Design and synthesis of 3-[3-(substituted phenyl)-4-piperidin-1-ylmethyl/- 4-morpholin-4-ylmethyl-4,5-dihydro-isoxazol-5-yl]-1H-indoles as potent anti-inflammatory agents

Article abstract of DOI:10.1007/s00044-009-9194-8

The synthesis of new indole derivatives bearing isoxazoline moiety (3a-d and 4a-d) has been described. IR, ¹H NMR, and mass spectral data supported the structures of synthesized compounds. The compounds were tested in vivo for their anti-inflam

Full text of DOI:10.1007/s00044-009-9194-8

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2010) 19:299–310
DOI 10.1007/s00044-009-9194-8
ORIGINAL RESEARCH
Design and synthesis of 3-[3-(substituted phenyl)-4-
piperidin-1-ylmethyl/-4-morpholin-4-ylmethyl-4,5-
dihydro-isoxazol-5-yl]-1H-indoles as potent
anti-inflammatory agents
Mohammad Amir Æ Sadique Akhtar Javed Æ
Harish Kumar
Received: 14 November 2008 / Accepted: 17 March 2009 / Published online: 23 April 2009
¨
Ó Birkhauser Boston 2009
Abstract The synthesis of new indole derivatives bearing isoxazoline moiety (3a–
d and 4a–d) has been described. IR, 1H NMR, and mass spectral data supported the
structures of synthesized compounds. The compounds were tested in vivo for their
anti-inflammatory activity by carrageenin-induced rat paw edema method. The
compounds that showed good anti-inflammatory activity were screened for their
ulcerogenic and lipid peroxidation activities. The most active compound of this
series is 3-[3-(4-methoxyphenyl)- 4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-
5yl]-1H-indole 4d.
Keywords Indole ꢀ Isoxazoline ꢀ Anti-inflammatory ꢀ Ulcerogenicity ꢀ
Lipid peroxidation
Introduction
Almost all nonsteroidal anti-inflammatory drugs (NSAIDs) under current clinical
usage have aryl acetic acid pharmacophore and are highly acidic in nature.
Therefore, long-term use of these drugs is associated with gastrointestinal
ulceration, bleeding, and nephrotoxicity (Langman et al., 1994), thus indicating a
clear need to develop nonacidic nonsteroidal anti-inflammatory agents. Two
NSAIDs, indomethacin and tenidap, which are derivatives of heterocyclic indole
nucleus (Fig. 1), are used clinically for the treatment of different anti-inflammatory
disorders and are known to induce erosions and ulcers in the gastrointestinal tract
(Hardman and Limbird, 2001; Moore et al., 1996; Yoshikawa et al., 1993). Indole
derivatives have been reported to possess a wide variety of biological properties
M. Amir (&) ꢀ S. A. Javed ꢀ H. Kumar
Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Hamdard University,
New Delhi 110 062, India
e-mail: mamir_s2003@yahoo.co.in

300
Med Chem Res (2010) 19:299–310
Fig. 1 Indomethacin and
tenidap are known to induce
erosions and ulcers in the
gastrointestinal tract
O
Cl
COOH
O
S
N
N
O
O
O
NH2
Tenidap
Cl
Indomethacin
viz., anti-inflammatory (Amir and Kumar, 2005; Amir et al., 2008; Singh et al.,
2008), analgesic (Radwan et al., 2007), anticonvulsant (El-Gendy et al., 2008), and
antibacterial (Gurkok et al., 2009; Yamamoto and Kurazono, 2007). Furthermore,
substitution of heterocyclic moiety at 3-position of indole ring has been shown to
improve anti-inflammatory activity (Radwan et al., 2007; Rani et al., 2004).
Moreover, isoxazoline derivatives have been reported to possess potent protein
tyrosine phosphatase inhibitor, and anti-inflammatory activity (Basappa et al., 2004;
Habeeb et al., 2001; Maurya et al., 2008). Recently, m-terphenyl amines, which do
not have traditional arylacetic acid moiety, have been reported to possess potent
cyclooxygenase-inhibiting activity (Bauer et al., 2007). Encouraged by these
observations and also in continuation of our search for potent anti-inflammatory
molecules (Amir et al,. 2007a, b), we report the synthesis of new 3-substituted
indole derivatives possessing isoxazoline moiety to get compounds of higher
biological significance.
Results and discussion
Chemistry
In the present work, a series of eight new compounds were synthesized. Scheme 1
illustrates the synthetic route for the preparation of target compounds. 1-(Substituted
phenyl)-3-(1H-indol-3-yl)-2-propen-1-ones 1a–d were prepared by the method
reported in literature (Amir et al., 2008). 3-[3-(Substituted phenyl)-4,5-dihydro-
isoxazol-5-yl]-1H-indoles 2a–d were obtained by cyclization of 1a–d by treating
with hydroxylamine hydrochloride in the presence of NaOH in absolute ethanol.
The IR spectrum of compound 2b showed absorption peaks at 1356 and 1652 cm-1
due to C–O–N and C=N stretching vibrations. The NH stretching vibration was
obtained at 3310 cm-1. The structure was further supported by its 1H NMR
spectrum, which showed two double doublets at d 3.59 and d 3.74 for CH2 protons
of isoxazoline ring. The CH proton at C-5 of isoxazoline was obtained as triplet at d
6.08. Thus, disappearance of signals of the olefinic protons and appearance of CH2
and CH proton signals in the spectrum confirmed the formation of isoxazoline ring.
The NH proton of indole nucleus was obtained as a singlet at d 8.17. The mass
spectrum of the compound 2b showed molecular ion peak M? at m/z 297 and
M??2 at m/z 299 corresponding to molecular formula C17H13ClN2O. The

Med Chem Res (2010) 19:299–310
301
Scheme 1 Protocol for the
synthesis of 3-[3-(substituted
phenyl)-4- piperidin-1-
CHO
R
O
+
ylmethyl/-4-morpholin-4-
ylmethyl-4,5-dihydro-isoxazol-
5-yl]-1H-indoles
N
H
2% NaOH
Ethanol
R
O
1a-d
NH2OH.HCl
NaOH
Ethanol
N
H
R
HN
N
O
Mannich reaction
HCHO
Morpholine
Mannich reaction
HCHO
Piperidine
2a-d
O
R
R
N
N
HN
HN
N
N
O
O
4a-d
3a-d
R = a: H, b: 4-Cl, c: 4-CH3, d: 4-OCH3
indolylisoxazolines 2a–d on treatment with piperidine in the presence of formal-
dehyde in ethanol gave corresponding 3-[3-(substituted phenyl)-4-piperidin-1-
ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indoles 3a–d. The IR spectrum of com-
pound 3b showed characteristic absorption peak at 3272 (NH), 1686 (C=N),
1362 cm-1 (C–O–N). The structure of the compound was further confirmed by its
1H NMR spectrum, which showed a doublet at d 5.58 for –CH2–N proton. The six
protons (3 CH2) of piperidine ring were obtained as a multiplet at d 1.37–1.55,
whereas the N–(CH2)2 protons of piperidine ring were observed as a multiplet at d
2.51–2.56. The mass spectrum of compound 3b showed molecular ion peak M? at
m/z 394 and M??2 at m/z 396 corresponding to molecular formula C23H24ClN3O.
3-[3-(substituted phenyl)-4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indoles 4a–d were prepared by treating 2a–d with morpholine in the presence of
formaldehyde in ethanol. The IR spectrum of compound 4b showed characteristic
absorption peak at 3323 (NH), 1689 (C=N), 1359 (C–O–N). The structure of the
compound was further confirmed by its 1H NMR spectra, which showed a doublet at
d 5.53 for –CH2–N proton. The four protons (CH2–N–CH2) of morpholine ring were
obtained as a triplet at d 2.52, whereas the (CH2–O–CH2) protons of morpholine
ring were observed as a triplet at d 3.73. The mass spectrum of compound 4b

302
Med Chem Res (2010) 19:299–310
showed molecular ion peak M? at m/z 396 and M??2 at m/z 398 corresponding to
molecular formula C22H22ClN3O2.
Pharmacology
All the newly synthesized compounds (3a–d and 4a–d) were evaluated for their
anti-inflammatory activity against carrageenin-induced paw edema method in rats,
and compared with the reference drug indomethacin. Percent of the edema
inhibition was calculated both after 3 and 4 h of carrageenin treatment. Both
readings are reported in Table 1, but because % inhibition was found to be more
significant after 4 h, the discussion is based on this reading. The tested compounds
showed anti-inflammatory activity ranging from 45.45–55.55% (Table 1), whereas
standard drug indomethacin showed 76.5% inhibition after 4 h. The anti-inflam-
matory activity of compounds 3a–d having a piperidin-1-ylmethyl group at fourth
position of isoxazoline ring was found in the range of 48.48–53.53%. Compound 3b
having 4-chlorophenyl group at third position of isoxazoline ring showed maximum
activity (53.53%). Replacement of 4-chlorophenyl group by phenyl (3a), 4-
methylphenyl (3c), and 4-methoxyphenyl (3d) groups resulted in a slight decrease in
anti-inflammatory activity (48.48%, 51.51%, and 50.5% respectively). The anti-
inflammatory activity of compounds 4a–d having a morpholin-4-ylmethyl group at
fourth position of isoxazoline ring was found between 45.45% and 55.55%. The
highest activity (55.55%) was found in compound 4d having 4-methoxyphenyl
group at third position of isoxazoline ring. Replacement of this group by phenyl
Table 1 Anti-inflammatory, ulcerogenic, and lipid peroxidation activity of compounds 3a–d and 4a–d
Compound Dose
(mg kg-1
Anti-inflammatory activity
(% inhibition SEM)a
Dose
Ulcerogenic
nmol MDA
)
(mg kg-1) activity (severity content SEM/
index SEM)a 100 mg tissuea
After 3 h (s) After 4 h (s)
3a
70
34.31 2.36 48.48 2.56*
–
–
–
3b
70
70
70
70
70
70
70
35
45.09 1.96 53.53 2.02* 210
41.16 2.14 51.51 1.56* 210
45.08 1.96 50.5 1.86* 210
0.583 0.2*
0.5 0*
4.38 0.1*
4.28 0.09*
3c
3d
0.75 0.25** 4.51 0.07*
4a
35.29 2.14 45.45 2.21*
44.1 2.51 50.5 1.86* 210
38.22 2.51 48.48 2.59*
44.11 55.55 2.55* 210
65.15 1.51 76.5 1.82 105
–
–
–
4b
0.75 0.25** 4.39 0.08*
4c
–
–
–
4d
2
0.25 0.11*
2.33 0.17
0
4.16 0.14*
9.29 0.33
3.23 0.05
IND
Controlb
1 ml 0.5%
CMC
–
–
1 ml 0.5%
CMC
IND indomethacin, CMC carboxymethyl cellulose
* P \ 0.0001; ** P \ 0.001
a
Relative to standard and data were analyzed by Student’s t test for n = 6
b
The group was given orally 1 ml of 0.5% aqueous carboxymethyl cellulose solution

Med Chem Res (2010) 19:299–310
303
(4a), 4-chlorophenyl (4b), and 4-methylphenyl (4c) groups resulted in decrease of
anti-inflammatory activity (45.45%, 50.5%, and 48.48% respectively).
The compounds 3b–d, 4b, and 4d, which showed more than 50% anti-
inflammatory activity, were further tested for their acute ulcerogenic activity. The
tested compounds showed a significant reduction in ulcerogenic activity with the
severity index ranging from 0.25 0.11 to 0.75 0.25, whereas the standard drug
indomethacin showed a high severity index of 2.33 0.17. The maximum
reduction in ulcerogenic activity (0.25 0.11) was found in compound 4d having
4-methoxyphenyl group at position 3, and a morpholin-4-ylmethyl group at position
4 of isoxazoline ring. Rest of the tested compounds also showed better
gastrointestinal safety profile compared with indomethacin (Table 1). It has been
reported that compounds showing less ulcerogenic activity also show reduced
malondialdehyde (MDA) content—a byproduct of lipid peroxidation (Pohle et al.,
2001). Therefore, an attempt was made to correlate the decrease in ulcerogenic
activity of the compounds with that of lipid peroxidation. All the compounds
screened for ulcerogenic activity were analyzed for their effect on lipid peroxida-
tion. Lipid peroxidation is measured as nanomoles of malondialdehyde (MDA) per
100 mg of gastric mucosa tissue. Indomethacin exhibited maximum tissue lipid
peroxidation 9.29 0.33, whereas the control group showed 3.23 0.05. It was
found that all the isoxazoline derivatives showing less ulcerogenic activity also
showed reduction in lipid peroxidation (Table 1). Thus, these studies showed that
the synthesized compounds have inhibited the induction of gastric mucosal lesions
and the results further suggested that their protective effect might be related to the
inhibition of lipid peroxidation in the gastric mucosa.
Conclusions
Various indolylisoxazoline derivatives were prepared with the objective of
developing better anti-inflammatory molecules with minimum ulcerogenic activity.
It was noted that five compounds—3b–d, 4b, and 4d—were found to have more
than 50% anti-inflammatory activity compared with standard drug (indomethacin
76.5%), against carrageenin induced paw edema in rats. It was observed that
indolylisoxazolines having a piperidin-1-ylmethyl group (3a, 3b, 3c) showed
slightly improved anti-inflammatory activity than their respective isoxazoline
derivatives having a morpholin-4-ylmethyl group (4a, 4b, 4c), except in compound
4d for which the activity was found to be more (55.55%) than compound 3d
(50.5%). These compounds were tested for ulcerogenic activity and showed a
significant reduction in severity index compared with standard reference drug. From
these studies compound 3-[3-(4-methoxyphenyl)-4-morpholin-4-ylmethyl)-4,5-
dihydro-isoxazol-5yl]-1H-indole 4d has emerged as a lead compound, which
showed maximum anti-inflammatory activity along with maximum reduction in
ulcerogenic activity and lipid peroxidation. Thus, the series provided a new
opportunity for possible modification of pharmacophoric requirements and future
exploitation.

304
Med Chem Res (2010) 19:299–310
Experimental
Chemistry
Chemicals were purchased from E Merck (Germany), S. D. Fine Chemicals (India),
and Qualigens (India). Melting points were determined in open capillary tubes and
are uncorrected. Purity of the compounds was checked on TLC plates (silica gel G)
and spots were located under iodine vapors or UV light. IR (KBr) spectra were
recorded on a Nicolet, 5PC FTIR spectrometer (mmax in cm-1) and 1H NMR spectra
were recorded in CDCl3 on a Bruker DRX-300 (300 MHz FT NMR) spectrometer
using TMS as internal reference (chemical shift in d ppm). FAB mass spectra were
recorded on a JEOL SX 102/DA-6000 mass spectrometer. Elemental analyses were
performed on a Carlo-Erba-1108 CHN elemental analyzer.
General Procedure for the Preparation of 3-[3-(substituted phenyl)-4,5-dihydro-
isoxazol-5-yl]-1H-indoles (2a–d). To a solution of compounds 1a–d (0.01 mol) in
absolute ethanol (50 ml), hydroxylamine hydrochloride (0.01 mol) and solid NaOH
(0.4 g) were added. The reaction mixture was refluxed for 4–6 h. The excess solvent
was distilled off and crude product poured into ice water. The solids thus separated
were filtered, washed with water, dried, and recrystallized from ethanol.
3-[3- phenyl-4,5-dihydro-isoxazol-5-yl]-1H-indole (2a)
Yield 56%; m.p. 166–167°C. IR (KBr, cm-1): 3290 (NH), 1640 (C=N), 1350 (C–O–
N). 1H NMR (CDCl3, 300 MHz) d: 8.15 (1H, s, NH of indole), 6.97–7.63 (10H, m,
ArH), 5.98 (1H, t, J = 9.7 Hz, CH), 3.71 (1H, dd, J = 11.3, 5.7 Hz, CH), 3.56 (1H,
dd, J = 9.2, 7.6 Hz, CH); MS: m/z 262 (M?). Elemental analysis: Calc. for
C17H14N2O: C, 77.84; H, 5.38; N, 10.68; found: C, 77.63; H, 5.16; N, 10.54%.
3-[3-(4-Chlorophenyl)-4,5-dihydro-isoxazol-5-yl]-1H-indole (2b)
Yield 72%; m.p. 186–188°C. IR (KBr, cm-1): 3310 (NH), 1652 (C=N), 1356 (C–O–
1
N). H NMR (CDCl3, 300 MHz) d: 8.17 (1H, s, NH of indole), 7.09–7.68 (9H, m,
ArH), 6.08 (1H, t, J = 9.9 Hz, CH), 3.74 (1H, dd, J = 11.1, 5.4 Hz, CH), 3.59 (1H,
dd, J = 9.0, 7.5 Hz, CH); MS: m/z 297 (M?), 299 (M??2). Elemental analysis:
Calc. for C17H13ClN2O: C, 68.81; H, 4.42; N, 9.44; found: C, 68.59; H, 4.31; N,
9.31%.
3-[3-(4- Methylphenyl)-4,5-dihydro-isoxazol-5-yl]-1H-indole (2c)
Yield 69%; m.p. 207–208°C. IR (KBr, cm-1): 3284 (NH), 1646 (C=N), 1343 (C–O–
1
N). H NMR (CDCl3, 300 MHz) d: 8.14 (1H, s, NH of indole,). 6.96–7.62 (9H, m,
ArH), 5.95 (1H, t, J = 9.7 Hz, CH), 3.73 (1H, dd, J = 11.4, 5.8 Hz, CH), 3.55 (1H,
dd, J = 9.4, 6.9 Hz, CH), 2.23 (3H, s, CH3); MS: m/z 276 (M?). Elemental
analysis: Calc. for C18H16N2O: C, 78.24; H, 5.84; N, 10.14; found: C, 78.06; H,
5.72; N, 10.03%.

Med Chem Res (2010) 19:299–310
305
3-[3-(4-Methoxyphenyl)-4,5-dihydro-isoxazol-5-yl]-1H-indole (2d)
Yield 57%; m.p. 174–176°C. IR (KBr, cm-1): 3297 (NH), 1650 (C=N), 1353 (C–O–
1
N). H NMR (CDCl3, 300 MHz) d: 8.12 (1H, s, NH of indole), 6.93–7.58 (9H, m,
ArH), 5.92 (1H, t, J = 9.9 Hz CH), 3.82 (3H, s, OCH3), 3.69 (1H, dd, J = 11.6,
6.1 Hz, CH), 3.51 (1H, dd, J = 8.9, 7.2 Hz, CH). MS: m/z 292 (M?). Elemental
analyses: Calc. for C18H16N2O2: C, 73.95; H, 5.52; N, 9.58; found: C, 73.78; H,
5.41; N, 9.43%.
General procedure for the preparation of 3-[3-(substituted phenyl)-4-piperidin-
1-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indoles (3a–d). Compounds 2a–d
(0.001 mol) were dissolved in a mixture of ethanol and dioxane (2:1). Then
formaldehyde (40%, 1.5 ml) and piperidine (0.001 mol) in ethanol were introduced
into the solution. The reaction mixture was stirred for 2 h and then refluxed for 4 h
on a water bath. The resulting solution was concentrated, cooled, and poured into
crushed ice. The solids thus separated were filtered, washed with water, dried, and
recrystallized from ethanol.
3-[3- Phenyl-4-piperidin-1-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indole (3a)
Yield 69%; m.p. 105–107°C. IR (KBr, cm-1): 3263 (NH), 2989 (C–H), 1680
(C=N), 1350 (C–O–N). 1H NMR (CDCl3, 300 MHz) d: 8.23 (1H, bs, NH of indole),
6.91–7.47 (10H, m, ArH), 5.87 (1H, d, J = 6.9 Hz, CH), 5.51 (2H, d, J = 6.3 Hz,
CH2–N), 3.43–3.51 (1H, m, CH–CH2–N), 2.43–2.47 (4H, m, CH2–N–CH2), 1.29–
1.48 (6H, m, 3CH2); MS: m/z 359 (M?). Elemental analysis: Calc. for C23H25N3O:
C, 76.85; H, 7.01; N, 11.69; found: C, 76.62; H, 6.87; N, 11.58%.
3-[3-(4- Chlorophenyl)-4-piperidin-1-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indole (3b)
Yield 60%; m.p. 76–77°C. IR (KBr, cm-1): 3272 (NH), 2993 (C–H), 1686 (C=N),
1362 (C–O–N). 1H NMR (CDCl3, 300 MHz) d: 8.41 (1H, bs, NH of indole). 7.08–
7.67 (9H, m, ArH), 5.9 (1H, d, J = 7.0 Hz, CH), 5.58 (2H, d, J = 6.2 Hz, CH2–N),
3.46–3.56 (1H, m, CH–CH2-N), 2.51–2.56 (4H, m, CH2–N–CH2), 1.37–1.55 (6H, m,
3CH2); MS: m/z 394 (M?), 396 (M??2). Elemental analysis: Calc. for
C23H24ClN3O: C, 70.13; H, 6.14; N, 10.67; found: C, 69.84; H, 5.97; N, 10.83%.
3-[3-(4-Methylphenyl)-4-piperidin-1-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indole
(3c)
Yield 63%; m.p. 124–126°C. IR (KBr, cm-1): 3306 (NH), 3107 (C–H), 1684
(C=N), 1352 (C–O–N). 1H NMR (CDCl3, 300 MHz) d: 8.32 (1H, bs, NH of indole).
6.71–7.48 (9H, m, ArH), 5.86 (1H, d, J = 7.1 Hz, CH), 5.36 (2H, d, J = 6.2 Hz,
CH2–N), 3.42–3.54 (1H, m, CH–CH2–N), 2.47–2.53 (4H, m, CH2–N–CH2), 1.31–
1.45 (6H, m, 3CH2), 1.25 (2H, s, CH3), MS: m/z 373 (M?). Elemental analysis:
Calc. for C24H27N3O: C, 77.18; H, 7.29; N, 11.25; found: C, 76.97; H, 7.16: N,
11.39%.

306
Med Chem Res (2010) 19:299–310
3-[3-(4-Methoxyphenyl)-4-piperidin-1-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indole (3d)
Yield 56%; m.p. 112–114°C. IR (KBr, cm-1): 3298 (NH), 3011 (C–H), 1681
(C=N), 1356 (C–O–N). 1H NMR (CDCl3, 300 MHz) d: 8.39 (1H, bs, NH of indole).
6.93–7.51 (9H, m, ArH), 5.89 (1H, d, J = 6.9 Hz, CH), 5.51 (2H, d, J = 6.0 Hz,
CH2–N), 3.81 (3H, s, OCH3), 3.45–3.59 (1H, m, CH–CH2–N), 2.49–2.57 (4H, m,
CH2–N–CH2), 1.32–1.49 (6H, m, 3CH2); MS: m/z 389 (M?). Elemental analysis:
Calc. for C24H27N3O2: C, 74.01; H, 6.99; N, 10.79; found; C, 73.83; H, 6.87: N,
10.65%.
General procedure for the preparation of 3-[3-(substituted phenyl)-4-morpholin-
4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indoles (4a–d). Compounds 2a–d
(0.001 mol) were dissolved in a mixture of ethanol and dioxane (2:1). Then
formaldehyde (40%, 1.5 ml) and morpholine (0.001 mol) in ethanol were
introduced into the solution. The reaction mixture was stirred for 2 h and then
refluxed for 4 h on a water bath. The resulting solution was concentrated, cooled,
and poured into crushed ice. The solids thus separated were filtered, washed with
water, dried, and recrystallized from ethanol.
3-[3-(Phenyl)-4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-indole (4a)
Yield 69%; m.p. 106–107°C. IR (KBr, cm-1): 3295 (NH), 3016 (C–H), 1683
(C=N), 1344 (C–O–N): 1H NMR (CDCl3, 300 MHz) d: 7.98 (1H, bs, NH of indole),
7.13–7.74 (9H, m, ArH), 5.87 (1H, d, J = 6.8 Hz, CH), 5.49 (2H, d, J = 6.2 Hz,
CH2–N), 3.61 (4H, t, J = 6.3 Hz, CH2–O–CH2), 3.29–3.51 (1H, m, CH–CH2–N),
2.46 (4H, t, J = 4.6 Hz, CH2–N–CH2); Mass: m/z 361 (M?). Elemental analysis:
Calc. for C22H23N3O2: C, 73.11; H, 6.41; N, 11.63; found: C, 72.89; H, 6.32: N,
11.46%.
3-[3-(4-Chlorophenyl)-4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indole (4b)
Yield 49%; m.p. 92–93°C. IR (KBr, cm-1): 3323 (NH), 3026 (C–H), 1689 (C=N),
1359 (C–O–N): 1H NMR (CDCl3, 300 MHz) d: 8.41 (1H, bs, NH of indole), 7.16–
7.78 (9H, m, ArH), 5.92 (1H, d, J = 6.9 Hz, CH), 5.53 (2H, d, J = 6.2 Hz, CH2–
N), 3.73 (4H, t, J = 6.6 Hz, CH2–O–CH2), 3.32–3.55 (1H, m, CH–CH2–N), 2.52
(4H, t, J = 4.5 Hz, CH2–N–CH2); Mass: m/z 396 (M?), 398 (M??2). Elemental
analysis: Calc. for C22H22ClN3O2: C, 66.75; H, 5.60; N, 10.61; found: C, 66.49; H,
5.48: N, 10.54%.
3-[3-(4-Methylphenyl)-4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indole (4c)
Yield 58%; m.p. 78–79°C. IR (KBr, cm-1): 3246 (NH), 3012(C–H), 1690 (C=N),
1350 (C–O–N): 1H NMR (CDCl3, 300 MHz) d: 8.13 (1H, bs, NH of indole). 7.13–
7.72 (9H, m, ArH), 6.02 (1H, d, J = 7.0 Hz, CH), 4.85 (2H, d, J = 6.3 Hz, CH2–

Med Chem Res (2010) 19:299–310
307
N), 3.65 (4H, t, J = 6.5 Hz, CH2–O–CH2), 3.43–3.51 (1H, m, CH–CH2–N), 2.25
(4H, t, J = 4.4 Hz, CH2–N–CH2), 1.26 (3H, s, CH3), Mass: m/z 375 (M?).
Elemental analysis: Calc. for C23H25N3O2: C, 73.57; H, 6.71; N, 11.19; found: C,
73.41; H, 6.63: N, 11.07%.
3-[3-(4-Methoxyphenyl)-4-morpholin-4-ylmethyl)-4,5-dihydro-isoxazol-5yl]-1H-
indole (4d)
Yield 64%; m.p. 104–106°C. IR (KBr, cm-1): 3291 (NH), 2987 (C–H), 1679
(C=N), 1346 (C–O–N): 1H NMR (CDCl3, 300 MHz) d: 8.26 (1H, bs, NH of indole).
7.14–7.79 (9H, m, ArH), 5.93 (1H, d, J = 6.9 Hz, CH), 5.46 (2H, d, J = 6.0 Hz,
CH2–N), 3.82 (3H, s, OCH3), 3.67 (4H, t, J = 6.9 Hz, CH2–O–CH2), 3.49-3.57
(1H, m, CH–CH2–N), 2.43 (4H, t, J = 4.8 Hz, CH2–N–CH2), Mass: m/z 391 (M?).
Elemental analysis: Calc. for C23H25N3O3: C, 70.57; H, 6.44; N, 10.73; found: C,
70.38; H, 6.31; N, 10.61%.
Pharmacology
Animals
Adult Wistar strain rats of either sex, weighing 150–200 g, were used for anti-
inflammatory, ulcerogenic, and lipid peroxidation activities. The animals were
allowed food and water ad libitum except during the experiments. They were
housed in a room at 25 2°C, and 50 5% relative humidity with 12-h light/dark
cycle. The animals were randomly allocated into groups at the beginning of all
experiments. The experimental protocol was approved by the animal ethics
committee of Hamdard University. All test compounds and the reference drug were
administered orally, suspended in 0.5% carboxymethyl cellulose (CMC) solution.
Anti-inflammatory activity
The synthesized compounds were evaluated for their anti-inflammatory activity
using the carrageenin induced paw edema method of Winter et al. (1962). The
animals were randomly divided into groups of six and were fasted for 24 h before
the experiment, with free access to water. Control group received 1 ml of 0.5%
aqueous CMC. Each group was given the test compounds (70 mg/kg) and reference
drug indomethacin (35 mg/kg) orally as a uniform suspension in 1 ml of 0.5%
aqueous CMC according to the procedure of Radwan et al. (2007); 0.1 ml of
carrageenin (1% solution in normal saline) was injected subcutaneously into the
subplantar region of the right hind paw of each rat, 1 h after the administration of
the test compounds and reference drug. The right hind paw volume was measured
before and after 3 and 4 h of carrageenin treatment by means of a plethysmometer.
The percent edema inhibition was calculated from the mean effect in the control and
treated animals according to the following equation:
Percent edema inhibition ¼ ðVc ꢁ Vt=VcÞ ꢂ 100

308
Med Chem Res (2010) 19:299–310
where, Vt represents the mean increase in paw volume in rats treated with test
compounds and Vc represents the mean increase in paw volume in control group of
rats.
Acute ulcerogenicity
Acute ulcerogenicity was determined according to method of Cioli et al. (1979).
The animals were allocated into different groups of six each. Ulcerogenic activity
was evaluated after oral administration of the test compounds at an oral dose of
210 mg/kg. The reference drug indomethacin was administered orally at a dose of
105 mg/kg. Control group received 1 ml of 0.5% CMC solution. Food, but not
water, was removed 24 h before administration of the test compounds. After the
drug treatment, the rats were fed with normal diet for 17 h and then killed. The
stomach was removed and opened along the greater curvature, washed with distilled
water, and cleaned gently by dipping in normal saline. The mucosal damage was
examined by means of a magnifying glass. For each stomach, the mucosal damage
was assessed according to the following scoring system: 0.5: redness; 1.0: spot
ulcers; 1.5: hemorrhagic streaks; 2.0: ulcers [3 but B5; 3.0: ulcers [5. The mean
score of each treated group minus the mean score of control group was regarded as
severity index of gastric mucosal damage.
Lipid peroxidation
Lipid peroxidation in the gastric mucosa was determined according to the method of
Ohkawa et al. (1979). After screening for ulcerogenic activity, the gastric mucosa
was scraped with two glass slides, weighed (100 mg), and homogenized in 1.8 ml of
1.15% ice cold KCl solution. The homogenate was supplemented with 0.2 ml of
8.1% sodium dodecyl sulphate (SDS), 1.5 ml of acetate buffer (pH 3.5), and 1.5 ml
of 0.8% thiobarbituric acid (TBA). The mixture was heated at 95°C for 60 min.
After cooling the reactants were supplemented with 5 ml of the mixture of n-butanol
and pyridine (15:1v/v), shaken vigorously for 1 min, and centrifuged for 10 min at
4000 rpm. The supernatant organic layer was taken out and absorbance was
measured at 532 nm on UV spectrophotometer. The results were expressed as
nanomol MDA per 100 mg tissue, using molar absorption coefficient
1.56 9 105 cm-1 M-1
.
Statistical analysis
All values are expressed as mean SEM. Statistical significance was determined
using Student’s t test.
Acknowledgements Sadique Akhtar Javed is grateful to the University Grant Commission-New Delhi,
India, for financial assistance. The authors thank Mrs. Shaukat Shah, in-charge animal house, and Jamia
Hamdard for providing Wistar rats.

Med Chem Res (2010) 19:299–310
309
References
Amir M, Kumar S (2005) Anti-inflammatory and gastro sparing activity of some new indomethacin
derivatives. Arch Pharm 338:24–31. doi:10.1002/ardp.200400891
Amir M, Kumar H, Javed SA (2007a) Noncarboxylic analogues of naproxen: design, synthesis and
pharmacological evaluation of some 1,3,4-oxadiazole/thiadiazole and 1,2,4-triazole derivatives.
Arch Pharm 340:577–585. doi:10.1002/ardp.200700065
Amir M, Kumar H, Javed SA (2007b) Synthesis and pharmacological evaluation of condensed
heterocyclic 6-substituted-1,2,4-triazolo[3, 4-b]- 1,3,4-thiadiazole derivatives of naproxen. Bioorg
Med Chem Lett 17:4504–4508. doi:10.1016/j.bmcl.2007.06.003
Amir M, Javed SA, Kumar H (2008) Synthesis and biological evaluation of some 4-(1H-indol-3-yl)-6-
phenyl-1, 2, 3, 4-tetrahydropyrimidine-2-ones/thiones as potent anti-inflammatory agents. Acta
Pharm 58:467–477. doi:10.2478/v10007-008-0028-x
Basappa, Kumar MS, Swamy SN, Mahendra M, Prasad JS, Viswanath BS, Rangappa KS (2004) Novel
d2-isoxazolines as group II phospholipase A2 inhibitors. Bioorg Med Chem Lett 14:3679–3681. doi:
10.1016/j.bmcl.2004.05.012
Bauer JD, Foster MS, Hugdahl JD, Burns KL, May SW, Pollock SH, Cutler HG, Cutler SJ (2007)
Synthesis and pharmacological evaluation of m-terphenyl amines as cyclooxygenase inhibitors. Med
Chem Res 16:119–129. doi:10.1007/s00044-007-9015-x
Cioli V, Putzolu S, Rossi V, Barcellona PS, Corradino C (1979) The role of direct tissue contact in the
production of gastro-intestinal ulcers by anti-inflammatory drugs in rats. Toxicol Appl Pharmacol
50:283–289. doi:10.1016/0041-008X(79)90153-4
El-Gendy AA, Said MM, Ghareb N, Mostafa YM, El-Ashry El SH (2008) Synthesis and biological
activity of functionalized indole-2-carboxylates, triazino- and pyridazino-indoles. Arch Pharm
341:294–300. doi:10.1002/ardp.200700161
Gurkok G, Altanlar N, Suzen S (2009) Investigation of antimicrobial activities of indole-3-aldehyde
hydrazide/hydrazone derivatives. Chemotherapy 55:15–19. doi:10.1159/000166999
Habeeb AG, Praveen Rao PN, Knaus EE (2001) Design and synthesis of 4,5-diphenyl-4-isoxazolines:
novel inhibitors of cyclooxygenase-2 with analgesics and anti-inflammatory activity. J Med Chem
44:2921–2927. doi:10.1021/jm0101287
Hardman JG, Limbird LE (2001) Goodman and Gilman’s the pharmacological basis of therapeutics, 10th
edn. McGraw-Hill, New York, p 705
Langman MJ, Weil J, Wainwright P, Lawson DH, Rawlins MD, Logan RF, Murphy M, Vessey MP,
Colin-Jones DG (1994) Risks of bleeding peptic ulcer associated with individual non-steroidal anti-
inflammatory drugs. Lancet 343:1075–1078. doi:10.1016/S0140-6736(94)90185-6
Maurya R, Gupta P, Ahmad G, Yadav DK, Chand K, Singh AB, Tamarkar AK, Srivastava AK (2008)
Synthesis of 3,5-disubstituted isoxazolines as protiern tyrosine phosphatase 1B inhibitors. Med
Chem Res 17:123–136. doi:10.1007/s00044-007-9043-6
Moore PF, Larson DL, Otterness IG, Weissman A, Kadin SB, Sweeney FJ, Eskara JD, Nagahisa A,
Sakakibara M, Carty TJ (1996) Tenidap, a structurally novel drug for the treatment of arthritis: anti-
inflammatory and analgesic properties. Inflamm Res 45:54–61. doi:10.1007/BF02265116
Ohkawa H, Ohishi N, Yagi K (1979) Assay for lipid peroxides in animal tissues by thiobarbituric acid
reaction. Anal Biochem 95:351–358. doi:10.1016/0003-2697(79)90738-3
Pohle T, Brzozowski T, Becker JC, Vander Voort IR, Markmann A, Konturek SJ, Moniczewski A,
Domschke W, Konturek JW (2001) Role of reactive oxygen metabolites in aspirin-induced gastric
damage in humans, gastroprotection by vitamins. Aliment Pharmacol Ther 15:677–687. doi:
10.1046/j.1365-2036.2001.00975.x
Radwan MAA, Ragab EA, Sabry NM, El-Shenawy SM (2007) Synthesis and biological evaluation of
new 3-substituted indole derivatives as potential anti-inflammatory and analgesic agents. Bioorg
Med Chem 15:3832–3841. doi:10.1016/j.bmc.2007.03.024
Rani P, Srivastava VK, Kumar A (2004) Synthesis and anti-inflammatory activity of heterocyclic indole
derivatives. Eur J Med Chem 39:449–452. doi:10.1016/j.ejmech.2003.11.002
Singh N, Bhati SK, Kumar A (2008) Thiazolyl/oxazolyl formazanyl indoles as potent anti-inflammatory
agents. Eur J Med Chem 43:2597–2609. doi:10.1016/j.ejmech.2007.12.024
Winter CA, Risley EA, Nuss GW (1962) Carrageenin-induced edema in hind paw of the rat as an assay
for anti-inflammatory drugs. Proc Soc Exp Biol Med 111:544–547

310
Med Chem Res (2010) 19:299–310
Yamamoto Y, Kurazono M (2007) A new class of anti-MRSA and anti-VRE agents: preparation and
antibacterial activities of indole-containing compounds. Bioorg Med Chem Lett 7:1626–1628. doi:
10.1016/j.bmcl.2006.12.081
Yoshikawa T, Naito Y, Kishi A, Tomii T, Kaneko T, Inuma S, Ichikawa H, Yasuda M, Takahashi S,
Kondo M (1993) Role of active oxygen, lipid peroxidation and antioxidants in the pathogenesis of
gastric mucosal injury induced by indomethacin in rats. Gut 34:732–737. doi:10.1136/gut.34.6.732