Design, synthesis and in-vivo hypoglycemic evaluation of novel non - TZD'S in a type - 2 diabetic model

Article abstract of DOI:10.2174/157340613804488468

With a view to develop novel non-TZD anti-diabetic compounds, series of isoxazolidinediones were designed to target the PPAR-γ receptors. Docking studies were performed on co-crystallized protein structure of rosiglitazone with PPAR-γ receptor obtained from Protein Data Bank (2PRG). Interactions similar to that of rosiglitazone were observed for three molecules; 3a, 3b and 3c which were further synthesized and subjected to in vivo hypoglycemic, total cholesterol (CHL) and triglyceride (TG) evaluation. 14 days treatment revealed significant reduction in blood glucose levels but did not portray desirable results in terms of total CHL and TG lowering effect. The blood glucose reduction observed for 3a, 3b and 3c at 20 mg/kg/day was 53.96 %, 61.35%, 61.32% respectively as against 59.95% of the standard pioglitazone at 10mg/kg/day.

Full text of DOI:10.2174/157340613804488468

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104
Medicinal Chemistry, 2013, 9, 104-111
Design, Synthesis and In-vivo Hypoglycemic Evaluation of Novel Non -
TZD’S in a Type - 2 Diabetic Model
Rupali Jadhav, Ranu Gupta-Rajoria, Tanushree Paland Ramaa Chelakara Subramanian*
Bharati Vidyapeeth’s College of Pharmacy, Sector-8, C.B.D., Belapur, Navi Mumbai, Maharashtra, 400614, India
Abstract: With a view to develop novel non-TZD anti-diabetic compounds, series of isoxazolidinediones were designed
to target the PPAR-ꢀ receptors. Docking studies were performed on co-crystallized protein structure of rosiglitazone with
PPAR-ꢀ receptor obtained from Protein Data Bank (2PRG). Interactions similar to that of rosiglitazone were observed for
three molecules; 3a, 3b and 3c which were further synthesized and subjected to in vivo hypoglycemic, total cholesterol
(CHL) and triglyceride (TG) evaluation. 14 days treatment revealed significant reduction in blood glucose levels but did
not portray desirable results in terms of total CHL and TG lowering effect. The blood glucose reduction observed for 3a,
3b and 3c at 20 mg/kg/day was 53.96 %, 61.35%, 61.32% respectively as against 59.95% of the standard pioglitazone at
10mg/kg/day.
Keywords: 4-benzylidene-3,5-isoxazolidinedione, docking, HFD- low STZ model, hypoglycemic activity , Knoevenagel
condensation.
INTRODUCTION
ity in STZ-induced diabetic animal model showed compara-
ble results with respect to the standard used viz. pioglita-
zone.
Thiazolidinediones (TZDs) also known as ‘Glitazones’
have been extensively acknowledged for their anti-diabetic
activity [1]. This category of molecules demonstrates promi-
nent interactions with the Peroxisome Proliferator- Activity
Receptors (PPARs), a subfamily of nuclear receptors (NRs).
The NR family is one of the largest families of transcription
factors. Three PPAR subtypes, i.e., PPARꢁ, PPARꢀ, and
PPAR ꢁ, have been identified in humans, and their structures
and functions are well known. PPARs play critical roles in
the regulation of cellular differentiation and development
and are, therefore, therapeutic targets in metabolic disorders
such as obesity, type 2 diabetes, atherosclerosis, and cancer
[2]. Glitazones are the representative family of ligands of
PPARꢀ. Ciglitazone and Troglitazone were the initial candi-
dates of the TZD class that were marketed in the 1990’s as
anti-diabetic agents. Rosiglitazone and pioglitazone followed
suit. The glitazones however portrayed discernible side-
effects like hepatotoxicity and cardiovascular toxicity. The
basis for toxicity profile was unclear but previous references
suggested that modifications other than the pharmacophore
TZD were accountable for its toxicity. As also, there were
evidences implying that acidic nature of the TZD is impor-
tant for insulin-sensitizing activity and hypoglycemic activ-
ity [3, 4].
But, a recent literature survey provided evidence that the
TZD ring of the troglitazone may be partially responsible for
its hepatotoxicity due to formation of toxic reactive metabo-
lites (RM). Five RM-GSH conjugates of troglitazone have
already been identified in vitro using human liver micro-
somes and in vivo using Sprague–Dawley rats. One of the
RMs is related to the quinone metabolite and four of the
other metabolites arose from the TZD ring. The quinone me-
tabolite however is not reported to be as cytotoxic as the
parent drug in human and porcine hepatocytes. Hence, the
authors tried an isosteric replacement of the TZD ring and
evaluated troglitazone and the isosteric derivative on normal
human hepatocytes cell line THLE-2. The study thus gave an
insight into the drawback attributed to the TZD ring [6]. This
gave us an impetus to modify the TZD ring based on the
bioisosterism theory stating the reduction of adverse effects
and optimization of pharmacokinetics in a view to build a
new series of lead molecules containing isoxazolidinedione
as the pharmacophore [7]. Isoxazolidinedione is a more
acidic heterocycle (pka=1.86) as compared to TZD (pka=
6.82). Hence, we designed our molecules using the Molecu-
lar Operating Environment (MOE.2009.10) software. In this
study, various structures of the desired compounds com-
plexed with the PPARꢀ Ligand Binding Domain 2PRG were
modeled and their interactions were analyzed. Three mole-
cules were synthesized and further screened for hypoglyce-
mic and hypolipidemic activity.
With the aforementioned inference, in our previous work
[5], we synthesized two novel moieties wherein we retained
the TZD ring; the pharmacophore and modified the non-
pharmacophoric component by introducing amide linkage
with a hope to alter the metabolism and thus the toxicity.
Molecules screened for antidiabetic and hypolipidemic activ
MATERIALS AND METHODS
Materials
Address correspondence to this author at the Bharati Vidyapeeth’s College
of Pharmacy, Sector-8, C.B.D., Belapur, Navi Mumbai, Maharashtra, India-
400614, India; Tel: 022-27571122; Fax: 022-27571182;
E-mail: sinharamaa@yahoo.in
2-Amino-5-methyl thiazole was received as a gift sample
from Ramdev Chemicals. 3-Aminobenzotrifluoride, 2-
aminobenzothiazole and chloroacetyl chloride were pur-
1875-6638/13 $58.00+.00
© 2013 Bentham Science Publishers

Design, Synthesis and In-vivo Hypoglycemic Evaluation
Step 1: Synthesis of 1 (Knoevenagel condensation)
CHO
Medicinal Chemistry, 2013, Vol. 9, No. 1 105
O
H2C
OCH3
OCH3
+
HO
O
Dimethyl malonate
i
4-Hydroxybenzaldehyde
O
O
ii
O
OCH3
NH
HO
O
HO
O
OCH3
Compoundsꢀaꢀbꢀc
1
R
2-(4-Hydroxybenzylidene)
malonic acid dimethyl ester
4-(4-Hydroxybenzylidene)
isoxazolidine-3,5-dione
Step 2: Synthesis of 2a-2c (Chloroacetylation)
O
O
iii
Cl
Cl
R
NH2
+
R
HN
2a-2c
Cl
Chloroacetyl chloride
Step-3: Synthesis of 3a-3c (Condensation of 2a-2c and 1)
O
O
R
O
O
iv
HN
Cl
O
NH
+
O
R
HN
O
NH
HO
O
O
2a-2c
1
3a-3c
(i)Piperidinebenzoate, toluene, reflux, (ii)NH2OH;HCl,Na2CO3 and THF:Methanol (1:1), (iii)K2CO3 and Chloroform (iv)K2CO3 and DMF
CF3
S
S
CH3
N
N
a
b
c
Fig. (1). Experimental scheme for synthetic work.
chased from Alfa Aesar. Piperidine was supplied by S.D
Fine Chem. Ltd. Benzoic acid and anhydrous potassium car-
bonate were procured from Research lab. Fine Chem. Indus-
tries, while p-hydroxybenzaldehyde from CDH Laboratories.
Streptozotocin (STZ) was purchased from Sigma-Aldrich,
USA. Pioglitazone hydrochloride was obtained from Amoli
Ltd., India as a gift sample. Sprague-Dawley female rats
were gifted by Glenmark Pharmaceuticals Ltd. Solvents used
were of LR grade. The animal feed ingredients were of vet-
erinary grade. The Software MOE.2009.10 used for docking
studies was procured from Chemical Computing Group,
Canada.
ronment (MOE 2009.10) as computational software. MOE
identifies favorable poses of flexible ligands in rigid binding
sites of macromolecules, typically proteins. MOE offers dif-
ferent routines for conformational sampling, placement and
scoring.
The co-crystallized protein structure of rosiglitazone with
PPAR-ꢀ receptor was obtained from the Protein Data Bank
(2PRG). The Rosiglitazone bound to the receptor was de-
leted. The crystal structure was then, protonated and the en-
ergy was minimized using AMBER99 forcefield. The active
site was generated using atom selector and was labeled as the
‘binding site’. All the designed molecules were built, energy
minimized and used as ligands for the docking studies using
the software. Docking was performed using Alpha PMI
placement method with London dG scoring. The Alpha PMI
method generates poses by aligning ligand conformations'
principal moments of inertia to a randomly chosen subset of
alpha sphere dummies in the receptor site. This method is
preferred for tight pockets.
METHODS
Docking Studies
All computations for docking studies were carried out on
a Pentium 2.67 GHz workstation, 256 MB memory with
Windows operating system and Molecular Operating Envi-

106 Medicinal Chemistry, 2013, Vol. 9, No. 1
Jadhav et al.
Table 2. Composition and Calorie Value of Cafeteria Diet
Different poses were scrutinized on the basis of suitable
interactions and the best fitting moieties were considered on
further studies.
Calorie Value
(kcal/100g)
Ingredients
Chemistry
Condensed Milk
Bread
335
230
550
360
320
80
2-(4-Hydroxybenzylidene) malonic acid dimethyl ester
was prepared by Knoevenagel condensation, wherein the
aryl aldehydes were refluxed with dimethylmalonate in pres-
ence of a base; piperidinium benzoate. Then the condensed
product was refluxed with hydroxylamine hydrochloride and
sodium carbonate using methanol and tetrahydrofuran mix-
ture to synthesize 4-(4-hydroxybenzylidene) isoxazolidine
3,5-dione [1]. The common intermediate 1 was further con-
densed at room temperature with various chloroacetylated
amines to yield the desired molecules (3a-3c) (Fig. 1) [3,8].
Chocolate
Biscuits
Cheese
Boiled potato
Dried coconut
660
BIOLOGICAL EVALUATION
Animals
COLLECTION OF BLOOD AND ANALYTICAL
METHODS
For blood glucose estimation, rats were given mild ether
anesthesia followed by collection of blood from the retro
orbital plexus. Blood was collected in sodium fluoride anti-
coagulant containing vials and glucose was analyzed by glu-
cose oxidase method. For triglycerides (TG) and cholesterol
(CHL) analysis, blood was collected in plain eppendorf’s
tubes to obtain serum and estimation was carried out using
standard biochemical kits.
Female Sprague-Dawley rats with an average body
weight of 180-200 g were selected for the study. Animals
were housed in polypropylene cages at an ambient tempera-
ture of 25 20C and 45-55% relative humidity with standard
12 hours light and dark cycle. They had free access to feed
and water. Animals were examined properly for infection
and metabolic disorders. CPCSEA guidelines for the use and
care of laboratory animals were followed throughout the
experiment and prior permission was sought from the institu-
tional ethics committee for conducting the study.
Biological Activity
Acute toxicity study for compounds 3a, 3b and 3c was
performed before testing the hypoglycemic activity of these
compounds. Different doses upto 500mg/kg were tried. Hy-
poglycemic and hypolipidemic activities were carried out
using a combination of high fat diet (HFD) and low dose of
STZ-treated female Sprague – Dawley rats. The animals
were fed HFD for a period of 14 days. After 14 days of die-
tary manipulation, rats were injected intraperitoneally (i.p.)
with low dose of STZ (35mg/kg), while respective control
rats were administered the vehicle; 1% Na-CMC (2ml/kg,
p.o.). Blood glucose (BG) was estimated just before the
study, 7 days after STZ injection and at the end of 14 days
treatment. Rats with non-fasting plasma glucose level more
than 250mg/dl were considered diabetic and selected for the
study. For hypolipidemic activity, CHL and TG estimations
were carried out prior to study, after 14 days of HFD and at
the end of drug treatment.
Experimental Design
Table 1. Experimental Design for Animal Study
Groups
Administration of chemicals
No. of animals
Normal Control- received only
vehicle
Group1
6
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Normal +3a
Diabetic +3a
Normal +3b
Diabetic +3b
Normal +3c
6
6
6
6
6
6
6
Diabetic rats were randomly divided into five groups.
The diabetic rats were either treated with test compound
(20mg/kg/day for 14 days) or with pioglitazone hydrochlo-
ride (equivalent to 10 mg/kg/day for 14 days).
Diabetic +3c
Diabetic +STD*
Diabetic Control –received only
vehicle
Group9
6
RESULT AND DISCUSSION
Design of Novel Molecules
*STD –Standard drug Pioglitazone HCl
Development of High Fat Diet (HFD) Fed and STZ
Treated Type 2 Diabetic Rats
The TZD ring of troglitazone has been reported to show
liver toxicity in human via reactive metabolite formation (6).
Acidity of the TZD moiety has been accounted for its insulin
sensitizing effect [3, 4]. Hence, various research groups have
attempted replacement of the TZD ring with different acidic
Except normal control animals rest all animals were
given HFD for a period of two weeks. The composition of
HFD is given below [9]:

Design, Synthesis and In-vivo Hypoglycemic Evaluation
Medicinal Chemistry, 2013, Vol. 9, No. 1 107
CENTRAL ARYL GROUP
LIPOPHILIC GROUP
O
H
N
PHARMACOPHORE
O
NH
O
Ar
O
O
LINKER
ALKOXY LINKER
Fig. (2). Generalized structure of novel benzylidene isoxazolidinediones.
drogen bonding with amino acid residues His323, Tyr327
and His449 as reported in the literature [12, 13].
MOE.2009.10 was successful in reproducing the binding
position for rosiglitazone, showing a Root Mean Square de-
viation of 1.5 A° in comparison with the experimental ge-
ometry when this TZD is co-crystallized in the PPAR-ꢀ.
groups. One of the attempts included, designing compounds
containing malonate as a substitute for TZD which have
been reported to possess antihyperglycemic activity similar
to the thiazolidinediones [4, 10]. However, the free malonate
group is susceptible to unspecific ester hydrolysis, convert-
ing it to malonic acid which is responsible for its toxicity
[11]. To circumvent these negative aspects related to the
molecule, cyclization of malonate moiety to isoxazolidinedi-
one ring, a more acidic bioisostere of TZD was considered.
The binding profile of the 4-(4-hydroxybenzylidine)
isoxazolidine-3,5-dione derivatives were compared with that
of the rosiglitazone molecule. Interactions were distinct in
the case of ligand N-(Benzo[d]thiazol-2-yl)-2-[4-[(3,5-
dioxoisoxazolidin-4-ylidene) methyl] phenoxy] acetamide
(3a). The nitrogen atom of the isoxazolidinedione ring dem-
onstrates hydrogen bonding with Tyr327 residue having 34%
binding and bond length of 2.1 A° and the oxygen of car-
bonyl group exhibits hydrogen bonding with Ser289 having
25% binding and bond length 1.8 A°. As also, the amino acid
residue Arg288 shows arene interactions with benzothiazole
moiety and a hydrogen bond of percent binding 14%, bond
length of 2 A° with oxygen of amide linker is also seen (Fig.
3A and 3B). The ligand 2-[4-[(3,5-dioxoisoxazolidin-4-
ylidene) methyl] phenoxy]-N-[(3-trifluoromethyl) phenyl]
acetamide (3b) shows significant interactions with His449
and the Ser289 amino acid residues, forming hydrogen
bonds with the two oxygen atoms of the carboxyl groups of
the isoxazolidinedione ring. The percentage binding and the
distance from the residues were found to be 12 %, 1.7 A°
and 47 %, 2.7 A° respectively (Fig. 3C and 3D). Similarly,
the ligand 2-[4-[(3,5-dioxoisoxazolidin-4-ylidene) methyl]
phenoxy]-N-(5-methylthiazol-2-yl) acetamide (3c), interacts
mainly with PPAR-ꢀ through the formation of hydrogen
bonds between the two oxygen of the carbonyl groups of the
isoxazolidinedione ring and the Tyr473 and the Ser289 resi-
dues, respectively (Fig. 3E and 3F). The percent binding
with the amino acid residues Tyr473 and Ser289 and the
distance through which binding occurs are 38%, 3.2 A° and
35%, 2.3 A° respectively.
The non-pharmacophoric fraction of our previously men-
tioned TZD molecules were retained as they were expected
to improve the lipophilicity of the molecules [5]. The novel
lipophilic moiety was attached to isoxazolidinedione ring by
using alkoxy linker and a benzylidine hydrophobic trunk.
(Schematic representation in Fig. 2).
Docking Studies
Structurally, PPARꢀ is composed of a DNA-binding do-
main (DBD), a hinge region, and a ligand-binding domain
(LBD). Agonist binding to PPARꢀ regulates activity by caus-
ing conformational changes to the LBD, which is composed
of approximately 250 amino acids near the C-terminal end of
the protein. The LBD of PPARꢀ is a large, T-shaped cavity
with a volume of approximately 1440 °A3 which can easily
accommodate many different ligands due to the dynamics of
the ligand-binding pocket.
PPARꢀ agonists typically possess a small polar region
and a hydrophobic region that form hydrogen bonds and
hydrophobic interactions, respectively, within the LBD. Hy-
drogen bonding typically occurs between His323, Tyr473,
Ser289 and His449 of the PPARꢀ LBD and carbonyl oxygen
of the ligand. Hydrogen bonding of the ligand to Tyr473 is
the key to the stabilization of the AF-2 region. The hydro-
phobic moiety interacts with other residues in the cavity,
such as Leu465, Leu469, and Ile472, establishing hydropho-
bic interactions to stabilize the domain. Using the structural
details of the receptor and the interactions we designed our
new chemicals and docked them into the active pockets of
the receptor.
BIOLOGICAL ACTIVITY
Statistics
The results were calculated with the help of GraphPad
InStat using one-way ANOVA and Dunetts’ test and are
expressed as mean SEM. The percent reduction was calcu-
lated using the equation: [1 - (TT/OT)/(TC/OC)] x 100,
where TT is test day treated, OT is zero day treated, TC is
test day control, and OC is zero day control.
Initiation of the study was done using rosiglitazone for
which the binding mode has been determined experimen-
tally. According to the crystal structure of rosiglitazone in
the LBD of PPAR-ꢀ (PDB code-2PRG), the thiazolidinedi-
one ring exhibits several specific interactions with neighbor-
ing amino acids of the LBD. These interactions include hy-

108 Medicinal Chemistry, 2013, Vol. 9, No. 1
Jadhav et al.
Fig. (3). Ligand interactions of 3a, 3b and 3c with PPARꢀ (PDB 2PRG): Pink dashed lines represent bonding interactions between the hy-
drogens of the residue and the oxygen of ligand and green circles represent arene - arene interaction between aromatic rings of residue and
ligand. [A] 3D interaction of 3a with 2PRG. [B] 2D interaction of 3a with 2PRG. [C] 3D interaction of 3b with 2PRG. [D] 2D interaction of
3b with 2PRG. [E] 3D interaction of 3c with 2PRG. [F] 2D interaction of 3c with 2PRG.
To study the insulin resistance in humans which is char-
acterized by obesity, mild hyperglycemia and hypercho-
lestrimia “HFD fed, low dose of STZ model” is used. Hypo-
glycemic activity in STZ-induced HFD fed rats was carried
out in a 14 days study for compounds 3a, 3b and 3c and they
showed 53.96, 61.35 and 61.32% blood glucose reduction,
respectively, when compared with disease control. Efficient
control of Triglyceride (TG) and Cholesterol (CHL) level in
type-2 diabetes mellitus (T2DM) is essential as diabetes is
also associated with accelerated atherosclerotic macrovascu-
lar diseases such as myocardial infarction, stroke and limb
amputation [10]. Thus, the drugs which effectively control
TG and CHL levels would be more suitable candidates for
further development for treatment of T2DM. In our study all
three compounds were evaluated for its TG and CHL lower-
ing activity. Slight alterations were observed in data during
the study but the results are not significant as compared to
disease control group, since observed TG and CHL lowering
effect of pioglitazone hydrochloride are not reported in clini-
cal trials [5].

Design, Synthesis and In-vivo Hypoglycemic Evaluation
Medicinal Chemistry, 2013, Vol. 9, No. 1 109
Table 3. Body Weight in Grams Before and After HFD
Table 6. Statistical Analysis of Blood Glucose Mg/Dl After 2
Weeks of Drug Treatment
Group
Normal rats
HFD rats
Groups
0th day
14th day
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Group9
177.5 3.35
186.6 3.57
135.8 3.27
177.5 4.95
169.16 10.6
181.66 5.72
182.5 4.33
181.66 4.94
192.5 3.09
195.83 6.63
197.5 3.09
200 4.90
Group 1
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Group9
128.66
123.5
130.67 7.07
259.16 3.68
119.17 1.99
267.33 2.91
137.15 7.96
268 4.04
111.5 3.97
116.83 3.79
104.5 1.41
101.16 6.57
108.83 8.93
103.5 5.02
100.5 2.90
260.16 10.83
192.5 4.78
190 4.47
195.8 2.71
193.33 5.19
265.6 3.73
265.67 3.24
Table 4. Cholesterol in mg/dl Before and After HFD
Table 7. Percent Reduction Values
Groups
Normal rats
HFD rats
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Group9
74.33 4.94
80.16 3.42
81.83 4.57
73.33 5.81
73.165.02
72 5.79
81.66 6.17
89.33 6.88
86.33 4.60
82.33 9.17
83.33 4.8
81.5 6.14
87 6.48
Blood Glucose
Triglyceride
% max
Cholesterol
Compound
% Max
No.
% max
Reduction
Reduction
Reduction
3a
3b
53.96**
61.35**
61.32**
59.95**
1N.S
4 N.S
5.9 N.S
8.14 N.S
0.049 N.S
9.52 N.S
3c
14.6 N.S
3.2 N.S
STD
75.66 5.73
80.83 4.97
‘% max reduction’ is the maximum achieved reduction relative to diabetic control
group. ** represents p < 0.01 (significant). N.S: Not significant
91.8 6.46
using TMS as internal standard. Chemical shift values are
reported in ꢀ, ppm. All reactions as well as column chroma-
tography were followed by TLC using Merck pre-coated
silica gel 60 F254 plates and spots were visualized by ob-
serving in UV cabinet under short UV. IR spectra were re-
corded on FTIR-8400S instrument with KBr pellets and only
the principal absorption levels (cm-1) have been listed. All
reagents were used as received unless otherwise stated.
Table 5. Triglyceride In mg/dl Before and After HFD
Groups
Normal rats
HFD rats
Group2
Group3
Group4
Group5
Group6
Group7
Group8
Group9
63.66 7.27
67.33 4.63
62.33 4.91
70.83 4.30
60.16 4.19
65.83 7.38
72.83 7.32
69.83 6.94
69.83 8.61
74.5 3.01
72.83 4.55
78.66 4.26
66.16 5.38
67.5 6.43
77.83 6.72
79 6.65
Synthesis of the 4-(4-hydroxybenzylidine) Isoxazolidin-
edione-3,5-dione (1)
4-(4-Hydroxybenzylidine) isoxazolidine-3,5-dione was
synthesized in two steps. First step was Knoevenagel con-
densation reaction wherein 4-hydoxybenzaldehyde (3.0 g,
5.0 mmol) and dimethyl malonate (2.82 ml, 5.0 mmol) in
presence of 20 ml toluene and catalytic quantity of piperidi-
nium benzoate were refluxed together to yield 2-(4-
hydroxybenzylidine) malonic acid dimethyl ester with con-
tinuous removal of water. Next step was cyclization of 2-(4-
hydroxybenzylidine) malonic acid dimethyl ester (4.3 g, 25.0
mmol) with hydroxylamine hydrochloride (1.8 g, 25.0
mmol) in presence of sodium carbonate (1.57 g, 5.0 mmol)
using solvent THF: methanol (1:1) under reflux of 5-6 hrs.
The cyclisation product of Knoevenagel condensation was
unstable [4].
The order of drug molecules showing blood sugar lower-
ing activity is 3b ~ 3c > 3a with significant result. Thus, in-
troducing the isoxazolidinedione ring may serve as a useful
strategy in drug design in the area of antidiabetic non-TZDs.
EXPERIMENTAL SECTION
1H NMR and 13C NMR spectra were recorded in DMSO-
d6 on Bruker 400 MHz and Varian 300 MHz instrument

110 Medicinal Chemistry, 2013, Vol. 9, No. 1
Jadhav et al.
Synthesis of N-(Benzo[d]thiazol-2-yl)-2-chloro Acetamide
(2a)
Synthesis
of
2-[4-[(3,5-dioxoisoxazolidin-4-ylidene)
methyl] Phenoxy]-N-[3-(trifluoromethyl) Phenyl] Aceta-
mide (3b)
2-Aminobenzothiazole (3 .0g, 1.0mol), anhydrous K2CO3
(4.14g, 1.73mol) and 25 ml of chloroform were taken in
RBF. Reaction mixture was cooled between 2-5°C and to
this chloroacetyl chloride (4.12ml, 2.6mol) was added drop
wise. The reaction mixture was allowed to stir overnight at
room temperature. After completion of reaction, water was
added to reaction mixture and organic layer was separated.
Then, it was passed through anhydrous Na2SO4. Chloroform
layer was distilled out and off white crystals were collected.
Yield 92.10%, white crystals, mp 143-144°C IR [KBr ꢁ cm-
Anhydrous K2CO3 (1.51g, 1.5mol) was stirred in DMF
for 10-15 min. To this 4-(4-hydroxybenzylidine) isoxa-
zolidine-3,5-dione (1.5g, 1.0mol) and chloroacetylated 3-
aminobenzotrifluoride (1.73g, 1.0mol) were dissolved sepa-
rately in DMF and added to the reaction mixture. The reac-
tion was continued to stir at room temperature till its comple-
tion. Reaction was monitored by TLC. Water was added to
reaction mixture and further stirred for 30 minutes. Product
was then extracted in chloroform and organic layer was
passed through anhydrous Na2SO4. Chloroform layer was
then distilled out to get crude product. Eluent for column
chromatography: Benzene: Methanol (2:0.48). Yield 30%,
color pale yellow colour powder, mp 96°C. IR [KBr ꢁ cm-1]:
3344 (N-H), 1724 (C=O), 1693 (C=O), 1158, 1128 (C-O).
1H NMR (300 MHz, ꢀ, ppm, DMSO-d6): 4.96 (2H, s, -CH2-),
7.43 (1H, d, aromatic), 7.55-7.60 (3H, m, aromatic and ben-
zylidene proton), 7.69-7.78 (2H, m, aromatic protons), 8.03-
8.0 (3H, m, aromatic protons), 10.58 (1H, s, -NH-CO). 13C
NMR (100 MHz, DMSO-d6, ꢀ): 167.00 (C=O), 166.15
(C=O), 138.73, 133.22, 132.62, 131.27, 130.64, 130.32,
129.49, 129.27, 128.23, 128.05, 125.39, 122.98, 120.33,
119.98, 116.27, 62.83.
1
1]: 3181 (N-H), 1660 (C=O). H NMR [300 MHz, ꢀ, ppm,
DMSO-d6]: 4.22 (2H, s), 7.35 (1H, t), 7.46 (1H, t), 7.77 (1H,
d, J = 8.1Hz), 8.01 (1H, d, J = 6.0 Hz), 12.79 (1H, s).
Synthesis of N-(Benzo[d]thiazol-2-yl)-2-[4-[(3,5-dioxoiso-
xazolidin-4-ylidene) Methyl] Phenoxy] Acetamide (3a)
Anhydrous K2CO3 (0.42g, 1.5mol) was stirred in DMF
for 10-15 min. To this 4-(4-hydroxybenzylidine) isoxa-
zolidine-3,5-dione (0.43g, 1.0mol) and chloroacetylated 3-
aminobenzthiazole (0.47g, 1.0mol) were dissolved separately
in DMF and added to the reaction mixture. The reaction was
allowed to stir at room temperature till its completion. Reac-
tion was monitored by TLC. Water was added to reaction
mixture and further stirred for 30 minutes. Product was then
extracted in chloroform and organic layer was passed
through anhydrous Na2SO4. Chloroform layer was then dis-
tilled out to get crude product. Eluent used for column chro-
matography: Chloroform: Methanol (2:0.36). Yield 24.10%,
off white powder, mp 234-239°C. IR [KBr ꢁ cm-1]: 3235 (N-
Synthesis of 2- Chloro -N-(5-methlthiazol-2-yl) Aceta-
mide (2c)
2-Amino-5-methylthiazole (3.0g, 1.0mol), anhydrous
K2CO3 (6.0g, 1.5mol) and 25ml of chloroform were taken in
RBF. Reaction mixture was cooled between 2-5 °C and to
this chloroacetyl chloride was added drop wise (5.0 ml,
1.5mol). The reaction mixture was allowed to stir overnight
at room temperature. Water was added to reaction mixture
and organic layer was separated. It was then passed through
anhydrous Na2SO4 layer. Chloroform layer was distilled out
and off white colour powder was collected. Yield 82%,
white needle shaped crystals, mp 191-192°C. IR [KBr ꢁ cm-
1
H), 1705 (C=O), 1602 (C=O), 1182, 1132 (C-O). H NMR
(400 MHz, ꢀ, ppm, DMSO-d6): 5.12 (2H, s, -CH2-), 7.31-
7.35 (1H, m, benzothiazole proton), 7.48-7.44 (1H, m, ben-
zothiazole proton), 7.59 (2H, t, phenyl proton), 7.71 (1H, t,
benzylidene proton), 7.78 (1H, d, J= 6Hz, benzothiazole pro-
ton), 8.00 (1H, d, J= 6Hz, benzothiazole proton), 8.06 (2H,
d, J= 6Hz, phenyl protons), 12.74 (1H, s, -NH-CO-). 13C
NMR (100 MHz, DMSO-d6, ꢀ): 167.26 (C=O), 165.89
(C=O), 134.28, 129.95, 129.35, 126.70, 124.22, 122.27,
121.16, 63.15.
1
1]: 3186 (N-H), 1704 (C=O), 1560 (C=N). H NMR [300
MHz, ꢀ, ppm, DMSO-d6]: 2.43 (3H, s), 4.24 (2H, s), 7.5 (1H,
s), 11.0 (1H, bs).
Synthesis
of
2-[4-[(3,5-dioxoisoxazolidin-4-ylidene)
Synthesis of 2-Chloro-N-[3-(trifluoromethyl) Phenyl]
Acetamide (2b)
methyl] Phenoxy]-N-(5-methylthiazol-2-yl) Acetamide
(3c).
Anhydrous K2CO3 (1.01g, 1.5mol) was stirred in DMF
for 5-10 min. To this 4-(4-hydroxybenzylidine) isoxa-
zolidine-3,5-dione (1.0g, 1.0mol) and chloroacetylated 2-
amino-5-methylthiazole (0.92g, 1.0mol) were dissolved in
DMF separately and added to the reaction mixture. The reac-
tion was continued to stir at room temperature till its comple-
tion. Reaction was monitored by TLC. Water was added to
reaction mixture and further stirred for 30 minutes. Product
was then extracted in chloroform and organic layer was
passed through anhydrous Na2SO4. Chloroform layer was
then distilled out to get crude product. It was further purified
by column chromatography. Yield 33.33%, buff colour pow-
der, mp 130°C. IR [KBr ꢁ cm-1]: 2922 (N-H), 1726 (C=O),
3-Aminobenzotrifluoride (2.31ml, 1.0mol), anhydrous
K2CO3 (3.85g, 1.5mol) and 25 ml of chloroform were taken
in RBF. Reaction mixture was cooled between 2-5°C and to
this drop wise (2.20ml, 1.5mol) of chloroacetyl chloride was
added. The reaction mixture was allowed to stir overnight at
room temperature. After completion of reaction water was
added to reaction mixture and organic layer was separated.
Then, it was passed through anhydrous Na2SO4. Chloroform
layer was distilled out and white crystals were collected.
Yield 67.0%, white crystalline, mp 129-130°C. IR [KBr ꢁ
cm-1]: 3305 (N-H), 1683 (C=O), 1070, 1124 (C-F). 1H NMR
[300 MHz, ꢀ, ppm, DMSO-d6]: 4.38(2H, s), 7.30-7.44 (2H,
m), 7.95 (1H, d), 8.2 (1H, s), 10.05 (1H, s).

Design, Synthesis and In-vivo Hypoglycemic Evaluation
Medicinal Chemistry, 2013, Vol. 9, No. 1 111
1
REFERENCES
1697 (C=O), 1595 (C=N), 1166, 1122 (C-O). H NMR (400
MHz, ꢀ, ppm, DMSO-d6): 2.34 (3H, s, -CH3), 5.02 (2H, s, -
CH2-), 7.15 (1H, s, thiazole proton), 7.55-7.62 (2H, m,
phenyl protons), 7.71 (1H, t, benzylidene proton), 8.03 (2H,
d, J=8Hz, phenyl protons), 12.27 (1H, bs, -NH-CO-). 13C
NMR (100 MHz, DMSO-d6, ꢀ): 165.90 (C=O), 156.09
(C=O), 135.20, 134.22, 129.91, 129.42, 129.32, 127.07,
62.98, 11.56.
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CONFLICT OF INTEREST
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OECD, SIDS Initial Assessment Report for SIAM 20 Paris, France,
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The author(s) confirm that this article content has no con-
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ACKNOWLEDGEMENTS
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zolidinedione yet weakly adipogenic. J. Pharm. Exp. Ther., 2003,
306(2), 763-771.
This work was supported by University Grant (University
of Mumbai). We are grateful to The Director - Board of Col-
lege and University Development, University of Mumbai.
We are immensely gratified by the help extended by all the
organizations that gave us chemicals and animals as gift
samples for our current research.
Received: September 14, 2011
Revised: May 10, 2012
Accepted: May 12, 2012