Synthesis and biological evaluation of novel pyrazolopyrimidines derivatives as anticancer and anti-5-lipoxygenase agents

Article abstract of DOI:10.1016/j.bioorg.2016.05.001

A novel series of 6-Aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 3a-h were synthesized in a single step via condensation of carboxamide 2 with some aromatic aldehydes (presence of iodine). Treatment of aminopyrazole 1a with acetic anhydri

Full text of DOI:10.1016/j.bioorg.2016.05.001

Bioorganic Chemistry 66 (2016) 160–168
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and biological evaluation of novel pyrazolopyrimidines
derivatives as anticancer and anti-5-lipoxygenase agents
Ameur Rahmouni a, Sawssen Souiei a, Mohamed Amine Belkacem a,b, Anis Romdhane a, Jalloul Bouajila b,
,
⇑
Hichem Ben Jannet a,
⇑
a Laboratoire de Chimie Hétérocyclique, Produits Naturels et Réactivité, Equipe: Chimie Médicinale et Produits Naturels, Faculté des Sciences de Monastir,
Université de Monastir, Avenue de l’Environnement, 5019 Monastir, Tunisia
b Université de Toulouse, Faculté de pharmacie de Toulouse, Laboratoire des Interactions Moléculaires et Réactivité Chimique et Photochimique UMR CNRS 5623,
Université Paul-Sabatier, 118 route de Narbonne, F-31062 Toulouse, France
a r t i c l e i n f o
a b s t r a c t
Article history:
A novel series of 6-aryl-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 3a-h were synthe-
sized in a single step via condensation of carboxamide 2 with some aromatic aldehydes (presence of
iodine). Treatment of aminopyrazole 1a with acetic anhydride afforded pyrazolopyrimidines 4 which
on treatment with ethyl chloroacetate in refluxing dry DMF furnished a single product identified as ethyl
2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl) acetate 5. On the other hand,
esterification of compound 6 with different alcohol, led to the formation of new esters linked pyrazolo
[3,4-d]pyrimidinones hybrids 7a-f. The reaction of compound 2 with 3-propargyl bromide gave the com-
pound 8 used as a dipolarophile to access to triazoles (4- and 5-regioisomers (9a-e) and (10a-e), respec-
tively) via the 1,3-dipoar cycloaddition reaction. Finally, condensation reaction of aminopyrazole 1b with
Received 19 March 2016
Revised 5 May 2016
Accepted 6 May 2016
Available online 7 May 2016
Keywords:
Pyrazoles
Pyrimidines
Pyrazolopyrimidines
Triazoles
a
-cyanocinnamonitiles gave the new pyrazolo[1,5-a]pyrimidine-3,6-dicarbonitriles 11a-e. Structures of
compounds were established on the basis of 1H/13C NMR and ESI-HRMS. Compounds were screened
for their cytotoxic (HCT-116 and MCF-7) and 5-lipoxygenase inhibition activities. The structure-
activity relationship (SAR) was discussed.
Anticancer
Anti-5-lipoxygenase
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
several decades, these compounds are reported to encompass
pharmacological potentials as antiinflammatory, antitumor
The pyrazole scaffold is a highly versatile drug-like template
that is being used extensively in the design of cancer therapies
and cellular apoptosis [1–3]. These compounds are capable to exert
remarkable anticancer effects by the inhibition of different types of
enzymes, proteins and receptors which play critical roles in cell
division [4]. On the other hand, the synthesis and study of
pyrazolo-fused compounds have been also emerged as an impor-
tant heterocyclic system due to their wide range of biological activ-
ities [5–7], as well as synthetic applications in medicinal chemistry
[8]. In particular, pyrazolo[3,4-d]pyrimidine has been a fruitful
source of inspiration for medicinal chemists for many years and
attracted their attention due to their numerous activities [9].
Pyrazolo[3,4-d]pyrimidines were identified as a general class of
adenosine receptors [10]. There is no big difference in the basic
structures of pyrazolopyrimidines and purines [11]. In the last
antituberculostatic, antimicrobial, antifungal, herbicidal and
antiviral [12–18].
Recently, it was reported that many pyrazolo[3,4-d] pyrimidine
and their derivatives exhibit anticancer activity via the interaction
with different enzymes and receptors [19,20]. For example, Abd El
Hamid and collaborators reported that pyrazolo[3,4-d]pyrimidines
were identified as potential anti-breast cancer agents (Fig. 1I) [21].
In 2011, Kumar et al. noted that pyrazolo[3,4-d]pyrimidines
possessed cytotoxic activity towards Src kinase and human ovarian
adenocarcinoma (SK-Ov-3), breast carcinoma (MDA-MB-361), and
colon adenocarcinoma (HT-29) (Fig. 1II) [22].
In addition, in 2012, Kim et al. described the SAR and synthesis
of pyrazolopyrimidines as potent and selective p70S6K inhibitors
(Fig. 1III) [23].
Also a careful literature survey reveals that pyrazolo[1,5-a]
pyrimidine derivatives were reported to exhibit a broad spectrum
of biological activities including antitumor, antimicrobial, antiin-
flammatory and cytotoxic [24–27]. Furthermore, numerous syn-
thetic strategies have been outlined for the synthesis of pyrazolo
⇑
Corresponding authors.
E-mail addresses: jalloul.bouajila@univ-tlse3.fr (J. Bouajila), hich.benjannet@
yahoo.fr (H. Ben Jannet).
http://dx.doi.org/10.1016/j.bioorg.2016.05.001
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
2. Results and discussion
161
2.1. Chemistry
According to the previously reported method [31] the starting
materials 5-aminopyrazole-4-carbonitriles 1a,b were prepared.
The treatment of compound 1b in ethanol by a solution of sodium
hydroxide
affords
the
5-amino-1-phenyl-1H-pyrazole-4-
carboxamide 2. Thus, the latter readily underwent cyclization
when heated in acetonitrile with various substituted aromatic
aldehydes in the presence of equimolar molecular iodine used as
a mild Lewis acid, to give a new series of pyrazolo[3,4-d]pyrimidi-
nones derivatives 3a-h in good yields (60–78%) (Scheme 1).
The structures of these compounds were confirmed according
to their spectral data. In fact, the 1H NMR spectrum of compound
3c, as an example, showed a singlet for NH proton at dH 12.4. Fur-
thermore, the same spectrum showed in addition of the signals
corresponding to the protons introduced by the pyrazole moiety,
an AB quartet peak at the aromatic region, confirming the presence
of the para-substituted aromatic ring and the occurrence of
heterocyclization.
FTIR and High Resolution Mass Spectrometry (HRMS) data of all
the formed derivatives were also in agreement with the proposed
structures.
Refluxing of compound 1b in acetic anhydride in the
presence of acetic acid afforded a white product identified as
3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one 4
Fig. 1. Example of pyrazolo[3,4-d]pyrimidines as anticancer agents.
[1,5-a]pyrimidines, some of the synthetic methods are the conden-
sation of arylhydrazone derivatives with 3-aminopyrazoleand
the one-pot reaction of ketones, aromatic aldehydes and
3-aminopyrazoles [28,29].
Taking all the above findings into consideration and by contin-
uing researches for new biaocative hybride molecules [30], we
report herein the synthesis of new series of pyrazolo[3,4-d]pyrim-
idinones, new esters and triazoles linked to pyrazolo[3,4-d]pyrim-
idinones as well as the synthesis of new series of pyrazolo[1,5-a]
pyrimidine derivatives. Most of the prepared compounds were
evaluated for their cytotoxic and anti-5-lipoxygenase activities
and the structure-activity relationship (SAR) were discussed.
Scheme 1. Synthetic route of compounds 2–7 and 11a-e.

162
A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
(Scheme 1). Its structure was ascertained by its 1H NMR spectrum
showing two characteristic singlet signals at dH 2.06 and 8.14 due
to methyl protons H9 and NH proton, respectively. The 13C NMR
spectrum revealed in particular the disappearance of the signal rel-
ative to the CN function (dC 114.7).
(0–20%). The proportions of the obtained regioisomers 9 and
10 were determined after their separation by column
chromatography.
The structure of compounds 9 and 10 were characterized by
spectroscopic methods. The formation of 1,4-triazoles 9a-e were
further confirmed by their 1H NMR spectra showing a singlet at
When compound 4 was treated with ethyl acetate chloride in
refluxing dry DMF using potassium carbonate as a base, it fur-
nished a single product identified as ethyl 2-(3,6-dimethyl-4-
oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl) acetate 5.
Its 1H NMR spectrum showed the absence of the signal related to
the mobile proton (NH) and the observation of two doublets at
dH 3.51 (J = 17.1 Hz) and at dH 4.53 (J = 17.1 Hz) relative to the
methylene group (NACH2) and the presence of signals relative to
the ester group. The 13C NMR spectrum reinforced this structure
by the appearance of the ester function signal at dC 169.6, two
methylene carbons signals at dC 49.1 and 61.8 corresponding to
C10 and C12, respectively, and the appearance of a signal at dC
14.0 attributed to the methyl carbon C13. The mass spectrum
recorded in ES-HRMS showed a pseudo-molecular ion peak [M
+H]+ at m/z 326.1379 which is consistent with its molecular for-
mula C17H18N4O3.
0
dH 7.95–8.05 attributable to the H5 of the triazole and new signals
at dH 7.02–7.20 consequent to the protons of the aromatic ring
attached to the triazole system. 13C NMR spectra provide addi-
tional support for the compounds by showing lactone carbonyl,
0
0
ANACH2 and 1,4 triazole carbons C5 and C4 resonances at dC
169.3–169.8, 42.6–43.4, 121.2–122.5 and 141.9–143.2, respec-
tively. The resulting 1,4-regioisomers were evidenced from the
0
0
NOEs between H5 /H10 and H5 /Harom, and the absence of any NOE
between H10/Harom
.
0
In fact, the H5 triazole proton in the obtained 1,4-regioisomers
1
0
is shifted than H4 in 1,5-regiosomers. The analysis of H NMR spec-
tra of 1,5-regioisomers showed clearly the presence a singlet at dH
0
8.15–8.20 attributable to the H4 of the triazole ring.
The 1,5-regiochemistry thus adopted was confirmed by the
0
NOESY experiment showing the NOEs between H4 /H10 and
0
In another hand, treatment of compound 5 with a solution
of NaOH (5%) in ethanol followed by acidification produces
the 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-
5(4H)-yl) acetic acid 6. The 1H NMR spectrum showed the disap-
pearance of the characteristic signals of the ester group and the
appearance of a new broad singlet at dH 12.70 corresponding to
the acidic proton, indicating that the pyrazolopyrimidinone 5
evolved into a carboxylic acid.
Esterification of compound 6 with different alcohols using
iodine as catalyst, led to the formation of new esters 7a-f linked
pyrazolo[3,4-d]pyrimidinones. The desired new compounds were
obtained in yields ranging from 40 to 83% (Scheme 1).
absence of NOE between H4 /Harom
.
The mass spectrometry analysis of all cycloadducts also rein-
forces the proposed structures (Scheme 2).
Pyrazolo [1,5-a]pyrimidine derivatives has shown an enormous
synthetic value in the preparation of various drugs and bio-active
molecules [32]. Stimulated by the successful applications of
pyrazolo[1,5-a]pyrimidines as anticancer and antitumor agents
[33], our objective was to synthesize a new class of these
compounds. In this context, we reacted 5-aminopyrazole 1a with
a
-cyanocinnamonitiles in acetonitrile in the presence of a catalytic
amount of piperidine to produce the corresponding pyrazolo[1,5-a]
pyrimidine-3,6-dicarbonitriles 11a-e (Scheme 1).
Interestingly, compound 4 seemed to be a useful candidate for
further chemical transformations. Thus, the required acetylenic
dipolarophile 8 was synthesized by the reaction of substituted
The 1H NMR spectra of compound 11a-e, displayed the signal
of the NH2 function at dC 9.18–9.40 and aromatic proton at dH
7.28–7.98 confirming the presence of an aromatic ring and the
occurrence of heterocyclization. The mass spectra of all prepared
compounds were compatible with the proposed structures.
pyrazolopyrimidinone
4 with 3-bromoprop-1-yne employing
NaH in dry DMF, which was then subjected to a reaction of
1,3-dipolar cycloaddition (Scheme 2).
The cycloaddition reaction of arylazides and dipolarophile 8,
under conventional conditions by refluxing in toluene in the pres-
ence of a catalytic amount of triethylamine for 8 h leads to two
regioisomers; the major one was the 1,4-disubstituted triazole 9
(45–75%) while that 1,5-isomer 10 was obtained in a small amount
2.2. Biological
2.2.1. Cytotoxic activity
For evaluation of cytotoxic activity of new synthesized com-
pounds (4, 5, 7a-f, 8, 9a-e and 11a-e), two human cancer cell lines
Scheme 2. Synthetic route of the 1,4- and 1,5-triazolyl derivatives 9 and 10.

A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
163
Table 1
Table 2
Cytotoxic activity of certain pyrazolo[3,4-d]pyrimidine-4(5H)-ones and pyrazolo[1,5-
a]pyrimidines.
5-Lipoxygenase inhibition capacity of certain pyrazolo[3,4-d]pyrimidine-4(5H)-ones.
Compounds
5-Lipoxygenase
Compounds
HCT-116
MCF-7
M)
% inhibition (concentration = 100 lM)
% inhibition (concentration = 100
l
3a
3b
3c
3d
3e
3f
3g
3h
4
14.5 1.0
naa
4
5
47.6 3.2
51.3 9.0
30.1 1.0
60.5 2.2
49.4 2.4
54.8 2.0
59.1 2.2
41.7 5.1
49.1 6.5
60.7 2.9
59.8 0.3
63.2 1.3
75.4 8.9
61.4 1.0
naa
25.0 3.8
31.8 2.1
14.2 1.0
31.3 6.8
33.6 0.5
28.4 1.8
48.9 1.5
16.8 2.1
56.4 1.0
42.0 3.2
67.3 7.4
59.6 2.1
72.0 4.8
58.1 0.1
na
22.5 1.1
25.5 1.0
68.0 3.3
72.2 0.4
43.8 0.1
43.9 1.0
6.1 1.0
28.5 0.6
10.6 0.6
12.4 1.5
13.9 1.7
33.9 1.5
22.7 1.2
24.5 1.1
21.8 1.4
8.6 0.6
na
7a
7b
7c
7d
7e
7f
5
8
7a
7b
7c
7d
7e
7f
8
9a
9b
9c
9a
9b
9c
9d
9e
11a
11b
11c
11d
11e
na
na
na
na
na
na
na
na
9.9 0.8
NDGA
98.0 0.1
Tamoxifen
0.36 0.03b
0.38 0.03b
a
Not active.
a
Not active.
IC50 (lM).
b
exerted by the substituent in para position of the aromatic ring
attached to the triazole moiety does not seem to influence in a con-
siderable way the cytotoxic activity.
The study of the structure-activity relationship (SAR) has noted
that the triazole moiety attached to the pyrazolopyrimidine 4 via
the methylene spacer link could be the origin of the significant
anticancer activity of compounds 9.
The results showed that none of the compounds 11a-e has
demonstrated cytotoxic activity on both HCT-116 and MCF-7 cell
lines.
were used: HCT-116 (colon carcinoma cell line) andMCF-7 (breast
carcinoma cell line). Results are given in Table 1 at 100 M.
l
The results showed that the pyrazolopyrimidinone 4 exhibited a
moderate cytotoxic activity against HCT-116 (% inhibi-
tion = 47.6 3.2%) but the MCF-7 cell line does not seem sensitive
to this compound (% inhibition = 25.0 3.8%). The esters 5 and 7a-f
displayed variable cytotoxic activities against HCT-116 and MCF-7
cell lines (% inhibition = 30.1–60.5% and 14.2–48.9%, respectively).
It has been found that the most active esters towards HCT-116
were compounds 7b (isobutyl) and 7e (n-butyl) with percentage
inhibition values of 60.5 2.2 and 59.1 2.2%, respectively. This
finding showed the importance of the structure of the alkyl chain
of the alcohol used in this activity against this cell line. Moreover,
we noticed that the presence of two free vicinal hydroxyl groups in
compound 7f has not improved its activity towards the same cell
line. Similarly, the co-existence of the two ester functions in com-
pound 7a did not improve this activity against HCT-116 by com-
parison to the rest of the esters. On the other hand, only
compound 7e (n-butyl) exhibited moderate effect against MCF-7
cell line (% inhibition = 48.9 1.5%). This finding showed the selec-
tivity of the cell lines used towards the tested compounds. The
results showed that the propargylation of compound 4 to afford
8 improved the cytotoxicity only against MCF-7 cell line, indeed,
the percent inhibition is more than doubled, it rose from 25.0 to
56.4%.
2.2.2. 5-Lipoxygenase inhibition
A series of prepared compounds have been tested for their abil-
ity to inhibit 5-lipoxygenase enzyme activity. The results revealed
that compounds 3e (4-OH-3-OCH3ꢀPh) and 3f (4-OH-3-OC2H5ꢀPh)
were found to be the most active against 5-lipoxygenase with inhi-
bition percentages of 68.0% and 72.2% at 100 lM, respectively
(Table 2). The slightly higher activity of compound 3f compared
to that of its analog 3e suggests that the longer the alkyl chain of
the ether group in 3 position of the aryl system is long more the
better activity. On the other hand, compounds 3g (NA(CH3)2ꢀPh)
and 3h (C5H4N) showed moderate activity with percentage
inhibition values of 43.8% and 43.9%, respectively. The remaining
compounds from the series 3 showed weak inhibitory effects.
These findings demonstrated the impact of the nature of the sub-
stituent(s) attached to the phenyl ring on the anti-5-lipoxygenase
activity. Indeed, the presence of the hydroxyl function and the
ether group (OC2H5 or OCH3) at 4 and 3 positions of the phenyl ring
respectively were in favor of this activity. On the other hand, com-
pounds 4, 5, 7a-f, 8 and 9a-c tested did not exhibit any interesting
Interestingly, the prepared 1,2,3-triazole-pyrazolopyrimidinone
hybrids 9a-e showed a moderate to good cytotoxic activity against
HCT-116. The variation of this activity is easily explained by the
nature of the aromatic system attached to the triazole ring. In fact,
triazole 9d bearing a 4-ClꢀPhat N-10 was found to be the most
active (% inhibition = 75.4 8.9%) followed by 9c (4-CH3OꢀPh) (%
inhibition = 63.2 1.3%), while compound 9a with a Ph attached
at N-10 (% inhibition = 60.7 2.9%) displayed towards the same cell
line a comparable activity to that of 9b (% inhibition = 59.8 0.3%)
bearing a 4-CH3ꢀPh.
activity at the concentration of 100 lM.
3. Conclusion
In conclusion, we have synthesized a series of 6-aryl-3-methyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones 3a-h via conden-
sation of carboxamide 2 with some arylaldehydes. Then, we have
described the successful access to new esters 7a-f linked pyrazolo
[3,4-d]pyrimidinones hybrids. Also, we designed and synthesized
novel triazole derivatives bearing pyrazolopyrimidines-based
From the estimation of the cytotoxic activity on the human
breast MCF-7 cancer cell line, the most active compounds were
found to be 9b (4-CH3ꢀPh) and 9d (4-ClꢀPh) with % inhibition values
of 67.3% and 72.0%, respectively. In this case the electronic effect

164
A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
moiety. Most of the newly prepared compounds have been tested for
their cytotoxic (HCT-116 and MCF-7 cell lines) and 5-lipoxygenase
inhibition activities. In this context, we noticed that the HCT-116
colon cell line more sensitive than MCF-7 cell line to the various
products tested. The nature of the alcohols used in the preparation
of esters 7a-f, the propargylation of the nitrogen atom at position
N-5 in compound 8 and the nature of the aromatic ring attached to
the triazole moiety in compounds 9a-e were the origins of the cyto-
toxic activity. On the hand, it was found that the co-existence of the
hydroxyl function and the alkyl fragment of the ether group (OC2H5
or OCH3) at 4 and 3 positions of the phenyl ring respectively led to
considerable anti-5-lipoxygenase activity.
4.1.1.4.
3-Methyl-6-(4-nitrophenyl)-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3d). Yellow solid, yield: 60%, mp.: 350–
352 °C; IR (KBr, cmꢁ1
) t: 3330 (ANHA), 1670 (C@O), 1412
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.39 (s, 3H, H8), 7.20–
8.82 (m, 9H, Harom), 12.63 (s, 1H, ANHA). 1H NMR (75 MHz,
DMSO-d6) dC: 13.1, 104.3, 122.7, 124.1, 126.3, 129.1, 129.6, 136.2,
139.5, 143.2, 148.7, 148.2, 158.4, 159.8. HRMS [M+H]+ calcd for
(C18H14N5O3)+ 348.1001, found 348.1011.
4.1.1.5.
6-(4-Hydroxy-3-methoxyphenyl)-3-methyl-1-phenyl-1H-
pyrazolo[3,4-d]pyrimidin-4(5H)-one (3e). White solid, yield: 62%,
mp.: 316–318 °C; IR (KBr, cmꢁ1
) t: 3328 (ANHA), 1675 (C@O),
1410 (N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.54 (s, 3H, H8),
4. Material and methods
3.89 (s, 3H, OACH3), 5.06 (s, 1H, AOHA), 6.91–8.15 (m, 8H, Harom),
11.04 (s, 1H, ANHA). 1H NMR (75 MHz, DMSO-d6) dC: 13.2, 55.9,
103.5, 112.2, 115.6, 121.1, 122.1, 122.7, 126.1, 129.0, 138.6,
145.8, 147.8, 150.9, 153.1, 155.8, 160.1. HRMS [M+H]+ calcd for
(C19H17N4O3)+ 349.1299, found 349.1301.
4.1. Chemistry
All reactions were monitored by TLC using aluminum sheets of
Merck silica gel 60 F254, 0.2 mm. Melting temperatures were
determined on an electrothermal 9002 apparatus and were
reported uncorrected. NMR spectra were recorded on a Bruker
AC-300 spectrometer at 300 MHz (1H) and 75 MHz (13C). All
chemical shifts were reported as d values (ppm) relative to resid-
ual non deuterated solvent. ESI-HRMS were acquired with a LCT
Premier XE (Waters, ESI technique, positive mode) mass
spectrometer.
4.1.1.6. 6-(3-Ethoxy-4-hydroxyphenyl)-3-methyl-1-phenyl-1H-pyra-
zolo[3,4-d]pyrimidin-4(5H)-one (3f). White solid, yield: 60%, mp.:
310–312 °C; IR (KBr, cmꢁ1
) t: 3323 (ANHA), 1672 (C@O), 1413
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 1.31 (t, 3H, J = 7.1 Hz,
OACH2ACH3), 2.56 (s, 3H, H8), 4.21 (q, 2H, J = 7.1 Hz, OACH2ACH3),
5.10 (s, 1H, AOHA), 6.94–8.17 (m, 8H, Harom), 11.60 (s, 1H, ANHA).
1H NMR (75 MHz, DMSO-d6) dC: 13.1, 14.5, 64.5, 103.4, 112.8,
115.6, 121.1, 122.0, 122.5, 126.4, 129.1, 138.5, 145.8, 146.8,
152.9, 155.1, 155.7, 159.8. HRMS [M+H]+ calcd for (C20H19N4O3)+
363.1412, found 363.1421.
4.1.1. General procedure for the synthesis of 6-aryl-3-methyl-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (3a-h)
To a mixture of carboxamide 2 (0.2 mmol) and various arylalde-
hydes (0.2 mmol) in dry acetonitrile (15 ml), molecular iodine
(0.050 g) was added. After the reaction was completed, the mixture
was cooled to room temperature. A solution of sodium thiosul-
phate (5%) was added and the resulted solid was filtered off and
washed with water. The crude product was recrystallized from
ethanol.
4.1.1.7. 6-(4-(Dimethylamino)phenyl)-3-methyl-1-phenyl-1H-pyra-
zolo[3,4-d]pyrimidin-4(5H)-one (3g). Pink solid, yield: 78%, mp.:
320–322 °C; IR (KBr, cmꢁ1
) t: 3330 (ANHA), 1670 (C@O), 1415
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.55 (s, 3H, H8), 3.03 (s,
6H, NA(CH3)2), 6.79–8.19 (m, 9H, Harom), 11.90 (s, 1H, ANHA).
1H NMR (75 MHz, DMSO-d6) dC: 13.1, 40.7, 103.2, 111.3, 118.3,
121.1, 125.9, 128.9, 129.0, 138.6, 145.8, 152.8, 153.4, 156.1,
159.9. HRMS [M+H]+ calcd for (C20H20N5O)+ 346.1620, found
346.1633.
4.1.1.1. 6-(4-Methoxyphenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3a). White solid, yield: 60%, mp.: 324–
326 °C; IR (KBr, cmꢁ1
) t: 3325 (ANHA), 1670 (C@O), 1410
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.53 (s, 3H, H8), 3.60 (s,
3H, AOCH3), 7.19–8.07 (m, 9H, Harom), 12.40 (s, 1H, ANHA). 13C
NMR (75 MHz, DMSO-d6) dC: 13.4, 54.5, 103.5, 117.5, 123.4,
125.2, 126.6, 129.2, 131.4, 139.7, 145.6, 149.8, 158.1, 159.9,
161.2. HRMS [M+H]+ calcd for (C19H17N4O2)+ 333.1310, found
333.1319.
4.1.1.8.
3-Methyl-1-phenyl-6-(pyridin-3-yl)-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3h). Pink solid, yield: 75%, mp.: 322–
324 °C; IR (KBr, cmꢁ1
) t: 3335 (ANHA), 1675 (C@O), 1411
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.58 (s, 3H, H8), 7.35–
9.31 (m, 9H, Harom), 12.53 (s, 1H, ANHA). 1H NMR (75 MHz,
DMSO-d6) dC: 13.2, 103.5, 121.3, 123.4, 126.4, 128.2, 129.0, 135.6,
139.2, 146.8, 149.1, 150.1, 151.1, 158.7, 160.1. HRMS [M+H]+ calcd
for (C17H14N5O)+ 304,1154, found 304,1164.
4.1.1.2. 6-(4-Bromophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3b). Brown solid, yield: 72%, mp.: 294–
296 °C; IR (KBr, cmꢁ1
) t: 3335 (ANHA), 1670 (C@O), 1414
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.46 (s, 3H, H8), 7.21–
8.10 (m, 9H, Harom), 11.38 (s, 1H, ANHA). 13C NMR (75 MHz,
DMSO-d6) dC: 13.2, 105.1, 122.3, 124.1, 125.2, 126.6, 128.4, 129.2,
131.2, 139.4, 144.2, 149.1, 158.7, 159.5. HRMS [M+H]+ calcd for
(C18H14BrN4O)+ 381.0350, found 381.0353.
4.1.2. General procedure for the synthesis of 3,6-dimethyl-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (4)
A solution of 5-aminopyrazole-4-carbonitrile 1b (0.02 mol) in
glacial acetic acid (10 mL) and acetic anhydride (20 mL) was
heated for 4 h, and then left to cool overnight at ambient temper-
ature. The solid formed was filtered, dried, and recrystallized from
4.1.1.3.
6-(4-Chlorophenyl)-3-methyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one (3c). White solid, yield: 63%, mp.: 346–
348 °C; IR (KBr, cmꢁ1
)
t
: 3320 (ANHA), 1675 (C@O), 1415
ethanol. White crystals, yield 78%, mp.: 277–279 °C; IR (KBr, cmꢁ1
)
(N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.58 (s, 3H, H8), 7.35–
8.56 (m, 9H, Harom), 12.40 (s, 1H, ANHA). 13C NMR (75 MHz,
DMSO-d6) dC: 13.4, 105.2, 123.7, 126.6, 128.1, 128.9, 129.2, 131.2,
135.3, 139.4, 145.2, 150.1, 158.2, 159.5. HRMS [M+H]+ calcd for
(C18H14ClN4O)+ 337.0855, found 337.0857.
t
: 3240 (NH), 1680 (C@O). 1H NMR (300 MHz, CDCl3) dH: 2.06 (s,
3H, H9), 2.51 (s, 3H, H8), 7.40–7.51 (m, 5H, ArAH), 8.14 (s, 1H,
ANHA). 13C NMR (75 MHz, CDCl3) dC: 13.0, 22.4, 113.1, 123.8,
127.8, 130.7, 137.1, 139.7, 151.9, 160.2, 169.0. HRMS [M+H]+ calcd
for.(C13H13N4O)+ 241.1083, found 241.1089.

A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
165
4.1.3. General procedure for the synthesis of 2-(3-methyl-4-oxo-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)acetate (5)
169.5. HRMS [M+H]+ calcd for (C19H23N4O3)+ 355.1768, found
355.1770.
A
solution of 3,6-dimethyl-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-4(5H)-one 4, ethyl chloroacetate and anhydrous potas-
sium carbonate was stirred and refluxed at 80 °C for 2 h in dry
DMF. After completion of reaction, the mixture was cooled and
poured into cold water. The obtained solid was filtered, washed
with water, dried and recrystallized from ethanol. White solid,
4.1.5.3. Sec-butyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-5(4H)-yl)acetate (7c). White solid, yield: 57%, mp.: 82–
84 °C (EtOH); IR (KBr, cmꢁ1 : 1665 (C@O), 1409 (N@N). 1H
) t
NMR (300 MHz, CDCl3) dH: 0.91 (t, 3H, ACH3ACH2A, J = 6.3 Hz),
1.34 (d, 3H, ACH3ACHA, J = 6.1 Hz), 1.55 (m, 2H, ACH3ACH2-
ACHA), 2.06 (s, 3H, H9), 2.48 (s, 3H, H8), 3.57 (d, NACH2(a),
yield: 90%, mp.: 108–110 °C; IR (KBr, cmꢁ1
) t: 1679 (C@O), 1410
(N@N). 1H NMR (300 MHz, CDCl3) dH: 1.28 (t, 3H, ACH3ACH2AOA,
J = 7.2 Hz), 2.15 (s, 3H, H9), 2.48 (s, 3H, H8), 3.51 (d, NACH2(a),
J = 17.1 Hz), 4.22 (q, 2H, ACH3ACH2AOA, J = 7.2 Hz), 4.53 (d,
NACH2(b), J = 17.1 Hz), 7.44–7.55 (m, 5H, Harom). 13C NMR
(75 MHz, CDCl3) dC: 13.1, 14.0, 21.5, 49.1, 61.8, 112.0, 123.5,
126.4, 129.9, 130.1, 136.9, 143.6, 152.5, 167.1, 169.6. HRMS [M
+H]+ calcd for (C17H19N4O3)+ 327.1450, found 327.1460.
J = 17.1 Hz), 4.10 (m, 1H, ACH3ACHACH2A), 4.53 (d, NACH2(b),
J = 17.1 Hz), 7.45–7.56 (m, 5H, Harom). 13C NMR (75 MHz, CDCl3)
dC: 12.3, 13.1, 19.8, 22.4, 28.5, 49.1, 73.7, 112.0, 123.5, 126.4,
129.7, 129.9, 136.8, 144.6, 153.5, 167.1, 169.4. HRMS [M+H]+ calcd
for (C19H23N4O3)+ 355.1769, found 355.1774.
4.1.5.4. Isopropyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-5(4H)-yl)acetate (7d). White solid, yield: 83%, mp.: 103–
4.1.4. General procedure for the synthesis of 2-(3-methyl-4-oxo-1-
phenyl-1H-pyrazolo[3,4-d]pyrimidin-5(4H)-yl)acetic acid (6)
A solution of compound 5 (10 mmol) and NaOH 5% (10 mL) was
refluxed in ethanol (25 mL) for 2 h. After cooling, the mixture was
acidified with H2SO4 and the precipitate dark yellow solid was col-
lected by filtration, dried and recrystallized from ethanol.
105 °C (EtOH); IR (KBr, cmꢁ1 : 1660 (C@O), 1410 (N@N). 1H NMR
) t
(300 MHz, CDCl3) dH: 1.26 (d, 6H, ACH(CH3)2), J = 6.1 Hz), 2.15 (s,
3H, H9), 2.48 (s, 3H, H8), 3.52 (d, NACH2(a), J = 17.1 Hz), 4.45 (d,
NACH2(b), J = 17.1 Hz), 5.09 (m, 1H, ACH(CH3)2), 7.45–7.55 (m,
5H, Harom). 13C NMR (75 MHz, CDCl3) dC: 13.1, 21.5, 29.6, 49.4,
69.8, 112.1, 123.5, 125.7, 128.8, 129.9, 136.9, 143.7, 152.5, 166.6,
169.5. HRMS [M+H]+ calcd for (C18H21N4O3)+ 341.1610, found
341.1615.
Yellow solid, yield: 55%, mp.: 156–158 °C; IR (KBr, cmꢁ1
) t:
1679 (C@O), 1415 (N@N), 1735 (C@O acid), 3330 (OH). 1H NMR
(300 MHz, DMSO-d6) dH: 2.14 (s, 3H, H9), 2.49 (s, 3H, H8), 3.53 (d,
NACH2(a), J = 17.1 Hz), 4.51 (d, NACH2(b), J = 17.1 Hz), 7.42–7.54
(m, 5H, Harom), 12.7 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6) dC:
13.2, 22.3, 47.9, 111.9, 123.7, 126.2, 129.5, 129.8, 136.8, 144.4,
152.3, 167.0, 169.4. HRMS [M+H]+ calcd for (C15H15N4O3)+
299.1140, found 299.1147.
4.1.5.5. Butyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-5(4H)-yl)acetate (7e). White solid, yield: 72%, mp.: 62–
64 °C (EtOH); IR (KBr, cmꢁ1 : 1664 (C@O), 1413 (N@N). 1H
) t
NMR (300 MHz, CDCl3) dH: 0.83 (t, 3H, ACH3ACH2ACH2ACH2AOA,
J = 7.5 Hz), 1.33 (m, 2H, ACH3ACH2ACH2ACH2AOA), 1.53 (m, 2H,
ACH3ACH2ACH2ACH2AOA), 2.05 (s, 3H, H9), 2.39 (s, 3H, H8),
3.45 (d, NACH2(a), J = 17.1 Hz), 4.09 (t, 2H, ACH3ACH2ACH2ACH2-
4.1.5. General procedure for preparation of esters (7a-f)
A mixture of acid 6 (3 mmol) and the appropriate alcohol (8 mL)
was refluxed in the presence of a catalytic amount of di-iodine for
8 h. After removal of the excess of alcohol, the residue was
extracted with EtOAc and the combined organic layer was dried
over sodium sulfate, concentrated in vacuo and chromatographed
(column, silica gel, CH2Cl2/EtOAc, 8:2) to give esters 7a-f.
AOA, J = 6.9 Hz), 4.44 (d, NACH2(b), J = 17.1 Hz), 7.36–7.46 (m, 5H,
Harom). 13C NMR (75 MHz, CDCl3) dC: 12.6, 13.1, 18.4, 21.0, 29.9,
48.1, 65.2, 111.5, 123.2, 125.9, 128.9, 129.4, 136.4, 143.1, 152.0,
166.7, 169.1. HRMS [M+H]+ calcd for (C19H23N4O3)+ 355.1767,
found 355.1774.
4.1.5.6.
pyrazolo[3,4-d]pyrimidin-5(4H)-yl)acetate (7f). White solid, yield:
: 1670 (C@O),
2,3-Dihydroxypropyl-2-(3,6-dimethyl-4-oxo-1-phenyl-1H-
4.1.5.1. Ethyl-2-(2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]
pyrimidin-5(4H) yl)acetoxy)propanoate (7a). Yellowish liquid,
40%, mp.: 133–135 °C (EtOH); IR (KBr, cmꢁ1
) t
yield: 60%; IR (CCl4, cmꢁ1 : 1659 (C@O), 1410 (N@N). 1H NMR
) t
1419 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.27 (s, 3H, H9), 2.40
(s, 3H, H8), 3.51(dd, 1H, ACHACH2AOH, J = 12.4 Hz, J = 7.9 Hz),
3.66 (dd, 1H, AOACH2ACHA, J = 6.9 Hz, J = 12.5 Hz), 3.70 (dd, 1H,
ACHACH2AOH, J = 12.4 Hz, J = 7.9 Hz) 3.96 (d, NACH2(a),
J = 17.1 Hz), 3.99 (m, 1H, AOHACH2ACHACH2A), 4.31 (dd, 1H,
(300 MHz, CDCl3) dH: 1.25 (t, 3H, ACH3ACH2AOA, J = 6.1 Hz),
1.55 (d, 3H, ACH3ACHA, J = 5.9 Hz) 2.04 (s, 3H, H9), 2.48 (s, 3H,
H8), 3.57 (d, NACH2(a), J = 17.1 Hz), 3.90 (d, NACH2(b),
J = 17.1 Hz), 4.18 (q, 2H, ACH3ACH2AOA, J = 6.1 Hz), 4.90 (q, 1H,
ACH3ACHA, J = 5.9 Hz) 7.42–7.53 (m, 5H, Harom). 13C NMR
(75 MHz, CDCl3) dC: 13.1, 14.3, 16.5, 22.1, 48.8, 60.5, 71.4, 112.1,
123.5, 126.3, 129.4, 129.7, 136.9, 144.6, 152.7, 167.3, 169.0,
169.5. HRMS [M+H]+ calcd for (C20H23N4O5)+ 399.1664, found
399.1668.
AOACH2ACHA, J = 12.5 Hz, J = 6.9 Hz), 4.49 (d, NACH2(b),
J = 17.1 Hz), 7.41–7.54 (m, 5H, Harom). 13C NMR (75 MHz, CDCl3)
dC: 12.7, 20.9, 49.2, 62.4, 66.0, 69.1, 111.5, 123.0, 126.1, 128.3,
129.4, 136.2, 143.3, 152.1, 166.7, 169.6. HRMS [M+H]+ calcd for
(C18H21N4O5)+ 373.1501, found 373.1508.
4.1.5.2. Isobutyl 2-(3,6-dimethyl-4-oxo-1-phenyl-1H-pyrazolo[3,4-d]
4.1.6. General procedure for N-propargylation of 3,6-dimethyl-1-phenyl-
1H-pyrazolo[3,4-d]pyrimidin-4(5H)-ones (4)
pyrimidin-5(4H)-yl)acetate (7b). White solid, yield: 78%, mp.: 92–
94 °C (EtOH); IR (KBr, cmꢁ1 : 1666 (C@O), 1418 (N@N). 1H
) t
To a solution of pyrazolopyrimidinone 4 (0.02 mol) in dry DMF
(15 mL), sodium hydride (0.02 mol) and 3-bromoprop-1-yne
(0.02 mol) was added. The resultant mixture was stirred at 20 °C
for 1 h. The mixture was diluted with water and extracted using
EtOAc. The organic layer was dried over anhydrous MgSO4. After
evaporation of the solvent, the residual was purified by column
chromatography, to give the desired dipolarophile 8.
NMR (300 MHz, CDCl3) dH: 0.92 (d, 6H, ACH(CH3)2, J = 6.5 Hz),
1.95 (m, 1H, ACH2ACH(CH3)2), 2.13 (s, 3H, H9), 2.47 (s, 3H, H8),
3.57 (d, NACH2(a), J = 17.1 Hz), 3.93 (d, 2H, OACH2ACH(CH3)2,
J = 7.0 Hz), 4.53 (d, NACH2(b), J = 17.1 Hz), 7.44–7.54 (m, 5H,
H
arom). 13C NMR (75 MHz, CDCl3) dC: 13.1, 18.9, 21.5, 27.5, 49.0,
71.7, 112.0, 123.5, 126.1, 129.2, 129.9, 136.9, 143.6, 152.5, 167.2,

166
A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
4.1.6.1. 3,6-Dimethyl-1-phenyl-5-(prop-2-yn-1-yl)-1H-pyrazolo[3,4-
4.1.7.5. 3,6-Dimethyl-5-((1-(naphthalen-1-yl)-1H-1,2,3-triazol-4-yl)
methyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9e).
d]pyrimidin-4(5H)-one (8). White solid, yield: 80%, mp.: 125–
127 °C; IR (KBr, cmꢁ1
)
t
: 1675 (C@O), 3370 (CH propynyl). 1H
Creamy white, yield: 48%, mp.: 134–136 °C; IR (KBr, cmꢁ1
) t:
1678 (C@O), 1413 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.00 (s,
3H, H9), 2.37 (s, 3H, H8), 4.27 (d, NACH2(a), J = 15.0 Hz), 5.32 (d,
NMR (300 MHz, CDCl3) dH: 2.01 (s, 3H, H9), 2.30 (t, 1H, AC„CAH,
J = 2.4 Hz), 2.49 (s, 3H, H8), 4.10–4.16 (dd, NACH2AC„CAH,
NACH2(b), J = 15.0 Hz), 7.36–7.90 (m, 12H, Harom), 7.96 (s, 1H,
J = 17.4 Hz,
J = 2.4 Hz),
4.58–4.64
(dd,
NACH2AC„CAH,
H
H5 ). 13C NMR (75 MHz, CDCl3) dC: 13.2, 22.0, 43.4, 111.9, 122.5,
0
J = 17.4 Hz, J = 2.4 Hz), 7.31–7.47 (m, 5H,
arom). 13C NMR
123.1, 126.0, 126.3, 126.5, 126.8, 128.4, 129.1, 130.2, 131.2,
133.2, 137.0, 137.4, 141.9, 143.7, 152.2, 169.8. HRMS [M+H]+ calcd
for (C26H22N7O)+ 448.1855, found 448.1860.
(75 MHz, CDCl3) dC: 13.30, 22.2, 37.1, 67.9, 92.8, 111.9, 123.0,
128.8, 129.3, 129.8, 137.0, 143.2, 152.3, 168.8. HRMS [M+H]+ calcd
for (C16H15N4O)+ 279.1246, found 279.1247.
4.1.7.6. 3,6-Dimethyl-1-phenyl-5-((1-(p-tolyl)-1H-1,2,3-triazol-5-yl)
4.1.7. General procedure for synthesis triazolyl derivatives (9a-e)
To alkyne 8 (3 mmol) in refluxing toluene, the appropriate ary-
lazides, regenerated in situ [34], in the presence of triethylamine
was added and the mixture was heated for 6 h. The resulting mix-
ture was washed with water, extracted twice with dichloro-
methane and then dried over Na2SO4. After removing the solvent
under reduced pressure, the residue thus obtained was purified
by column chromatography on silica gel eluted with mixture hex-
ane/ethyl acetate (9/1), affording the triazoles 9 and 10 as white
solids.
methyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (10b). White solid,
yield: 20%, mp.: 121–123 °C; IR (KBr, cmꢁ1
) t: 1675 (C@O), 1410
(N@N). 1H NMR (300 MHz, CDCl3) dH: 2.03 (s, 3H, H9), 2.30
(PhACH3), 2.43 (s, 3H, H8), 4.28 (d, NACH2(a), J = 15.1 Hz), 5.16
(d, NACH2(b), J = 15.1 Hz), 7.14–7.45 (m, 9H, Harom), 8.15 (s, 1H,
H4 ). 13C NMR (75 MHz, CDCl3) dC: 13.1, 20.8, 21.7, 43.5, 111.4,
0
119.1, 120.2, 121.2, 122.1, 123.2, 128.0, 128.7, 129.3, 130.7,
137.1, 142.1, 143.2, 152.4, 169.6. HRMS [M+H]+ calcd for
(C23H22N7O)+ 412.1880, found 412.1886.
4.1.7.1.
3,6-Dimethyl-1-phenyl-5-((1-phenyl-1H-1,2,3-triazol-4-yl)
4.1.7.7. 5-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-5-yl)methyl)-3,6-
methyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9a). White solid,
dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(10c).
: 1670
yield: 75%, mp.: 138–140 °C; IR (KBr, cmꢁ1
) t: 1670 (C@O), 1415
White solid, yield: 13%, mp.: 129–131 °C; IR (KBr, cmꢁ1
)
t
(N@N). 1H NMR (300 MHz, CDCl3) dH: 2.08 (s, 3H, H9), 2.41 (s,
3H, H8), 4.35 (d, NACH2(a), J = 15.0 Hz), 5.25 (d, NACH2(b),
(C@O), 1414 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.10 (s, 3H,
H9), 2.38 (s, 3H, H8), 3.84 (PhAOCH3), 4.30 (d, NACH2(a),
J = 15.0 Hz), 7.41–7.72 (m, 10H, Harom), 8.05 (s, 1H, H5 ). 13C NMR
0
J = 15.4 Hz), 5.24 (d, NACH2(b), J = 15.4 Hz), 7.05–7.66 (m, 9H,
(75 MHz, CDCl3) dC: 12.7, 21.5, 42.6, 111.4, 120.3, 121.7, 123.2,
126.2 128.4, 128.8, 129.2, 129.3, 136.4, 142.1, 143.3, 151.6, 169.3.
HRMS [M+H]+ calcd for (C22H20N7O)+ 398.1720, found 398.1729.
0
H
arom), 8.18 (s, 1H, H4 ). 13C NMR (75 MHz, CDCl3) dC: 12.5, 21.8,
43.0, 54.9, 110.9, 115.1, 122.4, 122.9, 123.5, 126.2, 129.5, 129.9,
131.4, 136.5, 142.9, 143.1, 152.1, 159.8, 169.5. HRMS [M+H]+ calcd
for (C23H22N7O2)+ 428.1830, found 428.1835.
4.1.7.2. 3,6-Dimethyl-1-phenyl-5-((1-(p-tolyl)-1H-1,2,3-triazol-4-yl)
methyl)-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one (9b). White solid,
4.1.7.8.
5-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-5-yl)methyl)-3,6-
yield: 45%, mp.: 120–122 °C; IR (KBr, cmꢁ1
) t: 1675 (C@O), 1410
dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(10d).
(N@N). 1H NMR (300 MHz, CDCl3) dH: 2.00 (s, 3H, H9), 2.33
(PhACH3), 2.47 (s, 3H, H8), 4.28 (d, NACH2(a), J = 15.0 Hz), 5.16
(d, NACH2(b), J = 15.0 Hz), 7.19–7.47 (m, 9H, Harom), 7.95 (s, 1H,
White solid, yield: 17%, mp.: 116–118 °C; IR (KBr, cmꢁ1
)
t
: 1672
(C@O), 1410 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.18 (s, 3H,
H9), 2.47 (s, 3H, H8), 4.33 (d, NACH2(a), J = 15.0 Hz), 5.24 (d,
H5 ). 13C NMR (75 MHz, CDCl3) dC: 13.2, 21.3, 22.0, 43.2, 111.9,
0
0
NACH2(b), J = 15.0 Hz), 7.46–7.70 (m, 9H, Harom), 8.20 (s, 1H, H4 ).
13C NMR (75 MHz, CDCl3) dC: 13.1, 22.4, 42.3, 111.6, 121.5, 121.9,
122.1, 123.1, 128.7, 129.0, 129.4, 130.6, 132.7, 136.7, 142.1,
144.1, 152.6, 169.9. HRMS [M+H]+ calcd for (C22H19 ClN7O)+
432.1338, found 432.1345.
119.3, 120.3, 121.4, 121.7, 123.2, 128.4, 128.7, 129.3, 129.7,
136.9, 142.5, 143.8, 152.1, 169.8. HRMS [M+H]+ calcd for
(C23H22N7O)+ 412.1880, found 412.1886.
4.1.7.3. 5-((1-(4-Methoxyphenyl)-1H-1,2,3-triazol-4-yl)methyl)-3,6-
dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(9c).
: 1668
4.1.7.9. 3,6-Dimethyl-5-((1-(naphthalen-1-yl)-1H-1,2,3-triazol-5-yl)
White solid, yield: 50%, mp.: 132–134 °C; IR (KBr, cmꢁ1
)
t
methyl)-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(10e).
(C@O), 1412 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.08 (s, 3H,
H9), 2.41 (s, 3H, H8), 3.87 (PhAOCH3), 4.34 (d, NACH2(a),
Creamy white, yield: 15%, mp.: 133–135 °C; IR (KBr, cmꢁ1
) t:
1675(C@O), 1410 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.20 (s,
3H, H9), 2.47 (s, 3H, H8), 4.23 (d, NACH2(a), J = 15.1 Hz), 5.30 (d,
NACH2(b), J = 15.1 Hz), 7.01–7.96 (m, 12H, Harom), 8.20 (s, 1H,
J = 15.2 Hz), 5.26 (d, NACH2(b), J = 15.2 Hz), 7.00–7.62 (m, 9H,
0
H
arom), 7.97 (s, 1H, H5 ). 13C NMR (75 MHz, CDCl3) dC: 12.7, 21.5,
H4 ). 13C NMR (75 MHz, CDCl3) dC: 13.4, 22.6, 42.7, 110.8, 122.0,
42.8, 55.1, 111.4, 114.9, 122.0, 122.3, 123.7, 126.1, 129.1, 129.7,
131.1, 136.4, 141.9, 143.3, 151.6, 159.7, 169.3. HRMS [M+H]+ calcd
for (C23H22N7O2)+ 428.1830, found 428.1835.
0
123.1, 125.8, 126.3, 126.7, 127.3, 128.4, 129.4, 130.2, 132.4,
133.4, 136.5, 138.7, 142.3, 144.1, 153.4, 169.6. HRMS [M+H]+ calcd
for (C26H22N7O)+ 448.1855, found 448.1860.
4.1.7.4.
5-((1-(4-Chlorophenyl)-1H-1,2,3-triazol-4-yl)methyl)-3,6-
dimethyl-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4(5H)-one
(9d).
: 1674
4.1.8. General procedure for the preparation of pyrazolo[1,5-a]
pyrimidines (11a-e) from the reaction of 5-aminopyrazole 1a with a-
cyanocinnamonitriles
White solid, yield: 53%, mp.: 114–116 °C; IR (KBr, cmꢁ1
)
t
(C@O), 1416 (N@N). 1H NMR (300 MHz, CDCl3) dH: 2.10 (s, 3H,
H9), 2.42 (s, 3H, H8), 4.30 (d, NACH2(a), J = 15.0 Hz), 5.28 (d,
A mixture of 5-aminopyrazle-4-carbonitrile 1a (0.01 mol) and
0
NACH2(b), J = 15.0 Hz), 7.44–7.71 (m, 9H, Harom), 8.05 (s, 1H, H5 ).
a-cyanocinnamonitriles derivatives (0.01 mol) in acetonitrile
13C NMR (75 MHz, CDCl3) dC: 12.7, 21.4, 42.6, 111.4, 120.1, 121.2,
122.2, 123.2, 128.4, 128.7, 129.3, 129.7, 132.5, 136.4, 142.5,
143.2, 151.6, 169.4. HRMS [M+H]+ calcd for (C22H19 ClN7O)+
432.1338, found 432.1345.
(20 mL) in the presence of piperidine (2 mL) were refluxed for
3 h. The solid product which formed after evaporation of the sol-
vent was collected by filtration and recrystallized from ethanol
to give 11a-e.

A. Rahmouni et al. / Bioorganic Chemistry 66 (2016) 160–168
167
4.1.8.1. 7-Amino-2-methyl-5-phenylpyrazolo[1,5-a]pyrimidine-3,6-
and read spectrophotometrically at 500 nm. Tamoxifen (HCT116
and MCF-7) was used as positive control.
dicarbonitrile (11a). Yellow solid, yield: 65%, mp.: 310–312 °C; IR
(KBr, cmꢁ1
) t: 3310–3450 (NH2), 2858 (CH stretching), 2215
(CN), 1410 (N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.63 (s, 3H,
4.2.2. 5-Lipoxygenase inhibition
The antisoybean 5-lipoxygenase activity of the compounds was
determined as described by Khlifi et al. [36] with some modifica-
CH3), 7.28–7.98 (m,5H, Harom), 9.30 (s, 2H, NH2). 13C NMR
(75 MHz, DMSO-d6) dC: 13.7, 78.1, 83.1, 115.2, 116.0, 127.1,
128.3, 129.1, 135.3, 150.1, 151.3, 159.1, 162.4. HRMS [M+H]+ calcd
for (C15H11N6)+ 275,1040, found 275,1042.
tions. Various concentrations of 20
mixed individually with sodium phosphate buffer (pH 7.4) con-
taining soybean 5-lipoxygenase and 60 of linoleic acid
(3.5 mM). However, the blank does not contain the substrate, but
will be added 30 L of buffer solution. All compounds were re-
lL of each compound were
lL
4.1.8.2. 7-Amino-2-methyl-5-(p-tolyl)pyrazolo[1,5-a]pyrimidine-3,6-
dicarbonitrile (11b). Yellow solid, yield: 70%, mp.: 314–316 °C; IR
l
(KBr, cmꢁ1
) t: 3330–3430 (NH2), 2853 (CH stretching), 2217
suspended in the DMSO followed by dilution in the buffer so that
the DMSO does not exceed 1%. The mixture was incubated at
25 °C for 10 min, and the absorbance was determined at 234 nm.
The absorption change with the conversion of linoleic acid to 13-
hydroperoxyoctadeca-9,11-dienoate was flowed for 10 min at
25 °C. Nordihydroguaiaretic acid (NDGA) was used as positive
control.
(CN), 1417 (N@N). 1H NMR (300 MHz, DMSO-d6) dH: 2.17 (s, 3H,
CH3), 2.49 (s, 3H, PhACH3), 7.66 (d, 2H, Harom, J = 8.5 Hz), 7,80 (d,
2H, Harom,
J = 8.5 Hz), 9.20 (s, 2H, NH2). 13C NMR (75 MHz,
DMSO-d6) dC: 13.8, 22.2, 78.2, 83.7, 115.6, 116.1, 128.1, 129.1,
135.2, 135.4, 150.5, 151.1, 158.1, 162.1. HRMS [M+H]+ calcd for
(C16H13N6)+ 289.1204, found 289.1210.
4.1.8.3. 7-Amino-5-(4-ethylphenyl)-2-methylpyrazolo[1,5-a]pyrim-
idine-3,6-dicarbonitrile (11c). Yellow solid, yield: 68%, mp.: 309–
References
311 °C; IR (KBr, cmꢁ1
) t: 3325–3450 (NH2), 2840 (CH stretching),
2217 (CN), 1410 (N@N). 1H NMR (300 MHz, DMSO-d6) dH: 1.20 (t,
2H, ACH2ACH3, J = 7.2 Hz), 2.25 (s, 3H, CH3), 2.59 (q, 3H, ACH2-
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4.1.8.4.
pyrimidine-3,6-dicarbonitrile (11d). Yellow solid, yield: 75%, mp.:
316–318 °C; IR (KBr, cmꢁ1
: 3310–3440 (NH2), 2860 (CH stretch-
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t
3.38 (s, 3H, CH3), 3.78 (s, 3H, OCH3), 7.60 (d, 2H, Harom
,
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320 °C; IR (KBr, cmꢁ1
)
t
: 3320–3445 (NH2), 2843 (CH stretching),
2210 (CN), 1410 (N@N). 1H NMR (300 MHz, DMSO-d6) dH: 3.37
(s, 3H, CH3), 7.60 (d, 2H, Harom, J = 8.4 Hz), 7.84 (d, 2H, Harom
,
J = 8.4 Hz), 9.40 (s, 2H, NH2). 13C NMR (75 MHz, DMSO-d6) dC:
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151.0, 158.0, 161.3. HRMS [M+H]+ calcd for (C15H9ClN6)+
309.0611, found 309.0617.
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