Design, synthesis and anti-ulcerogenic effect of some of furo-salicylic acid derivatives on acetic acid-induced ulcerative colitis

Article abstract of DOI:10.1016/j.ejmech.2010.05.071

Ulcerative colitis is a chronically recurrent inflammatory bowel disease of unknown origin. The present work describes design and synthesis of 3-aminofurosalicylic acid 4, azo-conjugates with aniline 2a, 4-ASA 2b or sulphapyridine 2c as well as N-arylsulphonamido 5, chlorosulphonyl 6, aminosulphonyl 7 and N-arylaminosulphonyl derivatives 8 (positional isosters of 5). All the synthesized compounds were evaluated for their anti-ulcerogenic effect on acetic acid-induced ulcerative colitis in rats. It was noticed that oral treatment with sulphasalazine (a reference drug) and the tested compounds 2a, 2c, 4 and 5c in equimolar doses significantly reduced the intensity of lesion score, ulcer area, ulcer index and wet weight/length ratio compared to the control group. On the other hand, compounds 2b, 5a, 5b and 7 had a lower anti-ulcerogenic efficacy. Also, the antimicrobial activity of the synthesized compounds was screened in vitro using the agar diffusion assay technique. In addition, docking of the tested compounds into cycloxygenase II using molecular operating environment (MOE) was performed in order to rationalize the obtained biological results and their mechanism of action. The present work describes design and synthesis of 3-aminofurosalicylic acid 4, azo-conjugates 2a-c, N-arylsulphonamido 5, chlorosulphonyl 6, aminosulphonyl 7 and N-arylaminosulphonyl derivatives 8. All the synthesized compounds and sulphasalazine (a reference) were evaluated for their anti-ulcerogenic effect on acetic acid-induced ulcerative colitis in rats.

Full text of DOI:10.1016/j.ejmech.2010.05.071

European Journal of Medicinal Chemistry 45 (2010) 4104e4112
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Design, synthesis and anti-ulcerogenic effect of some of furo-salicylic acid
derivatives on acetic acid-induced ulcerative colitis
,
b
Ghaneya S. Hassan a , Gamal A. Soliman
*
a Pharmaceutical Chemistry Department, Faculty of Pharmacy, Cairo University, El-Kasr El-Aini Street, Cairo 11562 Cairo, Egypt
b Pharmacology Department, Faculty of Pharmacy; Al-Kharj, King Saud University, Riyadh, Saudi Arabia
a r t i c l e i n f o
a b s t r a c t
Article history:
Ulcerative colitis is a chronically recurrent inflammatory bowel disease of unknown origin. The present
work describes design and synthesis of 3-aminofurosalicylic acid 4, azo-conjugates with aniline 2a, 4-
ASA 2b or sulphapyridine 2c as well as N-arylsulphonamido 5, chlorosulphonyl 6, aminosulphonyl 7 and
N-arylaminosulphonyl derivatives 8 (positional isosters of 5). All the synthesized compounds were
evaluated for their anti-ulcerogenic effect on acetic acid-induced ulcerative colitis in rats. It was noticed
that oral treatment with sulphasalazine (a reference drug) and the tested compounds 2a, 2c, 4 and 5c in
equimolar doses significantly reduced the intensity of lesion score, ulcer area, ulcer index and wet
weight/length ratio compared to the control group. On the other hand, compounds 2b, 5a, 5b and 7 had
a lower anti-ulcerogenic efficacy. Also, the antimicrobial activity of the synthesized compounds was
screened in vitro using the agar diffusion assay technique. In addition, docking of the tested compounds
into cycloxygenase II using molecular operating environment (MOE) was performed in order to ratio-
nalize the obtained biological results and their mechanism of action.
Received 15 December 2009
Received in revised form
26 May 2010
Accepted 28 May 2010
Available online 8 June 2010
Keywords:
Anti-ulcerogenic effect
Furo-salicylic acid derivatives
Acetic acid-induced ulcerative colitis
Antimicrobial activity
MOE
Ó 2010 Elsevier Masson SAS. All rights reserved.
1. Introduction
used to establish, and importantly, maintain remission of IBD.
Azathiopurine or 6-mercaptopurine and 5-aminosalicylates are co-
Inflammatory bowel disease (IBD) is
a
chronic relapsing
prescribed in UC [10]. Recently, mesalamine and the non-patho-
genic probiotic Escherichia coli Nissle 1917 are co-prescribed and
effective for treatment of UC [11].
inflammation afflicting any part of the entire bowel wall which can
take place anywhere in the gastrointestinal tract (GIT) between the
mouth and anus. IBD is the result of a deregulated, aberrant and
even inappropriate over activation of mucosal response in the
intestinal wall due to the defects in the barrier function of the
intestinal epithelium and mucosal immune system [1,2]. Two
specific IBD subtypes based on the site and extent of inflammation
are Crohn’s disease and ulcerative colitis. Crohn’s disease was first
described as a disease in the distal ileum, however, it may be found
anywhere in the GIT. Ulcerative colitis (UC) invariably affects the
rectum and may extend proximally in a confluent pattern to involve
a part of or the entire colon [3,4]. There is no complete cure for IBD
(other than colectomy in ulcerative colitis) and the major goal is to
decrease the relapse episodes and to increase the patient quality of
life [5]. Aminosalicylates [6] and glucocorticoids [7] are the drugs of
choice for treatment of the active phase of IBD. Immunosuppres-
sants (such as methotrexate), folate antagonists [8], and infliximab,
an anti-tumor necrosis factor (anti-TNF) alpha agent [9], are usually
OH
OH
COOH
COOH
H
N
SO2
NH2
N
N
N
Mesalamine
(5-ASA)
Sulphasalazine
OH
OH
N
COOH
COOH
O
CH2CH2COOH
C
OH
N
N
N
H
N
COOH
Olsalazine
Balsalazide
Mesalamine (5-aminosalicylic acid, 5-ASA) is a drug used for
treating UC. The mechanism of action of mesalamine is believed to
be by reducing inflammation in the colon. UC and other
* Corresponding author. Tel.: þ20 22517933, 0101811962; fax: þ20 23628426.
E-mail address: ghanmoom@yahoo.com (G.S. Hassan).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.05.071

G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
4105
inflammatory diseases cause excessive production of chemicals,
for example, prostaglandins that produce inflammation in the
colon. Prostaglandins are produced by the enzymes, cyclo-
oxygenase and lipoxygenase. These enzymes are over-active in
individuals with ulcerative colitis. Mesalamine may work by
blocking the activity of cyclooxygenase and lipoxygenase, thereby,
reducing the production of prostaglandins. Reduced production of
prostaglandins decreases inflammation in the colon and the
symptoms associated with UC. Available forms of mesalamine
differ in their route of administration and how often they are
administered, generally available as rectal enema [12]. Sulphasa-
lazine, an azo conjugate of 5-ASA with sulphapyridine, is a drug
that is used primarily for treating UC. It is a colon-specific prodrug
that is broken down by bacteria in the colon into 5-aminosalicylic
acid (5-ASA), and sulphapyridine. More importantly, 5-ASA was
shown to be the active therapeutic moiety while sulphpyridine
serves simply as a carrier [13]. Olsalazine [14] and Balsalazide [15]
also release 5-ASA by the bacteria in the large intestine. Thus they
are believed to act as delivery systems for the active agent (5-ASA)
to the large intestine, the active site of UC. Very recently the focus
of IBD research seems to have shifted from 5-ASA to 4-ASA (4-
aminosalicylic acid, a second line anti-tuberculosis agent) with
few of its prodrugs. Research studies showed that 4-ASA provides
a stable, inexpensive alternative to 5-ASA for topical treatment of
UC [16]. So, induction and maintenance of remission, mucosal
healing, the avoidance of surgical intervention, and decreasing the
likelihood of cancer developing are the primary therapeutic goals
in UC [17e19].
the appropriate benzene diazonium chloride in presence of
sodium hydroxide. Mono 7-nitro derivative 3 was prepared by
controlled mild nitration of compound 1 using nitric acid/acetone
and the temperature did not exceed 5 ꢀC to avoid the formation of
3,7-dinitro derivative [27]. The 7-amino furo-salicylic acid deriv-
ative 4 was prepared by two methods. The first method was
reductive cleavage of the azo compounds
2 using sodium
dithionite. The second was reduction of 7-nitro derivative 3 using
sodium dithionite. The NH2 group was elucidated through IR
spectrum, which offered two absorption bands at
n 3450 and
3350 cmꢁ1 and a broad signal at
d
3.26e3.43 ppm in H NMR
spectrum. The sulphonamides 5aec were prepared by reacting
compound 4 with aryl sulphonyl chlorides in acetone and pyridine
as an acid acceptor. IR spectra of 5aec exhibit absorption bands at
n
3368e3399 cmꢁ1 (NH) and 1378e1416 and 1156e1189 (SO2) and
1HNMR spectra showed exchangeable signals at
d 5 ppm (NH). The
chlorosulphonation of furo-salicylic acid derivative 1 with excess
chlorosulphonic acid afforded 7-sulphonyl chloride 6. IR spectrum
of the latter compound was characterized by two strong absorp-
tion bands at
Subsequent conversion of arylsulphonyl chloride 6 to the respec-
tive arylsulphonamide (by treatment with concentrated
n
1399 and 1138 cmꢁ1 arising from the SO2 group.
7
ammonia solution or with ammonium carbonate) or the
substituted arylsulphonamides 8aee (by reaction with the
appropriate amino compounds) was done. The OH group at
position-6 was clearly shown free and was proved by IR, 1H NMR
spectra and a positive ferric chloride test.
In the light of the above consideration, the aim of this study is
designing and synthesis of furo-salicylic acid derivatives, where 4
and 5-positions of salicylic acid nucleus are incorporated in furan
ring (benzofurans) and their evaluation for treatment of UC.
2.2. Pharmacology
All the new synthesized compounds and sulphasalazine as
a reference were initially screened in acetic acid-induced colitis in
rats, a well-established and a reproducible experimental model for
acute ulcerative colitis [28e32]. Data and parameters of anti-
ulcerogenic effect are listed in Table 1.
Benzofuran derivatives exhibit
a promising gastro-protective
activity [20,21] and show dual cycloxygenase (COX-2) and 5-lip-
oxygenase (5-LOX) inhibition, so they are potential candidates as
anti-inflammatory agents [22,23]. In addition, it is known that
bacterial infections often produce pain and inflammation, there-
fore, the antimicrobial activities of benzofurans have been exten-
sively investigated [24,25].
The present work describes design and synthesis of 3-amino-
furosalicylic acid 4, azo-conjugates with aniline 2a, 4-ASA 2b or
sulphapyridine 2c as well as N-arylsulphonamido 5, chlor-
osulphonyl 6, aminosulphonyl 7 and N-arylaminosulphonyl deriv-
atives 8 (positional isosters of 5). The effects of the new synthesized
compounds on acetic acid-induced UC in rats’ model and their
antimicrobial activities were evaluated.
Since, UC is an inflammation disease of the lining of the colon, or
large intestine and since the clinically used drugs for UC treatment
e.g. mesalamine may work by blocking the activity of cyclo-
oxygenase, molecular docking studies using molecular operating
environment (MOE) and murine COX enzymes were performed in
order to rationalize the obtained biological results and their
mechanism of action.
2.2.1. Statistical analysis
Statistical analysis was performed using SPSS 14.0 statistical
software. Differences among groups were examined using para-
metric one-way analysis of variance (ANOVA). Results are
expressed as the mean ꢂ SEM. The minimal level of significance
was identified at P<0.05 and P<0.01.
Table 1
Effects of sulphasalazine and the new synthesized compounds on the macroscopic
parameters of ulcerative colitis induced by acetic acid in rats. (n ¼ 5).
Groups
Lesion score Ulcer area
Ulcer index
Wet W/L
Ratio
(0e4)
(cm2)
Sham
0.0 ꢂ 0.0
0.0 ꢂ 0.0
0.0 ꢂ 0.0
66.4 ꢂ 3.17
123.7 ꢂ 8.01
79.0 ꢂ 6.32**
84.5 ꢂ 3.95**
99.4 ꢂ 6.86*
95.2 ꢂ 6.14**
90.0 ꢂ 7.24**
99.1 ꢂ 6.48*
98.6 ꢂ 7.66**
86.4 ꢂ 5.90**
112.0 ꢂ 7.40
Control (DMSO) 3.8 ꢂ 0.20
5.3 ꢂ 0.27
9.1 ꢂ 0.34
Sulphasalazine
1.6 ꢂ 0.24** 2.1 ꢂ 0.10** 3.7 ꢂ 0.30**
2.2 ꢂ 0.20** 2.6 ꢂ 0.24** 4.8 ꢂ 0.20**
2a
2b
2c
4
5a
5b
5c
6
3.0 ꢂ 0.31*
4.4 ꢂ 0.23*
7.4 ꢂ 0.39**
2.8 ꢂ 0.20** 3.9 ꢂ 0.33** 6.7 ꢂ 0.20**
2.2 ꢂ 0.20** 3.3 ꢂ 0.20** 5.5 ꢂ 0.38**
2. Results and discussion
3.0 ꢂ 0.44*
3.0 ꢂ 0.31*
4.4 ꢂ 0.24*
4.3 ꢂ 0.18*
7.4 ꢂ 0.50**
7.6 ꢂ 0.52**
2.1. Chemistry
2.0 ꢂ 0.31** 2.6 ꢂ 0.24** 4.6 ꢂ 0.40**
3.4 ꢂ 0.24
3.0 ꢂ 0.15*
3.4 ꢂ 0.24
3.4 ꢂ 0.24
3.4 ꢂ 0.24
3.4 ꢂ 0.24
3.6 ꢂ 0.24
4.7 ꢂ 0.40
4.4 ꢂ 0.24*
5.1 ꢂ 0.33
4.7 ꢂ 0.20
4.8 ꢂ 0.37
4.8 ꢂ 0.43
5.1 ꢂ 0.38
8.1 ꢂ 0.56
7
7.4 ꢂ 0.18** 103.4 ꢂ 7.18*
6-Hydroxy-4-methoxybenzofuran-5-carboxylic acid
a starting material was prepared via oxidative alkaline cleavage of
-pyranone ring of the naturally isolated 4-methoxy-7-methyl-
1
as
8a
8b
8c
8d
8e
8.5 ꢂ 0.31
8.1 ꢂ 0.40
8.2 ꢂ 0.37
8.2 ꢂ 0.52
8.7 ꢂ 0.60
119.9 ꢂ 6.90
111.0 ꢂ 6.20
112.0 ꢂ 7.23
110.0 ꢂ 6.89
120.1 ꢂ 7.30
g
5H-furo[3,2-g][1]benzopyran-5-one “visnagin” according to the
literature procedure [26]. It was convenient to use the highest
electron density on position 7 provided by the adjacent OH group
to prepare the azo compounds 2aec by coupling compound 1 with
W/L ¼ Weight/Length, *denote significant difference vs. control groups at P<0.05
and **P<0.01.

4106
G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
Table 2
2.2.2. LD50
In vitro antibacterial activity using agar diffusion assay technique.
LD50 figures are frequently used as a general indicator of
a substance’s acute toxicity.
Compound
Diameter of zone of growth inhibition (mm)
Oral administration of the tested compounds in doses less than
220 mg kgꢁ1 body weight (b.wt) failed to kill mice within 24 h. LD50
was 410 mg kgꢁ1 b.wt for compounds 2a, 2c, 8c and 8d and
400 mg kgꢁ1 b.wt for compounds 5a, 5b and 7. On the other hand,
LD50 for compounds 4, 8a, 8b and 8e was 396 mg kgꢁ1 b.wt. Finally,
LD50 of compounds 2b, 5c and 6 was 390 mg kgꢁ1 b.wt. Thus the
tested compounds are considered safe, since substances possessing
LD50 equal to 50 mg or less per kilogram of body weight when
administered orally to albino rats, are contemplated as toxic [33].
Staphylococcus
aureus
Bacillus
subtilis
Escherichia
coli
Candida
albicans
Control (DMF)
Sulphasalazine
Amphotercin B
1
2a
2b
2c
3
6
30
e
e
8
10
10
e
8
e
e
e
8
6
25
e
6
8
6
7
20
8
18
25
22
e
e
e
e
10
12
12
e
10
10
12
e
4
8
10
e
8
5a
5b
5c
6
10
10
12
8
8
12
e
10
15
8
20
14
15
10
12
8
2.2.3. Anti-ulcerogenic effect (Table 1)
e
Intra-rectal administration of 4% acetic acid in rats (control
group) significantly increased the colon weight and markedly
decreased the colon length, leading to elevation in wet weight/
length (W/L) ratio. Also, macroscopic examination of the colon of
control rats revealed hyperemia, edema, erosion, and ulceration in
their mucosa. No pathological changes were observed in sham
group suggesting that handling procedure had no interference with
the experimental outputs. It was noticed that oral treatment with
sulphasalazine (5 mg kgꢁ1 b.wt ¼ 12.5 mmol kgꢁ1 b.wt) as
a reference drug and the tested compounds 2a, 2c, 4 and 5c in
equimolar doses significantly reduced the intensity of lesion score,
ulcer area, ulcer index and wet weight/length ratio (P<0.01)
compared to the control group (Table 1). On the other hand,
compounds 2b, 5a, 5b and 7 had a lower anti-ulcerogenic efficacy
(P<0.05). Other compounds 6, 8a, 8b, 8c, 8d and 8e failed to
decrease the intensity of lesion score, ulcer area, ulcer index and
wet weight/length ratio.
From the previous results, it is clear that 3-aminofurosalicylic
acid 4 has a significant effect in correcting ulcerative colitis. Its azo-
conjugates with aniline 2a or with sulphapyridine 2c also have
a significant anti-ulcerogenic effect and their advantage over
compound 4 is believed to be the capacity to deliver the active
agent 4 past the small intestine and into the large intestine, the
active site of UC. Also, the azo conjugate with PAS 2b exhibited
a mild anti-ulcerogenic.
e
8
8
7
8
e
8a
8b
8c
8d
8e
e
8
12
12
e
e
e
e
e
e
10
e
8
Staphylococcus aureus (MTCC 3160) and Bacillus subtilis (MTCC
1456), two Gram-positive bacteria namely E. coli (ATCC 8739) and
Pseudomonas aeruginosa (MTCC 3541) and the fungus Candida
albicans (ATCC 10231). The antimicrobial activity is reflected as
zone of growth inhibition of the tested microorganisms (measured
in mm). Azo derivatives 2a, 2b and 2c exhibited antibacterial
activity against both Gram-positive and Gram-negative bacteria.
Compounds 5a, 5b, 5c, 8a and 8d showed antibacterial activity
against Gram-positive bacteria B. subtilis while, compounds 4, 8a,
8b and 8c showed antibacterial activity against Gram-negative
bacteria E. coli. All the tested compounds displayed inhibitory
activity against the fungus C. albicans except 1, 3, 4, 8d and 8e. Azo
derivatives 2ae2c and chlorosulphonyl compound 6 showed
a promising antifungal activity closely related to that of the stan-
dard, amphotercin B. The antifungal activity of compound 2b (azo
derivative of PAS) was better than that of amphotercin B.
It is also clear that compound 5c proved to be the most phar-
macologically active among the newly synthesized compounds. It
could be easily broken in the body by hydrolysis e decomposition,
a process which proceeds more quickly in presence of acids [34],
with the liberation of the parent pharmacologically active
compound 4. It is also interesting to observe that compound 5c (the
sulphonamido derivative of 4) proves to be more active than 4
(3-aminofurosalicylic acid). Hydrolysis of N-arylsulphonamido
derivatives 5 is attributed to the electronic contribution of the
substituents: electron withdrawing groups, for example chloride of
5c or electron donating groups, for example methyl of 5b. N-aryl-
sulphonamido derivatives 5a and 5b exhibited moderate anti-
ulcerogenic effect for UC as well as aminosulphonyl derivative 7. On
the other hand, both of chlorosulphonyl derivative 6 and N-aryla-
minosulphonyl derivatives 8 (positional isosters of 5) exhibited no
pharmacologic effect. This may be attributed to the fact that the
intact compounds 8aee and their hydrolytic products (furo-sali-
cylic acid nucleus substituted at 3-position with sulphonic acid
rather than amino group) were pharmacologically inactive.
2.4. Molecular docking study (Table 3)
Molecular docking studies of compounds 4e8 using MOE and
murine COX enzymes were performed to rationalize the obtained
biological results and their mechanism of action (Fig. 1). Besides,
molecular docking studies helped in understanding the various
interactions between the ligands and enzyme active site in detail.
Since it is highly probable that the investigated compounds 2aec
undergo in vivo enzymatic cleavage of azo moiety to afford the
corresponding amino analogue, molecular docking study for the
amino derivative 4 was performed.
Docking studies of the inhibitors were performed using
Molecular Operating Environment (MOE 2008.10; Chemical
Computing Group, Canada) [36] using murine COX-2 co-crystal-
lized with celecoxib (PDB: 6COX) as a template (Fig. 2). We per-
formed 100 docking iterations for each ligand and the top-scoring
configuration of each of the ligandeenzyme complexes was
selected on energetic grounds. Docking of compounds 2aec, 4,
5aec, 8a, 8c and d showed that the ligand was oriented so that the
carboxylate moiety was in the vicinity of Arg120 residue forming
a hydrogen bond interaction (side chain acceptor) the NH2 group of
2.3. Antimicrobial screening (Table 2)
All the newly synthesized compounds 2e8, sulphsalazine and
amphotercin B (as standards) were tested in vitro using the agar
diffusion assay technique [35]. The antimicrobial activity was
ꢀ
guanidine side chain (distance 2.22e2.84 A, score 11e31%), Table 3.
A hydrogen bond interaction between Arg120 and OH of 5-ASA or
carboxylate of salicylic acid (SA) was observed, while in case of
screened
against
two
Gram-positive
bacteria
namely

G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
4107
Table 3
From the abovementioned data, the molecular docking studies
of the examined compounds 4e8 showed that they bind to the
COX-2 active site with position and orientation very close to that
resulting from the crystal structure of celecoxib complex with COX-
2 and docking studies of the standard compounds SA, 5-ASA and
indomethacin. Consequently, these observations provided a good
explanation for the assumed inhibitory activity of COX enzyme with
compounds 2b, 2c, 4 and 5c.
Molecular Docking data using MOE.
Compound
Celecoxib
Bond
number
Amino acid
(bond length A,
score %)
Docking energy
(kcal./mole)
ꢀ
3
Arg120 4
ꢁ111.0077
Arg513 / (3.15, 17),
His90 / (2.69, 20).
Tyr355 / (2.57, 11),
Glu524 ) (1.30, 34).
Arg120 / (2.91, 13),
Tyr355 / (2.74, 35).
Arg120 / (2.84, 14),
Arg513 / (2.32, 23),
Leu352 ) (1.94, 30).
Arg120 / (2.85, 25),
Arg120 / (2.23, 30),
Tyr355 / (2.34, 56).
Arg120 4
Indomethacin
Salicylic acid
5-ASA
2
2
3
ꢁ110.9509
ꢁ39.7747
ꢁ55.6055
2.5. Evaluation of lipophilicity
Partition coefficients (Log P) of sulphasalazine and the synthe-
sized compounds were calculated using ChemDraw Ultra V 8.0. In
this study, the pharmacological activity is not attributed to the
value of Log P. Although, the pharmacologically active compounds
2a, 2c and 5c and sulphasalazine are considered to be lipophilic
(Log P are 3.12, 2.91, 1.65 and 3.42, respectively), the active parent
compound 4 is hydrophilic (Log P is 0.11).
2a
2b
3
7
ꢁ76.4459
ꢁ62.9665
Gly526 4
Arg513 / (2.82, 24),
Arg513 / (2.59, 15),
Tyr355 / (3.13, 20),
Val523 / (1.87, 36),
Glu524 / (1.30, 65).
Arg120 4
3. Conclusion
The data generated as an outcome of this work demonstrates
that four new synthesized compounds: 3-aminofurosalicylic acid 4,
its azo conjugate with aniline 2a, or with sulphapyridine 2c and N-
(p-chloro)sulphonamido derivative 5c have a remarkable anti-
ulcerogenic effect on acetic acid-induced ulcerative colitis in rats.
SAR:The presence of aminosalicylic acid structure is essential for
biological activity. Substitution of salicylic acid with amino group
either at position 5, 4, or 3 are biologically equivalents. For activity;
the amino group should be unsubstituted or have a substituent that
is removed readily in vivo.
2c
4
3
3
3
3
3
ꢁ76.8431
ꢁ57.9414
ꢁ64.3142
ꢁ88.2576
ꢁ60.1337
Arg120 4
Arg120 / (3.39, 13).
Arg120 / (3.08, 13),
Arg120 / (2.29, 31),
Tyr355 / (2.35, 68).
Arg120 / (2.33, 17),
Arg513 / (2.80, 60),
Tyr355 / (2.72, 41).
Arg120 / (2.71, 27),
Tyr355 / (2.36, 60),
Tyr355 / (2.81, 38).
Arg120 4
Ser530 / (2.73, 31),
Ser530 / (2.93, 27).
Tyr355 / (2.52, 47).
Tyr355 / (2.83, 15),
Tyr355 ) (3.24, 19).
Arg120 / (2.22, 31),
Arg120 / (3.13, 12),
Tyr355 / (2.45, 90).
Arg513 / (2.39, 53)
Arg120 / (2.73, 28),
Tyr355 / (2.73, 57),
Tyr355 / (2.86, 34).
Arg120 / (2.41, 15),
Arg120 / (2.66, 29),
Tyr355 / (2.35, 65).
Arg120 / (3.22, 11),
Val523 ) (2.03, 32).
5a
5b
5c
4. Experimental
6
7
1
2
ꢁ82.0727
ꢁ65.1178
4.1. Chemistry
8a
3
ꢁ104.8033
Melting points are uncorrected and determined in one end open
capillary tubes using Gallen Kamp melting point apparatus MFB-
595-010M (GallenKamp, London, England). Microanalysis was
carried out at the microanalytical unit, Faculty of Science, Cairo
University. IR spectra were determined using KBr discs (cmꢁ1) on
Shimadzu Infrared Spectrometer IR-435 (Shimadzu, Kyoto, Japan),
PerkineElmer FT-IR 1650 (PerkineElmer, Waltham, Massachusetts
02451, USA) and Mattson Genesis II FTIRÔ Spectrometer (Mattson,
8b
8c
1
3
ꢁ86.5446
ꢁ97.1818
8d
8e
3
2
ꢁ107.2287
ꢁ81.3963
Madison, WI, USA). 1H NMR (DMSO-d6, D2O)
d ppm spectra were
determined using Joel NMR Varian Gemini 200 MHz Spectrometer
(Joel, Tokyo, Japan) and Varian Mercury VX-300 MHz NMR Spec-
trometer (Varian, Oxford, England). Mass spectra were recorded
using Hewlett Packard Varian (Varian, Polo, USA) and Shimadzu
Gas Chromatograph Mass spectrometer-QP 1000 EX (Shimadzu,
Kyoto, Japan). TLC were carried out using Art. DC-Plastikfolien,
Kieselgel 60 F254 sheets (Merck, Darm-stadt, Germany), the devel-
oping solvents were CCl4/CH3COOC2H5 (9:1) or (4:1) and the spots
were visualized at 366, 254 nm by UV Vilber Lourmat 77 202
(Vilber, Marne La Vallee, France).
N.B. / means the bond is a side chain acceptor.
) is a side chain donor.
4 is a hydrophobic.
celecoxib the interaction with Arg120 was a hydrophobic bond.
Furthermore, a hydrogen bond interaction between the OH group
of Tyr355 and the ligand carboxylate and/or methoxy group was
ꢀ
observed (distance 2.35e3.24 A, score 15e90%) through docking
studies of all the new synthesized compounds e except 5c and 8b e
and the standard compounds: indomethacin and SA. The amino
group of guanidine residue of Arg513 interacts with OCH3 group of
4.1.1. Synthesis of 6-hydroxy-4-methoxybenzofuran-5-carboxylic
acid 1 (Scheme 1)
ꢀ
compounds 5a and 8b (distance 2.72, 2.39 A, score 60, 53%
respectively). The ligand 5c p-chlorophenyl moiety formed
a hydrophobic bond with Arg120 and two hydrogen bonds between
of the NH group of Ser530 and carboxylate and OCH3 (distance 2.73,
A compound 1 was prepared according to the literature proce-
dure [26].
ꢀ
2.93 A, score 31, 27% respectively). Docking energy (kcal/mole) of
4.1.2. Synthesis of 7-arylazo-6-hydroxy-4-methoxybenzofuran-5-
carboxylic acid 2aec (Scheme 1)
The appropriate aniline derivative (54 mmol) “aniline, p-ami-
nosalicylic acid or sulphapyridine” was dissolved in a mixture of
the new synthesized compounds showed that their interaction is
more stable than that of SA or 5-ASA and some of them are closely
related to that of celecoxib and indomethacin (8a and 8d).

4108
G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
Fig. 1. Docking of compound 4 in the active site of murine COX-2 (3D Contact Style).
concentrated hydrochloric acid (16 ml) and water (16 ml) and
cooled to 0e5 ꢀC. Sodium nitrite (4.0 g, 58 mmol) was dissolved in
water (16 ml) and cooled. The cold sodium nitrite solution was
added in small volumes to the cold aniline hydrochloride solution,
and the mixture was kept well shaken (solution A). The tempera-
ture was not allowed to rise above 10 ꢀC. A solution of the salicylic
acid derivative 1 (11.2 g, 54 mmol) in 10% NaOH (45 ml) was cooled
to 5 ꢀC (solution B). Solution A was added very slowly to solution B;
a dark-red solution was produced. When all the diazonium salt
solution had been added, the mixture was allowed to stand in an ice
bath for 30 min with occasional stirring. The dark-red precipitate
was filtered off, washed well with water, dried and crystallized
from acetic acid/water 1:1.
4.1.2.1. 6-Hydroxy-4-methoxy-7-(2-phenylazo) benzofuran-5-carbox-
ylic acid 2a. Yield 80%, mp 152e154 ꢀC. IR (KBr, cmꢁ1): 3200, 2850
(2OH), 1697 (C]O), 1590 (N]N). 1H NMR (DMSO-d6, D2O)
d ppm:
4.01 (s, 3H, OCH3), 6.73 (d, 1H, furan H, J: 2 Hz), 7.10e7.79 (m, 5H,
ArH), 7.92 (d, 1H, furan H, J: 2.1 Hz), 12.98 (s, 1H, OH exch.), 14.85 (s,
1H, COOH exch.). 13C NMR (DMSO-d6): 60.1 (OCH3), 92.7 (C5), 105.3
(C3), 109.3 (C3a), 119.5 (C2Ar, C6Ar), 123.3 (C7), 128.6 (C3Ar, C5Ar),
139.2 (C4Ar), 143.7 (C7a), 150.8 (C6), 154.6 (C1Ar), 156.9 (C4), 164.7
(C]O). Anal. Calcd. For C16H12N2O5 (312.28): C, 61.54; H, 3.87; N,
8.97. Found: C, 61.36; H, 4.05; N, 8.67. Log P: 3.12.
4.1.2.2. 7-[2-(4-Carboxy-3-hydroxy phenyl)azo]-6-hydroxy-4-methoxy
benzofuran-5-carboxylic acid 2b. Yield 90%, mp 170e172 ꢀC. IR (KBr,
cmꢁ1): 3156 (2OH), 2964 (2COOH), 1691, 1675 (2C]O), 1608 (N]N).
1H NMR (DMSO-d6, D2O)
d ppm: 4.14 (s, 3H, OCH3), 6.71 (d, 1H, furan
H, J: 2.1 Hz), 7.05e7.40 (m, 3H, ArH), 7.73 (d, 1H, furan H, J: 2.4 Hz),
12.0 (s, 1H, OH exch.), 14.0 (s, 1H, COOH exch.). Anal. Calcd. For
C17H12N2O8 (372.29): C, 54.85; H, 3.25; N, 7.52. Found: C, 54.90; H,
3.23; N, 7.72. Log P: 2.29.
4.1.2.3. 6-Hydroxy-4-methoxy-7-[2-(2-pyridinylsulphamoyl)
phe-
nylazo] benzofuran-5-carboxylic acid 2c. Yield 90%, mp 148e150 ꢀC.
IR (KBr, cmꢁ1): 3300 (NH), 3128 (OH), 2925 (COOH), 1693 (C]O),
1674 (NH bending), 1633 (N]N), 1373, 1145 (SO2). 1H NMR (DMSO-
d6, D2O)
d ppm: 4.01 (s, 3H, OCH3), 6.6e6.9 (m, 3H, furan H and
2ArH), 7.1e7.3 (m, 3H, 2ArH and py. C4eH), 7.6e7.9 (m, 2H, py. C3eH
and py. C5eH), 8.1e8.3 (m, 2H, furan H and py. C6eH), 9.5 (s, 1H, NH
Fig. 2. Docking of compound 4 in the active site of murine COX-2 (2D Contact Style).

G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
4109
OCH3
COOH
OCH3
2a R1 = H, R2 = H
COOH
O
OH
2b R1 = OH, R2 = COOH
a
N
O
OH
N
H
N
1
O
S
R1
2c R1 =
, R2 = H
N
2a-c
R2
O
b
c
OCH3
OCH3
COOH
d
COOH
O
OH
O
OH
NO2
NH2
3
4
Scheme 1. Reagents and solvents: a: Diazonium salt, 10% NaOH, 5 ꢀC. b: HNO3, acetone, 0 ꢀC. c: Sodium dithionite, ethanol, reflux. d: Sodium dithionite, 5% NaOH, heat.
exch.), 11.5 (s, 1H, OH exch.), 14.0 (s, 1H, COOH exch.). 13C NMR
(DMSO-d6): 60.1 (OCH3), 92.7 (C5), 105.2 (C3), 109.8 (C3a, C3py),
113.9 (C5py), 120.1 (C2Ar, C6Ar), 128.8 (C3Ar, C5Ar), 140.2 (C4py),
143.2 (C4Ar), 146.6 (C2), 148.0 (C6py), 151.4 (C6, C7a), 153.1 (C2py),
154.9 (C1Ar), 157.1 (C4), 168.7 (C]O). Anal. Calcd. For C21H16N4O7S
(468.44): C, 53.84; H, 3.44; N, 11.96. Found: C, 53.87; H, 3.78; N,
11.95. Log P: 2.91.
(DMSO-d6, D2O) d ppm: 3.43 (broad, 2H, NH2 exch.), 4.09 (s, 3H,
OCH3), 7.14 (d, 1H, furan H, J: 2.4 Hz), 7.84 (d, 1H, furan H, J: 2.4 Hz),
12.90 (s, 1H, OH exch.), 13.01 (s, 1H, COOH exch.).
4.1.5. Synthesis of 7-arylsulphonamido-6-hydroxy-4-
methoxybenzofuran-5-carboxylic acid 5aec (Scheme 2)
The amino derivative 4 (2.23 g, 10 mmol) was dissolved in
a mixture of dry acetone (20 ml) and pyridine (few drops), and the
appropriate benzene sulphonyl chloride derivative (10 mmol),
benzene sulphonyl chloride (1.77 g), p-toluene sulphonyl chloride
(1.91 g), or p-chlorobenzenesulphonyl chloride (2.11 g), was added.
The reaction mixture was refluxed for 3 h and left at room temper-
ature overnight with stirring. The separated solid was filtered off; by
diluting the filtrate with double volume water, a further crop is
obtained. The total product was recrystallised from acetone.
4.1.3. Synthesis of 6-hydroxy-4-methoxy-7-nitrobenzofuran-5-
carboxylic acid 3 (Scheme 1)
A solution of the salicylic acid derivative 1 (1 g) in acetone
(20 ml) was treated at 0e5 ꢀC with nitric acid (10 ml, obtained by
mixing 50 g nitric acid “d 1.43” with 35 ml water). The reaction
mixture was allowed to stand for 30 min. The yellow precipitate
was filtered off and crystallized from ethanol.
Yield 75%, mp 110e112 ꢀC. IR (KBr, cmꢁ1): 3207 (OH), 2961
(COOH), 1688 (C]O), 1522, 1334 (NO2). 1H NMR (DMSO-d6, D2O)
4.1.5.1. 6-Hydroxy-4-methoxy-7-(phenylsulphonamido) benzofuran-
5-carboxylic acid 5a. Yield 70%, mp 158e160 ꢀC. IR (KBr, cmꢁ1):
3368 (NH), 3100 (OH), 2900 (COOH), 1697 (C]O), 1675 (NH
d
ppm: 3.85 (s, 3H, OCH3), 7.40 (d, 1H, furan H, J: 1.9 Hz), 8.03 (d, 1H,
furan H, J: 1.9 Hz), 8.95 (s, 1H, OH exch.), 11.00 (s, 1H, COOH exch.).
Anal. Calcd. For C10H7NO7 (253.17): C, 47.44; H, 2.79; N, 5.53. Found:
C, 46.96; H, 3.12; N, 5.02. Log P: 3.12.
bending), 1400, 1189 (SO2). 1H NMR (CDCl3, D2O)
d ppm: 4.37 (s, 3H,
OCH3), 4.71 (s, 1H, NH exch.), 6.86 (d, 1H, furan H, J: 1.8 Hz),
7.40e7.60 (m, 5H, ArH), 7.96 (d, 1H, furan H, J: 1.8 Hz), 11.63 (s, 1H,
OH exch.), 12.20 (s, 1H, COOH exch.). 13C NMR (DMSO-d6): 56.3
(OCH3), 92.6 (C5), 105.2 (C3), 109.2 (C7), 111.6 (C3a), 127.22 (C2Ar,
C6Ar), 129.6 (C3Ar, C5Ar), 133.5 (C4Ar), 139.5 (C1Ar), 141.0 (C6),
143.4 (C2), 150.7 (C7a), 154.6 (C4), 164.8 (C]O). Mass spectrum
showed m/z 365 (Mþþ2). Anal. Calcd. For C16H13NO7S (363.35): C,
52.89; H, 3.61; N, 3.85. Found: N, 3.88. Log P: 1.09.
4.1.4. Synthesis of 7-amino-6-hydroxy-4-methoxybenzofuran-5-
carboxylic acid 4 (Scheme 1)
Method A: A suspension of the azo derivative 2a (1.56 g, 5 mmol)
in hot ethanol (60 ml) was treated gradually with a solution of
sodium dithionite (4.2 g, 20 mmol) in water (15 ml). The mixture
was refluxed for 1 h, cooled and filtered. The filtrate was diluted
with water to 2e3 volumes and the solid was filtered off and
crystallized from methanol.
4.1.5.2. 6-Hydroxy-4-methoxy-7-[(p-methylphenyl) sulphonamido]
benzofuran-5-carboxylic acid 5b. Yield 65%, mp 148e150 ꢀC. IR (KBr,
cmꢁ1): 3395 (NH), 3063 (OH), 2923 (COOH), 1700 (C]O), 1619 (NH
Yield 60%, mp 164e166 ꢀC. IR (KBr, cmꢁ1): 3456, 3327 (NH2),
3097 (OH), 2962 (COOH), 1693 (C]O), 1675 (NH bending). 1H NMR
(DMSO-d6, D2O)
d ppm: 3.26 (broad, 2H, NH2 exch.), 4.06 (s, 3H,
OCH3
OCH3
OCH3), 7.12 (d, 1H, furan H, J: 2.0 Hz), 7.90 (d, 1H, furan H, J: 2.1 Hz),
10.27 (s, 1H, OH exch.), 10.35 (s, 1H, COOH exch.). Anal. Calcd. For
C10H9NO5 (223.18): C, 53.82; H, 4.06; N, 6.28. Found: C, 54.09; H,
3.89; N, 6.48. Log P: 0.11.
COOH
COOH
a
O
O
OH
5a R = H
OH
O
Method B: A solution of the nitro derivative 3 (1.26 g, 5 mmol) in
NaOH (10 ml, 5%), was treated with a solution of sodium dithionite
(4.2 g, 20 mmol) in water (15 ml). The mixture was refluxed for
30 min, cooled and filtered. The solid was washed several times
with water, dried and crystallized from methanol.
5b R = CH3
5c R = Cl
NH2
HN
S
4
O
R
5a-c
Yield 60%, mp 163e165 ꢀC. IR (KBr, cmꢁ1): 3450, 3350 (NH2),
3090 (OH), 2950 (COOH), 1693 (C]O), 1677 (NH bending). 1H NMR
Scheme 2. Reagents and solvents. a: ClSO2C6H4R, dry acetone, pyridine, reflux.

4110
G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
bending), 1378, 1156 (SO2). 1H NMR (DMSO-d6, D2O)
d
ppm: 2.27 (s,
(287.25): C, 41.81; H, 3.16; N, 4.88. Found: C, 42.26; H, 2.83; N, 4.83.
Log P: 0.57.
3H, CH3), 4.01 (s, 3H, OCH3), 4.20 (s, 1H, NH exch.), 6.07 (d, 1H, furan
H, J: 1.8 Hz), 7.40 (d, 2H, ArH, J: 7.4 Hz), 7.53 (d, 2H, ArH, J: 7.4 Hz),
8.05 (d, 1H, furan H, J: 1.8 Hz), 9.00 (s, 1H, OH exch.), 11.20 (s, 1H,
COOH exch.). Anal. Calcd. For C17H15NO7S (377.37): C, 54.12; H, 4.01;
N, 3.71. Found: C, 54.00; H, 4.00; N, 3.89. Log P: 1.58.
Method B: A mixture of sulphonyl chloride derivative 6 (1.5 g,
5 mmol) and anhydrous ammonium carbonate (0.77 g, 10 mmol) in
dry ether (50 ml) and pyridine (few drops) was refluxed for 3 h. The
solvent was evaporated and the residue was triturated with dilute
HCl, washed with small volume of water and crystallized from
water. Yield 60%, mp 250e252 ꢀC.
4.1.5.3. 7-[4-(p-Chlorophenyl) sulphonamido]-6-hydroxy-4-methox-
ybenzofuran-5-carboxylic acid 5c. Yield 75%, mp 120e122 ꢀC. IR
(KBr, cmꢁ1): 3399 (NH), 3131 (OH), 2927 (COOH), 1683 (C]O), 1604
4.1.8. Synthesis of 7-arylaminolsulphonyl-6-hydroxy-4-
methoxybenzofuran-5-carboxylic acid 8aee (Scheme 3)
A mixture of sulphonyl chloride derivative 6 (1.5 g, 5 mmol) and
the appropriate amine (5 mmol) in dry ether (50 ml) and pyridine
(few drops) was refluxed for 3 h. The solvent was evaporated and
the residue was triturated with dilute HCl, washed with water and
crystallized from methanol/water.
(NH bending), 1416, 1157 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm:
4.08 (s, 3H, OCH3), 5.00 (s, 1H, NH exch.), 6.90 (d, 1H, furan H, J:
2.0 Hz), 7.62 (d, 2H, ArH, J: 7.6 Hz), 7.75 (d, 2H, ArH, J: 7.6 Hz), 8.03
(d, 1H, furan H, J: 2.0 Hz), 10.5 (s, 1H, OH exch.), 11.00 (s, 1H, COOH
exch.). Anal. Calcd. For C16H12ClNO7S (397.79): C, 48.31; H, 3.04; N,
3.52. Found: C, 48.31; H, 3.21; N, 3.33. Log P: 1.65.
4.1.6. Synthesis of 7-(chlorosulphonyl)-6-hydroxy-4-
methoxybenzofuran-5-carboxylic acid 6 (Scheme 3)
4.1.8.1. 6-Hydroxy-4-methoxy-7-(phenylaminolsulphonyl)
benzo-
furan-5-carboxylic acid 8a. Yield 55%, mp 179e181 ꢀC. IR (KBr,
cmꢁ1): 3365 (NH), 3123 (OH), 2939 (COOH), 1669 (C]O), 1606 (NH
Furo-salicylic acid derivative 1 (2.08 g, 5 mmol) was added to
chlorosulphonic acid (5.8 g, 3.41 ml, 50 mmol), magnetically stirred
at 0 ꢀC, over 30 min period. The stirring was continued for a further
period of 60 min at room temperature and the mixture poured on
to crushed ice. The obtained solid was filtered off, dried and crys-
tallized from water/methanol.
bending), 1307, 1154 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm: 4.08 (s,
3H, OCH3), 4.04 (s, 1H, NH exch.), 7.08 (d, 1H, furan H, J: 1.8 Hz),
7.25e7.62 (m, 5H, ArH), 8.08 (d, 1H, furan H, J: 1.8 Hz), 11.30 (s, 1H,
OH exch.), 12.65 (s, 1H, COOH exch.). Mass spectrum showed m/z
365 (Mþþ2). Anal. Calcd. For C16H13NO7S (363.35): C, 52.89; H,
3.61; N, 3.85. Found: C, 53.10; H, 3.81; N, 4.49. Log P: 1.09.
Yield 65%, mp 318e320 ꢀC. IR (KBr, cmꢁ1): 3200 (OH), 2925
(COOH), 1625 (C]O), 1399, 1138 (SO2). 1H NMR (DMSO-d6, D2O)
d
ppm: 4.36 (s, 3H, OCH3), 6.89 (d, 1H, furan H, J: 2.0 Hz), 7.69 (d, 1H,
4.1.8.2. 6-Hydroxy-4-methoxy-7-[(p-methylphenyl) aminosulphonyl]
benzofuran-5-carboxylic acid 8b. Yield 55%, mp 200e202 ꢀC. IR
(KBr, cmꢁ1): 3363 (NH), 3186 (OH), 2925 (COOH), 1660 (C]O), 1600
furan H, J: 2.0 Hz), 10.0 (s, 1H, OH exch.), 12.00 (s, 1H, COOH exch.).
Mass spectrum showed m/z 307 (Mþ). Anal. Calcd. For C10H7ClO7S
(306.68): C, 39.16; H, 2.30. Found: C, 39.28; H, 2.50. Log P: 0.42.
(NH bending), 1314, 1129 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm:
2.11 (s, 3H, CH3), 4.08 (s, 3H, OCH3), 5.30 (s, 1H, NH exch.), 6.88 (d,
2H, ArH, J: 8.2 Hz), 7.09 (d, 1H, furan H, J: 2.0 Hz), 7.31 (d, 2H, ArH,
J:8.0 Hz), 8.09 (d, 1H, furan H, J: 1.8 Hz), 11.40 (s, 1H, OH exch.),12.50
(s, 1H, COOH exch.). Anal. Calcd. For C17H15NO7S (377.37): C, 54.12;
H, 4.01; N, 3.71. Found: C, 54.22; H, 3.90; N, 4.11. Log P: 1.58.
4.1.7. Synthesis of 6-hydroxy-4-methoxy-7-(aminosulphonyl)
benzofuran-5-carboxylic acids 7 (Scheme 3)
Method A: To concentrated ammonia solution (20 ml, d 0.88),
sulphonyl chloride derivative 6 (2 g) was added. The mixture was
heated with occasional swirling to just below the boiling point for
15 min. The suspension was cooled in ice, and acidified with acetic
acid. The product was filtered, dried and crystallized from water.
Yield 80%, mp 252e254 ꢀC. IR (KBr, cmꢁ1): 3400e3200 (NH2 and
OH), 2901 (COOH), 1652 (C]O), 1372, 1164 (SO2). 1H NMR (DMSO-
4.1.8.3. 7-[4-(p-Chlorophenyl) aminosulphonyl]-6-hydroxy-4-methox-
ybenzofuran-5-carboxylic acid 8c. Yield 60%, mp 144e146 ꢀC. IR (KBr,
cmꢁ1): 3366 (NH), 3150 (OH), 2923 (COOH), 1660 (C]O), 1600 (NH
bending), 1319, 1151 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm: 4.03 (s,
d6, D2O)
d
ppm: 4.47 (s, 3H, OCH3), 4.83 (broad, 2H, NH2), 6.91 (d,
3H, OCH3), 5.40 (s, 1H, NH exch.), 6.81 (d, 2H, ArH, J: 8.7 Hz), 7.10 (d,
1H, furan H, J: 2.0 Hz), 7.38 (d, 2H, ArH, J: 8.7 Hz), 7.96 (d, 1H, furan H,
J: 2.0 Hz),11.5 (s,1H, OH exch.),12.50 (s,1H, COOH exch.). Anal. Calcd.
For C16H12ClNO7S (397.79): C, 48.31; H, 3.04; N, 3.52. Found: C,
48.06; H, 3.34; N, 3.33. Log P: 1.65.
1H, furan H, J: 2.0 Hz), 7.82 (d, 1H, furan H, J: 2.0 Hz),11.25 (s, 1H, OH
exch.), 13.80 (s, 1H, COOH exch.). Anal. Calcd. For C10H9NO7S
OCH3
COOH
OCH3
COOH
4.1.8.4. 7-[4-(2,4-Dichlorophenyl)
aminosulphonyl]-6-hydroxy-4-
methoxybenzofuran-5-carboxylic acid 8d. Yield 65%, mp 160e162 ꢀC.
IR (KBr, cmꢁ1): 3389 (NH), 3177 (OH), 2923 (COOH), 1615 (C]O),1600
a
O
OH
O
S
Cl
OH
(NH bending),1303,1058 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm: 3.99
O
O
b
1
(s, 3H, OCH3), 5.29 (s,1H, NH exch.), 6.75 (d,1H, furan H, J: 1.8 Hz), 6.91
(d,1H, ArH, J: 8.1 Hz), 7.20 (s,1H, ArH), 7.36 (d,1H, ArH, J: 7.8 Hz), 7.85
(d, 1H, furan H, J: 2.0 Hz), 11.5 (s, 1H, OH exch.), 12.50 (s, 1H, COOH
exch.). Anal. Calcd. For C16H11Cl2NO7S (432.24): C, 44.46; H, 2.57; N,
3.24. Found: C, 44.31; H, 2.61; N. Log P: 2.21.
6
c
OCH3
COOH
O
OH
OCH3
COOH
S
HN
O
O
R2
4.1.8.5. 6-Hydroxy-7-[4-(p-methoxyphenyl) aminosulphonyl]-4-methox-
ybenzofuran-5-carboxylic acid 8e. Yield 55%, mp 190e192 ꢀC. IR (KBr,
cmꢁ1): 3321 (NH), 3150 (OH), 2955 (COOH), 1660 (C]O), 1599 (NH
8a R1 = H,
R2 = H
O
OH
8b R1 = CH3, R2 = H
S
NH2
O
O
8c R1 = Cl,
8d R1 = Cl,
R2 = H
R2 = Cl
bending), 1307, 1130 (SO2). 1H NMR (DMSO-d6, D2O)
d ppm: 4.02 (s, 3H,
R1
OCH3), 4.08 (s, 3H, OCH3), 5.00 (s, 1H, NH exch.), 6.98 (d, 1H, furan H, J:
2.0 Hz), 7.23 (d, 2H, ArH, J: 8.7 Hz), 7.43 (d, 2H, ArH, J: 8.7 Hz), 8.10 (d,
1H, furan H, J: 2.0 Hz), 11.5 (s, 1H, OH exch.), 12.50 (s, 1H, COOH exch.).
13C NMR (DMSO-d6): 55.4 (OCH3), 61.58 (OCH3), 99.5 (C5), 104.3 (C7),
8e R1 = OCH3, R2 = H
8a-e
7
Scheme 3. Reagents and solvents. a: ClSO3H, 0e5 ꢀC, b: Conc NH3, reflux or anhydrous
(NH4)2CO3, dry ether, pyridine, reflux. c:R1R2C6H3NH2, dry ether, pyridine, reflux.

G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
4111
109.8 (C3), 112.1 (C3a), 114.7 (C3Ar, C5Ar), 122.0 (C2Ar, C6Ar), 128.4
(C1Ar), 146.5 (C2), 151.6 (C4Ar), 153.1 (C7a), 156.8 (C4), 164.2 (C6), 176.6
(C]O). Anal. Calcd. For C17H15ClNO8S (393.38): C, 51.91; H, 3.84; N,
3.56. Found: C, 52.00; H, 3.83; N, 3.89. Log P: 0.97.
administered the test compounds 2aec, 4, 5aec, 6, 7 and 8aee in
a dose of 12.5 mmol kgꢁ1 (2.8e5.8 mg kgꢁ1 b.wt.). All medications
were administered intragastrically via the aid of an orogastric
cannula for 5 successive days and the last dose was administered
2 h before colitis induction.
4.2. Pharmacology
4.2.3.1. Induction of ulcerative colitis. Rats were fasted for 36 h with
access to water ad libitum after which they were lightly anes-
thetized with ether. A flexible plastic rubber catheter with an
outside diameter of 2 mm was inserted 8 cm into the colon through
the anus. Ulcerative colitis was induced by injecting glacial acetic
acid (4%) into the colon of all rats (except the sham group) through
the rubber catheter [29] in a dose of 5 ml kgꢁ1. Injected rats were
maintained in a head-down position for 2 min to prevent solution
leakage. In sham group, equivolume of normal saline was injected
into the colon instead of diluted glacial acetic acid. After 24 h of
colitis induction, all rats were sacrificed using ether anesthesia.
4.2.1. Materials and methods
4.2.1.1. Animals. Adult male albino rats (180e200 g) and mice of
both sexes (25e30 g) were used. Rats were housed singly in wire
bottomed cages, while mice were housed randomly in groups in
polypropylene cages. All animals were kept under uniform and
controlled conditions of temperature and light/dark (12/12 h)
cycles, fed with standard rodent diet and water ad libitum. Animals
were allowed to adapt to the laboratory environment for one week
before experimentation. The experimental tests on animals have
been performed in accordance with the Institutional Ethical
Committee approval.
4.2.3.2. Assessment of colon macroscopic damage. The tissue of
colon, 8 cm in length and 3 cm proximal to the anus was excised,
opened longitudinally and washed in saline buffer. The specimens
were weighted and wet weight/length ratio was measured for all
the rats. Macroscopic damage of the colon was recorded according
to a previously validated scoring system from 0 to 4 [28].
The lesion scores were: 0 ¼ no ulcer, 1 ¼ mucosal erythema only,
2 ¼ mild mucosal edema, slight bleeding or slight erosion,
3 ¼ moderate edema, bleeding ulcers or erosions, 4 ¼ severe
ulceration, erosions, edema and tissue necrosis. Ulcer area was
measured using plane glass square. Each cell on the glass square
was 1 mm2 in area and the number of cells was counted and the
ulcer area was determined for each colon. Ulcer index was the last
parameter, measured by summing the lesion score and the ulcer
area for each tissue specimen [39]. The results were listed in Table 1.
4.2.1.2. Preparation of the tested chemical compounds. All the tested
chemicals and the reference drug (sulphasalazine) were dissolved
in DMSO before oral administration to the experimental animals.
4.2.1.3. Doses. In this investigation, sulphasalazine was given orally
in a dose of 12.5 mmol kgꢁ1 (¼5 mg kgꢁ1 b.wt). This dose was
calculated by converting the therapeutic dose that used in human
to rat’s dose [37]. Other groups were administered the test
compounds 2aec, 4, 5aec, 6,
7 and 8aee in a dose of
12.5 mmol kgꢁ1 (2.8e5.8 mg kgꢁ1 b.wt). Glacial acetic acid was
diluted in distilled water to be 4% and injected into the colon of rats
through a rubber catheter at the dose of 5 ml kgꢁ1
.
4.2.2. Determination of LD50
LD50 of each new compound was determined according to Fin-
ney’s method [38]. For this purpose, albino mice (25e30 g) were
divided into groups each was composed of 5 animals. Preliminary
experiments were done for each compound to determine the
minimal dose that kills all mice as well as the maximal dose that
fails to kill any animal. Consequently, several oral doses at equal
intervals were chosen in between these doses. Animals were kept
under observation for 24 h during which symptoms of toxicity and
rate of mortality in each group were recorded.
4.3. Antimicrobial activity (in vitro)
Antimicrobial susceptibilities of the tested compounds were
determined by agar-diffusion methods following the general
recommendations of the Clinical and Laboratory Standards Insti-
tute (CLSI) (formerly the National Committee for Clinical Labora-
tory Standards).
4.3.1. Medium
Calculations were performed using the following formula:
A solid medium, namely Mullere Hinton agar (MHA; beef infu-
sion 300 g/L, casein acid hydrate 17.5 g/L, starch 1.5 g/L, agaragar
17 g/L, and distilled water 1000 ml, adjusted to pH 7.4).
Percent Mortality ðPMÞ ¼ ðI ꢁ SÞ ꢃ 100=I:
where I am the initial number of animals and S is the number of
survivors.
4.3.2. Test microorganisms
Two Gram-positive bacteria namely S. aureus (MTCC 3160) and
B. subtilis (MTCC 1456). Two Gram-negative bacteria namely E. coli
(ATCC 8739) and P. aeruginosa (MTCC 3541) and the fungus C.
albicans (ATCC 10231).
4.2.2.1. Data analysis.
1. The percent mortality was calculated using the formula listed
above.
4.3.3. Primary screening
2. The concentration of the tested compound was plotted versus
the percent mortality on a graph.
Primary screening of 16 compounds 1, 2aec, 3, 4, 5aec, 6, 7 and
8aec was done by the agar diffusion assay technique [35]. Twenty-
four-hour-old bacterial cultures of all test microorganisms were
used as inoculums, which was adjusted to 0.5 McFarland Standard.
The stock solutions of all the test compounds (10 mg/ml) were
prepared by dissolving 10 mg of the test compound in DMF (1 ml).
Sulphasalazine and DMF were used as positive and negative
controls, respectively. Twenty milliliters of molten and cooled MHA
and 500 ml of each test bacterial culture were mixed (separate
flasks were used for each bacterial culture) and poured in sterilized
and labeled Petri plates. The wells of 6 mm were punched in the
3. The value corresponding to LD50 was obtained from the graph.
4.2.3. Anti-ulcerogenic effect
Eighty five male albino rats weighing 190e200 g were divided
into 17 equal groups. The first (sham) and the second (control)
groups were given the solvent only in a dose of 5 ml kgꢁ1. Animals
in the third group (reference) were given sulphasalazine in a dose
of 12.5 mmol kgꢁ1 (¼5 mg kgꢁ1 b.wt). Other groups were

4112
G.S. Hassan, G.A. Soliman / European Journal of Medicinal Chemistry 45 (2010) 4104e4112
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A
with MMFF94X force-
ꢀꢁ1
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The X-ray crystallographic structure of murine COX-2 complexes
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the ligandeenzymes interactions at the active site, Table 3.
Acknowledgement
The author wishes to offer her deep gratitude to Dr. Tamer
Essam, Department of Microbiology, Faculty of Pharmacy, Cairo
University, Egypt.
Appendix. Supplementary data
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