Multistep Synthesis of Fluorine-Substituted Pyrazolopyrimidine Derivatives With Higher Antibacterial Efficacy Based on In Vitro Molecular Docking and Density Functional Theory

Article abstract of DOI:10.1002/jhet.2923

Some new fluorine-substituted pyrazolopyrimidine derivatives (1.3–1.5) have been synthesized by the multistep reactions, starting from reaction of 4-fluorophenyl malanonitrile (1) with guanidine, followed by ring closure reaction with hydrazine to give 2,

Full text of DOI:10.1002/jhet.2923

Month 2017
Multistep Synthesis of Fluorine-Substituted Pyrazolopyrimidine
Derivatives With Higher Antibacterial Efficacy Based on In Vitro
Molecular Docking and Density Functional Theory
Salman A. Khan, Abdullah M. Asiri, R. M. Rahman,a Shaaban A. Elroby,a,c Faisal M. S. Aqlan,d
a
a,b
*
Mohmmad Y. Wani,e and Kamlesh Sharmaf
aChemistry Department, Faculty of Science, King Abdulaziz University, P.O. Box 80203, Jeddah 21589, Saudi Arabia
bCenter of Excellence for Advanced Materials Research (CEAMR), King Abdulaziz University, P.O. Box 80203, Jeddah
21589, Saudi Arabia
cChemistry Department, Faculty of Science, Benisuief University, Benisuief, Egypt
dChemistry Department, Faculty of Science, Jeddah University, Jeddah, Saudi Arabia
eTexas Therapeutics Institute, Brown Foundation Institute of Molecular Medicine, The University of Texas Health Science
Center at Houston, 1881 East Road, 77054, Texas, USA
fDepartment of Applied Science, School of Engineering and Technology, ITM University, Sector 23A, Gurgaon 122017,
India
*
E-mail: sahmad_phd@yahoo.co.in
Received January 13, 2017
DOI 10.1002/jhet.2923
Published online 00 Month 2017 in Wiley Online Library (wileyonlinelibrary.com).
Some new fluorine-substituted pyrazolopyrimidine derivatives (1.3–1.5) have been synthesized by the
multistep reactions, starting from reaction of 4-fluorophenyl malanonitrile (1) with guanidine, followed by
ring closure reaction with hydrazine to give 2,4-diamino-6-aryl-pyrimidine-5-carbonitrile (1.2). Structure
of the compounds was conformed by the spectral and elemental analyses. The antibacterial activity of these
compounds was tested in vitro by the disk diffusion assay against two Gram-positive and two Gram-negative
bacteria, and then the minimum inhibitory concentration was determined with reference to the standard drug
chloramphenicol. The results showed that compound 1.5 is better at inhibiting growth as compared with
chloramphenicol of both types of bacteria (gram-positive and gram-negative). Molecular orbital calculations
were carried out by using the density functional theory. The B3LYP/6-311 + G** level of theory was
employed to explore LUMO–HOMO gap energy and charge distribution of compounds 1.1 to 1.5. Docking
studies were performed on bacterial glucosamine-6-phosphate synthetase enzyme. Density functional theory
calculations and docking studies support the in vitro findings.
J. Heterocyclic Chem., 00, 00 (2017).
INTRODUCTION
resistant, resulting in a potential global health crisis.
There is already evidence that antibacterial resistance is
associated with an increase in mortality [5]. Frequently, it
is recommended to use new antibacterial agents with
enhanced broad-spectrum potency. Therefore, recent
efforts have been directed toward exploring novel
antibacterial agents. Nitrogen-containing heterocyclic
compounds with special reference of pyrimidine are
prominent, and therefore, various procedures have been
worked out for their synthesis [6]. Several pyrimidine
derivatives have been found to possess considerable
biological activities, which stimulated research activity in
this field. Their prominent effects are, for example,
antimicrobial [7], central nervous system [8], and
immunosuppressive [9] activities. There are several
substituted pyrazolines that have bleaching property or
Gram-positive and Gram-negative pathogens such as
Staphylococcus aureus, Staphylococcus pyogenes,
Salmonella typhimurium, and Escherichia coli are
causing food poisoning, rheumatic fever, salmonellosis,
diarrhea, and many other infectious diseases [1]. These
are serious health problems worldwide. More than 50
million people worldwide are infected, and up to 150 000
die every year due to these bacterial infections [2].
Amoxicillin, norfloxacin, and ciprofloxacin are the
principal drugs of choice in the treatment of bacterial
infection [3]. But several side effects such as nausea,
metallic taste, dizziness, hypertension, and resistance
have been reported [4]. After years of misuse and
overuse of antibiotics, bacteria are becoming antibiotic
© 2017 Wiley Periodicals, Inc.

S. A. Khan, A. M. Asiri, R. M. Rahman, S. A. Elroby, F. M. S. Aqlan, M. Y. Wani, and K. Sharma Vol 000
act as luminescent and fluorescent agents [10]. They are
also useful as biodegradable agrochemicals [11]. Bicyclic
pyrazolopyrimidine derivatives dramatically increase the
biological activates such as antibacterial [12], analgesic
[13], anti-inflammatory [14], antiviral [15,16], antifungal
[17], antiarthritic [18], cerebroprotective effect [19], and
antidepressant [20]. In addition, introduction of fluorine
atoms to heterocyclic nitrogen systems enhances and
improves their pharmacological properties such as
enhancing the electrostatic force and hydrophobic binding
stability against metabolic transformations [21–23].
Encouraged by these facts and in continuation with the
work related to the synthesis, spectral studies, and
biological properties of pyrazolopyrimidine, the synthesis
of some novel pyrazolopyrimidine is reported here.
Furthermore, the geometric structures of synthesized
compounds were also computationally calculated by
using density functional theory with RB3LYP method
and interaction of the compounds with their probable
target was established by docking studies.
RESULTS AND DISCUSSION
Chemistry.
In the present work, pyrazolopyrimidine
derivatives (1.3, 1.4, and 1.5) were prepared by the
multistep reaction [24]. The synthetic route of compounds
is outlined in Scheme 1. The chemical structures of the
synthesized compounds were established by spectroscopic
(FT-IR, 1H-NMR, 13C-NMR, and mass) and elemental
analysis. Based on these facts, the present work reports an
easy and efficient route for the synthesis of fluorine-
substituted pyrazolopyrimidines in view of their
antibacterial effects. Thus, cycloaddition of arylidene
malononitrile 1.1 with guanidine hydrochloride in refluxing
absolute ethanol-anhydrous K2CO3 yielded the 2,4-
diamino-6-(4-fluorophenyl)pyrimidin-5-carbonitrile 1.2.
The structure of 1.2 was deduced from that spectral data.
IR spectrum showed the absorption bands at v 3289,
2225, and 1250 cmÀ1 for NH, CN, and C─F function
groups, while 1H-NMR spectrum recorded resonated
signals at δ 9.97 and 9.73 ppm attributed to two NH2
Scheme 1. Synthesis of fluorine-substituted pyrazolopyrimidine derivatives. [Color figure can be viewed at wileyonlinelibrary.com]
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Fluorine-Substituted Pyrazolopyrimidine Derivatives With Higher Antibacterial
Efficacy
protons. Also, 13C-NMR exhibited resonated signals at δ
168.91, 50.74, and 24.35 ppm for NH═C, C─F, and
C─N. It is interesting that a simple nucleophilic attack of
hydrazine hydrate to a more electrophilic carbon of CN
in refluxing absolute ethanol afforded 4-(4-fluorophenyl)-
1H-pyrazolo[3,4-D]pyrimidine-3,6-diamine (1.3) via loss
1 mol of NH3.
E. coli. In the disk-diffusion method, sterile paper discs
(0.5 mm) impregnated with compound dissolved in
dimethyl sulfoxide (DMSO) at concentration of
100 μg/mL were used. Then, the paper discs impregnated
with the solution of the compound tested were placed on
the surface of the media inoculated with the
microorganism. The plates were incubated at 35°C for
24 h. After incubation, the growth inhibition zones are
shown in Table 1. The compounds were further checked
by MIC method. The results are presented in Table 2.
The former structure of 1.3 was established from the 1H-
NMR, which showed δ at 8.62, 8.61, and 8.56 ppm due to
the presence of NH2, NH2, and NH. Also, IR spectrum
showed the lacks of CN group. The full fluorinated
pyrazolo[3,4-b]pyrimidine derivatives 1.4 and 1.5 have
been obtained from fluorinated acylation and/or
benzeylation of compound 1.3 via warming with
trifluoroacetamide and/or 4-fluorobeneyl chloride in
acetic acid or dry C6H6-TEA (Scheme 1).
Molecular orbital calculations.
B3LYP/6-311 + G**
level of theory was employed to investigate the electronic
c structure of the compounds 1.1 to 1.5. Highest
occupied molecular
orbitals
(HOMOs),
lowest
unoccupied molecular orbitals (LUMOs), and Frontier
molecular orbitals (FMOs) can be used to characterize the
chemical reactions. The HOMO energy characterizes the
ability to donate an electron, and LUMO energy
characterizes the ability to obtain an electron. The energy
gap (LUMO–HOMO) is also used for determining the
molecular chemical stability and biological activity.
The energies of the FMOs of the five studied
compounds, HOMO, LUMO, and energy gap were
calculated at B3LYP/6-311 + G(d,p) theory level to
explore the relation between the activity and electronic
structure. HOMO, LUMO, energy gap, and the optimized
geometric structure of the studied compounds (1.1–1.5)
are presented in Table 3 and Figure 1, which display the
electron densities of the FMOs (HOMO and LUMO) for
the five molecules.
As can be seen from Figure 1, The LUMO is mainly
delocalized among all the atoms for all molecules, except
compounds 1.4 and 1.5. On other hand, the HOMOs are
localized on the nitrogen atoms, except in compounds 1.1
and 1.5. The FMOs for compound 1.5 have unique
electron density distribution properties than other
compounds. Besides these distributions, the LUMO
energy value (2.96 eV) for compound 1.5 is high. The
mean value of the energy gap for the studied compounds
The fine structure of compounds 1.4 and 1.5 was
deduced from corrected elemental analysis and their
1
spectral data. H-NMR of 1.4 showed resonated signals
at δ 7.82 and 7.53 ppm for the NH, while 13C-NMR
recorded resonated signals at δ 176.40 and 169.24 ppm
for two C═O carbons, in addition at δ 25.47, 25.42, and
24.35 ppm for their C─F. The IR spectrum of 1.4
exhibited the absorption bands at v 1637 cmÀ1 and
attributed ─CONH-functional groups.
Finally, the former structure of 1.5 was supported from
its 13C-NMR, which showed δ at 170.73 and 167.14 ppm
for two CO–CF3 and 165.45 ppm for CF3. 1H-NMR
spectrum exhibited resonated signals at δ 8.14, 8.11, and
7.89 ppm for three NH groups. In addition, mass
fragmentation measurement recorded the M+ +1 at 438
by 56% with a base peak at 95 with 100%.
In vitro screening: disc-diffusion and microdilution
assay.
The compounds (1.1–1.5) were tested for their
antibacterial activities by disk-diffusion method by using
nutrient broth medium [contained (g/L): beef extract 3 g;
peptone 5 g; pH 7.0] [25,26]. The Gram-positive bacteria
and Gram-negative bacteria utilized in this study
consisted of S. aureus, S. pyogenes, S. typhimurium, and
Table 1
Antibacterial activity of synthesized compounds (1.1–1.5), positive control chloramphenicol (chlora.), and negative control (DMSO) measured by the
Halo Zone Test (unit, mm) and their cytotoxicity.
Corresponding effect on microorganisms
Cytotoxicity
activity IC50 (in
Compounds
S. aureus
S. pyogenes
S. typhimurium
E. coli
mM/mL) (L123 cell)
1.1
1.2
1.3
1.4
1.5
Chlora.
DMSO
8.8 0.3
11.2 0.2
14.2 0.3
16.2 0.3
19.6 0.2
17.0 0.5
8.6 0.2
9.2 0.4
15.4 0.4
17.4 0.5
18.8 0.3
18.2 0.4
9.0 0.3
10.2 0.3
14.8 0.4
16.2 0.4
16.8 0.4
17.2 0.8
8.2 0.4
10.6 0.2
13.4 0.4
18. 2 0.1
22.4 0.4
20.0 0.2
50
>100
50
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S. A. Khan, A. M. Asiri, R. M. Rahman, S. A. Elroby, F. M. S. Aqlan, M. Y. Wani, and K. Sharma Vol 000
Table 2
1 mg/mL in serum-free medium) was added and incubated
Minimum inhibition concentration (MIC) of synthesized compounds
for 4 h at 37°C. After 4 h of incubation, the plate was
inverted on tissue paper to remove the MTT reagent. To
solubilize formazan crystals in the wells, 100 mL of 100%
DMSO was added to each well. The optical density was
measured by microtiter plate reader at 590 nm. Compound
concentration (mM) is required to reduce the viability of
mock-infected cells by 50% as determined by MTT
method, which is summarized in Table 1. The results of
MTT assay are mentioned on the cell viability based on the
ability to metabolize the compounds. The results show that
compound 1.4 is comparatively more cytotoxic than 1.3
and 1.5, as these derivatives showed IC50 values more than
100 mM/mL. It means that 4-(4-fluorophenyl)-1H-pyrazolo
(1.1–1.5) products, positive control: chloramphenicol.
MIC (μg mLÀ1) compound
Positive
Bacterial strain
1.1
1.2
1.3
1.4
1.5
control
S. aureus
512
512
512
512
512
256
512
256
128
64
256
128
64
32
64
64
16
16
32
32
32
32
32
32
S. pyogenes
S. typhimurium
E. coli
is found around 4.25 eV. This value makes these
compounds chemically quite stable and reactive as
antibacterial, especially compound 1.5. In conclusion,
compound 1.5 has low gap energy and high LUMO
energy value, which makes this structure as a good
antibacterial. These results are in good agreement with
the experimental results.
[3,4-D]pyrimidine-3,6-diamine
(1.3)
and
3,6-di
(trifluoroacetamide)-4-(4-fluorophenyl)-1H-pyrazolo[3,4-
D]pyrimidine (1.5) derivative showed higher cytotoxicity
that is dose-related.
Molecular docking. Docking of the target compound
1.5 into GlmS active site revealed that several molecular
interactions were responsible for the observed affinity. The
best docking energy model and most possible interaction
mode between ligand (1.5) and GlmS are shown in
Figure 2. It was observed that the compound mainly
interacts with glucosamine-6-phosphate synthase by
binding at active sites SER-303, SER-349, GLN-349, and
THR-352. The binding affinity values of the docked target
compound (1.5) were found to be À7.3 kcal molÀ1. The
results reveal that the nitrogen atoms of the pyrazole ring
show strong hydrogen bonding interaction with the amino
acid residues of the protein, which clearly advocates its
better antibacterial efficacy. From these results, it can be
inferred that the compounds probably show their
antibacterial activity by inhibiting the glucosamine-6-
phosphate synthetase enzyme, which is an important
enzyme for bacterial cell wall formation. Therefore, these
preliminary results indicate that pyrazole derivatives can
be the most promising therapeutic agents against bacterial
infections, which needs further optimization. The strategy
presented here therefore has potential for the discovery of
novel GlmS inhibitors as antibacterial agents.
EXPERIMENTAL
All chemicals and solvents used for this work were
obtained from Merck (Germany) and Aldrich chemical
company (USA). The melting points of the synthesized
compounds were determined in open-glass capillaries on
Stuart-SMP10 melting point apparatus (Staffordshire, UK)
and are uncorrected. IR absorption spectra were recorded
on Shimadzu FTIR-8400s (Kyoto, Japan) by using KBr
pellets in the range of 4000–400 cmÀ1. 1H-NMR and 13C-
NMR spectra were recorded on BRUCKER-AVANCE-III
600-MHz spectrophotometer (Karlsruhe, Germany) and
1
TMS (tetramethylsilane) as an internal standard. The H-
NMR and 13C-NMR chemical shifts were reported as parts
per million (ppm) downfield from TMS (Me4Si). The
splitting patterns are designated as follows: s, singlet; d,
doublet; m, multiplet. Mass spectra were recorded on EI-
MS spectrometer. IR, 1H-NMR, 13C-NMR, and mass
spectra were consistent with the assigned structures.
Elemental analyses (C, H, and N) were done on a CHN rapid
analyzer. All the new compounds gave C, H, and N analyses
within 0.03% of the theoretical values. Purity of the
compounds was checked by thin-layer chromatography
(TLC) on Merck silica gel 60F254 precoated sheets in
chloroform/methanol mixture, and spots were developed
by using iodine vapors/ultraviolet light as visualizing agent.
Cytotoxicity against L123 (human lung cells).
Cyto-
toxicity was performed by MTT assay method [27]. A 96-
well flat bottom tissue culture plate was seeded with
2 × 103 cells in 0.1 mL of MEM medium supplemented
with 10% FBS and allowed to attach for 24 h. After 24 h of
incubation, the cells were treated with test compounds to
obtain concentrations of 5, 10, 20, 50, and 100 mM/mL
incubated for 48 h. The cells in the control group received
only the medium containing the 0.2% DMSO. Each
treatment was performed in duplication. After the
treatment, drug-containing media were removed and
washed with 200 mL of phosphate-buffered saline. To each
well of the 96-well plate, 100 mL of MTT reagent (stock:
4-Fluorobenzylidene-propanedinitrile (1.1).
A mixture
of 4-fluorobenzylaldehyde
3
g
(0.024 mol) and
malononitrile 1.59 g (0.024 mol) in anhydrous ethanol
(15 ml), in the presence of a few drops of pyridine, was
refluxed at 80°C for 3 h with continuous stirring. The
progress of the reaction was monitored by TLC. After
completion of the reaction, the solution was cooled. The
heavy precipitate thus obtained was collected by filtration
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Table 3
Optimized geometric structures and FMO energies of compounds 1.1 to 1.5 calculated by using DFT/RB3LYP/6-311 + G** level of theory.
HOMO
LUMO
eV
Energy gap
Structures
À7.452
À3.345
À2.130
4.107
À6.429
4.298
À5.965
À1.857
4.108
À6.813
À2.319
4.494
À7.385
À2.964
4.421
and purified by recrystallization from a mixture of
methanol and chloroform.
25.3, 24.3; EI-MS m/z (rel. int.%): 173 (56) [M + 1]+; IR
(KBr) vmax cmÀ1: 3256 (Ar─H), 2951 (C─H), 1597 (C═C),
1508 (HC═N). Anal. Calcd. for C10H5N2: C, 69.77, H,
2.93, N, 16.27. Found: C, 69.74, H, 2.89, N, 16.23.
Dark orange solid (chloroform); yield: 78%; mp 112°C;
1H-NMR (DMSO-d6) (δ/ppm): 7.92 (s, CH═C), 7.80 (d,
CH aromatic, J = 7.6 Hz), 7.78 (d, CH aromatic,
J = 7.8 Hz), 6.96 (d, CH aromatic, J = 7.8 Hz), 6.82 (d, CH
aromatic, J = 7.6 Hz); 13C-NMR (DMSO-d6) (δ/ppm):
166.9, 165.2, 158.2, 154.4, 133.9, 127.3, 119.7, 114.7,
6-Amino-4-(4-fluorophenyl)-2-imino-1,2-dihydropyrimidine-
5-carbonitrile (1.2).
propanedinitrile (1)
A mixture of 4-fluorobenzylidene-
(0.0116 mol) guanidine
2
g
hydrochloride (0.0120 mol) in anhydrous ethanol (15 mL),
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S. A. Khan, A. M. Asiri, R. M. Rahman, S. A. Elroby, F. M. S. Aqlan, M. Y. Wani, and K. Sharma Vol 000
Figure 1. The electronic densities of HOMO and LUMO for compounds 1–5 using B3LYP/6-311 + G** level of theory. [Color figure can be viewed at
wileyonlinelibrary.com]
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Figure 2. Compound (1.5) showing interaction with SER-303, SER-349, GLN-349, and THR-352 residues of the bacterial G-6-P synthetase protein
(different poses of the ligand protein interaction are shown in cartoon and surface view). Compound is represented by sticks in gray color. Hydrogen bonds
represented by using dashed lines (red). Images are visualized by using PYMOL. [Color figure can be viewed at wileyonlinelibrary.com]
in the presence of K2CO3, was refluxed at 80°C for 6 h with
continuous stirring. The progress of the reaction was
monitored by TLC. After completion of the reaction, the
solution was cooled. The heavy precipitate thus obtained
was collected by filtration and purified by recrystallization
from a mixture of methanol and chloroform.
Dark orange solid (chloroform); yield: 72%; mp 128°C;
1H-NMR (DMSO-d6) (δ/ppm): 9.97 (s, NH2), 9.73 (s,
NH2), 7.32–7.02 (m, 4H, CH aromatic); 13C-NMR
(DMSO-d6) (δ/ppm): 168.9, 165.3, 162.6, 159.5, 151.2,
132.4, 131.9, 130.7, 129.0, 116.9, 50.7, 24.3; EI-MS m/z
(rel. int.%): 231(62) [M + 1]+; IR (KBr) vmax cmÀ1: 3289
(N─H), 2923 (C─H), 2225 (CN), 1571 (C═C), 1507
(C═N), 1250 (C─F), 1155 (C─N). Anal. Calcd. for
C11H8FN5: C, 57.64, H, 3.52, N, 30.55. Found: C, 57.61,
H, 3.45, N, 30.48.
4-(4-Fluorophenyl)-1H-pyrazolo[3,4-d]pyrimidine-3,6-diamine
(1.3). A mixture of 6-amino-4-(4-fluorophenyl)-2-imino-
1,2-dihydropyrimidine-5-carbonitrile
(2)
1.5
g
(0.0065 mol) was refluxed with hydrazine hydrate
(0.0070 mol) in dry EtOH (30 mL) and was heated at 80°
C for 4 h. The progress of the reaction was monitored by
TLC. After completion of the reaction, the solvent was
removed under reduced pressure and the residue obtained
was purified by column chromatography (20:80, diethyl
ether: petroleum ether). The obtained solid was
crystallized from EtOH to yield compound no. 3. dark
brown solid.
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Dark orange solid (chloroform); yield: 65%; mp 154°C;
1H-NMR (DMSO-d6) (δ/ppm): 8.62 (s, NH2), 8.61 (s,
NH2), 8.56 (s, NH), 7.38–6.90 (m, 4H, CH aromatic); 13C-
NMR (DMSO-d6) (δ/ppm): 168.9, 165.3, 160.9, 141.9,
132.4, 130.6, 127.8, 114.3, 50.7, 25.8, 15.1; EI-MS m/z
(rel. int.%): 246 (65) [M + 1]+; IR (KBr) vmax cmÀ1: 3388
(NH2), 3251 (N─H), 3221 (Ar─H), 2951 (C─H), 1558
(C═N), 1153 (C─N). Anal. Calcd. for C11H9FN6: C, 54.10,
H, 3.71, N, 34.41. Found: C, 54.07, H, 3.67, N, 34.33.
McFarland protocol in saline solution to produce a
suspension of about 105 cfu mLÀ1. Ten microliters of this
suspension was mixed with sterile antibiotic agar (10 mL)
at 40°C and poured onto an agar plate in a laminar flow
cabinet. Five paper disks (6.0 mm diameter) were fixed
onto a nutrient agar plate. Ten milligrams of each test
compound was dissolved in DMSO (100 μL) to prepare
stock solution, and from the stock solution, different
concentrations of 10 (1 μL of stock solution + 9 μL of
solvent), 20 (1 μL of stock solution + 4 μL of solvent), 25
(1 μL of stock solution + 3 μL of solvent), 50 (1 μL of
stock solution + 1 μL of solvent), and 100 μg/μL of each
test compound were prepared. These compounds of
different concentrations were poured over a disk plate.
Chloramphenicol (30 μg) was used as standard drug
(positive control). A DMSO-wetted disk was used as
negative control. The susceptibility of the bacteria to the
test compounds was determined by the formation of an
inhibitory zone after 18 h of incubation at 36°C. Table 1
reports the inhibition zones (mm) of each compound, and
the controls in this experiment were repeated two times for
each compound and found the same results.
3,6-Di(4-fluorobenzamide)-4-(4-fluorophenyl)-1H-pyrazolo[3,4-
d]pyrimidine (1.4). A mixture of 4-(4-fluorophenyl)-1H-
pyrazolo[3,4-D]pyrimidine-3,6-diamine (1.3) (3) (0.3 g,
0.0012 mol) and 4-fluorobenzoyl chloride (0.0028 mol)
in 15 mL of DMF in the presence of a few drops of TEA
was refluxed at 80°C for 8 h with continuous stirring.
The progress of the reaction was monitored by TLC.
After the completion of the reaction, the reaction mixture
was poured into ice water to obtained precipitated solid
and was filtered and recrystallized in methanol.
Dark orange solid (chloroform); yield: 75%; semisolid 1H-
NMR (DMSO-d6) (δ/ppm): 7.82 (s, NH), 7.53 (s, NH), 7.28–
6.20 (m, 12H, CH aromatic); 13C-NMR (DMSO-d6) (δ/
ppm): 176.4, 169.2, 165.0, 163.6, 162.2, 131.8, 131.8,
130.6, 130.6, 129.9, 128.9, 116.8, 116.2, 116.1, 115.9,
115.7, 114.2, 78.6, 48.9, 44.6, 25.5, 25.4, 24.3, 22.6. EI-MS
Theoretical method. The computational calculation of
compounds 1.1–1.5 was performed by using SPARTAN'08
Windows graphical software with density functional
theory, DFT/6-31G* basis set [28]. This method has been
previously used successfully for the small molecule’s
calculations [29]. The fundamental frequencies of
optimized structure were also calculated and assigned as
minima (no negative frequencies).
m/z (rel. int.%): 490 (76) [M + 1]+; IR (KBr) vmax cmÀ1
:
3262 (N─H), 2951(C─H), 1637 (C═O), 1597 (C═C), 1529
(C═N), 1150 (C─N). Anal. Calcd. for C25H15F3N6O2: C,
61.48, H, 3.10, N, 17.21. Found: C, 61.38, H, 3.07, N, 17.16.
3,6-Di(trifluoroacetamide)-4-(4-fluorophenyl)-1H-pyrazolo[3,4-
d]pyrimidine (1.5). A mixture of 4-(4-fluorophenyl)-1H-
Docking studies.
The 3D structures of the target
pyrazolo [3,4-D]pyrimidine-3,6-diamine (1.3) (3) (0.3 g,
0.0012 mol) and trifluoroacetyl chloride (0.0028 mol) in
15 mL of acetic acid was refluxed at 80°C for 8 h with
continuous stirring. The progress of the reaction was
monitored by TLC. After the completion of the reaction,
the reaction mixture was poured into ice water. The
obtained precipitated solid was filtered and recrystallized
in methanol. Dark orange solid (chloroform); yield: 78%;
compound (1.5) were created by CHEM DRAW ULTRA 8.0
and converted to the pdb file format. Ligand preparation
was conducted by assigning Gastegier charges, merging
nonpolar hydrogens, and saving it in pdbqt file format by
using AUTODOCK TOOLS 4.2. The crystal structure of the
glucosamine-6-phasphate synthase was downloaded from
Protein Data Bank (http: //www.rcsb.org/pdb. Code:
2VF4). AUTODOCK used the local search to search for the
optimum binding site of small molecules to the protein.
1
mp 166°C; H-NMR (DMSO-d6) (δ/ppm): 8.14 (s, NH),
8.11 (s, NH), 7.89 (s, NH), 7.42–7.02 (m, 4H, CH
aromatic); 13C-NMR (DMSO-d6) (δ/ppm): 170.7, 167.1,
165.4, 132.8, 132.8, 125.6, 125.6, 115.8, 115.7, 115.7,
115.6, 125.5, 115.3, 45.9, 8.6. EI-MS m/z (rel. int.%):
438 (56) [M + 1]+; IR (KBr) vmax cmÀ1: 3319 (N─H),
2948 (C─H), 1672 (C═O), 1558 (C═C), 1524 (C═N),
1128 (C─N). Anal. Calcd. for C15H7F7N6O2: C, 41.30,
H, 1.62, N, 19.26. Found: C, 41.25, H, 1.56, N, 19.19.
Organism culture and in vitro screening. Antibacterial
activity was assayed by the disk-diffusion method with
minor modifications. S. aureus, S. pyogenes,
S. typhimurium, and E. coli were subcultured in Brain Heart
Infusion medium and incubated for 18 h at 37°C, and then
the bacterial cells were suspended according to the
The active site was defined by
a grid box of
80 × 60 × 88 points and spacing of 0.375 Å with the
ligand binding site as the center. The final structure was
then saved in pdbqt format. Molecular docking
calculations were carried out with AUTODOCK VINA [30].
The conformation with the lowest binding free energy
was used for analysis. All molecular docked models were
prepared by using PYMOL viewer.
CONCLUSION
Based on the DFT/RB3LYP model, some heterocyclic
compounds were synthesized and screened for the
Journal of Heterocyclic Chemistry
DOI 10.1002/jhet

Month 2017
Fluorine-Substituted Pyrazolopyrimidine Derivatives With Higher Antibacterial
Efficacy
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pyrimidine-5-carbonitrile (1.2). The antibacterial activity
of these compounds was examined and the results showed
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compared to the reference drug. Among all the five
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showed better antibacterial activity on both types of
bacteria than the reference drug chloramphenicol. It was
observed theoretically that the compound with lower
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substituted derivatives play an important role in
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immense difference in the activity of the drug. This
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infective disease. The field is further open for
pharmacokinetics and clinic studies to establish these
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Acknowledgments. The authors are thankful to the Chemistry
Department at King Abdulaziz University for providing research
facilities.
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Journal of Heterocyclic Chemistry
DOI 10.1002/jhet