Synthesis of the Deacetoxytubuvaline Fragment of Pretubulysin and its Lipophilic Analogues for Enhanced Permeability in Cancer Cell Lines

Abstract

In the last two decades, tubulysins have emerged as alternatives to microtubule depolymerizing agents such as colchicine and vinblastine, which are well-established anticancer agents. However, the complex structure of tubulysins has always posed a challenge for synthetic chemists to scale up the production of these compounds. We report a new strategy for the practical gram-scale synthesis of a (4R)-4-[(tert-butoxycarbonyl)amino]-5-methylhexanoic acid through regioselective cleavage of a chiral aziridine ring with a vinyl Grignard reagent to afford tert-butyl [(1R)-1-isopropylbut-3-en-1-yl]carbamate, which was subjected to regioselective hydroboration–oxidation with 9-BBN. The resulting (4R)-4-[(tert-butoxycarbonyl)amino]-5-methylhexanoic acid was successfully transformed into the deacetoxytubuvaline fragment of pretubulysin or its highly lipophilic methyl-substituted thiazole and oxazole analogues for incorporation into pretubulysins. Increasing the lipophilicity of tubulysin or pretubulysin molecules should enhance their cell permeability and cytotoxicity in cancer cell lines.

Key words

amino acid - deacetoxytubuvaline - tubulysin - pretubulysin - medicinal chemistry

• References and Notes

• 1 Mukhtar E, Adhami VM, Mukhtar H. Mol. Cancer Ther. 2014; 13: 275
• 2 Sasse F, Steinmetz H, Heil J, Höfle G, Reichenbach H. J. Antibiot. 2000; 53: 879
• 3 Sandmann A, Sasse F, Müller R. Chem. Biol. 2004; 11: 1071
• 4 Wang Z, McPherson PA, Raccor BS, Balachandran R, Zhu G, Day BW, Vogt A, Wipf P. Chem. Biol. Drug Des. 2007; 70: 75
• 5 Rath S, Liebl J, Fürst R, Ullrich A, Burkhart JL, Kazmaier U, Herrmann J, Müller R, Günther M, Schreiner L, Wagner E, Vollmar AM, Zahler S. Br. J. Pharmacol. 2012; 167: 1048
• 6 Staben LR, Yu S.-F, Chen J, Yan G, Xu Z, Del Rosario G, Lau JT, Liu L, Guo J, Zheng B, dela Cruz-Chuh J, Lee BC, Ohri R, Cai W, Zhou H, Kozak KR, Xu K, Phillips GD. L, Lu J, Wai J, Polson AG, Pillow TH. ACS Med. Chem. Lett. 2017; 8: 1037
• 7 Murray BC, Peterson MT, Fecik RA. Nat. Prod. Rep. 2015; 32: 654
• 8 Herrmann J, Elnakady YA, Wiedmann RM, Ullrich A, Rohde M, Kazmaier U, Vollmar AM, Müller R. PLoS One 2012; 7: e37416
• 9 Braig S, Wiedmann RM, Liebl J, Singer M, Kubisch R, Schreiner L, Abhari BA, Wagner E, Kazmaier U, Fulda S, Vollmar AM. Cell Death Dis. 2014; 5: e1001
• 10 Eirich J, Burkhart JL, Ullrich A, Rudolf GC, Vollmar A, Zahler S, Kazmaier U, Sieber SA. Mol. BioSyst. 2012; 8: 2067
• 11 Truebenbach I, Gorges J, Kuhn J, Kern S, Baratti E, Kazmaier U, Wagner E, Lächelt U. Macromol. Biosci. 2017; 17: 1600520
• 12 Ullrich A, Herrmann J, Müller R, Kazmaier U. Eur. J. Org. Chem. 2009; 6367
• 13 Nicolaou KC, Erande RD, Yin J, Vourloumis D, Aujay M, Sandoval J, Munneke S, Gavrilyuk J. J. Am. Chem. Soc. 2018; 140: 3690
• 14 Brindisi M, Maramai S, Grillo A, Brogi S, Butini S, Novellino E, Campiani G, Gemma S. Tetrahedron Lett. 2016; 57: 920
• 15 Colombo R, Wang Z, Han J, Balachandran R, Daghestani HN, Camarco DP, Vogt A, Day BW, Mendel D, Wipf P. J. Org. Chem. 2016; 81: 10302
• 16 Trabocchi A, Guarna F, Guarna A. Curr. Org. Chem. 2005; 9: 1127
• 17 Ordóñez M, Cativiela C. Tetrahedron: Asymmetry 2007; 18: 3
• 18 Menche D, Hassfeld J, Li J, Rudolph S. J. Am. Chem. Soc. 2007; 129: 6100
• 19 Stark M, Assaraf YG. Oncotarget 2017; 8: 49973; DOI: 10.18632/oncotarget.18385
• 20 Wipf P, Takada T, Rishel MJ. Org. Lett. 2004; 6: 4057
• 21 Shankar PS, Sani M, Saunders FR, Wallace HM, Zanda M. Synlett 2011; 1673
• 22a Kitir B, Baldry M, Ingmer H, Olsen CA. Tetrahedron 2014; 70: 7721
• 22b Ye W, Leow D, Goh SL. M, Tan C.-T, Chian C.-H, Tan C.-H. Tetrahedron Lett. 2006; 47: 1007
• 23 Methyl 2-{(3R)-3-[(tert-Butoxycarbonyl)(methyl)amino]-4-methylpentyl}-1,3-thiazole-4-carboxylate (1) Anhyd DMF (2 mL) was added to the thiazole ethyl ester 11 (0.080 g, 0.22 mmol) in a 10 mL round-bottomed flask equipped with a magnetic stirrer bar. MeI (0.05 mL, 0.89 mmol) was then added, and the mixture was cooled to 0 °C. NaH (0.013 g, 0.55 mmol) was added portionwise over 15 min with constant stirring, and the mixture was stirred at 25 °C under N₂ for a further 12 h. When 11 was completely consumed (TLC), the reaction was quenched with sat. aq NH₄Cl (10 mL). The aqueous layer was extracted with EtOAc (3 × 25 mL), and the combined organic extracts were washed with brine (3 × 10 mL), dried (Na₂SO₄), filtered, and concentrated under reduced pressure. The resulting crude residue was purified by column chromatography [silica gel, hexane–EtOAc (80:20)] to give a colorless liquid; yield: 70.0 mg (87%); TLC: R_f = 0.25 (hexane–EtOAc, 70:30). [α]_D ²⁰ –20.4 (c 0.1, CH₂Cl₂). IR (CH₂Cl₂): 2963, 2920 (C–H), 1722, 1694 (C=O), 1685 (C=N), 1484, 1392 (C–H), 1170 (C–O), 1155 (S–O), 867 (=C–H), 775, 720 (C–H) cm^–1. ¹H NMR (400 MHz, CDCl₃, 25 °C): δ = 8.08 (s, 1 H, rotamer 1), 8.06 (s, 1 H, rotamer 2), 3.95 (s, 3 H, rotamer 1), 3.94 (s, 3 H, rotamer 2), 2.99–2.95 (m, 4 H, rotamers 1 + 2), 2.70 (s, 3 H, rotamer 1), 2.65 (s, 3 H, rotamer 2), 2.18–2.11 (m, 2 H, rotamers 1 + 2), 1.89–1.81 (m, 2 H, rotamers 1 + 2), 1.70–1.67 (m, 4 H, rotamers 1 + 2), 1.46 (s, 9 H, rotamer 1), 1.43 (s, 9 H, rotamer 2), 0.96 (t, J = 6.0 Hz, 6 H, rotamer 1), 0.85 (d, J = 6.5 Hz, 6 H, rotamer 2). ¹³C NMR (100 MHz, CDCl₃, 25 °C): δ = 171.9, 162.0, 156.6, 146.5, 127.2, 79.6, 60.3, 52.5, 30.73, 30.5, 30.0, 29.7, 28.4, 20.2, 19.9. Diagnostic signals of minor rotamer ¹³C NMR (100 MHz, CDCl₃, 25 °C): δ = 171.5, 161.9, 146.4, 79.2, 52.4, 20.1, 19.6. HRMS (ESI): m/z [M + H]⁺ calcd for C₁₇H₂₉N₂O₄S: 379.1662; found: 379.1675.