Synthesis and biological activities of ferrocenyl derivatives of paclitaxel

Damian Plażuk; Anna Wieczorek; Andrzej Błauż; Błażej Rychlik

doi:10.1039/C2MD00315E

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DOI: 10.1039/C2MD00315E (Concise Article) Med. Chem. Commun., 2012, 3, 498-501

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Synthesis and biological activities of ferrocenyl derivatives of paclitaxel†

Damian Plażuk *^a, Anna Wieczorek ^a, Andrzej Błauż ^b and Błażej Rychlik ^b
^aDepartment of Organic Chemistry, Faculty of Chemistry, University of Łódź, Tamka 12, 91-403 Łódź, Poland. E-mail: damplaz@uni.lodz.pl; Fax: +48 42 6786583; Tel: +48 42 6355760
^bCytometry Lab, Department of Molecular Biophysics, Faculty of Biology and Environmental Protection, University of Łódź, 12/16 Banacha St., 90-237 Łódź, Poland

Received 22nd December 2011 , Accepted 9th February 2012

First published on 10th February 2012

Abstract

A series of ferrocenyl taxoids were prepared by acylation of paclitaxel and docetaxel with ferrocenecarboxylic acid and 3-ferrocenoylpropionic acid in good yield. The prepared compounds showed high activities against multidrug-resistant colon adenocarcinoma cell lines.

The antineoplastic agents paclitaxel 1 (Taxol^®) and docetaxel 2 (Taxotere^®) (Fig.1) promote and stabilise microtubule polymerisation and, consequently, the death of a cell.¹ These drugs are widely used in therapies for many types of cancer disease. The structural modification of taxoids (e.g., paclitaxel) has influence on their particular anticancer activities, and this has subsequently generated great interest.^2–4 It has been reported that the replacement of the phenyl group at the C-10 position in the Taxol^® by an alkenyl or alkyl group brings about 1–2 orders of magnitude higher potency against drug-resistant cancer cell lines.^5,6 Bradley et al.⁷ have studied the influence of conjugation of natural fatty acids to paclitaxel. They demonstrated that conjugation of paclitaxel with acids increases antitumor activity in mice when compared to paclitaxel. Subsequent studies by Ojima et al.⁸ showed that polyunsaturated acids (such as docosahexaenoic acid, linolenic acid, and linoleic acid) linked to the C-2′ position of Taxol^® have exhibited strong activity against drug-resistant colon cancer and drug-sensitive ovarian cancer in mice.


	Fig. 1 Structures of paclitaxel 1 and docetaxel 2.

Organometallic compounds are among the promising drug-candidates with strongly enhanced anticancer activities. Ferrocene, one of the metallocenes, is the most promising organometallic compound. It is a redox active, stable in biological media, and nontoxic molecule. Ferrocene has shown only a little cytotoxic activity. Ferrocene, when conjugated to biologically active compounds (e.g., adenine,^9,10 peptides,^11,12 nucleoside and nucleobases,^13,14 vitamins^15,16 and phenols¹⁷), provides some promising anticancer agents. In recent years, many ferrocenyl compounds have been prepared which exhibit significant cytotoxicity^18–24 and antimalarial activities.²⁵ The mechanism of biological activity of ferrocenyl compounds (e.g., ferrocifene), proposed by Jaouen et al., is based on the oxidation of the ferrocene moiety in the living cell to the cytotoxic ferrocenium cation.^16,17,19 However, introduction of a ferrocenyl unit into a biologically active compound will not always produce cytotoxic compounds.²⁶

Herein, we describe the synthesis and biological properties of the first example of ferrocenylated taxanes (derivatives of paclitaxel and docetaxel). The desired compounds were prepared by acylation of 1 and 2 with ferrocenic acids. A representative example appears in Scheme 1.


	Scheme 1 Synthesis of the ferrocenylated paclitaxel 3.

A series of four ferrocenyl esters of Taxol^® and docetaxel were prepared this way. The reactions of ferrocenecarboxylic acid and 3-ferrocenoylpropionic acid with 1 or 2 were carried out at 0 °C in the presence of 2 eq. of DIC and DMAP, and produced a good yield of the corresponding esters. Due to the higher reactivity of the hydroxyl group at the C-2′ position in 1, we observed formation of the desired compounds as the main or sole products. The structures of the new taxoids were confirmed by NMR spectroscopy and MS spectra (¹H and ¹³C and MALDI spectra appear in the ESI†). Isolated yields of the products are as follow: 67%, 3, 69%, 4, 66%, 5, and 69%, 6. Structures of the prepared compounds are presented in Fig. 2.


	Fig. 2 Structures of the prepared ferrocenyl taxoids 3–6.

As taxanes are good substrates of ABCB1 protein (AKA MDR1 or Pgp), we decided to check the activity of newly synthesised compounds against drug-sensitive and differentially ABCB1 expressing cells. We chose a panel of multidrug-resistant (MDR) cell lines originating from the human colon adenocarcinoma SW620 line. The MDR cell lines were obtained by the stepwise drug selection of the sensitive mother cell line with four different chemotherapeutics, i.e., doxorubicin (D), etoposide (E), methotrexate (M) and vincristine (V). The MDR cell lines were fully characterised with regards to drug cross-resistance, different ABC transporter protein expression, and their subcellular localisation and activity (Błauż et al., in preparation). The pattern of ABCB1 overexpression amongst the employed cell lines is as follows: SW620V ≥ SW620D > SW620E ≫ SW620M = SW620 (ibidem). Experimental details appear in the ESI†. The summarised cytotoxic activities of the prepared compounds are listed in Table 1.

Table 1 Cytotoxic activities (IC₅₀ [μM], extrapolated values are given in parentheses whenever possible to calculate) of ferrocenyl taxanes 3–6 against the colon adenocarcinoma SW620 cell line panel (SW620D—doxorubicin-resistant, SW620E—etoposide-resistant, SW620M—methotrexate-resistant, and SW620V—vincristine resistant)

Compound	SW620	SW620D	SW620E	SW620M	SW620V
Paclitaxel 1	8.69	>30 (2180)	>30 (247)	6.38	>30 (3040)
3	3.07	>30 (172)	>30 (358)	5.96	NA
4	0.581	NA	NA	0.701	NA
Docetaxel 2	0.283	35.16	21.27	0.259	20.32
5	0.670	16.5	>30 (47.7)	3.35	>30 (108)
6	0.388	>30 (544)	1.56	2.81	>30 (200)

The results of viability of SW620 panel cell lines in the presence of 1 and 3 and 4 are shown in Fig. 3. The introduction of a ferrocenyl group into the C-2′ position for 1 increased the cytotoxic activity against the SW620 cancer cell line 2.8 fold for 3, and 15 fold for 4, compared to paclitaxel. The results of viability of the SW620 and SW620M cell lines in the presence of 3 and 4 are shown in Fig. 4. Moreover, in the case of the doxorubicin-resistant SW620D cell line, the activity of 5 was 13 fold higher compared to paclitaxel and 2.1 times higher when compared to docetaxel. Whereas, paclitaxel 3 and 4 were not active against the etoposide-resistant SW620E cell line, docetaxel showed a moderate activity at 21.27 μM, while 6 (IC₅₀ = 1.56 μM) is 13 times more active than docetaxel and 158 times more active than paclitaxel. The results of viability for the SW620D and SW620E cell lines in the presence of 2, 5 and 6 are detailed in the ESI† (Fig. S7 and S8).


	Fig. 3 Viability of the SW620 cell line panel in the presence of 1, 3 and 4. Data are mean ± SEM, n = 3.


	Fig. 4 Viability of cell lines in the presence of 3 and 4: (a) SW620 and (b) SW620M. Data are mean ± SEM, n = 3.

Fig. 3 and 4 indicated that compounds 3 and 4 were most active against SW620 and SW620M, i.e. ABCB1 non-expressing cancer cell lines, whereas, the ABCB1 positive cell lines were almost insensitive to 3 and 4. Little activity for 3 was observed in the case of the SW620V cell line (Fig. S1, ESI†). Compounds 5 and 6 showed similar, although lower than docetaxel, activity on the SW620 cell line (Fig. S2, ESI†). It was interesting to note that only 5 (IC₅₀ = 16.5 μM) is 2.1 times more active against the SW620D line, whereas, 6 (IC₅₀ > 30 (544) μM) was non-active up to 1 μM concentration (viability of cells was higher than 95%) (Fig. S3, ESI†). The positive impact of 5 on ABCB1-expressing cells was probably diminished by the presence of ABCG2 (another MDR protein), the expression of which was much higher in SW620V than SW620D cells. Summarised results for the viability of cancer cells appear in the ESI†.

We also checked the affinity of the ferrocenyl taxanes to porcine brain tubulin. The preliminary results indicated that although the mechanism for cytotostatic action of ferrocenyl compounds was the inhibition of microtubule depolymerisation, their affinity to tubulin was much lower compared to paclitaxel and docetaxel.

In conclusion, taxanes bearing ferrocenyl moiety coupled to the C-2′ position have been prepared. The ferrocenyl group was introduced by selective esterification of paclitaxel and docetaxel by ferrocenecarboxylic acid and 3-ferrocenoylpropionic acid. Prepared compounds exhibited cytotoxicity much higher than paclitaxel against SW620 and methotrexate-resistant SW620M cell lines and higher than docetaxel in the case of doxorubicine-resistant SW620D and etoposide-resistant SW620E cell lines. Compound 4 exhibited at least one order of magnitude higher in toxicity towards ABCB1-naive cells than paclitaxel. Compound 5 seems to be able to overcome the ABCB1-based drug resistance mechanism, whereas, the other compounds were not active. It is worth noting that 6 was the only active compound against the moderately ABCB1-expressing SW620E cells, at a concentration as low as 1.56 μM. A more detailed study to determine the mechanism of biological activity of ferrocenyl taxanes is currently under way.

The authors are grateful for the financial support of the Polish Ministry of Science and Higher Education (grant Iuventus Plus—financial support for years 2010–2011). The SW620 drug resistance cell panel was developed within MEDITRANS, an integrated project funded by the European Commission under the Sixth Framework Programme (NMP4-CT-2006-026668).

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c2md00315e

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