Reduction of Cisplatin and Carboplatin Pt(IV) Prodrugs by Homocysteine: Kinetic and Mechanistic Investigations

Article abstract of DOI:10.1002/kin.21107

Pt(IV) anticancer active complexes are commonly regarded as prodrugs, and the reduction of the prodrugs to their Pt(II) analogs is the activation process. The reduction of a cisplatin prodrug cis-[Pt(NH₃)₂Cl₄] and a carboplatin prodrug cis,trans-[Pt(cbdca)(NH₃)₂Cl₂] by dl-homocysteine (Hcy) has been investigated kinetically in a wide pH range in this work. The reduction process follows overall second-order kinetics: ?d[Pt(IV)]/dt = k′[Hcy]_tot[Pt(IV)], where [Hcy]_tot stands for the total concentration of Hcy and k′ pertains to the observed second-order rate constants. The k′ versus pH profiles have been established for both prodrugs. Spectrohotometric titrations reveal a stoichiometry of Δ[Pt(IV)]:Δ[Hcy]_tot = 1:2; homocystine is identified as the major oxidation product of Hcy by high-resolution mass spectrometry. A reaction mechanism has been proposed, which involves all the four protolysis species of Hcy attacking the Pt(IV) prodrugs in parallel. Moreover, these parallel attacks are the rate-determining steps, resulting in a Cl⁺ transfer from the Pt(IV) prodrugs to the attacking sulfur atom. Rate constants of the rate-determining steps have been derived, indicating that the two prodrugs are reduced with a very similar rate in spite of the difference between the coordination ligands in their equatorial positions. The reactivity analysis in the case of cis,trans-[Pt(cbdca)(NH₃)₂Cl₂] unravels that one species of Hcy (form III) is almost exclusively responsible for the reductions at the physiological pH (7.4), although it is existing only 5.2% of the total Hcy. On the other hand, the dominant existing form II of Hcy virtually does not make a contribution to the overall reactivity at pH 7.4.

Full text of DOI:10.1002/kin.21107

Reduction of Cisplatin and
Carboplatin Pt(IV) Prodrugs
by Homocysteine: Kinetic
and Mechanistic
Investigations
HONGWU TIAN,1 JINGRAN DONG,1 XUERU CHI,1 LIYAO XU,1 HONGMEI SHI,2 TIESHENG SHI1
1College of Chemistry and Environmental Science and the MOE Key Laboratory of Medicinal Chemistry and Molecular
Diagnostics, Hebei University, Baoding, 071002, People’s Republic of China
2School of Public Health, Hebei Medical University, Shijiazhuang, 050017, People’s Republic of China
Received 3 May 2017; revised 22 June 2017; accepted 22 June 2017
DOI 10.1002/kin.21107
Published online in Wiley Online Library (wileyonlinelibrary.com).
ABSTRACT: Pt(IV) anticancer active complexes are commonly regarded as prodrugs, and the
reduction of the prodrugs to their Pt(II) analogs is the activation process. The reduction of a
cisplatin prodrug cis-[Pt(NH3)2Cl4] and a carboplatin prodrug cis,trans-[Pt(cbdca)(NH3)2Cl2] by
DL-homocysteine (Hcy) has been investigated kinetically in a wide pH range in this work. The re-
duction process follows overall second-order kinetics: −d[Pt(IV)]/dt = kꢀ[Hcy]tot[Pt(IV)], where
[Hcy]tot stands for the total concentration of Hcy and kꢀ pertains to the observed second-order
rate constants. The kꢀ versus pH profiles have been established for both prodrugs. Spectrohoto-
metric titrations reveal a stoichiometry of ꢀ[Pt(IV)]:ꢀ[Hcy]tot = 1:2; homocystine is identified
as the major oxidation product of Hcy by high-resolution mass spectrometry. A reaction mech-
anism has been proposed, which involves all the four protolysis species of Hcy attacking the
Pt(IV) prodrugs in parallel. Moreover, these parallel attacks are the rate-determining steps,
resulting in a Cl+ transfer from the Pt(IV) prodrugs to the attacking sulfur atom. Rate constants
Correspondence to: Hongmei Shi; e-mail: hongmshi@163.com.
Tiesheng Shi; e-mail: transition state@126.com.
Contract grant sponsor: You Bo Program of Hebei University.
Contract grant number: YB201403.
Contract grant sponsor: Natural Science Foundation of Hebei
University.
Contract grant number: 2014–11.
Contract grant sponsor: National Natural Science Foundation of
China.
Contract grant number: 81273128 (to HS).
Supporting Information is available in the online issue at
www.wileyonlinelibrary.com.
ꢁC
2017 Wiley Periodicals, Inc.

2
TIAN ET AL.
of the rate-determining steps have been derived, indicating that the two prodrugs are reduced
with a very similar rate in spite of the difference between the coordination ligands in their
equatorial positions. The reactivity analysis in the case of cis,trans-[Pt(cbdca)(NH3)2Cl2] unrav-
els that one species of Hcy (form III) is almost exclusively responsible for the reductions at the
physiological pH (7.4), although it is existing only 5.2% of the total Hcy. On the other hand, the
dominant existing form II of Hcy virtually does not make a contribution to the overall reactivity
ꢁC
at pH 7.4.
2017 Wiley Periodicals, Inc. Int J Chem Kinet 1–9, 2017
INTRODUCTION
O
O
NH3
NH3
Cl
Cl
H3N
H3N
Pt
Pt
Platinum(II)-based anticancer drugs, represented by
cisplatin, carboplatin, and oxaliplatin that are in clin-
ical use worldwide, have led the chemotherapeutic
regimes [1,2]. It is also well known that adverse
side effects are involved in these drugs: nephrotoxi-
city, neurotoxicity/neuropathy, ototoxicity, hepatotox-
icity, myelosuppression, as well as acquired/inherent
drug resistance due to repeated doses [1,2]. In the de-
velopment of new generation of Pt-based anticancer
drugs that are more efficacious with fewer adverse
side effects, much attention has been paid to Pt(IV)
compounds [1–4]. Pt(IV) compounds with additional
ligands in their axial positions are generally sub-
stitution inert, thus conferring decreased interaction
with plasma proteins [1–4]. Indeed, several Pt(IV)
compounds have entered into clinical trials includ-
ing ormaplatin (tetraplatin), iproplatin, satraplatin, and
LA-12 [3,5–7]. Their structures are shown in Figure S1
in Supporting Information. Moreover, these clinical tri-
aled Pt(IV) anticancer drugs represent three prototypes
of structural configurations.
In contrast to their substitution properties, Pt(IV)
anticancer drugs can be readily reduced to their Pt(II)
counterparts by small reductants and proteins in plasma
en route to cancerous cells [1–4]. Therefore, Pt(IV) an-
ticancer drugs are commonly regarded as prodrugs and
the reduction is thus the activation process of the pro-
drugs [1–4]. Although the prodrug nature of Pt(IV)
compounds has been exploited extensively in the drug
delivery studies [8], the intense need for gaining exact
control over drug activation still remains unresolved
due largely to so many reductants involved in the bio-
logical systems [2].
O
O
Cisplatin
Carboplatin
Cl
Cl
O
Cl
Cl
O
Pt
O
NH3
NH3
H3N
H3N
Pt
O
Cl
Cl
cis-[Pt(NH3)2Cl4]
cis,trans-[Pt(cbdca)(NH3)2Cl2]
Figure 1 Structures of the cisplatin, its prodrug cis-
[Pt(NH3)2Cl4], carboplatin, and its prodrug cis,trans-
[Pt(cbdca)(NH3)2Cl2]. [Color figure can be viewed at wi-
leyonlinelibrary.com]
clinical studies of the relations between the Hcy lev-
els in plasma and CVD [11–16]. Moreover, Hcy is also
believed to be a pathological metabolic marker [15,16].
We have investigated kinetically and mecha-
nistically the reduction reactions of two inter-
esting prodrugs, which are a cisplatin prodrug
cis-[Pt(NH3)2Cl4] [17] and a carboplatin prodrug
cis,trans-[Pt(cbdca)(NH3)2Cl2] (cbdca = cyclobutane-
1,1-dicarboxylate) [18] by Hcy; their structures are
given in Fig. 1. In fact, many Pt(IV) prodrugs, which
possess configurations similar to ormaplatin, have been
developed [19–21]. Our goals of the present work were
to compare the reduction rates of the two prodrugs and
to provide a reactivity analysis of the Hcy protolysis
species in the reduction process.
Low molecular weight reductants of high abun-
dance in human plasma include ascorbic acid, glu-
tathione, L-cysteine, DL-homocysteine (Hcy), and a
dipeptide Gly-Cys [9,10]. Of these reductants, Hcy is
a nonprotein amino acid; it is involved in conversions
of methionine and cysteine catalyzed by enzymes. It
has been established that elevated Hcy levels in human
plasma are correlated with high risk of cardiovascular
disease (CVD) [11–16]. As a matter of fact, similar
correlations have been observed essentially in all the
EXPERIMENTAL
Instrumentation
An Accumet Basic AB15 Plus pH meter, equipped
with an Accumet AccutupHꢁR combination pH elec-
trode (Fisher Scientific, Pittsburgh, PA), was used to
measure the pH values of buffer solutions. Each time,
the electrode was calibrated using the standard buffers
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

REDUCTION OF CISPLATIN AND CARBOPLATIN PT(IV) PRODRUGS BY HOMOCYSTEINE
3
of pH 4.00, 7.00, and 10.00 immediately before the
pH measurements. ESI high-resolution mass spectra
were recorded on an Apex Ultra 7.0T FT-ICR mass
spectrometer (Bruker Daltonik, Germany) with elec-
trospray ionization (ESI) in the positive mode. UV–
visible (UV–Vis) spectra were recorded on a TU-1900
spectrophotometer (Beijing Puxi, Beijing, People’s Re-
public of China) using 1.00 cm quartz cells. Rapid scan
spectra and kinetic measurements were performed on
an Applied Photophysics SX-20 stopped-flow spec-
trometer (Applied Photophysics, Leatherhead, UK).
in the buffers was to minimize possible catalytic ef-
fects by traces of metal ions such as Cu(II) and Fe(III)
during thiol autooxidation [23–25], while that of NaCl
was to repress the hydrolysis of cis-[Pt(NH3)2Cl4] and
cis,trans-[Pt(cbdca)(NH3)2Cl2].
Kinetic Measurements
Stock solutions of Hcy were prepared by adding
the needed amount of Hcy in a buffer of specific
pH and were then flushed for 10 min with nitro-
gen. Stock solutions of 1.0 mM [Pt(NH3)2Cl4] or
[Pt(cbdca)(NH3)2Cl2] were prepared by dissolving the
desired amount of each of the complexes in a solu-
tion containing 0.90 M NaClO4, 0.09 M NaCl, and
0.01 M HCl; these solutions were only used for a
couple of hours. Solutions of Pt(IV) and Hcy for ki-
netic measurements were prepared by dilution of the
above stock solutions with the same pH buffer and
were flushed 10 min with nitrogen before loading onto
the stopped-flow spectrometer. Rapid scan spectra and
kinetic traces were run by mixing equal volumes of
the Pt(IV) and Hcy solutions directly in the stopped-
flow machine. All the kinetic studies were carried out
under pseudo–first-order conditions, namely a 10-fold
excess of [Hcy]tot over [Pt(IV)], where [Hcy]tot de-
notes the total concentration of Hcy. In this work, the
pseudo–first-order rate constants, kobsd, were the aver-
age values from five to seven duplicate runs; standard
deviations were usually less than 5%.
Materials
DL-Homocysteine (Hcy) and cis-[Pt(NH3)2Cl4] were
purchased from Sigma-Aldrich (St. Louis, MO). Car-
boplatin, cis-[Pt(cbdca)(NH3)2], was obtained from
Tokyo Chemical Industry. The prodrug cis,trans-
[Pt(cbdca)(NH3)2Cl2] was synthesized according to
the published procedure [18], but starting from a
larger scale (0.40 g carboplatin) and yielding 0.37 g
of [Pt(cbdca)(NH3)2Cl2]·⅓DMF. Elemental analysis,
theoretical: C, 18.01%; H, 3.07%; N, 7.01%. Found:
C, 18.20%; H, 3.21%; N, 7.16%. 1H NMR (600 MHz,
1
2
DMSO-d6) δ 5.96 (6H, m, JN-H = 51.6 Hz, JH-Pt
=
50.4 Hz), 2.52 (4H, t, largely shielded by the solvent
peak), 1.81 (2H, p, JH-H = 8 Hz). 13C NMR (150
3
MHz, DMSO-d6) δ 176.4, 55.8, 31.8, 15.6. In addi-
tion, the associated DMF with the synthesized Pt(IV)
1
compound was also confirmed by both H and 13C
NMR spectra: δ 2.73 (s, 3H), δ 2.87 (s, 3H), and
δ 7.95 (s, 1H); δ 162.3, 35.3, and 30.7 [22]. Acetic acid,
sodium acetate, sodium chloride, hydrochloric acid,
sodium dihydrogen-phosphate, disodium hydrogen-
phosphate, sodium bicarbonate, sodium carbonate,
trisodium phosphate, and sodium perchlorate were ob-
tained as the purest forms available either from Alfa
Aesar (Shanghai, Peoples’ Republic of China) or from
Fisher Scientific (Beijing, Peoples’ Republic of China)
and were used without further purifications. For pH
meter calibrations, standard buffers of pH 4.00, 7.00,
and 10.00 were also purchased from Fisher Scientific.
Doubly distilled water was utilized to prepare all the
solutions.
Mass Spectra
To identify the oxidation product(s) of Hcy, ESI high-
resolution mass spectra were recorded for a fresh so-
lution of 6 mM Hcy in 10 mM acetic acid and a re-
action mixture containing 6 mM Hcy and 1 mM cis-
[Pt(NH3)2Cl4] in 10 mM acetic acid after a reaction
time of 5 min.
RESULTS AND DISCUSSION
Kinetic Traces and Rapid Scan Spectra
The kinetic traces recorded for the reactions of the
Pt(IV) prodrugs with Hcy under the pseudo–first-
order conditions were generally biphasic, a rapid
decrease in absorbance was followed by a slow
increase. Fig. 2 and Fig. S2 (in the Supporting Informa-
tion) exemplify typical kinetic traces for the reactions
of [Pt(cbdca)(NH3)2Cl2] and [Pt(NH3)2Cl4], respec-
tively. The rapid decreases can be assigned to the re-
duction of [Pt(cbdca)(NH3)2Cl2] to carboplatin and of
Reaction Media
Combinations of HOAc/NaOAc, NaH2PO4/Na2HPO4,
NaHCO3/Na2CO3, and Na2HPO4/Na3PO4 (concentra-
tions of 0.15–0.25 M) were employed to prepare a se-
ries of buffers, covering a pH range of 3.22–11.24.
All the buffers that contained 2 mM EDTA and 0.10
M NaCl were converted to an ionic strength (μ) of
1.0 M by use of sodium perchlorate. The role of EDTA
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

4
TIAN ET AL.
Figure 2 Kinetic trace obtained at 245 nm for the reaction
between cis,trans-[Pt(cbdca)(NH3)2Cl2] and Hcy under a
set of conditions: [Pt(IV)] = 0.20 mM, [Hcy]tot = 2.00 mM,
phosphate buffer of pH 6.27, 25.0°C, and μ = 1.0 M. Insert:
an enlarged scale for the kinetic trace between 0–3 s. [Color
figure can be viewed at wileyonlinelibrary.com]
[Pt(NH3)2Cl4] to cisplatin, respectively, whereas the
slow increases are ascribed to the substitution reac-
tions, namely the substitutions of the coordinated cbdca
in carboplatin and of coordinated chlorides in cisplatin
by the excess Hcy in the reaction mixtures [26]. In the
present work, our attention was mainly paid to the re-
duction reactions which are also the activation process
of these prodrugs.
Rapid scan spectra recorded by the stopped-flow
technique for the reduction of [Pt(NH3)2Cl4] by Hcy
in a phosphate buffer of pH 6.27 are given in Fig. 3a;
the kinetic traces taken from the rapid-scan spectra at
several wavelengths were well simulated by Eq. (1) as
Figure 3 (a) Rapid scan spectra for the reduction of cis-
[Pt(NH3)2Cl4] by Hcy under the reaction conditions: [Pt(IV)]
= 0.20 mM, [Hcy]tot = 2.00 mM, pH 6.23 phosphate buffer,
25.0°C, and μ = 1.0 M. The spectra were obtained at 0.003,
0.05, 0.10, 0.20, 0.30, 0.50, 0.70, 1.0, 1.2, 1.5, 1.8, 2.0, 2.5,
and 3.0 s. (b) Kinetic traces at several wavelengths for the
above reaction (data points are from the absorption read-
ings in Fig. 3a). The solid lines were obtained from the
curve fits of Eq. (1) to the experimental data by nonlinear
least-squares routine. [Color figure can be viewed at wiley-
onlinelibrary.com]
At = (A0 − A ) exp (−kobsdt) + A
(1)
∞
∞
shown in Fig. 3b. In Eq. (1), At, A0, and Aꢀ stand
for absorbances at time t, zero, and infinity, respec-
tively, and kobsd pertains to the pseudo–first-order rate
constants [27]. Moreover, kobsd values evaluated from
the simulations were identical within the experimen-
tal errors. This character suggests that the reduction of
[Pt(NH3)2Cl4] to cisplatin is a simple process and is
first order with respect to [Pt(NH3)2Cl4]. Similarly, the
kinetic traces for reduction of [Pt(cbdca)(NH3)2Cl2]
by Hcy are also simulated perfectly by Eq. (1), cf. the
insert of Fig. 2.
Second-Order Kinetics
A large number of kobsd values as a function of [Hcy]tot
and of pH were collected; sufficiently large variations
of [Hcy]tot were made within the buffer capacities.
Plots of kobsd versus [Hcy]tot are linear and passing
through the origin shown in Figs. 4 and 5, demonstrat-
ing that the reduction is also first order in [Hcy]tot.
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

REDUCTION OF CISPLATIN AND CARBOPLATIN PT(IV) PRODRUGS BY HOMOCYSTEINE
5
Figure 5 kobsd as a function of [Hcy]tot for the reduction of
cis,trans-[Pt(cbdca)(NH3)2Cl2] by Hcy in several buffers of
different pH values at 25.0°C and μ = 1.0 M. [Color figure
can be viewed at wileyonlinelibrary.com]
Figure 4 kobsd as a function of [Hcy]tot for the reduction of
cis-[Pt(NH3)2Cl4] by Hcy in several buffers of different pH
values at 25.0°C and μ = 1.0 M. [Color figure can be viewed
at wileyonlinelibrary.com]
reductions of the Pt(IV) prodrugs by Hcy; Fig. S3 in
the Supporting Information shows the titration results.
Clearly, the data points in Fig. S3 follow two cross-
ing straight lines for each of the Pt(IV) prodrugs. The
consumed concentration ratios of ꢀ[Pt(IV)]:ꢀ[Hcy]tot
Eq. (2) expresses the overall second-order kinetics,
where kꢀ stands for the observed second-order rate con-
stants:
= 1:1.93
0.05 and 1:1.90
0.05 were de-
rived from the intersection points for [Pt(NH3)2Cl4]
and [Pt(cbdca)(NH3)2Cl2], respectively. Undoubtedly,
these concentration ratios suggest a 1:2 stoichiometry
within the experimental errors. The 1:2 stoichiometric
reaction can be expressed by Eq. (3) implying that Hcy
was oxidized to homocystine (Hcy-S-S-Hcy).
ꢀ
ꢁ
− [Pt (IV)] /dt = kobsd [Pt (IV)] = kꢀ Hcy tot [Pt (IV)]
(2)
Values of kꢀ evaluated as a function of pH are sum-
marized in Table SI in the Supporting Information; the
variations of kꢀ with pH are also displayed in Fig. 6
(data points are in the logarithmic scale).
Pt (IV) + 2 HOOCCH (NH2) CH2CH2SH → Pt (II) + 2H+ + 2Cl−
+HOOCCH (NH2) CH2CH2S-SCH2CH2CH (NH2) COOH (Hcy-S-S-Hcy)
(3)
Stoichiometry and Oxidation Product
To justify homocystine as the oxidation product
of Hcy, ESI high-resolution mass spectra recorded
for a solution of 6 mM Hcy in 10 mM acetic acid
Spectrophotometric titration experiments were carried
out to find out the reaction stoichiometries for the
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

6
TIAN ET AL.
tolysis species of Hcy (I–IV), which are able to re-
duce the Pt(IV) prodrugs in parallel as described by
k1 – k4, all being the rate-determining steps. In these
rate-determining steps, thiol and thiolate forms of Hcy
attack one of the trans-coordinated chlorides in the
Pt(IV) prodrugs and then undergo a Cl+ transfer from
the Pt(IV) centers to the sulfur atom [28–30]. Tran-
sient chlorothiol and sulfenyl chloride species are thus
generated in the rate-determining steps and are then
rapidly trapped by the excess Hcy [9,28], leading to
the formation of Hcy-S-S-Hcy.
Evaluation of Rate Constants
Eq. (4) is the rate expression derived from Scheme 1:
k1a3H + k2Ka1a2 + k3Ka1Ka2aH + k4Ka1Ka2Ka3
aH3 + Ka1a2H + Ka1Ka2aH + Ka1Ka2Ka3
− d [Pt (IV)] /dt =
H
ꢀ
ꢁ
×
Hcy tot [Pt (IV)]
(4)
In the equation, aH represents the proton activity,
which exactly corresponds to the pH measurements. A
comparison of Eq. (4) with Eq. (2) imparts
k1a3H + k2Ka1aH2 + k3Ka1Ka2aH + k4Ka1Ka2Ka3
kꢀ =
aH3 + Ka1aH2 + Ka1Ka2aH + Ka1Ka2Ka3
(5)
Protolysis constants of Hcy were reported to be
pKa1 = 2.27, pKa2 = 8.66, and pKa3 = 10.55 at 25.0°C
and μ = 0.3 M [31]. Eq. (5) was employed to simulate
the kꢀ – pH data in Fig. 6 by use of a weighted nonlin-
ear least-squares routine [29]. In the simulations, the
protolysis constants of Hcy were used as the direct in-
puts and k1 – k4 were treated as tunable parameters.
The simulation results showed that k1 is too small to
be evaluated with a confident accuracy. Thus k1 is not
observed and the k1 term in Eq. (5) can be neglected
leading to Eq. (6):
Figure 6 Second-order rate constants kꢀ as a function of
pH at 25.0°C and μ = 1.0 M for the reduction of cis-
[Pt(NH3)2Cl4] and cis,trans-[Pt(cbdca)(NH3)2Cl2] by Hcy
(data points). The solid curves were obtained by fitting
Eq. (6) to the experimental data using a weighted nonlin-
ear least-squares method. [Color figure can be viewed at
wileyonlinelibrary.com]
and for a reaction mixture containing 6 mM Hcy
and 1 mM cis-[Pt(NH3)2Cl4] in 10 mM acetic acid
are shown in Fig. 7. Assignments of major peaks
are given as m/z 136.0427/136.0426 for Hcy·H+, m/z
158.0247 for Hcy·Na+, m/z 180.0066 for {Hcy – H+ +
2Na+}, m/z 271.0783 for (2Hcy)·H+, m/z 293.0602 for
(2Hcy)·Na+, and m/z 269.0624 for (Hcy-S-S-Hcy)·H+.
It is clear from the peak assignments that homocystine
is the dominant oxidation product of Hcy, which is
consistent with the above stoichiometric results.
k2Ka1a2H + k3Ka1Ka2aH + k4Ka1Ka2Ka3
kꢀ =
(6)
aH3 + Ka1aH2 + Ka1Ka2aH + Ka1Ka2Ka3
Eq. (6) was then utilized to simulate the kꢀ – pH
data in Fig. 6, and the simulations resulted in good fits,
affording simultaneously the values of k2 – k4 which
are listed in Table I.
Reaction Mechanism
Reactivity Analysis
A plausible mechanism reconciling with the above ex-
perimental observations is proposed as delineated in
Scheme 1. The mechanism involves all the four pro-
The collection of the kinetic data in a broad pH range
enabled us to derive the rate constants of the rate-
determining steps in Table I. Apparently, the fully
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

REDUCTION OF CISPLATIN AND CARBOPLATIN PT(IV) PRODRUGS BY HOMOCYSTEINE
7
Figure 7 High-resolution ESI mass spectra for a solution of 6 mM Hcy in 10 mM acetic acid (top) and for a reaction mixture
comprising 5 mM Hcy and 1 mM cis-[Pt(NH3)2Cl4] in 10 mM acetic acid after a reaction time of 5 min (bottom).
k1
+
HS(CH2)2CH(NH3+)COOH
Cl
HS(CH2)2CH(NH3+)COOH
_
I
Pt(II) + Cl
Ka1
k2
_
Cl
Pt
+
_
HS(CH2)2CH(NH3+)COO
Cl
HS(CH2)2CH(NH3+)COO
_
II
fast
Pt(II) + Cl
IV
Hcy-S-S-Hcy
+
Ka2
_
k3
Cl + nH+
Hcy
_
_
_
_
ClS(CH2)2CH(NH3+)COO
S(CH2)2CH(NH3+)COO
Cl
_
III
Pt(II) + Cl
Ka3
k4
_
_
ClS(CH2)2CH(NH2)COO
S(CH2)2CH(NH2)COO
_
IV
Pt(II) + Cl
Pt(IV) = [Pt(NH3)2Cl4] or [Pt(cbdca)(NH3)2Cl2]; Pt(II) = [Pt(NH3)2Cl2] or [Pt(cbdca)(NH3)2]
Scheme 1 The proposed reaction mechanism.
protonated form of Hcy has a negligible reactivity to-
ward the reduction of both prodrugs; similarly, the fully
protonated forms of the thiol-containing compounds
were found to be inactive toward the Pt(IV) anticancer
model complex, trans-[PtCl2(CN)4]2− [28]. It is note-
worthy for these two prodrugs that the thiolate forms
of Hcy (III and IV) are > 105 more reactive than
the thiol form II due to the deprotonation of thiol,
highlighting a drastic electronic effect. On the other
hand, species IV is only about three to four times more
reactive than species III, which is caused by the de-
+
protonation of the electron-withdrawing group –NH3
(becoming an electron-donating group –NH2). In this
case, the electronic effect is, however, attenuated by
the distance between the two functional groups S− and
–NH3+/–NH2. The values of k2 – k4 between the two
International Journal of Chemical Kinetics DOI 10.1002/kin.21107

8
TIAN ET AL.
Table I Values of Rate Constants Obtained for the
Rate-Determining Steps in the Reductions of
cis-[Pt(NH3)2Cl4] and cis,trans-[Pt(cbdca)(NH3)2Cl2] by
Hcy at 25.0°C and μ = 1.0 M
Pt(IV)
km
Value (M−1s−1
)
[Pt(NH3)2Cl4]
k1
k2
k3
k4
Not observed
0.60 0.05
(1.43 0.06) × 105
(4.86 0.09) × 105
Not observed
[Pt(cbdca)(NH3)2Cl2]
k1
k2
k3
k4
0.70 0.05
(1.23 0.05) × 105
(5.07 0.15) × 105
prodrugs are surprisingly very close. Thus, when the
two coordinated chlorides in the equatorial positions
of cis-[Pt(NH3)2Cl4] are replaced by cbdca becoming
cis,trans-[Pt(cbdca)(NH3)2Cl2], the replacement has
essentially no influence on the reduction rate.
A straightforward way of examining the reactivity
of the Hcy species under the physiological pH (7.4) is
to construct the reactivity versus pH distribution dia-
gram [10,32,33]. The Hcy species versus pH distribu-
tion diagram is displayed in Fig. 8 (top part); compar-
atively, the reactivity versus pH distribution diagram
was calculated in the case of [Pt(cbdca)(NH3)2Cl2] and
is illustrated in Fig. 8 (bottom part). The diagrams re-
veal explicitly that II is the dominant existing species
at pH 7.4 (94.8%), but it only contributes 0.01% to
the total reactivity. On the other hand, III is a minor
existing species (5.2%), its contribution to the total
reactivity amounts to 99.7%. The reactivity versus pH
distribution diagram for the reduction of [Pt(NH3)2Cl4]
by Hcy is almost identical to Fig. 8 (bottom part).
Figure 8 (Top): Hcy species versus pH distribution dia-
gram at 25.0°C and μ = 1.0 M, which was calculated using
pKa1 = 2.27, pKa2 = 8.66, and pKa3 = 10.55. (Bottom): Re-
activity versus pH distribution diagram for the Hcy species in
the reduction of cis,trans-[Pt(cbdca)(NH3)2Cl2]; the above
pKa values and rate constants in Table I were employed in
the calculation. [Color figure can be viewed at wileyonlineli-
brary.com]
to the overall reactivity at pH 7.4. Undoubtedly, the
mechanistic and reactivity analyses provide insights
into the detail activation process of this type of Pt(IV)
anticancer prodrugs.
CONCLUSION
The reduction of the cisplatin and carboplatin Pt(IV)
prodrugs by Hcy has been characterized kinetically and
mechanistically using the stopped-flow and mass spec-
trometric techniques. The proposed reaction mecha-
nism is convincing as it can account for all the ex-
perimental observations. All the rate constants of the
rate-determining steps have been derived, and, surpris-
ingly, the values of k2 – k4 between the two prodrugs
are very close. Species III of Hcy is almost exclusively
responsible for the reduction of these prodrugs at phys-
iological pH although it is existing only 5.2% of the
total Hcy. On the other hand, the dominant existing
form II of Hcy virtually does not make a contribution
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