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inorganics Review Review

Anticancer Anticancer Applications Applications and and Recent Recent Investigations Investigations of of Metallodrugs Based on Gallium, Tin and Titanium Metallodrugs Based on Gallium, Tin and Titanium 1,2 Sanjiv Prashar 11 and Santiago Gómez-Ruiz1,1, * Younes Younes Ellahioui Ellahioui 1,2,, Sanjiv Prashar and Santiago Gómez-Ruiz * 1

Departamento de Biología y Geología, Física y Química Inorgánica, Universidad Rey Juan Carlos, Departamento de Biología y Geología, Física y Química Inorgánica, Universidad Rey Juan Carlos, Calle Calle Tulipán, s/n, E-28933 Móstoles (Madrid), Spain; [email protected] (Y.E.); Tulipán, s/n, E-28933 Móstoles (Madrid), Spain; [email protected] (Y.E.); [email protected] (S.P.) [email protected] (S.P.) 2 Faculté des Sciences, Département de Chimie, Université Abdelmalek Essâadi, 93030 Tétouan, Morocco 2 Faculté des Sciences, Département de Chimie, Université Abdelmalek Essâadi, 93030 Tétouan, Morocco * Correspondence: [email protected]; Tel.: +34-914-888-507 * Correspondence: [email protected]; Tel.: +34-914-888-507 Academic Editor: Luigi Messori Academic Editor: Luigi Messori Received: 5 December 2016; Accepted: 5 January 2017; Published: date Received: 5 December 2016; Accepted: 5 January 2017; Published: 12 January 2017 1

metal complexes have been extensively usedused in therapy and since Formore morethan than100 100years years metal complexes have been extensively in therapy and Abstract: For the discovery of cisplatin the research in this field has expanded exponentially. The scientific since the discovery of cisplatin the research in this field has expanded exponentially. The community is always in search of new alternatives to platinum compounds and a wide variety of metallodrugs based based on other metals have been reported with excellent therapeutic results. results. This short metallodrugs review focuses on the work that our research group has carried out since 2007 in collaboration with others and centers on the preparation of organogallium(III) compounds, organotin(IV) derivatives, derivatives, and titanocene(IV) complexes together with the study study of of their their cytotoxic cytotoxic anticancer anticancer properties. properties. nanostructures Keywords: metallodrugs; cisplatin; gallium; tin; titanium; cancer; nanostructures

1. Introduction Metals have been used in medicinal applications for more than 500 years [1]. For example, the Egyptians medicines in Arabia Arabia and and China, China, Egyptians used copper to sterilize water, water, gold gold was used in a variety of medicines and various iron remedies were used in Egypt around 1500 BC. At about the same time zinc was discovered to promote the healing of wounds. In the Renaissance era, mercury chloride was used as a diuretic the the nutritional essentiality of ironof wasiron discovered. However, in the last 100 the diureticand and nutritional essentiality was discovered. However, in years, the last medicinal of inorganic compounds has compounds been developed a rational manner. in the early 100 years, activity the medicinal activity of inorganic has in been developed in a Thus, rational manner. 1900s was used for treating and various Sb compounds forSb leishmaniasis. Thus, K[Au(CN) in the early2 ]1900s K[Au(CN) 2] wastuberculosis used for treating tuberculosis and various compounds In the antibacterial of various gold salts and arsenic compounds were used for foraddition, leishmaniasis. In addition, activity the antibacterial activity of various gold salts and arsenic compounds treating various diseases [2]. diseases [2]. were used for treating various In the century, a very important therapeutic activity activity of metal complexes was discovered, thetwentieth twentieth century, a very important therapeutic of metal complexes was namely their application for the treatment of cancer.ofRosenberg’s serendipitous discovery of the discovered, namely their application for the treatment cancer. Rosenberg’s serendipitous discovery anti-cancer action ofaction cisplatin 1) Figure in the 1960s a widespreada of the anti-cancer of (cis-[Pt(NH cisplatin (cis-[Pt(NH 3)2Cl2], 1) in precipitated the 1960s precipitated 3 )2 Cl2 ], Figure search for related similarwith or better activity [3].activity The observation of the cellofdivision widespread searchcomplexes for related with complexes similar or better [3]. The observation the cell suppression by this compound was crucial for its development. division suppression by this compound was crucial for its development.

Figure 1. 1. Structure of cisplatin. cisplatin. Figure Structure of

Until the turn of the century, cisplatin had been the most used drug in the world in therapy of Until the turn of the century, cisplatin had been the most used drug in the world in therapy of cancer, administered alone or combined with other compounds. Researchers still had the expectation cancer, administered alone or combined with other compounds. Researchers still had the expectation to to develop alternative drugs to improve the potential and the effectiveness against cancer, and develop alternative drugs to improve the potential and the effectiveness against cancer, and especially Inorganics Inorganics 2017, 2017, 5, 5, 4; 4; doi:10.3390/inorganics5010004 doi:10.3390/inorganics5010004

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especially to overcome the effects cisplatin, such neurotoxicity, to overcome undesirable effects of cisplatin, as nephrotoxicity, neurotoxicity, ototoxicity, especially to the overcome the undesirable undesirable effects of ofsuch cisplatin, such as as nephrotoxicity, nephrotoxicity, neurotoxicity, ototoxicity, nausea and vomiting [4]. nausea and vomiting [4]. ototoxicity, nausea and vomiting [4]. In In this this context, an extensive study of other metal complexes with similar anti-cancer action was was In this context, context, an an extensive extensive study study of of other other metal metal complexes complexes with with similar similar anti-cancer anti-cancer action action was carried out by the scientific community. Thus, the first non-platinum complex to enter clinical trials carried out by the scientific community. Thus, the first non-platinum complex to enter clinical trials carried out by the scientific community. Thus, the first non-platinum complex to enter clinical trials was budotitane although its applications were limited due to its low solubility and liver toxicity [5]. was was budotitane budotitane although although its its applications applications were were limited limited due due to to its its low low solubility solubility and and liver liver toxicity toxicity [5]. [5]. The cytotoxicity of cisplatin originates from its binding to DNA and the formation of covalent The cytotoxicity of cisplatin originates from its binding to DNA and the formation of covalent cross-links. cross-links. The The 1,2-intrastrand 1,2-intrastrand d(GpG) d(GpG) cross-link cross-link is is the the major major adduct. adduct. Binding Binding of of cisplatin cisplatin to to DNA DNA 1,2-intrastrand d(GpG) cross-link is Binding causes significant distortion of the helical structure and results in inhibition of DNA replication causes significant distortion of the helical structure and results in inhibition of DNA replication and and 2+ unit covalently binds to deoxyribonucleic acid (DNA), transcription (Figure Pt transcription [6].[6]. The The Pt2+ unit binds tobinds deoxyribonucleic acid (DNA),acid particularly transcription (Figure (Figure2) 2) 2) [6]. The Pt2+ covalently unit covalently to deoxyribonucleic (DNA), particularly the of (G) adenine in AG to the N7 ofto guanine (G)guanine or adenine in the (A) nucleotide sequencessequences GG and GG AG to form particularly toeither the N7 N7 of either either guanine (G) or or(A) adenine (A) in the the nucleotide nucleotide sequences GG and and AG to form interstrand cross-links [7] The so-formed cisplatin-DNA unit activates a new cellular pathway interstrand cross-links [7] The so-formed cisplatin-DNA unit activates a new cellular pathway which to form interstrand cross-links [7] The so-formed cisplatin-DNA unit activates a new cellular pathway which to inhibition, cell-cycle arrest, DNA and finally [8]. leads transcription inhibition, cell-cycle arrest, DNA and finally [8]. whichtoleads leads to transcription transcription inhibition, cell-cycle arrest,repair, DNA repair, repair, and apoptosis finally apoptosis apoptosis [8].

Figure Figure 2. 2. DNA DNA adduct adduct formation formation with with cisplatin cisplatin moiety. Figure 2. DNA adduct formation with cisplatin moiety.

Immediately after the the initial initial elucidation elucidation of of the Immediately after the cell cell death death mechanism mechanism of of cisplatin, cisplatin, other other platinum platinum analogues such as carboplatin [9] and oxaliplatin [10] (Figure 3) were synthesized and approved by analogues (Figure 3) 3) were synthesized andand approved by the analogues such such as ascarboplatin carboplatin[9] [9]and andoxaliplatin oxaliplatin[10] [10] (Figure were synthesized approved by the for as In some other such FDA for use anticancer drugs. drugs. In addition, some other compounds such as nedaplatin, lobaplatin, the FDA FDA forasuse use as anticancer anticancer drugs. In addition, addition, some other compounds compounds such as as nedaplatin, nedaplatin, lobaplatin, and (Figure 3) in trial heptaplatin, and satraplatin (Figure 3) are currently incurrently clinical trial phase [11]. lobaplatin, heptaplatin, heptaplatin, and satraplatin satraplatin (Figure 3) are are currently in clinical clinical trial phase phase [11]. [11]. H3 N H3 N H3 N H3 N

Cl Cl

Pt Pt

Cl Cl

NH2 NH2

Pt Pt

NH2 NH2

O O O O

Lobaplatin Lobaplatin

O O

NH2 NH2 NH2 NH2

O O

O O

NH2 NH2

Pt Pt

O O

O O

O O

O O

Heptaplatin Heptaplatin

Pt Pt

O O O O

O O

O O

O O

O O Cl Cl Cl Cl O O

O NH O NH33 Pt Pt NH3 O NH3 O Nedaplatin Nedaplatin

Oxaplatin Oxaplatin

Carboplatin Carboplatin

Cisplatin Cisplatin

NH2 NH2

O H3 N H3N Pt O H3N Pt O O H3N

O O NH3 NH3 Pt Pt NH NH22 O O Satraplatin Satraplatin

Figure 3. Anticancer platinum metallodrugs (FDA-approved and investigated in clinical trials). Figure and investigated investigated in in clinical clinical trials). trials). Figure 3. 3. Anticancer Anticancer platinum platinum metallodrugs metallodrugs (FDA-approved (FDA-approved and

One One of of the the main main disadvantages disadvantages of of cisplatin cisplatin is is that, that, in in many many cases, cases, cancer cancer cells cells acquire acquire resistance resistance One of the main disadvantages of cisplatin is that, inTo many cases,this, cancer cells acquire resistance to this drug, deactivating its effect against damaged cells. overcome cisplatin to this drug, deactivating its effect against damaged cells. To overcome this, cisplatin can can be be combined combined to this drug, deactivating its effect against damaged cells. To overcome this, cisplatin can be combined with with other other chemotherapeutics chemotherapeutics agents agents like like 5-fluorouracil, 5-fluorouracil, for for example example [12]. [12]. with Thus, other cisplatin chemotherapeutics agents like 5-fluorouracil, for example [12]. Thus, cisplatin and and its its derivatives derivatives have have been been used used for for many many years years as as chemotherapy chemotherapy agents agents in in the the treatment of cancer with excellent results against a wide variety of cell lines and tumors. However, treatment of cancer with excellent results against a wide variety of cell lines and tumors. However, because because of of the the induction induction of of drug-resistance drug-resistance of of the the tumors tumors after after treatment treatment with with cisplatin, cisplatin, the the sideside-

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Thus, cisplatin and its derivatives have been used for many years as chemotherapy agents in the treatment of cancer with excellent results against a wide variety of cell lines and tumors. However, because of the induction of drug-resistance of the tumors after treatment with cisplatin, the side-effects, the intrinsic toxicity of platinum, the limited bioavailability, and the solubility in physiological media of cisplatin-like compounds, it is of upmost importance to find alternative agents based on non-platinum metals-based systems with fewer side effects and improved cytotoxic and anticancer properties. Thus, a wide variety of preclinical and clinical studies using anticancer metallodrugs have been reported using different elements such as gallium, titanium, palladium, gold, cobalt, ruthenium, and tin. In this review, we describe the synthetic methods and preclinical studies in anticancer tests that our research group has carried out in the search for alternatives to cisplatin-like materials. As our work has mainly been based on the use of gallium, tin, and titanium compounds, we have divided this manuscript into three main parts, which cover specifically these metal complexes. In addition, a short section describes the latest results from our group using metallodrugs of other elements. 2. Gallium-Based Metallodrugs Among the p-block metals, gallium has shown some clinical activity in the treatment of soft tissue tumors. Gallium(III) complexes present a special activity in anticancer therapy due to the analogy of the Ga(III) ion with the Fe(III) ion in ionic radius, electron affinity, electronegativity, coordination geometry, and Lewis base affinity [13,14]. These similarities suggest that the Ga(III) ion may follow an analogous biochemical pathway to that observed in iron metabolism. Gallium(III) is stable under biological conditions, while the oxidation state 2+ in gallium is energetically unfavorable and too reactive under physiological conditions to be stable. Hence, redox chemistry is therefore not possible for Ga(III) in biological media. This phenomenon enables the utilization of gallium(III) as a potential therapeutic agent and facilitates its study in biological conditions [15]. The literature has described some interesting results of gallium(III) compounds in phase II clinical trials in the treatment of lymphomas and bladder carcinoma. In addition, the combination with other agents in the treatment of metastatic carcinoma of the urethelium and cisplatin-resistant ovarian cancer has delivered promising results [16]. The mechanism of action of gallium(III) complexes in anticancer chemotherapy has been briefly studied. Ga3+ ions usually compete with Fe3+ for binding to transferrin to reach the intracellular medium. In this way, a cellular uptake of large amounts of gallium is achieved [17]. Analyzing all the biochemical pathways of the gallium(III) ion, it seems clear that the enzyme ribonucleotide reductase is its biological target [18]. Furthermore, the induction of apoptosis through activation of the proapoptotic factor BAX and caspase-3 can be considered as a possible mechanism of cell death. However, proteasome inhibition should not be ruled out [19]. The simplest and most familiar gallium compound used as an anticancer drug is gallium nitrate. However, this compound is readily hydrolyzed in biological medium to give non-soluble gallium oxides which are able to block the absorption and membrane permeation of the gallium ion reducing its effectivity in cancer treatments. Other gallium(III) compounds containing caboxylato, thiolato, and alkoxo ligands have been tested as anticancer agents. In general, the cellular acquisition of gallium mainly occurs by transferrin-mediated uptake followed by accumulation in endosomes. After transport into the cytosol, gallium(III) binds to and inhibits the functioning of ribonucleotide reductase (RR, an enzyme recognized as the most significant intracellular target for antiproliferative activity of gallium). DNA replication activates cell cycle arrest and results finally in apoptosis through the mitochondrial pathway, from which gallium is liberated during, or before, passage across the intestinal epithelium to become in part bound to transferrin in blood (Figure 4) [20].

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Figure 4. Schematic representationofofthe themode modeof ofaction action of of gallium. gallium. Abbreviations: Figure 4. Schematic representation Abbreviations:TfTf= =transferrin; transferrin; NDP = nucleoside diphosphate; dNDP = desoxynucleoside diphosphate; =Tfa =proapoptotic NDP = nucleoside diphosphate; dNDP desoxynucleoside BAX BAX = a proapoptotic protein Figure 4. Schematic representation of =the mode of action ofdiphosphate; gallium. Abbreviations: transferrin; protein (Adapted from Ref.permission 20 with permission The Royaldiphosphate; Society of Chemistry). NDP = from nucleoside diphosphate; dNDP = desoxynucleoside BAX = a proapoptotic (Adapted Ref. 20 with from Thefrom Royal Society of Chemistry). protein (Adapted from Ref. 20 with permission from The Royal Society of Chemistry).

The metallodrug, KP46 (tris(8-hydroxyquinolinato)gallium(III)) (Figure 5), contains the metal The metallodrug, KP46 (tris(8-hydroxyquinolinato)gallium(III)) (Figure[21]. 5), contains thewellmetal chelating agent 8-hydroxyquinoline, which itself has anticancer properties Due tothe its The metallodrug, KP46 (tris(8-hydroxyquinolinato)gallium(III)) (Figure 5), contains metal chelating agent 8-hydroxyquinoline, which itself has anticancer properties [21]. Due to its well-defined defined toxicological and pharmacokinetic advantages, KP46 not only enables higher well chelating agent 8-hydroxyquinoline, which itself has anticancer properties [21]. Due to and its welltoxicological and gallium pharmacokinetic advantages, KP46 notbut only enables higher and well tolerable tolerable tissue concentrations to be established, also inhibitory effects on cell growth defined toxicological and pharmacokinetic advantages, KP46 not only enables higher and well tissue gallium concentrations be established, but also inhibitory effects on cell growth proliferation proliferation in gallium vitro and intovivo superior gallium salts IC50 values typically in the low tolerable tissue concentrations to betoestablished, but(with also inhibitory effects on cell growth in vitro and in vivo superior to gallium salts (with IC values typically in the low micromolar range). micromolar range). In addition, an oral formulation of KP46 (IT-235 from the companies Niikipharma 50 salts (with IC50 values typically in the low proliferation in vitro and in vivo superior to gallium In micromolar addition, an oral formulation KP46 (IT-235 from the(IT-235 companies Niikipharma and aIntezyne and Intezyne Technologies) showed aformulation novel pattern of cytotoxicity with synergism broad range). In addition, anoforal of KP46 from the companiesacross Niikipharma range of antitumor agents targeting the endoplasmic reticulum in multiple tumor types [22]. Technologies) showed a novel pattern of cytotoxicity with synergism across a broad range of antitumor and Intezyne Technologies) showed a novel pattern of cytotoxicity with synergism across a broad agents the endoplasmic reticulum in multiple tumor types [22]. tumor types [22]. rangetargeting of antitumor agents targeting the endoplasmic reticulum in multiple N O ON N

N Ga Ga O

O O N N

O KP46

KP46 and used in clinical trials. Figure 5. KP46 which was formulated

Figure5.5.KP46 KP46which which was was formulated formulated and Figure andused usedininclinical clinicaltrials. trials.

Similar gallium(III) complexes to KP46, including the ligand 7-chloroquinoline, were synthesized other groups and showed not only a very high activity in vitro butwere also Similar by gallium(III) complexes to KP46, including the cytotoxic ligand 7-chloroquinoline, Similar gallium(III) complexes to KP46, including the ligand 7-chloroquinoline, were synthesized antimalarial properties [23]. Bearing in mind the promising properties of gallium compounds our synthesized by other groups and showed not only a very high cytotoxic activity in vitro but also byantimalarial other groups and showed not only a very high cytotoxic activity in vitro but also antimalarial group embarked on the[23]. preparation gallium compounds withofdifferent Thus, as properties Bearing of in several mind the promising properties gallium ligands. compounds our properties [23]. Bearing in mind the promising properties of gallium compounds our group embarked the literature had shown an interesting and synergistic relation between gallium complexes and group embarked on the preparation of several gallium compounds with different ligands. Thus, as aminoacid derivatives such asinteresting glycine and DL-alanine cancer cell death, Thus, we decided toliterature synthesize onthe the literature preparation several compounds withindifferent ligands. as the had hadofshown angallium and synergistic relation between gallium complexes and two gallium complexes based on N-phthaloyl derivatives of neutral aminoacids, namely [Me 2Ga(µshown an interesting and synergistic relation between gallium complexes and aminoacid derivatives aminoacid derivatives such as glycine and DL-alanine in cancer cell death, we decided to synthesize O2as CCH 2N(CO) 2C6H4)] 2 (1) and RS-[Me 2Ga(µ-O 2CCHMeN(CO) 2C 4)]2 (2) (Figure 6). The[Me formation such glycine and cancer cell death, we decided to6H synthesize twonamely gallium complexes two gallium complexes based in on N-phthaloyl derivatives of neutral aminoacids, 2Ga(µDL -alanine of2CCH aon single diastereoisomer RS was observed in2CCHMeN(CO) the crystal structure determined by X-ray diffraction based N-phthaloyl of neutral aminoacids, namely Ga(µ-O CCH N(CO) C O 2N(CO)2C6H4derivatives )]2 (1) and RS-[Me 2Ga(µ-O 2[Me C6H24)] 2 (2) (Figure 6). The formation 2 2 2 6 H4 )]2 (1) studies. The high solubility and stability of both compounds in DMSO and mixtures of DMSO/water, and C6 H4 )]2 (2) (Figure 6).structure The formation of a single diastereoisomer ofRS-[Me a single2 Ga(µ-O diastereoisomer RS was 2observed in the crystal determined by X-ray diffraction 2 CCHMeN(CO) make them candidates for anticancer tests.compounds The good high and stability of both in diffraction DMSO and studies. mixturesThe of DMSO/water, RSstudies. was observed insolubility the crystal structure determined by X-ray high solubility make themofgood forin anticancer tests. and stability bothcandidates compounds DMSO and mixtures of DMSO/water, make them good candidates for anticancer tests.

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N N

O O

N N

O O

O O

O O

R R

CH O CH 3 O O Ga 3 H3 C O Ga CH3 H3C Ga O CH3 Ga O O H C H33C O

1 1

R= R=

2 2

R R

Me Me

Gallium(III) carboxylate Gallium(III) carboxylate complexes complexes

OMe OMe 3 3

OMe OMe 4 4

S S

5 5

Me Me Me Me

O O

Me Me

6 6

O O O O

7 7

Figure 6. Gallium(III) carboxylate compounds synthesized by our group. Figure Figure 6. 6. Gallium(III) Gallium(III) carboxylate carboxylate compounds compounds synthesized synthesized by by our our group. group.

Compounds 1 and 2 were tested as anticancer agents against four human tumor cell lines: 8505C Compounds 1 and 2 were tested as anticancer agents against four human tumor cell lines: 8505C Compounds 2 were tested as and anticancer agents against four human tumor cell lines:ovarian 8505C anaplastic thyroid1 and cancer, A253 head neck carcinoma, A549 lung carcinoma, A2780 anaplastic thyroid cancer, A253 head and neck carcinoma, A549 lung carcinoma, A2780 ovarian anaplastic thyroid cancer, A253 head and neck carcinoma, A549 lung carcinoma, A2780 ovarian cancer, and DLD-1 colon carcinoma. Comparing the results of cytotoxicity with gallium(III) nitrate, cancer, and DLD-1 colon carcinoma. Comparing the results of cytotoxicity with gallium(III) nitrate, cancer, and DLD-1 colon2 carcinoma. the results of cytotoxicity gallium(III) nitrate, the the compounds 1 and presented aComparing higher antiproliferative effect. Bothwith complexes present similar the compounds 1 and 2 presented a higher antiproliferative effect. Both complexes present similar compounds and 2 presented a higher antiproliferative effect. Both complexes present similar IC50 IC50 values in1 all the studied cell lines (Table 1) [24]. IC50 values in all the studied cell lines (Table 1) [24]. values all the studied cell lines (Table 1) [24]. Inin a second study, additional organometallic gallium(III) compounds (3–7) containing phenyl, In a second study, additional organometallic gallium(III) compounds (3–7) containing phenyl, In a second study, additional organometallic gallium(III) phenyl, thiophenyl, furane, and benzodioxane carboxylato ligands compounds (Figure 6) (3–7) werecontaining synthesized and thiophenyl, furane, and benzodioxane carboxylato ligands (Figure 6) were synthesized and thiophenyl, furane, benzodioxane 6) were synthesized characterized. characterized. Theand cytotoxic studycarboxylato of all ofligands these (Figure compounds showed a and dose-dependent characterized. The cytotoxic study of all of these compounds showed a dose-dependent The cytotoxic study all of these compounds showed dose-dependent antiproliferativ antiproliferative effectoftowards different cancer cell lines asuch as 8505C, A253, A549, A2780,effect and antiproliferative effect towards different cancer cell lines such as 8505C, A253, A549, A2780, and towards different cancer cell lines such 8505C,compounds A253, A549, was A2780, andhigher DLD-1. Thethat cytotoxic activity DLD-1. The cytotoxic activity of all theasstudied much than presented by DLD-1. The cytotoxic activity of all the studied compounds was much higher than that presented by of all the studied compounds wasreported much higher than that presented by gallium(III) nitrate. From all gallium(III) nitrate. From all the complexes, 7 (containing the benzodioxane carboxylate gallium(III) nitrate. From all the reported complexes, 7 (containing the benzodioxane carboxylate the reported complexes, 7 (containing the benzodioxane carboxylate ligand) presented highest ligand) presented the highest cytotoxicity against A253 cells with the lowest IC50thevalue of ligand) presented the highest cytotoxicity against A253 cells with the lowest IC50 value of cytotoxicity 6.6 ± 0.2 µM against [25]. A253 cells with the lowest IC50 value of 6.6 ± 0.2 µM [25]. 6.6 ± 0.2 µM [25]. After the cytotoxic studies using the gallium(III) carboxylate complexes, our research group After the cytotoxic studies using the gallium(III) carboxylate complexes, our research group prepared dinuclear and tetranuclear organometallic gallium(III) compounds containing heterocyclic prepared dinuclear and tetranuclear organometallic gallium(III) compounds containing heterocyclic thiolato ligands ligands (Figure (Figure 7). 7).These Thesecompounds compoundswere weresynthesized synthesizedbyby a simple protonolysis reaction a simple protonolysis reaction of thiolato ligands (Figure 7). These compounds were synthesized by a simple protonolysis reaction of of trimethylgallium and the thiol group of mercapto-substituted imidazole, tetrazole, benzothiazole trimethylgallium and the thiol group of mercapto-substituted imidazole, tetrazole, benzothiazole or trimethylgallium and the thiol group of mercapto-substituted imidazole, tetrazole, benzothiazole or or phenyl-oxadiazol heterocycles (Figure thecompounds compoundswere werecharacterized characterizedby by NMR, NMR, IR, IR, phenyl-oxadiazol heterocycles (Figure 7).7). AllAllthe phenyl-oxadiazol heterocycles (Figure 7). All the compounds were characterized by NMR, IR, and UV–Vis UV–Vis spectroscopy spectroscopy and and X-ray X-ray diffraction diffraction studies confirmed the formation of dinuclear or and UV–Vis spectroscopy and X-ray diffraction studies confirmed the formation of dinuclear or tetranuclear complexes. tetranuclear complexes. N Me Me GaN Ga S S Me Me

Me Me S S Ga Ga Me N Me N

Gallium(III) thiolato Gallium(III) thiolato complexes complexes S S N N

S = N S = N

S S N N

8 8

N N N N N N N N 9 9

N N 10 10

S S

S S

Ph Ph Me Me Me N N O Me Ga N O S Ga N S Me O Me O S Ph S N Me Ph Ga Me N N Ga N N Ga Me N Me Ga N N Ph S Ph Me S O Me S O S N O N Ga Me O N Ga Me N Me Ph 11Me Ph 11

Figure 7. Heterocyclic thiolate polynuclear gallium(III) derivatives with improved anticancer anticancer activity. Figure7. 7.Heterocyclic Heterocyclicthiolate thiolatepolynuclear polynuclear gallium(III) gallium(III) derivatives derivatives with with improved improved Figure anticancer activity. activity.

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Table 1. IC50 (µM) after 96 h of action of gallium compounds, gallium(III) nitrate and cisplatin on different cancer cell lines. IC50 ± SD

Compound 1 2 3 4 5 6 7 8 9 10 11 Ga(NO3 )3 cisplatin

8505C

A253

A249

A2780

DLD-1

HN

Cal27

Cal33

FaDu

14.12 ± 3.74 18.24 ± 4.28 17.50 ± 2.40 22.10 ± 4.10 20.50 ± 0.30 15.20 ± 2.10 14.50 ± 3.60 9.80 ± 1.74 8.09 ± 0.94 9.60 ± 2.56 7.88 ± 2.66 95.4 ± 10.1 5.02 ± 0.23

5.72 ± 0.29 6.59 ± 0.34 7.40 ± 0.90 8.90 ± 0.30 7.70 ± 0.30 7.90 ± 0.30 6.60 ± 0.20 13.74 ± 1.53 12.55 ± 1.27 23.04 ± 2.35 10.75 ± 0.78 33.9 ± 0.3 0.81 ± 0.2

26.31 ± 8.31 25.58 ± 4.73 31.00 ± 1.30 32.00 ± 1.40 26.90 ± 7.00 26.80 ± 6.40 22.80 ± 3.80 5.45 ± 0.61 3.97 ± 0.44 5.68 ± 0.39 6.07 ± 0.23 >100 1.51 ± 0.02

13.97 ± 0.74 15.88 ± 0.36 14.00 ± 0.40 13.30 ± 0.30 12.00 ± 0.40 14.90 ± 0.30 14.00 ± 0.40 5.15 ± 0.35 5.92 ± 0.14 7.05 ± 0.26 4.73 ± 0.62 32.0 ± 1.1 0.55 ± 0.03

15.58 ± 0.11 17.94 ± 0.23 13.80 ± 0.30 14.60 ± 0.50 12.40 ± 0.10 13.50 ± 0.90 20.20 ± 2.50 13.59 ± 0.06 11.11 ± 0.04 32.90 ± 1.09 5.49 ± 0.16 >100 5.14 ± 0.12

12.37 ± 2.73 15.28 ± 1.42 16.27 ± 1.41 13.81 ± 2.16 15.31 ± 1.36 10.77 ± 1.93 7.88 ± 0.22 10.92 ± 2.04 21.98 ± 2.56 14.03 ± 2.52 >100 1.54 ± 0.06

12.10 ± 1.18 14.98 ± 1.62 15.25 ± 1.81 11.63 ± 0.88 13.58 ± 1.23 10.62 ± 0.80 4.63 ± 0.36 9.18 ± 0.14 19.14 ± 2.88 12.79 ± 1.78 >100 4.61 ± 0.11

16.34 ± 1.47 18.52 ± 1.59 20.35 ± 3.61 14.19 ± 1.41 19.38 ± 3.31 13.86 ± 1.48 4.62 ± 0.38 11.41 ± 1.26 29.92 ± 1.91 17.43 ± 1.42 >100 2.48 ± 0.20

15.74 ± 0.13 18.43 ± 0.63 15.88 ± 0.88 15.37 ± 0.16 16.35 ± 0.24 14.27 ± 0.12 4.33 ± 0.11 12.04 ± 0.11 25.55 ± 3.25 19.37 ± 0.66 >100 1.33 ± 0.39

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Thiolate complexes 8–11 were tested against the same cancer cell lines used on carboxylate gallium compounds (1–7) and again a dose-dependent antiproliferative effect on all cancer cell lines (Table 1) was observed. The cytotoxicity of gallium(III) heterocyclic thiolato complexes is much higher than that of gallium nitrate, while being in the same range as that of cisplatin. An especially high cytotoxic activity was observed for 11 with an IC50 value against DLD-1 of 5.49 ± 0.16 µM which is similar to that observed for cisplatin (5.14 ± 0.12 µM). After selectivity tests of gallium compounds 8–11 and cisplatin on WWO70327 human fibroblasts, gallium(III) complexes were shown to be much more selective to cancer cells than cisplatin, indicating, therefore their potential applicability in anticancer therapy [26]. In the apoptosis studies, after 24 h exposure to IC90 concentrations of compounds 8–11, typical DNA ladders in DLD-1 cell line were observed which indicated the induction of apoptosis promoted by the gallium compounds. In addition, compounds 8–11 showed binding affinity to FS-DNA (confirmed by UV spectroscopy in simulated physiological medium) but not to plasmid pBR322 DNA. Following the interesting results observed for carboxylate and thiolate gallium(III) complexes (1–11), additional biological studies were carried out on a series of cancer cell lines, HN (soft palate), Cal27 and Cal33 (tongue), FaDu (hypopharynx), and A253 (Submandibular duct) (Table 1) [27]. Gallium(III) complexes 3, 6, and 8 induced cell death mediated apoptosis. Cal27 and FaDu cells were treated for 24 h with IC90 concentration of the complexes and DNA appeared as characteristic ladder-like fragments suggesting an apoptotic cell death promotion. In contrast to the Cal27 cell line, there was a slight translation of FaDu cells from the G1 phase to the apoptotic phase (Sub-G1) after treatment with compounds 3, 6, and 8, which indicates that apoptosis caused by these compounds on FaDu cells may be due to interference caused in the G1 phase of the cell cycle [28]. Finally, gallium(III) complexes 1, 3–8, and 11 were also tested against CT26CL25, HCT116, and SW480 colon cancer cell lines using CV and MTT assays. Compounds 1 and 3–8 affect mitochondrial function, while gallium(III) complex 11 activates different cell death pathways and presents an activity 1.7–3.0 times higher than the other organogallium(III) complexes. In addition, 11 induces caspase independent apoptosis with a strong blockage of first and second division inhibition of CT26CL25 cell proliferation [29]. In view of the biological tests carried out for the organogallium(III) compounds reported by our group, one can envisage that these compounds may be suitable alternatives to KP46 which finished phase I trials with the outcome of promising tolerability and evidence of clinical activity in renal cell carcinoma. However, we have observed that gallium(III) complexes present a limited selectivity on cancer cells. Only in some studies have we observed selectivity when comparing their action against cancer cells with fibroblasts. Thus, the research in this area should be directed to the preparation of new gallium(III) compounds with recognizable fragments to different overexpressed targets in cancer cells to improve the selectivity and cancer cell uptake. In addition, as gallium(III) compounds present water solubility issues, formulation of these compounds with encapsulating agents (such as chitosan or analogues) may increase the solubility or dispersability in water and the cell permeation ability, and should, therefore, be of current interest for the application of these compounds in animal tests. Finally, bearing in mind that our group has not carried out in vivo studies, a complete investigation on the toxicity in animals should be undertaken to determine their potential use in humans. 3. Tin-Based Metallodrugs The therapeutic properties of triphenyltin acetate in mice tumors was observed in the early 1970s [30], and this discovery triggered a very wide study of other organotin compounds against different cancer cells [31,32]. In this context, a recent study carried out by our group using very simple tricyclohexyltin(IV) compounds demonstrated the potential of tin compounds to overcome multidrug resistance as these metallodrugs are not substrates of the Pgp protein in K562 (leukemia), PANC-1 (pancreatic carcinoma), LN-229 and U87 (multiform glioblastoma) [33]. Our research group prepared a series of Sn(IV) compounds namely [SnPh3 (3-MPA)] (12), [SnPh3 (4-MPA)] (13), [SnPh3 (DMFU)] (14), [SnPh3 (BZDO)] (15), [SnPh2 (3-MPA)2 ] (16), [SnPh2 (4-MPA)2 ]

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(17), [SnPh bybythe thecarboxylic carboxylicacids acids MPA)[SnPh 2] (17), [SnPh2(DMFU) (18), and [SnPh 2(BZDO) 2] (19) thereaction reaction of the 32 (DMFU) 2 ] (18),2] and 2 (BZDO) 2 ] (19) 3-methoxyphenylacetic acid (3-MPAH), 4-methoxyphenylacetic acid (4-MPAH), 2,5-dimethyl-3-furoic methoxyphenylacetic acid (3-MPAH), 4-methoxyphenylacetic acid (4-MPAH), acid (DMFUH) or 1,4-benzodioxane-6-carboxylic 1,4-benzodioxane-6-carboxylic acid (BZDOH) with triphenyltin(IV) chloride or diphenyltin(IV) dichloride, respectively, respectively,in inthe thepresence presenceof oftriethylamine triethylamine(Scheme (Scheme1). 1). R

SnPh3Cl + RCOOH + NEt3

O

Toluene

O Sn

-NHEt3Cl

R = 3-MPA (12); 4-MPA (13); DMFU (14); BZDO (15)

R

SnPh2Cl2 + 2 RCOOH + 2 NEt3

O

Toluene

O Sn

-2NHEt3Cl

O

R O

R = 3-MPA (16); 4-MPA (17); DMFU (18); BZDO (19)

O

O

OH

OH

MeO OMe 4-MPAH

3-MPAH

O

O O O HO DMFUH

O OH

BZDOH

Scheme 1. 1. Synthesis Synthesis of of tin(IV) tin(IV) complexes complexes 12–19. 12–19. Scheme

All the tin(IV) compounds 12–19 were characterized by multinuclear NMR spectroscopy, mass All the tin(IV) compounds 12–19 were characterized by multinuclear NMR spectroscopy, mass spectrometry, and IR, and were tested against human adenocarcinoma (HeLa), human myelegenous spectrometry, and IR, and were tested against human adenocarcinoma (HeLa), human myelegenous leukemia (K562), and human malignant melanoma (Fem-x) using MTT-based assays. The carboxylic leukemia (K562), and human malignant melanoma (Fem-x) using MTT-based assays. The carboxylic acids showed no antipoliferative effect under physiological conditions, however, tin(IV) compounds acids showed no antipoliferative effect under physiological conditions, however, tin(IV) compounds (12–19) showed a dose-dependent antipoliferative effect toward all cell lines and on human PBMC (12–19) showed a dose-dependent antipoliferative effect toward all cell lines and on human PBMC and and stimulated PBMC (Table 2). The cytotoxic activity of the compounds was several times higher stimulated PBMC (Table 2). The cytotoxic activity of the compounds was several times higher than that than that of cisplatin. Notably, compound 14 presented from 30 to 112 times higher activity than that of cisplatin. Notably, compound 14 presented from 30 to 112 times higher activity than that recorded recorded for cisplatin. In this study, we observed that triphenyltin(IV) derivatives presented lower for cisplatin. In this study, we observed that triphenyltin(IV) derivatives presented lower IC50 values IC50 values against all the studied cancer cell lines than their corresponding diphenyltin(IV) against all the studied cancer cell lines than their corresponding diphenyltin(IV) counterparts [34]. counterparts [34]. In addition to this study, an analogous triphenyltin(IV) compound containing the In addition to this study, an analogous triphenyltin(IV) compound containing the 2,62,6-dimethoxynicotinate ligand was tested against HeLa, K562, Fem-x, and on human peripheral dimethoxynicotinate ligand was tested against HeLa, K562, Fem-x, and on human peripheral blood blood mononuclear cells (PMBC) showing a high activity against all evaluated cancer cell lines with a mononuclear cells (PMBC) showing a high activity against all evaluated cancer cell lines with a moderate selectivity on K562 compared to unstimulated PBMC [35]. moderate selectivity on K562 compared to unstimulated PBMC [35]. Following the work on tin(IV) compounds, additional thiolate complexes containing α,α’dimercapto-o-xylene ligand were synthesized (Scheme 2). The compounds 20 and 21 showed a good

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Table 2. IC50 (µM) after 96 h of action of tin compounds 13–21 and cisplatin on different cancer cell lines. IC50 ± SD

Compound 13 14 15 16 17 18 19 20 21 cisplatin

HeLa

K562

Fem-x

0.17 ± 0.02 0.15 ± 0.01 0.22 ± 0.02 1.18 ± 0.05 1.04 ± 0.09 1.57 ± 0.23 1.23 ± 0.01 2.48 ± 0.22 0.23 ± 0.04 4.4 ± 0.3

0.075 ± 0.002 0.051 ± 0.004 0.170 ± 0.005 0.90 0.53 ± 0.07 0.85 0.96 1.02 ± 0.08 0.14 ± 0.01 5.7 ± 0.3

0.083 ± 0.007 0.074 ± 0.004 0.163 ± 0.001 0.93 0.63 ± 0.03 0.80 ± 0.06 0.82 ± 0.03 4.7 ± 0.3

MDA-MB-453 LS174 4.08 ± 0.21 0.28 ± 0.06 13.0 ± 1.7

>5 0.25 ± 0.02 -

Inorganics 2017, 5, 4

PBMC

PBMC + PHA

>0.2 0.20 ± 0.01 0.24 ± 0.02 >20 1.27 ± 0.08 >20 >20 1.87 ± 0.03 0.60 ± 0.01 33.6

0.16 ± 0.02 0.15 ± 0.02 0.17 ± 0.02 >20 0.98 ± 0.09 >20 >20 1.54 ± 0.28 0.44 ± 0.14 26 ± 6 9 of 23

Following the work on tin(IV) compounds, additional thiolate complexes containing α,α’-dimercaptoo-xylene ligand different were synthesized 2). IC The compounds 209.7 and 21 and showed good activity against cancer cell(Scheme lines, with 50 values between ± 0.2 21.1 a ± 1.1 µMactivity (Table against different cancer cell lines, with IC50 values between 9.7 ± 0.2 and 21.1 ± 1.1 µM (Table 2). 2). Ph

S Sn

Ph SnPh2Cl2 + 2 NEt3

S 20

- 2NHEt3Cl SH SH 2 SnPh3Cl + 2 NEt3 Ph - 2NHEt3Cl Ph

Ph Sn S S Sn

Ph

Ph

Ph 21

Scheme 2. 2. Synthesis Synthesis of of Compounds Compounds 20 20 and and 21. 21. Scheme Table 2. IC50 (µM) after 96 h of action of tin compounds 13–21 and cisplatin on different cancer cell lines.

The dinuclear tin(IV) compound 21 is more cytotoxic than 20. This result was expected as 50 ± SD compound 21 presents two SnPh3 units which are IC normally associated with increase of cytotoxic Compound HeLa K562 Fem-x MDA-MB-453 LS174 DNA.PBMC PBMC +activity PHA + activity due to the interaction of SnPh3 moieties with protein kinases The cytotoxic 13 0.17 ± 0.02 0.075 ± 0.002 0.083 ± 0.007 >0.2 0.16 ± 0.02 of 20 and 21 was lower than that reported for carboxylate tin(IV) complexes (12–19) [36]. However, 14 0.15 ± 0.01 0.051 ± 0.004 0.074 ± 0.004 0.20 ± 0.01 0.15 ± 0.02 a more 15 in depth study of compound 21 against HeLa and Fem-x cell showed the induction 0.22 ± 0.02 0.170 ± 0.005 0.163 ± 0.001 0.24 ± 0.02 0.17 ± 0.02of an 16 cell death 1.18 ± [37]. 0.05 0.90 0.93 >20 >20 apoptotic 17 1.04 ± 0.09 0.53 ± 0.07 0.63tetraorganotin(IV) ± 0.03 1.27 ± 0.08cyclopentadienyl 0.98 ± 0.09 In another study, our group prepared compounds containing 18 1.57 ± 0.23 0.85 0.80 ± 0.06 >20 >20 ligands19(22–25)1.23which were prepared by the simple transmetallation reaction of>20lithium ± 0.01 0.96 0.82 ± 0.03 >20 cyclopentadienide [38]. All the isolated 20 2.48 ±derivatives 0.22 1.02 ±with 0.08 SnPh3 Cl - (Scheme 4.083) ± 0.21 >5 compounds 1.87 ± 0.03were 1.54 ± 0.28 as 21 0.23 ±though 0.04 0.14 0.01 - double 0.28 ± 0.06 ± 0.02 the 0.60formation ± 0.01 0.44 single isomers even the ±position of the bonds makes0.25 possible of a± 0.14 mixture cisplatin 4.4 ± 0.3 5.7 ± 0.3 4.7 ± 0.3 13.0 ± 1.7 33.6 26 ± 6 of positional isomers. Tin(IV) complexes (22–25) were tested in vitro against 8505C, A253, A549, A2780, and DLD-1 The dinuclear compound 21 ismicroculture more cytotoxic than 20. assay This result expected as cell lines by using tin(IV) the sulforhodamine-B colorimetric (Tablewas 3) [39]. All the compound 21 presents two SnPh3 units which are normally associated of cytotoxic compounds showed a dose-dependent antiproliferative effect toward cell with lines increase and presented lower 3+ moieties with protein kinases DNA. The cytotoxic activity of activity due to the interaction of SnPh IC50 values than those observed for cisplatin against the same cell lines. From all the series of 20 and 21 was lower than that reported for carboxylate tin(IV) complexes (12–19) [36]. However, a more in depth study of compound 21 against HeLa and Fem-x cell showed the induction of an apoptotic cell death [37]. In another study, our group prepared tetraorganotin(IV) compounds containing cyclopentadienyl ligands (22–25) which were prepared by the simple transmetallation reaction of lithium cyclopentadienide derivatives with SnPh3Cl (Scheme 3) [38]. All the compounds were

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cyclopentadienyl-substituted tin compounds, 24 (which contains the tetramethylcyclopentadienyl moiety) presented the highest cytotoxic activities against all the studied cancer cell lines with IC50 values between 0.037 and 0.085 µM (from 17 to 104 times higher than cisplatin). Compounds 22 and 23 presented similar activities (0.042–0.103 µM and 0.061–0.119 µM) while 25 had a lower cytotoxicity Inorganics 5, 4 between 0.163 and 0.384 µM. 10 of 23 with IC502017, values

R

R +

SnPh3Cl

Sn

-LiCl

Li R = But (22); CMe2(CH2)2CH=CH2 (23)

R

R + SnPh3Cl

Sn

-LiCl

Li R = H (24);SiMe3 (25)

Scheme compounds 22–25. 22–25. Scheme 3. 3. Synthesis Synthesis of of cyclopentadienyl-substituted cyclopentadienyl-substituted tin(IV) tin(IV) compounds

Tin(IV) complexes (22–25) were tested in vitro against 8505C, A253, A549, A2780, and DLD-1 Table by 3. ICusing after 96 h of action of tin compounds 22–30colorimetric and cisplatin on different cancer lines. cell lines sulforhodamine-B microculture assay (Table 3) cell [39]. All the 50 (µM)the compounds showed a dose-dependent antiproliferative effect toward cell lines and presented lower IC50 ± same SD IC50Compound values than those observed for cisplatin against the cell lines. From all the series of cyclopentadienyl-substituted tin compounds, 24 (which contains the tetramethylcyclopentadienyl 8505C A253 A249 A2780 DLD-1 moiety)22presented 0.103 the highest cytotoxic activities against all the studied cancer cell lines with IC50 ± 0.015 0.077 ± 0.012 0.079 ± 0.002 0.042 ± 0.004 0.044 ± 0.007 values between 0.037 and (from±17 to 104 times higher cisplatin). 22 and 23 0.110 ± 0.085 0.011 µM 0.118 0.028 0.108 ± 0.018 than0.061 ± 0.002Compounds 0.119 ± 0.004 24 ± 0.007(0.042–0.103 0.045 ±µM 0.004 0.038 ± 0.004 0.03725 ± had 0.007a lower 0.048 ± 0.002 23 presented similar0.085 activities and 0.061–0.119 µM) while cytotoxicity 25 0.343 ± 0.046 0.351 ± 0.045 0.384 ± 0.021 0.163 ± 0.002 0.309 ± 0.003 with IC50 values between 0.163 and 0.384 µM.

26 0.129 ± 0.004 0.093 ± 0.003 0.102 ± 0.004 0.121 ± 0.002 0.103 ± 0.004 27 0.179 ± 0.003 0.139 ± 0.005 0.152 ± 0.003 0.170 ± 0.002 0.165 ± 0.003 Table after ± 960.058 h of action0.238 of tin±compounds 22–30 cisplatin on different cell±lines. 283. IC50 (µM)0.241 0.002 0.236 ±and 0.011 0.130 ± 0.003 cancer 0.210 0.006 29 0.132 ± 0.010 0.081 ± 0.003 0.094 ± 0.013 0.060 ± 0.001 IC50 ± SD 30 0.172 ± 0.003 0.100 ± 0.014 0.129 ± 0.014 0.178 ± 0.002 Compound 8505C A249 A2780 DLD-1 cisplatin 5.02 ± 0.23 0.81 A253 ± 0.2 1.51 ± 0.02 0.55 ± 0.03 5.14 ± 0.12 22 0.103 ± 0.015 0.077 ± 0.012 0.079 ± 0.002 0.042 ± 0.004 0.044 ± 0.007 23 0.110 ± 0.011 0.118 ± 0.028 0.108 ± 0.018 0.061 ± 0.002 0.119 ± 0.004 In a subsequent study, ionic triphenyltin(IV) chloride carboxylate complexes (26–28, 24 0.085 a± series 0.007 of rare 0.045 ± 0.004 0.038 ± 0.004 0.037 ± 0.007 0.048 ± 0.002 Figure 8) was synthesized and tested against 8505C, A253, A549, A2780, and DLD-1 cell lines (Table 3). 25 0.343 ± 0.046 0.351 ± 0.045 0.384 ± 0.021 0.163 ± 0.002 0.309 ± 0.003 All the ionic activities 50 times more active 26 tin(IV) compounds 0.129 ± 0.004presented 0.093anticancer ± 0.003 0.102 ± 0.004up to0.121 ± 0.002 0.103 ±than 0.004those 27 (for example 0.179in± DLD-1 0.003 complex 0.139 ± 0.005 0.170 ± 0.002 0.165 ± 0.003 of cisplatin 26 has 0.152 an IC±500.003 value of 0.103 µM compared to that of 0.241 ± 0.058 0.238 ± 0.002 0.236 to ± 0.011 0.130 ± 0.003for future 0.210applications ± 0.006 cisplatin of285.14 µM). Therefore, from this series, 26 seems be very promising ± 0.010 0.081 ± 0.003 0.013 0.060 ± 0.001 cell in clinical 29 trials due to0.132 its high solubility, high activity0.094 and ±its capacity to induce a clean apoptotic 30 0.172 ± 0.003 0.100 ± 0.014 0.129 ± 0.014 0.178 ± 0.002 death. This compound affected the G1 and G2/M phases of the cell cycle. Its apoptotic action seems to cisplatin 5.02 ± 0.23 0.81+ ± 0.2 1.51 ± 0.02 0.55 ± 0.03 5.14 ± 0.12

be related to the interaction between SnPh3 moieties with protein kinases and DNA [40]. In addition, the apoptotic properties of compound 26 and the interaction with caspases 2, 3, and 8 were studied in In a subsequent study, a series of rare ionic triphenyltin(IV) chloride carboxylate complexes (26– DLD-1 cells with only caspase 8 being found to be upregulated after 4 h. However, cells treated for 6 h 28, Figure 8) was synthesized and tested against 8505C, A253, A549, A2780, and DLD-1 cell lines showed an additional activation of caspase 2 and 8, which was in contrast with the results observed (Table 3). All the ionic tin(IV) compounds presented anticancer activities up to 50 times more active when treating the cells with cisplatin which only showed activation of caspase 8 and 9. These results than those of cisplatin (for example in DLD-1 complex 26 has an IC50 value of 0.103 µM compared to suggest that 26 promotes a faster activation of apoptosis and that this was achieved in the DLD-1 cell that of cisplatin of 5.14 µM). Therefore, from this series, 26 seems to be very promising for future line in a different way to that observed for cisplatin. Respectively, cisplatin promotes apoptosis by applications in clinical trials due to its high solubility, high activity and its capacity to induce a clean apoptotic cell death. This compound affected the G1 and G2/M phases of the cell cycle. Its apoptotic action seems to be related to the interaction between SnPh3+ moieties with protein kinases and DNA [40]. In addition, the apoptotic properties of compound 26 and the interaction with caspases 2, 3, and 8 were studied in DLD-1 cells with only caspase 8 being found to be upregulated after 4 h. However,

achieved in the DLD-1 cell line in a different way to that observed for cisplatin. Respectively, cisplatin promotes apoptosis by both intrinsic (mitochondrial pathway, caspase 9 dependent pathway) and external signals (extrinsic or death receptor pathway), while 26 induces apoptosis only via extrinsic receptor pathway [40]. Further studies of the in vitro activity of 26 and 28 against 518A2 (melanoma), FaDu (head and Inorganics 2017, 5, 4 11 of 23 neck carcinoma), HT-29 (colon cancer), MCF-7 (breast carcinoma), and SW1736 (thyroid cancer) cell lines showed the potent cytotoxic activity of 26 and 28 which induce apoptosis. These results were confirmed by(mitochondrial the observation of membrane translocation of phosphatidylserine, DNA both intrinsic pathway, caspaseblebbing, 9 dependent pathway) and external signals (extrinsic fragmentation, and accumulation in the Sub-G1 only phasevia [41]. or death receptor pathway), while of 26cells induces apoptosis extrinsic receptor pathway [40]. O

O O

NHEt3

O

SnPh3Cl

N R O R = H (26); Me (27)

NHEt3

O O

O

SnPh3Cl

O 28

Figure with improved improved cytotoxic cytotoxic activity. activity. Figure 8. 8. Ionic Ionic triphenyltin(IV) triphenyltin(IV) chloride chloride carboxylate carboxylate complexes complexes with

Our group prepared two different 1D-polymeric triphenyltin(IV) carboxylate derivatives, based Further studies of the in vitro activity of 26 and 28 against 518A2 (melanoma), FaDu (head and on the reaction of SnPh3Cl with mesitylthioacetic acid and xylythioactic acid. The 1D-chains neck carcinoma), HT-29 (colon cancer), MCF-7 (breast carcinoma), and SW1736 (thyroid cancer) cell [{SnPh3(O2CCH2SXyl)}͚] (29) (Xyl = 3,2-Me2C6H3) and [{SnPh3(O2CCH2SMes)}͚] (30) (Mes = 2,4,6lines showed the potent cytotoxic activity of 26 and 28 which induce apoptosis. These results were Me3C6H2) were tested in vitro against 8505C, A253, A549, and DLD-1 cell lines observing that they confirmed by the observation of membrane blebbing, translocation of phosphatidylserine, DNA present higher activity (from 8 to 85 times higher) than those of cisplatin (Table 3) and between 285 fragmentation, and accumulation of cells in the Sub-G1 phase [41]. and 2520 times higher than their gallium(III) and titanocene(IV) analogues, respectively [42]. In Our group prepared two different 1D-polymeric triphenyltin(IV) carboxylate derivatives, based addition, these studies showed that compounds 29 and 30 interacted with DNA by classical on the reaction of SnPh3 Cl with mesitylthioacetic acid and xylythioactic acid. The 1D-chains electrostatic interactions with intrinsic binding constants of 1.68 × 105 and 1.02 × 105 M−1, respectively. [{SnPh3 (O2 CCH2 SXyl)}∞ ] (29) (Xyl = 3,2-Me2 C6 H3 ) and [{SnPh3 (O2 CCH2 SMes)}∞ ] (30) (Mes = Thus, one can conclude from our results that tin compounds show potential due to the high 2,4,6-Me3 C6 H2 ) were tested in vitro against 8505C, A253, A549, and DLD-1 cell lines observing that cytotoxicity that they present in vitro, the possibility of overcoming multidrug resistance [33], and they present higher activity (from 8 to 85 times higher) than those of cisplatin (Table 3) and between the wide variety of cancer cells that they can treat [43]. In addition, in the age of nanotechnology, 285 and 2520 times higher than their gallium(III) and titanocene(IV) analogues, respectively [42]. their medicinal applications are being enhanced by simple conjugations with silica-based In addition, these studies showed that compounds 29 and 30 interacted with DNA by classical nanomaterials. For example, our group is now working on the support of novel organotin(IV) electrostatic interactions with intrinsic binding constants of 1.68 × 105 and 1.02 × 105 M−1 , respectively. compounds onto nanostructured silica [44]. Our latest results showed excellent in vitro [45] and in Thus, one can conclude from our results that tin compounds show potential due to the high vivo [44] behavior of the new encapsulated systems which have the potential to be used in the future cytotoxicity that they present in vitro, the possibility of overcoming multidrug resistance [33], and the in phase I clinical trials. wide variety of cancer cells that they can treat [43]. In addition, in the age of nanotechnology, their medicinal applications are being enhanced by simple conjugations with silica-based nanomaterials. 4. Titanium-Based Metallodrugs For example, our group is now working on the support of novel organotin(IV) compounds onto 3+ can exist in aqueous media, the aqueous chemistry of titanium is dominated by Although Ti nanostructured silica [44]. Our latest results showed excellent in vitro [45] and in vivo [44] behavior of oxidation state +4 and the tendency of free Ti4+ to hydrolyze precipitate, ultimately forming the new encapsulated systems which have the potential to be usedand in the future in phase I clinical trials. insoluble TiO2, is very high. However, hydrolytic reactions can be minimized by surrounding the 4. Titanium-Based Metallodrugs metal with the appropriate ligands which decreases the rate of the hydrolysis reactions. Thus, the titanium β-diketonate complex, budotitane, was first non-platinum metal complex to enter 3+ Although Ti can exist in aqueous media, thethe aqueous chemistry of titanium is dominated by clinical trials for treatment of cancer. In this context, cyclopentadienyl ligands are also ideal 4+ oxidation state +4 and the tendency of free Ti to hydrolyze and precipitate, ultimately forming candidatesTiO for ,improving the hydrolytic stability of titanium(IV) with potential anticancer properties insoluble 2 is very high. However, hydrolytic reactions can be minimized by surrounding the of titanocene dihalide derivatives being observed by Köpf-Maier andhydrolysis Köpf in thereactions. 1980s [46]. metal with the appropriate ligands which decreases the rate of the Thus, the The preclinical trials of titanium compounds indicated their potential as therapeutic titanium β-diketonate complex, budotitane, was the first non-platinum metal complex to enter clinical metallodrugs against different tumors [47]. The main biological target of titanium-based trials for treatment of cancer. In this context, cyclopentadienyl ligands are also ideal candidates for metallodrugs is the inhibition of DNA synthesis, triggering apoptosis [48]. Some additional recent improving the hydrolytic stability of titanium(IV) with potential anticancer properties of titanocene studies have reported the observed inhibitionbyofKöpf-Maier the enzymeand topoisomerase II by [46]. titanocene dichloride and dihalide derivatives being Köpf in the 1980s this, The therefore, may be an alternative cell death induction pathway [49]. preclinical trials of titanium compounds indicated their potential as therapeutic metallodrugs Titanocene was The also main studied in phase target I clinical in 1993 [50] metallodrugs and later in phase II against differentdichloride tumors [47]. biological of trials titanium-based is the clinical trials [51,52] and became very important in the field of antitumor metallodrugs. Although the inhibition of DNA synthesis, triggering apoptosis [48]. Some additional recent studies have reported results of phase clinical trials were not satisfactory becausedichloride of the lackand of activity against the studied the inhibition ofIIthe enzyme topoisomerase II by titanocene this, therefore, may be an alternative cell death induction pathway [49]. Titanocene dichloride was also studied in phase I clinical trials in 1993 [50] and later in phase II clinical trials [51,52] and became very important in the field of antitumor metallodrugs. Although the results of phase II clinical trials were not satisfactory because of the lack of activity against the studied

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tumors, the excellent research on titanium compounds published by Tacke, Meléndez, McGowan, Baird, Valentine, and and Tshuva, Tshuva, reignited reignited the the interest interest in in novel novel titanium titanium compounds compounds with with anticancer anticancer properties properties [53–60]. [53–60]. Since 2007 our our research research group group has has synthesized synthesized different different titanocene titanocene derivatives derivatives which have have demonstrated high high activity activityagainst againsta aseries series of cancer cell lines. In our first study, titanocene and of cancer cell lines. In our first study, titanocene and ansaansa-titanocene compounds different alkyl and alkenyl ligands(Figure (Figure99compounds compounds 31–42) 31–42) were titanocene compounds withwith different alkyl and alkenyl ligands prepared and characterized. Most of the compounds were active against all the studied prepared and characterized. studied cancer cancer cell cell lines and the activity was dependent on the substituent at the Cp ring or at the ansa-bridge [61,62]. Of special special interest were the the alkenyl-substituted alkenyl-substituted compounds 38, 39, and 42 which which showed showed improved improved cytotoxic activity against the studied cell lines HeLa, K562, and Fem-x (Table 4). activity against the studied cell lines HeLa, K562, and Fem-x (Table 4). Me

Si

Cl

Ti

Ge

Cl

Cl

Si

Ti

Cl

Ti

Si

Cl

Ti

35

Ti

Cl

Cl

Si

Cl

Ti

Cl

34

But H Ph

Cl

Si

Cl

Ti

36

Cl Cl

Si

Ti

Si

Cl

Ti

37

Si

Cl

Cl

33

Pri H Ph

But

Si

H

32

31

Si

Cl

Ti

Cl

38

Cl

Si

Cl

Ti

Cl Cl

Si

39

40

Ti

41

Cl Cl

42

Figure 9. Titanocene and ansa-titanocene ansa-titanocene complexes complexes 31–42. 31–42.

Thus, a subsequent study with an alkenyl monosubstituted titanocene complex (43) and its 9Thus, a subsequent study with an alkenyl monosubstituted titanocene complex (43) and its BBN hydroboration product (44, Scheme 4) were synthesized and characterized. Both compounds 9-BBN hydroboration product (44, Scheme 4) were synthesized and characterized. Both compounds were tested against HeLa, K562, and MBA-MB-361 cell lines [63] and showed a dose-dependent were tested against HeLa, K562, and MBA-MB-361 cell lines [63] and showed a dose-dependent antiproliferative effect towards all cell lines and on human PBMC and stimulated PBMC (Table 4). antiproliferative effect towards all cell lines and on human PBMC and stimulated PBMC (Table 4).

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Table 4. IC50 (µM) of titanium(IV) compounds on different cancer cell lines. IC50 ± SD

Compound 31 32 33 34 38 39 40 42 43 44 55 56 57 58 71 72 73 74 [Ti(η5 -C2017, 5 H5 )Cl Inorganics 5, 42 ] Inorganics 2017, 5, 4

HeLa

K562

Fem-x

MDA-MB-453

135 ± 6 154 ± 4 109 ± 9 117 ± 3 84 ± 9 79 ± 7 189 ± 13 86 ± 3 149.2 ± 2.9 166.3 ± 7.4 142.2 ± 5.8 139.4 ± 12.7 107.2 ± 6.9 117.4 ± 8.1 171.2 ± 4.1 127.5 ± 1.5 22.4 ± 1.2 32.9 ± 0.4 >200

66 ± 6 73 ± 1 59 ± 8 88 ± 4 24 ± 3 64 ± 9 155 ± 9 66 ± 4 96.6 ± 3.4 155.6 ± 5.5 86.8 ± 0.3 78.2 ± 0.7 87.9 ± 3.6 72.2 ± 1.7 176.5 ± 3.2 83.7 ± 0.2 33.2 ± 1.5 32.8 ± 11.5 >200

96 ± 4 106 ± 5 116 ± 9 101 ± 9 89 ± 9 134 ± 18 >200 99 ± 6 133.6 ± 9.4 167.9 ± 4.2 164.9 ± 9.4 191.4 ± 5.5 90.2 ± 6.8 123.0 ± 5.2 179.8 ± 6.5 154.3 ± 2.4 36.4 ± 2.8 27.1 ± 3.4 177.7 ± 4.9

>200 161.1 ± 0.1 37.7 ± 1.5 48.9 ± 0.8 >200

PBMC

PBMC + PHA

112 ± 7 101 ± 6 72 ± 2 83 ± 10 55 ± 8 140 ± 1 >200 96 ± 3 149.8 ± 3.1 >200 146.2 ± 3.8 162.0 ± 3.7 104.6 ± 5.3 132.9 ± 0.6 >200

77 ± 6 83 ± 11 87 ± 2 82 ± 12 70 ± 6 151 ± 10 195 ± 5 101 ± 5 142.5 ± 0.9 >200 148.0 ± 1.3 156.1 ± 7.4 116.8 ± 11.7 127.3 ± 1.4 199.8 ± 9.9 13 of 23 13 of 23

B B 9-BBN 9-BBN

Cl Cl Cl Cl

Ti Ti

Ti Ti

43 43

Cl Cl Cl Cl

44 44

Scheme 4. Hydroboration reaction of an alkenyl-substituted titanocene derivative. Scheme 4. Hydroboration reaction of an alkenyl-substituted titanocene derivative.

The alkenyl-substituted alkenyl-substituted complex complex 44, 44, presented presented good activity against K562 (IC 50 96.6 ± 3.4 µM) The 3.4 µM) presented good good activity activity against against K562 K562 (IC (IC50 50 96.6 ± ± 3.4 50 149.2 ± 2.9 µM) and Fem-x (IC50 133.6 ± 9.4 µM), while complex and moderate activity on HeLa (IC and moderate 9.4µM), µM), while while complex 149.2± ± 2.9 µM) and Fem-x Fem-x (IC (IC50 50 133.6 ± ± 9.4 moderate activity activityon onHeLa HeLa(IC (IC5050 149.2 43 presented presented only only moderate moderate activity activity on K562, HeLa ,and Fem-x (Table 4). 43 activity on on K562, K562, HeLa HeLa ,and ,and Fem-x Fem-x (Table (Table4). 4). Subsequently, a a series series of of naphthyl-substituted naphthyl-substituted titanocene compounds compounds (45–48) were also also Subsequently, naphthyl-substituted titanocene (45–48) were synthesized and characterized by our group (Figure 10) [64]. The molecular structure of 46 was synthesized and characterized characterized by our group (Figure 10) [64]. The molecular structure of 46 was established by by single-crystal single-crystal X-ray X-ray diffraction diffraction studies. established diffraction studies. studies.

Cl Cl Cl Cl

Ti Ti

Ti Ti

45 45

Cl Cl Cl Cl

Me Me33Si Si

46 46

Ti Ti

Cl Cl Cl Cl

Me Me33Si Si

Ti Ti

Cl Cl Cl Cl

48 48

47 47

Figure 10. Naphthyl-substituted titanocene(IV) dichloride complexes. Figure 10. Naphthyl-substituted titanocene(IV) dichloride complexes. Table 4. IC50 (µM) of titanium(IV) compounds on different cancer cell lines. Table 4. IC50 (µM) of titanium(IV) compounds on different cancer cell lines. Compound Compound 31 31 32 32

HeLa HeLa 135 ± 6 135 ± 6 154 ± 4 154 ± 4

K562 K562 66 ± 6 66 ± 6 73 ± 1 73 ± 1

Fem-x Fem-x 96 ± 4 96 ± 4 106 ± 5 106 ± 5

IC50 ± SD IC50 ± SD MDA-MB-453 MDA-MB-453 -

PBMC PBMC 112 ± 7 112 ± 7 101 ± 6 101 ± 6

PBMC + PHA PBMC + PHA 77 ± 6 77 ± 6 83 ± 11 83 ± 11

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In anticancer tests against 8505C, A549, A2780, DLD-1, and FaDu, the titanocene(IV) complexes (45–48) showed a significant cytotoxic activity with IC50 values (Table 5) lower than titanocene dichloride. Compound 48 was the most active of all the tested compounds, with IC50 values between 35.65 ± 4.95 and 69.02 ± 1.67 µM. The improvements in cytotoxic activity of 48 were due to the presence of the trimethylsilyl group. Table 5. IC50 (µM) of titanocene compounds (43–48 and 59–70) on different cancer cell lines. IC50 ± SD

Compound 8505C

A253

A549

A2780

DLD-1

43 103.3 ± 2.4 89.6 ± 0.5 96.0 ± 2.9 70.6 ± 1.7 45 194.51± 4.80 191.72 ± 2.54 72.41 ± 7.52 161.34 ± 3.48 46 105.22 ± 1.51 114.20 ± 2.88 71.60 ± 1.23 116.22 ± 2.31 47 124.57 ± 3.24 104.20 ± 3.07 54.25 ± 2.35 97.95 ± 4.03 48 45.19 ± 1.26 53.38 ± 1.43 35.65 ± 4.95 61.31± 6.08 59 182.3 ± 2.5 182.6 ± 2.0 192.5 ± 1.1 151.2 ± 4.2 60 190.8 ± 2.2 131.2 ± 0.5 144.6 ± 2.9 115.7 ± 2.9 61 77.44 ± 1.61 62 82.14 ± 8.95 63 56.88 ± 6.06 64 78.19 ± 16.59 44.50 ± 3.11 - 14 of 23 Inorganics 2017,655, 4 66 64.44 ± 4.45 67 53.87 ± 2.68 In anticancer tests against 8505C, A549, A2780, DLD-1, and FaDu, the titanocene(IV) complexes 68 40.54 ± 4.39 (45–48) showed a significant -cytotoxic activity with IC50 values (Table 69 59.335)± lower 2.31 than titanocene dichloride. Compound 48 was -the most active- of all the tested- compounds, 70 57.66 with ± 4.78IC50 values- between 5 -C H )Cl ] ± 6.36 167.62 ± 3.31 activity 124.78 ±of 4.36 35.65[Ti(η ± 4.95 ± 1.67>200 µM. The 188.71 improvements in cytotoxic 48 were >200 due to the 5and 5 69.02 2

presence of the trimethylsilyl group. In In addition, addition, several several carbon carbon and and silicon-bridged silicon-bridged ansa-titanocene(IV) ansa-titanocene(IV) derivatives derivatives were were synthesized synthesized (Figure 11) 11) and and tested tested against against different different tumor tumor cell cell lines, lines, namely namely murine murine melanoma melanoma B16, B16, human human (Figure melanoma A375, colon cancer HCT116 and SW620, prostate cancer LNCaP and DU145, and mouse melanoma A375, colon cancer HCT116 and SW620, prostate cancer LNCaP and DU145, and mouse colon CT26CL25. colon cancer cancer CT26CL25.

Ti

Cl

Ti

Cl

Cl

Ti

Ph

Ti

Cl Ph

Cl

52

Ti

Si

Cl Cl

51

50

49

Si

Cl

Cl Cl

Ph

53

Si

Ti

Cl Cl

54

Figure 11. ansa-Titanocene derivatives. 5. IC50 (µM) of titanocene compounds (43–48 and 59–70) on different cancer cell lines. The Table C-bridged ansa-titanocene derivatives showed an increase in cytotoxic activity with an ethylene bridge (complex 49) and poor activity with methylene IC50 ± SD (complexes 50 and 51) while the Compound incorporation of a phenyl ring attached directly to the bridging atom decreases the viability DLD-1 of the cancer 8505C A253 A549 A2780 43 103.3 ± 2.4 (51), 89.6 ± 0.5 96.0viability ± 2.9 - cells in silicon-bridged 70.6 ± 1.7 cells in carbon-bridged compounds but increases the of cancer 45 and 54) (Table 194.51± 4.80 most cytotoxic 191.72 ± 2.54 72.41 7.52 54 were 161.34 ± 3.48 systems (53 6). The titanocene complexes 49± and also tested 46 47 48 59 60 61

105.22 ± 1.51 124.57 ± 3.24 45.19 ± 1.26 182.3 ± 2.5 190.8 ± 2.2 -

182.6 ± 2.0 131.2 ± 0.5 -

114.20 ± 2.88 104.20 ± 3.07 53.38 ± 1.43 192.5 ± 1.1 144.6 ± 2.9 -

71.60 ± 1.23 54.25 ± 2.35 35.65 ± 4.95 77.44 ± 1.61

116.22 ± 2.31 97.95 ± 4.03 61.31± 6.08 151.2 ± 4.2 115.7 ± 2.9 -

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Inorganics 5, 4 against 2017, primary

15 of of 23 mouse keratinocynates and lung fibroblasts while observing a large viability both primary cells and that 54 was nontoxic to primary cells [65]. In addition, 49 and 54 showed accumulation hypodiploid in compartment subG compartment inresistant cisplatin resistant HCT116 accumulation ofofhypodiploid cellscells in subG in cisplatin HCT116 and SW620. and SW620.

Table 6. IC50 (µM) of ansa-titanocene compounds (49–54) on different cancer cell lines. Table 6. IC50 (µM) of ansa-titanocene compounds (49–54) on different cancer cell lines. Compound Compound 4949 5050 5151 5252 5353 54 54 [Ti(η5 -C 5 H5 )Cl2 ] 5H5)Cl2] [Ti(η5-C

A375 B16 A375 B16 ± 36 7 7 124 ± 36 8686± ± 152 ± 182± ± ± 77 182 11 127 ± 15 178 178± ± ± 15 1919 170 17 >200 170 ± ± 17 >200 181 ± 9 >200 181 ± 9 >200 105 ± 29 43 ± 4 105 ± 29 43 ± 4 161 ± 1 >200 161 ± 1 >200

IC50 ± SD ± SD IC50 SW620 CT26CL25 CT26CL25 SW620

HCT116 HCT116 9898 ± 19 8787± ± 2 2 ± 19 148 >200 148 ± 1± 1 >200 144 ± 25 132 132± ± 144 ± 25 1818 >200 158 >200 158 ± ± 1 1 160 ± 1 199 ± 160 ± 1 199 ± 2 2 68 ± 6 75 ± 1 68 ± 6 75 ± 1 >200 141 ± 1 >200 141 ± 1

119 33 119±± >200 >200 148±± 148 2424 163 ± 163 ± 1 1 >200 >200 62 ± 13 62 ± 13 154 ± 1 154 ± 1

DU145 DU145 93 93±±3434 175±±1 1 175 117±±4343 117 142±±1 1 142 156±±1 1 156 83 ± 24 83 ± 24 163 ± 36 163 ± 36

LnCap LnCap 100 2020 100± ± >200 >200 163± ± 163 1111 197 197 ± ± 11 >200 >200 66 ± 19 66 ± 19 >200 >200

described titanocene and ansa-titanocene compounds were chloride derivatives, All the thepreviously previously described titanocene and ansa-titanocene compounds were chloride however, a complete study of the substitution of the chlorido by carboxylato ligands was carried out derivatives, however, a complete study of the substitution of the chlorido by carboxylato ligands was using mesitylthioacetic acid and different cyclopentadienyl ligands (Figure 12) [66]. carried out using mesitylthioacetic acid and different cyclopentadienyl ligands (Figure 12) [66].

R

O

S O

O R

Ti

OO

O O

Ti

S

S

R = H, R' = H (55); R = Me, R' = Me (56); R = H, R' = SiMe3 (57)

O S O O

S

58

Figure Figure 12. 12. Titanium Titanium carboxylate carboxylate complexes complexes containing containing the the mesitylthioaceticatato mesitylthioaceticatato ligand. ligand.

In addition, two new alkenyl-substituted titanocene(IV) carboxylate complexes containing the In addition, two new alkenyl-substituted titanocene(IV) carboxylate complexes containing mesitylthioacetato and the xilylthioacetato ligands (59 and 60, respectively, Scheme 5) were the mesitylthioacetato and the xilylthioacetato ligands (59 and 60, respectively, Scheme 5) were synthesized and characterized. The comparison of cytotoxic activities of the titanocene(IV) synthesized and characterized. The comparison of cytotoxic activities of the titanocene(IV) carboxylate carboxylate and titanocene(IV) dichloride against 8505C, A253, A549, and DLD-1, showed that and titanocene(IV) dichloride against 8505C, A253, A549, and DLD-1, showed that titanocene(IV) titanocene(IV) carboxylates (59 and 60) are less active against all the studied cells than their carboxylates (59 and 60) are less active against all the studied cells than their corresponding dichloride corresponding dichloride counterpart (43) (Table 5), indicating that the effect of the carboxylato counterpart (43) (Table 5), indicating that the effect of the carboxylato ligands on the cytotoxicity is ligands on the cytotoxicity is not synergistic but negative in the case of alkenyl-substituted titanocene not synergistic but negative in the case of alkenyl-substituted titanocene compounds [67]. This was compounds [67]. This was confirmed in the DNA interaction tests, where complex 43 showed a confirmed in the DNA interaction tests, where complex 43 showed a higher intrinsic binding constant higher intrinsic binding constant than 59 and 60. than 59 and 60. This study on the influence of carboxylato ligands on the cytotoxic activity of titanocene This study on the influence of carboxylato ligands on the cytotoxic activity of titanocene complexes was completed by the preparation of a wide variety of titanocene carboxylate derivatives complexes was completed by the preparation of a wide variety of titanocene carboxylate derivatives of the type [Ti(η5-C 5H5)2(OOC-L)2] and [Ti(η5-C5H 4Me)2(OOC-L)2] with different carboxylato ligands 5 of the type [Ti(η -C5 H5 )2 (OOC-L)2 ] and [Ti(η5 -C5 H4 Me)2 (OOC-L)2 ] with different carboxylato such as 3-methoxyphenylacetato, 4-methoxyphenylacetato, 1,4-benzodioxane-6-carboxylato, 2,5ligands such as 3-methoxyphenylacetato, 4-methoxyphenylacetato, 1,4-benzodioxane-6-carboxylato, dimethyl-3-furoato, and 4-(4-morpholinyl)benzoato (Scheme 6). All the carboxylate compounds 61– 2,5-dimethyl-3-furoato, and 4-(4-morpholinyl)benzoato (Scheme 6). All the carboxylate compounds 70 showed a higher cytotoxic activity than [Ti(η5-C5H55)2Cl2] or [Ti(η5-C5H4Me) 2Cl2] against ovarian 61–70 showed a higher cytotoxic activity than [Ti(η -C5 H5 )2 Cl2 ] or [Ti(η5 -C5 H4 Me)2 Cl2 ] against cell line (A2780), with IC50 values from 40.54 ± 4.39 to 82.14 ± 8.95 µM (Table 5). In addition, the DNA ovarian cell line (A2780), with IC50 values from 40.54 ± 4.39 to 82.14 ± 8.95 µM (Table 5). In addition, binding studies carried out in simulated body fluid showed the weak interaction of the titanocene the DNA binding studies carried out in simulated body fluid showed the weak interaction of the compounds with DNA [68]. titanocene compounds with DNA [68].

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16 of 23 16 of 23 16 of 23 O O

OH OH

S S

+2 +2 Me Me

+ 2 NEt + 2 NEt33

Me Me

O O

Me Me

O O

- 2 NHEt Cl - 2 NHEt33Cl

S S

O O

Ti Ti O O

S S

Me Me Me Me

Me Me Ti Ti

59 59

Cl Cl Cl Cl

O O +2 +2

43 43

S S

Me Me

OH OH Me Me

+ 2 NEt + 2 NEt33

Me Me

O O

Me Me

O O

Ti Ti

O O

- 2 NHEt Cl - 2 NHEt33Cl

O O

Me Me

S S Me Me Me S Me S

Me Me

Me Me 60 60

Scheme 5. 5. Synthesis Synthesis of of alkenyl-substituted alkenyl-substituted titanocene(IV) titanocene(IV) derivatives derivatives with with carboxylato carboxylato ligands. ligands. Scheme with carboxylato ligands. R R

R R Ti Ti

Cl Cl Cl Cl

Et3N N 22 Et 3 2 R-COOH ++ 2 R-COOH - 2 NHEt Cl - 2 NHEt 3Cl

Ti Ti

3

R' R'

-H -H -Me -Me -H -H

OMe OMe MeO MeO

-Me -Me

Compound Compound

R R

61 61

-H -H

62 62

-Me -Me

63 63

-H -H

64 64

-H -H

O O

65 65

-Me -Me

O O

66 66

-Me -Me

R' R' R' R'

O R O R

R R R R

O O

R' R' O O

Compound Compound 67 67 68 68 69 69

N O N O

70 70

Scheme 6. Synthesis Synthesis of alkenyl-substituted alkenyl-substituted titanocene(IV) derivatives derivatives with with carboxylato carboxylato ligands. ligands. Scheme Scheme 6. 6. Synthesis of of alkenyl-substituted titanocene(IV) titanocene(IV) derivatives with carboxylato ligands.

Our group group also also synthesized synthesized titanocene titanocene compounds compounds containing containing the the α,α’-dimercapto-o-xylene α,α’-dimercapto-o-xylene as as Our Our group also synthesized titanocene compounds containing the α,α’-dimercapto-o-xylene as a thiolato ligand ligand (71 (71 and and 72, 72, Figure Figure 13) 13) and and tested tested their their efficacy efficacy against against human human tumor tumor cell cell lines lines HeLa, HeLa, aa thiolato thiolato ligand(Table (71 and Figure 13)the andbiological tested their efficacy against human cell lines of HeLa, Fem-x, K562 K562 4) 72, [36]. When activity was analyzed, antumor improvement the Fem-x, (Table 4) [36]. When the biological activity was analyzed, an improvement of the 5 5 Fem-x, K562 (Table 4) [36]. When the biological activity was analyzed, an improvement of the cytotoxic cytotoxic activity activity was was observed observed compared compared with with [Ti(η [Ti(η5-C -C55H H55))22Cl Cl22]] and and [Ti(η [Ti(η5-C -C55H H44Me) Me)22Cl Cl22]] against against the the cytotoxic 5 -C H ) Cl ] and [Ti(η5 -C H Me) Cl ] against the cell lines activity was observed compared with [Ti(η 5higher 5 2 cytotoxicity 2 572 4and a2 slight 2 cell lines tested K562, HeLa, and Fem-x with a of preference against cell lines tested K562, HeLa, and Fem-x with a higher cytotoxicity of 72 and a slight preference against tested K562, 4) HeLa, K562 (Table (Table [36]. and Fem-x with a higher cytotoxicity of 72 and a slight preference against K562 K562 4) [36]. (Table 4) [36].

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17 of 23 17 of 23 17 of 23

S S Ti Ti S S

Me Me S S Ti Ti S S

Me Me

72 72

71 71

Figure 13. Titanium(IV) complexes with dmox as ligand. Figure 13. Titanium(IV) complexes with dmox as ligand.

Our group also synthesized two titanium(IV) complexes complexes anchored by a tripodal tripodal diamine diamine anchored by Our group also synthesized two titanium(IV) complexes anchored a bis(phenolate) ligands (73 and 74) which showed hydrolytic stability and a high cytotoxic activity bis(phenolate) ligands ligands(73 (73and and which showed hydrolytic stability high cytotoxic bis(phenolate) 74)74) which showed hydrolytic stability and a and high acytotoxic activity against against HeLa, HeLa, K562, K562, Fem-x and MDA-MB-453 with IC50ICvalues between 22.4 1.2 and activity and MDA-MB-453 between22.4 22.4±±±1.2 1.2 and against HeLa, K562, Fem-x Fem-x and MDA-MB-453 withwith IC50 values between 50 values 48.9 ±±0.8 µM [69]. 0.8µM µM[69]. [69]. 48.9 ± 0.8 After the extensive study study of our group on the biological applications of titanocene derivatives After the extensive extensive After study of our group on the biological applications of titanocene derivatives with different substituents either at the Cp ring or directly bound to titanium (31–72), we observed with different substituents either at the Cp ring or directly bound to titanium (31–72), we observed that most the synthesized compounds (especially those containing thiolato or carboxylato ligands) that most of of the the synthesized synthesized compounds compounds (especially (especially those those containing containing thiolato thiolato or or carboxylato carboxylato ligands) ligands) show a low hydrolytic stability. Therefore, the anticancer anticancer action of these compounds is usually due show a low hydrolytic stability. Therefore, the action of these compounds is usually due to decomposition products which are soluble ininwater and/or DMSO and thatthat are formed after the decomposition products productswhich whichare aresoluble solublein water and/or DMSO formed to decomposition water and/or DMSO andand that are are formed afterafter the elimination of one or more ligands [70]. Thus, we focused on the formulation of these titanium the elimination of one or more ligands [70]. Thus, we focused on the formulation of these titanium elimination of one or more ligands [70]. Thus, we focused on the formulation of these derivatives via functionalizationof of mesoporoussilica-based silica-based nanostructured materials such as MCMmaterials such as as MCM-41, derivatives via functionalization functionalization ofmesoporous mesoporous silica-basednanostructured nanostructured materials such MCM41, SBA-15, MSU-2, alumina or hydroxyapatite [71–74] in order to overcome the problems associated SBA-15, MSU-2, alumina or hydroxyapatite [71–74] in order to overcome the problems associated with 41, SBA-15, MSU-2, alumina or hydroxyapatite [71–74] in order to overcome the problems associated with the low hydrolytic stability of titanocene compounds. the low stability of titanocene compounds. with thehydrolytic low hydrolytic stability of titanocene compounds. During these studies and in an effort to encapsulate titanocene compounds on KIT-6, additional in an an effort effort to to encapsulate encapsulate titanocene titanocene compounds compounds on on KIT-6, KIT-6, additional During these studies and in alkenyl substituted (75–80) [75] and ether-substituted (81–84) [76] titanocene(IV) dichloride alkenyl substituted [75][75] and ether-substituted (81–84) [76] titanocene(IV) dichloride compounds substituted(75–80) (75–80) and ether-substituted (81–84) [76] titanocene(IV) dichloride compounds were synthesized (Figure 14), characterized and tested in vitro against a wide variety of were synthesized (Figure 14), (Figure characterized and tested in vitro against a wide variety of cancer compounds were synthesized 14), characterized and tested in vitro against a wide varietycell of cancer cell lines (Table 7). We observed a very high cytotoxicity (IC 50 values in the range of those lines (Table 7). We observed a very high a cytotoxicity values in the range of those described in cancer cell lines (Table 7). We observed very high (IC cytotoxicity (IC 50 values in the range of those 50 described in the literature for thecytotoxic most active cytotoxic titanocene such compounds such as titanocene-Y the literature for the most active titanocene compounds as titanocene-Y synthesized described in the literature for the most active cytotoxic titanocene compounds such as titanocene-Y synthesized by Tacke) with high selectivity towards cancer cell lines. by Tacke) with selectivity towards cancer cell lines. synthesized by high Tacke) with high selectivity towards cancer cell lines. R' R' Ti Ti

Cl Cl Cl Cl

R R

R' R' R R

Ti Ti

(75) R = H, R' = Me (75) (76) R R= = H, H, R' R' = = Me Ph (76) (81) R R= = H, Me,R'R'= =PhMe (81) (82) R R= = Me, Me, R' R' = = Me Ph (82) R = Me, R' = Ph

Cl Cl Cl Cl

R R

R' R' R R

Me Si Me33Si

R R

R' R R' R (77) R = H, R' = Me (77) (78) R R= = H, H, R' R' = = Me Ph (78) (83) R R= = H, Me,R'R'= =PhMe (83) (84) R R= = Me, Me, R' R' = = Me Ph (84) R = Me, R' = Ph

R R

Ti Ti

OMe OMe OMe OMe Cl Cl Cl Cl

(85) R = Me (85) (86) R R= = Me Ph (86) R = Ph

R R

Ti Ti

Ti Ti

Cl Cl Cl Cl

R R R R

(79) R = H, R' = Me (79) (80) R R= = H, H, R' R' = = Me Ph (80) R = H, R' = Ph

OMe OMe OMe OMe Cl Cl Cl Cl OMe OMe OMe OMe

R R (87) R = Me (87) (88) R R= = Me Ph (88) R = Ph

Figure 14. Titanocene(IV) complexes with alkenyl- or ether-substituents at the Cp rings. Figure Cp rings. rings. Figure 14. 14. Titanocene(IV) Titanocene(IV) complexes complexes with with alkenylalkenyl- or or ether-substituents ether-substituents at at the the Cp

Finally, after the incorporation of the compounds in KIT-6, a higher Ti-uptake by the treated Finally, after the incorporation of the compounds in KIT-6, a higher Ti-uptake by the treated cancer cells (from 4% to 23% of the initial amount of Ti) was observed when compared with the “free” cancer cells (from 4% to 23% of the initial amount of Ti) was observed when compared with the “free”

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Table 7. IC50 (µM) of titanocene compounds (75–87) on different cancer cell lines. IC50 ± SD

Compound 75 76 77 78 79 80 81 82 83 84 85 86 87 [Ti(η5 -C5 H5 )Cl2 ]

A549

A2780

DLD-1

MCF-7

Hek-293

HCT-118

78 ± 7 45 ± 13 123 ± 13 167 ± 4

100.07 ± 7.85 7.49 ± 1.06 65.67 ± 12.25 56.23 ± 11.20 7.83 ± 1.54 26.57 ± 1.12 28.46 ± 4.66 25.72 ± 5.47 41.42 ± 6.86 16.91 ± 1.66 124.78 ± 4.36

250.32 ± 17.20 96.67 ± 6.89 152.75 ± 33.48 97.65 ± 10.45 29.67 ± 3.55 136.33 ± 13.97 46.60 ± 5.11 37.44 ± 5.65 69.62 ± 8.54 41.06 ± 1.94 >200

143.80 ± 16.39 103.13 ± 19.42 62.15 ± 28.40 90.19 ± 10.95 12.55 ± 2.79 170.33 ± 32.61 8.84 ± 1.09 16.45 ± 3.64 10.80 ± 2.44 5.92 ± 1.00 65 ± 8 56 ± 10 198 ± 33 -

293.90 ± 8.42 232.67 ± 21.75 183.70 ± 15.76 157.41 ± 28.98 326.50 ± 23.22 134.43 ± 10.86 174.30 ± 41.78 79.92 ± 8.49 95.39 ± 15.20 70.77 ± 10.96 -

54 ± 12 64 ± 9 150 ± 18 -

Finally, after the incorporation of the compounds in KIT-6, a higher Ti-uptake by the treated cancer cells (from 4% to 23% of the initial amount of Ti) was observed when compared with the “free” titanocene compounds giving clear insights on the positive effect of the encapsulation with nanostructured silica. 5. Metallodrugs Based on Other Metals As previously explained in the introduction of this review, non-platinum compounds are being considered as an alternative to cisplatin-like compounds because their preclinical trials indicate that they might be capable of reducing the relatively high number of side effects associated with platinum treatments. In recent years, promising results have been obtained using other metallodrugs of main group or transition metal complexes, however, less attention has been paid to lanthanide and actinide compounds [77]. In this context, our research group synthesized a series of metal complexes of Y(III), La(III), Ce(III), Nd(III), Sm(III), and Yb(III) with p-substituted-cinnamate and p-substituted phenylacetate ligands. The toxicity of these compounds against immunocompetent cells (mice macrophages and erythrocytes) was tested. In addition, the cytotoxicity against specific human cancer cell lines such as HL60 (human promyelocytic leukemia), K562 (human erythromyeloblastoid leukemia), and MCF7 (breast cancer) was studied. All the lanthanide compounds tested showed a dose-dependent toxic activity which began to be significant from 400 µM. The cytotoxic activities of all the compounds synthesized were very low with IC50 values between 542.7 and >750 µM. This indicates that the studied lanthanide complexes with cinnamate and/or phenylacetate ligands were not appropriate for cancer therapy [78]. In spite of the discouraging results with lanthanides, recently, our group studied the cytotoxic properties of a novel Dy-based metallodrug [DyNa(ampy)4 ]n (88), a metalorganic framework prepared from 5-aminopyridine-2-carboxilic acid (Hampy) as ligand [79]. The structure of this compound was determined by X-ray crystallography. [DyNa(ampy)4 ]n (88) was tested against colon carcinoma cells, HT-29, DLD-1, and Caco-2 (Table 8) showing a moderate cytotoxicity especially against DLD-1. More interestingly, we observed that the combination of treatment with the dysprosium compound with a short exposure to a magnetic field led to a reduction of proliferation in all the cell lines. In addition, after short exposure to a magnetic field the multidrug-resistant properties of this 1D-MOF changed. Thus, our multidisciplinary preliminary study relating magnetic properties with cytotoxicity of MOFs is a very interesting starting point for further studies of different magnetic lanthanides or actinides in cancer therapy.

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Table 8. Cytotoxic results of [DyNa(ampy)4 ]n (88) on HT-29, DLD-1, and Caco-2. Compound

[DyNa(ampy)4 ]n (88)

Cell Line

IC50 (µM)

Log IC50

SD LogIC50

HT-29 DLD-1 Caco-2

87.1 174.9 248.8

1.940 2.243 2.396

0.201 0.125 0.117

6. Conclusions The use of metallodrugs is still a field of upmost interest for the scientific community and a high number of reports on this topic are being published. In this context our group has carried out basic scientific work in the field of metallodrugs of gallium, tin, and titanium and demonstrated a structure activity relationship which may be of interest for drug-design. It seems clear that the limitations of the metallodrugs regarding side-effects, low solubility, and low bioavailability in the human body due to their low hydrolytic stability are very difficult to address from a monodisciplinary point of view. However, these problems associated with metallodrugs can be overcome by the use of a mixed metallodrug-nanotechnological approaches such as encapsulation of metal-based drugs in different nanostructured materials. For example, the use of liposomes, lipid nanocapsules, human proteins, ceramic materials, carbon nanotubes, and metal or metal oxide nanoparticles with anticancer metallodrugs should be of great interest. Thus, by combining nanomaterials and metallodrugs to obtain more potent and reliable formulations, we can predict that a bright future still lies ahead for metal-based drugs in anticancer chemotherapy. Acknowledgments: We would like to thank to all the people involved in the work of our group during the last 10 years. In addition, we would like to thank Ministerio de Economía y Competitividad, Spain (Grant No. CTQ2015-66164-R) and the Universidad Rey Juan Carlos-Banco de Santander (Excellence Group QUINANOAP) for their support. Author Contributions: Younes Ellahioui, Sanjiv Prashar and Santiago Gómez-Ruiz contributed in the search, explanation and discussion of the cited literature references. Conflicts of Interest: The authors declare no conflict of interest.

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