Synthesis, X-ray structure and anticancer activity evaluation of a binuclear La(III) complex with anthranilic acid

Abstract A binuclear La(III) complex {[La2(HA)4(H2O)4(C2H5OH)2Cl2]Cl4 (C1)} with 2-aminobenzoic acid (HA) was prepared from the ligand and heptahydrated lanthanum chloride. The complex was characterised by X-ray crystallography that revealed anti-prismatic geometry around both of the lanthanum. In the complex, the four 2-aminobenzoic acid ligands are zwitter ionic and the two lanthanum(III) ions net charge is only counterbalanced by chloride ions. The complex cytotoxicity was determined against human breast (MDA-MB-231), prostate (PC-3) and bladder (T-24) cancer cells. This complex afforded cytotoxicity towards the T-24 bladder cancer cells with an IC50 value of 383.5 µg/mL (319 µM). In contrary, activities by the lanthanum complex with IC50 values of 1124 µg/mL (934 µM) and 739 µg/mL (614 µM) were, respectively, shown against the MDA-MB-231 and PC-3 cancer cells. This means the complex is more cytotoxic against the T-24 cells, despite that its activity is less compared with activities shown by classical drugs. Graphical Abstract


Introduction
The long history of cancer has recently been cleared by the discovery of remnants of a more than 4200 years old Egyptian woman infected by this disease (Tauxe 2015).In 2021, the American Cancer Society reported new cancer cases of about 1,898,160 and cancer associated deaths of about 608,570 people in the United States (5200 new cancer cases and 1667 deaths due to cancer per day) (Siegel et al. 2021).Nevertheless, a dramatic increment is anticipated regarding this death rate because of the presence of more than 200 types of cancer and the difficulty in the discovery of many cancer types at early stages (Aly et al. 2022b).Breast cancer is known as the primary reason for women death with neoplasia in the industrialised countries causing one fourth of non-accidental deaths among 35-54 years old women (Aly et al. 2022b).The breast cancer incidence rate is reported to rise fast up to the menopause but the rise in the breast cancer incidence is slower afterwards (Guinee et al. 2000;Caba et al. 2012).Prostate cancer is a very common men related cancer in the world being only next to the lung cancer to cause death by cancer in men (Wang et al. 2009).The obstacle in achieving feasible prostate cancer chemotherapeutics is primarily due to the absence of targeted delivery to the prostate (Barve et al. 2014;Tao et al. 2019).Bladder cancer, a malignant tumor common in the urogenital system, has invaded 430,000 people in the world in 2012 (Antoni et al. 2017).Tumor resection is regarded as the main treatment of this type of cancer; however, the bladder cancer recurrence rate remains very high (Li et al. 2015).
This research describes the preparation and molecular structure of a new binuclear complex of lanthanum as a rare earth metal ion with anthranilic acid in addition to exploration of the cytotoxic activity of this lanthanum complex against human cancer cells of breast (MDA-MB-231), prostate (PC-3) and bladder (T-24).Anthranilic acid, being the ligand in this study, was selected depending on well reported biological features for this acid and its derived compounds (Hosny et al. 2019).Anthranilic acid is extremely significant in the biosynthesis of the amino acid 'tryptophan' that is of vital roles in several biological and medicinal aspects, however several anthranilic acid derivatives have been shown to possess great antibacterial, anti-malarial, anti-inflammatory and antineoplastic effects (Goel et al. 1999;Sarrafi et al. 2009;Borozan and Stojanovic 2013;Shou et al. 2014).Regarding the metal, lanthanum is biologically considered as an antagonist for calcium involved in a variety of cellular reactions and lanthanum compounds have been widely studied regarding their anatomical barriers, membrane structure and subcellular transport systems (e.g. the calcium pathway) (He et al. 2005).

Synthesis and characterisation
The ligand 2-aminobenzoic acid (HL) was purchased in high purity from Sigma-Aldrich.For the preparation of the lanthanum complex, 2-aminobenzoic acid (2 millimoles) solution in 100 mL of ethanol was prepared.To the ligand solution, the heptahydrated lanthanum chloride salt (1 mmole) dissolved in a minimum volume of water was added.The reaction medium was boiled with stirring for 5 min before further stirring at ambient room conditions for 60 min.After any solid particles present in the reaction mixture were discarded, it was left at room temperature for ten days.Single crystals of the lanthanum complex C1 were recovered in a moderate yield of 48%.This lanthanum complex was subjected to elemental (carbon, hydrogen and nitrogen) analysis in addition to Fourier transform infrared, UV-Visible, hydrogen and carbon nuclear magnetic resonance and single crystal X-ray spectral analyses.The lanthanum complex dissolves in DMSO and DMF and shows air stability and light insensitivity.The complex CHN analysis and proton NMR data (discussed below) revealed its excellent purity as the complex structure was determined by X-ray single crystal diffraction studies.
The molecular structure of the lanthanum complex is displayed in Figure 1, while Figure 2 gives the complex packing along the b-axis.Crystallographic and refinement parameters for the lanthanum complex are listed in Table S1.Further, selected bond lengths and angles in addition to the parameters for H-bonding interaction (Table S2) in this complex are listed.The X-ray diffraction analysis of the lanthanum complex (0.218 Â 0.180 Â 0.072 mm) cleared its binuclear structure, where each lanthanum atom exhibits anti-prismatic coordination geometry.This lanthanum complex crystallised in the space group P 21/n of the monoclinic crystal system.Both of the lanthanum atoms in this complex exhibit the coordination number of eight.The four anthranilic acid ligand molecules exhibit bidentate mode of binding only forming bonds with lanthanum via the carboxylate group two oxygen atoms.In the complex structure, all the carboxylate groups act as bridges connecting both of the lanthanum atoms (e.g.La1 is bound to O1, O2 i , O3 and O4 i from the four anthranilic acid ligand molecules).Therefore, the anthranilic acid ligands are involved in a pair of centrosymmetric eight membered rings each with a La 2 C 2 O 4 set of atoms.The coordination number of eight of this complex is achieved by the formation of other bonds with a pair of oxygen atoms from water molecules (La1 is bound to O5 and O6), an oxygen atom (O7) from ethanol and a chlorine atom (Cl1).Interesting in this structure is that although of deprotonation of all carboxylic acid groups of the ligands, these ligand molecules are neutral exhibiting Zwitter ionic configuration resulted from protonation of the ligand amine groups in the complex.Hence, this lanthanum complex is cationic and the complex ion charge (þ4) in this case is compensated by four chloride ions in attraction with the complex coordination sphere.In this lanthanum complex, the distance of 4.622 Å was determined between the two lanthanum atoms.Furthermore, the La-O (anthranilic acid) bond distances range from 2.445 (1) Å (O1-La1) to 2.4942(1) Å (O2 i -La1).These values are considered as shorter bonds comparing with the bonds between the lanthanum atoms and the coordinated water molecules f2.503(1)-2.577(1)Åg.In turn, the latter bonds are shorter than the lanthanum-ethanol (O7) bonds and all the La-O bond distances are generally shorter than the La-Cl bond f2.9018(4)Åg.Despite all the amine groups in the anthranilic acid ligand molecules do not directly contribute in the coordination with lanthanum, these amine groups in addition to the water and alcohol molecules serve as donors to establish an extensive hydrogen bonds' net to stabilise the solid state structure of the complex.
Both of the lanthanum complex and the ligand were pressed in KBr pellets and their FT-IR spectral data were determined.As indicated in literature (Yildirim 2018;Hosny et al. 2019), the anthranilic acid spectrum exhibits a broad peak due to the OH group stretching vibration at 3584 cm À1 and two peaks related to symmetric and asymmetric stretching vibrations of the amine moiety at 3315 and 3376 cm À1 , respectively.Moreover, the carboxylate moiety also displays both symmetric and asymmetric vibrations, respectively, at 1427 and 1670 cm À1 (Yildirim 2018;Hosny et al. 2019).In the complex spectrum, the symmetric and asymmetric vibrations in the carboxylate moiety (COO -) were found, respectively, at 1390 and 1507 cm À1 .This small difference between both of these stretching vibrations provides a proof on the bidentate donation manner of the carboxylate moiety in the complex (C ¸akir 2002; Hosny and Shallaby 2007;Hosny 2009;Sherif and Hosny 2014).Furthermore, the complex spectrum exhibits a broad band at around 3590 cm À1 and another band at 543 cm À1 , respectively, attributed to t(H 2 O) and t(La-O) vibrations in the lanthanum complex (Hosny 2007(Hosny , 2010;;Hosny et al. 2015).
The proton NMR spectrum for complex C1 was determined in DMSO-d 6 (Figure S1) and displayed multiple bands in the 6.47-7.66ppm range due to the ring protons in addition to a broad band centered at 8.54 ppm integrated for the protons of both of the amino group and the carboxylic acid moiety.Indeed, the bands in the 1 H-NMR spectrum of the complex exist at almost the same chemical shifts reported for the free ligand (Portada et al. 2018).On the other hand, the presence of ethanol in the complex was confirmed by two sharp peaks at 1.06 and 3.46 ppm and a broad one at 4.32 ppm that these peaks are integrated for 3, 2 and 1 protons, respectively.These peaks for ethanol in the complex exist at almost the same chemical shifts reported for free ethanol measured in DMSO-d 6 (Gottlieb et al. 1997) and this may suggest displacement of the ethanol ligands with DMSO-d 6 in the complex during the NMR measurement.Finally, there is a band at 3.34 ppm in the complex spectrum that is very intense to be integrated for the water in the complex and normally this band is assigned to residual water in the DMSO-d 6 solvent (Gottlieb et al. 1997), but we assume this band may interfere with a band for the water molecules in the complex.

Evaluation of anticancer activities
Through the MTT assay method, we here investigated the in vitro cytotoxic behavior of this lanthanum complex against human cancer cells of breast (MDA-MB-231), bladder (T-24) and prostate (PC-3).We examined the cytotoxic effect of various concentrations of the complex (0-1000 mg/mL) in DMSO and recorded the respective surviving fraction of each cell in addition to the complex dosages that afforded 50% inhibition of the cancer cells (IC 50 ).Indeed, the ligand (2-aminobenzoic acid) and several derivatives have been biologically estimated for their anti-proliferative activities (Abdel-Rahman 2016;Kim 2016;Niu 2019;Aly et al. 2022a).But the novelty of lanthanum complexes of close structures incorporated as anticancer agents (particularly against PC-3, T-24 and MDA-MB-231 cells) in the literature afforded difficultly for reasonable comparison between the given anti-proliferative activities of this lanthanum complex with data obtained from similar complexes.
As shown in Figure S2, the lanthanum complex induced more enhanced cytotoxic effect against the bladder transitional carcinoma (T-24) with an IC 50 value of 383.5 mg/ mL (319 mM).Furthermore, the lanthanum complex afforded weak cytotoxic behavior with an IC 50 value of 1124 mg/mL (934 mM) against the breast MDA-MB-231 cancer cells and with an IC 50 value of 739 mg/mL (614 mM) against the prostate PC-3 cancer cells.Comparing these results with other data obtained from a recent study on a dimeric binuclear complex of copper with a substituted 2-aminobenzoic acid ligand (i.e.N-acetylanthranilic acid) indicates much insignificant activities by the lanthanum complex against the breast MDA-MB-231 and prostate PC-3 cancer cells (i.e. the copper complex exhibits IC 50 values of 131.2 mg/mL (127 mM) against MDA-MB-231 cells and of 200.7 mg/mL (195 mM) against PC-3 cells) (Aly et al. 2022b).On the other hand, the anti-proliferative activity given by the lanthanum complex against the bladder T-24 cells is close to that shown by the copper complex which gave an IC 50 value of 300.3 mg/mL (291 mM) against these cells (Aly et al. 2022b).
In the literature, a great inhibition of MDA-MB-231 cells by cisplatin was reported (IC 50 values of 1.3 ± 0.55 lg/mL and 0.9 ± 0.31 lg/mL after 24 and 48 h, respectively) (Fani et al. 2016).Moreover, the same drug (22 lM) inhibited the prostatic PC-3 cells completely (100% death) after exposure for 3 days (Borner et al. 1995).Gallic acid was also shown to possess excellent inhibition of the bladder cancer cells 'T-24' after 24, 48 and 72 h with IC 50 values of 21.73, 18.62 and 11.59 mg/mL, respectively (Zeng et al. 2020).Hence, we can conclude unsuitability of this complex as an effective anticancer agent against the studied cancer cells fMDA-MB-231, T-24 and PC-3g due to its weak anti-proliferative character in comparison to other compounds and classical drugs.Nevertheless, possible bioactivity profile of this lanthanum complex may be explored in the future via other researches based on other cell lines or animal models.

Chemicals and instrumentation
Lanthanum chloride heptahydrate (Alfa Aesar) and anthranilic acid (Sigma-Aldrich) in addition to absolute ethanol were used as received for the complex preparation.Data for carbon, hydrogen and nitrogen content in the lanthanum complex were investigated with an elementar analysensysteme GmbHvario EL III element analyser.The Fourier transform infrared vibrational data for anthranilic acid and complex C1 in KBr pellets were recorded in the 400 À 4000 cm À1 wavenumber range with a Nicolet iS10 spectrophotometer. 1 H-NMR spectral data of the complex in deuterated DMSO have been collected on an NMR Bruker spectrometer (400 MHz) involving tetramethylsilane as a standard.UV-Visible spectra of the free ligand and its lanthanum complex in DMSO were measured with a Perkin Elmer Lambda 40 UV/VIS spectrometer.The X-ray intensity results of complex C1 were recorded at 120(2) K on a Bruker D8 Venture Kappa DUO system equipped with a multilayer mirror monochromator and a MoKa rotating anode X-ray tube (k ¼ 0.71073 Å).Integration of the frames was done with the use of the Bruker SAINT software package (BrukerSAINT 2012).Correction of data for absorption effects was performed using the Multi-Scan method (SADABS) (Sheldrick 1996).The structure was solved and refined using the Bruker SHELXTL Software Package (Sheldrick 2015).All C-bound hydrogen atoms have been calculated in ideal geometry riding on their parent atoms, but the N-and O-bound hydrogen atoms have been refined freely.The crystallographic figures have been drawn with ORTEP-3 at the 50% ellipsoid probability level (Farrugia 2012).Crystallographic data have been deposited with the Cambridge Crystallographic Data Centre, CCDC, 12 Union Road, Cambridge CB21EZ, UK.Copies of the data can be obtained free of charge on quoting the depository number CCDC-2117292 (C1) (https://www.ccdc.cam.ac.uk/structures/).

Preparation of the lanthanum complex
A solution of 2-aminobenzoic acid (2 mmol, 274 mg) in 100 mL of absolute ethanol was prepared.Under vigorous stirring, LaCl 3 .7H 2 O (1 mmol, 371 mg) dissolved in a minimum volume of water was added.After boiling and stirring this mixture for 5 min, stirring was continued at the room temperature for 60 min.Settling of the obtained clear solution undisturbed at ambient room temperature for 10 days resulted in recovery of single crystals that were filtered, washed with ethanol and dried in an anhydrous calcium chloride desiccator.

Evaluation of anticancer activities
PC-3 (ATCC CRL-1435 TM ) human prostate cancer cells, MDA-MB-231 (ATCC HTB-26 TM ) human breast cancer cells and T-24 (ATCC HTB-4 TM ) human transitional cell carcinoma from the American Tissue Culture Collection (ATCC) were obtained.These cancer cells were cultured in Dulbecco's Modified Eagle Medium (DMEM) of low glucose content and supplemented with inactivated fetal bovine serum (FBS, 10%) and penicillin/ streptomycin (1%).After incubation in CO 2 incubator at 37 C and 5% CO 2 , the cancer cells were seeded into 96-well plates (10,000 cells/well).After 24 h, various concentrations (0-1 mg/mL) of complex C1 dissolved in DMSO were added to the cells and left for incubation in CO 2 incubator for one day.All cell viability values were then determined following the MTT (Sigma-Aldrich, St. Louis, MO, USA) method by adding 10 lL (5 mg/mL) to each well.After four hours of final incubation, dimethyl sulfoxide (100 mL) was added to each well and the absorbance was detected at 570 nm by the help of an ELISA microplate reader (Molecular Devices, Downingtown, PA, USA).

Conclusions
This article gave synthetic procedures of a complex dimer of lanthanum ([La 2 (HA) 4 (H 2 O) 4 (C 2 H 5 OH) 2 Cl 2 ]Cl 4 ) with 2-aminobnzoic benzoic acid as its structure was determined by X-ray crystallography.This analysis also revealed cationic character of the complex and the exhibition of anti-prismatic geometry around each of the lanthanum atoms.This lanthanum complex gave anti-proliferative activities against three cancer cells fMDA-MB-231, T-24 and PC-3g and the most enhanced activity was against the T-24 bladder cancer cells with an IC 50 value of 383.5 mg/mL.But classical drugs were documented to exhibit much enhanced anti-proliferative activities in comparison to this lanthanum complex against the three studied cell lines.

Figure 2 .
Figure 2. Packing of the lanthanum complex along b-axis.