Synthesis, structures, and insulin-like activity of oxidovanadium(V) complexes derived from 2-chloro-N′-(3-ethoxy-2-hydroxybenzylidene)benzohydrazide

Abstract Three new oxidovanadium(V) complexes, [VOLL′] (L = 2-chloro-N′-(3-ethoxy-2hydroxybenzylidene)benzohydrazide, L′ = acetohydroxamate for 1, methylmaltolate for 2, and ethylmaltolate for 3), have been prepared. The complexes have been characterized by physicochemical methods and single-crystal X-ray determination. Vanadium in each complex is coordinated by the NOO donor set of L, the OO donor set of L′, and one oxido, forming octahedral coordination. The complexes were administered intragastrically to both normal and alloxan-diabetic mice for two weeks. The biological activities show that the complexes at doses of 10.0 and 20.0 mg V·kg−1 can significantly decrease the blood glucose level in alloxan-diabetic mice, but the blood glucose level in the treated normal mice was not altered.


General methods and materials
Starting materials, reagents, and solvents were purchased from commercial suppliers with aR grade and used without purification. elemental analyses were performed on a Perkin-elmer 240C elemental analyzer. IR spectra were recorded on a Jasco FT/IR-4000 spectrometer as KBr pellets from 4000 to 400 cm −1 . uV-vis spectra were recorded on a Perkin-elmer Lambda 900 spectrometer. 1 H NMR spectra were recorded on a Bruker spectrometer at 300 MHz. HRMS data were obtained with eSI (electrospray ionization) mode. H 2 L was prepared according to the literature method [25].

Synthesis of the complexes
Complexes 1, 2, and 3 were prepared by the method described here. a methanolic solution (30 mL) of VO(acac) 2 (0.27 g, 1.0 mmol) was added to a methanolic solution (20 mL) of H 2 L (0.32 g, 1.0 mmol) and the bidentate ligands (1.0 mmol each), with stirring. The mixtures were stirred at room temperature for 30 min to give deep brown solution. The resulting solution was allowed to stand in air for a few days until three-quarters of the solvent evaporated. Brown block-shaped single crystals of the complexes, suitable for X-ray single-crystal diffraction, were formed at the bottom of the vessel. The crystals were

X-ray crystallography
diffraction intensities for the complexes were collected at 298(2) K using a Bruker SMaRT 1000 CCd area-detector diffractometer with MoKα radiation (λ = 0.71073 Å). The collected data were reduced with SaINT [26], and multi-scan absorption correction was performed using SadaBS [27]. Structures of the complexes were solved by direct methods and refined against F 2 by full-matrix least-squares using SHeLXTL [28]. all of the non-hydrogen atoms were refined anisotropically. The amino H in 1 was located from a difference Fourier map and refined isotropically, with N-H distance restrained to 0.90(1) Å. The remaining hydrogens were placed in calculated positions and constrained to ride on their parent atoms. Crystallographic data for the complexes are summarized in table 1. Selected bond lengths and angles are given in table 2.

Glucose-lowering assay
Male Kunming mice, weighing about 25-32 g, were obtained from the experimental animal Center, Shandong Lukang Pharmaceutical Co., Ltd. of China, and maintained on a light/dark cycle. all animals were allowed free access to food and water. Temperature and relative humidity were maintained at 24 °C and 50%. Mice were acclimatized for seven days prior to induction of diabetes. diabetes was induced by a single intra-peritoneal injection of freshly prepared alloxan (200 mg·kg −1 body weight) in 0.9% saline. The control mice were injected with an equal volume of vehicle. after seven days, blood was collected from the tail vein and serum samples were analyzed for blood glucose. animals showing fasting (12 h) blood glucose higher than 11.1 mmol·L −1 were considered to be diabetic and used for the study.
The experimental animals were randomly divided into six groups with six mice each according to the blood glucose. Group 1, normal control group: normal mice treated with 0.5% carboxymethyl cellulose (CMC). Groups 2-4, treated normal group: normal mice treated with 20 mg V·kg −1 vanadium complexes. Group 5, diabetic control group: alloxan diabetic mice treated with 0.5% CMC. Groups 6-11, treated diabetic group: alloxan diabetic mice treated with vanadium complexes at dose of 10 and 20 mg V·kg −1 ig. The complexes were administered as suspensions in 0.5% CMC. The substances were administered intragastrically once a day at the volume of 10 mL·kg −1 for two weeks.
Throughout the experimental period, the body weight of mice was monitored daily. Blood samples were obtained from the tail vein of the mice and blood glucose levels were determined with an accu-Chek blood glucose monitor (Roche diagnostics GmbH, Mannheim, Germany).

Chemistry
The aroylhydrazone, 2-chloro-N′-(3-ethoxy-2-hydroxybenzylidene)benzohydrazide, was readily prepared by condensation of 3-ethoxysalicylaldehyde with 2-chlorobenzohydrazide in methanol. The complexes were prepared by reaction of equimolar quantities of the aroylhydrazone and VO(acac) 2 with acetohydroxamic acid, methylmaltol, and ethylmaltol, respectively, in methanol. Crystals of the complexes are stable in open air at room temperature. elemental analyses are in agreement with the chemical formulas proposed for the compounds. It should be pointed out that the vanadium in the starting materials is V(IV), but it appears to be V(V) in the complexes, indicating that it was oxidized by air during the reaction.

IR and UV-vis spectra
The medium and broad absorption at 3443 cm −1 in the spectrum of the aroylhydrazone substantiates the presence of phenol group, which is absent in the complexes. The sharp bands indicative of N-H vibrations are located at 3175 and 3210 cm −1 , respectively, in the spectra of H 2 L and 1. The intense band indicative of the C=O vibration at 1663 cm −1 in the spectrum of the aroylhydrazone is absent in the complexes, indicating the enolization of the amide functionality and subsequent proton replacement by V. The strong absorption bands at 1612, 1602, and 1603 cm −1 for 1, 2, and 3, respectively, are assigned to the azomethine ν(C=N) [37], which are shifted to lower wavenumbers when compared with that of the free aroylhydrazone (1620 cm −1 ). The absorptions at 970, 973, and 978 cm −1 can be assigned to the V=O vibration [35]. electronic spectra of the complexes were recorded at 10 −5 mol·L −1 in acetonitrile from 200 to 800 nm. In the uV-vis region, the complexes show bands at approximately 350 nm and weak bands centered at 450 nm for 1 and 475 nm for 2 and 3. The weak bands are attributed to intramolecular charge transfer transitions from the p π orbital on the nitrogen and oxygen to the empty d orbitals of the metal [38]. The intense bands observed at 215 and 280 nm for the complexes are assigned to intraligand π-π* transitions [38].

Effects of complex on blood glucose in both normal and alloxan-diabetic mice
The complexes were administered intragastrically to both normal and alloxan-diabetic mice for two weeks. The results (table 3) showed that the complexes had blood glucose-lowering effect at doses of 10.0 and 20.0 mg V·kg −1 , which can significantly decrease the blood glucose level in alloxan-diabetic mice, but the blood glucose level in the treated normal mice (20.0 mg V·kg −1 ig for two weeks) was not altered as compared with the untreated normal mice (p > 0.05). The alloxan-diabetic mice exhibited significant hyperglycemia. after two-week administration with the complexes, the blood glucose level was decreased compared with the diabetic control group (p < 0.05). It is obvious that the order of the glucose-lowering effect of the complexes is 2 ≈ 3 > 1. during the experiment, the mean body weight in alloxan-diabetic mice was lower than normal mice. Two-week administration of the complexes had no effect on the body weight in the diabetic group, compared with the diabetic control group (table 4). The results agree with the conclusion that vanadium complexes with Schiff base ligands bearing Cl possess increased anti-diabetic properties [39]. In general, the glucose-lowering abilities of the complexes are similar to vanadium complexes reported [39,40].

Conclusion
The present study reports synthesis, characterization, and crystal structures of three new oxidovanadium(V) complexes derived from 2-chloro-N′-(3-ethoxy-2hydroxybenzylidene)benzohydrazide and similar bidentate ligands. Methylmaltol, ethylmaltol, and acetohydroxamic acid as co-ligands readily coordinate to V through the carbonyl and deprotonated phenol groups. The complexes have effective insulin-like activity on alloxan-diabetic mice.

Disclosure statement
No potential conflict of interest was reported by the authors.