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  • br Results and discussion br Synthesis

    2019-09-24


    2. Results and discussion
    2.1. Synthesis and characterization
    As the ester linkage was more susceptible to Ethylmalonyl Coenzyme A and esterase, it could be cleaved to release active biological species in cancer cells, NBDHEX as a GSTs targeting moiety was modified by reacting with succinic anhydride to generate compound 2 (Scheme 1) that was characterized by ESI-MS and 1H NMR. As shown in Fig. S1, compound 2 gives two pseudo-molecular ion peaks at m/z = 396.08 and 793.18, assigned to the [M − H]− or [2M − H]− peak, respectively. In the 1H NMR spectrum (CDCl3), the multiple-peak of alkyl groups in the re-sulting succinic acid could be obviously observed at 2.61–2.73 ppm.
    Oxoplatin (cis, cis, trans-diammine-dichlorido-dihydroxido-platinum (IV)), a Pt(IV) precursor of cisplatin containing two axial hydroxyl groups confers the ability to attach functional or targeting groups. Oxoplatin was obtained through the oxidation of cisplatin by hydrogen peroxide in water. In brief, treatment of a yellow-orange suspension of cisplatin in water with excess H2O2 at 60 °C gave rise to the target
    The reaction of oxoplatin and compound 2 in a 1:1.1 M ratio in DMF afforded complex 1 which was characterized by ESI-MS, 1H, 13C and 195Pt NMR spectra together with elemental analysis. As shown in Fig. S2, the pseudo-molecular ion peaks [M − H]− (m/z = 712.08) and [M + Cl]− (m/z = 748.06) were found, accompanied with isotopic peaks derived from platinum atom in the mass spectra. In addition to the peaks of compound 2 in the 1H NMR spectrum, multiply peaks appeared
    at 5.82–6.03 ppm (DMSO-d6) were assigned to the two amino groups of complex 1. The 195Pt NMR signal of complex 1 was found at 1024.67 ppm, which provided information on the oxidation state of the metal atom and the nature of the coordinated ligands. The multiplet peaks arising from 1J (14N-195Pt) coupling and temperature have influ-ences on the linewidth, which was observed in the 195Pt NMR spectrum. Moreover, the purity of complex 1 was detected by HPLC technique (Fig. S3), which could meet the requirement for further study.
    2.2. Stability and released ability
    It has been reported that Pt(IV) complexes are essentially chemical inert and can only perform anticancer activity after reduced by biolo-gical reducing agents such as ascorbic acid (VC) and GSH to release toxic Pt(II) species and the axial ligands [30]. Thus, HPLC technique was used to study the stability and released ability of complex 1. As illustrated in Fig. 2, complex 1 was stable both in methanol/phosphate buffered saline (PBS, pH = 7.4) solution (v:v = 1:4) and in cell culture medium RPMI-1640 at room temperature for 48 h, but a little decom-position was observed in methanol/PBS (pH = 5.0) solution (v:v = 1:4). It was noted that a small amount of NBDHEX was released from complex 1 or compound 2 under the slightly acidic environment (Fig. S4), that means the ester linkage will be broken under acidic condition. As proved in many researches, the tumor acidic micro-environment and abundant esterases in the cells would make the ester linkage more prone to breakage. Complex 1 gradually began to de-compose and release compound 2 in the presence of VC (Fig. S5). It was noted that cisplatin moiety was not observed due to its weak chromo-phore under the ultraviolet detecting condition in HPLC chromato-grams. The results confirmed that complex 1 can be reduced by a biomolecular agent to release the active Pt(II) species and the axial li-gand. Dyson et al. ever reported a Pt(IV) complex, named as ethacra-platin containing two molecules of EA, attempting to overcome cis-platin-resistance by inhibiting GSTs [25], but it was found that ethacraplatin could not readily release a Pt(II) species to exert the cy-totoxic effect, consistent with our recent study on an axially bi-modified Pt(IV) complex that is much more difficult to be reduced than a mono-modified Pt(IV) complex [31].