Synthesis, structure, and physiological effects of peroxovanadium(V) complexes containing amino acid derivatives as ancillary ligands

Article abstract of DOI:10.1246/cl.2012.377

We have synthesized and structurally characterized novel peroxovanadium(V) compounds containing amino acid derivatives as ancillary ligands; N-(4-imidazolylmethyl)-L-alanate (imala) and N-(4-imidazolylmethyl)-L- phenylalanate (imphe). The structure of the imala complex was determined using X-ray crystallography. These compounds stimulated the proliferation of H4IIEC3 rat hepatoma cells at low doses but were cytotoxic at high doses. They also exhibited insulin-mimetic effects.

Full text of DOI:10.1246/cl.2012.377

doi:10.1246/cl.2012.377
Published on the web March 24, 2012
377
Synthesis, Structure, and Physiological Effects of Peroxovanadium(V) Complexes
Containing Amino Acid Derivatives as Ancillary Ligands
1
2,3
4
5
Hironori Sugiyama, Seiichi Matsugo,* Tetsuya Konishi, Toshinari Takamura,
5
1
1
1
Shuichi Kaneko, Yukari Kubo, Kyouhei Sato, and Kan Kanamori*
Advanced Nano-BioScience, Graduate School of Innovative Life Science, University of Toyama,
190 Gofuku, Toyama 930-8555
1
3
2
School of Natural System, College of Science and Engineering, Kanazawa University,
Kakuma-machi, Kanazawa, Ishikawa 920-1192
CREST, Japan Science and Technology Agency (JST), Chiyoda-ku, Tokyo 102-0075
3
4
Niigata University of Pharmacy and Applied Life Sciences, 265-1 Higashijima, Akiha-ku, Niigata 956-8603
5
Department of Disease Control and Homeostasis, Graduate School of Medical Science,
Kanazawa University, 13-1 Takara-machi, Kanazawa, Ishikawa 920-8641
(
Received December 22, 2011; CL-111221; E-mail: kanamori@sci.u-toyama.ac.jp)
We have synthesized and structurally characterized novel
peroxovanadium(V) compounds containing amino acid deriva-
tives as ancillary ligands; N-(4-imidazolylmethyl)-L-alanate
(
imala) and N-(4-imidazolylmethyl)-L-phenylalanate (imphe).
The structure of the imala complex was determined using X-ray
crystallography. These compounds stimulated the proliferation
of H4IIEC3 rat hepatoma cells at low doses but were cytotoxic
at high doses. They also exhibited insulin-mimetic effects.
Figure 1. Structures of the amino acid derivative ligands imala
(
N-(4-imidazolylmethyl)-L-alanate; left) and imphe (N-(4-imi-
dazolylmethyl)-L-phenylalanate; right).
Vanadium forms a variety of peroxovanadium(V) com-
plexes (pVs) of chemical interest. In addition, the physiological
properties of these complexes have attracted much recent
attention because they have been found to have antitumor and
insulin-mimetic effects in higher animals.1 The mechanisms
underlying the pV insulin-mimetic effect have been previously
2
investigated; activation of the tyrosine kinase activity of the
3
insulin receptor (IR) appears to be responsible for the effect.
Various ancillary ligands have been combined with pVs to create
compounds with insulin-mimetic activity; however, the relation-
ship between pV ligand structure and the physiologic properties
of the pV compound is unclear. Furthermore it would be likely
that non-peroxo vanadium complexes in the cell might react
with reactive oxygen species such as peroxide. Thus, the study
of peroxovanadium complexes in vitro will provide an insight
into the metabolic pathways of vanadium complexes adminis-
trated in vivo.
Figure 2. ORTEP drawing of pV(imala).
Future development of pV compounds for use in clinical
applications will require further investigation into the ligand
property­activity relationship as it relates to the mechanism of
the insulin-mimetic effects. Ancillary ligands commonly used in
previous studies of stable pV compounds include 2,2¤-bipyridine
yl)-L-phenylalanate (imphe) (Figure 1). After synthesizing and
structurally characterizing these pV compounds [pV(imala) and
pV(imphe), respectively], we investigated their physiologic
effects in H4IIEC3 rat hepatoma cells.
The pV(imala) and pV(imphe) compounds were prepared
4
5
6
9
(
bpy), 1,10-phenanthroline (phen), 2-picolinate (pic), and 2,6-
according to standard methods. The structure of pV(imala) was
7
10
dipicolinate (dipic). Typically, these pV compounds exhibit
high cytotoxicity. The high cytotoxicity, which may be related
determined using X-ray crystallography; a molecular model of
8
this compound is shown in Figure 2. The vanadium atom in this
compound is seen to adopt a heptacoordinate, pentagonal-
bipyramidal structure that is typical for a monoperoxovana-
dium(V) compound with a heteroligand. Although we were
unable to obtain crystals of pV(imphe), its structure can be
reasonably assumed to be similar to that of pV(imala), as the
imphe and imala backbones are identical.
to the use of ligands that do not resemble biological substances,
poses a significant barrier to the use of pV compounds in
curative medicine.
In the present study, we attempted to decrease the
cytotoxicity of pV compounds by replacing the above non-
biological ancillary ligands with amino acid derivatives. These
derivatives, which differ only in their side arms, were N-(4-
imidazolylmethyl)-L-alanate (imala) and N-(4-imidazolylmeth-
We used the MTT method to evaluate the cytotoxic activity
11
of pV(imala) and pV(imphe) in H4IIEC3 rat hepatoma cells.
Chem. Lett. 2012, 41, 377­379
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

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78
Table 1. Results of the cytotoxic examination
Cell viability control/%
Dose
¯mol L
¹1
[VO(O2)(imala)(H2O)]¢
H2O
[VO(O2)(imphe)]¢
/
2
0.5H2O
3
0
0
00
00
00
00
00
108.09
112.43
111.14
104.73
92.16
93.75
10.71
1.23
106.53
112.54
104.14
93.64
28.38
2.09
1
3
1
2
3
5
8
0.81
0.93
Since the present peroxovanadium complexes are fairly stable at
physiological pH at least in vitro, we believe that they have
sufficient lifetimes to interact with some biological materials. As
summarized in Table 1, low-dose treatment of the cells with
pV(imala) or pV(imphe) stimulated cell proliferation, with the
number of cells reaching its maximum value for both com-
pounds at a concentration of 10 ¯M. Both compounds exhibited
significant cytotoxicity at higher doses, although their cytotox-
icity thresholds differed. Whereas pV(imphe) treatment at
Figure 3. Effects of pV(imala) and pV(imphe) on insulin-
related signaling. Cells were grown in 12-well plates until they
reached 90­100% confluence, starved for 1 h, and exposed to
pV(imala), pV(imphe), insulin (positive control), or vehicle
200 ¯M reduced cell viability by approximately 28%, pV(imala)
treatment at 200 ¯M only slightly reduced cell viability. No
significant reduction of the cell viability (11%) was observed for
pV(imala) until it reached a concentration of 500 ¯M. Notably,
this large difference in cytotoxicity was caused by a small
modification of the amino acid ligand: the substitution of a
methyl group [in pV(imala)] for a phenyl group [in pV(imphe)].
The reason for this large difference is unclear at present, but
lipophilicity is likely to be an important factor; the higher
lipophilicity of the phenyl group would facilitate passage of
pV(imphe) through the cell membrane.
(
negative control). After 30 min, the cells were harvested in lysis
buffer and sonicated. Equal amounts of total protein were
resolved by sodium dodecyl sulfate­polyacrylamide gel electro-
phoresis and transferred to poly(vinylidene difluoride) mem-
branes for immunoblot analysis. Blots were visualized with GE
ECL Plus western blotting detection reagent according to the
manufacturer’s instructions.
The physiologic effects of some pV compounds have been
investigated in other cell lines.1
2­15
In RINm5F rat pancreatic
rylation of several cytosolic IR substrates, and the activation of
two key signaling pathways. One of these pathways exerts
mitogenic and growth-promoting effects via phosphorylation of
extracellular signal-regulated protein kinase 1/2 (ERK1/2). The
second pathway, which involves phosphorylation of AKT
(protein kinase B), affects glucose metabolism and protein
synthesis. The multiple physiologic effects of insulin are thus
mediated by ERK1/2 and AKT phosphorylation of various
factors.
¹
cancer cells, [VO(O2)2(1,10-phen)] stimulates cell proliferation
at low doses but is cytotoxic at higher doses, like pV(imala) and
pV(imphe) in our case. Exposure of the RINm5F cells to 1 or
¯M [VO(O2)2(1,10-phen)] for 48 h increased cell viability by
approximately 15% and 20%, respectively. On the other hand,
treatment at 100 ¯M reduced cell viability by approximately
0%. A similar phenomenon has also been observed in PC12
¹
3
14
1
5
9
13
rat adrenal pheochromocytoma cells.
At low doses, the effects of diperoxo(1,10-phenanthro-
To further examine the possibility that low-cytotoxicity pV
compounds might be used at low doses in the treatment
of diabetes, we investigated the effects of pV(imala) and
pV(imphe) on insulin-related intracellular signaling. Specifi-
cally, we examined the effects of these compounds on the
phosphorylation of AKT and ERK1/2 in H4IIEC3 cells using
western blotting (Figure 3). Cells treated with insulin or vehicle
were used as positive and negative controls, respectively. As
shown in Figure 3, exposure of the cells to 30 or 100 ¯M
pV(imala) and pV(imphe) induced the phosphorylation of both
AKT and ERK1/2 in a dose-dependent manner, demonstrating
that both compounds activate insulin signaling pathways in
H4IIEC3 cells.
line)oxovanadate(V) [bpV(phen)] on cell growth are similar to
those of pV(imala) and pV(imphe).1
3,14
However, the concen-
tration at which bpV(phen) begins to inhibit cell viability is
approximately 1/10 that of pV(imala) and pV(imphe). Thus, the
latter compounds are much less cytotoxic than bpV(phen),
possibly because their ligands are derived from biological
substances. Thus, unlike bpV(phen), pV(imala), and pV(imphe)
might be therapeutically effective for treatment of diabetes at a
dose that is only weakly cytotoxic.
The effects of insulin on target cells are initiated by the
binding of insulin to the insulin receptor (IR) on cell
1
6,17
membranes.
This binding enhances the intrinsic protein
tyrosine kinase activity of the IR ¢-subunit, leading to
autophosphorylation of the ¢-subunit, the subsequent phospho-
It was considered that pV(imala) and pV(imphe) induced the
phosphorylation of the insulin receptor which is the upstream
Chem. Lett. 2012, 41, 377­379
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/

3
79
of AKT and ERK1/2. On the other hand it was reported that
the vanadate inhibits the protein tyrosine phosphatase 1B
1 mL of water. The mixture was stirred while heating. After
the resulting clear solution was cooled in an ice bath,
Himala¢2HCl¢EtOH¢1.5H2O (0.63 g, 2.0 mmol) was added
and the solution was stirred for 1 h. Then, 10% hydrogen
peroxide (0.68 g, 2.0 mmol) was added slowly to the
solution. The pH of the resulting red solution was adjusted
to 4.3 by the addition of 1 M hydrochloric acid. Storage of
this solution in a refrigerator for 2 days resulted in the
deposition of red columnar crystals. Yield: 66% (0.21 g)
Anal. Calcd for [VO(O )(imala)(H O)]¢2H O = C H -
1
8
(PTP1B). We have clarified the dual effects of hydrogen
peroxide (ROS) on the insulin signaling and also examined the
vanadate effect on insulin signaling. It was found that the
enhanced insulin signaling (pAKT activation and PTP1B
inhibition) was observed when vanadate and low concentration
of hydrogen peroxide were coadministered.18 Since pV(imala)
and pV(imphe) consist of pentavalent vanadium atom as in
vanadate and peroxide, they are reasonably considered to exhibit
similar effects to the above case. Therefore the insulin signaling
was transmitted in the cells.
The phosphorylation-inducing effect of pV(imala) and
pV(imphe) on the AKT substrate was weaker than that of
insulin, whereas their phosphorylation-inducing effect on the
ERK1/2 substrate was stronger than that of insulin. This
observation indicates that pV compounds are more potent than
insulin as promoters of cell differentiation and cell growth, thus
raising the possibility that they also pose an increased risk of
carcinogenesis relative to insulin. However, the extent of AKT
and ERK1/2 phosphorylation induced by treatment of the cells
with the pV compounds at 3 ¯M was less than that induced by
insulin treatment, indicating that the insulin-mimetic effects of
the pV compounds was dependent on their concentration.
2
2
2
7
16
N3O8V: C, 26.18; H, 5.03; N, 13.09%. Found: C, 25.64;
H, 4.84; N, 12.68%. The pV(imphe) compound was
prepared by a similar method. Yield: 39% Anal. Calcd for
[VO(O2)(imphe)]¢0.5H2O = C13H15N3O5.5V: C, 44.33; H,
4.29; N, 11.93%. Found: C, 44.49; H, 4.45; N, 12.04%.
10 X-ray structure data: Rigaku AFC7R diffractometer, ½
scans, Mo K¡ radiation (­ = 0.71070 ¡), graphite mono-
chrometer, T = 200(2) K, structure solution with direct
method (SHELX97: G. M. Sheldrick, Program for Crystal
Structure Solution from Diffraction Data, University of
Göttingen, Göttingen, Germany, 1997), refinement against
2
F
for all non-hydrogen atoms. Data collection: crystal
3
dimension 0.50 © 0.10 © 0.10 mm , monoclinic, space
group C2 (No. 5), a = 24.001(3), b = 6.351(2), c =
3
8
.449(3) ¡, ¢ = 100.78(2)°, V = 1265.1(6) ¡ , Z = 4,
Dcalcd = 1.686 g cm , ® = 0.825 mm¹1, F(000) = 664, data
collected 1609, unique data 1572 (Rint = 0.012), 199 refine
parameters, GOF = 1.077, final R indices (I > 2·(I)) (R1 =
­««Fo« ¹ «Fc««/­«Fo«, wR2 = [­w(Fo ¹ Fc )/­w(Fo )]1/2):
R = 0.0248, wR = 0.0664. The crystallographic data have
¹3
In this study, we synthesized and structurally characterized
novel pV compounds containing imala and imphe as ancillary
ligands. An assessment of their physiologic effects in vitro
demonstrated low toxicity and insulin-mimetic activity at
concentrations less than 100 ¯M. Therefore, these compounds
are expected to be useful in the treatment of diabetes at low
doses.
2
2
2
1
2
been deposited with the Cambridge Crystallographic Data
Centre (no. CCDC-855902). Copies of the data can be
obtained free of charge on application to CCDC, 12, Union
Road, Cambridge, CB2 1EZ, U.K. (fax: +44 1223 336033);
e-mail: deposit@ccdc.cam.ac.uk.
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measured at 595 nm by a microplate reader. The cell viability
was calculated by normalizing the absorbance to the
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Imala and imphe were prepared by a reaction of alanine and
phenylalanine, respectively, with 4-imidazolecarbaldehyde
using NaBH4 as a reductant. For synthesis of pV(imala),
2
.0 mL of 4 M potassium hydroxide (2.0 mmol) was added to
a suspension of vanadium pentoxide (0.18 g, 1.0 mmol) in
Chem. Lett. 2012, 41, 377­379
© 2012 The Chemical Society of Japan
www.csj.jp/journals/chem-lett/