Design, synthesis and in vitro PDE4 inhibition activity of certain quinazolinone derivatives for treatment of asthma

Article abstract of DOI:10.1007/s00044-011-9846-3

In this study, a novel series of quinazolinone derivatives analogue to nitraquazone structure were synthesized. The compounds tested for their inhibitory activity against phosphodiesterase 4B revealed that compound 6d shows promising inhibitory activity comparable to that of Rolipram, whereas compounds 6a and 6c exhibited moderate inhibitory activity. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9846-3

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3327–3335
DOI 10.1007/s00044-011-9846-3
SYMPOSIUM PAPER
Design, synthesis and in vitro PDE4 inhibition activity of certain
quinazolinone derivatives for treatment of asthma
•
Afaf K. Elansary Hanan H. Kadry
•
•
Eman M. Ahmed Amr Sayed Motawi Sonousi
Received: 6 May 2011 / Accepted: 25 October 2011 / Published online: 19 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, a novel series of quinazolinone
derivatives analogue to nitraquazone structure were syn-
thesized. The compounds tested for their inhibitory activity
against phosphodiesterase 4B revealed that compound 6d
shows promising inhibitory activity comparable to that of
Rolipram, whereas compounds 6a and 6c exhibited mod-
erate inhibitory activity.
major classes of compounds are currently used in the
treatment of asthma, including bronchodilators (particu-
ˆ
larly a-adrenoceptor agonists), immunosuppressive agents
(corticosteroids), antiallergic agents for prophylactic use
and xanthines (e.g. theophylline) (Vassallo and Lipsky,
1998), which appear to possess bronchodilator and antiin-
flammatory as well as immunomodulator properties. Newer
drugs include leukotriene antagonists such as montelukast
(Reques and Rodriguez, 1999). To date, much research has
been directed towards the discovery of new antiasthmatic
agents with high selectivity and efficacy and a reduced
side-effect profile.
Keywords Phosphodiesterase 4 (PDE4) inhibitors ꢀ
Quinazolinone ꢀ Asthma ꢀ Synthesis
Introduction
Phosphodiesterase enzymes (PDEs) (Butcher and Suth-
erland, 1962), involved in the intracellular degradation of
cAMP and cGMP to their corresponding 50-monophosphate
counterparts, have received a considerable amount of
attention as molecular targets for the treatment of asthma.
Intracellular cyclic AMP (cAMP) and cyclic GMP (cGMP)
are ubiquitous intracellular second messengers which play a
prominent role in the regulation of important cellular func-
tions such as secretion, contraction, metabolism and growth.
PDEs have been classified into 11 major families (PDE1-11)
with respect to their substrate sensitivity, Ca2/calmodulin
requirement and inhibitor selectivity (Torphy, 1998; Fisher
et al., 1998; Crocker and Townley, 1999; Juilfs et al., 1999;
Lanfear and Robas, 1999). PDE4 is a cAMP-specific enzyme
localized in airway smooth muscles in immune and inflam-
matory cells. Thus, the elevation of cAMP levels by PDE4
inhibition represents a useful strategy for the development of
new antiasthmatic and antiinflammatory drugs.
Asthma is one of the most common chronic diseases
(Barnes, 1992) worldwide and antiasthmatic medications
are widely prescribed. Despite advancements in treatment,
the incidences of asthma, asthma-related deaths and hos-
pitalizations for asthma have increased significantly during
the past decade. Asthma is characterized by a reversible
airway obstruction, ongoing cellular inflammation and
nonspecific hyper-responsiveness to a variety of chal-
lenges. Both acute and long-term manifestations of asthma
are believed to be a consequence of various inflammatory
mediators released by activated inflammatory and immune
cells (Holgate, 1988; Reed, 1988; Adelroth et al., 1990;
Djukanovic et al., 1990). Therefore, the ability to suppress
activation of these cells is an essential property for a
compound to have a therapeutic effect on asthma. Four
From a structural point of view, selective PDE4 inhib-
itors in the public domain can be divided into three classes:
structural analogues of rolipram, structural analogues of
nitraquazone and structures related to xanthines (Crespo
et al., 1998) (Fig. 1).
A. K. Elansary ꢀ H. H. Kadry ꢀ E. M. Ahmed (&) ꢀ
A. S. M. Sonousi
Department of Organic Chemistry, Faculty of Pharmacy,
Cairo University, Cairo 11562, Egypt
e-mail: dr_eman2001@hotmail.com
123

3328
Med Chem Res (2012) 21:3327–3335
O
Fig. 1 Compounds
representative of the three
chemical classes of PDE4
inhibitors: Rolipram,
nitraquazone and xanthine
derivatives (denbufylline)
O
N
O
O
O
N
N
N
N
O
O
N
O
N
H
NO2
Rolipram
Nitraquazone
Denbufylline
In 1984, nitraquazone, the first selective PDE type 4
having quinazolin-2,4-dione, was synthesized (Glaser and
Traber, 1984). Nitraquazone appears to be very attractive
model for the synthesis of novel PDE4 inhibitors poten-
tially useful for the treatment of asthma and chronic
obstructive pulmonary disease. Structural analogues of
nitraquazone could be devoid of the central side-effects
(nausea, vomiting and headache) of the archetypal Roli-
pram which hampered its development as a drug (Piaz and
Giovannoni, 2000).
Although the number of studies claiming different
chemical classes of PDE4 inhibitors is increasing in recent
years, only few detailed studies evaluated the PDE4 inhi-
bition of structural analogues of nitraquazone. On this
basis, the rational design of our compounds was based on a
hybrid structure of nitraquazone and denbufylline. A
common pharmacophore using flexible alignment module
implemented in MOE software was done for nitraquazone
and denbufylline (Fig. 2), which simultaneously searches
the conformation space and the space of alignments of
those molecules using stochastic search procedure. There-
fore, compounds obeying this pharmacophore, containing a
flat aromatic area of quinazoline, hydrogen bond acceptor
at position 4, hydrophobic part at position 3, aromatic
region at positions 1 or 2 of the quinazoline-4-one nucleus,
were synthesized; hoping that the new derivatives will have
potent PDE4 inhibition.
Fig. 2 Flexible alignment of nitraquazone and denbufylline using
MOE 2008.10 software
(Germany) or Bruker UXNMR/XWIN-NMR (300 MHz)
instruments. Chemical shifts (d) are reported in ppm
downfield from internal TMS standard. EI-MS Hewlett
Packard 5988 spectrometer, Micro analytical Center, Cairo
University, Egypt. ESI–MS Quadrupole VG Quattro
Institute of Pharmacy & Molecular Biotechnology in
Neuenheimer Field 364 69120 Heidelberg Germany. Ele-
mental analyses were carried out at the Micro analytical
Center, Cairo University, Egypt. Melting points were
determined with an Electro thermal melting point appara-
tus, and were uncorrected. 2-Amino-N-butylbenzamide (1)
(Clark and Wagner, 1944) and 3-butyl-2-hydrazinoquina-
zolin-4(3H)-one (4) (Kottke et al., 1990) was synthesized
according to reported procedures. 3-Butyl-2-thioxo-2,
3-dihydroquinazolin-4(1H)-one (3) (Shafik et al., 1979),
was synthesized by a new procedure rather than the
reported one.
Methods and materials
Chemistry
All chemicals and reagents were obtained from Aldrich
(Sigma-Aldrich, St. Louis, MO, USA) and Lancaster (Alfa
Aesar, Johnson Company, Ward Hill, MA, USA), and
were used without further purification. Reactions were
monitored by TLC, performed on silica gel glass plates
containing 60 GF-254 and visualization on TLC was
achieved by UV light or iodine indicator. IR spectra
were determined on Shimadzu IR 435 spectrophotometer
2-Aryl-3-butyl-2,3-dihydroquinazolin-4(1H)-ones
(2a–2f)
1
(KBr, cm-1). HNMR spectra were recorded on Gemini
Equimolar amount of 2-amino-N-butylbenzamide (1)
(0.57 g, 0.003 mol) and an appropriate aromatic aldehyde
Varian-VXR-unity (200 MHz), Gemini Varian 500 MHz
123

Med Chem Res (2012) 21:3327–3335
3329
(0.003 mol) in toluene (40 ml) containing p-toluenesul-
phonic acid (0.05 g, 0.2 mmol) was refluxed under Dean-
Stark trap for 4 h. The solution was filtered while hot and
the filtrate was evaporated under reduced pressure and the
residue was crystallized from benzene.
NCH2), 2.96 (s, 6H, N(CH3)2), 3.86–3.96 (m, 1H, NCH2),
4.42 (s, 1H, NH, D2O exchangeable), 5.68 (s, 1H, C2H
quinazoline), 6.50–7.96 (m, 8H, ArHs). EIMS (% rel.
abundance): 323 (M?, 60.19%), 324.10 (M ? 1, 15.15%),
121.10 (100%). Anal. Calcd for C20H25N3O: C, 74.27; H,
7.79; N, 12.99; Found: C, 74.45; H, 7.69; N, 13.04.
3-Butyl-2-(3-hydroxyphenyl)-2,3-dihydroquinazolin-4(1H)-
one (2a)
3-Butyl-2-(3-nitrophenyl)-2,3-dihydroquinazolin-4(1H)-
one (2e)
Yield: 84%; mp. 176–178°C. IR (cm-1): 3311 (NH), 3280
(OH) 3066, 3057 (CH aromatic), 2962, 2926 (CH ali-
phatic), 1616 (C=O). 1HNMR (300 MHz, DMSO); 0.85 (t,
3H, CH3), 1.22–1.30 (m, 2H, CH2CH3), 1.44–1.54 (m, 2H,
CH2CH2CH3), 2.69–2.74 (m, 1H, NCH2), 3.80–3.87 (m,
1H, NCH2), 5.72 (s, 1H, C2H quinazoline), 6.61–7.65 (m,
9H, ArHs ? NH, D2O exchangeable), 9.44 (s, 1H, OH,
D2O exchangeable). Anal. Calcd for C18H20N2O2: C,
72.95; H, 6.80; N, 9.45; Found: C, 73.10; H, 6.71; N, 9.41.
Yield: 87%; mp. 114–116°C. IR (cm-1): 3300 (NH), 3089,
3066 (CH aromatic), 2968, 2937 (CH aliphatic), 1627
1
(C=O), 1537, 1350 (NO2). HNMR (500 MHz, CDCl3):
0.90 (t, 3H, CH3), 1.31–1.39 (m, 2H, CH2CH3), 1.55–1.66
(m, 2H, CH2CH2CH3), 2.75–2.81 (m, 1H, NCH2),
4.08–4.13 (m, 1H, NCH2), 4.90 (s, 1H, NH, D2O
exchangeable), 5.85 (s, 1H, C2H quinazoline), 6.60 (d, 1H,
C8H of quinazoline), 6.88 (dd, 1H, C6H of quinazoline),
7.25 (dd, 1H, C7H of quinazoline), 7.52 (dd, 1H, C5H–3-
ArNO2), 7.71 (d, 1H, C5H of quinazoline), 7.95 (d, 1H,
C6H–3-ArNO2), 8.19 (d, 1H, C4H–3-ArNO2), 8.22 (s, 1H,
C2H–3-ArNO2). 13CNMR (CDCl3): 13.76 (CH3), 20.10
(CH2), 29.92 (CH2), 45.01 (CH2–N3), 70.83 (C2),
114.89–148(Ar-C), 162.75 (C=O). Anal. Calcd for
C18H19N3O3: C, 66.45; H, 5.88; N, 12.91; Found: C, 66.75;
H, 5.99; N, 12.86.
3-Butyl-2-(3-fluorophenyl)-2,3-dihydroquinazolin-4(1H)-
one (2b)
Yield: 80%; mp. 130–132°C. IR (cm-1): 3300 (NH), 2956,
2937 (CH aliphatic), 1627 (C=O) 1HNMR (300 MHz,
CDCl3): 0.91 (t, 3H, CH3), 1.27–1.43 (m, 2H, CH2CH3),
1.54–1.65 (m, 2H, CH2CH2CH3), 2.72–2.81 (m, 1H,
NCH2), 3.97–4.06 (m, 1H, NCH2), 4.73 (s, 1H, NH, D2O
exchangeable), 5.72 (s, 1H, C2H quinazoline), 6.52–7.96
(m, 8H, ArHs). Anal. Calcd for C18H19FN2O: C, 72.46; H,
6.41; N, 9.39; Found: C, 72.65; H, 6.38; N, 9.35.
3-Butyl-2-(4-hydroxy-3-methoxyphenyl)-2,
3-dihydroquinazolin-4(1H)-one (2f)
Yield: 78%; mp. 142–144°C. IR (cm-1): 3396 (NH), 3160
(OH), 2958, 2927 (CH aliphatic), 1625 (C=O). HNMR
1
3-Butyl-2-(4-fluorophenyl)-2,3-dihydroquinazolin-4(1H)-
one (2c)
(300 MHz, CDCl3): 0.88 (t, 3H, CH3), 1.27–1.38 (m, 2H,
CH2CH3), 1.45–1.54 (m, 2H, CH2CH2CH3), 2.74–2.83 (m,
1H, NCH2), 3.81 (s, 3H, OCH3), 3.84–3.97 (m, 1H,
NCH2), 4.71 (s, 2H, NH ? OH, D2O exchangeable), 5.69
(s, 1H, C2H quinazoline), 6.52–7.95 (m, 7H, ArHs). EIMS
(% rel. abundance): 326.15 (M?, 15.59%), 327.05
(M ? 1, 3.65%), 203.10 (100%). Anal. Calcd for
C19H22N2O3: C, 69.92; H, 6.79; N, 8.58; Found: C, 70.03;
H, 6.65; N, 8.31.
Yield: 75%; mp. 170–172°C. IR (cm-1): 3304 (NH), 3070,
3001 (CH aromatic), 2956, 2939 (CH aliphatic), 1631
1
(C=O). HNMR (200 MHz, CDCl3): 0.77 (t, 3H, CH3),
1.10–1.28 (m, 2H, CH2CH3), 1.36–1.55 (m, 2H,
CH2CH2CH3), 2.55–2.75 (m, 1H, NCH2), 3.78–3.94 (m,
1H, NCH2), 4.74 (s, 1H, NH, D2O exchangeable), 5.65 (s,
1H, C2H quinazoline), 6.45–7.84 (m, 8H, ArHs). EIMS (%
rel. abundance): 298.10 (M?, 14.11%), 299 (M ? 1,
3.14%), 203.10 (100%). Anal. Calcd for C18H19FN2O: C,
72.46; H, 6.41; N, 9.39; Found: C, 72.70; H, 6.41; N, 9.35.
3-Butyl-2-thioxo-2,3-dihydroquinazolin-4(1H)-one (3)
A solution of 2-amino-N-butylbenzamide (1) (9.61 g,
0.05 mol) in ethanol (50 ml) was treated with KOH
(3.36 g, 0.06 mol) in water (10 ml) and CS2 (7.56 g,
6.0 ml, 0.1 mol). The solution was heated under reflux for
8 h. After reflux, the reaction mixture was cooled, poured
into diluted HCl and the solid product was crystallized
from aqueous ethanol. Yield: 6.0 g (51%); mp. 175–176°C
(Shafik et al., 1979).
3-Butyl-2-[4-(dimethylamino)phenyl]-2,
3-dihydroquinazolin-4(1H)-one (2d)
Yield: 80%; mp. 176–178°C. IR (cm-1): 3305 (NH), 2958,
2933 (CH aliphatic), 1629 (C=O). 1HNMR (300 MHz,
CDCl3): 0.87 (t, 3H, CH3), 1.26–1.31 (m, 2H, CH2CH3),
1.52–1.57 (m, 2H, CH2CH2CH3), 2.73–2.84 (m, 1H,
123

3330
Med Chem Res (2012) 21:3327–3335
2-[2-(Arylidene)hydrazinyl]-3-butyl-quinazolin-4(3H)-
ones (5a–5d)
3-Butyl-2-[2-(4-hydroxy-3-methoxybenzylidene)
hydrazinyl]quinazolin-4(3H)-one (5d)
A solution of 3-butyl-2-hydrazinoquinazolin-4(1H)-one (4)
(0.51 g, 0.0022 mol), appropriate aromatic aldehyde
(0.0022 mol) in absolute ethanol (20 ml) and few drops of
acetic acid was refluxed for 30 h. The reaction mixture was
concentrated; few drops of water were added and cooled.
The precipitate formed was filtered, dried and crystallized
from aqueous ethanol.
Yield: 55%; mp. 170–172°C. IR (cm-1): 3417 (OH), 3367
(NH), 3062 (CH aromatic), 2958, 2931 (CH aliphatic),
1
1681 (C=O), 1627 (C=N). HNMR (300 MHz, DMSO):
0.82 (t, 3H, CH3), 1.20–1.23 (m, 2H, CH2CH3), 1.35–1.40
(m, 2H, CH2CH2CH3), 3.73 (s, 3H, OCH3), 3.86 (t, 2H,
NCH2), 5.61(s, 1H, OH, D2O exchangeable), 6.80–7.95 (m,
7H, ArHs), 8.80 (s, 1H, =CH), 9.25 (s, 1H, NH, D2O
exchangeable). EIMS (% rel. abundance): 366.20 (M?,
50.70%), 367.20 (M ? 1, 12.36%), 174.10 (100%). Anal.
Calcd for C20H22N4O3: C, 65.56; H, 6.05; N, 15.29; Found:
C, 65.55; H, 6.12; N, 15.08.
3-Butyl-2-[2-(3-hydroxybenzylidene)hydrazinyl]
quinazolin-4(3H)-one (5a)
Yield: 60%; mp. 220–222°C. IR (cm-1): 3377 (OH), 3354
(NH), 2966, 2951 (CH aliphatic), 1670 (C=O), 1612
1
(C=N). HNMR (300 MHz, CDCl3): 0.98 (t, 3H, CH3),
1-Aryl-4-butyl [1,2,4]triazolo[4,3-a] quinazolin-5(4H)-
ones (6a–6d)
1.41–1.49 (m, 2H, CH2CH3), 1.70–1.83 (m, 2H,
CH2CH2CH3), 4.24 (t, 2H, NCH2), 5.79 (s, 1H, OH, D2O
exchangeable), 6.8–8.1 (m, 8H, ArHs), 8.48 (s, 1H,
N=CH), 9.39 (s, 1H, NH, D2O exchangeable). EIMS m/z
(% rel. abundance): 337.30 (M ? 1, 100%). Anal. Calcd
for C19H20N4O2: C, 67.84; H, 5.99; N, 16.66; Found: C,
68.09; H, 6.03; N, 16.52.
To a solution of the appropriate schiffs bases 5a–5d
(0.001 mol) in ethanol (20 ml) was added a solution of
ferric chloride (2 M, 1 ml) and the mixture was refluxed
for 30 min. After reflux, the solution was left overnight at
room temperature then poured onto cold water. The solid
separated was filtered, washed with water, dried and finally
crystallized from aqueous ethanol.
3-Butyl-2-{2-[4-(dimethylamino)benzylidene]hydrazinyl}
quinazolin-4(3H)-one (5b)
4-Butyl-1-(3-hydroxyphenyl)[1,2,4]triazolo
[4,3-a]quinazolin-5(4H)-one (6a)
Yield: 80%; mp. 140–142°C. IR (cm-1): 3402 (NH), 2954
(CH aliphatic), 1670 (C=O), 1612 (C=N). 1HNMR
(300 MHz, CDCl3): 0.99 (t, 3H, CH3), 1.44–1.51 (m, 2H,
CH2CH3), 1.67–1.72 (m, 2H, CH2CH2CH3), 3.08 (s, 6H,
N(CH3)2), 3.70 (t, 2H, NCH2), 6.7–8.2 (m, 8H, ArHs), 8.89
(s, 1H, N=CH), 9.40 (s, 1H, NH, D2O exchangeable). Anal.
Calcd for C21H25N5O: C, 69.39; H, 6.93; N, 19.27; Found:
C, 69.65; H, 6.80; N, 19.00.
Yield: 90%; mp. 238–240°C. IR (cm-1): 3425 (OH), 3070,
3043 (CH aromatic), 2951, 2939 (CH aliphatic), 1685
1
(C=O), 1597 (C=N). HNMR (300 MHz, CDCl3): 1.00 (t,
3H, CH3), 1.47–1.54 (m, 2H, CH2CH3), 1.81–1.96 (m, 2H,
CH2CH2CH3), 4.39 (t, 2H, NCH2), 4.70 (s, 1H, OH, D2O
exchangeable), 7.02–8.40 (m, 8H, ArHs). EIMS (% rel.
abundance): 334.15 (M?, 24.72%), 335.05 (M ? 1, 6.15%),
277 (100%). Anal. Calcd for C19H18N4O2: C, 68.25; H,
5.42; N, 16.76; Found: C, 68.40; H, 5.41; N, 16.67.
3-Butyl-2-[2-(3-nitrobenzylidene)hydrazinyl]quinazolin-
4(3H)-one (5c)
4-Butyl-1-[4-(dimethylamino)phenyl][1,2,4]triazolo
[4,3-a]quinazolin-5(4H)-one (6b)
Yield: 75%; mp. 168–170°C. IR (cm-1): 3452 (NH), 3070
(CH aromatic), 2954, 2931 (CH aliphatic), 1670 (C=O),
1635 (C=N), 1531, 1350 (NO2). 1HNMR (300 MHz,
CDCl3): 0.97 (t, 3H, CH3), 1.42–1.50 (m, 2H, CH2CH3),
1.69–1.75 (m, 2H, CH2CH2CH3), 4.24 (t, 2H, NCH2),
7.3–8.6 (m, 8H, ArHs), 8.61 (s, 1H, N=CH), 9.40 (s, 1H,
NH, D2O exchangeable). EIMS (% rel. abundance): 365.05
(M?, 37.66%), 366.20 (M ? 1, 12.37%), 186.95(100%).
Anal. Calcd for C19H19N5O3: C, 62.46; H, 5.24; N, 19.17;
Found: C, 62.65; H, 5.30; N, 18.94.
Yield: 87%; mp. 180–182°C. IR (cm-1): 2954 (CH ali-
phatic), 1697 (C=O), 1625 (C=N). 1HNMR (300 MHz,
CDCl3): 0.93 (t, 3H, CH3), 1.34–1.39 (m, 2H, CH2CH3),
1.81–1.88 (m, 2H, CH2CH2CH3), 3.08 (s, 6H, N(CH3)2),
4.28 (t, 2H, NCH2), 6.81–8.48 (m, 8H, ArHs). EIMS (%
rel. abundance): 361 (M?, 3.26%), 63 (100%). Anal. Calcd
for C21H23N5O: C, 69.78; H, 6.41; N, 19.38; Found: C,
70.07; H, 6.40; N, 19.19.
123

Med Chem Res (2012) 21:3327–3335
3331
4-Butyl-1-(3-nitrophenyl)[1,2,4]triazolo[4,3-a]quinazolin-
5(4H)-one (6c)
Table 1 Inhibitory effects of the compounds and rolipram on
PDE4B2 enzyme
Compound (10 lM)
Mean % inhibition
Yield: 85%; mp. 180–182°C. IR (cm-1): 2962, 2931 (CH
aliphatic), 1705 (C=O), 1627 (C=N), 1523, 1354 (NO2).
1HNMR (300 MHz, CDCl3): 0.94 (t, 3H, CH3), 1.33–1.42
(m, 2H, CH2CH3), 1.83–1.91 (m, 2H, CH2CH2CH3), 4.27
(t, 2H, NCH2), 7.39–8.69 (m, 8H, ArHs). EIMS (% rel.
abundance): 363 (M?, 57.71%), 363.95 (M ? 1, 17.98%),
307(100%). Anal. Calcd for C19H17N5O3: C, 62.80; H,
4.71; N, 19.27; Found: C, 63.10; H, 4.84; N, 19.16.
No compound
0
90
7
Rolipram
2d
2e
2f
15
15
11
19
44
6
5a
5b
6a
6b
6c
6d
4-Butyl-1-(4-hydroxy-3-methoxyphenyl)[1,2,4]triazolo
[4,3-a]quinazolin-5(4H)-one (6d)
44
62
Yield: 85%; mp. 190–192°C. IR (cm-1): 3410 (OH), 2958,
2931 (CH aliphatic), 1693 (C=O), 1627(C=N). 1HNMR
(300 MHz, CDCl3): 0.92 (t, 3H, CH3), 1.33–1.40 (m, 2H,
CH2CH3), 1.82–1.89 (m, 2H, CH2CH2CH3), 4.01 (s, 3H,
OCH3), 4.28 (t, 2H, NCH2), 6.03 (s, 1H, OH, D2O
exchangeable), 7.08–8.49 (m, 7H, ArHs). Anal. Calcd for
C20H20N4O3: C, 65.92; H, 5.53; N, 15.38; Found: C, 66.20;
H, 5.61; N, 15.30.
Results and discussion
To prepare the target quinazoline derivatives, reactions
presented in Scheme 1 were performed. The starting
material 2-amino-N-butylbenzamide (1) was prepared as
reported procedure (Clark and Wagner, 1944). Reacting
1 with appropriate substituted benzaldehyde in the
presence of p-toluenesulphonic acid as
a catalyst
using Dean-Stark to trap water formed in the reaction
afforded 2-aryl-3-butyl-2,3-dihydroquinazolin-4(1H)-one
2a–2f).
Biological activities
Materials
1HNMR proved the presence of a singlet peak around d
5.7 ppm assignable to C2H of the quinazolinone nucleus. In
addition, an interesting observation was the appearance of
geminal hydrogens of NCH2 at different chemical shifts
(2.75 and 3.94) (Chen et al., 2008). Geminal hydrogens at
NCH2 was proved by COSY and HMQC spectra of com-
pound (2e) which showed that these two hydrogens are
coupled to each other (Fig. 3) and are attached to the same
carbon (Fig. 4).
PDE assay kit is used in the assay (Catalog number 60300).
PDE4B inhibition activity assay
100 lM solutions of the test compounds were prepared
with 10% DMSO in assay buffer and 5 ll of the solution
was added to a 50 ll reaction so that the final concen-
tration of DMSO is 1% in all of the reactions. The
enzymatic reactions were conducted at room temperature
for 60 min in a 50 ll mixture containing PDE assay
buffer, 100 nM FAM-cAMP, PDE4B2 and the test com-
pound. After the enzymatic reaction, 100 ll of a binding
solution (1:100 dilution of the binding agent with the
binding agent diluent) was added to each reaction and the
reaction was performed at room temperature for 60 min.
Fluorescence intensity was measured at an excitation of
470 nm and an emission of 528 nm using a Tecan Infinite
M1000 microplate reader. The percent activity in the
presence of the compound was calculated according to the
following equation: % activity = (FP - FPb)/(FPt - FPb) 9
100%, where FP is the fluorescence polarization in the
presence of the compound. The % PDE4B inhibition of
the synthesized compounds was consequently calculated
(Table 1; Fig. 7).
This observation may be resides on the fact that the two
hydrogens are diastereomeric protons due to the presence
of asymmetric center (C2). Moreover, the limited free
rotation of N3–C bond results in different environments for
the protons (Rodrıguez-Franco et al., 2000; Tormena et al.,
2002; Elmaaty and Castle, 2005). The restricted rotation
was supported by constructing models of these compounds
using MOE builder (Fig. 5) and plotting the conforma-
tional energy for each dihedral angle through N3–C bond
using dihedral energy plot module implemented in MOE.
The result showed that this conformer lies in a deep valley
in which high energy barriers must be overcome in order to
rotate (i.e. change its dihedral angle) the N3–C bond
(Fig. 6).
On the other hand, compound 1 was allowed to react
with carbon disulphide to give 3-butyl-2-thioxo-2,
123

3332
Med Chem Res (2012) 21:3327–3335
O
O
O
O
N
N
O
a
H
N
c
d
NH2
N
H
S
NH2
N
H
O
N
N
H
1
3
4
b
e
O
O
N
N
N
N
N
H
N
H
R
(a- )
5
d
(a- )
2
f
R
f
R
:
-
2a 3 OH
O
:
:
-
2b
2c
3 F
-
4 F
N
:
:
:
)
- (
2d 4 N
H
C
3
2
N
N
-
2e
2f
3 NO2
N
,
-
-
3 OCH3 4 OH
R
(a- )
6
d
R
:
:
:
6a
6b
-
3 OH
-
4 N
H
C
(
)
3 2
6c
6d
-
3 NO2
:
,
-
-
3 OCH3 4 OH
Scheme 1 Synthetic protocol for title compounds. (a) butyl amine; (b) substituted benzaldehydes, p-toluenesulphonic acid; (c) CS2, KOH;
(d) NH2NH2, reflux 30 h; (e) substituted benzaldehydes, ethanol, acetic acid; (f) FeCl3, ethanol
3-dihydroquinazolin-4(1H)-one (3) which upon treatment
with hydrazine hydrate gave 3-butyl-2-hydrazinoquinazo-
lin-4(3H)-one (4). Condensation of compound 4 with the
respective aromatic aldehyde was conducted in ethanol
containing a catalytic amount of glacial acetic acid to give
the novel Schiff’s base 5a–5d. The long duration of
reaction (30 h) required might be because of the presence
of bulky butyl group at position three which might have
reduced the reactivity of quinazoline ring system at C-2
1
position. HNMR revealed, in each case, three character-
istic signals in the regions d 0.97–4.24, 8.48–8.89
and 9.25–9.40 ppm corresponding to the butyl, azome-
thine (–N=CH–) and hydrazone (–NH–N=C) protons,
Fig. 3 Correlation spectroscopy (COSY) of compound (2e)
respectively.
123

Med Chem Res (2012) 21:3327–3335
3333
In addition to this, oxidative cyclization of the schiffs
base 5a–5d using ferric chloride yielded 1-aryl-4-butyl
1
[1,2,4]triazolo[4,3-a] quinazolin-5(4H)-one (6a–6d). HNMR
spectra showed the absence of both the azomethine proton
(–N=CH) and hydrazone –NH–N=C proton signals. Both
elemental analysis and mass spectrum of each compound
revealed that it has two hydrogens less than the respective
hydrazone.
Molecular docking studies of the synthesized com-
pounds were performed in order to rationalize the obtained
biological results (Fig. 7) as well as to help us in under-
standing the various interactions between the ligands and
enzyme active site in details.
The X-ray crystallographic structure of PDE4B com-
plexed with Rolipram (PDB: 1RO6) was used in our
docking studies. All water molecules in the experimental
structure were removed. Hydrogen atoms were added and
the protonation states of the amino acid residues were
assigned using the Protonate 3D algorithm. Ligand mole-
cules were modelled using MOE builder, and the structures
were energy minimized using the MMFF94x force field.
Validation of the function implemented in MOE was done
by docking of the native ligand into its binding site. The
docked results were compared to the crystal structure of the
bound ligand–protein complex. The RMSD of the docked
Fig. 4 Heteronuclear multiple quantum coherence (HMQC) of
compound (2e)
˚
ligand was 0.32 A as it seems exactly superimposed on the
native bound one (Fig. 8a). These results indicated the high
accuracy of the MOE simulation in comparison with the
biological methods. Although PDE4 enzyme crystal
structure with nitraquazone co-crystallized was absent, a
prediction of the binding model for nitraquazone was done
by docking nitraquazone in the active site gorge of PDE4B
(Fig. 8b). This binding model was useful in our interpre-
tation to the activities our synthesized compounds.
Fig. 5 3D model of the most stable conformer of compound 2e
Next, we performed docking studies to our synthesized
compound and the final docked complexes of ligand–
enzyme were selected according to the criteria of interaction
energy combined with geometrical matching quality. The
presence of a Zn binding group was important in the lead
compounds, 6a, 6c and 6d (Fig. 9), which showed high
Fig. 6 Dihedral energy plot of N3–C bond of compound 2e. This was
obtained using the dihedral energy plot implemented in MOE
2008.10. The dihedral angle energy of kcal/mol was based on the
energy of the most stable conformation
Fig. 7 Graph for the inhibitory effects of the compounds on PDE4B2
enzyme
123

3334
Med Chem Res (2012) 21:3327–3335
˚
Fig. 8 a The docked Rolipram ligand into PDE4B seems superimposed on the native Rolipram ligand, RMSD: 0.32 A. b Predicted binding
model of nitraquazone
inhibition against PDE4. The absence of Zn binding group in
6b was therefore responsible of its low inhibitory activity.
Although the aryl moieties of 2a–2f contain groups that
can bind with Zn2? or form hydrogen bonds with amino
acid residues (Asp392, His234 and His278). Unfortunately,
these aryls were not oriented towards the Zn2? and the
amino acids residues (Asp392, His234 and His278) but, on
the contrary, they were directed in the opposite direction
and partly exposed to the active site surface. This finding
gives a reasonable explanation to the poor PDE4 activities
of these derivatives (Fig. 10).
Conclusions
From the common pharmacophore for nitraquazone related
compounds, a series of novel heteroaromatic compounds
have been designed, synthesized and evaluated as PDE4
inhibitors. Rolipram was selected as a reference standard.
The biological results reported in Table 1 confirm that
compound 6d showed promising inhibitory activity com-
parable to that of Rolipram, whereas compound 6a and 6c
exhibited moderate inhibitory activity. Finally, the rest of
the tested compounds exhibited poor inhibition.
Fig. 9 a Proposed binding mode of 6d in PDE4B active site generated by MOE docking. b Simplified 2D ligand interaction of 6d
123

Med Chem Res (2012) 21:3327–3335
3335
Fig. 10 a Docked poses of 2e in PDE4B binding site generated by MOE docking. b Simplified 2D ligand interaction of 2e
Acknowledgment The authors are thankful to Dr Henry Zhu, Dr.
Yiming Chen, BPS Bioscience Inc. 6044 Cornerstone Court West,
Ste. E, San Diego, CA 92121, USA. for their effort in performing the
biological assay.
Glaser T, Traber J (1984) TVX 2706—a new phosphodiesterase
inhibitor with anti-inflammatory action. Agents Actions 15:
341–348
Holgate ST (1988) Inflammatory cells and their mediators in the
pathogenesis of asthma. Postgrad Med J 64:S82–S95
Juilfs DM, Soderling S, Burns F, Beavo JA (1999) Cyclic GMP as
substrate and regulator of cyclic nucleotide phosphodiesterases
(PDEs). Rev Physiol Biochem Pharmacol 135:67–104
Kottke K, Kuhmstedt H, Grafe I, Knoke D, Schleuder M (1990)
Synthesis of compounds having an aminoguanidine structure.
Part 17. Hydrazinolysis of 3-alkyl- and 3-aryl-substituted 4-oxo-
2-thioxo-1,2,3,4-tetrahydroquinazolines. Pharmazie 45:30–33
Lanfear J, Robas NM (1999) Mammalian cyclic nucleotide phospho-
diesterases and their cDNA sequences. Eur Pat ApplEP 967284 A1
Piaz VD, Giovannoni MP (2000) TVX 2706-a new phosphodiesterase
inhibitor with anti-inflammatory action. Eur J Med Chem
35:463–480
Reed CE (1988) Basic mechanisms of asthma. Role of inflammation.
Chest 94:175–177
Reques FG, Rodriguez JLE (1999) Tolerability of leukotriene
modifiers in asthmas. Biodrugs 11:385–394
Rodrıguez-Franco MI, Dorronsoro I, Castro A, Martınez A (2000)
Hindered inversion/rotation in diheteroaryl alkyl amines with a
N-(1-pyrazolyl) group: dynamic NMR and molecular modelling
studies. Tetrahedron 56:1739–1743
References
Adelroth E, Rosenhall L, Johansson SA, Linden M, Venge P (1990)
Inflammatory cells and eosinophilic activity in asthmatics
investigated by bronchoalveolar lavage: the effects of antiasth-
matic treatment with budesonide or terbutaline. Am Rev Respir
Dis 142:91–99
Barnes PJ (1992) Frontiers in medicine: new aspects of asthma.
J Intern Med 231:453–461
Butcher RW, Sutherland EW (1962) Adenosine 30,50-phosphate in
biological materials. J Biol Chem 237:1244–1250
Chen J, Wu D, He F, Liu M, Wu H, Ding J, Su W (2008) Gallium(III)
triflate-catalyzed one-pot selective synthesis of 2,3-dihydroqui-
nazolin-4(1H)-ones and quinazolin-4(3H)-ones. Tetrahedron Lett
49:3814–3818
Clark RH, Wagner EC (1944) Isatoic anhydride. I. Reactions with
primary and secondary amines and with some amides. J Org
Chem 9:55–67
Crespo MI, Pages L, Vega A, Segarra V, Lopez M, Domenech T,
Miralpeix M, Beleta J, Ryder H, Palacios JM (1998) Design,
synthesis, and biological activities of new thieno[3,2-d]pyrim-
idines as selective type 4 phosphodiesterase inhibitors. J Med
Chem 4:4021–4035
Crocker CI, Townley RG (1999) Therapeutic potential of phosphodi-
esterase 4 inhibitors in allergic diseases. Drugs Today 35:519–535
Djukanovic R, Roche WR, Wilson JW, Beasley CRW, Twentyman
OP, Howarth PH, Holgate ST (1990) Mucosal inflammation in
asthma. Am Rev Respir Dis 142:434–457
Shafik RM, Hazzaa AAB, Habib NS (1979) 2-(Mono- and dinitro-
phenylthio)-3-substituted-4(3H)-quinazolinones: potential seda-
tive-hypnotics agents. Pharmazie 34:148–150
Tormena CF, Freitas MP, Rittner R, Abraham RJ (2002) A 1H NMR
and molecular modelling investigation of diastereotopic meth-
ylene hydrogen atoms. Magn Reson Chem 40:279–283
Torphy JT (1998) Phosphodiesterase isozymes molecular targets for
novel antiasthma agents. Am J Respir Crit Care Med 157:
351–370
Vassallo R, Lipsky JJ (1998) Theophylline: recent advances in the
understanding of its mode of action and uses in clinical practice.
Mayo Clin Proc 73:346–354
Elmaaty TA, Castle LW (2005) Chromate oxidation of a-nitro
alcohols to a-nitro ketones: significant improvements to a classic
method. Molecules 10:1458–1461
Fisher DA, Smith JF, Pillar JS, Denis SH, Cheng JB (1998) Isolation
and characterization of PDE 8A, a novel human cAMP specific
phosphodiesterase. Biochem Biophys Res Commun 246:570–577
123