Lupinacidin C, an inhibitor of tumor cell invasion from micromonospora lupini

Article abstract of DOI:10.1021/np100779t

A new anthraquinone derivative, lupinacidin C (1), was isolated from the endophytic actinomycete Micromonospora lupini. The structure was elucidated on the basis of spectroscopic analyses, and the absolute configuration was determined by total synthesis.

Full text of DOI:10.1021/np100779t

NOTE
pubs.acs.org/jnp
Lupinacidin C, an Inhibitor of Tumor Cell Invasion
from Micromonospora lupini
Yasuhiro Igarashi,*,† Saeko Yanase,† Kohei Sugimoto,‡ Masaru Enomoto,‡ Satoshi Miyanaga,†
Martha E. Trujillo,§ Ikuo Saiki,^ and Shigefumi Kuwahara‡
†Biotechnology Research Center, Toyama Prefectural University, 5180 Kurokawa, Imizu, Toyama 939-0398, Japan
‡Graduate School of Agricultural Science, Tohoku University, Tsutsumidori-Amamiyamachi, Aoba-ku, Sendai 981-8555, Japan
§Departmento de Microbiologia y Genetica, Edificio Departamental Lab. 205, Campus Miguel de Unamuno, Universidad de Salamanca,
37007 Salamanca, Spain
^Department of Bioscience, Institute of Natural Medicine, University of Toyama, 2630 Sugitani, Toyama 930-0194, Japan
S Supporting Information
b
ABSTRACT: A new anthraquinone derivative, lupinacidin C (1), was isolated from the endophytic
actinomycete Micromonospora lupini. The structure was elucidated on the basis of spectroscopic
analyses, and the absolute configuration was determined by total synthesis. Lupinacidin C (1) exhibited
the most potent inhibitory effects among the congeners on the invasion of murine colon carcinoma cells
into the reconstituted basement membrane.
ndophytic actinomycetes are known to inhabit the phyllo-
sphere and the rhizosphere, and they are especially abundant
appropriate for C21H22O5 (calcd for C21H23O5 m/z 355.1540),
corresponding to the addition of a methylene fragment (CH2)
to lupinacidin A (2). The 13C NMR and HMQC spectra revealed
the three aromatic carbons (C-6, C-7, and C-8) each bearing
protons (H-6, H-7, and H-8). Contiguous couplings of these
protons (H-6/H-7/H-8) were confirmed by the 1H-1H COSY
spectrum. In addition, two carbonyl carbons, three hydroxylated
aromatic carbons (C-1, C-3, and C-5), a singlet methyl, and
E
in root tissues.1 In particular, similar to rhizobia in legume plants,
the genus Frankia is involved in root nodule formation and nitro-
gen fixation in nonlegume plants, establishing a close relationship
with host plants. Different from this symbiont, biological and
ecological functions of most of the endophytic actinomycetes in
plants are not comprehensively understood.1c,1d However, these
endophytes are currently of significant interest as an untapped
resource of novel bioactive small molecules because their metabo-
lites are speculated to affect the physiological conditions of host
plants such as growth and disease resistance.1d,2 Micromonospora is
a common genus in terrestrial and aquatic environments, but our
current knowledge on its inhabitation in plant tissues is limited.3
Recently, legume root nodules have been found to commonly
harbor this species, and a series of new species were recovered
from this previously ignored habitat.4 Our investigation on the
secondary metabolites from legume-derived new species resulted in
the discovery of new anthraquinones, lupinacidins A (2) and B (3)
from M. lupini isolated from Lupinus angustifolius.5 These com-
pounds inhibited the tumor cell invasion into the reconstituted
basement membraneina concentration-dependentmanner at non-
cytotoxic concentrations. Further chemical investigation of the
culture extract from M. lupini Lupac 08 led to the identification of
a new minor congener, lupinacidin C (1).
1
six quaternary sp2 carbons were observed. In the H NMR
spectrum, two hydrogen-bonded phenolic hydroxyl protons
were observed at δH 13.0 and 14.2. All these protons and carbons
attributable to the anthraquinone skeleton were almost identical
with those for 2 and 3. Comparison of the 1H and 13C NMR data
for 1 with those for 2 and 3 indicated the difference in the alkyl
substituent at C-4. The COSY spectrum showed three small spin
systems: H2-12/H2-13, H-14/H3-17, and H2-15/H3-16. These
fragments were combined on the basis of HMBC correlations
from H3-16 to C-14 and C-15 and from H3-17 to C-13, C-14, and
C-15, thereby establishing the 3-methylpentyl group. HMBC
correlations from H2-12 to C-3 and C-4 connected this alkyl
substituent to C-4.
To determine the absolute configuration of the stereogenic
center on the side chain, the S-enantiomer of 1 was synthesized
according to the protocol previously employed for the synthesis
of lupinacidins A (2) and B (3) (Scheme 1).6 L-Leucine (4) was
converted into the bromo acid 5,7 which was further treated with
LiAlH4 to give alcohol 6.8 The optically active alcohol 6 was
transformed into the corresponding iodide 7, and an enone 8 was
alkylated with 7.9 The resulting product 9 obtained as a ca. 1:1
The 1-butanol extract of the whole culture broth of M. lupini
Lupac 08 was subjected to normal- and reversed-phase column
chromatographies and HPLC purification to yield lupinacidin
C (1) as optically active orange needles ([R]D þ4.6, CHCl3).
The UV spectrum was closely similar to that for lupinacidins A
(2) and B (3) (λmax 212, 235, 253, 276, 445 nm).5 The high-
resolution FABMS gave an [M þ H]þ peak at m/z 355.1543
Received: October 27, 2010
Published: January 12, 2011
r
Copyright
2011 American Chemical Society and
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dx.doi.org/10.1021/np100779t J. Nat. Prod. 2011, 74, 862–865
American Society of Pharmacognosy
|

Journal of Natural Products
NOTE
Scheme 1. Synthesis of (S)-1
congeners, 1 displayed the most potent anti-invasive activity,
with an IC50 value of 0.019 μg/mL (0.054 μM), indicating that
the hydrophobicity of the alkyl side chain is involved in potency.
On the basis of these findings, chemical synthesis of additional
lupinacidin analogues is in progress for evaluation as a candi-
date for antimetastatic agent.
’ EXPERIMENTAL SECTION
General Experimental Procedures. Melting points were de-
termined with a Yanaco MP-J3 apparatus and are uncorrected. Optical
rotations were measured on a Jasco DIP-1030 or a Jasco DIP-371
polarimeter. UV spectra were recorded on a Hitachi U-3210 spectro-
photometer. IR spectra were collected on a Perkin-Elmer Spectrum 100.
NMR spectra were recorded on a Jeol JMN-LA400 or a Varian MR-400
spectrometer, using the signals of the residual solvent protons and
carbons as internal standard. The HRFABMS was measured using a
Jeol JMS-HX110A. HREIMS were recorded on a Jeol JMS-700. Di-
chloromethane and THF used for chemical synthesis were distilled
from CaH2 and Na/benzophenone, respectively.
Figure 1. 1H-1H COSY and key HMBC correlations for 1.
diastereomeric mixture was converted into silyl enol ether 10,
which was then subjected to the Diels-Alder reaction with
sulfinyl quinone 11 to afford tetracyclic intermediate 12 via in
situ liberation of p-toluenesulfenic acid from an intermediary
Diels-Alder adduct. Heating of 12 in toluene brought about the
elimination of ethylene to afford an anthraquinone intermediate,
which was then treated with aqueous HF in one pot to give 13.
Finally, deprotection of both the methoxy and acetyl groups of
Microorganism. The actinomycete strain Micromonospora lupini
Lupac 08 was isolated from the root nodule of Lupinus angustifolius
collected in Saelices el Chico (Salamanca, Spain) in 2003. Isolation
and characterization were described in the previous paper.4b
Fermentation and Isolation. Seed and production fermenta-
tions of strain Lupac 08 were carried out in V-22 and A-3 M medium,
respectively, as described previously.5 The combined 1-butanol extract
from 80 ꢀ 100 mL fermentation (23 g) was subjected to silica gel
column chromatography purification (Kanto Chemical Co., Inc., 63-
210 mesh, 60 ꢀ 400 mm) eluting with solvent mixtures of CHCl3/
MeOH (1:0, 20:1, 10:1, 4:1, 2:1, and 1:1 v/v), successively. Fractions 3
and 4 (10:1 and 4:1), containing lupinacidins, were concentrated and
refractionated by C-18 reversed-phase MPLC (Nacalai Tesque, Inc.,
silica gel 60-C18, 250-350 mesh, 55 ꢀ 250 mm) with a step gradient
of MeCN/0.15% KH2PO4 buffer solution (pH 3.5)/MeOH as fol-
lows: fractions 1-7, MeCN/KH2PO4 buffer solution (2:8, 3:7, 4:6,
5:5, 6:4, 7:3, and 8:2 v/v); and fraction 8, 100% MeOH. Evaporation
of fraction 8 in vacuo left a wet residue, which was taken up in EtOAc,
providing 500 mg of dark brown solid. Final purification was achieved
by repeated C-18 RP HPLC (Nacalai Tesque Inc., Cosmosil 5C18-
AR-II, 20 ꢀ 250 mm) with 85% MeCN in 0.15% KH2PO4 buffer
solution to afford 1 (5.8 mg).
1
13 with BBr3 furnished (S)-1, the H and 13C NMR spectra
of which were identical with those of natural 1. The specific
rotation of (S)-1, [R]24D þ5.3 (c 0.23, CHCl3), showed a good
agreement with that of the natural product, [R]24D þ4.6 (c 0.10,
CHCl3). Therefore, the absolute configuration of 1 was deter-
mined to be S.
Compound 1 was tested for anti-invasive activity using mu-
rine colon 26-L5 carcinoma cells, as compounds 2 and 3 were
previously tested in the same assay and showed moderately
strong activities (IC50 values: 0.21 μM for 2; 0.92 μM for 3).5
Prior to invasion assay, cytotoxicity of 1 was assessed at con-
centrations from 0.1 to 3 μg/mL. 1 was inactive to the cells at
less than 0.3 μg/mL, and thus the inhibitory effect on invasion
was assessed at concentrations less than 0.1 μg/mL. Among the
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Journal of Natural Products
NOTE
1
Table 1. H and 13C NMR Data for Lupinacidin C (1) in
CDCl3
quenched with saturated aqueous NH4Cl and extracted with hexane.
The extract was successively washed with saturated aqueous NaHSO3,
water, and brine, dried (MgSO4), and concentrated in vacuo. The
residue was purified by SiO2 column chromatography using hexane as
eluent to give 7 (7.40 g, 46.0% from 4) as a colorless oil: [R]27D þ15.8 (c
0.815, CHCl3); IR (ATR) νmax 2959, 2925, 2873, 1460, 1178 cm-1; 1H
NMR (CDCl3, 400 MHz) δ 3.25 (1H, ddd, J = 9.5, 8.6, 5.9 Hz), 3.17
(1H, ddd, J = 9.5, 8.3, 7.1 Hz), 1.88 (1H, m), 1.64 (1H, m), 1.48 (1H, m),
1.36 (1H, m), 1.18 (1H, m), 0.88 (3H, t, J = 7.6 Hz), 0.87 (3H, d, J = 6.6
Hz); 13C NMR (CDCl3, 100 MHz) δ 40.5, 35.4, 28.7, 18.2, 11.1, 5.4;
HREIMS m/z 212.0063 (calcd for C6H13I, 212.0062).
(R/S)-3-Methoxy-2-methyl-6-[(S)-3-methylpentyl]-2-cyclohexen-1-
one (9). To a stirred solution of LDA [prepared by treating a solution of
diisopropylamine (0.42 mL, 3.0 mmol) in THF (1.5 mL) with butyl-
lithium (1.6 M in hexane, 1.9 mL, 3.0 mmol) at -10 ꢀC for 20 min] was
added dropwise a solution of 8 (280 mg, 2.00 mmol) in THF (3.5 mL)
at -78 ꢀC. After 2 h, HMPA (1.0 mL, 5.7 mmol) and a solution of 7
(212 mg, 1.00 mmol) in THF (4 mL) were successively added at the
same temperature, and the resulting mixture was gradually warmed to
room temperature over 4 h. The reaction mixture was quenched with
saturated aqueous NH4Cl and extracted with Et2O. The extract was
successively washed with water and brine, dried (MgSO4), and con-
centrated in vacuo. The residue was chromatographed over SiO2
(hexane/EtOAc, 5:1) to give 107 mg (47.7 mmol, 48%) of 9 as a white
a
position
δC
δH mult (J in Hz)b
HMBCb,c
1
162.6d, qC
117.6, qC
159.7, qC
128.3, qC
128.1, qC
162.5d, qC
124.3, CH
136.0, CH
118.3, CH
133.1, qC
186.9, qC
110.8, qC
190.4, qC
117.1, qC
8.4, CH3
2
3
4
4a
5
6
7
7.27 (dd, 8.3, 1.0)
7.63 (dd, 8.3, 7.6)
5a, 9
6, 9a
8
8a
9
7.79 (dd, 7.6, 1.0)
5a, 7, 10
9a
10
10a
11
12
2.26 (s)
1, 2, 3
24.5, CH2
3.21 (dt, 11.2, 4.4)
3.16 (dt, 11.2, 4.4)
1.56 (m)
3, 4, 13, 14
3, 4, 13, 14
13
35.4, CH2
solid: mp 55.5-56.5 ꢀC; IR (ATR) νmax 1619, 1602, 1378 cm-1
;
1.40 (m)
1H NMR (CDCl3, 400 MHz) δ 3.80 (3H, s), 2.63 (1H, m), 2.48
(1H, m), 2.04-2.16 (2H, m), 1.69-1.89 (2H, m), 1.68 (3H, t, J =
1.6 Hz), 1.24-1.41 (4H, m), 1.09-1.19 (2H, m), 0.83-0.88 (6H, m);
13C NMR (CDCl3, 100 MHz) δ 200.9, 170.3, 114.1, 54.9, 44.8/44.7,
34.61/34.56, 34.1/33.9, 29.5/29.1, 27.2/27.1, 25.7/25.5, 23.5/23.4,
19.2/19.1, 11.4/11.3, 7.56; HREIMS m/z 224.1779 (calcd for
C14H24O2, 224.1776).
14
15
35.3, CH
1.58 (m)
29.3, CH2
1.49 (m)
1.28 (m)
16
11.4, CH3
19.1, CH3
0.95 (t, 7.4)
1.05 (d, 6.3)
14.2 (s)
14, 15
13, 14, 15
1, 2
17
1-OH
6-OH
13.0 (s)
5a, 6, 7
5-Acetoxy-3-hydroxy-1-methoxy-2-methyl-4-[(S)-3-methylpentyl]-
9,10-anthracenedione (13). To a stirred solution of LDA [prepared
by treating a solution of diisopropylamine (0.18 mL, 1.3 mmol) in THF
(0.84 mL) with a solution of butyllithium (1.57 M in hexane, 0.74 mL,
1.2 mmol) at -10 ꢀC for 20 min] was added a solution of 9 (95 mg,
0.42 mmol) in THF (0.84 mL) at -78 ꢀC. After 2 h, TESOTf (0.25 mL,
1.1 mmol) was added, and the resulting mixture was stirred at -78 ꢀC
for 2 h. To the mixture containing 10 was added dropwise a solution
of 11 (177 mg, 0.5 mmol) in CH2Cl2 (5 mL), and the mixture was stirred
at -78 ꢀC for 12 h. The mixture was quenched with MeOH/AcOH
(3:1) at -78 ꢀC and then gradually warmed to 0 ꢀC before being diluted
with saturated aqueous NH4Cl and extracted with Et2O. The extract was
successively washed with saturated aqueous NaHCO3 and brine, dried
(MgSO4), and concentrated in vacuo. The residue was partially puri-
fied by SiO2 column chromatographed (hexane/EtOAc, 10:1) to give a
mixture (120 mg) containing 12 as the major component. The product
was dissolved in toluene (2 mL) and stirred at 95 ꢀC for 9 h. To the
resulting solution were successively added MeCN (3 mL) and several
drops of hydrofluoric acid (46 wt % in water) at 0 ꢀC. The mixture was
stirred overnight at room temperature, diluted with water (10 mL), and
extracted with Et2O. The extract was washed with brine, dried (MgSO4),
and concentrated in vacuo. The residue was chromatographed over SiO2
(hexane/EtOAc, 10.1-5:1) to give 13 (81 mg, 47% from 9) as a yellow
solid: mp 116.0-117.0 ꢀC; [R]27D þ6.61 (c 0.880, CHCl3); IR (ATR)
a Recorded at 100 MHz. b Recorded at 400 MHz. c HMBC correlations
are from proton(s) stated to the indicated carbon. d Interchangeable.
Lupinacidin C (1):. orange needles; mp 198-200 ꢀC; [R]24D þ4.6 (c
0.10, CHCl3); UV (MeOH) λmax (log ε) 212 (4.41), 235 (4.17), 253
(4.21), 276 (4.21), 445 (3.97); IR (ATR) νmax 3430, 1595 cm-1; H
1
and 13 NMR data, see Table 1; HRFABMS [M þ H]þ 355.1543 (calcd
for C21H23O5, 355.1540).
(S)-1-Iodo-3-methylpentane (7). To a stirred suspension of L-iso-
leucine 4 (10.0 g, 76.2 mmol) in water (24 mL) was added aqueous HBr
(48 wt %, 52 mL) at -10 ꢀC, and the resulting mixture was gradually
warmed to 10 ꢀC over 1 h. To the mixture was added dropwise a solution
of NaNO2 (10.5 g, 152 mmol) in water (20 mL) at -10 ꢀC over 15 min.
The heavy, brown gas released from the reaction was scrubbed by 20%
NaOH solution (30 mL). After being stirred at -10 ꢀC for 30 min, the
reaction mixture was gradually warmed to room temperature over 4 h
and extracted with Et2O. The extract was successively washed with
saturated aqueous Na2S2O3, water, and brine, dried (MgSO4), and con-
centrated in vacuo to give crude 5 (14.9 g). The crude bromide (14.9 g)
was dissolved in THF (20 mL) and added to a stirred suspension of
lithium aluminum hydride (2.40 g 76.2 mmol) in THF (60.0 mL) at
0 ꢀC. After being stirred for 1 day at room temperature and for an
additional day at reflux, the reaction mixture was quenched with water at
0 ꢀC, and the resultant white precipitate was dissolved with 2 N H2SO4
(50 mL). The mixture was extracted with Et2O, and the extract was
successively washed with water and brine, dried (MgSO4), and con-
centrated in vacuo to give crude 6 (6.40 g), which was taken up in THF
(80 mL). To the solution were successively added imidazole (6.40 g,
94.0 mmol), triphenylphosphine (18.1 g, 69.0 mmol), and iodine (19.1 g,
75.3 mmol) at 0 ꢀC. The resulting mixture was stirred at 0 ꢀC for 1 h
and then gradually warmed to room temperature over 5 h before being
1
νmax 3451, 1772, 1670, 1596, 1552, 1190 cm-1; H NMR (CDCl3,
400 MHz) δ 8.07 (1H, dd, J = 7.9, 1.2 Hz), 7.67 (1H, t, J = 7.9 Hz), 7.29
(1H, dd, J = 7.9, 1.2 Hz), 5.73 (1H, br s, OH), 3.88 (3H, s), 2.96 (1H, m),
2.85 (1H, m), 2.42 (3H, s), 2.25 (3H, s), 1.64 (1H, m), 1.44-1.58 (3H,
m), 1.28 (1H. m), 1.04 (3H, d, J = 6.2 Hz), 0.95 (3H, t, J = 7.3 Hz);
13C NMR (CDCl3, 100 MHz) δ 185.7, 181.7, 169.8, 158.4, 158.3, 148.3,
136.3, 134.0, 133.8, 127.8, 127.1, 126.6, 124.5, 124.0, 120.0, 61.6, 35.8,
864
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Journal of Natural Products
NOTE
35.3, 29.2, 24.8, 21.1, 19.0, 11.5, 9.1; HREIMS m/z 411.2174 (calcd for
C25H31O5, 411.2171).
P.; Rodríguez, R.; Carro, L.; Cerda, E.; Alonso, P.; Martinez-Molina, E.
ISME J. 2010, 4, 1265–1281.
(5) Igarashi, Y.; Trujillo, M. E.; Martinez-Molina, E.; Yanase, S.;
Miyanaga, S.; Obata, T.; Sakurai, H.; Saiki, I.; Fujita, T.; Furumai, T.
Bioorg. Med. Chem. Lett. 2007, 17, 3702–3705.
1,3,5-Trihydroxy-2-methyl-4-[(S)-3-methylpentyl]-9,10-anthracene-
dione [(S)-1]. To a stirred solution of 13 (30.7 mg, 74.8 mmol) in
CH2Cl2 (3 mL) was added BBr3 (1.0 M in CH2Cl2, 0.40 mL, 0.40
mmol) at -78 ꢀC. The mixture was gradually warmed to 0 ꢀC over 4 h
and then quenched with saturated aqueous NaHCO3. The resulting
mixture was stirred overnight at room temperature before being
extracted with CH2Cl2. The extract was successively washed with
saturated aqueous NH4Cl, water, and brine, dried (MgSO4), and
concentrated in vacuo. The residue was chromatographed over SiO2
(hexane/EtOAc, 10:1) to give (S)-1 (17.6 mg, 67%) as an orange solid:
(6) Sugimoto, K.; Enomoto, M.; Kuwahara, S. Tetrahedron Lett.
2010, 51, 4570–4572.
(7) Li, B.-F.; Hughes, R. M.; Le, J.; McGee, K.; Gallagher, D. J.;
Gross, R. S.; Provencal, D.; Reddy, J. P.; Wang, P.; Zegelman, L.; Zhao,
Y.; Zook, S. E. Org. Process Res. Dev. 2009, 13, 463–467.
(8) Koppenhoefer, B.; Weber, R.; Schurig, V. Synthesis 1982, 316–
318.
(9) Katoh, N.; Nakahata, T.; Kuwahara, S. Tetrahedron 2008, 64,
9073–9077.
(10) (a) Saito, K. I.; Oku, T.; Ata, N.; Miyashiro, H.; Hattori, M.;
Saiki, I. Biol. Pharm. 1997, 20, 345–348. (b) Miyanaga, S.; Obata, T.;
Onaka, H.; Fujita, T.; Saito, N.; Sakurai, H.; Saiki, I.; Furumai, T.;
Igarashi, Y. J. Antibiot. 2006, 59, 698–703.
mp 198.0-198.5 ꢀC; [R]24 þ5.3 (c 0.23, CHCl3); IR (ATR) νmax
D
1
3458, 1618, 1605, 1240, 1192 cm-1; H NMR (CDCl3, 400 MHz) δ
14.2 (1H. s, OH), 13.0 (1H, s, OH), 7.79 (1H, dd, J = 7.5, 1.1 Hz), 7.62
(1H, dd, J = 8.4, 7.5 Hz), 7.26 (1H, dd, J = 8.4, 1.1 Hz), 5.67 (1H, s, OH),
3.12-3.27 (2H, m), 2.26 (3H. s), 1.53-1.62 (2H, m), 1.47 (1H, m),
1.40 (1H, m), 1.28 (1H, m), 1.05 (3H, d, J = 6.4 Hz), 0.95 (3H, t, J = 7.4
Hz); 13C NMR (CDCl3, 100 MHz) δ 190.3, 186.9, 162.6, 162.4, 159.6,
136.0, 133.0, 128.2, 128.0, 124.3, 118.3, 117.5, 117.0, 110.7, 35.4, 35.2,
29.3, 24.5, 19.1, 11.4, 8.4; HREIMS m/z 355.1550 (calcd for C21H23O5,
355.1545).
’ NOTE ADDED AFTER ASAP PUBLICATION
This paper was published on the Web on Jan 12, 2011, with an
error in the fourth paragraph. The value of the anti-invasive
activity for compound 1 was corrected to 0.54 μM. The corrected
version was reposted on Feb 10, 2011.
Biological Assays. Cytotoxic assay and invasion assay were car-
ried out according to the procedures previously described.10
’ ASSOCIATED CONTENT
Supporting Information. 1H and 13C NMR spectra of
S
b
lupinacidin (1) and synthetic (S)-1, 7, 9, and 13. This material
available is free of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*To whom correspondence should be addressed. Tel: þ81-766-
56-7500. Fax: þ81-766-56-2498. E-mail: yas@pu-toyama.ac.jp.
’ ACKNOWLEDGMENT
We acknowledge Dr. H. Sakurai at University of Toyama for
assistance with cytotoxic and anti-invasion assays.
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