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B604505g.dvi

www.rsc.org/obc | Organic & Biomolecular Chemistry Inotilone and related phenylpropanoid polyketides from Inonotus sp.
and their identification as potent COX and XO inhibitors

Hilaire V. Kemami Wangun,a Albert H¨artl,a Trinh Tam Kietb and Christian Hertweck*a,c
Received 28th March 2006, Accepted 9th May 2006
First published as an Advance Article on the web 24th May 2006
DOI: 10.1039/b604505g
By bioassay-guided isolation, phenylpropanoid-derived polyketides, including an unusual5-methyl-3(2H)-furanone derivative (inotilone) with potent cyclooxygenase (COX) and xanthoneoxidase (XO) inhibitory activities were obtained from the fruiting body of the mushroom Inonotus sp.
Introduction
Arthritis is a general term for severe inflammatory processes
in joints or joint tissue. Nonsteroidal anti-inflammatory drugs
(NSAIDs), such as diclofenac and indomethacin, have emerged
as the most commonly used anti-inflammatory agents for the
therapy of rheumatoid arthritis.1 Many of these drugs target
cyclooxygenases (COX), which catalyze the first two steps in the
biosynthesis of the prostaglandins from the substrate arachidonic
acid.2,3 In this context, the selective inhibition of enzyme subtypes,
COX-1 and COX-2, has become an important goal.4 In contrast
to rheumatoid arthritis, gouty arthritis is mediated by the crys-
tallisation of uric acid (UA) in the joints.5,6 Gout can be treated
with drugs that either increase the urinary excretion of UA, or with
xanthine oxidase (XO) inhibitors that block the terminal step of
UA biosynthesis.7,8 The purine analogue allopurinol is currently
the only XO inhibitor in clinical use. Unfortunately, it seems to be
associated with an infrequent but severe hypersensitivity.9 Thus,
the search for new potent inhibitors of these enzymes, which
could be useful as lead structures for new anti-inflammatory and
anti-arthritic therapeutics, plays a pivotal role. Here we report
on the isolation, structural elucidation and biological evaluation
of natural anti-inflammatory COX and XO inhibitors from the
mushroom Inonotus sp.
Results and discussion
Extracts from the fruiting body Inonotus sp. exhibited significantinhibitory activities against key enzymes involved in inflammatoryprocesses: 3a-HSD, COX and xanthine oxidase. Bioassay-guidedseparation of the combined crude ethanolic and CHCl /MeOH Structures of Inonotus sp. metabolites and model for their extracts of the fruiting body using open column and preparative biosynthesis. Key HMBC and NOESY correlations of 11.
HPLC yielded several phenolic compounds 11 (4 mg), 9 (20 mg),
5 (4 mg) together with the known compounds 4 (500 mg) and 7 (6
mg) (Scheme 1).
The main product from Inonotus sp. was identified as the known metabolite hispidin (4) by comparison of MS, IR and NMR data.10
aDept. Biomolecular Chemistry, Leibniz-Institute for Natural Products In addition to 4, another compound 5 with the same molecular
Research and Infection Biology, Beutenbergstr. 11a, 07745, Jena, Ger- formula (C H O ) was isolated. Also the 1H NMR spectrum of many. E-mail: christian.hertweck@hki-jena.de; Fax: INT+3641-656705; 5 showed signals similar to those of 4.10 However, the 13C NMR
spectrum, which showed a signal for a conjugated carbonyl at Centre of Biotechnology, Vietnam National University, 144 Xuan Thuy d 179.1, clearly established 5 as the tautomeric c-pyrone (iso-
cFriedrich-Schiller-University, Jena This journal is The Royal Society of Chemistry 2006 Org. Biomol. Chem., 2006, 4, 2545–2548 | 2545
The molecular formula of the second main product (9) was
Inhibitory activities of 4, 5, 7, 9, and 11 against 3-aHSD, COX-1,
determined as C H O based on HR-EIMS and its 13C NMR spectrum. Similar to 4 and 5, the 1H-NMR spectrum showed
signals attributable to the ABX spin coupling system of a trisubstituted phenyl moiety at d 6.77 (1H, d, J = 8.1 Hz, H- 12), d 7.02 (1H, dd, J = 8.2, 1.8 Hz, H-13), d 7.07 (1H, d, J = 1.8 Hz H-9), a trans disubstituted double bond at d 7.45 (1H, d, J = 15.8 Hz, H-7) and d 6.50 (1H, d, J = 15.8 Hz, H-6), and two exchangeable phenolic hydroxyl protons at d 9.15 and 9.65. In addition, a chelated proton at d 15.20 was detected. Analyses of Indomethacin
13 C, DEPT 135 and HMQC NMR spectra of 9 showed 14 carbon
Allopurinol
signals including six sp2 methines, four quaternary sp2 carbons(three of which are oxygenated), one methylene carbon at d 45.6, The structures of compounds 5, 9 and 11, as well as the isolation
a methoxy carbon at d 51.8, a carbonyl carbon at d 191.8, and a of the known 4 and 7 suggest that all metabolites share the same
carboxyl carbon at d 167.9. HMBC NMR spectra proved to be biosynthetic origin. All compounds represent linear or cyclized very helpful in defining their connectivities. The correlation of the polyketides derived from caffeyl-CoA (1). While 7 appears to
H-9 (d 7.07) with C-7 (d 141.0), C-8 (d 126.2), C-10 (d 145.6), and be a shunt product resulting from a premature release from the C-11 (d 148.4), the correlation of H-12 (d 6.77) with H-8, H-10, polyketide synthase, 4, 5, 9 and 11 are the result of two rounds
H-11, and H-13 and the correlation of H-13 (d 7.02) with C-7, C-8, of elongation. The structurally unusual 11 could be the product
C-9, C-11 and C-12, revealed an ortho substitution of the phenolic of a decarboxylation-radical ring closure sequence via the known hydroxyl protons. Other important information was obtained from metabolite hispolon 10.12 A related sequence could be involved
the observed correlation of the methylene protons (H-2) with C-1 in the formation of the tri- and tetrahydroxyaurone aglycones of (d 167.9), C-3 (d 191.8) and C-4 (d 100.3). Structural deductions sulfurein and cernuosides.13,14
from NMR data were supported by the IR spectrum of 9, which
All compounds were evaluated for their inhibitory activities in showed absorption bands for hydroxyl groups at 3183 cm−1, a hydroxysteroid dehydrogenase (3a-HSD), COX-1, COX-2 and XO conjugated carbonyl (1632 cm−1) a carboxyl group at 1733 cm−1, enzyme assays according to previously documented procedures.
and aromatic rings (1567, 1513 and 1435 cm−1). Consequently, 9
Their inhibitory potencies, expressed as IC represents the methyl ester of the open chain derivative of 4 or 5,
in Table 1 and are compared with those of the references, and was named inonotic acid methyl ester.
indomethacin and allopurinol. The results in the present study The molecular formula of compound 11 was determined as
demonstrated that the phenolic compounds exhibit strong COX C H O based on HR-EIMS and 13C NMR data. Similar to inhibitory effects with a prevalence for COX-2 in the case of and 9, the 1H NMR spectrum of 11 showed signals attributable to
the compounds 4, 7, 9 and 11. It should be highlighted that
the ABX spin coupling system of a trisubstituted phenyl moiety.
hispidin (4) and the novel inotilone (11) selectively inhibit COX-
Two olefinic protons at d 6.49 (1H, s, H-6), d 5.82 (1H, d, J = 2 at concentrations as low as those of the marketed selective 0.6 Hz, H-4) and a methyl group at d 2.39 (3H, s, H-13) were also inhibitors meloxicam and nimesulide.3 In all cases, except for
observed. Two proton signals were attributable to the phenolic compound 11, strong 3a-HSD inhibitory effects were noted, as
exchangeable hydroxyl protons. The 13C NMR and DEPT 135 well as moderate inhibitory effects toward XO, except hispidin spectra of 11 showed 11 sp2 carbon signals including five methines
(4), which exhibited an inhibitory activity at a level comparable
and five quaternary oxygenated carbons including one carbonyl.
with that of the standard allopurinol. As far as the tautomeric The occurrence of the carbonyl moiety was confirmed by the 13C compounds 4 and 5 are concerned, it seems that the a-pyrone is
spectrum, which showed one signal at d 186.6. The protonated carbons and their corresponding protons and the full connection In summary, we have isolated and characterized three new of compound 11 were established using HMQC and HMBC
phenylpropanoid polyketides with potent COX and XO inhibitory experiments, respectively. The correlation of the methyl proton activities from the mushroom Inonotus sp. Apart from their potent d 2.39 (3H, s, H-13) with C-2 (d 180.4), and C-3 (d 105.4), anti-arthritic activities, these metabolites represent new members and the correlation of the olefinic proton H-3 (d 5.82) with C- of caffeyl derived polyketides, out of which the structure of 4 (carbonyl moiety) and C-5 (d 144.3) unambiguously revealed a disubstituted dihydrofuranone moiety. The correlation of theolefinic proton H-6 (d 6.49) with C-4 (d 186.6), C-5 (d 144.3),C-7 (d 122.9), C-8 (d 117.9) and C-12 (d 124.7) enabled us to Experimental
connect the dihydrofuranone moiety with the rest of the molecule.
General experimental procedures
The configuration of the C-5 double bond was established basedon molecular modeling and NOESY, which showed a correlation IR spectra (film) were recorded on a JASCO FT/IR-4100 spec- between H-6 (d 6.49) and H-3 (d 5.82) and the correlation between trometer equipped with an ATR device. UV spectra were measured the protons H-8 (d 7.35) and H-12 (d 7.17) with the methyl with a Spericord 200 Carl Zeiss spectrometer. High-resolution protons H-13 (d 2.39). Thus the structure was established as 2- electron impact mass spectra (HR-EIMS) were recorded on an (3,4-dihydroxybenzylidene)-5-methyfuran-3-one, named inotilone AMD 402 double-focussing mass spectrometer with BE geometry.
(11). Only recently, related 5-methyl-3(2H)-furanone metabolites
NMR spectra were recorded on a Bruker Avance 500 DRX have been reported from Phellinus igniarius.11
spectrometer at 300.133 MHz for 1H and 75.475 MHz for 13C 2546 | Org. Biomol. Chem., 2006, 4, 2545–2548
This journal is The Royal Society of Chemistry 2006 in DMSO-d6. Chemical shifts are given in ppm relative to TMS 245 [M − H]−; HR-EIMS (found [M − H]−): 245.0464 calcd. for as internal standard. HSQC and NOESY (mixing time 0.7 s) data were obtained in the phase-sensitive mode TPPI. Columnchromatography was performed using silica gel (60, Merck; 0.063– Inonotic acid methyl ester (9).
0.2 lm) and Sephadex LH-20. HPLC was performed using a open column chromatography on Sephadex LH 20 using CHCl – Gilson binary gradient HPLC system equipped with a UV detector MeOH (v/v = 90 : 10) as eluent. Further purification was achieved (UV/VIS-151)(370 nm) using a preparative reverse phase C by HPLC using a water–acetonitrile gradient (95 : 5 to 5 : 95; (7 lm) column. TLC was carried out with silica gel 60 F 30 min) R = 20.5 min; UV (MeOH) k Spots were visualized by spraying with vanilline/H SO followed 3094, 1733, 1632, 1567, 1513, 1435, 1282, 1022, 974 cm−1; 1HNMR by heating. All solvents used were spectral grade or distilled prior (DMSO-d , 300 MHz) data see Table 2; 13C NMR (DMSO-d , 75 MHz) data see Table 2; m/z 277 [M − H]−; HR-EIMS (found[M − H]−): 277.0682 calcd. for C H O : 277.0707).
Inotilone (11).
Was obtained as a yellow oil by open column chromatography on Sephadex LH 20 using CHCl –MeOH (v/v = The fruiting body of Inonotus sp. was collected in Vietnam.
85 : 15) as eluent. Further purification was achieved by HPLC Its identity was verified by Prof. Trinh Tam Kiet from the using a water–acetonitrile gradient (95 : 5 to 5 : 95; 30 min); R = Mycological Research Center, Hanoi State University, Vietnam, 1588, 1435, 1287, 1014, 951 cm−1; 1HNMR (DMSO-d , 300 MHz) data see Table 2; 13C NMR (DMSO-d , 75 MHz) data see Table 2; Extraction and isolation
m/z 217 [M − H]−; HR-EIMS (found [M − H]−): 217.0495, calcd.
The fruiting body of Inonotus sp. (25 g dry weight) was cut into small species, dried and crushed. The resulting powder wasextracted three times with ethanol (2 L) and chloroform–methanol Biological assays
(1 : 1) (3 × 2 L, 3 days each). The extracts were subjected to silicagel chromatography (silica gel 60, Merck, 0.063∼0.1 mm, column The 3a-hydroxy steroid dehydrogenase assay (3-aHSD) was mea- 4 × 60 cm), using stepwise CHCl –MeOH (9 : 1, 8 : 2, 1 : 1 v/v) sured spectrophotometrically, and conducted according to the as eluent. Final purification was achieved by preparative HPLC method described by Penning.15 The inhibitory activities of the
(Spherisorb ODS-2 RP , 5 lm (Promochem), 250 × 25 mm, test compounds are indicated in terms of IC . Indomethacin was acetonitrile–H O (83 : 17 v/v), at a flow rate of 10 ml min−1 and UV detection at 372 nm). Yields: 500 mg of The peroxidative activity of cyclooxygenases I and II was 4, 4 mg of 5, 6 mg of
measured using luminol as a specific chemiluminescent substrate 7, 20 mg of 9, and 4 mg of 11.
according to the method described by Forghani et al.16 The
iso-Hispidin (5).
inhibitory activities of the test compounds are given in terms of chromatography on Sephadex LH20 using CHCl –MeOH 80 : 20 IC . Indomethacin was used as reference.
as eluent. Further purification was done by HPLC using gradient The xanthine oxidase activity was measured using lucigenin as (water–acetonitrile 95 : 5 to 5 : 95; 30 min) R = 14 min; UV the chemiluminescence substrate, and conducted according to the 248, 361 nm; IR (film) 3059, 1649, 1590, 1494, 1411, method described by Pierce et al.17 The inhibitory activities of the
1276, 1202, 1050, 1000 cm−1; 1H NMR (DMSO-d , 300 MHz) data test compounds are indicated in terms of IC . Allopurinol was see Table 2; 13C NMR (DMSO-d , 75 MHz) data see Table 2; m/z 1 H and 13C NMR dataa for compounds 5, 9, and 11
N
a Recorded in DMSO-d6.
This journal is The Royal Society of Chemistry 2006 Org. Biomol. Chem., 2006, 4, 2545–2548 | 2547
Acknowledgements
6 H. K. Choi, D. B. Mount and A. M. Reginato, Ann. Intern. Med., 2005, 143, 499.
This project has been financially supported by the European 7 G. Rastelli, L. Costantino and A. Albasini, J. Am. Chem. Soc., 1997, 119, 3007.
Community in the FP5 EUKETIDES programme. We thank 8 S. Ishibuchi, H. Morimoto, T. Oe, T. Ikebe, H. Inoue, A. Fukunari, M.
Mrs Schwinger and Mrs R ¨ohrig for their excellent assistance in Kamezawa, I. Yamada and Y. Naka, Bioorg. Med. Chem. Lett., 2001, 9 K. R. Hande, R. M. Noone and W. J. Stone, Am. J. Med., 1984, 76, 47.
10 L. R. Brady and R. G. Benedict, J. Pharm. Sci., 1972, 61, 318.
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2548 | Org. Biomol. Chem., 2006, 4, 2545–2548
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