Syntheses, crystal structures and in vitro anticancer activities of oxovanadium(IV) complexes of amino acid Schiff base and 1,10-phenanthroline ligands

Abstract

Four oxovanadium(IV) complexes, namely [VO(desa-met)(phen)]·MeOH·2H₂O (1) (desa-met = Schiff base derived from 4-(diethylamino)salicylaldehyde and dl-methionine, phen = 1,10-phenanthroline), [VO(o-van-met) (phen)]·MeOH·CH₂Cl₂·3H₂O (2) (o-van-met = Schiff base derived from o-vanillin and dl-methionine), [VO(dtbs-napa)(phen)]·2H₂O (3) (dtbs-napa = Schiff base derived from 3,5-di-tert-butyl salicylaldehyde and 3-(1-naphthyl)-l-alanine) and [VO(hyna-napa)(phen)]·1.5H₂O (4) (hyna-napa = Schiff base derived from 2-hydroxy-1-naphthaldehyde and 3-(1-naphthyl)-l-alanine), were synthesized and characterized by IR, HRMS, UV–vis spectra, molar conductance and single-crystal X-ray diffraction (XRD). X-ray structural analysis showed that the V(IV) atoms in all four complexes are six-coordinated in a distorted octahedral environment. In the crystals of complexes 1 and 2, π–π stacking interactions together with hydrogen bonds connect the molecular units into 2D networks. Meanwhile, CH–π stacking interactions are observed between the aromatic rings in the crystals of 1 and 4, while the π–π stacking interactions between aromatic rings in the crystals of 2 and 3 are arranged with a face-to-face mode. The in vitro anticancer activities of these complexes against A-549 and HeGp2 cells were tested by MTT assay.

Introduction

Cisplatin was the first platinum-based complex to be used as an effective anticancer drug [1, 2]. Cisplatin and related Pt(II) complexes exhibit remarkable anticancer activities; however, they suffer from problems related to severe side effects in normal tissue, nephrotoxicity, drug resistance and cost per patient. These drawbacks limit the use and efficiency of cisplatin and related Pt(II) complexes in cancer therapy [3–5]. These considerations have stimulated the search for new non-platinum anticancer agents that possess reduced side effects, lower resistance, lower cost and greater efficacy toward a wider range of cancers [6, 7]. Hence, in the past few decades, transition metal complexes have been extensively studied as anticancer and antibacterial agents [8, 9]. In particular, oxovanadium salts are less expensive than K₂PtCl₄, RuCl₃ or VCl₃ and are therefore attractive for vanadium-based research and development of inexpensive drugs. Current research suggests that transformed cells maintain an abnormal redox homeostasis that helps them to obtain a high level of genetic instability, which is conducive to cancer progression [10–12]. Vanadium’s intrinsic redox activity can generate reactive oxygen species (ROS). This enhanced level of ROS can disorganize the redox homeostasis in neoplastic cells and impart redox stress to the cell, either by cleaving DNA or by promoting mitochondrial membrane permeabilization [13]. Moreover, a growing body of evidence reveals that vanadium complexes can act as potent anticancer agents [14]. Although inorganic vanadium salts have relatively high toxicity and poor biological activity, the complexation of vanadium with organic ligands can minimize adverse effects while enhancing its benefits [15]. Therefore, it is important to synthesize novel oxovanadium complexes and investigate their medical applications.

Organic ligands containing Schiff bases, considered privileged structures, have attracted considerable attention due to their facile synthesis and variable coordination behavior toward metal ions, as well as their wide applications in various fields (e.g., as chemosensors [16], catalysts [17], anticancer agents [18, 19], adsorbents [20] and bactericidal agents [21, 22]). Efforts have been made to synthesize and characterize amino acid Schiff base complexes of transition metals [23, 24]. Recently, our research group, and others, reported some vanadium complexes with anticancer activities [15, 19]. In this work, four new amino acid Schiff base oxovanadium(V) complexes have been synthesized and characterized (Fig. 1). Moreover, their anticancer activities have been investigated against A-549 and HeGp2 cell lines by the MTT assay (laboratory test and standard colorimetric assay).

Fig. 1

The molecular structure of oxovanadium(IV) complexes 1, 2, 3 and 4

Experimental

Materials and instrumentation

All chemicals and reagents obtained from commercial sources were of AR grade and used without further purification. HRMS (ESI–MS) spectra were recorded using a time-of-flight Micromass LCT Premier XE spectrometer. Elemental (C, H, N) analyses were obtained with a Vario EL III elemental analyzer. IR spectra were recorded on KBr pellets, with a Vary FT-IR 1000 spectrophotometer in the range 400–4000 cm⁻¹. Molar conductivity measurements were obtained using a QCJD-2010 (China) conductivity meter.

Synthesis of complexes 1 and 2

Complexes 1 and 2 were prepared by a one-pot synthetic procedure in which a mixture of dl-methionine (0.14 g, 1.0 mmol) and KOH (0.06 g, 1.0 mmol) in 15 mL hot methanol–water (v:v = 1:1) was added to a solution of 4-(diethylamino)-salicylaldehyde (0.21 g, 1.0 mmol) or o-vanillin (0.15 g, 1.0 mmol) in ethanol (5 mL). The mixture was refluxed for 2 h, followed by addition of a solution of vanadyl sulfate (0.16 g, 1.0 mmol) in water (5 mL). The reaction was continued for another 1 h, followed by addition of a solution of 1,10-phenanthroline (0.20 g, 1.0 mmol) in 5 mL methanol. The resulting solution, after further refluxing for 1 h, gave a red precipitate. The solid was isolated by filtration, washed with small amounts of water, methanol and diethyl ether and dried. Single crystals of 1 and 2 suitable for X-ray diffraction were obtained over one week via slow evaporation of MeOH-CH₂Cl₂ (v:v = 1:1) solutions at room temperature.

Complex 1, [VO(desa-met)(phen)]·MeOH·2H₂O: Yield: 72.8 %. Anal. Calcd for C₂₈H₃₀N₄O₄SV·CH₃OH·2H₂O: C 54.6, H 6.0, N 8.8. Found: C 54.8, H 6.0, N 8.6. HRMS (ESI–MS) calculated for C₂₈H₃₀N₄O₄SVNa (M + Na)⁺: 593.1359, found m/z: 593.1310. FTIR: 3469(s), 3416(s), 3241(w), 2975(m), 2904(w), 1657(s), 1616(s), 1598(m), 1515(w), 1399(m), 953(m), 879(w), 728(w), 601(w), 484(w). UV–vis in DMSO:H₂O (v:v = 1:1), 2.0 × 10⁻⁶ mol/L, (λ _max/nm) 265, 354. Λ _m = 4.7 Ω⁻¹ mol⁻¹ cm² in DMSO: H₂O (v:v) = 1:1 at 25 °C.

Complex 2, [VO(o-van-met)(phen)]·MeOH·CH₂Cl₂·3H₂O: Yield: 75.3 %. Anal.Calcd for C₂₅H₂₃N₃O₅SV·CH₃OH·CH₂Cl₂·3H₂O: C 46.4, H 5.0, N 6.0. Found: C 46.5, H 5.1, N 6.1. HRMS (ESI–MS) calculated for C₂₅H₂₃N₃O₅SVNa (M + Na)⁺: 551.0668, found m/z: 551.0696. FTIR: 3469(s), 3415(s), 3231(m), 3018(w), 2920(w), 1638(s), 1617(s), 1535(w), 1516(w), 1400(m), 960(m), 850(m), 726(m), 618(w), 476(w). UV–vis in DMSO:H₂O (v:v = 1:1), 2.0 × 10⁻⁶ mol/L, (λ _max/nm) 262, 390, 410. Λ _m = 5.7 Ω⁻¹ mol⁻¹ cm² in DMSO:H₂O (v:v = 1:1) at 25 °C.

Synthesis of complexes 3 and 4

To a mixture of 3-(1-Naphthyl)-l-alanine (0.22 g, 1.0 mmol) and potassium hydroxide (0.06 g, 1.0 mmol) in 15 mL hot methanol–water (v:v = 1:1) was added to a solution of 3,5-di-tert-butyl salicylaldehyde (0.23 g, 1.0 mmol) or 2-hydroxy-1-naphthaldehyde (0.17 g, 1.0 mmol) in ethanol (5 mL). The mixture was refluxed for 2 h, followed by addition of a solution of vanadyl sulfate (0.16 g, 1.0 mmol) in water (5 mL). The resulting solution was then heated to reflux for another 1 h, followed by the addition of a solution of 1,10-phenanthroline (0.20 g, 1.0 mmol)] in methanol (5 mL). The resulting solution was refluxed for a further refluxing 1 h, giving a yellow precipitate. The solid was isolated, washed with small amounts of water, methanol and diethyl ether and dried. Single crystals of complexes 3 and 4 suitable for X-ray diffraction were obtained over one week via slow evaporation of MeOH-CH₂Cl₂ (v:v = 1:1) solutions at room temperature.

The data of 3, [VO(3,5-ditbsal-3-1-Naph-l-ala)(phen)]·2H₂O: Yield: 76.3 %. Anal. Calcd for C₄₀H₃₉N₃O₄V·2H₂O: C 67.4, H 6.1, N 5.9. Found: C 67.5, H 6.1, N 5.8. HRMS (ESI–MS) calculated for C₄₀H₃₉N₃O₄VNa (M + Na)⁺: 699.2278, found m/z: 699.2235. FTIR: 3468(s), 3421(s), 1636.7(s), 1617(s), 1544(w), 1466(w), 1400 (m), 958(m), 848(w), 727(w). UV–vis in DMSO:H₂O (v:v = 1:1), 2.0 × 10⁻⁶ mol/L, (λ _max/nm) 264, 369. Λ _m = 3.1 Ω⁻¹ mol⁻¹ cm² in DMSO:H₂O (v:v = 1:1) at 25 °C.

The data of 4, [VO(2-hyd-1-nade-3-1-Naph-l-ala)(phen)]·1.5H₂O: Yield: 78.6 %. Anal.Calcd for C₃₆H₂₅N₃O₄V·1.5H₂O: C 67.4, H 4.4, N 6.5. Found: C 67.3, H 4.3, N 6.4. HRMS (ESI–MS) calculated for C₃₆H₂₅N₃O₄VNa (M + Na)⁺: 637.1182, found m/z: 637.1173. FTIR: 3550(s), 3473(s), 3416(s), 3236(w), 1637(s), 1617(s), 1540(w), 1510(w), 1456(w), 1399(m), 1095(w), 963(m), 727(w), 617(w), 477(w). UV–vis in DMSO:H₂O (v:v = 1:1), 2.0 × 10⁻⁶ mol/L, (λ _max/nm) 265, 355. Λ _m = 6.4 Ω⁻¹ mol⁻¹ cm² in DMSO: H₂O (v:v) = 1:1 at 25 °C.

Crystal Structure Determination

Single-crystal XRD data were collected using a Rigaku diffractometer with a mercury charge-coupled device area detector (Mo Kα; λ = 0.71073 Å) at room temperature. Empirical absorption corrections were applied to the data using the Crystal Clear program [25]. The structure was solved by direct methods and refined by the full-matrix least-squares method on F ² using the SHELXTL-97 program [26]. Metal atoms were located from E-maps, and other non-hydrogen atoms were located in successive difference Fourier syntheses. All non-hydrogen atoms were refined anisotropically. The organic hydrogen atoms were positioned geometrically, and those in water molecules were located using the difference Fourier method and refined freely.

PLATON/SQUEEZE was used to remove the heavily disordered water molecules. Crystallographic data and other pertinent information for complexes 1–4 are summarized in Table 1. Selected bond distances and angles are listed in Tables S2–S5†. Bond lengths and angles of hydrogen bonds are listed in Tables S6† and S7†.

Table 1 Selected crystallographic and refinement data for 1–4 ^a

In vitro anticancer activities

A549 cells (human lung carcinoma cell line) and HepG2 cells (human hepatoma cell line) were purchased from ATCC. A549 cells were grown in DMEM/HIGH GLUCOSE(1X) (Dulbecco’s modified eagle’s medium), and HepG2 cells were maintained in modified Roswell Park Memorial Institute 1640 (RPMI-1640) which were supplemented with 10 % fetal bovine serum and 1 % penicillin streptomycin solution in a humidified atmosphere of 5 % CO₂, 95 % air at 37 °C. The passage number range for both cell lines was maintained between 10 and 20. The cells were cultured in 25 cm² cell culture flasks. For experimental purposes, A549 and HepG2 cells were cultured in 96-well plates, respectively (5 × 10⁴ cells/mL, 100 mL/well). Cells were allowed to attach for 24 h before treatment with the test complexes. A stock solution of each complex was prepared in DMSO and filtered with Minisart filters (0.45 μm). Each stock solution was diluted with serum-free medium to different concentrations (0–120 μM). Cell monolayers were washed with PBS, and the test complexes were added within a range of concentrations from 0 to 120 μM for 24 h. The concentration range for the complexes 1–4 and the exposure times have been selected based on preliminary studies performed in our laboratory.

The MTT assay is based on the protocol originally described by Mossmann [19]. The assay was optimized for the cell lines used in this experiment. Briefly, at the end of the incubation time, cells were incubated for 4 h with 0.5 mg/mL of MTT, dissolved in serum-free medium (DMEM or RPMI-1640 for A459 and HepG2 cells, respectively). Washing with PBS (100 μL) was followed by the addition of DMSO (200 μL) and gentle shaking for 10 min. The absorbance was recorded at 490 nm using Multiskan Spectrum. The cell viability ratio was calculated by the following formula:

$$ {\text{Inhibitory}}\;{\text{ratio}}(\% ) = 1 - \left( {{\text{OD}}_{\text{treated}} - {\text{OD}}_{\text{black}} } \right)/\left( {{\text{OD}}_{\text{control}} - {\text{OD}}_{\text{black}} } \right) \times 100\;\% $$

(1)

Cytotoxicity is expressed as the concentration of the complex inhibiting cell growth by 50 % (IC₅₀ value see Table 1).

Results and Discussion

Synthesis and Characterization

Complexes 1–4 have been prepared in high yields from a one-pot synthetic procedure in which vanadyl sulfate is reacted with the required dianionic α-amino acid Schiff base ligand and 1,10-phenanthroline in aqueous methanol.

The complexes were characterized by their IR spectra, UV–vis spectra and HRMS (ESI–MS) mass spectral data (Figures S1–S9†). All four complexes have small molar conductivity values in 50 % DMSO-H₂O at 25 °C, indicating that they are non-electrolytes [19, 27, 28].

Complexes 1–4 gave satisfactory C, H and N analyses. The HRMS (ESI–MS) mass spectra of complexes 1–4 are shown in Figures S1–S4†. The parent ion peaks are observed at m/z 593.1310 for 1, 551.0668 for 2, 699.2235 for 3 and 637.1173 for 4. In addition, the dominant features in the HRMS spectra, namely cationic fragment peaks at m/z 203.0588 to 203.0608, can be associated with the fragment [phen + Na⁺]⁺, indicating that all four complexes possess a phen ligand.

The FTIR spectra of complexes 1–4 are shown in Figures S5–S8†. Bands are observed at 953 cm⁻¹ for 1, 960 cm⁻¹ for 2, 958 cm⁻¹ for 3 and 963 cm⁻¹ for 4, assigned to the υ_(V=O) stretching vibration, which is typical for oxovanadium complexes [27, 28]. The very sharp absorptions at 1657–1636 cm⁻¹ are characteristic of the imine group, υ_(C=N), in the complexes [27, 28]. Weak bands at 848–728 cm⁻¹ are attributed to the ring-stretching frequencies [υ_(C=C) and υ_(C=N)] of 1,10-phenanthroline [27]. Two moderate absorptions at 1616–1617 cm⁻¹ and 1399–1400 cm⁻¹ can be assigned to the asymmetric and symmetric stretching vibrations of the $\upsilon_{{\left( {{\text{CO}}_{2}^{ - } } \right)}}$ group [29], respectively. The frequency separation (Δυ) is greater than 200 cm⁻¹, suggesting unidentate bonding for the carboxyl group [30]. Weak peaks in the low wavenumber region 400–650 cm⁻¹ may be attributed to υ_(V–O) and υ_(V–N) bonds in the complexes [28].

The UV–visible spectra of 1–4 are shown in Figure. S9†. All four complexes display a ligand-centered band at ca. 260 nm, assignable to the π → π* transition. 1,10-Phenanthroline with its respective quinoxaline moiety exhibits an additional band near 350 nm, assignable to the n → π* transition [28].

Description of crystal structures

The molecular structures of complexes 1–4 with the atom numbering schemes are shown in Figs. 2, 3, 4 and 5 and selected bond lengths and angles are given in Table S2–S4†, respectively. The complexes are similar in each of the structures, with the general formulation [VO₂L(Phen)]. All four complexes are mononuclear with distorted octahedral geometry. The V(IV) centers are six-coordinated, by two N atoms from one phen ligand, one O atom from the VO²⁺ moiety, two O atoms and another N atom from the tridentate Schiff base ligand (L). The terminal vanadyl V=O distances are 1.600(14) Å for 1, 1.588(2) Å for 2, 1.579(5) Å for 3 and 1.573(6) Å for 4, which are all in the 1.56–1.76 Å range reported for other vanadyl complexes [19, 31, 32]. The V1–O1 (phenolate oxygen) bond distances are 1.952(13) for 1, 2.00(2) for 2, 1.908(5) for 3 and 1.920(5) for 4, which are as expected from related VO Schiff base complexes [27, 32]. The V1–O2 (carboxylate oxygen) bond lengths at 1.978(14) for 1, 1.951(2) for 2, 2.021(5) for 3 and 1.992(5) for 4 are in the range found for other VO Schiff base complexes [33, 34]. The V(1)–N(3) amine bond lengths are 2.0333(15) Å for 1, 2.048(3) Å for 2, 2.0105(56) for 3 and 2.1294(68) Å for 4. The V(1)–N(2)(phen) bond trans to the V=O group is significantly longer [2.3603(16)–2.3955(70) Å] than the other V(1)–N(1)(phen) distances [2.1294(68)–2.1587 (60) Å]; the respective average length of 2.38 and 2.14 Å is similar to the values reported in the literature [27, 32–34]. In addition, the asymmetric unit contains one solvate methanol molecule in structures 1 and 2, and one solvate dichloromethane molecule is also found in structure 2 (Fig. 3).

Fig. 2

Molecular structure of 1 with thermal ellipsoids at 45 % probability (the solvent molecules were omitted)

Fig. 3

Molecular structure of 2 with thermal ellipsoids at 45 % probability (the solvent molecules were omitted)

Fig. 4

Molecular structure of 3 with thermal ellipsoids at 45 % probability

Fig. 5

Molecular structure of 4 with thermal ellipsoids at 45 % probability

In the supramolecular structures of complexes 1 and 4, CH–π stacking interactions between the phen and Schiff base ligands are observed. The H atom of one aromatic ring and another aromatic ring centroid are arranged in an edge-to-face mode with the distances of 2.7447(6) and 2.6998(4) Å, respectively (Figs. 6, 7). In addition, some classic hydrogen bonds are observed in structure 1. These intramolecular hydrogen bonds expand the structure into a supramolecular structure. The π–π stacking interactions together with the hydrogen bonds in the structure form a 2D network (Fig. 8). The lengths and angles of the hydrogen bonds of 1 are given in Table S5†.

Fig. 6

The CH–π stacking interactions in 1

Fig. 7

The CH–π stacking interactions in 4

Fig. 8

The 2D network of 1 by the hydrogen bonds interaction

The supramolecular structures of complexes 2 and 3 show π–π stacking interactions between the aromatic rings. Two aromatic rings are arranged in a face-to-face mode with center of mass distance of 3.4055(35) and 3.4993(43) Å, respectively (Figs. 9, 10).

Fig. 9

The π–π stacking interactions in 2

Fig. 10

The π–π stacking interactions in 3

Hydrogen bonds between the O–H groups of the solvent MeOH molecules and the uncoordinated carboxylate O atoms extend the structure of 2 into a 2D layer architecture, along with π–π stacking interactions (Fig. 11). The bond lengths and angles of the hydrogen bonds of 2 are given in Table S6†.

Fig. 11

The 2D network of 2 by the hydrogen bonds interaction

In vitro anticancer activities

The anticancer properties of these complexes and of cis-Pt against A-549 and HeGp2 cell lines were tested by the MTT assay. Cytotoxicity is expressed as the concentration of the complex inhibiting cell growth by 50 % (Table 2).

Table 2 In vitro anticancer activities of the complexes 1–4 ^a

Complexes 2–4 were found to have moderate anticancer activities toward A549 (human lung carcinoma cell line) and HepG2 (human hepatoma cell line), with IC₅₀ valves of 27.0–46.3 μmol L⁻¹. Although the activities of these complexes are lower than the controls of cis-Pt and VO(acac)₂ [32], they may serve as a starting point for further research and development.

Conclusion

Four new amino acid Schiff base oxovanadium(IV) complexes have been synthesized and characterized. The crystal structures of all four complexes show that their V(IV) atoms have distorted octahedral coordination geometries. Complexes 2–4 have moderate anticancer activities toward human lung carcinoma and human hepatoma cell lines.