Synthesis and antidiabetic activity of morpholinothiazolyl-2,4- thiazolidindione derivatives

Article abstract of DOI:10.3109/14756366.2011.594047

We report the synthesis and the in vitro insulin releasing and glucose uptake activity of the morpholino thiazolyl-2,4-thiazolidinediones (1-15). Compounds 5, 1115 (at lower concentration; 0.001mg/ml) were able to increase insulin release in the presence

Full text of DOI:10.3109/14756366.2011.594047

Journal of Enzyme Inhibition and Medicinal Chemistry, 2012; 27(3): 419–427
© 2012 Informa UK, Ltd.
ISSN 1475-6366 print/ISSN 1475-6374 online
DOI: 10.3109/14756366.2011.594047
RESEARCH ARTICLE
Synthesis and antidiabetic activity of
morpholinothiazolyl-2,4-thiazolidindione derivatives
Melis Ezer1, Leyla Tatar Yıldırım2, Ornela Bayro3, Eugen J. Verspohl3, and Oya Bozdag Dundar1
1Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Ankara University, Tandogan, Ankara, Turkey,
2Department of Physics Engineering, Hacettepe University, Beytepe, Ankara, Turkey, and 3Department of Pharmacology,
Institute of Medicinal Chemistry, Munster University, Munster, Germany
Abstract
We report the synthesis and the in vitro insulin releasing and glucose uptake activity of the morpholino thiazolyl-2,4-
thiazolidinediones (1-15). Compounds 5, 11–15 (at lower concentration; 0.001mg/ml) were able to increase insulin
release in the presence of 5.6 mmol/l glucose. The compounds, except derivative 3 show an increase of glucose
uptake. Various compounds are interesting potential antidiabetic leads showing pancreatic and extrapancreatic
effects.
Keywords: Antidiabetic drugs, 2;4-thiazolidinediones, thiazolyl-2;4-thiazolidinediones, insulinotropic activity,
glucose uptake
Introduction
ere is a greater need to develop a safe and effective
One of the most serious metabolic diseases worldwide
is diabetes mellitus. Insulin binds to its receptor and
increases the content of the GLUT 4 leading to enhanced
glucose uptake1. One of the problems in diabetes is that
the glucose uptake in peripheral tissues in response
to insulin is not sufficient, elevated blood levels of glu-
cose are the consequence2. A typically feature of type 2
diabetes is the insulin resistance; many organs such as
liver or muscle may become resistant to the action of
the hormone. Another consequence is an increased not
inhibited glucose output from the liver3.
It is known that peroxisome proliferator-activated
receptor γ (PPAR-γ) plays an important role in the regu-
lation of genes involved in glucose metabolism, insulin
signal transduction and lipid storage4. PPAR γ is the tar-
get for the treatment of insulin resistance using thiazoli-
dinediones (TZDs) (insulin sensitizer)5.
insulin sensitizier for type 2 diabetes. For these reasons,
significant efforts are ongoing to develop the novel TZDs,
which retain their insulin-sensitizing activity and are
devoid of activities that cause adverse effects. e struc-
tural characteristic common to all TZDs is a thiazolidin-
edione ring, to which divergent molecular moieties are
attached.
In the last few years, we reported the synthesis and
insulin releasing activity of flavonyl-TZDs7–11, chromonyl-
TZDs12–14 and thiazolyl-TZDs15,16. A significant insulino-
tropic effect was seen with those TZD compounds.
Herein, in our screening program to search for antidi-
abetic compounds, the 2,4-TZD N-acetic acid, acetic acid
ethyl ester, benzyl and phenacyl derivatives containing
morpholinothiazole ring were synthesized and their
insulin releasing activities in INS-1 cells and glucose
uptake activities were evaluated. It can be derived from
structure-activity data that, instead of imidic hydrogen
on the TZD ring at N-3 position carboxylic acid, carboxy-
lic acid ester groups and benzyl or phenacyl groups are
important for increasing the insulin releasing activity;
moreover it can be evaluated whether the compounds
Despite the clear clinical benefit of TZDs as a treat-
ment for type 2 diabetes, the use of the current genera-
tion of thiazolidinedione is associated with side effects of
clinical importance, such as fluid retention and possibly
heart failure6.
Address for Correspondence: Prof. Dr. Oya Bozdağ Dündar, Department of Pharmaceutical Chemistry, Faculty of pharmacy, Ankara
University, 06100-Tandogan, Ankara, Turkey. Tel: + 90(312)2033077; Fax: + 90(312)2131081. E-mail: bozdag@pharmacy.ankara.edu.tr
(Received 05 May 2011; revised 27 May 2011; accepted 27 May 2011)
419

420 M. Ezer et al.
possessing insulin releasing activity show glucose uptake
activity or not.
Synthesis of compounds 1,2,4-15
A mixture of 4-chloro-2-(morpholin-4-yl)-thiazole-5-
carbaldehyde (III) (0.001 mol) and I/IVa-f/Va-f/VI
(0.001 mol) was heated at 100–110°C in the presence of
0.5 ml acetic acid glacial and sodium acetate (0.001 mol)
for 5 h. e reaction mixture was extracted with CHCl3
(3 × 50 ml) and the organic layer was washed with
water, dried over anhydrous Na2SO4 and evaporated
to dryness. e residue was purified by column chro-
matography silica gel 60 (230–400 mesh ASTM) using
hexane:dichloromethane (1:2) as eluant.
Experimental
Chemistry
Melting points were measured on an electrothermal 9100
type apparatus (Electrothermal Engineering, Essex, UK)
and uncorrected. All instrumental analyses were per-
formed in Central Lab. of Pharmacy Faculty of Ankara
University.1HNMRspectraweremeasuredwithaVARIAN
Mercury 400 FT-NMR spectrometer (Palo Alto, CA) in
CDCl3 and DMSO-d6. All chemical shifts were reported
as δ (ppm) values. Elementary analyses were determined
on a Leco CHNS 932 analyzer (Leco, St. Joseph, MI) and
satisfactory results 0.4% of calculated values (C, H, N)
were obtained. For the chromatographic analysis Merck
Silica Gel 60 (230–400 mesh ASTM = American Society
for Testing and Materials) was used. e chemical
reagents used in synthesis were purchased from E. Merck
(Darmstadt, Germany) and Aldrich (Milwaukee, MI).
2,4-TZD (I17), 2,4-dichlorothiazole-5-carbaldehyde (II18),
4-chloro-2-(morpholin-4-yl)-thiazole-5-carbaldehyde
(III19), ethyl 2,4-dioxothiazolidine-3-ylacetate VI20, sub-
stituted benzyl-2,4-TZDs (IVa, c, e, f21, IVb17, IVd22) and
substituted phenacyl-2,4-TZDs (Va, c, f23, Vb24, Vd25, Ve21)
were synthesized according to the literature.
Crystals suitable for X-ray analysis were obtained
by recrystalization of compound 2 from ethylacetate:
n-hexane (4:1) e data collection was performed on a
CAD-4 diffractometer employing graphite-monochro-
mated CuKα radiation (λ= 1.54184Å). ree standard
reflections were measured every two hours. e structure
was solved by direct methods. e refinement was made
with anisotropic temperature factors for all non-hydro-
gen atoms. e hydrogen atoms were generated geo-
metrically. An empirical Ψ scan absorption correction
was applied. Crystallographic data (excluding structure
factors) for compound 2 have been deposited with the
Cambridge Crystallographic Data Centre as supplemen-
tary publication no CCDC 804860 Copies of the data can
be obtained, free of charge, on application to CCDC, 12
Union Road, Cambridge CB2 1EZ, UK (fax: +44–1223–
336033; e-mail: depositcdc.cam.ac.uk)
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)methylidene)
thiazolidine-2,4-dione (1). Yield: 43.0%, m.p.: 304–306°C,
IR (KBr): C=O (cm−1): 1759, 1687; 1H NMR, δ, ppm
(DMSO-d6): 3.56 (t, 4H, NCH2), 3.72 (t, 4H, OCH2),
7.65 (s, 1H, = CH), 12.59 (s, 1H, TZD-NH); Anal. for
C11H10ClN3O3S2: Calc. C: 39.82, H: 3.04, N: 12.66, S: 19.33.
Found C: 40.16, H: 2.93, N: 12.49, S: 19.34.
(Z)-Ethyl 2-(5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)
methylidene)-2,4-dioxothiazolidin-3-yl)acetate (2). Yield:
54.0%, m.p.: 187°C, IR (KBr): C=O (cm−1): 1729, 1676;
1H NMR, δ, ppm (DMSO-d6): 1.21 (t, 3H, CH3), 3.59 (t,
4H, NCH2), 3.73 (t, 4H, OCH2), 4.17 (q, 2H, CH2CH3),
4.46 (s, 2H, CH2COOEt), 7.82 (s, 1H, = CH); Anal. for
C15H16ClN3O5S2: Calc. C: 43.16, H: 3.87, N: 10.06, S: 15.33.
Found C: 43.00, H: 3.94, N: 9.96, S: 15.05.
3-Benzyl-5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)
methylidene) thiazolidine-2,4-dione (4). Yield: 33.0%,
m.p.: 205°C, IR (KBr): C=O (cm−1): 1729, 1682; 1H NMR, δ,
ppm (DMSO-d6): 3.57 (t, 4H, NCH2), 3.72 (t, 4H, OCH2),
4.81 (s, 2H, TZD-NCH2), 7.29–7.31 (m, 3H, Ar-o,p-H),
7.33–7.35 (m, 2H, Ar-m-H), 7.80 (s, 1H, = CH); Anal. for
C18H16ClN3O3S2. 0.5 H2O: Calc. C: 50.22, H: 3.98, N: 9.76, S:
14.86. Found C: 50.19, H: 3.59, N: 9.72, S: 14.59.
5-((4- chloro -2-(morpholin-4-yl)-1, 3-thiazol-5-yl)
methylidene)-3-(4-fluorobenzyl)-thiazolidine-2,4-dione (5).
Yield: 64.0%, m.p.: 197.6°C, IR (KBr): C=O (cm−1): 1734,
1
1684; H NMR, δ, ppm (DMSO-d6): 3.57 (t, 4H, NCH2),
3.72 (t, 4H, OCH2), 4.79 (s, 2H, TZD-NCH2), 7.15–7.20
(m, 2H, Ar-o-H), 7.34–7.37 (m, 2H, Ar-m-H), 7.79 (s,
1H, = CH); Anal. for C18H15ClFN3O3S2: Calc. C: 49.14, H:
3.44, N: 9.55, S: 14.58. Found C: 48.94, H: 3.33, N: 9.55, S:
14.69.
Synthesis of 4-chloro-2-(morpholin-4-yl)-thiazole-5-
carbaldehyde (III)
To a stirred suspension of 2,4-dichlorothiazole-5-car-
baldehyde (II) (1.0 g, 5.5 mmol) and sodium carbonate
(0.583 g, 5.5 mmol) in acetonitrile (25 ml) was added
morpholine (0,5 ml, 5.5 mmol), followed by stirring for
12 h at room temperature. e crude product was purified
by column chromatography using silica gel 60 (230–400
mesh ASTM) as adsorbent and chloroform: ethyl acetate
(5:1) as eluant. Yield: 1.3 g, 93.0%, m. p.: 200°C (Ref. 19,
200°C).
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)methyl-
idene)-3-(4-chlorobenzyl)-thiazolidine-2,4-dione(6). Yield:
83.0%, m.p.: 179.4°C, IR (KBr): C=O (cm−1): 1738, 1682;
1H NMR, δ, ppm (DMSO-d6): 3.54 (t, 4H, OCH2), 3.69
(t, 4H, NCH2), 4.77 (s, 2H, TZD-NCH2), 7.30 (d, 2H,
Ar-o-H), 7.39 (d, 2H, Ar-m-H), 7.76 (s, 1H, =CH); Anal. for
C18H15C12N3O3S2: Calc. C: 47.37, H: 3.31, N: 9.21, S: 14.05.
Found C: 47.42, H: 3.09, N: 9.13, S: 13.76.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antidiabetic activity of morpholinothiazolyl-2,4-thiazolidindione 421
5-((4- chloro -2-(morpholin-4-yl)-1, 3-thiazol-5-yl)
methylidene)-3-(4-bromobenzyl)-thiazolidine-2,4-dione
(7). Yield: 86.0%, m.p.: 192.6°C, IR (KBr): C=O (cm−1):
1738, 1680; 1H NMR, δ, ppm (DMSO-d6): 3.57 (t, 4H,
NCH2), 3.72 (t, 4H, OCH2), 4.78 (s, 2H, TZD-NCH2), 7.26
(d, 2H, Ar-o-H), 7.54 (d, 2H, Ar-m-H), 7.79 (s, 1H, = CH);
Anal. for C18H15BrClN3O3S2: Calc. C: 43.17, H: 3.02, N: 8.39,
S: 12.81. Found C: 43.02, H: 3.07, N: 8.40, S: 12.47.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2-(4-bromophenyl)-2-oxoethyl)-thiazolidine-
2,4-dione (13). Yield: 54.0%, m.p.: 285–286°C, IR (KBr):
1
C=O (cm−1): 1734, 1688, 1680; H NMR, δ, ppm (CDCl3):
3.63 (t, 4H, NCH2), 3.84 (t, 4H, OCH2), 5.11 (s, 2H, TZD-
NCH2), 7.67 (d, 2H, Ar-m-H), 7.85 (d, 2H, Ar-o-H), 8.05
(s, 1H, = CH); Anal. for C19H15BrClN3O4S2: Calc. C: 43.15,
H: 2.86, N: 7.95, S: 12.13. Found C: 43.14, H: 2.72, N: 8.14,
S: 12.30.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2,4-dichlorobenzyl)-thiazolidine-2,4-dione (8).
Yield: 76.0%, m.p.: 238.4°C, IR (KBr): C=O (cm−1): 1717,
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)methy-
lidene)-3-(2-(2,4-dichlorophenyl)-2-oxoethyl)-thiazolidine-
2,4-dione (14). Yield: 55.0%, m.p.: 216°C, IR (KBr): C=O
1
1699; H NMR, δ, ppm (DMSO-d6): 3.58 (t, 4H, NCH2),
1
3.73 (t, 4H, OCH2), 4.86 (s, 2H, TZD-NCH2), 7.31 (d,
1H, Jo = 8.40Hz, Ar-6’-H), 7.40 (dd, 1H, Jo = 8.40Hz,
Jm = 2.00Hz, Ar-5’-H), 7.67 (d, 1H, Jm = 2.00Hz, Ar-3’-H),
7.81 (s, 1H, = CH); Anal. for C18H14Cl3N3O3S2: Calc. C:
44.05, H: 2.88, N: 8.56, S: 13.07. Found C: 43.71, H: 2.69,
N: 8.57, S: 12.72.
(cm−1): 1733, 1683; H NMR, δ, ppm (CDCl3): 3.63 (t, 4H,
NCH2), 3.84 (t, 4H, OCH2), 5.08 (s, 2H, TZD-NCH2), 7.38
(dd, 1H, Jo = 8.80Hz, Jm = 2.00Hz, Ar-5’-H), 7.51 (d, 1H,
Jm = 2.00Hz, Ar-3′-H), 7.71 (d, 1H, Jo = 8.40Hz, Ar-6′-H),
8.05 (s, 1H, = CH); Anal. for C19H14Cl3N3O4S2: Calc. C:
43.98, H: 2.72, N: 8.10, S: 12.36. Found C: 43.80, H: 2.65,
N: 8.22, S: 12.44.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(4-nitrobenz yl)-thiazolidine-2,4- dione
(9). Yield: 81.0%, m.p.: 253.1°C, IR (KBr): C=O (cm−1):
1725, 1666; 1H NMR, δ, ppm (CDCl3): 3.62 (t, 4H, NCH2),
3.83 (t, 4H, OCH2), 4.96 (s, 2H, TZD-NCH2), 7.59 (d, 2H,
Ar-o-H), 8.04 (s, 1H, = CH), 8.19 (d, 2H, Ar-m-H); Anal. for
C18H15ClN4O5S2: Calc. C: 46.30, H: 3.24, N: 12.00, S: 13.73.
Found C: 46.67, H: 3.26, N: 11.76, S: 13.35.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2-(4-nitrophenyl)-2-oxoethyl)-thiazolidine-2,4-
dione (15). Yield: 70.0%, m.p.: 243°C, IR (KBr): C=O
1
(cm−1): 1728, 1700, 1683; H NMR, δ, ppm (DMSO-d6):
3.60 (t, 4H, NCH2), 3.74 (t, 4H, OCH2), 5.40 (s, 2H, TZD-
NCH2), 7.84 (s, 1H, = CH), 8.32 (d, 2H, Ar-H), 8.40 (d, 2H,
Ar-H); Anal. for C19H15ClN4O6S2: Calc. C: 46.15, H: 3.06, N:
11.34, S: 12.94. Found C: 46.03, H: 2.89, N: 11.32, S: 12.91.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2-oxo-2-phenylethyl)-thiazolidine-2,4-dione
(10). Yield: 40.0%, m.p.: 283.6°C, IR (KBr): C=O (cm−1):
1738, 1684; 1H NMR, δ, ppm (DMSO-d6): 3.51 (t, 4H,
NCH2), 3.68 (t, 4H, OCH2), 5.30 (s, 2H, TZD-NCH2),
7.59–7.63 (m, 2H, Ar-m-H), 7.68–7.73 (m, 2H, Ar-o-H),
8.07–8.09 (m, 1H, Ar-p-H), 8.18 (s, 1H, = CH); Anal. for
C19H16ClN3O4S2: Calc. C: 50.72, H: 3.58, N: 9.34, S: 14.25.
Found C: 50.72, H: 3.74, N: 9.76, S: 14.62.
Synthesisof(Z)-2-(5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-
5-yl) methylidene)-2,4-dioxothiazolidin-3-yl)acetic acid (3).
A mixture of acetic acid ester compound 2 (0.075 g, 0.18
mmol), glacial acetic acid (4 mL) and HCl 12 N (1 mL)
was refluxed for 2 h. After evaporation in vacuo, the
residue was refluxed again with glacial acetic acid (4mL)
and HCl 12 N (1 mL) for 2 h. After evaporation to dryness
in vacuo, the crude solid was crystallized from ethanol
providing pure carboxylic acid 3.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2-(4-fluoro-phenyl)-2-oxoethyl)-thiazolidine-2,4-
dione (11). Yield: 74.0%, m.p.: 210.3°C, IR (KBr): C=O
Yield: 52 mg, 74.0%, m.p.: 269°C, IR (KBr): C=O
1
(cm−1): 1729, 1676; H NMR, δ, ppm (DMSO-d6): 3.59 (t,
4H, NCH2), 3.73 (t, 4H, OCH2), 4.35(s, 2H, CH2COOH),
7.81 (s, 1H, = CH), 13.45 (broad s, 1H, COOH); Anal. for
C13H12ClN3O5S2: Calc. C: 40.05, H: 3.10, N: 10.78, S: 16.45.
Found C: 39.66, H: 3.03, N: 10.79, S: 16.45.
1
(cm−1): 1729, 1700, 1681; H NMR, δ, ppm (DMSO-d6):
3.60 (t, 4H, NCH2), 3.74 (t, 4H, OCH2), 5.30 (s, 2H, TZD-
NCH2), 7.42-7.46 (m, 2H, Ar-m-H), 7.83 (s, 1H, = CH),
8.16–8.19 (m, 2H, Ar-o-H); Anal. for C19H15ClFN3O4S2. 0.1
H2O: Calc. C: 48.63, H: 3.26, N: 8.96, S: 13.63. Found C:
48.28, H: 3.20, N: 8.92, S: 13.46.
Biological activity studies
Insulin releasing activity
Cell culture of INS-1 cells. INS-1 cells, generously pro-
vided by Dr. C. Wollheim, Geneva, Switzerland26, were
grown in plastic culture bottles or micro-wells for 4–6
days (half confluence: 1–2 × 106 cells per ml) in RPMI
medium supplemented with 10% (v/v) fetal calf serum,
100 U of penicillin per ml and 0.1 mg of streptomycin per
ml. Cells were seeded at a density of 5 × 105 cells/ml. e
medium was changed every 5 days, and the cells were
detached from the culture flask with trypsin 1 week after
seeding, centrifuged and reseeded as described above.
5-((4-chloro-2-(morpholin-4-yl)-1,3-thiazol-5-yl)meth-
ylidene)-3-(2-(4-chlorophenyl)-2-oxoethyl)-thiazolidine-2,4-
dione (12). Yield: 73.0%, m.p.: 236.5°C, IR (KBr): C=O
1
(cm−1): 1733, 1695, 1683; H NMR, δ, ppm (DMSO-d6):
3.60 (t, 4H, NCH2), 3.74 (t, 4H, OCH2), 5.31 (s, 2H, TZD-
NCH2), 7.68 (d, 2H, Ar-m-H), 7.83 (s, 1H, = CH), 8.10
(d, 2H, Ar-o-H); Anal. for C19H15Cl2N3O4S2. 0.5 H2O: Calc.
C: 46.34, H: 3.27, N: 8.54, S: 12.99. Found C: 46.15, H: 3.18,
N: 8.72, S: 12.71.
© 2012 Informa UK, Ltd.

422 M. Ezer et al.
Prior to the experiment cells were washed two times and
then incubated in Krebs-Ringer buffer containing 10 mM
HEPES and 0.5% bovine serum albumin (KRBH).
After 4 d of preincubation the supernatant was col-
lected and the cells were treated with or without 50 nM
insulin for 30 min. en the cells were incubated with
1 mM 2-NBDG for 5 min. e 2-NBDG uptake reaction
was stopped by removing the incubation medium and
washing the cells three times with cold phosphate buff-
ered saline (PBS). All substances were checked for adher-
ing to the plastic wells and can easily be drained away.
Fluorescence intensity of 2-NBDG was recorded using
FLUOstar Galaxy, a multifunctional microplate reader.
Insulin release. To measure insulin secretion, half-con-
fluent cells in micro-wells were incubated for 90 min.
at 37°C in the aforementioned KRBH buffer. Insulin
released into the medium was assayed with a radioim-
munoassay using rat insulin (Novo Nordisk, Bagsvaerd,
Denmark) as a standard, (mono 125I-Tyr A14)-porcine
insulin as the labelled compound (Sanofi-Aventis,
Germany) and anti-insulin antibodies from Linco (St.
Louis, MO). Each compound had been checked for
non-interference with the insulin radioimmunoassay.
e data were corrected for the effects of solubilizing
compounds (ethanol, DMSO).
Results and discussion
Chemistry
iazolyl-2,4-thiazolidinedione compounds 1-15 were
synthesized according to the synthetic pathway described
in Scheme 1. 2,4-dichlorothiazole-5-carbaldehyde (II)
wasobtainedwith2,4-TZD17andN,N-dimethylformamide
in phosphoryl chloride18. 4-chloro-2-(morpholin-4-yl)-
thiazole-5-carbaldehyde (III) was synthesized with 2,4-
dichlorothiazole-5-carbaldehyde (II) and morpholine in
sodium carbonate/acetonitrile19. Ethyl 2,4-dioxothiazo-
lidine-3-ylacetate (VI) was prepared by N-alkylation of
2,4-TZD with ethyl bromoacetate in THF/NaH20.
Substituted benzyl-2,4-thiazolidinediones IVa-f were
obtained with 2,4-TZD and appropriate benzyl halide
derivatives in NaOH/ethanol. Substituted phenacyl-2-
,4-thiazolidinediones Va-f were synthesized by reacting
potassium 2,4-thiazolidinedione with appropriate phen-
acylbromide derivatives in hot methanol.
e condensation of 4-chloro-2-(morpholin-4-yl)-
thiazole-5-carbaldehyde (III) with 2,4-thiazolidine-
dione I, ethyl 2,4-dioxothiazolidine-3-ylacetate VI,
substituted benzyl-2,4-thiazolidinediones IVa-f and
phenacyl-2,4-thiazolidinediones Va-f in the presence
of sodium acetate/acetic acid glacial by Knoevenagel
reaction, led to morpholino thiazolyl-2,4-thiazolidine-
diones 1, 2,4-thiazolidinedione acetic acid ethyl ester
2, morpholino thiazolyl-substituted benzyl-2,4-thiazo-
lidinediones 4-9 and morpholino thiazolyl-substituted
phenacyl-2,4-thiazolidinediones 10–15, respectively.
e acidic hydrolysis of 2 provided corresponding car-
boxylic acid 3.
Glucose uptake activity
Reagents. Dulbecco modified Eagle medium high glu-
cose (DMEM) and fetal calf serum was purchased from
PAA (COlbe, Germany). Rosiglitazone was isolated from
Avandia®. 2-NBDG (2-[N-(7-nitrobenz-2-oxa-1,3-diazol-
4-yl)amino]-2-deoxyglucose), as the fluorescence probe,
was obtained from Invitrogen (Darmstadt, Germany).
TNF-α and bovine Insulin was purchased from Sigma-
Aldrich (Steinheim, Germany).
Cell culture and treatment. HepG2 cells were obtained
from Boehringer Ingelheim (Biberach, Germany). e
human hepatocellular liver carcinoma cells were grown
at 37°C in a 5% CO2 humidified atmosphere in Dulbecco’
modified Eagle’s medium (DMEM) supplemented
with 10% heat-inactivated fetal calf serum (FCS), 100 U
of penicillin per ml and 0.1 mg of streptomycin per ml,
4 mM Glutamine. Medium was changed every third day
and was split after reaching 80% of confluence.
Glucose uptake measurement by fluorescence microplate
reader. e method used was first described by Zou
et al2. It uses the fluorescent probe 2-NBDG for the direct
measurement of glucose uptake followed by the detec-
tion of the fluorescence within the cells. 2-NBDG is a
fluorescent derivate of glucose modified with a 2-[N-(7-
nitrobenz-2-oxa-1,3-diazol-4-yl) amino group at the
C-2 position. is indicator was excited at 467 nm and
showed fluorescence at 542 nm27.
HepG2 cells were seeded at 1.5 × 104 cells /well into
96-well plates, and after the cells adhere on the bottom
of the well the supernatant was collected. en the cells
were treated with or without 100 µM Rosiglitazone as a
positive control, with or without 1 nM TNF-α to induce
insulin resistance and the compounds were added to
reverse the insulin resistance. Also their direct effect
was measured. All substances except insulin and TNF-α
were diluted in DMSO (final concentration in the well:
1% DMSO). To show if the compounds or Rosiglitazone
reverse the TNF-α effect we combined Rosiglitazone and
the compounds with TNF-α and insulin.
In our previous paper, the Z configuration of the meth-
yne proton was confirmed via X-ray diffractometric anal-
ysis and it was resonated at lower field than that of the
1
E configuration in H NMR. Additionally, the calculated
values of the methyne protons of Z and E isomeric form
of the compound were seen as a singlet at 8.05 ppm and
7.34 ppm, respectively28. In E isomers, due to the lesser
deshielding effect of 1-S of the TZD ring, such a proton
should resonate at lower chemical shift values29. In this
study, only one isomer of the synthesized compounds
was obtained. Furthermore, the X-ray diffractometric
analysis of compound 2 unambiguously confirmed the
Z configuration at the chiral axis (Figure 1). Methyne
proton of 2 was observed as a singlet at 7.82 ppm. As for
methyne proton of the compound 3 which was obtained
Journal of Enzyme Inhibition and Medicinal Chemistry

Antidiabetic activity of morpholinothiazolyl-2,4-thiazolidindione 423
Scheme 1. (a) NaH/THF; (b) Ethanol/NaOH; (c) Methanol; (d) POCl3/DMF; (e) morpholin; (f) CH3COOH/CH3COONa; (g) CH3COOH/HCl.
Figure 1. e molecular structure and atomic labeling scheme of compound 2.
by acidic hydrolysis of ester compound 2, was seen as a
singlet at 7.81 ppm. Methyne protons of the compounds
1–15 were seen at 7.65–8.18 ppm as a singlet.
Biological activity
Derivatives of morpholino thiazolyl TZD compounds
1-15 were tested comparing with glibenclamide for
© 2012 Informa UK, Ltd.

424 M. Ezer et al.
their insulinotropic activities in INS-1 cells at two differ-
ent concentrations (Table 1). Compounds 5, 11–15 (at
lower concentration; 0.001 mg/ml) were able to increase
insulin release in the presence of 5.6 mmol/l glucose.
Compounds 3-6, 8-10, 12–15 (at higher concentration;
0.01 mg/ml) were able to increase insulin release. In this
series, the most potent compounds are 12 and 14 which
are having phenacyl chloride and dichloride at N-3 posi-
tion of TZD ring, respectively.
Insulin and rosiglitazone show an increase of glucose
uptake by the HepG2 cells. TNF-α is able to reverse the
stimulatory effect of insulin. Rosiglitazone is able to
reverse the inhibitory effect of TNF-α and increases the
glucose uptake (Figure 2).
Different concentrations of Compound 4 were tested
to find out the optimal concentration for glucose uptake
(Figure 3). We choose the highest tested concentration of
0.1 mg/ml for further experiments.
Table 1. Effects of various compounds on glucose-mediated insulin release from INS-1 cells*.
Compound [concentration]
Glucose [3.0 mM]
Glucose [5.6 mM]
Plus 1 [1μg/ml]
Insulin release (%)
74.10 4.78
100
Concentration
Insulin release (%)
37.83 12.33
76.97 10.24
79.23 9.76
84.50 4.97
101.4 9.41
92.85 7.56
87.31 10.33
70.87 12.93
91.53 4.83
96.95 18.91
104.9 12.00
121.0 21.80
102.8 3.12
122.3 11.34
109.8 22.65
160.4 18.04
93.88 9.68
47.03 7.88
91.19 6.18
122.0 25.01
145.5 32.58
143.3 20.16
143.3 33.42
97.51 10.81
106.8 18.89
146.1 26.69
139.0 39.47
94.46 6.51
107.7 24.86
116.3 7.89
129.4 26.18
127.5 3.25
Plus 1 [10μg/ml]
Plus 2 [10μg/ml]
Plus 3 [10μg/ml]
Plus 4 [10μg/ml]
Plus 5 [10μg/ml]
Plus 6 [10μg/ml]
Plus 7 [10μg/ml]
Plus 8 [10μg/ml]
Plus 9 [10μg/ml]
Plus 10 [10μg/ml]
Plus 11 [10μg/ml]
Plus 12 [10μg/ml]
Plus 13 [10μg/ml]
Plus 14 [10μg/ml]
Plus 15 [10μg/ml]
Plus 2 [1μg/ml]
Plus 3 [1μg/ml]
Plus 4 [1μg/ml]
Plus 5 [1μg/ml]
Plus 6 [1μg/ml]
Plus 7 [1μg/ml]
Plus 8 [1μg/ml]
Plus 9 [1μg/ml]
Plus 10 [1μg/ml]
Plus 11 [1μg/ml]
Plus 12 [1μg/ml]
Plus 13 [1μg/ml]
Plus 14 [1μg/ml]
Plus 15 [1μg/ml]
Plus glibenclamide (1μg/ml)
DMSO 0.01%
DMSO 0.1%
104.5 13.86
*INS-1 cells in multi-wells were washed three times and incubated in KRBH buffer for 90min at 5.6mM glucose. e results are expressed
as percent insulin release at 5.6mM glucose alone. Values obtained in the presence of 3.0mM glucose (substimulatory concentration)
and glibenclamide (1μg/ml) served as negative and positive controls. e final concentration of the solvent DMSO was either 0.01 or
0.1%; a DMSO control (even at 1%) had no effect as shown in the table data. Each value represents the mean SEM of six independent
experiments.
Figure 2. Effect of insulin, rosiglitazone and TNF-α on glucose uptake by HEP G2 cells. Rosiglitazone and TNF-α were added for four days,
insulin for the final 30 minutes and the labeled glucose 2-NBDG for 5min. Mean SEM three experiments.
Journal of Enzyme Inhibition and Medicinal Chemistry

Antidiabetic activity of morpholinothiazolyl-2,4-thiazolidindione 425
e compounds except compound number 3 show an
increase of glucose uptake (Figure 4).
pathophysiological situation HepG2 cells were rendered
resistant by TNF-α.
Not in all experiments TNF-α was able to inhibit the
insulin stimulatory effect, but when it was possible the
compounds were able to reverse this effect and increase
the glucose uptake in the cells (Table 2).
Only compounds 3,4,5,6,9 and 10 have been tested
which had positive effects with respect to insulin
release. Compounds 4,5,6,9 and 10 also increased
glucose uptake by themselves. To simulate the
e compounds were able to reverse this effect.
Various compounds are interesting antidiabetic drugs in
that they possess a dual effect.
In our previous studies7–16, we showed that 2,4-TZD
N-acetic acid, acetic acid ethyl ester, benzyl and phen-
acyl derivatives were more potent than unsubstituted
TZDs with regard to their insulin releasing activities. As
seen in this study, 2,4-TZD-N-substituted derivatives
Figure 3. Concentration-response curve of compound 4. Mean SEM three experiments.
Figure 4. Effect of insulin and tested compounds on glucose uptake. Mean SEM two to four experiments.
© 2012 Informa UK, Ltd.

426 M. Ezer et al.
Table 2. Effect of insulin, a combination of TNF-α plus insulin and the addition of various compounds. Mean SEM three experiments.
Glucose uptake
Compound
Insulin
TNF-α plus Insulin
0.5776μg Glucose/well
1.0902μg Glucose/well
0.5292μg Glucose/well
0.5299μg Glucose/well
1.0902μg Glucose/well
0.5776μg Glucose/well
TNF-α plus Insulin plus Compound
0.8664μg Glucose/well
8.2828μg Glucose/well
1.6584μg Glucose/well
1.4202μg Glucose/well
7.1834μg Glucose/well
3.5378μg Glucose/well
3
1.2094μg Glucose/well
1.4488μg Glucose/well
0.9747μg Glucose/well
0.9747μg Glucose/well
1.4488μg Glucose/well
1.2094μg Glucose/well
4
5
6
9
10
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had also effect on insulin releasing activity in INS-1 cells.
According to these results, it should be pointed out that
compared with imidic hydrogen carboxylic acid, car-
boxylic acid ester, benzyl or phenacyl groups on the TZD
ring at N-3 position played a noticeable role for increas-
ing the insulin releasing activity in INS-1 cells. On the
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are the most potent compounds in terms of glucose
uptake activity. As a result, we can say that instead of
imidic hydrogen on the TZD ring at N-3 position benzylic
groups are important for increasing the glucose uptake
and the insulin releasing activity.
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of some new flavone derivatives. 2nd communication: 4’-flavonyl-
2,4-thiazolidinediones. Arzneimittelforschung 2000;50:539–543.
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Conclusion
We report the synthesis and the in vitro insulin releas-
ing and glucose uptake activity of the morpholino
thiazolyl-2,4-thiazolidinediones 1-15. Only com-
pounds 3,4,5,6,9 and 10 have been tested for glucose
uptake activity which had positive effects with respect
to insulin release. Compounds 4,5,6,9 and 10 also
increased glucose uptake by themselves. To simulate
the pathophysiological situation HepG2 cells were ren-
dered resistant by TNF-α. e compounds were able
to reverse this effect. In conclusion, we can say that
compounds 4,5,6,9 and 10 are interesting antidiabetic
drugs in that they possess pancreatic and extrapancre-
atic effects.
4th
communication:
6-flavonyl-2,4-thiazolidinediones.
Arzneimittelforschung 2001;51:623–627.
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M, Waheed A, Verspohl EJ et al. Synthesis and hypoglycemic
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SarikayaMetal.Synthesisandbiologicalactivityofsomenewflavonyl-
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12. Bozdag-Dündar O, Verspohl EJ, Waheed A, Ertan R. Synthesis
and antidiabetic activity of some new furochromonyl-2,4-
thiazolidinediones. Arzneimittelforschung 2003;53:831–836.
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Insulinotropic activity of chromonyl-2,4-thiazolidinediones. Med
Chem Res 2009;18:665–670.
Acknowledgement
is work was supported by Research Organization
of Ankara University, Turkey (No: 09B3336003) and
Hacettepe University Syntific Research Department
(Project number: 04A602004). e skillful help under-
graduate Ummu Duzgun.
15. Bozdag-Dündar O, Ceylan-Unlüsoy M, Verspohl EJ, Ertan R.
Synthesis and antidiabetic activity of novel 2,4-thiazolidinedione
derivatives containing
2006;56:621–625.
16. Bozdag-Dündar O, Mentese A, Verspohl EJ. Synthesis and
antidiabetic activity of some new thiazolyl-2,4-thiazolidinediones.
Arzneimittelforschung 2008;58:131–135.
a thiazole ring. Arzneimittelforschung
Declaration of interest
e authors report no conflicts of interest.
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Synthesis and antimicrobial activity of chlorobenzyl benzylidene
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2H-pyrazolo[3,4-d]thiazole and isoxazole [3,4-d]thiazole from
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