C6-C8 bridged epothilones: consequences of installing a conformational lock at the edge of the macrocycle

Abstract

A series of conformationally restrained epothilone analogues with a short bridge between the methyl groups at C6 and C8 was designed to mimic the binding pose assigned to our recently reported EpoA-microtubule binding model. A versatile synthetic route to these bridged epothilone analogues has been successfully devised and implemented. Biological evaluation of the compounds against A2780 human ovarian cancer and PC3 prostate cancer cell lines suggested that the introduction of a bridge between C6-C8 reduced potency by 25-1000 fold in comparison with natural epothilone D. Tubulin assembly measurements indicate these bridged epothilone analogues to be mildly active, but without significant microtubule stabilization capacity. Molecular mechanics and DFT energy evaluations suggest the mild activity of the bridged epo-analogues may be due to internal conformational strain.

Figure 1

Structures of natural epothilones A-D.

Figure 2

Structures of C6-C8 bridged epothilone analogs.

Figure 3

Docking poses of C6-C8 bridged epothilone analogs in the electron crystallography-determined tubulin binding site: (A) Docking poses of 1 (yellow) and 5 (cyan); (B) Docking poses of 1 (yellow) and 6 (blue). The shortest H---H contact between ligand and protein is 2.5 Å, an acceptable, though minimal, van der Waals separation.

Figure 4

Selected epothilone analogs with modification around C6-C8.

Figure 5

Tubulin assembly and microtubule cold stabilizing activity of bridged epothilones and PTX ( ), 5a ( ), 5 ( ), 6 ( ), 7 ( ), 8 ( ), 36 ( ), 37 ( ), 38 ( ). Tubulin (10 μM) was incubated with 50 μM of a bridged epothilone or 10 μM PTX in the presence of 1 mM GTP and 4% DMSO in PME. Assembly was monitored in terms of apparent absorption at 350 nm. The arrow indicates temperature drop from 37 °C to 4 °C. For reference, a tubulin sample in the absence of promoter (–) was also included.

Scheme 1

Retrosynthetic Analysis of C6-C8 Bridged EpoA 5 by Olefin Metathesis.

Scheme 2

Model Study. a) pivaldehyde, Et₂O/THF, −100 °C, 70%, dr > 95%. b) t-BuOOH, VO(acac)₂ (cat.), CH₂Cl₂, 88%; or mCPBA, CH₂Cl₂, 84%. c) CH₂=CH(CH₂)₃MgBr, CuCN (cat.), Et₂O, −60 → 0 °C, 89%. d) TBSOTf, 2,6-lutidine, CH₂Cl₂, −78°C, 85%. e) (COCl)₂, Et₃N, DMSO, CH₂Cl₂, −78 °C → RT, quant. f) 4-nitrobenzoyl chloride, pyridine, DAMP, THF, RT, 16%.

Scheme 3

a) i) O₃, CH₂Cl₂, −78 °C, then PPh₃, RT; ii) ethylene glycol, PTSA (cat.), benzene, reflux; iii) HF/Pyridine, THF, 0 °C → RT, 53% (3 steps). b) (COCl)₂, Et₃N, DMSO, CH₂Cl₂, −78°C → RT, quant.

Scheme 4

a) i) HF/Pyridine, THF, 0 °C → RT, 88%; ii) (COCl)₂, Et₃N, DMSO, CH₂Cl₂, −78 °C α RT, 94%. b) 16, THF, −78 °C, then H₂O₂, NaHCO₃, 40 °C, 92%, dr > 20:1. c) t-BuOOH, VO(acac)₂ (cat.), CH₂Cl₂, 93%, dr > 20:1 d) CH₂=CH(CH₂)₃MgBr (8–9 equiv.), CuCN (cat.), Et₂O, −55 → 0 °C, 90%. e) TBSOTf, 2,6-lutidine, CH₂Cl₂, −78 °C, 85%. f) (COCl)₂, Et₃N, DMSO, CH₂Cl₂, −78 °C → RT, 99%.

Scheme 5

a) TFA, CH₂Cl₂, −20 → 0 °C, 78%. b) t-BuOOH, VO(acac)₂ (cat.), CH₂Cl₂, 0 °C → RT, 89%, dr = 10:1 c) Ac₂O, DMAP, CH₂Cl₂, 0 °C → RT, 93%. d) i) n-Bu₄NHSO₄ (cat.), CH₃CN/H₂O, 50 °C; ii) NaIO₄, THF, RT, iii) NaClO₄, NaH₂PO₄, 2-methyl-2-butene, t-BuOH/H₂O, 45% (3 steps).

Scheme 6

a) EDCI, DMAP, CH₂Cl₂, RT, 58%; or 2,4,6-trichlorobenzoylchloride, DMAP, Et₃N, toluene, −78 → 0 °C, 86%. b) RCM, see text. c) DBU, CH₂Cl₂, 0 °C → RT, 96%.

Scheme 7

a) i) (ClCO₂CO)₂, DMAP, pyridine, CH₂Cl₂, 0 °C, quant.; ii) NaIO₄/H₅IO₆, THF/H₂ O; iii) NaClO₄, NaH₂PO₄, 2-methyl-2-butene, t-BuOH/H₂O, 72% (2 steps). b) 2,4,6-trichlorobenzoylchloride, DMAP, Et₃N, toluene, −78 → −35 °C, 49%. c) RCM, cat. 42, CH₂Cl₂, RT, 77%. d) NH₄OH/MeOH, 0 °C then NH₃/MeOH, 0 °C, 57%.

Scheme 8

Retrosynthesis of C6-C8 Bridged EpoA/B by Suzuki Coupling.

Scheme 9

a) See Lit. [19]. b) 16, THF, −78 °C, then H₂O₂, NaHCO₃, 40 °C, 96%, dr > 20:1. c) t-BuOOH, VO(acac)₂ (cat.), CH₂Cl₂, 93%, dr > 20:1 d) AllylMgBr (8.0 equiv.), CuCN (cat.), Et₂O, −55 → 0 °C, 85%. e) TBSOTf, 2,6-lutidine, CH₂Cl₂, −78 °C, 97%. f) (COCl)₂, Et₃N, DMSO, CH₂Cl₂, −78 °C → RT, 85%. g) See Lit. [39c, 42].

Scheme 10

a) 9-BBN, Cs₂CO₃, AsPh₃, PdCl₂(dppf), DMF/H₂ O, RT, 46: 92%, 47: 57%. b) (DHQD)₂PHAL, K₂CO₃, K₃Fe(CN)₆, CH₃SO₂NH₂, K₂OsO₄ 2H₂O, t-BuOH/H₂O, 0 °C → RT, 56: 42% (86%, BRSM), dr = 5:1; 57: 42% (87%, BRSM), dr = 4:1. c) i) NaIO₄, THF/H₂ O, 0 °C; ii) NaClO₄, NaH₂PO₄, 2-methyl-2-butene, t-BuOH/H₂O, 58: 78% (2 steps), 59: 58% (2 steps). d) TBAF, THF, 0 °C → RT, 60: 95%, 61: 90%. e) 2,4,6-trichlorobenzoylchloride, Et₃N, DMAP, toluene, RT, 62: 51%, 63: 60%. f) TFA, CH₂Cl₂, −20 → 0 °C, 7: 88%, 8: 91%. g) DMDO, CH₂Cl₂, −50 → −30 °C, 5: 84%, dr = 2:1, 6: 52%, dr > 20:1.