Novel quinazolin-4(3H)-one/Schiff base hybrids as antiproliferative and phosphodiesterase 4 inhibitors: Design, synthesis, and docking studies

Article abstract of DOI:10.1002/ardp.201400083

A novel series of quinazolin-4(3H)-one/Schiff base hybrids was rationally designed and synthesized. The prepared compounds were evaluated for in vitro activity to inhibit phosphodiesterase 4 (PDE4), where several of them showed good-to-moderate activity c

Full text of DOI:10.1002/ardp.201400083

650
Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Full Paper
Novel Quinazolin-4(3H)-one/Schiff Base Hybrids as
Antiproliferative and Phosphodiesterase 4 Inhibitors:
Design, Synthesis, and Docking Studies
Hamdy M. Abdel-Rahman1, Mohamed Abdel-Aziz2, Joshua C. Canzoneri3, Bernard D. Gary3,
and Gary A. Piazza3
1
Faculty of Pharmacy, Medicinal Chemistry Department, Assiut University, Assiut, Egypt
Faculty of Pharmacy, Medicinal Chemistry Department, Minia University, Minia, Egypt
USA Mitchell Cancer Institute, University of South Alabama, Mobile, AL, USA
2
3
A novel series of quinazolin-4(3H)-one/Schiff base hybrids was rationally designed and synthesized. The
prepared compounds were evaluated for in vitro activity to inhibit phosphodiesterase 4 (PDE4), where
several of them showed good-to-moderate activity compared to rolipram. Compound 7 showed potent
PDE4 inhibition in this series, with an IC50 of 1.60 mM. Compounds that showed PDE4 inhibition were
further assessed for antiproliferative activity using different human tumor cell lines. Compound 10
exhibited significant antiproliferative activity with IC50 values of 140, 79, and 320 nM in breast, lung,
and colon tumor cells, respectively. Docking of compound 7 in the active site of PDE4B illustrates its
possible binding mode and provides insight for further optimizations of this novel scaffold for
inhibiting PDE4.
Keywords: Antiproliferative / Docking / Hybrids / PDE4B inhibitors / Quinazolin-4(3H)-one
Received: March 3, 2014; Revised: April 25, 2014; Accepted: May 5, 2014
DOI 10.1002/ardp.201400083
Introduction
compounds while diminishing the negative side effects. PDE4
has also been shown to play a role in the proliferation,
angiogenesis, and motility of multiple cancer types [6–8].
Furthermore, inhibition of PDE4 has been proven to have
chemotherapeutic potential against different forms of cancer,
including oral [9], brain [10], and pancreatic [11] cancer types.
Thus, the discovery of novel selective PDE4B inhibitors
possessing antineoplastic activity is of great interest [12, 13].
Theophylline (Fig. 1), a non-selective PDE inhibitor, while
used for treatment of asthma and COPD, suffers from a
narrow therapeutic index [14]. Many synthetic analogs of
theophylline, mostly with bicyclic heterocyle scaffolds, have
been synthesized and tested as selective PDE4 inhibitors [3].
Additionally, quinazolines [15], quinazolindiones (as nitra-
quazone, Fig. 1) [16, 17], pyrazolopyridines [18], and the 1,7-
naphthyridines (Fig. 1) [19] are of particular interest in this
field. Furthermore, N-acylhydrazones are also potent PDE
inhibitors [20, 21]; substitution of the N-acylhydrazone
scaffold to quinazoline scaffold proved to be an effective tool
for lead optimization strategy as shown in Fig. 2 [21, 22].
Based on the above-mentioned studies, the present work
aims to increase the rigidity of the N-acylhydrazones scaffold
The phosphodiesterase (PDE) enzyme superfamily, consisting
of 11 unique subfamilies, is responsible for the degradation
of the second messengers, cyclic adenosine monophosphate
(cAMP) and cyclic guanosine monophosphate (cGMP).
PDEs are attractive drug targets owing to their significant
therapeutic potential [1, 2]. Specifically, PDE4, a cAMP specific
PDE isozyme family, has become a relevant drug target for the
treatment of respiratory inflammatory diseases such as
asthma and chronic obstructive pulmonary disease (COPD) [3].
Roflumilast (Fig. 1) was the first PDE4 inhibitor granted FDA
approval for treating COPD [4, 5]. However, dose-limiting side
effects such as nausea and emesis have limited the clinical
development of rolipram and other PDE4 inhibitors [3]. Of the
four PDE4 isoforms, PDE4A, B, C, and D, selective inhibition of
the PDE4B maintains the anti-inflammatory effect of these
Correspondence: Mohamed Abdel-Aziz, Faculty of Pharmacy, Medicinal
Chemistry Department, Minia University, Minia 61519, Egypt.
E-mail: abulnil@hotmail.com
Fax: þ20862369075
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Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Quinazolin-4(3H)-one/Schiff Base Hybrids as PDE4 Inhibitors
651
N
O
NH
Cl
Cl
HN
O
O
H
N
N
N
N
O
O
O
OCH3
OCHF2
Roflumilast
Theophylline
Rolipram
COOH
COOH
O
N
N
N
N
O
N
N
NO2
N
O
X
N
Nitraquazone
1,7-Naphthyridine compounds
Figure 1. Chemical structures of selected
PDE4 inhibitors.
Figure 2. Design of quinazolin-4(3H)-one/Schiff base hybrids as PDE4 inhibitors.
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H. M. Abdel-Rahman et al.
Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
by molecular hybridization with the quinazoline scaffold to
produce selective PDE4 inhibitors (Fig. 2). Toward this goal, we
describe here the rational design, synthesis, and characteri-
zation of a novel class of substituted quinazolin-4(3H)-one/
Schiff base hybrid PDE4 inhibitors. The prepared compounds
were evaluated for PDE4 inhibition activity. The prepared
compounds were also evaluated for their antiproliferative
activity against human MDA-MB-231 breast cancer, A549
lung cancer, and HT29 colon cancer cell lines. Additionally,
molecular modeling studies were also carried out to identify
their possible binding mode within the catalytic domain of
PDE4B.
aromatic aldehydes to afford the corresponding arylmethyl-
idine-hydrazides (Schiff bases) of substituted 3H-quinazoline-
4-ones 7–20, respectively (Scheme 1). The structures of the
prepared compounds were characterized by IR, 1H NMR,
13C NMR, and high resolution mass spectra.
PDE inhibition activity
The synthesized compounds 7–20 were evaluated for their in
vitro inhibition of recombinant human PDE4B. Rolipram
(Fig. 1) was used as a positive reference for PDE4 inhibition.
Initial screening of the compounds was done at 10 mM in
triplicate (Fig. 3). Precise IC50 values were determined for
rolipram and all lead compounds (PDE4 inhibition >25% at
10 mM) (Table 1). Compound 7 showed good PDE4B inhibitory
activity of 68.8% at 10 mM, while compounds 9 and 10
exhibited moderate PDE4B inhibitory activity of 26.5 and
27.1%, respectively, at 10 mM.
These results in Table 1 and Fig. 3 revealed that the presence
of the hydroxyl-substituted phenyl of Schiff base part is crucial
for inhibitory activity. Thus, the trihydroxyphenyl Schiff base 7
showed the highest PDE4B inhibitory activity in this series
with an IC50 of 1.60 mM, while Schiff base 10 showed moderate
inhibitory activity with IC50 of 29.3 mM. The methoxyphenyl-
containing Schiff bases 12 and 19 were the least active in this
series, suggesting the importance of the free OH groups on the
Schiff base aryl ring. Furthermore, the 5-methyl substitution
(compounds 15–20) did not improve the inhibitory activity of
the synthesized compounds.
Results and discussion
Chemistry
The reaction sequence employed for synthesis of the 3H-
quinazolin-4-ones 7–20 is outlined in Scheme 1. Reaction of
anthranilic acid or 6-methyl anthranilic acid with benzoyl
chloride yielded the corresponding 2-phenyl-3H-benzoxazin-4-
one 3 and 5-methyl-2-phenyl-3H-benzoxazin-4-one 4 by N-
acylation via dehydrative cyclization mechanism [23]. Subse-
quently, heating at reflux of compounds 3 or 4 with hydrazine
monohydrate in alcoholic medium afforded the correspond-
ing 3-amino-3H-quinazolin-4-ones 5 and 6, respectively, in a
good yield [24]. A series of 3-(benzylideneamino)-2-phenyl-3H-
quinazolin-4-ones were synthesized by condensation of 3-
amino-3H-quinazolin-4-ones 5 or 6 with different substituted
R4
O
R3
R2
R5
R1
R1
R1
O
N
R1
O
N
COOH
NH2
N
NH2
c
a
b
N
O
N
N
3, 4
1, 2
5, 6
7−20
7: R1 = R5 = H. R2 = R3 = R4 = OH.
8: R1 = R4 = R5 = H, R2 = R3 = OH.
9: R1 = R2 = R5 = H, R3 = R4 = OH.
1, 3, 5: R1 = H
2, 4, 6: R1 = CH3
10: R1 = R3 = R5 = H, R2 = R4 = OH.
11: R1 = R2 = R4 = R5 = H, R3 = OH.
12: R1 = R2 = H. R3 = R4 = R5 = OCH3.
13: R1 = R2 = R5 = H. R3 = R4 = OCH3.
14: R1 = R2 = R3 = R5 = H. R4 = OCH3.
15: R1 = CH3. R2 = R3= R4 = OH. R5 = H.
16: R1 = CH3. R2 = R4 = OH. R3 = R5 = H.
17: R1 = CH3. R3 = R4 = OH. R2 = R5 = H.
18: R1 = CH3. R2 = R3 = OH. R4 = R5 = H.
19: R1 = CH3. R3 = R4 = R5 = OCH3. R2 = H.
20: R1 = CH3. R3 = R4 = OCH3. R2 = R5 = H.
Scheme 1. Synthesis of the quinazolin-4(3H)-one/Schiff base hybrids. Reagents and conditions: (a) C6H5COCl, pyridine, heat; (b) H2NNH2,
C2H5OH, reflux; and (c) substituted benzaldehyde, C2H5OH, reflux.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Quinazolin-4(3H)-one/Schiff Base Hybrids as PDE4 Inhibitors
653
100
Table 2. Lead compound growth inhibition IC50 values.
Growth inhibition IC50 (mM)
75
50
25
0
MDA-MB-231
breast cancer
cell line
A549
lung cancer
cell line
HT29 colon
cancer
cell line
Compound
7
9
10
1.6
3.6
17
0.08
3.13
0.40
1.6
0.32
2.75
0.39
0.14
0.11
Doxorubicin
sensitivity of each cell line to a given compound, or the
involvement of a growth inhibitory pathway additional to
that of PDE4 inhibition.
Figure 3. Initial screen for PDE4B inhibition at 10 mM drug
concentration. Rolipram tested at 150 nM.
Molecular modeling study
Molecular docking studies of compounds 7 and 12 were
performed in order to rationalize the obtained biological
results and understand its likely interactions with PDE4B
enzyme active site. Docking studies were performed by
Molecular Operating Environment (MOE) [25] using PDE4B
co-crystallized with a thiopyrano[3,2-d]pyrimidine ligand
(PDB ID: 3W5E) [26] as a template. MOE alpha site finder
was used for the active site search in the enzyme structure
using all default items. The site of the enzyme containing the
Gln 443 was selected as docking site according to reported
data [17, 26].
About 100 dockings were performed for the interaction of
the ligand with the enzyme active site and the top-scoring
configuration of the ligand–enzyme complexes was selected
on energetic grounds. The molecular modeling of compound
7 showed a group of H-bond interactions between the ligand
and PDE4B (Fig. 4). The N-1 atom of the quinazolin-4(3H)-one
part formed a strong hydrogen bond interaction with the NH
group of Gln 443 (distance ¼ 2.71 Å). Additionally, the three
OH groups of Schiff base part formed a strong hydrogen bond
interaction with the His 324 (distance ¼ 2.57 Å), and weak
hydrogen bond interaction with His 238 (distance ¼ 3.42 Å)
and Asp 392 (distance ¼ 3.26 Å). These observations were in
agreement with the reported binding of the thiopyrano[3,2-d]
pyrimidine ligand to PDE4B (PDB ID: 3W5E) that showed a
hydrogen bonding network to enzyme active site via water
molecules [26]. Therefore, the free OH groups could be
considered in lieu of water molecules forming a network of
hydrogen bonds between compound 7 and the enzyme.
Furthermore, compound 7 is oriented within the PDE4B
hydrophobic region comprising Met 347, Leu 393, Trp 406,
Phe 414, Phe 446, and Phe 506.
Cell growth inhibition
Lead compounds 7, 9, and 10 were further assayed for their
possible antineoplastic potential. Compounds 7, 9, and 10
were tested against human MDA-MB-231 breast cancer, A549
lung cancer, and HT29 colon cancer cell lines (Table 2). The
results in Table 2 revealed that the lead compounds 7, 9, and
10 inhibited cancer cell growth, with compound 10 being the
most potent yielding an IC50 of 140, 80, and 320 nM in the
breast, lung, and colon lines, respectively. Compound 7
inhibits the growth of MDA-MB-231 breast cancer cells at the
same IC50, 1.6 mM, as is observed for its PDE4B inhibition. This
trend does not hold true for the other compounds and cell
lines tested. The lack of correlation between the PDE4
inhibition and growth inhibitory potential may indicate a
lack of cell permeability of the tested compound, varying
Table 1. PDE4B inhibitory activity (at 10 mM) and IC50 values for
compounds 7–20 compared to rolipram at 150 nM.
PDE4B2 activity
Compound
% Inhibition at 10 mM
IC50 (mM)
7
8
9
10
11
12
13
14
16
17
19
20
68.8
16.8
26.5
27.1
8.00
0.22
6.82
4.48
5.21
8.00
1.40
17.7
50.1
1.60
NT
>50
29.3
NT
NT
NT
NT
NT
NT
NT
NT
0.145
To give structural insights to highlight important factors
for inhibitor binding, the weak inhibitor 12 was docked in
the active site of PDE4B enzyme where it has weak docking
Rolipram at 150 nM
NT, not tested.
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Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Figure 4. Docking pose of compound 7 with PDE4B.
affinity score ¼ ꢀ6.6447 kcal/mol compared to ꢀ9.3276 kcal/
mol for compound 7.
phenyl Schiff base 7 was the most potent inhibitor of PDE4B
in this series with an IC50 of 1.6 mM. Molecular docking
analysis of compound 7 revealed that this compound bound
to the PDE4B active site and provided insight into its high
PDE inhibitory activity. Compound 10 inhibited cancer cell
growth with an IC50 of 140, 80, and 320 nM in the breast, lung,
and colon lines, respectively. Compound 7 also potently
inhibited the growth of breast, lung, and colon cancer cells.
Comparatively, compound 9 moderately inhibited the growth
of breast cancer cells with an IC50 of 390 nM. The mechanism
underlying this unexpectedly potent growth inhibition will
be a priority of future work.
Although the presence of free OH groups on the Schiff base
part is important for enzyme binding, their position and
orientation is also very important. In addition, free OH groups
are not the only site responsible for enzyme binding. For this
reason, compounds lacking the free OH groups may have
weak inhibition because they bind to the enzyme active site.
The above observations show that compound 7 is tightly
bound to the PDE4B active site.
Conclusion
A novel class of 3H-quinazolin-4-one/Schiff base hybrids
scaffold were designed, synthesized, and characterized by
different spectroscopic techniques. The prepared compounds
retain good-to-moderate PDE4B inhibitory activity and further
possess substantial antiproliferative activity. The trihydroxy-
Experimental
Chemistry
Melting points were determined on Stuart electrothermal
melting point apparatus and were uncorrected. IR spectra were
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Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Quinazolin-4(3H)-one/Schiff Base Hybrids as PDE4 Inhibitors
655
recorded on Nicolet iS5 FT-IR spectrometer at Minia University.
NMR spectra were carried out using a Bruker Advance 300 MHz
NMR spectrometer, using TMS as internal reference. Chemical
shifts (d) values are given in parts per million (ppm) relative to
CDCl3 (7.29 for proton and 76.9 for carbon), CD3OD (3.31 and 4.87
for protons and 49.00 for carbon) or DMSO-d6 (2.50 for proton and
39.50 for carbon), and coupling constants (J) in Hertz. Splitting
patterns are designated as follows: s, singlet; d, doublet; t, triplet;
q, quartet; dd, doublet of doublet; m, multiplet. High-resolution
mass spectra (HRMS) were obtained on Bruker BioTOF III (ESI-TOF)
and were reported as mass/charge (m/z) with percent relative
abundance in the Genomic Research Center, Academia Sinica,
Taiwan. The progress of reactions and the purity of the prepared
compounds were monitored by thin-layer chromatography (TLC)
using Merck 9385 pre-coated aluminum plate silica gel (Kieselgel
60) 5 cm ꢁ 20 cm plates with a layer thickness of 0.2 mm. The
spots were detected by exposure to UV-lamp at l ¼ 254 nm.
127.14, 128.62, 129.07, 132.27, 132.82, 134.53, 139.42, 139.65,
147.71, 148.60, 151.13, 164.62; HRMS (ESI-TOF) calcd. for
C
21H15N3O4 [MþH]þ 374.1135, found 374.1139.
3-[(2,3-Dihydroxybenzylidene)amino]-2-phenyl-3H-quin-
azolin-4-one 8: Grayish white powder (77% yield); m.p. 227°C;
FT-IR (cmꢀ1): 3215 (OH), 1648 (C O), 1637 (C C), 1602, 1588
–
–
–
–
–
(C N); 1H NMR (300 MHz, DMSO-d6, d ¼ ppm) d ¼ 6.75 (t, 1H,
–
J ¼ 6.00 Hz, Ar–H), 6.88 (d, 1H, J ¼ 6.00 Hz, Ar–H), 7.01 (d, 1H,
J ¼ 6.00 Hz, Ar–H), 7.28 (t, 1H, J ¼ 6.00 Hz, Ar–H), 7.56–7.65 (m, 4H,
Ar–H), 7.92–7.97 (m, 3H, Ar–H), 8.57 (d, 1H, J ¼ 6.30 Hz, Ar–H), 8.72
(s, 1H, Ar–H), 9.34 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6,
d ¼ ppm) d ¼ 117.63, 118.89, 119.31, 119.89, 120.15, 121.23,
123.26, 127.13, 128.69, 129.00, 132.20, 132.85, 134.49, 139.46,
139.55, 145.72, 146.27, 149.78, 164.68; HRMS (ESI-TOF) calcd. for
C21H15N3O3 [MþH]þ 358.1186, found 358.1192.
3-[(3,4-Dihydroxybenzylidene)amino]-2-phenyl-3H-quin-
azolin-4-one 9: Yellowish white powder (76% yield); m.p. 228–
General procedure for the synthesis of 2-phenyl-3H-
benzoxazin-4-ones 3 and 4
229°C; FT-IR (cmꢀ1): 3222 (OH), 1647 (C O), 1636 (C C), 1600,
–
–
–
–
(Un)Substituted anthranilic acid (0.1 M) was dissolved in 60 mL of
pyridine by adding benzoyl chloride (0.2 M) in a dropwise manner
with constant stirring at 0–5°C over the period of 1 h. After
completion of the addition, the reaction mixture was stirred for
half an hour at room temperature and treated with 10% NaHCO3
(15 mL). At the end of the reaction, a solid mass of the 2-phenyl-3H-
benzoxazin-4-one was obtained. It was filtered, washed succes-
sively with 10% NaHCO3, dried, and recrystallized from ethanol.
1
–
1587 (C N); H NMR (300 MHz, DMSO-d , d ¼ ppm) d ¼ 6.80 (d, 1H,
–
6
J ¼ 6.30 Hz, Ar–H), 6.95–6.97 (m, 1H, Ar–H), 7.25–7.28 (m, 2H,
Ar–H), 7.57–6.63 (m, 4H, Ar–H), 7.89 (d, 1H, J ¼ 5.70 Hz, Ar–H), 7.96
(d, 2H, J ¼ 5.10 Hz, Ar–H), 8.28 (s, 1H, Ar–H), 8.60 (d, 1H,
J ¼ 6.00 Hz, Ar–H), 9.40 (s, 2H, OH). 13C NMR (75 MHz, DMSO-d6,
d ¼ ppm) d ¼ 112.84, 115.66, 120.36, 120.87, 121.03, 123.12,
125.48, 127.07, 128.55, 129.01, 132.19, 132.55, 132.65, 134.44,
139.42, 145.84, 148.37, 149.76, 164.73; HRMS (ESI-TOF) calcd. for
C21H15N3O3 [MþH]þ 358.1186, found 358.1192.
General procedure for the synthesis of 3-amino-3H-
quinazolin-4-ones 5 and 6
3-[(2,4-Dihydroxybenzylidene)amino]-2-phenyl-3H-quin-
Equimolar quantities of (un)substituted-2-phenyl-3H-benzoxazin-
4-one 3 or 4 and hydrazine monohydrate in ethanol (50 mL)
were heated under reflux for 6 h. The reaction mixture was
concentrated and allowed to cool. The solid product obtained was
filtered off, washed with distilled water, and dried. Recrystalliza-
tion from ethanol afforded the desired solid compounds 5
(m.p. 181–182°C) and 6 (m.p. 212–214°C) in 82 and 84% yield,
respectively.
azolin-4-one 10: Yellowish white powder (76% yield); m.p. 245–
246°C; FT-IR (cmꢀ1): 3205 (OH), 1648 (C O), 1631 (C C), 1603,
–
–
–
–
1
–
1586 (C N); H NMR (300 MHz, DMSO-d , d ¼ ppm) d ¼ 6.33–6.38
–
6
(m, 2H, Ar–H), 7.27 (t, 1H, J ¼ 5.70 Hz, Ar–H), 7.36 (d, 1H,
J ¼ 6.00 Hz, Ar–H), 7.57–7.65 (m, 4H, Ar–H), 7.89 (d, 1H, J ¼ 6.00 Hz,
Ar–H), 7.91–7.96 (m, 2H, Ar–H), 8.54 (s, 1H, Ar–H), 8.55–8.58 (m,
1H, Ar–H), 10.03 (s, 1H, OH). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm)
d ¼ 102.44, 102.66, 107.90, 110.55, 120.17, 121.08, 123.17, 127.07,
128.56, 128.99, 131.15, 132.18, 132.65, 134.46, 139.36, 149.89,
159.53, 161.07, 164.68; HRMS (ESI-TOF) calcd. for C21H15N3O3
[MþH]þ 358.1186, found 358.1191.
General procedure for the synthesis of 3-substituted-
benzylideneamino)-5-(un)substituted-2-phenyl-3H-
quinazoline-4-ones 7–20
3-[(3-Hydroxybenzylidene)amino]-2-phenyl-3H-quinazo-
A mixture of 3-amino-2-phenyl-3H-quinazoline-4-one 5 or 6 and
different aldehydes in equimolar quantities in ethanol in
presence of few drops of glacial acetic acid was heated under
reflux for 8 h. The solution was cooled and the precipitated solid
was filtered off, washed with distilled water dried in vacuum, and
recrystallized from ethanol.
lin-4-one 11 [24]: White powder in (72% yield); m.p. 237°C; FT-
IR (cmꢀ1): 3240 (OH), 1647 (C O), 1601, 1586 (C N); 1H NMR
–
–
–
–
(300 MHz, DMSO-d6, d ¼ ppm) d ¼ 8.26–6.85 (m, 13H, ArH), 9.20 (s,
1H, CH N), 10.26 (s, 1H, OH); 13C NMR (75 MHz, DMSO-d6,
–
–
d ¼ ppm) d ¼ 115.80, 119.00, 120.20, 123.00, 124.00, 126.10,
126.70, 126.90, 128.00, 128.50, 130.20, 130.90, 133.20, 146.20,
154.20, 158.00, 158.10, 161.20, 168.20; HRMS (ESI-TOF) calcd. for
C21H15N3O2 [MþH]þ 342.1237, found 342.1243.
3-[(2,3,4-Trihydroxybenzylidene)amino]-2-phenyl-3H-quin-
azolin-4-one 7: White powder in (78% yield); m.p. 213–214°C;
FT-IR (cmꢀ1): 3481 (OH), 1646 (C O), 1636 (C C), 1601, 1587
–
–
–
–
(C N); 1H NMR (300 MHz, DMSO-d6, d ¼ ppm) d ¼ 6.41 (d, 1H,
3-[(3,4,5-Trimethoxybenzylidene)amino]-2-phenyl-3H-
–
–
quinazolin-4-one 12: Yellowish white powder (77% yield); m.p.
J ¼ 6.60 Hz, Ar–H), 6.82 (d, 1H, J ¼ 6.60 Hz, Ar–H), 7.27 (t, 1H,
J ¼ 5.40 Hz, Ar–H), 7.56–7.64 (m, 4H, Ar–H), 7.90 (d, 1H, J ¼ 5.70 Hz,
Ar–H), 7.94–7.96 (m, 2H, Ar–H), 8.49 (s, 1H, Ar–H), 8.59 (d, 1H,
J ¼ 5.70 Hz, Ar–H). 13C NMR (75 MHz, DMSO-d6, d ¼ ppm) d
¼ 107.83, 110.92, 120.60, 121.04, 121.20, 123.25, 127.00,
1
217°C; FT-IR (cmꢀ1): 1648 (C O), 1602 (C N); H NMR (300 MHz,
–
–
–
–
DMSO-d6, d ¼ ppm) d ¼ 3.70 (s, 3H, OCH3), 3.83 (s, 6H, 2 OCH3), 7.03
(s, 2H, Ar–H), 7.27 (t, 1H, J ¼ 5.70 Hz, Ar–H), 7.55–7.64 (m, 4H,
Ar–H), 7.88 (d, 1H, J ¼ 5.10 Hz, Ar–H), 7.95 (d, 2H, J ¼ 5.10 Hz,
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Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Ar–H), 8.38 (s, 1H, Ar–H), 8.52 (d, 1H, J ¼ 6.30 Hz, Ar–H). 13C NMR
(75 MHz, DMSO-d6, d ¼ ppm) d ¼ 56.00, 60.16, 104.51, 120.93,
121.27, 123.26, 127.12, 128.72, 128.96, 129.54, 132.16, 132.58,
134.45, 139.21, 139.46, 149.15, 153.26, 164.65, 164.94; HRMS (ESI-
TOF) calcd. for C24H21N3O4 [MþH]þ 416.1605, found 416.1610.
3-[(2,3-Dihydroxybenzylidene)amino]-5-methyl-2-phenyl-
3H-quinazolin-4-one 18: White powder (77% yield); m.p. 191–
1
192°C; FT-IR (cmꢀ1): 3222 (OH), 1652 (C O), 1603 (C N); H NMR
–
–
–
–
(300 MHz, CD3OD, d ¼ ppm) d ¼ 2.44 (s, 3H, CH3), 6.68–6.78 (m, 1H,
Ar–H), 6.82–6.87 (m, 2H, Ar–H), 7.20 (d, 1H, J ¼ 5.40 Hz, Ar–H),
7.38–7.45 (m, 3H, Ar–H), 7.48–7.53 (m, 1H, Ar–H), 7.58 (d, 1H,
J ¼ 6.00 Hz, Ar–H), 7.78–7.85 (m, 2H, Ar–H), 8.28 (s, 1H, N CH). 13C
–
–
NMR (75 MHz, CD3OD, d ¼ ppm) d ¼ 19.96, 118.88, 119.36, 120.59,
122.49, 124.56, 128.53, 128.58, 129.12, 129.33, 129.73, 131.48,
133.17, 135.45, 137.80, 146.74, 147.43, 149.34, 152.28, 166.42;
HRMS (ESI-TOF) calcd. for C22H17N3O3 [MþH]þ 372.1343, found
372.1335.
3-[(3,4-Dimethoxybenzylidene)amino]-2-phenyl-3H-quin-
azolin-4-one 13 [27]: White powder (78% yield); m.p. 216°C;
FT-IR (cmꢀ1): 1656 (C O), 1600, 1588 (C N); 1H NMR (300 MHz,
–
–
–
–
DMSO-d6, d ¼ ppm) d ¼ 3.85 (s, 6H, 2OCH3), 7.33–8.28 (m, 12H,
–
Ar–H), 9.40 (s, 1H, N CH); HRMS (ESI-TOF) calcd. for C23H19N3O3
–
[MþH]þ 386.1499, found 386.1505.
3-[(3,4,5-Trimethoxybenzylidene)amino]-5-methyl-2-phen-
3-[(4-Methoxybenzylidene)amino]-2-phenyl-3H-quinazo-
yl-3H-quinazolin-4-one 19: White powder (78% yield); m.p.
lin-4-one 14 [28]: White powder (78% yield); m.p. 240°C; FT-IR
238–239°C; FT-IR (cmꢀ1): 1648 (C O), 1632 (C C), 1605, 1574
–
–
–
(cmꢀ1): 1647 (C O), 1599 (C N); 1H NMR (300 MHz, DMSO-d6,
–
–
–
–
–
1
–
(C N); H NMR (300 MHz, DMSO-d , d ¼ ppm) d ¼ 2.36 (s, 3H, CH ),
–
6
3
d ¼ ppm) d ¼ 3.84 (s, 3H, OCH3), 7.30–8.60 (m, 13H, Ar–H), 9.30 (s,
1H, N CH); HRMS (ESI-TOF) calcd. for C22H17N3O2 [MþH]þ
3.68 (s, 3H, OCH3), 3.68 (s, 6H, 2OCH3), 6.97 (s, 2H, Ar–H), 7.20 (d,
1H, J ¼ 7.00 Hz, Ar–H), 7.32–7.75 (m, 5H, Ar–H), 7.87 (d, 2H,
–
–
356.1394, found 356.1398.
J ¼ 7.00 Hz, Ar–H), 8.14 (s, 1H, N H). 13C NMR (75 MHz, DMSO-d6,
–
–
d ¼ ppm) d ¼ 19.67, 55.70, 55.92, 60.12, 104.22, 127.42, 127.53,
128.33, 128.53, 129.11, 131.49, 131.78, 134.35, 135.60, 136.02,
139.11, 147.24, 152.90, 153.18, 163.27, 165.68; HRMS (ESI-TOF)
calcd. for C25H23N3O4 [MþH]þ 430.1761, found 430.1767.
3-[(2,3,4-Trihydroxybenzylidene)amino]-5-methyl-2-phen-
yl-3H-quinazolin-4-one 15: White powder (77% yield); m.p.
193°C; FT-IR (cmꢀ1): 3216 (OH), 1646 (C O), 1632 (C C), 1604
–
–
–
–
1
–
(C N); H NMR (300 MHz, DMSO-d , d ¼ ppm) d ¼ 2.36 (s, 3H, CH ),
–
6
3
6.35 (d, 1H, J ¼ 6.60 Hz, Ar–H), 6.73 (d, 1H, J ¼ 6.60 Hz, Ar–H), 7.15–
3-[(3,4-Dimethoxybenzylidene)amino]-5-methyl-2-phenyl-
7.20 (m, 1H, Ar–H), 7.34–7.57 (m, 5H, Ar–H), 7.80–7.85 (m, 2H,
3H-quinazolin-4-one 20: White powder (77% yield); m.p.
–
–
Ar–H), 8.25 (s, 1H, N CH), 9.75 (s, 1H, OH), 9.97 (s, 2H, OH).
220°C; FT-IR (cmꢀ1): 1648 (C O), 1602 (C N); 1H NMR
–
–
–
–
13C NMR (75 MHz, DMSO-d6, d ¼ ppm) d ¼ 19.94, 108.71, 111.99,
123.51, 124.41, 128.46, 129.24, 129.24, 129.68, 131.30, 131.38,
133.14, 133.84, 135.32, 136.37, 137.73, 148.52, 150.32, 153.35,
166.03; HRMS (ESI-TOF) calcd. for C22H17N3O4 [MþH]þ 388.1292,
found 388.1284.
(300 MHz, CD3OD, d ¼ ppm) d ¼ 2.46 (s, 3H, CH3), 3.85 (s, 3H,
OCH3), 3.88 (s, 3H, OCH3), 6.95 (d, 1H, J ¼ 6.30 Hz, Ar–H), 7.12 (d,
1H, J ¼ 6.00 Hz, Ar–H), 7.24 (d, 1H, J ¼ 6.00 Hz, Ar–H), 7.40–7.52 (m,
3H, Ar–H), 7.54–7.67 (m, 3H, Ar–H), 7.87 (d, 2H, J ¼ 5.40 Hz, Ar–H),
8.05 (s, 1H, N CH); HRMS (ESI-TOF) calcd. for C24H21N3O3 [MþH]þ
–
–
400.1656, found 400.1662.
3-[(2,4-Dihydroxybenzylidene)amino]-5-methyl-2-phenyl-
3H-quinazolin-4-one 16: Yellowish white powder (76%
PDE4B inhibition activity
yield); m.p. 236°C; FT-IR (cmꢀ1): 3214 (OH), 1649 (C O), 1629
–
–
PDE4B inhibitory activity was measured using the IMAP
fluorescence polarization PDE assay (Molecular Devices as
previously described [29]).
(C C), 1605, 1588 (C N); 1H NMR (300 MHz, CD3OD, d ¼ ppm)
–
–
–
–
d ¼ 2.44 (s, 3H, CH3), 6.28–6.36 (m, 2H, Ar–H), 7.15 (d, 1H,
J ¼ 6.00 Hz, Ar–H), 7.22 (d, 1H, J ¼ 5.70 Hz, Ar–H), 7.40–7.48 (m, 3H,
Ar–H), 7.52–7.56 (m, 1H, Ar–H), 7.61 (d, 1H, J ¼ 6.00 Hz, Ar–H),
7.80–7.87 (m, 2H, Ar–H), 8.20 (s, 1H, N CH). 13C NMR (75 MHz,
Cell growth inhibition
–
–
Tissue culture microtiter 96-well plates were seeded at a density of
5000 cells per well. Cells were incubated for 18–24 h, treated with
the specified compound or vehicle control, and incubated for an
additional 72 h. Doxorobicin was used as a positive control. The
inhibition of cell growth caused by treatment was determined
using the luminescent CellTiter-Glo Assay (Promega), which
measures viable cells based on ATP content. The assay was done
according to the manufacturer’s specifications with a maximum
DMSO concentration of 0.2%. Luminescence was measured using
a Synergy H4 (BioTek) plate reader.
CD3OD, d ¼ ppm) d ¼ 19.92, 103.81, 108.96, 111.12, 124.47,
128.53, 129.28, 129.65, 129.77, 131.33, 133.19, 133.44, 135.52,
136.4, 137.80, 149.40, 152.65, 161.47, 162.85, 166.13; HRMS
(ESI-TOF) calcd. for C22H17N3O3 [MþH]þ 372.1343, found
372.1349.
3-[(3,4-Dihydroxybenzylidene)amino]-5-methyl-2-phenyl-
3H-quinazolin-4-one 17: Yellowish white powder (78%
yield); m.p. 217–218°C; FT-IR (cmꢀ1): 3222 (OH), 1649 (C O),
–
–
1
–
1602 (C N); H NMR (300 MHz, CD OD, d ¼ ppm) d ¼ 2.45 (s, 3H,
–
3
CH3), 6.76 (d, 1H, J ¼ 5.70 Hz, Ar–H), 6.99–7.02 (m, 1H, Ar–H), 7.22
(d, 1H, J ¼ 5.70 Hz, Ar–H), 7.31 (s, 1H, Ar–H), 7.37–7.56 (m, 4H,
Ar–H), 7.64 (d, 1H, J ¼ 6.30 Hz, Ar–H), 7.77–7.88 (m, 2H, Ar–H), 7.97
Molecular modeling studies
All the molecular modeling studies were carried out on an Intel
Core i3 processor, 3 GB memory with Windows 7 operating
system using Molecular Operating Environment (MOE 2008,
Chemical Computing Group, Canada) [25] as the computational
software. All the minimizations were performed with MOE until
a RMSD gradient of 0.05 kcal molꢀ1 Åꢀ1 with MMFF94X force-field
(s, 1H, N CH). 13C NMR (75 MHz, CD3OD, d ¼ ppm) d ¼ 19.93,
–
–
114.23, 116.23, 122.88, 124.33, 126.94, 128.53, 129.24, 129.77,
129.81, 131.30, 131.60, 133.21, 135.52, 136.57, 137.78, 146.88,
149.93, 151.61, 166.90; HRMS (ESI-TOF) calcd. for C22H17N3O3
[MþH]þ 372.1343, found 372.1347.
ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com

Arch. Pharm. Chem. Life Sci. 2014, 347, 650–657
Quinazolin-4(3H)-one/Schiff Base Hybrids as PDE4 Inhibitors
[14] P. J. Barnes, Pharmaceuticals 2010, 3, 725–747.
657
and the partial charges were automatically calculated. The X-ray
crystallographic structure of PDE4B complexed with pyrano[3,2-
d]pyrimidine derivative (PDB ID: 3W5E) [26] was obtained from
the Protein Data Bank. The enzyme was prepared for docking
studies where: (i) ligand molecule was removed from the enzyme
active site; (ii) hydrogen atoms were added to the structure with
their standard geometry; (iii) MOE Alpha Site Finder was used for
the active sites search in the enzyme structure and dummy atoms
were created from the obtained alpha spheres; (iv) the obtained
model was then used in predicting the ligand–enzyme inter-
actions at the active site.
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ß 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.archpharm.com