1  

Journal Club 2014.12.18 Yuzuru Kanda (YUK) Synthesis and Biological Evaluation of QRSTUVWXYZA′ Domains of Maitotoxin

Kyriacos. C. Nicolaou, Philipp Heretsch, Tsuyoshi Nakamura, Anna Rudo, Michio Murata, and Keiichi Konoki J. Am. Chem. Soc. 2014, 136, 16444–16451.

§1. Introduction 1–1. Maitotoxin (MTX) 1976: First isolation from Ctenochaetus striatus (a kind of fish) by T. Yasumoto 1988: Examination of chemical properties by A. Yokoyama, M. Murata,Y. Oshima, T. Iwashita and T. Yasumoto 1996: Determination of complete structure by T. Nonomura, M.Sasaki, N. Matsumori, M. Murata, K. Tachibana,

Y. Kishi and T. Yasumoto

 Figure 1. Structure of MTX

1–2. Hypothetical Bioactivities (*Exact mechanism is still under investigation) ♦ Sinkins’ Hypothesis[1]…MTX converts the Ca2+ pump into the Ca2+–permeable nonselective cation channel.

• The structurally related marine toxin, Palytoxin, binds to the Na+–K+–ATPase and converts the Na+ pump into a nonselective cation channel (Figure 2).

• Ca2+–ATPase overexpressed insect cells and human kidneys cells were subjected to MTX and showed increase in MTX–induced whole cell membrane current.

♦ Murata’s Hypothesis[2]…W–F’ domain works as an anchor that binds to the α–helix active site

• Yessotoxin’s Ladder–Shaped Polyether structure, which is similar to W–F’ domain of MTX, binds to the α–helix peptide active site of Ca2+–ATPase.

• The average distance of ether oxygen atoms on one side matches the α-helix pitch of the active site (Figure 4).

? MTX binds to active site of Ca2+–ATPase with W–F’ domain and converts it to Ca2+–permeable nonselective cation channel.

AB C D E F

G IH J K

L M

NO

PQRST

UVW

XY

A'

Z

B'C'D'E'F'

2007: K. C. Nicolaou

2008: K. C. Nicolaou1996: K. C. Nicolaou

2008: T. Nakata

2008: T. Nakata

2008: T. Nakata

2010: K. C. Nicolaou

2010: K. C. Nicolaou

2011: K. C. Nicolaou

2011: K. C. Nicolaou

1996: K. C. Nicolaou

1996: K. C. Nicolaou

O

OO

H

MeOH

O

O

O

O

O

O

O

O O

O

O

OO

O

O

OO

OO

OO

OO

OO

O

O

O

OHO

NaO3SO

Me Me

HO OH

HOOHMe

OH H H

HOH

Me

H

H Me

H

H

H

OH

H OHOHH H H H OH

NaO3SOOHH H H OH

H H

H OH

OH

H

HOH

H

HO

OH

H OH

OHH

H H

OH

HO

H

H

OH

HMeHMe

HH

H

HMe

H Me H MeOH

MeMe

HH

H

H

HMeMeMe

Me MeHO

H H H H H HMe

Me

MeH

OH

OH

2014: K. C. Nicolaou(This Work)

✔ The largest secondary metabolite (3422 g/mol) ✔ The most potent neurotoxins (50 ng/kg) ✔ 32 rings and 98 asymmetric carbons

 Figure 2. (a) Free Na+–K+–ATPase NKA (b) Complex with Palytoxin

Palytoxin

Ca2+

(a)

(b)

Q–F' domainrich in ladder Poly Ether

F'

E'

D'

Z

A'

B'

C'

Y

X

W

HydrophobicLSP

α-helix

Figure 4. Possible structure of MTX interacting with active site

Figure 3. YTX, Names of rings correspond to similar rings of MTX.

OSO3NaO

O

OO

OO

O

O

O

O

HH

HH

Me

Me

HMeH

H H H H H HH

H

HH

WX

Y

A'

Z

B'C'D'E'F'HO Me

OH

MeOSO3Na

  2  

1–3. Remaining Challenges and This Work ✗ Total synthesis has not yet been achieved ✗ Mechanism of bioactivity is unknown §2. Results and Discussion (*Some parts are omitted accordingly for clarity and space limitation) 2–1. Synthesis of WXYZA’ domain, Synthesis of WXYZA’ Ketophosphonate 13

Scheme 1. Synthesis of 1–7,  Cascade Takai-Lombardo Olefination/Ring Closing Metathesis and its essence  

 

Scheme 2. Synthesis of 9–11, hydroxydithioketal cyclization methylation sequence and its essence [3]

     

O

OTBSO Me Me

Me

HHOTBDPSOPMB

1) TBAF2) TsCl3) TBSOTf4) KCN

1) DIBAL-H2) NaBH43) TBDPSCl4) DDQ

A' ZO

O

CN

A'O

OMe

OHA' Z

TBDPSO1

previously synthesized2 3

Z

Me

OPMB

Me Me

4, MNBA, Et3N,DMAP (cat.)

O

O Me

Me

OA' Z

OHO

OOTES

Me Me

OBn

OTBDPS

WO

OOR

Me Me

OBnW

1) p-TsOH•H2O (2 eq)

MeOH:DCM = 3:1, 0 ºC, 40 min

5: R = TES6: R = H

4previously synthesized

O

OA' Z

OTBDPS

O

Me

O

OR

MeMe

Y

WH

5 7

Cascade Takai-Lombardo Olefination/Ring Closing Metathesis

Me

Me2) TiCl4 (1.0 M in DCM, 50eq)TEMEDA (285 eq), Zn (110 eq)PbCl2 (5 eq), CH3CHBr2 (50 eq)

THF, 0 → 65 °C, 1.5 h3

Me Br

Br

TiCl2

Me

Me

Me

OZ

O

5

Me

Me

OZ

O

TiCl2

Me

Me

OZ

O

TiCl2Z

O

Me

Y

H7

TiCl2

Me

Ti Carbene Complexe

EssenceZn, TiCl4

IntramorecularTakai-Lombardo Reaction

ZO

Me

Y

H OTiCl2

OHOH OH OH

TES group was removed in advacenot to hinder this olefination

W W W

W

W

✔ Regeneration of intramolecular Ti Carbene Complex✔ Generation of 7 membered ring by intramolecular steleoselective Taki–Utimoto reaction

1) TMSOTf

2) CyBH23) DMP

7: R = H8: R = TMS

9

O

OA' Z

OO

OTMS

Me

Y

W

O

H10a: α-SEt10b: β-SEt(α:β ca 1:2)

O

OA' Z

OO

H

Me

Y

WH

OSEt

X

Hydroxydithioketal Cyclization Methylation Sequence

1) m-CPBA (4 eq)2,6-di-t-Bu-4-methylpyridine (5 eq)DCM, –78 → –10 °C, 20 min

2) Me3Al (30 eq), –78 → 0 °C, 1 hDCM

11

O

OA' Z

O

Me

O

H

Me

Y

WH H

OMe

X

Me Me

Me

Me Me Me

TBDPSOMe Me

1) p-TsOH•H2O (0.1 eq)MeOH:DCM = 1:10 °C, 30 min

2) Zn(OTf)2 (5 eq)EtSH:DCM = 4:1 (0.011 M)25 °C, 1.5 h

7

=> Fragments coupling entails huge importance to accomplish total synthesis => Q–A’ domain contains the fragment promisingly binds to the active site  => Understanding bioactivity provides insights into anticancer drug discovery  

OO

H

Me

Y

WH

O

S

X

Me

m–CPBA

OOEt

OO

H

Me

Y

WH

OX

Me

=MeAlMe

AlMe3

Essence

9 OO

OH

Me

O

HMe

Y

W

OO

OH

MeH

Me

Y

W

Zn(OTf)2

EtSH

O

OO

OH

MeH

Me

Y

W

OSEt

10

OO

H

Me

Y

WH

OSEt

X

Me

O OOMeMe

X

MeAlMe

Me

11O

H

Me

Y

WH

OMe

X

Me

OO

OH

MeH

Me

Y

W

SEtSEt

Hydroxydithioketal

Zn(OTf)2

EtSH

OO

OH

MeH

Me

Y

W

SEtSEt

H

Deprotection

TsOH/H2O

MeOH:CH2Cl2 = 1:10 °C, 30 min Cyclization was supressed

by low RXN Temp and short RXN time

OO

OH

Me

O

HMe

Y

W

X

"S" is more nucleofilic than "O"

✔ Genration of hydroxydithioketal intermediate then stereoselective ring formation✔ Stereoseletive methylation

MeO

"S(O)2Et" is better leaving group than "OH"

  3  

Scheme 3.  Synthesis of 12–13, completion of W–A’ domain  

 

2–2. Synthesis of QRSTU Aldehyde 18, Fragment Coupling and Completion of QRSTUVWXYZA’ Domain Scheme 4. Synthesis of 14–20, coupling of Q–U domain and W–A’ domain by Honor–Wadsworth Emmons Coupling

 

 

 

  Scheme 5. Synthesis of 21–24, reductive hydroxyketone ring closure and its essence [4]  

 

1) TPAP (cat.), NMO2) (MeO)2P(O)CH2Li3) DMP

13overall yield 8.1%

O

OTBSO Me

HH

A' Z

TBDPSO

O

Me

O

H

MeMe

OBnY

WH H

OMe

X

O

P(O)(OMe)2

1) DIBAL-H2) TBDPSCl

12O

OA' Z

OO

HO

Y

W

OX

MeMe

Me MeMe

11

OBnO

OO

O

OMe

HMeHH

H Me H MeH

Me

OBn

QR

STUO

SiO

t-But-Bu

OBn14

previously synthesized

OHO

OO

O

O

Me

Me Me

Me

OBn

QR

STU

OH15

Pd/C (cat.), H2

1) TEMPO (cat.),PhI(OAc)22) Ph3P=CH2Me

OHO

OO

O

O

Me

Me Me

Me

OBn

QR

STU

16

Me

1) TBAF2) TESOTf3) PPTS OTES

O

OO

O

OMe

Me

Me Me

Me

QR

STUTESO

X

1) TPAP cat.NMO2) 13 (1 eq)Ba(OH)2•8H2O (1.5 eq)then 18

THF: H2O = 6:125 °C, 4.5 h

17: X = O, H18: X = O

O

OO

O

OMe

Me

Me Me

Me

QR

STU

TESO

O

H

Me Me

OBn

HO

O

O

O

O

MeMeMe

TBSO

TBDPSO

HH

HA'Z

Y X W

19

Horner–Wadsworth–Emmons Coupling

17

O

OO

O

OMe

Me

Me MeH

Me

QR

STU

TESO

OMe MeO

O

O

O

O

MeMeMe

TBSO

TBDPSO

A'Z

Y X W

20

1) TBAF2) TESOTf[(PPh3)CuH]6

OTESO

OO

O

OMe

Me

Me Me

Me

QR

STUOMe Me

O

O

O

O

O

MeMeMe

TESO

TESO

A'Z

Y X W

21

TESO

BiBr3 (0.5 M in MeCN, 3 eq)TESH (50 eq)

MeCN:DCM = 4:1, –10 °C, 2 h

Me

O

OO

O

MeMe

Me

HO

HO

A'Z

Y X W

22OH

O

OO

O

OMe

Me

Me Me

Me

OBn

QR

STUO O

Me V

H

H

H

Reductive Hydroxyketone Ring Closure

Confirmed by NOE and 13C NMR

BiBr3

2HBr

H2O

BrBiO

U

O

O

W

21

Et3Si

O HEt3Si

HO

W

V

Br-

O H

HO

W

V

O HW

V

Br-

Et3SiBr

Et3SiBr

Et3SiOH

HBr

H2O

O HW

V

Et3SiOH

Et3SiOSiEt3

Et3SiH

Essence

✔ Stereoselective ring formation✔ TES deprotection, taking advantage of a byproduct HBr

22

U

U

U

U H

MeH

O

OO

O

MeMe

Me

HH

HA'Z

Y X W

23overall yield 31.3%

OHO

OO

O

OMe

HMeH

Me H MeH

Me

OBn

QR

STUO O

OBn

Me V

H

H

H

Me2C(OMe)2, CSA (cat.)

O

O

HMeMe

Pd(OH)2/C (cat.),

H

MeH

O

OO

O

MeMe

Me

HO

HO

HH

HA'Z

Y X W

24overall yield 37.8%

OHO

OO

O

OMe

HMeH

Me H MeH

Me

OH

QR

STUO O

OH

Me V

H

H

H

Me

  4  

2–3. Biological Evaluation • 19 different fragments, including previously synthesized A–E, A–G, Q–U, Q–A’, W–A’ and C’–F’ domains and its

analogs, were subjected to rat glioma C6 (a kind of cancer) cells (Figure 5) and human tumor cells. => Compound 24, 25 (Q–A’) and 26 (C’–F’) (Figure 6) gave a positive reaction to the Ca2+ influx examination. => Compound 24 exhibited significant growth inhibition against 10 different humane tumor cells.    

Others, A–E, A–G, Q–U domains and its analogs were completely inactive or slightly active.

Figure 5.  Process of the biological evaluation  

 

Figure 6.  Partial structures of MTX that induced Ca2+ influx

✔ Some domains bound to the active site, while all domains did not induce Ca2+ influx. => Binding domains and Ca2+ influx inducing domains are not the same. => More than 2 domains play a role of converting Ca2+–ATPase to Ca2+–permeable nonselective cation channel. ✔ Q–A’ domain and C’–F’ domain effectively bound to the active sites (consistent with Murata’s hypothesis). ✔ W–A’ domain nor S–U domain were not active (inconsistent with Murata’s Hypothesis). => Both S–U domain and W–A’ domain are necessary to bind to active sites. §3. Conclusion ✔ Succeeded in synthesizing QRSTUVWXYZA’ domains, which was remarkable advance towards total synthesis ✔ Evaluated bioactivity of different 19 domains and got new insight into the mechanism of bioactivity of MTX ✔ Promising anticancer activity was observed

§4. References [1] W. G. Sinkins et al. Am. J. Physiol. Cell Physiol. 2009, 297, C1533–C1543. [2] M. Murata et al. Bull. Chem. Soc. Jpn. 2008, 81, 307–319. [3] K. C. Nicolaou et al. J. Am. Chem. Soc. 1989, 111, 5321–5330. [4] P. A. Evans et al. J. Am. Chem. Soc. 2003, 125, 11456–11457. Abbreviations: MNBA = 2,6-methylnitrobenzoyl anhydride; TPAP = tetra-n-propylammonium perruthenate; NMO = N-methylmorpholine-N-oxide; PPTS = pyridineium p-toluene sulfonate; CAS = (±)-camphor-10-sulfonic acid

Rat glioma C6 cell1 mL (25000

cells/mL)

Compound of interest in MeOH

incubation buffer (250 mL)13 min

binding site "blocked" cell

45CaCl2 (50 µL, 6 µCi/mL)MTX

12 min

activecompound

inactivecompound

normal cell

binding site "blocked" cellRadio inactive

45Ca2+ containing cellRadioactive

Ca2+X

Ca2+

45CaCl2 (50 µL, 6 µCi/mL)MTX

12 min

Ca2+

O

O

OO

OO

OO

OO

O

MeHMe

HH

H

HMe

H Me H MeOH

MeMe

HH

H

H

HMe

Me

MeH

OH

QRST

UVW

XY

A'

Z

OH

O

O

MeMe

Me

O

O

O

OTBDPSO

MeMeMe

H H H H

C'D'E'F'OH

26previously synthesized

25This Work