Synthesis, biological evaluation and molecular docking studies of novel quinazolinones as antitubercular and antimicrobial agents

Article abstract of DOI:10.1016/j.bioorg.2020.104611

In the present study, a series of novel quinazolinone hybrids, viz. triazepino-quinazolinones 4, thiazolo-triazolo-quinazolinones 7 and triazolo-quinazolinones 8 have been synthesized from the key intermediate 3-(substituted phenyl)-2-hydrazinoquinazolin-4(3H)-ones 3. All the newly synthesized compounds were characterized by means of spectral (IR, ¹H NMR, ¹³C NMR) and elemental analysis. The target compounds were biologically screened for their in vitro antimicrobial and antitubercular activities against pathogenic strain. The results of bioassay demonstrated that some of the compounds exhibited pronounced antimicrobial activity comparable to that of standard drugs tested under similar conditions. Compounds 4c, 4e, 7e and 8b showed relatively very good inhibitory activity against pathogenic bacteria with minimum inhibitory concentration (MIC) of 2.6 μg/mL, 5.2 μg/mL, while the rest of the compounds showed moderate activity. Compounds 4c and 8b were found to be nearly equipotent with ciprofloxacin against P. aeruginosa with MIC 5.2 μg/mL, while compound 8b was more potent against pathogenic bacteria S. aureus. It is very remarkable that four compounds, 4c, 4e, 7e and 8b showed pronounced antifungal activity against selected pathogenic fungi, A. niger, C. albicans with MIC 2.6 μg/mL and 5.2 μg/mL. The antitubercular activity of synthesized compounds reveal that compound 8b showed better activity than the other compounds with a MIC of 5.2 μg/mL against M. tuberculosis (H₃₇Rv). Molecular docking studies of the compounds were performed to rationalize the inhibitory properties of these compounds and results showed that these compounds have good binding energy and better binding affinity within the active pocket, thus these compounds may be considered as potent inhibitors towards selective targets.

Full text of DOI:10.1016/j.bioorg.2020.104611

Bioorganic Chemistry 108 (2021) 104611
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis, biological evaluation and molecular docking studies of novel
quinazolinones as antitubercular and antimicrobial agents
,
Sarvesh Kumar Pandeya *, Umesh Yadavab, Anjali Upadhyaya, M.L. Sharmac
a Department of Chemistry, D.D.U. Gorakhpur University, Gorakhpur-273009, UP, India
b Department of Physics, D.D.U. Gorakhpur University, Gorakhpur-273009, UP, India
c Central Department of Chemistry, Tribhuvan University, Kirtipur- 44618, Kathmandu, Nepal
A R T I C L E I N F O
A B S T R A C T
Keywords:
In the present study, a series of novel quinazolinone hybrids, viz. triazepino-quinazolinones 4, thiazolo-triazolo-
quinazolinones 7 and triazolo-quinazolinones 8 have been synthesized from the key intermediate 3-(substituted
phenyl)-2-hydrazinoquinazolin-4(3H)-ones 3. All the newly synthesized compounds were characterized by
means of spectral (IR, 1H NMR, 13C NMR) and elemental analysis. The target compounds were biologically
screened for their in vitro antimicrobial and antitubercular activities against pathogenic strain. The results of
bioassay demonstrated that some of the compounds exhibited pronounced antimicrobial activity comparable to
that of standard drugs tested under similar conditions. Compounds 4c, 4e, 7e and 8b showed relatively very
Quinazolinones
Spectral characterization
Antimicrobial
Antitubercular
Molecular docking
good inhibitory activity against pathogenic bacteria with minimum inhibitory concentration (MIC) of 2.6
5.2 g/mL, while the rest of the compounds showed moderate activity. Compounds 4c and 8b were found to be
nearly equipotent with ciprofloxacin against P. aeruginosa with MIC 5.2 g/mL, while compound 8b was more
potent against pathogenic bacteria S. aureus. It is very remarkable that four compounds, 4c, 4e, 7e and 8b
showed pronounced antifungal activity against selected pathogenic fungi, A. niger, C. albicans with MIC 2.6 g/
mL and 5.2 g/mL. The antitubercular activity of synthesized compounds reveal that compound 8b showed
better activity than the other compounds with a MIC of 5.2
μg/mL,
μ
μ
μ
μ
μ
g/mL against M. tuberculosis (H37Rv). Molecular
docking studies of the compounds were performed to rationalize the inhibitory properties of these compounds
and results showed that these compounds have good binding energy and better binding affinity within the active
pocket, thus these compounds may be considered as potent inhibitors towards selective targets.
1. Introduction
patients [3–5]. Many of the currently available drugs are toxic, produces
recurrence due to fungistatic and not fungicidal in nature. As known, not
Although antibiotics are among the most prescribed drugs in the
world today and their development and commercialization have saved
countless lives, but the unmet need of efficacious drugs against bacteria
is still high. Several bacterial infections such as diarrhoea, food
poisoning, rheumatic, extra intestinal and intestinal wall infections are
caused by multi-drug resistant bacteria [1,2]. This rapid rise in bacterial
resistance towards available antibiotics is becoming a major threat to
human health. Therefore, design of new class of compounds, with novel
and distinct mode of action, from those of well-known classes, is of
prime interest. Since the fungal infections are caused by eukaryotic or-
ganism, for this reason, they generally present more difficult therapeutic
problems than do bacterial infections. Fungal infections have emerged
as a major cause of morbidity in immunocompromised and debilitated
only the biochemical similarity of human cell and fungi forms a hand-
icap for selective activity, but also, they easily gained resistance, is the
main problem encountered in developing safe and efficient antifungal
agents. Therefore, there is general consensus that new chemical entities
with potential antifungal properties, which could possibly have different
modes of activity or affect different sites in fungal cells, are urgently
needed for the management of fungal infections.
The 4-quinazolinone ring is a prominent structure motif found not
only in many of its synthetic pharmaceutically active derivatives but
also in several naturally occurring alkaloids isolated from animals,
plants kingdoms and microorganisms [6–8]. The 4-quinazolinone nu-
cleus is a ubiquitous feature of pharmacological interest and has been
proven to be a fertile source of bioactive agents such as antifungal [9],
* Corresponding author.
E-mail address: skpandey1982@gmail.com (S. Kumar Pandey).
https://doi.org/10.1016/j.bioorg.2020.104611
Received 13 October 2020; Received in revised form 11 December 2020; Accepted 28 December 2020
Available online 5 January 2021
0045-2068/© 2021 Elsevier Inc. All rights reserved.

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
antibacterial [10], antitumor [11], anti-inflammatory [12], anti-oxidant
[13], anticancer [14], anti-leishmanial [15], antitubercular [16]. In
addition, poly(ADP-ribose) polymerase-I(PARP-1) inhibitors[17], inhi-
bition of mammalian dihydrofolate reductase and molecular modelling
studies were also used to assess the fit of these compounds within the
active site of human DHFR [18].
disubstituted derivatives, we wish to incorporate thiazolo-triazole,
1,2,4-triazole and 1,2,4-triazepine nuclei in between N-1 and C-2 posi-
tions of quinazolinone ring to potentiate the biological activities of novel
fused systems 1,2,4-triazipeno-[3,4-a]-quinazolin-9(3H)-one, thiazolo-
[3,4-a]-1,2,4-triazolo-[2,3,-a]-quinazolin-9(3H)-one and 1,2,4-triazolo-
[4,3-a]-quinazolin-7(3H)-ones. Various groups on phenyl ring have been
introduced to investigate the influence of such structural variations on
anticipated biological activities.
Thiazole ring system has been employed in the compounds that exert
antibacterial, antitubercular, anti-HIV [19,20], anti-inflammatory [21],
anticancer activities [22] and as agricultural chemicals [23,24]. Among
them, Ritonavir is a well-known anti-HIV drug. In addition, thiazole ring
plays an important role in nature, e.g., the thiazolium ring present in
vitamin B1 serve as an electron sink and its co-enzyme form is important
The above observations and our earlier work [30–34] coupled with
our desire to develop efficacious antimicrobial and antitubercular agents
and to device an efficient and convenient synthetic method of hitherto
unknown and novel title compounds 1,2,4-triazipeno-[3,4-a]-quinazo-
lin-9-one, thiazolo-[3,4-a] 1,2,4-triazolo-[2,3,-a]-quinazolin-9-one and
1,2,4-triazolo-[4,3-a]-quinazolin-7-one. Molecular docking studies were
also performed to examine their inhibitory properties, possible in-
teractions and binding patterns with the proteins in relation to their
activity, which may improve our knowledge in understanding the
inhibitory actions in detail.
for decarboxylation of
α-keto acids [25]. It also acts as adenosine re-
ceptors antagonist [26].
Similarly, derivatives of triazoles and 1,2,4-triazepine have been
found to possess wide spectrum of pharmaceutical, medicinal and bio-
logical properties [27–29].
In light of the above facts and following the rationale that most of the
biologically active quinazolinones are either C-2 or N-3 mono and/ or
Scheme 1. Synthetic route of intermediate and final compounds.
2

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
2. Results and discussion
appearance of ethylenic proton (–CH = C–) at δ 5.3–5.6 and disap-
pearance of signals of –NH proton suggests the cyclodehydration of
compounds 6 into 7. The formation of compounds 8 were confirmed by
the appearance of a new singlet at δ 3.1 due to thiol (-SH). Signals at δ
8.2–8.4, δ 8.1–8.3 and δ 3.1 were exchanged with D2O and disappeared.
Carbon skeleton of compounds have been confirmed by 13C NMR.
Thirteen signals were observed in the 13C NMR spectrum of compound
8a with carbonyl and imine carbons at δ 180.3, 165.6 ppm and 160.2
ppm respectively, while that of aromatic carbon atoms resonate at δ
123.5–139.2 ppm. In the 13C NMR spectrum of compound 4a, 16 signals
were observed. Signals at δ 190.3 and δ 182.3 ppm are due to carbonyl
carbon of seven and six membered rings, respectively. Signals at δ 166.7
and 158.1 ppm are due to imine carbons of triazepine ring and δ
119.4–142.2 ppm for aromatic carbons. Signals at δ 42.0 ppm and δ 20.1
ppm are due to methylene and methyl carbons. The 13C NMR spectrum
of compound 7a shows 19 signals. Signal at δ 181.9 ppm due to carbonyl
carbon and δ 153.9 ppm due to imine carbon of quinazolinone ring.
Signal at δ 147.2 is due to fused carbon of triazolo-thiazole and at δ 89.2
ppm and δ 80.1 ppm are due to thiazole ring carbons. Aromatic carbons
are in the range of δ 120.2–160.9 ppm.
2.1. Chemistry
The reaction sequence employed for the synthesis of title compounds
is shown in Scheme 1. The required starting material 3-(substituted-
phenyl)-2-mercapto-quinazolin-4(3H)-ones 1 were synthesized from
anthranilic acid and substituted phenyl isothiocynates. Methylation of 1
with methyl iodide in the presence of fused sodium acetate in ethanol
afforded 3-(substituted-phenyl)-2-mercaptomethyl-quinazolin-4(3H)-
ones 2 in good yields. Nucleophilic displacement of mercaptomethyl
group of 2 by hydrazine hydrate yielded the desired key intermediates 3
in excellent yields.
The acid catalyzed condensation reaction between key intermediate
3 and ethylacetoacetate in methanol furnished 7-methyl-8-substituted-
5-oxo-1,2,4-triazipeno-[3,4-a]-quinazolin-9(3H)-ones 4, while conden-
sation reaction between appropriate ketone and key intermediate 3 in
methanol afforded Schiff base 5. The cycloaddition reaction between 5
and mercapto acetic acid under [3 + 2] strategy yielded compound 6,
which on cyclodehydration in cold concentrated H2SO4 yielded 2,2,8-
trisubstituted-[1,3]-thiazolo-[3,4-a][1,2,4]-triazolo-[2,3-a]-quinazolin-
9(3H)-ones 7. The reaction of intermediate 3 with CS2 and KOH afforded
3-mercapto-5-substituted-phenyl-[1,2,4]-trizolo-[4,3,a]-quinazolin-7
(3H)-ones 8.
2.3. Biological activity
2.3.1. Antibacterial studies
The newly synthesized compounds 4a-e, 7a-h and 8a-c were
screened for their in vitro antibacterial activity against Gram-positive
bacteria; namely, S. pneumoniae, B. subtilis, S. aureus and Gram-
negative bacteria; namely, E. coli, P. aeruginosa by disc diffusion
method [35].
2.2. Spectral analysis
2.2.1. IR studies
The IR spectra of compounds 1 exhibited characteristic bands at
1670–1680 cmꢀ 1 (>C O), 1620–1630 cm (>C N) and 2645–2650
The antibacterial screening data (Table 1) revealed that all the tested
compounds showed moderate to strong activity. The compounds 4c, 4e,
7e and 8b showed comparatively very good inhibitory activity against
ꢀ 1
–
–
–
–
cmꢀ 1 (-SH). The disappearance of bands at 2645–2650 cmꢀ 1 and
appearance of new bands at 1217–1221 cmꢀ 1 (C
– –
S C) in IR spectra of
compounds2 clearly indicates the methylation at sulfur atom. The bands
of these thio ethers disappeared in IR spectra of compounds 3. The
appearance of broad bands at 3425–3433 cmꢀ 1 (–NH and –NH2) are
more evident for the displacement of -SCH3 group of compounds 2 with
hydrazine hydrate. The IR spectra of compounds 4 revealed the presence
both Gram-positive and Gram-negative bacteria with MIC 2.6 μg/mL,
5.2
with MICs 10.4
standard drug Ciprofloxacin (MIC = 1.56
mL). Compounds 4c and 8b were found to be nearly equipotent with
Ciprofloxacin against P. aeruginosa with MIC 5.2 g/mL, while com-
μ
g/mL whereas the rest of the compounds showed moderate activity
g/mL, 20.8 g/mL and 41.7 g/mL as compared to
g/mL, 2.6 g/mL and 5.2 g/
μ
μ
μ
μ
μ
μ
of absorption bands at 1685–1675 cmꢀ 1 (>C O six membered ring)
μ
–
–
and 1651–1655 cmꢀ 1 (>C O seven membered ring). The formation of
pound 8b was more potent against pathogenic bacteria S. aureus. We
then turned our attention toward exploring the structure–activity rela-
tionship (SAR), this better activity is attributed probably due to the
presence of pharmacologically active mercapto-triazole segment along
with groups like 4-chloro, 4-ethoxy, and 4-fluoro present at the N-3
phenyl ring of the tested compounds. The fusion of different heterocyclic
rings in between N-1 and C-2 of parent quinazolinone viz. 1,2,4-triaze-
pine, thiazolo-1,2,4-triazole and 1,2,4-triazole ring exert a significant
influence on the antibacterial activity. The fusion of mercapto-triazole
segment on parent quinazolinone showed greater activity than that of
other fused segments, indicates that the presence of mercapto-triazole
moiety is additive towards antibacterial activity in this class of com-
pounds. From these results it is clear that substituent group play a def-
inite role in the biological activity of these types of molecules.
–
–
Schiff bases 5 was confirmed by the disappearance of band at 3433 cmꢀ 1
and appearance of a new band at 1620–1635 cmꢀ 1 (C N). The
–
–
appearance of new bands at 1660–1667 cmꢀ 1 may be ascribed to five
membered ring carbonyl group of compounds 6, which provide evi-
dence in support of the cycloaddition reaction between compounds 5
and mercapto acetic acid. The disappearance of these carbonyl bands
and appearance of a new bands at 1570–1575 cmꢀ 1 (C N, exo) in IR
–
–
spectrum of compounds 7 indicates the cyclodehydration of 6 into 7.
The IR spectra of compounds 8 showed prominent bands at 1603–1608
cmꢀ 1 (>C N) and at 2523–2530 cmꢀ 1 (-SH).
–
–
2.2.2. NMR studies
The structures of all the newly synthesized compounds were also
confirmed by 1H NMR and 13C NMR spectral studies. The 1H NMR
spectra of compounds 1 showed multiplet at δ 7.2–8.5 ppm for aromatic
and singlet at δ 2.9–3.3 ppm for -SH protons, respectively. The appear-
ance of new singlet in the 1H NMR spectra of compounds 2 at δ 2.4–2.8
ppm for (-SCH3) proton clearly indicates the methylation at sulphur
atom. The 1H NMR spectra of compounds 3 displayed a triplet and a
doublet at δ 8.2–8.4 ppm and δ 4.5–4.8 ppm corresponding to protons of
hydrazine functionality. The disappearance of signals of these protons
and appearance of singlet at δ 2.0–2.3 ppm (–CH3) and δ 2.5–2.6
(–CH2CO) clearly support the formation of compounds 4.
2.3.2. Antifungal studies
The encouraging results from the antibacterial studies impelled us to
go for the preliminary screening of the title compounds for their anti-
fungal activity against C. albicans, A. fumigatus, A. flavus and A. niger
(recultured) in DMSO by serial plate dilution methods[36].
The antifungal screening data (Table 2) of titled compounds showed
moderate to good activity. It is very interesting that four compounds,4c,
4e, 7e and 8b showed pronounced antifungal activity against selected
pathogenic fungi with MIC 2.6
4c, 7e and 8b showed very promising activity against A. niger with MIC
2.6 g/mL. The compounds 4c and 7e displayed good activity against
C. albicans with MIC 5.2 g/mL. The results showed also some important
structure–activity relationships (SARs) for this quinazolinone hybrids.
μg/mL and 5.2 μg/mL, while compounds
In the 1H NMR spectra of compounds 5, a broad new signal at δ
8.1–8.3 ppm was due to one –NH proton, in addition to other signals.
The formation of compounds 6 were confirmed by the presence of a new
singlet at δ 3.7–4.0 due to cyclic methylene protons. In compounds 7,
μ
μ
3

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
Table 1
Antibacterial activity of compounds 4a-c, 7a-h, 8a-c.
Comp. No.
R
R’
R’’
MICs (
E. coli
>50
μ
g/mL)a
P. aeruginosa
S. pneumoniae
B. subtilis
S. aureus
4a
H
–
–
50 ± 0.0
50 ± 0.0
33.3 ± 11.8
16.7 ± 5.9
8.33 ± 2.9
16.7 ± 5.9
5.2 ± 1.5
41.7 ± 11.8
33.3 ± 11.8
10.4 ± 2.9
33.3 ± 11.8
5.20 ± 1.5
>50
4b
4-CH3
4-Cl
–
–
20.8 ± 5.9
8.3 ± 2.9
25 ± 0.0
20.8 ± 5.9
2.6 ± 0.7
16.7 ± 5.9
20.8 ± 5.9
16.7 ± 5.9
16.7 ± 5.9
16.7 ± 5.9
16.7 ± 5.9
5.2 ± 1.5
50 ± 0.0
16.7 ± 5.9
10.4 ± 2.9
33.3 ± 11.8
10.4 ± 2.9
50 ± 0.0
4c
–
–
4d
4-OCH3
4-OC2H5
H
–
–
4e
–
–
10.4 ± 2.9
>50
7a
H
4-F-C6H4
16.7 ± 5.9
16.7 ± 5.9
33.3 ± 11.8
16.7 ± 5.9
2.6 ± 0.7
7b
H
CH3
4-F-C6H4
16.7 ± 5.9
33.3 ± 11.8
33.3 ± 11.8
5.2 ± 1.5
16.7 ± 5.9
16.7 ± 5.9
33.3 ± 11.8
33.3 ± 11.8
5.2 ± 1.5
33.3 ± 11.8
1.56 ± 0.0
33.3 ± 11.8
16.7 ± 5.9
33.3 ± 11.8
10.41 ± 2.9
33.3 ± 11.8
16.7 ± 5.9
16.7 ± 5.9
33.3 ± 11.8
2.6 ± 0.7
33.3 ± 11.8
33.3 ± 11.8
16.7 ± 5.9
5.2 ± 1.5
7c
H
CH(CH3)2
2-F-(4-CH3)C6H3
7d
H
CH2CH3
2-F-(4-CH3)C6H3
7e
4-OC2H5
4-OC2H5
4-OC2H5
4-OC2H5
H
H
4-F-C6H4
7f
CH3
4-F-C6H4
41.7 ± 11.8
16.7 ± 5.9
16.7 ± 5.9
33.3 ± 11.8
5.2 ± 1.5
33.3 ± 11.8
16.7 ± 5.9
33.3 ± 11.8
>50
7g
CH(CH3)2
2-F-(4-CH3)C6H3
33.3 ± 11.8
50 ± 0.0
7h
CH2CH3
2-F-(4-CH3)C6H3
8a
–
–
–
–
–
–
–
–
>50
8b
4-OC2H5
4-CH3
–
2.6 ± 0.7
16.7 ± 5.9
2.6 ± 0.7
2.6 ± 0.7
8c
16.7 ± 5.9
5.2 ± 1.5
33.3 ± 11.8
1.56 ± 0.0
33.3 ± 11.8
2.08 ± 0.7
Ciprofloxacin
a
Data are represented in the form of mean of MICs ± S.D.
Table 2
Antifungal activity of compounds 4a-c, 7a-h, 8a-c.
Comp. No.
R
R’
R’’
MICs (
μ
g/mL)a
C. albicans
A. fumigatus
A. flavus
A. niger
4a
H
–
–
>50
33.3 ± 11.8
16.7 ± 5.9
8.3 ± 2.9
>50
33.3 ± 11.8
16.7 ± 5.9
4.2 ± 1.5
16.7 ± 5.9
5.2 ± 1.5
16.7 ± 5.9
25 ± 0.0
4b
4-CH3
4-Cl
–
–
33.3 ± 11.8
16.7 ± 5.9
16.7 ± 5.9
5.2 ± 1.5
50 ± 0.0
33.3 ± 11.8
33.3 ± 11.8
25 ± 0.0
5.2 ± 1.5
50 ± 0.0
25 ± 0.0
41.7 ± 11.8
>50
16.7 ± 5.9
33.3 ± 11.8
16.7 ± 5.9
20.8 ± 5.9
33.3 ± 11.8
16.7 ± 5.9
25 ± 0.0
4c
–
–
4d
4-OCH3
4-OC2H5
H
–
–
33.3 ± 11.8
10.4 ± 2.9
16.7 ± 5.9
6.25 ± 0.0
16.7 ± 5.9
16.7 ± 5.9
10.4 ± 2.9
12.5 ± 0.0
16.7 ± 5.9
12.5 ± 0.0
25 ± 0.0
4e
–
–
7a
H
4-F-C6H4
7b
H
CH3
4-F-C6H4
7c
H
CH(CH3)2
2-F-(4-CH3)C6H3
16.7 ± 5.9
12.5 ± 0.0
2.6 ± 0.7
25 ± 0.0
7d
H
CH2CH3
2-F-(4-CH3)C6H3
25 ± 0.0
7e
4-OC2H5
4-OC2H5
4-OC2H5
4-OC2H5
H
H
4-F-C6H4
5.2 ± 1.5
33.3 ± 11.8
16.7 ± 5.9
33.3 ± 11.8
25 ± 0.0
7f
CH3
4-F-C6H4
7g
CH(CH3)2
2-F-(4-CH3)C6H3
25 ± 0.0
7h
CH2CH3
2-F-(4-CH3)C6H3
16.7 ± 5.9
50 ± 0.0
8a
–
–
–
–
–
–
–
–
8b
4-OC2H5
4-CH3
–
12.5 ± 0.0
>50
20.8 ± 5.9
25 ± 0.0
5.2 ± 1.5
33.3 ± 11.8
3.12 ± 0.0
2.6 ± 0.7
25 ± 0.0
8c
Fluconazole
1.56 ± 0.0
5.2 ± 1.5
1.56 ± 0.0
a
Data are represented in the form ofmean of MICs ± S.D.
First, the nature of substituents at the N-3 phenyl group have substantial
influence on the antifungal activity and second, the segment between N-
1 and C-2 of parent quinazolinone. Substitution of the electron deficient
aromatic ring at the N-3 position conferred far greater activity in com-
parison with the substitution of the electron rich aromatic ring at the N-3
position. It is notable that all these four compounds incorporate either a
4-chloro with 4-ethoxy or combination of 4-ethoxy with 4-fluoro groups
in their structures. The rest of the compounds showed moderate anti-
microdilution technique[37]. The results of antitubercular activity were
listed in Table 3. Biological results of the synthesized compounds reveal
that compound 8b showed better activity than the other compounds
with a minimum inhibitory concentration (MIC) value 5.2 μg/mL. The
structure-activity relationship of the tested compounds indicates that
compound 8b containing ethoxy group at 5-substituted phenyl ring of
quinazolinone hybrid and triazole nucleus in between N-1 and C-2 along
with mercapto moiety which suggests that the presence of ethoxy group
and heterocyclic nucleus containing mercapto group might be one of the
factors to enhance the activity against M. tuberculosis among all the
compounds tested.
fungal activity with MIC 10.4, 20.8 and 41.7 μg/mL as compared to
standard drug Fluconazole tested under similar condition. The data also
revealed that a minor alteration in the molecular configuration of
investigated compounds have pronounced effect on antifungal activity.
Thus, compounds 4a, 7a and 8a having no substituent at the phenyl ring
and compounds 4b, 7b and 8c having a methyl group at the phenyl ring
are less active as compared to the compounds having 4-chloro, 4-ethoxy
and 4-fluoro substituents. Compounds having fused thiazolo-1, 2, 4-tri-
azole segment between N-1 and C-2 positions of parent quinazolinone,
were found more potent antifungal agents than those having mercap-
totriazole or triazepine segments fused at these positions.
2.4. Molecular docking studies
2.4.1. Induced fit docking
Docking study of the most active compounds and standard drugs
used were performed to validate the structure-activity relationship
(SAR) of similar compounds. The obtained docking score was correlated
with the biological activities. To understand the mode of binding, mo-
lecular modeling and induced fit docking studies of the most potent
antibacterial inhibitors 7e and 8b within the active site of tyrosyl-tRNA
synthetase (PDB ID: 1JIJ); most potent antifungal inhibitors 4e and 7e
within the active site of Candida albicans dihydrofolate reductase (PDB
2.3.3. Antitubercular activity
The synthesized compounds were screened for their antitubercular
activity against Mycobacterium tuberculosis (H37Rv) by Agar
4

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
binding energies as obtained through docking have large variations in
different structures. The five best docked poses of each ligands with
corresponding receptors, as obtained through induced fit docking, are
depicted in Table 4. In the best docking poses, the non-covalent
hydrogen bonding interactions and mode of binding of ligands are
shown in Fig. 1.
Table 3
Antitubercular activity of compounds 4a-c, 7a-h, 8a-c.
Comp.
No
R
R’
R’’
MICs (
μ
g/mL)a
Mycobacterium tuberculosis
(H37Rv strain)
4a
4b
4c
4d
4e
H
–
–
–
–
–
–
–
–
–
–
>12.5
4-CH3
4-Cl
4-OCH3
4-
>12.5
Docking simulation of most potent antibacterial inhibitors 8b and 7e
with tyrosyl-tRNA synthetase (PDB ID: 1JIJ) indicated that the glide
score as well as the binding energy of compound 8b is better than that of
7e. From the interactions study, it has been observed that compound 8b
>12.5
>12.5
10.41 ± 2.9
OC2H5
H
–
exhibit weak intermolecular hydrogen bonding (C H… O) interactions
7a
7b
7c
H
4-F-C6H4
4-F-C6H4
2-F-(4-
>12.5
>12.5
>12.5
H
CH3
with GLY193 and ASP40 having hydrogen-acceptor distance values
H
CH
2.250 Å and 1.901 Å respectively, while compound 7e displayed
(CH3)2
CH2CH3
CH3)C6H3
2-F-(4-
–
hydrogen bonding interactions (C H..O) with residues LYS84 and
7d
7e
7f
H
>12.5
>12.5
>12.5
>12.5
>12.5
ASP145 having hydrogen-acceptor distance values 2.203 and 1.828 Å
CH3)C6H3
4-F-C6H4
4-
H
respectively. Ligand 8b also demonstrated aromatic π-π interaction with
OC2H5
4-
the residue HIS50, arised due to electrostatic interaction. It is also clear
from the table that van der Waals interaction play important role in
ligand protein interaction. Electrostatic interactions are also present in
the docking complexes which are stronger interactions. Polar in-
teractions exist between ligands and the residues of the active site
involving HIS17, HIS50, THR75, GLN196, GLN174, GLN190 and SER82
residues of the binding site. The residues which form hydrophobic
pocket within the active site of 1JIJ are PHE54, ALA39, TYR170, TYR36,
CYS37, PRO53, ILE200, VAL191, LEU223, VAL224, PRO220, LEU52,
ILE221, PRO53 etc.
CH3
4-F-C6H4
OC2H5
4-
7g
7h
CH
2-F-(4-
CH3)C6H3
2-F-(4-
CH3)C6H3
–
OC2H5
4-
(CH3)2
CH2CH3
OC2H5
H
8a
8b
–
–
>12.5
4-
–
5.2 ± 1.5
OC2H5
4-CH3
–
8c
–
–
–
–
>12.5
Isoniazid
0.75 ± 0.0
a
The induced fit docking of molecules 4e and 7e within the active site
of Candida albicans dihydrofolate reductase (PDB ID:1AI9) showed
improved activity of 4e over 7e. Binding energy (76.461 kcal/mol) and
glide energy (ꢀ 7.868) of 4e are much larger than the binding energy
(22.211 kcal/mol) and glide score (ꢀ 6.641 kcal/mol) respectively of 7e.
In the best docking poses, weak intermolecular hydrogen bonding in-
Data are represented in the form of mean of MICs ± S.D.
ID:1AI9) and most potent antitubercular inhibitors 4e and 8b within the
active site of alpha-sterol demethylase (CYP51) from M. tuberculosis
(PDB ID: 1EA1) have been carried out. These enzymes were chosen for
the docking studies because these enzymes catalyse reactions in the
metabolic pathways of Staphylococcus aures, Candida albicans and
Mycobacterium tuberculosis that are responsible for bacterial, fungal and
tubercular infections respectively and the studied compounds have been
evaluated for their biological activities against these microbes. The
–
teractions (C H… O) were exhibited by both ligands 4e and 7e
involving residue ALA11 with d(H… O) = 2.296 and 2.246 Å respec-
–
tively. Molecule 7e also exhibited classical O H… O hydrogen bonding
with SER61 (H… O = 1.740 Å). There exist aromatic
π-π stacking
Table 4
Induced fit docking results of potent compounds and drugs with corresponding targets.
Targets(PDBIDs)
Compounds
G-score(kcal/mol)
E-lipo(kcal/mol)
E-vdw(kcal/mol)
E-coul(kcal/mol)
E-glide(kcal/mol)
1JIJ
8b
ꢀ 5.763
ꢀ 5.726
ꢀ 5.547
ꢀ 5.455
ꢀ 4.324
ꢀ 4.082
ꢀ 4.046
ꢀ 4.012
ꢀ 7.496
ꢀ 7.495
ꢀ 7.868
ꢀ 6.384
ꢀ 6.243
ꢀ 6.235
ꢀ 6.641
ꢀ 5.171
ꢀ 4.738
ꢀ 4.715
ꢀ 6.087
ꢀ 6.072
ꢀ 5.457
ꢀ 5.454
ꢀ 5.425
ꢀ 5.390
ꢀ 4.972
ꢀ 4.851
ꢀ 4.669
ꢀ 4.467
ꢀ 5.892
ꢀ 5.844
ꢀ 1.852
ꢀ 1.844
ꢀ 1.583
ꢀ 1.574
ꢀ 2.505
ꢀ 2.433
ꢀ 2.451
ꢀ 2.396
ꢀ 1.966
ꢀ 1.967
ꢀ 1.824
ꢀ 2.056
ꢀ 1.800
ꢀ 1.542
ꢀ 3.720
ꢀ 2.920
ꢀ 2.635
ꢀ 2.630
ꢀ 1.648
ꢀ 1.652
ꢀ 1.630
ꢀ 1.558
ꢀ 1.975
ꢀ 2.426
ꢀ 1.150
ꢀ 1.678
ꢀ 1.533
ꢀ 1.178
ꢀ 1.280
ꢀ 1.282
ꢀ 17.160
ꢀ 17.452
ꢀ 17.148
ꢀ 17.062
ꢀ 15.262
ꢀ 14.631
ꢀ 14.738
ꢀ 14.566
ꢀ 39.815
ꢀ 39.825
ꢀ 8.161
ꢀ 6.623
ꢀ 6.006
ꢀ 6.415
ꢀ 6.740
ꢀ 1.225
ꢀ 0.847
ꢀ 0.552
ꢀ 0.654
ꢀ 9.534
ꢀ 9.535
ꢀ 68.300
ꢀ 8.660
ꢀ 9.128
ꢀ 9.823
ꢀ 9.166
ꢀ 7.651
ꢀ 5.231
ꢀ 4.820
ꢀ 5.161
ꢀ 5.493
ꢀ 4.810
ꢀ 6.196
ꢀ 5.448
–2.956
–23.783
–23.459
–23.563
–23.801
ꢀ 16.488
ꢀ 15.478
ꢀ 15.289
ꢀ 15.220
ꢀ 49.384
ꢀ 49.361
ꢀ 76.461
ꢀ 19.649
ꢀ 18.696
ꢀ 20.582
–22.211
ꢀ 21.141
ꢀ 20.648
ꢀ 20.954
ꢀ 37.327
ꢀ 37.758
ꢀ 16.543
ꢀ 17.420
ꢀ 17.456
ꢀ 15.051
ꢀ 19.463
ꢀ 19.731
ꢀ 21.857
ꢀ 16.760
ꢀ 25.316
ꢀ 25.308
7e
Ciprofloxacin
1AI9
4e
ꢀ 10.989
ꢀ 9.568
ꢀ 10.760
ꢀ 13.045
ꢀ 13.490
ꢀ 15.417
ꢀ 16.135
–32.166
–32.265
ꢀ 11.733
ꢀ 11.223
ꢀ 12.008
ꢀ 12.095
ꢀ 11.060
ꢀ 9.328
7e
Fluconazole
1EA1
8b
4e
ꢀ 8.403
ꢀ 10.403
ꢀ 11.536
ꢀ 7.087
ꢀ 3.807
ꢀ 3.829
ꢀ 10.322
ꢀ 9.673
Isoniazid
ꢀ 21.509
ꢀ 21.478
5

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
Fig. 1. The best docking poses, the non-covalent hydrogen bonding interactions and mode of binding of Compound 7e, (A), 8b, (B) within the active site of tyrosyl-
tRNA synthetase; Compound 4e, (C) 7e, (D) within the active site of Candida albicans dihydrofolate reductase, Compound 4e, (E), 8b, (F) within the active site of
alpha-sterol demethylase (CYP51) from M. tuberculosis.
interaction exhibited by 4e and residue PHE36. Residues forming hy-
drophobic pocket are ILE112, VAL52, LEU69, PRO70, ILE62, PHE66,
PHE36, MET25, ILE19, TYR118, ALA11, VAL10, ILE9, ILE33, TRP27,
VAL10 etc. The residues which are responsible for polar interactions in
the active site are THR58, THR147 and SER69.
of former is better. However, glide docking energy of 4e is slightly more
–
–
negative than that of 8b. Weak C H… N and C H… O hydrogen
bonding interactions are exhibited by 8b and 4e involving residues
THR260 and LEU321 respectively of the active site. Binding pocket
residues MET79, VAL434, PHE78, ILE385, PRO349, PRO319, LEU324,
PRO386, PRO320, PROLEU321, CYS394, VAL395, LEU100, PHE255
and PHE85 form hydrophobic enclosure.
The experimentally determined most potent antitubercular in-
hibitors 4e and 8e, when docked into the active site of alpha-sterol
demethylase (CYP51) from M. tuberculosis (PDB ID: 1EA1), showed
that molecule 8b bind with extra affinity compared to 4e as glide score
Analysis of the experimental and predicted activities was performed
by means of molecular docking to establish the predictive capability of
6

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
this approach. The docking models demonstrate reasonably good pre-
dictive power, as indicated by the correlation between the experimental
and predicted binding energy, docking score and hydrogen bonding
interactions of the potent inhibitors.
4.2. Synthesis
4.2.1. General procedure for the preparation of 3-(substituted-phenyl)-2-
hydrazino-quinazolin-4-ones 3 [30]
Our modelling results revealed that the mercapto-triazole segment in
between N-1 and C-2 of parent quinazolinone, 8b and electron releasing
group at para position of phenyl ring had an important influence on the
interactions of the protein–ligand complex and were crucial to the po-
tency of TyrRS inhibitory activity. The compound 8b also showing good
inhibitory action within the active site of alpha-sterol demethylase
(CYP51) from M. tuberculosis and may be due to aforesaid interaction.
The compound 4e, substituting a triazepine ring in between N-1 and C-2
and para ethoxy group for the phenyl group located at a N-3 position of
the parent quinazolinone, this may be causes that the enzyme inhibitory
activity of 4e within the active site of Candida albicans dihydrofolate
reductase. Presence of thiazolo-1,2,4-triazole skeleton between N-1 and
C-2 along with the para ethoxy group at N-3 phenyl ring and para fluoro
phenyl ring of thiazole nucleus leads to an increase in inhibitory actions
of the compound, which is evidenced by a significant docking scores i.e.,
binding energy and hydrogen bonding interactions. Docking studies of
approved antimicrobial, antifungal and antitubercular compounds have
also been done and are in good agreement with their experimental
inhibitory actions.
The starting material 1, 2 and 3 were prepared according to our
reported method [30]. A mixture of 3-(substituted-phenyl)-2-mercap-
tomethyl-quinazolin-4-one 2 (0.05 mol) and hydrazine hydrate (0.10
mol) in dioxane (50 mL) was reflux for 6 h. The solvent was removed and
residue was poured into ice cold water. The compound thus precipitated
was filtered, washed with water, dried in vacuum and crystallized from
aqueous ethanol.
4.3. General procedure for the preparation of 7-methyl-8-substituted-
phenyl-5-oxo-[1,2,4]-triazipeno-[3,4-a]-quinazolin-9(3H)-ones 4
A mixture of 3-phenyl-2-hydrazino-4-quinazolone (0.005 mol), eth-
ylacetoacetate (0.005 mol) and glacial acetic acid (2–3 drops) in dioxane
(50 mL) was refluxed for 6 h. The solvent was removed under reduced
pressure and residue was poured into ice cold water. The compound thus
precipitated was filtered, washed with water, dried in vacuum and
crystallized from aqueous ethanol.
4.3.1. 7-Methyl-8-phenyl-5-oxo-[1,2,4]-triazipeno-[3,4-a]-quinazolin-9
(3H)-one 4a
ꢀ 1
Yield 68%; mp 121 ◦C; IR (KBr) cm : 1685(C O six membered
–
3. Conclusion
–
–
–
–
ring), 1653(C O seven membered ring), 1572 (C N), 1556, 1525,
–
1
–
In the present investigation, novel quinazolinones were synthesized
and in-vitro screened for their potential use as antimicrobial and anti-
tubercular agents. The biological screening results of this investigation
revealed that, compounds having fused mercapto-1,2,4-triazole moiety
are more potent antibacterial agents, while compounds having fused
thiazolo-triazoles segment are more potent antifungal agents. It is to be
noted that compound 8b has highest activity against all tested bacteria
of this investigation. Most of the titled compounds were found to have
strong antifungal activities on A. niger than other tested species of fungi.
One of the target compounds, 8b showed better antitubercular profile
than other compounds against M. tuberculosis (H37Rv). Molecular
docking studies demonstrated that compounds 8b, 4e and 8b respec-
tively have better binding capabilities towards target enzymes. In
conclusion, it can be inferred that fusion of triazole nucleus (mercap-
totriazole or thiazolo-triazole) in between N-1 and C-2 positions of
quinazolinone ring works better for antimicrobial activity of this series
of compounds than others. The importance of such work lies in the
possibility that the new compounds could be more efficacious antimi-
crobial agents and can be used for further optimization in designing
more potent antimicrobial agents.
1440 (C C); H NMR (DMSO‑d ): δ 7.2–7.9 (m, 9H, Ar-H), δ 2.6(s, 2H,
–
–CH2CO), δ 2.0(s, 3H, –CH3); 613C NMR (DMSO‑d6):δ 190.3, 182.3,
166.7, 158.1, 140.2, 134.3, 133.1, 132.9, 131.8, 129.7 (2C), 127.4 (2C),
126.5, 125.8, 119.4, 42.2, 20.1. Anal. Calcd. for C18H14N4O2: C, 67.91;
H, 4.93; N, 17.60 Found: C,67.64; H,4.16; N,17.24.
4.3.2. 7-Methyl-8-(4-methylphenyl)-5-oxo-[1,2,4]-triazipeno-[3,4-a]-
quinazolin-9(3H)-one 4b
ꢀ 1
Yield 69%; mp 170 ◦C; IR (KBr) cm : 1685(C O six membered
–
–
–
–
–
ring), 1650(C O seven membered ring), 1551, 1515, 1425 (C C),
–
1
–
1570 (C N); H NMR (DMSO‑d ): δ 7.2–7.9 (m, 8H, Ar-H), δ 2.5(s, 2H,
–
6
–CH2CO), δ 2.3(s, 3H, –CH3), δ 2.0(s, 3H, –CH3); 13C NMR (DMSO‑d6):δ
190.4, 182.2, 166.5, 158.3, 140.3, 134.5, 133.6, 132.5, 134.7, 129.8
(2C), 128.0 (2C), 126.4, 126.0, 120.2, 42.3, 22.3, 20.2. Anal. Calcd. for
C
19H16N4O2: C, 68.66; H, 4.85; N, 16.86 Found: C,68.25; H,4.52;
N,16.62.
4.3.3. 7-Methyl-8-(4-chlorophenyl)-5-oxo-[1,2,4]-triazipeno-[3,4-a]-
quinazolin-9(3H)-one 4c
ꢀ 1
Yield 63%; mp 241 ◦C; IR (KBr) cm : 1680(C O six membered
–
–
–
–
–
ring), 1651(C O seven membered ring), 1540, 1476, 1410 (C C),
–
1
–
4. Experimental
1571 (C N); H NMR (DMSO‑d ):δ 7.5–7.9 (m, 8H, Ar-H), δ 2.5(s, 2H,
–
–CH2CO), δ 2.0(s, 3H, –CH3); 613C NMR (DMSO‑d6):δ 190.5, 182.0,
166.0, 158.4, 140.5, 134.6, 133.4, 132.7, 131.2 (2C), 129.8 (2C), 127.8,
126.6, 126.2, 119.9, 42.1, 20.5. Anal. Calcd. for C18H13N4O2Cl: C,
61.28; H, 3.71; N, 15.88 Found: C,60.55; H,3.47; N,15.59.
4.1. Materials
All reagents were purchased from Aldrich, solvents used were extra
dried. Melting points were taken in open capillaries and are uncorrected.
IR spectra were recorded in KBr on a Simadzu 8201 PC spectropho-
tometer (υmax in cmꢀ 1), 1H NMR and 13C NMR spectra in DMSO‑d6 on a
Bruker DRX-300 (300 MHz) spectrometer using TMS as an internal
reference (chemical shifts in δ ppm), Elemental analysis were performed
on elemental Vario EL III Carlo Erba 1105 CHN analyzer. Elemental (C,
H, N) analysis indicated that calculated and observed values were within
acceptable limit. The purity of compounds checked by thin layer chro-
matography on silica gel plate using ether/hexane and ethyl acetate as
solvent system. Iodine chamber was used as developing chamber. The
general synthesis of target compounds is depicted in Scheme 1. Molec-
ular docking studies of the compounds have been performed with the
suitable targets using Schrodinger software package (Glide, 2010).
4.3.4. 7-Methyl-8-(4-methoxyphenyl)-5-oxo-[1,2,4]-triazipeno-[3,4-a]-
quinazolin-9(3H)one 4d
ꢀ 1
Yield 61%; mp 249 ◦C; IR (KBr) cm : 1672(C O six membered
–
–
–
–
–
ring), 1651(C O seven membered ring), 1540, 1488, 1429 (C C),
–
1
–
1573 (C N); H NMR (DMSO‑d ): δ 7.2–7.9 (m, 8H, Ar-H), δ 3.9(s, 3H,
–
6
–OCH3), δ 2.5(s, 2H, –CH2O), δ 2.1(s, 3H, –CH3); 13C NMR (DMSO‑d6):δ
190.2, 182.4, 166.5, 158.2, 140.2, 134.2, 133.1, 132.0, 131.9, 129.8,
128.0, 126.8, 126.0 (2C), 120.5 (2C), 56.2, 42.2, 20.5. Anal. Calcd. for
C
19H16N4O3: C, 65.51; H, 4.63; N, 16.08 Found: C,6514; H,4.61;
N,15.08.
7

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
1
–
–
–
–
4.3.5. 7-Methyl-8-(4-ethoxyphenyl)-5-oxo-[1,2,4]-triazipeno-[3,4-a]-
1632 (exo C N), 1621 (endo C N); 1540, 1431, 1440 (C C); H NMR
–
–
quinazolin-9(3H)-one4e
(DMSO‑d6): δ 8.3(br, s, 1H, –NH), δ 7.2–7.8 (m, 12H, Ar-H), δ 2.3(q, 3H,
–CH3), Anal. Calcd. for C24H21N4O2F: C, 69.22; H, 5.08; N, 13.45 Found:
C,68.94; H,4.78; N,13.16.
ꢀ 1
Yield 59%; mp192 ◦C; IR (KBr) cm : 1675(C O six membered
–
–
–
–
–
–
ring), 1655(C O seven membered ring), 1610 (C N), 1535, 1478,
1
–
–
1410 (C C); H NMR (DMSO‑d ): δ 7.3–7.9 (m, 8H, Ar-H), δ 4.14(s, 2H,
6
–CH2), δ 2.5(s, 2H, –CH2O), δ 2.0(s, 3H, –CH3), δ1.2(t, 3H, –CH3), 13
C
4.4.7. 3-(4-Ethoxyphenyl)-2-[N’-1-(2-fluoro-4-methylphenyl)-2-
NMR (DMSO‑d6):δ 190.2, 182.3, 166.8, 158.3, 140.1, 134.5, 133.3,
132.9, 131.8, 129.9, 127.8, 126.9, 125.4 (2C), 120.2 (2C), 65.2, 41.3,
20.3, 15.2. Anal. Calcd. for C20H18N4O3: C, 66.29; H, 5.01; N, 15.46
Found: C,65.96; H,4.76; N,15.12.
methylpropylidene]-hydrazino-quinazolin-4-one 5g
ꢀ 1
Yield 65%; mp 152 ◦C; IR (KBr) cm : 3340(–NH), 1651(C O),
–
–
1
–
–
–
–
–
–
1635 (exo C N), 1620 (endo C N); 1531, 1470, 1415 (C C); H NMR
(DMSO‑d6): δ 8.1(br, s, 1H, –NH), δ 7.3–7.9 (m, 11H, Ar-H), δ 4.0(q, 2H,–
CH2), δ 1.4(m, 1H,–CH), δ 1.3(t, 3H, –CH3), 1.0(d, 6H, 2CH3); Anal.
Calcd. for C27H27N4O2F: C, 70.72; H, 5.94; N, 12.22 Found: C,70.51;
H,4.85; N,12.01.
4.4. General procedure for the preparation of 3-(substituted-phenyl)-2
[N’-[disubstituted-methylene]-hydrazino]-quinazolin-4-ones5
4.4.8. 3-(4-Ethoxyphenyl)-2-[N’-1-(2-fluoro-4-methylphenyl)
Equimolar quantities of 3-(substituted phenyl)-2-hydrazino-
quinazolin-4-one 3 (0.01 mol) and appropriate carbonyl compound
(0.01 mol) in methanol (20 mL) and glacial acetic acid (2 drops) were
refluxed for 6 h. The solvent was removed and reaction mixture was
poured into ice cold water, the crude product thus obtained was filtered,
dried in vacuum and crystallized from aqueous ethanol.
propylidene]-hydrazino]-quinazolin-4-one 5h
ꢀ 1
Yield 66%; mp 81 ◦C; IR (KBr) cm : 3350(–NH), 1654(C O), 1638
–
–
(exo C N), 1622 (endo C N), 1564, 1546, 1476 (C C); 1H NMR
–
–
–
–
–
–
(DMSO‑d6): δ 8.2(br, s, 1H, –NH), δ 7.2–7.8 (m, 11H, Ar-H), δ 3.8(q, 2H,–
OCH2), δ 1.5(q, 2H,- CH2), δ 2.1(S, 3H,–CH3), δ 1.3(t, 3H,–CH3), δ 1.0(t,
3H,–CH3), Anal. Calcd. for C26H25N4O2F: C, 70.25; H, 5.67; N, 12.60
Found: C,69.42; H,5.11; N,12.52.
4.4.1. 3-(Phenyl)-2[N’-(fluorophenyl-methylene)-hydrazino]-quinazolin-
4-one 5a
ꢀ 1
Yield 71%; mp 111 ◦C; IR (KBr) cm : 3340(–NH), 1660(C O),
4.5. General procedure for the preparation of 3-(substitutedphenyl)-2-{2,
–
–
1
–
–
–
–
–
–
1630 (exo C N), 1610 (endo C N), 1510, 1444, 1400 (C C); H NMR
2 disubstituted-4-oxo-thiazolidin-3-ylamino)-quinazolin-4-ones 6
(DMSO‑d6): δ 8.3(br, s, 1H, –NH), δ 7.2–8.0 (m, 13H, Ar-H), δ 6.4(s, 1H,
–CH). Anal. Calcd. for C21H15N4OF: C, 70.38; H, 4.22; N, 15.63 Found:
C,70.11; H,3.98; N,15.16.
A mixture of 3-(substituted-phenyl)-2[N’-[disubstituted-methylene]-
hydrazino]-quinazolin-4-ones 5 (0.002 mol) and mercapto acetic acid
(0.0022 mol) in dioxane (20 mL) was refluxed for 6 h.. The solvent was
removed and reaction mixture was poured into ice cold water. It was
treated with aqueous sodium bicarbonate to remove untreated mercapto
acetic acid. The crude product thus obtained was filtered, dried in
vacuum and crystallized from dioxan:water.
4.4.2. 3-(Phenyl)-2[N’-(4-fluorophenyl)ethylidene-hydrazino]-quinazolin-
4-one 5b
ꢀ 1
Yield 69%; mp 110 ◦C; IR (KBr) cm ; 3345(–NH), 1650(C O),
–
–
1
–
–
–
–
–
–
1620 (exo C N), 1600 (endo C N), 1570, 1445, 1400 (C C); H NMR
(DMSO‑d6): δ 8.1(br, s, 1H, NH), δ 7.2–8.0 (m, 13H, Ar-H), δ 2.3(s, 3H,
=CCH3). Anal. Calcd. for C22H17N4OF: C, 70.96; H, 4.60; N, 15.04
Found: C,69.94; H,3.98; N,15.15.
4.5.1. 3-(Phenyl)-2-[2-(4-fluorophenyl)-4-oxo-thiazolidin-3-ylamino]-
quinazolin-4-one 6a
ꢀ 1
Yield 75%; mp 86 ◦C; IR (KBr) cm : 3350(–NH), 1682 (C O six
–
–
–
–
–
4.4.3. 3-(Phenyl)-2[N’-(2-fluoro-4-methylphenyl)2-methylpropylidene-
membered ring), 1665 (C O five membered ring), 1608 (C N), 1542,
–
1
–
hydrazino]-quinazolin-4-one 5c
1486, 1400 (C C); H NMR (DMSO‑d ): δ 8.3(br, s, 1H, –NH), δ 7.2–8.8
–
6
ꢀ 1
Yield 70%; mp 89 ◦C; IR (KBr) cm : 3342(–NH), 1655(C O), 1622
(m, 13H, Ar-H), δ 5.1(s, 1H, –CH), δ 3.8(S, 2H,–CH2CO). Anal. Calcd. for
–
–
(exo C N), 1620 (endo C N), 1540, 1432, 1419 (C C); 1H NMR
C
23H17N4O2SF: C, 63.88; H, 3.96; N, 12.96 Found: C,63.54; H,3.58;
–
–
–
–
–
–
(DMSO‑d6): δ 8.2(br, s, 1H, –NH), δ 7.1–7.9 (m, 12H, Ar-H), 2.29 (s,3H,–
N,12.56.
CH3), δ 1.4(m, 1H,–CH(CH3)2), δ 1.1(d, 6H, 2CH3), Anal. Calcd. for
C
25H23N4OF: C, 72.45; H, 5.59; N, 13.52 Found: C,72.12; H,5.25;
4.5.2. 3-(Phenyl)-2-[2-(4-fluorophenyl)-2-methyl-4-oxo-thiazolidin-3-
N,14.86.
ylamino]-quinazolin-4-one 6b
ꢀ 1
Yield 70%; mp 92 ◦C; IR (KBr) cm : 3355(–NH), 1683 (C O six
–
–
–
4.4.4. 3-(Phenyl)-2[N’-1-(2-fluoro-4-methylphenyl)propylidene]-
membered ring), 1662 (C O five membered ring), 1531, 1470, 1415
–
1
–
–
–
hydrazino-quinazolin-4-one 5d
(C C), 1612 (C N); . H NMR (DMSO‑d ): δ 8.2(br,s, 1H,–NH), δ
–
6
ꢀ 1
Yield 56%; mp 94 ◦C; IR (KBr) cm : 3344(–NH), 1660(C O), 1625
7.1–7.8 (m, 13H, Ar-H), δ 3.8(s, 2H, –CH2CO), δ 2.4(s,3H,–CH3), Anal.
Calcd. for C24H19N4O2SF: C, 64.56; H, 4.29; N, 12.55 Found: C,64.25;
H,4.11; N,12.19.
–
–
(exo C N), 1621 (endo C N), 1545, 1430, 1415 (C C); 1H NMR
–
–
–
–
–
–
(DMSO‑d6): δ 8.2(br, s, 1H, –NH), δ 7.1–7.9 (m, 12H, Ar-H), δ 1.5(q, 2H,
–CH2), 1.0(t, 3H, –CH3). Anal. Calcd. for C24H21N4OF: C, 71.98; H, 5.29;
N, 13.99 Found: C,71.10; H,4.96; N,13.25.
4.5.3. 3-(Phenyl)-2-[2-(2-fluoro-4-methylphenyl-2-isopropyl)-4-oxo-
thiozolidin-3-ylamino]-quinazolin-4-one 6c
ꢀ 1
Yield 65%; mp 104 ◦C; IR (KBr) cm : 3353(–NH), 1681 (C O six
–
–
4.4.5. 3-(4-Ethoxyphenyl)-2-[N’-(4-fluorophenyl)methylene]-hydrazino-
–
quinazolin-4-one 5e.
membered ring), 1660 (C O five membered ring), 1540, 1465, 1405
–
ꢀ 1
Yield 68%; mp 97 ◦C; IR (KBr) cm : 3350(–NH), 1662(C O), 1630
(C C), 1610 (C N); 1H NMR (DMSO‑d6): δ 8.3(br, s, 1H, –NH), δ
–
–
–
–
–
–
(exo C N), 1623 (endo C N); 1564, 1444, 1430(C C); 1H NMR
7.2–7.9 (m, 12H, Ar-H), δ 3.9(s, 2H, CH2CO), δ 2.3(s,3H,–CH3), δ 1.4(m,
1H,–CHCH3), δ 1.0(d, 6H, 2-CH3), Anal. Calcd. for C27H25N4O2SF: C,
66.37; H, 5.16; N, 11.47 Found: C,66.10; H,4.90; N,11.10.
–
–
–
–
–
–
(DMSO‑d6): δ 8.3(br, s, 1H, –NH), δ 7.2–8.0 (m, 12H, Ar-H), δ 6.3(s, 1H,–
CH),
δ 4.0(q, 2H,–CH2), δ 1.2(t, 3H,–CH3); Anal. Calcd. for
C
23H19N4O2F: C, 68.65; H, 4.76; N, 13.92 Found: C,68.24; H,4.58;
N,13.56.
4.5.4. 3-(Phenyl)-2-[2-(ethyl-2-fluoro-4-methylphenyl)-4-oxo-thiazolidin-
3-ylamino]-quina zolin-4-one 6d
ꢀ 1
Yield 62%; mp 94 ◦C; IR (KBr) cm : 3356(–NH), 1685 (C O six
–
–
4.4.6. 3-(4-Ethoxyphenyl)-2-[N’-1-(4-fluorophenyl)ethylidene]-
–
hydrazino-quinazolin-4-one 5f
membered ring), 1665 (C O five membered ring), 1542, 1472, 1418
–
ꢀ 1
Yield 70%; mp 130 ◦C; IR (KBr) cm : 3346(–NH), 1659(C O),
(C C), 1615 (C N); 1H NMR (DMSO‑d6): δ 8.4(br, s, 1H, –NH), δ
–
–
–
–
–
–
8

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
–
7.1–7.9 (m, 12H, Ar-H), δ 3.8(s, 2H, –CH2CO), δ 2.2(s, 3H, –CH3), 2.1(q,
2H,–CH2), δ 1.1(t,3H,–CH3), δ 0.9(t, 3H, CH2CH2CH3), Anal. Calcd. for
C26H23N4O2SF: C, 65.81; H, 4.89; N, 11.81 Found: C,65.52; H,4.61;
N,11.56.
1515, 1442, 1400 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.9 (m, 13H, Ar-
–
H), δ 5.4(s, 1H, –CH = C), δ 2.3(s, 3H, CH3), 13C NMR (DMSO‑d6), δ
181.2, 160.8, 153.5, 147.3, 142.4, 136.5, 134.3, 132.8, 131.3, 128.7,
127.3, 126.9, 125.8 (2C), 125.0 (2C), 124.8 (2C), 121.3 (2C), 120.3,
90.5, 80.3, 21.2. Anal. Calcd. for C24H17N4OSF: C, 67.29; H, 4.00; N,
13.08 Found: C,66.95; H,3.68; N,12.79.
4.5.5. 3-(4-Ethoxyphenyl)-2-[2-(4-fluorophenyl)-4-oxo-thiazolidin-3-
ylamino]-quinazolin-4-one 6e
ꢀ 1
Yield 71%; mp 204 ◦C; IR (KBr) cm : 3365(–NH), 1690 (C O six
4.6.3. 2-(2-Fluoro-4-methylphenyl)-2-isopropyl-8-phenyl-[1,3]-thiazolo-
–
–
–
membered ring), 1667 (C O five membered ring), 1448, 1450, 1545
[3,4-a] [1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7c
–
ꢀ 1
(C C), 1610 (C N); 1H NMR (DMSO‑d6): δ8.3(br, s,1H, –NH), δ
Yield 69%; mp 107 ◦C; IR (KBr) cm : 1680 (C O), 1572 (C N),
–
–
–
–
–
–
–
–
–
6.7–7.9 (m, 12H, Ar-H), δ 5.1(s,1H,–CH), δ 4.0(q, 2H, –OCH2), δ 3.8(s,
2H, –CH2CO), 1.1(t,3H,–CH3) ; Anal. Calcd. for C25H21N4O3SF: C, 63.01;
H, 4.44; N, 11.7 Found: C,64.25; H,4.16; N,11.56.
1548, 1445, 1410 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.8 (m, 12H, Ar-
–
H), δ 5.3(s, 1H, –CH = C), δ 2.3(s, 3H, CH3), δ 1.2(m, 1H, CH), δ 1.1
(d, 6H, 2CH3); 13C NMR (DMSO‑d6), δ 181.2, 160.8, 153.9, 147.8, 142.5,
136.5, 134.2, 132.8, 132.3, 130.2, 129.9, 129.4, 127.2, 126.8, 126.5,
126.0, 125.8 (2C), 120.8 (2C), 119.7, 90.2, 80.7, 42.2, 21.5, 15.6 (2C).
Anal. Calcd. for C27H23N4OSF: C, 68.92; H, 4.93; N, 11.91 Found:
C,68.59; H,4.62; N,11.76.
4.5.6. 3-(4Ethoxyphenyl)-2-[2-(4-fluorophenyl)-2-methyl-4-oxo-
thiazolidin-3-ylamino]–quinazolin-4-one 6f
ꢀ 1
Yield 71%; mp 204 ◦C; IR (KBr) cm : 3360(–NH), 1690 (C O six
–
–
–
membered ring), 1665 (C O five membered ring), 1541, 1472, 1420
–
(C C), 1620 (C N); 1H NMR (DMSO‑d6): δ8.2(br, s, 1H, –NH), δ
4.6.4. 2-Ethyl-2(2-fluoro-4-methylphenyl)-8-phenyl-[1,3]-thiazolo-[3,4-
–
–
–
–
7.7–7.9 (m, 12H, Ar-H), δ 4.0(q, 2H, OCH2), δ 3.7(s, 2H, –CH2CO), δ 2.4
(s,3H,–CH3), δ 1.3(t, 3H, –CH3); Anal. Calcd. for C26H23N4O3SF: C,
63.66; H, 4.73; N, 11.42 Found: C,63.14; H,4.50; N,11.20.
a] [1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7d
ꢀ 1
Yield 60%; mp 109 ◦C; IR (KBr) cm : 1683 (C O), 1575 (C N),
–
–
–
–
1550, 1547, 1415 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.8 (m, 12H, Ar-
–
–
H), δ 5.4(s, 1H, –CH = C), δ 3.9(q,2H,–CH2), δ 2.3(s, 3H, CH3), δ 1.3(t,
3H, –CH3), 13C NMR (DMSO‑d6), δ 181.3, 160.9, 153.7, 147.9, 142.6,
136.9, 134.1, 132.5, 132.2, 130.1, 129.8, 129.3, 127.5, 126.3, 126.9,
126.2, 125.8 (2C), 120.7 (2C), 119.8, 90.3, 80.4, 42.5, 35.8, 16.2. Anal.
Calcd. for C26H21N4OSF: C, 68.40; H, 4.64; N, 12.27 Found: C,68.10;
H,4.25; N,11.96.
4.5.7. 3-(4-Ethoxyphenyl)-2-[2-(2-fluoro-4-methyl)-2-methyl-4-oxo-
thiazolidin-3-ylamino]-quinazolin-4-one 6g
ꢀ 1
Yield 65%; mp 231 ◦C; IR (KBr) cm : 3356(–NH), 1685 (C O six
–
–
–
–
membered ring), 1662 (C O five membered ring), 1545, 1488, 1425
(C C), 1622 (C N); 1H NMR (DMSO‑d6):δ8.9(br, s, 1H, –NH), δ
–
–
–
–
6.9–7.8 (m, 11H, Ar-H), δ 4.0(q, 2H, –OCH2), δ 3.8(s, 2H, –CH2CO), δ 2.3
(s,3H,–CH3), δ 1.3(t, 3H, –CH3), δ 1.5(m, 1H,–CHCH3), δ 1.0(d, 6H, 2-
CH3); Anal. Calcd. for C29H29N4O3SF: C, 65.39; H, 5.49; N, 10.52 Found:
C,65.01; H,5.24; N,10.10.
4.6.5. 2-(4-Fluorophenyl)-8-(4-ethoxyphenyl)-[1,3]-thiazolo-[3,4-a]
[1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7e
ꢀ 1
Yield 62% ;mp 102 ◦C; IR (KBr) cm : 1690 (C O), 1572 (C N),
–
–
–
–
1543, 1526, 1441 (C C); 1H NMR (DMSO‑d6): δ 7.2–7.8 (m, 12H, Ar-
–
–
4.5.8. 3-(4-Ethoxyphenyl)-2-[2-ethyl-(2-fluro-4-methyl)-4-oxo-
H), δ 5.6(s, 1H, –CH = C), δ 3.7(s, 1H, S-CH-N), δ 3.9(q, 2H, –CH2), δ
1.3(t, 3H, –CH3), 13C NMR (DMSO‑d6), δ 181.3, 160.8, 153.7, 147.2,
142.1, 136.1, 134.2, 132.9, 132.3, 129.4, 129.0, 127.3, 126.6 (2C),
126.0 (2C), 125.8 (2C), 120.8 (2C), 119.9, 90.2, 80.7, 63.2, 15.3. Anal.
Calcd. for C25H19N4O2SF: C, 65.49; H, 4.18; N, 12.22 Found: C,65.13;
H,4.03; N,12.01.
thiazoildin-3-ylamino]-quinazolin-4-one 6h
ꢀ 1
Yield 62%; mp 119 ◦C; IR (KBr) cm : 3354(–NH), 1685 (C O six
–
–
–
membered ring), 1660 (C O five membered ring), 1550, 1488, 1430
–
1
–
–
–
–
(C C), 1630 (C N); H NMR (DMSO‑d ): δ8.3(br, s, 1H –HN), δ 6.9–7.9
6
(m, 11H, Ar-H), δ 4.0(q, 2H, –OCH2), δ 3.9(s, 2H, –CH2CO), δ 2.3(s, 3H,
–CH3), δ 2.1(q, 2H, –CH2), δ 1.3(t, 3H, –OCH2CH3), δ 1.0(t, 3H,
–CH2CH3); Anal. Calcd. for C28H27N4O3SF: C, 64.85; H, 5.2; N, 10.80
Found: C,64.58; H,5.10; N,10.56.
4.6.6. 2-(4-Fluorophenyl)-2-methyl-8-(4-ethoxyphenyl)-[1,3]-thiazolo-
[3,4-a][1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7f
ꢀ 1
Yield 63%; mp 178 ◦C; IR (KBr) cm : 1688 (C O), 1570 (C N),
–
–
–
–
1542, 1526, 1541 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.8 (m, 12H, Ar-
–
–
4.6. General procedure for the preparation of 2,2,8,(trisubstituted-
phenyl)-[1,3]-thiazolo-[3,4-a][1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-
ones 7
H), δ 5.6(s, 1H, –CH = C), δ 3.9(q, 2H, –CH2), δ 2.4(s, 3H, –CH3), δ
2.3(t, 3H, –CH3); 13C NMR (DMSO‑d6), δ 181.3, 160.8, 153.2, 148.0,
142.1, 136.1, 134.5, 132.7, 132.0, 129.9, 129.3, 127.5, 126.4 (2C),
126.1 (2C), 125.8 (2C), 120.2 (2C), 119.6, 90.4, 80.7, 63.4, 21.3, 15.4.
Anal. Calcd. for C26H21N4O2SF: C, 66.69; H, 4.48; N, 11.86 Found:
C,65.81; H,4.21; N,11.64.
A slurry of compound 6 (0.001 mol) in conc. H2SO4 (1.5 mL) was
made below 15 ◦C and stirred for 2 h. The mixture was then treated with
crushed ice The well stirred solution was neutralized with sodium bi-
carbonate. The compound thus obtained was filtered, dried in vacuum
and crystallized from aqueous ethanol.
4.6.7. 2-(2-Fluoro-4-methylphenyl)-2-isopropyl-8-(4-ethoxyphenyl)-[1,3]-
thiazolo-[3,4-a][1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7g
ꢀ 1
Yield 58%; mp 240 ◦C; IR (KBr) cm : 1675 (C O), 1560 (C N),
–
–
–
–
4.6.1. 2-Ethyl-2-(4-fluorophenyl)-8-phenyl-[1,3]-thiazolo-[3,4-a][1,2,4]-
1550, 1445, 1400 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.8 (m, 11H, Ar-
–
–
triazolo-[2,3-a]-quinazolin-9(3H)-one 7a
ꢀ 1
Yield 72%; mp 204 ◦C; IR (KBr) cm : 1682 (C O), 1574 (C N),
–
–
–
–
H), δ 5.6(s, 1H, –CH = C), δ 3.9(q, 2H, –OCH2), δ 1.3(t, 3H, –CH3), δ
2.3(s, 3H, –CH3), δ 1.2(m, 1H, –CH(CH3)2), δ 1.1(d, 6H, 2-CH3); 13C
NMR (DMSO‑d6), δ 181.9, 160.8, 153.4, 147.5, 142.3, 136.2, 134.2,
132.9, 132.3, 130.4, 129.8, 129.5, 127.3, 126.6, 126.4, 126.0, 125.8
(2C), 120.8 (2C), 119.9, 90.3, 80.7, 64.8, 42.5, 21.6, 16.4, 15.3 (2C).
Anal. Calcd. for C29H27N4O2SF: C, 67.68; H, 5.29; N, 10.89 Found:
C,67.35; H,5.11; N,10.61.
1510, 1440, 1400 (C C); 1H NMR (DMSO‑d6): δ 7.2–7.9 (m, 13H, Ar-
–
–
H), δ 4.8(s, 1H, –CH), δ 5.3(s, 1H, –CH = C); 13C NMR (DMSO‑d6), δ
181.2, 160.9, 153.9, 147.2, 142.1, 136.2, 134.3, 132.9, 131.0, 128.5,
127.2, 126.8, 126.0 (2C), 125.8 (2C), 124.9 (2C), 121.0 (2C), 120.2,
89.2, 80.1. Anal. Calcd. for C23H15N4OSF: C, 66.55; H, 3.65; N, 13.52
Found: C,66.13; H,3.21; N,13.12.
4.6.8. Ethyl-2-(2-fluoro-4-methylphenyl)-8-(4-ethoxyphenyl)-[1,3]-
4.6.2. 2-(4-Fluorophenyl)-2-methyl-8-phenyl-[1,3]-thiazolo-[3,4-a]
thiazolo-[3,4-a][1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7h
[1,2,4]-triazolo-[2,3-a]-quinazolin-9(3H)-one 7b
ꢀ 1
ꢀ 1
Yield 61%; mp 192 ◦C; IR (KBr) cm : 1678 (C O), 1562 (C N),
Yield 62%; mp 117 ◦C; IR (KBr) cm : 1682 (C O), 1570 (C N),
–
–
–
–
–
–
–
–
9

S. Kumar Pandey et al.
Bioorganic Chemistry 108 (2021) 104611
1555, 1450, 1410 (C C); 1H NMR (DMSO‑d6): δ 6.9–7.8 (m, 11H, Ar-
compound that result no visible growth on plates were recorded in
Table 1. DMSO was used as a solvent control to insure that solvent had
no effect on bacterial growth [39]. Ciprofloxacin was designated as
standard drug in our experiment
–
–
H), δ 4.6(s, 1H, –CH = C), δ 4.0 (q, 2H, –OCH2), δ 2.4(q, 2H, –CH2), δ
2.2(s, 3H, –CH3), δ 1.2(t, 3H, –OCH2CH3), δ 1.0(t, 3H, –CH2CH3); 13C
NMR (DMSO‑d6), δ 181.8, 160.7, 153.9, 147.6, 142.5, 135.6, 134.8,
132.9, 132.4, 130.5, 130.0, 129.8, 127.5, 126.7, 126.2, 126.0, 125.9
(2C), 120.7 (2C), 119.4, 90.4, 80.5, 64.9, 35.9, 21.5, 16.6, 15.2. Anal.
Calcd. for C28H25N4O2SF: C, 67.18; H, 5.03; N, 11.09 Found: C,66.90;
H,4.81, N,10.90.
4.8.2. Antifungal screening
Serial plate dilution method was used to screen the antifungal ac-
tivity of all the titled compounds against the C. albicans, A. fumigatus, A.
flavus and A. niger (recultured) in DMSO. Test compound (5 mg) were
dissolved in 1 mL of dimethyl sulfoxide (DMSO) and solution was
diluted with water (9 mL). Further progressive dilution with melted
Muller Hinton agar were performed to obtain required concentrations of
4.7. General procedure for preparation of 3-mercapto-5-substituted
phenyl-[1,2,4]-triazolo-[4, 3-a]-quinazolin-7(3H)-ones 8
50 μg/mL, 25 μg/mL, 12.5 μg/mL, 6.25 μg/mL, 3.12 μg/mL and 1.56 μg/
A mixture of 3-substituted phenyl-2-hydrazino-4-quinazolone 3
(0.005 mol), carbondisulfide (0.007 mol) and potassium hydroxide
(0.007 mol) in methanol (20 mL) was refluxed for 4 h. The solvent was
removed; residue was poured into ice cold water and treated with con
HCl. The compound thus precipitated, was filtered, washed with water,
dried in vacuum and crystallized from aqueous ethanol.
mL. Petridishes were inoculated with 1.5 × 10ꢀ 4 colony forming units
(CFU) and incubated at 37 ◦C for 26 h and the growth was monitored
visually and spectrophotometrically. The lowest concentration required
to arrest the growth of bacteria termed as minimum inhibitory con-
centration (MIC in μg/mL). To insure that solvent has no effect on fungal
growth a control test was performed with DMSO. Fluconazole was used
as a standard drug in this investigation. The results are incorporated in
Table 2.
4.7.1. 3-Mercapto-5-phenyl [1,2,4]-triazolo-[4,3-a]-quinazolin-7(3H)-
ones 8a [38]
ꢀ 1
Yield 64%; mp 129 ◦C; IR (KBr) cm : 2523(-SH), 1675 (C O), 1603
–
–
1
–
–
–
–
– –
N
4.8.3. Antitubercular activity
(C N), 1541, 1486, 1400 (C C), 1109(C
C); H NMR (DMSO‑d6):
δ 7.2–7.9 (m, 9H, Ar-H), δ 3.1(s, 1H, -SH); 13C NMR (DMSO‑d6): δ 180.3,
165.6, 160.2, 139.2, 135.8, 134.9, 132.3, 129.7, 129.4, 127.2, 126.5,
125.8 (2C), 123.5 (2C). Anal. Calcd. for C15H10N4OS: C, 61.21; H, 3.42;
N, 19.04 Found: C,60.95; H,3.10; N,18.76.
Drug susceptibility and determination of MIC of the test compounds/
drugs against M. tuberculosis H37Rv were performed by agar micro
dilution method where serial twofold dilutions of each test compound
were added into 7H10 agar, and M. tuberculosis H37Rv was used as test
organism. MIC is the concentration of the compound that completely
inhibits the growth and colony forming ability of M. tuberculosis. In a 24-
well plate, 3 mL Middlebrook 7H11 agar medium with OADC supple-
ment is dispensed in each well. The test compound is added to the
Middlebrook medium agar before in duplicate so that final concentra-
4.7.2. 3-Mercapto-5-(4-ethoxyphenyl)-[1,2,4]-triazolo-[4,3-a]-
quinazolin-7(3H)-one 8b
ꢀ 1
Yield 72%; mp 222 ◦C; IR (KBr) cm : 2520(SH), 1672 (C O),
–
–
–
–
– –
N
1608C = N), 1540, 1488, 1429 (C C), 1132(C
C); 1H NMR
tion of test compound in each well is 12.5, 6.25, 3.12, and 1.56 μg/mL,
(DMSO‑d6): δ 7.0–7.9 (s, 8H, Ar-H), δ 3.1(s, 1H, -SH), δ 4.0(q,2H,–
OCH2), δ1.3(t, 3H, –CH3), 13C NMR (DMSO‑d6): δ179.5, 164.2, 160.1,
152.2, 136.2, 134.0, 133.1, 129.8, 129.0, 126.8, 126.0, 124.1 (2C),
123.1 (2C), 66.3, 15.2. Anal. Calcd. for C17H14N4O2S: C, 60.34; H, 4.17;
N, 16.56 Found: C,60.10; H,3.91; N,16.21.
respectively. The known CFU of H37Rv culture was dispensed on top of
agar in each well in negative pressure biosafety hood. The plates are
then incubated at 37 ◦C/5% CO2 incubator. The concentrations at which
complete inhibition of growths were observed and taken as MICs of
tested compounds. Isoniazid was used as a standard drug.
4.7.3. 3-Mercapto-5-(4-methylphenyl)-[1,2,4]-triazolo-[4,3-a]-
4.8.4. Statistical analysis
quinazolin-7(3H)-one 8c
ꢀ 1
Yield 64%; mp 136 ◦C; IR (KBr) cm : 2530(-SH), 1665 (C O), 1615
–
All the tested compounds exhibited substantial activity and the MIC
values were calculated statistically by following the values of standard
deviation. All the assays were done in triplicate and the results were
given as mean of MICs ± S.D. (Standard Deviation). Data were analyzed
and the values were considered as statistically significant.
–
1
–
–
–
–
– –
N
(C N), 1535, 1478, 1410 (C C), 1129(C
C); H NMR (DMSO‑d6):
δ 6.9–7.9 (s, 8H, Ar-H), δ 3.1(s, 1H, -SH), δ 2.4(s, 3H, –CH3), 13C NMR
(DMSO‑d6): δ 179.0, 162.2, 160.0, 141.2, 136.2, 135.1, 133.3, 128.7,
128.0, 127.2, 126.4, 125.8 (2C), 123.4 (2C), 20.8. Anal. Calcd. for
C
16H12N4OS: C, 62.32; H, 3.92; N, 18.17 Found: C,62.03; H,3.60;
4.8.5. In silico molecular docking studies
N,18.17.
Molecular docking studies of the best two compounds for each
category and standard drugs, screened from MICs values, have been
performed with the suitable targets using Schrodinger software package
(Glide, 2010). The initial structures of the targets (proteins) were
retrieved from protein data bank (pdb ID: 1JIJ, 1AI9 and 1EA1; www.
pdb.org). These crystallographic structures were prepared using pro-
tein preparation wizard of the package, where hydrogens were added
and subsequently refinements of structures were carried out. The water
molecules were deleted in all structures as they were no critical to the
functioning of protein ligand interactions and bond orders were reas-
signed. Missing residues were added through the Prime application. The
systems were then minimized to an RMSD of 0.30 Å.
4.8. Biological activity
4.8.1. Antibacterial screening
The newly synthesized compounds 4a-e, 7a-h and 8a-c were
screened for their in vitro antibacterial activity against Gram-positive
bacteria: S. pneumoniae, B. subtilis, S. aureus and Gram-negative bacte-
ria: E. coli, P. aeruginosa by disc diffusion method. The bacterial strains
were subcultured in broth agar and incubated for 18 h at 37 ◦C and then
freshly prepared bacterial cells were spread onto nutrient agar plate in
laminar flow cabinet. Sterilized paper disc (6.0 mm in diameter) were
placed on nutrient agar plates. Test compounds (5 mg) were dissolved in
1 mL of dimethyl sulfoxide (DMSO) separately to prepare stock solution.
Geometries of all the ligands were optimized through B3LYP density
functional in conjunction with 6-31G** basis set using GAUSSIAN 03.
The optimized ligands were prepared using Ligprep module which
produced structures with various ionization states, stereochemistries,
tautomers and ring conformations which were subjected to full mini-
mization in the gas phase with optimized potential for liquid simulation
(OPLS2005) force field [40]. The prepared ligands and proteins were
From stock solution, different concentration 50
μ
g/mL, 25
μg/mL, 12.5
μ
g/mL, 6.25 μg/mL, 3.12 μg/mL and 1.56 μg/mL of each compound
were prepared. Thus, proper amounts of different concentrations of
compounds were piped on blank disc, which were placed on the plates.
The plates were incubated at 37 ◦C for 24 h. The minimum inhibitory
concentrations (MICs), the lowest concentration (μg/mL) of the test
10

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Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
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Acknowledgments
The authors are thankful to the Head, Department of Chemistry, DDU
Gorakhpur University, Gorakhpur, for providing necessary facilities.
SAIF, CDRI, Lucknow, for providing spectral data. Authors are also
thankful to Department of Biotechnology, DDU Gorakhpur University,
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