Biomimetic Synthesis of Chaxine and its Related Compounds

Article abstract of DOI:10.1021/acs.joc.9b03482

The highly oxidized natural products chaxine B and BB have been synthesized from ergosterol in eight steps according to a route inspired by their proposed biosynthesis; key steps were an oxidative cascade from a furan intermediate to an enol ester using m-chloroperbenzoic acids (MCPBA), followed by diastereoselective epoxidation and acyloxy migration. This concise synthesis resulted in the revision of the structures of chaxine B and its naturally occurring analogs and syntheses of the unnatural analogues of these natural products for biological investigations.

Full text of DOI:10.1021/acs.joc.9b03482

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Article
A Biomimetic Synthesis of Chaxine and its Related Compounds
Misaki Niki, Yushi Hirata, Atsuo Nakazaki, Jing Wu, Hirokazu Kawagishi, and Toshio Nishikawa
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.9b03482 • Publication Date (Web): 24 Feb 2020
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Manuscript for J. Org. Chem.
Dec. 21, 2019
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A Biomimetic Synthesis of Chaxine and its Related Compounds
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Misaki Niki,a Yushi Hirata,a Atsuo Nakazaki,a Jing Wu,b Hirokazu Kawagishi,b and Toshio
Nishikawa a*
aGraduate School of Bioagricultural Sciences, Nagoya University,
Chikusa, Nagoya, 464-8601, Japan
bResearch Institute of Green Science and Technology, Shizuoka University,
836, Ohya, Suruga-ku, Shizuoka, 422-8529, Japan
e-mail: nisikawa@agr.nagoya-u.ac.jp
Abstract: The highly oxidized natural products chaxine B and BB have been synthesized from
ergosterol in eight steps according to a route inspired by their proposed biosynthesis; key steps
were an oxidative cascade from a furan intermediate to an enol ester using MCPBA, followed
by diastereoselective epoxidation and acyloxy migration. This concise synthesis resulted in the
revision of the structures of chaxine B and its naturally occurring analogs, and syntheses of the
unnatural analogues of these natural products for biological investigations.
Keywords: chaxine, natural product, biosynthesis, electrocyclic reaction, Baeyer-Villiger
oxidation, oxidative cascade
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Introduction
Chaxine B (1) and its analogs were isolated from the Chinese edible mushroom Agrocybe
chaxingu by Kawagishi1,2 and are unprecedented oxidized steroidal compounds, wherein the
A ring is connected to the CD ring moiety through an ester linkage, and the B ring is absent
(Figure 1). The relative stereochemistry of the A ring was deduced by NOE experiments, but
its absolute stereochemistry was not determined. Preliminary biological studies established
these natural products to inhibit osteoclast formation1,2 and induce fruiting body formation in
several mushrooms,3 but, the further details could not be clarified due to their very limited
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availability (only 2.1, 1.6, 0.5 and 0.4 mg of chaxines B, C, D and E respectively were isolated
from 1.5 kg of the dried fruiting bodies of the mushroom).4 Interestingly, the related compound
BB (7) was isolated together with chaxine B (1) from Penicilinim sp. These two compounds
were found to accelerate neural stem cell propagation, but the stereochemistry of the A ring
moiety of 7 was not reported.5 Detailed studies of the biological activity of BB (7) are also
impossible, again due to its very limited availability. We were inspired to pursue total syntheses
of these natural products by their interesting biological activities and unique chemical structures,
to establish the absolute stereochemistry of the A ring, and support future biological
investigations. We disclose herein full details of our synthetic efforts towards the chaxine
family of natural products.6
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Figure 1. Initially proposed structures of chaxine B and its related natural products
Results and Discussion
Speculation of a biosynthetic route of chaxines and a synthetic plan based on this
biosynthetic proposal
One possible retrosynthesis for chaxines B from demethylincisterol A3 (5)7 is depicted
in Scheme 1, but was quickly discarded due to the extreme difficulties associated with
esterification between a tertiary alcohol of the A ring moiety 8 and the carboxylic acid of the
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CD ring moiety 9, considered likely to exist as hemiacetal 5 (demethylincisterol A3).
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Scheme 1. A possible retrosynthesis of chaxines B
Instead, we considered the most plausible biosynthetic route of chaxine B (1) as shown in
Scheme 2.8 Ergosterol (10) is a possible starting material having an identical side chain to that
of the chaxine B, and is known to be abundant in many mushrooms. To construct the A ring
structure of chaxine B from ergosterol (10), we proposed cleavage of the two C-C bonds of the
B ring by an 6-electrocyclic reaction and Baeyer-Villiger oxidation of the ketone group at the
C-6 position after several oxidations. In the biosynthesis of vitamin D2, the photoinduced
electrocyclic reaction of ergosterol is known to give pre-vitamin D2 (11) and/or tachysterol
(12),9 which might be further oxidized through endoperoxide 13 to -hydroxy enone 14. The
Baeyer-Villiger oxidation of 14 occurs regioselectively to give enol ester 15,10 which is
epoxidized, and anticipated to undergo acyloxy rearrangement under acidic conditions to give
-acyloxy ketone 17. Finally, oxidation of the hydroxy group of 17 at the C-4 position would
give either chaxine B, or its 2’-epimer. Dehydration of chaxine B would give chaxine C.
Hydrolysis of the esters of 1 or 3 gives demethylincisterol A3 (5).11 Since the relative
stereochemistry of the A ring of chaxine B is as shown in Figure 1, our biosynthetic proposal
suggests the absolute stereochemistry of chaxine B to be 18 as shown in Scheme 2. We would
synthesize chaxines according to this proposed biosynthetic route.
Prior to the initiation of experiments, we considered three potential issues with the
biomimetic synthesis: (i) regioselectivity of the Baeyer-Villiger oxidation of 14; (ii)
diastereofacial selectivity of epoxidation of enol ester 15; and (iii) the stereochemistry of the
acyloxy migration of the enol ester epoxide 16. In the Baeyer-Villiger oxidation of 14, we
anticipated the A ring alkene would preferentially migrate due to the higher alkyl substitution.12
The stereochemistry of the epoxidation of enol ester 15 could be controlled by directed
epoxidation of homoallylic alcohol with a metal catalyst for syn selective epoxidation, or by
steric hindrance of protected homoallylic hydroxy group for anti selective epoxidation.13 Since
the acyloxy migration of a similar enol ester epoxide was reported to have occurred in a highly
stereoselective manner,14 we expected that two diastereomeric epoxides of 16 would give two
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different keto products, one of which would be identical to chaxine B after oxidation of hydroxy
group at the C-4 position. Accordingly, determination of the absolute stereochemistry of the A
ring of chaxine B and its analogs should be possible.
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Scheme 2. A proposed biosynthetic pathway of chaxine B, chaxine C and
demethylincisterol (5) from ergosterol (10)
Synthesis of chaxine B and BB
Our initial attempts to synthesize 14 from pre-vitamin D2 (11) (easily obtainable from
ergosterol (10)) failed due to its thermal instability. Accordingly, we sought to use tackysterol
(12), a regioisomer of 11, instead. However, the gram scale preparation of tachysterol 12
proved difficult. The photoinduced 6-electrocyclic reaction of ergosterol (10) gave a mixture
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of three isomers; pre-vitamin D2 (11), takysterol (12), and lumisterol, a epimer at C-10 of
ergosterol. Irradiation of 10 using a shorter wave-length of UV (254 nm) was reported to yield
tachysterol (12) as a major product,15 however, tachysterol was not obtained using a
photoreactor equipped with quartz glass and a low-pressure mercury lamp. After extensive
experimentation, we found that the quartz glass of the reactor interfered the electrocyclic
reaction. Direct irradiation of a solution of ergosterol in tert-BuOMe and a small amount of
BHT (3,5-di-tert-butyl-4-hydroxytoluene) using a low pressure-mercury light under argon16
gave a separable mixture (by flash column chromatography) of tachysterol (12) and pre-
vitamin D2 (11) in ca. 3:1 ratio; although the lability of tachysterol to oxygen and acids
necessitated the use of neutralized silica gel, solvents containing Et3N (3%), and nitrogen gas
(Scheme 3). Nevertheless, we were finally able to prepare tachysterol (12) from ergosterol
(10) on a gram-scale.
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Scheme 3. Preparation of tachysterol (12) from ergosterol (10) and synthesis of endoperoxide
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With sufficient 12 in hand, the synthesis of endoperoxide 19 from 12 using singlet oxygen could
be investigated. Conventional conditions (light, oxygen gas, and the photosensitizer TPP
(tetraphenylporphyrin)) resulted in a poor yield (< 30%) of 19, presumably due to the lability
of both starting material and the product to light and oxygen. Accordingly, we turned to NEP
(1,4-dimethylnapthtalene endoperoxide, 20),17 which generates singlet oxygen quantitatively
(t1/2 = 5 h at rt), as the source of singlet oxygen. Carbon tetrachloride was selected as the
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solvent; the half-life of O2 is much longer in CCl4 than CH2Cl2;18 and being non-polar, it
allowed residual NEP to be easily removed by filtration through a plug of silica gel without
evaporation of the solvent. A small amount of BHT was added to the reaction to prevent
autoxidation, although its effect was not quantified. Under these conditions, and using 3
equivalents of NEP, 19 was obtained in 57% yield as a single diastereomer judged by 1H NMR
spectra (Scheme 3). The configurations of the newly generated stereocenters were determined
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by NOESY, a correlation being observed between the proton at C-9 and the 18-methyl group.
The high diastereoselectivity is presumably due to the steric hindrance of the angular methyl
group (18-CH3). On a gram scale, 19 was obtained in a yield of 24% of two steps from 10,
where 12 was used without purification to avoid its decomposition during column
chromatography.
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Cleavage of the peroxy structure of 19 with DBU gave -hydroxy enone 21 in 81%
yield (Scheme 4). The selective deprotonation of the proton at the C-6 position is probably due
to its higher acidity than the proton at the C-9 position, being double allylic in nature. Initial
attempts at the Baeyer-Villiger oxidation of 21 were less successful: treatment with MCPBA
in CH2Cl2 resulted in epoxidation of the alkene of the A ring and the side chain unsaturation
without Baeyer-Villiger oxidation. This indicates that the A ring alkene and the ketone of 21
are not fully conjugated, because steric hindrance between the methyl group and the enone
precludes their coplanarity. In order to avoid the epoxidation, 21 was treated with (TMSO)2 and
TMSOTf, conditions reported to effect Baeyer-Villiger oxidation without alkene epoxidation,19
to give demethylincisterol A3 (5) and furan 23. From the formation of 5, we infer that the
Baeyer-Villiger oxidation of 21 occurred with the desired selectivity to give 15 (in Scheme 2).
However, hydrolysis of the ester 15 followed by oxidation of the C-9 position gave 5 under the
reaction conditions – perhaps by lactonization of product 15 of the Baeyer-Villiger reaction,
hydrolysis of the lactone and then oxidation of the hydroxy group. In order to prevent this, we
could have attempted Baeyer-Villiger oxidation of 2-ene-1,4-dione 24 (obtainable from 21),
although the prediction of the site and regioselectivity of the Baeyer-Villiger oxidation of 24
would be difficult12 as it incorporates two ketones conjugated through the same alkene, and
therefore there are four possible Baeyer-Villiger products. To the best of our knowledge, the
Baeyer-Villiger oxidation of such a complex substrate has not been reported.12 Although the
regioselectivity of the Baeyer-Villiger oxidation depends on many factors such as electron
density, steric of the substituent and stereoelectronic effect, we optimistically anticipated the
Baeyer-Villiger oxidation of 24 would take place preferentially at the ketone at C-6 position,
and that the A ring alkene would preferentially migrate due to its increased substitution; as the
rate-determining step of the Baeyer-Villiger oxidation is migration after addition of peroxide
to a ketone,12 the migratory aptitude of the substituents of the ketone determines the selectivity
– and of the four substituents of the two ketones of 24, the A ring alkene is tetrasubstituted with
three alkyl groups, while the alkene between the two ketones is electron deficient.
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Scheme 4. Synthesis of 21 and attempted Baeyer-Villiger oxidation of 21
However, preparation of 24 from 21 also proved problematic, even though production of 24
was observed when 21 was kept in a freezer for several months. Oxidation of 21 with MnO2
gave a complex mixture of products, and the attempted protection of the hydroxy group of the
A ring with TBSOTf and 2,6-lutidine in anticipation of the selective oxidation of the C-9
hydroxy group resulted in furan 23 (Paal-Knorr furan synthesis). At this point, we realized that
2-ene-1,4-dione 24, the desired substrate of the Baeyer-Villiger oxidation, could be prepared
by oxidation of furan 23. Synthesis of furan 23 from 21 was best carried out by with TMSOTf
in the presence of molecular sieves AW-300 at – 40 °C to give 23, an unstable product that
was used without purification (Scheme 5). Fortunately, upon treatment of 23 with MCPBA,
we observed production of desired 24 as well as enol ester 25, the desired product of the
Baeyer-Villiger oxidation of 24. Thus, the consecutive oxidation of furan 23 to give 24 and
then 25 could be carried out in one-pot using MCPBA. Optimized conditions for this sequence
were found to be 2.1 equivalent of MCPBA in CH2Cl2 at -40 °C, giving 25 in 38% yield in
three steps from 21. Two byproducts, epoxide 26 and spiro-product 27, were also obtained, the
both yields are 10%. and their structures were elucidated by NMR and X-ray analyses20 (For
details see the Supporting Information). Epoxide 26 is hypothesized to arise from 24 by
MCPBA. A possible but unvalidated mechanism for the formation of spiro compound 27 is
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depicted in Scheme 6, based on the observation that 27 was not formed by treating purified
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Scheme 5. Synthesis of enol ester 25 by oxidative cascade of furan 23
Scheme 6. A proposed mechanism for 27 from 23
We then turned our attention to stereoselective epoxidation of enol ester 25 (Scheme 7).
Epoxidation of 15 with MCPBA was first attempted with view to a development of an
oxidative cascade from furan 23, but an inseparable mixture of the products was obtained due
to competitive epoxidation of the side chain. However, the selective, directed epoxidation of
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the A ring alkene was accomplished using TBHP and catalytic VO(acac)2 in the presence of
Na2CO3, MS4A and WX-R (4,4’-thiobis-(6-tert-butyl-3-methylphenol)).22 to give epoxide
28a as a single diastereomeric product, which was exposed to silica gel without any
purification to give ester 29 as a single product in 67% yield over two steps from 25. The NMR
spectra of 29 was identical to that of BB, the stereochemistry of the A ring of which has not
been reported. X-ray crystallographic analysis of 29 clarified its structure and definitively
established it to incorporate a different ester linkage to that of 7 (in Figure 1). Since the relative
stereochemistry of the A ring of BB (29) is the same as that of the proposed structure of chaxine
B, the relative stereochemistry of the A ring of chaxine B (1) must be revised.
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The highly stereoselective conversion of -epoxide 28a to BB (29) is rationalized by the
mechanism shown in Scheme 8, and is consistent with a literature report of the stereochemical
outcome of another, similar reaction.23 We therefore hypothesized that chaxine B should be
accessible by the action of silica gel on α-epoxide 28b, a synthesis of which (from 25) was
now sought. Extensive exploration of the conditions for this reaction led us to conclude that
DMDO (dimethyldioxirane) was the best oxidant; epoxidation with DMDO in acetone gave a
mixture of 28a and 28b in ca. 3:5 ratio (by 1H-NMR spectra). Exposure of this mixture to silica
gel provided BB (29) and 30 in yields of 21 and 40% over two steps from 25, respectively. As
the NMR data of 30 was identical to those of natural chaxine B, the structure of chaxine B was
revised as 30, an epimer at the C-2’ position of BB (29). Since chaxine B is noncrystalline, the
structure of 30 was confirmed by spectroscopic analysis of the products of its reduction with
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NaBH4 and hydrolysis products (vide infra). Thus, an eight-step synthesis of chaxine B and
BB from ergosterol was accomplished.
To scale up the synthesis of chaxine B (30) and BB (29) (from 25) for their biological
evaluation, the epoxidation of 25 with DMDO generated in situ-from Oxone® and acetone was
investigated in biphasic solvent of aqueous NaHCO3 and several organic solvents.25
Interestingly, the choice of organic solvent was found to influence the stereoselectivity of the
epoxidation; the stereoselectivity () was 2.5:1 in t-BuOMe, 2.0:1 in AcOEt, 0.8:1 in benzene,
and 0.6:1 in CH2Cl2, by analysis of the 1H-NMR spectra. This observation is thought to be the
result of hydrogen bonding between polar solvents such as t-BuOMe, acetone, and AcOEt and
the allylic hydroxy group of the substrate which shields the -face, resulting in preferential -
epoxidation.26 On the other hand, nonpolar solvents such as CH2Cl2 and benzene do not
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significantly interact with the hydroxy group, and therefore epoxidation proceeds by hydrogen
bonding between DMDO and the homoallylic alcohol to give -epoxide preferentially. Thus,
576 mg of chaxine B (30) was obtained by the DMDO epoxidation of 25 in aqueous t-BuOMe
followed by treatment of silica gel.
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Scheme 7. Synthesis of chaxine B and BB
Scheme 8. A proposed mechanism for rearrangement of 28a to BB (29)
Synthesis of analogs of chaxine B and BB
Several analogues of chaxine B and BB were also prepared (Scheme 9). Dehydration of
synthetic chaxine B (30) and BB (29) with MsCl/Et3N gave 31 and 32, respectively.
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Comparison of the NMR spectra of 31 and 32 with those of chaxine C led to the conclusion
that 31 was chaxine C.24
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Scheme 9. Synthesis of chaxine C (31) and its epimer 32
Demethylincisterol A3 (5) was obtained by hydrolysis of synthetic chaxine B (30) and BB (29).
Alkaline hydrolysis of chaxine B (30) with aqueous lithium hydroxide gave demethylincisterol
A3 (5) and cyclohexenone 33 as a volatile compound27 (Scheme 10).28 Acid hydrolysis with
p-TsOH in aqueous THF of chaxine B (30) gave a mixture of 5 and 34; and of BB (29) gave 5
and 35 (Scheme 11). NMR analyses of 34 and 35 also confirmed the stereostructures of the A
rings of chaxine B (30) and BB (29), both of which bear a methyl group at the equatorial
position. Interestingly, the conformations of 34 and 35 are the opposite of those of the
corresponding ester BB (29) and chaxine B (30).
Scheme 10. Alkaline hydrolysis of chaxine B (30)
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Scheme 11. Acid hydrolysis of chaxine B (30) and BB (29) and conformations of the A ring
moieties
To undertake a SAR study of the side-chains of chaxine B and BB, 17-nor-chaxine D (46) and
its 2’-epimer 45 were synthesized from 7-dehydrocholesterol (36) in a similar manner to that
of chaxine B (Scheme 12; unoptimized conditions). Since the side chain of enol ester 42
does not contain an alkene in the side chain, we anticipated that the oxidative cascade from
furan 41 could be expanded to effect epoxidation of enol ester 42. Unfortunately, treatment of
41 with an excess amount of MCPBA resulted in an inseparable mixture of two diastereomers
45 and 46 along with two byproducts 43 and 44 after treatment of silica gel. Instead, we
synthesized 17-nor-chaxines D (46) and its epimer 45 by DMDO oxidation of 42, which was
prepared from furan 40 in a similar manner to chaxine B and BB. Dehydration of 46 with MsCl
and triethylamine gave 17-nor-chaxine E (47) in 86% yield. Alkaline hydrolysis of 46 with
aqueous LiOH gave 17-nor-chaxine A (48).
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Scheme 12. Synthesis of 17-nor-chaxine D (46) and its analogs from 7-dehydrocholesterol
(36) (The conditions were not optimized.)
Conclusion
The total syntheses of chaxine B and BB have been accomplished in eight steps starting
from ergosterol and proceeding through a common intermediate in a manner inspired by their
proposed biosynthesis. The key step is an oxidative cascade using MCPBA, which includes
oxidation of the furan 23 followed by Bayer-Villiger oxidation of the resulting enedione 24.
The enol ester of the cascade product 25 was the common intermediate; its -epoxidation gave
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chaxine B, and -epoxidation gave BB after stereoselective rearrangement of the epoxide with
silica gel. We believe that ergosterol is the precursor of the chaxines and the Baeyer-Villiger
oxidation of enedione 24 and the epoxidation of 25 closely resemble the biosynthesis of these
natural products. These two key reactions of the proposed biosynthesis are partially revised
from those reactions shown in Scheme 2 during this investigation. This study also resulted in
the revision of the stereostructures of the chaxines and BB; chaxine B and BB are 2’-epimers.
Thus, stereostructures of the A ring of chaxines D and E should be identical to those of chaxines
B and C, respectively. The scalable and concise nature of our synthesis enabled the synthesis
of natural and unnatural analogs of chaxine B; biological studies of these products are ongoing,
and results will be reported in due course.29
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Experimental
General Experimental: Melting points (MP) were recorded on a Yanaco MP-S3 melting
point apparatus and are not corrected. Optical rotations were measured on a JASCO DIP-370
digital polarimeter. Infrared spectra (IR) were recorded on a JASCO FT/IR-4100
spectrophotometer and are reported in wave number (cm-1). Proton nuclear magnetic resonance
(1H NMR) spectra were recorded on a Bruker AVANCE-400 (400 MHz) spectrometer. NMR
samples were dissolved in CDCl3 or C6D6, and chemical shifts are reported in ppm relative to
1
the residual undeuterated solvent (CDCl3 as  = 7.26 ppm, C6D6 as  = 7.16 ppm). H NMR
data are reported as follows; chemical shift, integration, multiplicity (s = singlet, d = doublet, t
= triplet, q = quartet, br = broadened, m = multiplet), coupling constant, and assignment.
Carbon nuclear magnetic resonance (13C{1H} NMR) spectra were recorded on a Bruker
AVANCE-400 (100 MHz) spectrometer. The samples were dissolved in CDCl3 or C6D6, and
chemical shifts are reported in ppm relative to the deuterated solvent (CDCl3 as  = 77.1 ppm,
C6D6 as  = 128.0 ppm). 1H NMR and 13C{1H} NMR spectra were measured at 300 K unless
otherwise noted. Elemental analyses were performed at the Analytical Laboratory of Graduate
School of Bioagricultural Sciences, Nagoya University. High resolution mass spectra (HRMS)
were recorded on an Applied Agilent 6220 Time of Flight (TOF) or an LTQ Orbitrap mass
spectrometer (Thermo Fisher Scientific) equipped with an electro spray ionization probe and
are reported in m/z. Reactions were monitored by thin-layer chromatography (TLC) on 0.25
mm silica gel coated glass plate 60 F254 (Merck, #1.05715). Silica gel 60 (spherical, particle
size 40-50 m, Kanto Chemical Co., Inc.) was used for flash-column chromatography. Unless
otherwise noted, non-aqueous reactions were carried out in flame-dried glassware under
nitrogen or argon. Dry CH2Cl2, THF, and Et2O were purchased from Kanto Chemical Co., Inc.
Et3N and DBU were distilled from CaH2. All other commercially available reagents were used
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as received. Activated AW-300, MS4A and dry silica gel (Merck, #1.07734.9025, 0.063-0.200
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nm) were prepared by heating at 250 °C in vacuo with a Kugelrohr apparatus for 3 h.
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Tachysterol (12): A flat dish (ф 18 cm, depth 3.6 cm, polypropylene) charged with ergosterol
(10) (3.08 g, 7.77 mmol), BHT (168 mg, 0.766 mmol) and a magnetic stirrer bar, was placed
on a magnetic stirrer. t-BuOMe (400 mL) was added, and three mercury lamps (GL 15, 15 W,
43.6 cm x  2.6 cm, Toshiba Corp. Ltd.) were placed on the flat dish. The whole apparatus was
wrapped with a vinyl bag, into which N2 gas was flowed. The flat dish was irradiated at room
temperature with stirring. t-BuOMe (ca. 50 mL) was added at 30 min intervals to maintain the
solvent volume. After 5 h, the solution was concentrated in vacuo at ice-bath temperature. The
residue was purified by flash column chromatography (neutral silica gel 90 g, 3% Et3N in
Et2O/hexane 5:9) to give tachysterol (12) (1.76 g, 57%) as a white amorphous solid.
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Endoperoxide 19: A three-necked flask connected to a three-way stopcock was charged with
tachysterol (12) (2.28 g, 5.75 mmol) and BHT (670 mg, 3.05 mmol), and then filled with argon
gas. CCl4 (180 mL) was degassed by freeze-thaw cycles (x3) in a separate flask and transferred
to the reaction vessel with a syringe. 1,4-Dimethylnapthalene endoperoxide (NEP) (20) (2.14
g, 11.4 mmol) was added, the reaction vessel was wrapped with aluminum foil. After stirring
at room temperature under argon atmosphere for 21 h, NEP (20) (1.05 g, 5.59 mmol) was added.
Stirring was continued for 17 h, and then the solution was directly subjected to flash column
chromatography (neutral silica gel 80 g, CH2Cl2 to AcOEt/hexane 1:3) to give 19 (1.41 g, 57%)
as a white amorphous solid.
Synthesis of endoperoxide 19 from 10 without purification of 19: Two flat dishes (ф18 cm,
depth 3.6 cm, polypropylene) were each charged with ergosterol (10) (3.88 g, 9.80 mmol and
3.66 g, 9.24 mmol), BHT (210 mg, 0.953 mmol and 171 mg, 0.776 mmol) and a magnetic
stirrer bar. t-BuOMe (450 mL) was added, and three mercury lamps were placed on the flat
dish. The whole apparatus was wrapped with a vinyl bag, and N2 gas was flowed into the bag.
The flat dishes were irradiated with three lamps at room temperature for 5.5 h, with stirring.
During this reaction, t-BuOMe (ca. 50 mL) was added at 30 min intervals to maintain the
solvent volume. The solutions of the dishes were combined and evaporated at an ice-bath
temperature. The residue was used for next reaction without purification. A three-necked flask
connected to a three-way stopcock was charged with the crude product (7.55 g) and BHT (1.38
g, 6.26 mmol) and then filled with argon gas. CCl4 (400 mL) was degassed by freeze-thaw
cycles (x3) in a separate flask and transferred to the vessel with a syringe. NEP (20) (4.74 g,
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25.2 mmol) was added, the reaction vessel was wrapped with aluminum foil. After stirring at
room temperature under argon atmosphere for 14 h, NEP (20) (1.16 g, 6.17 mmol) was added.
Stirring was continued for further 23 h, the solution was directly subjected to flash column
chromatography (neutral silica gel 210 g, CH2Cl2 to AcOEt/hexane 1:5) to give 19 (1.92 g, 24%
in 2 steps) as a white amorphous solid.
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1,4-Dimethylnaphthalene endoperoxide (20): A flask (1 L) was charged with 1,4-dimethyl
naphthalene (9.64 g, 61.7 mmol), methylene blue (44.7 mg, 0.139 mmol) and CH2Cl2 (1000
mL). LED light (10 W×100, a irradiation device consisted of 100 LED lights (70 pcs: red, 20
pcs: blue, 8 pcs: white, 1 pcs: UV, 1 pcs: IR)) was placed next to the flask as close as possible.
Irradiation was continued at 0 °C for 5 h with O2 bubbling. During the reaction, CH2Cl2 was
added at 30 min interval to maintain the solvent volume. The solution was evaporated at 0 °C
to ca. 10 mL of the solvent, which was filtered through a glass filter packed with silica gel (20
g) under vacuum to remove methylene blue. The filtrate (250-300 mL) was concentrated to
dryness in vacuo to provide orange crystals, which were washed with pentane to give NEP (20)
(6.02 g, 32.0 mmol, 52%) as white crystals. The filtrate was concentrated at 0 °C, the residue
was crystallized in the same way as already described to give NEP (20) (1.03 g, 5.48 mmol,
9%). The mother liqueur was recovered after decolorization with activated charcoal and
evaporated to give starting material, 1,4-dimethyl naphthalene (3.38 g, 90% recovery yield).
Unsaturated ketone 21: DBU (4.70 mL, 31.9 mmol) was added to a stirred solution of 19 (3.03
g, 7.08 mmol) in dry CH2Cl2 (80 mL) at -20 °C. After stirring at the same temperature for 24
h, the reaction was quenched with saturated aqueous NH4Cl (50 mL) and H2O, and the mixture
was allowed to warm to room temperature. After partitioning, the aqueous layer was extracted
with CH2Cl2 (15 mL x3), and the combined organic layer was washed with H2O (x1) and brine
(x1). The solution was passed through a short column packed with anhydrous Na2SO4 and
concentrated to dryness in vacuo. The residue was purified by flash column chromatography
(silica gel 120 g, Et2O/hexane 5:1 to Et2O) to give 21 (2.44 g, 81%) as a white amorphous solid.
Diepoxide 22: To a stirred solution of 21 (16.2 mg, 0.0379 mmol) in CH2Cl2 (1.5 mL) was
added MCPBA (27.4 mg, 0.159 mmol) at -10 °C. After stirring at the same temperature for 17
h, the reaction was quenched with saturated aqueous Na2SO3 (2 mL). The mixture was extracted
with CH2Cl2 (x3). The combined organic layer was washed with saturated aqueous NaHCO3
(x2) and brine (x1), dried over anhydrous Na2SO4, and evaporated under reduced pressure. The
residue was purified by column chromatography (AcOEt/hexane 3:1) to give 22 (9.7 mg, 56%)
as a white amorphous solid. 22: [α]D25 -47 (c 0.49, C6H6). IR (KBr) νmax 3420, 2961, 2874,
1673, 1615, 1461, 1375, 1203, 1015, 970, 922, 903 cm-1. HRMS (ESI) for C28H44O5Na
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[M+Na]+, calcd 483.3081, found: 483.3082. 1H and 13C{1H} NMR spectra (see the pdf of SI.)
show disappearance of the side chain unsaturation, remaining of the unsaturated ketone, and
four diastereomeric mixture.
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Synthesis of 23 and demethylincisterol A3 (5) from 21: TMSOTf (23 L, 0.13 mmol) and
(TMSO)2 (0.21 mL, 0.48 M in CH2Cl2, dried over AW-300) were added to a stirred solution of
21 (21.5 mg, 0.050 mmol) in CH2Cl2 (1.0 mL) at -40 °C. After stirring at the same temperature
for 2 h, additional (TMSO)2 (0.21 mL, 0.48 M in CH2Cl2, dried over AW-300) was added. After
stirring for 80 min, the reaction was quenched with Na2SO3 and the mixture extracted with
EtOAc (x4). The combined organic layer was washed with brine (x1), dried over anhydrous
Na2SO4, and evaporated under reduced pressure. The residue was purified by column
chromatography (Et2O/hexane 1:2 to 1:1) to give 23 (3.2 mg, 15%) and 5 (4.0 mg, 24%).
Synthesis of enol ester 25, 26 and 27 from 21: A suspension of 21 (2.18 g, 5.05 mmol) and
activated AW-300 (1.08 g) in dry CH2Cl2 (50 mL) was stirred under argon atmosphere at -
40 °C for 30 min. TMSOTf (2.20 mL, 12.6 mmol) was added at the same temperature, and after
20 min the reaction was quenched with saturated aqueous NaHCO3 (20 mL). The mixture was
diluted with H2O and allowed to warm to room temperature. To remove AW-300, the mixture
was filtered through a cotton plug. After partitioning, the aqueous layer was extracted with
CH2Cl2 (15 mL x3). The combined organic layer was washed with H2O (x1) and brine (x 1),
and filtered through a short column packed with anhydrous Na2SO4. The solvent was
evaporated to dryness in vacuo, and the residue was used for the next reaction without
purification. MCPBA (1.67 g, 10.1 mmol) was added to a solution of this crude product (1.96
g) in dry CH2Cl2 (50 mL) at -40 °C under an argon atmosphere. After stirring for 40 min, the
reaction was quenched with saturated aqueous Na2SO3 (15 mL) at -40 °C. H2O was added and
the mixture was allowed to warm to room temperature with stirring. After partitioning, the
aqueous layer was extracted with CH2Cl2 (15 mL x3). The combined organic layer was washed
with saturated aqueous NaHCO3 (x1) and brine (x1), and passed through a short column packed
with anhydrous Na2SO4. The solvent was evaporated to dryness in vacuo. The residue was
purified by flash column chromatography (neutral silica gel 90 g, AcOEt/hexane 2:3) to give
25 (857 mg, 38%) as a yellow oil, 26 (230 mg, 10%) as a yellow solid, and 27 (289 mg, 10%)
as a white solid. 26: Mp 135-138 °C. [α]D27 -14.8 (c 0.41, CHCl3). IR (KBr) νmax 3550, 2952,
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2872, 1712, 1651, 1459, 1419, 1368, 1290, 1265, 1237, 966, 741, 707 cm-1. H NMR (400
MHz, CDCl3) δ 5.26 (1H, dd, J = 15, 7.5 Hz, H-23), 5.15 (1H, dd, J = 15, 8.5 Hz, H-22), 4.03
(1H, m, H-4), 3.90 (1H, s, H-7), 2.86 (1H, brd, J = 16 Hz), 2.56-2.47 (2H, m), 2.41 (1H, dt, J =
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19, 6 Hz), 2.32 (1H, dd, J = 12, 7.5 Hz), 2.25-2.07 (4H, m), 2.04-1.96 (1H, m), 1.91 (3H, s, H-
19), 1.89-1.77 (3H, m), 1.75-1.61 (2H, m), 1.57-1.36 (4H, m), 1.31- 1.20 (1H, m), 1.15 (3H, s,
CH3-18), 1.04 (3H, d, J = 7 Hz, CH3-21), 0.91 (3H, d, J = 7 Hz, CH3-28), 0.83 (3H, d, J = 7
Hz, CH3-26 or 27), 0.81 (3H, d, J = 7 Hz, CH3-26 or 27). 13C{1H} NMR (100 MHz, CDCl3) δ
205.4, 199.6, 147.2, 134.8, 133.0, 128.5, 69.1, 66.0, 62.4, 55.7, 52.8, 45.5, 43.0, 39.9, 39.0,
37.2, 33.7, 33.2, 31.4, 29.8, 28.8, 21.7, 21.2, 20.3, 20.1, 19.8, 17.7, 12.2. HRMS (ESI) for
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C28H42O4Na [M+Na]+ , calcd 465.2975, found: 465.2954. 27: Mp 182-190 °C. [α]D -3.8 (c
0.52, CHCl3). IR (KBr) νmax 3359, 2956, 2870, 2076, 1666, 1456, 1141, 1104, 1028, 967, 827,
720, 709 cm-1. 1H NMR (400 MHz, CDCl3) δ 6.51 (1H, br, OH), 5.78 (1H, d, J = 2 Hz, H-7),
5.23 (1H, dd, J = 15, 7.5 Hz, H-23), 5.14 (1H, dd, J = 15, 8 Hz, H-22), 4.35 (1H, brd, J = 9 Hz,
OH), 3.98 (1H, br, H-4), 3.76 (1H, br, OH), 2.70 (1H, brt, J = 9 Hz, H-14), 2.47 (1H, td, J =
13, 4 Hz, H-11), 2.26 (1H, dd, J = 15, 3.5 Hz), 2.14-1.77 (8H, m), 1.74-1.63 (2H, m), 1.51-1.33
(6H, m), 1.26 (3H, s, CH3-19), 1.01 (3H, d, J = 7 Hz, CH3-21), 0.91 (3H, d, J = 7 Hz, CH3-28),
0.83 (3H, d, J = 7 Hz, H-26 or 27), 0.81 (3H, d, J = 7 Hz, H-26 or 27), 0.65 (3H, s, CH3-18).
13C{1H} NMR (100 MHz, CDCl3) δ 195.0, 162.9, 135.0, 132.8, 120.5, 93.6, 88.2, 74.6, 65.9,
55.9, 52.8, 48.1, 43.0, 40.4, 39.2, 38.4, 36.1, 33.2, 31.6, 30.8, 28.7, 24.1, 21.1, 20.9, 20.1, 19.8,
17.7, 12.1. HRMS (ESI) for C28H44O5Na [M+Na]+ , calcd 483.3061, found: 483.3081.
BB (29): A flask was charged with enol ester 25 (92.6 mg, 0.209 mmol), dried MS4A (101
mg), WX-R (3.8 mg, 10 mol), powdered Na2CO3 (133.7 mg, 1.26 mmol) and VO(acac)2 (10.8
mg, 42 mol), then filled with argon gas. Dry CH2Cl2 (4.5 mL) was added, and the reaction
mixture was stirred at 0 °C for 30 min under argon atmosphere. TBHP (3.0 M in toluene, 205
L, 0.628 mmol) was added at the same temperature, and the solution color turned to red from
green. After stirring for 4 h, TBHP (210 mL, 0.628 mmol) was added. Stirring was continued
for further 4 h, additional VO(acac)2 (5.0 mg, 19 mol) and TBHP (210 mL, 0.628 mmol) were
added. After 16 h, the reaction was quenched with saturated aqueous Na2SO3 (10 mL) at 0 °C
and then allowed to warm to room temperature. After partition, the aqueous layer was extracted
with EtOAc (6 mL x4), the combined organic layer was washed with H2O and brine (x1), and
passed through a short column packed with anhydrous Na2SO4. The solvents were removed by
evaporation. The residue was used for the next reaction without purification. To a stirred
solution of the crude product in dry CH2Cl2 (5.0 mL) was added dry silica gel (1.0 g) at room
temperature. Being stirred for 1.5 h at the same temperature, the reaction mixture was filtered
through a short column packed with cotton to remove the silica gel, which was eluted with
Et2O. The eluate was evaporated to dryness in vacuo. The residue was purified by flash
column chromatography (neutral silica gel 15 g, Et2O/hexane 3:1 to 5:1) to give 29 (63.8 mg,
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67% in 2 steps) as a colorless crystal.
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Chaxine B (30) and BB (29): NaHCO3 (8.69 g), H2O (13 ml) and acetone (6.5 mL) were added
to a solution of 25 (885 mg, 2.00 mmol) in t-BuOMe (13 mL) at 0 °C. A solution of Oxone®
(6.18 g, 20.0 mmol) in H2O (25.0 mL) was added dropwise over 1 h. The mixture was
vigorously stirred for 6 h at 0 °C. The reaction was quenched with saturated aqueous Na2CO3
(30 mL) and CH2Cl2 (10 mL) was added. After separation of the organic layer, the aqueous
layer was extracted with CH2Cl2 (10 mL x3). The combined organic layer was passed through
a short column packed with anhydrous Na2SO4, and concentrated to dryness in vacuo to give a
mixture of α & β epoxide 28a, 28b and enol ester 25 (2:5:1.2 ratio by 1H NMR). The mixture
(933 mg) was dissolved in dry CH2Cl2 (100 mL) and then silica gel (9.30 g) was added at room
temperature. After being stirred vigorously for 1 h, the reaction mixture was filtered through a
cotton plug, and then rinsed with AcOEt. The filtrate was concentrated and purified by flash
column chromatography (neutral silica gel 25 g, AcOEt/hexane 2:5) to give a mixture (576 mg)
of chaxine B (30) and BB (29) and recovered starting material 25 (76 mg, 9%). Further
purification by flash column chromatography (neutral silica gel 40 g, CHCl3/THF/pentane
9:1:3) gave chaxine B (30) (403 mg, 44% in 2 steps) and BB (29) (166 mg, 18% in 2 steps) as
a white amorphous solid respectively.
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Alkaline hydrolysis of Chaxine B (30) with LiOH: An aqueous solution of LiOH (1.2 mL;
0.05 M) was added to a solution of chaxine B (30) (19.3 mg, 42.0 µmol) in THF (0.6 mL) at
room temperature. After stirring at the same temperature for 3 h, the reaction was quenched
with 1N HCl (5 mL) and H2O. After separation of the organic layer, the aqueous layer was
extracted with CH2Cl2 (3 mL x3). The combined organic layer was passed through a short
column packed with anhydrous Na2SO4, and the solvent was removed by flowing argon gas.
The residue was purified by flash column chromatography (neutral silica gel 3 g, Et2O/pentane
1:1) to give demethylincisterol A3 (5) (12.1 mg, 86%) as a white solid, and 33 (1.8 mg, 34%) as
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volatile compound. Demethylincisterol A3 (5): Mp 141-142 °C (lit. 144-145 °C). [α]D
+189.6 (c 0.57, CHCl3). (lit. [α]D +169.72 (c 0.58, CHCl3)).6 IR (KBr) νmax 3304, 2957, 2925,
2870, 1760, 1727, 1661, 1456, 1446, 1433, 1114, 980, 970, 947, 901, 853 cm-1. 1H NMR (400
MHz, CDCl3) δ 5.63 (1H, d, J = 2 Hz, H-2), 5.25 (2H, dd, J = 15, 8 Hz, H-16), 5.16 (2H, dd, J
= 15, 8 Hz, H-15), 3.10 (1H, s, OH), 2.65 (1H, m, H-8), 2.28 (1H, ddd, J = 14, 4, 2.5 Hz, H-5),
2.11-2.01 (1H, m), 2.01-1.94 (1H, m), 1.94-1.81 (3H, m), 1.77-1.68 (1H, m), 1.65 (1H, dd, J =
13.5, 4 Hz), 1.61-1.56 (1H, m), 1.55-1.46 (4H, m), 1.04 (3H, d, J = 7 Hz, CH3-14), 0.92 (3H,
d, J = 7 Hz, CH3-21), 0.84 (3H, d, J = 7 Hz, CH3-19 or 20), 0.82 (3H, d, J = 7 Hz, CH3-19 or
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20), 0.61 (3H, s, CH3-12).13C{1H} NMR (100 MHz, CDCl3) δ 171.2, 170.8, 134.8, 133.0, 112.4,
105.0, 55.5, 50.5, 49.0, 43.0, 40.3, 35.4, 35.2, 33.2, 29.0, 21.5, 21.2, 20.1, 19.8, 17.8, 11.9.
HRMS (ESI) for C21H31O3 [M-H]- calcd, 331.2268, found: 331.2284. Cyclohexenone 33: 1H
NMR (400 MHz, CDCl3) δ 6.96 (1H, m, H-5’), 6.05 (1H, ddd, J = 10, 2.5, 1.5 Hz, H-6’), 3.65
(1H, s, OH), 2.52-2.45 (2H, m, H-4’), 2.13 (1H, m, H-3’), 2.05 (1H, ddd, J = 13, 10, 7 Hz, H-
3’), 1.33 (3H, s, H-7’). 13C{1H} NMR (100 MHz, CDCl3) δ 202.9, 151.1, 126.6, 73.3, 35.8,
25.6, 24.2.
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Acid hydrolysis of chaxine B (30): p-TsOH·H2O (40.8 mg) was added to a solution of chaxine
B (30) (6.9 mg, 15 µmol) in THF-H2O (3:1) (0.8 mL) at room temperature. After stirring at the
same temperature for 44 h, the reaction was quenched with 1.46 M Pyridine-THF solution (0.4
mL), and the solvent was evaporated to dryness in vacuo. The residue was purified by flash
column chromatography (neutral silica gel 1 g, CH2Cl2/MeOH 20:1) to give demethylincisterol
A3 (5) (6.4 mg, quant.) and 34 (1.8 mg, 80%) as a white solid respectively. 34: Mp 118-120 °C.
[α]D27 +63.2 (c 0.095, CHCl3). 1H NMR (400 MHz, CDCl3) δ 4.50 (1H, m, H-5’), 2.79 (1H, dd,
J = 14.5, 3.5 Hz, H-6’), 2.61 (1H, brd, J = 14.5 Hz, H-6’), 2.22-2.13 (1H, m), 2.01-1.91 (3H,
m), 1.41 (3H, s, H-7’). 13C{1H} NMR (100 MHz, CDCl3) δ 212.5, 76.3, 70.0, 45.4, 35.9, 30.1,
24.8. HRMS (ESI) for C7H12O3Na [M+Na]+ , calcd 167.0678, found: 167.0680.
Acid hydrolysis of BB (29): TsOH-H2O (40.0 mg) was added to a solution of BB (29) (7.5 mg,
16 µmol) in THF-H2O (3:1) (0.8 mL) at room temperature. After stirring at the same
temperature for 19 h, the reaction was quenched with 1.46 M pyridine-THF solution (0.4 mL),
and the solvent was evaporated to dryness in vacuo. The residue was purified by flash column
chromatography (neutral silica gel 1 g, CH2Cl2/MeOH 20:1) to give demethylincisterol A3 (5)
(5.2 mg, 96%) as a white solid and 35 (2.5 mg, quant) as an oil. 35: [α]D26 -23 (c 0.11, CHCl3).
1H NMR (400 MHz, CDCl3) δ 3.96 (1H, tt, J = 11, 5 Hz, H-5’), 2.86 (1H, ddd, J = 13.5, 5, 2.5
Hz, H-6’), 2.64 (1H, dd, J = 13.5, 11 Hz, H-6’), 2.14 (1H, m), 2.08 (1H, dt, J = 13.5, 3.5 Hz),
1.77 (1H, m), 1.58 (1H, td, J = 13, 3.5 Hz), 1.42 (3H, s, H-7’). 13C{1H} NMR (100 MHz,
CDCl3) δ 211.1, 75.7, 70.7, 46.9, 35.6, 32.0, 25.1. HRMS (ESI) for C7H12O3Na [M+Na]+ , calcd
167.0678, found: 167.0677.
Purification of 7-dehydrocholesterol (36): 7-Dehydrocholesterol (36) (5.69 g, supplier: nacalai
tesque) was suspended in MeOH (10 mL). The mixture was stirred for 1 h at room temperature
and filtered. The filtride washed with MeOH to give purified 7-dehydrocholesterol (35) (2.93
g, recovered 52%) as a white solid.
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Endoperoxide 39: Two flat-dishes (ф 18 cm, depth 3.6 cm, polypropylene) charged with 7-
dehydrocholesterol (36) (2.84 g, 7.38 mmol and 2.92 g, 7.59 mmol respectively), BHT (166
mg, 0.754 mmol, 182 mg, 0.827 mmol) and a magnetic stirrer bar, were placed on magnetic
stirrers. t-BuOMe (450 mL) was added, and three mercury lamps were placed on the flat dishes.
The whole apparatus was wrapped with a vinyl bag, and nitrogen gas was flowed into the bag.
The flat-dishes were irradiated with the lamps at room temperature for 5.5 h. During this
reaction, t-BuOMe (ca. 50 mL) was added at 30 min intervals to maintain the solvent volumes.
The solutions of the dishes were combined and evaporated at an ice-bath temperature to give a
mixture of 38, 36 and 37 (2:1:1 ratio by 1H NMR). The residue was used for the next reaction
without purification. A part of the crude product was purified by flash column chromatography
(Et2O/hexane 1:4) for spectral analysis of 38. A three-necked flask connected to a three-way
stopcock was charged with the crude product (5.75 g) and BHT (1.66 g, 7.54 mmol) and then
filled with argon gas. CCl4 (300 mL) was degassed by freeze-thaw cycles (x3) in a separate
flask and transferred to the reaction vessel through a syringe. NEP (20) (4.41 g, 23.5 mmol)
was added, the reaction vessel was wrapped with aluminum foil. After stirring at room
temperature under argon atmosphere for 15 h, NEP (20) (1.11 g, 5.90 mmol) was added. Stirring
was continued for further 32 h, the solution was directly subjected to flash column
chromatography (neutral silica gel 210 g, AcOEt / hexane 1:5) to give 39 (1.19 g, 19% in 2
steps) as a white amorphous solid. 38: [α]D27 -24.7 (c 0.94, CHCl3). IR (KBr) νmax 3346, 2951,
2867, 2830, 1635, 1466, 1429, 1376, 1366, 1348, 1098, 1062, 1037, 954 cm-1. 1H NMR (400
MHz, CDCl3) δ 6.72 (1 H, d, J = 16 Hz, H-6), 6.01 (1H, d, J = 16 Hz, H-7), 5.70 (1H, brd, J =
3 Hz, H-9), 3.97 (1H, br, H-3), 2.55 (1H, brd, J = 16.5 Hz), 2.30-2.15 (4H, m), 2.09 (1H, dd, J
= 16.5, 7 Hz), 2.04-1.92 (3H, m), 1.86 (1H, m), 1.79 (3H, s, CH3-19), 1.66-1.47 (4H, m), 1.45-
1.30 (6H, m), 1.26-1.08 (4H, m), 1.07-0.98 (1H, m), 0.96 (3H, d, J = 6.5 Hz, CH3-21), 0.87
(3H, d, J = 6.5 Hz, CH3-26 or 27), 0.86 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.69 (3H, s, CH3-
18). 13C{1H} NMR (100 MHz, CDCl3) δ 138.4, 131.6, 128.0, 125.6, 125.1, 124.8, 67.7, 54.2,
50.3, 43.0, 39.7, 36.3, 36.2, 36.1, 34.7, 31.3, 31.2, 28.8, 28.2, 25.2, 24.5, 24.0, 23.0, 22.7, 19.1,
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19.1, 11.4. Anal. Calcd for C27H44O: C, 84.31; H, 11.53. Found: C, 84.31; H, 11.51. 39: [α]D
+32.2 (c 1.22, CHCl3). IR (KBr) νmax 3444, 2950, 2932, 2870, 1640, 1467, 1441, 1056, 1036
cm-1. 1H NMR (400 MHz, C6D6) δ 5.40 (1H, m, H-7), 5.24 (1H, q, J = 2.5 Hz, H-6), 5.09 (1H,
m, H-9), 3.77 (1H, m, H-3), 3.03 (1H, m), 2.30-2.15 (2H, m), 1.95-1.77 (3H, m), 1.69-1.50 (5H,
m), 1.47(3H, s, CH3-19), 1.45-1.31 (6H, m), 1.30-1.15 (6H, m), 1.02 (2H, m), 0.94 (3H, d, J =
6.5 Hz, CH3-26 or 27), 0.93 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.83 (3H, d, J = 6.5 Hz, CH3-
21), 0.65 (3H, s, CH3-18). 13C{1H} NMR (100 MHz, C6D6) δ 142.0, 129.4, 117.7, 78.3, 76.8,
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67.4, 57.3, 48.2, 41.3, 39.9, 36.3, 36.2, 35.6, 35.6, 31.5, 31.0, 29.3, 28.4, 24.4, 23.8, 23.3, 23.0,
22.8, 18.6, 18.5, 18.5. HRMS (ESI) for C27H44O3Na [M+Na]+ , calcd 439.3182, found:
439.3186.
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Unsaturated ketone 40: DBU (2.00 mL, 12.8 mmol) was added to a stirred solution of 39 (1.10
g, 2.64 mmol) in dry CH2Cl2 (30 mL) at -20 °C. After stirring at the same temperature for 24
h, the reaction was quenched with saturated aqueous NH4Cl (30 mL) and H2O, and the mixture
was allowed to warm to room temperature. After partitioning, the aqueous layer was extracted
with CH2Cl2 (5 mL x3), and the combined organic layer was washed with H2O (x1) and brine
(x1). The solution was passed through a short column packed with anhydrous Na2SO4 and
concentrated to dryness in vacuo. The residue was purified by flash column chromatography
(silica gel 30 g, Et2O/hexane 5:1 to Et2O) to give 40 (737 mg, 67%) as a white amorphous solid.
40: [α]D28 -73.6 (c 1.13, CHCl3). IR (KBr) νmax 3397, 2869, 1609, 1467, 1444, 1380, 1366, 1265,
1237, 1215, 1158, 1068, 1055, 1016, 965, 922, 907 cm-1. 1H NMR (400 MHz, CDCl3) δ 5.99
(1H, d, J = 2 Hz, H-7), 5.04 (1H, brs, H-9), 4.00 (1H, br, H-3), 2.75-2.67 (2H, m), 2.61 (1H,
brs), 2.32 (1H, dt, J = 18, 5.5 Hz), 2.25-2.07 (2H, m), 2.00-1.72 (6H, m), 1.89 (3H, s, CH3-19),
1.70-1.61 (2H, m), 1.57-1.44 (3H, m), 1.44-1.28 (5H, m), 1.21-1.09 (3H, m), 1.07-0.97 (1H,
m), 0.93 (3H, d, J = 6 Hz, CH3-21), 0.87 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.86 (3H, d, J = 6.5
13
Hz, CH3-26 or 27), 0.59 (3H, s, CH3-18). C{1H} NMR (100 MHz, CDCl3) δ 197.3, 160.5,
141.1, 131.6, 124.6, 66.6, 64.8, 56.6, 51.2, 46.1, 39.6, 36.2, 36.1, 35.4, 34.8, 31.3, 30.5, 29.8,
28.2, 27.7, 24.0, 23.0, 22.7, 22.4, 21.6, 18.9, 12.4. HRMS (ESI) for C27H44O3Na [M+Na]+ ,
calcd 439.3182, found: 439.3180.
Synthesis of enol ester 42, 43 and 44 from 40: A suspension of 40 (195 mg, 0.468 mmol) and
activated AW-300 (94 mg) in dry CH2Cl2 (4.5 mL) was stirred under argon atmosphere at -
40 °C for 30 min. TMSOTf (0.20 mL, 1.1 mmol) was added at the same temperature and the
reaction was quenched with saturated aqueous NaHCO3 (10 mL) after stirring for 20 min. The
mixture was diluted with H2O, allowed to warm to room temperature. To remove AW-300, the
mixture was filtered through a cotton plug. The filtrate was partitioned, and the aqueous layer
was extracted with CH2Cl2 (5 mL x3). The combined organic layer was washed with H2O (x1)
and brine (x1), and filtered through a short column packed with anhydrous Na2SO4. The solvent
was evaporated to dryness in vacuo to give furan 41 (184 mg) as a white amorphous solid, a
sample of which was purified by flash column chromatography (CH2Cl2/hexane 5:1) for
spectral analysis. The residue was used for the next reaction without purification. The crude
product (185 mg) was dissolved in dry CH2Cl2 (4.5 mL) and then MCPBA (157 mg, 0.910
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mmol) was added at -40 °C under argon atmosphere. After stirring for 40 min, the reaction was
quenched with saturated aqueous Na2SO3 (10 mL) at -40 °C. H2O was added and the mixture
was allowed to warm to room temperature. After partitioning, the aqueous layer was extracted
with CH2Cl2 (5 mL x3). The combined organic layer was washed with saturated aqueous
NaHCO3 (x2) and brine (x1), and passed through a short column packed with anhydrous
Na2SO4. The solvent was evaporated to dryness in vacuo. The residue was purified by flash
column chromatography (neutral silica gel 10 g, AcOEt/hexane 2:3 to 1:2) to give 42 (88.5 mg,
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44%) as a yellow oil and a mixture of 43 and 44 (1:1 ratio by H NMR, 61.7 mg, calculated
15% yield, respectively) as a solid. A part of the mixture was purified by flash column
chromatography (AcOEt:CH2Cl2 1:5) for spectral analysis of 43 and 44. 41: [α]D27 +15 (c 0.73,
C6H6). IR (KBr) νmax 3385, 2952, 2931, 2868, 1698, 1623, 1466, 1445, 1377, 1366, 1331, 1098,
1052 cm-1. 1H NMR (400 MHz, C6D6) δ 6.18 (1H, s, H-7), 3.78 (1H, m, H-3), 2.79 (1H, brd, J
= 16.5 Hz), 2.72-2.56 (2H, m), 2.56-2.46 (1H, m), 2.38 (1H, ddd, J = 16, 7.5, 2 Hz), 2.15-1.90
(3H, m), 1.99 (3H, s, CH3-19), 1.89-1.81 (1H, m), 1.67-1.33 (10H, m), 1.28-1.15 (5H, m), 1.07
(1H, m), 0.95 (9H, d, J = 6.5 Hz, CH3-21 and 26 and 27), 0.62 (3H, s, CH3-18). 13C{1H} NMR
(100 MHz, C6D6) δ 154.0, 148.3, 129.1, 121.2, 120.0, 107.3, 66.9, 54.0, 47.8, 44.1, 39.9, 37.1,
36.7, 36.5, 35.8, 31.7, 31.4, 30.0, 28.4, 24.5, 23.9, 23.1, 22.8, 21.8, 21.6, 19.1, 11.0. Anal. Calcd
for C27H42O2: C, 81.35; H, 10.62. Found: C, 81.49; H, 10.79. 42: [α]D28 +86.9 (c 1.45, CHCl3).
IR (KBr) νmax 3437, 2953, 2932, 2869, 1731, 1704, 1645, 1467, 1446, 1382, 1326, 1236, 1159,
1146, 1132, 1114, 1039 cm-1. 1H NMR (400 MHz, C6D6) δ 5.61 (1H, d, J = 2.5 Hz, H-7), 3.83
(1H, m, H-3), 2.67 (1H, brd, J = 16 Hz), 2.54-2.39 (2H, m), 2.34 (1H, ddd, J = 15, 5, 2 Hz),
2.04-1.89 (2H, m), 1.83-1.61 (3H, m), 1.65 (3H, brs, CH3-19), 1.60-1.47 (4H, m), 1.39-1.05
(12H, m), 0.93 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.92 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.73
(3H, d, J = 6.5 Hz, CH3-21), 0.57 (3H, s, CH3-18). 13C{1H} NMR (100 MHz, C6D6) δ 202.1,
164.2, 155.9, 139.4, 120.5, 117.7, 66.9, 57.2, 55.5, 46.1, 39.9, 38.9, 37.9, 36.7, 36.2, 36.0, 30.8,
28.9, 28.4, 27.4, 24.3, 23.0, 22.8, 22.0, 18.7, 16.0, 11,6. HRMS (ESI) for C27H42O4Na [M+Na]+
, calcd 453.2975, found: 453.2964. 43: Mp 91-96 °C. [α]D27 -16.7 (c 1.13, CHCl3). IR (KBr)
νmax 3520, 3441, 2953, 2932, 2869, 1723, 1645, 1467, 1419, 1378, 1366, 1237, 1055 cm-1. 1H
NMR (400 MHz, CDCl3) δ 4.03 (1H, m, H-3), 3.91 (1H, s, H-7), 2.85, (1H, brd, J = 15.5 Hz),
2.60-2.48 (2H, m), 2.41 (1H, dt, J = 19, 6 Hz), 2.32 (1H, dd, J = 12, 7 Hz), 2.28-2.13 (2H, m),
2.05-1.95 (2H, m), 1.91 (3H, brs, CH3-19), 1.86-1.76 (2H, m), 1.75-1.62 (3H, m), 1.57-1.43
(4H, m), 1.41-1.23 (4H, m), 1.18-1.07 (2H, m), 1.14 (3H, s, CH3-18), 1.03 (1H, m), 0.95 (3H,
d, J = 6 Hz, CH3-21), 0.86 (3H, d, J = 6.5 Hz, CH3-26 or 27), 0.85 (3H, d, J = 6.5 Hz, CH3-26
or 27). 13C{1H} NMR (100 MHz, CDCl3) δ 205.5, 199.7, 147.2, 128.5, 69.1, 66.0, 62.4, 55.9,
52.8, 45.6, 39.5, 39.0, 37.3, 36.0, 35.6, 33.7, 31.4, 29.8, 28.6, 28.1, 23.9, 22.9, 22.7, 21.7, 20.3,
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18.8, 11.9. HRMS (ESI) for C27H42O4Na [M+Na]+ , calcd 453.2975, found: 453.2972. 44: Mp
180-183 °C. [α]D28 +8.68 (c 0.95, CHCl3). IR (KBr) νmax 3462, 2956, 2868, 1670, 1642, 1464,
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1444, 1381, 1297, 1234, 1137, 1106, 1084, 1025, 1006, 970, 954, 914 cm-1. H NMR (400
MHz, CDCl3) δ 6.25 (1H, br, OH), 5.79 (1H, d, J = 2 Hz, H-7), 4.27 (1H, brd, 10 Hz, OH), 3.98
(1H, m, H-3), 3.07 (1H, br, OH), 2.70 (1H, t, J = 10 Hz, H-14), 2.51 (1H, td, J = 14, 4 Hz), 2.26
(1H, dd, J = 15, 4 Hz), 2.10 (1H, m), 2.05-1.89 (5H, m), 1.74-1.66 (2H, m), 1.56-1.46 (3H, m),
1.43-1.32 (6H, m), 1.29 (3H, s, CH3-19), 1.18-1.09 (3H, m), 1.06-0.97 (1H, m), 0.92 (3H, d, J
= 6 Hz, CH3-21), 0.87 (3H, d, J = 6 Hz, CH3-26 or 27), 0.86 (3H, d, J = 6 Hz, CH3-26 or 27),
0.65 (3H, s, CH3-18). 13C{1H} NMR (100 MHz, CDCl3) δ 195.1, 162.6, 120.6, 93.6, 88.4, 74.3,
66.0, 56.1, 52.7, 48.2, 39.6, 39.2, 38.3, 36.2, 36.0, 31.5, 30.9, 28.5, 28.1, 24.0, 23.8, 22.9, 22.7,
20.9, 18.8, 11.8. HRMS (ESI) for C27H44O5Na [M+Na]+ , calcd 471.3081, found: 471.3071.
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Synthesis of 17-nor-chaxine D (46) and 17-nor-2’-epi-chaxine D (45): Compound 42 (104
mg, 0.242 mmol) was dissolved in t-BuOMe (3 mL), NaHCO3 (1.04 g), H2O (3 mL) and
acetone (1.5 mL) at 0 °C. After stirring at the same temperature, a solution of Oxone® (718 mg,
2.33 mmol) in H2O (3 mL) was added dropwise over 20 min. The mixture was vigorously
stirred at 0 °C for 8 h. The reaction was quenched with saturated aqueous Na2SO3 (15 mL) and
AcOEt (5 mL). After separation of the organic layer, the aqueous layer was extracted with
AcOEt (5 mL x3). The combined organic layer was washed with H2O (x1) and brine (x1), and
filtered through a short column packed with anhydrous Na2SO4, and concentrated to dryness in
vacuo to give a mixture of α & β epoxide. The ratio of α/β epoxide was determined to be ca.
5:2 by 1H NMR spectra. To a stirred solution of the crude product (103 mg) in dry CH2Cl2 (10
mL) was added silica gel (1.02 g) at room temperature. Being stirred for 1 h, the reaction
mixture was filtered through a cotton plug, and then eluted with AcOEt. The filtrate was
concentrated and purified by flash column chromatography (neutral silica gel 10 g,
AcOEt/hexane 2:5) to give a mixture (65.7 mg) of 17-nor-chaxine D (46) and 17-nor-2’-epi-
chaxine D (45) and recovered 42 (4.2 mg). Further purification by flash column
chromatography (neutral silica gel 10g, CHCl3/THF/pentane 9:1:3) to give 17-nor-chaxine D
(46) (44.3 mg, 41% in 2 steps) and 17-nor-epi chaxine D (45) (17.2 mg, 16% in 2 steps) as a
white amorphous solid respectively. 17-nor-chaxine D (46): [α]D25 +68 (c 0.67, CHCl3). IR
(KBr) νmax 3427, 2869, 1711, 1644, 1467, 1456, 1446, 1377, 1331, 1285, 1258, 1166, 1143,
1127, 1075 cm -1. 1H NMR (400 MHz, CDCl3) δ 5.57 (1H, d, J = 2 Hz, H-2), 3.95 (1H, tt, J =
9, 5 Hz, H-5’), 2.87 (1H, dd, J = 13, 9 Hz), 2.74-2.63 (2H, m), 2.55-2.45 (2H, m), 2.33 (1H, td,
J = 15, 5 Hz), 2.25 (1H, ddd, J = 13, 7, 2 Hz), 2.10-2.01 (1H, m), 1.99-1.86 (2H, m), 1.74-1.60
(3H, m), 1.59-1.47 (4H, m), 1.44 (3H, s, CH3-7’), 1.43-1.30 (4H, m), 1.21-1.08 (3H, m), 1.02
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= 6.5 Hz, CH3-19 or 20), 0.86 (3H, s, CH3-12). C{1H} NMR (100 MHz, CDCl3) δ 204.7,
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203.8, 165.4, 156.2, 117.4, 82.7, 70.3, 57.6, 55.7, 47.9, 46.5, 39.5, 39.0, 38.0, 35.9, 35.9, 33.4,
29.7, 28.9, 28.1, 23.9, 22.9, 22.7, 22.1, 20.4, 18.8, 12.0. HRMS (ESI) for C27H42O5Na [M+Na]+,
calcd 469.2924, found: 469.2921. 17-nor-epi chaxine D (45): [α]D25 +69 (c 0.43, CHCl3). IR
(KBr) νmax 3435, 2083, 1729, 1698, 1640, 1468, 1451, 1377, 1329, 1163, 1124, 1090, 957, 862
cm -1. 1H NMR (400 MHz, CDCl3) δ 5.52 (1H, d, J = 2 Hz, H-2), 4.46 (1H, br, H-5’), 3.10 (1H,
dd, J = 13.5, 3.5 Hz), 2.68 (1H, ddd, J = 15, 13, 7 Hz), 2.55-2.46 (2H, m), 2.39 (1H, dd, J = 13,
4 Hz), 2.26 (1H, brdd, J = 13, 7 Hz), 2.15 (1H, m), 2.10-1.97 (3H, m), 1.75-1.60 (4H, m), 1.57-
1.47 (3H, m), 1.45 (3H, s, CH3-7’), 1.42-1.30 (4H, m), 1.18-0.98 (4H, m), 0.94 (3H, d, J = 6.5
Hz, CH3-14), 0.87 (3H, s, CH3-12). 0.87 (3H, d, J = 6.5 Hz, CH3-19 or 20), 0.86 (3H, d, J = 6.5
Hz, CH3-19 or 20). 13C{1H} NMR (100 MHz, CDCl3) δ 205.6, 204.0, 165.5, 154.9, 117.5, 84.5,
70.5, 57.5, 55.7, 46.6, 46.4, 39.5, 38.9, 38.0, 35.9, 34.7, 29.8, 28.9, 28.2, 28.1, 23.9, 22.9, 22.7,
22.1, 20.5, 18.8, 12.0. HRMS (ESI) for C27H42O5Na [M+Na]+, calcd 469.2924, found: 469.2935.
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Oxidative cascade of 41 with MCPBA: MCPBA (34.0 mg, 0.198 mmol) was added to a
solution of 41 (25.4 mg, 63.8 µmol) in dry CH2Cl2 (1.0 mL) at -40 °C. The reaction mixture
was allowed to warm to room temperature gradually and stirred under argon atmosphere for 15
h. The reaction was quenched with saturated aqueous Na2SO3 (3 mL) and CH2Cl2 was added.
After separation of the organic layer, the aqueous layer was extracted with CH2Cl2 (3 mL x3).
The combined organic layer was filtered through a short column packed with anhydrous
Na2SO4 and concentrated to dryness in vacuo. The residue was purified by flash column
1
chromatography (neutral silica gel 5 g, CHCl3/THF/pentane 9:1:3) to give a 3:4:1:2 (by H
NMR) mixture of 17-nor-chaxine D (46), 17-nor-2’epi-chaxine D (45), 43 and 44. Separation
of the products was difficult because of the similar polarity.
17-nor-chaxine E (47): MsCl (25 µL, 32 µmol) and NEt3 (135 µL, 0.969 mmol) were added
to solution of 17-nor-chaxine D (46) (14.2 mg, 31.8 µmol) in dry CH2Cl2 (1 mL) at 0 °C. The
ice bath was removed and the reaction mixture was stirred for 45 min. Then the reaction was
quenched with saturated aqueous NaHCO3 (3 mL). The organic layer was separated and the
aqueous layer was extracted with CH2Cl2 (3 mL x3). The combined organic layer was washed
with H2O (x1) and brine (x1), and filtered through a short column packed with anhydrous
Na2SO4, and concentrated to dryness in vacuo. The residue was purified by preparative TLC
(AcOEt/Hexane 1: 4) to give 17-nor-chaxine E (47) (11.8 mg, 86%) as a white solid. 17-nor-
chaxine E (47): Mp 84-86 °C. [α]D28 +134 (c 0.59, CHCl3). IR (KBr) νmax 2954, 2869, 1729,
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1699, 1649, 1466, 1427, 1384, 1328, 1193, 1164, 1120, 1090, 1001, 866, 813 cm-1. 1H NMR
(400 MHz, CDCl3) δ 6.89 (1H, m, H-5’), 6.03 (1H, dd, J = 10, 2 Hz, H-6’), 5.64 (1H, d, J = 2
Hz, H-2), 2.93 (1H, td, J = 12, 6 Hz), 2.72 (1H, ddd, J = 16, 13, 7 Hz), 2.57-2.37 (4H, m), 2.23
(1H, ddd, J = 13, 7, 2 Hz), 2.10-1.98 (2H, m), 1.70-1.27 (9H, m) 1.47 (3H, s, CH3-7’), 1.23-
1.06 (3H, m), 1.01 (1H, m), 0.92 (3H, d, J = 6.5 Hz, CH3-14), 0.87 (3H, d, J = 6.5 Hz, CH3-19
or 20), 0.85 (3H, d, J = 6.5 Hz, CH3-19 or 20), 0.85 (3H, s, CH3-12). 13C{1H} NMR (100 MHz,
CDCl3) δ 204.9, 195.9, 164.4, 156.5, 149.0, 128.3, 117.5, 81.3, 57.9, 55.7, 46.8, 39.5, 39.2,
38.2, 35.9, 35.9, 32.2, 28.9, 28.1, 24.9, 23.9, 22.9, 22.7, 22.0, 21.8, 18.8, 11.9. HRMS (ESI) for
C27H40O4Na [M+Na]+, calcd 451.2819, found: 451.2808.
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17-nor-Chaxine A (48): To a solution of 17-nor-chaxine D (46) (9.0 mg, 20 µmol) in THF
(0.50 mL) was added 0.05 N LiOH (0.60 mL) at room temperature. After stirring at the same
temperature for 3 h, the reaction was quenched with 1 N HCl (3 mL) and H2O. After separation
of the organic layer, the aqueous layer was extracted with CH2Cl2 (3 mL x3). The combined
organic layer was passed through a short column packed with anhydrous Na2SO4, and
concentrated to dryness by spraying argon gas. The residue was purified by flash column
chromatography (neutral silica gel 3 g, Et2O/pentane 1:1) to give 17-nor-chaxine A (48) (5.7
mg, 94%) as a white solid and 33 (1.0 mg, 35%) as a volatile compound. 17-nor-chaxine A
(48): Mp 101-103 °C. [α]D28 +165 (c 0.29, CHCl3). IR (KBr) νmax 3413, 2955, 2930, 2869, 1742,
1662, 1467, 1381, 1177, 1128, 1048, 957, 934, 904, 853 cm-1. 1H NMR (400 MHz, CDCl3) δ
5.62 (1H, d, J = 2 Hz, H-2), 3.21 (1H, br, OH), 2.64 (1H, ddd, J = 12, 7, 2 Hz, H-8), 2.27 (1H,
ddd, J = 14, 4, 2.5 Hz), 2.07 (1H, m), 2.01 (1H, ddd, J = 13.5, 4.5, 2.5 Hz), 1.86 (1H, td, J = 14,
4.5 Hz), 1.75 (1H, m), 1.66- 1.57 (1H, m), 1.57-1.30 (6H, m), 1.20-1.01 (4H, m), 0.94 (3H, d,
J = 6 Hz, CH3-14), 0.87 (3H, d, J = 6.5 Hz, CH3-19 or 20), 0.86 (3H, d, J = 6.5 Hz, CH3-19 or
20), 0.59 (3H, s, CH3-12). 13C{1H} NMR (100 MHz, CDCl3) δ 171.2, 170.9, 112.4, 105.0, 55.7,
50.4, 49.2, 39.6, 36.1, 35.9, 35.4, 35.3, 28.8, 28.1, 23.9, 22.9, 22.7, 21.5, 18.8, 11.6. HRMS
(ESI) for C20H31O3 [M-H]-, calcd 319.2268, found: 319.2276.
Supporting information
The Supporting Information is available free of charge on the ACS Publications website at DOI:
10.1021/acs.joc.XXXXXXX.
An alternative biosynthetic route, X-ray crystallographic data for compound 27, setup apparatus
for electrocyclic reaction of ergosterol, and spectral data for all new compounds (PDF)
Crystal data for compound 27 (CIF)
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Acknowledgement: The authors acknowledge Dr. H. Onodera (Kyowa Hakko Kirin Co., Ltd.)
for providing NMR spectra of BB, Prof. M. Ojika (Nagoya University) for valuable discussion
on NMR analysis of the degradation products of 30. This work was supported by a Grant-in-
Aid for Scientific Research (B) (No.16H04915) and a Grants-in-Aid on Innovative Areas
‘‘Frontier Research on Chemical Communication’’ (No.17H06406) from MEXT, by the
Yamada Science Foundation.
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References
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(8) For an alternative biosynthetic proposal, see SI.
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(16) See the experimental for the details
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(20) CCDC-1966100 contains the supplementary crystallographic data for this paper. These
data can be obtained free of charge from The Cambridge Crystallographic Data Centre
via www.ccdc.cam.ac.uk/data_request/cif.
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Chem. Soc. 1973, 95, 6136-6137.
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(±)‐Indoxamycin B. Angew. Chem. Int. Ed. 2012, 51, 3474-3477.
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Silica‐Catalysed and Highly Stereoselective Convergent and Nonconvergent
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(24) The detail was described in ref. 6.
(25) For an example of in situ generation of DMDO: Cheshev, P.; Marra, A.; Condoni, A.
Direct epoxidation of D-glucal and D-galactal derivatives with in situ generated DMDO.
Carbohydr. Res. 2006, 341, 2714-2716.
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A. K. Solvent effects in the dimethyldioxirane oxidation of allylic alcohols: evidence for
hydrogen bonding in the dipolar transition state of oxygen transfer. Tetrahedron 1995,
51, 13039-13044. (b) Murray, R. W.; Singh, M.; Williams, B. L.; Moncrieff H. M.
Diastereoselectivity in the Epoxidation of Substituted Cyclohexenes by
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are not matched. See: Dai, Y.; Zhang, B.-B.; Liao, Z.-X. Chemical constituents of
Caryopteris tanguitica. Natural Product Research 2012, 26, 643-647.
(28) Alkaline hydrolysis of BB (29) was also carried out under the identical conditions,
however, enone 36 could not be isolated because of the volatility in the experiment.
(29) For a preliminary report of the biological activities: Wu, J.; Takahashi, M.; Ridwan, Y.
A.; Mori, T.; Choi, J.-H.; Hirai, H.; Niki, M.; Hirata, Y.; Nishikawa, T.; Harada, E.;
Kobori, H.; Toda, M.; Alessandro-Gabazza, C. D. Yasuma, T.; Gabazza, E.; Kawagishi,
H. Search for Immune Inhibitors from Mushrooms. Symposium papers of the 61th
Symposium on the Chemistry of Natural Products (Hiroshima, Japan), p 487-492, 2019.
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