Evaluation and structure-activity relationship analysis of a new series of 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-amines as potential antibacterial agents

Article abstract of DOI:10.1016/j.molstruc.2017.05.050

The synthesis of pyrazolo[3,4-d]pyrimidine derivatives is important due to their presence in various biologically active compounds such as anticancer, antimicrobial, antiparasitic, anti-inflammatory and antidiabetic agents. In this project, a new and efficient approach for the synthesis of some novel 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-amines from reaction of 5-amino-pyrazole-4-carbonitrile with various hydrazides in ethanolic sodium ethoxide medium was reported. Antimicrobial activities of all synthesized derivatives were evaluated against eight Gram-positive and five Gram-negative pathogenic bacteria. The moderate to good inhibitory effects were observed based on inhibition zone diameter (IZD), minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values. In order to determine the reasonable relationship between antibacterial activities and physiochemical properties of the derivatives, computational studies were carried out in terms of geometry optimization, short-range van der Waals forces, dipole moments, atomic charges and frontier orbital energies. It was found that both short-range forces and covalent bonds are important in the observed inhibitory effects of the molecules. The results suggested that pyrazolo[3,4-d]pyrimidine derivatives prefer a soft nucleophilic attack on bio-macromolecular targets. Furthermore, our models proposed that the antibacterial activities of these derivatives can be improved by substituting large electron donating groups on the 6-phenyl rings.

Full text of DOI:10.1016/j.molstruc.2017.05.050

Accepted Manuscript
Evaluation and structure-activity relationship analysis of a new series of 4-imino-5H
-pyrazolo[3,4-d]pyrimidin-5-amines as potential antibacterial agents
Hamid Beyzaei, Reza Aryan, Mohammadreza Moghaddam-Manesh, Behzad
Ghasemi, Pouya Karimi, Hojat Samareh Delarami, Mahmood Sanchooli
PII:
S0022-2860(17)30634-8
10.1016/j.molstruc.2017.05.050
MOLSTR 23791
DOI:
Reference:
To appear in:
Journal of Molecular Structure
Received Date:
Revised Date:
Accepted Date:
09 January 2017
10 May 2017
11 May 2017
Please cite this article as: Hamid Beyzaei, Reza Aryan, Mohammadreza Moghaddam-Manesh,
Behzad Ghasemi, Pouya Karimi, Hojat Samareh Delarami, Mahmood Sanchooli, Evaluation and
structure-activity relationship analysis of a new series of 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-
amines as potential antibacterial agents, Journal of Molecular Structure (2017), doi: 10.1016/j.
molstruc.2017.05.050
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form.
Please note that during the production process errors may be discovered which could affect the
content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Evaluation and structure-activity relationship analysis
of a new series of 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-amines as potential
antibacterial agents
Hamid Beyzaeia,*, Reza Aryana, Mohammadreza Moghaddam-Maneshb, Behzad Ghasemic, Pouya
Karimia, Hojat Samareh Delaramia, Mahmood Sanchoolia
a Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran
b Department of Chemistry, Faculty of Science, Kerman Branch, Islamic Azad University, Kerman,
Iran
c Young Researchers and Elite Club, Neyshabur Branch, Islamic Azad University, Neyshabur,
Iran

ACCEPTED MANUSCRIPT
Highlights
 Five novel 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-amine derivatives were synthesized
via a new and efficient method.
 The in vitro antimicrobial properties of all synthesized pyrazolo[3,4-d]pyrimidines were
evaluated against eight Gram-positive and five Gram-negative pathogenic bacteria.
 The inhibitory activities of derivatives as IZD, MIC and MBC values were determined by
disk diffusion and broth microdilution methods.
 Structural parameters such as dipole moment, atomic charge, molecular volume, frontier
orbital energy, molecular surface area, hardness, softness and electrophilicity were
calculated.
 The relatively strong relationships were found between hardness, softness,
electrophilicity, amount of negative charge on N-3 atom and the sum of atomic charges
on two heterocyclic rings of derivatives and the observed antibacterial activities.

ACCEPTED MANUSCRIPT
Evaluation and structure-activity relationship analysis of a new series of 4-imino-5H-
pyrazolo[3,4-d]pyrimidin-5-amines as potential antibacterial agents
Hamid Beyzaeia,*, Reza Aryana, Mohammadreza Moghaddam-Maneshb, Behzad Ghasemic,
Pouya Karimia, Hojat Samareh Delaramia, Mahmood Sanchoolia
a Department of Chemistry, Faculty of Science, University of Zabol, Zabol, Iran
b
Department of Chemistry, Faculty of Science, Kerman Branch, Islamic Azad University,
Kerman, Iran
c
Young Researchers and Elite Club, Neyshabur Branch, Islamic Azad University, Neyshabur,
Iran
∗Corresponding author: Hamid Beyzaei; E-mail: hbeyzaei@yahoo.com and hbeyzaei@uoz.ac.ir;
Tel.: +98 543 123 2186; Fax: +98 543 123 2180.
E-mail addresses: hbeyzaei@yahoo.com and hbeyzaei@uoz.ac.ir (H. Beyzaei),
rezaaryanchemist@yahoo.com (R. Aryan), mrm.manesh@gmail.com (M. Moghaddam-Manesh),
behzad.ghasemi99@gmail.com (B. Ghasemi), kahrobaieasby@yahoo.com (P. Karimi),
hsd102012@gmail.com (H. Samareh Delarami), sanchooli52@yahoo.com (M. Sanchooli).
1

ACCEPTED MANUSCRIPT
Abstract
The synthesis of pyrazolo[3,4-d]pyrimidine derivatives is important due to their presence in various biologically
active compounds such as anticancer, antimicrobial, antiparasitic, anti-inflammatory and antidiabetic agents. In this
project, a new and efficient approach for the synthesis of some novel 4-imino-5H-pyrazolo[3,4-d]pyrimidin-5-
amines from reaction of 5-amino-pyrazole-4-carbonitrile with various hydrazides in ethanolic sodium ethoxide
medium was reported. Antimicrobial activities of all synthesized derivatives were evaluated against eight Gram-
positive and five Gram-negative pathogenic bacteria. The moderate to good inhibitory effects were observed based
on inhibition zone diameter (IZD), minimum inhibitory concentration (MIC) and minimum bactericidal
concentration (MBC) values. In order to determine the reasonable relationship between antibacterial activities and
physiochemical properties of the derivatives, computational studies were carried out in terms of geometry
optimization, short-range van der Waals forces, dipole moments, atomic charges and frontier orbital energies. It was
found that both short-range forces and covalent bonds are important in the observed inhibitory effects of the
molecules. The results suggested that pyrazolo[3,4-d]pyrimidine derivatives prefer a soft nucleophilic attack on bio-
macromolecular targets. Furthermore, our models proposed that the antibacterial activities of these derivatives can
be improved by substituting large electron donating groups on the 6-phenyl rings.
Keywords Pyrazolo[3,4-d]pyrimidine, Hydrazide, New efficient synthesis, Antibacterial activity, Computational
study, Quantitative structure-activity relationship.
Introduction
Pyrazolo[3,4-d]pyrimidines as purine analogues are important class of fused heterocyclic compounds due to their
various pharmacological and biological activities. Commercial drugs or those are still under investigation such as
allopurinol and its metabolite oxypurinol are known as inhibitors of xanthine oxidase. These drugs are being used
for the treatment or prevention of hyperuricemia, acute uric acid nephropathy, gout, renal calculi and congestive
heart failure. Pyrazolo[3,4-d]pyrimidine cores were also found in several natural products. Phidolopine is a purine
derivative that was isolated from the bryozoan Phidolopora pacifica and is known as antifungal and antialgal agent
[1]. A number of pyrazolo[3,4-d]pyrimidine derivatives were synthesized and studied for their anticancer [2],
antiviral [3], antibacterial [4], antifungal [5], antiparasitic [6], herbicidal [7], radioprotective [8], anti-neoplastic [9],
anti-inflammatory and analgesic [10] activities as well. Apoptotic effects of some synthesized pyrazolo[3,4-
d]pyrimidines were proven on large granular lymphocyte leukemia cells via reduction of Fyn phosphorylation [11].
They were also used to treat hypertension [12], pyrexia and nociception [13]. The broad therapeutic scope for
pyrazolo[3,4-d]pyrimidine derivatives has attracted a wide range of researches in the field of organic chemistry
toward the design and synthesis of new derivatives. The synthetic strategies usually include reaction of pyrazole or
pyrimidine derivatives with formamide [14, 15], N,N-dimethylphosgeniminium chloride [16], acetic acid [17],
triethyl orthoalkanoate and ammonia [18], aldehydes [19], 1-benzyl-4-hydrazinylpiperidine dihydrochloride [20],
alkyl carbazates [21] and hydrazines [22]. 5-Amino-pyrazole-4-carbonitriles are important synthetic precursors
which were used as starting materials in the synthesis of these fused heterocycles [14, 16-18, 23] as well as
pyrazolotriazines [24], azo dyes [25], azaindenes [26], pyrazolo[3,4-b]pyridine-5,6-dicarboxylates [27],
2

ACCEPTED MANUSCRIPT
pyrazolo[3,4-d]pyridazines [28], pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines, pyrazolyl-tetrazoles [29] and
pyrazolo[4,3-e]-1,2,4-triazolo[1,5-c]pyrimidines [30].
Nowadays, computational approaches are widely used for the assessment of structure-activity relationships. Some
pyrazolo[3,4-d]pyrimidinones as antidiabetic agents were synthesized and their interactions with the DPP-IV
enzyme were studied using surflex dock module of SYBYL X-2.0 [31]. The anti-inflammatory and neuroprotective
properties of the synthesized pyrazolo[3,4-d]pyrimidine 4(5H)-ones were evaluated and the molecular operating
environment module was used for docking experimentation [32]. The inhibitory activities of some pyrazolo[3,4-
d]pyrimidine derivatives were proved against Bcr-Abl T315I mutant through both in vitro and in vivo tests and
docking studies [33]. The antimicrobial properties of a series of pyrazolo[3,4-d]pyrimidine-6-thiols were studied and
compared to the reference drugs via molecular modeling [34]. In another study, pyrazolo[1,5-c]pyrimidine-3-
carboxylates were synthesized and the mechanism of formation, NBO analysis, Fukui functions and energies of
HOMO and LUMO orbitals were calculated using B3LYP/6-31G (d,p) and B3LYP hybrid functional methods [35].
The wide range of pharmaceutical and biological properties of pyrazolo[3,4-d]pyrimidines encouraged us to a novel
approach for the synthesis of these compounds. The in vitro antimicrobial activities of newly synthesized derivatives
were evaluated against a number of pathogenic bacteria including Gram-positive and -negative standard strains of
various genera, and the results were reported as IZD, MIC and MBC values. Finally, the correlation between
biological activities and molecular properties of the novel derivatives were computationally studied.
Material and Methods
Chemicals
All chemicals and solvents were obtained from Merck and Sigma-Aldrich, were used without further purification.
Antibiotics were purchased from Sigma-Aldrich. All yields refer to isolated products. Melting points were recorded
on a Kruss type KSP1N melting point meter with no further correction. The reaction progress and the purity of the
products were affected by aluminium TLC plates pre-coated with silica gel 60 using CH2Cl2:CH3OH (8:2, v/v) as
the desired mobile phase. The result TLC plates were visualized with iodine vapour. The IR spectra of the products
were recorded on a Bruker Tensor-27 FT-IR spectrometer using KBr disks. The 1H and 13C-NMR spectra of DMSO-
d6 solutions were recorded on a Bruker FT-NMR Ultra Shield-400 spectrometer (400 and 100 MHz, resp.).
Elemental analyses were performed for C, H and N on a Thermo Finnigan Flash EA microanalyzer. The
concentration of bacterial suspension was determined using Jenway 6405 UV-Vis spectrophotometer. 5-Amino-3-
methyl-1-phenyl-1H-pyrazole-4-carbonitrile (4) was prepared according to the previous literature report [36].
General procedure for the preparation of 6-substituted 4-imino-3-methyl-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-
d]pyrimidin-5-amines 6a-e
Clean metallic sodium (10 mmol, 0.23 g) was added in a few portions to 20 mL of stirred and ice-water cooled
absolute ethanol. Then, pyrazole derivative 4 (10 mmol, 1.98 g) and hydrazides 5a-e (5a: 0.60 g, 5b: 1.36 g, 5c: 1.50
g, 5d: 1.81 g, 5e: 1.50 g; 10 mmol) were added and the mixture was heated to reflux for 10 h. The resulting solution
was cooled to room temperature. A precipitate was crashed out upon addition of 20 mL of 0.5 M acetic acid. The
3

ACCEPTED MANUSCRIPT
solid was filtered off, washed sequentially with water/acetone (2:1, v/v; 10 mL) and ethanol (10 mL), which then
dried in an oven at 80 oC to afford pure compounds 6a-e.
4-Imino-3-methyl-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine (6a)
o
1
Yield 75%; mp 268-270 C; IR (KBr) ν 3419, 3244, 2973, 1558, 1436, 1222, 1174, 924, 584 cm-1; H NMR
(DMSO-d6, 400 MHz) 훿 2.69 (3H, s, CH3), 7.50 (3H, m, H-3,4,5 Ph), 8.11 (1H, s, H-6), 8.16 (2H, d, J = 7.9 Hz, H-
2,6 Ph), 8.44 (2H, s, NH2), 12.00 (1H, s, C=NH); 13C NMR (DMSO-d6, 100 MHz) 훿 151.93 (C-4), 149.48 (C-3),
148.89 (C-6), 144.46 (C-7a), 140.30 (C-1 Ph), 129.91 (C-3,5 Ph), 125.08 (C-4 Ph), 124.40 (C-2,6 Ph), 113.93 (C-
3a), 13.05 (CH3). Anal Calcd. for C12H12N6 (%): C, 59.99; H, 5.03; N, 34.98. Found (%): C, 59.96; H, 5.02; N,
35.02.
4-Imino-3-methyl-1,6-diphenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine (6b)
o
1
Yield 61%; mp 228-230 C; IR (KBr) ν 3353, 3180, 1680, 1600, 1552, 1445, 1287, 1142, 912, 540 cm-1; H NMR
(DMSO-d6, 400 MHz) 훿 2.63 (3H, s, CH3), 7.47-7.63 (6H, m, H-3,4,5 Ph-1,6), 8.24 (2H, d, J = 7.6 Hz, H-2,6 Ph-1),
8.48 (2H, m, H-2,6 Ph-6), 8.74 (2H, s, NH2), 11.87 (1H, s, C=NH); 13C NMR (DMSO-d6, 100 MHz) 훿 151.33 (C-4),
149.36 (C-6), 148.28 (C-3), 145.66 (C-7a), 139.69 (C-1 Ph-1), 129.90 (C-4 Ph-6), 129.36 (C-3,5 Ph-1), 128.99 (C-
3,5 Ph-6), 128.10 (C-1 Ph-6), 127.59 (C-2,6 Ph-6), 125.66 (C-4 Ph-1), 123.59 (C-2,6 Ph-1), 112.78 (C-3a), 13.05
(CH3). Anal Calcd. for C18H16N6 (%): C, 68.34; H, 5.10; N, 26.56. Found (%): C, 68.31; H, 5.08; N, 26.61.
4-Imino-3-methyl-1-phenyl-6-(p-tolyl)-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine (6c)
o
1
Yield 73%; mp 219-221 C; IR (KBr) ν 3370, 3291, 1575, 1414, 1221, 1102, 967, 501 cm-1; H NMR (DMSO-d6,
400 MHz) 훿 2.46 (3H, s, CH3-Ph), 2.69 (3H, s, CH3-pyrazole), 7.39 (2H, d, J = 7.4 Hz, H-3,5 Ph-6), 7.50 (3H, m, H-
3,4,5 Ph-1), 7.54 (2H, d, J = 7.4 Hz, H-2,6 Ph-6), 8.20 (2H, d, J = 8.0 Hz, H-2,6 Ph-1), 8.56 (2H, s, NH2), 11.55
(1H, s, C=NH); 13C NMR (DMSO-d6, 100 MHz) 훿 150.15 (C-4), 149.71 (C-3), 146.57 (C-6), 144.85 (C-7a), 140.00
(C-4 Ph-6), 139.71 (C-1 Ph-1), 131.34 (C-2,6 Ph-6), 129.90 (C-3,5 Ph-1), 128.04 (C-3,5 Ph-6), 125.68 (C-4 Ph-1),
124.39 (C-1 Ph-6), 122.27 (C-2,6 Ph-1), 112.66 (C-3a), 20.96 (CH3-Ph), 13.05 (CH3-pyrazole). Anal Calcd. for
C19H18N6 (%): C, 69.07; H, 5.49; N, 25.44. Found (%): C, 69.12; H, 5.46; N, 25.42.
4-Imino-3-methyl-6-(4-nitrophenyl)-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine (6d)
o
1
Yield 77%; mp 264-266 C (decomp.); IR (KBr) ν 3361, 3224, 2925, 1678, 1593, 1504, 1125, 869, 577 cm-1; H
NMR (DMSO-d6, 400 MHz) 훿 2.69 (3H, s, CH3), 7.50 (2H, m, H-3,4,5 Ph-1), 8.04 (2H, d, J = 8.8 Hz, H-2,6 Ph-6),
8.13 (2H, d, J = 7.6 Hz, H-2,6 Ph-1), 8.33 (2H, d, J = 8.8 Hz, H-3,5 Ph-6), 8.63 (2H, s, NH2), 12.20 (1H, s, C=NH);
13C NMR (DMSO-d6, 100 MHz) 훿 156.05 (C-6), 151.13 (C-4), 150.52 (C-4 Ph-6), 149.92 (C-3), 145.30 (C-7a),
139.41 (C-1 Ph-1), 135.81 (C-1 Ph-6), 130.34 (C-2,6 Ph-6), 129.73 (C-3,5 Ph-1), 125.86 (C-4 Ph-1), 124.39 (C-3,5
Ph-6), 122.97 (C-2,6 Ph-1), 112.66 (C-3a), 13.05 (CH3). Anal Calcd. for C18H15N7O2 (%): C, 59.83; H, 4.18; N,
27.13. Found (%): C, 59.80; H, 4.19; N, 27.17.
6-Benzyl-4-imino-3-methyl-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amine (6e)
4

ACCEPTED MANUSCRIPT
o
1
Yield 74%; mp 212-214 C; IR (KBr) ν 3386, 3192, 1543, 1448, 1129, 865, 686 cm-1; H NMR (DMSO-d6, 400
MHz) 훿 2.69 (3H, s, CH3), 3.40 (CH2), 7.15 (1H, m, H-4 Ph-6), 7.17 (2H, m, H-2,6 Ph-6), 7.19 (2H, m, H-3,5 Ph-6),
7.44 (3H, t, J = 7.5 Hz, H-3,4,5 Ph-1), 8.26 (2H, d, J = 8.0 Hz, H-2,6 Ph-1), 8.55 (2H, s, NH2), 11.52 (1H, s,
C=NH); 13C NMR (DMSO-d6, 100 MHz) 훿 150.68 (C-4), 148.91 (C-3), 147.23 (C-6), 143.82 (C-7a), 140.37 (C-1
Ph-1), 136.18 (C-1 Ph-6), 129.80 (C-3,5 Ph-1), 129.39 (C-2,6 Ph-6), 128.44 (C-3,5 Ph-6), 125.11 (C-4 Ph-1), 124.55
(C-4 Ph-6), 123.26 (C-2,6 Ph-1), 113.73 (C-3a), 36.41 (CH2), 13.12 (CH3). Anal Calcd. for C18H14ClN5 (%): C,
64.38; H, 4.20; N, 20.86; Cl, 10.56. Found (%): C, 64.32; H, 4.13; N, 20.90; Cl, 10.65. Anal Calcd. for C19H18N6
(%): C, 69.07; H, 5.49; N, 25.44. Found (%): C, 69.08; H, 5.52; N, 25.40.
In vitro antibacterial evaluation
In vitro inhibitory activity of heterocyclic compounds and antibiotics were evaluated against thirteen pathogenic
bacteria including Gram-negative strains Shigella flexneri (PTCC 1234), Proteus mirabilis (PTCC 1776), Proteus
vulgaris (PTCC 1079), Salmonella enterica subsp. enterica (PTCC 1709), Salmonella typhi (PTCC 1609) and
Gram-positive strains Streptococcus pyogenes (PTCC 1447), Streptococcus agalactiae (PTCC 1768), Streptococcus
equinus (PTCC 1445), Streptococcus pneumoniae (PTCC 1240), Listeria monocytogenes (PTCC 1297),
Staphylococcus epidermidis (PTCC 1435), Bacillus thuringiensis subsp. kurstaki (PTCC 1494) and Rhodococcus
equi (PTCC 1633). All strains were prepared from the Persian Type Culture Collection (PTCC), Tehran, Iran. The
antibacterial activities were determined by employing broth microdilution and disk diffusion methods, according to
CLSI (Clinical and Laboratory Standards Institute) guidelines M07-A9, M26-A and M02-A11 [37]. The stock
solutions of all derivatives and antibiotics were respectively prepared in 10% DMSO and double-distilled water at
concentrations of 9011 and 17.6 μg/mL. The IZD values were measured at these initial concentrations. All
antibiogram tests were performed at least three times independently, and the results were reported as mean values ±
SD. No standard deviation was observed at mean MIC and MBC values.
Computational studies
All geometries were optimized without any symmetry constraint at the B3LYP level of theory in conjunction with 6-
311++G** basis set using Gaussian 09 program package [38]. Frequency calculations were performed at the
aforementioned level of theory and the results showed that all geometries were corresponded to true minima with no
imaginary frequency. The population analysis has been performed by the natural bond orbital (NBO) method at the
B3LYP/6-311++G** level using NBO program implemented under Gaussian 09 program package [39].
Results and Discussion
Synthesis and characterization of pyrazolo[3,4-d]pyrimidines
In this study, a new and efficient two step procedure was proposed for the synthesis of 6-substituted 4-imino-3-
methyl-1-phenyl-1,4-dihydro-5H-pyrazolo[3,4-d]pyrimidin-5-amines 6a-e (Scheme 1). 5-Amino-pyrazole-4-
carbonitrile 4 as starting material was prepared in one-pot procedure from the reaction of malononitrile (1), triethyl
5

ACCEPTED MANUSCRIPT
orthoacetate (2), phenylhydrazine (3) and a few drops of acetic acid in ethanolic solution after refluxing for 12 h.
The cyclocondensation of pyrazole 4 with hydrazides 5a-e in 0.5 M NaOEt/EtOH medium produced pyrazolo[3,4-
d]pyrimidines 6a-e in moderate yields.
Scheme 1 Synthesis of pyrazolo[3,4-d]pyrimidines 6a-e
The reaction proceeded only in the presence of sodium ethoxide as catalyst. It seems that the nucleophilicity of the
amino group was increased due to the formation of a strong hydrogen bonding with anionic oxygen of the ethoxide
(Scheme 2). The proposed mechanism was based on the same transformation observed previously [40].
Scheme 2 The proposed mechanism for the formation of products 6a-e
The chemical structure of all compounds were deduced from their spectral and analytical data. In 1H-NMR spectra,
signals attributed to H-3,4,5 and H-1,6 of 1-phenyl ring appear within 7.44-7.50 and 8.13-8.26 ppm. In addition, the
spectra showed singlet signals due to 3-CH3, 5-NH2 and 4-NH protons at 2.63-2.69, 8.44-8.74, 11.52-12.20 ppm. 13
C
NMR spectra exhibited signals belonging to C-3, C-3a, C-4, C-6 and C-7a at 147.23-149.92, 112.66-113.93, 150.15-
151.93, 146.57-156.05 and 143.82-145.66 ppm. The absorption bands appeared within ν = 3180-3291 and 3353-
3419 cm-1 corresponded to stretching vibrations of NH and NH2 groups. The purity of all derivatives also confirmed
by the microanalytical data.
Interpretation of antibacterial activities
The in vitro antibacterial activities of the newly synthesized derivatives were evaluated against a wide range of
Gram-positive and -negative pathogenic bacteria, which then compared with ceftriaxone and cefazolin antibiotics
and reported as IZD, MIC and MBC values in Table 1.
Table 1 IZD, MIC and MBC values of pyrazolo[3,4-d]pyrimidines 6a-e
Products
Bacteria
6a
6b
6c
6d
6e
Ceftriaxone
Cefazolin
IZD
MIC
MBC
IZD
MIC
MBC
IZD
-
-
-
9.54±0.24
13.22±0.11
-
-
-
-
-
-
25.88±0.63
21.64±0.53
128
256
-
-
-
64
128
0.5
1
8.27±0.26
4
8
9.26±0.32
512
2048
8.16±0.19
1024
-
-
-
-
-
7.13±0.31
256
1024
10.61±0.38
1024
7.48±0.41
512
2048
9.75±0.57
512
21.43±0.65
8
8
8
8
-
11.37±0.43
1024
21.51±0.28
2
MIC
-
6

ACCEPTED MANUSCRIPT
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
2048
-
-
-
-
-
-
7.90±0.16
512
1024
4096
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
4096
8.43±0.17
256
512
7.52±0.40
1024
2048
7.83±0.29
512
1024
8.64±0.55
2048
4096
8.18±0.38
512
1024
7.06±0.13
512
2048
-
-
-
-
-
-
7.06±0.17
2048
4096
2
-
33.91±0.44
0.063
17.38±0.49
0.25
2
-
-
1
-
-
-
-
-
-
-
7.41±0.26
2048
4096
-
8.24±0.54
8
32
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
32.64±0.37
23.25±0.22
-
-
-
-
-
-
-
-
2
8
-
-
2
2
-
10.36±0.29
36.08±0.67
256
512
-
-
-
-
8
8
-
34.08±0.26
21.88±0.61
2
4
2
2
1024
8.53±0.49
10.76±0.14
30.43±0.64
25.14±0.56
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
IZD
MIC
MBC
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
-
2048
4096
1024
2048
7.87±0.12
2048
4096
7.55±0.33
1024
2048
9.64±0.25
1024
-
-
-
-
-
-
-
-
-
-
-
0.063
0.125
8.09±0.48
0.063
0.125
21.18±0.24
-
-
-
-
-
-
-
-
-
8
16
-
-
16
64
-
-
-
-
18.54±0.23
25.90±0.37
0.5
2
1
2
2048
IZD (mm), MIC (μg/mL), MBC (μg/mL)
(-): Indicates no noticeable antibacterial effect at initial concentrations
The derivatives showed moderate to good inhibitory activities against thirteen Gram-positive and -negative bacterial
strains of various genera. The inhibitory effects against all tested Streptococcus and Proteus species were observed
from at least one heterocyclic compound. Pyrazolo[3,4-d]pyrimidine 6d was known as the most broad-spectrum
antibacterial agent due to inhibitory properties on twelve bacterial strains. Structural study of compound 6d
indicated that the presence of nitro moiety at position 4 of 6-phenyl ring as antimicrobial functional group was the
main reason for the observed difference in the inhibitory activity [41]. To the best of our knowledge, compound 6c
showed the highest inhibitory effect against Streptococcus pyogenes with MIC and MBC values of 64 and 128
μg/mL in comparison to others. There for, it is considered as the most effective antibacterial agent.
Theoretical calculations
Quantum mechanical studies were performed to shed some light on the correlation between antibacterial activities
and the molecular properties of fused heterocycles 6a-e. A general structure of heterocycles 6a-e is illustrated in Fig.
1. The results in Table 2 reflect the calculated parameters including energy, orbital energy, dipole moment,
molecular volume and surface area.
7

ACCEPTED MANUSCRIPT
Fig. 1 Typical structure of the pyrazolo[3,4-d]pyrimidines
Table 2 Quantum mechanical parameters of pyrazolo[3,4-d]pyrimidines
molecule
Energya
Dipole
HOMOa
LUMOa Molecular Molecular
momentb
volumec
surface
aread
6a
6b
6c
6d
6e
-793.187
-1024.294
-1063.622
-1228.847
-1063.615
0.631
1.652
2.264
0.965
6.052
-0.23174 -0.05577
-0.22389 -0.06622
-0.22168 -0.06371
-0.23354 -0.12399
-0.22149 -0.05288
702.18
912.13
962.50
975.48
953.39
432.75
541.81
565.86
579.46
567.80
a, b, c and d are in Hartree, Debye, Å3, and Å2, respectively.
Good structure-activity relationships were observed between the IZD values and molecular volumes/surface areas
respectively within r = 0.97 and r = 0.95 values, except for the highly active molecule 6a.
Fig. 2 Correlation between the IZD values and molecular volumes (in Å3) and surface areas (in Å2)
The molecular volumes/surface areas were directly proportional to the IZD values. No significant relationship was
observed between dipole moments and the IZD values. These results implied that dispersion forces play a
considerable role in inhibitory activity of the molecules 6b-e. Nevertheless, molecule 6a with lowest volume and
surface area showed the highest antibacterial activity. Such observations may imply that other interactions, beside
the van der Waals dispersion forces, could be involved. Recent studies indicate that reactive nucleophilic or
electrophilic species cause discrete types of injuries to microbial cells because of redox stress [42]. Accordingly, our
8

ACCEPTED MANUSCRIPT
attempts were focused on the construct a correlation between orbital energies and the IZD values of the molecules.
In reality, we have not found a straightforward correlation between HOMO, LUMO and HOMO-LUMO energy
gaps with the IZD values.
More investigations revealed that the IZD values can be correlated to the inverse of HOMO and LUMO values
pretty well, as shown in Fig. 3. It can be deduced that beside short-distance dispersion forces, covalent bonding
might be involved in the action mechanism of the antibacterial activity of compounds 6a-e.
Fig. 3 Correlation between antibacterial effects and the inverse of HOMO and LUMO values
It is difficult to interpret the interaction of probable chemical bonding according to the inverse of frontier orbital
energies. Many drug-receptor chemical interactions such as SN2, Schiff base formation and Michael addition
involve reaction of electrophiles with nucleophilic sites on biological macromolecules. The significant degree of
selectivity that occurs in electrophile-nucleophile interactions was predicted by Pearson's Hard and Soft, Acids and
Bases (HSAB) theory [43-45]. This principle implies that electrophilic molecules will preferentially react with
similar hard or soft biological targets. Hardness (HD), softness (SOF) and electrophilicity (EPH) are three important
electronic descriptors obtained from HOMO and LUMO energies. They describe stability, reactivity and a measure
of energy lowering due to maximal electron flow between the donor and acceptor [44]. These electronic features
can be calculated as HD = (LUMO-HOMO)/2, SOF = 1/HD, EPH = -(LUMO+HOMO)2/(8HD) [46-48]. These
chemical indices were calculated for all molecules 6a-e, as shown in Table 3.
Table 3 Chemical descriptors including
hardness, softness, and electrophilicity
Molecule
HDa
SOFb
EPHc
6a
6b
6c
6d
6e
0.088
0.158
0.158
0.110
0.169
11.37
6.34
6.33
9.13
5.93
-0.117
-0.067
-0.064
-0.146
-0.056
aHardness (in Hartree), bsoftness (in Hartree-1), celectrophilicity (in Hartree)
According to Figures 4 and 5, there are relatively strong relationships between the aforementioned descriptors and
the non-zero IZD values. The IZD values correlate reasonably with EPH, SOF, and HD within linear regression
coefficients of 0.90, 0.91 and 0.96, respectively. In addition to, direct and inverse correlations between softness and
hardness parameters and antibacterial effects are quite obvious.
9

ACCEPTED MANUSCRIPT
Fig. 4 Relationship between the IZDs and absolute values of electrophilicity
Fig. 5 Correlation of the IZD values with hardness (in Hartree) and softness (in Hartree-1) coefficients
The obtained results illustrated that the antibacterial activities of molecules 6a-e wererelated to redox interactions as
well as van der Waals dispersion forces. To find more verification on chemical interactions, charges on the most
important atoms in molecules were calculated. It was found that an increase in negative charge on N-3 atom of the
molecules, except for molecule 6b, was correlated to the increase of the IZD values (Fig. 6). Therefore, it would be
plausible to consider the N-3 atom connected to the amino group contributes to the electron flow between donors
and acceptors. Furthermore, sum of the atomic charges on the rings A and B were calculated for the molecules 6a-e
(Fig. 7). The inhibitory properties of molecules (except for molecule 6b) were improved by increasing negative
atomic charges on these two rings. It seems that charges flow from nucleophilic site especially N-3 atom to
electrophilic centers on the biological macromolecules. It can be concluded that the covalent bonding occurs when
the electron density flows between the reactive species of nucleophiles and electrophiles via their frontier molecular
orbitals (HOMO and LUMO). Therefore, based on HSAB theory, the nucleophilic molecules 6a-e (due to electron
rich N-3 atom) react preferentially with biological sites with similar hardness or softness. Due to the increase in
antibacterial activities of nucleophiles 6a-e with their softness, they prefer to interact with soft electrophilic
receptors.
10

ACCEPTED MANUSCRIPT
Fig. 6 The IZD values versus charge on N-3 atom in the pyrazolo[3,4-d]pyrimidines
Fig. 7 Relationship between the IZD values and the sum of atomic charges on the rings A and B
Conclusions
Some new pyrazolo[3,4-d]pyrimidines were synthesized through a novel and efficient method. Antibacterial
activities of the synthesized derivatives were evaluated against thirteen pathogenic bacteria and reported as IZD,
MIC and MBC values. Theoretical studies such as energy data and NBO analysis were carried out to determine
structure-activity relationships. Some plausible theoretical evidences proved that both physical (short-distance
forces) and chemical (covalent bonding) interactions are responsible in the observed antibacterial activities of the
pyrazolo[3,4-d]pyrimidine derivatives. Except for the smallest molecule 6a with the highest inhibitory activity, it is
concluded that van der Waals interactions might contribute to the action mechanism of these molecules. The direct
relationship between absolute values of the EPH and the inhibitory activities accentuates the role of the charge flow.
It appeared that the charge flows from nucleophilic N-3 atom on the molecules 6a-e to electrophilic sites on
biological macromolecule. Since the IZD values of the nucleophiles 6a-e increase with their softness, they interact
preferably with soft electrophilic receptors. Our models suggest that the soft pyrazolo[3,4-d]pyrimidines derivatives
with the higher electrophilicity should be considered for the synthesis of antibacterial potentials in the future.
Consequently, large electron donating substitutions on both rings A and B are recommended for the higher
biological activities.
Acknowledgment
11

ACCEPTED MANUSCRIPT
This study was funded by the University of Zabol [research project number 95-1].
Conflict of Interest
The authors declare that they have no conflict of interest.
References
1. Ayer SW, Andersen RJ, He CH, Clardy J (1984) J Org Chem 49:3869-3870, doi: 10.1021/jo00194a053.
2. Ismail NSM, Ali EMH, Ibrahim DA, Serya RAT, El Ell DAA (2016) Future J Pharm Sci 2:20-30, doi:
10.1016/j.fjps.2016.02.002.
3. Kasula M, Samunuri R, Chakravarty H, Bal C, Baba M, Jhab AK, Sharon A (2016) Nucleosides
Nucleotides Nucleic Acids 35:43-52, doi: 10.1080/15257770.2015.1114126.
4. Rostamizadeh S, Nojavan M, Aryan R, Sadeghian H, Davoodnejad M (2013) Chin Chem Lett 24:629-632,
doi: 10.1016/j.cclet.2013.04.035.
5. Vicentini CB, Romagnoli C, Andreottie E, Mares D (2007) J Agric Food Chem 55:10331-10338, doi:
10.1021/jf072077d.
6. Ma S, Zhou S, Lin W, Zhang R, Wu W, Zheng K (2016) RSC Adv 6:100772-100782, doi:
10.1039/C6RA20277B.
7. Liu H, Wang H-Q, Liu Z-J (2007) Bioorg Med Chem Lett 17:2203-2209, doi: 10.1016/j.bmcl.2007.01.083.
8. Ghorab MM, Ragab FA, Alqasoumi SI, Alafeefy AM, Aboulmagd SA (2010) Eur J Med Chem 45:171-
178, doi: 10.1016/j.ejmech.2009.09.039.
9. Antonelli A, Bocci G, La Motta C, Ferrari SM, Fallahi P, Corrado A, Fioravanti A, Sartini S, Orlandi P,
Piaggi S, Corti A, Materazzi G, Galleri D, Ulisse S, Fontanini G, Danesi R, Da Settimo F, Miccoli P (2014)
Mol Cell Endocrinol 393:56-64, doi: 10.1016/j.mce.2014.06.002.
10. Kadry HH (2014) Med Chem Res 23:5269-5281, doi: 10.1007/s00044-014-1079-9.
11. Laurenzana I, Caivano A, La Rocca F, Trino S, De Luca L, D'Alessio F, Tintori C, Simeon V, Traficante
A, Teramo A, Zambello R, Semenzato G, Schenone S, Botta M, Musto P, Del Vecchio L (2015) Blood
126:3254, doi: 10.18632/oncotarget.11496.
12. El-Hamouly WS, El-Khamry A-MA, Abbas EMH (2006) Indian J Chem 45B:2091-2098.
13. Agrebi A, Allouche F, Fetoui H, Chabchoub
10.13171/mjc.3.2.2014.13.05.23.
F (2014) Mediterr J Chem 3:864-876, doi:
14. Kumar A, Ahmad I, Chhikara BS, Tiwari R, Mandal D, Parang K (2011) Bioorg Med Chem Lett 21:1342-
1346, doi: 10.1016/j.bmcl.2011.01.047.
15. Niswender CM, Lebois EP, Luo Q, Kim K, Muchalski H, Yin H, Conn PJ, Lindsley CW (2008) Bioorg
Med Chem Lett 18:5626-5630, doi: 10.1016/j.bmcl.2008.08.087.
16. Schenone S, Brullo C, Bruno O, Bondavalli F, Mosti L, Maga G, Crespan E, Carraro F, Manetti F, Tintori
C, Botta M (2008) Eur J Med Chem 43:2665-2676, doi: 10.1016/j.ejmech.2008.01.034.
12

ACCEPTED MANUSCRIPT
17. Rashad AE, Hegab MI, Abdel-Megeid RE, Fathalla N, Abdel-Megeid F (2009) Eur J Med Chem 44:3285-
3292, doi: 10.1016/j.ejmech.2009.02.012.
18. Gupta S, Rodrigues LM, Esteves AP, Oliveira-Campos AM, Nascimento MS, Nazareth N, Cidade H,
Neves MP, Fernandes E, Pinto M, Cerqueira NM, Bras N (2008) Eur J Med Chem 43:771-780, doi:
10.1016/j.ejmech.2007.06.002.
19. Bakavoli M, Bagherzadeh G, Vaseghifar M, Shiri A, Pordel M, Mashreghi M, Pordeli P, Araghi M (2010)
Eur J Med Chem 45:647-650, doi: 10.1016/j.ejmech.2009.10.051.
20. Gilbert AM, Nowak P, Brooijmans N, Bursavich MG, Dehnhardt C, Santos ED, Feldberg LR, Hollander I,
Kim S, Lombardi S, Park K, Venkatesan AM, Mallon R (2010) Bioorg Med Chem Lett 20:636-639, doi:
10.1016/j.bmcl.2009.11.051.
21. Soth M, Abbot S, Abubakari A, Arora N, Arzeno H, Billedeau R, Dewdney N, Durkin K, Frauchiger S,
Ghate M, Goldstein DM, Hill RJ, Kuglstatter A, Li F, Loe B, McCaleb K, McIntosh J, Papp E, Park J,
Stahl M, Sung ML, Suttman R, Swinney DC, Weller P, Wong B, Zecic H, Gabriel T (2011) Bioorg Med
Chem Lett 21:3452-3456, doi: 10.1016/j.bmcl.2011.03.098.
22. Chauhan M, Kumar R (2013) Bioorg Med Chem 21:5657-5668, doi: 10.1016/j.bmc.2013.07.027.
23. Liu J, Zhang X-W, Wang Y, Chen Y, Zhang M-R, Cai Z-Q, Zhou Y-P, Xu L-F (2015) Synth Commun
45:1009-1017, doi: 10.1080/00397911.2014.996296.
24. Moyano EL, Colomer JP, Yranzo GI (2008) Eur
10.1002/ejoc.200701109.
J
Org Chem 2008:3377-3381, doi:
25. Salaheldin AM, Oliveira-Campos AMF, Rodrigues LM (2007) Tetrahedron Lett 48:8819-8822, doi:
10.1016/j.tetlet.2007.10.079.
26. Elnagdi MH, Sadek KU, Galil FMA, Hassan SME (1988) Arch Pharm 321:851-854, doi:
10.1002/ardp.19883211205.
27. Tominaga Y, Yoshioka N, Kataoka S, Aoyama N, Masunari T, Miike A (1995) Tetrahedron Lett 36:8641-
8644, doi: 10.1016/0040-4039(95)01862-C.
28. Elkholy A, Al-Qalaf F, Elnagdi MH (2008) Arkivoc 2008:124-131, doi: 10.3998/ark.5550190.0009.e14.
29. dos Santos Faiões V, Leon LL, Canto-Cavalheiro MM, Torres-Santos EC, Bernardino AM, Vegi PF, dos
Santos MS (2014) Chem Biol Drug Des 83:272-277, doi: 10.1111/cbdd.12235.
30. Romdhane A, Ben Said A, Cherif M, Ben Jannet H (2016) Med Chem Res 25:1358-1368, doi:
10.1007/s00044-016-1576-0.
31. Sagar SR, Agarwal JK, Pandya DH, Dash RP, Nivsarkar M, Vasu KK (2015) Bioorg Med Chem Lett
25:4428-4433, doi: 10.1016/j.bmcl.2015.09.015.
32. Youssef AM, Neeland EG, Villanueva EB, White MS, El-Ashmawy IM, Patrick B, Klegeris A, Abd-El-
Aziz AS (2010) Bioorg Med Chem 18:5685-5696, doi: 10.1016/j.bmc.2010.06.018.
33. Radi M, Tintori C, Musumeci F, Brullo C, Zamperini C, Dreassi E, Fallacara AL, Vignaroli G, Crespan E,
Zanoli S, Laurenzana I, Filippi I, Maga G, Schenone S, Angelucci A, Botta M (2013) J Med Chem
56:5382-5394, doi: 10.1021/jm400233w.
13

ACCEPTED MANUSCRIPT
34. Eweas AF, Swelam SA, Fathalla OA, Fawzy NM, Abdel-Moez SI (2012) Med Chem Res 21:3848-3857,
doi: 10.1007/s00044-011-9911-y.
35. Önal Z, Yildirim I, Kandemirli F, Arslan T (2010) Struct Chem 21:809-816, doi: 10.1007/s11224-010-
9615-9.
36. Allouche F, Chabchoub F, Carta F, Supuran CT (2013) J Enzyme Inhib Med Chem 28:343-349, doi:
10.3109/14756366.2012.720573.
37. Balouiri M, Sadiki M, Ibnsouda SK (2016) J Pharm Anal 6:71-79, doi: 10.1016/j.jpha.2015.11.005.
38. Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE, Robb MA, Cheeseman JR, Scalmani G, Barone V,
Mennucci B, Petersson GA, Nakatsuji H, Caricato M, Li X, Hratchian HP, Izmaylov AF, Bloino J, Zheng
G, Sonnenberg JL, Hada M, Ehara M, Toyota K, Fukuda R, Hasegawa J, Ishida M, Nakajima T, Honda Y,
Kitao O, Nakai H, Vreven T, Montgomery JA, Peralta JE, Ogliaro F, Bearpark M, Heyd JJ, Brothers E,
Kudin KN, Staroverov VN, Kobayashi R, Normand J, Raghavachari K, Rendell A, Burant JC, Iyengar SS,
Tomasi J, Cossi M, Rega N, Millam JM, Klene M, Knox JE, Cross JB, Bakken V, Adamo C, Jaramillo J,
Gomperts R, Stratmann RE, Yazyev O, Austin AJ, Cammi R, Pomelli C, Ochterski JW, Martin RL,
Morokuma K, Zakrzewski VG, Voth GA, Salvador P, Dannenberg JJ, Dapprich S, Daniels AD, Farkas O,
Foresman JB, Ortiz JV, Cioslowski J, Fox DJ (2009) Gaussian 09, Revision A 02 Gaussian Inc.,
Wallingford CT.
39. Glendening ED, Reed AE, Carpenter JE, Weinhold F NBO Version 3.1.
40. Tyurin RV, Vorob’ev EV, Minyaeva LG, Krasnikov VV, Mezheritskii VV (2005) Russ J Org Chem
41:916-921, doi: 10.1007/s11178-005-0266-z.
41. Strauss MJ (1979) Ind Eng Chem Prod Res Dev 18:158-166, doi: 10.1021/i360071a002.
42. Valko M, Morris H, Cronin MTD (2005) Curr Med Chem 12:1161-1208, doi: 10.2174/0929867053764635.
43. LoPachin RM, Barber DS, Gavin T (2008) Toxicol Sci 104:235-249, doi: 10.1093/toxsci/kfm301.
44. LoPachin RM, Gavin T, Petersen DR, Barber DS (2009) Chem Res Toxicol 22:1499-1508, doi:
10.1021/tx900147g.
45. Pearson RG (1990) Coord Chem Rev 100:403-425, doi: 10.1016/0010-8545(90)85016-L.
46. Parr RG, Chattaraj PK (1991) J Am Chem Soc 113:1854-1855, doi: 10.1021/ja00005a072.
47. Pearson RG (1990) in Theoretical Models of Chemical Bonding Part II, Maksic ZB (Ed.), Springer, Berlin,
p. 45.
48. Thanikaivelan P, Subramanian V, Raghava Rao J, Unni Nair B (2000) Chem Phys Lett 323:59-70, doi:
10.1016/S0009-2614(00)00488-7.
14