TETRODOTOXIN

O
HO
H O
N O
OH
H2 N
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

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 TTX: Structure
O
HO
H O
N O
OH
H2N
H N HO
OH
OH

O O
O HO
H O H OH
N O O
N OH
H2N H2N
H N 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
, SnCl4 H MsCl H
Me Me Me
MeCN, rt Et3N
Me
(83%) (quant.)
N O Me O Me O
OH N OH N Ms

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

2
 TTX: Kishi Synthesis
Towards Tetrodamine

H H H
O H H O H
O
H Me H Me 1) Al OCHMe2 3 H Me
HO (90% in 2 steps) O MPV-reduction O
H NH O 2) O(COCH3)2, pyr. O NH
NH OAc
O O (95% in 2 steps)
Ac Ac 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
H CH2OAc mCPBA CH2OAc KOAc, AcOH
O CH2Cl2, rt O O 90 oC, 2h
(quant.) (quant.)
O O
NH H NH H
AcO OAc AcO OAc
Ac Ac

AcO H
O 1) O(COCH3)2, pyr. O
H H H H
OH o
2) pyrolysis, 300 C, vacuum OAc
OAc CH 2 OAc 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
OAc CH2OAc OAc CH2OAc OAc CH2OAc
CH2Cl2 120 oC, 12h C
AcHN H H2N H AcHN N H
O (92%) 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
NH2 NH2
o OAc CH2OAc OAc CH2OAc
1) NaIO4, THF, 0 C C OsO4 C
TTX 2) NH4OH, MeOH
AcHN N
AcO
O
H
THF, -20 oC
AcHN N
AcO
O
H

(25% from mono- H H

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 O Oi-Pr
TMS

(61% in 4 steps) Pd(OAc)2,

OH OH OH
Ph3P, CuI,
OAc I
O Et3N, PhH
(available in 2 steps from )
(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

H2SO4 1) TBS-OTf OTBS 1) TBAF

H
MeOH O OMe Et3N, lutidine O OMe THF H2O
O
and then 5 2) TBS-Cl BzO 2) Cl3CCOCl
HgO (cat.) imid., DMF DMAP, pyr.
TBDPSO OHOBz O
(82%) O (74% in 4 steps)
OTBS
OTBDPS

OTBS OH 1) NaBH4, O
O OH MeOH, rt O OH
O OMe
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
3) NaClO2, NaH2PO4, COOMe CH2Cl2 COOMe
BzO Me2C=CHMe-H2O HO 2) Et3N, MeOH H2N O
4) TMS-CHN2 (99% in 2 steps)
OBOM (78% in 4 steps) OBOM OBOM
OTBDPS OTBDPS OTBDPS

O O
O
O O
1) t-BuOK, THF 1) Boc2O, Et3N
O NH OMMTr Boc
2) LiBH4, THF DMAP, THF
HO NH OMMTr
3) MMTrCl, pyr. 2) LiOH, MeOH
(88% in 3 steps) (CH2Cl)2-H2O
OBOM OBOM
OTBDPS (84% in 2 steps)
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 Me 1) TFA, MeOH
H
O OAc O O 2) CAN
Me O Me O
O O MeCN-H2O
BocHN BocHN 3) HgCl2 NHBoc
O (96% in 4 steps) O
OBz OBz Et3N-DMF
BOM OH Ac OAc (53% in 3 steps) BocN SMe

OBz OBz
AcO AcO
1) NaIO4
BocHN HO O H O
O MeOH-H2O Boc O
HO N OH
2) TFA, MeOH N
BocN N N O (50% in 3 steps)
O (90% in 2 steps) H
H OBz OBz
Ac Ac
OAc OAc
OBz
AcO
Ac Me O
Ac N O
O
N N O
Ac OBz
Ac
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 N O
MeOH-H2O H2N
N
+ H2N
N
(85%) O O
Ac OBz H H OH H H OH
Ac
OAc OH OH
(TTX-anhydride)

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

TTX: Du Bois Synthesis

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

stereospecific C-H
amination

Me Me
HO O O
Me Me
OH O O
HO OH OR2 N2 OR2
H
OHC OH O O Rh-catalyzed O O
H
HO CO2H C-H insertion
1
R O 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 O Me
Me 1) H2 (1200 psi), Rh-C,
1) Rh2(tpacam)4, CCl4 O O
OTBS CF3CO2H-MeOH O
2) NH3 BH3, CH2Cl2-MeOH Me
2) p-TsOH, 2,2-DMP;
(75% in 2 steps) Me
O O then t-BuCOCl, pyr., THF O
O
(77% in 2 steps) H
t-BuO2C O O CO2t-Bu

TTX: Du Bois Synthesis

Construction of Lactone Bicycle

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

Me Me
O Me O Me
O 1) t-BuCO2H O
HO O Me O
PhCl, 200 oC Me
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 Me O Me O
O Me OH Me O O
O O O
O O O
O Me O NH2
H H
O
Me O (66% in 3 steps)
O O
O O
H Me Me Cl
OH Me Me

Rh2(tfacam)4, PhI(OAc)2, Me O Me O
Me Me
MgO, DCE, 50 oC O O
O
O O
OH
O O O
(55% with 38% reactant) NH (59% in 4 steps) NHBoc
O O
O Cl O
Me 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 O O O O NHBoc
O H2O, 110 oC O BocHN SMe O
NHBoc NH2 N NHBoc
(95%) HgCl2, Et3N,
O O O
O O MeCN-CH2Cl2 O
Me Me (80%) Me
Me Me Me

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

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

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 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.

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 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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