Ligand role on insulin-mimetic properties of vanadium complexes. Structural and biological studies

Article abstract of DOI:10.1016/j.ica.2020.120135

The syntheses of 16 vanadium(IV and V) complexes with ONO tridentate Schiff base ligands are described. The vanadium(IV) compounds were synthesized with 1,10-phenantroline as a co-ligand. In the case of five complexes of vanadium(V), the solvent molecule

Full text of DOI:10.1016/j.ica.2020.120135

Inorganica Chimica Acta xxx (xxxx) xxx
Contents lists available at ScienceDirect
Inorganica Chimica Acta
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Research paper
Ligand role on insulin-mimetic properties of vanadium complexes.
Structural and biological studies
,
Janusz Szklarzewicza *, Anna Jurowskaa, Maciej Hodorowicza, Grzegorz Kazekb,
Monika Głuch-Lutwinb, Jacek Sapab
a
´
Jagiellonian University, Faculty of Chemistry, Gronostajowa 2, 30-387 Krakow, Poland
b
´
Jagiellonian University Medical College, Faculty of Pharmacy, Medyczna 9, 30-001 Krakow, Poland
A R T I C L E I N F O
A B S T R A C T
Keywords:
The syntheses of 16 vanadium(IV and V) complexes with ONO tridentate Schiff base ligands are described. The
vanadium(IV) compounds were synthesized with 1,10-phenantroline as a co-ligand. In the case of five complexes
of vanadium(V), the solvent molecule in form of ethanol or ethoxy ligand fill the coordination sphere of V(V) to
six. In the crystal structure of selected complexes the mixture of isomers in 1:1 ratio in the unit cell is observed as
the ligand is not symmetrical and forms both 5- and 6-membered rings in complex. All complexes were char-
acterized also by elemental analysis, IR and UV–Vis spectroscopy, magnetic and cyclic voltammetry measure-
ments. The stability of the complexes in DMSO-H2O mixture was performed at pH = 7.0 and 2.0. The relationship
between phosphatase inhibition and cytotoxicity of the compounds and the key role of organic ligands in
enhancing biological activity of vanadium complexes was also discussed.
Insulin-mimetic
Schiff base
Spectroscopy
Structure
Vanadium
1. Introduction
produced significant decrease in blood glucose level and improved the
function of liver and kidney after two weeks of daily use [21]. In our last
Schiff base ligands as well as their metal complexes represent one of
the most extensive long standing topics of interest. They owe this to the
ease of creation and the possibility of obtaining a variety of structures
with interesting physicochemical and catalytic properties. The investi-
gation of such compound is crucial to understand the relationship be-
tween structure and property and to apply them in a wide range of fields
as for example supramolecular chemistry, nanoscience, magnetism,
catalysis or biochemistry [1–9]. Here we concentrate on the biological
activity of the vanadium Schiff base complexes which can be potentially
used as insulin-mimetic compounds. Vanadium complexes having
organic ligands in the coordination sphere of the VO+2 , VO2+ or VO+
cation were extensively investigated for they antidiabetic potential in
last years [10–15]. Organic ligands provide the ability to precisely
match the vanadium compound in terms of stability, gastrointestinal
uptake (when administered orally), tissue cell targeting and internali-
zation, and toxicity [16]. The most intensively studied compound in this
field is bis(maltolato)oxidovanadium(IV) (BMOV) and its derivative as
bis(ethylmaltolato)oxidovanadium(IV) (BEOV) [17–19] and these
complexes were used in clinical tests [20]. In terms of vanadium Schiff
base complexes the pharmacological data showed that these compounds
publications, we have shown, that vanadium complexes with tridentate
ligands are stable enough to prevent formation of VO2+ ions in biolog-
ical systems and their activity is thus very specific and depends on ligand
used. In many cases they are much more active as insulin-mimetic
compounds than BMOV [22–24].
In this study we described the synthesis, structural and physico-
chemical characterization of vanadium(IV/V) complexes with ONO
Schiff base ligands derived from 3-hydroxysalicylaldehyde, 3,5-
dichloro-salicylaldehyde or 3,5-dibromo-4-methoxy-salicylaldehyde
and several aromatic hydrazides. The use of such tridentate ligand as
well as 1,10-phenantroline (phen) as a co-ligand (for V(IV) complexes)
increase the complex stability which is very important from the point of
view of the biological activity of these compounds. The vanadium
complexes with ONO donor ligands (also based on N-salicylidenhy-
drazones and hydrazides) were studied earlier in literature [25–28],
prompting us for more extended studies. We have shown, in our earlier
publications, that the biological activity of vanadium complexes with
tridentate ligands cannot be explained only by presence of vanadium
and its content, but strongly depends on type of ONO ligand type
[22–24]. The elemental analysis as well as the magnetic, structure and
* Corresponding author.
E-mail address: szklarze@chemia.uj.edu.pl (J. Szklarzewicz).
https://doi.org/10.1016/j.ica.2020.120135
Received 4 October 2020; Received in revised form 9 November 2020; Accepted 9 November 2020
Available online 12 November 2020
0020-1693/© 2020 Elsevier B.V. All rights reserved.
Please cite this article as: Janusz Szklarzewicz, Inorganica Chimica Acta, https://doi.org/10.1016/j.ica.2020.120135

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
spectroscopy measurements confirm the coordination of the Schiff base
and oxido ligand and phen (for V(IV)) or EtOH molecule (for V(V)). In
this paper we also described the biological activity of selected complexes
and we proved that among the tested vanadium complexes there are
compounds that have greater anti-diabetic efficacy at non-toxic con-
centrations than VOSO4 and BMOV. These two last compounds were
used as standards of vanadium activity references, as typically in such
studies.
other vanadium complexes [22,31].
Myocytes C2C12 cell line (ATCC CRL-1772), subclone of myoblasts
from mouse muscles were cultured according to standard protocol in
DMEM supplemented with 10% fetal calf serum, 100 IU/ml penicillin
and 100 µg/ml streptomycin at 37 ◦C in 5% CO2. Cells were pleated on
96 well microplate and after reach confluency were differentiated in
medium with 2% horse serum. Differentiation medium was changed
every 72 h.
Adipocytes 3 T3-L1 cell line (ATCC CRL-11605) derived from fi-
broblasts from mouse embryo tissue were cultured according to stan-
dard protocol in DMEM medium supplemented with 10% bovine calf
serum, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37 ◦C in 5%
CO2. Cells were seeded in 96-well poly-D-lysine coated plates and
cultured to reach confluency. The medium was then switched to dif-
ferentiation medium and changed every 48 h.
2. Experimental
2.1. Materials and methods
[VO(acac)2], VOSO4aq, [V(acac)3], 1,10-phenanthroline (phen), 3-
hydroxysalicylaldehyde, 3,5-dichlorosalicylaldehyde, 3,5-dibromo-4-
methoxysalicylaldehyde, benzhydrazide, 2-hydroxybenzhydrazide, 4-
hydroxybenzhydrazide, 4-chlorobenzhydrazide, phenylacetic acid hy-
drazide, 3-hydroxy-2-naphtoic acid hydrazide were of analytical grade
(Aldrich or Alfa Aesar) and were used as supplied. Ethanol (98%) of
pharmaceutical grade was from Polmos and used as supplied. All other
solvents were of analytical grade (Aldrich) and were used as supplied.
BaSO4 were of spectroscopic grade (Japan). Bu4NPF6 was synthesized
from Bu4NBr and KPF6 by a standard method and recrystallized from
acetone [29]. Microanalysis of carbon, hydrogen, nitrogen and sulphur
were performed using Elementar Vario MICRO Cube elemental analyzer.
IR spectra were recorded on a Bruker EQUINOX 55 FT-IR spectropho-
tometer in KBr pellets. The electronic absorption spectra were recorded
in 1 cm cuvette with Shimadzu UV-3600 UV–Vis-NIR spectrophotometer
equipped with a CPS-240 temperature controller. Diffuse reflectance
spectra were measured in BaSO4 pellets with BaSO4 as a reference on
Shimadzu 2101PC equipped with an ISR-260 integrating sphere
attachment. The magnetic susceptibility measurements were performed
on a SHERWOOD SCIENTIFIC magnetic susceptibility balance. Cyclic
voltammetry measurements were carried out in DMSO with [Bu4N]PF6
(0.1 M) as the supporting electrolyte, using Pt working and counter and
Ag/AgCl as reference electrodes on an AUTOLAB/PGSTAT 128 N
Potentiostat/Galvanostat. E1/2 values were calculated from the average
anodic and cathodic peak potentials, E1/2 = 0.5(Ea + Ec). The redox
potentials were calibrated versus ferrocene (0.440 V versus SHE), which
was used as an internal potential standard for measurements in organic
solvents to avoid the influence of a liquid junction potential; the final
values are reported versus the standard hydrogen electrode (SHE).
Human hepatocytes HepG2 cell line (ATCC HB-8065) were cultured
according to standard protocol in DMEM supplemented with 10% fetal
bovine serum, 100 IU/ml penicillin and 100 µg/ml streptomycin at 37 ◦C
in 5% CO2. Cells were pleated on 96 well microplate.
2.4. Cells viability and cytotoxicity tests
After differentiation of C2C12 myocytes medium was changed for
medium with 0.2% bovine serum albumin, 100 IU/ml penicillin, 100
µg/ml streptomycin and after 2 h incubation medium was changed for
fresh medium containing vanadium complexes. After 48 h incubation of
3T3-L1 adipocytes medium was changed for standard medium with
human recombinant insulin and tested vanadium complexes. Hepato-
cytes HepG2 was used for experiments after seeding for 18–24 h and
after medium exchange. Vanadium complexes was tested at final con-
centration 50 µM. After 24 h incubation with tested compounds at 37 ◦C
in 5% CO2 10 μl of PrestoBlue Cell Viability Reagent (Thermo Fisher
Scientific) was added to each microplate well, and the microplate was
placed on an orbital shaker for content mixing. Plates were incubated for
20 min, and the fluorescence intensity at 560 nm excitation and 590 nm
emission was determined using a multi-mode microplate reader
POLARstar Omega (BMG Labtech, Germany). The results were normal-
ized to the untreated control (cells with solvent only), wherein the in-
tensity of fluorescence was taken as 100%. Incubations vanadium
complexes with cells were performed in triplicates and each compound
was tested in three to four independent experiments. Similarly, cyto-
toxicity tests based on assessment of integration HepG2 hepatocytes
membranes were performed using ToxiLight Cyotoxicity Kit (Lonza)
according to manufacturer protocol.
2.2. Inhibition of human tyrosine phosphatases
For determination the ability of tested compounds to inhibition of
tyrosine phosphatases human recombinant protein were used. To the
2.5. Radiolabeled glucose transport in myocytes and adipocytes
solution of the tested complexes in DMSO-H2O (20 μl + 3 ml, respec-
Myocytes C2C12 after 8 days differentiation and adipocytes 3T3-L
after 11 days of differentiation were cultured and maintained as
described above but pleated on 96 well microplate coated with solid
phase scintillator (ScintiPlate, PerkinElmer, USA).
tively) on black 384-well microplate wells (PerkinElmer) an equal vol-
ume of a test solution of phosphatase was added in a reaction buffer (25
mM of 3-(N-morpholino) propanesulfonic acid (MOPS), 50 mM NaCl, 1
mM dithiothreitol (DTT) and 0.05% Tween-20, pH 7.0). The final con-
centration of each of phosphatase was as follows: 50 ng/ml, SHP1 400
ng/ml, SHP2 50 ng/ml and LAR 5 ng/ml. After 10 min, a solution of
phosphate 6,8-difluoro-4-methyl (DiFMUP) was added until its final
concentration was 0.1 mM. After 20 min of incubation at room tem-
perature, the fluorescence intensity (excitation 355 and emission 560
nm) was measured on a multifunctional plate reader PTP1B POLARstar
Omega (BMG Labtech, Germany). Assays were performed in triplicates.
The results were expressed as per cent of inhibition of untreated control
(enzyme with solvent only) [30].
Myocytes were washed and medium was changed for medium with
0.5% BSA instead serum. After 24 h incubation tested complexes were
added at final concentration 50 µM. After 2 h incubation at 37 ◦C in 5%
CO2 medium was changed for low glucose medium (1000 mg/l), tested
complexes were added again and further incubation for 4 h was con-
ducted. Next, cells were washed three times with Krebs-Ringer buffer
(KRB) without glucose (1 mM MgSO4, 1 mM CaCl2, 136 mM NaCl, 4.7
mM KCl and 10 mM HEPES; pH 7.4) and KRB with human recombinant
insulin at final concentration 100 nM (Sigma-Aldrich) was added for 15
min. Wortmannin at final concentration 200 nM (Sigma-Aldrich) was
used as negative control. After that, cells were washed three times with
cold KRB and cytochalasin B (10 µM final) was added for several wells as
“no uptake” control and proceeded as shown below.
2.3. Cell models
The cell models and methods used in this study have been previously
validated by the authors and used to study the biological activity of
Adipocytes were washed and medium was changed for basal me-
dium. After 24 h incubation tested complexes were added at final
2

J. Szklarzewicz et al.
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concentration 50 µM. After 2 h incubation at 37 ◦C in 5% CO2 medium
was changed for DMEM medium without glucose, with 0.1% BSA free
from fatty acid, 200 mM L-glutamine and 100 mM pyruvate. Tested
complexes were added again and further incubation for 4 h was con-
ducted. Next, cells were washed three times with Krebs-Ringer buffer
(KRB) without glucose and KRB with human recombinant insulin at final
concentration 100 nM (Sigma-Aldrich) was added for 15 min. Wort-
mannin at final concentration 200 nM (Sigma-Aldrich) was used as
negative control. After that, cells were washed three times with cold KRB
and cytochalasin B (10 µM final) was added for several wells as “no
uptake”.
Calc. for C28H26N4O6V: C, 59.47; H, 4.63; N, 9.91%. Found: C, 59.78; H,
4.29; N, 9.58%. The complex is paramagnetic µeff = 1.51 µB. FT-IR (KBr,
cmꢀ 1): 3410 (m), 3029 (w), 1607 (vs), 1551 (s), 1519 (vs), 1454 (vs),
1394 (w), 1350 (s), 1299 (m), 1269 (m), 1226 (m), 1142 (w), 1071 (w),
1054 (w), 1026 (w), 961 (s), 868 (m), 846 (m), 786 (w), 737 (w), 727
(s), 707 (s), 649 (w), 616 (w), 568 (w), 464 (w), 427 (w).
2.6.3. Synthesis of [VO(L2)(phen)]⋅3H2O 2
3-Hydroxysalicylaldehyde (210.5 mg, 1.5 mmol), 2-hydroxybenzhy-
drazide (227.9 mg, 1.5 mmol) and EtOH (25 ml) were refluxed under Ar
for 10 min. Then VOSO4aq (334.5 mg, 1.5 mmol) was added and
mixture was refluxed for additional 25 min. To the dark yellow solution
phen (270.2 mg, 1.5 mmol) was added and mixture was heated for next
3 min. Then it was cooled and the product was filtered off, washed with
EtOH and dried. Yield 0.293 g. MW = 571.43. Anal. Calc. for
For myocytes and adipocytes proceeded as above 2-deoxy-D-[U-
14C]-glucose solution in KRB with total activity 0.03 µCi was added to
each well. After 1 h incubation at 37 ◦C and 5% CO2 uptake was blocked
by adding cytochalasin B (10 µM final). Radioactivity of samples was
measured in scintillation counter MicroBeta Trilux 1450 (PerkinElmer,
USA). Non-specific radioactivity was subtracted from each result (cpm).
Two independent experiments with triplicates were conducted. Final
results were expressed as per cent of control contained solvent only
instead tested compound.
C
26H24N4O8V: C, 54.65; H, 4.23; N, 9.80%. Found: C, 54.61; H, 3.64; N,
9.78%. The complex is paramagnetic µeff = 1.52 µB. FT-IR (KBr, cmꢀ 1):
3392 (w), 3048 (w), 1602 (vs), 1560 (m), 1518 (vs), 1485 (s), 1450 (m),
1423 (s), 1389 (w), 1351 (s), 1300 (w), 1258 (m), 1220 (s), 1160 (w),
1145 (w), 1106 (w), 1052 (w), 1031 (w), 960 (s), 870 (w), 843 (m), 757
(m), 718 (s), 676 (w), 649 (w), 616 (w), 578 (w), 455(w).
2.6. Synthesis
2.6.4. Synthesis of [VO(L3)(phen)]⋅1.5H2O 3
3-Hydroxysalicylaldehyde (203.4 mg, 1.5 mmol), 4-hydroxybenzhy-
drazide (224.5 mg, 1.5 mmol) and EtOH (50 ml) were refluxed under Ar
for 10 min. The light yellow milky solution was formed and then solid
[VO(acac)2] (396.0 mg, 1.5 mmol) was added and mixture was refluxed
for additional 20 min. Then phen (274.2 mg, 1.5 mmol in 10 ml of EtOH)
was added and mixture was heated for additional 8 min. The next day
formed crystals were filtered off, washed with small amount of cold
EtOH and dried in air. Yield 0.521 g. MW = 544.41. Anal. Calc. for
In Table 1 the Schiff base ligand notation and ligand components are
listed.
2.6.1. Synthesis of [VO(L1)(phen)]⋅H2SO4⋅3H2O 1a
3-Hydroxysalicylaldehyde (210.8 mg, 1.5 mmol), benzhydrazide
(208.3 mg, 1.5 mmol) and EtOH (25 ml) were refluxed under Ar for 12
min. Then VOSO4aq (333.7 mg, 1.5 mmol) was added and mixture was
refluxed for additional 48 min. As no precipitation was observed on
cooling, phen (272.0 mg, 1.5 mmol) was added and mixture was heated
for additional 5 min. The mixture was left cooling, product was filtered
off, washed with small amount of EtOH and dried in air. Yield 0.377 g.
MW = 653.52. Anal. Calc. for C26H26N4O11SV: C, 47.78; H, 4.01; N,
8.57; S, 4.91%. Found: C, 48.10; H, 3.85; N, 8.63; S, ≈ 3.7%. The
complex is paramagnetic µeff = 1.56 µB. FT-IR (KBr, cmꢀ 1): 3392 (m),
1606 (s), 1541 (m), 1516 (w), 1491 (w), 1421 (m), 1393 (m), 1341 (w),
1314 (w), 1271 (w), 1221 (m), 1114 (s), 1065 (m), 963 (s), 872 (w), 853
(m), 786 (w), 740 (w), 722 (m), 703 (w), 653 (w), 620 (w), 586 (w), 538
(w).
C26H23N4O6.5V: C, 57.36; H, 3.89; N, 10.29%. Found: C, 57.02; H, 3.66;
N, 9.69%. The complex is paramagnetic µeff = 1.32 µB. FT-IR (KBr,
cmꢀ 1): 3448 (m), 3231 (w), 1605 (vs), 1552 (m), 1501 (vs), 1444 (s),
1420 (m), 1393 (w), 1359 (s), 1278 (m), 1217 (s), 1167 (m), 1103 (w),
1056 (w), 968 (s), 934 (m), 867 (w), 834 (s), 755 (w), 723 (s), 644 (w),
617 (w), 539 (w), 509 (w), 455 (w), 425 (w).
2.6.5. Synthesis of [VO(L4)(phen)]⋅H2O 4
The synthesis was performed as synthesis of 3. The hydrazide used
was 4-chlorobenzhydrazide (253.8 mg, 1.5 mmol). Yield 0.407 g. MW =
553.85. Anal. Calc. for C26H19ClN4O5V: C, 56.38; H, 3.46; N, 10.12%.
Found: C, 56.29; H, 3.48; N, 9.92%. The complex is paramagnetic µeff
=
1.49 µB. FT-IR (KBr, cmꢀ 1): 3539 (m), 1602 (s), 1551 (s), 1514 (s), 1487
(m), 1441 (vs), 1423 (m), 1388 (m), 1350 (s), 1300 (m), 1265 (w), 1219
(s), 1140 (w), 1091 (w), 1050 (w), 1014 (w), 962 (s), 915 (w), 872 (w),
843 (s), 743 (m), 723 (s), 685 (w), 642 (w), 586 (m), 487 (w), 434 (w).
2.6.2. Synthesis of [VO(L1)(phen)]⋅EtOH⋅H2O 1b
To the filtrate, obtained in synthesis of 1a, water was added and
precipitation of light yellow solid was observed. This was filtered off,
washed with water and dried in air. Yield 0.356 g. MW = 565.48. Anal.
Table 1
2.6.6. Synthesis of [VO(L5)(EtO)] 5
List of ligands and ligand components.
3-Hydroxysalicylaldehyde (207.5 mg, 1.5 mmol), phenylacetic acid
hydrazide (227.5 mg, 1.5 mmol) and EtOH (50 ml) were refluxed under
Ar for 10 min. Then [VO(acac)2] (398.2 mg, 1.5 mmol) was added and
mixture was refluxed for additional 41 min giving green-black trans-
parent solution. This was evaporated in part to ca 25 ml giving black oil
liquid. This was left for next day and the black precipitate was filtered
off, washed with EtOH and dried in air. Yield 0.187 g. MW = 380.27.
Anal. Calc. for C17H17N2O5V: C, 53.69; H, 4.51; N, 7.37%. Found: C,
53.53; H, 3.79; N, 8.08%. The complex is diamagnetic. FT-IR (KBr,
cmꢀ 1): 3448 (m), 1608 (vs), 1566 (w), 1545 (w), 1527 (s), 1495 (w),
1449 (w), 1426 (w), 1378 (w), 1347 (w), 1319 (w), 1272 (m), 1221 (w),
1171 (w), 1088 (w), 1059 (w), 1031 (w), 996 (s), 962 (w), 876 (m), 788
(m), 728 (s) 697 (w), 612 (m), 544 (w), 503 (w), 472 (w).
Ligand
Aldehyde
Hydrazide
notation
H2L1
H2L2
H2L3
H2L4
H2L5
3-hydroxysalicylaldehyde
benzhydrazide
2-hydroxybenzhydrazide
4-hydroxybenzhydrazide
4-chlorobenzhydrazide
phenylacetic acid hydrazide
H2L6
H2L7
H2L8
H2L9
3,5-dichlorosalicylaldehyde
4-hydroxybenzhydrazide
4-chlorobenzhydrazide
phenylacetic acid hydrazide
3-hydroxy-2-naphtoic acid
hydrazide
H2L10
H2L11
H2L12
H2L13
3,5-dibromo-4-
4-hydroxybenzhydrazide
4-chlorobenzhydrazide
phenylacetic acid hydrazide
3-hydroxy-2-naphtoic acid
hydrazide
methoxysalicylaldehyde
2.6.7. Synthesis of [VO(L6)(phen)]⋅H2O 6
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dichlorosalicylaldehyde (287.2 mg, 1.5 mmol) and the
3

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Inorganica Chimica Acta xxx (xxxx) xxx
hydrazide used was 4-hydroxybenzhydrazide (226.1 mg, 1.5 mmol).
Yield 0.649 g. MW = 588.29. Anal. Calc. for C26H18Cl2N4O5V: C, 53.08;
H, 3.08; N, 9.52%. Found: C, 52.89; H, 3.15; N, 9.33%. The complex is
paramagnetic µeff = 1.32 µB. FT-IR (KBr, cmꢀ 1): 3431 (m), 1608 (s),
1519 (w), 1495 (s), 1424 (s), 1375 (m), 1353 (m), 1301 (w), 1278 (w),
1210 (m), 1183 (m), 1167 (m), 1145 (w), 1104 (w), 1035 (w), 954 (s),
908 (w), 845 (m), 964 (m), 725 (m), 695 (w), 646 (w), 618 (w), 559 (w),
511 (w), 448 (w).
2.6.12. Synthesis of [VO(L8)(EtO)(EtOH)] 11
The synthesis was performed as synthesis of 3, but without addition
of phen ligand. The aldehyde used was 3,5-dichlorosalicylaldehyde
(287.2 mg, 1.5 mmol) and the hydrazide used was phenylacetic acid
hydrazide (227.4 mg, 1.5 mmol). Yield 0.305 g. MW = 479.23. Anal.
Calc. for C19H21Cl2N2O5V: C, 47.62; H, 4.42; N, 5.85%. Found: C, 47.27;
H, 4.27; N, 5.82%. The complex is diamagnetic. FT-IR (KBr, cmꢀ 1): 3488
(w), 2979 (w), 1608 (s), 1541 (s), 1494 (w), 1442 (s), 1378 (m), 1350
(w), 1294 (m), 1215 (w), 1190 (w), 1088 (m), 1048 (m), 1029 (m), 976
(s), 956 (m), 902 (w), 877 (w), 864 (w), 795 (w), 777 (m), 727 (s), 693
(w), 635 (w), 606 (m), 571 (w), 536 (w), 511 (w), 474 (w), 429 (w).
2.6.8. Synthesis of [VO(L7)(phen)] 7
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dichlorosalicylaldehyde (286.9 mg, 1.5 mmol) and the hydra-
zide used was 4-chlorobenzhydrazide (250.9 mg, 1.5 mmol). Yield
0.688 g. MW = 588.72. Anal. Calc. for C26H15Cl3N4O3V: C, 53.04; H,
2.57; N, 9.52%. Found: C, 52.90; H, 2.58; N, 9.39%. The complex is
paramagnetic µeff = 1.39 µB. FT-IR (KBr, cmꢀ 1): 3370 (m), 1605 (vs),
1579 (w), 1518 (m), 1504 (s), 1485 (m), 1435 (s), 1425 (s), 1394 (w),
1375 (m), 1358 (s), 1297 (m), 1208 (m), 1177 (s), 1141 (m), 1105 (w),
1087 (m), 1031 (w), 1015 (w), 955 (vs), 903 (w), 867 (w), 840 (s), 762
(m), 745 (w), 721 (s), 703 (w), 678 (w), 644 (w), 618 (w), 581 (m), 526
(w), 509 (w), 490 (w), 431 (w).
2.6.13. Synthesis of [VO(L10)(phen)] 12
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dibromo-4-methoxysalicylaldehyde (464.9 mg, 1.5 mmol) and
the hydrazide used was 4-hydroxybenzhydrazide (255.6 mg, 1.5 mmol).
Yield 0.736 g. MW = 689.21. Anal. Calc. for C27H18Br2N4O5V: C, 47.05;
H, 2.63; N, 8.13%. Found: C, 46.95; H, 2.77; N, 7.92%. The complex is
paramagnetic µeff = 1.92 µB. FT-IR (KBr, cmꢀ 1): 3456 (m), 1611 (s),
1592 (s), 1575 (w), 1499 (vs), 1455 (w), 1423 (s), 1402 (w), 1377 (w),
1351 (s), 1280 (w), 1239 (w), 1191 (s), 1166 (m), 1107 (w), 1067 (w),
1025 (w), 974 (w), 959 (s), 842 (m), 777 (w), 755 (w), 720 (m), 637 (w),
555 (w), 509 (w), 433 (w).
2.6.9. Synthesis of [VO(L8)(phen)] 8
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dichlorosalicylaldehyde (287.5 mg, 1.5 mmol) and the hydra-
zide used was phenylacetic acid hydrazide (226.1 mg, 1.5 mmol). Yield
0.641 g. MW = 568.31. Anal. Calc. for C27H18Cl2N4O3V: C, 57.06; H,
3.19; N, 9.86%. Found: C, 57.05; H, 3.20; N, 9.83%. The complex is
paramagnetic µeff = 1.55 µB. FT-IR (KBr, cmꢀ 1): 3443 (w), 3044 (w),
1605 (s), 1529 (vs), 1491 (w), 1436 (m), 1427 (m), 1381 (w), 1354 (w),
1301 (w), 1219 (w), 1179 (m), 1109 (w), 1042 (w), 1012 (w), 964 (s),
884 (w), 861 (w), 841 (m), 795 (w), 753 (m), 722 (s), 701 (w), 648 (w),
612 (w), 541 (w), 526 (w), 500 (w), 432 (w).
2.6.14. Synthesis of [VO(L11)(phen)] 13
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dibromo-4-methoxysalicylaldehyde (464.7 mg, 1.5 mmol) and
the hydrazide used was 4-chlorobenzhydrazide (254.6 mg, 1.5 mmol).
Yield 0.860 g. MW = 707.65. Anal. Calc. for C27H17Br2ClN4O4V: C,
45.83; H, 2.42; N, 7.92%. Found: C, 45.91; H, 2.56; N, 7.82%. The
complex is paramagnetic µeff = 1.46 µB. FT-IR (KBr, cmꢀ 1): 3453 (m),
1605 (s), 1579 (w), 1504 (s), 1491 (s), 1423 (vs), 1350 (s), 1297 (w),
1238 (m), 1196 (s), 1141 (w), 1086 (w), 1069 (w), 1035 (w), 1012 (w),
981 (w), 956 (s), 869 (w), 842 (m), 772 (w), 745 (w), 726 (m), 713 (m),
644 (w), 623 (w), 579 (m), 528 (w), 494 (w), 427 (w).
2.6.10. Synthesis of [VO(L9)(EtO)] 9
3,5-Dichlorosalicylaldehyde (287.0 mg, 1.5 mmol) and 3-hydroxy-2-
naphthoic acid hydrazide (303.0 mg, 1.5 mmol) were dissolved in EtOH
(90 ml) under Ar flow on a magnetic stirrer. The mixture was refluxed
for 10 min with constant stirring and the temperature was raised to
100 ◦C. Then VOSO4aq (333.0 mg, 1.5 mmol) and 10 ml of ethanol was
added and heterogenic mixture was refluxed for ca 180 min. After that,
the volume of the reaction mixture was concentrated to final volume of
ca 20 ml and cooled to the room temperature. The precipitate was
filtered off, washed with ethanol and dried in air. Yield 0.442 g. MW =
485.19. Anal. Calc. for C20H15Cl2N2O5V: C, 49.51H, 3.12; N, 5.77%.
Found: C, 50.06; H, 2.99; N, 6.14%. The complex is diamagnetic. FT-IR
(KBr, cmꢀ 1): 3047 (m), 1640 (s), 1621 (w), 1561 (m), 1517 (w), 1452
(m), 1393 (w), 1355 (w), 1345 (w), 1291 (w), 1222 (m), 1177 (m), 1150
(w), 1110 (w), 1079 (w), 1052 (w), 972 (m), 906 (w), 875 (m), 849 (w),
792 (w), 761 (w), 740 (m), 666 (w), 601 (m), 471 (w), 447 (w).
2.6.15. Synthesis of [VO(L12)(phen)] 14
The synthesis was performed as synthesis of 3. The aldehyde used
was 3,5-dibromo-4-methoxysalicylaldehyde (465.3 mg, 1.5 mmol) and
the hydrazide used was phenylacetic acid hydrazide (227.4 mg, 1.5
mmol). Yield 0.848 g. MW = 687.23. Anal. Calc. for C28H20Br2N4O4V: C,
48.94; H, 2.93; N, 8.15%. Found: C, 48.95; H, 2.94; N, 8.11%. The
complex is paramagnetic µeff = 1.41 µB. FT-IR (KBr, cmꢀ 1): 3478 (m),
1601 (s), 1575 (m), 1526 (s), 1491 (m), 1455 (w), 1417 (s), 1358 (m),
1293 (w), 1239 (w), 1196 (s), 1143 (w), 1102 (w), 1067 (w), 1025 (w),
1006 (w), 967 (s), 871 (w), 851 (m), 779 (w), 758 (w), 724 (m), 673 (w),
643 (w), 535 (w), 460 (w), 425 (w).
2.6.16. Synthesis of [VO(L12)(EtO)(EtOH)] 15
The synthesis was performed as synthesis of 3, but without addition
of phen ligand. The aldehyde used was 3,5-dibromo-4-methoxysalicylal-
dehyde (465.3 mg, 1.5 mmol) and the hydrazide used was phenylacetic
acid hydrazide (227.1 mg, 1.5 mmol). Yield 0.485 g. MW = 598.16.
Anal. Calc. for C20H23Br2N2O6V: C, 40.16; H, 3.88; N, 4.68%. Found: C,
40.06; H, 3.82; N, 4.61%. The complex is diamagnetic. FT-IR (KBr,
cmꢀ 1): 3487 (m), 2973 (w), 1605 (s), 1579 (w), 1534 (m), 1461 (w),
1419 (m), 1358 (w), 1301 (w), 1239 (m), 1204 (m), 1159 (w), 1095 (m),
1065 (w), 1046 (m), 976 (s), 955 (w), 904 (w), 871 (w), 791 (w), 751
(w), 730 (m), 700 (w), 671 (w), 635 (w), 545 (w), 519 (w), 444 (w).
2.6.11. Synthesis of [VO(L7)(EtO)(EtOH)] 10
3,5-Dichlorosalicylaldehyde (1610.0 mg, 8.009 mmol), 4-chloro-
benzhydrazide (1091.0 mg, 8.013 mmol) and EtOH (200 ml) were
refluxed under Ar for 15 min yielding a transparent yellow solution.
Then solid [V(acac)3] (2791.0 mg, 8.011 mmol in 22 ml of EtOH) was
added and mixture was refluxed for additional 50 min. The solution was
cooled to ꢀ 10 ◦C for 20 min. The product was filtered off, washed with
EtOH and dried in air. Yield 1.694 g. MW = 499.65. Anal. Calc. for
C
18H18Cl3N2O5V: C, 43.27; H, 3.63; N, 5.61%. Found: C, 43.06; H, 3.25;
N, 5.79%. The complex is diamagnetic. FT-IR (KBr, cmꢀ 1): 3465 (m),
1609 (s), 1537 (m), 1507 (w), 1490 (m), 1437 (m), 1396 (w), 1279 (m),
1215 (m), 1194 (w), 1141 (w), 1087 (s), 1050 (w), 1026 (m), 976 (m),
942 (vs), 905 (m), 877 (w), 863 (w), 836 (w), 778 (m), 736 (m), 703 (w),
680 (w), 629 (m), 601 (s), 512 (w), 488 (w), 462 (w), 440 (w).
2.6.17. Synthesis of [VO(L13)(EtO)] 16
The synthesis was performed as synthesis of 9. The aldehyde used
was 3,5-dibromo-4-methoxysalicylaldehyde (465.0 mg, 1.5 mmol) and
the hydrazide used was 3-hydroxy-2-naphtoic acid hydrazide (303 mg,
1.5 mmol). Yield 0.686 g. The filtrate was collected and after few days
the crystals of the compound suitable for x-ray analysis appeared in the
4

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
mother liquor. MW = 605.12. Anal. Calc. for C21H16Br2N2O6V: C, 41.75;
H, 2.84; N, 4.64%. Found: C, 41.47; H, 2.88; N, 4.67%. The complex is
diamagnetic. FT-IR (KBr, cmꢀ 1): 3442 (m), 2980 (w), 1636 (s), 1599
(vs), 1578 (m), 1521 (vs), 1464 (s), 1417 (s), 1342 (s), 1303 (s), 1235
(vs), 1218 (m), 1205 (w), 1146 (w), 1088 (m), 1066 (m), 1031 (m), 999
(s), 909 (m), 874 (w), 797 (w), 737 (s), 722 (m), 700 (w), 648 (m), 610
(w), 538 (m), 478 (m), 434 (w).
3. Results and discussion
3.1. General comments regarding the syntheses
The reaction of in situ formed Schiff base ligand with vanadium(IV)
substrate in EtOH results in formation of hexa-coordinated vanadium
with tridentate ONO Schiff base. All syntheses were performed under
restricted anaerobic conditions but, as complexes had to be handled in
air for biological applications, crystallization and complex isolation
were performed in air. This caused the oxidation of V(IV) to V(V). Such
complexes were found to be very sensitive to dioxygen and oxidation
(prior crystallization) to V(V) was observed. Moreover, we confirmed
that phen stabilizes +4 oxidation state of vanadium atom. We showed
also, that vanadium(IV) complexes can by synthesized using different
substrates – VOSO4aq or [VO(acac)2]. In this first case, we observed for
some complexes that products contains sulphur. In for example 1a, in
crystals, the H2SO4 molecule was trapped. In our previous studies, we
observed dimer formation with SO4 bridges (when phen as a co-ligand
was not used). This was not a case in 1a, as vanadium has no “free”
coordination places for other ligands. Presenting synthesis of 1b we
proved, that water can remove this molecule in solution and isolated 1b
does not contain the sulphur. The H2SO4 molecule in 1a probably is
trapped in crystals and is bonded via hydrogen bonds, similarly as water
of hydration. We used very different bidentate ligands, including not
only phen, but also 2,2′-bipyridine, picolinic acid, maltol and its de-
rivatives, ethylenediamine, salicylic acid, oxalic acid and 2-aminopyri-
dine. None of them gave crystalline products, in most cases oil like
products, contaminated with substrates, were isolated. This is why, in
2.7. Crystallographic data collection and structure refinement.
Diffraction intensity data for single crystal of four new compounds
were collected at 293 K on the Oxford Diffraction Super Nova diffrac-
tometer using monochromatic Mo Kα radiation, λ = 0.71073 Å. Cell
refinement and data reduction were performed using firmware [32–34].
Positions of all of non-hydrogen atoms were determined by direct
methods using SHELXL-2017/1 [35,36]. All non-hydrogen atoms were
refined anisotropically using weighted full-matrix least-squares on F2.
Refinement and further calculations were carried out using SHELXL-
2017/1 [35,36]. All hydrogen atoms joined to carbon atoms were
positioned with an idealized geometries and refined using a riding
model with Uiso(H) fixed at 1.2 Ueq (Carom). The positions of all
hydrogen atoms were constrained for all compounds using AFIX
(SHELXL) commands. CCDC 2016920, 2016848, 2016847, 2016921
contain the supplementary crystallographic data for 3, 10, 11 and 16
respectively. These data can be obtained free of charge from The Cam-
bridge Crystallographic Data Centre via www.ccdc.cam.ac.uk/data_re
quest/cif. The crystal data and structure refinement parameters are
summarized in Table 2.
Table 2
Crystal data and structure refinement parameters for 3, 10, 11 and 16.
3
10
11
16
Empirical formula
Formula weight
Temperature [K]
Wavelength [Å]
Crystal system
Space group
C
26H18N4O5V
C18H18Cl3N2O5V
499.63
C19H21Cl2N2O5V
479.22
C23H23Br2N2O7V
650.19
517.38
293(2)
293(2)
293(2) K
293(2)
0.71073
Triclinic
P ꢀ 1
0.71073
Triclinic
P ꢀ 1
0.71073
0.71073
Orthorhombic
P n a 21
Monoclinic
P 21/c
Unit cell
a
12.1230(5)
8.1530(4)
38.045(5)
27.288(5)
dimensions
[Å]
b
12.9770(5)
15.1730(5)
11.6680(5)
12.6640(5)
7.608(5)
7.305(5)
[Å]
c
15.221(5)
24.980(5)
[Å]
α
[◦]
95.023(2)
105.084(2)
92.833(2)
2289.44(15)
4
72.274(2)
73.739(3)
70.938(2)
1062.56(8)
2
90.000(5)
90.000(5)
90.000(5)
4406(3)
8
90.000(5)
91.925(5)
90.000(5)
4977(4)
8
β [◦]
γ [◦]
Volume [Ǻ3]
Z
Density (calculated) [mg/
1.501
1.562
1.445
1.736
m3]
Absorption coefficient
0.481
0.876
0.724
3.656
[mmꢀ 1
]
F(000)
1060
508
1968
2592
Crystal size [mm3]
Theta range for data
collection [o]
0.300 × 0.250 × 0.200
2.796 to 27.508
0.220 × 0.090 × 0.060
2.698 to 27.570
0.400 × 0.100 × 0.050
3.681 to 28.59
0.420 × 0.300 × 0.100
3.164 to 28.840
Index ranges
ꢀ 15 ≤ h ≤ 15–16 ≤ k ≤ 16–19 ≤ l
ꢀ 10 ≤ h ≤ 10–15 ≤ k ≤ 15–16 ≤ l
ꢀ 50 ≤ h ≤ 31–10 ≤ k ≤ 5 20 ≤ l ≤
ꢀ 37 ≤ h ≤ 37–9 ≤ k ≤ 9–17 ≤ l ≤
≤ 19
≤ 16
14
33
Reflections collected
Independent reflections
Completeness to theta [%]
Absorption correction
Refinement method
Data/restraints/
18,233
18,842
15,258
34,152
10,426 [R(int) = 0.0362]
4884 [R(int) = 0.0824]
8535 [R(int) = 0.0248]
99.4
11,814 [R(int) = 0.0602]
99.7
99.7
99.8
None
None
Semi-empirical from equivalents
Full-matrix least-squares on F2
8535/2/535
Semi-empirical from equivalents
Full-matrix least-squares on F2
11,814/4/653
Full-matrix least-squares on F2
10,426/1/665
Full-matrix least-squares on F2
4884/0/287
parameters
Goodness-of-fit on F2
1.030
1.002
0.904
1.053
Final R indices [I > 2
σ
(I)]
R1 = 0.0599 wR2 = 0.1401
R1 = 0.0969 wR2 = 0.1589
0.767 and ꢀ 0.548
R1 = 0.0463 wR2 = 0.0995
R1 = 0.0910 wR2 = 0.1153
0.335 and ꢀ 0.366
R1 = 0.0379 wR2 = 0.0719
R1 = 0.0675 wR2 = 0.0771
0.253 and ꢀ 0.220
R1 = 0.0856 wR2 = 0.1800
R1 = 0.1726 wR2 = 0.2080
2.526 and ꢀ 0.617
R indices (all data)
Largest diff. peak and hole
[e/Ǻ3]
5

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
spite of phen cytotoxicity, we decided to use this co-ligand.
Table 3
Selected bond distances [Å] and angles [◦] in 3, 10, 11 and 16.
The crystals taken directly form solution were transferred for X-ray
structure determination, this is a reason, why in structures of 10, 11 and
16 we were able to see EtOH molecule coordinated to vanadium center.
But, when complex was dried, typically very weakly bonded EtOH
molecule was released (see analysis in 16) and vanadium remained
penta-coordinated. In elemental analysis of 5, 9 and 16 only EtOꢀ anion
remained while in 10, 11 and 15 also by elemental analysis we were able
to see both EtOꢀ and EtOH molecules. In case of 3, when water is of
hydration type, crystals were much more stable, thus crystals for X-ray
structure measurement were taken many days after synthesis. This is a
reason, why in structure of 3 water of hydration is not present.
Complex
Bond length [Å]
Bond angle [◦]
3
V(1)-O(12)
V(1)-O(5)
V(1)-O(4)
V(1)-N(9)
V(1)-N(7)
V(1)-N(10)
1.610(2)
1.938(2)
2.013(2)
2.052(2)
2.149(2)
2.312(2)
O(12)-V(1)-N(9)
O(5)-V(1)-N(9)
O(4)-V(1)-N(9)
O(12)-V(1)-N(7)
O(5)-V(1)-N(7)
O(4)-V(1)-N(7)
N(9)-V(1)-N(10)
N(7)-V(1)-N(10)
103.91(11)
86.53(10)
76.43(9)
93.83(10
95.33(9)
95.22(9)
88.56(9)
73.52(9)
10
11
16
V(1)-O(25)
V(1)-O(19)
V(1)-O(7)
V(1)-O(12)
V(1)-N(9)
V(1)-O(22)
1.584(2)
O(25)-V(1)-O(19)
O(19)-V(1)-O(7)
O(19)-V(1)-O(12)
O(19)-V(1)-O(22)
O(25)-V(1)-N(9)
O(7)-V(1)-N(9)
101.86(10)
99.93(9)
97.98(9)
82.22(9)
95.81(10)
82.85(8)
73.90(8)
79.83(8)
1.7545(19)
1.8688(19)
1.9529(18)
2.139(2)
3.2. Description of the structures
2.326(2)
The structures of 3, 10, 11 and 16 complexes with marked poly-
hedrons are presented in Fig. 1, while in Table 3 selected bond distances
and angles are collected. The more detailed data can be obtained from
cif files.
O(12)-V(1)-N(9)
N(9)-V(1)-O(22)
V(1)-O(14)
V(1)-O(131)
V(1)-O(107)
V(1)-O(112)
V(1)-N(109)
V(1)-O(121)
1.565(3)
1.764(2)
1.858(3)
1.950(3)
2.139(3)
2.308(3)
O(14)-V(1)-O(131)
O(14)-V(1)-O(107)
O(14)-V(1)-O(112)
O(14)-V(1)-N(109)
O(131)-V(1)-O(121)
O(107)-V(1)-O(121)
O(112)-V(1)-O(121)
N(109)-V(1)-O(121)
101.20(13)
99.92(16)
96.63(15)
95.44(13)
82.70(12)
82.36(14)
79.59(13)
80.27(12)
In all complexes vanadium is octahedral with the oxygen of V=O
group in apical position and ONO ligand in square plane. Also in all
complexes the additional EtOH and EtOꢀ molecules are in cis position,
only in 3 phen molecule exchanges the ethanol molecules. As it was
found earlier, the addition of phen during syntheses results in stabili-
zation of V(IV) oxidation state, and only 3 is paramagnetic. The vana-
dium charge is compensated by ONO ligand L2ꢀ , O2ꢀ and EtOꢀ
molecules. To fill the coordination sphere, additional EtOH molecule in
complexes 10, 11 and 16 is coordinated, EtOH molecule is always in
trans position versus O2ꢀ ion. As the complexes 10, 11 and 16 are very
similar, the V=O are expected to be almost identical, but this is not the
case here. The V=O distances are 1.584 Å for 10, 1.565 Å and 1.569 Å
for 11, and 1.576 Å and 1.598 Å for 16, respectively. The differences in
this distance influence the V-O one (to EtOꢀ ligand) which are: 2.326 Å
for 10, 2.308 Å and 2.297 Å for 11, and 2.350 Å and 2.389 Å for 16,
respectively. As typical, short V=O bond results in long V-O one and vice
versa. In described series of complexes (10, 11 and 16) additional effect
is observed - the increase of V=O bond distance coming from 10 to 16 is
V(1)-O(32)
V(1)-O(29)
V(1)-O(23)
V(1)-O(13)
V(1)-N(15)
V(1)-O(26)
1.598(6)
1.753(5)
1.859(5)
1.966(5)
2.143(6)
2.389(6)
O(32)-V(1)-O(29)
O(32)-V(1)-O(23)
O(32)-V(1)-O(13)
O(32)-V(1)-N(15)
O(29)-V(1)-O(26)
O(23)-V(1)-O(26)
O(13)-V(1)-O(26)
N(15)-V(1)-O(26)
102.7(3)
99.4(3)
97.0(3)
98.3(3)
81.6(2)
83.5(2)
78.6(2)
76.8(2)
accompanied by an increase of V-OEt bond distance, contrary to ex-
pected. This suggests that additional factors, as intermolecular in-
teractions, influence those bonds, as described later in this section.
Due to short V=O distance octahedrons are distorted with vanadium
Fig. 1. Structures of complexes 3, 10, 11 and 16. 30% probability ellipsoids, hydrogens are omitted for clarity.
6

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
atom shifted out off square plane towards O2ꢀ ion. The distance of va-
nadium from square plane is not high and equal to 0.272 Å (for 10),
0.275 Å (for 11) and 0.280 Å (for 16), respectively. The ONO ligand is
not symmetric, and form 5- and 6-membered rings, this inequality is
responsible for additional deformation of square plane. The selected
angles are collected in Table 3.
supported). In the IR spectra for V(IV) complexes the intense band
located at a range 955–968 cmꢀ 1 is associated with a νV=O vibration and
for V(V) complexes this band is observed at 972–999 cmꢀ 1 range (5,
9–11, 15, 16). The νC=N vibration for a Schiff base ligand is located at ca.
1600 cmꢀ 1. For complexes with phen as a ligand the characteristic
intense bands at ca. 1490, 1425, 725 cmꢀ 1 are observed.
As it was mentioned in our previous publications, in case of un-
symmetrical ONO Schiff base ligands, complexes are a 1:1 mixture of
two isomers with distinct difference in 5- and 6-memebered ring posi-
tion of ONO ligand [37]. Analogous mixtures of isomers are present in
all studied complexes.
The UV–Vis spectra were measured to check the complexes solubility
in common solvents (the spectra present the saturated solutions in each
solvent, except DMSO and DMF due to relatively high solubility in these
last two solvents) to choose the best solvent for biological studies. Only
DMSO and DMF had a sufficiently high solubility to be used, and DMSO,
as the most relevant, was chosen. The spectra in different solvents were
measured also to check the complex stability and to ensure that observed
bands are of complexes origin and not of the decomposition products.
The MeCN was used to see the high energy bands in UV part, as a sol-
ubility in MeCN resemble that in DMSO. The reflectance spectra of
complexes were measured not only to detect the d-d transitions for V(IV)
complexes, but also to compare them with the solvent ones. The UV–Vis
spectra in a solid state as well as in the organic solvents are dominated
by the charge-transfer transitions, connected with the presence of ONO
Schiff base ligand (at 400–500 nm). In the UV part of the spectra the
intense bands of co-ligands are observed. For V(IV) complexes the band,
visible in reflectance spectra, above 700 nm can be assigned to the d-
d transition. This band is not visible in solution spectra due to its low
intensity (confirming its d-d character) and low solubility of complexes
in all solvents studied. Bands in this region are not present for d0 V(V)
complexes, confirming the vanadium oxidation state. The short period
stability (up to several dozen minutes) of complexes in solvents was
studied by spectral changes observation. In solvents, where no spectral
changes were observed, the spectra were measured also the next day,
but also no spectral changes, indicating complex decomposition, were
observed. Due to very fine crystals, the suspension formation was often
observed and spectral changes indicate on slow clarification of the so-
lutions (decrease of the initially high background absorption). In most
cases the increase of the band intensity is connected with a very slow
dissolution of the crystals, without band shift. As we would like to see
spectral changes at the beginning of the solution formation we did not
filtrate the solutions prior measurements as use of G4 or paper filters did
not give clear solutions. As DMSO was chosen for biological study
(where DMSO solution of complexes were used and mixed with other
The hydrogen bond network is presented in Fig. 2 (for 3 and 10) and
3 (for 11 and 16) while the long distance packing scheme of 3 and 10 or
11 and 16 are presented in Figs. 4 and 5, respectively. The intermo-
lecular interactions, responsible for packing of molecules in structure,
can be divided into hydrogen bonds and other weak interactions. In
general, two different hydrogen bond network are observed in com-
plexes studied. In 10 and 16 hydrogen bonds are present only between
two adjacent complexes, forming something like separated hydrogen
dimmers. In 3 and 11 net of hydrogen bonds binds all molecules
together. In 11 1D chain is formed, while in 3 all hydroxyl groups of L
ligand are involved in hydrogen bonds with nitrogen atoms and with
oxido ligand, as seen in Fig. 2. The complicated net of these bonds results
in 3D structure without separated layers. The other intermolecular in-
teractions additionally bond molecules together, they are mainly the
phen-phen interactions. In 11 the hydrogen bond is present only be-
tween nitrogen of ONO ligand and hydroxyl group of EtOH ligand and
–
bonds two isomers in intermolecular interactions, with N O distance of
2.861 Å and angle of 170.95◦. The formed 1D chains do not form any
other hydrogen bonds and only weak intermolecular interactions be-
tween chains can be observed. In 10 there is only one type of hydrogen
bonds, intermolecular between hydrogen of EtOH molecule and nitro-
gen N(110) of ONO ligand with bond distance of 2.820 Å and
donor–acceptor angle of 164.32◦. For 11, when only one hydrogen bond
is present, both V=O and V-OEt distances are the shortest ones. The net
of hydrogen bonds is much more complicated in 3. All protons of hy-
droxyl groups of ONO ligand, vanadyl oxygen and nitrogen of ONO
ligand participate in intermolecular hydrogen bonds. In 16 both inter-
and intra-molecular hydrogen bonds are present. The nitrogen atom of
ONO ligand as well as phenolic oxygen of hydrazide part of ONO ligands
form intramolecular hydrogen bond with 2.562 Å distance and angle of
146.29◦ indicating weak interaction. Presented intermolecular in-
teractions as a whole, can be responsible for observed differences in
V=O and V-OEt distances discussed earlier. When net of hydrogen bonds
and intermolecular interactions are stronger, thus shifting part of the
electrons towards vanadium, its coordination sphere, and thus V=O and
V-OEt distances, increases.Fig. 3.
solutions resulting in final DMSO-H2O ratio as 20
important was to study the stability in DMSO-H2O mixture. The spectral
changes of the complexes, dissolved in DMSO-H2O mixtures (20 L + 3
μl + 3 ml), the most
μ
ml respectively) in different conditions (pH = 7 and 2) versus time (up to
ca 340 min, time much longer that needed for vanadium passage
through digestion system of humans), were measured. Due to results
obtained, the complexes can be divided into several groups – the com-
plexes stable at pH = 7 and 2 (2, 7, 8, 9, 12, 13 and 16), the complexes
stable at pH = 7, but unstable at pH = 2 (1a, 1b, 3, 4, 6, 11, 14 and 15)
and the complex unstable at pH = 7 and 2 (5). The cyclic voltammetry
measurements are very similar to that described by us in the previous
publications [22,23,37] and show irreversible processes for vanadium
redox system and reversible ones for co-ligands (see Figs in the
3.3. Spectroscopic characteristics
The spectroscopic characterization of the complexes (IR, UV–Vis) as
well as the cyclic voltammetry measurements are presented in the
experimental part and in the Supplementary materials (all spectra are
Fig. 2. Hydrogen bonds in 3 (left) and 10 (right).
7

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
Fig. 3. Hydrogen bonds in 11 (left) and 16 (right).
Fig. 4. Crystal packing scheme of 3 (left side, 010 direction) and 10 (right side, 100 direction).
Therefore, the ability to inhibit the four most important phosphatases in
anti-diabetic effects: PTP1B, LAR, SHP1 and SHP2 was tested in this
study.
The tested complexes show diverse activity in inhibiting tyrosine
phosphatases (Table S1 in the Supplementary materials). The weakest
inhibitors among those are 12 and 13, whose activity is clearly lower
compared to VOSO4 and BMOV for PTP1B and LAR. Inhibition of SHP1
and SHP2 is for them the lowest among all tested complexes, but not
much lower than for VOSO4 and BMOV. In relation to these compara-
tors, 4 and 6 complexes have clearly weaker inhibition, however, this
only applies to PTP1B. The 1a, 3 and 8 complexes are the strongest
inhibitors, with no clear differences in the inhibition of particular tested
phosphatases. The remaining complexes are characterized by a greater
degree of SHP1 and SHP2 inhibition than PTP1A and LAR, however the
observed differences are too small to show the selectivity. The differ-
ences between the inhibition potency may be due to the structural
similarities of these phosphatase pairs.
3.5. Cytotoxicity and cells viability
The toxicity of vanadium compounds has been demonstrated in
numerous preclinical studies in cell and animal models, and the com-
plexes being less toxic than inorganic compounds. Phase I and phase IIa
human clinical trials have been completed with the bis(ethylmaltolato)
oxovanadium(IV) (BEOV), ethyl analogue of bis(maltolato)oxovana-
dium(IV) (BMOV). Despite the absence of adverse effects in several
studies and in the phase I clinical trial, kidney toxicity has been reported
in the phase II clinical trial [20,40]. The lack of significant cytotoxicity is
a condition for the possible use of vanadium complexes in clinical ap-
plications, and by modifying the structure of ligands it is possible to
reduce the cytotoxicity and even obtain a protective effect with main-
taining pharmacological activity [41–43].
Fig. 5. Crystal packing scheme of 11 (up, 100 direction) and 16 (down,
100 direction).
Supplementary materials. For complexes with phen as a co-ligand the
reversible process, in negative part of the voltammogram at ca. ꢀ 1.3 V
(assigned to the phen redox processes), is observed.
3.4. Inhibition of human tyrosine phosphatases
Phosphatases, especially tyrosine phosphatases are a well-described
biological target for vanadium activity. Inhibiting their activity is a
potential therapeutic strategy in many areas of pharmacotherapy,
including the pharmacotherapy of metabolic diseases [38,39].
Tests based on assessment of integration HepG2 hepatocytes mem-
branes did not show a cytotoxic effect for any of the complexes at all
8

J. Szklarzewicz et al.
Inorganica Chimica Acta xxx (xxxx) xxx
tested concentrations (Tables S2 and S3). In the case of tests using the
cytoplasm reducing power of living cells (viability), all tested complexes
these relationships, it is necessary to conduct a panel study of a larger
number of complexes with a more diverse structure. Differences in
cytotoxic and pharmacological effects depend on the type of cells, as
well as the method studying the biological mechanism. This indicates
the need to use various models and methods in the assessment of bio-
logical activity of vanadium complexes. It is important that among the
tested vanadium complexes there are compounds that have greater anti-
diabetic efficacy at non-toxic concentrations than VOSO4 and BMOV.
Presented investigations indicate that the biological activity of the
described vanadium complexes can be related to their stability in the
studied biological systems. When, in solution, the complexes are un-
stable and ligand release is observed, complexes show activity typical for
BMOV. Such activity is typical for unstable vanadium complexes (or
vanadium complexes with bidentate ligands) and was related in litera-
ture to presence of VO2+ ion. When complexes with ONO ligand are
stable, in biological study conditions, much higher insulin-mimetic
properties are observed. This indicates that complex as a whole mole-
cule, and not as VO2+, is active. In all studied by us vanadium complexes
(ca 200), including data presented here, in general V(IV) was found a
little more active and less toxic than V(V) complexes. Among selected
(basing on in vitro tests) 20 complexes of high activity studied in vivo, ca
80% was of V(IV). For complexes presented in manuscript, no differ-
ences were observed in the inhibitory activity of tyrosine phosphatases
depending on the valence of vanadium. They differ significantly in the
degree of PTP1B inhibition with the same V(IV) oxidation state. Also,
the hydrazone component of ONO ligand does not have itself a differ-
ential effect on the inhibition of this phosphatase, contrary to aldehyde
in several cases presented here. These results indicate that it is not
possible to clearly identify one structural factor that determines the
degree of activity. Rather, biological activity seems to be determined by
both structural components. Further study are in progress.
at a concentration of 50
μM showed a cytotoxic effect greater than
VOSO4 and BMOV used in these studies as activity comparators. For
most complexes at the concentration of 10 M, the cytotoxic effect was
μ
clearly reduced to the level accepted in cell model studies. None of the
tested complexes showed a decrease in the viability of C2C12 myocytes,
with the majority of them showing an increase of cytoplasmic reducing
activity, which can be explained by the stimulation of metabolic pro-
cesses by these complexes (Table S4). Observed values were much
higher than for the control and VOSO4 and BMOV. Only the 5 and 11 did
not increase the measured values. Both of these complexes have vana-
dium oxidation state V and, unlike the others, do not have phenan-
throline as a co-ligand. Comparison of the structure and activity of 8
(+phen) with 11 (-phen) seems to explain this relationship. Similarly, in
the case of viability of 3T3-L1 adipocytes, 8 (+phen) shows an increase
in the metabolic activity of the cytoplasm as opposed to 11 (ꢀ phen)
(Table S5). Except for 8 (+phen), none of the tested complexes showed
significant differences in 3T3-L1 adipocyte viability relative to control.
This indicates that the activity varies with different cell types.
3.6. Radiolabeled glucose analogue transport
All tested complexes showed the ability to increase glucose transport
to C2C12 myocytes and 3T3-L1 adipocytes, which is the basic cellular
parameter determining antidiabetic activity (Tables S6 and S7). All
complexes at 50 μM showed significantly higher activity than VOSO4
and BMOV in C2C12 myocytes. It was significant, that the activity of
these complexes (except for 13) was also clearly higher at a concen-
tration of 10 μM at which no significant cytotoxic effects were observed.
Similar to cytotoxicity studies, the effect of complex structure on their
activity is observed. 8 (+phen) has significantly higher activity than 11
(ꢀ phen). Another example could be the type of hydrazide component in
8 (phenylacetic acid hydrazide) and 14 (4-chlorobenzhydrazide) com-
plexes for which different levels of glucose transport intensification are
observed for both types of cells.
Declaration of Competing Interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
4. Conclusions
Syntheses of 16 new vanadium(IV and V) complexes with ONO tri-
dentate Schiff base ligands are described. 11 of them were synthesized
with phen as co-ligand, with typical octahedral vanadium(IV). In case of
5 others, solvent molecules (EtOH and EtOꢀ ) fill the coordination sphere
of vanadium(V) to six. Thus, independent on ligands both V(IV) and V
(V) were octahedral. The EtOH molecule trans to V=O bond is very
weakly bonded and in a solid state decrease of coordination number to 5
is observed on solvent release. As the ligand is not symmetrical and
forms both 5- and 6-membered rings in complex, mixture of isomers in
1:1 ratio in unit cell is observed. Due to a very low solubility in water, for
biological applications DMSO-H2O mixtures have to be introduced, thus
the thermal stability of such a solutions versus time was studied. The
spectroscopic study of solution stability was performed at pH = 7.0
(typical rather for in vitro buffers) and at pH 2.0 (typical for situation
when oral admission of complex in in vivo studies results in complex
passing through digestion system).
Acknowledgement
This work was partly financed by the European Regional Develop-
ment Fund under the Innovative Economy Programme 2007–2013
(WND POIG.01.03.01-174/09).
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ica.2020.120135.
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