSe),11.20 (bs,
1H, eNHeCOe), 8.59 (s,1H, eNeN¼CHe), 8.20 (s,1H, ¼ NeNeCHe),
7.93 (t, J ¼ 2.0 Hz,1H, ArH), 7.53 (ddd, J ¼ 7.9 Hz, J ¼ 2.0 Hz, J ¼ 1.3 Hz,
1H, ArH), 7.43 (t, J ¼ 7.9 Hz, 1H, ArH), 7.32 (ddd, J ¼ 7.9 Hz, J ¼ 2.0 Hz,
J ¼ 1.3 Hz, 1H, ArH), 3.89 Hz (s, 3H, CH3). 13C NMR (DMSO-d6, ppm):
180.13, 163.56, 141.02, 139.99, 135.26, 133.30, 130.91, 126.68, 124.64,
123.72, 116.07, 39.68. Calcd. for C12H11ClN4OS: C, 48.90; H, 3.76; N,
19.01; S, 10.88; Found: C, 48.77; H, 3.85; N, 18.89; S, 10.95%.
4.1.3.1. 1-Methyl-N-(4-methyl-3-phenylthiazol-2(3H)-ylidene)-1H-
pyrazole-4-carboxamide (6a). Yield 63%, mp 167e168 ꢂC. IR (cmꢀ1):
1616 (C¼O). 1H NMR (DMSO-d6, ppm): 7.78 (s, 1H, eNeN¼CHe),
7.64e7.56 (m, 3H, ArH), 7.48 (s, 1H, ¼ NeNeCHe), 7.45e7.42 (m,
2H, ArH), 6.75 (s, 1H, ¼ CH), 3.78 (s, 3H, CH3), 1.98 (s, 3H, CH3). 13
C
NMR (DMSO-d6, ppm): 169.31, 168.45, 139.92, 137.14, 134.29,
132.78, 129.42, 128.31, 122.16, 117.95, 104.18, 38.75, 14.61. Calcd. for
C15H14N4OS: C, 60.38; H, 4.73; N, 18.78; S, 10.75; Found: C, 60.49; H,
4.69; N, 18.83; S, 10.66%.
4.1.1.5. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(2,4-dichloro-
phenyl)-thiourea (4h). Yield 72%, mp 189e190 ꢂC. IR (cmꢀ1): 1668
(C¼O). 1H NMR (DMSO-d6, ppm): 12.50 (bs, 1H, eNHeCSe), 11.20
(bs, 1H, eNHeCOe), 8.60 (s, 1H, eNeN¼CHe), 8.22 (s,
1H, ¼ NeNeCHe), 8.05 (d, J ¼ 8.8 Hz, 1H, ArH), 7.74 (d, J ¼ 2.4 Hz,
1H, ArH), 7.48 (dd, J ¼ 8.8 Hz, J ¼ 2.4 Hz, 1H, ArH), 3.89 (s, 3H, CH3).
13C NMR (DMSO-d6, ppm): 181.09, 163.74, 141.11, 135.38, 135.24,
131.96, 130.24, 129.83, 129.55, 127.99, 115.89, 39.70. Calcd. for
C12H10Cl2N4OS: C, 43.78; H, 3.06; N, 17.02; S, 9.74; Found: C, 43.87;
H, 3.21; N, 17.11; S, 9.61%.
4.1.3.2. N-(3-(4-chlorophenyl)-4-methylthiazol-2(3H)-ylidene)-1-
methyl-1H-pyrazole-4-carboxamide (6b). Yield 65%, mp 238 ꢂC. IR
(cmꢀ1): 1587 (C¼O). 1H NMR (DMSO-d6, ppm): 7.79 (s, 1H,
eNeN¼CHe), 7.56 (d,
J
¼
8.6 Hz, 2H, ArH), 7.49 (s,
1H, ¼ NeNeCHe), 7.37 (d, J ¼ 8.6 Hz, 2H), 6.81 (s, 1H, ¼ CH), 3.79 (s,
3H, CH3), 1.38 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 171.18,170.26,
140.19, 137.06, 133.29, 132.21, 130.99, 128.61, 121.75, 119.07, 90.61,
38.68, 25.98. Calcd. for C15H13ClN4OS: C, 54.13; H, 3.94; N, 16.83; S,
9.63; Found: C, 54.22; H, 4.08; N, 16.77; S, 9.60%.
4.1.1.6. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(2,5-dichloro-
phenyl)-thiourea (4i). Yield 70%, mp 191e193 ꢂC. IR (cmꢀ1): 1672
(C¼O). 1H NMR (DMSO-d6, ppm): 12.50 (bs, 1H, eNHeCSe), 11.20
(bs, 1H, eNHeCOe), 8.59 (s, 1H, eNeN¼CHe), 8.28 (d, J ¼ 2.6 Hz,
1H, ArH), 8.23 (s, 1H, ¼ NeNeCHe), 7.62 (d, J ¼ 8.5 Hz, 1H, ArH),
7.39 (dd, J ¼ 8.5 Hz, J ¼ 2.6 Hz, 1H, ArH), 3.90 (s, 3H, CH3). 13C NMR
(DMSO-d6, ppm): 180.91, 163.76, 141.14, 137.21, 135.43, 131.74,
131.42, 128.28, 127.74, 127.43, 115.86, 39.71. Calcd. for
C12H10Cl2N4OS: C, 43.78; H, 3.06; N, 17.02; S, 9.74; Found: C, 43.69;
H, 2.97; N, 17.30; S, 9.81%.
4.1.4. General synthesis procedure for compounds 7aec
A solution of benzoyl chloride (10 mmol) in anhydrous acetone
(30 mL) is added to a solution of ammonium thiocyanate (10 mmol)
and refluxed for 1 h. The ammonium chloride is removed by
filtration and the suitable pyrazole amine (10 mmol) is added while
stirring. The mixture is heated under reflux for 1 h and then poured
into ten times its volume of cold water. The compounds were
recrystallized from isopropanol.
4.1.1.7. N-[(1-methyl-1H-pyrazole-4-yl)carbonyl]-N0-(2,6-dichloro-
phenyl)-thiourea (4j). Yield 66%, mp 186e187 ꢂC. IR (cmꢀ1): 1666
(C¼O). 1H NMR (DMSO-d6, ppm): 12.21 (bs, 1H, eNHeCSe), 11.60
(bs, 1H, eNHeCOe), 8.59 (s, 1H, eNeN¼CHe), 8.21 (s,
4.1.4.1. N-Benzoyl-N0-(5-methyl-1H-pyrazol-3-yl)thiourea
(7a). Yield 79%, mp 207e209 ꢂC. IR (cmꢀ1): 1662 (C¼O). 1H NMR
(DMSO-d6, ppm): 13.10 (s, 1H, ¼ NeNHe), 12.47 (s, 1H, eCSeNHe),

G.M. Nitulescu et al. / European Journal of Medicinal Chemistry 45 (2010) 4914e4919
4919
11.56 (s, 1H, eCOeNHe), 7.97 (d, J ¼ 7.4 Hz, 2H, ArH), 7.66 (t,
J ¼ 7.4 Hz, 1H, ArH), 7.53 (t, J ¼ 7.4 Hz, 2H, ArH), 6.87 (s, 1H, PyrzH),
2.25 (s, 3H, CH3). 13C NMR (DMSO-d6, ppm): 176.51, 168.68, 146.84,
138.36, 133.17, 132.07, 128.72, 128.43, 97.37, 10.71. Calcd. for
C12H12N4OS: C, 55.37; H, 4.65; N, 21.52; S, 12.32; Found: C, 55.44; H,
4.87; N, 21.40; S, 12.21%.
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 is calculated from
[(Ti ꢀ Tz)/Tz] ꢃ 100 ¼ ꢀ50. Values are calculated for each of these
three parameters if the level of activity is reached; however, if the
effect is not reached or is exceeded, the value for that parameter is
expressed as greater or less than the maximum or minimum
concentration tested.
4.1.4.2. N-Benzoyl-N0-(3-methyl-1-phenyl-1H-pyrazol-5-yl)-thiourea
(7b). Yield 76%, mp 166e168 ꢂC. IR (cmꢀ1): 1672 (C¼O). 1H NMR
(DMSO-d6, ppm): 12.46 (s, 1H, eCSeNHe), 11.80 (s, 1H, eCOeNHe),
7.91 (d, J ¼ 7.4 Hz, 2H, ArH), 7.65 (tt, J ¼ 7.4 Hz, J ¼ 2.2 Hz, 1H, ArH),
7.55 (tt, J ¼ 7.2 Hz, J ¼ 2.2 Hz, 2H, ArH), 7.51 (dt, J ¼ 7.1 Hz, J ¼ 2.3 Hz,
2H, ArH), 7.51 (tt, J ¼ 7.1 Hz, J ¼ 2.3 Hz, 2H, ArH), 7.38 (tt, J ¼ 7.1 Hz,
J ¼ 2.3 Hz, 1H, ArH), 6.58 (s, 1H, PyrzH), 2.26 (s, 3H, CH3). 13C NMR
(DMSO-d6, ppm): 180.24, 168.02, 147.82, 138.23, 136.74, 133.33,
131.72, 129.20, 128.78, 128.46, 127.58, 123.84, 102.87, 13.87. Calcd.
for C18H16N4OS: C, 64.27; H, 4.79; N, 16.65; S, 9.53; Found: C, 64.21;
H, 4.81; N, 16.77; S, 9.51%.
Acknowledgements
The authors wish to espress their gratitude to the members of
the Developmental Therapeutics Program at National Cancer
Institute (http://dtp.cancer.gov/) for the in vitro anticancer
evaluation.
References
4.1.4.3. N-Benzoyl-N0-(3-(4-bromophenyl)-1H-pyrazol-5-yl)-thio-
urea (7c). Yield 73%, mp 223e224 ꢂC. IR (cmꢀ1): 1665 (C¼O). 1H
NMR (DMSO-d6, ppm): 13.37 (s, 1H, NH), 13.17 (s, 1H, eCSeNHe),
11.65 (s, 1H, eCOeNHe), 7.99 (dd, J ¼ 7.2 Hz, J ¼ 1.5 Hz, 2H, ArH),
7.73e7.67 (m, 4H, ArH), 7.67 (tt, J ¼ 7.3 Hz, J ¼ 1.4 Hz, 1H, ArH), 7.54
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13.96; S, 7.99; Found: C, 51.03; H, 3.28; N, 13.88; S, 8.11%.
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[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢃ 100 for concentrations for which Ti ꢄ Tz.
[(Ti ꢀ Tz)/Tz] ꢃ 100 for concentrations for which Ti < Tz.
Three dose response parameters are calculated for each exper-
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[(Ti ꢀ Tz)/(C ꢀ Tz)] ꢃ 100 ¼ 50, which is the drug concentration
resulting in a 50% reduction in the net protein increase (as
measured by sulforhodamine B staining) in control cells during the
drug incubation. The drug concentration resulting in total growth
inhibition (TGI) is calculated from Ti ¼ Tz. The LC50 (concentration