De Novo Synthesis of Possible Candidates for the Inagami-Tamura Endogenous Digitalis-like Factor

Article abstract of DOI:10.1021/acs.joc.7b01640

De novo synthesis of possible candidates for the Inagami-Tamura endogenous digitalis-like factor (EDLF) was achieved to validate a previously proposed structure. Our synthetic approach involves a highly regio- and diastereoselective Mizoroki-Heck reaction and a Friedel-Crafts-type cyclodehydration to construct steroidal tetracycle 14 as a versatile common intermediate leading to seven 2,14β-dihydroxyestradiol analogues 1a-c, 2a-c, and 3 as possible candidates. By comparing the potency of inhibitory activity against Na⁺/K⁺-ATPase between the synthesized candidates and the EDLF, it was found that the proposed structure is not likely to be a true structure of the Inagami-Tamura EDLF.

Full text of DOI:10.1021/acs.joc.7b01640

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Article
De Novo Synthesis of Possible Candidates for the
Inagami-Tamura Endogenous Digitalis-Like Factor (EDLF)
Atsuo Nakazaki, Keiko Hashimoto, Ai Ikeda, Takahiro Shibata, and Toshio Nishikawa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.7b01640 • Publication Date (Web): 08 Aug 2017
Downloaded from http://pubs.acs.org on August 8, 2017
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De Novo Synthesis of Possible Candidates for the Inagami-Tamura
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Endogenous Digitalis-Like Factor (EDLF)
Atsuo Nakazaki,* Keiko Hashimoto, Ai Ikeda, Takahiro Shibata, and Toshio Nishikawa
Graduate School of Bioagricultural Sciences, Nagoya University, Furoꢀcho, Chikusa,
Nagoya 464ꢀ8601, Japan.
ABSTRACT: The de novo synthesis of possible candidates for the InagamiꢀTamura
endogenous digitalisꢀlike factor (EDLF) was achieved in order to validate a previously
proposed structure.
Our synthetic approach involves a highly regioꢀ and
diastereoselective Mizoroki–Heck reaction and a Friedel–Craftsꢀtype cyclodehydration
to construct steroidal tetracycle 14 as a versatile common intermediate leading to seven
2,14βꢀdihydroxyestradiol analogues 1aꢀc, 2aꢀc, and 3 as possible candidates. By
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comparing the potency of inhibitory activity against Na /K ꢀATPase between the
synthesized candidates and the EDLF, it was found that the proposed structure is not
likely to be a true structure of the InagamiꢀTamura EDLF.
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INTRODUCTION
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Cardiotonic steroids are originally isolated from plants, toads, snakes, and insects, and
they cause an increase in calcium concentration through the inhibition of
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Na /K ꢀATPase, leading to contraction of cells. Cardiotonic steroids possess common
structural features: (1) a βꢀhydroxy group at C14 in a steroidal scaffold bearing
cisꢀfused AB and CD rings, (2) a βꢀconfigured 5ꢀ or 6ꢀmembered unsaturated lactone
moiety at C17 (cardenolides or bufadienolides, respectively), and (3) the presence of a
variable number of sugar residues at C3. Lactones are considered to be the most
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essential functional group for biological activity of these molecules. A variety of the
related steroids has also been isolated from mammalian tissues and fluids. These still
unidentified compounds are referred to as the endogenous digitalisꢀlike factor (EDLF)
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(
Figure 1). Ouabain, the most wellꢀknown EDLF candidate, has been isolated from
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a
5b
human plasma and bovine hypothalamus and structurally characterized by FABMS
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and H NMR. Although EDLF plays a role in various disorders, most EDLF structures
remain ambiguous because the obtained amounts are typically quite small.
A candidate for EDLF was isolated from bovine adrenal by Inagami and Tamura in
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1987.
This compound, named InagamiꢀTamura EDLF, exhibited all of the
representative properties of an EDLF: (1) potent inhibitory activity against
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Na /K ꢀATPase (postulated IC50: 9.9 ~ 182 nM) comparable to ouabain (IC50: 23 nM);
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(2) competitive displacement activity against [ H]ꢀouabain bound to the enzyme; (3)
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inhibitory activity for
Rb uptake into intact human erythrocytes; and (4)
Overall, this compound
crossꢀreactivity with antiꢀdigoxinꢀspecific antibody.
functioned in a manner similar to that of ouabain. FABꢀLRMS spectra showed an m/z
value of 336, and biological evaluation suggested that it possesses a steroidal scaffold
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rather than a lipid or peptide composition. NMR spectra could not be acquired due to
the insufficient amount isolated (multiꢀmicrograms), and therefore, the structure has not
been elucidated.
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In 1996, Sakakibara and Uchida proposed a 2,14βꢀdihydroxyestradiol analogue
having two hydroxy groups, compound A, as a structural candidate for the
InagamiꢀTamura EDLF based on this limited information and their own deductions
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(
Figure 2). Compound A possesses a unique structural motif not found in other
reported EDLF candidates such as ouabain, owing to its unsaturated Aꢀring moiety.
Sakakibara and Uchida envisaged that the positions and stereochemistries of the
residual hydroxy groups on the dihydroxyestradiol basic scaffold could be determined
by comparison of the inhibitory activity of synthetic compounds. However, two
possible candidates semiꢀsynthesized from estradiol, 1a and 1b, exhibited low
contractile activity of isolated rat aorta and guinea pig left atrium, indicating insufficient
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potency for the InagamiꢀTamura EDLF. The validity of the basic scaffold, as well as
the positions and the stereochemistries of two hydroxy groups, have not been explored
further. In order to validate the structure proposed by Sakakibara, we embarked on the
synthesis of proposed 2,14βꢀdihydroxyestradiol analogues and an evaluation of their
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biological activities. In this report, we describe the de novo synthesis of seven
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possible candidates and their inhibitory activity of Na /K ꢀATPase. Our synthetic
approach involves a highly regioꢀ and diastereoselective Mizoroki–Heck reaction and a
Friedel–Craftsꢀtype cyclodehydration to construct a steroidal tetracycle as a common
intermediate. This steroidal intermediate allows facile construction of the proposed
2,14βꢀdihydroxyestradiol analogues such as 1aꢀc, 2aꢀc, and 3 (Figure 2) since this
intermediate possesses an alkene on the Bꢀ and Cꢀrings for the introduction of hydroxy
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groups. Furthermore, our de novo approach offers a new and facile entry to a variety
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of other estradiol analogues with different functionalities.
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HO
OH
ouabain
R'
OH
19ꢀnorbufalin: R, R' = H
telocinobufagin: R = Me, R' = OH
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OH
H
H
H
H
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HO
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H
O
O
H
OH
O
H
O
O
O
H
O
H
H
H
HO
OH
marionobufagenin
digoxin
Figure 1. Structures of the reported EDLF candidates.
Figure 2. Proposed structure A of the InagamiꢀTamura EDLF and its possible
candidates 1aꢀc, 2aꢀc and 3.
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SYNTHETIC PLAN
Our synthetic plan is depicted in Scheme 1. In contrast to Sakakibara’s semiꢀsynthesis
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of 1a and 1b from estradiol, we decided to adopt the de novo synthesis of the possible
candidates 1aꢀc, 2aꢀc and 3 from a steroidal tetracycle like B as a common intermediate
in order to introduce hydroxy groups into the Bꢀ and Cꢀrings. Tetracycle B would be
synthesized by the Friedel–Craftsꢀtype cyclodehydration of lactol C. An aryl group in
lactone D, a precursor of lactol C, would be stereoselectively introduced by means of
the Mizoroki–Heck reaction of lactone 8 with bromoarene E from the convex face of
the tricyclic lactone system. Tricyclic lactone 8 bearing a cisꢀhydroindane scaffold
would in turn be furnished by a newly designed intramolecular Diels–Alder reaction of
enol ester 5 and subsequent optical resolution.
Scheme 1. Synthetic plan for candidates 1aꢀc, 2aꢀc, and 3.
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RESULTS AND DISCUSSION
Synthesis of lactone 8. In order to obtain enantioenriched tricyclic lactone 8 at the
outset, we synthesized enol ester 5, a substrate of intramolecular Diels–Alder reaction,
from commercially available ethyl sorbate (Scheme 2). Thus, isomerization of ethyl
sorbate was carried out under conventional conditions to obtain β,γꢀunsaturated ester 4a,
which was then hydrolyzed to afford corresponding carboxylic acid 4b.
Transformation of 4b into an acid chloride and subsequent treatment with
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2ꢀmethylꢀ1,3ꢀcyclopentanedione in the presence of Et N afforded enol ester 5 in good
overall yield. Intramolecular Diels–Alder reaction of 5 was conducted at 200 °C with
BHT in 1,2,4ꢀtrichlorobenzene to provide the desired endoꢀ6 in 66% as a major
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diastereomer along with exoꢀ6 in 19% yield.
A decagramꢀscale synthesis of endoꢀ6
could be performed from carboxylic acid 4b (34% in 3 steps). The ketone carbonyl
group in endoꢀ6 was then reduced with NaBH in MeOH to afford alcohol (±)ꢀ7 in 82%
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yield. The relative stereochemistry of endoꢀ6 and (±)ꢀ7 was determined by NOESY
analysis as shown in Figure 3, which revealed that the reduction took place
preferentially from the convex face of the scaffold. With racemic alcohol (±)ꢀ7 in
hand, optical resolution was next conducted. After several attempts at kinetic
resolution with various enzymes, it was found that the resolution with lipase MYꢀ30
(Meito Sangyo Co. Ltd., Japan) in the presence of vinyl acetate and subsequent
recrystallization afforded the enantiomerically pure acetate 8 and alcohol 7 in good
yield on a multigram scale. The absolute configuration of 8 was unambiguously
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established by singleꢀcrystal Xꢀray diffraction analysis by using the Bijvoet method.
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Scheme 2. Synthesis of enantioenriched acetate 8 and alcohol 7.
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Figure 3. Diagnostic NOESY correlations of endoꢀ6 and alcohol (±)ꢀ7.
Synthesis of tetracycle 14, a common intermediate. We next investigated a
Mizoroki–Heck reaction using acetate 8 (Scheme 3). The Mizoroki–Heck reaction of
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acetate 8 and bromoarene 9 with palladacycle 10 as a catalyst, reported by Herrmann
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and coꢀworkers, resulted in the formation of the desired product 11a in 70% yield
along with its regioisomer 12a in 20% yield. Both compounds were obtained as a
single diastereomer. Alcohol (±)ꢀ7 also underwent the related transformation with 9 to
afford 11b and 12b in 62% and 28% yields, respectively.
The observed
regioselectivity could be attributed to steric hindrance by the buttressing effect of the
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angular methyl group. The relative stereochemistry of alcohols 11b and 12b was
confirmed by NOESY correlations, as shown in Figure 4. The relative stereochemistry
of acetates 11a and 12a was confirmed by derivatizations from 11b and 12b,
respectively. The stereochemical outcomes indicate that the Mizoroki–Heck reactions
took place from the convex face of the tricycic lactone system, as anticipated. Thus,
the present Mizoroki–Heck reaction enables stereoselective installation of the Aꢀring
fragment into 11a with a proper C9ꢀstereochemistry for construction of a transꢀBC ring
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junction in a steroidal tetracyclic system 14.
The second key reaction leading to the steroid scaffold was next examined. Aryl
adduct 11a was converted into lactol 13 by DIBAL reduction, and subsequent
Friedel–Craftsꢀtype cyclodehydration of 13 provided tetracycle 14, a common and
indispensable intermediate, in good yield. A combination of Sc(OTf)
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/MeOH was
other systems such as Sc(OTf) /CH Cl
, and BF ·OEt /CH Cl only furnished a complex
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crucial for this transformation;
pꢀTsOH/(CH Cl) , TFA/(CH Cl)
mixture instead of the desired 14.
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Scheme 3. Synthesis of tetracycle 14 through Mizoroki–Heck reaction and
Friedel–Craftsꢀtype cyclodehydration.
BnO
BnO
Br
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BnO
BnO
OR
OR
9
OR
H
palladacycle 10
9
H
NaOAc
BnO
BnO
1
3
+
O
H
O
O
DMF, 130 °C
O
O
O
8
: R = Ac (99% ee)
(dr = >95:<5)
(dr = >95:<5)
(±)ꢀ7: R = H
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1a: R = Ac 70%
1b: R = H 62%
12a: R = Ac 20%
12b: R = H 28%
R
R
O
P
O
Pd
Pd
O
O
P
R
R
palladacycle 10
R = oꢀtolyl)
(
OH
OH
H
H
DIBAL
Sc(OTf)3
BnO
BnO
BnO
BnO
11a
H
O
H
OH
CH2Cl2
MeOH
rt to reflux
85%
–78 °C
OH
1
3 90%
tetracycle 14
(dr = ca. 80:20)
Figure 4. Diagnostic NOESY correlations of 11b and 12b.
Synthesis of possible candidates 1a-c from tetracycle 14. With key steroidal
intermediate 14 in hand, we moved on to the synthesis of possible candidates 1aꢀc for
the InagamiꢀTamura EDLF, as depicted in Scheme 4. Hydrogenation of the C=C
bonds in tetracycle 14 with the Crabtree’s catalyst under a hydrogen atmosphere (1 atm)
provided 15 in 65% yield. Oxidation of the secondary alcohol in 15 led to ketone 16
3
which was treated with TMSOTf and Et N to provide the silyl enol ether with a
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TMSꢀprotected tertiary hydroxy group. Subsequent ItoꢀSaegusa reaction of the
resulting silyl enol ether led to enone 17a in 88% yield (2 steps). After deprotection of
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the TMS group, treatment of the resulting enone 17b with NaBH
MeOH at rt afforded 18 in 80% yield and epiꢀ18 in 9% yield. Dihydroxylation of the
resulting allylic alcohol 18 using OsO /NMO provided known compounds 19a and 19b
4 3 2
and CeCl ·7H O in
4
as a separable mixture in 17% and 42% yields, respectively. It is worth noting that
®
diols 19a and 19b were directly separated by using Chromatorex ꢀDIOL (Fuji Silysia
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Chemical Ltd., Japan), whereas in the previous report they were separated after
derivatization into the corresponding acetates. Finally, deprotection of the benzyl
groups in 19a and 19b was successfully carried out with Pd/BaSO
4
under a hydrogen
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1
atmosphere (1 atm) to afford the corresponding 1a and 1b, respectively. The H and
1
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C NMR spectra of synthesized compounds 19a, 19b, 1a and 1b were in agreement
with those reported; however, subtle differences in chemical shifts were observed.
Therefore, we identified 19a and 19b after acetylation to triacetates 20a and 20b. The
1
13
H and C NMR spectra of 20a and 20b were identical to those previously reported.
In order to obtain 15,16ꢀtransꢀdiol 1c, epoxidation of allylic alcohol 18 under the
1
7
2
Sharpless conditions [VO(acac) , TBHP] was followed by ringꢀopening of the
1
8
resulting epoxide 21 by using PhCO
2
Na at 150 °C to provide transꢀdiol 22.
Finally,
deprotection of the benzyl groups by the same manner afforded the corresponding 1c in
good overall yields. The newly generated stereogenic centers of 22 were determined
by NOESY correlation between the C9 and C15 protons, as shown in Figure 5.
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Scheme 4. Synthesis of 15,16ꢀdiols 1a and 1b and 15,16ꢀtransꢀdiol 1c from tetracycle
14.
OH
OH
O
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Crabtree cat.
H2 (1 atm)
H
H
H
BnO
BnO
BnO
BnO
IBX
BnO
BnO
H
OH
H
OH
H
OH
CH2Cl2, rt
DMSO
rt
87%
65%
tetracycle 14
15
16
O
OH
1
. TMSOTf, Et3N
CH2Cl2, 0 °C
NaBH4
CeCl3—7H2O
H
H
16
BnO
BnO
BnO
H
OR
15
H
OH
2
. Pd(OAc)2
O2 (1 atm)
DMSO
MeOH/THF
rt
BnO
1
1
7a: R = TMS
7b: R = H
epiꢀ
18 9%
18 80%
TBAF, THF
°C, 97%
rt to 60 °C
88% (2 steps)
0
OR2
OR2
H
OR2
H
H
OR2
1
1
R O
R O
OsO4, NMO
+
H
OH
OR2
OH
OR2
1
1
acetone/tꢀBuOH
R O
R O
/H2O, rt
9a: R = Bn, R2 = H 17%
20a: R = Bn, R2 = Ac
1
19b: R1 = Bn, R2 = H 42%
20b: R1 = Bn, R2 = Ac
1b: R , R2 = H
1
Pd/BaSO4
H2 (1 atm)
EtOAc, rt
quant.
Pd/BaSO4
H2 (1 atm)
EtOAc, rt
quant.
1
1
2
1
1a: R , R = H
OH
OH
TBHP
VO(acac)2
H
H
OH
BnO
BnO
PhCO Na
RO
O
2
H
OH
H
OH
OH
benzene, rt
DMFꢀH2O
150 °C
RO
9
0%
21
22: R = Bn
c: R = H
79%
Pd/BaSO4
H2 (1 atm)
EtOAc, rt
77%
1
Figure 5. Diagnostic NOESY correlations of 15,16ꢀdiol 22.
Synthesis of possible candidates 2a-c from tetracycle 14. Tetracycle 14 was
then converted into 6,7ꢀdiols 2aꢀc (Scheme 5). The buttressing effect of the angular
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2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
methyl group in 14 was expected to allow discrimination of the two alkenes at the
4
C6ꢀC7 and C11ꢀC12. Indeed, oxidation of 14 with OsO and NMO afforded desired
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
6,7ꢀcisꢀdiol 24 in 27% yield as a minor product and hydroxy ketone 23 in 50% yield.
Hydroxy ketone 23 was not an expected product but a useful intermediate to obtain
4
6,7ꢀtransꢀdiol 25. Reduction of 23 with NaBH afforded a separable mixture of
6,7ꢀtransꢀdiol 25 and 6,7ꢀcisꢀdiol 24. Deprotection of the benzyl groups and
hydrogenation of the remaining C11ꢀC12 alkene in the resulting 24 and 25 were
performed under the same conditions [Pd/BaSO , H (1 atm), EtOAc] leading to the
candidates 2a and 2b, respectively. On the other hand, Prevost reaction of tetracycle
4 provided the corresponding dibenzoate, which was then hydrolyzed to afford the
other 6,7ꢀtransꢀdiol 26. Final deprotection and hydrogenation also succeeded to obtain
c in the same manner. The relative stereochemistry of 24 and 26 were determined by
NOESY analysis (Figure 6).
4
2
1
2
Scheme 5. Synthesis of 6,7ꢀdiol 2aꢀc from common intermediate 14.
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14
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21
22
23
24
25
26
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34
35
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40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Figure 6. Diagnostic NOESY correlations of 24 and 26.
Synthesis of possible candidate 3 from tetracycle 14. Final candidate 3 was
synthesized from tetracycle 14 (Scheme 6). Regioselective hydrogenation of the
sterically less congested alkene in 14 was carried out with Wilkinson’s catalyst in 82%
1
9
yield. Subsequent dihydroxylation with trimethylamine Nꢀoxide resulted in the
exclusive formation of βꢀcisꢀdiol 28 in moderate yield. The relative stereochemistry of
2
8 was determined by NOESY analysis, as depicted in Figure 7. Final deprotection of
the benzyl groups in 28 furnished 11,12ꢀcisꢀdiol 3 in 42% yield.
Scheme 6. Synthesis of 11,12ꢀdiol 3 from common intermediate 14.
OH
OH
12
1
1
(Ph3P)3RhCl
H2 (3 atm)
H
H
BnO
BnO
BnO
BnO
H
OH
CH Cl , rt
H
OH
2
2
82%
tetracycle 14
27
OH
H
OH
HO
RO
RO
OsO4, Me3N–O
H
OH
acetone/tꢀBuOH/H2O
rt
40%
28: R = Bn
: R = H
Pd/BaSO4
H2 (1 atm)
EtOAc, rt
3
42%
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2
2
2
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3
3
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3
3
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3
3
3
4
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1
2
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4
5
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9
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1
2
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5
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 7. Diagnostic NOESY correlations of 11,12ꢀdiol 28.
EVALUATION OF INHIBITORY ACTIVITY
We investigated the inhibitory activity of seven synthesized analogues 1aꢀc, 2aꢀc,
+
+
and 3 against Na /K ꢀATPase from porcine cerebral cortex by colorimetric phosphate
quantitation using malachite green (Figure 8). Ouabain (OUA) was used as a positive
control. The inhibitory activities of the seven compounds were lower than that of
OUA at a final concentration of 3 µM (Figure 7, a). At a final concentration of 300
µM, some of the synthesized compounds such as 1aꢀc and 3 showed inhibitory activities
comparable to that of ouabain at 3 µM (Figure 7, b). In this study, the synthesis and
evaluation of the inhibitory activities were not examined for all possible dihydroxylated
regioꢀ and stereoisomeric analogues. However, based on the general trend observed
here for the structure and activity, other 2,14βꢀdihydroxyestradiol analogues seem to
exhibit less potent inhibitory activities than that of OUA as well, even by changing
position and stereochemistry of the hydroxy groups. From the view point of the potent
6
inhibitory activity reported by Inagami and Tamura, the structure proposed by
Sakakibara is not likely to be a true structure of the InagamiꢀTamura EDLF and the
basic scaffold may be incorrect.
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9
(
a)
(b)
1
00
100
90
80
70
60
50
40
30
20
10
0
9
0
0
0
8
7
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
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41
42
43
44
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47
48
49
50
51
52
53
54
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58
59
60
60
5
4
3
2
0
0
0
0
10
0
1a
1b
1c
2a
2b
2c
3
OUA
1a
1b
1c
2a
2b
2c
3
OUA
3 ꢁM
300 ꢁM
3 ꢁM
Figure 8. Inhibitory activity of synthesized compounds 1aꢀc, 2aꢀc, and 3. (a) At a
final concentration of 3 µM. (b) At a final concentration of 300 µM for synthesized
compounds and 3 µM for OUA (ouabain). The results shown are the means of three
independent experiments ± S.D (error bars).
CONCLUSION
We have developed the de novo synthesis of seven dihydroxylated
2,14βꢀdihydroxyestradiol analogues to validate Sakakibara’s proposed structure for the
InagamiꢀTamura EDLF. A newly designed intramolecular Diels–Alder reaction of
enol ester 5 enables construction of functionalized tricyclic lactone endoꢀ6 on a
decagram scale. Optical resolution of alcohol (±)ꢀ7 with lipase MYꢀ30 and subsequent
recrystallization provided enantiomerically pure acetate 8 in good yield. Two key
transformations, a highly regioꢀ and stereoselective Mizoroki–Heck reaction using
Herrmann palladacycle 10 and a Friedel–Craftsꢀtype cyclodehydration with
Sc(OTf)
Finally, tetracycle 14, a versatile intermediate, was successfully converted into seven
,14βꢀdihydroxyestradiol analogues 1aꢀc, 2aꢀc, and 3. We are hopeful that this newly
3
/MeOH, allowed for synthesis of steroidal tetracycle 14 in good overall yield.
2
developed de novo approach will provide a facile entry to a wide variety of other
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2
2
2
2
2
2
2
2
3
3
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6
estradiol analogues with functionalities on the Bꢀ and Cꢀrings, which are otherwise
9
difficult to obtain. As all of these synthetic analogues of Sakakibara’s proposed
+
+
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
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9
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1
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1
2
3
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7
8
9
0
1
2
3
4
5
6
7
8
9
0
structure exhibited low inhibitory activity against Na /K ꢀATPase, basic scaffold of the
proposed structure may be incorrect. It is reasonable to expect that further
confirmation of the structure for the InagamiꢀTamura EDLF would eventually be
achieved through reisolation and characterization based on NMR studies of the
compound.
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EXPERIMENTAL SECTION
General Techniques. Infrared spectra (IR) were recorded on a JASCO FT/IRꢀ4100
–1
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
type A spectrophotometer and are reported in wave number (cm ). Proton nuclear
1
magnetic resonance ( H NMR) spectra were recorded on a Varian Geminiꢀ2000 (300
MHz), a Bruker Avanceꢀ400 (400 MHz), or a Bruker ARXꢀ400 (400 MHz) spectrometer.
Chemical shifts of all compounds are reported in ppm relative to the residual
undeuterated solvent (CDCl as
δ
= 7.26, CD OD as
δ
= 3.31, DMSOꢀd as
δ
= 2.50,
3
3
6
acetoneꢀd as δ = 2.05). Data were reported as follows: Chemical shift, integration,
6
multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br =
broadened), coupling constant(s), and assignment. Carbon nuclear magnetic resonance
13
(
C NMR) spectra were recorded on a Varian Geminiꢀ2000 (75 MHz), a Bruker
Avanceꢀ400 (100 MHz), a Bruker ARXꢀ400 (100 MHz) or a Bruker Avanceꢀ600 (150
MHz) spectrometer. Chemical shifts of all compounds are reported in ppm relative to
the solvent (CDCl
3 3 6 6
as δ = 77.0, CD OD as δ = 49.0, DMSOꢀd as δ = 39.5, acetoneꢀd as
δ
= 29.8). All NMR were measured at 300 K. Highꢀresolution mass spectra (HRMS)
were recorded on an Applied Biosystems Mariner ESIꢀTOF spectrometer for ESIꢀMS,
and reported in m/z. Elemental analyses were performed by the Analytical Laboratory of
Graduate School of Bioagricultural Sciences, Nagoya University. Melting points (mp)
were recorded on a Yanaco MPꢀS3 melting point apparatus and are not corrected.
Absorbance was measured on a SPECTRAmax 250 microplate spectrophotometer.
All reactions were monitored by thin layer chromatography (TLC) on 0.25 mm silica
gel coated glass plates 60F254 (Merck, #1.05715.0001). Visualization was achieved by
using UV light (254 nm) and appropriate reagent (ethanolic phosphomolybdic acid or
pꢀanisaldehyde solution in H
2
SO
4
/AcOH/EtOH), followed by heating. Silica gel 60
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2
2
2
2
2
2
2
2
3
3
3
3
3
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3
3
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3
4
4
4
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5
5
5
5
5
5
5
5
5
5
6
(
particle size 0.063ꢀ0.200 mm, Merck, #1.07734.9025) was used for silica gel open
column chromatography. Silica gel 60 (spherical, particle size 0.04ꢀ0.05 mm, Kanto,
#37562ꢀ84) was used for silica gel flash column chromatography. Silica gel 60N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(neutral, particle size 0.063ꢀ0.200 mm, Kanto, #37563ꢀ79) was used for neutral silica gel
®
open column chromatography. Chromatorex ꢀDIOL (particle size MB100ꢀ75/200, Fuji
Silysia Chemical Ltd. HU50711) was used for Chromatorex DIOL silica gel open column
®
chromatography. Chromatorex ꢀDIOL (particle size MB100ꢀ40/75, Fuji Silysia
Chemical Ltd. HU50710) was used for Chromatorex DIOL silica gel flash column
chromatography. Preparative TLC separations were carried out on 0.5 mm silica gel
plates 60F254 (Merck). DIOL Preparative TLC separations were carried out on 0.25 mm
DIOL silica gel plates (Fuji Silysia Chemical Ltd.). Dry THF and CH
2
Cl
2
were
N,
2
. Celite (Hyflo SuperꢀCel
purchased from Kanto Chemical Co., Inc. Dry benzene, DMPU, DMF, toluene, Et
3
DME, DMSO and diisopropylamine, were distilled from CaH
®
Celite ) was purchased from Nacalai tesque Co., Inc. Florisil was purchased from
®
Kanto Chemical Co., Inc. DISMIC ꢀ13JP (disposable membrane filter unit, pore size
0
.20 ꢁm), purchased from Toyo Roshi Kaisha, Ltd., was used for 0.20 ꢁm membrane
filter. Lipase MYꢀ30 was purchased from Meito Sangyo Co., Inc. All other
commercially available reagents were used as received.
Ester 4a. A solution of LDA was prepared by slow addition of nꢀBuLi (1.60 M
solution in hexane, 255 mL, 408 mmol) to a solution of dry diisopropylamine (55.0 mL,
4
16 mmol) in dry THF (682 mL) at –78 °C. To this solution was added dropwise DMPU
(54.0 mL, 447 mmol) at –78 °C. After being stirred at –78 °C for 2 h, ethyl sorbate (47.8
g, 341 mmol) was added to this mixture. After being stirred at –78 °C for 2 h, the
reaction mixture was allowed to warm up to room temperature. The reaction was
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quenched with 10% acetic acid (700 mL, v/v). The aqueous layer was extracted with
pentane. The combined organic layer was washed with H
aqueous solution of NaHCO (1x), dried over Na SO , and concentrated carefully under
reduced pressure. The residue was purified by distillation to afford ester 4a (31.4 g, bp
2
O (4x) and a saturated
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
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55
56
57
58
59
60
3
2
4
1
13
6
9ꢀ71 °C/15 mmHg, 66% yield) as a paleꢀyellow oil. H NMR, C NMR and IR data
2
0
–1
were identical to those reported for this compound.
IR (KBr) vmax 1736, 1653 cm .
1
H NMR (300 MHz, CDCl3)
C=O), 4.15 (2H, q, J = 7 Hz, CH
=CH), 5.16 (1H, d, J = 17 Hz, CH
CH=CHCH C=O), 6.14 (1H, dd, J = 17, 15.5 Hz, CH=CHCH
δ 1.26 (3H, t, J = 7 Hz, CH CH OC=O), 3.11 (2H, d, J = 7
3 2
Hz, CH
2
3
CH
=CH), 5.79 (1H, dt, J = 15.5, 7 Hz,
C=O), 6.34 (1H, ddd, J =
C NMR (75 MHz, CDCl3) δ 14.2, 38.0, 60.7, 116.9,
2
OC=O), 5.06 (1H, d, J = 10 Hz,
CH
A
H
B
A B
H
2
2
13
1
7, 17, 10 Hz, CH H =CH).
A
B
1
25.7, 134.2, 136.3, 171.4.
Carboxylic acid 4b. To a solution of 4a (31.4 g, 224 mmol) and EtOH (3 mL) in dry
THF (450 mL) was added lithium hydroxide (1.0 M solution in H O, 270 mL, 269 mmol)
2
at 0 °C. The reaction mixture was stirred at 0 °C for 3 h. The reaction was quenched
with 1.0 N HCl. The aqueous layer was extracted with EtOAc (2x). The combined
2 4
organic layer was washed with brine (1x), dried over Na SO , and concentrated under
reduced pressure. The residue was purified by neutral silica gel open column
chromatography (hexane:EtOAc = 14:1 to 3:1) to afford carboxylic acid 4b (24.8 g, 99%
1
13
yield) as a yellow oil. H NMR, C NMR and IR data were identical to those reported
2
1
–1
1
for this compound.
IR (KBr) vmax 3021, 1709 cm . H NMR (400 MHz, CDCl
3.17 (2H, d, J = 7 Hz, CH C=O), 5.09 (1H, d, J = 10.5 Hz, CH =CH), 5.19 (1H, d, J
17 Hz, CH =CH), 5.77 (1H, dt, J = 15.5, 7 Hz, CH=CHCH ), 6.17 (1H, dd, J = 15.5,
), 6.34 (1H, ddd, J = 17, 10.5, 10.5 Hz, CH
3
)
δ
2
A B
H
=
A
H
B
2
1
3
1
0.5 Hz, CH=CHCH
2
A
H
B
=CH).
C NMR
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4
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4
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5
5
5
5
5
6
(
100 MHz, CDCl3)
δ
37.6, 117.4, 124.5, 134.9, 136.1, 178.1.
Enol ester 5. Carboxylic acid 4b (1.00 g, 8.92 mmol) was dried azeotropically with
toluene and dissolved in dry CH Cl (50 mL). To this solution was added DMF (1 drop)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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2
3
4
5
6
7
8
9
0
2
2
followed by oxalyl chloride (1.30 mL, 15.2 mmol) at room temperature. The reaction
mixture was stirred at room temperature for 1.5 h and concentrated under reduced
pressure. The residue was diluted with dry CH
to a solution of 2ꢀmethylꢀ1,3ꢀcyclopentanedione (770 mg, 6.86 mmol) and dry Et
mL, 7.55 mmol) in dry CH Cl (30 mL) at room temperature immediately via cannula.
After being stirred at room temperature for 1 h, the reaction mixture was washed with
O (1x), a saturated aqueous solution of NaHCO (1x) and brine (1x), dried over
Na SO , and concentrated under reduced pressure. The residue was purified by neutral
Cl
2 2
(50 mL). This solution was added
3
N (1.10
2
2
H
2
3
2
4
silica gel open column chromatography (hexane:EtOAc = 5:1) to afford enol ester 5 (1.18
g, 84% in 2 steps) as a yellow oil. IR (KBr) vmax 1768, 1707, 1668, 1319, 1184, 1105
–1
1
cm . H NMR (400 MHz, CDCl
CH x2), 2.82 (2H, m, CH x2), 3.34 (2H, d, J = 7.5 Hz, CH=CHCH
0 Hz, CH =CH), 5.22 (1H, d, J = 17 Hz, CH =CH), 5.79 (1H, dt, J = 15, 7.5 Hz,
CH=CHCH C=O), 6.23 (1H, dd, J = 15, 10 Hz, CH=CHCH
0, 10 Hz, CH
3
)
δ
1.62 (3H, t, J = 2 Hz, CH
3
CC=O), 2.52 (2H, m,
2
C=O), 5.12 (1H, d, J =
1
A
H
B
A B
H
2
2
C=O), 6.35 (1H, ddd, J = 17,
1
3
1
A
H
B
=CH).
C NMR (100 MHz, CDCl
3
)
δ
6.6, 27.0, 34.3, 37.8, 117.9,
+
123.5, 126.3, 135.6, 135.9, 167.2, 175.6, 205.9. HRMS (ESI) [M+Na] calcd for
C
12
H
14
O
3
Na, 229.0835; found, 229.0843.
Intramolecular DielsꢀAlder reaction of 5. A solution of 5 (140 mg, 0.676 mmol) and
BHT (5.6 mg, 0.027 mmol) in 1,2,4ꢀtrichlorobenzene (33 mL) was stirred at 200 °C for 4
h. The reaction mixture was concentrated under reduced pressure and the residue was
purified by silica gel open column chromatography (hexane to EtOAc). This mixture
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was purified by silica gel flash column chromatography (hexane:EtOAc = 4:1 to 3:1 to
2:1) to afford endoꢀ6 (92.0 mg, 66% yield) as a white solid and exoꢀ6 (25.9 mg, 19%
yield) as a white solid. Relative stereochemistry of compound endoꢀ6 was determined
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
–
1
1
by NOESY correlations (Figure 3). endoꢀ6: IR (KBr) vmax 1782, 1742 cm . H NMR
(
400 MHz, CDCl3) 1.14 (3H, s, CH CC=O), 1.89 (1H, ddd, J = 14, 7, 4 Hz,
δ
3
CH
A
H
B
CH
CH
A B
C=O), 2.71 (1H, dd, J = 16.5, 7 Hz, CHCH H
2
C=O), 2.11ꢀ2.17 (2H, m, CH=CHCH
C=O), 2.43ꢀ2.50 (2H, m, CH CH C=O), 2.55 (1H, dd, J = 16.5, 14.5 Hz,
C=O), 3.06 (1H, m,
), 5.94 (1H, ddd, J = 9.5,
2
), 2.22 (1H, dd, J = 14, 10.5 Hz,
CH
A
H
B
2
A
H
B
2
CHCH
CHCH
A
H
B
A
H
B
C=O), 5.73 (1H, ddd, J = 9.5, 7, 3.5 Hz, CH=CHCH
2
13
4.5, 2 Hz, CH=CHCH
2
).
C NMR (100 MHz, CDCl
3
)
δ
15.2, 26.7, 32.6, 33.5, 35.8,
+
3
8.3, 50.2, 94.2, 124.9, 127.9, 175.1, 216.8. HRMS (ESI) [M+Na] calcd for
o
C
12
H
14
O
3
Na, 229.0835; found, 229.0845. mp: 98ꢀ99 C. exoꢀ6: IR (KBr) vmax 1774,
–1
1
1743, 1237 cm . H NMR (400 MHz, CDCl3)
J = 17.5, 4 Hz, CH=CHCH ), 2.08 (1H, m, CH=CHCH
CH CH C=O), 2.31 (1H, ddd, J = 14, 9, 3 Hz, CH CH C=O), 2.41 (1H, dd, J =
7.5, 10.5 Hz, CHCH C=O), 2.48ꢀ2.57 (2H, m, CH CH
7.5, 10 Hz, CHCH C=O), 2.95 (1H, m, CHCH
δ 1.11 (3H, s, CH CC=O), 1.96 (1H, dd,
3
A
H
B
A B
H ), 2.17 (1H, m,
H
A B
2
A
H
B
2
1
A
H
B
A
H
B
2
C=O), 2.85 (1H, dd, J =
1
H
A B
H
A B
C=O), 5.75ꢀ5.78 (2H, m,
13
CH=CHCH
A
H
B
).
C NMR (100 MHz, CDCl
3
)
δ
15.3, 31.66, 31.69, 33.9, 35.6, 38.1,
+
50.7, 91.6, 124.0, 125.6, 174.8, 216.3. HRMS (ESI) [M+Na] calcd for C12
H
14
O
3
Na,
o
229.0835; found, 229.0836. mp: 87ꢀ89 C.
Decagramꢀscale synthesis of endoꢀ6 from carboxylic acid 4b. To a solution of 4b
24.8 g, 221 mmol) in dry CH Cl (1.5 L) was added DMF (0.05 mL) followed by oxalyl
(
2
2
chloride (32.0 mL, 376 mmol) at room temperature. The reaction mixture was stirred at
room temperature for 1.5 h and concentrated under reduced pressure. The residue was
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5
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5
5
5
5
6
diluted with dry CH
2ꢀmethylꢀ1,3ꢀcyclopentanedione (19.1 g, 170 mmol) and dry Et
in dry CH Cl (850 mL) at room temperature immediately via cannula. After being
stirred at room temperature for 1 h, the reaction mixture was washed with H
saturated aqueous solution of NaHCO (1x) and brine (1x), dried over Na
2
Cl
2
(100 mL). This solution was added to a solution of
3
N (26.1 mL, 187 mmol)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2
2
2
O (1x), a
SO , and
3
2
4
concentrated under reduced pressure. The residue was passed through a column with
neutral silica gel (hexane:EtOAc = 4:1) to afford enol ester 5 (27.6 g, contained about 8%
of carboxylic acid 4b) as a yellow oil. A solution of 5 (27.6 g, 134 mmol) and BHT (1.20
g, 5.45 mmol) in 1,2,4ꢀtrichlorobenzene (1340 mL) was stirred at 155 °C for 10 h. The
reaction mixture was concentrated under reduced pressure at 83 °C (7 mmHg). The
residue was purified by silica gel open column chromatography (hexane to EtOAc) to
give an 83:17 mixture of endoꢀ6 and exoꢀ6. This mixture was purified by
recrystallization from hexane/EtOAc and silica gel open column chromatography
(hexane:EtOAc = 2:1) to afford endoꢀ6 (16.4 g, 34% yield in 3 steps) as a white solid and
exoꢀ6 (3.68 g, 8% yield in 3 steps) as a white solid.
Reduction of endoꢀ6. To a solution of endoꢀ6 (8.87 g, 43.0 mmol) in MeOH (717
mL) was added NaBH
min, the reaction was quenched with acetic acid and the reaction mixture was
concentrated under reduced pressure. The residue was diluted with H O and the aqueous
layer was extracted with EtOAc (2x). The combined organic layer was washed with
brine (1x), dried over Na SO , and concentrated under reduced pressure. The residue
4
(3.38 g, 89.3 mmol) at 0 °C. After being stirred at 0 °C for 30
2
2
4
was purified by silica gel flash column chromatography (hexane:EtOAc = 3:2) to afford
major isomer (±)ꢀ7 (7.33 g, 82% yield) as a white solid and minor isomer (±)ꢀepiꢀ7 (788
mg, 9% yield) as a white solid. Relative stereochemistry of (±)ꢀ7 was determined by
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NOESY correlations (Figure 3). (±)ꢀ7: IR (KBr) vmax 3433, 1773, 1010 cm .
NMR (400 MHz, CDCl 1.12 (3H, s, CH CCOH), 1.38 (1H, m,
CH CH CHOH), 1.56 (1H, m, CH CHOH), 1.73 (1H, dd, J = 18.5, 2.5
Hz, CH=CHCH ), 2.02ꢀ2.14 (2H, m, CH
Hz, CHCH
6, 7 Hz, CHCH
J = 9, 8 Hz, CHOH), 5.77ꢀ5.85 (2H, m, CH=CHCH
H
3
)
δ
3
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
H
H
H
CH
H
A
B
A
B
A
B
A
B
A
H
B
A
H
B
CH
A
H
B
CHOH), 2.43 (1H, dd, J = 16, 14
), 2.61 (1H, dd, J =
C=O), 4.13 (1H, dd,
A
H
B
C=O), 2.54 (1H, ddd, J = 18.5, 4, 2 Hz, CH=CHCH
A B
C=O), 2.95 (1H, ddd, J = 14, 7, 2 Hz, CHCH H
A B
H
1
A
H
B
13
A
H
B
).
C NMR (100 MHz,
CDCl
3
)
δ
19.0, 29.5, 29.9, 31.4, 32.7, 39.6, 46.9, 78.2, 96.1, 124.1, 130.0, 176.5.
+
HRMS (ESI) [M+Na] calcd for C12
H
16
O
3
Na, 231.0992; found, 231.1002. mp: 93ꢀ94
o
–1
1
C. (±)ꢀepiꢀ7: IR (KBr) vmax 3485, 1754 cm .
3
H NMR (400 MHz, CDCl )
δ
1.16 (3H, s, CH CCOH), 1.76ꢀ1.89 (3H, m, CH x3), 1.99ꢀ2.11 (3H, m, CH x3), 2.44
3
(
1H, dd, J = 16, 14.5 Hz, CHCH
A
H
B
C=O) 2.64 (1H, dd, J = 16, 7 Hz, CHCH
A
H
B
C=O),
2.96 (1H, m, CHCH
A
H
B
C=O), 3.78 (1H, d, J = 4 Hz, CHOH), 5.74 (1H, m, CH=CHCH
2
).
1
3
5.84 (1H, ddd, J = 10, 4, 2.5 Hz, CH=CHCH
2
).
C NMR (100 MHz, CDCl
3
)
δ
16.2,
+
3
1.1, 31.7, 33.1, 38.4, 39.3, 48.3, 81.3, 97.2 124.3, 129.3, 175.9. HRMS (ESI) [M+Na]
o
calcd for C12
H
16
O
3
Na, 231.0992; found, 231.0983. mp: 112ꢀ113 C.
Optical resolution of alcohol (±)ꢀ7. To a solution of racemic alcohol (±)ꢀ7 (6.24 g,
3
0.0 mmol) in dry DME/toluene (278 mL, 1:4) were added vinyl acetate (52.3 g, 608
mmol) and lipase MYꢀ30 (11.35 g) at room temperature. After being stirred at room
temperature for 13 h, to the resulting mixture was added lipase MYꢀ30 (11.33 g) at room
temperature. After being stirred at room temperature for 10.5 h, to the resulting
mixture was added lipase MYꢀ30 (11.09 g) at room temperature. After being stirred at
room temperature for 16 h, to the resulting mixture was added lipase MYꢀ30 (11.02 g) at
room temperature. After being stirred at room temperature for 7.5 h, the reaction
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2
2
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3
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4
4
4
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4
4
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4
4
5
5
5
5
5
5
5
5
5
5
6
mixture was filtered through a pad of Celite, and the filtrate was concentrated under
reduced pressure. The residue was purified by silica gel open column chromatography
(hexane:EtOAc = 2:1) to afford acetate 8 (3.50 g, 47% yield, 90% ee) as a white solid and
alcohol 7 (3.10 g, 50% yield, 89% ee) as a white solid. Recrystallization from
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
2 2
hexane/CH Cl afforded 8 (1.92 g, 99% ee, 55% yield) as a white solid.
Recrystallization from hexane/EtOAc afforded 7 (77%, >99% ee) as a white solid.
Enantiomeric excess was determined by chiral HPLC analysis. Chiral HPLC analysis
for 8 (CHIRALPAK IB column, hexane/2ꢀpropanol = 90/10, flow rate = 0.5 mL/min,
detection 220 nm light, 30 °C) t
Chiral HPLC analysis for 7 (CHIRALPAK IB column, hexane/2ꢀpropanol = 90/10, flow
rate = 0.5 mL/min, detection 220 nm light, 30 °C) t = 20.9 min (minor isomer), 22.1
min (major isomer). Absolute stereochemistry of 8 was determined by Xꢀray
R
= 17.7 min (major isomer), 19.1 min (minor isomer).
R
–1
1
crystallographic analysis. acetate 8: IR (KBr) vmax 1772, 1734, 1064, 1022 cm .
NMR (400 MHz, CDCl 1.09 (3H, s, CH CCCH ), 1.40 (1H, m, CH), 1.60 (1H, ddd, J
14.5, 9, 8 Hz, CH), 1.84 (1H, dq, J = 19, 3 Hz, CH), 2.05 (3H, s, CH C=O), 2.10 (1H, m,
H
3
)
δ
3
2
=
3
CH), 2.30 (1H, m, CH), 2.43 (1H, dd, J = 16, 14.5 Hz, CH), 2.48 (1H, m, CH), 2.61 (1H,
dd, J = 16, 7 Hz, CH), 2.94 (1H, m, CH), 5.01 (1H, t, J = 8.5 Hz, CHOAc), 5.74ꢀ5.86 (2H,
1
3
m, CH=CH).
C NMR (100 MHz, CDCl
3
)
δ
18.9, 21.0, 27.0, 29.9, 32.8, 33.1, 39.1,
: C, 67.18; H,
46.2, 80.1, 95.0, 124.0, 129.3, 170.6, 175.9. Anal. Calcd for C14
18 4
H O
2
D
9
7.25. Found: C, 67.16; H, 7.50. mp: 123ꢀ124 °C. [α]
3
+5.3 (c 0.17, CHCl ).
24
D
alcohol 7: [α]
3
+4.1 (c 0.85, CHCl ).
12
Mizoroki–Heck reaction of 8. To a solution of 8 (2.00 g, 7.94 mmol) and 9 (4.41 g,
11.9 mmol) in dry DMF (40 mL) was added NaOAc (725 mg, 8.84 mmol) under Ar
atmosphere. The resulting mixture was degassed by three freezeꢀthaw cycles, and the
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flask was filled with Ar. After being heated to 50 °C, to this solution was added
palladacycle 10 (133 mg, 0.142 mmol). After being heated to 130 °C, the resulting
mixture was stirred for 2 h. The reaction mixture was cooled to room temperature and
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
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33
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
filtered through Florisil. The filtrate was washed with H
2
O (1x) and brine (1x), and
dried over Na SO , and concentrated under reduced pressure. The residue was purified
2
4
by silica gel flash column chromatography (hexane:EtOAc = 5:1 to 3:1 to 1:1) to afford
1
1
1a (2.99 g, 70% yield, dr = >95:<5 determined by H NMR analysis) as a yellow solid
1
and 12a (842 mg, 20% yield, dr = >95:<5 determined by H NMR analysis) as a yellow
1
solid. Relative stereochemistry of 11a was determined by comparison of H NMR
spectra of lactol 13 prepared from alcohol 11b whose relative stereochemistry was
determined by NOESY correlations (Figure 4). Relative stereochemistry of 12a was
1
determined by comparison of H NMR spectra of acetate 12a prepared from alcohol
12b whose relative stereochemistry was determined by NOESY correlations (Figure 4).
–
1
1
11a: IR (KBr) vmax 1781, 1740, 1509, 1069, 1020 cm . H NMR (400 MHz, CDCl
3
)
δ
1.17 (3H, s, CH CCCH), 1.43 (1H, m, CH), 1.69 (1H, m, CH), 2.03ꢀ2.36 (5H, m, CH
3
x5), 2.11 (3H, s, CH
3
C=O), 3.26 (1H, d, J = 10 Hz, CH), 5.08 (1H, t, J = 8.5 Hz,
of Bn), 5.17 (2H, s, CH of Bn), 5.66 (1H, dd, J = 10.5, 1.5
CHOAc), 5.16 (2H, s, CH
2
2
Hz, CH=CH), 5.78 (1H, dd, J = 10.5, 2.5 Hz, CH=CH), 6.64 (1H, br s, aryl Hꢀ2), 6.66
(1H, dd, J = 8, 2 Hz, aryl Hꢀ6), 6.89 (1H, d, J = 8 Hz, aryl Hꢀ5), 7.28ꢀ7.48 (10H, m, Ph
13
of Bn).
3
C NMR (100 MHz, CDCl ) δ 17.9, 21.0, 26.9, 28.1, 32.1, 44.7, 44.8, 48.9,
71.3, 71.6, 79.4, 92.3, 115.1, 115.2, 120.3, 127.3, 127.4, 127.9, 128.0, 128.5, 130.9,
+
131.0, 134.6, 137.1, 137.2, 148.4, 148.8, 170.5, 175.0. HRMSꢀESI (m/z): [M+Na]
29
calcd for C34
H
34
O
6
Na, 561.2248; found, 561.2230. mp: 42ꢀ44 °C. [α]
D
+83.2 (c
–1
1
0.87, CHCl
3
). 12a: IR (KBr) vmax 1748, 1511, 1024 cm . H NMR (400 MHz,
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2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
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4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
CDCl3)
m, CH x2), 2.02 (3H, s, CH
2 Hz, CH), 2.98 (1H, dd, J = 17, 6 Hz, CH), 3.38 (1H, ddd, J = 11, 7.5, 6.5 Hz, CH),
.05 (1H, dd, J = 7.5, 7 Hz, CHOAc), 5.13 (1H, d, J = 12 Hz, CH of Bn), 5.14 (2H,
s, CH of Bn), 5.18 (1H, d, J = 12 Hz, CH of Bn), 5.84 (1H, d, J = 1.5 Hz,
C=CHC=O), 6.74 (1H, dd, J = 8, 2 Hz, aryl Hꢀ6), 6.76 (1H, br s, aryl Hꢀ2), 6.86 (1H, d,
δ 0.66 (3H, s, CH CCCH), 1.59 (1H, m, CH), 1.80 (1H, m, CH), 1.89ꢀ1.97 (2H,
3
3
C=O), 2.34ꢀ2.51 (2H, m, CH x2), 2.86 (1H, ddd, J = 17, 7.5,
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
5
A B
H
2
A B
H
1
3
J = 8 Hz, aryl Hꢀ5), 7.27ꢀ7.47 (10H, m, Ph of Bn).
20.4, 21.1, 28.8, 30.9, 34.4, 37.2, 38.0, 51.5, 71.3, 71.7, 83.3, 95.3, 114.8, 115.0, 115.8,
19.7, 127.25, 127.31, 127.85, 127.89, 128.49, 128.51, 137.1, 137.7, 147.9, 148.7,
C NMR (100 MHz, CDCl3)
δ
1
+
1
70.3, 172.0. HRMSꢀESI (m/z): [M+Na] calcd for C34
H
34
O
6
Na, 561.2248; found,
29
D
561.2275. mp: 44ꢀ46 °C. [α]
3
–101 (c 0.71, CHCl ).
Mizoroki–Heck reaction of alcohol (±)ꢀ7. To a solution of alcohol (±)ꢀ7 (315 mg,
1.51 mmol, racemate) and 9 (899 mg, 2.43 mmol) in dry DMF (7.6 mL) was added
NaOAc (138 mg, 1.68 mmol) under Ar atmosphere. The resulting mixture was degassed
by three freezeꢀthaw cycles, and the flask was filled with Ar. After being heated to 50 °C,
to this solution was added palladacycle 10 (155 mg, 0.165 mmol). After being heated to
1
30 °C, the resulting mixture was stirred for 5 h. The reaction mixture was cooled to
room temperature and then filtered through Florisil. The filtrate was washed with H
(1x) and brine (1x), and dried over Na SO , and concentrated under reduced pressure.
The residue was purified by silica gel flash column chromatography (hexane:EtOAc =
2
O
2
4
3:1 to 2:1 to 1:1) to afford major product 11b (462 mg, 62% yield, dr = >95:<5
1
determined by H NMR analysis) as a white solid and minor product 12b (208 mg, 28%
1
yield, dr = >95:<5 determined by H NMR analysis) as a white solid. Relative
stereochemistry of compounds 11b and 12b was determined by NOESY correlations
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(
Figure 4). 11b: IR (KBr) vmax 3446, 1776, 1509, 1011 cm . H NMR (400 MHz,
CDCl
3
)
δ
1.19 (3H, s, CH
CHOH), 1.63 (1H, ddd, J = 14.5, 9, 8 Hz, CH
CH CHOH, CHCH C=O), 3.27 (1H, br d, J = 9.5 Hz,
ArCH), 4.16 (1H, dd, J = 8, 8 Hz, CHOH), 5.16 (2H, s, CH of Bn), 5.17 (2H, s, CH of
Bn), 5.67 (1H, dd, J = 10, 1.5 Hz, CHCH=CH), 5.82 (1H, dd, J = 10, 2 Hz, CHCH=CH),
3
), 1.43 (1H, dddd, J = 12.5, 11, 9, 8 Hz,
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
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56
57
58
59
60
CH
A
H
CH
H
A B
H
A B
A B
CH H CHOH),
B
2
.02ꢀ2.30 (5H, m, CH
A
H
B
A
H
B
2
2
2
6
.65 (1H, br s, aryl Hꢀ6), 6.66 (1H, dd, J = 8.5, 2 Hz, aryl Hꢀ2), 6.89 (1H, d, J = 8.5 Hz,
1
3
aryl Hꢀ5), 7.28ꢀ7.48 (10H, m, Ph of Bn).
3
C NMR (100 MHz, CDCl ) δ 17.7, 27.8,
2
9.6, 32.1, 44.9, 45.1, 49.5, 71.3, 71.5, 78.0, 93.2, 115.1, 115.2, 120.3, 127.3, 127.4,
1
27.8, 127.9, 128.5, 130.5, 130.9, 134.8, 137.1, 137.2, 148.2, 148.8, 175.6.
+
HRMSꢀESI (m/z): [M+Na] calcd for C32
32
H O
5
Na, 519.2142; found, 519.2159.
–1
1
mp:148ꢀ149 °C. 12b: IR (KBr) vmax 3447, 1747, 1512, 1258 cm . H NMR (400
MHz, CDCl3)
1.73ꢀ1.93 (2H, m, CH
ArCHCH CCH
.5, 1.5 Hz, ArCHCH
1H, m, ArCH), 4.11 (1H, t, J = 7.5 Hz, CH
1H, d, J = 12 Hz, CH of Bn), 5.17 (1H, d, J = 12 Hz, CH
δ
0.46 (3H, s, CH ), 1.67 (1H, dd, J = 15, 5.5 Hz, ArCHCH H CCH ),
3
A
B
3
H
A B
CH
A
H
B
CHOH), 2.00 (1H, dd, J = 15, 5 Hz,
CH CHOH), 2.80 (1H, ddd, J = 16,
C=C), 3.09 (1H, dd, J = 16, 4 Hz, ArCHCH
CCHOH), 5.13 (2H, s, CH
H
A B
3
), 2.23ꢀ2.45 (2H, m, CH
H
A B
A B
H
7
A
H
B
A
H
B
C=C), 3.44
(
(
3
2
of Bn), 5.13
A
H
B
A
H
B
of Bn), 5.83 (1H, d, J
= 1.5 Hz, C=CHC=O), 6.73 (1H, dd, J = 8, 1.5 Hz, aryl Hꢀ6), 6.77 (1H, d, J = 1.5 Hz,
1
3
aryl Hꢀ2), 6.83 (1H, d, J = 8 Hz, aryl Hꢀ5), 7.27ꢀ7.47 (10H, m, Ph of Bn).
100 MHz, CDCl3) 18.9, 29.8, 30.1, 32.6, 35.6, 37.3, 52.0, 71.3, 71.5, 81.7, 96.0,
14.77, 114.81, 115.3, 119.4, 127.28, 127.31, 127.78, 127.80, 128.45, 128.51, 137.17,
C NMR
(
δ
1
1
+
37.21, 137.6, 147.5, 148.5, 170.8, 172.3. HRMSꢀESI (m/z): [M+Na] calcd for
C
32
H
32
O
5
Na, 519.2142; found, 519.2154. mp:152ꢀ153 °C.
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5
5
6
Lactol 13. Lactone 11a (1.83 g, 3.40 mmol) was dried azeotropically with
2 2 2 2
toluene/CH Cl (1:1) and dissolved in dry CH Cl (85 mL). To this solution was added
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
dropwise DIBAL (1.00 M solution in toluene, 8.50 mL, 8.50 mmol) at –78 °C. After
being stirred at –78 °C for 1 h, the reaction was quenched with 1 N HCl. The aqueous
layer was extracted with EtOAc (2x). The combined organic layer was washed with a
saturated aqueous solution of NaHCO
3 2 4
(1x) and brine (1x), dried over Na SO , and
concentrated under reduced pressure. The residue was purified by silica gel open
2 2
column chromatography (CH Cl :THF = 4:1) to afford lactol 13 (1.51 g, 90% yield, dr =
1
8
0:20 determined by H NMR analysis) as a white solid. IR (KBr) vmax 3427, 1638
–1
1
cm . H NMR (400 MHz, DMSOꢀd
6
)
δ
3 3
0.99 (2.4H, s, CH ), 1.05 (0.6H, s, CH ),
1
.12ꢀ1.37 (1.2H, m), 1.43 (0.8H, td, J = 12, 6 Hz), 1.59ꢀ2.02 (5H, m), 3.20 (0.2H, br d, J
=
11 Hz), 3.26 (0.8H, br d, J = 10.5 H), 3.88 (1H, m), 4.80 (1H, d, J = 5 Hz), 5.07 (1H,
of Bn), 5.09 (2H, s, CH of Bn), 5.13 (1H, d, J = 12 Hz, CH of
d, J = 12 Hz, CH
A
H
B
2
A B
H
Bn), 5.23 (1H, m), 5.49 (0.8H, d, J = 10.5 Hz, CH=CH), 5.52 (0.2H, d, J = 10.5 Hz,
CH=CH), 5.70 (0.8H, dd, J = 10.5, 1.5 Hz, CH=CH), 5.74 (0.2H, dd, J = 10.5, 1.5 Hz,
CH=CH), 5.84 (0.2H, d, J = 6 Hz, OH), 6.19 (0.8H, d, J = 4 Hz, OH), 6.65ꢀ6.73 (1H, m,
aryl Hꢀ6), 6.84 (0.8H, d, J = 1.5 Hz, aryl Hꢀ2), 6.88 (0.2H, d, J = 1.5 Hz, aryl Hꢀ2), 6.99
(
0.8H, d, J = 8 Hz, aryl Hꢀ5), 7.01 (0.2H, d, J = 7.5 Hz, aryl Hꢀ5), 7.27ꢀ7.48 (10H, m,
1
3
Ph of Bn).
C NMR (100 MHz, DMSOꢀd6) δ 18.2, 18.3, 28.9, 29.2, 29.7, 29.9, 35.9,
42.8, 44.7, 44.9, 46.3, 49.4, 49.6, 70.2, 70.3, 77.4, 77.5, 88.2, 89.5, 95.7, 97.8, 114.4,
14.6, 114.7, 119.8, 127.5, 127.65, 127.70, 128.30, 128.35, 130.3, 131.8, 132.2, 136.8,
1
1
37.0, 137.3, 137.4, 137.5, 146.9, 148.07, 148.13. Anal. Calcd for C32
7.08; H, 6.87. Found: C, 76.73; H, 6.83. mp: 62ꢀ63 °C.
34 5
H O : C,
7
Tetracycle 14. To a solution of 13 (1.35 g, 2.71 mmol) in MeOH (45 mL) was
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added Sc(OTf)
3
(269 mg, 0.547 mmol) at room temperature. The reaction mixture was
stirred at room temperature for 15 min, and then heated to reflux. After being stirred at
reflux for 45 h, the reaction mixture was cooled to room temperature. The reaction was
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
quenched with a saturated aqueous solution of NaHCO
extracted with EtOAc (4x). The combined organic layer was washed with brine (1x),
dried over Na SO , and concentrated under reduced pressure. The residue was purified
by silica gel flash column chromatography (hexane:EtOAc = 1:1) to afford tetracycle 14
3
. The aqueous layer was
2
4
–1
1
(1.10 g, 85% yield) as a white solid. IR (KBr) vmax 3397, 1505, 1275, 1012 cm .
H
NMR (400 MHz, CDCl3) 1.12 (3H, s, CH ), 1.38 (1H, ddd, J = 20.5, 10.5, 10.5 Hz,
δ
3
CH
CH
A
H
B
CH
A
H
B
CHOH), 1.56 (1H, m, CH
CHOH), 2.26 (1H, br d, J = 15 Hz, COHCH), 3.25 (1H, br d, J = 15 Hz,
CCHOH), 5.14 (2H, s, CH of Bn), 5.16 (1H, d, J = 12
of Bn), 5.21 (1H, d, J = 12 Hz, CH of Bn), 5.78 (1H, dd, J = 10.5, 2.5
CCH=CH), 6.20 (1H, d, J = 10.5 Hz, CH CCH=CH), 6.23 (1H, d, J = 9.5 Hz,
A B A B
H CH H CHOH), 2.02ꢀ2.19 (2H, m,
A
H
B
A B
CH H
COHCHCH), 4.18 (1H, m, CH
3
2
Hz, CH
A
H
B
A B
H
Hz, CH
3
3
COHCHCH=CH), 6.51 (1H, dd, J = 9.5, 2.5 Hz, COHCHCH=CH), 6.78 (1H, s, aryl
13
Hꢀ4), 7.00 (1H, s, aryl Hꢀ1), 7.27ꢀ7.49 (10H, m, Ph of Bn).
CDCl3)
C NMR (100 MHz,
δ
19.7, 30.0, 30.8, 39.6, 42.6, 50.7, 71.7, 72.0, 78.9, 81.0, 111.5, 114.2, 125.1,
127.3, 127.4, 127.5, 127.7, 127.77, 127.84, 128.3, 128.45, 128.48, 128.9, 130.7, 130.8,
+
137.4, 147.4, 148.2. HRMSꢀESI (m/z): [M+Na] calcd for C32
H
32
O
4
Na, 503.2193;
28
D
found, 503.2181. mp: 57ꢀ59 °C. [α]
3
–63.9 (c 0.86, CHCl ).
Alcohol 15. To a solution of 14 (1.07 g, 2.24 mmol) in dry CH
2
Cl (22.4 mL) was
2
added Crabtree catalyst (174 mg, 0.216 mmol) at room temperature. The reaction
mixture was stirred under atmosphere of hydrogen (1 atm) at room temperature for 20 h,
and then filtered through Florisil. The resulting mixture was concentrated under reduced
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