3-Methyl-2-phenyl-1-substituted-indole derivatives as indomethacin analogs: Design, synthesis and biological evaluation as potential anti-inflammatory and analgesic agents

Article abstract of DOI:10.3109/14756366.2015.1022174

In a new group of 3-methyl-2-phenyl-1-substituted-indole derivatives (10a-f), the indomethacin analogs were prepared via the Fisher indole synthesis reaction of propiophenone with appropriately substituted phenylhydrazine hydrochloride. This is followed by the insertion of the appropriate benzyl or benzoyl fragment. All the synthesized compounds were evaluated for their anti-inflammatory (in vitro and in vivo) and analgesic activities. The methanesulphonyl derivatives 10d, e and f showed the highest anti-inflammatory (in vitro and in vivo) and analgesic activities. In addition, molecular docking studies were performed on compounds 10a-f and the results were in agreement with that obtained from the in vitro COX inhibition assays. The significant anti-inflammatory and analgesic activities exhibited by 10d and 10e warrant continued preclinical development as potential anti-inflammatory and analgesic agents.

Full text of DOI:10.3109/14756366.2015.1022174

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J Enzyme Inhib Med Chem, Early Online: 1–7
2015 Informa UK Ltd. DOI: 10.3109/14756366.2015.1022174
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RESEARCH ARTICLE
3-Methyl-2-phenyl-1-substituted-indole derivatives as indomethacin
analogs: design, synthesis and biological evaluation as potential
anti-inflammatory and analgesic agents
1
Khaled R. A. Abdellatif , Phoebe F. Lamie , and Hany A. Omar
1
2,3
1
2
Department of Pharmaceutical Organic Chemistry and Department of Pharmacology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, Egypt,
3
and Department of Pharmacology, Sharjah Institute for Medical Research, College of Pharmacy, University of Sharjah, Sharjah, UAE
Abstract
Keywords
In a new group of 3-methyl-2-phenyl-1-substituted-indole derivatives (10a–f), the indometh-
acin analogs were prepared via the Fisher indole synthesis reaction of propiophenone with
appropriately substituted phenylhydrazine hydrochloride. This is followed by the insertion of
the appropriate benzyl or benzoyl fragment. All the synthesized compounds were evaluated for
their anti-inflammatory (in vitro and in vivo) and analgesic activities. The methanesulphonyl
derivatives 10d, e and f showed the highest anti-inflammatory (in vitro and in vivo) and
analgesic activities. In addition, molecular docking studies were performed on compounds
Anti-inflammatory activity, cyclooxygenase,
indomethacin, molecular modeling
History
Received 30 December 2014
Revised 12 February 2015
Accepted 12 February 2015
Published online 23 March 2015
1
0a–f and the results were in agreement with that obtained from the in vitro COX inhibition
assays. The significant anti-inflammatory and analgesic activities exhibited by 10d and 10e
warrant continued preclinical development as potential anti-inflammatory and analgesic
agents.
9
,10
Introduction
resulted in the withdrawal of rofecoxib and valdecoxib
Figure 1).
Indomethacin (6) is one of the most potent NSAIDs, which are
(
Non-steroidal anti-inflammatory drugs (NSAIDs) produce their
activity via inhibition of cyclooxygenase (COX) catalyzed
biotransformation of arachidonic acid to pro-inflammatory pros-
taglandins (PGs) and thromboxanes (TXs) . Cyclooxygenase
enzyme exists in two distinct isoforms, a constitutive form (COX-
effective against rheumatoid arthritis, ankylosing spondylitis,
osteoarthritis of large joints and other types of inflammatory
1
–3
11
diseases . At the same time, it is also one of the most ulcerogenic
NSAIDs because of its high COX-1 selectivity and the acidic
1
) and an inducible form (COX-2); the constitutive COX-1
12
nature of the drug . From our point of view, there are two main
is responsible for the maintenance of physiological functions,
such as protection of gastric mucosa, vascular homeostasis and
platelet aggregation . Unlike COX-1, the COX-2 isoform is
induced in response to mitogenic and pro-inflammatory stimuli
and it is responsible for the progression of inflammation . Thus,
selective COX-2 inhibitor drugs (coxibs) as celecoxib (1),
rofecoxib (2) and valdecoxib (3) were more useful for the
treatment of inflammation and inflammation-associated disorders
than the non-selective traditional NSAIDs as aspirin (4), ibupro-
fen (5) and indomethacin (6) (Figure 1). Unfortunately, some
cardiovascular side effects including the increased incidences of
high blood pressure and myocardial infarction accompanied the
highly selective COX-2 inhibitors were attributed to the bio-
approaches to prevent and/or treat gastroenteropathy associated
with indomethacin as an example of traditional NSAIDs; the first
4
–6
comprises synthesis of prodrugs, where
a nitric oxide
7
,8
(NO)-donating moiety is covalently attached to NSAID (indo-
methacin). This moiety should reduce the gastrointestinal toxicity
through the protective effect of locally released NO on the gastric
mucosa. In this regard, we previously reported some prodrugs of
indomethacin and other NSAIDs, which showed higher safety
13,14
profiles than of the parent NSAIDs
. The second strategy is
based on maintaining the potency of the indomethacin by keeping
the main scaffold of the drug with trials to increase COX-2
selectivity via the modifications of the side groups. Accordingly,
the current work described the synthesis, molecular modeling
studies, in vitro evaluation as COX-1/COX-2 inhibitors, and the
in vivo anti-inflammatory (AI) activity of a new series of
N-substituted indole derivatives as indomethacin analogs 10a–f in
9
,10
chemical changes in the COX pathway . These adverse effects
which; (i) the chlorobenzoyl moiety of indomethacin in position
1
tained in 10b, 10e, replaced with benzoyl in 10a, 10d or replaced
with benzyl in 10c, 10f, (ii) the methyl group in position 2 was
replaced with the phenyl group and this replacement is expected
Address for correspondence: Prof. Khaled R. A. Abdellatif, PhD,
Department of Pharmaceutical Organic Chemistry, Faculty of
Pharmacy, Beni-Suef University, Beni-Suef 62514, Egypt. Tel: (002)-
15
, which is important for anti-inflammatory activity , is main-
0100-2535444. Fax: (002)-082-2317958. E-mail: khaled.ahmed
@pharm.bsu.edu.eg

2
K. R. A. Abdellatif et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 1. Chemical structures of some selective cyclooxygenase-2 (COX-2) inhibitor drugs (1–3) and some traditional non-selective NSAIDs (4–6).
Figure 2. Chemical structures of the trad-
itional NSAID indomethacin (6) and the
designed indomethacin analogs 10a–f.
to maximize the interaction with the hydrophobic residues within yield (42–58%). All the prepared compounds have been
1
13
COX-2 active site and to enhance COX-2 selectivity, since the characterized by IR, H NMR, C NMR, mass spectra and
proposed compounds will be too large to fit into the smaller COX- elemental analyses.
1 active site, (iii) the –CH COOH moiety in position 3 was
2
replaced with a methyl group and this replacement is expected to Biological evaluation
decrease the acidity of the new compounds, which will conse-
quently decrease the local action exerted by the direct contact of
Anti-inflammatory activity
the new compounds with gastric mucosa, and (iv) the methoxy In vitro cyclooxygenase inhibition assay. In vitro structure–
group in position 5 is replaced with H in 10a–c or with COX-2 activity relationships acquired by this group of indomethacin
pharmacophore methanesulfonyl moiety (SO Me) in 10d–f to derivatives (10a–f) showed that they are relatively weak inhibi-
2
study the effect of SO Me moiety on COX selectivity and anti- tors of COX-1 (IC50 ¼ 7.78–42.36 mM) when compared with
2
inflammatory activity (Figure 2).
the reference drug indomethacin (IC ¼ 0.63 mM). However,
5
0
they are moderately-to-highly potent inhibitors of COX-2
(IC50 ¼ 1.65 to 8.58 mM range) in comparison with indomethacin
Results and discussion
(
IC50 ¼ 11.63 mM) (Table 1). Compounds having a SO Me moiety
2
Chemistry
(COX-2 pharmacophore) were generally more potent inhibitors of
The synthesis of the target 1-substituted-2-phenyl-3-methyl indole COX-2 than the corresponding analogs free of SO Me (10e410b;
2
derivatives (10a–f) was accomplished in accordance with the 10f410c; but 10a and 10d were approximately of the same
sequence of reactions depicted in Scheme 1. Reaction of potency). In general, all compounds were more selective to COX-
propiophenone (7) with either phenylhydrazine hydrochloride 2 enzyme with selectivity index (S.I.) ¼ 0.90 to 25.67 compared to
(
8a) or 4-methylsulfonylphenylhydrazine hydrochloride (8b) in indomethacin (COX-2 S.I. ¼ 0.055). The selectivity could be
ethanol under the Fisher indole synthesis reaction conditions attributed to the size of compounds (10a–f), which is too large to
afforded the respective 2-phenyl-3-methylindole derivative 9a fit into the smaller COX-1 active site. Moreover, the presence of
(79%) or 9b (82%). Replacement of the chlorine atom present COX-2 pharmacophore in some analogs (10d–f) increased
in benzoyl chloride, 4-chlorobenzoyl chloride or benzyl chlor- the selectivity of COX-2 isozyme. The most potent COX-2
ide by the indole derivatives (9a–b) in DMF under basic inhibitor (10e) with IC50 ¼ 1.65 mM and S.I. ¼ 25.65 had indo-
conditions (NaH) afforded the target products (10a–f) in moderate methacin as a chlorobenzoyl moiety in addition to SO Me moiety
2

DOI: 10.3109/14756366.2015.1022174
3-Methyl-2-phenyl-1-substituted-indole derivatives
3
Scheme 1. Reagents and conditions: (a) glacial acetic acid, reflux, 10h; (b) benzoyl chloride, 4-chlorobenzoyl chloride or benzyl chloride, NaH, DMF,
RT, overnight.
Table 1. In vitro COX-1 and COX-2 inhibition of compounds 10a–f, and
reference drug (indomethacin).
Table 2. Anti-inflammatory activities of compounds 10a–f,
and reference drug (indomethacin) in carrageenan-induced
rat paw edema assay.
COX Inhibition (IC50 mM)*
Anti-inflammatory activity (%)
Selectivity
indexy
Compounds
COX-1
COX-2
Compounds
1 h
3 h
6 h
1
1
1
1
1
1
0a
0b
0c
0d
0e
0f
10.39 ± 1.36
18.72 ± 1.64
7.78 ± 2.01
32.23 ± 2.36
42.36 ± 2.45
17.65 ± 2.79
0.63 ± 0.02
6.54 ± 1.61
8.21 ± 2.10
8.58 ± 2.14
7.98 ± 1.91
1.65 ± 1.02
2.36 ± 1.58
11.36 ± 1.6
1.58
2.28
0.90
13.65
25.67
7.47
10a
10b
10c
10d
10e
10f
Indomethacin
26.9
17.3
5.8
73.1
73.5
67.9
71.2
16.2
17.4
8.8
80.1
71.8
66.8
86.3
21.2
15.7
6.1
78.8
71.8
57.9
88.4
Indomethacin
0.055
*
Values are expressed as mean ± SEM (n ¼ 3).
All test compounds (10a–f, indomethacin) were adminis-
tered (10 mg/kg) 30 min prior to testing. The anti-
inflammatory activity % is expressed according to the
following equation:
ySelectivity index (COX-1 IC50/COX-2 IC50).
AI (%) ¼ (V
c
ꢀ V
t
/V
c
) ꢁ 100, where V
c
represents the paw
(
COX-2 pharmacophore) which was 467 times more COX-2
t
volume of control group of animals and V represents the
selective than indomethacin (COX-2 IC50 ¼ 11.36 mM,
paw volume in drug treated animals.
S.I. ¼ 0.055).
methanesulfonyl (SO Me) moiety (in compounds 10d, e and f)
2
In vivo anti-inflammatory activity. The anti-inflammatory
activity of the test compounds compared to indomethacin was
tested using the carrageen-induced rat paw edema test, which
is widely used as a primary test for the screening of new
anti-inflammatory agents. Results indicated that after 1 h, indo-
methacin derivatives 10a, b and c showed percentage anti-
inflammatory activity of about 5.8–26.9%, while indomethacin
increases the anti-inflammatory activity for this class of com-
pounds, and (ii) maintaining chlorobenzoyl moiety of indometh-
acin as in compound 10e or its replacement with benzoyl moiety
(
compound 10d) maintains or increases the anti-inflammatory
activity, while its replacement with benzyl moiety (compound
0f) sharply decreased the anti-inflammatory activity (Table 2).
1
derivatives with SO Me 10d, e and f showed higher percentage of
2
Analgesic activity
anti-inflammatory activity (67.9–73.5%). After 3 h, compounds
1
8
inflammatory activity of 66.8–80.1%. Similarly after 6 h, com-
pounds 10a, b and c showed percentage activity of 6.1–21.2%,
while compounds 10d, e and f showed percentage anti-inflam-
matory activity of 57.9–78.8%. 1-Benzyl-3-methyl-2-phenyl-1H-
indole (10c) displayed the lowest anti-inflammatory activity at
all-time intervals (5.8–8.8%), while (4-chlorophenyl)-(5-metha-
0a, b and c showed percentage anti-inflammatory activity of
.8–17.4% and compounds 10d, e and f showed percentage anti-
The analgesic activity of the target compounds (10a–f) and
indomethacin as a reference drug was also investigated using
acetic acid-induced writhing test. Acetic acid-induced writhing
was significantly reduced in mice receiving SO Me containing
2
compounds 10d, e and f, and the degree of inhibition of the
writhing response by these compounds was 49.1, 65.1 and 59.3%,
respectively, compared to the vehicle-treated control group. The
analgesic activity of SO Me free compounds (10a, b, and c) was
2
nesulfonyl-3-methyl-2-phenyl-indol-1-yl)-methanone
(10e)
relatively low (33.0, 30.2 and 18.9%, respectively) (Figure 3).
From these results, the analgesic activities of these compounds
are clearly parallel to their anti-inflammatory activities.
showed the highest anti-inflammatory activity (73.5%) at one-
hour interval (more than indomethacin, 71.2%). The benzoyl
derivative (10d) showed good anti-inflammatory activity at three-
and six-hour intervals (80.1 and 78.8%, respectively) comparable
to that of indomethacin (86.3 and 88.4%, respectively) and it was
Molecular modeling
the most potent among the six compounds 10a–f at these time To know the plausible mode of interactions of the prepared
intervals. These data indicate that; (i) presence of compounds 10a–f within the COX-1 and COX-2 isozymes,

4
K. R. A. Abdellatif et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
Figure 3. Analgesic effect of test compounds
0a–f compared to indomethacin using acetic
1
acid-induced writhing in mice. Data repre-
sent the mean value ± SD of four mice per
group. Statistical comparisons between basal
and post-drug values were analyzed for
statistical significance using the one-way
ANOVA followed by Dunnett’s test and
denoted by *p50.05, #p50.01.
Table 3. Molecular modeling data for compounds 10a–f, indomethacin and valdecoxib during docking in the COX-1 (PDB:ID 4COX) and COX-2
PDB:ID 2AW1) active sites.
(
COX-1
COX-2
Distance
(in A) from main
residue
˚
˚
Distance (in A)
from main residue
Affinity
(kcal/mol)
Functional
group
Affinity
(kcal/mol)
Functional
group
Compound
1
1
0a
0b
ꢀ5.5167
ꢀ9.0055
2.66
–
Arg120
–
–CO
–
ꢀ14.5783
ꢀ11.4647
2.70
2.76
Asn67
Asn67
Asn62
–
His94
Thr199
Thr199
His94
Thr199
Thr199
His94
Thr199
–
–CO
–CO
–CO
–
3
.03
–
1
1
0c
0d
ꢀ12.1998
ꢀ5.6238
–
2.70
–
Arg120
–
–CO
ꢀ15.6805
ꢀ20.8529
2.90
–SO
–SO
–SO
–SO
–SO
–SO
–SO
–SO
–
2
2
2
2
2
2
2
2
3
2
.05
.53
1
1
0e
0f
ꢀ5.0689
–
–
–
–
–
ꢀ20.7351
2.93
2
2
.96
.83
ꢀ4.7538
ꢀ17.0967
–
–
ꢀ21.7493
–
3.06
2
.90
–
Indomethacin
Valdecoxib
2.96
Tyr385
Arg120
–
–N
–CO
–
2
.76
–
ꢀ17.0929
3.28
His94
Thr199
Thr199
–NH
–NH
2
2
2
.73
.80
2
–SO
2
molecular docking experiments were performed using X-ray, hydrogen bonding interactions between the amino acid residues
crystal structure data for COX-1 and COX-2 were obtained from and functional groups of the compounds are summarized in
1
6,17
the protein data bank
1
. Indomethacin (6) – as a selective COX- Table 3. For COX-1, all six compounds 10a–f indicated lower
inhibitor – was used as a ligand for COX-1 isoform while, binding interactions (affinity in kcal/mol ranges from ꢀ5.5167 to
valdecoxib (3) was used as a ligand for COX-2 isoform. The main ꢀ12.1998 with no or one hydrogen bonding interaction) than
interactions of indomethacin with COX-1 resulted in two main indomethacin (ꢀ17.0967 with two hydrogen bonding inter-
residues Tyr385 and Arg120 amino acids, sequentially forming actions). In case of COX-2, while compounds 10a–c showed
two hydrogen bonding interactions with –N and –CO of appreciable binding interactions (affinity in kcal/mol ranges from
˚
indomethacin at a distance equal to 2.96 and 2.76 A, respectively. ꢀ11.4647 to ꢀ15.6805 with one or two hydrogen bonding
On the other hand, docking valdecoxib into COX-2 isozyme interactions), compounds 10d–f (have SO Me as COX-2 pharma-
2
afforded three hydrogen bonding interactions; (i) NH with His94 cophore) showed excellent binding interactions (affinity in kcal/
2
˚
˚
(
3.28 A), (ii) NH with Thr199 (2.73 A), and (iii) SO with mol ranges from ꢀ20.7351 to ꢀ21.7493 with two or three
2
2
˚
Thr199 (2.80 A).
hydrogen bonding interactions) in comparison with valdecoxib
The docking results including the energy associated with (ꢀ17.0929 with three hydrogen bonding interactions). All the
intermolecular interactions (affinity in kcal/mol) obtained upon docked compounds were in response to COX-2 rather than COX-1
computational docking for all compounds (10a–f, indomethacin and that is consistent with their experimentally observed potent
and valdecoxib) within COX-1 and COX-2 active sites and the COX-2 inhibition (Figure 4).

DOI: 10.3109/14756366.2015.1022174
3-Methyl-2-phenyl-1-substituted-indole derivatives
5
Hertz (Hz) and chemical shifts were recorded in ppm on ꢀ scale.
Mass spectra (MS) were recorded on a Hewlett Packard 5988
spectrometer (Palo Alto, CA). Microanalyses for C, H and N were
carried out on Perkin-Elmer 2400 analyzer (Perkin-Elmer,
Norwalk, CT) at the Micro analytical unit of Cairo University,
Egypt, and all compounds were within ± 0.4% of the theoretical
values. All other reagents, purchased from the Aldrich Chemical
Company (Milwaukee, WI), were used without further purifica-
tion. 4-Methylsulfonylphenylhydrazine hydrochloride (8b) was
1
8
prepared according to a previous procedure .
General procedure for synthesis of 3-methyl-2-phenylindole
derivatives 9a–b
A mixture of propiophenone (7, 1.3 g, 0.01 mol) and the
appropriate phenyl hydrazine hydrochloride (8a or 8b, 0.01 mol)
in glacial acetic acid (30 mL) was heated under reflux for 10 h.
After cooling, the reaction mixture was poured into ice-cold
water. The separated solid was filtered, dried and crystallized
from acetone to give pure compounds 9a and 9b. Physical and
spectral data are listed below.
Figure 4. Binding of the most active compounds 10d and e inside COX-2
active site. (A) The proposed binding mode inside the active site of COX-
2 resulting from docking, the most important amino acids are shown
together with their respective numbers. (B) 2D interaction.
19
-Methyl-2-phenyl-1H-indole (9a). Buff solid ; Yield 79%;
3
m.p. 136–138 C; IR (KBr) 3331 (NH), 3023 (CH aromatic),
ꢂ
ꢀ1 1
2
924 (CH aliphatic) cm ; H NMR (DMSO-d ) ꢀ 2.44 (s, 3H,
6
CH ), 7.04 (t, 1H, J ¼ 7.2 Hz, phenyl H-4), 7.12–7.16 (m, 1H,
3
indole H-5), 7.35–7.42 (m, 2H, indole H-6, H-7), 7.50–7.56 (m,
Conclusion
3
Six new compounds 10a–f, as indomethacin derivatives, were phenyl H-2, H-6), 11.19 (s, 1H, NH, D O exchangeable);
H, phenyl H-3, H-5 and indole H-4), 7.70 (d, 2H, J ¼ 7.6 Hz,
2
1
3
C
synthesized for biological evaluation as anti-inflammatory and NMR (DMSO-d ) ꢀ 10.30, 107.22, 111.48, 118.88, 119.03,
6
analgesic activities. The in vitro anti-inflammatory activity 122.01, 127.43, 127.95, 129.16, 129.85, 133.55, 134.19, 136.38;
+
studies showed that all compounds were more COX-2 selective EIMS (m/z) 208 (M + 1, 17.88%), 207 (M , 100%), 206 (M-1,
(
COX-2 S.I. ¼ 0.90 to 25.67) than indomethacin (COX-2 89.24%). Anal. Calcd for C H N: C, 86.92; H, 6.32; N, 6.76.
1
5 13
S.I. ¼ 0.055), especially compound 10e (467 times more COX-2 Found: C, 86.79; H, 6.22; N, 7.01.
selective than the reference drug indomethacin). While, the
in vivo anti-inflammatory activity studies indicated that com- 5-Methanesulfonyl-3-methyl-2-phenyl-1H-indole (9b). Brown
ꢂ
pounds 10d, e and f (have SO Me as COX-2 pharmacophore) solid; Yield 82%; m.p. 197–199 C; IR (KBr) 3413 (NH), 3048
2
displayed good anti-inflammatory activity (57.9–80.1%) compar- (CH aromatic), 2912, 2862 (CH aliphatic), 1307, 1158 (SO )
2
ꢀ
1 1
able to that of indomethacin (71.2–88.4%), especially 10d (73.1– cm ; H NMR (DMSO-d
0.1%) and 10e (71.8–73.5%). In addition, the docking experi- SO CH
), 7.42 (t, 1H, J ¼ 7.6 Hz, phenyl H-4), 7.53–7.59 (m, 3H,
ments showed that all the docked compounds were fitted to COX- phenyl H-3, H-5 and indole H-7), 7.65–7.72 (m, 3H, phenyl H-2,
) ꢀ 2.47 (s, 3H, CH ), 3.19 (s, 3H,
6
3
8
2
3
2
rather than COX-1 and compounds 10d–f (having SO Me as H-6 and indole H-6), 8.16 (s, 1H, indole H-4), 11.81 (s, 1H, NH,
2
13
COX-2 pharmacophore) showed excellent binding interactions
D
2
O exchangeable); C NMR (DMSO-d
CH ), 108.84, 112.02, 119.23, 120.19, 128.21, 128.23,
bonding interactions in comparison with the COX-2 selective drug 129.18, 129.32, 131.51, 132.57, 136.95, 138.44; EIMS (m/z)
6 3
) ꢀ 10.08 (CH ), 45.02
(
ꢀ20.7351 to ꢀ21.7493 kcal/mol) with two or three hydrogen (SO
2
3
+
(
valdecoxib, ꢀ17.0929 kcal/mol and three hydrogen bonding 286 (M + 1, 24.21%), 285 (M , 100%). Anal. Calcd for
C H15NO S: C, 67.34; H, 5.30; N, 4.91. Found: C, 67.55; H,
16 2
interactions), which was in parallel correlation with the in vitro
COX inhibition evaluation. Also, the analgesic activities of these 5.58; N, 4.69.
compounds 10a–f are clearly parallel to their anti-inflammatory
activities. Because of their relatively potent anti-inflammatory
and analgesic activities, the compounds 10d and 10e warrant
continued preclinical development as potential anti-inflammatory
and analgesic agents.
General procedure for synthesis of 3-methyl-2-phenyl-1-substi-
tuted-indole derivatives (10a–f)
To a solution of the respective 2-phenyl indole derivative 9a or 9b
(2.5 mmol) in dry DMF (5 mL), NaH (0.11 g, 4.5 mmol) was
slowly added with stirring at room temperature for 30 min. Then,
the reaction flask was cooled down on a bath of ice followed by a
slow addition for a solution of the appropriate alkyl or acyl halide
Experimental
Chemistry
(
2.5 mmol) in DMF (5 mL) and the mixture was stirred at room
Melting points were determined on a Thomas-Hoover capillary
apparatus and are uncorrected. Infrared (IR) spectra were
recorded as films on NaCl plates using a Nicolet 550 Series II
temperature overnight. The reaction mixture was poured into ice-
cold water and extracted with hexane (4 ꢁ 10 mL). The organic
layer was dried and crystallized from methanol/chloroform
mixture (4:1) to give pure compounds 10a–f. Physical and
spectral data are listed below.
1
13
Magna FT-IR spectrometer (Middleton, WI). H NMR and
NMR spectra were measured on a Bruker Avance III 400 MHz
C
1
Bruker BioSpin AG, F a¨ llanden, Switzerland) for H and
(
1
00 MHz for C with BBFO Smart Probe and Bruker 400
3
1
AEON Nitrogen-Free Magnet, Faculty of Pharmacy, Beni-Suef (3-Methyl-2-phenyl-indol-1-yl)-phenyl-methanone (10a). Buff
ꢂ
University, Egypt, in DMSO-d6 with TMS as the internal solid; Yield 54%; m.p. 300–302 C; IR (KBr) 3273 (CH
ꢀ
1
1
standard, where J (coupling constant) values are estimated in aromatic), 2924 (CH aliphatic), 1675 (C¼O) cm ; H NMR

6
K. R. A. Abdellatif et al.
J Enzyme Inhib Med Chem, Early Online: 1–7
(
DMSO-d ) ꢀ 2.50 (s, 3H, CH ), 7.04 (t, 1H, J ¼ 7.2 Hz, phenyl 1-Benzyl-5-methanesulfonyl-3-methyl-2-phenyl-1H-indole
6
3
ꢂ
H-4), 7.11–7.15 (m, 1H, indolyl H-5), 7.34–7.42 (m, 2H, indolyl (10f). Buff solid; Yield 42%; m.p. 235–237 C; IR (KBr) 3031
H-6, H-7), 7.45 (m, 7H, phenyl H-2, H-3, H-5, H-6, indolyl H-4 (CH aromatic) 2921, 2861 (CH aliphatic), 1343, 1182 (SO )
and benzoyl H-3, H-5), 7.68–7.95 (m, 3H, benzoyl H-2, H-4, H- cm ; H NMR (DMSO-d ) ꢀ 2.47 (s, 3H, CH ), 3.19 (s, 3H,
6
6
1
1
2
ꢀ1 1
3
1
3
); C NMR (DMSO-d ) ꢀ 9.63 (CH ), 113.82, 116.35, 119.78, SO CH ), 5.40 (s, 2H, CH ), 6.83 (d, 2H, J ¼ 7.6 Hz, benzyl H-2,
6
3
2
3
2
23.29, 124.89, 127.93, 128.27, 128.46, 129.61, 129.77, 130.10, H-6), 7.19–7.21 (m, 3H, benzyl H-3, H-4, H-5), 7.41–7.65 (m,
31.19, 132.43, 133.22, 135.37, 136.95, 169.47 (C¼O); EIMS 7H, phenyl protons and indole H-6, H-7), 8.18 (s, 1H, indole H-4);
+
+
13
(m/z) 312 (M + 1, 3.43%), 311 (M , 13.64%), 105 (C H O ,
7
C NMR (DMSO-d ) ꢀ 10.06 (CH ),45.03 (SO CH ), 47.35
6 3 2 3
5
1
Found: C, 85.13; H, 5.56; N, 4.38.
00%). Anal. Calcd for C H NO: C, 84.86; H, 5.50; N, 4.50. (CH ), 110.55, 111.59, 112.04, 119.38, 120.19, 127.66, 128.08,
2
2
2
17
128.23, 129.00, 129.12, 129.32, 130.73, 130.93, 132.16, 138.12,
1
+
38.72; EIMS (m/z) 376 (M + 1, 13.95%), 375 (M , 47.96%), 91
þ
(C7H , 100%). Anal. Calcd for C H NO S: C, 73.57; H, 5.64;
N, 3.73. Found: C, 73.42; H, 5.53; N, 3.81.
7
23 21
2
(
(
(
(
4-Chlorophenyl)-(3-methyl-2-phenyl-indol-1-yl)-methanone
ꢂ
10b). Buff solid; Yield 57%; m.p. 99–101 C; IR (KBr) 3061
ꢀ1 1
CH aromatic), 2992 (CH aliphatic), 1685 (C¼O) cm ; H NMR
Biological evaluation
DMSO-d ) ꢀ 2.40 (s, 3H, CH ), 7.10–7.34 (m, 8H, 5 phenyl
6
3
Animals
protons and indolyl H-5, H-6, H-7), 7.52–7.85 (m, 5H, indolyl H-
1
3
4
1
1
1
2
7
and 4 benzoyl protons); C NMR (DMSO-d ) ꢀ 9.64 (CH ),
6 3
Swiss albino mice (20–25 g) and Wistar rats (150–175 g) were
obtained from the National Research Center, Cairo, Egypt. They
were used throughout the study and were kept at controlled
14.05, 116.66, 119.81, 123.55, 125.15, 127.84, 128.51, 128.86,
30.21, 130.52, 131.95, 132.27, 134.19, 136.17, 136.89, 137.76,
+
68.63 (C¼O); EIMS (m/z) 347 (M + 2, 9.70%), 345 (M ,
ꢂ
conditions (temperature 23 ± 2 C, humidity 60 ± 10%) at a 12/
1
+
5.82%), 139 (C H ClO , 100%). Anal. Calcd for C H NO: C,
6.41; H, 4.66; 10.25; N, 4.05. Found: C, 76.35; H, 4.49; N, 4.23.
7
4
22 17
2 h light/dark cycle. All procedures relating to animal care and
treatments were conducted in accordance with protocols approved
by the Research Ethical Committee of Faculty of Pharmacy Beni-
Suef University (2014-Beni-Suef, Egypt).
2
-Benzyl-3-methyl-2-phenyl-1H-indole (10c). Yellow solid ;
0
1
Yield 49%; m.p. 110–112 C; IR (KBr) 3059, 3027 (CH
ꢂ
ꢀ
1 1
aromatic), 2923, 2859 (CH aliphatic) cm ; H NMR (DMSO-
COX-1/COX-2 inhibition colorimetric assay
d ) ꢀ 2.23 (s, 3H, CH ), 5.31 (s, 2H, CH ), 6.83 (d, 2H, J ¼ 6.8 Hz,
6
3
2
The in vitro inhibition of COX-1/COX-2 was measured using
colorimetric COX (ovine) Inhibitor Screening Assay Kit (Cayman
Chemical, Ann Arbor, MI) according to manufacturer’s instruc-
tions. This assay directly measures PGF2a that was produced by
stannous chloride reduction of COX-derived PGH2 by
enzyme immunoassay. All assays were conducted in triplicates
and IC50 values are the average of three determinations for each
compound.
benzyl H-2, H-6), 7.08–7.21 (m, 5H, benzyl H-3, H-4, H-5 and
indole H-5, H-6), 7.33 (d, 1H, J ¼ 7.6 Hz, indole H-7), 7.39–7.51
1
3
(
m, 5H, phenyl protons), 7.57 (d, 1H, J ¼ 7.6 Hz, indole H-4);
C
NMR (DMSO-d ) ꢀ 9.76 (CH ), 47.06 (CH ), 108.76, 110.89,
6
3
2
1
1
19.13, 119.65, 122.23, 126.43, 127.40, 128.52, 128.76, 128.87,
29.05, 130.71, 131.84, 136.81, 137.70, 138.87; EIMS (m/z) 298
+
þ
(M + 1, 12.57%), 297 (M , 53.35%), 91 (C7H , 100%). Anal.
Calcd for C H N: C, 88.85; H, 6.44; N, 4.71. Found: C, 88.93;
H, 6.56; N, 4.63.
7
2
2 19
Carrageenan-induced rat paw edema assay
(5-Methanesulfonyl-3-methyl-2-phenyl-indol-1-yl)-phenyl-metha-
none (10d). Buff solid; Yield 55%; m.p. 151–153 C; IR (KBr)
Rats were administered with vehicle, test compounds, indometh-
acin or celecoxib at a dose of 10 mg/kg by oral gavages.
Immediately thereafter, the rats received 100 mL of vehicle or
carrageenan (1% in saline) s.c. on the plantar surface of the left
hind paw under mild anesthesia, essentially, as reported before .
The paw edema was evaluated by measuring paw-volume changes
at 1, 3 and 6 h after carrageenan injection using a plethysmometer.
The right hind paw served as a reference of non-inflamed paw for
comparison. Results are expressed as paw-volume change (mL).
ꢂ
3
1
061 (CH aromatic), 2925, 2859 (CH aliphatic), 1677 (C¼O),
ꢀ
2 6
1
1
321, 1178 (SO ) cm ; H NMR (DMSO-d ) ꢀ 2.41 (s, 3H,
21
CH ), 3.19 (s, 3H, SO CH ), 7.21–7.72 (m, 11H, phenyl protons,
3
2
3
benzoyl protons and indolyl H-7), 7.76 (d, 1H, J ¼ 7.2 Hz, indolyl
1
3
H-6), 8.29 (s, 1H, indolyl H-4); C NMR (DMSO-d ) ꢀ 10.08
6
(CH ), 45.02 (SO CH ), 108.84, 112.03, 116.31, 119.60, 120.20,
3 2 3
1
1
0
27.33, 128.23, 128.64, 129.18, 129.31, 129.49, 130.35, 130.82,
31.60, 132.57, 138.79, 169.56 (C¼O); EIMS (m/z) 390 (M + 1,
+
þ
2
.27%), 389 (M , 0.93%), 285 (C16H15NSO , 100%). Anal. Calcd
Acetic acid-induced writhing test
for C H NO S: C, 70.93; H, 4.92; N, 3.60. Found: C, 80.13; H,
3
2
3
19
2
2
The writhing tests were carried as described before . Briefly,
mice were administered orally vehicle, test compounds, indo-
methacin or celecoxib at a dose of 10 mg/kg 30 min prior to
intraperitoneal administration of 0.7% v/v acetic acid solution
5
.04; N, 3.81.
(4-Chlorophenyl)-(5-methanesulfonyl-3-methyl-2-phenyl-indol-1-
yl)-methanone (10e). Buff solid; Yield 58%; m.p. 204–206 C;
ꢂ
(
10 mL/kg body weight). The number of writhes (i.e. abdominal
IR (KBr) 3047 (CH aromatic), 2943, 2914 (CH aliphatic), 1672
1
ꢀ
1
constriction followed by dorsiflexion and extension) occurring
during a 20-min period beginning 5 min after acetic acid injection
was measured. The results are expressed as the number of writhes
per 20-min period.
(
3
C¼O), 1317, 1175 (SO ) cm ; H NMR (DMSO-d ) ꢀ 2.33 (s,
2
6
H, CH ), 3.27 (s, 3H, SO CH ), 7.23–7.29 (m, 5H, phenyl
3 2 3
protons), 7.37 (d, 2H, J ¼ 8.0 Hz, benzoyl H-3, H-5), 7.56 (d, 2H,
J ¼ 8.0 Hz, benzoyl H-2, H-6), 7.81 (d, 2H, J ¼ 8.8 Hz, indolyl H-
7
), 7.86 (d, 2H, J ¼ 8.8 Hz, indolyl H-6), 8.29 (s, 1H, indolyl H-4);
C NMR (DMSO-d ) ꢀ 9.51 (CH ), 44.58 (SO CH ), 114.61,
1
3
Statistical analysis
6
3
2
3
1
1
16.61, 119.58, 123.30, 128.40, 128.69, 129.02, 130.24, 130.31, Comparisons among the groups were analyzed for statistical
31.48, 132.19, 133.35, 135.82, 138.42, 138.51, 139.00, 168.56 significance using the one-way ANOVA followed by Dunnett’s
+
(
(
C¼O); EIMS (m/z) 425 (M + 2, 4.52%), 423 (M , 11.15%), 139 test. Data were represented as mean ± standard deviation (SD).
+
C H ClO , 100%). Anal. Calcd for C H ClNO S: C, 65.17; H, Differences were considered significant at *p50.05, and
7
4
23 18
3
4
.28; N, 3.30. Found: C, 65.32; H, 4.41; N, 3.21.
**p50.01, respectively.

DOI: 10.3109/14756366.2015.1022174
3-Methyl-2-phenyl-1-substituted-indole derivatives
7
Molecular modeling and docking
9. Scheen AJ. Withdrawal of rofecoxib (Vioxx): what about cardio-
vascular safety of COX-2 selective non-steroidal anti-inflammatory
drugs? Rev Med Liege 2004;59:565–9.
The crystal structures of indomethacin bound at COX-1 (PDB: ID
1
7
4
COX) and valdecoxib bound at the COX-2 (PDB:ID 2AW1) 10. Dogne JM, Supuran CT, Pratico D. Adverse cardiovascular effects of
1
8
the coxibs. J Med Chem 2005;48:2251–7.
active sites [obtained from the protein data bank at Research
Collaboration for Structural Bioinformatics (RSCB) Protein
Database (PDB)]. Docking of the co-crystallized ligand should
be carried out to study the scoring energy(s), root mean standard
deviation (RMSD) and amino acid interactions. The RMSD,
1
1
1. Kaur J, Bhardwaj A, Huang Z, Knaus EE. N-1 and C-3 substituted
indole Schiff bases as selective COX-2 inhibitors: synthesis and
biological evaluation. Bioorg Med Chem Lett 2012;22:2154–9.
2. Bandgar BP, Sarangdhar RJ, Viswakarma S, Ahamed FA. Synthesis
and biological evaluation of orally active prodrugs of indomethacin.
J Med Chem 2011;54:1191–201.
˚
which is a measure of superposing was 0.50 A for the lead
compounds. Docking was performed using London dG force and 13. Abdellatif KR, Chowdhury MA, Dong Y, et al. Dinitroglyceryl and
diazen-1-ium-1,2-diolated nitric oxide donor ester prodrugs of
refinement of the results was done using force field energy.
aspirin, indomethacin and ibuprofen: synthesis, biological evalu-
Preparation of the synthesized compounds for docking was
ation and nitric oxide release studies. Bioorg Med Chem Lett 2009;
achieved via their 3D structure built by Molecular Operating
19:3014–18.
4. Huang Z,
Environment (MOE, Version 2005.06, Chemical Computing
Group Inc., Montreal, Quebec, Canada). Certain procedures
were taken before docking, which include: 3D protonation of the
structures, running conformational analysis using systemic search,
selecting the least energetic conformer and applying the same
docking protocol used with ligands. Docking for the synthesized
compounds was applied. Amino acid interactions and the
hydrogen bond lengths are summarized in Table 3.
1
Velazquez
CA,
Abdellatif
KR,
et
al.
Ethanesulfohydroxamic acid ester prodrugs of nonsteroidal anti-
inflammatory drugs (NSAIDs): synthesis, nitric oxide and nitroxyl
release, cyclooxygenase inhibition, anti-inflammatory, and ulcero-
genicity index studies. J Med Chem 2011;54:1356–64.
1
5. Chowdhury MA, Huang Z, Abdellatif KR, et al. Synthesis and
biological evaluation of indomethacin analogs possessing
a
N-difluoromethyl-1,2-dihydropyrid-2-one ring system: a search for
novel cyclooxygenase and lipoxygenase inhibitors. Bioorg Med
Chem Lett 2010;20:5776–80.
Declaration of interest
16. Kurumbail RG, Stevens AM, Gierse JK, et al. Structural basis for
selective inhibition of cyclooxygenase-2 by anti-inflammatory
agents. Nature 1996;384:644–8.
The authors have declared no conflict of interest.
1
7. Di Fiore A, Pedone C, D’Ambrosio K, et al. Carbonic anhydrase
inhibitors: valdecoxib binds to a different active site region of the
human isoform II as compared to the structurally related
cyclooxygenase II ‘‘selective’’ inhibitor celecoxib. Bioorg Med
Chem Lett 2006;16:437–42.
8. Abdellatif KR, Chowdhury MA, Dong Y, et al. Diazen-1-ium-1,2-
diolated nitric oxide donor ester prodrugs of 5-(4-hydroxymethyl-
phenyl)-1-(4-aminosulfonylphenyl)-3-trifluoromethyl-1H-pyrazole
and its methanesulfonyl analog: synthesis, biological evalu-
ation and nitric oxide release studies. Bioorg Med Chem 2008;16:
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