Design and Synthesis of Novel 1-substituted-3-(3-(3-nitrophenyl)-4-oxo-3,4-dihydrobenzopyrimidin-2-yl amino) Isothioureas for their Anti-HIV, Antibacterial Activities, Graph Theoretical Analysis, Insilico Modeling, Prediction of Toxicity and Metabolic Stu

Article abstract of DOI:10.1055/a-0991-7617

In the present study, we have placed the substituted thiosemicarbazide moiety at the C-2 position and 3-nitrophenyl group at N-3 position of benzopyrimidines and studied their antitubercular, anti-HIV and antibacterial activities against selected gram pos

Full text of DOI:10.1055/a-0991-7617

Published online: 2020-06-19
Thieme
Original Article
Narendhar B et al. Design and synthesis of… Drug Res 2019;
00: 00–00
Design and Synthesis of Novel 1-substituted-3-(3-(3-nitrophenyl)-
4-oxo-3,4-dihydrobenzopyrimidin-2-yl amino) Isothioureas for
their Anti-HIV, Antibacterial Activities, Graph Theoretical Analysis,
Insilico Modeling, Prediction of Toxicity and Metabolic Studies
Authors
Bandi Narendhar1, Veerachamy Alagarsamy1, Chitra Krishnan2
Supporting Information for this article is available online at
Affiliations
http://www.thieme-connect.de/products
1
Medicinal Chemistry Research Laboratory, MNR College
of Pharmacy, Sangareddy, Gr. Hyderabad, India
Faculty of Pharmacy, Sri Ramachandra Institute of Higher
Education and Research (Deemed to be University),
Porur, Chennai, India
ABStrACt
2
In the present study, we have placed the substituted thiosemi-
carbazide moiety at the C-2 position and 3-nitrophenyl group at
N-3 position of benzopyrimidines and studied their antituber-
cular, anti-HIV and antibacterial activities against selected gram
positive and negative bacteria. The target compounds 1-substi-
tuted-3-(3-(3-nitrophenyl)-4-oxo-3,4-dihydrobenzopyrimi-
din-2-ylamino) isothioureas (PTS1 – PTS15 ) were obtained by
the reaction of 2-hydrazino-3-(3-nitrophenyl) benzopyrimi-
din-4(3H)-one (5) with different alkyl/aryl isothiocyanates fol-
lowed by methylation with dimethyl sulphate. All synthesized
compounds were screened for their antitubercular, anti-HIV
and antibacterial activity against selective gram positive and
gram negative bacteria by agar dilution method. Among the
series, compound 2-methyl-3-(3-(3-nitrophenyl)-4-oxo-3,4-
dihydrobenzopyrimidin-2-ylamino)-1-(3-chlorophenyl)iso-
thiourea (PtS14) shown most potent activity against Klebsiella
pneumoniae, Proteus vulgaris and Staphylococcus aureus; PtS14
exhibited the antitubercular activity at the minimum micro-
gram of 1.56µg/mL and anti-HIV activity at 0.96µg/mL against
HIV1 and HIV2 and offers potential for further optimization and
development to new antitubercular and anti-HIV agents. The
results obtained from this study confirm that the synthesized
and biologically evaluated benzopyrimidines showed promis-
ing antimicrobial, antitubercular and anti-HIV activities and are
Key words
condensed pyrimidine, graph theoretical analysis,
substituted thiosemicarbazide, antibacterial activity,
antitubercular activity, anti-HIV activity
received
accepted
30.06.2019
05.08.2019
Bibliography
DOI https://doi.org/10.1055/a-0991-7617
Published online: 19.6.2020
Drug Res 2020; 70: 348–355
© Georg Thieme Verlag KG Stuttgart · New York
ISSN 2194-9379
Correspondence
Veerachamy Alagarsamy
Medicinal Chemistry Research Laboratory
MNR College of Pharmacy
Sangareddy – 502 294
Gr. Hyderabad
India
new scaffolds for antimicrobial activity.
Tel.: + 91/8455/230 690, Fax: + 91/8455/230 555
drvalagarsamy@gmail.com;
profvalagarsamy@gmail.com
Introduction
of TB disease, among that 1.2 million (11%) people living with HIV,
there were an estimated 456 000 deaths from HIV-associated TB
[1–4]. Although exhaustive efforts to prevent and treat TB is taken
the problem still continues due to multi-drug-resistant (MDR-TB)
to isoniazid, rifampicin, quinolones and aminoglycosides. Recent-
ly TB threat has an additional challenge with the emergence of both
multi-drug-resistant TB (MDR-TB) and extensively drug-resistant
TB (XDR-TB) strains. It clearly highlights the urgent need to devel-
Tuberculosis (TB) is an opportunistic infection that occurs more
often or is more severe in people with weakened immune systems
than in people with healthy immune systems. Human immunode-
ficiency virus (HIV) weakens the immune system, increasing the
risk of TB in people with HIV. Co-infection with TB and HIV poses a
tremendous challenge to TB control, particularly in resource-lim-
ited treatment option. In 2015, it was estimated 10.4 million cases
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
348

op novel “druggable” molecules for the co-infection treatment and
strains of MDR-TB and XDR-TB.
tained from Merck, SD fine chemicals, Aldrich (USA), Lancaster
(USA), or Spectrochem (India) and were used without further pu-
rification.
Recent year’s pyrimidines and condensed pyrimidines gained
much attention to medicinal chemists and pharmacologist due to
their potential druggable behaviour [5]. Among that antimicrobial
activities of 2,3-disubstituted benzopyrimidines are encouraging
for further development. Recent literature evident that the 2,3-dis-
ubstituted benzopyrimidines nucleus showed significant antitu-
bercular activity [5, 6].
General procedure for the synthesis of compound
Synthesis of 3-(3-nitrophenyl)-2-thioxo-2,3-dihydro
benzopyrimidin-4-one (3)
A solution of the 3-nitro aniline (0.02 mol) in dimethyl sulfoxide
(10 mL) was stirred vigorously. Carbon disulphide (1.6 ml) and
aqueous 20 M sodium hydroxide (1.2 mL) was added drop wise to
the above 3-nitro aniline solution during 30min. Gradually dime-
thyl sulphate (0.02 mol) was added by keeping the reaction mix-
ture stirring in freezing mixture for 2h. The obtained reaction mix-
ture was poured into ice cold water. The solid obtained was filtered,
washed with water, dried and recrystallized from ethanol.
To the above prepared N-(3-nitrophenyl)-methyl dithiocarba-
mic acid (0.02mol), methyl anthranilate (0.02mol) was added and
dissolved in ethanol (20 mL). Anhydrous potassium carbonate
(100mg) was added to the above mixture and refluxed for the 22h.
Using ice the reaction mixture was cooled and the solid separated
was filtered. The obtained product (3) was purified by dissolving in
10 % alcoholic sodium hydroxide solution and re-precipitated by
treating with dilute hydrochloric acid. The solid obtained was fil-
tered, washed with water, dried and recrystallized from ethanol.
Yield: 86 %; mp: 281–282 °C; IR (KBr) cm− 1: 3355 (NH), 3062 (Ar-
CH), 1729 (C = O), 1607 (C = C), 1520 & 1346 (NO2), 1251 (C = S);
1H NMR (CDCl3): δ6.73–6.75 (m, 2H, Ar-H), 7.26 (d, J=7.5Hz, 1H,
Ar-H), 7.52–7.54 (m, 2 H, Ar-H), 7.71 (d, J = 2.0 Hz, 1 H, Ar-H),7.95
(d, J=7.5Hz, 1H, Ar-H), 8.01 (d, J=2.0Hz, 1H, Ar-H), 8.09 (m, 1H,
Ar-H),9.16 (brs, 1 H, CSNH); MS (m/z): 299 [M+ ]; Anal. Calcd.
for(C14H9N3O3S): C, 56.18; H, 3.03; N, 14.04. Found: C, 56.31; H,
3.02; N, 14.00.
Thiosemicarbazones have been explored for various medicinal
chemistry properties due to their widespread pharmacological ac-
tivities including antineoplastic, antimycobacterial, antibacterial,
antifungal, antiviral, and antimalarial. Thiosemicarbazones bind
great coordination with target sites because of its versatility due
to nitrogen and sulfur atoms. Thiosemicarbazone is also known as
an iron-chelating agent, bonding the sulfur and azomethine nitro-
gen atoms. The complexes of nitrogen and sulfur atoms with metal
ions may be considered prospective pharmacological behaviour.
In recent literature, many thiosemicarbazone derivatives have been
synthesized and evaluated for their antibacterial activity. The cur-
rent use of these agents in bacterial infections has led to the devel-
opment of novel antibacterial and antitubercular agent [7–15].
The present work is a continuation of our efforts towards devel-
oping potent antitubercular and antimicrobial agents by a phar-
macophore hybrid approach using the benzopyrimidine scaffold.
In this approach a hybrid molecule was created by merging two or
more pharmacophores. Therefore, a hybrid molecule containing
more than one pharmacophore, each pharmacophores may be ad-
dressing the active site of targets and offer the opportunity to se-
lectivity, further it can also reduce unwanted side effects [16]. In
the present study, we have placed the substituted phenyl moiety
at the N-3 position and substituted thiosemicarbazide group at C-2
position of benzopyrimidines ring ▶Supporting Information Fig.
1S and ▶Fig. 1 [16–18] and studied their anti-HIV, antitubercular
and antibacterial activities against selected gram positive and neg-
ative bacteria.
Synthesis of 2-methylthio-3-(3-nitrophenyl)
benzopyrimidin-4-one (4)
The 3-(3-nitrophenyl)-2-thioxo-2,3-dihydro benzopyrimidin-4-one
(3) (0.01mol) was dissolved in alcoholic sodium hydroxide solution
(25 mL). To this dimethyl sulphate (0.01mol) was added drop wise
with stirring and stirring was continued for 1h after complete ad-
dition of dimethyl sulphate. The reaction mixture was then poured
into ice cold water. The solid obtained was filtered, washed with
water, dried and recrystallized from ethanol. Yield: 91%; mp: 181–
182 °C; IR (KBr) cm− 1: 3059 (Ar-CH), 2957 (CH3-CH), 1715 (C= O),
1668 (C=N), 1612 (C=C), 1534 & 1326 (NO2), 672 (C-S-C); 1H NMR
(CDCl3) δ: 2.20 (s, 3 H, SCH3), 7.08–7.10 (m, 2 H, Ar-H), 7.26 (d,
J = 7.5 Hz, 1 H, Ar-H), 7.52–7.54 (m, 2 H, Ar-H), 7.71 (d, J = 2.0 Hz,
1 H, Ar-H), 7.90 (d, J= 7.5 Hz, 1 H, Ar-H), 7.92 (d, J= 2.0 Hz, 1 H, Ar-
H); MS (m/z): 313 [M+ ]; Anal. Calcd. for(C15H11N3O3S): C, 57.50;
H, 3.54; N, 13.41. Found: C, 57.67; H, 3.55; N, 13.38.
Materials and Methods
General
The molecular docking of test compounds was studied by using
Schrodinger, LLC, New York, version 11 (2016) GLIDE program. In
open capillaries melting points (mp) were measured on a Thomas
Hoover melting point apparatus (Thomas Hoover, USA) and are un-
corrected. Using potassium bromide disks the IR spectra (ν, cm–1
)
were recorded on Bruker FT-IR spectrometer (Bruker, USA). Using
Bruker FT-NMR spectrometer (Bruker, USA) the 1H-NMR spectra
were recorded in CDCl3 at 300MHz. The chemical shifts are report-
ed as parts per million (δ, ppm) using tetramethylsilane (TMS) as
an internal standard. Using fast atom bombardment (FAB positive)
mass spectra were obtained on a JEOL-SX-102 instrument (JEOL,
Japan). Perkin Elmer 2400 CHN analyzer (Perkin Elmer, USA) was
used to perform elemental analysis and values were within the ac-
ceptable limits of the calculated values ( ±0.4%). Readymade silica
gel plates (Merck, Norway) are used to monitor the progress of the
reaction. All chemicals and reagents used in the synthesis were ob-
Synthesis of 2-hydrazino-3-(3-nitrophenyl)
benzopyrimidin-4-one (5)
The 2-methylthio-3-(3-nitrophenyl) benzopyrimidin-4-one (4)
(0.01 mol) was dissolved in ethanol (25 mL). To this mixture 99 %
hydrazine hydrate (0.1 mol) and anhydrous potassium carbonate
(100 mg) was added and refluxed for 33 h. The reaction mixture
was cooled and poured into ice cold water. The solid so obtained
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
349

Thieme
Original Article
was filtered, washed with water, dried and recrystallized using eth-
anol to afford the compound ((5). Yield: 83%; mp: 200–201 °C; IR
(KBr) cm− 1: 3371 & 3286 (NH), 3035 (Ar-CH), 1748 (C = O), 1650
(C = N), 1621 (C = C), 1519 & 1353 (NO2); 1H NMR (CDCl3) δ: 5.72
(brs, 1 H, Ar-NH), 6.17 (brs, 2 H, NH2), 6.73–6.75 (m, 2 H, Ar-H),
6.94 (d, J = 7.5 Hz, 1 H, Ar-H), 7.42–7.44 (m, 2 H, Ar-H), 7.61 (d,
J=2.0Hz, 1H, Ar-H), 7.95 (d, J=7.5Hz, 1H, Ar-H), 8.18 (d, J=2.0Hz,
1 H, Ar-H); MS (m/z): 297 [M+ ]; Anal. Calcd. for (C14H11N5O3): C,
56.56; H, 3.73; N, 23.56. Found: C, 56.45; H, 3.75; N, 23.64.
10 % (v/v) heat inactivated fetal calf serum and 20 μg/mL gen-
tamicin (E. Merck, Darmstadt, Germany). From the culture super-
natant of HIV-1 infected MT-4 cell lines HIV-1 (III B) and HIV-2 (ROD)
were obtained and the virus stocks were stored at −70°C until used.
Microtiter plates was used to perform antiHIV assay by filled with
100 μL of medium and 25 μL volumes of compounds in triplicate
so as to allow simultaneous evaluation of their effects on HIV and
mock infected cells. 50 μL of HIV at 100 CCID50 medium was added
to either infected or mock infected part of microtiter tray. At 37°C
the cell cultures were incubated in a humidified atmosphere of 5%
CO2 in air. By the MTT method after five days of infection spectro-
photometrically examined the viability of mock and HIV infected
cells. The effective dose of compound achieving 50% protection of
MT-4 cells against the cytopathic effect (Virus cause cell degenera-
tion or cell death, which can be seen by microscopical examination
of cultures. Cell degeneration is manifested by certain pathologi-
cal changes) of HIV (EC50) and the cytotoxic dose of compound, re-
quired to reduce the viability of normal uninfected MT-4 cells by
50 % (CC50) were calculated.
Synthesis of 3-(3-nitrophenyl)-2-(substituted
thiosemicarbazido) benzopyrimidin-4-one (6A–6O)
A mixture of compound 5 (0.01 mol) and alkyl/ aryl isothiocyanate
(0.01mol) in dioxane (25mL) was refluxed for 6h. The reaction mix-
ture was concentrated and the product obtained was filtered, dried
and recrystallised from dioxane.
Refer details of 6A–6O experimental characterization in
Supporting Information.
1-Substituted-3-(3-(3-nitrophenyl)-4-oxo-3,4-
dihydrobenzopyrimidin-2-ylamino) isothioureas (PtS1
– PtS15)
Antibacterial activity
Agar dilution method was used to evaluate antibacterial activity of
compounds [23, 24]. From the American type culture collection
(ATCC), Rockville, USA the standard strains were procured and from
the department of microbiology, MNR medical college, Sangareddy,
India the pathological strains were procured. The antibacterial activ-
ity of the test analogs was screened against the following bacterial
strains: E. coli ATCC 25922, P. vulgaris ATCC 9484, S. typhimurium ATCC
33068, K. pneumoniae ATCC 13883, P. aeruginosa ATCC 2853, B. subti-
lis ATCC 6051, S. aureus ATCC25923, M. luteusATCC 10240, S. epider-
midis ATCC 35984, S. albus ATCC 17900. Muller–Hinton Agar (Hi-me-
dia) plates (37 °C, 24h) were used for bacterial growth and the mini-
mum inhibitory concentration (MIC) was considered to be the lowest
concentration that completely inhibited the growth on agar plates,
disregarding a single colony or faint haze caused by the inoculums.
Ciprofloxacin was used as reference drug for comparison of the MIC of
the test compounds. The values are mentioned in ▶table 1 calculat-
ed from at least three different experiments in duplicate.
The compound 6 (0.01 mol) was dissolved in alcoholic sodiumhy-
droxide solution (20 mL; 0.01 mol). To this mixture dimethyl sul-
phate (0.01 mol) was added drop wise with stirring. Stirring was
continued for 3h and poured in to ice water. The solid obtained was
filtered, dried and recrystallized from ethanol.
Refer details of PtS1–PtS15 experimental characterization in
Supporting Information.
Pharmacology
Antitubercular activity
Into Middle brook 7H11 agar slants 10 fold serial dilutions of each
test compound/drug were incorporated with OADC growth sup-
plement. Fresh Middle brook 7H11 agar slants with OADC growth
supplement was used to prepare inoculums of M. tuberculosis
H37RV and adjusted to 1 mg/mL in Tween 80 (0.05 % W/V) saline
diluted to 10–2 to give a 107 cfu/mL concentrate approximately.
Into 7H11 agar tubes containing 10 fold serial dilutions of drug per
ml a 5µL amount of bacterial suspension was spotted. At 37 ºC the
tubes were incubated, and after 28 days the final readings were re-
corded. Control tubes where medium alone was incubated with
H37RV were used to compare tubes having the compounds. Active
concentration of test compound was taken as the concentration at
which complete inhibition of colonies occurred. The minimum con-
centration of compound required to give complete inhibition of
bacterial growth was taken as MIC [19–21]. Against reference drug
isoniazide, rifampicin and etahmbutol, the MIC of the test com-
pounds was compared.
Graph theoretical analysis
Kyoto encyclopedia of genes and genomes database was utilized
for the study of graph theoretical analysis. The influential proteins
was recognized and chosen by network of protein interaction (mtu:
00770) in homo sapiens[25–28].
Density functional theory
Gauss view molecular visualized program [29] and Gaussian 03W
package [30] was used to perform computational calculations of
the test analogs. Using B3LYP functional with 6–311 G (d,p) basis
set density functional theory (DFT) optimizes the ground state mo-
lecular structures of synthesized compounds. From the optimized
structures the frontier molecular orbital energies [lowest unoccu-
pied molecular orbital (ELUMO), highest occupied molecular orbital
(EHOMO)and their energy gap (Eg)] of all molecules were derived.
Gauss view molecular visualized program is used to visualize the
obtained molecular orbital energy diagrams of the synthesized de-
rivatives.
Anti-HIV activity
In MT-4 cells anti-HIV activity of the compounds (PtS1 – PtS15)
were tested against replication of HIV-1 (III B) and HIV-2 (ROD) [22].
The MT-4 cells were grown in RPMI-1640 DM (Dutch modification)
medium (Flow laboratories, Irvine, Scotland), supplemented with
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
350

Molecular docking
GLIDE program is used to perform the molecular docking studies
of compounds. Based on the report of network analysis 3IVX was
chosen as the target proteins. In this study the research collabora-
tory for structural bioinformatics (RCSB) protein data bank was
used to retrieve the X-ray crystal structures of target proteins 3IVX
[Pantothenatesynthetase (panC)] along with literature based tar-
get proteins 1BVR [2-trans-enoyl-ACP reductase (InhA)] and 3KRD
[Proteasome]. Extra precision (XP) mode of GLIDE program is used
for performing all docking calculations. GLIDE score (G score) is
used to select the greatest docked structure.
ADME & Toxicity studies
In clinical trials poor absorption, distribution, metabolism, and ex-
cretion (ADME) properties is the major reason for failing of most of
drug candidates. Thus avoiding of compounds not having drug like-
liness and good ADME property is an important aspect of drug dis-
covery. The QikProp module of Schrodinger is a quick and accurate
tool so that its easy-to-use to identify ADME prediction program
design to produce certain descriptors related to ADME. Both phys-
icochemical significant descriptors and relevant properties of phar-
macokinetics are predicted by the QikProp module. Based on Li-
pinski’s rule of five ADME properties determines the drug-like ac-
tivity of ligand molecules. Qikprop module of Schrodinger 2016
(https://www.schrodinger.com/qikprop) was used to study the
ADME/T property of the test compounds.
SOM prediction using SMARTCyp
SMARTCyp software is a SOM predictor, released by Rydberg [31].
The drug molecule was uploaded in SMILES strings format repre-
senting the molecule. SMARTCyp contains a database of ligands
metabolized by CYP450 and energies were pre-calculated by the
activation of DFT. The database contains smiles arbitrary, target
specification based fragments and it was used by SMARTCyp com-
bination with an accessibility descriptor to obtain a ranking of SOM.
Results and Discussion
Chemistry
By using new innovative route the key intermediate 3-(3-
nitrophenyl)-2-thioxo-2,3-dihydro-1H- benzopyrimidin-4-one (3)
was prepared (▶Fig. 1). In which 3-nitro aniline (1) treated with
carbon disulphide and sodium hydroxide in dimethyl sulphoxide to
give sodium dithiocarbamate, which was methylated with dime-
thyl sulphate to afford the dithiocarbamic acid methyl ester. The
dithiocarbamic acid methyl ester on reflux with methyl anthranilate
(2) in ethanol yielded the desired 3-(3-nitrophenyl)-2-thioxo-2,3-
dihydro-1H-benzopyrimidin-4-one (3) via the thiourea intermedi-
ate in good yield (86%). Rate of reaction is enhanced by the use of
DMSO as the reaction solvent and due to less solvation hydrolysis
of the intermediate are prevented by the use of alkali in higher con-
centration. The product obtained was cyclic and not an open chain
thiourea. The IR spectrum of 3 show intense peaks at 3355cm− 1 for
cyclic thiourea (NH), 1729cm− 1 for carbonyl (C=O) and 1251cm− 1
1
showed multi-
for thioxo (C=S) stretching. H NMR spectrum of 3
plet due to aromatic (8 H) protons at δ 6.73–8.09 ppm and a sin-
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
351

Thieme
Original Article
Compound -R
Compound -R
Compound -R
-C2H5
PTS 1
PTS 4
-CH3
-CH2CH = CH2
PTS 2
PTS 5
PTS 3
PTS 6
-CH2CH2CH3
-C6H5
PTS 7
PTS 8
PTS 9
OCH3
NO2
-CH2
CH3
PTS 10
PTS 13
PTS 11
PTS 14
PTS 12
PTS 15
CH2CH2
Br
Cl
Cl
F
▶Fig. 1 Synthesis of 1-substituted-3-(3-(3-nitrophenyl)-4-oxo-3,4-dihydrobenzopyrimidin-2-ylamino)isothioureas. Reagents and conditions: a CS2,
NaOH, DMSO, 30min; b Dimethyl sulphate, 2h; c Methyl anthranilate, Anhydrous K2CO3, EtOH reflux, 22h; d 2% alcoholic sodium hydroxide solu-
tion. Dimethyl sulphate, 1h; e Hydrazine hydrate, Anhydrous potassium carbonate, Ethanol reflux, 33h; f alkyl/aryl isothiocyanates, Ethanol reflux,
6h; g NaOH, Dimethyl sulphate.
glet at δ 9.16ppm indicating the presence of NH. Data from the el-
emental analyses have been found to be in conformity with the as-
signed structure. Further, the molecular ion recorded in the mass
spectrum is also in agreement with the molecular weight of the
compound.
(8 H) protons. Data from the elemental analyses have been found
to be in conformity with the assigned structure. Further the mo-
lecular ion recorded in the mass spectrum is also in agreement with
the molecular weight of the compound.
The compounds 1-(3-(3-nitrophenyl)-4-oxo-3H-dihydroquina-
zolin-2-yl)-4-(substituted) thiosemicarbazides (6A – 6O) were ob-
tained by the condensation of amino group of 3-(3-nitrophenyl)-
2-hydrazino-3H-benzopyrimidin-4-one (5) with a variety of alkyl/
aryl isothiocyanates. The formation of desired products is indicat-
ed by the disappearance of peak due to NH, NH2 of the starting ma-
terial in IR and 1H NMR spectrum of all the compounds 6A–6O. The
IR and 1H NMR spectrum of these compounds showed the presence
of peaks due to thiosemicarbazides, carbonyl (C= O), NH and aryl
groups. Data from the elemental analyses have been found to be
in conformity with the assigned structure. The mass spectrum is
also in agreement with the molecular weight of the compound.
The title compounds 1-substituted-3-(3-(3-nitrophenyl)-
4-oxo-3,4-dihydrobenzopyrimidin-2-ylamino)isothioureas (PtS1
– PtS15,) were obtained by the methylation of the thiosemicar-
bazides (6A–6O) using dimethyl sulphate. The formation of title
product is indicated by the disappearance of peak due to NH and
C = S of the starting material in IR and 1H NMR spectrum of all the
compounds PtS1 – PtS15. The IR and 1H NMR spectrum of these
compounds showed the presence of peaks due to methylthiosure-
as, carbonyl (C = O), NH and aryl groups. The mass spectra of the
title compounds showed molecular ion peaks corresponding to
The 2-methylsulfanyl-3-(3-nitrophenyl)-3H-benzopyrimidin-
4-one 4 was obtained by dissolving 3in 2 % alcoholic sodium hy-
droxide solution and methylating with dimethyl sulphate with stir-
ring at room temperature. The IR spectrum of 5 showed disappear-
ance of NH and C=S stretching signals of cyclic thiourea. It showed
a peak for carbonyl (C = O) stretching at 1715 cm− 1. The 1H NMR
spectrum of compound 4 showed singlet at δ 2.20 for SCH3 group;
and a multipletfor aromatic (8H) protons observed at δ 7.08–7.92
Data from the elemental analyses and molecular ion recorded in
the mass spectrum further confirmed the assigned structure.
Nucleophilic displacement of –SCH3 group of 4 with hydrazine
hydrate was carried out using ethanol as solvent to afford 2-hydrazi-
no-3-(3-nitro phenyl)-3H-benzopyrimidine 5. The long duration of
reaction (33h) required might be due to the presence of bulky butyl
group at position 4, which might have reduced the reactivity of
quinazoline ring system at C-2 position. The formation of 5 was
confirmed by the presence of NH and NH2 signals at 3371–
3286 cm− 1 in the IR spectrum. It also showed a peak for carbonyl
(C = O) at 1748 cm− 1. The 1H NMR spectrum of the compound 5
showed singlets δ 5.72 and 6.17ppm due to NH2 and NH respec-
tively, a multiplet at δ 6.94–8.18 ppm was observed for aromatic
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
352

their molecular formulae. In mass spectra of compounds PtS1 –
PtS15, a common peak at m/z 144 corresponding to quinazolin-
4-one moiety appeared in all mass spectrum of the compounds.
Elemental (C, H, N) analysis satisfactorily confirmed elemental com-
position and purity of the synthesized compounds.
more attention because of its interaction between TB proteins
which is cause of spreading disease. From the study it was found
that pan C is a significant target for TB to identify active quinazo-
line moiety.
Density functional theory
Antitubercular activity
In bioactivities the frontier-orbital energies of a compound play an
important role. EHOMO is one among that which is a rough measure
of electron donating ability of a compound. In general, biological
activity is increasing with increased value of EHOMO, whereas the
ELUMO acts in reverse. Chemical reactivity of the molecule and bio-
activity of compounds is reflected by energy values of the ELUMO and
EHOMO and their ΔE. EHOMO energy falls over at the PtS7 of parent ring
but ELUMO energy falls on parent PtS14 in an equal ratio to EHOMO
value, hence for all the synthesized compounds energy gap value are
in the same range and there is no much difference ▶Supporting
Information table 3S. Results suggests substitution of electron
withdrawing group at parent ring PtS11 – PtS15 can further in-
crease EHOMO energy and reduce the ELUMO energy, thus there was
an increase in energy gap and it turns the stability and bioactivity
of compounds.
Against M. tuberculosis strain H37RV the synthesized compounds
(▶table 1) were screened for their in vitro antimycobacterial activ-
ity. The results are expressed in terms of Minimum Inhibitory Con-
centration (MIC). The results of antimycobacterial activity depict-
ed in ▶table 1, indicates that the test compounds inhibited the
growth of Mycobacterium in varying degree. Compounds with ali-
phatic substituents showed lesser antitubercular activity over the
aryl and heteroaryl substituents. The compounds with electron
withdrawing substituent on the aryl ring showed better activity
over the unsubstituted or electron donating substituent on the aryl
ring. Among the test compounds, 2-methyl-3-(3-(3-nitrophenyl)-
4-oxo-3,4-dihydrobenzopyrimidin-2-ylamino)-1-(3-chlorophenyl)
isothiourea (PtS14) exhibited most potent antitubercular activity
at the minimum microgram (1.56 µg/mL) concentration.
Anti-HIV activity
Molecular docking
Mild to moderate anti-HIV activity was exhibited by all compounds;
whereas compounds PtS14 containing electron withdrawing
groups exhibited anti-HIV activity at 0.96µg/mL against HIV1 and
HIV2. Compounds with aliphatic substituents showed lesser an-
titubercular activity over the aryl substituted (expect PtS9 and
PtS10). Aryl compounds with electron withdrawing substituents
like chloro and nitro showed better activity over the unsubstitut-
ed. While the test compounds with other substituent’s showed
moderate anti-HIV activity against HIV1 and HIV2 with the MIC in
the range of 2.91 to 100µg/mL.
In the G-score form the scoring function of GLIDE docking program
is presented. The most straight forward method of evaluating the
accuracy of a docking procedure is to determine how closely the low-
est energy pose (binding conformation) predicted by the object scor-
ing function. In this study, from the G-score by removing the inhib-
itor compound with selected proteins has been analyzed for vali-
dation of extra precision GLIDE docking procedure. In ▶Supporting
Information table 4S the docking result of these ligands are sum-
marized. For each minimized complex the interaction energy in-
cludes van der Waals energy, electrostatic energy, as well as inter-
molecular hydrogen bonding were calculated and presented in
▶Supporting Information tables 4S and 5S. Towards panC pro-
tein all fifteen designed ligand molecules showed potent interac-
tion. The docking score was ranging from − 4.590 to − 8.039 for
the tested series PtS1 – PtS15 with panC protein ▶Supporting
Information Fig. 4S. These synthesized analogs displayed low scor-
ing against the literature based chosen other InhA and Proteasome
targets. By hydrophobic and hydrophilic interaction all the test mol-
ecules bound to protein, which is shown in ▶Supporting Informa-
tion Figs. 4S and ▶5S. From the in silico results, it was found that
towards panC protein the designed test compounds showed po-
tent and selective action. Thus it proves that, to find potential an-
alogue towards antiTB drug development the network based tar-
get selection was effective and most significant. antiTB potency of
the test analogs was proved form observed results. Among various
tested analogs, the synthesized analogues PtS 11 – PtS15 were
found to be more potent and selective in action against panC pro-
tein.
Antibacterial activity
Among the different substituents at N-3 position of the quinazo-
lin-2-yl, aryl substituents exhibited better activity over the aliphat-
ic cyclic substituents (▶table 1). Compounds with electron with-
drawing substituents like chloro and nitro showed better activity
over the unsubstituted and electron donating substituents. Among
the series, compound 2-methyl-3-(3-(3-nitrophenyl)-4-oxo-3,4-
dihydrobenzopyrimidin-2-ylamino)-1-(3-chlorophenyl) isothiou-
rea (PtS14) shown most potent activity against K. Pneumonia P.
Vulgaris and S. aureus; while the compound PtS11 and PtS12
showed most potent activity against P. Vulgaris. Compounds PtS14,
PtS11 and PtS 12 were emerged as the most active compounds
of the series.
Graph theoretical analysis
The pathway was renewed to a graph with proteins as nodes and
interactions as edges and it was represented in ▶Supporting In-
formation Figs. 2S and 3S, ▶tables 1S and 2S. The network was
carries 61 nodes and 68 edges. Parameters like degree centrality,
betweenness centrality, eigenvector centrality, radialitycentrality,
stress centrality and closeness centrality from the network was cal-
culated to identify the significance of proteins. The target was iden-
tified as pan C based on its threshold values. In addition pan C has
Ligand based toxicity prediction
QikProp module of Schrodinger uses toxicity properties to catego-
rize the drug-like activity of the ligand molecule. QikProp module
of Schrodinger was used to study the toxicity property of synthe-
sized molecules and the obtained results were shown in ▶Support-
Narendhar B et al. Synthesis of Benzopyrimidine Isothiourea… Drug Res 2020; 70: 348–355
353

Thieme
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of five. In addition all predicted properties of test analogs were in
the range of 95% of known oral drugs.
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SMARTCyp was used to predict the test compounds primary site of
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Conclusion
In summary, synthesis of new series of 1-substituted-3-(3-(3-
nitrophenyl)-4-oxo-3,4-dihydrobenzopyrimidin-2-ylamino)isothi-
oureas PtS1 – PtS15 have been described. These derivatives have
exhibited significant antibacterial activity against the various gram
positive and gram negative bacteria including M. tuberculosis; and
moderate activity against HIV1 and HIV2 strains. The substituents
at the thiosemicarbazide shown varied antimicrobial activity, aryl
substituents with electron withdrawing group showed most po-
tent and the allyl, alkyl/aryl substituents showed moderate activ-
ity and the aryl substituents with electron donating groups showed
the least activity, among the series, compound 2-methyl-3-(3-(3-
nitrophenyl)-4-oxo-3,4-dihydrobenzopyrimidin-2-ylamino)-
1-(3-chlorophenyl)isothiourea (PtS14) shown most potent activ-
ity against K. Pneumonia P. Vulgaris and S. aureus; PtS14 exhibited
the antitubercular activity at the minimum microgram (MIC:
1.56 µg/ml) and antiHIV activity at 0.96 µg/mL against HIV1 and
HIV2 and offers potential for further optimization and development
to new antitubercular and anti-HIV agents.
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Conflict of Interest
[19] Sriram D, Yogeeswari P, Basha JS et al. Synthesis and antimycobacterial
evaluation of various 7-substituted ciprofloxacin derivatives. Bioorg
Med Chem 2005; 13: 5774–5778
The authors report no conflicts of interest. The authors alone are
responsible for the content and writing of the paper.
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