V(V)-Schiff base species induce adipogenesis through structure-specific influence of genetic targets

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V(V)-Schiﬀ base species induce adipogenesis

through structure-specific influence of genetic

targets†

Cite this: New J. Chem., 2019,

43, 17872

a

b

O. Tsave,

E. Halevas,

M. P. Yavropoulou,

E. Yovos,

c

d

e

f

A. Hatzidimitriou,

A. Salifoglou

V. Psycharis,

K. Ypsilantis,

P. Stathi

and

a

*

Vanadium has been known to exhibit numerous functions in biological systems, projecting exogenous

activity linked to regulatory roles in cell metabolism and insulin mimesis. Poised to probe into the

vanadium adipogenic potential, reflecting on eﬃcient anti-diabetic drugs, research was launched in our

labs to pursue the (a) synthesis of diversely structured Schiﬀ base ligands, as vanadium chelating binders,

containing a variably configured o-vanillin core with a specified common organic o-aminophenol tether,

(b) synthesis of a family of well-defined soluble vanadium-Schiﬀ base compounds, bearing the above

mentioned Schiﬀ ligands (and in one case 4,4⁰-bipyridine), (c) study of their toxicity profile and

adipogenic activity in 3T3-L1 fibroblasts toward mature adipocytes, and (d) determination of molecular

biological targets linked to the vanadium-induced cell diﬀerentiation process, thereby unravelling factors

impacting signaling pathways influencing insulin mimesis. The results suggest that (a) all emerging

vanadospecies contain V(^V) mononuclear centers bound to Schiﬀ bases (and 4,4⁰-bipyridine), (b) a well-

defined (solid-state and solution) physicochemical profile of all species justifies their selection in

biological studies, (c) the vanadium toxicity profile is strongly related to the form of V(^V) (complexed

forms, substrate-ligand nature), (d) there is a structure-specific behavior of vanadium influencing

adipogenesis, and (e) molecular target loci are also influenced by vanadoforms in a structure-specific

fashion, thereby collectively projecting an interwoven role of factors emanating from vanadium and

impacting cell diﬀerentiation and insulin mimetic activity. The so far accrued knowledge constitutes the

basis for further development of biomarker-driven structure-specific vanadodrugs, contributing through

insulin biomimicry to therapeutic technologies in Diabetes mellitus.

Received 16th May 2019,

Accepted 15th October 2019

DOI: 10.1039/c9nj02520k

rsc.li/njc

1. Introduction

Diabetes mellitus (DM) is one of the most common endocrine

disorders. It is estimated that in 2018 there were more than

500 million certified cases of type 2 diabetes worldwide, with

^aLaboratory of Inorganic Chemistry and Advanced Materials, Department of

Chemical Engineering, Aristotle University of Thessaloniki, Thessaloniki 54124,

Greece. E-mail: salif@auth.gr; Fax: +30-2310-996-196; Tel: +30-2310-996-179

^bDivision of Clinical and Molecular Endocrinology, 1st Department of Internal

Medicine, AHEPA, University Hospital, Aristotle University of Thessaloniki,

Thessaloniki 54124, Greece

^cLaboratory of Inorganic Chemistry, Department of Chemistry,

Aristotle University of Thessaloniki, Thessaloniki 54124, Greece

^dInstitute of Nanoscience and Nanotechnology, NCSR ‘‘Demokritos’’,

Aghia Paraskevi 15310, Attiki, Greece

^eDepartment of Chemistry, University of Ioannina, Ioannina 45110, Greece

^fLaboratory of Physical Chemistry of Materials & Environment,

Department of Physics, University of Ioannina, Ioannina 45110, Greece

† Electronic supplementary information (ESI) available. CCDC 1914116 (1),

1898796 (2), 1898797 (3), and 1898798 (4). For ESI and crystallographic data in

CIF or other electronic format see DOI: 10.1039/c9nj02520k

the onset of the disease expected to rise dramatically in the

upcoming years.¹DM can be divided into several clinically

distinct types. Broadly, there are two major types, type 1 diabetes

(T1D) and type 2 diabetes (T2D), according to the etiopathology

of the disorder. T2D appears to be the most common form of

diabetes (90% of all diabetic patients), mainly characterized by

insulin resistance, whereas T1D is an autoimmune disorder.²In

all cases, insulin serves as the key factor with abnormal secre-

tion and/or action. Insulin deficiency results in elevated blood

glucose levels (hyperglycemia) and impaired metabolism of

carbohydrates, fat, and proteins, among others, and is strongly

related to usually severe secondary complications (cardiovascular

disease, diabetic neuropathy, etc.). Current treatment pathways of

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DM are based either on exogenous insulin and/or hypoglycemic bases. They are simple organic ligands, emulating a significant

agents.^3,4Although these therapeutic approaches are well- number of natural substrates of variable mass and (bio)chemical

established, they have several limitations, including significant reactivity, while concurrently possessing a diverse spectrum of

side-eﬀects and elevated levels of patient stress. To this end, structural features, enabling them to pursue eﬀective interactions

extensive eﬀorts have been made over the past few decades to with metal ions.^18–20Suffice to state that they have been studied

investigate the insulin mimetic eﬀect of a plethora of agents extensively as compounds exhibiting a variable spectrum of biolo-

(natural products, metal ions, organic extracts, etc.) that could gical activities linked to industrial, antifungal, antibacterial, anti-

enhance or even displace insulin use in treating or alleviating DM cancer, antidiabetic, antiviral and herbicidal applications.^21–31

symptoms.^5–7

Guided by the idea that appropriately designed Schiﬀ bases

The first report on therapeutic applications of vanadium might eﬀectively bind V(^IV,V) ions, thereby formulating the

compounds in humans appeared very early in 1899. However, it biological profile of those metal ions with respect to insulin mimesis,

was not until 1980 that the insulin-mimetic eﬀect of vanadium eﬀorts were launched to (a) design and synthesize diversely

was discovered.^8,9The eﬀect pertains to the ability of vanadium composed Schiﬀ bases, bearing the common 2-aminophenol

salts and vanadium complexes to lower blood glucose levels by component, promoting vanadium complexation through dis-

triggering cellular glucose uptake for further catabolism. It is tinctly structured anchor groups, (b) pursue the synthesis of

worth mentioning that although simple vanadium salts, such binary and/or ternary complex assemblies of such Schiﬀ base

as NaVO₃and VOSO₄, can lower blood glucose levels in rats and ligands with vanadium and ancillary binders, (c) investigate

humans, numerous studies have shown that hybrid vanadium- their insulin mimetic potency toward cellular diﬀerentiation of

organic substrate complexes are less toxic and several times premature fibroblasts toward mature fibroblasts capable of

more eﬀective in lowering blood glucose levels.^10,11What was taking up glucose, (d) study the interactions of the arising

considered a critical step toward the use of vanadium as a vanadium species with cellular molecular targets intimately

potent anti-diabetic agent in those early studies was the development linked to insulin-induced signaling pathways promoting glucose

of vanadium complexes that would have increased bioavailability, uptake, and finally (e) configure biological profiles, thus allowing

such that lower doses could improve eﬃcacy and safety. As such, identification of structure-specific speciation leading to eﬃcient

considerable research has gone into studying the active form of insulin mimetic processes. On these grounds, the herein described

insulin-mimetic vanadium complexes, their pharmacological proper- research pertains to (a) systematic eﬀorts leading to binary–ternary

ties and their transport to target sites in the body by the blood vanadium(^V)-Schiff base complex compounds and their physico-

stream. Vanadium complexes show insulin-mimetic eﬀects in three chemical characterization, and (b) molecular biological studies

oxidation states [V(^III), V(IV) and V(V)]. Vanadium(V)-oxido anions and exemplifying biochemical profiles enabling meaningful correlations

complexes are known to inhibit protein tyrosine phosphatases with structural features, collectively contributing to the under-

(PTPs).¹²Inhibition of PTPs keeps the insulin receptor phos- standing of vanadium insulin-mimesis and its potential use

phorylated, allowing glucose transport into the cell. Under through metallodrugs in the treatment of diabetes.

physiological conditions, vanadium complexes can interconvert

between V(^IV) and V(V), and V(V) can inhibit PTPs.¹³Moreover,

V(^IV) can activate the recruitment of glucose transporters to the

2. Results and discussion

2.1 Synthesis

cell membrane, thereby increasing glucose cell uptake and

lowering blood glucose levels.¹⁴

Synthetic exploration of the binary V(^V)-Schiﬀ base systems in this

Given the fact that insulin exerts its properties in multiple

work followed carefully designed approaches. The employed

ways (cell proliferation, diﬀerentiation, growth, gene expression,

ligands L₁H₂–L₃H₂were synthesized through Schiff base conden-

sation reactions involving o-vanillin and 2-aminophenol (L₁H₂),

etc.) insulin mimesis of candidate agents can also be studied

toward these actions.¹⁵Adipogenesis is an intimately linked

salicylaldehyde and 2-aminophenol (L₂H₂), and 2-hydroxy-1-

part of metabolism, defined as a highly complex physiological

naphthaldehyde and 2-aminophenol (L₃H₂).^32–34Subsequently,

process, by which precursor stem cells undergo diﬀerentiation

to form mature adipocytes.¹⁶In mammals, energy is mainly

the [VO(L₁)(OCH₃)(CH₃OH)] (1) complex was synthesized in a facile

fashion from simple reagents in alcoholic solutions. In a typical

stored in the form of fat in the adipose tissue, which serves as a

source on-demand.¹⁷Adipose tissue is characterized as a highly

reaction, [VO(C₅H₇O₂)₂] reacted with the Schiff base L₁H₂using

methanol as a solvent. The overall stoichiometric reaction leading

to 1 is shown schematically below:

metabolic endocrine organ and hence adipose tissue is consid-

ered as a key component in the investigation of such relevant

(patho)physiologies in insulin resistance and hence Diabetes

mellitus II (T2D).

Taking into account that antidiabetic candidate drugs should (a)

exhibit atoxicity, (b) optimal insulin mimetic activity, and (c)

enhanced ability to cross membrane barriers in their eﬀort to

deliver their active cargo, organic substrates appear to play a

significant role, thereby attracting considerable interest as potential

V(^IV,v) binders. One such family of organic substrates includes Schiﬀ

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Similarly, [VO(C₅H₇O₂)₂] reacted with Schiﬀ base L₁H₂2.2 Physicochemical characterization

Paper

in methanol, in the presence of 4,4⁰-bipyridine. The stoi-

chiometric reaction leading to the formation of compound

[VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)(CH₃O)] (2) is shown below:

2.2.1 X-ray crystallographic structures. The X-ray crystal

structures of 1–4 reveal discrete solid-state lattices. Crystal

structure information is provided in Table 1, with molecular

structures of 1–4 given in Fig. 1–4, respectively; selected bond

distances and angles are listed in Table 2. Compound 1 crystallizes

in the monoclinic space group P2₁/a. In the asymmetric unit, there

exist [VO(L₁)(OCH₃)(CH₃OH)] assemblies with the molecular struc-

ture presented in Fig. 1. The vanadium centers in the aforemen-

tioned assemblies are six coordinate, with an NO₅environment

generating a distorted octahedral coordination geometry. Two of

the oxygen atoms are provided by the deprotonated L₁ligand and

three are from the methoxy, methanol and oxido ligands. The axial

V–O(22) bond (trans to the VQO bond) is longer than the

equatorial bonds due to the trans effect.³⁵In the equatorial plane,

the Schiff base is coordinated to the V center through five- and six-

membered chelate rings, with bite angles 77.41 (O(1)–V–N) and

83.51 (O(2)–V–N), respectively. The other oxygen-containing bond

lengths follow the trend observed in similar compounds with an

NO₅environment around vanadium.³⁶The complex assemblies in

1 through the O(22)–H(22)OꢀꢀꢀO(1)⁰hydrogen bonds (O–H: 0.73,

HꢀꢀꢀO(1)⁰: 2.08, O(22)ꢀꢀꢀO(1)⁰: 2.752 Å and O(22)–H(22)OꢀꢀꢀO(1)⁰:

1531; symmetry code: x ꢁ 1/2, ꢁy + 1/2, z) form chains parallel to

In an analogous reaction, [VO(C₅H₇O₂)₂] reacted with Schiﬀ

base L₂H₂in methanol. The stoichiometric reaction leading to

the formation of [VO(L₂)(OCH₃)(CH₃OH)] (3) is shown below:

In a similar reaction, [VO(C₅H₇O₂)₂] reacted with Schiﬀ base the a crystallographic axis (Fig. S1, ESI†). These chains interact

L₃H₂in methanol, in the presence of sodium hydroxide. The

through shifted p–p interactions of [L₁]^2ꢁligands, thus forming

stoichiometric reaction leading to the formation of compound

[{VO₂}(L₃)]Na (4) is shown below:

layers parallel to the (001) crystallographic plane (Fig. S1, ESI†).

The centroid–centroid distanses take on values of 3.778 and

4.048 Å (indicated with dark red and orange dashed lines,

respectively, in Fig. S1, ESI†).

Compound 2 crystallizes in the triclinic space group

%

P1. In the unit cell there exist dinuclear complex

[VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)(CH₃O)] assemblies, bearing a unit

molecular structure presented in Fig. 2. The complexes are centro-

symmetric, with the inversion center located at the middle of the

distance of the sigma bond in the 4,4⁰-bipyridine ligand.

Vanadium (V(^V)) cations are again six coordinate, exhibiting

an O₄N₂environment and a distorted octahedral geometry. Two

of the oxygen atoms are provided by the doubly deprotonated

[L₁]^2ꢁligand, with an additional two oxygens originating from

the methoxy and oxido ligands. One of the nitrogen atoms

(N(1)) is from [L₁]^2ꢁ, with the second one (N(2)) belonging to

the 4,4⁰-bipyridine ligand. The axial bond length V–N(2) is

2.4772(19) Å, considerably longer than the rest of the equatorial

bonds due to the trans effect (trans to the VQO(3) bond). The

coordination geometry of the 4,4⁰-bipyridine ligand is strange,

as the metal cation is shifted from the ligand mean plane by

0.864 Å and the angle V–N(2)–C(17) is 161.861. The doubly

deprotonated Schiff base is coordinated to the vanadium center

V(^V) in a chelate tridentate mode through one five-membered

and one six-membered ring, with bite angles of 77.081 (O(1)–

V(1)–N(1)) and 83.031 (O(2)–V(1)–N(1)), respectively. The rest of

the vanadium–oxygen bond lengths are quite similar to those

observed in analogous complexes.³⁷No hydrogen bonding or

other type of intermolecular or intramolecular interaction was

found in the crystal structure.

Diethyl ether was added as a precipitating solvent to the

reaction mixture in the first and second reactions described

above. The third and fourth reaction mixtures were allowed to

evaporate slowly. Brown crystalline materials emerged in all

four reactions described above, the analytical composition of

which was consistent with the formulation in 1–4, respectively

(vide supra). Positive identification of the crystalline products

was achieved by elemental analysis, FT-IR spectroscopic methods

and X-ray crystallographic determination for isolated single

crystals from 1–4. All complexes are stable in the crystalline form,

in air, for fairly long periods of time. All four species are readily

dissolved in water and DMSO, less soluble in CH₃OH and

acetone, sparingly soluble in i-PrOH, and insoluble in acetonitrile

and chloroform at room temperature.

The common feature of all members in the Schiﬀ base series

was the o-aminophenol moiety, ultimately providing vanadium

binders L₁H₂, L₂H₂, and L₃H₂. Progressive modification of ligand

nature led to synthetic eﬀorts in methanolic reaction mixtures

under specific conditions, guided by reagent molecular stoichio-

metries, finally aﬀording isolable crystalline materials 1–4.

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Table 1 Crystallographic data for compounds [VO(L₁)(CH₃O)(CH₃OH)] (1), [VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)(CH₃O)] (2), [VO(L₂)(CH₃O)(CH₃OH)] (3), and

[VO₂(L₃)Na(H₂O)₂VO₂(L₃)Na(H₂O)₃]₂ꢀH₂O (4)

1

2

3

4

Compound

Chemical formula

M_r

Crystal system

Space group

C

₁₆H₁₈NO₆V

C₂₀H₁₈N₂O₅V

417.31

Triclinic

C₁₅H₁₆NO₅V

341.24

Monoclinic

P2₁/n

C₆₈H₆₆N₄Na₄O₂₇V₄

1667.01

Monoclinic

P2₁/n

371.25

Monoclinic

P2₁/a

¯

Pı

Temperature (K)

a (Å)

b (Å)

c (Å)

a (1)

b (1)

160

295

299

7.1228 (1)

22.3349 (5)

10.4326 (2)

90

100.566 (1)

90

7.9698 (8)

10.5539 (11)

11.3860 (12)

98.204 (3)

99.184 (3)

90.805 (4)

935.09 (10)

2

7.2356 (4),

21.3942 (13)

19.5771 (13)

90

100.594 (4)

90

7.3508 (5),

21.9513 (15)

21.7346 (17)

90

92.504 (4)

90

g (1)

V (Å³)

1631.55 (5)

4

2978.9 (3)

8

3503.7 (4)

2

Z

Radiation type

Cu Ka

5.38

Mo Ka

0.57

Mo Ka

0.69

Mo Ka

0.63

m (mm^ꢁ1

)

T_min, T_max

0.549, 1.000

0.91, 0.92

0.86, 0.91

0.97, 0.97

No. of reflections

Measured

10 727

2642

2100

0.050

0.588

11 781

7021

3498

0.025

0.770

46 827

5992

4139

0.057

0.626

58 024

7682

5051

0.016

0.648

Independent

Observed [I 4 2.0s(I)]

R_int

(sin y/l)_max(Å^ꢁ1

)

Refinement

R[F²4 2s(F²)]

R_w(F²)

0.051

0.134

1.05

0.044

0.106

1.00

0.052

0.112

1.00

0.049

0.097

1.00

S

No. of parameters

253

0.51, ꢁ0.48

253

0.60, ꢁ0.38

397

0.67, ꢁ0.42

487

0.50, ꢁ0.71

Dr_max, Dr_min(e Å^ꢁ3

)

Fig. 1 Partially labeled plot of the molecular structure of compound 1.

Compound 3 crystallizes in the monoclinic space group P2₁/n.

Fig. 2 Partially labeled plot of the molecular structure of 2.

In the asymmetric unit there are two slightly diﬀerent complex equatorial plane, the Schiff base is coordinated to the V(^V) center

assemblies bearing the formula [VO(L₂)(CH₃O)(CH₃OH)]. Fig. 3 through five and six-membered chelate rings, with bite angles

presents the molecular structure of one of the two molecules. It is 76.901 and 77.691 (O(1)–V(1)–N(1) and O(6)–V(2)–N(2)), respectively,

evident that the structures of compounds 1 and 3 are nearly and 84.691 and 84.271 for O(2)–V(1)–N(1) and O(7)–V(2)–N(2),

similar. Bond lengths and angles are listed in Table 2. Vanadium respectively. All bond lengths follow the trend observed in

(V(^V)) cations are six coordinate, similar to the corresponding similar compounds exhibiting an O_xN_y(where x + y = 6)

center in 1, with the same O₅N chromophore and a distorted environment around the vanadium center.³⁷Hydrogen bonds

octahedral geometry. Two of the oxygen atoms are provided by arise from the coordinated methanol oxygen atoms to phenolic

the doubly deprotonated [L₂]^2ꢁligand, with an additional three oxygen atoms from neighboring molecules, thus forming

oxygens originating from the methoxy, methanol and oxido chains parallel to the a crystallographic axis (Fig. S2 in cyan

ligands. The axial, V–O(5) bond (trans to the VQO(3) bond), is lines, ESI†). These chains interact through shifted p–p inter-

longer than the equatorial bonds due to the trans effect. In the actions of [L₂]^2ꢁligands, thus forming layers parallel to the

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with similar structures.³⁷The bond lengths of the oxido ligands are

slightly unequal, as V(1)QO(4) and V(2)QO(8) bonds are found

to be 1.643(3) Å and 1.644(3) Å, whereas V(1)QO(3) and

V(2)QO(7) are found to be 1.623(3) Å and 1.592(3) Å, respectively.

This bond diﬀerentiation could be attributed to the fact that O(8)

is also coordinated to two diﬀerent sodium cations (Na(1) and

Na(2)), bridging them together and also bridging both to the V(2)

cation, whereas O(4) is singly bridging V(1) to Na(1). The atoms

O(3) and O(7) are axially coordinated atoms, placed on top of the

two tetragonal pyramids forming around the vanadium centers.

In the basal plane, the Schiﬀ base is coordinated to the vanadium

center through five and six-membered chelate rings exhibiting

bite angles 76.421 and 76.991 (for O(1)–V(1)–N(1) and O(5)–V(2)–

N(2), respectively), and 81.541 and 81.671 (for O(2)–V(1)–N(1) and

O(6)–V(2)–N(2), respectively). The six-membered coordination

sphere around Na(1) also comprises two more bridging oxygen

atoms from the phenolic moieties of two different [L₃]^2ꢁligands

and two bound water molecules (O(9) and O(10)). The coordina-

tion geometry around Na(1) can be described as highly distorted

trigonal prismatic, with O(1), O(4), O(10) and O(5), O(8), O(9)

forming the trigonal bases. The second sodium cation, Na(2), was

found to be five coordinate, with four bound water molecules

(O(11), O(12), O(12)⁰and (O13)) and O(8) as already described.

The atoms Na(2), O(12), Na(2)’ and O(12)⁰form a parallelogram,

with its center being the center of symmetry of the assembled

octanuclear bimetallic complex.

Fig. 3 Partially labeled plot of the molecular structure of compound 3.

A hydrogen-bonding network of twelve interactions is present

in the crystal structure of the compound. All water molecules,

coordinated or free, participate in these mainly intermolecular

(eight out of the twelve) interactions, thereby creating molecular

chains parallel to the a crystallographic axis and forming

ultimately a 1D lattice (Fig. S3, ESI†).

Worth emphasizing in all of the above structures of derived

materials 1–4 was the fact that: (a) all compounds involved

mononuclear vanadium complexes, and (b) in all cases, the

vanadium oxidation state was V(^V). Confirmation of that con-

tention, beyond X-ray crystallography, was achieved through

EPR spectroscopy, with silent spectra observed in all cases (not

Fig. 4 Partially labeled plot of the molecular structure of compound 4.

(001) crystallographic plane (Fig. S2, ESI†). The centroid– shown), (c) the molecular stoichiometry of V(^V) : ligand was 1 : 1,

centroid distanses take up values 3.806 and 3.909 Å (indicated and (d) the deprotonation state of the ligand bound to vanadium

by violet lines in Fig. S2, ESI†).

was 2-. Moreover, quite interesting was the synthetic introduction

Compound 4 crystallizes in the monoclinic space group P2₁/n. of 4,4⁰-bipy in the coordination sphere of vanadium. Albeit quite

The asymmetric unit contains one centrosymmetric bimetallic successfully pursued in the case of compound 2 with ligand [L₁]^2ꢁ

octanuclear complex bearing the formula C₆₈H₆₄N₄Na₄O₂₆V₄(a derivative of compound 1), reflecting replacement of the bound

and one lattice water molecule. Fig. 4 shows the structure of methanol molecule in 1 by a 4,4⁰-bipy moiety, analogous reactions

[VO₂(L₃)Na(H₂O)₂VO₂(L₃)Na(H₂O)₃]₂ꢀH₂O. Bond lengths and of vanadium with ligands L₂H₂and L₃H₂did not afford tractable

angles are listed in Table 2. Vanadium (V(^V)) cations as parts crystalline materials (in the case of 3 and 4) amenable to further

+

of the dioxidovanadium(^V) (VO₂) units were found to be five physicochemical characterization. Quite distinct, however, was the

coordinate with an O₄N chromophore. The trigonality index correlation of the chemical reactivity of vanadium with the bulk

t values are equal to 0.101 and 0.187 (close to zero) for V(1) and nature of the ligand employed. In that respect, with the starting

V(2), respectively. This indicates a distorted tetragonal pyramidal material in all cases being vanadium acetylacetonate (V(^IV)),

geometry around the vanadium centers. Two of the oxygen atoms employment of 2-hydroxy-1-naphthaldehyde led to the isolation

in the coordination sphere of the vanadium cations are provided of crystalline material 4 bearing the [VO₂]⁺unit in the complex

by the doubly deprotonated [L₃]^2ꢁligands, with another two oxygens assembly. Inevitably, therefore, a characteristic difference between

coming from oxido ligands. All bond lengths around the vanadium complexes 1–3 and 4 was the fact that the former species all

centers are similar to those found in dioxidovanadium compounds contain V(^V) in a [VO]³⁺core moiety, with the corresponding core

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Table 2 Bond lengths [Å] and angles [deg] of [VO(L₁)(CH₃O)(CH₃OH)] (1), [VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)(CH₃O)] (2), [VO(L₂)(CH₃O)(CH₃OH)] (3), and

[VO₂(L₃)Na(H₂O)₂VO₂(L₃)Na(H₂O)₃]₂ꢀH₂O (4)

1

2

3

4

Distances (Å)

1.9064(17)

1.8602(15)

1.5975(17)

1.7760(16)

2.1603(19)

2.4772(19)

V–O

1.588(3)

1.769(3)

1.860(2)

1.939(2)

2.166(3)

2.295(3)

V(1)–O(1)

V(1)–O(2)

V(1)–O(3)

V(1)–O(5)

V(1)–N(1)

V(1)–N(2)

V(1)–O(1)

V(1)–O(2)

V(1)–O(3)

V(1)–O(4)

V(1)–O(5)

V(1)–N(1)

V(2)–O(6)

V(2)–O(7)

V(2)–O(8)

V(2)–O(9)

V(2)–O(10)

V(2)–N(2)

1.921(4)

1.860(4)

1.578(5)

1.762(4)

2.392(4)

2.158(5)

1.931(4)

1.850(4)

1.586(4)

1.759(4)

2.388(4)

2.147(5)

V(1)–O(1)

V(1)–O(3)

V(1)–O(4)

V(1)–N(1)

V(2)–O(5)

V(2)–O(6)

V(2)–O(7)

V(2)–O(8)

1.906(3)

1.623(3)

1.644(3)

2.189(3)

1.918(3)

1.904(3)

1.592(3)

1.643(3)

2.160(3)

2.275(3)

2.537(3)

2.347(3)

2.406(4)

2.333(4)

2.648(4)

2.442(4)

2.417(4)

2.393(4)

2.258(4)

2.143(5)

V–O(21)

V–O(2)

V–O(1)

V–N

V–O(22)

V(2)–N(2)

Na(1)–O(1)

Na(1)–O(4)

Na(1)–O(5)

Na(1)–O(8)

Na(1)–O(9)

Na(1)–O(10)

Na(2)–O(12)ⁱ

Na(2)–O(8)

Na(2)–O(11)

Na(2)–O(12)

Na(2)–O(13)

Angles (8)

O–V–O(21)

O–V–O(2)

O(21)–V–O(2)

O–V–O(1)

O(21)–V–O(1)

O(2)–V–O(1)

O–V–N

O(21)–V–N

O(2)–V–N

102.10(13)1

98.15(13)1

101.19(11)1

99.75(12)1

92.88(11)1

154.33(11)1

92.60(12)1

163.65(12)1

83.53(11)1

77.43(11)1

173.06(12)1

84.82(11)1

79.68(11)1

80.36(11)1

80.62(11)1

O(1)–V(1)–O(2)

O(1)–V(1)–O(3)

O(2)–V(1)–O(3)

O(1)–V(1)–O(5)

O(2)–V(1)–O(5)

O(3)–V(1)–O(5)

O(1)–V(1)–N(1)

O(2)–V(1)–N(1)

O(3)–V(1)–N(1)

O(5)–V(1)–N(1)

O(1)–V(1)–N(2)

O(2)–V(1)–N(2)

O(3)–V(1)–N(2)

O(5)–V(1)–N(2)

N(1)–V(1)–N(2)

150.40(8)1

102.26(8)1

99.89(8)1

93.14(8)1

101.92(8)1

100.43(9)1

77.08(7)1

83.03(7)1

91.35(8)1

166.13(8)1

79.91(7)1

77.65(7)1

177.49(8)1

80.62(8)1

87.91(7)1

O(1)–V(1)–O(2)

O(1)–V(1)–O(3)

O(2)–V(1)–O(3)

O(1)–V(1)–O(4)

O(2)–V(1)–O(4)

O(3)–V(1)–O(4)

O(1)–V(1)–O(5)

O(2)–V(1)–O(5)

O(3)–V(1)–O(5)

O(4)–V(1)–O(5)

O(1)–V(1)–N(1)

O(2)–V(1)–N(1)

O(3)–V(1)–N(1)

O(4)–V(1)–N(1)

O(5)–V(1)–N(1)

O(6)–V(2)–O(7)

O(6)–V(2)–O(8)

O(7)–V(2)–O(8)

O(6)–V(2)–O(9)

O(7)–V(2)–O(9)

O(8)–V(2)–O(9)

O(6)–V(2)–O(10)

O(7)–V(2)–O(10)

O(8)–V(2)–O(10)

O(9)–V(2)–O(10)

O(6)–V(2)–N(2)

O(7)–V(2)–N(2)

O(8)–V(2)–N(2)

O(9)–V(2)–N(2)

153.3(2)1

101.4(2)1

98.8(2)1

92.94(19)1

99.5(2)1

O(1)–V(1)–O(2)

O(1)–V(1)–O(3)

O(2)–V(1)–O(3)

O(1)–V(1)–O(4)

O(2)–V(1)–O(4)

O(3)–V(1)–O(4)

O(1)–V(1)–N(1)

O(2)–V(1)–N(1)

O(3)–V(1)–N(1)

O(4)–V(1)–N(1)

O(5)–V(2)–O(6)

O(5)–V(2)–O(7)

O(6)–V(2)–O(7)

O(5)–V(2)–O(8)

O(6)–V(2)–O(8)

O(7)–V(2)–O(8)

O(5)–V(2)–N(2)

O(6)–V(2)–N(2)

O(7)–V(2)–N(2)

O(8)–V(2)–N(2)

143.47(13)1

106.17(14)1

105.99(13)1

91.25(13)1

93.35(13)1

110.56(15)1

76.42(11)1

81.54(11)1

99.66(12)1

149.55(14)1

142.05(14)1

106.86(16)1

106.68(15)1

91.06(14)1

94.34(14)1

109.09(16)1

76.99(11)1

81.67(12)1

97.29(14)1

153.30(14)1

102.9(2)1

79.06(17)1

78.73(18)1

172.7(2)1

84.30(19)1

76.90(19)1

84.69(19)1

93.2(2)1

162.5(2)1

79.79(17)1

153.97(19)1

100.2(2)1

99.5(2)1

92.56(18)1

99.60(18)1

102.8(2)1

80.01(15)1

78.74(17)1

174.10(19)1

83.04(17)1

77.69(17)1

84.27(17)1

93.28(19)1

162.49(19)

O(1)–V–N

O–V–O(22)

O(21)–V–O(22)

O(2)–V–O(22)

O(1)–V–O(22)

N–V–O(22)

in 4 being [VO₂]⁺(vide infra). This observation should further be in the [VO]³⁺group. As a consequence of such an arrangement,

investigated to confirm or disprove the association of the bulky the protonated methanol moiety in the axial position seemed to

substituent with the specific core-containing vanadium center. be the one that could be replaced by another ligand. In that

The composition of the coordination sphere of vanadium in respect, methanol in complex 1 was easily replaced by 4,4⁰-

all four materials was reflected in the nature of the ligand used bipyridine, with (a) methanol being the solvent of the synthesis

in their synthesis and crystallization. In that sense, the first reaction, and (b) retention of the coordination environment in

three complexes all had vanadium residing at the center of an 2 upon entry of 4,4⁰-bipyridine in the axial position of the

octahedral assembly. A common feature in such an assembly coordination sphere of vanadium in 1.

involved: (a) the doubly deprotonated Schiﬀ-base ligand occupying

Employment of the bulkier 2-hydroxy-1-naphthaldehyde

three sites in the equatorial plane, (b) the methoxido ligand Schiﬀ-base in the synthetic process led to the emergence of the

occupying the fourth equatorial site, and (c) the solvent methanol [VO₂]⁺-containing complex assembly, distinctly diﬀerentiating the

moiety occupying the axial position trans to the oxido ligand nature of the complex emerging upon crystallization. Specifically,

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the newly formulated coordination sphere of vanadium was (Fig. S7, ESI†), with the assembly retaining its metal : ligand

composed of (a) the doubly deprotonated Schiﬀ ligand occupying stoichiometry of 1 : 1. The collective spectra provide ample clarifica-

the three apices of the base of a tetragonal pyramid, (b) one of the tion on the nature of species emerging upon dissolution of the

oxido ligands occupying the fourth apex of the same tetragonal original crystalline materials in aqueous media, thereby lending

base of the pyramid, and (c) the second oxido ligand being at credence to the ensuing biological studies.

the apex of the pyramid, collectively giving rise to a pyramidal

2.2.4 UV-Vis spectroscopy. The electronic spectra of 1–4

coordination environment for vanadium. Therefore, the employ- (Fig. S8, ESI†) were obtained in MeOH at a concentration of

ment of the bulky Schiﬀ base in the synthesis diﬀerentiated the 10^ꢁ4M, at room temperature. In all cases, the spectra show two

coordination environment of vanadium from octahedral in the intense bands and one relatively weak band in the 227–467 nm

first three species to tetragonal pyramidal in the fourth case. Quite region. The intense band at 227 nm (e = 3880 mol^ꢁ1cm^ꢁ1) for 1,

interesting in that respect was also the observation that as a result 231 nm (e = 4020 mol^ꢁ1cm^ꢁ1) for 2, 231 nm (e = 4010 mol^ꢁ1cm^ꢁ1

)

of such a change in the synthetic process, the first three for 3, and 231 nm (e = 4020 mol^ꢁ1cm^ꢁ1) for 4, respectively,

materials 1–3 involved molecular species bearing zero charge, can be assigned to the p - p* transitions of the aromatic rings,

whereas the fourth species was negatively charged (ꢁ1), thereby with the less intense band at 371 nm (e = 1150 mol^ꢁ1cm^ꢁ1

)

requiring the presence of a counter ion to achieve neutralization. for 1, 370 nm (e = 1190 mol^ꢁ1cm^ꢁ1) for 2, 372 nm (e =

Ostensibly, the presence of sodium and thus the inorganic base 1540 mol^ꢁ1cm^ꢁ1) for 3, and 371 nm (e = 1580 mol^ꢁ1cm^ꢁ1

)

in the reaction mixture was involved in the formulation of the for 4, respectively, attributed to the p - p* and n - p* transi-

coordination sphere of vanadium, with employed organic bases tions of the CQN moiety.⁴⁰The very intense band at 431 nm (e =

failing to play that role (data not shown). Regardless, however, of 2330 mol^ꢁ1cm^ꢁ1) for 1, 421 nm (e = 2330 mol^ꢁ1cm^ꢁ1) for 2,

the nature of the Schiff-base ligand employed in the synthesis, 467 nm (e

= ) for 3, and 445 nm

6670 mol^ꢁ1cm^ꢁ1

development of the reaction mixture under aerobic conditions (e = 5110 mol^ꢁ1cm^ꢁ1) for 4, respectively, can be correlated with

was the most noted signature of the investigated reactivity, the O(p) - V(d) ligand-to-metal charge transfer (LMCT) transition,

initially involving V(^IV) and ultimately affording V(V)-containing thereby providing strong evidence that [VO(OR)]²⁺is coordinated

species. On an equal footing, exploration of the exclusion of to the phenolato oxygen atom of the ligand.⁴¹Here, too, the

oxygen from the reactivity mentioned is a factor warranting electronic spectral features were analogous to those previously

investigation, currently probed into in our lab.

observed and support the notion of complex species present in

2.2.2 FT-IR spectroscopy. The infrared spectra of 1–4 show solution.⁴⁰

active n(OH) and n(CQN) vibrations at 3380 cm^ꢁ1and 1605 cm^ꢁ1

2.2.5 Thermal studies. The thermal decomposition of com-

for 1, 3441 cm^ꢁ1and 1638 cm^ꢁ1for 2, 3430 cm^ꢁ1and 1617 cm^ꢁ1pounds 1–4 was studied by TGA under aerobic conditions (Fig. S9,

for 3, and 3443 cm^ꢁ1and 1606 cm^ꢁ1for 4, respectively. Binding ESI†). Compound 1 is thermally stable up to 45 1C. From that

of the azomethine nitrogen is revealed by lowering of the point on, a fairly broad heat process points to the release of the

resonance frequency (10–23 cm^ꢁ1). The characteristic n(VQO) coordinated methanol molecule of 1 between 45 1C and 329 1C. In

stretch appears as a medium-strong band at 967 cm^ꢁ1for 1, fact, a 8.4% drop in weight, from the start of the thermal treatment

949 cm^ꢁ1for 2, 982 cm^ꢁ1for 3, and 962 cm^ꢁ1for 4, respectively, until 329 1C, is consistent with the release of the methoxy molecule

which is in line with the literature.^38,39The absorption bands at in the coordination sphere of vanadium, consistent with the

1602 cm^ꢁ1and 1039 cm^ꢁ1for 2 are indicative of the n(C–N) of theoretically expected value of 8.6%. Between 329 1C and 442 1C,

the 4,4⁰-bipy moiety. The individual characteristic spectral a further weight loss is observed, in line with the decomposition of

features observed are analogous to those previously observed the organic structure of the molecule. No clear plateaus are

and reported.^38,39

reached in this temperature range, suggesting that the formed

2.2.3 ESI-MS spectrometry. ESI-MS spectrometry was employed products are unstable and decompose further. A plateau in the

under optimal experimental conditions to probe the behavior of the decomposition of 1 is reached at 442 1C, with no further loss up to

title compounds in solution. The ESI-MS spectrum of 1 in methanol 550 1C, in line with the thesis that the product at that temperature

reveals the presence of fragments m/z = 355.02 with z = 1, reflecting and beyond (550 1C) is V₂O₅. The total weight loss of B76.0% is in

species [VO₂(L₁)(CH₃O)]⁺(Fig. S4, ESI†). The spectrum of 2 in water agreement with the theoretical value of B75.5%, according to the

reveals the presence of fragments m/z = 265.12 with z = 1 reflecting following equation:

species [(L₁) + 1](Na⁺), and m/z = 324.01 with z = 1, reflecting

species [VO₂(L₁)], thereby attesting to the vanadium:ligand stoi-

4[VO(C₁₄H₁₁NO₃)(CH₃O)(CH₃OH)] + 79O₂

chiometry of 1 : 1 (Fig. S5, ESI†). Dissociation of 4,4⁰-bipyridine

from the compound molecule was attested to in the positive mode

of ionization, whereby the presence of the 4,4⁰-bipyridinium ion

- 2V₂O₅+ 64CO₂+ 4NO₂+ 36H₂O

Compound 2 is thermally stable up to 167 1C. Between

[C₁₀H₉N₂]⁺was observed, with m/z = 157.07 and z = 1. In the case of 167 1C and 513 1C, a weight loss is observed, in line with the

compound 3, the spectrum suggests the presence of m/z = 293.99 decomposition of the organic structure of the molecule. No

with z = 1 species, thus reflecting the [VO₂(L₂)] moiety (Fig. S6, clear plateaus are reached in this temperature range, suggesting

ESI†), with the complex retaining the metal : ligand stoichiometry that the formed products are unstable and decompose further.

of 1 : 1. The spectrum of compound 4 in water shows the presence A plateau in the decomposition of 2 is reached at 513 1C, with no

of m/z = 344.01 with z = 1 species, reflecting the [VO₂(L₃)] moiety further loss up to 550 1C, in line with the thesis that the product

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at that temperature and beyond (550 1C) is V₂O₅. The total at 200 mM ( p o 0.05) in the presence of L₁H₂. L₂H₂and L₃H₂are

weight loss of B78.7% is in agreement with the theoretical completely atoxic at all concentrations tested ( p 4 0.05).

value of B78.2%, according to the following equation:

2.3.2 V(^V) compound toxicity in 3T3-L1 pre-adipocytes for

24 h. To examine whether the newly-synthesized V(^V) compounds

(1–4) affect cell survival rates, 3T3-L1 pre-adipocytes were treated

with 1, 10, 50, and 100 mM of the title compounds for 24 h.

Fig. 5A–D shows that all four compounds affect cell survival in a

concentration-dependent fashion under the employed experi-

mental conditions. More specifically, cell survival, in the presence

of 1, stands at 106.7% at 1 mM, 99.1% at 10 mM, 96.0% at 50 mM,

and 71.3% at 100 mM (p 4 0.05). In the case of 2, cell survival

stands at 96.5% at 1 mM, 95.1% at 10 mM, 74.3% at 50 mM

(p 4 0.05), and 28.2% at 100 mM (p o 0.001). Similarly, cell survival

stands at 79.4%, 71.6%, 40.2% and 6.4% at 1, 10, 50, and 100 mM,

respectively, following exposure to 3, and 98.3%, 80.7, 64.8, and

51.6% in the case of 4, and for the same concentration range.

2.3.3 V(^V) compound toxicity in 3T3-L1 pre-adipocytes for

48 h. Similarly, cell survival was also assessed in the same

concentration range (1–100 mM), following treatment with 1–4

for 48 h, since cells, during induction of the differentiation

process, are exposed to the title compounds for more than 24 h.

The results in Fig. 6A–D show that the tested compounds affect

cell survival in a concentration-dependent manner. It should be

mentioned, however, that for longer incubation times (48 h),

V(^V)-compounds exerted a proliferative effect in most cases. Cell

viability stands at 179.6%, 132.0%, 103.6% and 101.2% follow-

ing treatment with 1 at 1, 10, 50, and 100 mM, respectively. In the

case of 2, cell viability stands at 96.9%, 140.0%, 147.5%, and

42.1% for the same concentration range. Compound 3 was

completely atoxic at low concentrations (103.1% and 95.4% at

1 and 10 mM, respectively), whereas at higher concentrations

cell survival was significantly reduced (5.2% and 0.5% at 50 mM

and 100 mM, respectively). Compound 4 projected a cytotoxic

behavior similar to 3. More specifically, cell survival amounts to

102.8%, 110.7%, 52.4%, and 3.7% in the range 1–100 mM

compared to the control (untreated cells).

2{[VO(C₁₄H₁₁NO₃)(CH₃O)](C₁₀H₈N₂)[VO(C₁₄H₁₁NO₃)(CH₃O)]} + 101O₂

- 2V₂O₅+ 80CO₂+ 8NO₂+ 36H₂O

Compound 3 is thermally stable up to 244 1C. Between

244 1C and 513 1C, a weight loss is observed, in line with the

decomposition of the organic structure of the molecule. No

clear plateaus are reached in this temperature range, suggesting

that the resultant products undergo further decomposition. A

plateau in the decomposition of 3 is reached at 542 1C, with no

further loss up to 550 1C, in line with the product at that

temperature and beyond (550 1C) being V₂O₅. The total weight

loss of B73.0% is in agreement with the theoretical value of

B73.4%, according to the following equation:

4[VO(C₁₃H₉NO₂)(CH₃O)(CH₃OH)] + 75O₂

- 2V₂O₅+ 60CO₂+ 4NO₂+ 32H₂O

Compound 4 is thermally stable up to 269 1C. Between

269 1C and 504 1C, a weight loss is observed, in line with the

decomposition of the organic structure of the molecule. No

clear plateaus are reached in this temperature range, suggesting

that the resultant products undergo further decomposition. A

plateau in the decomposition of 3 is reached at 504 1C, with no

further loss up to 550 1C, in line with the product at that

temperature and beyond (550 1C) being NaVO₃. The total weight

loss of B71.0% is in agreement with the theoretical value of

B70.7%, according to the following equation:

{[VO₂(C₁₇H₁₁NO₂)]₂[Na₂(H₂O)₅]}₂ꢀH₂O + 81O₂

- 4NaVO₃+ 68CO₂+ 4NO₂+ 33H₂O

The described tentative assignments are consistent with

previously reported results on thermogravimetric analysis.^37,42

The collective physicochemical characterization of all four

species (elemental analysis, FT-IR, EPR, UV-Visible spectroscopy,

and X-ray crystallography) was consistent with the formulation

unraveled by the structural characterization of the materials.

Further investigation of the solution behavior of the four com-

plexes through ESI-MS spectrometry led to the formulation of

their profile in solution, thereby lending credence to their

employment in the subsequent biological studies.

In previous studies,³⁷it had been shown that V(V), in the form

of KVO₃, aﬀects 3T3-L1 cell survival in a concentration-dependent

manner for concentrations higher than 1 mM. To this end, it

becomes evident that ligation alters the cytotoxic behavior of the

complex vanadoforms. More specifically, in the present work,

ligation enhances the bioavailability of the title compounds and

limits the cytotoxic profile under the employed experimental

conditions. Taken together, all four compounds can further be

used to examine the potential insulin-mimetic properties towards

adipogenesis.

2.3 Cytotoxicity studies

2.3.4 Cell migration–cell morphology. Since the specific

2.3.1 Ligand toxicity in 3T3-L1 pre-adipocytes. To investigate cell line exerts cell migration properties, in an effort to investigate

the potential cytotoxic eﬀects of the organic ligands-substrates the potential effects of the title compounds on the endogenous cell

L_iH₂(i = 1–3) on 3T3-L1 pre-adipocytes, cells were treated with 1, migration of 3T3-L1 pre-adipocytes, an in vitro scratch assay was

10, 100, 200 mM of L₁H₂, L₂H₂, and L₃H₂, respectively, for 24 h performed. The physiological migrating activity of cells, grown in

(Fig. S10A–C, ESI†). The results show that all three ligands the presence of only DMEM, was considered as control. Cells were

do not affect cell survival under the employed experimental treated with 10 mM (a concentration with mild toxicity for all

conditions. L₁H projects a slight concentration-dependent cyto- compounds) of each V(^V)-compound (1–4) for 24 and 48 h,

toxic profile ( p 4 0.05). In particular, cell survival amounts to respectively (10ꢂ 5ꢂ magnification). As shown in Fig. 7, cells

112.1% at 1 mM, 108.6% at 10 mM, 94.7% at 100 mM, and B90% exhibited full inhibition of cell migration in the case of 1 and 4,

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Fig. 5 Percent of cell survival in 3T3-L1 pre-adipocytes following treatment with various concentrations (1–100 mM) of 1 (A), 2 (B), 3 (C), and 4 (D) for 24 h. Sodium

deoxycholate has been used as a positive control. Values represent the mean value of several (n = 3) independent experiments. Vertical bars represent SEMs.

*p o 0.05 (significant), **p o 0.01 (highly significant), ***p o 0.001 (extremely significant) and ****p r 0.0001 (extremely significant) or non-significant (p 4 0.05).

whereas cell migration was slightly inhibited in the presence of insulin compared to control (Fig. 8H), but it was significantly

2 and 3 after 24 h compared to control. Similar results were also lower compared to positive control. In an analogous fashion, 3

obtained following treatment for 48 h (data not shown).

and 4 were also employed to induce cell differentiation of 3T3-L1

fibroblasts into mature adipocytes either by fully replacing insu-

lin or in combination with it. In both cases, the effect was

2.4 Insulin-mimetic/enhancing studies

2.4.1 V(^V)-compound oil red O staining results. In an eﬀort comparable to the differentiation effect induced by insulin

to investigate the adipogenic and thus insulin mimetic and/or (Fig. 8I–N). Oil red O staining is a qualitative assay, assessing

enhancing activity of the newly synthesized materials, the title the potential adipogenic activity of various agents. Thus, no

compounds were employed to induce cell diﬀerentiation of quantitative results can be obtained. However, the assay can be

3T3-L1 fibroblasts into mature adipocytes, following a standard used to investigate the synergistic/enhancing or inhibiting effect

diﬀerentiation protocol. Oil red O staining was performed on of such agents. In the present study, all compounds tested

the 8^thday of the diﬀerentiation process to examine the lipid induced adipogenesis either alone or in combination with

content of mature adipocytes diﬀerentiated in the presence of insulin. The concentrations used were selected based on the

1–4. As assessed through staining, 1 induced differentiation of cytotoxicity results for 24 and 48 h. To further validate and

pre-adipocytes into mature adipocytes at all concentrations quantitatively evaluate the adipogenic potential of 1–4, the

tested (1 and 20 mM), compared to control (untreated cells, relative mRNA expression of closely linked biomarkers (PPAR-g,

Fig. 8A), whereas no synergistic or inhibiting effect was GLUT4, ADIPOQ, INS-R, and RETN) was also investigated.

observed when cells were treated in combination with insulin

2.4.2 Relative mRNA expression of related biomarkers

(20 mM of 1 with insulin) (Fig. 8C–E) compared to positive (PPAR-c, GLUT4, ADIPOQ, INS-R, and RETN). On the 8^thday

control (insulin) (Fig. 8B). In the case of 2, cell differentiation of the diﬀerentiation process, total mRNA was extracted and

was achieved when the compound was used alone in a slight RT-PCR was run to examine the relative mRNA expression of

concentration-dependent manner (Fig. 8F and G). The effect selected molecular targets. In that sense, the relative mRNA

was clearly enhanced when 2 was used in combination with concentration was determined for PPAR-g, GLUT4, ADIPOQ,

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Fig. 6 Percent of cell survival in 3T3-L1 pre-adipocytes following treatment with various concentrations (1–100 mM) of 1 (A), 2 (B), 3 (C), and 4 (D) for

48 h. Sodium deoxycholate has been used as a positive control. Values represent the mean value of several (n = 3) independent experiments. Vertical bars

represent SEMs. *p o 0.05 (significant), **p o 0.01 (highly significant), ***p o 0.001 (extremely significant) and ****p r 0.0001 (extremely significant) or

non-significant (p 4 0.05).

INS-R, and RETN. Initially, the expression of PPAR-g was insulin the fold increase is 6.3. The results indicate a slight

investigated, which serves as a key transcription factor during concentration-dependent effect and a synergistic/enhancing

adipogenesis, influencing more than 2000 genes involved in the effect on insulin compared to positive control (Fig. 9C). When

specific process.^43,44Cells were treated with 1–4 at 1 mM and cells were differentiated in the presence of 4, relative mRNA

20 mM or in combination with insulin (20 mM with insulin). expression of PPAR-g was 2.6 and 4.2 at 1 mM and 20 mM,

Again, untreated cells were considered as negative control of respectively. When cells were treated with 4 in combination with

the assay, whereas insulin alone (standard differentiation insulin, relative mRNA expression was 1.8, indicating a potential

protocol) as the positive control. Insulin alone projected a inhibiting effect toward insulin action under the employed

4-fold increase (Fig. 9). In the case of 1, PPAR-g projects a experimental conditions considering this gene (Fig. 9D).

10.5 fold increase at 1 mM and 8.5 fold increase at 20 mM. When

Similarly, the relative mRNA expression of GLUT4 was also

1 is used in combination with insulin, PPAR-g is slightly determined, in view of the fact that the specific gene is only

expressed (B1.9 fold increase), thus indicating a potential expressed in mature adipocytes, serving as a biomarker of

inhibiting effect toward insulin action under the employed successful diﬀerentiation. In the case of 1, relative mRNA

experimental conditions for this gene (Fig. 9A). By the same expression exhibited a 6.8- and 6.6-fold increase at 1 mM and

token, in the case of 2, PPAR-g projected a 7-fold increase at 20 mM, respectively, compared to control, whereas in combination

1 mM and 1.1-fold increase at 20 mM. When 2 was used in with insulin, 1 projected a 4.8-fold increase (Fig. 10A). The results

combination with insulin, the relative mRNA expression of PPAR- indicate that there is no significant diﬀerence between the two

g was almost the same as that of insulin alone (B3.9) (Fig. 9B). concentrations employed. Moreover, 1 has no eﬀect on insulin

Given the fact that 2 has almost no effect, when used alone at action compared to positive control (B5 fold increase). In the case

20 mM and the expression levels in combination with insulin are of 2, the relative mRNA expression of GLUT4 is 2.4 at 1 mM and 1.8

equal to that of insulin alone, the obtained results indicate that the at 20 mM (Fig. 10B). When 2 is used in combination with insulin,

observed effect is due to insulin and 2 has no effect (either the fold increase is B6, indicating a slight insulin-enhancing

enhancing or inhibiting) on insulin action (Fig. 9B). In the case eﬀect. In the case of 3, the relative mRNA expression is 3.8 at

of 3, the expression of PPAR-g is 1.2 and 2 at 1 mM and 20 mM, 1 mM, 15.7 at 20 mM, and 3.7 when 3 is used in combination with

respectively, whereas when 3 is used in combination with insulin (Fig. 10C). The results indicate a concentration-dependent

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Fig. 7 Cell migration of 3T3-L1 (A) control (t = 0), (B) control after 24 h, (C) cells treated with 10 mM of 1, (D) 2, (E) 3, and (F) 4 using an in vitro standard

scratch assay.

eﬀect when 3 is used alone, yet interestingly, 3 exhibits an with 20 mM of 2). The specific result indicates a potential

inhibiting/competitive eﬀect towards insulin action. Similarly, antagonism between 2 and insulin rather than a slight synergistic

when cells are diﬀerentiated in the presence of 4, the mRNA eﬀect (Fig. 11B). In an analogous fashion, 3 and 4 exhibit a

expression of GLUT4 is 4.5 at 1 mM, 9.7 at 20 mM, and B5 when 4 concentration-dependent expression of ADIPOQ (4.8 and 6.6 for

is used in combination with insulin (Fig. 10D).

3 and 4.9 and 7.3 for 4 at 1 and 20 mM, respectively) (Fig. 11C

It is well-established that adipose tissue is capable of secreting and D). The combination of 3 and insulin exhibits a 6.9-fold

several adipocytokines closely related to the metabolic status increase, whereas in the case of 4 the fold increase is 5.6. It is

among others.⁴⁵Besides GLUT4, adiponectin (ADIPQ) also serves worth pointing out that only 1 projects higher expression levels

as a valid biomarker of successful adipogenesis.⁴⁶In that sense, in all concentrations/cases used.

the relative mRNA expression of adiponectin was examined at the

Furthermore, expression levels for resistin were also investigated

end of the diﬀerentiation process. Again, untreated cells were (Fig. 12). Resistin, also known as the adipose tissue-specific

considered as control and diﬀerentiation with insulin alone as secretory factor (ADSF) or C/EBP–epsilon-regulated myeloid-

the positive control (7.6-fold increase) (Fig. 11). When cells are specific secreted cysteine-rich protein (XCP1), is a cysteine-rich

diﬀerentiated in the presence of 1, the relative expression is adipose-derived peptide hormone, which in humans is encoded

10.3 and B9 at 1 and 20 mM, respectively. When cells are by the RETN gene. Although the role of resistin in obesity is still

diﬀerentiated in combination of 1 and insulin, the expression questionable, it is well-established that the specific adipokine is

of ADIPOQ is 18.7, indicating an insulin enhancing eﬀect of 1 secreted by white adipose tissue and could be used as a biomarker

toward insulin (Fig. 11A). In the case of 2, the expression of successful adipogenesis. The mature adipocytes differentiated

pattern is concentration-dependent (B3 and 8.7 at 1 and in the presence of insulin project a 3-fold increase compared to

20 mM, respectively), whereas combination of 2 and insulin control (untreated cells) (Fig. 12). In the case of 1, the expression of

shows an 8.5-fold increase (equal to that of cells treated only resistin is 6.6 and 2.6, when cells are differentiated with 1 and

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Fig. 8 Representative micrographs of 3T3-L1 pre-adipocyte diﬀerentiation, as assessed by oil red O staining. (A) Untreated cells, (B) mature adipocytes

diﬀerentiated in the presence of insulin (10 ng mL^ꢁ1), mature adipocytes diﬀerentiated in the presence of 1, 20 mM, and 20 mM, in combination with insulin

of 1 (C–E), 2 (F–H), 3 (I–K), and 4 (L–N).

20 mM, respectively. When 1 is used in combination with insulin, vanadium coordination was driven toward appropriately con-

the relative expression is 5.2 (Fig. 12A). In the case of 2, the relative figured Schiﬀ bases and the oxidation state of vanadium being

expression of resistin is 2.6 and 4.0 at 1 and 20 mM, respectively, V(^V). The latter oxidation state is one of the biologically relevant

whereas when cells are treated with 2 in combination with insulin oxidation states of vanadium in biological fluids. The Schiﬀ

the relative expression level is 4.8 (Fig. 12B). Similarly, in the case base series relied on a common 2-aminophenol moiety attached

of 3, resistin projects a 5.4 and 4.1-fold increase at 1 and 20 mM, to an o-vanillin core, with the latter structure sequentially

respectively, and 4.2 when cells are treated in combination of 3 modified through removal of the methoxy substituent on the

and insulin (Fig. 12C). Finally, in the case of 4, the expression aromatic ring and the concurrent derivatization of the core

levels are B4 and 6.6 when cells are treated with 1 and 20 mM, through annexation of yet another aromatic substituent, thereby

respectively, and 7.6 when cells are treated in combination with enhancing the bulk and hydrophobicity of the Schiﬀ base. In

insulin, thus indicating a synergistic effect of 4 toward insulin almost all cases investigated so far, the expression pattern of

(Fig. 12D). The relative mRNA expression of the insulin receptor GLUT4 follows the PPAR-g expression pattern. This observation

(INS-R) was also assessed on the 8^thday of the differentiation is consistently in line with the literature.⁴⁸

process, to examine any potential variation of the expression

pattern between premature and mature adipocytes.⁴⁷The results

show that there is no significant fold increase expression (a)

between pre-mature and mature adipocytes (pre-adipocytes vs.

treated ones with insulin), and (b) when cells are treated with

3. Experimental section

3.1 Materials and methods

All experiments were carried out under aerobic conditions.

1–4 or in combination with insulin (data not shown).

The structures of the ligands used are listed in Scheme 1. The

Inevitably, the complex forms of vanadium developed over

following starting materials were purchased from commercial

the years have involved parameters including its oxidation

sources (Sigma, Fluka) and were used without further purification:

2-aminophenol, o-vanillin, 2-hydroxy-1-naphthaldehyde, salicyl-

aldehyde, 4,4⁰-bipyridine (4,4⁰-bipy) and vanadyl acetylacetonate

state, ligand nature, charge, and hydrophilicity, among others.

As a result of such features having been taken into consideration,

the synthesized vanadium complexes have exhibited variable

(VO(C₅H₇O₂)₂); solvents: ultrapure water, methanol, and diethyl

physicochemical properties linked to their ultimate insulin

ether. The isolated complexes, dried under vacuum at room

mimetic activity. In view of the fact that such biological activity

temperature, are air-stable.

applies to diﬀerent phases of the cellular functions leading to

glucose uptake, the present investigation revolved around the

design of the vanadium coordination environment that could

3.2 Physical measurements

influence adipocyte diﬀerentiation processes intimately linked FT-Infrared spectra were recorded on a PerkinElmer 1760X

to insulin mimesis. To that end, the choice of ligands formulating FT-infrared spectrometer. A ThermoFinnigan Flash EA 1112

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Fig. 9 Relative concentration of mRNA expression for PPAR-g in mature

adipocytes treated with insulin or 1 (A), 2 (B), 3 (C), and 4 (D) compared to

pre-adipocytes on the 8^thday of the diﬀerentiation process. Values

represent the mean value of several (n = 3) independent experiments.

Vertical bars represent SEMs; non-significant (p 4 0.05); *: p o 0.05;

**: p o 0.01; ***: p o 0.001 vs. control.

Fig. 10 Relative concentration of mRNA expression for GLUT4 in mature

adipocytes treated with insulin or 1 (A), 2 (B), 3 (C), and 4 (D) compared to

pre-adipocytes on the 8^thday of the diﬀerentiation process. Values

represent the mean value of several (n = 3) independent experiments.

Vertical bars represent SEMs; non-significant (p 4 0.05); *: p o 0.05;

**: p o 0.01; ***: p o 0.001 vs. control.

CHNS elemental analyser was used for the simultaneous deter-

mination of carbon, hydrogen, and nitrogen (%). The analyser sample (at 1800 1C) followed by reduction, trapping, complete

operation is based on the dynamic flash combustion of the GC separation and detection of the products. The instrument is

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Fig. 12 Relative concentration of mRNA expression for RETN in mature

adipocytes treated with insulin or 1 (A), 2 (B), 3 (C), and 4 (D) compared to

pre-adipocytes on the 8^thday of the diﬀerentiation process. Values

represent the mean value of several (n = 3) independent experiments.

Vertical bars represent SEMs; non-significant (p 4 0.05); *: p o 0.05;

**: p o 0.01; ***: p o 0.001 vs. control.

Fig. 11 Relative concentration of mRNA expression for ADIPOQ in

mature adipocytes treated with insulin or 1 (A), 2 (B), 3 (C), and 4 (D)

compared to pre-adipocytes on the 8^thday of the diﬀerentiation process.

Values represent the mean value of several (n

= 3) independent

experiments. Vertical bars represent SEMs; non-significant (p 4 0.05);

*: p o 0.05; **: p o 0.01; ***: p o 0.001 vs. control.

(a) fully automated and controlled by PC via the Eager 300 gaseous substances. Electron paramagnetic resonance (EPR)

dedicated software, and (b) capable of handling solid, liquid or spectra were recorded at liquid N₂temperature (77 K) on a

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Scheme 1 Schematic representation of the ligands (L₁H₂, L₂H₂, L₃H₂) used in this study.

Bruker ER200D spectrometer, equipped with an Agilent 5310A Subsequently, a solution of [VO(C₅H₇O₂)₂] (0.27 g, 1.0 mmol) in

frequency counter. The spectrometer runs under home-made CH₃OH (10 mL) was added under continuous stirring. The resulting

software based on LabView. Typically, adequate signal-to-noise ratio slightly insoluble brownish reaction mixture was refluxed for an

was obtained after 3–5 scans. EPR samples were prepared in 5 mm additional 2 h and then cooled to room temperature. Subsequently,

suprasil (Willmad Co.) quartz tubes with no further treatment.

the insoluble material was removed by filtration, the reaction flask

with the clear reaction solution was placed at 4 1C, and diethyl

ether was added. One week later, a brownish crystalline material

3.3 UV-Visible measurements

UV-Visible (UV-Vis) measurements were carried out on a Hitachi precipitated at the bottom of the flask. The product was isolated

U-2001 spectrophotometer, in the range from 190 to 1000 nm.

by filtration and dried in vacuo. Yield: 0.22 g (58%). Anal. calcd

for 1, [VO(L₁)(OCH₃)(CH₃OH)] (1). (C₁₆H₁₈NO₆V M_r371.25): C,

51.76; H, 4.89; N, 3.77. Found: C, 51.68; H, 4.79; N, 3.74.

3.6.3 Preparation of complex [VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)-

3.4 Thermal studies

A PerkinElmer, Pyris 1, system was used to run the simultaneous

Thermogravimetric Analysis (TGA) experiments. The instrument (CH₃O)] (2). To a solution of o-vanillin (0.15 g, 1.0 mmol) in CH₃OH

mass precision is 1 mg. About 2–5 mg of compounds 1–4 was (10 mL), 2-aminophenol (0.11 g, 1.0 mmol) was added and the

placed in an open alumina sample pan for each experiment. High resulting red solution was refluxed for 2 h under continuous

purity air was used at a constant flow rate of 30 mL min^ꢁ1

,

stirring. Then, it was cooled down to room temperature. Subse-

depending on the conditions required for running the experiments. quently, a solution of [VO(C₅H₇O₂)₂] (0.27 g, 1.0 mmol) in CH₃OH

During the experiments, the sample weight loss and rate of weight (10 mL) was added under continuous stirring. The resulting slightly

loss were recorded continuously under dynamic conditions, as a insoluble brownish reaction mixture was refluxed for an additional

function of time or temperature, in the range 30–550 1C. Prior 2 h and then cooled to room temperature. To that, a solution of 4,4⁰-

to activating the heating routine program, the entire system bipyridine (0.16 g, 1.0 mmol) in CH₃OH (2 mL) was added under

was purged with the appropriate gas for 10 min, at a rate of continuous stirring. The resulting insoluble brownish reaction

30 mL min^ꢁ1, to ensure that the desired environment had been mixture was refluxed for an additional 2 h and then cooled to

established.

room temperature. Subsequently, the insoluble material was

removed by filtration, the reaction flask with the clear reaction

solution was placed at 4 1C, and diethyl ether was added. Two

3.5 ESI-MS measurements

The electron spray ionization mass spectrum was obtained on weeks later, a brownish crystalline material precipitated at the

an Agilent Technology LC/MSD trap SL instrument and a bottom of the flask. The product was isolated by filtration and

Thermo Scientific, LTQ Orbitrap XLt high resolution system. dried in vacuo. Yield: 0.14 g (33%). Anal. calcd for 2,

The spectra of all compounds 1–4 were optimally run in the [VO(L₁)(CH₃O)](C₁₀H₈N₂)[VO(L₁)(CH₃O)] (2). 2 ꢂ (C₂₀H₁₈N₂O₅V),

negative mode of ionization in water, with the exception of M_r2 ꢂ 417.31: C, 57.56; H, 4.35; N, 6.71. Found: C, 57.50; H,

compounds 1 and 2, the spectra of which were run in both 4.33; N, 6.73.

negative and positive modes in methanol and/or water. In the

3.6.4 Preparation of complex [VO(L₂)(CH₃O)(CH₃OH)] (3).

case of compound 1, 0.01% formic acid was added to the To a solution of salicylaldehyde (0.11 mL, 1 mmol) in CH₃OH

sample run in the positive mode in methanol.

(10 mL), 2-aminophenol (0.11 g, 1.0 mmol) was added and the

resulting red solution was refluxed for 2 h. Then, it was cooled

down to room temperature. Subsequently, a solution of

3.6 Synthesis

3.6.1 Preparation of ligands L_iH₂(i = 1–3). Ligands L₁H₂–L₃H₂[VO(C₅H₇O₂)₂] (0.27 g, 1.0 mmol) in CH₃OH (10 mL) was added

were prepared according to the literature.^32,34

under continuous stirring and the resulting slightly insoluble

3.6.2 Preparation of complex [VO(L₁)(CH₃O)(CH₃OH)] (1). brownish solution was refluxed for an additional 2 h. Then, it

To a solution of o-vanillin (0.15 g, 1.0 mmol) in CH₃OH (10 mL), was cooled down to room temperature. Subsequently, the

2-aminophenol (0.11 g, 1.0 mmol) was added under stirring. insoluble material was removed by filtration and the reaction

The resulting red solution was refluxed for two hours under flask with the clear reaction solution was placed at 4 1C to

continuous stirring and then it was cooled to room temperature. evaporate slowly. Ten days later, a brown crystalline material

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precipitated at the bottom of the flask. The product was the numerical method (SADABS) based on crystal dimensions.⁵²

isolated by filtration and dried in vacuo. Yield: 0.19 g (55%). Data refinement (full-matrix least-squares methods on F²) and all

Anal. calcd for 3, [VO(L₂)(CH₃O)(CH₃OH)] (3). (C₁₅H₁₆NO₅V, M_rsubsequent calculations were performed using the Crystals version

341.24): C, 52.80; H, 4.73; N, 4.10. Found: C, 52.75; H, 4.70; 14.61 build 6236 program package⁵³The structures were solved by

N, 4.12.

the SUPERFLIP method.⁵⁴Molecular illustrations were drawn

3.6.5 Preparation of complex [VO₂(L₃)Na(H₂O)₂VO₂(L₃)- by Diamond 3.1c, CAMERON and Mercury crystallographic

Na(H₂O)₃]₂ꢀH₂O (4). To a solution of 2-hydroxy-1-naphthaldehyde packages.⁵⁵In all four structures 1–4, all non-hydrogen atoms

(0.17 g, 1.0 mmol) in CH₃OH (10 mL), 2-aminophenol (0.11 g, were refined anisotropically. Further experimental crystallo-

1.0 mmol) was added and the resulting red solution was refluxed graphic details for 2: y_max= 33.1781; R/R_w(for all data),

for 2 h at 70 1C. Then, it was cooled down to room temperature. 0.0549/0.1113; for 3: y_max= 26.4231; R/R_w(for all data), 0.0810/

Subsequently, a solution of [VO(C₅H₇O₂)₂] (0.27 g, 1.0 mmol) in 0.1332; for 4: y_max= 27.4421; R/R_w(for all data), 0.0846/0.1183.

CH₃OH (10 mL) was added under continuous stirring and the Crystallographic details for 1–4 are summarized in Table 1.

resulting slightly insoluble brownish solution was refluxed for an

3.8 Cell cultures and biological tests

additional 2 h. Then, it was cooled down to room temperature.

Subsequently, sodium hydroxide (0.08 g, 2.0 mmol) was added to

3.8.1 Cell culture. Since the aim of the present study was to

the reaction mixture under continuous stirring and the resulting assess the adipogenic potential of the title V(^V)-complexes, the

clear, brownish solution was refluxed for an additional 2 h. established murine in vitro model 3T3-L1 (pre-adipocytes) was

Subsequently, the reaction flask was placed at 4 1C and allowed used. The specific cell line is insulin-sensitive and a common/

to evaporate slowly. One week later, a brown crystalline material ideal model of chemically induced diﬀerentiation to mature

precipitated at the bottom of the flask. The product was isolated by adipocytes. Both pre- and mature adipocytes were cultured in

filtration and dried in vacuo. Yield: 0.19 g (45%). Anal. calcd for 4, 75 cm²cell culture flasks under appropriate conditions (5%

[VO₂(L₃)Na(H₂O)₂VO₂(L₃)Na(H₂O)₃]₂ꢀH₂O (4). (C₆₈H₆₆N₄Na₄O₂₇V₄, CO₂at 37 1C and standard humidity) in Dulbecco’s modified

M_r1667.01): C, 48.99; H, 3.99; N, 3.36. Found: C, 49.01; H, Eagle’s medium, DMEM (Sigma, Steinheim, Germany). Culture

3.96; N, 3.38.

media were supplemented with 10% Fetal Bovine Serum (FBS)

(Biochrom, Berlin, Germany) and 1% penicillin–streptomycin

(Biochrom, Berlin, Germany) prior to use. The specific cell line

3.7 X-ray crystal structure determination

X-ray quality crystals of compounds 1 and 2 were grown from a was tested and found free of mycoplasma contamination using

mixture of methanol-diethyl ether, whereas crystals of compounds a MycoAlert^sKit (Promega). All experiments were run at least in

3 and 4 were grown from methanol solutions. A crystal of 1 (0.03 ꢂ triplicate employing cells with a low passage number (P5–P9).

0.05 ꢂ 0.30 mm) was recovered from the mother liquor and

3.8.2 Chemically induced adipogenesis in vitro. Adipogenesis

immediately cooled to ꢁ113 1C. Diﬀraction measurements were (maturation of pre-adipocytes into lipid droplet-containing adipo-

made on a Rigaku R-AXIS SPIDER Image Plate diﬀractometer cytes) was achieved by adding an appropriate adipogenic hormone

using graphite monochromated Cu Ka radiation. Data collection cocktail (dexamethazone, isobutyl-methyl-xanthine, and insulin).

(o-scans) and processing (cell refinement, data reduction and 3T3-L1 fibroblasts were diﬀerentiated into mature adipocytes

empirical absorption correction) were performed using the Crystal- according to a standard diﬀerentiation protocol⁵⁶as previously

Clear program package.⁴⁹The structures were solved by direct described. Briefly, two days post 70% confluency (day 0), cells

methods using SHELXS-97 and refined by full-matrix least-squares were treated with 0.5 mM 3-isobutyl-1-methylxanthine (Sigma,

methods on F²with SHELXL2014/6.⁵⁰Further experimental Germany), 1.0 mM dexamethazone (Sigma, Steinheim, Germany),

crystallographic details for 1: 2y_max= 130.001; 253 parameters and 10 ng mL^ꢁ1of insulin as final concentrations in DMEM. Two

refined; (D/s)_max= 0.008; (Dr)_max/(Dr)_min= 0.51/ꢁ0.48 e Å^ꢁ3. The days later (day 2), the cells were cultured in maintenance medium

hydrogen atoms were refined isotropically at positions located treated only with insulin (main adipogenic factor) and the

either from diﬀerence Fourier maps or at calculated positions medium was serially changed every two days. All experiments

using a riding model. All non-hydrogen atoms were refined were performed on the 8^thday of the diﬀerentiation process.

anisotropically.

3.8.3 Cell viability assay. To examine cell survival rates

Crystals of 2, 3, and 4 suitable for X-ray diﬀraction, with post-stimulation with the well-characterized V(^V)-complex species,

dimensions 0.39 ꢂ 0.16 ꢂ 0.14 mm, 0.26 ꢂ 0.22 ꢂ 0.13 mm, both 3T3-L1 pre-adipocytes and mature adipocytes, diﬀerentiated

and 0.53 ꢂ 0.14 ꢂ 0.06 mm, respectively, were taken from the with insulin as mentioned above, were seeded in 96-multi-well

mother liquor and mounted, at room temperature, on a Bruker plates (2500 cells per well) and incubated with the title compounds

Kappa 2 APEX diﬀractometer equipped with a triumph mono- for 24 and 48 h in DMEM. The employed assay relies on the

chromator using Mo Ka radiation. Unit cell dimensions were quantitation of ATP present, thereby confirming the presence of

determined and refined by using the angular settings of at least metabolically active cells. The reagent (Promega kit Cell Titer-

100 high intensity reflections (410s(I)) in the range 15 o 2y o 401. Glo^s) is added to the cell culture (volumetric reagent/super-

Intensity data were recorded using f and o scans. All crystals natant ratio 1 : 1) without removing the supernatant medium.

exhibited no decay during data collection. The frames collected The luminescence of each sample was measured on a GloMax^s

were integrated with the Bruker SAINT software package⁵¹using a 96 Microplate Luminometer (Promega Corporation, WI USA).

narrow-frame algorithm. Data were corrected for absorption using Results were expressed in relative percent change of luminescence

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units (RLU). Cell viability was additionally tested via Trypan blue

mGLUT4 forward 5⁰-AACCAGCATCTTCGAGTCGG-3⁰and

assay. Sodium deoxycholate (known cytotoxic agent) was used as reverse 5⁰-TAAGAGCACCGAGACCAACG-3⁰;

positive control of the assay at a final concentration of 1 mg mL^ꢁ1

.

mPPAR-g forward 5⁰-GTCAGCGACTGGGACTTTTC-3⁰and

3.8.4 Cell migration assay–cell morphology. Since the specific reverse 5⁰-CGAGGACATCCAAGACAACC-3⁰.

cell line exemplifies migrating characteristics, inhibition of the

The sequence of each primer was run on BLAST to exclude

endogenous migrating capacity of 3T3-L1 pre-adipocytes was also those that would amplify undesired genes. Customized primers

assessed through an in vitro scratch assay.^57,58For this purpose, were also used from Qiagen for adiponectin (Mm_Adipoq_1_SG,

cells were seeded in 35 mm cell culture dishes in DMEM. After NM_009605, Q01048047), resistin (Mm_Retn_1_SG, QT00093450)

achieving confluency (70–80%), (a) a scratch in the monolayer and insulin receptor (Mm_Insr_1_SG, NM_010568, QT00287903).

traversing the entire diameter of each culture dish was engraved

3.8.8 Statistical analysis. The data were presented as average

using a sterile pipette tip, and (b) cells were allowed to grow in the and standard error mean (SEM) values of multiple sets of

culture medium, in the presence of a final concentration range of independent measurements. Mean survival rates and SEMs were

1–20 mM of the title compounds. Cells were visualized using an calculated for each individual group. Absolute survival rates

Axio Observer Z1 microscope, with a 10ꢂ phase contrast objective were calculated for each control group and one way analysis of

(Carl Zeiss, GmbH Jena, Germany). Images were captured using an variance (ANOVA) was performed for all pair comparisons,

AxioCam Hc camera, at distinct time points (24 and 48 h) after the followed by post hoc analyses (Tukey) and Student t test in the

scratch had been made. Potential cytotoxic effects, in the presence case of RT-PCR experiments using GraphPad Prism v.6. Degrees

of the materials tested, were also investigated with respect to of significance were assessed by three diﬀerent rating values:

cell morphology. Cells were regularly examined with respect to *p o 0.05 (significant), **p o 0.01 (highly significant), ***p o

shape, appearance, color, confluency, etc., to further confirm 0.001 (extremely significant) and ****p r 0.0001 (extremely

any aberration from the healthy status at several time points significant) or non-significant ( p 4 0.05).

(prior and post treatment).

3.8.5 Induction of adipogenesis with V(^V)-complexes. 3T3-

L1 pre-adipocytes were diﬀerentiated into mature adipocytes

4. Conclusions

following the standard diﬀerentiation protocol (vide supra) as

Synthetic exploration of appropriately designed Schiﬀ base

described elsewhere.⁵⁹3T3-L1 fibroblast-like cells were treated

ligands, extending from o-vanillin to salicylaldehyde and 2-hydroxy-

with 10 ng mL^ꢁ1of insulin and/or selected well-defined vanadium

1-naphthaldehyde cores and encompassing 2-aminophenolate

complexes (1–20 mM). Vanadium complexes were dissolved in

tethers, with vanadyl acetylacetonate in methanolic solutions, led

DMEM (10% FBS, 1% penicillin/streptomycin), followed by sterile

to crystalline materials 1–4, bearing distinct molecular metal–ligand

filtration. In all experiments run, the insulin group was used as

ratios, vanadium oxidation state and coordination composition as

positive control. A control group with no treated cells (without

well as geometries. The physicochemical characterization in the

insulin or vanadium complexes) was also included. On the 8^thday

solid state and in solution revealed the solid state–solution correla-

of the differentiation process, cell differentiation was assessed

tion profile, based on which further molecular biological studies,

with oil red O staining and Real-Time PCR for relative expression

targeting their insulin mimetic potential in adipocyte differentiation,

of closely related biomarkers. All tests were carried out at least

were pursued. The results (a) portray a well-described structure-

in triplicate.

specific behavior of vanadoforms inducing cellular differentiation,

3.8.6 Oil red O staining. Successful cell diﬀerentiation into

thus projecting the importance of that metal ion in adipogenesis,

mature adipocytes was evaluated through oil red O staining,

and (b) generate a well-defined molecular biological profile

which was performed on the 8^thday of the diﬀerentiation

through genetic biomarkers pointing to potential metallopharm-

process. For that purpose, cells were washed with PBS (1ꢂ,

aceutical applications toward therapeutic interventions in

pH 7.4) and fixed with 4% formalin for 20 min. Then, cells were

Diabetes mellitus II (T2D).

washed with sterile doubly deionized water (ddH₂O) and treated

with oil red O working solution for 15 min at room temperature.

Subsequently, cells were washed with sterile ultrapure water and

stained with hematoxylin for 1 min at room temperature. Cells

Abbreviations

were visualized using an Axio Observer Z1 microscope, with a acac

10ꢂ and 40ꢂ phase contrast objective (Carl Zeiss, GmbH Jena, 4,4⁰-bipy

Germany). Images were captured on an AxioCam Hc camera. All DMEM

Acetylacetonato

4,4⁰-Bipyridine

Dulbecco’s modified Eagle’s medium

Electron paramagnetic resonance

Electron spray ionization-mass spectrometry

Fetal bovine serum

Maltolato

Adiponectin

tests were carried out at least in triplicate.

EPR

3.8.7 RT-PCR assay. Total RNA was extracted from cells on ESI-MS

the 8^thday of the diﬀerentiation process, using Trizol (Life Science, FBS

Chemilab, Berkeley, California). Synthesis of cDNA was performed malto

with the iScript cDNA synthesis kit (BioRad), according to the ADIPOQ

manufacturer instructions. RT-PCR was run on a Rotor Gene Q ANOVA

(Qiagen) using the QuantiTect SYBR Green PCR kit and appro- DM

One way analysis of variance

Diabetes mellitus

priate reagents (Qiagen). The primers used were the following:

GLUT4

Glucose transporter 4

17888 | New J. Chem., 2019, 43, 17872--17890 This journal is ©The Royal Society of Chemistry and the Centre National de la Recherche Scientifique 2019

View Article Online

Paper

NJC

20 D. C. Crans, J. T. Koehn, S. M. Petry, C. M. Glover,

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21 E. M. Zayed and M. A. Zayed, Spectrochim. Acta, Part A, 2015,

143, 81–90.

RETN

INS-R

PBS

PPAR-g

SEM

Resistin

Insulin receptor

Phosphate buﬀer saline

Peroxisome proliferator-activated receptor gamma

Standard error mean

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V(V)-Schiff base species induce adipogenesis through structure-specific influence of genetic targets

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