A new insulin-enhancing agent: [N,N′-bis(4-hydroxysalicylidene)-o-phenylene-diamine]oxovanadium(IV) and its permeability and cytotoxicity

Article abstract of DOI:10.1016/j.ejmech.2010.02.010

A new insulin-enhancing agent: [N,N′-bis(4-hydroxysalicylidene)-o-phenylene-diamine] oxovanadium(IV) (BPOV) was synthesized and characterized by X-ray crystallography. BPOV was administered intragastrically to STZ-diabetic rats for 4 weeks. The results sh

Full text of DOI:10.1016/j.ejmech.2010.02.010

European Journal of Medicinal Chemistry 45 (2010) 2327e2335
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
A new insulin-enhancing agent: [N,N0-bis(4-hydroxysalicylidene)-o-phenylene-
diamine]oxovanadium(IV) and its permeability and cytotoxicity
,
b
c
d
e
b
f
Ming-jin Xie a , Ling Li , Xiao-Da Yang , Wei-ping Liu , Shi-ping Yan , Yan-fen Niu , Zhao-hui Meng
*
a Department of Chemistry, Yunnan University, Cuihu North Road No.2, Kunming 650091, Yunnan, China
b Yunnan Pharmacological Laboratories of Natural Products, Kunming Medical College, Kunming 650031, China
c Department of Chemical Biology, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China
d Kunming Institute of Precious Metals, Kunming 650221, China
e Department of Chemistry, Nankai University, Tianjin 300071, China
f Kunming Hospital of Population and Health, Kunming 650204, China
a r t i c l e i n f o
a b s t r a c t
A new insulin-enhancing agent: [N,N0-bis(4-hydroxysalicylidene)-o-phenylene-diamine] oxovanadium
(IV) (BPOV) was synthesized and characterized by X-ray crystallography. BPOV was administered
intragastrically to STZ-diabetic rats for 4 weeks. The results showed that BPOV could significantly
decrease the blood glucose level and ameliorated impaired glucose tolerance in STZ-diabetic rats. BPOV
has been further tested on insulin, glycogen and serum lipid studies. The results suggested BPOV has
glucose-lowering activity in diabetic rats, as well as improved the disorder of lipid metabolism in dia-
betes. BPOV had permeability above 10ꢀ5 cm sꢀ1. It was suggested good lipophilic properties. The
cytotoxicity of BPOV on Caco-2 cells was measured by MTT assay which suggested BPOV have higher
effect on impairment of cellular associated with lower level capacity of cellular accumulation.
Ó 2010 Elsevier Masson SAS. All rights reserved.
Article history:
Received 29 September 2009
Received in revised form
29 January 2010
Accepted 2 February 2010
Available online 10 February 2010
Keywords:
Insulin-enhancing
Salen-type ligands
Caco-2 cells
1. Introduction
ligand with less or no toxicity is to be great interest. A lot of organic
vanadium complexes have been synthesized and demonstrated to
Vanadium is a trace element that plays an essential role of its
physiological effects and medical applications. Vanadium
compounds, as a nonspecific phosphotyrosine phosphatase inhib-
itor, have been known to possess insulin-mimetic and/or enhancing
effects both in vitro and in vivo [1e3]. Studies testing compounds in
animal model systems [4e8] and in human beings [9e11] show
that simple vanadium salts and V-complexes alleviate the symp-
toms of diabetes. Several studies have shown that vanadium
compounds improve not only hyperglycemia in human subjects
and animal models of type 1 diabetes but also glucose homeostasis
in type 2 diabetes [12,13].
be effective. A large class of compounds based on V(IV) chelate
complexes [20] have been extensively studied mainly by the
Sakurai and Orvig/McNeill groups, respectively [21e23]. In 1992,
bis(maltolato)oxovanadium(IV) BMOV was reported two to three
times more effective acutely than vanadyl sulfate as a glucose-
lowering agent, was better tolerated than inorganic vanadium salts,
and resulted in reliable glucose-lowering in all animal models of
diabetes [24e27]. Furthermore, the first Phase II clinical trial of
a
designed vanadium-based pharmaceutical agent (bis(ethyl-
maltolato)oxovanadium(IV), BEOV), was completed by Medeval
Ltd., Manchester, UK [28]. Several vanadium complexes of the Schiff
base sal2en {N,N0-ethylenediaminebis(salicylideneiminato)} and
related ligands have been proposed as insulin-enhancing agents,
and for the treatment of obesity and hypertension [29]. At present,
only [VIVO(sal2en)] has been tested in vivo for insulin-mimetic
activity.
In our work, salen-type ligands were study with it present
versatile steric, electronic and lipophilic properties. A new insulin-
enhancing vanadyl complex [N,N0-bis(4-hydroxysalicylidene)-o-
phenylene-diamine]oxovanadium(IV) (BPOV) has been synthesized,
ensuring a balanced lipophilicity and hydrophilicity for orally
available for vanadium-based pharmaceuticals and its insulin-
mimetic activity investigated. Our studies have shown BPOV could
Vanadium salts, at doses ranging from 0.1 to 0.7 mM kgꢀ1 dꢀ1
[14e16], in a variety of animal models of diabetes [17], normalized
blood glucose and lipid levels, corrected thyroid hormone defi-
ciency, improved insulin sensitivity, and prevented or reversed
secondary complications, such as cardiomyopathy, cataract devel-
opment, impaired antioxidant status and excessive food and fluid
intake [18,19]. In order to reduce the toxicity and improve the
absorption, tissue uptake and efficiency of vanadium, the organic
* Corresponding author.
E-mail address: xmj7193@yahoo.com.cn (M.-j. Xie).
0223-5234/$ e see front matter Ó 2010 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2010.02.010

2328
M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
H2N
CHO
+
OH
NH2
OH
O
V
VOSO4.3H2O
N
N
N
N
O
O
OH
HO
OH
OH
OH
OH
Scheme 1. Synthesis procedure of the complex BPOV.
significantly decrease the blood glucose level and ameliorated
impaired glucose tolerance in STZ-diabetic rats. Furthermore, we
have taken on insulin, glycogen and serum lipid studies of the
complex, attempted to know the in vivo anti-diabetes activity
relationships of BPOV with VO(O2N2) coordination mode.
To investigate the factors leading to the different bioavailability
of vanadium compounds and their relation to the cytotoxicity, the
mechanism of permeation and effects on Caco-2 and the cell
monolayer were studied with vanadium compounds [30,31]. In this
paper, the permeability and toxicity of BPOV were assessed using
the Caco-2 cell monolayer to explore its absorption, distribution,
metabolism, elimination, and toxicity (ADME/T) properties.
diamine, 2,4-dihydroxybenzaldehyde and vanadyl sulfate hyrate
(VOSO4$3H2O,) were purchased from Aldrich (purity ꢁ 98%). The
complex [N,N0-bis(4-hydroxysalicylidene)-o-phenylene-diamine]
oxovanadium(IV) (BPOV) was synthesized by 2,4-dihydrox-
ybenzaldehyde and o-phenylenediamine with vanadyl sulfate,
produced a monomeric VIV complex. The reaction sequences are
outlined in Scheme 1. The block shaped well defined green colour
crystals of BPOV suitable for X-ray studies were obtained from the
mother liquor.
2.1. Crystallographic data collection and structure analysis
2. Chemistry
X-ray structure determinations Intensity data for single
crystals of complex was collected on a BRUKER SMART 1000 CCD
detector with graphite-monochromatized Mo Ka radiation
Chemicals and solvents were reagent grade and were used
without further purification unless otherwise noted. O-phenylene-
(
l
¼ 0.071073 nm). For BPOV, a total of 8035 independent reflec-
tions were collected, of which 5595 [R(int) ¼ 0.0345] were
considered as observed [I > 2 (I)]. The structure was solved by
s
Table 1
direct method using the program SHELXS-97 [32] and subsequent
Fourier difference techniques, and refined anisotropically by full-
matrix least-squares on F2 using SHELXL-97 [33]. Crystal data and
structural refinements of complex and selected bond lengths and
angles are shown in Tables 1 and 2. Crystallographic data of the
complex has been deposited at the Cambridge Crystallogrphic Data
Center, CCDC Nos. 252952. Any queries relating to the data can be e-
mailed to deposit@ccdc.cam.ac.uk.
Crystal data and structural refinements for the complex.
Empirical formula
Fw
C24H30N2O9S2V
605.56
Temperature/K
Wavelength/nm
Crystal system
Space group
293(2)
0.71073
Triclinic
P-1
ꢂ
Unit cell dimensions (nm,
)
a ¼ 9.325(3)
a
¼ 78.464(5)
b ¼ 12.312(4)
c ¼ 12.829(4)
1187.3(11)
2
b
¼ 72.067(6)
¼ 85.072(6)
g
Volume/nm3
Z
Density (calc.)/g cmꢀ3
1.465
0.567
Table 2
m
/mmꢀ1
Bond lengths [A] and angles [ꢂ] around the vanadium atom in [N,N0-1,3-propyl-bis
F (000)
630
(salicyladimine)]oxovanadium (IV), BPOV.
Crystal size/mm3
0.20 ꢃ 0.16 ꢃ 0.12
q
range/ꢂ
2.16e26.46
V(1)eO(5)
V(1)eO(3)
V(1)eO(1)
V(1)eN(1)
V(1)eN(2)
1.592 (3)
1.915(3)
1.926 (2)
2.049(3)
2.045(3)
O(5)eV(1)eO(3)
O(5)eV(1)eO(1)
O(3)eV(1)eO(1)
O(5)eV(1)eN(2)
O(3)eV(1)eN(2)
O(1)eV(1)eN(2)
O(5)eV(1)eN(1)
O(3)eV(1)eN(1)
O(1)eV(1)eN(1)
N(1)eV(1)eN(2)
O(5)eV(1)eN(1)eC7
110.44 (13)
108.63 (12)
84.26 (11)
104.61 (12)
88.37 (12)
146.46 (11)
106.62 (13)
142.71 (12)
87.97 (11)
78.35 (12)
93.4 (3)
Limiting indices
Reflection collected
Independent reflection
Max. and min. transmission
Refinement method
ꢀ8 ꢄ h ꢄ 11, ꢀ15 ꢄ k ꢄ 15, ꢀ16 ꢄ l ꢄ 13
8035
5595 [R(int) ¼ 0.0345]
0.790 and 0.745
Full-matrix least-squares on F2
5595/0/349
Data/restraints/parameters
Goodness-of-fit on F2
1.001
Final R indices [I > 2
R indices (all data)
Largest diff. peak and hole/e$A
s
(I)]
R1 ¼ 0.0608, wR2 ¼ 0.1138
R1 ¼ 0.1314, wR2 ¼ 0.1364
0.562 and ꢀ0.348
ꢀꢀ3

M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
2329
A
400
300
200
100
0
Normal rats
Normal rats +BPOV
STZ rats
STZ rats+BPOV 5mg
STZ rats+BPOV 10mg
STZ rats+BPOV 20mg
c
bc
bc
bc
bc
bc
c
c
c
c
bc
c
c
c
ac
c
c
ac
c
c
0
1
2
3
4
WEEK
c
300
200
100
0
B
Normal rats
Normal rats +BPOV
STZ rats
c
ac
c
ac
c
c
ac
c
c
c
c
c
ac
c
c
STZ rats+BPOV 5mg
STZ rats+BPOV 10mg
STZ rats+BPOV 20mg
1
2
3
4
WEEK
C
c
c
1000
750
500
250
0
c
c
ac
Normal rats
Normal rats +BPOV
STZ rats
STZ rats+BPOV 5mg
STZ rats+BPOV 10mg
STZ rats+BPOV 20mg
c
c
c
ac
c
c
ac
c
c
c
ac
1
2
3
4
WEEK
Fig. 1. Effects of complex BPOV on body weight (A), food (B) and water (C) intake in normal and STZ-induced diabetic rats (n ¼ 9e10). The diabetic rats were induced by a single
intraperitoneal injection of STZ 50 mg kgꢀ1. CON: normal control group, 0.9% NaCl solution ig; CON þ BPOV: treated normal group, BPOV 3 10 mg V kgꢀ1 ig; DM: diabetic group, 0.9%
NaCl solution ig; DM þ BPOV L, M, H: treated diabetic group, BPOV 5, 10, 20 mg V kgꢀ1 ig, respectively. Values are mean ꢅ SD. aP < 0.05, bP < 0.01, vs. normal control; cP < 0.01, vs. DM
control (Dunnett's test).
3. Pharmacology
administration of BPOV (20.0 mg V kgꢀ1 ig) significantly reduced
the body weight in the diabetic group, compared with the diabetic
control group (P < 0.05) (Fig. 1).
In present study, the blood glucose level in normal control group
was maintained in the normal range. When normal rats received
3.1. Effects of BPOV on blood glucose in both normal and STZ-
induced diabetic rats
In this experiment, the STZ-diabetic rats exhibited a significant
increase in the fluid and food consumption, compared with the
normal rats. After administration, BPOV markedly reduced the
intake of fluid in STZ-diabetic rats but not in normal rats. The food
intake was transitory decreased in the BPOV-treated STZ-diabetic
rats, compared with the STZ-diabetic rats. The mean body weight in
STZ-diabetic rats was lower than that in normal rats. After
administration of BPOV, the tendency of decrease in the body
weight was observed both in the diabetic and normal rats. 4 Weeks
a daily oral administration of the complex at a dose of
10.0 mg V kgꢀ1 for 4 weeks, the blood glucose level did not decrease
as compared with the untreated normal rats (P > 0.05), suggesting
that BPOV did not affect the blood glucose level of normal rats.
The STZ-diabetic rats exhibited significant hyperglycemia. After
four-weeks administration with the complex, the blood glucose
level was significantly decreased in a dose-dependent manner,
compared with the diabetic control group (P < 0.05, 0.01). It was
suggested that the complex has hypoglycemic effect on STZ-

2330
M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
Table 3
Effects of intragastric BPOV on blood glucose levels in both normal and STZ-diabetic rats.
Group
Dose (mg V kgꢀ1
)
Blood glucose (mmol Lꢀ1
)
0
1
2
3
4 (Week)
Normal rats
Saline
10
Saline
5
10
20
3.97 ꢅ 0.26
4.06 ꢅ 0.46
3.52 ꢅ 0.23
3.56 ꢅ 0.29
3.44 ꢅ 0.33
3.22 ꢅ 0.42
3.98 ꢅ 0.28
3.93 ꢅ 0.33
4.14 ꢅ 0.27
3.79 ꢅ 0.52
Normal rats þ BPOV
STZ-rats
11.25 ꢅ 0.42c
11.67 ꢅ 0.70c
11.85 ꢅ 1.52c
11.24 ꢅ 1.29c
11.52 ꢅ 1.04c
11.30 ꢅ 0.86c
10.51 ꢅ 1.44c
11.05 ꢅ 0.75c
12.48 ꢅ 0.53c
13.41 ꢅ 1.01c
10.66 ꢅ 1.02bc
10.88 ꢅ 1.06bc
12.41 ꢅ 0.88c
12.33 ꢅ 0.91c
11.34 ꢅ 0.64ac
10.25 ꢅ 1.56bc
16.09 ꢅ 1.58c
13.77 ꢅ 1.92c
12.67 ꢅ 1.57bc
11.46 ꢅ 2.43bc
STZ-rats þ BPOV
The diabetic rats were induced by a single intraperitoneal injection of STZ 50 mg kgꢀ1. Data were expressed as mean ꢅ standard deviations for 10 rats in each group. aP < 0.05
or less vs. normal rats; bP < 0.05 or less vs. SZT-diabetic rats (Dunnett's test).
diabetic rats (Table 3). Moreover, it was also suggested that the
hypoglycemic effect was achieved maybe by increasing the sensi-
tivity of insulin and not by enhancing the excretion of insulin.
serum insulin was significantly decreased in the non-diabetic rats
treated with BPOV at the end of experiment. The STZ-induced
diabetic rats exhibited significant hyperglycemia and lower serum
insulin concentration. After administration, BPOV markedly
decreased the blood glucose concentrations in a dose-dependent
manner, compared with diabetic rats (P < 0.05 and 0.01). However,
the serum insulin concentration did not increase along with the
decrease of blood glucose and even decreased in the diabetic rats
treated with BPOV 20.0 mg V kgꢀ1 body weight. Moreover, this
suggested BPOV has insulin-mimetic activities or increased sensi-
tivity of insulin-accepter. This may suggest that BPOV has glucose-
lowing effect because it itself has insulin-mimetic activities or
increased sensitivity of insulin-accepter, which it did not excrete
3.2. Oral glucose tolerance test (OGTT) [34]
OGTT was performed to investigate the effect of BPOV on
glucose tolerance. One hour after treatment with the loaded
glucose, the blood glucose increased to the peak value
(4.86 mmol Lꢀ1) and decreased to the fasting blood glucose level
after 2 h in normal control group. When normal rats received BPOV
at a dose of 10.0 mg V kgꢀ1, the oral glucose tolerance curve was not
significantly changed as compared with normal control group
(Fig. 2). It was suggested that BPOV did not influence glucose
tolerance in normal rats.
In diabetic control group, the blood glucose levels at 0, 15, 30, 60
and 120 min were much higher than that in normal control group,
and reached the peak value with 21.63 mmol Lꢀ1 at 30 min after
glucose loading. Even at 120 min, the blood glucose still was high to
17.97 mmol Lꢀ1. After BPOV administration for 4 weeks, the oral
glucose tolerance curve became flattened in a dose-dependent
manner. BPOV at 10 and 20 mg V kgꢀ1 marked lowered the blood
glucose levels, compared with diabetic group (P < 0.05 or P < 0.01)
(Fig. 3). It showed that the complex could accelerate the glucose
clearance and improve glucose tolerance in STZ-diabetic rats.
insulin via enhancing injured insulin
b cells (Fig. 3.)
Glycosylated hemoglobin was found to increase in patients
with diabetes mellitus and the amount of increase is directly
proportional to blood glucose concentration [35]. Therefore, gly-
cosylated hemoglobin is used to indicate blood glucose concen-
tration of diabetes in a long time (4e10 weeks) and not affected by
the temporary fluctuation of blood glucose concentration.
Administration of BPOV complex could decrease the increased
glycosylated hemoglobin in STZ-diabetic rats, suggesting that the
complex controlled the glycation of hemoglobin. While there was
no changes in BFOV-treated non-diabetic rats. The results further
manifested that BPOV has glucose-lowering activity in diabetic
rats.
Fig. 4 demonstrated the content of glycogen in liver and dia-
phragmatic muscle of experimental rats after 4 weeks adminis-
tration of BPOV. A significant increase was observed in the hepatic
and muscle glycogen of BPOV-treated diabetic rats, but not in
treated non-diabetic rats, compared with diabetic rats. Moreover,
the hepatic glycogen contents in diabetic rats were higher than that
in non-diabetic rats. Thus it suggests that BPOV can regulate serum
lipid and ameliorate metabolism turbulence on the lipid of diabetic
animals. Administration of BPOV increased hepatic and muscle
glycogen contents, which was beneficial to reduce blood glucose
concentration. But it was not clear that this was a result of
enhanced glycogenesis or inhibited glycogenolysis.
3.3. Insulin, glycogen and serum lipid studies
As shown in Fig. 3, normal rats received a daily oral adminis-
tration of BPOV at dose of 10.0 mg V kgꢀ1 for 4 weeks, whereas the
Normal rats
Normal rats +BPOV
STZ rats
STZ rats+BPOV 5mg
STZ rats+BPOV 10mg
STZ rats+BPOV 20mg
30
bc
bc
bc
20
10
bc
bc
bc
c
c
c
c
c
c
c
45
a
c
c
Normal rats
c
c
Normal -rats+ BPOV 10 mg
STZ-rats
a
a
30
a
0
STZ-rats 5 mg
STZ-rats 10 mg
ab
0
30
60
min
90
120
STZ-rats 20 mg
15
Fig. 2. Effect of BPOV on glucose tolerance in normal rats and STZ-induced diabetic
rats. The diabetic rats were induced by a single intraperitoneal injection of STZ
50 mg kgꢀ1. CON: normal control group, 0.9% NaCl solution ig; CON þ BPOV: treated
normal group, BPOV 10 mg V kgꢀ1 ig; DM: diabetic group, 0.9% NaCl solution ig;
DM þ BPOV L, M, H: treated diabetic group, BPOV 5, 10, 20 mg V kgꢀ1 ig, respectively.
n ¼ 9 or 10 rats in each group. Mean ꢅ SD. aP < 0.05, bP < 0.01, vs. DM; cP < 0.01, vs.
CON (Dunnett's test).
0
Fig. 3. Effect of intragastric BPOV for 4 weeks on serum insulin levels in normal rats
and STZ-rats. Normal rats, STZ-rats, BPOV. Values are meanTS.E. for seven rats in each
group. aP < 0.05 vs. Normal rats; bP < 0.05 vs. STZ-rats (ANOVA).

M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
2331
A
ab
40
30
20
10
0
Normal rats
Normal -rats+ BPOV 10 mg
STZ-rats
STZ-rats 5 mg
STZ-rats 10 mg
STZ-rats 20 mg
a
a
a
B
4.0
3.5
3.0
2.5
2.0
1.5
1.0
0.5
0.0
ab
Normal rats
Normal -rats+ BPOV 10 mg
STZ-rats
STZ-rats 5 mg
STZ-rats 10 mg
STZ-rats 20 mg
ab
Fig. 4. Changes of the hepatic (A) and muscle glycogen (B) content in normal rats and STZ-rats. Normal rats, STZ-rats, BPOV. Values are meanTS.E. for seven rats in each group.
aP < 0.05 vs. Normal rats; bP < 0.05 vs. STZ-rats (ANOVA).
Table 4 shows the changes in the concentration of serum lipid of
control and experimental rats. There was a significant increase of
the serum total cholesterol, triglycerides and low-density lip-
oproteinecholesterol in STZ-diabetic rats. Administration of BPOV
to diabetic rats markedly reversed the changes and increased
serum high-density lipoproteinecholesterol concentration. No
significant alterations were observed in BPOV-treated non-diabetic
rats.
Changes in plasma lipid concentration are a frequent compli-
cation in patients with diabetes mellitus [36,37], which contributes
to the development of vascular disease. In the present study, STZ-
diabetic rats exhibited hypertriglyceridemia and hypercholester-
olemia, while BPOV treatment significantly decreased serum total
cholesterol and triglycerides concentration as well as increased
serum high-density lipoproteinecholesterol concentrations in
diabetic rats, suggesting that BPOV could improve the disorder of
lipid metabolism in diabetes.
side, and the intracellular accumulation of BPOV are shown in Table
5. BPOV showed transmembrane permeability of 3.7 ꢃ 10ꢀ5 cm sꢀ1
.
The Papp for BPOV was independent of the corresponding
compound concentrations (Fig. 5). The ratio of Papp in the apical to
basolateral direction (A / B) to the reversed direction (B / A) was
0.5e0.8. BPOV had permeability above 10ꢀ6 cm sꢀ1, suggesting
BPOV has better gastrointestinal stress for oral administration.
The Papp constants for BPOV were found to be concentration
independent (Fig. 5). The permeability of BPOV in the apical to
basolateral (Papp, A to B) was about twice that of the basolateral to
apical directions (Papp, B to A). The experimental results illuminate
that BPOV passes through Caco-2 cell monolayer via the simple
passive diffusion. The proof includes: (i). The rates of BPOV
increased linearly with the concentration and did not show satu-
ration up to 0.5 mM (Fig. 5), indicating that this permeation process
is driven by the concentration gradient. (ii) No efflux was observed
for BPOV. The AP to BL permeability is larger than the reversed
direction. A ratio of w2 for A to B permeability over that of B to A is
unexpected for the simple diffusion process. The reasons might be
the differentiated Caco-2 cells are polarized, the microvilli
membrane side has a relatively larger surface and thus possesses
a much higher binding capacity than the basolateral membrane
side. When B to A transport is carried out, more binding of
3.4. Permeation of vanadium complexes across Caco-2 monolayer
Within 1 h, the vanadium complex BPOV permeated through
the Caco-2 cell monolayer. The apparent permeability coefficients
(Papp), the bidirectional transport between apical and basolateral
Table 4
Effects of 4 weeks of intragastric of BPOV on the concentration of serum lipid in normal rats and STZ-rats.
Treatment
Dose (mg V kgꢀ1 dꢀ1
)
Serum lipid (mmol Lꢀ1
TC
)
TG
LDL
HDL
Normal rats
Saline
10.0
Saline
5.0
10.0
20.0
2.34 ꢅ 0.14
2.50 ꢅ 0.46
2.91 ꢅ 0.36a
3.05 ꢅ 0.48a
2.19 ꢅ 0.25b
2.35 ꢅ 0.33b
0.71 ꢅ 0.12
0.50 ꢅ 0.06a
1.03 ꢅ 0.13a
0.86 ꢅ 0.11b
0.71 ꢅ 0.12b
0.71 ꢅ 0.16b
1.09 ꢅ 0.24
1.20 ꢅ 0.22
1.67 ꢅ 0.33a
1.55 ꢅ 0.41b
1.10 ꢅ 0.14b
0.83 ꢅ 0.12b
0.76 ꢅ 0.15
0.79 ꢅ 0.17
0.66 ꢅ 0.11a
0.66 ꢅ 0.07a
0.80 ꢅ 0.09b
0.85 ꢅ 0.10b
Normal rats þ BPOV
STZ-rats
STZ-rats þ BPOV
Normal control, STZ-rats, BPOVd[N,N0-bis(4-hydroxysalicylidene)-o-phenylene-diamine] oxovanadium(IV). Values are meanTS.E. for seven rats in each group. TCdtotal
cholesterol, TGdtriglycerides, LDHdlow-density lipoproteinecholesterol. HDLdhigh-density lipoproteinecholesterol. a < 0.05 vs. Normal control. b < 0.05 vs. STZ-rats.

2332
M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
Table 5
AP——BL
BL—— AP
The apparent permeability coefficients of BPOV.
a
Complex
Papp (10ꢀ5 cm sꢀ1
)
2.5
A / B (Papp aeb
1.96 ꢅ 0.1
)
B / A (Papp bea
)
Paeb/Pbea
BPOV
0.53 ꢅ 0.1
3.7
2.0
1.5
1.0
0.5
0.0
a
Each data point was the average of three assays.
vanadium complex to the microvilli membrane could result in less
vanadium permeation across monolayer than that in the reverse
transport direction. (iii) The fact that the Papp was independent of
vanadium concentration (Fig. 5) did not support involvement of an
active transport mechanism.
The experimental results above suggested that simple diffusion
could be the major pathway for organic vanadium compounds in
oral administration. It is widely accepted that lipophilic drugs have
better cellular permeability and absorption profiles than hydro-
philic drugs. Therefore, lipophilicity could be one of the major
determining factors for the oral bioavailability of vanadium
compounds. The experimental results indicate that BPOV with
salen-type ligands has good lipophilic properties.
0
100
200
300
400
500
Vanadium concentration (µ M)
Fig. 5. Plot of apparent permeability coefficient, Papp of BPOV vs. the concentration in
the absorptive (APeBL) direction or the exsorptive (BLeAP) direction.
4.2. Spectra
3.5. The cytotoxicity and of vanadium complex BPOV
BPOV exhibits a magnetic moment at room temperature and
110 K in the solid state. With an electron configuration of [Ar]3d1, V
(IV) has one unpaired electron for which the spin-only formula
predicts a magnetic moment of 1.73 BM. BPOV has an effective
magnetic moment (meff) of 1.75 BM at 300 K, and this is a typical
value for a V(IV) complex with S ¼ 1/2. This confirmed that BPOV is
in the mononuclear vanadyl state. The ESR spectrum of BPOV at
room temperature exhibited the characteristric widely spaced
eight-lined pattern due to the coupling of the unpaired electron
with large moment of the ~100% abundant 51V nucleus (I ¼ 7/2),
indicated only one mononuclear vanadium (IV) species predomi-
nates in the complex solution examined.
The cytotoxicity of the vanadium complex BPOV on Caco-2 cells
was measured by decrease of cell viability using the MTT assay as
described in the Experimental protocols Section. The data of cell
viability decrease was fitted to the Hill model and the calculated
IC50 value was 1.52 ꢃ10ꢀ5 mmol Lꢀ1
.
Our data showed that the IC50 value of BPOV was higher to the
neutral charge organic complexes, e.g., bis(maltolato) oxovanadium
([VO(ma)2]) [38]. It suggested BPOV has effect on impairment of
cellular associated with lower level capacity of cellular
accumulation.
In the visible absorption spectrum of the complex, four
absorption bands at 265, 332, 415 and 695 nm were observed.
4. Results and discussion
Absorption bands at 265 and 332 nm can be assigned to
p / p*
Structure of BPOV is based on elemental analyses, spectroscopic
(IR, UV/vis, EPR) data and X-ray diffraction analyses.
transition based on the ligand. Generally, vanadyl complexes with
lower symmetry than C4n usually give rise to three ded bands, due
to the splitting of the degenerate dxz and dyz [44]. Two or three
absorption bands assigned to ded transitions [dxy / (dxz,dyz),
4.1. Single crystal X-ray diffraction studies
dxy / dx2ey2, and dxy / dz2 in the order of decreasing wavelength]
were observed in the visible region [45]. Absorption bands at
695 nm can be assigned to a dxy / (dxz,dyz) transition and the
absorption bands at 415 nm can be assigned to a dxy / dx2ey2
transition. The increase of the ligand field strength is responsible
for a blue shift of the absorption bands. Therefore, the excitation to
the orbital occurs at 350e400 nm and can be masked by intraligand
or charge transfer bands. The IR absorption band due to V]O
The asymmetric unit of BPOV consists of a monomeric vanadium
(IV) complex, two dimethyl sulfoxide (DMSO) molecules and two
water molecules(Fig. 6). The VIV atom is five-coordinate in a dis-
torted square-pyramidal environment. The basal square plane is
constituted by the N,N0-bis(salicylidene)-o-phenylene-diamine
molecule, which acts as a tetradentate ligand through its o-phe-
nylene-diamine N atoms and its deprotonated phenol O atoms. The
V atom is located 0.5997 (2) Å above the mean plane defined by
atoms N1/N2/O1/O3. This distance is very similar to those observed
for VO(acac)2 (0.55 Å) [39] and [VO(acen)] [acen is N,N-ethylenebis
(acetylacetoneiminate); 0.58 Å] [40]. The apical position is occu-
pied by the oxo ligand; the VeOoxo bond distance of 1.592 (3) Å is
typical for five-coordinate vanadyl species. Similar to other VO
(salen) [N,N0-ethylenebis(salicylaldiminato)]oxovanadium(IV)-type
compounds [41,42], the title complex is best described as having
stretching frequency was found at 905 cmꢀ1
.
5. Conclusions
A newly prepared complex [N,N0-bis(4-hydroxysalicylidene)-
o-phenylene-diamine] oxovanadium(IV) (BPOV) is found to be
a potent insulin-enhancing and antidiabetic vanadyl compound.
It was characterized by elemental analysis, IR, UVevis, EPR and X-
ray crystallographic analysis. The VIV atom is five-coordinate in
a distorted square-pyramidal environment. Based on in vivo
evaluations, a 4 weeks administration with BPOV resulted in
a decrease of the blood glucose levels in STZ-diabetic rats.
purely square-pyramidal geometry, because of its
z value of 0.06.
The parameter was introduced by Cornman [43] to measure
z
the distortion of a square-pyramidal structure toward trigonal-
bipyramidal.

M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
2333
Fig. 6. A view of BPOV molecule with the atomic labelling scheme. Displacement ellipsoids are shown at the 30% probability level. The water and DMSO molecules have been
omitted.
Furthermore, OGTT demonstrated that the impaired glucose
tolerance in STZ-diabetic rats was ameliorated by BPOV treat-
ment. As tested on insulin, glycogen and serum lipid studies with
BPOV, BPOV has proved its antidiabetic potency due to its blood
glucose-lowering and glycogenesis actions, its abilities to
improve lipid metabolism. BPOV had permeability above
10ꢀ5 cm sꢀ1. It was suggested BPOV with salen-type ligands has
better gastrointestinal stress for oral administration and good
lipophilic properties. The cytotoxicity of BPOV on Caco-2 cells
was measured by decrease of cell viability using the MTT assay
which implyed salen-type ligands for vanadium complexes have
higher effect on impairment of cellular associated with lower
level capacity of cellular accumulation.
6.2. Chemistry
Elemental analyses were carried out using a Carlo-Erba 1106
Elemental analyzer. IR spectra were recorded on a Nicolet 170SXFT-
IR using a KBr disk technique. UVevis spectra were recorded on
a Shimadzu UV-2000 at room temperature. HPLC spectra were
recorded on a Shimadzu LC-10A at room temperature. Biological
activity experiments were measured on CL-770 Shimadzu Clinic
Spectrometer, BS Semi-Automation Biochemistry analyzer which
was purchased from Beijing Biochem Analyze Ltd., MP4R and
OM8464 US-IEC freezing centrifugation instrument.
6.2.1. Perparation of [N,N0-bis(4-hydroxysalicylidene)-o-phenylene-
diamine] oxovanadium(IV) (BPOV)
A methanol solution (10 mL) of o-phenylene-diamine (0.309 g,
3.0 mmol) was added to a methanol solution (10 mL) of 2,4-dihy-
droxybenzaldehyde (0.828 g, 6.0 mmol) and the resulting mixture
was refluxed for 1 h. The ligands were not isolated from the
methanol solution and the solution mixture was added first to an
aqueous solution of VOSO4$3H2O (0.783 g, 3.0 mmol), then to an
Et3N (0.602 g, 6.0 mmol) solution, and refluxed for 2 h. The green
precipitate was collected by filtration. Crystals of suitable quality
for X-ray analysis were obtained by slow evaporation of a dimethyl
sulfoxide solution. C24H30N2O9S2V, M ¼ 605.56. Anal. Found: C,
47.42; H, 4.57; N, 4.16. Calc.: C, 47.55; H, 4.95; N, 4.65%. UVevis
6. Experimental protocols
6.1. General
Streptozotocin (STZ) was purchased from Sigma (purity ꢁ 98%).
Glucose reagent kit was a product of Shanghai institute of Biological
Products (purity ꢁ 98%).
Caco-2 cells were obtained from Cell Culture Center of Peking
Union Medical Science. Fetal bovine serum and non-essential
amino acid mixture were purchased from Hyclone. Dulbecco_s
modified eagle medium (DMEM, high glucose) was purchased from
Gibco. Hank's balance salt solution (HBSS), Earlier's balance salt
solution (EBSS), phosphate buffer saline (PBS), MTT, N-2-hydroxy-
ethylpiperazine-ethylpiperazine-N0-2-ethanesulfonic acid (HEPES),
(DMSO) lmax ¼ 265 (
e
¼ 2136 Mꢀ1 cmꢀ1), 332 (
e
¼ 1683 Mꢀ1 cmꢀ1),
415 (
e
¼ 43 Mꢀ1 cmꢀ1) nm, 695 (
e
¼ 31 Mꢀ1 cmꢀ1) nm. IR (KBr disk):
n
(cmꢀ1) ¼ 905(nv]o), 523nveo). ESR (RT, DMSO): 8-Line pattern, g
0 ¼ 2.01 ꢅ0.001, A 0 ¼ 101.6 ꢅ 0.1 ꢃ10ꢀ4 cmꢀ1. Magnetic moment
(solid): 1.75 BM (one unpaired electron). Purity: ꢁ96% (HPLC: used
a C18 column, methanolewater (5/95,V/V) as the mobile phase at
25 ꢂC with detection wavelength at 210 nm).
20,70-dichlorofluorescein
diacetate
(DCFH-DA),
ethyl-
enediaminetetra-acetate (EDTA), and trypsin were purchased from
Sigma. Transwells were purchased from Corning Costar (Cam-
bridge, MA, USA). Solutions of BPOV were freshly prepared by
dissolving the compounds in HBSS (pH 7.2) or EBSS (pH 6.5) buffer
to 1.0 mM and immediately diluting to the desired concentrations.
DCFH-DA solution was prepared by dissolving in dimethyl sulfoxide
(DMSO) and diluting with PBS buffer to the final concentration of
6.3. Glucose-lowering studies
Male Sprague-Dawley rats weighing 200e250 g were obtained
from Experimental Animal Center, Kunming Medical Collage
(Grade II, Certificate number 2001034). Rats were allowed free
10
mM. MTT was also prepared in DMSO at the working concen-
tration of 0.5 mg mlꢀ1
.

2334
M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
access to standard solid food for laboratory animals and tap water.
Diabetes was induced by a single intraperitoneal injection of STZ
50 mg kgꢀ1 in 0.1 mol Lꢀ1 citrate buffer (pH 4.4). Seven days after
STZ injection, blood samples for analysis of blood glucose were
obtained from the tail vein of the rats and the blood glucose was
measured by the glucose oxidase method. The STZ-rats with the
blood glucose level S11.1 mmol Lꢀ1 were considered as diabetic
rats. Normal rats were injected with 0.1 M citrate buffer alone.
Rats,1 week after STZ treatment, were used for the experiments.
STZ-rats were allowed free access to standard solid food for labo-
ratory animals and tap water. Body weights of STZ-rats were
measured weekly during the experiments. Intakes of solid food and
drinking water in each rat were checked every day throughout the
experiments. At concentration 5, 10, 20 mg V kgꢀ1, BPOV was dis-
solved in water (solution were prepared fresh daily) and adminis-
tered to STZ-rats for 4 weeks by ig. During the experiments, serum
insulin of rats treated by BPOV was measured at 9:00 a.m. daily.
The experimental animals were randomly divided into six
groups with ten rats each according to the blood glucose. Normal
control group: normal rats treated with 0.9% saline; treated normal
group: normal rats treated with 10 mg V kgꢀ1 BPOV; diabetic
control group: STZ-diabetic rats treated with 0.9% saline; treated
diabetic group: STZ-diabetic rats treated with BPOV 5, 10,
20 mg V kgꢀ1 ig. The substances above were administered intra-
gastrically once a day at the volume of 10 mL kgꢀ1 for 2 weeks. Once
per week the blood samples were collected from the tail vein of the
rats for the blood glucose assay.
reach confluence and differentiation. The trans-epithelial electric
resistance (TEER) of the monolayer was measured with a Millicell
electrode resistance system (Millipore) and TEER values of
>700
U
cm2 were routinely observed.
6.7. Transport of complex BPOV across caco-2 monolayer
The filter inserts upon which caco-2 monolayer grew were
rinsed twice with HBSS (pH 7.4). Transport experiment was initi-
ated by adding 0e500 mM of the vanadium complexes to either the
apical side (vanadium compound dissolved in EBSS, pH 6.0) or the
basolateral side (vanadium compound dissolved in HBSS, pH 7.4).
Then the samples were shaken (37 ꢂC 50 rpm) and 100
mL aliquots
were taken from the receiver side at desired time intervals of 0e1 h.
Samples were determined by inductively coupled plasma mass
spectrum (ICP-MS). The apparent permeability coefficient (Papp
)
across the caco-2 cell monolayers was calculated with the following
equation: Papp ¼ ðdQ=dtÞ=ðA$C0Þ;
where dQ/dt is the flux of drug across the monolayer (mol
transported/s), A (cm2) is the surface area of the inserts, and C0 (M)
is the initial drug concentration in the donor compartment. To
measure the cellular uptake of vanadium complex in the Caco-2
monolayer, the Caco-2 cells were washed three times with 1 mL of
ice-cold 5 mM of EDTA/PBS (pH 7.2) solution after the transport
studies described above to remove residual medium and surface-
bound metal ions. Then the cells were collected and completely
digested with 0.5 mL ultrapure nitric acid. The amount of vanadium
in the collected samples was determined with ICP-MS (Leeman
Labs Inc., USA). To investigate the effects of ion pair reagents, trie-
thanolamine hydrochloride or tetrabutylammonium chloride, was
added to a final concentration of 1.0 mM at the donor side of the
transwell. Then the transport experiments were carried out as
described above.
6.4. Oral glucose tolerance test [34]
Normal rats and STZ-diabetic rats were prepared and randomly
divided into six groups as described in Glucose-Lowering Studies.
After the drugs were administered intragastrically in both normal
and STZ-induced diabetic rats for 4 weeks, Oral Glucose Tolerance
Test was undertaken. The experimental rats were fasted for 14 h
and given an oral glucose challenge at a dose of 2.0 g kgꢀ1 body
weight [34]. Blood glucose levels were measured at 0, 15, 30, 60,
120 min after glucose loading.
6.7.1. Measurement of TEER (trans-epithelial electrical resistance)
of Caco-2 cell monolayer
The Caco-2 monolayer was incubated with 0e0.5 mM of vana-
dium complex in EBSS on the apical side for 60 min at 37 ꢂC. Then
the sample was shaken at 50 rpm. The integrity of the monolayer
was monitored by measuring the trans-epithelial electrical resis-
tance (TEER) using an epithelial voltohmmeter (World Precision
Instrument, Sarasota, FL, USA).
6.5. Insulin, glycogen and serum lipid studies
STZ-rats were prepared and randomly divided into as described
as above. After drug administered to STZ-rats for 4 weeks, blood
was collected from the tail vein of the rats following fasting 12 h.
Then centrifuged (3000 rpm) for 10 min, the blood serum were
collected into refrigeratory at ꢀ20 ꢂC. The insulin was measured by
125I radioimmunoassay. Cholesterol (CHO) and Triglyceride (TG)
were measured by enzyme colorimetry .The high-density lipid
(HDL) and the low-density lipid (LDL) were measured by poly-
ethylene sulfuric acid precipitation method and phospho-tungstic
acid magnesium precipitation method, respectively. The result was
6.8. Cytotoxicity assay
The Caco-2 cells were seeded on 96-well microplate at
5 ꢃ104 cells/well and cultured for 48 h. The cells were washed with
DMEM once, then incubated with various concentrations of vana-
dium complexes from 0 to 2.7 mM in 100
Afterwards, the medium was removed and 100
(0.5 mg mlꢀ1 in PBS) was added to each well and incubated for 4 h.
The MTT medium was subsequently removed and 200 L DMSO
mL DMEM for 24 h.
mL
MTT
done by student
s
test.
m
was added to dissolve the formazan formed. The absorption at 570
and 630 nm was measured 15 min later on plate reader (TECAN
SUNRISE, Switzerland). The 50% inhibition concentration (IC50) was
calculated by plotting the cell viability vs. vanadium concentrations
and fitted to the Hill model using a MicroCale Origin program (Lab
Corporation, USA).
6.6. Cell culture and monolayer preparation
Caco-2 cells were cultured in Dulbecco's Modified Eagle's
Medium (DMEM) containing 4.5 gꢀl -glucose and 3.7 gꢀl NaHCO3,
D
supplemented with 10% Fetal bovine serum, 1% non-essential
amino acids, 100 U mlꢀ1 penicillin and 100 g mLꢀ1 streptomycin
m
[46]. The cells were incubated at 37 ꢂC in a humidified atmosphere
of 5% CO2. Cells between passage 45 and 60 were used in the
studies. The cell monolayer was prepared as the method described
previously [46]. Briefly, Caco-2 cells were seeded on Transwell
6.9. Statistical analysis
Statistics values were presented as means ꢅ standard devia-
tions. Statistical analysis was performed by one-way analysis of
variance followed by Dunnett's multiple comparison tests using
Prism version 4.0 software.
insert filter (surface area ¼ 1.0 cm2, pore size ¼ 3.0
mm) at a density
of about 1 ꢃ105 cells cmꢀ2. Cells were left to grow for 21 days to

M.-j. Xie et al. / European Journal of Medicinal Chemistry 45 (2010) 2327e2335
2335
[19] S. Dai, K.H. Thompson, J.H. McNeill, Pharmacol. Toxicol. 74 (1994) 101e109.
[20] H. Sakurai, K. Fujii, H. Watanabe, H. Tamura, Biochem. Biophys. Res. Commun.
214 (1995) 1095e1101.
Acknowledgements
This work was supported by National Natural Science Founda-
tion of China (30260118) and Yunnan Natural Science Foundation
(2002C0019R) and (2009CD006) and Yunnan University Science
Foundation (2005Q002A).
[21] K.H. Thompson, J.H. McNeill, C. Orvig, Chem. Rev. 99 (1999) 2561e2572.
[22] H. Yasui, A. Tamura, T. Takino, H. Sakurai, J. Inorg. Biochem. 91 (2002)
327e328.
[23] K. Fukui, Y. Fujisawa, H. Ohya-Nishiguchi, H. Kamada, H. Sakurai, J. Inorg.
Biochem. 77 (1999) 215e224.
[24] C. Orvig, K.H. Thompson, M. Battell, J.H. McNeill, Met. Ions Biol. Syst. 31 (1995)
575e594.
[25] P. Caravan, L. Gelmini, N. Glover, F.G. Herring, H. Li, J.H. McNeill, S.J. Rettig, I.
A. Setyawati, E. Shuter, Y. Sun, A.S. Tracey, V.G. Yuen, C. Orvig, J. Am. Chem. Soc.
117 (1995) 12759e12770.
[26] V.G. Yuen, C. Orvig, J.H. McNeill, Can. J. Physiol. Pharmacol. 71 (1993) 263e269.
[27] K.H. Thompson, V.G. Yuen, J.H. McNeill, C. Orvig, Chemical and pharmaco-
logical studies of a new class of antidiabetic vanadium complexes. in: A.
S. Tracey, D.C. Crans (Eds.), Vanadium Compounds. Chemistry, Biochemistry,
and Therapeutic Applications, ACS Symposium Series, vol. 711. American
Chemical Society, Washington, DC, 1998, pp. 329e343.
[28] K.H. Thompson, C. Orvig, Dalton Trans. (2006) 761e764.
[29] N. Durai, G. Saminathan, J. Clin. Biochem. Nutr. 22 (1997) 31e39.
[30] I.J. Hidalgo, T.J. Raub, R.T. Borchardt, Gastroenterology 96 (1989) 736e749.
[31] S. Yamashita, T. Furubayashi, M. Kataoka, T. Sakane, H. Sezaki, H. Tokuda, Eur. J.
Pharm. Sci. 10 (2000) 195e204.
[32] G.M. Sheldrick, SHELXS97 and SHELXL97. University of Gottingen, Germany,
1997.
[33] G.M. Sheldrick, SHELXTL. Version 6.10. Bruker AXS Inc., Madison, Wisconsin,
USA, 2000.
[34] P. Basnet, S. Kadota, M. Shimizu, Y. Takata, M. Kobayashi, T. Namba, Planta
Med. 61 (1995) 402e405.
[35] R.J. Koenig, C.M. Peterson, R.L. Jones, C. Saudek, M. Lehrman, A. Cerami, N.
Engl. J. Med. 295 (1976) 417e420.
Appendix. Supplementary data
Supplementary data associated with this article can be found in
the online version, at doi:10.1016/j.ejmech.2010.02.010.
References
[1] D.C. Crans, J.J. Smee, E. Gaidamauskas, L. Yang, Chem. Rev. 104 (2004) 849e902.
[2] D. Rehder, Inorg. Chem. Commun. 6 (2003) 694e697.
[3] A.K. Srivastava, M.Z. Mehdi, Diabet. Med 22 (2004) 2e13.
[4] H. Sakurai, K. Tsuchiya, M. Nukatsuka, M. Sofue, J. Kawada, J. Endocrinol. 126
(1990) 451e459.
[5] J.H. McNeil, V.G. Yuen, H.R. Hoveyda, C. Orvig, J. Med. Chem. 35 (1992)
1489e1491.
[6] V.G. Yuen, P. Caravan, L. Gelmini, N. Glover, J.H. McNeill, I.A. Setyawati, Y. Zhou,
C. Orvig, J. Inorg. Biochem. 68 (1997) 109e116.
[7] B.A. Reul, S.S. Amin, J.-P. Buchet, L.N. Ongemba, D.C. Crans, S.M. Brichard, Br. J.
Pharmacol. 126 (1999) 467e477.
[8] D.C. Crans, L. Yang, J.A. Alfano, L.-H. Chi, W. Jin, M. Mahroof-Tahir, K. Robbins,
M.M. Toloue, L.K. Chan, A.J. Plante, R.Z. Grayson, G.R. Willsky, Coord. Chem.
Rev. 237 (2003) 13e22.
[36] B.V. Howard, P.J. Savage, L.J. Bennion, P.H. Bennett, Atherosclerosis 30 (1978)
153e162.
[9] G. Boden, X. Chen, J. Ruiz, G.D. van Rossum, S. Turco, Metabolism 4 (5) (1996)
1130e1135.
[37] E.A. Nikkila, M. Kekki, Metabolism 22 (1973) 1e22.
[38] X.G. Yang, X.D. Yang, L. Yuan, K. Wang, D.C. Crans, Pharm. Res. 21 (2004)
1026e1033.
[39] R.P. Dodge, D.H. Templeton, A. Zalkin, J. Chem. Phys. 35 (1961) 55e67.
[40] D. Bruins, D.L. Weaver, Inorg. Chem. 9 (1970) 130e135.
[41] .M. Pasquali, Marchetti, C. Floriani, M. Cesari, Inorg. Chem. 19 (1980) 1198.
[42] P.E. Riley, V.L. Pecoraro, C.J. Carrano, J.A. Bonadies, K.N. Raymond, Inorg. Chem.
25 (1986) 154e160.
[10] M. Halberstam, N. Cohen, P. Shlimovich, L. Rossetti, H. Shamoon, Diabetes 45
(1996) 659e666.
[11] A.K. Goldfine, M.-E. Patti, L. Zuberi, B.J. Goldstein, R. LeBlanc, E.J. Landaker, Z.
Y. Jiang, G.R. Willsky, C.R. Kahn, Metabolism 49 (2000) 400e410.
[12] V.G. Yuen, E. Vera, M.L. Battell, W.M. Li, J.H. McNeill, Diabetes Res. Clin. Pract.
43 (1999) 9e19.
[13] J. Meyerovitch, P. Rothenberg, Y. Shechter, S. Bonner-Weir, C.R. Kahn, J. Clin.
Invest. 87 (1991) 1286e1294.
[14] M. Bendayan, D. Gingras, Diabetologia 32 (1989) 561e567.
[15] R.A. Pederson, S. Ramanadham, A.M. Buchan, J.H. McNeill, Diabetes 38 (1989)
1390e1395.
[43] C.R. Cornman, K.M. Geisre-Bush, S.R. Rowley, P.D. Boyle, Inorg. Chem. 36
(1997) 6401e6408.
[44] N.D. Chasteen, R.L. Belford, I.C. Paul, Inorg. Chem. 8 (1969) 408e418.
[45] C.J. Ballhausen, H.B. Gray, Inorg. Chem. 1 (1962) 111e122.
[46] J. Gao, E.D. Hugger, M.S. Bech-Westermeyer, R.T. Borchardt, in: S.J. Enna,
M. Williams, J.W. Ferkany, T. Kenakin, R.D. Porsolt, J.P. Sullivan (Eds.), Current
Protocols in Pharmacology, Wiley, New York, 2000, pp. 7.2.1e7.2.23.
[16] S.M. Brichard, C.J. Bailey, J.-C. Henquin, Diabetes 39 (1990) 1326e1332.
[17] S.M. Brichard, A.M. Pottier, J.C. Henquin, Endocrinology 125 (1989) 2510e2516.
[18] K.H. Thompson, J.H. McNeill, Res. Commun. Chem. Pathol. Pharmacol. 80
(1993) 187e200.