Melanocyte-Directed enzyme prodrug therapy (MDEPT): Development of second generation prodrugs for targeted treatment of malignant melanoma
Evaluation of second generation prodrugs for MDEPT, by oximetry, has highlighted structural properties that are advantageous and disadvantageous for efficient oxidation using mushroom tyrosinase. In particular, a sterically undemanding prodrug bis-(2-chloroethyl)amino-4-hydroxyphenylaminomethanone was synthesised and found to be oxidised by mushroom tyrosinase at a superior rate to tyrosine methyl ester, the carboxylic acid of which is the natural substrate for tyrosinase.
Introduction
Malignant melanoma continues to be a serious clinical problem on account of its increasing incidence, the relatively young population that it affects and the poor prognosis of the disseminated disease.1 The high mortality rate is a reflection of the failure of melanoma cells to respond to current cytotoxic treatment in the form of radiation and chemotherapy. Thus, there is an urgent need for improved treatment.
A selective strategy towards the treatment of malignant melanoma in the form of a melanocyte-directed enzyme prodrug therapy (MDEPT) has previously been reported by our group.2 Based on the unique occurrence of tyrosinase expression in melanocytes,3 MDEPT offers a highly selective drug delivery system. Therefore, with increased selectivity in the delivery of cytotoxic agents, intolerable side effects and the need for administration of large amounts of drugs should be minimised. Initial studies into MDEPT were carried out using prodrug 1 (Scheme 1). The proposed drug release mechanism, mediated by tyrosinase, is outlined in scheme 1.
Prodrug 1 can be viewed as a three component system, with a dopamine moiety acting as the tyrosinase substrate, a drug such as phenyl mustard, as the cytotoxic agent and a carbamate linkage coupling the two components together. In order to further develop this strategy, we have now prepared a more extensive range of compounds and examined their ability to be oxidised by mushroom tyrosinase. Since oxidation by tyrosinase is the first step in our proposed drug release mechanism, it is essential that good substrates for tyrosinase are identified at an early stage of our programme.
Three different cytotoxic agents were incorporated within the prodrugs, namely, phenyl mustard,4 a bis-ethyl amine mustard5 and daunomycin.6 All three of these cytotoxic agents have been previously used as anti-cancer drugs in the clinic.
Herein, we describe the synthesis and analysis of the prodrugs, with particular emphasis on their ability to act as substrates for tyrosinase, as determined by oximetry.
Section snippets
Synthesis of phenyl mustard prodrugs
The general protocol for the synthesis of the various prodrugs was based on the premise of forming a reactive carbonate that was prone to nucleophilic attack by a primary or secondary amine. Prodrugs with a carbamate linkage were synthesised as previously reported, and required access to the reactive carbonate intermediate 6. Facile preparation of p-hydroxyphenyl mustard hydrochloride Scheme 2, Scheme 3 was achieved by treating benzyloxyaniline 2 with ethylene oxide to give diol 3.7 Conversion
Conclusions
A second generation of prodrugs has been synthesised and the abilities of the individual prodrugs to act as substrates for tyrosinase have been examined. Prodrugs that closely resemble natural tyrosinase substrates were readily oxidised making them suitable MDEPT candidates. Functional group transformation, that is, carbamate to thiocarbamate resulted in a decrease in tyrosinase oxidation over 25 min incubation; Graph 1, PD(12) versus PD(25). Removal of steric bulk from the substrate's oxidative
Experimental
All NMR spectra were recorded on a Bruker WM250, Bruker AC250, Bruker Avance DPX 250, Bruker AMX400 or Jeol AX400 spectrometer, using CHCl3 as an internal standard unless stated otherwise (7.26 ppm for 1H NMR, 77.0 ppm for 13C NMR). 13C spectra were recorded using Distortionless Enhancement by Polarisation Transfer. Mass spectra were recorded on a Fisons VG Autospec. Infra-red spectra were recorded on a Perkin–Elmer Paragon 1000 FT-IR spectrometer. Optical activities were determined using a
Acknowledgements
We gratefully acknowledge Phairson Medical Ltd (Post-doctoral fellowship to H.M.), the BBSRC (Post-doctoral fellowship to A.M.J., grant no. 45/SBDO7534) and the Royal Society for their financial support of this work. We also gratefully acknowledge the Drug Synthesis & Chemistry Branch, Development Therapeutics Program, Division of Cancer Treatment, National Cancer Institute for the donation of daunomycin.
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Present address: Ribotargets Ltd, Granta Park, Abington, Cambridge CB1 6GB, UK.