The β-glucuronyl-based prodrug strategy allows for its application on β-glucuronyl-platinum conjugates

Article abstract of DOI:10.1016/j.bmcl.2004.06.015

The use of platinum drugs in antitumour therapy is well established. An important drawback of these chemotherapeutics is the lack of selectivity for tumour cells, usually resulting in severe toxic side effects. A glucuronyl-platinum conjugate was designed and synthesised to test the compatibility of platinum compounds with β-glucuronidase-based prodrug therapy. Instantaneous cleavage of the β-glucuronic bond in the glucuronyl-platinum conjugate was observed upon addition of β-glucuronidase resulting in Pt^II(dach)(4-hydroxybenzylmalonate) and glucuronic acid.

Full text of DOI:10.1016/j.bmcl.2004.06.015

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4273–4276
The b-glucuronyl-based prodrug strategy allows for its
application on b-glucuronyl-platinum conjugates
Reynier A. Tromp, Stella S. G. E. van Boom, C. Marco Timmers, Steven van Zutphen,
Gijsbert A. van der Marel, Herman S. Overkleeft, Jacques H. van Boom and Jan Reedijk*
Leiden Institute of Chemistry, Gorlaeus Laboratories, Leiden University, PO Box 9502, 2300 RA Leiden, The Netherlands
Received 22 January 2004; revised 12 May 2004; accepted 2 June 2004
Available online 2 July 2004
Abstract—The use of platinum drugs in antitumour therapy is well established. An important drawback of these chemotherapeutics
is the lack of selectivity for tumour cells, usually resulting in severe toxic side effects. A glucuronyl-platinum conjugate was designed
and synthesised to test the compatibility of platinum compounds with b-glucuronidase-based prodrug therapy. Instantaneous
cleavage of the b-glucuronic bond in the glucuronyl-platinum conjugate was observed upon addition of b-glucuronidase resulting in
PtII(dach)(4-hydroxybenzylmalonate) and glucuronic acid.
ꢀ 2004 Elsevier Ltd. All rights reserved.
Platinum cytostatics are recognised as an important
class of drugs in cancer chemotherapy.1;2 An important
drawback of these chemotherapeutics is the lack of
selectivity for tumour cells, usually resulting in severe
toxic side effects. Since the discovery of the antitumour
activity of cisplatin,3 a plethora of platinum derivatives
have been developed in the quest for a platinum drug
that, in comparison with cisplatin, has a broader activity
spectrum, can overcome drug resistance, has less toxic
side effects, and is better soluble in water. A number of
these platinum compounds overcome some of the limi-
tations of cisplatin.2;4 Most of the well-known platinum
anticancer complexes have the general formula cis-
PtX2(NHR2)2, in which R is an organic fragment and X
is a leaving group, such as chloride, or (chelating bis)-
carboxylate.5 Currently three drugs are used in routine
clinical treatments: cisplatin, carboplatin and oxalipla-
tin.6 Several others are under investigation in Phase-I
and Phase-II clinical trials.
example of a platinum complex designed for ADEPT, a
platinum-cephalosporine prodrug in combination with
prokaryotic b-lactamase, has been reported.11
In this study, the compatibility of platinum compounds
towards b-glucuronidase-based prodrug therapy has
been investigated. b-Glucuronidase has proven to be a
versatile activating enzyme in prodrug therapy.12 As an
endogenous enzyme, b-glucuronidase will not elicit
immune responses, which may appear upon application
of exogenous enzymes in ADEPT. Additionally, extra-
cellular b-glucuronidase is predominantly present in
necrotic areas in high local concentrations.13
The design of b-glucuronyl-platinum conjugate 12 is
based on the following considerations. The attachment
of a glucuronide unit to a platinum derivative greatly
enhances the solubility of the platinum compound in
aqueous media and probably leads to a reduced cell-
membrane permeability of the prodrug. Release of the
polar glucuronic acid moiety by b-glucuronidase affords
a neutral platinum species that can enter the tumour cell.
As diamine dicarboxylate complexes are potent plati-
num drugs,14–16 a functionalised platinum(II)(dach)
malonate complex was selected as the releasable moiety.
To avoid ineffective enzymatic hydrolysis as a result
of a too small distance between the glucuronide
and the platinum centre,17 an aromatic linker was
installed between the platinum and the glucuronic acid
moieties.
Selective generation of a cytotoxic drug from an inactive
prodrug is feasible with antibody-directed enzyme pro-
drug therapy (ADEPT), or with prodrug monotherapy
(PMT).7–10 To the best of our knowledge, only one
Keywords: Platinum; Anticancer drugs; Prodrug therapy; b-Glucuron-
idase; Glucuronide prodrugs; ADEPT; PMT.
* Corresponding author. Tel.: +31-71-5274459; fax: +31-71-5274671;
e-mail: reedijk@chem.leidenuniv.nl
0960-894X/$ - see front matter ꢀ 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.06.015

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R. A. Tromp et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4273–4276
Scheme 1. Reagents and conditions: (i) BzCl, pyridine, 97%; (ii) NBS, AIBN, CC14, reflux, 50%; (iii) diethyl malonate, KOtBu, 18-crown-6, THF,
67%; (iv) KOtBu, ethanol, 56%; (v) 3,3-dimethyldioxirane, acetone/CH2C12 (4/1, v/v); (vi) 5, KOtBu, 18-crown-6, i-PrOH/THF (1/1, v/v), 61% over
two steps; (vii) 20% Pd/C, H2, t-BuOH/H2O (1/1, v/v), 73%; (viii) 1 M NaOH, dioxane; (ix) NaBr, TEMPO, NaOC1, H2O, pH 10.8, 76% over two
steps; (x) PtII(dach)(NO3)2, H2O, 33%.
The assembly of glucuronyl-platinum conjugate 12 is
depicted in Scheme 1. The aglycone 5 was prepared from
4-cresol (1) in four steps. Benzoylation of 4-cresol (1)
gave compound 2 (97%). Reaction of 2 with N-bromo-
succinimide and a catalytic amount of AIBN in reflux-
ing tetrachloromethane yielded bromide 3 in 50% yield.
Dropwise addition of bromide 3 to the anion of dieth-
ylmalonate, prepared in situ with 1 equiv KOtBu in the
presence of a catalytic amount of 18-crown-6 in dry
THF, afforded malonate 4 in 67% yield. Removal of the
benzoyl protective group was effected with EtOH/KOt-
Bu to give aglycone 5 in 56% yield. Coupling of glucal 6
with glycosyl acceptor 5 was accomplished using a
modification of the two-step procedure for stereoselec-
tive generation of aryl b-glycosides described by Dani-
shefsky et al.18 Thus, a-1,2-anhydroglucoside 719
prepared by stereoselective oxidation of glucal 6 with
3,3-dimethyldioxirane20 was added to a solution of
aglycone 5, KOtBu (1 equiv) and 18-crown-6 (1 equiv) in
isopropanol/THF (1/1, v/v) to afford b-aryl glucoside 8
(61% over two steps). Use of KOtBu in the presence of
isopropanol resulted in transesterification, yielding iso-
propylester 8, instead of the corresponding ethyl ester.
The nature of the base and solvent in the glycosylation
reaction proved to be important, since the coupling did
not proceed with K2CO3 in refluxing acetone, as de-
scribed for other b-aryl glucosides.18
TEMPO (2,2,6,6-tetramethyl-1-piperidinyloxy, free
radical)21–23 with sodium hypobromite as the regenerat-
ing oxidant to give 11. b-Glucuronide 11 was purified by
gel filtration on a Fractogel TSK HW40 (S) column and
isolated in 34% overall yield, starting from glucal 6.
In the next stage, the target compound 12 was assembled
by reaction of PtII(dach)(NO3)2 with the malonate
function of glucuronide 11. Although chromatographic
purification of platinum complexes has been reported to
be problematic due to potential hydrolysis and side-
product formation in the dilute aqueous solutions used
as the mobile phase,24 PtII(glucuronide) complex 12 was
successfully purified by gel filtration on a Fractogel TSK
HW40 (S) column. The appropriate fractions were lyo-
philised immediately after collection. The structure of 12
was established by 1H and 195Pt NMR spectroscopy and
mass spectrometry.25
The enzymatic cleavage of the b-glucuronyl-platinum
conjugate (12) by b-glucuronidase (Scheme 2) was car-
ried out in the NMR tube26 at 37 ꢁC and pD 7.2. Com-
pound 12 was dissolved in a deuterated phosphate buffer
1
(pD 7.2) at 37 ꢁC. After recording the H NMR spec-
trum at 37 ꢁC, a b-glucuronidase buffer solution was
added. The H NMR spectrum was taken immediately
1
after addition. This revealed the virtually instantaneous
formation of glucuronic acid and platinum compound
13 (Fig. 1). The identity of the products formed after
enzymatic hydrolysis was confirmed by comparison with
glucuronic acid and a separately prepared synthetic
standard of platinum complex 13.27
Deprotection of 8 was effected by hydrogenolysis of the
benzyl groups and subsequent saponification of the
isopropyl ester functions to provide 10. The resulting
glucoside 10 was selectively oxidised at C-6 using

R. A. Tromp et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4273–4276
4275
glucuronide conjugates as prodrugs. Currently, we are
working on the next step towards clinically interesting
platinum prodrugs through developing b-glucuronyl-
platinum conjugates based on highly potent platinum
drugs in combination with traceless linkers.
Acknowledgements
We thank Dr. Daan Noort (TNO, The Netherlands) for
valuable suggestions and Ing. Hans van den Elst for
technical assistance. This work was supported by the
Netherlands Council for Chemical Research with finan-
cial aid from the Netherlands Organisation for Scientific
Research (NWO). We also thank Johnson & Matthey
(Reading, UK) for their generous loan of K2PtCl4. This
work has been performed under the auspices of the joint
BIOMAC Research Graduate School of Leiden Uni-
versity and Delft University of Technology.
Scheme 2. Reagents and conditions: Deuterated phosphate buffer
(pD 7.2), b-glucuronidase, 37 ꢁC.
References and notes
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83, 67.
11. Hanessian, S.; Wang, J. Can. J. Chem. 1993, 71, 896.
12. For a review on b-glucuronide-mediated drug release: de
Graaf, M.; Boven, E.; Scheeren, H. W.; Haisma, H. J.;
Pinedo, H. M. Curr. Pharm. Des. 2002, 8, 1391.
13. Bosslet, K.; Straub, R.; Blumrich, M.; Czech, J.; Gerken,
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Donner, A.; Williams, L.; Greene, G.; Burchenal, J. H.;
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42, 4831.
Figure 1. 1H NMR spectra at 37 ꢁC and pD 7.2 depicting (A) b-glu-
curonyl-platinum conjugate 12 in the absence of b-glucuronidase, (B)
b-glucuronidase-mediated release of platinum compound 13 with re-
17. de Groot, F. M.; Damen, E. W.; Scheeren, H. W. Curr.
Med. Chem. 2001, 8, 1093.
18. Dushin, R. G.; Danishefsky, S. J. J. Am. Chem. Soc. 1992,
114, 3471.
19. Halcomb, R. L.; Danishefsky, S. J. J. Am. Chem. Soc.
1989, 111, 6661.
lease of glucuronic acid ( ) and (C) glucuronic acid.
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20. Adam, W.; Bialas, L.; Hadjiarapoglou, L. Chem. Ber.
1991, 124, 2377.
21. Davis, N. J.; Flitsch, S. L. Tetrahedron Lett. 1993, 34, 1181.
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In conclusion, we have demonstrated for the first time,
that the b-glucuronidase-based prodrug therapy is fea-
sible for platinum complexes. The virtually instanta-
neous cleavage of the synthesised conjugate (12) by
b-glucuronidase demonstrates the potential of platinum-
23. de Nooy, A. E. J.; Besemer, A. C. Tetrahedron 1995, 51, 8023.

4276
R. A. Tromp et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4273–4276
24. Roberts, J. D.; Schmidt, W. L.; Tong, W. P.; Hacker, M.
P. Inorg. Chim. Acta 1988, 153, 123.
(6.78 mg, 50 · 10ꢁ3 mmol) in D2O, lyophilised three times
and dissolved in 2 mL of D2O. To 0.4 mg (ꢂ3720 units) b-
glucuronidase (9300 units mgꢁ1 solid for phenolphthalein
glucuronide) was added 0.3 mL of deuterated phosphate
buffer (pH 7.2, 50 mM). Glucuronyl-platinum complex 12
(1.74 mg, 2.44 · 10ꢁ3 mmol) was dissolved in 400 lL deu-
terated phosphate buffer (50 mM, pH 7.2) at 37 ꢁC. After
taking the 1H NMR spectrum at 37 ꢁC, 180 lL b-glucu-
ronidase buffer solution was added. The 1H NMR
spectrum was taken immediately.
25. Purification of platinum complex 12 was performed by gel
filtration on a Fractogel TSK HW40 (S) column, by elution
with triethylammonium hydrogencarbonate buffer (0.15 M
in methanol/water, 1/9, v/v) at a flow rate of 1.5 mL/min.
To avoid side product formation, the volatile mobile phase
was removed by lyophilisation to give 12 in 33% yield). 1H
NMR (300 MHz, deuterated phosphate buffer, pD 7.06) d
(relative to TMS) ¼ 1.09–1.25 (m, 4H, cyclohexane CH2),
1.51–1.53 (m, 2H, cyclohexane CH2), 1.96–2.00 (m, 2H,
cyclohexanyl Heq-3, -6), 2.17–2.35 (m, 2H, cyclohexane H-
1, H-2), 3.55–3.66 (m, 5H, H-2, H-3, H-4, CH2CH), 3.70–
3.76 (m, 1H, CH2CH), 3.78–3.88 (m, 1H, H-5), 5.06 (d, 1H,
J ¼ 6:8, H-1), 7.08 (d, 2H, J ¼ 8:6, CH arom), 7.27 (d, 2H,
J ¼ 8:6, CH arom). 195Pt NMR (300 MHz, D2O) d (relative
to K2PtCl4, external ref.) ¼ )1895. Calculated Mass: 714.
FAB: (M+H)þ ¼ 715, (M+Na)þ ¼ 737.
27. Reference compound 13 was prepared by reaction of
PtII(dach)(NO3)2 with bis-sodium 4-hydroxybenzylmalo-
1
nato (deprotected 5) in 41% yield. H NMR (deuterated
d
phosphate buffer, pH 7.06, 37 ꢁC)
(relative to
TMS) ¼ 1.01–1.29 (m, 4H, cyclohexane CH2), 1.49–1.63
(m, 2H, cyclohexane CH2), 1.92–2.08 (m, 2H, cyclohexa-
nyl Heq-3, -6), 2.22–2.42 (m, 2H, cyclohexane H-1, H-2),
3.52–3.62 (m, 2H, CH2 CH), 3.68–3.76 (m, 1H, CH2CH),
6.90 (d, 2H, J ¼ 7:1, CH arom), 7.24 (d, 2H, J ¼ 7:1, CH
arom). 195Pt NMR (300 MHz, deuterated phosphate
buffer) d (relative to K2PtCl4, external ref.) ¼ ꢁ1879.
Calculated Mass: 517. ESI/MS: (M+Na)þ ¼ 540.
26. b-Glucuronidase (EC 3.2.1.31 from Escherichia coli (type
X-A)) was purchased from Sigma Chemical Company.
Deuterated phosphate buffer was prepared by dissolving
Na2HPO4 (7.06 mg, 50 · 10ꢁ3 mmol) and KH2PO4