Pt(IV) Bifunctional Prodrug Containing 2-(2-Propynyl)octanoato Axial

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Pt(IV) bifunctional prodrug containing 2-(2-propynyl)octanoato axial ligand: induction of immunogenic cell death on colon cancer Maurizio Sabbatini, Ilaria Zanellato, Mauro Ravera, Elisabetta Gabano, Elena Perin, Beatrice Rangone, and Domenico Osella J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01860 • Publication Date (Web): 16 Mar 2019 Downloaded from http://pubs.acs.org on March 17, 2019

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Journal of Medicinal Chemistry

Pt(IV) bifunctional prodrug containing 2-(2propynyl)octanoato axial ligand: induction of immunogenic cell death on colon cancer.# Maurizio Sabbatini,‡ Ilaria Zanellato,‡ Mauro Ravera, Elisabetta Gabano, Elena Perin, Beatrice Rangone, Domenico Osella* Dipartimento di Scienze e Innovazione Tecnologica, Università del Piemonte Orientale, Viale Michel 11, 15121 Alessandria, Italy.

KEYWORDS Pt(IV) prodrug, epigenetic, antiproliferative activity, oxaliplatin, immunogenic cell death, CD8, colon cancer.

#

partially presented at the XVIII Workshop on PharmaBiometallics, Arezzo (Italy), 22-24 March

2019. 1 ACS Paragon Plus Environment

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ABSTRACT. The synthesis, characterization and in vitro activity of cyclohexane-1R, 2R-diaminebased Pt(IV) derivative containing the histone deacetylase inhibitor rac-2-(2-propynyl)octanoato, namely

(OC-6-44)-acetatodichlorido(cyclohexane-1R,2R-diamine)(rac-2-(2-propynyl)octanoato)-

platinum(IV), is reported together its isomers containing enantiomerically-enriched axial ligands. These Pt(IV) complexes showed comparable activity, of two orders of magnitude higher than reference drug oxaliplatin on three human (HCT 116, SW480 and HT-29) and on one mouse (CT26) colon cancer cell lines. In vivo experiments were carried out on immunocompetent BALB/c mice bearing the same syngeneic tumor. The complex (OC-6-44)-acetatodichlorido(cyclohexane-1R,2Rdiamine)(rac-2-(2-propynyl)octanoato)platinum(IV) showed higher tumor mass Pt accumulation than oxaliplatin, due to its higher lipophilicity, with negligible nephro- and hepato-toxicity when administered by iv. A remarkable tumor mass invasion by cytotoxic CD8+ T lymphocytes, following the Pt(IV) treatment, indicated a strong induction of immunogenic cell death (ICD).

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INTRODUCTION Even in the era of targeted- and immune stimulation- anticancer therapy, the conventional cytotoxic (DNA-damaging) drugs are still very useful, especially against very aggressive tumors. In this context Pt(II) drugs (cisplatin, carboplatin and oxaliplatin, Chart 1) play an important role, especially in combination therapy, being present in about 50% of anticancer protocols, according to Martindale’s Complete Drug Reference.1 Other complexes based on different metals have also been studied as antitumor drugs.2-10 Remaining in the Pt-drug area, Pt(IV) conjugates can provide a great advantage in the combinatory approach. Indeed, the well-established Pt(IV) chemistry allows to design multifunctional prodrugs, often called “combo”.11, 12 One or two adjuvant/synergistic agents (coordinated to the metal via carboxylic functionalities) can be added to the parent cytotoxic Pt(II) square-plane scaffold in axial position. These Pt(IV) derivatives are quite inert to ligand substitution or hydrolysis, and reach intact tumor cells, minimizing the off-target reactions and the side-effects typical of the more reactive Pt(II) progenitors.

Chart 1. Pt(II) drugs worldwide approved for clinical use

Finally, Pt(IV) prodrugs can be reduced in the hypoxic intracellular milieu of tumor cells to the corresponding cytotoxic Pt(II) metabolites with the simultaneous loss of the adjuvant or synergistic agents from axial positions (activation by reduction, Scheme 1).13-17 Moreover, the coordination of 3 ACS Paragon Plus Environment


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the amphiphilic carboxylates to the Pt core neutralizes their negative charges, whereas their organic chains protrude towards the exterior, thus generating very lipophilic conjugates. This combo drug enters cells via passive diffusion (the main if not the only mechanism of cellular uptake) more efficiently than the free components (synergistic cellular accumulation).18

Scheme 1. Scheme of the 2e- reduction of a Pt(IV) complex containing two am(m)ine (A), two chloridos or a dicarboxylato (X) as equatorial ligands and two carboxylatos (L) axial ligands.

Most of the multi-functional Pt(IV) prodrugs bear epigenetic agents, such as the histone deacetylase inhibitors (HDACi) 4-phenylbutyric acid, valproic acid, and the very active 2-(2-propynyl)octanoic acid, POA.18-22 HDACi increase the acetylation level of histones in chromatin, thus weakening the histone-DNA interactions and inducing chromatin relaxation, thus promoting tumor growth suppression and apoptosis induction by DNA-damaging chemotherapeutics.23-26 Moreover, HDACi alter the activity of several genes, modulating the expression and functions of apoptosis-related, DNA-repair proteins.27, 28 HDACi also modulate genes involved in the so-called immunoescape, the mechanism that allows cancer cells not to be found by the immune system,29,

30

leading to its

reversion.31 As a result, HDACi-based combination therapies have gained much attention in cancer treatment.27, 28 Very recently, the cisplatin-based combo (OC-6-44)-acetatodiamminedichlorido(2-(2propynyl)octanoato)platinum(IV) was reported to exhibit excellent in vitro antiproliferative activity on a large set of human tumor cell lines and in vivo anticancer activity on mice bearing Lewis tumor.20 Nowadays, dozens of such antitumor Pt(IV) prodrugs have been designed, synthesized and tested. The majority is based on cisplatin scaffold, whereas the oxaliplatin framework is less employed. 4 ACS Paragon Plus Environment

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Importantly, oxaliplatin (Eloxatin™) is used for treatment of colorectal cancer (where cisplatin and carboplatin exhibit minor activity), being FOLFOX (5-FU, leucovorin, and oxaliplatin) and CAPEOX (capecitabine and oxaliplatin) among the most used combination regimens in clinics.32,

33

The

anticancer effect of oxaliplatin on colon cancer cells is not simply related to the DNA damaging activity, but also relies in the reversion of the immune evasion mechanisms.34 The reversion of immunoescape consists into activation of immunitary T cells able to recognize and kill cancer cells. Considering the ability of immunitary cells to establish a specific immunological memory, T cells activation can ensure a protection against the specific malignant cell transformation and invasion, promoting a long-lasting remission. Therefore, stimulation of the antitumor T cell immunity is nowadays considered the forefront strategy for the next-generation of anticancer therapies.35, 36 Thus, a suitable Pt(IV) conjugate, able to produce after reduction i) the desired, electrophilic (alkylating) agent [Pt(dach)]2+ (dach = cyclohexane-1R,2R-diamine), and ii) the very active HDACi POA

has

been

designed.

The

dichlorido

analogue

of

oxaliplatin

(i.e.

(SP-4-2)-

dichlorido(cyclohexane-1R,2R-diamine)platinum(II), [PtCl2(dach)], Chart 1), might be a good synthon for the design of an efficient Pt(IV) combo, specifically directed towards colon cancer and endowed of increased immunogenic activity. This Pt(II) complex was originally tested along with oxaliplatin, but it was discarded because of its very poor water solubility and quick hydrolysis (aquation).37 However, [Pt(dach)]2+ exhibited good anticancer activity when gradually released from block copolymer micelles or Lipiodol suspensions in a phase I clinical trial to treat hepatocellular carcinoma.38 A transformation in a Pt(IV) complex may be a way to slow down its activation.37, 39-41 In this work, the in vitro activity on colon cancer cells of the dach-based (OC-6-44)acetatodichlorido(cyclohexane-1R,2R-diamine)(rac-2-(2-propynyl)octanoato)platinum(IV), 1, was compared to that of its isomers (OC-6-44)-acetatodichlorido(cyclohexane-1R,2R-diamine)(2R-(2propynyl)octanoato)platinum(IV),

1R,

and

(OC-6-44)-acetatodichlorido(cyclohexane-1R,2R-

diamine)(2S-(2-propynyl)octanoato)platinum(IV), 1S, containing enantiomerically-enriched POA (Scheme 2). Moreover, complex 1 was administered on BALB/c mice bearing CT26 syngeneic mouse 5 ACS Paragon Plus Environment


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colon carcinoma both per os and iv to verify its anticancer and immunogenic activity. The comparison between the two administration routes was also a specific focus of the present work, in order to verify the better administration of complex 1 in term of efficacy/toxicity balancing. Indeed, epigenetic drugs are gaining increasing attention for the therapy of colorectal cancer.42


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Scheme 2. Synthetic pathways for the synthesis of compounds 1, 1R, and 1S: i) H2O2 in CH3COOH (rt, 3-4 h); ii) oxalyl dichloride in CH2Cl2, DMF cat. (rt, overnight); iii) in acetonitrile + pyridine (microwave-assisted heating, 55 °C, 1 h). The numbering scheme used for the assignment of the NMR 7 ACS Paragon Plus Environment


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signals is also reported (see Experimental Section). The cisplatin-based derivative 2 was added for comparison purposes.

RESULTS AND DISCUSSION Synthesis and characterization of the complexes. The synthesis of the Pt(IV) complexes started with the oxidation of [PtCl2(dach)] with H2O2 in acetic acid, to give the intermediate 1-OH (Scheme 1).43 This synthon was turned into the corresponding dicarboxylato complex 1 by reaction with POA. For this synthesis, a commercial racemic mixture of POA (rac-POA) was converted into its acyl chloride using oxalyl dichloride in the presence of a catalytic amount of DMF.20 The resulting acyl chloride reacted with 1-OH, in the presence of pyridine, by microwave-assisted heating to give 1. Such a reaction of the enantiomerically pure 1-OH with rac-POA generated two diastereoisomers in 1:1 molar ratio, namely 1R and 1S, distinguishable by HPLC (see Supporting Information, Figure S1) However, instead of employing a solvent- and time-consuming preparative HPLC separation, 1R and 1S were synthesized starting from the corresponding enantiomerically-enriched isomers of POA, namely R-POA and S-POA, obtained from rac-POA by the classical organic separation (see Experimental section).44 The isomers R-POA and S-POA were obtained with 99.8% purity and about 80% enantiomeric excess (ee). The ee was evaluated by the literature data for the enantiomerically pure compounds45 and the values were confirmed by the areas of the chromatographic peaks. The optical rotatory power of all the ligands and complexes was measured. The dach-based complexes 1R and 1S showed much higher rotation angles with respect those of the parent ligands (see Supporting Information, Table S1), but their diastereomeric excess (de) is the same of the ee of the parent ligands. To ensure that the results obtained with the 1 derivatives are not artifacts, UV-vis spectra of 1R and 1S were run allowing to verify that no strong visible absorption occurred near to 8 ACS Paragon Plus Environment

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the sodium D line ( = 598 nm).46 For comparison purpose, the optical rotatory power of similar dach-containing complexes (namely [PtCl2(dach)], (OC-6-44)-acetatodichlorido(cyclohexane1R,2R-diamine)eptanoatoplatinum(IV)

and

(OC-6-33)-dichlorido(cyclohexane-1R,2R-diamine)-

dieptanoatoplatinum(IV))47 was also measured, confirming that the main contribution to the optical rotatory power of the final complexes came from the coordinated dach. All the complexes were characterized by HPLC-MS and multinuclear NMR spectroscopy. As reported above, 1R and 1S diastereoisomers showed different retention time in HPLC and different NMR signals (see Supporting Information, Figures S1-S14) as expected. The low solubility in water of the three Pt(IV) compounds made hard the study of their stability in physiological conditions. Therefore, they were challenged with water solutions containing 80% v/v ethanol, at room temperature: all the compounds remained intact (>95%) within 5 d.

In vitro activity. The newly synthesized complexes 1, 1R, and 1S, along with the reference compounds rac-POA, cisplatin, oxaliplatin, [PtCl2(dach)] and the racemic cisplatin-based derivative (OC-6-44)-acetatodiamminedichlorido(2-(2-propynyl)octanoato)platinum(IV), namely 2 (Scheme 2)20 were assayed on three different human colon cancer cell lines, namely HCT 116, SW480 and HT-29, having different histologic history ranging from D (more aggressive) to B (less tumorigenic) respectively, in the “old” Dukes’ classification of colon cancer grade. Additionally, all the compounds were tested on CT26 mouse colon carcinoma cell line in the prospect of in vivo experiments (Table 1). The results showed that the activity of the enriched isomers 1R and 1S was almost identical within the experimental error to that of 1, as already observed for the cisplatin-based analogue 2.20 As expected, the lipophilic Pt(IV) conjugates 1, 1R, 1S and 2 were more active than the Pt(II) reference models of one-two orders of magnitude. Importantly, the free ligand POA is almost inactive within the same range of concentrations (IC50 > 100 µM), likely due to its scarce propensity to enter cells being in form of amphiphilic anion at physiological pH. 9 ACS Paragon Plus Environment


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Table 1. Antiproliferative activity (IC50, M) obtained after 72 h of continuous treatment of human colon carcinomas HCT 116, HT-29, and SW490, and mouse colon carcinoma CT26 cell lines. Data are means ± sem of at least three independent replicates.

Compound

IC50 [M] HCT 116

HT-29

SW480

CT26

POA

(4.4±0.2)×10-4 (9.7±0.8)×10-4 (4.7±0.9)×10-4 (2.0±0.2)×10-4

Cisplatin

(2.3±0.3)×10-6 (2.7±0.1)×10-6 (2.6±0.2)×10-6 (4.1±0.6)×10-7

Oxaliplatin

(1.2±0.1)×10-6 (9.2±0.9)×10-7 (4.7±0.2)×10-7 (1.5±0.5)×10-6

[PtCl2(dach)] (2.7±0.1)×10-7 (2.4±0.2)×10-7 (1.8±0.1)×10-7 (4.6±0.3)×10-7 1

(1.0±0.1)×10-8 (1.5±0.6)×10-8 (8.1±0.8)×10-9 (7.1±0.6)×10-9

1R

(9.1±0.6)×10-9 (1.5±0.1)×10-8 (7.4±0.6)×10-9 (8.8±0.6)×10-9

1S

(1.1±0.7)×10-8 (1.3±0.4)×10-8 (8.2±0.7)×10-9 (6.3±0.8)×10-9

2

(2.9±0.9)×10-8 (8.0±0.8)×10-8 (3.7±0.6)×10-8 (4.4±0.3)×10-8

It is widely recognized that the cellular transmembrane permeability of non-electrolytes correlates with their logarithm of the octanol/water partition coefficient (LogP) derived from the traditional shake-flask method. On the other hand, this procedure may be not completely reliable in the case of molecules that are extremely lipophilic or hydrophilic. For this reason, shake-flask method is often replaced by HPLC measurements, where the retention is due to partitioning between C18 chains of the stationary phase (representing the cellular membrane) and aqueous eluent (representing the water inside and outside cells).48, 49 The calculation of the capacity factor k’ (k’ = (tR - t0) / t0, where tR = retention time of the compound under investigation, and t0 = column dead time) gives a parameter closer to LogP and, although the scale-interval of log k’ is different from that of LogP, the meaning is the same (i.e. higher the values, higher the lipophilicity). Therefore, 1R and 1S (log k’ = 0.58 and 0.62, respectively) are more lipophilic than all Pt(II) drugs (log k’ = -0.38 for cisplatin and -0.28 for both oxaliplatin and [PtCl2(dach)]). This higher lipophilicity is due to the presence of the one 10 ACS Paragon Plus Environment

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lipophilic POA (log k’ = 0.67) as axial ligand.20, 21, 50 These data are in tune with the Pt cellular uptake, expressed as accumulation ratio (AR), being the ratio between the internal and the external Pt concentration, experimentally verified by inductively coupled plasma-mass spectrometry (ICP-MS) on cells treated with the Pt complexes (Figure 1). As expected by the log k’ values, the AR of 1, 1R and 1S are very similar to each other (within experimental error), and by far higher than those of the reference drugs. Based on the above reported in vitro results, a set of in vivo experiments was planned simply employing 1, since its activity was comparable with the pure diastereoisomers 1R and 1S.

Figure 1. Accumulation ratio (AR) of 1, 2, [PtCl2(dach)], cisplatin, and oxaliplatin, measured on HCT 116 colon cancer cells treated for 4 h with the compounds. Data are means±sem of at least three independent replicates and were compared to 1 by means of a one-way ANOVA-Turkey test (no indication = not significant; * p 500 nm that may interfere with the resazurin assay. In each experiment, cells were challenged with the drug candidates at different concentrations and the final data were calculated from at least three replicates of the same experiment performed in triplicate. The fluorescence of 8 wells containing medium without cells were used as a blank. Fluorescence data were normalized to 100% cell viability for nontreated cells. Half inhibiting concentration (IC50), defined as the concentration of the drug reducing cell viability by 50%, was obtained from the dose-response sigmoid using Origin Pro (version 8, Microcal Software, Inc., Northampton, MA, USA). Hoechst 33342 staining. HCT 116 cells (2×105) were seeded on Nunc™ Lab-Tek™ 4-chamber slides and allowed to attach for 24 h. The following day, the treatment was performed to a final concentration of 5 mM POA, 1 µM complex 1 and [PtCl2(dach)]. After 24 h, the medium was replaced with the staining solution, consisting of 5 ng mL-1 Hoechst 33342 in PBS. Cells were incubated in the 26 ACS Paragon Plus Environment

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dark for 5 min, washed with PBS and immediately observed using a standard DAPI filter set (excitation  = 350 nm, emission  = 461 nm) of a fluorescence microscope (Zeiss Axiolab), equipped with a digital photo camera (Nikon digital Sights, DS-U3). Pictures were taken at 40× magnification. Cellular accumulation. Around 5106 HCT 116 cells were treated for 4 h with the complexes under investigations (10 M for cisplatin and oxaliplatin, 1 M for 1 and 2). At the end of the exposure, cells were washed three times with phosphate buffered saline (PBS), detached from the Petri dishes using 0.05% Trypsin 1X + 2% EDTA (HyClone, Thermo Fisher) and harvested in fresh complete medium. An automatic cell counting device (Countess®, Life Technologies) was used to measure the number and the mean diameter from every cell count. Moreover, 100 µL of medium were taken out from each sample at time zero to check the extracellular Pt concentration. Cells were transferred into a borosilicate glass tube and centrifuged at 1100 rpm for 5 min at room temperature. The supernatant was carefully removed by aspiration, while about 200 µL of the supernatant were left in order to limit the cellular loss. Cellular pellets were stored at -80 °C until mineralization. Platinum content determination was performed by ICP-MS (Thermo Optek X Series 2). Instrumental settings were optimized to yield maximum sensitivity for platinum. For quantitative determination, the most abundant isotopes of platinum and indium (used as internal standard) were measured at m/z 195 and 115, respectively. Mineralization was performed by addition of 70% w/w HNO3 to each sample (after defrosting), followed by incubation for 1 h at 60 °C in an ultrasonic bath. Before the ICP-MS measurement, the HNO3 was diluted to a final 1% concentration. To calculate the Pt cellular concentration, the total cellular volume of each sample was obtained considering the mean cell diameter and cell number. The ratio between the internal and the external cell Pt concentration, namely the Accumulation Ratio (AR), was computed as previously reported.77 In vivo experiments. A total of 18 BALB/c male mice (Envigo RMS srl, San Pietro al Natisone, Udine, Italy) 5 weeks old at weight of 20-25 g were fed ad libitum with standard diet (Special Diets Services Ltd, Witham, Essex, England). During the entire period of the study the animals were maintained in conditioned and at limited access environments (mean temperature: 22°C; lighting: 27 ACS Paragon Plus Environment


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controlled to give a daily 12 h photoperiod). All the procedures involving the animals were conducted according to the national and international laws on experimental animal (L.D. 26/2014; Directive 2010/63/EU) and to the approved experimental protocol procedure (Authorization no. 229/2016-PR). No validated non-animal alternatives are known to meet the objectives of the study. After a period of acclimation (5 d) tumor induction was performed as follows. CT26 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 IU mL-1 penicillin and 100 µg mL-1 streptomycin. After two washes with PBS, cells (1.5106) were suspended in 0.2 mL of serum-free medium and injected subcutaneously in the right flank of each mouse. Tumors were let grow approximately 10 d until the tumor become visible in each animal (100-150 mm3). Then, mice were randomly assigned to 6 experimental groups (3 animals per group) and the chemotherapeutic treatments were started. For each administration route (i.e. per os and iv) each group received 1, oxaliplatin, or vehicle. The reference drug oxaliplatin was immediately dissolved before use in double distilled water and the resulting solution was protected from light, whereas 1 was dissolved in vehicle solution (20% v/v Cremophor EL, 20% v/v PEG400 and 60% v/v saline solution). Schedules of two parallel experiments are described in results and discussion. Body weight was recorded every two days during the observation period. Animals obviously in pain or showing signs of severe distress were sacrificed. Criteria for making the decision to sacrifice severely suffering animals, and guidance on the recognition of predictable or impending death, are the subject of an OECD Guidance Document. Tumor growth was followed by electronic caliper measurement every two days. Tumor volume (mm3) was calculated according to the formula (W2  L)/2, where W and L are the maximum width and length of the tumor. Quantitative evaluation of body weight and tumor volume were performed. The antitumor effect was evaluated by comparing the tumor size among groups. Magnetic Resonance Imaging (MRI) acquisitions were performed at the end of observation period on one representative animal for each experimental group. Mice were anesthetized under 0.5-2% isoflurane gas anesthesia (O2 95.0%). MRI was performed on a 7 Tesla MRI scanner (Pharmascan MRI 7T, Bruker, Germany) 28 ACS Paragon Plus Environment

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using Rare sequences. The T2-weighted images were acquired with the optimized parameters for high-resolution images with a good contrast to detect the tumor mass. Organs from each animal of experimental groups (tumor, kidneys, spleen, liver, heart, and lungs) were collected at sacrifice and divided in two equal parts: one part was formalin-fixed and paraffin-embedded and the other one was stored at -20 °C for quantitative determination the platinum content. The Pt accumulation was measured on dried samples of 5-20 mg of each organ in triplicate after microwave-assisted digestion (microwave power = 1200 W, temperature = 200 °C, digestion time = 45 min, Milestone Start D, Milestone Srl, Sorisole, Italy) in a mixture containing 80% v/v of HNO3 (70% w/w) and 20% v/v of H2O2 (30% w/w) and following dilution with 1% v/v HNO3 by means of ICP-MS analysis. Slices (5 µm thick) were obtained from the paraffin embedded organs by rotative microtome and stained by haematoxylin/eosin procedure for microanatomical examination to identify tissue suffering and alteration. CD8 and CD68 immunohistochemistry was performed on tumor tissue slides of several experimental groups to identify cytotoxic lymphocytes (namely CD8+ T cells) and macrophages (namely CD68+ cells); for both rabbit polyclonal anti-mouse (Abcam: ab203035 and ab125212, Cambridge, UK) was used as primary antibody, revealed using biotinylated secondary antibody and avidin/biotinylated enzyme complex (Vectastain Elite kit, Vector Laboratories, Burlingame, CA, USA) together with 3,3-diaminobenzidine as chromogen (DAB chromogen kit Vector Laboratories, Burlingame, CA, USA). The Oxidative Stress Detection Kit (OxyIHC, S7450; Millipore USA), was used to perform immunohistochemical evidences about 1-induced oxidative stress in liver, in according with manufacturer instructions.

ASSOCIATED CONTENT Supporting Information. HPLC, ESI-MS, NMR characterization, and optical activity of complexes 1, 1R, and 1S; mean body weight of mice treated with 1, images illustrating the 29 ACS Paragon Plus Environment


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alterations in the organ anatomy and structure, the increase of carbonyl modified proteins as a consequence of direct ROS action, and the distribution of CD8+ positive lymphocytes and CD68+ macrophages in spleen and tumor tissues. Molecular Formula Strings with IC50. AUTHOR INFORMATION Corresponding Author *e-mail: [email protected]. Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. ‡These authors contributed equally. ACKNOWLEDGMENT This research is original and has a financial support of the Università del Piemonte Orientale. We acknowledge Euro-BioImaging (http://www.eurobioimaging.eu) for providing access to imaging technologies and services via the Italian Multi-sited Multi-Modal Molecular Imaging (MMMI, Torino, Italy) Node. We were indebted to Inter-University Consortium for Research on the Chemistry of Metals in Biological Systems (CIRCMSB, Bari, Italy) for providing opportunities of stimulating discussion. Thanks are due to Prof. Giorgio Pelosi (University of Parma, Italy) for the measurements of rotatory power and Dr. Flavia Capri (University of Piemonte Orientale, Alessandria, Italy) for the preparation of microanatomical slides. ABBREVIATIONS AR, accumulation ratio; dach, cyclohexane-1R,2R-diamine; DAPI, 4',6-diamidine-2'-phenylindole dihydrochloride; HDACI, histone deacetylase inhibitors; ICD, immunogenic cell death; ICP-MS, inductively coupled plasma-mass spectrometry; k', HPLC capacity factor; POA, 2-(230 ACS Paragon Plus Environment

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Graphic for the Table of Contents 175x48mm (300 x 300 DPI)

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Pt(II) drugs worldwide approved for clinical use 76x67mm (300 x 300 DPI)

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Accumulation ratio (AR) of 1, 2, [PtCl2(dach)], cisplatin, and oxaliplatin, measured on HCT 116 colon cancer cells treated for 4 h with the compounds. Data are means±sem of at least three independent replicates and were compared to 1 by means of a one-way ANOVA-Turkey test (no indication = not significant; * p

Pt(IV) Bifunctional Prodrug Containing 2-(2-Propynyl)octanoato Axial

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