Potential insulinominetic agents of zinc(II) complexes with picolinamide derivatives: Preparations of complexes, in vitro and in vivo studies

Article abstract of DOI:10.1248/cpb.50.337

Following the finding of in vitro insulinominetic activities of new prepared Zn(II) complexes with amide ligands (2-picolinamide (pa-a) and 6-methyl-2-picolinmethylamide (6mpa-ma)) in isolated rat adipocytes treated with epinephrine in terms of inhibition of free fatty acid release, their blood glucose normalizing effects were observed on daily intraperitoneal injections for 14 d in a type 2 diabetes mellitus model animal, KK-A^y mice. The blood glucose levels of KK-A^y mice were maintained in a normal range during the administration of both complexes. After the administration of each complex for 14 d, the improvement of glucose metabolism was confirmed as judged by the glucose tolerance test.

Full text of DOI:10.1248/cpb.50.337

March 2002
Chem. Pharm. Bull. 50(3) 337—340 (2002)
337
Potential Insulinominetic Agents of Zinc(II) Complexes with Picolinamide
Derivatives: Preparations of Complexes, in Vitro and in Vivo Studies
Eriko UEDA,a Yutaka YOSHIKAWA,a Yoshio ISHINO,b Hiromu SAKURAI,c and Yoshitane KOJIMA*,a
Department of Chemistry, Graduate School of Science, Osaka City University,a 3–3–138 Sugimoto, Sumiyoshi-ku, Osaka
558–8585, Japan, Department of Organic Chemistry, Osaka Municipal Technical Research Institute,b 1–6–50 Morinomiya,
Joto-ku, Osaka 536–8553, Japan, and Department of Analytical and Bioinorganic Chemistry, Kyoto Pharmaceutical
University,c 5 Nakauchi-cho, Misasagi, Yamashina-ku, Kyoto 607–8414, Japan.
Received October 3, 2001; accepted December 21, 2001
Following the finding of in vitro insulinominetic activities of new prepared Zn(II) complexes with amide lig-
ands (2-picolinamide (pa-a) and 6-methyl-2-picolinmethylamide (6mpa-ma)) in isolated rat adipocytes treated
with epinephrine in terms of inhibition of free fatty acid release, their blood glucose normalizing effects were ob-
served on daily intraperitoneal injections for 14 d in a type 2 diabetes mellitus model animal, KK-Ay mice. The
blood glucose levels of KK-Ay mice were maintained in a normal range during the administration of both com-
plexes. After the administration of each complex for 14 d, the improvement of glucose metabolism was confirmed
as judged by the glucose tolerance test.
Key words Zinc(II) complex; picolinamide derivative; type 2 diabetes mellitus; insulinominetic activity
of the rats given Zn(II) alone.12) Furthermore, previous stud-
ies have demonstrated that zinc acts on adipocytes and pro-
motes the induction of leptine, and also acts on the pancreas,
therefore it helps insulin to combine with insulin receptor, re-
sulting in improvement of the conditions of type 2 DM.16,17)
On the basis of these observations, Zn(II) is expected to be
less toxic than other metal ions. Generally, it is known that
the complexation of free metal ions lowers the toxicity of the
metal ions and promotes their absorption into the blood.18,19)
We indicated that Zn(II) complexes with maltol, picolinic
acid and amino acids have higher insulinomimetic activity
than ZnSO4 in in vitro experiments, and the administrations
of the Zn(II) complexes to KK-Ay mice, a type 2 DM model
animal, were found to show normoglycemic activity.20—23)
However, such Zn(II) complexes were all molecular com-
plexes. In recent years, many researchers have proposed
metal-containing therapeutic agents of cationic complexes
such as BBR3364 of cisplatin derivatives24) and 99mTc-tetro-
fosfin complex of diagnostic radiopharmacuetics.25) In this
paper, we have planned to synthesize new cationic Zn(II)
complexes with picolinamide and its derivative and to esti-
mate both in vitro insulinomimetic activities and in vivo
blood glucose normalizing effects in KK-Ay mice. Four
Zn(II) complexes, Zn(pa-a)3Cl2 1, Zn(pa-a)3(ClO4)2 2, Zn-
(6mpa-ma)2Cl2 3, and Zn(6mpa-ma)2SO4 4 of 2-picolinamide
(pa-a) and 6-methyl-2-picolinemethylamide (6mpa-ma) lig-
ands exhibited higher insulinomimetic activities than those of
VOSO4 and ZnSO4 as standard.
Diabetes mellitus (DM), one of the life-style related dis-
eases, is a metabolic disease which shows the symptom of
hyperglycemia and causes many complications.1,2) Recently,
DM is becoming a serious social problem not only in Japan
but also around the world, and the number of people suffer-
ing from DM has increased to over 14 million including the
estimate of potential patients in Japan. Many researchers
have enthusiastically studied the development of antidiabetic
agents. However, according to reports, many therapeutics
have side effects, therefore new antidiabetic agents with high
effectiveness and low side effects are eagerly sought all over
the world.
During the endeavor, a very important fact was revealed
that several metal ions show insulinomimetic activity.3—12) For
example, Schwarz and Mertz. reported that the chromium ion
stimulates insulin function and improves glucose tolerance.3)
Tuvemo et al.4) and Paolisso et al.5) proposed that hypomag-
nesemia is related to DM and thus the insulin sensitivity is
improved by magnesium administration. Since the 1980s it
has been recognized that vanadium has normoglycemic activ-
ity in streptozotocin-induced type 1 DM rats (STZ rats),6—9)
and the investigations on vanadium are energetically ex-
tended. In 1980, Coulston and Dandona found that Zn(II),
one of the essential elements in animals and humans, stimu-
lates lipogenesis in rat adipocytes similarly to the action of
insulin.10)
There is a close relation between Zn(II) and insulin, be-
cause Zn(II) is essential for the stabilization of the insulin
precursor (proinsulin) and taken into the pancreas and se-
creted to the blood with insulin.13) In addition, it was ob-
served that Zn(II) concentration in the fingernails of the pa-
tients with DM decreases14) and consequently the excretion
of Zn(II) into the urine increases.15) Therefore, the study on
the relationship between Zn(II) and DM is very important
and many researchers have investigated insulinomimetic ac-
tivity of Zn(II).11,12) Shisheva et al. reported that Zn(II) ions
administered orally to STZ rats reduce the blood glucose lev-
els as much as 50%.11) Song et al. observed that when STZ
rats are given drinking water containing Zn(II) with
Experimental
Materials Zinc sulfate (ZnSO4·7H2O), NEFA-C test Wako, and acacia
were purchased from Wako Pure Chemicals (Osaka, Japan). D-(ϩ)-Glucose
was obtained from Nakalai Tesque Inc. (Kyoto, Japan). (Ϯ)-Epinephrine hy-
drochloride, collagenase and bovine serum albumin (BSA) were from Sigma
Chemical Co. (St. Louis, U.S.A.). All other reagents were of analytical
reagent quality and were used without further purification. Purity of ZnSO4·
7H2O was determined by chelatometry using Cu-Pan (Cu-1-(2-pyridyl-azo-
2-naphthol) (Dojindo, Kumamoto, Japan) as indicator.
Instruments Elemental analyses were carried out on a Perkin-Elmer
240C Elemental Analyzer (MA, U.S.A.). Fourier transform (FT)-IR spectra
1
were recorded on a Jasco FT/IR-420 (Tokyo, Japan) spectrophotometer. H-
NMR spectra were recorded on a JEOL LA-300 WB FT-NMR spectrometer
cyclo(His-Pro), the blood glucose levels were lower than those (Tokyo, Japan). FAB-MS was obtained with a JEOL AX-500 (Tokyo,
To whom correspondence should be addressed. e-mail: kojima@sci.osaka-cu.ac.jp © 2002 Pharmaceutical Society of Japan

338
Vol. 50, No. 3
male rat adipocytes (1.0ϫ106 cells/ml) prepared as described in ref. 26 were
preincubated at 37 °C for 30 min with various concentrations (10Ϫ4—10Ϫ3 M)
of Zn(II) complexes in KRB buffer (120 mM NaCl, 1.27 mM CaCl2, 1.2 mM
MgSO4, 4.75 mM KCl, 1.2 mM KH2PO4, 24 mM NaHCO3: pH 7.4) containing
2% BSA. A 10Ϫ4 M epinephrine was then added to the reaction mixtures and
the resulting solutions were incubated at 37 °C for 3 h. The reactions were
stopped by soaking in ice water and the mixtures were centrifuged at
3000 rpm for 10 min. For the outer solutions of the cells, FFA levels were
determined with NEFA-C test Wako.
Blood Glucose Lowering Effects of Zn(II) Complexes, 1 and 4, in KK-
Ay Mice KK-Ay mice (4 weeks old : CREA Japan Inc., Tokyo, Japan)
were kept in the laboratory for 4 weeks. The 8 weeks old KK-Ay mice with a
type 2 DM model received daily intraperitoneal (i.p.) injections (5 mice in a
group) of 1 and 4 dissolved in 5% acacia vehicle at about 10 : 30 a.m. after
the determination of their blood glucose levels for 14 d. The blood sample
for the analysis of glucose levels was obtained from the tail vein of each
mouse and measured with Glucocard. Body weights of KK-Ay mice who
were allowed free access to solid food (CREA Japan Inc.) and tap water
were measured daily during the administration of 1 and 4. In addition, in-
takes of solid food and drinking water in each mouse were checked daily
throughout the experiments. The dose of 1 and 4 were 4.0 mg Zn (61.2 mmol
for 1 and 4)/kg body weight. The blood samples for the analyses of BUN,
GOT, GPT, TCHO, and FFA were withdrawn from the cavernous sinus with
capillary under anesthesia with ether.
Japan). Melting points were taken with Yanako MP-J3 Micro Point Aparatus
(Kyoto, Japan). Hemoglobin A1c (HbA1c) levels were measured with
DCA2000 System (Bayer Sankyo Co. Ltd., Tokyo, Japan). The blood glu-
cose levels were measured with a Glucocard (Arkray Co. Ltd., Kyoto,
Japan). The serum concentrations of blood urea nitrogen (BUN), glutamic-
oxaloacetic transaminase (GOT), glutamic-pyruvic transaminase (GPT), and
total cholesterol (TCHO) were determined by a Fuji Dry Chem (Tokyo,
Japan).
Preparation of 6-Methyl-2-picolinmethylamide (6mpa-ma) Twenty-
five milliliters of 98% H2SO4 was added by drops to 6-methyl-2-picolinic
acid (5.48 g, 40 mmol) in 100 ml of methanol in an ice bath. The reaction
mixture was stirred at 50 °C for 3 d. After the reaction mixture was poured
into saturated NaHCO3 and extracted with CHCl3 three times, the CHCl3
layer was dried over anhydrous Na2SO4, and evaporated to give methyl 6-
methyl-2-picolinate (4.95 g, 82.0%). A 40% CH3NH2 (25.5 g, 328 mmol) in
methanol was added to methyl 6-methyl-2-picolinate (4.95 g, 32.8 mmol).
The mixture was stirred at room temperature for 3 d, and then evaporated.
The residue was purified as a pale brown oil by sephadex LH-20 using
methanol (4.55 g, 92%). Anal. Calcd for C8H10N2O·0.8H2O: C, 58.38; H,
1
7.10; N, 17.02. Found: C, 58.20; H, 6.61; N, 16.98. H-NMR (CDCl3) d:
2.56 (3H, s), 3.03 (3H, d, Jϭ5.1 Hz), 7.27 (1H, d, Jϭ7.7 Hz), 7.72 (1H, dd,
Jϭ7.7, 7.9 Hz), 8.00 (1H, d, Jϭ7.7 Hz), 8.08 (1H, br). IR (neat) cmϪ1: 1667
for nCϭO. FAB-MS: m/z: 151 (MϩH)ϩ.
Preparation of Zn(pa-a)3Cl2 1 To a methanol solution of pa-a (0.37 g,
3.0 mmol), a methanol solution of ZnCl2 (0.14 g, 1.0 mmol) was added and
stirred for 3 h at room temperature. 1 was obtained as the residue by remov-
ing the solvent. Yield: 0.50 g (98%). Anal. Calcd for C18H18N6O3Cl2Zn·
0.4H2O: C, 42.40; N, 16.48; H, 3.72. Found: C, 42.64; N, 16.40; H, 3.67. IR
(KBr): 1667 cmϪ1 for nCϭO. Mp; 112—130 °C.
Oral Glucose Tolerance Test (OGTT) After daily i.p. injection of 1
and 4 for 14 d, the mice were fasted for 14 h and the blood glucose levels (0,
30, 60, 90, 120 min) were measured after gastric gavage of 1 g glucose/kg
body weight for each mouse. The blood samples obtained from a tail vein
were measured with Glucocard.
Preparation of Zn(pa-a)3(ClO4)2
2
To an aqueous solution of pa-a
(0.49 g, 4.0 mmol), an aqueous solution of ZnSO4·7H2O (0.58 g, 2.0 mmol)
and Ba(ClO4)2·3H2O (0.78 g, 2.0 mmol) were added and stirred overnight at
room temperature. After the filtration of BaSO4 and removal of the solvent,
2 was recrystallized from a small amount of hot water. Yield: 0.23 g (27%).
Anal. Calcd for C18H18N6O11Cl2Zn·0.8H2O: C, 33.51; N, 13.13; H, 3.06.
Found: C, 33.53; N, 13.02; H, 3.10. IR (KBr): 1671 cmϪ1 for nCϭO. Mp;
265—270 °C.
Preparation of Zn(6mpa-ma)2Cl2 3 The complex 3 was prepared by
referring to the method of 1. Yield: 0.39 g (96%). Anal. Calcd for
C16H20N4O2Cl2Zn·0.8H2O: C, 42.40; N, 12.42; H, 4.83. Found: C, 42.72; N,
12.24; H, 4.88. IR (KBr): 1645 cmϪ1 for nCϭO. Mp; Ͼ300 °C.
Preparation of Zn(6mpa-ma)2SO4 4 To an aqueous solution of 6mpa-
ma (0.33 g, 2.0 mmol), an aqueous solution of ZnSO4·7H2O (0.29 g,
1.0 mmol) was added and following stirred for 3 h at room temperature. 4
was obtained as the residue by removing the solvent. Yield: 0.50 g (94%).
Anal. Calcd for C16H20N4O6SZn·4.3H2O: C, 35.64; N, 10.39; H, 5.35.
Found: C, 35.68; N, 10.36; H, 5.25. IR (KBr): 1643 cmϪ1 for nCϭO. Mp;
Ͼ300 °C.
Results and Discussion
In Vitro Insulinomimetic Activity of Zn(II) Complexes;
1—4 In vitro insulinomimetic activities of four Zn(II) com-
plexes were examined with regard to inhibition of FFA re-
lease from isolated rat adipocytes treated with epinephrine.
Complexes 1—4 were confirmed to act dose-dependently in
the concentration of 10Ϫ4, 5ϫ10Ϫ4 and 10Ϫ3 M of the Zn(II)
complexes (Fig. 1). The apparent IC50 values, the 50% in-
hibitory concentration of the Zn(II) complexes on the FFA
release, were 0.70, 0.71, 0.95, and 0.97 mM for 1, 2, 3, and 4,
respectively (Table 1). These Zn(II) complexes showed
higher insulinomimetic activities than the standards VOSO4
and ZnSO4. The IC50 values of Zn(II) complexes with differ-
ent types of counter anion (ClϪ and ClO4 for Zn(pa-a)32ϩ,
Ϫ
ClϪ and SO42Ϫ for Zn(6mpa-ma)22ϩ) were not significant.
Inhibition Effects of Zn(II) Complexes on Free Fatty Acid (FFA) Re-
lease from Isolated Rat Adipocytes Treated with Epinephrine Isolated From these results, the effect of counter anions of Zn(II)
Fig. 1. Inhibitory Effects of VOSO4 (VS), ZnSO4 (ZS) and Zn(II) Complexes (1—4) on FFA Release from Isolated Rat Adipocytes Treated with Epineph-
rine (EP)
Rat adipocytes were prepared as reported ref. 26. Each column is expressed as the meanϮS.D. for 3 experiments. Blank: cells only; control: cells plus 10Ϫ5 M EP. In each system,
adipocytes (1.0Ϯ106 cells/ml) were treated with 10Ϫ4, 5ϫ10Ϫ4, 10Ϫ3 M (3—17 column) of the compound in each numerical order, respectively, for 30 min and then incubated with
10Ϫ5 M EP for 3 h at 37 °C.

March 2002
339
Table 1. Estimated IC50 Values of Zn(II) Complexes (1—4)
Complex
IC50 (mM)
VOSO4
1.00Ϯ0.08
ZnSO4
1.58Ϯ0.05
1
2
3
4
0.70Ϯ0.04**
0.71Ϯ0.05**
0.95Ϯ0.05*
0.97Ϯ0.04*
Significance at pϽ0.01 vs. ZnSO4. Significance at pϽ0.005 vs. ZnSO4.
Fig. 3. OGTT for Control KK-Ay Mice (—᭿—) and KK-Ay Mice Treated
with Zn(II) Complex 1 (—᭺—) and 4 (—᭹)
After i.p. injections of 1 and 4 for 14 d, the mice were fasted for 14 h and the blood
glucose levels (0, 30, 60, 90, 120 min) were measured after gastric gavage of 1 g glu-
cose/kg body weight. The blood samples obtained from a tail vein were measured with
Glucocard. Each symbol is expressed as the meanϮS.D. for 5 mice. Significance at
pϽ0.05 vs. untreated KK-Ay mice.
Significance at pϽ0.01 vs. untreated KK-Ay
mice.
Table 2. Hemoglobin A1c (HbA1c) Levels of KK-Ay Mice before and after
the i.p. Injections of 5% Acacia (Control) and Zn(II) Complexes 1 and 4 for
14 d
HbA1c (%)
Fig. 2. Changes of Blood Glucose Levels before and after i.p. Injections
for 14 d
Treatment
Before the treatment
After the treatment
Hyperglycemic KK-Ay mice received daily i.p. injection of 5% acacia (control)
(nϭ5) and Zn(II) complexes of 1 and 4 at a dose of 4.0 mg Zn/kg body weight (nϭ5)
for 14 d. Each column is expressed as the meanϮS.D. for 5 mice. Significance at
pϽ0.05 vs. before the i.p. injections. Significance at pϽ0.001 vs. before the i.p. in-
Control
7.6Ϯ0.5
7.0Ϯ0.8
7.4Ϯ0.8
8.3Ϯ0.3
6.4Ϯ0.8*
6.1Ϯ0.5**
1
4
jections.
Significance at pϽ0.0001 vs. before the i.p. injections. # Significance at
pϽ0.001 vs. after the i.p. injections of control.
Values are meanϮS.D. for 5 or 4 mice (1; 5 mice and control and 4; 4 mice). Sig-
nificance at pϽ0.005 vs. after the i.p. injections of control KK-Ay mice. Significance
at pϽ0.001 vs. after the i.p. injections of control KK-Ay mice.
complexes was not observed in the insulinomimetic activi-
ties.
Blood Glucose Normalizing Effects of Zn(II) Com- creased slowly afterwards. The changes of the blood glucose
plexes with Amides in KK-Ay Mice We tested in vivo levels of the group treated with 4 for 2 h at 30 min intervals
blood glucose normalizing effects of 1 and 4 in KK-Ay mice were significantly lower compared with the untreated KK-Ay
receiving daily i.p. injections for 14 d. A complex 1 with ClϪ, mice (0 min: at the time of the administration of glucose). It
which exists in animals and humans, was administered to was observed that the glucose metabolism of KK-Ay mice
KK-Ay mice. A complex 4 was injected to KK-Ay mice to was improved by i.p. administration of Zn(II) complex 4. In
compare with 1. Figure 2 presents the changes of blood glu- comparison with the blood glucose levels of the group
cose levels of KK-Ay mice before and after i.p. administra- treated with 4, the group treated with 1 was slightly higher,
tions. When 1 and 4 were administered at a dose of 4.0 mg but the state of DM in both groups treated with 1 and 4 was
(61.2 mmol for 1 and 4) Zn/kg body weight, the blood glu- confirmed to be improved.
cose levels dropped down to approximately 200 mg/dl (11.1
HbA1c and the Serum Parameters Furthermore, we
mM) after 24 h and the same dose was given to the mice to measured HbA1c, which shows the number of glucose mole-
maintain the blood glucose level at around 200 mg/dl. The ef- cules attached to hemoglobin, in red blood cells and indi-
fectiveness of 1 on the blood glucose level was almost as cated average blood glucose levels over a long period. In the
good as 4. During the treatment of 1 and 4 for 14 d, the body untreated KK-Ay mice, the HbA1c levels increased from
weights of KK-Ay mice steadily increased from 39.6Ϯ1.3 7.6Ϯ0.5 to 8.3Ϯ0.3 (%) before and after the examination, re-
and 40.1Ϯ1.5 to 41.9Ϯ2.1 and 42.3Ϯ2.0 g, respectively. The spectively (Table 2). In contrast, HbA1c levels of the KK-Ay
decrease of body weight associated with toxicity was not ob- mice treated with 1 and 4 were decreased from 7.0Ϯ0.8 and
served in KK-Ay mice treated 1 and 4.
7.4Ϯ0.8 to 6.4Ϯ0.8 and 6.1Ϯ0.5 (%), respectively, indicat-
Oral Glucose Tolerance Test After the daily i.p. admin- ing that the blood glucose normalizing effects of Zn(II) com-
istrations for 14 d, we examined the oral glucose tolerance plexes are long-term.
test (OGTT) to examine if the glucose metabolism of KK-Ay
The serum parameters, GOT, GPT, TCHO, and BUN lev-
mice was improved or not. As shown in Fig. 3, when the KK- els of KK-Ay mice, untreated and treated with 1 and 4 after
Ay mice were given orally 1 g glucose/kg body weight after 14 d administrations are summarized in Table 3. The GPT
they were fasted for 14 h, the blood glucose levels of KK-Ay and TCHO levels were not altered between the untreated and
mice treated with 5% acacia (untreated KK-Ay mice) went up the treated KK-Ay mice with Zn(II) complexes (Table 3). The
to a maximum of 317 mg/dl (17.6 mM) in 30 min and de- GOT levels of KK-Ay mice treated with 1 and 4 were higher

340
Vol. 50, No. 3
Table 3. Serum Parameters of KK-Ay Mice after the i.p. Injections of 5% Acacia (Control) and Zn(II) Complexes 1 and 4 for 14 d
Treatment
BUN (mg/dl)
GOT (U/l)
GPT (U/l)
TCHO (mg/dl)
FFA (mEq/l)
Control
32.9Ϯ2.4
25.1Ϯ4.3
23.9Ϯ2.7*
26.5Ϯ2.3
61Ϯ15
99Ϯ28*
111Ϯ36*
86Ϯ24
22Ϯ6
24Ϯ6
25Ϯ4
23Ϯ5
181Ϯ27
140Ϯ37
154Ϯ19
89Ϯ11
1.75Ϯ0.07
1.27Ϯ0.10**
0.89Ϯ0.20**
—
1
4
C57/black
Values are meanϮS.D. for 5 mice. Significance at pϽ0.05 vs. control KK-Ay mice. Significance at pϽ0.001 vs. control KK-Ay mice.
than that of the untreated KK-Ay mice, because some serum
samples of KK-Ay mice treated with 1 and 4 were partially
hemolytic. The BUN levels of KK-Ay mice treated with 4
were lower than those of untreated KK-Ay mice. C57/black
mice, non-diabetic mice and the same series as KK-Ay mice,
received 5% acacia daily i.p. injections for 14 d and blood
samples were taken to compare with the serum parameters of
KK-Ay mice. The BUN levels of KK-Ay mice were higher
than those of C57/black mice. The average BUN level of 10
weeks old C57/black for 3 mice was 26.5Ϯ2.3 (mg/dl). The
BUN levels of KK-Ay mice treated with 4 were not different
from those of C57/black. From these results, we consider that
the function of the kidney of KK-Ay mice given Zn(II) com-
plexes are not damaged, suggesting that Zn(II) complexes
with amides, 1 and 4, appear to be essentially non-toxic to
the hepatic and renal functions. Furthermore, we measured
the FFA levels of KK-Ay mice after 14 d administrations.
KK-Ay mice administered Zn(II) complexes exhibited signif-
icantly lower FFA levels compared with the control mice. It
was reported that increasing in the FFA levels give rise to the
insulin resistance,27) thus, it was suggested that the adminis-
trations of Zn(II) complex 1 and 4 improve the insulin resis-
tance of KK-Ay mice.
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Acknowledgement The authors are grateful to the members of the Ana-
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