Design, synthesis, and biological activity of certain quinazolinedione derivatives as potent phosphodiestrase4 inhibitors

Article abstract of DOI:10.1007/s00044-011-9892-x

In this study, a series of 3-βutylquinazolinedione linked with different substituent to N1 of quinazoline nucleus have been synthesized. Some of the new final compounds tested in vitro for their inhibitory activity against phosphodiestrase 4B which is the enzyme responsible for the hydrolysis of cyclic adenosine mono phosphate, the second messenger involved in the regulation of important cell functions. Compound 7f (100%) showed inhibition better than rolipram (90%), while the other tested compounds showed moderate activity. Docking study has been done to rationalize the obtained biological results. Springer Science+Business Media, LLC 2011.

Full text of DOI:10.1007/s00044-011-9892-x

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:3557–3567
DOI 10.1007/s00044-011-9892-x
ORIGINAL RESEARCH
Design, synthesis, and biological activity of certain
quinazolinedione derivatives as potent phosphodiestrase4
inhibitors
•
Afaf K. Elansary Hanan H. Kadry
•
•
Eman M. Ahmed Amr Sayed Motawi Sonousi
Received: 8 June 2011 / Accepted: 8 November 2011 / Published online: 25 November 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this study, a series of 3-butylquinazolinedi-
one linked with different substituent to N1 of quinazoline
nucleus have been synthesized. Some of the new final
compounds tested in vitro for their inhibitory activity
against phosphodiestrase 4B which is the enzyme respon-
sible for the hydrolysis of cyclic adenosine mono phos-
phate, the second messenger involved in the regulation of
important cell functions. Compound 7f (100%) showed
inhibition better than rolipram (90%), while the other tes-
ted compounds showed moderate activity. Docking study
has been done to rationalize the obtained biological results.
that c-AMP play a central role in regulating the function of
airway smooth muscle (Torphy, 1998), inflammatory cells
(Souness et al., 2000), and immune cells and the c-AMP
specific PDE4 is the predominant isoenzyme found in pro-
inflammatory cells associated with a number of airways
disorders (Barnes, 1999).
There are at least two main reasons for the basis of the
rapid development of the chemical, pharmacological and
biochemical research in the therapeutic utility of selective
PDE4 inhibitors. First, there is a general conviction that the
mixed anti-inflammatory and bronchodilator profile of
PDE4 inhibitors could allow, through the optimization of
first generation prototypes, the discovery of new agents
able to compete and, perhaps, to replace corticosteroids
which represent the basis of the therapeutic management of
asthma. Moreover, PDE4 inhibitors may be beneficial in
the treatment of chronic obstructive pulmonary disease
(COPD), a major respiratory disease for which pharmaco-
logical treatment is still inadequate (Norman, 1999).
Second, new and promising therapeutic applications of
PDE4 inhibitors in certain unmet autoimmune diseases,
e.g. rheumatoid arthritis, multiple sclerosis, and type 2
diabetes have emerged in recent years (Burnouf et al.,
1998).
Keywords Synthesis Á
Phosphodiestase4B (PDE4b) inhibitor Á Quinazolindiones Á
Docking study
Introduction
Phosphodiestrases (PDEs) are enzymes responsible for the
hydrolysis of cyclic adenosine mono phosphate (c-AMP)
and cyclic guanosine mono phosphate (c-GMP) which are
second messengers involved in the regulation of important
cell functions such as secretion, contraction, metabolism,
and growth (Potter, 1990). The inhibition of PDEs activity
increases cellular levels of the key second messengers
c-AMP and c-GMP, thereby activating specific protein
phosphorylation cascade that elicit a variety of functional
response (Palacios et al., 1995). Strong evidence suggests
The PDE4 family is comprised of four primary gene
products (PDE4A, PDE4B, PDE4C, and PDE4D) and is
highly expressed in neutrophils and monocytes, CNS tis-
sue, and smooth muscles of the lung (Bender and Beavo,
2006; McKenna and Muller, 2006; Zhang et al., 2005).
Knockout studies have revealed that PDE4B ablation
suppresses TNF-a production, and PDE4D may be
responsible for the occurrence of nausea and emesis
(Robichaud et al., 2002; Zhang et al., 2005), heart failure,
and the risk of arrhythmias (Lehnart et al., 2005).
A. K. Elansary (&) Á H. H. Kadry Á E. M. Ahmed Á
A. S. M. Sonousi
Department of Organic Chemistry, Faculty of Pharmacy, Cairo
University, Cairo 11562, Egypt
e-mail: hanankadry2005@yahoo.com
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Med Chem Res (2012) 21:3557–3567
From a structural point of view, selective PDE4 inhib-
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 (KBr,
cm-1). 1H-NMR, C13-NMR spectra were recorded on
Gemini Varian-VXR-unity (200 MHz), Gemini Varian
500 MHz (Germany) or Bruker UXNMR/XWIN-NMR
(300 MHz) instruments. Chemical shifts (d) are reported in
ppm downfield from internal TMS standard. EI-MS Hew-
lett 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.
Elemental analyses were carried out in the Micro analytical
Center, Cairo University, Egypt. Melting points were
determined with an electro thermal melting point appara-
tus, and were uncorrected. On the other hand, 2-amino-N-
butylbenzamide (1) (Clark and Wagner, 1944) was
synthesized according to reported procedures. While
3-butylquinazoline-2,4(1H, 3H)-dione (3) was synthesized
by a new procedure rather than the reported one (Staiger
and Wagner, 1953).
itors in the public domain can be divided into three classes:
structural analogues of rolipram, structural analogues of
nitraquazone, and structures related to xanthines (Fig. 1).
In fact rolipram, since its discovery as a potent and
selective PDE 4 inhibitor (Schneider et al., 1986), has
represented a useful pharmacological tool for the charac-
terization of this isoenzyme in different tissues, as well as
the main template for the synthesis of novel inhibitors.
However, severe limiting side effects (nausea, vomiting,
headache) precluded the development of rolipram and
many promising candidates have been discontinued.
Although a number of studies claiming different chemical
classes of PDE 4 inhibitors are increasing in recent years,
only few detailed studies evaluated the PDE4 inhibition of
structural analogues of nitraquazone. These compounds
could be devoid of the central side effects of the archetypal
rolipram which hampered its development as a drug (Piaz
and Giovannoni, 2000).
On this basis, this work was directed to synthesize a
hybrid structure containing the quinazoline-2,4-dione
nucleus of nitraquazone along with the n-butyl side chain
of denbufylline. This scaffold is linked with different
substituents at N1, which were chosen by selecting the ones
with highest scores in a virtual screening study. Moreover,
this paper describes the synthesis, PDE4B inhibition eval-
uation and docking studies for series of quinazolinones
structures. Docking results were compared to biological
data with the aim of obtaining useful information for the
rational design of new PDE4B antagonist.
2-Ethoxycarbonylamino-N-butylbenzamide (2)
A
mixture containing 2-amino-N-butylbenzamide (1)
(1.92 g, 0.01 mol) and ethyl chloroformate (28.5 g, 25 ml,
0.26 mol) was heated over steam for 3 h. The reaction
mixture was evaporated under reduced pressure. The solid
formed crystallized from ethanol/water mixture.
Yield: 80% mp.: 60°C. IR tmax (cm-1) (KBr): 3323
(2 NH), 2958, 2933 (CH aliphatic), 1737 (carbamate C=O),
1662 (C=O). 1HNMR (500 MHz, CDCl3-) dppm: 0.90(t, 3H,
CH3), 1.31(t, 3H, J = 7.5 Hz, OCH2CH3), 1.38–1.46(m, 2H,
CH2CH3), 1.58–1.64(m, 2H, CH2CH2CH3), 3.42(q, 2H,
N3CH2), 4.20(q, 2H, J = 7.5 Hz, OCH2CH3), 6.24(s, 1H,
NH, D2O exchangeable), 7.01(dd, 1H, Ar–C5H), 7.40–7.49
(m, 2H, Ar–C3H ? Ar–C4H), 8.38(d, 1H, Ar–C6H), 10.40(s,
1H, NH). 13C NMR (CDCl3) dppm: 13.7 (CH3), 14.5 (CH3),
20.2 (CH2), 31.5 (CH2), 39.7 (CH2–NH), 61.0 (OCH2),
Methods and materials
Chemistry
All chemicals and reagents were obtained from Aldrich
(Sigma-Aldrich, St. Louis, MO, USA), Lancaster (Alfa
Aesar, Johnson Company, Ward Hill, MA, USA) and
were used without further purification. Reactions were
Fig. 1 Compounds
O
representative of the three
chemical classes of PDE 4
inhibitors: rolipram,
nitraquazone, and xanthine
derivatives (denbufylline)
O
O
O
N
N
N
N
N
O
O
O
N
O
N
H
NO2
Rolipram
Nitraquazone
Denbufylline
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Med Chem Res (2012) 21:3557–3567
3559
3-Butyl-1-[2-(pyrrolidin-1-yl)ethyl]quinazoline-
114.7–139.9 (Ar–C), 153.9 (C=O), 168.8 (C=O). ESIMS m/z
(% rel. abundance): 265.21(M ? 1, 100%). Anal. calcd. for
C14H20N2O3: C, 63.62; H, 7.63; N, 10.60; Found C, 63.92; H,
7.34; N, 10.90.
2,4(1H,3H)-dione (5a)
Yield: 75% mp.: 75°C (ethanol). IR tmax (cm-1) (KBr): 2958,
2931, 2872(CHaliphatic),1699(C=O),1654(C=O).1HNMR
(300 MHz, CDCl3-) dppm: 0.96(t, 3H, CH3), 1.35–1.47(m,
2H, CH2CH3), 1.62–1.72(m, 2H, CH2CH2CH3),
1.80–1.84(m, 4H, C3 & C4 of pyrrolidine), 2.64–2.68(m, 4H,
3-Butylquinazoline-2,4(1H,3H)-dione (3)
A mixture of 2-ethoxycarbonylamino-N-butylbenzamide
(2) (7.92 g, 0.03 mol) and KOH (3.36 g, 0.06 mol) in
absolute ethanol (150 ml) was refluxed over steam for 4 h.
The reaction mixture was evaporated under reduced pres-
sure. The residue obtained was dissolved in a minimum
amount of water, which was adjusted to pH 7–8 with acetic
acid. The precipitated product was crystallized from etha-
nol/water mixture. Yield: 5.5 g (84%) mp.: 156–157°C (as
reported).
C2
& C5 of pyrrolidine), 2.79(t, 2H, J = 7.5 Hz,
N1CH2CH2N), 4.08(t, 2H, N3CH2), 4.29(t, 2H, J = 7.5 Hz,
N1CH2), 7.21–7.29(m, 2H, C6H & C8H of quinazoline),
7.65(dd, 1H, C7H of quinazoline), 8.28(d, 1H, C5H of qui-
nazoline). ESIMS m/z (% rel. abundance): 316.30 (M ? 1,
100%). Anal. calcd for C18H25N3O2: C, 68.54; H, 7.99; N,
13.32; Found C, 68.60; H, 7.92; N, 12.96.
3-Butyl-1-[2-(morpholin-4-yl)ethyl]quinazoline-
2,4(1H,3H)-dione (5b)
3-Butyl-1-(2-chloroethyl) quinazoline-2,4(1H,3H)-
Yield: 70% mp.: 102–104°C (ethanol). IR tmax (cm-1) (KBr):
2962, 2931(CHaliphatic),1697(C=O),1651(C=O).1HNMR
(500 MHz, CDCl3-) dppm: 0.95(t, 3H, CH3), 1.37–1.44(m,
2H, CH2CH3), 1.64–1.70(m, 2H, CH2CH2CH3),
2.47–2.53(m, 4H, C3 & C5 of morpholine), 2.75(t, 2H,
J = 7.5 Hz, N1CH2CH2N), 3.65–3.82(m, 4H, C2 & C6 of
morpholine), 4.08(t, 2H, N3CH2), 4.29(t, 2H, J = 7.5 Hz,
N1CH2), 7.22–7.27(m, 2H, C6H & C8H of quinazoline),
7.62(dd, 1H, C7H of quinazoline), 8.25(d, 1H, C5H of qui-
nazoline). ESIMS m/z (% rel. abundance): 332.27 (M ? 1,
100%). Anal. calcd for C18H25N3O3 : C, 65.23; H, 7.60; N,
12.68; Found C, 65.19; H, 7.56; N, 12.49.
dione (4)
A ternary mixture of 3-butylquinazoline-2,4(1H,3H)-dione
(3) (2.18 g, 0.01 mol), 1-bromo-2-chloroethane(7.17 g,
4.16 ml, 0.05 mol) and anhydrous K2CO3 (6.90 g,
0.05 mol) in dry DMF (30 ml) was stirred at room tem-
perature over night. The mixture was poured onto water
and the formed precipitate was filtered, dried, and crys-
tallized from ethanol/water mixture.
Yield: 64% mp.: 83°C. IR tmax (cm-1) (KBr): 2954,
2931, 2870 (CH aliphatic), 1701 (C=O), 1662 (C=O).
1HNMR (500 MHz, CDCl3-) dppm: 0.95(t, 3H, CH3),
1.37–1.45(m,
2H,
CH2CH3),
1.64–1.70(m,
2H,
CH2CH2CH3), 3.80(t, 2H, J = 7.0 Hz, CH2Cl), 4.08(t, 2H,
N3CH2), 4.45(t, 2H, J = 7.0 Hz, N1CH2), 7.23–7.29(m,
2H, C6H & C8H of quinazoline), 7.58(dd, 1H, C7H of
quinazoline), 8.24(d, 1H, C5H of quinazoline). 13C NMR
(CDCl3) dppm: 13.8 (CH3), 20.2 (CH2), 29.9 (CH2), 39.6
(CH2Cl), 41.8 (CH2–N3), 44.9 (CH2–N1), 113.2–139.6
(Ar–C), 150.7 (C=O), 161.4 (C=O). Anal. calcd. for
C14H17ClN2O2 : C, 59.89; H, 6.10; N, 9.98; Found C,
60.19; H, 6.12; N, 9.93.
3-Butyl-1-[2-(piperidin-1-yl)ethyl]quinazoline-
2,4(1H,3H)-dione (5c)
Yield: 78% mp.: 98–100°C (ethanol). IR tmax (cm-1) (KBr):
2931, 2862 (CH aliphatic), 1697 (C=O), 1654(C=O). 1HNMR
(300 MHz, DMSO) dppm: 0.89(t, 3H, CH3), 1.26–1.58(m,
10H, 2CH2 ?3CH2 of C3, C4, C5 piperidine), 2.39–2.52(m,
6H, CH2 ? 2CH2 of C2, C6 piperidine), 3.94 (t, 2H, N3CH2),
4.20(t, 2H, N1CH2), 7.27(dd, 1H, C6H of quinazoline), 7.45
(d, 1H, C8Hofquinazoline),7.75(dd,1H, C7H of quinazoline),
8.04(d, 1H, C5Hofquinazoline), Anal. calcd forC19H27N3O2 :
C, 69.27;H, 8.26; N, 12.76;FoundC, 69.49;H, 8.25; N, 12.77.
General procedure for the synthesis of compounds 5a–e
To the solution of 3-butyl-1-(2-chloroethyl) quinazoline-
2,4(1H,3H)-dione (4) (0.84 g, 0.003 mol) in dry acetoni-
trile (20 ml), anhydrous K2CO3 (2.07 g, 0.015 mol) and
few specs of KI were added and the mixture was heated
under reflux for 30 min. Appropriate amine (0.009 mol)
was then added slowly into the reaction mixture. The
resulting mixture was heated under reflux for 15 h, cooled
and poured onto ice-cold water. The separated solid was
filtered, dried, and crystallized from a suitable solvent.
3-Butyl-1-[2-(4-phenylpiperazin-1-yl)
ethyl]quinazoline-2,4(1H,3H)-dione (5d)
Yield: 69% mp.: 78–84°C (hexane/ethanol). IR tmax
(cm-1
)
(KBr): 2954, 2927, 2831 (CH aliphatic),
1697(C=O), 1647 (C=O). 1HNMR (300 MHz, CDCl3-
)
dppm: 0.95(t, 3H, CH3), 1.35–1.47(m, 2H, CH2CH3),
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Med Chem Res (2012) 21:3557–3567
1.63–1.73(m, 2H, CH2CH2CH3), 2.75–2.81(m, 6H,
N1CH2CH2N ?2CH2 piperazine), 3.22–3.25(m, 4H, 2CH2
piperazine), 4.09(t, 2H, N3CH2), 4.36(t, 2H, N1CH2),
6.85–8.25(m, 9H, ArHs). EIMS m/z (% rel. abundance):
406.10 (M?, 22.62%), 407.00 (M ? 1, 7.31%),
175.05(100%). Anal. calcd for C24H30N4O2: C, 70.91; H,
7.44; N, 13.78. Found C, 70.86; H, 7.35; N, 13.66.
extracted with chloroform. The chloroform layer was
washed with water, dried over anhydrous sodium sulfate,
and evaporated. The oily product was dissolved in acetone
and treated with ethereal hydrochloride. The separated
solid was refrigerated for 48 h, filtered and dried.
3-Butyl-1-[3-(piperidin-1-yl)propyl]quinazoline-
2,4(1H,3H)-dione hydrochloride (7a)
3-Butyl-1-[2-(diethylamino)ethyl]quinazoline-
Yield 40% mp.: 210°C. IR tmax (cm-1) (KBr): 3421(NH),
2951, 2808 (CH aliphatic), 1701(C=O), 1654 (C=O).
1HNMR (300 MHz, DMSO-) dppm: 0.90(t, 3H, CH3),
2,4(1H,3H)-dione (5e)
Yield: 60% mp.: 46–48°C (ethanol). IR tmax (cm-1) (KBr):
2966, 2931 (CH aliphatic), 1701(C=O), 1654 (C=O).
1HNMR (300 MHz, CDCl3) dppm: 0.96(t, 3H, CH3),
1.05(t, 6H, J = 7 Hz, NCH2CH3) 1.35–1.47(m, 2H,
CH2CH3), 1.63–1.73(m, 2H, CH2CH2CH3), 2.64(q, 4H,
J = 7 Hz, NCH2CH3), 2.73(t, 2H, J = 7.5 Hz,
N1CH2CH2N), 4.09(t, 2H, N3CH2), 4.22(t, 2H, J = 7.5 Hz,
N1CH2), 7.23–7.27(m, 2H, C6H & C8H of quinazoline),
7.65(dd, 1H, C7H of quinazoline), 8.24(d, 1H, C5H of
quinazoline). Anal. calcd for C18H27N3O2 : C, 68.11; H,
8.57; N, 13.24; Found C, 68.23; H, 8.38; N, 13.02.
1.27–1.34(m,
2H,
CH2CH3),
1.54–1.59(m,
2H,
CH2CH2CH3), 1.63–1.74(m, 6H, CH2 of C3,C4,C5 of
piperidine), 2.05–2.10(m, 2H, N1CH2CH2CH2N), 2.82(t,
2H, N1CH2CH2CH2N), 3.02–3.12(m, 4H, C2,C6 of piperi-
dine), 3.94(t, 2H, N3CH2), 4.17(t, 2H, N1CH2), 7.30(dd,
1H, C6H of quinazoline), 7.55 (d, 1H, C8H of quinazoline),
7.77(dd, 1H, C7H of quinazoline), 8.07(d, 1H, C5H of
quinazoline), 9.94(s, 1H, NH of HCl salt). Anal. calcd for
C20H29N3O2.HCl. C, 63.22; H, 7.95; N, 11.06; Found C,
63.12; H, 7.87; N, 11.10.
3-Butyl-1-(3-chloropropyl) quinazoline-2,4(1H,3H)-
3-Butyl-1-[3-(4-phenylpiperazin-1-yl)propyl]
dione (6)
quinazoline-2,4(1H,3H)-dione hydrochloride (7b)
A mixture of 3-butylquinazoline-2,4(1H,3H)-dione (3)
(2.18 g, 0.01 mol), 1-bromo-3-chloropropane (4.72 g,
2.968 ml, 0.03 mol) and anhydrous K2CO3 (6.90 g,
0.05 mol) in dry DMF (30 ml) was stirred over night at
room temperature. The mixture was poured onto cold water
and the formed precipitate was filtered, dried, and crys-
tallized from ethanol/water mixture.
Yield 55% mp.: 180°C. IR tmax (cm-1) (KBr): 3487(NH),
2981, 2866 (CH aliphatic), 1697(C=O), 1654 (C=O).
1HNMR (200 MHz, DMSO-) dppm:0.94(t, 3H, CH3), 1.24–
1.44(m, 2H, CH2CH3), 1.54–1.70(m, 2H, CH2CH2CH3),
2.00–2.32(m, 2H, N1CH2CH2CH2N), 3.10–3.34(m, 4H,
2CH2 of piperazine) 3.40(t, 2H, N1CH2CH2CH2N),
3.70–3.92(m, 4H, 2CH2 of piperazine), 3.98(t, 2H, N3CH2),
4.24(t, 2H, N1CH2), 6.88–8.09(m, 4H, ArHs), 11.38(s, 1H,
NH of HCl salt, D2O exchangeable). EIMS m/z (% rel.
abundance): 420 (M?, 30.36%), 421 (M ? 1, 10.19%), 175
(100%). Anal. calcd for C25H32N4O2.HCl. C, 65.70; H, 7.27;
N, 12.25; Found C, 65.67; H, 7.24; N, 12.22.
Yield: 68% mp.: 90°C. IR tmax (cm-1) (KBr): 2960,
2933, 2873, 2862 (CH aliphatic), 1699 (C=O), 1658
1
(C=O). HNMR (200 MHz, CDCl3-) dppm: 0.92(t, 3H,
CH3), 1.31–1.46(m, 2H, CH2CH3), 1.45–1.75(m, 2H,
CH2CH2CH3), 2.16–2.29(m, 2H, CH2CH2Cl), 3.69(t, 2H,
CH2Cl), 4.08(t, 2H, N3CH2), 4.26(t, 2H, N1CH2),
7.17–8.26(m, 4H, ArHs). EIMS m/z (% rel. abundance):
294 (M?, 22.94%), 296 (M ? 2, 9.64%), 132 (100%).
Anal. calcd for C15H19ClN2O2: C, 61.12; H, 6.50; N, 9.50;
Found: C, 61.20; H, 6.40; N, 9.30.
3-Butyl-1-{3-[4-(4-methoxyphenyl)piperazin-1-yl]
propyl}quinazoline-2,4(1H,3H)-dione hydrochloride
(7c)
Yield (50%) mp.: 152°C. IR tmax (cm-1) (KBr): 3485(NH),
2980, 2858 (CH aliphatic), 1701 (C=O), 1654 (C=O). 1HNMR
(300 MHz, DMSO) dppm: 0.89(t, 3H, CH3), 1.26–1.35(m, 2H,
CH2CH3), 1.52–1.62(m, 2H, CH2CH2CH3), 2.10–2.19(m, 2H,
N1CH2CH2CH2N), 2.90–3.15(m, 6H, 2CH2 of pipera-
zine ? N1CH2CH2CH2N), 3.49–3.57(m, 4H, 2CH2 of piper-
azine), 3.68(s, 3H, OCH3), 3.94(t, 2H, N3CH2), 4.19(t, 2H,
N1CH2), 6.9–6.82 (dd, 4H, ArHs), 7.30(dd, 1H, C6H of qui-
nazoline), 7.56(d, 1H, C8H of quinazoline), 7.77(dd, 1H, C7H
General procedure for the synthesis of compounds 7a–f
To a solution of 3-butyl-1-(3-chloropropyl) quinazoline-
2,4(1H, 3H)-dione (6) (0.88 g, 0.003 mol) in dry acetoni-
trile (20 ml), few specs of KI and anhydrous K2CO3
(2.07 g, 0.015 mol) were added. The resulted mixture was
refluxed for 30 min. Appropriate amine (0.009 mol) was
then added slowly into the reaction mixture and refluxed
for 15 h. The mixture was cooled, diluted with water, and
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Med Chem Res (2012) 21:3557–3567
3561
of quinazoline), 8.08(d, 1H, C5H of quinazoline), 10.72(s, 1H,
NH of HCl salt, D2O exchangeable). EIMS m/z (% rel. abun-
dance):450 (M?, 100%), 451 (M ? 1, 29.68%). Anal. calcd for
C26H34N4O3.HCl C, 64.11; H, 7.24; N, 11.50; Found C, 64.13;
H, 7.13; N, 11.46.
CH2 ofC3, C5 ofmorpholine andN3CH2), 4.25(t, 2H, N1CH2),
5.39(s, 1H, NH of HCl salt, D2O exchangeable), 7.23(dd, 1H,
C6H of quinazoline), 7.36 (d, 1H, C8H of quinazoline),
7.68(dd, 1H, C7H of quinazoline), 8.18(d, 1H, C5H of qui-
nazoline), Anal. calcd for C19H27N3O3.HCl C, 59.75; H, 7.39;
N, 11.00; Found C, 59.63; H, 7.42; N, 11.10.
3-Butyl-1-[3-(diethylamino)propyl]quinazoline-
2,4(1H,3H)-dione hydrochloride (7d)
1-Benzyl-3-butylquinazoline-2,4(1H,3H)-dione (8)
Yield (42%) mp.: 127°C. IR tmax (cm-1) (KBr): 3417(NH),
2954, 2870 (CH aliphatic), 1701 (C=O), 1654 (C=O).
1HNMR (300 MHz, DMSO) dppm: 0.90(t, 3H, CH3),
1.20(t, 6H, J = 7 Hz, NCH2CH3), 1.24–1.37(m, 2H,
CH2CH2CH3), 1.49–1.61(m, 2H, CH2CH2CH3), 1.99–
2.09(m, 2H, N1CH2CH2CH2N), 3.05(q, 4H, J = 7 Hz,
NCH2CH3), 3.15(t, 2H, N1CH2CH2CH2N), 3.94(t, 2H,
N3CH2), 4.18(t, 2H, N1CH2), 7.30(dd, 1H, C6H of qui-
nazoline), 7.61(d, 1H, C8H of quinazoline), 7.76(dd, 1H,
C7H of quinazoline), 8.07(d, 1H, C5H of quinazoline),
10.76(s, 1H, NH of HCl salt). ESIMS m/z (% rel.
abundance): 332.39(M ? 1, 100%). Anal. calcd for
C19H29N3O2.HCl C, 62.02; H, 8.21; N, 11.42; Found C,
62.12; H, 8.43; N, 11.41.
A mixture of 3-butylquinazoline-2,4(1H,3H)-dione (3)
(0.218 g, 0.001 mol), benzyl chloride (0.25 g, 0.002 mol),
anhydrous K2CO3 (0.69 g, 0.005 mol) and few specs of KI
in dry acetone (15 ml) was stirred and refluxed for 7 h. The
reaction mixture was cooled and poured onto cold water.
The formed precipitate was filtered, dried and crystallized
from ethanol.
Yield: 0.25 g (81%) mp.: 109–111°C. IR tmax (cm-1
)
(KBr): 2958 (CH aliphatic), 1701(C=O), 1654(C=O).
1HNMR (300 MHz, CDCl3-) dppm: 0.99(t, 3H, CH3),
1.38–1.51(m,
2H,
CH2CH3),
1.66–1.79(m,
2H,
CH2CH2CH3), 4.16 (t, 2H, N3CH2), 5.38(s, 2H, N1CH2),
7.10–8.25(m, 9H, ArHs). Anal. calcd for C19H20N2O2 C,
74.00; H, 6.54; N, 9.08; Found C, 74.21; H, 6.43; N, 9.11.
3-Butyl-1-[3-(pyrrolidin-1-yl)propyl]quinazoline-
General procedure for the synthesis of compounds 9a–c
2,4(1H,3H)-dione hydrochloride (7e)
A mixture of 3-butylquinazoline-2,4(1H,3H)-dione (3)
(0.218 g, 0.001 mol), appropriate phenacyl bromide
(0.001 mol) and anhydrous K2CO3 (0.69 g, 0.005 mol) in
dry DMF (15 ml) was stirred and refluxed for 3 h The
reaction mixture was cooled, poured onto cold water and
the solid formed was filtered, dried, and crystallized from
ethanol.
Yield (39%) mp.: 142°C. IR tmax (cm-1) (KBr): 3412(NH),
2956, 2872 (CH aliphatic), 1701 (C=O), 1654 (C=O).
1HNMR (500 MHz, CDCl3-) dppm: 0.91(t, 3H, CH3),
1.30–1.38(m, 2H, CH2CH2CH3), 1.56–1.63(m, 2H,
CH2CH2CH3), 1.99–2.07(m, 2H, C3H of pyrrolidine),
2.13–2.22(m, 2H, C4H of pyrrolidine), 2.32–2.38(m, 2H,
N1CH2CH2CH2N), 2.80(t, 2H, N1CH2CH2CH2N),
3.20–3.24(m, 2H, C2H of pyrrolidine), 3.74–3.77(m, 2H,
C5H of pyrrolidine), 4.00(t, 2H, N3CH2), 4.22(t, 2H,
N1CH2), 7.20(dd, 1H, C6H of quinazoline), 7.32 (d, 1H,
C8H of quinazoline), 7.64 (dd, 1H, C7H of quinazoline),
8.18(d, 1H, C5H of quinazoline), 12.42(s, 1H, NH of HCl
salt). EIMS m/z (% rel. abundance): 329 (M?, 8.05%),
330 (M ? 1, 1.97%), 84 (100%). Anal. calcd for
C19H27N3O2.HCl C, 62.36; H, 7.71; N, 11.48; Found C,
62.18; H, 7.49; N, 11.46.
3-Butyl-1-(2-oxo-2-phenylethyl)quinazoline-
2,4(1H,3H)-dione (9a)
Yield: (65%) mp.: 110°C. IR tmax (cm-1) (KBr): 2956,
2931(CH aliphatic), 1695(C=O), 1625–1665(C=O).
1HNMR (200 MHz, CDCl3-) dppm: 0.96(t, 3H, CH3),
1.36–1.47(m,
2H,
CH2CH3),
1.63–1.72(m,
2H,
CH2CH2CH3), 4.11(t, 2H, N3CH2), 5.62(s, 2H, N1CH2),
6.79–8.28(m, 9H, ArHs). EIMS m/z (% rel. abundance):
336.20 (M?, 12.88%), 337.20 (M ? 1, 3.13%),
105.05(100%). Anal. calcd for C20H20N2O3: C, 71.41; H,
5.99; N, 8.33; Found C, 71.71; H, 5.79; N, 8.23.
3-Butyl-1-[3-(morpholin-4-yl)propyl]quinazoline-
2,4(1H,3H)-dione hydrochloride (7f)
Yield (45%) mp.: 130°C. IR tmax (cm-1) (KBr): 3425(NH),
2970, 2870 (CH aliphatic), 1697(C=O), 1654 (C=O). 1HNMR
(300 MHz, CDCl3-) dppm: 0.93(t, 3H, CH3), 1.31–1.43(m,
2H, CH2CH2CH3), 1.57–1.67(m, 2H, CH2CH2CH3),
2.34–2.39(m, 2H, N1CH2CH2CH2N), 3.14–3.20(m, 6H, C2,
C6 of morpholine and N1CH2CH2CH2N), 3.94–4.06(m, 6H,
3-Butyl-1-[2-(4-chlorophenyl)-2-oxoethyl]quinazoline-
2,4(1H,3H)-dione (9b)
Yield: (68%) mp.: 212°C. IR tmax (cm-1) (KBr): 2958,
2935(CH aliphatic), 1701(C=O), 1650–1675(C=O). 1HNMR
123

3562
Med Chem Res (2012) 21:3557–3567
(200 MHz, CDCl3-) dppm: 0.96(t, 3H, CH3), 1.36–1.43(m,
2H, CH2CH3), 1.63–1.73(m, 2H, CH2CH2CH3), 4.10(t, 2H,
N3CH2), 5.57(s, 2H, N1CH2), 6.79–8.28(m, 8H, ArHs). EIMS
m/z (% rel. abundance): 370.15 (M?, 7.64%), 371.20 (M ? 1,
2.18%), 372.20 (M ? 2, 2.86%), 132.10(100%). Anal. calcd
for C20H19ClN2O3 C, 64.78; H, 5.16; N, 7.55; Found C, 63.78
H, 5.00; N, 7.25.
The percent activity in the presence of the compound was
calculated according to the following equation: % activ-
ity = (FP - FPb)/(FPt - FPb) 9 100%, where FP is the
fluorescence polarization in the presence of the compound.
The fluorescence polarizations of PDE4B were then mea-
sured in the presence of the synthesized compounds and
consequently their % inhibition were calculated.
1-[2-(4-Bromophenyl)-2-oxoethyl]-3-butylquinazoline-
Molecular docking
2,4(1H,3H)-dione (9c)
All the molecular modeling studies were carried out on an
Intel Pentium 1.6 GHz processor, 512 MB memory with
Windows XP operating system using Molecular Operating
Environment (MOE 2008.10; Chemical Computing Group,
Canada) (MOE 2008.10) as the computational software.
All the minimizations were performed with MOE until a
Yield: (71%) mp.: 223°C. IR tmax (cm-1) (KBr):
2956, 2935 (CH aliphatic), 1703 (C=O), 1635–1670
1
(C=O). HNMR (500 MHz, CDCl3-) dppm: 0.95(t, 3H,
CH3), 1.38–1.45(m, 2H, CH2CH3), 1.66–1.72(m, 2H,
CH2CH2CH3), 4.12(t, 2H, N3CH2), 5.59(s, 2H, N1CH2),
6.82(d, 1H, C8H of quinazoline), 7.26 (dd, 1H, C6H of
quinazoline), 7.57(dd, 1H, C7H of quinazoline), 7.65(dd,
J = 8 Hz, 2H, C3,C5 of 4-Br-ph), 7.95(dd, 2H, J = 8 Hz,
C2,C6 of 4-Br-ph), 8.35(d, 1H, C5H of quinazoline). 13C
NMR (CDCl3) dppm: 13.79 (CH3), 20.21 (CH2), 29.88
(CH2), 41.96 (CH2–N3), 49.57 (CH2–N1), 113.08–139.81
(Ar–C), 151.11(C=O), 161.54(C=O), 191.20(Ph–C=O).
Anal. calcd for C20H19BrN2O3 C, 57.84; H, 4.61; N, 6.75;
Found C, 58.14; H, 4.36; N, 6.68.
-1
RMSD gradient of 0.05 kcal mol-1
with MMFF94x
˚
A
force-field and the partial charges were automatically
calculated.
The X-ray crystallographic structure of phosphodies-
terase 4B complexed with rolipram (PDB ID: 1RO6) was
obtained from the protein data bank available at the RCSB
˚
Protein Data Bank, http://www.pdb.org) with a 2.00 A
resolution
1. Enzyme structures were checked for missing atoms,
bonds and contacts.
2. Water molecules were manually deleted.
3. Hydrogens and partial charges were added to the
system using Protonate3D application.
Biological activity
4. The active site was generated using the residues within
˚
5 A near to the rolipram atoms.
Evaluation of PDE4B inhibitory activity
5. The ligand molecules were constructed using the
builder module and were energy minimized using the
MMFF94x force field.
With several new analogs now in hand, we evaluated their
inhibitory potency against PDE4B via a purified enzyme
fluorescence polarization assay. PDE activity assays were
performed in duplicate at 10 lM. Fluorescence intensity is
converted to fluorescence polarization using the Magellan6
software. The fluorescence polarization data were analyzed
using the computer software, Graphpad Prism. 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 concentration 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 compound. After the enzy-
matic reaction, 100 ll of a binding solution (1:100 dilution
of the binding agent with the binding agent diluents) 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.
6. All antagonist structures were docked into the active
site by using the MOE Dock tool. This method is
divided into a number of stages:
(a) Conformational analysis of ligands: the algorithm
generated conformations from a single 3D con-
formation by conducting a systematic search. In
this way, all combinations of angles were created
for each ligand.
(b) Placement: a collection of poses was generated
from the pool of ligand conformations using
Triangle Matcher placement method. Poses were
generated by superposition of ligand atom triplets
and triplets of points in the receptor binding site
in a systematic way
(c) Scoring: poses generated by the placement
methodology were scored using London dG
scoring function implemented in MOE, which
123

Med Chem Res (2012) 21:3557–3567
3563
estimates the free energy of binding of the ligand
Result and discussion
from a given pose. The top 30 poses for each
ligand were output in a MOE database. Each
resulting ligand pose was then subjected to
MMFF94x energy minimization until the RMS
The starting compound 2-amino-N-butylbenzamide 1 was
prepared as the reported procedure (Clark and Wagner, 1944).
Formylation of 1 with ethyl chloroformate afforded 2-eth-
oxycarbonylamino-N-butylbenzamide 2 which was cyclized
to the desired key compound 3. We report here a new appli-
cation of the method described by Gadekar et al. (Gadekar
et al., 1964) for the preparation of 3-butylquinazoline-2,4(1H,
gradient of the potential energy was less than
0.05 kJ mol mol-1 -1. The minimized docking
A
˚
conformations were then re-scored using London
dG scoring methods.
O
O
O
O
NH
NH
a
b
N
H
O
NH2
OEt
NH
1
2
O
c
O
N
N
H
O
3
O
d
N
f
N
O
4
Cl
O
N
e
N
O
O
6
C l
N
R1 R2
g
N
O
N
5a :
5b :
N
N
O
N
R1
R2
O
N
R1 R2
5c :
5d :
5 (a-e)
N
N
7 :
a
N
N
.HCl
O
C H
C H
3
3
N
N
N
7 :
b
N
5e :
7 :
N
N
O
CH
c
3
CH
R2
3
3
N
7 :
7 (a-f)
d
CH
R1
7 :
N
N
e
O
7 :
f
Scheme 1 Synthetic protocol for title compounds 5a–e and
7a–f. Reagents and conditions: (a) butyl amine (b) ethyl chlorofor-
mate, heated over steam, 3 h (c) KOH, ethanol, heated over steam,
4 h (d) 1-bromo-2-chloroethane, anhydrous K2CO3, dry DMF, rt,
overnight (e) HNR1R2 (f) 1-bromo-3-chloropropane, anhydrous
K2CO3, dry DMF, rt, overnight (g) (1) HNR1R2 (2) ethereal HCl
123

3564
Med Chem Res (2012) 21:3557–3567
3H)-dione 3 by heating the intermediate compound 2 with
ethanolic potassium hydroxide. Treatment of quinazolinedi-
one 3 with either 1-bromo-2-chloro ethane or 1-bromo-3-
chloropropane using standard reaction conditions (Blizzard
et al., 1989) afforded the intermediate compounds 4 and 6,
respectively. Nucleophilic displacement of the chlorine atom
on the substituted quinazoline ring with different secondary
amines afforded compounds 5a–e and 7a–f. On the other
hand, Scheme 2 describes alkylation of the key compound 3
with either benzyl chloride or substituted phenacyl chloride to
give the desired compounds 8 and 9a–c. Structures of the new
compounds 2, 4, 5a–e, 7a–f, 8, and 9a–c were confirmed by
elemental analysis and spectral data. The synthetic routes for
the preparation of the new compounds are outlined in
Schemes 1 and 2.
Table 1 Inhibition data for derivatives investigated in the present
paper and standard rolipram against PDE4B
Compound (10 lM)
Mean% inhibition
No compound
0
Rolipram
90
17
11
25
8
5b
5c
5d
5e
7a
7b
7d
7f
11
13
9
100
56
22
8
8
9a
9b
Results of PDE4B inhibition activity assessement are
very promising as most of the compounds in the present
series had PDE4B inhibition activity (Table 1). Compound
7f is the most active one, it demonstrated inhibitory activity
(100%) better than rolipram (90%), while 5d (25%), 8
(56%), and 9a (22%) showed moderate activity. The rest of
the compounds showed mild inhibitory activity (8–13%).
The structural feature of the highly active compounds was
found to be significantly different as compound 7f has a
propyl spacer linked to the morpholine nucleus, while
compound 5d has an ethyl spacer linked to phenyl piper-
azine nucleus and compound 8 has a methylene spacer so a
docking study was very important to find an explanation to
these biological results.
Molecular docking studies of the synthesized com-
pounds were performed in order to rationalize the obtained
biological results as well as to help us in understanding the
various interactions between the ligands and enzyme active
site in details.
The X-ray crystallographic structure of PDE4B complexed
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
Protonate3D algorithm. Ligand molecules were modeled
Scheme 2 Synthetic protocol
for title compounds. Reagents
and conditions:
O
N
(a) benzylchloride, dry acetone,
reflux, 7 h (b) p-substituted
phenacyl bromide, anhydrous
K2CO3, dry DMF, reflux, 3 h
N
H
O
3
a
b
O
N
O
N
N
N
O
O
O
R
9a: H
8
9b: Cl
9c: Br
R
9(a-c)
123

Med Chem Res (2012) 21:3557–3567
3565
using MOE builder, and the structureswere energyminimized
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.
nitraquazone was done by docking nitraquazone in the active
site gorge of PDE4B (Fig. 2b). This binding model was useful
in our interpretation to the activities of our synthesized
compounds.
Then, we performed docking studies to our synthesized
compound and the final docked complexes of ligand–
enzyme were selected according to the criteria of interac-
tion energy combined with geometrical matching quality.
The saved pose for the ligand–enzyme complex of the
most active compound 7f (Fig. 3) revealed that several
molecular interactions were considered to be responsible
˚
The RMSD of the docked ligand was 0.32 A as it seems
exactly superimposed on the native bound one (Fig. 2a).
These results indicated the high accuracy of the MOE simu-
lation in comparison with the biological methods. Although
PDE4 enzyme crystal structure with nitraquazone co-crys-
tallized was absent, a prediction of the binding model for
˚
Fig. 2 a The docked rolipram ligand into PDE4B seems superimposed on the native rolipram ligand, RMSD: 0.32 A. b Predicted binding model
of nitraquazone
Fig. 3 a Docked conformation alignment of 7f and its original co-crystallized ligand in PDE4B binding site generated by MOE docking. b
Simplified structure of 7f docked at PDE4B active site showing hydrogen bonds and metal coordination
123

3566
Med Chem Res (2012) 21:3557–3567
˚
bounded within the active site (distance = 2.37 A). This
for the observed affinity: (i) The quinazoline nucleus was
sandwiched between the side chains of Phe446 and Ile410
showing great overlapping of benzo of quinazoline with the
phenyl ring of rolipram. (ii) Quinazolinone’s 4-oxo forms
hydrogen bond with the side-chain NH2 of Gln443 (dis-
metal coordination seemed to be crucial for the high
potency of 7f.
Comparing the active 7f, with a propyl spacer, to its less
active congener 5b, with an ethyl spacer, shows similar
binding interactions except of Zn2? binding and His234
hydrogen bond, which were lost in 5b as the ethyl spacer
was not long enough to allow morpholine’s oxygen to
reach the Zn2? and His234 in the active site. Although
compounds 5d, 8, and 9a showed less inhibitory action to
PDE4B than 7f which was rationalized by the fact that they
were unable to bind to Zn2?, these compounds still binds to
His234 by p-cation interaction. This explained why com-
pounds 5d, 8, and 9a achieved better inhibitory activity to
PDE4B (Fig. 5) than the rest of our compounds which lost
both Zn2? and His234 bindings.
˚
tance = 2.71 A). (iii) Unlike nitraquazone N-ethyl group,
the N-butyl group of 7f was too bulky to be embedded to
the rolipram’s methoxy group small pocket made up of
Tyr403, Tyr233, Thr407, Pro396, Gln443, Asn395, Ile410,
and Trp406, instead quinazoline ring of 7f adopted a flip-
ped position so that its n-butyl occupies the rolipram’s
cyclopentoxy group hydrophobic pocket surrounded by
Phe414, Phe446, Met411, Met431, Ser442, and Gln443
(Fig. 4). (iv) Moreover, the morpholine oxygen was
weakly hydrogen bond to imidazole’s NH of His234 (dis-
2?
˚
tance = 3.27 A) and metal coordinated with the Zn
Fig. 4 Predicted binding model
of nitraquazone and the docked
pose of 7f showing its flipped
quinazoline nucleus
Fig. 5 Schematic view of a compound 5d, b compound 8 and c compound 9a docking conformations within PDE4B active site showing p-cation
interaction with His234
123

Med Chem Res (2012) 21:3557–3567
3567
Soc 4666–4668. http://pubs.rsc.org/en/content/articlelanding/1964/
jr/jr9640004633
Conclusion
Lehnart SE, Wehrens XHT, Reiken S, Warrier S, Belevych AE,
Harvey RD, Richter W, Jin SLC, Conti M, Marks AR (2005)
Phosphodiesterase 4D deficiency in the ryanodine-receptor
complex promotes heart failure and arrhythmias. Cell 123:25–35
McKenna JM, Muller GW (2006) Cyclic nucleotide phosphodiester-
ases. In: Beavo JA, Francis SH, Houslay MD (eds) Health and
disease. CRC Press, pp 667–670
Molecular Operating Environment (MOE 2008.10); C.C.G., Inc.,
1255 University St., Suite 1600, Montreal, Quebec, Canada H3B
3X3. 2005. http://www.chemcomp.com
We observed that the bulkiness of N3 substituent is
important in the orientation of quinazoline nucleus in the
active site. Metal coordination with the Zn2? bound within
the active site greatly increases activity and may lead to
potent PDE4 inhibition. Hydrogen bonds to aminoacids
residues (Asp392 and His234) or p-cation interaction with
His234 increases activity. Moreover quinazolinone’s 2-oxo
seems to be unessential for PDE4 binding. These pre-
liminary encouraging results of biological screening of the
tested compounds could offer an excellent framework in
this field that may lead to discovery of potent PDE4B
inhibitors which can be used for treatment of several
diseases.
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Acknowledgments 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
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