Two mixed-NH3/amine platinum (II) anticancer complexes featuring a dichloroacetate moiety in the leaving group

Two mixed-NH3/amine platinum (II) complexes of 3-dichoroacetoxylcyclobutane-1, 1-dicarboxylate have been prepared in the present study and characterized by elemental analysis and IR, HPLC-MS and 1H, 13C-NMR. The complexes exist in equilibrium between two position isomeric forms and undergo hydrolysis reaction in aqueous solution, releasing the platinum pharmacophores and dichloroacetate which is a small-molecular cell apoptosis inducer. Both complexes were evaluated for in vitro cytotoxic profile in A549, SGC-7901 and SK-OV-3 caner cells as well as in BEAS-2B normal cells. They exhibit markedly cytoxicity toward cancer cells by selectively inducing the apoptosis of cancer cells, whereas leaving normal cells less affected. They have also the ability to overcome the resistance of SK-OV-3 cancer cells to cisplatin. Our findings offer an alternative novel way to develop platinum drugs which can both overcome the drug resistance and selectively target tumor cells.

Two mixed-NH 3 /amine platinum (II) complexes of 3-dichoroacetoxylcyclobutane-1, 1-dicarboxylate have been prepared in the present study and characterized by elemental analysis and IR, HPLC-MS and 1 H, 13 C-NMR. The complexes exist in equilibrium between two position isomeric forms and undergo hydrolysis reaction in aqueous solution, releasing the platinum pharmacophores and dichloroacetate which is a small-molecular cell apoptosis inducer. Both complexes were evaluated for in vitro cytotoxic profile in A549, SGC-7901 and SK-OV-3 caner cells as well as in BEAS-2B normal cells. They exhibit markedly cytoxicity toward cancer cells by selectively inducing the apoptosis of cancer cells, whereas leaving normal cells less affected. They have also the ability to overcome the resistance of SK-OV-3 cancer cells to cisplatin. Our findings offer an alternative novel way to develop platinum drugs which can both overcome the drug resistance and selectively target tumor cells. P latinum-based drugs represented by cisplatin, carboplatin and oxaliplatin have become fundamental components of standard chemotherapy regiments, and are widely used in antitumor therapy of testicular/ ovarian tumors and lung/colorectal carcinomas 1,2 . Despite the therapeutic benefits, the efficacy of platinum-based treatment regiments is considerably compromised not only by severe side-effects but also by insurmountable cross-resistance 3 . Therefore efforts to circumvent platinum resistance continue to play an important role in the development of novel platinum drugs. Although a number of mechanisms are involved in drug resistance, failure to achieve cell death after the formation of platinum-DNA adduct might be an important factor 3,4 . One effective way of combating the resistance, as illustrated by recent studies [5][6][7][8][9] , is to combine the platinum pharmacophores with small-molecular cell apoptosis inducers to form dual-functional compounds so as to enhance the drug sensitivity and decrease the resistance via a synergistic effect between the two active components.
Dichloroacetate (DCA) is a small molecule which has been used for years to treat patients with mitochondrial diseases 10 . It was shown by recent reports [11][12][13] to be able to trigger apoptosis through selectively targeting the mitochondria of cancer cells resistant to the anticancer drugs. Furthermore, unlike other anticancer drugs, DCA does not appear to affect normal cells 14 . This unique action of DCA gives a clue on the development of novel platinum drugs which are able to overcome the acquired resistance to cisplatin in certain type of cancer and avoid toxicity towards the normal cells at the meanwhile. The first platinum complex containing DCA moiety is mitaplatin reported in 2009 by Dhar and Lippard who demonstrated, mitaplatin, as a Pt (IV) compound with two DCA moieties directly coordinated to platinum in the axial positions, displayed a dual-killing mode and was considerably effective in cisplatin-resistant cells 9 . More recently, Dr Haihua described another Pt (IV) complex bearing an axial DCA ligand which also showed enhanced cytotoxicity 15 .
Inspired by this innovative research, we designed and synthesized a series of platinum (II) complexes featuring a dichloroacetate moiety tethered to the leaving group via ester bond (Fig. 1, 2). We expected that a synergistic anticancer action would be realized by combining the effects of platinum pharmacophore with DCA produced by hydrolysis in cancer cells. Among these complexes, we found that two mixed-NH 3 /amine (amine 5 cyclopentylamine, cyclohexylamine) platinum (II) complexes exhibited potent anticancer properties. In this paper, we report synthesis and cytotoxic profiles of the two complexes (1a, 2a).

Results
Synthesis and characterization. Cis-[Pt(NH 3 )(RNH 2 )I 2 ], as an important intermiediate for mixed-NH 3 /amine platinum(II) complexes, was prepared according to a well-known method 16 . The target complexes 1a and 2a were synthesized following the routes shown in Fig. 3 with a yield of 50-55% based on the final reaction between 4 and 7. Similar to cycloplatam 17 , both complexes existed in equilibrium between two isomeric forms due to 3-dichloroacetoxyl on the cyclobutane ring in either the cis or trans orientations in relation to NH 3 (Fig. 4). The position isomeric phenomenon was confirmed by HPLC-Mass measurements in our studies. Under proper chromatographic conditions, two peaks developed at a different retention time, in addition to the peaks of the solvent, in the chromatograms (see Supplementary  Fig. S1 and S2). They had nearly the same shape and area, and displayed exactly the same pattern of UV-visible absorption spectra and MS-ESI 1 spectra with M 1 at m/e 567 for 1a and 581 for 2a, the calculated molecular weights. The complexes also showed characteristic cluster ions in their mass spectra due to the isotopic peaks of platinum and chloride ion. Platinum has five total isomers out of which three have major abundance with 33% for 194 Pt, 34% for 195 Pt and 25% for 196 Pt, whereas there are two 35 Cl (76%) and 37 Cl (24%) isotopes for chlorine. For a complex containing one platinum atom and two chlorine atoms 18 , the calculated main value distribution for the molecular ion cluster is 725735100547558. The most prominent ion observed in the mass spectra of both 1a and 2a was the [M 1 H] 1 cluster. The isotopic peak distribution was 8.5% at m/z 56558.6% at m/ z 566512% at m/z 56755.5% at m/z 56858.7% at m/z 569 for 1a, and was 21% at m/z 579523% at m/z 580531% at m/z 581524% at m/z 582522% at m/z 583 for 2a, roughly in agreement with the calculated values. We tried to separate the two isomeric forms but failed, for the complexes do not have a sufficient solubility in water/methanol solvent.
The chemical structures were characterized by elemental analysis, IR, ESI 1 -mass and 1 H, 13 C NNR. All the data and spectra (see Supplementary Fig. S3-S10) agreed well with the proposed structures. The introduction of COCHCl 2 moiety to complexes 1a and 2a was evidenced by the appearance of C5O stretch band at 1748 cm 21 in the infrared spectra and C signals at 164 ppm in 13 C NNR spectra. It is important to note that most of the carbons had two signals because of two isomeric forms co-existing in the complexes.
Both complexes were nearly insoluble in water or ethanol (<0.1 mg/ml) and slightly soluble in methanol and DMSO, but soluble in polyethyleneglycol 400 which was chosen as the solvent to make the stock solutions of the complexes for the following biological testing. The hydrolysis rate of the complexes in 1510 (v/v) solution of water and methanol determined by HPLC was less than 2% within 4 hours and the hydrolysis products were, as expected, dichloroacetate and 1b for complex 1a, or 2b for complex 2a. Complexes 1b and 2b were also synthesized for comparison in HPLC measurements as well as in biological tests. As 1b and 2b were found very soluble in water (.25 mg/ml) compared with 1a or 2a, another synthetic reaction was employed to produce the desired complexes. Accordingly complexes 1b and 2b also have two positional isomeric forms. All the complexes used in our studies were, in fact, a mixture of two isomeric forms.
Cytotoxic profile. Selective cytotoxicity of the target complexes 1a and 2a on cancer cells. The cytotoxicity of 1a and 2a was determined by MTS assay along with DCA, carboplatin, cisplatin, 1b and 2b (hydrolysis products of 1a and 2a) as well as a mixture of 1b and DCA against three human cancer cell lines representing three tumor entities: non-small cell lung carcinoma (A549), gastric carcinoma (SGC-7901) and ovarian carcinoma (SK-OV-3), and the normal lung epithelial cell line (BEAS-2B) as well. As shown in Table 1, DCA alone did not show any significant anticancer activity with the IC 50 exceeding 200 mM, however the target complexes 1a and 2a bearing a DCA group exhibited obvious cytotoxicity against A549,  SGC-7901 and SK-OV-3 cancer cells (IC 50 , 11.54-21.88 mM), only slightly less than cisplatin, but much greater than carboplatin and the corresponding hydrolysis products 1b and 2b. Noteworthy, 1a was also much more active than the 151 molar mixture of 1b and DCA, implying that a synergistic anticancer action had achieved between 1b and DCA released from the hydrolysis of 1a. Moreover, by comparing the IC 50 values of the tested compounds towards cancer cell lines with those towards the normal lung epithelial cells BEAS-2B, 1a and 2a showed selective cytotoxicity on the cancer cells, whereas cisplatin, carboplatin and other compounds exhibited general cytotoxicity towards all the cells tested.
Selective apoptosis induced by 1a in A549 cancer cells over non-cancerous cells. Cell apoptosis was analyzed by Annexin-V-FITC/ Propidium iodide staining and flow cytometry. Apoptosis induced and the quantified ratios of apoptotic cells under the treatment of 1a or cisplatin at indicated dosages are shown in Fig. 5 and Table 2 respectively. Similar to cisplatin, 1a significantly induced apoptosis of A549 cells in a dose-dependent manner with a apoptosis ratio of 18.93% at the concentration of 12.5 mM. Surprisingly, it had little effect on normal human lung bronchial epithelial cells even at the concentration of 100 mM (apotoic ratio, 2.88), indicating that 1a may have a tumor cell-selective apoptosis-inducing property. On the contrary, obvious apoptosis induced by cisplatin was observed in both A549 cancer cells and the BEAS-2B normal human lung bronchial epithelial cells when the concentration is over 25 mM.
Cytotoxicity of the complexes 1a and 2a in cisplatin-resistant SK-OV-3 cell line. Cross-resistance profile of the complexes 1a and 2a was evaluated. Resistance Index was defined as the ratio of IC 50 value of resistant cells to that of sensitive cells. SK-OV-3 and cisplatinresistant SK-OV-3 cells (SK-OV-3/DDP) cell lines were chosen for MTS assay because drug resistance frequently occurred in the chemotherapy of ovarian tumors. As expected, 1a and 2a displayed similar cytotoxic activity against both sensitive and cisplatin-resistant human ovarian cancer cells with the resistance index being nearly equal to 1, indicating that they have the potential to overcome the resistance of cancer cells to cisplatin ( Table 3). The results may be attributed to the synergistic effect between the platinum pharmacophores and DCA released by the hydrolysis of 1a and 2a in the cells.

Discussion
Development of new platinum anti-cancer drugs remains an important field in medicinal chemistry and attracts extensive interests from both academic institutions and pharmaceutical industry. In the past ten years much effort has been devoted to non-classical platinum complexes but without success. No any new platinum complex has been approved for clinical use since the year of 2001. Therefore, direct structural modification of classical platinum drugs by introducing bioactive groups could still be an effective way to develop new-generation drugs.   In the present study, two mixed-NH 3 /amine platinum (II) complexes of 3-dichoroacetoxylcyclobutane-1,1-dicarboxylate have been prepared, characterized and evaluated for their cytotoxicity profile. Both complexes exist in equilibrium between two isomeric forms due to 3-dichloroacetoxyl on the cyclobutane ring in either the cis or trans orientations in relation to NH 3 . They undergo hydrolysis in water, releasing two active species, the platinum pharmacophores and dichloroacetate. The two complexes exhibit markedly cytotoxicity in cancer cells by selectively inducing apoptosis of cancer cells, and exert little effect on normal human cells. They have the potential to overcome the resistance of cancer cells to cisplatin, probably due to the synergistic effect between the platinum pharmacophores and dichloroacetate. These two complexes, as neutral and relatively lipophilic molecules, are also expected to enter the cells more easily and bring more dichloroacetate into cells than free dichloroacetate alone which exists as anion in physiological. PH condition. These results offer an alternative novel way to develop platinum drugs which can both overcome the drug resistance and target tumor cells selectively.

Methods
Chemistry. General. K[Pt(NH 3 )Cl 3 ], a commercially available platinum compound, was purchased from Alfa Aesar and 3-hydroxy-1,1-cyclobutanedicarboxylic acid 3 3 was prepared as previously described 19,20 Composition analyses for C, H and N were performed with a Carlo-Ebra instrument, whereas the content of platinum was determined according to the method in EP6.5. LC/MS measurements were carried out on a Waters Acquity Xevo TQ-S in ESI 1 mode using MeOH/H 2 O as the solvent. FT-IR spectra were measured in KBr pellets with a Perkin Elmer 880 spectrometer. 1 H, 13 C NMR spectra were recorded in DMSO on Brucker DRX-500 MHz relative to TMS (tetramethylsilane) as an external standard. A VG Autospec was also used to measure FAB 1 spectra with glycerol (Gly) as the matrix.