Design, synthesis, and in vitro anticancer screening of novel pyrazolinyl-pyrazole/1, 2, 3-triazole hybrids

Article abstract of DOI:10.1002/jhet.4076

Novel hybrids, pyrazolinylpyrazoles, and pyrazolinyltriazoles were designed, synthesized, and screened for their anticancer activity. Eleven compounds were selected by the National Cancer Institute (NCI)/USA for anticancer screening at single high dose (10^?5 M) in full NCI 60 cell panels. Two compounds: 4-aminopyrazole-5-carbonitrile, 5c (NSC-747630/1) and ethyl 4-aminopyrazole-5- carboxylate, 7d (NSC-747634/1) were the most active candidates of the series and were selected for further evaluation at five dose screening as they displayed significant growth inhibition with full panel median growth inhibition (GI₅₀) 5.47 and 2.24 μM, respectively. Both 5c and 7d hybrids exhibited broad spectrum antitumor activity; however 7d showed moderate selectivity (selectivity ratio 3.6) toward leukemia.

Full text of DOI:10.1002/jhet.4076

Design, synthesis, and in-vitro anticancer screening of novel pyrazolinyl-
pyrazole/-1, 2, 3-triazole hybrids
Magda M. F. Ismail1, Hanan G. Abdulwahab1, Mohamed H. Elnagdi2
1Pharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr
City, 11754 , Cairo, Egypt
2Department of Chemistry, Faculty of Science, University of Kuwait, Safat, 13060, Kuwait
Corresponding author: m.elalfy101@gmail.com
Phone: 01063370137
orcid.org/0000-0002-3915-9786
Address: 69 Ahmad El-Sawy St., Makram Ebeid| Nasr City, Cairo| Egypt
Abstract: Novel hybrids, pyrazolinylpyrazoles and pyrazolinyltriazoles were designed, synthesized
and screened for their anticancer activity. Eleven compounds were selected by the National Cancer
Institute (NCI)/USA for anticancer screening at single high dose (10-5 M) in full NCI 60 cell panel.
Two compounds: 4-aminopyrazole-5-carbonitrile, 5c (NSC-747630/1) and ethyl 4-aminopyrazole-
5- carboxylate, 7d (NSC-747634/1) were the most active candidates of the series and were selected
for further evaluation at five dose screening as they displayed significant growth inhibition with full
panel median growth inhibition (GI50) 5.47 and 2.24 μM, respectively. Both 5c and 7d hybrids
exhibited broad spectrum antitumor activity; however 7d showed moderate selectivity (selectivity
ratio 3.6) toward leukemia.
Keywords: synthesis, pyrazolinyl-pyrazole, pyrazolinyl-triazole,hybrids, anticancer activity/ NCI
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1. Introduction
Cancer is one of the most challenging diseases in the modern world. According to
World Health Organization, one person among three is affected and a quarter of all deaths in the
developed world is caused by cancer, during the year 2000. After heart diseases, it is the largest
cause of death [1]. Development of resistance against the existing anticancer drugs keeps
discovering new anticancer agents critically important. Therefore, the search for new anticancer
agents continues to be the target of various scientific efforts.
Pyrazoline template is one of the privileged structure fragments in modern medicinal chemistry
considering its broad pharmacological spectrum and affinity for various biotargets.
Numerous pyrazoline derivatives have been found to possess considerable biological activities such
as antimicrobial [2, 3], antifungal [4], anti-inflammatory, analgesic [5], antidepressant [6],
anticonvulsant [7] and anticancer activities [8-11]. It also possesses a selective biological activity
like Nitric oxide synthase (NOS) inhibitor and Cannabinoid CB1 receptor antagonist activity [12].
On the other hand, over the past few years, much interest has been given to the
chemotherapeutic activity of pyrazoles [13-15] and triazoles [16] as potential anticancer agents. It
has been reported that some pyrazoles have antiproliferative effect on certain cell lines in addition to
their pronounced effect on cyclin dependent kinase (CDK) enzyme [17-21] and extracellular
regulated kinase (ERK) pathway [22-24] which play a key role during cell division. The biological
profiles of these new generations of pyrazolinyl-pyrazole hybrids would represent a productive
matrix for further advancement of better anticancer specialists.
Pyrazinolyl-pyrazole hybrids I (Fig. 1) [25, 26] has displayed remarkable in vitro growth inhibitory
activity pattern against leukemia CCRF-CEM, MOLT-4, lung cancer HOP-92, colon cancer HCC-
2998, CNS cancer SNB-75, melanoma SK-MEL-2, ovarian cancer IGROV1, renal cancer 786-0,
RXF 393, breast cancer HS 578T and T-47D (GI50: 1.00–2.71 µM). Similar hybrid II (Fig. 1)
exhibited better inhibition profile against hCA II (Ki = 0.17 nM). [27] Additionally, triazole is
considered to be an attractive scaffold possessing anticancer activity, [28]
In view of these facts, and as a part of our ongoing research [29] in the discovery of new active
anticancer compounds, we are aiming to synthesize pyrazoline scaffold linked to pyrazole or
triazole moiety (Fig. 1) and evaluate their anticancer activity of these hybrids hoping to have
synergistic effect. In addition, the SARs study of substitution at 3-, 4- and 5- positions of pyrazole
and 5- position of triazole will be explored.
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Ph
N
N
N
H
N
N
COCH3
Cl
COCH3
Cl
N
N
N
N
O
SO2NH2
(II)
(I)
Ph
N
N
O
R
N
X
N
NH2
New prepared Hybrids
X=C, N
O
R'
R= --,CN
R'= NH2, OEt, Ph
Fig 1: Reported compounds and Design of new prazinolyl-pyrazole/1,2,3-triazole hybrids
2. Results and discussion
2.1. Chemistry
Coupling of the prepared 3-oxonitriles, 2a, b with 4-aminoantipyrine diazonium salt, 3
produced new key intermediates, 4a-d. The IR spectra of the latter compounds showed the
characteristic band of the NH at the region of 3425-3157 and a well defined band at 2223-2181 cm-1
1
corresponding to the CN groups. Moreover, H NMR spectra revealed a singlet at the range of: δ
12.56-14.30 ppm corresponding to the NH proton, the CH3CO protons of 4c appeared at δ 3.1, and
the triplet-quartet pattern of the ethyl ester protons of 4b at δ 1.2 and 4.2 ppm respectively. The
reaction of 4a-d with chloroacetonitrile or ethyl chloroacetate in DMF / TEA solution afforded the
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pyrazole-5-carbonitriles 5a-c, (Scheme 1) and the pyrazole-5-carboxylates, 7a-d (Scheme 2)
respectively in fair yields. The 1H NMR spectra of 5a-c and 7a-d revealed the disappearance of the
deshielded NH singlet of the parent, it was replaced by a singlet at the region of δ 6.08-6.60 and
5.48-5.64 ppm respectively, corresponding to the NH2 protons, in addition to the usual ethyl ester
protons of 7a-d appeared as triplet and quartet at their characteristic chemical shifts (see
supplementary document SI-1).
Reflux of 5b, c in acetic anhydride furnished the corresponding 4-acetamido pyrazole
carbonitrile derivatives 6a, b (Scheme 1). The infrared spectrum of the latter compounds showed
one band in the region 3441-3338 representing the NH group, and another band in the region 2239-
1
2231 cm-1 attributed to CN group. Their H NMR spectra showed two singlets at δ 2.30 and 10.02
ppm corresponding to CH3CO and NH protons, respectively (see supplementary document SI-1).
On the other hand, the hydrazones 4a, b reacted with hydroxylamine hydrochloride in
ethanolic sodium acetate to give amidoximes 8a, b in a good yield. Their IR spectra revealed the
absence of CN band of the parent, and new bands at the region of 3494-2850 and 3494-3156 cm-1
1
were observed corresponding to OH and NH, NH2 respectively. In addition, their HNMR spectra
showed a singlet at the region δ 6.31-6.69 ppm corresponding to NH2 protons and another singlet of
OH proton at the region δ 10.18 - 10.28 ppm. Following the procedure reported by Elnagdi et al.
[30], 1,2,3-triazole derivatives 9a-c (Scheme 2) were obtained directly by refluxing 4a, b, d with
hydroxylamine hydrochloride in presence of anhydrous sodium acetate using DMF as solvent,
(Scheme 2). Their 1H NMR spectra showed a new singlet at δ 5.40 - 5.76 ppm corresponding to the
NH2 protons. Unfortunately, attempts to prepare 9a, b by refluxing amidoximes, 8a,b in acetic
13
anhydride failed. Mass spectra and elemental analyses for all compounds, in addition to, C NMR
spectra for some compounds verified the proposed structures (see supplementary document SI-1).
2.2. In vitro anticancer screening
2.2.1. Primary in vitro one dose 60-cell line assay
Eleven compounds (5a-c, 6a, 7a-d, and 9a-c) were selected by National Cancer Institute (NCI)
to be evaluated for their anticancer activity in 60 cell line panel screening.
Primary in vitro one dose-anticancer assay was performed in full NCI 60 cell panel representing
leukemia, melanoma and cancers of lung, colon, brain, breast, ovary, kidney and prostate in
accordance with the protocol of the NCI, USA. The compounds were added at a single
concentration (10-5 M) and the culture was incubated for 48 h. Results for each compound were
reported as a mean graph of the percent growth of the treated cells when compared to the untreated
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control cells. The screening results of one dose-anticancer assay are summarized in Table 1 and
represented in Figures 2, 3 (see supplementary documents SI-2, 3).
Examining NCI results of 4-amino-3-substituted-pyrazolinylpyrazole-5-carbonitriles (5a-c &
6a) revealed that, compound 5c bearing lipophilic group at position 3 (R=Ph), exhibited a
significant growth inhibitory activity while its counterparts (5a, b & 6a) having polar groups
(R=NH2, OEt) lack anticancer activity (Table 1). Similarly, compound 7d (R=Ph) was the most
active antitumor agent among its ethyl ester series 7; it exhibited potent growth inhibitory activity
against all cell lines particularly Leukemia, Renal cancer and Melanoma cell lines (Table 1).
Additionally, the bioisosteric replacement of pyrazolyl- with 1, 2, 3-triazolyl- moiety afforded
compounds 9a-c with cytotoxic activity almost affected directly by lipophilicity order. As an
example; the most polar analog, (9a, R=NH2) was inactive, where 9b (R=OEt) with moderate
lipophilicity demonstrated anticancer activity against 3 subpanels namely, Non-Small Cell Lung
cancer, Ovarian cancer and Renal cancer, while the highly lipophilic analog, 9c (R=Ph) elicited
enhanced cytotoxicity against 4 subpanels, Non-Small Cell Lung cancer, Ovarian cancer, Breast
cancer and CNS cancer, (Table 1).
2.2.2. In vitro 5-dose 60-cell line assay
According to (NCI) protocols, the two pyrazole derivatives, 5c and 7d with satisfied pre-
determined threshold inhibition criteria were selected for NCI full panel five-dose assay. All the cell
lines (about 60), representing nine tumor subpanels, were incubated at five different concentrations
(0.01, 0.1, 1, 10 &100 mM). The outcomes were used to create log concentration Vs % growth
inhibition curves and three response parameters (GI50, TGI and LC50) were calculated for each cell
line. The GI50 value (growth inhibitory activity) corresponds to the concentration of the compound
causing 50% decrease in net cell growth, the TGI value (cytostatic activity) is the concentration of
the compound resulting in total growth inhibition and LC50 value (cytotoxic activity) is the
concentration of the compound causing net 50% loss of initial cells at the end of the incubation
period of 48 h. The screening results of five dose anticancer assay are summarized in Table 2 (see
supplementary documents SI- 4, 5).
Compound 5c (NSC-747630/1) exhibited remarkable anticancer activity against most of the
tested cell lines representing the nine different subpanels with GI50 MG-MID value 5.47 µM (Table
3). Regarding the sensitivity against some individual cell lines, it showed high activity against
leukemia SR, colon cancer HCT-15, CNS cancer SF-255, renal cancer CAKl-1, UO-31 and breast
cancer NCI/ADR-RES cell lines with GI50 values less than 1 µM (Table 2). On the other hand, the
pyrazole-5-carboxylate, 7d (NSC-747634/1) showed significant growth inhibition against most of
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the tested cell lines with GI50 MG-MID value 2.24 µM (Table 3). The obtained data revealed
obvious sensitivity profile towards leukemia subpanels with GI50 values ranging from 0.5 to 2.03
µM (Table 2). Leukemia CCRF-CEM, colon cancer HCT-15, CNS cancer SF-255, renal cancer
CAKl-1 and breast cancer NCI/ADR-RES cell lines were highly sensitive to compound 7d with
GI50 values ≤ 0.5 µM (Table 2).
The selectivity criteria of a compound depend upon the ratio obtained by dividing the full panel
MID (the average sensitivity of all cell lines towards the test agent) by their individual subpanel
MID (the average sensitivity of all cell lines of a particular subpanel towards the test agent). Ratios
3–6 refer to moderate selectivity; ratios greater than 6 indicate high selectivity towards the
corresponding cell line, while compounds not meeting either of these criteria rated non-selective
[31]. Concerning the evaluation of the active compounds in the present study, 5c and 7d were found
to be non-selective with broad-spectrum antitumor activity against the nine tumor subpanels tested.
Their selectivity ratios are ranging between 0.67–1.75 and 0.44–3.6 at the GI50 level, respectively,
particularly, compound 7d showed moderate selectivity toward the leukemia subpanel with
selectivity ratio SR=3.6 at GI50 level, (Table 3).
NCI web-resources allow the comparison of the selectivity patterns of tested compounds with
standard anticancer agents. Successful application of COMPARE algorithm could provide the
preliminary information regarding growth inhibition and cell killing mechanism. COMPARE [32]
analysis was performed for the active compounds at GI50 level, however, obtained correlation
coefficients (r) didn’t allow the explanation of the cytotoxicity mechanism of tested compounds
with high probability.
3. Conclusion
A close examination of the structures and the obtained results of the active compounds revealed
that, the anticancer activity of the pyrazolinylpyrazoles is sensitive to the nature of substituent in
position 3 of the pyrazole nucleus, as e.g. lipophilic substituent (R=Ph for 5c & 7d) enhanced
antitumor activity. Replacement of the cyano group in compound 5c with ethyl carboxylate group in
7d increased the antitumor activity especially on the leukemia subpanel (moderate selectivity).
Bioisosteric replacement of pyrazole nucleus with 1, 2, 3-triazole either abolished or reduced
activity.
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Table (1) the mean growth percent's, delta values and the growth percent's of compounds 5a-c, 6a, 7a-d and
9a-c.
Mean
NSC-
Number
Cpd
No
growth
percent
111.45
Delta
Panel
Subpanel cell lines (Growth %)
NCI-H522 (33.12).
5a
747628/1
78.33 Non-Small Cell Lung
Cancer
5b
5c
747629/1
747630/1
117.11
32.72
36.39 ــــa
ــــ
52.81 Non-Small Cell Lung
Cancer
A549/ATCC (24.14), EKVX (30.26), HOP-62
(38.43), HOP-92(45.1), NCI-H322M (29.98), NCI-
H460 (18.68), NCI-H522 (-0.67).
Colon Cancer
COLO 205 (34.68), HCC-2998 (38.85), HCT-116
(15.91), HCT-15(30.02), HT29 (41.43), KM12
(44.51), SW-620 (34.18).
Breast Cancer
Ovarian Cancer
Leukemia
MCF7 (34.35), MDA-MB-435 (48.96), MDA-MB-468
(30.89), NCI/ADR-RES (7.4), T-47D (47.23).
OVCAR-3 (21.71), OVCAR-4 (43.91), OVCAR-8 (-
15.35), SK-OV-3 (44.75).
CCRF-CEM (8.48), HL-60(TB) (10.54), MOLT-4
(15.58), RPMI-8226 (-20.09).
Renal Cancer
Melanoma
786-0 (21.38), ACHN (20.88), CAK1-1 (-2.83), TK-
10 (35.82), UO-31 (-8.31).
LOX IMVI (33.88), M14 (28.86), MALME-3M (26.02),
SK-MEL-28 (49.94), SK-MEL-5 (35.87), UACC-257
(4.07), UACC-62 (16.13).
Prostate Cancer
CNS Cancer
PC-3 (46.21).
SF-268 (37.18), SF-295 (17.92), SF-539 (28.8),
SNB-19 (77.99), U251 (28.2).
6a
7a
7b
747638/1
747631/1
747632/1
109.40
108.23
109.97
24.48 ــــ
14.73 ــــ
60.23 Non-Small Cell Lung
Cancer
ــــ
ــــ
NCI-H522(49.74)
7c
7d
747633/1
747634/1
113.53
31.54
57.87 ــــ
42.54 Non-Small Cell Lung
Cancer
ــــ
A549/ATCC (35.56), EKVX (27.88), HOP-62
(17.58), HOP-92 (44.88), NCI-H322M (48.71), NCI-
H460 (11.37), NCI-H522 (30.28).
Colon Cancer
COLO 205 (32.19), HCC-2998 (39.55), HCT-116
(14.67), HCT-15(30.85), HT29 (44.26), KM12
(38.21), SW-620 (33.47).
Breast Cancer
Ovarian Cancer
Leukemia
MCF7 (26.04), MDA-MB-435 (45.59), MDA-MB-468
(20.42), NCI/ADR-RES (5.97), T-47D (46.7).
IGROV1 (41.87), OVCAR-3 (32.99), OVCAR-4
(31.41), OVCAR-8 (19.06), SK-OV-3 (47.65).
CCRF-CEM (8.77), HL-60(TB) (3.85),K-562 (15.33),
MOLT-4 (10.04), RPMI-8226 2.39), SR (3.77).
786-0 (-11), AA98 (4.46), ACHN (15.24), TK-10
(46.35), UO-31 (9.94).
Renal Cancer
Melanoma
LOX IMVI (17.06), M14 (8.65), SK-MEL-28 (38.53),
SK-MEL-5 (1.98), UACC-257 (46.33), UACC-62
(3.95).
Prostate Cancer
CNS Cancer
PC-3 (50.14).
SF-268 (39.02), SF-295 (8.97), SF-539 (5.83), SNB-
19 (47.18), U251 (18.34).
9a
9b
747635/1
747636/1
108.15
110.05
22.36 ــــ
49.19 Non-Small Cell Lung
Cancer
ــــ
NCI-H522(62.9)
Ovarian Cancer
Renal Cancer
OVCAR-4(60.86)
CAKI-1(63.85)
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9c
747637/1
101.23
58.43 Non-Small Cell Lung
Cancer
NCI-H522(42.8)
Breast Cancer
Ovarian Cancer
CNS Cancer
MCF7(73.65)
OVCAR-4(64.19)
SNB-75(71.00)
a: inactive
Table (2): Median growth inhibitory concentrations (GI50, μM) of in vitro subpanel tumor cell lines and GI50 (μM) full
panel mean-graph mid-points (MG-MID) of compounds 5c & 7d
Subpanel tumor cell linesa
Cpd.
No.
GI50 MG-
MIDb
I
II
III
IV
V
VI
VII VIII
IX
3.15
0.62
5.76 3.59 3.49 8.16 4.09 5.45 7.34
2.28 1.97 1.06 5.07 2.03 1.38 2.39
8.19
3.37
5.47
2.24
5c
7d
a) I: Leukemia; II: non-small cell lung cancer; III: colon cancer; IV: CNS cancer; V: melanoma; VI: ovarian
cancer; VII: renal cancer; VIII: prostate cancer; IX: breast cancer.
b) GI50 full panel mean-graph midpoint (µM).
Table (3): Selectivity ratios for compounds 5c & 7d towards the nine tumor cell lines
Subpanel tumor cell linesa
Cpd.
No.
I
II
III
IV
V
VI
VII
VIII
0.75
0.94
IX
5c
7d
1.73
3.60
0.95
0.98
1.53
1.14
1.57
2.11
0.67
0.44
1.34
1.11
1.00
1.62
0.67
0.66
a) For sub-panel tumor cell lines see footnote (a) of Table (2).
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Fig. 2: one-dose mean graph of compound 5c
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Fig. 3: One-dose mean graph of compound 7d
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4. Experimental
4.1. Chemistry:
All the commercials used were laboratory grade and obtained from Sigma Co. Melting points
were measured in open capillary tubes using Griffin apparatus and were uncorrected. Elemental
microanalyses were carried out at Microanalytical Unit, Cairo University & were also obtained by
means of a LECO CHNS-932 Elemental Analyzer at Chemistry Department, Kuwait University.
The infrared (IR) spectra were recorded using potassium bromide disc technique on a Schimadzu
435 IR spectrophotometer at Micro analytical Unit, Cairo University and a Perkin Elmer 2000 FT-
IR system at Chemistry Department, Faculty of Science, Kuwait University.The proton nuclear
magnetic resonance (1H NMR) spectra were performed on a Varian Mercury VX-300 NMR
spectrophotometer 300 MHz at Faculty of Science, Cairo University and a Bruker DPX 400 MHz
superconducting spectrophotometer at Chemistry Department, Faculty of Science, Kuwait
University, using tetramethylsilane (TMS) as internal standard. Chemical shift values (δ) are given
using parts per million scales (ppm). Mass spectra were recorded on a DI-50 unit of Shimadzu GC/
MS-QP 5050A spectrometer at the Regional Centre for Mycology and Biotechnology, Al-Azhar
University, a Hewlett Packard 5988 Spectrometer at Micro analytical Unit, Cairo University and a
VG-Autospec-Q high performance tri-sector GC/MS/MS spectrometer at Chemistry Department,
Faculty of Science, Kuwait University. All reactions were monitored by TLC using precoated
Aluminium sheets silica gel Merck 60 F 254 and were visualized by UV lamp. 3-Oxobutanenitrile,
o
2c (53%), is an oil and 3-oxo-3-phenylpropanenitrile, 2d (70%), m.p. 79-80 C according to the
reported procedure [33].
2-(2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)hydrazono)-3-oxo-3-
substituted- propanenitriles (4a-d):
General procedure: 4-Aminoantipyrine (0.01 mol) was dissolved in a mixture of water (6 ml) and
hydrochloric acid (0.03 mol, 3 ml of 37 % w/v solution). A solution of sodium nitrite (0.01 mol) in
ice-cold water (least amount) was added dropwise with stirring to the cooled amine solution (0-
5oC). The diazonium salt solution, 3 so formed was then added dropwise to a solution of
cyanoacetamide, ethylcyanoacetate, or intermediates 2a,b (0.01 mol) and sodium acetate (0.03 mol)
in ethanol–water mixture with continuous stirring and cooling. After adding the diazonium salt, the
reaction mixture was stirred and cooled for additional 30 min. The precipitated hydrazone product
was filtered, washed with water, dried and crystallized from ethanol.
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2-(2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)hydrazono)-cyanoacetamide (4a):
o
Yield (87.9%); m.p. 235-237 C. IR (cm−1): 3368, 3214, 3184 (NH2), 2194 (CN), 1658, 1630
(CON); 1H NMR (400MHz, DMSO-d6): 2.28 (s, 3H, CH3), 3.13 (s, 3H, N-CH3), 7.32-7.56 (m, 5H,
Ph-H), 7.80 (s, 1H, NH2), 7.99 (s, 1H, NH2), 13.72 (s, 1H, NH); MS (m/z %): 298 (M+, 83.0), 254
(17.0), 229 (5.0), 202 (58.0), 56 (100.0); Anal. Calcd for C14H14N6O2: C, 56.37; H, 4.73, Found: C,
56.21; H, 4.92.
Ethyl 2-cyano-2-(2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-yl)hydrazono) acetate
(4b): Yield (94.8%); m.p. 177-178 oC. IR (cm−1): 3157 (NH), 2223 (CN), 1686 (COO),1665 (CON);
1H NMR (400MHz, DMSO- d6): 1.20 (t, 3H, CH3 CH2), 2.29 (s, 3H, CH3), 3.14 (s, 3H, NCH3), 4.22
13
(q, 2H, CH3CH2), 7.30-7.70 (m, 5H, Ph-H), 12.56 (s, H, NH); C NMR (100 MHz, DMSO- d6):
10.70 (CH3), 13.80 (CH3CH2), 35.39 (NCH3), 61.70 (CH2O), 103.0, 111.80, 115.87, 124.50 (2C),
127.30, 129.30 (2C), 134.30, 145.40, 158.40 (CON), 161.40 (COO); MS (m/z %): 327 (M+, 92.0),
282 (3.0), 254 (22.0), 229 (8.0), 202 (92.0), 83 (100.0); Anal. Calcd for C16H17N5O3: C, 58.71; H,
5.23, Found: C, 58.37; H, 5.25.
3-Cyano-3-(2- (1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-yl) hydrazono) acetone
(4c): Yield (51%); m.p. 268-271 oC. IR (cm−1): 3250 (NH), 2220 (CN), 1660 (CON); 1H NMR (400
MHz, DMSO- d6): 2.68 (s, 3H, CH3), 3.10 (s, 3H, CH3CO), 3.40 (s, 3H, NCH3), 7.30-7.60 (m, 5H,
Ph-H), 14.30 (s, H, NH); MS (m/z %): 296 (M+-1, 3.6), 281 (7.1), 253 (5.3), 226 (3.8), 202 (3.7),
149 (33.3), 55 (100.0); Anal. Calcd for C15H15N5O2: C, 60.6; H, 5.09, Found: C, 60.20; H, 4.98.
N`-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazole-4-yl)-2-oxo-2-phenyl-acetohydrazonyl
o
cyanide (4d): Yield (98%); m.p. 225-228 C. IR (cm−1): 3425 (NH), 2181 (CN), 1648 (CO), 1638
(CON); 1H NMR (400MHz, DMSO- d6): 2.15 (s, 3H, CH3), 3.33 (s, 3H, NCH3), 7.31-7.43 (m, 10H,
Ph-H), 12.50 (s, H, NH); MS (m/z %): 381(M++1.25 H2O, 3.0), 359 (M+, 5.0), 332 (2.0), 254 (2.0),
227 (2.0), 226 (4.0), 202 (5.0), 197 (18.5), 105 (100.0); Anal. Calcd for C20H17N5O2.1.25 H2O: C,
62.90; H, 5.15, Found: C, 62.70; H, 4.81.
4-Amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-substituted-1H-
pyrazole-5-carbonitriles (5a-c):
General procedure: Chloroacetonitrile (0.10 mol) was added to a solution of 4a,b,d (0.05 mol) in a
mixture of N,N-dimethylformamide (2 mL) and triethylamine (10 mL), the reaction mixture was
refluxed for 45 min, and then poured onto ice-cold water / HCl, the solid product so formed was
filtered off and crystallized from ethanol.
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4-Amino-5-cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-pyrazole-3-
o
carboxamide (5a): Yield (84.8%); m.p. 224-227 C. IR (cm−1): 3421, 3374 (NH2), 3312, 3200
(NH2), 2218 (CN), 1660 (CON); 1H NMR (300MHz, DMSO-d6): 2.27 (s, 3H, CH3), 3.28 (s, 3H, N-
CH3), 6.01 (s, 2H, NH2), 7.36-7.59 (m, 5H, Ph-H), 7.65 (s, 2H, CONH2); MS (m/z %): 337 (M+,
11.4), 336 (M-1, 18.2), 293 (19.0), 120 (8.7), 90 (12.0), 56 (100.0); Anal. Calcd for C16H15N7O2: C,
56.97; H, 4.48; N, 29.07, Found: C, 56.77; H, 4.77; N, 28.82.
Ethyl
4-amino-5-cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
pyrazole-3-carboxylate (5b): Yield (94.8%); m.p. 318-321oC. IR (cm−1): 3427 (br NH2), 2231 (CN),
1
1640 (br COO, CON); H NMR (400MHz, DMSO-d6): 1.31 (t, 3H, CH3CH2), 2.27 (s, 3H, CH3),
3.32 (s, 3H, N-CH3), 4.30 (q, 2H, CH3CH2), 6.08 (s, 2H, NH2), 7.39-7.57 (m, 5H, Ph-H); MS (m/z
%): 366 (M+, 100.0), 321 (5.0), 293 (90.0), 266 (7.0), 250 (1.0); Anal. Calcd for C18H18N6O3: C,
59.01; H, 4.95; N, 22.94, Found: C, 59.32; H, 4.93; N, 23.10.
4-Amino-3-benzoyl-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-pyrazole-5-
carbonitrile (5c): Yield (94.8%); m.p. charring >300oC. IR (cm−1): 3482 (br NH2), 2186 (CN), 1622
(br CO, CON); 1H NMR (400MHz, DMSO- d6): 2.15 (s, 3H, CH3), 3.36 (s, 3H, N-CH3), 6.60 (s,
2H, NH2), 7.30-7.75 (m, 10H, Ph-H); MS (m/z %): 398 (M+, 7.0), 380 (12.8), 293 (13.8), 274 (0.8),
266 (1.5), 248 (0.9), 105 (19.9), 56 (100.0); Anal. Calcd for C22H18N6O2: C, 66.32; H, 4.55; N,
21.09, Found: C, 66.11; H, 4.80; N, 20.91
4-Acetamido derivatives (6a, b)
General procedure: 0.01 mol of 5b, c was refluxed in acetic anhydride (20 ml) for 4 h. The solvent
was evaporated under vacuum and the crude product was collected and crystallized from ethanol.
Ethyl
4-acetamido-5-cyano-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
o
pyrazole-3-carboxylate (6a): Yield (37%); m.p. 222 – 225 C. IR (cm−1): 3338 (NH) , 2231 (CN) ,
1
1701 (COO), 1673 (2 CON); H NMR (400MHz, DMSO- d6): 1.31 (t, 3H, CH3CH2), 2.15 (s, 3H,
CH3), 2.30 (s, 3H, CH3CO), 3.30 (s, 3H, N-CH3), 4.34 (q, 2H, CH3CH2), 7.38-7.60 (m, 5H, Ph-H),
10.02 (s, 1H, NH); MS (m/z %):409 (M++1), 408 (M+, 25.1), 393 (2.0), 366 (23.8), 320 (2.7), 293
(32.6), 266 (5.5), 241 (1.7), 214 (2.3), 202 (8.2), 56 (100.0); Anal. Calcd for C20H20N6O4: C, 58.52;
H, 4.94; N, 20.58, Found: C, 58.65; H, 5.07; N, 20.91.
4-Acetamido-3-benzoyl-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
pyrazole-5-carbonitrile (6b): Yield (40%); m.p. 122 - 123oC. IR (cm−1): 3441 (NH), 2239 (CN),
1675 (br, CO & 2 CON); 1H NMR (300MHz, CDCl3): 2.29 (s, 3H, CH3), 2.39 (s, 3H, CH3CO), 3.35
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(s, 3H, N-CH3), 7.41-7.79 (m, 10H, Ph-H), 10.05 (s, 1H, NH); MS (m/z %): 440 (M+, 0.2), 425
(0.3), 398 (0.3), 380 (100.0), 335 (0.8), 322 (2.3), 293 (1.1), 266 (1.0), 202 (17.6); Anal. Calcd for
C24H20N6O3: C, 65.45; H, 4.58; N, 19.08, Found: C, 65.20; H, 4.78; N, 19.23.
Ethyl 4-amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-3-substituted-1H-
pyrazole-5-carboxylates (7a-d):
General procedure: Ethyl chloroacetate (0.10 mmol ) was added to a solution of 4a-d (0.05 mol) in a
mixture of N,N-dimethylformamide (2 ml) and triethylamine (10 ml ), the mixture was refluxed for
45 min, and then poured onto ice-cold water and HCl, the solid product so formed was filtered off
and crystallized from ethanol.
Ethyl
4-amino-3-carbamoyl-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
pyrazole-5-carboxylate (7a): Yield (84.8%); m.p. 224 - 227oC. IR (cm−1): 3476, 3378, 3284 (2NH2),
1
1722 (COO), 1647 (CON); H NMR (400MHz, DMSO- d6): 1.17 (t, 3H, CH3CH2), 2.17 (s, 3H,
CH3), 3.21 (s, 3H, N-CH3), 4.17 (q, 2H, CH3CH2), 5.67 (s, 2H, NH2), 7.37-7.57 (m, 5H, Ph-H), 7.63
(s, 2H, NH2CO); MS (m/z %): 385 (M+1, 40.0), 384 (M+, 100.0), 340 (23.0), 312 (2.5), 295 (20.0),
266 (63.0), 241 (7.0); Anal. Calcd for C18H20N6O4: C, 56.24; H, 5.24; N, 21.86, Found: C, 56.53; H,
5.34; N, 22.00.
Diethyl 4-amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-pyrazole-3,5-
dicarboxylate (7b): Yield (77.4%); m.p. 200-202oC. IR (cm−1): 3485, 3380 (NH2), 1725 (COO),
1
1708 (COO), 1673 (CON); H NMR (400MHz, DMSO-d6): 1.16 (t, 3H, CH3CH2, J=7.2),1.32 (t,
3H, CH3, J=7.2 ), 2.17 (s, 3H, CH3), 3.30 (s, 3H, NCH3), 4.17 (q, 2H, CH3CH2, J=7.2), 4.32 (q, 2H,
13
CH2, J=7.2), 5.64 (s, 2H, NH2), 7.39-7.55 (m, 5H, Ph-H); C NMR (100 MHz, DMSO-d6): 10.65
(CH3), 14.39 (CH3CH2), 14.67 (CH3CH2), 35.72 (NCH3), 60.73 (CH2O), 60.76 (CH2O), 111.37,
117.90, 124.50 (2C), 127.50, 129.73 (2C), 129.9, 135.00, 140.90, 152.50, 159.00 (CON), 160.71
(COO), 162.70 (COO); MS (m/z %): 414 (M+1, 60.0), 413 (M+, 100.0), 368 (12.0), 340 (18.0), 311
(2.0), 294 (20.0), 266 (62.0), 241 (22.0), 214 (52.0) 56 (87.5) ; Anal. Calcd for C20H23N5O5: C,
58.10; H, 5.61; N, 16.94, Found: C, 57.64; H, 5.67; N, 17.30.
Ethyl
3-acetyl-4-amino-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
o
pyrazole-5-carboxylate (7c): Yield (60%); m.p. 294 – 298 C. IR (cm−1): 3309, 3202 (NH2), 1708
1
(COO), 1626 (CON); H NMR (400MHz, DMSO- d6): 1.20 (t, 3H, CH3CH2), 2.19 (s, 3H, CH3),
2.68 (s, 3H, CH3CO), 3.30 (s, 3H, NCH3) , 4.20 (q, 2H, CH3CH2), 5.48 (s, 2H, NH2), 7.42-7.55 (m,
5H, Ph-H); MS (m/z %): 383 (M+, 0.8), 340 (1.33), 338 (2.0), 311 (1.44), 310 (1.4), 301 (7.6), 295
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(0.8), 266 (1.5), 51 (100.0); Anal. Calcd for C19H21N5O4: C, 59.52; H, 5.52; N, 18.27; Found: C,
59.40; H, 5.87; N, 18.01.
Ethyl
4-amino-3-benzoyl-1-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-1H-
pyrazole-5-carboxylate (7d): Yield (95%); m.p. 292-294oC. IR (cm−1): 3157 (NH), 1686 (COO),
1665 (CON); 1H NMR (400MHz, DMSO-d6): 1.20 (t, 3H, CH3CH2), 2.16 (s, 3H, CH3), 3.34 (s, 3H,
NCH3), 4.22 (q, 2H, CH3CH2), 5.30 (s, 2H, NH2), 7.30-7.70 (m, 10 H, Ph-H); MS (m/z %): 445
(M+, 80.0), 340 (24.0), 294 (20.0), 266 (40.0), 105 (70.0), 56 (100.0); Anal. Calcd for C24H23N5O4:
C, 64.71; H, 5.20; N, 15.72, Found: C, 64.52; H, 4.90; N, 16.01.
Amidoximes (8a,b):
General procedure: A mixture of 4a or 4b (0.01 mole), hydroxylamine hydrochloride (0.01 mole),
and sodium acetate (0.01 mole) in ethanol (20 ml) was refluxed for 4-6 h. After cooling the mixture
was poured onto ice-water. The solid, so formed, was collected by filtration and crystallized from
ethanol.
2-[2-(1,5-Dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4yl)hydrazin-1-ylidene]-2-(N'
hydroxycarbamidoyl)acetamide (8a): Yield (82%); m.p. 217 - 220oC. IR (cm−1): 3494-2853 (OH),
1
3494, 3451, 3337, 3156 (2NH2, NH), 1666, 1636 (CON), 1621 (C=N); H NMR (400 MHz,
DMSO- d6): 3.01 (s, 3H, CH3), 3.60 (s, 3H, NCH3), 6.69 (s, 2H, NH2), 7.22-7.54 (m, 5H, Ph-H),
13
7.86 (s, 2H, NH2CO), 10.18 (s, 1H, OH), 13.18 (s, 1H, NH); C NMR (100 MHz, DMSO-d6):
11.61 (CH3), 37.10 (NCH3), 115.40, 120.30, 121.34, 123.72, 126.80, 129.60, 135.20, 143.80,
150.80, 159.59 (C=NOH), 166.07 (CON), 168.60 (CON); MS (m/z %): 331 (M+, 10.0), 313 (100.0),
269 (66.0), 253 (2.0), 216 (16.1), 202 (18.0); Anal. Calcd for C14H17N7O3: C, 50.75; H, 5.17; N,
29.59, Found: C, 50.41; H, 5.50; N, 29.23.
Ethyl
2-[2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)hydrazin-1-ylidene]-2-(N'
hydroxycarbamidoyl) acetate (8b): Yield (79%); m.p. 187 - 190oC. IR (cm−1): 3489-2850 (OH),
3489, 3374, 3255 (NH2, NH), 1680 (COO), 1643 (CON), 1620 (C=N); 1H NMR (400MHz, DMSO-
d6): 1.28 (t, 3H, CH3CH2), 3.02 (s, 3H, CH3), 3.33 (s, 3H, NCH3), 4.20 (q, 2H, CH3CH2), 6.31 (s,
2H, NH2), 7.53-7.55 (m, 5H, Ph-H), 10.28 (s, 1H, OH), 12.94 (s, 1H, NH); MS (m/z, %): 360 (M+,
7.0), 342 (100.0), 327 (4.0), 298 (7.0), 254 (4.0), 202 (15.0); Anal. Calcd for C16H20N6O4: C, 53.33;
H, 5.59; N, 23.32, Found: C, 53.70; H, 5.90; N, 23.10.
2-(1,5-Dimethyl-2-phenyl-2,3-dihydro-3-oxo-1H-pyrazol-4-yl)-5-substituted-2H-1,2,3-triazol-
4-ylamines (9a-c):
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General procedure: A mixture of 4a,b,d (0.01 mole), and hydroxyl amine hydrochloride (0.01 mole)
was refluxed in N, N-dimethylformamide (20 ml) in presence of anhydrous sodium acetate (2 g) for
8 h. After cooling, the mixture was poured onto ice-water. The solid so-formed was collected by
filtration and crystallized from ethanol.
5-Amino-2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2H-1,2,3-triazole-4
1
carboxamide (9a): Yield (52%); m.p. 139 -141oC. IR (cm−1): 3380, 3314 (NH2), 1661 (CON); H
NMR (400MHz, DMSO- d6): 2.26 (s, 3H, CH3), 3.23 (s, 3H, N-CH3), 5.76 (s, 2H, NH2), 7.36-7.57
(m, 5H, Ph-H), 7.65 (s, 2H, NH2CO); MS (m/z %): 314 (M++1, 42.0), 313 (M+, 100.0), 269 (100.0),
241 (3.0), 217 (60.0), 202 (2.5); Anal. Calcd for C14H15N7O2: C, 53.67; H, 4.93; N, 31.29, Found: C,
53.28; H, 4.63; N, 31.01.
Ethyl 5-amino-2-(1,5-dimethyl-3-oxo-2-phenyl-2,3-dihydro-1H-pyrazol-4-yl)-2H-1,2,3-triazole-4-
carboxylate (9b): Yield (25%); m.p. 125-127. IR (cm−1): 3360, 3320 (NH2), 1689 (COO), 1660
(CON); 1H NMR (300MHz, CDCl3): 1.39 (t, 3H, CH3CH2), 2.49 (s, 3H, CH3), 3.11 (s, 3H, N-CH3),
4.36 (q, 2H, CH3CH2), 5.40 (s, 2H, NH2), 7.35-7.49 (m, 5H, Ph-H); MS (m/z %): 342 (M+, 100.0),
328 (77.3), 280 (72.7), 256 (50.0), 231 (45.5), 202 (45.5); Anal. Calcd for C16H18N6O3: C, 56.13; H,
5.30; N, 24.55, Found: C, 55.91; H, 5.03; N, 24.31.
5-Benzoyl-2-(1,5-dimethyl-2-phenyl-3-oxo-2,3-dihydro-1H-pyrazol-4-yl)-2H-1,2,3-triazol-4-
ylamines (9c): Yield (21%); m.p. 137-139oC. IR טּmax: 3451, 3321 (NH2), 1660 (br. conjugated CO,
CON); 1H NMR (300MHz, CDCl3): 2.26 (s, 3H, CH3), 3.25 (s, 3H, N-CH3), 5.70 (s, 2H, NH2),
7.31-7.62 (m, 10H, Ph-H); MS (m/z %): 374 (M+, 34.0), 359 (0.4), 268 (14.8), 253 (1.0), 228 (0.4),
226 (0.9), 216 (25.6), 202 (1.4), 56 (100.0); Anal. Calcd for C20H18N6O2: C, 64.16; H, 4.85; N,
22.45, Found: C, 64.35; H, 4.76; N, 22.30.
4.2. In vitro antitumor screening
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium
containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells
are inoculated into 96-well microtiter plates in 100 µL at plating densities ranging from 5.000 to
40.000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the
microtiter plates are incubated at 37°C, 5 % CO2, 95 % air and 100 % relative humidity for 24 h
prior to addition of experimental drugs.
After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the
cell population for each cell line at the time of drug addition. Experimental drugs are solubilized in
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dimethylsulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior
to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice
the desired final maximum test concentration with complete medium containing 50µg/mL
gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five
drug concentrations plus control. Aliquots of 100 µl of these different drug dilutions are added to
the appropriate microtiter wells already containing 100 µl of medium, resulting in the required final
drug concentrations.
Following drug addition, the plates are incubated for an additional 48 h at 37°C, 5 % CO2, 95 % air,
and 100 % relative humidity. For adherent cells, the assay is terminated by the addition of cold
TCA. Cells are fixed in situ by the gentle addition of 50 µl of cold 50 % (w/v) TCA (final
concentration, 10 % TCA) and incubated for 60 minutes at 4°C. The supernatant is discarded, and
the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100
µl) at 0.4 % (w/v) in 1 % acetic acid is added to each well, and plates are incubated for 10 minutes
at room temperature. After staining, unbound dye is removed by washing five times with 1 % acetic
acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base,
and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension
cells, the methodology is the same except that the assay is terminated by fixing settled cells at the
bottom of the wells by gently adding 50 µl of 80 % TCA (final concentration, 16 % TCA).
Acknowledgement
We are grateful to the authority of National Cancer Institute (Bethesda, Maryland, U.S.A.) for
carrying out the in vitro anticancer screening.
References:
1. G. L. Patrick, An Introduction to Medicinal Chemistry, 3rd ed., Oxford University Press Inc.,
New York, 2005, pp. 489 – 553.
2. P. Patel, S. Koregaokar, M. Shah, H. Parekh, Farmaco 1996, 51, 59.
3. V.
H.
Babu,
C.H.
Sridevi,
A.
Joseph,
K.K.
Srinivasan
Indian J Pharm. Sci. 2007, 69, 470.
4. S. B. Jadhav, R. A. Shastri, K. V. Gaikwad, S. V. Gaikwad, E-J. Chem. 2009, 6(S1), S183.
5. S.K. Sahu, M. Banerjee, A. Samantray, C. Behera, M.A. Azam, Trop. J Pharm. Res. 2008 7, 961.
6. Y. R. Prasad, A. L. Rao, L. Prasoona, K. Murali, P. R. Kumar, Bioorg. Med. Chem. Lett. 2005,
15, 5030.
This article is protected by copyright. All rights reserved.

7. Z. Ozdemir, H. B. Kandilici, B. Gumusel, U. Calis, A. A. Bilgin, Eur. J Med. Chem. 2007 42,
373.
8. A. Mansour, M.M. Eid, N. S. A. M. Khalil, Molecules 2003, 8, 744.
9. M. Shaharyar, M. M. Abdullah, M. A. Bakht, J. Majeed, Eur. J Med. Chem. 2010, 45, 114.
10. M. Shaharyar, A.A. Siddiqui, M.A. Ali, V. Murugan, R. Chandrashekhar. J. Chin.Chem. Soc.
2007, 54, 81.
11. M. Johnson, B. Younglove, L. Lee, R. LeBlanc, H. Holt, Jr., P. Hills, H. Mackay, T. Brown, S.L.
Mooberry, M. Lee, Bioorg. Med. Chem. Lett. 2007, 17, 5897.
12. K. Suresh, B. Sandhya, D. Sushma, K. Rajiv, G. Himanshu, Recent Pat. Anti-Infect. Drug
Discov. 2009, 4, 154.
13. A. M. Farag, A. S. Mayhoub, S. E. Barakat, A. H. Bayomi, Bioorg. Med. Chem. 2008, 16, 881.
14. F. Wei, B. Zhao, B. Huang, L. Zhang, C. Sun, W. Dong, D. Shinc, J. Miaob, Bioorg. Med. Chem.
Lett. 2006, 16, 6342.
15. H. Naito, M. Sugimori, I. Mitsui, Y. Nakamura, M. Ishii, M. Iwahana, K. Hirotani, E. Kumazawa,
A. Ejima, Chem. Pharm. Bull. 2005, 53, 153.
16. K. Odlo, J. Hentzen, J.F. dit Chabert, S. Ducki, O. A. B. S. M. Gani, I. Sylte, M. Skrede, V. Ann
Flّrenesd, T.V. Hanse, Bioorg. Med. Chem. 2008, 16, 4829.
17. J. Tang, L. M. Shewchuk, H. Sato, M. Hasegawa, Y. Washioa, N. Nishigakia, Bioorg. Med.
Chem. Lett. 2003, 13, 2985.
18. P. Pevarello, M. G. Brasca, R. Amici, P. Orsini, G. Traquandi, L. Corti, C. Piutti, P. Sansonna, M.
Villa, B.S. Pierce, M. Pulici, P. Giordano, K. Martina, E. L. Fritzen, R. A. Nugent, E. Casale, A.
Cameron, M. Ciomei, F. Roletto, A. Isacchi, G. P. Fogliatto, E. Pesenti, W. Pastori, A. Marsiglio,
K. L. Leach, P. M. Clare, F. Fiorentini, M. Varasi, A. Vulpetti, M. A. Warpehoski, J. Med. Chem.
2004, 47, 3367.
19. P. Pevarello, M. G. Brasca, R. Amici, P. Orsini, G. Traquandi, L. Corti, C. Piutti, P. Sansonna, M.
Villa, B.S. Pierce, M. Pulici, P. Giordano, K. Martina, E. L. Fritzen, R. A. Nugent, E. Casale, A.
Cameron, M. Ciomei, F. Roletto, A. Isacchi, G. P. Fogliatto, E. Pesenti, W. Pastori, A. Marsiglio,
K. L. Leach, P. M. Clare, F. Fiorentini, M. Varasi, A. Vulpetti, M. A. Warpehoski, J. Med. Chem.
2005, 48, 2944 .
This article is protected by copyright. All rights reserved.

20. V. Krystof, P. Cankar, I. Frysova, J. Slouka, G. Kontopidis, P. Dzubak., M.ch Hajdu, J. Srovnal,
W. F. de Jr. Azevedo, M. Orsag, M. Paprskarova, J. Rolcık, A. Latr, P. M. Fischer, M. Strnad, J.
Med. Chem. 2006, 49, 6500.
21. T. Persson, C. W. Yde, J. E. Rasmussen, T. L. Rasmussen, B. Guerra, Issinger, J. Nielsen Org.
Biomol. Chem. 2007, 5, 3963.
22. M. Ohori, T. Kinoshita, M. Okubo, K. Sato, A. Yamazaki, H. Arakawa, S. Nishimura, N.
Inamura, H. Nakajima, M. Neya, H. Miyake, T. Fujii, Biochem. Biophys. Res. Commun. 2005,
336, 357.
23. A. M. Aronov, C. Baker, G.W. Bemis, J. Cao, G. Chen, P. J. Ford, U. A. Germann, J. Green, M.
R. Hale, M. Jacobs, J. W. Janetka, F. Maltais, G. Martinez-Botella, M. N. Namchuk, J. Straub, Q.
Tang, X Xie, J. Med. Chem. 2007, 50, 1280.
24. H. Park, Y. J. Bahn, D. G. Jeong, E. J. Woob, J. S. Kwon, S. E. Ryu, Bioorg. Med. Chem. Lett.
2008, 18, 5372.
25. S.S. Abd El-Karim; M.M. Anwar; N.A. Mohamed; T. Nasr; S.A. Elseginy, Bioorg. Chem. 2015,
63, 1.
26. K. Karrouchi, S. Radi, Y. Ramli, J. Taoufik, N. Y. Mabkhot, F.A. Al-aizari and M. Ansar
Molecules 2018, 23, 134.
27. P. Khloya; M. Ceruso; S. Ram; C.T. Supuran; P.K.Sharma, Bioorg. Med. Chem. Lett. 2015, 25,
3208.
28. B. Morak-Młodawska, K. Pluta, M. Latocha, M. Jeleń, and D. Kuśmierz, Molecules. 2019, 24,
4388.
29. K. M. Amin, M. M. F. Ismail, E. Noaman, D. H. Soliman, Y. A. Ammar, Bioorg. Med. Chem.
2006, 14, 6917.
30. S. A. Ghozlan, I.A. Abdelhamid, H. M. Ibrahim, M. H. Elnagdi, ARKIVOC 2006, xv, 53.
31. S.A.F. Rostom, Bioorg. Med. Chem. 2006, 14, 6475.
32. <http://dtp.nci.nih.gov>.
33. K. Drauz, A. Kleemann, E. Wolf-Heuss, U.S. Patent 4,728,743, 1988; through
http://patft.uspto.gov.
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O
R
O
R'
R'
N
R
O
R
a
2 R;a=CH3
1 a CH3 C2H5
b C6H5 CH3
NH2
b=C6H5
O
3
b
N N
Ph
O
O
O
N
NH2
NHCOCH3
R
R
R
N
N
N
CN
CN
O
NH
c
N
N
d
O
O
N
N
N
N
N
N
Ph
Ph
Ph
4 a R = NH2
5 a R = NH2
6a R = OC2H5
= C6H5
b
c
d
= OC2H5
= CH3
= C6H5
b
c
= OC2H5
= C6H5
b
Scheme I
Reagents and conditions: a: CH3CN / NaH Toluene, reflux, b: HCl / NaNO2 / 00
c: ClCH2CN/Et3N /DMF, reflux 45 min, d: Ac2O / Reflux 4h
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O
NH2
O
OH
O
N
R
N
R
N
N
NH
b
NH
O
N
N
N
N
a
4a R = NH2
8a R = NH2
Ph
Ph
b
c
d
= OC2H5
b
= OC2H5
=
=
CH3
C6H5

c
d
O
O
NH2
NH2
R
R
N
N
N
COOC2H5
O
N
N
O
7a R = NH2
9a R = NH2
N
N
N
N
b
c
d
= OC2H5
b
c
= OC2H5
= C6H5
Ph
Ph
=
=
CH3
C6H5
Scheme 2
Reagents and conditions; a:ClCH2COOC2H5/Et3N / DMF, reflux 45 min, b: H2NOH.HCl /
NaOAc/ EtOH, reflux 4-6h; c: Ac2O/reflux 9h, d:H2NOH.HCl / NaOAc/DMF, reflux 8h
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Design, synthesis, and in-vitro anticancer screening of novel pyrazolinyl-
pyrazole/1,2,3-triazole hybrids
Magda M. F. Ismail1, Hanan G. Abd Al-Wahab1, Mohamed H. Elnagdi2
1Pharmaceutical Chemistry Department, Faculty of Pharmacy (Girls), Al-Azhar University, Nasr
City, 11754 , Cairo, Egypt..
2Department of Chemistry, Faculty of Science, University of Kuwait, Safat, 13060, Kuwait
O
O
NH2
NH2
N
N
COOC2H5
CN
N
N
O
O
N
N
N
N
Ph
Ph
7d
5c
Novel hybrids of pyrazolinlpyrazole and pyrazolinltriazole were designed and synthesized. Eleven
compounds were selected by the National Cancer Institute (NCI)/USA for anticancer screening.
Among them, two compounds: 4-aminopyrazole-5-carbonitrile, 5c (NSC-747630/1) and ethyl 4-
aminopyrazole-5- carboxylate, 7d (NSC-747634/1) were the most active candidates displaying
significant growth inhibition with full panel median growth inhibition (GI50) 5.47 and 2.24 μM,
respectively. Both 5c and 7d exhibited broad spectrum antitumor activity; however 7d showed
selectivity ratio 3.6 toward leukemia.
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