Electron-transfer-driven trans-ligand labilization: A novel activation mechanism for Pt(IV) anticancer complexes [5]

Full text of DOI:10.1021/ja980393q

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J. Am. Chem. Soc. 1998, 120, 8253-8254
8253
ethylenediamine (peak b, 3.380 ppm). The second multiplet
(3.037 ppm at pH 7 (Figure 1A, peak 2a′)) was overlapped with
the â-CH2 resonances of the cystine moiety at pH 3 and 276 and
298 K (Figure 1B,C). After 3 h at 276 K, only signals for free
GSSG and free ethylenediamine were observed, suggesting that
the disulfide product does not coordinate to Pt. At later stages,
however, broad peaks appeared at 4.1, 3.9, 2.7, and 2.4 ppm.
Analogous 1H NMR spectral changes were observed for the
reactions of N-Ac-Cys with complex 1.6
Electron-Transfer-Driven Trans-Ligand Labilization:
A Novel Activation Mechanism for Pt(IV) Anticancer
Complexes
Nicole A. Kratochwil,† Zijian Guo,†
Piedad del Socorro Murdoch,† John A. Parkinson,†
Patrick J. Bednarski,‡ and Peter J. Sadler*,†
Department of Chemistry, The UniVersity of Edinburgh
King’s Buildings, West Mains Road, Edinburgh EH9 3JJ, U.K.
The Institut fu¨r Pharmazie, UniVersita¨t Regensburg
93040 Regensburg, Germany
The reduction of the labeled complex ([15N]1) was investigated
by 2D [1H, 15N] HSQC and 2D [1H, 15N] HSQC-TOCSY NMR.4,7
The kinetic courses of the reactions with the reducing agents at
a 1:2 molar ratio (1 mM [15N]1) were studied only at pH 3 and
276 and 298 K because of the poor solubility of ([15N]1) at pH
7. At 276 K the 15N/1H shifts for ([15N]1) were 15.20/6.99 ppm
(1b). A new cross-peak at -26.92/5.56 ppm (peak 2b) was
observed in the 2D [1H, 15N] HSQC NMR spectrum 9 min after
the addition of GSH (or N-Ac-Cys). Cross-peak 2b disappeared
after 3 h. The 2D [1H, 15N] HSQC-TOCSY spectrum showed
that the inequivalent CH2 groups of the ethylenediamine moiety
are part of the same spin system. They (2a and 2a′) are coupled
to the cross-peak 2b and to one another (evidence from 2D COSY
data, data not shown). At later stages of the reaction at 298 K,
cross-peaks with 15N/1H shifts of -10.82/5.13 and -8.34/4.99
ppm, compatible with ethylenediamine NH2 trans to S in Pt(II)
complexes, and a cross-peak assignable to free [15N]en (15N/1H
shifts at 8.23/7.83 ppm) were observed in the 2D [1H, 15N] HSQC
NMR spectrum.
ReceiVed February 4, 1998
The first orally active platinum drug, cis,trans,cis-[PtCl2(OAc)2-
NH3(c-C6H11NH2)] (JM216), has now entered phase II clinical
trials,1 and octahedral Pt(IV) complexes in general offer new
strategies for the design of platinum-based antitumor agents.
Because of their inertness to substitution, Pt(IV) antitumor
complexes are thought to be prodrugs activated in vivo by
reduction to their Pt(II) analogues by biological reductants such
as glutathione, with loss of two axial ligands.2 However, there
is only scant information in the literature concerning the mech-
anism of reduction of antitumor Pt(IV) diamines by biologically
important thiols. We report here the unexpected detection of a
long-lived chelate-ring-opened Pt(II) complex generated from a
chelated cytotoxic Pt(IV) complex under biologically relevant
conditions. Electron-transfer-driven trans-ligand labilization reac-
tions may therefore provide novel activation mechanisms for Pt-
(IV) complexes in vivo.
The inequivalence of the two CH2 groups (2a and 2a′) of
coordinated ethylenediamine in the 1D 1H and 2D [1H, 15N]
HSQC-TOCSY NMR spectra and the observation of only one
15N/1H cross-peak 2b (-26.92/5.56 ppm) in the 2D spectra of
both reactions of GSH and N-Ac-Cys suggest that ring-opened
Pt(II) ethylenediamine complexes8 are formed during the above
reduction reactions. This is also argued on the basis of chemical
shifts in the 1D 1H spectrum (Figure 1), in which 2a is similar to
b for free ethylenediamine and 2a′ is similar to 1a for complex
We have studied the reduction of the Pt(IV) complex trans,-
cis-[Pt(en)(OH)2I2] (1) (en ) ethylenediamine), a complex which
is active against a wide variety of cancer cells in vitro and a
member of a class of photoactivatable Pt(IV) complexes.3
Reactions of 1 (100 µM) with glutathione (GSH, γ-Glu-Cys-Gly)
1
+
1. The H/15N cross-peak for the NH3 group of monodentate
[15N]enH+ is likely to be broadened beyond detection due to NH
exchange with the solvent. Siebert and Sheldrick9 have reported
that a ring-opened Pt(II) species is formed during reaction of [Pt-
(en)(H2O)2]2+ with methionine-containing di-and tripeptides and
at pH 2.4 gave rise to two 1H NMR CH2 multiplets for
monodentate ethylenediamine, which have shifts similar to those
observed here. The observation of only free disulfide in reactions
of either GSH or N-Ac-Cys with complex 1 indicates that the
ring-opened intermediate 2 does not contain bound disulfide. At
later stages, the ring-opened Pt(II) species undergoes further
substitution reactions. In the case of N-Ac-Cys and pH 7, the
final products of the reaction of complex 1 with N-Ac-Cys were
or N-acetyl-L-cysteine (N-Ac-Cys) in a molar ratio of 1:2 were
1
first investigated by H NMR spectroscopy4 at pHs 3 and 7 and
276 or 298 K for a period of 24 h. For the GSH reactions at pH
7 and 276 K (Figure 1A), signals for complex 1 (CH2 2.776 ppm,
1a) and those for the free thiol (e.g., g2) decreased in intensity
within 5 min, and two new multiplets at 3.524 (peak 2a) and
3.037 (peak 2a′) ppm and resonances for free disulfide (GSSG,
peaks g2′) were observed. Peaks 2a and 2a′ reached their
maximum intensity after 11 min and were detectable for over 6
h. At this pH, no release of ethylenediamine was observed during
the reaction.5 At pH 3 and 276 K (Figure 1, B), the multiplet at
3.494 ppm (peak 2a) was clearly detectable after 11 min, together
with signals assignable to free disulfide (peaks g2′) and free
(4) 1H, 2D [1H, 15N] HSQC, and 2D [1H, 15N] HSQC-TOCSY NMR spectra
(TOCSY mixing time of 50 ms) were recorded on a Bruker DMX 500 (1H,
500 MHz; 15N, 50.7 MHz) NMR spectrometer using procedures similar to
those described previously: Berners-Price, S. J.; Frey, U.; Ranford, J. D.;
Sadler, P. J. J. Am. Chem. Soc. 1993, 115, 8649-8659. All experiments
described in the present paper were carried out in the absence of light.
(5) At pH 7, en CH2 peak at 3.263 ppm (298 K), 3.260 ppm (276 K).
(6) Reactions of [Pt(en)I2] (3) and N-Ac-Cys gave rise to Pt(II)-(N-Ac-
1
† The University of Edinburgh.
Cys) complexes, which have H (Figure S3) and 2D [1H, 15N] NMR spectra
‡ Universita¨t Regensburg.
(Figure S4) comparable to those of the end products observed for the reaction
of complex 1 with N-Ac-Cys (Figures S1 and S2A,B).
* To whom correspondence should be addressed.
(1) Beale, P. J.; Kelland, L. R.; Judson, I. R. Expert. Opin. InVest. Drugs
(7) The 15N chemical shifts of square-planar Pt-am(m)ine are diagnostic
of the trans ligand: Berners-Price, S. J.; Sadler, P. J. Coord. Chem. ReV. 1996,
151, 1-40.
1996, 5 (6), 681-693.
(2) (a) Bose, R. N.; Weaver, E. L. J. Chem. Soc., Dalton Trans. 1997,
1797-1799. (b) Talman, E. G.; Bru¨ning, W.; Reedijk, J.; Spek, A. L.;
Veldman, N. Inorg. Chem. 1997, 36, 854-861. (c) Shi, T.; Berglund, J.;
Elding, L. I. Inorg. Chem. 1996, 35, 3498-3503. (d) Gibbons, G. R.; Wyrick,
S.; Chaney, S. G. Cancer Res. 1989, 49, 1402-1407.
(3) Kratochwil, N. A.; Zabel, M.; Range, K. J.; Bednarski, P. J. J. Med.
Chem. 1996, 39, 2499-2507.
(8) The possibility that the intermediate is still a Pt(IV) species could be
ruled out because of the 15N shift in the 2D spectra indicating a Pt(II) species
and the immediate observation of disulfide (2e- oxidation step).
(9) Siebert, A. F. M.; Sheldrick, W. S. J. Chem. Soc., Dalton Trans. 1997,
1
385-393. [Pt(Hen-κN)(gly-met-κ3N,N′,S)]2+ at pH 2.4 has H NMR en CH2
signals at 2.92 and 3.15 ppm.
S0002-7863(98)00393-X CCC: $15.00 © 1998 American Chemical Society
Published on Web 08/01/1998

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8254 J. Am. Chem. Soc., Vol. 120, No. 32, 1998
Communications to the Editor
Figure 2. Platination of calf thymus DNA with trans,cis-[Pt(en)(OH)2I2]
(1, 7.5 µM) in the presence of glutathione (15 µM) (2). Conditions: 10
mM PIPES (pH 6.8), 0.250 mg L-1 calf thymus DNA, at 310 K in the
dark. Platinum bound to DNA was determined by AAS.3 No platination
was observed in the absence of GSH. In contrast, platination by [Pt(en)-
I2] (3) ([) is significantly slower.
In conclusion, our data enable us to postulate the existence of
a new mechanism for the reduction of complex 1 by biologically
important thiols such as GSH involving the formation of an
unexpected chelate ring-opened Pt(II) complex, even at pH 7.
Such a reduction produces Pt species which are capable of forming
DNA-Pt adducts in the presence of GSH much more rapidly
than [Pt(en)I2] (3) (Figure 2), which might be expected to be the
major product from reduction of complex 1 if the reaction simply
involved the loss of two axial ligands. Moreover, the Pt(IV)
complex 1 is highly cytotoxic to tumor cells in contrast to [Pt-
(en)I2] (3).3 The only report of a related reaction appears to be
that of Beattie et al.,14 who postulated that the ring-opened
complex [Pt(pn)(pnH)Cl]2+ (pn ) propylenediamine) was a
product from inner-sphere two-electron reduction of the Pt(IV)
complex cis-[Pt(pn)2Cl2]2+ by [Cr(H2O)6]2+, although the nature
of the product was investigated only by ion-exchange chroma-
tography and titration studies. Vorob’ev-Desyatovskii and Kuku-
shkin15 reported that during the reverse reaction (oxidation of Pt(II)
to Pt(IV)) certain electrophiles can oxidize a coordinated ligand
giving rise to a large trans effect and expulsion of the trans ligand.
The introduction of electron-transfer-driven trans effects into Pt
complexes should allow the generation in vivo of Pt(II) complexes
which are not simple analogues of the parent Pt(IV) prodrugs
and provide a novel concept for metallodrug design.
1
Figure 1. 500-MHz H NMR spectra recorded during the reduction of
trans,cis-[Pt(en)(OH)2I2] (1) by GSH in a 1:2 molar ratio: (A) pH 7, 276
K, after 5 min (200 µM GSH); (B) pH 3, 276 K, after 11 min (2 mM
GSH); (C) pH 3, 298 K, after 11 min (2 mM GSH). The two multiplets
at 3.037 (peak 2a′) and 3.524 ppm (peak 2a) in part A are assignable to
the en CH2 of a ring-opened intermediate (2). In B and C peak 2a′ is
overlapped with g2′; free en is labeled b, and the peak for 1 is at 2.8
ppm (peak 1a).
Scheme 1. Proposed Mechanism for the Reduction of
trans,cis-[Pt(en)(OH)2I2] (1) by Biologically Important Thiolsa
Acknowledgment. We thank the EC (Marie Curie Research Training
Grant No. ERB4001GT963865 to N.A.K. and COST Actions D1 and
D8), BBSRC, EPSRC, and DFG (Grant Be 1287/1-2) for their support
for this work.
Supporting Information Available: 500-MHz 1H and 2D [1H, 15N]
HSQC NMR spectra of reactions of complex 1 and [Pt(en)I2] (3) with
N-Ac-Cys and HPLC chromatogram and 2D NMR spectra of the final
products (4 pages, print/PDF). See any current masthead page for
ordering information and Web access instructions.
a The nature of the ligands Y in complex 2 is unknown, but strong
candidates are H2O/OH.
characterized by HPLC, 2D [1H, 15N] HSQC NMR, and APCI
mass spectroscopy, giving rise to complex 3 and the Pt(II) dimer,
[{Pt(en)(µ-SCys-N-Ac)}2]2+ (4) (Figures S1-S4).6 At pH 3,
however, ethylenediamine is easily released during the reduction
reaction and the later substitution products were not further
characterized, being of no biological relevance.
JA980393Q
(10) (a) Sulfenyliodides are very reactive and well-known in organic sulfur
chemistry: Field, L.; Vanhorne, J. L.; Cunningham, L. W. J. Org. Chem. 1970,
35, 3267-3273. (b) The complex {[(en)2Co(SCH2CH2NH2)]2I}5+ has been
characterized by X-ray analysis, can be viewed as containing a derivative of
a coordinated sulfenyliodide, and reacts with thiols to form disulfides: Nosco,
D. L.; Heeg, M. J.; Glick, M. D.; Elder, R. C.; Deutsch, E. J. Am. Chem. Soc.
1980, 102, 7786-7787.
On the basis of these results, a mechanism for the reduction
of complex 1 by GSH or N-Ac-Cys at neutral pH can be proposed
(Scheme 1), consisting of initial attack of the thiol on an iodide
ligand of the Pt(IV) complex forming a five-coordinated
transition state,10,11 which then undergoes a ring-opening reaction.
Attack on the highly reactive sulfenyliodide by another glutathione
molecule would give rise to the disulfide. The ring-opened Pt-
(II) species 2 can then undergo further ring-closure reactions and
react with the released I-, forming [Pt(en)I2] (3), and with RSH
or RSSR,12 giving [{Pt(en)(µ-SR)}2]2+(4).13 At pH 3, ethylene-
diamine is released, but at pH 7, no free ethylenediamine is
observed.
(11) (a) Goldberg, K. I.; Yan J.; Breitung, E. M. J. Am. Chem. Soc. 1995,
117, 6889-6896. (b) van Beek, J. A. M.; van Koten, G., Smeets, W. J. J.;
Spek, A. L. J. Am. Chem. Soc. 1986, 108, 5010-5011. (c) Byers, P. K.; Canty,
A. J., Crespo, M.; Puddephatt, R. J.; Scott, J. D. Organometallics 1988, 7,
1363-1367. Chelate ring opening on Pt(IV) prior to the reduction by the
thiol could also be proposed as an alternative mechanism to the Scheme 1
but seems rather unlikely.11a
(12) There is precedent for the reduction of disulfide bonds by Pt(II):
Lempers, E. L. M.; Inagaki, K.; Reedijk, J. Inorg. Chim. Acta 1980, 152,
201-207. Ohta, N.; Inagaki, K., Muta, H.; Yotsuyanagi, T.; Matsuo, T. Int.
J. Pharmaceutics 1998, 161 (1), 15-21.
(13) Appleton, T. G. Coord. Chem. ReV. 1997, 166, 313-359.
(14) Beattie, J. K.; Starink, J. Inorg. Chem. 1975, 14, 996-999.
(15) Vorob’ev-Desyatovskii, N. V.; Kukushkin, Yu. N. Zh. Obshch. Khim.
1991, 61, 2613-2623.