Lorneic acids, trialkyl-substituted aromatic acids from a marine-derived actinomycete

Article abstract of DOI:10.1021/np900353y

A marine-derived actinomyces strain (NPS554) isolated from a marine sediment sample collected from Miyazaki Harbor, Japan, at a depth of 38 m yielded two trialkyl-substituted aromatic acids, lorneic acid A (1) and lorneic acid B (2). The structures of the

Full text of DOI:10.1021/np900353y

2046
J. Nat. Prod. 2009, 72, 2046–2048
Lorneic Acids, Trialkyl-Substituted Aromatic Acids from a Marine-Derived Actinomycete
Fumie Iwata,* Seizo Sato, Takako Mukai, Shoichi Yamada, Jiro Takeo, Akihisa Abe, Takaaki Okita, and Hiroyuki Kawahara
Central Research Laboratory, Nippon Suisan Kaisha, Ltd, 559-6 Kitano-machi Hachioji, Tokyo 192-0906, Japan
ReceiVed June 18, 2009
A marine-derived actinomyces strain (NPS554) isolated from a marine sediment sample collected from Miyazaki Harbor,
Japan, at a depth of 38 m yielded two trialkyl-substituted aromatic acids, lorneic acid A (1) and lorneic acid B (2). The
structures of the lorneic acids, which were elucidated by spectroscopic analysis, differed only in the side-chain, which
contained either a conjugated double bond or a benzylic alcohol. Their structural differences affected inhibition activities
against phosphodiesterase 5.
Actinomycetes from natural sources are widely recognized to
produce secondary metabolites, including many antimicrobials such
as streptomycin, erythromycin, and tetracycline, with original and
ingenious structures and potent biological activities.1 Therefore,
actinomycetes are considered to be a potent resource for new lead
compounds in drug development. Analysis of secondary metabolites
produced by a Streptomyces strain (NPS554) with low sequence
homology (16S rDNA 98.0%) resulted in the isolation of two
trialkyl-substituted aromatic acids, lorneic acids A (1) and B (2).
This report describes the isolation, structural elucidation, and
biological activities of lorneic acids.
The strain NPS554 was cultured in an artificial seawater-based
KG medium.2 At the end of the culture period, the culture
supernatant was partitioned between EtOAc and H2O, and the H2O
phase was further extracted with n-BuOH. In addition, the mycelia
were extracted with MeOH, and the MeOH extract was partitioned
between EtOAc and H2O. The combined crude extracts (EtOAc
extract and n-BuOH extract) were subjected to C-18 reversed-phase
flash chromatography and silica gel chromatography to yield lorneic
acid A (1) and lorneic acid B (2).
13C NMR spectrum of 1, including edited gs-HSQC experimental
data,4 showed one carboxylic acid carbon, six aromatic carbons
including three quaternary carbons, four olefinic carbons, and six
additional carbon signals between δ 14.3 and 43.9. 1H-1H COSY
cross-peaks allowed assignment of the side-chain parts of the
molecule (H-2 to H-4, H-11 to H-16) and the adjacent aromatic
ring proton (H-6 to H-7). In the HMBC experiment (Figure 1), the
H-12 signal correlated with C-10, and H-11 correlated with C-5,
C-9, and C-10, suggesting a connection of C-10/C-11 and C-5/C-
10/C-9. The H-4 signal correlated with both C-10 and C-6, and
H-3 correlated with C-5, suggesting a connection of C-4/C-5/C-
10/C-9 and C-4/C-5/C-6/C-7. The remaining structure was deter-
mined by the correlation from H-17 to C-7, C-8, and C-9, which
established a 3,4-disubstituted toluene structure. The H-2 signal
correlated with a carboxyl carbon (C-1), indicating an unsaturated
carboxylic group. Finally, a large coupling constant (J ) 15.6 Hz)
was observed between H-3 and H-4 and between H-11 and H-12,
suggesting that both C-3/C-4 and C-11/C-12 double bonds have
the E-geometry.
Lorneic acid B (2) was obtained as a colorless oil, which had
strong IR absorption bands at 3393 and 1671 cm-1, indicative of
hydroxy and carboxylic acid functional groups, respectively. The
molecular formula was established as C17H24O3 (HRESITOFMS:
[M - H]- m/z 275.1655) in the negative mode. Treatment with
trimethylsilyldiazomethane afforded the methyl ester 2a, confirming
1
the presence of a carboxylic acid functionality. The H and 13C
NMR spectra differed from that of 1 at the quaternary aromatic
carbon (C-10), methine carbon (C-11), and methylene carbons (C-
12, C-13), indicating the presence of a benzylic hydroxy group at
C-11 (δH 4.97, δC 70.9). This substitution was confirmed by 1H-1H
COSY correlations between H-11 and H-12 along with the HMBC
correlations from H-12 to C-10 and C-11 and from H-11 to C-5,
C-9, C-12, and C-13.
Lorneic acid A (1) was obtained as a colorless oil. The molecular
formula was established as C17H22O2 by high-resolution electro-
spray-ionization time-of-flight mass spectrometry (HRESITOFMS:
[M - H]- m/z 257.1505) in the negative mode. The IR spectrum
exhibited an absorption band at 1636 cm-1, indicative of a carboxyl
functional group. The presence of a carboxylic acid functionality
was confirmed by methylation of 1 with trimethylsilyldiazomethane3
to yield the methyl ester 1a, which produced a new 1H NMR signal
at δ 3.73. In addition, the signal correlated with the carbon signal
The lorneic acids possess a common skeleton to the known
lorneamides from a marine actinomycete (MSTMA190).5 For the
lorneic acids and the lorneamides, the side-chain substitution
(carboxylic acid or carboxyamide functionality) is different.
The biological activity of lorneic acid A (1) is currently being
examined in diverse bioassays. In initial screenings at 10 µM,
lorneic acid A displayed significant activity in phosphodiesterase
(PDE) inhibition experiments. It is interesting to note that lorneic
acid A showed selectivity for PDEs (Figure 2.) Selective inhibition
was further examined in detail with both lorneic acids A and B.
As a consequence, PDE5 inhibitory activity of lorneic acid B (2)
was weaker than that of 1 (estimated IC50 values: 1, 12.6 µM; 2,
87.1 µM). These results suggested that the conjugated double bond
(C-11 to C-12) of the side-chain part of a molecule might be
important for PDE5 inhibition.
1
at δ 174.1 in the HMBC experiment. The H NMR spectrum in
methanol-d4 (Table 1) showed three aromatic methines, four olefinic
methines, two allylic methylenes, two aliphatic methylenes, a singlet
methyl, and a triplet methyl. Here, the two olefinic methines (H-4,
δ 6.68; H-11, δ 6.68) overlapped in methanol-d4. Thus, structure
of lorneic acid A (1) was elucidated by an NMR spectrum in
methanol-d4 and chloroform-d (H-4, δ 6.75; H-11, δ 6.59). The
* Corresponding author. Tel: +81-42-656-5192. Fax: +81-42-656-5188.
E-mail: iwata@nissui.co.jp.
10.1021/np900353y CCC: $40.75  2009 American Chemical Society and American Society of Pharmacognosy
Published on Web 10/26/2009

Notes
Journal of Natural Products, 2009, Vol. 72, No. 11 2047
Table 1. NMR Spectroscopic Data (400 MHz, CD3OD) for Lorneic Acids A (1) and B (2)
lorneic acid A (1)
lorneic acid B (2)
δH (J in Hz)
a
b
a
b
no.
δC mult.
δH (J in Hz)
HMBC
δC mult.
HMBC
1
180.2, qC
179.8, qC
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
43.9, CH2
128.3, CH
129.9, CH
134.2, qC
127.3, CH
128.7, CH
137.6, qC
127.7, CH
137.0, qC
129.2, CH
133.7, CH
34.1, CH2
32.9, CH2
23.4, CH2
14.3, CH3
21.2, CH3
3.12, d (7.2)
1, 3, 4
2, 5
2, 6, 10
43.4, CH2
128.3, CH
129.4, CH
134.0, qC
127.2, CH
128.7, CH
137.7, qC
127.0, CH
143.2, qC
70.9, CH
39.7, CH2
26.7, CH2
32.9, CH2
23.7, CH2
14.4, CH3
21.4, CH3
3.13, d (7.2)
6.24, dt (15.6, 7.2)
6.73, d (15.6)
1, 3
1, 2, 5
2, 5, 6
6.26, dt (15.6, 7.2)
6.68,c d (15.6)
7.34, d (8.0)
6.97, d (8.0)
4, 8, 10
5, 17
7.36, d (8.0)
7.01, d (8.0)
4, 8, 10
5, 9, 17
7.17, s
5, 7, 17
7.26, s
5, 7, 17
6.68,d d (15.6)
6.05, dt (15.6, 6.8)
2.26, dt (6.8, 6.8)
1.50, tt (7.2, 6.8)
1.44, tq (7.2, 7.2)
0.98, t (7.2)
5, 9, 10, 13
10, 13
11, 12, 14, 15
12, 13, 15, 16
14, 16
4.97, t (6.4)
1.68, dt (6.8, 6.4)
1.39, m
1.32, m
1.33, m
5, 9, 10, 12, 13
10, 11, 13, 14
13, 14, 16
14, 15
7, 8, 9
14, 15
7, 8, 9
0.90, t (6.8)
2.33, s
2.30, s
a 100 MHz. b Assignments by edited gs-HSQC experiments. c δH 6.75 (CDCl3). d δH 6.59 (CDCl3).
pH was adjusted to 6.95 before autoclaving) while shaking at 200 rpm
at 28 °C for 6 days.
Extraction and Isolation of Lorneic Acids. The culture was
centrifuged (2000g, 10 min), and the upper layer was partitioned with
EtOAc and n-BuOH. The mycelia were extracted with MeOH, the
solvent was removed, and the residue was partitioned between H2O
and EtOAc. Removal of the solvents (EtOAc and n-BuOH) provided
3.5 g of dry extract per 5 L of culture solution. The extract (3.5 g) was
subjected to C-18 reversed-phase column chromatography purification
eluting with H2O/MeOH (3:1), H2O/MeOH (1:1), H2O/MeOH (1:3),
and MeOH, successively. The MeOH eluting fraction was fractionated
by silica gel column chromatography eluting with hexane to EtOAc.
The hexane/EtOAc (1:2) fraction was further subjected to preparative
TLC purification with CHCl3/MeOH (3:1) to obtain pure lorneic acid
A (1, 3.4 mg) [Rf value 0.51; solvent CHCl3/MeOH (3:1)]. The H2O/
MeOH (1:3) fraction was purified by silica gel column chromatography
eluting with CHCl3 to MeOH. Final purification of the CHCl3/MeOH
(9:1) fraction by preparative TLC with the eluent CHCl3/MeOH (9:1)
afforded pure lorneic acid B (2, 3.1 mg) [Rf value 0.41; solvent CHCl3/
MeOH (9:1)].
1
Figure 1. Key H-1H COSY and HMBC correlations of lorneic
acid A (1).
Lorneic acid A (1): colorless oil; UV (MeOH) λmax (log ꢀ) 240 nm
(4.59), 265 nm (4.48); IR (KBr) 2954, 2927, 2856, 1636, 1577, 1457,
1428, 1396, 1372, 1247, 1192, 1099, 965, 934, 826 cm-1; NMR data,
1
see Table 1 (CD3OD); H NMR (400 MHz, CDCl3) δ 7.30 (1H, d, J
Figure 2. Phosphodiesterase inhibitory activities of lorneic acid A
(1) at 10 µM.
) 8.0 Hz, H-6), 7.18 (1H, s, H-9), 6.99 (1H, d, J ) 8.0 Hz, H-7), 6.75
(1H, d, J ) 15.6 Hz, H-4), 6.59 (1H, d, J ) 15.6 Hz, H-11), 6.09 (1H,
dt, J ) 15.6, 6.8 Hz, H-3), 6.04 (1H, dt, J ) 15.6, 6.8 Hz, H-12), 3.31
(2H, d, J ) 6.8 Hz, H-2), 2.31 (3H, s, H-17), 2.23 (2H, q, J ) 6.8 Hz,
H-13), 1.46 (2H, m, H-14), 1.38 (2H, m, H-15), 0.93 (3H, t, J ) 7.2
Hz, H-16); 13C NMR (100 MHz, CDCl3) δ 177.7 (C-1), 137.3 (C-8),
136.1 (C-10), 133.7 (C-12), 131.9 (C-4), 131.7 (C-5), 127.8 (C-7), 127.4
(C-11), 127.0 (C-9), 126.4 (C-6), 121.8 (C-3), 38.3 (C-2), 33.0 (C-
13), 31.6 (C-14), 22.3 (C-15), 21.2 (C-17), 13.9 (C-16); HRESITOFMS
[M - H]- m/z 257.1505, calcd for C17H22O2, 257.1540.
Experimental Section
General Experimental Procedures. Optical rotations were measured
with a JASCO DIP-370 digital polarimeter. UV spectra were measured
with a JASCO V-560 UV/vis spectrophotometer. IR spectra were
obtained on a JASCO FTIR VALOR-III spectrophotometer. NMR
spectra were recorded on a Bruker AVANCE DPX 400 spectrometer
with methanol-d4 and chloroform-d, the chemical shift of which was
used as internal standard. High-resolution ESITOFMS spectra were
measured on a Waters LCT-Premier XE using an electrospray ioniza-
tion, time-of-flight mass analyzer.
Isolation of the NPS 554 Strain and Cultivation. Streptomyces
strain NPS554 was isolated on a modified HV agar6 (1.0 g of humic
acid, 0.5 g of Na2HPO4, 1.71 g of KCl, 0.05 g of MgSO4 ·7H2O, 0.01 g
of FeSO4 ·7H2O, 0.02 g of CaCO3, 0.5 mg each of thiamine-HCl,
riboflavin, niacin, pyridoxine-HC1, inositol, Ca-pantothenate, and
p-aminobenzoic acid, 0.25 mg of biotin, 50 mg of cycloheximide, 18 g
of agar, 18 g of Daigo’s artificial seawater, and 1 L of distilled water)
from marine sediment collected at a depth of 38 m near Miyazaki
Harbor, Japan. The 16S rDNA sequence of the NPS554 strain (1503
base pairs) was deposited in the DDBJ Genbank (DDBJ accession
number AB515328). This strain shared 98.0% 16S rDNA sequence
identity with Streptomyces sp. CNQ-233_SD01_MAR2. The strain was
cultured in 50 × 100 mL volumes of KG medium (25 g of glucose,
6 g of soytone, 2 g of yeast extract, 4 g of CaCO3, 18 g of Daigo’s
artificial seawater, 800 µL of 2 M NaOH, and 1 L of distilled water;
Lorneic acid B (2): colorless oil, [R]D -22 (c 0.2, MeOH); UV
(MeOH) λmax (log ꢀ) 252 nm (4.00); IR (KBr) 3393, 2956, 2930, 2859,
1718, 1671, 1577, 1457, 1389, 1255, 1163, 1057, 969 cm-1; HRES-
ITOFMS: [M - H]- m/z 275.1655, calcd for C17H24O3, 275.1645.
Lorneic Acid A Methyl Ester (1a). To a solution of lorneic acid A
(1) (1.0 mg, 3.9 µmol) in MeOH (0.5 mL) was added trimethylsilyl-
diazomethane (0.12 mL, 0.23 mmol, 2.0 M in hexane solution) at room
temperature. After stirring at room temperature for 20 min, the reaction
mixture was concentrated under reduced pressure to give the methyl
1
ester 1a as a colorless oil: H NMR (400 MHz, CD3OD) δ 7.30 (1H,
d, J ) 8.0 Hz, H-6), 7.20 (1H, s, H-9), 7.00 (1H, d, J ) 8.0 Hz, H-7),
6.76 (1H, d, J ) 15.6 Hz, H-4), 6.64 (1H, d, J ) 15.6 Hz, H-11), 6.10
(1H, dt, J ) 15.6, 7.2 Hz, H-3), 6.06 (1H, dt, J ) 15.6, 7.2 Hz, H-12),
3.73 (3H, s, OMe), 3.29 (2H, dd, J ) 7.2, 1.6 Hz, H-2), 2.32 (3H, s,
H-17), 2.26 (2H, dt, J ) 7.2, 7.2 Hz, H-13), 1.50 (2H, m, H-14), 1.42
(2H, m, H-15), 0.98 (3H, t, J ) 7.2 Hz, H-16); 13C NMR (100 MHz,
CD3OD) δ 174.1 (C-1), 138.3 (C-8), 137.3 (C-10), 134.3 (C-12), 133.3
(C-5), 132.6 (C-4), 129.0 (C-11), 128.8 (C-7), 128.0 (C-9), 127.4 (C-

2048 Journal of Natural Products, 2009, Vol. 72, No. 11
Notes
6), 124.0 (C-3), 52.4 (OMe), 39.0 (C-2), 34.1 (C-13), 32.8 (C-14), 23.4
(C-15), 21.2 (C-17), 14.3 (C-16); HRESITOFMS [M - H]+ m/z
273.1829, calcd for C18H25O2, 273.1855.
was removed and counted to determine the amount of [3H]guanosine
formed. All test compounds were assayed in duplicate.
Acknowledgment. We wish to thank Dr. M. Aoki for culturing
microbes and Dr. C. Aoyama and Ms. W. Seki for performing the
biological tests. We are grateful to Dr. M. Kuramoto for his valuable
advice throughout this investigation.
Lorneic Acid B Methyl Ester (2a). Lorneic acid B (2) (1.0 mg)
was converted to the corresponding methyl ester 2a by the same
experimental procedure as described for methyl ester 1a: colorless oil;
1H NMR (400 MHz, CD3OD) δ 7.32 (1H, d, J ) 8.0 Hz, H-6), 7.28
(1H, s, H-9), 7.04 (1H, d, J ) 8.0 Hz, H-7), 6.84 (1H, d, J ) 16.0 Hz,
H-4), 6.09 (1H, dt, J ) 16.0, 4.0 Hz, H-3), 4.91 (1H, dd, J ) 6.0, 4.8
Hz, H-11), 3.73 (3H, s, OMe), 3.29 (2H, dd, J ) 6.8, 1.6 Hz, H-2),
2.34 (3H, s, H-17), 1.68 (2H, m, H-12) 1.48-1.25 (6H, m, H-13, H-14,
and H-15), 0.90 (3H, t, J ) 6.8 Hz, H-16); 13C NMR (100 MHz,
CD3OD) δ 174.0 (C-1), 143.4 (C-10), 138.4 (C-8), 133.4 (C-5), 132.2
(C-4), 128.8 (C-7), 127.3 (C-6), 127.3 (C-9), 124.2 (C-3), 71.3 (C-
11), 49.9 (OMe), 39.7 (C-12), 39.7 (C-2), 32.9 (C-14), 26.7 (C-13),
23.7 (C-15), 21.4 (C-17), 14.4 (C-16); HRESITOFMS [M - H]+ m/z
291.1952, calcd for C18H27O3, 291.1960.
PDE5 Assay. Human platelet phosphodiesterase (PDE5) is used.
Test compound and vehicle were preincubated with 35 µg/mL enzyme
in Tris-HCl buffer (pH 7.5) for 15 min at 25 °C. The reaction was
initiated by the addition of 1 µM cGMP and 0.01 µM [3H]cGMP for
another 20 min incubation period and terminated at 100 °C. The
resulting [3H]GMP is converted to [3H]guanosine by the addition of
snake venom nucleotidase and separated by AG1X2 resin. An aliquot
Supporting Information Available: 1H and 13C NMR and UV
1
spectra of 1 and 2 and H and 13C NMR spectra of 1a and 2a are
available free of charge via the Internet at http://pubs.acs.org.
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NP900353Y