Discovery of novel glitazones incorporated with phenylalanine and tyrosine: Synthesis, antidiabetic activity and structure-activity relationships

Article abstract of DOI:10.1016/j.bioorg.2012.08.002

We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and absence of insulin. Six of the most active compounds

Full text of DOI:10.1016/j.bioorg.2012.08.002

Bioorganic Chemistry 45 (2012) 12–28
Contents lists available at SciVerse ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Discovery of novel glitazones incorporated with phenylalanine and tyrosine:
Synthesis, antidiabetic activity and structure–activity relationships q
B.R. Prashantha Kumar a, Nasir R. Baig b, Sai Sudhir b, Koyal Kar a, M. Kiranmai c, M. Pankaj c,
Nanjan M. Joghee c,
⇑
a Department of Pharmaceutical Chemistry, JSS College of Pharmacy, Mysore 570 015, India
b Department of Organic Chemistry, Indian Institute of Science, Bangalore 560 012, India
c TIFAC CORE, JSS College of Pharmacy, Ootacamund 643 001, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 19 March 2012
Available online 23 August 2012
We report a series of new glitazones incorporated with phenylalanine and tyrosine. All the compounds
were tested for their in vitro glucose uptake activity using rat-hemidiaphragm, both in presence and
absence of insulin. Six of the most active compounds from the in vitro screening were taken forward
for their in vivo triglyceride and glucose lowering activity against dexamethazone induced hyperlipid-
emia and insulin resistance in Wistar rats. The liver samples of rats that received the most active com-
pounds, 23 and 24, in the in vivo studies, were subjected to histopathological examination to assess
their short term hepatotoxicity. The investigations on the in vitro glucose uptake, in vivo triglyceride
and glucose lowering activity are described here along with the quantitative structure–activity
relationships.
Keywords:
CoMSIA
Dexamethazone
Glitazones
Glucose uptake activity
Insulin resistance
Thiazolidinediones
Ó 2012 Elsevier Inc. All rights reserved.
1. Introduction
In the year 1999, troglitazone, a new class of drug called ‘glitaz-
one’ or thiazolidinedione (TZD) was approved by the US FDA for
Type 2 diabetes mellitus, considered today as a life-style dis-
ease, is usually associated with urbanization, mechanization,
change in life style habits and caloric imbalance. Over 90% of the
diabetes mellitus patients are of type 2 patients across the world
[1]. Type 2 diabetes is a chronic disease characterized by hypergly-
cemia due to insulin resistance in the liver and peripheral tissues,
or due to a defect in pancreatic b-cells or both [2,3]. The insulin
resistance state at the peripheral level causes impaired glucose uti-
lization leading to hyperglycemia, which is believed to play a role
in the etiology of a wide spectrum of metabolic disorders such as
obesity, hypertension, atherosclerosis, neuropathy, nephropathy,
and retinopathy [4,5]. The conventional therapy of type 2 diabetes
mellitus using sulphonylureas and biguanides has not been satis-
factory as it is not successful in treating associated cardiovascular
risk factors, a major cause of morbidity. Traditional treatment
strategies include several oral hypoglycemic agents but practically
none of these agents maintain glucose levels indefinitely. Many of
the patients, therefore, require exogenous insulin therapy. The
current trend is, therefore, to make therapy better by choosing
appropriate combination of available drugs.
the treatment of type 2 diabetes. It was, however, withdrawn later
due to severe hepatotoxicity. Rosiglitazone and pioglitazone intro-
duced later were marketed under regular monitoring and account
for 21% of oral antidiabetic drugs used in the USA [6,7]. Glitazones
correct hyperglycemia by enhancing tissue sensitivity to insulin.
Glitazones are not associated with dangerous hypoglycemic inci-
dents that are observed with conventional sulfonylureas and insu-
lin therapy. Glitazones, however, are known to have cardiovascular
risks in association with fluid retention and weight gain [8–13].
Identification and development of newer drugs from this class is,
therefore, the need of the hour.
Glitazones are the agonists for the peroxisome proliferator acti-
vating receptors-
insulin-responsive genes involved in the control of glucose produc-
tion, transport and utilization [14]. PPAR- is a member of the sub-
c (PPAR-c), which regulate the transcription of
c
family, which belongs to a 48 member nuclear receptor super
family [15–17]. A large number of insulin sensitizers have been
synthesized and tested for their antidiabetic activity. Many of the
candidate compounds have reportedly entered clinical studies.
Some of these belong to the glitazone class, but others have differ-
ent chemical structures with clofibrate and tyrosine moieties, etc.
[18–22]. The majority of TZD containing antidiabetic agents
reported in literature have a polar TZD ring system as a head fol-
lowed by hydrophobic benzyloxy moiety as their trunk which is
in turn connected to the hydrophobic tail via a two carbon linker
q
Author contributions: Authors have equally contributed for the science and in
editing this manuscript.
⇑
Corresponding author. Fax: +91 4232447135.
E-mail address: mjnanjan@gmail.com (N.M. Joghee).
0045-2068/$ - see front matter Ó 2012 Elsevier Inc. All rights reserved.
http://dx.doi.org/10.1016/j.bioorg.2012.08.002

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
13
Two carbon
linker
2. Chemistry
S
X
H
Considering the above structural features of glitazones and with
the objective of further probing the structure–activity relation-
ships (SAR), we designed structures of some novel glitazones incor-
porated with phenylalanine and tyrosine residues. The hypothesis
here is that, the amino acid moiety will increase the polarity and
acidity near the thiazolidine ring due to the freely exposed
carboxyl group and is also relatively safe with respect to its metab-
olism in the body. The synthesis of the title compounds incorpo-
rated with amino acids, phenylalanine and tyrosine were,
therefore, carried out according to Scheme 1 to obtain compounds
5–20 and Scheme 2 to obtain compounds 23–28.
We have selected two water soluble hydrophobic amino acids
such as phenylalanine and its counterpart, tyrosine, as building
blocks (Scheme 1). The amino acid was first converted to the cor-
responding ammonium salt and then reacted with carbon disulfide
to yield the corresponding dithiocarbamates (1 and 2). The
N
O
HN
O
Polar,
acidic
head
Hydrophobic
tail
Hydrophobic
trunk
X = O, thiazolidinedione
X= S, rhodanine
Fig. 1. Traditional pharmacophore structural part of glitazones.
(Fig. 1) [23,24]. Though the majority of TZD containing glitazones
possess a methylene group bridged to the TZD and phenyl group,
it has been shown by us and others independently that compounds
with 5-benzylidene TZDs can also act as insulin sensitizers. Fur-
ther, replacing TZD head with its bioisostere rhodanine ring has
also been shown to retain activity to considerable levels [24,25].
S
NH
4OOC
COOH
NH2
S
S
S
O
O
S
N
N
H
SNH4
[b]
ONa
NH
[a]
HOOC
[c]
OOC
Na
R
3, R = H
R
4, R = OH
1, R = H
R = H, OH
2, R = OH
R
R
[d]
S
N
S
R1
R
COOH
5-12, R1 = H
13-20, R1 = OH
O
Scheme 1. Synthesis of 2-thioxo-thiazolidine-4-one derivatives incorporated with phenylalanine and tyrosine residues: (a) H2O, 0–5 °C, aqueous ammonia solution 25–30%,
CS2, stir for 10 h; (b) 5–10 °C, sodium chloroacetate solution; (c) 6 N HCl, two drops POCl3, 60–70 °C for 3 h; (d) toluene, subst aldehyde, piperidine or N-methyl piperazine,
acetic acid, molecular sieves, reflux, 110 °C for 15 h or MW irradiation 700 W, 30 min.
O
H
O
O
Cl
[a]
R2
[b]
N
H
R
Cl
NH2
R
Cl
O
O
21
NH
R
22
[c]
S
S
N
O
O
O
R2
HOOC
NH
23-27, R2= OCH3
28, R2= H
23-28
R
OH
Scheme 2. Synthesis of glitazones 4–18: (a) CHCl3, Et3N, 0–5 °C to rt, stirred for 6 h; (b) acetone, vanillin, K2CO3, stirred at rt 36 h; (c) toluene, 4, N-methyl piperazine,
ammonium acetate, molecular sieves, reflux at 110 °C for 15 h or MW irradiation 700 W, 30 min.

14
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
dithiocarbamates were then reacted with sodium chloroacetate
followed by cyclization under acidic conditions in presence of
phosphorous oxychloride to yield products in which the N-termi-
nal of the amino acid is converted to the corresponding 2-thioxo-
thiazolidine-4-ones, 3 and 4. Compounds 3 and 4 were then
subjected to the Knoevenagel condensation with aryl aldehydes,
using N-methyl piperazine and ammonium acetate in refluxing tol-
uene, to yield compounds 5–12 and 13–20 as listed in Table 1.
Knoevenagel condensation was performed on 3 and 4, using both
conventional and microwave methods without the aid of Dean–
Stark apparatus.
Apart from good yields, we have not obtained the same results
contrary to the results reported by Wang et al. and Hardej et al.
[26]. The present method is simple and easy. Here, we have pre-
pared ammonium salt of dithiocarbamate rather sodium salt and
the reaction completes at room temperature itself without heating.
We also observed that the dithiocarbamates are relatively more
stable under cold conditions. Phosphorous oxychloride along with
HCl was found to be useful in the cyclization step, as it enhances
the yields. The rate of Knoevenagel condensation with aldehydes
over the active methylene group has been shown to increase when
N-methyl piperazine and ammonium acetate are used, rather than
piperidine and acetic acid mixture, under similar conditions [27].
The tail part was first synthesized by connecting various aro-
matic/alicyclic amines to the two carbon linker (Scheme 2). The
acylated amines 21 were then connected to the p-hydroxy group
of vanillin to obtain 23–27 and to p-hydroxy benzaldehyde to ob-
tain 28. Vanillin was used because it is a natural product and there
are not many reports in literature about glitazones incorporated
with a natural product like vanillin as part of their trunk [24].
The trunk, which is connected with the hydrophobic tail via the
two carbon linker, was then connected to the head group, namely,
2-thioxo-thiazolidine-4-one or rhodanine ring system of 4 by
adopting the Knoevenagel condensation reaction as described un-
der Scheme 1.
Windows (San Diego, California, USA). In the first set, group 1 that
served as the control, was compared with groups (3–27) that are
studied in the absence of insulin including the analogous standard
drug (rosiglitazone). In the second set group 2 that served as the
control, was compared with groups (3–27) that are studied in the
presence of insulin. We neither compared set one versus set two
groups nor all the groups with each other, as it is well known bio-
logically that insulin enhances the glucose uptake. The focus was,
therefore, to compare the differences made by the presence of
compounds. A reasonable correlation between these two set of
groups indicate some degree of correlation for the compounds with
respect to their glucose uptake enhancement in the absence and
presence of insulin. The results of the in vitro glucose uptake study
indicate that all the compounds enhance the glucose uptake by the
tissue from weak to moderate and from moderate to significant
glucose levels. The results also reveal one key aspect, namely, the
compounds significantly enhance the glucose uptake in the pres-
ence of insulin rather than in the absence of external insulin. It is
evident, therefore, that this class of compounds tends to sensitize
the tissue to take up insulin which enhances the glucose utilization
by the tissue cells [31,32].
We have also investigated the structure–activity relationships
based on the results obtained. Compounds 3 and 4 (which lack
substitution at the 5th position of thiazolidine ring system) do
not exhibit good activity, indicating that substitution at the 5th po-
sition of thiazolidine ring system is essential. Compounds 6 and 9
exhibit reasonably good glucose uptake activity, whereas the rest
of the compounds incorporated with phenylalanine do not. Inter-
estingly, compounds which are incorporated with tyrosine exhibit
significant activity, especially, compounds 15, 17, 23, 24, 25, 27
and 28 (P < 0.01). These compounds exhibit comparable glucose
uptake activity when compared to the standard drug (rosiglitaz-
one). Compounds 23, 24, 25, 27 and 28 contain vanillin as their
trunk, followed by two carbon acyl linker and hydrophobic tail in
their structures. Compound 26 (P < 0.05) with one carbon linker
exhibits moderate activity.
The structures of the synthesized compounds were confirmed
by IR, Mass and NMR analysis. The peak at around 7.6 d ppm in
1H NMR spectra and the signal between 130 and 135 d ppm in
13C NMR spectra confirm the @CH at the fifth position of thiazoli-
dine ring system. The reason for this deshielding is attributed to
the cis-position of the carbonyl function of 2-thioxo-thiazolidine-
4-one ring to the @CH and hence the Z configuration [27,28]. Cis
positioning is due to a high degree of thermodynamic stability of
these compounds because of the intramolecular hydrogen bonding
that are formed between the hydrogen atom of the @CH and the
oxygen atom in the 2-thioxo-thiazolidine-4-one ring [29].
3.2. In vivo antihyperglycemic activity
Serum glucose and triglyceride lowering activity of the six most
active compounds and the standard drug (rosiglitazone), were
tested at a dose of 10 mg/kg, p.o. against dexamethasone induced
hyperlipidemia and insulin resistance in Wistar rats [33]. Dexa-
methazone being a glucocorticoid, is well known for inducing
obesity and insulin resistance in both animals and human beings
[34,35]. On the day 11 of treatment, blood was collected for the
estimation of serum glucose and triglyceride levels. The results of
in vivo antihyperglycemic activity of the test compounds 15, 23,
24, 25, 27 and 28 are shown in Table 2.
3. Results and discussion
3.1. Glucose uptake by in vitro rat hemi-diaphragm
3.2.1. Effect on serum glucose level
Except compound 28, all the compounds at a dose of 10 mg/kg,
p.o. significantly decrease the dexamethasone induced elevation of
serum glucose. Standard rosiglitazone also significantly (P < 0.01)
decrease the dexamethasone induced elevation of serum glucose.
Among the six compounds tested, compound 23 show good anti-
hyperglycemic activity superior to rosiglitazone. Compounds 24,
27 and 25 also show significant glucose lowering activity
(P < 0.01) in the descending order. Compound 15 shows good activ-
ity (P < 0.05). Compound 28 shows weak activity.
The antidiabetic activities of the 24 synthesized compounds
were measured using glucose uptake by rat hemi-diaphargm
according to the method described by Walaas and Chattopadhyay
[30,24]. The institutional animal ethics committee (IAEC) of JSS
College of Pharmacy, Ootacamund, India, approved the proposal.
Rat diaphragm was selected because the striated muscle is quanti-
tatively the most important tissue for glucose disposal in the ani-
mal body. The glucose content was measured and the glucose
uptake was calculated as the difference between the initial and
final glucose content. Data were expressed as mean standard er-
ror of mean (SEM) and shown in Table 1.
3.2.2. Effect on serum triglyceride level
Statistical comparisons between the groups were performed in
two sets using one-way ANOVA followed by Dunnet’s multiple
comparison post-test using graphPad Prism 4.0 software for
Out of the six compounds and rosiglitazone tested, rosiglitaz-
one, compounds 25, 23, 27 and 24 show significant triglyceride
lowering activity (P < 0.01) as compared to the dexamethasone

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
15
Table 1
Glitazones and their effect on glucose uptake by the isolated rat hemi-diaphragm.
Cpd. no. (group no.)
R
Glucose uptake (mg/g/45 min)
CoMSIA
No insulin
With insulin
Àln MGU
Pred. act
3 (3,28)
4 (4,29)
–
–
9.86 0.97
10.40 0.96
9.90 0.82
26.82 1.80
28.03 0.92
27.77 2.32
2.3492
2.3604
2.6749a
2.38
2.332
2.672a
5 (5,30)
6 (6/31)
7 (7,32)
Cl
10.04 1.04
10.06 1.34
29.32 1.66
27.51 1.71
2.6427
2.5962
2.63
N
2.612
10.70 0.50
10.32 0.91
9.04 0.93
28.84 1.70
29.28 1.25
26.16 1.18
2.5914
2.612
2.619
2.592
2.618
2.603
8 (8,33)
HO
9 (9,34)
H3CO
10 (10,35)
O
9.69 1.15
9.33 0.91
27.79 0.88
26.30 1.53
2.6285
2.8103
2.619
2.798
11 (11,36)
12 (12,37)
OH
Cl
Cl
10.55 0.91
10.72 1.00
11.56 2.15
29.87 1.73
30.39 1.47
33.95 2.11**
2.5563a
2.6237
2.5412
2.668a
2.661
2.535
13 (13,38)
14 (14,39)
15 (15,40)
Cl
HO
O
10.55 0.98
28.80 1.00
2.7555
2.749
16 (16,41)
Cl
Cl
10.86 1.40
10.63 1.14
32.60 1.26**
31.07 1.80
2.5439
2.6074
2.543
2.61
17 (17,42)
18 (18,43)
H3CO
O2N
10.37 1.02
28.59 1.03
2.6848
2.68
19 (19,44)
Cl
10.81 1.61
12.55 1.03
30.11 1.30
2.5891a
2.7765
2.612a
2.780
20 (20,45)
23 (21,46)
HO
36.98 0.87**
O
H
N
O
O
H3CO
11.02 1.28
33.80 0.73**
2.8913
2.885
O
NO2
H
24 (22,47)
N
O
O
(continued on next page)

16
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
Table 1 (continued)
Cpd. no. (group no.)
R
Glucose uptake (mg/g/45 min)
CoMSIA
Àln MGU
2.8318
No insulin
With insulin
Pred. act
2.833
11.76 1.54
33.28 1.04**
O
25 (23,48)
H
N
N
O
O
O
11.00 0.91
11.52 1.54
31.61 0.98*
2.8022
2.804
O
26 (24,49)
27 (25,50)
34.55 1.29**
2.7926a
2.713a
O
H
N
O
O
O
O
12.01 1.08
11.26 0.85
33.88 1.47**
36.60 1.24**
2.7575
2.762
–
H
N
28 (26,51)
Std (27,52)
–
–
Group 1: 7.80 0.56, group 2: 25.56 0.63.
Std: Standard drug (rosiglitazone).
a
Test set compounds.
P < 0.05.
P < 0.01.
*
**
Table 2
Effect of glitazones on dexamethasone induced elevation of serum glucose and triglyceride levels.
Group
Treatment
Serum glucose (mg/dL)
Serum triglyceride (mg/dL)
1 (Normal)
2 (Positive control)
3 (Standard drug)
4
5
6
7
8
9
Vehicle (10 mL/kg, p.o.)
118.8 2.04**
238.0 6.67**
147.8 3.80**
209.8 6.53*
132.3 3.14**
171.7 5.62**
185.2 4.90**
177.3 9.31**
222.3 12.51
92.67 5.88**
270.0 7.58**
177.5 8.67**
242.3 12.63*
184.5 8.119**
194.5 6.428**
186.5 11.60**
193.0 9.438**
268.8 12.73
Dexamethasone (10 mg/kg, s.c.) + vehicle (10 mL/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + rosiglitazone (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 15 (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 23 (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 24 (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 25 (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 27 (10 mg/kg, p.o.)
Dexamethasone (10 mg/kg, s.c.) + compound 28 (10 mg/kg, p.o.)
Values mean SEM, n = 6.
s.c.: subcutaneously.
*
P < 0.05.
P < 0.01.
**
alone treated group. Compound 15 show good activity (P < 0.05).
Compound 28 shows weak activity.
3.3. Histopathology of the liver
It is well known that insulin resistance in type II diabetes is not
only associated with hyperglycemia but also with hyperlipidemia
and atherosclerosis. Drugs that simultaneously ameliorate insulin
resistance and hyperlipidemia, therefore, facilitates better man-
agement of type II diabetes [36,37]. The present study thus aims
towards the development of such a drug. All the six compounds
screened show both serum glucose and triglyceride lowering activ-
ity except compound 28. Compounds 24 and 23 that emerge as the
most active compounds in in vitro studies also show more or less
similar trends in in vivo studies. Compounds 24, 25 and 27 which
are active in the in vitro study appear to be more or less same in
the in vivo study as well. The results reveal some degree of corre-
lation between serum glucose lowering activity and the serum tri-
glyceride lowering activity in compounds 15, 23, 24, 27, 28 and
rosiglitazone. Surprisingly, compound 28 (analogous to 27 and
23) lacking vanillin as its trunk and methoxy group over the termi-
nal aromatic ring (hydrophobic tail) fails to exhibit good activity.
PPAR ligand-induced hepato toxicity could be influenced by any
number of mechanisms that may or may not be receptor-depen-
dent. However, it is tempting to suggest that a specific PPAR ligand
would cause toxicity solely through a receptor-mediated mecha-
nism. This may not always be true and in any case not clearly
understood. Liver, being an organ known to facilitate the first pass
drug metabolism and detoxification process, thus gets itself dam-
aged due to the release of toxic metabolites. Troglitazone was re-
moved from the market after reports of some deaths because of
the rare but severe hepatotoxicity. It also led to the discontinuation
of ciglitazone and englitazone after the phase II clinical trials.
Reported cases involving the newer TZDs, rosiglitazone and pioglit-
azone, have been fewer in number and less severe in consequence.
Pharmacovigilance for hepatotoxicity is probably still required
from the point of liver safety [38–40].
Eventhough there is a need for separate long term study on the
liver toxicity of these compounds, we have made an attempt to

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
17
study the liver samples of the rats that received glitazones to study
the effect of glitazones on dexamethasone induced elevation of
serum glucose and triglyceride levels. After the sacrifice of animals,
the rat liver samples from the selected groups of in vivo study
subjected to the histopathological examination under light micros-
copy. The microscopic slides are shown in Table 3. Histopatholo-
gical examinations of liver samples of vehicle treated group
reveal no abnormalities (group 1, slides 1 and 2). Dexamethazone
treated rat liver samples also show no abnormalities (group 2),
possibly due to beneficial effect of dexamethazone as an anti-
inflammatory agent at the dose of 10 mg/kg [41].
The standard drug, rosiglitazone, treated liver samples (group 3)
were normal and no abnormalities were observed indicating that
rosiglitazone reduces triglyceride levels to keep the liver cells
almost normal [42].
Compound 23 treated rats (group 5) show vacuolar degenera-
tion with focal necrobiotic changes (slide 10a), distortion of hepa-
tocytes with multifocal infiltration of inflammatory cells (slide
10b) and cytoplasmic rarification of hepatocytes in liver paren-
chyma (slide 12). It also shows necrosis, severe infiltration of
inflammatory cells and fibrous tissue proliferation at the periphery
(slide 11). These observations indicate that compound 23 is prone
to cause hepatotoxicity.
P, steric and electrostatic contributions are given in Table 4. Steric,
electrostatic, hydrophobicity, hydrogen bond donors and acceptors
contributions were found to be 18.1%, 19.2%, 28.3%, 23.2% and
11.2%, respectively. The developed model was further validated
by predicting the activities of the compounds of the test set.
3.5. Structure–activity relationship
The structure–activity relationships drawn from the above con-
tour plots of the steric, electrostatic, hydrophobic, hydrogen bond
acceptor and donor contributions of the generated CoMSIA model
are, the superimposed common part of the structures, namely, po-
lar rhodanine head followed by polar tyrosine or phenylalanine
moiety at the third position and benzylidene moiety at the fifth
position of the rhodanine ring seems to be the pharmacophore,
as most of the compounds possesses this common substructure
(Figs. 3–5). This indicates the necessity of relatively more polar
acidic groups nearer the thiazolidine head.
The hydrophobic benzyloxy trunk, which in turn is connected to
the two carbon acyl linker in association with the amide bond, also
seems to be the important structural components for the com-
pounds 23–28 to exhibit significant antidiabetic activity when
compared to the rest of the compounds (Figs. 3–5). Regarding the
hydrophobic trunk part, it appears vanillin is doing extremely good
as many of the active compounds in the series contain the same.
Compound 28 alone lacks the vanillin moiety from the series and
fails to produce good activity. The two carbon acyl linker, in the
form of amide bridge (CH2CONH) in these compounds, appears to
be an important requirement for their significant activity. The
hydrophobic tail part which differs in most of the compounds is
of major interest. Compound 23 with anisidine moiety is the most
active compound from this series, even when compared with the
standard drug (rosiglitazone). Compound 24 with o-nitro aniline
as its tail also shows equally good activity.
Rats that received compound 24 show no such severe abnor-
malities (group 6) in comparison to compound 23. However, one
of the animals shows eosinophilic degenerative area in liver paren-
chyma cells.
No sign of carcinoma, adenoma or preneoplastic lesion are ob-
served in all the samples. These results are just preliminary and
a separate long term toxicity study in the absence of dexamethaz-
one is, however, required to bring in clarity with respect to toxicity
profiles.
3.4. CoMSIA study
The yellow contour over the phenylalanine or tyrosine part of
the structure indicates that further extension is unfavorable for
the glucose uptake activity (Fig. 3). Small blue contour for the elec-
tronegative groups near the acidic carboxyl group in Fig. 3 and
orange contour for the hydrogen bond donor in the same region
in Fig. 4, indicate that they will favor glucose uptake activity as
evidenced by the yellow contour at the same region in Fig. 4 indi-
cating, hydrogen bond acceptors which are the components of
hydrogen bond donors at the similar positions are also essential
for the activity.
An oxygen atom, connected directly to the aromatic ring
(hydrophobic trunk) in the form of ether, is essential for the activ-
ity, as there is a blue contour near the red colored oxygen atoms in
Fig. 3. A two carbon atom linker between the oxygen atom and ter-
minal alicyclic or aromatic ring is ideal for the activity, as some of
the compounds not containing the two carbon atom linker fail to
show significant activity. Extension of carbon linker chain between
the benzyl moiety and the terminal heterocyclic ring may pull the
terminal ring towards the yellow region and decrease the activity.
A prominent yellow contour in that region indicates that there
should not be any substitution over those two carbon atoms.
In Fig. 4, the magenta colored contour after the benzoxy group
(hydrophobic trunk) and over the two carbon linker indicates that
introducing hydrogen bond donor in that region will reduce the
activity. Compound 8 with phenolic OH group at the same position
fails to produce good glucose uptake activity. However, compound
15 with a vanillin moiety as its trunk produces reasonably good
glucose uptake activity. This could probably be due to the presence
of OCH3 group in vanillin structure apart from the hydrogen bond
donor group (OH).
The 3D-QSAR models were derived employing the CoMSIA
method with 20 compounds in the training set. The remaining four
compounds, which are congeneric to training set compounds, con-
stituted the test set. All the molecules were processed through the
regular protocol of conformational analysis, energy minimization
and denoting charges as described in Section 5. The alignment rule
was optimized by using the appropriate common substructure,
2-(4-oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic acid. The
aligned training set is shown in Figs. 2–4. Field fit was used to opti-
mize the alignment of molecules to a previously calculated steric,
electrostatic, hydrophobicity, hydrogen bond donors and acceptors
fields [43]. The PLS analyses on 20 training set compounds yielded
a q2 value (by LOO method) of 0.623 at the optimal number of com-
ponents 4. The statistical data are shown in Table 4. Since the value
of q2 was reasonably good, no compound was removed as an out-
lier. Further, exclusion of compounds of a particular congeneric
series may affect the predictivity of the model. The developed
model show good predicted activities for the training set with
low residual values as shown in Table 1 and Fig. 2. The predictive
ability of the model was checked by predicting the activities of
the test set compounds under different models. The model corre-
lating the training set compounds with respect to steric, electro-
static, hydrophobic, hydrogen bond donor and hydrogen bond
acceptor fields seems to be of better statistical significance when
compared to all other models as shown in Table 4. The predicted
activities of the training and test set compounds reported in this
article are all from this optimized model.
Contour plots depicting the steric, electrostatic, hydrophobicity,
hydrogen bond donor and hydrogen bond acceptor fields of the
compounds generated based on the contributions towards the
PLS model is shown in Fig. 2. Statistical parameters like q2, r2, F,
The cyan colored contours over the thiazolidine ring system,
near the two carbon linker and after the hydrophobic tail, indicates

18
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
Table 3
Histopathological results of some liver samples.
Slide no.
1
Group no.
1
Treatment
Microscopic observations and pictures
Vehicle (10 mL/kg, p.o.)
No abnormalities detected (H&E 45Â)
2
1
-do-
No abnormality detected (H&E 100Â)
3
1
-do-
No abnormalities detected (H&E 45Â)
4
2
Vehicle (10 mL/kg, p.o.) + dexamethasone
(10 mg/kg, s.c.)
No abnormalities detected (H&E 45Â)
5
2
-do-

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
19
Table 3 (continued)
Slide no.
Group no.
Treatment
Microscopic observations and pictures
No abnormalities detected (H&E 45Â)
6
7
2
3
-do-
Rosiglitazone (10 mg/kg, p.o.)
No abnormalities detected (H&E 45Â)
8
3
-do-
No abnormality detected (H&E 100Â)
9
3
-do-
No abnormalities detected (H&E 45Â)
(continued on next page)

20
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
Table 3 (continued)
Slide no.
10a
Group no.
Treatment
Microscopic observations and pictures
5
Compound 23 (10 mg/kg, p.o.)
Note vacuolar degeneration with focal necrobiotic changes (H&E 100Â)
10b
5
-do-
Distortion of hepatocytes with multifocal infiltration of inflammatory cells (H&E 100Â)
11
5
-do-
Note central area of necrosis with severe infiltration of inflammatory
cells and fibrous tissue proliferation at the periphery (H&E 45Â)
12
5
-do-
Cytoplasmic rarefication of hepatocytes in liver parenchyma (H&E 100Â)

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
21
Table 3 (continued)
Slide no.
13
Group no.
Treatment
Microscopic observations and pictures
6
Compound 24 (10 mg/kg, p.o.)
No abnormalities detected (H&E 45Â)
14
6
-do-
Note eosinophilic degenerative area in liver parenchyma (H&E 100Â)
15
6
-do-
No abnormalities detected (H&E 45Â)
NAD: No abnormality detected.
that introducing some hydrophobic groups at these positions will
reduce the activity (Fig. 5). The small white contour near the benz-
oxy group (hydrophobic trunk) in Fig. 5 indicates that replacing OH
group with OCH3 group will enhance the activity (compounds 17
and 23).
Finally, the developed CoMSIA model indicates that thiazolidin-
edione ring system with tyrosine substitution at the third position
and a hydrophobic trunk connected to the hydrophobic tail via a
two carbon linker at the fifth position is essential for these to exhi-
bit significant activity.
glucose uptake activity, glucose lowering activity and triglyceride
lowering activity. Compounds 23 and 24 seem to be the candidate
compounds to investigate further for their efficacy and safety.
5. Experimental section
5.1. Chemistry
The melting points were determined in open capillaries using
Veego VMP-1 melting point apparatus and are uncorrected. The
temperatures were expressed in °C. The IR spectra were recorded
on Shimadzu infrared-283 FTIR spectrometer by KBr pellet tech-
nique and are expressed in cmÀ1. NMR spectra were recorded on
a Bruker Avance 500 and 400 spectrometers with TMS as internal
standard (500 and 400 MHz for 1H NMR, 125 and 100 MHz for
13C) and DMSO-d6 as solvent. The chemical shifts are expressed
in d ppm and the following abbreviations are used; s = singlet,
4. Summary and conclusion
In summary, we report here a simple, efficient method to syn-
thesize some novel rhodanines incorporated with amino acids such
as phenylalanine and tyrosine. We have thus found a new series of
glitazones with different body make up having promising levels of

22
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
3
2.8
2.6
2.4
2.2
2
2
2.2
2.4
2.6
2.8
3
Observed activity
Fig. 4. Aligned glitazones CoMSIA SD x coefficient contour plot: Orange contours
indicate regions where hydrogen bond donor is favorable and magenta contours
indicate regions where hydrogen bond donor is not favored; yellow contours
indicate regions where hydrogen bond acceptors increase activity and purple
contours indicate regions where hydrogen bond acceptors decrease activity. (For
interpretation of the references to color in this figure legend, the reader is referred
to the web version of this article.)
Fig. 2. Correlation between the observed and predicted activities of the developed
CoMSIA model: j indicates the training set compounds and d indicates the test set
compounds.
allowed to stand for 2 h in a refrigerator to yield dithiocarbamate
of the corresponding amino acid (1 and 2). Sodium chloroacetate
solution was prepared, by dissolving chloroacetic acid (0.003 M)
and NaOH (0.003 M) each in 3 mL of water, separately. Both were
then mixed and stirred for 30 min. Solid sodium carbonate was
then added slowly until the reaction mixture became basic to
litmus. Sodium chloroacetate solution was added to a flask con-
taining dithiocarbamate solution slowly at 5–10 °C under stirring.
The reaction mixture was allowed to stir for 1 h. The uncyclized
N-substituted rhodanine formed was cyclized by adding 10 mL of
6 N HCl, 2 or 3 drops of phosphorous oxychloride and heated at
60–70 °C for about 3 h. The reaction was monitored through TLC
using 20% methanol in chloroform as the mobile phase. The spots
on TLC were detected using alcoholic dodeca-phosphomolybdinum
staining and heating, apart from using Ninhydrin reagent. The
reaction mixture was allowed to cool. The solid formed was filtered
and recrystallised with diethyl ether to obtain 3 and 4. Compounds
5–12 and 13–20 were prepared by subjecting 3 and 4 to the
Knoevenagel condensation reaction with different substituted
aldehydes.
Fig. 3. Aligned glitazones CoMSIA SD x coefficient contour plot: Green contours
indicate regions where steric bulk is favorable and yellow contours indicate regions
where steric bulk is not favored; Blue contours indicate regions where electroneg-
ative groups increase activity and red contours indicate regions where electroneg-
ative groups decrease activity. (For interpretation of the references to color in this
figure legend, the reader is referred to the web version of this article.)
bs = broad singlet, d = doublet, bd = broad doublet, t = triplet,
bt = broad triplet, q = quartlet, m = multiplet, bm = broad multiplet.
Attempt has also been made to identify the peaks in 13C NMR spec-
tra based on the relative chemical environments of the carbon
atoms in the structure. Mass spectra were recorded using high
resolution mass spectrometer (HRMS) and liquid chromatography
mass spectrometer (LCMS) under electro spray ionization tech-
nique, using time of flight and triple quadrupole mass analyzers,
respectively. All the compounds synthesized showed 1H and 13C
NMR, IR and Mass spectra consistent with the proposed structures.
Column chromatography was done with Merck silica gel 60
(0.0634, 200 mm). When reactions were performed in anhydrous
conditions, the mixtures were maintained under nitrogen. TLC
was performed using 4% methanol in chloroform as mobile phase
on aluminum plates which are precoated with silica gel GF, unless
otherwise stated. Compounds were named following the IUPAC
rules.
5.1.1.1. ( )-2-(4-Oxo-2-thioxothiazolidin-3-yl)-3-phenylpropanoic
acid (3). Pale yellow amorphous solid, mp 188–190 °C, Yield 86%.
FTIR (KBr, cmÀ1): 3400–2700 (O–H), 3028 (Ar–H), 2924 (AliC–H),
1741 (C@O), 1708 (C@O), 1494 (ArC@C), 1222 (C@S), 1201 (C–O),
1174 (C–N). 1H NMR (300 MHz, DMSO-d6, d ppm): 3.54 (t,
J = 6.0 Hz, 2H, CH2), 3.82 (s, 2H, CH2), 5.74 (bm, 1H,
a-hydrogen,
CH), 7.14–7.40 (m, 5H, ArH). 13C NMR (500 MHz, DMSO-d6, d
ppm): 33.41 (CH2), 35.16 (CH2), 58.51 (CH), 127.12 (@C), 128.73
(2ArC), 129.51 (2ArC), 137.16 (1ArC), 169.33 (C@O), 174.43
(C@O), 203.11 (C@S). HRMS (ES-TOF) m/z found 304.0068
(M+Na), calculated 304.0078 (M+Na).
5.1.1.2. ( )-2-((Z)-5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-3-
(4-hydroxyphenyl)propanoic acid (4). Yellow crystals, mp 210–
212 °C, Yield 58%. FTIR (KBr, cmÀ1): 3500–2900 (O–H), 3022
(Ar–H), 2960 (AliC–H), 1707 (C@O), 1608 (C@O), 1512 (ArC@C),
1267 (C@S), 1244 (C–O), 1153 (C–N). 1H NMR (400 MHz, DMSO-
d6, d ppm): 3.37 (bt, 2H, CH2), 5.43 (bm, 1H, CH), 6.55 (d,
J = 8.5 Hz, 2H, ArH), 6.85 (d, J = 8.5 Hz, 2H, ArH), 7.45–7.62 (m,
5H, ArH), 7.64 (s, 1H, @CH), 8.91 (bs, 1H, OH). 13C NMR
(400 MHz, DMSO-d6, d ppm): 34.08 (CH2), 61.22 (CH), 115.57
(1ArC), 115.79 (2ArC), 125.70 (@C), 129.71 (1ArC), 130.02 (1ArC),
130.33 (1ArC), 130.39 (1ArC), 130.69 (2ArC), 130.91 (1ArH),
5.1.1. General procedure followed for the preparation of 3 and 4
Water (10 mL) and ammonia solution (30 mL, 25–30%) were
transferred into a conical flask. The conical flask was placed in
ice bath and the temperature was maintained at 0–5 °C. Carbon
disulfide (0.0031 M) was then added. ( )-Tyrosine or ( )-phenylal-
anine (0.003 M) was added slowly to this solution for a period of
30 min with continuous stirring. Cotton plug was put to the flask
and the reaction mixture was continued to stir for 10 h and

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
23
Table 4
PLS statistics of the CoMSIA models.
PLS statistics parameters
CoMSIA
SE
SHE
SED
SEA
SEDA
SEHD
SEHA
SEHDA
q2
0.520
0.798
0.163
105.76
4
0.558
0.873
0.122
119.40
4
0.527
0.806
0.117
124.29
4
0.540
0.855
0.089
137.60
4
0.545
0.857
0.078
141.55
4
0.585
0.928
0.041
170.25
4
0.609
0.942
0.036
182.86
4
0.623
0.989
0.018
193.99
4
r2
SEE
F
PLS components
P value
0
0
0
0
0
0
0
0
Steric
0.479
0.521
–
–
–
0.285
0.314
0.401
–
0.246
0.455
–
0.299
–
0.359
0.491
–
0.212
0.325
–
0.303
0.160
0.487
0.145
0.272
0.358
0.225
–
0.203
0.279
0.382
–
0.136
0.553
0.181
0.192
0.283
0.232
0.112
0.570
Electrostatic
Hydrophobic
H donor
H acceptor
Pred r2 for test set
–
–
0.150
0.470
0.385
0.435
0.397
0.528
SEE = Standard error of estimate, S = steric field, E = electrostatic field, H = hydrophobic field, D = hydrogen bond donor, A = hydrogen bond acceptor.
tals, mp 282–286 °C, Yield 75%. FTIR (KBr, cmÀ1): 3350–2500 (O–
H), 3028 (Ar–H), 2945 (AliC–H), 1701 (C@O), 1673 (C@O), 1525
(ArC@C), 1280 (C@S), 1226 (O–H), 1172 (C–N). 1H NMR
(400 MHz, DMSO-d6, d ppm): 3.50 (bt, 2H, CH2), 3.04 (s, 6H,
2 Â CH3), 5.50 (bm, 1H, CH), 6.82 (d, J = 9.04 Hz, 2H, ArH), 7.05–
7.18 (m, 5H, ArH), 7.40 (d, J = 9.04 Hz, 2H, ArH), 7.53 (s, 1H,
@CH). HRMS (ES-TOF) m/z found 435.0803 (M+Na), calculated
435.1007 (M+Na).
5.1.2.3. ( )-2-((Z)-5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-3-
phenylpropanoic acid (7). Pale yellow crystals, mp 236–239 °C,
Yield 78%. FTIR (KBr, cmÀ1): 3000–2500 (O–H), 3005 (Ar–H),
2939 (AliC–H), 1697 (C@O), 1593 (ArC@C), 1271 (C@S), 1232
(C–O), 1172 (C–N). 1H NMR (400 MHz, DMSO-d6, d ppm): 3.54
(bt, 2H, CH2), 5.49 (bm, 1H, CH), 7.07–7.20 (m, 5H, ArH), 7.50–
7.60 (m, 5H, ArH), 7.69 (s, 1H, @CH). 13C NMR (400 MHz, DMSO-
d6, d ppm): 34.32 (CH2), 61.99 (CH), 121.68 (@C), 125.96 (2ArC),
128.06 (2ArC), 128.56 (2ArC), 129.41 (1ArC), 130.48 (2ArC),
130.71 (1ArC), 131.91 (1ArC), 132.97 (1ArC), 139.04 (@CH),
166.88 (C@O), 168.67 (C@O), 194 (C@S). HRMS (ES-TOF) m/z found
392.0402 (M+Na), calculated 392.0391 (M+Na).
Fig. 5. Aligned glitazones CoMSIA SD x coefficient contour plot: Cyan contours
indicate regions where hydrophobicity is unfavorable and white contours indicate
regions where hydrophobicity favored. (For interpretation of the references to color
in this figure legend, the reader is referred to the web version of this article.)
131.02 (1ArC), 131.29 (1ArH), 156.98 (@CH), 170.82 (C@O), 171.42
(C@O), 190.77 (C@S). HRMS (ES-TOF) m/z found 408.0345 (M+Na),
calculated 408.1046 (M+Na).
5.1.2. General procedure for the Knoevenagel condensation reaction
Equimolar amounts of the substrate, 3 or 4, 22 (0.002 M) and
20–30 mL of dry toluene, were transferred to a flask. N-methyl
piperazine (0.0001 M), ammonium acetate (0.0001 M) and molec-
ular sieves (0.5 g) were then added. The reaction mixture was stir-
red for 5 min and then heated under reflux at 110 °C with
occasional stirring for about 15 h. After the completion of the reac-
tion, the reaction mixture was allowed to cool and the precipitated
solid was filtered and recrystallized using methanol to obtain the
pure compound [24].
5.1.2.4. ( )-2-((Z)-5-(4-hydroxybenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-phenylpropanoic acid (8). Yellow amorphous solid, mp
297–300 °C, Yield 73%. FTIR (KBr, cmÀ1): 3250–2500 (O–H), 3005
(Ar–H), 2943 (AliC–H), 1695 (C@O), 1633 (C@O), 1579 (ArC@C),
1267 (C@S), 1219 (C–O), 1168 (C–N). 1H NMR (400 MHz, DMSO-
d6, d ppm): 3.52 (bt, 2H, CH2), 5.53 (bm, 1H, CH), 6.90 (d,
J = 8.72 Hz, 2H, ArH), 7.05–7.18 (m, 5H, ArH), 7.42 (d, J = 8.68 Hz,
2H, ArH), 7.55 (s, 1H, @CH), 9.76 (bs, 1H, OH). 13C NMR
(400 MHz, DMSO-d6, d ppm): 34.35 (CH2), 61.63 (CH), 116.71
(2ArC), 123.55 (@C), 125.96 (1ArC), 128.03 (2ArC), 128.61 (2ArC),
128.83 (1ArC), 132.80 (1ArC), 133.05 (2ArH), 133.98 (@CH),
161.16 (1ArC), 167.03 (C@O), 169.34 (C@O), 193.63 (C@S). HRMS
(ES-TOF) m/z found 408.0344 (M+Na), calculated 408.0340 (M+Na).
5.1.2.1. ( )-2-((Z)-5-(4-chlorobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-phenylpropanoic acid (5). Yellow crystals, mp 250–
252 °C, Yield 76%. FTIR (KBr, cmÀ1): 3400 (O–H), 3014 (Ar–H),
2943 (AliC–H), 1705 (C@O), 1641 (C@O), 1585 (ArC@C), 1274
(C@S), 1238 (C–O), 1166 (C–N), 675 (C–Cl). 1H NMR (400 MHz,
DMSO-d6, d ppm): 3.53 (bt, 2H, CH2), 5.50 (bm, 1H, CH), 7.07–
7.62 (m, 9H, ArH), 7.70 (s, 1H, @CH). 13C NMR (400 MHz, DMSO-
d6, d ppm): 34.19 (CH2), 61.69 (CH), 122.30 (@C), 126.05 (2ArC),
128.09 (2ArC), 128.59 (2ArC), 129.47 (1ArC), 130.72 (1ArC),
131.83 (1ArC), 132.12 (2ArC), 135.39 (1ArC), 135.77 (@CH),
166.75 (C@O), 168.82 (C@O), 193.33 (C@S). HRMS (ES-TOF) m/z
found 426.0004 (M+Na), calculated 426.0001 (M+Na).
5.1.2.5. ( )-2-((Z)-5-(4-methoxybenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-phenylpropanoic acid (9). Yellow crystlas, mp 264–
267 °C, Yield 81%. FTIR (KBr, cmÀ1): 3100–2500 (O–H), 3005
(Ar–H), 2941 (AliC–H), 1687 (C@O), 1635 (C@O), 1583 (ArC@C),
1265 (C@S), 1220 (C–O), 1170 (C–N). 1H NMR (400 MHz, DMSO-
d6, d ppm): 3.52 (bt, 2H, CH2), 3.82 (s, 3H, CH3), 5.50 (bm, 1H,
CH), 7.05–7.20 (m, 7H, ArH), 7.47 (s, 1H, @CH), 7.55 (d, J = 8.8 Hz,
2H, ArH). 13C NMR (400 MHz, DMSO-d6, d ppm): 34.31 (CH2),
55.51 (CH3), 61.86 (CH), 115.07 (2ArC), 125.57 (@C), 125.93
(2ArC), 128.03 (2ArC), 128.56 (2ArC), 130.12 (1ArC), 132.01
(1ArC), 132.69 (2ArH), 133.09 (@CH), 161.29 (C@O), 168.62
5.1.2.2. ( )-2-((Z)-5-(4-(dimethylamino)benzylidene)-4-oxo-2-thi-
oxothiazolidin-3-yl)-3-phenylpropanoic acid (6). Orange red crys-

24
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
(C@O), 194.27 (C@S). HRMS (ES-TOF) m/z found 422.0498 (M+Na),
calculated 422.0961 (M+Na).
1041 (C–N). 1H NMR (500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H,
CH2), 3.83 (s, 3H, CH3), 5.56 (bm, 1H, CH), 6.50–7.0 (m, 7H, ArH),
7.64 (s, 1H, @CH), 9.14 (bs, 1H, OH), 9.21 (bs, 1H, OH). LCMS (LC-
MSD-Trap-SL) m/z found 432.0 (M+1), calculated 432.0 (M+1).
5.1.2.6. ( )-2-((Z)-5-((furan-2-yl)methylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl)-3-phenylpropanoic acid (10). Black crystals, mp 221–
224 °C, Yield 64%. FTIR (KBr, cmÀ1): 3450–2700 (O–H), 3000 (Ar–
H), 2949 (AliC–H), 1705 (C@O), 1668 (C@O), 1556 (ArC@C), 1239
(C@S), 1211 (C–O), 1082 (C–N). 1H NMR (400 MHz, DMSO-d6, d
ppm): 3.50 (bt, 2H, CH2), 5.58 (bm, 1H, CH), 7.18–7.78 (m, 8H,
ArH) 7.70 (s, 1H, @CH). 13C NMR (400 MHz, DMSO-d6, d ppm):
34.33 (CH2), 61.75 (CH), 121.41 (@C), 128.87 (2ArC), 129.48
(1ArC), 129.67 (2ArC), 137.24 (1ArC), 138.35 (1ArC), 133.05
(@CH), 139.78 (1ArC), 143.2 (1ArC), 148.0 (1ArC), 169.37 (C@O),
174.39 (C@O), 198.76 (C@S). HRMS (ES-TOF) m/z found 382.118
(M+Na), calculated 382.2016 (M+Na).
5.1.2.12. ( )-2-((Z)-5-(2,4-dichlorobenzylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl)-3-(4-hydroxyphenyl)propanoic acid (16). Yellow amor-
phous solid, mp > 300 °C, Yield 59%. FTIR (KBr, cmÀ1): 3400–2500
(O–H), 3022 (Ar–H), 2960 (AliC–H), 1716 (C@O), 1683 (C@O),
1512 (ArC@C), 1244 (C@S), 1215 (C–O), 1111(C–N), 574 (C–Cl).
1H NMR (500 MHz, DMSO-d6, d ppm): 3.38 (bt, 2H, CH2), 5.75
(bm, 1H, CH), 6.52–7.6 (m, 7H, ArH), 7.85 (s, 1H, @CH), 9.25 (bs,
1H, OH). LCMS (LC-MSD-Trap-SL) m/z found 454.0 (M+1), calcu-
lated 454.0 (M+1).
5.1.2.13. ( )-2-((Z)-5-(4-methoxybenzylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl)-3-(4-hydroxyphenyl)propanoic acid (17). Yellow crys-
tals, mp 223–225 °C, Yield 61%. FTIR (KBr, cmÀ1): 3500–2500 (O–
H), 3026 (Ar–H), 2960 (AliC–H), 1732 (C@O), 1707 (C@O), 1589
(ArC@C), 1230 (C@S), 1170 (C–O), 1012 (C–N). 1H NMR
(500 MHz, DMSO-d6, d ppm): 3.40 (t, 2H, CH2), 3.88 (s, 3H, CH3),
5.62 (bm, 1H, CH), 6.65 (d, J = 8.4 Hz, 2H, ArH), 6.70 (d, J = 8.5,
2H, ArH), 6.94 (d, J = 8.4 Hz, 2H, ArH), 7.03 (d, J = 8.5 Hz, 2H,
ArH), 7.84 (s, 1H, @CH), 9.28 (bs, 1H, OH), 10.34 (bs, 1H, OH). 13C
NMR (500 MHz, DMSO-d6, d ppm): 36.00 (CH2), 56.07 (CH3),
59.13 (CH), 115.35 (2ArC), 115.51 (2ArC), 125.93 (@C), 130.22
(1ArC), 130.77 (2ArC), 130.90 (2ArC), 133.47 (@CH), 156.22
(1ArC), 156.92 (1ArC), 157.64 (1ArC), 170.90 (C@O), 175.78
(C@O), 196.0 (C@S). LCMS (LC-MSD-Trap-SL) m/z found 416.8
(M+1), calculated 415.0 (M+1).
5.1.2.7. ( )-2-((Z)-5-(2-hydroxybenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-phenylpropanoic acid (11). Yellow crystals, mp 272–
275 °C, Yield 62%. FTIR (KBr, cmÀ1): 3460–3050 (O–H), 3042 (Ar–
H), 2965 (AliC–H), 1701 (C@O), 1677 (C@O), 1589 (ArC@C), 1228
(C@S), 1228 (C–O), 1155 (C–N). 1H NMR (400 MHz, DMSO-d6, d
ppm): 3.52 (bt, 2H, CH2), 5.50 (bm, 1H, CH), 7.08–7.58 (m, 9H,
ArH), 7.71 (s, 1H, @CH), 9.62 (bs, 1H, OH). HRMS (ES-TOF) m/z
found 408.2018 (M+Na), calculated 408.3009 (M+Na).
5.1.2.8. ( )-2-((Z)-5-(2,4-dichlorobenzylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl)-3-phenylpropanoic acid (12). Light yellow crystals, mp
201–204 °C, Yield 64%. FTIR (KBr, cmÀ1): 3300–2600 (O–H), 3026
(Ar–H), 2951 (AliC–H), 1718 (C@O), 1676 (C@O), 1560 (ArC@C),
1274 (C@S), 1201 (C–O), 1155 (C–N), 653 (C–Cl). 1H NMR
(400 MHz, DMSO-d6, d ppm): 3.55 (bt, 2H, CH2), 5.50 (bm, 1H,
CH), 7.08–7.60 (m, 7H, ArH), 7.68 (s, 1H, ArH), 7.73 (s, 1H, @CH).
HRMS (ES-TOF) m/z found 461.2013 (M+Na), calculated 461.1820
(M+Na).
5.1.2.14. ( )-2-((Z)-5-(3-nitrobenzylidene)-4-oxo-2-thioxothiazolidin-
3-yl)-3-(4-hydroxyphenyl)propanoic acid (18). Pale yellow crystals,
mp 260–262 °C, Yield 59%. FTIR (KBr, cmÀ1): 3500–2500 (O–H),
3022 (Ar–H), 2929 (AliC–H), 1705 (C@O), 1700 (C@O), 1512
(ArC@C), 1244 (C@S), 1174 (C–O), 1014 (C–N). 1H NMR
(500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 5.75 (bm, 1H,
CH), 6.80–8.50 (m, 8H, ArH), 7.97 (s, 1H, @CH), 9.20 (bs, 1H, OH).
13C NMR (500 MHz, DMSO-d6, d ppm): 32.75 (CH2), 59.38 (CH),
115.41 (1ArC), 115.61 (2ArC), 122.39 (@C), 125.79 (1ArC), 127.02
(1ArC), 128.07 (1ArC), 130.33 (2ArC), 130.88 (1ArC), 131.52
(1ArC), 134.73 (@CH), 148.80 (1ArC), 156.21 (1ArC), 169.09
(C@O), 172.79 (C@O), 188 (C@S). LCMS (LC-MSD-Trap-SL) m/z
found 431.5 (M+1), calculated 431.0 (M+1).
5.1.2.9. ( )-2-((Z)-5-benzylidene-4-oxo-2-thioxothiazolidin-3-yl)-3-
(4-hydroxyphenyl)propanoic acid (13). Yellow crystals, mp 210–
212 °C, Yield 58%. FTIR (KBr, cmÀ1): 3500–2900 (O–H), 3022
(Ar–H), 2960 (AliC–H), 1707 (C@O), 1608 (C@O), 1512 (ArC@C),
1267 (C@S), 1244 (C–O), 1153 (C–N). 1H NMR (400 MHz, DMSO-
d6, d ppm): 3.37 (bt, 2H, CH2), 5.43 (bm, 1H, CH), 6.55 (d,
J = 8.5 Hz, 2H, ArH), 6.85 (d, J = 8.5 Hz, 2H, ArH), 7.45–7.62 (m,
5H, ArH), 7.64 (s, 1H, @CH), 8.91 (bs, 1H, OH). 13C NMR
(400 MHz, DMSO-d6, d ppm): 34.08 (CH2), 61.22 (CH), 115.57
(1ArC), 115.79 (2ArC), 125.70 (@C), 129.71 (1ArC), 130.02 (1ArC),
130.33 (1ArC), 130.39 (1ArC), 130.69 (2ArC), 130.91 (1ArH),
131.02 (1ArC), 131.29 (1ArH), 134.98 (@CH), 170.82 (C@O),
171.42 (C@O), 190.77 (C@S). HRMS (ES-TOF) m/z found 408.0345
(M+Na), calculated 408.0841 (M+Na).
5.1.2.15. ( )-2-((Z)-5-(2-chlorobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-(4-hydroxyphenyl)propanoic acid (19). Brown crystals,
mp 229–231 °C, Yield 55%. FTIR (KBr, cmÀ1): 3500–2500 (O–H),
3039 (Ar–H), 2960 (AliC–H), 1728 (C@O), 1700 (C@O), 1514
(ArC@C), 1244 (C@S), 1091 (C–O), 1041 (C–N), 574 (C–Cl). 1H
NMR (500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 5.73 (bm,
1H, CH), 6.60–7.60 (m, 8H, ArH), 7.90 (s, 1H, @CH), 9.28 (bs, 1H,
OH). LCMS (LC-MSD-Trap-SL) m/z found 420.0 (M+1), calculated
420.0 (M+1).
5.1.2.10. ( )-2-((Z)-5-(4-chlorobenzylidene)-4-oxo-2-thioxothiazoli-
din-3-yl)-3-(4-hydroxyphenyl)propanoic acid (14). Cream colored
crystals, mp 237–240 °C, Yield 65%. FTIR (KBr, cmÀ1): 3500–2800
(O–H), 3024 (Ar–H), 2906 (AliC–H), 1707 (C@O), 1637 (C@O),
1521 (ArC@C), 1300 (C@S), 1210 (C–O), 1099 (C–N), 534 (C–Cl).
1H NMR (400 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 5.40
(bm, 1H, CH), 6.60 (d, J = 8.3 Hz, 2H, ArH), 6.85 (d, J = 8.3 Hz, 2H,
ArH), 7.57–7.63 (m, 4H, ArH), 7.68 (s, 1H, @CH), 7.93 (bs, 1H,
OH). HRMS (ES-TOF) m/z found 442.0615 (M+Na), calculated
442.1075 (M+Na).
5.1.2.16. ( )-2-((Z)-5-(4-hydroxybenzylidene)-4-oxo-2-thioxothiaz-
olidin-3-yl)-3-(4-hydroxyphenyl)propanoic acid (20). Yellow crys-
tals, mp 278–281 °C, Yield 61%. FTIR (KBr, cmÀ1): 3500–2500 (O–
H), 3022 (Ar–H), 2960 (AliC–H), 1700 (C@O), 1707 (C@O), 1512
(ArC@C), 1244 (C@S), 1176 (C–O), 1041 (C–N). 1H NMR
(500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 5.64 (bm, 1H,
CH), 6.50 (d, J = 8.5 Hz, 2H, ArH), 6.85 (d, J = 8.5, 2H, ArH), 6.95
(d, J = 8.5 Hz, 2H, ArH), 7.45 (d, J = 8.5 Hz, 2H, ArH), 7.69 (s, 1H,
@CH), 9.15 (bs, 1H, OH), 9.20 (bs, 1H, OH). LCMS (LC-MSD-Trap-
SL) m/z found 400.0 (MÀ1), calculated 400.0 (MÀ1).
5.1.2.11. ( )-2-((Z)-5-(4-hydroxy-3-methoxybenzylidene)-4-oxo-2-
thioxothiazolidin-3-yl)-3-(4-hydroxyphenyl)propanoic
acid
(15). Orange crystals, mp 282–286 °C, Yield 76%. FTIR (KBr,
cmÀ1): 3400–2500 (O–H), 3039 (Ar–H), 2929 (AliC–H), 1701
(C@O), 1637 (C@O), 1512 (ArC@C), 1244 (C@S), 1176 (O–H),

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
25
5.1.3. Procedure for the preparation of compounds 23–28
(500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 3.89 (s, 3H, CH3),
4.95 (s, 2H, CH2), 5.60 (bm, ( hydrogen) 1H, CH), 6.60 (d,
Alicyclic or aromatic amine (0.1 M) and triethylamine (0.1 M)
along with dry chloroform (80 mL) were transferred into a flask fit-
ted with a guard tube. The mixture was allowed to stir under ice
cold conditions (0–5 °C). Chloroacetyl chloride (0.11 M) was added
drop wise for a period of 30 min. The reaction mixture was contin-
ued to stir to reach room temperature. The reaction mixture was
again stirred for about 6 h at 30–35 °C. The reaction was monitored
through TLC using 20% ethyl acetate in pet ether as a mobile phase.
After the completion of the reaction, the solvent was evaporated.
The product obtained was washed with water and dried to obtain
the acylated amines 21 (90–99%).
a
J = 8.5 Hz, 1H, ArH), 6.96 (t, J = 8.5 Hz, 1H, ArH), 7.12–8.05 (m,
8H, ArH), 7.46 (s, 1H, @CH), 8.34 (d, J = 8.5 Hz, 1H, ArH), 9.16 (bs,
1H, OH), 10.50 (s, 1H, NH). 13C NMR (500 MHz, DMSO-d6, d
ppm): 38.77 (CH2), 56.10 (CH3), 67.0 (CH2), 68.02 (CH), 110.34
(1ArC), 113.24 (1ArC), 115.36 (@C), 120.32 (1ArC), 122.91 (1ArC),
126.05 (1ArC), 128.40 (1ArC), 130.16 (1ArC), 134.57 (@CH),
138.87 (2ArC), 148.62 (2ArC), 149.75 (1ArC), 150.19 (1ArC),
151.72 (1ArC), 135.47 (1ArC), 140.15 (1ArC), 149.24 (1ArC),
156.12 (1ArC), 167.41 (C@O), 173.18 (C@O), 191.87 (C@S). LCMS
(LC-MSD-Trap-SL) m/z found 566.0 (M+1), calculated 566.0 (M+1).
A mixture of acylated amine 21 (0.09 M), finely powdered anhy-
drous K2CO3 (0.125 M) and vanillin (0.12 M) along with 80 mL of
dry acetone was stirred at 35–40 °C for about 36 h. The progress
of the reaction was monitored by TLC using 15% ethyl acetate in
pet ether as a mobile phase. The reaction mixture was then poured
into water and extracted with ethyl acetate. The ethyl acetate layer
was washed thrice with 10% aqueous NaOH solution, washed once
with brine solution and then finally dried over anhydrous Na2SO4.
The ethyl acetate layer was evaporated to obtain compound 22
(65–80%). Compounds 23–28 were prepared by subjecting the
substituted aldehyde 22 to the Knoevenagel condensation reaction
with tyrosine incorporated rhodanine 4.
5.1.3.4. ( )-2-((Z)-5-(4-(benzyloxy)-3-methoxybenzylidene)-4-oxo-2-
thioxo-thiazolidin-3-yl)-3-(4-hydroxyphenyl)propanoic acid (26). Yel-
low amorphous solid, mp 247–251 °C, Yield 69%. FTIR (KBr,
cmÀ1): 3400–2500 (O–H), 3022 (Ar–H), 2960 (AliC–H), 1712
(C@O), 1508 (ArC@C), 1267 (C@S), 1244 (C–O), 1138 (C–N). 1H
NMR (500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 3.84 (s, 3H,
CH3), 5.22 (s, 2H, CH2), 5.80 (bm, (
a hydrogen) 1H, CH), 6.50–
7.60 (m, 12H, ArH), 7.53 (s, 1H, @CH), 9.22 (bs, 1H, OH), 10.45 (s,
1H, NH). LCMS (LC-MSD-Trap-SL) m/z found 522.9 (M+1), calcu-
lated 522.0 (M+1).
5.1.3.5. ( )-2-((Z)-5-(4-((phenylcarbamoyl)methoxy)-3-methoxyben-
zylidene)-4-oxo-2-thioxo-thiazolidin-3-yl)-3-(4-hydroxyphenyl)prop-
anoic acid (27). Yellow solid, mp 283–286 °C, Yield 61%. FTIR (KBr,
cmÀ1): 3500–3000 (O–H), 3207 (N–H), 3014 (Ar–H), 2929 (AliC–H),
1738 (C@O), 1697 (C@O), 1512 (C@N), 1504 (ArC@C), 1280 (C@S),
1232 (C–O), 1139 (C–N). 1H NMR (500 MHz, DMSO-d6, d ppm):
3.40 (bt, 2H, CH2), 3.91 (s, 3H, CH3), 4.92 (s, 2H, CH2), 5.65 (bm,
1H, CH), 6.60–8.20 (m, 12H, ArH), 7.48 (s, 1H, @CH), 9.10 (bs, 1H,
OH), 9.87 (s, 1H, NH), 10.90 (s, 1H, OH). 13C NMR (500 MHz,
DMSO-d6, d ppm): 37.90 (CH2), 56.19 (CH3), 56.50 (CH), 68.03
(CH2), 110.64 (1ArC), 113.70 (2ArC), 115.64 (@C), 124.25 (1ArC),
125.46 (1ArC), 125.87 (2ArC), 125.89 (2ArC), 130.27 (@CH),
131.21 (1ArC), 132.12 (1ArC), 135.41 (2ArC), 140.31 (2ArC),
149.91 (1ArC), 152.41 (1ArC), 156.28 (1ArC), 167.23 (C@O), 169.1
(C@O), 172.0 (C@O), 191.88 (C@S). HRMS (ES-TOF) m/z found
587.1063 (M+Na), calculated 587.0907 (M+Na).
5.1.3.1. ( )-2-((Z)-5-(4-((4-methoxyphenylcarbamoyl)methoxy)-3-
methoxybenzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl)-3-(4-hydroxy-
phenyl)propanoic acid (23). Yellow crystals, 262–265 °C, Yield 62%.
FTIR (KBr, cmÀ1): 3500–2500 (O–H), 3205 (N–H), 3043 (Ar–H),
2933 (AliC–H), 1693 (C@O), 1662 (C@O), 1508 (ArC@C), 1284
(C@S), 1234 (C–O), 1145 (C–N). 1H NMR (500 MHz, DMSO-d6, d
ppm): 3.40 (bt, 2H, CH2), 3.72 (s, 3H, CH3), 3.87 (s, 3H, CH3), 4.82
(s, 2H, CH2), 5.65 (bm, 1H, CH), 6.60–7.60 (m, 11H, ArH), 7.53 (s,
1H, @CH), 9.10 (bs, 1H, OH), 9.85 (s, 1H, NH), 10.08 (s, 1H, OH).
13C NMR (500 MHz, DMSO-d6, d ppm): 55.65 (2 Â CH3), 56.12
(CH2), 56.50 (CH), 68.21 (CH2), 110.63 (@C), 113.31 (1ArC),
114.39 (4ArC), 115.37 (1ArC), 115.50 (2ArC), 121.49 (4ArC),
130.76 (1ArC), 131.1 (2ArC), 130.88 (@CH), 131.93 (1ArC), 149.81
(1ArC), 153.31 (1ArC), 156.0 (1ArC), 165.79 (C@O), 172.0 (C@O),
191.88 (C@S). HRMS (ES-TOF) m/z found 617.1109 (M+Na), calcu-
lated 617.1805 (M+Na).
5.1.3.6. ( )-5-(4-((phenylcarbamoyl)methoxy)benzylidene)-4-oxo-2-
thioxo-thiazolidin-3-yl)-3-(4-hydroxyphenyl)propanoic acid (28). Pale
yellow solid, mp 248–251 °C, Yield 63%. FTIR (KBr, cmÀ1): 3500–
2500 (O–H), 3205 (N–H), 3022 (Ar–H), 2929 (AliC–H), 1707
(C@O), 1680 (C@O), 1589 (ArC@C), 1244 (C@S), 1215 (C–O), 1111
(C–N). 1H NMR (500 MHz, DMSO-d6, d ppm): 3.40 (bt, 2H, CH2),
4.85 (s, 2H, CH2), 5.70 (bm, 1H, CH), 6.60–7.90 (m, 13H, ArH),
7.77 (s, 1H, @CH), 9.23 (bs, 1H, OH), 9.88 (s, 1H, NH), 10.20 (s,
1H, OH). 13C NMR (500 MHz, DMSO-d6, d ppm): 37.90 (CH2),
56.50 (CH), 68.03 (CH2), 110.64 (1ArC), 113.70 (2ArC), 115.64
(@C), 124.25 (1ArC), 125.46 (1ArC), 125.87 (2ArC), 125.89 (2ArC),
130.27 (@CH), 131.21 (1ArC), 132.12 (1ArC), 135.41 (2ArC),
140.31 (2ArC), 149.91 (1ArC), 152.41 (1ArC), 156.28 (1ArC),
167.23 (C@O), 172.0 (2 Â C@O), 191.88 (C@S). HRMS (ES-TOF)
m/z found 557.0092 (M+Na), calculated 557.0901 (M+Na).
5.1.3.2.
( )-2-((Z)-5-(4-((2-nitrophenylcarbamoyl)methoxy)-3-
methoxybenzylidene)-4-oxo-2-thioxo-thiazolidine-3-yl)-3-(4-hydroxy-
phenyl)propanoic acid (24). Green crystals, 270–273 °C, Yield 60%.
FTIR (KBr, cmÀ1): 3300–2800 (O–H), 3306 (N–H), 3018 (Ar–H),
2933 (AliC–H), 1703 (C@O), 1685 (C@O), 1585 (ArC@C), 1504
(N@O), 1278 (C@S), 1219 (C–O), 1145 (C–N). 1H NMR (500 MHz,
DMSO-d6, d ppm): 3.40 (bt, 2H, CH2), 3.90 (s, 3H, CH3), 4.86 (s,
2H, CH2), 5.60 (bm, 1H, CH), 7.17–7.41 (m, 6H, ArH), 7.60 (s, 1H,
@CH), 7.77 (t, 1H, ArH), 8.10 (d, J = 9.5 Hz, 2H, ArH), 8.20 (d,
J = 9.5 Hz, 2H, ArH), 9.20 (s, 1H, OH), 9.42 (bs, 1H, NH), 10.83 (s,
1H, OH). 13C NMR (500 MHz, DMSO-d6, d ppm): 36.0 (CH2), 56.23
(CH3), 57.0 (CH), 68.15 (CH2), 114.36 (1ArC), 114.76 (2ArC), 116.0
(@C), 124.16 (2ArC), 125.0 (2ArC), 124.41 (1ArC), 125.42 (1ArC),
125.91 (2ArC), 127.76 (1ArC), 132.20 (@CH), 132.21 (1ArC),
135.47 (1ArC), 140.15 (2ArC), 149.24 (1ArC), 149.84 (1ArC),
167.41 (C@O), 169.0 (2 Â C@O), 196.54 (C@S). LCMS (LC-MSD-
Trap-SL) m/z found 610.1 (M+1), calculated 610.0 (M+1).
5.2. Biological methods
The methods used to evaluate the biological activity of com-
pounds have been reported by us earlier [24,33].
5.1.3.3.
( )-2-((Z)-5-(4-((pyridin-2-ylcarbamoyl)methoxy)-3-meth-
oxybenzylidene)-4-oxo-2-thioxo-thiazolidin-3-yl)-3-(4-hydroxyphenyl)
propanoic acid (25). Pale yellow amorphous solid, mp 279–284 °C,
Yield 63%. FTIR (KBr, cmÀ1): 3400–2500 (O–H), 3205 (N–H), 3039
(Ar–H), 2960 (AliC–H), 1708 (C@O), 1676 (C@O), 1535 (C@N),
1508 (ArC@C), 1282 (C@S), 1244 (C–O), 1139 (C–N). 1H NMR
5.2.1. Glucose uptake measurement
Six well microtitre plates with each well capacity of 5 mL (n = 4)
were selected for the study. Plates were divided into following
groups; Group 1: 2 mL of Tyrode solution with 2000 mg/L glucose.

26
B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
Group 2: 2 mL of Tyrode solution with 2000 mg/L glucose and reg-
ular insulin (Nova Nardisk, 40 IU/mL) 5 L containing 0.2 units of
water) for a duration of about 90 h. The tissue samples were
embedded in paraffin blocks using the standard procedure. Histo-
l
insulin. Groups 3–26: 2 mL of Tyrode solution with 2000 mg/L
glucose and 2 mg of the test compound 3–20, 23–28. Group 27:
2 mL of Tyrode solution with 2000 mg/L glucose and 2 mg of rosig-
litazone (standard). Groups 28–51: 2 mL of Tyrode solution with
2000 mg/L glucose, regular insulin 5 lL containing 0.2 units of
insulin and 2 mg of the test compound 3–20, 23–28. Group 52:
chemical staining method applied to the 4 lm sections obtained
from liver blocks was examined for general tissue composition,
by hematoxyline–eosin (H&E). All slides were covered with Canada
balsam, and viewed under light microscope with an inbuilt camera
attachment for the presence of carcinomas, adenomas or preneo-
plastic lesions using established criteria. The results are shown in
Table 3 [44].
2 mL of Tyrode solution with 2000 mg/L glucose, regular insulin
5 lL containing 0.2 units of insulin and 2 mg of rosiglitazone (stan-
dard). Wistar rats of either sex were maintained on a standard pel-
let diet, water ad libitum, and fasted overnight. The animals were
killed by decapitation and diaphragms were taken out swiftly
avoiding trauma and divided into two halves. The hemi-dia-
phragms were then rinsed in cold Tyrode solution (without
glucose) to remove any blood clots and transferred to the respec-
tive wells. The plates were closed with the lids and incubated for
45 min at 21 °C with shaking at 60 cycles per min. Following the
incubation, the glucose content of the incubated wells was mea-
sured by GOD/POD enzymatic method using Merckotest glucose
kit and Merck-Microlab 200 analyser.
5.2.5. CoMSIA study
The glucose uptake activities of the title compounds were taken
forward for the CoMSIA study to define the structure–activity
relationships. The test and training set compounds were chosen
manually such that low-, moderate-, and high-activity compounds
were present in approximately equal proportions in both the sets.
The glucose uptake activities were converted into natural log molar
antihyperglycemic activity data by dividing the original values by
their respective molecular weights and taking natural logarithms,
as it would give numerically larger values for the active com-
pounds than those of the inactive compounds (ÀlnMGU). The
training set was designed in such a way so as to account for all
the structural and activity variations among the compounds.
Twenty compounds were taken in the training set such that they
represent each congeneric series of compounds, which spans the
24 compounds. The rest, four compounds constituted the test set
or external set to validate the model generated by the CoMSIA
study. The common structures of each congeneric series of
compounds along with the structure of each compound and their
biological activity values are given in Table 1.
The molecular modeling software SYBYL 6.7 installed on a Sili-
con graphics work station with IRIX 6.5 operating system was used
for the three-dimensional structure generation and molecular
modeling studies [45]. All the compounds were built from frag-
ments in the SYBYL database. Each structure was fully geometry-
optimized using the standard Tripos force field with a distance-
dependent dielectric function until a root mean square (rms) devi-
ation of 0.001 kcal/mol Å was achieved [46]. The partial atomic
charges, required for the electrostatic interactions, were computed
using Gasteiger–Marsili method as implemented in the SYBYL [47].
The conformational search was performed using systematic
search protocol. The rotatable bonds in all molecules were
searched from 0° to 360° in 10° increments. The minimum energy
conformation thus obtained was subsequently used in the
analyses.
5.2.2. Serum glucose and triglyceride lowering activity
Male Wistar rats weighing between 150 and 200 g were pro-
cured from in-house animal facility of J.S.S. college of Pharmacy,
Ootacamund. The animals were housed under standard conditions
of temperature (22 3 °C) and relative humidity (30–70%) with a
12:12 light:dark cycle. The animals were fed with standard pellet
diet (3–5% fat, 68–70% carbohydrate, 18–20% protein, 0.5–1% vita-
mins and minerals) and water ad libitum. The Institutional Animal
Ethics Committee (IAEC) of J.S.S. College of pharmacy approved the
proposal. Dexamethasone sodium phosphate was obtained as a gift
sample from M/S. Strides Arcolabs, Bangalore, India. Triglyceride
and glucose estimation kits were from Ecoline, Merck Ltd. Mumbai,
India. All the other reagents and chemicals used in the study were
of analytical grade. The six compounds that showed activity in
in vitro screening and rosiglitazone were suspended in 0.5%w/v so-
dium carboxymethyl cellulose (CMC) at the concentration of 1 mg/
mL. These suspensions were administered at a dose volume of
10 mL/kg body weight. As a preliminary study all the synthesized
compounds and rosiglitazone were tested at a single dose level
of 10 mg/kg, per oral (p.o.). The animals were divided into nine
groups, each consisting of six rats, to study the dexamethasone in-
duced insulin resistance and hyperlipidemia (Table 2). All the ani-
mals received their assigned treatment daily for a period of
10 days. Rats of groups 2–9 were daily fasted overnight before
the dexamethasone treatment [21]. No death of animal was ob-
served in the experimental period. On day 11 the animals were
anesthetized with ether and blood was collected from reteroorbital
plexus. Serum was then separated for the estimation of glucose
and triglyceride using Ecoline triglyceride and glucose estimation
kits.
The important requirement for CoMSIA technique is that the 3D
structures of the molecules to be analyzed be aligned according to
a suitable conformation template, which is assumed to adopt a
‘‘bioactive conformation’’ [48]. The molecules in the database were
aligned using the ‘‘Database Align’’ routine available in SYBYL. The
compounds were fitted to the template molecule 24, one of the
most active molecules that correlated with the binding affinity to-
wards PPAR-c receptor. The alignment rule was optimized by using
5.2.3. Statistics
the appropriate common substructure (phenylalanine connected
to the rhodanine ring structure) for the alignment. The aligned
training set is shown in Figs. 3–5. Field fit was used to optimize
the alignment of molecules to previously calculated fields. These
aligned molecules were stored as train aligned and test aligned
databases, respectively.
All the results were expressed as mean SEM and the data was
analyzed using one-way ANOVA followed by Dennett’s multiple
comparison post-test using GraphPad Prism 4 software. P values
<0.05 were considered significant.
5.2.4. Histopathology of the liver samples
For all steps, the default Sybyl settings were used unless other-
wise noted. CoMSIA steric, electrostatic, hydrophobicity, hydrogen
bond donor and hydrogen bond acceptor interaction fields were
calculated at each 3D cubic lattice intersection point of a regularly
spaced grid 2.0 Å in x, y and z directions to encompass the aligned
molecules. The steric term (Lennard–Jones 6–12 potential) and
electrostatic (Coulomb potential) field interactions were calculated
After collecting blood on day 11, animals were sacrificed under
ether anaesthesia and each liver was examined for any grossly vis-
ible lesions. Three representative liver samples, each from groups
1, 2, 3, 5 and 6 (Table 2), were selected for the histopathological
examination. All the removed-livers were fixed in a formaldehyde
solution (which consists of 10 mL formaldehyde, 90 mL distilled

B.R. Prashantha Kumar et al. / Bioorganic Chemistry 45 (2012) 12–28
27
using the sp3 carbon probe atom (1.52 Å van der Waals radius) car-
rying +1 charge, with a distance dependant dielectric at each lattice
point. The charges were determined using Gasteiger–Marsili meth-
od. The energy calculation was performed for all grid points such
that all energies were constrained to be between À30 and
30 kcal/mol.
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Partial least squares (PLS) analysis was done to derive the
3D-QSAR models, utilizing the CoMSIA standard scaling for
the molecular fields [47]. The activity data (-lnMGA) was used as
the dependant variable and the predictive value of the model, rep-
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cross validation method. Column filtering was used to exclude
the columns with a variance smaller than 2.0 units, to speed up cal-
culations and to reduce the noise between the calculated columns.
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the standard error of prediction at different component levels
and q2 values. The conventional r2 values for training set were also
calculated under different combinations of CoMSIA models. Boot-
strapping analysis for 100 runs was performed, to further assess
the robustness and statistical confidence of the derived models.
Bootstrapping involves the generation of many new datasets from
the original dataset and is obtained by randomly choosing samples
from the original dataset. The statistical calculation is performed
on each of these bootstrapping samplings [49]. The difference be-
tween the parameters calculated from the original dataset and
the average of the parameters calculated from the many bootstrap-
ping samplings is a measure of the bias of the original calculations.
Models with a crossvalidation (q2) value above 0.3 were sought,
since at this value the probability of chance correlation is less than
5% [43]. The activities of the training set and test set compounds
were predicted, to validate and to ensure the developed CoMSIA
model. The actual and predicted values of the compounds are listed
in Table 1.
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The 3D-QSAR models were established for our structures.
Graphical examination of the best developed model reveal the
areas around the molecules that contain a greater or lesser amount
of electronegative groups, electropositive groups, hydrophobic
groups, hydrogen bond donors (OH or NH groups), hydrogen bond
acceptors (N, O, F) and whether sterics were an important factor for
glucose uptake activity.
B.R. Prashantha Kumar, B.R. Mukesh Soni, S. Santhosh Kumar, Kuldeep Singh,
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Acknowledgment
(b) D. Hardej, C.R. Ashby Jr., N.S. Khadtare, S.S. Kulkarni, S. Singh, T.T. Talele,
The synthesis of phenylalanine-derived c5-substituted rhodanines and their
activity against selected methicillin-resistant Staphylococcus aureus strains,
Eur. J. Med. Chem. 45 (2010) 5827–5832.
We acknowledge AICTE, New Delhi, for funding this project un-
der research promotion scheme.
[27] B.R. Prashantha Kumar, M.D. Karvekar, L. Adhikary, M.J. Nanjan, B. Suresh,
Microwave assisted syntheses of thiazolidine-2,4-dione motif and microwave
induced efficient synthesis of 5-benzylidene-thiazolidine-2,4-dione and 5-
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