Article

Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX [Link]/jnp

Prenylated Acetophloroglucinol Dimers from Acronychia trifoliolata:

Structure Elucidation and Total Synthesis
Katsunori Miyake,† Chihiro Morita,‡ Airi Suzuki,‡ Natsuko Matsushita,‡ Yohei Saito,‡ Masuo Goto,§
David J. Newman,⊥ Barry R. O’Keefe,∥,# Kuo-Hsiung Lee,§,¶ and Kyoko Nakagawa-Goto*,‡,§
†
Tokyo University of Pharmacy and Life Sciences, Hachioji, Tokyo 192-0392, Japan
‡
School of Pharmaceutical Sciences, College of Medical, Pharmaceutical and Health Sciences, Kanazawa University, Kanazawa,
920-1192, Japan
§
Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel
See [Link] for options on how to legitimately share published articles.

Hill, North Carolina 27599-7568, United States

⊥
NIH Special Volunteer, Wayne, Pennsylvania 19087, United States
Downloaded via UNIV OF NEW ENGLAND on September 25, 2019 at 01:43:17 (UTC).

∥
Natural Products Branch, Developmental Therapeutics Program, Division of Cancer Treatment and Diagnosis, National Cancer
Institute, NCI at Frederick, Frederick, Maryland 21702-1201, United States
#
Molecular Targets Program, Center for Cancer Research, National Cancer Institute, NCI at Frederick, Frederick, Maryland
21702-1201, United States
¶
Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung, 40447, Taiwan
*
S Supporting Information

ABSTRACT: The isolation of 12 secondary metabolites, including seven new acetophenone monomers, from the 50%
CH3OH/CH2Cl2 extract (N089419-L/6) of Acronychia trifoliolata was reported previously. In the present work, three new
prenylated acetophenone dimers (1−3) and five known dimers (4−8) were isolated, and their structures were elucidated by
using various NMR spectroscopic techniques and HRMS. Among the new dimers, an unprecedented 4-isobutyl-3-
isopropyltetrahydro-2H-pyran ring was observed in the structure of 1. This study is the first to report the formation of a 2H-
pyran ring between two prenylated acetophloroglucinols. Only four related dimers have been reported before, and they were
formylated phloroglucinol dimers from the family Eucalypteae. Compounds 2 and 3 are acrovestone-like dimers, and the
structure of 3 was confirmed by total synthesis. The evaluation of the antiproliferative activity of isolated and synthesized
acrovestone-like dimers indicated that a double bond in the prenyl-like moiety as found in the more active compounds might be
important for mediating activity, while the pendant isobutyl group seems to be less important.

T he genus Acronychia belongs to the family Rutaceae in the

major group Angiosperms (flowering plants). Among 49
species,1 only half of these have been investigated phytochemi-
which two prenylated acetophenones are connected by a
methylene with a pendant isobutyl group. Seven dimers,
acrovestone,12 acrovestenol,13 acrofolione A,7 acrofolione B,7
cally and pharmacologically, even though many species are acropyrone,14 acropyranol A,14 and acropyranol B,14 have been
used in traditional folk medicine for the treatment of diarrhea, isolated, and all of them exhibit some cytotoxicity against
cough, asthma, sores, ulcers, itchy skin, pain, and rheuma- cancer cells.
tism.2,3 Thus, plants in this genus merit further phytochemical Our previous phytochemical research led to the identi-
research for the discovery of potential new bioactive natural fication of new noncytotoxic prenylated acetophenone
products. monomers from A. trifoliolata Zoll. & Moritzi,11 a rainforest
Prenylated acetophenones3−11 are characteristic secondary
metabolites of the family Rutaceae. Especially, the genus Received: June 28, 2019
Acronychia is known to biosynthesize dimeric compounds, in
© XXXX American Chemical Society and
American Society of Pharmacognosy A DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Figure 1. Isolated compounds 1−8 from Acronychia trifoliolata and the structure of sideroxylonal C.

plant distributed from Java and Christmas Island to the phenyl rings. These signals were very similar to those of
Solomon Islands. In the present work, a 50% CH3OH/CH2Cl2 sideroxylonal C, isolated from Eucalyptus albens15 and E.
extract of A. trifoliolata (N089419-L/6), obtained from the melliodora,16 except for the two pairs of prenyl groups and two
National Cancer Institute (NCI), displayed significant acetyl groups present instead of four formyl groups as well as
antiproliferative activity against NCI60 panels of human the two methoxy groups replacing two hydroxy groups.
tumor cell lines (Figure S36). Subsequent isolation work led Furthermore, compound 1 showed a protonated molecular
to the characterization of three new prenylated acetophlor- ion peak at m/z 637.3744 in the HRFABMS, corresponding to
oglucinol dimers, 1−3. the molecular formula C38H53O8, which suggested that this

■ RESULTS AND DISCUSSION

A 50% CH3OH/CH2Cl2 extract of the bark of A. trifoliolata
compound is a dimeric derivative of 1-[2,4-dihydroxy-6-
methoxy-3,5-bis(3-methyl-2-buten-1-yl)phenyl]ethanone, pre-
viously isolated from the same extract.11 The planar structure
(4.9 g) was fractionated with ethyl acetate and water to of 1 was confirmed by 2D NMR experiments, including the
1
provide ethyl acetate- and water-soluble fractions. The ethyl H−1H COSY, HMQC, HMBC, and NOESY spectra (Figures
acetate-soluble fraction was separated by a combination of 2 and 3). Data from the 1H−1H COSY experiment confirmed
medium-pressure liquid chromatography (MPLC), silica gel the presence of two prenyl groups and an aliphatic 10-carbon
column chromatography, and preparative TLC techniques to unit. The connections of the two phenyl rings with the 10-
give three new prenylated acetophloroglucinol dimers, carbon unit were deduced from HMBC correlations. The
acrotrifoliolane A (1), acrofolione C (2), and acrofolional NOESY correlations of the methoxy group methyl protons (δH
(3), together with five known compounds, acrovestone (4),3 3.75) with the methylene protons of a prenyl moiety (δH 3.22)
acropyrone (5),13 acropyranols A (6)7 and B (7),14 and and the acetyl methyl protons (δH 2.73) indicated the latter
acrovestenol (8), of which the latter three were isolated as two groups and the methoxy group to be close in space.
racemates (Figure 1). Similarly, the NOESY correlations of the other methoxy
The 1H NMR spectrum of acrotrifoliolane A (1) showed methyl protons (δH 3.77) with the methylene protons of a
signals assignable to two olefins (δH 5.24, 5.05), a deshielded
prenyl moiety (δH 3.34) and the acetyl methyl protons (δH
oxymethine (δH 5.14), two methoxy group methyls (δH 3.77,
2.71) as well as of the acetyl methyl protons (δH 2.71) with a
3.75), two deshielded methylenes (δH 3.34, 3.22), a deshielded
methine (δH 3.29), two acetyl methyls (δH 2.73, 2.71), three phenolic proton (δH 13.68) determined the arrangement of the
methines (δH 2.26, 1.71, 1.48), a methylene (δH 1.33, 1.43), substituents on the phenyl ring. The relative configuration of 1
four tertiary methyls (δH 0.96, 0.91, 0.87, 0.79), and three was determined from the NOESY data (Figure 3). The
hydrogen-bonded hydroxy protons (δH 13.68, 13.29, 7.96) correlations between H-1⁗, H-5⁗, and the OH at C-2‴
(Table 1). The 13C NMR spectrum of 1 (Table 1) indicated indicated that the phenyl ring at C-1⁗ and an isopropyl group
38 carbons including two ketone carbonyl carbons (δC 204.4, at C-2⁗ face in opposite directions (i.e., are trans oriented). In
203.9), two pairs of olefinic carbons (δC 132.0, 131.9, 122.5, addition, the isopropyl and isobutyl groups were also trans
123.0), 12 aromatic carbons (δC 161.6, 161.4, 161.3, 161.1, oriented based on NOESY correlations between H-2′, H-2⁗,
160.4, 158.7, 117.9, 116.3, 115.4, 110.2, 111.0, 108.7), and an and the OH at C-4‴. The assigned relative configuration was
oxymethine (δC 79.1), which suggested the presence of two supported by a comparison of the H-1′, H-1⁗, and H-
B DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Table 1. 1H and 13
C NMR Spectroscopic Data of 1−3 2⁗coupling constants with those of the related phloroglucinol
dimers, sideroxylonals A, B, and C.15−17
3
1 (CDCl3) 2 (DMSO-d6) (CDCl3) It should be noted that sideroxylonals A, B, and C15−17 as
well as loxophlebal A,18 four related phloroglucinol dimers
δH (J in δH (J in
position δH (J in Hz)a δC Hz)a δC Hz)b with a 4-isobutyl-3-isopropyltetrahydro-2H-pyran unit, were
1 111.0 N.D.c
isolated only from the genus Eucalyptus15−21 in the family
2 160.4 159.0d Myrtaceae. They were all racemic with the same planar
3 117.9 113.8e structure derived from formylated phloroglucinols. Therefore,
4 161.6 161.5 compound 1 is the first prenylated acetophloroglucinol dimer
5 116.3 114.3 identified from a plant other than in the Myrtaceae. As
6 158.7 159.0 proposed before,17,18 the 2H-pyran ring could be formed
1′ 3.29, m 27.1 5.24, brt 26.7 4.70, t biosynthetically by a Diels−Alder cyclization between the
(7.8) (7.2) related o-quinone methide as a diene with the styrene
2′ 1.33, m, 49.3 1.60, m, 40.7 2.15, m monomer as a dienophile (Figure 4).
1.43, m 2.09, m
The 1H NMR spectrum (Table 1) of 2 was closely related to
3′ 1.48, m 25.4 1.38, m 25.8 1.40, m
that of known acrofolione B, previously isolated as a racemate
4′ 0.87, d (6.0) 22.9 0.81, d 22.1 0.89, brs
(6.6) from A. trifoliolata.7 Some signals were broadened in the 1H
5′ 0.91, d (6.0) 23.0 0.86, d 23.1 0.89, brs and 13C NMR spectra because of freely rotatable aryl moieties
(6.6) in 2, making the structural elucidation difficult. Although the
1″ 3.22, t (6.0) 22.8 3.17, brs 22.4 3.30, brs connectivities could not be determined from the spectra in
2″ 5.05, m 122.5 5.09, brt 123.5 5.18, brs (CD3)2SO, confirmation was provided by measuring the
(6.6)
spectra in several deuterated solvents, such as C6D6 and
3″ 132.0 130.3
(CD3)2CO (Figure 2). The resultant planar structure
4″ 1.50, brs 17.5 1.69, brs 17.8 1.77, brs
established was identical to that of acrofolione B. Acrofolione
5″ 1.57, brs 25.6 1.62, brs 25.4 1.70, brs
B was originally elucidated by using 1H NMR, DQFCOSY, and
1‴ 108.7 100.3
NOE difference spectra, and the assigned fusion position of the
2‴ 161.1 161.6
dihydrofuran ring was not conclusive. In another study,14 this
3‴ 110.2 105.3
4‴ 161.4 161.8
position was determined by comparison of the NOE
5‴ 115.4 107.3e
correlations between acrofoliones A and B with reference to
6‴ 161.3 161.2d
acropyranols A and B. Although 2D NMR analysis indicated
1⁗ 5.14, d (10.2) 79.1 2.97, m, 27.0 10.16, s
that 2 and acrofolione B have the same planar structure, the
2.99, m chemical shift patterns of the two compounds were different
2⁗ 2.26, dt 51.9 4.71, dd 91.2 (Table S1, Supporting Information). Thus, compound 2
(10.2, 2.4) (9.0, 9.0) (acrofolione C) was proposed as a diastereomer of acrofolione
3⁗ 1.71, m 28.4 70.0 B.
4⁗ 0.79, d (6.6) 16.6 1.11, d 25.7 Compound 3 was isolated in a limited quantity, insufficient
(2.4)
5⁗ 0.96, d (7.2) 21.0 1.20, brs 25.6
to obtain a clear 13C NMR spectrum. However, the 1H NMR
1″‴ 3.34, dd (7.8, 22.6
spectrum of 3 resembled that of 4 except for the absence of
6.6) signals for a prenyl group and presence of a signal for an
2″‴ 5.24, dd (7.8, 123.0 aldehyde proton at 10.16 ppm (Figure S32, Supporting
6.6) Information). Based on the correlations in 1H−1H COSY
3″‴ 131.9 and NOESY data, compound 3 (acrofolional) was proposed as
4″‴ 1.79, brs 18.0 a carbaldehyde derivative of acrovestone-type acetophenone
5″‴ 1.72, brs 25.7 dimers. Its structure was confirmed by total synthesis.
COCH3-1 2.73, s 31.4 2.58, s 30.8 2.73, s To synthesize 3, acetophloroglucinol (9) was converted
COCH3-1 204.4 202.4 initially to the prenylated acetophenone 10.22 The preparation
COCH3-1‴ 2.71, s 31.2 2.62, s 30.8 2.74, s
of acetophenone homodimers was previously reported23,24
COCH3-1‴ 203.9 203.3
using a Friedel−Craft reaction of electron-rich aryl groups with
OCH3-6‴ 3.75, s 63.0 3.65, s 62.5 3.73, s
alkyl aldehydes (Scheme 1). Although heterodimer syntheses
OCH3-6‴ 3.77, s 62.7
have not been reported, we performed the Friedel−Craft
OH 13.29, s (OH- 8.32, s 8.73, brs
2) reaction of acetophenone 10 with isovaleraldehyde in the
OH 13.68, s (OH- 9.37, brs 10.88, presence of AlCl3 under heating at 120 °C. The reaction time
2‴) brs was controlled carefully to avoid the production of homodimer
OH 7.96, s (OH- 10.69, brs 14.48, 13. After the disappearance of starting material by TLC, the
4‴) brs
aryl derivative 9 was added to the reaction mixture. The
OH 14.17, brs 16.11,
brs desired heterodimer 12 was obtained in 19% yield together
OH 14.67, brs with homodimer 13 (22%) and 10 (11%). The treatment of 12
under Vilsmeier−Haack conditions produced 3 in 28% yield.
The 1H NMR (Figure S38, Supporting Information), thin-
layer chromatography (TLC), and high-performance liquid
a
Measured at 600 MHz. bMeasured at 400 MHz. cNot detected. chromatography (HPLC) data were identical to those of the
d,e
Signals are interchangeable. isolated compound.
C DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Figure 2. Selected HMBC correlations (arrows; black: in CDCl3; red: in (CD3)2SO; green: in C6D6; blue: in (CD3)2CO), 1H−1H COSY
connectivities (bold lines), and NOESY correlations (dotted arrows) for 1−3.

Figure 3. Key NOESY correlations (blue arrows) for 1.

Figure 4. Proposed biosynthetic 2H-pyran ring formation by Diels−Alder reaction.

Scheme 1. Total Synthesis of Compound 3

To determine the structure−activity relationship of the antiproliferative activity against four tumor cell lines (Table
pendant isobutyl group, the related derivatives 15 and 16 were 2). Among them, compounds 4, 5, 12, 15, and 16 inhibited the
also synthesized through dimethylamine 14 produced by tumor cell growth with IC50 values of around 5 μM against all
Eschenmoser salt and 10 (Scheme 2). tested cell lines, whereas 8 and 13 showed lower activities.
The isolated acetophenone dimers 2−8 as well as synthetic Since compounds 2, 6, and 7 were less potent, a double bond
derivatives 12, 13, 15, and 16 were evaluated for in the prenyl-like moiety as found in the more active
D DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

Scheme 2. Synthesis of Acetophenone Dimer Derivatives

Table 2. Antiproliferative Activity of the Isolated and Institution, Washington, DC. A voucher extract (N089419-L/6) was
Synthesized Compounds deposited at the NCI and at Kanazawa University.
Extraction and Isolation. A 50% CH3OH/CH2Cl2 extract of the
cell linea (IC50 μM)b bark of A. trifoliolata (4.9 g), provided by NCI, was dissolved into
compound A549 MDA-MB-231 KB KB-VIN water and partitioned with EtOAc to yield EtOAc-soluble (3.8 g) and
H2O-soluble portions. The EtOAc-soluble fraction (3.7 g) was
2, 3, 6, 7 >10 >10 >10 >10 dissolved in EtOAc to remove precipitated material and then was
4 6.1 6.4 4.9 4.4 subjected to silica gel MPLC (KP-Sil 100 g), with a CH3OH/CHCl3
5 5.9 5.4 4.9 4.3 gradient system [CHCl3 (5 fractions) → 2% CH3OH/CHCl3 (5
8 >10 10 6.0 6.5 fractions) → 10% CH3OH (5 fractions) → CH3OH (1 fraction);
12 4.9 5.8 5.2 5.0 collection per 100 mL], to yield 16 fractions.
13 >10 >10 7.6 8.4 Fraction 2 (750 mg) was subjected to silica gel MPLC (SNAP HP-
15 5.6 6.9 5.2 5.1 Sil 25 g), with the eluents EtOAc/n-hexane [1:19, 12 column volumes
16 6.4 5.9 5.5 5.5 (CVs) and 1:4, 5 CVs)], followed by CH3OH, to yield 18
paclitaxel (nM) 6.3 10.7 7.4 4531
subfractions. Subfraction 4 (50.6 mg) was purified by reversed-
a
phase MPLC (KP-C18-HS 12 g) with 60% acetone/H2O, reversed-
A549 (lung carcinoma), MDA-MB-231 (triple-negative breast phase preparative TLC with CH3OH and 90% acetone/H2O, and
cancer), KB (identical to AV-3 as a cervical carcinoma HeLa preparative TLC with toluene to yield 1 (0.5 mg) and acropyrone (6,
derivative), KB-VIN (P-gp-overexpressing MDR subline of KB). 1.6 mg). Subfractions 6−8 were combined (114 mg), then were
b
Antiproliferative activity expressed as IC50 values for each cell line, purified by Si gel MPLC (SNAP Ultra 10 g) with toluene and
the concentration of compound that caused 50% reduction relative to preparative TLC with 10% EtOAc/toluene, to yield 2 (9.8 mg).
untreated cells determined by the SRB assay. cNot determined. Subfractions 10−12 were combined (57.1 mg), then were purified by
preparative TLC with 10% EtOAc/toluene to yield acrovestone (4,
11.5 mg). Subfractions 13−15 were combined (97.8 mg) and then
compounds might be important for the antiproliferative purified by reversed-phase MPLC (KP-C18-HS 12 g), with an
activity. The comparison between 12 and 15 as well as 13 acetone/H2O stepwise gradient system [50%, 70%, 90%, and 100%; 6
and 16 indicated that a pendant isobutyl moiety is not crucial CVs each], reversed-phase preparative TLC with 80% acetone/H2O,
and preparative TLC with 5% acetone/toluene, to yield acrovestenol
for this type of biological activity. (5, 2.9 mg). Subfractions 16 and 17 were combined (72.0 mg) and
In this study, eight prenylated acetophenone dimers were were purified by Si gel MPLC (SNAP Ultra 10 g) with toluene
isolated, including three new compounds, acrotrifoliolane A followed by 10% EtOAc/toluene, reversed-phase preparative TLC
(1), acrofolione C (2), and acrofolional (3), from a rainforest with 80% acetone/H2O, and preparative TLC with 20% acetone/n-
plant, A. trifoliolata. Their chemical structures were charac- hexane, to yield acropyranol A (7, 0.5 mg) and acropyranol B (8, 1.1
terized by spectroscopic and spectrometric data. All com- mg). Subfraction 18 (251 mg) was purified by reversed-phase MPLC
pounds were isolated as racemates similarly to acetophenone (KP-C18-HS 12 g) with a CH3OH/H2O stepwise gradient system
monomers and dimers purified from other Acronychia species. [70%, 80%, 90%, 100%; 6 CVs each], preparative TLC with 50%
The total synthesis of 3 was accomplished to confirm its EtOAc/n-hexane, reversed-phase preparative TLC with acetone/
structure. Finally, the antiproliferative activities of isolated H2O/CH3COOH (80:19:1), and preparative TLC with 25% EtOAc/
toluene, to yield 3 (0.5 mg).
compounds were also evaluated.

■
Acrotrifoliolane A (1): yellow, amorphous solid; [α]22D +0.24 (c
0.03, CH3OH); IR νmax (CHCl3) 3396, 2956, 2927, 1739, 1613, 1455,
EXPERIMENTAL SECTION 1416, 1366, 1268; 1192, 1109, 1038, 978 cm−1; 1H and 13C NMR,
General Experimental Procedures. Optical rotations were Table 1; FABMS m/z 637, HRFABMS m/z 637.3744 [M + H]+
recorded on a JASCO P-2200 digital polarimeter. Infrared spectra (calcd for C38H53O8, 637.3740).
(IR) were measured with a Shimadzu FTIR-8700 instrument for Acrofolione C (2): yellow, amorphous solid; [α]21D −2.77 (c 0.06,
samples in CHCl3. NMR spectra were taken on a JEOL JMN- CH3OH); IR νmax (CHCl3) 3260, 2956, 2929, 2868, 1615, 1454,
ECA600 spectrometer and JMN-ECS400 spectrometer with tetrame- 1366, 1321; 1270, 1190, 1097, 975 cm−1; 1H and 13C NMR, Table 1;
thylsilane as an internal standard, and chemical shifts are expressed as FABMS m/z 571, 319; HRFABMS m/z 571.2900 [M + H]+ (calcd
δ values. High-resolution mass spectrometric data were obtained on a for C32H43O9, 571.2907).
JEOL JMS-700 (FAB) or a JMS-T100TD (DART) mass Acrofolional (3): yellow, amorphous solid; [α]17D +0.48 (c 0.03,
spectrometer. Analytical and preparative TLC was carried out on CHCl3); IR νmax (CHCl3) 3366, 2956, 2925, 1614, 1469, 1367; 1320,
precoated silica gel 60F254 and RP-18F254 plates (0.25 or 0.50 mm 1214, 1100, 974 cm−1; 1H NMR, Table 1; FABMS m/z 515, 319;
thickness; Merck). MPLC was performed with silica gel and C18 HRFABMS m/z 515.2247 [M + H]+ (calcd for C28H35O9, 515.2281).
cartridges (Biotage, Uppsala, Sweden). 1-{3-[1-(3-Acetyl-2,4,6-trihydroxyphenyl)-3-methylbutyl]-2,4-di-
Plant Material. The bark of Acronychia trifoliolata was collected in hydroxy-6-methoxy-5-(3-methylbut-2-en-1-yl)phenyl}ethanone
Bali, Indonesia, at an altitude of 1200 m on July 5, 1994, by Dr. Djaja (12). To a solution of 10 (32.4 mg, 0.13 mmol) in anhydrous toluene
D. Soejarto and identified by the taxonomist, A. McDonald. A (1.0 mL) was added anhydrous AlCl3 (2.7 mg, 0.02 mmol) at room
voucher specimen (#U44Z-4773) is housed at the Smithsonian temperature. After stirring for 15 min, isovaleraldehyde (14 μL, 0.13

E DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

mmol) was added and refluxed for 1 h. After consumption of 10 54%) as a pale yellow oil along with the recovered starting material 16
(TLC), the mixture was cooled to room temperature and 9 (22.1 mg) (6.2 mg, 42%): 1H NMR (600 MHz, CDCl3) δ 15.5 (2H, s), 9.58
was added. After refluxing for 9 h, the stirring was continued for 16 h (2H, brs), 5.21−5.19 (2H, m), 3.85 (2H, s), 3.71 (6H, s), 3.30 (4H,
at 80 °C. The mixture was extracted with.CH2Cl2, and the organic d, J = 6.6 Hz), 2.71 (6H, s), 1.77 (6H, s), 1.69 (6H, s); 13C NMR
layer was washed with brine, dried over Na2SO4, and concentrated in (150 MHz, CDCl3) δ 204.0, 162.2, 160.3, 160.1, 131.9, 122.9, 116.3,
vacuo. The residue was chromatographed on silica gel with EtOAc/n- 109.4, 108.2, 62.7, 30.6, 25.7, 23.0, 18.0, 16.4; HRFABMS m/z
hexane (1:5−1:2) to afford the target compounds 12 (11.7 mg, 19%) 513.2468 [M + H]+ (calcd for C29H37O8, 513.2488).
and 13 (8.8 mg, 22%) as pale yellow oils along with recovered starting Antiproliferative Activity Assay. The compounds were
material 10 (3.5 mg, 11%): [α]21D +1.74 (c 0.30, MeOH); 1H NMR evaluated for antiproliferative activity using the sulforhodamine B
(600 MHz, CDCl3) δ 15.7 (1H, brs), 15.6 (1H, brs), 9.95 (1H, brs), (SRB) assay, as described previously.11 Briefly, test compounds were
9.15 (1H, brs), 5.88 (1H, brs), 5.21−5.18 (1H, m), 4.71 (1H, t, J = added to freshly harvested cells seeded on 96-well microtiter plates at
7.8 Hz), 3.71 (3H, s), 3.30 (2H, brs), 2.71 (3H, s), 2.68 (3H, s), 2.24 a density of 4000−11 000 cells per well and cultured for 72 h. Cells
(1H, brs), 2.08 (1H, brs), 1.77 (3H, s), 1.70 (3H, s), 1.41 (1H, 7, J = were fixed in 10% trichloroacetic acid and then stained with 0.04%
6.0 Hz), 0.88−0.87 (6H, m); 13C NMR (100 MHz, CDCl3) δ 204.2, SRB. The absorbance of Tris base-solubilized protein-bound dye (10
160.2, 122.9, 62.7, 32.5, 30.7, 28.2, 27.0, 25.7, 23.1, 22.7, 22.4, 18.0; mM) was measured at 515 nm using a microplate reader (ELx800,
HRFABMS m/z 486.2210 [M]+ (calcd for C27H34O8, 486.2254). BioTek) operated by Gen5 software (BioTek). No less than three
Synthesis of Acrofolional (3). To a solution of dimethylforma- independent experiments in duplicate (n = 6) were conducted, and
mide (DMF, 0.1 mL) in anhydrous CH2Cl2 (0.18 mL) was added IC50 data were calculated statistically (MS Excel). The cells were
POCl3 (0.12 mL) at 0 °C under Ar, and the mixture was stirred for 20 cultured in RPMI-1640 medium supplemented with 2 mM L-
min to generate the Vilsmeier reagent. A solution of 12 (6.5 mg, 0.01 glutamine and 25 mM HEPES (Corning), containing 10% fetal
mmol) in anhydrous CH2Cl2 (0.6 mL) was added to the Vilsmeier bovine serum (Specialty Media), 100 μg/mL streptomycin, and 100
reagent (0.05 mL) at 0 °C under Ar and stirred for 1 h. The mixture IU penicillin (Corning). KB-VIN stock cells, provided by Professor
was quenched with water (1.0 mL), and stirring was continued for 30 Y.-C. Cheng, Yale University, were maintained in the presence of 100
min at room temperature. The mixture was extracted three times with nM vincristine. The remaining human tumor cell lines in this study
EtOAc, and the combined organic layers were washed with brine, were obtained from the Lineberger Comprehensive Cancer Center
dried over Na2SO4, and concentrated in vacuo. The crude product (UNC-CH) or from ATCC (Manassas, VA, USA). The experimental
control compound used was paclitaxel.

■
was purified using preparative TLC with EtOAc to afford 3 (1.9 mg,
28%) as a yellow, amorphous solid: [α]21D −0.48 (c 0.08, CHCl3); 1H
NMR (400 MHz, CDCl3) δ 17.1 (1H, brs), 16.1 (1H, brs), 14.5 (1H, ASSOCIATED CONTENT
brs), 10.2 (1H, brs), 5.20−5.17 (1H, m), 4.70 (1H, t, J = 10.4 Hz), *
S Supporting Information
3.73 (3H, s), 3.30 (2H, brs), 2.74 (3H, s), 2.73 (3H, s), 1.77 (3H, s), The Supporting Information is available free of charge on the
1.70 (3H, s), 1.44−1.36 (1H, m), 0.88 (6H, brs); 13C NMR (150
ACS Publications website at DOI: 10.1021/[Link]-
MHz, CDCl3) δ 193.5, 62.7, 32.3, 29.7, 26.9, 25.7, 22.5, 18.0;
HRFABMS m/z 515.2234 [M + H]+ (calcd for C28H35O9, 515.2281). prod.9b00596.
1-{3-[(Dimethylamino)methyl]-2,4-dihydroxy-6-methoxy-5-(3- NMR spectra and HFFABMS data for 1−3, NCI60
methylbut-2-en-1-yl)phenyl}ethenone (14). To a solution of 10 panel screening data of plant extract, and 1H NMR of
(281.9 mg, 1.13 mmol) in CHCl3 (20 mL) was added Eschenmoser’s synthesized 3 (PDF)

■
salt (289.9 mg, 1.57 mmol), and the mixture was refluxed for 30 min
under N2. The mixture was cooled to room temperature, quenched
with water (20 mL), and then extracted with CHCl3. The combined AUTHOR INFORMATION
organic layers were washed with brine, dried over Na2SO4, and Corresponding Author
concentrated in vacuo. The residue was chromatographed on silica gel *Tel: +81-76-264-6305. E-mail: kngoto@[Link].
with EtOAc/n-hexane (1:2−10:0) to afford 14 (332.2 mg, 96%) as a
pale yellow oil: 1H NMR (600 MHz, CDCl3) δ 13.8 (1H, brs), 5.21− ORCID
5.19 (1H, m), 3.79 (2H, s), 3.72 (3H, s), 3.27 (2H, d, J = 6.0 Hz), Masuo Goto: 0000-0002-9659-1460
2.67 (3H, s), 2.38 (6H, s), 1.77 (3H, s), 1.70 (3H, s); 13C NMR (150 Barry R. O’Keefe: 0000-0003-0772-4856
MHz, CDCl3) δ 203.0, 166.3, 161.6, 160.7, 131.5, 123.5, 114.7, 107.4, Kuo-Hsiung Lee: 0000-0002-6562-0070
103.5, 62.6, 55.1, 44.2, 30.8, 25.7, 22.5, 17.9; HRFABMS m/z Kyoko Nakagawa-Goto: 0000-0002-1642-6538
307.1798 [M]+ (calcd for C17H25NO4, 307.1784).
1,1′-{(3-Methylbutane-1,1-diyl)bis[2,4-dihydroxy-6-methoxy-5- Notes
(3-methylbut-2-en-1-yl)-3,1-phenylene]}bis(ethanone) (15). To a The authors declare no competing financial interest.
solution of 14 (32.1 mg, 0.10 mmol) in toluene (7.0 mL) were added
9 (32.3 mg, 0.19 mmol) and toluene (8.0 mL) at room temperature,
and the mixture was refluxed for 1.0 h under N2. The mixture was
cooled to room temperature and concentrated in vacuo. The residue
■ ACKNOWLEDGMENTS
We appreciate critical comments, suggestions, and editing on
was chromatographed on silica gel with EtOAc/n-hexane (1:6−1:4− the manuscript by Dr. S. L. Morris-Natschke (UNC-CH). This
1:2) to afford the target 15 (14.2 mg, 32%) as a pale yellow solid, study was supported by a Grant-in-Aid from the Ministry of
along with the recovered starting material 14 (10.3 mg, 32%): 1H Education, Culture, Sports, Science and Technology (MEXT
NMR (600 MHz, CDCl3) δ 9.36 (2H, brs), 5.91 (1H, s), 5.21−5.19 KAKENHI, Japan), awarded to K.N.G. (grant number
(1H, m), 3.79 (2H, s), 3.71 (3H, s), 3.30 (2H, d, J = 5.4 Hz), 2.71 25293024). This work was also supported by NIH grant
(3H, s), 2.69 (3H, s), 2.67 (3H, s), 1.78 (3H, s), 1.70 (3H, s); 13C CA177584 from the National Cancer Institute awarded to K.-
NMR (150 MHz, CDCl3) δ 204.2, 203.9, 160.3, 159.9, 132.1, 122.7, H.L., as well as partial support from the Eshelman Institute for
116.2, 109.8, 108.3, 96.5, 62.7, 32.4, 30.6, 25.7, 23.0, 18.0, 15.9; Innovation (Chapel Hill, NC, USA) awarded to M.G. This
HRFABMS m/z 431.1705 [M + H]+ (calcd for C23H27O8, 431.1706). project has been funded in whole or in part with federal funds
1,1′-[Methylenebis(2,4-dihydroxy-6-methoxy-5-(3-methylbut-2-
en-1-yl)-3,1-phenylene)]bis(ethanone) (16). A solution of 14 (15.2 from the National Cancer Institute, National Institutes of
mg, 0.05 mmol) and 10 (14.9 mg, 0.06 mmol) in toluene (2.0 mL) Health (NIH), under contract HHSN261200800001E. The
was refluxed for 18 h. The mixture was cooled to room temperature content of this publication does not necessarily reflect the
and concentrated in vacuo. The residue was purified using preparative views or policies of the Department of Health and Human
TLC, with EtOAc/n-hexane (1:3) as eluent, to afford 16 (13.7 mg, Services, nor does the mention of trade names, commercial
F DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX
Journal of Natural Products Article

products, or organizations imply endorsement by the U.S.

Government. This project has also been supported in part by
the Intramural Research Program of the NIH, National Cancer
Institute, Center for Cancer Research.

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G DOI: 10.1021/[Link].9b00596
J. Nat. Prod. XXXX, XXX, XXX−XXX