Multiaction Pt(IV) Carbamate Complexes Can Codeliver Pt(II) Drugs and Amine Containing Bioactive Molecules

Tomer Babu, Amrita Sarkar, Subhendu Karmakar, Claudia Schmidt, and Dan Gibson*

ABSTRACT:

Multiaction Pt(IV) prodrugs can overcome resistance associated with the FDA approved Pt(II) drugs like cisplatin. Intracellular reduction of the octahedral Pt(IV) derivatives of cisplatin releases cisplatin and the two axial ligands. When the released axial ligands act synergistically with cisplatin to kill the cancer cells, we have multiaction prodrugs. Most Pt(IV) multiaction prodrugs have bioactive ligands possessing a carboxylate that is conjugated to the Pt(IV) because breaking the Pt(IV)−ligand bond releases the active moiety. As many drugs that act synergistically with cisplatin do not have carboxylates, a major challenge is to prepare multiaction Pt(IV) complexes with drugs that have amino groups or hydroxyl groups such that following reduction, the drugs are released in their active form. Our objective was to prepare multiaction Pt(IV) prodrugs that release bioactive molecules having amino groups. Because we cannot conjugate amino groups to the axial position of Pt(IV), we developed a novel and efficient approach for the synthesis of Pt(IV)−carbamato complexes and demonstrated that following reduction of the Pt(IV), the released carbamates undergo rapid decarboxylation, releasing the free amine, as in the case of the PARP-1 inhibitor 3aminobenzamide and the amino derivative of the HDAC inhibitor SAHA. Pt(IV)−carbamato complexes are stable in cell culture medium and are reduced by ascorbate. They are reduced slower than their carboxylato and carbonato analogues. We believe that this approach paves the way for preparing novel classes of multiaction Pt(IV) prodrugs with amino containing bioactive molecules that up to now were not accessible.

■ INTRODUCTION

Divalent Pt(II) complexes such as cisplatin, carboplatin, and complexes are stable outside the cancer cell and activated by oxaliplatin (Figure 1) are widely used by clinicians to treat reductive elimination inside the cell. The two-electron several forms of cancer, primarily in combination with other reduction results in the breaking of the bonds between the drugs.1,2 Pt(II) drugs are administered intravenously; after platinum and the axial ligands, releasing the two axial ligands as entering the cancer cell, they lose their nonam(m)ine ligands, well as the original Pt(II) drug (Figure 1). triggering cellular responses that result in apoptosis.3,4 Although they are quite effective, like most chemotherapeutic agents, they suffer from two major problems: acquired or inherent resistance to the drugs and the dose-limiting side effects.5−7 Clinicians try to overcome resistance to a given drug, and to diminish side effects, by treating patients with a combination of drugs that have different modes of action and fferent cellular targets.8−10 One approach to overcome resistance and minimize side effects is to use prodrugs that inside the cancer cell while targeting agents can increase selectivity and uptake to cancer cells. In addition, the axial positions can be used as linkers to polymers, nanoparticles, proteins, or other delivery agents.12−14 The axial ligands can be approved drugs, inhibitors of key enzymes, pathway activators or suppressors, antimetabolites, epigenetic modifiers, etc. that attack different cellular targets and work in synergy with Pt(II) drugs to overcome resistance.15 and the cis-[Pt(Am)2]2+ moiety binds to two adjacent guanines The axial ligands of Pt(IV) complexes can fulfill different on the same strand of the DNA, distorting its structure and functions. Lipophilic moieties enhance passive cellular uptake simultaneously release several antiproliferative agents that act in synergy with each other. Octahedral Pt(IV) complexes are particularly suitable to act as prodrugs.11 Pt(IV) prodrugs are generally prepared by oxidizing the square planar Pt(II) drugs such as cisplatin with H2O2, yielding the dihydroxido complex ctc-[Pt(NH3)2(OH)2Cl2]. The axial OH ligands can subsequently be modified to incorporate ligands designed to
The axial hydroxido ligand in Pt(IV)−OH is a nucleophile that can react with the carbonyl carbon (electrophile) of acid chlorides, anhydrides, or activated esters to yield the carboxylic link. Reaction between the axial OH and isocyanates yields the carbamate link (Scheme 1A).16 When the axial ligands are lipophilic moieties, targeting agents, or delivery systems, the nature of the linkage to the Pt(IV) is not usually important because the axial ligands do not possess biological activity and are not designed to play a role in the actual killing of the cancer cell. When the axial ligand is a bioactive ligand, the nature of the linkage is important because the bioactive moiety needs to be released in its native, unmodified active form. Therefore, in the majority of Pt(IV) multiaction drugs reported to date, the bioactive ligands contain a carboxylate through which they are tethered to the Pt(IV) because the breaking of the Pt(IV)−O bond immediately releases the active moiety. Using only bioactive ligands with carboxylates significantly limits the scope of Pt(IV) multiaction drugs, especially if we consider that many of the FDA approved drugs that are coadministered in the clinic with platinum drugs (such as gemcitabine, taxol, doxorubicin, irinotectan, 5-FU, etc.) do not possess carboxylates. Recently, we described a method for conjugating molecules with an OH group to Pt(IV) via a carbonate linkage, such that following reduction, the native form of the molecule is released.17 Herein, we demonstrate that when amino groups are conjugated to the Pt(IV) via a carbamate linkage, reduction of these complexes is followed by rapid decarboxylation of the carbamate, leading to the release of the free amines. This paves the way for the preparation of novel classes of multiaction prodrugs with bioactive molecule that have amines.

■ EXPERIMENTAL SECTION

Materials and Methods. All the chemicals and solvents were procured from authentic commercial sources and used without further purification.
The newly synthesized Pt compounds were characterized by 1H NMR, 13C NMR, 195Pt NMR, electrospray ionization mass spectometry (ESI-MS), and elemental analysis. Progress of reactions was monitored by analytical RP-HPLC system (Thermo Scientific UltiMate 3000) with a reverse-phase C18 column (Phenomenex Kinetex, Length 250 mm, Internal dia 4.60 mm, Particle size 5 μm, Pore size 100 Å − Column I) or (Phenomenex Kinetex, Length 100 mm, Internal dia 4.60 mm, Particle size 2.6 μm, Pore size 100 Å − Column II). The purity and retention time (RT) of synthesized compound reported here were measured with the same analytical RPHPLC system with either water/acetonitrile gradient or TFA (0.1% in water)/acetonitrile gradient at the flow rate of 1 mL/min. Reaction mixtures were purified on a preparative RP-HPLC system (Thermo Scientific UltimaMate 3000 station) equipped with a reverse-phase C18 column (Phenomenex Luna 250 × 21.2 mm, 10 μm, 100 Å) with a similar type of mobile phase used with the flow rate of 15 mL/min. UV detection was set at 220 nm in both the HPLC systems. The fractions were combined and lyophilized to get the pure compounds. All NMR data were collected on a Bruker AVANCE III HD 500 MHz spectrometer. The data were processed using either MestreNova or Bruker TopSpin 3.6.0 software. 1H and 13C NMR chemical shifts were referenced with the individual solvent residual peaks of respective NMR solvents used. 195Pt NMR chemical shifts were reported with respect to chemical shift of standard K2PtCl4 in water at −1624 ppm. ESI-MS was done using a Thermo Scientific triple quadrature mass spectrometer (Quantum Access) by positive mode electrospray ionization. Elemental analyses reported were performed using a Thermo Scientific FLASH 2000 element analyzer.
Stability Studies in Cell Culture Medium. The stability of the synthesized Pt(IV) complexes were tested in RPMI medium (pH 7.4, without adding the serum, Roswell Park Memorial Institute Medium) at 37 °C in the dark and were monitored by analytical HPLC. A 1:1 mixture of RPMI and MeOH was used for the Pt(IV) complexes 1−4 and 7−10. The stability studies of Pt(IV) complexes 5, 6, and 11 were done in RPMI medium containing 1% DMSO and 19% MeCN.
Reduction Studies. All reduction studies of reported Pt(IV) complexes were performed in the presence of 10 equiv of ascorbic acid and were performed at pH 7.0 at 37 °C in the dark and monitored by analytical HPLC. Samples (1−4 and 7−10) were dissolved depending on their solubility either in MeOH/phosphate buffer (100 mM, pH 7.0) mixture or only MeOH, in which the pH was adjusted using sodium hydroxide to pH 7.0. The reduction of Pt(IV) complexes 5, 6, and 11 were done in phosphate buffer (100 mM, pH 7.0) containing 1% DMSO and 19% MeCN.
NMR reduction study of 3 was performed with 10 equiv of ascorbic acid in DMSO-d6 (pH 7.0), whereas a 1:1 mixture of MeOD and 100 mM phosphate buffer of pH 7.0 (prepared in D2O) was used for the ascorbic acid reduction study of 7.
The half-lives (t1/2) of all compounds were obtained after linear fitting of ln(At/A0) vs time (t) by considering a pseudo first order rate equation (At = A0e−kt), where A0 and At are the integrated areas of HPLC peaks of the respective complexes at t = 0 and at time t, respectively, and k (slope) is the rate constant. Then, the t1/2 was calculated by utilizing the equation t1/2 = 0.693/k.
In Vitro Study. Stock solutions of the ligands and Pt(IV) complexes in DMSO were prepared just before the experiment, and a calculated amount of these drug solutions was added to the respective cell growth medium to a final DMSO concentration of 0.1% which had no discernible effect on cell killing. Cisplatin was dissolved in PBS buffer. Cisplatin and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) were obtained from Sigma Chemical Co., St Louis, USA.
A2780 and A2780cisR human ovarian carcinoma cells, A375 human malignant melanoma cells, and PC9 human nonsmall cell lung carcinoma cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD). Cell lines were maintained in the logarithmic phase at 37 °C under a 5% carbon dioxide atmosphere and were passaged twice a week. A2780, A2780cisR, and A375 were cultured in RPMI 1640 (no glutamine) supplemented with heat inactivated fetal bovine serum (south American origin) (10% v/v) gentamycin sulfate solution (1% v/v) and L-glutamine solution 200 nM (1% v/v). PC9 cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, high glucose, no glutamine/sodium pyruvate) supplemented with L-glutamine solution 200 nM (1% v/ v), 10% (v/v) heat inactivated fetal bovine serum (south American origin, European grade) and 1% (v/v) penicillin−streptomycin solution (containing penicillin G sodium salt: 10 000 units/mL and streptomycin sulfate: 10 mg/mL). All reagents were purchased at Biological Industries. A2780cisR was also treated once a week with 50 μL of 100 mM cisplatin in 0.9% NaCl solution to maintain their resistance to cisplatin.
MTT Assay. The growth inhibitory effect toward the human cell line was evaluated by means of MTT (tetrazolium salt reduction) assay. Dependent upon the growth characteristics of the cell line, A357 melanoma cells (4000 cells/well), A2780 ovarian carcinoma cells (5000 cells/well), A2780cisR cisplatin resistant ovarian carcinoma cells (7000 cells/well), and PC9 small lung cell carcinoma cells (4000 cells/well) were seeded in the respective cell culture medium (100 μL per well) in flat-bottom 96-well plates and incubated at 37 °C/5% CO2 for 24 h.
After 24 h, the medium was removed and replaced with a fresh one containing the compounds to be studied at appropriate graded concentrations as described below. Quadruplicate cultures were established for each treatment. After 72 h, each well was treated with 100 μL of a 0.5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide) saline solution, and after a 1 h additional incubation, the solution was removed, and 100 μL of DMSO was added to each well for 20 min while shaking. Afterward, the inhibition of cell growth induced by the tested complexes was detected by measuring the delta of absorbance of each well at 570 and 690 nm using Cytation 3 Cell Imaging Multi-Mode Reader by BioTek Instruments, Inc. The mean absorbance for each drug dose was expressed as a percentage absorbance of the control (untreated wells, set as 100%) and plotted vs drug concentration. IC50 values represent the drug concentrations that reduce the mean delta absorbance to 50% of those in the untreated control wells. Results were calculated as the mean values of three independent experiments.

■ RESULTS AND DISCUSSION

Synthesis of Pt(IV) Carbamate Complexes (Primary, Secondary, and Anilinic). The initial synthesis of three Pt(IV) carbamate complexes was described over 20 years ago by Giandomenico.16 They reacted oxoplatin, ctc-[Pt(NH3)2(OH)2Cl2], with an excess of isocyanates for 12 h. Sixteen years later, Wilson et. al reported on the synthesis and cytotoxicity of eight Pt(IV) complexes with aryl and alkyl carbamates (Scheme 1A).20 They used the same synthetic approach and reacted oxoplatin with 4 equiv of the isocyanates overnight in DMF. In both cases, they obtained symmetric biscarbamato complexes of the type ctc-[Pt(Am)2(O2CNHR)2Cl2], where Am = NH3 or isopropylamine. The same synthetic approach was later used by Kowol to link Pt(IV) complexes to albumin.21
Herein, we describe new approaches to the synthesis of Pt(IV) carbamate complexes. Rather than purchase or synthesize isocyanates, we embarked on two novel synthetic approaches for conjugating primary, secondary, or anilinic amines to Pt(IV). The first approach involves reaction of the axial OH ligand of Pt(IV) with DSC, [bis(2,5-dioxopyrrolidin1-yl)carbonate], to form the electrophilic [Pt(Am)2(MSC)(OH)L′2], where MSC is [mono(2,5-dioxopyrrolidin-1-yl)carbonate], that can react with nucleophiles such as primary or secondary aliphatic amines and with anilinic amines to yield the carbamates (Schemes 1B, 1C). The second approach involves the activation of the amine with DSC, followed by reaction with the axial OH ligand of Pt(IV) (Scheme 1D). To explore the applicability of our approaches for the preparation of Pt(IV)−carbamates, we selected 3-phenyl-1-propylamine and diethylamine as models for primary and secondary aliphatic amines, respectively, and aniline as a model for aromatic amines.
Synthesis of ctc-[Pt(NH3)2(O2CNH(CH2)3Ph)2Cl2]. Oxoplatin was reacted for 2 h with 2.4 equiv of DSC in DMF to yield ctc-[Pt(NH3)2(MSC)2Cl2], which was isolated by precipitation with ether and characterized by 1H, 13C, and 195 (NH3)2(MSC)2Cl2] was stirred with 4 equiv of 3-phenyl-1propylamine at 4 °C in DMF, and the progress of the reaction was monitored by RP-HPLC. After 1 h, the reaction was complete, and we obtained the symmetric ctc-[Pt(NH3)2(O2CNH(CH2)3Ph)Cl2] in high yield (Scheme 1B). At higher temperatures, additional peaks appeared in the HPLC reducing the yield. The second approach of first activating the amine with DSC and then reacting it with oxoplatin (Scheme 1D) proved inferior due to longer reaction times and lower yields.
Synthesis of ctc-[Pt(NH3)2(O2CNHR)(L)Cl2], L = OH, O2CR′. There are several approaches for the preparation of monocarboxylatomonocarbamato ctc-[Pt(NH3)2(O2CNHR)(O2CR′)Cl2] complexes. The first is to prepare the monocarboxylatomonohydroxido ctc-[Pt(NH3)2(OH)(O2CR′)Cl2] and then activate the axial OH with DSC to obtain ctc-[Pt(NH3)2(MSC)(O2CR′)Cl2]. This approach can only be used when the O2CR′ moiety does not contain unprotected functional groups such as hydroxides or amines that can react with DSC. The activation of ctc-[Pt(NH3)2(OH)(O2CR′)Cl2] with DSC is a very efficient reaction, and already after 0.5 h, the product is obtained in high yield (over 80%). The formation of the activated Pt(IV) was confirmed by a new peak in the 195Pt NMR at ∼1200 ppm. The activated Pt(IV) complex can be isolated and used directly or stored for future use at room temperature. The activated Pt(IV) complex reacts readily with primary and secondary aliphatic amines to obtain the required Pt(IV)−carbamato complexes (Scheme 1C).
For example, ctc-[Pt(NH3)2(MSC)(PhB)Cl2] was reacted with 2 equiv of 3-phenyl-1-propylamine, and after 1 h at RT, we obtained a 95% yield. The reaction of the activated Pt(IV) with analinic amines proceeds slower and often needs to be stirred overnight at 45 °C. This can be ascribed to the lower nucleophilicity of the analinic nitrogen compared with primary and secondary amines. When we used the second strategy and activated the primary amine with DSC and reacted it with 3 equiv of ctc-[Pt(NH3)2(OH)(PhB)Cl2] at 45 °C, after 48 h, we obtained only 27% yield.
Another approach is to react oxoplatin with 0.7 equiv of DSC to obtain the activated monohydroxido complex ctc[Pt(NH3)2(OH)(MSC)Cl2]. The reaction was carried out in DMF at RT and monitored by 195Pt NMR, and after 30 min and the appropriate workup, we obtained ctc-[Pt(NH3)2(OH)(MSC)Cl2] in good yield. Reaction of ctc-[Pt(NH3)2(OH)(MSC)Cl2] with an amine yielded after ∼60 min ctc-[Pt(NH3)2(OH)(O2CNHR)Cl2] that can be modified with the desired anhydrides or activated esters to obtain ctc[Pt(NH3)2(O2CR′)(O2CNHR)Cl2]. The synthetic details and the characterization of the various compounds are described in the experimental section.
The carbamate compounds we prepared for this study are depicted in Figure 2. All compounds were purified by RPHPLC and characterized by 1H, 13C, and 195Pt NMR.
To sum up the synthetic approaches, we see that compared to the procedure reported by Wilson and Lippard for the preparation of carbamates,20 our method of activating the Pt(IV) with DSC has several advantages: (a) it uses cheaper, widely available nontoxic amine ligands, (b) both primary and secondary amines can be conjugated to the Pt(IV), and (c) the reactions are faster and with higher yields. To the best of our knowledge, this is the first example where the conjugation is done by direct activation of the Pt(IV).
Characterizing the Reduction Products of Pt(IV) Complexes with Axial Carbamate Linkages. To find out whether Pt(IV)−carbamate linkages are prodrugs that can release free amines in addition to the Pt(II), we need to investigate the fate of the axial carbamate ligands following reduction. Interestingly, although Pt(IV)−carbamate complexes were prepared as potential prodrugs and their reduction potentials were measured, neither the fate of the axial ligands nor the reduction kinetics were reported.20 To explore this issue, we prepared three Pt(IV)−carbamato complexes with bioactive ligands with amino groups in addition to the four model compounds (1−4). The first is 3-aminobenzamide (3ABA) that is a PARP-1 inhibitor (see compounds 5 and 6). The second is a derivative of SAHA (also known as vorinostat), a potent FDA approved HDAC inhibitor, to which an amino group was added in the trans position (see SAHA-NH2 compound 7). We synthesized the seven complexes depicted in Figure 2 and studied their reduction with excess ascorbate. To be able to identify the reduction products, we reacted each of the compounds with 10 equiv of ascorbic acid in phosphate buffer in pH 7 and 37 °C and characterized the products by analytical RP-HPLC. While it is clear that following reduction the carbamate, −O2CNHR, is released into solution, the question is whether it undergoes decarboxylation and releases the amine or whether it stays intact. Under the conditions of the RP-HPLC analysis, we expect intact carbamates to have shorter retention times than the original amines due to increased hydrophilicity and charge compared to the amines. Moreover, if the carbamate is stable and stays intact, we should not see the peaks of the free amines in the chromatogram. As can be seen in Figure 3A and 3B, 12 min following the addition of ascorbate to compounds 5 and 6, the peak of 3-ABA at 2.8 min is clearly visible attesting to the rapid decarboxylation of the carbamate. The reduction in these cases is rapid and is complete in less than 2 h. In the case of compound 3, where the diethylamine cannot be observed by HPLC, we followed the reduction by 1H NMR in DMSO-d6 to which 10 equiv of ascorbic acid was added. We only observed the peaks of the free PhB (4-phenyl butyrate) and diethylamine, suggesting that the carbamate underwent rapid decarboxylation, releasing the free amine (Figure 3C). This is reminiscent of the rapid decarboxylation we observed when the monoester of carbonic acids are released following reduction of Pt(IV).17
Pastorin and Ang described a study of ratiometric delivery of cisplatin and doxorubicin (Figure 4A) using tumor-targeting carbon nanotubes. Because doxorubicin has no carboxylates, they used its only amino groups to tether the doxorubicin to the Pt(IV). They prepared the dual action Pt(IV) derivative of cisplatin where one axial ligand was benzoate and the other was doxorubicin that was tethered to the Pt(IV) via a succinate linker that formed an amide linkage with the amino group (Figure 4B).22 Because amide bonds are stable, it is reasonable to assume that following the reduction of the benzoatePt(IV)−succinate−doxorubicin conjugate, the moiety released inside the cell was not doxorubicin but rather the succinate− doxorubicin conjugate.
To clarify this point, we followed their example and tethered 3-ABA to Pt(IV) via a succinate linker to obtain OAc-Pt-Suc-3ABA (Figure 4C, compound 11) to see whether reduction of the Pt(IV) will yield 3-ABA in its active form or whether the succinate will remain linked to the amino group. Following reduction with 10 equiv of ascorbic acid, we did not observe the peak of the free 3-ABA at 2.8 min in the HPLC chromatogram. ESI-MS of the reaction mixture revealed peaks corresponding to the succinated 3-ABA (Figure S79), confirming that there is no rapid hydrolysis of the amide bond and release of 3-ABA. In aggregate, when the amine is tethered to the Pt(IV) via a succinate, we get rapid release of the succinated amine due to the stability of the amide bond (Figure 4D), while in contrast, when the amine is linked via a carbamate, there is a release of the free amine due to rapid decarboxylation of the carbamate (Figure 4E).
Stability in Cell Culture Medium and Rates of Reduction. After having demonstrated that carbonate linkages can release OH-containing molecules17 and that carbamate linkages can release amine-containing molecules, we thought it worthwhile to compare the stability and rates of reduction of carboxylate vs carbonate vs carbamate linkers. Toward this end, we synthesized the symmetric bis-carbonates, biscarboxylates, and bis-carbamates (Figure 5) and compared their rates of reduction and their stability in cell culture medium. The half-lives of the reduction and the stability in cell culture medium are presented in Table 1.
The carbamates with primary or secondary aliphatic amines (1−3) are very stable in cell culture medium with half-lives (t1/2) of 309, 202, and 281 h, respectively. Interestingly, compound 2, which has an OH trans to the carbamate, is significantly less stable than compounds 1 and 3 that have a carboxylate (PhB) trans to the carbamate. Compound 10 with two trans carbamates has a half-life similar to those of 1 and 3 (325 h), suggesting that the OH group in 2 might adversely affect the stability relative to the bis compounds. That being said, compound 2 is stable with regard to the timetable of cytotoxicity studies (72 h). The carbamates with anilinic amines are significantly less stable than their counterparts with aliphatic amines. Compounds 4−6 have half-lives of 83, 21, and 85 h, respectively. Direct comparison of bis-carboxylates (t1/2 = 265 h), bis-carbonates (t1/2 = 54 h), and bis-carbamates (t1/2 = 325 h) indicates that the carbonates are significantly less stable than the carboxylates and carbamates.
We also looked at the relative rates of reduction by ascorbate (Table 1). All the reduction studies were carried out at 37 °C and in phosphate buffer at pH 7. The concentration of the complexes was 1 mM and that of the ascorbate was 10 mM.
First, we compared reduction rates between the three bisPt(IV) complexes with the different linkages; carboxylate (8), carbonate (9), and carbamate (10). The bis-carbamate complex is reduced slowly with a t1/2 of 11 h while the biscarboxylate complex has a moderate reduction rate with a t1/2 of 4.4 h, and the bis-carbonate complex is reduced rapidly with a t1/2 of 0.5 h.
Interestingly, the bis-carboxylate (8) was reduced 2.5× faster than the bis-carbamate (10), while the monohydroxidomonocarboxylato, ctc-[Pt(NH3)2(OH)(O2C-(CH2)3Ph)Cl2], was reduced 1.3-fold faster than its carbamate analogue ctc[Pt(NH3)2(OH)(O2CNH-(CH2)3Ph)Cl2] (2), having halflives of 2.5 (data not shown here) and 3.2 h, respectively. These results can be rationalized by assuming that the rate of reduction depends on two main parameters: the rate of electron transfer from the ascorbate to the Pt(IV) and the ease of breaking the bond between the platinum and the oxygen of the axial ligand. The faster reduction of the monohydroxido complexes 2 and ctc-[Pt(NH3)2(OH)(O2C-(CH2)3Ph)Cl2] compared with the bis compounds 10 and 8, is probably due to the faster electron transfer from ascorbate to the Pt(IV) via an inner sphere mechanism where the ascorbate forms a bridge with the OH.23 The similar half-lives of 2 and ctc[Pt(NH3)2(OH)(O2C-(CH2)3Ph)Cl2] suggest that in this case, the rate-determining step is the electron transfer to the platinum, and assuming similar rates of electron transfer to both compounds, the differences between the carbamate complex (2) and the carboxylate, ctc-[Pt(NH3)2(OH)(O2C(CH2)3Ph)Cl2], are probably due to differences in the bond strengths between the Pt(IV) and the axial ligands.
Compounds 1, 3, and 4 have a carboxylate linkage (PhB) in one axial position and a primary, secondary, or anilinic carbamate, respectively, in the other axial position. Interestingly, there is quite a difference in the rates of reduction between the anilinic carbamate (t1/2 = 1.5 h) and the primary and secondary aliphatic carbamates (t1/2 = 8.9 and 7.3 h, respectively). Moreover, the reduction half-life of the anilinic carbamate is even shorter than the monohydroxidomonocarbamato derivative of the primary amine, ctc-[Pt(NH3)2(OH)(O2CNH-(CH2)3Ph)Cl2] (t1/2= 3.2 h) that is reduced by an inner sphere mechanism. Because compounds 4- 7 are stable (t1/2 = 21−101 h), it is not likely that the fast reduction rates (t1/2 < 0.2−1.5 h) are due to rapid hydrolysis of the axial carbamate ligands followed by inner sphere reduction by ascorbate. Therefore, we suggest that the electronic properties of the aryl substituents on the carbamate ligand that are weaker electron donors compared to the alkyl substituents destabilize the +4 oxidation state facilitating the fast reduction.
Cytotoxicity. Compounds 5−7 were prepared as potential dual- and triple-action anticancer agents. Compounds 5 and 6 are prodrugs designed to release in cancer cells cisplatin as well as the poly(ADP-ribose) polymerase inhibitor (PARPi), 3aminobenzamide (3-ABA), and in the case of 5, also the histone deacetylase inhibitor (HDACi) PhB. PARP inhibitors such as olaparib, rucaparib, and niraparib have been approved for the treatment of cancer.24 Interestingly, there are reports on synergistic effects between PARP inhibitors and platinum drugs.25−27 Specifically, the PARP-1 inhibitor 3-aminobenzamide was reported to suppress cell growth and migration and to enhance the potency of cisplatin in resistant cell lines.28−30 The cytotoxicity data against ovarian cancer cell lines (A2780) and its cisplatin resistance line (A2789cisR), as well as nonsmall cell lung carcinoma (PC9) and the melanoma (A375), are reported in Table 2.
The PARP-1i, 3-ABA itself has no significant cytotoxicity against all cell lines tested (IC50 > 80 μM). The dual-action prodrug (6) has moderate activity against A2780 and A375 (IC50 = 4.85 and 20.67 μM, respectively) but was significantly less potent against A2780cisR and PC9 (IC50 = 40.71 and 41.12, respectively). While it displayed good activity against the sensitive ovarian cancer cells it was not able to overcome the acquired cisplatin resistance. As the dual action compound (6) is less potent than cisplatin in all cell lines it seems that combining cisplatin with 3-ABA was rather counterproductive. The triple-action compound (5) displayed good cytotoxicity and was more potent than 6 with low μM IC50 values in all cell lines with partial ability to overcome acquired resistance. Compound 5 was comparable to cisplatin in A2780, PC9, and A375 and 4-fold better than cisplatin against A2780cisR. The better potency of 5 compared to 6 might be due to the activity of the PhB. As the cytotoxicity data are not very encouraging, and primarily because the main goal of this work was to demonstrate that amine containing molecules can be conjugated to Pt(IV) and be released intact following reduction, we did not explore cell uptake, DNA binding, or enzyme inhibition.
We and others have reported on Pt(IV) prodrugs with axial HDAC inhibitors such as valproate or 4-phenylbutyrate.34−36 ctc-[Pt(NH3)2(PhB)2Cl2] is a very potent cytotoxic agent having nanomolar IC50 values in all 9 cancer cell lines tested.32 It was 20-, 115-, and 25-fold more potent than cisplatin against A2780, A2780cisR, and A375, respectively. The high potency was ascribed to the simultaneous action of cisplatin (DNA platination) and PhB (HDAC inhibition). PhB, however, is not a very potent HDAC inhibitor compared with the FDA approved HDAC inhibitors vorinostat and belinostat that have a hydroxamic acid moieties (Figure 6).
Hydroxymic acids are strong chelators, and vorinostat and belinostat inhibit HDAC activity by binding the Zn in the active site of the enzyme. The phenyl ring of vorinostat protrudes out of the catalytic pocket of HDAC.37 In view our ability to prepare multiaction Pt(IV) prodrugs with molecules having amino groups, we decided to make just a minor modification to vorinostat by adding an amino group to the phenyl ring trans to the amide substituent to obtain SAHA-NH2 (Figure 6) in the hope that this small modification will not interfere with the binding to the active site of HDAC. Comparison of the literature IC50 values of SAHA and those of SAHA-NH2 shows that in A2780 and PC9 the values are similar while in A2780cisR and A375 SAHA outperforms SAHA-NH2. The IC50 values of the triple action prodrug combining PhB and SAHA-NH2 with cisplatin (7) were not as impressive as we hoped (2−15 μM) and not significantly better than SAHA (1.77−5.32 μM) or SAHA-NH2 itself (7− 20 μM) or cisplatin (1−22 μM). It is, however, interesting to compare the cytotoxicity of ctc-[Pt(NH3)2(PhB)2Cl2] that is comprised of cisplatin and two equivalents of a weak HDAC inhibitor with ctc-[Pt(NH3)2(SAHA-NH2)(PhB)Cl2] comprised of cisplatin and one weak and one presumably very strong HDAC inhibitor. Replacing one PhB (weak HDACi) with SAHA-NH2 (strong HDACi) results in a drastic reduction in potency as ctc-[Pt(NH3)2(PhB)2Cl2] is 15-, 128-, and 62-fold more cytotoxic than 7 in A2780, A2780cisR, and A375, respectively.35 Here again, no further biological studies were undertaken.

■ CONCLUSIONS

We developed a novel synthetic approach for the synthesis of Pt(IV)−carbamato complexes. This approach is based on the activation of the axial OH of Pt(IV) with DSC followed by reaction with primary, secondary or aromatic amines. This approach is superior to the prior art because it bypasses the need to prepare isocyanates that are not commercially available, allows for preparation of secondary carbamates, and requires shorter reaction times and provides high yields. To the best of our knowledge, this is the first example where the axial hydroxido of a Pt(IV) complex was functionalized with DSC, activating it to direct attack by amines. We demonstrated that reduction of the Pt(IV)−carbamato complexes results in rapid decarboxylation of the released carbamate to obtain the free amine, allowing for the design of multiaction prodrugs with bioactive molecules having amino groups. Pt(IV)−carbamato complexes are very stable in cell culture medium. We found that the half-life for the reduction of the bis-carbamato derivatives of cisplatin is about threefold longer than that for the bis-carboxylato analogue and 20-fold longer than the bis-carbonato analogue. We prepared three multiaction prodrugs with the PARP-1 inhibitor 3-ABA and a derivative of the HDAC inhibitor SAHA. Although the cytotoxicity data in these specific cases did not show synergistic effects between the 3-ABA/SAHA-NH2 and cisplatin, we believe that this approach paves the way for preparing novel classes of multiaction Pt(IV)-based prodrugs with other more potent amino containing bioactive molecules, that up to now were not accessible.

■ REFERENCES

(1) Muggia, F. M.; Bonetti, A.; Hoeschele, J. D.; Rozencweig, M.; Howell, S. B. Platinum Antitumor Complexes: 50 Years Since Barnett Rosenberg’s Discovery. J. Clin. Oncol. 2015, 33, 4219−26.
(2) Dasari, S.; Tchounwou, P. B. Cisplatin in cancer therapy: molecular mechanisms of action. Eur. J. Pharmacol. 2014, 740, 364− 378.
(3) Siddik, Z. H. Cisplatin: mode of cytotoxic action and molecular basis of resistance. Oncogene 2003, 22, 7265−79.
(4) Jung, Y. W.; Lippard, S. J. Direct cellular responses to platinuminduced DNA damage. Chem. Rev. 2007, 107, 1387−1407.
(5) Galluzzi, L.; Senovilla, L.; Vitale, I.; Michels, J.; Martins, I.; Kepp, O.; Castedo, M.; Kroemer, G. Molecular mechanisms of cisplatin resistance. Oncogene 2012, 31, 1869−83.
(6) Oun, R.; Moussa, Y. E.; Wheate, N. J. The side effects of platinum-based chemotherapy drugs: a review for chemists. Dalton Trans 2018, 47, 6645−6653.
(7) Florea, A. M.; Busselberg, D. Cisplatin as an anti-tumor drug: cellular mechanisms of activity, drug resistance and induced side effects. Cancers 2011, 3, 1351−71.
(8) Gustavsson, B.; Carlsson, G.; Machover, D.; Petrelli, N.; Roth, A.; Schmoll, H. J.; Tveit, K. M.; Gibson, F. A review of the evolution of systemic chemotherapy in the management of colorectal cancer. Clin. Colorectal Cancer 2015, 14, 1−10.
(9) Boulikas, T.; Vougiouka, M. Recent clinical trials using cisplatin, carboplatin and their combination chemotherapy drugs (review). Oncol. Rep. 2004, 11, 559−95.
(10) Mahlberg, R.; Lorenzen, S.; Thuss-Patience, P.; Heinemann, V.; Pfeiffer, P.; Mohler, M. New Perspectives in the Treatment of Advanced Gastric Cancer: S-1 as a Novel Oral 5-FU Therapy in Combination with Cisplatin. Chemotherapy 2016, 62, 62−70.
(11) Gibson, D. Platinum(IV) anticancer prodrugs – hypotheses and facts. Dalton Trans 2016, 45, 12983−12991.
(12) Kenny, R. G.; Marmion, C. J. Toward Multi-Targeted Platinum and Ruthenium Drugs-A New Paradigm in Cancer Drug Treatment Regimens? Chem. Rev. 2019, 119, 1058−1137.
(13) Gibson, D. Multi-action Pt(IV) anticancer agents; do we understand how they work? J. Inorg. Biochem. 2019, 191, 77−84.
(14) Mayr, J.; Heffeter, P.; Groza, D.; Galvez, L.; Koellensperger, G.; Roller, A.; Alte, B.; Haider, M.; Berger, W.; Kowol, C. R.; Keppler, B. K. An albumin-based tumor-targeted oxaliplatin prodrug with distinctly improved anticancer activity in vivo. Chem. Sci. 2017, 8, 2241−2250.
(15) Ravera, M.; Gabano, E.; McGlinchey, M. J.; Osella, D. A view on multi-action Pt(IV) antitumor prodrugs. Inorg. Chim. Acta 2019, 492, 32−47.
(16) Giandomenico, C. M.; Abrams, M. J.; Murrer, B. A.; Vollano, J. F.; Rheinheimer, M. I.; Wyer, S. B.; Bossard, G. E.; Higgins, J. D. Carboxylation of Kinetically Inert Platinum(Iv) Hydroxy Complexes an Entree into Orally-Active Platinum(Iv) Antitumor Agents. Inorg. Chem. 1995, 34, 1015−1021.
(17) Yempala, T.; Babu, T.; Karmakar, S.; Nemirovski, A.; Ishan, M.; Gandin, V.; Gibson, D. Expanding the Arsenal of Pt-IV Anticancer Agents: Multi-action Pt-IV Anticancer Agents with Bioactive Ligands Possessing a Hydroxy Functional Group. Angew. Chem. 2019, 131, 18386.
(18) Petruzzella, E.; Sirota, R.; Solazzo, I.; Gandin, V.; Gibson, D. Triple action Pt(IV) derivatives of cisplatin: a new class of potent anticancer agents that overcome resistance. Chem. Sci. 2018, 9, 4299− 4307.
(19) Zhang, J. Z.; Bonnitcha, P.; Wexselblatt, E.; Klein, A. V.; Najajreh, Y.; Gibson, D.; Hambley, T. W. Facile Preparation of Mono-, Di- and Mixed-Carboxylato Platinum(IV) Complexes for Versatile Anticancer Prodrug Design. Chem. – Eur. J. 2013, 19, 1672− 1676.
(20) Wilson, J. J.; Lippard, S. J. Synthesis, Characterization, and Cytotoxicity of Platinum(IV) Carbamate Complexes. Inorg. Chem. 2011, 50, 3103−3115.
(21) Pichler, V.; Mayr, J.; Heffeter, P.; Domotor, O.; Enyedy, E. A.; Hermann, G.; Groza, D.; Kollensperger, G.; Galanksi, M.; Berger, W.; Keppler, B. K.; Kowol, C. R. Maleimide-functionalised platinum(IV) complexes as a synthetic platform for targeted drug delivery. Chem. Commun. 2013, 49, 2249−2251.
(22) Chin, C. F.; Yap, S. Q.; Li, J.; Pastorin, G.; Ang, W. H. Ratiometric delivery of cisplatin and doxorubicin using tumourtargeting carbon-nanotubes entrapping platinum(IV) prodrugs. Chem. Sci. 2014, 5, 2265−2270.
(23) Zhang, J. Z.; Wexselblatt, E.; Hambley, T. W.; Gibson, D. Pt(IV) analogs of oxaliplatin that do not follow the expected correlation between electrochemical reduction potential and rate of reduction by ascorbate. Chem. Commun. 2012, 48, 847−849.
(24) Benafif, S.; Hall, M. An update on PARP inhibitors for the treatment of cancer. OncoTargets Ther. 2015, 8, 519−28.
(25) Yasukawa, M.; Fujihara, H.; Fujimori, H.; Kawaguchi, K.; Yamada, H.; Nakayama, R.; Yamamoto, N.; Kishi, Y.; Hamada, Y.; Masutani, M. Synergetic Effects of PARP Inhibitor AZD2281 and Cisplatin in Oral Squamous Cell Carcinoma in Vitro and in Vivo. Int. J. Mol. Sci. 2016, 17, 272.
(26) Michels, J.; Vitale, I.; Senovilla, L.; Enot, D. P.; Garcia, P.; Lissa, D.; Olaussen, K. A.; Brenner, C.; Soria, J. C.; Castedo, M.; Kroemer, G. Synergistic interaction between cisplatin and PARP inhibitors in non-small cell lung cancer. Cell Cycle 2013, 12, 877−83.
(27) Oei, A. L.; van Leeuwen, C. M.; Ahire, V. R.; Rodermond, H. M.; Ten Cate, R.; Westermann, A. M.; Stalpers, L. J. A.; Crezee, J.; Kok, H. P.; Krawczyk, P. M.; Kanaar, R.; Franken, N. A. P. Enhancing synthetic BRD3308 lethality of PARP-inhibitor and cisplatin in BRCA-proficient tumour cells with hyperthermia. Oncotarget 2017, 8, 28116−28124.
(28) Zheng, Y. D.; Xu, X. Q.; Peng, F.; Yu, J. Z.; Wu, H. The poly(ADP-ribose) polymerase-1 inhibitor 3-aminobenzamide suppresses cell growth and migration, enhancing suppressive effects of cisplatin in osteosarcoma cells. Oncol. Rep. 2011, 25, 1399−405.
(29) Zhang, J.; Kan, Y.; Tian, Y.; Wang, Z.; Zhang, J. Effects of poly (ADP-ribosyl) polymerase (PARP) inhibitor on cisplatin resistance & proliferation of the ovarian cancer C13* cells. Indian J. Med. Res. 2013, 137, 527−32.
(30) Nguewa, P. A.; Fuertes, M. A.; Cepeda, V.; Alonso, C.; Quevedo, C.; Soto, M.; Perez, J. M. Med. Chem. 2006, 2, 47−53.
(31) Griffith, D.; Morgan, M. P.; Marmion, C. J. A novel anti-cancer bifunctional platinum drug candidate with dual DNA binding and histone deacetylase inhibitory activity. Chem. Commun. 2009, 44, 6735−6737.
(32) Chen, M. C.; Chen, C. H.; Wang, J. C.; Tsai, A. C.; Liou, J. P.; Pan, S. L.; Teng, C. M. The HDAC inhibitor, MPT0E028, enhances erlotinib-induced cell death in EGFR-TKI-resistant NSCLC cells. Cell Death Dis. 2013, 4, No. e810.
(33) Gatti, L.; Sevko, A.; De Cesare, M.; Arrighetti, N.; Manenti, G.; Ciusani, E.; Verderio, P.; Ciniselli, C. M.; Cominetti, D.; Carenini, N.; Corna, E.; Zaffaroni, N.; Rodolfo, M.; Rivoltini, L.; Umansky, V.; Perego, P. Histone deacetylase inhibitor-Temozolomide co-treatment inhibits melanoma growth through suppression of Chemokine (C-C motif) ligand 2-driven signals. Oncotarget 2014, 5, 4516−28.
(34) Yang, J.; Sun, X.; Mao, W.; Sui, M.; Tang, J.; Shen, Y. Conjugate of Pt(IV)-histone deacetylase inhibitor as a prodrug for cancer chemotherapy. Mol. Pharmaceutics 2012, 9, 2793−800.
(35) Alessio, M.; Zanellato, I.; Bonarrigo, I.; Gabano, E.; Ravera, M.; Osella, D. Antiproliferative activity of Pt(IV)-bis(carboxylato) conjugates on malignant pleural mesothelioma cells. J. Inorg. Biochem. 2013, 129, 52−7.
(36) Raveendran, R.; Braude, J. P.; Wexselblatt, E.; Novohradsky, V.; Stuchlikova, O.; Brabec, V.; Gandin, V.; Gibson, D. Pt(iv) derivatives of cisplatin and oxaliplatin with phenylbutyrate axial ligands are potent cytotoxic agents that act by several mechanisms of action. Chem. Sci. 2016, 7, 2381−2391.
(37) Finnin, M. S.; Donigian, J. R.; Cohen, A.; Richon, V. M.; Rifkind, R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 1999, 401, 188−93.