Synthesis of new naphthoisoxazole amide derivatives and study of their biological evaluations

Article abstract of DOI:10.1007/s00044-014-0961-9

A series of naphthoisoxazole amide derivatives 4a-h have been synthesized from naphthoisoxazole acetic acid 3. 2-Acetyl-1-naphthol-1 on reaction with pulverized sodium and diethyl carbonate gave 4-hydroxy naphthopyrone 2 which on Posner reaction gave naphthoisoxazole acetic acid 3. The cytotoxic activity study for inhibiting melanoma cell survival was evaluated on a series of melanoma cell lines. The anticonvulsant activity of these compounds has been evaluated in Wistar rats. A compound called 4h has been found to have anticonvulsant activity comparable to that of the standard drug phenytoin. Springer Science+Business Media 2014.

Full text of DOI:10.1007/s00044-014-0961-9

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res
DOI 10.1007/s00044-014-0961-9
ORIGINAL RESEARCH
Synthesis of new naphthoisoxazole amide derivatives and study
of their biological evaluations
•
Shubhangi S. Soman Jigar N. Soni
•
Timir B. Patel
Received: 1 July 2013 / Accepted: 15 February 2014
Ó Springer Science+Business Media New York 2014
Abstract A series of naphthoisoxazole amide derivatives
4a–h have been synthesized from naphthoisoxazole acetic
acid 3. 2-Acetyl-1-naphthol-1 on reaction with pulverized
sodium and diethyl carbonate gave 4-hydroxy naphthopy-
rone 2 which on Posner reaction gave naphthoisoxazole
acetic acid 3. The cytotoxic activity study for inhibiting
melanoma cell survival was evaluated on a series of mel-
anoma cell lines. The anticonvulsant activity of these
compounds has been evaluated in Wistar rats. A compound
called 4h has been found to have anticonvulsant activity
comparable to that of the standard drug phenytoin.
zonisamide (Stiff and Zemaitis, 1990) which is also used as
antiepileptic agent. Substitution on benzene ring leads to increase
in reactivity and also increase in neurotoxicity, while change in
sulfonamide group leads to decrease in activity (Masuda et al.,
1998).
{1-[3-(6-Fluoro-l,2-benzisoxazole-3-yl)propyl]-4-(2-
oxo-l-benzimidazolinyl)} piperidine, haloperidol, and thiorida-
zine exhibit dopamine-blocking properties (Fielding et al.,
1983). Naphtho[2,3-d]isoxazole-4,9-dione-3-carboxylates have
been reported as potent cytoprotective agents (Santos et al.,
2009). Some isoxazole derivatives have been reported as pro-
drugs for the treatment of cancer (Jain and Kwon, 2003). Furo-
benzisoxazole derivatives have been reported to possess
uricosuric and diuretic properties for treatment of hyperuricemia,
edema, and hypertension (Sato et al., 1988 and Anthony and
Plattner, 1984). Trifluoromethyl benzisoxazole derivatives have
shown affinity and potency for PPARa as well as better affinity
for PPARc (Adams et al., 2003).
Keywords Angular isoxazole ꢀ Amide ꢀ
Anticonvulsant activity
Introduction
Literature search reveals that all reported isoxazole
derivatives have linear ring fusion with benzene or naphtha-
lene but not a single report documented isoxazole derivatives
with angular ring fusion. Substitution on naphthalene ring
may increase neurotoxicity (Masuda et al., 1998). Based on
standard drugs phenytoin and zonisamide, we have designed
new naphthoisoxazole derivatives as shown in Fig. 1. We
report herein investigation on isoxazole derivatives (Patel and
Soman, 2009), detailing the synthesis and biological activity
of a series of naphthoisoxazole derivatives.
Derivatives of benzisoxazole are known to possess biological
activities such as antimicrobial (Lamani et al., 2009) and herbi-
cidal activities (Andreani et al., 1991). The most extensively
studied isoxazole-containing drug is the anticonvulsant drug,
Electronic supplementary material The online version of this
article (doi:10.1007/s00044-014-0961-9) contains supplementary
material, which is available to authorized users.
S. S. Soman (&) ꢀ J. N. Soni
Department of Chemistry, Faculty of Science, The M.
S. University of Baroda, Vadodara 390002, India
e-mail: shubhangiss@rediffmail.com
Results and discussion
J. N. Soni
e-mail: jigarsoni84@yahoo.com
Chemistry
T. B. Patel
Faculty of Pharmacy, Dharmsinh Desai University, Nadiad,
India
2-Acetyl-1-naphthol 1 on reaction (Boyd and Robertson,
1948) with diethyl carbonate in the presence of pulverized
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Med Chem Res
Fig. 1 Design of
naphthoisoxazole derivatives
O
O
O
R
N
O
S
N
NH2
HN
O
O
R'
NH
N
O
phenytoin
Naphthoisoxazole
zonisamide
sodium gave 4-hydroxy-2H-benzo[h]chromen-2-one 2
(Bhargava et al., 1975). The IR of compound 2 showed
cyclic amines like piperidine (4e), pyrrolidine (4f), mor-
pholine (4g), and open chain secondary amine i.e., diethyl
amine (4h), the IC50 values were [40 lM indicating less
toxicity of these compounds. Since IC50 values were
[20 lM, further detail study of anticancer activity against
various cancer cell lines and its comparison with standard
drugs was not carried out.
bands at 3423 cm-1 for hydroxyl group and 1604 cm-1 for
1
carbonyl group. The H NMR showed singlet at d 5.83 for
C-3 proton, broad peak at d 11.94 for –OH proton, and all
aromatic protons were observed between d 7.60 and 8.53
confirmed formation of 2. Posner reaction (Posner and
Hess, 1913 and Gianella et al., 1971) of 2 with hydroxyl-
amine hydrochloride in the presence of sodium bicarbonate
gave 2-(naphtho[2,1-d]isoxazole-3-yl) acetic acid 3. The
IR of compound 3 showed bands at 3435 cm-1 for
hydroxyl group, 1732 cm-1 for carbonyl group, and
1641 cm-1 for C=N stretching frequency. In 1H NMR of 3
singlet observed at d 4.17 for methylene proton, broad peak
at d 12.92 for carboxylic acid proton. The disappearance of
peak at d 5.83 confirmed the absence of lactone ring. All
aromatic protons were appeared between d 7.75 and 8.38
confirmed formation of 3. The isoxazole acetic acid 3
converted into corresponding amides (Muijlwijk-Koezen
et al., 2001) by reaction with different amines using
dicyclohexylcarbodiimide (DCC) as promoter and N,N-
dimethyl amino pyridine (DMAP) as catalyst (Scheme 1).
Anticonvulsant activity study
Since the isoxazole amide derivatives can show anticon-
vulsant activity, the anticonvulsant activity study of all
synthesized compounds was tested by the MES method
(Tandon and Gupta, 2005) in Wistar rats, and the results
are summarized in Table 2. The experiments were carried
out on male Wistar rats (150–180 g). Animals were housed
in plastic cages at a constant temperature of 20–28 °C with
natural light–dark cycles. The animals had free access to
standard pellet diet and water, and were used after a min-
imum of 3 days of acclimatization to the housing condi-
tions. Control and experimental groups consisted of six
animals each. All the procedures used followed the NIH
Animal Care and Use Committee guidelines and were
approved by the Ethics Committee at the Dharmsinh Uni-
versity, Nadiad, India. The examined compounds were
administered as solution in 0.1 ml DMSO via IP route at a
constant dose of 5, 10, and 15 mg kg-1 of body weight.
Different phases of activity were observed. In case of
compound 4c and 4h i.e., amines used were p-bromo ani-
line (4c) and N,N-diethyl amine (4h), the Flexion phase
was found to be very comparable to standard drug phe-
nytoin. For compound 4f i.e., amine used was pyrrolidine
(4f), the Extension phase was found to be very low. In case
of compound 4h i.e. amine used was N,N-diethyl amine
(4h), the Clonus phase was found to be comparable with
standard drug as shown in Fig. 2.
All the compounds were characterized by IR, 1H NMR, 13
C
NMR, Elemental analyses, and Mass spectra. Purity of
compounds was checked by reverse phase HPLC.
Biological evaluation
Cytotoxicity studies
The IC50 values of all synthesized compounds were
determined by in vitro screening on a series of various
melanoma cell lines Melan-A, WM3211, WM278,
UACC903, and 1205Lu using a MTS assay to establish the
efficacy of the compound for alter cell survival (Sharma
et al., 2009). The values are given in Table 1. All com-
pounds exhibited low toxicity with IC50 values[20 lM. In
the series 4a–h, when the amines used were p-toluidine
(4a), aniline (4b), showed IC50 values 24.31, 34.89 against
WM3211 cell line, respectively. When amine used was p-
bromoaniline (4c), showed IC50 value 25.51 against
WM278 cell line. When the amines used were secondary
Conclusion
We have concluded that angular isoxazole amide deriva-
tives could be a good scaffold for CNS activities. Since all
123

Med Chem Res
Br
O
O
O
N
N
O
O
N
O
N
N
N
H
4c
4f
4h
Fig. 2 Structure activity relationship of naphthoisoxazole amide derivatives
O
OH
O
O
O
N
O
b
a
OH
OH
1
3
2
c
O
N
O
R
Br
3'
4'
2'
4
O
5'
1'
NH
N
O
R = 4a= NH
N
4e=
6'
1
3
2
NH
4b=
4f= N
4g= N
O
NH
NH
Br
4c=
4h= N
4d=
HO
Scheme 1 Reagents and conditions: a pulverized sodium, diethyl carbonate, 30 min; b NH2OHꢀHCl, NaHCO3, methanol, reflux, 15 h; c DCC,
DMAP, amine, ethyl acetate, room temperature, 12 h
compounds have IC50 values more than 20 lM in Cyto-
toxicity study we cannot use them as anticancer agent, but
we have studied anticonvulsant activity of all compounds.
Compound 4h showed excellent anticonvulsant activity as
compare with the standard drug Phenytoin in all phases.
Compound 4c has low Flexion phase and 4f has low
extension phase as compare to the standard drug which
indicated that such scaffolds can be useful in the treatment
of various CNS diseases.
Experimental section
Reagent grade chemicals and solvents were purchased
from commercial supplier and used without purification.
TLC was performed on silica gel F254 plates (Merck).
Melting points are uncorrected and were measured in open
capillary tubes, using a Rolex melting point apparatus. IR
spectra were recorded as KBr pellets on Perkin Elmer RX 1
1
spectrometer. H NMR and 13C NMR spectral data were
123

Med Chem Res
recorded on Bruker Advance 300 spectrometer (300 MHz)
and Advance 400 spectrometer (400 MHz) with CDCl3 or
DMSO-d6 as solvent and TMS as internal standard. J val-
ues are in Hz. Mass spectra were determined by ESI/MS,
using a Shimadzu LCMS 2020 apparatus. Purity of com-
pounds was checked by reverse phase HPLC using W2996
PDA detector at 254 nm wavelength using Acetonitrile:
Water as solvent. CHN elemental analyses were recorded
on Thermosinnigan Flash 11–12 series EA.
added to decompose the unreacted sodium. The reaction mass
was then poured into water (250 ml) and the aqueous layer
washed twice with Pet. Ether (50 ml). Concentrated hydro-
chloric acid was slowly added to the aqueous layer until pH 2,
and the solid obtained was collected by filtration. The crude
product was crystallized from ethanol as light-yellow solid
(1.66 g, 96 %). m.p. 283–285 °C [Lit. 284 °C (Bhargava
et al., 1975)]; IR (KBr) cmax: 3423 (OH), 1604, 1561 (C=O)
cm-1; 1H NMR (400 MHz, DMSO-d6): d = 5.83 (1H, s, C-3
proton), 7.60–7.76 (3H, m, ArH), 7.86–7.94 (2H, m, ArH),
8.48–8.53 (1H, m, ArH), 11.94 (1H, s, OH); 13C NMR
(400 MHz, DMSO-d6): d = 91.08 (C-3), 111.62 (C-5),
119.43 (C-13), 122.15 (C-7), 122.66 (C-11), 124.06 (C-12),
127.80 (C-8), 128.57 (C-9), 129.25 (C-10), 135.27 (C-14),
151.12 (C-6), 162.31 (C-4), 167.16 (C-2,[C=O).
Synthetic part
4-Hydroxy-2H-benzo[h]chromen-2-one (2)
A solution of 1-acetyl-2-naphthol-1 (2 g, 1.07 mmol) in
diethyl carbonate (30 ml) was slowly added to pulverized
sodium (4 g, 17.39 mmol) under anhydrous conditions.
Highly exothermic reaction was observed. It was then
allowed to cool to room temperature. Ethanol (50 ml) was
2-(Naphtho[2,1-d]isoxazole-3-yl)acetic acid (3)
To a solution of compound 2 (5 g, 23.6 mmol) in methanol
(50 ml), hydroxylamine hydrochloride (5.736 g, 82.5 mmol)
and sodium bicarbonate (6.935 g, 82.5 mmol) were added,
and the reaction mixture was refluxed for 15 h. Excess
methanol was distilled off, and the reaction mass was dis-
solved in 10 % sodium bicarbonate solution (200 ml) and
filtered. Filtrate was acidified with concentrated hydrochloric
acid till pH 2; the solid obtained was filtered off and washed
with 5 % hot ethyl acetate in Pet. Ether gave off white solid
(2.29 g, 43 %). m.p. 189–192 °C; IR (KBr) cmax: 3435(OH),
Table 1 IC50 value of each compound in lM concentration
Cell lines
IC50 in lM
Compound
name
Melan- WM3211b WM278b UACC903c 1205Luc
Aa
4a
4b
4c
4d
4e
4f
4g
4h
a
[50
[50
[50
[50
[50
[50
[50
[50
24.31
34.89
[50
[50
[50
25.51
[50
[50
[50
[50
44.87
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
[50
;
1732 (C=O) cm-1 1H NMR (300 MHz, DMSO-d6):
[50
d = 4.17 (2H, s, CH2), 7.75–7.80 (3H, m, ArH), 7.86 (1H, d,
J = 8.72 Hz, ArH), 8.14–8.17 (1H, m, ArH), 8.36 (1H, d,
J = 3.28 Hz, ArH), 12.92 (1H, s, COOH); 13C NMR
(400 MHz, DMSO-d6): d = 31.27 (CH2), 117.52 (C-12),
118.78 (C-10), 119.26 (C-6), 121.53 (C-4), 125.18 (C-11),
128.07 (C-7), 128.93 (C-8), 129.08 (C-9), 133.92 (C-13),
154.79 (C-3), 160.92 (C-5), 170.49 ([C=O); ESI/MS m/z
227.8 [M?1]?.
95.05
91.05
[50
80.40
Mouse melanocyte (normal cells)
Early stage melanoma cell lines
Metastatic melanoma cell lines
b
c
Table 2 Anticonvulsant
activity of compounds by MES
method in Wistar rats
Phases (s)
Comp. no.
Flexion
Extension
Clonus
No. of jerks
Stupor phase
4a
1.86 0.08
9.16 0.34
0.51 0.07
1.02 0.07
2.0 0.05
6.91 0.09
3.0 0.06
0.41 0.07
2.98 0.09
0.4 0.15
10.41 0.33
1.91 0.06
10.58 0.14
10.05 0.15
11.03 0.14
0.33 0.09
3.18 0.12
2.96 0.08
13.45 0.18
0.38 0.17
10.3 0.32
30.25 0.6
8.93 0.2
15 0.57
7.33 0.39
17.53 0.43
16.51 0.47
15.11 0.26
31.58 0.26
13.11 0.32
61.35 2.73
4.63 0.32
28.46 1.18
2.76 0.27
4b
91.33 0.36
4c
7
0.36
4d
15.28 0.24
33.3 0.21
34.63 0.42
21.98 0.26
3.9 0.2
25.33 0.76
65.5 1.61
84.5 1.14
42.66 0.88
5.83 0.94
18 0.57
4e
4f
4g
4h
DMSO
Phenytoin
22.98 0.55
3.26 0.18
1.83 0.3
123

Med Chem Res
General procedure for compounds (4a–h)
N-(4-Bromophenyl)-2-(naphtho[2,1-d]isoxazole-3-yl)acet-
amide (4c) This compound was obtained as brownish
white solid (0.19 g, 22 %). m.p. 267–269 °C; IR (KBr)
c
To a solution of 3 (0.5 g, 0.2 mmol) in ethyl acetate
(50 ml), DMAP (0.050 g, 10 %), primary amine/secondary
amine (0.22 mmol), and DCC (0.546 g, 0.26 mol) were
added, and the reaction mixture was allowed to stir at room
temperature for 12 h. Excess solvent was evaporated under
reduced pressure to give solid which was treated with
20 ml saturated sodium bicarbonate solution and then with
20 ml 10 % conc. HCl (20 ml brine solution in case of
secondary amine only). Product was purified by recrystal-
lization with ethanol.
1
max: 3345 (NH), 1684 (C=O) cm-1; H NMR (400 MHz,
DMSO-d6): d = 4.19 (2H, s, CH2), 7.38 (2H, d, J = 8.
76 Hz, ArH), 7.58 (2H, d, J = 8.8 Hz, ArH), 7.68–7.73
(3H, m, ArH), 7.82 (1H, d, J = 8.68 Hz, ArH), 7.98–8.04
(1H, m, ArH), 8.34–8.41 (1H, m, ArH), 10.34 (1H, s, NH);
13C NMR (400 MHz, DMSO-d6): d = 33.09 (CH2), 115.
08 (naphthalene carbon), 117.05 (naphthalene carbon),
118.27 (naphthalene carbon), 118.84 (naphthalene carbon),
120.99 (C-20 and C-60), 121.11 (C-40), 124.62 (naphthalene
carbon), 127.51 (naphthalene carbon), 128.38 (naphthalene
carbon), 128.52 (naphthalene carbon), 131.54 (C-30 and
C-50), 133.4 (C-10), 138.12 (naphthalene carbon), 154.67
(C-3), 160.43 (naphthalene carbon), 166.08 ([C=O);
HPLC Purity 99.73 %; Ele. Ana. calcd. (%) for
C19H13BrN2O2: C, 59.59; H, 3.53; N, 7.63. Found: C, 59.
86; H, 3.44; N, 7.35.
2-(Naphtho[2,1-d]isoxazole-3-yl)-N-p-tolylacetamide (4a)
This compound was obtained as off light brown solid (0.
4 g, 57 %). m.p. 257–259 °C; IR (KBr) cmax: 3291 (NH),
1656 (C=O) cm-1; 1H NMR (300 MHz, CDCl3): d = 2.29
(3H, s, CH3), 4.19 (2H, s, CH2), 7.11 (2H, d, J = 8.26 Hz,
ArH), 7.37 (2H, d, J = 8.38 Hz, ArH), 7.67–7.76 (4H, m,
ArH), 7.98–8.05 (2H, m, ArH and NH), 8.43 (1H, d, J = 2.
00 Hz, ArH); 13C NMR 400 MHz (DMSO-d6): d = 29.73
(CH3), 34.74 (CH2), 117.64 (naphthalene carbon), 119.20
(naphthalene carbon), 120.15 (naphthalene carbon), 121.76
(naphthalene carbon, C-20 and C-60), 125.47 (naphthalene
carbon), 127.41 (naphthalene carbon), 128.51 (naphthalene
carbon), 128.54 (naphthalene carbon), 129.50 (C-30 and
C-50), 134.18 (C-10), 134.46 (C-40), 134.79 (naphthalene
carbon and C-3), 164.87 ([C=O); ESI/MS m/z 316.6 [M]?
and 317.7 [M?1]?; HPLC Purity 100 %; Ele. Ana. calcd.
(%) for C20H16N2O2: C, 75.32; H, 5.26; N, 8.40. Found: C,
75.53; H, 5.10; N, 8.66.
N-(2-Hydroxyphenyl)-2-(naphtho[2,1-d]isoxazole-3-yl)acet-
amide (4d) This compound was obtained as brown solid
(0.33 g, 47 %). m.p. 208–210 °C; IR (KBr) cmax: 3323
1
(OH) 1763 (C=O) cm-1; H NMR (400 MHz, DMSO-d6):
d = 4.36 (2H, s, CH2), 6.77 (1H, t, J = 6.8, 1.2 Hz, ArH),
6.89–6.98 (2H, m, ArH), 7.75–7.89 (3H, m, ArH), 8.14–8.
17 (1H, m, ArH), 8.39 (1H, d, J = 4.00, 2.00 Hz, ArH), 9.
77 (1H, s, NH), 9.89 (1H, s, OH); 13C NMR 400 MHz
(DMSO-d6): d = 47.99 (CH2), 115.80 (naphthalene car-
bon), 117.62 (Naphthalene carbon), 118.82 (naphthalene
carbon), 119.34 (C-30), 119.46 (C-50), 121.57 (C-20), 122.
85 (C-60), 125.10 (naphthalene carbon), 125.28 (naphtha-
lene carbon), 126.38 (naphthalene carbon), 128.06 (naph-
thalene carbon), 128.93 (naphthalene carbon), 129.09
(naphthalene carbon), 133.95 (C-10), 148.44 (C-3), 155.57
(C-40), 160.92 (naphthalene carbon), 166.66 ([C=O);
HPLC Purity 96.74 %.
2-(Naphtho[2,1-d]isoxazole-3-yl)-N-phenylacetamide (4b)
This compound was obtained as off light brown solid (0.
514 g, 77 %). m.p. 250–252 °C; IR (KBr) cmax: 3289
(NH), 1657 (C=O) cm-1; 1H NMR (400 MHz, DMSO-d6):
d = 4.19 (2H, s, CH2), 7.06 (1H, t, J = 7.32, 7.44 Hz,
ArH), 7.28 (2H, t, J = 7.92, 7.72 Hz, ArH), 7.62–7.73 (5H,
m, ArH), 7.84 (1H, d, J = 8.72 Hz, ArH), 8.01 (1H, d,
J = 5.32, ArH), 8.37 (1H, d, J = 5.28, 4.12, ArH), 10.20
(1H, s, NH); 13C NMR 400 MHz (DMSO-d6): d = 33.59
(CH2), 117.58 (naphthalene carbon), 118.78 (naphthalene
carbon), 119.34 (naphthalene carbon), 119.74 (naphthalene
carbon), 121.54 (C-20 and C-60), 124.13 (naphthalene car-
bon), 125.21 (naphthalene carbon), 128.11 (C-40), 128.97
(C-30 and C-50), 129.07 (naphthalene carbon), 129.31
(naphthalene carbon), 133.93 (naphthalene carbon), 139.22
(C-10), 155.37 (C-3), 160.95 (naphthalene carbon), 166.47
([C=O); ESI/MS m/z 302.7 [M]? and 300.8 [M-2]?;
HPLC Purity 100 %; Ele. Ana. calcd. for C19H14N2O2: C,
75.69; H, 4.73; N, 9.53. Found: C, 75.48; H, 4.67; N, 9.27.
2-(Naphtho[2,1-d]isoxazole-3-yl)-1-(piperidine-1-yl)eth-
anone (4e) This compound was obtained as light brown
solid (0.42 g, 64 %). m.p. 213–215 °C; IR (KBr) cmax
:
3052, 2925, 2851 (CH, CH2), 1689 (C=O) cm-1; 1H NMR
(400 MHz, DMSO-d6): d = 1.25–1.38 (2H, m, CH2), 1.
62–1.67 (2H, m, CH2), 1.75–1.79 (2H, m, CH2), 1.90 (2H,
d, CH2), 1.98 (2H, d, CH2), 4.20 (2H, s, CH2), 7.67–7.74
(4H, m, ArH), 7.88 (1H, d, J = 8.00 Hz, ArH), 8.0 (1H, d,
J = 2.12 Hz, ArH), 8.37 (1H, d, J = 5.8 Hz, ArH); 13C
NMR 400 MHz (DMSO-d6): d = 24.84 (C-30), 25.54 (C-
20), 25.81 (C-40), 31.77 (C-10), 32.15 (C-50), 50.21 (CH2),
117.65 (naphthalene carbon), 118.81 (naphthalene carbon),
119.35 (naphthalene carbon), 121.52 (naphthalene carbon),
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Med Chem Res
125.04 (naphthalene carbon), 128.06 (naphthalene carbon),
128.89 (naphthalene carbon), 129.08 (naphthalene carbon),
133.88 (naphthalene carbon), 155.19 (C-3), 160.83 (naph-
thalene carbon), 165.27 ([C=O); HPLC purity 97.7 %.
carbon), 155.19 (C-3), 160.39 (Naphthalene carbon), 165.
80 ([C=O); HPLC Purity 98.24 %.
Biological evaluation
2-(Naphtho[2,1-d]isoxazole-3-yl)-1-(pyrrolidin-1-yl)etha-
none (4f) This compound was obtained as light brown solid
(0.59 g, 95 %). m.p. 198–200 °C; IR (KBr) cmax: 3053, 2927,
Procedure to assess the effect of the isoxazole derivatives
on melanoma cell survival using the MTS
1
2852 (CH, CH2), 1659 (C=O) cm-1; H NMR (400 MHz,
Ninety-six well plates were plated with 100 ll Media
(DMEM ? 10 % Fetal bovine serum and L-glutamine)
containing , cells/well. Stock of 20 mM solution was pre-
pared for compounds to get a series of concentration
ranging from 50 to 0.625 lM. 100 ll of these compounds
were added to the 96-well plates. These 96-well plates
were incubated at 37 °C in humidified incubator under 5 %
CO2 atmosphere for 24, 48, and 72 h.
DMSO-d6): d = 1.60–1.65 (2H, m, CH2), 1.77–1.81 (2H, m,
CH2), 1.87–1.90 (2H, m, CH2), 1.96–1.98 (2H, m, CH2), 4.18
(2H, s, CH2), 7.68–7.72 (3H, m, ArH), 8.03 (1H, d, J = 2.
52 Hz, ArH), 8.30 (1H, d, J = 7.76 Hz, ArH), 8.34–8.36 (1H,
m, ArH); 13C NMR 400 MHz (DMSO-d6): d = 25.54 (C-20),
25.81 (C-30), 31.77 (C-10), 32.15 (C-50), 50.21 (CH2), 117.65
(naphthalene carbon), 118.81 (naphthalene carbon), 119.33
(naphthalene carbon), 121.52 (naphthalene carbon), 125.04
(naphthalene carbon), 128.06 (naphthalene carbon), 128.89
(naphthalene carbon), 129.08 (naphthalene carbon), 133.88
(naphthalene carbon), 155.19 (C-3), 160.83 (naphthalene
carbon), 165.27 ([C=O); HPLC Purity 97.6 %.
In vitro inhibitory efficacy of cancer cell lines repre-
senting different cancer types following treatment with
compounds was measured using the 3-(4,5-dimethylthia-
zol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-
2H-tetrazolium (MTS) assay (Promega, Madison, WI). In
brief, 5 9 103 cells per well in 100 ll of DMEM con-
taining 10 % FBS were grown in a 96-well plate for 24 h
and treated with either control DMSO vehicle or increasing
concentrations (0.625–50 lM) of these compounds for 24,
48, and 72 h. The proportion of viable cells compared to
control DMSO-treated cells were determined using MTS
assay and IC50 values calculated using GraphPad Prism,
version 4.01 (GraphPad software, San Diego, CA). The
IC50 value for each compound was determined by at least
three independent experiments and represented with a
standard error. IC50 in lM concentration of all compounds
is given in Table 1.
1-Morpholino-2-(naphtho[2,1-d]isoxazole-3-yl)ethanone
(4g) This compound was obtained as light brownish
white solid (0.33 g, 51 %). m.p. [260 °C; IR (KBr) cmax
:
3071, 2927, 2853 (CH, CH2), 1636 (C=O) cm-1; 1H NMR
(400 MHz, DMSO-d6): d = 1.54–0.156 (2H, m, CH2), 1.
66 (2H, d, CH2), 1.82 (2H, m, CH2), 3.94 (2H, s, CH2), 7.
65 (3H, m, ArH), 7.77 (1H, d, J = 9.72 Hz, ArH), 7.95
(1H, m, ArH), 8.33 (1H, m, ArH); 13C NMR 400 MHz
(DMSO-d6): d = 24.93 (C-10), 25.61 (C-60), 32.74 (C-20),
32.79 (C-50), 48.34 (CH2), 117.50 (naphthalene carbon),
118.78 (naphthalene carbon), 119.45 (naphthalene carbon),
121.52 (naphthalene carbon), 125.00 (naphthalene carbon),
128.05 (naphthalene carbon), 128.91 (naphthalene carbon),
129.08 (naphthalene carbon), 133.91 (naphthalene carbon),
155.70 (C-3), 160.84 (naphthalene carbon), 166.36 ([C=
O); HPLC Purity 96.92 %.
Procedure of MES test for anticonvulsant activity
Wistar rats weighing 150–180 g were used for the MES
test. Rats were divided in ten groups with six rats in each
group for synthesized compounds, standard drug, and
control. 15 mg kg-1 ml-1 of synthesized compound in
DMSO was given via IP route to each rat and after 30 min
150 lV AC current for 0.2 s was given for convulsion.
Phenytoin was used as standard drug. Different phases
were observed as given in Table 2.
N,N-Diethyl-2-(naphtho[2,1-d]isoxazole-3-yl)acetamide
(4h) This compound was obtained as light brown solid
(0.36 g, 58 %). m.p. 236–238 °C; IR (KBr) cmax: 3071,
2927, 2853 (CH, CH2), 1637 (C=O) cm-1 1H NMR
;
(400 MHz, DMSO-d6): d = 1.11–1.25 (6H, m, CH3), 1.
63–1.67 (2H, m, CH2), 1.79 (2H, d, CH2), 3.88 (2H, s,
CH2), 7.60–7.65 (3H, m, ArH), 7.93–7.96 (2H, m, ArH), 8.
26, 8.29 (1H, m,0ArH); 13C NMR (400 MHz, DMSO-d6):
Acknowledgments The authors are thankful to The Head,
Department of Chemistry, Faculty of Science, The M. S. University
of Baroda for providing laboratory facility. The authors are also
thankful to Dr. Gavin P. Robertson and Dr. Gajanan S. Inamdar,
Department of Pharmacology, The Pennsylvania State University
College of Medicine, Hershey, Pennsylvania, USA for in vitro
screening of compounds against melanoma cancer cell lines. One of
them, JNS, is thankful to UGC New Delhi (RFSMS) for financial
support.
0
0
d = 24.43 (CH2 ), 25.29 (CH2 ), 32.36 (CH3 ), 33.31
0
(CH3 ), 47.84 (CH2), 117.04 (Naphthalene carbon), 118.35
(Naphthalene carbon), 118.95 (Naphthalene carbon), 121.
04 (Naphthalene carbon), 121.48 (Naphthalene carbon),
127.51 (Naphthalene carbon), 128.36 (Naphthalene car-
bon), 128.55 (Naphthalene carbon), 133.43 (Naphthalene
123

Med Chem Res
imidazo[2,1-b][1,3,4]thiadiazole derivatives. Eur J Med Chem
44:2828
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