Total Synthesis and Biological Assessment of Cyclopropane-Based Epothilone Analogues - Modulation of Drug Efflux through Polarity Adjustments

Abstract

Benzimidazole-based analogues of epothilones containing a cyclopropane ring in place of the natural epoxide moiety have been prepared based on the selective cyclopropanation of homo­allylic alcohol intermediates as one of the key steps. In contrast to the epoxide-containing parent compounds the cyclopropane analogues are not or only minimally susceptible to P-gp170-mediated drug efflux.

antitumor agents - epothilones - total synthesis - natural products - stereoselectivity

References and Notes

<A NAME="RG03606ST-1">1</A>

Current address: Prestwick Chemical, Illkirch, France.

<A NAME="RG03606ST-2">2</A>

Current address: AMCIS AG, Bubendorf, Switzerland.

For recent reviews on the chemistry and biology of epothilones, see, for example:

<A NAME="RG03606ST-3A">3a</A> Höfle G. Reichenbach H. In Anticancer Agents from Natural Products   Cragg GM. Kingston DGI. Newman DJ. CRC Press LLC; Boca Raton FL: 2005.  p.413-450  

<A NAME="RG03606ST-3B">3b</A> Altmann K.-H. Curr. Pharm. Des.  2005,  11:  1595 

<A NAME="RG03606ST-3C">3c</A> Altmann K.-H. Org. Biomol. Chem.  2004,  2137 

<A NAME="RG03606ST-3D">3d</A> Borzilleri RM. Vite GD. Drugs Future  2003,  27:  1149 

<A NAME="RG03606ST-3E">3e</A> Harris CR. Danishefsky SJ. J. Org. Chem.  1999,  64:  8434 

<A NAME="RG03606ST-3F">3f</A> Nicolaou KC. Roschangar F. Vourloumis D. Angew. Chem. Int. Ed.  1998,  37:  2014 

<A NAME="RG03606ST-4">4</A> Bollag DM. McQueney PA. Zhu J. Hensens O. Koupal L. Liesch J. Goetz M. Lazarides E. Woods CM. Cancer Res.  1995,  55:  2325 

<A NAME="RG03606ST-5">5</A> Hardt IH. Steinmetz H. Gerth K. Sasse F. Reichenbach H. Höfle G. J. Nat. Prod.  2001,  64:  847 

For some recent examples, see:

<A NAME="RG03606ST-6A">6a</A> Nicolaou KC. Pratt BA. Arseniyadis S. Wartmann M. O’Brate A. Giannakakou P. ChemMedChem  2006,  1:  41 

<A NAME="RG03606ST-6B">6b</A> Yoshimura F. Rivkin A. Gabarda AE. Chou T.-C. Dong H. Sukenick G. Morel FF. Taylor RE. Danishefsky SJ. Angew. Chem. Int. Ed.  2003,  42:  2518 

<A NAME="RG03606ST-6C">6c</A> Nicolaou KC. Sasmal PK. Rassias G. Reddy MV. Altmann K.-H. Wartmann M. O’Brate A. Giannakakou P. Angew. Chem. Int. Ed.  2003,  42:  3515 

<A NAME="RG03606ST-6D">6d</A> Biswas K. Lin H. Njardson JT. Chappell MD. Chou T.-C. Guan Y. Tong WP. He L. Horwitz SB. Danishefsky SJ. J. Am. Chem. Soc.  2002,  124:  9825 

<A NAME="RG03606ST-7A">7a</A> Cachoux F. Isarno T. Wartmann M. Altmann K.-H. ChemBioChem  2006,  7:  54 

<A NAME="RG03606ST-7B">7b</A> Cachoux F. Isarno T. Wartmann M. Altmann K.-H. Angew. Chem. Int. Ed.  2005,  44:  7469 

<A NAME="RG03606ST-7C">7c</A> Altmann K.-H. Bold G. Caravatti G. Flörsheimer A. Guagnano V. Wartmann M. Bioorg. Med. Chem. Lett.  2000,  10:  2765 

<A NAME="RG03606ST-8">8</A>

The clogP values for the most relevant compounds are as follows: Epo A, 3.256; Epo B, 3.703; 3, 2.871; 1, 4.154 (calculated with the Molinspiration Desktop Property Calculator from Molinspiration Cheminformatics: http://www.molinspiration.com/docu/mipc/index.html).

<A NAME="RG03606ST-9A">9a</A> Nicolaou KC. Namoto K. Ritzen A. Ulven T. Shoji M. Li J. D’Amico G. Liotta D. French CT. Wartmann M. Altmann K.-H. Giannakakou P. J. Am. Chem. Soc.  2001,  123:  9313 

<A NAME="RG03606ST-9B">9b</A> Nicolaou KC. Namoto K. Li J. Ritzen A. Ulven T. Shoji M. Zaharevitz D. Gussio R. Sackett DL. Ward RD. Hensler A. Fojo T. Giannakakou P. ChemBioChem  2001,  2:  69 

<A NAME="RG03606ST-9C">9c</A> Johnson J. Kim SH. Bifano M. DiMarco J. Fairchild C. Gougoutas J. Lee F. Long B. Tokarski J. Vite G. Org. Lett.  2000,  2:  1537 

<A NAME="RG03606ST-10">10</A> Charette AB. Francoeur S. Martel J. Wilb N. Angew. Chem. Int. Ed.  2000,  39:  4539 

<A NAME="RG03606ST-11A">11a</A> Mende U. Radüchel B. Skuballa W. Vorbrüggen H. Tetrahedron Lett.  1975,  9:  629 

<A NAME="RG03606ST-11B">11b</A> Kottwitz J. Vorbrüggen H. Synthesis  1975,  636 

<A NAME="RG03606ST-12A">12a</A> Lorenz JC. Long J. Yang Z. Xue S. Xie Y. Shi Y. J. Org. Chem.  2004,  69:  327 

<A NAME="RG03606ST-12B">12b</A> Yang Z. Lorenz JC. Shi Y. Tetrahedron Lett.  1998,  39:  8621 

<A NAME="RG03606ST-13">13</A> For other examples of stereoselective cyclopropanations of homoallylic alcohols see: Mohr P. Tetrahedron Lett.  1995,  36:  7221 

<A NAME="RG03606ST-14">14</A>

The isomer ratio was determined by 400 MHz ¹H NMR spectroscopy in benzene-d ₆ and is based on the ratio of integrals observed for the signals of the isolated aromatic proton of the benzimidazole moiety (i.e., the proton ortho to the unsubstituted nitrogen of the imidazole ring). No signal splitting was observed for any other well-resolved signal.

The difference in biological activity between 1 and 1a provides a reasonably solid basis for the stereochemical assignment of the cyclopropane moiety. For cis-epoxide-based epothilone analogues, we have always observed the natural epoxide stereochemistry to be associated with substantially higher biological activity (unpublished data; see also ref. 15a). In addition, (12S,13S)-trans-Epo A (stereochemistry corresponding to 2) is >500-fold more potent than the corresponding (12R,13R)-isomer.^15b Lastly, Nicolaou et al. have reported the synthesis of the cyclopropane-based analogue of (12R,13R)-trans-Epo A,^15c which likewise showed poor biological activity.

<A NAME="RG03606ST-15A">15a</A> Nicolaou KC. Vourloumis D. Li T. Pastor J. Winssinger N. He Y. Ninkovic S. Sarabia F. Vallberg H. Roschangar F. King NP. Finlay MR. Giannakakou P. Verdier-Pinard P. Hamel E. Angew. Chem., Int. Ed. Engl.  1997,  36:  2097 

<A NAME="RG03606ST-15B">15b</A> Altmann K.-H. Bold G. Caravatti G. Denni D. Flörsheimer A. Schmidt A. Rihs G. Wartmann M. Helv. Chim. Acta  2002,  85:  4086 

<A NAME="RG03606ST-15C">15c</A> Nicolaou KC. Finlay MR. Ninkovic S. King NP. He Y. Li T. Sarabia F. Vourloumis D. Chem. Biol.  1998,  5:  365 

<A NAME="RG03606ST-16">16</A> Inanaga J. Hirata K. Saeki H. Katsuki T. Yamaguchi M. Bull. Chem. Soc. Jpn.  1979,  52:  1989 

<A NAME="RG03606ST-17">17</A> Confer, for example: Paterson I. De Savi C. Tudge M. Org. Lett.  2001,  3:  3149 

<A NAME="RG03606ST-18">18</A>

Preparation of 11.
To a solution of Et₂Zn (3.38 mmol) in 5 mL of CH₂Cl₂ and 3.38 mL of hexane (obtained through addition of 3.38 mL of 1 M Et₂Zn in hexane to 5 mL of CH₂Cl₂), CF₃CO₂H (0.259 mL, 3.38 mmol) in 2 mL of CH₂Cl₂ was added dropwise at -13 °C over a period of 10 min. The reaction mixture was stirred at -13 °C for 15 min followed by dropwise addition of a solution of CH₂I₂ (0.273 mL, 3.38 mmol) in 2 mL of CH₂Cl₂. After additional 30 min at -13 °C, a solution of 10 (0.28 g, 0.375 mmol) in 2 mL of CH₂Cl₂ was added dropwise and the reaction mixture was stirred for 20 min at -13 °C. The reaction was then quenched with sat. aq NH₄Cl, the organic layer was separated, and the aqueous solution was extracted with three 20 mL portions of CH₂Cl₂. The combined organic extracts were dried (MgSO₄) and concentrated in vacuo. Purification by flash column chromatography (hexane-acetone 1:1) afforded 220 mg (77%) of the title compound.
¹H NMR (400 MHz, CD₃OD): δ = 7.58 (s, 1 H, H_arom), 7.42 (d, 1 H, H_arom), 7.30 (d, 1 H, H_arom), 4.67 (m, 1 H, CHOH), 4.38 (m, 1 H, CHOTBS), 3.81 (s, 3 H, CO₂CH ₃), 3.77 (m, 1 H, CHOTBS), 3.64 (s, 3 H, NCH ₃), 3.30 (m, 1 H), 3.20 (m, 1 H, CH₃CH), 2.70 [s, 3 H, CH ₃ (benzimidazole)], 2.45 (m, 1 H, CH ₂CO₂Me), 2.25 (m, 1 H, CH ₂CO₂Me), 1.60 (m, 2 H), 1.40 (m, 4 H), 1.22 (s, 3 H, gem-CH ₃), 1.07 (s, 3 H, gem-CH ₃), 1.05 (d, 3 H, CH ₃CHCO), 0.92 (d, 3 H, CH ₃CH), 0.91 (s, 18 H, t-BuSi), 0.32 (m, 2 H, H-cyclopropane), 0.15 (s, 3 H, CH ₃Si), 0.08 (s, 3 H, CH ₃Si), 0.05 (s, 3 H, CH ₃Si), 0.02 (s, 3 H, CH ₃Si). ESI-MS: m/z = 759.2 [M + H]⁺. HRMS (MALDI): m/z calcd for C₄₂H₇₄N₂O₆Si₂: 759.5188; found: 759.5142 [M + H]⁺.
Only one set of signals was observed in the NMR spectrum of 11 either in CD₃OD or C₆D₆. It is not clear, however, whether the presence of a mixture of isomers would have been reflected in the doubling of at least some of the NMR peaks at this stage. Nevertheless, 11 can be inferred to contain at most trace amounts of the undesired cyclopropane isomer based on the NMR analysis of the derived cyclization products 2 as well as its protected precursor [2-(TBS)₂], for both of which the occurrence of separate sets of signals for the two cyclopropane isomers had been established in the cyclopropanation experiments with 5 (Scheme [1] ). Only one set of signals was observed for either 2 or 2-(TBS)₂.

<A NAME="RG03606ST-19">19</A>

Preparation of 2.
To a solution of 2-(TBS)₂ (70 mg, 0.096 mmol) in 4 mL of MeCN in a Teflon tube was added 1 mL of HF·pyridine (70:30) at r.t. and the reaction mixture was stirred at r.t. for 2 h. It was then washed with 5% aq NaHCO₃ (pH 5), followed by extraction with three 15 mL portions of EtOAc. The combined organic extracts were dried (MgSO₄) and the solvent was evaporated to give 40 mg (83%) of crude 2. This material was further purified by preparative HPLC to afford 20 mg (42%) of pure title compound.
¹H NMR (400 MHz, CD₃OD): δ = 7.60 (s, 1 H, H_arom), 7.38 (m, 1 H, H_arom), 7.28 (m, 1 H, H_arom), 5.97 (m, 1 H, PhCHO), 4.30 (m, 1 H, CHOH), 3.92 (m, 1 H, CHOH), 3.70 (s, 3 H, NCH ₃), 3.30 (m, 1 H, CH₃CH), 2.60 [s, 3 H, CH ₃ (benzimidazole)], 2.42 (m, 1 H, CH ₂CO₂), 2.10 (m, 1 H, CH ₂CO₂H), 1.60 (m, 2 H), 1.40 (m, 1 H), 1.37 (s, 3H, gem-CH ₃), 1.20 (d, 3 H, CH ₃CHCO), 1.01 (s, 3 H, gem-CH ₃), 0.99 (d, 3 H, CH ₃CH), 0.80 (m, 1 H), 0.60 (m, 1 H), 0.22 (m, 2 H, H-cyclopropane). ESI-MS: m/z = 498.6 [M + H]⁺. HRMS (MALDI): m/z calcd for C₂₉H₄₂N₂O₅: 499.3167; found: 499.3159 [M + H]⁺.

<A NAME="RG03606ST-20">20</A>

The IC₅₀ values ± SD (three independent experiments) for compounds 1 and 2 are as follows: KB-31 cells: 1, 0.48 ± 0.03 nM; 2, 0.17 ± 0.04 nM. KB-8511 cells: 1, 0.96 ± 0.06 nM; 2, 0.13 ± 0.02 nM. The IC₅₀-values for 3 and 4 against KB-31/KB-8511 cells are 0.59/6.62 nM and 0.21/1.02 nM, respectively.^7a The corresponding values for 1a are 26.9 ± 5.3 nM (KB-31) and 35.9 ± 6.9 nM (KB-8511).