COX-1/COX-2 inhibition assays and histopathological study of the new designed anti-inflammatory agent with a pyrazolopyrimidine core

Article abstract of DOI:10.1016/j.bioorg.2019.01.031

Four pyrazolopyrimidine series were prepared with a substitution at position- 4 by Schiff base, triazole, oxadiazole and pyrazole moieties (7a-f, 8a,b, 9a-f, 10a,b and 13a,b), respectively. All the synthesized compounds were evaluated in vitro against COX-2 and in vivo against carrageenan-induced rat paw edema as anti-inflammatory agents. Regarding the anti-inflammatory activity (AI) compounds 7c, 7f, 8a, and 9a showed higher activity with respect to celecoxib. Compounds 9a, 7d, and 7f were closely selective to celecoxib. Also, 7c and 7d were safer than indomethacin and similar to celecoxib as resulted from the histopathological study. In addition, the docking study that showed the binding mode of prominent pyrazolopyrimidine compounds inside the COX-2 receptor. Formation of unexpected pyrazole 13a and 13b was briefly discussed using 2D NMR.

Full text of DOI:10.1016/j.bioorg.2019.01.031

Accepted Manuscript
COX-1/COX-2 inhibition assays and histopathological study of the new de-
signed anti-inflammatory agent with a pyrazolopyrimidine core
Eman K.A. Abdelall, Phoebe F. Lamie, Amira K.M. Ahmed, EL-Shaymaa EL-
Nahass
PII:
S0045-2068(18)31338-5
DOI:
https://doi.org/10.1016/j.bioorg.2019.01.031
YBIOO 2735
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
21 November 2018
31 December 2018
20 January 2019
Please cite this article as: E.K.A. Abdelall, P.F. Lamie, A.K.M. Ahmed, E-S. EL-Nahass, COX-1/COX-2 inhibition
assays and histopathological study of the new designed anti-inflammatory agent with a pyrazolopyrimidine core,
Bioorganic Chemistry (2019), doi: https://doi.org/10.1016/j.bioorg.2019.01.031
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COX-1/COX-2 inhibition assays and histopathological study of the new designed anti-
inflammatory agent with a pyrazolopyrimidine core.
a*
a
a
b
Eman K. A. Abdelall , Phoebe F. Lamie , Amira K.M. Ahmed , EL-Shaymaa EL-Nahass
a
Department of Pharmaceutical Organic Chemistry, Faculty of Pharmacy, Beni-Suef University,
Beni-Suef 62514, Egypt
b
Department of Pathology, Faculty of Veterinary Medicine, Beni-suef University, 62511, Egypt
Abstract
Four pyrazolopyrimidine series were prepared with a substitution at position- 4 by Schiff base,
triazole, oxadiazole and pyrazole moieties (7a-f, 8a,b, 9a-f, 10a,b and 13a,b), respectively. All
the synthesized compounds were evaluated in vitro against COX-2 and in vivo against
carrageenan-induced rat paw edema as anti-inflammatory agents. Regarding the anti-
inflammatory activity (AI) compounds 7c, 7f, 8a, and 9a showed higher activity with respect to
celecoxib. Compounds 9a, 7d, and 7f were closely selective to celecoxib. Also, 7c and 7d were
safer than indomethacin and similar to celecoxib as resulted from the histopathological study. In
addition, the docking study that showed the binding mode of prominent pyrazolopyrimidine
compounds inside the COX-2 receptor. Formation of unexpected pyrazole 13a and 13b was
briefly discussed using 2D NMR.
Key words: Anti-inflammatory; Celecoxib; COX-2 inhibitors; Pyrazolo[3,4-d]
pyrimidine;
Corresponding author: Tel: +002 01141021524 fax: +002 2319397;
e-mail: eman.ahmed@pharm.bsu.edu.eg, emanabdelall70@yahoo.com
SO Me pharamacophores
2
*

1
.
Introduction
The inflammation process involves sequential activation of signaling molecules, pro-
inflammatory mediators, such as prostaglandins, leukotrienes, and oxygen free radicals [1]. Two
cyclooxygenases enzymes were responsible for activation of the production of prostaglandins
(
PG1s) and other inflammatory mediators. Cyclooxygenase-1 (COX-1) is a constitutive form
which regulates the gastrointestinal cytoprotection and maintains the renal functions [2]. The
other cyclooxygenase-2 isozyme (COX-2) catalyses the (PG1s) formation and is responsible for
pain, fever and other inflammatory symptoms [3,4]. Traditional NSAIDs as ibuprofen, aspirin or
indomethacin, suffered from many adverse effects including gastric ulceration, renal injury, and
cardiotoxicity [5]. These drawbacks are due to non-selective inhibition of both COX-1 and COX-
2
isozymes [6,7]. Selective COX-2 inhibitors like coxibs have been developed to limit those
adverse effects. In spite of coxibs safety, they are still suffering from cardiovascular side effects.
Regarding that, there is a continual need for more selective and safer COX-2 inhibitors. The
selective COX-2 inhibitors, coxibs family and its members celecoxib (I), rofecoxib (II) and
valdecoxib (III) (Fig.1), all candidates contain a pyrazole core with a diaryl substituted with a
sulphamoyl or methanesulphonyl group as selective COX-2 pharmacophores [8,9].
[
Please insert Fig.1]
Also, the pyrazole and their fused pyrazolopyrimidine drug cores are of increasing interest.
These drugs containing molecules based on the pyrazolo[3,4-d]pyrimidine ring system exhibited
a multitude of wide pharmacological properties including anti-inflammatory agent [10-11],
anticancer [12-14], tuberculostatic [15,16] and antimicrobial [17,18]. Recently, several novel

series of pyrazolo[3,4-d]pyrimidine derivative (IV) have been prepared and showed a
comparable anti-inflammatory activity (AI) to that of ketorolac against carrageenan-induced rat
paw edema [10]. Also, the sulfamoyl pyrazolopyrimidine derivative (V) exhibited good anti-
inflammatory activity compared to celecoxib (dose = 25mg/kg) [19]. Furthermore, compound
(
VI) with an amino substitution at position-4 was reported to inhibit selectively and potently
COX-2 activity in human monocytes (IC50 = 0.9 nM for COX-2 vs. IC50 = 59.6 nM for COX-1)
with an anti-angiogenic activity as well [20]. Moreover, different pharmacophores such as
pyrazole [21,22], triazole [23-25], oxadiazole [26,27] and Schiff base [28,29] were Found to
have anti-inflammatory activity. Due to the presence of additional side pocket at COX-2 active
site increase its volume to accommodate more bulky structures. Guided by the previously
mentioned studies and to a continuation of previous work [30-33], our strategy is to synthesize
new pyrazolo[3,4-d]pyrimidine derivatives aiming to be more selective COX-2 inhibitors. The
presence of an additional side pocket on COX-2 active site increases its volume to accommodate
more bulky structures. So the design of the synthesized compounds depends on the presence of a
more bulky pyrazolopyrimidine core than pyrazole, central ring of coxibs in a way to increase
fitting with larger COX-2 receptor site. This pyrazolopyrimidine central ring substituted with
phenyl ring or p-methylsulfonyl (COX-2 pharmacophore) phenyl at N1. Another amino
substitution at position- 4 was placed to increase the incidence of hydrogen bonds with receptor.
In addition, this amino group was decorated with different moieties such as Schiff bases (7a-f),
triazoles (8a, 8b, and 9a-f), oxadiazoles (10a and 10b) and pyrazoles (13a and 13b). The
resulted compounds were evaluated against COX-1\COX-2. Also, carrageenan-induced rat paw
edema model and histopathological study was operated to determine their AI and gastric safety.
In addition, docking study was performed to predict the mode of action of the target compounds

inside the COX-2 active site aiming to get new anti-inflammatory compounds with improved
activity and minor drawbacks.
2
.
Results and discussion
2
.1.
Chemistry
In a way, to synthesize the new pyrazolopyrimidine series, the precursor 5-Amino-pyrazole-4-
carbonitrile 2a and 2b were prepared through the reaction of ketene dithioacetal 1 [34] with
either PhNHNH2 or p-methylsulfonyl phenylhydrazine, respectively. Elemental analysis and
spectral data confirmed the chemical structure of compound 2b. The IR spectrum showed an
absorption band at 3332, 3444 cm-1 refer to (NH
due to the (C≡N) group. Its 1H NMR revealed two singlet signals at δ 2.52 and 3.27 ppm
corresponding to (SCH ) and (SO CH ), in sequent. Moreover, an additional singlet signal
appeared at δ 7.12 ppm, which is exchangeable with D O due to (NH ) protons. This key
2
) group and another band detected at 2214 cm-
1
3
2
3
2
2
intermediates 2a and 2b were heated and fused with formic acid to get one reported
pyrazolo[3,4-d]pyrimidine 3a [35] and the other new one 3b respectively. The IR spectrum of 3b
showed an absorption band at 3333 cm-1 refer to (NH) group and another strong band for (C=O)
group at 1678 cm-1. The 1H NMR spectrum of 3b showed singlet signal at δ 8.23 ppm
characteristic to the pyrimidine 6-H, in addition to a D O exchangeable singlet signal appeared at
2
δ 12.61 ppm due to (NH) proton. Subsequently, chlorination of 3a and 3b with phosphorus
oxychloride yielded the chloro derivative 4a [35] and 4b. The structure of 4b was illustrated via
elemental analysis and the spectral data. IR spectrum of 4b exhibited the disappearance of both
absorption bands due to (NH) and (C=O) groups in the parent compound 3b. The mass spectrum
of 4b showed the molecular ion peak at m/z 355 (M+., 17.42%) and m/z 357 (M+.+2, 6.04%)
due to the natural abundance of chlorine and sulfur isotopes in the 4b molecule. The ester

derivatives 5a and 5b were accomplished through induction of glycine ester moiety to the chloro
compounds 4a and 4b. The latter 4a and 4b underwent a nucleophilic displacement with glycine
ethyl ester hydrochloride. The structure of 5a and 5b was confirmed by their elemental analysis
1
and spectral data. The H NMR spectrum of 5a showed the pronounced ethyl ester pattern as
triplet signal at δ 1.22 ppm due to (OCH
for (OCH CH ) protons. Additionally, a doublet signal at δ 4.32 ppm due to (NCH
Also, a D O exchangeable triplet signal appeared at δ 7.32 ppm corresponding to (NH) proton of
a. Finally, hydrazinolysis of the ester group in compounds 5a and 5b using hydrazine hydrate
2
CH
3
) protons and another quartet signal at δ 4.15 ppm
2
3
2
) protons.
2
5
yielded the key intermediates acetohydrazide 6a and 6b. The structure of 6a and 6b was
1
established on the basis of elemental analysis and spectral data. The H NMR spectrum of 6a
showed the absence of the ethyl ester protons and the appearance of a singlet signal at δ 4.28
ppm for (NH
2
) protons. Another one was appeared at δ 9.24 ppm due to (NH) proton, both of
them were exchanged with D
2
O (scheme1).
[
Please insert scheme 1]
The acetohydrazide intermediates 6a and 6b were reacted with a series of various aldehydes to
1
yield the hydrazones 7a-f. The H NMR spectrum of 7a showed a singlet signal at δ 8.41 ppm
attributed to azomethine proton (N=CH). Also, condensation of the acetohydrazide 6a and 6b
1
with ethyl isothiocyanate furnished triazole-3-thiol derivatives 8a and 8b, respectively. The H
NMR spectrum of 8a showed triplet and quartet signals belong to an ethyl group at δ 1.25 and
4
.10 ppm with a coupling constant 7.2 Hz. Besides the appearance of a singlet signal at δ 13.57
1
3
attributed to thiol proton (SH). The DEPTQ C NMR spectrum of 8a showed the appearance of a
signal at δ13c 166.96 ppm corresponding to (C-SH) carbon. Similarly, compounds 9a-f were
prepared by the reaction of acetohydrazide 6a and 6b with phenyl isothiocyanate derivatives.

1
The obtained data established the chemical structure of compounds 9a-f. The H NMR spectrum
of 9a revealed the absence of signals for (NH ) and (NH) protons as in the parent compound 6a,
2
the appearance of a singlet signal at δ 13.80 ppm attributed to thiol (SH) proton and additional
multiplet signal at δ 7.35-7.55 ppm due to phenyl protons in 9a (Scheme 2).
[
Please insert Scheme 2]
Moreover, heating of the acid hydrazide 6a and 6b with an equivalent amount of CS and KOH
2
gave the new pyrazolo [3,4-d]pyrimidine derivatives 10a and 10b bearing oxadiazole scaffold at
position 4. From 10a NMR spectrum data an exchangeable singlet signal appeared at δ 14.55
ppm that attributed to a thiol (SH) proton. Additionally, oxadiazole C-5H and C-2H appeared at
1
3
δ
13c 161.89 and 178.16 ppm, sequentially in C NMR spectrum of 10b. Finally, on the way to get
pyrazolo-5-one (11) via cyclization of the acid hydrazide 6a or 6b with ethylacetoacetate
afforded the ethoxy pyrazole derivatives 13a or 13b, respectively. Elemental analysis and
1
spectral data confirmed the structure of 13a and 13b. Thus, the H NMR spectrum of 13a
exhibited triplet and quartet peaks due to ethyl ether protons at δ 1.21 and 4.14 ppm. No
1
3
evidence for the presence of THE (CH
spectrum of 13a confirmed the construction of the pyrazole ring rather than pyrazolone. Thus,
pyrazole C-4H appeared at δ13c 89.35 ppm and the ether carbons (CH ) and (CH ) at δ13c 15.35
2
) pyrazolone ring in structure (11). Also, C NMR
3
2
and 61.11 ppm, respectively (Scheme 3). The mechanism of formation 13a and 13b is illustrated
in (Fig.2).
[
Please insert scheme 3]
The pyrazole cyclization of compounds formation13a and 13b could have occurred through two
1
different pathways through the formation of two intermediate 14 or 15. All spectral data (IR, H

1
3
NMR and C NMR) were not enough to illustrate which intermediate is formed either 12 or 13.
Additional spectral NMR experiment [NOESY] (Fig.3) is operated for elucidation of structure
1
3a. Upon 3D studying of two regioisomers 12 and 13, it was found that there is a proton-
proton correlation between CH of pyrazole and CH of glycinyl moiety in form 12 while
regioisomer 13 don’t show this NOE correlation. The NOESY experiment revealed that no NOE
correlation existed between CH and CH in 13a that confirmed its structure. 13a regioisomer
was achieved through the condensation reaction of NH of hydrazide 6a with the carbonyl group
3
2
3
2
2
of acetate moiety followed by another condensation of NH of 6a with the carbonyl of ethyl ester
moiety belong to ethylacetoacetate molecule as illustrated in (Fig.2).
[
Please insert Fig.3].
2
.2.
Biological anti-inflammatory activity
2
.2.1. COX-1 and COX-2 inhibition assays
The tested compounds were subjected to in vitro inhibition of ovine COX-1/COX-2 to determine
their selectivity through the determination of the minimum inhibitory dose causing 50% activity
(
IC50). In addition the COX-2 selectivity index (S.I.) that defines as IC50 (COX-1)/IC50 (COX-2)
/
/
were determined. These values were monitored by using N, N, N , N -tetramethyl-p-
phenylenediamine at 590 nm and the enzyme immunoassay (EIA) kit [4]. The data calculated
and compared with standard drugs (celecoxib, diclofenac sodium and indomethacin).
Compounds 7a-f - 13a and 13b were evaluated and the obtained results were listed in Table 1
and represented in Fig. 4. The results revealed that all the tested compounds showed inhibitory
activity on COX-2 enzyme with IC50 range (IC50 = 0.10 – 0.38 µM) more than that on COX-1
isoform (IC50 = 5.28 – 13.11 µM). The most active compounds on COX-2 were 7d, 7f, 9a, 13a

and 7c (IC50 = 0.10 – 0.11 µM range) if compared to celecoxib (IC50 = 0.049 µM). The COX-2
selectivity index for all the tested compounds (S.I. = 14.84 – 131.10) was higher than that of both
indomethacin (S.I. = 0.080) and diclofenac sodium (S.I. = 4.52). The results revealed that
Schiff's derivatives 7c-f were weak COX-1enzyme inhibitors (IC50 = 10.24 – 12.31 µM range) in
comparison with celecoxib (IC50 = 8.1 µM) and showed high potency against COX-2 enzyme
(
IC50 = 0.10 – 0.12 µM range). Among them the appreciated thienyl 7c and 7f and the furyl 7d
with p-methyl sulfonyl substituted phenyl ring. Also the triazole derivative 9a with a COX-1
inhibitory activity (IC50 = 13.11 µM), a high potency against COX-2 (IC50 = 0.10 µM), and was
the most selective (SI = 131.10) close to celecoxib (SI = 165.30). On the other hand compound,
9
c with a 4-chlorophenyl triazole moiety was the unsuccessful choice that showed a lower
selectivity index as the COX-2 inhibitor (SI = 14.84).
[
Please insert Table 1, Fig.4]
2
.3. In vivo AI assay.
Carrageenan-induced rat paw edema method [30] was carried out to evaluate synthesized
compounds AI activity. The change in edema volume was measured after 1, 3 and 5 h compared
to standard drugs celecoxib and indomethacin (Table.2 and Fig.5).the results revealed that after
one hour, compound 10a (oxadiazole derivative) and 13a (pyrazole derivative) showed
inhibitory activity (59% and 82%), respectively that more than that of celecoxib (50%). After
three hours, 7c-f, 10b, and 13a showed AI (75 – 94% range). The long-lasting anti-inflammatory
activity was observed in Schiff's compounds 7c (with a thienyl derivative), 7d-f (bearing
SO
2
CH group), ethyl triazole derivative 8a, phenyl triazole derivative 9a and pyrazole
3
derivative 13a in the range 88 - 96%. For most of the tested compounds, % of anti-inflammatory
activity was increased after five hours than three hours and one hour except compound 13a.

[
Please insert Table 2 and Fig.5]
2
.4.
Histopathological study
Examination of histopathological lesions was proceeded to evaluate the ulcerogenic effect for the
most active compounds (7c-f, 9a and 13a) on both glandular and non-glandular portions of the
stomach and to compare the severity of lesions with those induced by celecoxib and
indomethacin as a standard (Table 3). The control negative group received 2.5% tween 80,
normal histological structure of the stomach, including glandular and non-glandular portions. In
the former, normal mucosal lining, submucosa and mucosa layers (Fig. 6 (Ia)). In the later,
normal histological structure could be Found (Fig. 6 (Ib)). Administrations of indomethacin were
associated with severe pathological lesions in the form of degenerative change and necrotic
changes in the glandular and non-glandular stomach. The former portion showed erosive and
ulcerative changes associated with lymphocytic infiltration, edema and congestion in the
submucosal layer. The muscular layer showed severe hyalinosis and diffuse leuckocyte
infiltration (Fig. 6 (IIa)). The later portion (non-glandular stomach) was suffering from severe
hyperkeratosis accompanied by focal erosive and ulcerative lesions (Fig. 6 (IIb)).
Administrations of celecoxib showed mild to moderate lesions in the glandular stomach, mainly
degenerative changes of the mucosal lining and mild leuckocyte infiltration in the submucosal
layer (Fig. 6 (IIIa)). Additionally, the mucosal lining of the non-glandular stomach portion was
suffering from mild hyperkeratosis (Fig. 6 (IIIb)). For Schiff bases, 7c (H substituted, 2-thienyl)
and 7d (-SO
2
CH substituted, 2-furyl) administrations showed similar histopathological lesions
3
in the stomach to celecoxib. Moderate degenerative changes of the mucosal lining of the

glandular portion of the stomach in both treatments could be Found associated with mild necrotic
changes in the treatment 7c and moderate lesions in that treated with 7d (Fig. 6 (IVa, Va)). The
submucosal layer showed moderate congestion, leuckocytic infiltration, and edema in the
submucosal layer. In addition to, mild hyalinosis and leuckocytic infiltration of the muscular
layer of the glandular stomach. The non-glandular stomach showed more or less normal
histological structure except for mild focal hyperkeratosis in treatment 7d (Fig. 6 (IVb, Vb)).
[Please insert Fig.6 and 7]
For 9a (N4 substituted, phenyltriazole) administrations revealed the presence of multifocal areas
of degeneration in the lining epithelium in association with moderate necrotic changes. The
submucosal layer showed moderate to severe congestion, leukocytic infiltration, hyalinosis and
moderate leuckocytic infiltration in the muscular layer (Fig. 7 (VIa)). Moderate hyperkeratosis of
the non-glandular stomach could be detected and absence of any erosive or ulcerative lesions
(
Fig. 7 (VIb)). For 7e (Schiff base -SO
2
CH substituted, 2-pyridyl) and 13a (H substituted,
3
pyrazole) administrations were associated with severe degenerative changes, necrosis of the
glandular portion of the stomach in certain areas (Fig. 7 (VIIa, VIIIa)), massive leuckocytic
infiltration and congestion of the muscular layer which showed moderate hyalinosis and the
presence of focal areas of hyperkeratosis and degenerative changes in the non-glandular stomach
(
Fig. 7 (VIIb, VIIIb)). For 7f (Schiff base -SO
2
CH substituted, 2-thienyl) administration
3
revealed the presence of the highest pathological lesions in the form of severe degenerative
changes, necrosis and ulceration in the glandular stomach which accompanied by congestion,
leuckocytic infiltration in the submucosal and mucosal layer (Fig. 7 (IXa)). Moderate erosive
and ulcerative changes and hyperkeratosis could be detected in the non-glandular stomach (Fig.
7
(IXb)).

[
Please Insert Table 3 ]
2
.4. Docking study
The docking study was performed for 7c, 7d, 7f, 9a and 13a as the most in vivo active
compounds in a way to illustrate the possible binding mode of the newly synthesized
pyrazolopyrimidines within the binding site of COX-2. The Molecular Operating Environment
(
MOE) version 2008.10 modeling software was used in this study. The crystal structure of SC-
58 bound at COX-2 active site was deposited in the protein data bank with code (PDB: 1CX2)
36]. It was reported that the presence of an additional side pocket on COX-2 active site increase
5
[
its volume to accommodate the bulky structures. This pocket allows more interaction with
Arg513 that replaced by His513 in COX-1[37]. It was observed that the ligand SC-558
(
bromocelecoxib) bound to the COX-2 active site by forming two hydrogen bonds (HBs)
between (His90 and Arg513) amino acids and –SO CH group in a distance of 2.35 and 2.47 A°,
2
3
respectively (Fig.8). In addition the energy score was -13.39 Kcal/mol. Regarding the tested
compounds, Schiff bases 7c, 7d, 7f, triazole derivative 9a and pyrazole containing compound
1
3a were docked inside the COX-2 active site (Fig. 9, 10, 11, 12 and 13, in the order). All the
tested compounds exhibited good selectivity toward COX-2 enzyme by forming 1 – 4 hydrogen
bonds with different amino acids, namely, His90, Arg513 (as with SC-558), Arg120, Ser530 and
Tyr355 with energy scores between - 13.46: -16.71 Kcal/mol. The results of docking were in
accordance with that of both in vivo anti-inflammatory evaluation and COX-2 inhibitory activity.
Thus, the most active compounds 7c and 7d showed % of anti-inflammatory activity 4, 81 and 96
%
in 7c and 2, 79 and 88 % for 7d if compared to celecoxib (50, 73 and 89 %) after 1, 3 and 5
hours, sequentially. Moreover, IC50 for 7c and 7d toward COX-2 was 0.11 and 0.10 µM,
respectively, and S.I equal to 94.54 and 123.1, in the order. Also, the histopathological study

confirmed the above results as, both 7c and 7d showed more or less normal glandular stomach
layers (mucosa, submucosa, and muscolosa) and also in the non-glandular stomach if compared
to the reference celecoxib. It is important to observe that His90 and Arg513 exhibited two
hydrogen bonds with C=O (Schiff base moiety) in 7c and -SO
2
CH group in 7d, while,
3
additional amino acids Ser530 made an additional hydrogen bond with C=O (Schiff base
moiety). The same amino acids His90 and Arg513 were the target amino acids for the ligand SC-
5
58 inside the COX-2 active site. All the obtained data (amino acids, energy scores and HB
lengths) were listed in Table 4.
[
Please Insert Table 4, Fig.8 and 9]
Conclusion
In this research, several anti-inflammatory agents with pyrazolo[3,4-d]pyrimidine cores were
synthesized. Such choice of large core than regular pyrazole was valid as a proper modification.
That most of the prepared compounds showed excellent AI activity. In addition , substitution in
position-4 with a variety of active phramacophores exhibited a good activity. Pyrazole 13a its
result was more potent and significant than celecoxib. The long lasting anti-inflammatory
activity was observed in Schiff's derivatives especially that bearing SO
moreover the triazole derivative showed a good inhibitory activity when compared to celecoxib
For gastric safety, many compounds were safe as celecoxib as obtained from the
histopathological study.
2
CH pharmacophores .
3
.
3
.
Experimental
3
.1.
Chemistry

The Griffin apparatus was used to determine the melting points and were uncorrected. Shimadzu
IR-435 spectrophotometer was used for recording IR spectra using KBr discs and the obtained
-1
1
data was represented in cm . Bruker spectrophotometer was used to carry out H NMR spectra
1
3
(
at 400 MHz) and C NMR spectra (at 100 MHz) at (Faculty of pharmacy, Beni-Suef
University, Egypt). DMSO-d and D O with TMS as an internal standard, chemical shift was
6
2
recorded in ppm on δ scale while the coupling constant (J) values were estimated in Hertz (Hz).
Mass spectra (MS) were run on a Hewlett Packard 5988 spectrophotometer (Palo Alto, CA).
Microanalysis for C, H, N (within + 0.4% of the theoretical value), was proceeded at the regional
center for Mycology and Biotechnology (Al-Azhar University, Egypt). All reactions were
monitored by thin layer chromatography (TLC) using a UV lamp. All other reagents and
compound 1 were purchased from Acros Chemical Company.
3
.1.1. General procedure for preparation of 2a and 2b
A mixture of ketene dithioacetal 1 (0.9 g, 0.005 mol), appropriate phenylhydrazine hydrochloride
0.005 mol) of each and sodium acetate (0.6 g, 0.005 mol) was dissolved in methanol or ethanol
5%. The reaction mixture was heated under reflux for 4-6 h. After cooling, the formed
precipitate was filtered, dried and crystallized from methanol to give crystals of 2a [34] or 2b.
.1.1.1. 5-Amino-3-(methylthio)-1-(4-methylsulphonylphenyl)-1H-pyrazole-4-carbonitrile
2b). yellow crystals; 90% yield; mp: 182-184°C; IR: 3444, 3332 (NH ), 3008 (aromatic C-H),
); H NMR: δ 2.52 (s, 3H, SCH ), 3.27 (s,
O ), 7.82 (d, J = 8.4 Hz, 2H, phenyl H-3, H-
), 8.07 (d, J = 8.4 Hz, 2H, phenyl H-2, H-6); C NMR : δ 13.58 (SCH ), 43.98 (SO CH ), 74.38
(
9
3
(
2
1
2
3
5
924 (aliphatic C-H), 2214 (C≡N), 1384, 1145 (SO
H, SO CH ), 7.12 (br s, 2H, NH , exchange with D
2
3
2
3
2
2
1
3
3
2
3
(
(
pyrazole C-4), 114.15(C≡N), 124.53 (phenyl C-2, C-6), 128.97 (phenyl C-3, C-5), 139.63
phenyl C-4), 141.83 (phenyl C-1), 150.72 (pyrazole C-3), 153.30 (pyrazole C-5); MS (m/z, %):

+
.
3
08 [(M) , 100%]; Anal.Calcd for C12
H, 4.15; N, 18.43.
.1.2. General procedure for preparation of 3a and 3b
H
12
N
4
O
2
2
S : C, 46.74; H, 3.92; N, 18.17. Found: C, 46.86;
3
A solution of the appropriate pyrazole derivative 2a or 2b (0.01 mol) of each in formic acid (30
ml, 85%) was heated under reflux for 10 h. The reaction mixture was cooled and poured into ice-
cooled water. The separated solid was filtered off, dried and crystallized from ethanol 95% to
give crystals of 3a [35] or 3b.
3
.1.2.1.
3-Methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4(5H)-
one (3b).White crystals; 74% yield; mp: 312-314°C; IR: 3333 (NH), 3044 (aromatic C-H), 2924
1
(
aliphatic C-H), 1678 (C=O), 1369, 1149 (SO
SO CH
.4 Hz, 2H, phenyl H-2, H-6), 12.61 (br s, 1H, NH, exchange with D
SCH ), 44.13 (SO CH ), 106.53 (pyrazolopyrimidine C-3a), 121.13 (phenyl C-2, C-6), 128.89
phenyl C-3, C-5), 138.32 (phenyl C-4), 142.34 (pyrazolopyrimidine C-7a), 147.44 (phenyl C-1),
2
); H NMR: δ 2.78 (s, 3H, SCH
3
), 3.26 (s, 3H,
2
3
), 8.07 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 8.23 (s, IH, pyrimidine C-H), 8.36 (d, J =
1
3
8
2
O ); C NMR : δ 13.17
(
(
3
2
3
1
50.52 (pyrazolopyrimidine C-6), 154.32 (pyrazolopyrimidine C-3), 157.26 (C=O); MS (m/z,
+
.
%
): 336 [(M) , 100%]; Anal.Calcd for C13
H
12
N O
4 3
2
S : C, 46.42; H, 3.60; N, 16.66. Found: C,
4
6.76; H, 3.74; N, 16.98.
.1.3. General procedure for preparation of 4a and 4b
3
The appropriate pyrazolopyrimidinone derivative 3a or 3b (0.24 mol) of each was suspended in
phosphorusoxychloride (50 ml). The mixture was heated under reflux for 4 h. The excess
phosphorusoxychloride was removed under reduced pressure. The obtained residue was
triturated with ice-cold water (250 ml). The aqueous suspension was extracted with chloroform

(
3 × 60 ml). The solvent was evaporated and the residue obtained was dried and crystallized
from methylene chloride/methanol (8:2) to obtain pure crystals of 4a [35] or 4b.
.1.3.1. 4-Chloro-3-methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-
3
d]pyrimidine (4b). Buff crystals; 92% yield; mp: 363-365°C; IR: 3048 (aromatic C-H), 2924
1
(
(
aliphatic C-H), 1354, 1145 (SO
2
); H NMR: δ 2.78 (s, 3H, SCH
3
), 3.26 (s, 3H, SO
2
CH ), 8.07
3
d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 8.23 (s, IH, pyrimidine C-H), 8.36 (d, J = 8.4 Hz, 2H,
1
3
phenyl H-2, H-6); C NMR : δ 13.17 (SCH
a), 121.13 (phenyl C-2, C-6), 128.81 (phenyl C-3, C-5), 138.32 (phenyl C-4), 142.34
pyrazolopyrimidine C-7a), 147.44 (phenyl C-1), 150.52 (pyrazolopyrimidine C-6), 154.32
3
), 44.13 (SO
2
CH
3
), 106.53 (pyrazolopyrimidine C-
3
(
(
+
.
pyrazolopyrimidine C-3), 158.36 (pyrazolopyrimidine C-4); MS (m/z, %): 355 [(M) , 17.42%],
3
3
3
48 [100%]; Anal.Calcd for C13
.30; N, 16.12.
H
11Cl N
4
O
2
S : C, 44.00; H, 3.12; N, 15.79. Found: C, 43.89; H,
2
.1.4. General procedure for preparation of 5a and 5b
To a mixture of the appropriate chloro-derivative 4a or 4b (0.01 mol) of each and glycine ethyl
ester hydrochloride (1.39 g, 0.01 mol) in absolute ethanol (30 ml), a catalytic amount of
triethylamine was added. The reaction mixture was heated under reflux for 5-6 h. The reaction
mixture was evaporated under reduced pressure. The solid obtained was crystallized from
ethanol 95% to afford compound 5a or 5b.
3
.1.4.1.
Ethyl-2-[3-(methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-4-
ylamino]acetate (5a). Yellow crystals; 86% yield; mp: 175-177°C; IR: 3387 (NH), 2928
1
(
aliphatic C-H), 1736 (C=O); H NMR: δ 1.22 (t, J = 6.8 Hz, 3H, OCH
2
CH
), 4.32 (d, J = 4.8 Hz, 2H, NHCH ), 7.32 (t, J = 4.8
O ), 7.35 (t, J = 7.2 Hz, 1H, phenyl H-4), 7.54 (t, J = 7.2 Hz, 2H,
3
), 2.74 (s, 3H,
SCH
3
), 4.15 (q, J = 6.8 Hz, 2H, OCH
2
CH
3
2
Hz, 1H, NH, exchange with D
2

1
3
phenyl H-3, H-5), 8.17 (d, J = 7.6 Hz, 2H, phenyl H-2, H-6), 8.39 (s, IH, pyrimidine C-H); C
NMR : δ 14.54 (SCH ), 15.38 (CH CH ), 42.87 (NHCH ), 61.10 (CH CH ), 101.15
pyrazolopyrimidine C-3a), 120.99 (phenyl C-2, C-6), 126.62 (phenyl C-4), 129.59 (phenyl C-3,
C-5), 138.54 (phenyl C-1), 141.55 (pyrazolopyrimidine C-7a), 153.50 (pyrazolopyrimidine C-3),
3
2
3
2
2
3
(
1
54.45 (pyrazolopyrimidine C-4), 156.69 (pyrazolopyrimidine C-6), 169.58 (C=O); MS (m/z,
+
.
%
): 343 [(M) , 25.65%], 258 [100%]; Anal.Calcd for C16
H
17
N
5
2
O S: C, 55.96; H, 4.99; N, 20.39.
Found: C, 56.23; H, 5.08; N, 20.61.
.1.4.2. Ethyl-2-[3-(methylthio)-1-(4-methylsulphonyphenyl)-1H-pyrazolo[3,4-
3
d]pyrimidine-4-ylamino]acetate (5b). Buff powder; 85% yield; mp: 365-367°C; IR: 3422 (NH),
1
2
928 (aliphatic C-H), 1736 (C=O), 1377, 1296 (SO
OCH CH ), 2.78 (s, 3H, SCH ), 3.26 (s, 3H, SO CH
d, J = 5.6 Hz, 2H, NHCH ), 7.47 (t, J = 5.6 Hz, 1H, NH, exchange with D
Hz, 2H, phenyl H-3, H-5), 8.46 (s, IH, pyrimidine C-H), 8.52 (d, J = 8.8 Hz, 2H, phenyl H-2, H-
2
); H NMR: δ 1.21 (t, J = 7.2 Hz, 3H,
2
3
3
2
3
), 4.16 (q, J = 7.2 Hz, 2H, OCH
2
CH ), 4.31
3
(
2
2
O ), 8.09 (d, J = 8.8
1
3
6
); C NMR : δ 14.55 (SCH
CH CH ), 101.41 (pyrazolopyrimidine C-3a), 120.42 (phenyl C-2, C-6), 128.95 (phenyl C-3, C-
), 137.66 (phenyl C-4), 142.74 (phenyl C-1), 143.64 (pyrazolopyrimidine C-7a), 155.34
3
), 14.90 (CH
2
CH
3
), 42.93 (NHCH
2
), 44.18 (SO
2
CH
3
), 61.15
(
2
3
5
(
pyrazolopyrimidine C-3), 156.68 (pyrazolopyrimidine C-4), 157.51 (pyrazolopyrimidine C-6),
+
.
1
70.12 (C=O); MS (m/z, %): 421 [(M) , 69.87%], 348 [100%]; Anal.Calcd for C17
H
19
N
5
O
4
2
S :
C, 48.44; H, 4.54; N, 16.62. Found: C, 48.67; H, 4.72; N, 16.89.
.1.5. General procedure for preparation of 6a and 6b
3
A mixture of compound 5a or 5b (0.01 mol) of each and hydrazine hydrate (99.9%) (2.5 ml, 0.05
mol) in absolute ethanol (30 ml) was heated under reflux for 5 h. The solid formed was filtered
while hot, dried and crystallized from dioxane to obtain compounds 6a or 6b.

3
.1.5.1.
2-[3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidine-4-
ylamino]acetohydrazide (6a). White powder; 85% yield; mp: 210-212°C; IR: 3446, 3368 (NH ),
2
1
3
3
318, 3217 (2NH), 3024 (aromatic C-H), 2928 (aliphatic C-H), 1686 (C=O); H NMR: δ 2.74 (s,
H, SCH
3
), 4.15 (d, J = 5.6 Hz, 2H, NHCH
, exchange with D O ), 7.34 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.55 (t, J =
.6 Hz, 2H, phenyl H-3, H-5), 8.16 (d, J = 7.6 Hz, 2H, phenyl H-2, H-6), 8.38 (s, IH, pyrimidine
2
), 4.28 (br s, 2H, NH
2
, exchange with D
2
O ), 7.17 (t,
J = 5.6 Hz, 1H, NHCH
2
2
7
1
3
C-H), 9.24 (br s, 1H, NH, exchange with D
2
O ); C NMR : δ 15.64 (SCH
3
), 42.75 (NHCH ),
2
1
01.50 (pyrazolopyrimidine C-3a), 120.93 (phenyl C-2, C-6), 126.57 (phenyl C-4), 129.61
(
phenyl C-3, C-5), 139.04 (phenyl C-1), 141.57 (pyrazolopyrimidine C-7a), 154.36
pyrazolopyrimidine C-3), 156.50 (pyrazolopyrimidine C-4), 157.31 (pyrazolopyrimidine C-6),
(
+
.
1
5
3
68.36 (C=O); MS (m/z, %): 329 [(M) , 99.92%], 298 [100%]; Anal.Calcd for C14
H
15
7
N OS: C,
1.05; H, 4.59; N, 29.77. Found: C, 51.32; H, 4.32; N, 29.85.
.1.5.2.
2-[3-(Methylthio)-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidine-4-
ylamino]acetohydrazide (6b). White powder; 85% yield; mp: 220-222°C; IR: 3446, 3368 (NH
2
2
),
);
3
318, 3217 (2NH), 3024(aromatic C-H), 2928 (aliphatic C-H), 1686 (C=O), 1380, 1142 (SO
1
H NMR: δ 2.77 (s, 3H, SCH
O ), 7.20 (br s, 1H, NHCH
phenyl H-3, H-5), 8.44 (s, IH, pyrimidine C-H), 8.54 (d, J = 6.6 Hz, 2H, phenyl H-2, H-6), 9.24
3
), 3.26 (s, 3H, SO
2
CH
3
), 4.16 (s, 2H, NHCH
2
), 4.37 (br s, 2H, NH
2
,
exchange with D
2
2
, exchange with D
2
O ), 8.10 (d, J = 6.6 Hz, 2H,
1
3
(
(
br s, 1H, NH, exchange with D
SO CH ), 101.65 (pyrazolopyrimidine C-3a), 120.25 (phenyl C-2, C-6), 128.88 (phenyl C-3, C-
), 137.50 (phenyl C-4), 142.77 (phenyl C-1), 143.66 (pyrazolopyrimidine C-7a), 155.14
pyrazolopyrimidine C-3), 156.62 (pyrazolopyrimidine C-4), 157.55 (pyrazolopyrimidine C-6),
2
O ); C NMR : δ 14.50 (SCH
3
), 42.92 (NHCH ), 44.20
2
2
3
5
(

+
.
1
4
3
68.31 (C=O); MS (m/z, %): 407 [(M) , 9.26%], 348 [100%]; Anal.Calcd for C15
4.21; H, 4.21; N, 24.06. Found: C, 44.49; H, 4.30; N, 24.34.
.1.6. General procedure for preparation of 7a-f
H
17
N
7
O
3
2
S : C,
A mixture of acid hydrazide derivative 6a or 6b (0.005 mol) of each and the appropriate
aromatic aldehyde (0.005 mol) in absolute ethanol (30 ml) containing a catalytic amount of
glacial acetic acid (0.2 ml) was heated under reflux for 3 h. The solid precipitated on hot was
filtered off, dried and crystallized from acetic acid 96% to give compounds 7a-f.
/
3
.1.6.1.
N -(furyl-2-ylmethylene)-2-[3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine-4-ylamino]acetohydrazide (7a). white powder; 79% yield; mp: 233-235°C; IR:
1
3
3
441, 3362 (2NH), 3047 (aromatic C-H), 2934 (aliphatic C-H), 1682 (C=O); H NMR: δ 2.76 (s,
H, SCH
3
), 4.65 (s, 2H, NHCH
2
), 6.64 (br s, 1H, NHCH
2
, exchange with D O ), 6.93 (t, J = 3.6
2
Hz, 1H, furyl H-4), 7.19 (d, J = 3.6 Hz, 1H, furyl H-3), 7.35 (t, J = 6.8 Hz, 1H, phenyl H-4), 7.55
t, J = 6.8 Hz, 2H, phenyl H-3, H-5), 7.85 (s, IH, pyrimidine C-H), 7.94 (d, J = 3.6 Hz, 1H, furyl
H-5), 8.19 (d, J = 7.2 Hz, 2H, phenyl H-2, H-6), 8.41 (s, 1H, N=CH azomethine), 11.65 (br s,
(
1
3
1
H, NH, exchange with D
2
O ); C NMR : δ 15.49 (SCH
3
), 42.65 (NHCH
2
), 101.26
(
pyrazolopyrimidine C-3a), 112.63 (furyl C-4), 114.36 (furyl C-3), 120.92 (phenyl C-2, C-6),
1
1
26.58 (phenyl C-4), 129.59 (phenyl C-3, C-5), 137.15 (N=CH azomethine), 139.00 (phenyl C-
), 141.43 (pyrazolopyrimidine C-7a), 145.56 (furyl C-5), 149.40 (furyl C-2), 154.34
(
pyrazolopyrimidine C-3), 156.67 (pyrazolopyrimidine C-4), 157.35 (pyrazolopyrimidine C-6),
+
.
1
5
3
70.22 (C=O); MS (m/z, %): 407 [(M) , 28.37%], 298 [100%]; Anal.Calcd for C19
H
17
N
7
2
O S: C,
6.01; H, 4.21; N, 24.06. Found: C, 56.43; H, 4.32; N, 24.38.
/
.1.6.2.
N -(pyridyl-4-ylmethylene)-2-[3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine-4-ylamino]acetohydrazide (7b). White powder; 85% yield; mp: 234-236°C; IR:

1
3
3
358, 3239 (2NH), 3048 (aromatic C-H), 2941 (aliphatic C-H), 1702 (C=O); H NMR: δ 2.76 (s,
H, SCH ), 4.76 (d, J = 5.2 Hz, 2H, NHCH ), 7.25 (t, J = 5.2 Hz, 1H, NHCH , exchange with
O ), 7.35 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.56 (t, J = 7.6 Hz, 2H, phenyl H-3, H-5), 7.69 (d, J
5.2 Hz, 2H, pyridyl H-3, H-5), 8.04 (s, IH, pyrimidine C-H), 8.19 (d, J = 8 Hz, 2H, phenyl H-
3
2
2
D
2
=
2
1
, H-6), 8.41 (s, 1H, N=CH azomethine), 8.67 (d, J = 5.2 Hz, 2H, pyridyl H-2, H-6), 11.94 (br s,
1
3
H, NH, exchange with D
2
O ); C NMR : δ 15.54 (SCH
3
), 42.67 (NHCH ), 101.26
2
(
pyrazolopyrimidine C-3a), 121.10 (phenyl C-2, C-6), 121.32 (pyridyl C-3, C-5), 126.74 (phenyl
C-4), 129.67 (phenyl C-3, C-5), 138.96 (phenyl C-1), 141.57 (pyridyl C-4), 142.09 (N=CH
azomethine), 150.72 (pyridyl C-2, C6) 151.31 (pyrazolopyrimidine C-7a), 154.43
(
pyrazolopyrimidine C-3), 156.93 (pyrazolopyrimidine C-4), 157.39 (pyrazolopyrimidine C-6),
+
.
1
70.22 (C=O); MS (m/z, %): 418 [(M) , 100%]; Anal.Calcd for C20
H
18
8
N OS: C, 57.40; H, 4.34;
N, 26.78. Found: C, 57.78; H, 4.41; N, 26.96.
/
3
.1.6.3.
N -(thienyl-2-ylmethylene)-2-[3-(Methylthio)-1-phenyl-1H-pyrazolo[3,4-
d]pyrimidine-4-ylamino]acetohydrazide (7c). Yellow powder; 85% yield; mp: 235-237°C; IR :
1
3
3
5
457, 3368 (2NH), 3080 (aromatic C-H), 2930 (aliphatic C-H), 1679 (C=O); H NMR: δ 2.76 (s,
H, SCH
3
), 4.65 (d, J = 5.2 Hz, 2H, NHCH
2
), 7.15 (t, J = 3.6 Hz, 1H, thienyl H-4), 7.20 (t, J =
.2 Hz, 1H, NHCH
2
, exchange with D O ), 7.35 (t, J = 7.6 Hz, 1H, phenyl H-4), 7.46 (d, J = 3.6
2
Hz, 1H, thienyl H-3), 7.56 (t, J = 7.6 Hz, 2H, phenyl H-3, H-5), 7.66 (d, J = 3.6 Hz, 1H, thienyl
H-5), 8.19 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 8.23 (s, IH, pyrimidine C-H), 8.42 (s, 1H, N=CH
1
3
azomethine), 11.66 (br s, 1H, NH, exchange with D
NHCH ), 102.26 (pyrazolopyrimidine C-3a), 121.01 (phenyl C-2, C-6), 126.64 (N=CH
azomethine), 128.44 (phenyl C-4), 129.07 (thienyl C-4), 129.64 (thienyl C-5), 131.15 (phenyl C-
, C-5), 139.05 (phenyl C-1), 139.66 (thienyl C-3), 141.55 (thienyl C -2), 148.67
2
O ); C NMR : δ 15.56 (SCH ), 42.50
3
(
2
3

(
pyrazolopyrimidine C-7a), 154.46 (pyrazolopyrimidine C-3), 156.85 (pyrazolopyrimidine C-4),
+
.
1
58.21 (pyrazolopyrimidine C-6), 170.01 (C=O); MS (m/z, %): 423 [(M) , 22.98%], 298
OS : C, 53.88; H, 4.05; N, 23.15. Found: C, 53.94; H, 3.98; N,
[
100%]; Anal.Calcd for C19
H
17
N
7
2
2
3
2.82.
/
.1.6.4.
N -(furyl-2-ylmethylene)-2-[3-(Methylthio)-1-(4-methylsulphonylphenyl)-1H-
pyrazolo[3,4-d]pyrimidine-4-ylamino]acetohydrazide (7d). Buff powder; 73% yield; mp: 259-
2
1
61°C; IR : 3458, 3374 (2NH), 3119 (aromatic C-H), 2926 (aliphatic C-H), 1684 (C=O), 1294,
1
142 (SO
2
); H NMR: δ 2.75 (s, 3H, SCH
3
), 3.26 (s, 3H, SO
2
CH ), 4.65 (d, J = 5.2 Hz, 2H,
3
NHCH
Hz, 1H, NH, exchange with D
H), 8.11 (d, J = 8.8 Hz, 2H, phenyl H-3, H-5), 8.48 (s, 1H, N=CH azomethine), 8.56 (d, J = 8.8
2
), 6.64 (t, J = 3.6 Hz, 1H, furyl H-4), 7.94 (d, J = 3.6 Hz, 1H, furyl H-3), 7.23 (t, J = 5.2
2
O ), 7.85 (d, J = 3.6 Hz, 1H, furyl H-5), 7.93 (s, IH, pyrimidine C-
1
3
Hz, 2H, phenyl H-2, H-6), 11.64 (br s, 1H, NH, exchange with D
2.72 (NHCH ), 44.22 (SO CH ), 101.60 (pyrazolopyrimidine C-3a), 112.63 (furyl C-4), 114.32
furyl C-3), 120.39 (phenyl C-2, C-6), 128.99 (phenyl C-3, C-5), 134.62 (N=CH azomethine),
2
O); C NMR : δ 15.10 (SCH
3
),
4
2
2
3
(
1
5
1
37.75 (phenyl C-4), 142.82 (phenyl C-1), 143.56 (pyrazolopyrimidine C-7a), 145.60 (furyl C-
), 149.45 (furyl C-2), 155.34 (pyrazolopyrimidine C-3), 156.79 (pyrazolopyrimidine C-4),
+
.
57.80 (pyrazolopyrimidine C-6), 170.11 (C=O); MS (m/z, %): 485 [(M) , 7.78%], 335 [100%];
Anal.Calcd for C20
H
19
N
7
O
4
2
S : C, 49.47; H, 3.94; N, 20.19. Found: C, 49.19; H, 4.01; N, 20.52.
/
3
.1.6.5.
N -(pyridyl-4-ylmethylene)-2-[3-(Methylthio)-1-(4-methylsulphonylphenyl)-1H-
pyrazolo[3,4-d]pyrimidine-4-ylamino]acetohydrazide (7e). White powder; 82% yield; mp: 268-
2
1
70°C; IR : 3446, 3373 (2NH), 2932 (aromatic C-H), 2781 (aliphatic C-H), 1693 (C=O), 1397,
1
146 (SO
2
); H NMR: δ 2.81 (s, 3H, SCH
3
), 3.26 (s, 3H, SO
2
CH
3
), 4.76 (d, J = 4.8 Hz, 2H,
NHCH
2
), 7.21 (t, J = 4.8 Hz, 1H, NHCH
2
, exchange with D O), 7.69 (d, J = 5.2 Hz, 2H, pyridyl
2

H-3, H-5), 8.04 (s, IH, pyrimidine C-H), 8.12 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 8.48 (s, 1H,
N=CH azomethine), 8.56 (d, J = 8.4 Hz, 2H, phenyl H-2, H-6), 8.67 (d, J = 5.2 Hz, 2H, pyridyl
1
3
H-2, H-6), 11.94 (br s, 1H, NH, exchange with D
NHCH ), 44.20 (SO CH ), 101.60 (pyrazolopyrimidine C-3a), 120.46 (pyridyl C-3, C-5),
21.31 (phenyl C-2, C-6), 128.98 (phenyl C-3, C-5), 137.72 (phenyl C-4), 141.53 (pyridyl C-4),
42.10 (N=CH azomethine), 143.63 (phenyl C-1), 148.01 (pyrazolopyrimidine C-7a), 150.75
2
O); C NMR : δ 15.07 (SCH ), 42.74
3
(
2
2
3
1
1
(
(
pyridyl C-2, C6), 155.34 (pyrazolopyrimidine C-3), 156.92 (pyrazolopyrimidine C-4), 157.82
+
.
pyrazolopyrimidine C-6), 170.01 (C=O); MS (m/z, %): 496 [(M) , 5.18%], 376 [100%];
Anal.Calcd for C21
H
20
N
8
O
3
2
S : C, 50.79; H, 4.06; N, 22.57. Found: C, 50.91; H, 4.09; N, 22.81.
/
3
.1.6.6.
N -(thienyl-2-ylmethylene)-2-[3-(Methylthio)-1-(4-methylsulphonylphenyl)-1H-
pyrazolo[3,4-d]pyrimidine-4-ylamino]acetohydrazide (7f). Yellow powder; 85% yield; mp: 245-
2
1
47°C; IR : 3374, 3250 (2NH), 3119 (aromatic C-H), 2926 (aliphatic C-H), 1682 (C=O), 1300,
1
149 (SO
2
); H NMR: δ 2.80 (s, 3H, SCH
3
), 3.26 (s, 3H, SO
2
CH
3
), 4.63 (d, J = 5.2 Hz, 2H,
, exchange with
O), 7.46 (d, J = 3.2 Hz, 1H, thienyl H-3), 7.66 (d, J = 4.8 Hz, 1H, thienyl H-5), 8.11 (d, J = 8.8
Hz, 2H, phenyl H-3, H-5), 8.23 (s, IH, pyrimidine C-H), 8.48 (s, 1H, N=CH azomethine), 8.53
NHCH
2
), 7.14 (t, J = 4.8 Hz, 1H, thienyl H-4), 7.22 (t, J = 5.2 Hz, 1H, NHCH
2
D
2
1
3
(
d, J = 8.8 Hz, 2H, phenyl H-2, H-6), 11.66 (br s, 1H, NH, exchange with D
2
O); C NMR : δ
1
5.08 (SCH ), 42.56 (NHCH ), 44.21 (SO CH ), 101.58 (pyrazolopyrimidine C-3a), 120.37
3
2
2
3
(
(
(
(
phenyl C-2, C-6), 128.44 (thienyl C-4), 129.08 (phenyl C-3, C-5), 131.15 (thienyl C-5), 137.71
phenyl C-4), 139.09 (thienyl C-2), 139.70 (thienyl C-3), 142.55 (N=CH azomethine), 142.81
phenyl C-1), 143.57 (pyrazolopyrimidine C-7a), 155.32 (pyrazolopyrimidine C-3), 156.81
pyrazolopyrimidine C-4), 157.80 (pyrazolopyrimidine C-6), 169.90 (C=O); MS (m/z, %): 501

+
.
[
(M) , 0.76%], 376 [100%]; Anal.Calcd for C20
C, 47.81; H, 3.73; N, 19.38.
.1.7. General procedure for preparation of 8a and 8b.
A mixture of acid hydrazide derivative 6a or 6b (0.003 mol) of each and ethyl isothiocyanate
0.26 g, 0.003 mol) in absolute ethanol (30 ml) containing a catalytic amount of TEA (3dps) was
H
19
N
7
O
3
3
S : C, 47.89; H, 3.82; N, 19.55. Found:
3
(
heated under reflux for 3 h. The precipitate formed on hot was filtered off, dried and crystallized
from ethanol 95% to give compounds 8a or 8b.
3
.1.7.1.
4-Ethyl-5-{[3-methylthio-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]-
methyl}-4H-[1,2,4]triazole-3-thiol (8a). White powder; 75% yield; mp: 256-258°C; IR : 3350
1
(
NH), 3106 (aromatic C-H), 2935 (aliphatic C-H), 2347 (SH); H NMR: δ 1.25 (t, J = 7.2 Hz,
3
H, CH
NHCH ), 7.35 (t, J = 6.8 Hz, 1H, NH, exchange with D
H-5), 8.17 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 8.41 (s, IH, pyrimidine C-H), 13.57 (s, 1H,
2
CH
3
), 2.74 (s, 3H, SCH
3
), 4.10 (q, J = 7.2 Hz, 2H, CH
2
CH ), 4.90 (d, J = 5.2 Hz, 2H,
3
2
2
O), 7.53-7.57 (m, 3H, phenyl H-3, H-4,
1
3
triazole SH); C NMR : δ 13.81 (SCH
3
), 15.36 (CH
2
CH
3
), 36.34 (CH
2
CH
3
), 38.79 (NHCH ),
2
1
01.29 (pyrazolopyrimidine C-3a), 121.09 (phenyl C-2, C-6), 128.70 (phenyl C-4), 129.63
(
(
(
phenyl C-3, C-5), 138.95 (phenyl C-1), 141.58 (pyrazolopyrimidine C-7a), 150.14
pyrazolopyrimidine C-3), 154.54 (triazole C-5), 156.69 (pyrazolopyrimidine C-4), 157.17
+
.
pyrazolopyrimidine C-6), 166.96 (triazole C-3); MS (m/z, %): 398 [(M) , 100%]; Anal.Calcd
: C, 51.24; H, 4.55; N, 28.12. Found: C, 51.55; H, 4.49; N, 28.41.
4-Ethyl-5-{[3-methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-
d]pyrimidin-4-ylamino]-methyl}-4H-[1,2,4]triazole-3-thiol (8b). Buff powder; 64% yield; mp:
for C17
H
18
N S
8 2
3
.1.7.2.
3
35-337°C; IR : 3350 (NH), 3106 (aromatic C-H), 2935 (aliphatic C-H), 2630 (SH), 1300, 1145
1
(
SO
2
); H NMR: δ 1.25 (t, J = 6.8 Hz, 3H, CH
2
CH
3
), 2.77 (s, 3H, SCH
3
), 3.25 (s, 3H, SO
2
CH ),
3

4
.09 (q, J = 6.8 Hz, 2H, CH
2
CH
3
), 4.88 (d, J = 4.8 Hz, 2H, NHCH ), 7.63 (t, J = 4.8 Hz, 1H, NH,
2
exchange with D O), 8.10 (d, J = 8.4 Hz, 2H, phenyl H-2, H-6), 8.46 (s, IH, pyrimidine C-H),
2
1
3
8
1
.52 (d, J = 8.4 Hz, 2H, phenyl H-3, H-5), 13.59 (s, IH, triazole SH); C NMR : δ 13.81 (SCH
3
),
4.93 (CH CH ), 36.41 (CH CH ), 38.79 (NHCH ), 44.21 (SO CH ), 101.59
2
3
2
3
2
2
3
(
pyrazolopyrimidine C-3a), 120.42 (phenyl C-2, C-6), 128.94 (phenyl C-3, C-5), 137.75 (phenyl
C-4), 142.71 (phenyl C-1), 143.61 (pyrazolopyrimidine C-7a), 150.02 (pyrazolopyrimidine C-3),
1
1
55.40 (triazole C-5), 156.60 (pyrazolopyrimidine C-4), 157.50 (pyrazolopyrimidine C-6),
+
.
66.98 (triazole C-3); MS (m/z, %): 477 [(M) , 1.25%], 368 [100%]; Anal.Calcd for
: C, 45.36; H, 4.23; N, 23.51. Found: C, 45.62; H, 4.39; N, 23.79.
.1.8. General procedure for preparation of 9a-f.
C
18
H
20
N
8
O S
2 3
3
A mixture of acid hydrazide derivative 6a or 6b (0.003 mol) of each and the appropriate phenyl
isothiocyanate derivatives (0.003 mol) in absolute ethanol (30 ml) containing a catalytic amount
of TEA (3dps) was heated under reflux for 3 h. The solid separated on hot was filtered off, dried
and crystallized from ethanol 95% to give compounds 9a-f.
3
.1.8.1.
5-{[3-Methylthio-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]-methyl}-4-
phenyl-4H-[1,2,4]triazole-3-thiol (9a). White powder; 75% yield; mp: 262-264°C; IR : 3332
1
(
NH), 3090 (aromatic C-H), 2924 (aliphatic C-H), 2347 (SH); H NMR: δ 2.71 (s, 3H, SCH
3
),
O), 7.34 (t, J =
.6 Hz, 1H, phenyl H-4), 7.47-7.55 (m, 7H, pyrazolpyrimidine phenyl H-2, H-3, H-4, H-5, H-6
phenyl H-3, H-5), 8.15 (d, J = 8 Hz, 2H, phenyl H-2, H-6), 8.33 (s, IH, pyrimidine C-H),
4
7
.65 (d, J = 5.6 Hz, 2H, NHCH
2
), 7.31 (t, J = 5.6 Hz, 1H, NH, exchange with D
2
&
1
3
1
3.81 (s, 1H, triazole SH); C NMR : δ 15.49 (SCH
3
), 36.69 (NHCH ), 101.24
2
(
(
pyrazolopyrimidine C-3a), 121.10 (pyrazolopyrimidine phenyl C-2, C-6), 126.73
pyrazolopyrimidine phenyl C-4), 128.56 (pyrazolopyrimidine phenyl C-3, C-5), 129.64 (phenyl

C-3, C-5), 129.82 (phenyl C-4), 129.92 (phenyl C-2, C-6), 133.82 (pyrazolopyrimidine phenyl
C-1), 138.91 (phenyl C-1), 141.51 (pyrazolopyrimidine C-7a), 150.26 (pyrazolopyrimidine C-3),
1
1
4
3
54.36 (triazole C-5), 156.36 (pyrazolopyrimidine C-4), 157.01 (pyrazolopyrimidine C-6),
+
.
68.46 (triazole C-3); MS (m/z, %): 446 [(M) , 100%]; Anal.Calcd for C21
.06; N, 25.09. Found: C, 56.54; H, 3.95; N, 24.97.
H
18
N
8
2
S : C, 56.48; H,
.1.8.2.
5-{[3-Methylthio-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]-methyl}-4-
(4-florophenyl)-4H-[1,2,4]triazole-3-thiol (9b). Buff powder; 75% yield; mp: 260-262°C; IR :
1
3
332 (NH), 3090 (aromatic C-H), 2924 (aliphatic C-H), 2367 (SH); H NMR: δ 2.74 (s, 3H,
SCH
phenyl H-4 & NH (exchange with D
pyrazolpyrimidine phenyl H-3, H-5), 8.14 (d, J = 8 Hz, 2H, pyrazolopyrimidine phenyl H-2, H-
3
), 4.66 (s, 2H, NHCH
2
), 7.33-7.38 (m, 4H, florophenyl H-3, H-5, pyrazolopyrimidine
2
O)), 7.54-7.59 (m, 4H, florophenyl H-2, H-6 &
1
3
6
), 8.31 (s, IH, pyrimidine C-H), 13.80 (s, 1H, triazole SH); C NMR : δ 15.43 (SCH
3
), 36.62
(
(
(
(
NHCH ), 101.19 (pyrazolopyrimidine C-3a), 116.57 (florophenyl C-3, C-5), 121.05
2
pyrazolopyrimidine phenyl C-2, C-6), 126.66 (pyrazolopyrimidine phenyl C-4), 129.60
pyrazolopyrimidine phenyl C-3, C-5), 130.98 (florophenyl C-2, C-6), 138.94
pyrazolopyrimidine phenyl C-1), 141.48 (florophenyl C-1), 150.33 (pyrazolopyrimidine C-7a),
1
1
4
54.35 (pyrazolopyrimidine C-3), 156.28 (triazole C-5), 156.94 (pyrazolopyrimidine C-6),
61.41 (pyrazolopyrimidine C-4), 163.86 (florophenyl C-4), 168.60 (triazole C-3); MS (m/z, %):
+
.
64 [(M) , 9.91%], 433 [100%]; Anal.Calcd for C21
H17FN
8
2
S : C, 54.30; H, 3.69; N, 24.12.
Found: C, 54.62; H, 3.84; N, 24.37.
.1.8.3. 5-{[3-Methylthio-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]-methyl}-4-
3
(4-chlorophenyl)-4H-[1,2,4]triazole-3-thiol (9c). White powder; 75% yield; mp: 265-267°C; IR :
1
3
375 (NH), 3063 (aromatic C-H), 2928 (aliphatic C-H), 2376 (SH); H NMR: δ 2.71 (s, 3H,

SCH
.4 Hz, 1H, pyrazolpyrimidine phenyl H-4), 7.47-7.51 (m, 4H, pyrazolopyrimidine phenyl H-2,
H-3, H-5, H-6), 7.56 (d, J = 8 Hz, 2H, chlorophenyl H-3, H-5), 8.14 (d, J = 8 Hz, 2H,
3
), 4.69 (d, J = 4.8 Hz, 2H, NHCH
2
), 7.30 (br s, 1H, NH, exchange with D O), 7.34 (t, J =
2
8
1
3
chlorophenyl H-2, H-6), 8.31 (s, IH, pyrimidine C-H), 13.87 (s, 1H, triazole SH); C NMR : δ
5.38 (SCH ), 36.59 (NHCH ), 101.13 (pyrazolopyrimidine C-3a), 121.05 (pyrazolopyrimidine
phenyl C-2, C-6), 126.64 (pyrazolopyrimidine phenyl C-4), 129.60 (chlorophenyl C-2, C-6),
29.76 (chlorophenyl C-3, C-5), 130.56 (pyrazolopyrimidine phenyl C-3, C-5), 132.79
1
3
2
1
(
chlorophenyl C-4), 134.54 (pyrazolopyrimidine phenyl C-1), 138.95 (chlorophenyl C-1), 141.49
pyrazolopyrimidine C-7a), 150.25 (pyrazolopyrimidine C-3), 154.32 (triazole C-5), 156.20
pyrazolopyrimidine C-4), 156.90 (pyrazolopyrimidine C-6), 168.37 (triazole C-3); MS (m/z,
(
(
+
.
%
): 480 [(M) , 100%]; Anal.Calcd for C21
H17ClN
8
2
S : C, 52.44; H, 3.56; N, 23.30. Found: C,
5
2.78; H, 3.80; N, 23.13.
.1.8.4. 5-{[3-Methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
3
ylamino]-methyl}-4-phenyl-4H-[1,2,4]triazole-3-thiol (9d). White powder; 68% yield; mp: 275-
2
77°C; IR : 3386 (NH), 3116 (aromatic C-H), 2933 (aliphatic C-H), 2349 (SH), 1295, 1146
1
(
SO
2
); H NMR: δ 2.75 (s, 3H, SCH
3
), 3.25 (s, 3H, SO
2
CH
3
), 4.65 (d, J = 5.6 Hz, 2H, NHCH
2
),
7
.39 (t, J = 5.6 Hz, 1H, NH, exchange with D
2
O), 7.46-7.53 (m, 5H, phenyl-H), 8.08 (d, J = 8.8
Hz, 2H, pyrazolopyrimidine phenyl H-3, H-5), 8.39 (s, IH, pyrimidine C-H), 8.50 (d, J = 8.8 Hz,
1
3
2
H, pyrazolopyrimidine phenyl H-2, H-6), 13.85 (s, 1H, triazole SH); C NMR : δ 15.03
(
(
(
SCH ), 36.74 (NHCH ), 44.20 (SO CH ), 101.54 (pyrazolopyrimidine C-3a), 120.47
3
2
2
3
pyrazolopyrimidine phenyl C-2, C-6), 128.58 (phenyl C-4), 128.97 (phenyl C-3, C-5), 129.81
pyrazolopyrimidine phenyl C-3, C-5), 129.89 (phenyl C-2, C-6), 133.83 (pyrazolopyrimidine
phenyl C-4), 137.77 (pyrazolopyrimidine phenyl C-1), 142.72 (phenyl C-1), 143.59

(
pyrazolopyrimidine C-7a), 150.17 (pyrazolopyrimidine C-3), 155.26 (triazole C-5), 156.34
pyrazolopyrimidine C-4), 157.37 (pyrazolopyrimidine C-6), 168.49 (triazole C-3); MS (m/z,
(
+
.
%
): 524 [(M) , 2.03%], 335 [100%]; Anal.Calcd for C22
H
20
N
8
O
2
3
S : C, 50.36; H, 3.84; N, 21.36.
Found: C, 50.07; H, 3.91; N, 21.44.
.1.8.5. 5-{[3-Methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
3
ylamino]-methyl}-4-(4-florophenyl)-4H-[1,2,4]triazole-3-thiol (9e). Buff powder; 65% yield;
mp: 280-282°C; IR : 3386 (NH), 3116 (aromatic C-H), 2933 (aliphatic C-H), 2380 (SH), 1288,
1
1
149 (SO
NHCH
4.8 Hz, J = 8.4 Hz, 2H, florophenyl H-2, H-6), 8.08 (d, J = 8.8 Hz, 2H, pyrazolopyrimidine
phenyl H-3, H-5), 8.34 (s, IH, pyrimidine C-H), 8.48 (d, J = 8.8 Hz, 2H, pyrazolopyrimidine
2
); H NMR: δ 2.72 (s, 3H, SCH
3
), 3.25 (s, 3H, SO
2
CH
3
), 4.65 (d, J = 5.2 Hz, 2H,
2
), 7.29-7.36 (m, 3H, florophenyl H-3, H-5 & NH (exchange with D
2
O)), 7.50, 7.51 (dd, J
=
1
3
phenyl H-2, H-6), 13.86 (s, 1H, triazole SH); C NMR : δ 14.92 (SCH
3
), 36.64 (NHCH
2
), 44.19
(
(
(
SO
2
CH
3
), 101.43 (pyrazolopyrimidine C-3a), 116.81 (florophenyl C-3, C-5), 120.35
pyrazolopyrimidine phenyl C-2, C-6), 128.91 (pyrazolopyrimidine phenyl C-3, C-5), 130.96
florophenyl C-2, C-6), 137.65 (pyrazolopyrimidine phenyl C-4), 142.68 (florophenyl C-1),
1
43.56 (pyrazolopyrimidine phenyl C-1), 150.24 (pyrazolopyrimidine C-7a), 155.17
(
(
[
pyrazolopyrimidine C-3), 156.21 (triazole C-5), 157.25 (pyrazolopyrimidine C-6), 161.40
pyrazolopyrimidine C-4), 163.86 (florophenyl C-4), 168.57 (triazole C-3); MS (m/z, %): 542
+
.
(M) , 4.58%], 335 [100%]; Anal.Calcd for C22
H19FN
8
O
2
3
S : C, 48.70; H, 3.53; N, 20.65. Found:
C, 48.97; H, 3.65; N, 20.88.
.1.8.6. 5-{[3-Methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
3
ylamino]-methyl}-4-(4-chlorophenyl)-4H-[1,2,4]triazole-3-thiol (9f). White powder; 65% yield;
mp: 282-284°C; IR : 3368 (NH), 3105 (aromatic C-H), 2932 (aliphatic C-H), 2380 (SH), 1287,

1
1
149 (SO
2
); H NMR: δ 2.73 (s, 3H, SCH
3
), 3.25 (s, 3H, SO
2
CH ), 4.69 (d, J = 4 Hz, 2H,
3
NHCH
2
), 7.33 (t, J = 4 Hz, 1H, NH, exchange with D
2
O), 7.45-7.51 (m, 4H, chlorophenyl H-2,
H-3, H-5, H-6), 8.07 (d, J = 8.4 Hz, 2H, pyrazolopyrimidine phenyl H-3, H-5), 8.35 (s, IH,
1
3
pyrimidine C-H), 8.47 (d, J = 8.4 Hz, 2H, pyrazolopyrimidine phenyl H-2, H-6); C NMR : δ
1
4.92 (SCH ), 36.61 (NHCH ), 44.18 (SO CH ), 101.41 (pyrazolopyrimidine C-3a), 120.38
3
2
2
3
(
(
pyrazolopyrimidine phenyl C-2, C-6), 128.92 (chlorophenyl C-3, C-5), 129.75
pyrazolopyrimidine phenyl C-3, C-5), 130.53 (chlorophenyl C-2, C-6), 132.76 (chlorophenyl C-
4
), 134.54 (pyrazolopyrimidine phenyl C-4), 137.64 (chlorophenyl C-1),
142.71
150.13
(
(
(
pyrazolopyrimidine phenyl C-1), 143.57 (pyrazolopyrimidine C-7a),
pyrazolopyrimidine C-3), 155.16 (triazole C-5), 156.15 (pyrazolopyrimidine C-4), 157.21
+
.
pyrazolopyrimidine C-6), 168.39 (triazole C-3); MS (m/z, %): 558 [(M) , 2.82%], 464 [100%];
: C, 47.26; H, 3.43; N, 20.04. Found: C, 47.03; H, 3.60; N,
Anal.Calcd for C22
H19ClN
8
O S
2 3
2
0.19.
.1.9. General procedure for preparation of 10a and 10b.
3
A mixture of acid hydrazide derivative 6a or 6b (0.003 mol) of each, carbon disulphide (0.68 g,
0
3
.009 mol) and KOH (0.17 g, 0.003 mol) in absolute ethanol (30 ml) was heated under reflux for
h. The reaction mixture was allowed to cool, then poured into ice-cold water. The obtained
solution was acidified with dil. HCl (1 ml, 33%). The obtained precipitate was filtered off,
washed with water, dried and crystallized from ethanol 95%to obtain compounds 10a or 10b.
3
.1.9.1.
5-{[3-Methylthio-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]-methyl}-
[
1,3,4]oxadiazole-2-thiol (10a). Yellow powder; 87% yield; mp: 215-217°C; IR : 3333 (NH),
1
2
994 (aromatic C-H), 2920 (aliphatic C-H), 2376 (SH); H NMR: δ 2.73 (s, 3H, SCH
3
), 4.86 (d,
J = 5.6 Hz, 2H, NHCH
2
), 7.34 (t, J = 5.6 Hz, 1H, NHCH
2
, exchange with D
2
O), 7.53 (t, J = 8

Hz, 2H, phenyl H-3, H-5), 7.73 (t, J = 8 Hz, 1H, phenyl H-4), 8.12 (d, J = 8 Hz, 2H, phenyl H-
1
3
2
3
, H-6), 8.41 (s, IH, pyrimidine C-H), 14.55 (s, 1H, oxadiazole SH); C NMR : δ 14.45 (SCH
3
),
6.44 (NHCH ), 101.39 (pyrazolopyrimidine C-3a), 121.22 (phenyl C-2, C-6), 126.78 (phenyl
2
C-4), 129.58 (phenyl C-3, C-5), 138.89 (phenyl C-1), 141.64 (pyrazolopyrimidine C-7a), 154.48
(
pyrazolopyrimidine C-3), 156.44 (pyrazolopyrimidine C-4), 156.85 (pyrazolopyrimidine C-6),
+
.
1
61.87 (oxadiazole C-5), 178.25 (oxadiazole C-2); MS (m/z, %): 371 [(M) , 20.42%], 336
[
100%]; Anal.Calcd for C15
H
13
N
7
OS : C, 48.50; H, 3.53; N, 26.40. Found: C, 48.82; H, 3.61; N,
2
2
6.74.
3
.1.9.2.
5-{[3-Methylthio-1-(4-methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-
ylamino]-methyl}-[1,3,4]oxadiazole-2-thiol (10b). Yellow powder; 85% yield; mp: 225-227°C;
1
IR : 3333 (NH), 2994 (aromatic C-H), 2920 (aliphatic C-H), 2380 (SH), 1299, 1140 (SO
NMR: δ 2.73 (s, 3H, SCH ), 3.25 (s, 3H, SO CH ), 4.83 (d, J = 5.6 Hz, 2H, NHCH
.6 Hz, 1H, NH, exchange with D O), 8.04 (d, J = 8.8 Hz, 2H, phenyl H-3, H-5), 8.42 (s, IH,
pyrimidine C-H), 8.46 (d, J = 8.8 Hz, 2H, phenyl H-2, H-6), SH (not observed); C NMR: δ
4.92 (SCH ), 36.48 (NHCH ), 44.21 (SO CH ), 101.63 (pyrazolopyrimidine C-3a), 120.52
2
); H
3
2
3
2
), 7.71 (t, J =
5
2
1
3
1
3
2
2
3
(
(
phenyl C-2, C-6), 128.98 (phenyl C-3, C-5), 137.85 (phenyl C-4), 142.70 (phenyl C-1), 143.64
pyrazolopyrimidine C-7a), 154.48 (pyrazolopyrimidine C-3), 156.47 (pyrazolopyrimidine C-4),
1
57.50 (pyrazolopyrimidine C-6), 161.89 (oxadiazole C-5), 178.16 (oxadiazole C-2); MS (m/z,
+
.
%
): 449 [(M) , 0.83%], 64 [100%]; Anal.Calcd for C16
H
15
N
7
O
3
3
S : C, 42.75; H, 3.36; N, 21.81.
Found: C, 43.01; H, 3.49; N, 22.05.
.1.10. General procedure for preparation of 13a and 13b.
3

A mixture of acid hydrazide derivative 6a or 6b (0.003 mol) of each and ethylacetoacetate (0.39
g, 0.003 mol) in absolute ethanol (30 ml) was heated under reflux for 10 h. The reaction mixture
was evaporated under reduced pressure. The solid obtained was crystallized from ethanol 95% to
get compounds 13a or 13b.
3
.1.10.1.
1-(5-Ethoxy-3-methyl-1H-pyrazol-1-yl)-2-[3-methythio-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4-ylamino]ethanone (13a). Yellow needles; 75% yield; mp: 256-
1
2
1
7
7
58°C; IR : 3387 (NH), 3102 (aromatic C-H), 2924 (aliphatic C-H), 1740 (C=O); H NMR: δ
.21 (t, J = 7.2 Hz, 3H, CH
.2 Hz, 2H, CH CH ), 4.30 (d, J = 5.6 Hz, 2H, NHCH
.6 Hz, 1H, phenyl H-4), 7.40 (t, J = 5.6 Hz, 1H, NHCH
2
CH
3
), 2.09 (s, 3H, pyrazole CH
), 5.21 (s,1H, pyrazole H-4), 7.34 (t, J =
, exchange with D O), 7.55 (t, J = 7.6
3
), 2.74 (s, 3H, SCH
3
), 4.14 (q, J =
2
3
2
2
2
Hz, 2H, phenyl H-3, H-5), 8.15 (d, J = 7.6 Hz, 2H, phenyl H-2, H-6), 8.39 (s, IH, pyrimidine C-
1
3
H); C NMR : δ 11.63 (pyrazole CH
3
), 14.55 (SCH
), 89.35 (pyrazole C-4), 101.13 (pyrazolopyrimidine C-3a), 120.78 (phenyl C-2, C-6),
26.67 (phenyl C-4), 129.62 (phenyl C-3, C-5), 138.94 (phenyl C-1), 141.56 (pyrazole C-3),
48.31 (pyrazolopyrimidine C-7a), 154.46 (pyrazolopyrimidine C-3), 156.71
3
), 15.35 (CH
2
CH
3
), 42.87 (NHCH ), 61.11
2
(
CH
2
CH
3
1
1
(
(
pyrazolopyrimidine C-4), 157.16 (pyrazolopyrimidine C-6), 162.53 (pyrazole C-5), 170.22
+
.
C=O); MS (m/z, %): 423 [(M) , 0.94%], 343 [100%]; Anal.Calcd for C20
H
21
N
7
2
O S: C, 56.72;
H, 5.00; N, 23.15. Found: C, 56.52; H, 5.23; N, 23.40.
.1.10.2. 1-(5-Ethoxy-3-methyl-1H-pyrazol-1-yl)-2-[3-methythio-1-(4-
methylsulphonylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-ylamino]ethanone
3
(13b).
Yellow
needles; 74% yield; mp: 272-274°C; IR : 3387 (NH), 3102 (aromatic C-H), 2924 (aliphatic C-
1
H), 1740 (C=O), 1377, 1141 (SO
2
); H NMR: δ 1.21 (t, J = 6.8 Hz, 3H, CH
2
CH
3
), 2.08 (s, 3H,
CH ),
pyrazole CH ), 2.76 (s, 3H, SCH
3
3
), 3.25 (s, 3H, SO
2
CH
3
), 4.13 (q, J = 6.8 Hz, 2H, CH
2
3