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Progress Toward the Total Synthesis of Tubulysin D and Its Analogues

Progress Toward the Total Synthesis of Tubulysin D and Its Analogues

Progress Toward the Total Synthesis of Tubulysin D and its Analogues

Michael J. Rishel Wipf Lab

University of Pittsburgh May 22, 2004

Mike Rishel @ Wipf Group 1 5/22/04 A General Look at the

• Cell cycle is divided into four main parts

– G1 or Gap 1 phase – S or Synthesis phase

– G2 or Gap 2 period – M or Mitosis phase • Cell cycle contains two major control points

– G1/S is the point at which cells commit to replicate genetic material, enter quiescence (G0), or terminally differentiate and die

– G2/M is the point at which cells commit to division – inhibitors typically cause treated cells to http://www.biology.arizona.edu/cell_bio/tutorials/ cell_cycle/cells2.html accumulate at the G2/M checkpoint

Mike Rishel @ Wipf Group 2 5/22/04 A Brief Introduction to Tubulin

• Tubulin exists as a heterodimeric structure of the a and b tubulin proteins. – tubulin proteins are highly conserved structures in eukaryotes – the a and b-subunits are of similar secondary and tertiary structure – each subunit is ca. 55 kD in mass – the heterodimeric structure is tightly bound together and dissociates only under denaturing conditions

• Each tubulin monomer is capable of binding a molecule of GTP () – a-tubulin binds GTP and retains it in the heterodimer – b-tubulin binds a molecule of GTP and hydrolyses it to GDP during or shortly following the incorporation of the heterodimer into a protofilament

Mike Rishel @ Wipf Group 3 5/22/04 Structure of the ab- a-tubulin Tubulin Dimer from Electron Crystallography

• Purple subunits represent bound GTP ( a-tubulin), and GDP (b-tubulin) each bound within a Rossman fold. b-tubulin • Subtle differences between intradimeric and interdimeric binding contacts remain somewhat ambiguous.

Nature 1998, 391, 199-203.

Mike Rishel @ Wipf Group 4 5/22/04 Introduction to

• Microtubules exist as helical entities consisting protofilaments – microtubules are “3-step” helices consisting of 13 protofilaments each – standard microtubules are 25 nm in diameter and vary in length depending on envionmental stimuli (dynamic instability) – protofilaments are polar entities consisting of a “+” (at b-tubulin) and “-” (at a-tubulin) • growth of the occurs at the “+” end • degredation of the microtubule occurs at “-” end

Mike Rishel @ Wipf Group 5 5/22/04 The Basics of Tubulin Polymerization

Annu. Rev. Cell Dev. Biol. 1997, 13, 83-117.

Mike Rishel @ Wipf Group 6 5/22/04 Dynamic Instability

http://www.ch.ic.ac.uk/local/projects/a_abowath/Tubulin.html

Mike Rishel @ Wipf Group 7 5/22/04 Microtubule Decomposition and Growth in Real Time

J. Cell Sci.1992, 103, 965-976.

Mike Rishel @ Wipf Group 8 5/22/04 A Survey of Tubulin Inhibitors – Colchicine Domain

• Alkaloid produced by the meadow saffron ( Colchicum O autumnale) . MeO • Inhibits microtubule-dependant N cellular processes by strongly H MeO binding to b-tubulin, thus interfering with polymerization MeO of tubulin protomers. O • Arrests mitotic cycle in plants Cholchicine and animals at metaphase. OMe • Commonly used in the treatment of Gout. • The agent is not selectively toxic to cancer cell lines.

(a) Pharmac. Ther., 1991, 51, 377. (b) Alkaloids 1992, 41, 125. (c) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 9 5/22/04 A Survey of Tubulin Inhibitors – Colchicine Domain

• Obtained as an extract from the plant. Podophyllum peltatum OH • Binding to tubulin is more rapid O and reversible than colchicine O derivitaves. O H • Behaves as a competitive inhibitor O of colchicine binding to tubulin. • Inhibits tubulin dependent GTP MeO OMe hydrolysis (vide supra) thus OH suppressing the dynamic instability Podophillotoxin properties of microtubules.

(a) Pharmac. Ther., 1993, 59, 163. (b) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 10 5/22/04 A Survey of Tubulin Inhibitors – Colchicine Domain

• Isolated from the marine cyanobacterium Lynbya majuscula. • Inhibitor of mitosis, tubulin Me polymerization, and a competative OMe inhibitor to colchicine binding. H N • Interaction with tubulin characterized S Me by rapid binding, slow dissociation, and induction of GTP hydrolysis H H Curacin A uncoupled from normal tubulin assembly. • Although an inhibitor of normal tubulin polymerization, treatment with curacin A often results in the formation of abnormal tubulin polymers.

(a) J. Org. Chem. 1994, 59, 1243. (b) Tetrahedron Lett. 1995, 34, 110. (c) Med. Res. Rev. 1996, 16, 207-231. (d) J. Org. Chem. 1996, 61, 6556-6562. (e) J. Med. Chem. 2002, 45, 1901-1917. Mike Rishel @ Wipf Group 11 5/22/04 A Survey of Tubulin Inhibitors – Vinca Site

• Isolated From the Madagascan periwinkle Catharanus roseus (formerly Vinca rosea). • Inhibits of microtubule polymerization through binding to the OH OAc vinca binding site on b- tubulin. N OMe N • Characteristically aberrant tubulin H polymerization reactions result OH O N following in vivo treatment with R Vinca alkaloids. N H O • The alkaloids strongly inhibit tubulin- MeO O Me dependent GTP hydrolysis, and weakly inhibits the binding of GTP Vinblastine R = CH3 and GDP at the exchangeable Vincristine R = CHO nucleotide site. • Widely used in combination , often in conjunction with DNA damaging agents like (a) Pharmac. Ther., 1991, 51, 257. (b) bleomycin. Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 12 5/22/04 A Survey of Tubulin Inhibitors – Vinca Site

• Isolated from fermentation cultures of the fungus Rhizopus chinensis. Me • Reversibly binds to tubulin at 37 °C H O O Rhizoxin • Agent demonstrates plant, fungal, H and cellular toxicity. H Me O • Under clinical evaluation for HO H N Me O treatment in human cancers. O O Me • Binding of radiolabled rhizoxane H O was competitively inhibited by OMe vinblastine (Ki = 3 mM). • Noncompetitive inhibition of Rhizoxin was observed with phomopsin A.

(a) J. Antibiot. 1984, 37, 354. (b) Cancer Res. 1992, 52, 2894. (c) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 13 5/22/04 A Survey of Tubulin Inhibitors – Vinca Domain

• A toxic secretion from the fungus Phomopsis leptostomiformis. O • Strong inhibitor of tubulin OH polymerization. HN O • The agent behaves as a OH O O HO noncompetitive inhibitor in the O NH Cl O binding of [3H] vincristine to tubulin (K = 2.8 mM), and as a competative N i HN inhibitor to dolostatin 10 / tubulin O binding. HO O N • Inhibits tubulin-dependent GTP NH H Phomopsin A Me hydrolysis, nucleotide exchange, and formation of the cys12 – cys 201/211 cross-link. • Phomopsin strongly stabilizes tubulin conformation.

(a) J. Chem. Soc. Chem. Commun. 1983, 1259. (b) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 14 5/22/04 A Survey of Tubulin Inhibitors – Vinca Domain

• An unusual peptide isolated from the shell-less mollusk Dolabella auricularia • Agent causes mitotic arrest in vivo, with visible degredation of intracellular microtubules.

• The agent behaves as a O H noncompetitive inhibitor in the N N O N N N 3 binding of [ H] vincristine to tubulin O OMe O N MeO H S (Ki = 1.4 mM), and as a competative Me inhibitor to radio labled phomopsin Dolostatin 10 A– tubulin binding. • Inhibits tubulin-dependent GTP hydrolysis, nucleotide exchange, and formation of the cys12 – cys 201/211 cross-link.

(a) Tetrahedron, 1993, 9151-9179. (b) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 15 5/22/04 A Survey of Tubulin Inhibitors – Vinca Domain

• Isolated from the marine OH Spirastrella spinispirulifera and Hyrtios altum.. Cl OAc • Highly toxic to a variety of OH O O Me human cancer lines in culture. HO O O OH (IC50 = 20 pM for L1210 O Me murine leukemia cells). HO O • Noncompetitively inhibits the OH Me AcO binding of both dolostatin 10 Me O and vinblastine to tubulin. Me O • The agent is a strong inhibitor O

of GDP nucleotide exchange. Spongistatin 1 OCH3

(a) J. Org. Chem. 1993, 58, 1302. (b) Tetrahedron Lett. 1993, 34, 2795. (c) Mol. Pharmacol. 1993, 44, 757. (d) Med. Res. Rev. 1996, 16, 207-231. (e) Angew. Chem. Int. Ed. 1998, 37, 187-192. (f) Angew. Chem. Int. Ed. 1998, 37, 192-196.

Mike Rishel @ Wipf Group 16 5/22/04 Microtubule Stabilizing Agents – Taxol

• Originally isolated from the plant Taxus brevifolia as a cytotoxic agent. • The agent was the first to derive its mechanism of action from the OAc stabilization of microtubules rather HN O O than by the inhibition of their Me OH synthesis. O OH O

• Binding of Taxol occurs only with the HO H O O O tubulin polymer, and in a roughly 1:1 Taxol AcO stoichiometry. • Taxol binds to the polymer in a

reversible fashion with a KD = 1 mM.

(a) J. Am. Chem. Soc., 1971, 93, 2325. (b) Nature, 1979, 277, 665-667. (c) J. Biol. Chem. 1988, 263, 1342-1346. (d) J. Med. Chem. 1993, 36, 1918-1922. (e) Med. Res. Rev. 1996, 16, 207-231. (f) Khalil, M.-W. M. Dissertation 1999, Technical University of Braunschweig.

Mike Rishel @ Wipf Group 17 5/22/04 Microtubule Stabilizing Agents –

• Isolated from cultures of the myxobacterium Sorangium cellulosum. R O • Epothilones were demonstrated S to induce tubulin Me OH polymerization in vitro, in the N absence of added GTP, and in a O concentration dependent O OH O manner. A R = H • The epothilones inhibited the Epothilone B R = Me binding of radiolabled to microtubules.

(a) Höfle, G.; Bedorf, N.; Gerth, K.; Reichenbach, G. H. 1993, (GBF) DE-4138042; Chem. Abstr. 1993, 120, 52841. (b) Cancer Res. 1995, 55, 2325-2333. (c) Angew. Chem. Int. Ed. 1996, 35, 1567-1569. (d) J. Antibiot. 1996, 49, 560-563. (e) Med. Res. Rev. 1996, 16, 207-231.

Mike Rishel @ Wipf Group 18 5/22/04 Microtubule Stabilizing Agents – Discodermolide

• Isolated from the marine sponge Discodermia dissoluta. • Arrests the cell cycle of treated cells in G2/M phase. Me Me Me • The agent was more potent in HO microtubule stabilization than O O OH O O Me Me Taxol under a variety of OH NH reaction conditions. Me Me 2 OH • Discodermolide stabilized Discodermolide microtubules were morphologically distinct (shorter) from those resulting from their Taxol stabilized counterparts.

(a) J. Org. Chem. 1990, 55, 4912-4915. (b) J. Org. Chem. 1991, 56, 1346. (c) Chem. Biol. 1994, 1, 67-71. (d) Med. Res. Rev. 1996, 16, 207-231. (e) Biochemistry, 1996, 35, 243-250.

Mike Rishel @ Wipf Group 19 5/22/04 Tubulysins: Potent Antimitotic Agents from Archangium gephyra, and Angiococcus disciformis

R'

O OAc H O Me N N OH N N N H Me O S O O

O R

Tubulysin A R = CH2CH(CH3)2 R’ = OH Tubulysin B R = CH(CH3)2 R’ = OH Tubulysin C R = CH2CH3 R’ = OH Tubulysin D R = CH2CH(CH3)2 R’ = H Tubulysin E R = CH2CH3) R’ = H Tubulysin F R = CH2CH3 R’ = H

Sesse, F.; Steinmetz, J.; Heil, J.; Höfle, G. J. Antibiot. 2000, 53, 879-885.

Mike Rishel @ Wipf Group 20 5/22/04 Cytotoxicities of Various Selected Tubulin Inhibitors

Cytotoxic activity of tubulin inhibitors for sensitive and resistant cell lines (IC50 ng/mL) Cell Line Compound L929a K562b KB–3.1c KB–VId Taxol 80 10 10 150 Vinblastine 15 6 7 120 Epothilone B 0.7 0.3 0.6 0.3 Dolostatin 10 0.1 0.1 0.2 1.2 Tubulysin A 0.2 0.07 0.2 0.4 Tubulysin B 0.4 0.2 0.3 1.0 Tubulysin D 0.03 0.02 0.2 0.08 Tubulysin E 0.1 0.05 0.03 0.1 1 Fibroblast cell line from connective tissue of a mouse’ b Human myelogenous leukemia cell line. c Human cervix carcinoma cell line. d Multidrug-resistant KB clone.

Pure Appl. Chem. 2003, 75, 167-178.

Mike Rishel @ Wipf Group 21 5/22/04 Degradation Studies: Total Acidic Hydrolysis of Tubulysin D

6N HCl

O OAc H O CH3 N N OH N N N H O S O O H O

CH3 OH OH N H2N O O O H2C O H2N (R)-N-methyl OH formaldehyde OAc O tubuphenylalanine pipecolinic acid N (1:1 mixture) H N + 2 1' OH H S OH (S)-isoleucine tubuvaline O 3-methylbutyric acid

Pure Appl. Chem. 2003, 75, 167-178 Mike Rishel @ Wipf Group 22 5/22/04 Determination of the Absolute Stereochemistry of the Tubuvaline Subunit

O OAc H O CH3 N N OH N N N H O S O O

O

1. 6N HCl 2. TFAA, pyr. dilute HCl 3. EtOH, H+ 4. TPAP O H N 3' N O O O O 1. TMSOTf, NEt3 1' 2. O , N N 3 O O F3C N 1' OEt 3. NaOH, H O N H 2 2 S S H H2N OH cyclotubulysin D S-valine

Pure Appl. Chem. 2003, 75, 167-178

Mike Rishel @ Wipf Group 23 5/22/04 Determination of the Absolute Stereochemistry of the Tubuphenylalanine Subunit

1. LiAlH4 2. Swern oxidation + 3. Ph P=C(CH )COOMe BocHN OH 3 3 BocN BocHN OMe O H3C O O CH3

1. H2, Pd/C 2. LiOH, H2O2 3. TFA / CH2Cl2

O OAc O H 4 4 N N 60% H2NNH2 N N N H2N H2N H 2 2 O S OH OH OH O H3C H3C H3C H O O O O

Pure Appl. Chem. 2003, 75, 167-178

Mike Rishel @ Wipf Group 24 5/22/04 The Absolute Stereochemistry of the Tubulysins

O OAc H O CH3 N N OH N N N H O S O O H O

CH3 OH OH N H2N O O O H2C O (R)-N-methyl H2N (2S, 4R)-tubuphenylalanine OH formaldehyde OAc O pipecolinic acid N H N + 2 1' OH H S OH (S)-isoleucine (1’R,3’R)-tubuvaline O 3-methylbutyric acid

Pure Appl. Chem. 2003, 75, 167-178

Mike Rishel @ Wipf Group 25 5/22/04 Dolostatin 10 and Tubulysin D: An Interesting Comparison

O H N N O N N N O OMe O N MeO H S Me Dolostatin 10

O OAc H O N N N N N H O S OH O H3C H O O

Tubulysin D

(a) Pure Appl. Chem. 2003, 75, 167-178. (b) J. Nat. Prod. 2001, 64, 907-910.

Mike Rishel @ Wipf Group 26 5/22/04

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