Synthesis and SAR studies of potent H+/K+-ATPase inhibitors of quinazolinone-Schiff's base analogues

Article abstract of DOI:10.1016/j.bioorg.2016.07.001

A series of quinazolinone derived Schiff base derivatives 7–36 were synthesized and characterized by analytical and spectroscopic techniques. The synthesized analogues were screened for their in vitro H⁺/K⁺-ATPase inhibition. Most of the compounds showed excellent activity, compared to that of omeprazole, a reference drug. In particular, hydroxy and methoxy derivatives 13–24 were the most active compounds possessing a significant increase for different substituents on the benzene ring thus, contributing positively to gastric H⁺/K⁺-ATPase inhibition. Preliminary structure-activity relationship revealed that the compounds 13–24 with electron donating moiety (OH, OCH₃) were found to be excellent activity and compounds 9–12 and 25–36 with electron withdrawing moiety (Cl, F, NO₂ and Br) were found to be least antiulcer agents.

Full text of DOI:10.1016/j.bioorg.2016.07.001

Bioorganic Chemistry 68 (2016) 1–8
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and SAR studies of potent H+/K+-ATPase inhibitors of
quinazolinone-Schiff’s base analogues
b
c
K.P. Rakesh a, , C.S. Shantharam , H.M. Manukumar
⇑
a Department of Studies in Chemistry, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India
b Department of Chemistry, Pooja Bhagavath Memorial Mahajana Education Centre, Mysuru 570016, Karnataka, India
c Department of Studies in Biotechnology, University of Mysore, Manasagangotri, Mysuru 570 006, Karnataka, India
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of quinazolinone derived Schiff base derivatives 7–36 were synthesized and characterized
by analytical and spectroscopic techniques. The synthesized analogues were screened for their in vitro
H+/K+-ATPase inhibition. Most of the compounds showed excellent activity, compared to that of omeprazole,
a reference drug. In particular, hydroxy and methoxy derivatives 13–24 were the most active compounds
possessing a significant increase for different substituents on the benzene ring thus, contributing posi-
tively to gastric H+/K+-ATPase inhibition. Preliminary structure-activity relationship revealed that the
compounds 13–24 with electron donating moiety (OH, OCH3) were found to be excellent activity and
compounds 9–12 and 25–36 with electron withdrawing moiety (Cl, F, NO2 and Br) were found to be least
antiulcer agents.
Received 26 April 2016
Revised 1 July 2016
Accepted 4 July 2016
Available online 5 July 2016
Keywords:
Quinazolinones
Schiff’s bases
Electronic effect
H+/K+-ATPase activity
Ó 2016 Elsevier Inc. All rights reserved.
1. Introduction
to develop agrochemicals and medicines, including fungicide [10],
bactericide [11], antivirals [12], antioxidants [13], antiproliferative
Gastric and duodenal ulcers are commonly occurring diseases.
Ulcers are believed to result from an imbalance between the
aggressive (acid, pepsin) and defensive forces (bicarbonate,
mucous) in the stomach and duodenum. Reduction of acid secre-
tion, especially by antagonism of the H2 receptor, has proven to
be a useful means for promoting the healing of ulcers, particularly
those of the duodenal mucosa [1]. Recently, agents have been iden-
tified that completely suppress acid secretion by inhibition of the
gastric proton pump H+/K+-ATPase. Such inhibition leads to a pro-
found and prolonged achlorhydria, which results in ulcer healing
rates substantially more rapid than those achievable by H2 antag-
onists. H+/K+-ATPase catalyzes the terminal step in gastric acid
whereas histamine is but one of three key messengers which stim-
ulate acid secretion (the other two being acetylcholine and gas-
trin). As a result, inhibition of H+/K+-ATPase can provide an
intrinsically greater reduction in gastric acid secretion [2–7].
Although several efficient drugs are available for their treatment
with different mechanisms of action there is always a need for
new agents with even better efficacy and safety profiles [8].
The Schiff’s base family is composed of natural products with
critical pharmacophores [9]. It can be used as ideal lead structures
[14] and antimicrobial drug [15]. Various natural alkaloids with
critical pharmacophores contain quinazolinone groups. For exam-
ple, febrifugine, isofebrifugine, thiabutazide, (ꢀ)-benzomalvin A,
2-(4-hydroxybutyl) quinazolin-4-one, and luotonin F were found
in the plants, animals, and microorganisms [16,17]. Moreover,
the quinazolinone nucleus and its derivatives have been exten-
sively studied because of their wide range of pharmacological
activities. As medicines, many of them display antitubercular
[18], anti-inflammatory [19], anticonvulsant [20], antidepressant
[21], antiulcer [22] and analgesic [23] activities. Some of the
reported antiulcer activities of Schiff’s bases are shown in Fig. 1
[24–26].
In medicinal chemistry quinazolinone derivatives also play a
biological importance [27–31]. Based on the above facts and in
continuation of our drug development program [32–34], recently
we have reported antioxidants and anti-inflammatory activity of
quinazolinone derivatives [35]. On this basis we have designed
and synthesized two quinazolinone analogues and these are
built-in into the different aromatic aldehydes containing electron
donating and withdrawing groups. This quinazolinone-Schiff’s
base derivative shows potent antioxidant and anti-inflammatory
agents. Further we have inspired by our research group earlier
work on urea/thiourea derivatives as potent H+/K+ ATPase inhibi-
tors [36], we would like to test synthesized compounds against
⇑
Corresponding author.
E-mail address: rakeshasg@gmail.com (K.P. Rakesh).
http://dx.doi.org/10.1016/j.bioorg.2016.07.001
0045-2068/Ó 2016 Elsevier Inc. All rights reserved.

2
K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
O
S
H
3C
N
N
H
O
H
N
Cl
O
H
N
Cl
N
Cl
HO
O
O
NH
N
NH
N
NH
NH
O
O
OH
OH
OMe
Fig. 1. Structures of antiulcer Schiff’s bases.
H+/K+ ATPase inhibition activity using same methodology and we
have got good results and searching for new antiulcer agents.
The obtained results are compared with our earlier research group
work in conclusion part.
Table 1
Antiulcer activity of the synthesized compounds.
Entry
Antiulcer activitya IC50 Entry
M)
Antiulcer activitya IC50
M)
(l
(l
1
2
3
4
5
6
7
8
Inactive
Inactive
Inactive
Inactive
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
29 1.02
24 0.31
22 0.16
14 0.49
15 0.28
10 0.48
170 1.23
160 0.98
180 1.69
169 1.03
146 1.09
131 0.91
129 1.32
142 0.45
151 0.56
139 1.16
160 1.65
144 1.07
2. Results and discussion
248 2.68
241 1.67
210 1.58
197 1.69
152 1.44
134 1.24
140 1.88
127 0.81
60 0.59
51 0.98
54 1.32
41 2.31
37 1.34
32 1.27
2.1. Chemistry
Syntheses of the desired compounds were achieved according
to the steps illustrated in Scheme. Anthranilamide reacts with suc-
cinic anhydride in benzene-1,4-dioxane mixture for 2 h in rt to get
3-(4-Oxo-3,4-dihydroquinozolin-2-yl)propanoic acid (1) and
Anthranilamide reacts with glutaric anhydride in benzene-1,4-
dioxane mixture for 2 h in rt to get 4-(4-oxo-3,4-dihydroquinozo
lin-2-yl)butanoic acid (2) [37–39]. Compounds 1 and 2 were
methylated using trimethylsilylchloride (TMS-Cl) and methanol
at room temperature, which upon reaction with excess of hydra-
zine hydrate afforded the corresponding quinazolinone hydrazides
(5 and 6). The Schiff’s bases (7–36) were obtained by reacting 5 and
6 with different aromatic aldehydes in presence of catalytic
amount of glacial acetic acid. All the derivatives were obtained in
high yield and the methods employed are very simple. The struc-
tures of all the newly synthesized compounds including intermedi-
ates were confirmed by IR, 1H NMR, 13C NMR and mass spectral
analysis. The formation of methyl esters (3 and 4) were confirmed
by the appearance of a singlet at 3.71 d for OCH3 and absence of
COOH proton peak at 12.19 d in 1H NMR spectrum. In IR spectra,
bands at 3335 and 3220 cmꢀ1 for NH2ANH groups indicates the
conversion of methyl esters into hydrazides. The formation of
Schiff’s bases were confirmed by the presence of absorption at
1605–1630 for imines i.e., AN@CHA in IR spectra. The presence
of all requisite peaks and absence of extraneous peaks in 1H NMR
and 13C NMR confirms the structures.
9
10
11
12
13
14
15
16
17
18
Omeprazole 76 1.24
Omeprazole
76 1.24
a
Values are mean of three determinations, the ranges of which are <5% of the
mean in all cases.
one methoxy group substituents on the phenyl ring showed
good activity compared to standard omeprazole IC50 = 76
Therefore, the effect of substituents on the phenyl ring was further
investigated. Compounds 17 (IC50 = 37 M) and 18 (IC50 = 32 M)
both hydroxy and methoxy group was placed on the phenyl ring,
derivatives exhibited highly potent activity. Compounds 19
lM.
l
l
(IC50 = 29
methoxy group and 21 (IC50 = 22
group, 23 (IC50 = 15 M) and 24 (IC50 = 10
l
M), 20 (IC50 = 24
l
M) containing 2 hydroxy and 1
M), 22 (IC50 = 14 M) 3 methoxy
M) 3 hydroxy group on
l
l
l
l
phenyl ring showed excellent activity. Compounds 9–12 and 25–
36 containing electron withdrawing (Cl, F, NO2 and Br) groups
showed least antiulcer activity compared to the standards. This
result revealed that electronic effect plays an important role in
the activity. From the above results, some broad generalizations
could be drawn. Due to electron releasing methoxy and hydroxy
derivatives, an increase in the activity was witnessed. Among
electron withdrawing groups, chloro, fluoro, nitro and bromo
derivatives showed least antiulcer activity. The number of hydroxy
and methoxy groups increases on phenyl ring the activity also
increases.
2.2. Biological activity
2.2.1. In vitro antiulcer activity
All the synthesized compounds were evaluated for gastric
H+/K+-ATPase activity. The results are tabulated in Table 1.
Omeprazole was used as the reference compound. Among the
derivatives, compounds 13 (IC50 = 60
one hydroxy and compounds 15 (IC50 = 54
l
M) and 14 (IC50 = 51
l
l
M)
M)
lM) and 16 (IC50 = 41

K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
3
acidified with acetic acid (10 mL). The resulting precipitate was fil-
tered under vacuum and washed with water to get compound 1
(Scheme 1).
3. Conclusion
In the present investigation we have tested in vitro H+/K+
ATPase activity of quinazolinone-Schiff’s base derivative results
were compared with another in vitro H+/K+ ATPase activities of
urea/thiourea derivatives of glutamic acid conjugated benzisothia-
zole analogues. We have synthesized quinazolinone-Schiff’s base
activity results are nearly equal potent to urea/thiourea derivatives
of glutamic acid conjugated benzisothiazole analogues. Chemical
modification of both structures is different. In our most active com-
IR: 1628, 1640, 1680 (C@O), 3320 (NH); 1H NMR (DMSO-d6) d
ppm: 12.10 (1H, s, OH), 8.37–7.08 (4H, m, ArH), 7.03–7.02 (1H, s,
NH), 2.89–2.87 (2H, t, CH2), 2.50–2.46 (2H, s, NH2), 2.32–2.30
(2H, t, CH2); 13C NMR (DMSO-d6) d ppm: 173.7, 171.8, 170.6,
137.6, 131.4, 128.6, 127.5, 124.6, 123.8, 30.4, 29.6; Mass (M+1):
219.1080; M.P.: 206–208 (lit 208–209).
pounds, activity range is 10–29
taining Schiff’s base of electron donating (OH and OCH3) groups
most favour the antiulcer agents and activity range 127–157
lM and these quinazolinone con-
4.2.3. Synthesis of 5-(2-carbamoylphenylamino)-5-oxopentanoic acid
(b)
lM
To
a
solution of dihydro-3H-pyran-2,6-dione (5.00 g,
are the electron withdrawing group (Cl, NO2, F and Br) are least
active agents. Whereas urea/thiourea derivatives of glutamic acid
conjugated benzisothiazole analogues, most active compounds
43.85 mmol) in ether (40 mL) was added solution of anthranil-
amide (5.96 g, 43.85 mmol) in benzene-1,4-dioxane mixture
(60 mL, 2:1) in a drop wise fashion with constant stirring at room
temperature. The reaction mixture was further stirred for 2 h and
the formed precipitate was filtered under vacuum and washed
with ether (50 mL). The obtained compound 3 was used for next
step without further purification.
IR: 1630 (C@O), 1674 (C@O, amide), 1690 (C@O, amide), 3310
(NH), 3562 (OH); 1H NMR (DMSO-d6) d ppm: 12.12 (1H, s, OH),
8.32–7.10 (4H, m, ArH), 7.02–7.00 (1H, s, NH), 2.88–2.87 (2H, t,
CH2), 2.55–2.54 (2H, s, NH2), 2.30–2.29 (2H, t, CH2); 2.15–2.14
(2H, m, CH2); 13C NMR (DMSO-d6) d ppm: 177.8, 175.6, 171.4,
137.8, 131.4, 127.8, 126.6, 124.5, 122.8, 33.7, 30.8, 21.8; Mass (M
+1): 251.2314; M.P.: 209–211 (Lit 210–212).
range is 5.4–19
lM which containing electron donating (OCH3)
groups in the molecule and electron withdrawing group (Cl, F
and Br) less active (44–80 lM). Finally we have synthesized simple
and small active analogues which shown potent H+/K+ ATPase
activity. Further studies on the relevant mechanism of action and
the toxicity studies of these compounds are in progress.
4. Experimental
4.1. Materials
All chemicals and reagents obtained from Merck (India) and
Avra Synthesis (India) were used without further purification.
Melting points were determined on a Superfit melting point appa-
ratus (India) and are uncorrected. FT-IR was performed using a
Jasco spectrometer (Japan) using Nujol media. 1H NMR
(400 MHz) and 13C NMR (100 MHz) spectra were recorded on a
Agilent Technologies (USA) using DMSO (d6) as solvent. High reso-
lution mass spectroscopic analysis was performed on a Bruker
MicroTOF QII mass spectrometer in positive mode. Progress of
the reaction was monitored by TLC using silica gel coated on glass
plates with the solvent system comprising chloroform/methanol/
acetic acid in the ratio 98:02:03 (Raf ) and 95:05:03 (Rfb) and the
compounds on the TLC plates were detected by iodine vapors.
4.2.4. Synthesis of 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid
(2)
A mixture of compound 3 (5.00 g, 20 mmol) in 5% NaOH (50 mL)
and EtOH (25 mL) was heated to reflux for 30 min, whereupon the
solution was allowed to cool for 15 min and then acidified with
acetic acid (5 mL). The resulting precipitate was filtered under vac-
uum and washed with water to get compound 2 (Scheme 2).
IR: 1637 (C@O), 1680 (C@O, amide), 3325 (NH), 3516 (OH); 1H
NMR (DMSO-d6) d ppm: 12.10 (2H, s, OH and NH), 8.05–7.39 (4H,
m, ArH), 2.62–2.58 (2H, t, CH2), 2.30–2.26 (2H, t, CH2), 1.90–1.89
(2H, m, CH2); 13C NMR (DMSO-d6) d ppm:174.4, 162.1, 157.2,
134.6, 127.2, 126.3, 126.1, 121.3, 33.9, 33.2, 22.2; Mass (M+1):
233.1265; M.P.: 225–226 (Lit 225–227) [26,27].
4.2. Chemistry
4.2.5. Experimental procedure for the synthesis of 3 and 4
To a solution of QZN 1 (0.02 mol, 4.36 g) and QZN 2 (0.02 mol,
4.64 g) separately in methanol (40 mL), trimethylsilylchloride
(0.02 mol, 3.80 mL) was added slowly. The reaction mixture was
stirred for 4 h to complete the reaction (monitored by TLC). The
solvent was removed under reduced pressure and the resultant
precipitate was washed with ice cold water and filtered to yield
the desired products 3 and 4 respectively (Scheme 3).
4.2.1. Synthesis of 4-(2-carbamoylphenylamino)-4-oxobutanoic
acid (a)
To a solution of succinic anhydride (8.00 g, 80 mmol) in ether
(50 mL) was added
a solution of anthranilamide (10.88 g,
80 mmol) in benzene-1,4-dioxane mixture (75 mL, 2:1) in a drop-
wise fashion with constant stirring at room temperature. The reac-
tion mixture was further stirred for 2 h and the formed precipitate
was filtered under vacuum and washed with ether (50 mL). The
obtained compound 1 was used for next step without further
purification.
4.2.6. Methyl 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoate (3)
Yield 91.41%, Raf = 0.66, Rfb = 0.70, m.p. 184–185 °C, IR KBr
(cmꢀ1): 2990, 1768, 1635; 1H NMR (DMSO-d6) d ppm: 11.31 (s,
1H, Het-NH), 8.15–7.59 (m, 4H, ArAH), 3.68 (s, 3H, AOCH3),
3.11–3.08 (t, J = 6.8 Hz, 2H, CH2), 3.00–2.97 (t, J = 6.8 Hz, 2H,
CH2); 13C NMR (DMSO-d6) d ppm: 173.21, 161.09, 156.27, 146.76,
133.71, 129.23, 126.39, 126.14, 121.33, 51.89, 30.34, 29.15; HRMS
m/z: 232.5918.
IR: 1628, 1640, 1680 (C@O), 3320 (NH); 1H NMR (DMSO-d6) d
ppm: 12.10 (1H, s, OH), 8.37–7.08 (4H, m, ArH), 7.03–7.02 (1H, s,
NH), 2.89–2.87 (2H, t, CH2), 2.50–2.46 (2H, s, NH2), 2.32–2.30
(2H, t, CH2); 13C NMR (DMSO-d6) d ppm: 173.7, 171.8, 170.6,
137.6, 131.4, 128.6, 127.5, 124.6, 123.8, 30.4, 29.6; Mass (M+1):
237.2164; M.P.: 196–197 (lit 197–198).
4.2.2. Synthesis of 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic
acid (1)
A mixture of compound 1 (10.00 g, 42.37 mmol) in 5% NaOH
(100 mL) and EtOH (50 mL) was heated to reflux for 30 min,
whereupon the solution was allowed to cool for 15 min and then
4.2.7. Methyl 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoate (4)
Yield 92.36%, Raf = 0.64, Rfb = 0.69, m.p. 180–182 °C, IR KBr
(cmꢀ1): 3210, 1745, 1640; 1H NMR (DMSO-d6) d ppm: 11.20 (s,
1H, Het-NH), 8.17–7.20 (m, 4H, ArAH), 3.68 (s, 3H, AOCH3),
3.19–3.17 (t, J = 7.6 Hz, 2H, CH2), 3.01–2.97 (q, J = 7.6 Hz 2H,

4
K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
Scheme 1. Synthesis of 3-(4-oxo-3,4-dihydroquinazolin-2-yl)propanoic acid (a) Et2O/C6H6/1,4-dioxane (2:2:1), rt, 2 h (b) 5% NaOH/EtOH.
Scheme 2. Synthesis of 4-(4-oxo-3,4-dihydroquinazolin-2-yl)butanoic acid (a) Et2O/C6H6/1,4-dioxane (2:2:1), rt, 2 h (b) 5% NaOH/EtOH.
Scheme 3. Synthesis of title compounds 7–36.
CH2), 2.70–2.68 (t, J = 7.6 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm:
172.69, 162.29, 156.17, 146.25, 133.21, 128.13, 126.87, 126.44,
121.43, 51.60, 30.344, 29.10, 28.45; HRMS m/z: 246.6434.
4.2.8. Experimental procedure for the synthesis of 5 and 6
To a solution of 3 (0.015 mol, 4.02 g) and 4 (0.015 mol, 4.23 g)
separately in ethanol (40 mL), hydrazine hydrate (0.020 mol,

K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
5
0.97 mL) was added. The reaction mixture was refluxed for 16 h for
completion of the reaction (monitored by TLC). The solvent was
removed under reduced pressure and cooled by adding ice cold
water. The resulting precipitate was filtered, washed with cold
water and recrystallized from ethanol to get the desired com-
pounds 5 and 6 respectively.
157.27, 146.16, 143.23, 134.79, 134.68, 130.33, 129.23, 127.15,
126.36, 126.20, 126.14, 121.33, 30.64, 29.25; HRMS m/z:
354.5013 [M+], 356.1265 [M+1].
4.2.15. N0-(4-chlorobenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-2-
yl)butanehydrazide (10)
Yield 87.01%, Raf = 0.62, Rfb = 0.68, m.p. 229–230 °C, IR KBr
(cmꢀ1): 3220, 1780, 1672, 1616; 1H NMR (DMSO-d6) d ppm:
12.16 (s, 1H, NH), 11.39 (s, 1H, Het-NH), 8.09–7.35 (m, 8H, ArAH),
7.90 (s, 1H, CH), 2.71–2.67 (q, J = 7.2 Hz, 2H, CH2), 2.27–2.25 (t,
J = 7.2 Hz, 2H, CH2), 2.04–2.00 (t, J = 7.2 Hz, 2H, CH2); 13C NMR
4.2.9. 3-(4-Oxo-3,4-dihydroquinazolin-2-yl)propanehydrazide (5)
Yield 83.33%, Raf = 0.32, Rfb = 0.36, m.p. 220–221 °C, IR KBr
(cmꢀ1): 3310, 3217, 3210, 1745, 1628; 1H NMR (DMSO-d6) d
ppm: 11.20 (s, 1H, Het-NH), 8.99 (s, 1H, NH), 8.02–7.38 (m, 4H,
ArAH), 4.14 (s, 2H, NH2), 2.79–2.77 (t, J = 7.8 Hz, 2H, CH2), 2.48–
2.46 (t, J = 7.8 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.27,
162.19, 156.77, 146.14, 133.61, 128.23, 127.10, 126.80, 121.23,
30.14, 29.45; HRMS m/z: 232.1435.
(DMSO-d6)
d ppm: 174.44, 162.21, 157.43, 149.33, 144.96,
141.63, 134.67, 129.28, 129.00, 128.60, 127.29, 126.37, 126.11,
121.32, 34.21, 31.57, 22.15; HRMS m/z: 368.1636 [M+], 370.2364
[M+1].
4.2.10. 4-(4-Oxo-3,4-dihydroquinazolin-2-yl)butanehydrazide (6)
Yield 84.01%, Raf = 0.33, Rfb = 0.38, m.p. 224–226 °C, IR KBr
(cmꢀ1): 3310, 3251, 3216, 1766, 1643; 1H NMR (DMSO-d6) d
ppm: 11.10 (s, 1H, Het-NH), 8.89 (s, 1H, NH), 8.12–7.56 (m, 4H,
ArAH), 4.44 (s, 2H, NH2), 3.16–3.14 (t, J = 7.8 Hz, 2H, CH2), 2.86–
2.84 (q, J = 7.8 Hz 2H, CH2), 2.60–2.58 (t, J = 7.8 Hz, 2H, CH2); 13C
NMR (DMSO-d6) d ppm: 173.57, 162.09, 156.13, 146.24, 133.51,
127.23, 127.80, 126.20, 121.43, 30.14, 29.45, 28.14; HRMS m/z:
246.1265.
4.2.16. N0-(4-nitrobenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-2-yl)
propanehydrazide (11)
Yield 85.53%, Raf = 0.61, Rfb = 0.68, m.p. 235–238 °C, IR KBr
(cmꢀ1): 3326, 1677, 1627, 1610; 1H NMR (DMSO-d6) d ppm:
12.22 (s, 1H, NH), 11.31 (s, 1H, Het-NH), 8.14–7.39 (m, 8H, ArAH),
7.97 (s, 1H, CH), 3.17–3.14 (t, J = 6.8 Hz, 2H, CH2), 2.93–2.89 (t,
J = 6.8 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.66, 162.16,
157.27, 149.21, 146.16, 143.22, 134.71, 134.68, 129.25, 127.23,
126.45, 126.20, 126.14, 121.33, 30.64, 29.12; HRMS m/z: 365.4231.
4.2.11. General procedure for the synthesis of Schiff’s bases (7–24)
An equimolar amount of 5 (1 mmol) and 6 (1 mmol) was dis-
solved separately in ethanol (10 mL/g of compound) and treated
with appropriate aldehydes (1 mmol) in the presence of catalytic
amount of glacial acetic acid. The reaction mixtures were refluxed
for 7–8 h and the completion of reaction was monitored by TLC.
After completion of the reaction, the solvent was removed under
reduced pressure and cooled by adding ice cold water. The result-
ing precipitate was filtered, washed with water and recrystallized
from ethanol to obtain the desired Schiff’s bases (Scheme 3) (7–
36).
4.2.17. N0-(4-nitrobenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-2-yl)
butanehydrazide (12)
Yield 91.60%, Raf = 0.62, Rfb = 0.66, m.p. 244–246 °C, IR KBr
(cmꢀ1): 3245, 1774, 1660, 1619; 1H NMR (DMSO-d6) d ppm:
12.17 (s, 1H, NH), 11.63 (s, 1H, Het-NH), 8.25–7.40 (m, 8H, ArAH),
7.88 (s, 1H, CH), 2.75–2.71 (t, J = 7.2 Hz, 2H, CH2), 2.68–2.65 (q,
J = 7.2 Hz, 2H, CH2), 2.05–2.02 (t, J = 7.2 Hz, 2H, CH2); 13C NMR
(DMSO-d6)
d ppm: 174.80, 162.21, 157.38, 149.31, 148.17,
143.79, 140.58, 134.67, 128.28, 127.84, 127.29, 126.38, 124.43,
121.31, 34.14, 31.50, 22.07; HRMS m/z: 379.1245.
4.2.18. N0-(4-hydroxybenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-2-
yl)propanehydrazide (13)
4.2.12. N0-benzylidene-3-(4-oxo-3,4-dihydroquinazolin-2-yl)
propanehydrazide (7)
Yield 87.55%, Raf = 0.55, Rfb = 0.64, m.p. 239–241 °C, IR KBr
(cmꢀ1): 3518, 3221, 1769, 1660, 1623; 1H NMR (DMSO-d6) d
ppm: 12.20 (s, 1H, NH), 11.04 (s, 1H, Het-NH), 8.73 (s, 1H, OH),
8.04–6.75 (m, 8H, ArAH), 7.86 (s, 1H, CH), 3.12–3.09 (t, J = 6.8 Hz,
2H, CH2), 2.90–2.87 (t, J = 6.8 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.16, 161.88, 159.65, 156.94, 146.72, 143.73, 134.73,
129.17, 127.18, 126.50, 126.42, 125.72, 121.22, 116.09, 30.65,
29.72; HRMS m/z: 336.3465.
Yield 89.18%, Raf = 0.58, Rfb = 0.64, m.p. 242–244 °C, IR KBr
(cmꢀ1): 3322, 1731, 1674, 1610; 1H NMR (DMSO-d6) d ppm:
12.22 (s, 1H, NH), 11.31 (s, 1H, Het-NH), 8.14–7.39 (m, 9H, ArAH),
7.97 (s, 1H, CH), 3.17–3.14 (t, J = 6.8 Hz, 2H, CH2), 2.93–2.89 (t,
J = 6.8 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.09, 162.16,
157.27, 146.16, 143.22, 134.79, 134.71, 130.14, 129.23, 127.39,
127.15, 126.36, 126.14, 121.33, 30.64, 29.25; HRMS m/z: 320.4152.
4.2.13. N0-benzylidene-4-(4-oxo-3,4-dihydroquinazolin-2-yl)
butanehydrazide (8)
4.2.19. N0-(4-hydroxybenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-2-
yl)butanehydrazide (14)
Yield 92.13%, Raf = 0.57, Rfb = 0.63, m.p. 206–207 °C, IR KBr
(cmꢀ1): 3315, 1738, 1662, 1608; 1H NMR (DMSO-d6) d ppm:
12.16 (s, 1H, NH), 11.29 (s, 1H, Het-NH), 8.18–7.16 (m, 9H, ArAH),
7.90 (s, 1H, CH), 3.15–3.13 (t, J = 7.4 Hz, 2H, CH2), 2.76–2.73 (q,
J = 7.4 Hz, 2H, CH2), 2.53–2.51 (t, J = 7.4 Hz, 2H, CH2); 13C NMR
Yield 85.20%, Raf = 0.57, Rfb = 0.63, m.p. 237–238 °C, IR KBr
(cmꢀ1): 3522, 3244, 1770, 1770, 1608; 1H NMR (DMSO-d6) d
ppm: 12.25 (s, 1H, NH), 11.14 (s, 1H, Het-NH), 8.90 (s, IH, OH),
8.116.89 (m, 8H, ArAH), 7.80 (s, 1H, CH), 3.19–3.16 (t, J = 7.2 Hz,
2H, CH2), 2.91–2.88 (q, J = 7.2 Hz, 2H, CH2), 2.58–2.56 (t,
J = 7.2 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.11, 161.25,
159.25, 156.16, 146.14, 143.23, 133.73, 129.14, 127.78, 126.93,
126.16, 125.36, 121.64, 116.26, 34.62, 31.88, 22.44; HRMS m/z:
350.6130.
(DMSO-d6)
d ppm: 172.59, 161.89, 156.24, 146.33, 144.20,
134.18, 133.81, 130.18, 129.17, 127.19, 127.10, 126.86, 126.32,
121.14, 30.25, 29.16, 27.88; HRMS m/z: 334.5617.
4.2.14. N0-(4-chlorobenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-2-
yl)propanehydrazide (9)
4.2.20. N0-(4-methoxybenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-
2-yl)propanehydrazide (15)
Yield 84.40%, Raf = 0.64, Rfb = 0.71, m.p. 221–223 °C, IR KBr
(cmꢀ1): 3206, 1739, 1680, 1624; 1H NMR (DMSO-d6) d ppm:
12.20 (s, 1H, NH), 11.30 (s, 1H, Het-NH), 8.04–7.39 (m, 8H, ArAH),
7.86 (s, 1H, CH), 3.12–3.08 (t, J = 7.0 Hz, 2H, CH2), 2.90–2.87 (t,
J = 7.0 Hz, 2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.66, 162.15,
Yield 87.16%, Raf = 0.56, Rfb = 0.62, m.p. 195–197 °C, IR KBr
(cmꢀ1): 3270, 1739, 1680, 1614; 1H NMR (DMSO-d6) d ppm:
12.13 (s, 1H, NH), 11.15 (s, 1H, Het-NH), 8.14–6.79 (m, 8H, ArAH),
7.88 (s, 1H, CH), 3.78 (s, 3H, AOCH3), 3.16–3.14 (t, J = 6.8 Hz, 2H,

6
K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
CH2), 2.90–2.88 (t, 2H, J = 6.8 Hz, CH2); 13C NMR (DMSO-d6) d ppm:
173.18, 161.91, 161.60, 156.16, 146.22, 143.17, 133.26, 129.17,
128.51, 127.61, 126.16, 126.11, 121.43, 115.10, 51.45, 30.85,
29.11; HRMS m/z: 350.2687.
4.2.26. 3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N0-
(3,4,5trimethoxybenzylidene) propanehydrazide (21)
Yield 85.98%, Raf = 0.61, Rfb = 0.68, m.p. 198–201 °C, IR KBr
(cmꢀ1): 3215, 1738, 1678, 1611; 1H NMR (DMSO-d6) d ppm:
12.20 (s, 1H, NH), 11.48 (s, 1H, Het-NH), 8.06–6.89 (m, 6H, ArAH),
7.87 (s, 1H, CH), 3.81 (s, 9H, 3-OCH3), 3.15–3.14 (t, J = 6.0 Hz, 2H,
CH2), 2.76–2.74 (t, J = 6.0 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.64, 162.10, 156.94, 153.60, 149.22, 146.18, 143.14,
134.60, 130.25, 127.18, 126.42, 126.18, 121.34, 104.45, 60.56,
56.38, 30.67, 29.04; HRMS m/z: 410.9165.
4.2.21. N0-(4-methoxybenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-
2-yl)butanehydrazide (16)
Yield 92.70%, Raf = 0.55, Rfb = 0.63, m.p. 234–236 °C, IR KBr
(cmꢀ1): 3310, 1755, 1670, 1610; 1H NMR (DMSO-d6) d ppm:
12.11 (s, 1H, NH), 11.10 (s, 1H, Het-NH), 8.19–6.84 (m, 8H, ArAH),
7.92 (s, 1H, CH), 3.81 (s, 3H, AOCH3), 3.12–3.09 (t, J = 6.8 Hz, 2H,
CH2), 2.95–2.93 (q, J = 6.8 Hz, 2H, CH2), 2.64–2.62 (t, J = 6.8 Hz,
2H, CH2); 13C NMR (DMSO-d6) d ppm: 173.78, 161.31, 160.30,
156.26, 146.12, 144.17, 133.86, 129.57, 128.11, 127.21, 126.11,
126.01, 121.43, 115.13, 52.36, 34.65, 31.61, 22.08; HRMS m/z:
364.2456.
4.2.27. 4-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N0
(3,4,5trimethoxybenzylidene) butanehydrazide (22)
Yield 88.55%, Raf = 0.60, Rfb = 0.66, m.p. 204–206 °C, IR KBr
(cmꢀ1): 3251, 1766, 1674, 1612; 1H NMR (DMSO-d6) d ppm:
12.12 (s, 1H, NH), 11.32 (s, 1H, Het-NH), 8.05–6.84 (m, 6H, ArAH),
7.82 (s, 1H, CH), 3.80 (s, 9H, 3-OCH3), 2.68–2.64 (t, J = 6.4 Hz 2H,
CH2), 2.27–2.26 (q, J = 6.4 Hz, 2H, CH2), 2.03–2.02 (t, J = 6.4 Hz,
2H, CH2); 13C NMR (DMSO-d6) d ppm: 174.35, 162.30, 157.36,
153.52, 149.18, 146.43, 142.96, 134.68, 130.20, 127.11, 126.39,
126.08, 121.14, 104.27, 60.52, 56.37, 34.28, 31.38, 22.08; HRMS
m/z: 424.6513.
4.2.22. N0-(4-hydroxy-3-methoxybenzylidene)-3-(4-oxo-3,4-
dihydroquinazolin-2-yl)propanehydrazide (17)
Yield 87.06%, Raf = 0.52, Rfb = 0.57, m.p. 222–224 °C, IR KBr
(cmꢀ1): 3566, 3298, 1762, 1654, 1628; 1H NMR (DMSO-d6) d
ppm: 12.19 (s, 1H, NH), 11.10 (s, 1H, Het-NH), 9.44 (s, 1H, OH),
8.05–6.77 (m, 7H, ArAH), 7.85 (s, 1H, CH), 3.77 (s, 3H, AOCH3),
3.15–3.11 (t, J = 6.8 Hz, 2H, CH2), 2.82–2.89 (t, J = 6.8 Hz, 2H,
CH2); 13C NMR (DMSO-d6) d ppm: 173.12, 162.08, 157.37, 149.21,
148.38, 146.75, 143.73, 134.73, 127.24, 126.35, 126.18, 126.14,
122.35, 121.36, 116.03, 109.97, 55.96, 30.68, 29.75; HRMS m/z:
366.9615.
4.2.28. 3-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N0-(3,4,5-
trihydroxybenzylidene) propanehydrazide (23)
Yield 90.63%, Raf = 0.32, Rfb = 0.37, m.p. 229–231 °C, IR KBr
(cmꢀ1): 3560, 3278, 1768, 1653, 1608; 1H NMR (DMSO-d6) d
ppm: 12.20 (s, 1H, NH), 11.21 (s, 1H, Het-NH), 8.95 (s, 3H, 3OH),
8.06–6.60 (m, 6H, ArAH), 7.85 (s, 1H, CH), 3.11–3.08 (t, J = 7.0 Hz,
2H, CH2), 2.91–2.88 (t, J = 7.0 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.13, 162.16, 156.94, 149.19, 146.58, 144.21, 136.00,
134.73, 127.24, 126.40, 126.15, 125.02, 121.26, 106.62, 30.63,
29.05; HRMS m/z: 368.1698.
4.2.23. N0-(4-hydroxy-3-methoxybenzylidene)-4-(4-oxo-3,4-
dihydroquinazolin-2-yl) butanehydrazide (18)
Yield 87.82%, Raf = 0.54, Rfb = 0.59, m.p. 238–240 °C, IR KBr
(cmꢀ1): 3610, 3315, 1746, 1648, 1604; 1H NMR (DMSO-d6) d
ppm: 12.13 (s, 1H, NH), 11.15 (s, 1H, Het-NH), 9.14 (s, 1H, OH),
8.15–6.79 (m, 7H, ArAH), 7.89 (s, 1H, CH), 3.82 (s, 3H, AOCH3),
3.13–3.11 (t, J = 7.0 Hz, 2H, CH2), 2.90–2.87 (q, J = 7.0 Hz, 2H,
CH2), 2.57–2.55 (t, J = 7.0 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.17, 162.58, 158.98, 156.21, 146.38, 143.75, 135.73,
131.73, 128.84, 128.35, 127.18, 126.44, 122.35, 121.16, 112.03,
109.14, 56.96, 34.28, 31.15, 22.10; HRMS m/z: 380.2456.
4.2.29. 4-(4-Oxo-3,4-dihydroquinazolin-2-yl)-N0-(3,4,5-
trihydroxybenzylidene) butanehydrazide (24)
Yield 89.88%, Raf = 0.29, Rfb = 0.35, m.p. 210–212 °C, IR KBr
(cmꢀ1): 3547, 3245, 1742, 1634, 1615; 1H NMR (DMSO-d6) d
ppm: 12.16 (s, 1H, NH), 11.05 (s, 1H, Het-NH), 9.07 (s, 2H, 2OH),
8.53 (s, 1H, 1OH), 8.05–6.56 (m, 6H, ArAH), 7.81 (s, 1H, CH),
2.67–2.62 (q, J = 6.8 Hz, 2H, CH2), 2.24–2.22 (q, J = 6.8 Hz, 2H,
CH2), 2.02–2.01 (t, J = 6.8 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.84, 162.27, 157.50, 147.13, 146.58, 144.08, 136.00,
134.71, 127.27, 126.42, 126.13, 125.09, 121.25, 106.65, 34.37,
31.71, 22.03; HRMS m/z: 382.6310.
4.2.24. N0-(3,4-dihydroxy-5-methoxybenzylidene)-3-(4-oxo-3,4-
dihydroquinazolin-2-yl)propanehydrazide (19)
Yield 89.60%, Raf = 0.51, Rfb = 0.57, m.p. 238–240 °C, IR KBr
(cmꢀ1): 3542, 3246, 1766, 1645, 1626; 1H NMR (DMSO-d6) d
ppm: 12.19 (s, 1H, NH), 11.07 (s, 1H, Het-NH), 8.94 (s, 2H, 2OH),
8.07–6.72 (m, 6H, ArAH), 7.94 (s, 1H, CH), 3.76 (s, 3H, AOCH3),
3.14–3.11 (t, J = 7.0 Hz, 2H, CH2), 2.93–2.90 (t, J = 7.0 Hz, 2H,
CH2); 13C NMR (DMSO-d6) d ppm: 173.29, 162.11, 157.01, 148.91,
146.44, 146.34, 144.05, 136.87, 134.73, 127.28, 126.39, 126.19,
125.08, 121.37, 108.64, 102.88, 56.34, 30.70, 29.07; HRMS m/z:
382.5941.
4.2.30. N-(4-Fluorobenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-2-
yl)propane hydrazide (25)
Yield 88.0%, Raf = 0.49, Rfb = 0.55, m.p. 205 °C, IR KBr (cmꢀ1):
1619 (AN@CH), 1640 (C@O), 1698 (C@O), 3349 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.77 (2H, t, J = 7.8 Hz, CH2), 3.15
(2H, t, J = 8.2 Hz, CH2), 7.11–8.13 (8H, m, ArAH), 7.85 (1H, s,
AN@CH), 11.32 (1H, s, NH), 12.12 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm = 29.62, 30.21, 115.63, 120.88, 126.36, 126.79,
127.23, 129.36, 130.88, 134.41, 144.14, 148.09, 156.44, 161.90,
165.23, 169.70, HRMS (m/z) [M+1]: 339.3012.
4.2.25. N0-(3,4-dihydroxy-5-methoxybenzylidene)-4-(4-oxo-3,4-
dihydroquinazolin-2-yl)butanehydrazide (20)
Yield 91.47%, Raf = 0.49, Rfb = 0.54, m.p. 212–213 °C, IR KBr
(cmꢀ1): 3533, 3245, 1765, 1633, 1624; 1H NMR (DMSO-d6) d
ppm: 12.16 (s, 1H, NH), 11.15 (s, 1H, Het-NH), 8.90 (s, 2H, 2OH),
8.16–6.85 (m, 6H, ArAH), 7.82 (s, 1H, CH), 3.78 (s, 3H, AOCH3),
3.14–3.12 (t, J = 7.2 Hz 2H, CH2), 2.81–2.78 (q, J = 7.2 Hz, 2H,
CH2), 2.24–2.22 (t, J = 7.2 Hz, 2H, CH2); 13C NMR (DMSO-d6) d
ppm: 173.14, 161.77, 157.01, 148.22, 146.23, 145.16, 144.47,
136.22, 134.17, 128.28, 127.39, 126.29, 125.15, 121.16, 107.98,
103.37, 56.18, 34.89, 31.65, 22.77; HRMS m/z: 396.4527.
4.2.31. N-(4-Fluorobenzylidene)-3-(4-oxo-3,4-dihydroquinazolin-2-
yl)butane hydrazide (26)
Yield 94.0%, Raf = 0.50, Rfb = 0.58, m.p. 202–205 °C, IR KBr (cmꢀ1):
1619 (AN@CH), 1621 (C@O), 1710 (C@O), 3358 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.23 (2H, t, J = 6.2 Hz, CH2), 2.79
(2H, m, CH2), 3.17 (2H, t, J = 7.8 Hz, CH2), 6.89–7.56 (8H, m, ArAH),
7.58 (1H, s, AN@CH), 11.39 (1H, s, NH), 11.90 (1H, s, QZN-NH), 13
NMR (DMSO-d6, 100 MHz) d ppm = 24.5, 28.54, 30.18, 115.54,
120.76, 125.28, 126.76, 127.31, 130.02, 131.82, 134.18, 143.27,
C

K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
7
148.10, 156.24, 161.92, 164.50, 168.66, HRMS (m/z) [M+1]:
353.1200.
(2H, m, J = 7.8 Hz, CH2), 3.19 (2H, t, J = 7.6 Hz, CH2), 6.92–7.95
(7H, m, ArAH), 7.89 (1H, s, AN@CH), 11.18 (1H, s, NH), 12.06
(1H, s, QZN-NH), 13C NMR (DMSO-d6) d ppm: 22.18, 28.23, 29.35,
118.12, 119.89, 120.59, 124.02, 126.88, 126.97, 127.10, 127.18,
134.14, 143.48, 143.87, 148.77, 156.14, 161.74, 165.19, 168.44,
HRMS (m/z) [M+1]: 398.1265.
4.2.32. N0-(2,4-difluorobenzyliden)-3-(4-oxo-3,4-dihydroquinazolin-
2-yl)propane hydrazide (27)
Yield 82.0%, Raf = 0.43, Rfb = 0.46, m.p. 179–178 °C, IR KBr (cmꢀ1):
1620 (AN@CH), 1644 (C@O), 1760 (C@O), 3350 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.85 (2H, t, J = 7.6 Hz, CH2), 3.13
(2H, t, J = 7.6 Hz, CH2), 7.10–8.04 (7H, m, ArAH), 7.80 (1H, s,
AN@CH), 11.08 (1H, s, NH), 12.01 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm: 28.56, 29.17, 111.10, 112.68, 113.26, 120.89,
126.64, 127.19, 127.80, 1328.24, 133.55, 142.10, 146.88, 156.99,
161.41, 162.06, 163.70, 168.22, HRMS (m/z) [M+1]: 357.4567.
4.2.38. N0-(2,3-dibromobenzyliden)-3-(4-oxo-3,4-dihydroquinazolin-
2-yl)propane hydrazide (33)
Yield 85.0%, Raf = 0.47, Rfb = 0.55, m.p. 210–212 °C, IR KBr (cmꢀ1):
1607 (AN@CH), 1638 (C@O), 1756 (C@O), 3322 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.81 (2H, t, J = 7.2 Hz, CH2), 3.16
(2H, t, J = 7.6 Hz, CH2), 6.98–7.99 (7H, m, ArAH), 7.85 (1H, s,
AN@CH), 11.05 (1H, s, NH), 12.13 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm: 27.89, 30.14, 120.48, 125.33, 126.01, 126.19,
126.84, 126.98, 127.77, 128.23, 133.14, 136.21, 137.65, 143.88,
148.56, 156.01, 161.46, 168.88, HRMS (m/z) [M+1]: 479.2654,
481.2364 [M+3].
4.2.33. N0-(2,4-difluorobenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-
2-yl)butanehydrazide (28)
Yield 91.0%, Raf = 0.48, Rfb = 0.54, m.p. 184–185 °C, IR KBr (cmꢀ1):
1617 (AN@CH), 1660 (C@O), 1745 (C@O), 3328 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.16 (2H, t, J = 7.6 Hz, CH2), 2.85
(2H, m, J = 8.6 Hz, CH2), 3.15 (2H, t, J = 7.2 Hz, CH2), 6.90–8.18
(7H, m, ArAH), 7.86 (1H, s, AN@CH), 11.12 (1H, s, NH), 121.10
(1H, s, QZN-NH), 13C NMR (DMSO-d6) d ppm: 22.45, 28.18, 29.39,
111.79, 112.78, 119.26, 120.16, 1264.17, 126.88, 127.10, 132.24,
133.12, 144.14, 147.18, 156.12, 161.01, 162.06, 164.71, 168.12,
HRMS (m/z) [M+1]: 371.1265.
4.2.39. N0-(2,3-dibromobenzyliden)-4-(4-oxo-3,4-dihydroquinazolin-
2-yl)butanehydrazide (34)
Yield 84.0%, Raf = 0.48, Rfb = 0.53, m.p. 178–180 °C, IR KBr (cmꢀ1):
1614 (AN@CH), 1660 (C@O), 1780 (C@O), 3345 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.16 (2H, t, J = 7.0 Hz, CH2), 2.79
(2H, m, J = 8.2 Hz, CH2), 3.15 (2H, t, J = 7.8 Hz, CH2), 6.98–7.89
(7H, m, ArAH), 7.85 (1H, s, AN@CH), 11.14 (1H, s, NH), 12.10
(1H, s, QZN-NH), 13C NMR (DMSO-d6) d ppm: 22.48, 29.21, 30.31,
120.14, 125.14, 126.02, 126.28, 126.77, 126.98, 127.02, 128.18,
134.14, 136.48, 137.46, 143.09, 148.88, 156.14, 161.88, 167.28,
HRMS (m/z) [M+1]: 493.15, 495.3245 [M+3].
4.2.34. N0-(2,4-dichlorobenzyliden)-3-(4-oxo-3,4-dihydroquinazolin-
2-yl)propane hydrazide (29)
Yield 86.0%, Raf = 0.46, Rfb = 0.52, m.p. 212–214 °C, IR KBr (cmꢀ1):
1609 (AN@CH), 1648 (C@O), 1768 (C@O), 3314 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.80 (2H, t, J = 7.6 Hz, CH2), 3.17
(2H, t, J = 7.6 Hz, CH2), 6.90–7.86 (7H, m, ArAH), 7.90 (1H, s,
AN@CH), 11.16 (1H, s, NH), 12.28 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm: 27.14, 29.56, 111.10, 112.68, 113.26, 120.89,
126.64, 127.19, 127.80, 132.24, 133.55, 142.10, 146.88, 156.99,
161.41, 162.06, 163.70, 168.22, HRMS (m/z) [M+1]: 389.4567,
391.2364 [M+3].
4.2.40. N0-(2-Chloro-6-fluorobenzylidene)-3-(4-oxo-3,4-
dihydroquinazolin-2-yl)propane hydrazide (35)
Yield 95.0%, Raf = 0.52, Rfb = 0.59, m.p. 215–216 °C, IR KBr (cmꢀ1):
1615 (AN@CH), 1638 (C@O), 1730 (C@O), 3314 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.90 (2H, t, J = 8.4 Hz, CH2), 3.13
(2H, t, J = 10.5 Hz, CH2), 6.92–7.56 (7H, m, ArAH), 8.05 (1H, s,
AN@CH), 11.46 (1H, s, NH), 12.10 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm: 29.14, 30.24, 113.57, 118.60, 120.81, 125.78,
126.36, 126.78, 127.34, 133.46, 134.40, 135.35, 143.53, 146.94,
156.44, 161.03, 167.87. HRMS (m/z) [M+1]: 373.1120, 375.1236
[M+3].
4.2.35. N0-(2,4-dichlorobenzylidene)-4-(4-oxo-3,4-dihydroquinazolin-
2-yl)butanehydrazide (30)
Yield 87.0%, Raf = 0.42, Rfb = 0.49, m.p. 190–192 °C, IR KBr (cmꢀ1):
1616 (AN@CH), 1650 (C@O), 1760 (C@O), 3320 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.20 (2H, t, J = 6.4 Hz, CH2), 2.76
(2H, m, J = 7.6 Hz, CH2), 3.16 (2H, t, J = 7.6 Hz, CH2), 6.92–7.95
(7H, m, ArAH), 7.99 (1H, s, AN@CH), 11.10 (1H, s, NH), 12.20
(1H, s, QZN-NH), 13C NMR (DMSO-d6) d ppm: 23.48, 28.04, 29.13,
112.12, 112.89, 113.16, 120.02, 126.45, 126.99, 127.10, 132.18,
133.14, 143.10, 146.77, 156.02, 161.40, 163.06, 163.89, 168.01,
HRMS (m/z) [M+1]: 403.1654, 405.2645 [M+3].
4.2.41. N0-(2-Chloro-6-fluorobenzylidene)-3-(4-oxo-3,4-
dihydroquinazolin-2-yl)butane hydrazide (36)
Yield 94.0%, Raf = 0.54, Rfb = 0.61, m.p. 216–218 °C, IR KBr (cmꢀ1):
1610 (AN@CH), 1630 (C@O), 1734 (C@O), 3320 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.01 (2H, t, J = 8.6 Hz, CH2), 2.29
(2H, m, J = 14.4 Hz, CH2), 2.63 (2H, m, CH2), 7.17–8.34 (7H, m,
ArAH), 8.02 (1H, s, AN@CH), 11.42 (1H, s, NH), 12.12 (1H, s,
QZN-NH), 13C NMR (DMSO-d6, 100 MHz) d ppm: 21.98, 31.51,
33.69, 115.75, 118.61, 120.80, 121.29, 126.07, 126.66, 127.24,
133.66, 134.65, 135.75, 143.83, 149.31, 157.32, 161.99, 168.70,
HRMS (m/z) [M+1]: 387.2264, 389.2274 [M+3].
4.2.36. N0-(2-fluoro-5-nitrobenzyliden)-3-(4-oxo-3,4-
dihydroquinazolin-2-yl)propane hydrazide (31)
Yield 89.0%, Raf = 0.40, Rfb = 0.48, m.p. 193–195 °C, IR KBr (cmꢀ1):
1620 (AN@CH), 1660 (C@O), 1770 (C@O), 3329 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.88 (2H, t, J = 6.8 Hz, CH2), 3.20
(2H, t, J = 7.8 Hz, CH2), 7.10–7.98 (7H, m, ArAH), 7.83 (1H, s,
AN@CH), 11.26 (1H, s, NH), 12.08 (1H, s, QZN-NH), 13C NMR
(DMSO-d6) d ppm: 27.88, 30.54, 118.10, 119.68, 120.48, 124.33,
126.01, 126.19, 127.77, 127.23, 133.14, 142.14, 143.88, 148.56,
156.01, 161.46, 163.06, 168.88, HRMS (m/z) [M+1]: 384.1256.
4.3. Biological activity
4.3.1. Gastric H+/K+-ATPase activity
4.3.1.1. Isolation of parietal cells from sheep stomach. The fundic sto-
mach portion of sheep soon after sacrificing was collected and was
rinsed with Krebs ringer buffer (250 mM sucrose, 2 mM MgCl2,
1 mM EGTA and 2 mM Hepes-Tris of pH 7.4). The upper layer
was pinned with the help of needles on the dissection table. Muco-
sal scrapings were suspended in 10 volumes of Krebs ringer buffer
(pH = 7.4) containing 250 mM sucrose and homogenized with 20 S
of a mortar driven Teflon pestle homogenizer. The tissues were
4.2.37. N0-(2-fluoro-5-nitrobenzyliden)-4-(4-oxo-3,4-
dihydroquinazolin-2-yl)butanehydrazide (32)
Yield 84.0%, Raf = 0.39, Rfb = 0.46, m.p. 212–214 °C, IR KBr (cmꢀ1):
1626 (AN@CH), 1668 (C@O), 1774 (C@O), 3317 (NH); 1H NMR
(DMSO-d6, 400 MHz) d ppm: 2.17 (2H, t, J = 6.8 Hz, CH2), 2.82

8
K.P. Rakesh et al. / Bioorganic Chemistry 68 (2016) 1–8
discarded and the filtrate was subjected to sub cellular fractiona-
tion. The pellets so obtained were dissolved in 2 mL of sucrose-
EGTA buffer and was used as enzyme sample.
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working standard solution (40
and dry test tubes in the volume of 0, 0.2, 0.4, 0.6, 0.8 and
1.0 mL; 5 L and 10 L of the enzyme sample were taken in test
lg/mL) were added into 8 clean
l
l
tube 7th and 8th respectively. The volume in the test tube was
made up to 8.6 mL using 10% TCA. Ammonium molybdate (1 mL)
and ANSA reagent (0.4 mL) were added to each test tube. The tubes
were allowed to stand for 8 min at room temperature. The color
developed was read at its kmax of 660 nm.
5. ATPase activity
ATPase activity was determined as described by Blakeman and
McGrath [42]. Basal Mg2+ dependent ATPase activity was measured
in 1.0 mL of the reaction medium consisting of 2 mmol/L ATP and
50 mmol/L Tris-HCl buffer (pH = 7.5). K+-stimulated and HCOꢀ3
stimulated ATPase activity was defined as the activity in the basal
medium. The ATPase reaction was started by the addition of the
substrate (ATP), carried out at 37 °C for 15 min and stopped with
1 mL ice cold 20% TCA. Liberated inorganic phosphate from ATP
was estimated by Fiske Subbarow’s method [41].
Activity of the enzyme sample in the presence of ATP is
0.066
Activity of the enzyme sample in the absence of ATP is
0.042 moles.
100% activity of the enzyme is 0.066–0.042
0.024 moles.
lmoles.
l
lmoles =
l
[39] J. Fournier, C. Bruneau, H. Dixneuf, S. Lécolier, J. Org. Chem. 56 (1991) 4456–
4458.
Acknowledgments
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265–275.
[41] C.H. Fiske, Y. Subbarow, J. Biol. Chem. 66 (1925) 375–400.
[42] W.B. Im, J.C. Sih, D.P. Blakeman, J.P. McGrath, J. Biol. Chem. 260 (1985) 4591–
4597.
The authors gratefully acknowledge Center with Potential for
Excellence in a Particular Area (CPEPA), UGC, New Delhi for the
financial assistance.