Synthesis of new pyrazole and antibacterial pyrazolopyrimidine derivatives

Article abstract of DOI:10.3906/kim-1303-20

3-Substituted-1-phenyl-1H-pyrazolo[3,4-d]pyrimidin-4-amines 2a-c were synthesized by treating 5-aminopyrazole- 4-carbonitriles 1a-c with formamide. The reactivity of compounds 1a-c towards some cyclic anhydrides was studied. The condensation of 5-aminopyrazole-4-carbonitrile 1b with triethylorthoformate gives imidate 7b, which reacts with a series of primary amines and leads to pyrazolo[3,4-d]pyrimidine-4-amines 9 and 10. The reaction of imidate 7b with ammonia and hydroxylamine afforded pyrazolopyrimidine 2b and pyrazolo[3,4-d]pyrimidin-5-(4H) -ol 11, respectively. The synthesized compounds were completely characterized by ¹H NMR, ¹³C NMR, IR, and HRMS. The antibacterial activity of some new synthesized compounds was evaluated and appeared to be signiflcant. Tubitak.

Full text of DOI:10.3906/kim-1303-20

Turk J Chem
Turkish Journal of Chemistry
(2014) 38: 210 – 221
¨
http://journals.tubitak.gov.tr/chem/
˙
c
⃝ TUBITAK
doi:10.3906/kim-1303-20
Research Article
Synthesis of new pyrazole and antibacterial pyrazolopyrimidine derivatives
Ameur RAHMOUNI1, Anis ROMDHANE1, Abderrahim BEN SAID1,
Kaouther MAJOULI2, Hichem BEN JANNET1,∗
1Laboratory of Heterocyclic Chemistry, Natural Products and Reactivity, Team: Medicinal Chemistry and
Natural Products, Monastir Faculty of Science, University of Monastir, Monastir, Tunisia
2Laboratory of Biochemistry, Molecular Mechanisms, and Pathologies, Faculty of Medicine,
University of Monastir, Monastir, Tunisia
Received: 09.03.2013
•
Accepted: 18.08.2013
•
Published Online: 14.03.2014
•
Printed: 11.04.2014
Abstract: 3-Substituted-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amines 2a–c were synthesized by treating 5-aminopy-
razole-4-carbonitriles 1a–c with formamide. The reactivity of compounds 1a–c towards some cyclic anhydrides was
studied. The condensation of 5-aminopyrazole-4-carbonitrile 1b with triethylorthoformate gives imidate 7b, which
reacts with a series of primary amines and leads to pyrazolo[3,4-d]pyrimidine-4-amines 9 and 10. The reaction of
imidate 7b with ammonia and hydroxylamine afforded pyrazolopyrimidine 2b and pyrazolo[3,4-d]pyrimidin-5-(4H)-ol
11, respectively. The synthesized compounds were completely characterized by 1 H NMR, 13 C NMR, IR, and HRMS.
The antibacterial activity of some new synthesized compounds was evaluated and appeared to be significant.
Key words: Aminopyrazoles, pyrimidines, maleic anhydride, pyrazolo[3,4-d]pyrimidines, Dimroth rearrangement,
antibacterial activity
1. Introduction
Examples of natural products containing a pyrazole nucleus are very scarce, but many synthetic pyrazoles
are biologically active,1 and some have shown pharmacological utility as antipyretic, analgesic, and anti-
inflammatory agents,1−3 as well as for their antimicrobial properties, especially antibacterial4 and antifungal
activities.5 Moreover, this heterocyclic moiety is present within the core structure of numerous drugs, including
Celebrex, Viagra, and Zaleplon.6
Our literature survey showed that the chemistry of fused pyrazolo[3,4-d]pyrimidine derivatives has drawn
great attention due to their pharmacological importance7,8 and their structural resemblance to purines.9 In
fact, several pyrazolo[3,4-d]pyrimidine derivatives demonstrated significant antimicrobial10,11 and cytotoxic
activities.12
On the other hand, the literature13−15 reveals that several methods have been described for the elabora-
tion of substituted pyrazolo[3,4-d]pyrimidines. Among the already known routes to the fused pyrazolopyrimidine
scaffold, the most commonly used strategy involves a preliminary transformation of aminopyrazole-carbonitrile
derivatives into the corresponding imidates followed by a subsequent ring-closure into pyrazolo[3,4-d]pyrimidines
upon treatment with hydrazine.16
Therefore, taking into account all these above-described data, we report here our recent work on the
∗Correspondence: hichem.benjannet@yahoo.fr
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RAHMOUNI et al./Turk J Chem
synthesis of a new family of fused heterocyclic compounds using a nucleophilic addition-cyclization reaction on
5-aminopyrazole-4-carbonitriles with formamide and a series of cyclic anhydrides.
Prompted by the varied biological activities of pyrazolo[3,4-d]pyrimidine derivatives, we also described the
synthesis of pyrazolo[3,4-d]pyrimidine-4-amines by condensation of imidate 7b with various aromatic primary
amines. Some new synthesized compounds were evaluated for their antibacterial activity using microdilution
tests against some strains of bacteria.
2. Results and discussion
According to the previously reported method,17 we synthesized the starting material 5-aminopyrazole-4-
carbonitriles 1a–c and we subjected them to a reaction with formamide18,19 to give pyrazolo[3,4-d]pyrimidines
2a–c (Scheme 1). The structure of these compounds was confirmed based on their spectral data. The IR
spectra of obtained products 2a–c showed large bands between 3200 and 3400 cm−1 , assignable to NH2 , but
no absorption frequency in the CN region. 1 H NMR spectra showed that NH2 -protons appeared as broad
singlets in the 3.34–8.20 ppm region, while H6 resonated in the 8.26–8.35 ppm region. The 13 C NMR spectra
of these compounds revealed essentially the appearance of the signals of C4 and C6 carbons at δ 156.4–157.2
and 158.1–158.9 ppm, respectively. Moreover, we note the disappearance of the signal due to the carbon of the
–CN function.
The literature20−23 shows that aminopyrazole type 1a–c is often used as synthon for preparing some
novel fused pyrazoles. Therefore, it was considered of interest to synthesize some new heterocyclic systems
incorporating pyrazole fragments. In this context, we reacted the intermediates 1a–c with a series of cyclic
anhydrides in refluxing glacial acetic acid to produce the corresponding imides 3a–c and 4a–c (Scheme 1).
When we used phthalic and tricyclic anhydrides we did not observe any side product and the reaction led to the
corresponding imides 3a–c and 4a–c, respectively. In the case of the use of the maleic and succinic anhydrides
in the same conditions the reaction afforded only the corresponding imides 5a–c and 6a–c. The cyclic side chain
in the phthalic and tricylic anhydrides may favor intramolecular cyclization in the case of 3a–c and 4a–c, while
free rotation at the C7 –C8 band in compounds 5a–c and 6a–c may not favor this intramolecular cyclization.
The IR spectra of the obtained imides revealed the presence of characteristic absorption bands at ν
2210–2220 and at 1695–1710 cm−1 assignable to cyano and carbonyl groups, respectively. In addition, 1 H
NMR spectra of these compounds showed the absence of signals related to the mobile protons. The formation
of these products was also confirmed by 13 C NMR data with the observation of signals of carbons introduced
by cyclic anhydrides. Furthermore, the mass spectra (ES+) of compounds 3a–c and 4a–c showed molecular
ion peaks in good agreement with the assigned structures. Under the same conditions, maleic and succinic
anhydrides reacted with the appropriate aminopyrazoles 1a–c to generate the corresponding carboxy-amides
5a–c and 6a–c, respectively, in good yields (Scheme 1). The structure of the obtained carboxy-amides was
appropriately established by spectroscopic NMR and IR data. The IR spectra of compounds 5a–c showed 2
strong absorption bands at ν 1650–1660 and 1730–1745 cm−1 assignable to amide and acid carbonyl groups,
respectively, and other important bands revealed at 3244 cm−1 and 3334 cm−1 were attributed to the amide
NH and OH carboxylic acid, respectively. The 1 H NMR spectrum of compound 5c as an example exhibits
2 doublets at δ 6.65 ppm (J = 16 Hz) assigned to the olefinic hydrogens and a broad singlet at 12.70 ppm
corresponding to the acidic proton.
Furthermore, the 5-amino pyrazole-4-carbonitrile 1b was reacted with triethyl orthoformate to give
the corresponding ethoxymethylene amino derivative 7b (Scheme 2), often used as a synthon key to access
211

RAHMOUNI et al./Turk J Chem
H2N
4
R
5
6
3
N
3a
N
N
2
7a
N
HCONH2
7
1
2a-c
O
R
CN
O
O
N
N
O
N
O
3a-c
O
7'
a
R
CN
CN
R
4
3
O
O
6'
8
'
5'
-
'
6
1'
N
N
2
O
5
N
1
N
2'
3'
a
-
'
NH2
N
2
4'
O
O
4a-c
O
R
CN
4
3
O
7
9
O
6
N
H
N
2
5
1a-c
O
N
8
1
10
HO
a, R = H
b, R = Me
c, R = Et
O
5a-c
O
R
CN
O
O
N
N
H
N
OH
O
6a-c
Scheme 1. Synthetic route of compounds 2–6.
pyrazolo[3,4-d]pyrimidines.24 It seemed of interest to react the intermediate 7b with a series of amines (Scheme
2). In this case, we considered that the presence of amidine moiety may ensure the possibility of closure of the
pyrimidine ring, resulting in novel derivatives of pyrazolo[3,4-d]pyrimidine of significant biological interest since
such compounds are substituted analogues of the well-known drug allopurinol.25 We selected some aromatic and
aliphatic primary amines, the more basic ammonia and hydroxylamine hydrochloride, to study their reactions
with the imidate 7b.
212

RAHMOUNI et al./Turk J Chem
The imidate 7b reacted with both their electrophilic sites with aliphatic amines to yield the new pyra-
zolopyrimidines type 9a–c in 2 steps. In the first step, the condensation of 7b with aliphatic amines in ethanol
in the presence of a catalytic amount of acetic acid led to the intermediate 8a–c by the nucleophilic attack of the
NH2 motif on imidic carbon. In the second step, the isolable amidine 8a–c was heated in toluene in the presence
of a few drops of piperidine to provide the novel pyrazolopyrimidines 9a–c via Dimroth rearrangement.26,27
The isomerization of 8’a–c into thermodynamically more stable pyrazolopyrimidines derivative 9a–c (Scheme 2)
seems to occur through base-catalyzed tandem ring opening and ring closure. This rearrangement is consistent
with those reported in some earlier reports.16,27
The alternative structure 8a was excluded based on NMR data. The 1 H NMR spectrum showed the
disappearance of signals related to the ethoxy group and the appearance of signals introduced by the amine
used (R = Ph), and a characteristic signal at δ 8.38 ppm, assignable to the proton –N=CH. The analysis of
the 13 C NMR spectrum revealed that the signal of the CN group appeared at δ 117.4 ppm.
In addition, a whole set of linkages confirming the molecular structure of compounds 8a–c was deduced
from 2D NMR experiments. As an example, the spreading of the 1 H-1 H COSY spectrum of compound 8a
shows the correlation of both methylenic and ethylenic protons with that of the –NH– function.
The 1 H NMR spectra of compounds 9a–c showed the presence of a singlet at δ 8.34–8.50 ppm attributable
to the aromatic proton H6 . The disappearance of the cyano signal in the 13 C NMR spectra of compounds
9a–c was in favor of an intramolecular cyclization but it was not sufficient to differentiate between structures
of the nonconversion products 8’a–c and the rearranged ones 9a–c of which the structures were confirmed by
2D NMR experiments.
The 1 H-1 H COSY spectrum of compound 9a as an example showed a clear correlation peak between
–NH– (br s, 5.70 ppm) and the 2 methylenic protons (d, 4.90 ppm, J = 5.7 Hz), which was in favor of the
Dimroth rearrangement of the nonisolable intermediate 8’a, which leads to 9a. The reaction of 7b with some
aromatic amines led to the N -aryl-3-methyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidine-4-amines 10a–d via the
Dimroth rearrangement of the intermediates 7’a–d.
The structure of compounds 10a–d was confirmed by 2D NMR experiments.
The HMBC spectrum of compound 10b as an example showed a correlation peak between the –NH–
proton (br s, 8.01 ppm) and the quaternary (δc 116.0 ppm) and tertiary (δc 118.4 ppm) aromatic carbons,
reinforcing the structure of 10b.
Hydroxyiminolysis of iminoether 7b in ethanol in the presence of triethylamine provides pyrazolo[3,4-
d]pyrimidine 11 (Scheme 2). The formation of compound 11 was established by the presence of vibration bands
corresponding to the OH and NH functions near 3500 and 3400 cm−1 , respectively. In the 1 H NMR spectrum
of compound 11 measured in dimethyl-d6 sulfoxide, we observed the signal of a CH3 group at δ 2.78 ppm, the
signal of an OH group at δ 4.08 ppm, and aromatic proton signals and NH at 7.28–8.19 ppm.
Finally the imidate 7b was added to methanol saturated with ammonia at 0 ◦ C for 1 h (Scheme 2). The
reaction mixture was then warmed to room temperature and stirred for 6 h to produce pyrazolo[3,4-d]pyrimidine
2b, which was already synthesized by the addition of formamide to 5-aminopyrazole-4-carbonitrile 1b (Scheme
1). The 1 H NMR spectrum of compound 2b exhibited 2 singlets at δ 7.30 and 8.40 ppm, representing the
protons of pyrimidine and NH2 , respectively.
The mass spectra of all prepared compounds were compatible with the proposed structures.
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RAHMOUNI et al./Turk J Chem
NH2
NH
OH
H3C
H3C
N
N
N
N
N
N
N
N
2b
11
NH3
NH2OH(HCl)
H3C
R
HN
N
H3C
NH
CN
N
N
R
H3C
HC(OC2H5)3
Dimroth
Rearrangement
N
N
H
R-NH2
EtOH
N
N
1b
N
C
N
OEt
N
N
a, R = Ph
10a-d
b, R = o.Me-Ph
7b
c, R = p.Et.Ph
7'a-d
R-CH2-NH2
EtOH
d, R = naphtalen-1-yl
R
HN
a
NH
4
R
-
H3C
2
CN
3
3
H3C
H3C
5
N
R
N
HN
Dimroth
N
6
Toluene
N
N
N
N
a
-
7
N
N
N
N
1
Rearrangement
piperidine
7
H
9a-c
a, R = Ph
8a-c
8'a-c
b, R = -CH2-Ph
c, R = o.Cl-Ph
Scheme 2. Synthetic route of compounds 8–11 and 2b.
3. Antibacterial activity
The incorporation of another heterocyclic moiety in pyrazole, in the form of a substituent or as a fused
component, changes its properties and converts it into an altogether new and important heterocyclic derivative.
Pyrimidines have attracted particular interest over the last few decades due to the use of such a ring system as
the core nucleus in various drugs.28 They are well known for their popular pharmacological activities.29,30
Considering the importance of pyrazolopyrimidine derivatives for their biological activity, it was thought
worthwhile to test most of our prepared compounds (3a–c, 4a–c, 5a–c, 6a–c, 9a–c, 10a–c, 11, and 2b) for their
antibacterial activity against some bacteria, namely Escherichia coli, Pseudomonas aeruginosa, Staphylococcus
aureus, and Enterococcus faecalis.
The minimum inhibitory concentrations (MICs) were ascertained by the broth dilution method (microdi-
lution using 96-well microplates).31 The results presented in the Table showed that 9a is the most active towards
Pseudomonas aeruginosa. We also noted that adding a CH2 to the fragment R decreases this activity.
Compounds 10a and 10b were the most active against E. coli. The presence of the chlorine atom in the
ortho position in 10c significantly reduced this activity. The remaining compounds were found to have slight
or moderate activity against the tested organisms and some of the compounds were found to be inactive (MIC
> 4).
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RAHMOUNI et al./Turk J Chem
Table. Antibacterial activity of some synthesized compounds: 2b, 9a–c, 10a–c, and 11.
Staphylococcus
aureus
Pseudomonas Enterococcus
Escherichia coli
Acinetobacter
aeruginosa
faecalis
Compounds
MIC (mg/mL)
2b
9a
9b
9c
10a
10b
10c
11
4
2
> 4
2
4
1
2
1
0.62
2
4
2
0.5
2
2
1
1
> 4
2
1
2
4
2
> 4
> 4
2
2
-
4
> 4
> 4
> 4
2
> 4
> 4
0.5
0.5
> 4
> 4
1.25
2
> 4
> 4
-
Ampicillin
1.25
Compounds 3a–c, 4a–c, 5a–c, and 6a–c did not show any significant antibacterial activity against the
strains used (MIC > 100).
4. Experimental section
General. All melting points were determined on a Kofler-type microscope and are uncorrected. IR spectra
were recorded on a PerkinElmer FT-IR spectrophotometer (4000–400 cm−1) using KBr pellets. 1 H NMR and
13 C NMR spectra were recorded at room temperature (rt) in CDCl3 and dimethylsulfoxide (DMSO-d6) at 300
MHz and at 75 MHz, respectively, using residual nondeuterated solvent peaks as internal reference. Coupling
constants are given in hertz. HRMS spectra were acquired with an electrospray-time-of-flight (ESI-TOF, LCT
Premier XE, Waters) mass spectrometer in positive ion mode.
General procedure for 3-substituted-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4 amines 2a–c
A mixture of compound 1 (10 mmol) and formamide (10 mmol) was heated under reflux for 2 h, and
then left to cool overnight at ambient temperature. The solid formed was filtered, dried, and recrystallized from
ethanol.
1-Phenyl -1H -pyrazolo[3,4-d]pyrimidin-4-amine (2a):
Gray solid, yield: 80%, mp: 215–217 ◦ C (EtOH); IR (KBr, cm−1)ν : 3200–3400 (NH2). 1 H NMR
(DMSO-d6 , 300 MHz): δ (ppm) = 7.28–8.19 (m, 5H, H-arom), 7.81 (s, 2H, NH2), 8.29 (s, 1H, H-3), 8.35 (s,
1H, H-6). 13 C NMR (DMSO-d6 , 75 MHz): δ (ppm) = 101.9 (C-3a), 121.0, 126.5, 129.6, 134.5, (C-arom),
140.7 (C-3), 153.7 (C-7a), 157.2 (C-4), 158.8 (C-6). HRMS [M + H]+ calcd for (C11 H10 N5)+ 212.0936, found
212.0945.
3-Methyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (2b):
Gray solid, yield: 85%, mp: 217–218 ◦ C (EtOH); IR (KBr, cm−1)ν : 3250–3450 (NH2), 1 H NMR
(DMSO-d6 , 300 MHz): δ (ppm) = 2.60 (s, 3H, CH3), 7.28–7.80 (m, 5H, H-arom), 8.20 (s, 2H, NH2), 8.35
(s, 1H, H-6). 13 C NMR (DMSO-d6 , 75 MHz): δ (ppm) = 14.5 (CH3), 100.9 (C-3a), 120.1, 126.1, 129.4,
139.0 (C-arom), 141.7 (C-3), 153.5 (C-7a), 156.8 (C-4) 158.1 (C-6). HRMS [M + H]+ calcd for (C12 H12 N5)+
226.1011, found 226.1027.
3-Ethyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (2c):
Gray solid, yield: 80%, mp: 222–224 ◦ C (EtOH); IR (KBr, cm−1)ν : 3200–3490 (NH2), 1 H NMR
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RAHMOUNI et al./Turk J Chem
(DMSO-d6 , 300 MHz): δ (ppm) = 1.28 (t, 3H, J = 7.5 Hz, –CH2 –CH3), 3.05 (q, 2H, J = 7.5 Hz, –CH2 –
CH3), 3,34 (s, 2H, NH2) 7.23–8.18 (m, 5H, H-arom), 8.26 (s, 1H, H-6). 13 C NMR (DMSO-d6 , 75 MHz): δ
(ppm) = 13.5 (–CH2 –CH3), 21.9 (–CH2 –CH3), 100.0 (C-3a), 120.8, 126.0, 129.4, 139.5 (C-arom), 144.5 (C-3),
154.8 (C-7a), 156.4 (C-4) 158.9 (C-6). HRMS [M + H]+ calcd for (C13 H14 N5)+ 240.1145, found 240.1150.
General procedure for 5-(1,3-dioxoisoindolin-2-yl)-3-substituted-1-phenyl-1H - pyrazole-4-
carbonitriles 3a–c and 4a–c
Phthalic or bicyclic anhydride (0.1 mol) was completely dissolved at room temperature in glacial acetic
acid (20 mL) or THF, and then aminopyrazoles 1a–c (0.1 mol) were added to the solution; the resulting mixture
was stirred under reflux for 3 h. The reaction mixture was allowed to cool to room temperature, and then was
poured into water; the precipitate formed was filtered off, washed with water, dried, and crystallized from
ethanol to give the imides 3 and 4.
5-(1,3-Dioxoisoindolin-2-yl)-1-phenyl-1H -pyrazole-4-carbonitrile (3a):
Green solid, yield: 75%, mp: 157–159 ◦ C (EtOH); IR (KBr, cm−1)ν : 1680 (C=O, imide), 2210 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 7.30–8.01 (m, 9H, H-arom), 8.30 (s, 1H, H-3). 13 C NMR (CDCl3 ,
75 MHz): δ (ppm) = 78.1 (C-4), 116.8 (CN), 120.8, 126.4, 127.7, 129.2, 132.3, 133.3, 139.1 (C-arom), 152.8
(C-3), 156.3 (C-5), 168.6 (C=O). HRMS [M + H]+ calcd for (C18 H11 N4 O2)+ 315.0810, found 315.0813.
5-(1,3-Dioxoisoindolin-2-yl)-3-methyl-1-phenyl-1H -pyrazole-4-carbonitrile (3b):
Green solid, yield: 80%, mp: 160–162 ◦ C (EtOH); IR (KBr, cm−1)ν : 1695 (C=O, imide), 2220 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 2.69 (CH3), 7.39–8.10 (m, 9H, H-arom). 13 C NMR (CDCl3 , 75
MHz): δ (ppm) = 13.6 (CH3), 78.7 (C-4), 117.1 (CN), 120.7, 125.9, 128.1, 129.2, 132.2, 132.8, 139.1 (C-arom),
153.0 (C-3), 158.3 (C-5), 167.6 (C=O). HRMS [M + H]+ calcd for (C19 H13 N4 O2)+ 329.1035, found 329.1039.
5-(1,3-Dioxoisoindolin-2-yl)-3-ethyl-1-phenyl-1H -pyrazole-4-carbonitrile (3c):
Green solid, yield: 80%, mp: 164–166 ◦ C (EtOH); IR (KBr, cm−1)ν : 1710 (C=O, imide), 2217 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 1.32 (t, 3H, J = 7.1 Hz, –CH2 –CH3), 2.90 (q, 2H, J = 7.2 Hz,
–CH2 –CH3), 7.20–8.05 (m, 9H, H-arom). 13 C NMR (CDCl3 , 75 MHz): δ (ppm) = 14.5 (–CH2 –CH3), 21.9
(–CH2 –CH3), 79.1 (C-4), 117.0 (CN), 120.2, 126.4, 128.1, 129.2, 132.2, 132.0, 138.7 (C-arom), 153.5 (C-3),
157.6 (C-5), 167.7 (C=O). HRMS [M + H]+ calcd for (C20 H15 N4 O2)+ 343.1017, found 343.1024.
5-(1,3-Dioxopyrrolidino[3,4-d]hept[2.2.1]-5-en-2-yl)-1-phenyl-1H -pyrazole-4carbonitrile (4a):
White solid, yield: 70%, mp: 170–172 ◦ C (EtOH); IR (KBr, cm−1)ν : 1700 (C=O, imide), 2210 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 1.50–1.68 (m, 2H, H-7’), 3.36 (m, 2H, H-3’, H-6’), 3.48 (m, 2H,
H-6’a, H-2’a), 5.40 (s, 2H, H-4’, H-5’), 7.50–7.55 (m, 5H, H-arom), 8.10 (s, 1H, H-3). 13 C NMR (CDCl3 ,
75 MHz): δ (ppm) = 45.1 (C-6’a, C-2’a), 46.7 (C-7’), 52.6 (C-3’, C-6’), 82.1 (C-4), 114.4 (CN), 124.1, 126.9,
127.7, 129.5 (C-arom), 135.3 (C-4’, C-5’), 150.0 (C-5), 152.1 (C-3), 174.1 (C=O). HRMS [M + H]+ calcd for
(C19 H15 N4 O2)+ 331.1138, found 331.1143.
5-(1,3-Dioxopyrrolidino[3,4-b]hept[2.2.1]-5-en-2-yl)-3-methyl-1-phenyl-1H -pyrazole4-carbo-
nitrile (4b):
White solid, yield: 77%, mp: 174–176 ◦ C (EtOH); IR (KBr, cm−1)ν : 1710 (C=O, imide), 2215 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 1.53–1.70 (m, 2H, H-7’), 2.45 (CH3), 3.32 (m, 2H, H-3’, H-6’), 3.50
(m, 2H, H-6’a, H-2’a), 5.43 (s, 2H, H-4’, H-5’), 7.48–7.54 (m, 5H, H-arom). 13 C NMR (CDCl3 , 75 MHz): δ
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RAHMOUNI et al./Turk J Chem
(ppm) = 12.5 (CH3), 44.6 (C-6’-a, C-2’a), 46.2 (C-7’), 51.8 (C-3’, C-6’), 82.0 (C-4), 114.1 (CN), 124.1, 126.9,
127.7, 129.5 (C-arom), 134.7 (C-4’, C-5’), 150.1 (C-5), 152.0 (C-3), 173.9 (C=O). HRMS [M + H]+ calcd for
(C20 H17 N4 O2)+ 345.1350, found 345.1356.
5-(1,3-Dioxopyrrolidino[3,4-d]hept[2.2.1]-5-en-2-yl)-3-ethyl-1-phenyl-1H -pyrazole-4-carbo-
nitrile (4c):
White solid, yield: 75%, mp: 181–183 ◦ C (EtOH); IR (KBr, cm−1)ν : 1705 (C=O, imide), 2220 (CN).
1 H NMR (CDCl3 , 300 MHz): δ (ppm) = 1.25 (t, 3H, J = 7.1 Hz, –CH2 –CH3), 3.05 (q, 2H, J = 7.4
Hz, –CH2 –CH3), 1.51–1.67 (m, 2H, H-7’), 3.33 (m, 2H, H-3’, H-6’), 3.53 (m, 2H, H-6’a , H-2’a), 5.46 (s,
2H, H-4’, H-5’), 7.39–7.45 (m, 5H, H-arom). 13 C NMR (CDCl3 , 75 MHz): δ (ppm) = 13.5 (–CH2 –CH3),
21.4 (–CH2 –CH3), 45.3 (C-6’a, C-2’a), 46.7 (C-7’), 52.7 (C-3’, C-6’), 81.5 (C-4), 114.6 (CN), 124.1, 126.9,
127.7, 129.5 (C-arom), 134.5 (C-4’, C-5’), 150.1 (C-5), 151.7 (C-3), 174.5 (C=O). HRMS [M + H]+ calcd for
(C21 H19 N4 O2)+ 359.1458, found 359.1462.
General procedure for 4-(4-cyano-3-substituted-1-phenyl-1H -pyrazol-5-ylamino)-4-oxocar-
boxylic-acids 5a–c and 6a–c
Maleic or succinic anhydride (0.1 mol) was completely dissolved at room temperature in glacial acetic
(20 mL), and then aminopyrazoles 1a–c (0.1 mol) were added to the solution; the resulting mixture was stirred
under reflux for 3 h. The reaction mixture was allowed to cool to room temperature and then was poured into
water; the precipitate formed was filtered off, washed with water, dried, and crystallized from ethanol to afford
the N -cyclic maleamic or succinic acids.
(E)-4-(4-Cyano-1-phenyl-1H -pyrazol-5-ylamino)-4-oxobut-2-enoic acid (5a):
Yellow solid, yield: 75%, mp: 260–262 ◦ C (EtOH); IR (KBr, cm−1)ν : 1650 (C=O, amide), 1730 (C=O,
acid), 3244 (–NH–), 3334 (OH). 1 H NMR (DMSO-d6 , 300 MHz): δ (ppm) = 6.81 (d, 1H,J = 16 Hz, H-9), 7.20
(d, 1H, J = 16 Hz, H-8), 7.21–8.10 (m, 6H, NH + H-arom), 8.40 (s, 1H, H-3), 12.87 (s, 1H, OH). 13 C NMR
(DMSO-d6 , 75 MHz): δ (ppm) = 79.1 (C-4), 117.1 (CN), 121.7, 126.5, 129.6, 139.5 (C-arom), 132.8 (C-9),
138.7, (C-8), 152.3 (C-3), 153.2 (C-5), 159.9 (C-7), 168.6 (C-10). HRMS [M + H]+ calcd for (C14 H11 N4 O3)+
283.0853, found 283.0855.
(E)-4-(4-Cyano-3-methyl-1-phenyl-1H -pyrazol-5-ylamino)-4-oxobut-2-enoic acid (5b):
Yellow solid, yield: 80%, mp: 265–267 ◦ C (EtOH); IR (KBr, cm−1)ν : 1660 (C=O, amide), 1735 (C=O,
acid), 3240 (–NH–), 3330 (OH). 1 H NMR (DMSO-d6 , 300 MHz): δ (ppm) = 3.07 (CH3), 6.86 (d, 1H, J =
16 Hz, H-9), 7.38 (d, 1H, J = 16 Hz, H-8), 7.20–8.05 (m, 6H, NH + H-arom), 12.97 (s, 1H, OH). 13 C NMR
(DMSO-d6 , 75 MHz): δ (ppm) = 14.5 (CH3), 80.1 (C-4), 116.0 (CN), 122.7, 127.4, 130.6, 139.7 (C-arom),
130.1 (C-9), 136.1 (C-8), 151.8 (C-3), 154.0 (C-5), 159.9 (C-7), 169.7 (C-10). HRMS [M + H]+ calcd for
(C15 H13 N4 O3)+ 297.0988, found 297.0988.
(E)-4-(4-Cyano-3-ethyl-1-phenyl-1H -pyrazol-5-ylamino)-4-oxobut-2-enoic acid (5c):
Yellow solid, yield: 85%, mp: 267–269 ◦ C (EtOH); IR (KBr, cm−1)ν : 1650 (C=O, amide) 1730 (C=O,
acid), 3235 (–NH–), 3330 (OH). 1 H NMR (DMSO-d6 , 300 MHz): δ (ppm) = 1.32 (t, 3H, J = 7.0 Hz, –
CH2 –CH3), 2.91 (q, 2H, J = 7.0 Hz, –CH2 –CH3), 6.65 (d, 1H, J = 16 Hz, H-9), 7.02 (d, 1H, J = 16 Hz,
H-8), 7.20–8.05 (m, 6H, NH + H-arom), 12.70 (s, 1H, OH). 13 C NMR (DMSO-d6 , 75 MHz): δ (ppm) = 13.3
(–CH2 –CH3), 21.6 (–CH2 –CH3), 78.9 (C-4), 114.0 (CN), 121.8, 126.8, 129.6, 139.7 (C-arom), 130.0 (C-9),
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RAHMOUNI et al./Turk J Chem
135.7 (C-8), 151.9 (C-3), 153.5 (C-5), 158.3 (C-7), 167.7 (C-10). HRMS [M + H]+ calcd for (C16 H15 N4 O3)+
311.1144, found 311.1150.
4-(4-Cyano-1-phenyl-1H -pyrazol-5-ylamino)-4-oxobutanoic acid (6a):
White solid, yield: 65%, mp: 230–232 ◦ C (EtOH); IR (KBr, cm−1)ν : 1660 (C=O, amide), 1740 (C=O,
acid), 3240 (–NH–), 3330 (OH). 1 H NMR (DMSO-d6 , 300 MHz): 2.30 (t, 2H,J = 6.01 Hz, H-8), 2.47 (t, 2H, J
= 6.01 Hz, H-9), 7.21–7.50 (m, 5H, H-arom), 8.29 (s, 1H, H-3), 8.40 (s, 1H, –NH–), 12.55 (s, 1H, OH). 13 C NMR
(DMSO-d6 , 75 MHz): δ (ppm) = 27.4 (C-8), 34.9 (C-9), 83.8 (C-4), 117.1 (CN), 120.7, 126.3, 129.6, 139.8
(C-arom), 152.3 (C-3), 152.5 (C-5), 169.9 (C-7), 171.9 (C-10). HRMS [M + H]+ calcd for (C14 H13 N4 O3)+
285.1001, found 285.1003.
4-(4-Cyano-3-methyl-1-phenyl-1H -pyrazol-5-ylamino)-4-oxabutanoic acid (6b):
White solid, yield: 68%, mp: 237–239 ◦ C (EtOH); IR (KBr, cm−1)ν : 1650 (C=O, amide), 1740 (C=O,
acid), 3235 (–NH–), 3330 (OH). 1 H NMR (DMSO-d6 , 300 MHz): δ (ppm) = 2.40 (t, 2H, J = 5.9 Hz, H-8),
2.46 (t, 2H, J = 5.9 Hz, H-9), 2.58 (s, 3H, CH3), 7.30–7.62 (m, 5H, H-arom), 8.60 (s, 1H, –NH–), 12.40 (s,
1H, OH). 13 C NMR (DMSO-d6, 75 MHz): δ (ppm) = 14.7 (CH3) 28.5 (C-8), 32.0 (C-9), 79.2 (C-4), 116.6
(CN), 120.2, 126.3, 129.6, 139.4 (C-arom), 152.2 (C-3), 153.8 (C-5), 170.8 (C-7), 173.7 (C-10). HRMS [M +
H]+ calcd for (C15 H15 N4 O3)+ 299.1053, found 299.1055.
4-(4-Cyano-3-ethyl-1-phenyl-1H -pyrazol-5-ylamino)-4-oxobutanoic acid (6c):
White solid, yield: 68%, mp: 240–242 ◦ C (EtOH); IR (KBr, cm−1)ν : 1655 (C=O, amide), 1735 (C=O,
acid), 3230 (–NH–), 3334 (OH). 1 H NMR (DMSO-d6 , 300 MHz): δ (ppm) = 1.30 (t, 3H, J = 7.0 Hz, –CH2 –
CH3), 2.37 (t, 2H, J = 5.9 Hz, H-8), 2.46 (t, 2H, J = 5.9 Hz, H-9), 2.60 (q, 2H, J = 7.1 Hz, –CH2 –CH3),
7.20–7.52 (m, 5H, H-arom), 8.35 (s, 1H, –NH–), 12.34 (s, 1H, OH). 13 C NMR (DMSO-d6 , 75 MHz): δ (ppm) =
13.5 (–CH2 –CH3), 21.7 (–CH2 –CH3), 29.9 (C-8), 31.6 (C-9), 81.0 (C-4), 116.0 (CN), 121.2, 126.2, 129.3, 138.3
(C-arom), 152.0 (C-3), 153.3 (C-5), 173.8 (C-7), 177.7 (C-10). HRMS [M + H]+ calcd for (C16 H17 N4 O3)+
313.1220, found 313.1235.
General procedure for 3-methyl-N -benzyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amines
9a–c
Firstly to a solution of imidate 7 (0.1 mol) in ethanol (20 mL) in the presence of a few drops of acetic
acid was added aliphatic amines (0.01 mol). The reaction was heated under reflux for 4 h. After cooling, the
product was collected and recrystallized from ethanol to afford 8a–c as colorless needles.
Secondly a solution of amidines 8a–c in toluene in the presence of a few drops of piperidine was heated
under reflux for 2 h. The residue obtained in each case after removing the solvent in vacuo was chromatographed
on silica gel using chloroform as a mobile phase to yield compounds 9a–c.
N-Benzyl-3-methyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (9a):
Yellow solid, yield: 70%, mp: 180–182 ◦ C (EtOH); IR (KBr, cm−1)ν : 3450 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 2.68 (s, 3H, CH3), 4.90 (d, 2H, J = 5.7 Hz, –CH2 – Ph), 5.70 (br s, 1H, –NH–), 7.28–8.13
(m, 10H, H-arom), 8.50 (s, 1H, H-6). 13 C NMR (CDCl3 , 75 MHz): δ (ppm) = 15.4 (CH3), 45.2 (–CH2 –Ph),
103.8 (C-3-a), 121.8, 124.4, 126.7, 128.2, 129.3, 137.3, 139.1, 141.7 (C-arom), 150.3 (C-3), 154.3 (C-7-a), 156.6
(C-6) 157.5 (C-4). HRMS [M + H]+ calcd for (C19 H18 N5)+ 316.1480, found 316.1492.
3-Methyl-N-phenethyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (9b):
Yellow solid, yield: 68%, mp: 183–185 ◦ C (EtOH); IR (KBr, cm−1)ν : 3420 (NH). 1 H NMR (CDCl3 ,
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RAHMOUNI et al./Turk J Chem
300 MHz): δ 2.29 (s, 3H, CH3), 2.92 (t, 2H, J = 7.1 Hz, –NH–CH2 –CH2 –Ph), 3.88 (q, 2H, J = 7.0 Hz, –NH–
CH2 –CH2 –Ph), 5.16 (br s, 1H, –NH–), 7.00–7.93 (m, 10H, H-arom), 8.34 (s, 1H, H-6). 13 C NMR (CDCl3 , 75
MHz): δ (ppm) = 14.5 (CH3), 35.3 (–NH–CH2 –CH2 –Ph), 45.2 (–CH2 –CH2 –Ph), 104.1 (C-3-a), 121.9, 126.9,
127.4, 129.0, 129.3, 138.5, 139.1, 139.7 (C-arom), 144.7 (C-3), 150.5 (C-7-a), 156.1 (C-6), 157.1 (C-4). HRMS
[M + H]+ calcd for (C20 H20 N5)+ 330.1624, found 330.1632.
3-Methyl-N-(2-chlorobenzyl)-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (9c):
Yellow solid, yield: 70%, mp: 179–181 ◦ C (EtOH); IR (KBr, cm−1)ν : 3445 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 2.60 (s, 3H, CH3), 4.01 (d, 2H, J = 7.0 Hz, –NH–CH2 –2.Cl.Ph), 5.80 (br s, 1H, –NH–),
7.19–8.35 (m, 9H, H-arom), 8.41 (s, 1H, H-6). 13 C NMR (CDCl3 , 75 MHz): δ (ppm) = 14.4 (CH3), 39.7
(–NH–CH2 –2.Cl.Ph), 103.2 (C-3-a), 121.1, 125.3, 126.9, 128.5, 128.3, 129.3, 133.0, 137.3, 139.1, 141.3 (C-arom),
144.1 (C-3), 153.2 (C-7-a), 156.8 (C-6) 157.5 (C-4). HRMS [M + H]+ calcd for (C19 H17 ClN5)+ 350.8773,
found 350.8780.
General procedure for the synthesis of 3-methyl-N -aryl-1-phenyl-1H -pyrazolo[3, 4-d]pyri-
midin-4-amines 10a–d
The appropriate primary aromatic amine (0.001 mol) was added to the suitable imidate 7b (0.01 mol),
and the mixture was stirred at reflux in toluene (20 mL) for 6 h. After cooling, the precipitated solid was
filtered, washed with cold ether and dried, and then recrystallized from ethanol to give compounds 10a–d.
3-Methyl-N,1-diphenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (10a):
Yellow solid, yield: 60%, mp: 171–173 ◦ C (EtOH); IR (KBr, cm−1)ν : 3440 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 2.37 (s, 3H, CH3), 6.99–7.66 (m, 11H, NH + H-arom), 8.85 (s, 1H, H-6). 13 C NMR
(CDCl3 , 75 MHz): δ (ppm) = 13.5 (CH3), 101.9 (C-3-a), 116.0, 119.1, 123.1, 127.1, 129.3, 139.1, 140.1 (C-
arom), 141.8 (C-3), 150.5 (C-7a), 155.8 (C-4), 156.0 (C-6). HRMS [M + H]+ calcd for (C18 H16 N5)+ 302.1405,
found 302.1410.
3-Methyl-1-pheny-N-o-tolyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (10b):
Yellow solid, yield: 65%, mp: 190–192 ◦ C (EtOH); IR (KBr, cm−1)ν : 3450 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 2.33 (s, 3H, Ph–CH3), 2.46 (s, 3H, CH3), 6.82–7.79 (m, 9H, H-arom), 8.01 (br s, 1H,
NH), 8.93 (s, 1H, H-6). 13 C NMR (CDCl3 , 75 MHz): δ (ppm) = 13.6 (CH3), 21.8 (Ph–CH3), 103.9 (C-3a),
116.1, 118.4, 121.1, 126.1, 127.1, 128.0, 129.4, 129.7, 139.1, 141.4 (C-arom), 143.4 (C-3), 150.0 (C-7a), 155.1
(C-4), 156.1 (C-6). HRMS [M + H]+ calcd for (C19 H18 N5)+ 316.1562, found 316.1567.
N -(4-ethylphenyl) 3-methyl-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (10c):
Yellow solid, yield: 75%, mp: 187–189 ◦ C (EtOH); IR (KBr, cm−1)ν : 3450 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 1.27 (t, 3H, J = 6.9 Hz, Ph–CH2 –CH3), 2.60 (s, 3H, CH3), 2.67 (q, 2H, J = 7.0
Hz, Ph–CH2 –CH3), 6.82–8.01 (m, 10H, NH + H-arom), 8.10 (s, 1H, H-6). 13 C NMR (CDCl3 , 75 MHz): δ
(ppm) = 15.5 (Ph–CH2 –CH3), 16.0 (CH3), 21.7 (Ph–CH2 –CH3), 103.2 (C-3a), 116.1, 121.2, 126.2, 127.1,
128.8, 129.5, 129.5, 138.1, 139.7 (C-arom), 142.5 (C-3), 151.0 (C-7a), 154.3 (C-4), 156.6 (C-6). HRMS [M +
H]+ calcd for (C20 H20 N5)+ 330.1718, found 330.1720.
3-Methyl-N -(naphtalen-1-yl)-1-phenyl-1H -pyrazolo[3,4-d]pyrimidin-4-amine (10d):
Yellow solid, yield: 75%, mp: 190–192 ◦ C (EtOH); IR (KBr, cm−1)ν : 3430 (NH). 1 H NMR (CDCl3 ,
300 MHz): δ (ppm) = 2.79 (s, 3H, CH3), 6.82–8.04 (m, 12H, H-arom + NH + H-6). 13 C NMR (CDCl3 , 75
MHz): δ (ppm) = 13.5 (CH3), 104.2 (C-3a), 110.1, 117.9, 120.1, 120.8, 123.3, 124.1, 125.1, 126.1, 126.8, 127.8,
219

RAHMOUNI et al./Turk J Chem
127.3, 129.5, 132.1, 138.6, 140.2 (C-arom), 143.8 (C-3), 150.2 (C-7a), 153.7 (C-4), 157.0 (C-6). HRMS [M +
H]+ calcd for (C22 H18 N5)+ 352.2219, found 352.2221.
General procedure for the synthesis of 4-imino-3-methyl-1-phenyl-1H,4H pyrazolo[3,4-
d]pyrimidin-5-ol 11
A mixture of 7 (10 mmol) and hydroxylamine hydrochloride (10 mmol) in ethanol (20 mL) containing
triethylamine (5 mL) was refluxed for 5 h. The reaction mixture was then cooled and poured into cold water.
The formed precipitate was filtered off, washed with water, dried, and recrystallized from ethanol.
4-Imino-3-methyl-1-phenyl-1H ,4H -pyrazolo[3,4-d]pyrimidin-5-ol (11):
Yellow solid, yield: 88%, mp: 215–217 ◦ C (EtOH); IR (KBr, cm−1)ν : 3400 (NH), 3500 (OH). 1 H NMR
(DMSO-d6 , 300 MHz): δ (ppm) = 2.78 (s, 3H, CH3), 4.08 (s, 1H, NH), 7.48–8.18(m, 5H, H-arom), 8.92 (s,
1H, H-6), 9.00 (s, 1H, OH). 13 C NMR (DMSO-d6 , 75 MHz): δ (ppm) = 14.5 (CH3), 103.1 (C-3a), 121.2,
126.5, 129.6, 139.6 (C-arom), 143.6 (C-7a), 146.9 (C-3), 147.4 (C-6), 159.5 (C-4). HRMS [M + H]+ calcd for
(C12 H12 N5 O)+ 242.1042, found 242.1046.
5. Antibacterial activity
5.1. Microorganisms
The antibacterial activity was tested against 5 microorganisms, including reference strains consisting of gram-
negative rods: Escherichia coli (ATCC 25922) and Pseudomonas aeruginosa (ATCC 27853); gram-positive
cocci: Staphylococcus aureus (ATCC 25923) and Enterococcus faecalis (ATCC 29212); and clinical strains:
Acinetobacter sp. The bacterial strains were cultured overnight at 37 ◦ C in Mueller–Hinton agar.
5.2. Micro-well dilution assay
The MIC was defined as the lowest concentration able to inhibit any visible bacterial growth. MIC values
were determined by a microtiter plate dilution method dissolving the sample in 10% DMSO solution. Sterile
10% DMSO solution (100 µL) was pipetted into all wells of the microtiter plate before transferring 100 µL of
stock solution to the microplate. Serial dilutions were made to obtain concentrations ranging from 10 to 0.0775
mg/mL. Finally, 50 µL of 106 colony forming units (cfu/mL) (according to McFarland turbidity standards)
of standard microorganism suspensions were inoculated onto microplates and incubated at 37 ◦ C for 24 h. At
the end of the incubation period, the plates were evaluated for the presence or absence of growth. MIC values
were determined as the lowest concentration of the sample at which the absence of growth was recorded. All
the samples were screened 3 times against each microorganism. Ampicillin was used as antibacterial positive.
The final concentration of DMSO in the well had no effect on bacterial growth.
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