Synthesis, structure, solution behavior, reactivity and biological evaluation of oxidovanadium(iv/v) thiosemicarbazone complexes

Article abstract of DOI:10.1039/c8dt01668b

The synthesis and characterization of an oxidovanadium(iv) [V^IVO(L)(acac)] (1) and of two dioxidovanadium(v) [V^VO₂(L′)] (2) and [V^VO₂(L)] (2a) complexes of the Schiff base formed from the reaction of 4-(p-fluorophenyl) thiosemicarbazone with pyridine-2-aldehyde (HL) are described. The oxidovanadium(iv) species [V^IVO(L)(acac)] (1) was synthesized by the reaction of V^IVO(acac)₂ with the thiosemicarbazone HL in refluxing ethanol. The recrystallization of [V^IVO(L)(acac)] (1) in DMF, CH₃CN or EtOH gave the same product i.e. the dioxidovanadium(v) complex [V^VO₂(L)] (2a); however, upon recrystallization of 1 in DMSO a distinct compound [V^VO₂(L′)] (2) was formed, wherein the original ligand L^- is transformed to a rearranged one, L′^-. In the presence of DMSO the ligand in complex 1 is found to undergo methylation at the carbon centre attached to imine nitrogen (aldimine) and transformed to the corresponding V^VO₂-species through in situ reaction. The synthesized HL and the metal complexes were characterized by elemental analysis, IR, UV-Vis, NMR and EPR spectroscopy. The molecular structure of [V^VO₂(L′)] (2) was determined by single crystal X-ray crystallography. The methylation of various other ligands and complexes prepared from different vanadium precursors under similar reaction conditions was also attempted and it was confirmed that the imine methylation observed is both ligand and metal precursor specific. Complexes 1 and 2 show in vitro insulin-like activity against insulin responsive L6 myoblast cells, higher than V^IVO(acac)₂, with complex 1 being more potent. In addition, the in vitro cytotoxicity studies of HL, and of complexes 1 and 2 against the MCF-7 and Vero cell lines were also done. The ligand is not cytotoxic and complex 2 is significantly more cytotoxic than 1. DAPI staining experiments indicate that an increase in the time of incubation and an increase of concentration of the complexes lead to the increase in cell death.

Full text of DOI:10.1039/c8dt01668b

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Tiekink and R. Dinda, Dalton Trans., 2018, DOI: 10.1039/C8DT01668B.
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Synthesis, structure, solution behavior, reactivity and biological evaluation of
oxidovanadium(IV/V) thiosemicarbazone complexes.
Saswati,a Pedro Adão,b Sudarshana Majumder,a,c Subhashree P. Dash,a,d Satabdi Roy,a,e Maxim L. Kuznetsov,b João
Costa Pessoa,b,* Clara S. B. Gomes,b Manasi R. Hardikar,f Edward R. T. Tiekinkg and Rupam Dinda*a
aDepartment of Chemistry, National Institute of Technology, Rourkela 769008, Odisha, India
bCentro de Química Estrutural, Instituto Superior Técnico, Universidade de Lisboa, Av. Rovisco Pais, 1049‐001
Lisboa, Portugal
cDarmstadt University of Technology, Clemens‐Schöpf Institute of Organic Chemistry and Biochemistry, Alarich‐
Weiss Str. 4, 64287 Darmstadt, Germany.
dDepartment of Basic Sciences, Parala Maharaja Engineering College, Sitalapalli, Brahmapur, Odisha 761003, India
eDepartment of Chemistry, Indian Institute of Technology, Kanpur 208016, Uttar Pradesh, India.
fBiometry and Nutrition Group, Agharkar Research Institute, G.G. Agrakar Road, Pune 411004
gResearch Centre for Crystalline Materials, School of Science and Technology, Sunway University, Bandar Sunway,
Selangor Darul Ehsan 47500, Malaysia
_____________________________________________________________________________________
Abstract
The synthesis and characterization of an oxidovanadium(IV) [VIVO(L)(acac)] (1) and of two dioxidovanadium(V)
[VVO2(L´)] (2) and [VVO2(L)] (2a) complexes of the Schiff base formed from the reaction of 4‐(p‐fluorophenyl)
thiosemicarbazone with pyridine‐2‐aldehyde (HL) is described. The oxidovanadium(IV) species [VIVO(L)(acac)] (1)
was synthesized by the reaction of VIVO(acac)2 with the thiosemicarbazone HL in refluxing ethanol. The
recrystallization of [VIVO(L)(acac)] (1) in DMF, CH3CN or EtOH gave the same product i.e. the dioxidovanadium(V)
complex [VVO2(L)] (2a); however, upon recrystallization of 1 in DMSO a distinct compound [VVO2(L')] (2) was
formed, wherein the original ligand L is transformed to a rearranged one, L´. In the presence of DMSO the ligand
in complex 1 is found to undergo methylation at the carbon centre attached to imine nitrogen (aldimine) and
transformed to the corresponding VVO2‐ species through in situ reaction. The synthesized HL and the metal
1

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complexes were characterized by elemental analysis, IR, UV–Vis, NMR and EPR spectroscopy. The molecular
structure of [VVO2(L')] (2) was determined by single crystal X–ray crystallography. The methylation of various other
ligands and complexes prepared from different vanadium precursors under similar reaction conditions was also
attempted and it was confirmed that the imine methylation observed is both ligand and metal precursor specific.
Complexes 1 and 2 show in vitro insulin‐like activity against insulin responsive L6 myoblast cells, higher than
VIVO(acac)2, with complex 1 being more potent. In addition, the in vitro cytotoxicity studies of HL, and of complexes
1 and 2 against the MCF–7 and Vero cell lines were also done. The ligand is not cytotoxic and complex 2 is
significantly more cytotoxic than 1. DAPI staining experiments indicate that increase in time of incubation as well
as increase of concentration of the complexes lead to increase in cell death.
_____________________________________________________________________________________
Introduction
Reports of dimethylsulfoxide (DMSO) acting as a methylating agent in aromatic hydrocarbons1a and in N‐
methylation of amines1b,c have attracted significant attention, but DMSO is a comparatively stable molecule and
has very few applications as reactant in organic reactions.1d The methylation of the C5 carbon of deoxycytosine
nucleotide was reported to be brought about by DMSO in the presence of Fenton’s reagent, where a methyl
radical generated from DMSO is responsible for the methylation.1e Another example is the methylation of aromatic
1a
hydrocarbons by DMSO reported to take place via the methylsulfinyl carbanion generated from DMSO. DMSO
was also reported to be a methylating agent for amines under catalyst free conditions.1b
The coordination chemistry of vanadium has attracted increasing interest, due to its biological properties, 2–9 and
for the use of oxidovanadium complexes in oxidation and oxo transfer reactions catalysis,10‐16 and potential
medicinal applications.17‐21 Vanadium is one of the trace bio‐elements existing in the human body whose
17,19–
complexes have been found to show antibacterial, antitumor, insulin‐enhancing and anti‐parasitic effects.
21a,22‐25,26 Vanadium complexes could also suppress the growth and spread of existing tumours by inhibiting tumour
2

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cell proliferation, inducing apoptosis and limiting the invasion and metastatic potential of neoplastic cells. 19,20,25,27–
28
Inorganic VIVO‐ and VVO2‐compounds, as well as some vanadium(IV)/(V) compounds, are insulin‐enhancing
17,19,29‐33
agents
and the discovery of the in vivo insulin mimesis per os of oxidovanadates(V),34 of the
oxidovanadium(IV)‐ (VIVO‐) precursor, VIVOSO4,35 and of the more potent bis(maltolato)‐oxidovanadium(IV) (BMOV)
30,32,36,37
encouraged exploration of vanadium complexes for the treatment of type II diabetes, as well as many
attempts to understand the mechanisms of action. 30,32,37‐41 Thus, several different approaches have been used to
develop orally active vanadium containing insulin enhancing agents.32,33,41,42,43
Thiosemicarbazones are an important class of compounds that have considerable pharmacological interest due to
their antibacterial, anti‐viral, anti‐malarial, anti‐leprotic, anti‐tumor and anti‐cancer activities.44–47 Metal complexes
of thiosemicarbazone ligands have shown variable bonding properties and structural diversity, along with
promising biological applications and ion sensing abilities.48–59
Keeping these factors in mind and in applying our expertise in oxidovanadium chemistry, 60 herein we report the
formation of an oxidovanadium(IV) complex [VIVO(L)(acac)] (1) (acac = acetylacetonato) derived from the Schiff
base formed by the condensation of 4‐(p‐fluorophenyl)thiosemicarbazone with pyridine‐2‐aldehyde (HL), which
gradually oxidizes to the dioxidovanadium(V) complex [VVO2(L´)] (2) upon standing in DMSO solution, with an
unprecedented methylation of the aldimine carbon in the ligand backbone (Scheme 1). No such methylation
occurs upon recrystallization of 1 in other solvents such as DMF, CH3CN, EtOH, where [VIVO(L)(acac)] (1) undergoes
oxidation to [VVO2(L)] (2a), with no modification in the ligand.
The synthesized ligand and the metal complexes were characterized by elemental analysis, IR, UV–Vis, NMR and
EPR spectroscopy. The molecular structure of [VVO2(L´)] (2) was also determined by single crystal X–ray diffraction
crystallography (XRD). The outline of the process of formation of the methylated oxidovanadium(V) complex 2 is
tentatively explained and further supported by DFT calculations. In addition, from the comparative study of various
other ligands and complexes prepared from different vanadium precursors in similar reaction conditions, it was
fortified that the imine methylation observed is both ligand and metal precursor specific for this system.
Complexes 1 and 2 were assayed for their in vitro insulin‐like activity against insulin responsive L6 myoblast cells.
Results indicate both complex 1 and 2 to exhibit considerable insulin‐like activity, higher than VIVO(acac)2, with 1
3

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being more potent than 2. In addition, the in vitro toxicity studies of 1 and 2 against MCF–7 (breast cancer cells)
and Vero cells (noncancerous mammalian cells) were also assayed and complex 1 was found to have higher
cytotoxicity towards both cell lines. Results of DAPI staining experiments carried out indicated that increase in time
of incubation and/or increase of concentration of complexes 1 and 2 lead to increase in cell death.
Experimental Section
Materials and methods
[VIVO(acac)2], where acac is acetylacetonate, was prepared as described in the literature.61 Reagent grade solvents were dried
and distilled prior to use. All other chemicals were of reagent grade, available commercially and used as received. HPLC grade
DMSO and CH3CN were used for spectroscopic and electrochemical studies, and ethanol or methanol were used for synthesis of
ligands and metal complexes. Commercially available TEAP (tetraethylammonium perchlorate) was properly dried and used as a
supporting electrolyte for recording cyclic voltammograms of the complexes. Elemental analyses were made on a Vario ELcube
1
CHNS Elemental analyzer. IR spectra were recorded on a Perkin‐Elmer Spectrum RXI spectrometer. H and 13C NMR spectra
were recorded with a Bruker Ultrashield 400 MHz spectrometer using SiMe4 as an internal standard and 51V NMR spectra were
obtained on a Bruker Avance III 400 MHz spectrometer and chemical shift 51V values (δV) were referenced relative to neat
VVOCl3 as external standard. Electronic spectra were recorded on a Lamda 25, Perkin Elmer spectrophotometer.
Electrochemical data were collected using PAR Versastat‐II instrument driven by E‐chem software (PAR) at 298 K in a dry
nitrogen atmosphere. Cyclic voltammetry experiments were carried out with Pt working and auxiliary electrodes and Ag/AgCl as
reference electrode. EPR spectra were recorded with a Bruker EMX X‐band spectrometer either at 77 K or at room
temperature. X‐band EPR measurements were carried out on a JEOL JES‐FA 200 and Bruker EMX EPR Spectrometer. The mass
spectrometer used is an ion trap equipped with an ESI ion source (Thermo Scientific). The mass spectrometer was operated in
the ESI positive and negative ion modes, with the following optimized parameters: ion spray voltage, + 4/ ‐5.3 kV; capillary
voltage, 5/‐20 V; tube lens offset, 63/‐124; sheath gas (N2), 20 arbitrary units; capillary temperature 275 ºC. The spectra,
typically correspond to the average of 20‐30 scans, and were normally recorded in the range 100‐2000 Da. MCF–7 cells (a
human breast adenocarcinoma cell line) and Vero cells (noncancerous mammalian cells, to check the effect of the complexes
on the noncancerous cells) were obtained from the National Centre of Cell Science (NCCS), Pune, India and were maintained in
minimal essential medium supplemented with 10% fetal bovine serum (FBS), penicillin‐streptomycin solution and incubated at
37°C in 5% CO2 and 95% humidified incubator.
Synthesis of ligand (HL)
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The thiosemicabazide was prepared from distilled p‐fluoroaniline by a method reported earlier.62 The Schiff base ligand, 4‐(p‐
fluorophenyl) thiosemicarbazone of pyridine‐2‐aldehyde, was prepared in 80–90% yield by stirring an equimolar ratio of the
thiosemicabazide with pyridine‐2‐aldehyde in methanol medium by standard procedures.63 The resulting compound was
filtered, washed thoroughly with methanol and dried over fused CaCl2.
HL: Yield: 85%. Anal. calc. for C13H11N4SF: C, 56.92; H, 4.04; N, 20.42. Found: C, 56.95; H, 4.00; N, 20.41. Main IR peaks (KBr,
cm1, atom numbering is depicted in Scheme 1): ν(N(1)–H), 3311 s; ν(N(2)–H), 2967 s; ν(C(8)–H), 2825 m; ν(C=C), 1638 s; ν(C(7)–
S(1)), 836 s. 1H NMR (DMSO–d6, 400 MHz, ppm): δH: 12.08 (s, 1H, N(1)H), 10.29 (s, 1H, N(2)H), 8.59 (s, 1H, C(8)H), 8.58–7.20 (m,
8H, C6H4). 13C NMR (DMSO‐d6, 100 MHz, ppm): δc: 177.25, 161.46, 159.05, 153.58, 149.82, 143.62, 136.98, 135.77, 128.86,
128.77, 124.76, 121.12, 115.37.
Synthesis of [VIVO(L)(acac)] (1)
To a solution of HL (0.274 g, 0.100 mmol) in hot ethanol, VIVO(acac)2 (0.265 g, 1.0 mmol) was added. The mixture was refluxed
for 2 h, yielding a dark green color crystalline precipitate of [VIVO(L)(acac)] (1): Yield: 75%. Anal. calc. for C18H17FN4VO3S: C,
49.21; H, 3.90; N, 12.75. Found: C, 49.24; H, 3.87; N, 12.73. Main solid state IR peaks (KBr, cm1): ν(N(1)–H), 3317 s; ν(C=C), 1602
s; ν(V=O), 949 s; ν(C(7)–S(1)), 762 s. Spin Hamiltonian parameters determined from EPR spectra at 77 K: gx,y =1.978, gz=1.952,
|Ax,y| = 51.9×104 cm1, |Az| = 153.2×104 cm1, if the spectrum is simulated as having tetragonal symmetry. Assuming the x
and y axis are not equivalent: gx,y =1.977 , gx,y =1.981, gz=1.95, |Ax,y| =51.1 ×104 cm1, |Ax,y| = 52.6×104 cm1, |Az| =
152.9×104 cm1.
Synthesis of [VVO2(L´)] (2)
The dark green crystalline [VIVO(L)(acac)] (1) was dissolved in DMSO at 60⁰C (ca. 25 mM) and allowed to evaporate at room
temperature (ca. 22 – 27 ⁰C) for recrystallization. Reddish brown crystals were obtained from DMSO after 15 days.
[VVO2(L´)] (2): Yield: 65%. Anal. calc. for C14H12FN4VO2S: C, 45.41; H, 3.27; N, 15.13. Found: C, 45.43; H, 3.25; N, 15.16. Main IR
peaks (KBr, cm1): ν(N(1)–H), 3268 s; ν(C=C), 1604 s; ν(V=O), 937, 925 s; ν(C(7)–S(1)), 765 s. 1H NMR (DMSO–d6, 400 MHz, ppm):
δH: 10.20 (s, 1H, N(1)H), 8.72–7.18 (m, 8H, C6H4), 2.66 (s, 3H, C(8)H3). 13C NMR (DMSO‐d6, 100 MHz, ppm): δc: 154.34, 153.80,
143.72, 141.34, 137.23, 130.68, 129.12, 126.94, 125.35, 122.48, 122.40, 115.88, 115.65, 67.87. 51V NMR (DMSO–d6, ppm) δv: ‐
434, ‐577.
Synthesis of [VVO2(L)] (2a)
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The dark green crystalline residue of [VIVO(L)(acac)] (1) was dissolved in EtOH (ca. 25 mM) for recrystallization and allowed to
evaporate at room temperature (ca. 22 – 27 ⁰C). A yellow residue of the complex was obtained after few days. Similar results
were obtained when recrystallization of 1 was done in CH3CN and DMF.
[VVO2(L)] (2a): Yield: 70%. Anal. calc. for C13H10FN4VO2S: C, 43.83; H, 2.83; N, 15.73. Found: C, 43.80; H, 2.85; N, 15.76. Main IR
peaks (KBr, cm1): ν(N(1)–H), 3256 s; ν(C(8)–H), 2834 m; ν(C=C), 1589 s; ν(V=O), 934, 922 s; ν(C(7)–S(1)), 775 s. 1H NMR (DMSO–
d6, 400 MHz, ppm): δH 10.29 (s, 1H, N(1)H), 8.79 (s, 1H, C(8)H), 8.78–7.16 (m, 8H, C6H4). 13C NMR (DMSO‐d6, 100 MHz, ppm): δc
166.16, 160.28, 159.10, 156.73, 150.24, 149.31, 138.11, 137.27, 125.45, 119.96, 119.88, 116.29, 116.06. 51V NMR (DMSO–d6,
ppm) δv: ‐434.
[N‐(4‐fluorophenyl)‐5‐(pyridin‐2‐yl)‐1,3,4‐thiadiazol‐2‐amine] (3)
The compound [VVO2(L)] (2a) was dissolved in DMSO (ca. 25 mM) and the solution kept at 60 ⁰C. There was no change in colour
1
but there was a significant alteration to the H NMR spectrum after 5 days under these conditions (Figs. 10 and S15). After
cooling the reaction mixture and letting it stand at room temperature, a crystalline material slowly formed after approximately
a week. The obtained crystalline product corresponds to a cyclized and oxidized compound 3 (Fig. S1), which was characterized
by single‐crystal x‐ray diffraction.
Computational Details
The full geometry optimization of the molecular structures was carried out at the DFT level of theory using B3P86 functional64
with the help of the Gaussian‐09 program package.65 This functional was found to be sufficiently accurate for the theoretical
studies of structural parameters and 51V NMR chemical shifts of various V complexes.66 No symmetry operations were applied
for any of the structures calculated. The geometry optimization was carried out using a relativistic Stuttgart pseudopotential
which describes 10 core electrons and the appropriate contracted basis set (8s7p6d1f)/[6s5p3d1f] for the vanadium atom67 and
the 6‐31G* basis set for other atoms. The Hessian matrix was calculated analytically for the optimized structures to prove the
location of correct minima (no imaginary frequencies) and to estimate the thermodynamic parameters, the latter being
calculated at 25 °C.
The total energies corrected for solvent effects Es were estimated at the single‐point calculations on the basis of gas‐
phase geometries at the IEFPCM‐B3P86/6‐311+G*//gas‐B3P86/6‐31G* level of theory using the polarizable continuum model in
the IEFPCM version68 with DMSO as the solvent. The UAKS model was applied for the molecular cavity with dispersion,
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repulsion and cavitation contributions. The entropic term in DMSO solution (Ss) was calculated according to the procedure
described by Wertz69a and Cooper and Ziegler69b using eqn (1)–(4):
ΔS RlnVs
Vm,gas
(1)
(2)
m,liq
1

s
ΔS RlnV
V
m
m,liq
2
,s
,s
S
liq (S gas RlnVs
Vm,gas)
m,liq
α 
(3)
,s
(S gas RlnVs
Vm,gas
)
m,liq
Ss = Sg + Ssol = Sg + [S1 + (Sg + S1) + S2] =
Sg + [(–11.60 cal/mol∙K) – 0.27(Sg – 11.60 cal/mol∙K) + 5.25 cal/mol∙K]
(4)
where Sg is the gas‐phase entropy of the solute, ΔSsol is the solvation entropy, S°,sliq, S°,sgas and Vsm,liq are the standard entropies
and molar volumes of the solvent in the liquid or gas phases (188.8 and 306.7 J/mol∙K and 71.03 mL/mol, respectively, for
DMSO), Vm,gas is the molar volume of the ideal gas at 25 °C (24450 mL/mol), V°m is the molar volume of the solution that
correspond to the standard conditions (1000 mL/mol). The enthalpies and Gibbs free energies in solution (Hs and Gs,
respectively) were estimated using equations 5 and 6
Hs = Es(6‐311+G*) + Hg(6‐31G*) – Eg(6‐311+G*)
Gs = Hs – TSs
(5)
(6)
where Es and Eg are the total energies in solution and the gas phase and Hg is the gas‐phase enthalpy calculated at the
corresponding level.
Magnetic shielding was calculated for the equilibrium geometries using the GIAO method70 at the IEFPCM‐B3P86/6‐
311+G(2d,p)//6‐31G(d) level. The 51V chemical shifts were estimated relative to VVOCl3, calculated at the same level of theory.
The 51V hyperfine coupling constants were estimated at the single‐point calculations using the BHandHLYP functional and 6‐
311G* basis set for all atoms on the basis of the equilibrium geometry obtained at the B3P86/6‐31G*(V‐ECP) level of theory.
The anisotropic 51V hyperfine coupling constants Ax, Ay, and Az were estimated as the sum of the isotropic Fermi contact term
and corresponding dipolar hyperfine interaction term.71 It must be emphasized that for a VIV‐species the hyperfine coupling
constant (A) values may be negative, but in the literature normally it is their absolute value that is reported. This simplification
was also adopted in this work. Previously, it was found that half‐and‐half functionals such as BHandHLYP demonstrate better
performance in the calculations of EPR parameters than many other hybrid functionals due to more accurate estimates of the
Fermi contact term.71a‐b Atomic charges were calculated using the natural bond orbital (NBO) partitioning scheme. 72
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X‐ray crystallography
Intensity data for and 2 (295 K) were measured on an Agilent Technologies SuperNova Dual CCD with an Atlas detector fitted
with Mo K radiation ( = 0.71073 Å). Data processing and absorption corrections were accomplished with CrysAlis PRO.73
Intensity data for 3 (293 K) were collected on a Bruker AXS‐KAPPA APEX II diffractometer fitted with Mo Kα radiation (λ =
0.71073 Å). Data processing was carried out with SMART and SAINT,74a and the absorption correction was accomplished with
SADABS.74b The structures were solved by direct methods with SHELXS‐97 (2)75a and SIR2014 (3),75b and refinement (anisotropic
displacement parameters (ADP’s) with C‐bound H atoms in the riding model approximation and a weighting scheme of the form
2
w = 1/[2(Fo ) + (aP)2 + bP] for P = (Fo2 + 2Fc2)/3) was on F2 by means of SHELXL‐2014/7.75c The N‐bound H atoms were refined
with the distance constraint N–H = 0.88(1) Å. For 2, disorder was evident in the fluorobenzene ring and this was modeled over
two positions of equal weight; the dihedral angle between the two orientations is 4(1)°. Each benzene ring was constrained to
be an ideal hexagon and, due to the near‐overlap of the atoms, equivalent pairs were constrained to have identical ADP’s. The
data for 3 was less than optimal but, the structure determined unambiguously without any unusual features. Crystallographic
data and final refinement details are given in Table 1. The molecular structure diagrams were drawn using ORTEP‐3 for
Windows76a at the 35% probability level and the remaining crystallographic figures were drawn with DIAMOND76b using
arbitrary spheres. Data manipulation and interpretation were with WinGX76a and PLATON.77
Table 1 Crystal data and structure refinement details for 2 and 3.
Compound
Formula
2
3
C14H12FN4O2SV
370.28
C13H9FN4S
272.30
Formula weight
Color, habit
Yellow, prism
0.05 × 0.05 × 0.25
Monoclinic
C2/c
Orange, prism
0.10 x 0.12 x 0.20
Monoclinic
P21/c
Size mm
Crystal system
Space group
Unit cell dimensions
a (Å)
b (Å)
c (Å)
a (º)
 (º)
g (º)
19.600(3)
14.7000(8)
14.922(2)
90
10.8207(16)
17.650(3)
13.3819(19)
90
134.83(3)
90
99.691(5)
90
8

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Volume (Å3)
Z/Zꞌ
3049.1(17)
8/1
2519.3(7)
8/2
Calculated density (g cm‐3)
Radiation
1.613
2519.3(7)
Mo K
Mo K
1.436
Absorption coefficient (mm‐1) 0.812
F(000)
1504
1120
Theta range for data
collection (º)
2.7 – 29.4
2.3 – 25.7
Reflections collected / unique 12030 / 3723
43705 / 4590
Rint
0.040
0.131
Data (I>2 (I)) / restraints/
2197 / 38 / 210
2246 / 2 / 349
parameters
Goodness‐of‐fit on F2
1.01
1.03
Final R, wR indices [I>2σ(I)]
Final R, wR indices [all data]
a, b wght scheme
0.053, 0.121
0.101, 0.149
0.059, 3.139
‐0.27, 0.41
0.110, 0.305
0.204, 0.385
0.156, 12.576
‐0.47, 0.56
Residual electron density
peaks (eÅ‐3)
In vitro Glucose uptake assay on L6 myotubes.
L6 myoblast were cultured in DMEM containing 10% FBS and penicillin (100 U/mL) and streptomycin (100 µg/mL) in a
humidified 5% CO2 incubator at 37°C. To differentiate to myotubes, the myoblast cells (5x104) were seeded in 24 well plates in
DMEM containing 2% FBS. The myoblast cells (5x104) were grown for 11 days in 0.4 mL of 2% FBS/DMEM to allow the
formation of myotubes. The medium was changed every 2 days. On the 11th day, the cells were washed and incubated in Kreb’s
bicarbonate buffer (KRBB) for 2 h. Myotubes were then further cultured in KRBB containing 25 mM glucose along with 25, 50,
100 µM of vanadium compounds ([VIVO(L)(acac)] (1), [VVO2(L´)] (2) and VIVO(acac)2) for 4 h. Insulin (2 µM) was taken as positive
control.78a The residual glucose in the buffer aliquots remaining in each well after 4 h was estimated using hexokiase method by
Randox autoanalyser.
Cytotoxicity studies.
The effect of complexes 1 and 2 on the MCF–7 cells (a human breast adenocarcinoma cell line) and Vero cells (noncancerous
mammalian cells) was studied using the MTT (3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide) dye reduction
assay, by measuring the optical density at 595 nm using a micro plate reader spectrophotometer. The cells were maintained in
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minimal essential medium supplemented with 10% FBS, penicillin‐streptomycin solution and incubated at 37°C in 5% CO2 and
95% humidified incubator. After the cells being grown for 24 h to a subconfluent state (~ 60–70%), they were treated with
different concentrations of [VIVO(L)(acac)] (1) and [VVO2(L´)] (2) (5, 50, 100, 200 μM) in culture medium. The complexes were
dissolved in DMSO and working solutions were prepared by diluting the stock of 5 mM of each compound with plain DMEM.
Final working concentration of DMSO in assay was less than 2%. The samples were tested in triplicates. After 24, 48 and 72 h
the effect of the compound on cell viability was examined by the MTT test. Optical density was measured at 595 nm using an
automatic microplate reader.
DAPI staining.
DAPI (4´,6‐diamidino‐2‐phenylindole dihydrochloride) staining was carried out to examine the morphology of the nuclei after
treatment. The cells were grown in the 96–well plates. After reaching approximately 90% confluency the cells were treated
with [VIVO(L)(acac)] (1) and [VVO2(L´)] (2) at different concentrations (5, 50 100, 200 µM), and were incubated for 24, 48 and 72
h. Cells were observed with inverted fluorescence microscope after treatment to check for morphological changes, during cell
death. Then the cells were washed twice with PBS (pH 7.4) and 3.7% of paraformaldehyde was added and incubated for 15 min.
The cells were again washed twice with PBS and treated with 0.2% triton–X 100 and 2% BSA in PBS for 30 seconds. Again the
cells were washed twice with PBS and DAPI was added and kept for 30 min in the dark. Finally, the cells were washed twice with
PBS and imaged under fluorescence microscope (FLoiD, Life technologies).
Results and discussion
Synthesis
Scheme 1 illustrates the formation of the oxidovanadium(IV) complex, [VIVO(L)(acac)] (1), and the
dioxidovanadium(V) complexes [VVO2(L´)] (2) and [VVO2(L)] (2a). The spectroscopic data are consistent
with the proposed formulations. The purity of these compounds was confirmed by elemental analyses.
All complexes were soluble in EtOH, CH3CN, DMF and DMSO.
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O
V
O
O
N
N
reflux, 2 h
ethanol
N
S
C
HC
N
H
+
VO(acac)2
N
C
H
N
S
HN
C
HN
[V(IV)O(L)(acac)] (1)
F
HL
F
recrystallization
13
14
12
11
1
O
O
V
4
2
O
S
O
S
N
N
N
V
10
1
1
C
8
N
C
3
N
C
H3C
N
C
H
1
2
5
2
9
7
3
1
HN
HN
4
6
F
F
[V(V)O2(L')] (2)
[V(V)O2(L)] (2a)
Scheme 1 Schematic representation of HL and outline of the processes to obtain [VIVO(L)(acac)] (1),
[VVO2(L´)] (2) and [VVO2(L)] (2a).
The processes take place in two steps: firstly, the formation of [VIVO(L)(acac)] (1) from the reaction of
[VIVO(acac)2] with HL {4‐(p‐fluorophenyl)thiosemicarbazone of pyridine‐2‐aldehyde}. Then, in presence
of DMSO, there is aerial oxidation of [VIVO(L)(acac)] (1) to form the VVO2‐complex [VVO2(L´)] (2), which
contains a rearranged ligand L´ where methylation of an aldimine carbon has taken place. However, in
the presence of other solvents such as DMF, CH3CN and EtOH no such ligand change is observed and the
corresponding VVO2‐compound [VVO2(L)] (2a) is formed from [VIVO(L)(acac)] (1). The reaction of 2a with
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DMSO was also carried out at 60 °C; however, in that case there was no change in colour of the reaction
mixture or methylation at the aldimine C atom.
Plausible pathways for the alkylation of complex 1 in the presence of DMSO
Scheme 2 depicts pathways A and B that can be proposed for the formation of the methylated product
[VVO2(L´)] (2) from the VIVO‐complex [VIVO(L)(acac)] (1).
O
S
O
S
O
S
- H+
CH3
Eq. (i)
H3C
DMSO
H3C
CH2
DMSO
H3C
CH2
A:
O
V
O
V
O
O
N
N
N
O
O
H3C
S
H
H2C
C
N
C
S
H+
S
CH2
N
H
O
N
C
F
C
O
V
N
H
S
H3C
N
O
O
HN
F
O
Eqs. (ii)
'
N
C
Ia
[VIVO(L)(acac)] (1)
O
H3C
S
CH3SOH
F
N
C
HN
B:
O
V
Ib
O
O
O
N
N
V
O
H
N
C
N
C
H3C
S
H
S
H
CH2
N
S
N
H
C
C
H3C
O
HN
F
HN
CH2
F
S
Ia
O
O
O
N
N
N
O
O
O
V
Eq. (iii)
+ O2
V
4
C
+ 4 Hacac
F
C
H3C
N
4
S
+ 2 H2O
S
H3C
N
N
C
C
HN
F
HN
Ib
[VVO2(L´)] (2)
Scheme 2. Processes that are relevant for the formation of [VVO2(L’)] (2) from [VIVO2(L)(acac)] (1). Eqs.
(ii) correspond to the proposed possible routes A and B for the alkylation of 1 by DMSO at 60 ⁰C.
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Pathway A:
A transient nucleophilic dimsyl species might be formed by the loss of one proton from DMSO as
depicted in Eq. (i) of Scheme 2. The proton may be taken up by the aldimine N‐atom or, in case a
transient ‐oxido bridged or hydroxido‐VIVOL or VVOL species is formed, the highly basic ‐oxido or
hydroxido ligands may bind the proton. The deprotonation of DMSO by either a V‐bonded hydroxido or
‐oxido ligand appears also likely, given that the pKa values of water and DMSO in DMSO are 31.4 and
35.1.78b‐d In fact, DMSO can be deprotonated by bases weaker than dimsyl such as hydroxide or t‐
butoxide (pKa of t‐butanol: 32.2).78e,f The deprotonation could also be further facilitated by the increased
acidity of the DMSO C‐H bonds upon coordination to the oxophilic VIV or VV moieties. Once formed, the
dimsyl species attacks the most electrophilic CH moiety nearby (Scheme 2, Eq. ii, A) and forms an
intermediate species Ia , which loses CH3SOH78g‐i species to give rise to another intermediate species Ib.
’
Finally, the aerial oxidation of Ib and loss of a Hacac fragment leads to the formation of [VVO2(L´)] (2) (Eq.
iii; here the source of the proton might be from the DMSO or the moisture present in the DMSO.)
Pathway B:
This does not require the formation of a dimsyl anion and involves the direct attack of the carbon atom
of DMSO to the imine C atom of 1, (Scheme 2, eq. ii, B) with the formation of the intermediate species
Ia. This intermediate species then forms CH3SOH and Ib, which upon oxidation forms 2 (Eq. iii).
The proposed pathways are supported by the Natural Bond Orbital (NBO) atomic charge distribution in
complex 1 (Fig. 1a). Among all CH groups, the carbon undergoing methylation has the highest charge
together with one of the C atoms of the pyridine ring. There are other C atoms with even higher charges
but due to steric and electronic constraints we do not expect there methylation. In addition, the NBO
charges at the C atoms of DMSO are strongly negative, particularly in the dimsyl species (Fig. 1b).
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a)
b)
-0.23
0.08
-0.79
H3C
-0.79
O
-0.77
-1.13
–
-0.15
-0.20
CH3
N
H3C
CH2
O
O
0.50
-0.49
0.53
1.04
0.16
V
S
S1.16
S
N
C
H
0.08
N
0.30
O
-1.01
-0.22
-1.14
C
O
-0.56
-0.27
HN
0.13
0.37
F
-0.24
-0.27
Fig. 1 Diagrams depicting NBO atomic charges at the carbon atoms (a) of 1 and (b) of neutral DMSO
(left) and the dimsyl anion (right).
The structures of intermediates Ia´ and Ia shown in Scheme 2 for pathways A and B were optimized using
–
the DFT (B3P86) method. The addition of the dimsyl anion, CH3S(=O)CH2 , to complex 1 giving
intermediate Ia´ is highly exoergonic (by –35.3 kcal/mol). In turn, structure Ia was found to be 0.3
kcal/mol more stable than the initial complex 1 plus DMSO (in terms of G in DMSO solution), which is
quite reasonable for a transient reaction species. The free Gibbs energy of the complete reaction (ii) in
Scheme 2 (i.e., 1 + DMSO  Ib + CH3SOH) is –20.7 kcal/mol that indicates the thermodynamic feasibility
of this reaction (either pathway A or B). The calculated Gs value of reaction (iii) is –70.2 kcal/mol.
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Therefore, both pathways A and B are plausible from the thermodynamic point of view, the relative
importance of A and B depending on the temperature, on the pH of the medium and efficacy of basic
species (e.g. a ‐oxido or hydroxido vanadium species) in removing a proton from DMSO to yield the
–
CH3S(=O)CH2 anion. Meanwhile, detailed investigation of the mechanistic features and confirmation of
the kinetic feasibility of the proposed mechanism should be the subject of a separate work.
IR spectroscopy
The IR spectrum of HL exhibits bands due to ν(N(1)–H) and ν(N(2)–H) moieties in the 3311–2967 cm–1
region, but complexes 1, 2 and 2a do not exhibit the ν(N(2)–H) band. This indicates that the ligand
coordinates to the metal centre in the anionic form. The sharp band at 836 cm–1, due to ν(C(7)–S(1))
stretching in the ligand, is lowered to 775–762 cm–1 in the complexes, indicating participation of the
thione sulfur in the coordination.78j,k Fig. S2 shows the IR spectra of complexes 1 and 2 in the 1100‐800
cm1 range. The presence of a strong sharp band at 949 cm–1 in 1 suggests the presence of oxido
(V=O)79, whereas two peaks in the range 937–922 cm–1 in 2 and 2a are assigned to dioxido (V=O)
stretchings.80 The IR data are summarized in Table 2.
Table 2 IR and electronic spectral data for complexes 1, 2 and 2a.
Complex
V−O str,a cm‐1
949
UV−Visb λmax, nm (, dm3mol‐1cm‐1)
[VIVO(L)(acac)] (1)
272 (20703), 332 (25043), 401 (21818), 444 (18182),
603 (120), 761 (45)
[VVO2(L´)] (2)
[VVO2(L)] (2a)
937, 925
934, 922
<257 (24048), 431 (19919)
260 (26127), 428 (19919)
aIn KBr pellet. bSolutions in CH3OH.
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UV−Vis spectroscopy
The electronic spectra of 1, 2 and 2a were recorded in CH3OH solution and part of the spectra of
complexes 1 and 2 are depicted in Fig. 2. In all complexes two strong absorption bands are observed in
the wavelength range 431–257 nm. The lower energy absorptions at 430‐400 nm are ascribable to
ligand to metal charge transfer (LMCT) transitions,80a whereas the higher energy absorptions are likely to
be due to ligand centred transitions.80b Two weak absorptions at 761 nm (max = 45 dm3mol1cm1) and
603 nm (max = 120 dm3mol1cm1) observed for 1 (a VIV complex) are assigned to d−d transitions. 79‐81a
The UV−Vis data are summarized in Table 2.
a)
b)
1200
1000
800
600
400
200
0
25000
20000
15000
10000
5000
0
431
603
761
500
600
700
800
900
400
600
800
Wavelength (nm)
Wavelength (nm)
Fig. 2. Part of the electronic absorption spectra of methanolic solutions of (a) [VIVO(L)(acac)] (1.8104
M) (1) and (b) [VVO2(L´)] (2) (1.5105 M).
EPR spectroscopy
The EPR spectrum of 1 was measured in frozen solution of DMF at 77 K (Fig. 3. The spectrum displays a
slight rhombicity in the g= 1.95‐1.85 region, so it was simulated assuming that the x and y axis are not
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equivalent. The experimental spin Hamiltonian parameters obtained are: gx,y =1.977 , gx,y =1.981, gz=1.95,
|Ax,y| =51.1 ×104 cm1, |Ax,y| = 52.6×104 cm1, |Az| = 152.9×104 cm1. The obtained z‐component spin
Hamiltonian parameters are consistent with a [Nim, Npy, Oacac, SRS] equatorial donor atom set.79,81b‐e The
|Az| value calculated by DFT procedures (see above), after the structural optimization of complex 1, is
154.610–4 cm–1, reasonably correlating with the experimental value. The DFT calculated |Ax| (58.910–4
cm–1) and |Ay| (56.210–4 cm–1) also reasonably agree with the values obtained by simulation of the
experimental spectrum.
Fig. 3. First derivative of the frozen solution X‐Band EPR spectrum of 1 in DMF (0.0025 M) recorded at 77
K.
NMR spectroscopy
1
The H NMR spectrum of HL (Fig. S3) was recorded in DMSO‐d6. It exhibits two singlets in the range
12.08 to 10.29 ppm due to two NH moieties {N(1)H and N(2)H}, whereas in the spectra of complexes 2
and 2a (Figs. S4 and S5) there is only one peak in the range 10.29 to 10.20 ppm for one NH moiety
{N(1)H}, indicating coordination of the ligand to the metal centre in anionic form. The peak due to C(8)H
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appears as a singlet at 8.59 ppm for the ligand and at 8.79 ppm for 2a. The presence of a methyl peak at
2.66 ppm in the spectrum of [VVO2(L´)] (2) and absence of a C(8)H peak indicates the methylation at
imine carbon C(8) and formation of a new ligand. The detailed NMR data is included in the experimental
section.
The reaction of HL with DMSO in the absence and presence of VIVOSO4 or VVO(OiPr)3 was tested further
1
with distinct conditions of temperature and period of reaction, this being monitored by H NMR
experiments (Figs. S6−S11). No alkylaꢀon reacꢀon is noꢀceable since the CH=N resonance at ca. 8.75
ppm did not subside. These results reinforce the assessment that the methylation reaction may be
confined to complex 1.
Moreover, the reaction of 1 with DMSO at 60⁰C was monitored by 1H and 51V NMR. A saturated solution
of 1 in DMSO was heated to 60⁰C till all solid dissolved (ca. 10 minutes). A gradual colour change from
green to brown was observed. The solution rested at room temperature for another 2 h before NMR
analysis. It was observed that the CH=N resonance at δ 8.75 ppm nearly disappeared (Figs. 4 and S12).
1
Fig. 4. Low field region of the H NMR spectrum of 1 (ca. 0.19 M) in DMSO after heating for 10 min at
60⁰C and cooling down for 2 h to room temperature. The resonance at 8.75 ppm is assigned to the imine
proton (CH=N).
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51
The respective V NMR spectrum (Figs. 5 and S13) shows two broad but faint resonances at ca. δV = ‐
434 ppm and δV = ‐577 ppm. There are still some VIVO‐species in solution and the resonances are
tentatively assigned to a VVO2‐species and a µ‐oxido bridged VVO‐species, respectively. The chemical
shift δV calculated by DFT (δVcalc) for 2a is ‐433 ppm, thus supporting the assignment of the experimental
calc
resonance at ‐434 ppm; for complex 2 the δV is ‐449 ppm. The above observations indicate that the
reaction proceeds swiftly at 60 ⁰C.
Fig. 5. Close‐up of the 51V NMR spectrum of 1 (ca. 0.19 M) in DMSO after heating for 10 min at 60⁰C and
cooling down for 2 h to room temperature.
1
The reaction of compound 2a with DMSO at 60 ⁰C was also monitored by H and 51V NMR using the
same procedure as for 1. In this case there was no change in color or apparent alteration of the 1H NMR
spectrum after 24 h: the CH=N resonance at  8.75 ppm remained present with an integration indicative
of 1 proton (Fig. S14). Other experiments showed that no alteration occurred after 24 h at 60 ⁰C in
DMSO (Fig. S14, B). Only after 5 days under these conditions the CH=N resonance disappeared, but the
obtained end product revealed to be a cyclized and oxidized compound 3 (Figs. 9, S1, S15 and S26; see
Table S1 for selected bond lengths and angles). The driving force for this intramolecular cyclisation
comes from the highly nucleophilic character of the thiolate moiety. Upon dissociation from the
vanadium center, the thiolate attacks the electrophilic imine carbon, forming a cyclic thioaminal. The
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aromatisation of this heterocycle to a nitrogenated thiophene favors the subsequent oxidation step,
most likely by vanadate(V) formed upon solvolysis of the complex.
The 51V NMR spectrum obtained from the experiments at 24 h showed only one broad but faint
resonance at ca. V = ‐434 ppm, which may be assigned to the cis‐VO2 species 2a. Species at higher field,
specifically at V ‐530 ppm, only appeared after 5 days at 60 ⁰C, but by then decomposition of the
complex had already occurred. It may be due to an inorganic vanadate species soluble in DMSO, but
considering that the ligand undergoes significant transformations it may also be a not characterized and
unknown VV‐species. These experiments indicate that the cis‐dioxidovanadium(V) complex 2a is unable
to mediate the intramolecular methylation of the ligand with DMSO.
NMR spectra upon methylation attempts of other imine ligands
To test the scope of applicability of methylation reactions in DMSO, several other –C=N containing
compounds (L1‐L10) (Chart S1), along with some VIVO complex (for example C1, Chart S1), were
subjected to the same conditions as those used for 1. Compounds L1‐L10 were prepared in situ by
mixing equimolar amounts of an aldehyde or ketone and amine in DMSO. After 5 min of stirring at room
temperature, an equimolar amount of VIVO(acac)2 or VVO(OiPr)3 was added to the reaction mixture.
After complete dissolution of the metal precursor the mixture was heated to 60 ⁰C in DMSO for 30 min
1
to 1 h, depending on the ligand precursor used. The H NMR spectra of the obtained solutions were
measured and compared, when possible, to those obtained for the expected ketimine products (Figs.
S16−S22). Somewhat broadened 1H NMR spectra were obtained in most cases even when a VV precursor
was used. This is due to the significant presence of paramagnetic VIV‐species, which in the case of the VV
precursors, could have been formed by the oxidation of the in situ ligand. In some instances, such as
when L4 and L5 were used, significant precipitation of solids precluded the measurement of adequate
NMR spectra. Notwithstanding, despite the broadened spectra obtained in most cases, no alkylation
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could be detected with any of the compounds L1‐L10 and complexes tested. Importantly, the ligand HL
was not alkylated when the metal precursor was VVO(OiPr)3 (Fig. 6). Therefore, these results indicate
that the alkylation reaction is confined to the specific case of 1 and, for this system in particular, a VIVO‐
species appears to be a requisite for the reaction to occur.
Fig. 6. (A) 1H NMR spectra of a 1:1 mixture of HL and VVO(OiPr)3 in DMSO after 10 min. at 60⁰C; (B) 1:1
mixture of HL and VVO(OiPr)3 in DMSO after 30 min. at 60⁰C; (C) 1:1: mixture of HL, VVO(OiPr)3 and H2O
in DMSO after 10 min. at 60⁰C; (D) 1:1: mixture of HL, VVO(OiPr)3 and H2O in DMSO after 30 min. at 60⁰C.
No alkylation of HL may be detected from the analysis of these spectra. Reagent concentration was ca.
90 mM.
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ESI‐MS studies
The negative and positive ESI‐MS spectra obtained for compound 1 (Figs. S23 and S24) are of great
complexity due to aggregation, but the major peaks were identified. The sample was diluted in
methanol in an attempt to minimise aggregation. The observation of methanesulfenic acid or
methanesulfenate anion is difficult under the conditions used, given its high reactivity. Instead, we
sought to observe methanesulfonic acid (MW = 96.1), as it is the most likely decomposition product of
the sulfenic acid. Most of our MS data are for m/z > 100, but nevertheless in the rare ESI‐MS spectra
recorded with data below m/z of 100, we detected a peak in negative ISI mode at m/z = 95.4 (ca. 85%),

(Fig. S25), which may be tentatively attributed to CH3SO3 , which might have resulted from the CH3SOH
formed (eq. (ii) in Scheme 2).
The ESI‐MS spectrum in the negative mode shows two identifiable major species at m/z = 355 and 385
which are assigned respectively to the free methylated ligand (HMeL) [(MeL)∙2H2O∙MeOH] (m/z= 355)
and the complex {[VIVO(MeL)]OMe}(m/z=385). As for the positive mode ESI‐MS spectrum, three major
peaks were identified: (1) the peak at m/z = 242 is assigned to {[VIVO(HMeL)]∙4MeOH}2+ (m/z= 483/2 =
241.5), whereas (2) the peak at m/z = 321 is assigned to [(H2MeL)∙MeOH]+, and (3) the peak at m/z = 778
is tentatively assigned to a mixed valence ‐oxido bridged species, {Na[VVO(MeL)OMe]‐O[VIVO(MeL)]}+.
Lower mass peaks are present in both positive and negative spectra, which are indicative of
fragmentation. In the positive mode the weak peak at 439.97 could be assigned to the molecular ion
1∙H+. Despite the complexity of the obtained MS spectra, these clearly show that a reaction between 1
and DMSO took place with the formation of methylated products.
Electrochemical properties
The electrochemical properties of 1 and 2 were examined in CH3CN solution (0.1 M TEAP) by cyclic
voltammetry using a platinum working electrode, platinum auxiliary electrode, and Ag/AgCl reference
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electrode. The potential data are listed in Table 3. The voltammogram pattern for 1 includes both
oxidation and reduction processes (Fig. 7(a)); the redox couple at E1/2 value 0.62 V corresponds to VIV/VV
oxidation, whereas E1/2 at –1.13 V corresponds to VIV/VIII reduction couple. The voltammogram pattern
for 2 includes only a quasi‐reversible redox couple at E1/2 = –0.73 V corresponding to VV/VIV reduction
(Fig. 7(b)). The redox characteristics of 2a were also examined by measuring the corresponding
voltammogram; as expected it is very similar to that of complex 2.
Fig. 7. Cyclic voltammograms of [VIVO(L)(acac)] (1) (a) and [VVO2(L´)] (2) (b) in CH3CN solution.
Table 3 Cyclic voltammetric[a] data for [VIVO(L)(acac)] (1) and [VVO2(L´)] (2) at 298 K.
Complex
VIV/VV
VIV/VIII
VV/VIV
Ea1/2 (V), ΔEa (mV)
Ec1/2 (V), ΔEcP(mV)
Ec1/2 (V), ΔEcP (mV)
P
[VIVO(L)(acac)] (1)
[VVO2(L´)] (2)
0.62, 450
–
–1.13,200
–
–
–0.73, 120
[a]In CH3CN at a scan rate 100 mV/s. E1/2 = (Epa + Epc)/2, where Epa and Epc are anodic and cathodic peak
potentials vs. Ag/AgCl, respectively. ΔEP = Epa – Epc.
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X‐ray diffraction crystallography.
It was not possible to obtain X‐ray diffraction crystals of 1 with adequate quality but, there are two
closely related structures in the literature. These feature very similar atom donor sets, geometries and
with oxido and acac ligands as in 1 but, with 2‐acetylpyridine‐4‐phenylthiosemicarbazato82 and
dimethylthiosemicarbazonato83 as the tridentate ligands.
A view of the molecular structure of 2 is shown in Fig. 8 and selected geometric parameters are
collected in Table 4. In this structure, the V(V) atom is bonded to the S, N, N‐donors of the uninegative
tridentate ligand, as for 1, and two oxido‐O atoms. The length of the C–S bond [1.730(3) Å] is suggestive
that the ligand is coordinating as a thiolate, a conclusion also supported by the short C7–N2 [1.305(4) Å]
and C8–N3 [1.306(4)] bonds. The V1,S1,N2,N3,C7 and V,N3,N4,C8,C10 five‐membered chelate rings are
each planar, having r.m.s. deviations of 0.018 and 0.015 Å, respectively, and form a dihedral angle of
2.6(1)°. The pyridyl ring forms dihedral angles of 4.26(17) and 4.0(5)° with the adjacent chelate ring and
terminal fluorophenyl ring, respectively, indicating that the entire tridentate ligand is approximately
planar. The N2O2S donor set defines a highly distorted coordination geometry as seen in the value of the
trigonality index,  = 0.43, which is intermediate between square pyramidal ( = 0.0) and trigonal
bipyramidal ( = 1.0).84 To a first approximation, the observed molecular structure of 2 matches that
reported for the N‐bound methyl derivative which differs in geometric parameters. In the latter the V–S
[2.3759(9) Å], V–O [1.631(2) Å] and N3–C8 [1.323(4) Å] bond lengths are marginally longer than in 2;28
the coordination geometry is closer to square pyramidal ( = 0.22).
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Fig. 8. Ortep representation (35%) showing a view of the molecular structure of 2; one of the disorder
components of the fluorophenyl ring was omitted for reasons of clarity.
Table 4 Selected geometric parameters (Å, °) for 2
Atoms
Parameter
Atoms
Parameter
Complex 2
V–S1
V–O2
V–N4
C7–N2
2.3653(13)
1.596(3)
2.086(3)
V–O1
V–N3
C7–S1
N2–N3
1.607(2)
2.143(3)
1.730(3)
1.374(4)
1.305(4)
C8–N3
1.306(4)
S1–V–O1
S1–V–N4
O1–V–O2
101.93(10)
77.58(7)
110.14(16)
S1–V–O2
S1–V–N4
98.93(11)
151.34(10)
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Fig. 9. Ortep representation (35%) showing a view of the molecular structure of 3, first independent
molecule.
Two independent molecules comprise the asymmetric unit of 3, Figs. 9 and S26 (a), and these adopt very
similar conformations as seen in the overlay diagram in Fig. S26 (b). The structure features a 1,3,4‐
thiadiazolyl ring with 4‐fluorophenylamine and 2‐pyridyl substituents at the 2‐ and 5‐positions. The
molecular packing of compounds 2 and 3 are discussed in the ESI section (Figs. S26‐S29).
In vitro insulin‐like activity. In vitro insulin mimetic activity. Vanadium compounds are known to have
insulin enhancing effect17,19,30,32,33,36,37,41,42a,b,d,43a,c both in vitro and in vivo. In this study [VIVO(L)(acac)] (1)
and [VVO2(L´)] (2) were tested for this feature in an in vitro glucose uptake assay. In L6 myotubes, a cell
line of skeletal muscle origin, glucose uptake in response to insulin stimulation plays a key role in the
whole body glucose homeostasis. Glucose transporter GLUT4 is the major glucose transporter of muscle
exquisitely regulated by insulin through posttranslational events.85 The L6 myotubes differentiated from
L6 myoblast have the ability to respond to insulin via insulin receptor signaling and translocation of
GLUT4 to the plasma membrane. The hyperglycemic condition was created in cell culture by incubating
the cells in 25 mM glucose. Complex 1 at 100 µM concentration showed glucose uptake stimulating
activity in the myotubes which was found to be comparable to that of 2 µM of insulin (Fig. 10). Complex
2 at 100 µM concentration also showed considerable insulin‐like activity, whereas in the conditions of
our experiments VIVO(acac)2 showed negligible insulin enhancing effects at the concentrations tested. In
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case of 1, the enhancement of the insulin mimetic activity, with respect to 2, might be related to the
difference in the oxidation state of the complexes as vanadium(IV) complexes; similar differences
between the effects of VIV‐ and VV‐compounds has been often mentioned.86 Noteworthy is the
observation that in our in vitro data both 1 and 2 have significantly higher insulin‐like activity than
VIVO(acac)2, a compound previously shown to be insulin enhancing both in vitro and in vivo.34,35
Fig. 10. Effect of complexes (1 and 2) on glucose uptake in cultured L6 myotubes under high glucose
conditions (25 mM Glucose). Three concentrations 25 µM, 50 µM and 100 µM were tested for each
complex. The data is indicated as mean ± SEM (n=3). Student’s t–Test was used where bars with asterisk
show a significant difference (*P< .05) with respect to the untreated control.
Cytotoxicity Studies. Vanadium compounds have increasingly being considered to have potential as
anti‐cancer drugs,19,20,21b so to evaluate the potentiality of these compounds as drugs for the treatment
of cancer, in the present work in vitro cytotoxicity studies of complexes 1 and 2 was assayed by
determining the viability of MCF–7 (breast cancer cells) and Vero cells (noncancerous mammalian cells)
using the MTT assay. The ligand HL and VIVO(acac)2 gave high IC50 values at 24 h higher than 200 μM,
whereas [VIVO(L)(acac)] (1) and [VVO2(L´)] (2) gave lower IC50 values that are depicted in Table 5. It was
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observed that, upon increase in time of incubation from 24 to 72 h, the IC50 values decrease, being lower
for the Vero cells, but they do not differ much for 48 and 72 h. In comparison to the IC50 values
obtained, cisplatin a well‐established chemotherapeutic drug, has an IC50 value of 286 μM at 72 h.87
The cytotoxicity of some VIVO‐complexes of salicylaldimines and polypyridyl ligands, here designated as
[VIVO(sal‐AMA)(NN)] were recently also studied against MCF‐7 cells,87 where the IC50 values varied
between 5.9 ± 1.4 to 40.9 ± 7.5 μM at 24 h and between 2.8 ± 1.3 to 8.4 ± 2.4 μM at 72 h. The present
compounds depict comparable IC50 values at 24 h to those of this previous report, but with somewhat
higher IC50 values; thus complexes 1 and 2 are less toxic against MCF‐7 cells than the tested [VIVO(sal‐
AMA)(NN)] compounds.87 Noteworthy, 1 and 2 are more toxic for the Vero cells, used here as reference
for normal cells, thus their selectivity does not appear adequate, at least when solely compared against
this cell line.
Table 5 IC50 (µM) values in MCF–7 and Vero cell lines for complexes 1 and 2
Cell Line
MCF‐7
Time / h
IC50 values (µM)
1
2
24
48
72
28.5  0.45
26.3  0.36
23.2  0.38
115.6 1.02
90.6 0.97
84.0 0.93
Vero cells
24
48
72
41.6  0.54
3.1 0.25
3.2 0.28
120.6  1.12
30.1  0.31
29.4 0.38
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The higher IC50 values of HL (and of VIVO(acac)2) in comparison to the metal complexes 1 and 2 indicate
that the incorporation of vanadium in the ligand environment increases the cytotoxicity towards these
cells. It is plausible that the permeation of the vanadium complexes through the lipid layer of the cell
membrane is favoured by coordination, the polarity of the ligand and the central metal ion being
globally decreased through charge equilibration.88,34a Bovine serum albumin (BSA) is present in relatively
high concentration in FBS, which was added in 10% to the incubation medium. Albumins, e.g. BSA may
serve as vehicles of uptake of drugs, namely metal‐based drugs34b by the cells, thus it is also possible
that the higher toxicity of 1 and 2 when compared to the ligand, might be due to distinct binding to the
albumin leading to a more efficient uptake in the case of complexes 1 and 2. Similarly with VIVO(acac)2
which binds albumins rather weakly.35
It may be concluded from the present in vitro data that the metal complexes exhibit greater biological
activities than the free ligands, which is also in accordance with previous reports.89,90 Apart from the
possible higher uptake of complexes when compared with the corresponding ligands, or synergistic
effects operating, vanadium ions by themselves present some toxicity.91 Comparing the activity of the
two complexes, it is observed that the toxicity of [VIVO(L)(acac)] (1) is greater than that of [VVO2(L´)] (2),
which is reflected from their IC50 values with dose dependency illustrated in Fig. 11 and Table 5, which
can either be correlated with the difference in the oxidation states of the complexes and/or to different
uptake efficiency by the cells.
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