Synthesis, antimicrobial evaluation, and docking studies of novel 4-substituted quinazoline derivatives as DNA-gyrase inhibitors

Article abstract of DOI:10.1002/ardp.201000065

Quinazoline derivatives are reported to have anti-inflammatory, analgesic, antibacterial, and anticancer activities. The incorporation of "OCH ₂CONH₂" (oxymethylcarbamide) at 4^th position of the quinazoline ring was found

Full text of DOI:10.1002/ardp.201000065

570
Arch. Pharm. Chem. Life Sci. 2010, 10, 570–576
Article
Synthesis, Antimicrobial Evaluation, and Docking Studies of
Novel 4-Substituted Quinazoline Derivatives as DNA-Gyrase
Inhibitors
Shireesha Boyapati, Umasankar Kulandaivelu, Srinivas Sangu and Malla Reddy Vanga
Medicinal Chemistry Research Division, Vaagdevi College of Pharmacy, Warangal, A.P, India
Quinazoline derivatives are reported to have anti-inflammatory, analgesic, antibacterial, and
anticancer activities. The incorporation of ‘‘OCH2CONH2’’ (oxymethylcarbamide) at 4th position of
the quinazoline ring was found to influence the biological activities of the molecules. With this
rationale, some new oxadiazolyl methyloxy quinazolines, pyrazolyl acetoxy methyl quinazolines,
triazolylmethyloxy quinazolines were synthesized from anthranilic acid through quinazolyl
oxyacetylhydrazide intermediates. All the compounds were characterized by IR, 1H-NMR, EI-MS,
and C, H, N analyses and evaluated for their antimicrobial activity. Docking studies on the DNA-
gyrase enzyme (1KZN) show their role in the antimicrobial activity of the molecules and explain the
higher potency of compounds 6a, 6b, 8a, 8b based on ReRanking scores and binding poses of the
molecules.
Keywords: DNA-Gyrase inhibitors / Antimicrobial activity / 4-Substituted quinazolines
Received: March 2, 2010; Accepted: April 16, 2010
DOI 10.1002/ardp.201000065
Introduction
agents as DNA gyrase inhibitors [16]. DNA gyrase is a pro-
karyotic type II topoisomerase and a major target of quino-
lone antibacterials.
Quinazolines and condensed quinazolines are found to pos-
sess potent anticancer [1], antihistaminic [2], and CNS activi-
ties like analgesic and anti-inflammatory [3–6], sedative-
hypnotic and anticonvulsant [7] activities. Quinazolinones
with substitution of ‘‘OCH2CONH2’’ (oxymethylcarbamide)
group at the 4th position have been reported to enhance
the biological activities [8]. Quinazolines were also found
to have antibacterial activity [9–15]. Antibiotic resistance
in the community is a growing public health concern due
to the continued emergence of multi drug resistant bacterial
strains. In view of this fact, the design and synthesis of newer
antibacterials are of immense significance and continue to
attract the attention of numerous medicinal chemists.
The 4-quinolones such as ciprofloxacin, ofloxacin, lome-
floxacin, gatifloxacin, are established synthetic antibacterial
Bacterial DNA gyrase belongs to a superfamily of ATPases,
known as GHKL (gyrase, Hsp90, histidine, kinase, MutL),
which consists of several important targets for infection
and cancer treatments. It is involved in the vital processes
of DNA replication, transcription, and recombination [17]. It
has the ability to catalyze the ATP-dependent introduction of
negative supercoils into bacterial DNA and to unknot DNA.
This enzyme consists of two subunits, A and B, of molecular
masses 97 and 90 kDa, respectively, with the active enzyme
being an A2B2 complex. The A-subunit of DNA gyrase is
involved in DNA breakage and reunion, whereas the B-sub-
unit catalyzes the hydrolysis of ATP [18]. A majority of
mutations conferring resistance to quinolones arise within
the quinolone resistance-determining region of GyrA close to
the active site (Tyr122) where DNA is bound and cleaved.
However, some quinolone resistance mutations are known
to exist in GyrB and these residues lie at a considerable
distance from the quinolone resistance-determining region.
The mutations exert their effect by decreasing the amount of
Correspondence: Dr. Shireesha Boyapati, M. Pharm. PhD, Associate
Professor Medicinal Chemistry Research Division, Vaagdevi College of
Pharmacy, Hanamkonda, Warangal-506001, India.
E-mail: sirimedchem@yahoo.co.in; sirimedchem@gmail.com
Fax: þ91 870 254-4949
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Arch. Pharm. Chem. Life Sci. 2010, 10, 570–576
4-Substituted Quinazolines as DNA-Gyrase Inhibitors
571
quinolone bound to a gyrase-DNA complex and GyrB residues Results and Discussion
form part of a quinolone binding pocket that includes DNA
and the quinolone resistance-determining region in GyrA.
Large conformational changes during the catalytic cycle of
the enzyme allow these regions to come into close proximity
[19, 20].
Chemistry
Quinazolines with substitutions on the 2nd and 4th positions
and either halogens or electron-rich substituents on the 6th
position were known to promote activity against bacterial
cultures. The synthesis of designed 4-substituted quinazoline
derivatives was achieved following the steps outlined in
Scheme 1. Anthranilic acid 1 underwent cyclization with
benzoyl chloride in pyridine at 0–58C to give 2-phenyl-(4H)-
3,1-benzoxazin-4-one 2 by benzoylation followed by dehy-
dration. Compound 2, on reaction with formamide, results
in the more stable 2-phenyl-4(3H) quinazolinone 3.
Compounds 2 and 3 were identified by comparing them
with earlier reports [26–28] and the physical data of the
compounds are given in Table 1.
Substituted quinazolines were reported as isosters of
quinolones to show a variety of antibacterial activities
with DNA gyrase inhibition [21, 22]. In continuation of
our efforts to synthesize quinazoline derivatives [23–25],
we report the synthesis of novel 2,6-substituted quinazoline
derivatives with ‘‘OCH2CONH2’’ substitution at the 4th
position along with their antimicrobial activities. The
possibility of an involvement of the DNA-gyrase enzyme
in the activity of quinazolines was confirmed from docking
studies.
Scheme 1. Synthesis of 4-substituted quinazolines.
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Arch. Pharm. Chem. Life Sci. 2010, 10, 570–576
Table 1. Physical data of benzoxazin-4(3H)-ones 2 and quinazolin-
4(3H)-ones 3.
were comparable with the reported values [31]. Perusal of
the activity data shows that compound 8b was more
potent and compounds 7a, 7b were inactive against all
the strains. The presence of a bromine at the 6th position
of the quinazoline system was found to influence the anti-
bacterial activity in relation to the unsubstituted derivatives:
6a, 6b and 8a, 8b. Compounds 6b and 8b with diazolyl,
oxadiazolyl substitutions on the 4th position, respectively,
were found to be relatively potent against Gram-negative
bacteria with a potency comparable to that of the standard
ciprofloxacin. The orders of activity was found to be
8b > 6b > 8a > 9b > 6a, 9a and 8b, 6b > 9a > 6a, 8a
against Gram-negative and -positive strains, respectively.
Compound
X
R
m. p. (8C)
Yield (%)
2a
2b
3a
3b
H
Br
H
Ph
Ph
Ph
Ph
122
182
226
230
82
80
84
80
Br
Compound 3 on alkylation with ethyl chloroacetate in dry
acetone over anhydrous potassium carbonate yielded exclu-
sively its O-alkylated product: ethyl-[(2-phenyl-4-quinazolinyl)-
oxy]-acetate 4. This is attributed not only to the basic medium
but also steric factors as the bulky phenyl group is in
the 2nd position [29]. Compound 4 was reacted further with
hydrazine hydrate (80%) to obtain the corresponding 2-
Antifungal activity
All the synthesized quinazoline derivatives were evaluated
for antifungal activity against two fungal strains: Candida
albicans (NCIM 3471) and Aspargillus flavus (NCIM 555). The
activity of all molecules was compared with an antifungal
drug, amphoterecin-B as presented in Table 2. The minimum
inhibitory concentrations of amphoterecin-B against both
strains were compared with literature reports [32, 33]. The
antifungal activity of the test compounds was not significant.
Only compound 8a was found to inhibit C. albicans with MIC,
24 mg/mL and A. flavus with MIC, 22 mg/mL.
phenyl-4-quinazolinyloxyacetyl hydrazides
5
[30]. The
hydrazide 5 on reaction with ethylacetoacetate/triethyl
orthoformate/carbon disulfide and potassium hydroxide/
potassium thiocyanide in hydrochloric acid led to the for-
mation of pyrazoles 6a, 6b, oxadiazoles 7a, 7b, oxadiazol-2-
thiones 8a, 8b, and triazol-2-thiones 9a, 9b, respectively. All
the compounds synthesized were characterized based on
their physical and spectral data.
Antimicrobial evaluation
Antibacterial activity
Docking studies
Compounds 6a, 6b, 7a, 7b, 8a, 8b, and 9a, 9b were screened
for their in-vitro antibacterial activity against two Gram-nega-
tive strains, i. e., Escherichia coli (NCIM 2068) and Klebsiella
pneumonia (NCIM 2957) and two Gram-positive strains, i. e.,
Bacillus subtilis (NCIM 2921) and Staphylococcus aureus (NCIM
2079), and their MIC values are presented in Table 2. The
antibacterial activity of the molecules was compared with a
standard drug, ciprofloxacin. Minimum inhibitory concen-
trations (MIC values) of ciprofloxacin in various strains
In the present investigation, an attempt was made to under-
stand the ligand-receptor interactions of quinazoline deriva-
tives 6–9 against bacterial DNA gyrase as a target enzyme, by
performing docking studies using molegro Virtual Docker
(MVD) [34] version 2008.3.2.1, installed on an Intel Pentium 4
Machine (Intel Corporation, Santa Clara, CA, USA), probably
the most accurate predictive tool of binding geometry, today.
All the ligand structures were constructed using Chem 3D
ultra 8.0 software (Molecular Modeling and Analysis;
Table 2. Antibacterial activity of 4-substituted quinazolines 6–9.
Test compound
Antibacterial activity MIC (mg/mL)
Antifungal activity MIC (mg/mL)
E. coli
NCIM 2068
K. pneumonia
NCIM 2957
S. aureus
NCIM 2079
B. subtilis
NCIM 2921
C. albicans
NCIM 3471
A. flavus
NCIM 555
6a
6b
7a
7b
8a
8b
9a
9b
45
4
–
–
20
2
45
25
0.05
–
43
12
–
–
25
8
50
30
0.06
–
35
5
270
330
40
4
27
26
0.5
–
50
4
275
375
34
5
27
24
1
30
100
300
–
24
31
244
278
–
50
215
–
–
22
34
150
231
–
Ciprofloxacin
Amphoterecin-B
–
5
4
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4-Substituted Quinazolines as DNA-Gyrase Inhibitors
573
Table 3. MolDock score and ReRank scores of all quinazoline
derivatives.
Ligand MolDock Score
ReRank Score
HBond
6a
6b
7a
7b
8a
8b
9a
9b
ꢁ83.80
ꢁ109.99
ꢁ67.58
ꢁ88.84
ꢁ94.59
ꢁ129.09
ꢁ96.09
ꢁ89.80
ꢁ65.09
ꢁ90.12
ꢁ20.59
ꢁ32.54
ꢁ67.09
ꢁ90.99
ꢁ9.88
ꢁ0.1017
ꢁ5.6533
ꢁ0.5073
ꢁ0.9796
ꢁ1.0123
ꢁ0.9502
0.0794
ꢁ51.78
ꢁ0.6498
Figure 2. Snap shot of superimposed quinazolines in the binding
cavity of 1KZN.
scores and ReRank scores of molecules are presented in
Table 3. Perusal of Table 3 shows ReRank scores of ꢁ90.12,
ꢁ90.99 for compounds 6b and 8b, respectively, which are in
correlation with the experimental values. The obtained pose
showed four hydrogen-bond interactions. Atoms of protein
(1KZN) and ligand 8b involved in hydrogen-bond interactions
are presented along with their bond distance in Table 4,
indicating that the docking method was most appropriate
for clarifying the binding mode of 4-substituted quinazolines
as DNA-gyrase inhibitors, as illustrated in Figs. 1 and 2.
Experimental
Figure 1. Snapshot of compound 8b and its hydrogen-bond inter-
actions with 1KZN.
Chemistry
Melting points were recorded on Casia-siamia (VMP-AM) melting
point apparatus and are uncorrected. IR spectra were recorded
on a Perkin-Elmer FT-IR 240-C spectro photometer using KBr
optics (Perkin-Elmer. USA). 1H-NMR spectra were recorded on
Gemini Varian (Varian, USA) 200 MHz, Bruker (Bruker
Bioscience, USA) AV 300 MHz, and Unity (Varian) 400 MHz spec-
trometer in DMSO-d6 or CDCl3 using TMS as an internal standard.
Electron impact (EI) and chemical ionization mass spectra were
recorded on a VG 7070 H instrument (Micromass, UK) at 70 eV.
All reactions were monitored by thin layer chromatography
(TLC) on precoated silica gel 60 F254 (mesh); spots were visualized
with UV light. Merck silica gel (100–200 mesh; Merck, Germany)
was used for chromatography. CHN analyses were recorded on a
Vario EL analyzer.
Cambridge Soft Corporation, USA, 2004), and then these
structures were energetically minimized by using MOPAC
(semi-empirical quantum mechanics), Jop Type with 100 iter-
ations and minimum RMS gradient of 0.01, and saved as MDL
molFile (ꢀ.mol). The crystal structure of the target protein
isomerase (1KZN) is the crystal structure of E. coli 24-kda
domain in complex with clorobiocin [35] being retrieved
from the Protein Data Bank (www.rcsb.org/pdb/welcome.-do).
Docking studies were performed to identify the nature and
amount of interactions of the synthesized quinazoline
derivatives with DNA-gyrase enzyme (1KZN). The MolDock
Table 4. Amino acid residues and atoms of ligand 8b involved in hydrogen bonding and their bond lengths.
˚
Distance (A)
Residues Atom ID
Pdb Atom Name
Ligand atom
Asp 76
Glu 50
Asn 46
Gly 77
464
266
243
466
N (Donor)
N (Donor)
O (Donor)
N (Acceptor)
N (Acceptor) oxadiazolyl
O (Acceptor) etherial oxygen
N-2 (Acceptor) quinazolyl
N-1 (Donor) quinazolyl
3.57
3.10
3.02
2.15
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(t, 2H, Ar-H), 8.5 (s,1H, Ar-H), 8.6 (s, 1H, pyrazole-H), 9.1–9.3 (s, 1H,
N–H); EI-MS: 360 [Mþ] (20%). Anal. calcd. for C20H16N4O3: C, 66.66;
H, 4.48; N, 15.55. Found: C, 66.45; H, 4.31; N, 15.40.
Synthesis of 1,3-benzoxazin-4-ones 2a, 2b [26–28]
The physical data of the compounds are presented in Table 1 and
compared with the reported values.
[(2-Phenyl-6-bromo-4-quinazolinyl)-oxy]acetyl-3-methyl
pyrazol-5-one 6b
Synthesis of 4-quinazolinones 3a, 3b [26–28]
The physical data of the compounds are presented in Table 1 and
compared with the reported values.
Yield: 50% (219.5 mg); m. p.: 1858C; IR (KBr, cmꢁ1): 1720 (C O),
–
–
1
–
–
1628 (C N), 3345 (N–H); H-NMR (CDCl , ppm) d: 2.2 (s, 3H, CH ),
3
3
4.6 (s, 2H, OCH2), 7.6–7.7 (m, 3H, Ar-H), 7.8–7.9 (t, 2H, Ar-H),
8.3–8.4 (t, 2H, Ar-H), 8.5 (s, 1H, Ar-H), 8.6 (s, 1H, pyrazole-H),
9.1–9.3 (s, 1H, NH); EI-MS: 439 [Mþ] (65%). Anal. calcd.
for C20H15BrN4O3: C, 54.69; H, 3.44; N, 12.75. Found: C, 54.52;
H, 3.19; N, 12.57.
Synthesis of quinazolinyl-4-oxy-ethylacetate 4a, 4b [30]
Compound 3a or 3b (0.03 mol), ethyl chloroacetate (0.03 mol),
potassium carbonate (0.03 mol) were taken up in 10 mL of dry
acetone and the mixture was heated at reflux for a period of 24 h.
Completion of the reaction was monitored by TLC. After the
reaction, the mixture was added to about 100 mL of ice-cold
water and the solution was kept in a refrigerator overnight. The
resultant compound was extracted with ether; the removal of
ether in vacuo yielded the product. The product ester thus
obtained was used in the next step of synthesis without further
purification.
General procedure for synthesis of 4-oxadiazolylmethoxy-
2-phenylquinazolines 7a, 7b
Compound 5a or 5b (0.01 mol) and triethyl orthoformate
(0.01 mol) were taken in 10 mL of ethanol and heated at reflux
on a water bath for 18 h. Completion of the reaction was moni-
tored by TLC. The solvent was evaporated in vacuo and the residue
was washed with a small amount of ethanol and filtered. The
product was dried and purified by column chromatography.
Synthesis of quinazolinyl-4-oxy-acetyl hydrazine 5a, 5b
Compound 4a or 4b (0.001 mol) and hydrazine hydrate
(0.001 mol) were taken up in ethanol (10 mL) and heated on
a water bath at reflux for 6 h. The solid that precipitated on
cooling was filtered, dried, and purified by column
chromatography.
4-(1,3,4-Oxadiazol-2-ylmethoxy)-2-phenylquinazoline 7a
Yield: 52% (158 mg); m. p.: 1768C; IR (KBr, cmꢁ1): 1326 (C–O–C);
1H-NMR (CDCl3, ppm) d: 4.4 (s, 2H, OCH2), 7.4–7.6 (m, 4H, Ar-H),
7.8–7.9 (t, 2H, Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.4 (s, 1H, Ar-H), 8.5 (s,
1H, oxadiazole-H); EI-MS: 305 [M þ 1] (95%). Anal. calcd.
for C17H12N4O2: C, 67.10; H, 3.97; N, 18.41. Found: C, 66.95; H,
3.82; N, 18.24.
[(2-Phenyl-4-quinazolinyl)oxy]-acetyl hydrazine 5a
Yield: 68% (395 mg); m. p.: 2108C; IR (KBr, cmꢁ1): 1290 (C–O–C),
1
1616 (C N), 1762 (C O), 3296 (N–H), 3056, 2918 (C–H); H-NMR
–
–
–
–
(CDCl3, ppm) d: 4.2 (s, 2H, OCH2), 4.4 (s, 2H, NH2), 7.4–7.6 (m, 4H,
Ar-H), 7.8–7.9 (t, 2H, Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.4 (s,1H, Ar-H), 9.3
(s, 1H, N–H); EI-MS: 294 [Mþ] (19%). Anal. calcd. for C16H14N4O2: C,
65.30; H, 4.79; N, 19.04. Found: C, 65.19; H, 4.72; N, 19.01.
6-Bromo-4-(1,3,4-oxadiazol-2-ylmethoxy)-2-phenyl-
quinazoline 7b
Yield: 51% (158 mg); m. p.: 1848C; 1H-NMR (CDCl3, ppm) d: 4.5 (s,
2H, OCH2), 7.5–7.6 (m, 3H, Ar-H), 7.8–7.9 (t, 2H, Ar-H), 8.2–8.3 (t,
2H, Ar-H), 8.5 (s, 1H, Ar-H), 8.6 (s, 1H, oxadiazole-H); EI-MS: 384
[M þ 1] (65%). Anal. calcd. for C17H11BrN4O2: C, 53.28; H, 2.89; N,
14.62. Found: C, 53.13; H, 2.76; N, 14.47.
[(6-Bromo-2-phenyl-4-quinazolinyl)oxy]-acetyl hydrazine 5b
Yield: 55% (205 mg); m. p.: 1958C; IR (KBr, cmꢁ1): 1768 (C O),
–
–
1620 (C N), 3285 (N–H), 3080, 2945 (C–H); H-NMR (CDCl , ppm)
1
–
–
3
d: 4.3 (s, 2H, OCH2), 4.7 (s, 2H, NH2), 7.5–7.7 (m, 3H, Ar-H), 7.8–7.9
(t, 2H, Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.5 (s,1H, Ar-H), 9.4 (s,1H, N-H);
EI-MS: 373 [Mþ] (19%). Anal. calcd. for C16H13BrN4O2: C, 51.49; H,
3.51; N, 15.01. Found: C, 51.35; H, 3.42; N, 14.92.
Synthesis of 5-{[(2-phenylquinazolin-4-yl)oxy]methyl}-
1,3,4-oxadiazol-2(3H)-thione derivatives 8a, 8b
Compounds 5a
& 5b (0.001 mol) and carbon disulphide
(0.003 mol) were taken in ethanolic solution of KOH (0.1 mol,
15 mL) and heated at reflux on a water bath for 20 h. Completion
of the reaction is monitored by TLC. The solvent was evaporated
under reduced pressure and the residue was triturated with cold
water. Neutralization of the solution with acetic acid yielded a
precipitate which was filtered, dried, and purified using column
chromatography.
General procedure for synthesis of [(2-phenyl-4-
quinazolinyl)-oxy]acetyl-3-methyl pyrazol-5-ones 6a, 6b
Ethyl acetoacetate (0.001 mol) and compound 5a or 5b
(0.01 mol) were taken up in ethanol (15 mL) along with 4 to
5 drops of HCl and heated on a water bath at reflux for 5 h.
Completion of the reaction was monitored by TLC. Ethanol was
removed in vacuo and the residue was triturated with cold
water. The solid obtained was filtered under pressure, dried,
and purified by using column chromatography.
5-{[(2-Phenylquinazolin-4-yl)oxy]methyl}-1,3,4-oxadiazol-
2(3H)-thione 8a
Yield: 61% (205 mg); m. p.: 1988C; IR (KBr, cmꢁ1): 3448 (N-H), 1204
1
[(2-Phenyl-4-quinazolinyl)-oxy]acetyl-3-methyl pyrazol-5-
one 6a
–
–
(C S), 1632 (C N), 3051, 2851 (C–H); H-NMR (CDCl , ppm) d: 4.3
–
–
3
(s, 2H, OCH2), 7.3 (s, 1H, N-H), 7.4–7.6 (m, 4H, Ar-H), 7.8–7.9 (t, 2H,
Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.5 (s, 1H, Ar-H); EI-MS: 336 [Mþ] (100%).
Anal. calcd. for C17H12N4O2S: C, 60.70; H, 3.60; N, 16.66. Found: C,
60.65; H, 3.72; N, 16.75.
Yield: 57% (205 mg); m. p.: 1968C; IR (KBr, cmꢁ1): 1707 (C O),
–
–
1
–
1625 (C N), 3320 (N–H); H-NMR (CDCl , ppm) d: 2.2 (s, 3H, CH ),
–
4.4 (s, 2H, OCH2), 7.4–7.6 (m, 4H, Ar-H), 7.8–7.9 (t, 2H, Ar-H), 8.2–
3
3
8.3
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Arch. Pharm. Chem. Life Sci. 2010, 10, 570–576
4-Substituted Quinazolines as DNA-Gyrase Inhibitors
575
Ciprofloxacin was used as the standard for antibacterial activity
and amphoterecin-B for antifungal activity, while keeping DMSO
as control.
5-{[(6-Bromo-2-phenylquinazolin-4-yl)oxy]methyl}-1,3,4-
oxadiazol-2(3H)-thione 8b
Yield: 52% (215 mg); m. p.: 2048C; IR (KBr, cmꢁ1): 3450 (N–H),
1
–
–
1220 (C S), 1630 (C N), 2865 (C-H); H-NMR (CDCl , ppm) d: 4.4 (s,
–
–
3
In-silico studies
2H, OCH2), 7.3 (s, 1H, N–H), 7.6–7.8 (m, 4H, Ar-H), 7.8–7.9 (t, 2H, Ar-
H), 8.2–8.3 (t, 2H, Ar-H), 8.3–8.5 (s, 1H, Ar-H); EI-MS: 415 [M þ 1]
(60%). Anal. calcd. for C17H11BrN4O2S: C, 49.17; H, 2.67; N, 13.49.
Found: C, 49.15; H, 2.43; N, 13.56.
The protein structure of 1KZN was imported in MVD, and miss-
ing bond orders, hybridization states, and angles were then
assigned. To obtain better potential binding sites in the protein,
a maximum of five cavities were detected using parameters such
as molecular surface (expanded van der Waals), maximum num-
ber of cavities (n ¼ 5), minimum cavity volume (10), probe size
(1.20), maximum number of ray checks (n ¼ 16), minimum
number of ray hits (n ¼ 12), and grid resolution (0.80). The
chosen cavity was further refined using side-chain minimization
by selecting the add-visible option at a maximum of steps per
residue (10 000) and a maximum of global steps (10 000). The
setup for side-chain flexibility by selection of the add-visible
option, the setting for the selected flexible side chain during
the docking option, and other parameters, all were kept in
default.
Synthesis of 5-{[(2-phenylquinazolin-4-yl)oxy]methyl}-2,4-
dihydro-3H-1,2,4-triazole-3-thiones 9a, 9b
A mixture of compound 5a or 5b (0.01 mol) and potassium
thiocyanate (0.01 mol) was taken up in ethanolic solution of
sodium hydroxide (0.1 mol) and the resulting solution was
heated at reflux on a water bath for 24 h. Completion of the
reaction was monitored by TLC. The solvent was removed in vacuo
and the residue was triturated with ice-cold water.
Neutralization of the solution using 5% hydrochloric acid
solution yielded a product which was filtered and dried. The
product was purified by column chromatography.
All docking calculations were carried out using the grid-based
MolDock score (GRID) function with a grid resolution of 0.30 A.
The binding site on the receptor was defined as extending in X, Y,
and Z directions around the Dock molecule with a radius of
5-{[(2-Phenylquinazolin-4-yl)oxy]methyl}-2,4-dihydro-3H-
1,2,4-triazol-3-thione 9a
˚
Yield: 60% (200 mg); m. p.: 848C; IR (KBr, cmꢁ1): 3450 (NH), 1320
approximately 10 to 17 A. The MolDock optimization search
1
algorithm with a maximum of ten runs was used through the
calculations, with all other parameters kept as defaults. One pose
per run was retained based on root mean square division cluster-
–
(C-O), 1230 (C S); H-NMR (CDCl , ppm) d: 4.3 (s, 2H, OCH ), 7.4–
–
3
2
7.6 (m, 4H, Ar-H), 7.8–7.9 (t, 2H, Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.3 (s,
1H, Ar-H), 11.1 (s, 2H, N-H); EI-MS: 336 [M þ 1] (80%). Anal. calcd.
for C17H13N5OS: C, 60.88; H, 3.91; N, 20.88. Found: C, 60.84; H,
3.87; N, 20.73.
˚
ing using a heavy atom threshold set at 2.0 A and an energy
penalty of 100. All the poses were examined manually and the
best poses were retained.
5-{[(6-Bromo-2-phenylquinazolin-4-yl)oxy]methyl}-2,4-
dihydro-3H-1,2,4-triazol-3-thione 9b
Optimization of the parameter for suitable docking
To obtain better potential binding sites in the protein (1KZN), a
maximum of three cavities was detected using default
parameters. Out of the detected cavities, cavity number 1 (cavity
Yield: 53% (219 mg); m. p. 938C; IR (KBr, cmꢁ1): 3426 (NH), 1315
1
–
–
(C-O), 1224 (C S); H-NMR (CDCl , ppm) d: 4.6 (s, 2H, OCH ), 7.6–
3
2
7.7 (m, 3H, Ar-H), 7.8–7.9 (t, 2H, Ar-H), 8.2–8.3 (t, 2H, Ar-H), 8.4 (s,
1H, Ar-H), 11.2 (s, 2H, N-H); EI-MS: 415 [M þ 1] (60%). Anal. calcd.
for C17H12BrN5OS: C, 49.29; H, 2.92; N, 16.90. Found: C, 49.22; H,
2.95; N, 16.76.
˚
volume, 58 894 A) was selected for further studies. The chosen
cavity was further refined using side-chain minimization by
selection of an add-visible option set at a maximum of 10 000
steps per residue and at a maximum of 10 000 global steps. The
side-chain flexibility was set by selecting the add-visible option.
The same was selected during docking, and the remaining
parameters were kept as fixed variables. Furthermore, the dock-
ing simulation was run and the best pose for the set derivatives
was selected on the basis of the MolDock score and ReRank score.
Antimicrobial activity
All the compounds were screened for their in-vitro antibacterial
activity against two Gram-negative strains, i. e., Escherichia coli
(NCIM 2068) and Klebsiella pneumonia (NCIM 2957) and two Gram-
positive strains, i. e., Bacillus subtilis (NCIM 2921) and Staphylococcus
aureus (NCIM 2079), and for their antifungal activity against two
fungal strains Candida albicans (NCIM 3471) and Aspergillus flavus
(NCIM 555). The specified stains of organisms were procured
from the National Chemical Laboratory, Pune, India, and were
used for the evaluation of the test compounds by broth dilution
method [36]. Cultures of test organisms were maintained on
nutrient agar slants and were subcultured in nutrient broth
prior to testing. The inoculum size was approximately 10 colony
forming units (CFU/mL). The media used was nutrient agar for
bacterial strains and sabouraud dextrose agar medium for
Candida albicans and czapexs dox agar medium for Aspergillus
flavus procured from Himedia Laboratories, Mumbai, India. All
the compounds were dissolved in dimethyl sulfoxide to give a
concentration of 1 mg/mL. The test compounds were prepared in
different concentrations from 5 mg/mL to 500 mg/mL in DMSO.
Authors are thankful to the Secretary, Viswambhara Educational Society
for providing the facilities.
The authors have declared no conflict of interest.
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