Synthesis and antinociceptive activity of pyrazolyl isoxazolines and pyrazolyl isoxazoles

Article abstract of DOI:10.1016/j.bmcl.2009.05.055

Pyrazolyl isoxazolines and isoxazoles were synthesised in moderate to good yields using 1,3-dipolar cycloaddition of pyrazole derived nitrile oxide with various dipolarophiles such as N-substituted maleimide, diethylacetylene dicarboxylate and phenylacety

Full text of DOI:10.1016/j.bmcl.2009.05.055

Bioorganic & Medicinal Chemistry Letters 19 (2009) 3370–3373
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and antinociceptive activity of pyrazolyl isoxazolines and pyrazolyl
isoxazoles
K. Karthikeyan a, T. Veenus Seelan b, K. G. Lalitha b, P. T. Perumal a,
*
a Organic Chemistry Division, Central Leather Research Institute, Adyar, Chennai 600 020, India
b Department of Pharmaceutical Chemistry, Ultra College of Pharmacy, Madurai 625020, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Pyrazolyl isoxazolines and isoxazoles were synthesised in moderate to good yields using 1,3-dipolar
cycloaddition of pyrazole derived nitrile oxide with various dipolarophiles such as N-substituted malei-
mide, diethylacetylene dicarboxylate and phenylacetylene. The synthesized compounds were evaluated
for antinociceptive activities. The 3-pyrazolyl-4,5-dicarbethoxy isoxazoles (9a–c) exhibited the maxi-
mum antinociceptive activity.
Received 4 April 2009
Revised 12 May 2009
Accepted 14 May 2009
Available online 18 May 2009
Ó 2009 Elsevier Ltd. All rights reserved.
Keywords:
1,3-Dipolar cycloaddition
Pyrazolyl isoxazoline
Pyrazolyl isoxazole
Antinociceptive activity
1,3-Dipolar cycloaddition is a versatile synthetic strategy for the
construction of five-membered heterocycles.1 The 1,3-dipolar
cycloaddition of nitrile oxides to alkenes and alkynes affords isox-
azolines and isoxazoles respectively.2,3 Isoxazolines (4,5-dihyd-
roisoxazoles) are versatile intermediates in the synthesis of
various natural products.4 The reductive cleavage of isoxazoline
ring can lead to many synthetically important compounds, such
to treat both acute and chronic pain with limited effects is one of
the prominent goals in biomedical research.
Isoxazolines exhibit important biological activities such as
antibacterial,9 antiplatelet,10 antiviral,11 anticonvulsant,12 immu-
nostimulatory13 and antinociceptive.14,15 Pyrazole nucleus has pro-
nounced pharmacological applications as anti-anxiety,16,17
antipyretic, antinociceptive18 and anti-inflammatory drugs.19–21
Certain alkyl pyrazoles show significant bacteriostatic, bactericidal
as b-hydroxy ketones,
a,b-unsaturated ketones or c-amino alco-
hols.5 The general and most widely used method for the synthesis
of isoxazolines is the 1,3-dipolar cycloaddition of nitrile oxides to
activated double bond of alkenes; aliphatic nitrile oxides are pre-
dominantly generated in situ from primary nitro compounds in a
Mukaiyama reaction,6 while their aromatic counterparts are pre-
pared by dehydrohalogenation of hydroximoyl chlorides.7 In most
cases, 1,3-dipolar cycloadditions of nitrile oxides to alkenes pro-
ceed with high regioselectivity and the relative configuration on
the 4- and 5-carbon atoms of the isoxazoline ring depends on the
geometry of the alkene.
Pain is widely accepted to be one of the most important deter-
minations of quality of life. A study reported by the World Health
Organization demonstrated that individuals who live with persis-
tent pain suffer fourfold more from depression (or) anxiety com-
pared to healthy subjects.8 The identification of compounds able
NH2OH.HCl
NaHCO3
N
N
N
N
Ethanol: H2O
CHO
7 : 3
rt
N
OH
R1
H
R1
1a-e
2a-e
rt
NCS, CH2Cl2
N
N
N
OH
Cl
3a-e
R1
* Corresponding author. Tel.: +91 44 24913289; fax: +91 44 24911589.
E-mail addresses: ptperumal@gmail.com, sibi.gb@gmail.com (P.T. Perumal).
Scheme 1. Preparation of pyrazolyl hydroximoyl chlorides.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.05.055

K. Karthikeyan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3370–3373
3371
Table 2
Synthesis of isoxazoles
O
N
OH
N
N
R2
Entry
R1
Alkyne
Producta
Yieldb (%)
N
R2
O
N
Cl
O
1
2
3
4
5
6
7
Br
OMe
Cl
Br
OMe
Cl
6
6
6
9a
9b
9c
10a
10b
10c
10d
81
78
76
80
81
76
78
5a-c
O
N
N
5
O
1
4
3
3a-e
N
7a R3 = Ph
7a R3 = Ph
7a R3 = Ph
7b R3 = (CH2)3–CH3)
2
R1
rt
8a-i
Et3N, CH2Cl2
COOEt
COOEt
Cl
R1
a
All the reactions took place in 30 min.
Isolated yield after column chromatography.
EtOOC
COOEt
6
b
+
-
N
N
N
N
N
O
O
N
9a-c
4a-e
triethylamine facilitated the in situ formation of nitrile oxides.
Addition of N-substituted maleimide 5a–c as the dipolarophile to
the reaction mixture resulted in the formation of isoxazolines
8a–i (Scheme 2). The mixture was stirred at room temperature
for 15–30 min for completion. (Table 1) The generality of this reac-
tion was demonstrated by employing dipolarophiles of diverse
structures (Table 2). The structure of the compounds was assigned
based on the NMR and mass spectral data.25
In the present study, two different experimental models tested
the potential antinociceptive activity of the synthesized com-
pounds.26 In the Tail Flick test the acute thermal stimulus was ap-
plied and the abdominal constriction test, in which a painful
chemical stimulus (acetic acid) was used.
R1
R1
R3
7
R3
N
O
N
N
10a-d
R1
Scheme 2. Synthesis of Isoxazolines and Isoxazoles.
The oral administration 15 mg/kg po and 30 mg/kg po of syn-
thetic compounds induced antinociception in rat Tail Flick test.
The effect appeared 15 min after administration, peaked after
60 min and then slowly diminished (Table 3). Antinociceptive effi-
cacy of the synthesized compounds was comparable to that of
standard drug pentazocine (5 mg/kg). All compounds at a dose of
15 mg/kg po and 30 mg/kg po exhibited the antinociceptive activ-
ity in a dose dependent manner. Among the 16 compounds, com-
pounds 9a–c showed the maximum antinociceptive effect at
30 mg/kg po which was found to be comparable with that of the
standard drug. The compounds 8a, 8c, 8d, 8e, 8f, 8g, 8h, 10c, and
10d showed moderate antinociceptive effect at 30 mg/kg po. Final-
ly the compounds 8b, 8i, 10a, 10b have minimal antinociceptive ef-
fect at a dose of 30 mg/kg po.
In acetic acid induced abdominal constriction assay all the syn-
thesized compounds were able to reduce the number of abdominal
constrictions and showed a potency comparable to that of refer-
ence standards (aspirin). In particular 9a, 9b and 9c were able to
exhibit a good antinociceptive activity reducing by more than
50% the number of abdominal constrictions with respect to con-
trols (Table 4). Antinociceptive effect induced by the above men-
tioned compounds was comparable to that shown by standard
drug aspirin. Compounds 8a, 8c, 8e, 8f, 8i, 10c, and 10d reduced
by around 50% the number of abdominal constrictions and finally
compounds 8b, 8d, 8g, 8h, 10a, and 10b had the least protection
in the abdominal constriction test.
For Structure–Activity Relationship (SARs), the data obtained
in both the methods clearly suggest that the activity of the ser-
ies does not depend on the diphenyl pyrazolyl fused system.
When the diphenyl pyrazolyl fused system is linked with the
isoxazole 4,5-dicarboxylicacid diethyl ester, good results were
obtained with different functional groups at position 4 of the
phenyl ring.
In summary, we have reported the synthesis of novel pyrazolyl
isoxazolines and isoxazoles. Based on the inherent biological activ-
ity of pyrazole and isoxazole units we identified a group of antin-
ociceptive agents that are active in the Tail Flick test and acetic
acid induced abdominal constriction assay method with an efficacy
comparable to that of standard drugs.
and fungicidal activities.22 In continuation of our work on the
biological activities of pyrazole derivatives23 and products from
1,3-dipolar cycloaddition reactions,24 we herein report the synthe-
sis and screening results of the antinociceptive activities of pyraz-
olyl isoxazoles and pyrazolyl isoxazolines.
We have investigated the 1,3-dipolar cycloaddition of pyrazole
derived nitrile oxides with both alkenes and alkynes. Pyrazolyl
nitrile oxides were prepared from pyrazolyl oxime, N-chloro-
succinimide and triethylamine in dichloromethane at room
temperature.
Treatment of pyrazolyl oximes 2a–e with an equimolar
amount of N-chlorosuccinimide (NCS) in dichloromethane at
room temperature gave the corresponding hydroximoyl chlorides
3a–e. The reaction occured instantaneously upon mixing the sub-
strates as indicated by the appearance of yellow colour in the
reaction mixture. Completion of the reaction was finally assessed
by TLC. The generality of this reaction was demonstrated by the
synthesis of hydroximoyl chlorides with different functionalities
(Scheme 1).
Reaction of pyrazolyl oximes 2a–e with N-chlorosuccinimide in
dichloromethane at room temperature and subsequent addition of
Table 1
Synthesis of isoxazolines
Entry
R1
R2
Producta
Yieldb (%)
1
2
3
4
5
6
7
8
9
H
H
Ph
Me
Ph
Me
Ph
Me
Ph
Me
8a
8b
8c
8d
8e
8f
8g
8h
8i
85
82
78
79
82
81
76
80
77
Br
Br
OEt
OEt
Cl
Cl
Cl
p-Me-Ph
a
All the reactions took place in 30 min.
Isolated yield after column chromatography.
b

3372
K. Karthikeyan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3370–3373
Table 3
Antinociceptive activity of synthetic compounds on tail Flick method in rats
Groups
Treatments
Dose mg/kg
Tail Flick latency in seconds (mean SEM)
At time
0 min
30 min
60 min
120 min
180 min
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Control (saline)
Pentazocine
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
9c
10a
10b
10c
10d
1 ml
5 mg
3.33 0.21
3.80 0.19a
3.33 0.21a
3.16 0.16a
3.16 0.16a
3.33 0.21a
3.16 0.16a
3.33 0.21a
3.16 0.16a
3.16 0.16a
3.16 0.16a
3.33 0.21a
3.33 0.21a
3.16 0.16a
3.33 0.21a
3.16 0.21a
3.33 0.16a
3.16 0.16a
3.33 0.21
9.16 0.50a
3.33 0.57a
3.33 0.21a
3.16 0.16a
3.33 0.21a
3.16 0.16a
3.83 0.16a
3.33 0.16a
3.33 0.21a
3.33 0.21a
3.33 0.21a
3.83 0.16a
3.83 0.16a
3.33 0.21a
3.33 0.16a
3.83 0.21a
3.33 0.28a
3.16 0.16
9.33 0.30a
6.83 0.16a
5.16 0.16a
7.00 0.00a
7.16 0.16a
6.83 0.16a
6.16 0.16a
6.16 0.16a
6.66 0.21a
5.33 0.21a
8.16 0.16a
7.83 0.16a
8.66 0.21a
5.83 0.16a
6.00 0.00a
6.83 0.21a
6.83 0.16a
3.33 0.21
9.30 0.50a
6.66 0.21a
5.00 0.00a
6.83 0.21a
7.00 0.00a
6.16 0.21a
6.00 0.00a
6.00 0.00a
6.50 0.22a
5.16 0.16a
8.00 0.00a
7.66 0.21a
8.33 0.21a
5.66 0.21a
5.83 0.16a
6.60 0.90a
6.60 0.21a
3.16 0.16
8.00 0.90a
5.66 0.21a
4.83 0.16a
6.50 0.22a
6.50 0.22a
5.83 0.21a
5.83 0.16a
5.50 0.22a
5.50 0.22a
4.50 0.22a
7.50 0.22a
7.00 0.00a
7.50 0.22a
5.16 0.16a
5.33 0.21a
6.16 0.33a
6.16 0.33a
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
N = 6.
Data were analysed by one way ANOVA followed by Dunnett’s test.
Values are expressed as mean SEM.
a
P < 0.01 versus control.
Table 4
Antinociceptive activity of synthesized compounds on acetic acid induced abdominal constriction in MICE
Groups
Treatments
Dose (mg/kg)
No. of abdominal constriction (per 10 min)
% Production
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
Control (saline)
Aspirin
8a
8b
8c
8d
8e
8f
8g
8h
8i
9a
9b
5 mg/kg
100
42.50 0.428
16.00 0.365a
22.33 0.422a
29.50 0.428a
21.66 0.258a
25.33 0.223a
21.33 0.333a
22.33 0.760a
24.16 0.577a
32.66 0.333a
24.33 0.333a
18.83 0.477a
18.00 0.447a
19.00 0.421a
29.16 0.307a
29.33 0.210a
21.66 0.330a
22.33 0.341a
—
62.35
47.39
30.58
49.04
40.40
49.51
47.46
43.15
23.15
42.75
56.87
57.65
55.29
31.39
30.99
49.04
47.09
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
30 mg
9c
10a
10b
10c
10d
N = 6.
Data were analysed by one way ANOVA followed by Dunnett’s test.
Values are expressed as mean SEM.
a
P < 0.01 versus control.
2. (a) Caramella, P.; Grunanger, P.. In 1,3-Dipolar Cycloaddition Chemistry; Padwa,
A., Ed.; Wiley: New York, NY, 1984; Vol. 1, pp 291–392. Chapter 3; (b) Kudyba,
I.; Jozwik, J.; Romanski, J.; Raczko, J.; Jurczak, J. Tetrahedron: Asymmetry 2005,
16, 2257.
3. (a) Torssell, K. B. G.. Nitrile Oxides. In Nitrones and Nitronates in Organic
Synthesis; VCH: Weinheim, 1988; (b) Grunanger, P.; Vita-Finzi, P.. Isoxazoles—
Part 1. In Chemistry of Heterocyclic Compounds; Wiley: New York, NY, 1991;
Vol. 49.
Acknowledgement
One of the authors K.K.K. thanks the Council of Scientific and
Industrial Research, New Delhi, India for the research fellowship.
Supplementary data
4. (a) Kozikowski, A. P.; Chen, Y. Y.; Wang, B. C.; Xu, Z. B. Tetrahedron 1984, 40,
2345; (b) Martin, S. F.; Colapret, J. A.; Dappen, M. S.; Dupre, B.; Murphy, C. J. J.
Org. Chem. 1989, 54, 2209; (c) Smith, A. L.; Pitsinos, E. N.; Hwang, C. K.; Mizuno,
Y.; Saimoto, H.; Scarlato, G. R.; Suzuki, T.; Nicolaou, K. C. J. Am. Chem. Soc. 1993,
115, 7612; (d) Bode, J. W.; Carreira, E. M. J. Org. Chem. 2001, 66, 6410; (e) Kim,
D.; Lee, J.; Shim, P. J.; Lim, J. I.; Jo, H.; Kim, S. J. Org. Chem. 2002, 67, 764.
5. (a) Padwa, A. Angew. Chem. 1976, 88, 131; (b) Huisgen, R. Angew. Chem. 1963,
75, 604.
6. Mukaiyama, T.; Hoshino, T. J. Am. Chem. Soc. 1960, 82, 5339.
7. (a) Huisgen, R.; Mack, W. Tetrahedron Lett. 1961, 2, 583; (b) Yamamoto, H.;
Wantanbe, S.; Kadotani, K.; Hasegawa, M.; Noguchi, M.; Kanemasa, S.
Tetrahedron Lett. 2000, 41, 3131; (c) Kumar, V.; Kaushik, M. P. Tetrahedron
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmcl.2009.05.055.
References and notes
1. (a) Jiang, H.; Zhao, J.; Han, X.; Zhu, S. Tetrahedron 2006, 62, 11008; (b) Coutouli-
Argyropoulou, E.; Lianis, P.; Mitakou, M.; Giannoulis, A.; Nowak, J. Tetrahedron
2006, 62, 1494; (c) Gomes, P. J. S.; Nunes, C. M.; Pais, A. A. C. C.; Pinho e Melo, T.
M. V. D.; Arnaut, L. G. Tetrahedron Lett. 2006, 47, 5475.

K. Karthikeyan et al. / Bioorg. Med. Chem. Lett. 19 (2009) 3370–3373
3373
Lett. 2006, 47, 1457; (d) Shih, M.-H. Tetrahedron 2002, 58, 10437; (e) Dondoni,
A.; Giovannini, P. P. Synthesis 2002, 1701; (f) Parhi, A. K.; Franck, R. W. Org. Lett.
2004, 6, 3063; (g) Shing, T. K. M.; Wong, W. F.; Cheng, H. M.; Kwok, W. S.; So, K.
H. Org. Lett. 2007, 9, 753; (h) Jiang, H.; Yue, W.; Xiao, H.; Zhu, S. Tetrahedron
2007, 63, 2315; (i) Mishiky, N.; Asaad, F. M.; Ibrahim, Y. A.; Girgis, A. S. J. Chem.
Res. 1997, 438; (j) Nair, V.; Radhakrishnan, K. V.; Sheela, K. C.; Rath, N. P.
Tetrahedron 1999, 55, 14199; (k) Kumar, R. R.; Perumal, S. Tetrahedron 2007, 63,
12220.
(50% ethyl acetate/petroleum ether); IR (cmÀ1): 3449, 3060, 1727, 1596, 1498,
1372, 1189; 1H NMR (500 MHz, DMSO-d6): d 5.19 (d, 1H, J = 9.9 Hz), 5.68 (d,
1H, J = 9.2 Hz), 7.25 (d, 2H, J = 7.7 Hz), 7.37–7.41 (m, 2H), 7.46 (t, 2H, J = 8.4 Hz),
7.55 (t, 2H, J = 7.7 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.73 (d, 2H, J = 8.5 Hz), 7.87 (d,
2H, J = 8.4 Hz), 9.11 (s, 1H); 13C NMR (125 MHz, DMSO-d6): d 57.6, 80.5, 109.5,
119.3, 122.6, 127.6, 127.8, 129.3, 129.5, 130.3, 131.4, 131.5, 131.9, 132.1, 132.7,
139.2, 147.5, 150.4, 171.4, 172.6; MS m/z = 513 M++1, 515 M++3; Anal. Calcd for
C26H17BrN4O3 (512.05): C, 60.83; H, 3.34; N, 10.91. Found: C, 60.72; H, 3.31; N,
10.99.
3-[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-isoxazole-4,5-dicarboxylic acid
diethyl ester (9a): Pale green solid; mp 98–100 °C; Rf: 0.55 (30% ethyl acetate/
petroleum ether); IR (cmÀ1): 3429, 3062, 1723, 1670, 1514, 1361, 1181; 1H
NMR (500 MHz, CDCl3): d 1.14 (t, 3H, J = 6.9 Hz), 1.41 (t, 3H, J = 6.9 Hz), 3.97 (q,
2H, J = 6.9 Hz), 4.46 (q, 2H, J = 6.9 Hz), 7.34 (t, 1H, J = 7.7 Hz), 7.47–7.51 (m, 6H),
7.77 (d, 2H, J = 7.7 Hz), 8.34 (s, 1H); 13C NMR (125 MHz, CDCl3): d 13.7, 14.0,
62.1, 63.0, 107.6, 115.9, 119.4, 122.8, 127.4, 129.6, 129.7, 130.5, 131.2, 131.9,
139.4, 150.7, 155.5, 156.1, 160.3, 160.4; MS m/z = 510 M++1, 512 M++3; Anal.
Calcd for C24H20BrN3O5 (509.06): C, 56.48; H, 3.95; N, 8.23. Found: C, 56.56; H,
3.97; N, 8.41.
8. Gureje, O.; Von Knoff, M.; Simon, G. E.; Gater, R. J. Am. Med. Assoc. 1998, 280,
147.
9. Kang, Y. K.; Shin, K. J.; Yoo, K. H.; Seo, K. J.; Hong, C. Y.; Lee, C.; Park, S. Y.; Kim,
D. J.; Park, S. W. Bioorg. Med. Chem. Lett. 2000, 10, 95.
10. Xue, C.; Roderick, J.; Mousa, S.; Olson, R. E.; DeGrado, W. F. Bioorg. Med. Chem.
Lett. 1998, 8, 3499.
11. Diana, G. D.; McKinlay, M. A.; Brisson, C. J.; Zalay, E. S.; Miralles, J. V.; Salvador,
U. J. J. Med. Chem. 1985, 28, 748.
12. Lepage, F.; Tombert, F.; Cuvier, G.; Marivain, A.; Gillardin, J. M. Eur. J. Med.
Chem. 1992, 27, 581.
13. Ryng, S.; Machon, Z.; Wieczorek, Z.; Zimecki, M.; Mokrosz, M. Eur. J. Med. Chem.
1998, 33, 831.
3-[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenylisoxazole
(10a):
14. Badio, B.; Garraffo, H. M.; Plummer, C. V.; padgett, W. L.; Daly, J. W. Eur. J.
Pharmacol. 1997, 26, 89.
15. Ivy Carroll, F. Bioorg. Med. Chem. Lett. 2004, 14, 1889.
Colourless solid; mp 140–142 °C; Rf: 0.61 (30% ethyl acetate/petroleum
ether); IR (cmÀ1): 3430, 3055, 1585, 1501, 1456, 1364, 1065; 1H NMR
(500 MHz, CDCl3): d 6.40 (s, 1H), 7.34 (t, 1H, J = 6.9 Hz), 7.43–7.51 (m, 5H),
7.57 (d, 2H, J = 8.4 Hz), 7.64 (d, 2H, J = 8.4 Hz), 7.75 (dd, 2H, J = 7.6, 1.5 Hz), 7.79
(d, 2H, J = 7.7 Hz), 8.38 (s, 1H); 13C NMR (125 MHz, CDCl3): d 99.2, 110.7, 119.3,
122.9, 125.9, 127.3, 127.9, 129.0, 129.6, 130.3, 130.4, 131.5, 131.7, 139.5, 150.4,
156.5, 170.1; MS m/z = 442 M++1, 444 M++3; Anal. Calcd for C24H16BrN3O
(441.05): C, 65.17; H, 3.65; N, 9.50. Found: C, 65.21; H, 3.66; N, 9.55.
26. Materials and methods for the antinociceptive activity: Male albino rats
(wistar strain, 150–200 g, Tail Flick method) and Swiss albino mice (25–30 g,
Acetic acid induced constriction assay), were used as experimental models. The
animals were given food and water ad libitum. The animals were housed under
standard conditions of 12 h light and 12 h dark cycle at ambient temperature
(35–36 °C). The experiments were carried out during light cycle. The animal
care and experimental protocols were in accordance with International Animal
Ethical Committee (IAEC), and the experimental proposal number is UCP/IAEC/
2008/036.
16. Haufel, J.; Breitmaier, E. Angew. Chem. 1974, 13, 604.
17. Wustrow, D. J.; Capiris, T.; Rubin, R.; Knobelsdorf, J. A.; Akunne, H.; Davis, M.
D.; MacKenzie, R.; Pugsley, T. A.; Zoski, K. T.; Heffner, T. G.; Wise, L. D. Bioorg.
Med. Chem. Lett. 1998, 8, 2067.
18. (a) Souza, F. R.; Fighera, M. R.; Lima, T. T. F.; Bastiani, J.; Barcellos, I. B.; Almeida,
C. E.; Oliveira, M. R.; Bonacorso, H. G.; Flores, A. E.; Mello, C. F. Pharm. Biochem.
Behav. 2001, 68, 525; (b) Godoy, M. C. M.; Fighera, M. R.; Souza, F. R.; Flores, A.
E.; Rubin, M. A.; Oliveira, M. R.; Zanatta, N.; Martins, M. A. P.; Bonacorso, H. G.;
Mello, C. F. Eur. J. Pharmacol. 2004, 496, 93; (c) Souza, F. R.; Ratzlaff, V. T.;
Borges, L. P.; Oliveira, M. R.; Bonacorso, H. G.; Zanatta, N.; Martins, M. A. P.;
Mello, C. F. Eur. J. Pharmacol. 2002, 451, 141; (d) Tomazetti, J.; Ávila, D. S.;
Ferreira, A. P. O.; Martins, J. S.; Souza, F. R.; Royer, C.; Rubin, M. A.; Oliveira, M.
R.; Bonacorso, H. G.; Martins, M. A. P.; Zanatta, N.; Mello, C. F. J. Neurosci. Meth.
2005, 147, 29.
19. Eid, A. I.; Kira, M. A.; Fahmy, H. H. J. Pharm. Belg. 1978, 33, 303.
20. Menozzi, G.; Mosti, L.; Fossa, P.; Mattioli, F.; Ghia, M. J. Heterocycl. Chem. 1997,
34, 963.
21. Penning, T. D.; Talley, J. J.; Bertenshaw, S. R.; Carter, J. S.; Collins, P. W.; Docter,
S.; Graneto, M. J.; Lee, L. F.; Malecha, J. W.; Miyashiro, J. M.; Rogers, R. S.; Rogier,
D. J.; Yu, S. S.; Anderson, G. D.; Burton, E. G.; Cogburn, J. N.; Gregory, S. A.;
Koboldt, C. M.; Perkins, W. E.; Seibert, K.; Veenhuizen, A. W.; Zhang, Y. Y.; Isak-
son, P. C. J. Med. Chem. 1997, 40, 1347.
22. (a) Rich, S.; Horsfall, J. G. Phytopathology 1952, 42, 457; (b) Potts, K. T.. In
Comprehensive heterocyclic Chemistry; Pergamon: Oxford, 1986; Vol. 5,. Part 4A.
23. (a) Sridhar, R.; Perumal, P. T.; Etti, S.; Shanmugam, G.; Ponnusamy, M. N.;
Prabavathy, V. R.; Mathivanan, N. Bioorg. Med. Chem. Lett. 2004, 14, 6035; (b)
Sivaprasad, G.; Perumal, P. T.; Prabavathy, V. R.; Mathivanan, N. Bioorg. Med.
Chem. Lett. 2006, 16, 6302.
Tail Flick test: Tail Flick latency was assessed by analgesiometer.27,28 In this
method the animals (rats) were selected by preliminary screening. Those in
variation of more than one second between two reaction times at 15 min
interval (or) more than three seconds from the group mean were discarded. A
cut-off reaction time fixed at 10 s was maintained to avoid damage. The
reaction time of animals on the Tail Flick apparatus was recorded at 30, 60,
120, and 180 min after the administration of the drug. The rats were divided in
to 34 groups, each group consisting of 6-animals. The test drugs administered
at a dose of 15 mg/kg po and 30 mg/kg po and standard drug pentazocine a
dose of 5 mg/kg ip and aspirin 100 mg/kg po.
Abdominal constriction test: Mice were injected ip with a 0.6% solution of acetic
acid (10 ml/kg), according to the procedure of modified Seigmund
technique.29,30 The number of stretching movements was counted for
10 min, starting 5 min after acetic acid injection.
24. Karthikeyan, K.; Perumal, P. T.; Etti, S.; Shanmugam, G. Tetrahedron 2007, 63,
10581.
Statistical analysis: All experimental results are given as the mean S.E.M. The
data were statistically analyzed by one-way ANOVA followed by Dunnet’s test.
P value of less than 0.01 were considered significant.
Drugs: The following drugs were used Aspirin (Apex, India), Pentazocine
(Ranbaxy, India). Other chemicals were of highest quality commercials
available. All drugs were dissolved in isotonic (NaCl 0.9%) saline (or)
dispersed in sodium carboxy methyl cellulose 1%. Drug concentrations were
prepared in such a way that the necessary dose could be administered in a
volume of 0.5 ml/dose for mice and 1.0 ml/dose for rats.
25. A mixture of pyrazolyl oxime (0.5 mmol), N-chlorosuccinimide (0.5 mmol) in
dichloromethane was stirred at room temperature for 5 min. After formation of
hydroximoyl chlorides indicated by TLC, N-substituted maleimide (0.6 mmol)
or Phenyl acetylene (2 mmol) or diethyl acetylene dicarboxylate (2 mmol) and
Et3N (0.75 mmol) was added to the reaction mixture. The reaction mixture was
stirred at the same temperature for additional 30 min. The reaction mixture
was diluted by addition of water (20 mL) and extracted with CH2Cl2
(2 Â 20 mL). The combined organic layers were dried over anhydrous
Na2SO4, concentrated in vacuum. The crude was purified by column
chromatography on silica gel (Merck, 100–200 mesh, ethyl acetate–
petroleum ether (20:80) to afford pure isoxazolidine and ethyl acetate–
petroleum ether (8:92) to afford pure isoxazoles.
27. Kulkarni, S. K., 2nd ed. In Hand Book of Experimental Pharmacology; Vallabh
Prakashan: Delhi, 1993; Vol. 43.
28. Turner, R. A. In Screening Methods in Pharmacology; Academic Press: New York
and London, 1965; Vol. 112,.
29. Seigmund, E.; Cadmus, R.; Lu, G. Proc. Soc. Exp. Biol. Med. 1957, 95,
729.
3-[3-(4-Bromophenyl)-1-phenyl-1H-pyrazol-4-yl)-5-phenyl-3a,6a-dihydro-
pyrrolo[3,4-d]isoxazole-4,6-dione (8c): Colourless solid; mp 224–226 °C; Rf: 0.50
30. Koster, R.; Anderson, N.; De Beer, E. J. Fed. Proc. 1959, 18, 412.