TETRODOTOXIN

O HO H O O N OH H2N H N HO OH OH

Presentation by Neil Vasan

Baran Lab Group Seminar August 13, 2003

TTX: Background

• Toxic venom component of puffer fish or fugu (Spheroides rubripes), a Japanese delicacy (1 fish = ~$400) • First isolated in 1909 and named after puffer fish order Tetraodontidae • Structure first elucidated in 1964 by Woodward (confirmed by Kishi in 1965) • First synthesis by Kishi, et. al. in 1972 • Toxicity attributed to selective blockage of Na+ channels of skeletal muscles • Lethal dose for adult human = .001 mg • Upon ingestion, one feels tingling and lightheadedness but is lucid; paralysis and death ensue within 6-24 hours • 70-100 deaths each year, mostly in rural Japan • No known antidote exists

1 TTX: Structure

O HO H O O N OH H2N H N HO OH OH

O O O HO H O H OH O N N O H N OH 2 N H2N H HO H N HO OH OH OH OH

Equilibrium mixture among ortho ester, anhydride, and lactone forms

TTX: Kishi Synthesis

Synthesis of Cyclohexane chiral core

O O O H H Me , SnCl4 Me MsCl Me MeCN, rt Et N Me 3 (83%) (quant.) N O Me O Me O OH N OH N Ms

H O OH H H H O o Me Me H Me H2O, 100 C NaBH4 m-CPBA Beckmann MeOH camphorsulfonic acidHO (96%) (75%) (61%) NH NH H NH O O O Ac Ac Ac

2 TTX: Kishi Synthesis

Towards Tetrodamine

H H H H H O O H O H Me H Me H Me 1) Al OCHMe2 3 HO (90% in 2 steps) O MPV-reduction O 2) H NH O NH O(COCH3)2, pyr. O NH O O OAc Ac Ac (95% in 2 steps) Ac

H H O H O H H CH2OAc 1. o-DCB, reflux H CH2OAc AcOH, rt (76% in 6 steps)EtO O 2. m-CPBA, K2CO3 O (70% from CH2Cl2 EtO diethyl ketal) EtO NH H O NH H OAc OAc Ac Ac

TTX: Kishi Synthesis

Towards Tetrodamine

H H O H H O H mCPBA H CH2OAc CH2OAc KOAc, AcOH o O CH2Cl2, rt O O 90 C, 2h (quant.) (quant.) O O NH H NH H AcO OAc AcO OAc Ac Ac

AcO H O 1) O(COCH ) , pyr. O H H 3 2 H H OH 2) pyrolysis, 300 oC, vacuum OAc OAc CH2OAc OAc CH2OAc (80% in 2 steps) AcHN H AcHN H O O (acetylated AcO AcO H H tetrodamine) O O

3 TTX: Kishi Synthesis

Tetrodamine to Tetrodotoxin

O O SEt O H H H H H H OAc OAc OAc BF3 Et2O AcN SEt SEt CH2OAc CH2OAc CH2OAc OAc OAc o OAc CH2Cl2 120 C, 12h C AcHN H H N H AcHN N H O (92%) 2 O O AcO AcO AcO H H H O O O (acetylated tetrodamine) (20% in 3 steps from tetrodamine)

HO O O HO H H H H OAc OAc NH NH 2 OAc CH2OAc 2 OAc CH2OAc 1) NaIO , THF, 0 oC C OsO C 4 AcHN N H 4 AcHN N H TTX O o O 2) NH4OH, MeOH AcO THF, -20 C AcO H H (25% from mono- O O acetylguanidine)

TTX: Isobe Synthesis Retrosynthesis

4 TTX: Isobe Synthesis

Sonogashira Coupling and Claisen Rearrangement

OH OTBS OTBS O Oi-Pr O Oi-Pr TMS O Oi-Pr

(61% in 4 steps) Pd(OAc)2, OH OH OH Ph3P, CuI, OAc I Et N, PhH (available in 2 steps from O ) 3 (99%) AcO OAc TMS OAc

OTBS OTBS OTBS O Oi-Pr O Oi-Pr O Oi-Pr OMe K2CO3 PPTS/THF o-DCB BzO O (50% in 6 steps) 5 150 oC (89%) Me O O (94%) OTBDPS TMS TMS

TTX: Isobe Synthesis

Cyclohexenone and Exoolefin Synthesis

1) TBS-OTf OTBS 1) TBAF H2SO4 H O OMe Et N, lutidine O OMe THF H O MeOH O 3 2 5 and then 2) TBS-Cl BzO 2) Cl3CCOCl HgO (cat.) imid., DMF TBDPSO OH O DMAP, pyr. (82%) OBz O OTBS (74% in 4 steps) OTBDPS

OTBS OH 1) NaBH4, O MeOH, rt O OMe O OH O OH 2) Me2C(OMe)2, BzO BzO CSA, acetone BzO 3) PPTS, MeOH (67% in 6 steps) O OBOM (93% in 3 steps) OBOM OTBDPS OTBDPS OTBDPS

5 TTX: Isobe Synthesis

Previous Overman Rearrangement Problem with Overman Rearrangement

TTX: Isobe Synthesis

Installation of Nitrogen Functionality: Conjugate Carbamate Addition

O 1) DIBAL-H O O O OH 2) TEMPO, NCS O 1) Cl3CCONCO O O COOMe COOMe 3) NaClO2, NaH2PO4, CH2Cl2 BzO HO O Me2C=CHMe-H2O 2) Et3N, MeOH H2N 4) TMS-CHN2 (99% in 2 steps) OBOM (78% in 4 steps) OBOM OBOM OTBDPS OTBDPS OTBDPS

O O O O 1) t-BuOK, THF 1) Boc O, Et N O 2 3 Boc 2) LiBH , THF O NH OMMTr DMAP, THF 4 HO NH OMMTr 3) MMTrCl, pyr. 2) LiOH, MeOH (88% in 3 steps) (CH2Cl)2-H2O OBOM OBOM (84% in 2 steps) OTBDPS OTBDPS

6 TTX: Isobe Synthesis Construction of Lactone Bicycle

O O Boc HO NH OMMTr (70% in 7 steps) OBOM OTBDPS

TTX: Isobe Synthesis Introduction of Guanidine

HO O AcO OBz Me 1) TFA, MeOH H Me O OAc O O 2) CAN Me O Me O O O MeCN-H2O BocHN BocHN 3) HgCl O (96% in 4 steps) O 2 NHBoc OBz OBz Et N-DMF BOM Ac 3 OH OAc (53% in 3 steps) BocN SMe

OBz OBz AcO AcO 1) NaIO4 BocHN HO O H O MeOH-H2O Boc O HO O N OH 2) TFA, MeOH N N N BocN O O (50% in 3 steps) H (90% in 2 steps) H OBz Ac OBz Ac OAc OAc OBz AcO Ac Me O Ac O N O N N O Ac Ac OBz OAc

7 TTX: Isobe Synthesis

Deprotection and Final Conversion

OBz O O AcO HO O Ac Me O H Me O H O Ac O Et3N O O N O N O N N MeOH-H2O H2N + H2N N O N O N O Ac (85%) H OH H OH Ac OBz H H OAc OH OH (TTX-anhydride)

O HO H O O TFA-d/D2O N OH (-) TTX H2N H N HO OH OH (TTX-ortho ester)

TTX: Du Bois Synthesis

Retrosynthesis OH HO O O OH OH HO HO OH OH O O OH HO OH HN N OH OH OHC OH O H H OH H NH2 HO CO2H

stereospecific C-H amination

Me Me HO O O Me Me OH O O HO OH 2 2 OR N2 OR H OH OHC O O O O H Rh-catalyzed HO CO H C-H insertion 2 R1O O R1O O

8 TTX: Du Bois Synthesis

Rh-carbene C-H insertion

O Me BnO2C OH Me O Me Me OH O O O O O O CO2Bn O COOt-Bu (64% in 5 steps) H HO OH OTBS (53% in 4 steps) TBSO OH O N2

Me Me O Me Me O 1) H2 (1200 psi), Rh-C, 1) Rh2(tpacam)4, CCl4 O O OTBS CF3CO2H-MeOH 2) NH BH , CH Cl -MeOH O Me 3 3 2 2 2) p-TsOH, 2,2-DMP; (75% in 2 steps) then t-BuCOCl, pyr., THF O Me O O O (77% in 2 steps) H CO t-Bu t-BuO2C O O 2

TTX: Du Bois Synthesis

Construction of Lactone Bicycle

Me Me Me Me Me O O O Me O O O O O O Me Me Ph2Se2, PhIO2 O Me o Me (56% in 3 steps) Me 100 C, pyr., PhCl O O O O Me H H (70%) H Me2NOC Me NOC CO t-Bu CO t-Bu 2 O 2 2 CO2t-Bu

Me Me O Me O Me O 1) t-BuCO H O HO 2 O Me o O Me PhCl, 200 C O (77% in 2 steps) O Me 2) NaOMe, THF-MeOH O Me H O H Me2N (78% in 2 steps) OCOt-Bu OH O

9 TTX: Du Bois Synthesis

Rh-nitrene C-H insertion

Me Me O O Me Me O Me OH Me O O O O O O O O O O Me H NH O H 2 (66% in 3 steps) O Me O O O O H Me Me Cl OH Me Me

Rh2(tfacam)4, PhI(OAc)2, Me O Me O Me Me o O OH MgO, DCE, 50 C O O O O O O O (55% with 38% reactant) NH (59% in 4 steps) NHBoc O O O O Me Cl Me Me Me

TTX: Du Bois Synthesis

Guanidine Insertion and Final Steps

Me O Me O Me O Me Me NBoc Me OH OH OH O O NHBoc O O o O O O H2O, 110 C O BocHN SMe O NHBoc NH N (95%) 2 HgCl , Et N, NHBoc O O 2 3 O O O MeCN-CH2Cl2 O Me Me (80%) Me Me Me Me

OH OH H O-CF CO H O O O O 2 3 2 HO OH O OH 5 days O3, Me2S, (4:1 mixture) (-) TTX CH2Cl2-MeOH; HO OH OH HN HN (65% from then H2O-CF3CO2H N OH N OH H H after deprotection) (1:1 mixture) H2N H2N

([-] TTX-ortho ester) ([-] TTX-anhydride)

10 Comparisons and Contrasts Chemist KISHI ISOBE DU BOIS Date Sept. 1972 Jan. 2003 June 2003 Type Racemic Asymmetric Asymmetric Diels-Alder synthesis Claisen Rhodium-catalyzed Highlights of skeleton; 6 chiral rearrangement; carbene and nitrene centers established Sonogashira C-H insertions;

on cyclohexane core coupling; Ph2Se2-promoted ring; Only ketal and Intramolecular allylic oxidation; acetyl protecting carbamate-ester Shows power of groups; Common conjugate addition; organometallic reagents employed All OH groups chemistry in total to carry out elegant differently protected synthesis chemistry for future analog synthesis # of Steps 29 67 32 Yield 0.66% 1.22% 0.49%

TTX: References

Structure: Woodward, R. B. Pure. Appl. Chem. 1964, 9, 49-74. Tsuda, K., et. al. Chem. Pharm. Bull. 1964, 12, 1357-1374. Goto, T.; Kishi, Y.; Takahashi, S.; Hirata, Y. Tetrahedron 1965, 21, 2059-2088.

Absolute stereochemistry: Furusaki, A., et. al. Bull. Chem. Soc. Jpn. 1970, 43, 3332-3341.

Bioorganic studies: Narahashi, T.; Moore, J. W.; Scott, W. R. J. Gen. Physiol. 1964, 47, 965-974. Hucho, F. Angew. Chem, Int. Ed. Engl. 1995, 34, 39-50. Numa, S.; Noda, M. Ann. N.Y. Acad. Sci. 1986, 479, 338-355.

Other analog syntheses: Nishikawa, T., et. al. Angew. Chem., Int. Ed. 1999, 38, 3081-3084. Asai, M., et. al. Tetrahedron 2001, 57, 4543-4558. Nishikawa, T., et. al. J. Am. Chem. Soc. 2002, 124, 7847-7852.

11 TTX: References

Kishi synthesis: Kishi, Y., et. al. Tetrahedron Lett. 1970, 59, 5127-5128. Kishi, Y., et. al. Tetrahedron Lett. 1970, 59, 5129-5132. Kishi, Y., et. al. J. Am. Chem. Soc. 1972, 94, 9217-9219. Kishi, Y., et. al. J. Am. Chem. Soc. 1972, 94, 9219-9221.

Isobe synthesis: Isobe, M., et. al. J. Am. Chem. Soc. 2003, 125, 8798-8805.

Overman Rearrangement: Overman, L. E. Acc. Chem. Res. 1980, 13, 218-224. Sonogashira Coupling: Sonogashira, K., et. al. Tetrahedron Lett. 1975, 16, 4467-4470. α-hydroxyl Lactone: Corey, E. J.; Ghosh, A. K. Tetrahedron Lett. 1988, 26, 3205-3206.

Du Bois Synthesis: Hinman, A.; Du Bois, J., personal communication. (to be published in JACS)

Rh-carbene C-H insertion: Espino, C. G.; Du Bois, J. Angew. Chem., Int. Rd. Engl. 2001, 40, 598-600. Rh-nitrene C-H insertion: Guthikonda, K.; Du Bois, J., J. Am. Chem. Soc. 2002, 124, 13672-13673.

Ph2Se2 allylic oxidation: Barton, D. H. R.; Crich, D. Tetrahedron 1985, 41, 4359-4364.

TTX: Acknowledgements

• Prof. Justin Du Bois, Stanford University

• The Baran Laboratory, TSRI

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