Synthesis, in vitro antimicrobial and cytotoxic activities of some new pyrazolo[1,5-a]pyrimidine derivatives

Article abstract of DOI:10.3390/molecules24061080

A new series of pyrazole 4–7 and pyrazolo[1,5-a]pyrimidine 8–13 were synthesized by using a simple, efficient procedure, and screened for their in-vitro antimicrobial and antitumor activities. Symmetrical and asymmetrical 3,6-diarylazo-2,5,7-triaminopyraz

Full text of DOI:10.3390/molecules24061080

molecules
Article
Synthesis, In Vitro Antimicrobial and Cytotoxic
Activities of Some New
Pyrazolo[1,5-a]pyrimidine Derivatives
Ahmed M. Fouda 1, Hebat-Allah S. Abbas 1,2,
*
, Eman H. Ahmed 1, Ali A. Shati 3,
Mohammad Y. Alfaifi 3 and Serag Eldin I. Elbehairi 3,4
1
Chemistry Department, Faculty of Science, King Khalid University, Abha 9004, Saudi Arabia;
amfouda@hotmail.com (A.M.F.); em1433an@gmail.com (E.H.A.)
Photochemistry Department, National Research Centre, Dokki, Cairo 12622, Egypt
Biology Department, Faculty of Science, King Khalid University, Abha 9004, Saudi Arabia;
2
3
aaalshati@kku.edu.sa (A.A.S.); alfaifi@kku.edu.sa (M.Y.A.); serag@kku.edu.sa (S.E.I.E.)
Cell Culture Lab, Egyptian Organization for Biological Products and Vaccines (VACSERA Holding
4
Company), 51 Wezaret El-Zeraa St., Agouza, Giza 12311, Egypt
*
Correspondence: hebanrc@yahoo.com or hsabas@kku.edu.sa; Tel.: +96-650-288-4583 or +20-100-521-1407
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Academic Editors: Richard A. Bunce, Philippe Belmont, Wim Dehaen and Eugene Babaev
Received: 13 February 2019; Accepted: 13 March 2019; Published: 19 March 2019
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: A new series of pyrazole 4–7 and pyrazolo[1,5-a]pyrimidine 8–13 were synthesized by
using a simple, efficient procedure, and screened for their in-vitro antimicrobial and antitumor
activities. Symmetrical and asymmetrical 3,6-diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine were
synthesized by the conventional method and also subjected to microwave irradiation and under
ultrasound conditions. The biological results revealed that most of the tested compounds proved to
be active as antibacterial and antifungal agents. The antitumor activity of the synthesized compounds
was evaluated against human cancer cell lines, MCF-7, HCT-116, and HepG-2, as compared with
Doxorubicin as a control.
Keywords: pyrazole; 3,5-diaminopyrazole; pyrazolo[1,5-a]pyrimidine; microwave; antimicrobial;
antitumor activities
1. Introduction
Pyrazole, a heterocyclic five-membered ring system, is one of the most significant heterogeneous
compounds. Pyrazole derivatives have been found to possess a broad spectrum of biological properties
including antitumor activity [
their high antibacterial and antifungal values [
anti-HIV [ ], antiviral [ ], anti-diabetic [ ], and anti-tubercular activities [8
1
,
2
]. Pyrazoles have attracted much attention from researchers due to
]. In addition, pyrazoles exhibit anti-inflammatory [ ],
]. It gained great attention
3
4
5
6
7
since the privileged structure was commonly found as an active constituent in commercial drugs
(Figure 1), such as lonazolac nonsteroidal anti-inflammatory drug (NSAID), pyrazofurin (anticancer),
difenamizole (analgesic), and deracoxib (NSAID). A great deal of interest has been paid to pyrazole
derivatives in the last few years in agrochemical and chemical industries [9], as some pyrazole
derivatives have exhibited insecticidal [10] and herbicidal activities [11]. The great importance
of pyrazole is due to its ability to construct fused pyrazole compounds that have been used as
intermediates for the synthesis of dyestuffs mainly for heterocyclic azo pyrazole disperse dyes [12].
Also, it has gained considerable attention from researchers in the past few decades since it is a major
component in some marketing drugs such as Ocinaplon and Lorediplon [12]. Based on the above facts,
the purpose of this paper is to design and synthesize new compounds containing pyrazole moieties
and study their antimicrobial activity.
Molecules 2019, 24, 1080; doi:10.3390/molecules24061080
www.mdpi.com/journal/molecules

Molecules 2019, 24, 1080
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Figure 1. Several commercial drugs containing pyrazole ring.
2. Results and Discussion
2.1. Chemistry
2-Arylazomalononitriles 2a–c were synthesized by diazotization of aniline derivatives 1a–c
followed by coupling with malononitrile according to the reported method [13] (Scheme 1).
The structure of 2-[4-(trifluoromethyl) phenylazo]malononitrile (2b) was established by analytical and
−1
spectral data. The IR spectrum of (2b) revealed the presence of absorption bands at 3141 and 2227 cm
characteristic for NH and CN groups, respectively. The 1H-NMR spectrum revealed two doublets at
δ
7.64 and 7.76 ppm assignable to the aromatic protons and a broad signal at
presence of the NH2 group, please find more detailed data in the Supplementary Materials.
Refluxing of arylazomalononitrile 2a with piperidine or morpholine in ethanol afforded the
δ 10.36 ppm indicating the
amino analogs 3a
compounds 3a
,
b
(Scheme 1). The structures for 3a
,
b
were con−fi1rmed by spectral data. IR spectra−o1f
,
b
showed the appearance of NH at 3301–3303 cm and CN stretch at 2179–2184 cm
The 1H and 13C-NMR spectrum of compound 3b revealed the presence of two characteristic signals
aliphatic groups N-(CH2)2 and O-(CH2)2 of morpholine moiety.
.
Scheme 1. Synthetic route for 2-arylazomalononitrile derivatives 2a
–
c
and 3a
,
b
.
Reagents
and Conditions: (
a
)◦NaNO2/HCl/stirring at 0–5 ◦C 1 h; (
b) CH2(CN)2/CH3COONa/50% eq.
EtOH/stirring at 0–5 C 2 h; (c) 2a/piperidine or morpholine/EtOH/reflux 1 h.

Molecules 2019, 24, 1080
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Several publications in recent years reported that various pyrazole derivatives were afforded
by the reaction of arylazomalononitriles with different hydrazines [14]. The reactivity of the
arylazomalononitriles 2a
were investigated. Thus, treatment of arylazomalononitriles 2a
derivatives—namely, nicotinohydrazide or benzohydrazide in ethanol—provided the corresponding
3,5-diaminopyrazole derivatives 4a or N-substituted diaminopyrazole derivatives 5a , respectively
,
b
with hydrazine hydrate, phenylhydrazine, and acid hydrazide derivatives
,
b
with hydrazine hydrate or hydrazide
,
b
,b
(Scheme 2). Furthermore, condensation of 2-[4-(trifluoromethyl)phenylazo]malononitrile (2b) with
phenylhydrazine in refluxing ethanol furnished the corresponding 3,5-diamino-N-phenyl-4-[4-
(trifluoromethyl)phenylazo]-1H-pyrazole (6) (Scheme 2). Spectral data confirmed the assignment
of 3,5-diamino-4-[4-(trifluoromethyl)phenylazo]-1H-pyrazole (4b). The IR spectrum shows the absence
of the characteristic absorption band of CN and the appearance of absorption bands at 3465, 3389, and
3296 corresponding to NH2 groups. Its 1H-NMR revealed the presence of NH2 signal at
δ 6.29 ppm,
a pair of doublets at
δ 7.71 and 7.83 ppm corresponding to aromatic protons, and NH signal at δ
10.87 ppm. Also, the mass spectrum showed a molecular ion peak at m/z 270 (M+, 100%) which
confirmed its molecular formula C10H9F3N6.
Scheme 2. Synthetic route for 3,5-diaminopyrazole derivatives
N2H4 H2O/EtOH-pyridine/reflux 2 h; ( ) EtOH-pyridine/reflux 15 h; (
pyridine/reflux 3 h; (d) AcOH/reflux 10 h.
4
–
7
. Reagents and Conditions: (
a)
·
b
c)
2b/PhNHNH2/EtOH-
Acetylation of 3,5-diamino-1H-pyrazole derivatives 4a
corresponding 4-arylazo-3,5-diacetamidopyrazole derivatives 7a
1H-pyrazole derivatives 7a
were supported by elemental and spectral data. The 1H-NMR spectrum
for compound 7a exhibited two singlet signals at 2.12 and 2.17 ppm corresponding to the presence of
methyl protons and two broad signals at 6.56 and 10.27 ppm related to two (NH) protons, while its
mass spectrum revealed a molecular ion peak at m/z 304 (M+, 100%).
3,5-Diaminopyrazoles was employed as a key intermediate for the synthesis of several
poly substituted fused pyrazolopyrimidines. Pyrazolo[1,5-a]pyrimidines are of considerable
,b
in refluxing acetic acid afforded the
,b; (Scheme 2). The structures of
,b
δ
δ
4

Molecules 2019, 24, 1080
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pharmacological important as purine analogs [15
–17]. Thus, it was important to study the reactivity
of diaminopyrazole derivatives 4a
3,6-diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine (8a
hydrazone 2a 13 18] with hydrazine hydrate in the molar ratio 2:1 in ethanol containing 0.5 mL of
pyridine under reflux for 4–5 h, or via the reaction of 3,5-diaminopyrazole 4a with the equivalent from
the hydrazone derivatives 2a (Scheme 3). Asymmetrical 2,5,7-triaminopyrazolo[1,5-a]pyrimidine
9a were synthesized by the cyclization of diaminopyrazole 4a with equimolar of different
hydrazone derivatives 2a in ethanol containing 0.5 mL of pyridine under reflux for 6–7 h (Scheme 3).
,
b
toward a variety of nucleophilic reagents. Symmetrical
,b) were synthesized by the reaction of
,b [ ,
,
b
,b
,b
,b
,
b
Scheme 3. Synthetic route for symmetrical and asymmetrical 3,6-diarylazo-pyrazolo[1,5-a]
pyrimidine derivatives 8a and 9a . Reagents and Conditions: ( ) 2-arylazomalononitrile 2a
N2H4 H2O (2:1)/EtOH-pyridine/reflux 5 h; (
4a
,
b
,b
a
,b:
·
b
) 2-arylazomalononitrile 2a
,
b
: 5-diaminopyrazoles
◦
,b
(1:1)/EtOH-pyridine/reflux 4–7 h or heated under MW at 140 C 2 min or sonicated at room
temperature 1 h.
Moreover, the synthesis of pyrazolopyrimidine derivatives 8a,b and 9a,b were achieved under
microwave irradiation and ultrasound reactions conditions. TLC monitored the progress of the
reactions, and the yields of the products were noted. Comparisons between conventional, microwave,
and ultrasound techniques are given in (Table 1).
Table 1. Comparison between methods for the synthesis of compounds 8a,b and 9a,b.
Reflux
MW
Yield%
US
Compound No.
Time
Yield%
Time
Time
Yield%
8a
8b
9a
9b
300 min.
240 min.
360 min.
420 min.
62
60
63
67
2 min.
2 min.
2 min.
2 min.
74
71
76
78
60 min.
60 min.
60 min.
60 min.
52
40
60
43
The analytical and spectral data of the newly synthesized compounds 8a,b and 9a,b were
compatible with their structures. For instance, IR spectrum for 6-[(4-trifluoromethyl)phenylazo]-3-(4-
fluorophenylazo)-2,5,7-triaminopyrazolo[1,5-a] pyrimidine (9a) showed absorption bands at 3474,
1
3276, and 3123 cm−1, representing the presence of the amino groups, while H-NMR spectrum

Molecules 2019, 24, 1080
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showed signals at
δ
6.98 and 8.22 ppm referring to two amino groups, and its mass spectrum
showed a molecular ion peak at m/z 458 (M+, 100%), which corresponds with its molecular formula
C19H14F4N10.
Sulfonamide group can be considered as the main component in some drugs used
in clinical treatment.
as antibacterial, antifungal, antihypertensive, anticancer, antimalarial, and antiprotozoal
agents [13]. Thus, the reaction of 3,5-diaminopyrazole 4a with several hydrazones
It is reported that many of the sulfonamide derivatives act
,
b
of sulfa drugs (N-[4-(N-butylsulfamoyl)phenyl]carbonohydrazonoyl dicyanide, N-{4-[N-(4,6-
dimethylpyrimidin-2-yl)sulfamoyl]phenyl}carbono-hydrazonoyl dicyanide, and N-[4-(piperidin-
1-ylsulfonyl)phenyl]carbono-hydrazonoyl dicyanide) in ethanol under reflux in the presence of a
catalytic amount of pyridine afforded pyrazolo[1,5-a]pyrimidine derivatives 10a
(Scheme 4). Aiming to modify the yields and reduce the reactions time, we applied microwave
irradiation conditions to resynthesize the targeted compounds 10a 12a , and the comparison of
,b–12a,b, respectively
,b–
,b
the reactions times and yields between the conventional and microwave irradiation conditions are
summarized in Table 2.
The structures of 10a,b–12a,b were confirmed by their elemental analysis and spectral data (MS,
IR, 1H-NMR, and 13C-NMR). The IR spectrum revealed an absorption band at 3474–3207 cm−1 that
corresponds to NH and NH2 groups and at 2960–2835 and 1369–1347 cm−1 characteristic to aliphatic
1
CH2 and SO2 groups, respectively. H-NMR spectrum of (12b), as an example, revealed signals at
δ
1.36, 1.55, and 2.91 which refer to the presence of (3CH2) and (2NCH2) groups, respectively, while its
13C-NMR spectrum exhibited signals representing the presence of piperidine protons at
δ 22.85, 24.66,
and 46.58. Mass spectrum of (12b), as an example, gives the molecular ion peak at m/z 587 (M+; 44%),
corresponding to the molecular formula C24H24FN11O2S.
Also, 6-(thiazol-2-yl-azo)-3-(4-(trifluoromethyl)phenylazo)-2,5,7-triamino pyrazolo [1,5-
a]pyrimidine (13) was synthesized via the treatment of 3,5-diamine-4-[4-(trifluoromethyl)phenylazo]-
1H-pyrazole (4b) with N-(thiazol-2-yl) carbonohydrazonoyl dicyanide in ethanol under reflux or
microwave irradiation (Scheme 4).
Table 2. Comparison between methods for the synthesis of compounds 10a,b–13.
Reflux
MW
Compound No.
Time
Yield%
Time
Yield%
10a
10b
11a
11b
12a
12b
13
540 min.
480 min.
360 min.
420 min.
420 min.
420 min.
720 min.
70
78
88
72
73
75
69
5 min.
5 min.
3 min.
3 min.
3 min.
7 min.
8 min.
76
84
92
78
80
81
78
1H-NMR spectrum of 13 showed singlet signals at
protons of thiazole ring, as well as signals at 7.11 and 8.24 ppm for amino groups. The mass spectrum
revealed a molecular ion peak at 447 (M+; 100%), which confirms its molecular formula C16H12F3N11S.
δ 7.47 and 7.76 ppm due to the presence of two
δ

Molecules 2019, 24, 1080
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Scheme 4. Synthetic route for 2,5,7-triaminopyrazolo[1,5-a]pyrimidine derivatives 10–13. Reagents and Conditions: (a) EtOH-pyridine/reflux 7–12 h or heated under
◦
MW at 140 C 3–8 min.

Molecules 2019, 24, 1080
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2.2. Biological Evaluation
2.2.1. In Vitro Antimicrobial Evaluation
The newly synthesized compounds were evaluated for their antimicrobial activity toward six
bacterial strains, three Gram-positive (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus mutants),
and three Gram-negative (Enterococcus faecalis, Proteus vulgaris, and Escherichia coli), using the standard
antibiotic Gentamycin (5 mg/mL) as a reference. Also, their antifungal activity was evaluated against
three fungal strains (Aspergillus fumigates, Aspergillus flavus, and Candida albicans) to determine the zone
of inhibition using the standard antibiotic Ketoconazole (5 mg/mL). Their antibacterial activities were
tested for their activities at a concentration of (5 mg/mL) using inhibition zone diameter in mm as a
criterion for the antimicrobial activity [19,20], and the results are shown in (Table 3).
Compounds 2a, 2c, and 11b demonstrated more potent significant activity against tested
Enterococcus faecalis and Proteus vulgaris with a diameter of inhibition zone between 25 and 40 mm;
further, compound 2a showed high antimicrobial activity on Bacillus subtilis with a zone of inhibition
of 40 mm, and these compounds had moderate antimicrobial activity on other bacterial strains with an
inhibition zone ranging from 13 to 25 mm. Moreover, compound 2a has significant antifungal activity
towards three fungal strains, and compound 11b showed a moderate effect on fungal strains, while
compound 2c was not active on fungal strains. Also, compound 4b had a moderate effect on bacterial
strains with an inhibition zone of 20–24 mm diameter, as well as on fungal strains with an inhibitory
region ranging from 13 to 20 mm.
Furthermore, compounds 5a
, 7a, 7b, and 11a showed mild antibacterial activity on the tested
Gram-positive and Gram-negative bacterial strains with an inhibition zone of 9 to 18 mm, except
compound 5a was NA on Streptococcus mutants strain. Meanwhile, compounds 5a, 7b, and 11a had no
antifungal activity towards the three fungal strains.
Compounds 8a
,
8b
,
9a,
9b
,
10a
,
10b
,
12a
, 12b, and 13 were NA on all evaluated bacterial and
fungal strains, except for compounds 8a
,
8b, and 9b, which exhibited low antibacterial activity on
Escherichia coli (10 mm), Bacillus subtilis (8 mm), and Staphylococcus aureus (10 mm), respectively.

Molecules 2019, 24, 1080
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Table 3. Inhibition zone in (mm) as a criterion of antimicrobial activity of some newly synthesized compounds.
Microorganism Inhibition Zone Diameter (mm)
Gram +ve Bacteria
Gram -ve Bacteria
Fungi
Compound No.
Staphylococcus
aureus
Bacillus
subtilis
Strepotococcus
mutants
Enterococcus
faecalis
proteus
vulgaris
Escherichia
coli
Aspergillus
fumigates
Aspergillus
flavus
Candida
Albicans
2a
2c
4b
25
22
23
40
24
23
22
18
24
35
25
22
40
26
23
28
14
20
24
NA
20
20
NA
13
21
NA
15
5a
5b
12
18
8
25
NA
20
11
22
8
28
12
20
NA
15
NA
10
NA
16
7a
7b
8a
8b
9a
9b
10a
10b
11a
11b
12a
12b
13
14
15
16
17
NA
8
NA
NA
NA
NA
12
14
19
13
18
20
16
16
13
10
NA
NA
NA
NA
NA
11
15
9
11
NA
NA
NA
NA
NA
NA
NA
NA
20
NA
NA
NA
NA
NA
NA
NA
NA
17
NA
NA
NA
NA
NA
NA
NA
NA
15
NA
NA
NA
10
NA
NA
18
NA
NA
NA
NA
NA
NA
9
NA
NA
NA
NA
NA
NA
13
NA
NA
NA
NA
NA
NA
9
22
23
29
25
26
13
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
NA
Gentamycin
Ketoconazole
24
-
26
-
20
-
26
-
25
-
30
-
-
17
-
16
-
20
NA = not active; Diameter of hole = 6 mm; data are expressed in the form of mean ± SD.

Molecules 2019, 24, 1080
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2.2.2. In Vitro Cytotoxic Screening
The antitumor activity of the target compounds was screened against three human cancer
cell lines—breast adenocarcinoma (MCF-7), hepatocellular carcinoma (HepG2), and human colon
carcinoma (HCT-116). The IC50 values of the tested compounds are listed in Table 4 and Figure 2.
From the obtained results, compounds 2a,b, 4b, 5b, 8b, and 12b showed the most potent cytotoxic
profile on cancer cells (MCF-7, HepG2, and HCT-116) with IC50 values ranging from 0.3 to 3.4 µg while
the compound 8b is most effective among all of them on all types of cancer cells.
Compounds 2c
with IC50 values ranging from 2.7 to 9.8
HCT 116 with IC50 of 1.2 g and 2.4, respectively. Moreover, the compounds 4a
moderate effects on cancer cells with IC50 values ranging from 6.7 to 12.9 g, while the compound
8a was the most significant on HePG2 cells with IC50 of 4.9 g; on the other hand, the effect of the 4a
compound was low on MCF-7 cells. In addition, the compounds 3a 5a 7a 9b 11a, and 12a had
slightly significant effects with IC50 values ranging from 14.1 to 38.5 g, the compound 3a indicated a
slightly noticeable effect on HePG2 cells with IC50 of 6.1 g, and the compound 12a had weak toxicity
on HCT 116 cells with IC50 of 46.1 g. Furthermore, the 10b compound had a low cytotoxic effect on
MCF-7 and HePG2 cells with IC50 of 43.9 and 85.9 g, respectively, and a moderate effect with IC50 of
29.4 g. Compound 9a showed very low cellular toxicity with IC50 higher than 100 g compared to all
other compounds on cancer cells.
,
11b, and 13 have demonstrated a significant effect on MCF-7 and HePG2 cells
g, and 2c and 11b compounds significantly increased on
8a, and 10a showed
µ
µ
,
µ
µ
,
b,
,
,
b,
,
µ
µ
µ
µ
µ
µ
Table 4. The IC50 (µg) of the new tested chemicals compounds against different tumor cell lines.
IC50 (µg/mL)
Compound
MCF-7
HepG2
HCT-116
2a
2b
2c
3a
3b
4a
4b
5a
5b
7a
7b
8a
8b
9a
9b
10a
1.3 ± 0.7
0.5 ± 0.04
5.9 ± 0.2
18.8 ± 1.5
19.4 ± 2.6
32.1 ± 1.8
1.7 ± 0.2
24.6 ± 3.2
2.8 ± 0.7
16.3 ± 0.6
20.8 ± 1.7
10.7 ± 0.3
0.3 ± 0.01
106 ± 4.3
38.5 ± 0.5
10.1 ± 2.6
43.9 ± 1.1
19.8 ± 1.9
4.5 ± 0.4
18.8 ± 1.7
1.4 ± 0.03
4.2 ± 0.2
1.2 ± 0.2
0.5 ± 0.2
1.1 ± 0.3
9.8 ± 0.5
6.1 ± 1.1
19.3 ± 5.7
6.7 ± 0.9
1.1 ± 0.1
73.6 ± 3.1
0.9 ± 0.1
14.3 ± 2.6
21.3 ± 3.1
4.9 ± 0.6
0.6 ± 0.2
226.9 ± 3.2
18.9 ± 0.8
7.5 ± 0.7
85.9 ± 3.4
26.6 ± 4.4
3.9 ± 0.4
31.4 ± 2.7
3.4 ± 0.6
4.1 ± 0.3
0.9 ± 0.3
1.2 ± 0.1
0.5 ± 0.2
1.2 ± 0.12
19.1 ± 3.1
27.8 ± 2.6
11.5 ± 1.1
1.1 ± 0.08
24.1 ± 0.7
3.7 ± 0.7
14.1 ± 1.9
15.7 ± 1.7
11.1 ± 0.5
0.4 ± 0.02
103.8 ± 5.1
31.1 ± 2.5
12.9 ± 1.6
29.4 ± 2.4
25.9 ± 1.7
2.7 ± 0.6
10b
11a
11b
12a
12b
46.1 ± 5.9
2.4 ± 0.4
13
5.3 ± 0.8
Doxorubicin
1.6 ± 0.2

Molecules 2019, 24, 1080
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Figure 2. The differences of IC50 % of new tested chemicals compounds effects on different tumor cells
(MCF-7, HePG2, and HCT 116 cells).
3. Materials and Methods
3.1. General Information
All melting points were determined with a Stuart Digital Melting Point apparatus SMP10 and are
uncorrected. Elemental analyses were performed on a Perkin-Elmer 240 microanalyser, PE 2400 Series
II CHNS/O Analyzer, carried out at the regional center for mycology and biotechnology, Al-Azhar
University, cairo, Egypt. IR spectra were determined as KBr pellets on a Thermo Nicolet apparatus
(Thermo Scientific, Madison, WI, USA) at Postgraduate campus for Girls at Lassan, King Khalid
University, Abha, Saudi Arabia. The NMR spectra were recorded on a Bruker NMR spectrometer
(Bruker, Billerica, MA, USA) in DMSO-d6, as solvent at 300, 500 MHz for 1H-NMR and 75, 125 MHz
for 13C-NMR at Faculty of Science, Cairo University, Egypt and King Khalid University, Abha,
respectively. The chemical shifts (δ) are reported in parts per million (ppm). Mass spectra were
measured on GC/MS-QP5 spectrometer at regional center for mycology and biotechnology, Al-Azhar
University, Egypt. Antimicrobial activity was measured at the regional center for mycology and
biotechnology, Al-Azhar University, Egypt. Follow-up of the reactions and checking the purity of
the compounds were carried out using TLC on silica gel-precoated aluminum sheets (Fluorescent
indicator 254 nm, Fluka, Germany) and the spots were detected by exposure to UV lamp at
λ 254/366
nm for a few seconds or under iodine vapor. Compounds 2-(4-fluorphenylazo)malononitrile 2a
and 3,5-diamino-4-(4-fluorophenylazo)-1H-pyrazole 4b were prepared according to the reported
procedure [21–23].
3.1.1. General Procedure for the Synthesis of 2-Arylazomalononitrile 2a–c:
To a solution of aniline derivatives 1a–c (0.01 mol) in hydrochloric acid (6 mL), a solution of
sodium nitrite (0.72 g, 0.0105 mol) in water (3 mL) was added portion wise with stirring at 0–5 ◦C
for 1 h. The clear diazonium salt was added to a stirred solution of malononitrile (0.66 g, 0.01 mol)
and sodium acetate (4.6 g) in aqueous ethanol 50% (50 mL) with continued stirring at 0–5 ◦C for 2 h.
The reaction mixture was allowed to stand overnight at room temperature, then the precipitate that
formed was filtered off, washed several times with water, dried, and recrystallized from ethanol to
afford 2-arylazomalononitrile 2a–c.
◦
2-[4-(Trifluoromethyl)phenylazo]malononitrile (2b): Yield: 93%; (orange powder): mp 179–180 C; IR (KBr)
1
ν
in cm−1: 3141 (NH), 2227 (C
≡
N), 1618 (C=N) H-NMR (500 MHz, DMSO-d6):
δ
7.64 (d, 2H,
max
Ar–H, J = 6.9 Hz), 7.76 (d, 2H, Ar–H, J = 7.1 Hz), 10.36 (s, br, 1H, NH); 13C-NMR (125 MHz, DMSO-d6):

Molecules 2019, 24, 1080
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δ
86.98 (C(CN)2), 109.52 (C
≡
N), 113.88, 116.69 (Ar–C), 123.03, 125.19 (q, C–F3, J = 270 Hz), 126.72,
127.35, 144.52 (Ar–C); MS (m/z), 239 (M+ + 1; 6%), 238 (M+; 10%), 94 (95%), 67 (100%). Anal. Calcd. for
C10H5F3N4 (238.17): C, 50.43; H, 2.12; N, 23.52%. Found C, 50.47; H, 2.34; N, 23.64%.
2-(4,5-Dimethyl-2-nitrophenylazo)malononitrile (2c): Yield: 45%; (yellow powder): mp 160–162 ◦C; IR
1
(KBr)
(500 MHz, DMSO-d6):
br, 1H, NH); 13C-NMR (125 MHz, DMSO-d6):
ν
in cm−1: 3231 (NH), 2989, 2954 (C-H aliphatic), 2215 (C
≡
N), 1617 (C=N); H-NMR
max
δ
2.30, 2.36 (2s, 6H, 2CH3), 7.60 (s, 1H, Ar–H), 7.98 (s, 1H, Ar–H), 9.88 (s,
18.52, 19.69 (CH3), 89.05 (C(CN)2), 109.05 (C N),
113.34, 118.97, 125.61, 134.62, 135.11, 146.70 (Ar–C); MS (m/z), 244 (M+ + 1; 11%), 243 (M+; 25%), 61
(100%). Anal. Calcd. for C11H9N5O2 (243.23): C, 54.32; H, 3.73; N, 28.79%. Found C, 54.19; H, 3.59; N,
28.86%.
δ
≡
3.1.2. General Procedures for the Synthesis of 2-[(4-Substituted)arylazo] malononitrile 3a,b:
A mixture of 2-arylazomalononitrile 2a (0.01 mol) and piperidine or morpholine (0.01 mol) in
ethanol (30 mL) was refluxed for 1 h and then left to cool to room temperature. The reaction mixture
was poured onto cooled water with continuous stirring. The precipitate that formed was filtered, dried,
and recrystallized from toluene to afford 2-[(4-substituted)arylazo] malononitrile 3a,b.
◦
2-[4-(Piperidino)phenylazo]malononitrile (3a): Yield: 81%; (yellow plates): mp 197–199 C; IR (KBr)
ν
in cm−1: 3301 (NH), 2942, 2856 (C-H aliphatic), 2184 (C N), 1621 (C=N); 1H-NMR (300 MHz,
≡
max
DMSO-d6):
2H Ar–H + NH); 13C-NMR (75 MHz, DMSO-d6):
115.12, 115.41 (C
N), 116.95, 121.75, 121.86 (Ar–C); MS (m/z), 253 (M+; 7%), 242 (88%), 72 (100%).
δ
1.64 (m, 6H, 3CH2), 3.55 (m, 4H, 2N–CH2), 7.13 (d, 2H, Ar–H, J = 6.9 Hz), 7.50 (m, 3H,
δ
23.61, 25.62 (CH2), 49.31 (N–CH2), 92.58 (C(CN)2),
≡
Anal. Calcd. for C14H15N5 (253.31): C, 66.38; H, 5.97; N, 27.65%. Found C, 66.33; H, 5.80; N, 27.54%.
◦
2-(4-Morpholinophenylazo)malononitrile (3b): Yield: 81%; (yellow crystals): mp 166–167 C; IR (KBr)
ν
max in cm−1: 3303 (NH), 2934, 2861 (C–H aliphatic), 2179 (C N), 1618 (C=N); 1H-NMR (300 MHz,
≡
DMSO-d6):
Ar–H, J = 5.4 Hz), 7.69 (s, 1H, NH); 13C-NMR (75 MHz, DMSO-d6):
92.64 (C(CN)2), 115.19, 115.48 (C
N), 116.87, 121.91, 122.02 (Ar–C); MS (m/z), 255 (M+; 21%), 111
(56%), 43 (100%). Anal. Calcd. for C13H13N5O (255.28): C, 61.17; H, 5.13; N, 27.43%. Found C, 61.28; H,
5.22; N, 27.48%.
δ
3.60 (t, 4H, 2N–CH2), 3.70 (t, 4H, 2O–CH2), 7.15 (d, 2H, 2 Ar–H, J = 4.8 Hz), 7.51(d, 2H, 2
δ
48.75 (N–CH2), 65.99 (O–CH2),
≡
3.1.3. General Procedure for the Synthesis of 4-Arylazo-3,5-diaminopyrazole 4a,b
To a solution of 2-arylazomalononitrile 2a,b (0.01 mol) in ethanol (30 mL), hydrazine hydrate
(0.01 mol) and pyridine (0.5 mL) were added. The reaction mixture was heated under reflux for 2
h (monitored by TLC). After completion of the reaction, the precipitate that formed was filtered off,
dried, and recrystallized from ethanol to afford 3,5-diamino-4-(arylazo)pyrazole 4a,b.
3,5-Diamino-4-[4-(trifluoromethyl)phenylazo]-1H-pyrazole (4b): Yield: 98%; (orange crystals): mp
231–232 ◦C; IR (KBr)
ν
max
in cm−1: 3465, 3389, 3296 (NH2,NH), 1622 (C=N), 1421 (N=N); 1H-NMR
(500 MHz, DMSO-d6):
δ
6.29 (br, 4H, 2NH2), 7.71 (d, 2H, Ar–H, J = 8.5 Hz), 7.83 (d, 2H, Ar–H, J = 8.0 Hz),
10.87 (br, 1H, NH); 13C-NMR (125 MHz, DMSO-d6):
δ 115.61, 120.62, 120.94 (Ar–C), 123.47, 125.63 (q,
C–F3, J = 270 Hz), 125.80, 125.83, 127.79 (Ar–C), 156.37 (C=N); MS (m/z), 271 (M+ + 1; 14%), 270 (M+;
100%). Anal. Calcd. for C10H9F3N6 (270.22): C, 44.45; H, 3.36; N, 31.10%. Found C, 44.39; H, 3.23; N,
31.19%.
3.1.4. General Procedure for the Synthesis of 4-Arylazo-3,5-diamino-N-substitutedpyrazole 5a,b
To a solution of 2-arylazomalononitrile 2a,b (0.01 mol) in ethanol (30 mL), hydrazide derivatives,
namely nicotinohydrazide or benzohydrazide (0.01 mol) and pyridine (0.5 mL), were added.
The reaction mixture was heated under reflux for 15 h (monitored by TLC). After completion of

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the reaction, the precipitate that formed was filtered off, dried, and recrystallized from ethanol to
afford N-substituted-3,5-diamino-4-(arylazo)pyrazole 5a,b.
{3,5-Diamino-4-[(4-fluorophenyl)azo]-1H-pyrazol-1-yl}(pyridin-3-yl)methanone (5a): Yield: 69%; (orange
plates): mp >3 00 ◦C; IR (KBr)
ν
in cm−1: 3477, 3274, 3127 (NH2), 1672 (C=O), 1593 (C=N), 1423
max
(N=N); 1H-NMR (500 MHz, DMSO-d6):
δ
6.92 (s, 2H, NH2), 7.28–7.32 (m, 4H, Ar–H), 7.72–7.75 (m,
107.82,
2H, Ar–H), 7.95–7.98 (m, 2H, Ar–H), 8.08 (br, s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6):
δ
112.32, 120.54, 121.24, 125.82, 125.89, 126.92, 127.16, 128.35, 129.02, 131.04 (Ar–C), 153.15, 157.93 (C=N),
160.52 (C–F), 166.38 (C=O); MS (m/z), 327 (M+ + 2; 3.3%), 325 (M+; 12%), 95 (100%). Anal. Calcd. for
C15H12FN7O (325.31): C, 55.38; H, 3.72; N, 30.14%. Found C, 55.28; H, 3.68; N, 30.24%.
{3,5-Diamino-4-[(4-(trifluoromethyl)phenylazo]-1H-pyrazol-1-yl}(phenyl)methanone (5b): Yield: 71%;
◦
(orange powder): mp 284–286 C; IR (KBr)
(C=N), 1411 (N=N); 1H-NMR (500 MHz, DMSO-d6):
7.77–7.86 (m, 7H, Ar–H+NH2); 13C-NMR (125 MHz, DMSO-d6):
ν
in cm−1: 3506, 3353, (NH2), 1689 (C=O), 1618
max
δ
7.02 (s, 2H, NH2), 7.67–7.73 (m, 4H, Ar–H),
107.46, 121.02, 121.16, 121.65 (Ar–C),
δ
123.63, 125.81 (q, C–F3, J = 272.5 Hz), 126.15, 127.02, 127.68, 149.08 (Ar–C), 150.11 (C=N), 167.31 (C=O);
MS (m/z), 375 (M+ + 1; 5%), 374 (M+; 12%), 145 (100%). Anal. Calcd. for C17H13F3N6O (374.33): C,
54.55; H, 3.50; N, 22.45%. Found C, 54.75; H, 3.58; N, 22.35%.
3.1.5. Synthesis of 3,5-Diamino-N-phenyl-4-[4-(trifluoro-methyl)phenylazo]-1H-pyrazole (6)
Phenylhydrazine (0.98 mL, 0.01 mol) and pyridine (0.5 mL) were added to a solution
of 2-arylazomalononitrile 2b (2.7 g, 0.01 mol) in ethanol (30 mL). The reaction mixture
was heated under reflux for 3 h (monitored by TLC). After completion of the reaction,
the precipitate that formed was filtered off, dried, and recrystallized from ethanol to give
3,5-diamino-1-phenyl-4-[4-(trifluoro-methyl)phenylazo]-1H-pyrazole 6. Yield: 71%; (yellow crystals):
mp >300 ◦C; IR (KBr)
(500 MHz, DMSO-d6):
J = 8.0 Hz), 7.53–7.76 (m, 5H, Ar–H), 7.95 (br, s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6):
ν
max in cm−1: 3400, 3304, 3197 (NH2, NH), 1616 (C=N), 1423 (N=N); 1H-NMR
6.99 (br, s, 2H, NH2), 7.33 (d, 2H, Ar–H, J = 7.5 Hz), 7.50 (d, 2H, Ar–H,
101.00,
δ
δ
121.07, 121.26, 122.26 (Ar–C), 123.42, 125.59, 125.95 (q, C–F3, J = 270 Hz), 126.16, 126.41, 129.27, 149.07
(Ar–C), 156.07 (C=N). Anal. Calcd. for C16H13F3N6 (346.32): C, 55.49; H, 3.78; N, 24.27%. Found C,
55.68; H, 3.91; N, 24.22%.
3.1.6. Synthesis of 4-Arylazo-3,5-diacetamido-1H-pyrazole 7a,b
A solution of 3,5-diamine-4-arylazo-1H-pyrazole 4a,b (0.01 mol) in acetic acid (30 mL) was heated
under reflux for 10 h (monitored by TLC). The reaction mixture was left to cool to room temperature,
and then poured onto cooled water (50 mL) portion wise with continuous stirring for 1 h. The separated
yellow compound was filtered off, washed with water, and, finally, dried and recrystallized from
ethanol to afford 3,5-diacetamio-4-arylazo-1H-pyrazole 7a,b.
3,5-Diacetamido-4-[(4-fluorophenyl)azo]-1H-pyrazole (7a): Yield: 86%; (yellow powder): mp 234–236 ◦C;
IR (KBr)
DMSO-d6):
ν
in cm−1: 3277, 3144 (NH), 1698 (C=O), 1605 (C=N), 1418 (N=N); 1H-NMR (300 MHz,
δ 2.12, 2.17 (2s, 6H, 2CH3), 6.56 (br, 1H, NH), 7.77–7.92 (m, 5H, Ar–H+NH), 10.27 (br, 1H,
max
NH); 13C-NMR (75 MHz, DMSO-d6):
δ 23.12 (CH3), 115.82, 116.08, 120.56, 122.74, 122.85, 123.68, 123.80
(Ar–C), 149.12 (C=N), 161.04 (C–F), 169.24 (2C=O): MS (m/z), 305 (M+ + 1; 28%), 304 (M+; 100%), 42
(76%). Anal. Calcd. for C13H13FN6O2 (304.29): C, 51.31; H, 4.31; N, 27.62%. Found C, 51.38; H, 4.41; N,
27.53%.
3,5-Diacetamido-4-[4-(trifluoromethyl)phenylazo]-1H-pyrazole (7b): Yield: 80%; (Yellow plates): mp
◦
1
236–238 C; IR (KBr)
ν
in cm−1: 3238 (NH), 1678 (C=O), 1595 (C=N), 1426 (N=N); H-NMR
max
(300 MHz, DMSO-d6):
δ
2.24 (s, 6H, 2CH3), 7.82–7.96 (m, 5H, Ar–H+NH), 8.40, 10.11 (2br, s, 2H, 2NH);
23.38 (CH3), 100.33, (Ar–C), 123.10, 125.26 (q, C–F3, J = 270 Hz),
126.07, 126.24, 126.26, 128.75, 129.00 (Ar–C), 155.61 (C=N), 167.33 (C=O): MS (m/z), 354 (M+; 19%), 312
13C-NMR (125 MHz, DMSO-d6):
δ

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(72%), 70 (100%). Anal. Calcd. for C14H13F3N6O2 (354.29): C, 47.46; H, 3.70; N, 23.72%. Found C, 47.57;
H, 3.79; N, 23.76%.
3.1.7. General Procedure for the Synthesis of 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine
derivatives 8a,b
A. Under reflux Condition:
Method A: A mixture of 2-arylazomalononitrile 2a,b (0.02 mol) and hydrazine hydrate (0.01 mol)
in ethanol (30 mL) in the presence of pyridine (0.5 mL) was heated under reflux for 5 h, (monitored
by TLC). After completion of the reaction, the precipitates those formed were filtered off, dried, and
recrystallized from 1,4-dioxane to afford 8a,b.
Method B: A mixture of 4-arylazo-3,5-diaminopyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile
2a,b (0.01 mol) in ethanol (30 mL) and in the presence of pyridine (0.5 mL) was heated under reflux
for 4–5 h, (monitored by TLC). After completion of the reaction, the precipitates those formed were
filtered off, dried, and recrystallized from 1,4-dioxane to afford 8a and 8b, respectively.
B. Under Microwave Irradiation:
A mixture of 5-diamino-4-arylazopyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile 2a,b
(0.01 mol) was grinded carefully in a porcelain mortar using a pestle and transferred to a pyrex test
tube; then, ethanol (4 mL) was added, followed by pyridine (0.5 mL). The reaction mixture was heated
under microwave irradiation at 50% power for 2 min at 140 ◦C, monitored by TLC. After completion
of the reaction, the reaction mixture was cooled to room temperature and the precipitated solid was
filtered off, washed with MeOH, and recrystallized from dioxane to afford 8a and 8b, respectively.
C-Under Ultrasound Condition:
To a solution of 4-arylazo-3,5-diaminopyrazole 4a
,b (0.01 mol) in ethanol (30 mL),
2-arylazomalononitrile 2a (0.01 mol) and pyridine (0.5 mL) was added. The reaction mixture
,b
was sonicated for 1 h at room temperature, (monitored by TLC). After completion of the reaction,
the precipitate product was filtered off, dried, and recrystallized from dioxane to afford 8a and
8b respectively.
3,6-Bi◦s[(4-fluorophenyl)azo]-2,5,7-triaminopyrazolo[1,5-a]pyrimidine (8a): Yield: 74%; (orange plates): mp
1
>300 C; IR (KBr)
ν
in cm−1: 3422, 3274 (NH2), 1615 (C=N), 1420 (N=N); H-NMR (500 MHz,
max
DMSO-d6):
δ
6.92 (s, 2H, NH2), 7.29–7.32 (m, 4H, Ar–H), 7.72–7.75 (m, 4H, Ar–H), 7.96 (s, br, 2H, NH2),
107.81, 115.11, 115.66, 115.70, 115.84, 115.86,
8.10 (s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6):
δ
122.34, 122.40, 123.72, 123.79, 147.22, 149.08, 149.10 (Ar–C), 150.05, 152.31 (C=N), 160.52, 161.18 (C–F);
MS (m/z), 409 (M+ + 1; 30%), 408 (M+; 100%), 363 (54%), Anal. Calcd. for C18H14F2N10 (408.38): C,
52.94; H, 3.46; N, 34.30%. Found C, 52.83; H, 3.52; N, 34.22%.
3,6-Bis[4-(trifluoromethyl)phenylazo]-2,5,7-triaminopyrazolo[1,5-a]pyrimidine (8b): Yield: 71%; (orange
◦
crystals): mp >300 C; IR (KBr)
ν
in cm−1: 3460, 3259, 3103 (NH2), 1602 (C=N), 1417 (N=N);
δ 7.06 (s, 2H, NH2; exchangeable with D2O), 7.77–7.86 (m, 10H,
max
1H-NMR (500 MHz, DMSO-d6):
Ar–H+NH2; exchangeable with D2O), 8.19 (s, 2H, NH2; exchangeable with D2O); 13C-NMR (125 MHz,
DMSO-d6):
δ 108.98, 116.57, 121.82, 122.25 (Ar–C), 123.19, 125.35 (q, C–F3, J = 287.5 Hz), 125.49, 125.99,
126.02, 126.17, 126.20, 126.83, 127.08, 127.86, 128.11, 148.05 (Ar–C), 152.38, 154.71 (C=N); MS (m/z), 509
(M+ + 1; 25%), 508 (M+; 100%). Anal. Calcd. for C20H14F6N10 (508.39): C, 47.25; H, 2.78; F, 22.42; N,
27.55%. Found C, 47.34; H, 2.71; N, 27.44%.

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3.1.8. General Procedure for the Synthesis of 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine
Derivatives 9a,b
A. Under Reflux Condition:
To a solution of 4-arylazo-3,5-diaminopyrazole 4a
,b (0.01 mol) in ethanol (30 mL),
2-arylazomalononitrile 2a (0.01 mol) and pyridine (0.5 mL) were added. The reaction mixture
,b
was heated under reflux for 6–7 h (monitored by TLC). After completion of the reaction, the formed
precipitates were filtered off, dried, and recrystallized from ethanol to afford 9a,b.
B. Under Microwave Irradiation:
A mixture of 5-diamino-4-(arylazo)pyrazole 4a,b (0.01 mol) and 2-arylazomalononitrile 2a,b
(0.01 mol) was grinded carefully in a porcelain mortar using a pestle and transferred to a pyrex test
tube, and ethanol (4 mL) was added, followed by pyridine (0.5 mL). The reaction mixture was heated
under microwave irradiation at 50% power for 2 min at 140 ◦C, monitored by TLC. After completion
of the reaction, the reaction mixture was cooled to room temperature and the precipitated solid was
filtered off, washed with MeOH, and recrystallized from ethanol to afford 9a,b.
C-Under Ultrasound Condition:
To 5-diamino-4-(arylazo)pyrazole 4a,b (0.01 mol) in ethanol (30 mL), 2-arylazomalononitrile 2a,b
(0.01 mol) and pyridine (0.5 mL) was added. The reaction mixture was sonicated for 1 h at room
temperature (monitored by TLC). After completion of the reaction, the precipitate was filtered off,
dried, and recrystallized from ethanol to afford 9a,b.
6-[(4-Trifluoromethyl)phenylazo]-3-[(4-fluorophenyl)azo]-2,5,7-triaminopyrazolo [1,5-a]pyrimidine (9a): Yield:
76%; (orange plates): mp >300 ◦C; IR (KBr) max in cm−1: 3474, 3276, 3123 (NH2), 1616 (C=N), 1423
ν
(N=N); 1H-NMR (500 MHz, DMSO-d6):
δ 6.98 (s, 2H, NH2), 7.31–7.35 (m, 4H, Ar–H), 7.76–7.82 (m, 6H,
Ar–H+NH2), 8.22 (s, 2H, NH2); MS (m/z), 408 (45%), 458 (M+; 100%), 459 (M+ + 1; 22%). Anal. Calcd.
for C19H14F4N10 (458.13): C, 49.79; H, 3.08; N, 30.56. Found C, 49.65; H, 3.19; N, 30.45%.
3-[(4-Trifluoromethyl)phenylazo]-6-[(4-fluorophenyl)azo]-2,5,7-triaminopyrazolo [1,5-a]pyrimidine (9b): Yield:
78%; (orange plates): mp >300 ◦C; IR (KBr) max in cm−1: 3468, 3266, 3124 (NH2), 1594 (C=N), 1419
ν
(N=N); 1H-NMR (500 MHz, DMSO-d6):
J = 7.5 Hz), 8.10 (s, br, 2H, NH2), 8.13 (s, 2H, NH2), 7.84 (d, 2H, Ar–H, J = 7.5 Hz); 13C-NMR (125 MHz,
DMSO-d6): 108.98, 116.48, 121.82, 122.25 (Ar–C), 123.19, 125.35 (q, C–F3, J = 270 Hz), 125.52, 125.99,
δ 7.06 (s, 2H, NH2), 7.28–7.32 (m, 4H, Ar–H), 7.80 (d, 2H, Ar–H,
δ
126.02, 126.17, 126.20, 126.90, 127.68, 128.11, 148.05 (Ar–C), 152.83, 154.90 (C=N), 161.27 (C–F). Anal.
Calcd. for C19H14F4N10 (458.13): C, 49.79; H, 3.08; N, 30.56%. Found C, 49.50; H, 3.21; N, 30.46%.
3.1.9. General Procedure for the Synthesis of 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a] pyrimidine
Derivatives 10a,b
A. Under reflux Condition:
To a solution of 4-arylazo-3,5-diaminopyrazole 4a,b (0.01 mol) in ethanol (30 mL),
N-[4-(N-butylsulfamoyl)phenyl]carbonohydrazonoyl dicyanide (3.05 g, 0.01 mol) and pyridine
(0.5 mL) was added. The reaction mixture was heated under reflux for 8–9 h (monitored by TLC).
After completion of the reaction, the precipitated product that formed was filtered off, dried, and
recrystallized from toluene to afford 3-arylazo-2,5,7-triaminopyrazolo[1,5-a] pyrimidine 10a,b.
B. Under Microwave Irradiation:
A
mixture of 5-diamino-4-(arylazo)pyrazole 4a
,
b
(0.01 mol) and N-[4-(N-
butylsulfamoyl)phenyl]carbonohydrazonoyl dicyanide (3.05 g, 0.01 mol) was grinded carefully in a
porcelain mortar using a pestle and transferred to a pyrex test tube, and ethanol (4 mL) was added,

Molecules 2019, 24, 1080
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followed by pyridine (0.5 mL). The reaction mixture was heated under microwave irradiation at 50%
power for 5 min at 140 ◦C, monitored by TLC. After completion of the reaction, the reaction mixture
was cooled to room temperature and the precipitated solid was filtered off, washed with MeOH, and
recrystallized from ethanol to afford 10a,b.
N-Butyl-4-{[2,5,7-triamino-3-(4-fluorophenylazo)pyrazolo[1,5-a]pyrimidin-6-yl]azo}benzenesulfonamide (10a):
Yield: 76%; (orange plates): mp >300 ◦C; IR (KBr)
ν
in cm−1 3474, 3279 (NH2), 2932, 2870 (C–H
max
aliphatic), 1611 (C=N), 1420 (N=N), 1369 (SO2); 1H-NMR (500 MHz, DMSO-d6):
δ 0.80 (t, 3H, CH3),
1.22–1.27 (m, 2H, CH2), 1.34–1.37 (m, 2H, CH2), 2.75–2.77 (t, 2H, CH2), 6.94 (s, 2H, NH2), 7.29–7.33
(m, 4H, Ar–H), 7.60 (br, s, 1H, NH), 7.74–7.76 (m, 4H, Ar–H), 7.83 (s, 2H, NH2), 8.18 (s, 2H, NH2);
13C-NMR (125 MHz, DMSO-d6):
δ 13.43 (CH3), 19.19, 31.02 (CH2), 42.20 (N–CH2), 108.95, 115.28,
115.73, 115.91, 116.65, 120.81, 122.06, 122.43, 122.50, 123.91, 127.44, 127.68, 139.29, 147.51, 150.01 (Ar–C),
152.58, 154.48 (C=N), 160.63 (C–F); MS (m/z), 525 (M+; 11%), 526 (M+ + 1; 4%), 403 (100%). Anal. Calcd.
for C22H24FN11O2S (525.57): C, 50.28; H, 4.60; N, 29.32; S, 6.10%. Found C, 50.39; H, 4.78; N, 29.39; S,
6.01%.
N-Butyl-4-{[2,5,7-triamino-3-((4-(trifluoromethyl)phenyl)azo)pyrazolo[1,5-a]pyrimidin-6-yl]azo}benzene-
sulfonamide (10b): Yield: 84%; (reddish brown powder): mp 289–291 ◦C; IR (KBr)
3315 (NH2) 2960, 2870 (C-H aliphatic), 1611 (C=N), 1412 (N=N), 1366 (SO2); H-NMR (500 MHz,
ν
max in cm−1: 3446,
1
DMSO-d6):
7.07 (s, 2H, NH2), 7.61 (br, s, 1H, NH), 7.82–7.89 (m, 8H, Ar–H), 8.21 (br, s, 4H, 2NH2); 13C-NMR
(125 MHz, DMSO-d6): 13.40 (CH3), 19.19, 31.01 (2CH2), 42.19 (N–CH2), 109.06, 116.58, 120.72, 120.89,
δ 0.79 (t, 3H, CH3), 1.23–1.26 (m, 2H, CH2), 1.34–1.37 (m, 2H, CH2), 2.76–2.77 (t, 2H, CH2),
δ
121.03 (Ar–C), 123.32, 125.48 (q, C–F3, J = 270 Hz), 126.22, 126.85, 127.10, 127.45, 127.69, 139.47, 148.04
(Ar–C), 152.40, 154.41 (C=N); MS (m/z), 576 (M+ + 1; 3%), 575 (M+; 9%), 398 (100%). Anal. Calcd. for
C23H24F3N11O2S (575.58): C, 48.00; H, 4.20; N, 26.77%. Found C, 48.24; H, 4.14; N, 26.86%.
3.1.10. General Procedure for the Synthesis of 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a] pyrimidine
Derivatives 11a,b
A. Under Reflux Condition:
To a solution of 5-diamino-4-(arylazo)pyrazole 4a,b (0.01 mol) in ethanol (30 mL), N-{4-[N-(4,6-
dimethylpyrimidin-2-yl)sulfamoyl]phenyl} carbonohydrazonoyl dicyanide (3.55 g, 0.01 mol) and
pyridine (0.5 mL) were added. The reaction mixture was heated under reflux for 6–7 h, (monitored by
TLC). After completion of the reaction, the precipitated product that formed was filtered off, dried,
and recrystallized from toluene to afford 3-arylazo-2,5,7-triamino pyrazolo[1,5-a]pyrimidine 11a,b.
B. Under Microwave Irradiation:
A
mixture of 5-diamino-4-(arylazo)pyrazole 4a
,b
(0.01 mol) and N-{4-[N-(4,6-
dimethylpyrimidin-2-yl)sulfamoyl]phenyl}carbonohydrazonoyl dicyanide (3.55 g, 0.01 mol)
was grinded carefully in a porcelain mortar using a pestle and transferred to a pyrex test tube, and
ethanol (4 mL) was added, followed by pyridine (0.5 mL). The reaction mixture was heated under
microwave irradiation at power 50% for 3 min at 140 ◦C, monitored by TLC. After completion of the
reaction, the reaction mixture was cooled to room temperature and the precipitated solid was filtered
off, washed with MeOH, and recrystallized from ethanol to afford 11a,b.
N-(4,6-Dimethylpyrimidin-2-yl)-4-{[2,5,7-triamino-3-(4-fluorophenylazo)pyrazolo
yl]azo}benzenesulfonamide (11a): Yield: 92%; (reddish-brown powder): mp > 300 C; IR (KBr)
in cm−1: 3403, 3307 (NH2), 2959 (C–H aliphatic), 1616 (C=N), 1421 (N=N), 1347 (SO2); H-NMR
[1,5-a]pyrimidin-6-
◦
ν
max
1
(500 MHz, DMSO-d6):
δ 2.26 (s, 6H, 2CH3), 6.75 (s, 1H, CH–pyrimidine), 6.94 (s, 2H, NH2), 7.20–7.32
(m, 5H, Ar–H+NH), 7.73–7.76 (m, 4H, Ar–H), 8.03, 8.13 (2br, s, 4H, 2NH2); MS (m/z), 575 (M+; 41%),
408 (100%). Anal. Calcd. for C24H22FN13O2S (575.59): C, 50.08; H, 3.85; N, 31.64. Found C, 50.27; H,
3.97; N, 31.51%.

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N-(4,6-Dimethylpyrimidin-2-yl)-4-{[2,5,7-triamino-3-(4-(trifluoromethyl) phenylazo)pyrazolo[1,5-a]pyrimidin-6-
yl]azo}benzenesulfonamide (11b): Yield: 78%; (reddish orange powder): mp >300 ◦C; IR (KBr)
max in
cm−1: 3418, 3324 (NH2), 1600 (C=N), 1418 (N=N), 1355 (SO2); 1H-NMR (500 MHz, DMSO-d6):
2.26 (s,
ν
δ
6H, 2CH3), 6.75 (s, 1H, CH–pyrimidine), 7.07 (s, 2H, NH2), 7.80 (d, 2H, Ar–H, J = 8.5 Hz), 7.85 (d, 2H,
Ar–H, J = 8.5 Hz), 8.04 (d, 2H, Ar–H, J = 7.5 Hz), 8.14 (d, 2H, ArvH, J = 7.5 Hz), 8.24 (br, 4H, 2NH2), 9.5
(br, 1H, NH); 13C-NMR (125 MHz, DMSO-d6):
δ 22.73 (CH3), 109.08, 113.25, 116.52, 120.70, 121.03,
121.36, 122.26 (Ar–C), 123.32, 125.48 (q, C–F3, J = 270 Hz), 126.20, 126.22, 126.86, 127.11, 127.64, 128.98,
139.73, 148.03 (Ar–C), 152.40, 154.50, 155.80, 156.08 (C=N). Anal. Calcd. C25H22F3N13O2S (625.60): C,
48.00; H, 3.54; N, 29.11. Found C, 48.18; H, 3.46; N, 29.08%.
3.1.11. General Procedure for the Synthesis of 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine
Derivatives 12a,b
A. Under Reflux Condition:
N-[4-(Piperidin-1-ylsulfonyl)phenyl]carbonhydrazonoyl dicyanide (3.17 g, 0.01 mol) and pyridine
(0.5 mL) were added to a solution of 4-arylazo-3,5-diaminopyrazole 4a,b (0.01 mol) in ethanol (30 mL).
The reaction mixture was heated under reflux for 7 h (monitored by TLC). After completion of the
reaction, the precipitates which formed were filtered off, dried, and recrystallized from ethanol to
afford 3-arylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine 12a,b.
B. Under Microwave Irradiation:
A
mixture of 4-arylazo-3,5-diaminopyrazole 4a,b (0.01 mol) and N-[4-(piperidin-1-
ylsulfonyl)phenyl]carbonhydrazonoyl dicyanide (3.17 g, 0.01 mol) was grinded carefully in a
porcelain mortar using a pestle and transferred to a pyrex test tube, and ethanol (4 mL) was added,
followed by pyridine (0.5 mL). The reaction mixture was heated under microwave irradiation at power
50% for 3–7 min at 140 ◦C (monitored by TLC). After completion of the reaction, the reaction mixture
was cooled to room temperature and the precipitated solid was filtered off, washed with MeOH,
and recrystallized from ethanol to afford 12a,b.
3-[(4-Fluorophenyl)azo]-6-[4-(piperidin-1-ylsulfonyl)phenylazo]-2,5,7-triamino pyrazolo[1,5-a]pyrimidine
◦
(
12a): Yield: 80%; (orange plates): mp >300 C; IR (KBr)
2835 (C-H aliphatic), 1611 (C=N), 1420 (N=N), 1371 (SO2); 1H-NMR (500 MHz, DMSO-d6):
(m, 2H, CH2), 1.54–1.55 (m, 4H, 2CH2), 2.91–2.93 (m, 4H, 2NCH2), 6.95 (s, 2H, NH2), 7.29–7.33 (m,
4H, Ar–H), 7.74–7.77 (m, 6H, Ar–H+NH2), 8.21 (br, s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6):
ν
in cm−1: 3404, 3266 (NH2), 2940,
max
δ
1.36–1.37
δ
22.85 (CH2), 24.67 (CH2), 46.58 (N–CH2), 109.17, 115.30, 115.74, 115.92, 120.88, 122.14, 122.45, 122.51,
128.38, 128.67, 134.07, 147.54, 149.99 (Ar–C), 152.62, 154.97 (C=N), 160.64 (C–F). Anal. Calcd. for
C23H24FN11O2S (537.58): C, 51.39; H, 4.50; N, 28.66. Found C, 51.32; H, 4.73; N, 28.49%.
6-[(4-Piperidin-1-ylsulfonyl)phenylazo]-3-[4-(trifluoromethyl)phenylazo]-2,5,7-triaminopyrazolo[1,5-
a]pyrimidine (12b): Yield: 81%; (reddish brown crystals): mp > 300 ◦C; IR (KBr)
ν
1
in cm−1: 3439,
max
3273 (NH2), 2941, 2855 (C-H aliphatic), 1607 (C=N), 1409 (N=N), 1361 (SO2); H-NMR (500 MHz,
DMSO-d6): 1.36 (m, 2H, CH2), 1.55 (m, 4H, 2CH2), 2.91 (m, 4H, 2N–CH2), 7.07 (s, 2H, NH2), 7.76–7.87
(m, 10H, Ar–H+NH2), 8.23 (br, s, 2H, NH2); 13C-NMR (125 MHz, DMSO-d6):
22.85 (CH2), 24.66
δ
δ
(CH2), 46.58 (N–CH2), 109.27, 116.59, 120.62, 120.97, 121.06, 122.24 (Ar–C), 123.33, 125.50, (q, C–F3,
J = 271.2 Hz), 126.22, 126.25, 126.87, 127.13, 128.38, 128.66, 134.28, 148.10 (Ar–C), 152.43, 154.90 (C=N);
MS (m/z), 588 (M+ + 1; 21%), 587 (M+; 44%), 442 (100%), 54 (99%). Anal. Calcd. for C24H24F3N11O2S
(587.59): C, 49.06; H, 4.12; N, 26.22. Found C, 49.21; H, 4.32; N, 26.41%.

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3.1.12. Synthesis of 6-(Thiazol-2-yldiazenyl)-3-(4-(trifluoro methyl)phenylazo)-2,5,7-
triaminopyrazolo[1,5-a] pyrimidine (13)
A. Under reflux Condition:
N-(Thiazol-2-yl)carbonohydrazonoyl dicyanide (1.77 g, 0.01 mol) and pyridine (0.5 mL) were
added to a solution of 3,5-diamino-4-[4-(trifluoromethyl)phenylazo]-1H-pyrazole (4b) (2.7 g, 0.01 mol)
in ethanol (30 mL). The reaction mixture was heated under reflux for 12 h (monitored by TLC).
After completion of the reaction, the precipitate which formed was filtered off, dried, and
recrystallized from ethanol to afford 6-(thiazol-2-yldiazenyl)-3-(4-(trifluoromethyl)phenylazo)-2,5,7-
triaminopyrazolo[1,5-a] pyrimidine (13).
B. Under Microwave Irradiation:
A mixture of 3,5-diamino-4-[4-(trifluoromethyl)phenylazo]-1H-pyrazole (4b) (2.7 g, 0.01 mol) and
N-(thiazol-2-yl)carbonohydrazonoyl dicyanide (1.77 g, 0.01 mol) was grinded carefully in a porcelain
mortar using a pestle and transferred to a pyrex test tube, and ethanol (4 mL) was added, followed by
pyridine (0◦.5 mL). The reaction mixture was heated under microwave irradiation at 50% power for 8
min at 140 C, monitored by TLC. After completion of the reaction, the reaction mixture was cooled to
room temperature and the precipitated solid was filtered off, washed with MeOH, and recrystallized
from ethanol to give 13.
◦
Yield: 78%; (brown powder): mp > 300 C; IR (KBr)
ν
in cm−1: 3415, 3309 (NH2), 1608 (C=N), 1419
max
(N=N); 1H-NMR (500 MHz, DMSO-d6):
7.65 (d, 1H, CH–thiazol, J = 3.5 Hz), 7.77–7.89 (m, 6H, Ar–H+NH2), 8.24 (br, s, 2H, NH2); 13C-NMR
(125 MHz, DMSO-d6): 109.10, 109.20, 116.78, 119.63, 121.15, 121.34, 122.43 (Ar–C), 123.30, 125.46 (q,
δ 7.11 (s, 2H, NH2), 7.46 (d, 1H, CH–thiazole, J = 3.5 Hz),
δ
C–F3, J = 270 Hz), 126.04, 126.24, 126.27, 142.47 (Ar–C), 152.76, 155.58, (2C=N), 167.33 (C=N thiazole);
MS (m/z), 448 (M+ + 1; 24%), 447 (M+; 100%). Anal. Calcd. for C16H12F3N11S (447.40): C, 42.95; H,
2.70; N, 34.44. Found C, 42.81; H, 2.49; N, 34.38%.
3.2. Biological Evaluation
3.2.1. In Vitro Antimicrobial Evaluation
Antimicrobial activities were carried out against highly pathogenic strains—three strains of
Gram-positive bacteria (Staphylococcus aureus, Bacillus subtilis, and Staphylococcus mutants), three strains
of Gram-negative bacteria (Enterococcus faecalis, proteus vulgaris, and Escherichia coli) using the standard
antibiotic Gentamycin (5 mg/mL) as reference drugs, and fungi (Aspergillus fumigates, Aspergillus flavus,
and Candida Albicans) using the standard antibiotic Ketoconazol (5 mg/mL). Antimicrobial activity of
the tested samples was determined by using the agar diffusion method using Mueller–Hinton agar
medium for bacteria and Sabouraud’s agar medium for fungi [19,20].
The tested microorganisms were obtained from the Regional Center for Mycology and
Biotechnology (RCMP), Al-Azhar University. The assayed collection included Gram-positive
(Staphylococcus aureus, Bacillus subtilis, and Staphylococcus mutants), Gram-negative bacteria (Enterococcus
faecalis, proteus vulgaris, and Escherichia coli) using the standard antibiotic Gentamycin (5 mg/mL) as
reference drugs, and fungi (Aspergillus fumigates, Aspergillus flavus, and Candida Albicans) using the
standard antibiotic Ketoconazole (5 mg/mL). The mean zone of inhibition in mm
±
standard deviation
beyond the diameter (6 mm) was determined using a 5 g/mL concentration of the tested compounds.
µ
The inhibitory effects of the synthetic compounds against these organisms are listed in Table 3.
3.2.2. In Vitro Cytotoxic Screening
Cell culture: Human hepatocellular carcinoma cell line (HepG-2), colorectal adenocarcinoma cell
line (HCT-116), and breast adenocarcinoma cell line (MCF-7 cell) were obtained from the American
type culture collection (ATCC). Cells were maintained in RPMI-1640 supplemented with (100 µg/mL),

Molecules 2019, 24, 1080
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penicillin (100 units/mL), and heat-inactivated fetal bovine serum (10% v/v) in a humidified, 5% (v/v)
CO2 atmosphere at 37 ◦C [24].
Cytotoxicity assessment: The cytotoxicity of different compounds was tested against human
tumor cells using Sulphorhodamine B assay (SRB). Healthy growing cells were cultured in a 96-well
tissue culture plate (3000 cells/well) for 24 h before treatment with the synthesized compounds to
allow attachment of the cells to the plate. Cells were exposed to the five different concentrations of
tested compounds (0.01, 0.1, 1, 10, and 100 µM/mL); untreated cells (control) were added. Triplicate
wells were incubated with the different concentrations for 72 h and subsequently fixed with TCA (10%
◦
w/v) for 1 h at 4 C. After several washing, cells were stained by 0.4% (w/v) SRB solution for 10 min in
a dark place. Excess stain was washed with 1% (v/v) glacial acetic acid. After drying overnight, the
SRB-stained cells were dissolved with tris–HCl and the color intensity was measured in a microplate
reader at 540 nm. The linear relation between viability percentage of each tumor cell line and extracts
concentrations were analyzed to get the IC50 (dose of the drug which reduces survival to 50%) using
Sigma Plot 12.0 software [25].
4. Conclusions
In conclusion, 4-arylazo-3,5-diaminopyrazole
for the synthesis of pyrazolopyrimidine derivatives such as 3,6-diarylazo-2,5,7-triaminopyrazolo[1,5-
a]pyrimidine, and , 3,6-Diarylazo-2,5,7-triaminopyrazolo[1,5-a]pyrimidine 10–12 and 13. The
4–6 was synthesized and used as a building block
8
9
structures of newly synthesized compounds were confirmed by spectral data (IR, 1H-NMR, 13C-NMR,
and mass spectra) and elemental analysis. Most of the newly synthesized compounds were assessed
as antimicrobial agents against a number of Gram-positive, Gram-negative, and fungal strains. Some
of the tested compounds showed excellent antimicrobial activity compared to the standard antibiotics.
The obtained data reflected that compound 2b exhibited the best antimicrobial activity against most
tested microorganisms and the rest of the compounds were moderately active or inactive for all the
microorganisms. Most of the synthesized compounds exhibited good cytotoxic activity towards the
examined three cell lines. From the obtained results, compounds 2a
,b, 4b, 5b, 8b, and 12b showed
the most potent cytotoxic profile on cancer cells (MCF-7, HepG2, and HCT-116) with IC50 values
ranging from 0.3 to 3.4
cancer cells.
µg, while the compound 8b is most effective among all of them on all types of
Supplementary Materials: The Supplementary Materials are available online.
Author Contributions: The listed authors contributed to this work as described in the following. E.H.A. carried
out the synthetic work; E.H.A., A.M.F. and H.-A.S.A. gave the concepts of the work, interpreted the results, and
prepared the manuscript. A.A.S., M.Y.A. and S.E.I.E. carried and interpreted the results of the cytotoxic activities.
All authors read and approved the final manuscript.
Funding: This research received no external funding.
Acknowledgments: The authors would like to gratefully acknowledge for King Abdulaziz City for Science and
Technology to support this research.
Conflicts of Interest: The authors declare no conflict of interest.
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©
2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).