Kinetics and mechanism for reduction of the anticancer prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by thiols.

Article abstract of DOI:10.1021/ic991351l

The reduction of the platinum(IV) prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by L-cysteine, DL-penicillamine, DL-homocysteine, N-acetyl-L-cysteine, 2-mercaptopropanoic acid, 2-mercaptosuccinic acid, and glutathione has been investigated at 25 degrees C in a 1.0 M aqueous perchlorate medium with 6.8 < or = pH < or = 11.2 using stopped-flow spectrophotometry. The stoichiometry of Pt(IV):thiol is 1:2, and the redox reactions follow the second-order rate law -d[Pt(IV)]/dt = k[Pt(IV)][RSH]tot, where k denotes the pH-dependent second-order rate constant and [RSH]tot the total concentration of thiol. The pH dependence of k is ascribed to parallel reductions of JM335 by the various protolytic species of the thiols, the relative contributions of which change with pH. Electron transfer from thiol (RSH) or thiolate (RS-) to JM335 is suggested to take place as a reductive elimination process through an attack by sulfur at one of the mutually trans chloride ligands, yielding trans-[Pt(OH)2(c-C6H11NH2)(NH3)] and RSSR as the reaction products, as confirmed by 1H NMR. Second-order rate constants for the reduction of JM335 by the various protolytic species of the thiols span more than 3 orders of magnitude. Reduction with RS- is approximately 30-2000 times faster than with RSH. The linear correlation log(kRS) = (0.52 +/- 0.06)-pKRSH--(2.8 +/- 0.5) is observed, where kRS denotes the second-order rate constant for reduction of JM335 by a particular thiolate RS- and KRSH is the acid dissociation constant for the corresponding thiol RSH. The slope of the linear correlation indicates that the reactivity of the various thiolate species is governed by their proton basicity, and no significant steric effects are observed. The half-life for reduction of JM335 by 6 mM glutathione (40-fold excess) at physiologically relevant conditions of 37 degrees C and pH 7.30 is 23 s. This implies that JM335, in clinical use, is likely to undergo in vivo reduction by intracellular reducing agents such as glutathione prior to binding to DNA. Reduction results in the immediate formation of a highly reactive platinum(II) species, i.e., the bishydroxo complex in rapid protolytic equilibrium with its aqua form.

Full text of DOI:10.1021/ic991351l

1728
Inorg. Chem. 2000, 39, 1728-1734
Kinetics and Mechanism for Reduction of the Anticancer Prodrug
trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by Thiols
Kelemu Lemma,†,‡ Tiesheng Shi,§ and Lars I. Elding*,†
Inorganic Chemistry 1, Chemical Center, Lund University, P.O. Box 124, SE-221 00 Lund, Sweden,
Department of Chemistry, University of California, Riverside, California 92521, and
Department of Chemistry, Addis Ababa University, P.O. Box 1176, Addis Ababa, Ethiopia
ReceiVed NoVember 22, 1999
The reduction of the platinum(IV) prodrug trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM335) by L-cysteine,
DL-penicillamine, DL-homocysteine, N-acetyl-L-cysteine, 2-mercaptopropanoic acid, 2-mercaptosuccinic acid, and
glutathione has been investigated at 25 °C in a 1.0 M aqueous perchlorate medium with 6.8 e pH e 11.2 using
stopped-flow spectrophotometry. The stoichiometry of Pt(IV):thiol is 1:2, and the redox reactions follow the
second-order rate law -d[Pt(IV)]/dt ) k[Pt(IV)][RSH]tot, where k denotes the pH-dependent second-order rate
constant and [RSH]tot the total concentration of thiol. The pH dependence of k is ascribed to parallel reductions
of JM335 by the various protolytic species of the thiols, the relative contributions of which change with pH.
Electron transfer from thiol (RSH) or thiolate (RS-) to JM335 is suggested to take place as a reductive elimination
process through an attack by sulfur at one of the mutually trans chloride ligands, yielding trans-[Pt(OH)2-
(c-C6H11NH2)(NH3)] and RSSR as the reaction products, as confirmed by 1H NMR. Second-order rate constants
for the reduction of JM335 by the various protolytic species of the thiols span more than 3 orders of magnitude.
Reduction with RS- is ∼30-2000 times faster than with RSH. The linear correlation log(kRS ) ) (0.52 ( 0.06)-
-
-
pKRSH - (2.8 ( 0.5) is observed, where kRS denotes the second-order rate constant for reduction of JM335 by
a particular thiolate RS- and KRSH is the acid dissociation constant for the corresponding thiol RSH. The slope
of the linear correlation indicates that the reactivity of the various thiolate species is governed by their proton
basicity, and no significant steric effects are observed. The half-life for reduction of JM335 by 6 mM glutathione
(40-fold excess) at physiologically relevant conditions of 37 °C and pH 7.30 is 23 s. This implies that JM335, in
clinical use, is likely to undergo in vivo reduction by intracellular reducing agents such as glutathione prior to
binding to DNA. Reduction results in the immediate formation of a highly reactive platinum(II) species, i.e., the
bishydroxo complex in rapid protolytic equilibrium with its aqua form.
Introduction
substituted by more sterically demanding ligands such as quino-
line, cyclohexylamine, and iminoethers. The rationale behind
the design of such complexes is that the structural features are
expected to result in a binding to DNA distinct from that of
cisplatin.9,10
Trans platinum(IV) dihydroxo complexes represent a new
group of such drugs. The most widely studied compound of
this type is trans,trans,trans-[PtCl2(OH)2(c-C6H11NH2)(NH3)]
(JM335), which exhibits antitumor activity in both murine and
human subcutaneous cell tumor models.8 The mechanism for
the antitumor activity is not entirely known, but JM335 is
reported to be efficient in forming interstrand cross-links to DNA
and to be able to cause single-strand breaks.11 In view of the
fact that platinum(IV) complexes are coordinatively saturated
and generally substitution-inert,12 reduction of Pt(IV) by intra-
cellular reducing agents, such as glutathione or similar reagents,
The clinical efficacy of cisplatin, cis-[PtCl2(NH3)2], is limited
by toxic side effects, in particular a dose-limiting nephrotoxicity,
by drug resistance in the tumor cells, and by a narrow range of
activity.1,2 With the second-generation drug carboplatin, cis-
[Pt(cyclobutane-1,1-dicarboxylate)(NH3)2], toxicity is reduced,
but drug resistance due to reduced platinum accumulation,
increased cytoplasmic detoxification, and enhanced DNA-
platinum adduct removal still presents limitations.3 To circum-
vent these problems, there is a current interest in platinum
complexes with a trans geometry,4-9 in which ammonia is
* To whom correspondence should be addressed: Professor Lars I.
Elding, Inorganic Chemistry 1, Chemical Center, Lund University, P.O.
Box 124, SE-221 00 Lund, Sweden. Fax: +46-46-2224439. E-mail:
LarsI.Elding@inorg.lu.se.
† Lund University.
‡ Addis Ababa University.
§ University of California.
(6) Farrell, N.; Kelland, L. R.; Roberts, J. D.; van Beusichem, M. Cancer
Res. 1992, 52, 5065.
(7) Coluccia, M.; Nassi, A.; Loseto, F.; Boccareli, A.; Mariggio, M. A.;
Giordano, D.; Intini, F. P.; Caputo, P.; Natile, G. A. J. Med. Chem.
1993, 36, 510.
(8) Kelland, L. R.; Barnard, C. F. J.; Mellish, K. J.; Jones, M.; Goddard,
P. M.; Valenti, M.; Bryant, A.; Murrer, B. A.; Harrap, K. R. Cancer
Res. 1994, 54, 5618.
(1) For recent general reviews on platinum-based antitumor drugs, see,
for example: (a) Wong, E.; Giandomenico, C. M. Chem. ReV. 1999,
99, 2451. (b) Jamieson, E. R.; Lippard, S. J. Chem. ReV. 1999, 99,
2467. (c) Reedijk, J. Chem. ReV. 1999, 99, 2467.
(2) von Hoff, D. D.; Schilsky, R.; Reichert, C. M.; Reddick, R. L.;
Rozencweig, M.; Young, R. C.; Muggia, F. M. Cancer Treat. Rep.
1979, 63, 1527.
(3) Kelland, L. R. Crit. ReV. Oncol. Hematol. 1993, 15, 191.
(4) Farrell, N.; Ha, T. T. B.; Souchard, J. P.; Wimmer, F. L.; Cros, S.;
Johnson, N. P. J. Med. Chem. 1989, 32, 2240.
(9) Natile, G.; Coluccia, M. Top. Biol. Inorg. Chem. 1999, 1, 73.
(10) Farrell, N. Met. Ions Biol. Syst. 1996, 32, 603.
(11) Mellish, K. J.; Barnard, C. F. J.; Murrer, B. A.; Kelland, L. R. Int. J.
Cancer 1995, 62, 717.
(5) Van Beusichem, M.; Farrell, N. Inorg. Chem. 1992, 31, 634.
10.1021/ic991351l CCC: $19.00 © 2000 American Chemical Society
Published on Web 03/25/2000

Reduction of an Anticancer Prodrug by Thiols
Inorganic Chemistry, Vol. 39, No. 8, 2000 1729
to the more reactive Pt(II) analogue is assumed to take place
before binding to DNA. For example, reduction of JM335 by
glutathione has been observed,13 and cis-[PtCl2(c-C6H11NH2)-
(NH3)] (JM118) has been identified as the major metabolite in
samples from patients treated with cis,trans,cis-[PtCl2(OAc)2-
(c-C6H11NH2)(NH3)] (JM216).14 Furthermore, it has been
concluded from DNA unwinding experiments that Pt(IV)
complexes do not form bifunctional adducts with DNA unless
they are reduced to divalent species after addition of glu-
tathione.15,16
Chart 1
A knowledge of the kinetics and mechanism for reduction
of Pt(IV) compounds by biologically relevant reductants is thus
important for an understanding of the mechanism of activity in
vivo and for the design of new drugs. It has been shown that
reduction of the model compound trans-[PtCl2(CN)4]2- by
biologically relevant thiols is rapid already in acidic media,17
and mechanisms for reduction of platinum(IV) complexes by
cysteine and methionine,18 ascorbate,19,20 and glutathione21 have
been studied recently. We report here the kinetics and mecha-
nism for reduction of JM335 by thiols of disparate electronic
and steric properties. Reduction of the cis congener of JM335,
viz., cis,trans,cis-[PtCl2(OH)2(c-C6H11NH2)(NH3)] (JM149), was
not observed under the conditions used for JM335. This is an
interesting observation as JM335 is reported to exhibit an in
vitro cytotoxicity against human ovarian carcinoma cells greater
than that of JM149.8 Structures of JM335 and JM149 and the
protonated forms of the thiols used are shown in Chart 1.
readings. UV/Vis spectra were recorded with Milton Roy 3000 diode
array and Cary 300 Bio UV/Vis spectrophotometers using 1.00-cm
quartz Suprasil cells. Proton NMR spectra were recorded on a Varian
Unity 300 MHz spectrometer with D2O as the solvent and with the
residual solvent signal as the reference at constant temperature, pH,
and ionic medium.
The compound trans-[Pt(OD)2(c-C6H11NH2)(NH3)] was prepared by
treating a solution of 2 mM trans-[PtCl2(c-C6H11NH2)(NH3)] (JM334)
in D2O with 2 equiv of silver nitrate at room temperature for 24 h.
After removal of the AgCl precipitate by centrifugation, the pH of the
solution was raised to ∼7, and the proton NMR spectrum was recorded
(Figure 2c below).
Experimental Section
Chemicals. JM335 and JM149 were kindly supplied as a loan by
the Johnson Matthey Technology Centre (Reading, Berkshire, U.K.).
JM334 was a generous gift from Dr. Nicholas Farrell. L-Cysteine (ICN
Biomedicals Inc.), glutathione (Merck), 2-mercaptopropanoic acid
(Acros), DL-homocysteine (Sigma), DL-penicillamine (Janssen), DL-
mercaptosuccinic acid (Acros), and N-acetyl-L-cysteine (Janssen) were
used as received. The quality of the thiols was assessed with Ellman’s
reagent.22,23 All other chemicals used were of analytical grade. TRIS-
3-
HCl, HCO3-/CO32-, and HPO42-/PO4 buffers containing 2-3 mM
Kinetic Measurements. Reduction of JM335 by the thiols was
investigated by UV/VIS spectrophotometry using an Applied Photo-
physics Bio-Sequential SX-18MV stopped-flow ASVD spectropho-
tometer. Kinetic measurements were made at 25 °C over the region
6.80 e pH e 11.22. The pH of stock solutions of the thiols was tested
and adjusted by addition of a few drops of strong base when different
from the desired value. Na2H2(edta) (2-3 mM) was present in all
buffers in order to sequester trace transition metal ions such as Cu(II)
and Fe(III) that could catalyze autoxidation of the thiols.24-27 Buffer
solutions were flushed with argon for ∼30 min before use in order to
remove dissolved oxygen. Fresh stock solutions of the thiols (5-40
mM) prepared in buffer were used for each kinetic run. Sample solutions
of JM335 were prepared by diluting 1.0-1.5 mL of stock solutions (2
mM JM335 in 100 mM sodium chloride) with buffer to 10 mL. The
ionic strength of all solutions was adjusted to 1.00 M with NaClO4.
All kinetic measurements were performed under pseudo-first-order
conditions with excess thiol by monitoring the decrease in absorbance
at 272 nm where the thiols and reaction products are practically
transparent. Reactions were followed for at least 4 half-lives with 4-6
repetitive runs. Single-exponential kinetic traces were obtained in all
cases. Pseudo-first-order rate constants kobsd were obtained from an
online nonlinear least-squares analysis of the absorbance-time data
using an Applied Photophysics software package.28
Na2H2(edta) and 100 mM sodium chloride were used to maintain
constant pH in the studied region where 6.80 e pH e 11.22. Water
was doubly distilled from quartz.
Physical Measurements. The pH of the buffers was measured at
25 °C with a Metrohm 632 digital pH meter equipped with a
combination glass electrode. Standard buffers of pH 7.0 and 9.0,
obtained from Merck, were used to calibrate the electrode. Oxonium
ion activities aH ) 10-pH were obtained directly from the pH meter
(12) Roundhill, D. M. ComprehensiVe Coordination Chemistry; Wilkinson,
G., Gillard, R. D., McCleverty, J. A., Eds.; Pergamon Press: Oxford,
U.K., 1987; Vol. 5, p 351.
(13) Talman, E. G.; Bruning, W.; Reedijk, J.; Spek, A. L.; Veldman, N.
Inorg. Chem. 1997, 36, 854.
(14) Poon, G. K.; Raynaud, F. I.; Mistry, P.; Odell, D. E.; Kelland, L. R.;
Harrap, K. R.; Barnard, C. F. J.; Murrer, B. A. J. Chromatogr., A
1995, 712, 61.
(15) Keck, M. V.; Lippard, S. J. J. Am. Chem. Soc. 1992, 114, 3386.
(16) Ellis, L. T.; Er, H. M.; Hambley, T. W. Aust. J. Chem. 1995, 48, 793.
(17) Shi, T.; Berglund, J.; Elding, L. I. Inorg. Chem. 1996, 35, 3498.
(18) (a) Chen, L.; Lee, P. F.; Ranford, J. D.; Vittal, J. J.; Wong, S. Y. J.
Chem. Soc., Dalton Trans. 1999, 1209. (b) Shi, T.; Berglund, J.; Elding,
L. I. J. Chem. Soc., Dalton Trans. 1997, 2073.
(19) Choi, S.; Filotto, C.; Bisanzo, M.; Delaney, S.; Lagasee, D.; Witworth,
J. L.; Jusko, A.; Li, C.; Wood, N. A.; Willingham, J.; Schwenker, A.;
Spaulding, K. Inorg. Chem. 1998, 37, 2500.
(20) Lemma, K.; Sargeson, A. M.; Elding, L. I. J. Chem. Soc., Dalton Trans.
2000, 1167.
(21) Lemma, K.; Berglund, J.; Farrell, N.; Elding, L. I. J. Biol. Inorg. Chem.
2000, 5, in press.
(22) Ellman, G. L. Arch. Biochem. Biophys. 1959, 82, 70.
(23) Riddles, P. W.; Blakeley, R. L.; Zerner, B. Anal. Biochem. 1979, 94,
75.
(24) Taylor, J. E.; Yan, J. F.; Wang, J.-L. J. Am. Chem. Soc. 1966, 88,
1663.
(25) Bridgart, G. J.; Wilson, I. R. J. Chem. Soc., Dalton Trans. 1973, 1281.
(26) York, M. J.; Beilharz, G. R.; Kuchel, P. W. Int. J. Pept. Protein Res.
1987, 29, 638.
(27) Ehrenberg, L.; Harms-Ringdahl, M.; Fedorcak, I.; Granath, F. Acta
Chem. Scand. 1989, 43, 177.

1730 Inorganic Chemistry, Vol. 39, No. 8, 2000
Lemma et al.
Figure 1. Proton NMR spectra at 25 °C of (a) the oxidation product
for the reaction between 2 mM JM335 and 4 mM glutathione (GSH)
in a 1:2 JM335:GSH molar mixture at pH ∼9, (b) 2 mM GSSG at pH
∼5, and (c) 4 mM glutathione at pH ∼9. Oxidation of GSH to GSSH
is confirmed by a disappearance of the multiplet peak at 3 ppm assigned
to the Cys-âCH2 group of GSH (c), and the apperance of the doublet
of doublets at 3.06 and 3.36 ppm for GSSG (a and b).
Results and Discussion
Stoichiometry. The stoichiometry of [Pt(IV)]:[RSH] for
reduction of trans-[PtCl2(CN)4]2- by a series of thiols has been
established to be 1:2.17 An analogous spectrophotometric
determination is not feasible in the present cases because of
interference from slow subsequent substitution processes at the
Pt(II) product.29-32 However, glutathione was quantitatively
oxidized to the disulfide GSSG in a JM335:GSH 1:2 molar
mixture at 25 °C and pH ∼9, consistent with the assumed 1:2
stoichiometry in the present cases (cf. spectra in Figure 1).
Proton NMR spectra of reactants and products (Figure 2) show
that JM335 was reduced to trans-[Pt(OH)2(c-C6H11NH2)(NH3)].
Kinetics. Observed rate constants are independent of the
initial concentration of Pt(IV), and the plots of kobsd vs [RSH]tot
at constant pH are linear with zero intercept. Figure 3 shows
four examples of such plots. Thus, the redox reactions are first-
order with respect to both JM335 and thiol, according to the
experimental rate law defined by eq 1.
Figure 2. Proton NMR spectra at 25 °C of (a) JM335, (b) reaction
product for the redox reaction between JM335 (in slight excess) and
penicillamine at pH ∼9, (c) trans-[Pt(OD)2(c-C6H11NH2)(NH3)] at pH
∼7, and (d) trans-[PtCl2(c-C6H11NH2)(NH3)]. The two broad bands at
∼2.4 and 1.8 ppm in spectra b-d due to the protons of the
cyclohexylamine moiety are largely different for the bishydroxo (c)
and bischloro (d) complexes. The peak in the spectrum of the product
mixture (b) at ∼2.22 ppm is due to residual Pt(IV). The similarity
between spectra b and c indicates that the bishydroxo complex is the
reaction product.
-d[Pt(IV)]/dt ) kobsd[Pt(IV)] ) k[RSH]tot[Pt(IV)] (1)
The pH-dependent second-order overall rate constants k,
obtained as slopes of plots of kobsd vs [RSH]tot, are summarized
in Supporting Information Table S1. The fact that the redox
reactions are first-order with respect to [RSH]tot implies that
the second molecule/ion of the thiols reacts in a subsequent rapid
step (vide infra). The pH dependence of k is attributed to the
displacement of protolytic equilibria involving the various an-
ionic species of the thiols, cf. Schemes 1-4. JM335 is not ex-
pected to be involved in such equilibria in the pH region studied.
Figure 3. Plots of xkobsd as a function of [RSH]tot for the reduction of
JM335 by N-acetyl-L-cysteine (2, x ) 1.25), L-cysteine (4, x ) 1),
2-mercaptopropanoic acid ([, x ) 0.6), and DL-penicillamine (0, x )
1) at pH 9.52 and 25 °C.
The anionic species of the thiols reduce JM335 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 thiol species. Only those
protolytic species whose concentrations are significant in the
pH region 6.8-11.2 are included in the schemes. Second-order
rate constants ki (i ) 2, 3, 4, or 5) were calculated by least-
squares fitting of equations derived from Schemes 1-4 to the
experimental data using acid dissociation constants from the
(28) Applied Photophysics Bio Sequential SX-18 MV, Sequential Stopped-
Flow ASVD Spectrofluorimeter software, Applied Photophysics Ltd.,
Leatherhead, Surrey, U.K.
(29) cis- and trans-[PtCl2(NH3)2] are known to undergo substitution
reactions with cysteine and glutathione.30-32 The reaction conditions
chosen here (i.e., pseudo-first-order with excess of thiol) suggest that
similar reactions will occur.
(30) Appleton, T. G.; Connor, J. W.; Hall, J. R.; Prenzler, P. D. Inorg.
Chem. 1989, 28, 2030.
(31) Berners-Price, S. J.; Kuchel, P. W. J. Inorg. Biochem. 1990, 38, 305.
(32) Odenheimer, B.; Wolf, W. Inorg. Chim. Acta 1982, L41-L43.

Reduction of an Anticancer Prodrug by Thiols
Inorganic Chemistry, Vol. 39, No. 8, 2000 1731
Scheme 1. Model for Reduction of JM335 by Cysteine,
Penicillamine, and Homocysteine
Scheme 2. Model for Reduction of JM335 by N-Acetyl-L-
cysteine and 2-Mercaptopropanoic Acid
Scheme 3. Model for Reduction of JM335 by
Mercaptosuccinic Acid
Scheme 4. Model for Reduction of JM335 by Glutathione
Figure 4. Plots of the second-order rate constants k as a function of
pH for the reduction of JM335 with L-cysteine, DL-penicillamine, and
DL-homocysteine. The solid lines represent the fits of eq 6 to
experimental data.
electron transfer involves reductive elimination through nucleo-
philic attack by the reductant on a halide coordinated trans to
a good leaving group. Reductive elimination reactions of
platinum(IV) compounds via the halide-bridged activated
complex are formally equivalent to a transfer of X+ (X ) Cl,
Br) from the oxidizing Pt(IV) center to the reducing nucleophile,
followed by loss of the trans ligand.17,18b,39-43 The detection of
a BrCN intermediate for reduction of trans-[PtBr(CN)4(OH)]2-
by CN- has been presented as strong support for such a
mechanism.39 Reduction of biscarboxylato platinum(IV) com-
plexes by methionine and cysteine was recently proposed to
take place by a similar reductive elimination mechanism.18a For
these trans biscarboxylato complexes, however, an outer-sphere
reaction might also be feasible.20
literature as constants (cf. Appendix).33-37 The pH profiles, i.e.,
the plots of k vs pH, for all of the thiols used are displayed in
Figures 4-6, and the derived second-order rate constants ki are
summarized in Table 1.
Reaction Mechanism. Because platinum(IV) compounds are
generally substitution-inert,38 electron transfer by a substitution-
controlled inner-sphere mechanism is unlikely. Previous mecha-
nistic studies on reductions of platinum(IV) halide complexes
by inorganic39-43 and biological17-21 reductants have shown that
An important question regarding the detailed mechanism for
reduction of JM335 is whether the reaction takes place via attack
(39) Wilmarth, W. K.; Fanchiang, Y.-T.; Byrd, J. E. Coord. Chem. ReV.
1983, 51, 141.
(40) Wilmarth, W. K.; Dooly, M. M.; Byrd, J. E. Coord. Chem. ReV. 1983,
51, 125.
(33) Connet, P. H.; Wetterhahn, K. E. J. Am. Chem. Soc. 1986, 108, 1842.
(34) Reid, R. S.; Rabenstein, D. L. Can. J. Chem. 1981, 59, 1505.
(35) Reuben, D. M. E.; Bruice, T. C. J. Am. Chem. Soc. 1976, 98, 114.
(36) Smith, R. M.; Martell, A. E. Critical Stability Constants - Other
Organic Ligands; Plenum Press: New York, 1977.
(37) Rabenstein, D. L. J. Am. Chem. Soc. 1973, 95, 2797.
(38) Mason, W. R. Coord. Chem. ReV. 1972, 7, 241.
(41) Chandayot, P.; Fanchiang, Y.-T. Inorg. Chem. 1985, 24, 3532.
(42) Chandayot, P.; Fanchiang, Y.-T. Inorg. Chem. 1985, 24, 3535.
(43) (a) Elding, L. I.; Gustafson, L. Inorg. Chim. Acta 1976, 19, 165. (b)
Elding, L. I.; Gustafson, L. Inorg. Chim. Acta 1977, 24, 239. (c)
Drougge, L.; Elding, L. I. Inorg. Chim. Acta 1986, 121, 175. (d)
Berglund, J.; Voigt, R.; Fronaeus, S.; Elding, L. I. Inorg. Chem. 1994,
33, 3346. (e) Shi, T.; Elding, L. I. Inorg. Chim. Acta 1998, 282, 55.

1732 Inorganic Chemistry, Vol. 39, No. 8, 2000
Lemma et al.
Chart 2
-
Figure 7. Correlation between the second-order rate constants kRS for
the reduction of JM335 by thiolates RS- and the acid dissociation
constants KRSH. The numbers 1-7 denote the thiolates of DL-pen-
icillamine, L-cysteine, glutathione, DL-homocysteine, N-acetyl-L-cys-
teine, 2-mercaptopropionic acid, and DL-mercaptosuccinic acid, respec-
tively.
Figure 5. Plots of the second-order rate constants k as a function of
pH for the reduction of JM335 with N-acetyl-L-cysteine and 2-mer-
captopropanoic acid. The solid lines represent the fits of eq 7 to the
experimental data.
the product analysis, which shows that the trans bishydroxo
complex is the reduction product. Further characterization of
the product by 195Pt NMR is not possible because of interference
from subsequent processes, presumably substitution of the
hydroxide/aqua ligands of the platinum(II) product by the excess
thiol.
It is noteworthy that the cis, trans, cis isomer, JM149, which
has been reported to have a cytotoxicity lower than that of
JM335,8 does not undergo observable reduction under the
conditions used for reduction of JM335. The chloride ligands
in JM149 are coordinated trans to the strongly bound ammonia
and cyclohexylamine ligands (cf. Chart 1), and therefore, the
reductive elimination pathway via attack on chloride is energeti-
cally unfavorable. However, thiol reduction of JM149 may occur
on a much longer time scale than that observed for JM335. Thus,
Sadler and co-workers recently observed thiol reduction of
cis,trans,cis-[Pt(en)(OH)2I2].44,45
The intermediate oxidation products, RSCl, undergo the rapid
subsequent reactions 2-4, leading to the final products RSSR
(cf. Figure 1).46
RSCl + H2O f RSOH + Cl- + H+
RSOH + RSH f RSSR + H2O
(2)
(3)
RSOH + RS- f RSSR + OH-
(4)
Figure 6. Plots of the second-order rate constants k as a function of
pH for the reduction of JM335 with DL-mercaptosuccinic acid and
glutathione. The solid lines represent the fits of eqs 8 and 9 to the
experimental data for DL-mercaptosuccinic acid and glutathione,
respectively.
The fact that the thiols attack coordinated chloride through the
sulfur atom is inferred from the large difference between the
reactivity of the thiols (RSH) and that of the thiolates (RS-);
deprotonation of the thiol group -SH increases the reduction
rate by a factor between ∼30 and more than 2000 (Table 1).
Other substituents affecting the electron distribution at the sulfur
on chloride or on hydroxide. The ammonia and the cyclohexyl-
amine are not good bridging or leaving ligands.38 Although
hydroxide as well as chloride is capable of bridging, electron
transfer is expected to take place by a reductive attack by thiol
or thiolate on the chloride, as it is much easier to produce Cl+
than OH+ thermodynamically. Thus, reduction of JM335 is
expected to take place via a chloride-bridged activated complex
of the type shown in Chart 2. This conclusion is confirmed by
(44) Kratochwil, N. A.; Guo, Z.; del Socco Murdoch, P.; Parkinson, J. A.;
Bednarski, P. J.; Sadler, P. J. J. Am. Chem. Soc. 1998, 120, 8253.
(45) Kratochwil, N. A.; Ivanov, A. I.; Patriarca, M.; Parkinson, J. A.;
Gouldsworthy, A. M.; del Socco Murdoch, P.; Sadler, P. J. J. Am.
Chem. Soc. 1999, 121, 8193.
(46) Allison, W. S. Acc. Chem. Res. 1976, 9, 293.

Reduction of an Anticancer Prodrug by Thiols
Inorganic Chemistry, Vol. 39, No. 8, 2000 1733
product, is inactive.11,50 Because the present experiments show
that reduction of JM335 results in formation of trans-[Pt(OH)2-
(c-C6H11NH2)(NH3)], and not formation of the trans bischloro
complex as assumed,11,50 a study of the clinical properties of
the trans bishydroxo platinum(II) complex is highly desirable,
in particular because this compound is in rapid protolytic
equilibrium with highly reactive aqua complexes (pKa values
for the analogous trans-[Pt(H2O)2(NH3)2] complex are 4.35 and
4.70 at 298 K).51
Table 1. Second-Order Rate Constants ki for Reduction of JM335
by Protolytic Species of the Thiols at 25 °C and an Ionic Strength
of 1.00 M
thiol species
ki, M-1 s-1
-
HSCH2CH(NH3+)CO2
k2, 1.77 ( 0.03
k3, 56.4 ( 0.3
k4, 184 ( 1
k2, 0.37 ( 0.03
k3, 88.5 ( 0.4
k4, 197 ( 1
k2, 0.74 ( 0.01
k3, 19.3 ( 0.1
k4, 136 ( 1
k2, 0.40 ( 0.02
k3, 138.6 ( 0.7
k2, 0.122 ( 0.006
k3, 290 ( 1
-SCH2CH(NH3+)CO2
-
-SCH2CH(NH2)CO2
-
HSCH2CH2CH(NH3+)CO2
-
-SCH2CH2CH(NH3+)CO2
-
-SCH2CH2CH(NH2)CO2
-
HSC(CH3)2CH(NH3+)CO2
-
-SC(CH3)2CH(NH3+)CO2
-
Acknowledgment. Financial support from the Swedish
National Science Research Council (NFR) and a grant for K.L.
from the Swedish International Development Cooperation
Agency (SIDA) within the SAERC sandwich program are
gratefully acknowledged. We thank Johnson Matthey Technol-
ogy Centre and Dr. C. F. J. Barnard for the generous loan of
the compounds JM149 and JM335, Professor N. Farrell for a
loan of trans-[PtCl2(c-C6H11NH2)(NH3)], and Professor A. M.
Sargeson for constructive criticism of the manuscript.
-SC(CH3)2CH(NH2)CO2
-
HSCH2CH(CO2-)NHCOCH3
-SCH2CH(CO2-)NHCOCH3
-
HS(CH3)CHCO2
-S(CH3)CHCO2
-
-O2CCH2CH(SH)CO2
k3, 1.283 ( 0.013
k4, 362 ( 1
-
-O2CCH2CH(S-)CO2
-
-O2CCH(NH3+)(CH2)2CONHCH(CH2SH)CONHCH2CO2 k3, 0.48 ( 0.01
-
-O2CCH(NH3+)(CH2)2CONHCH(CH2S-)CONHCH2CO2
k4, 111 ( 1
k5, 76.0 ( 0.4
-
-O2CCH(NH2)(CH2)2CONHCH(CH2S-)CONHCH2CO2
-
Supporting Information Available: Second-order rate constants
k as a function of pH for reduction of JM335 by thiols at 25 °C and an
ionic strength of 1.00 M (Table S1). This material is available free of
charge via the Internet at http://pubs.acs.org.
atom are also expected to have a large influence on the
reactivities. On the other hand, steric factors and deprotonation
of the carboxylic acid groups appear to have little effect on the
rates.
The kinetics data in Table 1 fit well to the Brønsted
Appendix: Calculation of Rate Constants
-
correlation defined by eq 5, where kRS denotes the second-order
Equation 6, derived from Scheme 1, was fit to the experi-
mental data for reduction of JM335 with cysteine, penicillamine,
and homocysteine. The contribution of the term containing k3′
to the overall rate of reduction is assumed to be insignificant
compared to that of the term containing k3 on the basis of the
fact that the thiolate (RS-) is much more reactive than the
protonated species (RSH).17
rate constant for reduction of JM335 by a thiolate species RS-
and KRSH is the acid dissociation constant for the corresponding
thiol. It can be seen from Figure 7 that the reactivity of the
thiolate species is directly related to their proton basicities
log kRS- ) (0.52 ( 0.06)pKRSH - (2.8 ( 0.5)
(5)
k2aH2 + k3KAaH + k4KAKC
aH2 + (KA + KB)aH + KAKC
and that glutathione, which is a bulky thiol, and penicillamine,
which has a much larger cone angle,47 do not deviate signifi-
cantly from the correlation. This is in line with a mechanism in
which the thiols attack a coordinated ligand rather the Pt(IV)
center where steric effects are expected to be much more
significant.
k )
(6)
In eq 6, KA, KB, and KC are the microscopic acid dissociation
constants of the thiols corresponding to the reported values of
pKA ) 8.40, pKB ) 8.85, and pKC ) 10.05 at 25 °C for
cysteine;33,34 pKA ) 8.05, pKB ) 8.61, and pKC ) 10.29 at 25
°C for penicillamine;33,34 and pKA ) 9.02, pKB ) 9.04, and
pKC ) 9.71 at 30 °C for homocysteine.35
Equation 7, derived from Scheme 2, was fit to the data
obtained for reduction with the diprotic thiols N-acetyl-L-
cysteine and 2-mercaptopropanoic acid. The parameter Ka2
denotes the acid dissociation constant for the sulfhydryl protons
of the thiols. The values for Ka2 used in eq 7
It is also noteworthy that the reduction of JM335 by thiols is
several orders of magnitude slower than that of trans-[PtCl2-
(CN)4]2- 17
. This large difference can be attributed to stabilization
of JM335 in the tetravalent state by the hydroxo ligands,48 as
well as to stabilization of the platinum(II) product formed from
the platinum(IV) cyanide complex by the π-acceptor properties
of the cyanide ligands.
Conclusion
k2aH + k3Ka2
aH + Ka2
The present results show that reduction of JM335 by
biologically relevant thiols in a moderately alkaline aqueous
perchlorate medium is fairly rapid. The half-life for reduction
by 6 mM glutathione (40-fold excess) at physiologically rele-
vant conditions of pH 7.30 and 37 °C was determined to be 23
s (Supporting Information Table S1). Thus, it is likely that
JM335 undergoes rapid in vivo reduction by intracellular
reducing agents before binding to DNA. Glutathione appears
to be the most likely reductant in the cytoplasm as its
concentration ranges from 5 to 10 mM.49 JM335 exhibits in
vivo anticancer activity,8 whereas trans-[PtCl2(c-C6H11NH2)-
(NH3)] (JM334), which has been presumed to be the reduction
k )
(7)
are 9.55 at 25 °C for N-acetyl-L-cysteine33 and 9.93 at 25 °C
for 2-meracptopropanoic acid.36
Equation 8, derived from Scheme 3, was fit to the data
obtained for reduction with mercaptosuccinic acid, which is a
triprotic thiol with the acid dissociation constants corresponding
to pKa1 ) 3.13, pKa2 ) 4.63, and pKa3 ) 10.26 (SH) at 25
°C.34 The fully deprotonated trianionic species and the dianionic
species in which only the carboxylic groups are deprotonated
are the redox active species; reduction by the monoanionic
(47) Shi, T.; Elding, L. I. Inorg. Chem. 1996, 35, 5941.
(48) Beattie, J. K.; Basolo, F. Inorg. Chem. 1967, 6, 2069.
(49) Lempers, E. L. M.; Inagaki, K.; Reedijk, J. Inorg. Chim. Acta 1988,
152, 201.
(50) Goddard, P. M.; Orr, R. M.; Valenti, M. R.; Barnard, C. F. J.; Murrer,
B. A.; Kelland, L. R.; Harrap, K. R. Anticancer Res. 1996, 16, 33.
(51) Appleton, T. G.; Bailey, A. J.; Barnham, K. J.; Hall, J. R. Inorg. Chem.
1992, 31, 3077.

1734 Inorganic Chemistry, Vol. 39, No. 8, 2000
Lemma et al.
species was not observed in the pH region used.
RSH and RS-.17 The microscopic acid dissociation constants
used in eq 9 correspond to pKAA ) 8.97, pKBB ) 9.17, and
pKCC ) 9.35 at 25 °C.37
k3Ka2aH + k4Ka2Ka3
k )
(8)
aH2 + Ka2aH + Ka2Ka3
k3aH2 + k4KAAaH + k5KAAKCC
aH2 + (KAA + KBB)aH + KAAKCC
k )
(9)
Equation 9, derived from Scheme 4, was fit to the data
obtained for reduction of JM335 by glutathione. In the derivation
of this equation, the term containing k4′ can be neglected because
of the large difference between the reactivities of the species
IC991351L