J. Med. Chem. 1998, 41, 2701-2708
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Synthesis and in Vitro Efficacy of Transferrin Conjugates of the Anticancer Drug Chlorambucil Ulrich Beyer,‡ Thomas Roth,‡ Peter Schumacher,‡ Gerhard Maier,‡ Anuschka Unold,‡ August W. Frahm,§ Heinz H. Fiebig,‡ Clemens Unger,‡ and Felix Kratz*,‡ Department of Medical Oncology, Clinical Research, Tumor Biology Center, Breisacher Strasse 117, 79106 Freiburg, Germany, and Chair of Pharmaceutical Chemistry, Faculty of Chemistry and Pharmacy, University of Freiburg, Hermann-Herder-Strasse 9, 79104 Freiburg, Germany Received July 16, 1997
One strategy for improving the selectivity and toxicity profile of antitumor agents is to design drug carrier systems employing soluble macromolecules or carrier proteins. Thus, five maleimide derivatives of chlorambucil were bound to thiolated human serum transferrin which differ in the stability of the chemical link between drug and spacer. The maleimide ester derivatives 1 and 2 were prepared by reacting 2-hydroxyethylmaleimide or 3-maleimidophenol with the carboxyl group of chlorambucil, and the carboxylic hydrazone derivatives 5-7 were obtained through reaction of 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde with the carboxylic acid hydrazide derivative of chlorambucil. The alkylating activity of transferrin-bound chlorambucil was determined with the aid of 4-(4nitrobenzyl)pyridine (NBP) demonstrating that on average 3 equivalents were protein-bound. Evaluation of the cytotoxicity of free chlorambucil and the respective transferrin conjugates in the MCF7 mammary carcinoma and MOLT4 leukemia cell line employing a propidium iodide fluorescence assay demonstrated that the conjugates in which chlorambucil was bound to transferrin through non-acid-sensitive linkers, i.e., an ester or benzaldehyde carboxylic hydrazone bond, were not, on the whole, as active as chlorambucil. In contrast, the two conjugates in which chlorambucil was bound to transferrin through acid-sensitive carboxylic hydrazone bonds were as active as or more active than chlorambucil in both cell lines. Especially, the conjugate in which chlorambucil was bound to transferrin through an acetaldehyde carboxylic hydrazone bond exhibited IC50 values which were approximately 3-18fold lower than those of chlorambucil. Preliminary toxicity studies in mice showed that this conjugate can be administered at higher doses in comparison to unbound chlorambucil. The structure-activity relationships of the transferrin conjugates are discussed with respect to their pH-dependent acid sensitivity, their serum stability, and their cytotoxicity. Introduction Chlorambucil (Leukeran) is a nitrogen mustard which is used clinically against chronic lymphatic leukemia, lymphomas, and advanced ovarian and breast carcinomas.1 The clinical application of this anticancer drug, which exhibits its cytotoxicity due to its alkylating properties, is, however, limited by its toxic side effects such as nausea, myelotoxicity, and neurotoxicity.2 One approach to overcome the toxicity of anticancer drugs to normal tissue is to attach cytotoxic drugs to suitable carrier proteins which accumulate in tumor tissue. Due to our interest in the role which human plasma proteins play in the in vivo distribution of anticancer drugs,3,4 we have developed chlorambucil conjugates of the serum protein transferrin, the iron(III) transport protein. Transferrin exhibits a significant uptake in tumor tissue due to high amounts of specific transferrin receptors (150 000-1 000 000/cell) on the cell surface of tumor cells.5,6 Antigenic heterogeneity or modulation is not present (in comparison to * To whom correspondence should be addressed. Tel: 0049-7612062176. Fax: 0049-761-2061899. E-mail:
[email protected]. ‡ Tumor Biology Center. § University of Freiburg.
tumor-associated antigens) since the transferrin receptor is essential for cell growth.7 Furthermore, transferrin has been used as a delivery system for toxins and DNA8 and is a stable, commercially available protein, which has been intensively studied and characterized.6 Chlorambucil was one of the first anticancer agents which was used to prepare antibody conjugates.9-11 However, in these reports9,10 chlorambucil was either physically adsorbed or chemically linked to the carrier by mere incubation or by direct coupling using Nhydroxysuccinimide/N,N′-dicyclohexylcarbodiimide (DCC).11 These direct methods have the disadvantage that during preparation polymeric products are likely to be formed and the resulting conjugates are chemically not well-defined with respect to the chemical link between drug and carrier protein. We therefore wanted to improve the coupling technique in order to prepare better defined protein conjugates of chlorambucil in which the stability of the bond between carrier protein and chlorambucil could also be varied. An effective method of preparing protein conjugates is to introduce a maleimide group into the drug, which is then able to bind selectively to sulfhydryl groups of carrier proteins through its carbon double
S0022-2623(97)00466-4 CCC: $15.00 © 1998 American Chemical Society Published on Web 06/25/1998
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Journal of Medicinal Chemistry, 1998, Vol. 41, No. 15
Scheme 1
bond.12 Recently, we developed a number of maleimide compounds for this purpose.13 Chlorambucil is a suitable agent for chemical modification due to the presence of only one carboxyl group in the molecule, and thus we synthesized five maleimide derivatives of chlorambucil which differed in the stability of the chemical link (aliphatic and aromatic ester or carboxylic hydrazone bond) between the drug and the spacer group. The rationale for varying the stability of the chemical link was to assess the significance of the pH-dependent stability of the link between drug and carrier for in vitro and in vivo activity. Transferrin is taken up by the cell through receptor-mediated endocytosis. During internalization the pH is reduced from 7.4 to 5.5-5.0, and this pH change can be exploited through acid cleavage of a predetermined breaking point so that the drug can be released inside the tumor cell.14 In this paper we report on the synthesis and characterization of chlorambucil-transferrin conjugates, their antiproliferative efficacy, and the relationship between pH-dependent stability, serum stability, and cytotoxicity. Chemistry Synthesis of Maleimide Derivatives of Chlorambucil. The route of preparing the maleimide ester derivatives of chlorambucil (1 and 2) is depicted in Scheme 1. The aliphatic and aromatic esters of chlorambucil were synthesized by reacting chlorambucil with an excess of 2-hydroxyethylmaleimide or 3-maleimidophenol in CH2Cl2 and addition of DCC or N-cyclohexyl-N′-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate and catalytic amounts of (dimethylamino)pyridine. The carboxylic hydrazone derivatives 5-7 were obtained by reaction of the acid hydrazide of chlorambucil
Beyer et al.
(prepared by reacting the acid chloride of chlorambucil with tert-butyl carbazate and subsequent cleavage with CF3COOH) with 2-maleimidoacetaldehyde, 3-maleimidoacetophenone, or 3-maleimidobenzaldehyde (see Scheme 2). Characteristic peaks of the introduced maleimide groups are singlets in the range from 6.8 to 7.2 ppm for the proton signals of the double bond in the 1H NMR spectra and at 134-135 and 169-170 ppm for the carbon atoms of the double bonds and carbonyl groups in the 13C NMR spectra. Furthermore, the proton signal of the carboxyl group (12.1 ppm in chlorambucil) is not present in the spectra of 1, 2, and 5-7. In the 1H NMR and 13C NMR spectra of 5-7 (recorded in CDCl3) only one set of signals is observed showing the presence of only one steresoisomer. From a steric point of view the E-isomer is favored, and we therefore tentatively suggest that this stereoisomer is present which is in accordance with studies on simple hydrazones.15 The molecular ion peak of 1, 2, and 5-7 was observed in the mass spectra (EI/CI, FAB) indicating that the chlorine atoms were not lost or substituted during synthesis which is also confirmed by elemental analysis (see the Experimental Section). In addition, we determined the alkylating activity of our derivatives with the aid of a nitrobenzylpyridine (NBP)-based assay according to Epstein et al.16 This reagent produces a violetcolored complex (565 ) 15 100 M-1 cm-1) with alkylating agents under basic conditions. Compounds 1, 2, and 5-7 exhibited 97-100% alkylating activity when compared to pure chlorambucil, which was set as the standard. Results and Discussion pH-Dependent Stability of 8-12. To assess the significance of pH-dependent stability of the chemical link between drug and protein for subsequent in vitro studies, we wanted to determine the stability of the ester and hydrazone links realized in 1, 2, and 5-7. Unfortunately, due to the rapid hydrolysis (detection of a number of peaks within a few hours with the aid of HPLC17) of the chlorine atoms of chlorambucil, aqueous stability studies of 1, 2, and 5-7 with respect to the chemical link between the maleimide spacer and chlorambucil could not be carried out. We therefore synthesized the model compounds 8-12 in which chlorambucil was substituted by 4-phenylbutyric acid and the maleimide spacer by ethanol, phenol, phenylacetaldehyde, acetophenone, and benzaldehyde, respectively18 (see Chart 1). 8 and 9 were synthesized according to the literature19 and 10-12 in analogy to that of 5-7 (synthesis and characterization data are available as Supporting Information). The pH-dependent stability of 8-12 was studied at pH values of 5.0 and 7.4 on a reverse-phase C18 HPLC column. The decrease of the peak areas of 8-12 in the chromatograms was used to calculate the respective half-lives. Whereas the half-lives of 8 and 9 were not reached after 4 days at either pH 5.0 or 7.4 (2.5 HS groups/protein and less than 10% of chlorambucil with respect to the amount of protein present demonstrating that chlorambucil does not couple to thiolated proteins under these experimental conditions. (22) Stability studies carried in 10% FCS did not differ significantly from those performed in 10% human serum. (23) Grunicke, H.; Doppler, W.; Hofmann, J.; Lindner, H.; Maly, K.; Oberhuber, H.; Ringsdorf, H.; Roberts, J. J. Plasma Membrane as Target of Alkylating Agents. Adv. Enzyme Regul. 1985, 24, 247-261. (24) We confirmed a pH-dependent stability of the transferrin conjugates through the following experiment: T-1, T-2, T-5, T-6, and T-7 were incubated at pH 7.4 and 5, and their timedependent stability was determined by HPLC (Biosil SEC 250 column) over a period of 6 days. Chlorambucil or hydrolysis products such as chlorambucil hydrazide are seen on this column at around 10.5-11.0 min (detection at 260 nm). When the acidsensitive conjugates T-5-T-7 were incubated at pH 5.0, they showed a distinct peak at 10.5 min with time which steadily increased in size during the course of the experiment in the order T-5 > T-6 > T-7. At pH 7.4, however, only very small peaks were observed after 6 days of incubation. The appearance of the peak at 10.5 min at pH 5 was especially pronounced in T-5 which is the conjugate with the highest in vitro activity. The conjugates T-1 and T-2 showed only a small peak at 10.5-11.0 min at both pH-values. (25) Greenfield, R. S.; Kaneko, T.; Firestone, R. A. (6-Maleimidocaproyl)hydrazone of Doxorubicin - a New Derivative for the Preparation of Immunoconjugates of Doxorubicin. Bioconjugate Chem. 1993, 4, 521-527. (26) Kratz, F.; Beyer, U.; Kru¨ger, M.; Schumacher, P.; Zahn, H.; Roth, T.; Fiebig, H. H.; Unger, C. Synthesis of New Maleimide Derivatives of Daunorubicin and Biological Activity of Acid Labile Transferrin Conjugates. Biorg. Med. Chem. Lett. 1997, 7, 617-622. (27) Fichtner, I.; Beyer, U.; Falken, U.; Meyer, G.; Schumacher, P.; Unger, C.; Kratz, F. Efficacy of Acid Labile Transferrin and Albumin Doxorubicin Conjugates in In Vitro and In Vivo Breast Carcinoma Systems. 2nd International Symposium on Polymer Therapeuticals, Kumamoto, Japan, 1997. (28) Kratz, F.; Beyer, U.; Roth, T.; Tarasova, N.; Collery, P.; Lecheneault, F.; Cazabat, A.; Schumacher, P.; Unger, C.; Falken, U. J. Pharm. Sci. 1998, 87, 338-346. (29) Ellmann, G. L. Tissue Sulfhydryl Groups. Arch. Biochem. Biophys. 1959, 82, 70-77. (30) Dengler, W. A.; Schulte, J.; Berger, D. P.; Mertelsmann, R.; Fiebig, H. H. Development of a Propidium Iodide Fluorescence Assay for Proliferation and Cytotoxicity Assays. Anti-Cancer Drugs 1995, 6, 522-532.
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