http://www.chemistrymag.org/cji/2008/104021pe.htm

Apr. 1, 2008  Vol.10 No.4 P.21 Copyright cij17logo.gif (917 bytes)

Synthesis of a novel anti-cancer prodrug analogue PtCl2(S2COC2H5)2 and its reduction by L-cysteine

Zhang Haibing, Shen Shigang, Shi Hongmei, Huo Shuying, Xie Mingshu, Song Changying, Zhang Suna
(College of Chemistry and Environmental Science, Hebei Uinversity, Baoding 071002, Hebei Province, China)

Abstract A novel platinum xanthate complex (PtCl2(S2COC2H5)2) has been synthesised and characterized by IR. Reduction of the trans-dichlorobis(O-ethyldithiocarbonato) platinum(IV) by L-cysteine has been investigated at 298.15 K in the rang 3 ≤ pH ≤ 7 using the conventional spectrophotometry. The reaction display an overall second-order rate law: -d[Pt(IV)]/dt = k'[Pt(IV)][L-cysteine]tot, where [L-cysteine]tot is the total concentration of L-cysteine. The pH dependence of k' is ascribed to parallel reductions of trans-dichlorobis(O-ethyldithiocarbonato) platinum(IV) by various protolytic species of L-cysteine, the relative contributions of which change with pH. Electron transfer from thiol to Pt(IV) complex is suggested to take place as a reductive elimination process through an attack by sulfur at one of the mutually trans chloride ligands. It was found that the pseudo-first order rate constant kobs increased with increasing concentration of 1/[-S2COC2H5], which involves a pre-equilibria. The reaction mechanism was proposed can explain all the experimental observations.
Keywords L-cysteine, Platinum xanthate complex, Reduction, Kinetics and mechanism, Synthesis

1. INTRODUCTION
Platinum complexes are now a well-established class of cancer chemotherapy agents. Cisplatin is widely used in treatment of several human cancers including those of tests, ovaries and bladder[1-4]. However, its spectrum of antitumor activity is narrow, and its clinical use is limited by severe dose toxicities such as nephrotoxicity, ototoxicity, neutotoxicity, nausea, vomiting, and myelosuppression [1,3,5-9 ]. Therefore, researchs in many laboratories around the world have been actively engaged in synthesizing and studying cisplatin and its analogues, hoping to discover a better antitumor drug that is less toxic, has better antitumor activity, and is fairly soluble in water. The result was carboplatin and 254-S, which have been approved for clinical use and several others are on clinical trials [3, 5, 10, 11].
    A considerable part of the effort in the developing a new generation of platinum-based anticancer drugs is to find novel platinum(IV ). Indeed, many platinum(IV) complexes have been found to be anticancer active [12-14 ]; cis-[Pt(NH3)2Cl4], iproplatin, tetraplatin, JM335 and JM216 (cis,cis-,trans-[Pt(Cl2(NH3)(cyclo-C6H11NH2)(CH3COO)2]) can be regarded as the represe- ntatives for this developing process [1,15-20]. Iproplatin(cis,trans,cis-[PtCl2(OH)2(isopropylamin- e)2]) was selected from a range of platinum(IV) complexes synthesized by Tobe and coworkers for its high solubility [21]. Iproplatin was sufficiently well tolerated to enter phase II and III clinical trials，but was ultimately found to be less active than cisplatin and so has not entered widespread clinical use [22]. Tetraplatin show great promise in preclinical studies but caused severe neurotoxicity in treated patients and the trials were subsequently abandoned at the phase I level. JM216 is a rationally designed drug which was in phase III trials, but trials were abandoned due to variability in drug uptake [23].
    Recently a platinum complex based on sulfur as complex-forming atoms, bis(O-ethyldithiocarbonato)platinum(II), named thioplatin, with antitumoral activity against a number of human tumor lines was described. It was found that thioplatin displayed significantly higher cytotocicity when tumor cells were cultivated in media of pH of 6.8 compared to media of pH 7.4. Because in solid tumors a pH of 6.8 and lower has been frequently observed [25], an improved therapeutic index with thioplatin could be expected. Indeed, thioplatin displayed antitumoral activity on human tumors xenotransplanted in nude mice, which was comparable to cispaltin. Yet a significantly lower toxicity on kidney, small intestines, and white blood cell count was encountered [24]. Twenty different bis(O-ethyldithiocarbonato)platinum(II), complexes were synthesized and tested for cytotoxic activity in a panel of six human tumor lines by Wolfgang Friebolin and his coworkers. Derivatives with up to 7-fold increased activity compared to thioplatin and up to 25-fold more activity than cisplatin were identified. Bis(O-alkyldithiocarto)platinum(II) complexes with short n-alkyl chains were superior to compounds with long n-alkyl chain [26].
    Conversion of platinum(II) complexes to platinum(IV) analogues is a approach to moderate the toxicity of platinum(II) complexes [27]. In fact, much has been done to develop more stable Pt(IV) analogues [28] that would facilitate clinical evaluation and also from the view point of modulating favorable interactions with target DNA [29], increase the spectrum of antitumor activity. The reduction potentials of Pt(IV) complexes are dependent on the nature of the axial and equatorial ligands, but the axial ligands generally exert the stronger influence, reduction occurs most readily when the axial ligands are chloro [8]. In this paper, PtCl2(S2COC2H5)2, as shown in Chart 1, that a novel anti-cancer prodrug thioplatin's analogue was synthesized and characterized. In present study, we report here the kinetics and mechanism studies of its reduction by L-cysteine in vitro. The purpose of this study is to have a better understand of the reduction process and mechanism and to give some information about design of new drug

Chart 1 PtCl2(S2COC2H5)2

2. EXPERMENTAL SECTION
2.1 Chemicals and solutions              
L-cysteine was obtained from Shanghai zhengxiang Chemical Reagent Company. K2PtCl4 was purchased from Alfa Aesar. NaCl, NaClO4, HClO4, isopropyl alcohol and potassium ethyl xanthate were obtained either from Beijing Chemical Reagent Company (Beijing, China) or from Tianjin Chemical Reagent (Tianjin, China). All the above reagents were of either analytical grade or reagent grade and used as received without further purification. All solutions were prepared with doubly distilled water.
2.2. Synthesis and Characterization of PtCl2(S2COC2H5)2
The Pt(S2COC2H5)2 was synthesized in accordance to the literature [26]. Postassium ethyl xanthate was reacted with dipotassium tetrachloroplatinate (II). A solution of 0.6mmol potassium ethyl xanthate in 5mL of water was added to 0.6mmol of K2PtCl4 dissolved in 10mL of water. Immediate precipitation of a yellow solid could be observed. The reaction progressed slowly. The mixture was stirred at room temperature for 4 h. Then Cl2 was blowed into the mixture for 3 h, followed by purging with nitrogen for 2 h in order to remove dissolved chlorine. The mixture was filtered, washed three times with distilled water, and was crystallized from acetone. The product was dried in vacuo (yield: 145.4mg, 47.7% ). IR (KBr): 3419 (m), 2983 (w), 2935 (w), 1632 (w), 1397 (w), 1195 (s), 1060 (w), 879 (w), 597 (w).
2.3. Spectral and kinetic measurements
UV-visible spectra were recorded on a TU-1901 spectrophotometer (Beijing, China) and quartz cells with a 1.00 cm optical pathlength were used. The spectrophotometer was equipped with a cell compartment which was thermostated by circulation of water from a thermostat (BG-chiller E10, Beijing Biotect Inc., Beijing). Temperature of solutions in cells can be controlled to ±0.2℃ when cells are put in the compartment. Two reaction solutions, one containing known concentrations of PtCl2(S2COC2H5)2, KS2COC2H5, NaCl, (CH3)2CHOH, and the other containing desired concentrations of L-cysteine, HClO4 and NaClO4, were thermostated for at least 20 min before mixing each other. The function of NaClO4 was to adjust the ionic strength (m) in the reaction solutions and m= 0.5 M was kept for all the kinetic measurements in this work. The HClO4 was used to adjust the pH. The pH of the system was measured at 298.15 K with a Metrohn 632 digital pH meter equipped with a combination glass electrode. All platinum xanthates were found to be insoluble in water. Thus, (CH3)2CHOH was added to enhance the product's solubility. Kinetic traces were followed at 262 nm by the same spectrophotometer mentioned above. Pseudo first-order reaction conditions were fulfilled by making [L-cysteine] ≥ 50[Pt(IV)]. Reactions were followed for at least 8 half-life with 3 repetitive runs.

3. RESULT AND DISCUSSION
3.1 Kinetic                                
The kinetic traces for reduction of Pt(IV) to Pt(II) can be described by single exponentials and variation of [Pt(IV)] and added [Cl-] in the condition of our experimental system do not affect the rate constants (Table 1,2), suggesting that the reduction is first-order in platinum(IV) complexes.

Table 1  103kobs/s-1 varying with different concentrations of [Pt(IV)] at 298.15K. Reaction conditions: [ClO-4] = 0.5 M, [Cl-] = 3 mM, [L-cysteine] = 5.6 mM，[-S2COC2H5]= 0.1 mM, [(CH3)2CHOH] = 1.95 M, pH = 4.

105c/M

2.8

1.4

4.2

5.6

7.0

103kobs/s-1

9.79

9.82

9.81

9.82

9.78

Table 2 103 kobs/s-1 varying with different concentrations of [Cl-] at 298.15K. Reaction conditions: [ClO-4] = 0.5 M, [Pt(IV)] = 0.056 mM, [L-cysteine] = 5.6 mM，[-S2COC2H5] = 0.1 mM, [(CH3)2CHOH] = 1.95 M, pH= 4.

c/M

0.001

0.002

0.003

0.004

0.005

103kobs/s-1

9.97

10.36

9.97

9.64

9.99

    Plots of kobs versus [L-cysteine] are linear and pass through the origin at different temperature, as shown in Fig. 1, proving that the reduction is also first-order in L-cysteine.
    Thus, the rate law is described by Equation (1), where [L-cysteine] represents the total concentration of reductant.
= kobs[Pt(IV)] = k' [L-cysteine] [Pt(IV)] (1)
    Kinetics and mechanism for oxidation of L-cysteine by Pt(IV) was investigated under the condition of 298.15K and pH 4, and the oxidation reaction also follow an overall second-order rate law. Keeping other condition constant, plots of kobs versus 1/[-S2COC2H5] (0.1-0.5 mM) are linear at different temperature as shown in Fig. 2, kobs increases with increasing 1/[ -S2COC2H5 ]. The values of kobs versus [(CH3)2CHOH] and pH have been collected and displayed in Fig. 3 and Table 3.

Fig.1 Pseudo first-order rate constants, kobs, as a function of [L-cysteine]. Reaction conditions: [Pt(IV)] = 0.056 mM, [ClO-4] = 0.5 M, [-S2COC2H5] = 0.1 mM, [(CH3)2CHOH] = 1.95 M, pH = 4, [Cl-] = 3 mM.


3.2 Protolytic equilibria
Under the reaction conditions in the present work, we keep pH 4 constant during the reaction course. In aqueous solutions, the ionization of L-cysteine depends on pH and can be described as follows [30]

Chart 2

    It can be calculated from those protolysis data that more than 99.17% of amino acid is existing in the form of H3N+CH(CH2SH)COO- in pH 4. Based on the above equilibrium constants, calculations reveal that under our experimental conditions H3N+CH(CH2SH)COO- is the predominant species whereas the H3N+CH(CH2SH)COOH and H3N+CH(CH2S-)COO- make a very minor contribution to the total [L-cysteine] speciation are very trifling and negligible. Thus, the total concentration of [L-cysteine], [L-cysteine]tot , can be expressed by:
[L-cysteine]tot = [H3N+CH(CH2SH)COO-] +[H3N+CH(CH2SH)COOH]+[H3N+CH(CH2S-)COO-]≈[H3N+CH(CH2SH)COO-] (2)
[H3N+CH(CH2SH)COO-]= (3)

3.3 Reaction mechanism                            
The kobs-pH values shown in Table 3 clearly demonstrate that the deprotonated L-cysteine species are more reactive than the protonated ones. kobs increases with the increasing pH.

Table 3  pseudo first-order rate constants kobs with pH at 298.15K. Reaction conditions: [Pt(IV)] = 0.056 mM, [ClO-4] = 0.5 M, [Cl-] = 3 mM, [L-cysteine] = 5.6 mM，[-S2COC2H5] = 0.1 mM, [(CH3)2CHOH] = 1.95 M.

pH

3

4

5

6

7

103kobs/s-1

8.87

9.67

10.02

10.55

11.86

    The dramatic change of kobs with pH has also been described in the reduction of trans- [Pt(CN)4Cl2]2- by T. Shi [31]. The pH dependence of kobs is attributed to the displacement of protolytic equilibria involving the various anionic species of the L-cysteine as shown in Chart 2. The anionic species of L-cysteine reduce PtCl2(S2COC2H5)2 in parallel reactions in which the contribution of each pathway to the overall reduction depends on the relative concentration and reducing power of the various L-cysteine species. It can be calculated from those protolysis data as shown in Chart 2 in pH 4 that H3N+CH(CH2SH)COO- is the predominant species, others is very trifling and negligible.
    It is expected that initial substitution of the coordinated chlorides by L-cysteine is unlikely, since the platinum(IV) is essentially substitution inert and variation of chloride concentration does not affect the reduction rate. Previous mechanistic studies on reductions of platinum (IV) halide complexes by inorganic [32-34] and biological [31,35] reductant have been shown that electron transfer involves reductive elimination through nucleophilic attack by the reductant on a halide coordinated trans to a good leaving group. Reductive elimination reaction of platinum(IV) compounds via the halide-bridged activated complex are formally equivalent to a transfer of Cl+ from the oxidizing Pt(IV) center to the reducing nucleophile, followed by loss of the trans ligand[31-34] as follow (where RSH = L-cysteine).

Chart 3

    The intermediate oxidation products, RSHCl+ and RSCl, undergo the rapid subsequent reaction (4)-(6), leading to the final products RSSR [8, 36].
RSHCl+ +H2O →RSOH + Cl+ + 2H+ (4)
RSCl + H2O → RSOH + Cl- + H+ (5)
RSOH + RSH → RSSR + H2O (6)
RSOH +RS- → RSSR + OH- (7)
    RSHCl+ and RSCl will hydrolyze in a fast subsequent step according to Equations (4) and (5) ,and RSOH formed will trapped by RSH and RS- according to Equations (6) and (7), respectively [31].
    The fact that thiols attack coordinated chloride through the sulfur atom is described by Kelemu and his colleagues, the deprotonation of the thiol group-SH increases the reduction rate. The fact is consistent with our experimental data, RSH produces more RS- with pH increasing that will increase the reduction rate.
    It is clear from the Fig.2 that kobs decrease with [-S2COC2H5] increasing, [-S2COC2H5], this kind of reaction trend has been interpreted in terms of a pre-equilibrium in which PtCl2(S2COC2H5)2 equilibrates with [PtCl2(CH3)2CHOHS2COC2H5]. It displays in equation (8).

Fig. 2 Pseudo first-order rate constants kobs as a function of [-S2COC2H5] at four temperatures. Reaction conditions: [Pt(IV)] = 0.056 mM, [ClO-4] = 0.5 M, [(CH3)2CHOH] = 1.95 M, pH = 4, [Cl-] = 3 mM, [L-cysteine] = 5.6 mM.

PtCl2(S2COC2H5)2+2(CH3)2CHOH[PtCl2((CH3)2CHOH)2S2COC2H5]+-S2COC2H5 (8)
(9)
    Moreover, it was assumed that [PtCl2((CH3)2CHOH)2S2COC2H5] was the reactive species whereas PtCl2(S2COC2H5)2 had less reactive.
    It is noteworthy that the kobs increase rapidly with increasing [(CH3)2CHOH], the plots kobs versus [(CH3)2CHOH]2 are linear, as shown in Fig. 3,indicating that [PtCl2((CH3)2CHOH)2S2COC2H5] is the reactive species.

Fig. 3 Pseudo first-order rate constants kobs as a function of [(CH3)2CHOH]2 for the reduction of Pt(IV) by L-cysteine. Reaction conditions : [Pt(IV)] = 0.056 mM, [ClO-4] = 0.5 M, pH = 4, [Cl-] = 3 mM, [L-cysteine] = 5.6 mM，[-S2COC2H5] = 0.1 mM.

    With PtCl2(S2COC2H5)2 and PtCl2((CH3)2CHOH)2S2COC2H5 acting in the two parallel pathways, reactions (8), (10) and (11) describe the present systems.
PtCl2(S2COC2H5)2 + RSH RSHCl+ + Cl- + Pt(S2COC2H5)2 (10)
[PtCl2((CH3)2CHOH)2S2COC2H5]+RSHRSHCl+ + Cl-+ [Pt((CH3)2CHOH)2S2COC2H5] (11)

    The rate law is then given by Equation (12),where
[L-cysteine]tot= and [Pt(IV)] = [PtCl2(S2COC2H5)2]+ [PtCl2((CH3)2CHOH)2S2COC2H5].
= kobs[Pt(IV)]
= k' [L-cysteine]tot [Pt(IV)]


    The PtCl2((CH3)2CHOH)2S2COC2H5 was the reactive species, k2 >>1, K << 1, thus K[(CH3)2CHOH]2 can be neglected in the rate law, it is confirmed by our calculation . Reaction (11) is the rate-determining step.
(12)
Or (13)
    From Equation (13), the plots of kobs versus [(CH3)2CHOH]2 are straight line and increase with [(CH3)2CHOH]2 increasing, the value of kobs versus 1 / [-S2COC2H5] is line and increase with increasing 1 / [-S2COC2H5], [Cl-] does not affect the rate constant, plots of kobs versus [L-cysteine] are linear, and increase with increasing pH. These are consistent with our experiments result.

4 CONCLUSION
Undoubtedly, the proposed reaction mechanism can explain convincingly all the experimental observations. The reactions display an overall second-order behavior: first-order with respect to both Pt(IV) and L-cysteine. The kobs-pH demonstrate that the deprotonated L-cysteine species are more reactive than the protonated ones. The reduction is strongly pH dependent, being related to the protonation of the amino acid. The reduction of Pt(IV) involves halide-mediated reductive-elimination reactions of platinum(IV)-halogen complexes involving various reducants have been suggested to take place via an attack by reductant on co-ordinated halide. The present results show that reduction of PtCl2(S2COC2H5)2 by L-cysteine in a acidic aqueous perchlorate medium is not fast, which increase the chance of Pt(II) arriving at the target site intact[8,33]. Our next step is to study its antitumor activity in vivo.

Acknowledgement   Financial support of this work in part by a grant from the Natural Science Foudation of Hebei Province (B2006000962) is gratefully acknowledged.

Appendix A. Supplementary material  Supporting tables S1-S .summarize the pseudo first-order rate constants measured under various reaction conditions.

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新型四价铂抗癌药物PtCl2(S2COC2H5)2的合成以及与L-半胱氨酸的作用机理的研究
张海兵，申世刚，石红梅，霍树营，谢明树，宋常英，张素娜
(河北大学化学与环境科学学院，河北，保定，071002)
摘要 本文合成了一种新型抗癌药物的二氯二乙基黄原酸合铂，利用红外进行了表征。我们在298.15K，3 ≤ pH ≤ 7，0.1mM条件下，用双光束的紫外分光光度计对其与L-半胱氨酸的还原反应进行了研究。研究表明整个反应是二级反应，-d[Pt(IV)]/dt = k'[Pt(IV)][L-cysteine]tot, [L-cysteine]tot是L-半胱氨酸的总浓度。pH对k' 的影响，主要是pH变化使L-半胱氨酸不同的质子化形式与Pt（IV）反应。电子从硫醇到Pt（IV）的转移过程，表明发生了还原消去反应，通过反位的氯进攻硫而实现的。我们研究发现准一级反应速率常数kobs随1 / [-S2COC2H5]增加而增加，其涉及一个前期平衡反应。我们提出的反应机理，很好的解释了实验现象。
关键词 L-半胱氨酸；黄酸合铂；还原；动力学及机理；合成

　

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