Systemic study on the biogenic pathways of Yezo'otogirins: Total synthesis and antitumor activities of (±)-Yezo'otogirin C and its structural analogues

Article abstract of DOI:10.1021/jo502267g

A systematic study of the biomimetic pathways to yezootogirin C under aerobic and anaerobic conditions has been investigated, and both are found to be feasible pathways to the natural product depending on the physiological conditions. Because of the lower activation energy, the aerobic process would be more favorable when the in vivo oxygen level is high. In the course of this study, a highly efficient synthetic route to (±)-yezootogirin C has been established in four steps (31% overall yield) from a readily available compound without using any protecting groups. The natural product and its structural analogues exhibited antitumor activities against several human cancer cell lines and appeared to arrest cell cycles in different phases.

Full text of DOI:10.1021/jo502267g

Article
pubs.acs.org/joc
Systemic Study on the Biogenic Pathways of Yezo’otogirins: Total
Synthesis and Antitumor Activities of ( )-Yezo’otogirin C and Its
Structural Analogues
Wei Yang,† Jingming Cao,† Mengxun Zhang,† Rongfeng Lan,‡ Lizhi Zhu,† Guangyan Du,†
Shuzhong He,*,§ and Chi-Sing Lee*,†
†Laboratory of Chemical Genomics, School of Chemical Biology and Biotechnology, Peking University Shenzhen Graduate School,
Shenzhen University Town, Xili, Shenzhen 518055, China
‡Institute of Research and Continuing Education, Hong Kong Baptist University, Shenzhen 518057, China
§School of Pharmaceutical Sciences, Guizhou University, Guiyang, Guizhou 550025, China
S
* Supporting Information
ABSTRACT: A systematic study of the biomimetic pathways
to yezo’otogirin C under aerobic and anaerobic conditions has
been investigated, and both are found to be feasible pathways
to the natural product depending on the physiological
conditions. Because of the lower activation energy, the aerobic
process would be more favorable when the in vivo oxygen level
is high. In the course of this study, a highly efficient synthetic
route to ( )-yezo’otogirin C has been established in four steps
(31% overall yield) from a readily available compound without
using any protecting groups. The natural product and its
structural analogues exhibited antitumor activities against
several human cancer cell lines and appeared to arrest cell
cycles in different phases.
yezoense.5 Preliminary in vitro assays showed that these natural
INTRODUCTION
■
products are noncytotoxic against L1210 murine leukemia.
Plants of the Hypericum genus are popular folk medicine to
However, no further study on their biological activities has been
relieve pain, swelling, inflammation, burns, and symptoms of
reported probably due to the limited supply of these natural
various kinds of neurological disorders.1 In particular, St. John’s
products.
wort (H. perforatum L.)2 is a well-known medicinal herb for its
The biosynthesis of the yezo’otogirins has not been fully
anti-inflammatory and antidepression properities.3 In order to
search for plants with high biological values in the Hypericum
genus, the extracts of many related species have been surveyed
for different bioactivities including antiviral, antibacterial,
elucidated. A known coisolated hyperforin derivative 46 (Figure
1) was hypothesized as a potential biosynthetic precursor of
yezo’otogirin A (1).5 On the basis of this biogenic proposal,
two feasible biomimetic pathways to the yezo’otogirins could be
antifungal, antitumor, anti-inflammatory, and antioxidant
considered. As shown in Scheme 1, single-electron oxidative 5-
activities.4 The tricyclic terpenoid yezo’otogirins A−C (Figure
exo-trig radical cyclization of precursors I could produce the cis-
hydrindanes of II,7 which could undergo cationic cyclization via
1), which contain a rare bowl-shape skeleton with four to five
stereogenic centers, were isolated from the shoots of H.
another single-electron oxidation to form the tricyclic
intermediate (IV) under anaerobic conditions (pathway a).
Subsequent deprotonation and enolization would afford the
yezo’otogirins. An alternative pathway would be the trapping of
II by a molecular oxygen under aerobic conditions7,8 (pathway
b) forming the peroxy-bridged compounds (V). Reduction of V
followed by elimination could also provide the yezo’otogir-
ins.8a,9
In the course of a systematic study on these two potential
biogenic pathways to the yezo’otogirins, we have successfully
Figure 1. Yezo’otogirins A−C (1−3) and the coisolate hyperforin
derivative (4).
Received: October 3, 2014
© XXXX American Chemical Society
A
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The Journal of Organic Chemistry
Article
under aerobic and anaerobic conditions by using β-keto ester 5
as the cyclization precursor of the model study and diketone 6
as the biomimetic cyclization precursor of yezo’otogirin C (3)
(Scheme 2b).
Scheme 1. Potential Biomimetic Pathways for the
Yezo’otogirins
RESULTS AND DISCUSSION
■
1. Preparation of β-Keto Ester 5 and Diketone 6. The
synthesis of 5 and 6 began with a common starting material
(12), which was obtained from 1,3-cyclohexanedione in three
steps (90% total yield in decagram scales) with known
procedures.12 As shown in Scheme 3, α′-methylation of 12
by LDA/CH3I gave compound 13 in good yield with excellent
diastereoselectivity, which is presumably due to the flat
structure of the enolate intermediate and the encumbrance of
the prenyl group. β-Keto ester 5 was readily obtained as a
mixture of diastereomers by installing the 4-methylpent-3-en-1-
yl side chain via conjugation addition and trapping the enolate
intermediate with methyl cyanoformate.13 Diketone 6 was
prepared in a similar way via trapping of the enolate
intermediate with isobutyraldehyde and oxidation of inter-
mediate 14 (a single diastereomer).14 Compounds 5 and 6
were prepared efficiently from a known enone (12) in only two
to three steps with overall yields of 60−72%, respectively. The
high diastereoselectivity of the conjugate addition reactions
could be attributable to the encumbrance of the prenyl group.
2. Model Study with β-Keto Ester 5 as the Substrate.
a. Anaerobic Oxidative Free-Radical Cyclization of 5. As β-
keto esters are more active substrates for oxidative free-radical
cyclizations,15 β-keto ester 5 was employed as the model
cyclization precursor for the biomimetic study. Under anaerobic
conditions (Scheme 1, pathway a), oxidative free-radical
cyclization of 5 using Mn(OAc)3·2H2O in acetic acid did not
produce the expected cyclization product (7) in any significant
yield, but a variety of unidentified elimination side products
(Table 1, entry 1). A change of solvent to ethanol increased the
yield to 13% (entry 2). Encouraged by this result, the effects of
other single-electron oxidants8 including Cu(OAc)2·H2O,
employed β-keto ester 5 as the cyclization precursor of a model
study and found that pathway b (oxidative free-radical
cyclization under aerobic conditions) is the more favorable
synthetic route to the tricyclic core of 7, which can be
converted to ( )-yezo’otogirin C (3) in four steps.10 During
the preparation of this manuscript, the group of George
reported the attempts of oxidative free-radical cyclization of 4
and epi-6-4.11 Interestingly, they found that the stereogenic
center at C6 has great influence on the cyclization and only 4
can provide yezo’otogirin A (1) in only modest yield under
anaerobic conditions at very high reaction temperature
(Scheme 2a). We herein report the details of the systematic
study on the potential biogenic pathways of yezo’otogirin C (3)
Scheme 2. Study of the George Group and the Overall Objective of This Study
B
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Scheme 3. Preparation of β-Keto Ester 5 and Diketone 6
a
a
Table 1. Anaerobic Oxidative Free-Radical Cyclization of 5
Table 2. Aerobic Oxidative Free-Radical Cyclization of 5
b
yield (%)
b
temp
yield
(%)
temp
entry
1
oxidant
co-oxidant
solvent
AcOH
(°C)
entry
oxidant/co-oxidant
Mn(OAc)3·2H2O/−
Cu(OAc)2·H2O/−
CAN/−
FeCl3/−
Mn(OAc)2·4H2O/−
equiv
(°C)
8
5
Mn(OAc)3·
2H2O
rt
<3
13
22
20
1
2
3
4
5
6
2.0/−
2.0/−
2.0/−
2.0/−
2.0/−
2.0/0.2
rt
rt
rt
rt
rt
rt
20
<3
<3
65
82
78
2
3
4
Mn(OAc)3·
2H2O
EtOH
EtOH
EtOH
rt
Mn(OAc)3·
2H2O
Cu(OAc)2·
H2O
rt
44
55
Mn(OAc)2·4H2O/Mn(OAc)3·
2H2O
Mn(OAc)3·
2H2O
Cu(OAc)2·
H2O
reflux
a
7
8
9
Mn(OAc)2·4H2O/KMnO4
Mn(OAc)2·4H2O/Pb(OAc)4
Mn(OAc)2·4H2O/Cu(OAc)2·
H2O
2.0/0.2
2.0/0.2
2.0/0.2
rt
rt
rt
36
31
35
The general procedures for the oxidative free-radical cyclization
b
under anaerobic conditions were followed. Isolated yields (%) after
silica gel flash column chromatography.
10
11
12
13
14
15
16
17
Mn(OAc)2·4H2O/CrO3
Mn(OAc)2·4H2O/CAN
Mn(OAc)2·4H2O/FeCl3
Mn(OAc)2·4H2O/−
Mn(OAc)2·4H2O/−
Mn(OAc)2·4H2O/−
Mn(OAc)2·4H2O/−
2.0/0.2
2.0/0.2
2.0/0.2
1.0/−
0.5/−
0.1/−
rt
rt
rt
rt
rt
rt
50
rt
27
42
20
38
35
35
35
44
CAN, and FeCl3 were studied. Unfortunately, these oxidants
did not give any of the expected cyclization product (data not
shown). The combination of Mn(OAc)3·2H2O/Cu(OAc)2·
H2O in ethanol successfully increased the yield of 7 to 22%
(entry 3). However, increasing the reaction temperature
resulted in a slightly lower yield (entry 4). Addition of a
variety of bases led to slow decomposition of the substrate (5)
at room temperature probably due to the hydrolysis of the ester
moieties (data not shown). The necessity for the formation of
the highly strained intermediate (IV in Scheme 1) may be the
cause of the inefficacy of this cyclization process.7g
b. Aerobic Oxidative Free-Radical Cyclization of 5. The
aerobic oxidative free-radical cyclization 5 (Scheme 1, pathway
b)7,8 at room temperature were studied with a variety of
oxidants. Using Mn(OAc)3·2H2O in ethanol afforded 20% yield
of the peroxy-bridged compound 8 (Table 2, entry 1) together
with a number of unidentifiable side products. Compound 8
was found to be a single diastereomer with its structure
characterized by X-ray crystallography.16 Similar to the case in
the anaerobic process, other single-electron oxidant, such as
17
14
c
0.1/−
0.1/0.1
Mn(OAc)2·4H2O/Mn(OAc)3·
2H2O
18
19
Mn(OAc)2·4H2O/KMnO4
Mn(OAc)2·4H2O/Cu(OAc)2·
H2O
0.1/0.1
0.1/0.1
rt
rt
11
38
54
27
20
Mn(OAc)2·4H2O/CAN
0.1/0.1
rt
27
13
a
The general procedures for the oxidative free-radical cyclization
under aerobic conditions were followed. Isolated yields (%) after
silica gel flash column chromatography. Reaction time = 1 d.
b
c
Cu(OAc)2·H2O, CAN, or FeCl3 only produced a trace amount
of desirable products with 65−82% of recovered starting
materials (entries 2−4). Interestingly, Mn(OAc)2·4H2O (which
generated Mn(III) in the presence of oxygen) afforded 44%
C
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Scheme 4. Analysis of the Molecular Conformation of the Reaction Intermediates
yield of 8 (entry 5). With this encouraging result in hand, the
effects of a variety of co-oxidants with Mn(OAc)2·4H2O were
investigated (entry 6−12).8b,c The optimal results were
obtained by using Kurosawa and Nishino’s combination of
oxidants (Mn(OAc)2·4H2O/Mn(OAc)3·2H2O),8b which af-
forded the 55% yield of 8 (entry 6), and the formation of
compound 7 was not observed.
After a survey of the effects on the oxidant loading, we found
that using a catalytic amount (0.1 equiv) of Mn(OAc)2·4H2O
only caused a minor drop in the yield of 8 (35−38%, entries
13−15) and the cyclization rate. The use of 0.1 equiv of
Mn(OAc)2·4H2O resulted in incomplete reaction (entry 15).
Although the reaction can be forced to completion at alleviated
temperature (50 °C) for 2 days, the isolated yield cannot be
improved because of the thermal instability of 8 (entry 16).
Addition of 0.1 equiv of co-oxidant (Mn(OAc)3·2H2O or
Cu(OAc)2·H2O) afforded 8 in 44 and 38% yield (entries 17
and 19, respectively).
The results of the above model study could be rationalized
by detail analysis of the molecular conformation of the reaction
intermediates in both biogenic pathways. The radical
intermediate generated from single-electron oxidation of 5
could adopt four possible half-chair conformations prior to the
5-exo-trig cyclization (Scheme 4). According to the Beckwith’s
transition state model,17 endo-VI/VI′ are considered more
favorable than exo-VI/VI′. It could also be rationalized by the
encumbrance generated by the manganese (not shown for
clarity) complexed to both the ketone and the ester carbonyls
with the gem-substituted alkene in exo-VI/VI′. Cyclization of
the less congested endo-VI′ requires a higher energy twist-boat
transition state, while the endo-VI could cyclize via a generally
more favorable chairlike transition state, but a strong 1,3-diaxial
interaction between the axial methyl and the incoming alkene
may disfavor this reaction path. Thus, both endo-VI and VI′
could undergo cyclization and lead to VII (chair). Cyclization
of exo-VI/VI′ gives radical VII′ containing β-i-Pr and leads to
various side products as cyclization with the ketone cannot take
place. Radical VII (chair) possesses the proper conformation
for cyclization with a molecular oxygen to form the peroxy-
bridged compound 8. On the other hand, VII (chair) needs to
undergo conformational changes to the less stable VII (boat) or
VII (chair) in order to cyclize with the ketone. The resultant
tricyclic cation VIII is highly strained and can undergo either
deprotonation/enolization to afford 7 or ring opening to give a
variety of side products.
Although the experimental results indicated that the
anaerobic process for the formation of the tricyclic compound
7 from 5 is a possible pathway, this conformational analysis
suggested that the activation energy of the anaerobic process
(pathway a) is much higher than that of the aerobic process
(pathway b) due to the conformational changes and the
formation of highly strained intermediate during the reaction.
Thus, the aerobic process (pathway b) for the formation of the
peroxy-bridged compound (8) is the more favorable pathway
under aerobic conditions. This is also supported by the fact that
the formation of 7 was not observed under the aerobic
conditions.
3. Conversion of 8 to ( )-Yezo’otogirin C (3). With 8 in
hand, a number of conditions for reduction of the peroxy-
bridge moiety were studied.8a,9 As shown in Table 3, PPh3 in
refluxing CH2Cl2 or zinc dust in hot ethanol did not give any
expected product (entries 1 and 2). Switching the solvent to
acetic acid with zinc dust resulted in 28% yield of 7 (entry 3).
Using CuCl/CH3CN and hydrogenation with Pd/C provided
only a modest yield of 7 (entries 4 and 5). Thiourea in acetic
acid at 60 °C gave 7 in 68% yield (entry 6). Switching the
solvent to methanol improved the yield to 80%. The conditions
were finally optimized by using thiourea in refluxing
methanol,8a which afforded 7 in 92% yield (entry 8). The
NMR data of 7 from the reduction of 8 are identical to those
for the compound obtained from 5 under anaerobic conditions.
Compound 7 was expected to be transformed to
( )-yezo’otogirin C (3) in one pot upon treatment of i-
PrMgBr or i-PrLi.18 However, when excess i-PrMgBr was used
D
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the optimal conditions of the aerobic oxidative free-radical
cyclization of 5 were employed for study of the cyclization of
6.7,8 However, single-electron oxidation of diketone 6 turned
out to be much more difficult than that of β-keto ester 5. After
a survey of different oxidative conditions, we found that
cyclization of 6 at room temperature led to no reaction or only
a trace amount of the peroxy-bridged compound 9 (Table 4,
Table 3. Reduction of Peroxy-Bridged Compound 8
a
entry
reductant
solvent
temp (°C)
yield (%)
1
2
3
4
5
6
7
8
PPh3
CH2Cl2
EtOH
reflux
60
a
Table 4. Aerobic Oxidative Free-Radical Cyclization of 6
Zn
Zn
AcOH
CH3CN
MeOH
AcOH
MeOH
MeOH
60
28
40
47
68
80
92
CuCl
rt
Pd/C, H2
thiourea
thiourea
thiourea
70
70
70
reflux
a
Isolated yield (%) after silica gel flash column chromatography.
c
temp
yield
(%)
entry
1
oxidant
co-oxidant
base
(°C)
Mn(OAc)3·
2H2O
rt
<3
at room temperature, no reaction was observed. Elevation of
the reaction temperature resulted in the decomposition of
compound 7. Also, no product was formed by using 1 equiv of
i-PrLi at −78 °C to ambient temperature. Instead, decarbox-
ylation of 7 was observed when a large amount of i-PrLi was
used and resulted in 50−80% yield of 15 at 0 °C or room
temperature (Scheme 5). Switching to basic conditions for
hydrolysis of the methyl ester or Lewis acidic conditions for
direct conversion of the methyl ester to the corresponding
Weinreb amide also resulted in no reaction. Eventually,
( )-yezo’otogirin C (3) was obtained via DIBAL reduction
of 7 followed by TPAP/NMO oxidation, i-PrLi addition to the
resultant aldehyde (17), and a subsequent Dess−Martin
oxidation. NMR spectral data of the final product are identical
to those reported in the literature.5
The formation of the decarboxylation side-product 15 would
be resulted from either a double i-PrLi addition/retro-aldol
sequence or a Krapcho-type decarboxylation.19 To study the
mechanism of this unusual decarboxylation process, ( )-ye-
zo’otogirin C (3) was submitted to a large excess of i-PrLi from
0 °C to room temperature. Interestingly, ( )-yezo’otogirin C
(3) was found to be stable with i-PrLi. This result indicated that
double addition of i-PrLi to the ester moiety of 7 is not feasible,
and the decarboxylation side product 15 would be most likely
formed via the Krapcho-type decarboxylation mechanism.
4. Systematic Study of the Biomimetic Pathways to
( )-Yezo’otogirin C (3). a. Aerobic Oxidative Free-Radical
Cyclization of 6. Encouraged by the results of the model study,
2
Mn(OAc)2·
4H2O
rt
<3
3
4
5
6
Cu(OAc)2·H2O
CAN
rt
rt
rt
rt
d
d
FeCl3
d
b
Mn(OAc)2·
4H2O
base
<3
b
b
7
Mn(OAc)2·
4H2O
base
base
55
3−7
e
8
Mn(OAc)2·
4H2O
reflux
rt
9
Mn(OAc)2·
4H2O
Mn(OAc)3·
2H2O
iPr2NH
iPr2NH
iPr2NH
iPr2NH
<3
15
9
10
11
12
Mn(OAc)2·
4H2O
Mn(OAc)3·
2H2O
40
Mn(OAc)2·
4H2O
Mn(OAc)3·
2H2O
55
Mn(OAc)2·
4H2O
Mn(OAc)3·
2H2O
reflux
e
a
The general procedures for oxidative free-radical cyclization under
aerobic conditions were followed. Base = pyridine, DBU, Et3N,
nBuNH2, piperidine, Et2NH or iPr2NH. Isolated yield (%) after silica
gel flash column chromatography. No reaction. Compound 6 was
b
c
d
e
consumed.
entries 1−6). The reaction proceeded much faster under high
reaction temperature (55 °C) with an amine additive (entry 7).
Although further increasing the reaction temperature enhanced
the cyclization rate, the peroxy-bridged product 9 was found to
Scheme 5. Conversion of 7 to ( )-Yezo’otogirin C (3)
E
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be unstable under refluxing temperature and resulted in
decomposition. The reaction temperature was then optimized
by balancing the rates for the formation of 9 and its
decomposition (entry 9−12). Although cyclization of 6 can
be proceeded at a temperature as low as 40 °C, the reaction
afforded only 15% yield (a single diastereomer) due to the
decomposition of 9 (entry 10). There was no formation of the
natural product under these conditions. The structure of 9 was
characterized by X-ray crystallography.16 Despite the disap-
pointing results, ( )-yezo’otogirin C (3) was eventually
obtained via reacting 9 with thiourea in refluxing methanol
(Scheme 6).
Table 5. Anaerobic Oxidative Free-Radical Cyclization of
Diketone 6.
a
c
entry
additive
base
temp
rt
yield (%)
1
<3
<3
6
b
2
base
rt
3
pyridine
DBU
reflux
reflux
reflux
reflux
reflux
reflux
reflux
40 °C
reflux
reflux
reflux
reflux
reflux
Scheme 6. Conversion of 9 to ( )-Yezo’otogirin C (3)
4
22
30
34
34
47
52
<3
36
8
5
Et3N
6
n-BuNH2
piperidine
Et2NH
7
8
9
i-Pr2NH
i-Pr2NH
i-Pr2NH
i-Pr2NH
i-Pr2NH
i-Pr2NH
10
11
12
13
14
15
Cu(OAc)2·H2O
AcOH
Cl2HCCO2H
TsOH
20
10
10
L-proline
a
The general procedures for the oxidative free-radical cyclization
b
under anaerobic conditions were followed. Base = pyridine, DBU,
Et3N, nBuNH2, piperidine, Et2NH or iPr2NH. Isolated yield (%) after
c
b. Anaerobic Oxidative Free-Radical Cyclization of 6. On
the basis of the above results, both amine additives and high
reaction temperature are beneficial to the radical initiation of
diketone 6. The amine base would also promote the late-stage
deprotonation and enolization of the tricyclic cation
intermediate (IV, Scheme 1). Thus, the effects of a variety of
amines under different reaction temperature were studied
under anaerobic conditions. As shown in Table 5, Mn(OAc)3·
2H2O in ethanol provided only a trace amount of
( )-yezo’otogirin C (3) at room temperature (entry 1).
Addition of a base resulted in similar results (entry 2).
Increasing the reaction temperature generally enhanced the rate
and the yields of the reaction, which is consistent with the
results under aerobic conditions. With pyridine as the base in
refluxing ethanol, the reaction gave 6% yield of ( )-yezo’oto-
girin C (3) as a single diastereomer (entry 3). Switching to a
stronger base, such as DBU or triethylamine, greatly increased
the yields to 22 and 30% respectively (entries 4 and 5). Both
primary and secondary amines are able to enhance the
efficiency of the cyclization process and afforded 34−52%
yield of the natural product (entry 6−9). Lowering the reaction
temperature to 40 °C (the optimal temperature for the aerobic
pathway) led to very poor efficiency (entry 10). Addition of a
co-oxidant (entry 11) or Brønsted acid (entry 12−15) did not
improve the yields of the reaction. Finally, the conditions were
optimized by using Mn(OAc)3·2H2O with diisopropylamine in
refluxing ethanol (entry 9), which afforded 52% yield of
( )-yezo’otogirin C (3) as a single diastereomer. The NMR
data of ( )-yezo’otogirin C (3) from diketone 6 are identical to
those in the model study and the literature.5
silica gel flash column chromatography.
However, the initiation of the anaerobic process required
refluxing temperature of ethanol (78 °C), which is an unusual
physiological condition. On the other hand, the temperature
required for the aerobic process is much lower (40 °C), and the
anaerobic process cannot be initiated at this temperature. These
results suggested that the aerobic process would be the more
favorable pathway when the in vivo oxygen level is high. This is
also supported by the fact that the direct formation of the
natural product was not observed under the aerobic conditions.
c. Study on the Effects of Bases and Temperature. During
the study on the cyclization of diketone 6 under the anaerobic
conditions, epimerization of the stereogenic center at C6 of
diketone 6 was observed. Therefore, the effects of the base and
reaction temperature on the stereogenic center at C6 were
studied. As showed in Figure 2, diisopropylamine at room
temperature did not lead to epimerization at C6 (entry 1). On
the other hand, the stereogenic center at C6 epimerized slowly
upon heating in ethanol (entry 2). Using i-Pr2NEt in refluxing
ethanol also led to the formation of epi-6-6 (entry 3−5). The
ratio of 6/epi-6-6 obtained after 3 days of heating with i-Pr2NEt
(entry 5) is similar to that without i-Pr2NEt (entry 2). In the
presence of Mn(OAc)3·2H2O, the disappearance rate of epi-6-6
is much higher than that of 6 (entry 6). The results of this
study indicated that the C6 stereogenic center of 6 would be
epimerized under the high reaction temperature conditions,
and epi-6-6 is a more active substrate for the anaerobic process.
This observation is also consistent with that reported by the
group of George.11 The epi-6-6 is more likely to be the
bioprecursor of ( )-yezo’otogirin C (3) for its higher reactivity
and the same configuration at C6 of the hyperforin derivative 4.
5. Antitumor Activities of ( )-Yezo’otogirin C (3) and
Its Structural analogues (7−9 and 15). The antitumor
activity of ( )-yezo’otogirin C (3) and its structural analogues
Contrary to the results of the model study, diketone 6
preferentially underwent oxidative free-radical cyclization under
anaerobic conditions and directly led to the natural product (3)
in one pot with good yields. This result suggested that the
anaerobic process is a possible biogenic pathway of
( )-yezo’otogirin C (3) when the in vivo oxygen level is low.
F
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CONCLUSION
■
In summary, the aerobic and anaerobic biomimetic pathways of
( )-yezo’otogirin C (3) have been studied, and both are found
to be possible pathways to the natural product depending on
the physiological conditions. The aerobic process would be the
more favorable biogenic pathway when the in vivo oxygen level
is high due to its lower activation energy. In the course of this
study, a highly efficient synthetic route to ( )-yezo’otogirin C
has been established in four steps (31% overall yield) from a
readily available compound (12) without using any protecting
groups. An asymmetric synthesis could be readily obtained by
employing enantiomerically enriched 12.14,20 The natural
product (3) and its analogues (7−9 and 15) showed anticancer
activity against several human cancer cell lines with IC50 values
up to 9.54 μM. These compounds appeared to arrest cell cycles
in different phases. We are currently preparing a library of
structural analogues using this biomimetic strategy for a detail
structure−activity relationship study, and exploring the
potential applications of this highly convergent oxidative free-
radical cyclization strategy on other classes of natural products,
such as picrotoxanes.21
EXPERIMENTAL SECTION
■
General Information for Synthesis. Unless otherwise stated, all
air- and water-sensitive reactions were performed under inert
atmosphere (N2 or Ar) and anhydrous conditions with dry solvents.
Reactions were monitored by thin-layer chromatography (TLC)
performed on 0.25 mm thick silica gel plates (60 F254) under 254 nm
ultraviolet irradiation or via p-anisaldehyde staining (150 mL ethanol
of 5.00 mL of concentrated H2SO4, 1.50 mL of glacial HOAc, and 3.70
mL of anisaldehyde). Flash column chromatography was carried out
on silica gel (200−300 mesh). All commercial chemicals were used
without further purification. Anhydrous THF was distilled over
powdered sodium and benzophenone. Anhydrous toluene was distilled
from powdered sodium. Anhydrous CH3CN and CH2Cl2 were
Figure 2. NMR study on the effects of base and temperature.
(7−9 and 15) toward a number of human cancer cell lines was
studied by MTT assays. All four compounds exhibited cell
inhibition activities against human cervical, liver, and gastric
cancer cells (Table 6). For gastric cancer MGc80-3,
Table 6. IC50 of the Cell Growth Inhibitory Effects of
( )-Yezo’otogirin C (3) and Its Structural Analogues (7−9
a
and 15) by MTT Assays
1
IC50 (μM)
distilled over calcium hydride. H NMR and 13C NMR spectra were
recorded using a 300, 400, or 500 MHz spectrometer. The NOESY
experiments were carried out using a 400 or 500 MHz spectrometer.
The multiplicities of 1H NMR were designated as s (singlet), d
(doublet), t (triplet), q (quartet), m (multiplet), br (broad). High-
resolution mass spectra were obtained on an electrospray ionization
time-of-flight (ESI-TOF) mass spectrometer. Melting points were
uncorrected and obtained with a micromelting point meter. Crystallo-
graphic data were achieved using a single-crystal X-ray diffractometer.
IR spectra (shown as wavenumbers, cm−1) were measured using an
FTIR spectrometer.
cell lines
HeLa
3
7
8
9
15
32.82
20.41
9.54
G2
88.33
57.78
27.15
G2
32.15
34.22
9.98
G1
310.64
101.87
62.88
28.88
23.85
12.05
G2
SMMC-7721
MGc80−3
cell arrested at
G1, S
a
The general procedures for the biological assays were followed.
General Information for Biological Assays. Human cervical
cancer cell HeLa were maintained in DMEM medium; Human gastric
cancer cell MGc80-3 and human liver cancer cell SMMC-7721 were
cultured in RPMI-1640 medium; both medium were supplemented
with 10% fetal bovine serum and antibiotics. For cell viability assay
(MTT), cells were seeded in 96-well plates and were exponentially
growth. Then cells were dosed with chemicals and treated for 36 h.
Live cell population were analyzed using the MTT assay, by addition
of equal amounts of 3-(4,5-dimethylthiazol-2-yl)-2 and 5-diphenylte-
trazolium bromide (0.5 mg/mL) for 4 h to produce formazan in living
cells. The reaction products were dissolved by addition of dimethyl
sulfoxide, and the absorbance of the solutions was measured in
microplate reader. Reactions were performed in triplicate for each
concentration of the chemicals. For flow cytometry assay (cell cycle),
cells were harvested by trypsinization, centrifuged, rinsed in 1× PBS
twice, and then fixed in cold 70% ethanol for 120 min. Before analysis,
propidium iodide (20 μg/mL) and RNaseA (0.1 mg/mL) were added
to the cells for nucleic acid staining and RNA digestion. Samples were
analyzed in a flow cytometry analyzer.
( )-yezo’otogirin C (3) and the peroxy-bridged ester analogue
(8) exhibited good antitumor activity (IC50 = 9.54 and 9.98
μM, respectively). The deisobutrylated analogue (15) also
showed good antitumor activity (IC50 = 12.05 μM). The ester
analogue (7) showed a moderate activity, and its peroxy-
bridged analogue (9) showed the lowest IC50 value. In the flow
cytometry cell cycle analyses, ( )-yezo’otogirin C (3), its ester
analogue (7), and the deisobutrylated analogue (15) mainly
caused G2 phase arrest of cell cycles. In particular, the
deisobutrylated analogue (15) induced a 6.6-fold increment of
G2 cell cycle arrestment (43.79% to 6.62%) in a concentration-
dependent manner (Table S1, Supporting Information) and
also a massive cell death in 60 μM. On the other hand, peroxy-
bridged compounds 8 and 9 inhibit cell grow mainly in G1
phase in a concentration-dependent manner (from 10 to 50
μM), and 9 showed inhibition in S phase in high concentration
(60 μM).
3-Methyl-4-(3-methylbut-2-en-1-yl)cyclohex-2-enone (12).12 To a
stirred solution of cyclohexane-1,3-dione (50.00 g, 0.45 mol) in freshly
G
DOI: 10.1021/jo502267g
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distilled toluene (1 L) under argon were added 2-methylpropan-1-ol
(99.11 g, 1.34 mol) and TsOH (0.38 g, 2.23 mmol). The solution was
heated under reflux, and water was removed with a Dean−Stark trap
for 4 h. The solution was then concentrated under reduced pressure.
Silica gel column chromatography (EtOAc/hexanes 1:5) of the crude
mixture afforded a yellow oil (71.25 g, 0.42 mol) as the product (10).
The THF (200 mL) solution of compound 10 (71.25 g, 0.42 mol) was
added dropwise to LDA (233.26 mL, 0.47 mol) in THF (1 L) at −78
°C. After 30 min, 1-bromo-3-methylbut-2-ene (69.53 g, 0.47 mol) was
added slowly at −78 °C. The solution was stirred at −78 °C for 0.5 h
and 0 °C until the full consumption of starting 10. A saturated aqueous
NH4Cl solution was added to quench the reaction. Ethyl acetate (500
mL × 3) was used to extract the aqueous layer. The combined organic
solution was treated by MgSO4, filtered, and concentrated. Purification
of the crude mixture by flash chromatography (EtOAc/hexanes 1:20)
gave a yellow oil (100 g, 0.42 mol) as the product (11). MeLi (0.39 L,
0.51 mmol) was added dropwise to a solution of 11 (100 g, 0.42 mol)
in THF (1 L) at 0 °C. The resulting solution was stirred at ambient
temperature for 1 h and then treated with a 4 N aqueous HCl (160
mL) slowly at 0 °C. After the reaction was stirred at ambient
temperature for 30 min, ethyl acetate (500 mL × 3) was used to
extract the aqueous layer. The combined organic solution was washed
using aqueous NaHCO3 solution and aqueous NaCl solution and
dried by MgSO4. After filtration and concentration under reduced
pressure, silica gel flash column chromatography (EtOAc/hexanes
1:20) of the crude mixture gave a yellow oil (72.2 g, 0.41 mol, 90% in
dropwise to the flask at −20 °C. The resulting solution was stirred at
−20 °C for 30 min and 0 °C overnight. Saturated NaCl aqueous
solution was added to quench the reaction, and ethyl acetate (100 mL
× 3) was used to extract the aqueous layer. The combined organic
solvent was treated with MgSO4. After filtration and concentration
under reduced pressure, flash chromatography (EtOAc/hexanes =
1:50) of the crude mixture provided a colorless oil (5.57 g, 16.67
mmol, 80%) as the desired product. 5 (a mixture a high enolizable β-
1
keto esters): H NMR (500 MHz, CDCl3) δ 5.17−5.03 (m, 2H),
3.70−3.56 (m, 3H), 2.67−2.42 (m, 1H), 2.56−2.22 (m, 1H), 2.16−
2.10 (m, 1H), 2.02−1.83 (m, 3H), 1.81−1.73 (m, 5H), 1.68−1.65 (m,
5H), 1.63−1.60 (m, 4H), 1.57−1.49 (m, 2H), 1.32−1.12 (m, 3H),
1.09−1.02 (m, 4H); 13C NMR (125 MHz, CDCl3) δ 210.6, 207.3,
169.3, 169.0, 133.1, 132.9, 131.8, 131.4, 124.2, 123.6, 123.4, 122.8,
63.0, 60.4, 51.5, 44.5, 44.5, 42.9, 39.9, 39.7, 38.3, 37.2, 34.5, 33.6, 33.2,
27.0, 25.8, 25.8, 25.7, 25.6, 22.7, 21.6, 21.3, 18.2, 17.9, 17.9, 17.6, 17.4,
14.4; IR (neat, cm-1): 3457, 2966, 2927, 2881, 1755, 1713, 1633,
1597, 1436, 1378, 1339, 1213, 1133, 1007, 846; HRMS (ESI/[M +
H]+) calcd for C21H35O3 335.2586, found 335.2581.
1-Hydroxy-2-methylpropyl)-3,6-dimethyl-4-(3-methylbut-2-en-1-
yl)-3-(4-methylpent-3-en-1-yl)cyclohexanone (14) (Single Diaster-
eomer). To a stirred mixture of mashed magnesium (0.25 g, 10.42
mmol) and iodine (0.01 g) in THF (12 mL) under argon was added a
portion (0.3 mL) of a solution of 5-bromo-2-methyl-2-pentene (1.36
g, 8.3 mmol) in dried THF (2 mL). The suspension was heated gently
to initiate the reaction. Then the rest of the solution was added slowly
to the suspension. The resulting suspension was refluxed for 1.5 h and
cooled to ambient temperature. This solution was transferred to a
THF (5 mL) solution of CuBr·Me2S (54 mg, 0.26 mmol) at −20 °C
slowly using a gastight syringe. After the solution was stirred for 30
min, 13 (1 g, 5.2 mmol) dissolved in THF (2 mL) was added slowly to
the flask at −20 °C. After the mixture was stirred at −20 °C for 20
min, a solution of isobutyraldehyde (0.61 mL, 6.8 mmol) in THF (2
mL) was added to the suspension slowly at −78 °C. The suspension
was stirred at −78 °C until full consumption of compound 13. A
saturated NH4Cl aqueous solution was added to quench the reaction,
and ethyl acetate (20 mL × 3) was used to extract the aqueous layer.
The combined organic solvent was treated by MgSO4. After filtration
and concentration under reduced pressure, flash chromatography
(EtOAc/hexanes = 1:40) of the crude mixture provided a colorless oil
(1.4 g, 4.1 mmol, 78%). 14: 1H NMR (500 MHz, CDCl3) δ 5.08−5.07
(m, 2H), 3.72 (d, J = 11.0 Hz, 1H), 3.36 (dd, J = 11.0, 9.0 Hz, 1H),
2.88 (s, 1H), 2.50−2.45 (m, 1H), 2.18−2.14 (m, 1H), 2.07−1.94 (m,
2H), 1.84−1.77 (m, 2H), 1.76−1.74 (m, 1H), 1.73−1.68 (m, 8H),
1.66−1.63 (m, 1H), 1.62 (s, 3H), 1.61 (s, 3H), 1.57−1.55 (m, 1H),
1.23 (d, J = 7.5 Hz, 3H), 1.01 (d, J = 7.0 Hz, 3H), 0.98 (s, 3H), 0.85
(d, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 221.7, 132.6,
131.5, 123.8, 123.2, 75.7, 51.0, 46.5, 45.6, 37.8, 36.8, 35.0, 34.2, 27.6,
25.8, 25.7, 21.2, 20.2, 19.8, 18.3, 17.9, 17.7; IR (neat, cm-1): 3520,
2981, 2930, 2868, 1689, 1456, 1385, 1201, 1121, 994, 832; HRMS
(ESI/[M + H]+) calcd for C23H41O2 349.3107, found 349.3089.
2-Isobutyryl-3,6-dimethyl-4-(3-methylbut-2-en-1-yl)-3-(4-methyl-
pent-3-en-1-yl)cyclohexanone (6) (Single Diastereomer). Com-
pound 14 (1.4 g, 4.0 mmol) and NaHCO3 (1.35 g, 16.1 mmol)
were dissolved in CH2Cl2 (30 mL), followed by the addition of Dess−
Martin periodinate (2.1 g, 4.8 mmol) at 0 °C. The resulting
suspension was stirred at ambient temperature until TLC analysis
showed the full consumption of 14. Diethyl ether (30 mL) and
saturated Na2S2O3 aqueous solution were added sequentially to the
reaction at 0 °C to quench the reaction. After the solution turned clear,
ethyl acetate (30 mL × 3) was used to extract the aqueous layer. The
combined organic solvent was treated by MgSO4. After filtration and
concentration under reduced pressure, flash chromatography (EtOAc/
hexanes = 1:70) of the crude mixture provided a colorless oil (1.18 g,
1
three steps from cyclohexane-1,3-dione) as the product. 12: H NMR
(500 MHz, CDCl3) δ 5.84 (s, 1H), 5.10−5.12 (m, 1H), 2.35−2.47 (m,
1H), 2.21−2.31 (m, 3H), 2.10−2.20 (m, 1H), 1.94−2.05 (m, 4H),
1.78−1.90 (m, 1H), 1.72 (s, 3H), 1.62 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 199.5, 165.7, 133.9, 126.9, 121.8, 39.9, 34.0, 29.7, 26.5, 25.8,
23.0, 17.8; IR (neat, cm−1): 3032, 2964, 2930, 2873, 1685, 1625, 1458,
1374, 1223, 874; HRMS (ESI/[M + H]+) calcd for C12H19O
179.1436, found 179.1428.
3,6-Dimethyl-4-(3-methylbut-2-en-1-yl)cyclohex-2-enone (13). n-
BuLi (13.88 mL, 33.30 mmol) was added dropwise to a stirred THF
(100 mL) solution of i-Pr2NH (4.88 mL, 34.69 mmol) at 0 °C. The
resulting mixture was allowed to stir at room temperature for 30 min
and then cooled to −78 °C. A THF (20 mL) solution of compound
12 (4.94 g, 27.75 mmol) was added slowly to the flask at −78 °C.
After the solution was stirred for 0.5 h, MeI (3.46 mL, 55.51 mmol)
was added slowly to the solution at −78 °C. Then the mixture was
allowed to stir at −78 °C for 0.5 h and 0 °C until the full consumption
of 16. A saturated aqueous NH4Cl solution was added to quench the
reaction. Ethyl acetate (100 mL × 3) was used to extract the aqueous
layer. The combined organic solution was washed using aqueous NaCl
solution and treated with MgSO4. After filtration and concentration
under reduced pressure, flash chromatography (EtOAc/hexanes 1:20)
of the crude mixture afforded a yellow oil (4.53 g, 23.59 mmol, 85%)
1
as the desired product. 13: H NMR (500 MHz, CDCl3) δ 5.79 (s,
1H), 5.15 (t, J = 6.5 Hz, 1H), 2.49−2.42 (m, 1H), 2.31−2.17 (m, 3H),
1.97−1.93 (m, 4H), 1.78−1.72 (m, 4H), 1.63 (s, 3H), 1.10 (d, J = 6.5
Hz, 3H); 13C NMR (125 MHz, CDCl3) δ 201.7, 164.4, 133.9, 126.2,
122.3, 40.1, 36.2, 34.9, 29.8, 25.7, 22.8, 17.8, 15.4; IR (neat, cm−1):
3027, 2970, 2927, 2872, 1674, 1628, 1448, 1374, 1213, 878, 854;
HRMS (ESI/[M + H]+) calcd for C13H21O 193.1592, found 193.1588.
Methyl 2,5-Dimethyl-3-(3-methylbut-2-en-1-yl)-2-(4-methylpent-
3-en-1-yl)-6-oxocyclohexanecarboxylate (5). To a stirred suspension
of mashed magnesium (2.5 g, 104.2 mmol) and iodine (0.1 g) in THF
(80 mL) under argon was added a portion (3 mL) of a solution of 5-
bromo-2-methyl-2-pentene (10.2 g, 62.5 mmol) in THF (15 mL). The
suspension was heated gently to initiate the reaction. Then the rest of
the solution was added slowly to the suspension. The resulting
suspension was refluxed for 1.5 h and cooled to ambient temperature.
This solution was transferred slowly to a THF (10 mL) solution of
CuBr·Me2S (1.1 g, 5.2 mmol) at −20 °C via a gastight syringe. After
the the solution was stirred for 0.5 h, a THF (10 mL) solution of 13 (4
g, 20.84 mmol) was added dropwise to the mixture at −20 °C. After
the solution was stirred at −20 °C for 20 min, HMPA (12.8 mL, 72.9
mmol) and methyl cyanoformate (5.8 mL, 72.9 mmol) were added
1
3.4 mmol, 85%). 6: H NMR (500 MHz, CDCl3) δ 5.07−5.06 (m,
1H), 5.00 (t, J = 6.5 Hz, 1H), 4.00 (s, 1H), 2.68−2.61 (m, 1H), 2.55−
2.47 (m, 1H), 2.16−2.13 (m, 1H), 2.07−2.01 (m, 1H), 1.89−1.81 (m,
3H), 1.75−1.70 (m, 5H), 1.70 (s, 3H), 1.62 (s, 3H), 1.59 (s, 3H),
1.46−1.42 (m, 2H), 1.24 (d, J = 7.5 Hz, 3H), 1.08−1.05 (m, 9H); 13C
NMR (125 MHz, CDCl3) δ 212.8, 211.2, 132.8, 131.7, 123.6, 123.1,
H
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64.2, 45.3, 43.2, 43.0, 38.0, 37.8, 33.8, 26.8, 25.8, 25.6, 22.0, 18.0, 18.0,
17.9, 17.8, 17.6, 17.5; IR (neat, cm−1): 3466, 2971, 2928, 2869, 1726,
1699, 1460, 1380, 1092, 1048, 836; HRMS (ESI/[M + H]+) calcd for
C23H39O2 347.2950, found 347.2938.
2, 2, 4a, 7-Tetramethyl-5 -(3-methylbut-2-en-1-yl )-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan (15). To a stirred
THF (2 mL) solution of compound 7 (106 mg, 0.32 mmol) was
added isopropyllithium (0.96 mL, 0.96 mmol) dropwise at 0 °C under
argon. The resulting solution was stirred at 0 °C for 10 min, followed
by the addition of NH4Cl aqueous solution. Ethyl acetate (5 mL × 3)
was used to extract the aqueous layer. The combined organic solvent
was treated by MgSO4. After filtration and concentration under
reduced pressure, silica gel column chromatography (EtOAc/hexanes
1:50) of the crude mixture provided a pale yellow oil (70 mg, 0.25
General Procedure for Anaerobic Oxidative Free-Radical
Cyclization Reaction. To a stirred solution of 5 or 6 (∼50 mg, 0.15
mmol) in EtOH (10 mL) was added the appropriate amount and
combination of oxidant (2 equiv), co-oxidant (1 equiv), base (1
equiv), and acid (1 equiv). The mixture was then frozen and degassed
under vacuum ( × 3). The solution was stirred at the selected reaction
temperature under argon for 2 days or until TLC analysis showed the
full consumption of 5 or 6. A saturated NH4Cl aqueous solution was
added to quench the reaction, and ethyl acetate (10 mL × 3) was used
to extract the aqueous layer. The combined organic solvent was treated
by MgSO4. After filtration and concentration under reduced pressure,
the product was obtained by flash chromatography (EtOAc/hexanes
1:100 to 1:40) of the crude mixture.
1
mmol, 80%) as the product. 15: H NMR (500 MHz, CDCl3) δ 5.13
(t, J = 7.3 Hz, 1H), 2.57 (d, J = 6.5 Hz, 1H), 2.28 (m, 1H), 2.05−2.02
(m, 1H), 1.90−1.83 (m, 1H), 1.78−1.67 (m, 7H), 1.60−1.56 (m, 6H),
1.53−1.47 (m, 1H), 1.34 (s, 3H), 1.30−1.24 (m, 4H), 1.22−1.17 (m,
1H), 1.00 (s, 3H); 13C NMR (125 MHz, CDCl3) δ 150.0, 131.7,
124.9, 100.6, 84.2, 54.1, 52.2, 46.5, 44.3, 39.8, 32.2, 30.1, 29.2, 26.1,
25.8, 24.5, 17.8, 15.6, 15.5; IR (neat, cm−1): 2971, 2932, 2884, 2855,
1718, 1645, 1446, 1378, 1269, 1162, 1099, 909, 866, 749; HRMS
(ESI/[M + H]+) calcd for C19H31O 275.2375, found 275.2372.
2, 2, 4a, 7-Tetramethyl-5 -(3-methylbut-2-en-1-yl )-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan-2a1-yl)methanol
(16). DIBAL (0.83 mL, 1.2 M in toluene, 0.99 mmol) was added
slowly to a stirred solution of compound 7 (55 mg, 0.17 mmol) in
DCM (4 mL) at −78 °C under argon. The reaction was stirred at −78
°C for 2 h, followed by the slow addition of methanol (0.5 mL) and
NaCl aqueous solution (10 mL) at 0 °C. After being stirred at ambient
temperature for 30 min, ethyl acetate (10 mL × 3) was used to extract
the aqueous layer. The combined organic solvent was treated by
MgSO4. After filtration and concentration under reduced pressure,
silica gel column chromatography (EtOAc/hexanes 1:50) of the crude
mixture afforded a white solid (49 mg, 0.16 mmol, 93%) as the
product. 16: 1H NMR (500 MHz, CDCl3) δ 5.11 (t, J = 6.5 Hz, 1H),
3.70−3.62 (m, 2H), 2.21 (t, J = 8.0 Hz,1H), 2.02−1.99 (m, 1H),
1.88−1.84 (m, 2H), 1.82−1.76 (m, 1H), 1.74−1.70 (m,4H), 1.65 (s,
3H), 1.60 (s, 3H), 1.58−1.55 (m, 2H), 1.41 (s, 3H), 1.39−1.33 (m,
1H), 1.27−1.23 (m, 1H), 1.19 (s, 3H), 1.07 (s, 3H); 13C NMR (125
MHz, CDCl3) δ 149.60, 131.95, 124.87, 107.62, 83.96, 65.43, 61.27,
55.09, 46.50, 46.05, 41.68, 31.85, 30.23, 29.76, 25.79, 25.50, 25.36,
18.32, 17.83, 16.00; IR (neat, cm−1): 3461, 2976, 2927, 2859, 1718,
1645, 1451, 1381, 1259, 1164, 1101, 1053, 861; HRMS (ESI/[M +
H]+) calcd for C20H33O2 305.2481, found 305.2473.
2, 2, 4a, 7-Tetramethyl-5 -(3-methylbut-2-en-1-yl )-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan-2a1-carbalde-
hyde (17). Compound 16 (50 mg, 0.16 mmol), NMO (77 mg, 0.008
mmol), and 4 Å molecular sieves (20 mg) were dissolved in DCM (10
mL) followed by the addition of TPAP (130 mg, 0.32 mmol) at
ambient temperature. The reaction was stirred at ambient temperature
for 3 h and then treated with brine (10 mL) at 0 °C. Ethyl acetate (10
mL × 3) was used to extract the aqueous layer. The combined organic
solvent was treated by MgSO4. After filtration and concentration under
reduced pressure, flash chromatography (EtOAc/hexanes 1:50) of the
crude mixture afforded a colorless oil (41 mg, 0.14 mmol, 83%) as the
desired product. 17: 1H NMR (500 MHz, CDCl3) δ 9.36 (s, 1H), 5.11
(t, J = 7.0 Hz, 1H), 2.94 (dd, J = 10.0, 7.5 Hz, 1H), 2.00−1.96 (m,1H),
1.94−1.91 (m, 1H), 1.87−1.78 (m, 3H), 1.72 (s, 3H), 1.71 (s,3H),
1.69−1.65 (m, 1H), 1.63−1.54 (m, 4H), 1.40−1.34 (m, 1H), 1.26−
1.24 (m, 4H), 1.22 (s, 3H), 0.90 (s, 3H); 13C NMR (125 MHz,
CDCl3) δ 200.9, 146.7, 132.5, 124.0, 107.2, 84.9, 72.4, 51.6, 49.0, 45.6,
41.2, 32.3, 29.6, 29.3, 25.8, 25.3, 25.3, 19.9, 17.8, 16.2; IR (neat, cm−1):
2971, 2932, 2859, 2711, 1715, 1446, 1383, 1262, 1169, 1101, 866, 722;
HRMS (ESI/[M + H]+) calcd for C20H31O2 303.2324, found
303.2315.
Methyl 2,2,4a,7-Tetramethyl-5-(3-methylbut-2-en-1-yl)-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan-2a1-carboxylate
(7) from 5. The general procedure for anaerobic conditions was
followed with 5 (47 mg, 0.14 mmol) as the substrate in EtOH (10
mL) using Mn(OAc)3·2H2O (151 mg, 0.56 mmol) and Cu(OAc)2·
H2O (56 mg, 0.28 mmol) at room temperature for 2 days. A white
solid (10 mg, 0.03 mmol, 22%) was obtained by flash chromatography
1
(EtOAc/hexanes = 1:40). 7: mp = 65.7−66.8 °C; H NMR (400
MHz, CDCl3) δ5.10 (t, J = 6.8 Hz, 1H), 3.67 (s, 3H), 3.02 (dd, J =
10.8, 7.2 Hz,1H), 1.98−1.75 (m, 5H), 1.71−1.69 (m, 4H), 1.67 (s,
3H), 1.64−1.49 (m,5H), 1.44−1.36(m, 1H), 1.26 (s, 3H), 1.21(s, 3H),
0.91 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 175.0, 147.7, 132.2,
124.4, 107.0, 67.0, 56.2, 51.9, 48.7, 46.9, 41.1, 31.5, 29.7, 29.3, 25.8,
25.8, 25.2, 19.6, 17.8, 16.0; IR (neat, cm−1): 2971, 2932, 2859, 1721,
1449, 1381, 1237, 1160, 1024, 870, 730; HRMS (ESI/[M + H]+) calcd
for C21H33O3 333.2430, found 333.2425.
General Procedure for Aerobic Oxidative Free-Radical
Cyclization Reaction. To a stirred solution of 5 or 6 (∼50 mg,
0.15 mmol) in a solvent (10 mL) was added the appropriate amount
and combination of oxidant (2 equiv), co-oxidant (0.2 equiv), and base
(1 equiv). The mixture was stirred at the appropriate temperature
under oxygen for 2 days or until full consumption of 5 or 6. A
saturated NaCl aqueous solution was added to quench the reaction,
and ethyl acetate (10 mL × 3) was used to extract the aqueous layer.
The combined organic solvent was treated with MgSO4. After filtration
and concentration under reduced pressure, the product was obtained
by flash chromatography (EtOAc/hexanes 1:20) of the crude mixture.
Methyl 8a-Hydroxy-3,3,5a,8-tetramethyl-6-(3-methylbut-2-en-1-
yl)decahydroindeno[7,1-cd][1,2]dioxine-3a1-carboxylate (8). The
general procedures under aerobic conditions were followed with 5
(48 mg, 0.14 mmol) as the substrate using Mn(OAc)2·4H2O (73 mg,
0.30 mmol) and Mn(OAc)3·2H2O (8 mg, 0.03 mmol) at room
temperature for 2 days. A white solid (30 mg, 0.08 mmol, 55%) was
achieved as the desired compound 8 by flash chromatography
(EtOAc/hexanes 1:10). 8: mp = 101.5−102.2 °C; 1H NMR (400
MHz, CDCl3) δ 5.10 (t, J = 6.4 Hz, 1H), 3.82 (s, 1H), 3.69 (s,3H),
3.17 (d, J = 7.2 Hz, 1H), 2.99−2.90 (m, 1H), 2.07−1.87 (m, 4H),
1.81−1.70 (m,5H), 1.60(s, 3H), 1.55−1.47 (m, 2H), 1.44(s, 3H),
1.39−1.31 (m, 1H), 1.27 (s, 3H), 1.02 (d, J = 7.2 Hz, 3H), 0.97 (s,
3H); 13C NMR (100 MHz, CDCl3) δ174.5, 132.3, 123.7, 102.1, 80.5,
64.0, 51.3, 50.0, 48.9, 42.1, 41.3, 32.2, 31.9, 30.4, 29.6, 26.7, 25.8, 24.1,
21.9, 17.8, 14.9; IR (neat, cm−1): 3471, 2966, 2939, 2881, 1725, 1638,
1453, 1385, 1237, 1082; HRMS (ESI/[M + Na]+) calcd for
C21H34NaO5 389.2304, found 389.2298.
Methyl 2,2,4a,7-Tetramethyl-5-(3-methylbut-2-en-1-yl)-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan-2a1-carboxylate
(7) from 8. Thiourea (11 mg, 0.14 mmol) was added in one portion to
a solution of 8 (44 mg, 0.12 mmol) in MeOH (2 mL). The resulting
mixture was heated under refluxed and stirred for 10 h. Concentration
and flash chromatography (EtOAc/hexanes = 1:40) of the crude
afforded a white solid (36 mg, 0.105 mmol, 92%) as the product. 7:
The characterization data of the white solid are identical to those for
the compound prepared from 5.
2-Methyl-1-(2,2,4a,7-tetramethyl-5-(3-methylbut-2-en-1-yl)-
2,2a,2a1,3,4,4a,5,6-octahydroindeno[7,1-bc]furan-2a1-yl)propan-
1-ol (18). Isopropyllithium (0.25 mL, 1 M in hexanes, 0.25 mmol) was
added dropwise at 0 °C to the stirred solution of 17 (15 mg, 0.05
mmol) in THF (2.5 mL) under an argon atmosphere. The solution
was stirred at 0 °C for 5 min, followed by treatment using saturated
NaCl aqueous solution (5 mL). Ethyl acetate (10 mL × 3) was used to
I
DOI: 10.1021/jo502267g
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
extract the aqueous layer, and the combined organic solution was
treated by MgSO4.
ASSOCIATED CONTENT
* Supporting Information
■
S
After filtration and concentration under reduced pressure, flash
chromatography (EtOAc/hexanes 1:100) of the crude mixture
afforded provided a pale yellow oil (16 mg, 0.047 mmol, 93%) as
X-ray structures of compounds 8 and 9, MTT assays on human
cancer cells and flow cytometry cell cycle analysis of Hela cells
for compound 3, 7−9, and 15, and H NMR and 13C NMR
1
1
the desired compound 18. 18 (mixture of diastereomers): H NMR
spectroscopic data for the new compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
(500 MHz, CDCl3) δ 5.11 (t, J = 7.0 Hz, 1H), 3.49 (dd, J = 10.5, 1.5
Hz, 1H), 2.76 (t, J = 7.5 Hz, 1H), 2.11−2.07 (m, 1H), 1.97−1.78 (m,
6H), 1.72 (s, 3H), 1.61 (s,3H), 1.60 (s, 3H), 1.55−1.51 (m, 2H), 1.47
(s, 3H), 1.44−1.39 (m, 1H), 1.13 (s, 3H), 1.01 (s, 3H), 0.99 (d, J = 7.0
Hz, 3H), 0.88 (d, J = 7.0 Hz, 3H); 13C NMR (125 MHz, CDCl3) δ
149.4, 132.0, 124.7, 109.7, 84.3, 79.7, 63.9, 55.9, 47.0, 44.4, 43.7, 32.9,
31.0, 30.3, 28.2, 25.8, 25.0, 24.7, 23.0, 19.3, 17.8, 16.3, 16.0; IR (neat,
cm−1): 3491, 2971, 2923, 2869, 1721, 1643, 1451, 1381, 1242, 1099,
997, 853; HRMS (ESI/[M + H]+) calcd for C23H39O2 347.2950,
found 347.2941.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: she48@wisc.edu.
*E-mail: lizc@pkusz.edu.cn.
Notes
The authors declare no competing financial interest.
( )-Yezo’otogirin C (3) from 18. To the stirred solution of 18 (16
mg, 0.046 mmol) in DCM (3 mL) were added NaHCO3 (16 mg,
0.185 mmol) and Dess−-Martin periodinate (23 mg, 0.055 mmol) in
one portion at 0 °C. The reaction was stirred at ambient temperature
for 1 h, followed by treatment with saturated Na2S2O3 aqueous
solution. Ethyl acetate (10 mL × 3) was used to extract the aqueous
layer. The combined organic solution was treated by MgSO4. After
filtration and concentration under reduced pressure, flash chromatog-
raphy (EtOAc/hexanes 1:100) of the crude mixture provided a
colorless oil (13 mg, 0.039 mmol, 84%) as the natural product.
ACKNOWLEDGMENTS
■
We gratefully thank the NSFC (National Natural Science
Foundation of China, No. 21272012), the SSTIC (Shenzhen
Science and Technology Innovation Committee,
KQTD201103 and ZDSY20120614144410389), and Peking
University Shenzhen Graduate School for financial support. We
also thank Prof. Hon-Wah Michael Lam (City University of
Hong Kong) for proofreading this manuscript and Prof. Tao
Wang (Southern University of Science and Technology) for
solving the X-ray structures of 8 and 9.
1
( )-Yezo’otogirin C (3): H NMR (400 MHz, CDCl3) δ 5.12 (t, J =
6.8 Hz, 1H), 3.19 (t, J = 9.6 Hz, 1H), 2.96 (m, 1H),1.99−1.92(m,
2H), 1.89−1.87 (m, 1H), 1.85−1.80 (m, 1H), 1.78−1.76 (m,1H), 1.73
(s, 3H), 1.71 (s, 3H), 1.61 (s, 3H), 1.56−1.52 (m, 2H), 1.40−1.35 (m,
1H), 1.24−1.21 (m, 1H), 1.18 (s, 3H), 1.15 (s, 3H), 1.03 (d, J = 2.4
Hz, 3H), 1.01 (d, J = 2.8 Hz, 3H), 0.75 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 217.5, 149.4, 132.5, 124.2, 107.5, 83.4, 73.6, 54.9, 48.5, 47.0,
41.5, 37.9, 32.8, 29.6, 29.5, 25.9, 25.4, 25.4, 21.5, 19.6, 18.3, 17.8, 16.3;
IR (neat, cm−1): 3466, 2971, 2932, 2876, 2850, 1715, 1694, 1643,
1468, 1451, 1381, 1259, 1162, 1104, 1026, 802; HRMS (ESI/[M +
H]+) calcd for C23H37O2 345.2794, found 345.2788.
REFERENCES
■
(1) (a) Dostalek, M.; Pistovcakova, J.; Jurica, J.; Tomandl, J.; Lin-
hart, I.; Sulcava,
́
A.; Hadasova, E. Life Sci. 2005, 78, 239−244.
(b) Beerhues, L. Phytochemistry 2006, 67, 2201−2207.
(2) Bystrov, N. S.; Chernov, B. K.; Dobrynin, V. N.; Kolosov, M. N.
Tetrahedron Lett. 1975, 32, 2791−2794.
(3) Chatterjee, S. S.; Bhattacharya, S. K.; Wonnemann, M.; Singer,
A.; Muller, W. E. Life Sci. 1998, 63, 499−510.
̈
1-(8a-Hydroxy-3,3,5a,8-tetramethyl-6-(3-methylbut-2-en-1-yl)-
decahydroindeno[7,1-cd][1,2]dioxin-3a1-yl)-2-methylpropan-1-one
(9). The general procedure under aerobic conditions was followed with
6 (50 mg, 0.14 mmol) as the substrate using Mn(OAc)2·4H2O (71
mg, 0.29 mmol), Mn(OAc)3·2H2O (8 mg, 0.03 mmol), and i-Pr2NH
(0.02 mL, 0.15 mmol) at 40 °C. After purification using flash
chromatography, a white solid (9 mg, 0.019 mmol, 15%) was achieved
(4) Guedes, A. P.; Franklin, G.; Fernandes-Ferreira, M. Phytochem.
Rev. 2012, 11, 127−152.
(5) Tanaka, N.; Kakuguchi, Y.; Ishiyama, H.; Kubota, T.; Kobayashi,
J. Tetrahedron Lett. 2009, 50, 4747−4750.
(6) Shan, M. D.; Hu, L. H.; Chen, Z. L. J. Nat. Prod. 2001, 64, 127−
130.
(7) For comprehensive reviews of radical cyclization reactions, see:
(a) Iqbal, J.; Bhatia, B.; Nayyar, N. K. Chem. Rev. 1994, 94, 519−564.
(b) Radicals in Organic Synthesis; Renaud, P., Sibi, M. P., Eds.; Wiley-
VCH: Weinheim, 2001. (c) Majumdar, K. C.; Basu, P. K.;
Mukhopadhyay, P. P. Tetrahedron 2005, 61, 10603−10642. (d) Baralle,
1
as the desired product. 9: mp = 88.1−90.2 °C; H NMR (400 MHz,
CDCl3) δ 5.09 (t, J = 7.2 Hz, 1H), 3.52 (s, 1H), 3.09−3.00 (m, 2H),
2.96−2.86 (m, 1H), 2.05−2.00 (m, 1H), 1.97−1.89 (m, 2H), 1.88−
1.80 (m, 1H), 1.76−1.70 (m, 4H), 1.63−1.59(m, 5H), 1.50−1.43 (m,
4H), 1.36−1.29 (m, 4H), 1.15 (d, J = 6.8 Hz, 3H), 1.07 (d, J = 6.4 Hz,
3H), 0.98 (d, J = 7.2 Hz, 3H), 0.84 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 216.9, 132.2, 123.6, 102.8, 80.3, 67.9, 50.8, 47.6, 40.6, 40.5,
36.7, 32.2, 31.9, 30.2, 29.9, 26.9, 25.8, 24.5, 23.1, 21.2, 19.5, 17.8, 14.6;
IR (neat, cm−1) 3493, 2968, 2930, 2875, 1709, 1645, 1463, 1379, 1265,
1100, 1041, 805; HRMS (ESI/[M + Na]+) calcd for C23H38NaO4
401.2668, found 401.2659.
̂
A.; Baroudi, A.; Daniel, M.; Fensterbank, L.; Goddard, J.-P.; Lacote, E.;
Larraufie, M.-H.; Maestri, G.; Malacria, M.; Ollivier, C. In Encyclopedia
of Radicals in Chemistry, Biology and Materials; Studer, A.,
Chatgilialoglu, C., Eds.; John Wiley & Sons: Chichester, 2012; Vol.
2, pp 729−766. For reviews of Mn(III)-promoted radical cyclization
reactions, see: (e) Snider, B. B. Chem. Rev. 1996, 96, 339−363. For
selected examples of Mn(OAc)3-promoted radical reactions, see:
(f) Heiba, E. I.; Dessau, R. M. J. Org. Chem. 1974, 39, 3456−3457.
(g) Kates, S. A.; Dombroski, M. A.; Snider, B. B. J. Org. Chem. 1990,
55, 2427−2436. (h) Curran, D. P.; Morgan, T. M.; Schwartz, C. E.;
Snider, B. B.; Dombroski, M. A. J. Am. Chem. Soc. 1991, 113, 6607−
6617. (i) Snider, B. B. Tetrahedron 2009, 65, 10738−10744. (j) Taber,
D. F.; Nelson, C. G. J. Org. Chem. 2011, 76, 1874−1882. For reviews
of CAN-promoted radical cyclization reactions, see: (k) Nair, V.;
Mathew, J.; Prabhakaran, J. Chem. Soc. Rev. 1997, 26, 127−132.
(l) Nair, V.; Deepthi, A. Chem. Rev. 2007, 107, 1862−1891. For
selected examples of AIBN-promoted radical reactions, see:
(m) George, J. H.; Hesse, M. D.; Baldwin, J. E.; Adlington, R. M.
Org. Lett. 2010, 12, 3532−3535.
( )-Yezo’otogirin C (3) from 9. Thiourea (11 mg, 0.14 mmol) was
added in one portion to the stirred MeOH (2 mL) solution of 9 (44
mg, 0.12 mmol). The solution was refluxed for 10 h. Concentration
and flash chromatography (EtOAc/hexanes = 1:40) of the crude
mixture afforded a white solid (33 mg, 0.97 mmol, 85%) as the natural
product. ( )-yezo’otogirin C (3): The characterization data of the
white solid are identical to those for the compound prepared from 18.
( )-Yezo’otogirin C (3) from 6. The general procedures under
anaerobic conditions was followed using 6 (50 mg, 0.14 mmol) as the
substrate with Mn(OAc)3·2H2O (78 mg, 0.29 mmol) and i-Pr2NH
(0.02 mL, 0.15 mL), and a colorless oil (25 mg, 0.073 mmol, 52%) was
obtained as the product.( )-yezo’otogirin C (3): The character-
ization data of the white solid are identical to those for the compound
prepared from 19.
(8) (a) Yoshida, J.-i.; Nakatani, S.; Sakaguchi, K.; Isoe, S. J. Org.
Chem. 1989, 54, 3383−3389. (b) Qian, C. Y.; Yamada, T.; Nishino,
J
DOI: 10.1021/jo502267g
J. Org. Chem. XXXX, XXX, XXX−XXX

The Journal of Organic Chemistry
Article
H.; Kurosawa, K. Bull. Chem. Soc. Jpn. 1992, 65, 1371−1378.
(c) Yamada, T.; Iwahara, Y.; Nishino, H.; Kurosawa, K. J. Chem.
Soc., Perkin Trans. 1 1993, 609−616. (d) Yin, J.; Wang, C.; Kong, L.;
Cai, S.; Gao, S. Angew. Chem., Int. Ed. 2012, 51, 7786−7789.
(e) Krabbe, S. W.; Do, D. T.; Johnson, J. S. Org. Lett. 2012, 14, 5932−
5935.
(9) For a review of reduction of peroxy-bridge compounds, see:
(a) Balci, M. Chem. Rev. 1981, 81, 91−108. For selected examples of
reduction conditions, see: (b) Gupta, D.; Soan, R.; Dev, S. Tetrahedron
1982, 38, 3013−3018. (c) Yoshioka, M.; Oka, M.; Ishikawa, Y.;
Tomita, H.; Hasegawa, T. J. Chem. Soc., Chem. Commun. 1986, 639−
640. (d) Chowdhury, F. A.; Kajikawa, S.; Nishino, H.; Kurosawa, K.
Tetrahedron Lett. 1999, 40, 3765−3768.
(10) He, S.; Yang, W.; Zhu, L.; Du, G.; Lee, C.-S. Org. Lett. 2014, 16,
496−499.
(11) Lam, H. C.; Kuan, K. K. W.; George, J. H. Org. Biomol. Chem.
2014, 12, 2519−2522.
(12) Spessard, S. J.; Stoltz, B. M. Org. Lett. 2002, 4, 1943−1946.
(13) (a) Aubin, Y.; Audran, G.; Monti, H. Tetrahedron Lett. 2006, 47,
3669−3671. (b) Saeki, M.; Toyota, M. Tetrahedron Lett. 2010, 51,
4620−4622.
(14) Kuramochi, A.; Usuda, H.; Yamatsugu, K.; Kanai, M.; Shibasaki,
M. J. Am. Chem. Soc. 2005, 127, 14200−14201.
(15) Nishino, H. Top. Heterocycl. Chem. 2006, 6, 39−76.
(16) CCDC-924074 (8) and CCDC-1014815 (9) 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.
(17) Beckwith, A. L. J.; Phillipou, C.; Serelis, A. K. Tetrahedron Lett.
1981, 22, 2811−2814.
(18) Mukaiyama, T. Tetrahedron 1981, 37, 4111−4119.
(19) (a) Krapcho, A. P.; Glynn, G. A.; Grenon, B. J. Tetrahedron Lett.
1967, 8, 215−217. (b) Krapcho, A. P.; Weimaster, J. F.; Eldridge, J.
M.; Jahngen, E. G. E., Jr.; Lovey, A. J.; Stephens, W. P. J. Org. Chem.
1978, 43, 138−147.
(20) Imai, M.; Hagihara, A.; Kawasaki, H.; Manabe, K.; Koga, K. J.
Am. Chem. Soc. 1994, 116, 8829−8830.
(21) Porter, L. A. Chem. Rev. 1967, 67, 441.
K
DOI: 10.1021/jo502267g
J. Org. Chem. XXXX, XXX, XXX−XXX