Isolation and synthesis of a new aromatic compound, brefelamide, from Dictyostelium cellular slime molds and its inhibitory effect on the proliferation of astrocytoma cells

Article abstract of DOI:10.1021/jo051352x

We have explored the diversity of secondary metabolites produced by cellular slime molds to examine the possible use of such cellular slime molds as a resource for novel drug development. A new aromatic amide, brefelamide (1), was isolated from methanol e

Full text of DOI:10.1021/jo051352x

Isolation and Synthesis of a New Aromatic Compound,
Brefelamide, from Dictyostelium Cellular Slime Molds and Its
Inhibitory Effect on the Proliferation of Astrocytoma Cells
Haruhisa Kikuchi,† Yoshinori Saito,† Jun’ichi Sekiya,† Yumiko Okano,† Masaki Saito,†
Norimichi Nakahata,† Yuzuru Kubohara,‡ and Yoshiteru Oshima*,†
Graduate School of Pharmaceutical Sciences, Tohoku University, Aoba-yama, Aoba-ku,
Sendai 980-8578, Japan, and Institute for Molecular and Cellular Regulation, Gunma University,
Maebashi 371-8512, Japan
oshima@mail.pharm.tohoku.ac.jp
Received June 29, 2005
We have explored the diversity of secondary metabolites produced by cellular slime molds to examine
the possible use of such cellular slime molds as a resource for novel drug development. A new
aromatic amide, brefelamide (1), was isolated from methanol extracts of the fruiting bodies of
Dictyostelium brefeldianum and D. giganteum. The structure of 1 was determined by spectral means
1
including EIMS and H and 13C NMR. The total synthesis of 1 was carried out to confirm the
structure and obtain sufficient samples for performing biological evaluation. Interestingly, compound
1 inhibited the cellular proliferation of 1321N1 human astrocytoma cells.
Introduction
been reported as development-regulating substances of
cellular slime molds. DIF-1 was also found to inhibit
proliferation and induce differentiation in mammalian
leukemia cell lines.5 Furthermore, an intriguing experi-
mental result suggesting that DIF-1 may induce dif-
ferentiation of vascular smooth muscle cells in vitro could
lead to novel strategies for the treatment of vascular
diseases.6 A chlorine-substituted aromatic compound,
AB0022A, was also isolated as an antibacterial sub-
stance.7 However, except for these compounds, there have
been no reports on secondary metabolites of cellular slime
molds. We have focused on the utility of cellular slime
molds as a resource for novel drug development and have
studied the diversity of cellular slime mold secondary
metabolites as well as their physiological and pharma-
cological activities. We have isolated R-pyronoids and
aromatics with unique structures;8,9 these compounds
The cellular slime mold Dictyostelium discoideum is
thought to be an excellent model organism for the study
of cell and developmental biology because of its simple
pattern of development.1 Vegetative cells of D. discoi-
deum grow as single amoebae by eating bacteria; how-
ever, when starved, they start a developmental program
of morphogenesis and gather to form a slug-shaped
multicellular aggregate. This aggregate then differenti-
ates into two distinct cell types, prespore and prestalk
cells, which are precursors of spores and stalk cells,
respectively. At the end of its development, the aggregate
forms a fruiting body consisting of spores and a multi-
cellular stalk.
Several small molecules including differentiation-
inducing factors (DIFs),2 discadenine,3 and cAMP4 have
* To whom correspondence should be addressed. Phone: +81-22-
795-6822. Fax: +81-22-795-6821.
(4) Konijn, T. M.; van de Meene, J. G.; Bonner, J. T.; Barkley, D. S.
Proc. Natl. Acad. Sci. U.S.A. 1976, 58, 1152-1154.
† Tohoku University.
‡ Gunma University.
(5) (a) Asahi, K.; Sakurai, A.; Takahashi, N.; Kubohara, Y.; Okamoto,
K.; Tanaka, Y. Biochem. Biophys. Res. Commun. 1995, 208, 1036-
1039. (b) Kubohara, Y. Biochem. Biophys. Res. Commun. 1997, 236,
418-422. (c) Kubohara, Y.; Kimura, C.; Tatemoto, K. Dev., Growth
Differ. 1995, 37, 711-716. (d) Kubohara, Y.; Saito, Y.; Tatemoto, K.
FEBS Lett. 1995, 359, 119-122.
(1) Dictyostelium, A Model System for Cell and Developmental
Biology; Frontiers Science Series no. 21; Maeda, Y., Inoue, K.,
Takeuchi, I., Eds.; Universal Academy Press, Inc.: Tokyo, 1997.
(2) (a) Morris, H. R.; Taylor, G. W.; Masento, M. S.; Jermyn, K. A.;
Kay, R. R. Nature 1987, 328, 811-814. (b) Morris, H. R.; Masento, M.
S.; Taylor, G. W.; Jermyn, K. A.; Kay, R. R. Biochem. J. 1988, 249,
903-906.
(6) Miwa, Y.; Sasaguri, T.; Kosaka, C.; Taba, Y.; Ishida, A.; Abumiya,
T.; Kubohara, Y. Circ. Res. 2000, 86, 68-75.
(7) Sawada, T.; Aono, M.; Asakawa, S.; Ito, A.; Awano, K. J. Antibiot
(Tokyo) 2000, 53, 959-966.
(3) Abe, H.; Uchiyama, M.; Tanaka, Y.; Saito, H. Tetrahedron Lett.
1976, 42, 3807-3810.
10.1021/jo051352x CCC: $30.25 © 2005 American Chemical Society
Published on Web 09/03/2005
8854
J. Org. Chem. 2005, 70, 8854-8858

Isolation and Synthesis of Brefelamide
TABLE 1. 13C and 1H NMR Spectral Data of
Brefelamide (1)a
13C
1H
1
126.4b
130.2
116.1
162.1
170.2
37.1
2,6
3,5
4
7.67 (2H, d, J ) 8.9 Hz)
6.80 (2H, d, J ) 8.9 Hz)
1′
3′
3.73 (2H, t, J ) 6.8 Hz)
3.30 (2H, t, J ) 6.8 Hz)
4′
39.9
5′
202.5
121.8
144.1
147.4
121.8
115.4
126.5b
150.6
121.0
117.2
154.9
1′′
2′′
3′′
4′′
6.77 (1H, dd, J ) 7.8, 1.3 Hz)
6.52 (1H, dd, J ) 8.2, 7.8 Hz)
7.57 (1H, dd, J ) 8.2, 1.3 Hz)
5′′
6′′
1′ ′′
2′ ′′,6′ ′′
3′ ′′,5′ ′′
4′ ′′
6.84 (2H, d, J ) 9.1 Hz)
6.76 (2H, d, J ) 9.1 Hz)
a 600 MHz for 1H and 150 MHz for 13C in CD3OD. b These
signals were indistinguishable.
FIGURE 1. Structure elucidation of brefelamide (1).
rings (δ 7.67 and 6.80 (each 2H, d, J ) 8.9 Hz) (Figure
1A), and δ 6.84 and 6.76 (each 2H, d, J ) 9.1 Hz)) (Figure
1C), and an ethylene group (δ 3.73 and 3.30 (each 2H, t,
J ) 6.8 Hz)). HMBC correlations for H-3 and H-3′ to C-1′
and H-3′, H-4′, and H-6" to C-5′ suggested that benzene
rings A and B were linked by an amide-containing unit
C1′-C5′ (Figure 1D). An amino group attached to a
benzene ring moves the chemical shifts of ortho and para
protons further to the upper field than a hydroxy or
alkoxy group. Thus, H-5" (δ 6.52) and H-4" (δ 6.77)
indicated that an amino group and a hydroxy or alkoxy
group were bonded at C-2′′ and C-3′′, respectively.
Fragment ion peaks at m/z 228 and 121 in EIMS
determined the place of benzene ring C, yielding the
structure of 1 (Figure 1E).
have demonstrated physiological activities such as control
of cellular slime mold development.10,11 This paper reports
the structure elucidation and synthesis of a new aromatic
compound, brefelamide (1). The inhibitory effect of 1 on
the proliferation of 1321N1 human astrocytoma cells is
also described.
Results and Discussion
Synthesis. The total synthesis of 1 was carried out to
confirm the structure and obtain a sufficient quantity of
sample for performing several biological evaluations
(Scheme 1). An SNAr reaction of commercially available
2-fluoronitrobenzene (2) and 4-(benzyloxy)phenol (3) with
cesium carbonate produced diaryl ether 4. Reduction of
4 to 5 was performed using an NaBH4-SnCl2 system12
in good yield. 7-Aryloxytryptamine 8 was synthesized by
modified Abramovitch-Shapiro tryptamine synthesis.13,14
The reaction of diazotized 5 with 2-piperidone-3-carbox-
ylic acid13 formed hydrazone 6, which was cyclized by
warming with formic acid to yield tetrahydro-â-carboline
7. Following debenzylation of 7, alkaline hydrolysis and
acidic decarboxylation provided tryptamine 8. Compound
10 was produced by selective acylation with 4-(meth-
oxymethoxy)benzoic acid15 and protection of the phenolic
hydroxyl group of 8. Oxidative cleavage of the indole
ring16 in 10 by NaIO4 and subsequent acidic hydrolysis
allowed us to complete the synthesis of brefelamide (1).
All of the spectral data on synthetic 1 were identical to
Isolation and Structure Elucidation. Fruiting bod-
ies (wet weight 335 g) of the cellular slime mold D.
brefeldianum were cultured on plates and extracted three
times with methanol at room temperature to yield an
extract (12 g), which was partitioned with ethyl acetate
and water. The ethyl acetate solubles (2.4 g) were
separated by repeated column chromatography over SiO2
and ODS to yield 1 (2.1 mg) as a yellow oil. Compound 1
(0.8 mg) was obtained from the fruiting bodies (wet
weight 411 g) of D. giganteum in a similar manner.
HREIMS (m/z 392.1396) and 1H and 13C NMR spectra
indicated that the molecular formula of 1 was C22H20N2O5
(Table 1). The 13C NMR spectrum showed 16 sp2 carbon
signals (>CdO × 2, dC-O (or N) × 5, >Cd × 2, and
-CHd × 7) and two sp3 methylene carbon signals,
indicating the presence of symmetrical benzene rings.
The 1H NMR spectrum indicated the presence of a 1,2,3-
trisubstituted benzene ring (δ 7.57 (1H, dd, J ) 8.2, 1.3
Hz), 6.77 (1H, dd, J ) 7.8, 1.3 Hz), and 6.52 (1H, dd, J )
8.2, 7.8 Hz)) (Figure 1B), two 1,4-disubstituted benzene
(12) Satoh, T.; Mitsuo, N.; Nishiki, M.; Inoue, Y.; Ooi, Y. Chem.
Pharm. Bull. 1981, 29, 1443-1445.
(13) So´ti, F.; Incze, M.; Kardos-Balogh, Z.; Kajta´r-Peredy, M.;
Sza´ntay C. Synth. Commun. 1993, 23, 1689-1698.
(14) Abramovitch, R. A.; Shapiro, D. J. Chem. Soc. 1956, 4589-
4592.
(15) Inouye, M.; Chiba, J.; Nakazumi, H. J. Org. Chem. 1999, 64,
8170-8176.
(16) Dolby, L. J.; Booth, D. L. J. Am. Chem. Soc. 1966, 88, 1049-
1051.
(8) Takaya, Y.; Kikuchi, H.; Terui, Y.; Komiya, J.; Furukawa, K.;
Seya, K.; Motomura, S.; Ito, A.; Oshima, Y. J. Org. Chem. 2000, 65,
985-989.
(9) Takaya, Y.; Kikuchi, H.; Terui, Y.; Komiya, J.; Maeda, Y.; Ito,
A.; Oshima, Y. Tetrahedron Lett. 2001, 42, 61-63.
(10) Maeda, Y.; Kikuchi, H.; Sasaki, K.; Amagai, A.; Sekiya, J.;
Takaya, Y.; Oshima, Y. Protoplasma 2003, 221, 185-192.
(11) Kikuchi, H.; Sasaki, K.; Sekiya, J.; Maeda, Y.; Amagai, A.;
Kubohara, Y.; Oshima, Y. Bioorg. Med. Chem. 2004, 12, 3203-3214.
J. Org. Chem, Vol. 70, No. 22, 2005 8855

Kikuchi et al.
SCHEME 1. Synthesis of Brefelamide (1)a
a Reagents and conditions: (a) Cs2CO3, DMSO, 80 °C (77%); (b) SnCl2‚2H2O, NaBH4, EtOH-EtOAc (3:1), 60 °C (87%); (c) (1) NaNO2,
4.5 M HCl-DMF (2:1), 0 °C; (2) 2-piperidone-3-carboxylic acid, AcONa, 0 °C (68%); (d) HCOOH, DMF, 50 °C (56%); (e) (1) H2, Pd(OH)2/C,
EtOAc-EtOH (1:1), rt; (2) 1 M KOH-EtOH (17:2), reflux; (3) concd H2SO4, reflux (53%); (f) 4-(methoxymethoxy)benzoic acid, WSC, DMF,
0 °C (65%); (g) MOMCl, N,N-diisopropylethylamine, CH2Cl2, 0 °C (52%); (h) NaIO4, H2O-MeCN (1:1), 60 °C (75%); (i) TFA, CH2Cl2, 0 °C
(67%).
FIGURE 2. Effects of brefelamide (1) on the proliferation of
1321N1 human astrocytoma cells. 1321N1 Human astrocy-
toma cells were incubated with various concentrations of 1 for
2 days in DMEM containing 5% FCS. MTT assays were
performed to determine the number of live cells. Each point
represents the mean ( SE from three determinations.
those of the natural compound, confirming the proposed
structure of 1 in Figure 1.
Biological Evaluation. Brefelamide (1) inhibited the
FIGURE 3. Effects of brefelamide (1) on the cell-cycle of
cellular proliferation of 1321N1 human astrocytoma cells
1321N1 human astrocytoma cells. 1321N1 Human astrocy-
in a concentration-dependent manner with an EC50 value
of approximately 10 µM (Figure 2). Flow cytometric
analysis demonstrated that treatment with 1 resulted in
cell-cycle inhibition through the accumulation of cells in
G2/M phase (Figure 3). These results suggest that 1
causes G2/M phase arrest and inhibits proliferation,
similar to asiatic acid17 and isoliquiritigenin.18
toma cells were incubated with 10 µM of 1 for 2 days in DMEM
containing 5% FCS. Cells were washed with PBS and fixed in
cold 70% ethanol for 1-2 h at 4 °C. Fixed cells were incubated
with RNase A for 20 min at 37 °C and stained with propidium
iodide for 10 min at room temperature. Cell-cycle profiles were
examined by flow cytometry.
afford 3-hydroxykynurenamine. This compound may be
linked with p-hydroxybenzoic acid and hydroquinone to
give 1. Compounds similar to 1 have been only rarely
described, as with erebusinone (12), a simple acetamide
Conclusions
We presume that brefelamide (1) is biosynthesized
from tryptophan (Scheme 2). 3-Hydroxykynurenine, a
known tryptophan metabolite,19 is decarboxylated to
(17) Hsu, Y. L.; Kuo, P. L.; Lin, L. T.; Lin, C. C. J. Pharmacol. Exp.
Ther. 2005, 313, 333-344.
(18) Hsu, Y. L.; Kuo, P. L.; Lin, C. C. Life Sci. 2005, 77, 279-292.
(19) Kaseda, H.; Noguchi, T.; Kido, R. J. Biochem. (Tokyo) 1973,
74, 127-133.
8856 J. Org. Chem., Vol. 70, No. 22, 2005

Isolation and Synthesis of Brefelamide
washed with H2O and brine, dried over sodium sulfate, and
evaporated. The residue was chromatographed over SiO2 and
eluted by n-hexane-EtOAc (39:1) to give 4 (12.4 g, 38.6 mmol,
77%). Data for 4: yellowish amorphous solid; 1H NMR (CDCl3,
600 MHz) δ 7.91 (1H, dd, J ) 8.1, 1.8 Hz), 7.44 (1H, td, J )
8.4, 1.8 Hz), 7.43 (2H, d, J ) 7.3 Hz), 7.39 (2H, t, J ) 7.3 Hz),
7.33 (1H, t, J ) 7.3 Hz), 7.12 (1H, ddd, J ) 8.4, 8.1, 1.1 Hz),
7.01 (2H, d, J ) 9.2 Hz), 6.98 (2H, d, J ) 9.2 Hz), 6.92 (1H,
dd, J ) 8.4, 1.1 Hz), 5.05 (2H, s); 13C NMR (CDCl3, 150 MHz)
δ 155.9, 151.8, 148.9, 140.7, 136.7, 134.0, 128.6 (2C), 128.1,
127.5 (2C), 125.7, 122.3, 121.1 (2C), 119.1, 116.2 (2C), 70.5;
EIMS m/z 321 [M]+, 91 (base); HREIMS m/z 321.1009 (321.1000
calcd for C19H15NO4).
SCHEME 2. Plausible Biosynthetic Pathway for
Brefelamide (1)
2-(4-Benzyloxyphenoxy)aniline (5). A solution of 4 (12.4
g, 38.6 mmol) and tin(II) chloride dihydrate (40.7 g, 180 mmol)
in 100 mL of EtOH-EtOAc (3:1) was heated at 60 °C for 1 h.
Sodium borohydride (730.1 mg, 19.3 mmol) in EtOH (10 mL)
was added to this solution over a period of 1 h with stirring at
60 °C. After being stirred for further 1 h, the reaction mixture
was cooled to room temperature and poured into H2O (20 mL).
The solvent was evaporated off, and the aqueous solution was
alkalinized with 1 M NaOH and then extracted with EtOAc
three times. The organic layers were combined, washed with
H2O and brine, dried over sodium sulfate, and evaporated. The
residue was chromatographed over SiO2 eluted by n-hexanes-
EtOAc (24:1) to give 5 (9.81 g, 33.7 mmol, 87%). Data for 5:
brownish amorphous solid; 1H NMR (CDCl3, 600 MHz) δ 7.42
(2H, d, J ) 7.3 Hz), 7.38 (2H, t, J ) 7.3 Hz), 7.32 (1H, t, J )
7.3 Hz), 6.93 (1H, ddd, J ) 7.9, 7.3, 1.5 Hz), 6.92 (4H, s), 6.79
(1H, dd, J ) 7.9, 1.5 Hz), 6.78 (1H, dd, J ) 8.0, 1.5 Hz), 6.67
(1H, ddd, J ) 8.0, 7.3, 1.5 Hz), 5.02 (2H, s), 3.81 (2H, br.s);
13C NMR (CDCl3, 150 MHz) δ 154.6, 151.0, 144.4, 138.1, 137.0,
128.6 (2C), 127.9, 127.5 (2C), 124.1, 118.9 (2C), 118.8, 118.6,
116.2, 115.9 (2C), 70.5; EIMS m/z 291 [M]+, 200, 91 (base);
HREIMS m/z 291.1258 (291.1258 calcd for C19H17NO2).
3-((2-(4-Benzyloxyphenoxy)phenyl)hydrazono)piper-
idin-2-one (6). 3-Carbethoxy-2-piperidone (6.07 g, 35.5 mmol)
was dissolved in 1 M KOH (40 mL) and stirred at room
temperature for 10 h. Then, 6 M HCl (4.0 mL) was added
dropwise to this solution at 0 °C. The solution was added
dropwise to a solution of diazotized 5, which was prepared
according to the next procedure. A solution of sodium nitrite
(2.49 g, 36.1 mmol) in H2O (9 mL) was added to a solution of
5 (9.53 g, 32.7 mmol) in 4.5 M HCl (67 mL) and DMF (33 mL)
during 10 min at 0 °C and stirred for an additional 15 min.
After combining the above solutions, sodium acetate (13.4 g,
164 mmol) in H2O (32 mL) was added (pH ∼4.5). The reaction
mixture was stirred at 0 °C for 5 h and extracted with EtOAc
three times. The organic layers were combined, washed with
H2O and brine, dried over sodium sulfate, and evaporated. The
residue was chromatographed over SiO2 eluted by n-hexanes-
EtOAc (7:3) to give 6 (8.96 g, 22.3 mmol, 68%). Data for 6:
yellowish brown amorphous solid; 1H NMR (CDCl3, 600 MHz)
δ 13.12 (1H, s), 7.60 (1H, d, J ) 7.9 Hz), 7.40 (2H, d, J ) 7.3
Hz), 7.36 (2H, t, J ) 7.3 Hz), 7.30 (1H, t, J ) 7.3 Hz), 7.04
(1H, td, J ) 7.9, 2.4 Hz), 6.94 (2H, d, J ) 9.2 Hz), 6.88 (2H, d,
J ) 9.2 Hz), 6.79 (1H, d, J ) 2.6 Hz), 6.76-6.80 (2H, m), 4.98
(2H, s), 3.17 (2H, td, J ) 6.0, 2.6 Hz), 2.64 (2H, t, J ) 6.0 Hz),
1.85 (2H, quint, J ) 6.0 Hz); 13C NMR (CDCl3, 150 MHz) δ
164.1, 154.6, 150.8, 143.5, 137.0, 135.6, 128.5 (2C), 127.8,
127.4, 127.4 (2C), 124.0, 120.4, 119.3 (2C), 118.2, 115.7 (2C),
113.0, 70.4, 41.6, 31.0, 22.5; EIMS m/z 401 [M]+ (base), 200,
91; HREIMS m/z 401.1743 (401.1738 calcd for C24H23N3O3).
of 3-hydroxykynurenamine.20 The isolation of novel classes
of compounds such as DIF-1,2 dictyopyrones,8 and 1
shows that cellular slime molds are promising sources
in natural product chemistry.
In addition, brefelamide (1) showed inhibitory effects
on the proliferation of 1321N1 human astrocytoma cells
caused by G2/M phase arrest. This result indicates that
1 may be utilized as a novel lead compound for anticancer
agents.
Experimental Section
Isolation of Brefelamide (1). The cultured fruiting bodies
(wet weight 335 g) of D. brefeldianum were extracted three
times with MeOH at room temperature to give an extract (12.0
g), which was then partitioned with EtOAc and H2O to yield
EtOAc solubles (2.4 g). The EtOAc solubles were chromato-
graphed over SiO2, and the column was eluted with n-hex-
anes-EtOAc solutions with increasing polarity. n-Hexanes-
EtOAc (1:3) eluent (130 mg) was further chromatographed over
SiO2 using CHCl3-MeOH (49:1) to give crude brefelamide. The
crude sample was purified by ODS column chromatography
(H2O-MeOH (1:1)) to give brefelamide (1) (2.1 mg). In a
similar manner, the cultured fruiting bodies of D. giganteum
(wet weight 411 g) yielded 1 (0.8 mg). Data for 1: yellow oil;
1H NMR and 13C NMR data are shown in Table 1; EIMS m/z
392 [M]+ (48%), 374 (3%), 254 (90%), 228 (28%), 121 (100%);
HREIMS m/z 392.1396 (392.1372 calcd for C22H20N2O5).
2-(4-Benzyloxyphenoxy)nitrobenzene (4). To a solution
of 2-fluoronitrobenzene (2) (8.91 g, 63.2 mmol) and the 4-ben-
zyloxyphenol (3) (10.0 g, 50.0 mmol) in DMSO (100 mL) was
added cesium carbonate (24.5 g, 75.1 mmol) over 1 h. After
being stirred at 80 °C for 12 h, the reaction mixture was cooled
to room temperature, poured into H2O (250 mL), and extracted
with EtOAc three times. The combined organic layers were
8-(4-Benzyloxyphenoxy)-1,2,3,4-tetrahydro-â-carbolin-
1-one (7). To a solution of 5 (3.32 g, 8.27 mmol) in DMF (8.0
mL) was added formic acid (32 mL) dropwise. After being
stirred for 15 h at 50 °C, the reaction mixture was cooled to
room temperature and evaporated. The residue was chromato-
graphed over SiO2 eluted by n-hexane-CHCl3 (1:4) to give 7
(1.78 g, 4.64 mmol, 56%). Data for 7: brownish-orange
1
amorphous solid; H NMR (CDCl3, 600 MHz) δ 10.07 (1H, s),
(20) Moon, B.; Park, Y. C.; McClintock, J. B.; Baker, B. J. Tetrahe-
dron 2000, 56, 9057-9062.
7.37 (2H, d, J ) 7.3 Hz), 7.34 (2H, t, J ) 7.3 Hz), 7.29 (1H, t,
J. Org. Chem, Vol. 70, No. 22, 2005 8857

Kikuchi et al.
J ) 7.3 Hz), 7.28 (1H, dd, J ) 7.9, 0.7 Hz), 7.00 (1H, t, J ) 7.9
Hz), 6.97 (1H, t, J ) 2.5 Hz), 6.95 (2H, d, J ) 9.2 Hz), 6.84
(2H, d, J ) 9.2 Hz), 6.74 (1H, dd, J ) 7.9, 0.7 Hz), 4.91 (2H,
s), 3.60 (2H, td, J ) 7.1, 2.5 Hz), 2.96 (2H, t, J ) 7.1 Hz); 13C
NMR (CDCl3, 150 MHz) δ 163.1, 154.7, 150.5, 144.2, 136.9,
129.4, 128.4 (2C), 127.8, 127.6, 127.4 (2C), 126.7, 120.5, 120.0,
119.5 (2C), 115.7 (2C), 115.0, 112.1, 70.3, 41.8, 20.8; EIMS m/z
384 [M]+ (base), 293, 91; HREIMS m/z 384.1463 (384.1473
calcd for C24H20N2O3).
diisopropylethylamine (700 µL, 4.02 mmol) and chloromethyl
methyl ether (200 µL, 2.63 mmol) at 0 °C. The mixture was
gradually warmed to room temperature. After being stirred
for 12 h, it was poured into 0.2 M HCl and extracted with
EtOAc three times. The organic layers were combined, washed
with H2O and brine, dried over sodium sulfate, and evaporated.
The residue was chromatographed over SiO2 eluted by n-hex-
anes-EtOAc (2:1 and 1:1) to give 10 (399 mg, 0.838 mmol,
52%) and recovered 9 (256 mg, 0.592 mmol, 37%). Data for
1
3-(2-Aminoethyl)-7-(4-hydroxyphenoxy)indole (8). A
solution of 7 (2.99 g, 7.78 mmol) in EtOAc (28 mL) was added
to a suspension of palladium hydroxide (20 wt % Pd on carbon)
(598 mg) in ethanol (28 mL) and stirred under H2 atmosphere
for 4 h. The reaction mixture was filtered through a Celite
pad, and the filter cake was washed with MeOH and EtOAc.
The combined filtrates were concentrated. The residue was
dissolved in 1 M KOH (34 mL) and EtOH (4 mL). After being
refluxed for 5 h, the mixture was evaporated, and the residue
was dissolved in H2O (40 mL). To this solution was added
concentrated H2SO4 (6.0 mL) dropwise at 0 °C. After being
refluxed for 5 h, the mixture was alkalinized with 5M KOH
under vigorous stirring, and extracted with EtOAc and n-
BuOH two times, respectively. The EtOAc layers were washed
with H2O and brine, dried over sodium sulfate, and evaporated.
The n-BuOH layers were also evaporated. Each residues were
combined and chromatographed over SiO2 eluted by EtOAc-
MeOH (4:1) and MeOH to give 8 (1.10 g, 4.10 mmol, 53%).
10: offwhite amorphous solid; H NMR (CDCl3, 600 MHz) δ
8.40 (1H, s), 7.63 (2H, d, J ) 8.6 Hz), 7.35 (1H, d, J ) 7.9 Hz),
6.96-7.00 (8H, m), 6.66 (1H, d, J ) 7.9 Hz), 6.27 (1H, t, J )
5.9 Hz), 5.17 (2H, s), 5.13 (2H, s), 3.76 (2H, td, J ) 6.6, 5.9
Hz), 3.48 (3H, s), 3.45 (3H, s), 3.06 (2H, t, J ) 6.6 Hz); 13C
NMR (CDCl3, 150 MHz) δ 166.9, 159.6, 153.3, 151.2, 143.5,
129.8, 128.5 (2C), 128.0, 128.0, 122.3, 119.9, 119.7 (2C), 117.6
(2C), 115.7 (2C), 113.7, 113.6, 109.5, 95.0, 94.1, 56.1, 55.9, 40.2,
25.4; EIMS m/z 476 [M]+, 295 (base); HREIMS m/z 476.1947
(476.1946 calcd for C27H28N2O6).
N-(3-(2-Formylamino-3-(4-(methoxymethoxy)phenox-
y)phenyl)-3-oxopropyl)-4-methoxymethoxybenzamide
(11). Sodium periodate (402 mg, 1.88 mmol) was added to a
solution of 10 (222 mg, 0.467 mmol) in acetonitrile (5 mL) and
H2O (5 mL). After being stirred for 36 h at 60 °C, the reaction
mixture was extracted with EtOAc three times. The organic
layers were combined, washed with H2O and brine, dried over
sodium sulfate, and evaporated. The residue was chromato-
graphed over SiO2 eluted by n-hexanes-EtOAc (1:1 and 2:3)
to give 11 (178 mg, 0.349 mmol, 75%). Data for 11: yellow oil;
1H NMR (acetone-d6, 600 MHz) δ 9.60 (1H, br.s), 8.34 (1H,
br.s), 7.88 (2H, d, J ) 9.0 Hz), 7.74 (1H, br.s), 7.40 (1H, br.s),
7.24 (1H, t, J ) 7.9 Hz), 7.06 (2H, d, J ) 9.2 Hz), 7.05 (2H, d,
J ) 9.0 Hz), 6.99 (1H, br.s), 6.98 (2H, d, J ) 9.2 Hz), 5.23 (2H,
s), 5.16 (2H, s), 3.75 (2H, td, J ) 6.2, 5.5 Hz), 3.43 (3H, s),
3.41 (3H, s), 3.32 (2H, br.s); 13C NMR (acetone-d6, 150 MHz)
δ 201.2, 166.8, 161.0, 160.5, 154.9, 151.6, 151.3, 137.4, 129.6
(2C), 129.6, 129.1, 126.8, 122.3, 121.0 (2C), 120.4, 118.6 (2C),
116.3 (2C), 95.5, 94.8, 56.1, 56.0, 41.1, 35.9; EIMS m/z 508
[M]+, 165, 45 (base); HREIMS m/z 508.1858 (508.1844 calcd
for C27H28N2O8).
1
Data for 8: a brownish powder; H NMR (CD3OD, 600 MHz)
δ 7.27 (1H, dd, J ) 7.9, 0.7 Hz), 7.07 (1H, s), 6.90 (1H, t, J )
7.9 Hz), 6.88 (2H, d, J ) 9.0 Hz), 6.76 (2H, d, J ) 9.0 Hz),
6.49 (1H, dd, J ) 7.9, 0.7 Hz), 2.96-2.98 (2H, m), 2.91-2.93
(2H, m); 13C NMR (CD3OD, 150 MHz) δ 154.6, 151.1, 145.6,
131.4, 129.7, 124.0, 121.0 (2C), 120.1, 117.0 (2C), 114.2, 113.9,
109.6, 43.0, 29.2; EIMS m/z 268 [M]+, 238 (base); HREIMS
m/z 268.1195 (268.1211 calcd for C16H16N2O2).
N-(2-(7-(4-Hydroxyphenoxy)indol-3-yl)ethyl)-4-(meth-
oxymethoxy)benzamide (9). To a solution of 8 (570 mg, 2.13
mmol) in DMF (10 mL) were added 4-(methoxymethoxy)-
benzoic acid15 (465 mg, 2.55 mmol) and N-(3-dimethylamino-
propyl)-N′-ethylcarbodiimide hydrochloride (WSC) (1.22 g, 6.38
mmol) at 0 °C. The mixture was gradually warmed to room
temperature. After being stirred for 2 h, it was poured into
0.3 M HCl and extracted with EtOAc three times. The organic
layers were combined, washed with H2O and brine, dried over
sodium sulfate, and evaporated. The residue was chromato-
graphed over SiO2 eluted by n-hexanes-EtOAc (1:1) to give 9
(599 mg, 1.39 mmol, 65%). Data for 9: offwhite amorphous
N-(3-(2-Amino-3-(4-hydroxyphenoxy)phenyl)-3-oxopro-
pyl)-4-hydroxybenzamide (Brefelamide (1)). To a solution
of 11 (12.7 mg, 0.025 mmol) in CH2Cl2 (900 µL) was added
trifluoroacetic acid (200 µL) dropwise at 0 °C. After being
stirred for 3 h, the mixture was evaporated. The residue was
chromatographed over aluminum oxide eluted by CHCl3-
MeOH (9:1) to give brefelamide (1) (7.0 mg, 0.018 mmol, 67%).
All spectral data of the synthetic product were identical with
those of the natural product.
1
solid; H NMR (CDCl3, 600 MHz) δ 8.17 (1H, s), 7.62 (2H, d,
J ) 9.0 Hz), 7.36 (1H, d, J ) 7.9 Hz), 7.05 (1H, s), 7.01 (2H, d,
J ) 9.0 Hz), 7.00 (1H, t, J ) 7.9 Hz), 6.96 (2H, d, J ) 9.0 Hz),
6.83 (2H, d, J ) 9.0 Hz), 6.67 (1H, d, J ) 7.9 Hz), 6.14 (1H, t,
J ) 6.0 Hz), 5.51 (1H, s), 5.20 (2H, s), 3.79 (2H, td, J ) 6.6,
6.0 Hz), 3.47 (3H, s), 3.09 (2H, t, J ) 6.6 Hz); 13C NMR (CDCl3,
150 MHz) δ 167.0, 159.7, 152.1, 150.0, 143.9, 129.8, 128.6 (2C),
128.1, 128.0, 122.2, 120.2 (2C), 120.1, 116.4 (2C), 115.9 (2C),
113.7, 113.6, 109.4, 94.2, 56.2, 40.2, 25.5; EIMS m/z 432 [M]+,
251 (base); HREIMS m/z 432.1678 (432.1684 calcd for C25H24N2-
O5).
Acknowledgment. This work was supported in part
by a Grant-in-Aid for Scientific Research (no. 15710153)
from the Ministry of Education, Science, Sports and
Culture of Japan.
Supporting Information Available: General experimen-
tal methods and NMR spectra for new compounds including
natural and synthetic brefelamide (1). This material is avail-
able free of charge via the Internet at http://pubs.acs.org.
4-(Methoxymethoxy)-N-(2-(7-(4-(methoxymethoxy)phe-
noxy)indol-3-yl)ethyl)benzamide (10). To a solution of 9
(699 mg, 1.62 mmol) in CH2Cl2 (8.0 mL) were added N,N-
JO051352X
8858 J. Org. Chem., Vol. 70, No. 22, 2005