Design and synthesis of 3,5-diarylisoxazole derivatives as novel class of anti-hyperglycemic and lipid lowering agents

Article abstract of DOI:10.1016/j.bmc.2009.05.033

We have designed 1,3-disubstituted-5-membered heteroaromatic ring system as a common core motif from known anti-hyperglycemic agents. Designed compounds were synthesized and screened for in vivo anti-hyperglycemic activity in sucrose loaded model (SLM), sucrose-challenged streptozotocin-induced diabetic rat model (STZ-S) as well as db/db mice model. Some of the synthesized compounds showed promising in vivo anti-hyperglycemic as well as moderate lipid lowering activity. Synthesized Compounds were screened in various in vitro models of type-2 diabeties such as DPP-4, PTP1B and PPARγ to know the mechanism of their anti-hyperglycemic action. None of the synthesized compounds showed DPP-4 inhibitory as well as PPARγ activity. These compounds have shown promising PTP-1B inhibitory activity there by revealing that compounds exhibit anti-diabetic activity by PTP1B pathway.

Full text of DOI:10.1016/j.bmc.2009.05.033

Bioorganic & Medicinal Chemistry 17 (2009) 5285–5292
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design and synthesis of 3,5-diarylisoxazole derivatives as novel class
of anti-hyperglycemic and lipid lowering agents q
a
a
a
b
b
Atul Kumar a, , Ram Awatar Maurya , Siddharth Sharma , Pervez Ahmad , A. B. Singh , A. K. Tamrakar ,
*
Arvind K. Srivastava b
a Medicinal and Process Chemistry Division, Central Drug Research Institute, Lucknow 226001, India
b Biochemistry Division, Central Drug Research Institute, Lucknow 226001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
We have designed 1,3-disubstituted-5-membered heteroaromatic ring system as a common core motif
from known anti-hyperglycemic agents. Designed compounds were synthesized and screened for
in vivo anti-hyperglycemic activity in sucrose loaded model (SLM), sucrose-challenged streptozotocin-
induced diabetic rat model (STZ-S) as well as db/db mice model. Some of the synthesized compounds
showed promising in vivo anti-hyperglycemic as well as moderate lipid lowering activity. Synthesized
Received 4 March 2009
Revised 12 May 2009
Accepted 13 May 2009
Available online 18 May 2009
Compounds were screened in various in vitro models of type-2 diabeties such as DPP-4, PTP1B and PPAR
to know the mechanism of their anti-hyperglycemic action. None of the synthesized compounds showed
DPP-4 inhibitory as well as PPAR activity. These compounds have shown promising PTP-1B inhibitory
activity there by revealing that compounds exhibit anti-diabetic activity by PTP1B pathway.
Ó 2009 Elsevier Ltd. All rights reserved.
c
Keywords:
3,5-Diarylisoxazole
Anti-hyperglycemic activity
Lipid lowering activity
PTP1B
c
1. Introduction
Several small molecules such as hydroxy phenyl thiazole deriva-
tive (Japan Tobacco I),25,26 II,27 III, IV, V, VI and VII28–32 have been
reported as potential anti-hyperglycemic agents. After a careful
observation of the structural frame work of these anti-hyperglycae-
mic agents, we found a very interesting structural resemblance
among these molecules (Fig. 1). The structural analysis of these mol-
ecules revealed that all these molecules have a common core motif
1,3-disubstituted-5-membered heteroaromatic ring. Although all
these structures have different aryl substitution basically due to
presence of heteroatom in the ring system, the relative position of
substituents is 1,3 in five membered heteroaromatic ring system.
Utilizing the identified core motif of compounds shown in Fig-
ure 1 for future drug design, we have designed and synthesized
3,5-diarylisoxazole moiety having 1,3-disubstituted 5-membered
heteroaromatic framework as potential anti-hyperglycemic agents.
Some of these design compounds have exhibited significant antidi-
abetic, triglyceride and cholesterol lowering activity along with
promising PTP-1B inhibitory activity.
Type-2 diabetes (T2D) is complex, chronic metabolic disorder
mainly associated with three basic pathophysiological abnormali-
ties: (1) insulin resistance in target tissue, (2) excessive hepatic
glucose production and (3) insulin resistance in skeleton muscle,
liver and adipose tissue.1–5 Diabetes mellitus has now treated as
epidemic disease and considered as one of the main threats to
human health. The primary therapy for type-2 diabetes is caloric
restriction and exercise.6 There are four major types of pharmaco-
logical agents available for the treatment of type-2 diabetes: (1)
insulin secretagogues sulfonylureas,7–9 like Glucotrol, Micronase,
Glynase and DiaBeta (2) bigunides10–14 like metformin which delay
gastrointestinal glucose absorption, (3) insulin sensitivity enhanc-
ers like glitazones15–19 and (4) acarbose.20–22 But none of these
agents are having final answer to type-2 diabetes and also carrying
undesirable side effects like hepatotoxicity, weight gain, edema,
etc.
Since current available therapies cannot prevent disease’s
progression, many research groups are focusing their efforts
towards the identification of novel approaches.23,24 Protein Tyro-
sine Phosphatase-1B (PTP-1B), Dipeptidyl Peptidase IV and Perox-
isome proliferator-activated receptor (PPAR) have been identified
as potential target of type-2 diabetes (T2D).
2. Results and discussion
2.1. Chemistry
The key intermediate 3,5-diarylisoxazole 5 having 1,3-disubsti-
tuted-5-membered heteroaromatic framework was synthesized
from 2,4-dihydroxy acetophenone 1 in four steps. 2,4-Dihydroxy
acetophenone 1 was treated with dimethyl sulphate and K2CO3
q
CDRI Comm. No. 7236.
* Corresponding author. Tel.: +91 522 2612411; fax: +91 522 2623405.
E-mail address: dratulsax@gmail.com (A. Kumar).
0968-0896/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2009.05.033

5286
A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
N
O
N
N
Br
Br
N
O
N
HO
HO
R3
S
I
CF3
RO
O
O
III
II
R1 R2
(PTP-1B inhibitor)
Japan Tobacco
HOOC
OC2H5
O
N
PPAR
dual
agonist
PPAR
dual
agonist
N
Core Motif
O
N
1,3-Disubstituted 5-membered
heteroaromatic framework
V
O
IV
O
DPP4 inhibitor
O
COOCH3
F
F
F
N
CN
N
N
N
O
NH2
N
H
VI
VII
Figure 1. Design or 1,3-disubstituted five-membered heteroaromatic framework (3,5-diaryl isooxazole) based on known antihyperglycemic agents.
in acetone to yield 2-hydroxy-4-methoxy acetophenone 2, which
on reaction with benzoylchloride in pyridine gave ortho-benzoylat-
ed derivative 3 in 88% yield. Benzoyl group of compound 3 was
migrated on treatment with KOH–pyridine resulting to the forma-
tion of diketone 4 via Baker Venkataraman rearrangement.33
Diketone 4, when refluxed with hydroxylamine hydrochloride
afforded 3,5-diaryl isoxazole 5 in 45% yields.
The isoxazole 5 on Williamson type O-alkylation reaction with
different 1-(2-chloro-alkyl) substituted amine hydrochloride gave
alkyl substituted amino diaryl isoxzoles (6–11). 3,5-Diarylisoxaz-
ole on Williamson type O-alkylation with diholoalkyl compound
gave compound 12, which on reaction with amines gave com-
pounds 13–17. 3,5-Diarylisoxazole 5 on reaction with haloalkyl
esters gave compounds 18–21. Compound 5 on reaction with
epichlrohydrine afforded compound 22 in very good yield.
Compound 22 on reaction with appropriate amines gave
compound 23–25 in good yields (Scheme 1).
2.2.1. Dose dependant anti-hyperglycemic effect of 7, 10, 11and
24 in sucrose challenged streptozotocin-induced diabetic rats
Dose dependency of compounds 7, 10, 11and 24 was studied by
administering different doses of test compound to streptozotocin-
induced diabetic rats (Table 2). Doses ranges from 7.5 to 100 mg/kg
were given and blood glucose level was measured at 30, 60, 90,
120, 180, 240, 300, 1440 min post administration of sucrose load
as described in sucrose challenged streptozotocin-induced diabetic
rats’ protocol. Compounds 7, 10, 11 and 24 showed dose depen-
dency and their ED50 were found to be 45.3 mg/kg, 54.8 mg/kg,
64.5 mg/kg and 80.2 mg/kg respectively.
Four compounds (7, 10, 11 and 24) were further tested for
their anti-hyperglycemic as well as lipid lowering activity (Table
3 and Figs. 4–6) in db/db mice model 6 days and 10 days proto-
col. These compounds (7, 10, 11 and 24) showed promising
in vivo anti-diabetic and anti-dyslipidemic activity in db/db
mice.
In order to know the mode of action for anti hyperglycemic
activity of compounds, we screened these compounds in protein-
tyrosine-phosphatase-1B inhibitory activity, DPP-4 enzyme inhibi-
2.2. Biological activity
All the synthesized compounds were evaluated for in vivo anti-
hyperglycemic activities are given in Table 1.
tion assay and tested for PPARc transactivation assay (Table 4).
Compounds 7–11, 16–18, 20 and 23–25 exhibited significant
PTP-1B inhibitory activity (>80%). Vanadate (Sodium ortho vana-
date) a non-selective PTPs inhibitor was taken as a control.
The structure–activity relationship studies of diarylisoxazole
revealed that compounds with two carbon side chains (7–11) have
shown better activity than compounds with three carbon side
chains (13–17 and 23–25).
Compounds (7, 8, 10, 11, 16, 17, 24) were also tested for DPP-4
enzyme inhibition assay and PPARc transactivation assay. None of
the compounds has shown any significant activity in DPP-4 as well
as PPAR assay.
Compoundswere screened in vivo antidiabetic activityin sucrose
loaded model (SLM) male albino rats for anti-hyperglycemic activ-
ity. Compounds 7 (29.9%), 8 (21.7%), 10 (36.9%), 11 (27.6%), 16
(21.2%), 17 (20.8%) and 24 (26.8%) were showing significant blood
glucose lowering activity in SLM model (Table 1 and Fig. 2). The com-
pounds which showed greater than 12.0% blood glucose lowering
activity in SLM model, were further tested for anti-hyperglycemic
activity in sucrose challenged streptozotocin (STZ-S) induced
diabetic rats. Compound 7, 10, 11 and 24 have shown promising
antidiabetic activity in STZ-S model (Table 1 and Fig. 3).

A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
5287
O
O
O
O
O
O
(d)
(a)
(b)
(c)
Ph
OH
HO
H3CO
OH
H3CO
H3CO
OH
1
2
3
4
O
N
Ph
N
O
R1H = Dimethyl amine 6
Diethyl amine 7
diisopropylamine 8
Morpholine 9
O
(e)
H3CO
OH
H3CO
O(CH2)2R1
5
Pyrrolidine 10
6-11
Piperidine 11
N
O
N
O
O
H3CO
O(CH2)3R1
(f )
(g)
13-17
Cl
H3CO
R1H = Dimethylamin e 13
Diethyl amine 14
n-Methylbutylamine 15
Pyrrolidine16
12
N
O
Piperidine 17
(h)
H3CO
O(CH2)nCOOR1
18-21
R
1 = C2H5, n = 1, 18
C2H5, n = 3, 19
H, n = 1, 20
N
O
H, n = 3, 21
N
O
O
OH
O
(j)
H3CO
(i)
23-25
R1
H3CO
22
O
1
23
R H = Diethyl amine
24
25
Pyrrolidine
Piperidine
Scheme 1. Reagent and condition: (a) (CH3)2SO4, K2CO3, acetone; (b) benzoylchloride, pyridine, 1 h, rt; (c) KOH, pyridine, 8 h, rt; (d) NH2OHÁHCl, pyridine; (e) K2CO3, acetone,
Cl(CH2)2R1ÁHCl, 8–10 h, reflux; (f) K2CO3, acetone, Cl(CH2)3Br, 6 h, reflux; (g) DMF, KI (catalytic), amine, heat, 8–10 h; (h) K2CO3, acetone, Cl (CH2)nCOOR1, 8–10 h, reflux; (i)
K2CO3, acetone, epichlorohydrin, 7 h, reflux; (j) DMF/ethanol, amine, heat, 8–10 h.
200 MHz spectrometer. Chemical shifts (d) are given in ppm rela-
tive to TMS, coupling constants (J) in hertz. Mass spectra were ob-
tained by the using a JEOL SX-102 (FAB) instrument. IR spectra
were taken on a VARIAN FT-IR spectrometer as KBr pellets. Ele-
mental analysis was preformed at a Perkin Elmer Autosystem XL
Analyzer. Melting points were measured on a COMPLAB melting
point apparatus and all the melting points were uncorrected. Reac-
tions were monitored by thin-layer chromatography (TLC) carried
out on 0.25 mm silica gel plates visualized with UV light.
3. Conclusion
In conclusion, we have designed and synthesized a series of 3,5-
diarylisoxazole derivatives as potential anti-hyperglycemic agents.
Compounds were screened for their in vivo anti-hyperglycemic
activity in sucrose loaded rat model (SLM), sucrose-challenged
streptozotocin-induced diabetic rat model (STZ-S) and db/db mice
model. Compounds (7, 10, 11 and 24) were showing promising
anti-hyperglycemic activity in SLM and STZ-S models. Four
compounds (7, 10, 11 and 24) were showing promising anti-hyper-
glycemic and lipid lowering activity in db/db mice model. In order
to know the mode of anti-hyperglycemic action of the compounds,
we screened them in various models such as DPP-4, PTP1B and
4.1.1. Benzoic acid 2-acetyl-5-methoxy-phenyl ester (3)
2-Hydroxy-4-methoxy acetophenone 2 (1.66 g, 10 mmol) was
taken in 10 ml of dry pyridine and benzoyl chloride (1.38 ml,
12 mmol) was added to it. The reaction mixture was stirred for
1 h. After completion the reaction mixture was poured in 100 ml
of water, extracted with ethyl acetate (50 Â 3 ml) and dried over
sodium sulphate. Organic layer was concentrated to yield 3. Yield:
(2.4 g, 81.48%); FAB MS: (m/z) = 271 (M+1). IR (KBr, cmÀ1): 2961,
2362, 1732.
PPARc. Compounds did not show any significant activity in PPAR
and DPP-4 assays. The compounds exhibited promising PTP-1B
inhibitory activity thereby revealing their mode of anti-hypergly-
cemic action via PTP1B inhibition.
4. Experimental
4.1. Chemistry
4.1.2. Typical experimental procedure for the synthesis of 1-(2-
hydroxy-4-methoxyphenyl)-3-phenylpropane-1,3-dione (4)
Compound 3 (1.35 g, 5 mmol), potassium hydroxide (0. 84 g,
15 mmol) in 25 ml of pyridine was stirred at 25 °C for 8 h followed
by T.L.C. monitoring. After completion reaction mixture was
All the chemical reagents were purchased from Sigma–Aldrich
Chemical Company and were used directly without further any
purification. NMR spectra were obtained using the Brucker DRX

5288
A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
Table 1
Table 2
In vivo antihyperglycemic activity in SLM, STZ-S and db/db mice models
Dose dependent anti-hyperglycemic effect of 7, 10, 11 and 24 in diabetic rats
Entry
Compound
% Anti-hyperglycemic activity in vivoa
STZ-Sb db/db-mice modelb,c
24 h
Compound
Dose (mg/kg)
% Glucose lowering
ED50 (mg/kg)
45.3
SLMb
7
7.5
15
25
50
100
10.3
15.5*
19.5**
21.7**
25.4**
5 h
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
9.8
ND
ND
—
16.8
29.9**
21.7*
2.8
15.3
26.9***
11.4
ND
27.7***
17.6*
ND
15.9
25.7***
9.7
—
21.0*
—
10
11
24
7.5
15
25
50
100
11.5
13.7
15.2*
18.5**
27.7**
54.8
64.5
80.2
ND
—
36.9**
27.6**
9.24
11.8
14.3
15.0
21.2*
20.8*
10.4
4.6
11.7
14.2
10.7
13.8
26.8**
17.0
—
28.7***
18.7**
ND
22.8*
18.5*
—
7.5
15
25
50
100
9.8
ND
ND
—
12.6
14.0*
16.7**
20.7**
7.8
9.2
—
12.6
13.4
14.7
ND
14.2
11.2
15.0
ND
—
—
—
—
7.5
15
25
50
100
8.4
ND
ND
—
11.6
13.8*
17.9*
18.5**
ND
ND
—
21
22
23
24
14.9
ND
14.2
ND
—
—
11.6
18.5**
15.8
—
11.4
17.6*
16.8
—
—
15.7
—
Values are expressed as mean SD, N = 5, p < 0.05 ( ), p < 0.01 ( ) and p < 0.001
**
*
25
(
) versus control. Statistical analysis was made by Dunnett’s test (Prism
***
Na2VO3
Metformin
Glybenclamide
Glyclazide
Software).
12.9
33.9
44.8
19.1
32.8
27.7
20.2
—
—
11.2
13.6
17.4
Table 3
a
Anti-hyperglycemic and lipid lowering activity in db/db mice model
Experiments were carried out at 100 mg/kg dose.
Values are expressed as mean SD, n = 5, *p < 0.05, **p < 0.01 and ***p < 0.001
b
Entry (Compd No.) Anti hyperglycemic and lipid lowering activity in db/db mice
(% efficacy, 6 and 10 days) (100 mg/kg)
versus vehicle treated control.
c
Three days protocol.
Anti-hyperglycemic
Lipid lowering
activity
6 Days
10 Days
TG
CHOL.
HDL
7
22.6**
21.4**
17.4*
15.9*
21.5**
21.6**
16.1*
15.5*
À12.0
À10.5
À7.5
À14.5*
À17.6*
À12.2
À9.8
+3.9
+3.0
+1.3
+1.8
10
11
24
À8.5
Values are expressed as mean SD, N = 5, p < 0.05( ), and p < 0.01( ) versus control.
*
**
Statistical analysis was made by Dunnett’s test (Prism Software).
poured in 150 ml of water and neutralized with 10% CH3COOH
solution. Reaction mixture was extracted with ethyl acetate
(50 Â 4 ml) washed with sodium bicarbonate solution and dried
over sodium sulphate. The ethyl acetate layer was concentrated
to give crude product. The crude product was purified by silica
gel column chromatography using hexane, ethylacetate (4:1) as
eluent to yield compound 4. Yield: (1.10 g, 81.48%), FAB MS: (m/
z) = 271 (M+1). IR: (KBr, cmÀ1): 2961, 2362, 1602. 1H NMR: CDCl3,
200 MHz) d: 3.73 (s, 3H, OCH3,), 6.35 (m, 2H, ArH), 6.60 (s, 1H,
Figure 2. Anti-hyperglycemic activity of compounds 7, 8, 10, 11, 16, 17 and 24 in
SLM model. Statistical analysis was made by Dunnet test (Prism Software 3). Values
were expressed as mean SD, n = 5.
Triglyceride lowering activity of Compounds
140
135
130
7.5%
8.5%
10.5%
125
120
115
110
105
100
12.0%
Control
7
10
11
24
Compound (100 mg/kg)
Figure 3. Anti-hyperglycemic activity of compounds 7, 8, 10, 11, 16, 17 and 24 in
STZ model. Statistical analysis was made by Dunnet test (Prism Software 3). Values
were expressed as mean SD, n = 5.
Figure 4. Triglyceride lowering activity of compounds 7, 10, 11 and 24 in db/db
mice model. Statistical analysis was made by Dunnet’s test (Prism Software 3).

A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
5289
enolic), 7.43 (m, 4H, ArH), 7.82 (m, 2H, ArH). Anal. Calcd for
C16H14O4: C, 71.10; H, 5.22. Found: C, 71.04; H, 5.15.
Cholesterol lowering activity of Compounds
260
240
220
200
180
160
140
120
100
9.8%
14.5%
*
12.2%
4.1.3. Typical experimental procedure for the synthesis of 5-
methoxy-2-(3-phenylisoxazol-5-yl)phenol (5)
17.6%
*
Compound 4 (2.7 g, 10 mmol) and NH2OHÁHCl (1.3 g, 13 mmol)
was taken in 30 ml of dry pyridine. Reaction mixture was stirred at
25 °C for 30 min. After completion (TLC), the reaction mixture was
neutralised with 6 M HCl. Then the reaction mixture was extracted
with ethylacetate (50 Â 5 ml) and dried over sodium sulphate.
Organic layer was concentrated to remove ethyl acetate to yield
a solid which was recrystallized with acetone to yield compound
5, Yield: (1.20 g, 44.94%); mp 235 °C (acetone); FAB MS (m/
z) = 268 (M+1). 1H NMR (DMSOd6, 200 MHz) d: 3.86 (s, 3H,
OCH3), 6.59 (s, 1H, ArH), 6.62 (m, 1H, ArH), 7.20 (s, 1H, isoxazole),
7.53 (m, 3H, ArH), 7.92 (d, J = 2.0 Hz, 1H, ArH), 7.94 (m, 2H, ArH).
Anal. Calcd for C16H113NO3: C, 71.90; H, 4.90; N, 5.24. Found: C,
71.84; H, 5.00; N, 5.16.
Control
7
10
11
24
Compound (100 mg/kg)
Figure 5. Cholesterol lowering activity of compounds 7, 10, 11 and 24 in db/db
mice model. Statistical analysis was made by Dunnet’s test (Prism Software 3).
4.1.4. Typical experimental procedure for the synthesis of
compounds (6–11)
HDL-cholesterol increasing activity of Compounds
39
38.5
38
3.9%
Compound 5 (1.34 g, 5 mmol), Cl(CH2)2R1.HCl (6 mmol) and
K2CO3 (2.07 g, 15 mmol) were taken in 30 ml of acetone. It was
refluxed for 8 h. After completion the reaction, K2CO3 was filtered
off and the filtrate was concentrated to yield crude product. The
crude product was subjected to column chromatography to yield
pure product 6–11.
3.0%
1.8%
1.3%
37.5
37
36.5
36
4.1.4.1. 2-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)-N,N-
dimethylethanamine (6). Yield (90.50%), mp 97 °C (ethyl ace-
tate/hexane). FAB MS (m/z) = 339 (M+H). 1H NMR (CDCl3,
200 MHz) d: 2.30 (s, 6H, NCH3), 2.85 (t, J = 5.8 Hz, 2H, NCH2),
3.86 (s, 3H, OCH3), 4.19 (t, J = 5.8 Hz, 2H, OCH2), 6.55 (s, 1H, ArH),
6.59 (m,1H, ArH), 7.26 (s, 1H, isoxazole), 7.47 (m, 3H, ArH), 7.89
(m, 3H, ArH). Anal. Calcd for C20H22N2O3: C, 70.99; H, 6.55; N,
8.28. Found: C, 71.10; H, 6.48; N, 8.19.
Control
7
10
Compound (100 mg/kg)
11
24
Figure 6. HDL-cholesterol increasing activity of compounds 7, 10, 11, 24 on plasma
lipid profiles in db/db mice, values are expressed as mean SD, n = 5, *p < 0.05
versus vehicle treated control db/db mice.
Table 4
4.1.4.2. Diethyl-{2-[5-methoxy-2-(3-phenyl-isoxazol-5-yl)-
phenoxy]-ethyl}-amine (7). Yield (84.42%), mp 58 °C. FAB MS (m/
z) = 367 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.07 (t, J = 7.2 Hz, 6H,
CH3), 2.60–2.78 (m, 4H, NCH2), 2.97 (t, J = 6.2 Hz, 2H, NCH2), 3.83
(s, 3H, OCH3), 4.16 (t, J = 6.8 Hz, 2H, OCH2), 6.56 (s, 1H, ArH), 6.62
(m, 1H, ArH), 7.17 (s, 1H, isoxazole), 7.46 (m, 3H, ArH), 7.89 (m,
3H, ArH). Anal. Calcd for C22H26N2O3: C, 72.11; H, 7.15; N, 7.64.
Found: C, 72.04; H, 7.11; N, 7.56.
PTP1B inhibitory activity for the active compounds of in vivo models
Entry
Compound
PTP-1B inhibitory activitya
Inhibtiona (%)
ÀTriton 100a,b
+Triton 100a,b
IC50 M)
IC50
(
l
M)
(l
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21.8
33.5
94.7
89.4
82.9
99.2
76.3
9.0
22.3
21.8
14.4
82.7
89.5
79.3
20.1
75.2
24.4
11.8
82.8
86.2
85.9
94.0
—
27.45
24.16
2.97
5.50
2.50
1.50
8.80
56.78
25.28
28.03
37.31
3.50
4.40
5.25
26.73
9.10
22.32
31.11
4.30
3.40
6.80
7.70
—
27.63
24.29
2.90
5.44
2.61
4.1.4.3. N-Isopropyl-N-(2-(5-methoxy-2-(3-phenylisoxazol-5-yl)
phenoxy)ethyl)propan-2-amine (8). Yield (86.40%), MS (m/
z) = 395 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.08 (d, J = 6.2 Hz,
12H, CH3), 2.60–2.74 (m, 2H, NCH2), 3.75–3.79 (m, 4H, NCH,
OCH2), 3.83 (s, 3H, OCH3), 6.56 (s, 1H, ArH), 6.60–6.62 (m, 1H,
ArH), 7.17 (s, 1H, isoxazole), 7.42–7.52 (m, 3H, ArH), 7.82–7.90
(m, 3H, ArH). Anal. Calcd for C24H30N2O3: C, 73.07; H, 7.66; N,
7.10. Found: C, 73.04; H, 7.54; N, 6.96.
1.51
9.00
56.89
25.34
28.16
37.46
3.46
4.42
5.35
26.71
9.30
22.36
31.32
6.89
4.1.4.4. 4-(2-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)
ethyl) morpholine (9). Yield (85.09%), mp 140 °C (ethyl acetate/
hexane). FAB MS (m/z) = 381 (M+H). 1H NMR (CDCl3, 200 MHz) d:
2.30–2.56 (m, 6H, NCH2), 3.60–3.76 (m, 4H, OCH2), 3.83 (s, 3H,
OCH3), 4.02 (t, J = 7.3 Hz, 2H, OCH2), 6.50 (s,1H, ArH), 6.54–6.59
(m, 1H, ArH), 7.18 (s, 1H, oxazole), 7.44–7.62 (m, 3H, ArH), 7.80–
7.91 (m, 3H, ArH). Anal. Calcd for C22H24N2O4: C, 69.46; H, 6.46;
N, 7.36. Found: C, 69.38; H, 6.40; N, 7.24.
21
22
23
24
4.20
3.31
25
Na 2VO3
Metformin
Glybenclamide
Glyclazide
—
—
—
—
—
—
—
a
Values are mean from three independent sets of experiments screened at 10
concentration, ND = not done.
lM
4.1.4.5. [4-Methoxy-2-(2-pyrrolidin-1-yl-ethoxy)-phenyl]-3-
phenyl-isoxazole (10). Yield (85.85%), mp 98 °C (ethyl acetate/
b
Compounds were tested with Triton X-100.

5290
A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
hexane). FAB MS (m/z) 365 (M+H), 1H NMR (CDCl3, 200 MHz) d:
1.78–1.88 (m, 4H, CH2), 2.62–2.78 (m, 4H, NCH2), 3.02 (t,
J = 5.8 Hz, 2H, NCH2), 3.85 (s, 3H, OCH3), 4.23 (t, J = 4.2 Hz, 2H,
OCH2), 6.53 (s, 1H, ArH), 6.56–6.59 (m, 1H, ArH), 7.24 (s, 1H, isox-
azole), 7.48 (m, 3H, ArH), 7.80–7.92 (m, 3H, ArH). Anal. Calcd for
C22H24N2O3: C, 72.50; H, 6.64; N, 7.69. Found: C, 72.58; H, 6.60;
N, 7.58.
CH3), 2.44–2.60 (m, 4H, NCH2), 3.80 (s, 3H, OCH3), 4.00 (t,
J = 7.1 Hz, 2H, OCH2), 6.50 (s, 1H, ArH), 6.60–6.63 (m, 1H, ArH),
7.19 (s, 1H, isoxazole), 7.42–7.54 (m, 3H, ArH), 7.79–7.90 (m, 3H,
ArH). Anal. Calcd for C24H30N2O3: C, 73.07; H, 7.66; N, 7.10. Found:
C, 72.96; H, 7.54; N, 7.00.
4.1.6.4. 5-(4-Methoxy-2-(3-(pyrrolidin-1-yl) propoxy) phenyl)-
3-phenylisoxazole (16). Yield (82.50%), FAB MS (m/z) = 379 (M+H).
1H NMR (CDCl3, 200 MHz) d: 1.68–1.84 (m, 4H, CH2), 1.89–2.00 (m,
2H, CH2), 2.46–2.74 (m, 6H, NCH2), 3.80 (s, 3H, OCH3), 4.02 (t,
J = 7.2 Hz, 2H, OCH2), 6.49 (s, 1H, ArH), 6.60–6.62 (m, 1H, ArH),
7.20 (s, 1H, isoxazole), 7.40–7.53 (m, 3H, ArH), 7.80–7.90 (m, 3H,
ArH). Anal. Calcd for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40. Found:
C, 72.90; H, 7.00; N, 7.30.
4.1.4.6. 1-{2-[5-Methoxy-2-(3-phenyl-isoxazole-5-yl)-phenoxy]-
ethyl}-piperidine (11). Yield (83.59%), mp 124 °C (ethyl acetate/hex-
ane). FAB MS (m/z) = 379 (M+H). 1H NMR (CDCl3, 200 MH2) d: 1.46 (m,
2H, CH2), 1.64–1.76 (m, 4H, CH2), 2.53–2.66 (m, 4H, NCH2 of piperi-
dine ring), 2.87 (t, J = 6.0 Hz, 2H, NCH2), 3.81 (s, 3H, OCH3), 4.20 (t,
J = 5.8 Hz, 2H, OCH2), 6.53 (s,1H, ArH), 6.56–6.59 (m, 1H, ArH), 7.18
(s, 1H, oxazole), 7.42–7.55 (m, 3H, ArH), 7.82–7.95 (m, 3H, ArH). Anal.
Calcd for C23H26N2O3: C, 72.99; H, 6.92; N, 7.40. Found: C, 73.08; H,
6.81; N, 7.30.
4.1.6.5. 5-(4-Methoxy-2-(3-(piperidin-1-yl) propoxy) phenyl)-3-
phenylisoxazole (17). Yield (80.35%), FAB MS (m/z) = 393 (M+H).
1H NMR (CDCl3, 200 MHz) d: 1.68–1.84 (m, 4H, CH2), 1.89–2.00
(m, 2H, CH2), 2.46–2.74 (m, 6H, NCH2), 3.80 (s, 3H, OCH3), 4.02
(t, J = 7.2 Hz, 2H, OCH2), 6.49 (s, 1H, ArH), 6.60–6.62 (m, 1H,
ArH), 7.20 (s, 1H, isoxazole), 7.40–7.53 (m, 3H, ArH), 7.80–7.90
(m, 3H, ArH). Anal. Calcd for C24H28N2O3: C, 73.44; H, 7.19; N,
7.14. Found: C, 73.49; H, 7.10; N, 7.02.
4.1.5. Typical experimental procedure for the synthesis of 5-(2-
(3-chloropropoxy)-4-methoxyphenyl)-3-phenyl isoxazole (12)
Compound 5 (1.34 g, 5 mmol), Cl(CH2)3Br (0.93 g, 6 mmol) and
K2CO3 (2.07 g, 15 mmol) were taken in 30 ml of acetone. The reac-
tion mixture was refluxed for 8 h. After completion the reaction,
K2CO3 was filtered off and the filtrate was concentrated to yield
crude product. The crude product was subjected to column chro-
matography to yield pure product 12. Yield (92.50%), FAB MS (m/
z) = 344 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.90–2.10 (m, 2H,
CH2), 3.50 (t, J = 7.2 Hz, 2H, ClCH2), 3.81 (s, 3H, OCH3), 4.10 (t,
J = 6.8 Hz, 2H, OCH2), 6.56 (s, 1H, ArH), 6.58–6.60 (m, 1H, ArH),
7.17 (s, 1H, isoxazole), 7.40–7.53 (m, 3H, ArH), 7.80–7.91 (m, 3H,
ArH). Anal. Calcd for C19H18ClNO3: C, 66.38; H, 5.28; N, 4.07.
Found: C, 66.45; H, 5.24; N, 3.94.
4.1.7. Typical experimental procedure for the synthesis of
compounds (18–21)
Compound 5 ((1.34 g, 5 mmol)), Cl(CH2)nCOOR1 (6 mmol) and
K2CO3, (2.07 g, 15 mmol) were taken in 30 ml of acetone. The reac-
tion mixture was refluxed for 8–10 h. After completion the reac-
tion, K2CO3 was filtered off and the filtrate was concentrated to
yield crude product. The crude product was subjected to column
chromatography to yield pure product 18–21.
4.1.6. General synthetic procedure for synthesis of compounds
(13–17)
4.1.7.1. Ethyl 2-(5-methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)
acetate (18). Yield (88.80%), FAB MS (m/z) = 354 (M+H). 1H NMR
(CDCl3, 200 MHz) d: 1.17 (t, J = 7.1 Hz, 3H, CH3), 3.80 (s, 3H,
OCH3), 4.05 (t, J = 7.1 Hz, 2H, OCH2), 5.10 (s, 2H, OCH2), 6.50 (s,
1H, ArH), 6.60–6.62 (m, 1H, ArH), 7.19 (s, 1H, isoxazole), 7.40–
7.53 (m, 3H, ArH), 7.78–7.90 (m, 3H, ArH). Anal. Calcd for
C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found: C, 67.86; H, 5.44;
N, 3.85.
Compound 12 (0.343 g, 1 mmol), amine (2 mmol) in 10 ml DMF
were taken. To it KI (0.1 g) was added and the reaction mixture was
refluxed for 8–10 h. After completion the reaction mixture was
cooled and poured in 100 ml of water and extracted with ethyl ace-
tate (50 ml). The ethyl acetate layer was dried over anhydrous
sodium sulphate and concentrated to yield crude product. The
crude product was purified by silica gel column chromatography
to yield compounds 13–17.
4.1.7.2. Ethyl 4-(5-methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)
butanoate (19). Yield (90.50%), FAB MS (m/z) = 382 (M+H). 1H
NMR (CDCl3, 200 MHz) d: 1.19 (t, J = 7.1 Hz, 3H, CH3), 2.16–2.36
(m, 4H, CH2), 3.81 (s, 3H, OCH3), 4.00–4.10 (m, 4H, OCH2), 5.10
(s, 2H, OCH2), 6.50 (s, 1H, ArH), 6.60–6.62 (m, 1H, ArH), 7.20 (s,
1H, isoxazole), 7.42–7.55 (m, 3H, ArH), 7.79–7.94 (m, 3H, ArH).
Anal. Calcd for C22H23NO5: C, 69.28; H, 6.08; N, 3.67. Found: C,
69.22; H, 6.00; N, 3.55.
4.1.6.1. 3-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)-N,N-
dimethylpropan-1-amine (13). Yield (80.58%), FAB MS (m/
z) = 353 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.94–2.02 (m, 2H,
CH2), 2.32 (s, 6H, NCH3), 2.76 (t, J = 5.8 Hz, 2H, NCH2), 3.79 (s, 3H,
OCH3), 4.02 (t, J = 6.9 Hz, 2H, OCH2), 6.50 (s, 1H, ArH), 6.60–6.63
(m, 1H, ArH), 7.18 (s, 1H, isoxazole), 7.40–7.54 (m, 3H, ArH),
7.80–7.93 (m, 3H, ArH). Anal. Calcd for C21H24N2O3: C, 71.57; H,
6.86; N, 7.95. Found: C, 71.48; H, 6.74; N, 8.08.
4.1.7.3. 2-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)acetic
acid (20). Yield (92.50%), FAB MS (m/z) = 326 (M+H). 1H NMR
(CDCl3, 200 MHz) d: 3.82 (s, 3H, OCH3), 5.00 (s, 2H, OCH2), 6.50
(s, 1H, ArH), 6.60–6.62 (m, 1H, ArH), 7.20 (s, 1H, isoxazole), 7.42–
7.55 (m, 3H, ArH), 7.79–7.94 (m, 3H, ArH). Anal. Calcd for
C18H15NO5: C, 66.46; H, 4.65; N, 4.31. Found: C, 69.22; H, 4.56;
N, 4.19.
4.1.6.2. N,N-Diethyl-3-(5-methoxy-2-(3-phenylisoxazol-5-yl)
phenoxy)propan-1-amine (14). Yield (78.80%), FAB MS (m/
z) = 381 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.07 (t, J = 7.2 Hz,
6H, CH3), 1.92–2.02 (m, 2H, CH2), 2.64–2.90 (m, 6H, NCH2), 3.80
(s, 3H, OCH3), 4.04 (t, J = 7.1 Hz, 2H, OCH2), 6.48 (s, 1H, ArH),
6.60–6.62 (m, 1H, ArH), 7.19 (s, 1H, isoxazole), 7.40–7.53 (m, 3H,
ArH), 7.78–7.90 (m, 3H, ArH). Anal. Calcd for C23H28N2O3: C,
72.60; H, 7.42; N, 7.36. Found: C, 72.56; H, 7.44; N, 7.24.
4.1.7.4. 4-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy) buta-
noic acid (21). Yield (90.65%), FAB MS (m/z) = 354 (M+H). 1H NMR
(CDCl3, 200 MHz) d: 2.12–2.30 (m, 4H, CH2), 3.81 (s, 3H, OCH3),
4.20 (s, 2H, OCH2), 6.50 (s, 1H, ArH), 6.60–6.62 (m, 1H, ArH), 7.20
(s, 1H, isoxazole), 7.42–7.55 (m, 3H, ArH), 7.79–7.94 (m, 3H,
ArH). Anal. Calcd for C20H19NO5: C, 67.98; H, 5.42; N, 3.96. Found:
C, 67.92; H, 5.54; N, 4.09.
4.1.6.3. N-(3-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)
propyl)-N-methylbutan-1-amine (15). Yield (83.70%), FAB MS (m/
z) = 395 (M+H). 1H NMR (CDCl3, 200 MHz) d: 0.98 (t, J = 6.7 Hz, 3H,
CH3), 1.32–1.46 (m, 4H, CH2), 1.90–2.02 (m, 2H, CH2), 2.30 (s, 3H,

A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
5291
4.1.8. Typical experimental procedure for the synthesis of 5-(4-
methoxy-2-(oxiran-2-ylmethoxy)phenyl)-3-phenyl isoxazole
(22)
erated in the reaction mixture. Sodium orthovanadate was taken
as standard in enzyme assay. The IC50 of the compounds were deter-
mined by constructing a dose-response curve and examining the ef-
fect of different concentrations of compounds.
Compound
5
(1.34 g, 5 mmol), epichlorohydrin (0.92 g,
10 mmol) and K2CO3 (2.07 g, 15 mmol) were taken in 30 ml of ace-
tone. The reaction mixture was refluxed for 7 h. After completion
the reaction, K2CO3 was filtered off and the filtrate was concen-
trated to yield crude product. The crude product was subjected
to column chromatography to yield pure product 22. Yield
(80.60%), FAB MS (m/z) = 324 (M+H). 1H NMR (CDCl3, 200 MHz)
d: 3.12 (s, 1H, CH), 3.42–3.56 (m, 2H, OCH2), 3.81 (s, 3H, OCH3),
4.00–4.20 (m, 2H, OCH2), 6.50 (s, 1H, ArH), 6.60–6.62 (m, 1H,
ArH), 7.20 (s, 1H, isoxazole), 7.42–7.55 (m, 3H, ArH), 7.79–7.94
(m, 3H, ArH). Anal. Calcd for C19H17NO4: C, 70.58; H, 5.30; N,
4.33. Found: C, 70.50; H, 5.24; N, 4.20.
4.2.2. Sucrose loaded rat model (SLM)35
Male albino rats of Charles Foster/Wistar strain of average body
weight 160 20 g were selected for this study. The blood glucose
level of each animal was checked by glucometer using glucostrips
(Boehringer Mannheim) after 16 h starvation. Animals showing
blood glucose levels between 3.33 and 4.44 mM (60–80 mg/dl)
were divided into groups of five to six animals in each. Animals
of experimental group were administered suspension of the
desired synthetic compound orally (made in 1.0% gum acacia) at
a dose of 100 mg/kg body weight. Animals of control group were
given an equal amount of 1.0% gum acacia. A sucrose load
(10.0 g/kg) was given to each animal orally exactly after 30 min
post administration of the test sample/vehicle. Blood glucose pro-
file of each rat was again determined at 30, 60, 90 and 120 min
post administration of sucrose by glucometer Food but not water
was withheld from the cages during the course of experimentation.
Quantitative glucose tolerance of each animal was calculated by
Area Under Curve (AUC) method (Prism Software). Comparing
the AUC of experimental and control groups determined the per-
centage anti-hyperglycemic activity.
4.1.9. Typical experimental procedure for the synthesis of
compounds (23–25)
Compound 22 (5 mmol) and amine (10 mmol) were taken in
10 ml of DMF and heated for 8–10 h. After completion, the reaction
mixture was poured in 100 ml of water and extracted with ethyl
acetate (50 ml  2). The ethyl acetate layer was dried over anhy-
drous sodium sulphate and concentrated to yield crude product.
The crude product was purified by column chromatography to tield
compound 23–25.
4.1.9.1. 1-(Diethylamino)-3-(5-methoxy-2-(3-phenylisoxazol-5-
yl)phenoxy)propan-2-ol (23). Yield (90.56%), FAB MS (m/z) = 397
(M+H). 1H NMR (CDCl3, 200 MHz) d: 1.10 (t, J = 7.2 Hz, 6H, CH3)
2.42–2.60 (m, 6H, CH2), 3.22 (s, 1H, OH), 3.84 (s, 3H, OCH3),
3.95–4.21 (m, 3H, OCH, OCH2), 6.50 (s, 1H, ArH), 6.60–6.62 (m,
1H, ArH), 7.20 (s, 1H, isoxazole), 7.42–7.55 (m, 3H, ArH), 7.79–
7.94 (m, 3H, ArH). Anal. Calcd for C23H28N2O4: C, 69.67; H, 7.12;
N, 7.07. Found: C, 69.55; H, 7.20; N, 6.91.
4.2.3. Sucrose-challenged streptozotocin-induced diabetic rat
model (STZ-S)35
Male albino rats of Sprague Dawley strain of body weight
160 20 g were selected for this study. Streptozotocin (Sigma,
USA) was dissolved in 100 mM citrate buffer pH 4.5 and calculated
amount of the fresh solution was injected to overnight fasted rats
(60 mg/kg) intraperitoneally. Blood glucose level was checked 48 h
later by glucostrips and animals showing blood glucose values
between 144 and 270 mg/dl (8–15 mM) were included in the exper-
iment and considered as diabetic. The diabetic animals were divided
into groups consisting of five to six animals in each group. Animals of
experimental groups were administered suspension of the desired
test samples orally (made in 1.0% gum acacia) at a dose of 100 mg/
kg body weight. Animals of 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. After 30 min
of post sucrose load blood glucose level was again checked by gluco-
strips at 30, 60, 90, 120, 180, 240, 300 min and at 24 h, respectively.
Animals not found diabetic after 24 h post treatment of the test sam-
ple were not considered and omitted from the calculations and
termed as non-responders. The animals, which did not show any fall
in blood glucose profile in a group while the others in that group,
showed fall in blood glucose profile were also considered as non-
responders. Food but not water was withheld from the cages during
the experimentation. Comparing the AUC of experimental and con-
trol groups determined the percent antihyperglycemic activity.
4.1.9.2.
1-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)-3-
(pyrrolidin-1-yl)propan-2-ol (24). Yield (89.70%), FAB MS (m/
z) = 395 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.10 (t, J = 7.2 Hz,
6H, CH3) 2.42–2.60 (m, 6H, CH2), 3.30 (s, 1H, OH), 3.84 (s, 3H,
OCH3), 3.95–4.21 (m, 3H, OCH, OCH2), 6.50 (s, 1H, ArH), 6.60–
6.62 (m, 1H, ArH), 7.20 (s, 1H, isoxazole), 7.42–7.55 (m, 3H, ArH),
7.79–7.94 (m, 3H, ArH). Anal. Calcd for C23H26N2O4: C, 70.03; H,
6.64; N, 7.10. Found: C, 69.95; H, 6.50; N, 6.90.
4.1.9.3. 1-(5-Methoxy-2-(3-phenylisoxazol-5-yl)phenoxy)-3-
(piperidin-1-yl)propan-2-ol (25). Yield (85.50%), FAB MS (m/
z) = 409 (M+H). 1H NMR (CDCl3, 200 MHz) d: 1.40–1.58 (m, 6H,
CH2) 2.40–2.68 (m, 6H, NCH2), 3.42 (s, 1H, OH), 3.80 (s, 3H,
OCH3), 4.00–4.19 (m, 3H, OCH & OCH2), 6.50 (s, 1H, ArH), 6.60–
6.62 (m, 1H, ArH), 7.20 (s, 1H, isoxazole), 7.42–7.55 (m, 3H, ArH),
7.79–7.94 (m, 3H, ArH). Anal. Calcd for C24H28N2O4: C, 70.57; H,
6.91; N, 6.86. Found: C, 70.48; H, 6.80; N, 6.78.
4.2. Pharmacology
4.2.4. Anti-hyperglycemic activity in db/db mice36
The db/db mouse is a well-characterised model of type 2 diabe-
tes. The background for the db/db mouse is the C57BL/Ks strain.
The optimal age of db/db mice used for experiments is from week
12 to 18 when they have developed NIDDM with diabetic dyslipi-
demia but still have functional b-cells in the pancreas. C57BL/
KsBom-db mice 12–18 weeks, 40–50 g bred in the animal house
of CDRI, Lucknow. Ten mice (5 males and 5 females) were used
in the experiments. The mice were housed in groups of 5 (same
sex) in a room controlled for temperature (23 2.0 °C) and 12/
12 h light/dark cycle (lights on at 6.00 am). Body weight was
measured daily from day 1 to day 10. All animals had free access
to fresh water and to normal chow except on the days of the post-
4.2.1. Protein-tyrosine-phosphatase-1B inhibitory assay
Protein tyrosine phosphatase inhibitory activity of compounds
was determined by modified method of Goldstein et al.34 The test
compounds were pre-incubated for 10 min with the enzyme in the
absence and presence of 0.01% Triton X–100. Assay was performed
in final volume of 1.0 ml in test mixture containing 10 mM of pNPP
in 50 mM HEPES buffer (pH 7.0) with 1 mM DTT and 2 mM EDTA.
Thereaction wasstopped after30 minof incubationat 37 °C byaddi-
tion of 500 ll of 0.1 N NaOH and the absorbance was determined at
410 nm. A molar extinction coeffcient of 1.78 Â 104 MÀ1 cmÀ1 was
utilized to calculate the concentration of p-nitrophenolate ion gen-

5292
A. Kumar et al. / Bioorg. Med. Chem. 17 (2009) 5285–5292
3. Cao, Y.; Lam, L. Drugs Today 2002, 38, 419.
prandial protocol day 6 and during the overnight fast before the
OGTT on day 10. The animals always had access to water during
experimental periods. Blood glucose was checked every morning
up till day 5. On day 6 postprandial protocol was employed, in this
method blood glucose was checked at À0.5 h and 0 h. Test drugs
were given to the treatment group whereas vehicle received only
gum acacia (1.0%); the blood glucose was again checked at 1, 2,
3, 4 and 6 h post test drug treatment. On day 8, blood was collected
for estimation of serum lipid parameters and finally on day 10 an
oral glucose tolerance test (OGTT) was performed after an over-
night fasting. Blood glucose was measured at À30.0 min and test
drugs were administered. The blood glucose was again measured
at 0.0 min post treatment and at this juncture glucose solution
was given at a dose of 3 gm/kg to all the groups including vehicle.
The blood glucose levels were checked at 30 min, 60 min, 90 min
and 120 min post glucose administration.
4. Ling, R.; Yoshida, M.; Mariano, P. S. J. Org. Chem. 1996, 61, 4439.
5. Mudaliar, S.; Henry, R. R. Annu. Rev. Med. 2001, 52, 239.
6. Zimmet, P.; Alberti, K. G.; Shaw, J. Nature 2001, 414, 782.
7. Haimoto, H.; Iwata, M.; Wakai, K.; Umegaki, H. Diabetes Res. Clin. Pract. 2008,
79, 350.
8. Prato, S. D. Metabolism 2006, 55, 435.
9. Perfetti, R.; Ahmad, A. Trends Endocrinol. Metab. 2000, 11, 218.
10. Turner, N. C.; Clapham, J. C. Prog. Drug Res. 1998, 51, 33.
11. Astrup, A.; Breum, L.; Toubo, S. Obesity 1995, 4, 537S.
12. Kelley, D. E. Diabetes Rev. 1995, 3, 366.
13. Babenko, A. P.; Aguilar-Bryan, L.; Bryan, J. Annu. Rev. Physiol. 1998, 60,
667.
14. Aguilar-Bryan, L.; Clement, J. P.; Gonzalez, G.; Kunjilwar, K.; Babenko, A. J.
Bryan Physiol. Rev. 1998, 78, 227.
15. Ram, V. J. Prog. Drug Res. 2003, 60, 93.
16. Diamant, M.; Heine, R. J. Drugs 2003, 63, 1373.
17. Einhorn, D.; Aroda, V. R.; Henry, R. R. Endocrinol. Metab. Clin. North America
2004, 33, 595.
18. Balfour, J. A.; Plosker, G. L. Drugs 1999, 57, 921.
19. Wagstaff, A. J.; Goa, K. L. Drugs 2002, 62, 1805.
20. Coniff, R.; Krol, A. Clin. Ther. 1997, 19, 16.
21. Cusi, K.; DeFronzo, R. A. Diabetes Rev. 1998, 6, 89.
22. Dunn, C. J.; Peters, D. H. Drugs 1995, 49, 721.
23. Moller, D. E. Nature 2001, 414, 821.
24. Klein, R. Diabetes Care 1995, 18, 258.
25. Huijsduijnen, R. H. V.; Bombrun, A.; Swinnen, D. Drug Discovery Today 2002, 7,
1013.
4.2.5. DPP4 enzyme inhibition essay
Enzyme inhibition essay was done as described in the litera-
ture.37 Enzymatic activity was determined at 37 °C by the cleavage
rate of a substrate, Gly-Pro-AMC (30 lM) (Sigma–Aldrich, USA).
Briefly, 10
lL of DPP-IV solution was added to each well of a 96-
26. Huijsduijnen, H. V.; Sauer, W. H. B.; Bombrun, A.; Swinnen, D. J. Med. Chem.
2004, 47, 4142.
well flat-bottomed microtiter plate, followed by the addition of
27. Malamas, M. S.; Sredy, J.; Gunawan, I.; Mihan, B.; Sawicki, D. R.; Seestaller, L.;
Sullivan, D.; Flam, B. R. J. Med. Chem. 2000, 43, 995.
28. Xie, J.; Seto, C. T. Bioorg. Med. Chem. 2007, 15, 458.
29. Kuhn, B.; Hilpert, H.; Benz, J.; Binggeli, A.; Grether, U.; Humm, R.; Maerki, H. P.;
Meyer, M.; Mohr, P. Bioorg. Med. Chem. Lett. 2006, 16, 4016.
30. Yu, J.; Tang, L.; Yang, Y.; Ji, R. Eur. J. Med. Chem. 2008, 43, 2428.
31. Kondo, T.; Sugimoto, I.; Nekado, T.; Ochi, K.; Ohtani, T.; Tajima, Y.;
Yamamoto, S.; Kawabata, K.; Nakai, H.; Toda, M. Bioorg. Med. Chem. Lett.
2007, 15, 2715.
32. Backes, B. J.; Longenecker, K.; Hamilton, G. L.; Stewart, K.; Lai, C.; Kopecka, H.;
von Geldern, T. W.; Madar, DJ.; Pei, Z.; Lubben, T. H.; Zinker, B. A.; Tian, Z.;
Ballaron, S. J.; Stashko, M. A.; Mika, A. K.; Beno, D. W. A.; Kempf-Grote, A. J.;
Schaefer, C.; Shama, B. H. L.; Trevillyana, J. M. Bioorg. Med. Chem. Lett. 2007, 17,
2005.
50
20
l
L of 60
l
L Gly-Pro-AMC, 10
l
L of 500 mM Tris–HCl (pH 7.4),
lL of distilled water, and 10
l
L of a test compound. The change
of fluorescence was monitored at 37 °C using a spectrofluorometer
(excitation at 355 nm/emission at 460 nm) (fmax, Molecular
Devices, USA). The initial rate of DPP-IV enzyme activity was calcu-
lated over the first 15 min of the reaction, with units/mL being
defined as the rate of increase in the fluorescence intensity (arbi-
trary units) under these conditions.
Acknowledgements
33. Jain, P. K.; Makrandi, J. K.; Grover, S. K. Synthesis 1982, 221.
34. Goldstein, B. J.; Bittner-Kowalezyk, A.; White, M. F.; Harbeck, M. J. Biol. Chem.
2000, 275, 4283.
35. Maurya, R.; Akanksha, Jayendra; Singh, A. B.; Srivastava, A. K. Bioorg. Med.
Chem. Lett. 2008, 18, 6534.
Ram Awatar Maurya, Siddharth Sharma, Pervez Ahmad and
Amar Bahadur Singh are thankful to CSIR New Delhi for financial
support. Authors also acknowledge CSIR Network Project on Diabe-
tes for financial support.
36. Kumar, A.; Ahmad, P.; Maurya, R. A.; Singh, A. B.; Srivastava, A. K. Eur. J. Med.
Chem. 2009, 44, 109.
37. Wright, S. W.; Ammirati, M. J.; Andrews, K. M.; Brodeur, A. M.; Danley, D. E.;
Doran, S. D.; Lillquist, J. S.; McClure, L. D.; McPherson, R. K.; Orena, S. J.; Parker,
J. C.; Polivkova, J.; Qiu, X.; Soeller, W. C.; Soglia, C. B.; Treadway, J. L.;
VanVolkenberg, M. A.; Wang, H.; Wilder, D. C.; Olson, T. V. J. Med. Chem. 2006,
49, 3068.
References and notes
1. Rotella, D. P. J. Med. Chem. 2004, 47, 4111.
2. Skyler, J. S. J. Med. Chem. 2004, 47, 4113.