Literture Seminor (M1 part)2011/9/6 (Tus)
Keiichi Kaneko (M1)

Total synthesis of Pinnatoxins A.

Contents
1. History & Introduction
2. Total Synthesisi Of Pinnatoxin A
2-1 Y.Kishi's Strategy
2-2 A.Zakarian's Strategy
3. Revision of the Mode of Action of Pinnatoxin A
4. Summary

History
1) Uemura and coworker isolated Pinnatoxin A from shellfish Pinna
muricata in 1995.
2) Total synthesis of Pinnatoxin A
Y. Kishi -----------------------------------K. Nagasawa ----------------------------M. Inoue and M. Hirama -------------S. Nakamura and S. Hashimoto --A. Zakarian --------------------------------

1998 (J. Am. Chem. Soc.)
2000 (J. Syn. Chem. Jap.)
2004 (Angew. Chem. Int. Ed.)2008 (Angew. Chem. Int. Ed.)2011 (J. Am. Chem. Soc.)

1/16

Introduction
Pinnatoxin A, a marine toxin isolated from Pinna muricata,
exhibits characteristec activity toward the Ca2+ channel.
HN A

B
HO

G
H
CO2OE
O
F OH

O

OC
O
D

(+)-Pinnatoxin A (PnTX A, 1)

Several unique structural features are(i) 27-membered carbocyclic framework,(ii) 19-membered polyether ring,(iii) 14 chiral centers,(iv) bis-spiroketal,(v) imminium and carboxylate functionality.

Proposed biosynthesis of pinnatoxin A

This unique structure, which includes a 6,7-spiro ring (A and G rings), can be explained by the plausible
biogenetic pathway shown here. This biogenesis does not involve the sequence of oxidation steps.

Biological activities of Pinnatoxins
It has been initially suggested that the mode of action of
pinnatoxins involves calcium-channel activation.
However, there is a growing body of evidence thatdifferent mechanism of activity may be operational.

2/16

Kishi's Strategy
Ni(II)/Cr(II)-mediated couplings between
vinyl iodides with C6 and C32 aldehydes.

Kishi's retrosynthetic analysis

HN

B

C

O
6

G
H
CO2OF
OE OH

O

HO O

H3N

A

H
O

O
25

HO O

O

CO2-

32

O

OH

O

D
Their retrosynthetic analysis of pinnatoxin A is based on
Uemura's
biosynthetic
proposal,entailinganintramolecular Diels-Alder reaction to construct the Gring as well as the macrocycle, followed by imine
formation to establish the 6,7-spiro-ring system.

Dithiane-based coupling toform the C25-C26 bond.

Construction of fragment 16
12 steps

OTBS

O

12

16

23

19

HO

HO

H

O

O

3
H

CSA, MeOH

O

H

12
16 O

HO

MgBr2

O
HO

O 19
23

H
4 (30%)O

HO
O

TBSOTf2,6-lutidine
95%

O
O
H

72%

O
OH
5 (51%)

H

OTBS
6
H

H

OPMB

OPMB

O

TBAF, rt,quantitative

O
H

O

HO

O

TBSO

OsO4, NMO;NaIO4, 85%

O

TBSO

1. 4-iodobutyl-p-methoxy-benzyl ether,t-BuLi, Et2O, -78 oC
2. Swern oxidation
3. PPh3CH3Br, n-BuLi, 0 oC

OTBS
8

1. 1,3-dithiane, t-BuLi,
10% HMPA/THF, 92%
I2, PPh3,oimidazole 2. TBAF, 70 C, 95%
92%

O
O

S

H

S
10

3/16

1. t-BuLi, 10% HMPA/THF,
H

H

OPMB

CH2OH

O
O

HO

I

O

OTBS
TBSO

O

HO
O

11 (71%)2. (CF3CO2)2IPh, CaCO3, 82%
3. DDQ, 85%

S

H

O

O

O

O

OTBS
H

S

TBSO
12

10

1. Dess-Martin oxidation, 90%
2.
I

O

O

H
NHAlloc

15

NHAlloc

13
HOI
CO2t-Bu
1% NiCl2/CrCl2, DMSO, 55%
3. HF pyridine, pyridine, THF, 91% 33% NiCl2/CrCl2, bispyridinyl
ligand, THF, 88%
4. Dess-Martin oxidation, 91%

O

O

O
O

O

O
CO2tBu

H

TBSO

O

OH

16

Ni(II)/Cr(II)-Mediated Coupling Reaction
H

H

NHAlloc

NHAlloc
O

HO

O

HO

O

I
O O
H

O
CHO
TBSO

O

O

O

O O

O

O

CO2tBuNiCl2/CrCl2, bispyridinyl ligand
THF

H

CO2tBu
TBSO

O

OH

16

Catalytic cycle1) Nickel(II) chloride is first reduced to nickel(0)with 2 equiv of chromium(II) chloride.
2) Oxidative addition of alkenyl triflates to thenickel(0) takes place.
3) Then transmetalation reaction between the
resulting alkenylnickel species andchromium(III) salt occurs to afford
alkenylchromium reagents, which react withaldehydes to produce the desired allylic
alcohols.
4/16

Bispyridinyl ligand

This process appears to involve the activation of the carbon-iodine bond via Ni(0)or Ni(I), the transmetalation of Ni to Cr, and the carbon-carbon bond formation via
the organochromium reagent. A catalytic cycle of Ni is required for this process tofunction efficiently, suggesting that a chiral ligand for the current purpose mustmeet with the condition that its capacity to form metal complexes should not be
too strong with Ni but should be sufficiently strong with Cr.

The homocoupling reaction was completely suppressed in thepresence of these ligands. With a 1:2 mixture of NiCl2 andCrCl2, the rate of coupling was dramatically enhanced, andreaction even at -20 oC became practical.
I

Me
R

CHO

NiCl2 /CrCl2

+
O

R=

OMe

O

TBS
TBS

Me

Me
OH

R

+

3

O
TBS

O

OH

R

OMe

4
O

OMe

Construction of (-)-Pinnatoxin A
H

H
O

HO

O

O
O O

NHAlloc

NHAlloc
1. TFA, CH2Cl2, H2O, 71% TESO
2. MsCl, TEA, -78 oC, 85%
3. TESOTf, 2,6-lutidine, 79%

O

O
O
O

CO2t-Bu

H

TBSO

O

O

O
O

H

OH

CO2t-Bu
OMs
OTBS
17

16
H2N
DABCO, TEA, benzene;
70 oC, 0.2 mM diene in 1. HF pyridine, pyridine, THF, 94%
dodecane, 78%
2. Pd(PPh3)4, AcOH, toluene, 82%

O
1. 200 oC, 1-2 Torr, 70%
CO2t-Bu 2. 1:1 TFA/CH2Cl2, 95%

H
O

O

O
HO

CO2-

H
O
O

HO

OH

O
19

HN
O

O

O
O

OH

Conclusion
Kishi's group have completed the first total synthesis of pinnatoxin A utilizing
a biomimetic intramolecular Diels-Alder reaction This synthesis has alsoestablished the absolute stereochemistry of pinnatoxin A as the antipode of
the structure 1.

(-)-PnTX A (1)
5/16Zakarian's Strategy
Zakarian's retrosynthetic analysis

Cl

Construction of fragment 6
Ph
BnO

O
+

HO

OPMB
O

8
46% overall yield, 9 steps from(S)-citronellic acid

BnO

Cl

Cl
O Cl
Et3N, DMAP, C6H6

O

TIPSO

OSEM OH
946% overall yield, 10 steps from
D-ribose

O

OPMB
O

84%

OSEM

O
Ph

OTIPS

O
10

A rapid and mild esterification by means of mixed anhydride

2,4,6-Trichlorobenzoyl chloride was proved to be themost satisfactory one in rate and in the yield of thealcoholysis.
Aromatic hydrocarbons, such as benzene or toluene,
were found to be the most suitable solvents for thealcoholysis step.

10(S)-11-CF3 or (S)-11-Ph
THF, -78 oC; Me3SiClO

OSiMe3
R

OBn

OPMB

OSEM
R= O
OTIPS
Ph O
20 oC, 12 h

OH
R

O

OBn

OPMB

then aqueous
work-up

LiAlH4, Et2O
BnO
OPMB

HO
O
Ph
O

H
OSEM
OTIPS

12
C5 center constructed.

6/16

Highly stereoselective enolization of the acyclic , -disubstituted ester.
A. Zakarian, SY NLETT , 2010, 11, 1717.
K.Koga, JACS, 1986, 108, 543.

i) Enantioselective deprotonation by chiral lithium amide bases
O(S)-11-Ph-78 oC, THF

RO

OLi
96% E with (R)-11-Ph"Zakarian, ester

RO

96% Z
[Characteristics of this chiral reagent]
R R1
R2
R3 4
X N
Li

It is shown that the degree of asymmetric induction ishighly dependent on the structures of chiral bases
used and also on the bulkyness of the alkyl groups incyclohexanones.
5-membered chelated structures are expected to beformed for the lithium amide as upward, where theisopropyl group on nitrogen should be exclusivelytrans to the bulky substituent on the chiral carbon forsteric reasons. This infers that the direction of thelone pair on chiral nitrogen to be used for
deprotonation is fixed.

For deprotonation to occur by synchronous proton
and lithium ion transfer, the carbonyl group in 1 will
coordinate to the lithium from the same side as thelone pair.

ii) Reductive enolate formation from bicyclic thioglycolate lactams

O
N

Me
Et
S

OLi
LiDBB, THF-78 oC

Et

N

Me
94% Z

H

J. L. Gleason, OL, 2009, 11, 1725.
J. L. Gleason, JACS, 2001, 123, 2092.

" Gleason, amide

H

Highest levels of stereocontrol are usuallyassociated with cyclic frameworks, while
control based on differential stericenvironments is less reliable.

SLi

They report a method for controlling enolate geometry in disubstituted amide enolates where the E/Z selectivity
is dependent only on the geometry and stereochemistry of the enolate precursor.
Model for stereoselective enolate genaration
The design utilizes a two-electron
reduction of , -dialkylated bicyclicthilglycolate lactams to provide
disubstituted amide enolates.
7/16

Reductive Enolization of Bycyclic Thioglycolate Lactams

Assuming thata) two alkyl groups (R1 and R2) are installed
stereoselectively at -position,b) the O-C-C-S dihedral angle is held as close to
90o as possible by the bycyclic system, andc) significant bond rotation does not occur about the
carbonyl-carbon/ -carbon bond during the twoelectron reduction process, the E/Z stereochemistryof the enolate should be controlled by the relative
position of R1 and R2 in the starting lactam.

The method affords both E- and Z-amide enolates without relying on a steric difference between the two
substituents.
iii) Stereocontrolled Alkylative Construction of Quaternary Carbon Centers
A. G. Myers, JACS, 2008, 130, 13231.

O
N
OH

OLi

LDA, LiCl
0 oC, THF
" Myers, amide

N
OLi
Z-isomer
They propose that in the favored pretransition state
assemblies the alkoxide side chain and base arepositioned on opposite faces of the incipient enolate, withthe -C-H bond aligned for deprotonation.

Proposed pretransition state assemblies leading to favored(Z) and disfavord (E) grometric enolate isomers by
sequential deprotonations of pseudoephedrine amide.
The electrophile is proposed to approach theenolate -face opposite the alkoxide side
chain. Benzylation of the Z-enolate is bothmore diastereoselective and more rapid thanbenzylation of the E-enolate.
Z- and E- -methyl- -ethyl disubstituted
pseudoephedirine amide enolates arealkylated preferentially from a common
diastereoface.

8/16

BnO

1. BzCl, Py.
2. DDQ, CH2Cl2-H2O
3. O3, N-methylmorpholine
N-oxide, CH2Cl2
4. NaBH4, EtOH

OPMB

HO
O
Ph
H
OSEM

O

BnO

O

77 %

OH

BzO
Ph
O

OH
H
OSEM

OTIPS
OTIPS
13

12

BnO

BnO
(COCl)2, DMSO,
Et3N, CH2Cl2

Bn2NH2+CF3CO2PhMe, 50 oC

BzO
Ph
O

swern oxidation

H
O
O
OSEM

O

fast (5 min )

Ph

BzO
O
H
OH
OSEM

O

OTIPS

OTIPS
14

15

BnO

-H2O
slow (20 h)
89 %1. NaBH4, EtOH
2. MOMCl, Bu4NI, i-Pr2NEt
3. n-Bu4NF, THF
4. (COCl)2, DMSO; NEt3
5. Ph3PCH3Br, KN(SiMe3)2

BzO
Ph
O
O

O

H
O
OSEM

BnO

Ph

BzO
O
O

87 %

H
OMOM
OSEM

OTIPS
16

17

BnO
O

1. LiAlH4, Et2O
2. (COCl)2, DMSO,
Et3N
87 %

Ph

O
O

H
OMOM
OSEM
6

16 % yield from (S)-cirronellic acid
(26 steps)

9/16

Synthesis of BCD-Bisketal Fragment 33

OAc O
Ot-Bu

O

Aminolipase PS-D,
vinylacetate, pentane
30 oC

OH
Ot-Bu
O

CO2t-BuO

18

19
44 %, > 95 % ee
OH O

HO

O

Ot-Bu

OH
21

33

49 %, > 95 % ee

Acylation of racemic alcohol 18 was terminated at 50 % conversion, and the products were separated bycolumn chromatography.
1. LiAlH4
2. Me2C(OMe)2
TsOH

21

80 %

O

O

O

OH O

88%

1. 10 % Pd/C, H2, EtOAc;
2. MeC CCH2CH2MgBr, THF

O

O

O

24

23

24 was not reliable due to difficulties in
efficient generation of the Grignard reagent.

1. 10 % Pd/C, H2, EtOAc
SiMe2Ph2.

O

1. Swern oxidation;
2. NaH,(EtO)2P(O)CH2CON(Me)OMe

91%

22

OMe
N
O

O

Sia2BH, THF;
NaOH, H2O2

SiMe2Pht-BuLi, Et2O

Li , THF

I

28

THPO

3. NaBH4, MeOH; 97%
4. TBSCl, imidazole; 99%O

SiMe2Ph

Ot-Bu
1. NIS, (CF3)2CHOH
2. 20, Pd(dppf)Cl2
Ph3As, Cs2CO3

90% from 23
TBSO

O

O

TBSO

O

O

29
OTHP

OAc O
Ot-Bu
19

1. DHP, TsOH
2. 9-BBN, THF

30

B
CO2t-Bu
20
10/16

THPO

THPO

O
1. AD-mix
2. n-Bu4NF
3. (COCl)2, DMSO

Ot-Bu

O
Ot-Bu
camphorsulfonicacid, MeOHO

HO

61% from 29
TBSO

O

O

31

30

HO

O

O

Ot-Bu

change solvent
to cyclohexane
25 oC, 48 h

Ot-Bu
HO

O

O

O

O

78%

O
HO

O

19

O

OH OH

O

OH

33
32

Hydrogen-bonding stabilization in spiroketal intermediates
19

19

15

Upon superficial analysis, isomer 33 fully stabilized byanomeric effect should be increasingly more favored
over its epimer 34 lacking anomeric stabilization at
C19 as the medium polarity

D-ringThe C19 epimer 34 can be stabilized by a hydrogen bond between the tertiary hydroxy group at C15 and the Dring oxygen, while the desired isomer is stabilized by long-range hydrogen bonding between the terminal ester andhydroxyl groups. The unusual long-range hydrogen bonding is supported by crystallographic studies.
SYNLET T, 1997, 18, 298.
The structure of 35 was unambiguously established by X-ray
analysis, which indicates the existence of an intermolecularhydrogen bond between the terminal diol groups. Thishydrogen bond must play an important role in stereoselective
formation of 35 in toluene.

11/16

Effect of Selective Silylation on Relative Stability of C19 Epimers

Upon extended reaction times, the desired diastereomer
33 is produced as a major product; however, the C19
epimer is formed initially and thus is favored underkinetic control.
19

19

15

D-ringIn order to further ascertain the effect ofhydrogen bonding on the selectivity ofspiroketalization,
They carried out equiliaration experiment withselectively silylated substrares. In all cases, the desiredconfiguration at C19 was favored, but the level ofpreference varied substantially.

As shown the Table, silylation of the tertiary hydroxyl group (Et3Si) increased the ratio significantly to 13:1 (entry 3).
These results indicate that when only the anomeric effect is operational in the fully silylated substrate(entry 3), the thermodynamic ratio of 8.3:1 is observed in toluene, and hydrogen bonding from both
tertialy and primary and primary hydroxyl groups has a significant effect on the position of theequilibrium.

Solvent Effect on the Spiroketalization
As the solvent polarity decreased, we obseved anincreased ratio of the desired diastereomer to its
C19 epimer. And the optimal selectivity (9.8:1) was
achieved when cyclohexane was used as thesolvent.

12/16

Construction of fragment 7
O

O
Ot-Bu

O

1. i-Pr3SiCl, ImH
2. Et3SiCl, ImH
3.i-Bu2AlH

O

HO

O

4. PMBOCH2CH2CH2Li
5. DMP, Py
6. Me3SiCH2Li
7. KN(SiMe3)2O
Et3SiO

OH

O

33

O

OTIPS

36

OPMB

I
1. DDQ, H2O, CH2Cl2
2. I2, Ph3P, imidazoleO
O

Et3SiO

76% from 33

O

O
O

Et3SiO

87%

OTIPS

O

OTIPS

7
13% overall yield, from acrolein(26 steps)

37

Completion of PnTX A
BnO

BnO

OH

O
Ph

O

Ph

H
OMOM
OSEM

O

t-BuLi, Et2O
-78 oC, 1 h

6

O

O

H
OMOM
OSEM

O
Et3SiOO

1. n-Bu4NF, THF
2. DMP, CH2Cl2
3. vinylmagnesium
bromide, THF

O

78%

78%

38

7
OTIPS
BnO

BnO

O

Ph

O

O
O
Et3SiO

O

H
OMOM
OSEM

Cl
Ru
Cl
O

O

Ph

15

Et3SiO

O

O
H
OMOM
OSEM

O
O
O

39

0.2 equiv.
75%

HO

O

NMe

MeN

OH

41
13/16

O
Ph

O
Et3SiO O

O

1. DDQ, H2O, CH2Cl2
2. Ts2O, Py, CH2Cl2
3. NaN3, DMF, 80 oC

BnO

1. DMP, Py, CH2Cl2
2. MeCu(CN)Li
BF3 Et2O

BnO

O

Ph O
73% from 41
O
H
O
OMOM
O
OSEM
Et3SiO O
O
41
O
OH
O

66%

H
OMOM
OSEM

N3
O

43
O

HN
CO2-

H
O

O

O
HO

O
O

OH

1. TEMPO, PhI(OAc)2
2. NaClO2, NaH2PO4
3. Me3SiCHN2
4. H2, Pd/CaCO35.
CO2-HNEt3+
6. LiOH, THF-H2O

O
H
OMOM
OSEM

O

N3

Et3SiO O
O

O
H
O

O

OH
OH

O
HO

Ph

O
O

37-43%

O

44

H
O

CO2-

LiBF4
i-PrOH
H2O
71%

4

1-(+)-PnTX A

Mode of Action of Pinnatoxin A
H3N
HN
H
O

O
HO

O

O
O
O

OH
HO

O
(+)-PnTXGi) PnTX A blocks human 7 and 4 2, and Torpedo 12

O

O

OH

O
PnTX AKnAChRs in oocytes

Expressing (a) human 7 (b) Terpedo 12
and (c)human 4 2 nAChRs, before (black tracing) and after(red tracing) the action of PnTX A.
[Characteristics]
PnTX A did not affect the rate of desensitization in
the verious subtypes.
Inhibitory potency of PnTX A is dependent on the
nAChR receptor subtype.
PnTX AKPnTX APnTX AK had no action on the different nAChR
subtypes. These results indicate that the spiroimine
component is an important structural factor forblocking nAChRs by PnTX A.
14/16

a: Oocytes expressed human neuronal 7 or 4 2
nAChR subtypes or were microtransplanted withmuscle-type 12
nAChR.
b: Mean values from concentration-response
curves were recorded on 46-50 oocytes for eachexperimental condition; 95% confidence intervals
are shown in parentheses.

ii) Binding Competition Experiments between PnTX A and Radiolabeled Ligands

Effect of PnTX A and AK on various nAChRs.
Inhibition of specific [125I] -bungarotoxin or ( )-[3H]epibatidine
binding by increasing concentrations of PnTX A or AK on(a) Torpedo and neuronal 7-5HT3 or(b) heteropentameric 3 2 and 4 2 nAChRs.
The results are expressed as the ratio of the specificradiotracer binding measured with (B) or without (B0)competitive ligands, expressed as a percentage.

PnTX A binds to these receptors with affinities innanomolar range, and its order of potency on thevarious nAChRs subtypes is 7-5HT3 > Torpedo >Studies on the binding potency of PnTX AK on
the nAChRs subtypes showed that this toxin
produces no significant displacement of theradioactive tracer, even at high concentrations.

Thus, disruption of the imine ring in PnTX A is
responsible for the drastic loss of affinity of thiscompound for the various nAChR subtypes.

15/16iii) Potential inhibitory activity of 1 M PnTX A or PnTX AK was evaluated on CHO cells stably
expressing the distinct human muscarinic acetylcholine receptor (mAChR) subtypes
Inhibition of interaction of [3H]NMS with five humanmAChR subtypes by various ligands.
BT is [3H]NMS total binding, and NS is nonspecificbinding in the presence of 50 M atropine. Note thatthe effect of PnTX A and PnTX AK was evaluated at
1 M concentration.

As shown here, both PnTX A and PnTX AK had no significant effect on 3H-NMS binding to
M1, M2, M3 and M4 mAChR subtypes, whereas PnTX A induced 35% radiotracer
displacement in M5 mAChR, indicating a low micromolar affinity for this receptor subtype.

Summary
Pinnatoxin A: Selective Inhibition of nAChRs
Indeed, until now, the mode of action of pinnatoxins was ascribed to action on calcium channels.However, in thisstudies, even at 10 M PnTX A, no significant binding activity was detected for calcium channels.
Toxicological profile is also seen with agents affecting nicotinic receptors.
[The reason of the inactivity of PnTX AK with nAChRs]
This inactivity can be explained by the existence of conformers strongly stabilized byan intramolecular ionic interaction between the ammonium and carboxylate groupsof PnTX AK in solution.

[The cyclic imine moiety represents the key feature in this family of toxin]1) In conjection with the C28 hydroxy groups of the bridged EF-ketal, it anchors
the ligand to the binding site through hydrogen bonds in a conformation ideally
positioned to optimize the interactions with neighboring residues.
2) Binding of the closed imino ring A in PnTX A appears to be more favorable,both sterically and energetically, than that of the corresponding open amino
ketone form in PnTX AK. The functional signature in the cyclic imine
phycotoxins, interacting with nicotinic receptors as has been shown for PnTX A.
Finally, aside from their clear effect on nAChRs, interaction of cyclic amine toxins with other targets of thecholinergic neurotransmission pathway, especially the muscarinic receptors (mAChRs), has also been reported
recently. Although no interaction was observed with M1-4 mAChRs subtypes, a weak effect of PnTX A wasmeasured for the M5 subtype, suggesting a low micromolar affinity of this toxin for the M5 receptor subtype.

16/16