An extremely high insulin-mimetic activity of bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-pyridinolato)zinc(II) complex

Article abstract of DOI:10.1246/cl.2002.114

Vanadyl and zinc(II) complexes with VO(O₂S₂) and Zn(O₂S₂) coordination mode, respectively, were synthesized. Among them, bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-pyridinolato)zinc(II) complex exhibited an extremely high insulin-mimetic activity (IC₅₀ = 0.04 mM when IC₅₀ value of a positive control, VOSO₄ was estimated to be 1.0 mM) compared to vanadyl and zinc(II) complexes reported previously.

Full text of DOI:10.1246/cl.2002.114

114
Chemistry Letters 2002
An Extremely High Insulin-Mimetic Activity of Bis(1,4-dihydro-2-methyl-1-phenyl-
4-thioxo-3-pyridinolato)zinc(II) Complex
Akira Katoh,ꢀ Takeshi Tsukahara, Ryota Saito, Kallol K. Ghosh, Yutaka Yoshikawa,y Yoshitane Kojima,y Asuka Tamurayy
and Hiromu Sakuraiyy
Department of Applied Chemistry, Faculty of Engineering, Seikei University, Musashino, Tokyo 180-8633
yDepartment of Chemistry, Graduate School of Science, Osaka City University, Sumiyoshi-ku, Osaka 558-8585
yyDepartment of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical University, Yamashina-ku, Kyoto 607-8413
(Received October 22, 2001; CL-011027)
Vanadyl and zinc(II) complexes with VO(O2S2) and
of Et3N in dry MeCN at room temperature (Scheme 1).
Zn(O2S2) coordination mode, respectively, were synthesized.
Among them, bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-
pyridinolato)zinc(II) complex exhibited an extremely high
insulin-mimetic activity (IC50 ¼ 0:04 mM when IC50 value of a
positive control, VOSO4 was estimated to be 1.0 mM) compared
to vanadyl and zinc(II) complexes reported previously.
Scheme 1.
Diabetes mellitus (DM) affects over 140 million people all
over the world.1 DM is generally classified into type I DM
(insulin-dependent) and type II DM (non-insulin-dependent). The
treatment of type II DM is essentially based on dietary measures,
exercise and stimulation of insulin sensitivity by oral chemo-
therapeutic agents. Such agents have some undesirable side
effects. On the other hand, daily injections of insulin are still
necessary for the treatment of type I DM.2 Much effort, therefore,
has been devoted to find less toxic and orally active drugs that
enhance the insulin sensitivity or insulin-mimetic activity. A
number of orally active insulin-mimetic vanadyl complexes have
been reported.3;4 Among them, a vanadyl complex, bis(1-oxy-2-
pyridinethiolato)oxovanadium(IV), with VO(O2S2) coordination
mode has been demonstrated to treat both types of DM in
experimental animals.5 Then, we have focused on developing
insulin-mimetic zinc(II) complexes with similar Zn(O2S2)
coordination mode, because zinc(II) is generally less toxic than
vanadyl.6 Further, the following points were taken into account
for the molecular design; 1) Zn(II) complex with a bidentate
ligand is superior to inorganic Zn(II) salt from the viewpoint of
efficacy of gastrointestinal absorption and 2) a bidentate ligand,
which can be easily synthesized and changed the hydrophobicity
by introducing a variety of substituents at N-1 position, should be
selected for the elucidation of the structure-activity relationship.
Previously, we proposed the first insulin-mimetic zinc(II)
complexes of maltol and 2-hydroxypyridine N-oxide with a
Zn(O4) coordination mode.7 In extending our studies on
heterocycles for application to chemotherapeutic agents,8 we
found an extremely high insulin-mimetic bis(1,4-dihydro-2-
methyl-1-phenyl-4-thioxo-3-pyrdinolato)zinc(II) complex. This
paper reports the first insulin-mimetic Zn(II) complexes with
Zn(O2S2) coordination mode.
Typical procedures for syntheses of vanadyl and zinc(II)
complexes are as follows (Scheme 2). To a solution of 3a
(155 mg, 1 mmol) in H2O (5 mL) was added dropwise a solution
of VOSO4ꢁ3H2O (109 mg, 0.5 mmol) in H2O (5 mL). The pH of
the solution was adjusted to 10.5 with 2 M KOH, and the mixture
was stirred overnight. The resulting precipitate was collected by
filtration, washed several times with H2O, and then dried over
anhydrous P2O5 in vacuo to give bis(1,4-dihydro-1,2-dimethyl-4-
thioxo-3-pyridinolato)oxovanadium(IV) (4a; 169 mg, 90%).11
To a solution of 3b (869 mg, 4 mmol) and LiOHꢁH2O (84 mg,
2 mmol) in H2O (40 mL) was added a solution of ZnSO4ꢁ7H2O
(575 mg, 2 mmol) in H2O (40 mL), and then the reaction mixture
was stirred overnight at room temperature. The resulting white
precipitate was collected by filtration, washed well with H2O, and
then dried over P2O5 in vacuo to give bis(1,4-dihydro-2-methyl-
1-phenyl-4-thioxo-3-pyridinolato) zinc (II) (5b; 905 mg, 91%).12
Scheme 2.
1,2-Dimethyl-3-hydroxy-(2a) and 3-hydroxy-2-methyl-1-
phenyl-4(1H)-pyridinone (2b) were prepared from a commer-
cially available maltol (1) and the corresponding amines
according to literature methods.9;10 1,2-Dimethyl-3-hydroxy-
(3a) and 3-hydroxy-2-methyl-1-phenyl-4(1H)-pyridinethione
(3b) were prepared by treatment of 2a,b with P2S5 in the presence
The insulin-mimetic activity of vanadyl and zinc(II) com-
plexes was evaluated by in vitro experiments, in which the
inhibition of the release of free fatty acid (FFA) from isolated rat
adipocytes treated with epinephrine was estimated by comparing
the activity of vanadyl sulfate (VS) as a positive control.13 The
effects of vanadyl and zinc(II) complexes were found to be dose-
Copyright Ó 2002 The Chemical Society of Japan

Chemistry Letters 2002
115
dependent in the concentration range from 5 ꢂ 10ꢃ5 to 1ꢂ
10ꢃ3 M (Figure 1), and the apparent IC50 value, which is a 50%
inhibition concentration of FFA release in each complex, was
estimated from these data, in which the IC50 values for VS are
normalized to 1.0 mM to compare with other complexes. The
obtained IC50 values for two vanadyl complexes (11.2 mM for 4a
and 2.45 mM for 4b) were shown to be higher than that of VOSO4
(IC50 ¼ 1:0 mM) as a positive control. On the other hand, zinc(II)
complex (5b) (IC50 ¼ 0:04 mM) exhibited an extremely high
insulin-mimetic activity than VOSO4 (IC50 ¼ 1:0 mM). The
partition coefficient (Coctanol/Cbuffer) of zinc(II) complex (5b) was
measured to be 1.62 by means of UV-vis spectroscopy, indicating
the importance of some degree of the hydrophobicity.3;8
Unfortunately, the activity of zinc(II) complex (5a) could not
be measured accurately owing to its low solubility under the
employed condition.
reported previously in terms of IC50 value, 2) zinc(II) complex
(5b) showed activity about 10 times higher than the first insulin-
mimetic zinc(II) complexes, bis(maltolato)zinc(II) and bis(2-
hydroxypyridine-N-oxide)zinc(II),7 and 3) zinc(II) complex (5b)
exhibited insulin-mimetic activity higher than the corresponding
vanadyl complex (4b). The result 3) is the first report concerning
the direct comparison of the activities between vanadyl and
zinc(II) complexes. On the basis of the results, we propose here
that zinc(II) complex (5b) is a potent insulin-mimetic complex.
Further investigation on in vivo activity as well as action
mechanism of the complex in relation to the insulin receptor
and glucose transporter is under way.
References and Notes
1
L. C. Y. Woo, V. G. Yuen, K. H. Thompson, J. H. McNeill, and C.
Orvig, J. Inorg. Biochem., 76, 251 (1999).
2
3
S. M. Brichard and J.-C. Henquin, TiPS, 16, 265 (1995).
H. Sakurai and A. Tsuji, in ‘‘Vanadium in the Environment, Part 2,’’ ed.
by J. O. Nriagu, John Wiley & Sons, New York (1998), p 297; H.
Sakurai, K. Fujii, S. Fujimoto, Y. Fujisawa, K. Takechi, and H. Yasui, in
‘‘Vanadium Compounds: Chemistry, Biochemistry, and Therapeutic
Applications,’’ ed. by A. S. Tracey and D. C. Crans, American
Chemical Society, Washington, D.C. (1998), p 344.
4
5
K. H. Thompson and C. Orvig, J. Chem. Soc., Dalton Trans., 2000,
2885.
H. Sakurai, H. Sano, T. Takino, and H. Yasui, Chem. Lett., 1999, 913; H.
Sakurai, H. Sano, T. Takino, and H. Yasui, J. Inorg. Biochem., 80, 99
(2000); S. Takeshita, I. Kawamura, T. Yasuno, C. Kimura, T.
Yamamoto, J. Seki, A. Tamura, H. Sakurai, and T. Goto, J. Inorg.
Biochem., 85, 179 (2001).
6
7
8
K. T. Smith and F. H. Nielsen, in ‘‘Trace Minerals in Foods,’’ ed. by
K. T. Smith, Marcel Dakker, New York (1988), p 209 and p 257.
Y. Yoshikawa, E. Ueda, K. Kawabe, H. Miyake, H. Sakurai, and Y.
Kojima, Chem. Lett., 2000, 874.
J. Ohkanda and A. Katoh, Rev. Heteroatom Chem., 18, 87 (1998) and
references cited therein; A. Katoh, K. Taguchi, H. Okada, M. Harata, Y.
Fujisawa, T. Takino, and H. Sakurai, Chem. Lett., 2000, 866.
B. L. Rai, L. S. Dekhordi, H. Khodr, Y. Jin, Z. Liu, and R. C. Hider, J.
Med. Chem., 41, 3347 (1998).
9
10 Z. Zhang, S. J. Rettig, and C. Orvig, Inorg. Chem., 30, 509 (1991).
11 Bis(1,4-dihydro-1,2-dimethyl-4-thioxo-3-pyridinolato)oxovanadium-
(IV)(4a): IR(KBr):962 cmꢃ1 (ꢀV=O); UV-vis:ꢁmax (c ¼ 5 ꢂ 10ꢃ3 M in
DMSO)/nm649("/dm3 molꢃ1 cmꢃ1 92) and 541(146). Anal. Found: C,
44.70; H, 4.11; N, 7.34%. Calcd for C14H16N2O3S2V: C, 44.80; H, 4.30;
N, 7.46%. Bis(1,4-dihydro-2-methyl-1-phenyl-4-thioxo-3-pyridinola-
to)-oxovanadium(IV) (4b): IR(KBr): 972 cmꢃ1 (ꢀV=O); UV-vis: ꢁmax
(c ¼ 5 ꢂ 10ꢃ3 M in DMSO)/nm 716 ("/dm3 molꢃ1 cmꢃ1 38) and 551
(160). Anal. Found: C, 57.33; H, 3.95; N, 5.39%. Calcd for
C24H20N2O3S2V: C, 57.71; H, 4.04; N, 5.61%.
12 Bis(1,4-dihydro-1,2-dimethyl-4-thioxo-3-pyridinolato)zinc(II) (5a):
IR(KBr): 1585, 1454, 1313, and 1199 cmꢃ1; UV-vis: ꢁmax (c ¼
2 ꢂ 10ꢃ5 M in DMSO)/nm 357 ("/dm3 molꢃ1 cmꢃ1 35800), 290
(8400), and 263 (38500); 1H NMR (400 MHz, DMSO-d6) ꢂ 2.47 (3H,
s), 3.91 (3H, s), 7.42 (1H,d, J ¼ 6:2 Hz), and 7.53 ppm (1H, d,
J ¼ 6:2 Hz); Anal. Found: C, 45.15; H, 4.27; N, 7.27%. Calcd for
C14H16N2O2S2Zn: C, 44.99; H, 4.31; N, 7.49%. Bis(1,4-dihydro-2-
methyl-1-phenyl-4-thioxo-3-pyridinolato)zinc(II) (5b): IR(KBr): 1610,
Figure 1. Inhibitory effects of vanadul (upper) and zinc(II) complexes
(bottom) on FFA release from rat adipocytes treated with epinephrine in
the presence of 0.1% glucose.13 B is blank without epinephrine and
complex, and C is control without complex. In the case of VS1-3, 4a-1-3,
1571, 1490, 1459, 1321, 1222, and 1170 cmꢃ1
; UV-vis: ꢁmax
and 4b-1-3, 1-3 stand for concentrations of the complexes: 1 ¼ 1 ꢂ 10ꢃ4
;
(c ¼ 2 ꢂ 10ꢃ5 M in DMSO)/nm 363 ("/dm3 molꢃ1 cmꢃ1 48500), 289
(9400), and 269 (37700); 1H NMR (400 MHz, DMSO-d6) ꢂ2.17 (3H, s),
7.51 (1H,d, J ¼ 6:3 Hz), 7.56 (1H, d, J ¼ 6:3 Hz), and 7.50-7.65 (5H,
m); Anal. Found: C, 57.89; H, 3.88; N, 5.51%. Calcd for
C24H20N2O2S2Zn: C, 57.89; H, 4.05; N, 5.63%.
2 ¼ 5 ꢂ 10ꢃ4
,
3 ¼ 1 ꢂ 10ꢃ3 M. 5a-1-4 and 5b-1-4 also stand for
concentrations: 1 ¼ 5 ꢂ 10ꢃ5; 2 ¼ 1 ꢂ 10ꢃ4; 3 ¼ 5 ꢂ 10ꢃ4; 4 ¼ 1ꢂ
10ꢃ3 M. In each system, adipocytes (2:3 ꢄ 0:6 ꢂ 106 cells/mL) were
treated with complexes for 30 min, and then incubated with epinephrine
(1 ꢂ 10ꢃ5 M) for 3 h at 37 ꢅC. Each column is expressed as the mean ꢄ SD
for three repeated experiments.
13 H. Sakurai, K. Fujii, H. Watanabe, and H. Tamura, Biochem. Biophys.
Res. Commun., 214, 1095 (1995); S. Fujimoto, K. Fujii, H. Yasui, R.
Matsushita, J. Takeda, and H. Sakurai, J. Clin. Biochem. Nutr., 23, 113
(1997); M. Nakai, H. Watanabe, C. Fujiwara, H. Kakegawa, T. Satoh, J.
Takada, R. Matsushita, and H. Sakurai, Biol. Pharm. Bull., 18, 719
(1995).
It was concluded that 1) bis(1,4-dihydro-2-methyl-1-phenyl-
4-thioxo-3-pyridinolato)zinc(II) (5b) exhibited the highest in-
sulin-mimetic activity among vanadyl and zinc(II) complexes