Total Synthesis and in Vitro Anti-Tumor-Promoting Activities of Racemic Acetophenone Monomers from Acronychia trifoliolata

Article abstract of DOI:10.1021/acs.jnatprod.6b00646

Six acetophenone derivatives, acronyculatins I (1), J (2), K (3), L (4), N (5), and O (6), were recently isolated from Acronychia trifoliolata, and the structure of the known acronyculatin B (7) was revised. Because of the limited quantities of isolated p

Full text of DOI:10.1021/acs.jnatprod.6b00646

Article
pubs.acs.org/jnp
Total Synthesis and in Vitro Anti-Tumor-Promoting Activities of
Racemic Acetophenone Monomers from Acronychia trifoliolata
Chihiro Morita,† Yukiko Kobayashi,† Yohei Saito,† Katsunori Miyake,† Harukuni Tokuda,‡
Nobutaka Suzuki,§ Eiichiro Ichiishi,⊥ Kuo-Hsiung Lee,∥,# and Kyoko Nakagawa-Goto*,†,∥
§
†School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, and Department of Complementary
and Alternative Medicine, Clinical R&D, Graduate School of Medical Science, Kanazawa University, Kanazawa, 920-1192, Japan
‡Organic Chemistry in Life Science, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University,
Kyoto 606-8502, Japan
⊥International Health & Welfare University Hospital, Nasushiobara, Tochigi 329-2763, Japan
∥Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel
Hill, North Carolina 27599-7568, United States
#Chinese Medicine Research and Development Center, China Medical University and Hospital, 2 Yuh-Der Road, Taichung, 40447,
Taiwan
S
* Supporting Information
ABSTRACT: Six acetophenone derivatives, acronyculatins I (1), J (2), K (3), L (4), N (5), and O (6), were recently isolated
from Acronychia trifoliolata, and the structure of the known acronyculatin B (7) was revised. Because of the limited quantities of
isolated products as well as their structure similarity, racemic acronyculatins I−L, N, O, and B (1−7) were synthesized to confirm
their structures and to obtain sufficient material for biological evaluation. Trihydroxyacetophenone was converted to the target
compounds by various sequences of hydroxy group protection, allylation or prenylation, and epoxidation followed by cyclization.
C-Prenylations were carried out by direct addition of a prenyl group or through 1,3- or 3,3-sigmatropic rearrangement. The
synthesized racemic compounds were evaluated in an anti-tumor-promoting assay using the Epstein−Barr virus early antigen
(EBV-EA) activation induced by 12-O-tetradecanoylphorbol-13-acetate in Raji cells. All tested compounds significantly inhibited
EBV-EA activation. Especially, racemic acronyculatin I (1) displayed the most potent inhibitory effects, with an IC50 value of 7.3
μM.
phytochemical study has been reported on this species.7 In the
renylated acetophenones are mainly distributed in specific
Pgenera of the Rutaceae, such as Acradenia, Bosistoa,
course of the discovery of unknown bioactive natural products
from rainforest plants, six new acetophenone monomers,
named acronyculatins I (1), J (2), K (3), L (4), M, N (5),
Melicope, Medicosma, and Acronychia.1 In rare cases, they are
found in the root bark of Derris indica2 and Brazilian propolis.3
and O (6), were isolated from a 1:1 CH3OH/CH2Cl2 extract of
this plant.9 In addition, the structure of acronyculatin B (7),
originally identified by Su et al.,10 was revised to be 1-[6-
hydroxy-2-(2-hydroxypropan-2-yl)-4-methoxy-7-(3-methylbut-
2-en-1-yl)-2,3-dihydrobenzofuran-5-yl]ethan-1-one based on
More than 50 prenylated acetophenones have been isolated
from the above species.1 The prenyl group(s) is sometimes
cyclized with a neighboring phenolic oxygen to form a pyran or
furan ring. Interesting dimeric acetophenones, such as acrove-
stone, acropyrone, and acropyraonols, are found only in
Acronychia.4−8
Acronychia trifoliolata Zoll. & Moritzi is distributed from Java
and Christmas Island to the Solomon Islands, and only one
Received: July 12, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00646
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
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a
Scheme 1. Total Synthesis of Racemic Acetophenone Monomers 1−5
a
Conditions: (a) MOMCl, DIPEA, CH2Cl2, rt; (b) prenyl-Br, K2CO3, acetone, reflux; (c) PhNEt2, microwave, 210 °C; (d) MeI, K2CO3, DMF,
reflux; (e) 3 N HCl/MeOH (1:10), reflux; (f) 3 N HCl/MeOH (1:5), reflux; (g) mCPBA, CH2Cl2, rt, 20 min, then montmorillonite K10 (M-K10),
30 min; (h) M-K10, CH2Cl2, microwave, 60 °C; (i) M-K10, CH2Cl2, 0 °C to rt; (j) Pd/C, H2, EtOH, rt; (k) prenyl-Br, DBU, THF, rt; (l) allyl-Br,
K2CO3, acetone, reflux; (m) 2% OsO4, NaIO4, 1,4-dioxane, rt. *rsm: recovery of starting material.
extensive NMR studies. Because the isolation was performed
on a limited amount (4.9 g) of extract provided by the U.S.
National Cancer Institute (NCI), and no more material was
available, small quantities of each compound were obtained.
However, all isolated compounds were successfully identified
using various NMR, HRMS, and IR techniques. Compounds
1−7 showed weak or no antiproliferative activity.9 However,
prior studies have indicated that acetophenone derivatives
display potent inhibition of tumor-promoting activities.11,12 In
addition, the importance of a prenyl-like functional group was
discussed in our previous reports.13−16 Thus, prenylated
acetophenones, such as the acronyculatins, could significantly
inhibit the tumor-promoting activities. To confirm the structure
elucidation as well as provide sufficient quantities of materials
for further bioassays, the total syntheses of racemic
acronyculatins 1−7 were performed. Herein, the synthesis
details and evaluation of the anti-tumor-promoting activities are
described.
B
DOI: 10.1021/acs.jnatprod.6b00646
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a
Scheme 2. Preparation of Racemic Acetophenone Monomers 6 and 7
a
Conditions: (a) prenyl-Br, 10% KOHaq, CH2Cl2, rt, 1.5 h, 46%; (b) TBSCl, imidazole, DMAP, DMF, 85 °C, 45 min, 31%; (c) Me2SO4, NaH, THF,
rt, 3.0 h, 91%; (d) TFA/H2O, THF, rt, 1.0 h, 92%; (e) Ac2O, DMAP, Py, rt, 1.0 h, 87%; (f) mCPBA, CH2Cl2, −70 °C, 30 min; (g) TBAF, HOAc,
THF, 0 °C to rt, 1 h, 27% for 29, 64% for 30; (h) 0.1 M Ba(OH)2, MeOH, 30 min, rt, 56% for 6, 53% for 7.
microwave-assisted 3,3-sigmatropic rearrangement of the allyl
ether produced 22. Deprotection of both MOM groups gave
23. Oxidative cleavage of the terminal olefinic bond was
followed by spontaneous hemiacetalization to provide 5.
As shown in Scheme 2, the prenylation of trihydroxyaceto-
phenone 8 in the presence of 10% KOH produced diprenylated
acetophenone 24, which was converted to 25 through a three-
step sequence, i.e., disilylation using tert-butyldimethylsilyl
chloride (TBSCl), methylation, and selective deprotection23 of
the TBS ether. Treatment of 25 with 1 molar equiv of mCPBA
at low temperature resulted in nonselective monoepoxidation
of the olefinic functional groups. The resulting two products
were probably the dihydrobenzofuran 31, which was sponta-
neously cyclized through 27a, and epoxide 27b. To avoid the
unfavorable cyclization, the phenolic moiety on 25 was
protected as the acetate to give 26. The monoepoxidation of
26 under the above conditions afforded 28a and 28b as an
RESULTS AND DISCUSSION
■
Since acetophloroglucinol is the core skeleton for all target
compounds, 2,4,6-trihydroxyacetophenone (8) was selected as
the starting material. Scheme 1 illustrates the synthesis of
acetophenone monomers 1−5. Two hydroxy groups of
trihydroxyacetophenone (8) were first protected as methox-
ymethyl (MOM) ethers to provide 9. Prenylation of the
hydrogen-bonded phenolic group, followed by microwave-
assisted para-Claisen rearrangement, produced 10,17 which was
methylated with MeI to generate 11. The selective removal of
only one MOM protecting group was achieved successfully by
controlled reaction conditions in 3 N HCl/MeOH (1:10 v/v)
solution. Treatment of 1218 with m-chloroperoxybenzoic acid
(m-CPBA) afforded an epoxide, and subsequent cyclization to
the chromane 14 was catalyzed by montmorillonite K10 clay.19
The removal of the remaining MOM group, prenylation of the
phenolic group, and 1,3-rearrangement of the prenyl group
using montmorillonite K1020 gave racemic acronyculatin L (4).
Racemic acronyculatin K (3) was synthesized similarly from 11.
Both MOM groups were removed by using more concentrated
acidic conditions [3 N HCl/MeOH (1:5 v/v)] than those
mentioned above for the selective removal of one MOM group.
Two subsequent reactions (mCPBA, montmorillonite K10
clay) on the resulting 1318,21 produced only 16, in which
cyclization had occurred via the non-hydrogen-bonded hydroxy
group. The O-prenylation and microwave-assisted para-Claisen
rearrangement of 16 gave racemic acronyculatin K (3).
Catalytic reduction of 3 produced unnatural compound 18, a
regioisomer of 2.
1
inseparable ca. 1:3 mixture by H NMR analysis. The removal
of the TBS group with tetra-n-butylammonium fluoride
(TBAF)/HOAc prompted cyclization to the separable
dihydrobenzofurans 29 and 30 in 27% and 64% yield,
respectively. Under the same conditions, no chromane type
of product was obtained, while the use of KF/18-crown-6/TFA
at −10 °C generated 32 as a minor product and
dihydrobenzofurans 29 and 30. Hydrolysis of the acetoxy
group of 29 and 30 by using Ba(OH)2 generated racemic
acronyculatins O (6) and B (7) in 56% and 53% yield,
respectively.
The structures of the compounds were defined via HRMS
and NMR data and were consistent with the assigned structures
of the compounds9 isolated from A. trifoliolata Zoll. & Moritzi.
As mentioned above, previous studies have indicated that
prenyl-like structures and acetophenone derivatives tend to
show potent effects on inhibition of 12-O-tetradecanoylphor-
bol-13-acetate (TPA)-mediated tumor-promoting activity.11−16
Thus, compounds 1−7, 18, 29, and 30 were evaluated for
cancer chemopreventive activity by means of the Epstein−Barr
virus early antigen (EBV-EA) activation stimulated by TPA in
Raji cells. It has been proven that inhibitory effects on EBV-EA
activation correlate well with anti-tumor-promoting activity in
Compound 8 was treated with prenyl bromide in the
presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU)22 to
generate monoprenylated acetophenone 19, which was
converted to 20 by MOM protection, methylation, and
deprotection of MOM groups. Dihydroxyacetophenone 20
was treated with mCPBA and montmorillonite K10 clay to
afford 21. Prenylation of the phenolic group and subsequent
1,3-rearrangement gave racemic acronyculatin I (1), which was
converted to racemic acronyculatin J (2) by catalytic reduction.
Racemic acronyculatin M (5) was prepared from di-MOM
ether 9. O-Allylation of the phenolic group and subsequent
C
DOI: 10.1021/acs.jnatprod.6b00646
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a
Table 3. Relative Ratio of EBV-EA Activation with Respect to Positive Control (100%)
percentage EBV-EA positive cells
b
concentration (mol ratio/TPA )
c
1000
500
100
10
IC50 (μM)
d
1
0.0 0.3 (60)
4.9 0.2 (60)
3.0 0.3 (60)
1.5 0.2 (60)
10.8 0.5 (60)
0.0 0.3 (60)
0.0 0.3 (60)
0.0 0.4 (60)
7.0 0.6 (60)
5.3 0.5 (60)
0.0 0.4 (60)
26.5 1.4
31.5 1.4
29.1 1.4
27.1 1.1
53.3 1.6
32.0 1.5
30.6 1.5
36.6 1.5
43.8 1.2
33.9 1.5
21.1 1.1
65.5 2.5
76.0 2.6
72.5 2.4
68.2 2.5
77.9 2.2
75.5 2.3
74.3 2.5
78.8 2.3
71.0 2.3
78.6 2.4
80.1 2.4
92.4 0.5
97.7 0.6
96.9 0.5
96.6 0.5
100 0.3
94.9 0.4
93.3 0.4
97.8 0.3
100 0.3
97.8 0.5
7.3
9.3
2
3
8.9
4
8.4
5
15.7
8.0
6
7
7.7
18
8.1
29
15.0
9.9
30
curcumin
100 0.1
12.1
a
b
c
Values represent percentages relative to the positive control value (100%). TPA concentration is 32 nM. The concentration of compound
d
required to inhibit 50% of the positive control activated with 32 nM TPA. Values in parentheses are viability percentages of Raji cells.
60F254 and RP-18F254 plates (0.25 or 0.50 mm thickness; Merck).
MPLC was performed with silica gel and C18 cartridges (Biotage,
Uppsala Sweden). Compounds 9−1316,17,20 and 1921 were obtained
previously.
vivo, as was previously reported for several natural product
derivatives, such as the analogues of dimethyl biphenyldicar-
boxylate,13 betulinic acid,15,24 and coumarins.16,25
As shown in Table 3, the tested compounds displayed low to
moderate cytotoxicity, as shown by high viability (60%) of Raji
cells, implying less than 40% growth inhibition, even at a high
concentration of TPA (32 nmol, a compound/TPA molar ratio
of 1000:1). Furthermore, all compounds significantly inhibited
TPA-mediated EBV-EA activation. Racemic acronyculatin I (1)
showed the most potent inhibitory activity, with 100%
inhibition of EBV-EA activation at the highest concentration
(1000 mol ratio/TPA) and 7.6% inhibition at the lowest tested
concentration (10 mol ratio/TPA). The IC50 value was 7.3 μM.
Among the phenolic compounds 1−7 and 18, compound 5,
devoid of a prenyl group, clearly exhibited reduced activity.
These data supported the previous observations that a prenyl or
prenyl-like group plays an important role in anti-tumor-
promoting effects. Interestingly, the position of functional
groups slightly affected the activity in the case of compounds
with a chromane skeleton (1 vs 3 and 2 vs 18), while no
difference was observed between the two compounds with a
dihydrobenzofuran skeleton (6 vs 7). In addition, with the
latter two compounds, the absence of a phenolic group led to
decreased activity (6 vs 29 and 7 vs 30).
1-[3-Hydroxy-7-methoxy-5-(methoxymethoxy)-2,2-
dimethylchroman-8-yl]ethanone (14). To a solution of 12 (27.4
mg, 0.09 mmol) in anhydrous CH2Cl2 (2.0 mL) was added 75% m-
CPBA (25.2 mg, 0.11 mmol) at 0 °C, and the mixture was stirred for
20 min at room temperature. After consumption of 12 (TLC),
montmorillonite K10 (27.3 mg) was added and stirring was continued
for 30 min at room temperature. The mixture was filtered and washed
with EtOAc. The organic layer was washed with saturated Na2CO3,
H2O, and brine, dried over Na2SO4, and concentrated in vacuo. The
residue was chromatographed on silica gel with EtOAc/n-hexane (1:2)
1
to afford 14 (20.0 mg, 69%) as a yellow oil: H NMR (600 MHz,
CDCl3) δ 6.33 (1H, s), 5.20 (2H, s), 3.79−3.76 (1H, m), 3.77 (3H, s),
3.49 (3H, s), 2.87 (1H, dd, J = 16.8, 5.4 Hz), 2.65 (1H, dd, J = 17.4,
6.0 Hz), 2.46 (3H, s), 1.75 (1H, d, J = 7.2 Hz), 1.33 (3H, s), 1.31 (3H,
s); 13C NMR (150 MHz, CDCl3) δ 201.7, 157.2, 156.3, 151.1, 114.3,
101.6, 94.4, 90.9, 77.5, 69.1, 56.2, 56.0, 32.6, 26.1, 24.6, 21.8; HRMS
(FAB) m/z 311.1499 [M + H]+ (calcd for C16H23O6, 311.1495).
1-(3,5-Dihydroxy-7-methoxy-2,2-dimethylchroman-8-yl)-
ethanone. To a solution of 14 (20.0 mg, 0.06 mmol) in anhydrous
MeOH (1.5 mL) was added 3 N HCl (0.3 mL), and the mixture was
refluxed for 1.0 h under N2. After cooling to room temperature, the
mixture was stirred for 15 min. The reaction was quenched with H2O
and extracted with EtOAc (3 × 10 mL). The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was chromatographed on silica gel with EtOAc/n-
hexane (1:1) to afford the deprotected compound (16.0 mg, 93%) as a
colorless solid: 1H NMR (600 MHz, CDCl3) δ 6.00 (1H, s), 5.28 (1H,
brs), 3.82−3.80 (1H, m), 3.70 (3H, s), 2.84 (1H, dd, J = 16.8, 4.8 Hz),
2.62 (1H, dd, J = 18.0, 6.6 Hz), 2.47 (3H, s), 1.91 (1H, d, J = 6.0 Hz),
1.34 (3H, s), 1.32 (3H, s); 13C NMR (150 MHz, acetone-d6) δ 199.3,
156.8, 156.1, 151.6, 112.8, 100.7, 91.4, 77.5, 68.7, 55.1, 31.8, 26.0, 25.1,
19.4; HRMS (FAB) m/z 267.1227 [M + H]+ (calcd for C14H19O5,
267.1232).
In summary, seven racemic acronyculatins isolated from A.
trifoliolata Zoll. & Moritzi. and the acetophenone monomer 18
were synthesized. The NMR spectra of the synthetic racemic
acronyculatins B and I−O were identical to those of the natural
products. Evaluation of anti-tumor-promoting activity revealed
that all tested acetophenones significantly inhibited EBV-EA
activation induced by TPA in Raji cells. Especially, racemic
acronyculatin I (1) displayed the most potent activity.
1-[3-Hydroxy-7-methoxy-2,2-dimethyl-5-(3-methylbut-2-
enyloxy)chroman-8-yl]ethanone (15). To a solution of 1-(3,5-
dihydroxy-7-methoxy-2,2-dimethylchroman-8-yl)ethanone (10.9 mg,
0.04 mmol) and K2CO3 (23.0 mg, 0.17 mmol) in acetone (1.0 mL)
was added prenyl bromide (0.07 mL, 0.06 mmol). The mixture was
heated under reflux for 2.0 h under N2. After cooling to room
temperature, the mixture was filtered, washed with EtOAc, and
concentrated in vacuo. The residue was chromatographed on silica gel
with EtOAc/n-hexane (1:2) to afford 15 (10.4 mg, 76%) as a yellow
oil: 1H NMR (600 MHz, CDCl3) δ 6.07 (1H, s), 5.46−5.44 (1H, m),
4.53 (2H, d, J = 6.6 Hz), 3.79 (3H, s), 3.79−3.76 (1H, m), 2.85 (1H,
dd, J = 16.8, 5.4 Hz), 2.63 (1H, dd, J = 17.4, 6.0 Hz), 2.47 (3H, s),
EXPERIMENTAL SECTION
■
General Experimental Procedures. Optical rotations were
recorded on a JASCO P-2200 digital polarimeter. Infrared spectra
(IR) were measured with a Shimadzu FTIR-8700 instrument for
samples in CHCl3. NMR spectra were acquired on JEOL JMN-
ECA600 and JMN-ECS400 spectrometers with tetramethylsilane as
internal standard, and chemical shifts are expressed as δ values. HRMS
data were obtained on a JMS-SX102A (FAB) or JMS-T100TD
(DART) mass spectrometer. Microwave irradiation experiments were
carried out in a dedicated Biotage Initiator 2.5 microwave apparatus.
Analytical and preparative TLC was carried out on precoated silica gel
D
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1.79 (3H, s), 1.75 (3H, s), 1.73 (1H, d, J = 7.2 Hz), 1.33 (3H, s), 1.30
(3H, s); 13C NMR (150 MHz, CDCl3) δ 201.7, 158.9, 156.5, 151.3,
138.0, 119.5, 113.3, 100.9, 89.0, 77.5, 69.1, 65.2, 56.0, 32.6, 26.1, 25.8,
24.6, 21.8, 18.3; HRMS (FAB) m/z 335.1860 [M + H]+ (calcd for
C19H27O5, 335.1858).
(rac)-Acronyculatin L (4). To a solution of 15 (10.4 mg, 0.03
mmol) in anhydrous CH2Cl2 (0.5 mL) was added montmorillonite
K10 (10.3 mg) at 0 °C. The mixture was heated in a microwave
instrument at 60 °C for 4.0 h. The mixture was filtered, washed with
EtOAc, and concentrated in vacuo. The residue was purified using
preparative TLC with EtOAc/n-hexane (3:5) to afford the target 4
[4.9 mg, 66% (based on recovery of starting material)] as a yellow oil:
1H NMR (600 MHz, CDCl3) δ 5.74 (1H, s), 5.23−5.20 (1H, m),
3.80−3.78 (1H, m), 3.71 (3H, s), 3.35 (2H, d, J = 7.2 Hz), 2.87 (1H,
dd, J = 16.8, 4.8 Hz), 2.65 (1H, dd, J = 16.8, 5.4 Hz), 2.50 (3H, s),
1.85 (3H, s), 1.79 (3H, s), 1.74 (1H, brs), 1.34 (3H, s), 1.32 (3H, s);
13C NMR (600 MHz, CDCl3) δ 202.2, 155.5, 154.5, 149.5, 136.3,
sealed and stirred at room temperature for 2.0 h under H2. The
mixture was filtered through Celite, washed with EtOAc, and
concentrated in vacuo. The residue was purified using preparative
TLC with CHCl3/MeOH (30:1) to afford the target 18 (5.9 mg, 72%)
as a yellow oil: 1H NMR (400 MHz, CDCl3) δ 13.6 (1H, s), 3.84 (1H,
q, J = 4.8 Hz), 3.72 (3H, s), 2.90 (1H, dd, J = 16.8, 5.4 Hz), 2.68 (1H,
dd, J = 17.4, 6.0 Hz), 2.68 (3H, s), 2.55−2.51 (2H, m), 1.63−1.60
(2H, m), 1.40−1.37 (1H, m), 1.38 (3H, s), 1.33 (3H, s), 0.97 (3H, s),
0.95 (3H, s); 13C NMR (150 MHz, CDCl3) δ 203.4, 161.9, 159.1,
158.1, 116.3, 108.6, 103.1, 78.1, 68.9, 62.9, 39.2, 30.9, 28.4, 25.9, 24.9,
22.6, 22.6, 22.1, 21.3; HRMS (FAB) m/z 337.1975 [M + H]+ (calcd
for C19H29O5, 337.2015).
1-[2-Hydroxy-4,6-bis(methoxymethoxy)-3-(3-methylbut-2-
enyl)phenyl]ethanone. To a solution of 19 (167.5 mg, 0.71 mmol)
in anhydrous CH2Cl2 (5.0 mL) was added N,N-diisopropylethylamine
(0.35 mL, 2.01 mmol) at 0 °C, and the mixture was stirred for 20 min
under Ar. MOMCl (0.13 mL, 1.71 mmol) was added, and stirring at
room temperature continued for 45 min. The reaction was quenched
with H2O and extracted with CH2Cl2 (3 × 10 mL). The combined
organic layers were washed with brine, dried over Na2SO4, and
concentrated in vacuo. The residue was chromatographed on silica gel
with CH2Cl2/n-hexane (1:1) to afford the target di-MOM ether (149.2
121.9, 118.2, 111.8, 103.6, 77.5, 69.1, 63.5, 32.7, 26.2, 25.9, 24.7, 22.8,
21.9, 18.0; HRMS (FAB) m/z 335.1850 [M + H]+ (calcd for
C19H27O5, 335.1858).
1-(3,5-Dihydroxy-7-methoxy-2,2-dimethylchroman-6-yl)-
ethanone (16). To a solution of 13 (48.2 mg, 0.19 mmol) in
anhydrous CH2Cl2 (3.0 mL) was added 75% m-CPBA (53.2 mg, 0.23
mmol) at 0 °C, and the mixture was stirred for 20 min at room
temperature. After complete consumption of 13 (TLC), montmor-
illonite K10 (48.3 mg) was added and the mixture was further stirred
for 30 min at room temperature. The mixture was filtered and washed
with EtOAc. The organic layer was washed with saturated
Na2CO3(aq), H2O, and brine, dried over Na2SO4, and concentrated
in vacuo. The residue was chromatographed on silica gel with EtOAc/
1
mg, 65%) as a pale yellow solid: H NMR (600 MHz, CDCl3) δ 13.8
(1H, s), 6.39 (1H, s), 5.25 (2H, s), 5.23 (2H, s), 5.21−5.18 (1H, m),
3.51 (3H, s), 3.47 (3H, s), 3.30 (2H, d, J = 7.2 Hz), 2.65 (3H, s), 1.78
(3H, s), 1.66 (3H, s); 13C NMR (150 MHz, CDCl3) δ 203.5, 163.5,
160.7, 158.8, 131.4, 122.5, 111.6, 106.9, 94.6, 93.9, 91.2, 56.7, 56.3,
33.2, 25.8, 21.6, 17.8; HRMS (FAB) m/z 325.1668 [M + H]+ (calcd
for C17H25O6, 325.1651).
1-[2-Methoxy-4,6-bis(methoxymethoxy)-3-(3-methylbut-2-
enyl)phenyl]ethanone. To a solution of di-MOM ether (132.6 mg,
0.41 mmol) in anhydrous DMF (5.0 mL) were added K2CO3 (360.0
mg, 2.61 mmol) and iodomethane (0.08 mL, 1.23 mmol). The mixture
was heated at reflux temperature for 2.5 h under N2. After cooling to
room temperature, the reaction was quenched with H2O (10.0 mL)
and extracted with EtOAc (3 × 15 mL). The combined organic layers
were washed with brine, dried over Na2SO4, and concentrated in
vacuo. The residue was chromatographed on silica gel with EtOAc/n-
hexane (1:5) to afford the methyl ether (104.6 mg, 76%) as a yellow
oil: 1H NMR (600 MHz, CDCl3) δ 6.70 (1H, s), 5.19 (2H, s), 5.17−
5.14 (1H, m), 5.14 (2H, s), 3.72 (3H, s), 3.47 (3H, s), 3.46 (3H, s),
3.29 (2H, d, J = 7.2 Hz), 2.52 (3H, s), 1.76 (3H, s), 1.67 (3H, s); 13C
NMR (150 MHz, CDCl3) δ 202.2, 157.2, 156.1, 153.0, 131.3, 123.0,
120.8, 118.3, 97.9, 95.1, 94.3, 63.1, 56.4, 56.2, 32.6, 25.7, 22.7, 17.8;
HRMS (FAB) m/z 339.1777 [M + H]+ (calcd for C18H27O6,
339.1808).
1-[4,6-Dihydroxy-2-methoxy-3-(3-methylbut-2-enyl)-
phenyl]ethanone (20). To a solution of 1-[2-methoxy-4,6-bis-
(methoxymethoxy)-3-(3-methylbut-2-enyl)phenyl]ethanone (54.7 mg,
0.16 mmol) in anhydrous MeOH (3.0 mL) was added 3 N HCl (0.6
mL), and the mixture was heated at reflux temperature for 1.5 h under
N2. The mixture was cooled to room temperature and stirred for 15
min, quenched with H2O (10.0 mL), and extracted with EtOAc (3 ×
15 mL). The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel with EtOAc/n-hexane (1:6) to afford the target
20 (31.2 mg, 77%) as a colorless solid: 1H NMR (600 MHz, CDCl3) δ
13.2 (1H, s), 6.53 (1H, s), 6.22 (1H, s), 5.23−5.21 (1H, m), 3.74 (3H,
s), 3.35 (2H, d, J = 7.2 Hz), 2.69 (3H, s), 1.82 (3H, s), 1.75 (3H, s);
13C NMR (150 MHz, CDCl3) δ 203.7, 164.1, 162.6, 161.4, 135.0,
1
n-hexane (1:2) to afford 16 (31.3 mg, 61%) as a colorless solid: H
NMR (600 MHz, CDCl3) δ 14.4 (1H, s), 5.90 (1H, s), 3.85−3.82
(1H, m), 3.83 (3H, s), 2.87 (1H, dd, J = 16.8, 4.8 Hz), 2.65 (1H, dd, J
= 16.8, 5.4 Hz), 2.61 (3H, s), 1.61 (1H, d, J = 6.6 Hz), 1.39 (3H, s),
1.33 (3H, s); 13C NMR (150 MHz, CDCl3) δ 203.2, 165.2, 161.5,
159.5, 105.6, 99.3, 91.1, 78.4, 69.1, 55.4, 32.9, 25.4, 24.8, 22.2; HRMS
(FAB) m/z 267.1241 [M + H]+ (calcd for C14H19O5, 267.1232).
1-[3-Hydroxy-7-methoxy-2,2-dimethyl-5-(3-methylbut-2-
enyloxy)chroman-6-yl]ethanone (17). To a solution of 16 (27.9
mg, 0.10 mmol) and K2CO3 (58 mg, 0.42 mmol) in acetone (2.0 mL)
was added prenyl bromide (0.02 mL, 0.15 mmol), and the mixture was
heated at reflux temperature for 20 h under N2. The mixture was
cooled to room temperature, filtered, washed with EtOAc, and
concentrated in vacuo. The residue was purified using preparative TLC
with CHCl3/MeOH (20:1) to afford 17 (28.3 mg, 81%) as a colorless
oil: 1H NMR (600 MHz, CDCl3) δ 6.21 (1H, s), 5.47−5.45 (1H, m),
4.35 (2H, d, J = 7.2 Hz), 3.81−3.79 (1H, m), 3.76 (3H, s), 2.92 (1H,
dd, J = 16.8, 4.8 Hz), 2.70 (1H, dd, J = 16.8, 5.4 Hz), 2.50 (3H, s),
1.77 (3H, s), 1.67 (3H, s), 1.65 (1H, s), 1.36 (3H, s), 1.32 (3H, s); 13C
NMR (150 MHz, CDCl3) δ 202.1, 156.5, 155.9, 155.2, 138.8, 119.8,
118.9, 105.4, 96.2, 91.1, 71.6, 69.3, 55.7, 32.6, 26.3, 25.8, 24.7, 22.0,
18.1; HRMS (FAB) m/z 335.1854 [M + H]+ (calcd for C19H27O5,
335.1858).
(rac)-Acronyculatin K (3). A solution of 17 (26.4 mg, 0.08 mmol)
in N,N-diethylaniline (0.5 mL) was heated in a microwave instrument
at 210 °C for 1.0 h. After cooling to room temperature, the mixture
was washed with aqueous 10% HCl, H2O, and brine, dried over
Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel with EtOAc/n-hexane (1:5) to afford 3 (18.5 mg,
1
70%) as a yellow oil: H NMR (600 MHz, CDCl3) δ 13.6 (1H, s),
121.9, 113.2, 109.5, 100.5, 62.8, 31.0, 25.8, 22.7, 18.0; HRMS (FAB)
5.15−5.13 (1H, m), 3.85−3.83 (1H, m), 3.71 (3H, s), 3.26 (2H, t, J =
5.4 Hz,), 2.91 (1H, dd, J = 17.4, 5.4 Hz), 2.68 (1H, dd, J = 17.4, 6.0
Hz), 2.69 (3H, s), 1.77 (3H, s), 1.68 (3H, s), 1.62 (1H, d, J = 7.2 Hz),
1.37 (3H, s), 1.32 (3H, s); 13C NMR (150 MHz, CDCl3) δ 203.4,
162.1, 159.1, 158.1, 131.1, 123.2, 115.0, 108.6, 103.2, 78.2, 68.9, 62.7,
30.9, 25.8, 25.7, 24.8, 22.5, 22.1, 18.0; HRMS (FAB) m/z 335.1852
[M + H]+ (calcd for C19H27O5, 335.1858).
1-(3,5-Dihydroxy-8-isopentyl-7-methoxy-2,2-dimethylchro-
man-6-yl)ethanone (18). To a solution of 3 (8.1 mg, 0.02 mmol) in
EtOH (0.5 mL) was added Pd/C (3.3 mg). The reaction mixture was
m/z 251.1290 [M + H]+ (calcd for C14H19O4, 251.1283).
1-(3,7-Dihydroxy-5-methoxy-2,2-dimethylchroman-6-yl)-
ethanone (21). To a solution of 20 (29.2 mg, 0.12 mmol) in
anhydrous CH2Cl2 (3.0 mL) was added 75% m-CPBA (31.7 mg, 0.14
mmol) at 0 °C, and the mixture was stirred for 20 min at room
temperature. After consumption of 20 (TLC), montmorillonite K10
(29.2 mg) was added and the mixture was stirred for 30 min at room
temperature. The mixture was filtered and washed with EtOAc. The
organic layer was washed with saturated Na2CO3, H2O, and brine,
E
DOI: 10.1021/acs.jnatprod.6b00646
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Journal of Natural Products
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1
dried over Na2SO4, and concentrated in vacuo. The residue was
83% based on recovery of starting material) as a pale yellow oil: H
chromatographed on silica gel with EtOAc/n-hexane (1:2) to afford 21
(26.4 mg, 85%) as a colorless solid: H NMR (600 MHz, CDCl3) δ
NMR (400 MHz, CDCl3) δ 13.9 (1H, s), 6.40 (1H, s), 5.98−5.90
(1H, m), 5.26 (2H, s), 5.23 (2H, s), 5.03−4.93 (2H, m), 3.52 (3H, s),
3.47 (3H, s), 3.38−3.36 (2H, m), 2.66 (3H, s).
1
13.0 (1H, s), 6.20 (1H, s), 3.83−3.82 (1H, m), 3.78 (3H, s), 2.95 (1H,
dd, J = 16.8, 5.4 Hz), 2.71 (1H, dd, J = 16.8, 6.6 Hz), 2.67 (3H, s),
1.99 (1H, d, J = 4.2 Hz), 1.37 (3H, s), 1.36 (3H, s); 13C NMR (150
MHz, CDCl3) δ 203.2, 163.7, 162.1, 160.2, 109.7, 104.9, 101.2, 78.1,
69.1, 61.5, 31.1, 26.0, 25.1, 21.8; HRMS (FAB) m/z 267.1228 [M +
H]+ (calcd for C14H19O5, 267.1232).
1-[2-Methoxy-3-(2-propenyl)-4,6-bis(methoxymethoxy)-
phenyl]ethanone. Iodomethane (0.023 mL, 0.37 mmol) was added
to a solution of 22 (43.1 mg, 0.15 mmol) and K2CO3 (104.3 mg, 0.75
mmol) in DMF (2.0 mL), and the mixture was heated under reflux for
3 h. The mixture was cooled to room temperature and quenched with
H2O (20 mL). The residue was extracted with EtOAc (3 × 15 mL),
and the combined organic layers were washed with brine, dried over
Na2SO4, and concentrated in vacuo. The residue was purified using
column chromatography on silica gel to obtain the product (39.6 mg,
0.13 mmol, 87% based on recovery of starting material) as a pale
1-[3-Hydroxy-5-methoxy-2,2-dimethyl-7-(3-methylbut-2-
enyloxy)chroman-6-yl]ethanone. To a solution of 21 (28.3 mg,
0.11 mmol) and K2CO3 (59.7 mg, 0.43 mmol) in acetone (2.0 mL)
was added prenyl bromide (0.02 mL, 0.17 mmol), and the mixture was
heated at reflux temperature for 12.5 h under N2. The mixture was
cooled to room temperature, filtered, washed with EtOAc, and
concentrated in vacuo. The residue was chromatographed on silica gel
with EtOAc/n-hexane (1:2) to afford the product (34.3 mg, 97%) as a
1
yellow oil: H NMR (400 MHz, CDCl3) δ 6.71 (1H, s), 6.00−5.91
(1H, m), 5.17 (2H, s), 5.14 (2H, s), 5.00−4.95 (2H, m), 3.12 (3H, s),
3.46 (3H, s), 3.46 (3H, s), 3.36−3.35 (2H, m), 2.51 (3H, s); 13C NMR
(150 MHz, CDCl3) δ 202.2, 157.3, 156.3, 153.3, 136.8, 120.8, 116.4,
114.6, 97.8, 95.0, 94.4, 63.4, 56.4, 56.3, 32.6, 27.7; HRMS (FAB) m/z
311.1491 [M + H]+ (calcd for C16H23O6, 311.1495).
1
colorless oil: H NMR (600 MHz, CDCl3) δ 6.21 (1H, s), 5.43−5.41
(1H, m), 4.45 (2H, d, J = 7.2 Hz), 3.82−3.79 (1H, m), 3.74 (3H, s),
2.91 (1H, dd, J = 16.8, 4.8 Hz), 2.68 (1H, dd, J = 13.8, 6.0 Hz), 2.50
(3H, s), 1.77 (3H, s), 1.69 (3H, s), 1.67 (1H, d, J = 6.6 Hz), 1.36 (3H,
s), 1.32 (3H, s); 13C NMR (150 MHz, CDCl3) δ 202.1, 156.8, 156.1,
155.2, 137.9, 119.3, 118.8, 105.0, 101.3, 97.2, 69.1, 65.5, 62.1, 32.6,
26.0, 25.7, 24.8, 21.9, 18.2; HRMS (FAB) m/z 335.1870 [M + H]+
(calcd for C19H27O5, 335.1858).
1-[2-Hydroxy-3-(2-propenyl)-4,6-dihydroxyphenyl]-
ethanone (23). A solution of 1-[2-methoxy-3-(2-propenyl)-4,6-
bis(methoxymethoxy)phenyl]ethanone (33.7 mg, 0.11 mmol) in
MeOH (1.0 mL) was treated with HCl (0.09 mL, 0.18 mmol), and
the mixture was heated under reflux for 7 h. The mixture was cooled to
room temperature, quenched with H2O (10 mL), and extracted with
EtOAc (3 × 10 mL). The combined organic layers were washed with
brine, dried over Na2SO4, and concentrated in vacuo. The residue was
purified with column chromatography on silica gel to obtain 23 (19.3
mg, 0.09 mmol, 90%) as a colorless oil: 1H NMR (400 MHz, CDCl3)
δ 13.2 (1H, s), 6.25 (1H, s), 6.09−6.02 (1H, m), 5.61 (1H, s), 5.21−
5.18 and 5.15−5.14 (2H, each m), 3.75 (3H, s), 3.44−3.41 (2H, m),
2.70 (3H, s); 13C NMR (150 MHz, CDCl3) δ 203.6, 164.4, 161.9,
161.9, 136.2, 116.4, 111.0, 109.8, 100.6, 63.1, 31.0, 27.8.
(rac)-Acronyculatin N (5). A solution of 23 (4.3 mg, 0.02 mmol)
in 1,4-dioxane (0.26 mL) was treated with 2% OsO4/tert-BuOH (50
μL, 0.004 mmol), and the mixture was stirred in the dark at room
temperature. After 30 min, 1.3% NaIO4/H2O (0.8 mL, 0.05 mmol)
was added dropwise. The reaction was quenched with H2O (5 mL)
and extracted with EtOAc (3 × 7 mL). The combined organic layers
were washed with 20% aqueous Na2S2O3, dried over Na2SO4, and
concentrated in vacuo. The residue was purified with column
chromatography on silica gel to obtain 5 (3.1 mg, 0.013 mmol,
65%) as a colorless solid. The physical data (1H NMR, 13C NMR,
HRMS) were essentially identical to the reported data for the natural
product.9
1-[2,4-Bis(tert-butyldimethylsilyloxy)-6-hydroxy-3,5-bis(3-
methylbut-2-enyl)phenyl]ethanone. To a solution of 24 (95.9
mg, 0.32 mmol) in anhydrous N,N-dimethylformamide (1.0 mL) were
added 4-dimethylaminopyridine (3.8 mg, 0.03 mmol) and imidazole
(68 mg, 1.00 mmol) at room temperature. The mixture was cooled to
0 °C, and then TBSCl (101.0 mg, 0.67 mmol) was added. The
resulting mixture was allowed to warm to room temperature and was
stirred for 45 min. The reaction mixture was adjusted to pH 1 by
addition of 1 N HCl and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was chromatographed on silica
gel with CH2Cl2/n-hexane (1:6) to afford the title compound (51.7
mg, 31%) as a yellow oil: 1H NMR (600 MHz, CDCl3) δ 12.0 (1H, s),
5.17−5.14 (2H, m), 3.26 (2H, d, J = 6.6 Hz), 3.20 (2H, d, J = 5.4 Hz),
2.58 (3H, s), 1.72 (3H, s), 1.68 (3H, s), 1.66 (3H, s), 1.64 (3H, s),
1.05 (9H, s), 1.01 (9H, s), 0.19 (6H, s), 0.04 (6H, s); 13C NMR (150
MHz, CDCl3) δ 204.5, 158.9, 158.0, 153.3, 131.5, 131.1, 124.3, 124.3,
123.2, 117.3, 114.7, 112.6, 31.4, 26.1, 25.9, 25.7, 25.4, 24.4, 23.5, 18.8,
18.2, 18.1, 18.0, −3.03, −4.19; HRMS (FAB) m/z 533.3443 [M + H]+
(calcd for C30H53O4Si2, 533.3482).
(rac)-Acronyculatin I (1). To a solution of 1-[3-hydroxy-5-
methoxy-2,2-dimethyl-7-(3-methylbut-2-enyloxy)chroman-6-yl]-
ethanone (34.3 mg, 0.10 mmol) in anhydrous CH2Cl2 (0.5 mL) was
added montmorillonite K10 (34.4 mg) at 0 °C, and the mixture was
stirred for 3.0 h at room temperature. The mixture was filtered, washed
with EtOAc, and concentrated in vacuo. The residue was purified using
preparative TLC with EtOAc/n-hexane (1:1) to afford 1 (18.8 mg,
55%) as a colorless solid. The physical data (1H NMR, 13C NMR,
HRMS) were essentially identical to the reported data for the natural
product.9
(rac)-Acronyculatin J (2). To a solution of 1 (7.6 mg, 0.02 mmol)
in EtOH (0.5 mL) was added Pd/C (3.6 mg). The reaction mixture
was sealed and stirred at room temperature for 2.0 h under H2. The
mixture was filtered through Celite, washed with EtOAc, and
concentrated in vacuo. The residue was purified using preparative
TLC with CHCl3/MeOH (30:1) to afford 2 (5.7 mg, 75%) as a
colorless oil. The physical data (1H NMR, 13C NMR, HRMS) were
essentially identical to the reported data for the natural product.9
1-[2-Allyloxy-4,6-bis(methoxymethoxy)phenyl]ethanone.
To a mixture of 9 (71.9 mg, 0.28 mmol) and K2CO3 (152.6 mg, 1.10
mmol) in acetone (3.0 mL) was added dropwise a solution of allyl
bromide (0.07 mL, 0.83 mmol) in acetone (0.9 mL). The reaction
mixture was stirred under reflux for 28 h. The mixture was cooled to
room temperature, filtered, and evaporated in vacuo. The residue was
extracted with CH2Cl2 (3 × 15 mL). The combined organic layers
were washed with saturated NaHCO3 and brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified with column
chromatography on silica gel to obtain the product (72.3 mg, 0.24
mmol, 86%) as a pale yellow oil: 1H NMR (400 MHz, CDCl3) δ 6.46
(1H, s, J = 2.0 Hz), 6.31 (1H, d, J = 2.0 Hz), 6.04−5.94 (1H, m),
5.40−5.35 and 5.28−5.25 (2H, each m), 5.14 (4H, d, J = 4.4 Hz),
4.53−4.51 (2H, m), 3.47 (6H, d, J = 5.6 Hz), 2.49 (3H, s); 13C NMR
(150 MHz, CDCl3) δ 201.6, 159.6, 156.8, 155.3, 132.6, 117.7, 116.2,
96.2, 95.0, 94.8, 94.4, 69.4, 56.3, 56.2, 32.5; HRMS (FAB) m/z
297.1338 [M + H]+ (calcd for C15H21O6, 297.1355).
1-[2-Hydroxy-3-(2-propenyl)-4,6-bis(methoxymethoxy)-
phenyl]ethanone (22). A solution of 1-[2-allyloxy-4,6-bis-
(methoxymethoxy)phenyl]ethanone (104.6 mg, 0.35 mmol) in N,N-
diethylaniline (0.5 mL) was irradiated in a microwave oven for 1 h at
210 °C. The mixture was cooled to room temperature and the reaction
quenched with ice-cooled 15% aquoeus HCl. The residue was
extracted three times with CH2Cl2 (3 × 15 mL). The combined
organic layers were washed with H2O, dried over Na2SO4, and
concentrated in vacuo. The residue was purified with column
chromatography on silica gel to obtain 22 (83.5 mg, 0.28 mmol,
1-[2,4-Bis(tert-butyldimethylsilyloxy)-6-methoxy-3,5-bis(3-
methylbut-2-enyl)phenyl]ethanone. A 60% NaH (10.0 mg)
solution was washed with n-hexane (0.5 mL) and dissolved in
anhydrous THF (0.5 mL). The mixture was cooled to 0 °C, and 1-
F
DOI: 10.1021/acs.jnatprod.6b00646
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Journal of Natural Products
Article
[2,4-bis(tert-butyldimethylsilyloxy)-6-hydroxy-3,5-bis(3-methylbut-2-
enyl)phenyl]ethanone (22.6 mg, 0.04 mmol) in anhydrous THF (0.5
mL) and dimethyl sulfate (0.02 mL, 0.19 mmol) were added under N2.
The mixture was allowed to warm to room temperature and stirred for
3.0 h. The reaction mixture was then adjusted to pH 8 by addition of
saturated aqueous NH4Cl and extracted with EtOAc (3 × 10 mL). The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was chromatographed on silica
gel with EtOAc/n-hexane (1:20) to afford the product (21.1 mg, 91%)
as a colorless oil: 1H NMR (600 MHz, CDCl3) δ 5.17−5.13 (2H, m),
3.65 (3H, s), 3.24 (4H, m), 2.50 (3H, s), 1.69 (3H, s), 1.66 (3H, s),
1.65 (3H, s), 1.63 (3H, s), 1.00 (9H, s), 0.99 (9H, s), 0.21 (6H, s),
0.07 (6H, s); 13C NMR (150 MHz, CDCl3) δ 202.7, 154.4, 154.0,
149.1, 131.1, 130.9, 123.9, 123.6, 121.7, 121.2, 62.7, 32.9, 26.1, 26.0,
25.6, 25.4, 24.8, 24.0, 18.7, 18.4, 18.1, 18.0, −3.20, −3.72; HRMS
(FAB) m/z 547.3659 [M + H]+ (calcd for C30H55O4Si2, 547.3639).
1-[4-(tert-Butyldimethylsilyloxy)-2-hydroxy-6-methoxy-3,5-
bis(3-methylbut-2-enyl)phenyl]ethanone (25). To a solution of
1-[2,4-bis(tert-butyldimethylsilyloxy)-6-methoxy-3,5-bis(3-methylbut-
2-enyl)phenyl]ethanone (14.8 mg, 0.03 mmol) in anhydrous THF
(0.2 mL) were added TFA (0.1 mL) and H2O (0.1 mL). The mixture
was stirred at room temperature for 1.0 h. The reaction mixture was
adjusted to pH 7 by addition of saturated aqueous NaHCO3 and
extracted with CH2Cl2 (3 × 5 mL). The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated in vacuo. The
residue was purified using preparative TLC with EtOAc/n-hexane
0.015 mmol), and the mixture was stirred at 0 °C for 10 min under Ar.
To the mixture was added p-TsOH (2.4 mg, 0.01 mmol), and the
mixture was stirred for 1.0 h. The mixture was adjusted to pH 7 by
addition of saturated aqueous NaHCO3 and extracted with EtOAc (3
× 5 mL). The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was purified
using preparative TLC with EtOAc/n-hexane (1:2) to afford 29 (1.3
mg, 27%) and 30 (2.9 mg, 64%) as yellow oils.
5-Acetyl-2-(2-hydroxypropan-2-yl)-6-methoxy-7-(3-methyl-
1
but-2-enyl)-2,3-dihydrobenzofuran-4-yl Acetate (29). H NMR
(600 MHz, CDCl3) δ 5.20−5.17 (1H, m), 4.68 (1H, t, J = 8.4 Hz),
3.70 (3H, s), 3.28 (2H, d, J = 7.8 Hz), 3.02−2.99 (2H, m), 2.51 (3H,
s), 2.25 (3H, s), 1.76 (3H, s), 1.70 (3H, s), 1.30 (3H, s), 1.19 (3H, s);
13C NMR (100 MHz, CDCl3) δ 168.6, 166.1, 160.8, 157.5, 153.2,
132.3, 121.7, 116.3, 115.3, 90.5, 77.2, 71.8, 63.4, 31.5, 28.4, 25.7, 25.6,
24.0, 23.0, 20.7, 17.8; HRMS (FAB) m/z 377.1975 [M + H]+ (calcd
for C21H29O6, 377.1964).
5-Acetyl-2-(2-hydroxypropan-2-yl)-4-methoxy-7-(3-methyl-
1
but-2-enyl)-2,3-dihydrobenzofuran-6-yl Acetate (30). H NMR
(600 MHz, CDCl3) δ 5.11−5.08 (1H, m), 4.66 (1H, t, J = 9.6 Hz),
3.86 (3H, s), 3.29−3.27 (2H, m), 3.16 (1H, dd, J = 15.0, 7.8 Hz), 3.10
(1H, dd, J = 14.4, 6.6 Hz) 2.46 (3H, s), 2.24 (3H, s), 1.72 (3H, s), 1.67
(3H, s), 1.35 (3H, s), 1.23 (3H, s); 13C NMR (100 MHz, CDCl3) δ
200.3, 169.5, 161.1, 152.8, 146.5, 132.2, 121.1, 114.0, 112.1, 90.1, 77.2,
71.6, 59.9, 31.8, 29.4, 26.0, 25.7, 24.3, 23.4, 20.7, 17.8; HRMS (FAB)
m/z 377.1958 [M + H]+ (calcd for C21H29O6, 377.1964).
(rac)-Acronyculatin O (6). To a solution of 29 (2.0 mg, 0.005
mmol) in anhydrous MeOH (0.1 mL) was added Ba(OH)2 (0.1 mL,
0.01 mmol, 0.1 M in MeOH), and the mixture was stirred for 30 min
at room temperature. The mixture was directly purified using
preparative TLC with CHCl3/MeOH (20:1) to afford 6 (1.1 mg,
56%) as a colorless solid. The physical data (1H NMR, 13C NMR,
HRMS) were essentially identical to the reported data for the natural
product.9
1
(1:12) to afford 25 (12.7 mg, 92%) as a yellow oil: H NMR (600
MHz, CDCl3) δ 13.2 (1H, s), 5.17−5.14 (1H, m), 5.13−5.11 (1H, m),
3.66 (3H, s), 3.26 (4H, t, J = 6.6 Hz), 2.68 (3H, s), 1.72 (3H, s), 1.71
(3H, s), 1.67 (3H, s), 1.66 (3H, s), 1.00 (9H, s), 0.21 (6H, s); 13C
NMR (150 MHz, CDCl3) δ 203.9, 161.7, 159.3, 158.8, 131.8, 131.1,
123.7, 122.8, 118.4, 117.3, 110.4, 62.5, 31.0, 26.1, 25.7, 25.5, 23.8, 23.5,
18.9, 18.0, −3.05; HRMS (FAB) m/z 433.2733 [M + H]+ (calcd for
C25H41O4Si, 433.2774).
(rac)-Acronyculatin B (7). To a solution of 30 (5.3 mg, 0.02
mmol) in anhydrous MeOH (0.2 mL) was added Ba(OH)2 (0.2 mL,
0.02 mmol, 0.1 M in MeOH), and stirring continued for 30 min at
room temperature. The mixture was directly purified using preparative
TLC with CHCl3/MeOH (20:1) to afford 7 (2.5 mg, 53%) as a
colorless solid. The physical data (1H NMR, 13C NMR, HRMS) were
essentially identical to the reported data for the natural product.9
In Vitro EBV-EA Activation Experiments. The anti-tumor-
promoting activities of compounds were assessed using the EBV-EA
activation assay in the presence of 32 pmol/mL TPA as described
before.26,27 The average EBV-EA induction of the test compound was
determined as a ratio relative to the control. The viability of treated
Raji cells was evaluated by trypan blue staining.
2-Acetyl-5-(tert-butyldimethylsilyloxy)-3-methoxy-4,6-bis(3-
methylbut-2-enyl)phenyl Acetate (26). To a solution of 25 (58.9
mg, 0.14 mmol) in pyridine (1.0 mL) were added DMAP (2.3 mg,
0.02 mmol) and Ac2O (0.06 mL, 0.68 mmol), and the mixture was
stirred at room temperature for 1.0 h. The reaction mixture was
adjusted to pH 4 by addition of 1 N HCl and extracted with EtOAc (3
× 10 mL). The combined organic layers were washed with brine, dried
over Na2SO4, and concentrated in vacuo. The residue was chromato-
graphed on silica gel with EtOAc/n-hexane (1:10) to afford 26 (56.4
mg, 87%) as a colorless oil: 1H NMR (600 MHz, CDCl3) δ 5.14−5.12
(1H, m), 5.02−4.99 (1H, m), 3.65 (3H, s), 3.30 (2H, d, J = 6.6 Hz),
3.19 (2H, d, J = 6.0 Hz), 2.52 (3H, s), 2.17 (3H, s), 1.71 (3H, s), 1.67
(3H, s), 1.67 (3H, s), 1.66 (3H, s), 1.00 (9H, s), 0.19 (6H, s); 13C
NMR (100 MHz, CDCl3) δ 201.5, 169.3, 155.6, 154.1, 144.9, 131.6,
131.5, 124.7, 123.0, 122.5, 122.4, 62.8, 31.3, 26.0, 25.6, 25.5, 24.5, 24.1,
20.8, 18.8, 18.0, 17.9, −3.11; HRMS (FAB) m/z 475.2862 [M + H]+
(calcd for C27H43O5Si, 475.2880).
2-Acetyl-5-[(tert-butyldimethylsilyl)oxy]-6-[(3,3-dimethylox-
iran-2-yl)methyl)]-3-methoxy-4-(3-methylbut-2-en-1-yl)phenyl
Acetate (28a) and 2-Acetyl-5-[(tert-butyldimethylsilyl)oxy]-4-
[(3,3-dimethyloxiran-2-yl)methyl]-3-methoxy-6-(3-methylbut-
2-en-1-yl)phenyl Acetate (28b). To a solution of 26 (12.9 mg, 0.03
mmol) in anhydrous CH2Cl2 (0.5 mL) was added 75% m-CPBA (6.2
mg, 0.03 mmol) at −70 °C, and the mixture was stirred for 30 min.
The reaction mixture was adjusted to pH 7 by addition of saturated
aqueous Na2CO3 and extracted with CH2Cl2 (3 × 5 mL). The
combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was chromatographed on silica
gel with EtOAc/n-hexane (1:5) to afford a mixture of the target
compounds 28a and 28b [6.1 mg, 77% (based on recovery of starting
material)] as a yellow oil.
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00646.
■
S
1H NMR/13C NMR for 1−7, 14−18, 20−23, and 25−
30, the experimental procedures for known compounds
9−13, 19, and 24 (PDF)
AUTHOR INFORMATION
Corresponding Author
*Tel (K. Nakagawa-Goto): +81-76-264-6305. E-mail: kngoto@
p.kanazawa-u.ac.jp.
■
Notes
5-Acetyl-2-(2-hydroxypropan-2-yl)-6-methoxy-7-(3-methyl-
but-2-en-1-yl)-2,3-dihydrobenzofuran-4-yl Acetate (29) and 5-
Acetyl-2-(2-hydroxypropan-2-yl)-4-methoxy-7-(3-methylbut-
2-en-1-yl)-2,3-dihydrobenzofuran-6-yl Acetate (30). To a
mixture of 28a and 28b (6.1 mg, 0.01 mmol) in anhydrous THF
(0.5 mL) were added HOAc (2.0 μL, 0.03 mmol) and TBAF (15.0 μL,
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We appreciate critical comments, suggestions, and editing of
the manuscript by Dr. S. L. Morris-Natschke (UNC-CH). This
G
DOI: 10.1021/acs.jnatprod.6b00646
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
work was supported by JSPS KAKENHI Grant Number
JP25293024 awarded to K.N.G. This study was also supported
in part by a grant from the National Cancer Institute/NIH
(CA177584), awarded to K.H.L.
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DOI: 10.1021/acs.jnatprod.6b00646
J. Nat. Prod. XXXX, XXX, XXX−XXX