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Chapter 3 Synthesis of C3′ and C4 modified paclitaxel analogs
Introduction to 2-methoxyestradiol (2ME2)
Methoxyestradiol (Figure 3.1) is an analog of the natural hormone estradiol with antitumor and antiangiogenic activity.1,2 It is a microtubule-targeting drug (MTD), in the same class as the taxanes and the vinca alkaloids, and is currently in phase II oncology clinical trials. Its binding location on β-tubulin is the same as that of colchicine,3 and it works as a microtubule depolymerizing agent, inducing mitotic arrest at the G2-M transition.4,5 Compared to other MTDs, 2ME2 is a promising drug with fewer side effects. It does not induce peripheral neuropathy and myelosuppression, which are the major limitations in the use of MTDs like paclitaxel. The reason for this is not quite clear, but recent research supports the idea that the insensitivity of β-tubulin isotype VI (Hβ1) to 2ME2 might contribute to that result.6 The fact that cells expressing Hβ1 are insensitive to 2ME2 also supports this idea. So far, at least seven classes of human β-tubulin isotypes have been characterized, but th function of them is not very clear.7,8 It is known that Hβ1 expression is confined to human hematopoietic tissues, especially those of peripheral blood and bone marrow.9,10,11 This suggests that its lack of interaction with Hβ1 may be the reason that 2ME2 is not myelosuppressive.
By comparing the difference between the structures of Hβ1 and those of other tubulin isotypes, it is believed that the change of amino acid 236 from Val to Ile is the key to its resistance to 2ME2. Val236 is a highly conserved residue for all tubulin isotypes except for class VI (Hβ1) tubulin, which instead has Ile236, and it resides in the 2ME2/colchicine binding site of tubulin. Besides Ile236, two other residues also have been changed in the 2ME2/colchicine binding site of Hβ1 tubulin, from Ile316 and Phe200 to Val316 and Tyr200. Our collaborator Dr. James Snyder has constructed a 3D model of the 2ME2/colchicine binding site, and this model shows that the substitution of these amino acids makes the 2ME2 binding pocket more sterically congested, reducing the binding ability of 2ME2 in Hβ1 tubulin.6,12
Design of new paclitaxel analogs to reduce paclitaxel’s myelosuppression
The purpose of our research is aimed at discovering paclitaxel analogs which can discriminate between Hβ1 tubulin and other β tubulin isotypes, enabling them to act as anticancer agents with reduced myelosuppressive activity.
Although paclitaxel is one of the most effective antitumor drugs, and has been widely used in treating breast and ovarian carcinomas,13,14 its use is limited by its adverse effects, including peripheral neuropathy and myelosuppression.15
Inspired by the example of 2ME2 explained above, we hypothesize that paclitaxel analogs may be designed which can discriminate between Hβ1 tubulin and other β tubulin isotypes, and thus do not induce myelosuppression. The paclitaxel binding pocket on β-tubulin has been modeled by Dr. Snyder, and it is known that Val23 and Ala231 in this pocket are replaced by Met23 and Leu231 in Hβ1 tubulin. In Dr. Snyder’s optimized model, paclitaxel’s benzamide phenyl ring fits snugly between Val23 and Ala231 in normal tubulin. But in Hβ1 tubulin, where these residues are replaced by the bulkier groups, Met23 and Leu231, there are additional steric constraints on the binding of paclitaxel (Figure 3.2). However, calculations provided by Dr. Snyder indicated that paclitaxel could still bind to Hβ1 tubulin because of the flexible nature of the binding site.6
Although paclitaxel does not discriminate between the different β tubulin isotypes, it is hypothesized that paclitaxel analogs with increased steric bulk will not bind as well to Hβ1 tubulin as to other tubulin isotypes.
Based on the model developed by Dr. Snyder, paclitaxel analogs with increase bulk at C3′ may contribute to a decreased ability to bind to Hβ1 tubulin. According to the SAR of paclitaxel,16 phenyl or a close analog is required at the C3′ position. However, Dr. Snyder’s calculations indicate that a benzofuran group may be as effective as a phenyl group for normal tubulins, but will decrease binding to Hβ1 tubulin. In addition, modification at the C4 position with bulkier acyl groups may also contribute to a reduction of the binding to Hβ1 tubulin without changing the binding to other tubulins. Based on these analyses, we decided to modify C3′ with benzofuran-2-yl and benzofuran-3-yl groups, and to modify C4 with longer chain acyl groups, such as pentanoyl and octanoyl groups.
Synthesis and biological evaluation of C3′ and C4 modified paclitaxel analogs
Synthesis of C3′ modified paclitaxel analogs (3.1a and 3.1b)
The first two paclitaxel analogs prepared were the C3′ modified analogs .
The retrosynthesis of compounds 3.1a and 3.1b is shown in Scheme 3.1. This synthesis uses Holton’s β-lactam method for attaching the side chain to the baccatin III core of paclitaxel, and it shows that two major fragments are necessary for the synthesis of 3.1a and 3.1b. One of them is the β-lactam part, and the other is the 7-triethylsilyl-10-acetyl-baccatin III.
The synthetic route for β-lactams are shown in Scheme 3.2 and 3.3. The synthesis of the β-lactams requires benzofuran-2-carbaldehyde or benzofuran-3-carbaldehyde as starting materials. Both benzofuran-2-carbaldehyde and benzofuran-3-carbaldehyde are commercially available, but the latter is expensive and not readily available. As a result, we elected to synthesize it from the commercially available compound 1-(2-hydroxyphenyl)ethanone by the literature method (Scheme 3.2).17
The β-lactams were then prepared by reaction of the aldehydes with p-anisidine to form imine intermediates, followed by reaction with acetoxyacetyl chloride to give the corresponding racemic β-lactams. These were resolved by treatment with PS-Amano lipase, which converts the racemic β-lactams to give the desired (+)-enantiomers (3.12a and 3.12b) and undesired (-)-alcohols. After separation of these products, the chiral β-lactams were hydrolyzed to secondary alcohols under basic conditions, followed by protection of the hydroxyl as its triisopropylsilyl ether. Cerium (IV) ammonium nitrate (CAN) was then used to deprotect the nitrogen, and finally the nitrogen was benzoylated to obtain the desired β-lactams 3.16a and 3.16 (Scheme 3.3).18
The synthesis of the other fragment, 7-triethylsilyl-10-acetyl-baccatin III, a known compound, started from the commercially available compound, 10-deacetyl-baccatin III. In the presence of the Lewis acid CeCl3, acetylation selectively occurred at the C10 position rather than the C7 position.19,20 Finally, protection of the C7 hydroxyl group as its triethylsilyl ether yielded the final product The coupling reactions between β-lactams (3.16a and 3.16b) and 7-triethylsilyl-10-acetyl-baccatin III (3.17) were carried out in the presence of th strong base LiHMDS at -45 oC. Deprotection of the silyl group by HF/pyridine yielded the final desired compounds 3.1a and 3.1b (Scheme 3.4).
Synthesis of the C3′ and C4 modified paclitaxel analogs 3.19a, 3.19b, 3.20a and 3.20b
The second group of compounds prepared composed the analogs 3.19a, 3.19b, 3.20a and 3.20b with modified substituents at the C3′ and C4 positions (Figure 3.4).
The synthesis of these compounds can be accomplished by the coupling of the β-lactams (3.16a and 3.16b) and suitable modified baccatin III derivatives. The synthetic routes are shown in Scheme 3.5.
The only difference between 3.26 and 3.30 is the nature of the substituted group at C4. Compound 3.26 has a pentanoyl group, while compound 3.30 has an octanoyl substitution. They were thus made by similar synthetic routes. The synthesis of 3.26 and 3.30 started from 10-deacetylbaccatin III. This was protected by triethylsilyl groups at C7, C10 and C13, followed protection at C1 by a dimethylsilyl ether group, and selective hydrolysis at C4 position by treatment with Red Al to give the 4-deacetyl derivative 3.22.21 Compound 3.22 was reacylated at C4 using either pentanoyl chloride or octanoyl chloride to yield the corresponding esters 3.23 and 3.27. Removal of the silyl groups occurred on the treatment with HF/pyridine. Selective acetylation at C10 as previously described and reprotection of the C7 hydroxyl group as its triethylsilyl ether gave the desired 4-acylbaccatin derivatives 3.26 and 3.30. The coupling of β-lactams (3.16a and 3.16b) and the baccatin cores (3.26 and 3.30) was accomplished by the treatment with LiHMDS. Deprotection with HF/pyridine then yielded the desired products 3.19a, 3.19b, 3.20a and 3.20b (scheme 3.6).
Abstract
Acknowledgment
1. Overview of Microtubules and Paclitaxel
1.1 Introduction to microtubules
1.2 Introduction to paclitaxel.
References
2. The design and synthesis of new antagonists for pro- inflammatory lipopolysaccharide ligands: C10-acyl-A-nor- paclitaxel derivatives
2.1 Introduction to inflammation
2.2 Lipopolysaccharide and inflammation
2.3 Paclitaxel and A-nor-paclitaxel, potential antagonists to LPS
2.4 Design and synthesis of C10 A-nor-paclitaxel analogs
2.5 Biological evaluations of all analogs and conclusion
2.6 Experimental section
References
3. Synthesis of C3′ and C4 modified paclitaxel analogs.
3.1 Introduction to 2-methoxyestradiol (2ME2)
3.2 Design of new paclitaxel analogs
3.3 Synthesis and biological evaluation of C3′ and C4 modified paclitaxel analogs
3.4 Biological evaluation of C3′ and C4 modified paclitaxel analogs
3.5 Experimental section
References
4. Appendix
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Synthesis of Paclitaxel Analogs
