# Kinetic Analysis of the Reduction Processes of a Cisplatin Pt(IV) Prodrug by Mesna, Thioglycolic Acid, and Thiolactic Acid

Authors: Yanqing Xia; Hongwu Tian; Yanlei Li; Xinru Yang; Jinming Liu; Chunli Liu; Li Zhou; Lincai Zhang; Tiejian Li; Tiesheng Shi
Journal: Journal of Chemistry
 (2020)

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## Abstract

Although Mesna is an FDA-approved chemotherapeutic adjuvant and an antioxidant based largely on its antioxidative properties, kinetic and mechanistic studies of its redox reactions are limited. A kinetic analysis of the reduction processes ofcis-diamminetetrachloroplatinum(IV) (cis-[Pt(NH3)2Cl4], a cisplatin Pt(IV) prodrug) by thiol-containing compounds Mesna, thioglycolic acid (TGA), and DL-thiolactic acid (TLA) was carried out in this work at 25.0°C and 1.0 M ionic strength. The reduction processes were followed under pseudo-first-order conditions and were found to strictly obey overall second-order kinetics; the observed second-order rate constant k′ versus pH profiles were established in a wide pH range. A general reaction stoichiometry of Δ[Pt(IV)] : Δ[Thiol]tot = 1 : 2 was revealed for all the thiols; the thiols were oxidized to their corresponding disulfides which were identified by mass spectrometry. Reaction mechanisms are proposed which involves all the prololytic species of the thiols attacking the Pt(IV) prodrug in parallel, designating as the rate-determining steps. Transient species chlorothiol and/or chlorothiolate are formed in these steps; for each particular thiol, these transient species can be trapped rapidly by another thiol molecule which is in excess in the reaction mixture, giving rise to a disulfide as the oxidation product. The rate constants of the rate-determining steps were elucidated, revealing reactivity enhancements of (1.4–8.9) × 105 times when the thiols become thiolates. The species versus pH and reactivity of species versus pH distribution diagrams were constructed, demonstrating that the species ‒SCH2CH2SO3‒ of Mesna largely governs the total reactivity when pH > 5; in contrast, the form of Mesna per se (mainly as HSCH2CH2SO3‒) makes a negligible contribution. In addition, a well-determined dissociation constant for the Mesna thiol group (pKa2 = 8.85 ± 0.05 at 25.0°C and μ = 1.0 M) is offered in this work, which was determined by both kinetic approach and spectrophotometic titration method.


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## Body

## 1. Introduction

Mesna (namely, sodium 2-mercaptoethanesulfonate) is an FDA-approved drug which has been used to reduce the risk of hemorrhagic cystitis (a condition that causes inflammation of the bladder and can result in serious bleeding) in people who receive ifosfamide or cyclophosphamide for cancer treatments [1–3]. These two anticancer drugs in vivo may be converted to urotoxic metabolites such as acrolein. The protecting mechanism of Mesna is assisting to detoxify these metabolites by reaction of its thiol group with the α,β-unsaturated carbonyl containing compounds including acrolein. Thus, Mesna is a chemotherapeutic adjuvant [1–3]. Moreover, it is also an antioxidant, and its FDA approval was based largely on its antioxidative properties [1–3]. On the other hand, the protonated form of Mesna is 2-mercaptoethanesulfonic acid which bears another name: coenzyme M [4–7]. Coenzyme M is the smallest known organic cofactor and serves as a methyl group carrier in key reactions within the pathway of methane formation from C1 precursors [6]. In the alkene metabolism pathway, it is involved in aliphatic epoxide carboxylation [4–7].Cisplatin (cis-[Pt(NH3)2Cl2]), the first Pt(II)-based antineoplastic drug, has played a central role in cancer chemotherapies covering a relatively wide spectrum including testicular, ovarian, cervical, breast, bladder, head and neck, esophageal and lung cancers, mesothelioma, brain tumors, and neuroblastoma [8–10]. Despite of its tremendous success, it has several severe side effects such as nephrotoxicity, ototoxicity, neurotoxicity, and gastrointestinal toxicity in addition to the acquired resistance to the drug [9, 10]. Two general approaches have been developed in order to overcome or minimize these detrimental side effects [11]: (a) the search of adjuvant or rescuing agents which are used together with cisplatin [12–14], and (b) the conversation of cisplatin to its Pt(IV) prodrugs by assumption that these prodrugs can be delivered to the tumor sites more efficiently [15–18]. The first approach has had a very limited success, as such, Mesna has been found to ameliorate significantly the severe side effects of cisplatin in certain types of cancers [19–23]. Furthermore, the combined use of cisplatin, ifosfamide, and Mesna has been exploited to the treatment of advanced ovarian carcinoma [24–26]. Whereas the second approach still remains elusive although large efforts have been attempted [11]. Also, it was shown that cisplatin Pt(IV) prodrugs can induce oxidative stress [18]; this might be not surprising since Pt(IV) prodrugs per se are oxidants.In contrast to the rich biomedical studies of Mesna, the kinetic and mechanistic aspect related to the redox reactions is scanty [27–29], which encompasses the interaction of Mesna with cisplatin [27] and the reduction reactions of bromate and bromine [28] and of the anticancer prodrug ruthenium(III) compound [29] by Mesna. We carried out a kinetic analysis of the reduction processes of cis-diamminetetrachloroplatinum(IV) (cis-[Pt(NH3)2Cl4]) by Mesna and its structurally related thiols thioglycolic acid (TGA) and DL-thiolactic acid (TLA), cf. their structures in Figure 1. cis-[Pt(NH3)2Cl4] per se is anticancer active [30, 31] and is also a prodrug of cisplatin since it is usually reduced to cisplatin. The essentially substitution-inertness property of cis-[Pt(NH3)2Cl4] enabled us to study its redox reactions in a wide pH range and to derive the reactivity of each protolytic species of reductants [32, 33]. We thus divulge the results from the kinetic analysis.Figure 1
Structures of thiol-containing compounds: (a) Mesna (sodium 2-mercaptoethane-sulfonate), (b) coenzyme M (2-Mercaptorthanesulfonic acid), (c) thioglycolic acid (TGA), (d) thiolactic acid (TLA), (e)cis-[Pt(NH3)2Cl4], and (f) cis-[Pt(NH3)2Cl2] (cisplatin).
(a)(b)(c)(d)(e)(f)


## 2. Materials and Methods

### 2.1. Chemicals

Mesna, thioglycolic acid, DL-thiolactic acid, the platinum(IV) compoundcis-[Pt(NH3)2Cl4], and cis-[Pt(NH3)2Cl2] (cisplatin) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphoric acid, acetic acid, sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, sodium perchlorate, perchloric acid, sodium chloride, and Na2EDTA in their purist forms available were obtained from either Fisher Scientific or Alfa Aesar which were used to prepare buffer solutions. All the solutions were prepared by use of doubly distilled water.


### 2.2. Instruments

UV-Vis spectra, absorption measurements, and time-resolved spectra were recorded on a TU-1900 spectrophotometer (Beijing Persee, Inc., Beijing, China) using 1.00 cm quartz cells. Kinetic measurements and rapid scan spectra were carried out on an Applied Photophysics SX-20 stopped-flow spectrometer (Applied Photophysics Ltd., Leatherhead, U.K.). Both spectrophotometer and spectrometer were equipped with a water bath circulation from a thermostat (Lauda Alpha RA8, Belran, NJ, USA); temperature could be controlled to ± 0.1°C. Mass spectra were recorded on an Agilent 1200/6310 ion trap mass spectrometer with electrospray ionization (ESI). An Accumet Basic AB15 Plus pH meter, equipped with an Accumet AccutupH® combination pH electrode (Fisher Scientific, Pittsburgh, PA), was used to measure the pH values of buffer solutions. Standard buffers of pH 4.00, 7.00, and 10.00, also from Fisher Scientific, were used for calibrations of the electrode just before pH measurements.


### 2.3. Buffer Solutions

Buffering pairs of H3PO4/NaH2PO4, HAc/NaAc, NaH2PO4/Na2HPO4, NaHCO3/Na2CO3, and Na2HPO4/Na3PO4 (0.15–0.2 M) were prepared to cover a pH range from 2.47 ≤ pH ≤ 11.97. The buffers contained 0.10 M of NaCl and 2.0 mM of EDTA; sodium perchlorate was used to adjust the ion strength (μ) of buffers to an ionic strength of 1.0 M. Details for the buffer preparations are delineated in our earlier works [32, 33]. The buffer pairs are listed in Table S1 in the Supplementary Materials (SM).


### 2.4. Kinetic Experiments

Stock solutions of 1.0 mM cis-[Pt(NH3)2Cl4] were daily prepared by dissolving the desired amount of cis-[Pt(NH3)2Cl4] in a solution containing 0.90 M NaClO4, 0.09 M NaCl, and 0.01 M HCl; these solutions were only used for a couple of hours. Stock solutions of thiols were prepared by adding the needed amount of the thiol to a buffer solution of specific pH. The solutions were flushed with highly pure nitrogen gas for 10 min. Solutions of Pt(IV) and thiol for kinetic measurements were prepared by dilution of the stock solutions with the same pH buffer and were then nitrogen-flushed for 10 min before loading onto the stopped-flow spectrometer. Equal volumes of Pt(IV) and thiol solutions were mixed directly in the stopped-flow instrument. Kinetic traces were followed between 265 and 280 nm; pseudo-first-order conditions were fulfilled with a 10-fold excess of [thiol]tot over [Pt(IV)], where [thiol]tot represents the total concentration of thiol.


### 2.5. Stoichiometric Measurements

For each of the thiols, a series of reaction mixtures were prepared in a phosphate buffer of pH 6.25 in which [Pt(IV)] = 0.30 mM was retained constant whereas [thiols]tot was changed from 0 to 1.50 mM. For each of the reaction mixtures, the absorption values at 265 nm were determined after a reaction time of 8–10 min at room temperature.


## 2.1. Chemicals

Mesna, thioglycolic acid, DL-thiolactic acid, the platinum(IV) compoundcis-[Pt(NH3)2Cl4], and cis-[Pt(NH3)2Cl2] (cisplatin) were purchased from Sigma-Aldrich (St. Louis, MO). Phosphoric acid, acetic acid, sodium acetate, sodium dihydrogen phosphate, disodium hydrogen phosphate, sodium carbonate, sodium bicarbonate, sodium perchlorate, perchloric acid, sodium chloride, and Na2EDTA in their purist forms available were obtained from either Fisher Scientific or Alfa Aesar which were used to prepare buffer solutions. All the solutions were prepared by use of doubly distilled water.


## 2.2. Instruments

UV-Vis spectra, absorption measurements, and time-resolved spectra were recorded on a TU-1900 spectrophotometer (Beijing Persee, Inc., Beijing, China) using 1.00 cm quartz cells. Kinetic measurements and rapid scan spectra were carried out on an Applied Photophysics SX-20 stopped-flow spectrometer (Applied Photophysics Ltd., Leatherhead, U.K.). Both spectrophotometer and spectrometer were equipped with a water bath circulation from a thermostat (Lauda Alpha RA8, Belran, NJ, USA); temperature could be controlled to ± 0.1°C. Mass spectra were recorded on an Agilent 1200/6310 ion trap mass spectrometer with electrospray ionization (ESI). An Accumet Basic AB15 Plus pH meter, equipped with an Accumet AccutupH® combination pH electrode (Fisher Scientific, Pittsburgh, PA), was used to measure the pH values of buffer solutions. Standard buffers of pH 4.00, 7.00, and 10.00, also from Fisher Scientific, were used for calibrations of the electrode just before pH measurements.


## 2.3. Buffer Solutions

Buffering pairs of H3PO4/NaH2PO4, HAc/NaAc, NaH2PO4/Na2HPO4, NaHCO3/Na2CO3, and Na2HPO4/Na3PO4 (0.15–0.2 M) were prepared to cover a pH range from 2.47 ≤ pH ≤ 11.97. The buffers contained 0.10 M of NaCl and 2.0 mM of EDTA; sodium perchlorate was used to adjust the ion strength (μ) of buffers to an ionic strength of 1.0 M. Details for the buffer preparations are delineated in our earlier works [32, 33]. The buffer pairs are listed in Table S1 in the Supplementary Materials (SM).


## 2.4. Kinetic Experiments

Stock solutions of 1.0 mM cis-[Pt(NH3)2Cl4] were daily prepared by dissolving the desired amount of cis-[Pt(NH3)2Cl4] in a solution containing 0.90 M NaClO4, 0.09 M NaCl, and 0.01 M HCl; these solutions were only used for a couple of hours. Stock solutions of thiols were prepared by adding the needed amount of the thiol to a buffer solution of specific pH. The solutions were flushed with highly pure nitrogen gas for 10 min. Solutions of Pt(IV) and thiol for kinetic measurements were prepared by dilution of the stock solutions with the same pH buffer and were then nitrogen-flushed for 10 min before loading onto the stopped-flow spectrometer. Equal volumes of Pt(IV) and thiol solutions were mixed directly in the stopped-flow instrument. Kinetic traces were followed between 265 and 280 nm; pseudo-first-order conditions were fulfilled with a 10-fold excess of [thiol]tot over [Pt(IV)], where [thiol]tot represents the total concentration of thiol.


## 2.5. Stoichiometric Measurements

For each of the thiols, a series of reaction mixtures were prepared in a phosphate buffer of pH 6.25 in which [Pt(IV)] = 0.30 mM was retained constant whereas [thiols]tot was changed from 0 to 1.50 mM. For each of the reaction mixtures, the absorption values at 265 nm were determined after a reaction time of 8–10 min at room temperature.


## 3. Results and Discussion

### 3.1. UV-Vis and Rapid-Scan Spectra

The UV-Vis spectra for the compounds employed in this work were recorded by use of their solutions freshly prepared in a buffer of pH 4.42. The obtained spectra for 0.20 mMcis-[Pt(NH3)2Cl4], 0.20 mM cis-[Pt(NH3)2Cl2], 1.0 mM Mesna, 1.0 mM TGA, and 1.0 mM TLA are shown in the upper panel of Figure S1. In addition, the UV-Vis spectra for the fresh solutions of 0.20 mM cis-[Pt(NH3)2Cl4] in buffers of pH 4.42, 6.67, and 10.98 were also recorded, and are given in the lower panel of Figure S1.In order to gain some insights into these reduction processes, the reduction ofcis-[Pt(NH3)2Cl4] by Mesna in a buffer of pH 6.25 was probed by recording the rapid scan spectra under a set of reaction conditions. The spectra were recorded between 200–350 nm and are shown in Figure 2(a). Clearly, the absorption band around 236 nm and absorption shoulder round 265 nm decreased concertedly as the reaction proceeded, and no new absorption bands emerged. The absorbance readings at 236 and 266 nm as a function of time are shown in Figure 2(b) (data points). The absorbance versus time curves or kinetic traces were fit by equation (1) [34], where kobsd represents of pseudo-first-order rate constant and(1)At=A0−A∞exp−kobsd+A∞,where At, A0, and A∞ pertain to absorbance at time t, zero, and infinity, respectively. The resulting fittings are excellent (Figure 2(b)) and moreover, the value of kobsd obtained at 236 nm equals to that acquired at 266 nm within the experimental errors. These kinetic attributes indicate that the reduction process is first-order in [Pt(IV)] and that the absorbance decrease in Figure 2 corresponds to the reduction of cis-[Pt(NH3)2Cl4] without other complications. As a matter of fact, the kinetic attributes are in good agreement with the nature of substitution inertness of Pt(IV) complexes in general [35, 36]. In the time-resolved spectra recorded for the reduction process of cis-[Pt(NH3)2Cl4] by TLA in an acidic medium, similar spectral and kinetic attributes were observed, cf., Figure S2 in the SM.Figure 2
(a): Rapid scan spectra acquired for the reduction ofcis-[Pt(NH3)2Cl4] by Mesna under a set of reaction conditions: [Pt(IV)] = 0.10 mM, [Mesna]tot = 1.00 mM, phosphate buffer of pH 6.25, 25.0°C, and μ = 1.0 M. The spectra were acquired at 0.10, 0.30, 0.50, 0.70, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, and 10.0 seconds after start of the reaction. (b): Kinetic traces at 236 and 265 nm (data points) from the rapid scan spectra; the solid-curves were the best fits of equation (1) to the experimental data by a nonlinear squares method, providing values of kobsd = 0.58 ± 0.02 s−1 at 236 nm and 0.61 ± 0.02 s−1 at 266 nm.
(a)(b)


### 3.2. Second-Order Kinetics and Data Collection

The effects of varying [thiol]tot on the reduction rates were studied, aiming at ascertaining the reaction order of the thiols. In most of buffer solutions, [thiol]tot was varied from 0.20 to 2.00 mM and the reaction medium had big enough buffering capacities so that the variation of [thiol]tot did not cause any pH changes in the medium. The kinetic traces were followed by the stopped-flow spectrometer in a wavelength region from 265 to 280 nm. Pseudo-first-order rate constant kobsd was acquired from the simulations of kinetic traces by equation (1). For each [thiol]tot, 3–5 duplicate runs were made, and the values of kobsd were the averages from the duplicated runs. Standard deviations are usually much less than 5%.Plots ofkobsd versus [thiol]tot in the case of Mesna are shown in Figure 3 for a series of buffers at 25.0°C and 1.0 M ionic strength (μ = 1.0 M). Similar plots for the reactions of TGA and TLA are shown in Figures S3 and S4 in the SM. Undoubtedly, all the plots in Figures 3, S3, and S4 are linear and do not have any significant intercepts. Thus, the reduction reactions are first-order in [thiol]tot; thus, an overall second-order rate law (2) is warranted, where k′ denotes the observed second-order rate constant and k′ = kobsd/[thiol]tot:(2)−dPtIVdt=kobsdPtIV=k′thioltotPtIV.Figure 3
Pseudo-first-order rate constantskobsd versus [Mesna]tot in buffer solutions of different pHs at 25.0°C and μ = 1.0 M.
(a)(b)(c)We collected a large body of kinetic data at 25.0°C andμ = 1.0 M covering a wide pH range. Values of k′ were calculated from the linear kobsd versus [thiol]tot plots for all the three thiols and are summarized in Tables S1–S3 in the SM. Alternatively, logk′ versus pH plots are illustrated in Figures 4 and 5 (data points).Figure 4
Second-order rate constantsk′ in logarithmic scale versus pH profiles for (a) TGA and (b) TLA (data points). The solid lines were obtained by simulations of equation (6) to the experimental data by use of a weighted nonlinear least-squares method.
(a)(b)Figure 5
Second-order rate constantsk' in logarithmic scale versus pH profile for Mesna (data points). The solid lines were obtained by simulations of equation (7) to the experimental data by use of a weighted nonlinear least-squares method.


### 3.3. Reaction Stoichiometry and the Oxidation Products

A spectrophotometric titration method was employed to find the stoichiometry for the redox reactions betweencis-[Pt(NH3)2Cl4] and the thiols [32–34]. The plots of absorption values at 265 nm versus the ratio [thiol]tot/[Pt(IV)] are displayed in Figure 6. For each thiol, the data points clearly follow two crossing straight lines affording an intersection point. The stoichiometric ratios were acquired from these intersection points: 1.81 ± 0.05 for TGA, 1.87 ± 0.05 for TLA, and 1.97 ± 0.05 for Mesna. These ratios, within the experimental errors, point virtually to a reaction stoichiometry of Δ[Pt(IV)] : Δ[Thiol]tot = 1 : 2. Stoichiometric reaction (3a) is thus suggested for the reactions of TGA (R = H) and TLA (R = Me), while the stoichiometric reaction (3b) is inferred for the Mesna reaction where the sulfonic acid groups are deprotonated due to its strong acidity (vide infra):(3a)PtNH32Cl4+2HSCHRCOOH⟶PtNH32Cl2+HOOCCHRSSCHRCOOH+2HCl(3b)PtNH32Cl4+2HSCH2CH2SO3−⟶PtNH32Cl2+−O3SCH2CH2SSCH2CH2SO3−+2HClFigure 6
Stoichiometric ratios determined by spectrophotometric titrations: absorption values at 265 nm as a function of the ratio [thiol]tot/[Pt(IV)]. The intersection points in figure give rise to the stoichiometric values. (a) TGA. (b) TLA. (c) Mensa.
(a)(b)(c)ESI mass spectra were recorded for reaction mixtures of 1 mM Pt(IV) with 8 mM TGA and of 1.0 mM Pt(IV) with 8 mM TLA in 10 mM HCl after a reaction time about 1 h; the obtained spectra are shown in FiguresS5 and S6 in the SM together with the peak assignments. From the peak assignments, formations of disulfides HOOCCH2S-SCH2COOH and HOOCCMeS-SCMeCOOH were confirmed for the reactions of TGA and TLA, respectively.


### 3.4. Reaction Mechanisms

The observed second-order rate constantsk′ increase several orders of magnitude (Table S1–S3 and Figures 4 and 5) when the reaction media are changed from acidic via neutral to basic for the three thiols, unveiling that the thiolate species of TGA, TLA, and Mesna are much more reactive than their corresponding thiol forms. On the other hand, cis-[Pt(NH3)2Cl4] possesses an octahedral configuration; substitution reactions on the Pt(IV) compounds are generally very sluggish [35, 36]. In contrast, the reduction processes of cis-[Pt(NH3)2Cl4] by the three thiols are much quicker, thus ruling out the possibility that the reduction reactions proceed via ligand substitution(s). This is consistent with the attributes found in the time-resolved spectra discussed above. If all the protolytic species of TGA, TLA, and Mesna are logically assumed to be able to reduce the Pt(IV) complex, the reaction mechanism delineated in Scheme 1 is suggested for the reactions of TGA and TLA [32, 33]. The reaction mechanism described in Scheme 2 is proposed analogously for the Mesna reaction. In the mechanisms, the reactions designated by k1–k3 are the rate-determining steps. Each of the steps is taking place via an attack on one of the two axially-coordinated chlorides by the sulfur atom of the thiols, forming chloride-bridged transitions states which in the case of Mesna are depicted conceivably as follows [37–39]:(4)Scheme 1
A reaction mechanism suggested for the reduction processes ofcis-[Pt(NH3)2Cl4] by TGA and TLA.Scheme 2
A reaction mechanism proposed for the reduction process ofcis-[Pt(NH3)2Cl4] by coenzyme M/Mesna.In the transition states, partial bond formation (or bridge formation) between Cl and S atom occurs whereas concurrently the bonds of Cl-Pt-Cl are partially broken [37–39]. The collapses of the transition states generate transient chlorothiol and/or chlorothiolate species [37–39]. For each particular thiol, these transient species can be trapped rapidly by another thiol molecule which is in excess in the reaction mixture, giving rise to a disulfide as the oxidation product [32, 33, 37].


### 3.5. Rate Constants of the Rate-Determining Steps

The reaction mechanisms outlined inSchemes 1 and 2 are very similar and a common rate expression can be derived as follows:(5)−dPtIVdt=k1aH2+k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2=thioltotPtIV.Equation (5) is equivalent virtually to equation (2), where aH is the proton activity and corresponds to the measured pH values by a relation: pH = −log(aH). A comparison of equations (2) and (5) renders(6)k′=k1aH2+k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2.Equation (6) was utilized to simulate the kinetic data in Figures 4 and 5. In the simulations, the rate constants k1–k3 were unknowns and treated as adjustable parameters. On the other hand, if the acid dissociation constants Ka1 and Ka2 for the thiol-containing compounds are available at the relevant conditions, they will be used as direct inputs, minimizing the number of adjustable parameters. Fortunately, the acid dissociation constants of TGA were reported to be pKa1 = 3.53 and pKa2 = 10.05 at 25.0°C and μ = 1.0 M [37]. When these pKa values were used direct inputs, equation (6) was employed to simulate the k′–pH dependence data by use of a weighted nonlinear least-squares method. The simulated result for TGA turned out to be good and is shown in Figure 4(a), concurrently providing values for the rate constants of k1–k3 which are listed in Table 1.Table 1
Values of rate constants derived for the rate-determining steps for reduction ofcis-[Pt(NH3)2Cl4] by the protolylic species of TGA, TLA, and Mesna at 25.0°C and μ = 1.0 M.
ThiolspKa valueskmValue (M−1s−1)TGApKa1 = 3.53k10.26 ± 0.05pKa2 = 10.05k26.8 ± 0.2k3(1.15 ± 0.05) × 106TLApKa1 = 3.38k10.19 ± 0.04pKa2 = 9.93k26.2 ± 0.2k3(8.8 ± 0.2) × 105MesnapKa1 = 1.2–2.0k1Not obsd.pKa2 = 8.86 ± 0.08ak20.37 ± 0.09pKa2 = 8.85 ± 0.05bk3(3.29 ± 0.09) × 105aObtained by kinetic approach. bDetermined by the spectrophotometric titration method.The acid dissociation constants of TLA were reported aspKa1 = 3.38 and pKa2 = 9.93 at 25.0°C and μ = 0.50 M [40] and were also utilized as direct inputs although the ionic strength is slightly differentiated. The simulation of equation (6) to the k′–pH dependence data revealed an essentially perfect fit, as shown in Figure 4(b). The acquired values of k1–k3 from the simulation are also listed in Table 1.A reliable value of the thiol dissociation constant for Mesna (i.e.pKa2 in Scheme 2) appears nonavailable in the literature under our experimental conditions except that some articles [2, 41] mentioned a value of 9.2 without specifying any conditions, which is in contrast to the fact that Mesna has been subjected to extensive medical studies as illustrated in the introduction section. The pKa1 value of coenzyme M in Scheme 2 was not found in the literature. Due to the biomedical importance of Mesna, acquiring a reliable value of the thiol dissociation constant is appealing. On the other hand, the pKa1 value of coenzyme M is anticipated to be close to that of ethanesulfonic acid (CH3CH2SO3H), which was reported to be pKa = 1.65 at 25.0°C [42].For the kinetic data analysis by equation (6), an initial value of pKa1 = 1.65 and a tunable pKa2 were tried for the simulations. The trial simulations indicated that the k1 value was indeterminate when pKa1 was varied from 1.2 to 2.0. Thus, the k1-term in equation (6) is negligible, leading to follows:(7)k′=k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2.Equation (7) was then employed for simulation of the k′-pH dependence data, and the simulated result is shown in Figure 5, conferring well-defined values for pKa2 = 8.86 ± 0.08 and k3 = (3.29 ± 0.09) × 105 M−1s−1 at 25.0°C and μ = 1.0 M. Furthermore, more simulations were performed with pKa1 values changed from 1.2 to 2.0 (the real pKa1 value is certainly in this region), yielding the robust values for pKa2 and k3 mentioned above. The obtained k2 values has a small variation, but only changed from 0.32 ± 0.03 to 0.42 ± 0.03 M−1s−1. The results are given in Table 1 where k2 value is taken as an average. Therefore, although the pKa1 value of coenzyme M is not known, we obtained the well-defined values for pKa2 and k3, and a reasonable value for k2.


### 3.6. Determination of the Thiol Dissociation Constant of Mesna

In order to further ascertain thepKa2 value of Mesna obtained by the above kinetic approach, we thus determined it by the spectrophotometric titration method [43–45]. A series of buffer solutions containing 0.12 mM Mesna at 1.0 M ionic strength were prepared covering a pH range from 6.77 to 11.97. Those solutions were flushed by nitrogen gas and concurrently thermoequilibated at 25.0°C for 10 min. For each pH, absorbance at 235 nm was measured with the corresponding buffer without Mesna as a reference. The measured absorption value as a function of pH is given in Figure 7 (data points). Equation (8) is a standard correction between the measured absorbance and pKa2 [43–45] in which ε2 and ε3 are the molar absorptivities for HSCH2CH2SO3‒ and ‒SCH2CH2SO3‒,(8)Abs235nm=Mesnatotε3+ε210pKa2−pH1+10pKa2−pH,respectively. The ε2 value was determined separately to be 59.0 ± 0.5 M−1cm−1 by use of three solutions of ca. 10 mM Mesna between pH 4 and 5. Equation (8) was then employed to simulate the data in Figure 7 using a nonlinear least-squares routine; the simulation resulted in a good fit, furnishing pKa2 = 8.85 ± 0.05 and ε3 = (5.6 ± 0.1) × 103 M−1cm−1 at 25.0°C and μ = 1.0 M. The excellent agreement between the pKa2 values obtained by the kinetic approach and by the spectreophotometric titration method emphasizes that we provide a reliable value for Mesna thiol dissociation and that the kinetic approach is a good method for acquisition of pKa values [46, 47].Figure 7
Absorbance at 235 nm of 0.12 mM Mesna solutions as function of pH at 25.0°C andμ = 1.0 M. The solid curve was obtained from the best fit of equation (8) to the experimental data.


### 3.7. Reactivity of Species the Three Thiols towards the Reduction ofcis-[Pt(NH3)2Cl4]

Our kinetic data collection in the wide pH range enables us to derive the reactivity of all the protolytic species of the three thiols towards the reduction ofcis-[Pt(NH3)2Cl4] and to make a comparison. The ratios of k1 : k2 : k3 were found to be 0.038 : 1 : 1.7 × 105 for TGA, 0.031 : 1 : 1.4 × 105 for TLA, and 0 : 1 : 8.9 × 105 for coenzyme M (Mesna), respectively. Clearly, the deprotonation of the carboxylic acids in TGA and TLA gives a reactivity enhancement of about 30 times which is caused by an inductive effect conferred by the deprotonations. When the thiols become thiolates after a further deprotonation, reactivity enhancements of (1.4–8.9) × 105 times are engendered for all the thiols. These huge reactivity enhancements account convincingly for the logk′ versus pH profiles in Figures 4 and 5.More straightforwardly, the species versus pH and the species reactivity versus pH distribution diagrams were constructed [33, 46], which are displayed in Figures S7–S9 in the SM. For all the thiols, species II dominate in the existing forms from pH 7.0 to 8.0 but make negligible contributions to their respect total reactivity. On the other hand, species III play dominant roles in determining the total reactivity whereas their populations are extremely low in the pH region from 7 to 8. In particular, the protolytic species ‒SCH2CH2SO3‒ largely dominates the total reactivity of Mesna from above pH 5; contrarily, the form of Mesna per se (mainly as HSCH2CH2SO3‒) makes a negligible contribution to the total reactivity when pH > 5, cf. Figure S10. It follows that the protolytic species ‒SCH2CH2SO3‒ but not HSCH2CH2SO3‒ of Mesna may play a leading role in some pharmacological processes of this drug.Mechanistically, the reduction processes ofcis-[Pt(NH3)2Cl4] and of the anticancer model compound trans-[PtCl2(CN)4]2‒ [37] by TGA are similar, rendering a possibility of reactivity comparison. The ratios of k2 ([PtCl2(CN)4]2‒)/k2 ([Pt(NH3)2Cl4]) and k3 ([PtCl2(CN)4]2‒)/k3 ([Pt(NH3)2Cl4]) were calculated to be 168 and 1.9 × 104, respectively, highlighting that the reduction of trans-[PtCl2(CN)4]2‒ is much faster than cis-[Pt(NH3)2Cl4]. This can be accounted for in terms of transition state stabilization which is ascribed to the strong σ-donor and π-acceptor properties of the cyanide ligands in [PtCl2(CN)4]2‒ [48].


## 3.1. UV-Vis and Rapid-Scan Spectra

The UV-Vis spectra for the compounds employed in this work were recorded by use of their solutions freshly prepared in a buffer of pH 4.42. The obtained spectra for 0.20 mMcis-[Pt(NH3)2Cl4], 0.20 mM cis-[Pt(NH3)2Cl2], 1.0 mM Mesna, 1.0 mM TGA, and 1.0 mM TLA are shown in the upper panel of Figure S1. In addition, the UV-Vis spectra for the fresh solutions of 0.20 mM cis-[Pt(NH3)2Cl4] in buffers of pH 4.42, 6.67, and 10.98 were also recorded, and are given in the lower panel of Figure S1.In order to gain some insights into these reduction processes, the reduction ofcis-[Pt(NH3)2Cl4] by Mesna in a buffer of pH 6.25 was probed by recording the rapid scan spectra under a set of reaction conditions. The spectra were recorded between 200–350 nm and are shown in Figure 2(a). Clearly, the absorption band around 236 nm and absorption shoulder round 265 nm decreased concertedly as the reaction proceeded, and no new absorption bands emerged. The absorbance readings at 236 and 266 nm as a function of time are shown in Figure 2(b) (data points). The absorbance versus time curves or kinetic traces were fit by equation (1) [34], where kobsd represents of pseudo-first-order rate constant and(1)At=A0−A∞exp−kobsd+A∞,where At, A0, and A∞ pertain to absorbance at time t, zero, and infinity, respectively. The resulting fittings are excellent (Figure 2(b)) and moreover, the value of kobsd obtained at 236 nm equals to that acquired at 266 nm within the experimental errors. These kinetic attributes indicate that the reduction process is first-order in [Pt(IV)] and that the absorbance decrease in Figure 2 corresponds to the reduction of cis-[Pt(NH3)2Cl4] without other complications. As a matter of fact, the kinetic attributes are in good agreement with the nature of substitution inertness of Pt(IV) complexes in general [35, 36]. In the time-resolved spectra recorded for the reduction process of cis-[Pt(NH3)2Cl4] by TLA in an acidic medium, similar spectral and kinetic attributes were observed, cf., Figure S2 in the SM.Figure 2
(a): Rapid scan spectra acquired for the reduction ofcis-[Pt(NH3)2Cl4] by Mesna under a set of reaction conditions: [Pt(IV)] = 0.10 mM, [Mesna]tot = 1.00 mM, phosphate buffer of pH 6.25, 25.0°C, and μ = 1.0 M. The spectra were acquired at 0.10, 0.30, 0.50, 0.70, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 5.0, 6.0, 8.0, and 10.0 seconds after start of the reaction. (b): Kinetic traces at 236 and 265 nm (data points) from the rapid scan spectra; the solid-curves were the best fits of equation (1) to the experimental data by a nonlinear squares method, providing values of kobsd = 0.58 ± 0.02 s−1 at 236 nm and 0.61 ± 0.02 s−1 at 266 nm.
(a)(b)


## 3.2. Second-Order Kinetics and Data Collection

The effects of varying [thiol]tot on the reduction rates were studied, aiming at ascertaining the reaction order of the thiols. In most of buffer solutions, [thiol]tot was varied from 0.20 to 2.00 mM and the reaction medium had big enough buffering capacities so that the variation of [thiol]tot did not cause any pH changes in the medium. The kinetic traces were followed by the stopped-flow spectrometer in a wavelength region from 265 to 280 nm. Pseudo-first-order rate constant kobsd was acquired from the simulations of kinetic traces by equation (1). For each [thiol]tot, 3–5 duplicate runs were made, and the values of kobsd were the averages from the duplicated runs. Standard deviations are usually much less than 5%.Plots ofkobsd versus [thiol]tot in the case of Mesna are shown in Figure 3 for a series of buffers at 25.0°C and 1.0 M ionic strength (μ = 1.0 M). Similar plots for the reactions of TGA and TLA are shown in Figures S3 and S4 in the SM. Undoubtedly, all the plots in Figures 3, S3, and S4 are linear and do not have any significant intercepts. Thus, the reduction reactions are first-order in [thiol]tot; thus, an overall second-order rate law (2) is warranted, where k′ denotes the observed second-order rate constant and k′ = kobsd/[thiol]tot:(2)−dPtIVdt=kobsdPtIV=k′thioltotPtIV.Figure 3
Pseudo-first-order rate constantskobsd versus [Mesna]tot in buffer solutions of different pHs at 25.0°C and μ = 1.0 M.
(a)(b)(c)We collected a large body of kinetic data at 25.0°C andμ = 1.0 M covering a wide pH range. Values of k′ were calculated from the linear kobsd versus [thiol]tot plots for all the three thiols and are summarized in Tables S1–S3 in the SM. Alternatively, logk′ versus pH plots are illustrated in Figures 4 and 5 (data points).Figure 4
Second-order rate constantsk′ in logarithmic scale versus pH profiles for (a) TGA and (b) TLA (data points). The solid lines were obtained by simulations of equation (6) to the experimental data by use of a weighted nonlinear least-squares method.
(a)(b)Figure 5
Second-order rate constantsk' in logarithmic scale versus pH profile for Mesna (data points). The solid lines were obtained by simulations of equation (7) to the experimental data by use of a weighted nonlinear least-squares method.


## 3.3. Reaction Stoichiometry and the Oxidation Products

A spectrophotometric titration method was employed to find the stoichiometry for the redox reactions betweencis-[Pt(NH3)2Cl4] and the thiols [32–34]. The plots of absorption values at 265 nm versus the ratio [thiol]tot/[Pt(IV)] are displayed in Figure 6. For each thiol, the data points clearly follow two crossing straight lines affording an intersection point. The stoichiometric ratios were acquired from these intersection points: 1.81 ± 0.05 for TGA, 1.87 ± 0.05 for TLA, and 1.97 ± 0.05 for Mesna. These ratios, within the experimental errors, point virtually to a reaction stoichiometry of Δ[Pt(IV)] : Δ[Thiol]tot = 1 : 2. Stoichiometric reaction (3a) is thus suggested for the reactions of TGA (R = H) and TLA (R = Me), while the stoichiometric reaction (3b) is inferred for the Mesna reaction where the sulfonic acid groups are deprotonated due to its strong acidity (vide infra):(3a)PtNH32Cl4+2HSCHRCOOH⟶PtNH32Cl2+HOOCCHRSSCHRCOOH+2HCl(3b)PtNH32Cl4+2HSCH2CH2SO3−⟶PtNH32Cl2+−O3SCH2CH2SSCH2CH2SO3−+2HClFigure 6
Stoichiometric ratios determined by spectrophotometric titrations: absorption values at 265 nm as a function of the ratio [thiol]tot/[Pt(IV)]. The intersection points in figure give rise to the stoichiometric values. (a) TGA. (b) TLA. (c) Mensa.
(a)(b)(c)ESI mass spectra were recorded for reaction mixtures of 1 mM Pt(IV) with 8 mM TGA and of 1.0 mM Pt(IV) with 8 mM TLA in 10 mM HCl after a reaction time about 1 h; the obtained spectra are shown in FiguresS5 and S6 in the SM together with the peak assignments. From the peak assignments, formations of disulfides HOOCCH2S-SCH2COOH and HOOCCMeS-SCMeCOOH were confirmed for the reactions of TGA and TLA, respectively.


## 3.4. Reaction Mechanisms

The observed second-order rate constantsk′ increase several orders of magnitude (Table S1–S3 and Figures 4 and 5) when the reaction media are changed from acidic via neutral to basic for the three thiols, unveiling that the thiolate species of TGA, TLA, and Mesna are much more reactive than their corresponding thiol forms. On the other hand, cis-[Pt(NH3)2Cl4] possesses an octahedral configuration; substitution reactions on the Pt(IV) compounds are generally very sluggish [35, 36]. In contrast, the reduction processes of cis-[Pt(NH3)2Cl4] by the three thiols are much quicker, thus ruling out the possibility that the reduction reactions proceed via ligand substitution(s). This is consistent with the attributes found in the time-resolved spectra discussed above. If all the protolytic species of TGA, TLA, and Mesna are logically assumed to be able to reduce the Pt(IV) complex, the reaction mechanism delineated in Scheme 1 is suggested for the reactions of TGA and TLA [32, 33]. The reaction mechanism described in Scheme 2 is proposed analogously for the Mesna reaction. In the mechanisms, the reactions designated by k1–k3 are the rate-determining steps. Each of the steps is taking place via an attack on one of the two axially-coordinated chlorides by the sulfur atom of the thiols, forming chloride-bridged transitions states which in the case of Mesna are depicted conceivably as follows [37–39]:(4)Scheme 1
A reaction mechanism suggested for the reduction processes ofcis-[Pt(NH3)2Cl4] by TGA and TLA.Scheme 2
A reaction mechanism proposed for the reduction process ofcis-[Pt(NH3)2Cl4] by coenzyme M/Mesna.In the transition states, partial bond formation (or bridge formation) between Cl and S atom occurs whereas concurrently the bonds of Cl-Pt-Cl are partially broken [37–39]. The collapses of the transition states generate transient chlorothiol and/or chlorothiolate species [37–39]. For each particular thiol, these transient species can be trapped rapidly by another thiol molecule which is in excess in the reaction mixture, giving rise to a disulfide as the oxidation product [32, 33, 37].


## 3.5. Rate Constants of the Rate-Determining Steps

The reaction mechanisms outlined inSchemes 1 and 2 are very similar and a common rate expression can be derived as follows:(5)−dPtIVdt=k1aH2+k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2=thioltotPtIV.Equation (5) is equivalent virtually to equation (2), where aH is the proton activity and corresponds to the measured pH values by a relation: pH = −log(aH). A comparison of equations (2) and (5) renders(6)k′=k1aH2+k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2.Equation (6) was utilized to simulate the kinetic data in Figures 4 and 5. In the simulations, the rate constants k1–k3 were unknowns and treated as adjustable parameters. On the other hand, if the acid dissociation constants Ka1 and Ka2 for the thiol-containing compounds are available at the relevant conditions, they will be used as direct inputs, minimizing the number of adjustable parameters. Fortunately, the acid dissociation constants of TGA were reported to be pKa1 = 3.53 and pKa2 = 10.05 at 25.0°C and μ = 1.0 M [37]. When these pKa values were used direct inputs, equation (6) was employed to simulate the k′–pH dependence data by use of a weighted nonlinear least-squares method. The simulated result for TGA turned out to be good and is shown in Figure 4(a), concurrently providing values for the rate constants of k1–k3 which are listed in Table 1.Table 1
Values of rate constants derived for the rate-determining steps for reduction ofcis-[Pt(NH3)2Cl4] by the protolylic species of TGA, TLA, and Mesna at 25.0°C and μ = 1.0 M.
ThiolspKa valueskmValue (M−1s−1)TGApKa1 = 3.53k10.26 ± 0.05pKa2 = 10.05k26.8 ± 0.2k3(1.15 ± 0.05) × 106TLApKa1 = 3.38k10.19 ± 0.04pKa2 = 9.93k26.2 ± 0.2k3(8.8 ± 0.2) × 105MesnapKa1 = 1.2–2.0k1Not obsd.pKa2 = 8.86 ± 0.08ak20.37 ± 0.09pKa2 = 8.85 ± 0.05bk3(3.29 ± 0.09) × 105aObtained by kinetic approach. bDetermined by the spectrophotometric titration method.The acid dissociation constants of TLA were reported aspKa1 = 3.38 and pKa2 = 9.93 at 25.0°C and μ = 0.50 M [40] and were also utilized as direct inputs although the ionic strength is slightly differentiated. The simulation of equation (6) to the k′–pH dependence data revealed an essentially perfect fit, as shown in Figure 4(b). The acquired values of k1–k3 from the simulation are also listed in Table 1.A reliable value of the thiol dissociation constant for Mesna (i.e.pKa2 in Scheme 2) appears nonavailable in the literature under our experimental conditions except that some articles [2, 41] mentioned a value of 9.2 without specifying any conditions, which is in contrast to the fact that Mesna has been subjected to extensive medical studies as illustrated in the introduction section. The pKa1 value of coenzyme M in Scheme 2 was not found in the literature. Due to the biomedical importance of Mesna, acquiring a reliable value of the thiol dissociation constant is appealing. On the other hand, the pKa1 value of coenzyme M is anticipated to be close to that of ethanesulfonic acid (CH3CH2SO3H), which was reported to be pKa = 1.65 at 25.0°C [42].For the kinetic data analysis by equation (6), an initial value of pKa1 = 1.65 and a tunable pKa2 were tried for the simulations. The trial simulations indicated that the k1 value was indeterminate when pKa1 was varied from 1.2 to 2.0. Thus, the k1-term in equation (6) is negligible, leading to follows:(7)k′=k2Ka1aH+k3Ka1Ka2aH2+Ka1aH+Ka1Ka2.Equation (7) was then employed for simulation of the k′-pH dependence data, and the simulated result is shown in Figure 5, conferring well-defined values for pKa2 = 8.86 ± 0.08 and k3 = (3.29 ± 0.09) × 105 M−1s−1 at 25.0°C and μ = 1.0 M. Furthermore, more simulations were performed with pKa1 values changed from 1.2 to 2.0 (the real pKa1 value is certainly in this region), yielding the robust values for pKa2 and k3 mentioned above. The obtained k2 values has a small variation, but only changed from 0.32 ± 0.03 to 0.42 ± 0.03 M−1s−1. The results are given in Table 1 where k2 value is taken as an average. Therefore, although the pKa1 value of coenzyme M is not known, we obtained the well-defined values for pKa2 and k3, and a reasonable value for k2.


## 3.6. Determination of the Thiol Dissociation Constant of Mesna

In order to further ascertain thepKa2 value of Mesna obtained by the above kinetic approach, we thus determined it by the spectrophotometric titration method [43–45]. A series of buffer solutions containing 0.12 mM Mesna at 1.0 M ionic strength were prepared covering a pH range from 6.77 to 11.97. Those solutions were flushed by nitrogen gas and concurrently thermoequilibated at 25.0°C for 10 min. For each pH, absorbance at 235 nm was measured with the corresponding buffer without Mesna as a reference. The measured absorption value as a function of pH is given in Figure 7 (data points). Equation (8) is a standard correction between the measured absorbance and pKa2 [43–45] in which ε2 and ε3 are the molar absorptivities for HSCH2CH2SO3‒ and ‒SCH2CH2SO3‒,(8)Abs235nm=Mesnatotε3+ε210pKa2−pH1+10pKa2−pH,respectively. The ε2 value was determined separately to be 59.0 ± 0.5 M−1cm−1 by use of three solutions of ca. 10 mM Mesna between pH 4 and 5. Equation (8) was then employed to simulate the data in Figure 7 using a nonlinear least-squares routine; the simulation resulted in a good fit, furnishing pKa2 = 8.85 ± 0.05 and ε3 = (5.6 ± 0.1) × 103 M−1cm−1 at 25.0°C and μ = 1.0 M. The excellent agreement between the pKa2 values obtained by the kinetic approach and by the spectreophotometric titration method emphasizes that we provide a reliable value for Mesna thiol dissociation and that the kinetic approach is a good method for acquisition of pKa values [46, 47].Figure 7
Absorbance at 235 nm of 0.12 mM Mesna solutions as function of pH at 25.0°C andμ = 1.0 M. The solid curve was obtained from the best fit of equation (8) to the experimental data.


## 3.7. Reactivity of Species the Three Thiols towards the Reduction ofcis-[Pt(NH3)2Cl4]

Our kinetic data collection in the wide pH range enables us to derive the reactivity of all the protolytic species of the three thiols towards the reduction ofcis-[Pt(NH3)2Cl4] and to make a comparison. The ratios of k1 : k2 : k3 were found to be 0.038 : 1 : 1.7 × 105 for TGA, 0.031 : 1 : 1.4 × 105 for TLA, and 0 : 1 : 8.9 × 105 for coenzyme M (Mesna), respectively. Clearly, the deprotonation of the carboxylic acids in TGA and TLA gives a reactivity enhancement of about 30 times which is caused by an inductive effect conferred by the deprotonations. When the thiols become thiolates after a further deprotonation, reactivity enhancements of (1.4–8.9) × 105 times are engendered for all the thiols. These huge reactivity enhancements account convincingly for the logk′ versus pH profiles in Figures 4 and 5.More straightforwardly, the species versus pH and the species reactivity versus pH distribution diagrams were constructed [33, 46], which are displayed in Figures S7–S9 in the SM. For all the thiols, species II dominate in the existing forms from pH 7.0 to 8.0 but make negligible contributions to their respect total reactivity. On the other hand, species III play dominant roles in determining the total reactivity whereas their populations are extremely low in the pH region from 7 to 8. In particular, the protolytic species ‒SCH2CH2SO3‒ largely dominates the total reactivity of Mesna from above pH 5; contrarily, the form of Mesna per se (mainly as HSCH2CH2SO3‒) makes a negligible contribution to the total reactivity when pH > 5, cf. Figure S10. It follows that the protolytic species ‒SCH2CH2SO3‒ but not HSCH2CH2SO3‒ of Mesna may play a leading role in some pharmacological processes of this drug.Mechanistically, the reduction processes ofcis-[Pt(NH3)2Cl4] and of the anticancer model compound trans-[PtCl2(CN)4]2‒ [37] by TGA are similar, rendering a possibility of reactivity comparison. The ratios of k2 ([PtCl2(CN)4]2‒)/k2 ([Pt(NH3)2Cl4]) and k3 ([PtCl2(CN)4]2‒)/k3 ([Pt(NH3)2Cl4]) were calculated to be 168 and 1.9 × 104, respectively, highlighting that the reduction of trans-[PtCl2(CN)4]2‒ is much faster than cis-[Pt(NH3)2Cl4]. This can be accounted for in terms of transition state stabilization which is ascribed to the strong σ-donor and π-acceptor properties of the cyanide ligands in [PtCl2(CN)4]2‒ [48].


## 4. Conclusions

The reduction processes of a cisplatin Pt(IV) prodrug cis-[Pt(NH3)2Cl4] by Mesna, TGA and TLA strictly follow overall second-order kinetics, and the k′ versus pH profiles have been established in a wide pH range. The proposed reaction mechanisms involve all the prololytic species of the thiols attacking the Pt(IV) in parallel, which are the rate-determining steps. The rate constants of these rate-determining steps have been elucidated revealing reactivity enhancements of (1.4–8.9) × 105 times when the thiols become thiolates. The constructed species versus pH and species reactivity versus pH distribution diagrams demonstrated that the species ‒SCH2CH2SO3‒ of Mesna largely governs the total reactivity when pH > 5; contrarily, the form of Mesna per se (mainly as HSCH2CH2SO3‒) makes a negligible contribution. In addition, a well-determined and reliable dissociation constant for the Mesna thiol group (pKa2 = 8.85 ± 0.05 at 25.0°C and μ = 1.0 M) is offered in this work.



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