Synthesis and anticancer activity of two highly water-soluble and ionic Pt(IV) complexes as prodrugs for Pt(II) anticancer drugs

Two new Pt(IV) complexes featuring mesylate as outer sphere anions, cis,trans,cis-[PtCl 2 (OH 2 ) 2 (NH 3 ) 2 ](CH 3 SO 3 ) 2 (SPt-1) and cis,trans,cis-[PtCl 2 (OH 2 ) 2 ((1R,2R-DACH)](CH 3 SO 3 ) 2 (SPt-2) were synthesized, and characterized by elemental analysis, 1 H- and 13 C-NMR,IR, and ESI-MS. Both complexes have excellent water-solubility and high molar conductivity as well as good water-stability. They exhibit an irreversible two-electron reduction event with the peak potentials (Ep) for the processes being − 0.39 V and − 0.64 V for SPt-1 and 0.09 V and − 0.52 V for SPt-2. The biological tests reveal that SPt-2 possesses high in vitro anticancer activity against three human cancer cell lines and its overall anticancer activity is slightly greater than that of oxaliplatin, whereas SPt-1 is less active than cisplatin. Moreover, the antitumor ecacy of SPt-2 on human colon carcinoma HCT-116 xenografts in nude mice is also greater than that of oxaliplatin, suggesting SPt-2 deserves further evaluation as a prodrug for oxaliplatin.


Introduction
In today's world, malignant tumors have become one of the most prevalent and serious diseases, and rank rst in human disease-related lethality. Chemotherapy is a central component in the ght against malignant tumors, and is based on different classes of anticancer drugs. Among them, platinum-based drugs represent an important class characterized by killing cancer cells primarily through cross-linking DNA and inhibiting transcription [1,2]. Platinum-based drugs now available for clinical options include cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin lobaplatin and miriplatin hydrate, and they have been successfully used in the treatment of solid tumors [3][4][5]. However, like other chemotherapy agents, the clinical applications of platinum-based drugs are largely restricted by side-effects as well as drug resistance [6][7][8]. As a result, the need for the development of new strategies to overcome these drawbacks is highlighted.
One of the strategies involves providing Pt(IV) complexes as prodrugs for Pt(II) anticancer drugs. Platinum(IV) complexes are almost always six-coordinated with octahedron geometries, the saturated, kinetically much more inert coordination sphere is less susceptible to ligand substitution reactions than four-coordinate platinum(II) centers, thus minimizing undesired side reactions with biomolecules prior to DNA binding [9]. In addition, the two extra axial ligands of low-spin d6 platinum(IV) centers provide a means to endow and ne-tune desired biological properties such as lipophilicity, redox stability, cancer-cell targeting, orthogonal or complementary bioactivity, and improved cellular uptake [10][11][12][13][14][15]. However, although platinum(IV) complexes can platinate DNA in their oxidized form, the formation of cytotoxic lesions by ligand substitution occurs on the scale of weeks [16], therefore, reduction of the platinum(IV) center to homologous platinum(II), accompanied by the loss of two axial ligands, is thought to be essential for these agents to exert anticancer activity [17].
So far, four Pt(IV) complexes, including iproplatin, satraplatin, tetraplatin and LA-12( Fig. 1), have undergone clinical trials, However, the outcomes of clinical trials are unsatisfactory as expected, none of these compounds has been approved for clinical application, because they can't exhibit overall effectiveness that surpassed that of their prototype Pt(II) anticancer drugs or have severe neurotoxicity, etc. [18][19][20][21].
Herein, we present two new Pt(IV) complexes, SPt-1 and SPt-2, as shown in Fig. 2. They belong to ionic complexes with high molar conductivity featuring with two axial aqua ligands in the inner coordination sphere and two mesylate ions in the outer coordination sphere. Both complexes have good water-solubility and water-stability compared with other Pt(II) and Pt(IV) anticancer complexes, Moreover, SPt-2 seems to be more active against colorectal cancer than its corresponding Pt(II) drug, oxailplatin.
Composition analyses for C, H, and N were performed with using a Carlo-Erba Instrument, whereas platinum content was determined according to the method in USP24. Electrospray ionization mass spectrometry (ESI-MS) measurements were acquired on a Agilent G6230 Spectrometry in the ESI + mode. FT-IR spectra were recorded in the 4,000-400 cm -1 region on a BRUKER Tensor-27 spectrometer with KBr pellets. 1 H and 13 C NMR spectra were recorded in deuterated oxide (D 2 O) on a Bruker AVANCE III 500 MHz spectrometer at 20℃. All NMR chemical shifts (δ) were reported in parts per million (ppm). 1 H and 13 C NMR spectra were referenced internally to residual solvent peaks and chemical shifts are expressed relative to TMS.
Cyclic voltammograms were obtained by using a BAS100 potentiostat at room temperature. A three electrode system was used consisting of a glassy carbon electrode as the working electrode, a Pt wire as the auxiliary electrode, and Ag/AgCl electrode as the reference electrode. Samples were prepared as 1 mM solutions in water with 0.05 mM Na 2 SO 4 as the supporting electrolyte. Reported values were peak potentials of the irreversible reduction event at a scan rate of 100 mV/s. Both constant pressure and initial pressure were 0.9 V. Sciences, Chinese Academy of Sciences (Shanghai, China), and were grown in DMEM or RPMI-1640 medium (Hyclone, USA) containing 10% fetal bovine serum. Both media were supplemented with 100 U/ml of penicillin and 100 μg/ml of streptomycin. Cells were maintained at 37℃ in a humidi ed incubator with an atmosphere of 5% CO 2 for 24 h, and then seeded at a density of 5×10 4 cells per well in 96-well microplates.

MTT assay
In vitro cytotoxic activity was determined by colorimetric MTT [3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide] assay. A 100 μL of cell suspension was seeded in 96-well cell culture plates and allowed to adhere overnight. The tested complexes were dissolved in 5% glucose solution just before the incubation with cancer cells, and diluted in culture media at the indicated concentrations. The cells were incubated with drugs for 72 h, and then a 20 μL of CellTiter 96® AQueous One Solution Reagent (Promega, Madison, USA) was added and the cells were further incubated at 37 °C for 1-2 h. Cell viability was measured by reading the absorbance at the wavelength of 490 nm. Concentrations of 50% inhibition of growth (IC 50 ) were calculated on the basis of the relative survival curve.

In vivo tests
Four-to ve-week-old female BALB/c-nude mice were purchased from Beijing Weitonglihua Experimental Animal Technology Co., Ltd. and were kept in a pathogen-free environment. Animal experiments were conducted by following a well-established and recognized methods. Every procedure with animals was done in a laminar air ow cabinet. 5×10 6 HCT-116 cells with 0.2ml per mice were implanted subcutaneously into the right axillary region of BALB/c mice. When tumor volumes reached 100-300 mm 3 , the mice were randomly assigned to control and treatment groups and the administration of drug or compound tested was started. Animals were given i.p. every other day (on day 0 to day 25) with SPt-2 (1.25μM/kg, 2.5μM/kg and 5μM/kg dissolved in 5% glucose) or oxaliplatin (2.5μM/kg and 5μM/kg in 5% glucose).
Animals in the control group received the same amount of 5% glucose solution. Tumor size was assessed regularly by vernier caliper measurement and tumor volume was expressed as (length × width 2 )/2. The relative tumor growth rate T/C, %) was used as an indicator to evaluate the in vivo ntitumor activity. Mice body weight was determined at baseline before the drug administration and recorded regularly during the experiment which was terminated on day 25.
All animal experiments were conducted in accordance with the Institutional Animal Care and Use Committee Guidelines of Kunming Medical University.

Results And Discussion
The synthesis of the platinum(IV) mesylate complexes was accomplished by treating cis,trans,cis-[PtCl 2 (OH) 2 (NH 3 ) 2 ] or cis,trans,cis-[Pt(IV)(1R,2R-DACH)(OH) 2 Cl 2 ] with methylsulfonic acid in water. Because two dihydroxy intermediates are largely insoluble in water, the progress of the reaction can be monitored visually by observing the conversion of the reaction mixture from suspension to homogenous solution. After the dihydroxy intermediate was dissolved in methylsulfonic acid, the solution was rotary evaporated to precipitate the desired product.
The chemical structures were well con rmed by elemental analysis and spectroscopic ( Supplementary Fig. S1-S8), which were in accordance with the expected structurues (Fig. 2). The compositions were in good agreement with the calculated values based on the molecular formula. A peaks of relative intensity developed at m/z 333 and 420 were corresponding to [M] 2+ of SPt-1 and SPt-2 respectively. The characteristic absorption bands arising from NH 3 groups of SPt-1 were apparent at 3190 and 3061 cm -1 , whereas NH 2 groups of SPt-2 were at 3503 and 3444 cm -1 . The ν as (SO 2 ) and ν s (SO 2 ) value of SPt-1 were 1208 and 1170 cm -1 compared with that of SPt-2 at 1218 and 1160 cm -1 . 1 H NMR was consistent with the protons of SPt-1 and SPt-2 both in terms of chemical shifts and integration. All the 13 C NMR signals could be assigned to the corresponding carbon atoms in the complex molecule. The chemical shift of carbon was nearly equal to that of CH 3 SO 3 Na, indicating that CH 3 SO 3 did not bind to platinum center in the coordination way.
The solubility of SPt-1 in water was 14 mg/ml and that of SPt-2 was 50 mg/ml, much larger than that of the corresponding platinum(II) complexes. The water-stability was judged by the changes in 1 H NNR of these compounds in D 2 O (10 mg/mL) with time. As seen from Fig. 3 and Fig. 4, there were no apparent changes of 1 H signals of SPt-1 and SPt-2 in D 2 O within 48 h at 20℃, implying that they have su cient water stability as drug candidates. The molar conductivity of SPt-1 and SPt-2 in water were determined to be 297.2 and 288.71 Ω -1 cm 2 mol -1 , respectively, at room temperature, similar to that of Ca(CH 3 SO 3 ) 2 , suggesting that they were ionic compounds, consistent with the proposed structures.

Cyclic voltammetry
The biological activity of platinum(IV) complexes was regulated by their redox chemistry. In most cases, unlike their platinum(II) progeny, platinum(IV) complexes did not bind directly to DNA or other biological nucleophiles. The redox potential of platinum(IV) complexes was therefore considered to be an important factor in their e cacy as antitumor agents. As illustrated in Fig. 5, the complexes exhibited an irreversible two-electron reduction event in the potential window of +1.0 to -1.0 V vs. Ag/AgCl. The peak potentials (Ep) were found to be -0.39 and -0.64 V for SPt-1, and 0.09 and -0.52 V for SPt-2, implying that both Pt(IV) complexes could be readily reduced to platinum(II) in the hypoxia environment of cancerous cells.
In vitro cytotoxic activity The cytotoxicity of SPt-1, SPt-2 along with their corresponding platinum(II) compounds, cisplatin and oxaliplatin, was tested by the means of MTT assay against three human cancer cell lines: colon carcinoma (HCT-116), non-small-cell lung carcinoma (A549) and gastric carcinoma (MKN-1) cells. The IC 50 values, de ned as the concentrations corresponding to 50% growth inhibition, were presented in Table 1. SPt-1 and SPt-2 showed signi cantly inhibiting effect against the growth of the three cancer cell lines in a good time-effect relationship manner. The overall anticancer activity of SPt-2 was greater than that of oxaliplatin, whereas SPt-1 was inferior to cisplatin. In particular, SPt-2 exhibited the highest cytotoxicity against HCT-116 cancer cells, which was distinctly higher than oxaliplatin, a rst-line chemotherapeutic agent for metastatic colorectal cancer. In vivo antitumor activity The in vivo antitumor activity of SPt-2 and oxaliplatin was compared on HCT-116 xenograft in nude mice. As seen from Table 2, at the dose of 1.25 μM kg -1 , 2.5 μM kg -1 , and 5 μM kg -1 , T/C after the treatment with SPt-2 was 53% (p 0.01), 40% (p 0.01) and 31% (p 0.001), respectively, which implied it could signi cantly inhibit the growth of tumor in a dosedependent manner, while obvious inhibition effect of oxaliplatin on the growth of tumor wasn't manifested until the dose was up to 5μM/kg and then T/C was 44%. In the meantime, the apparent change in mice body weights during the treatment of SPt-2 did not occur at low and medium doses. As a consequence, the curative effect of SPt-2 on HCT-116 xenograft in nude mice was superior to oxaliplatin at the same dose.

Conclusions
In this paper, we present two new water-soluble and stable platinum(IV) prodrugs featuring the ionic-type complexes. They exhibit an irreversible two-electron reduction event and can be reduced to active platinum(II) species. Both platinum(IV) prodrugs show cytotoxicity against the proliferation of three human cancer cell lines, especially SPt-2, whose in vitro cytotoxicity and in vivo antitumor e cacy were stronger than those of oxaliplatin, a rst-line chemotherapeutic agent for colorectal cancer, suggesting SPt-2 deserves further evaluation as a prodrug for oxaliplatin in the treatment of colorectal cancer. Figure 1 Chemical structures of platinum(IV) agents that have undergone clinical trials The chemical structures of SPt-1 and SPt-2.

Figures
Page 9/11  The change in 1H NMR of SPt-2 in D2O with the standing time Cyclic voltammogram for SPt-1 and SPt-2 measured in aqueous solution.