Bacterial cell leakage potential of newly synthesized quinazoline derivatives of 1,5-benzodiazepines analogue

Article abstract of DOI:10.1002/jhet.3879

Novel quinazoline embellished analogues of 1,5-benzodiazepine (6, 9, 13, 17, 21 and 24) were synthesized using one-pot domino approach. Structure of the compounds synthesized, were established with the help of IR, ¹H NMR, ¹³C NMR and

Full text of DOI:10.1002/jhet.3879

Received: 7 May 2019
DOI: 10.1002/jhet.3879
Revised: 11 December 2019
Accepted: 16 December 2019
A R T I C L E
Bacterial cell leakage potential of newly synthesized
quinazoline derivatives of 1,5-benzodiazepines analogue
Apoorva Misra1 | Jaya Dwivedi1
Swapnil Sharma2
| Shruti Shukla1 | Dharma Kishore1
|
1Department of Chemistry, Banasthali
Abstract
Vidyapith, Rajasthan, India
2Department of Pharmacy, Banasthali
Vidyapith, Rajasthan, India
Novel quinazoline embellished analogues of 1,5-benzodiazepine (6, 9, 13, 17,
21 and 24) were synthesized using one-pot domino approach. Structure of the
1
compounds synthesized, were established with the help of IR, H NMR, 13C
Correspondence
NMR and mass spectral data. Further, antibacterial activity, FE-SEM imaging
and cell leakage study was also performed.
Prof. Jaya Dwivedi, Department of
Chemistry,
Banasthali Vidyapith, Rajasthan 304022,
India.
Email: jayadwivedi@yahoo.co.in
Funding information
CURIE, Department of Science and
Technology (DST), New Delhi, Grant/
Award Number: SR/CURIE-III/01/2015
(G); Department of Science and
Technology
1 | INTRODUCTION
substrates in an ecological, economical, and also atom
economical manner[25]
.
Quinazoline derivatives have caused universal apprehen-
sion because of their broad and discrete biopharmaceuti-
cal properties. Quinazolines play a significant role in
various biological activities[1], which includes many bio-
logical properties including antihypertensive[2], antimi-
Its unique biological properties have drawn attention
toward the utilities of quinazoline incorporated heterocy-
clic structures in medicinal chemistry. Keeping this
in mind, the present paper describes the synthesis
of some novel quinazoline substituted analogues of
1,5-benzodiazepine using domino approach and explores
their antibacterial activities.
This paper focuses on the development of synthetic
strategies, which, in addition to being highly innova-
tive, were simple in operation, required inexpensive
materials, and are applicable to a wide range of
functionalized substrates, for the synthesis of
quinazoline condensed 1,5-benzodizepine derivatives
having extensively explored chemotherapeutic activities
and broad spectrum of biological properties. Our strat-
egy was based on the use of nitrile as a coupling part-
ner with an activated benzanilide (activation of which
is achieved by its reaction with Tf2O and then with
2-chloropyridine)[26]. The resulting nitrilium ion will
set the stage ready for its cyclocondensation with the
crobial[3]
,
antihyperlipidemic[4]
,
anti-inflammatory[5]
,
and anticonvulsant[6] activities. Besides, it also exhibits a
variety of therapeutic activities including antiviral[7]
,
antibacterial[8], antifungal[9], antimalarial[10], antican-
cer[11], antihypertensive[12], diuretic[13,14], anticonvul-
sant[15,16]
inhibitory activities[17]
, anti-inflammatory, analgesic, and COX-2
.
Erlotinib[18] Trimetrexate[19]
, ,
Vandetanib[20], Febrifugine[21], and Quazinone[22] are
some of the marketed drugs having quinazoline
pharmacophore.
Domino reaction has recently been used for the
diastereoselective and enantioselective construction of
complex molecules from simpler one in one pot
method[23] and is highly efficient[24] for the synthesis of
molecules which contain multiple bonds from simple
J Heterocycl Chem. 2020;1–14.
wileyonlinelibrary.com/journal/jhet
© 2020 Wiley Periodicals, Inc.
1

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MISRA ET AL.
SCHEME 1 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from ethyl cyanoacetate
SCHEME 2 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from ethyl cyanoacetate
SCHEME 3 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from pyruvonitrile
SCHEME 4 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from benzoylacetonitrile
π-electron system with the vinyl or arene part of the
benzamide to deliver the quinazoline nucleus in one
step[27]. The synthesis of quinazolino embellished ana-
logues of 1,5-benzodiazepines from the corresponding
nitrile derivatives is depicted in Schemes 1–6, and its
mechanism is depicted in Scheme 7.
2 | EXPERIMENTAL
2.1 | Materials
High purity solvent and reagent were purchased from
Sigma Aldrich, USA. TLC and CHNS (analyzer Perkin

MISRA ET AL.
3
SCHEME 5 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from acetylacetone
SCHEME 6 Synthesis of quinazoline derivatives of 1,5-benzodiazepine from malonic ester
SCHEME 7 Mechanism of formation of quinazoline derivative from the corresponding nitrile derivative
1
Elmer, USA) were employed to check purity and for ele-
mental analysis. Precoated silica gel 60F254 plates
(200 mm; Merck, Darmstadt, Germany) were used to
check purity of the compound and capillary apparatus for
melting point determination. Characterization of synthe-
and mass spectrometer. H NMR and 13C NMR spectra
were recorded on a Jeol Resonance/Bruker Ascend
400-MHz spectrometer. The chemical shift (parts per mil-
lion) was calculated by using TMS as standard reference.
KBr was employed for taking IR spectra on an Agilent
tech, Cary 660 FTIR spectrophotometer. Mass spectra
1
sized compound was done by IR, H NMR, 13C NMR,

4
MISRA ET AL.
were recorded on waters, Q-TOF micromass (LCMS),
mass spectrometer using Argon/Xenon (6 kV, 10 mB)
gas. Field emission scanning electron microscope (FE-
SEM) study was done on Tescan, Mira 3.
2.2.3 | Preparation of 4-(methylthio)-
3-(2-phenylquinazolin-4-yl)-1H-benzo[b]
[1,4] diazepin-2(5H)-one (6)
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol), and dichloromethane (50 mL) was stirred
for 30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reac-
tion mixture was kept in an ice water bath and warmed
to room temperature. Then, nucleophile (4) was added,
and reaction mixture was heated to 45ꢀC in an oil bath
for 5 hours. It was then cooled to room temperature,
and the trifluoromethane sulfonate salts were neutral-
ized by triethylamine. The volatiles were separated by
rotary evaporation, and the product was purified by
preparative TLC method to give quinazoline
derivative 6.
Obtained as a brown solid; yield: 69%; M.p.: 160ꢀC. IR
(KBr, ν/cm−1): 3348, 3223, 1707, 1509, 1512, 1166, 781.
1H NMR (δ, ppm in DMSO-d6): 7.82 (s, 1H) 7.65 (s, 3H),
7.23 to 7.19 (m, 4H), 7.18 to 6.75 (m, 6H), 4.15 (s,1H),
1.80 (s, 3H). 13C NMR (δ, ppm in DMSO-d6): 165.45,
160.62, 149.80, 135.45, 131.39, 127.97, 125.45, 123.26,
119.80, 81.79, 15.17. Anal. calc. for C24H18N4OS: C70.22,
H4.42, N13.65%. Found: C70.25, H4.39, N13.68%. m/z:
410.6676.
2.2 | Synthesis
Following the schemes depicted below, the targeted com-
pounds were synthesized.
2.2.1 | Preparation of ethyl-2-cyano-
3,3-bis(methylthio)acrylate (2)
A mixture of t-BuOK (1.34 g, 0.012 mol), dry toluene, and
DMF (3.0 mL) was well stirred in an ice bath, followed
by the addition of ethyl cyanoacetate 1 (0.006 mol). After
30-minute stirring, CS2 (1 mL, 0.006 mol) was added.
Stirring was continued for 2 hours, and then methyl
iodide (2 mL, 0.012 mol) was added with continuous stir-
ring and external cooling. Stirring continued for 2 hours
and then refluxed for 3.5 hours. The reaction mixture
was then poured into ice water. It was then extracted
with toluene, washed with water, dried (anhydrous
Na2SO4), and rotatory evaporator was used to remove sol-
vent yielding, 2.
Obtained as a yellow solid; yield: 64%; M.p.: 89ꢀC. IR
(KBr, ν/cm−1): 2979, 2200, 1688, 1647, 1247, 1017, 639.
1H NMR (δ, ppm in DMSO-d6): 4.18 to 4.14 (q, 2H), 1.89
(s, 6H), 1.36 to 1.32 (t, 3H). 13C NMR (δ, ppm in DMSO-
d6): 179.35, 167.40, 111.90, 79.35, 65.39, 17.64, 15.17.
Anal. calc. for C8H11NO2S2: C44.22, H5.10, N6.45%.
Found: C44.19, H5.10, N6.42%.
2.2.4 | Preparation of (Z)-ethyl-2-cyano-
3-(dimethylamino)acrylate (7)
The mixture of ethyl cyanoacetate 1 (0.01 mol) and DMF.
DMA (15 mL) was heated under reflux for 6 hours, con-
centrated, and the residue so obtained was triturated and
filtered by using hexane to give 7.
Obtained as a brown solid; yield: 78%; M.p.: 72ꢀC. IR
(KBr, ν/cm−1): 2976, 2197, 1729, 1641, 1321, 1255. 1H
NMR (δ, ppm in DMSO-d6): 7.23 (s, 1H), 4.19 to 4.14 (q,
2H), 3.26 (s, 6H), 1.26 to 1.23 (t, 3H). 13C NMR (δ, ppm in
DMSO-d6): 160.59, 151.67, 119.80, 81.79, 59.32, 39.68,
15.32. Anal. calc. for C8H12N2O2: C57.13, H7.19,
N16.66%. Found: C57.18, H7.15, N16.62%.
2.2.2 | Preparation of 4-methylsulfanyl-
2-oxo-2,5-dihydro-1H-benzo[b][1,4]
diazepine-3-carbonitrile (4)
O-phenylenediamine
(3)
(0.01 mol)
and
oxoketenedithioacetal derivative (2) (0.01 mol) was
refluxed in ethanol for 4 to 5 hours. Rotatory evaporator
was used to remove solvent, followed by addition of ice
cold water to the residue. Chloroform was used to extract
the product and dried by using Na2SO4 to give 4.
Obtained as a brown solid; yield: 67%; M.p.: 120ꢀC. IR
2.2.5 | Preparation of 2-oxo-2,5-dihydro-
1H-benzo[b][1,4]diazepine-
3-carbonitrile (8)
(KBr, ν/cm−1): 3321, 3163, 2974, 2190, 1623, 1563, 1469,
1
1138, 686. H NMR (δ, ppm in DMSO-d6): 7.48 (s, 1H),
7.23 to 6.75 (m, 4H), 4.14 (s, 1H), 1.77 (s, 3H). 13C NMR
(δ, ppm in DMSO-d6): 161.70, 137.40, 127.40, 125.45,
117.40, 111.90, 65.39, 15.64. Anal. calc. for C11H9N3OS:
C57.13, H3.92, N18.17%. Found: C57.15, H3.89, N18.21%.
O-phenylenediamine (3) (0.01 mol) and dimethyl-
aminomethylene ketone derivative (7) (0.01 mol) mixture
was refluxed in ethanol for 11 hours. Rotatory evaporator

MISRA ET AL.
5
was used to remove solvent, followed by addition of ice
cold water to the residue. Chloroform was used to extract
product from solvent and dried by using Na2SO4 to give 8.
Obtained as a yellow solid; yield: 59%; M.p.: 134ꢀC. IR
(KBr, ν/cm−1): 3409, 3188, 2903, 2218, 1695, 1645, 1451,
1246. 1H NMR (δ, ppm in DMSO-d6): 8.02 (s, 1H), 7.60 (s,
1H), 7.25 to 6.74 (m, 4H), 4.17 (s, 1H). 13C NMR (δ, ppm
in DMSO-d6): 164.26, 161.70, 137.90, 127.45, 125.70,
117.40, 111.80, 79.80. Anal. calc. for C10H7N3O: C64.86,
H3.81, N22.69%. Found: C64.84, H3.78, N22.67%.
to 7.40 (m, 5H), 6.64 to 6.60 (d, 1H). 13C NMR (δ, ppm in
DMSO-d6): 189.80, 160.62, 137.40, 135.45, 131.39, 115.45.
Anal. calc. for C10H7NO: C76.42, H4.49, N8.91%. Found:
C76.39, H4.52, N8.88%.
2.2.8 | Preparation of 4-phenyl-
4,5-dihydro-3H-benzo[b][1,4]diazepine-
2-carbonitrile (12)
A mixture of α,β-unsaturated ketone derivative (11)
(0.01 mol), o-phenylenediamine (3) (0.01 mol), ethanol,
and four to five drops of piperidine was refluxed for
6 hours. Completion of the reaction was checked by TLC
monitoring. About half of the solvent was removed by
rotary evaporation, and the resulting mixture was
allowed to stand at room temperature. The crystalline
compound thus formed was filtered, washed with cold
aqueous ethanol, and dried to give 12.
2.2.6 | Preparation of
3-(2-phenylquinazolin-4-yl)-1H-benzo[b]
[1,4]diazepin-2(5H)-one (9)
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol), and dichloromethane (50 mL) was stirred for
30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reac-
tion mixture was kept in an ice water bath and warmed
to room temperature. Then, nucleophile (8) was added,
and reaction mixture was heated to 45ꢀC in an oil bath
for 5 hours. It was then cooled to room temperature, and
the trifluoromethane sulfonate salts were neutralized by
triethylamine. The volatiles were separated by rotary
evaporation, and the product was purified by preparative
TLC method to give quinazoline derivative 9.
Obtained as a yellow solid; Yield: 63%; M.p.: 146ꢀC.
IR (KBr, ν/cm−1): 3220, 3024, 2204, 1500, 1449, 1613,
1
1220. H NMR (δ, ppm in DMSO-d6): 7.94 (m, 2H), 7.55
to 6.89 (m, 7H), 4.90 (s, 1H), 4.14 (s, 1H), 1.87 (s, 2H). 13
C
NMR (δ, ppm in DMSO-d6): 160.62, 141.67, 135.45,
131.39, 129.80, 125.45, 111.90, 119.80, 81.79, 51.70. Anal.
calc. for C16H13N3: C77.71, H5.30, N16.99%. Found:
C77.73, H5.32, N16.95%.
2.2.9 | Preparation of 2-phenyl-
4-(2-phenylquinazolin-4-yl)-2,3-dihydro-1H-
benzo[b] [1,4]diazepine (13)
Obtained as a brown solid; yield: 72%; M.p.: 185ꢀC. IR
(KBr, ν/cm−1): 3449, 3058, 1744, 1646, 1571 and 1384. H
1
NMR (δ, ppm in DMSO-d6): 8.18 (s, 1H), 8.02 (s, 1H),
7.60 to 7.52 (m, 5H), 7.29 to 6.75 (m, 8H), 4.19 (s, 1H).
13C NMR (δ, ppm in DMSO-d6): 163.26, 159.32, 151.67,
144.26, 135.64, 130.62, 128.86, 126.89, 123.26, 119.35.
Anal. calc. for C23H16N4O: C75.81, H4.43, N15.38%.
Found: C75.78, H4.41, N15.34%. m/z: 364.0477.
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol) and dichloromethane (50 mL) was stirred for
30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reac-
tion mixture was kept in an ice water bath and warmed
to room temperature. Then, nucleophile (12) was added,
and the reaction mixture was heated to 45ꢀC in an oil
bath for 5 hours. It was then cooled to room temperature,
and the trifluoromethane sulfonate salts were neutralized
by triethylamine. The volatiles were separated by rotary
evaporation, and the product was purified by preparative
TLC method to give quinazoline derivative 13.
Obtained as a yellow solid; yield: 77%; M.p.: 137ꢀC. IR
(KBr, ν/cm−1): 3565, 3026, 1688, 1449, and 1247. 1H NMR
(δ, ppm in DMSO-d6): 8.28 (s, 2H), 7.82 (s, 3H), 7.65 (s,
3H), 7.23 to 7.19 (m, 4H), 7.18 to 6.75 (m, 6H), 4.26 (s,
1H), 3.89 to 3.87 (t, 1H), 1.65 to 1.63 (d, 2H). 13C NMR (δ,
ppm in DMSO-d6): 167.40, 161.67, 156.89, 151.67, 144.35,
2.2.7 | Preparation of cinnamoyl
cyanide (11)
Pyruvonitrile 10 (0.01 mol), benzaldehyde (1.06 g,
0.01 mol), and fused sodium acetate (1.08 g, 0.015 mol)
were mixed in glacial acetic acid. The reaction mixture
was refluxed for 5 hours and then poured into ice water.
The resulting solid was filtered, washed with water, dried
(anhydrous sodium sulfate), and recrystallized from
aq. ethanol to give 11.
Obtained as a brown solid; yield: 62%; M.p.: 112ꢀC. IR
(KBr, ν/cm−1): 3136, 2976, 2197, 1729, 1641, 1451, 1255.
1H NMR (δ, ppm in DMSO-d6): 7.65 to 7.64 (d, 1H), 7.60

6
MISRA ET AL.
135.64, 131.67, 128.86, 126.89, 122.97, 119.17, 59.32,
28.86. Anal. calc. for C29H22N4: C81.66, H5.20, N13.4%.
Found: C81.68, H5.22, N13.10%. m/z: 426.0388.
the trifluoromethane sulfonate salts were neutralized by
triethylamine. The volatiles were separated by rotary
evaporation, and the product was purified by preparative
TLC method to give quinazoline derivative 17.
Obtained as a brown solid; yield: 57%; M.p.: 167ꢀC. IR
1
2.2.10 | Preparation of (Z)-2-benzoyl-
(KBr, ν/cm−1): 3445, 3223, 1509, 1483, 1264. H NMR (δ,
3-(dimethylamino)acrylonitrile (15)
ppm in DMSO-d6): 8.18 (s, 2H), 8.02 (s, 2H), 7.60 to 7.52
(m, 5H), 7.29 to 6.75 (m, 9H), 4.26 (s, 1H), 4.19 (s, 1H).
13C NMR (δ, ppm in DMSO-d6): 164.26, 149.80, 144.26,
135.45, 131.39, 127.97, 125.45, 123.26, 111.79, 81.79. Anal.
calc. for C29H20N4: C82.05, H4.75, N13.20%. Found:
C82.07, H4.72, N13.21%. m/z: 424.0385.
The mixture of benzoylacetonitrile 14 (0.01 mol) and
DMF.DMA (15 mL) was heated under reflux for 6 hours
and concentrated. The residue was triturated with hex-
ane, filtered, and washed with hexane to give 15.
Obtained as a yellow solid; yield: 68%; M.p.: 95ꢀC. IR
(KBr, ν/cm−1): 3063, 2254, 1674, 1645, 1540,1320. 1H
NMR (δ, ppm in DMSO-d6): 7.82 to 7.62 (m, 5H), 7.35 (s,
1H), 3.64 (s, 6H). 13C NMR (δ, ppm in DMSO-d6): 189.80,
160.62, 137.40, 135.45, 131.39, 129.80, 119.80, 81.79,
51.70. Anal. calc. for C12H12N2O: C71.98, H6.04,
N13.99%. Found: C71.95, H6.01, N13.94%.
2.2.13 | Preparation of 2,4-dimethyl-3H-
benzo[b][1,4]diazepine-7-carbonitrile (20)
3,4-Diaminobenzonitrile (19) (10 mmol), acetylacetone
(18) (10 mmol), and acetic acid (1 mL) in ethanol were
refluxed for 7 to 8 hours. The reaction mixture was cooled
by diluting with ice H2O, and the product precipitated by
means of HCl (12 N, 10 mL), and the whole mixture was
kept overnight at 0ꢀC. The crystals of benzodiazepenium
hydrochloride were filtered off, washed successively with
cold ethanol, ether, and dried. The crystals were rec-
rystallized from ethanol. The treatment of 50% aqueous
ethanolic solution of this hydrochloride with ammonia
solution afforded the benzodiazepine 20.
Obtained as a brown solid; yield: 63%; M.p.: 197ꢀC. IR
(KBr, ν/cm−1): 2830, 3023, 2219, 1604, 1507, 1278. 1H
NMR (δ, ppm in DMSO-d6): 7.53 to 7.51 (m, 3H), 3.26 (s,
1H), 1.82 (s, 6H). 13C NMR (δ, ppm in DMSO-d6): 161.70,
144.70, 135.45, 127.40, 125.45, 117.40, 111.90, 53.04,
28.86. Anal. calc. for C12H11N3: C73.07, H5.62, N2.30%.
Found: C73.04, H5.64, N21.31%.
2.2.11 | Preparation of 4-phenyl-1H-
benzo[b][1,4]diazepine-3-carbonitrile (16)
O-phenylenediamine
dimethylaminomethylene
(3)
ketone
(0.01 mol)
derivative
and
(15)
(0.01 mol) were refluxed in ethanol for 11 hours. Rota-
tory evaporator was used to remove solvent, and ice cold
water was poured into the residue, followed by extraction
with chloroform and dried (over Na2SO4) to give 16.
Obtained as a yellow solid; Yield: 64%; M.p.: 138ꢀC. IR
(KBr, ν/cm−1): 3321, 3074, 2190, 1623, 1563, 1317. 1H NMR
(δ, ppm in DMSO-d6): 7.94 to 7.80 (s, 2H), 7.55 to 6.89 (m,
7H), 5.36 (s, 1H), 4.14 (s, 1H). 13C NMR (δ, ppm in DMSO-
d6): 167.70, 161.40, 141.67, 135.45, 131.39, 129.80, 125.45,
117.40, 111.20, 79.20. Anal. calc. for C16H11N3: C78.35,
H4.52, N17.13%. Found: C78.33, H4.54, N17.13%.
2.2.14 | Preparation of 2,4-dimethyl-
7-(2-phenylquinazolin-4-yl)-3H-benzo[b]
[1,4] diazepine (21)
2.2.12 | Preparation of 4-phenyl-
3-(2-phenylquinazolin-4-yl)-1H-benzo[b]
[1,4]diazepine (17)
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol), and dichloromethane (50 mL) was stirred for
30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reac-
tion mixture was kept in an ice water bath and warmed
to room temperature. Then, nucleophile (20) was added,
and reaction mixture was heated to 45ꢀC in an oil bath
for 5 hours. It was then cooled to room temperature, and
the trifluoromethane sulfonate salts were neutralized by
triethylamine. The volatiles were separated by rotary
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol), and dichloromethane (50 mL) was stirred for
30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reac-
tion mixture was kept in an ice water bath and warmed
to room temperature. Then, nucleophile (16) was added,
and reaction mixture was heated to 45ꢀC in an oil bath
for 5 hours. It was then cooled to room temperature, and

MISRA ET AL.
7
evaporation, and the product was purified by preparative
TLC method to give quinazoline derivative 21.
NMR (δ, ppm in DMSO-d6): 7.84 (s, 2H), 7.65 (s, 2H),
7.24 to 7.19 (m, 4H), 7.18 to 6.76 (m, 6H), 3.51 (s, 2H).
13C NMR (δ, ppm in DMSO-d6): 168.85, 157.73, 146.04,
132.06, 128.86, 125.17, 123.26, 115.46, 67.40. Anal. calc.
for C23H16N4O2: C72.62, H4.24, N14.73%. Found: C72.64,
H4.21, N14.76%. m/z: 380.0261.
Obtained as a yellow solid; yield: 68%; M.p.: 170ꢀC. IR
1
(KBr, ν/cm−1): 3616, 3065, 1598, 1489, 1177. H NMR (δ,
ppm in DMSO-d6): 7.82 (s, 1H), 7.65 (s, 1H), 7.23 to 7.19
(m, 4H), 7.18 to 6.75 (m, 6H), 3.51 (s, 2H), 1.80 (s, 6H).
13C NMR (δ, ppm in DMSO-d6): 162.17, 155.45, 144.26,
135.45, 130.30, 127.32, 123.26, 53.04, 23.26. Anal. calc. for
C25H20N4: C79.76, H5.35, N14.88%. Found: C79.74,
H5.37, N14.89%. m/z: 376.0666.
2.3 | Antibacterial activity
In vitro, antibacterial activity was screened for the synthe-
sized compound against Staphylococcus aureus (MTCC
9886) and Escherichia coli (MTCC 433). Lyophilized bacte-
rial cultures were obtained from Microbial Type Culture
Collection, Chandigarh, India, which was further cultured
and maintained under optimum broth medium.
2.2.15 | Preparation of 2,4-dioxo-
2,3,4,5-tetrahydro-1H-benzo[b][1,4]
diazepine-7-carbonitrile (23)
A
mixture of malonic ester (22) (0.01 mol),
3,4-diaminobenzonitrile (19) (0.01 mol), ethanol, and four
to five drops of piperidine was refluxed for 6 hours. Comple-
tion of the reaction was checked by TLC monitoring. About
half of the solvent was removed by rotary evaporation, and
the resulting mixture was allowed to stand at room temper-
ature. The crystalline compound thus formed was filtered,
washed with cold aqueous ethanol, and dried to give 23.
Obtained as a brown solid; yield: 63%; M.p.: 142ꢀC. IR
(KBr, ν/cm−1): 3409, 3188, 2903, 2218, 1695, 1645, 1582,
2.3.1 | Determination of half maximal
inhibitory concentration (IC50)
Broth dilution assay method was used to test antibacterial
susceptibility, and IC50 value was determined for the test
compounds. Strains were subcultured for each experiment
at 37 2ꢀC for 24 hours. Nutrient broth, autoclaved for
15 to 20 minutes at 15 lbs pressure and at 121ꢀC, was used
to prepare culture media. DMSO was used to prepare dif-
ferently concentrated test compound and ampicillin, a
standard drug. Different culture tubes with broth media
were added with bacterial suspension (20 μL) having
approximately 109 cell/mL and with standard compound
for 18 hours at 37ꢀC incubation. Bacterial growth inhibi-
tion was then analyzed by employing UV-Visible spectro-
photometry at 600 nm. After getting three concurrent
reading, IC50 was determined as mean concentration.
1
1451, 1246. H NMR (δ, ppm in DMSO-d6): 8.02 (s, 1H),
7.60 to 7.57 (m, 3H), 3.26 (s, 1H). 13C NMR (δ, ppm in
DMSO-d6): 161.70, 144.56, 135.20, 127.28, 125.81, 117.57,
111.29, 53.11. Anal. calc. for C10H7N3O2: C59.70, H3.51,
N20.89%. Found: C59.68, H3.53, N20.87%.
2.2.16 | Preparation of
7-(2-phenylquinazolin-4-yl)-1H-benzo[b]
[1,4]diazepine-2,4(3H,5H)-dione (24)
A solution of benzanilide (0.01 mol), 2-chloropyridine
(0.02 mol), and dichloromethane (50 mL) was stirred for
30 minutes. The temperature of the reaction was
maintained to −78ꢀC (using dry ice and acetone), and
Tf2O (trifluoromethane sulfonic anhydride) (0.02 mol)
was added into it dropwise. After 30 minutes, the reaction
mixture was kept in an ice water bath and warmed to
room temperature. Then, nucleophile (23) was added,
and the reaction mixture was heated to 45ꢀC in an oil
bath for 5 hours. It was then cooled to room temperature,
and the trifluoromethane sulfonate salts were neutralized
by triethylamine. The volatiles were separated by rotary
evaporation, and the product was purified by preparative
TLC method to give quinazoline derivative 24.
2.3.2 | Field emission scanning electron
microscope (FE-SEM) study
Morphological changes by test compounds on bacterial
cell were done by FE-SEM and are compared with
untreated bacterial suspension. S. aureus and E. coli bac-
terial strains were treated and incubated with the test
compound at a concentration of 200 and 300 μg/mL
respectively, which were centrifuged at 1000 rpm for
20 minutes, resulting in the formation of pellets. Phos-
phate buffered saline was used to wash the pellets and
prefixed cells. Later on, the prefixed cells were dried with
2.5% glutaraldehyde for 20 minutes, and with 50%, 75%,
and 100% of ethanol, respectively. The fixed cells were
coated with palladium by using plasma sputter (Quorum,
USA) and were observed using FE-SEM (Tescan Mira 3).
Obtained as a yellow solid; yield: 67%; M.p.: 152ꢀC. IR
(KBr, ν/cm−1): 3237, 3069, 1675, 1454, 1315, 1054. 1H

8
MISRA ET AL.
2.3.3 | Leakage study
ethyl cyanoacetate (1), pyruvonitrile (10), and
benzoylacetonitrile (14) (Schemes 1–4). The formation of
compound 2 was established by the appearance of peaks at
1647 cm−1 (C═C str.) and 639 cm−1 (C-S str.) in the IR
spectrum of compound 2, which was further confirmed by
1H NMR spectra of compound 2, which showed a sharp
singlet at δ1.89 for 6 protons of CH3 group. Appearance of
peaks at 1321 cm−1 (C―N str.) and 1641 cm−1 (C═C of
α,β-unsaturated ketone) in the IR spectrum of 7 ascertained
the formation of compound 7 from 1. Structure of Com-
pound 15 was also established similarly. The formation of
compound 11 from 10 was established by the appearance
of peaks at 1641 cm−1 (C═C α,β-unsaturated str.),
3136 cm−1 (C―H Ar str.), and 1451 cm−1 (C═C ArH str.)
in the IR spectrum of compound 11.
Cell leakage analysis was done to confirm the deleterious
potential of test compound on cell membrane. S. aureus
and E. coli cell cultures were incubated overnight and cen-
trifuged at 10 000 rpm for 10 minutes and suspended in
sterile NaCl solution (0.9%). The bacterial cell cultures were
then exposed to test compound at its IC50 and further incu-
bated at 0, 4, 8, 12, 16, 20, and 24 hours, respectively. Cen-
trifugation was done again at 10 000 rpm for 30 minutes.
Supernatant obtained after centrifugation was analyzed
using UV-Visible spectrophotometer[28] at 320 nm.
3 | RESULTS AND DISCUSSION
3.1 | Chemistry
Cycloaddition of o-phenylenediamine 3 with the
nitrile bearing intermediates 2, 7, 11, and 15, gives the
corresponding intermediates 4, 8, 12, and 16, respectively.
The formation of benzodiazepine ring in compound
4 was confirmed on the basis of one upfield singlet at δ
4.14 for one proton of NH and a multiplet at δ 7.55 to 6.89
for benzene ring's protons. The infrared spectrum of com-
pound 4 exhibited a medium intensity band at 3321 cm−1
due to N―H stretching of primary amine; NH bending at
1469 cm−1 clearly indicated the incorporation of
The nitrile bearing oxoketene dithioacetal (2),
dimethylaminomethylene ketone (7, 15), and α,β- unsatu-
rated ketone (11) derivatives were synthesized from
TABLE 1 Antibacterial activities of synthesized quinazoline
derivatives of 1,5-benzodiazepine
1
1,5-benzodiazepine ring in compound 4. The H NMR
IC50 (μg/mL)
spectrum of compound 4 displayed characteristic signals
for the presence of nine protons in the molecule. Simi-
larly, the structures of compounds 11, 16, and 21 were
established.
S. No.
Compd.
S. aureus
200
E. coli
300
1
2
3
4
5
6
6
9
300
200
Schemes
5
and
6
show the formation of
13
17
21
24
400
300
1,5-benzodiazepines 20 and 23 (bearing a nitrile function
on 7 position) following the cyclocondensation of
3,4-diaminobenzonitrile (19) with acetylacetone (18) and
diethyl malonate (22), respectively.
200
200
200
200
200
400
FIGURE 1 FE-SEM
micrograph of E. coli. (A: control; B:
treated with compound 9)

MISRA ET AL.
9
FIGURE 2 FE-SEM
micrograph of E. coli. (A: control; B:
treated with compound 17)
FIGURE 3 FE-SEM
micrograph of E. coli. (A: control; B:
treated with compound 21)
FIGURE 4 FE-SEM
micrograph of S. aureus. (A: control;
B: treated with compound 6)

10
MISRA ET AL.
FIGURE 5 FE-SEM
micrograph of S. aureus. (A: control;
B: treated with compound 17)
FIGURE 6 FE-SEM
micrograph of S. aureus. (A: control;
B: treated with compound 21)
FIGURE 7 FE-SEM
micrograph of S. aureus. (A: control;
B: treated with compound 24)

MISRA ET AL.
11
FIGURE 8 Cell leakage
activity of test compound 6 against
(A) E. coli and (B) S. aureus
FIGURE 9 Cell leakage
activity of test compound 9 against
(A) E. coli and (B) S. aureus
FIGURE 10 Cell leakage
activity of test compound 13 against
(A) E. coli and (B) S. aureus
FIGURE 11 Cell leakage
activity of test compound
17 against (A) E. coli and
(B) S. aureus

12
MISRA ET AL.
FIGURE 12 Cell leakage
activity of test compound 21 against
(A) E. coli and (B) S. aureus
FIGURE 13 Cell leakage
activity of test compound 24 against
(A) E. coli and (B) S. aureus
The reaction of nitrile bearing 1,5-benzodiazepines (4,
3.2 | Antibacterial study
8, 12, 16, 20, and 23) with an activated benzanilide (activa-
tion of which is achieved by its reaction with Tf2O and
then with 2- chloropyridine). The resulting nitrilium ion
will set the stage ready for its cyclocondensation with the
π-electron system of the arene part of the benzanilide to
deliver the quinazoline derivatives (6, 9, 13, 17, 21, and 24)
in one step.
3.2.1 | Determination of half maximal
inhibitory concentration (IC50)
Gram-positive bacterium (S. aureus) and Gram-negative
bacterium (E. coli) were used to screen the minimum
inhibitory concentration (IC50). The test compounds
(6, 9, 13, 17, 21, and 24) showed dominant inhibitory
activity against S. aureus and E. coli. The IC50 values for
these compounds are shown in Table 1.
IR spectra of all quinazoline derivatives (6, 9, 13,
17, 21, and 24) reveal characteristic C═N and C―N in
between the regions of 1688 to 1454 cm−1 and 1384 to
1054 cm−1, respectively. Other prominent stretching
vibrations observed in the IR spectra of all these com-
pounds were the characteristic N―H vibrations around
3617 to 3231 cm−1, C―H (Ar) vibrations around 3223
to 3026 cm−1, and C═C (Ar) vibrations around 1571 to
1315 cm−1. Besides, these C═O stretching vibrations
were observed in the IR spectra of compounds 6, 9, and
24. Also, C―S stretching vibrations were observed in the
IR spectra of compounds 6.1H NMR spectra of compound
6 displayed two singlets at 7.82 and 4.15 ppm for proton
of two NH groups. All the aromatic protons present in
the molecule were observed as a singlet and two multi-
plets at 7.65, 7.23 to 7.19, and 7.18 to 6.75 ppm, respec-
tively. A singlet for three protons at 1.80 ppm clearly
indicated the presence of methyl group. Similarly, the
structure of compounds 9, 13, 17, 21, and 24 were
ascertained.
3.2.2 | Field emission scanning electron
microscopy (FESEM) study
To examine the effect of test compounds (6, 9, 13, 17,
21, and 24) on both Gram-positive (S. aureus) and Gram-
negative (E. coli) bacterium cell wall's morphology, FESEM
study was done. The result revealed that the cell surface of
E. coli showed normal morphological changes when it is not
treated with the test compound (Figures 1A, 2A, and 3A);
however, notable damage of cell wall, leading to disintegra-
tion of cell membrane, has been observed when treated with
the test compound (Figures 1B, 2B, and 3B). Similar results
obtained in case of S. aureus sample (Figures 4B, 5B, 6B, and
7B) when compared with control (untreated) bacterial cells
(Figures 4A, 5a, 6a, and 7a). Therefore, the test compound

MISRA ET AL.
13
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3.2.3 | Leakage study
The plot of optical density with respect to exposure time
at 320 nm confirms the cell nucleotide leakage, linearly
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4 | CONCLUSION
Potentially bio-active derivatives (6, 9, 13, 17, 21, and 24)
of 1,5-benzodiazepines have been synthesized by one-pot
synthesis using a greener approach. All the synthesized
compounds were screened for antibacterial activity,
mechanistic study, and cell leakage study. FESEM results
clearly indicated that the test compounds caused bacterial
lysis through its membrane damaging effects on both of
the bacterium sample. Cell leakage study showed that
rate of leakage of cell nucleotides increased with increase
in exposure time through cell membrane of treated bacte-
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ACKNOWLEDGEMENT
The authors are deeply thankful for financial support
that has been provided by the Department of Science and
Technology (DST), New Delhi under CURIE
(Consolidation of University Research for Innovation and
Excellence in Women Universities) Scheme and MHRD,
New Delhi, under Training and Research in Frontier
Areas of Science and Technology (FAST) Scheme.
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CONFLICT OF INTEREST
The authors declare that they have no conflict of interest.
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ORCID
Jaya Dwivedi https://orcid.org/0000-0002-1524-7714
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How to cite this article: Misra A, Dwivedi J,
Shukla S, Kishore D, Sharma S. Bacterial cell
leakage potential of newly synthesized quinazoline
derivatives of 1,5-benzodiazepines analogue.
J Heterocycl Chem. 2020;1–14. https://doi.org/10.
1002/jhet.3879
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