Synthesis, molecular docking, and in silico ADME prediction of some fused pyrazolo[1,5-a]pyrimidine and pyrazole derivatives as potential antimicrobial agents

Journal of the Iranian Chemical Society

https://doi.org/10.1007/s13738-021-02319-4

ORIGINAL PAPER

Synthesis, molecular docking, and in silico ADME prediction of some

fused pyrazolo[1,5‑a]pyrimidine and pyrazole derivatives as potential

antimicrobial agents

Ashraf S. Hassan¹· Nesrin M. Morsy¹· Hassan M. Awad²· Ahmed Ragab³

Received: 29 March 2021 / Accepted: 20 June 2021

Abstract

Twenty compounds of pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole derivatives as Schif bases 16a–j have been synthe-

sized by the reaction of 5-amino-pyrazole derivatives 9a, b with 2-(arylidene)malononitriles 10a–e or various aldehydes

15a–e, respectively. The biological activity of the pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole derivatives 16a–j was

evaluated and showed a variation in antimicrobial inhibitory activity. In particular, the three pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and four pyrazole Schif bases (16c, 16d, 16 h, and 16i) have possessed a broad spectrum of activity

with the inhibition zone range from 15 to 20 mm. Also, the structure–activity relationship study was done. Furthermore,

physicochemical properties, drug-likeness model score, bioactivity scores, ADME, and toxicity properties were predicted in

silico and some products possessed acceptable and a good results. Finally, the molecular docking studies performed inside the

active site of DNA gyrase (PDB: 2XCT), the secreted aspartic protease from C. albicans (PDB: 1ZAP), and exhibited lower

binding energy with diferent types of binding mode. Besides, the electrostatic potential maps were generated to determine

the charge distribution around a molecule and therefore determine the regions that could form hydrogen bonding donors

and acceptors that confrmed the binding mode in the docking study. The results suggested that these derivatives could act

as antimicrobial agents.

* Ashraf S. Hassan

ashraf_salmoon@yahoo.com

* Ahmed Ragab

Ahmed_ragab@azhar.edu.eg

1

Organometallic and Organometalloid Chemistry

Department, National Research Centre, Dokki, Cairo 12622,

Egypt

2

Chemistry of Natural and Microbial Products Department,

Pharmaceutical and Drug Industries Research Division,

National Research Centre, Dokki, Cairo 12622, Egypt

3

Department of Chemistry, Faculty of Science (Boys),

Al-Azhar University, Nasr City, Cairo 11884, Egypt

Vol.:(0123456789)

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Journal of the Iranian Chemical Society

Graphic abstract

Besides, pyrazole Schiff bases (pyrazole-azomethine)

are known to have a vast spectrum of biological activi-

ties such as antimicrobial, antitumor, immunomodula-

tory, analgesic, DNA binding, and DHFR/DNA gyrase

inhibitors [12–18]. In 2013, Malladi and co-workers have

described a synthesis of compound triazole-pyrazole

Schiff base 6 which exhibited promising antibacterial

activity against Staphylococcus aureus [19]. Recently, in

2020, Hassan et al., prepared two furan-pyrazole Schiff

bases 7 and 8 as Nitrofurantoin^®analogues which exhib-

ited antibacterial activities against Escherichia coli and

Salmonella typhimurium bacteria [20] (Fig. 1).

Introduction

The important tasks of the researchers in the medical,

pharmaceutical, and chemical felds are the design and

preparation of bioactive compounds to overcome some

health problems such as cancer and bacterial infectious

diseases. Therefore, there is a growing interest in the prep-

aration of pyrazolo[1,5-a]pyrimidines derivatives due to

their biological applications as promising antimicrobial,

anticancer, antitubercular, and enzyme inhibitors [1–6].

For example, pyrazolo[1,5-a]pyrimidine-3-carbonitr ile

derivative 1 was synthesized by Attia et al., as a promis-

ing anti-proliferative agent against the MCF-7 cell line [7].

The two water-soluble pyrazolo[1,5-a]pyrimidine deriva-

tives (2 and 3) were synthesized and characterized with

antibacterial activity against Escherichia coli [8]. Also,

from our previous studies, the two pyrazolo[1,5-a ]pyrimi-

dine derivatives {5,7-dimethylpyrazolo[1,5-a]pyrimidine

derivative 4 and 7-phenylpyrazolo[1,5-a]pyrimidine deriv-

ative 5} were prepared and displayed a promising anti-

microbial agent [9 ] and antitumor agent against HepG-2

cells [10], respectively (Fig. 1). Moreover, the pyrazolopy-

rimidine structure is present in drugs such as Dinaciclib,

Anagliptin, and Ocinaplon [11].

Because of the biological importance of p yrazolo[1,5-

a]pyrimidines, pyrazole Schif bases, and in continua-

tion of our program for synthesis bioactive compounds

[21–40]. In this work, we have synthesized 5-amino-

N -aryl-1H-pyrazole-4-carboxamides 9a, b [41] as start-

ing materials for the preparation of new 5-aryl-7-amino-

pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schif

bases 16a–j for evaluation of their antimicrobial properties

as well as the structure–activity relationship (SAR), the

molecular docking, the electrostatic potential maps (MEP),

and in silico properties prediction were studied (Fig. 2).

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Journal of the Iranian Chemical Society

Fig. 1 Biological applications of pyrazolopyrimidines 1–5 and pyrazole Schif bases 6–8

A series of 7-amino-pyrazolo[1,5-a]pyrimidines 14a–j

Result and discussion

were prepared via the reaction of 5-amino-N-aryl-1H-pyra-

zole-4-carboxamides 9a, b with 2-(arylidene)malononi-

triles 10a–e in refuxing ethanol (Scheme 1). This reaction

involves nucleophilic addition of amino group of 9a, b

to double bond of 2-(arylidene)malononitriles 10a–e to

produce the intermediate 5-((2,2-dicyano-1-phenylethyl)

amino)-1H-pyrazole derivative 11a–j. Furthermore, the

cyclic imino group in intermediate 1H-pyrazole deriva-

tive 11a–j acts as nucleophile to cyano group to obtain

7-imino-pyrazolo[1,5-a]pyrimidine-3-carboxamide

Chemistry

The target compounds, pyrazolo[1,5-a]pyrimidines 14a–j

and pyrazole Schif bases 16a–j, are shown in Schemes 1

and 2. The starting materials, 5-amino-pyrazole deriva-

tives 9a, b and 2-(arylidene)malononitriles 10a–e, were

prepared according to previously reported methods [42,

43] and are described in Scheme 3.

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Fig. 2 The target compounds (14a–j and 16a–j) and their studies

derivatives 12a–j that underwent proton shift and oxida-

tion to aford the corresponding 7-amino-pyrazolo[1,5-a]

pyrimidine derivatives 14a–j.

exchangeable singlet signals at δ 9.23 and 10.14 ppm cor-

responding to two NH groups.

The three aromatic rings (Figure S1, Supplementary

Material) were interpreted as the following:

The structure of 7-amino-pyrazolo[1,5-a]pyrimi-

dines structures 14a–j was confrmed by spectral data

and elemental analysis. The IR spectrum of 7-amino-

pyrazolo[1,5-a]pyrimidine derivative 14a was character-

ized by appearing new band for C≡N group at ν 2213 cm⁻¹

and two bands at ν 3428 and 3304 cm⁻¹for NH₂and

2NH groups. Also, the C=O amide group appeared at ν

1641 cm⁻¹. The mass spectrum of pyrazolo[1,5-a]pyrimi-

dine derivative 14a was exhibited a molecular ion pack at

m/z 489 with intensity 17.65% corresponding to the for-

mula C₂₈H₂₃N₇O₂(489.53) and the base peak at m/z 396

(100%). The ¹H NMR (δ/ppm) spectrum of 14a exhibited

two signals at δ 2.43 and 3.74 ppm assigned to the protons

of CH₃and OCH₃groups, respectively. Besides, the NH₂

group appeared as a broad signal at δ 9.00 ppm, and two

(i) Phenyl ring (fve protons) appeared as two triplets

at δ 7.09 ppm (1H, J=7.5 Hz) and δ 7.37 ppm (2H,

J = 7.8 Hz) and one doublet at δ 7.60 ppm (2H,

J=7.7 Hz).

(ii) 4-Methoxyphenyl ring (4 protons, AB-system)

appeared as two doublets at δ 6.90 (2H) and 7.84

(2H) ppm (each with J (coupling constant)=9.0 Hz).

(iii) 4-Methylphenyl ring (4 protons, AB-system) appeared

as two doublets at δ 7.44 (2H) and 7.90 (2H) ppm

(each with J (coupling constant)=8.2 Hz).

Additionally, the ¹³C NMR spectrum of 14a exhibited

characterized signals of CH₃and –OCH₃carbon at δ 21.19

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Scheme 1 Synthesis of pyrazolo[1,5-a]pyrimidines 14a–j

and 55.26 ppm, respectively. The rest of carbon atoms (26

carbon) of 7-amino-pyrazolo[1,5-a]pyrimidine derivative

14a have appeared ranged between δ 74.88–145.70 ppm

as well as signals at δ 149.49, 153.98, 156.32, 161.47, and

162.29 ppm corresponding to two C=N, carbonyl group,

carbon attached to methoxy (C–OMe), and carbon attached

to an amino group (C-NH₂), respectively.

at δ 8.97 ppm, and two signals at δ 9.11 and 10.04 ppm

refer to the protons of the amino group (NH₂) as well as two

NH groups, respectively. The ¹³C NMR spectrum of 14j

exhibited characterized signals of three methoxy carbons

at δ 55.12, 55.68, and 55.93 ppm. The rest of carbon atoms

(26 carbon) of 14j have appeared at δ 78.02, 88.84, 104.95,

113.21, 114.13, 115.17, 117.23, 118.96, 120.38, 126.91,

128.90, 133.25, 137.20, 145.72, 148.51, 150.65, 153.07,

153.91, 156.07, 159.97, 159.99, and 162.09 ppm. Also, the

structure of 7-amino-pyrazolo[1,5-a]pyrimidine derivatives

14a–j and reaction mechanism pathway was proved from

our previous study through X-ray single-crystal analysis of

analogues [44].

Moreover, the 5-(2,5-dimethoxyphenyl)-pyrazolo[1,5-a]

pyrimidine-3-carboxamide derivative 14j was another exam-

ple of the ¹H NMR spectra, exhibited three singlet signals

at δ 3.70, 3.75, and 3.78 ppm for the three methoxy groups.

The aromatics protons appeared at δ 6.86 (d, 2H, J=9.5 Hz,

p-methoxyphenyl ring), 7.10–7.17 (m, 3H, 2, 5-dimethoxy-

phenyl ring), 7.34 (d, 2H, J=9.2 Hz, p-chlorophenyl ring),

7.48 (d, 2H, J = 9.2 Hz, p-chlorophenyl ring), and 7.78

(d, 2H, J = 9.3 Hz, p-methoxyphenyl ring). Further, three

exchangeable singlet signals appeared as the broad peak

Schif bases 16a–j were prepared via the reaction of

5-amino-pyrazoles 9a, b with various aldehydes 15a–e in

refuxing absolute ethanol (Scheme 2). Schif bases structure

16a–j was confrmed by spectral analysis. The IR spectrum

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Scheme 2 Synthesis of Schif bases 16a–j

Scheme 3 Synthesis of starting materials (9a, b and 10a–e)

of azomethine derivative 16d displayed bands at ν 3431,

3235, 1635, and 1615 cm⁻¹characteristic for 3NH, carbonyl

(C=O), and imino (C=N) groups, respectively. The ¹H NMR

(δ/ppm) spectrum of 5-(4-chloro-3-nitrobenzylideneamino)-

1H-pyrazole-4-carboxamide derivative 16d exhibited one

signal at δ 3.70 ppm for the methoxy group. The aromatic

protons have appeared at δ 6.89 (d, 2H, J=8.3 Hz, p-meth-

oxyphenyl ring), 7.06 (t, 1H, J = 7.3 Hz, phenyl ring),

7.31–7.34 (m, 4H, phenyl ring), 7.67 (d, 2H, J = 7.9 Hz,

p-methoxyphenyl ring), 8.00 (d, 1H, J=8.3, 4-chloro-3-ni-

trophenyl ring), 8.30 (d, 1H, J=8.3, 4-chloro-3-nitrophenyl

ring), and 8.67 (s, 1H, 4-chloro-3-nitrophenyl ring). The

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Journal of the Iranian Chemical Society

methine proton (–CH=N–) has appeared at δ 8.76 ppm.

Besides, the three exchangeable signals appeared at δ 9.13,

9.79, and 12.80 ppm related to three NH protons.

comparable antibacterial activity of the Tetracycline drug

(IZ = 18 mm). Both the pyrazolopyrimidine derivatives

(14c and 14g–i) and pyrazole Schif bases (16c–h and 16j)

showed a good inhibition with range=15–17 mm.

Additionally, the IR spectrum of pyrazole Schif base

derivative 16f was characterized by bands for 3NH, C=O,

C=N, and C=C groups at ν 3434, 3229, 1656, 1612, and

1587 cm⁻¹, respectively. The ¹H NMR (δ/ppm) spectrum of

pyrazole derivative 16f exhibited two signals at δ 3.73 and

3.87 ppm for the protons of two methoxy groups. Besides,

the proton of –CH=N– function appeared at δ 8.66 ppm and

the protons of the three NH appeared as single signals at δ

9.03, 10.02, and 12.78 ppm. The spectrum has exhibited the

protons of the three aromatic rings (Figure S2, Supplemen-

tary Material) as.

In the case of Escherichia coli (G−) bacteria: all the

pyrazolo[1,5-a] pyrimidines 14a–j and pyrazole Schif bases

16a–j (IZ=13 to 16 mm) presented from good to an excel-

lent inhibition compared to the Novobiocin (IZ=16 mm).

Aspergillus niger (Filamentous fungi): Pyrazolopyrimi-

dine 14f and four pyrazole Schif bases (16c, 16d, 16h, and

16i) revealed IZ from (18 mm) to (20 mm) and confrmed

a robust inhibition impact, while, four pyrazolopyrimidines

(14d, 14e, 14g, and 14i) and three Schif bases (16a, 16e,

and 16j) with IZ=15 mm showed a reasonable inhibitory

activity compared to Clotrimazole (IZ=23 mm) as a posi-

tive control. It is worth mentioning that Aspergillus niger is

one of the fungal pathogens that can afect the respiratory

tract. A. niger is a causative agent of pulmonary diseases

inclusive of aspergillosis, bronchial asthma, and acute aller-

gic alveolitis. The fungus colonizes ancient tuberculosis or

bronchi static cavities, in which its varieties a large colony

(aspergilloma); or it can also without a doubt invade the lung

tissue to produce hemorrhagic and necrotizing pneumonia

[47].

(i) Two doublets appeared at δ 6.90 (2H, J=8.9 Hz) and

7.69 (2H, J=8.9 Hz) for the p-methoxyphenyl ring.

(ii) One doublet at δ 7.40 (4H) for the p-chlorophenyl

ring with J=8.9 Hz.

(iii) m-Methoxyphenyl ring; one proton as a doublet–dou-

blet (dd) at δ 7.23 (J=8.2 & 1.8 Hz), one proton as

a triplet at δ 7.53 (J=7.9 Hz), single signal at δ 7.59

for one proton, and one doublet for one proton at δ

7.62 (J=2.4 Hz).

In the case of Candida albicans (Yeast): the pyrazo-

lopyrimidines (14b, 14d, and 14f) and Schif base 16i (IZ

rang=17–20 mm) showed a good inhibition efect. Moreo-

ver, the pyrazolopyrimidines (14c, 14e, 14g, and 14i) and

Schif bases (16b, 16c, 16d, 16e, 16g, and 16h) showed

a moderate inhibition with inhibition zone (IZ) = 15 mm.

Further, the pyrazolopyrimidine 14a (IZ=13 mm) and two

Schif bases 16a (IZ=13 mm) and 16j (IZ=12 mm) showed

a weak efect in contrast to the antibacterial standard antibi-

otic Cyclohexamide (IZ=32 mm).

The ¹³C NMR spectrum of 16f exhibited characterized

signals of two methoxy carbons at δ 55.19 and 55.23 ppm.

The rest of 23 carbon atoms appeared at δ 92.61, 112.24,

114.39, 119.22, 119.53, 120.41, 122.48, 126.69, 128.34,

128.73, 130.28, 133.79, 136.17, 137.46, 151.21, 154.04,

159.74, 162.03, and 162.65 ppm.

Antimicrobial evaluation

From the above, the three pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and four pyrazole Schif bases (16c, 16d,

16h, and 16i) proved to be potent against the four microbes

tested, possessed a broad spectrum of activity, and in several

cases, their activities exceeded the reference drugs used.

Pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schif bases

16a–j were evaluated in vitro for their antimicrobial activity

against four microbes using the agar di fusion method [45,

46 ], and the results are listed in Table 1 and represented in

Fig. 3.

From the results obtained in Table 1 and Fig. 3, it was

found that most of the pyrazolo[1,5-a]pyrimidines 14a–j and

pyrazole Schif bases 16a–j have a reasonable antimicrobial

undertaking impact against most of the tested pathogens in

this study. Besides, most of the pyrazolopyrimidines 14a–j

and pyrazole Schif bases 16a–j confrmed a diverse inhibi-

tory efect against all pathogenic microorganisms used.

In the case of Bacillus subtilis (G+) bacteria: the

three pyrazolopyrimidines [14b, 14e (inhibition zone

(IZ) = 20 mm), and 14j (IZ = 21 mm)] and the pyrazole

Schif base 16i (IZ=19 mm) confrmed a strong inhibition

efect more than the antibacterial standard used (Tetracy-

cline, IZ=18 mm). Furthermore, pyrazolopyrimidine 14d

and the pyrazole Schif bases 16a (IZ = 18 mm) showed

Structure activity relationship study

Depending on the structure represented on Schemes 1, 3,

and Fig. 4, our synthesis strategy depends on the prepar a-

tion of two 2-cyano-N-(aryl)acetamide, and the aryl repre-

sented as phenyl or 4-chlorophenyl. The cyanoacetanilide

derivatives reacted with 4-methoxyphenyl isothiocyante and

fnally refuxed with hydrazine hydrate to produce 5-amino-

pyrazole derivatives 9a, b with two variable aryl groups.

The 5-amino-pyrazole derivatives 9a, b obtained used as

key starting materials to form pyrazolo[1,5-a]pyrimidine

derivatives 14a–j and azomethine pyrazole derivatives

16a–j. Generally, most of the target compounds 14a–j and

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Table 1 Antimicrobial activities represented by the mean of Inhibition Zone (IZ) in (mm) of pyrazolo[1,5-a]pyrimidines 14a–j, pyrazole Schif

bases 16a–j, and standard antibiotics using the agar difusion method

Compounds

Ar

Ar₁

Ar₂

Bacteria

Fungi

(G⁺)

(G⁻)

Filamentous

A. niger

NRRL-3

Unicellular

C. albicans

ATCC-10231

B. subtilis

ATCC-6633

E. coli

ATCC-25922

14a

14b

14c

14d

14e

14f

14g

14h

14i

14j

16a

16b

16c

16d

16e

16f

16g

16h

16i

16j

A

Ph

4-CH₃OC₆H₄

–

4-CH₃C₆H₄

3-CH₃C₆H₄

3-CH₃OC₆H₄

3-ClC₆H₄

2,5-di-CH₃OC₆H₃

4-CH₃C₆H₄

3-CH₃C₆H₄

3-CH₃OC₆H₄

3-ClC₆H₄

2,5-di-CH₃OC₆H₃

3-CH₃OC₆H₄

4-(CH₃)₂N C₆H₄

2,5-di-CH₃OC₆H₃

4-Cl-3-NO₂-C₆H₃

Thiophen-2-yl

3-CH₃OC₆H₄

4-(CH₃)₂N C₆H₄

2,5-di-CH₃OC₆H₃

4-Cl-3-NO₂-C₆H₃

Thiophen-2-yl

–

12

20

15

18

20

13

16

15

16

21

18

12

15

17

15

16

19

16

18

–

14

16

14

15

16

14

15

16

15

13

14

16

15

14

15

14

–

0

13

17

15

17

15

18

15

0

15

18

15

0

15

0

15

0

20

15

0

Ph

4-ClC₆H₄

Ph

4-ClC₆H₄

–

15

0

13

15

0

15

20

12

–

0

20

15

–

23

–

B

C

D

–

16

–

32

–

Antibacterial Standard; A=Tetracycline (30 µg); B=Novobiocine (30 µg), Antifungal Standard; C=Clotrimazole (50 µg); D=Cyclohexamide

(50 µg)

16a–j showed moderate to favorable activity against the

tested pathogens.

16–21 mm, while 5-(3-CH₃-C₆H₄)pyrazolo[1,5-a]pyrimi-

dine-3-carboxamide derivative 14g revealed slight decrease

with IZs 16 mm, and 15 mm against B. subtilis and E. Coli,

respectively. Additionally, it was found that the phenyl ring

in pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative 14d

had higher activity than the corresponding member incorpo-

rating chloro group as N-(4-chlorophenyl)-pyrazolo[1,5-a]

pyrimidine-3-carboxamide derivative 14j against B. subtilis

strain. Moreover, replacement N-phenyl by N-(4-chlorophe-

nyl) of 3-carboxamide group in pyrazolo[1,5-a]pyrimidine

derivatives 14h and 14j demonstrated no activity against

fungal strains. Besides, addition of the electron-withdrawn

group as chlorine atom in pyrazolo[1,5-a]pyrimidine-

3-carboxamide derivatives 14f and 14g displayed increased

the activity with IZ values from zero to 18 and 15 mm,

respectively.

Firstly, the 7-amino-6-cyano-2-((4-methoxyphenyl)

amino)-N-phenyl-5-(aryl)pyrazolo[1,5-a]pyrimidine-3-car-

boxamide 14a–e displayed good antibacterial results, moder-

ate antifungal activity, and the activity depends on the aryl

group at position fve and aromatic amide at position three

of pyrazolo[1,5-a]pyrimidine derivatives 14. The presence

of a dimethoxy group at positions two and fve in the aryl

group and meta methyl group in pyrazolo[1,5-a]pyrimidine

14e & 14b exhibited antibacterial activity with IZ ranged

between 16–20 mm compared with Tetracycline 18 mm and

Novobiocin 16 mm. Additionally, these two derivatives dis-

played moderate antifungal activity with IZs ranged between

15–17 mm in comparison with Clotrimazole (IZ=23 mm)

and Cycloheximide (IZ = 32 mm). Further, replacement

N-phenyl by N-(4-chlorophenyl) exhibited equipotent in

case of 5-(2,5-di-CH₃O-C₆H₃)pyrazolo[1,5-a]pyrimidine-

3-carboxamide derivative 14j with IZs ranged between

Similarly, the azomethine of 5-amino-pyrazole deriva-

tives 16a–j showed variation in activity related to the dif-

ferent substituents in both two-aryl groups at positions

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Fig. 3 Showed the antimicrobial activities (Inhibition Zone (IZ) in mm) of pyrazolo[1,5-a]pyrimidines 14a–j, pyrazole Schif bases 16a–j, and

standard antibiotics a bacteria, b fungi

three and fve in pyrazole derivatives 16a–j. Regarding the

series of 5-((substituted benzylidene)amino)-1H-pyrazole-

4-carboxamide derivatives 16a–j, changing N-(phenyl) or

N-(4-chlorophenyl) has little efect on antimicrobial activ-

ity. Furthermore, introducing more electron-withdrawing

group as N-(4-chlorophenyl) to pyrazole derivatives 16a–j

increase activity against B. subtilis from 12–18 mm for pyra-

zoles 16a–e to 15–19 mm for pyrazoles 16f–j. Additionally,

changing of 5-((substituted benzylidene)amino) by hetero-

cyclic aldehyde as 5-((thiophen-2-ylmethylene)amino) in

1H-pyrazole-4-carboxamide derivatives 16a–d and 16f–i not

cause signifcant enhancement in antimicrobial activity. The

most active azomethine pyrazole derivatives 16a–j among

the synthesized derivatives that having 5-((4-chloro-3-ni-

trobenzylidene)amino) and N-(4-chlorophenyl)-4-carbox-

amide derivative is pyrazole derivative 16i demonstrated

antimicrobial activity IZs ranging between 15–19 mm and

antifungal activity IZs 20 mm. Unfortunately, the pyrazole

derivatives 16b, 16f, and 16g are inactive against A. Niger

except for moderate activity for other pyrazole derivatives.

While the pyrazole derivative 16f showed no activity against

C. albicans.

Finally, in the general presence of the chloro atom

in amide as N-(4-chlorophenyl) in position three in

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Fig. 4 Illustrate the structure

of diferent substituent in both

pyrazolo[1,5-a]pyrimidine

derivatives 14a–j and pyrazole-

azomethine derivatives 16a–j

pyrazolo[1,5-a]pyrimidine derivatives 14a–j and position

four in pyrazole derivatives 16a–j causes increases the

activity with signifcant results against B. subtilis, while in

the case of E. Coli nearly results were obtained. Besides,

N-(phenyl)-1H-pyrazolo[1,5-a]pyrimidin-3-carboxamide

derivatives 14a–j and N-(phenyl)-1H-pyrazole-4-carbox-

amide derivatives 16a–j revealed abroad antifungal activ-

ity. Moreover, it can be concluded that the pyrazolo[1,5-a]

pyrimidine derivatives 14a–j more sensitive to antibacterial

activity while 5-((substituted benzylidene)amino)-1H-pyra-

zole-4-carboxamide derivatives 16a–j displayed better anti-

fungal activity.

each of Lipinski’s rule of fve and Veber’s rule by the viola-

tion in molecular weight and TPSA.

The rule for the drug-likeness model score estimation

is based on positive and negative values, when the drug-

likeness score was positive value; the compound was like

a drug. But, if the drug-likeness score was negative value;

the compound was non-drug [51].

Accordingly, all the tested compounds except 16c

showed positive values in the range from 0.05 to 0.44;

therefore, these compounds may be considered to be drug-

like. Figure 5 represents the drug-likeness model score of

the two pyrazolo[1,5-a]pyrimidines 14b and 14j.

The bioactivity scores prediction

In silico studies

The bioactivity scores of the pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and Schif bases (16c, 16d, 16h, and

16i) toward G protein-coupled receptor (GPCR) ligand,

ion channel modulator, a kinase inhibitor, nuclear recep-

tor ligand, protease inhibitors, and enzyme inhibitor were

predicted by using the Molinspiration bioactivity score

v2018.03 w eb (https://www.molinspiration.com/cgi-bin/

properties) [52 ]. The predicted results are written in

Table 3 .

Physicochemical properties and drug‑likeness

model score

The physicochemical properties and drug-likeness (Lipinski

and Veber rules) of the pyrazolo[1,5-a]pyrimidines (14b,

14e, and 14j) and Schif bases (16c, 16d, 16h, and 16i ) were

predicted by using the SwissAD ME web (http://swissadme.

ch/index.php#unde f ned) [48, 49]. Also, the drug-likeness

model score was predicte d by u sing the Molsoft web (https://

www.molsoft.com/mprop/) [50]. The predicted results are

listed in Table 2 .

The rule for the bioactivity scores estimation is the fol-

lowing: when the bioactivity score was more than 0.00; the

compound was considered active. While if the bioactivity

score in the range between −0.50 and 0.00; the compound

was moderately active. But if the bioactivity score was less

than −0.50, the compound was inactive.

From Table 2, the pyrazolo[1,5-a]pyrimidine 14b and

Schiff bases (16c, 16d, 16h, and 16i) agreement with

Lipinski’s rule of fve and with Veber’s rule. While the two

pyrazolo[1,5-a]pyrimidines (14e and 14j) do not match with

The following observations are concluded from Table 3:

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Table 2 Physicochemical properties and drug-likeness model score of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and pyrazole Schif

bases (16c, 16d, 16h, and 16i)

Physicochemical proper-

ties and drug-likeness

score

Pyrazolo[1,5-a]pyrimidines

14b 14e

Pyrazole Schif bases

16c 16d 16h

14j

16i

Molecular weight≤500

#H-bond acceptors≤10

#H-bond donors≤5

489.53 535.55

570.00

7

3

2.92

471.51 490.90 505.95

525.34

6

3

3.75

5

3

7

3

6

3

6

3

6

3

Lipophilicity

3.26

2.46

3.03

3.28

3.50

MLOGP≤4.15

Violations Lipinski’s rule Yes; 0 No; 2

No; 2

Yes; 0 Yes; 0 Yes; 1: MW>500 Yes; 1: MW>500

(Yes; 0 or 1)

MW>500, N or O>10 MW>500, N or O>10

#Rotatable bonds≤10

7

9

10

9

10

9

TPSA^a≤140

130.36 148.82

Yes; 0 No; 1:

148.82

No; 1: TPSA>140

109.86 137.22 109.86

Yes; 0 Yes; 0 Yes; 0

137.22

Yes; 0

Violations Veber’s rule

(Yes; 0)

TPSA>140

0.06

Drug-likeness model score 0.38

0.44

-0.23 0.05

0.22

0.15

(MolSoft)

^aTPSA Topological polar surface area

Fig. 5 Displayed the drug-likeness model score plotting of two pyrazolo[1,5-a]pyrimidines 14b and 14j. The green curve represents non-drug,

and the blue curve represents drug

Table 3 The bioactivity

The bioactivity scores

Pyrazolo[1,5-a]pyrimidines

Pyrazole Schif bases

scores prediction of the

pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and

pyrazole Schif bases (16c, 16d,

16h, and 16i)

14b

14e

14j

16c

16d

16h

16i

GPCR ligand^a

Ion channel modulator

Kinase inhibitor

Nuclear receptor ligand

Protease inhibitor

Enzyme inhibitor

−0.11

−0.32

0.45

−0.61

−0.42

−0.23

−0.10

−0.44

0.36

−0.62

−0.39

−0.26

−0.12

−0.52

0.30

−0.67

−0.41

−0.32

−0.61

0.07

−0.60

−0.37

−0.28

−0.44

−0.60

−0.02

−0.77

−0.56

−0.38

−0.31

−0.62

0.06

−0.59

−0.39

−0.29

−0.43

−0.61

−0.03

−0.75

−0.55

−0.37

^aG protein-coupled receptor (GPCR) ligand

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Journal of the Iranian Chemical Society

•

G protein-coupled receptor (GPCR) ligand: all the

pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and

Schif bases (16c, 16d, 16h, and 16i) were found to be

moderately active with the bioactivity scores in the range

from −0.10 to −0.44.

16i) were found to be inactive with the bioactivity scores

-0.56 and -0.55, respectively.

•

Enzyme inhibitor: all the pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and Schif bases (16c, 16d, 16h,

and 16i) were found to be moderately active (range from

−0.23 to −0.38).

Ion channel modulator: the two pyrazolo[1,5-a]pyrimi-

dines 14b and 14e were found to be moderately active

with the bioactivity scores − 0.32 and − 0.44, respec-

tively, while the other compounds were found to be inac-

tive.

From the above, the pyrazolo[1,5-a]pyrimidine 14b

almost shows the highest bioactivity score compared to the

other compounds.

Kinase inhibitor: the three pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and the two Schif bases (16c and

16h) were found to be active with bioactivity scores in

the range from 0.06 to 0.045. While the two Schif bases

(16d and 16i) were found to be moderately active with

the bioactivity scores −0.02 and −0.03, respectively.

Nuclear receptor inhibitor: all the pyrazolo[1,5-a]pyri-

midines (14b, 14e, and 14j) and Schif bases (16c, 16d,

16h, and 16i) were found to be inactive with the bioac-

tivity scores less than −0.50 (the range from −0.59 to

−0.77).

ADME and toxicity properties prediction

ADME properties and toxicity properties prediction of the

pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and Schif

bases (16c, 16d, 16h, and 16i ) were predicted b y using the

Pre-ADMET web (https://preadmet.bmdrc.kr/) [53]. The

predicted results are written in Table 4 .

•

The following observations are concluded from Table 4:

•

Cytochrome P450 isoforms determine the pharmacoki-

netic properties, bioactivation, and safety persistence of

drugs. In the case of all the pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and Schif bases (16c, 16d, 16h, and

16i) are an inhibitor of the two enzymes (CYP2C9 and

Protease inhibitor: the three pyrazolo[1,5-a]pyrimidines

(14b, 14e, and 14j) and the two Schif bases (16c and

16h) were found to be moderately active (range from

− 0.37 to − 0.42). But, the two Schif bases (16d and

Table 4 ADME and toxicity properties prediction of the pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and pyrazole Schif bases (16c, 16d,

16h, and 16i)

Pre-ADMET web

ADME and toxicity

properties prediction

Pyrazolo[1,5-a]pyrimidines

Pyrazole-Schiff bases

14b

14e

14j

16c

16d

16h

16i

CYP2C19 inhibition

CYP2C9 inhibition

CYP2D6 inhibition

CYP3A4 inhibition

P-glycoprotein inhibition

Plasma Protein Binding

Ames test (Mutagenicity)

Carcinogenicity (Mouse)

Carcinogenicity (Rat)

hERG inhibition

91.17

91.23

85.27

86.22

94.18

83.91

98.23

= Inhibitor, Non-mutagen, Negative carcinogenicity, and low risk

= Non-inhibitor, Mutagen, Positive carcinogenicity, and high risk

= Ambiguous,

= Medium risk

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Journal of the Iranian Chemical Society

CYP3A4). But all the compounds are non-inhibitor of

the two enzymes (CYP2C19 and CYP2D6).

active derivatives inside both DNA gyrase for bacterial

and secreted aspartic protease for fungal promising deriva-

tives using Molecular Operating Environmental (MOE)

software version 2008.10. The docking result is repre-

sented in Table 5 and Fig. 6a–d.

•

P-glycoprotein (P-gp) has two functions the frst is a bar-

rier for delivering drugs properly and it is responsible for

extruding toxins. All the compounds (14b, 14e, 14j, 16c,

16d, 16h, and 16i) were found to be efective.

The rule for the plasma protein binding (PPB) estimation

is the following: when the value was less than 90%; the

compound was low activity. While if the value was more

than or equal to 90%; the compound was high activity.

The two pyrazolo[1,5-a]pyrimidines (14b and 14e) and

two Schif bases (16d and 16i) had plasma protein bind-

ing (PPB) more than 90% (range from 91.17 to 98.23%,

highly active), which afects the excretion of the drugs

and its efciency.

Study the most active derivatives against bacterial

strains inside DNA gyrase

The most active derivatives 14b, 14e, 14j, and 16i against

Bacillus subtilis and Escherichia coli were docked inside

the active site of S. auras DNA gyrase (PDB: 2XCT)

[57, 58]. The co-crystallized ligand (Ciprofloxacin)

redoc ked inside S. aureus Gyrase’s active site revealed

binding energy S = − 9.87 kcal mol⁻¹. The ligand formed

one hydrogen bond donor between Tyr580 and nitrogen

of piperazine ring as well as hydrogen bond acceptor

between His1081 and oxygen of carboxylate group with

bond length 2.35, 2.30 Å, respectively. The most promis-

ing pyrazolo[1,5-a]pyrimidine-3-carboxamide derivative

14j with inhibition zone (IZ) ranged between 16-21 mm

showed binding energy S = − 25.35 kcal mol⁻¹, through

two hydrogen bond and one arene–cation interaction

between His1061 and pyrazole ring. The residue Asp512

formed a side-chain hydrogen bond donor with the amino

group of pyrimidine as well as a side-chain hydrogen bond

acceptor between Ser1028 and oxygen of methoxy group

with bond length and strength 2.83 Å (11%), 2.99 Å

(38%), respectively Fig. 6a.

•

Ames test (mutagenicity), the three pyrazolo[1,5-a]pyri-

midines (14b, 14e, and 14j) and three Schif bases (16c,

16d, and 16h) were predicted to be mutagens.

Carcinogenicity (mouse) test and the two Schif bases

(16d and 16i) were predicted as positive, while the three

pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and the

two Schif bases (16c and 16h) presented negatively.

Carcinogenicity (rat) test, the pyrazolo[1,5-a]pyrimidine

14j, and the three Schif bases (16d, 16h, and 16i) were

predicted as negative.

•

In the case of the hERG inhibition, the two compounds

14e and 16c showed high risk and the three compounds

14j, 16h, and 16i presented a medium risk. While com-

pounds 14b and 16d presented an ambiguous risk.

From the above, all compounds are an inhibitor of the

Furthermore, N-phenyl-5-(3-methylphenyl)pyrazolo[1,5-

a]pyrimidine-3-carboxamide derivative 14b exhibited bind-

ing afnity S =−23.05 kcal mol⁻¹, through two hydrogen

bonds from a side-chain. These two bonds created between

the Ser1028 with the cyano in pyrimidine ring (H-acceptor),

Ser1028 with the amino of pyrimidine (H-donor) with bond

length 2.89 Å, and 3.05 Å. Besides, arene–cation inter-

action between His1061 and pyrazole ring Fig. 6b. Simi-

larly, N-phenyl-5-(2,5-dimethoxyphenyl)-pyrazolo[1,5-a]

pyrimidine-3-carboxamide derivative 14e displayed bind-

ing energy S= − 22.76 kcal mol⁻¹, by one hydrogen bond

side-chain acceptor between Ser1028 and cyano group

attached to pyrimidine ring as well as one arene–cation

interaction between His1061 and pyrazole ring. Moreover,

N-(4-chlorophenyl)-1H-pyrazole-4-carboxamide 16i dis-

played one hydrogen bond donor between Asp510 and NH

of pyrazole ring with bond length 2.76 Å and strength 45%.

Finally, the active derivatives 14b, 14e, 14j, and 16i

showed a good binding afnity inside the pocket sites of

DNA gyrase ranged between −25.35 to −20.18 kcal mol⁻¹

with diferent binding modes and therefore suggested that

these derivatives act as gyrase inhibitors.

two cytochrome P450 isoforms (CYP2C9 and CYP3A4

enzymes) and P-glycoprotein (P-gp). Besides, some com-

pounds showed highly active toward plasma protein binding

(PPB), were predicted as negative against Carcinogenicity

(mouse and rat) test, and presented medium risk in the case

of the hERG inhibition.

Computational study of the binding mode

One of the most important tools in structure-based drug

discovery is molecular docking simulation. It can describe

newly designed derivatives’ behavior in the binding site of

target proteins [54]. Docking study plays an important role

and considers one of the most applied simulated screen-

ing methods due to its ability to predict and get insights

into a protein–ligand complex structure, which is useful

information for the successful development of drug target-

ing and lead optimization [55]. The preferring structure

selected from one to another in the docking study related

to the highest binding afnity between the ligand and the

protein [56]. Our study involved simulation of the most

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Table 5 Molecular modeling study of the most promising derivatives inside the active sites of S. aureus DNA gyrase (PDB: 2XCT) and secreted

aspartic protease from c. albicans (PDB: IZAP)

Cpd. No S (kcal mol⁻¹) Residue Interacting groups

Distance (Å) Strength (%) Hydrophobic interaction

Docking inside the S. aureus DNA gyrase (PDB: 2XCT)

14b

−23.05

Ser1028 CN of pyrimidine

Ser1028 NH2 of pyrimidine

His1061 Pyrazole ring

2.89

3.05

–

57

15

–

Arg1033, Val511, Ala1032, Asp510, Asp512, Glu435,

Tyr1150, Pro1080, Tyr580, Lys581

14e

14j

−22.76

−25.35

Ser1028 CN OF Pyrimidine

His1061 Pyrazole ring

Asp512 NH2 of pyrimidine

Ser1028 Oxygen of methoxy

His1061 Pyrazole ring

2.88

–

2.59

69

–

50

Arg1033, Val511, Asp512, Asp510, Pro1080, Lys581,

Tyr1150, Tyr580, Gly582, Lys581

Asp508, Asp510, Lys581,

Pro1080, Gly582, Glu585

16i

−20.18

−9.87

Asp510 NH of pyrazole

2.76

45

Ser438, Asp437, Gly436,

Glu435, Gly582, Lys581, Gly545, Tyr580, Tyr1150

Cip

His1081 Oxygen of carboxylate 2.30

Tyr580 Nitrogen of piprazine 2.35

37

48

Ser1028, Arg1033, Val511, Asp510, Pro1080,

Tyr1150, Lys581, Gly545

Docking inside the secreted aspartic protease from c. albicans (PDB: IZAP)

16c

−27.52

Asp218 NH of anisidine der

2.41

2.72

37

28

16

22

27

–

– –

24

12

39

13

–

Tyr225, Gly85, Thr221, Iie305, Gly220, Tyr84,

Asn301, Leu216, Glu193, Gly34, Thr13, Ile30,

Ile119

Asp32

Ser88

Gly34

Nitrogen of pyrazole

Oxygen of methoxy gr 3.20

NH of anisidine der

16d

−24.91

2.97

2.56

–

Gly83, Thr221, Glu193,

Ala303, Gly85, Asp86, Ile305, Asn301

Asp218 NH of pyrazole

Arg195 Phenyl of benzamide

Tyr225 Aryl of aldehyde der

– –

16h

16i

−27.66

−28.03

Asp32

Nitrogen of pyrazole

2.84

2.80

2.41

3.18

–

Gly34, Leu216, Asn301, Thr221, Ile305, Tyr225,

Ile119, Ile30, Thrr84, Asp86, Val12, Ser88, Asp32,

Gly85

Asp218 NH of anisidine der

Asn301 Oxygen of methoxy

Tyr84

Tyr225, Ile305, Gly85, Asp86, Asp218, Ser35, Gly34,

Asn131, Asp32, Thr221, Gly220, Gly83, Glu132,

Ile82, Ile133

Aryl of benzamide der

L.Nor −30.98

Thr221 NH of amide der

Asp218 OH group

3.05

2.31

22

23

Ile223, Tyr84, Leu216, Asp86, Gly220, Glu193,

Asp32, Ile123, Ala303, Gly85, Ile305, Asn301,

Tyr225, Thr222

Cip.=Ciprofoxacin; L.Nor.=Norleucinamide; (–) arene–cation interaction, (– –) arene–arene interaction

fungal strains 1H-pyrazole-4-carboxamide derivative 16i

Study the most active derivatives against fungal

strains inside secreted aspartic protease

exhibited binding energy S=−28.03 kcal mol⁻¹through one

side-chain hydrogen bond acceptor. The pyrazole derivative

16i indicated that a hydrogen bond was formed between the

oxygen of the methoxy group and the residue Asn301 (dis-

tance 3.18 Å and strength 13%). Meanwhile, an arene–arene

interaction was observed between the 4-chlorobenzamide

derivative and Tyr84 (Fig. 6c). (All docking study fgures in

supplementary material, Figures S3-S22).

As new possible antifungal drug goals, secreted aspartic

protease (Saps) obviously hold promise. Saps are important

virulence factors because it includes a wide variety of physi-

ological processes of fungal as well as the various aspects

of the interactions between host and fungal [59]. Similarly,

the most active four pyrazole derivatives 16c, 16d, 16h, and

16i against A. niger and C. albicans were docked inside the

active site of secreted aspartic protease (Saps) (PDB: 1ZAP)

[58]. The co-crystallized ligand (L-Norleucinamide deriva-

tive) showed binding energy S=−30.98 kcal mol⁻¹through

two hydrogen bond side-chain donor between the residue

Thr221 and NH of amide derivative as well as Asp218 with

hydroxyl group with bond length 3.05 Å (22%) and 2.31 Å

(23%), respectively. The most promising derivative against

Additionally, the NH of pyrazole and NH of anisidine

derivatives in pyrazole 16d could form two hydrogen

bonds donor between Asp218 and Gly34, respectively.

Besides, the aryl group of aldehyde derivative created an

arene–cation interaction with Tyr225. Further, the dock-

ing pose of 5-((2,5-dimethoxybenzylidene)amino)-3-((4-

methoxyphenyl)amino)-N-(4-chlorophenyl)-1H-pyra-

zole-4-carboxamide (16h) demonstrated three favorable

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Journal of the Iranian Chemical Society

Fig. 6 a 2D and 3D binding mode of pyrazolo[1,5-a]pyrimidine-

3-carboxamide derivative 14j inside the active site of S. auras DNA

gyrase (PDB: 2XCT). b 2D and 3D binding mode of pyrazolo[1,5-

a]pyrimidine-3-carboxamide derivative 14b inside the active site of

S. auras DNA gyrase (PDB: 2XCT). c 2D and 3D binding mode of

1H-pyrazole-4-carboxamide derivative 16i inside the active site of

secreted aspartic protease (Saps) (PDB: 1ZAP). d 2D and 3D binding

mode of 1H-pyrazole-4-carboxamide derivative 16c inside the active

site of secreted aspartic protease (Saps) (PDB: 1ZAP)

hydrogen bonds with binding energy S=−27.66 kcal mol⁻¹.

Two of them between the nitrogen of pyrazole derivative

with the oxygen of carboxylate and oxygen of carbonyl

of Asp32 (distance 2.81, 2.84 Å). Besides, one hydrogen

bond side-chain donor between the NH of anisidine moi-

ety and Asp218 with bond length 2.41 Å. Furthermore,

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Journal of the Iranian Chemical Society

Fig. 6 (continued)

N-phenyl-1H-pyrazole-4-carboxamide 16c displayed bind-

ing energy S=−27.52 kcal mol⁻¹with three hydrogen bonds

between Asp218, Asp32, and Ser88 with NH of anisidine,

the nitrogen of pyrazole, and oxygen of methoxy group,

respectively (Fig. 6d). (All docking study fgures in sup-

plementary material, Figures S3-S22).

were suggested to be DNA gyrase inhibitors with good bind

energy scores and diferent binding modes. Overall, these

derivatives are suggested to be inhibitors for bacterial and

fungal by a diferent mode of actions and thus may partici-

pate in antimicrobial activity.

Finally, it can be concluded that the docking results

revealed that the 1H-pyrazole-4-carboxamide derivative

16c, 16d, 16h, and 16i binding to the active site of secreted

aspartic protease (Saps) (PDB: 1ZAP) and may act as inhibi-

tors. Besides, the other derivatives 14b, 14e, 14j, and 16i

Molecular electrostatic potential (MEP) maps

Our work was extended to investigate the reasons behind a

good binding of the most promising derivatives inside the

two active pockets. Molecular electrostatic potential (MEP)

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Journal of the Iranian Chemical Society

maps were generated to determine the charge distribution

around a molecule. MEP maps provide information about

the regions with electron-rich regions (red) and electron

defciency regions, i.e., hydrophobic regions that responsi-

ble for forming interactions (hydrogen bonds, arene–arene,

and arene–cation interactions) with the residue inside the

active site. The MEP was performed using MOE 2008.10,

where the active derivatives’ geometry optimized using the

MMFF94x force feld according to the previously reported

method [60]. As represented in Fig. 7, the positive charge

(blue) was located on the nitrogen of pyrimidine and pyra-

zoles as well as amino, cyano, and NH of both benzamide

and anisidine derivatives. Simultaneously, the negative

charge (red) was displayed on the oxygen of carbonyl and

methoxy groups. Finally, the presence of nucleophiles (red)

and electrophile (blue) regions showed that these regions

could form hydrogen bonding donors and acceptors and

illustrate the binding mode in the docking study (All Molec-

ular Electrostatic Potential (MEP) maps fgures in supple-

mentary material, Figures S23-S29).

Fig. 7 Showed molecular electrostatic potential maps of most promising derivatives to determine the charge distribution around molecules

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Journal of the Iranian Chemical Society

temples for the development of a novel antibacterial and

antifungal therapeutic drug.

Conclusion

In summary, new bioactive pyrazolo[1,5-a]pyrimidines

14a–j and pyrazole Schif bases 16a–j have been designed

and synthesized by the reaction of 5-amino-pyrazoles 9a,

b with 2-(arylidene)malononitriles 10a–e or various alde-

hydes 15a–e, respectively. The structures of new bioac-

tive pyrazolo[1,5-a]pyrimidines 14a–j and pyrazole Schif

bases 16a–j were confrmed using diferent spectroscopic

techniques. The pyrazolo[1,5-a]pyrimidines 14a–j and

pyrazole Schif bases 16a–j were screened for their in vitro

antimicrobial activities against four microbes. The three

pyrazolo[1,5-a]pyrimidines (14b, 14e, and 14j) and four

pyrazole Schif bases (16c, 16d, 16h, and 16i) proved to be

potent against the four microbes tested, have a wide spec-

trum efect, and in several cases, their activities exceeded

that of the reference drugs used. The structure–activity

relationship (SAR) was performed. As well as, physico-

chemical properties, drug-likeness model score, bioactiv-

ity scores, ADME, and toxicity properties were predicted

in silico. The prediction result showed the fve compounds

(14b, 16c, 16d, 16h, and 16i) agreement with Lipinski’s

rule of fve and with Veber’s rule. Also, in the case of a

drug-likeness model score, all the tested compounds (14b,

14e, 14j, 16d, 16h, and 16i) except 16c showed positive

values (range 0.05 to 0.44). Therefore, these compounds

may be considered to be drug-like. The pyrazolo[1,5-a]

pyrimidine 14b almost shows the highest bioactivity score

compared to the other compounds. All compounds are an

inhibitor of CYP2C9, CYP3A4 enzymes, and P-glycopro-

tein (P-gp). Also, some compounds showed highly active

(≥ 90%) toward plasma protein binding (PPB), were pre-

dicted as negative against carcinogenicity (mouse and rat)

test, and presented medium risk in the hERG inhibition

test. Furthermore, molecular docking studies were per-

formed for the most active derivatives against S. auras

DNA gyrase (PDB: 2XCT) and secreted aspartic protease

(Saps) (PDB: 1ZAP) as antibacterial and antifungal tar-

gets, respectively, to determine the binding patterns. The

most active antibacterial derivatives 14b, 14e, 14j, and 16i

against DNA Gyrase (PDB:2XCT) showed binding energy

S = − 25.35 to − 20.18 kcal mol⁻¹. Besides, for promising

fungal derivatives 16c, 16d, 16h, and 16i displayed bind-

ing energy S = − 28.03 to − 24.91 kcal mol⁻¹, and both of

them showed diferent types of binding mode. The results

suggested that these derivatives could act as good anti-

microbial agents. MEP maps were performed to provide

information about the regions with electron-rich regions

(red) and electron defciency regions that responsible for

forming interactions with the residue inside the active site

and therefore confrmed the binding mode in the docking

study. In the future, these compounds are possibly suitable

Materials and methods

Chemistry

All melting points were measured on a Gallenkamp melting

point apparatus and are uncorrected. The IR spectra were

recorded (KBr disk) on a PerkinElmer 1650 FTIR instrument.

¹H NMR (500 or 400 MHz) and ¹³C NMR (125 or 100 MHz)

spectra were recorded on a JEOL spectrometer using DMSO-

d₆as solvent and tetramethylsilane (TMS) as an internal stand-

ard. Chemical shifts (δ-values) are reported in ppm. Mass

spectra were recorded on a Varian MAT 112 spectrometer at

70 eV. Elemental analyses were performed at the Micro Ana-

lytical Center, Cairo University, Egypt.

The progress of the reactions was monitored by thin-layer

chromatography (TLC) using aluminum sheets coated with

silica gel F₂₅₄(Merck), viewing under a short-wavelength UV

lamp efected detection. All evaporations were performed

under reduced pressure at 40 °C.

3-Methoxybenzaldehyde (15a), 4-(dimethylamino)benza-

ldehyde (15b), 2,5-dimethoxybenzaldehyde (15c), 4-chloro-

3-nitrobenzaldehyde (15d), and thiophene-2-carbaldehyde

(15e) were of Merck AR grade, Germany.

Synthesis of 5-amino-N-aryl-3-(4-methoxyphenylamino)-

1H-pyrazole-4-carboxamide (9a, b) [41] and 2-(arylidene)

malononitriles (10a–e) [42, 43] were prepared according to

the literature procedure.

Synthesis of 7‑amino‑6‑cyano‑5‑aryl‑2‑(4‑methoxyphenyl

amino)‑N‑aryl‑pyrazolo[1,5‑a]pyrimidine‑3‑carboxamides

(14a–j)

A mixture of 5-amino-N-aryl-1H-pyrazole-4-carboxamide

derivatives 9a, b (0.01 mol) with 2-(arylidene)malononi-

triles 10a–e (0.01 mol) as [2-(4-methylbenzylidene)malon-

onitrile (10a), 2-(3-methylbenzylidene)malononitrile (10b),

2-(3-methyoxybenzylidene)malononitrile (10c), 2-(3-chlo-

robenzylidene)malononitrile (10d), and 2-(2,5-dimethoxy-

benzylidene)malononitrile (10e)] and a catalytic amount of

triethylamine (four drops) in absolute ethanol (30 mL) was

refuxed for 6 h. After cooling, the solvent was concentrated

under reduced pressure and the solid obtained was collected

and recrystallized from ethanol to give the corresponding

pyrazolo[1,5-a]pyrimidines 14a–j.

1 3

Journal of the Iranian Chemical Society

7‑Amino‑6‑cyano‑2‑(4‑methoxyphenylamino)‑N‑phe‑

nyl‑5‑(4‑methylphenyl)pyrazolo[1,5‑a]pyrimidine‑3‑car‑

boxamide (14a)

ring), 7.85 (d, 2H, J=9.0 Hz, p-methoxyphenyl ring), 9.09

(s, br, 2H, NH₂exchangeable by D₂O), 9.23, 10.14 (2s, 2H,

2NH exchangeable by D₂O); ¹³C NMR (125 MHz, DMSO-

d₆, δ/ppm): 55.43, 56.11 (2C, 2OCH₃), 75.09, 89.19, 113.86,

114.24, 115.98, 116.90, 118.89, 119.06, 120.98, 123.51,

129.14, 129.87, 133.30, 137.95, 138.38, 145.57, 149.40,

154.00, 156.36, 159.26, 160.78, 162.17 (26C); MS (m/z,

%): 505 (M⁺, 41.43%), 93 (base peak, 100%); Anal. Calcd.

(%) for C₂₈H₂₃N₇O₃(505.53): C, 66.52; H, 4.59; N, 19.39.

Found: C, 66.40; H, 4.31; N, 19.65%.

Yellow crystals; M.p.:>300 °C; Yield (80%); IR (KBr) ν_max

/

cm⁻¹3428, 3304 (NH₂, 2NH), 2213 (C≡N), 1641 (C=O),

1

1597 (C=N), 1551 (C=C); H NMR (500 MHz, DMSO-

d₆, δ/ppm): 2.43 (s, 3H, CH₃), 3.74 (s, 3H, OCH₃), 6.90

(d, 2H, J = 9.0 Hz, p-methoxyphenyl ring), 7.09 (t, 1H,

J=7.5 Hz, phenyl ring), 7.37 (t, 2H, J=7.8 Hz, phenyl ring),

7.44 (d, 2H, J=8.2 Hz, p-methylphenyl ring), 7.60 (d, 2H,

J=7.7 Hz, phenyl ring), 7.84 (d, 2H, J=9.0 Hz, p-methoxy-

phenyl ring), 7.90 (d, 2H, J=8.2 Hz, p-methylphenyl ring),

9.00 (s, br, 2H, NH₂exchangeable by D₂O), 9.23, 10.14

(2s, 2H, 2NH exchangeable by D₂O); ¹³C NMR (125 MHz,

DMSO-d₆, δ ppm): 21.15 (C, CH₃), 55.26 (C, OCH₃), 74.88,

89.09, 114.26, 118.87, 119.06, 123.54, 123.78, 128.69,

129.24, 129.34, 133.40, 133.98, 138.46, 141.08, 145.70,

149.49, 153.98, 156.32, 161.47, 162.29 (26C); MS (m/z,

%): 489 (M⁺, 17.65%), 396 (base peak, 100%); Anal. Calcd.

(%) for C₂₈H₂₃N₇O₂(489.53): C, 68.70; H, 4.74; N, 20.03.

Found: C, 68.98; H, 4.57; N, 20.29%.

7‑Amino‑5‑(3‑chlorophenyl)‑6‑cyano‑2‑(4‑methoxyphenyl

amino)‑N‑phenylpyrazolo[1,5‑a]pyrimidine‑3‑carboxamide

(14d) [61]

Orange crystals; M.p.>300 °C; Yield (74%); IR (KBr) ν_max

/

cm⁻¹3442, 3299 (NH₂, 2NH), 2212 (C≡N), 1665 (C=O),

1632 (C=N), 1598 (C=C); ¹H NMR (400 MHz, DMSO-d₆,

δ/ppm) 3.78 (s, 3H, OCH₃), 6.89 (d, 2H, J=9.0 Hz, p-meth-

oxyphenyl ring), 7.07 (t, 1H, J=7.4 Hz, phenyl ring), 7.33 (t,

2H, J=8.3 & 7.5 Hz, phenyl ring), 7.60 (d, 2H, J=8.0 Hz,

phenyl ring), 7.62 (d, 1H, J=2.9 Hz, m-chlorophenyl ring),

7.76 (d, 2H, J=9.0 Hz, p-methoxyphenyl ring), 7.93–7.96

(m, 1H, m-chlorophenyl ring), 8.02 (s, 1H, m-chlorophenyl

ring), 8.06 (d, 1H, J= 2.8 Hz, m-chlorophenyl ring), 8.93

(s, br, 2H, NH₂exchangeable by D₂O), 9.28, 10.06 (2s, 2H,

2NH exchangeable by D₂O); ¹³C NMR (100 MHz, DMSO-

d_6,CDCl₃, δ/ppm): 55.45 (1C, OCH₃), 75.14, 89.98, 114.40,

115.97, 119.20, 119.29, 123.66, 127.23, 128.77, 129.21,

130.48, 130.86, 133.59, 134.16, 138.73, 138.78, 145.75,

149.60, 154.38, 157.00, 159.45, 162.53 (26C); MS (m/z, %):

511 (M⁺², 2.69), 509 (M⁺, 8.45), 368 (base peak, 100%);

Anal. Calcd. (%) for C₂₇H₂₀ClN₇O₂(509.95): C, 63.59; H,

3.95; N, 19.23. Found: C, 63.50; H, 4.00; N, 19.27%.

7‑Amino‑6‑cyano‑2‑(4‑methoxyphenylamino)‑N‑phe‑

nyl‑5‑(3‑methylphenyl)pyrazolo[1,5‑a]pyrimidine‑3‑car‑

boxamide (14b)

Yellow crystals; M.p.:>300 °C; Yield (77%); IR (KBr) ν_max

/

cm⁻¹3428, 3319 (NH₂, 2NH), 2216 (C≡N), 1656 (C=O),

1

1599 (C=N), 1554 (C=C); H NMR (500 MHz, DMSO-

d₆, δ/ppm): 2.48 (s, 3H, CH₃), 3.79 (s, 3H, OCH₃), 6.94

(d, 2H, J = 9.5 Hz, p-methoxyphenyl ring), 7.13 (d, 1H,

J = 7.7 Hz, m-methylphenyl ring), 7.39 (t, 2H, J= 8.2 Hz,

phenyl ring), 7.50 (d, 1H, J=7.9 Hz, m-methylphenyl ring),

7.56 (t, 1H, J=7.9 Hz, phenyl ring), 7.62 (d, 4H, J=8.6 Hz,

m-methylphenyl ring+phenyl ring), 7.86 (d, 2H, J=9.2 Hz,

p-methoxyphenyl ring), 9.12 (s, br, 2H, NH₂exchangeable

by D₂O), 9.23, 10.21 (2s, 2H, 2NH exchangeable by D₂O);

Anal. Calcd. (%) for C₂₈H₂₃N₇O₂(489.53): C, 68.70; H,

4.74; N, 20.03. Found: C, 68.53; H, 4.48; N, 20.27%.

7‑Amino‑6‑cyano‑5‑(2,5‑dimethoxyphenyl)‑2‑(4‑methoxy‑

phenylamino)‑N‑phenylpyrazolo[1,5‑a]pyrimidine‑3‑car‑

boxamide (14e)

Orange crystals; M.p. 210–212 °C; Yield (75%); IR (KBr)

ν

_max/cm⁻¹3429, 3321 (NH₂, 2NH), 2217 (C≡N), 1654

7‑Amino‑6‑cyano‑5‑(3‑methoxyphenyl)‑2‑(4‑methoxyphe

nylamino)‑N‑phenylpyrazolo[1,5‑a]pyrimidine‑3‑carboxa‑

mide (14c)

1

(C=O), 1595 (C=N), 1552 (C=C); H NMR (500 MHz,

DMSO-d₆, δ/ppm): 3.72, 3.82, 3.88 (3s, 9H, 3OCH₃), 6.90

(d, 2H, J = 7.5 Hz, p-methoxyphenyl ring), 7.07–7.22 (m,

4H, 2,5-dimethoxyphenyl ring +phenyl ring), 7.34 (t, 2H,

J=7.3 Hz, phenyl ring), 7.51 (d, 1H, J=7.8 Hz, 2,5-dimeth-

oxyphenyl ring), 7.65 (d, 2H, J=7.8 Hz, p-methoxyphenyl

ring), 7.85 (d, 1H, J =8.3 Hz, 2,5-dimethoxyphenyl ring),

8.68 (s, br, 2H, NH₂exchangeable by D₂O), 9.34, 10.06

(2s, 2H, 2NH exchangeable by D₂O); Anal. Calcd. (%) for

C₂₉H₂₅N₇O₄(535.55): C, 65.04; H, 4.71; N, 18.31. Found:

C, 65.24; H, 4.45; N, 18.65%.

Yellow crystals; M.p.: 258–260 °C; Yield (72%); IR (KBr)

ν_max/cm⁻¹3433, 3332 (NH₂, 2NH), 2212 (C≡N), 1652

1

(C=O), 1595 (C=N), 1547 (C=C); H NMR (500 MHz,

DMSO-d₆, δ/ppm): 3.74, 3.86 (2s, 6H, 2OCH₃), 6.90 (d, 2H,

J=9.0 Hz, p-methoxyphenyl ring), 7.09 (t, 1H, J=7.4 Hz,

phenyl ring), 7.18–7.21 (m, 1H, m-methoxyphenyl ring),

7.36 (t, 2H, J = 7.9 Hz, phenyl ring), 7.54–7.56 (m, 3H,

m-methoxyphenyl ring), 7.59 (d, 2H, J = 7.7 Hz, phenyl

1 3

Journal of the Iranian Chemical Society

7‑Amino‑N‑(4‑chlorophenyl)‑6‑cy‑

ano‑2‑(4‑methoxyphenylamino)‑5‑(4‑methylphenyl)

pyrazolo[1,5‑a]pyrimidine‑3‑carboxamide (14f)

C, 62.28; H, 4.11; N, 18.16. Found: C, 62.35; H, 4.05; N,

18.34%.

7‑Amino‑5‑(3‑chlorophenyl)‑N‑(4‑chlorophenyl)‑6‑cy‑

ano‑2‑(4‑methoxyphenylamino)pyrazolo[1,5‑a]pyrimi‑

dine‑3‑carboxamide (14i) [61]

Yellow crystals; M.p.: > 300 °C; Yield (76%); IR (KBr)

ν_max/cm⁻¹3439, 3296, 3234 (NH₂, 2NH), 2214 (C≡N),

1639 (C=O), 1595 (C=N), 1564 (C=C); ¹H NMR

(500 MHz, DMSO-d₆, δ/ppm): 2.38 (s, 3H, CH₃), 3.70

(s, 3H, OCH₃), 6.85 (d, 2H, J = 7.3 Hz, p-methoxyphe-

nyl ring), 7.36 (d, 4H, J = 7.5 Hz, ArH), 7.55 (d, 2H,

J = 6.2 Hz, ArH), 7.74 (d, 2H, J = 7.1 Hz, ArH), 7.83 (d,

2H, J = 7.7 Hz, p-methoxyphenyl ring), 8.76 (s, br, 2H,

NH₂), 9.06, 10.15 (2s, 2H, NH); MS (m/z, %): 525 (M⁺²,

25.41), 523 (M⁺, 76.85), 93 (base peak, 100%); Anal.

Calcd. (%) for C₂₈H₂₂ClN₇O₂(523.97): C, 64.18; H, 4.23;

N, 18.71. Found: C, 64.35; H, 4.05; N, 18.48.

Yellow crystals; M.p. 252–254 ºC; Yield (71%); IR (KBr)

ν_max/cm⁻¹3397, 3308 (NH₂, 2NH), 2211 (C≡N), 1652

1

(C=O), 1613 (C=N), 1547 (C=C); H NMR (400 MHz,

DMSO-d_6,CDCl₃, δ/ppm): 3.73 (s, 3H, OCH₃), 6.82 (d,

2H, J=8.8 Hz, ArH), 7.16–7.22 (m, 2H, ArH), 7.52 (d, 2H,

J = 7.8 Hz, ArH), 7.60 (d, 2H, J = 8.8 Hz, ArH), 7.66 (d,

1H, J=8.8 Hz, ArH), 7.81–7.91 (m, 3H, ArH), 8.63 (s, br,

2H, NH₂exchangeable by D₂O), 9.14, 10.02 (2s, 2H, 2NH

exchangeable by D₂O); ¹³C NMR (100 MHz, DMSO-d_6,

CDCl₃, δ/ppm): 55.52 (1C, OCH₃), 75.48, 89.92, 114.36,

115.87, 119.28, 120.51, 127.13, 127.89, 128.34, 129.01,

130.38, 131.00, 133.49, 134.24, 134.93, 137.49, 145.89,

149.54, 154.42, 156.98, 161.21, 163.20 (26C); Anal. Calcd.

(%) for C₂₇H₁₉Cl₂N₇O₂(544.39): C, 59.57; H, 3.52; N,

18.01. Found: C, 59.49; H, 3.60; N, 17.95%.

7‑Amino‑N‑(4‑chlorophenyl)‑6‑cy‑

ano‑2‑(4‑methoxyphenylamino)‑5‑(3‑methylphenyl)

pyrazolo[1,5‑a]pyrimidine‑3‑carboxamide (14g)

Yellow crystals; M.p.:>300 °C; Yield (71%); IR (KBr) ν_max

/

cm⁻¹3419, 3335 (NH₂, 2NH), 2216 (C≡N), 1651 (C=O),

1592 (C=N), 1554 (C=C); ¹H NMR (500 MHz, DMSO-d₆,

δ/ppm): 2.44 (s, 3H, CH₃), 3.74 (s, 3H, OCH₃), 6.89 (d, 2H,

J=8.9 Hz, p-methoxyphenyl ring), 7.40 (d, 2H, J=8.9 Hz,

p-chlorophenyl ring), 7.44 (d, 1H, J = 7.7 Hz, m-methyl-

phenyl ring), 7.49 (t, 1H, J=7.6 Hz, m-methylphenyl ring),

7.58 (d, 2H, J=8.8 Hz, p-chlorophenyl ring), 7.77 (d, 1H,

J=7.7 Hz, m-methylphenyl ring), 7.80 (s, 1H, m-methylphe-

nyl ring), 7.82 (d, 2H, J=8.9 Hz, p-methoxyphenyl ring),

9.00 (s, br, 2H, NH₂exchangeable by D₂O), 9.12, 10.21

(2s, 2H, 2NH exchangeable by D₂O); Anal. Calcd. (%) for

C₂₈H₂₂ClN₇O₂(523.97): C, 64.18; H, 4.23; N, 18.71. Found:

C, 64.02; H, 4.54; N, 18.52%.

7‑Amino‑N‑(4‑chlorophenyl)‑6‑cy‑

ano‑5‑(2,5‑dimethoxyphenyl)‑2‑(4‑methoxyphenylamino)

pyrazolo[1,5‑a]pyrimidine‑3‑carboxamide (14j)

Yellow crystals; M.p.:>300 °C; Yield (74%); IR (KBr) ν_max

/

cm⁻¹3446, 3310 (NH₂, 2NH), 2215 (C≡N), 1637 (C=O),

1

1596 (C=N), 1568 (C=C); H NMR (500 MHz, DMSO-

d₆, δ/ppm): 3.70, 3.75, 3.78 (3s, 9H, 3OCH₃), 6.86 (d, 2H,

J = 9.5 Hz, p-methoxyphenyl ring), 7.10–7.17 (m, 3H,

2,5-dimethoxyphenyl ring), 7.34 (d, 2H, J=9.2 Hz, p-chlo-

rophenyl ring), 7.48 (d, 2H, J=9.2 Hz, p-chlorophenyl ring),

7.78 (d, 2H, J=9.3 Hz, p-methoxyphenyl ring), 8.97 (s, br,

2H, NH₂exchangeable by D₂O), 9.11, 10.04 (2s, 2H, 2NH

exchangeable by D₂O); ¹³C NMR (125 MHz, DMSO-d_6,

δ/ppm): 55.12, 55.68, 55.93 (3C, 3OCH₃), 78.02, 88.84,

104.95, 113.21, 114.13, 115.17, 117.23, 118.96, 120.38,

126.91, 128.90, 133.25, 137.20, 145.72, 148.51, 150.65,

153.07, 153.91, 156.07, 159.97, 159.99, 162.09 (26C); Anal.

Calcd. (%) for C₂₉H₂₄ClN₇O₄(570.00): C, 61.11; H, 4.24; N,

17.20. Found: C, 60.90; H, 4.39; N, 17.48%.

7‑Amino‑N‑(4‑chlorophenyl)‑6‑cy‑

ano‑5‑(3‑methoxyphenyl)‑2‑(4‑methoxyphenylamino)

pyrazolo[1,5‑a]pyrimidine‑3‑carboxamide (14h)

Yellow crystals; M.p.:>300 °C; Yield (73%); IR (KBr) ν_max

/

cm⁻¹3436, 3307 (NH₂, 2NH), 2213 (C≡N), 1643 (C=O),

1603 (C=N), 1544 (C=C); ¹H NMR (500 MHz, DMSO-d₆,

δ/ppm): 3.74, 3.86 (2s, 6H, 2OCH₃), 6.90 (d, 2H, J=9.1 Hz,

p-methoxyphenyl ring), 7.18–7.20 (m, 1H, m-methoxyphe-

nyl ring), 7.42 (d, 2H, J = 8.8 Hz, p-chlorophenyl ring),

7.51–7.55 (m, 3H, J = 7.7 Hz, m-methoxyphenyl ring),

7.62 (d, 2H, J=8.9 Hz, p-chlorophenyl ring), 7.84 (d, 2H,

J = 9.0 Hz, p-methoxyphenyl ring), 8.99 (s, br, 2H, NH₂

exchangeable by D₂O), 9.17, 10.18 (2s, 2H, 2NH exchange-

able by D₂O); Anal. Calcd. (%) for C₂₈H₂₂ClN₇O₃(539.97):

Synthesis of Schif bases 16a‑j

A mixture of 5-amino-N-aryl-1H-pyrazole-4-carboxa-

mide derivatives 9a, b (0.01 mol) and various aldehydes

15a–e (0.01 mol) {namely: 3-methoxybenzaldehyde (15a),

4-(dimethylamino)benzaldehyde (15b), 2,5-dimethoxyben-

zaldehyde (15c), 4-chloro-3-nitrobenzaldehyde (15d), or

thiophene-2-carbaldehyde (15e)} with a catalytic amount

of glacial acetic acid (1 mL) in absolute ethanol (25 mL) was

1 3

Journal of the Iranian Chemical Society

refuxed for 2 h and then left to cool. The solid product was

fltered of, dried and fnally recrystallized from ethanol to

aford the corresponding Schif bases 16a–j.

p-methoxyphenyl ring), 8.69 (s, 1H, –CH=N–), 9.47, 10.18,

13.01 (3s, 3H, 3NH exchangeable by D₂O); Anal. Calcd.

(%) for C₂₆H₂₅N₅O₄(471.51): C, 66.23; H, 5.34; N, 14.85.

Found: C, 66.62; H, 5.17; N, 14.68%.

5‑(3‑Methoxybenzylideneamino)‑3‑(4‑methoxyphenylamin

o)‑N‑phenyl‑1H‑pyrazole‑4‑carboxamide (16a)

5‑(4‑Chloro‑3‑nitrobenzylideneamino)‑3‑(4‑methoxypheny

lamino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxamide (16d)

Yellow crystals; M.p.: 201–203 °C; Yield (75%); IR (KBr)

ν_max/cm⁻¹3434, 3229 (3NH), 1656 (C=O), 1612 (C=N),

1587 (C=C); ¹H NMR (500 MHz, DMSO-d₆, δ/ppm): 3.73,

3.88 (2s, 6H, 2OCH₃), 6.91 (d, 2H, J=8.3 Hz, p-methoxy-

phenyl ring), 7.08 (t, 1H, J = 7.4 Hz, phenyl ring), 7.23

(d, 1H, J = 7.5 Hz, m-methoxyphenyl ring), 7.36 (t, 2H,

J = 7.9 Hz, phenyl ring), 7.55 (t, 1H, J = 7.8 Hz, m-meth-

oxyphenyl ring), 7.62–7.64 (m, 4H, phenyl ring+m-meth-

oxyphenyl ring), 7.68 (d, 2H, J=8.0 Hz, p-methoxyphenyl

ring), 8.71 (s, 1H, –CH=N–), 9.05, 9.99, 12.80 (3s, 3H, 3NH

exchangeable by D₂O); ¹³C NMR (125 MHz, DMSO-d₆, δ/

ppm): 55.27, 55.34 (2C, 2OCH₃), 83.17, 112.33, 114.51,

118.98, 119.82, 123.30, 128.54, 129.08, 129.37, 130.53,

135.02, 138.54, 146.21, 152.25, 155.38, 156.89, 159.83,

161.70, 162.69 (23C); MS (m/z, %): 441 (M⁺, 25.67%),

83 (base peak, 100%); Anal. Calcd. (%) for C₂₅H₂₃N₅O₃

(441.48): C, 68.01; H, 5.25; N, 15.86. Found: C, 68.17; H,

5.06; N, 15.69%.

Buf crystals; M.p.: 252–254 °C; Yield (72%); IR (KBr)

ν_max/cm⁻¹3431, 3235 (3NH), 1653 (C=O), 1615 (C=N),

1513 (C=C); ¹H NMR (500 MHz, DMSO-d₆, δ/ppm): 3.70

(s, 3H, OCH₃), 6.89 (d, 2H, J = 8.3 Hz, p-methoxyphenyl

ring), 7.06 (t, 1H, J= 7.3 Hz, phenyl ring), 7.31–7.34 (m,

4H, phenyl ring), 7.67 (d, 2H, J=7.9 Hz, p-methoxyphenyl

ring), 8.00 (d, 1H, J=8.3, 4-chloro-3-nitrophenyl ring), 8.30

(d, 1H, J = 8.3, 4-chloro-3-nitrophenyl ring), 8.67 (s, 1H,

4-chloro-3-nitrophenyl ring), 8.76 (s, 1H, –CH=N–), 9.13,

9.79, 12.80 (3s, 3H, 3NH exchangeable by D₂O); ¹³C NMR

(125 MHz, DMSO-d₆, δ/ppm): 55.23 (1C, OCH₃), 96.78,

114.61, 118.92, 119.73, 121.51, 123.06, 125.26, 126.27,

128.87, 132.72, 133.81, 134.09, 138.39, 147.84, 152.02,

153.37, 157.62, 160.86, 162.42 (23C); Anal. Calcd. (%) for

C₂₄H₁₉ClN₆O₄(490.90): C, 58.72; H, 3.90; N, 17.12; Found:

C, 58.99; H, 3.79; N, 17.28%.

3‑(4‑Methoxyphenylamino)‑N‑phenyl‑5‑(thiophen‑2‑yl‑

methyleneamino)‑1H‑pyrazole‑4‑carboxamide (16e)

5‑(4‑(Dimethylamino)benzylideneamino)‑3‑(4‑methoxyphe

nylamino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxamide (16b) [62]

Yellow crystals; M.p.: 212–214 °C; Yield (67%); IR (KBr)

ν_max/cm⁻¹3433, 3272 (3NH), 1652 (C=O), 1594 (C=N),

1547 (C=C); ¹H NMR (500 MHz, DMSO-d₆, δ/ppm): 3.73

(s, 3H, OCH₃), 6.90 (d, 2H, J = 7.1 Hz, p-methoxyphenyl

ring), 7.09 (d, 1H, J=5.3 Hz, thiophene ring), 7.33–7.40 (m,

5H, phenyl ring), 7.72 (d, 2H, J=7.0 Hz, p-methoxyphenyl

ring), 7.91 (d, 1H, J=3.6 Hz, thiophene ring), 8.05 (s, 1H,

thiophene ring), 8.68 (s, 1H, –CH=N–), 9.19, 9.98, 12.50

(3s, 3H, 3NH exchangeable by D₂O); ¹³C NMR (125 MHz,

DMSO-d₆, δ/ppm): 55.79 (1C, OCH₃), 89.70, 115.01,

119.23, 123.68, 129.62, 129.74, 133.89, 134.14, 134.29,

136.99, 139.23, 141.83, 155.79, 156.45, 157.46, 159.54,

163.28 (21C); MS (m/z, %): 417 (M⁺, 18.85%), 186 (base

peak, 100%); Anal. Calcd. (%) for C₂₂H₁₉N₅O₂S (417.48):

C, 63.29; H, 4.59; N, 16.78; Found: C, 63.45; H, 4.39; N,

16.85%.

Yellow crystals; M.p.: 216–218 °C; Yield (87%); IR (KBr)

ν_max/cm⁻¹3445, 3292 (3NH), 1652 (C=O), 1614 (C=N),

1

1592 (C=C); H NMR (400 MHz, DMSO-d₆, δ/ppm):

3.01 (s, 6H, N(CH₃)₂), 3.66 (s, 3H, OCH₃), 6.83 (d, 4H,

J = 8.9 Hz, ArH), 7.02 (t, 1H, ArH), 7.33 (t, 2H, ArH),

7.48 (2H, ArH), 7.60 (d, 2H, J=8.4 Hz, ArH), 7.80 (d, 2H,

J=8.7 Hz, ArH), 8.67 (s, 1H, –N=CH–), 9.03, 10.07, 12.59

(3s, 3H, 3NH exchangeable by D₂O); ¹³C NMR (100 MHz,

DMSO-d₆, δ/ppm) δ 40.10 (2C, N (CH₃)₂under solvent

DMSO), 55.48 (OCH₃), 92.74, 112.34, 115.12, 120.08,

121.82, 126.58, 128.07, 128.43, 129.19, 132.02, 132.84,

135.21, 152.78, 153.40, 153.84, 154.45, 160.57, 162.06

(23C); Anal. Calcd. (%) for C₂₆H₂₆N₆O₂(454.52): C, 68.70;

H, 5.77; N, 18.49. Found: C, 68.75; H, 5.70; N, 18.55%.

5‑(2,5‑Dimethoxybenzylideneamino)‑3‑(4‑methoxyphenyla

mino)‑N‑phenyl‑1H‑pyrazole‑4‑carboxamide (16c)

N‑(4‑Chlorophenyl)‑5‑(3‑methoxybenzylideneamino)‑3‑(4‑

methoxyphenylamino)‑1H‑pyrazole‑4‑carboxamide (16f)

Orange crystals; M.p.: 234–236 °C; Yield (69%); IR (KBr)

ν

_max/cm⁻¹3389, 3320 (3NH), 1654 (C=O), 1594 (C=N),

Yellow crystals; M.p.: 205–207 °C; Yield (73%); IR

(KBr) ν_max/cm⁻¹3434, 3229 (3NH), 1656 (C=O), 1612

1

1552 (C=C); H NMR (500 MHz, DMSO-d₆, δ/ppm):

3.72, 3.83, 3.89 (3s, 9H, 3OCH₃), 6.91 (d, 2H, J=8.0 Hz,

p-methoxyphenyl ring), 7.08–7.35 (m, 7H, Ar–H), 7.50 (s,

1H, 2,5-dimethoxyphenyl ring), 7.65 (d, 2H, J = 8.3 Hz,

1

(C=N), 1587 (C=C); H NMR (500 MHz, DMSO-d₆, δ/

ppm): 3.73, 3.87 (2s, 6H, 2OCH₃), 6.90 (d, 2H, J=8.9 Hz,

p-methoxyphenyl ring), 7.21, 7.23 (dd, 1H, J=8.2, 1.8 Hz,

1 3

Journal of the Iranian Chemical Society

m-methoxyphenyl ring), 7.40 (d, 4H, J=8.9 Hz, p-chloro-

phenyl ring), 7.53 (t, 1H, J=7.9 Hz, m-methoxyphenyl ring),

7.59 (s, 1H, m-methoxyphenyl ring), 7.62 (d, 1H, J=2.4 Hz,

m-methoxyphenyl ring), 7.69 (d, 2H, J = 8.9 Hz, p-meth-

oxyphenyl ring), 8.66 (s, 1H, –CH=N–), 9.03, 10.02, 12.78

(3s, 3H, 3NH exchangeable by D₂O); ¹³C NMR (125 MHz,

DMSO-d₆, δ/ppm): 55.19, 55.23 (2C, 2OCH₃), 92.61,

112.24, 114.39, 119.22, 119.53, 120.41, 122.48, 126.69,

128.34, 128.73, 130.28, 133.79, 136.17, 137.46, 151.21,

154.04, 159.74, 162.03, 162.65 (23C); Anal. Calcd. (%) for

C₂₅H₂₂ClN₅O₃(475.93): C, 63.09; H, 4.66; N, 14.72; Found:

C, 63.34; H, 4.53; N, 14.43%.

134.18, 137.62, 153.41, 153.81, 154.87, 156.70, 156.90,

160.72, 162.88 (23C); Anal. Calcd. (%) for C₂₆H₂₄ClN₅O₄

(505.95): C, 61.72; H, 4.78; N, 13.84; Found: C, 61.90; H,

4.51; N, 13.98%.

5‑(4‑Chloro‑3‑nitrobenzylideneamino)‑N‑(4‑chlorophenyl

)‑3‑(4‑methoxyphenylamino)‑1H‑pyrazole‑4‑carboxamide

(16i)

Yellow crystals; M.p.: 280–282 °C; yield (71%); IR (KBr)

ν_max/cm⁻¹3440, 3280, 3230 (3NH), 1653 (C=O), 1615

1

(C=N), 1561 (C=C); H NMR (500 MHz, DMSO-d₆, δ/

ppm): 3.72 (s, 3H, OCH₃), 6.89 (d, 2H, J=8.7 Hz, p-meth-

oxyphenyl ring), 7.35 (d, 4H, J = 8.8 Hz, p-chlorophenyl

ring), 7.70 (d, 2H, J=8.8 Hz, p-methoxyphenyl ring), 7.96

(d, 1H, J = 8.4 Hz, 4-chloro-3-nitrophenyl ring), 8.27 (d,

1H, J = 8.3 Hz, 4-chloro-3-nitrophenyl ring), 8.26 (s, 1H,

4-chloro-3-nitrophenyl ring), 8.72 (s, 1H, –CH=N–), 9.14,

9.87, 12.94 (3s, 3H, 3NH exchangeable by D₂O); Anal.

Calcd. (%) for C₂₄H₁₈Cl₂N₆O₄(525.34): C, 54.87; H, 3.45;

N, 16.00; Found: C, 55.05; H, 3.28; N, 16.19%.

N‑(4‑Chlorophenyl)‑5‑(4‑(dimethylamino)benzylideneamin

o)‑3‑(4‑methoxyphenylamino)‑1H‑pyrazole‑4‑carboxamide

(16g) [62]

Yellow crystals; M.p.: 209–211 °C, Yield (86%); IR (KBr)

ν_max/cm⁻¹3431, 3275 (3NH), 1662 (C=O), 1609 (C=N),

1581 (C=C); ¹H NMR (400 MHz, DMSO-d₆, δ ppm): 3.38

(s, 6H, N(CH₃)₂), 3.71 (s, 3H, OCH₃), 6.85 (d, 2H, J=9 Hz,

p-dimethylaminophenyl ring), 6.88 (d, 2H, J = 8.9 Hz,

p-methoxyphenyl ring), 7.40 (d, 2H, J=8.6 Hz, p-chloro-

phenyl ring), 7.45 (d, 2H, J=8.5 Hz, p-chlorophenyl ring),

7.68 (d, 2H, J = 8.6 Hz, p-methoxyphenyl ring), 7.82 (d,

2H, J=8.5 Hz, p-dimethylaminophenyl ring), 8.66 (s, 1H,

–CH=N–), 8.74, 10.24, 12.24 (3s, 3H, 3NH exchangeable by

D₂O); ¹³C NMR (100 MHz, DMSO-d₆, δ ppm): 40.06 (2C,

N (CH₃)₂under solvent DMSO), 55.67 (1C, OCH₃), 92.48,

112.27, 114.78, 120.87, 121.08, 122.12, 127.29, 129.43,

132.02, 133.35, 135.00, 152.88, 153.70, 153.89, 154.25,

161.19, 163.66 (23C); Anal. Calcd. (%) for C₂₆H₂₅ClN₆O₂

(488.97): C, 63.86; H, 5.15; N, 17.19; Found: C, 63.95; H,

5.10; N, 17.25%.

N‑(4‑Chlorophenyl)‑3‑(4‑methoxyphenylamino)‑5‑(thiophe

n‑2‑ylmethyleneamino)‑1H‑pyrazole‑4‑carboxamide (16j)

Yellow crystals; M.p.: 221–223 °C; yield (68%); IR (KBr)

ν_max/cm⁻¹3432, 3278 (3NH), 1653 (C=O), 1598 (C=N),

1562 (C=C); ¹H NMR (500 MHz, DMSO-d₆, δ/ppm): 3.73

(s, 3H, OCH₃), 6.91 (d, 2H, J = 8.1 Hz, p-methoxyphenyl

ring), 7.39–7.45 (m, 5H, p-chlorophenyl ring + thiophene

ring), 7.74 (d, 2H, J=8.0 Hz, p-methoxyphenyl ring), 7.91

(d, 1H, J=2.3 Hz, thiophene ring), 8.05 (d, 1H, J=4.8 Hz,

thiophene ring), 8.64 (s, 1H, –CH=N–), 9.19, 10.04, 12.97

(3s, 3H, 3NH exchangeable by D₂O); Anal. Calcd. (%) for

C₂₂H₁₈ClN₅O₂S (451.93): C, 58.47; H, 4.01; N, 15.50;

Found: C, 58.58; H, 4.19; N, 15.21%.

N‑(4‑Chlorophenyl)‑5‑(2,5‑dimethoxybenzylideneamino)

‑3‑(4‑methoxyphenylamino)‑1H‑pyrazole‑4‑carboxamide

(16h)

In vitro antimicrobial evaluation

Orange crystals; M.p.: 250–252 °C; Yield (74%); IR (KBr)

ν_max/cm⁻¹3432, 3251 (3NH), 1653 (C=O), 1597 (C=N),

1544 (C=C); ¹H NMR (500 MHz, DMSO-d₆, δ/ppm): 3.73,

3.82, 3.88 (3s, 9H, 3OCH₃), 6.89, 6.91 (dd, 2H, J=2.8 &

9.5 Hz, p-methoxyphenyl ring), 7.18 (d, 1H, J = 2.8 Hz,

2,5-dimethoxyphenyl ring), 7.21 (d, 1H, J = 2.7 Hz,

2,5-dimethoxyphenyl ring), 7.38 (d, 2H, J=9.2 Hz, p-chlo-

rophenyl ring), 7.42 (d, 2H, J = 9.0 Hz, p-chlorophenyl

ring), 7.61 (s, 1H, 2,5-dimethoxyphenyl ring), 7.66, 7.68

(dd, 2H, J = 9.2, 2.7 Hz, p-methoxyphenyl ring), 8.64 (s,

1H, –CH=N–), 9.34, 10.13, 11.74 (3s, 3H, 3NH exchange-

able by D₂O); ¹³C NMR (125 MHz, DMSO-d₆, δ/ppm):

55.26, 55.59, 56.47 (3C, 3OCH₃), 92.55, 109.44, 114.21,

114.42, 119.00, 120.58, 121.94, 123.30, 126.66, 128.83,

Microorganisms

The microbes used in this study were Gram-positive bacte-

rial strain (Bacillus subtilis ATCC-6633), Gram-negative

bacterial strain (Escherichia coli ATCC-25922), yeast (Can-

dida albicans ATCC-10231), and Aspergillus niger NRRL-3

as fungi. These microorganisms were obtained from the

American Type Culture Collection (ATCC, Rockville, MD,

USA) and Northern Utilization Research and Development

Division, United State Department of Agriculture, Peoria,

Illinois, USA (NRRL). The bacterial strains were revived for

bonsai by sub-culturing fresh nutrient agar (NA) medium

(Merck, Darmstadt, Germany) for 24 h before the test, while,

fungi were cultured on potato dextrose agar (PDA) (Lab M.,

1 3

Journal of the Iranian Chemical Society

Bury, Lancashire, UK) for 7 days at 28 °C before the experi-

ment was carried out.

gradient of 0.05 kcal mol⁻¹Å as well as the partial charged

calculated. For promising antibacterial derivatives S. aureus

Gyrase complex with Ciprofoxacin (PDB:2XCT), and for

promising fungal derivatives secreted aspartic protease from

C. albicans (PDB: 1ZAP) were selected for this study [57,

58]. The enzyme with its co-crystallized ligand was retrieved

from the protein data bank. For DNA gyrase (PDB:2XCT),

the docking process performed using only one chain G chain

only according to the reported method. For fungal, the pro-

tein structure (PDB: 1ZAP) contains only one chain (A).

The enzyme was prepared according to default protocol by

removing the water molecules, hydrogen atom added. The

alpha site fnder was used to generate the active site. For

docking studies, the trigonal matcher placement and the

London dG scoring function were selected after redocking

the co-crystalized ligand in both enzymes.

Inoculum preparation

Stock cultures were maintained at 4 °C on slopes of nutrient

agar and potato dextrose agar. Active cultures for experi-

ments were prepared by transferring a lapful of cells from

the stock cultures to test tubes of Mueller–Hinton broth

(MHB) (Lab M Limited, Bury, Lancashire, UK) for bacteria

and Sabouraud dextrose broth (SDB) (Lab M., Bury, Lanca-

shire, UK) for fungi that were incubated without agitation

for 24 h at 37 °C and 25 °C, respectively. To 5 mL of MHB

and SDB, 0.2 mL of culture was inoculated and incubated

(or diluted) till it reached the turbidity equal to that of the

standard 0.5 McFarland solution at 625 nm (A=0.08 to 0.1)

which is equivalent to 1.5×10⁸cfu.mL⁻¹.

Supplementary Information The online version contains supplemen-

tary material available at https://doi.org/10.1007/s13738-021-02319-4.

Bioassay method

Acknowledgements The authors wish to express their thanks to the

Agar difusion technique as described by Perez et al. [45]

was used to determine the in vitro antibacterial activity of

the powder samples. A 0.1 mL aliquot of 18 h broth culture

of the above-mentioned bacteria that had been adjusted to

the turbidity equivalent of 0.5 McFarland standards [63] was

dispensed into sterile Petri dishes previously labeled with the

test bacteria. Molten sterile Muller-Hinton was aseptically

poured into the plates and gently rotated for the bacteria

to be homogeneously distributed in the medium. The agar

plates were allowed to solidify. The antibacterial screen-

ing bioassay was made by the agar well di fusion method

described by Jorgensen and Turnidge [64] using Muel-

ler–Hinton agar (Lab M Limited, Bury, Lancashire, UK),

then the plates allowed to difuse for two hours at 4 °C. The

experiment was conducted in triplicates. All plates were

incubated at 37 °C for 24 h for bacterial strains and 28–30 °C

for 48 h for fungal strain. Clearance zones around the wells

were noted and measured in millimeters [65]. Standard bac-

terial antibiotics as Tetracycline (30 µg) and Novobiocine

(30 µg) and fungal antibiotic as Clotrimazole (50 µg), and

Cyclohexamide (50 µg) were used as a positive control for

bacteria and fungi, respectively.

National Research Centre for the facilities provided.

Declarations

Conflict of interest The authors declare that they have no confict of

interest.

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Synthesis, molecular docking, and in silico ADME prediction of some fused pyrazolo[1,5-a]pyrimidine and pyrazole derivatives as potential antimicrobial agents

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