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Journal of Inorganic Biochemistry 76 (1999) 251–257

Vanadyl–biguanide complexes as potential synergistic insulin mimics

Lenny C.Y. Woo a, Violet G. Yuen b, Katherine H. Thompson a, John H. McNeill b,*,
Chris Orvig 1,a
a
Department of Chemistry, University of British Columbia, Vancouver, BC, V6T 1Z1, Canada
b
Faculty of Pharmaceutical Sciences, University of British Columbia, Vancouver, BC, V6T 1Z3, Canada

Received 28 April 1999; received in revised form 17 August 1999; accepted 23 August 1999

Abstract

Vanadium has well-documented blood-glucose-lowering properties both in vitro and in vivo. The design of new oxovanadium(IV)
coordination compounds, intended for use as insulin-enhancing agents in the treatment of diabetes mellitus, can potentially benefit from a
synergistic approach, in which the whole complex has more than an additive effect from its component parts. Biguanides, most importantly
metformin, are oral hypoglycemic agents used today to treat type 2 diabetes mellitus. In this study, biguanide, metformin, and phenformin,
all biguanides, were coordinated to oxovanadium(IV) to form potential insulin-enhancing compounds. Highly colored, air-stable,
bis(biguanidato)oxovanadium(IV), [VO(big)2], bis(N9,N9-dimethylbiguanidato)oxovanadium(IV), [VO(metf)2], and bis(b-phenethyl-
biguanidato)oxovanadium(IV), [VO(phenf)2], were prepared. Solvation with dimethylsulfoxide occurred with VO(metf)2 to form a six-
coordinate complex. Precursor ligands and oxovanadium(IV) coordination complexes were characterized by infrared spectroscopy, mass
spectrometry, elemental analyses, magnetic susceptibility, and, where appropriate, 1H NMR spectroscopy. Biological testing with VO(metf)2,
a representative compound, for insulin-enhancing potential included acute (72 h) administration, both by intraperitoneal (i.p.) injection and
by oral gavage (p.o.) in streptozotocin (STZ)-diabetic rats. VO(metf)2 administration resulted in significant blood-glucose lowering at doses
of 0.12 mmol kgy1 i.p. and 0.60 mmol kgy1 p.o. (previously established as ED50 doses for organically chelated oxovanadium(IV) complexes);
however, no positive associative effects due to the presence of biguanide in the complex were apparent. q1999 Elsevier Science Inc. All
rights reserved.

Keywords: Vanadium; Biguanide; Insulin mimic; Coordination complex

1. Introduction control with available therapies continues to be an elusive

Diabetes mellitus is a heterogeneous disorder, which
afflicts over 140 million people worldwide and can lead to
cardiovascular disease, blindness, kidney disease, and death.
It is characterized by hyperglycemia, alterations in carbohy-
drate and lipid metabolism, and vascular and neurological
complications. An underlying insufficiency of insulin results
from either inadequate pancreatic production (type 1 diabe-
tes) [1], or defective insulin utilization (type 2 diabetes)
[2]. This insufficiency can be partly corrected therapeuti-
cally, either by daily subcutaneous injections of insulin (most
common for type 1); by administration of one or more of the
currently available oral hypoglycemic agents (type 2); or,
not infrequently, by a combination therapy. Good diabetic
* Corresponding author. Phone: q1-604-822-9373; fax: q1-604-822-
8001; e-mail: jmcneill@unixg.ubc.ca
1
Also corresponding author. Tel.: q1-604-822-4449; fax: q1-604-822-
2487; e-mail: orvig@chem.ubc.ca
goal. Thus, there is a need to find effective, orally active drugs
that mimic or enhance the properties of insulin.
Discovery of vanadium’s profound inhibition of Naq–Kq
ATPase in vitro [3,4]piqued interest in a possible biological
role for vanadium. The oral insulin-mimetic properties of
inorganic vanadate in vivo were first reported in 1985 [5].
Since then, there has been great interest in understanding the
normal physiological role of vanadium, and also in devel-
oping new vanadium compounds as potential oral insulin
alternatives in diabetes treatment. Vanadate is a recognized
phosphate analog, and is believed to inhibit potently protein
tyrosine phosphatases by substituting for phosphate as a tran-
sition-state analog, although direct stimulation of protein
tyrosine kinases is also possible [6,7]. The therapeutic value
of inorganic vanadium, in the form of vanadate ([VO4]3y)
or vanadyl ([VO]2q), as an orally active agent against dia-
betes has been well documented [8,9]. The term ‘insulin
mimetic’ is something of a misnomer, since no vanadium

0162-0134/99/$ - see front matter q1999 Elsevier Science Inc. All rights reserved.
PII S 0 1 6 2 - 0 1 3 4 ( 9 9 ) 0 0 1 5 2 - X

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252 L.C.Y. Woo et al. / Journal of Inorganic Biochemistry 76 (1999) 251–257
compound can completely substitute for insulin, but the
actions of vanadium compounds are ‘insulin like’, or at least
insulin enhancing, for the most part. Poor absorption from
the gastrointestinal (GI) tract into the bloodstream and the
narrowness of the window of optimal effectiveness in vivo
are current limitations to administering vanadate to diabetic
patients; investigators are working to overcome these
constraints.
Vanadium complexes, such as bis(maltolato)oxo-
vanadium(IV), (BMOV) [10,11] (Scheme 1), have shown
increased potency over inorganic vanadium in STZ-diabetic
rat studies [12].
Complexation of vanadyl (VO2q) with maltol improved
GI absorption, and consequently lowered the vanadium dose
required for effective glucose lowering [13]. Instead of the
approved food additive maltol, in a different approach
reported herein, we have chelated the vanadium metal center
with an approved anti-diabetic drug, in the expectation that
additive or synergistic effects might ensue from administra-
tion of the resulting complex.
The biguanides (Scheme 2) belong to a class of com-
pounds that enhances insulin action without altering insulin
secretion [14,15]. They are very strong diacid bases (ligands
that have a basic character, but are also able to lose two
protons sequentially) characterized by strongly basic, as well
as strongly acidic, dissociation constants (pKa values ;11.5
and 2–3, respectively). Biguanides have a conjugated dou-
ble-bond system stabilized as a hydrogen-bonded intramo-
lecular six-membered ring [16]. In this class are metformin
(N9,N9-dimethylbiguanide hydrochloride) and phenformin
(b-phenethylbiguanide hydrochloride), synthetic oral glu-
cose-lowering agents introduced in the 1950s to treat type 2
diabetes mellitus. (The latter was later withdrawn due to
serious lactic acidosis side effects.) Metformin, with fewer
side effects, is used worldwide in monotherapy (and sold
under many trade names) and in combination treatment with
Scheme 1.
Scheme 2.
Thursday Nov 11 04:27 PM
sulfonylureas, another class of hypoglycemic drugs used to
treat type 2 diabetes.
Bidentate biguanide and substituted biguanide ligands
form brilliantly colorful, stable chelate compounds with
extensive p delocalization [16,17]. Coordination com-
pounds of biguanides with transition metals in various oxi-
dation states were reviewed many years ago; however, there
remains a paucity of detailed synthesis, characterization, and
reaction chemistry information [16].
The concept of combining vanadium and a biguanide to
form potentially synergistic compounds for diabetes treat-
ment is particularly attractive. The preparation and charac-
terization of oxovanadium(IV) combined with each of
biguanide, metformin, and phenformin to form VO(big)2,
VO(metf)2, and VO(phenf)2, respectively, are presented
herein. Results from preliminary acute STZ-diabetic rat stud-
ies with VO(metf)2 compared with BMOV are also reported.
2. Experimental
2.1. Chemicals and instrumentation
All solvents (Fisher, Aldrich) and chemicals were reagent
grade and used without further purification unless otherwise
specified: metformin (N9,N9-dimethylbiguanide hydrochlo-
ride, Sigma), b-phenethylamine hydrochloride (Aldrich),
dicyandiamide (Sigma), maltol (3-hydroxy-2-methyl-4-
pyrone, Sigma), and vanadyl sulfate trihydrate (Aldrich).
Water was deionized (Barnstead D8902 and D8904 car-
tridges) and distilled (Corning MP-1 Megapure Still) before
use. The yields are for analytically pure compounds and cal-
culations are based on vanadium. All complexation reactions
were carried out under argon unless noted. Melting points
were measured on a Mel-Temp apparatus and are
uncorrected.
The biguanides and their complexes were characterized by
infrared (IR) spectroscopy, mass spectrometry, elemental
analyses, and magnetic susceptibility. Where appropriate, 1H
NMR was used for further characterization. Infrared spectra
were recorded as KBr disks in the range 4000–400 cmy1 on
a Galaxy Series 5000 FTIR spectrometer. Mass spectra
(qion) were obtained with a Kratos MS 50 (electron-impact
ionization, EI), or a Kratos Concept II H32Q (Csq liquid
secondary ion mass spectrometry, LSIMS) instrument.
Room-temperature (293 K) magnetic susceptibilities were
measured on a Johnson Matthey balance, using
Hg[Co(NCS)4] as the susceptibility standard. Diamagnetic
corrections were estimated using Pascal’s constants [18]. All
C, H, N analyses were performed by Mr Peter Borda (Carlo
Erba analytical instrumentation). 1H NMR spectra of samples
in DMSO-d6 were recorded on a Bruker AC-200E instrument
at 200 MHz.
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2.2. Syntheses of biguanides
2.2.1. Biguanide sulfate, C2H7N5PH2SO4 (HbigPH2SO4)
This was prepared by a modification of a literature proce-
dure [19]. Dicyandiamide (5.00 g, 0.059 mol) and ammo-
nium chloride (8.00 g, 0.150 mol) were separately ground
to a fine state, mixed, and heated in an oil bath until a liquid
melt was obtained. This melt was maintained for 15 min at
160–1658C, with constant stirring. After it was cooled in air,
the solid was broken up and dissolved in 30 ml boiling water.
The resulting mixture was filtered and the precipitate (amme-
line) was washed with 2=5 ml hot water. Ammoniacal cop-
per(II) sulfate solution was added in excess to the filtrate,
precipitating red copper biguanide sulfate. This was filtered,
washed with water, and dissolved in 7 ml hot 10% sulfuric
acid solution. Upon cooling this acid solution in an ice bath,
crude biguanide sulfate precipitated, was filtered out and dis-
solved in boiling water; this solution was cooled in ice. The
white product of biguanide sulfate dihydrate was filtered,
washed with water, then ethanol, and dried at 1108C over-
night. The anhydrous yield obtained was 1.12 g (9.5%); m.p.
231–2328C. IR (cmy1, KBr disk) 3285, 3083 (nN–H); 1642,
1630 (nC_N). EIMS m/zs102 ([HLq1]q, [C2H8N5]q).
The solid-state diamagnetic susceptibility (x) was
y1.989=10y7 cm3 gy1. Anal. Calc. (found) for
C2H7N5PH2SO4: C, 12.06 (12.09); H, 4.55 (4.51); N, 35.16
(35.17)%.
2.2.2. Phenformin, C10H15N5PHCl (HphenfPHCl)
This was prepared by a modification of a literature proce-
dure [20]. b-Phenethylamine hydrochloride (5.00 g, 0.032
mol) and dicyandiamide (2.65 g, 0.032 mol) were heated
gradually with stirring in an oil bath up to 1308C. The mixture
was further heated for an hour at 145–1508C, initiating an
exothermic reaction as the mixture fused. When cool, the
solid product was recrystallized from isopropanol, filtered
out, and dried in vacuo overnight to obtain a yield of 4.21 g
(55%); m.p. 170–1728C. IR (cmy1, KBr disk) 3409, 3320,
3166 (nN–H) 1675, 1630 (nC_N). EIMS m/zs205 ([HL]q,
[C10H15N5]q). The solid-state diamagnetic susceptibility
(x) was y5.790=10y7 cm3 gy1. Anal. Calc. (found) for
C10H15N5PHCl: C, 49.69 (49.80); H, 6.67 (6.58); N, 28.97
(28.64)%.
2.3. Syntheses of vanadyl–biguanide complexes
2.3.1. Bis(biguanidato)oxovanadium(IV), VO(big)2P1.5H2O
Biguanide sulfate (0.46 g, 2.31 mmol) was dissolved in 5
ml hot water. VOSO4P3H2O (0.25 g, 1.15 mmol) in 3 ml
H2O was added, with stirring, under Ar, to the biguanide
solution to yield a clear blue solution. When NaOH (2 M,
0.28 g, 7.00 mmol) was added dropwise slowly; the solution
turned gray, then light brown, and finally green (pH;11).
The mixture was stirred for 1 h and the green precipitate was
filtered out under vacuum, washed with water followed by
diethyl ether, and dried overnight in vacuo. The yield was
Thursday Nov 11 04:27 PM
253
0.25 g (81% based on V). IR (cmy1, KBr disk) 942 (nV_O).
qLSIMS m/zs268 ([Mq1]q, [C4H13N10OV]q). The
solid-state magnetic moment was 1.70 BM. Anal. Calc.
(found) for C4H12N10OVP1.5H2O: C, 16.33 (16.52); H,
5.14 (4.81); N, 47.61 (47.15)%.
2.3.2. Bis(N9,N9-dimethylbiguanidato)oxovanadium(IV),
VO(metf)2PH2O
VOSO4P3H2O (1.00 g, 4.60 mmol) was dissolved in 5 ml
water and added slowly, with stirring and bubbling with Ar,
to a solution of 2 equivalents metformin (1.52 g, 9.20 mmol)
in 5 ml H2O. NaOH (2 M, 0.90 g, 23.0 mmol) was added
dropwise to bring the pH to ;12. Initial addition of base
resulted in brown hydroxide complex formation; however,
upon complete addition, a light green solid eventually pre-
cipitated. The solution was stirred for 2–3 h and the solid was
collected by vacuum filtration, washed with cold water fol-
lowed by ether, and dried overnight in vacuo. The yield was
0.90 g (56% based on V). IR (cmy1, KBr disk) 929 (nV_O).
EIMS m/zs323 (Mq, [C8H20N10OV]q). The solid-state
magnetic moment was 1.99 BM. Anal. Calc. (found) for
C8H20N10OVPH2O: C, 28.16 (28.44); H, 6.50 (6.59); N,
41.04 (41.26)%.
2.3.3. Bis(b-phenethylbiguanidato)oxovanadium(IV),
VO(phenf)2PH2O
VOSO4P3H2O (0.45 g, 2.07 mmol) was dissolved in ;3
ml water and added slowly to a solution of 2 equivalents
phenformin (1.00 g, 4.14 mmol) in 5 ml H2O (under Ar).
Dilute NaOH (0.17 g, 4.14 mmol in 3 ml H2O) was added
dropwise to bring the pH to ;11. Initial addition of base
resulted in light brown hydroxide formation; however, upon
complete addition and after stirring for 1 h, a light blue solid
eventually precipitated. The solution was stirred for an addi-
tional hour and the solid was collected by vacuum filtration,
washed with cold water followed by ether, and dried over-
night in vacuo. The yield was 0.72 g (37% based on V). IR
(cmy1, KBr disk) 953 (nV_O); qLSIMS m/zs476
([Mq1]q, [C20H29N10OV]q), 206 ([HLq1]q,
[C10H16N5]q). The solid-state magnetic moment was 1.94
BM. Anal. Calc. (found) for C20H28N10OVPH2O: C, 48.68
(48.64); H, 6.13 (5.80)%.
2.3.4. Bis(N9,N9-dimethylbiguanidato)(dimethyl-
sulfoxide)oxovanadium(IV), VO(metf)2(DMSO)PH2O
Dimethylsulfoxide (DMSO, 1 ml) was added to green
VO(metf)2 (0.020 g, 0.062 mmol) with stirring and bub-
bling with Ar. The solid turned light purple and dissolved
over a period of 2–3 days, resulting in a purple solution.
Before dissolution, the purple solid was collected by vacuum
filtration, washed with diethyl ether, and dried in vacuo over-
night. The yield was 0.007 g (28% based on V). IR (cmy1,
KBr disk) 952 (nV_O). EIMS m/zs323 ([M]q,
[C8H20N10OV]q); qLSIMS m/zs130 ([HLq1]q,
[C4H12N5]q). The solid-state magnetic moment was 1.93
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BM. Anal. Calc. (found) for C8H20N10OVP2DMSOPH2O:

C, 30.09 (29.42); H, 6.77 (6.70); N, 27.85 (27.90)%.

2.4. Glucose-lowering studies: materials and methods

Male Wistar rats weighing 190–220 g (Animal Care Unit,

UBC) were housed two per cage (polycarbonate), and were
allowed ad libitum access to food (Purina Lab Chow) and
water. Rats were on a 12 h light:dark schedule and were cared
for in accordance with the principles and guidelines of the
Canadian Council on Animal Care. The animals were accli-
matized for 7–14 days, then diabetes was induced by a single
intravenous injection of STZ (60 mg kgy1 in 0.9% NaCl, 1
ml kgy1) under light halothane anesthesia. Diabetes was
confirmed three days after STZ injection by tail-vein blood-
glucose determination (Ames Glucometer and Glucostix),
with blood-glucose levels over 13 mM being taken as dia-
betic. On day seven post-STZ, the animals were divided into
treatment groups: gum arabic alone, metformin, and
VO(metf)2. Using a similar protocol, animals were treated
with either BMOV or gum arabic alone. All drugs were Fig. 1. Plasma glucose levels for gum arabic (s, ns9), BMOV (j, ns11),
administered as suspensions in 3% gum arabic. Animals were metformin (d, ns5), and VO(metf)2 (h, ns5) following acute intra-
not fasted prior to drug administration. Blood, 50 ml, was peritoneal injection in STZ-diabetic rats at a dose of 0.12 mmol kgy1.
collected for glucose analysis immediately prior to drug
administration and at selected times up to 72 h after drug
administration. Blood was collected from the tail vein into
heparinized capillary tubes and centrifuged at 10 000g=15
min. The plasma was separated and analyzed immediately
for glucose levels using Boehringer Mannheim kits (glucose
oxidase method). At all time points the animals were
observed for signs of toxicity (e.g., diarrhea).

2.4.1. Intraperitoneal (i.p.) administration in STZ-diabetic

rats
Thirty diabetic rats were treated with gum arabic (ns9),
BMOV (ns11), metformin (ns5), or VO(metf)2 (ns5).
All drug candidates were administered by i.p. injection in a
volume of 5.0 ml kgy1 at a dose of 0.12 mmol kgy1. The
control groups (gum arabic) received an equivalent volume
of 3% gum arabic alone. Glucose-level results are shown in
Fig. 1.

2.4.2. Oral gavage of STZ-diabetic rats

Thirty-six diabetic rats were treated with gum arabic
(ns10), BMOV (ns14), metformin (ns6), or
VO(metf)2 (ns6). All drug candidates were administered
as suspensions in 3% gum arabic by oral gavage in a volume
of 5.0 ml kgy1 at a dose of 0.60 mmol kgy1. The control
groups (gum arabic) received an equivalent volume of 3%
gum arabic alone. Glucose-level results are shown in Fig. 2.
3. Results and discussion
3.1. Preparation and characterization of biguanides
The biguanides were readily synthesized by combining
dicyandiamide and the appropriate aryl- or alkyl-amine,
Fig. 2. Plasma glucose levels for gum arabic (s, ns10), BMOV (j,
ns14), metformin (d, ns6), and VO(metf)2 (h, ns6) following acute
oral gavage administration in STZ-diabetic rats at a dose of 0.6 mmol kgy1.
either by heating the reactants in aqueous solution in the
presence of copper(II) sulfate or by fusing the amine hydro-
chloride with dicyandiamide in a melt. The low yield of
biguanide sulfate is not unusual for syntheses that include
formation of a melt. Distinctive IR vibrations in the 3300–
3400 and 1600–1640 cmy1 regions are indicative of the
N–H and C_N stretching modes, respectively. The infrared
spectrum of guanylurea hydrochloride [21] was used as a
reference to assign the vibrations of the biguanides.

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The electron impact ionization mass spectra show the pro-
tonated biguanide as the parent mass. The broad peaks in the
1
H NMR spectra are characteristic of the hydrogens quickly
exchanging due to biguanide tautomerization. Experimental
elemental analyses correlate well with the calculated values
for each organic moiety. Diamagnetic susceptibilities were
obtained for later magnetic moment calculations of V(IV) in
the prepared complexes.
Most importantly, biguanides exist in a multitude of reso-
nance structures. The conjugated double-bond system is
stabilized as an intramolecularly hydrogen-bonded six-
membered ring, which influences the physiological properties
of biguanides [22]. Biguanides are already identified as
strongly chelating ligands that bind readily to a variety of
metals, such as copper, zinc, cobalt, and manganese, and non-
metals, such as methanesulfonic, benzenesulfonic, or 2-ami-
noethanesulfonic (taurine) acids [16]. The lone electron
pairs situated on the N atoms are capable of forming a delo-
calized p electron system whereby biguanide complexes with
transition metals.
3.2. Preparation and characterization of vanadyl–
biguanide complexes
The neutral oxovanadium(IV)–biguanide complexes
were prepared by the addition of vanadyl sulfate to alkaline
solutions of each biguanide, in a ratio of 1:2, with moderate
yields. A slight excess of ligand precursor was used to drive
the reaction to completion. Slow addition of dilute sodium
hydroxide to the reaction mixture was necessary, whereupon
vanadyl hydroxide initially precipitated. When a pH of ;11
was reached, the bis(biguanidato)oxovanadium(IV) com-
plex predominated and precipitated out of solution as light
green, green, and light blue solids, characterized as
VO(metf)2, VO(big)2, and VO(phenf)2, respectively.
Characteristic stretching frequencies of the V_O bond in
oxovanadium(IV) complexes generally occur in the region
930–1030 cmy1 [23]. The complexes VO(big)2,
VO(metf)2, and VO(phenf)2 exhibited low values of nV_O,
at 942, 929, and 953 cmy1, respectively, because of the strong
out-of-plane dp–pp bonding. The addition of dimethylsulf-
oxide slightly increased the nV_O of VO(big)2 and
VO(metf)2 due to the increased electron density on V from
the donating solvent, most likely coordinated trans to the
axial V_O. This enhanced the s and p donations from V to
the oxo-O, subsequently raising the nV_O frequency. Typical
N–H stretching vibrations were observed in all spectra.
Elemental analyses of the complexes were generally con-
sistent with the calculated values. Low nitrogen values in
some of the analyses were possibly due to the formation of
vanadium nitride during incomplete combustion of the sam-
ple. This is not unusual in complexes with metal–nitrogen
coordination; the biguanide ligands, in particular, are highly
nitrogen rich and each coordinate at two N sites to the metal.
After drying in vacuo at 608C for 24 h, residual water could
not be eliminated.
Thursday Nov 11 04:27 PM
255
The distinctive fragment peaks in the corresponding EI and
qLSI mass spectra support the suggested complex formu-
lations. Complexes containing water or other solvent show
the same HVOL2q peaks as the parent mass.
All the complexes prepared were paramagnetic in the solid
state. Room-temperature paramagnetic susceptibilities were
obtained. The diamagnetic susceptibilities of the biguanides
were corrected using Pascal’s constants and these diamag-
netic susceptibilities were used to calculate the effective mag-
netic moments of the complexes. With an electronic
configuration of [Ar]3d1, vanadium(IV) has one unpaired
electron for which the spin-only formula predicts a magnetic
moment of 1.73 BM. The experimental values are in the range
1.70–2.00 BM for the vanadyl complexes. Literature values
of 1.45–1.94 BM [24] for some vanadyl–biguanide com-
pounds are lower, possibly due to an exchange interaction
from solid-state dimer formation [16].
Solution equilibria and stability measurements of the van-
adyl–metformin system could not be undertaken because
hydrolysis predominates at approximately pH 4, with the
accompanying formation of vanadium(IV) hydroxides, most
notably VO(OH)2.
3.3. Properties of vanadyl–biguanide complexes
All of the vanadyl–biguanide complexes are air-stable
powders, exhibiting magnetic moments at room temperature
in the solid state. The complexes lack solubility in common
organic solvents; hence, they could not be recrystallized. In
water, hydrolysis is facile and a brown solution (of vanadyl
hydroxide) results. In DMSO, each complex turns a distinct
shade of purple and eventually dissolves; the amount of time
required is dependent on concentration.
Biguanides combine with many elements of the transition
series to give highly colored chelate compounds [16]. The
ligands are monodeprotonated in all cases discussed, a desir-
able feature because an overall neutral complex is favored
for increased lipophilicity and GI absorption in drug appli-
cations. When deprotonated, the bidentate biguanides act as
hard Lewis bases, which bind readily to VO2q, a hard Lewis
acid. The resonating deprotonated ligand coordinates through
the N donor atoms forming a rigid, planar, six-membered, p-
delocalized chelate ring with the metal, enhancing overall
thermodynamic stability. X-ray-quality crystals were, unfor-
tunately, not obtained. We expect a square pyramidal geom-
etry for this N4-type (as observed for BMOV [11]), in
contrast to the distorted octohedral coordination geometry
seen in N2O2 complexes [25].
Preliminary electron paramagnetic resonance spectro-
scopic data for VO(big)2 and VO(metf)2 showed the char-
acteristic eight-line pattern expected for V(IV); however, the
unresolved superhyperfine splitting (from four nitrogen
atoms) made it difficult to gain any information as to the
metal ion coordination sphere and its geometry. For both
compounds, formation of the solvated DMSO species can be
monitored by the noticeable color change to purple upon
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solvent addition during sample preparation. Possible sites for
solvent coordination are trans (T) or cis (C) to the oxo-O
[26], yielding six possible species in solution; a more
detailed understanding of the coordination environment
would be derived from an X-ray crystal-structure analysis.
3.4. Glucose-lowering studies in STZ-diabetic rats
Animal studies were performed sequentially using
VO(metf)2 as a representative biguanide coordination com-
plex and BMOV as a representative standard. The functional
end-point by which the novel complex was deemed to have
exceeded previously prepared insulin-enhancing agents was
the glucose-lowering capacity in vivo. Both i.p. and oral
gavage studies were carried out. Treated rats were considered
to have responded to treatment if their plasma glucose levels
declined to -9 mM (normal level). Two sets of diabetic
treatment groups were monitored: first, gum arabic and
BMOV; subsequently, gum arabic, metformin, and
VO(metf)2. (Gum arabic results have been combined, as
there were no differences between groups.) The compounds
were administered to STZ-diabetic rats at the dose previously
shown with BMOV to lower blood glucose to normal levels
in 50% of the animals tested (ED50) [12,27].
3.4.1. Intraperitoneal (i.p.) injection
Following treatment by acute i.p. injection at a dose of 0.12
mmol kgy1, 40% of the animals responded to VO(metf)2,
64% to BMOV, and none to metformin or to the control (Fig.
1). The effect of VO(metf)2 on plasma glucose levels was
not significantly lower at any of the time points measured
(compared with the zero time point); however, the glucose-
lowering effect of BMOV, compared 12 h after injection with
the zero time point, was highly significant (p-0.001, paired
t-test). This glucose-lowering effect persisted up to 72 h (Fig.
1). There was also a noticeable incidence of diarrhea in 50%
of the VO(metf)2-treated rats compared with no diarrhea
among the BMOV-treated animals. This may be due, at least
in part, to the high pH of the VO(metf)2/gum arabic suspen-
sion (11.5, compared with pH 4.3 for BMOV) administered
to the test animals. Testing at the lower i.p. dose suggested
recently [28] (0.025 mmol kgy1) may have circumvented
this difficulty.
3.4.2. Oral gavage
Following acute oral gavage at a dose of 0.60 mmol kgy1,
there was no difference in plasma glucose levels between the
animals treated with VO(metf)2 and those treated with
BMOV (Fig. 2). Both VO(metf)2 and BMOV rendered most
diabetic animals normoglycemic by 24 h (100% response for
VO(metf)2, 71% for BMOV); however, with VO(metf)2
plasma glucose returned to hyperglycemic levels (19.5"1.5
mM) for all rats within 72 h, a result similar to that seen with
VOSO4 treatment [12]. With BMOV, only 43% were hyper-
glycemic by 72 h, indicating a more sustained response. Mild
GI effects were noted in all groups except gum arabic
Thursday Nov 11 04:27 PM
(BMOV, 40%, 8 h; VO(metf)2, 67%, 20 h; metformin, 25%,
6 h). There was no glucose-lowering response to metformin,
indicating that the dose is likely too low to see any effect of
this commercial insulin-enhancing hypoglycemic drug. No
appreciable weight gain (or loss) was observed with
VO(metf)2 in either study.
4. Conclusions
The design of the vanadyl–biguanide complexes was
appealing because of the potential for associative effects with
complexation of vanadium to biguanide ligands. There is
considerable evidence for the orally effective glucose-low-
ering properties of V(V) and V(IV), and biguanides are
currently used worldwide for the treatment of type 2 diabetes
mellitus. However, vanadium alone, in the form of vanadyl
or vanadate, is effective in glucose lowering at a far lower
dose, on a mmol kgy1 basis, than is metformin alone. Thus,
metformin alone is taken up to a maximal daily human dosage
of over 2 g, in comparison with what would be milligram
treatments of vanadium. Simultaneous administration of bi-
guanides and vanadium in individually appropriate doses in
a combination treatment could prove effective, and could
possibly result in associative or synergistic effects, but com-
plexed vanadium and metformin (as shown here) has the
same pharmacological potency as an organically chelated
vanadium compound by itself. Increasing the VO(metf)2
dose in the hopes of observing synergy is unlikely to be
advantageous, due to the potential toxicity of high doses of
vanadium. From preliminary animal investigations with
VO(metf)2, it may be concluded that this compound is com-
parable to BMOV in lowering blood glucose in STZ-diabetic
rats; however, neither synergy nor additive effects were
observed.
Acknowledgements
The authors gratefully acknowledge the Medical Research
Council (MRC) of Canada for an operating grant, and both
Angiotech Pharmaceuticals, Inc., and Kinetek Pharmaceuti-
cals, Inc., for personnel support, as well as Barry Liboiron
for his work on the EPR spectra.
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