Design, synthesis and biological evaluation of novel quinazoline-based anti-inflammatory agents acting as PDE4B inhibitors

Article abstract of DOI:10.1248/cpb.c14-00737

A novel series of quinzoline based compounds (IIIa-d, VIa-f, IXa-f) were designed, synthesized and screened for their inhibitory activity towards the PDE4B isoform. The in vivo anti-inflammatory effect of the titled compounds (IIIa-d, VIa-f, IXa-f) as well as their effect on the level of tumor necrosis factor (TNF-α) were evaluated. Among all of the synthesized compounds, IXb, IXd and IXf, exhibited good inhibitory activity against PDE4B enzyme with inhibition percentages of 42, 62 and 68%, respectively. Most of the tested compounds showed potent anti-inflammatory activity compared to indomethacin with a marked decrease in TNF-α level. The ulcerogenic effect of the tested compounds was also examined. The gastric mucosa of the tested animals remained intact after oral administration of the hit compounds. Additionally, docking study was used to explore the possible binding mode of the active compounds on the PDE4B enzyme as well as to illustrate the selectivity of the active hits on the PDE4B isoform.

Full text of DOI:10.1248/cpb.c14-00737

102
Chem. Pharm. Bull. 63, 102–116 (2015)
Vol. 63, No. 2
Regular Article
Design, Synthesis and Biological Evaluation of Novel Quinazoline-Based
Anti-inflammatory Agents Acting as PDE4B Inhibitors
,a
Rabah Ahmed Taha Serya,* Abeer Hussin Abbas,a Nasser Saad Mohamed Ismail,a
Ahmed Esmat,b and Dalal Abdelrahman Abou El Ellaa
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Ain Shams University; ElKhalifa ElMaamoon St.,
b
11566 Abbasseya, Cairo, Egypt: and Department of Pharmacology and Toxicology, Faculty of Pharmacy, Ain Shams
University; ElKhalifa ElMaamoon St., 11566 Abbasseya, Cairo, Egypt.
Received October 23, 2014; accepted November 12, 2014
A novel series of quinzoline based compounds (IIIa–d, VIa–f, IXa–f) were designed, synthesized and
screened for their inhibitory activity towards the PDE4B isoform. The in vivo anti-inflammatory effect of the
titled compounds (IIIa–d, VIa–f, IXa–f) as well as their effect on the level of tumor necrosis factor (TNF-α)
were evaluated. Among all of the synthesized compounds, IXb, IXd and IXf, exhibited good inhibitory activ-
ity against PDE4B enzyme with inhibition percentages of 42, 62 and 68%, respectively. Most of the tested
compounds showed potent anti-inflammatory activity compared to indomethacin with a marked decrease in
TNF-α level. The ulcerogenic effect of the tested compounds was also examined. The gastric mucosa of the
tested animals remained intact after oral administration of the hit compounds. Additionally, docking study
was used to explore the possible binding mode of the active compounds on the PDE4B enzyme as well as to
illustrate the selectivity of the active hits on the PDE4B isoform.
Key words PDE4B inhibition; tumor necrosis factor-α (TNF-α); quinazoline; anti-inflammatory; docking
Inflammation is a self-protection that preserves the integrity side effects than already known non-subtype selective PDE4
of the organisms against physical, chemical, and infective in- inhibitors.7–9) These side effects, mainly nausea and emesis,
sults. However, the inflammatory response to several insults considerably limit the use of these drugs. PDE4 inhibitors
could erroneously lead to normal tissue damage while the are very promising compounds, which could bring new al-
body is trying to heal itself.1) The margination of leukocytes, ternatives for the treatment of chronic diseases like asthma,
primarily neutrophils, is one of the early cellular inflamma- COPD, rheumatoid arthritis and atopic dermatitis.10) The role
tory events.2) Tumor necrosis factor-α (TNF-α), a cytokine, of a PDE4B inhibitor is to inhibit c-AMP degradation, thus
plays an important role in inflammation because of its ability leading to an increase in c-AMP level thus suppresses TNF-α
to stimulate cytokine release from neutrophils and biosynthe- production in human monocytes.11) The first recognized non
sis of chemokines.3) Thus this biomedical pathway has been selective PDE inhibitor was the natural Mesembrine (1) which
targeted by the anti-inflammatory therapies used in different is an alkaloid present in Sceletium tortuosum (kanna)12) that
chronic inflammatory diseases as chronic obstructive pulmo- has been reported to have a weak PDE4 inhibition.13) Rolip-
nary disease (COPD) and rheumatoid arthritis.
ram (2) was the first synthetically developed first generation
In addition, cyclic AMP (cAMP) and cyclic GMP (cGMP) PDE4 inhibitor. Unfortunately, its narrow therapeutic index,
are key regulators in various signaling pathways in inflamma- emesis and other adverse effects limited its use.14–18) A break-
tion inducing numerous intracellular responses by transduc- through of PDE4 inhibitors as anti-inflammatory agents came
tion of different inflammatory stimuli among which is TNF-α up with second generation Roflumilast (3) Dexas® by Merck
regulation in human monocytes.4) High levels of cAMP have Sharp & Dohme, a selective, long-acting inhibitor of the en-
been reported to lead to suppress the function of proinflamma- zyme PDE4B.19–22) Continuous efforts in this era lead to the
tory and immunocompetent cells.5) Thus, one of the most im- discovery of Cilomilast (4) Ariflo®, the most advanced PDE4
portant anti-inflammatory targets is to inhibit degradation of inhibitor developed by GSK.10) Many quinoline based PDE4B
c-AMP leading to its accumulation and therefore TNF-α pro- inhibitors were also reported in literature among which SCH
duction will be suppressed.4) Phosphodiesterases (PDEs) have 351591(5) and SCH 365351 (6).23–25) Further optimization lead
been known for their ability to degrade cyclic nucleosides.6) to the discovery of the PDE4B inhibitor GSK256066 (7) with
There are 11 primary families designated (1–11) expressed improved potency optimized for inhaled administration.26) Not
throughout the human body and having multiple isoforms,7) only quinoline but also some quinazoline based derivatives
some of which are nonspecific that degrade both c-AMP and were reported to have potent PDE4B inhibition such as com-
c-GMP, others are specific and degrade either one of them.6,8,9) pounds (8–11) and (12–14) that have been recently identified
The isoform of interest in inflammation is PDE4, as it is in- to be selective PDE4B inhibitors in the in vitro assay.27,28) In
volved in regulation of TNF-α production.4) PDE4 isoform is addition, nitraquazone having good inhibitory activity with
comprised of 4 genotype products (PDE4A, PDE4B, PDE4C IC50=1.9 µM (15) was identified as promising (PDE4) inhibi-
and PDE4D) and are highly expressed in neutrophils, mono- tor.29) Moreover 4-substituted-6-nitro quinazoline derivatives
cytes, central nervous system (CNS) and smooth muscles of compound (16) had been reported to inhibit TNF-α production
the lung. Literature shows that PDE4B subtype could be a with unique anti-inflammatory effect.30) In 2013, 2,4-disubsti-
good target for new anti-inflammatory compounds with fewer tuted quinazoline derivative E6005 (17) was identified to be a
*To whom correspondence should be addressed. e-mail: rabahat2003@yahoo.com
© 2015 The Pharmaceutical Society of Japan

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Reagents and conditions: i: Chloroaceyl chloride in benzene, 1h; ii: R-NH2, K2CO3 in acetonilrile, 6h reflux.
Chart 1. Synthesis of Compounds IIIa–d
potent PDE4 inhibitor with minimal systemic side effects.31)
On investigation of the binding site of different isoforms, it
was clear that there is a great similarity in the binging site of
both PDE4B and PDE4D isoforms, having adenosine recogni-
tion site, metal binding pocket and solvent filled side pocket.32)
Although it was known for decades that the unwanted side
effects are mostly attributed to the binding of PDE4 inhibi-
tor to central PDE4D isoform,33–35) however, recent studies
showed that side effects such as nausea and emesis are more
dose dependent rather than binding to the central PDE4D
isoform.36) The challenge was the development of novel inhibi-
tors against the peripheral PDE4B isoform lacking the ability
to cross blood brain barrier aiming to decrease the CNS side
effects.
In the present study quinazoline scaffold was used as a
main pharmacophoric feature to design selective PDE4B in-
hibitors. In (Chart 1) series IIIa–d was designed using 4-quin-
azolinone scaffold as potential PDE4B inhibitors which is able
to form hydrophobic interactions with the binding pocket.27)
The design was based on the structural modifications of the
reference compound 10 (IC50=13 µM) as a quinazoline based
lead compound exhibiting a good PDE4B inhibitory activ-
ity.28) The modification strategy was based on the replacement
of thione moiety with polar amino group as hydrogen bond
donor element through one carbon atom spacer and eliminat-
ing 3 substituent moiety in reference compound (10) aiming to
minimize CNS side effects (Fig. 1).
Fig. 1. Design of Proposed Target Compounds IIIa–d Based on Mo-
lecular Modifications of Reference Compound 10
Nucleoside binding moiety is shown in rectangular shape, the conserved car-
bonyl group in oval dashed line shape and the hydrogen bond formation element
in circle shape.
The design of target compounds (VIa–f and IXa–f, Chart
2 and Chart 3, respectively) was based on the modification
of the potent quinazoline based PDE4B inhibitors (16)30) and
(17)31) as follows (Fig. 2):
a) The quinazoline scaffold was kept as a core which form
hydrophobic interactions with the binding pocket and
showed hydrogen bonding with Gln443 via the N1 of the
heterocyclic ring.37)
position number 4 in the quinazoline scaffold through the
amine functionality (NH-R) increasing also the polarity
of the target compounds as it was reported that aryl polar
scaffolds are known to act as selective PDE4B inhibi-
tors.23)
c) A nitro group was kept at position 6 of designed target
compounds VIa–f (Chart 2) as it is reported that the
presence of an electron withdrawing group at this posi-
tion increases the PDE4B inhibitory activity.38)
b) The hydrogen bond formation element was retained at the

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
105
Reagents and conditions: i: Triethyl orthoformate, reflux 16h, ii: R-NH2, gl.acetic acid, 1h.
Chart 2. Synthesis of Target Compounds VIa–f
Reagents and conditions: i: Diethyl oxalate, reflux 6h, ii: POCl3 in benzene, reflux 6h, iii: R-NH2 in ethanol, r.t., 24h.
Chart 3. Synthesis of Target Compounds IXa–f
azoline-2-carboxylate derivatives (IXa–f) (Chart 3). The reac-
Results and Discussion
Chemistry Quinazolines have been prepared by differ- tion was activated by the presence of the carboxylate moiety
ent methods in literature allowing variable substitutions at which has (–M) effect that facilitates the release of chlorine as
different positions.39–43) The intermediate compounds (II, V, good leaving group. Structural elucidation of the synthesized
VII and VIII) were prepared as reported and showed similar target compounds IIIa–d, VIa–f and IXa–f was confirmed by
analytical data.44–46)
different spectral and analytical data.
Cyclization of 2-(2-chloroacetamido)benzamide (II) with
Biological Evaluation. In Vivo Evaluation of the Newly
various different amines in acetonitrile in the presence of Synthesized Target Compounds The anti-inflammatory
anhydrous K2CO3 and catalytic amount of potassium iodide activity of the synthesized compounds (IIIa–d, VIa–f, IXa–f)
afforded 2-(aminomethyl) quinazolin-4(3H)-one derivatives was first investigated by rat paw edema model followed by
(IIIa–d) (Chart 1). Ring closure of intermediate compound measuring the percentage decrease in TNF- α level in the test-
V by the reaction with different amines in glacial acetic acid ed lab animals after oral administration of the test compounds
produced 6-nitro 4-substituted amino quinazolines (VIa–f) (IIIa–d, VIa–f, IXa–f) as stated in ref. 47.
(Chart 2). The reaction of compound VIII with different
Effect of Test Compounds on Carrageenan-Induced Rat Paw
amines in ethanol afforded ethyl 4-(substituted amino) quin- Edema Model: Most of the newly synthesized compounds

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Chem. Pharm. Bull.
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Fig. 2. Design of Proposed Target Compounds VIa–f and Target Compounds IXa–f Based on Hybridization between Reference Compounds 16 and
17
Nucleoside binding moiety is shown in rectangle dashed shape, the polar aryl group in oval dashed shape, hydrogen bond formation element in circle dashed shape and
the electron withdrawing group in circle solid line shape.
(IIIa–d, VIa–f, IXa–f) were tested for their anti-inflammato- that produced by indomethacin after 3h whereas compound
ry activity against carrageenan induced rat paw edema using VIa showed lower protection than that of indomethacin after
indomethacin (10mg/kg) as potent standard anti-inflammatory 3h.
drug and using carrageenan for induction of inflammation
The tested compounds IXa, IXb, IXd and IXf (Chart 3)
in the lab animals.48) The standard dose used of tested com- showed a good anti-inflammatory activity compared to indo-
pounds was (50mg/kg).49) Test compounds were given orally methacin with an inhibitory profile ranging between 68–78%
to lab animals followed by injection of the carrageenan, The after 3h of carrageenan injection. Compounds IXc and IXe
inhibitory activity of the tested compounds against inflamma- showed less anti-inflammatory activity than indomethacin
tion was determined by measuring the volume of injected rat with inhibitory profiles 25.57% and 51.6% after 3h, respec-
paw after 1, 2 and 3h of carrageenan injection using plethys- tively.
mometer (Table 1).
The Measurement of the Effect of Tested Compounds on
The results shown in Table 1 revealed that carragenan TNF-α Level in the Inflammatory Exudates in the Carra-
caused massive increase in the injected paw volume (about geenan Induced Rat Edema Model: Inflammatory exudates
double the volume of the untreated paw in animals of con- from rat paws were used to measure TNF-α released via
trol group). Indomethacin pretreatment (10mg/kg) produced sandwiched enzyme-linked immunosorbent assay (ELISA)
significant reduction of edema volume in carrageenan treated technique50) using TNF-α kits for rats from Raybiotech®
animals and inhibited edema by 40, 50.7 and 64.38% after 1, 2 (Parkway Lane, Norcross GA, U.S.A.).
and 3h, as compared to carrageenan group.
Carrageenan significantly increased the TNF-α level, as
The measurement of rat paw volumes and determination of compared to the control group. Moreover, most of the tested
percentage edema inhibition by each of the tested synthesized compounds caused significant reduction in the TNF-α concen-
compounds showed that most of them possess significant anti- tration (ranging between 75.4–107.7% reduction) compared to
inflammatory activity with percentage edema inhibition better that obtained by indomethacin (83.76% reduction).
than indomethacin after 3h. Among the tested target com-
Among all the tested compounds (IIIa–d, VIa–f, IXa–f),
pounds IIIa–d (Chart 1), compound IIId exhibited protection compound IXa showed the best inhibitory activity against
profile 17.14, 56.15 and 71.69% after 1, 2 and 3h, respectively TNF-α as it lowered the TNF-α concentration about four folds
which reveals that anti-inflammatory action appears after 2h than standard indomethacin. The results shown in (Table 2)
and increases by time. Regarding the tested target compounds revealed that carrageenan caused massive increase in the
VIa–f (Chart 2), compounds VIb, VIc, VId and VIe exhibited TNF-α level (about six folds the concentration in the exudates
higher protection compared to indomethacin after 3h. At the of the untreated paw in animals of control group). Indometha-
same time compound VIf showed comparable protection to cin pretreatment (10mg/kg) produced significant reduction

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
107
Table 1. The Effect of Tested Compounds on Rat Paw Volume in Carrageenan-Induced Rat Edema Model
1h
% Edema inhibition
2h
% Edema inhibition
3h
% Edema inhibition
Group
Paw vol. (mL)
Paw vol. (mL)
Paw vol. (mL)
b)
Control
0.66
0.073
—
—
0.487a,b) 0.051
0.703a,b) 0.104
—
0.453a,b) 0.043
0.818a,b) 0.108
0.583a,b) 0.067
0.633a,b) 0.063
0.673a,b) 0.046
0.707a,b) 0.161
0.557a,b) 0.069
0.617a,b 0.066
0.552a,b) 0.091
0.563a,b) 0.049
0.553a,b) 0.069
0.575a,b) 0.105
0.595a,b) 0.035
0.533b) 0.070
0.57 1b) 0.084
0.725a) 0.073
0.563b) 0.089
0.630b) 0.121
0.543b) 0.079
—
Carrageenin (Car.)
Car.+Indomethacin
Car.+IIIa
Car.+IIIb
Car.+IIIc
Car.+IIId
Car.+VIa
Car.+VIb
Car.+VIc
Car.+VId
Car.+VIe
Car.+VIf
Car.+IXa
Car.+IXb
Car.+IXc
Car.+IXd
Car.+IXe
Car.+IXf
0.718a) 0.144
0.695a,b) 0.046
0.715a,b) 0.036
0.712a,b) 0.056
0.715a,b) 0.069
0.708a,b) 0.055
0.703a,b 0.071
0.702a,b) 0.098
0.705a,b) 0.032
—
—
40%
0.593a,b) 0.084
50.70%
14.62%
13.08%
17.60%
56.15%
41.54%
58.46%
56.92%
46.15%
36.92%
31.54%
56.15%
39.23%
11.54%
25.38%
32.31%
33.08%
64.38%
50.68%
39.73%
30.59%
71.69%
55.25%
73.06%
69.86%
72.60%
66.67%
61.19%
78.08%
68.04%
25.57%
69.86%
51.60%
75.34%
a,b)
5.71%
0.672
0.675
0.057
0.059
a,b)
11.43%
5.71%
0.665a,b) 0.172
a,b)
17.14%
25.71%
28.57%
22.86%
20%
0.582
0.613
0.577
0.580
0.603
0.073
0.055
0.107
0.046
0.103
a,b
a,b)
a,b)
a,b)
0.707a,b) 0.071
a,b)
0.705
0.061
22.86%
17.14%
37.14%
17.14%
5.71%
0.623a,b) 0.1058
0.708a,b) 0.038
0.697 0.039
0.708 0.107
0.715 0.076
0.712 0.101
0.703 0.092
0.705 0.065
0.635
0.050
a,b)
0.583 0.078
0.618a,b) 0.092
0.678a) 0.045
0.648 0.045
0.633 0.065
0.632 0.072
11.43%
25.71%
22.86%
Data are presented as mean S.D. Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test. n=6. a) Statistically different from the cor-
responding control. b) Statistically different from carrageenan-treated group.
Table 2. The Effect of Tested Compounds on TNF-α Level in the Inflammatory Exudates in Carrageenan-Induced Rat Edema Model
% Decrease in the
TNF-α level
% Decrease in the
TNF-α level
Group
Control
TNF-α level
Group
TNF-α level
2.682b) 0.343
13.633a) 2.631
4.459a,b) 0.123
5.374a,b) 0.563
6.505a,b) 0.612
6.819a,b) 1.167
2.589b) 0.186
4.749a,b) 1.209
2.424b) 0.396
2.369b) 0.485
Car.+VId
Car.+VIe
Car.+VIf
Car.+IXa
Car.+IXb
Car.+IXc
Car.+IXd
Car.+IXe
Car.+IXf
2.289b) 0.420
2.306b) 0.450
2.695b) 0.290
1.835b) 0.235
2.068b) 0.296
7.783a,b) 1.038
2.344b) 0.153
3.062b) 0.369
3.346b) 0.613
103.58%
103.42%
99.87%
107.72%
105.60%
53.41%
103.07%
96.53%
93.93%
Carrageenin (Car.)
Car.+Indomethacin
Car.+IIIa
Car.+IIIb
Car.+IIIc
Car.+IIId
Car.+VIa
Car.+VIb
Car.+VIc
83.76%
75.41%
65.08%
62.21%
100.83%
81.12%
102.35%
102.85%
Data are presented as mean S.D. Statistical analysis was performed using one-way ANOVA followed by Tukey post hoc test. n=6. a) Statistically different from the cor-
responding control. b) Statistically different from carrageenan-treated group.
of in TNF-α level in carrageenan treated animals by about
Acute inflammation such as carrageenan-induced paw
three folds as compared to carrageenan group. The measure- edema involves the synthesis or release of mediators at the in-
ment TNF-α concentration and percentage decrease of TNF-α jured site. Carrageenan-induced paw edema is an experimen-
level by each of the tested synthesized compounds showed tal animal model for acute inflammation. In the present study,
that most of them exhibit significant anti-inflammatory activ- the rat paw edema model showed the ability of prepared target
ity with a decrease in TNF-α concentration better than indo- compounds with quinazoline scaffold to significantly reduce
methacin. Among the tested target compounds IIIa–d (Chart paw edema, indicating their potential anti-inflammatory activ-
1), compound IIId, which possess an aliphatic substitution ity. TNF-α is a pleiotropic cytokine that plays a critical role in
at 2-position of quinazoline4-one ring, caused a decrease in both acute and chronic inflammation.51) TNF-α promotes an
TNF-α concentration double that occurred by indomethacin. acute-phase reaction.52) Several inflammagens have the abil-
Nearly all tested target compounds VIa–f (Chart 2), com- ity to induce TNF-α synthesis. The formation of several small
pounds VIb, VIc, VId, VIe and VIf were more effective in molecular mediators of inflammation is linked with TNF-α
lowering TNF-α concentration with the exception to com- and thus contributes to the range of mediators that critically
pound VIa, with 2-methyl, p-nitro aniline substitution at 4-po- control inflammation.53) TNF-α facilitates inflammatory cell
sistion of quinazoline ring, that caused a comparable decrease infiltration by promoting the adhesion of neutrophils and lym-
in TNF-α concentration to that observed by indomethacin. For phocytes to endothelial cells.54) When the TNF-α effect is spe-
tested compounds of Chart 3, only compound IXc showed less cifically blocked, the severity of inflammation is reduced.55)
anti-inflammatory activity than indomethacin.
Based on the previous data, it was satisfactory to measure the

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Chem. Pharm. Bull.
Vol. 63, No. 2 (2015)
Fig. 3. Histological Section Showing Stomach Mucosa in Different Animal Groups Using Digital Video Camera Mounted at Light Microscope (A–E)
A: Intact stomach mucosa from control group. B: Ulcerated stomach from animals group administrated indomethacin. C: Stomach mucosa from animals group admin-
istrated titled compounds IIId. D: Stomach mucosa from animals group administrated titled compounds Vic. E: Stomach mucosa from animals group administrated titled
compounds IXb.
Table 3. Ulcer Scores Calculated for Testing Synthesized Compounds Together with Control and Indomethacin as a Reference Compound after Testing
on Laboratory Animal Groups
Indomethacin
(Reference drug)
Control
IIId
VIc
IXb
No. of ulcers/rat
0
0
0
0
0
0
0
9
1
2
1
13
2
3
1
9
1
3
1
12
2
2
2
10
2
2
1
12
1
2
2
Ulcer score S.D.
10.833 1.722
1.5 0.547
2.333 0.516
1.333 0.516
levels of TNF-α reduction in the inflammatory exudates in rat at the effective dose (50mg/kg) compared to indomethacin
paws.
administrated group at (10mg/kg) as shown in (Fig. 3). In-
Examination of the Effect of the Prepared Compounds on spection of the stained sections revealed obvious ulceration of
the Gut Mucosa “Ulcerogenisity Test and Ulcer Index Mea- stomach mucosa or animal group administrated indomethacin
surement”: Most of the anti-inflammatory medications espe- compared to the control group. The mucosa of animals in the
cially NSAIDS exhibit unwanted side effects such as gastric three groups administrated the prepared target compound
ulceration and bleeding especially with the prolonged use. showed no ulceration and almost remains intact as that of the
From here, there was a concern to discover the effect of the control animal group.
tested target compounds on the gastric mucosa. Histologically
The evaluation of the degree of ulceration was expressed
examined laboratory animals’ stomach showed no ulceration in terms of ulcer score, which was calculated by dividing the
after oral administration of the synthesized target compounds total number of ulcers in each group by the number of rats in

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
109
Table 4. The Results of Screened Compounds at Concentration of 10µM
over PDE4B Enzyme
Compound ID
% Inhib. 1
% Inhib. 2
Average inhib.
IIId
VIc
VIe
VIf
IXa
IXb
IXd
IXf
10
8
12
10
8
11
9
10
5
9
9
7
21
44
61
69
15
40
63
67
18
42
62
68
Fig. 4. Ulcer Scores Calculated for Testing Synthesized Compounds
together with Control and Indomethacin as a Reference Compound after
Testing on Laboratory Animal Groups
Fig. 5. Results of Screened Compounds at Concentration of 10µM over PDE4B Enzyme
that group.56) Titled compound IIId, VIc and IXb were chosen in the active site using GOLD and the docking pose was
as representative sample of the synthesized target compounds compared to the original crystal structure. RMSD calculation
and ulcer scores obtained were as shown in (Table 3, Fig. 4).
revealed a value of 0.3Å indicating the ability of the docking
In Vitro Evaluation of PDE4B Inhibitory Activity Gen- algorithm to accurately predict the binding mode of inhibi-
erally, it has been reported that the PDE4B inhibitory activity tors in this binding site. Structural analysis of many PDE4B
of the synthesized compounds could be evaluated at doses up inhibitor complexes has identified two common features.60)
to 100µM.57) The inhibitory activity of the newly synthesized The molecular modelling study showed two main features
compounds (IIId, VIc,e,f, IXa,b,d,f) which have shown a at the active site, the π–π interactions of planar quinazoline
good TNF-α levels reduction have been evaluated against scaffold with the Phe446 by defined as nucleoside binding
PDE4B enzyme at doses (10µM). The assay was conducted by region and the hydrogen bonding with the invariant glutamine
PerkinElmer, Inc. Discovery Services (MD, U.S.A.). A phos- residue (Gln443) that is very essential for activity. It is essen-
phodiesterase-specific compound trequistin was used as a ref- tial to mention that the loss of that hydrogen bonding in the
erence in the assay. Compounds (IXb, IXd and IXf) revealed PDE4B inhibitor diminishes the activity. Representation of
good inhibitory activity at (10µM) with inhibition percent 42, the 3D binding mode of GSK256066 (7) after docking using
62, and 68%, respectively (Table 4, Fig. 5).
GOLD is shown in (Suppl. 1). The molecular modeling study
Molecular Docking Study Molecular docking study was revealed that compounds (IXb, IXd and IXf) which exhib-
carried out to all newly synthesized target compounds (IIIa– ited high inhibition percent against PDE4B enzyme showed
d, VIa–f, IXa–f) using GOLD algorithm under the interface comparable Gold Fitness scores relative to the quinoline based
of accelry’s discovery studio 2.5. The docking study of new GSK256066 (7) (Table 5).
selective PDE4 inhibitors was facilitated by the published
Compounds IXb and IXd (Figs. 6, 7, respectively) showed
crystal structures of PDE4.32,58,59) The docking results showed π–π interaction with Ile410 in addition to the interactions with
good correlation between the enzymatic activity of our com- the essential Phe446. Also, the aryl amine at the 4-position
pounds and their binding interactions as well as their Gold of the quinazoline ring of compound IXb showed a third π–π
Fitness scores.
interaction with Met347. Regarding compound IXf, and extra
In the present study, quinoline lead GSK256066 (7) that hydrogen bonding was clear with Gln443 through the carbonyl
was co-crystallized with the 3D-structure obtained from the moiety at 2-position in addition to the original H-bonding with
protein data bank (Code: 3GWT)37) was used a reference N1 of the quinazoline ring (Fig. 8).
compound to evaluate the molecular modeling docking study
results.
Molecular Modelling Study of Selectivity of Hit Com-
pounds for PDE 4B Isoform In 2014,61) literature reported
In order to validate the docking algorithm on the target en- that ligands with binding modes similar to compound (18)
zyme, the co-crystallized ligand was extracted and re-docked (A33) would provide another level of specificity by only en-

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Chem. Pharm. Bull.
Vol. 63, No. 2 (2015)
Inhibition of PDE4B
Table 5. Goldscore Fitness of the Synthesized Compounds and the Percentage PDE4B Inhibition of Screened Compounds
Compound ID
Goldscore fitness
Inhibition of PDE4B
Compound ID
Goldscore fitness
Compound (7)
IIIa
43.6
37.1
32.5
31.4
39.2
41.5
39.1
37.4
38.1
n.t.
n.t.
n.t.
n.t.
11%
n.t.
n.t.
9%
n.t.
VIe
VIf
XIa
XIb
XIc
XId
XIe
XIf
38.5
39.2
44.2
44.7
35.4
45.6
42.8
44.1
9%
7%
IIIb
18%
42%
n.t.
IIIc
IIId
VIa
62%
n.t.
VIb
VIc
68%
VId
All the inhibitory activity shown at 10µM. n.t.: Not tested.
Fig. 6. 3D Diagram of Compound IXb at PDE4B Active Site (PDB
Code: 3GWT) Showing Interaction of Different Groups with Essential
Amino Acids for Inhibitory Activity
Fig. 8. 3D Diagram of Compound IXf at PDE4B Active Site (PDB
Code: 3GWT) Showing Interaction of Different Groups with Essential
Amino Acids for Inhibitory Activity
Fig. 9. 2D Interaction Diagram of Compound 18 (A33) with PDE4B
Active Site (PDB Code: 4MYQ)
Fig. 7. 3D Diagram of Compound IXd at PDE4B Active Site (PDB
Code: 3GWT) Showing Interaction of Different Groups with Essential
Amino Acids for Inhibitory Activity
catalytic domain and simultaneously with residues from CR3
gaging a specific subset of PDE4B-partner protein complexes. (Fig. 9).
Therefore molecular docking study was fulfilled to study the
Compound (18) (A33) binds the PDE4B catalytic domain
binding mode of compound (18) (A33) and compare it to syn- with its central pyrimidine ring and theinyl ring forming π
thesized and biologically evaluated compounds.
interactions with Phe618 and in position to hydrogen bond
Docking study using PDB coded (4MYQ) showed that to Gln615. The primary substituent responsible for PDE4B
compound (18) (A33) makes multiple interactions with the selectivity is arylcarboxymethyl substituents. This group spe-

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
111
cifically interacts by making hydrogen bond with amine of tion with Phe618 which is matched with the thiophene ring
Lys677 residue from CR3. The amine of Lys677 directly ties of compound A33 (18) making the same π interaction with
CR3 to the catalytic domain. This interaction is reported be Phe618, the N1 of quinazoline ring in IXf and IXd replaced
to have a significant effect on the overall binding energy of N of pyrimidine ring of (18) in making H-bond with Gln615
the closed complex (18/catalytic domain/CR3). Together, these
results show that compound (18) has good shape complemen-
tarity as an interface between the catalytic domain and CR3
and makes multiple hydrophobic and polar interactions with
both domains. This holds CR3 in a “closed conformation” that
blocks substrate binding.62)
Investigation of binding mode of our biologically active
compounds IXf (Fig. 10) and IXd (Fig. 11) which exhibited
good inhibitory activity against PDE4B enzyme with inhibi-
tion percent 62% and 68% to active site revealed that both
compounds have the same binding mode as compound A33
(18) (A33).
In both compounds the quinazoline ring makes a π interac-
Fig. 10. 2D Interaction Diagram of Compound IXf with PDE4B Active Site (PDB Code: 4MYQ)
Fig. 11. 2D Interaction Diagram of Compound IXd with PDE4B Active Site (PDB Code: 4MYQ)

112
Chem. Pharm. Bull.
Vol. 63, No. 2 (2015)
Fig. 12. 2D Interaction Diagram of Compound IXb with PDE4B Active Site (PDB Code: 4MYQ)
with extra hydrogen bonding with the same amino acid from
carbonyl moiety of ester group. Regarding the CR3, both
compounds have the same binding mode with Lys677 by H-
bonding with amide NH in IXf and aniline phenyl ring of IXd
respectively. Regarding compound IXb which exhibited 42%
enzyme inhibition, it showed slightly different binding pattern
than IXf and IXd as shown in (Fig. 12) which may explain
lower enzyme inhibition percentage. In IXb quinazoline ring
made π interaction with Phe618 and aniline phenyl ring made
π interaction with Lys677 but the interaction with Gln615 was
made by chlorine atoms rather by N1 of quinazoline ring in
IXf and IXd.
This binding mode of the selected hits was matched with
biological results and may indicate that they could have high
selectivity for PDE 4B.
Figure 13 shows alignment of compound IXf which has the
highest enzyme inhibition with compound (18) (A33) which
illustrate that quinazoline ring in IXf is perfectly aligned with
pyrimidine and thiophene ring in (A33). Moreover, aniline
ring of both compounds are perfectly aligned together.
Fig. 13. Alignment of IXf (with Line Shaped Bonds) with Compound
18 (with Stick Shaped Bonds)
Conclusion
Out of the prepared compounds, 4-substituted quinazoline
2-carboxylate derivatives (IXb, IXd and IXf) were found to quinazoline ring seems to play an important role in PDE4B
be the most active compounds. This was proved biologically inhibition.
via in vivo assays as Rat Paw edema test and measurement
of the level of decrease of TNF-α, the important cytokine re-
leased during inflammation. In vitro assay was carried out by
Experimental
Chemistry All the starting materials were purchased
measurement of the enzyme inhibition of our compounds and from Sigma-Aldrich (U.S.A.). Melting points were determined
they have shown remarkable inhibition. Also, the molecular on Stuart Scientific apparatus and uncorrected. Reactions
modeling study using GOLD was able to illustrate the good were monitored using thin layer chromatography (TLC), per-
inhibitory of the synthesized compounds as well as high se- formed on 0.255mm silica gel plates (Merck®), with visualiza-
lectivity of these active hits to PDE4B isoform. Additionally, tion under UV light (254nm). FT-IR spectra were recorded
1
the substitution at the 4-anilino moiety has a great influence on a Perkin-Elmer spectrophotometer. H-NMR spectra were
on the PDE4B inhibitory activity. The presence of p-methyl or recorded in δ scale on a Perkin-Elmer 300MHz spectrometer
o-carboxamido on the aniline moiety together with the pres- at Cairo University. EI-MS spectra were recorded on Finnigan
ence of the ethyl carboxylate moiety at the 2-position of the Mat SSQ 7000 (70eV) mass spectrometer, Cairo University.

Vol. 63, No. 2 (2015)
Chem. Pharm. Bull.
113
Elemental analyses were performed at the Microanalytical
Center, Cairo University.
2-((6-Nitroquinazolin-4-yl)amino)benzamide (VIb) : Y e l -
low crystals (70%); mp: 268–270°C; FT-IR (v− max, cm−1):
The intermediate compounds (II, V, VII and VIII) were 3416–3399 (NH, NH2), 1681 (C=O amide); 1H-NMR
prepared as reported and showed similar analytical data.44–46)
(300MHz, DMSO-d6) δ: 11.21 (s, 2H, NH2), 10.71 (s, 1H,
Preparation of 2-((4-Oxo-3,4-dihydroquinazolin-2-yl) NH), 8.44–8.26 (m, 2H, quinazoline Ar H), 8.73 (s, 1H, quin-
methyl) Amino Derivatives (IIIa–c) (General Procedure) azoline Ar-H), 8.49 (s, 1H, quinazoline-H), 7.23–6.90 (m,
A mixture of II (0.85g, 4.00mmol) and the respective amine 4H, Ar); MS: m/z (%) 309 [M+, (64.80%)]; Anal. Calcd for
(4.00mmol) in the presence of K2CO3 (6.00mmol) and KI C15H11N5O3: C, 58.25; H, 3.58; N, 22.64. Found: C, 58.39; H,
(0.20mmol) was refluxed in acetonitrile (10mL) for 6h. After 3.54; N, 22.58.
that, the solvent was evaporated under reduced pressure, and
N-(2,4-Dichlorophenyl)-6-nitroquinazolin-4-amine (VIc):
then the residue was triturated with cooled dil HCl (10%). The Yellow crystals (70%); mp: 198–200°C; FT-IR (v− max,
precipitate was then filtered and washed with diethyl ether cm−1): 3408 (N-H), 1575 and 1323 (NO2); H-NMR (300MHz,
1
and crystallized from ethylacetate to yield titled compounds DMSO-d6) δ: 10.61 (s, 1H, NH), 8.74–8.42 (m, 2H, quin-
IIIa–d.
azoline Ar H), 8.43 (s, 1H, quinazoline Ar-H), 8.38 (s, 1H,
2-(((2-Methyl-4-nitrophenyl)amino)methyl)quinazolin- quinazoline-H), 7.23–6.90 (m, 3H, Ar); MS: m/z (%) 335 [M+,
4(3H)-one (IIIa): Yellow crystals (60%); mp: 118–120°C; (7.58%)], Anal. Calcd for C14H8Cl2N4O2: C, 50.17; H, 2.41; N,
FT-IR (v− max, cm−1): 3466 (N-H), 1505 and 1382 (NO2); 16.72. Found: C, 50.36; H, 2.21; N, 16.45.
1H-NMR (300MHz, DMSO-d6) δ: 7.88–7.73 (m, 3H, Ar),
N-(3,4-Dichlorophenyl)-6-nitroquinazolin-4-amine (VId):
7.46–6.65 (m, 4H, Ar), 6.51 (s, 1H, NH), 5.35 (s, 1H, NH), Yellow crystals (70%); mp: 200–203°C; FT-IR (v− max,
3.50 (s, 2H, CH2-NH), 2.08 (s, 3H, CH3-Ar); MS: m/z (%) 310 cm−1): 3408 (N-H), 1497 and 1323 (NO2); H-NMR (300MHz,
1
[M+, (38.00%)]; Anal. Calcd for C16H14N4O3: C, 61.93; H, 4.55; DMSO-d6) δ: 10.69 (s, 1H, NH), 8.74–8.32 (m, 2H, quinazo-
N, 18.06. Found: C, 61.74; H, 4.56; N, 18.11.
line Ar H), 8.33 (s, 1H, quinazoline Ar-H), 8.48 (s, 1H, quin-
2-(((4-Oxo-3,4-dihydroquinazolin-2-yl)methyl)amino)benz- azoline-H), 7.23–6.77 (m, 3H, Ar); MS: m/z (%) 335.00 [M+,
amide (IIIb): White crystals (50%); mp: 125–128°C; FT-IR (16.04%)]; Anal. Calcd for C14H8Cl2N4O2: C, 50.17; H, 2.41; N,
(v− max, cm−1): 3347–3107 (N-H, N-H2 forked), 1710 (C=O 16.72. Found: C, 50.18; H, 2.31; N, 16.60.
1
amide), 1648 (C=O amide); H-NMR (300MHz, DMSO-d6)
Ethyl 4-((6-nitroquinazolin-4-yl)amino)benzoate (VIe):
δ: 8.90 (s, 2H, NH2), 8.10–6.81 (m, 8H, Ar), 6.74 (s, 1H, NH), Yellow crystals (70%); mp: 204–206°C; FT-IR (v− max, cm−1):
6.55 (s, 1H, NH), 4.31 (s, 2H, NH-CH2); MS: m/z (%) 294 3408 (N-H), 1771 (C=O ester), 1642 (C=N), 1572 and 1319
[M+, (23.42%)]; Anal. Calcd for C16H14N4O2: C, 65.30; H, 4.79; (NO2); H-NMR (300MHz, DMSO-d6) δ: 11.70 (s, 1H, NH),
1
N, 19.04. Found: C, 65.57; H, 4.61; N, 19.23.
8.76–8.40 (m, 2H, quinazoline Ar H), 8.73 (s, 1H, quinazoline
2-(((2,4-Dichlorophenyl)amino)methyl)quinazolin-4(3H)- Ar-H), 8.36 (s, 1H, quinazoline-H), 4.36 (q, J=7.11Hz, 2H,
one (IIIc): Yellow crystals (60%); mp: 220–223°C; FT-IR CH2-CH3), 1.38 (t, J=7.18Hz, 3H, CH3-CH2); 7.51 (dd, J=7.5,
1
(v− max, cm−1): 3340 (N-H), 1714 (C=O amide); H-NMR 2H, Ar-H), 7.59 (dd, J=7.5, 2H, Ar-H), MS: m/z (%) 338 [M+,
(300MHz, DMSO-d6) δ: 8.38–6.95 (m, 7H, Ar), 6.36 (s, 1H, (46.85%)]; Anal. Calcd for C17H14N4O4: C, 60.35; H, 4.17; N,
NH), 5.59 (s, 1H, NH), 4.19 (s, 2H, NH-CH2); MS: m/z (%) 16.56. Found: C, 60.57; H, 4.24; N, 16.61.
320 [M+, (13.00%); Anal. Calcd for C15H11Cl2N3O: C, 56.27; H,
3-(1H-Indol-3-yl)-2-((6-nitroquinazolin-4-yl)amino)propa-
noic acid (VIf): Yellow crystals (50%); mp: 185–186°C;
3.46; N, 13.12. Found: C, 56.51; H, 3.53; N, 13.06.
2-(((4-Oxo-3,4-dihydroquinazolin-2-yl)methyl)amino)acetic FT-IR (v− max, cm−1): 3409–3892 broad (OH carboxylic),
1
acid (IIId): White crystals (50%); mp: 158–160°C; FT-IR 3330 (N-H), 1747 (C=O acid), 1498 and 1316 (NO2); H-NMR
(v− max, cm−1): 3369–3410 (OH carboxylic), 3283–3197 (N-H), (300MHz, DMSO-d6) δ: 13.10 (s, 1H, OH), 11.07 (s, 1H, NH),
1
1682 (C=O acid), 1648 (C=O amide); H-NMR (300MHz, 10.90 (s, 1H, NH), 8.56–8.41 (m, 2H, quinazoline Ar H),
DMSO-d6) δ: 12.22 (s, 1H, OH), 11.90 (s, 1H, NH), 8.39–7.09 8.75 (s, 1H, quinazoline Ar-H), 8.46 (s, 1H, quinazoline-H),
(m, 4H, Ar), 4.91 (s, 1H, NH), 2.88 (s, 2H, CH2-NH), 3.50 7.64–7.15 (m, 4H, Ar-indole), 7.02 (s, 1H, C=CH-NH indole),
(s, 2H, CH2-COOH); MS: m/z (%) 233 [M+, (10.00%)]; Anal. 3.78 (m, 1H, NH-CH-COOH), 3.20 (d, J=7.90Hz, 2H, NH-
Calcd for C11H11N3O3: C, 56.65; H, 4.75; N, 18.02. Found: C, CH-CH2); MS: m/z (%) 377 [M+, (21.40%)]; Anal. Calcd for
56.56; H, 4.78; N, 18.17.
C19H15N5O4: C, 60.47; H, 4.01; N, 16.56. Found: C, 60.28; H,
Preparation of 6-Nitro 4-Substituted Amino Quinazo- 4.08; N, 16.33.
line Derivatives (VIa–f) (General Procedure) A mixture
Preparation of Ethyl 4-(Substituted-amino)quinazoline-
of V (1.01g, 5.00mmol) with the respective amine (5.00mmol) 2-carboxylate Derivatives (IXa–f) (General Procedure)
in glacial acetic acid (10mL) was refluxed for 1h. The reac- To a stirred ice cooled solution of the respective amine
tion mixture was then allowed to cool and the precipitate was (1.50mmol) in ethanol, a cooled solution of VIII (0.24g,
collected by filtration, washed with diethyl ether and crystal- 1.00mmol) in ethanol was added dropwise, the reaction mix-
lized from ethanol to afford titled compounds VIa–f.
ture was then allowed to stir for 24h, after that the solvent
N-(2-Methyl-4-nitrophenyl)-6-nitroquinazolin-4-amine was evaporated under reduced pressure, the precipitate was
(VIa): Yellow crystals (70%); mp: 190–192°C; FT-IR (v− filtered and washed with 10% HCl, diethyl ether and recrystal-
max, cm−1): 3330 (N-H), 1573 and 1314 (NO2); 1H-NMR lized from ethanol to yield titled compounds IXa–f.
(300MHz, DMSO-d6) δ: 9.16 (s, 1H, NH), 8.74–8.36 (m, 2H,
Ethyl 4-((2,4-Dichlorophenyl)amino)quinazoline-2-carbox-
quinazoline Ar H), 8.45 (s, 1H, quinazoline Ar-H), 2.58 (s, 3H, ylate (IXa): Yellow crystals (50%); mp: 185–186°C; FT-IR
1
Ar-CH3); 7.8–7.1 (m, 3H, Ar-H), 8.29 (s, 1H, quinazoline-H), (v− max, cm−1) : 3423 (N-H), 1685 (C=O ester); H-NMR
MS: m/z (%) 325 [M+, (4.58%)]; Anal. Calcd for C15H11N5O4: (300MHz, DMSO-d6) δ: 11.13 (s, 1H, NH), 7.90–6.59 (m, 7H,
C, 55.39; H, 3.41; N, 21.53. Found: C, 55.21; H, 3.36; N, 21.36. Ar), 4.3 (q, J=7.11Hz, 2H, CH2-CH3), 1.57 (t, J=7.13Hz, 3H,

114
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Vol. 63, No. 2 (2015)
CH2-CH3); MS: m/z (%) 362 [M+, (62.5%)], Anal. Calcd for group and the second one to be injected with carrageenan only.
C17H13Cl2N3O2: C, 56.37; H, 3.62; N, 11.60. Found: C, 56.11; Animals in the third group were given orally indomethacin as
H, 3.51; N, 11.65.
standard anti-inflammatory drug (10mg/kg). The remaining
Ethyl 4-((3,4-Dichlorophenyl)amino)quinazoline-2-carbox- groups were given orally the test compounds at (50mg/kg).
ylate (IXb): Yellow crystals (50%); mp: 212–213°C; FT-IR Dosing volume was kept constant (10mL/kg). Thirty minutes
(v− max, cm−1): 3381 (N-H), 1699 (C=O ester); δ 11.07 (s, after oral administration, rats in the first group were injected
1H, NH), 7.30–6.71 (m, 7H, Ar), 4.37 (q, J=7.11Hz, 2H, CH2- subcutaneously with 0.05mL saline, while animals in all
CH3), 1.31 (t, J=7.10Hz, 3H, CH2-CH3); MS: m/z (%) 362 [M+, other groups injected subcutaneously (s.c.) with 0.05mL car-
(14.29%)]; Anal. Calcd for C17H13Cl2N3O2: C, 56.37; H, 3.62; rageenan (1% solution in saline) on the plantar surface of the
N, 11.60. Found: C, 56.28; H, 3.64; N, 11.52.
right hind paw. The right hind paw volume was measured at
Ethyl 4-((4-(Ethoxycarbonyl)phenyl)amino)quinazoline-2- 1, 2 and 3h after carrageenan injection by saline displacement
carboxylate (IXc): White crystals (60%); mp: 187–190°C; using UGO-BASILE 7140 plethysmometer (Comerio, Italy).
FT-IR (v− max, cm−1): 3418 (N-H), 1718 (C=O ester, ke- The measured volumes were used to calculate % edema inhi-
1
tone); H-NMR (300MHz, DMSO-d6) δ: 11.35 (s, 1H, NH), bition for each compound at each dose level.63)
8.03–7.40 (m, 4H, quinazoline-Ar), 7.61 (dd, J=7.5, 2H, Ar-H),
Statistical Analysis: Data are presented as mean S.D. val-
7.57 (dd, J=7.5, 2H, Ar-H), 4.43 (q, J=7.10Hz, 4H, {CH2- ues. Statistical analysis was performed using one-way analysis
CH3}2), 1.36 (t, J=7.11Hz, 6H, {CH2-CH3}2); MS: m/z (%) 365 of variance followed by Tukey–Kramer multiple comparisons
[M+, (93.33%)]; Anal. Calcd for C20H19N3O4: C, 65.74; H, 5.24; tests. The 0.05 level of probability was used as the criterion
N, 11.50. Found: C, 65.80; H, 5.26; N, 11.66.
for significance. All statistical analyses were performed using
Ethyl 4-(p-Tolylamino)quinazoline-2-carboxylate (IXd): GraphPad (La Jolla, CA, U.S.A.) InStat software version 3.
Yellow crystals (60%); mp: 178–180°C; FT-IR (v− max, cm−1): Graphs were sketched using GraphPad Prism software version
1
3360 (N-H), 1726 (C=O ester); H-NMR (300MHz, DMSO- 4 (ISI_Software, La Jolla, CA, U.S.A.).
d6) δ: 10.90 (s, 1H, NH), 8.16–7.28 (m, 4H, quinazoline-Ar),
Determination of Rat TNF-α Concentration: The assay
7.21 (dd, J=7.5, 2H, Ar-H), 7.34 (dd, J=7.5, 2H, Ar-H), 4.41 was carried out according to the manufacturer recommenda-
(q, J=7.13Hz, 2H, CH2-CH3), 2.37 (s, 3H, CH3-Ar), 1.41 (t, tions using ELISA kit provided by RayBiotech systems. Brief-
J=7.12Hz, 3H, CH2-CH3); MS: m/z (%) 307 [M+, (58.80%)]; ly, Rat TNF-α level was determined by quantitative sandwich
Anal. Calcd for C18H17N3O2: C, 70.34; H, 5.58; N, 13.67. enzyme immunoassay technique. The enzyme reaction yielded
Found: C, 69.91; H, 5.16; N, 13.53.
a blue product that turned yellow when the stop solution was
Ethyl 4-(1H-Benzo[d]imidazol-1-yl)quinazoline-2-carbox- added. The intensity of the color measured was proportional
ylate (IXe): White crystals (60%); mp: 170–172°C; FT-IR to the amount of TNF-α bound in the initial step.
(v− max, cm−1): 1714 (C=O ester), 1630 (C=N); 1H-NMR
Induction of Gastric Ulcer: The animals were fasted for
(300MHz, DMSO-d6) δ: 8.61–8.17 (m, 4H, quinazoline- 36h in separate cages with raised wide wire mesh to avoid
Ar), 8.05–7.64 (m, 4H, Ar-benzimidazole), 8.11 (s, 1H, coprophagia,64) but with water given ad libitum. After 8h,
benzimidazole-H), 4.31 (q, J=7.10Hz, 2H, CH2-CH3), 1.32 (t, animals were sacrified, stomach removed and opened on
J=7.10Hz, 3H, CH2-CH3); MS: m/z (%) 318 [M+, (13.60%)]; greater curvature and examined for ulceration. Evaluation of
Anal. Calcd for C18H14N4O2: C, 67.91; H, 4.43; N, 17.60. Found: degree of ulceration was expressed in terms of ulcer score
C, 67.78; H, 4.58; N, 17.58.
which is calculated by dividing the total number of ulcers
Ethyl 4-((2-Carbamoylphenyl)amino)quinazoline-2-carbox- in each group by number of rats in that group.65) To get the
ylate (IXf) White crystals (60%); mp: 178–180°C; FT-IR histological data, representative stomach samples were taken
(v− max, cm−1): 3441–3403 (N-H, N-H2 forked), 1732 (C=O from different group and fixed in 10% formalin in saline for
ester), 1658 (C=O amide); 1H-NMR (300MHz, DMSO-d6) 24h. Washing was done in tap water then serial dilutions of
δ: 13.30 (s, 2H, NH2), 10.50 (s, 1H, NH), 8.05–7.15 (m, 8H, alcohol (methyl, ethyl and absolute ethyl) were used for dehy-
Ar), 4.33 (q, J=7.10Hz, 2H, CH2-CH3), 1.35 (t, J=7.10Hz, 3H, dration. Specimens were cleared in xylene and embedded in
CH2-CH3); MS: m/z (%) 336 [M+, (55.56%)], Anal. Calcd for paraffin at 56°C in hot air oven for twenty four hours. Paraffin
C18H16N4O3: C, 64.28; H, 4.79; N, 16.66. Found: C, 64.25; H, bees wax tissue blocks were prepared for sectioning at 4µm
4.99; N, 16.54.
thickness by slidge microtome. The obtained tissue sections
Biological Evaluation. In Vivo Evaluation of Anti-in- were collected on glass slides, deparaffinized, stained by he-
flammatory Activity Chemicals: Inflammatory-grade car- matoxylin and eosin stain then examination was done through
rageenan was purchased from FMC (Rockland, ME, U.S.A.). using digital video camera mounted at light microscope (CX2,
All other chemicals were of the highest available commercial OLYMPUS, Japan).66)
grade. All test compounds were dissolved in dimethyl sulf-
oxide (DMSO) (Sigma-Aldrich, U.S.A.) before being injected Prepared Compounds in Vitro Methodolgy: The assay
into the animals.
was done using the Caliper LabChip 3000 and a 12-sipper
Measurement of PDE4B Enzyme Inhibitory Activity of
Animals: Throughout the experiments, adult male LabChip. The separations are based on that the product and
Sprague–Dawley rats weighing 120–150g were used. They substrate are electrophoretically separated. The off-chip in-
were housed at a temperature of 23 2°C with free access to cubation mobility-shift phosphodiesterase assay uses a micro-
water and standard food pellets. Rats were acclimatized in our fluidic chip to measure the conversion of a fluorescent cyclic
animal facility for at least 1 week prior to the experiment.
AMP or GMP substrate to a 5′-AMP or 5′-GMP product. The
Measurement of Paw Volume in Carrageenan-Induced Rat reaction mixture, from a microtiter plate well, is introduced
Edema Model: Rats were equally divided into several groups through a capillary sipper onto the chip, where the fluorescent
(6 animals /group). The first group was to be used as control substrate and product are separated by electrophoresis and de-

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Chem. Pharm. Bull.
115
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tected via laser-induced fluorescence.
Materials and Conditions of the Assay: Materials used and
the assay conditions for screening of the target compounds
over PDE4B as got from PerkinElmer, Inc., Company were
to use PDE4B in 0.037nM and 100mM N-(2-hydroxyethyl)-
piperazine-N′-2-ethanesulfonic acid (Hepes) (pH=7.5) as
buffer, 0.010% Brij as detergent and peptide used was 2.0µM
iFluor 488-AHC-cAMP and trequinsin as reference com-
pound.
Conflict of Interest The authors declare no conflict of
interest.
Supplementary Materials The online version of this ar-
ticle contains supplementary materials.
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