Synthesis, Characterization, and Preliminary In Vitro Anticancer Activity of Zinc Complexes Containing Amino Acid-Derived Imidazolium-Based Dicarboxylate Ligands
Abstract
Coordination polymers containing zinc and imidazolium-based dicarboxylate ligands, [LR]−, were synthesized by reacting zinc acetate with HLR compounds, 1. The resulting complexes were characterized and structurally identified using single-crystal X-ray diffraction, revealing polymeric structures for the complexes [Zn(LR)2]n (R = Gly, 2a; βAla, 2b) and [Zn(LLeu)2(H2O)2]n (2c). In these structures, the [LR]− ligands adopt a bridging monodentate μ-κ1-O1,κ1-O3 coordination mode, resulting in distorted tetrahedral (2a, 2b) or octahedral (2c) geometries around the zinc center. When the synthesis was carried out in the presence of amino acids, mixed ligand complexes [Zn(LR)(aa)(H2O)]n (R = aa = Val, 2d, and R = aa = Ile, 2e) were formed. Complexes 2d–2e were also structurally characterized using single-crystal X-ray crystallography, revealing that the ligand [LR]− maintained the same coordination mode, while the zinc center adopted a five-coordinated geometry. The cytotoxic activity of complexes 2a–2e was evaluated against three cancer cell lines and one non-cancerous cell line. Remarkably, these complexes exhibited higher toxicity against cancer cells than against the non-cancerous cell line, and they showed greater selectivity than carboplatin, a commonly used chemotherapy drug. Although, in general, these complexes did not surpass the selectivity of gemcitabine, complex 2c stood out for exhibiting a selectivity index value similar to that of gemcitabine against melanoma cells. Among the series, compounds 2a–2c demonstrated the highest activity, with 2a being the only complex with some selective activity against lung cancer. Complex 2b was the most active, though with low selectivity, while complex 2c exhibited the highest selectivity for melanoma and bladder cancer (selectivity index of 3.0).
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Academic Editors: Samir Acherar and Sung-Kun (Sean) Kim Received: 4 March 2025 Revised: 27 March 2025 Accepted: 27 March 2025 Published: 30 March 2025 Citation: Carrasco, C.J.; Pastor, A.; Conejo, M.d.M.; Álvarez, E.; Calderón-Montaño, J.M.; López-Lázaro, M.; Galindo, A. Synthesis, Characterization, and Preliminary In Vitro Anticancer Activity of Zinc Complexes Containing Amino Acid-Derived Imidazolium-Based Dicarboxylate Ligands. Int. J. Mol. Sci. 2025,26, 3202. https://doi.org/10.3390/ ijms26073202 Copyright: © 2025 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/ licenses/by/4.0/). Article Synthesis, Characterization, and Preliminary In Vitro Anticancer Activity of Zinc Complexes Containing Amino Acid-Derived Imidazolium-Based Dicarboxylate Ligands Carlos J. Carrasco 1, Antonio Pastor 1, María del Mar Conejo 1, Eleuterio Álvarez 2, José Manuel Calderón-Montaño 3, Miguel López-Lázaro 3and Agustín Galindo 1,* 1Departamento de Química Inorgánica, Facultad de Química, Universidad de Sevilla, 41071 Sevilla, Spain; [email protected] (C.J.C.); [email protected] (A.P.); [email protected] (M.d.M.C.) 2Instituto de Investigaciones Químicas, CSIC-Universidad de Sevilla, Avda. Américo Vespucio 49, 41092 Sevilla, Spain; [email protected] 3Departamento de Farmacología, Facultad de Farmacia, Universidad de Sevilla, 41012 Sevilla, Spain; [email protected] (J.M.C.-M.); mlopezlazar[email protected] (M.L.-L.) *Correspondence: [email protected] Abstract: Coordination polymers containing zinc and imidazolium-based dicarboxylate ligands, [L R ] − , were synthesized by reacting zinc acetate with HL R compounds, 1. The resulting complexes were characterized and structurally identified using single-crystal X-ray diffraction, revealing polymeric structures for the complexes [Zn(L R ) 2 ] n (R = Gly, 2a; β Ala, 2b) and [Zn(L Leu ) 2 (H 2 O) 2 ] n (2c). In these structures, the [L R ] − ligands adopt a bridging monodentate µ - κ1 -O 1 , κ1 -O 3 coordination mode, resulting in distorted tetrahedral (2a,2b) or octahedral (2c) geometries around the zinc center. When the synthesis was carried out in the presence of amino acids, mixed ligand complexes [Zn(L R )(aa)(H 2 O)] n (R = aa = Val, 2d, and R = aa = Ile, 2e) were formed. Complexes 2d–2e were also structurally characterized using single-crystal X-ray crystallography, revealing that the ligand [L R ] − maintained the same coordination mode, while the zinc center adopted a five-coordinated geometry. The cytotoxic activity of complexes 2a–2e was evaluated against three cancer cell lines and one non-cancerous cell line. Remarkably, these complexes exhibited higher toxicity against cancer cells than against the non-cancerous cell line, and they showed greater selectivity than carboplatin, a commonly used chemotherapy drug. Although, in general, these complexes did not surpass the selectivity of gemcitabine, complex 2c stood out for exhibiting a selectivity index value similar to that of gemcitabine against melanoma cells. Among the series, compounds 2a–2c demonstrated the highest activity, with 2a being the only complex with some selective activity against lung cancer. Complex 2b was the most active, though with low selectivity, while complex 2c exhibited the highest selectivity for melanoma and bladder cancer (selectivity index of 3.0). Keywords: zinc; anticancer; selectivity; amino acid; imidazolium-dicarboxylate; X-ray 1. Introduction The development of transition metal complexes with anticancer potential continues to be a dynamic field of research [ 1 – 4 ]. A key goal in this area is to identify compounds with low toxicity toward non-cancerous cells, making metals such as copper [ 5 ] and zinc [ 6 , 7 ] particularly attractive for study due to their relatively low toxicity. Zinc, in particular, is an essential trace element in the human body because it is a part of numerous enzymes [ 8 , 9 ] and plays an important role in several biological processes [ 10 , 11 ]. The Int. J. Mol. Sci. 2025,26, 3202 https://doi.org/10.3390/ijms26073202
Int. J. Mol. Sci. 2025,26, 3202 2 of 13 coordination chemistry of Zn 2+ is rich and versatile, with a wide range of well-characterized complexes involving O-, Nand S-donor ligands [ 12 ]. Furthermore, its d 10 configuration supports various stable coordination geometries—such as tetrahedral, trigonal bipyramidal, and octahedral—and, in general, the resulting complexes are susceptible to easy ligand exchange [13]. Previously, we synthesized and characterized zinc complexes with imidazolium-based dicarboxylate amino acid-derived ligands [ 14 ], as well as related silver derivatives that exhibited antimicrobial activity [ 15 ]. Both systems showed water solubility and biocompatibility [ 14 ], making them promising candidates for further investigation in anticancer applications. Here, we expand this series to include new zinc complexes: [Zn(L R ) 2 ] n ( R = Gly ,2a, and β Ala, 2b), [Zn(L Leu ) 2 (H 2 O) 2 ] n (2c), and [Zn(L R )(aa)(H 2 O)] n ( R = aa = Val , 2d, and R = aa = Ile, 2e). Continuing our interest in amino acid-derived transition metal complexes [ 15 – 18 ] and their applications as antimicrobial [ 19 , 20 ] and anticancer agents [ 21 ], we now report on the anticancer activity of these zinc complexes (2a–2e) against three human cancer cell lines: melanoma cells (MeWo), lung adenocarcinoma (A549), and bladder cancer cells (T24). These cancers are among the most common in the world and have high mortality rates in advanced stages [ 22 ]. Lung cancer remains the leading cause of cancer-related mortality worldwide [ 22 ], with approximately 40% of patients diagnosed at the metastatic stage, where the five-year survival rate is below 10%. Bladder cancer, although associated with high survival rates in the early stages, has a dramatically reduced five-year survival rate of less than 9% once metastasized. Melanoma, while often treatable in the early stages, also shows a substantial drop in prognosis at the metastatic stage, with a five-year survival rate of approximately 35% [ 23 ]. Cytotoxic activity was evaluated in comparison with that of a human non-malignant cell line (skin cells, HaCaT) to determine therapeutic selectivity. This cell line, derived from adult tissue, is characterized by a rapid rate of cell proliferation [ 24 ]. Most cancer patients experience side effects during treatment, often necessitating dose reductions or even discontinuation. These side effects arise because most current cancer drugs target rapidly dividing cells, affecting both cancer cells and healthy cells. 2. Results 2.1. Syntheses and Characterization of Complexes 2a–2e The reaction of zinc acetate with HL R compounds, 1, produced complexes [Zn(L R ) 2 ] (R = Gly, 2a; β Ala, 2b; Leu, 2c). The isotropic nature of the Zn(II) d 10 configuration allowed the isolation of these compounds in the solid state as [Zn(L R ) 2 ] n (2a and 2b) or [Zn(L Leu ) 2 (H 2 O) 2 ] n (2c) polymeric complexes, featuring four-and six-coordinated zinc environments, respectively (Scheme 1). Complexes 2a–2c were obtained as air-stable crystalline white solids that are water-soluble but sparingly soluble in organic solvents. Infrared (FTIR) spectroscopy showed broad absorptions for the antisymmetric COO stretching vibrations of the carboxylate groups in the 1655–1600 cm −1 range, indicating coordination of the [L R ] − ligands to zinc (Figure S1). This coordination was evidenced by the shift to lower wavenumbers in comparison to those of the HL R ligand precursors, 1. Symmetric COO vibrations were observed in the 1375–1350 cm −1 range, and the difference values of ( ν COO asym - ν COO sym ) suggested the κ1 -O coordination of the carboxylate group [ 25 ], which was consistent with the structural characterization discussed later. Similar FTIR absorption patterns have been reported for analogous zinc derivatives [ 16 , 26 , 27 ]. The 1 H and 13 C{ 1 H} NMR spectra of 2a–2c (in deuterated water; see Figure S1) displayed signals corresponding to the [L R ] − ligands, with chemical shifts different from those of the HL R precursors, 1, due to zinc coordination [ 28 , 29 ]. The spectroscopic properties of complex 2a are similar to those reported in the bibliography [26,30,31].
Int. J. Mol. Sci. 2025,26, 3202 3 of 13 Int.J.Mol.Sci.2025,26,xFORPEERREVIEW3of14 precursors,1,duetozinccoordination[28,29].Thespectroscopicpropertiesofcomplex2a aresimilartothosereportedinthebibliography[26,30,31]. WhenzincacetatewasreactedwithHLR,1,inthepresenceofoneequivalentofan aminoacid,L‐valineorL‐isoleucine,complexes2dand2ewereobtained,respectively (Scheme2).Theywereidentifiedinthesolidstateaspolymericcomplexes [Zn(LR)(aa)(H2O)]n(R=aa=Val,2d,andR=aa=Ile,2e).Complexes2dand2ewerealso obtainedasair‐stablewater‐solublecrystallinewhitesolidsthataresparinglysolublein organicsolvents.TheirFTIRspectrashowedcarboxylatestretchingvibrations,againcon‐ sistentwithκ1‐Ocoordination(FigureS1),confirmedbythestructuralcharacterizationof 2dand2e(seebelow).The1Hand13C{1H}NMRspectraofthesecomplexes,indeuterated water,includedsignalsforboththe[LR]−ligandsandthecoordinatedaminoacid(Figure S1).Forexample,thesec‐butylgroupofthecoordinatedaminoacidIlein2ewasclearly identifiedbyitscharacteristicsignalsobservedintheNMRspectra(assignmentsdetailed intheSection3andFigureS1). Scheme1.Synthesisofcomplexes2a–2c(inEtOH/H2Oat70°Cfor1h). Scheme2.Synthesisofcomplexes2dand2e(inEtOH/H2Oat70°Cfor1h). 2.2.StructuralCharacterizationofComplexes2a–2e Theidentificationofcomplexes2ascoordinationpolymersinthesolidstatewascon‐ firmedusingX‐raycrystallography.Thestructureofcomplex2awassimilartothatpre‐ viouslydescribed[30,31]andisnotdiscussedindetailhere(seeFigureS6andTablesS1 andS2intheSupplementaryMaterials).Thecompound[Zn(LβAla)2]n,2b,crystallizesin themonoclinicCCspacegroup,formingaone‐dimensional(1D)coordinationpolymer thatextendsalongthebaxis(Figure1).Zincionsarelinkedbybridging[LβAla]–anions Scheme 1. Synthesis of complexes 2a–2c (in EtOH/H2O at 70 ◦C for 1 h). When zinc acetate was reacted with HL R ,1, in the presence of one equivalent of an amino acid, L-valine or L-isoleucine, complexes 2d and 2e were obtained, respectively (Scheme 2). They were identified in the solid state as polymeric complexes [Zn(L R )(aa)(H 2 O)] n (R = aa = Val, 2d, and R = aa = Ile, 2e). Complexes 2d and 2e were also obtained as air-stable water-soluble crystalline white solids that are sparingly soluble in organic solvents. Their FTIR spectra showed carboxylate stretching vibrations, again consistent with κ1 -O coordination (Figure S1), confirmed by the structural characterization of 2d and 2e (see below). The 1H and 13C{1H} NMR spectra of these complexes, in deuterated water, included signals for both the [L R ] − ligands and the coordinated amino acid ( Figure S1 ). For example, the sec-butyl group of the coordinated amino acid Ile in 2e was clearly identified by its characteristic signals observed in the NMR spectra (assignments detailed in the Section 3and Figure S1). Int.J.Mol.Sci.2025,26,xFORPEERREVIEW3of14 precursors,1,duetozinccoordination[28,29].Thespectroscopicpropertiesofcomplex2a aresimilartothosereportedinthebibliography[26,30,31]. WhenzincacetatewasreactedwithHLR,1,inthepresenceofoneequivalentofan aminoacid,L‐valineorL‐isoleucine,complexes2dand2ewereobtained,respectively (Scheme2).Theywereidentifiedinthesolidstateaspolymericcomplexes [Zn(LR)(aa)(H2O)]n(R=aa=Val,2d,andR=aa=Ile,2e).Complexes2dand2ewerealso obtainedasair‐stablewater‐solublecrystallinewhitesolidsthataresparinglysolublein organicsolvents.TheirFTIRspectrashowedcarboxylatestretchingvibrations,againcon‐ sistentwithκ1‐Ocoordination(FigureS1),confirmedbythestructuralcharacterizationof 2dand2e(seebelow).The1Hand13C{1H}NMRspectraofthesecomplexes,indeuterated water,includedsignalsforboththe[LR]−ligandsandthecoordinatedaminoacid(Figure S1).Forexample,thesec‐butylgroupofthecoordinatedaminoacidIlein2ewasclearly identifiedbyitscharacteristicsignalsobservedintheNMRspectra(assignmentsdetailed intheSection3andFigureS1). Scheme1.Synthesisofcomplexes2a–2c(inEtOH/H2Oat70°Cfor1h). Scheme2.Synthesisofcomplexes2dand2e(inEtOH/H2Oat70°Cfor1h). 2.2.StructuralCharacterizationofComplexes2a–2e Theidentificationofcomplexes2ascoordinationpolymersinthesolidstatewascon‐ firmedusingX‐raycrystallography.Thestructureofcomplex2awassimilartothatpre‐ viouslydescribed[30,31]andisnotdiscussedindetailhere(seeFigureS6andTablesS1 andS2intheSupplementaryMaterials).Thecompound[Zn(LβAla)2]n,2b,crystallizesin themonoclinicCCspacegroup,formingaone‐dimensional(1D)coordinationpolymer thatextendsalongthebaxis(Figure1).Zincionsarelinkedbybridging[LβAla]–anions Scheme 2. Synthesis of complexes 2d and 2e (in EtOH/H2O at 70 ◦C for 1 h). 2.2. Structural Characterization of Complexes 2a–2e The identification of complexes 2as coordination polymers in the solid state was confirmed using X-ray crystallography. The structure of complex 2a was similar to that previously described [ 30 , 31 ] and is not discussed in detail here (see Figure S6 and Tables S1 and S2 in the Supplementary Materials). The compound [Zn(L βAla ) 2 ] n ,2b, crystallizes in the monoclinic C C space group, forming a one-dimensional (1D) coordination polymer that extends along the baxis (Figure 1). Zinc ions are linked by bridging [L βAla ] − anions through carboxylate functionalities that adopt a µ - κ1 -O 1 , κ1 -O 3 coordination mode. This results in asymmetric C-O bond lengths within carboxylate groups (e.g., C(6)-O(1), 1.219(7) and C(6)-O(2), 1.268(7) Å). Other selected structural parameters are listed in Table S2. The
Int. J. Mol. Sci. 2025,26, 3202 4 of 13 zinc ion exhibits a tetrahedral coordination geometry, as occurred in related derivatives [Zn(L R ) 2 ] n (R = i Pr, CH 2 Ph) [ 16 ], with a four-coordinate geometry index of τ4 = 0.85. Noncoordinated C = O groups in carboxylates form weak non-classical C = O . . . H-C hydrogen bonds with adjacent 1D chains (Figure S2), resulting in the observed three-dimensional (3D) crystal packing arrangement (Figure S3). Int.J.Mol.Sci.2025,26,xFORPEERREVIEW4of14 throughcarboxylatefunctionalitiesthatadoptaμ‐κ1‐O1,κ1‐O3coordinationmode.This resultsinasymmetricC‐Obondlengthswithincarboxylategroups(e.g.,C(6)‐O(1), 1.219(7)andC(6)‐O(2),1.268(7)Å).OtherselectedstructuralparametersarelistedinTable S2.Thezincionexhibitsatetrahedralcoordinationgeometry,asoccurredinrelatedde‐ rivatives[Zn(LR)2]n(R=iPr,CH2Ph)[16],withafour‐coordinategeometryindexofτ4= 0.85.Non‐coordinatedC=Ogroupsincarboxylatesformweaknon‐classicalC=O…H‐C hydrogenbondswithadjacent1Dchains(FigureS2),resultingintheobservedthree‐di‐ mensional(3D)crystalpackingarrangement(FigureS3). Figure1.1Dcoordinationpolymerofcomplex2balignedalongthebaxiswithellipsoidsofthe ORTEPrepresentationdisplayedwith50%probability. Complex2ccrystallizesinthetrigonalspacegroupP3221,formingatwo‐dimensional (2D)metal–organicframeworkwherezincionsareinterconnectedbybridging[LLeu]–an‐ ionsthroughcarboxylatefunctionalities.Theasymmetricunitof2cconsistsofaZn2+ion, a[LLeu]–ligand,andonewatermolecule(Figure2a).Anintramolecularhydrogenbondis observedbetweenthewaterligandandoneoxygenatomofthecarboxylate(O(5)...O(2) distanceof2.649(3)Å).Thezinccenteradoptsadistortedoctahedralcoordinationgeom‐ etry,surroundedbysixoxygenatomsfrom[LLeu]–,waterligands,andtheirsymmetry‐ relatedcounterparts(Figure2b).Eachcarboxylategroupiscoordinatedtozincinamono‐ dentatemode,μ‐κ1‐O1,κ1‐O3,leadingtoasymmetricC‐Odistances(e.g.,C(5)‐O(1),1.254(3) andC(5)‐O(2),1.237(3)Å).Theasymmetryinthesecondcarboxylategroupislesspro‐ nounced(C(11)‐O(3),1.252(3)andC(11)‐O(4),1.248(3)Å)becausetheO(4)atompartici‐ patesinanintermolecularhydrogenbondwiththewaterligand(O(5)...O(4)#1distanceof 2.683(3)Å).TheotherselectedstructuralparametersaresummarizedinTableS2.The pseudo‐transarrangementofthecarboxylategroupsinthe[LLeu]–ligand(torsionangleCcar‐ boxy–Cchiral–C′chiral–C′carboxyofapproximately106°)facilitatesasquarelatticetopology(sql, FigureS4c),creatinga2Ddistributionoflamellarsheets(FigureS4a,b). Complexes2dand2ecrystallizeinthemonoclinicspacegroupsC2andP21,respec‐ tively.Theasymmetricunitsof2dand2econtainaZn(II)ion,theimidazoliumdicarbox‐ ylateligand([LVal]–for2dand[LIle]–for2e),abidentateaminoacidligand(valinefor2d andisoleucinefor2e),andonewatermolecule(Figure3).Anintramolecularhydrogen bondisobservedwithinthisunitbetweenthecoordinatedaminoacidNH2groupand oneoxygenatomofthecarboxylategroupof[LR]–(N(3)...O(1)distanceof2.962(4)Åfor2d andN(3)...O(3)distanceof2.993(11)Åfor2e).Thegrowthoftheasymmetricunitsforms 1Dpolymericstructures(FigureS5afor2d)inwhichthebridgingligands,[LVal]–and[LIle]– ,coordinatetozincionsinamonodentatemode,μ‐κ1‐O1,κ1‐O3,asoccurredin2a–2c.In bothcomplexes,thezinccenterexhibitsabipyramidaltrigonalgeometryinwhichthe equatorialpositionsareoccupiedbythreeoxygendonoratomsofwater,thecarboxylate Figure 1. 1D coordination polymer of complex 2b aligned along the baxis with ellipsoids of the ORTEP representation displayed with 50% probability. Complex 2c crystallizes in the trigonal space group P3 2 21, forming a two-dimensional (2D) metal–organic framework where zinc ions are interconnected by bridging [L Leu ] − anions through carboxylate functionalities. The asymmetric unit of 2c consists of a Zn 2+ ion, a [L Leu ] − ligand, and one water molecule (Figure 2a). An intramolecular hydrogen bond is observed between the water ligand and one oxygen atom of the carboxylate (O(5) . . . O(2) distance of 2.649(3) Å). The zinc center adopts a distorted octahedral coordination geometry, surrounded by six oxygen atoms from [L Leu ] − , water ligands, and their symmetry-related counterparts (Figure 2b). Each carboxylate group is coordinated to zinc in a monodentate mode, µ - κ1 -O 1 , κ1 -O 3 , leading to asymmetric C-O distances (e.g., C(5)-O(1), 1.254(3) and C(5)-O(2), 1.237(3) Å). The asymmetry in the second carboxylate group is less pronounced (C(11)-O(3), 1.252(3) and C(11)-O(4), 1.248(3) Å) because the O(4) atom participates in an intermolecular hydrogen bond with the water ligand (O(5) . . . O(4)#1 distance of 2.683(3) Å). The other selected structural parameters are summarized in Table S2. The pseudo-trans arrangement of the carboxylate groups in the [L Leu ] − ligand (torsion angle C carboxy –C chiral – C ′chiral –C ′carboxy of approximately 106 ◦ ) facilitates a square lattice topology (sql, Figure S4c), creating a 2D distribution of lamellar sheets (Figure S4a,b). Complexes 2d and 2e crystallize in the monoclinic space groups C 2 and P2 1 , respectively. The asymmetric units of 2d and 2e contain a Zn(II) ion, the imidazolium dicarboxylate ligand ([L Val ] − for 2d and [L Ile ] − for 2e), a bidentate amino acid ligand (valine for 2d and isoleucine for 2e), and one water molecule (Figure 3). An intramolecular hydrogen bond is observed within this unit between the coordinated amino acid NH 2 group and one oxygen atom of the carboxylate group of [L R ] − (N(3) . . . O(1) distance of 2.962(4) Å for 2d and N(3) . . . O(3) distance of 2.993(11) Å for 2e). The growth of the asymmetric units forms 1D polymeric structures (Figure S5a for 2d) in which the bridging ligands, [L Val ] − and [L Ile ] − , coordinate to zinc ions in a monodentate mode, µ - κ1 -O 1 , κ1 -O 3 , as occurred in 2a–2c. In both complexes, the zinc center exhibits a bipyramidal trigonal geometry in which the equatorial positions are occupied by three oxygen donor atoms of water, the carboxylate of isoleucine, and one of the carboxylate groups of the [L Val ] − or [L Ile ] − ligand. In 2d, the asymmetry of the three carboxylate C-O distances is clearly observed, while in 2e, the asymmetry is more pronounced in the isoleucine ligand (C(16)-O(5), 1.239(13)
Int. J. Mol. Sci. 2025,26, 3202 5 of 13 and C(16)-O(6), 1.264(13) Å) compared to the [L Ile ] − ligand. Other selected structural parameters are detailed in Table S2. The 3D packing occurs through intermolecular hydrogen bonds between the water ligand and one oxygen atom of the carboxylate group (for 2d: O(7) . . . O(5)#1 and O(7) . . . O(3)#2 distances of 2.610(4) and 2.727(4) Å, respectively; Figure S5b). Int.J.Mol.Sci.2025,26,xFORPEERREVIEW5of14 ofisoleucine,andoneofthecarboxylategroupsofthe[L Val ] – or[L Ile ] – ligand.In2d,the asymmetryofthethreecarboxylateC‐Odistancesisclearlyobserved,whilein2e,the asymmetryismorepronouncedintheisoleucineligand(C(16)‐O(5),1.239(13)andC(16)‐ O(6),1.264(13)Å)comparedtothe[L Ile ] – ligand.Otherselectedstructuralparametersare detailedinTableS2.The3Dpackingoccursthroughintermolecularhydrogenbondsbe‐ tweenthewaterligandandoneoxygenatomofthecarboxylategroup(for2d: O(7) ... O(5)#1andO(7) ... O(3)#2distancesof2.610(4)and2.727(4)Å,respectively;Figure S5b). (a)(b) Figure2.(a)ORTEPrepresentationoftheasymmetricunitof2c,withellipsoidsdisplayedwith50% probability.(b)Polymeric2cgrowthshowingtheoctahedralcoordinationofzinc. (a)(b) Figure3.ORTEPrepresentationofasymmetricunitsof2d(a)and2e(b),withellipsoidsdisplayed with50%probability. 2.3.EvaluationofAnticancerActivity Thecytotoxicityofcomplexes2wasevaluatedinvitrobydeterminingthehalfinhib‐ itoryconcentrations(IC 50 )againstfourhumancelllines:wild‐typeBRAFmelanoma (MeWo),lungadenocarcinoma(A549),bladdercancer(T24),andnon‐cancerousskin keratinocytes(HaCaT).TheIC 50 valueswerecalculatedfromcellviabilityvaluesobtained after72hofexposureofthedrugtocellsusingtheresazurinassay.Theviabilityresults, Figure 2. (a) ORTEP representation of the asymmetric unit of 2c, with ellipsoids displayed with 50% probability. (b) Polymeric 2c growth showing the octahedral coordination of zinc. Int.J.Mol.Sci.2025,26,xFORPEERREVIEW5of14 ofisoleucine,andoneofthecarboxylategroupsofthe[L Val ] – or[L Ile ] – ligand.In2d,the asymmetryofthethreecarboxylateC‐Odistancesisclearlyobserved,whilein2e,the asymmetryismorepronouncedintheisoleucineligand(C(16)‐O(5),1.239(13)andC(16)‐ O(6),1.264(13)Å)comparedtothe[L Ile ] – ligand.Otherselectedstructuralparametersare detailedinTableS2.The3Dpackingoccursthroughintermolecularhydrogenbondsbe‐ tweenthewaterligandandoneoxygenatomofthecarboxylategroup(for2d: O(7) ... O(5)#1andO(7) ... O(3)#2distancesof2.610(4)and2.727(4)Å,respectively;Figure S5b). (a)(b) Figure2.(a)ORTEPrepresentationoftheasymmetricunitof2c,withellipsoidsdisplayedwith50% probability.(b)Polymeric2cgrowthshowingtheoctahedralcoordinationofzinc. (a)(b) Figure3.ORTEPrepresentationofasymmetricunitsof2d(a)and2e(b),withellipsoidsdisplayed with50%probability. 2.3.EvaluationofAnticancerActivity Thecytotoxicityofcomplexes2wasevaluatedinvitrobydeterminingthehalfinhib‐ itoryconcentrations(IC 50 )againstfourhumancelllines:wild‐typeBRAFmelanoma (MeWo),lungadenocarcinoma(A549),bladdercancer(T24),andnon‐cancerousskin keratinocytes(HaCaT).TheIC 50 valueswerecalculatedfromcellviabilityvaluesobtained after72hofexposureofthedrugtocellsusingtheresazurinassay.Theviabilityresults, Figure 3. ORTEP representation of asymmetric units of 2d (a) and 2e (b), with ellipsoids displayed with 50% probability. 2.3. Evaluation of Anticancer Activity The cytotoxicity of complexes 2was evaluated in vitro by determining the half inhibitory concentrations (IC 50 ) against four human cell lines: wild-type BRAF melanoma (MeWo), lung adenocarcinoma (A549), bladder cancer (T24), and non-cancerous skin keratinocytes (HaCaT). The IC 50 values were calculated from cell viability values obtained after 72 h of exposure of the drug to cells using the resazurin assay. The viability results, summarized in Table 1(as µ M concentrations) and illustrated in Figure 4, also include the values of the selectivity index (SI). The SI was calculated by dividing the IC 50 value in the non-cancerous cells by that in the cancer cells [ 32 ]. The higher the SI value, the
Int. J. Mol. Sci. 2025,26, 3202 6 of 13 higher the selectivity of the compound against cancer cells. This parameter also allows for a comparison of the selectivity of the investigated compound with the selectivity of the standard anticancer drug. For comparison, the cytotoxicity of two clinical anticancer drugs, carboplatin and gemcitabine, as well as imidazolium dicarboxylate precursor ligands (HL R , 1) was evaluated under identical experimental conditions (see viability graphs in Figure 4 and Figure S7). Complexes 2demonstrated cytotoxic activity against the three cancer cell lines, with IC 50 values ranging from 75 to 312 µ M (Table 1), while the HL R precursor ligands, 1, did not show cytotoxicity (IC 50 > 3000 µ M, see Figure S7). Two noteworthy observations can be drawn from Table 1. First, although complexes 2exhibited lower selectivity compared to gemcitabine, they showed greater selectivity than carboplatin, a well-established clinical anticancer drug. Carboplatin is used for the treatment of ovarian cancer, lung cancer, bladder cancer, and head and neck cancer. Second, these zinc-based complexes were more cytotoxic to cancer cells than to non-malignant HaCaT cells, underscoring their potential therapeutic selectivity. Table 1. IC 50 values ( µ M) and selectivity indexes (SIs) of zinc complexes tested against human cell lines a. Complex IC50 (Mean ±SEM; pValue vs. HaCaT) (Selectivity Index, Mean ±SEM) HaCaT (Non-Malignant Keratinocyte) A549 (Lung Adenocarcinoma) MeWo (Melanoma) T24 (Bladder Cancer) 2a 260.2 ±20.0 156.3 ±3.7; 0.0214 (1.7 ±0.2) 126.5 ±1.9; 0.0071 (2.1 ±0.2) 124.4 ±5.5; 0.0101 (2.1 ±0.2) 2b 185.0 ±33.7 131.8 ±11.2; 0.1048 (1.4 ±0.1) 108.7 ±2.2; 0.1139 (1.7 ±0.3) 108.5 ±3.9; 0.1183 (1.7 ±0.3) 2c 231.4 ±7.4 202.4 ±7.6; 0.0965 (1.1 ±0.1) 75.7 ±1.9; 0.0001 (3.1 ±0.0) 83.2 ±3.8; 0.0001 (2.8 ±0.1) 2d 328.9 ±77.8 283.3 ±59.7; 0.1027 (1.1 ±0.1) 227.7 ±66.4; 0.1403 (1.6 ±0.5) 238.9 ±74.5; 0.2019 (1.6 ±0.5) 2e 343.9 ±12.5 311.7 ±3.2; 0.1299 (1.1 ±0.1) 162.3 ±19.3; 0.0032 (2.2 ±0.3) 194.6 ±37.7; 0.0157 (1.9 ±0.3) Carboplatin 35.9 ±3.8 42.9 ±1.4; 0.2188 (0.8 ±0.1) 100.4 ±17.4; 0.0182 (0.4 ±0.0) 22.1 ±2.6; 0.0130 (1.7 ±0.2) Gemcitabine (nM) 22.1 ±3.1 5.6 ±0.1; 0.0130 (4.0 ±0.5) 7.3 ±0.7; 0.0270 (3.3 ±0.8) 2.3 ±0.3; 0.0062 (9.3 ±0.4) a After 72 h of treatment, cell viability was measured using the resazurin assay. IC 50 values are given as the mean value obtained from at least three independent experiments ± standard error of the mean (SEM) (see Section 3for more details). The selectivity index (SI) values were calculated as the average of the IC 50 values in the HaCaT noncancerous cell line divided by the IC 50 value in the cancer cell line obtained in each independent experiment. For the calculation of the µ M concentration, the molecular weight of the monomer of the corresponding coordination polymer was used. For lung adenocarcinoma (A549), 2a demonstrated moderate activity with some selectivity, while the remaining complexes showed limited efficacy. The IC 50 values for 2a–2c are comparable to those reported for a telmisartan zinc derivative that contains only O-donor ligands (IC 50 75 µ M) [ 33 ]. In particular, other zinc complexes with Nor S-donor ligands show lower IC 50 values ≤ 10 µ M) [ 7 ], suggesting a ligand-dependent effect [ 34 – 36 ]. In the case of T24 bladder cancer cells, complexes 2a–2c exhibited IC 50 values between 83 and 124 µ M (Table 1), with 2c showing the highest selectivity (close to 3). These results are consistent with related zinc complexes [ 37 ]. A particularly strong IC 50 for T24 cells was
Int. J. Mol. Sci. 2025,26, 3202 7 of 13 previously reported for an oxoaporphine–zinc complex, although the free ligand itself also showed significant cytotoxicity (IC 50 9.12 µ M) [ 38 ]. In melanoma cells (MeWo), complexes 2a–2c achieved the best IC 50 values (~75 µ M for 2c, Table 1), but no comparable studies were identified in the literature involving this melanoma subtype [ 6 , 7 ]. Importantly, the selectivity of 2c against this cancer was high, surpassing carboplatin and approaching that of gemcitabine (see Figure S8). Interestingly, the viability of melanoma cells exposed to 145 µ M of 2c was reduced to less than 3%, while the viability of non-malignant cells remained high (~85%). On the contrary, even at the highest concentrations of gemcitabine tested, the cell viability of melanoma cells did not drop below 35%, while the viability of non-malignant cells decreased to 13% (Figure 4). The reduced anticancer activity of the 2d–2e complexes, compared to 2a–2c, can be attributed to the bidentate coordination of the amino acid ligand. Zinc release through dissociation would be more hindered due to the chelate effect than observed in complexes 2a–2c, which have only monodentate ligands. Previous studies suggested that cytotoxicity in zinc complexes, with a reduction in levels of the mutp53 protein, was associated with the release of zinc ions by dissociation [ 39 ]. The presence of chelating amino acid ligands in 2d–2e hinders this dissociation, thus reducing its efficacy. Our results on the anticancer activity of these compounds are preliminary and require in vivo studies to confirm their anticancer effects and reveal potential toxicities in normal cell types beyond keratinocytes. Nonetheless, the selectivity indices observed in our panel of cell lines are higher than those of several commonly used anticancer drugs (see Table S3), supporting the relevance of our in vitro findings. Int.J.Mol.Sci.2025,26,xFORPEERREVIEW7of14 InthecaseofT24bladdercancercells,complexes2a–2cexhibitedIC 50 valuesbetween83 and124μM(Table1),with2cshowingthehighestselectivity(closeto3).Theseresults areconsistentwithrelatedzinccomplexes[37].AparticularlystrongIC 50 forT24cellswas previouslyreportedforanoxoaporphine–zinccomplex,althoughthefreeliganditself alsoshowedsignificantcytotoxicity(IC 50 9.12μM)[38].Inmelanomacells(MeWo),com‐ plexes2a–2cachievedthebestIC 50 values(~75μMfor2c,Table1),butnocomparable studieswereidentifiedintheliteratureinvolvingthismelanomasubtype[6,7].Im‐ portantly,theselectivityof2cagainstthiscancerwashigh,surpassingcarboplatinand approachingthatofgemcitabine(seeFigureS8).Interestingly,theviabilityofmelanoma cellsexposedto145μMof2cwasreducedtolessthan3%,whiletheviabilityofnon‐ malignantcellsremainedhigh(~85%).Onthecontrary,evenatthehighestconcentrations ofgemcitabinetested,thecellviabilityofmelanomacellsdidnotdropbelow35%,while theviabilityofnon‐malignantcellsdecreasedto13%(Figure4).Thereducedanticancer activityofthe2d–2ecomplexes,comparedto2a–2c,canbeattributedtothebidentate coordinationoftheaminoacidligand.Zincreleasethroughdissociationwouldbemore hinderedduetothechelateeffectthanobservedincomplexes2a–2c,whichhaveonly monodentateligands.Previousstudiessuggestedthatcytotoxicityinzinccomplexes, withareductioninlevelsofthemutp53protein,wasassociatedwiththereleaseofzinc ionsbydissociation[39].Thepresenceofchelatingaminoacidligandsin2d–2ehinders thisdissociation,thusreducingitsefficacy.Ourresultsontheanticanceractivityofthese compoundsarepreliminaryandrequireinvivostudiestoconfirmtheiranticancereffects andrevealpotentialtoxicitiesinnormalcelltypesbeyondkeratinocytes.Nonetheless,the selectivityindicesobservedinourpanelofcelllinesarehigherthanthoseofseveralcom‐ monlyusedanticancerdrugs(seeTableS3),supportingtherelevanceofourinvitrofind‐ ings. Figure 4. Effect of complexes 2, carboplatin and gemcitabine, on the viability of human non-malignant cells (HaCaT) and human cancer cells (A549, MeWo, and T24). The cells were exposed to reagents for 72 h, and cell viability was measured using the resazurin assay. Data represent mean ± SEM from at least three independent experiments. For statistical analysis, the t-test (paired, two-tailed) was used. *p< 0.05, ** p< 0.01, and *** p<0.001 (similar representations for marks + or #).
Int. J. Mol. Sci. 2025,26, 3202 8 of 13 3. Materials and Methods 3.1. General All synthetic preparations and other operations were carried out under aerobic conditions. Solvents were purified and dried appropriately prior to use, using standard procedures. Cell culture reagents were purchased from Biowest (Nuaillé, France). Resazurin was purchased from Sigma, gemcitabine was obtained from Pfizer, and carboplatin was obtained from Teva. Other chemicals were obtained from commercial sources and used as supplied. Infrared spectra were recorded on a PerkinElmer FT-IR Spectrum Two spectrophotometer (Waltham, MA, USA) using the ATR technique. NMR spectra were recorded on Bruker AMX-300 or Avance III spectrometers (Billerica, MA, USA) at the Centro de Investigaciones, Tecnología e Innovación (CITIUS) of the University of Sevilla, with 1 H and 13 C{ 1 H} NMR shifts referenced to residual signals from deuterated solvents. All data are reported in ppm downfield from Si(CH 3 ) 4 . Elemental analyses (C, H, N) were conducted by the CITIUS of the University of Sevilla on an Elemental LECO CHNS 93 analyzer (LECO Corporation, St. Joseph, MI, USA). Bis-imidazolium precursors were prepared according to the literature experimental methods [ 28 , 29 , 40 ]. Complex 2a was prepared with slight differences from the procedure reported [30]. 3.2. Synthesis 3.2.1. Complexes [Zn(LR)2]n,2a and 2b A solution of Zn(AcO) 2· 2H 2 O (0.110 g, 0.5 mmol) in 10 mL of ethanol was added to HL Gly (0.184 g, 1 mmol) dissolved in the smallest amount of a 3:1 ethanol–water mixture. The resulting solution was stirred for 1 h at 70 ◦ C. The solvent was then removed under reduced pressure and an oil was obtained, which was recrystallized in a 1:1 dimethylformamide–water mixture. The compound [Zn(L Gly ) 2 ] n ,2a, was obtained as an off-white solid (0.59 g, 47 %). IR (ATR, cm−1): 3140 (w), 3081 (w), 3003 (w), 2876 (w), 2168 (w), 1652 (vs, νas COO), 1562 (s), 1433 (m), 1416 (m), 1379 (vs), 1362 (vs), 1350 (vs, νs COO), 1323 (m), 1290 (vs), 1207 (w), 1188 (m), 1167 (s), 1109 (m), 1035 (m), 976 (w), 870 (m), 802 (s), 674 (vs), 636 (s), 585 (vs), 496 (s), 442 (m), 431 (m). 1 H NMR (300 MHz, D 2 O): 8.68 (br t, 2H, CH im ), 7.38 (d, 3 J HH = 2 Hz, 4H, CH im ), 4.77 (s, 8H, CH 2 COO). 13 C{ 1 H} NMR (D 2 O, 75 MHz): 172.3 (COO), 137.2 (N-CH im -N), 123.2 (CH im ), 51.9 (CH 2 COO). Elemental Anal. Calc. for C 17 H 21 N 5 O 9 Zn (2a + DMF): C, 40.45; H, 4.19; N, 13.87. Found: C, 39.68; H, 3.48; N, 13.52%. Complex [Zn(L βAla ) 2 ] n ,2b, was prepared following the same method, using HL βAla (212 mg, 1 mmol) and Zn(AcO) 2· 2H 2 O (110 mg, 0.5 mmol); 2b was obtained as a pale white solid (0.096 g, 39 %). IR (ATR, cm −1 ): 3383 (m), 3142 (m), 3108 (m), 3007 (w), 2965 (w), 1604 (vs, νas COO), 1569 (vs), 1559 (vs), 1450 (m), 1395 (vs), 1372 (vs, νs COO), 1325 (s), 1300 (s), 1244 (m), 1229 (m), 1182 (s), 1150 (vs), 1100 (m), 1066 (m), 1023 (w), 988 (w), 943 (m), 864 (m), 825 (m), 775 (m), 755 (m), 650 (s), 634 (s), 604 (s), 559 (m), 532 (m), 519 (m), 434 (w), 418 (w). 1 H NMR (300 MHz, D 2 O): 8.70 (br t, 2H, CH im ), 7.44 (d, 3 J HH = 2 Hz, 4H, CH im ), 4.36 (t, 3 J HH = 7 Hz, 8H, CH 2 CH 2 COO), 2.72 (t, 3 J HH = 7 Hz, 8H, CH 2 CH 2 COO). 13 C{ 1 H} NMR (D 2 O, 75 MHz): 178.1 (COO), 136.0 (N-CH im -N), 122.2 (CH im ), 46.7 (CH 2 CH 2 COO), 37.1 (CH 2 CH 2 COO). Elemental Anal. Calc. for C 18 H 22 N 4 O 8 Zn (2b): C, 44.32; H, 4.55; N, 11.49. Found: C, 44.17; H, 4.65; N, 11.31%. 3.2.2. Complex [Zn(LLeu)2(H2O)2]n,2c The experimental method was similar to 2a,b, using HL Leu (296 mg, 1 mmol) and Zn(AcO) 2· 2H 2 O (109 mg, 0.5 mmol). From crystallization in a DMF:H 2 O 1:1 mixture 2c was obtained as a pale white solid with the formula [Zn(L Leu ) 2 (H 2 O) 2 ] n (0.165 g, 47 %). IR (ATR, cm −1 ): 3143 (w), 2956 (m), 2871 (w), 1621 (vs, νas COO), 1467 (w), 1435 (w), 1372 (vs,
Int. J. Mol. Sci. 2025,26, 3202 9 of 13 νs COO), 1284 (w), 1234 (w), 1156 (m), 900 (w), 827 (w), 738 (w), 698 (m), 651 (m), 618 (w), 501 (ws), 418 (w). 1 H NMR (300 MHz, D 2 O): 8.93 (br t, 1H, CH im ), 7.50 (d, 3 J HH = 1.3 Hz, 2H, CH im ), 4.86 (dd, 3 J HH = 10.2, 5.5 Hz, 2H, CHCOO), 1.95 (m, 4H, (CH 3 ) 2 CHCH 2 ), 1.24 (hp, 3 J HH = 6.6 Hz, 2H, (CH 3 ) 2 CHCH 2 ), 0.82 (d, 3 J HH = 6.6 Hz, 3H, (CH 3 ) 2 CHCH 2 ), 0.81 (d, 3 J HH = 6.6 Hz, 3H, (CH 3 ) 2 CHCH 2 CH). 13 C{ 1 H} NMR (D 2 O, 75 MHz): 174.9 (COO), 135.4 (N-CH im -N), 121.7 (CH im ), 63.57 (CHCOO), 40.67 ((CH 3 ) 2 CHCH 2 ), 24.5 ((CH 3 ) 2 CHCH 2 ), 21.99 ((CH 3 ) 2 CHCH 2 ), 20.29 ((CH 3 ) 2 CHCH 2 ). Elemental Anal. Calc. for C 30 H 50 N 4 O 10 Zn (2c): C, 52.06; H, 7.28; N, 8.10. Found: C, 54.94; H, 7.29; N, 8.05%. 3.2.3. Complexes [Zn(LR)(aa)(H2O)]n,2d and 2e [Zn(LVal)(Val)(H2O)]n,2d. Following the same method as in 2a, using the precursor HL Val (134 mg, 0.5 mmol) and Zn(AcO) 2· 2H 2 O (110 mg, 0.5 mmol) in the presence of L-valine (58 mg, 0.5 mmol). Complex 2d was obtained as a pale white solid after crystallization from a DMF:H 2 O 3:1 mixture. Yield: 0.135 g (56%). IR (ATR, cm −1 ): 3296 (w), 3260 (w), 3138 (w), 2970 (w), 2873 (w), 1607 (vs, νas COO), 1586 (vs), 1466 (m), 1414 (m), 1375 (vs, νs COO), 1330 (m), 1306 (m), 1263 (w), 1236 (m), 1181 (w), 1156 (m), 1121 (w), 1095 (m), 1064 (m), 1015 (w), 987 (w), 950 (w), 916 (w), 863 (w), 847 (w), 816 (w), 789 (w), 773 (m), 749 (s), 730 (m), 703 (s), 665 (m), 635 (s), 614 (s), 585 (m), 543 (w), 502 (m), 453 (w), 423 (w), 409 (w). 1 H NMR ( 300 MHz , D 2 O): 8.90 (t, 1H, 3 J HH = 1.8 Hz, CH im ), 7.52 (d, 3 J HH = 1.8 Hz, 2H, CH im ), 4.53 (d, 3JHH = 8.1 Hz , 2H, NCHCOO), 3.40 (br d, 1H, 3 J HH = 3 Hz, NCHCOO Val), 2.39 (hp, 2H, 3 J HH = 6.9 Hz, CH(CH 3 ) 2 ), 2.26 (m, 1H, CH(CH 3 ) 2 Val), 0.96–0.90 (m, 9H, CH(CH 3 ) 2 ), 0.84–0.79 (m, 9H, CH(CH 3 ) 2 ). 13 C{ 1 H} NMR (D 2 O, 75 MHz): 173.8 (COO), 135.7 (N-CH im - N), 122.0 (CH im ), 71.5 (NCHCOO), 59.5 (NCHCOO Val), 31.1 (CH(CH 3 )), 29.5 (CH(CH 3 ) Val), 18.6 (CH(CH 3 )), 18.4 (CH(CH 3 ) Val), 17.5 (CH(CH 3 )), 15.8 (CH(CH 3 ) Val). Elemental Anal. Calc. for C 18 H 31 N 3 O 7 Zn (2d): C, 46.31; H, 6.69; N, 9.00. Found: C, 46.70; H, 6.53; N, 8.81%. [Zn(LIle)(Ile)(H2O)]n,2e. Following the same method as in 2a, using the HL Ile precursor (149 mg, 0.5 mmol) and Zn(AcO) 2· 2H 2 O (110 mg, 0.5 mmol) in the presence of L-isoleucine (66 mg, 0.5 mmol). Complex 2e was obtained as a pale white solid after crystallization from a DMF:H 2 O 3:1 mixture. Yield: 0.11 g (42%). IR (ATR, cm −1 ): 3136 (w), 2968 (m), 2933 (w), 2881 (w), 1631 (vs, νas COO), 1615 (vs), 1584 (vs), 1512 (s), 1460 (m), 1421 (w), 1363 (vs, νs COO), 1329 (s), 1307 (m), 1256 (m), 1234 (w), 1155 (m), 1099 (w), 1062 (w), 1026 (w), 961 (w), 919 (w), 870 (w), 840 (w), 746 (s), 709 (m), 649 (m), 615 (m), 559 (s), 537 (s), 455 (s). 1 H NMR (300 MHz, D 2 O): 8.89 (t, 3 J HH = 2 Hz, 1H, CH im ), 7.51 (d, 3 J HH = 2 Hz, 2H, CH im ), 4.56 (d, 3 J HH = 8.1 Hz, 2H, NCHCOO), 3.42 (br, 1H, NCHCOO Ile), 2.17 (m, 2H, CH 3 CHCH 2 CH 3 ), 1.95 (m, 1H, CH 3 CHCH 2 CH 3 Ile), 1.25–1.11 (m, 6H, CH 3 CHCH 2 CH 3 ), 0.97-0.88 (m, 9H, CH 3 CHCH 2 CH 3 ), 0.82-0.76 (m, 9H, CH 3 CHCH 2 CH 3 ). 13 C{ 1 H}-NMR (D 2 O, 75 MHz): 173.9 (COO), 135.6 (N-CH im -N), 122.0 (CH im ), 70.5 (NCHCOO), 59.1 (NCHCOO Ile), 36.9 (CH 3 CHCH 2 CH 3 ), 36.5 (CH 3 CHCH 2 CH 3 Ile), 24.6 (CH 3 CHCH 2 CH 3 ), 23.8 (CH 3 CHCH 2 CH 3 Ile), 15.2 (CH 3 CHCH 2 CH 3 Ile), 15.1 (CH 3 CHCH 2 CH 3 ), 11.2 (CH 3 CHCH 2 CH 3 Ile), 10.3 (CH 3 CHCH 2 CH 3 ). Elemental Anal. Calc. for C 21 H 34 N 3 O 7 Zn (2e): C, 49.86; H, 6.77; N, 8.31. Found: C, 51.23; H, 7.50; N, 8.46%. 3.3. Cell Lines and Cell Viability Assays HaCaT cells (human keratinocytes) [ 24 ] A549 (human lung adenocarcinoma), MeWo (human melanoma), and T24 (human urinary bladder carcinoma) were purchased from Cell Lines Service (CLS). The cells were maintained in Dulbecco’s Modified Eagle Medium