Novel glitazones: Glucose uptake and cytotoxic activities, and structure-activity relationships

Article abstract of DOI:10.1007/s00044-011-9792-0

In this article, two series of novel glitazones are reported for their synthesis, in vitro glucose uptake activity by the rat hemi-diaphragm, and in vitro cytotoxic activity against human carcinoma HEp-2 (epidermoid carcinoma of the larynx) and A549 (human lung cancer cell line) cells. Some of the compounds exhibit good glucose uptake and cytotoxic activities. Compound 5 with 2,4-dichlorophenol moiety connected to TZD via the two carbon linker has exhibited good glucose uptake activity. One of the compounds exhibits both cytotoxic and glucose uptake activities (compound 4). Illustrations about their synthesis, and in vitro glucose uptake and cytotoxic activities are described along with their structure-activity relationships.

Full text of DOI:10.1007/s00044-011-9792-0

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2012) 21:2689–2701
DOI 10.1007/s00044-011-9792-0
ORIGINAL RESEARCH
Novel glitazones: glucose uptake and cytotoxic activities,
and structure–activity relationships
•
B. R. Prashantha Kumar S. Santhosh Kumar
•
•
Patel Viral Ashish Wadhwani R. Vadivelan
•
•
•
•
M. N. Satish Kumar K. Elango M. J. Nanjan
Received: 27 January 2011 / Accepted: 3 September 2011 / Published online: 21 September 2011
Ó Springer Science+Business Media, LLC 2011
Abstract In this article, two series of novel glitazones are
reported for their synthesis, in vitro glucose uptake activity
by the rat hemi-diaphragm, and in vitro cytotoxic activity
against human carcinoma HEp-2 (epidermoid carcinoma of
the larynx) and A549 (human lung cancer cell line) cells.
Some of the compounds exhibit good glucose uptake and
cytotoxic activities. Compound 5 with 2,4-dichlorophenol
moiety connected to TZD via the two carbon linker has
exhibited good glucose uptake activity. One of the com-
pounds exhibits both cytotoxic and glucose uptake activi-
ties (compound 4). Illustrations about their synthesis, and
in vitro glucose uptake and cytotoxic activities are
described along with their structure–activity relationships.
macrovascular disease, hypertension, cardiovascular dis-
ease, and strokes. Type 2 diabetes is a major threat today
affecting majority of the population (Carlisle et al., 2007).
In the course of investigations on the fibrate class of
hypolipidemic agents at Takeda Co., Japan, a series of
5-(4-alkoxybenzy1)-2,4-thiazolidinediones (TZDs) were
shown to reduce insulin resistance in genetically diabetic
and obese animals (Sohda et al., 1982a, b). Ciglitazone, the
prototype from this class, was found to reduce hypergly-
cemia, hyperlipidemia, and hypertriglyceridemia in insu-
lin-resistant animal models, but not in normoglycemic
animals (Fujita et al., 1983; Chang et al., 1983). Ciglitaz-
one was, therefore, taken for human trials but was with-
drawn later because of low potency and the appearance of
cataracts in animals receiving this drug for longer period of
time. Troglitazone, the first marketed TZD was also with-
drawn because of its increased risk of hepatotoxicity.
Approximately 1.9% of patients treated with troglitazone
during preapproval-controlled clinical trials showed an
increase in plasma levels of the liver enzyme alanine
aminotransferase (ALT) greater than three times the upper
limit of the normal range. In trials with pioglitazone and
rosiglitazone developed later, the incidence of significantly
elevated ALT was low and similar to that of placebo.
However, some reports say that regular monitoring of liver
enzymes was recommended while using these drugs
(Fu¨chtenbusch et al., 2000; Scheen, 2001). The safer
pioglitazone developed subsequently received marketing
approval in the United States and certain other countries.
Its long-term side effects were, however, not well known at
that time when compared with the other classes of antidi-
abetic agents.
Keywords Diabetes, glitazones Á Glucose uptake Á
PPAR-c, thiazolidinediones Á Cytotoxic activity
Introduction
Diabetes is a major threat to the health of the world. Type 1
diabetes is generally due to autoimmune destruction of
pancreatic b cells, whereas type 2diabetes is a heterogeneous
disorder predominantly characterized by peripheral resis-
tance to insulin action, increased hepatic glucose output, and
progressive b cell failure. Long-term complications include
retinopathy, nephropathy, painful peripheral neuropathy,
autonomic neuropathy, impotence, gastroparesis, diarrhea,
B. R. Prashantha Kumar (&) Á S. Santhosh Kumar Á P. Viral Á
A. Wadhwani Á R. Vadivelan Á M. N. Satish Kumar Á
K. Elango Á M. J. Nanjan
Relationship between the structure and antihyperglyce-
mic activity of the substituted TZDs and related com-
pounds has been a subject of intensive investigation. TZDs
JSS College of Pharmacy (A Constituent College of JSS
University, Mysore, India), Ootacamund 643 001, India
e-mail: prashantha@jsscpooty.org
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Med Chem Res (2012) 21:2689–2701
with significant antihyperglycemic activity are known for
their common structural feature, namely, acidic head group
connected to a lipophilic tail by a phenoxyalkyl linker
(Fig. 1) (Yoshioka et al., 1989).
2006, 2007, 2008). Glitazones incorporated with phenols
were synthesized according to Scheme 1. p-Hydroxy benz-
aldehyde was first connected to the two carbon linker (1,2
dibromo ethane) to give 4-(2-bromoethoxy)benzaldehyde 1.
The two carbon linker terminal to the different substituted
phenols under similar basic conditions. The various phenols
substituted 4-(2-bromoethoxy)benzaldehydes 2 were later
subjected to the Knoevenagel condensation with thiazoli-
dine-2,4-dione or its bioisostere 2-thioxo-thiazolidine-4-one
to obtain the corresponding final compounds 3–9 at moder-
ate-to-good yields.
Removal of the acidic function by N-methylation over
the acidic head group leads to loss of activity. TZD ring
can be replaced with heterocyclic ring like oxazolidinedi-
one (bioisostere of TZD) or a-substituted carboxylic acid
(Dow et al., 1991; Sohda et al., 1982a, b). In phenoxyalkyl
linker portion, phenoxyethyl group often connected with a
two carbon linker (n = 2) results in highly active com-
pounds. In the lipophilic tail, aromatic and heteroaromatic
groups produce good antidiabetic activity (Willson et al.,
1996).
The second series of title compounds (N-substituted
TZDs) were synthesized according to the Scheme 2.
2-Amino benzamide was first acylated with chloro acetyl
chloride to give 2-(2-chloroacetamido)-benzamide 10.
Thiazolidine-2,4-dione was converted to the corresponding
potassium salt 11 and fused with the 10 to obtain 12.
Compound 12 was later subjected to the Knoevenagel
condensation with various substituted aldehydes to obtain
compounds 13–21. N-Methyl piperazine or piperidine was
used as a basic catalyst for the Knoevenagel condensation.
N-Methyl piperazine gave better results in terms of yields
when compared to piperidine under both microwave and
conventional methods. All the reported compounds were
Keeping all these structural features in mind, it was
proposed to develop drugs from this class via rational drug
design. In continuation with our earlier reports (Kumar and
Nanjan, 2008, 2010; Kumar et al., 2006, 2007, 2008), the
present study thus reports some novel glitazones of bio-
logical interest. TZDs show remarkable antihyperglycemic
activity in addition to various other biological properties,
`
such as antiproliferative (Yanagisawa et al., 2000; Ottana
et al., 2005a, b; Li et al., 2007), antituberculosis (Babaoglu
`
et al., 2003), anti-inflammatory (Ottana et al., 2005a, b),
1
and antimicrobial activity (Salama et al., 1990; Bonde and
Gaikwad, 2004). Some reports have claimed potential
cytotoxic activity for the compounds containing thiazoli-
dine ring system (Zhou et al., 2008; Gududuru et al., 2005).
The titled compounds were, therefore, screened also for
their in vitro cytotoxicity against human carcinoma HEp-2
and A549cells.
characterized using IR, H-NMR, 13C-NMR, and LC–MS
spectral studies.
Pharmacology
Glucose uptake activity
The glucose uptake activity was measured for eleven
compounds 3–8, 14–16, 18, and 20 using rat hemi-dia-
phragm model as previously proposed by some authors
(Walaas and Walaas, 1952; Chattopadhyay et al., 1992).
Rat diaphragm was selected because striated muscle is
quantitatively the most important tissue for glucose dis-
Materials and methods
Chemistry
´
posal in the animal body (Giron et al., 2003; Ghosh et al.,
The title compounds were synthesized using two different
schemes. The TZD nucleus was prepared by reacting equi-
molar amounts of chloroacetic acid and thiourea under ice
cold conditions to yield 2-imino-thiazolidine-4-one. It was
then acidified with HCl and refluxed for 12 h to yield TZD
(90–94%) (Kumar and Nanjan, 2008, 2010; Kumar et al.,
2004; Sabu and Subburaju, 2002). Insulin stimulates glu-
cose uptake by causing translocation of the transporters
from an intracellular site to the plasma membrane. In
muscles, two types of glucose transporter iso-forms are
expressed: GLUT1 and GLUT4. The latter is quantitatively
S
N
S
N
O
O
O
N
O
N
H
O
N
O
B
A
H
Acidic head group Phenoxyalkyl linker
Liphophilic tail
Acidic head group
Phenoxyalkyl linker
Liphophilic tail
Fig. 1 Common structural components of pioglitazone A and rosiglitazone B
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Med Chem Res (2012) 21:2689–2701
2691
Scheme 1 Reagents and
O
O
H
conditions: a dry acetone,
anhydrous K2CO3, stir, reflux at
40°C for 72 h, 85–90%; b dry
acetone, substituted phenols,
anhydrous K2CO3, stir, reflux at
40°C for 36–48 h, 60–70%;
c dry toluene, TZD or
a
+
Br
HO
Br
H
Br
O
1
b
rhodamine, N-methyl piperazine
or piperidine, ammonium
R1
O
acetate, and molecular sieves.
Conventional: reflux at 110°C
for 12–15 h. Microwave:
O
H
O
700 W for 30–45 min, 90–92%
2
c
X
O
S
R1
HN
O
O
3-9
Scheme 2 Reagents and
O
O
conditions: a dry chloroform,
triethylamine, and chloroacetyl
chloride were added drop wise
for about 30–45 min, 0–5°C to
rt, stirred for 24–30 h, 90%;
b ethanol, KOH, stirred for 2 h,
98–100%; c DMF, 11, reflux at
80°C for 18 h, 60%; d dry
toluene, aldehyde, N-methyl
piperazine or piperidine,
O
NH2
NH
NH2
a
Cl
+
Cl
NH2
10
O
Cl
H
N
c
O
O
ammonium acetate, and
S
molecular sieves. Conventional:
reflux at 110°C for 12–15 h,
88–90%. Microwave: 420 W
for 20–40 min, 88–90%
O
NH2
O
H
b
N
N
S
O
O
12
K
N
O
O
d
S
O
NH2
R2
O
11
H
N
N
O
S
O
13-21
more abundant in adult rat muscle and is distributed among
intracellular compartments in the basal state, from where it
is rapidly translocated to the plasma membrane in response
to insulin. Glucose uptake is measured in mg/dl/45 min
according to the standard procedure. The results are
expressed as mean standard error of mean (SEM), as
shown in Table 1. Statistical comparisons between the
groups were performed in two sets using one-way ANOVA
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Med Chem Res (2012) 21:2689–2701
2695
and Dunnet’s multiple comparison post-test on graphPad
Prism 4.0 software for Windows (San Diego, California,
USA). In the first set, group 1 that served as a control was
compared to groups 3–12 including the analogous standard
drug (rosiglitazone). In the second set, group 2 that served
as control was compared to groups 14–23.
50 lg/ml, respectively. Compounds 4 and 18 exhibit sig-
nificant cytotoxic activity against A549 cells with CTC50
value of 28 and 40 lg/ml, respectively. Among the com-
pounds screened, compound 4, with 2-thioxo-thiazolidine-
4-one connected to the o-chloro phenoxy group via
benzylidenoxy group and two carbon linker is found to be
the most potent compound.
Cytotoxic activity
Nine of the synthesised compounds were screened for their
in vitro cytotoxic activity against HEp-2 cells and eleven
compounds against A549 cells (Table 1). Cell cultures
were obtained from the National Centre for Cell Sciences,
Pune. Sulforhodamine B (SRB) assay was carried out
according to the standard procedure. SRB is a dark pink
amino xanthenes dye with sulfonic groups. Under mild
conditions, SRB binds to the basic amino acid residues of
proteins in trichloro acetic acid (TCA) fixed cells to pro-
vide a sensitive index of cellular protein content that is
linear over a cell density range of at least two orders of
magnitude.
Conclusion
In conclusion, we have designed, synthesized seventeen
glitazones, and evaluated some of them for their in vitro
glucose uptake and cytotoxic activities. Compound 5 with
2,4-dichlorophenol moiety connected to TZD via the two
carbon linker seems to be the interesting compound, which
needs to be investigated further for its antidiabetic activity.
Compound 4 is the candidate compound worth investigat-
ing further for its dual cytotoxic and antidiabetic activities.
Experimental protocols
Results and discussion
TZDs or glitazones are prepared by keeping general
structure-activity relationships (SAR) of potent hypogly-
cemic agents via two different schemes in laboratory and
their physical and analytical data are recorded. Melting
points were measured on a VeegoVMP-1 apparatus and are
uncorrected. The infrared spectra of compounds were
recorded on Shimadzu-FTIR 8400S spectrophotometer
using KBr pellet technique and are expressed in cm-1. The
1H-NMR, 13C-NMR, and HOMO COSY Spectra recorded
on AV-III (400 MHz) spectrometer using DMSO-d6 as a
solvent, and trimethyl silane (TMS) as internal standard
(IISc, Bangalore, India). Mass spectra were recorded
on LC–MS (Shimadzu) under electrospray ionization
technique.
The results of the in vitro glucose uptake study indicate
that most of the screened compounds enhance the glucose
uptake by the tissue except compounds 3, 7, 8, and 16.
Compounds 5, 4, 15, 20, 18, 14, and 6 exhibit good-to-
moderate glucose uptake activity, especially in the pres-
ence of insulin when compared to the same in the absence
of insulin. Most of the compounds failed to exhibit good
glucose uptake activity in the absence of insulin. Com-
pound 5 was found to be the most potent compound
among all the reported compounds here as its glucose
uptake activity is comparable to that of standard drug
(rosiglitazone). Compound 5, was found to contain TZD
as its acidic head group, central phenoxy ethyl linker, and
highly hydrophobic 2,4-dichlorophenoxy group as its
lipophilic tail. Compound 6, with 2-thioxo-thiazolidine-4-
one as its acidic head showed moderate glucose uptake
activity. Modification of tail portion with o-chlorophen-
oxy in place of 2,4-dichlorophenoxy has led to loss of
activity (compound 3). Surprisingly, replacing the head
portion with 2-thioxo-thiazolidine-4-one and the tail por-
tion with o-chlorophenoxy has led to good activity
(compound 4). In contrast to the first series of com-
pounds, the second series of compounds 14, 15, 18, and
20 do not exhibit significant glucose uptake activity. This
is possibly due to the N-substitution of acidic head group
(TZD).
Synthetic procedures
Synthesis of thiazolidine-2,4-dione
Equimolar amounts of thiourea (0.6 M) and chloroacetic
acid (0.6 M) were taken separately in a 60 ml of water and
mixed under ice cold condition (0–5°C) with stirring for
20 min to form white precipitate of 2-imino-thiazolidine-
4-one. Conc. HCl (60 ml) was added and refluxed for
10–12 h. Reaction was monitored though TLC (ethyl
acetate: pet ether, 8: 2). Thiazolidine-2,4-dione as white
crystalline solid obtained after cooling was then subjected
to filtration, washed with water, and dried. The yield was
found to be 90–94%.
Compounds 4, 15, and 19 show significant cytotoxic
activity against HEp-2 cells with CTC50 of 36, 55, and
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Med Chem Res (2012) 21:2689–2701
Synthesis of Scheme 1 compounds
mixture wasallowed tocooland precipitatedsolidwas filtered
off. The corresponding product 3–9 was recrystallized with
ethyl acetate. The yield was found to be 90–92%.
Connecting dibromoethane to p-hydroxy benzaldehyde
One equivalent of p-hydroxy benzaldehyde, two equiva-
lents of dibromoethane along with 1.1 equivalent of
anhydrous K2CO3, and 20 ml of dry acetone were trans-
ferred to a round bottom flask (RBF). Stirred at 40°C for
72 h. Reaction was monitored through TLC (ethyl ace-
tate:pet ether 8:2). After completion of the reaction, solvent
was evaporated, and the reaction mixture was transferred to
the beaker containing water. Extraction with ethyl acetate:
Ethyl acetate layer was washed thrice with 10% sodium
hydroxide solution, washed with Brine solution, and finally
dried over anhydrous sodium sulfate to get a crude product.
Later, it was purified by subjecting it to a column chro-
matography using 5% ethyl acetate in pet ether as a mobile
phase. The white crystals of the pure product 4-(2-
bromoethoxy)benzaldehyde 1 were taken further for the
treatment with phenols. The yield was found to be 85–90%.
Microwave method
Thiazolidine-2,4-dione or 2-thioxo-thiazolidine-4-one (0.01 M)
in dry toluene was transferred to long necked microwave
flask. Then phenol incorporated aldehyde (0.01 M), N-methyl
piperazine or piperidine (0.5 M), acetic acid (4–5 drops) and
6–8 molecular sieves were added. The reaction mixture was
irradiated with microwaves at 700 W for 30–45 min. The
reaction was monitored though TLC. Upon completion of the
reaction, reaction mixture was allowed to cool and precipi-
tated solid was filtered off. The corresponding product 3–9
was recrystallized with ethyl acetate. The yield was found to
be 90–92%.
Synthesis of Scheme 2 compounds
Synthesis of 2-(2-chloroacetamido)-benzamide (10)
General procedure for connecting 4-(2-bromoethoxy)
benzaldehyde to the different phenols
2-(2-Chloroacetamido)-benzamide 10 was prepared
according to Scheme 2. 2-Aminobenzamide (0.2 M) and
triethylamine (0.2 M) along with dry chloroform (50 ml)
was transferred to RBF fitted with guard tube. The mixture
was allowed for stir under ice cold conditions 0–5°C to rt.
Chloroacetyl chloride (0.21 M) was added drop wise for a
period of about 30–45 min. The reaction mixture was
continued to stir for about 24–30 h. The reaction was
monitored through TLC using 20% ethyl acetate in pet
ether as a mobile phase. After the reaction was completed,
the solvent was evaporated. The crude product obtained
was washed with water and dried to get the brown colored
2-(2-chloroacetamido)benzamide. It was taken directly for
the next step without any further purification. The yield
was found to be 90%.
4-(2-Bromoethoxy) benzaldehyde 1 (1 equiv.) was treated
with the various substituted phenols (2 equiv.) by refluxing
at 40°C with anhydrous K2CO3 (2.2 equiv.) in a dry ace-
tone for 36–48 h. Reaction was monitored through TLC.
After completion of the reaction, the solvent was evapo-
rated and the reaction mixture transferred to the beaker
containing water. Extraction with ethyl acetate: Ethyl
acetate layer was washed thrice with 10% sodium
hydroxide solution, washed with Brine solution, and finally
dried over anhydrous sodium sulfate to get a pure product
2. The yield was found to be 60–70%. Later, it was sub-
jected to the Knoevenagel condensation to get final
compounds.
General procedure for Knovenagel condensation (3–9)
Potassium salt of thiazolidine-2,4-dione (11)
The phenol-incorporated p-hydroxy benzaldehyde via two
carbon linker was subjected to the Knoevenagel conden-
sation reaction to get the final compounds (3–9).
Potassium salt of TZD 11 was prepared. Thiazolidine-2,
4-dione (16 g) was dissolved in 25 ml of ethanol. To this,
solution of potassium hydroxide (8.4 g) in ethanol (20 ml)
was added. The mixture was stirred for 2 h. The white
crystalline solid was collected after filtration, washed with
ethanol and dried under vacuum to get potassium salt of
TZD 11 and yield was found to be 98–100%.
Conventional method
Thiazolidine-2,4-dione or 2-thioxo-thiazolidine-4-one (0.01 M)
in dry toluene was transferred to RBF. Then, phenol-incor-
porated aldehyde 2 (0.01 M), N-methyl piperazine or piperi-
dine (0.5 M), acetic acid (4–5 drops), and molecular sieves
were added. The reaction mixture was refluxed at 110°C with
occasional stirring for 12–15 h. The reaction was monitored
though TLC. Upon completion of the reaction, reaction
Synthesis of 2-(2-(2,4-dioxothiazolidin-3-
yl)acetamido)benzamide (12)
Potassium salt of 2,4-TZDs 11 (0.02 M) in DMF was taken
into RBF. Then 2-(2-chloroacetamido)benzamide 10 was
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Med Chem Res (2012) 21:2689–2701
2697
added slowly with stirring. After the addition was com-
plete, the mixture was allowed to stir and heated at 80°C
under reflux for 18 h. Reaction was monitored through
TLC. After completion of the reaction, mixture was poured
in water (500 ml) and product 12 precipitated as a solid
collected on a filter and dried. The yield was found to be
60%.
NH). 13C-NMR (d ppm, DMSO-d6) d: 66.58 (CH2), 67.38
(CH2), 114.20 (=C), 115.40 (2ArC), 121.42 (ArC), 121.80
(ArC), 122.5 (ArC), 126.17 (ArC), 128.30 (ArC), 129.96
(ArC), 130.27 (=CH), 131.80 (2ArC), 153.65 (ArC),
159.71 (ArC), 168.97 (C=O), 169.8 (C=O). ESI–MS m/
z found 374.13 (m - 1) peak, calcd 374.
General procedure for the Knoevenagel condensation
over 2-(2-(2,4-dioxothiazolidin-3-yl) acetamido)benzamide
(13–21).
(Z)-5-(4-(2-(2-Chlorophenoxy)ethoxy)benzylidene)-2-
thioxothiazolidin-4-one (4)
Brownish yellow amorphous solid, yield 68%, mp 200–
202°C. IR (KBr, cm-1): 3146 (N–H, stretch), 3036 (ArH,
stretch), 2945 (AliH, stretch), 1693 (C=O, stretch), 1508
(ArC=C, stretch), 1261 (C–O, stretch), 1236 (C=S, stretch),
1066 (C–N, stretch), 599 (C–Cl, stretch). 1H-NMR (d ppm,
DMSO-d6) d: 4.42 (s, 4H, 2CH2), 6.97 (t, J = 6.4 Hz, 1H,
ArH),7.16 (d, J = 8.80 Hz, 2H, ArH), 7.21 (d, J = 7.04 Hz,
1H, ArH), 7.31 (t, J = 6.71 Hz, 1H, ArH), 7.42 (d, J =
6.32 Hz, 1H, ArH), 7.56 (d, J = 8.88 Hz, 2H, ArH), 7.62 (s,
1H, =CH). 13C-NMR (d ppm, DMSO-d6) d: 66.66 (CH2),
67.36 (CH2), 114.20 (=C), 115.6 (2ArC), 121.42 (ArC),
121.82 (ArC), 125.90 (ArC), 128.30 (ArC), 129.96 (ArC),
131.09 (ArC), 131.41 (=CH), 132.52 (2ArC), 153.64 (ArC),
160.23 (ArC), 170.48 (C=O), 196.01 (C=S). ESI–MS m/
z found 390.21 (m - 1) peak, calcd 390.
Conventional method
2-(2-(2,4-Dioxothiazolidin-3-yl)acetamido)benzamide (0.01 M)
in dry toluene was transferred in RBF. Then aldehyde
(0.01 M), piperidine (0.5 M), acetic acid (4–5 drops) and
molecularsieves were added. The reaction mixture was
stirred 5 min and then heated under reflux at 110°C with
occasional stirring for 12–15 h. The reaction was monitored
through TLC. Upon completion of the reaction, reaction
mixture was allowed to cool and the precipitated solid fil-
tered off. The resultant solid 13–21 was recrystallized with
methanol. The yield was found to be 88–90%.
Microwave method
2-(2-(2,4-Dioxothiazolidin-3-yl)acetamido)benzamide
(0.01 M) in dry toluene was transferred in RBF. Then,
aldehyde (0.01 M), piperidine (0.5 M), acetic acid (4–5
drops), and molecularsieves were added. The reaction
mixture was stirred for 5 min, and then heated under
microwave irradiation (420 W) for 20–40 min. The reac-
tion was monitored through TLC. Upon completion of the
reaction, reaction mixture was allowed to cool, and the
precipitated solid was filtered off. The resultant solid 13–21
was recrystallized with methanol. The yield was found to
be 88–90%.
(Z)-5-(4-(2-(2,4-Dichlorophenoxy)ethoxy)benzylidene)
thiazolidine-2,4-dione (5)
Green solid, yield 60%, mp 153–155°C. IR (KBr, cm-1):
3101 (N–H, stretch), 3022 (ArH, stretch), 2887 (AliH,
stretch), 1780 (C=O, stretch), 1681 (C=O, stretch), 1512
(ArC=C, stretch), 1259 (C–O, stretch), 1182 (C–N,
1
stretch), 605 (C–Cl, stretch). H-NMR (d ppm, DMSO-d6)
d: 4.42 (s, 4H, 2CH2), 7.15–7.55 (m, 7H, ArH), 7.7 (s,
1H, =CH), 12.21 (bs, 1H, NH). ESI–MS m/z found 408.09
(m - 1) peak, calcd 408.
Physical and analytic data
(Z)-5-(4-(2-(2,4-Dichlorophenoxy)ethoxy)benzylidene)-2-
thioxothiazolidin-4-one (6)
(Z)-5-(4-(2-(2-Chlorophenoxy)ethoxy)benzylidene)
thiazolidine-2,4-dione (3)
Yellow amorphous solid, yield 80%, mp 164–165°C. IR
(KBr, cm-1): 3147 (N–H, stretch), 3022 (ArH, stretch),
2949 (AliH, stretch), 1703 (C=O, stretch), 1246 (C=S,
stretch), 1508 (ArC=C, stretch), 1292 (C–O, stretch), 678
Pale yellow amorphous solid, yield 85%, mp 210–212°C.
IR (KBr, cm-1): 3134 (N–H, stretch), 3037 (Ar C–H,
stretch), 2943 (Ali C–H, stretch), 1741 (C=O, stretch),
1689 (C=O, stretch), 1512 (Ar C=C, stretch), 1259 (C–O,
stretch), 1155 (C–N, stretch), 607 (C–Cl, stretch). 1H-NMR
(d ppm, DMSO-d6) d: 4.4 (s, 4H, 2CH2), 6.96(t,
J = 7.48 Hz, 1H, ArH), 7.14 (d, J = 8.80 Hz, 2H, ArH),
7.21 (d, J = 7.00 Hz, 1H, ArH), 7.31 (t, J = 6.76 Hz, 1H,
ArH), 7.42 (d, J = 6.32 Hz, 1H, ArH), 7.54 (d,
J = 8.84 Hz, 2H, ArH), 6.68 (s, 1H, =CH), 12.4 (s, 1H,
1
(C–Cl, stretch). H-NMR (d ppm, DMSO-d6) d: 4.42 (s,
4H, 2CH2), 7.14–7.55 (m, 7H, ArH), 7.54 (s, 1H, =CH).
13C-NMR (d ppm, DMSO-d6) d: 66.54 (CH2), 67.79 (CH2),
115.34 (=C), 115.57 (2ArC), 122.46 (ArC), 124.75 (ArC),
125.24 (ArC), 126.06 (ArC), 128.07 (ArC), 128.13 (ArC),
129.29 (ArC), 130.58 (ArC), 132.42 (2ArC), 171.31
(C=O), 196.42 (C=S). ESI–MS m/z found 424.10 (m - 1)
peak, calcd 424.
123

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Med Chem Res (2012) 21:2689–2701
(C–N, stretch), 1138 (C–N, stretch). 1H-NMR (d ppm,
DMSO-d6) d: 4.31 (s, 4H, 2CH2), 7.15 (t, J = 7.12 Hz, 1H,
ArH),7.49 (t, J = 7.24 Hz, 1H, ArH), 7.78 (bs, 1H, NH2),
7.82 (d, J = 6.8 Hz, 1H, ArH), 8.31 (bs, 1H, NH2), 8.36 (d,
J = 8.04 Hz, 1H, ArH). 13C-NMR (d ppm, DMSO-d6) d:
34.13 (CH2), 44.54 (CH2), 119.68 (ArC), 120.11 (ArC),
123.01 (ArC), 128.62 (ArC), 132.37 (ArC), 138.80 (ArC),
163.99 (C=O), 170.67 (C=O), 171.54 (C=O), 171.97
(C=O). ESI–MS m/z found 292.05 (m - 1) peak, calcd
292.
(Z)-5-(4-(2-(4-Nitrophenoxy)ethoxy)benzylidene)
thiazolidine-2,4-dione (7)
Greenish yellow amorphous solid, yield 72%, mp
220–223°C. IR (KBr, cm-1): 3039 (N–H, stretch), 3113
(ArC–H, stretch), 2956 (AliC–H, stretch), 1693 (C=O,
stretch), 1635 (C=O, stretch), 1510 (ArC=C, stretch), 1249
(C–O, stretch), 1180 (C–N, stretch), 1334 (Ar–N=O,
stretch). 1H-NMR (d ppm, DMSO-d6) d: 4.41 (s, 2H, CH2),
4.47 (s, 2H, CH2), 7.09 (d, J = 8.72 Hz, 2H, ArH), 7.19 (d,
J = 9.20 Hz, 2H, ArH), 7.50 (d, J = 7.36 Hz, 2H, ArH),
7.51 (s, 1H, =CH), 8.19 (d, J = 9.20 Hz, 2H, ArH). 13C-
NMR (d ppm, DMSO-d6) d: 66.23 (CH2), 67.27 (CH2),
115.08 (2ArC), 115.12 (2ArC), 125.82 (2ArC), 126.73
(=C),127.16 (2ArC), 131.32 (2ArC), 140.97 (=CH), 158.89
(ArC), 163.56 (ArC), 171.66 (C=O), 174.79 (C=O). ESI–
MS m/z found 385.08 (m - 1) peak, calcd 385.
2-(2-((E)-5-(4-Hydroxybenzylidene)-2,4-dioxothiazolidin-
3-yl)acetamido)benzamide (13)
Pale green amorphous solid, yield 60%, mp 274–276°C. IR
(KBr, cm-1): 3153 (N–H, stretch), 3068 (ArC–H, stretch),
2937 (AliC–H, stretch), 1741 (C=O, stretch), 1662 (C=O,
1
stretch), 1518 (ArC=C, stretch), 1143 (C–N, stretch). H-
(Z)-5-(4-(2-(4-Nitrophenoxy)ethoxy)benzylidene)-2-
thioxothiazolidin-4-one (8)
NMR (d ppm, DMSO-d6) d: 4.5 (s, 2H, CH2), 6.69 (d,
J = 8.6 Hz, 1H, ArH), 6.94 (d, J = 8.68 Hz, 2H, ArH),
7.2 (m, 1H, ArH), 7.5 (m, 1H, ArH), 7.8 (d, J = 6.68 Hz,
1H, ArH), 7.9 (s, 1H, =CH), 8.37 (d, J = 8.40 Hz, 2H,
ArH), 9.5 (s, 1H, OH), 10.4 (s, 2H, NH2), 12.1 (bs, 1H,
NH). ESI–MS m/z found 396.08 (m - 1) peak, calcd 396.
Brownish yellow amorphous solid, yield 45%, mp
194–196°C. IR (KBr, cm-1): 3306 (N–H, stretch), 3109
(ArC–H, stretch), 2933 (AliC–H, stretch), 1701 (C=O,
stretch), 1240 (C=S, stretch), 1508 (ArC=C, stretch), 1170
1
(C–N, stretch), 1336 (Ar–N=O, stretch). H-NMR (d ppm,
2-(2-((E)-5-(4-(Dimethylamino)benzylidene)-2,4-
dioxothiazolidin-3-yl)acetamido)benzamide (14)
DMSO-d6) d: 4.43 (s, 2H, CH2), 4.48 (s, 2H, CH2), 7.12 (d,
J = 8.84 Hz, 2H, ArH), 7.2 (d, J = 9.20 Hz, 2H, ArH),
7.37 (s, 1H, =CH), 7.5 (d, J = 8.84 Hz, 2H, ArH), 8.2 (d,
J = 9.24 Hz, 2H, ArH). 13C-NMR (d ppm, DMSO-d6) d:
66.30 (CH2), 67.25 (CH2), 115.10 (2ArC), 115.32 (2ArC),
125.83 (2ArC), 126.93 (2ArC), 127.59 (=C), 131.93
(2ArC), 140.98 (=CH), 159.28 (ArC), 163.55 (ArC),
176.55 (C=O), 199.09 (C=S). ESI–MS m/z found 401.16
(m - 1) peak, calcd 401.
Pale orange amorphous solid, yield 80%, mp 260–262°C.
IR (KBr, cm-1): 3416 (N–H, stretch), 3053 (ArC–H,
stretch), 2939 (AliC–H, stretch), 1722 (C=O, stretch), 1674
(C=O, stretch), 1523 (ArC=C, stretch), 1197 (C–N,
stretch). 1H-NMR (d ppm, DMSO-d6) d: 3.02 (s, 6H,
2CH3), 4.47 (s, 2H, CH2), 6.83 (d, J = 9.04 Hz, 2H, ArH),
7.14 (t, J = 6.72 Hz, 1H, ArH), 7.48 (d, J = 8.88 Hz, 2H,
ArH), 7.51 (t, J = 7.26 Hz, 1H, ArH), 7.77 (bs, 1H, NH2),
7.82 (d, J = 6.64 Hz, 1H, ArH), 7.84 (s, 1H, =CH), 8.28
(bs, 1H, NH2), 8.38 (d, J = 7.48 Hz, 1H, ArH), 12.15 (s,
1H, NH). 13C-NMR (d ppm, DMSO-d6) d: 44.74 (CH2),
112.05 (=C), 112.73 (ArC), 119.59 (ArC), 119.65 (ArC),
120.13 (ArC), 122.97 (ArC), 128.60 (ArC), 132.33 (=CH),
132.49 (2ArC), 134.66 (2ArC), 138.87 (ArC), 151.72
(ArC), 164.18 (C=O), 165.38 (C=O), 167.44 (C=O),
170.57 (C=O). ESI–MS m/z found 423.12 (m - 1) peak,
calcd 423.
(Z)-5-(4-(2-(4-Bromophenoxy)ethoxy)benzylidene)
thiazolidine-2,4-dione (9)
Pale brown amorphous solid, yield 42%, mp 156–158°C.
IR (KBr, cm-1): 3037 (N–H, stretch), 3015 (ArC–H,
stretch), 2941 (AliC–H, stretch), 1658 (C=O, stretch), 1510
(ArC=C, stretch), 1242 (C–O, stretch), 1176 (C–N,
1
stretch), 650 (C–Br, stretch). H-NMR (d ppm, DMSO-d6)
d: 4.31 (s, 2H, CH2), 4.34 (s, 2H, CH2), 5.64–7.66 (m, 8H,
ArH). ESI–MS m/z found 419.12 (m - 1) peak, calcd 419.
2-(2-((E)-5-(4-(Benzyloxy)-3-methoxybenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamido)benzamide (15)
2-(2-(2,4-Dioxothiazolidin-3-yl)acetamido)benzamide (12)
Pale brown amorphous solid, yield 65%, mp 192–194°C.
IR (KBr, cm-1): 3471 (N–H, stretch), 3205 (N–H, stretch),
3014 (ArC–H, stretch), 1768 (C=O, stretch), 1685 (C=O,
stretch), 1662 (C=O, stretch), 1525 (ArC=C, stretch), 1159
Pale green amorphous solid, yield 75%, mp 250–252°C. IR
(KBr, cm-1): 3165 (N–H, stretch), 3038 (ArC–H, stretch),
2987 (AliC–H, stretch), 1735 (C=O, stretch), 1689 (C=O,
stretch), 1666 (C=O, stretch), 1508 (ArC=C, stretch), 1232
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Med Chem Res (2012) 21:2689–2701
2699
(C–O, stretch), 1143 (C–N, stretch), 1174 (C–N, stretch).
1H-NMR (d ppm, DMSO-d6) d: 3.83 (s, 3H, CH3), 4.51 (s,
2H, CH2), 5.18 (s, 2H, CH2), 7.94 (s, 1H, =CH), 7.77 (bs,
1H, NH2), 7.82 (d, J = 6.9 Hz, 1H, ArH), 8.29 (bs, 1H,
NH2), 8.38 (d, J = 8.24 Hz, 1H, ArH),), 7.13–7.52 (m, 7H,
ArH), 12.17 (s, 1H, NH). 13C-NMR (d ppm, DMSO-d6) d:
44.83 (CH2), 55.65 (CH3), 69.89 (CH2), 113.61 (=C),
113.89 (ArC), 117.90 (ArC), 119.70 (ArC), 120.16 (ArC),
123.84 (2ArC), 127.99 (ArC), 128.44 (2ArC), 128.60
(=CH), 132.34 (ArC),133.95 (ArC), 136.45 (ArC), 138.82
(ArC), 149.27 (ArC), 150.12 (ArC), 163.99 (C=O), 165.26
(C=O), 167.22 (C=O), 170.56 (C=O). ESI–MS m/z found
516.05 (m - 1) peak, calcd 516.
(ArC), 132.40 (ArC), 133.56 (ArC), 133.68 (ArC), 134.46
(ArC), 138.80 (=CH), 148.26 (ArC), 164.0 (C=O), 165.31
(C=O), 167.23 (C=O), 170.56 (C=O). ESI–MS m/z found
410.10 (m - 1) peak, calcd 410.
2-(2-((E)-5-(2-Hydroxybenzylidene)-2,4-dioxothiazolidin-
3-yl)acetamido)benzamide (18)
Dark green amorphous solid, yield 65%, mp 160–162°C. IR
(KBr, cm-1): 3335 (O–H, stretch), 3184 (N–H, stretch),
3066 (ArC–H, stretch), 2928 (AliC–H, stretch), 1674 (C=O,
stretch), 1525 (ArC=C, stretch), 1249 (C–O, stretch). ESI–
MS m/z found 395.97 (m - 1) peak, calcd 396.
2-(2-((E)-5-(4-Hydroxy-3-methoxybenzylidene)-2,4-
dioxothiazolidin-3-yl)acetamido)benzamide (16)
2-(2-((5E)-2,4-Dioxo-5-((E)-3-
phenylallylidene)thiazolidin-3-yl)
acetamido)benzamide (19)
Pale greenish brown amorphous solid, yield 78%, mp
230–232°C. IR (KBr, cm-1): 3520 (O–H, stretch), 3400
(N–H, stretch), 3022 (ArC–H, stretch), 2927 (AliC–H,
stretch), 1741 (C=O, stretch), 1689 (C=O, stretch), 1512
(C=C, stretch), 1251 (C–O, stretch), 1145 (C–N, stretch).
1H-NMR (d ppm, DMSO-d6) d: 4.13 (s, 2H, CH2), 4.42 (s,
3H, CH3), 6.97 (t, J = 7.72 Hz, 2H, ArH), 7.14 (d,
J = 7.68 Hz, 1H, ArH), 7.16 (s, 1H, OH), 7.20 (bs, 1H,
NH2), 7.22 (s, 1H, ArH), 7.29 (d, J = 7.4 Hz, 1H, ArH),
7.33 (bs, 1H, NH2), 7.42 (d, J = 7.98 Hz, 1H, ArH), 7.55
(d, J = 8.8 Hz, 1H, ArH), 7.6 (s, 1H, =CH), 12.4 (bs, 1H,
NH). 13C-NMR (d ppm, DMSO-d6) d: 66.57 (CH2), 67.38
(CH3), 114.20 (=C), 115.4 (ArC), 121.41 (2ArC), 121.80
(ArC), 126.16 (ArC), 128.29 (2ArC), 129.96 (2ArC),
130.28 (=CH), 131.80 (2ArC), 153.65 (ArC), 159.71
(2C=O), 165.66 (2C=O). ESI–MS m/z found 426.08
(m - 1) peak, calcd 426.
Dark green amorphous solid, yield 60%, mp 243–245°C.
IR (KBr, cm-1): 3398 (N–H, stretch), 3030 (ArC–H,
stretch), 2947 (AliC–H, stretch), 1735 (C=O, stretch), 1689
(C=O, stretch), 1525 (ArC=C, stretch), 1147 (C–N,
stretch). 1H-NMR (d ppm, DMSO-d6) d: 4.47 (s, 2H, CH2),
7.06 (d, J = 11.34 Hz, 1H, ArH), 7.15 (t, J = 7.04 Hz,
2H, ArH), 7.36 (s, 1H, =CH), 7.40 (s, 1H, =CH), 7.42 (s,
1H, =CH), 7.50 (t, J = 7.16 Hz, 2H, ArH), 7.68–7.70 (m,
2H, ArH), 7.77 (bs, 1H, NH2), 7.82 (d, J = 11.9 Hz, 1H,
ArH), 8.29 (bs, 1H, NH2), 8.38 (d, J = 7.72 Hz, 1H, ArH),
12.16 (bs, 1H, NH). 13C-NMR (d ppm, DMSO-d6) d: 44.7
(CH2), 119.65 (=C), 120.13 (ArC), 121.95 (ArC), 123.0
(ArC), 123.19 (ArC), 127.94 (ArC), 128.60 (ArC), 128.90
(ArC), 129.90 (ArC), 132.34 (ArC), 134.01 (=CH), 135.45
(=CH), 138.82 (=CH), 144.59 (ArC), 164.01 (C=O),
164.66 (C=O) 167.00 (C=O), 170.56 (C=O). ESI–MS
m/z found 406.02 (m - 1) peak, calcd 406.
2-(2-((E)-5-(4-Methoxybenzylidene)-2,4-dioxothiazolidin-
3-yl)acetamido)benzamide (17)
2-(2-((E)-5-Benzylidene-2,4-dioxothiazolidin-3-
yl)acetamido)benzamide (20)
Dirty white amorphous solid, yield 70%, mp 272–274°C.
IR (KBr, cm-1): 3470 (N–H, stretch), 3012 (ArC–H,
stretch), 2929 (AliC–H, stretch), 1689 (C=O, stretch), 1678
(C=O, stretch), 1510 (C=C, stretch), 1265 (C–O, stretch),
1139 (C–N, stretch), 1176 (C–N, stretch). 1H-NMR (d
ppm, DMSO-d6) d: 3.73 (s, 3H, CH3), 3.83 (s, 2H, CH2),
6.92 (d, J = 8.64 Hz, 1H, ArH), 7.12 (d, J = 8.76 Hz,
1H, ArH), 7.27 (d, J = 8.60 Hz, 1H, ArH), 7.51 (t, J =
6.24 Hz, 1H, ArH), 7.62 (bs, 1H, NH2), 7.67 (d, J =
9.08 Hz, 1H, ArH), 7.80 (t, J = 7.24 Hz, 1H, ArH), 7.95
(s, 1H, =CH), 8.12 (d, J = 7.12 Hz, 1H, ArH), 8.20 (bs,
1H, NH2), 8.38 (d, J = 8.36 Hz, 1H, ArH), 12.07 (bs, 1H,
NH). 13C-NMR (d ppm, DMSO-d6) d: 59.11 (CH2), 63.32
(CH3), 113.89 (=C), 121.38 (ArC), 125.28 (ArC), 125.79
(ArC), 126.72 (ArC), 127.17 (ArC), 127.58 (ArC), 128.60
Pale brown amorphous solid, yield 62%, mp 248–250°C.
IR (KBr, cm-1): 3471 (N–H, stretch), 3061 (ArC–H,
stretch), 2935 (AliC–H, stretch), 1747 (C=O, stretch), 1527
1
(ArC=C, stretch), 1149 (C–N, stretch). H-NMR (d ppm,
DMSO-d6) d: 4.52 (s, 2H, CH2), 7.13–7.17 (m, 2H, ArH),
7.48–7.58 (m, 5H, ArH), 7.77 (bs, 1H, NH2), 7.79–7.83 (m,
5H, ArH), 8.0 (s, 1H, =CH), 8.29 (bs, 1H, NH2), 12.28 (bs,
1H, NH). 13C-NMR (d ppm, DMSO-d6) d: 63.01 (CH2),
121.42 (=C), 123.05 (ArC), 125.80 (ArC), 126.34 (ArC),
126.75 (ArC), 127.23 (ArC), 127.76 (ArC), 128.52 (ArC),
128.61 (ArC), 129.39 (ArC), 130.23 (ArC), 130.81 (ArC),
132.34 (ArC), 141.76 (=CH), 163.91 (C=O), 165.20 (C=O)
167.15 (C=O), 170.56 (C=O). ESI–MS m/z found 380.08
(m - 1) peak, calcd 380.
123

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Med Chem Res (2012) 21:2689–2701
2-(2-((E)-5-(2-Chlorobenzylidene)-2,4-dioxothiazolidin-3-
yl)acetamido)benzamide (21)
recorded every 24 h. After 72 h, the plates were subjected
for the SRB assay, 25 ll of 50% TCA was added to the
wells gently such that it forms a thin layer over the drug
dilution to form an overall concentration of 10%. The
plates were incubated at 4°C for 1 h. The plates were
flicked and washed five times with tap water to remove
traces of medium, test compound, and serum, and were
then air-dried. The air-dried plates were stained with 0.4%
SRB in 1% acetic acid for 30 min. Then, the unbound dye
was removed by rapidly washing four times with 1% acetic
acid, and the plates were air-dried. 100 ll of 10 mM Tris
base (pH 10.5) was then added to the wells to solubilize the
bound dye. The plates were shaken vigorously for 5 min.
The OD (optical density) value was read on a plate reader
at a wavelength of 540 nm. The ratio of the OD value of a
compound-treated culture to the OD value of a mock-
treated culture, expressed in percentage, was used to
quantify the cytotoxicity of a compound. Results were
expressed as CTC50 (the drug concentration that reduces
50% the absorbance in treated cells versus untreated cells).
Brown amorphous solid, yield 55%, mp 146–148°C. IR
(KBr, cm-1): 3381 (N–H, stretch), 3171 (ArC–H, stretch),
2924 (AliC–H, stretch), 1751 (C=O, stretch), 1689 (C=O,
stretch), 1300 (C–N, stretch), 761 (C–Cl, stretch). ESI–MS
m/z found 414.01 (m - 1) peak, calcd 414.
Determination of glucose uptake activity
Glucose uptake measurement: Six-well microtitre plates
were selected for study with each well capacity of 5 ml
(n = 2). Plates were divided into following groups: Group 1:
2 ml of Tyrode solution with 20,000 mg/l glucose; Group 2:
2 ml of Tyrode solution with 20,000 mg/l glucose and reg-
ular insulin (Nova Nardisk, 40 IU/ml), 5 ll containing 0.2
units of insulin; Groups 3–12: 2 ml of Tyrode solution with
20,000 mg/l glucose and 2 mg of the test compounds 2–6,
12–14, 16, and 18; Group 13: 2 ml of Tyrode solution with
20,000 mg/l glucose and 2 mg of rosiglitazone (standard);
Groups 14–23: 2 ml of Tyrode solution with 20,000 mg/l
glucose, regular insulin 5 ll containing 0.2 units of insulin
and 2 mg of the test compounds 2–6, 12–14, 16, and 18; and
Group 24: 2 ml of Tyrode solution with 20,000 mg/l glu-
cose, regular insulin 5 ll containing 0.2 units of insulin and
2 mg of rosiglitazone (standard). 24 Wistar rats of either sex
were maintained on a standard pellet diet, water ad libitum,
and fasted overnight. The animals were euthanized by
decapitation, and diaphragms were taken out swiftly avoid-
ing 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
respective 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 measured by GOD/POD enzymatic
method using Merckotest glucose kit and Merck-Microlab
200 analyzer.
Acknowledgments Mr. Santosh Kumar would like to thank Dr.
P. Vijayan and Mr. J. S. K. Nagarajan, JSS College of Pharmacy,
Ooty, India, for their support during this project.
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