Usimines A-C, bioactive usnic acid derivatives from the antarctic lichen Stereocaulon alpinum

Article abstract of DOI:10.1021/np070464b

Usimines A-C (1-3), three new usnic acid derivatives, have been isolated from a MeOH extract of the Antarctic lichen Stereocaulon alpinum by various chromatographic methods. The structures of 1-3 were determined by analysis of their spectroscopic data (NMR, UV, MS) and by chemical methods. The known compound usnic acid (4) was also obtained. Compounds 1-4 showed moderate inhibitory activity against therapeutically targeted protein tyrosine phosphatase 1B (PTP1B).

Full text of DOI:10.1021/np070464b

7
10
J. Nat. Prod. 2008, 71, 710–712
Usimines A-C, Bioactive Usnic Acid Derivatives from the Antarctic Lichen Stereocaulon
alpinum
†
†
‡
§
§
,†
Changon Seo, Jae Hak Sohn, Seong Min Park, Joung Han Yim, Hong Kum Lee, and Hyuncheol Oh*
College of Medical and Life Sciences, Silla UniVersity, Busan 617-736, Korea, Industry-UniVersity Cooperation Foundation,
Pukyong National UniVersity, Busan 608-739, Korea, and Korea Polar Research Institute, KORDI, Incheon 406-840, Korea
ReceiVed September 1, 2007
Usimines A-C (1–3), three new usnic acid derivatives, have been isolated from a MeOH extract of the Antarctic lichen
Stereocaulon alpinum by various chromatographic methods. The structures of 1–3 were determined by analysis of their
spectroscopic data (NMR, UV, MS) and by chemical methods. The known compound usnic acid (4) was also obtained.
Compounds 1–4 showed moderate inhibitory activity against therapeutically targeted protein tyrosine phosphatase 1B
(
PTP1B).
Lichens, comprising about 17 000 species, are symbiotic as-
sociations formed between fungi and algae and/or cyanobacteria.
groups, two phenolic OH groups, and an enolic OH group. The
NMR signals for the basic skeleton of 4 (i.e., the 9bH-dibenzo-
furandione moiety) and its substitution pattern, except for the
absence of one ketone functionality, were apparent in the NMR
spectra of 1–3. Therefore, the structure elucidation of these
compounds focused mainly on identification of the remaining parts
of the molecules and assignments of their absolute configurations.
1
They produce secondary metabolites that have been suggested to
1
,2
serve as antimicrobial or antiherbivore agents. The slow growth
of lichens and harsh conditions they encounter during growth are
presumed to make production of protective metabolites a necessity
2,3
for them. Several lichen extracts have been used in folk medicine,
and some lichen metabolites have been shown to have antibiotic,
Usimine A (1) had the molecular formula C24
H
25NO10, as
1
,4
antimycobacterial, antiviral, analgesic, or antipyretic properties.
13
+
deduced from C NMR and HRESIMS [m/z 488.1552 (M + H) ;
Thus, there is considerable interest in lichen metabolites as potential
∆
+ 0.5 mmu] data. This formula indicated 13 degrees of
5
sources of pharmacological agents.
1
13
unsaturation. Analysis of H and C NMR data, and comparison
with those of 4, enabled identification of the signals corresponding
to the 9bH-dibenzofurandione moiety. The numbering system
shown for 1 was chosen by analogy to that used previously for 4.
During the course of chemical studies of lichens from Antarctica
as potential sources of new bioactive secondary metabolites, three
new metabolites named usimines A-C (1–3), along with the well-
known lichen metabolite usnic acid (4), were isolated from a MeOH
extract of Stereocaulon alpinum. This report describes the isolation,
structure elucidation, and biological activities of compounds 1–4.
1
13
H and C NMR assignments for this portion of the molecule were
confirmed by analysis of HMQC and HMBC data. The most notable
13
differences relative to the data for 4 were the absence of a
C
NMR signal for the C-10 ketone functionality and chemical shift
changes for the methyl group R to the ketone moiety. This
observation suggested that the difference of 1 relative to 4 occurred
1
at C-10. Aside from the aforementioned NMR signals, the H and
1
3
C NMR spectra showed signals for three carbonyl carbons (one
of which was later identified to be involved in an imine group), a
methoxy group, and a -CH-CH -CH - unit. These structural
2
2
features accounted for 13 unsaturation equivalents, requiring linear
connection of the remaining structural unit to C-10. Analysis of
the COSY data revealed the spin system ranging from C-1′ to C-3′
in 1. The carbonyl carbon at δ
the basis of HMBC correlations of H-1′/C-5′ and H
HMQC data showed a correlation of the methyl group protons at
3.59 to δ 51.7, and an HMBC correlation of this signal to the
carbonyl carbon at δ 172.9 indicated the presence of a methyl
ester group. The methyl ester group was connected to C-3′ by
HMBC correlation of H -3′ with C-4′. Connection of the methyl
carbon (C-11) to the carbon resonating at δ 175.1 (C-10) and to
C
173.0 was connected to C-1′ on
2
-2′/C-5′. The
δ
H
C
C
2
C
2
,6
Usnic acid (4) was isolated from the S. alpinum extract as a
major component. The structure of 4 was readily established by
C-2 was established on the basis of HMBC correlations of H -11
3
to C-2 and C-10. An HMBC correlation of H-1′ with C-10, together
6
comparison of NMR and MS data with literature values. Com-
pounds 1–3 were recognized as derivatives of usnic acid by analysis
of their NMR data. The general spectroscopic characteristics of
usnic acid include the presence of H and/or C NMR signals
corresponding to a 1,3-keto-enolic moiety, four isolated methyl
with the presence of nitrogen in the molecule, and the chemical
shifts for C-10, C-1′, and C-5′ enabled the assignment of C-10 as
the carbon (CdN) of an imine group. The remaining OH group
was located at C-5′, thereby completing the gross structure of 1 as
shown.
1
13
It is noteworthy that the 13C NMR spectrum of 1 measured in
CDCl revealed only 19 distinctive carbon signals. Detailed analysis
3
of the NMR data (see Experimental Section) indicated that signals
corresponding to C-2, C-3, and C-10 were missing. These carbons
were presumed to be involved in imine-enamine tautomerism
*
To whom correspondence should be addressed. Tel: +82-51-999-5026.
Fax: +82-51-999-5176. E-mail: hoh@silla.ac.kr.
†
Silla University.
Pukyong National University.
Korea Polar Research Institute.
‡
§
1
0.1021/np070464b CCC: $40.75

2008 American Chemical Society and American Society of Pharmacognosy
Published on Web 02/21/2008

Notes
Journal of Natural Products, 2008, Vol. 71, No. 4 711
Experimental Section
involving the OH group at C-3. Similar spectroscopic behavior due
to imine-enamine tautomerism has been observed in the NMR
spectra of gymnodimine, a cyclic imine containing marine toxin
from Gymnodinium sp. The imine configuration of 1 was proposed
3
on the basis of NOESY data. A NOESY correlation of H -11 to
General Experimental Procedures. Optical rotations were recorded
on a Perkin-Elmer 341 digital polarimeter. UV spectra were recorded
on a Biochrom 1300 UV/visible spectrophotometer. ESIMS data were
obtained using a Mariner ESI-MS instrument (Perseptive Biosystem,
USA) at the Korean Basic Science Institute, Daejeon, Korea. NMR
7
H-1′ required C-11 and C-1′ to be cis with respect to the imine
group, and thus the E-geometry was assigned to the imine.
spectra (1D and 2D) were recorded in pyridine-d
5
1
and CDCl
3
using a
JEOL JNM ECP-400 spectrometer (400 MHz for H and 100 MHz for
The molecular formula of usimine B (2) was established as
1
3
C), and chemical shifts were referenced relative to tetramethylsilane
+
C
∆
23
H
23NO10 by analysis of its HRESIMS [m/z 474.1392 (M + H) ;
1
(
)
δ
H
/δ
C
) 0). HMQC and HMBC experiments were optimized for JCH
+0.8 mmu] and NMR data, which reflected a 14 mass unit (CH
2
)
n
140 Hz and JCH ) 8 Hz, respectively. Solvents for extractions and
1
13
decrease compared with that of 1. The H and C NMR data of 2
were almost identical with those of 1, except for the absence of
the methoxy group signals. Therefore, the methoxy group in 1 was
presumed to be replaced by an OH group in 2. Analysis of COSY,
HMQC, and HMBC data was consistent with the structure as
shown. The imine configuration of 2 was again proposed to have
open-column chromatography were reagent grade and used without
further purification. Solvents used for HPLC were analytical grade.
Flash column chromatography was carried out using Aldrich octadecyl-
functionalized silica gel (C18). HPLC separations were performed on a
Shiseido Capcell Pak C18 column (10 × 250 mm; 5 µm particle size)
with a flow rate of 2 mL/min. Compounds were detected by UV
absorption at 254 nm.
Specimen Collection and Identification. Stereocaulon alpinum was
collected and identified by one of us (J.H.Y.) from Barton Peninsula
around King Sejong Station (S 62°13.3′, W 58°47.0′) on King George
Island, Antarctica, in January 2003. Voucher specimens (reference L-5)
have been deposited in the Korea Polar Research Institute.
Isolation of Compounds 1-4. A dried sample of S. alpinum (50
g) was extracted with MeOH (1 L × 2) for 24 h. The resulting crude
MeOH extract (5.9 g) was subjected to C18-functionalized silica gel
flash column chromatography (3 × 15 cm), eluting with a stepwise
gradient of 20%, 40%, 60%, 70%, 80%, 90%, and 100% (v/v) MeOH
the E-geometry on the basis of a NOESY correlation of H
H-1′.
3
-11 to
The isomeric compound usimine B (3) was assigned the same
gross structure as 2 by analysis of its molecular formula,
+
C
23
H23NO10 [m/z 474.1393 (M + H) ; ∆ +0.7 mmu], and NMR
1
13
data. The H and C NMR data for 3 were almost identical with
those of 2. The only noticeable differences were slight changes in
the chemical shifts of the signals for C-2′, C-3′, C-4′, and C-5′.
Thus, 3 was suggested to be a geometrical isomer of 2, with the
imine group in 3 having a Z-geometry. This assignment was
consistent with the NOESY data, which showed no correlation
in H
was then subjected to semipreparative reversed-phase HPLC using a
gradient from 50 to 91% CH CN in H O (0.1% formic acid) over 49
min to yield 1 (7.8 mg; t ) 29.2 min). The fraction (56 mg) eluted
2
O (400 mL each). The fraction eluted with 70% MeOH (76 mg)
3
between H -11 and H-1′.
3
2
Usimines A-C are related to usnic acid (4), a well-known lichen
metabolite that is derived biosynthetically from oxidative phenolic
coupling. However, usimines 1–3 are unique usnic acid deriva-
tives possessing a nitrogen-bearing side chain that is presumably
derived from glutamic acid. Encountering secondary metabolites
with an imine group is interesting since this functional group is
R
with 60% MeOH in H
phase HPLC using a gradient from 40 to 69% CH
formic acid) over 29 min to yield 2 (3.6 mg; t ) 22.2 min) and 3 (2.0
mg; t
) 27.2 min). Application of open silica gel column (2.5 × 50
cm) chromatography utilizing stepwise gradient solution
(hexane-CH Cl -MeOH) on the fraction eluted at 100% MeOH from
the C18-functionalized silica gel flash column chromatography yielded
compound 4 (225.9 mg, eluted between 90% hexane-CH Cl and 100%
CH Cl ).
Usimine A (1): yellow gum; [R]
(CH OH) λ (log ꢀ) 297 (4.1), 216 (4.0); H NMR (pyridine-d , 400
2
O was subjected to semipreparative reversed-
2
,8
3
CN in H O (0.1%
2
R
R
a
9
2
2
generally relatively reactive and sensitive to hydrolysis. It is
unlikely that compounds 1–3 are produced artificially from usnic
acid and glutamic acid during the isolation process because HPLC
analysis of mixtures of usnic acid and glutamic acid that were
dissolved in MeOH or MeOH containing 0.1% formic acid, and
stirred at room temperature for 15 days, showed no chromatographic
changes.
2
2
2
2
25
D
2 2
+39 (c 0.77, CH Cl ); UV
1
3
max
5
MHz) δ 12.65 (OH-9), 6.15 (1H, s, H-4), 5.16 (1H, dd, J ) 13.6, 5.9
Hz, H-1′), 3.59 (3H, s, 4′-OCH ), 2.84 (3H, s, H -11), 2.79 (2H, m,
-3′), 2.66 (3H, s, H -13), 2.59 (2H, m, H -2′), 2.40 (3H, s, H -14),
, 100 MHz) δ 201.2 (C-12),
3
3
H
2
3
2
3
The absolute configurations of the chiral centers in 1–3 were
determined by comparison of optical rotations and Marfey’s
derivatization of the mixture from acid hydrolysis. Treatment of 1
with 3 N HCl in THF resulted in conversion of 1 to 4, as confirmed
by HPLC and H NMR analysis. The specific rotation of the
resulting sample of 4 (+125) was comparable with that of (+)-
usnic acid. Thus the absolute configuration of C-9b in 1 was
determined as R. The absolute configuration of the glutamic acid
residue was determined by acid hydrolysis (6 N HCl, 120 °C, 24 h)
of 1, followed by derivatization with Marfey’s reagent (1-fluoro-
1
3
1
1
1
3 5
.72 (3H, s, H -15); C NMR (pyridine-d
98.6 (C-1), 190.3 (C-3), 175.1 (C-10), 174.1 (C-4a), 173.0 (C-5′),
72.9 (C-4′), 163.9 (C-7), 158.9 (C-9), 156.5 (C-5a), 107.8 (C-8), 106.0
(
(
C-9a), 103.1 (C-2, C-4), 101.8 (C-6), 57.3 (C-9b), 57.0 (C-1′), 51.7
4′-OCH ), 31.8 (C-15), 31.2 (C-13), 30.0 (C-3′), 28.7 (C-2′), 19.2 (C-
1
3
5
11), 8.1 (C-14); HMBC data (pyridine-d , 400 MHz) H-4 f C-2, C-4a,
1
0
C-9b, C-15; H
C-8, C-9; H -15 f C-1, C-4a, C-9a, C-9b; H-1′ f C-10, C-2′, C-3′,
C-5′; H-2′ f C-1′, C-3′, C-5′; H-3′ f C-1′, C-2′, C-4′; 9-OH f C-8,
3 3 3
-11 f C-2, C-10; H -13 f C-6, C-12; H -14 f C-7,
3
1
C-9, C-9a; 4′-OCH
(OH-7), 11.67 (OH-9), 5.84 (1H, s, H-4), 4.69 (1H, m, H-1′), 2.67
3H, s, H -13), 2.65 (3H, s, H -11), 2.57 (2H, m, H –3′), 2.30 (2H, m,
-2′), 2.09 (3H, s, H -14), 1.71(3H, s, H -15); C NMR (CDCl , 100
MHz) δ 200.8 (C-12), 198.9 (C-1), 175.1 (C-4a), 172.6 (C-4′), 172.0
C-5′), 163.7 (C-7), 158.2 (C-9), 155.9 (C-5a), 108.4 (C-8), 104.9 (C-
3 3
f C-4′; H NMR (CDCl , 400 MHz) δ 13.33
1
1
2
,4-dinitrophnyl-5- L-alanine amide) and subsequent HPLC
(
H
3
3
2
analysis. By comparing the retention times of authentic standards
of L- and D-Glu, the hydrolysate was identified to contain L-Glu.
Thus, the absolute configuration of C-1′ in 1 was determined as S.
The absolute configurations of compounds 2 and 3 were presumed
to be analogous to that of 1.
Compounds 1–4 moderately inhibited the activity of PTP1B in
a dose-dependent manner, and their IC50 values were determined
as 15.0 ( 0.1, 27.7 ( 2.1, 23.2 ( 3.2, and 16.4 ( 0.4 µM,
respectively. A known phosphatase inhibitor, RK-682 (IC50 ) 4.5
1
3
2
3
3
3
(
9
3
a), 102.3 (C-4), 101.5 (C-6), 57.6 (C-9b), 55.3 (C-1′), 52.2 (OCH
2.0 (C-15), 31.4 (C-13), 29.3 (C-3′), 27.9 (C-2′), 18.9 (C-11), 7.6 (C-
, 400 MHz) H-4 f C-1, C-4a, C-9b, C-15;
-14 f C-7, C-8, C-9; H -15 f C-1, C-4a, C-9a,
C-9b; H-1′ f C-2′, C-5′; H-2′ f C-1′, C-3′, C-4′, C-5′; H-3′ f C-1′,
C-2′, C-4′; 7-OH f C-6, C-7, C-8; 9-OH f C-8, C-9; OCH f C-4′;
26NO10, 488.1557).
+159 (c 0.46, MeOH); UV
3
),
14); HMBC data (CDCl
-13 f C-6, C-12; H
3
H
3
3
3
3
+
1
2,13
HRESIMS m/z 488.1552 (M + H) (calc for C24H
25
(
0.5 µM), was employed as a positive control in the assay.
Usimine B (2): yellow gum; [R]
CH OH) λmax (log ꢀ) 297 (4.2), 216 (4.3); H NMR (pyridine-d
MHz) δ 6.08 (1H, s, H-4), 5.17 (1H, br dd, H-1′), 2.85 (2H, m, H
.83 (3H, s, H -11), 2.73 (2H, m, H -2′), 2.66 (3H, s, H -13), 2.37
(3H, s, H -14), 1.70 (3H, s, H -15); C NMR (pyridine-d , 100 MHz)
D
Inhibitors of PTP1B, a major nontransmembrane phosphotyrosine
phosphatase in human tissues and a negative regulator of the insulin-
1
(
3
5
, 400
-3′),
2
1
4
stimulated signal transduction pathway, are considered as potential
agents in efforts to develop new treatments for type 2 diabetes and
2
3
2
3
1
3
3
3
5
1
5–17
related metabolic syndromes.
δ 201.2 (C-12), 198.4 (C-1), 190.1 (C-3), 174.9 (C-10), 174.3 (C-4′),

7
12 Journal of Natural Products, 2008, Vol. 71, No. 4
Notes
1
73.9 (C-4a), 173.6 (C-5′), 164.1 (C-7), 158.9 (C-9), 156.4 (C-5a), 107.7
measured in a reaction mixture containing 2 mM p-nitrophenyl
phosphate (pNPP) in 50 mM citrate, pH 6.0, 0.1 M NaCl, 1 mM EDTA,
and 1 mM dithiothreitol (DTT). The reaction mixture was placed in a
30 °C incubator for 30 min, and the reaction was terminated by addition
of 1 N NaOH. The amount of p-nitrophenol produced was estimated
by measuring the increase in absorbance at 405 nm. The nonenzymatic
hydrolysis of 2 mM pNPP was corrected by measuring the increase in
(
C-8), 106.0 (C-9a), 103.1 (C-4), 102.9 (C-2), 101.7 (C-6), 57.6 (C-
1
′), 57.2 (C-9b), 31.8 (C-15), 31.4 (C-3′), 31.2 (C-13), 29.5 (C-2′),
9.3 (C-11), 8.0 (C-14); HMBC data (pyridine-d , 400 MHz) H-4 f
-11 f C-2, C-10; H -13 f C-6, C-12; H
-15 f C-1, C-4a, C-9a, C-9b; HRESIMS m/z
73.1392 (M + H) (calc for C23 24NO10, 474.1400).
Usimine C (3): yellow gum; [R]
CH OH) λmax (log ꢀ) 297 (4.2), 216 (4.3); H NMR (pyridine-d
MHz) δ 6.07 (1H, s, H-4), 5.16 (1H, br dd, H-1′), 2.93 (2H, m, H
.86 (3H, s, H -11), 2.68 (2H, m, H -2′), 2.66 (3H, s, H -13), 2.37
3H, s, H -14), 1.71 (3H, s, H , 100 MHz)
δ 201.1 (C-12), 198.4 (C-1), 190.1 (C-3), 175.1 (C-5′), 174.8 (C-10),
73.8 (C-4a), 173.5 (C-4′), 164.1 (C-7), 159.0 (C-9), 156.5 (C-5a), 107.7
C-8), 106.0 (C-9a), 103.1 (C-4), 103.0 (C-2), 101.7 (C-6), 57.6 (C-
1
5
C-2, C-4a, C-9b, C-15; H
3
3
3
-
1
4
4 f C-7, C-8, C-9; H
3
+
H
2
5
12,13
D
+162 (c 0.43, MeOH); UV
absorbance at 405 nm obtained in the absence of PTP1B enzyme.
1
(
3
5
, 400
-3′),
Acknowledgment. This work was supported by a grant from the
KOPRI Project (PE07050). Mass spectral data were kindly provided
by the Korea Basic Science Institute. We would like to thank Professor
J. B. Gloer of the University of Iowa for proofreading the manuscript
and for valuable suggestions.
2
2
(
3
2
3
1
3
3
3 5
-15); C NMR (pyridine-d
1
(
1
1
′), 57.2 (C-9b), 31.8 (C-15), 31.2 (C-13), 30.7 (C-3′), 29.3 (C-2′),
9.3 (C-11), 8.0 (C-14); HRESIMS m/z 473.1393 (M + H) (calc for
Supporting Information Available: 1D and 2D NMR spectra for
+
1
13
compound 1; H NMR and C NMR spectra for compounds 2 and 3.
This material is available free of charge on the Internet at http://
pubs.acs.org.
23
C H24NO10, 474.1400).
2
5
(
+)-Usnic acid (4): yellow needles; [R]
D
+300 (c 0.54, CH
2
Cl );
2
1H, C NMR data (CDCl
3
13
) comparable to published data; ESIMS m/z
6
3
+
45 (M + H) .
References and Notes
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dissolved in a mixture of THF (1 mL) and 3 N HCl (1.5 mL) and
heated (40 °C) for 12 h. After confirming the presence of 4 in the
reaction mixture by HPLC analysis, the reaction mixture was concen-
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CapcellPak C18 column, linear gradient from 30 to 60% CH CN in
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and D-Glu were derivatized with FDAA in the same manner as that of
. Retention times (min) of the FDAA amino acid derivatives used as
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O (1:1; 1 mL). An aliquot of this solution (20
(
(
(
[
3
H
2
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1
standards were as follows: L-Glu (32.7), D-Glu (33.9). The retention
time of the peak in the HPLC trace of the FDAA-derivatized hydrolysis
product of 1 was 32.4.
Assay Procedures. PTP1B (human, recombinant) was purchased
from BIOMOL Research Laboratories, Inc. The enzyme activity was
(
16) Dadke, S.; Chernoff, J. Curr. Drug Targets-Immune, Endocr. Metabol.
Disord. 2003, 3, 299–304.
(
17) Tonks, N. K. FEBS Lett. 2003, 546, 140–148.
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