Synthesis and biological evaluation of novel pyrazoles and pyrazolo[3,4-d]pyrimidines incorporating a benzenesulfonamide moiety

Article abstract of DOI:10.1002/ardp.200800178

Synthesis and biological evaluation of novel pyrazoles and pyrazolo[3,4-d]pyrimidines are reported. Fourteen compounds were selected by the NCI and tested for their preliminary in-vitro anticancer activity, whereas all the synthesized compounds were evalu

Full text of DOI:10.1002/ardp.200800178

238
Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Full Paper
Synthesis and Biological Evaluation of Novel Pyrazoles and
Pyrazolo[3,4-d]pyrimidines Incorporating a
Benzenesulfonamide Moiety
Hayam M. A. Ashour1 and Abeer E. Abdel Wahab2
1 Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Alexandria, Alexandria, Egypt
2 Genetic Engineering and Biotechnology Research Institute (GEBRI), Mubarak City for Scientific Research
and Technology Application, Borg El-Arab, Alexandria, Egypt
Synthesis and biological evaluation of novel pyrazoles and pyrazolo[3,4-d]pyrimidines are
reported. Fourteen compounds were selected by the NCI and tested for their preliminary in-vitro
anticancer activity, whereas all the synthesized compounds were evaluated for their in-vitro anti-
microbial activity. Compound 12a was proven to possess the highest anticancer activity with a
broad spectrum profile. It showed particular effectiveness towards leukemia HL-60 (TB), K-562,
non-small cell lung cancer NCI-H23, and colon cancer HT 29, KM 12 cell lines (GI50 = 6.59, 4.44,
1.37, 3.33, and 9.63 lM, respectively). Out of the synthesized compounds, thirteen derivatives
were found to display pronounced antimicrobial activity especially against P. aeruginosa. Com-
pounds 2c, 5b, 10, 11b, 17b, 18b, and 19 were proven to be the most active with a broad spec-
trum of activity. Compound 19 was found to be equipotent to ampicillin against B. subtilis,
whereas compounds 11b and 19 were four times superior to ampicillin against P. aeruginosa,
while compounds 5b and 18b were equipotent to ampicillin against the same organism. More-
over, compounds 2c, 10, and 11b were nearly equipotent to ampicillin against E. coli. On the
other hand, compounds 2c, 5b, 10, 11a, 17b, and 18b exerted nearly half the activity of clotrima-
zole against C. albicans.
Keywords: Antimicrobial activity / Anticancer activity / Benzenesulfonamide / Pyrazoles / Pyrazolo[3,4-d]pyrimidines /
Received: September 22, 2008; accepted: November 21, 2008
DOI 10.1002/ardp.200800178
anti-angiogenic properties [6, 7] of celecoxib and SC-236
Introduction
[8] (Fig. 1). Celecoxib has been proved to be effective for
the treatment of familial adenomatous polyposis and
ongoing clinical trials are currently assessing its poten-
tial therapeutic role in both prevention and treatment of
a diverse range of human cancers [9]. Moreover, pyrazole
derivatives acting as selective inhibitors of bacterial DNA
gyrase have also been reported [10–12]. In addition, fused
pyrazoles such as pyrazolopyrimidines and related fused
heterocycles have displayed an impressive array of phar-
macological activities. They have been identified as
adenosine antagonists, tyrosine kinase, and Src-kinase
inhibitors [13–19]. Purine derivatives such as olomou-
cine and roscovitine (Fig. 1); structurally related to pyra-
zolo[3,4-d]pyrimidine compounds were found to exhibit
moderate inhibitory activity but good selectivity for a
panel of cyclin-dependent kinases (CDK) [20] which play a
key role during cell division. Recently, it was reported
In the last few years, much attention has been focused on
pyrazoles due to their diverse biological activities. Pyra-
zole-containing structures have been found among natu-
rally occurring compounds such as the antibiotic pyrazo-
furin which has been documented to possess potent anti-
cancer, antimicrobial, and broad-spectrum antiviral
activity against many RNA and DNA viruses [1]. Recently,
extensive studies have been devoted to N-aryl pyrazole
derivatives [2–5] after the discovery of the antitumor and
Correspondence: Hayam M. A. Ashour, Ph. D., Lecturer of Pharmaceut-
ical Chemistry, Department of Pharmaceutical Chemistry, Faculty of
Pharmacy, University of Alexandria, Alexandria 21521, Egypt.
E-mail: hayamashour70@yahoo.com
Fax: +20-3-4873273
Abbreviations: mean-graph midpoint values (MG-MID); minimum bacte-
rial concentration (MBC); minimal inhibitory concentration (MIC)
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
239
Figure 1. The structure of some biolog-
ically active pyrazoles and pyrazolo[3,4-
d]pyrimidines.
that a pyrazolo[3,4-d]pyrimidin-4-one subunit is a signifi-
To ameliorate the pharmacological profile of the pyra-
cant antimicrobial activity enhancing group which zole derivatives it became worthwhile to introduce the
inhibits DNA polymerase III [21]. amidic pharmacophores which are believed to be respon-
On the other hand, antibacterial sulfonamides con- sible for the biological significance of some relevant nat-
tinue to play an important role in chemotherapy either ural and synthetic chemotherapeutic agents [29, 30].
alone or in combination with other drugs. Besides, classi- Moreover, Schiff' bases with their effective contribution
cal human carbonic anhydrase inhibitors characterized as potential chemotherapeutic agents [31, 32] were not
by the presence of aromatic or heteroaromatic sulfona- far of our attention. Regarding the pyrazolo [3,4-d] pyri-
mide scaffolds have gained much interest as possible midine derivatives, it was planned to introduce variable
antitumor agents [22–24] especially after the discovery substituents in position 4 of the pyrazolopyrimidine core
of overexpression of human carbonic anhydrase IX in structure. Such substituents were selected to confer
many cancer tissues when compared to normal cells [25].
different electronic and lipophilic characteristics to the
The above-mentioned findings, coupled with our inter- molecules. Moreover, owing to the well documented
est in the chemistry of pyrazoles [26–28] envisioned our therapeutic potential associated with triazoles [33, 34],
approach towards the investigation of novel pyrazoles we deemed it interesting to prepare compounds incorpo-
and pyrazolo[3,4-d]pyrimidines incorporating a benzene- rating the triazole ring fused with the pyrazolopyrimi-
sulfonamide moiety as anticancer and antimicrobial dine nucleus in order to explore the influence of such
agents. These compounds were designed to comprise as a structural variation on the anticipated biological activ-
general feature the benzenesulfonamide moiety ities.
attached to N-1 of the biodynamic pyrazole ring. Such
structural assembly was supposed to encourage the bio-
logical potential of this class of compounds. The aim of
the present study is the discovery of novel structure leads
Results and discussion
that might be of use in designing new compounds Chemistry
endowed with both anticancer and antimicrobial proper- Synthesis of the intermediate and target compounds was
ties.
performed according to the reactions outlined in
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240
H. M. A. Ashour and A. E. Abdel Wahab
Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Schemes 1, 2, and 3. The amino ester 2a was prepared as tive one-dose assay has been added to the NCI-60 cell
previously described [35]. Synthesis of carboxamides screen in order to increase compound throughput and
2b, c was achieved by refluxing a mixture of the selected reduce data-turnaround time to suppliers while main-
N-substituted-2-cyano-3-ethoxyprop-2-enamides 1b, c and taining efficient identification of active compounds
4-hydrazinobenzenesulfonamide hydrochloride in etha- [40, 41]. All compounds submitted to the NCI-60 cell
nol containing anhydrous sodium acetate. Heating the screen are now tested initially at a single high dose
amino ester 2a in hydrazine hydrate 80%, gave rise to the (10 lM) in the full NCI-60 cell panel including leukemia,
target acid hydrazide 3, which, upon treatment with ace- non-small cell lung, colon, CNS melanoma, ovarian,
tyl or benzoyl chloride in dry pyridine, resulted in the renal, prostate, and breast cancer cell lines. Only com-
corresponding acyl derivatives 4a, b. The arylidene deriv- pounds which satisfy pre-determined threshold inhibi-
atives 5a, b were prepared following previously reported tion criteria would proceed to the five-dose screen. The
reaction conditions [36]. Refluxing 5a, b with thioglycolic threshold inhibition criteria for proceeding to the five-
acid in dry dioxane afforded the respective thiazolidi- dose screen was designed to efficiently capture com-
nones 6a, b. Synthesis of oxadiazole-2-thiol
7 was pounds with anti-proliferative activity, and it is based on
achieved following the reaction conditions adopted for careful analysis of historical Development Therapeutic
the preparation of related compounds [37]. Reacting the Program (DTP) screening data. Data are reported as a
acid hydrazide 3 with an equimolar amount of acetyl ace- mean graph of the percent growth of treated cells, and
tone in refluxing ethanol, yielded the corresponding 3,5- presented as percentage growth inhibition (GI%) caused
dimethylpyrazole 8. The key intermediate thione 10 was by the test compounds (Table 1). Moreover, three
prepared from compound 9 [35] utilizing the same proce- response parameters (GI50, TGI, and LC50) were calculated
dure for the preparation of allied compounds [38]. The S- for each cell line for compound 12a (Table 2). The GI50
alkyl analogs 11a–d were prepared according to the pro- value corresponds to the compound concentration caus-
cedure reported by Tromp et al. [39]. Fusion of the methyl- ing 50% decrease in net cell growth. The TGI value is the
thio analog 11a with the selected amine at 160-1708C compound concentration resulting in total growth
afforded the respective substituted amino derivatives inhibition and the LC50 value is the compound concentra-
12a–d. On the other hand, refluxing 11a with 98% hydra- tion causing a net 50% loss of initial cells at the end of
zine hydrate in ethanol furnished the corresponding the incubation period (48 h). Subpanel and fullpanel
hydrazine derivative 13, which was employed as key mean-graph midpoint values (MG-MID) for certain agents
intermediate in Scheme 3. Thus, heating 13 with ethyl are the average of individual real and default GI50, TGI, or
chloroformate in dry pyridine gave rise to the ethyl car- LC50 values of all cell lines in subpanel and fullpanel,
boxylate derivative 14. Refluxing the hydrazine deriv- respectively [42].
ative 13 in glacial acetic acid yielded the target pyrazolo-
A deep insight into the percentage growth inhibition
triazolopyrimidine 15. Synthesis of arylidene derivatives of the test compounds (Table 1) revealed that some of the
16a, b was accomplished adopting the same procedure tested subpanel tumor cell lines exhibited pronounced
for preparation of compounds 5a, b. Cyclocondensation sensitivity profiles against most of the tested compounds.
of 13 with phenacyl or 4-chlorophenacyl cyanide gave Among these, the non-small cell lung cancer HOP-92 cell
rise to the requisite 5-aminopyrazolyl derivatives 17a, b. line exhibited a variable degree of sensitivity towards
Synthesis of N-substituted thiocarbamoylhydrazino nine out of the 14 compounds selected with particular
derivatives 18a, b was accomplished by stirring a mixture sensitivity towards compounds 12a and 18a (GI% = 82.2
of 13 and the proper aryl isothiocyanate in DMF at room and 78.9, respectively). Furthermore, the CNS cancer SF-
temperature. The proposed tricyclic analog 19 was pre- 295 cell line demonstrated remarkable sensitivity
pared by refluxing 13 with carbon disulfide in dry pyri- towards ten out of the 14 compounds selected, with par-
dine. It should be noted that compound 19 was unexpect- ticular sensitivity toward compounds 12a, 16a, and 19
edly separated on refluxing the hydrazine derivative 13 (GI% = 70.5, 70.2, and 87.1%, respectively). In addition,
with aryl isothiocyanates in dry dioxane.
growth of the renal cancer CAKI-1, RXF 393, and UO-31
cell lines was variably affected by ten out of the tested
compounds. Special high activity against CAKI-1 was
Preliminary in-vitro anticancer screening
Out of the newly synthesized compounds, 14 derivatives shown by compounds 11b and 11c (GI% = 73.7 and 92.2%,
namely: 2a, 8, 10, 11a–d, 12a–d, 16a, 18a, and 19 were respectively); whereas remarkable activity against the
selected by the National Cancer Institute (NCI) in-vitro dis- RXF 393 cell line was displayed by 12a, 12d, 16a, and 19
ease-oriented human cells screening panel assay to be (GI% = 89.1, 86.6, 118.8 and 82.2%, respectively). It should
evaluated for their in-vitro antitumor activity. An effec- be noted here that compound 16a showed lethal effect
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
241
Table 1. In vitro percentage growth inhibition (GI%) caused by the test compounds against some selected tumor cell lines at the
single dose assaya).
Compound
NSC No.
747468
Panel
Subpanel tumor cell lines (% growth inhibitory activity)
2a
Renal Cancer
UO-31 (35.2)
CNS Cancer
SNB-75 (21.9)
8
10
747472
742615
CNS Cancer
SNB-75 (21.9)
HOP – 92 (45.9)
IGROV1 (21.2)
HL-60(TB) (21.3)
Non-Small Cell Lung Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Prostate Cancer
CNS Cancer
CAKI-1 (52.6),RXF 393 (50.8)
PC-3 (13.5)
SF-295 (36)
11a
11b
742616
742617
Non-Small Cell Lung Cancer
Leukemia
Renal Cancer
HOP-92 (32.2)
HL-60 (TB)(30.3), K-562(23.4), MOLT-4(20)
CAKI-1 (61.2), RXF 393 (68)
SF-295 (41.2)
A549/ATCC (19.2), HOP-62(33), HOP-92(57).
CT 116 (17.3)
CNS Cancer
Non-Small Cell Lung Cancer
Colon Cancer
Leukemia
K-562 (25.24), MOLT-4 (49.7)
CAKI-1 (73.7)
Renal Cancer
Melanoma
Prostate Cancer
CNS Cancer
M14 (33), MALME-3M (17.2), SK-MEL-5(20.5), UACC-257(43.4).
DU-145 (21.9)
SF-295 (35.5).
11c
742618
Non-Small Cell Lung Cancer
Colon Cancer
Breast Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Melanoma
CNS Cancer
HOP – 92 (49)
HCT-116 (29.4), SW-620 (26.5)
MCF 7 (16.9), MDA-MB-435 (23.8).
OVCAR-3 (15.6), OVCAR-8 (16.5)
HL-60 (TB)(27.6), K-562(36.8), SR(42.9)
CAKI-1 (92.2), RXF 393 (65.8)
MALME-3M(30.9), SK-MEL-5(20.9), UACC-257(34.5)
SF-295 (45.5)
11d
12a
742619
742621
Non-Small Cell Lung Cancer
Renal Cancer
Melanoma
HOP-62 (18),HOP-92(42.2)
RXF 393 (63.7)
UACC-257 (20.5)
CNS Cancer
SF-295 (63) , SNB-19 (16.9)
A549/ATCC (24), HOP-92(82.2), NCI-H322M (28.1).
HCT-15 (27.6), HT29 (58.6), KM12 (24.5).
IGROV1 (43.3)
HL-60 (TB) (47.8), K-562 (61.8), MOLT-4(31.2), RPMI-8226 (47.3).
CAKI-1 (62), RXF 393 (89.1), TK-10(29.2), UO-31(84.7).
LOXIMVI(29.8), M14(30.4),SK-MEL-5(20.3), UACC-257(18.6)
PC-3 (20.5).
SF-295 (70.5), U251 (24.9).
HOP – 92 (23.4)
HCT-116 (19.9), HCT-15 (17.9), HT 29 (35.5).
MDA-MB-468 (25.4).
Non-Small Cell Lung Cancer
Colon Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Melanoma
Prostate Cancer
CNS Cancer
Non-Small Cell Lung Cancer
Colon Cancer
Breast Cancer
Leukemia
12b
747471
CCRF-CEM (46.7), HL-60(TB)(35.7),K-562(49.5),RPMI-8226(28.9).
RXF 393 (22.1), SN 12C (19.6), UO-31 (27).
M14 (16.6).
Renal Cancer
Melanoma
CNS Cancer
SNB-75 (17.4), U251 (18.6).
NCI-H322M (19.4).
MDA–MB–231/ATCC (17.2) , NCI / ADR-RES (30.1) ,T-47D (18.3)
IGROV1 (40.4).
12c
12d
742622
742623
Non-Small Cell Lung Cancer
Breast Cancer
Ovarian Cancer
Leukemia
K-562 (37.4), SR (57.5).
Renal Cancer
Melanoma
CAKI-1 (29.7), UO-31 (36.1).
LOX IMVI (22.6), SK-MEL-5 (24.1).
A549/ATCC (17), HOP-92 (52.5).
HT29 (18.1), KM12 (16.4).
T-47D (19.6).
Non-Small Cell Lung Cancer
Colon Cancer
Breast Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Melanoma
CNS Cancer
IGROV1 (22)
HL-60 (TB) (29.3), K-562 (45.5), MOLT-4 (27.4), SR (36.8).
CAKI-1 (60.4), RXF 393 (86.6), UO-31(99.1).
LOXI MVI (19.2), UACC-257(20.4).
SF-295 (51.1).
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242
H. M. A. Ashour and A. E. Abdel Wahab
Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Table 1. Continued.
Compound
NSC No.
Panel
Subpanel tumor cell lines (% growth inhibitory activity)
16a
742624
Non-Small Cell Lung Cancer
Colon Cancer
Ovarian Cancer
Leukemia
Renal Cancer
Melanoma
Prostate Cancer
CNS Cancer
Non-Small Cell Lung Cancer
Renal Cancer
Melanoma
CNS Cancer
A549/ATCC (48), NCI-H23 (19.4), NCI-H460 (33.6)
HCC-2998(25.3), HCT-116(16.8), HT 29 (19.1)
IGROV1 (23.5), OVCAR-4 (45.7), OVCAR-8 (44.5)
HL-60(TB) (16.9), K-562 (16.9), MOLT-4 (28.6), SR (49.2).
CAKI-1 (46.6), RXF 393 (118.8), UO-31 (31.2)
SK-MEL-2 (29.5), UACC-257(37.6).
DU-145(26.9), PC-3 (19.4).
SF-268(23.1), SF-295(70.2), SF-539(23.8), SNB-19 (19.6).
HOP-62 (24.4), HOP-92 (78.9).
CAKI-1 (66).
18a
19
742625
742627
M14 (20.3).
SF-295 (50.4).
HOP-92 (33.5).
IGROV1 (32)
Non-Small Cell Lung Cancer
Ovarian Cancer
Renal Cancer
RXF 393 (82.2).
Melanoma
UACC-257 (20.4).
CNS Cancer
SF-295 (87.1).
a)
The data obtained from NCI in-vitro disease-oriented human tumor cell screen at 10 lM concentration.
Table 2. Growth inhibitory action (GI50) of some selected in-vitro tumor cell lines (lM)a) for compound 12a (NCS 742621).
Panel
(Subpanel cell lines) (Cytotoxicity GI50 lM)
Leukemia
Lung Cancer
CCRF-CEM (17.70), HL-60 (TB) (6.59), K-562 (4.44), MOLT-4 (14.50), RPMI-8226 (15.30), SR (29.60)
A549 / ATCC (14.30), EKVX (41.40), HOP-62 (16.70), HOP-92 (23.60), NCI-H226 (46.40), NCI-H23 (1.37), NCI-
H322M (31.50), NCI-H460 (14.90), NCI-H522 (51.10)
Colon Cancer
COLO205 (21.10), HCC-2998 (16.60), HCT-116 (27.40), HCT-15 (19.10), HT-29 (3.33), KM 12 (9.63), SW-620
(30.30)
CNS Cancer
Melanoma
SF-268 (43.10), SF- 295 (16.90), SF-539 (36.20), SNB-75 (33.50), U251 (26.90)
LOX IMVI (14.90), MALME-3M (33.00), M14 (32.80), SK-MEL-2 (44.30), SK- MEL-28 (31.60), SK- MEL-5 (24.40),
UACC-257 (39.40), UACC-62 (42.80)
Ovarian Cancer
Renal Cancer
Prostate Cancer
Breast Cancer
OVCAR-3 (38.20), OVCAR-4 (32.10), OVCAR-5 (34.90), OVCAR-8 (18.30)
786-O (18.30), A498 (24.30), ACHN (38.50), CAKI-1 (23.70), RXF 393 (29.60), TK-10 (19.10), UO-31 (23.90)
PC-3 (35.10), DU-145 (35.60)
MCF7 (49.20), NCI / ADR-RES (19.50), MDA- MB- 231 / ATCC (15.0), HS 578T (33.80), MDA-MB-435 (46.10), BT-
549 (30.30), T-47D (24.20)
a)
Data obtained from NCI in-vitro disease-oriented human cell screen.
towards the RXF 393 cell line with 18.8%. However, can be concluded that the pyrazolopyrimidine deriv-
growth of the UO-31 cell line was affected by three com- atives showed considerable activity against the tested
pounds, namely: 2a, 12a, and 12d (GI% = 35.2, 84.7, and tumor cell lines, whereas the pyrazole derivatives
99.1, respectively). In addition, the colon cancer HT 29 showed very weak or no activity, and only compound 12a
was only reasonably inhibited by compound 12a (GI% = fulfilled the requirements of selection for five-dose assay.
58.6), whereas growth of ovarian cancer OVCAR-4 and
Further interpretation of the five-dose screening data
OVCAR-8 was inhibited by compound 16a (GI% = 45.7 and for compound 12a (Table 2) revealed that this compound
44.5, respectively). More importantly, appreciable activ- was proven to be the most active member in this study
ity against leukemia SR cell line was manifested by com- with a broad spectrum of activity against most of the
pounds 11c, 12c, and 16a (GI% = 42.9, 57.5, and 49.2, tested subpanel tumor cell lines with particular effective-
respectively), while moderate activity against leukemia K ness against leukemia K-562, lung cancer NCI-H23, and
562 cell line was shown by compounds 12a, b and d (GI% colon cancer HT 29 (GI50 = 4.44, 1.37, 3.33 lM and TGI =
= 61.8, 49.5, and 45.5, respectively). The rest of the tested 37.7, 23.8, and 15.7 lM, respectively). The compound also
tumor cell lines showed weak activity or no sensitivity showed significant activity against leukemia HL-60 (TB)
towards the tested compounds (GI% = 0–20). Overall, it and colon cancer KM 12 (GI50 = 6.59 and 9.63 lM, respec-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
243
Table 3. The MG-MID (GI50, lM) and the selectivity ratio of compound 12a (NSC 742621).
MG-MIDa)
Subpanel tumor cell linesb) GI50 MG-MID (lM) (SI)c)
I
II
III
IV
V
VI
VII
VIII
IX
28.93
14.7
26.8
18.2
31.3
32.9
44.7
25.3
35.3
31.2
(1.97)
(1.08)
(1.59)
(0.92)
(0.88)
(0.65)
(1.14)
(0.82)
(0.93)
a)
GI50: Full panel mean-graph midpoint (lM).
I: leukemia; II: non-small cell lung cancer; III: colon cancer; IV: CNS cancer; V: melanoma; VI: ovarian cancer; VII: renal cancer;
b)
VIII: prostate cancer; IX: breast cancer.
SI: selectivity index.
c)
Table 4. The inhibition zones (IZ) in mm diameter of the most
active compounds.
tively). In addition, it displayed moderate activity against
leukemia CCRF-CEM, MOLT-4, RPMI-8226, lung cancer A
549/ATCC, HOP-62, NCI-H 460, colon cancer HCC-2998,
HCT-15, CNS cancer SF 295, melanoma LOX IMVI, ovarian
cancer OVCAR-8, renal cancer 786-O, TK-10 and Breast
NCI/ADR-RES, MDA-MB-231/ATCC cell lines (GI50 range
from 14.3 to 19.5 lM). The LC50 (cytotoxicity values) were
A100 lM for all tested cell lines.
The ratio obtained by dividing the compound fullpanel
MG-MID (lM) by its individual subpanel MG-MID (lM) is
considered as a measure of compound selectivity
(Table 3). Ratios between 3 and 6 refer to moderate selec-
tivity, ratios >6 indicate high selectivity toward the corre-
sponding cell line, while compounds meeting neither of
these criteria are rated non-selective [40]. Accordingly,
compound 12a proved to be non-selective.
Compound S. aureus B. subtilis P. aeruginosa E. coli C. albicans
2c
5b
9
18
14
18
13
12
12
13
13
14
20
18
14
17
15
17
18
11
13
13
16
18
18
21
18
17
18
14
16
15
15
14
13
18
18
18
19
13
16
18
12
15
15
13
14
14
16
17
16
18
18
15
14
11
15
14
16
13
14
16
15
15
17
14
15
14
10
11a
11b
11c
12b
12c
13
17b
18b
19
Antimicrobial screening
potency of the active compounds against S. aureus, com-
All the newly synthesized compounds were evaluated for pounds 11c and 17b (MIC = 12.5 lg/mL) displayed nearly
their in-vitro antibacterial activity against Staphylococcus half the activity of ampicillin, whereas the remaining
aureus and Bacillus subtilis as Gram-positive bacteria, compounds showed mild to weak activity. With regard to
Escherichia coli and Pseudomonas aeruginosa as Gram-nega- the activity against B. subtilis, only compound 19 was
tive bacteria. They were also evaluated for their in-vitro found to be equipotent to ampicillin while compounds
antifungal potential against Candida albicans. Their 5b, 11b, 12b, 12c, 13, 17b, and 18b were four times less
inhibition zones using the cup-diffusion technique [43] active than ampicillin against the same organism. Inter-
were measured and further evaluation was carried out estingly, P. aeruginosa was proven to be the most sensitive
for compounds showing reasonable inhibition zones microorganism to most of the tested compounds. Com-
(A13 mm) to determine their minimal inhibitory concen- pounds 11b and 19 (MIC = 12.5 lg/mL) were four times
tration (MIC) and minimum bacterial concentration more active than ampicillin, whereas compounds 5b,
(MBC) using the twofold serial dilution method [44]. 12c, and 18b exerted the same level of activity as ampicil-
Ampicillin was used as standard antibacterial while clo- lin, while compounds 9, 11a, 11c, 12b, 13, and 17b
trimazole was used as antifungal reference. Dimethyl- showed half the activity of ampicillin. Furthermore, com-
sulfoxide (DMSO) was used as blank and showed no anti- pounds 2c, 10, and 11b (MIC = 12.5 lg/mL) were nearly
microbial activity.
equivalent to ampicillin against E. coli, meanwhile the
As revealed from Tables 4 and 5, thirteen compounds remaining compounds showed moderate to mild activ-
displayed promising inhibitory effects on the growth of ity. On the other hand, investigation of the antifungal
the tested Gram-positive and Gram-negative microorgan- activity of the tested compounds revealed that six ana-
isms with special high activity against P. aeruginosa. logs, namely 2c, 5b, 10, 11a, 17b, and 18b were able to
Moreover, they exhibited variable degrees of antifungal produce significant growth inhibition (MIC values =
activity against C. albicans. Concerning the antibacterial 12.5 lg/mL).
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Table 5. Minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (MBC) of the most active compounds in
lg/mL.
Compound
S. aureus
B. subtilis
P. aeruginosa
E. coli
MBC
C. albicans
MIC
MBC
MIC
MBC
MIC
MBC
MIC
12.5
100
50
12.5
50
12.5
MIC
12.5
12.5
50
12.5
12.5
50
MBC
2c
5b
9
10
11a
11b
50
100
100
50
50
50
50
100
200
50
50
50
100
50
100
100
100
50
100
50
100
100
100
50
200
50
100
200
100
200
50
100
200
200
25
12.5
100
50
50
50
25
25
50
50
25
12.5
12.5
50
11c
12b
12c
13
17b
18b
19
12.5
200
50
100
12.5
100
50
5
25
200
50
200
12.5
100
50
100
50
50
50
50
50
12.5
12.5
100
50
50
50
50
50
12.5
–
100
100
50
100
100
50
12.5
50
–
200
100
50
100
100
50
25
–
–
100
200
100
100
50
100
200
12.5
25
100
–
–
100
100
50
50
100
50
100
100
50
50
100
50
–
50
100
100
12.5
12.5
100
Ampicillin
Clotrimazole
–
–
10
–
–
–
–
–
–
5
According to the MIC and MBC limits derived from the However, cyclization of 5b to the thiazolidinyl counter-
latest National Committee on Clinical Laboratory Stand- part 6b completely abolished the antimicrobial activity.
ards (NCCLS), it can be determined whether the test com-
Regarding the pyrazolo[3,4-d]pyrimidine derivatives
pound is bactericidal or bacteriostatic to the test organ- (Schemes 2, 3), the results revealed that the key inter-
ism [42]. Accordingly, and as revealed from Table 5, com- mediate 9 displayed remarkable activity only against P.
pound 17b was bactericidal against S. aureus while com- aeruginosa. Bioisosteric replacement of 4-one moiety with
pound 19 was bactericidal against B. subtilis. Compounds 4-thione resulted in compound 10 with a noticeable
5b, 12c, and 18b were also bactericidal against P. aerugi- improvement in the antimicrobial profile. This might be
nosa. In addition, compounds 2c and 11b were bacterici- attributed to the change in the electronic character as
dal against E. coli, while compound 17b was fungicidal well as lipophilic properties of the molecule. Thus, a two-
against C. albicans. The remaining compounds were bac- fold increase in antimicrobial activity against S. aureus
teriostatic against the test organisms.
was observed (MIC = 50 lg/mL), whereas a remarkable
The activity of the test compounds could be correlated increase in activity against E. coli and C. albicans was
to the structural variations and modification. Within the shown (MIC = 12.5 lg/mL for both organisms). Substitu-
pyrazole series (Scheme 1), the nature of the substituent tion of the thione group with alkyl- or aralkylthio group
at position 4 seems to manipulate the antimicrobial resulted in three active compounds (11a–c). Among
activity. Although the phenyl pyrazolecarboxamide them, compound 11c (R = CH2CH2-N(CH2CH3)2) showed
derivative 2b was totally inactive against all the tested the highest activity against S. aureus (MIC = 12.5 lg/mL),
organisms, the 4-chlorophenyl analog 2c displayed sub- while compound 11b (R = CH2C6H5) demonstrated the
stantial antimicrobial activity against P. aeruginosa, E. best activity against both P. aeruginosa and E. coli (MIC =
coli, and C. albicans (MIC = 200, 12.5, 12.5 lg/mL, respec- 12.5 lg/mL); whereas compound 11a (R = CH3) exhibited
tively), however, it showed weak antimicrobial activity substantial antifungal activity against C. albicans (MIC =
against the remaining test organisms. The prototype 3 12.5 lg/mL). Substitution of the methylthio group of 11a
did not show any antimicrobial activity against the with 4-chlorophenylamino 12b or benzylamino group
tested organisms. Acetylation, benzoylation, or conden- 12c resulted in an altered spectrum of antimicrobial
sation to the corresponding oxadiazolyl and 3,5-dime- activity. Thus, compound 12b showed a twofold increase
thylpyrazolyl derivatives (4a, b, 7, 8) did not result in any in activity against B. subtilis, while compound 12c dis-
change in the microbiological profile. On the other played a twofold increase in activity against B. subtilis
hand, condensation of 3 with aromatic aldehydes and P. aeruginosa. However, both compounds demon-
afforded one active derivative which is the 4-chloro ana- strated a pronounced decrease in activity against E. coli
log 5b that exhibited significant activity against B. subti- and C. albicans. On the contrary, introduction of a phenyl
lis, P. aeruginosa, and C. albicans (MIC = 50, 50, 12.5 lg/mL). or cyclohexyl amino group (12a, 12d) abolished the activ-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
245
Reagents and conditions: i) = H2NSO2C6H4NHNH26HCl / CH3CO2Na / EtOH, reflux; ii) = NH2NH26H2O / EtOH, reflux;
iii) = RCOCl, pyridine, rt.; iv) = R'C6H4CHO / dioxane, reflux; v) = HSCH2COOH / dioxane, reflux; vi) = CS2 / KOH / EtOH,
reflux; vii) = CH3COCH2COCH3 / EtOH, reflux.
Scheme 1. Synthesis of the intermediate and target compounds.
ity. The results also revealed that the hydrazine deriv- bial activity against S. aureus and C. albicans (MIC =
ative 13 was found to possess considerable activity 12.5 lg/mL), while compound 18b demonstrated a two-
against B. subtilis and P. aeruginosa. Replacement of the fold increase in activity against P. aeruginosa (MIC = 50 lg/
hydrazino group with 5-amino-3-(4-chlorophenyl)pyra- mL) and an eightfold increase in activity against C. albi-
zolyl moiety 17b or derivatization to 4-chlorophenylthio- cans (MIC = 12.5 lg/mL). Cyclization of compound 13 to
carbamoylhydrazino 18b led to an increase in both the the corresponding pyrazolotriazolopyrimidine 19
antimicrobial potential and spectrum. Accordingly, com- resulted in a dramatic increase in the antimicrobial activ-
pound 17b displayed an eightfold increase in antimicro- ity against S. aureus, B. subtilis, and P. aeruginosa (MIC = 50,
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H. M. A. Ashour and A. E. Abdel Wahab
Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Reagents and conditions: i) = HCONH2, reflux; ii) = P2S5 / pyridine, reflux; iii) = RX / anhydrous K2CO3 / DMF, rt.; iv) = R'NH2, heat at
160-1708C; v) = NH2NH26H2O, EtOH, reflux.
Scheme 2. Synthesis of the intermediate and target compounds.
12.5, 12.5 lg/mL, respectively). Overall, compounds 2c, 6.59, 4.44, 1.37, 3.33, and 9.63 lM, respectively). On the
5b, 10, 11b, 17b, 18b, and 19 was proven to be the most other hand, thirteen of the newly synthesized com-
active antimicrobial compounds in this study.
pounds displayed promising antibacterial activity
against Gram-positive and Gram-negative bacteria with
special high activity against P. aeruginosa in addition to a
variable degree of antifungal activity against C. albicans.
Potential antibacterial and antifungal activities were
Conclusion
In conclusion, the results obtained from the preliminary confined mainly to the pyrazolopyrimidine derivatives
in-vitro anticancer screening revealed that the pyra- rather than the pyrazole ones. Compounds 2c, 5b, 10,
zolo[3,4-d]pyrimidine derivative 12a was found to be the 11b, 17b, 18b, and 19 were proven to be the most active
most active anticancer member in this study with a antimicrobial members in this investigation with a
broad spectrum of activity against most of the tested sub- broad spectrum of activity. Moreover, compounds 11b
panel tumor cell lines. According to the five-dose screen, and 19 emerged with the highest potential against P. aer-
compound 12a showed particular effectiveness towards uginosa as they were four times superior to ampicillin.
leukemia HL-60 (TB), K-562, non-small cell lung cancer The best antifungal activity was demonstrated by com-
NCI-H23, and colon cancer HT29, KM12 cell lines (GI50
=
pounds 2c, 5b, 10, 11a, 17b, and 18b which possessed half
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
247
Reagents and conditions: i) ClCOOEt, pyridine, reflux; ii) glacial CH3COOH, reflux; iii) R-C6H4CHO, dioxane, reflux; iv) R-C6H4COCH2CN,
CH3COOH, EtOH, reflux; v) R-C6H4NCS, DMF, rt.; vi) CS2, pyridine, reflux or R-C6H4NCS, dioxane, reflux.
Scheme 3. Synthesis of the intermediate and target compounds.
the activity of clotrimazole. The results also pointed out this type of compounds. Finally, these types of com-
that the 4-chloro derivatives 5b, 12b, 17b, and 18b eli- pounds could be used as a fruitful template for further
cited remarkable antimicrobial activity while their development of more potent and selective anticancer
unsubstituted phenyl analogs were devoid of antimicro- and / or antimicrobial agents.
bial activity against all the tested organisms. Thus, it
For physicochemical and analytical data and IR and 1H-
could be inferred that the presence of a chloro substitu- NMR spectra of compounds 2–19 see Tables 6 and 7,
ent imparts much towards the antibacterial power of respectively.
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248
H. M. A. Ashour and A. E. Abdel Wahab
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Table 6. Physicochemical and analytical data for compounds 2–19.
Compound
R, R1 or X
Yield (%)
M.p. (8C) (Crystallization
solvent)*
Molecular Formula
(Mol. weight)a)
2b
2c
3
4a
4b
5a
5b
6a
6b
NHC6H5
NH C6H4Cl(4)
–
CH3
C6H5
C6H5
C6H4Cl(4)
C6H5
C6H4Cl(4)
–
–
–
CH3
CH2C6H5
CH2 CH2N(C2H5)2
CH2 CH2-morpholino
62
73
82
75
65
78
80
50
51
84
86
70
75
78
69
73
65
67
74
62
75
68
74
72
75
63
65
80
84
73
315–316 (DMF / E)
319–320 (DMF / E)
296–297 (DMF / E)
280–281 (DMF / E)
277–278 (E)
300–301 (DMF / E)
307–308 (DMF / E)
257–258 (E)
273–274 (E)
268–270 (DMF / E)
244–245 (D)
>320 (DMF / W)
259–261 (D)
250–251 (D / W)
260–261 (D)
C16H15N5O3S (357.39)
C16H14ClN5O3S (391.84)
C10H12N6O3S (296.31)
C12H14N6O4S (338.34)
C17H16N6O4S (400.42)
C17H16N6O3S (384.42)
C17H15ClN6O3S (418.86)
C19H18N6O4S2 (458.52)
C19H17ClN6 O4S2 (492.96)
C11H10N6O3S2 (338.37)
C15H16N6O3S (360.39)
C11H9N5O2S2 (307.36)
C12H11N5O2S2 (321.38)
C18H15N5O2S2 (397.48)
C17H22N6O2S2 (406.53)
C17H20N6O3S2 (420.52)
C17H14N6O2S (366.41)
C17H13ClN6O2S (400.85)
C18H16N6O2S (380.43)
C17H20N6O2S (372.45)
C11H11N7O2S (305.32)
C14H15N7O4S (377.38)
C13H11N7O2S (329.34)
C18H15N7O2S (393.43)
C18H14ClN7 O2S (427.87)
C20H16N8O2S (432.46)
C20H15ClN8 O2S (466.91)
C18H16N8O2S2 (440.51)
C18H15ClN8O2S2 (474.95)
C12H9N7O2S2 (347.38)
7
8
10
11a
11b
11c
11d
12a
12b
12c
12d
13
236–237 (D)
C6H5
>320 (DMF / E)
>320 (DMF / E)
250-251 (D)
C6H4Cl(4)
CH2C6H5
cyclohexyl
–
–
–
C6H5
C6H4Cl(4)
C6H5
C6H4Cl(4)
C6H5
C6H4Cl(4)
–
263-264 (D)
253-254 (DMF / W)
>320 (DMF / E)
>320 (DMF / W)
>320 (DMF / E)
>320 (DMF / E)
306–307 (D)
14
15
16a
16b
17a
17b
18a
18b
19
>320 (D)
>320 (DMF / E)
>320 (DMF / E)
>320 (DMF / W)
* Crystallization solvent (s): DMF (N,N-dimethylformamide), E: ethanol, W: water, D: dioxane.
a)
The values found are within l 0.4% of the calculated values.
were performed at the microanalytical unit, Faculty of Science,
Cairo University, and at the central lab, Faculty of Pharmacy,
Alexandria University, Egypt and were found within l0.4% of
the theoretical values. Follow up of the reactions and checking
the purity of the compounds was made by thin layer chromatog-
raphy (TLC) on silica gel-precoated aluminium sheets (Type 60
GF254; Merck; Germany) and the spots were detected by exposure
to UV lamp at k254 nm for few seconds.
The authors are grateful to the staff of the department of
Health and Human Services, National Cancer Institute,
Bethesda, Maryland, USA for carrying out the anticancer
screening of the newly synthesized compounds.
The authors have declared no conflict of interest.
5-Amino-1-(4-sulfamoylphenyl)-N-(phenyl or 4-
Experimental
chlorophenyl)-1H-pyrazole-4-carboxamides 2b, c
A mixture of the selected N-substituted-2-cyano-3-ethoxyprop-2-
enamide (10 mmol), 4-hydrazinobenzenesulfonamide hydro-
chloride (2.24 g, 10 mmol) and anhydrous sodium acetate
(0.82 g, 10 mmol) in ethanol (10 mL) was heated under reflux for
4 h, then allowed to attain room temperature. The separated
solid product was filtered, washed with ethanol, dried, and crys-
tallized. Physicochemical and analytical data are recorded in
Tables 6 and 7.
Chemistry
Melting points were determined in open glass capillaries using
Stuart Capillary melting point apparatus (Stuart Scientific
Stone, Staffordshire, UK) and are uncorrected. Infrared (IR) spec-
tra were recorded on Perkin-Elmer 1430 infrared spectropho-
tometer (Perkin Elmer, Beaconsfield, UK). 1H-NMR spectra were
scanned on Jeol-500 MHz spectrometer (Jeol, Tokyo, Japan) using
tetramethylsilane (TMS) as internal standard and DMSO-d6 as the
solvent (chemical shifts are given in d ppm). Splitting patterns
were designated as follows: s: singlet; d: doublet; t: triplet; m:
multiplet. Mass spectra were carried out using a Schimadzu
GCMS-QP-1000EX mass spectrometer (Shimadzu, Tokyo, Japan)
at 70 eV, Faculty of Science, Cairo University. Elemental analyses
4-[5-Amino-4-(hydrazinocarbonyl)-1H-pyrazol-1-
yl]benzenesulfonamide 3
A suspension of 2a (6.2 g, 20 mmol) in hydrazine hydrate 80%
(30 mL) was heated under reflux for 8 h. The reaction mixture
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
249
Table 7. IR and 1H-NMR spectra for compounds 2–19.
IR (KBr, cm– 1
)
1H-NMR (DMSO-d6)
2b, c: 3454–3421, 3370–3354, 3277–3273, 2c: 6.72 (s, 2H, pyrazole NH2, D2O exchangeable), 7.35, 7.71 (2d, J = 8.8 Hz, 4H, chloro-
3180–3158 (NH2, NH), 1644–1626 (C = O), phenyl-H), 7.48 (s, 2H, SO2NH2, D2O exchangeable), 7.77, 7.93 (2d, J = 8.8 Hz, 4H, sulfa-
1595–1592 (C = N), 1350–1329, 1164–1162 moylphenyl C2,6-H & C3,5-H), 8.21 (s, 1H, pyrazole C3-H), 9.72 (s, 1H, NH, D2O exchange-
(SO2).
able).
3: 3348, 3174 (NH2, NH), 1616 (C = O), 1597
(C = N), 1328, 1159 (SO2).
3: 4.27 (s, 2H, hydrazine NH2, D2O exchangeable), 6.51 (s, 2H, pyrazole NH2, D2O ex-
changeable), 7.46 (s, 2H, SO2NH2, D2O exchangeable), 7.76–7.91 (2d, J = 8.03 Hz, 4H,
benzenesulfonamide C3,5-H & C2,6-H), 7.92 (s, 1H, pyrazole C3-H), 9.18 (s, 1H, NH, D2O ex-
changeable).
4a-c: 3377–3375, 3339–3330, 3168–3164
4b: (s, 2H, pyrazole NH2, D2O exchangeable), 7.47 (s, 2H, SO2NH2, D2O exchangeable),
(NH2, NH), 1675–1672, 1649–1647 (C = O),
7.48–7.58 (m, 3H, phenyl C3,4,5-H), 7.78 (d, J = 8.8 Hz, 2H, benzenesulfonamide C3,5-H),
1620–1619 (C = N), 1320–1317, 1158–1157 7.89 (d, J = 7.65 Hz, phenyl C2,6-H), 7.93 (d, J = 8.8 Hz, 2H, benzenesulfonamide C2,6-H),
(SO2).
8.07 (s, 1H, pyrazole C3-H), 9.96, 10.33 (2s, 2H, 2NH, D2O exchangeable).
5a, b: 3424, 3292, 3216, 3190 (NH2, NH),
1643, 1614 (C = O), 1338, 1163 (SO2).
5b: 6.74 (brs, 2H, NH2, D2O exchangeable), 7.48 (s, 2H, SO2NH2, D2O exchangeable), 7.49,
7.69 (2d, J = 8.4 Hz, 4H, chlorophenyl-H), 7.78, 7.93 (2d, J = 8.8 Hz, 4H, benzenesulfona-
mide C3,5-H & C2,6-H), 8.09 (s, 1H, pyrazole C3-H), 8.28 (s, 1H, N=CH), 11.38 (brs, 1H, NH,
D2O exchangeable).
6a, b: 3445, 3341 (NH2, NH), 1685, 1653
(C = O), 1604 (C = N), 1339, 1165 (SO2).
6b: 3.75, 3.92 (2d, J = 16.05 Hz, 2H, thiazolidine C5-H2), 5.84 (s, 1H, thiazolidine C2-H),
6.62 (s, 2H, pyrazole NH2, D2O exchangeable), 7.43–7.52 (m, 6H, chlorophenyl-H and
SO2NH2), 7.71, 7.90 (2d, J = 8.4 Hz, 4H, sulfamoylphenyl C2,6-H & C3,5-H), 7.84 (s, 1H, pyra-
zole C3-H), 10.15 (s, 1H, NH, D2O exchangeable).
7: 3397, 3318, 3291 (NH2), 2731 (SH), 1630,
1594 (C = N), 1522, 1296, 1092, 946 (NCS),
1328, 1174 (SO2).
7: 6.36 (s, 2H, pyrazole NH2, D2O exchangeable), 7.49 (s, 2H, SO2NH2, D2O exchangeable),
7.76 (d, J = 8.8 Hz, 2H, benzenesulfonamide C3,5-H), 7.91 (s, 1H, pyrazole C3-H), 7.93 (d, J
= 8.8 Hz, 2H, benzenesulfonamide C2,6-H), 14.59 (s, 1H, SH, D2O exchangeable).
8: 3441, 3345, 3187 (NH2), 1656 (C = O),
1611 (C = N), 1337, 1164 (SO2).
8: 2.20, 2.49 (2s, 6H, 2CH3), 6.15 (s, 1H, pyrazole C4-H), 7.16 (s, 2H, pyrazole NH2, D2O ex-
changeable), 7.50 (s, 2H, SO2NH2, D2O exchangeable), 7.76, 7.94 (2d, J = 8.8 Hz, 4H, ben-
zenesulfonamide C3,5-H & C2,6-H), 8.46 (s, 1H, pyrazole C3-H).
10: 3172, 3105 (NH2), 2766 (SH), 1635 (C = N), 10: 7.47 (s, 2H, SO2NH2), 7.98, 8.26 (2d, J = 8.4 Hz, 4H, benzenesulfonamide C3,5-H & C2,6
1539, 1301, 1093, 954 (NCS), 1337, 1154 (SO2). H), 8.32 (s, 1H, pyrazolopyrimidine C3-H), 8.48 (s, 1H, pyrazolopyrimidine C6-H), 13.95
(s, 1H, SH, D2O exchangeable).
-
11a-d: 3341–3198 (NH2), 1617–1593 (C = N), 11a: 2.69 (s, 3H, CH3), 7.47 (s, 2H, SO2NH2, D2O exchangeable), 8.0, 8.4 (2d, J = 8.4 Hz, 4H,
1337–1332, 1155–1150 (SO2), 1276–1272,
1095–1072 (C-S-C).
benzenesulfonamide C3,5-H & C2,6-H), 8.64 (s, 1H, pyrazolopyrimidine C3-H), 8.88 (s, 1H,
pyrazolopyrimidine C6-H). 11d: 2.43 (t, J = 6.9 Hz, 2H, SCH2CH2N-), 2.65 (t, J = 6.9 Hz, 2H,
SCH2CH2N), 3.52 (m, 8H, morpholine C3,5-H2 & C2,6-H2), 7.47 (s, 2H, SO2NH2, D2O ex-
changeable), 8.0, 8.4 (2d, J = 8.4 Hz, 4H, benzenesulfonamide C3,5-H & C2,6-H), 8.68 (s, 1H,
pyrazolopyrimidine C3-H), 8.89 (s, 1H, pyrazolopyrimidine C6-H).
12a-d: 3380–3345, 3327–3293 (NH2),
3198–3183 (NH), 1625–1615 (C = N), 1339,
1161–1155 (SO2).
12a: 7.13 (t, J = 7.27 Hz, 1H, phenyl C4-H), 7.35–7.43 (m, 2H, phenyl C2,6-H), 7.45 (s, 2H,
SO2NH2, D2O exchangeable), 7.82 (d, J = 7.27 Hz, 2H, phenyl C3,5-H), 7.99, 8.45 (2d, J =
8.77 Hz, 4H, benzenesulfonamide C3,5-H2 & C2,6-H2), 8.56 (s, 2H, pyrazolopyrimidine C3-
H & pyrazolopyrimidine C6-H), 10.29 (s, 1H, NH, D2O exchangeable). 12c: 4.76 (d, J = 6.1
Hz, 2H, CH2), 7.21–7.36 (m, 5H, benzyl-H), 7.42 (s, 2H, SO2NH2, D2O exchangeable), 7.96,
8.43 (2d, J = 9.15 Hz, 4H, benzenesulfonamide C3,5-H & C2,6-H), 8.41 (s, 1H, pyrazolopyri-
midine C3-H), 8.47 (s, 1H, pyrazolopyrimidine C6-H), 9.03 (t, J = 5.7 Hz, 1H, NH, D2O ex-
changeable).
13: 3313, 3207 (NH2, NH), 1584 (C = N),
1323, 1159 (SO2).
13: 4.95 (s, 2H, NH2, D2O exchangeable), 7.41 (s, 2H, SO2NH2, D2O exchangeable), 7.96 (d,
J = 8.4 Hz, 2H, benzenesulfonamide C3,5-H), 8.22 (s, 1H, pyrazolopyrimidine C3-H), 8.45
(d, J = 8.4 Hz, 2H, benzenesulfonamide C2,6-H), 8.61 (s, 1H, pyrazolopyrimidine C6-H),
9.41 (s, 1H, NH, D2O exchangeable).
14: 3331, 3256, 3216 (NH2, NH), 1724 (C = O), 14: 1.21 (t, J = 6.9 Hz, 3H, CH3), 4.1 (q, J = 6.9 ,Hz, 2H, CH2), 7.45 (s, 2H, SO2NH2, D2O ex-
1590 (C = N), 1321, 1163 (SO2), 1240, 1117,
1099 (C-O-C).
changeable), 7.98, 8.42 (2d, J = 8.4 Hz, 4H, sulfamoylphenyl C2,6-H & C3,5-H), 8.44 (s, 2H,
pyrazolopyrimidine C3-H & pyrazolopyrimidine C6-H), 9.49, 10.04 (2s, 2H, 2NH, D2O ex-
changeable).
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250
H. M. A. Ashour and A. E. Abdel Wahab
Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Table 7. Continued.
IR (KBr, cm– 1
)
1H-NMR (DMSO-d6)
15: 3288, 3186 (NH2), 1648 (C = N), 1337,
1162 (SO2).
15: 2.48 (s, 3H, CH3), 7.51 (s, 2H, SO2NH2, D2O exchangeable), 8.02, 8.34 (2d, J = 8.8 Hz,
4H, benzenesulfonamide C3,5-H & C2,6-H), 8.71 (s, 1H, pyrazolotriazolopyrimidine C5-H),
9.61 (s, 1H, pyrazolotriazolopyrimidine C9-H).
16a, b: 3361–3354, 3266–3211, 3202–3135 16a: 7.44 (s, 2H, SO2NH2, D2O exchangeable), 7.45–7.50 (m, 3H, phenyl C3,4,5-H), 7.79 (d,
(NH2, NH), 1612–1588 (C = N), 1334–1321,
1160–1155 (SO2).
J = 6.9 Hz, 2H, phenyl C2,6-H), 7.99 (d, J = 9.15 Hz, 2H, benzenesulfonamide C3,5-H), 8.27
(s, 1H, N=CH), 8.46 (d, J = 9.15 Hz, 2H, benzenesulfonamide C2,6-H), 8.50 (s, 1H, pyrazolo-
pyrimidine C3-H), 8.65 (s, 1H, pyrazolopyrimidine C6-H), 12.30 (s, 1H, NH, D2O ex-
changeable).
17a, b: 3443–3424, 3372–3341, 3216–3210, 17b: 5.94 (s, 1H, pyrazole C4-H), 7.44 (s, 2H, NH2, D2O exchangeable), 7.47 (s, 2H, SO2NH2,
3190–3175 (NH2, NH), 1643–1639, 1614–
1595 (C = N), 1338–1337, 1163–1161 (SO2).
D2O exchangeable), 7.48, 7.98 (2d, J = 7.25 Hz, 4H, chlorophenyl-H), 8.03, 8.44 (2d, J =
8.8 Hz, 4H, benzenesulfonamide C3,5-H & C2,6-H), 8.89 (s, 1H, pyrazolopyrimidine C3-H),
9.0 (s, 1H, pyrazolopyrimidine C6-H)
18a, b: 3318–3312, 3242–3229, 3140–3145 18a: 7.08–7.38 (m, 5H, phenyl-H), 7.44 (s, 2H, SO2NH2, D2O exchangeable), 7.99 (d, J =
(NH2, NH), 1592–1591 (C = N), 1329–1321,
1157–1151 (SO2), 1536–1516, 1274–1273,
1094–1093, 969–944 (NCS).
8.8 Hz, 2H, benzenesulfonamide C3,5-H), 8.35 (s, 1H, pyrazolopyrimidine C3-H), 8.45 (d, J
= 8.8 Hz, 2H, benzenesulfonamide C2,6-H), 8.55 (s, 1H, pyrazolopyrimidine C6-H), 10.19,
10.35, 10.52 (3s, 3H, 3NH, D2O exchangeable).
19: 3366, 3267 (NH2), 2792 (SH), 1593 (C = N), 7.51 (s, 2H, SO2NH2, D2O exchangeable), 8.02, 8.24 (2d, J = 8.4 Hz, 4H, benzenesulfona-
1520, 1293, 1098, 965 (NCS), 1335, 1157 (SO2). mide C3,5-H & C2,6-H), 8.66 (s, 1H, pyrazolotriazolopyrimidine C9-H), 9.03 (s, 1H, pyrazo-
lotriazolopyrimidine C5-H), 14.64 (brs, 1H, SH, D2O exchangeable).
was left overnight and the separated solid was filtered, washed
with ethanol, dried and recrystallized. Physicochemical and ana-
lytical data are recorded in Tables 6 and 7.
4-(4-Substituted amino-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)benzenesulfonamides 12a–d
A mixture of 11a (1 g, 3 mmol) and the selected amine (9 mmol)
was heated in an oil bath at 160–1708C for 6 h. The reaction
mixture was triturated with dilute hydrochloric acid, filtered,
washed with water, dried, and crystallized. Physicochemical and
analytical data are recorded in Tables 6 and 7.
4-{[4-(2-Acetyl or benzoylhydrazinocarbonyl)]-5-Amino-
1H-pyrazol-1-yl}benzensulfonamides 4a, b
A mixture of 3 (1 g, 3.38 mmol) and acetyl chloride or benzoyl
chloride (3.38 mmol) in dry pyridine (5 mL) was stirred at room
temperature for 6 h. The reaction mixture was poured onto
crushed ice and the separated solid was filtered off, washed with
water, dried, and crystallized. Physicochemical and analytical
data are recorded in Tables 6 and 7.
4-(4-Hydrazino-1H-pyrazolo[3,4-d]pyrimidin-1-
yl)benzenesulfonamide 13
To a suspension of 11a (10 g, 30 mmol) in ethanol (50 mL), hydra-
zine hydrate (12 g, 11.6 mL, 240 mmol) was added and the reac-
tion mixture was heated under reflux for 6 h. It was then left to
cool and the obtained product was filtered, washed with etha-
nol, dried, and crystallized. Physicochemical and analytical data
are recorded in Tables 6 and 7.
5-Amino-N-[4-oxo-2-(phenyl or 4-chlorophenyl)-3-
thiazolidin-3-yl]-1-(4-sulfamoylphenyl)-1H-pyrazole-4-
carboxamides 6a, b
Ethyl 2-[1-(4-sulfamoylphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-yl]hydrazinecarboxylate 14
A mixture of 5a, b (1 mmol) and thioglycolic acid (2.65 g, 2 mL,
29 mmol) in dry dioxane (30 mL) was heated under reflux for
18 h. The solvent was evaporated under reduced pressure and
then neutralized with 10% sodium carbonate solution. The solid
was filtered off, washed with water, dried, and crystallized. Phys-
icochemical and analytical data are recorded in Tables 6 and 7.
To a suspension of the hydrazine 13 (1 g, 3.3 mmol) in dry pyri-
dine (5 mL), ethyl chloroformate (0.43 g, 0.38 mL, 4 mmol) was
added dropwise while stirring in an ice bath. The reaction mix-
ture was left stirred at room temperature for 2 h, and then
heated under reflux for further 2 h. It was then allowed to cool,
poured onto crushed ice and the obtained product was filtered,
washed with water, dried, and crystallized. Physicochemical and
analytical data are recorded in Tables 6 and 7.
4-{5-Amino-4-[(3,5-dimethyl-1H-pyrazol-1-yl)carbonyl]-
1H-pyrazol-1-yl}benzenesulfonamide 8
A mixture of equimolar amounts of 3 (0.6 g, 2 mmol) and acetyl
acetone (0.2 g, 2 mmol) in absolute ethanol (15 mL) was refluxed
for 8 h, then allowed to attain room temperature. The separated
crystalline product was filtered, dried, and recrystallized. Physi-
cochemical and analytical data are recorded in Tables 6 and 7.
4-(3-Methyl-7H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-
c]pyrimidin-7-yl)benzenesulfonamide 15
A suspension of 13 (1 g, 3.3 mmol) in glacial acetic acid (8 mL)
was heated under reflux for 6 h then left to cool to room temper-
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Arch. Pharm. Chem. Life Sci. 2009, 342, 238–252
Pyrazoles and Pyrazolo[3,4-d]pyrimidines
251
ature. The separated product was filtered, washed with ethanol,
dried, and crystallized. Physicochemical and analytical data are
recorded in Tables 6 and 7.
agar was poured into sterile Petri dishes. Cups (8 mm in diame-
ter) were cut in the agar. Each cup received 0.1 mL of the 1 mg/
mL solution of the test compounds. The plates were then incu-
bated at 378C for 24 h or, in case of C. albicans, for 48 h. A control
using DMSO without the test compound was included for each
organism. Ampicillin was used as standard antibacterial, while
clotrimazole was used as antifungal reference. The resulting
inhibition zones are recorded (Table 4).
4-[4-(5-Amino-3-aryl-1H-pyrazol-1-yl)-1H-pyrazolo[3,4-
d]pyrimidin-1-yl]benzenesulfonamides 17a, b
To a suspension of 13 (1 g, 3.3 mmol) in ethanol / acetic acid mix-
ture (4 : 1) (20 mL), the appropriate phenacyl cyanide (3.3 mmol)
was added. The reaction mixture was heated under reflux for 6 h
during which a crystalline precipitate separated out. The reac-
tion mixture was cooled, filtered, washed with ethanol, and crys-
tallized. Physicochemical and analytical data are recorded in
Tables 6 and 7.
Minimal inhibitory concentration (MIC) measurement
The minimal inhibitory concentrations (MIC) of the most active
compounds were measured using the twofold serial broth dilu-
tion method [44]. The test organisms were grown in their suit-
able broth: 24 h for bacteria and 48 h for fungi at 378C. Twofold
serial dilutions of solutions of the test compounds were pre-
pared using 200, 100, 50, 25, and 12.5 lg/mL. The tubes were
then inoculated with the test organisms; each 5 mL received
0.1 mL of the above inoculum and were incubated at 378C for
48 h. Then, the tubes were observed for the presence or absence
of microbial growth. The MIC values of the prepared compounds
are listed in Table 5.
4-[4-(N-Substituted thiocarbamoylhydrazino)-1H-
pyrazolo[3,4-d]pyrimidin-1-yl]benzenesulfonamides
18a, b
To a solution of the hydrazine 13 (1 g, 3.3 mmol) in dry DMF
(5 mL), the appropriate arylisothiocyanate (3.3 mmol) was added
and the mixture was stirred at room temperature for 6 h. The
reaction mixture was poured onto crushed ice and the separated
solid product was filtered, washed with water, dried, and crystal-
lized. Physicochemical and analytical data are recorded in
Tables 6 and 7.
Minimal bactericidal concentration (MBC) measurement
MIC tests were always extended to measure the MBC as follows:
A loop-full from the tube not showing visible growth (MIC) was
spread over a quarter of Mꢀller–Hinton agar plate. After 18 h of
incubation, the plates were examined for growth. Again, the
tube containing the lowest concentration of the test compound
that failed to yield growth on subculture plates was judged to
contain the MBC of that compound for the respective test organ-
ism (Table 6).
4-(3-Sulfanyl-7H-pyrazolo[4,3-e][1,2,4]triazolo[4,3-
c]pyrimidin-7-yl)benzenesulfonamide 19
To a solution of 13 (0.6 g, 2 mmol) in dry pyridine (5 mL), carbon
disulfide (3.8 g, 3 mL, 50 mmol) was added and the mixture was
heated under reflux for 8 h. The reaction mixture was poured
onto crushed ice and the separated solid product was filtered,
washed with water, dried, and crystallized. Physicochemical and
analytical data are recorded in Tables 6 and 7.
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