Synthesis, analgesic and anti-inflammatory evaluation of some novel quinazoline derivatives

Article abstract of DOI:10.1016/j.ejmech.2010.07.067

Two series of some new 2,4,6-trisubstituted-quinazoline derivatives were prepared and screened for their analgesic, anti-inflammatory activity and acute toxicity. Four compounds were more potent analgesic agents than the reference drug Indomethacin and thirteen compounds showed significant anti-inflammatory activity. Seven compounds showed combined ability to inhibit both pain and inflammation. Compounds tested for acute toxicity showed no toxic symptoms or mortality rates 24 h post-administration implying their good safety margin.

Full text of DOI:10.1016/j.ejmech.2010.07.067

European Journal of Medicinal Chemistry 45 (2010) 4947e4952
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis, analgesic and anti-inflammatory evaluation of some novel quinazoline
derivatives
,
a
a
b
c
Ahmed M. Alafeefy a , Adnan A. Kadi , Omar A. Al-Deeb , Kamal E.H. El-Tahir , Nabila A. Al-jaber
*
a Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, Riyadh 11451, P.O. Box 2457, Saudi Arabia
b Department of Pharmacology, College of Pharmacy, King Saud University, Riyadh 11451, P.O. Box 2457, Saudi Arabia
c Department of Chemistry, College of Science, King Saud University, Riyadh 11451, P.O. Box 2455, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Two series of some new 2,4,6-trisubstituted-quinazoline derivatives were prepared and screened for their
analgesic, anti-inflammatory activity and acute toxicity. Four compounds were more potent analgesic
agents than the reference drug Indomethacin and thirteen compounds showed significant anti-inflam-
matory activity. Seven compounds showed combined ability to inhibit both pain and inflammation.
Compounds tested for acute toxicity showed no toxic symptoms or mortality rates 24 h post-administration
implying their good safety margin.
Received 11 February 2010
Received in revised form
24 July 2010
Accepted 29 July 2010
Available online 12 August 2010
Ó 2010 Elsevier Masson SAS. All rights reserved.
Keywords:
Quinazoline
Analgesic
Anti-inflammatory
Synthesis
suppression of proinflammtory cytokines levels and be beneficial
1. Introduction
for the treatment of the aforementioned diseases [8].
Tobe et al. [9,10] conducted a reporter gene-based screening,
with the reporter DNA having the binding sequence for NF-KB and
the luciferase gene, to find new structural class NF-KB activation
inhibitors. This effort led to the identification of quinazoline I as
Autoimmune diseases and allergies, such as rheumatoid
arthritis, septic shock, transplant rejection, asthma and psoriasis,
are considered to be caused by abnormalities of T cell immune
responses [1,2]. In particular, the activation of T cells initiates
a network of events that results in the overproduction of certain
transcription factors and proinflammtory cytokines [3,4]. Tran-
scription factors are DNA-binding proteins that regulate the
production of proinflammtory cytokines and a variety of other
cellular regulators.
Nuclear factor-kB (NF-KB) is a pivotal transcription factor that
functions to enhance the transcription of proinflammtory cytokines
which are thought to be important in the generation of acute
inflammatory responses [5,6]. Activation and regulation of NF-KB
are tightly controlled by members of a family of inhibitory proteins
referred to as I-KB.
a lead compound (IC50 ¼ 2.381
mM), Fig. 1.
In the present work, two new series of quinazoline compounds
of the general formula II and III were designed in such a way to
accommodate phenyl, 4-chlorophenyl at position 2, so as to modify
the electronic effects. 4-Methyl piperidine, 4-phenylpiperidine,
4-methylpiperazine, 4-phenylpiperazine, at position 4, to impart
structural similarity to compound I. Hydrogen, bromine, iodine
atom at position 6, to improve lipophylicity, aiming to identify new
candidates that may be of value as potent, selective and less toxic
analgesic and anti-inflammatory agents.
In the resting cells, NF-KB is sequestered in the cytoplasm
through its interaction with its inhibitor. However, a large variety of
inflammatory conditions, such as inflammatory cytokines, bacterial
and viral infections and certain chemical agents, induce NF-KB
activity [7]. The inhibition of NF-KB activation would lead to
2. Results and discussion
2.1. Chemistry
The synthesis of the quinazoline derivatives 6aem, 7ael and
8e13 was carried out following Schemes 1e3. Briefly, the appro-
priate acid chloride was allowed to react with the selected
anthranilic acid derivative in dichloromethane containing catalytic
* Corresponding author. Tel.: þ966 1 4673765; fax: þ966 1 4676220.
E-mail address: aalafeefy@yahoo.com (A.M. Alafeefy).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.07.067

4948
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 45 (2010) 4947e4952
C6H5
R
N
R
N
R
N
N
HN
N
Cl
N
X
X
X
X
R N
NH
N
N
N
N
N
N
N
N
N
5a-f
7a-l
I
II
Z
III
Z
Y
Y
Fig. 1.
7a: X=H.
7b: X=H.
7c: X=Br.
7d: X=Br.
7e: X=I.
Z=H. R= Me.
Z=H. R= Ph.
Z=H. R= Me.
Z=H. R= Ph.
Z=H. R= Me.
Z=H. R= Ph.
Z=Cl. R= Me.
Z=Cl. R= Ph.
Z=Cl. R= Me.
Z=Cl. R= Ph.
Z=Cl. R=Me.
Z=Cl. R= Ph.
amount of triethylamine to yield compounds 2aef. The corre-
sponding benzoxazone derivatives 3aef were obtained by boiling
2aef in acetic anhydride. The structures of compounds 3aef were
assigned by analytical and spectroscopic data. The IR and 1H NMR
spectra indicated the absence of both NH and OH groups. The
benzoxazone derivatives 3aef were then boiled with formamide to
afford the corresponding 3H-quinazolin-4-one derivatives 4aef.
Chlorination of the later derivatives was accomplished by fusion
using a mixture of phosphorus oxychloride and phosphorus pen-
tachloride to afford the 4-chloro derivatives 5aef. The IR spectra of
the compounds 5aef revealed the absence of both the NH and the
endocyclic carbonyl C]O groups of the parent compounds and no
signal for the NH groups were seen in the 1H NMR spectra. The
appropriate piperidine and/or piperazine derivatives were allowed
to react with the 4-chloro derivatives in acetone containing anhy-
drous potassium carbonate to afford the target 4-substituted
derivatives 6aem and 7ael respectively [11e14]. The structures of
these compounds were confirmed by IR, NMR and mass spectra.
Generally, the characteristic methylene protons of the piperidine
and piperazine nuclei and the carbons bearing them were detected
in the 1H NMR and 13C spectra as well. The carbohydrazide 8 was
prepared in 95% yield via prolonged heating of compound 6m with
hydrazine hydrate in the absence of solvent. The IR spectrum of
which revealed the presence of NH & NH2 absorption bands at 3310,
3290, 3250 cmꢀ1, also the carbonyl absorption band appeared at
1677 cmꢀ1 (in place of the ester carbonyl in 6m at 1745 cmꢀ1). Also,
the 1H NMR spectrum of 8 revealed the absence of the tri-
pletequartet pattern characteristic of ethyl ester (of 6m that
7f: X=I.
7g: X=H.
7h: X=H.
7i: X=Br.
7j: X=Br.
7k: X=I.
7l: X=I.
Scheme 2.
appeared at d 1.3 and 4.2 ppm respectively) and the presence of NH
& NH2 signals. The dithiocarbazic acid of the potassium dithio-
carbazate 9 was dehydrated by concentrated sulphuric acid to yield
the target 1,3,4-thiadiazolin-2-thione 10. The carbohydrazide 8 was
converted to the thiosemicarbazide derivative 11 through reaction
with potassium thiocyanate. Dehydration of 11 with sulphuric acid
yielded compound 12 in 51% yields. The 1,3,4-thiadiazol-2(3H)-one
13 was prepared by treating the amine derivative 12 with nitrous
acid. The spectral data of compound 13 revealed that it existed as
the lactam structure as proved by IR spectrum which showed
medium NH peak at 3197 cmꢀ1 in addition to C]O sharp peak at
1752 cmꢀ1. The 13C NMR spectrum showed the C-5 and C]O
carbons of 1,3,4-thiadiazole ring at
d 150.1 and 179.4 respectively.
2.2. Pharmacology
All the new compounds (6aem, 7ael, 8e13) were screened to
evaluate their analgesic and anti-inflammatory activities and acute
toxicity. Indomethacin was used as a reference standard. The
analgesic activity was performed by the heat-induced nociception
and the anti-inflammatory activity was evaluated by the formal-
dehyde-induced edema techniques (Table 1).
O
X
CO2H
NH
X
CO2H
NH2
X
Ac2O
Z
O
HCONH2
ArCOCl
N
3a-f
1a-c
O
Z
R
2a-f
S
HN
N
S
R
H
N
S
O
NHNH2
O
N
S
K
H
N
N
Cl
N
O
N
N
H
X
X
X
POCl3
PCl5
N
NH
N
N
N
N
CS2
KOH
N
N
N
98%
H2S04
NH2NH2
6-m
4a-f
5a-f
6a-m
Z
Z
Z
N
N
N
8
9
10
6a: X=H.
6b: X=H.
6c: X=Br.
6d: X=Br.
6e: X=I.
Z=H. R= Me.
Z=H. R= Ph.
Z=H. R= Me.
Z=H. R= Ph.
Z=H. R= Me.
Z=H. R= Ph.
Z=Cl. R= Me.
Z=Cl. R= Ph.
Z=Cl. R= Me.
Z=Cl. R= Ph.
Z=Cl. R=Me.
Z=Cl. R= Ph.
Z=H. R= CO2Et
KNCS
O
NH2
N
S
HN
H
N
S
N
S
O
N
N
6f: X=I.
N
NH2
H
6g: X=H.
6h: X=H.
6i: X=Br.
6j: X=Br.
6k: X=I.
6l: X=I.
NaNO2
HCl
98%
H2S04
N
N
N
N
N
N
N
N
6m:X=H.
12
Scheme 3.
13
11
Scheme 1.

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 45 (2010) 4947e4952
4949
Table 1
Analgesic and anti-inflammatory activity of the new compounds.
2.2.2. Anti-inflammatory activity
When the compounds were tested against formaldehyde
induced inflammation, 6 compounds were considered devoid of
Compounda
Analgesic activityb
Anti-inflammatory
activityc
activity, and
8 compounds showed weak anti-inflammatory
6a
6b
6c
6d
6e
6f
6g
6h
6i
6j
6k
6l
6m
7a
7b
7c
7d
7e
7f
7g
7h
7i
7j
7k
7l
10
12
13
88.0 þ 4.1**
69 þ 2.9**
activity. However, 13 compounds (6aej, 7b, 10, 12) showed signif-
icant inhibition, of which 6b and 6f were more potent than the
reference drug and compound 7b was almost as active as
indomethacin.
Generally, piperidine containing molecules were more active
than those with piperazine instead. Within the piperidine series,
halogenated derivatives were almost less active than halogen free
ones [6a, 6b, 6m, were relatively more active than 6iel and 7a, 7b
showed significant analgesic effect while compounds 7cel were
devoid of any analgesic effect]. However, halogen atom at the 6-
position of the quinazoline backbone has the same impact on
activity as that of the phenyl ring attached at 2-position.
Formaldehyde-induced edema is believed to be mediated via
release of various mediators such as prostaglandins, 5-hydroxy-
tryptamine, together with other mediators [18]. Thus, the observed
anti-inflammatory effect of the compounds in this study may result
from inhibition of any or all of these mediators. However, the
combined ability of compounds 6a, 6b, 6f, 6m, 7b, 10 and 12 to
inhibit both pain and inflammation greatly pinpoint to the
involvement of prostaglandins inhibition in the observed actions.
153.8 þ 3.7*
85.3 þ 3.1*
57.8 þ 1.6*
57.6 þ 1.7*
56.40 þ 2.8**
82.5 þ 1.2**
55.1 þ 2.3*
53.7 þ 1.2**
55.1 þ 3.5**
50.0 þ 1.8**
45.8 þ 1.3**
41.4 þ 1.8*
70.3 þ 3.3**
45.2 þ 1.9**
77.4 þ 1.9*
45.0 þ 3.0*
40.7 þ 1.0**
39.6 þ 2.5*
39.2 þ 2.3*
37.0 þ 1.8**
12.6 þ 3.0
e
89.8 þ 2.1*
85.7 þ 2.1**
110.2 þ 3.1**
e
87.3 þ 2.0**
85.2 þ 2.2**
106.1 þ 3.0**
82.3 þ 2.6**
e
98.5 þ 3.5**
86.2 þ 2.7**
107.3 þ 1.5*
e
e
e
e
e
e
e
20.0 þ 2.1
e
16.2 þ 1.0
e
17.0 þ 1.0
e
13.5 þ 2.6
88.0 þ 1.5**
83.2 þ 1.7*
81.5 þ 1.4*
97.5 þ 3.9*
71.3 þ 3.1**
73.0 þ 1.5*
12.7 þ 1.1*
78.3 þ 2.7a
2.2.3. Acute toxicity
Toxicological studies of the most promising analgesic and/or
anti-inflammatory active agents [6b, 6d, 6m, 7b, 10, 12, 13] were
performed using LD50 standard method in mice in 500, 750 and
1000 mg/kg (body weight), that is up to 5-fold of the used effective
dose. However, no toxic symptoms or mortality rates were
observed 24 h post-administration implying their good safety
margin.
Indomethacin
*Significantly different from control at p < 0.05.
**Significantly different from control at p < 0.01.
a
Dose was (100 mg/kg).
b
Percent increase in latency measured 60 min after drug administration.
Percent inhibition of formaldehyde-induced edema measured 2 h after form-
c
aldehyde injection.
Concerning other actions, compounds 6f, 6j and 7b induced
urination while compounds 7f, 7j and 12 induced sedation, calm-
ness, muscle relaxation and decreased respiration. However, no
toxic symptoms or mortality rates were observed after 24 h.
2.2.1. Analgesic activity
Treatment of mice with compounds 6c, 6g, 6l, 7cel did not affect
the heat-induced nociception to any degree. However, thirteen
compounds induced significant antinociceptive effects against
heat-induced pain (Table 1). Compounds 6b, 6f, 6j and 7b were
even more active than the reference drug. The antinociceptive
effect of compound 6b was at least one and half fold the activity of
the reference standard drug indomethacin. Compound 6m was as
potent as the reference drug. Nine compounds showed activity that
is comparable but less than that of the reference drug. It is worth to
mention that the potent analgesic effect of compound 13 without
any significant inhibition of inflammation and induction of S-sha-
ped tail suggests that this compound seemed to be an opioid
analgesic [15].
3. Conclusion
We have synthesized and tested two series of 2,4,6-trisubsti-
tuted-quinazoline derivatives for their analgesic and anti-inflam-
matory activities. Fifteen compounds namely induced significant
activity. The piperidine series showed better results than the
piperazine series and the halogenated derivatives were almost less
active than halogen free ones. Four compounds were more potent
analgesic agents than the reference drug; compound 13 seemed to
express an opioid activity. Seven compounds showed combined
ability to inhibit both pain and inflammation. Compounds tested
for acute toxicity showed good safety margin.
With regard to the duration of analgesia, it was observed that all
of these compounds exhibited analgesia for more than 3 h except
for compound 13 which showed potent analgesia for more than 4 h.
The process of pain generally involves sensitization of the
nociceptors, transmission of impulses to the spinal cord and then
the pain center in the thalamus followed by integration of the
sensation in the sensory cortex as pain [16]. Thus, the anti-
nociceptive action of the test compounds may be exerted either
peripherally at the site of the nociceptors or centrally in the spinal
cord or the brain.
The significant inhibitory effects of these compounds on the
hot-plate induced nociception may have involved peripheral and/
or cerebral mechanisms. This can only be pinpointed by investi-
gating the influence of the compounds on the main sensitizers of
the peripheral nociceptors, mainly the prostaglandins [17].
4. Experimental
4.1. Chemistry
4.1.1. General
All melting points (ꢁC, uncorrected) were determined on
a Stuart melting point apparatus (Stuart Scientific, Redhill, UK).
Elemental analyses (C, H, N) were performed on PerkineElmer
2400 analyzer (PerkineElmer, Norwalk, CT, USA) and were in full
agreement with the proposed structures within ꢂ0.4% of the
theoretical values. Infra red (IR) spectra (KBr) were recoded using
Pye Unicam SP 1000 IR spectrometer (Thermoelectron, Egelbach,
Germany) and expressed in wave number
were obtained on a Bruker AC 300 Ultra Shield NMR spectrometer
y
(cmꢀ1). NMR spectra

4950
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 45 (2010) 4947e4952
(Bruker, Munich, Germany) at 300 MHz for 1H and 75 MHz for 13C,
the chemical shifts are expressed in (ppm) downfield from tet-
2H, 30 & 50 piperidine), 2.52e2.56 (m, 2H, 30 & 50 piperidine),
2.62e2.65 (m, 1H, 40 piperidine), 2.71e2.73 (m, 2H, 20 & 60 piperi-
dine), 3.21e3.25 (m, 2H, 20 & 60 piperidine), 7.55e8.34 (m, 13H,
d
ramethylsilane (TMS). Splitting patterns were designated as
follows: s: singlet; d: doublet; t: triplet; m: multiplet. Electron
impact mass spectra were recorded on a Varian Mat 311-A70 eV
instrument (Varian, Fort Collins, USA). Chemicals used are supplied
from SigmaeAldrich (SigmaeAldrich Chemie GmbH, Steinheim,
Germany).
AreH). 13C NMR: 31.9, 32.1 (C 30 & 50 piperidine), 34.8 (C 40 piper-
d
idine), 52.7, 54.5 (C 20 & 60 piperidine), 120.5, 123.0, 125.4, 126.7,
127.8,129.5,130.1,131.5,135.2,147.5,154.7,172.6 (AreC). MS (EI): m/z
491 [Mþ, 9.6%]. Anal. (C25H22IN3) C, H, N. 6g: Yield, 79%; m.p.
201e203 ꢁC; 1H NMR (DMSO-d6)
d
: 0.98 (d, 3H, CH3, J ¼ 7.5 Hz),
1.64e1.67 (m,1H, 40 piperidine),1.70e1.74 (m, 2H, 30 & 50 piperidine),
1.91e1.93 (m, 2H, 30 & 50 piperidine), 2.21e2.24 (m, 1H, 40 piperi-
dine), 2.39e2.42 (m, 2H, 20 & 60 piperidine), 3.20e3.23 (m, 2H, 20 & 60
4.1.2. Compounds 1aec, 2aef, 3aef, 4aef and 5aef
These compounds were prepared, in our laboratory, according
to reported procedures [19,20].
piperidine), 7.14e8.35 (m, 8H, AreH). 13C NMR:
d 16.5 (CH3), 30.2,
30.7 (C 30 & 50 piperidine), 31.1 (C 40 piperidine), 51.0, 52.5 (C 20 & 60
piperidine),118.5,121.5,124.7,126.2,127.6,129.0,130.5, 132.6,135.8,
147.9, 155.2, 177.5 (AreC). MS (EI): m/z 340 [Mþ þ 2, 1.7%], 338 [Mþ,
5.3%]. Anal. (C20H20ClN3) C, H, N. 6h: Yield, 76%; m.p. 213e215 ꢁC; 1H
4.1.3. 2-(4-Substituted-phenyl)-4-(4-substituted piperidino)-6-
substituted quinazolines 6aem and 2-(4-substituted-phenyl)-4-(4-
substituted piperazino)-6-substituted quinazoline derivatives 7ael
General procedure: A mixture of 5aef (0.003 mol) and the
appropriate nucleophile (0.003 mol) in acetone (12 mL) in presence
of anhydrous potassium carbonate (0.5 g) was heated under reflux
for 3 h. The solvent was then evaporated under vacuum and the
separated solid was filtered, washed with water, dried and crys-
tallized from ethanol or dioxane to afford compounds 6aem and/or
compounds 7ael.
NMR (DMSO-d6) d
: 2.38e2.40 (m, 2H, 30 & 50 piperidine), 2.47e2.51
(m, 2H, 30 & 50 piperidine), 2.60e2.64 (m, 1H, 40 piperidine),
2.70e2.73 (m, 2H, 20 & 60 piperidine), 3.18e3.22 (m, 2H, 20 & 60
piperidine), 7.35e8.37 (m,13H, AreH).13C NMR: 31.8, 32.6 (C 30 & 50
d
piperidine), 35.3 (C 40 piperidine), 55.3, 57.5 (C 20 & 60 piperidine),
123.5,126.5,128.7,130.5,131.4,134.3,136.5,138.4,140.3,147.5,156.1,
174.5 (AreC). Anal. (C25H22ClN3) C, H, N. 6i: Yield, 74%; m.p.
6a: Yield, 74%; m.p. 192e194 ꢁC; 1H NMR (CDCl3):
d
1.05 (d, 3H,
270e272 ꢁC; 1H NMR (DMSO-d6)
d
: 0.99 (d, 3H, CH3, J ¼ 7.0 Hz),
CH3, J ¼ 7.0 Hz),1.66e1.73 (m,1H, 40 piperidine),1.72e1.85 (m, 2H, 30
& 50 piperidine),1.92e1.94 (m, 2H, 30 & 50 piperidine), 2.20e2.26 (m,
1H, 40 piperidine), 2.45e2.57 (m, 2H, 20 & 60 piperidine), 3.20e3.25
1.63e1.67 (m,1H, 40 piperidine),1.69e1.74 (m, 2H, 30 & 50 piperidine),
1.90e1.93 (m, 2H, 30 & 50 piperidine), 2.21e2.24 (m, 1H, 40 piperi-
dine), 2.38e2.42 (m, 2H, 20 & 60 piperidine), 3.20e3.23 (m, 2H, 20 & 60
(m, 2H, 20 & 60 piperidine), 7.68e8.12 (m, 9H, AreH). 13C NMR:
d
15.4
piperidine), 7.12e8.35 (m, 7H, AreH). 13C NMR:
d 16.6 (CH3), 30.1,
(CH3), 30.6, 30.8 (C 30 & 50 piperidine), 31.2 (C 40 piperidine), 51.6, 52.1
(C 20 & 60 piperidine), 120.5, 122.5, 125.4, 126.1, 127.0, 128.3, 128.8,
130.6, 132.3, 148.4, 156.5, 174.5 (AreC). MS (EI): m/z 303 [Mþ, 11%].
Anal. (C20H21N3) C, H, N. 6b: Yield, 86%; m.p. 232e234 ꢁC; 1H NMR
30.7 (C 30 & 50 piperidine), 31.0 (C 40 piperidine), 51.1, 52.5 (C 20 & 60
piperidine), 119.5, 121.5, 124.8, 126.1, 127.5, 129.2, 130.5, 132.7, 135.2,
147.9,155.4,174.5 (AreC). Anal. (C20H19BrClN3) C, H, N. 6j: Yield, 76%;
m.p. 213e215 ꢁC; 1H NMR (DMSO-d6) : 2.38e2.40 (m, 2H, 30 & 50
d
(CDCl3)
d
: 2.31e2.35 (m, 2H, 30 & 50 piperidine), 2.51e2.56 (m, 2H, 30
piperidine), 2.47e2.51 (m, 2H, 30 & 50 piperidine), 2.60e2.64 (m, 1H,
40 piperidine), 2.70e2.73 (m, 2H, 20 & 60 piperidine), 3.18e3.22 (m,
& 50 piperidine), 2.63e2.66 (m,1H, 40 piperidine), 2.70e2.73 (m, 2H,
20 & 60 piperidine), 3.20e3.25 (m, 2H, 20 & 60 piperidine), 7.60e8.32
2H, 20 & 60 piperidine), 7.35e8.37 (m, 12H, AreH). 13C NMR:
d 31.4,
(m,14H, AreH). 13C NMR:
d
31.6, 32.2 (C 30 & 50 piperidine), 33.7 (C 40
32.5 (C 30 & 50 piperidine), 35.3 (C 40 piperidine), 55.3, 57.4 (C 20 & 60
piperidine),122.5,126.5,128.4,130.1,132.4,134.3,136.6,138.4,141.3,
147.5, 156.2, 174.0 (AreC). Anal. (C25H21BrClN3) C, H, N. 6k: Yield,
piperidine), 53.0, 54.5(C 20 & 60 piperidine),119.5,123.5,125.9,126.6,
127.9,129.5,130.6,131.9,134.0,146.9,153.8,170.5 (AreC). MS (EI): m/
z 365 [Mþ, 17%]. Anal. (C25H23N3) C, H, N. 6c: Yield, 70%; m.p.
74%; m.p. 266e268 ꢁC; 1H NMR (DMSO-d6)
d: 0.98 (d, 3H, CH3,
245e247 ꢁC; 1H NMR (DMSO-d6)
d: 0.98 (d, 3H, CH3, J ¼ 7.0 Hz),
J ¼ 7.5 Hz),1.62e1.65 (m, 1H, 40 piperidine), 1.69e1.74 (m, 2H, 30 & 50
piperidine), 1.90e1.93 (m, 2H, 30 & 50 piperidine), 2.20e2.24 (m, 1H,
40 piperidine), 2.38e2.44 (m, 2H, 20 & 60 piperidine), 3.20e3.26 (m,
1.64e1.68 (m,1H, 40 piperidine),1.70e1.74 (m, 2H, 30 & 50 piperidine),
1.91e1.94 (m, 2H, 30 & 50 piperidine), 2.21e2.25 (m, 1H, 40 piperi-
dine), 2.39e2.43 (m, 2H, 20 & 60 piperidine), 3.20e3.24 (m, 2H, 20 & 60
2H, 20 & 60 piperidine), 7.45e8.35 (m, 7H, AreH). 13C NMR:
d 16.6
piperidine), 7.15e8.35 (m, 8H, AreH). 13C NMR:
d
16.5 (CH3), 30.4,
(CH3), 30.1, 30.8 (C 30 & 50 piperidine), 31.7 (C 40 piperidine), 51.0, 52.5
(C 20 & 60 piperidine), 119.6, 121.9, 124.8, 126.2, 127.5, 129.0, 130.5,
132.8, 135.3, 147.9, 155.7, 176.5 (AreC). MS (EI): m/z 466 [Mþ þ 2,
6.6%], 338[Mþ, 20.2%]. Anal. (C20H19ClIN3) C, H, N. 6l: Yield, 76%; m.p.
30.7 (C 30 & 50 piperidine), 31.0 (C 40 piperidine), 51.4, 52.6 (C 20 & 60
piperidine),116.5, 121.5, 124.8, 126.2, 127.6, 129.1, 130.5, 132.6, 135.3,
146.9,155.0,178.5 (AreC). MS (EI): m/z 384 [Mþ þ 2,11.5%], 382 [Mþ,
12.1%]. Anal. (C20H20BrN3) C, H, N. 6d: Yield, 78%; m.p. 226e228 ꢁC;
287e289 ꢁC; 1H NMR (DMSO-d6) : 2.28e2.31 (m, 2H, 30 & 50
d
1H NMR (DMSO-d6)
d
: 2.40e2.42 (m, 2H, 30 & 50 piperidine),
piperidine), 2.46e2.51 (m, 2H, 30 & 50 piperidine), 2.58e2.63 (m, 1H,
40 piperidine), 2.70e2.73 (m, 2H, 20 & 60 piperidine), 3.19e3.22 (m,
2.49e2.53 (m, 2H, 30 & 50 piperidine), 2.60e2.64 (m, 1H, 40 piperi-
dine), 2.71e2.73 (m, 2H, 20 & 60 piperidine), 3.18e3.23 (m, 2H, 20 & 60
2H, 20 & 60 piperidine), 7.25e8.37 (m, 12H, AreH). 13C NMR:
d 31.4,
piperidine), 7.35e8.28 (m,13H, AreH).13C NMR:
d
31.9, 32.9 (C 30 & 50
32.5 (C 30 & 50 piperidine), 35.3 (C 40 piperidine), 55.3, 57.4 (C 20 & 60
piperidine),122.5,126.5,128.4,130.1,132.4,134.3,136.6,138.4,141.3,
147.5, 156.2, 174.0 (AreC). MS (EI): m/z 528 [Mþ þ 2, 5.0%], 526 [Mþ,
15.6%]. Anal. (C25H21ClIN3) C, H, N. 6m: Yield, 67%; m.p. 173e175 ꢁC;
IR (KBr, cmꢀ1): 3096 (CH arom.), 2931 (CH aliph.), 1745 (C]O). 1H
piperidine), 35.3 (C 40 piperidine), 55.1, 57.5 (C 20 & 60 piperidine),
123.5,127.5,128.9,130.6,131.9,134.5,136.6,138.5,140.3,146.5,155.6,
172.6 (AreC). MS (EI): m/z 446 [Mþ þ 2, 6.2%], 444 [Mþ, 6.5%]. Anal.
(C25H22BrN3) C, H, N. 6e: Yield, 69%; m.p. 257e259 ꢁC; 1H NMR
(DMSO-d6)
d
: 0.99 (d, 3H, CH3, J ¼ 8.5 Hz), 1.71e1.73 (m, 1H, 40
NMR (DMSO-d6)
d
: 1.31 (t, 3H, CH3, J ¼ 8.0 Hz), 2.28e2.31 (m, 2H, 30 &
piperidine),1.72e1.85 (m, 2H, 30 & 50 piperidine),1.91e1.94 (m, 2H, 30
& 50 piperidine), 2.20e2.25 (m,1H, 40 piperidine), 2.45e2.66 (m, 2H,
20 & 60 piperidine), 3.20e3.28 (m, 2H, 20 & 60 piperidine), 7.60e8.33
50 piperidine), 2.46e2.51 (m, 2H, 30 & 50 piperidine), 2.58e2.63 (m,
1H, 40 piperidine), 2.70e2.73 (m, 2H, 20 & 60 piperidine), 3.19e3.22
(m, 2H, 20 & 60 piperidine), 4.08e4.15 (m, 2H, CH2), 7.22e8.36 (m, 9H,
(m, 8H, AreH).13C NMR:
d
15.5 (CH3), 30.0, 30.8 (C 30 & 50 piperidine),
AreH).13C NMR: 15.12 (C, CH3), 31.4, 32.5 (C 30 & 50 piperidine), 35.3
d
31.4 (C 40 piperidine), 51.4, 52.3 (C 20 & 60 piperidine), 121.5, 122.6,
125.2, 126.1, 127.2, 128.2, 128.8, 130.7, 133.5, 148.4, 154.5, 172.9
(AreC). MS (EI): m/z 429 [Mþ, 2.9%]. Anal. (C20H20IN3) C, H, N. 6f:
(C 40 piperidine), 55.3, 57.4 (C 20 & 60 piperidine), 62.27 (C, CH2),
122.5, 126.5, 128.4, 130.1, 132.4, 134.3, 136.6, 138.4, 141.3, 145.3147.5,
156.2 (AreC), 174.0 (C]O). MS (EI): m/z 361 [11%, Mþ]. Anal.
(C22H23N3O2) C, H, N. 7a: Yield, 70%; m.p. 207e209 ꢁC; 1H NMR
Yield, 80%; m.p. 232e234 ꢁC; 1H NMR (DMSO-d6)
d: 2.31e2.34 (m,

A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 45 (2010) 4947e4952
4951
(CDCl3)
d
: 1.05 (s, 3H, CH3), 2.47 (s, 4H, piperazineeH), 2.92 (s, 4H,
8: Yield, 95%; m.p. 262e264 ꢁC; IR (KBr, cmꢀ1): 3310, 3290, 3258
(NH, NH2), 3088 (CH arom.), 2915 (CH aliph.), 1677 (C]O). 1H NMR
piperazineeH), 7.31e8.45 (m, 9H, AreH).13C NMR:
d
15.9 (CH3), 55.1,
58.6 (piperazineeC), 118.8, 122.0, 124.7, 126.2, 127.2, 128.0, 129.5,
131.6, 132.9, 147.1, 155.5, 175.8 (AreC). MS (EI): m/z 304 [Mþ, 8.4%].
Anal. (C19H20N4) C, H, N [11]. 7b: Yield, 82%; m.p. 199e201 ꢁC; 1H
(DMSO-d6) d
: 2.22e2.25 (m, 2H, 30 & 50 piperidine), 2.31e2.35 (m,
2H, 30 & 50 piperidine), 2.60e2.63 (m, 1H, 40 piperidine), 2.68e2.71
(m, 2H, 20 & 60 piperidine), 3.27e3.31 (m, 2H, 20 & 60 piperidine),
4.25 (s, 2H, NH2, exchangeable), 7.17e7.90 (m, 9H, AreH), 8.94 (s,
NMR (CDCl3)
d: 2.42 (s, 4H, piperazineeH), 2.93 (s, 4H,
piperazineeH), 7.18e7.77 (m, 14H, AreH). 13C NMR: 55.5, 58.4
(piperazineeC), 120.5, 122.5, 125.4, 126.1, 127.0, 128.3, 128.8, 130.6,
132.3, 148.4, 156.5, 178.3 (AreC). MS (EI): m/z 366 [Mþ, 4.7%]. Anal.
(C24H22N4) C, H, N. 7c: Yield, 70%; m.p. 211e213 ꢁC; 1H NMR (DMSO-
1H, NH-exchangeable). C NMR: 30.1, 30.6 (C 30 & 50 piperidine),
13
33.1 (C 40 piperidine), 52.4, 53.7 (C 20 & 60 piperidine), 121.7, 125.6,
127.5, 129.0, 130.4, 132.7, 135.6, 137.6, 139.4, 147.5, 153.6, 175.0
(AreC). MS (EI): m/z 347 (Mþ, 13.5%). Anal. (C20H21N5O) C, H, N.
d6)
d
: 1.2 (s, 3H, CH3), 2.49 (s, 4H, piperazineeH), 2.92 (s, 4H,
piperazineeH), 7.35e8.52 (m, 8H, AreH).13C NMR:
d
15.7 (CH3), 55.7,
4.1.5. Potassium 2-(1-(2-phenylquinazolin-4-yl)piperidine-4-
carbonyl)-hydrazine carbodithioate 9
58.0 (piperazineeC), 119.7, 121.6, 124.0, 126.4, 127.5, 129.1, 131.5,
133.6, 135.5, 146.4, 156.0, 173.8 (AreC). MS (EI): m/z 385 [Mþ þ 2,
0.86%], 383 [Mþ, 0.82%]. Anal. (C19H19BrN4) C, H, N [12]. 7d: Yield,
Carbon disulphide (0.114 g, 0.0015 mol) was added dropwise to
a solution of 8 (0.471 g, 0.0015 mol) and potassium hydroxide
(0.084 g, 0.0015 mol) in ethanol (25 mL), and the mixture was
stirred at room temperature for 3 h. Dry ether (20 mL) was then
added to the mixture and the precipitated solid was filtered,
washed with ether and dried at 65 ꢁC for 1 h to yield compound 9 in
almost quantitative yield and used without further purification.
85%; m.p. 226e228 ꢁC; 1H NMR (DMSO-d6)
d: 2.45 (s, 4H,
piperazineeH), 2.94 (s, 4H, piperazineeH), 7.18e7.90 (m, 13H,
AreH). 13C NMR: 55.7, 58.1 (piperazineeC), 123.7, 125.9, 127.4, 129.1,
130.5,132.3,135.8,137.6,139.3,148.5,154.8,171.5 (AreC). MS (EI): m/
z 447 [Mþ þ 2, 2.1%], 445 [Mþ, 2.2%]. Anal. (C24H21BrN4) C, H, N. 7e:
Yield, 71%; m.p. 240e242 ꢁC; 1H NMR (DMSO-d6)
d: 1.03 (s, 3H, CH3),
2.40 (s, 4H, piperazineeH), 2.90 (s, 4H, piperazineeH), 7.31e8.43 (m,
4.1.6. 5-(1-(2-Phenylquinazolin-4-yl)piperidin-4-yl)-1,3,4-
thiadiazole-2(3H)-thione 10
8H, AreH). 13C NMR:
d
14.9 (CH3), 55.5, 58.5 (piperazineeC), 118.8,
121.5, 124.6, 126.1, 127.1, 128.0, 129.4, 131.5, 133.7, 147.4, 154.0, 173.5
Compound 9 (0.69 g, 0.005 mol) was added portionwise to 98%
sulphuric acid (15 mL) and the resulted clear solution was stirred at
room temperature for 24 h. The mixture was cautiously added to
crushed ice (20 g), stirred for 1 h, refrigerated for 2 h, and the
separated white precipitate was filtered, washed with water, dried
and crystallized from ethanol.
(AreC). MS (EI): m/z 430 [Mþ, 3.5%]. Anal. (C19H19IN4) C, H, N. 7f:
Yield, 85%; m.p. 183e185 ꢁC; 1H NMR (DMSO-d6)
d: 2.42 (s, 4H,
piperazineeH), 2.79 (s, 4H, piperazineeH), 7.25e7.61 (m, 13H,
AreH).13C NMR: 55.5, 58.6 (piperazineeC),120.2,122.5,124.4,125.9,
127.1,128.2,128.8,130.4,133.3,148.6,156.5,175.2 (AreC). MS (EI): m/
z 492 [Mþ, 5.4%]. Anal. (C24H21IN4) C, H, N. 7g: Yield, 75%; m.p.
10: Yield, 78%; m.p. 290e292 ꢁC; IR (KBr, cmꢀ1): 3288 (NH),
215e217 ꢁC; 1H NMR (DMSO-d6)
d
: 1.02 (s, 3H, CH3), 2.42 (s, 4H,
piperazineeH), 2.59 (s, 4H, piperazineeH), 7.35e8.54 (m, 8H, AreH).
13C NMR:
15.7 (CH3), 56.0, 58.4 (piperazineeC), 119.8, 121.6, 124.1,
3088 (CH arom.), 2915 (CH aliph.), 1265 (C]S). 1H NMR (DMSO-d6)
d
: 2.22e2.25 (m, 2H, 30 & 50 piperidine), 2.31e2.35 (m, 2H, 30 & 50
d
piperidine), 2.60e2.63 (m,1H, 40 piperidine), 2.68e2.71 (m, 2H, 20 &
60 piperidine), 3.27e3.31 (m, 2H, 20 & 60 piperidine), 6.62 (s, 1H, NH-
exchangeable), 7.17e7.90 (m, 9H, AreH). 13C NMR: 30.1, 30.6 (C 30 &
50 piperidine), 33.1 (C 40 piperidine), 52.4, 53.7 (C 20 & 60 piperidine),
121.7,125.6,127.5,129.0, 130.4,132.7,135.6,137.6,139.4,147.5,153.6,
175.0 (AreC). MS (EI): m/z 405 (Mþ, 20.6%). Anal. (C21H19N5S2) C, H,
N.
126.4, 127.6, 129.2, 131.5, 133.6, 136.3, 146.7, 154.8, 176.1 (AreC). MS
(EI): m/z339[Mþ þ 2, 2.4%] [Mþ, 7.5%]. Anal. (C19H19ClN4) C, H, N[13].
7h: Yield, 66%; m.p. 262e264 ꢁC; 1H NMR (DMSO-d6)
d: 2.43 (s, 4H,
piperazineeH), 2.57 (s, 4H, piperazineeH), 7.18e7.90 (m, 13H,
AreH).13C NMR: 54.8, 58.5 (piperazineeC),122.7,125.5,127.5,129.2,
130.4, 132.7, 135.6, 137.6, 139.8, 147.5, 154.9, 174.5 (AreC). Anal.
(C24H21ClN4) C, H, N. 7i: Yield, 71%; m.p. 279e281 ꢁC; 1H NMR
(DMSO-d6)
d
:1.01 (s, 3H, CH3), 2.44 (s, 4H, piperazineeH), 2.56 (s, 4H,
15.7 (CH3), 54.6,
4.1.7. 2-(1-(2-Phenylquinazolin-4-yl)piperidine-4-carbonyl)
hydrazine carbothioamide 11
piperazineeH), 7.31e8.54 (m, 7H, AreH).13C NMR:
d
58.4 (piperazineeC), 119.8, 121.6, 124.1, 126.4, 127.6, 129.2, 131.5,
133.6, 136.3, 146.7, 154.8, 176.1 (AreC). Anal. (C19H18BrClN4) C, H, N.
A suspension of 8 (0.471 g, 0.0015 mol), potassium thiocyanate
(0.135 g, 0.0015 mol) and concentrated hydrochloric acid (5 mL), in
water (25 mL), was heated under reflux for 2 h. On cooling, the
separated solid was filtered, washed with cold water, dried and
crystallized from ethanol.
7j: Yield, 69%; m.p. 269e271 ꢁC; 1H NMR (DMSO-d6)
d: 2.51 (s, 4H,
piperazineeH), 2.60 (s, 4H, piperazineeH), 7.17e7.90 (m, 12H,
AreH). 13C NMR: 56.2, 58.4 (piperazineeC),121.7,125.6,127.5,129.0,
130.4, 132.7, 135.6, 137.6, 139.5, 147.5, 153.7, 175.5 (AreC). Anal.
(C24H20BrClN4) C, H, N. 7k: Yield, 68%; m.p. 279e281 ꢁC; 1H NMR
11: Yield, 89%; m.p. 257e259 ꢁC; IR (KBr, cmꢀ1): 3432, 3347, 3236
(NH, NH2), 3064 (CH arom.), 2957, 2866 (CH aliph.), 1646 (C]O),
(DMSO-d6)
d
: 1.01 (s, 3H, CH3), 2.51 (s, 4H, piperazineeH), 2.60 (s, 4H,
1266 (C]S). 1H NMR (DMSO-d6) : 2.28e2.31 (m, 2H, 30 & 50 piper-
d
piperazineeH), 7.30e8.54 (m, 7H, AreH).13C NMR:
d
14.8 (CH3), 56.1,
idine), 2.46e2.51 (m, 2H, 30 & 50 piperidine), 2.58e2.63 (m, 1H, 40
piperidine), 2.70e2.73 (m, 2H, 20 & 60 piperidine), 3.19e3.22 (m, 2H,
20 & 60 piperidine), 5.0e5.11 (m, 2H, NH2-exchangeable), 7.20e8.03
58.7 (piperazineeC), 119.5, 121.4, 124.2, 126.4, 127.7, 129.2, 131.6,
133.7, 136.5, 146.4, 155.8, 176.8 (AreC). Anal. (C19H18ClIN4) C, H, N
[14]. 7l: Yield, 60%; m.p. 262e264 ꢁC; 1H NMR (DMSO-d6)
d: 2.53 (s,
(m, 9H, AreH) 8.75 (b s,1H, NH-exchangeable).13C NMR:
d 31.4, 32.5
4H, piperazineeH), 2.66 (s, 4H, piperazineeH), 7.17e7.90 (m, 12H,
AreH). 13C NMR: 55.8, 59.3 (piperazineeC),121.7,125.6,127.5,129.0,
130.4, 132.7, 135.6, 137.6, 139.4, 147.5, 153.6, 175.0 (AreC). Anal.
(C24H20ClIN4) C, H, N.
(C 30 & 50 piperidine), 35.3 (C 40 piperidine), 55.3, 57.4 (C 20 & 60
piperidine),122.5,126.5,128.4,130.1,132.4,134.3,136.6,138.4,141.3,
145.3147.5, 156.2 (AreC). MS (EI): m/z 406 (Mþ, 1.8%). Anal.
(C21H22N6OS) C, H, N.
4.1.4. 1-(2-Phenylquinazolin-4-yl)piperidine-4-carbohydrazide 8
A mixture of 6m (3.61 g, 0.01 mol) and 98% hydrazine hydrate
(15 mL) was heated under reflux with stirring for 15 h. On cooling,
cold water (150 mL) was added to the mixture and the separated
solid was filtered, washed with cold water, dried and crystallized
from ethanol.
4.1.8. 5-(1-(2-Phenylquinazolin-4-yl)piperidin-4-yl)-1,3,4-
thiadiazol-2-amine 12
98% Sulphuric acid (20 mL) was added to compound 11 (0.81 g,
0.002 mol), and the mixture was stirred for 24 h at room temper-
ature. The mixture was then poured onto crushed ice (200 g),
neutralized with concentrated ammonium hydroxide solution and

4952
A.M. Alafeefy et al. / European Journal of Medicinal Chemistry 45 (2010) 4947e4952
stirred for 20 min. The separated crude product was filtered,
washed with water, dried and crystallized from ethanol.
4.2.2. Anti-inflammatory activity
Anti-inflammatory activity was evaluated by the formaldehyde-
induced edema test in rats [22]. Indomethacin (as a reference drug
for comparison) and the test compounds were administered orally,
at a dose of 100 mg/kg once daily for 4 d. Rats in the control group
received the same volume of vehicle. Before formaldehyde injec-
tion, the right hind paw volume was measured with plethys-
mometer; 1 h after drug administration, formaldehyde 0.2 mL (10%,
w/v) was subcutaneously injected into the plantar surface of the
right hind paw. The paw volumes were measured 1 and 2 h after
injection. The anti-inflammatory effects (% inhibition) in animals
that received the test compounds were compared with those in the
control and indomethacin administered groups.
12: Yield, 51%; m.p. 201e203 ꢁC; IR (KBr, cmꢀ1): 3398, 3336
(NH2), 3048 (CH arom.), 2970, 2861 (CH aliph.). 1H NMR (DMSO-d6)
d
: 2.28e2.31 (m, 2H, 30 & 50 piperidine), 2.46e2.51 (m, 2H, 30 & 50
piperidine), 2.58e2.63 (m,1H, 40 piperidine), 2.70e2.73 (m, 2H, 20 &
60 piperidine), 3.19e3.22 (m, 2H, 20 & 60 piperidine), 7.60e8.03 (m,
9H, AreH) 8.45 (m, 2H, NH2-exchangeable). 13C NMR:
d 31.4, 32.5
(C 30 & 50 piperidine), 35.3 (C 40 piperidine), 55.3, 57.4 (C 20 & 60
piperidine), 122.5, 126.8, 128.1, 130.2, 132.7, 134.4, 136.0, 137.1, 138.7,
140.0, 141.6143.5, 159.4 (AreC). MS (EI): m/z 388 (Mþ, 7.6%). Anal.
(C21H20N6S) C, H, N.
4.1.9. 5-(1-(2-Phenylquinazolin-4-yl)piperidin-4-yl)-1,3,4-
thiadiazol-2(3H)-one 13
4.2.3. Acute toxicity study
10% Aqueous sodium nitrite solution (10 mL) was added drop-
wise to an ice-cooled suspension of the 2-amino-1,3,4-thiadiazole
12 (0.338 g, 0.001 mol) and hydrochloric acid (5 mL) in cold
dimethylformamide (10 mL), with continuous stirring over a period
of 20 min. The temperature was then allowed to rise to room
temperature and the mixture was heated to boiling for 10 min,
cooled and allowed to stand overnight. The separated crude
product was filtered, washed with water, dried and crystallized
from ethanol.
Approximate 50% lethal dose (ALD50) of the most promising
compounds was determined using standard procedures [23].
Albino mice of either sex weighing 25e35 g were divided in 12
groups of 6 mice each. The test compounds were dissolved in DMSO
in 500, 750, and 1000 mg/kg doses were injected. The toxic
symptoms and mortality rates in each group were recorded 24 h
after drug administration.
4.2.4. Statistical analysis
13: Yield, 61%; m.p. 165e167 ꢁC; IR (KBr, cmꢀ1): 3197 (NH), 3081
The results are expressed as means; One Way Analysis of Vari-
ance (ANOVA) test with Dunnestt’s comparison to controls and
unpaired Student’s t-test were used to determine Statistical
significance.
(CH arom.), 2978, 2863 (CH aliph.), 1752. 1H NMR (DMSO-d6)
d:
2.28e2.31 (m, 2H, 30 & 50 piperidine), 2.46e2.51 (m, 2H, 30 & 50
piperidine), 2.57e2.63 (m, 1H, 40 piperidine), 2.71e2.74 (m, 2H, 20 &
60 piperidine), 3.18e3.22 (m, 2H, 20 & 60 piperidine), 7.61e8.13 (m,
9H, AreH) 10.05 (b s, 1H, NH-exchangeable). 13C NMR:
d 30.5, 32.0
(C 30 & 50 piperidine), 35.5 (C 40 piperidine), 55.2, 57.6 (C 20 & 60
piperidine),122.4, 125.9, 128.0, 130.1,132.5, 134.3, 136.1,137.0,138.5,
140.0, 141.2150.1, 179.4 (AreC). MS (EI): m/z 389 (Mþ, 10.5%). Anal.
(C21H19N5OS) C, H, N.
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The synthesized compounds were evaluated for analgesic and
anti-inflammatory activities. The new compounds and the standard
drug were administered in the form of a suspension (1% carbox-
ymethyl cellulose as vehicle). Six groups each comprised six
animals were used in the assay. The first four groups received the
test compounds while the fifth and sixth groups, which served as
positive and negative controls, received 10 mg/kg indomethacin
and 5.0 mL vehicle, respectively. The animals were procured from
the Animal House Center, College of Pharmacy, King Saud Univer-
sity, Saudi Arabia, and were maintained in a colony cages at
25 ꢂ 2 ꢁC, relative humidity of 45e55%, under 12 h light and dark
cycle; they were fed standard animal feed. All animals were accli-
matized for a week before use. The Institutional Animal Ethics
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