Exploring the Hydrolytic Behavior of the Platinum(IV) Complexes with

Aug 3, 2017 - Synopsis. Platinum(IV) complexes with axial acetato ligands can undergo hydrolysis under biologically revelant conditions, resulting in ...
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Exploring the Hydrolytic Behavior of the Platinum(IV) Complexes with Axial Acetato Ligands Jian Zhao,†,‡,§ Zichen Xu,†,§ Jing Lin,†,‡ and Shaohua Gou*,†,‡ †

Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Southeast University, Nanjing 211189, China Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China

‡

S Supporting Information *

ABSTRACT: Platinum(IV) complexes are generally thought to be kinetically inert, and are expected to be stable enough to resist premature aquation before entering the cancer cells. Nevertheless, in this work, complex 2 with axial acetato ligands can hydrolyze relatively quickly under biologically relevant conditions with a half-life of 91.7 min, resulting in the loss of the equatorial chlorido ligand. Further study indicated that the fast hydrolysis of complex 2 may be attributed to the strong σ-donor ability of N-isopropyl-1R,2R-diaminocyclohexane, and an increasing σ-donor ability of the amine group can promote the hydrolysis rate of the corresponding platinum(IV) complex. The experiment results were proven by the corresponding DFT calculation. Our study can help to re-evaluate the aqueous properties of the platinum(IV) complexes with axial acetate, which may be less inert to hydrolysis than expected under biologically relevant conditions.

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INTRODUCTION The successful application of cisplatin, carboplatin, and oxaliplatin as anticancer drugs in the clinic has attracted much attention to discovering a new generation of platinum-based anticancer drugs,1 in which platinum(IV) complexes have been thought to be promising alternatives.2 Although the anticancer potential of platinum(IV) complexes was recognized much earlier, their clinical value as anticancer agents has been realized recently.2−7 To date, four platinum(IV) drugs were subjected to clinical trials (Figure 1), but none of them has been approved for clinical application. Satraplatin, the first orally administrable platinumbased anticancer agent, was thought to be the most promising platinum(IV) compound, but there was no significant improvement when it was used in combination with prednisone for phase-III trial against hormone-refractory prostate cancer.8 LA-12, a satraplatin analogue, is still under clinical trial, while iproplatin and tetraplatin have been abandoned due to low activity and severe side effects, respectively.9 Notably, among these platinum(IV) drugs, aliphatic amines like isopropylamine, cyclohexylamine, and adamantylamine were used as carrier ligands at the equatorial position of the six-coordinated octahedral geometry, and the asymmetric amine ligands seem to have profound effects on the biological activities of the compounds.10−13 © 2017 American Chemical Society

Although platinum(IV) complexes as antitumor agents have been widely studied,14−22 the underlying mechanism of action is still not fully understood.23−31 The design of antitumor platinum(IV) complexes is generally upon the assumption that the low spin octahedral d6 platinum(IV) complexes were kinetically inert and stable enough to resist premature aquation,32−35 but would be activated by bioreducing agents to release the cytotoxic parent platinum(II) species at the tumor site.36−38 Platinum(IV) complexes with axial acetato ligands were found to be relatively inert, and are stable enough to resist premature aquation and bind to essential plasma proteins.27,39 For example, negligible aquation was observed for ctc-[Pt(NH3)2(OAc)2Cl2] over 3−4 weeks.27 However, one or two equatorial chlorido ligands of satraplatin were observed to be replaced by OH− after oral administration to cancer patients while the axial OAc− ligands remained intact,8,40 and ctc-[Pt(NH3) (cyclohexylamine)(OAc)2(OH)2] may be the major metabolite in some cases.41,42 It has been reported that the hydration process of satraplatin is a kinetically forbidden reaction in equatorial position.34 Moreover, it ruled out the possibility that the metabolite was obtained by initial reduction Received: May 26, 2017 Published: August 3, 2017 9851

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry

Figure 1. Related antitumor platinum(IV) complexes in this paper.

of satraplatin and followed by oxidation.8 Thus, the observation seems to contradict the assumption on platinum(IV) complexes. Besides, the hydrolysis of the platinum(IV) complexes may alter their physicochemical properties such as lipophilicity, chemical reactivity, and reduction rates, which may further affect their pharmacological properties. Rational modification of the leading platinum(IV) compounds with improved pharmaceutical properties is still one of the important approaches for medicinal chemists to discover new clinically effective agents. In this work, three platinum(IV) complexes of N-isopropyl-1R,2R-diaminocyclohexane with different axial ligands were deliberately designed and synthesized (Figure 1), and were biologically evaluated as potential antitumor agents in addition to analyzed for their hydrolysis behaviors, aiming to further reveal their aquation/ hydrolysis mechanism.

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Figure 2. ORTEP plot of complex 2 with thermal ellipsoids at 50% probability.

RESULTS AND DISCUSSION Synthesis and Characterization. The starting platinum(II) complex (cis-[(1R,2R)-N-isopropyl-1,2-diaminocyclohexaneN,N]dichloroplatinum(II), SM) was synthesized according to our previous reports,43 and the platinum(IV) dihydroxo product 1 can be obtained by oxidation with hydrogen peroxide (Scheme S1). Complexes 2 and 3 were subsequently prepared by the substitution of acetate and dichloroacetate for the hydroxyl groups (Figure 1). The resulting platinum(IV) complexes were characterized by elemental analysis and 1H and 13C NMR spectroscopy with electrospray ionization mass (ESI-MS) spectroscopy. All the spectral data were in good conformity with the proposed molecular structures of complexes 1−3. Moreover, X-ray crystallographic data proved that complex 2 has the expected molecular structure (Figure 2 and Table S1). Stability Study. It has been reported that platinum(IV) complexes can overcome limitations of platinum(II) complexes by resisting premature aquation,32−34 except those with axial haloacetato ligands.7,18,44,45 The latter, reported by Gibson and co-workers,18,44 can undergo rapid hydrolysis by releasing the axial haloacetato ligands. Hence, the stability of complexes 1−3 was studied by HPLC in aqueous solution at 37.5 °C. Complexes 1 and 2 were found to be stable under the studied condition (data not shown), while the content of complex 3 decreased very quickly (Figure 3) in the first 20 min, and dichloroacetate (DCA) was observed by negative-ion ESI-MS (Figure S1), indicative of the departure of the axial dichloroacetato ligands.

In Vitro Cytotoxicity. The cytotoxic activities of complex 1−3 were investigated by MTT assay against three human cancer cell lines: HCT116 (colon), HepG-2 (hepatoma), and A549 (lung). Cisplatin, carboplatin, and oxaliplatin were used as positive agents. The results of cytotoxicity against tumor cells in vitro are expressed as IC50 values which were calculated from the plot of cell viability against compound concentrations. According to the IC50 values in Table 1, complexes 1−3 showed potent cytotoxicity against HCT-116 and HepG-2 cancer cell lines; however, a very different picture was observed in the A549 cancer cell line which showed relatively less responsiveness to complexes 1−3. The overall antitumor potency order of the platinum(IV) complexes is complex 3 > 1 ≈ 2, suggesting that axial ligands have an obvious influence on the anticancer activity of the complexes. Among them, complex 3 is the most active compound, which showed superior cytotoxicity to cisplatin and oxaliplatin against the tested cancer cell lines. It seemed that A549 cells were markedly less chemosensitive to complexes 1 and 2, but complex 3 exhibited extraordinary cytotoxicity with an IC50 value of 0.98 μM, which is 7.5-fold and 17.2-fold as potent as cisplatin (7.3 μM) and oxaliplatin (16.9 μM). Notably, replacement of the axial hydroxyl groups in complex 1 by DCA has resulted in great improvement of cytotoxicity against the tested cancer cell lines, demonstrating that DCA and platinum complex may play a dual-killing mode against cancer cells. 9852

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry

Figure 3. HPLC chromatograms of the hydrolysis of complex 3 at (a) t = 0 min, (b) t = 20 min, (c) t = 40 min, (d) t = 60 min, and (e) t = 120 min.

Table 1. In Vitro Cytotoxicity (IC50 μM) of Complexes 1−3 with Cisplatin, Carboplatin, and Oxaliplatin as Positive Controls IC50 (μM) compd cisplatin carboplatin oxaliplatin complex 1 complex 2 complex 3

HCT-116 9.7 55.5 2.1 5.6 2.6 1.9

± ± ± ± ± ±

a

0.3 1.7 0.2 0.3 0.1 0.1

HepG-2b 4.8 25.6 13.1 8.6 9.1 1.6

± ± ± ± ± ±

0.3 2.1 0.8 0.5 0.4 0.1

A549c 7.3 42.6 16.9 55.0 50.6 0.98

± ± ± ± ± ±

0.1 0.1 1.1 0.1 0.1 0.07

a

Human colorectal cancer cell line. bHuman hepatocellular carcinoma cell line. cHuman non-small-cell lung cancer cell line.

Apoptosis Studies. Apoptosis is a programmed cell death in multicellular organisms, which plays an important role for the maintenance of tissue homeostasis and the deletion of aberrant cells. Thus, the apoptotic analysis of complexes 1 and 3 against HepG-2 cells was performed by using an Annexin V-FITC/propidium iodide assay. The tested compounds were incubated with HepG-2 cells for 24 h at 20 μM. Detailed analysis of the data revealed that cisplatin and complexes 1 and 3 improved the apoptotic rate of the HepG-2 cells as compared with that of the untreated cells (Figure 4). However, the effect of complex 3 (53.4%) on the cancer cell apoptosis is stronger than that of cisplatin (20.2%). Moreover, the population of annexin V+/PI− cells is evident upon complex 3 treatments, but little is observed after incubation with complex 1. Notably, the relative order of inducing apoptosis against HepG-2 cells is 3 (53.4%) > cisplatin (20.2%) > 1 (10.8%), which is consistent with the results of cytotoxicity assays. Thus, the platinum(IV) complexes produce cancer cell death through an apoptotic pathway. Hydrolysis Studies. The hydrolytic behavior of complexes 1−3 was studied by a kinetic method at three different pH values (5.0, 7.4, and 9.0). The time evolution spectra for 2 and 3 at pH 5.0 were recorded every 10 min (Figure S2). No changes of the absorption bands of complex 2 were observed within 10 h, demonstrating that complex 2 was stable at pH 5.0. However, the absorption bands of complex 3 decreased with time, indicating that hydrolysis of 3 can occur at pH 5.0. Complex 1 was not studied at pH 5.0 due to the acid−base reaction of the axial hydroxyl groups. As for pH 7.4, the changes of the absorption bands of complex 1 are very small, which means 1 was relatively stable at pH 7.4 (Figure 5). Surprisingly, the absorbance spectra of complex 2 decreased gradually with time, demonstrating that the aquation occurred at pH 7.4, which is contradictory to the assumption that the platinum(IV) complex is stable under

Figure 4. Flow cytometry analysis for apoptosis of HepG-2 cells induced by cisplatin and complexes 1 and 3 at the same concentration of 10 μM for 24 h: lower left, living cells; lower right, early apoptotic cells; upper right, late apoptotic cells; upper left, necrotic cells. Inserted numbers in the profiles indicate the percentage of the cells present in this area.

biologically relevant conditions. In addition, the absorption bands of complex 3 decreased quite fast in the first 10 min, indicating that there is a rapid first reaction, followed by a relatively slower second reaction step. The time trace observed at 250 nm for the hydrolysis of complex 2 was fitted well to a double-exponential function (Figure S3).46,47 However, owing to the slowness of the second reaction step, it did not go to completion during the test time range. Hence, only the first reaction step was analyzed by discarding the inaccurate value obtained for the second step.48 The hydrolysis rate constants of complex 2, and half-lives, are listed in Table 2. It can be seen that complex 2 can hydrolyze quickly at pH 7.4 with a half-live of 91.7 min. The study indicated that complex 2 may be less inert to hydrolysis under biologically relevant conditions. As for pH 9.0, the hydrolysis rates of complexes 2 and 3 were greatly accelerated as compared with those of pH 7.4 (for representative spectra of complex 2, see the Supporting Information, Figure S4). Moreover, the changes of the absorption 9853

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry

Figure 5. Time-dependent UV−vis spectra recorded every 10 min for the hydrolysis reaction of 0.1 mM complexes 1−3 (2% MeOH/98% H2O) at 37.5 °C, pH 7.4 (hepes).

Because the hydrolysis reaction of complex 2 may proceed through either the axial acetato ligands or the equatorial chlorido ligands by water/hydroxyl groups, an ESI-MS technique was applied to determine the hydrolysis positions of 2. The study was conducted by dissolving complex 2 in unbuffered water under a basic condition with pH < 10, and the MS spectra were detected after 2 h. Surprisingly, only the equatorially aquated moiety, [Pt(N-isopropyl-1R,2R-DACH)trans(OAc)2(OH)2], was observed (Figure 6), indicating that two equatorial chlorido ligands were replaced by OH−, while two axial OAc− groups remained intact. On the basis of the above study, it has been proposed that the equatorial chlorido ligands were substituted by OH− (Scheme 1). Thus, the influence of the Cl− on the hydrolysis of complex 2 was studied as a function of chloride concentration at pH 7.4. Obviously, 2 can reach equilibrium quickly in the presence of Cl− (Figure 7A), and kobs1 and kobs2 were obtained (Table S2) by fitting the time trace to a doubleexponential function. A linear dependence on the concentration of Cl− was observed for both steps (Figure 7B). Upon the proposed mechanism in Scheme 1, the expression for the observed first order rate constant, kobs1 and kobs2, can be expressed by eqs 1 (kobs1= k1 + k−1[Cl−]) and 2 (kobs2 = k2 + k−2[Cl−]). The values of k1, k−1, k2, and k−2 calculated from these plots are summarized in Table S3. The rate constant for the first step (k1 = 0.000144 s−1) agreed reasonably well with the values obtained in the absence of Cl− (0.000126 s−1). The result further confirms our conjecture that the hydrolysis of complex 2 occurs in the equatorial position. The surprising result prompted us to further study the influence of the carrier ligands of the platinum(IV) complexes. Thus, ctc-[Pt(NH3)2(OAc)2Cl2] (4), [Pt(1R,2R-DACH)trans(OAc)2Cl2]

Table 2. Hydrolysis Data for Complexes 1−3 (0.1 mM in 2% MeOH/98% H2O) Determined by UV−Vis Spectroscopy at 37.5 °C, pH 7.4 (Hepes) pH = 7.4

a

compd

104 k1 (s−1)

t1/2 (min)

complex 1 complex 2 complex 3

n.d.a 1.26 ± 0.13 60.2 ± 5.5

n.d.a 91.7 1.9

n.d.= not determined.

bands were also observed for complex 1 (Figure S5), which means the hydrolysis of complex 1 could occur at pH 9.0. According to the values presented in Table 3, the k1 values for Table 3. Hydrolysis Data for Complexes 1−3 (0.1 mM, 2% MeOH/98% H2O) Determined by UV−Vis Spectroscopy at 37.5 °C, pH 7.4 (Hepes) pH = 9.0

a

compd

10 k1(s )

t1/2 (min)

104 k2 (s−1)

t1/2 (min)

complex 1 complex 2 complex 3

3.71 ± 0.25 45.4 ± 3.3 341.3 ± 43.2

31.1 2.54 0.34

n.d.a 9.85 ± 0.76 43.3 ± 5.5

n.d.a 11.7 2.67

4

−1

n.d.= not determined.

complex 2 increased by a factor of 36, and that of complex 3 was accelerated by a factor of 5.6 on going from pH 7.4 to pH 9.0. In general, this study indicated that the hydrolysis of the studied platinum(IV) complexes is pH-dependent, which proceeds much faster at basic pH.

Figure 6. Positive mass spectra of the solution resulting from the hydrolysis of complex 2 at 37.5 °C after 2 h. 9854

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry Scheme 1. Proposed Pathway for the Hydrolysis of Complex 2

Figure 7. (A) Absorbance−time trace and two-exponential fit obtained for the hydrolysis of 0.1 mM complex 2 (2% MeOH/98% H2O) in the presence of 3 M Cl−. (B) Plots of kobs versus [Cl−] concentration for complex 2. T = 37.5 °C, pH 7.4 (hepes).

studied here.28 For the latter case, the first dechloration step was assumed to occur in trans position to N-isopropyl amine due to the higher trans effect.34 The fully optimized geometries can be found in the Supporting Information (Table S4 and Figure 8), and the free energy profiles are shown in Figure 9. It is clear that the substitution of the equatorial chlorido ligand has to overcome a barrier of 26.8 kcal/mol from the previous minimum, while that for the axial acetato ligand is 34 kcal/mol, revealing that the equatorial substitution could occur prior to the axial one, which is in accordance with our experimental findings. The hydrolysis in the equatorial position of complexes 1−5 and satraplatin was also investigated by theoretical calculations. The calculated free energies of the minima and free energy barriers for hydrolysis of complexes 1−5 and satraplatin are collected in Table 5. It appears that the energy barrier values of complexes 1−3 are comparable to each other. As complexes 2, 4, 5, and satraplatin possess a similar structure except for the different carrier ligands, the calculated results show that the barriers for these four complexes are 4 > satraplatin > 5 > 2, which are also quite consistent with our experimental results, demonstrating that the increasing σ-donor ability of the carrier ligand can decrease the activation energy of the corresponding platinum(IV) complex in the hydrolysis reaction.

(5), and satraplatin were synthesized, and the hydrolysis behaviors of complexes 4 and 5 and satraplatin were studied as well. The time-dependent UV−vis spectra of 4, 5, and satraplatin at different pH values were presented in Figure S6. It appeared that complex 4 was stable at pH 7.4, but hydrolyzed at pH 9.0. However, the reaction was so slow that an induction time (about 3 h) was observed for the reaction to start (Figure S7).31 As for complex 5 and satraplatin, the hydrolysis could be observed at pH 7.4 and 9.0, and the hydrolysis rates at pH 9.0 were presented in Table 4 (Figure S8). Notably, the k1 values Table 4. Hydrolysis Data for Platinum(IV) Complexes Determined by UV−Vis Spectroscopy at 37.5 °C, pH 7.4 (Hepes) pH = 9.0

a

compd

10 k1 (s )

t1/2 (min)

104 k2 (s−1)

t1/2 (min)

complex 2 complex 4 complex 5 satraplatin

45.4 ± 3.3 n.d.a 8.76 ± 0.82 1.95 ± 0.15

2.5 n.d.a 13.2 59.2

9.85 ± 0.76 n.d.a 2.42 ± 0.29 0.42 ± 0.04

11.7 n.d.a 47.7 275.1

4

−1

n.d.= not determined (too slow).

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increased by a factor of 5.2, and the second step was accelerated by a factor of 4.2 on going from complex 5 to complex 2 at pH 9.0, indicating that the introduction of the isopropyl group to 1R,2R-diaminocyclohexane can promote the hydrolysis rates of the corresponding platinum(IV) complexes. Comparing the hydrolysis rates of complexes 2, 4, 5 and satraplatin bearing different carrier ligands, it is rational to conclude that increasing σ-donor ability of the carrier ligand (NH3 < NH2R < NHR2) can improve the hydrolysis rates of the corresponding complex (4 < satraplatin < 5 < 2). Taken together, the platinum(IV) complexes with axial acetato ligands may be less inert to hydrolysis than expected under biologically relevant conditions. This study can explain why the equatorially aquated species of satraplatin can be observed under biologically relevant conditions. Computational Study. DFT calculations were applied to elucidate the hydrolysis mechanism of complexes 1−5 and satraplatin. The hydrolysis of complex 2 may occur either in axial or equatorial positions. Thus, two potential pathways were

CONCLUSION In summary, complex 2 with axial acetato ligands can hydrolyze relatively quickly under biologically relevant conditions with a half-life of 91.7 min, resulting in the loss of the equatorial chlorido ligands. Further study indicated that the fast hydrolysis of complex 2 may be attributed to the strong σ-donor ability of N-isopropyl-1R,2R-diaminocyclohexane, and the increasing σ-donor ability of the amine group can promote the hydrolysis rate of the corresponding platinum(IV) complex. This study can help to re-evaluate the aqueous properties of the platinum(IV) complexes with axial acetato ligands, which is contradictory to the prevailing notion that platinum(IV) complexes are stable enough to resist premature aquation and bind to essential plasma proteins. As platinum(IV) complexes with axial acetato ligands like satraplatin and LA-12 have great potential in clinical 9855

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry

Figure 8. Optimized structures and selected structural parameters for the hydrolysis of complex 2: (a) hydrolysis at the axial position and (b) hydrolysis at the equatorial position. The indicated values are bond distances in angstroms. analyzed by Cell Quest software. UV−vis spectra and kinetic traces were recorded on a Shimadzu UV2600 instrument equipped with a thermostatically controlled cell holder. 1H and 13C NMR spectra were measured in DMSO-d6 with a Bruker 300 MHz spectrometer. Mass spectra were measured by an Agilent 6224 ESI/TOF MS instrument. Elemental analyses of C, H, and N used a Vario MICRO CHNOS elemental analyzer (Elementar). cis-[(1R,2R)-N-Isopropyl-1,2-diaminocyclohexane- N,N′]dichloroplatinum(II) (SM) was prepared according to our previous report.43 Procedure for Synthesis of Complex 1. cis-[(1R,2R)-N-Isopropyl1,2-diaminocyclohexane-N,N′]dichloroplatinum(II) (SM) (2.1 g, 5 mmol) was suspended in 10 mL of water, and 10 mL of 30% H2O2 was added. The mixture was stirred at 50 °C for 12 h in the dark. Then, the reaction mixture was concentrated to 5 mL and cooled to room temperature; the crude product was separated, washed with water, and dried in a vacuum-dryer. Yield: 1.78 g (82.1%), yellow powder. Anal. Calcd (%) for C9H22Cl2N2O2Pt: C 23.69, H 4.86, N 6.14. Found: C 23.60, H 4.91, N 6.05. ESI-MS: m/z [2M + H]+ = 913.15. 1H NMR (300 MHz, DMSO-d6) δ 1.06−1.13 (2H, m, CH2 of DACH), 1.31−1.33 (3H, d, 3JH,H = 6.9 Hz, CH3CHCH3), 1.41−1.43 (4H, d, 3JH,H = 6.9 Hz, CH3CHCH3 and CH2 of DACH), 1.62−1.65 (3H, m, CH2 of DACH), 2.08−2.12 (1H, m, CH2 of DACH), 2.20−2.24 (1H, m, CH2 of DACH), 2.85 (2H, m, CH2 of DACH), 4.36−4.41 (1H, m, CH3CHCH3), 6.09 (1H, m, NH), 6.78 (1H, m, NH), 7.40 (1H, m, NH), 10.22 (2H, m, OH) ppm. 13C NMR (75 MHz, DMSO-d6) δ 19.48, 24.34, 24.76, 31.69, 31.84, 55.24, 61.76, 67.18 ppm. General Procedure for Synthesis of Complexes 2 and 3. Acetic anhydride/dichloroacetic anhydride (3 mmol) was added to a solution of complex 1 (0.46 g, 1 mmol) in DMF (8 mL), and the reaction mixture was stirred at room temperature for 12 h. Diethyl ether was added to the mixture to precipitate the products, which were washed several times with diethyl ether and dried in vacuum. The precipitate was recrystallized from methanol at 50 °C. Complex 2.

Figure 9. Free energy profiles (at 310.15 K) for the first-step hydrolysis reaction of complex 2 at different positions.

Table 5. Computational Results of Hydrolysis of Complexes 1−5 and Satraplatin in Equatorial Positiona compd

RAb

TSc (ΔE⧧)

PAd

complex 1 complex 2 complex 3 complex 4 complex 5 satraplatin

6.4 6.3 5.8 4.4 5.8 4.2

26.3 26.8 26.7 32.1 28.4 30.1

15.3 17.0 15.6 14.3 17.8 15.9

a

All the values are in kcal/mol. bRelative free energies of formation of the first reaction adduct. cActivation energies. dRelative free energies of product.

applications, it is of much significance to explore the aqueous chemistry of platinum(IV) complexes with axial acetato ligands. Our study suggests that the hydrolysis of the platinum(IV) complexes should be considered when designing new platinum(IV) complexes as antitumor agents.

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EXPERIMENTAL SECTION

Materials and Measurements. All chemicals and solvents were of analytical reagent grade and used without further purification. Potassium tetrachloroplatinate(II) was purchased from a local chemical company (Lingfeng Chemical Ltd.). Apoptosis experiments were measured by flow cytometry (FAC Scan, Becton Dickenson) and

Yield: 0.33 g (61%). Pale yellow crystals. Anal. Calcd (%) for C13H26Cl2N2O4Pt: C 28.90, H 4.85, N 5.18. Found: C 28.79, H 4.91, N 4.89. ESI-MS: m/z [2M + H]+ = 1081.19 (100%), [M + H]+ = 9856

DOI: 10.1021/acs.inorgchem.7b01355 Inorg. Chem. 2017, 56, 9851−9859

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Inorganic Chemistry 541.10 (50%). 1H NMR (300 MHz, DMSO-d6) δ 1.22−1.24 (4H, d, 3 JH,H = 6.9 Hz, CH3CHCH3 and CH2 of DACH), 1.29−1.38 (6H, m, CH3CHCH3 and CH2 of DACH), 1.50 (2H, m, CH2 of DACH), 1.96−1.98 (6H, d, J = 4.5 Hz, CH3COO), 2.24−2.27 (1H, m, CH2 of DACH), 2.37 (1H, m, CH2 of DACH), 2.58−2.62 (1H, m, CH2 of DACH), 2.90−2.93 (1H, m, CH2 of DACH), 4.17−4.21 (1H, m, CH3CHCH3), 8.15 (1H, m, NH), 10.02 (1H, m, NH), 10.98−11.21 (1H, m, NH) ppm. 13C NMR (75 MHz, DMSO-d6) δ 18.08, 23.85, 23.99, 24.34, 32.62, 33.03, 53.34, 62.58, 65.61, 181.94, 182.40 ppm. Complex 3.

for complexes 1 and 2 and CH2CN/H2O (75:25) for complex 3. The flow rate was 1 mL/min−1 with detection at 230 nm. Complexes 1−3 were diluted to 0.1 mm with purified water and incubated at 37.5 °C; data were collected at different times by reversed-phase HPLC. UV−Vis Kinetics Experiments. Hydrolyses of complexes 1−5 and satraplatin were recorded on a Shimadzu UV2600 instrument equipped with a thermostatically controlled cell holder. A suitable wavelength for kinetic trace was determined by recording spectra of the reaction mixture over the wavelength range 200−400 nm, and 250 nm was selected for observation of all the kinetics measurements. Hepes buffer was selected to control the pH because it is sterically crowded and does not coordinate to platinum complexes.51 For pH 5.0 and 9.0, a small amount of phosphate was added to the buffer. All the reactions were studied at 37.5 ± 0.1 °C in the presence of hepes (0.1 M) and NaClO4 (0.1 M). Computational Details. All calculations were performed using the Gaussian 09 program package.52 All structures of reactants, products, transition states, and intermediates were optimized and characterized as minima or transition states at the M06-L/6-31G(d,p)//LanL2DZ level in the gas phase at 310.15 K and 1 atm.53,54 Transition states were further checked by intrinsic reaction coordinate (IRC) analysis.55 More accurate energies were refined by the M06-L/6-311++G(2df, 2pd)//LanL2DZ single-point calculations with solvation effects of water included and simulated by IEFPCM model. Apoptosis Study. HepG-2 cells were transferred to 6-well plates and cultured overnight in 5% CO2 at 37 °C. Platinum(IV) complexes were added, which were diluted to a concentration of 10 μM. After 24 h, the cells were digested with trypsin and washed twice with cold PBS. Then, cells were collected by centrifugation (5 min, 25 °C, 2000 rpm). The cells were then washed twice with cold water, resuspended in binding buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4), and incubated with annexin V-FITC (100 ng/mL) and then with propidium iodide (2 μg/mL) for 15 min in the dark at room temperature. At last, the fluorescence of cells was detected by an annexin V-FITC apoptosis detection kit (Roche) according to the manufacturer’s protocol, and cells were analyzed by a computer station running Cell Quest software. In Vitro Cytotoxicity Studies. Cytotoxicity of complexes 1−3, cisplatin, carboplatin, and oxaliplatin against HCT-116 (human colorectal cancer cell line), A549 (human non-small-cell lung cancer cell line), and HepG2 (human hepatocellular carcinoma cell line) was determined by means of the colorimetric assay MTT (3-(4,5dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide). The cells were plated in 96-well culture plates at a density of 3000 cells per well with culture medium and were incubated for 24 h at 37 °C in a water atmosphere (5% CO2). The compounds with desired concentrations were obtained by dissolving in DMF and diluting with culture medium (DMF final concentration