PH-metric chemical speciation modeling and studies of in vitro antidiabetic effects of bis[(imidazolyl)carboxylato]oxidovanadium(IV) complexes

Article abstract of DOI:10.1016/j.jinorgbio.2014.12.019

A range of bidentate N,O-donor ligands of the imidazolyl-carboxylate moiety, which partially mimic naturally occurring bioligands, were prepared and reacted with the oxidovanadium(IV) ion to form the corresponding bis-coordinated oxidovanadium(IV) complexes. The aqueous pH-metric chemical speciation was investigated using glass electrode potentiometry, which allowed for the determination of protonation and stability constants of the ligands and complexes, respectively. The species distribution diagrams generated from this information gave evidence that the bis[(imidazolyl)carboxylato]oxovanadium(IV) complexes possess a broad pH-metric stability. The complexes improved glucose uptake in cell cultures using 3T3-L1 adipocytes, C2C12 muscle cells and Chang liver cells. The PTP inhibition studies indicated that the mechanism underlying insulin-stimulated glucose uptake was possibly via the protein tyrosine phosphorylation through the inhibition of the protein tyrosine phosphatase 1B (PTP 1B). The vanadium compounds also demonstrated the inhibition of D-dimer formation, suggesting that these compounds could potentially relieve a hypercoagulative state in diabetic patients.

Full text of DOI:10.1016/j.jinorgbio.2014.12.019

pH-metric chemical speciation modeling and studies of in vitro antidiabetic
effects of bis[(imidazolyl)carboxylato]oxidovanadium(IV) complexes
Isaac Z. Gundhla, Ryan S. Walmsley, Vital Ugirinema, Nandipha O.
Mnonopi, Eric Hosten, Richard Betz, Carminita L. Frost, Zenixole R. Tshentu
PII:
DOI:
Reference:
S0162-0134(14)00312-2
doi: 10.1016/j.jinorgbio.2014.12.019
JIB 9642
To appear in:
Journal of Inorganic Biochemistry
Received date:
Revised date:
Accepted date:
25 March 2014
18 December 2014
19 December 2014
Please cite this article as: Isaac Z. Gundhla, Ryan S. Walmsley, Vital Ugirinema,
Nandipha O. Mnonopi, Eric Hosten, Richard Betz, Carminita L. Frost, Zenixole R.
Tshentu, pH-metric chemical speciation modeling and studies of in vitro antidiabetic ef-
fects of bis[(imidazolyl)carboxylato]oxidovanadium(IV) complexes, Journal of Inorganic
Biochemistry (2014), doi: 10.1016/j.jinorgbio.2014.12.019
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ACCEPTED MANUSCRIPT
pH-metric chemical speciation modeling and studies of in vitro antidiabetic
effects of bis[(imidazolyl)carboxylato]oxidovanadium(IV) complexes
Isaac Z. Gundhla,a Ryan S. Walmsley,a Vital Ugirinema,b Nandipha O. Mnonopi,c Eric Hosten,b
Richard Betz,b Carminita L. Frost,c,* Zenixole R. Tshentub,*
aDepartment of Chemistry, Rhodes University, P.O. Box 94, Grahamstown 6140, South Africa
bDepartment of Chemistry, Nelson Mandela Metropolitan University, P.O. Box 77000, Port
Elizabeth 6031, South Africa
cDepartment of Biochemistry and Microbiology, Nelson Mandela Metropolitan University, P.O.
Box 77000, Port Elizabeth 6031, South Africa
Abstract
A range of bidentate N,O-donor ligands of the imidazolyl-carboxylate moiety, which partially
mimic naturally occurring bioligands, were prepared and reacted with the oxidovanadium(IV) ion to
form the corresponding bis-coordinated oxidovanadium(IV) complexes. The aqueous pH-metric
chemical speciation was investigated using glass electrode potentiometry which allowed for the
determination of protonation and stability constants of the ligands and complexes, respectively. The
species distribution diagrams generated from this information gave evidence that the
bis[(imidazolyl)carboxylato]oxovanadium(IV) complexes possess a broad pH-metric stability. The
complexes improved glucose uptake in cell cultures using 3T3-L1 adipocytes, C2C12 muscle cells
and Chang liver cells. The PTP inhibition studies indicated that the mechanism underlying insulin-
stimulated glucose uptake was possibly via the protein tyrosine phosphorylation through inhibition
of the protein tyrosine phosphatase 1B (PTP 1B). The vanadium compounds also demonstrated the
inhibition of D-dimer formation, suggesting that these compounds could potentially relieve a
hypercoagulative state in diabetic patients.
Keywords: oxidovanadium(IV) complexes, speciation, anti-diabetic effects, anti-coagulative effects
* Emails: carminita.frost@nmmu.ac.za (C.L. Frost) and zenixole.tshentu@nmmu.ac.za (Z.R. Tshentu)
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1. Introduction
Diabetes mellitus, a chronic metabolic disorder, is becoming a major health concern worldwide, and
the cost of treatment for this burden is becoming an insurmountable undertaking for low and
middle-income countries [1,2]. Insulin, a pancreatic signaling hormone, is the principal treatment
for type 1 diabetes while type 2 diabetes can often be controlled by oral pharmaceuticals namely;
sulphonylureas such as glipizide, biguanides such as metformin, thiazolidinediones such as
pioglitazone, and meglitinides such as prandin. However, these available prescription drugs present
adverse side effects including hypoglycaemia for sulphonylureas, lactic acidosis for the biguanides,
and weight gain for thiazolidinediones. Therefore, there is a need for the development of another
class of oral pharmaceuticals that does not present undesirable side effects, and vanadium has
shown promise in this regard.
Vanadium is by no means the only metal that has therapeutic effects for the treatment of diabetes
mellitus. Metals or minerals such as copper, manganese, zinc and selenium exhibit increased
sensitivity to insulin due to their ability to relieve the oxidative stress caused by the reactive oxygen
species within cells [3,4]. At high doses, however, these metals may become pro-oxidant hence
supplementation is often restricted to a change of diet to include foods richer in minerals. The
supposed positive role of chromium in glucose metabolism has also resulted in the marketing of
chromium as a nutritional supplement with chromium picolinate being available over the counter in
a form of pills, sports drinks and nutrition bars [4]. However, Cefalu et al. (2010) have recently
carried out a comprehensive study on the metabolic and physiological response from chromium
supplementation and suggested that chromium is most likely to elicit a response to insulin resistant
individuals who have more elevated fasting glucose [5]. Vanadium compounds are also being
considered for mitigating insufficient insulin response in diabetes mellitus [6]. They cannot,
however, entirely substitute for lack of insulin (as in type 1 diabetes) but can reduce reliance on
exogenous insulin and/or perhaps substitute for other oral hypoglycaemic agents used in treatment
of type 2 diabetes [7,8]. Inorganic salts of vanadium have shown promising glucose metabolism
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action but the potential toxicity of vanadate (anionic form) and low absorption rate of
oxidovanadium(IV) (cationic form) pushed the focus towards designing new organovanadium
compounds [9,10]. Thus tailoring of the properties of these vanadium complexes can be achieved by
simple modification of the organic ligand.
Vanadate and oxidovanadium(IV) are the main species that can exist under physiological conditions
[6]. The physiological effects are in many cases a consequence of the good complexation behaviour
of the VO2+ ion with a carrier ligand, allowing it to be absorbed in a complexed form and then
released in the bloodstream to be shuttled by biological ligands such as transferrin to the cells [11].
Vanadium has been implicated in performing many functions of insulin such as inhibition of
lipolysis and gluconeogenesis as well as stimulating lipogenesis and cellular glucose uptake, hence
its compounds are referred to as insulin-mimetics [12-15]. The true form of the necessary vanadium
species under physiological conditions is a subject of much debate but a focal mechanism of action
which seems to enjoy a broad consensus is its involvement in the inhibition of phosphatases [12-
15]. The action of vanadium at an intracellular cellular level is due to the structural similarity
between phosphate and vanadate. This property leads to the inhibition of protein tyrosine
phosphatase 1B (PTP 1B) thereby maintaining the signal for glucose internalisation and subsequent
metabolism is achieved [16,17]. A few vanadium compounds seem to have insulin-enhancing
effects, and among others, bis(picolinato)oxidovanadium(IV) has proved to be orally active in
treating diabetes mellitus [18], and bis(maltolato)oxidovanadium(IV) (BMOV) and
bis(ethylmaltolato)oxidovanadium(IV) (BEOV) have also shown significant glucose lowering effect
[19], with the latter having undergone Phase IIa clinical trials [7].
The development of vanadium anti-diabetic metallo-pharmaceuticals is focused on oral agents for
lowering blood glucose levels, therefore the complexes must be thermodynamically stable in both
acidic and neutral environments. This stability is required because the vanadium complex must
survive the digestion process where the pH can be as low as 2 and must also remain intact under pH
conditions of the intestines and be delivered in significant doses into the bloodstream where the pH
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can be as high as 7.4. Therefore the determination of the thermodynamic stability of the vanadium
complexes as a function of pH is a necessary prerequisite for evaluating the usefulness of such
complexes to serve as glucose-lowering drugs. The ligands, therefore, act as shields that help to
stabilize vanadium complexes under the pH conditions of the digestive tract and intestines, and
assist with absorption of vanadium into the bloodstream. The latter renders the balance of
hydrophilicity and hydrophobicity of the complexes very important [19]. The pH-metric chemical
speciation of the vanadium compounds can be appropriately designed through the correct
combination of donor atoms involved in the bidentate coordination of the ligands in order to achieve
the requisite stabilization under the physiological pH range.
This study explores the use of imidazole derivatives as binding moiety to oxidovanadium(IV) since
imidazoles are known to play a significant role in a large variety of biological processes [20]. We
have shown that imidazolyl-carboxylates are simple and effective bidentate ligands for stabilizing
VIVO in biological systems. The syntheses and characterization of the oxidovanadium(IV)
complexes with imidazole-4-carboxylic acid (Im4COOH), imidazole-2-carboxylic acid
((Im2COOH) and 1-methylimidazole-2-carboxylic acid (MeIm2COOH) as coordinating ligands is
presented. The stability constants for the complexation of oxidovanadium(IV) to the ligands were
determined via potentiometric acid-base titrations. The anti-diabetic potential of these complexes
has been investigated via the in vitro glucose assay using 3T3-L1 adipocytes, C2C12 muscle cells
and Chang liver cells as well as the PTP 1B inhibition assay. In addition, both the extrinsic and
intrinsic coagulation pathways [21,22] were investigated in order to ascertain the potential of the
complexes in relieving blood coagulation.
2. Experimental Section
2.1 Materials and instrumentation
Oxidovanadium(IV) sulfate hydrate was obtained from BDH Limited (England).
1-
Methylimidazole (MeIm) (99%) and Im4COOH (1a) (98%) were obtained from Sigma-Aldrich
(USA). Imidazole-2-carboxaldehyde (97%) was obtained from Fluka (USA). All solvents were
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obtained from Merck Chemicals (SA) and were of reagent grade and used without further
purification. Other reagent grade chemicals were also obtained from commercial sources and used
as received. PTP 1B was purchased from Sigma-Aldrich (SA) and microtiter plates from Lasec
(SA).
The infrared spectra were recorded on a Perkin Elmer 2000 FTIR spectrometer in the mid-IR range
(4000 – 400 cm-1) as KBr pellets. 1H and 13C NMR spectra of all ligands were recorded on a Bruker
AMX 400 NMR MHz spectrometer and reported relative to tetramethylsilane (δ 0.00). Electronic
spectra were recorded on a Perkin Elmer Lambda 25 UV-Vis spectrophotometer using 1 cm quartz
cells and water as the solvent. Microanalysis was carried out using a Vario Elementar Microcube
ELIII. Potentiometric studies were performed using a Metrohm 794 Titrino equipped with a
Metrohm LL Ecotrode. A Bio-Tek KC4 powerwave XS microtiter plate reader was used to measure
absorbance for 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT), Glucose and
PTP 1B inhibition assays.
2.2 Preparative work
2.2.1 Im2COOH (1b)
An aqueous 30% H2O2 solution (10 g) was added dropwise to a stirred solution of imidazole-2-
carboxaldehyde (2.88 g 0.030 mol) in water (10 ml). The reaction was allowed to proceed at room
temperature for 72 h, following which the water was removed in vacuo at room temperature to
afford a white crystalline solid. This solid was washed with a stirred mixture of diethylether/water
(4:1) to remove the excess peroxide. Note: heating causes decarboxylation. Yield: 97.5%. Mp =
156-158oC. δH (400 MHz, D2O): 7.56 (2H, s, Im-H); δC (400 MHz, D2O): 158.86, 141.02, 120.49
ppm. IR ν (KBr): 3392(m), 3124(m), 2861(m), 1618(s), 1502(m), 1462(m), 1421(s), 1388(s),
1322(m), 1108(s), 925(s), 910(s), 819(m), 797(s), 774(m) cm-1. Anal. Calc. for C4H6N2O3 (130.11);
C, 36.92; H, 4.65; N, 21.53%. Found: C, 37.18; H, 4.94; N, 21.47%.
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2.2.2 MeIm2COOH (1c)
Firstly, 1-methylimidazole-2-carboxaldehyde was prepared according to the literature procedure
[23]. The synthesis procedure then followed that of Im2COOH (1b) above and the yield was also
quantitative after the removal of water under high vacuum (no washing with diethylether/water was
necessary). Note: heating causes decarboxylation. Yield: 100%. Mp = 99-101oC. δH (400 MHz,
D2O): 7.42, 7.39 (2H, s, Im-H) and 4.08 ppm (3H, s, NCH3); δC (400 MHz, D2O): 158.67, 139.68,
125.83, 118.46, 36.73 ppm. IR ν (KBr): 3347(m) 3119(m), 2663(w), 1641(s), 1683(m), 1507(s),
1449(m), 1388(s), 1338(s), 1285(s), 1173(m), 1123(s), 961(m) 910(m), 776(s), 685(s) cm-1. Anal.
Calc. for C5H8N2O3 (144.12); C, 41.67; H, 5.59; N, 19.44%. Found: C, 41.28; H, 5.23; N, 19.12%.
2.2.3 VO(im4COO)2.H2O (2a)
This complex was prepared according to a literature method but with slight modifications [24]. To a
stirred aqueous solution of Im4COOH (0.254 g, 2.26 mmol), was added 10% TMAOH (1.13
mmol). To this solution was added aqueous VOCl2 (1.13 mmol), prepared by the reaction of VOSO4
with BaCl2. The reaction was allowed to stir overnight, following which the light blue precipitate
was collected, washed with methanol and ether and dried in an oven (100oC). Yield: 64.6%.
Mp>300ºC. IR ν (neat): 3129(m), 2992(m), 2856(m), 1606(s), 1573(s), 1499(m), 1439(m), 1356(s),
1209(m), 1080(m), 1012(m), 981(s), 878(m), 819(s), 780(s) cm-1. Anal. Calc. for C8H8N4O6V
(307.11); C, 31.29; H, 2.63; N, 18.24%. Found: C, 31.20; H, 2.67; N, 18.14%. UV/Vis (solid
reflectance) λmax (nm): 733, 593, 346.
2.2.4 VO(im2COO)2.H2O (2b)
This complex was prepared in the same manner as 2a, except that an aqueous solution of VOSO4
was added to the ligand solution instead of aqueous VOCl2. Yield: 69.9%. Mp > 300ºC. IR ν (neat):
3448(m), 3129(w), 3008(m), 2906(m), 1625(s), 1558(m), 1487(m), 1398(s), 1338(m), 1277(m),
1171(m), 1112(s), 984(s), 908(m), 829(m), 769(s), 658(s) cm-1. Anal. Calc. for C8H8N4O6V
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(307.11); C, 31.29; H, 2.63; N, 18.24%. Found: C, 31.21; H, 2.53; N, 18.07%. UV/Vis (solid
reflectance) λmax (nm): 744, 592, 338.
2.2.5 VO(MeIm2COO)2.H2O (2c)
This complex was prepared similarly to 2a. However, upon stirring overnight a blue precipitate did
not form. The blue solution was then allowed to stand for 24 h and a light blue precipitate formed,
o
which was filtered and washed with methanol and then ether and dried in an oven at 100 C. Yield
62.3%. Mp = 218-220ºC. IR ν (neat): 3134 (m), 1628(s), 1487(s), 1426(s), 1327(s), 1286(m),
1180(s), 1166(s), 963(s), 837(m), 797(s), 769(s), 698(s) cm-1. Anal. Calc. for C10H12N4O6V
(335.17); C, 35.83; H, 3.61; N, 16.72%. Found: C, 36.01; H, 3.47; N, 16.67%. UV/Vis (solid
reflectance) λmax (nm): 736, 585, 362.
2.3 X-ray structure determination
X-ray diffraction analysis of VO(im4COO)2·H2O (2a) was performed at 200 K using a Bruker
Kappa Apex II diffractometer with a monochromated Mo Kα radiation (λ = 0.71073 Å). APEXII
[25] was used for data collection and SAINT [25] for cell refinement and data reduction. The
structure was solved by direct methods using SHELXS–2013 [26] and refined by least-squares
procedures using SHELXL-2013 [26] with SHELXLE [27] as a graphical interface. All non-
hydrogen atoms were refined anisotropically. Carbon-bound H atoms were placed in calculated
positions (C–H 0.95 Å for aromatic carbon atoms) and were included in the refinement in the riding
model approximation, with Uiso(H) set to 1.2Ueq(C). Nitrogen-bound H atoms were located on a
difference Fourier map and refined freely. Data were corrected for absorption effects using the
numerical method implemented in SADABS [25].
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2.4 Potentiometric studies
The protonation and stability constants for the ligands and oxidovanadium(IV) complexes were
determined by potentiometric titration of approximately 25 ml samples. All solutions were prepared
using freshly boiled and degassed deionized milli-Q water to ensure the removal of dissolved
oxygen and carbon dioxide. The ligand concentration was 1 mM and metal-to-ligand ratios of 1:1,
1:5 and 1:10 were used. Titrations were performed over the pH range of 2-11 under a continuous
flow of purified nitrogen using HCl and tetramethylammonium hydroxide (TMAOH). The
vanadium stock solution containing 0.10 M HCl was standardized by titration with permanganate.
The ionic strength of the titration solutions was kept constant at 0.10 M tetramethylammonium
chloride (TMACl). Titrations were controlled using Tiamo software, and the titration rate used was
0.01 ml/min and the pausing time was 60 s. The glass electrode was calibrated for a strong acid-base
reaction by the Gran-method [28] using the program GLEE [29], to determine the standard potential
Eo. The ionic product of water (pKw) of 13.83(1) at 25.0±0.1 oC in 0.10 M TMACl was used in all
calculations [30]. The hydrolysis model of a oxidovanadium(IV) system was included in the model;
[VO(OH)3]- (logβ10-3 = -18.0) and [(VO)2(OH)5]- (logβ20-5 = -22.0), while [VO(OH)]+ (logβ10-1 = -
5.94) and [(VO)2(OH)2]2+ (logβ20-2 = -6.95) [31] did not fit. The concentration stability constants
βpqr = [MpLqHr]/[M]p[L]q[H]r were calculated by using the computer program HYPERQUAD [32].
2.5 In vitro studies
2.5.1 Maintenance of cells lines
3T3-L1 preadipocytes, were maintained in Dulbecco’s modified eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS), while C2C12 mouse skeletal myoblasts and
Chang liver cells were maintained in RPMI-1640 medium (Sigma) supplemented with 10% FBS.
o
These were incubated at 37 C in a humidified incubator with 5% CO2. Cells were subcultured at
70% confluence and seeded at a density of 35 000 cells/ml (for 3T3-L1) and 25 000 cells/ml (for
Chang and C2C12) in 24-well culture plates.
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2.5.2 MTT cytotoxicity studies
Cells were seeded in 24 well plates (Nunc) at densities of 25 000 for C2C12 and Chang liver cells,
and 35 000 cells/mL for 3T3-L1. After overnight attachment, the culture medium was replaced with
medium containing the test compounds at a range of concentrations (0.5-100 μM). Cells were
incubated for 48 h at 37 °C, after which the MTT assay was performed [33].
2.5.3 Glucose uptake studies
The glucose uptake assay was performed using the GLUCOSE (Glu-cinet) kit (BAUER) [34].
Control cells were represented by untreated differentiated fat (3T3-L1), liver (Chang) and
undifferentiated muscle (C2C12) cells (Con) incubated in culture media. Positive control cells (Met)
were represented by untreated differentiated fat, liver and muscle cells exposed to metformin. Cells
were then exposed for 48 h to the test compounds. Thereafter glucose uptake was determined and
the cell number was normalized using the MTT assay [33].
2.5.4 PTP inhibition assay
PTP 1B was reconstituted according to the manufacturer’s instructions. It was used to establish a
standard curve (results not shown) ranging from 40 – 320 mU. A modified method from Zhang et
al. (2006) [35] was performed in a final volume of 200 µl. Forty microliters of PTP 1B (40 mU)
was added to 40 µl of assay buffer (50 mM HEPES, 2 mM EDTA, 3 mM DTT, 100 mM NaCl, pH
7.4). To this 120 µl of pNPP substrate (50 mM) was added and the absorbance was measured every
5 min for 60 min at 405 nm.
2.5.5 Anti-coagulation studies
The effect of the various vanadium compounds on blood coagulation was investigated using a CL
Coagulation Analyzer (Beckman). The assays used were the HemosIL® activated partial
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thromboplastin time (APTT), prothromin time (PT), Fibrinogen-C (Fib-C), and D-Dimer as per the
instructions which can be found on the manufacturers website [36]. The APTT assays were
performed by adding the test compounds, positive control or diluent, and APTT reagent to plasma.
This was incubated for 3 min at 37 °C followed by addition of CaCl2 to initiate coagulation. PT was
performed by incubating PT reagent and treatments followed by the addition of plasma. Fib-C was
performed by treating diluted plasma [1:4 (v/v)], incubating, and adding bovine thrombin. D-Dimer
was performed by adding treatments and reaction buffer to plasma, hereafter D-Dimer latex reagent
was added, the vial was mixed 1-2 times and the agglutination measured. Vanadium compounds
were used at 10 μM for the screening assays and 0.01 μM, 0.1 μM, and 10 μM for concentration
dependence studies, and the positive control was heparin at 0.1 U.mL-1.
2.5.6 Statistical analysis
Error bars indicate the standard error of the mean (SEM) unless specified otherwise (n = 3). The
two-tail paired test was used to determine significance of results (p<0.05) and (p<0.01).
3. Results and discussion
3.1 Synthesis and general considerations
The 1-R-imidazole-2-carboxylic acids were prepared from the corresponding imidazole-2-
carboxaldehydes by the hydrogen peroxide facilitated oxidation in water [37,38]. This reaction
requires no heating and water was the only by-product of oxidation, making it an environmentally
friendly option. The only purification necessary was the removal of residual water in vacuo, at room
temperature for 1-methylimidazole-2-carboxylic acid yielding a quantitative product but imidazole-
2-carboxylic acid crystallized out with a mole equivalent of hydrogen peroxide. This was removed
by washing with diethylether to prevent the oxidation of oxidovanadium(IV) to vanadium(V) during
the synthesis of the complexes.
The complexes were synthesized by addition of an aqueous oxidovanadium(IV) chloride or
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oxidovanadium(IV) sulfate solution to an aqueous solution of the corresponding ligand in a 1:2
molar ratio in the presence of a base [38]. The complexes which precipitated out of solution were
easily collected by filtration. The compounds (2a-c) are slightly soluble in water and tend to
decompose in organic solvents, such as acetonitrile, alcohols and N,N-dimethylformamide (DMF),
upon dissolution at elevated temperatures.
3.2 Spectroscopic characterization
In the IR spectra (Figs S1-S3) of the complexes (2a-c) there are strong absorption bands at 981, 984
and 963 cm-1 respectively, which are assigned to the V=O stretch and are within the range 930-1030
cm-1 reported in the literature [39]. In an unsubstituted imidazole ring, the (C=N) is usually
observed at about 1660 cm-1 [40]. The coordination-induced shift of this stretching frequency from
1618-1641 cm-1 in the 2-substituted free ligands to 1606-1628 cm-1 in the complexes was observed
[40,41]. It must be acknowledged that the broad and strong peaks in the region of the (C=N) are
also a representation of the asymmetric COO- stretching frequencies (usual range of 1540-1660 cm-
1). It has been exhibited that the lower the pKa value of the acid the higher wavenumber for the
as(COO-) [42], and that seems to also be observed in this work. However, confident assignment
was only possible for the spectrum of 2a since a split peak appeared (1573 and 1606 cm-1) which
could be assigned to the (C=N) and the as(COO-) respectively (Fig. S1). In the spectra of 2b and
2c (Figs S2 and S3), there was little or no splitting of the peaks but appeared broad and that can be
interpreted as a significant overlapping of the two bands. There is also evidence of coordination of
the carboxylate group on 2a-2c as evidenced by the slight shift of the as(COO-) to lower
wavenumbers (Figs S1-S3).
The electronic spectra of 2a-2c display three low intensity d-d transitions, in the range of 330-1000
nm, which are typical of square-pyramidal oxidovanadium(IV) complexes [43]. The coordination of
the sixth ligand to oxidovanadium(IV) does not cause significant shift on spectra of square planar
oxidovanadium(IV) complexes in the formation of distorted octahedral complexes.
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3.3 X-ray crystal structure
Single crystals of VO(im4COO)2·H2O (2a) which were suitable for X-ray analysis were obtained
from the mother liquor of this complex upon standing it at room temperature for two weeks. The
structure has been reported recently by other researchers [24] but in this account it has been re-
determined and the unit cell is presented in a standard form (with a space group of P21/c not P21/a)
which gives a slightly different unit cell volume as well as slightly different unit cell dimensions
and angles. Table 1 shows the crystallographic and structure refinement data for
VO(im4COO)2·H2O while table 2 contains the selected bond lengths and angles. An ORTEP
diagram of VO(im4COO)2·H2O is presented in Fig. 1.
The structure showed discrete molecules. The molecule has a distorted octahedral geometry with the
vanadium atom bound to two bidentate im4COO- ligands, a terminal oxido, and a water molecule.
This connectivity is in contrast to the known square-pyramidal VOL2 complex with L = imidazole-
4-acetic acid [44]. There is evidence of a long V–O bond trans to the oxidovanadium(IV) V=O
bond. The V=O bond length of 1.587(2) Å falls within the range (1.56-1.66 Å) observed for
oxidovanadium(IV) compounds containing the N2O2 donor set [45,46].
(Insert Fig.1 here)
(Insert Table 1 here)
(Insert Table 2 here)
3.4 pH-metric speciation studies
The protonation (log K) and oxidovanadium(IV) complex formation (log β) constants are listed in
Table 3 together with standard deviations. The ligands exhibit two protonation processes in the pH
range 2-11 for the carboxylate and imidazole groups respectively. The log K2 values for the
protonation of imidazole in all cases, Im4COOH (6.13), Im2COOH (6.44) and MeIm2COOH
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(6.75), are lower than those of free imidazole (6.95) [47] and free MeIm (7.20) [48] respectively.
The latter is slightly more basic probably due to the presence of the electron releasing methyl
substituent. The low log K1 values (2.70, 2.72 and 1.29) for Im4COOH, Im2COOH and
MeIm2COOH respectively suggest internal hydrogen bonding between the protonated imidazole
and a carboxylate. This suggests that these ligands exist in a zwitterion form in an aqueous medium
such as that observed for simple amino acids in water. The pyrrole sites in Im4COOH and
Im2COOH do not dissociate in the pH range measured.
(Insert Table 3 here)
The proposed interactions of these ligands in aqueous solutions with oxidovanadium(IV) are
represented by Scheme 1 below. The ligand Im2COOH shows similar interactions and stability with
oxidovanadium(IV) as Im4COOH. The species distribution diagrams, which were generated using
the program HYSS [51], are represented by Fig. 2 and Figs S4-S5 (in supplementary information)
for the (VIVO)-Im4COOH, (VIVO)-Im2COOH and the (VIVO)-MeIm2COOH systems, respectively.
(Insert Scheme 1 here)
The ligands adopt bidentate chelation. The first binding constants could be calculated with a 1:1
metal-to-ligand ratio and precipitation due to the formation of hydrolysis products was observed for
titrations with a 1:2 metal-to-ligand ratio. Extensive VIVO complexation was obtained with a high
ligand excess (10-fold), allowing for the determination of the second binding constant. The
hydroxido binding ([VOL2(OH)]-) could not be fitted in these solution chemistry models while the
assumption of the binary hydroxyl species [(VO)2(OH)5]- as well as [VO(OH)3]- in highly basic
solutions fitted well. The fitting of the titration data supports the formation of monomeric [VOL]+
and [VOL2] species as well as [VOLH]2+ for MeIm2COOH. For the MeIm2COOH system,
13

ACCEPTED MANUSCRIPT
however, the coordination of a protonated ligand is observed at low pH with log β111=14.85(7). This
suggests strong binding and the possibility of a symmetrically chelating carboxylate ion, with the
imidazole nitrogen protonated (see Scheme 1). The constant for the bidentate coordination of the
carboxylate to [VO(H2O)5]2+ can be calculated to be 8.10 if the protonation equilibrium (pK2 = 6.75)
is considered in the cumulative association constant of 14.85. The formation of
[VO(MeIm2COOH)]2+, with the chelating carboxylate, was confirmed by analyzing the titration
solution at pH 2.05 with IR spectroscopy (Fig. S6). The presence of a broad band at 1643 cm-1 is not
conclusive evidence of bidentate coordination since the as(COO-) could be occluded by the
(C=N). However, the monodentate coordination of the carboxylate group was proven to be at 1628
cm-1for the same ligand interacting with the vanadyl ion. A new peak, however, was also found at
1711 cm-1 and this could not be assigned confidently.
(Insert Fig. 2 here)
The overall stability constant for the (VIVO)-MeIm2COOH system (log β120 =15.49(9)) is far larger
than that for Im4COOH system (log β12 =11.38(8)) and Im2COOH system (log β12 =11.62(6)). This
can be explained by the fact that the imidazole nitrogen in MeIm2COOH is more basic. However,
the overall stability constant of the (VIVO)-Im4COOH and (VIVO)-Im2COOH systems are
comparable to that of (VIVO)-imidazole-4-acetic acid (im-4-acetic acid) (log β120 =10.70(1)) [49]
and that with mildly basic pyridine nitrogen of picolinic acid (log β120 =12.11(2)) [50] intimating
that the inclusion of an electron donating group in the 1-position of imidazole is important to effect
stability. In addition, six-membered ring chelate formation in the (VIVO)-imidazole-4-acetic acid
system [49] results in weaker binding compared with five-membered ring systems studied here.
For the formation of the bis-coordinated [VOL2] species, the very acidic carboxylic acid groups
interact with VO2+ at very low pH and the mildly basic imidazole is able to interact with
oxidovanadium(IV) at relatively higher pH. The result is the stabilization of these bis-coordinated
14

ACCEPTED MANUSCRIPT
oxidovanadium(IV) complexes in the relevant biological pH range. This, therefore, implies that the
compounds may survive the digestion process and be delivered in the bloodstream as the neat bis-
coordinated complexes. However, substitution by bioligands will be possible once the compounds
are exposed to the blood plasma matrix.
3.5 Biochemical studies
3.5.1 In vitro glucose uptake studies
The vanadium compounds [VO(Im4COO)2], [VO(Im2COO)2], [VO(MeIm2COO)2] and the simple
vanadium salt, VOSO4, showed no cytotoxicity (>80% cell viability) between 0.5- 1 µM in the 3T3-
L1, Chang and C2C12 cell lines tested as determined by an MTT-cell viability assay. At
concentrations from 10 μM and above, the vanadium compounds proved to be cytotoxic (<80% cell
viability) (see Figure S7 in the supplementary information section). Thus, the glucose uptake ability
of the vanadium compounds was screened at concentrations of 0.5, 1 and 10 μM. The results for the
1 μM concentrations are presented in Fig. 3, while the results for all concentrations tested can be
found in Table S1 (in supplementary information).
(Insert Fig. 3 here)
There were varying effects observed in the 3T3-L1 adipocytes (Fig. 3). Metformin, along with
oxidovanadium(IV) sulfate and [VO(Im2COO)2] improved glucose uptake. However for
[VO(Im4COO)2] and [VO(MeIm2COO)2], less than basal glucose uptake was observed. For the
Chang cells, enhanced glucose uptake was observed for all of the oxidovanadium(IV) compounds
besides [VO(Im2COO)2]. In the C2C12 cells, VOSO4 and [VO(Im4COO)2] enhanced glucose
uptake to a level comparable to Metformin. It should be noted that a higher level of increase in the
percentage of glucose uptake was expected for metformin within the Chang liver cells as this is
where metformin is most effective. The results are, however, significant and the difference in our
15

ACCEPTED MANUSCRIPT
earlier findings [52] could be due to biological variation between the various passages numbers of
cells used. When higher than 1 μM concentrations of the oxidovanadium(IV) compounds were
administered to the cells, the general trend was a slight drop in glucose uptake (Table S1), possibly
due to some cytotoxic effects at these conditions.
3.5.2 PTP inhibition studies
The PTP 1B inhibition studies were carried out for the complexes in order to validate mechanistic
understanding of the effects of vanadium compounds as insulin-enhancing agents [24,53]. For the
compounds tested both Lineweaver-Burk and Dixon plots (Fig. S8) were completed and the Ki
values were determined. VO(Melm2COO)2, VO(im2COO)2 and VOSO4 had Ki values of 60, 85,
and 18 nM respectively. A Ki value could not be obtained in the nanomolar range for
VO(Im4COO)2.
In recent years a variety of PTP-1B inhibitors with a large diversity of chemical structures have
been designed for various sites on PTP-1B. Inhibitors can either be designed against the catalytic
site (either competitive or non-competitive inhibitors), low affinity binding non-catalytic sites (e.g.
site B) or allosteric inhibitors [54-56]. Examples to be considered of both natural and synthetic
chemical compounds include vanadate, phenol compounds, oleanolic acid derivatives and
sulfathiazole-related compounds as examples as either allosteric or active site inhibitors [57-62].
Various studies on PTP1B inhibitors have found inhibitors, including vanadium-based inhibitors,
with Ki values ranging from 3.2 µM – 22nM [54,62]. There are a variety of vanadium inhibitors
such as the V(III), V(IV) and V(V) complexes [54]. Vanadate is indicated as a competitive inhibitor
(Ki = 0.38 µM) [59] and the mechanism of action has in recent times been proposed to be via
inhibition of PTPase, and thus counteracting dephosphorylation of the tyrosine phosphate residue of
the beta-subunit of the insulin receptor, in such a way restoring the signaling path for cellular
glucose intake. In this study we found vanadium salt (VOSO4) to have the lowest Ki (18 nM) and
all the other complexes to have Ki values within the nanomolar range and displaying competitive
16

ACCEPTED MANUSCRIPT
inhibition, with the exception of VO(im4COO)2. A possible explanation could be the structural
rearrangement/decomposition of this complex or that the slight structural difference has not allowed
for the optimal orientation of this complex in the active site to optimise the effective inhibition of
the hydrogen bonding to be an effect competitive inhibitor [54,56].
3.5.3 Anticoagulation studies
It would be beneficial to create anti-diabetic drugs which simultaneously improve glucose
metabolism as well as reduce the hypercoagulable state of diabetic patients. In this study both the
extrinsic and intrinsic coagulation pathways were investigated [21,22]. The PT test, APTT assay, D-
dimer assay, and the Fib-C test were used to investigate the anti-coagulative effects of the
oxidovanadium(IV) complexes [21,22]. The prothrombin time (PT) is the screening test for the
extrinsic coagulation pathway. In the PT test, the time required for plasma to clot following
conversion of prothrombin to thrombin upon addition of CaCl2 is measured. The activated partial
thromboplastin time (APTT) assay investigates the intrinsic pathway. In an APTT reagent,
negatively charged particles such as silica are mixed with phospholipids and buffers providing an
ideal environment for activation of intrinsic plasma proteins. After incubation of the APTT reagent
with plasma, CaCl2 is added which initiates multiple steps of the intrinsic pathway penultimately
leading to a fibrin clot. The time from addition of the calcium salt until the clot formation is
measured in seconds. In the D-Dimer assay, elevated levels of fibrin reflect a state of activated
coagulation and fibrinolysis (hypercoagulation state). The D-Dimer reagent contains latex particles
coated with monoclonal antibody specific for the D-Dimer domain of fibrin (soluble fibrin). These
D-Dimers are produced when cross-linked fibrin is degraded. In the presence of the D-Dimer
reagent these particles agglutinate and the degree of agglutination is directly proportional to the D-
Dimer concentration. The Fib-C screening test uses an excess of thrombin to convert fibrinogen to
fibrin in diluted plasma. High levels of fibrinogen are usually associated with an increased risk of
cardiovascular disease, and the results for the vanadium complexes tested in this study do not alter
17

ACCEPTED MANUSCRIPT
the fibrinogen levels and maintain the control levels therefore they would not potentially increase
cardiovascular complications. Despite the fact that diabetes mellitus sufferers are usually at a higher
risk of thrombotic associated fatalities there has been no investigation into the anti-coagulation
potential of the oxidovanadium(IV) complexes before this account.
For the APTT and PT screening tests, only heparin (positive control) significantly increased clotting
times relative to the control (Fig. 4). Thus, the oxidovanadium(IV) complexes did not affect either
the intrinsic or extrinsic pathways [21,22]. From the Fib-C screening test, it can be seen that
[VO(Im4COO)2] decreased fibrin formation slightly but this was within error of the control (solvent
vehicle) and certainly not as significant as the positive control, heparin. From these results,
therefore, it is evident that the complexes do not affect either the enzymes playing a role in the
intrinsic and extrinsic pathways (as evidenced by lack of inhibition in the PT and APPT tests). They
also did not affect the formation of fibrin as seen in the Fib-C test however, the crosslinking
required to form a solid clot was inhibited at the concentrations tested as noted by the D-Dimer test.
The oxidovanadium(IV) complexes could potentially reduce the hypercoagulation state. However,
these results suggested a potential role of vanadium in regulation of the coagulation-thrombolysis
process but need to be investigated further.
(Insert Fig. 4 here)
4. Conclusions
Three oxidovanadium(IV) complexes with bidentate imidazolyl-carboxylato ligands were
successfully prepared and characterized. Aqueous potentiometric titrations revealed that the
incorporation of the low pKa carboxylic acid group improved the stability of the complexes at low
pH values compared to the 2-(2ˊ-hydroxyphenyl)-1R-imidazoline ligands presented by us in the
literature [52], and other systems with relatively high pKa hydroxyl groups. The prepared complexes
exhibited minimal toxicity in the concentration range tested as well as notable glucose lowering
18

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activity in an in vitro assay using muscle, liver and fat cells. The findings from the PTP 1B
inhibition studies indicated competitive binding and it is could be speculated that the mechanism of
action of the oxidovanadium(IV) complexes could be similar to vanadate, however, this requires
further investigation since the complexes could be decomposing and eventually forming vanadate.
In addition, there was no significant effect of the complexes for any coagulation tests, with the
exception of inhibition of D-dimer formation. Based on the concentrations tested there appears to be
potential for the vanadium complexes tested to be able to selectively inhibit the formation of
crosslinking of fibrin during its formation from fibrinogen, however these results would need to be
completed in a future study using an in vivo model. The prepared oxidovanadium(IV) complexes
showed potential, and this warrant a further investigation into their anti-diabetic effects using an in
vivo diabetic rat model.
Acknowledgements
The authors are grateful for financial assistance from the National Research Foundation (NRF-TTK
grant) and Medical Research Council (MRC-SIR). We also thank Rhodes University (RU) and
Nelson Mandela Metropolitan University (NMMU) for the facilities to carry out this research. We
also wish to thank Rudi Nicholas (NMMU) for his assistance with the PTP 1B assays. IZG, RSW
and VU are grateful for bursaries received from the Andrew Mellon Foundation, Sasol (Pty) Ltd and
Rwanda Education Board, respectively. We thank Dr Adeniyi Ogunlaja for his assistance. We are
also grateful to some reviewers for constructive comments.
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Legend of figures
Fig. 1. An ORTEP diagram of VO(im4COO)2·H2O with ellipsoids drawn at 50% probability level.
Fig. 2. Speciation distribution curves for complexes formed in the (VIVO)-imidazole-4-carboxylic
acid system (M:L ratio is 1:4).
Fig. 3. The effects of metformin (Met), oxidovanadium(IV) sulfate (VOSO4), [VO(Im4COO)2],
[VO(Im2COO)2] and [VO(MeIm2COO)2] (at 1 μM concentration) on Chang, C2C12 and 3T3-L1
glucose uptake. The basal glucose uptake, is represented as 100% (Control). Error bars indicate
SEM (n=3), *(p <0.05) relative to the (Con).
Fig. 4. The effect of [VO(Im4COO)2], [VO(Im2COO)2] and [VO(MeIm2COO)2] (10 μM) on APTT
(A) and PT (B) clotting times, on fibrin formation (C) and D-Dimer formation (D). The control
represents untreated sample while the positive control represents the anticoagulant heparin (0.1
U.mL-1) (n = 3)
List of Schemes
Scheme 1. The stepwise formation of oxidovanadium(IV) complexes. LH and L- are omitted in the
complexation equilibria for clarity.
24

ACCEPTED MANUSCRIPT
Fig. 1.
100
80
60
40
20
0
[VO(im4COO)2]
[VO(im4COO)]+
[(VO)2(OH)5]-
[VO(OH)3]-
VO2+
3
5
7
9
11
pH
Fig. 2.
25

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180
160
140
120
100
80
Chang
C2C12
3T3-L1
*
*
*
*
*
*
*
*
60
40
20
0
]
]
]
4
rol
Cont
O
)
)
)
2
2
2
S
O
O
O
ormin
f
O
V
et
M
m2CO
eI
(Im4CO
(Im2CO
O
O
(M
V
[
V
[
O
V
[
Treatment (1x10-6 M)
Fig. 3.
26

ACCEPTED MANUSCRIPT
Control
Positive control
[VO(im4COO)2]
[VO(im2COO)2]
[VO(MeIm2COO)2]
60
50
40
30
20
10
0
25
20
15
10
5
(A)
(B)
0
400
300
200
100
0
350
300
250
200
150
100
50
(C)
(D)
0
Fig. 4.
List of Schemes
27

ACCEPTED MANUSCRIPT
Scheme 1. The stepwise formation of oxidovanadium(IV) complexes. LH and L- are omitted in the
complexation equilibria for clarity.
28

ACCEPTED MANUSCRIPT
List of Tables
Table 1. Crystallographic data and structure refinement for VO(im4COO)2·H2O
Compound
Empirical formula
Formula weight
Crystal color
Crystal system
Space group
Temperature
a, Å
VO(im4COO)2·H2O
C8H8N4O6V
307.12
Blue
Monoclinic
P21/c
200 K
13.2735(7)
12.0099(8)
7.0971(4)
90
b, Å
c, Å
α, deg
β, deg
98.082(3)
90
γ, deg
V, Å3
1120.14(11)
4
Z
ρcalc, g/cm3
Wavelength, Å
Total reflections
Unique reflections
R
1.821
0.71073
10700
2774
0.0431
Rw
0.1039
29