Synthesis and structure-activity relationship studies of pyrazole-based heterocycles as antitumor agents

Article abstract of DOI:10.1002/ardp.200900176

Several 4-cyano-1,5-diphenylpyrazoles attached to different heterocyclic ring systems at position 3 were synthesized starting from ethyl 4-cyano-1,5-diphenyl-1H-pyrazole-3-carboxylate 1. The newly synthesized compounds were tested in vivo for their anti-estrogenic effects and evaluated in vitro for their cytotoxic properties against estrogen-dependent tumors. 3-(5-Mercapto-1,3,4-oxadiazole-2-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 13 revealed the highest cytotoxic activity with a GI₅₀ value equal to 40 nM against the IGROVI ovarian tumor cell line. It also showed an anti-estrogen activity 1.6 more effective than the reference drug, in addition to a high tolerable dose. 3-(5-(Methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-diphenyl- 1H-pyrazole-4-carbonitrile 7 was found to have the highest anti-estrogenic activity, while 1,5-diphenyl-3-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]-1H- pyrazole-4-carbonitrile 11 showed the lowest activity. The oral LD₅₀ values revealed that most of the tested compounds are relatively nontoxic.

Full text of DOI:10.1002/ardp.200900176

384
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Full Paper
Synthesis and Structure-activity Relationship Studies of
Pyrazole-based Heterocycles as Antitumor Agents
Ahmad M. Farag1, Abdelrahman S. Mayhoub2, Taha M. A. Eldebss1, Abdel-Galil E. Amr3,
Korany A. K. Ali3, Naglaa A. Abdel-Hafez3, and Mohamed M. Abdulla4
1 Department of Chemistry, Faculty of Science, Cairo University, Giza, Egypt
2 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Al-Azhar University, Cairo, Egypt
3 Department of Applied Chemistry, National Research Center, Dokki, Giza, Egypt
4 Research Units, Hi-Care Pharmaceutical Co., Cairo, Egypt
Several 4-cyano-1,5-diphenylpyrazoles attached to different heterocyclic ring systems at position
3 were synthesized starting from ethyl 4-cyano-1,5-diphenyl-1H-pyrazole-3-carboxylate 1. The
newly synthesized compounds were tested in vivo for their anti-estrogenic effects and evaluated
in vitro for their cytotoxic properties against estrogen-dependent tumors. 3-(5-Mercapto-1,3,4-oxa-
diazole-2-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 13 revealed the highest cytotoxic activity
with a GI50 value equal to 40 nM against the IGROVI ovarian tumor cell line. It also showed an
anti-estrogen activity 1.6 more effective than the reference drug, in addition to a high tolerable
dose. 3-(5-(Methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 7 was
found to have the highest anti-estrogenic activity, while 1,5-diphenyl-3-[5-(phenylamino)-1,3,4-
thiadiazol-2-yl]-1H-pyrazole-4-carbonitrile 11 showed the lowest activity. The oral LD50 values
revealed that most of the tested compounds are relatively nontoxic.
Keywords: Anti-estrogenic activity / 1,5-Diphenylpyrazoles / 1,3,4-Oxadiazoles / 1,3,4-Thiadiazole / 1,2,4-Triazoles /
Received: July 24, 2009; Accepted: December 31, 2009
DOI 10.1002/ardp.200900176
of pelvic pain scores in women with refractory endome-
Introduction
triosis [4]. This class includes nonsteroidal derivatives
which have both estrogenic and anti-estrogenic proper-
ties such as clomifene [5, 6], cyclofenil, and the more
selective nonsteroidal anti-estrogens ormeloxifene [7]
and raloxifene [8, 9].
Estrogens control the development and maintenance of
the female sex organs, secondary sex characteristics, and
mammary glands, as well as certain functions of the ute-
rus and its accessory organs. They are also formed in the
placenta in late pregnancy, which increases the sponta-
neous activity of the uterine muscle and its response to
oxytocic drugs. Estradiol is the most active of the estro-
gens formed from androgen precursors in the ovarian fol-
licles of premenopausal women. In men and postmeno-
pausal women, estrogens are also formed in adipose tis-
sue from adrenal androgens.
Since some types of breast cancer and other cancers
were found to be estrogen-dependent, in which binding
of estrogen with its receptors activates transcription of
its target genes, the latter are responsible for cancer cell
proliferation in estrogen-dependent breast tumor [10].
Therefore, treatment of these conditions focuses on
decreasing estrogen, either by oophorectomy or by hor-
monal therapy [11–13]. The anti-estrogens, used in the
hormonal treatment of breast cancer, include the estro-
gen receptor antagonists tamoxifen [14–16], toremifene
[17, 18], and various aromatase cytochrome P450 (CYP19)
inhibitors, which catalyze the conversion of androstene-
dione and testostron to estradiol [19, 20], such as formes-
tane [21], anastrozole [22, 23], vorozole [24, 25], fadrozole
[26], testolactone [27], and letrozole [28, 29].
The class of anti-estrogens is used therapeutically in
several diseases such as malignant neoplasms of the
breast [1], gynaecomastia [2], mastalgia [3], and reduction
Correspondence: Prof. Ahmad M. Farag, Department of Chemistry,
Faculty of Science, Cairo University, Giza 12613, Egypt.
E-mail: afarag49@yahoo.com
Fax: +20 235 727-556
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
385
Figure 1. a) Celecoxib; b–d) general formulae of the synthesized
compounds.
The importance of 1,5-diphenylpyrazoles as anti-estro-
gens, recently became important after discovering the
effect of cyclooxygenase-2 (COX-2) inhibitors such as cele-
coxib (Fig. 1a) and meloxicam [30] on many types of
malignant tumors, especially breast cancer [31–37]. It is
proved that 1,5-diphenylpyrazoles exert this action via
inhibition of the aromatase enzyme [38].
In continuation of our recent work aiming at the syn-
thesis of heterocyclic systems with remarkable biological
importance [39–52], we report here on the synthesis of
some new 1,5-diphenylpyrazole derivatives with differ-
ent substituted triazole and triazole bioisosteres at posi-
tion 3 (Fig. 1b) as an important counterpart to fit with
the required enzyme (CYP19) [28]. The present study also
involves the anti-estrogenic effects of the synthesized
compounds in vivo and evaluating their cytotoxic proper-
ties in vitro against different breast and ovarian tumor
cell lines.
The structure-activity relationship (SAR) study of this
new promising group was carried out in order to get new
agents that could be optimized as potent antitumor
drugs. In a second line of chemical optimization, several
1,5-diphenylpyrazole derivatives were synthesized with
different fused triazole ring systems at position 3 (Fig.
1c). To achieve this goal, we utilized several intermedi-
ates such as carboxylic acid hydrazide 2 and thiosemi-
carbazide derivatives 3 and 5. Since the acid hydrazide 2
showed a promising anti-estrogenic activity as well as a
significant cytotoxic activity against certain types of
breast tumor cell lines, we decided to optimize its struc-
ture (Fig. 1d) in order to increase its selectivity towards
ovarian tumors.
Scheme 1. Synthetic pathway for the formation of compounds 2,
3, and 4.
[53] is an excellent building block for the synthesis of sev-
eral heterocyclic ring systems. Thus, when the latter com-
pound was treated with hydrazine hydrate in ethanol, it
afforded the corresponding 4-cyano-1,5-diphenyl-1H-pyr-
azole-3-carboxylic acid hydrazide 2 in high yield (Scheme
1). The structure of product 2 was established on the basis
of its elemental analysis and spectral data. For example,
its IR spectrum revealed absorption bands at 3309, 3160,
3111, 2237, and 1674 cm–1 corresponding to NH, NH2,
1
nitrile, and amide carbonyl groups, respectively. Its H-
NMR spectrum showed broad D2O-exchangeble signals at
d = 2.78 and 8.50 ppm corresponding to NH2 and NH pro-
tons, respectively, in addition to a multiplet at d = 7.21–
7.43 characteristic for aromatic protons. The mass spec-
trum of the same product showed a peak at m/z: 303 cor-
responding to its molecular ion.
Treatment of the acid hydrazide 2 with potassium thio-
cyanate and HCl, under reflux condition, afforded 1-(4-
cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)thiosemicarb-
azide 3. The 1H-NMR spectrum of the latter product dis-
played broad D2O-exchangeable signals at d = 11.30, 7.85,
and 4.34 ppm characteristic for two NH and NH2 protons,
respectively. Its mass spectrum showed a peak at m/z: 362
corresponding to its molecular ion. The structure of com-
pound 3 was further confirmed by its alternate synthesis
from the reaction of pyrazole-3-carboxylic acid ethyl
ester 1 with thiosemicarbazide in refluxing dioxan
(Scheme 1). When the pyrazole thiosemicarbazide 3 was
treated with potassium hydroxide in refluxing ethanol,
it afforded 3-(5-mercapto-4H-1,2,4-triazol-3-yl)-1,5-di-
All of the synthesized compounds were designed to
contain a nitrile group as it is common in different aro-
matase inhibitors such as anastrozole [22], fadrozole [24],
and letrozole [28].
Results and discussion
Chemistry
In the course of our investigation, we have found that
ethyl 4-cyano-1,5-diphenyl-1H-pyrazole-3-carboxylate
1
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A. M. Farag et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Scheme 2. Synthetic pathway for the formation of compounds 5, 6, 7, 8, 9, and 10.
1
phenyl-1H-pyrazole-4-carbonitrile 4 (Scheme 1). The H- characteristic for fifteen aromatic protons. The mass
NMR spectrum of the latter product revealed two D2O- spectrum of the same product showed a peak correspond-
exchangeable signals at d = 10.5 and 14.1 ppm character- ing to its molecular ion peak at m/z: 420 (see Experimen-
istic for NH and SH protons, respectively. Its mass spec- tal, section 4).
trum showed a peak at m/z: 344 corresponding to its
Treatment of compound 6 with an ethanolic solution of
molecular ion.
sodium ethoxide followed by the addition of an equimo-
Treatment of the acid hydrazide 2 with phenyl isothio- lar amount of methyl iodide, afforded a single product
cynate, in ethanol under reflux, afforded a single product identified as 3-(5-(methylthio)-4-phenyl-4H-1,2,4-triazol-3-
identified as 1-(4-cyano-1,5-diphenyl-1H-pyrazole-3-car- yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 7 (Scheme 2).
1
bonyl)-4-phenylthiosemicarbazide 5 (Scheme 2). The H- The1H-NMR spectrum of the latter product displayed a sin-
NMR spectrum of the product 5 displayed three D2O- glet signal at d = 3.45 ppm characteristic for S-CH3 protons.
exchangeble signals at d = 7.93, 8.74, and 11.34 ppm char- Its mass spectrum showed a peak corresponding to its
acteristic for three NH protons. Its mass spectrum molecular ion at m/z: 434. Treatment of a suspension of
showed a peak corresponding to its molecular ion at m/z: compound 7 with hydrazine hydrate under reflux condi-
438.
tion afforded 3-(5-hydrazino-4-phenyl-4H-1,2,4-triazol-3-
Heating of the thiosemicarbazide derivative 5 in potas- yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 8. The mass
sium hydroxide solution afforded a product identified as spectrum of the latter product revealed a peak corre-
3-(5-mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-diphenyl-
1H-pyrazole-4-carbonitrile 6 (Scheme 2). The IR spectrum
sponding to its molecular ion at m/z: 418.
The structure of compound 8 was further confirmed
of the latter product showed two characteristic bands at by its alternate synthesis from the reaction of 6 with
3265 and 2228 cm–1 corresponding to a SH group and a hydrazine hydrate in refluxing ethanol (Scheme 2).
1
nitrile function, respectively. Its H-NMR spectrum dis-
Treatment of compound 5 with phenacyl bromide
played broad D2O-exchangeable signals at d = 11.2 and derivatives 9a–c, in the presence of a catalytic amount of
14.16 ppm corresponding to NH and SH protons, respec- triethylamine, afford the corresponding 4-cyano-1,5-di-
tively, in addition to a multiplet at d = 6.84–7.47 ppm phenyl-1H-pyrazole-[4-aryl-3-phenyl-3H-thiazol-2-ylidene]-
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
387
Scheme 4. Synthetic pathway for the formation of compounds
17 and 19.
salt 12 (Scheme 3). Heating of the potassium salt inter-
mediate 12 in potassium hydroxide solution, afforded 3-
(5-mercapto-1,3,4-oxadiazole-2-yl)-1,5-diphenyl-1H-pyra-
zole-4-carbonitrile 13 (Scheme 3).
Treatment of the potassium salt 12 with hydrazine
hydrate under reflux condition, afforded a single prod-
uct identified as 3-(5-mercapto-4-amino-4H-1,2,4-triazole-
3-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitile 14 (Scheme
3). The IR spectrum of the latter product revealed absorp-
tion bands at 3294, 3120, and 2585 cm–1 characteristic
Scheme 3. Synthetic pathway for the formation of compounds
12, 13, 14, 15, and 16.
3-carboxylic acid hydrazide derivatives 10a–c (Scheme 2).
The IR spectra of the isolated products 10a–c showed, in
each case, one carbonyl absorption band in the region
1
for NH2 and SH groups, respectively. The H-NMR spec-
1
1680 to 1690 cm–1. The H-NMR spectrum of compound
trum of the same product displayed signals at d = 5.56
and 14.1 ppm (D2O-exchangable) characteristic for NH2
and SH protons, respectively. Its mass spectrum revealed
a peak at m/z: 359 corresponding to its molecular. The
same product was also obtained from the reaction of oxa-
diazole derivative 13 with hydrazine hydrate (Scheme 3;
see Experimental, section 4).
The pyrazole carboxylic acid hydrazide 2 reacts with
acetylacetone or with ethyl acetoacetate, in ethanol solu-
tion under reflux condition, to afford products 15 and
16, respectively (Scheme 4).
The 1H-NMR spectrum of compound 15 reveled two-sin-
glet signal at d = 1.95 and 2.06 ppm corresponding to two
methyl groups and a singlet signal at 5.32 ppm corre-
sponding to pyrazole-4H, whereas its mass spectrum
showed a peak at m/z: 367 corresponding to its molecular
ion. The IR spectrum of compound 16 exhibited two
bands at 1665 and 1651 cm–1 characteristic for two car-
bonyl groups.
Treatment of the mecaptotriazole 14 with phenacyl
bromide derivatives 9a, c in ethanol, in the presence of a
catalytic amount of triethylamine, at reflux tempera-
ture, afford the corresponding 3-(4-cyano-1,5-diphenyl-
1H-pyrazole-3-yl)-6-aryl-7H-1,2,4-triazolo[3,4-b]-1,3,4-thia-
diazine derivatives 17a, b (Scheme 4). The IR spectrum of
compound 17b, taken as a typical example, revealed a
10c, taken as a typical example of the prepared series, dis-
played signals at d = 3.68, 7.74, 6.51 ppm characteristic for
OCH3, NH, and CH of thiazole protons, respectively, in
addition to a multiplet at d = 7.15–7.37 ppm characteristic
for aromatic protons. Its mass spectrum showed a peak
corresponding to its molecular ion at m/z: 568 (see Experi-
mental, section 4).
Similarly, the thiosemicarbazid derivative 5 reacts with
chloroacetone 9d in the presence of a catalytic amount of
triethylamine to afford the corresponding 4-cyano-1,5-
diphenyl-1H-pyrazole-[4-methyl-3-phenyl-3H-thiazol-2-yli-
dene]-3-carboxylic acidhydrazide 10d (Scheme2).
When the thiosemicarbazide 5 was treated with sul-
phuric acid, it afforded 1,5-diphenyl-3-(5-(phenylamino)-
1,3,4-thiadiazol-2-yl)-1H-pyrazole-4-carbonitrile
11
(Scheme 2). The mass spectrum of the latter product
revealed a peak at m/z: 420 corresponding to its molecu-
lar ion. Its IR spectrum revealed an absorption band at
3190 cm–1 due to the NH group. The 1H-NMR spectrum of
the same product displayed a broad D2O-exchangable sin-
glet signal at d = 7.75 ppm characteristic for the NH pro-
ton.
When the acid hydrazide 2 was treated with carbon
disulphide and potassium hydroxide in ethanol at room
temperature, it afforded the corresponding potassium
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388
A. M. Farag et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
band at 2230 cm–1 characteristic for a nitrile function. Its
1H-NMR spectrum displayed a singlet signal at d = 3.86
ppm corresponding to OCH3 protons and a singlet signal
at d = 4.71 ppm corresponding to the CH2 protons of thia-
diazine, in addition to a multiplet at d = 7.15–7.37 ppm
due to aromatic protons.
The mecaptotriazole 14 reacts also with benzoic acid
and with phenylacetic acid, in the presence of POCl3, to
afford 6-phenyl-3-(4-cyano-1,5-diphenyl–1H-pyrazole-3-yl)-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole 19a and 6-benzyle-3-
(4-cyano-1,5-diphenyl-1H-pyrazole-3-yl)-1,2,4-triazolo-
[3,4-b]-1,3,4-thiadiazole 19b, respectively (Scheme 4). The
IR spectrum of compound 19b, taken as a typical example,
revealed an absorption band at 2237 cm–1 corresponding
to a nitrile function. Its 1H-NMR spectrum displayed a sin-
glet signal at d = 3.96 ppm corresponding to CH2 protons,
in addition to an aromatic multiplet at d = 7.04–7.77 ppm,
whereasitsmassspectrumshowedapeakatm/z:459corre-
spondingtoitsmolecularion.
Table 1. The mean anti-estrogenic activity and acute toxicity
(LD50) of the synthesized compounds and letrozole.
Compound
% Reduction
in uterine
weight*
Relative
potency to
Letrozole
LD50 (mg/kg)
Letrozole
2
4
5
6
7
8
10d
11
12
13
14
15
16
21.65
43.43
36.34
16.92
19.65
43.98
23.87
32.43
13.98
43.54
34.75
23.76
32.45
43.46
1.00
2.01
1.67
0.78
0.90
2.03
1.10
1.49
0.64
2.01
1.60
1.09
1.49
2.00
252.61 € 0.11
332.11 € 0.13
434.71 € 0.13
311.52 € 0.12
122.48 € 0.13
131.85 € 0.13
102.61 € 0.11
122.54 € 0.16
232.54 € 0.15
431.85 € 0.13
323.06 € 0.11
121.4 € 0.011
873.06 € 0.19
234.71 € 0.13
* Value represents mean of twelve rats.
Pharmacology and toxicology
Anti-estrogenic activity
The anti-estrogenic activity of the newly synthesized
compounds as well as letrozole (Femaram), as a reference
drug, were evaluated in the Pharmacology and Toxicol-
ogy Department, Research Units, Hi-Care Pharmaceutical
Co., Cairo, Egypt.
The method used is based on the increase of the ute-
rine weight in castrated female rats, induced by repeated
administration of estradiol as antagonized by anti-estro-
genic compounds. The test compounds (more than 99%
purity) in carboxymethylcellulose (CMC) were adminis-
tered orally by gavage to groups of immature ovarectom-
ized rats. On the 8th day, the animals were sacrificed and
the uterine weights were determined.
Figure 2. Anti-estrogenic potency of the test compounds relative
to letrozole.
The mean value of percent reduction in uterine weight
was calculated with regard to the control group, which
was treated with estradiol alone, and the relative potency
to the reference drug was calculated as depicted in
Table 1.
Figs. 2 and 3). Methyl substitution of the SH group of the
latter compound increases the activity by twofold com-
pared to the reference drug and decreases the safety of
the resultant molecule as shown in compound 7 (Table 1,
Figs. 2 and 3).
Acute toxicity (LD50)
Replacement of the mercapto moiety with a hydrazino
group showed a slight improvement in the anti-estro-
genic activity and decreased the safety to 0.4 of that of
letrozole as in case of compound 8 (Table 1, Figs. 2 and 3).
Substitution of N1 of the triazole moiety with a polar
group such as amino group, as in compound 14,
decreases the activity dramatically and increases the tox-
icity in comparison with the unsubstituted triazole deriv-
ative 4 (Table 1, Figs. 2 and 3).
The rats were dosed by oral gavage with different doses of
an aqueous suspensions of a very fine powder of the
tested compounds. Under these conditions, the test com-
pounds were of great safety for the albino rats compared
to letrozole as a reference drug, as illustrated in Table 1.
The anti-estrogenic activity of 5-mercaptotriazole 4
was found to be more active and safer than the reference
drug letrozole. Addition of a phenyl moiety to N1 of the
triazole ring decreases the anti-estrogenic activity and
increases the toxicity as shown in compound 6 (Table 1,
Replacement of the triazole ring with its bioisostere
oxadiazole has no effect for the pharmacological activity
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
389
Table 2. Growth inhibitory concentration (GI50, lM) of tested compounds on different breast cancer cell lines.
Cell lines
MDA
MCF-7
T-47D
NCI
HS-578T
MDA
MiDA-N
BT 549
MB-231
ATTC
ADR-RES
MB-435
2
4
5
6
7
8
10a
10b
10c
10d
11
12
13
14
15
16
17a
17b
19a
19b
61.2
6.27
I.A.§
I.A.§
I.A.§
I.A.§
7.36
28.0
I.A.§
I.A.§
5.84
4.90
38.4
I.A.§
6.21
23.8
13.2
5.49
I.A.§
52.1
85.8
I.A.§
99.8
I.A.§
68.0
I.A.§
4.72
40.8
I.A.§
79.8
8.53
I.A.§
38.0
23.4
I.A.§
19.8
19.8
I.A.§
I.A.§
41.6
N.T.#
6.68
45.6
I.A.§
36.3
I.A.§
62.5
22.2
I.A.§
26.5
18.0
63.4
72.0
39.9
2.45
54.8
9.08
I.A.§
I.A.§
42.6
N.T.#
2.48
I.A.§
I.A.§
I.A.§
I.A.§
7.4
N.T.#
I.A.
N.T.#
2.64
42.8
I.A.§
41.8
I.A.§
8.62
61.0
I.A.§
I.A.§
55.8
9.30
I.A.§
2.93
2.75
39.9
48.0
1.23
I.A.§
9.23
1.34
2.58
34.8
I.A.§
73.8
I.A.§
7.00
56.8
I.A.§
26.5
93.8
2.86
55.4
5.00
I.A.§
23.8
73.0
1.19
I.A.§
5.49
9.13
I.A.§
37.7
I.A.§
44.8
I.A.§
4.12
11.8
I.A.§
11.8
I.A.§
I.A.§
26.2
I.A.§
I.A.§
11.8
51.6
I.A.§
I.A.§
I.A.§
61.8
I.A.§
69.8
I.A.§
6.72
61.8
I.A.§
I.A.§
I.A.§
8.04
98.0
8.94
I.A.§
I.A.§
I.A.§
9.23
I.A.§
8.94
I.A.§
I.A.§
28.0
99.8
I.A.§
99.0
I.A.§
2.76
98.0
28.0
8.4
I.A.§
74.7
§ I.A.: inactive; GI50 value >100 lM; # N.T.: not tested.
mediates were also investigated for their anti-estrogenic
activity. Thus, it was found that both of the acid hydra-
zide 2 and the potassium salt of 12 are more active than
letrozole, while the phenylthiosemicarbazide 5 is less
active than the same drug (Table 1, Figs. 2 and 3).
In-vitro disease-oriented primary antitumor screening
All newly synthesized compounds were selected by the
National Cancer Institute (NCI), Bethesda, Maryland,
USA, for evaluation of their in-vitro antitumor activity
using 14 breast and ovarian cell lines.
Effect of the newly synthesized compounds on breast
cancer
Figure 3. LD50 of the tested compounds and letrozole.
We started the present study with the mercaptotriazole
derivative 4 which showed good activity against five
breast tumor cell lines, namely MDA/MB-231 ATTC, T-
47D, NCI/ADR-RES, MDA/MB-435, and MiDA-N (GI50 values
between 2.4 and 6.6 lM as depicted in Table 2). Increasing
the lipophilic character of such a compound by attaching
a phenyl moiety to the triazole ring renders the resultant
structure without any cytotoxic properties as in case of
compound 6 (Table 2). In contrast, replacement of the
phenyl moiety by a more hydrophilic group such as an
amino group showed dramatic improvement in the cyto-
toxic behavior as demonstrated in compound 14 which
revealed GI50 values against tumor cell lines MDA/MB-
and slightly decreases the toxicity of the resultant com-
pound as in the case of compound 13 (Table 1, Figs. 2 and
3). In contrast, the thiadiazole bioisostere 11 showed a
lower anti-estrogenic activity than both the reference
drug and compound 4 (Table 1, Figs. 2 and 3). Further-
more, the pyrazole derivative 15 was found to be 3.5
times less toxic than letrozole with an anti-estrogenic
activity 1.5 times that of the same drug. The pyrazolone
derivative 16 was found to be two times more potent
than letrozole with almost the same safety range (Table
1, Figs. 2 and 3). Moreover, some of the synthesized inter-
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A. M. Farag et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Table 3. Growth inhibitory concentration (GI50, lM) of the tested
compounds on different ovarian cancer cell lines.
435, MiDA-N, and HS-578T equal to 2.93, 5.00, and 8.94
lM, respectively (Table 2). On the other hand, methyla-
tion of the mercapto group of the triazole moiety
excerted only a little effect on the cytotoxic properties as
shown in compound 7 (Table 2). In addition, replacement
of the mercapto group with a hydrazino moiety renders
the molecule completely inactive against the tested
breast tumor cell lines as in case of compound 8 (Table 2).
In the second line of structural optimization, the tria-
zole ring was replaced with thiadiazole moiety which
improved the cytotoxic effect against MDA/MB-231-ATTC
and MCF-7 breast tumor cell lines (GI50 values are 5.84
and 8.53 ppm, respectively) as shown in compound 11
(Table 2). Whereas, the oxadiazole isostere was found to
be less effective as in the case of compound 13 (Table 2).
The pyrazole derivative 15 has significant cytotoxic
effects against T-47D, NCI/ADR-RES, MDA/MB-435, and
MDA/MB-231-ATTC breast cell lines (Table 2). Although
the pyrazolone derivative 16 revealed a good anti-estro-
genic activity in vivo, it has a moderate to weak cytotoxic
effect against breast tumor cell lines (Tables 1 and 2).
Since the carboxylic acid hydrazide derivative 2
showed a remarkable cytotoxic effect against two breast
tumor cell lines, we decided to modify its structure by
Cell lines
IGROVI OVCAR-3 OVCAR-5 OVCAR-8 OVCAR-4 SK-OV-3
2
4
5
6
7
8
92.8
53.4
91.5
I.A.§
I.A.§
I.A.§
I.A.§
I.A.§
93.8
63.8
8.94
5.19
13.3
94.5
7.66
91.9
I.A.§
N.T.#
I.A.§
I.A.§
99.8
58.9
93.0
I.A.§
93.8
I.A.§
31.8
3.94
I.A.§
31.8
9.23
I.A.§
7.66
94.4
5.12
98.0
61.8
N.T.#
20.3
61.8
I.A.§
94.0
I.A.a
7.94
I.A.§
8.04
71.8
9.94
I.A.§
I.A.§
9.23
7.66
9.12
I.A.§
91.0
I.A.§
81.8
N.T.#
I.A.§
61.8
61.8
I.A.§
61.8
3.23
61.8
9.33
33.8
12.8
38.4
5.89
5.49
7.66
7.26
32.4
60.1
38.4
73.8
N.T.#
23.0
63.8
18.4
94.0
63.8
3.49
7.66
4.49
26.8
56.8
36.8
I.A.§
I.A.§
9.72
9.22
10.8
36.4
36.8
26.8
N.T.#
I.A.§
36.8
23.8
I.A.§
36.8
I.A.§
9.72
I.A.§
10a 7.76
10b 80.0
10c 38.4
10d 81.0
11
12
13
14
15
16
91.8
I.A.§
0.040
93.8
95.8
I.A.§
17a 98.0
17b 9.24
19a 94.8
19b 9.34
§ I.A.: inactive; GI50 value >100 lM; # N.T.: not tested.
attaching different substituted five-membered heterocy- more, replacement of the thiadiazine 17 with the five-
clic rings to its N2 position. The first obtained compound membered thiadiazole ring gave a totally inactive com-
is phenylthiazolidene derivative 10a which showed GI50 pound as in the case of compound 19a, in addition to an
values against all breast tumor cell lines ranging between active compound 19b against MiDA-N, HS-578T, and
4.1 and 8.6 lM with the exception of T-47D (Table 2). The MDA/MB-435 tumor cell lines (GI50 values between 5.4
cytotoxic activity against the latter cell was slightly and 9.2 lM) as in case of compound 19b (Table 2).
improved by adding a chlorine atom at the para-position
Beside the designed compounds in the present study,
of phenyl ring as in compound 10b (Table 2). In contrast, all intermediates were tested for their antitumor activity.
replacement of the chlorine atom in the latter product Thus, the thiosemicarbazide derivative 5 was found to
with a methyl group renders the resultant compound have moderate to weak activity, while the potassium salt
completely inactive as shown in compound 10c (Table 2). 12 revealed good GI50 values between 2.8 and 9.3 lM
Furthermore, replacement of the phenyl moiety with a against MDA/MB-231 ATTC, MiDA-N, HS-578T, and MDA/
methyl group increased the cytotoxic activity against the MB-435 breast tumor cell lines (Table 2).
BT 549 breast tumor cell line as shown in compound 10d
(Table 2).
Effect of the newly synthesized compounds on ovarian
In the last line of structural optimization, utilizing cancer
both mercapto and amino groups in compound 14 for Ovarian OVCAR-5 and eight tumor cell lines were found
obtaining the corresponding fused ring systems, 17 and to be sensitive to triazolethiol derivative 4 (GI50 values are
19 showed high cytotoxic effects. Such modifications 3.94 and 9.94 lM, respectively) (Table 3). The more lipo-
afforded a remarkable improvement in the antitumor philic derivative 3-(5-mercapto-4-phenyl-4H-1,2,4-triazol-
properties of the resultant molecules. For example, the 3-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile 6 is more
GI50 value against the MiDA-N tumor cell line was active against the OVCAR-4 tumor cell line, while the
reduced from 5.0 to 1.1 lM in case of the triazolothiadi- more hydrophilic derivative 3-(5-mercapto-4-amino-4H-
azine derivative 17b (Table 2). The latter compound 1,2,4-triazol-3-yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile
showed also significant in-vitro cytotoxic activity against 14 has moderate, weak, or no activity (Table 3). Methyla-
MDA/MB-435 (GI50 value 1.23 lM; Table 2). In addition, its tion of compound 6 afforded a broad-spectrum active
GI50 values were below 10 lM against MDA/MB-231. ATTC, compound against all OVCAR types (GI50 ranging
NCI/ADR-RES, and HS-578T cell lines (Table 2). Further- between 5.4 to 9.2 lM) as shown for compound 7 (Table
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
391
3). Replacement of the mercapto group with a hydrazino vitro cytotoxic activity against all OVCAR ovarian tumor
moiety increases the activity against the SK-OV-3 ovarian cell lines (GI50 values below 10 lM) with excellent anti-
tumor cell line (GI50 value 9.72 lM) as shown in com- estrogen properties more than twofold compared to
pound 8 (Table 3).
letrozole. Unfortunately, these compounds were found
The oxadiazole derivative 13 revealed cytotoxic activity to be more toxic than letrozole.
in the nanomolar range against the IGROVI tumor cell
line (GI50 value 40 nM; Table 3). In contrast, thiadiazole
derivative 11 showed no promising cytotoxic results
Experimental
(Table 3).
General
Although the carboxylic acid hydrazide derivative 2
All melting points were measured on a Gallenkamp melting
showedmoderatetoweakactivity(Table3), itsphenylthia-
zolidene derivative 10a has a broad-spectrum activity
against all tested ovarian tumor cell lines (GI50 values are
10 € 3 lM; Table 3). In addition, introduction of a methyl
group in the para-position of the phenyl ring of the latter
product improves its antitumor properties against
OVCAR-3 and 5 cell lines as shown in compound 10c (Table
3). Replacement of the methyl group with chlorine atom
in the same compound decreases the cytotoxic activity
against all ovarian cell linesexcept SK-OV-3 (Table 3).
The fused triazole derivative 17a showed insignificant
cytotoxic results against the tested ovarian cell lines. Its
para-methoxy derivative 17b revealed remarkable antitu-
mor characteristics against OVCAR-8 and 4 with a GI50
value around 3.3 lM (Table 3). Finally, both triazolothia-
diazoles 19a and b are active against the OVCAR-4 ovarian
tumor cell line (GI50 values are 7.66 and 4.49 lM, respec-
tively; Table 3).
point apparatus (Weiss-Gallenkamp, London, UK). The infrared
spectra were recorded in potassium bromide disks on a pye Uni-
cam SP 3300 and Shimadzu FT IR 8101 PC infrared spectropho-
tometers (Pye Unicam Ltd. Cambridge, England and Shimadzu,
Tokyo, Japan, respectively). The NMR spectra were recorded on a
Varian Mercury VX-300 NMR spectrometer (Varian, Palo Alto,
CA, USA). 1H spectra were run at 300 MHz and 13C spectra were
run at 75.46 MHz in deuterated chloroform (CDCl3) or dimethyl
sulphoxide (DMSO-d6). Chemical shifts were related to that of the
solvent. Mass spectra were recorded on a Shimadzu GCMS-QP
1000 EX mass spectrometer (Shimadzu) at 70 eV. Elemental anal-
yses were carried out at the Micro-analytical Center of Cairo Uni-
versity, Giza, Egypt. Ethyl 4-cyano-1,5-diphenyl-1H-pyrazole-3-
carboxylate 1 was prepared following the procedures reported
in the literature [53].
Chemistry
4-Cyano-1,5-diphenyl-1H-pyrazole-3-carboxylic acid
hydrazide 2
Hydrazine hydrate (80%, 10 mL) was added to a stirred solution
of the 4-cyano-1,5-diphenyl-1H-pyrazole-3-carboxylate 1 (5 g, 16.5
mmol) in absolute ethanol (20 mL) and the reaction mixture was
stirred for 10 h. The precipitated white solid was collected by fil-
tration, washed with water, and crystallized from diluted etha-
nol to afford the corresponding acid hydrazide 2 in 78% yield;
m. p.: 148–1498C; IR (KBr) mmax/cm–1: 3309, 3160, 3111 (NH, NH2),
Conclusion
In this article, different triazole, oxadiazole, thiadiazole,
and pyrazole as well as the fused triazole derivatives of 4-
cyano-1,5-diphenylpyrazole were synthesized via the
intermediates 1, 2, 3, 5, and 12. All of the newly designed
compounds were tested in vitro for there cytotoxic prop-
erties against estrogen-dependent tumor cell lines (breast
and ovarian tumor types). In addition, the anti-estrogen
activity of thirteen selected compounds was determined
in vivo and their LD50 values were determined.
3-(5-Mercapto-1,3,4-oxadiazole-2-yl)-1,5-diphenyl-1H-py-
razole-4-carbonitrile 13 revealed the highest cytotoxic
activity in a nanomolar range against the IGROVI ovarian
tumor cell line with an anti-estrogen activity 1.6 times
that of letrozole and with a greater safety margin. 4-
Cyano-1,5-diphenyl-1H-pyrazole-[3,4-diphenyl-3H-thiazol-
(2E)-ylidene]-3-carboxylic acid hydrazide 10a showed sig-
nificant activity against both breast and ovarian tumor
cell lines.
1
2237 (CN), 1674 (C=O); H-NMR (DMSO-d6) d: 2.78 (br s, 2H, NH2
D2O-exchangeable), 7.21–7.43 (m, 10H, ArH's), 8.50 (br s, 1H, NH,
D2O-exchangeable); MS m/z (%): 303 [M+] (100), 180 (11), 141 (10.9),
77 (59.3) Anal. calcd. for C17H13N5O (303.32): C, 67.31; H, 4.32; N,
23.09. Found: C, 67.35; H, 4.40; N, 23.13.
1-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-
carbonyl)thiosemicarbazide 3
Method A: To a solution of the pyrazole carboxylic acid hydra-
zide 2 (6.06 g, 20 mmol) in methanol (50 mL), a solution of potas-
sium thiocyanate (2.91 g, 30 mmol) and hydrochloric acid (3 mL)
was added with constant stirring. The mixture was immediately
evaporated to dryness under reduced pressure and heated for an
additional one hour with another 50 mL of methanol. The result-
ing solid was treated with water and ethanol, and finally recrys-
tallized from ethanol to afford 1-(4-cyano-1,5-diphenyl-1H-pyra-
zole-3-carbonyl)thiosemicarbazide 3 in 78% yield.
Method B: To a solution of ethyl 4-cyano-1,5-diphenyl-1H-pyra-
zole-3-carboxlate 1 (9.69 g, 30 mmol) and thiosemicarbazide
(2.91 g, 30 mmol) in dioxan (30 mL) was added with a few drops
of piperidine; the reaction mixture was refluxed for 10 h and
then left to cool. The precipitated solid product was filtered off,
washed with ethanol, dried, and finally recrystallized from etha-
3-(5-(Methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-di-
phenyl-1H-pyrazole-4-carbonitrile 7 showed selective in-
i 2010 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
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392
A. M. Farag et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
nol to afford a product in all respects (m. p., mixture m. p., TLC,
IR and NMR spectra) identical with that obtained by method A
above.
metal (0.11 g, 5 mg/atom) in 20 mL ethanol). Then, methyl iodide
(0.28 g, 2 mmol) was added gradually with stirring to the result-
ing solution. The reaction mixture was refluxed for 2 h, concen-
trated, cooled, diluted with water, and left to stand overnight.
The formed precipitate was filtered off, washed with water,
dried, and recrystallized from ethanol to afford compound 7 in
M. p.: 185–1868C; IR (KBr) mmax/cm–1: 3350, 3200 (overlapped
NH, NH2), 2229 (CN), 1670 (C=O); 1H-NMR (DMSO-d6) d: 4.34 (br s,
2H, NH2 D2O-exchangeable), 7.35–7.47 (m, 10H, ArH's), 7.85,
11.30 (br., s, 2H, 2 NH, D2O-exchangeable); MS m/z: 362 [M+] (15.6),
272 (100), 244 (10.2), 77 (40.1) Anal. calcd. for C18H14N6OS
(362.41): C, 59.65; H, 3.89; N, 23.19; S, 8.84. Found: C, 59.61; H,
3.87; N, 23.16; S, 8.81.
1
80% yield; m. p.: 236–2388C; IR (KBr) mmax/cm–1: 2225 (CN); H-
NMR (DMSO-d6) d: 3.45 (s, 3H, SCH3), 7.31–7.65 (m, 15H, ArH's);
MS m/z (%): 434 [M+] (16.4), 401 (14.0), 77 (100). Anal. calcd. for C25
H18N6 S (434.52): C, 69.10; H, 4.17; N, 19.34; S, 7.37. Found: C,
69.16; H, 4.20; N, 19.30; S, 7.29.
3-(5-Mercapto-4H-1,2,4-triazol-3-yl)-1,5-diphenyl-1H-
3-(5-Hydrazino-4-phenyl-4H-1,2,4–triazol-3-yl)-1,5-
pyrazole-4-carbonitrile 4
A suspension of the thiosemicarbazide 3 (0.36 g, 1 mmol) in
potassium hydroxide solution (10 mL, 7%) was heated under
reflux for 3 h. The reaction mixture was allowed to cool and was
then adjusted to pH 6 with 10% hydrochloric acid. The formed
precipitate was filtered off, washed with water, dried, and
finally recrystallized from ethanol to afford the triazolethiol 4
in 68% yield; m.p.: 295–2978C; IR (KBr) mmax/cm–1: 3200 (NH), 2239
diphenyl-1H-pyrazole-4-carbonitrile 8
A mixture of 3-(5-(methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile 7 (1.3 g, 3 mmol) or 3-(5-mer-
capto 4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-diphenyl-1H-pyrazole-4-
carbonitrile 6 (1.26 g, 3 mmol) and hydrazine hydrate (80%, 5
mL) was refluxed for 5 h. The reaction mixture was evaporated
under reduced pressure to remove excess hydrazine hydrate and
was allowed to cool. The formed solid product were filtered off,
washed with ethanol, and recrystallized from ethanol to give
compound 8 in 65% yield; m. p.: 295–2978C; IR (KBr) mmax/cm–1:
3250, 3120, (NH, NH2), 2225 (CN); 1H-NMR (DMSO-d6) d: 2.49 (br s,
2H, NH2, D2O-exchangeable), 6.95 (br s, 1H, NH, D2O-exchange-
able), 7.21–7.43 (m, 15H, ArH's); MS m/z (%): 418 [M+] (49.8), 286
(35.8), 77 (100). Anal. calcd. for C24H18N8 (418.46): C, 68.88; H,
4.33; N, 26.78. Found: C, 68.90; H, 4.34; N, 26.75.
1
(CN); H-NMR (DMSO-d6) d: 7.21–7.43 (m, 10H, ArH's), 10.5 (s.1H,
NH, D2O-exchangeable), 14.1 (s, 1H, SH); MS m/z (%): 344 [M+]
21.2), 317 (61.2), 245 (10.0), 180 (59.9), 141 (28.1), 77 (100). Anal.
calcd. for C18H12N6S (344.40): C, 62.78; H, 3.51; N, 24.40; S, 9.31.
Found: C, 62.83; H, 3.48; N, 24.48; S, 9.33.
1-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-4-
phenylthiosemicarbazide 5
An equimolar quantity of the acid hydrazide 2 (6.06 g, 20 mmol)
and phenyl isothiocyanate (2.7 g, 20 mmol) in absolute ethanol
(40 mL) were refluxed for 3 h and then allowed to cool to room
temperature. Fine crystals of the thiosemicarbazide 5 were sepa-
rated out, filtered off, and recrystallized from ethanol to afford
the compound in 85% yield; m. p.: 1308C; IR (KBr) mmax/cm–1:
Reaction of 1-(4-cyano-1,5-diphenyl-1H-pyrazole-3-yl)-4-
phenylthiosemicarbazide (5) with phenacyl bromide
derivatives and chloroacetone: General procedure
To a solution of 1-(4-cyano-1,5-diphenyl-1H-pyrazole-3-carbonyl)-
4-phenyl thiosemicarbazide 5 (1.31 g, 3 mmol) and the appropri-
ate phenacyl bromide derivatives or chloroacetone (3 mmol) in
absolute ethanol (20 mL), a catalytic amount of triethylamine
(0.1 mL) was added. Then, the reaction mixture was heated
under reflux for 3 h, and was allowed to cool. The precipitated
product was filtered off, washed with ethanol, dried, and finally
recrystallized from ethanol. The synthesized compounds
together with their physical and spectral data are listed below.
1
3310, 3150 (3 NH, overlapped), 2233 (CN), 1667 (C=O); H-NMR
(DMSO-d6) d: 7.03–7.30 (m, 15H, ArH's), 7.93, 8.74, 11.34 (br s, 3H,
3 NH, D2O-exchangeable); MS m/z (%): 439 (57.1), 438 [M+] (100),
422 (7.3), 180 (10.0), 77 (56.7); Anal. calcd. for C24H18N6OS
(438.51): C, 65.74; H, 4.14; N, 19.16; S, 7.31. Found: C, 65.71; H,
4.13; N, 19.20; S, 7.34.
3-(5-Mercapto-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile 6
4-Cyano-1,5-diphenyl-1H-pyrazole-[3,4-diphenyl-3H-
The thiosemicarbazide 5 (4.38 g, 10 mmol) was refluxed in potas-
sium hydroxide solution (5%, 25 mL) for 3 h. The resulting solu-
tion was treated with charcoal, filtered, and cooled. The filtrate
was acidified with hydrochloric acid to pH = 5 and the formed
solid was filtered off, washed with water, dried, and recrystal-
lized from ethanol to afford the thiazole 6 in 85% yield; m. p.:
250–2528C; IR (KBr) mmax/cm–1: 3265 (NH), 2228 (CN); 1H-NMR
(DMSO-d6) d: 6.84–7.47 (m, 15H, ArHs), 11.2 (br s, 1H, NH, D2O-
exchangeable), 14.16 (br s, 1H, SH); MS m/z (%): 420 [M+] (47.3),
244 (4.9), 286 (35.8), 127 (3.3), 77 (100). Anal. calcd. for C24H16N6S
(420.50): C, 68.55; H, 3.84; N, 19.98; S, 7.62. Found: C, 68.61; H,
3.88; N, 19.93; S, 7.58.
thiazol-2-ylidene]-3-carboxylic acid hydrazide 10a
Yield: 68%; m.p.: 155–1568C; IR (KBr) mmax/cm–1: 3128 (NH), 2229
(CN), 1689 (C=O); 1H-NMR (DMSO-d6) d: 6.54 (s, 1H, thiazole-5 CH),
721–7.50 (m, 20H, ArH's), 11.01 (br s, 1H, NH, D2O-exchangeable);
MS m/z (%): 539 (25.7), 538 [M+] (56.5), 272 (42.4), 180 (26.2), 135
(25.5), 77 (100). Anal. calcd. for C32H22N6OS (538.63): C, 71.36; H,
4.11; N, 15.60; S, 5.95. Found: C, 71.40; H, 4.16; N, 15.68; S, 5.89.
4-Cyano-1,5-diphenyl-1H-pyrazole-[4-(4-chlorophenyl)-
3-phenyl-3H-thiazol-2-ylidene]-3-carboxylic acid hydrazid
10b
Yield: 68%; m. p.: 250–2528C; IR (KBr) mmax/cm–1: 3115 (NH), 2229
(CN), 1688 (C=O); 1H-NMR (DMSO-d6) d: 6.61 (s, 1H, thiazole-5 CH),
732–7.63 (m, 19H, ArH's), 10.82 (br s, 1H, NH, D2O-exchangeable);
13C-NMR (DMSO-d6) d: 112.86, 115.64, 120.67, 120.81, 123.21,
125.77, 125.87, 126.12, 126.21, 127.87, 128.57, 129.17, 129.33,
3-(5-(Methylthio)-4-phenyl-4H-1,2,4-triazol-3-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile 7
The mercaptotriazol 6 (2.1 g, 5 mmol) was dissolved in an etha-
nolic solution of sodium ethoxide – prepared from sodium
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
393
130.32, 130.42, 133.24, 137.26, 138.23, 144.68, 147.25, 149.70,
150.31, 150.46, 153.63, 158.51; MS m/z (%): 574 [M+] 18), 572 [M+]
(57), 555 (33), 285 (22), 180 (37), 141 (18), 77 (100). Anal. calcd. for
C32H21N6OSCl (573.07): C, 67.07; H, 3.69; N, 14.66; S, 5.59. Found:
C, 67.16; H, 3.66; N, 14.70; S, 5.75.
3-(5-Mercapto-1,3,4-oxadiazole-2-yl)-1,5-diphenyl-1H-
pyrazole-4-carbonitrile 13
Method A: A solution of potassium hydroxide (0.84 g, 15 mmol)
and the acid hydrazide 2 (3.03 g, 10 mmol) in absolute ethanol
(200 mL) were added (1.14 g, 15 mmol). This reaction mixture
was refluxed for 5 h, then diluted with water and acidified with
HCl. The precipitated solid was filtered off, washed with water,
dried, and finally recrystallized with ethanol to afford com-
pound 13 in 89% yield.
4-Cyano-1,5-diphenyl-1H-pyrazole[4-(4-methoxyphenyl)-
3-phenyl-3H-thiazol-2-ylidene]-3-carboxylic acid
Method B: A solution of potassium hydroxide (0.84 g, 15
mmol) and the potassium salt 12 (4.72 g, 10 mmol) in absolute
ethanol (200 mL) was refluxed for 4 h, till the evolution of H2S
ceased; then, it was diluted with water and acidified with HCl.
The precipitated solid was filtered off, washed with water, dried,
and was finally crystallized with ethanol to give compound 13
in 90% yield; m.p.: 1378C; IR (KBr) mmax/cm–1: 2235 (CN). 1H-NMR
(DMSO-d6) d: 6.48–7.47 (m, 10H, ArH's), 14.0 (s, 1H, SH, D2O
exchangeable); 13C-NMR (DMSO-d6) d: 115.49, 120.51, 120.54,
120.56, 125.62, 127.85, 128.19, 128.43, 128.75, 128.90, 129.94,
138.40, 143.94, 144.57; MS m/z (%): 345 [M+] (40.7), 256 (17.5), 180
(41.2), 77 (100). Anal. calcd for C18H11N5OS (345.38): C, 62.59; H,
3.21; N, 20.27; S, 9.28. Found: C, 62.51; H, 3.22; N, 20.23; S, 9.23.
hydrazide 10c
Yield: 65%; m.p.: 2068C; IR (KBr) mmax/cm–1: 3150 (NH), 2229 (CN),
1684 (C=O); 1H-NMR (CDCl3) d: 3.68 (s, 3H, OCH3), 6.51 (s, 1H, thia-
zole-5 CH), 7.15-7.37 (m, 19H, ArH's), 7.74 (br s, 1H, NH, D2O-
exchangeable); 13C-NMR (DMSO-d6) d: 55.10, 113.85, 120.14,
120.79, 122.99, 125.76, 125.78, 126.00, 126.08, 127.24, 128.50,
129.26, 129.37, 130.19, 130.86, 131.54, 137.68, 137.96, 139.21,
144.18, 150.00, 150.28, 150.40, 151.54, 159.11; MS m/z (%): 568
[M+] (28.6), 282 (100), 149 (25.8), 77 (70.7). Anal. calcd. for
C33H24N6O2S (568.59): C, 69.70; H, 4.25; N, 14.78; S, 5.64. Found: C,
69.65; H, 4.26; N, 14.80; S, 5.58.
4-Cyano-1,5-diphenyl-1H-pyrazole-[4-methyl-3-phenyl-
3H-thiazol-2-ylidene)-3-carboxylic acid hydrazide 10d
Yield: 68%; m.p.: 265–2578C; IR (KBr) mmax/cm–1: 3120 (NH), 2223
(CN), 1698 (C=O); 1H-NMR (CDCl3) d: 1.89 (s, 3H, CH3), 6.02 (s, 1H,
thiazole-5 CH), 7.11–7.40 (m, 15H, ArH's), 7.87 (br s, 1H, NH, D2O-
exchangeable); MS m/z (%): 476 [M+] (34.9), 190 (100), 77 (29.4).
Anal. calcd. for C27H20N6OS (476.55): C, 68.05; H, 4.23; N, 17.63; S,
6.75. Found: C, 67.99; H, 4.25; N, 17.60; S, 6.73.
3-(5-Mercapto-4-amino-4H-1,2,4-triazol-3-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile 14
Method A: The potassium salt 12 (4.72 g, 10 mmol) was sus-
pended in 70% hydrazine hydrate (5 mL), then refluxed for 3 h.
The formed white solid, which was filtered off, was washed with
water, dried, and finally recrystallized with ethanol/DMF to
afford compound 14 in 85% yield.
Method B: A solution of the oxadiazole 13 (3.45 g, 10 mmol) in
ethanol (20 mL) and 70% hydrazine hydrate (5 mL) was refluxed
for 3 h, then allowed to cool, diluted with cold water, and acidi-
fied with HCl. The precipitated solid was filtered, washed with
water, dried, and recrystallized with ethanol/DMF to give com-
pound 14 in 72% yield; m.p.: 235–2368C; IR (KBr) mmax/cm–1: 3294,
3120 (NH2), 2585 (SH), 2229 (CN); 1H-NMR (DMSO-d6) d: 5.56 (s, 2H,
NH2, D2O-exchangeable), 7.09–7.47 (m, 10H, ArH's), 14.1 (s, 1H,
SH); MS m/z (%): 359 [M+] (16.2), 327 (26.3), 269 (25.1), 180 (20.4),
126 (25.7), 90 (65.9), 77 (100). Anal. calcd. for C18H13N7S (359.41):
C, 60.15; H, 3.64; N, 27.28; S, 8.92. Found: C, 60.20; H, 3.58; N,
27.36; S, 8.84.
1,5-Diphenyl-3-[5-(phenylamino)-1,3,4-thiadiazol-2-yl]-
1H-pyrazole-4-carbonitrile 11
A mixture of the thiosemicarbazide 5 (1.31 g, 0.3 mmol) and con-
centrated sulphuric acid (10 mL) was stirred for 4 h, then, the
reaction mixture was poured into ice-cold water. The formed
precipitate was filtered off, washed with water several times,
dried, and recrystallized from ethanol. Yield: 70%; m. p.: 1858C;
1
IR (KBr) mmax/cm–1: 3190 (NH), 2229 (CN); H-NMR (DMSO-d6) d:
7.06–7.50 (m, 15H, ArH's), 7.75 (s, 1H, NH, D2O exchangeable); MS
m/z (%): 420 [M+] (43.8), 105 (31.5), 77 (100). Anal. calcd for
C24H16N6S (420.50): C, 68.55; H, 3.84; N, 19.99; S, 7.63. Found: C,
68.53; H, 3.82; N, 19.94; S, 7.65.
1,5-Diphenyl-3-(3,5-dimethylpyrazole-1-carbonyl)-1H-
Potassium salt of thiosemicarbazide derivative 12
To a cold stirred solution of the acid hydrazide 2 (3.03 g, 10
mmol) in absolute ethanol (100 mL) containing potassium
hydroxide (0.84 g, 15 mmol), carbon disulphide (1.14 g, 15
mmol) was added gradually. The reaction mixture was stirred at
room temperature for 8 h. A yellow precipitate of the corre-
sponding potassium salt 12 was separated. Then, dry ether (100
mL) was added to complete the precipitation of the formed salt
which was filtered off and washed with dry ether (100 mL). Yield:
95%; m.p.: >3008C; IR (KBr) mmax/cm–1: 3280, 3150 (2 NH), 2230
(CN) 1675 (CO); 1H-NMR (DMSO-d6) d: 7.37–7.49 (m, 10H, ArH's),
7.48 (s, 1H, NH, D2O exchangeable), 11.19 (s, 1H, NH, D2O
exchangeable). Anal. calcd. for C18H12KN5OS2 (417.55): C, 51.78;
H, 2.90; N, 16.77; S, 15.36. Found: C, 51.85; H, 2.91; N, 16.73; S,
15.39.
pyrazole-4-carbonitrile 15
To a mixture of the acid hydrazide 2 (3.03 g, 10 mmol) and acetyl-
acetone (1.0 g, 10 mmol) in ethanol (20 mL), a few drops of piper-
idine were added. The reaction mixture was refluxed for 6 h and
then allowed to cool. The precipitated solid was filtered off,
washed with water, dried, and recrystallized from dilute ethanol
to afford compound 15 in 67% yield; m. p.: 1988C; IR (KBr) mmax
/
cm–1: 2234 (CN), 1658 (C=O); 1H-NMR (CDCl3) d: 1.95 (s, 3H, CH3),
2.06 (s, 3H, CH3), 5.32 (s, 1H, CH), 7.06–7.60 (m, 10H, ArH's); 13C-
NMR (DMSO-d6) d: 15.61, 22.98, 112.96, 119.25, 120.77, 125.64,
125.92, 126.11, 128.85, 129.21, 129.28, 130.26, 137.82, 140.21,
140.36, 149.78, 156.93, 158.89; MS m/z (%): 367 [M+] (21.4), 338
(31.6), 272 (100), 180 (10.5), 141 (23.9), 95 (22.9), 77 (45.1). Anal.
calcd. for C22H17N5O (367.41): C, 71.92; H, 4.66; N, 19.06. Found:
C, 71.98; H, 4.70; N, 19.10.
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394
A. M. Farag et al.
Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
2 h. Then, the reaction mixture was cooled, poured gradually
with stirring into an ice cold sodium bicarbonate solution. The
separated product was filtered off, washed with water, dried,
and finally recrystallized from ethanol. The compounds pre-
pared by this method are listed below.
1,5-Diphenyl-3-(3-methylpyrazol-5-one-1-carbonyl)-
pyrazole-1H-4-carbonitrile 16
To a mixture of the acid hydrazide 2 (3.03 g, 10 mmol) and ethyl
acetoacetate (1.3 g, 10 mmol) in ethanol (20 mL), a few drops of
piperidine were added. The reaction mixture was refluxed for 8
h. The precipitated solid was filtered off, washed with water,
dried, and recrystallized from ethanol to afford compound 16 in
60% yield; m. p.: 180–1818C; IR (KBr) mmax/cm–1: 2237 (CN), 1665,
1651 (2 C=O); MS m/z (%): 369 [M+] (30.5), 272 (100), 244 (12.1), 77
(23.7). Anal. calcd. for C21H15N5O2 (369.37): C, 68.28; H, 4.09; N,
18.96. Found: C, 68.35; H, 4.11; N, 18.95.
6-Phenyl-3-(4-cyano-1,5-diphenyl-1H-pyrazole-3-yl)-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole 19a
1
Yield: 84%; m.p.: 2358C; IR (KBr) mmax/cm–1: 2230 (CN); H-NMR
(DMSO-d6) d: 7.11–7.59 (m, 15H, ArH's); MS m/z (%): 445 [M+] (14.1),
372 (74.2), 271 (21.3), 180 (14.1), 77 (100). Anal. calcd. for
C25H15N7S (445.51): C, 67.40; H, 3.39; N, 22.08; S, 7.20. Found: C,
67.42; H, 3.35; N, 22.01; S, 7.19.
General procedure for 3-(4-cyano-1,5-diphenyl-1H-
pyrazole-3-yl)-6-aryl-7H-1,2,4-triazolo[3,4-b]-1,3,4-
6-Benzyl-3-(4-cyano-1,5-diphenyl-1H-pyrazole-3-yl)-
thiadiazine 17a, b
To a solution of 3-(5-mercapto- 4-amino-4H-1,2,4-triazol-3-yl)-1,5-
diphenyl-1H-pyrazole-4-carbonitrile 14 (0.72 g, 2 mmol) and the
appropriate phenacyl bromide derivatives 9a, c (2 mmol) in abso-
lute ethanol (25 mL), a few drops of triethylamine were added.
The reaction mixture was heated under reflux for 5 h, then
cooled, adjusted to pH = 8 by the addition of cold saturated solu-
tion of sodium acetate, and left to stand overnight. The precipi-
tated product was filtered off, washed with water, dried, and
was finally recrystallized from ethanol. The synthesized com-
pounds together with their physical and spectral data are listed
below.
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazole 19b
1
Yield: 68%; m.p.: 2908C; IR (KBr) mmax/cm–1: 2237 (CN); H-NMR
(DMSO-d6) d: 3.96 (s, 2H, CH2), 7.04–7.77 (m, 15H, ArH's); 13C-NMR
(DMSO-d6) d: 41.33, 112.14 (pyrazole C4), 115.95 (CN), 120.50,
121.01, 124.76, 127.99, 128.00, 128.06, 128.76, 128.90, 129.02,
133.55, 134.07, 134.59, 144.23, 145.20, 149.07, 149.30 (ArC's),
160.66 (thiadiazole-C5); MS m/z (%): 459 [M+] (8.5), 401 (22.4), 271
(32.8), 180 (15.3), 141 (10.7), 77 (100). Anal. calcd. for C26H17N7S
(459.53): C, 67.95; H, 3.73; N, 21.33; S, 6.98. Found: C, 67.90; H,
3.80; N, 21.30; S, 7.00.
Pharmacology
Antagonism of uterus-weight increase due to estrogen
treatment (anti-estrogenic activity)
3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-6-phenyl-7H-
1,2,4-triazolo[3,4-b]-1,3,4-thiadiazine 17a
1
Yield: 70%; m. p.: 241–2428C; IR (KBr) mmax/cm–1: 2225 (CN); H-
Animals: Immature female Sprague-Dawley rats weighing about
55 g were obtained from Animal House Laboratory, Nile Com-
pany, Cairo, Egypt and acclimatized for one week in the animal
facility that has a 12 h light/dark cycle with the temperature
controlled at 21–238C. Normal rat chow and water were made
available. The animals were housed individually in stainless
steel cages in temperature-controlled and humidity-monitored
quarters. Test animals were provided with a continuous access
to tap water
NMR (CDCl3) d: 4.43 (s, 2H, CH2, thiadiazine) 7.02–7.27 (m, 15H,
ArH's); 13C-NMR (DMSO-d6) d: 40.004 (6CH2, thiadiazine), 105.138
(5C, thiadiazine), 115.661 (CN), 120.04, 121.070, 121.076, 122.38,
124.99, 125.94, 128.11, 129.10, 130.00, 130.135, 130.139, 132.51,
138.09, 138.25, 143.97, 148.40, 155.05 (17 ArC's); MS m/z (%): 459
[M+] (23.6), 370 (10.7), 270 (7.9), 77 (100). Anal. calcd. for C26H17N7S
(459.53): C, 67.95; H, 3.73; N, 21.33; S, 6.97. Found: C, 68.00; H,
3.71; N, 21.34; S, 6.99.
Procedure: Groups of 12 animals were daily injected subcuta-
neously for seven days a week with estradiol (0.03–0.05 lg per
animal) and various doses (0.0 to 0.06 lg per animal) of the test
compounds and letrozole or estradiol alone as reference stand-
ard.
The test compound was orally administered in a 0.5% solution
of carboxymethylcellulose. On the 8th day, the animals were sac-
rificed and the uterine weights were determined.
3-(4-Cyano-1,5-diphenyl-1H-pyrazole-3-yl)-6-(4-
methoxyphenyl)-7H-1,2,4-triazolo[3,4-b]-1,3,4-
thiadiazine 17b
1
Yield: 75%; m. p.: 2208C; IR (KBr) mmax/cm–1: 2230 (CN); H-NMR
(CDCl3) d: 3.86 (s, 3H, OCH3), 4.71 (s, 2H, CH2, thiadiazine) 7.15–
7.37 (m, 14H, ArH's); 13C-NMR (DMSO-d6) d: 37.73 (6CH2 thiadia-
zine), 55.43 (OCH3), 106.58 (5C, thiadiazine), 113.83 (CN), 120.65,
120.67, 120.68, 125.82, 126.96, 128.09, 128.83, 129.04, 130.15,
130.34, 130.55, 137.57, 138.20, 140.40, 144.14, 150.60, 153.37
(17 ArC's); MS m/z (%): 489 [M+] (30), 370 (26.7), 270 (28.3), 180
(15.9), 135 (73.4), 77 (100). Anal. calcd. for C27H19N7SO (489.56): C,
66.24; H, 3.91; N, 20.02; S, 6.55. Found: C, 66.25; H, 3.90; N, 19.99;
S, 6.60.
Evaluation: Mean values of each group are calculated and
expressed as percent reduction of uterine weight compared to
controls treated with estradiol alone.
Evaluation of acute toxicity following a single-dose
administration
Animals: Four hundreds adult mice of both sexes weighing 25 €
3 g were obtained from Animal House Laboratory, Nile Com-
pany, Cairo, Egypt and acclimatized for one week in the animal
facility that has a 12 h light/dark cycle with the temperature
controlled at 21–238C. Normal rat chow and water were made
available.
Reaction of 3-(5-(mercapto)-4-amino-4H-1,2,4-triazol-3-
yl)-1,5-diphenyl-1H-pyrazole-4-carbonitrile (14) with
carboxylic acids: General procedure
A mixture of compound 14 (1.07 g, 3 mmol) and the appropriate
carboxylic acid (benzoic or phenylacetic acids; 3 mmol) in phos-
phorus oxychloride (10 mL) was heated under reflux at 1008C for
Procedure: LD50 was measured on 30 mice. Animals were
fasted for 12 h prior to dosing. Rats were divided into six groups
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Arch. Pharm. Chem. Life Sci. 2010, 343, 384–396
Pyrazole-based Heterocycles as Antitumor Agents
395
with five animals in each group. Treated rats were dosed by oral
gavage, using a curved, balltipped stainless steel feeding needle,
with aqueous suspensions of very fine powder of the test com-
pounds. Animals in each group were given doses of 100, 160,
256, 409, 655, and 1050 mg/kg b.w. After 24 h the results were
recorded. The controls received tap water by gavage in the same
volume.
reader at a wavelength of 515 nm. For suspension cells, the
methodology was the same except that the assay was terminated
by fixing settled cells at the bottom of the wells by gently adding
50 mL of 80% TCA (final concentration, 16% TCA). The parameter
used here is GI50 which is the log10 concentration at which PG is
+ 50, was calculated for each cell line [56–58].
Observation: All animals were monitored continuously for
10 h after dosing for signs of toxicity. For the remainder of the
14 days study period, animals were monitored for mortality. At
the end of the study, the number of dead animals was expressed
in percentage and the LD50 value was calculated according to the
Weill method (1952; [54]).
The authors are very grateful to the staff members of the Department of
Health and Human Services, National Cancer Institute (NCI), Bethesda,
Maryland, USA, for carrying out the anticancer screening of the newly
synthesized compounds.
The authors have declared no conflict of interest.
Statistical analysis
The data were evaluated for homogeneity of variances and nor-
mality by Bartlett's test. Where Bartlett's test indicated homoge-
neous variances, treated and control groups were compared
using a one-way analysis of variance (ANOVA), followed by com-
parison of the treated groups with the control groups by Dun-
nett's t-test for multiple comparisons [55], where variances were
considered significantly different by Bartlett's test.
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