Synthesis of 3,5-disubstituted isoxazolines as protein tyrosine phosphatase 1B inhibitors

Article abstract of DOI:10.1007/s00044-007-9043-6

The protein tyrosine phosphatases (PTPase) are a group of regulatory enzymes that are critically important to a wide variety of cellular functions. PTPase 1B has recently been implicated in the pathogenesis of diabetes, neuronal disease, and autoimmune diseases. A number of these PTPase that act as negative regulators of the insulin signaling cascade have been identified as novel targets for the therapeutic enhancement of insulin action in insulin-resistant disease states like type II diabetes. Therefore, in the present work we describes a study of the synthesis and structure-activity relationship (SAR) of chromene and 2,4-dimethoxy benzaldehyde-based isoxazolines, which are structurally related to potent PTPase inhibitors. Compounds 5-7 and 10-19 were synthesized via 1,3-dipolar cycloaddition reaction and evaluated against PTPase enzyme in vitro. Compounds 10, 14, and 19 displayed significant inhibitory activity with IC₅₀ values of 69, 88, and 62.7 μM, respectively. Active compounds 10, 11, 14-16, and 19 were also tested in the STZ-S in vivo assay model, and compounds 10, 14, and 19 were found to be active.

Full text of DOI:10.1007/s00044-007-9043-6

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2008) 17:123–136
DOI 10.1007/s00044-007-9043-6
ORIGINAL RESEARCH
Synthesis of 3,5-disubstituted isoxazolines as protein
tyrosine phosphatase 1B inhibitors
Rakesh Maurya Æ Prasoon Gupta Æ Ghufran Ahmad Æ
Dinesh Kumar Yadav Æ Kailash Chand Æ
Amar Bahadur Singh Æ Akhilesh K. Tamrakar Æ
Arvind K. Srivastava
Received: 25 October 2007 / Accepted: 31 October 2007 / Published online: 22 December 2007
Ó Birkh a¨ user Boston 2007
Abstract The protein tyrosine phosphatases (PTPase) are a group of regulatory
enzymes that are critically important to a wide variety of cellular functions. PTPase
1
B has recently been implicated in the pathogenesis of diabetes, neuronal disease,
and autoimmune diseases. A number of these PTPase that act as negative regulators
of the insulin signaling cascade have been identified as novel targets for the ther-
apeutic enhancement of insulin action in insulin-resistant disease states like type II
diabetes. Therefore, in the present work we describes a study of the synthesis and
structure–activity relationship (SAR) of chromene and 2,4-dimethoxy benzalde-
hyde-based isoxazolines, which are structurally related to potent PTPase inhibitors.
Compounds 5–7 and 10–19 were synthesized via 1,3-dipolar cycloaddition reaction
and evaluated against PTPase enzyme in vitro. Compounds 10, 14, and 19 displayed
significant inhibitory activity with IC50 values of 69, 88, and 62.7 lM, respectively.
Active compounds 10, 11, 14–16, and 19 were also tested in the STZ-S in vivo
assay model, and compounds 10, 14, and 19 were found to be active.
Keywords Isoxazolines ꢀ Protein tyrosine phosphatases ꢀ
1
,3-Dipolar cycloaddition
This is CDRI communication no. 7148
R. Maurya (&) ꢀ P. Gupta ꢀ G. Ahmad ꢀ D. K. Yadav ꢀ K. Chand
Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226 001,
India
e-mail: mauryarakesh@rediffmail.com
A. B. Singh ꢀ A. K. Tamrakar ꢀ A. K. Srivastava
Biochemistry Division, Central Drug Research Institute, Lucknow 226 001, India

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Med Chem Res (2008) 17:123–136
Introduction
Diabetes is one of the most prevalent diseases in various parts of the world,
including India, with type II diabetes being the most common. A distinguishing
feature of type II diabetes is that patients are insulin resistant. The phosphorylation
state of the insulin receptor is controlled by a balance between the relative activities
of the insulin receptor kinase and cellular protein tyrosine phosphatase. Protein
tyrosine phosphatase 1B (PTP1B) plays an important role in insulin receptor
signaling (Saltiel and Kahn, 2001; Neel and Tonks, 1997). There is clear evidence
suggesting that PTP1B is primarily responsible for the dephosphorylation of the
insulin receptor and therefore acts to negatively regulate insulin signaling. Inhibitor
of this enzyme would be predicted to enhance insulin-stimulated glucose transport
and have potential for the treatment of type II diabetes (Kennedy and Ramachan-
dran, 2000). Since the identification of the crystal structure of protein tyrosine
phosphates (Barford et al., 1994), PTP1B has served as a general target for the
development of PTP inhibitor (Echerbly et al., 1999) and a novel target for the
treatment of obesity and type II diabetes. In recent years PTP1B inhibitors have
become the focus of effort aimed at the identification of clinical candidates for the
treatment of type II diabetes. Importantly, 2-(oxalylamino) benzoic acid (OBA) I
seems to be one of the potent minimal-unit phenyl phosphate mimetics identified so
far (Mc Cormack et al., 2000). A synthetic small molecule that selectively inhibits
PTP1B action is therefore expected to have a similar beneficial effect in humans.
Since N-phenyloxamic acid appears to be a potent non-phosphorus-containing PTyr
mimetic, a series of heterocyclic (isoxazole, isoxazoline) carboxylic acids (II–V)
were identified as potential N-phenyloxamic acid mimetics with reduced pKa in the
literature (Liu et al., 2003) (Fig. 1).
As part of our ongoing research program for the identification of antihypergly-
cemic compounds from plant sources, we have investigated Pongamia pinnata
fruits, which has led to the isolation of several compounds, including karanjin (VI,
Fig. 2) (Yadav et al., 2004). Karanjin displayed around 35% antihyperglycemic
activity in Streptozotocin-induced diabetic rats at a dose of 100 mg/kg and 15.8%
PTPase 1B inhibitory potential at a concentration of 100 lM. Recently, using
Br
COOH
COOH HO
NH
O
N
O
O
R
I.
II.
HOOC
HOOC
N
O
N
O
O
O
COOCH3
OH
NH
III.
IV. R=H
V. R=F
Fig. 1 Structures of the PTP1B inhibitors

Med Chem Res (2008) 17:123–136
125
karanjin as a starting material, we have investigated a series of new benzofuran
isoxazolines, some of which (compounds VII and VIII, Fig. 2) are several times
more active than the parent compound (Ahmad et al., 2006). These results inspired
us to further design and investigate small molecules as PTPase 1B inhibitors. Thus,
we have designed a synthetic strategy (Scheme 1 and 2) to assess the role of the
furan ring in PTPase 1B inhibitory activity. Therefore, in the present work, we
describe the synthesis of chromeno (5–7) and 2,4-dimethoxy benzaldehyde- based
(
10 –15) isoxazolines and their PTP 1B inhibitory activity.
Results and discussion
Chemistry
5
-Methoxy-2,2-dimethyl-2H-chromene-6-carbaldehyde oxime 4 was the key inter-
mediate of the series 5–7, synthesized in three steps starting from 2,4-dihydroxy
benzaldehyde 1 (Scheme 1). 5-Hydroxy-2,2-dimethyl-2H-chromene-6-carbalde-
hyde 2 was prepared by the reaction of 1 and 3-methyl-but-2-enal at 70% yield
(
Bandaranyake et al., 1971). Alkylation of 2 furnished 3 in quantitative yield.
Compound 3 on reaction with hydroxylamine hydrochloride in the presence of base
afforded 4 (as anti-isomer) at 87% yield (Sayer and Jencks, 1972). Furthermore,
compound 9 was prepared by the literature procedure (Sayer and Jencks, 1972;
Johnstone and Rose, 1979). The cycloaddition reaction of oxime 4, 9 with olefins
such as styrene, 4-methoxy styrene and 2-vinyl pyridine, 4-vinyl pyridine, 1-vinyl
imidazole, and trans methoxy cinnamate, in the presence of chloramine-T (Hassner
and Lokanatha, 1989), afforded isoxazolines 5–7 (Scheme 1) and 10–15 (Scheme 2)
in almost quantitative yields.
Product 15 proceeds regiospecifically to cis cycloadduct, as was confirmed by the
vicinal spin–spin coupling constant of the H-8 and H-9 protons (J = 7.0 Hz)
(Lukevics et al., 1998). All the synthesized compounds were characterized by
spectroscopic data and elemental analysis. Compounds 16–19 (Scheme 3) were
prepared using the known method of demethylation and acetylation.
Biological activity
Vanadate can normalize blood glucose level in diabetics (Sekar et al., 1996) and is a
specific inhibitor of phosphatases. Taking sodium-O-vanadate as a standard,
O
O
OCH3
O
O
O
OCH3
N O
OCH3
OCH3
N O
VI. 15.8%,
VII. 80.4%, IC50= 76 (µM)
VIII. 79.6%, IC50= 81 (µM)
Fig. 2 Structure of karanjin and its analogues

126
Med Chem Res (2008) 17:123–136
2
9
HO
OH
O
OR
O
OCH3
O
OCH3
a,b
c
d
10
5
12
R
3
8
CHO
CHO
1
N
N
O
OH
1
2: R = H
: R = CH3
4
5: R = phenyl
: R = 4-methoxy phenyl
: R = 2-pyridinyl
6
7
3
Scheme 1 Reagents and conditions: (a) 3-methyl-but-2-enal, pyridine reflux at 140°C, 11 h (b) CH
CO , dry acetone, reflux, 1 h (c) HCI.NH OH, aq. ethanol, 10% NaOH, reflux, 1 h (d) chloramine-T,
substituted alkene, ethanol, reflux
3
I,
K
2
3
2
H CO
3
OCH3
CHO
H CO
3
OCH3
H CO
3
2
1
OCH3
a
b
4
R1
1
2
R = R
8
R2
9
N
OH
N
O
8
9
10-15
Scheme 2 Reagents and conditions: (a) HCI.NH
ethanol, olefin, reflux
2
OH, aq. ethanol, 10% NaOH, reflux (b) chloramine-T,
1
OR2
N
Scheme 3 Reagents and
R O
conditions: (a) BBr
78°C; (b) Ac O/pyridine, room
temperature
3
, dry DCM,
a, b
–
2
1
0
O
1
6-19
compounds 5–7 and 10–19 were evaluated in vitro for PTPase 1B inhibitory activity
at a concentration of 100 lM; the results are summarized in Table 1. Compound 19
was the most active compound of the series with an IC50 value of 62.7 lM.
Compounds 10 and 14 were the next most active compounds of the series, showed a
similar order of activity with IC50 values of 69 and 88 lM, respectively.
Compounds 5–7, 11, 15, and 16 showed moderate activity with IC50 values of
70, 91, 87, 67, and 165 lM, respectively, however, compounds 12, 13, 17, and 18
were found to be inactive in this model.
The compounds that showed better PTPase 1B inhibition in vitro were further
evaluated in vivo in a streptozotosin-induced diabetic rat model using metformin as
standard drug. Compounds 10, 14, and 19 displayed a significant lowering in blood
glucose after 24 h observation (Table 2). These results revealed that compounds 10,
1
4, and 19 have potential antidiabetic as well as PTP1B inhibitory activity. The
ongoing improvement of the potency suggests selectivity induced by the introduc-
tion of the methoxy group in place of the furan ring (Fig. 2: compound VII and
VIII) (Ahmad et al., 2006).

Med Chem Res (2008) 17:123–136
127
Table 1 PTP1B inhibitory activity of compounds 5–7 and 10–19
Compound
R
1
R
2
Inhibtion (%)
IC50 (lM)
Ki (lM)
5
6
7
–
–
–
–
–
–
64.2 ± 2.1
57.7 ± 2.7
62.4 ± 2.0
88.5 ± 4.1
70
91
87
69
30
56
34
48
1
1
1
0
1
2
H
H
H
65.8 ± 3.9
9.8 ± 0.9
67
48
OCH3
N
485
170
1
1
3
4
H
H
11.5 ± 4.0
79.8 ± 3.2
275
88
150
63
N
N
N
15
CO
2
CH
3
46.5 ± 1.7
165
135
1
1
1
1
6
7
8
9
CH
CH
H
3
H
47.1 ± 3.2
28.8 ± 3.7
29.5 ± 1.9
91.6 ± 2.6
56.2
–
–
3
COCH
H
3
–
–
–
–
COCH
3
COCH
–
3
62.7
86.5
25
67
3 4
Na VO
–
Table 2 Blood glucose lowering activity in STZ-S in vivo model
Compound
Test dose mg/kg p.o.
Lowering of blood glucose (STZ-S) (%)
5
h
24 h
1
1
1
1
1
1
0
1
4
5
7
9
100
100
100
100
100
100
100
18.0
2.56
22.3
21.6
20.5
23.0
23.6
19.7
12.2
15.0
7.74
6.96
15.0
26.5
Metformin
Conclusion
It is clear from the in vitro activity profile (Table 1) that the isoxazolines 10, 14, and
9 show better PTP1B inhibition compared to the chromeno isoxazolines 5–7. The
1
structure–activity relationship study concluded that, when the furan ring was
replaced with a chromene ring (compounds 5–7), a loss in PTP1B inhibition was
seen as compared to compounds VII and VIII (Fig. 2). Further when the furan ring
was replaced with a methoxy group, a greater improvement in activity was
observed. The acetyl derivative 19 remarkably enhanced the activity profile as
compared to the reference compound sodium vanadate. In vivo results also

128
Med Chem Res (2008) 17:123–136
confirmed the activity of 10, 14, and 19. In conclusion, we have designed and
synthesized a series of novel isoxazolines analogs, among which compounds 10, 11,
1
4, and 19 displayed promising in vitro and in vivo results. This observation
suggests the possibility of further investigation for the development of potent and
selective PTP1B inhibitors.
Experimental procedure
Melting points (m.p.) were taken in open capillaries on an electrically heated
melting-point Complab apparatus and are uncorrected. Infrared (IR) spectra were
recorded on a Perkin-Elmer RX-1 spectrophotometer using either KBr pallets or
neat. The fast-atom bombardment mass spectrometry (FAB-MS) results were
recorded using a beam of argon (2–8 eV) on a Jeol SX 102/DA-6000 mass
spectrometer. The nuclear magnetic resonance (NMR) spectra were run on
AVANCE DPX 200 and Bruker DRX 300 Fourier transform (FT) NMR
spectrometers. The chemical shifts are reported in d (ppm) downfield from
tetramethylsilane (TMS), which was used as internal standard. Elemental analyses,
carbon, hydrogen and nitrogen (CHN) were obtained in a Carlo-Erba-1108 CHN
elemental analyzer. Silica gel (60–120 mesh) was used for column chromatography
while silica gel (230–400 mesh) was used for flash chromatography. Thin-layer
chromatography (TLC) was run either on precoated silica gel 60F254 and RP-18
F254 (Merck) or handmade plates. Detection of spots was done either by iodine
vapor and spraying with 1% cerric sulfate in 1 M H SO or spraying with 10%
2
4
methanolic sulfuric acid followed by heating at 110°C.
Preparation of 5-methoxy-2, 2-dimethyl-2H-chromene-6-carbaldehyde oxime
4) as a representative of 4, 9
(
The aldehyde 3 (2.18 g, 10 mmol) was dissolved in ethanol (40 mL), and
hydroxylamine hydrochloride (1.05 g, 15.12 mmol) was added. The reaction
mixture was basified to pH 10 by 10% aq. NaOH and refluxed for 1 h. The
mixture was allowed to cool and was extracted with ethyl acetate (100 mL 9 4);
the combined organic layer was washed with water (100 mL 9 2) and brine
(
100 mL), dried over anhydrous sodium sulfate, filtered, and concentrated. The
resulting residue was purified by column chromatography over silica gel (60– 120
mesh size), eluting with hexane and ethyl acetate (93:07 v/v) to give 4 (2.00 g, 87%
yield), viscous; IR (neat) mmax: 3287, 2975, 2933, 1595, 1477, 1370, 1280, 1216,
-
1 1
1
114, 1071, 986 cm ; H NMR (CDCl , 200 MHz) d: 8.33 (1H, s, CHNOH) 7.49
3
(
1H, d, J = 8.6 Hz, H-7), 6.60 (1H, d, J = 8.5 Hz, H-8), 6.57 (1H, d, J = 9.9 Hz, H-
1
), 5.64 (1H, d, J = 9.9 Hz, H-3), 3.77 (3H, s, OMe-4), 1.43 (2CH3); C NMR
3
4
(
(
CDCl , 50 MHz) d: 156.1 (C-9), 155.5 (C-5), 146.5 (CHNOH), 130.9 (C-3), 127.3
3
C-7), 117.9 (C-6), 116.9 (C-8), 115.1 (C-10), 113.7 (C-4), 80.3 (C-2), 63.5 (OMe-
+
), 28.3 (2CH ); FAB MS (+ve): m/z 233, 234.0 [M+H] for C H NO .
5
3
13 15
3

Med Chem Res (2008) 17:123–136
129
Preparation of 2,4-dimethoxy-benzaldehyde oxime (9)
The procedure for the synthesis of 4 was repeated with 2,4-dimethoxy benzaldehyde
(1.7 g, 10 mmol) and hydroxylamine hydrochloride (1.05 g, 15.12 mmol). The
8
crude product was purified by column chromatography over silica gel (60–120
mesh) eluting with hexane and ethyl acetate (93:07 v/v) to give 9 (1.5 g, 87% yield),
colorless crystals, mp 107–110°C (compared with literature value, 105–108°C); IR
(
KBr) mmax: 3286, 2943, 2835, 1608, 1506, 1464, 1416, 1288, 1215, 1158, 1120,
-
1 1
1
CHNOH) 7.53 (1H, d, J = 8.4 Hz, H-6), 6.49 (1H, dd, J = 8.4, 2.2 Hz, H-5), 6.45
035, 957, 845 cm ; H NMR (CDCl , 200 MHz) d: 9.23 (1H, s, OH), 8.37 (1H, s,
3
1
1H, d, J = 2.2 Hz, H-3), 3.84 (3H, s, OMe-2), 3.82 (3H, s, OMe-4); C NMR
3
(
(
(
CDCl , 50 MHz) d: 162.7 (C-4), 159.3 (C-2), 146.9 (CHNOH), 128.9 (C-6), 114.1
3
C-1), 105.7 (C-5), 98.8 (C-3), 55.9 (OMe-2), 55.8 (OMe-4); FAB MS (+ve): m/z
+
81, 182.0 [M+H] for C H NO .
1
9
11
3
3
-(5-Methoxy-2,2-dimethyl-2H-chromen-6-yl)-5 phenyl-4,5-dihydro-isoxazole
(
5)
A solution of oxime 4 (0.32 g, 1.37 mmol), styrene (0.21 g, 2.05 mmol), and
chloramine-T (0.46 g, 1.64 mmol) in absolute alcohol (30 mL) was refluxed with
stirring for 9 h. The reaction mixture was concentrated and the resulting crude
product was purified by column chromatography over silica gel (60–120 mesh)
using isocratic elution with hexane: acetone (95:05), affording 5 (0.41 g, 89 %);
3
1
0
viscous; [a]
-2.0 (c 0.35, CHCl ); IR (neat) m : 2975, 2933, 1635, 1597,
D
3
max
-1
1
458, 1372, 1279, 1217, 1115, 1058, 985, 888, 820 cm ; UV (CHCl ) kmax:
12 nm; H NMR (CDCl , 200 MHz) d: 7.48–7.36 (6H, m, H-2 to H-6 , H-7), 6.58
3
1
0
0
3
3
(
2H, dd, J = 9.7, 8.5 Hz, H-4, H-8), 5.67 (2H, dd, J = 10.0, 7.4 Hz, H-3, H-13),
.80 (1H, dd, J = 17.0, 10.7 Hz, H-12a), 3.68 (3H, s, OMe-5), 3.39 (1H, dd,
3
1
3
J = 17.0, 8.0 Hz, H-12b), 1.43 (2CH ); C NMR (CDCl , 50 MHz) d: 156.2 (C-
3
3
0
0
0
1
1
8
1), 155.2 (C-5, C-9), 141.6 (C-1 ), 131.1(C-3), 129.9 (C-7), 129.0 (C-3 , C-5 ),
0
0
0
28.4 (C-4 ), 126.2 (C-2 , C-6 ), 117.0 (C-8), 116.0 (C-6), 115.6 (C-10), 113.4 (C-4),
2.4 (C-13), 80.4 (C-2), 62.9 (OMe-5), 45.7 (C-12), 28.2 (2CH ); FAB MS (+ve):
3
+
m/z 335, 336.0 [M+H] . Elemental analysis: calc. for C H NO : C, 75.20; H, 6.31;
2
1
21
3
N, 4.18; found: C, 75.51, H, 6.19, N, 4.37 %.
3-(5-Methoxy-2,2-dimethyl-2H-chromen-6-yl)-5-(4-methoxy-phenyl)-4,5-
dihydro-isoxazole (6)
The procedure for the synthesis of 5 was repeated with 4 (0.35 g, 1.50 mmol), p-
methoxy styrene (0.30 g, 2.25 mmol) and chloramine-T (0.50 g, 1.80 mmol). The
crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with hexane: acetone (94:06), afforded 6 (0.43 g, 78
3
1
0
%
); brown crystals, m.p. 113–115°C; [a]
973, 2936, 2839, 1602, 1514, 1463, 1373, 1249, 1176, 1114, 1057, 985, 890,
-6.5 (c 0.26, CHCl ); IR (KBr) mmax:
D
3
2

130
Med Chem Res (2008) 17:123–136
-
1
1
8
25 cm ; UV (CHCl ) k : 266, 280 nm; H NMR (CDCl , 200 MHz) d: 7.47
3
max
3
0 0
(
1H, d, J = 8.6 Hz, H-7), 7.32 (2H, d, J = 8.6 Hz, H-3 , H-5 ), 6.89 (2H, d,
0
0
J = 8.6 Hz, H-2 , H-6 ), 6.59 (2H, dd, J = 9.7, 8.4 Hz, H-4, H-8), 5.63 (2H, dd,
J = 10.0, 8.5 Hz, H-3, H-13), 3.78 (3H, s, OMe-5), 3.78 (H-12a, merged with
0
methoxy signal), 3.71 (3H, s, OMe-4 ), 3.37 (1H, dd, J = 17.0, 8.3 Hz, H-12b), 1.44
1
2CH3); C NMR (CDCl , 50 MHz) d: 159.8 (C-4 ) 156.2 (C-11), 155.3 (C-9),
55.2 (C-5), 133.5 (C-1 ), 131.1(C-3), 129.8 (C-7), 127.6 (C-2 , C-6 ), 117.0 (C-8),
16.1 (C-6), 115.6 (C-10), 114.4 (C-3 , C-5 ), 113.4 (C-4), 82.3 (C-13), 80.2 (C-2),
3
0
(
3
0
0
0
1
1
6
0
0
0
3.0 (OMe-5), 55.7 (OMe-4 ), 45.4 (C-12), 28.2 (2CH ); FAB MS (+ve): m/z 366.0
3
+
M+H] . Elemental analysis: calc. for C H NO : C, 72.31; H, 6.34; N, 3.83;
[
found: C, 72.48, H, 6.19, N, 4.04 %.
2
2
23
4
2-[3-(5-Methoxy-2,2-dimethyl-2H-chromen-6-yl)-4,5-dihydro-isoxazol-5-yl]-
pyridine (7)
The procedure for the synthesis of 5 was repeated with 4 (0.38 g, 1.63 mmol), 2-
vinyl pyridine (0.25 g, 2.44 mmol) and chloramine-T (0.54 g, 1.95 mmol). The
crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with hexane:ethyl acetate (88:12), afforded 7 (0.34 g,
3
1
0
6
1
3
2 %); viscous; [a]
-5.4 (c 0.33, CHCl ); IR (neat) m : 2975, 2935, 1635,
D
3
max
-1
594, 1473, 1373, 1218, 1115, 1058, 985, 887, 819 cm ; UV (CHCl ) kmax:
03 nm; H NMR (CDCl , 200 MHz) d: 8.57 (1H, d, J = 3.7 Hz, H-3 ), 7.71–7.21
(4H, m, H-4 , H-5 , H-6 , H-7), 6.58 (2H, dd, J = 9.8, 8.4 Hz, H-4, H-8), 5.78 (1H,
brt, J = 3.8 Hz, H-13), 5.65 (1H, d, J = 10.0 Hz, H-3), 3.80 (1H, dd, J = 17.9,
3
1
0
3
0
0
0
1
1
5
1
6
0.6 Hz, H-12a), 3.68 (3H, s, OMe-5), 3.68 (H-12b, merged with methoxy signal),
1
3
0
.43 (2CH3); C NMR (CDCl , 50 MHz) d: 160.9 (C-1 ), 156.2 (C-11), 155.5 (C-
3
0
0
0
), 155.3(C-9), 149.7 (C-3 ), 137.2 (C-5 ), 131.0 (C-3), 129.9 (C-7), 123.1 (C-6 ),
0
20.7 (C-4 ), 117.0 (C-8), 115.7 (C-6, C-10), 113.3 (C-4), 82.4 (C-13), 80.6 (C-2),
+
2.9 (OMe-5), 44.1 (C-12), 28.2 (2CH ); FAB MS (+ve): m/z 337.0 [M+H] .
3
Elemental analysis: calc. for C H N O : C, 71.41; H, 5.99; N, 8.33; found: C,
2
0 20 2 3
7
1.69, H, 5.81, N, 8.55 %.
3
-(2,4-Dimethoxy-phenyl)-5-phenyl-4,5-dihydro-isoxazole (10)
The procedure for the synthesis of 5 was repeated with 9 (3.52 g, 19.44 mmol),
styrene (3.03 g, 29.16 mmol) and chloramine-T (6.55 g, 23.32 mmol). The crude
product was purified by column chromatography over silica gel (60–120 mesh)
using isocratic elution with hexane: acetone (95:05), afforded 10 (4.84 g, 88 %);
3
1
0
yellow crystals, mp 89–90 °C; [a]
-2.8 (c 0.31, CHCl ); IR (KBr) m : 2938,
-1
D
3
max
2
840, 1605, 1501, 1458, 1347, 1291, 1211, 1163, 1129, 1050, 888, 845 cm ; UV
1
CHCl ) kmax: 265, 298 nm; H NMR (CDCl , 200 MHz) d: 7.71 (1H, d,
J = 8.5 Hz, H-6), 7.41–7.27 (5H, m, H-2 to H-6 ), 6.48 (2H, dd, J = 8.5, 2.3 Hz, H-
, H-3), 5.61 (1H, dd, J = 10.6, 8.6 Hz, H-9), 3.80 (3H, s, OMe-2), 3.80 (H-8a
merged with methoxy signal), 3.78 (3H, s, OMe-4), 3.40 (1H, dd, J = 17.3, 8.5 Hz,
(
3
3
0
0
5

Med Chem Res (2008) 17:123–136
131
1
3
H-8b); C NMR (CDCl , 50 MHz) d: 162.9 (C-4), 159.3 (C-7), 155.8 (C-2), 141.8
(
3
0 0 0 0 0 0
C-1 ), 130.7(C-6), 129.0 (C-3 ,C-5 ), 128.3 (C-4 ), 126.4 (C-2 , C-6 ), 111.9 (C-1),
1
2
4
05.7 (C-5), 99.1 (C-3), 82.6 (C-9), 55.8 (2OMe), 46.2 (C-8); FAB MS (+ve): m/z
+
83, 284.0 [M+H] . Elemental analysis: calc. for C H NO : C, 72.07; H, 6.05; N,
.94; Found: C, 72.32, H, 5.88, N, 5.19 %.
1
7
17
3
3
-(2,4-Dimethoxy-phenyl)-5-(4-methoxy-phenyl)-4,5-dihydro-isoxazole (11)
The procedure for the synthesis of 5 was repeated with 9 (1.75 g, 9.66 mmol), p-
methoxy styrene (1.94 g, 14.49 mmol) and chloramine-T (3.25 g, 11.59 mmol).
The crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with hexane: acetone (93:07), afforded 11 (2.46 g, 81
3
1
D
0
%
); colorless crystals, m.p. 91–93°C; [a]
007, 2939, 2838, 1609, 1511, 1462, 1347, 1294, 1249, 1211, 1163, 1032, 831 cm
-2.7 (c 0.33, CHCl ); IR (KBr) m
:
max
3
-
3
1
1
;
J = 8.4 Hz, H-6), 7.32 (2H, d, J = 8.5 H-3 , H-5 ), 6.88 (2H, d, J = 8.6 Hz, H-2 , H-
UV (CHCl ) k : 268, 302 nm; H NMR (CDCl , 200 MHz) d: 7.71 (1H, d,
3
max
3
0
0
0
0
6 ), 6.53 (1H, d, J = 2.3 Hz, H-3), 6.51 (1H, d, J = 8.5 Hz, H-5), 5.56 (1H, dd,
J = 10.2, 9.1 Hz, H-9), 3.81 (3H, s, OMe-2), 3.81 (H-8a merged with methoxy
signal), 3.79 (3H, s, OMe-4), 3.78 (3H, s, OMe-4 ), 3.39 (1H, dd, J = 17.3, 8.8 Hz,
1
H-8b); C NMR (CDCl , 50 MHz) d: 162.8 (C-4), 159.8 (C-4 ), 159.2 (C-7), 155.9
0
3
0
3
0
0
0
0
0
(
C-2), 133.7 (C-1 ), 130.7 (C-6), 127.8 (C-2 ,C-6 ), 114.4 (C-3 , C-5 ), 112.0 (C-1),
0
1
MS (+ve): m/z 314.0 [M+H] . Elemental analysis: calc. for C H NO : C, 68.99;
05.6 (C-5), 99.1 (C-3), 82.5 (C-9), 55.8 (2OMe), 55.7 (OMe-4 ), 46.0 (C-8); FAB
+
1
8
19
4
H, 6.11; N, 4.47; found: C, 69.18, H, 5.94, N, 4.64 %.
2
-[3-(2,4-Dimethoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-pyridine (12)
The procedure for the synthesis of 5 was repeated with 9 (1.61 g, 8.89 mmol), 2-
vinyl pyridine (1.40 g, 13.33 mmol), and chloramine-T (3.00 g, 10.66 mmol). The
crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with hexane:ethyl acetate (84:16), afforded 12 (1.88 g,
3
1
0
7
4 %); viscous; [a]
-1.4 (c 0.54, CHCl ); IR (neat) m : 3008, 2941, 2840,
D
3
max
-1
607, 1507, 1465, 1348, 1289, 1212, 1161, 1051, 1029, 888, 839 cm ; UV
1
1
(CHCl ) kmax: 266, 296 nm; H NMR (CDCl , 200 MHz) d: 8.57 (1H, d,
J = 4.5 Hz, H-3 ), 7.71 (1H, d, J = 8.4 Hz, H-6), 7.69–7.56 (3H, m, H-4 , H-5 , H-
), 6.50 (1H, dd, J = 8.6, 2.1 Hz, H-5), 6.45 (1H, brs, H-3), 5.74 (1H, dd, J = 10.9,
.0 Hz, H-9), 3.94 (1H, dd, J = 17.4, 11.0 Hz, H-8a), 3.82 (3H, s, OMe-2), 3.80
3H, s, OMe-4), 3.68 (1H, dd, J = 17.3, 7.0 Hz, H-8b); C NMR (CDCl , 50 MHz)
3
3
3
0
0
0
0
6
7
1
3
(
0
0
0
d: 162.9 (C-4), 161.1 (C-1 ), 159.3 (C-7), 155.9 (C-2), 149.6 (C-3 ), 137.2 (C-5 ),
0
0
1
30.8 (C-6), 123.0 (C-6 ), 120.7 (C-4 ), 111.6 (C-1), 105.6 (C-5), 99.1 (C-3), 82.5
+
C-9), 55.8 (2OMe), 44.8 (C-8); FAB MS (+ve): m/z 284, 285.0 [M+H] . Elemental
(
analysis: calc. for C H N O : C, 67.59; H, 5.67; N, 9.85; found: C, 67.77, H, 5.46,
N, 10.07 %.
1
6 16 2 3

132
Med Chem Res (2008) 17:123–136
4
-[3-(2,4-Dimethoxy-phenyl)-4,5-dihydro-isoxazol-5-yl]-pyridine (13)
The procedure for the synthesis of 5 was repeated with 9 (1.45 g, 8.01 mmol), 4-
vinyl pyridine (1.26 g, 12.01 mmol), and chloramine-T (2.70 g, 9.61 mmol). The
crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with hexane:ethyl acetate (84:16), affording 13
(
1.76 g, 77 %); viscous; [a]31D -6.40 (c 0.34, CHCl3); IR (neat) mmax: 3018, 2968,
-1
1
2
7
608, 1507, 1463, 1418, 1347, 1294, 1215, 1162, 1031 cm ; UV (CHCl3) kmax:
0
0
62, 296 nm; 1H NMR (CDCl3, 200 MHz) d: 8.57 (2H, d, J = 5.8 Hz, H-3 , H-5 ),
.70 (1H, d, J = 8.5 Hz, H-6), 7.31 (2H, d, J = 5.7 H-2 , H-6 ), 6.51 (1H, d, J = 8.6,
0
0
Hz, H-5), 6.45 (1H, d, J = 1.8 Hz, H-3), 5.61 (1H, dd, J = 10.7, 7.5 Hz, H-9), 3.89
1H, dd, J = 17.3, 11.0 Hz, H-8a), 3.81 (3H, s, OMe-2), 3.79 (3H, s, OMe-4), 3.37
1H, dd, J = 17.2, 7.4 Hz, H-8b); 13C NMR (CDCl3, 50 MHz) d: 163.0 (C-4),
(
(
0
0
0
0
1
59.2 (C-7), 155.5 (C-2), 150.9 (C-1 ), 150.3 (C-3 , C-5 ), 130.6 (C-6), 120.9 (C-2 ,
0
C-6 ), 111.1 (C-1), 105.7 (C-5), 99.0 (C-3), 80.5 (C-9), 55.8 (2OMe), 45.9 (C-8);
FAB MS (+ve): m/z 284, 285.0 [M+H]+. Elemental analysis: calc. for
C16H16N2O3: C, 67.59; H, 5.67; N, 9.85; found: C, 67.79, H, 5.43, N, 10.05 %.
3
-(2,4-Dimethoxy-phenyl)-5-imidazol-1-yl-4,5-dihydro-isoxazole (14)
The procedure for the synthesis of 5 was repeated with 9 (1.56 g, 8.61 mmol), 1-
vinyl imidazole (1.21 g, 12.91 mmol), and chloramine-T (2.90 g, 10.33 mmol). The
crude product was purified by column chromatography over silica gel (60–120
mesh) using isocratic elution with ethyl acetate: methanol (9:1), afforded 14 (1.32 g,
3
1
0
5
1
2
6 %); viscous; [a]
-1.6 (c 0.31, MeOH); IR (neat) m : 3011, 2968, 1611,
D
max
-1
507, 1463, 1422, 1351, 1293, 1214, 1162, 1029, 931, 837 cm ; UV (CHCl ) kmax:
3
1
64, 297 nm; H NMR (CDCl , 200 MHz) d: 7.75 (1H, d, J = 8.5 Hz, H-6), 7.67
3
0
0
0
(
1H, brs, H-2 ), 7.07 (1H, brs, H-4 ), 7.00 (1H, brs, H-5 ), 6.56 (1H, dd, J = 8.5,
2
3
2
2
9
.2 Hz, H-5), 6.49 (1H, d, J = 2.1 Hz, H-3), 6.42 (1H, dd, J = 8.7, 2.9 Hz, H-9),
.92 (1H, dd, J = 18.4, 8.7 Hz, H-8a), 3.85 (6H, s, 2OMe), 3.73 (1H, dd, J = 18.3,
1
3
.9 Hz, H-8b); C NMR (CDCl , 50 MHz) d: 163.6 (C-4), 159.4 (C-7), 156.0 (C-
3
0
0
0
), 135.9 (C-2 ), 130.9 (C-6), 130.7 (C-5 ), 116.6 (C-4 ), 110.1 (C-1), 106.0 (C-5),
9.1 (C-3), 85.60 (C-9), 55.9 (2OMe), 44.9 (C-8); FAB MS (+ve): m/z 274.0
+
M+H] . Elemental analysis: calc. for C H N O : C, 61.53; H, 5.53; N, 15.38;
[
found: C, 61.76, H, 5.39, N, 15.66 %.
1
4 15 3 3
3-(2,4-Dimethoxy-phenyl)-5-phenyl-4,5-dihydro-isoxazole-4-carboxylic acid
methyl ester (15)
The procedure for the synthesis of 5 was repeated with 9 (1.82 g, 10.05 mmol), methyl
cinnamate (2.44 g, 15.07 mmol), and chloramine-T (3.38 g, 12.06 mmol). The crude
product was purified by column chromatography over silica gel (60–120 mesh) using
isocratic elution with hexane: acetone (92:08), affording 15 (2.45 g, 71 %); colorless
3
1
crystal, m.p. 84–86°C; [a] –3.6° (c 0.32, CHCl ); IR (KBr) m : 3002, 2949, 2837,
D
3
max
-
1
1
740, 1607 1502, 1459, 1279, 1207, 1169, 1028, 901, 832 cm ; UV (CHCl ) kmax:
3

Med Chem Res (2008) 17:123–136
1
133
2
68, 302 nm; H NMR (CDCl , 200 MHz) d: 7.84 (1H, d, J = 8.6 Hz, H-6), 7.40–7.35
3
0
0
(
5H, m, H-2 to H-6 ), 6.54 (1H, d, J = 8.6 Hz, H-5), 6.41 (1H, d, J = 1.9 Hz, H-3),
.75 (1H, d, J = 7.0 Hz, H-9), 4.63 (1H, d, J = 7.0 Hz, H-8), 3.82 (3H, s, OMe-2), 3.73
5
1
3
(
3H, s, OMe-4), 3.71 (3H, s, COOMe); C NMR (CDCl , 50 MHz) d: 170.6
3
0
(
COOMe), 163.1(C-4), 158.5 (C-7), 153.7 (C-2), 140.2 (C-1 ), 130.9 (C-6), 129.2 (C-
0
0 0 0 0
3
, C-5 ), 128.8 (C-4 ), 126.1 (C-2 , C-6 ), 111.6 (C-1), 106.1 (C-5), 98.9 (C-3), 86.6
C-9), 64.0 (C-8), 55.8 (OMe-2), 55.5 (OMe-4), 52.9 (COOMe); FAB MS (+ve): m/z
(
+
3
41, 342.0 [M+H] . Elemental analysis: calc. for C H NO : C, 66.85; H, 5.61; N,
19 19 5
4
.10; Found: C, 67.03, H, 5.44, N, 4.32 %.
5
-Methoxy-2-(5-phenyl-4,5-dihydro-isoxazol-3-yl)-phenol (16)
A solution of 10 (1.15 g, 4.06 mmol) was dissolved in dry dichloromethane (40 ml)
in a 100 ml RB flask, placed under nitrogen atmosphere. One molar solution of
BBr 8.5 ml (2.13 g, 8.52 mmol) in DCM was added drop wise with stirring at –
3
7
8°C and maintained it for 3 hour then the reaction mixture was brought about at
room temperature for 1 h. After completion of the reaction, the reaction mixture
was quenched by drop wise addition of water and poured the reaction mixture in to
the saturated solution of sodium bicarbonate (50 ml). Reaction mixture was
extracted with dichloromethane (5 9 50 ml), washed with brine (3 9 50 ml), dried
over anhydrous sodium sulphate and concentrated to get crude product. The crude
product was purified by column chromatography over silica gel (60–120 mesh) by
isocratic elution with hexane: acetone (95:05), afforded 16 (0.94 g, 86 %); colorless
3
1
D
0
crystals, mp 98–101 °C; [a]
-4.8 (c 0.31, CHCl ); IR (KBr) m : 2936, 2843,
-1
3
max
1
630, 1596, 1516, 1359, 1293, 1161, 1144, 1039, 937, 846, 809 cm ; UV (CHCl3)
1
kmax: 267, 296 nm; H NMR (CDCl , 200 MHz) d: 9.98 (OH), 7.34 (5H, brs, H-2
to H-6 ), 7.01 (1H, d, J = 8.6 Hz, H-3), 6.55 (1H, d, J = 2.2 Hz, H-6), 6.44 (1H, dd,
J = 8.6, 2.2 Hz, H-4), 5.59 (1H, dd, J = 10.6, 8.3 Hz, H-9), 3.76 (3H, s, OMe-5),
.76 (H-8a merged with methoxy signal), 3.33 (1H, dd, J = 16.6, 8.3 Hz, H-8b);
0
3
0
1
3
3
C
0
NMR (CDCl , 50 MHz) d: 163.0 (C-5), 159.6 (C-7), 158.4 (C-1), 140.6 (C-1 ),
3
0 0 0 0 0
1
29.9 (C-3), 129.3 (C-3 ,C-5 ), 128.9 (C-4 ), 126.4 (C-2 , C-6 ), 107.6 (C-2), 107.3
C-4), 101.9 (C-6), 81.5 (C-9), 55.8 (OMe-5), 43.8 (C-8); FAB MS (+ve): m/z 270.0
(
+
M+H] . Elemental analysis: calc. for C H NO : C, 71.36; H, 5.61; N, 5.20;
[
Found: C, 71.53, H, 5.38, N, 5.41 %.
1
6
15
3
Acetic acid 5-methoxy-2-(5-phenyl-4,5-dihydro-isoxazol-3-yl)-phenyl ester (17)
A mixture of 16 (0.24 g, 0.89 mmol) and acetic anhydride (0.10 g, 1.06 mmol) in
dry pyridine (5 ml) stirred for 12 h. The reaction mixture was evaporated to dryness
under reduced pressure and the residue was chromatographed on silica gel eluting
with hexane: ethyl acetate (93:7), afforded 17 (0.27 g, 97 %) as white crystals, mp
3
1
D
0
1
1
10–111 °C; [a]
611, 1508, 1459, 1369, 1348, 1210, 1184, 1111, 1013, 910, 812 cm ; UV
-3.4 (c 0.26, CHCl ); IR (KBr) m : 3019, 2978, 2933, 1759,
-1
3
max
1
(
CHCl ) k : 270 nm; H NMR (CDCl , 200 MHz) d: 7.41 (1H, d, J = 8.8 Hz, H-
), 7.37–7.33 (5H, m, H-2 to H-6 ), 6.81 (1H, dd, J = 8.7, 2.5 Hz, H-4), 6.68 (1H, d,
3
max
3
0 0
3

134
Med Chem Res (2008) 17:123–136
J = 2.5 Hz, H-6), 5.61 (1H, dd, J = 10.8, 8.4 Hz, H-9), 3.82 (3H, s, OMe-5), 3.73
1H, dd, J = 16.5, 10.8 Hz, H-8a), 3.30 (1H, dd, J = 16.5, 8.4 Hz, H-8b), 2.31 (3H,
(
1
3
s, OAc); C NMR (CDCl , 50 MHz) d: 170 (CO), 161.9 (C-5), 153.6 (C-7), 150.1
3
0
0
0
0
0
0
(
C-1), 141.1 (C-1 ), 130.8 (C-3), 129.1 (C-3 ,C-5 ), 128.6 (C-4 ), 126.4 (C-2 , C-6 ),
15.6 (C-2), 112.4 (C-4), 110.0 (C-6), 82.0 (C-9), 56.0 (OMe-5), 45.1 (C-8) 21.6
1
+
(
CH CO); FAB MS (+ve): m/z 312.0 [M+H] for C H NO .
3 18 17 4
4
-(5-Phenyl-4,5-dihydro-isoxazol-3-yl)-benzene-1,3-diol (18)
The procedure for the synthesis of 16 (see 4.2.12.) was repeated with 10 (1.32 g,
.66 mmol) and BBr 14.4 ml (3.61 g, 14.44 mmol) of 1 M solution in DCM. The
crude product was purified by column chromatography over silica gel (60–120
4
3
mesh) using isocratic elution with hexane: ethyl acetate (9:1), afforded 18 (1.13 g,
3
1
0
9
5 %); pink color crystals, mp 139–140 °C; [a]
-5.3 (c 0.3, CHCl ); IR (KBr)
D 3
-
1
mmax: 3058, 2933, 1629, 1602, 1512, 1369, 1288, 1164, 1131, 974, 909, 878 cm
UV (CHCl ) k : 316 nm; H NMR (CDCl , 200 MHz) d: 10.12 (hump of OH),
;
1
3
max
3
0
0
7
H-2), 6.41 (1H, d, J = 8.4 Hz, H-6), 5.65 (1H, dd, J = 10.6, 8.2 Hz, H-9), 3.81 (1H,
.37 (5H, brs, H-2 to H-6 ), 7.01 (1H, d, J = 8.4 Hz, H-5), 6.51 (1H, d, J = 2.2 Hz,
1
dd, J = 16.4, 10.7 Hz, H-8a), 3.37 (1H, dd, J = 16.5, 8.1 Hz, H-8b); C NMR
3
0
(
CDCl , 50 MHz) d: 159.4 (C-1, C-3), 158.3 (C-7), 140.5 (C-1 ), 130.2 (C-5), 129.2
3
0
0
0
0
0
(
C-3 ,C-5 ), 128.9 (C-4 ), 126.3 (C-2 , C-6 ), 108.0 (C-6), 107.7 (C-4), 104.0 (C-2),
+
1.5 (C-9), 43.8 (C-8); FAB MS (+ve): m/z 256.0 [M+H] . Elemental analysis: calc.
8
for C H NO : C, 70.58; H, 5.13; N, 5.49; found: C, 70.75, H, 4.96, N, 5.63 %.
1
5
13
3
Acetic acid 3-acetoxy-4-(5-phenyl-4,5-dihydro-isoxazol-3-yl)-phenyl ester (19)
The procedure for the synthesis of 17 (see 4.2.13.) was repeated with 18 (0.25 g,
0.98 mmol), acetic anhydride (0.21 g, 2.15 mmol) and pyridine (5 ml). The crude
product was purified by column chromatography over silica gel (60–120 mesh)
using isocratic elution with hexane:ethyl acetate (92:08), affording 19 (0.32 g, 96
3
1
D
%
) Pink crystals, mp 130–132°C; [a]
083, 2926, 1763, 1603, 1503, 1483, 1366, 1344, 1278, 1196, 1151, 1116, 1016,
–0.7° (c 0.54, CHCl ); IR (KBr) mmax:
3
3
9
7
2
3
-
1
1
16, 844, 827 cm ; UV (CHCl ) k
264 nm; H NMR (CDCl , 200 MHz) d:
3
max
3
0
0
.52 (1H, d, J = 8.5 Hz, H-3), 7.37 (5H, brs, H-2 to H-6 ), 7.07 (1H, dd, J = 8.5,
.3 Hz, H-4), 6.99 (1H, d, J = 2.2 Hz, H-6), 5.66 (1H, dd, J = 10.9, 8.3 Hz, H-9),
.74 (1H, dd, J = 16.6, 10.9 Hz, H-8a); 3.34 (1H, dd, J = 16.6, 8.3 Hz, H-8b), 2.30
+
6H, s, 2OAc); FAB MS (+ve): m/z 340.0 [M+H] for C H NO .
(
1
9
17
5
Biological experimental
In vitro assay
The effect of the test compounds on PTP1B (Sigma, USA) was studied by
preincubating the test compound with enzyme in the reaction system for 10 min and

Med Chem Res (2008) 17:123–136
135
determining the residual enzyme activity according to the Goldstein et al method
Goldstein et al., 2000) using p-nitrophenylphosphate (pNPP) as the substrate. The
.0-mL assay mixture contained 10 mM pNPP in 50 mM 4-(2-hydroxyethyl)-1-
(
1
piperazineethene sulphonic acid (HEPES) buffer (pH 7.0), with 1 mM ethylene-
diaminetetraacetic acid (EDTA) and 2 mM dithiothreitol (DTT), respectively. The
reaction was stopped by addition of the 500 ll of 0.1 N NaOH and the optical
density was measured at 410 nm. Control tubes omitting the enzyme were always
run in parallel to nullify the nonenzymic reaction. A molar extinction coefficient of
4
.78 9 10 was used to determine the concentration of p-nitrophenolate produced in
1
the system.
In vivo assay
For in vivo testing male albino Sprague–Dawley rats of body weight 160 ± 20 g
were selected. Streptozotocin (Bedoya et al., 1996) (Sigma, USA) was dissolved in
1
00 mM citrate buffer pH 4.5 and a calculated amount of the fresh solution was
injected to overnight-fasted rats (45 mg/kg) intraperitoneally. Blood glucose level
was checked 48 h later by glucostrips and animals showing blood glucose values of
1
44–270 mg/dl (8–15 mM) were included in the experiment and termed diabetic.
The diabetic animals were divided into groups consisting of five or six animals in
each group. Animals of experimental groups were administered the suspension of
the desired test samples orally (made in 1.0% gum acacia) at a dose of 100 mg/kg
body weight. Animals in the control group were given an equal amount of 1.0%
gum acacia. A sucrose load of 2.5 g/kg body weight was given after 30 min of
compound administration. Blood glucose level was checked again at 30, 60, 90,
1
20, 180, 240, and 300 min and at 24 h. Animals not found to be diabetic 24 h post
treatment of the test sample were not considered and omitted from the calculations
and termed nonresponders. Food but not water was withheld from the cages during
the experimentation. Comparing the AUC of experimental and control groups
determined the percentage anti-hyperglycemic activity. Details of the streptozotocin
induced low dose sucrose challenged diabetic rat (STZ-S) activity are given in
Table 2.
Acknowledgements The authors DKY and KC would like to thank CSIR and UGC, respectively, New
Delhi for JRFs.
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