Hilaire V. Kemami Wanguna, Albert Härtla, Trinh Tam Kietb and Christian Hertweck*ac
aDept. Biomolecular Chemistry, Leibniz-Institute for Natural Products Research and Infection Biology, Beutenbergstr. 11a, 07745, Jena, Germany. E-mail: christian.hertweck@hki-jena.de; Fax: INT+3641-656705; Tel: INT+3641-656700
bCentre of Biotechnology, Vietnam National University, 144 Xuan Thuy Street, Hanoi, Vietnam
cFriedrich-Schiller-University, Jena
First published on 24th May 2006
By bioassay-guided isolation, phenylpropanoid-derived polyketides, including an unusual 5-methyl-3(2H)-furanone derivative (inotilone) with potent cyclooxygenase (COX) and xanthone oxidase (XO) inhibitory activities were obtained from the fruiting body of the mushroom Inonotus sp.
The main product from Inonotus sp. was identified as the known metabolite hispidin (4) by comparison of MS, IR and NMR data.10 In addition to 4, another compound 5 with the same molecular formula (C13H10O5) was isolated. Also the 1H NMR spectrum of 5 showed signals similar to those of 4.10 However, the 13C NMR spectrum, which showed a signal for a conjugated carbonyl at δ 179.1, clearly established 5 as the tautomeric γ-pyrone (iso-hispidin).
The molecular formula of the second main product (9) was determined as C14H14O6 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 δ 6.77 (1H, d, J = 8.1 Hz, H-12), δ 7.02 (1H, dd, J = 8.2, 1.8 Hz, H-13), δ 7.07 (1H, d, J = 1.8 Hz H-9), a trans disubstituted double bond at δ 7.45 (1H, d, J = 15.8 Hz, H-7) and δ 6.50 (1H, d, J = 15.8 Hz, H-6), and two exchangeable phenolic hydroxyl protons at δ 9.15 and 9.65. In addition, a chelated proton at δ 15.20 was detected. Analyses of 13C, DEPT 135 and HMQC NMR spectra of 9 showed 14 carbon signals including six sp2 methines, four quaternary sp2 carbons (three of which are oxygenated), one methylene carbon at δ 45.6, a methoxy carbon at δ 51.8, a carbonyl carbon at δ 191.8, and a carboxyl carbon at δ 167.9. HMBC NMR spectra proved to be very helpful in defining their connectivities. The correlation of the H-9 (δ 7.07) with C-7 (δ 141.0), C-8 (δ 126.2), C-10 (δ 145.6), and C-11 (δ 148.4), the correlation of H-12 (δ 6.77) with H-8, H-10, H-11, and H-13 and the correlation of H-13 (δ 7.02) with C-7, C-8, C-9, C-11 and C-12, revealed an ortho substitution of the phenolic hydroxyl protons. Other important information was obtained from the observed correlation of the methylene protons (H-2) with C-1 (δ 167.9), C-3 (δ 191.8) and C-4 (δ 100.3). Structural deductions from NMR data were supported by the IR spectrum of 9, which showed absorption bands for hydroxyl groups at 3183 cm−1, a conjugated carbonyl (1632 cm−1) a carboxyl group at 1733 cm−1, and aromatic rings (1567, 1513 and 1435 cm−1). Consequently, 9 represents the methyl ester of the open chain derivative of 4 or 5, and was named inonotic acid methyl ester.
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Scheme 1 Structures of Inonotus sp. metabolites and model for their biosynthesis. Key HMBC and NOESY correlations of 11. |
The molecular formula of compound 11 was determined as C12H10O4 based on HR-EIMS and 13C NMR data. Similar to 4, 5 and 9, the 1H NMR spectrum of 11 showed signals attributable to the ABX spin coupling system of a trisubstituted phenyl moiety. Two olefinic protons at δ 6.49 (1H, s, H-6), δ 5.82 (1H, d, J = 0.6 Hz, H-4) and a methyl group at δ 2.39 (3H, s, H-13) were also observed. Two proton signals were attributable to the phenolic exchangeable hydroxyl protons. The 13C NMR and DEPT 135 spectra of 11 showed 11 sp2 carbon signals including five methines and five quaternary oxygenated carbons including one carbonyl. The occurrence of the carbonyl moiety was confirmed by the 13C spectrum, which showed one signal at δ 186.6. The protonated carbons and their corresponding protons and the full connection of compound 11 were established using HMQC and HMBC experiments, respectively. The correlation of the methyl proton δ 2.39 (3H, s, H-13) with C-2 (δ 180.4), and C-3 (δ 105.4), and the correlation of the olefinic proton H-3 (δ 5.82) with C-4 (carbonyl moiety) and C-5 (δ 144.3) unambiguously revealed a disubstituted dihydrofuranone moiety. The correlation of the olefinic proton H-6 (δ 6.49) with C-4 (δ 186.6), C-5 (δ 144.3), C-7 (δ 122.9), C-8 (δ 117.9) and C-12 (δ 124.7) enabled us to connect the dihydrofuranone moiety with the rest of the molecule. The configuration of the C-5 double bond was established based on molecular modeling and NOESY, which showed a correlation between H-6 (δ 6.49) and H-3 (δ 5.82) and the correlation between the protons H-8 (δ 7.35) and H-12 (δ 7.17) with the methyl protons H-13 (δ 2.39). Thus the structure was established as 2-(3,4-dihydroxybenzylidene)-5-methyfuran-3-one, named inotilone (11). Only recently, related 5-methyl-3(2H)-furanone metabolites have been reported from Phellinus igniarius.11
The structures of compounds 5, 9 and 11, as well as the isolation of the known 4 and 7 suggest that all metabolites share the same biosynthetic origin. All compounds represent linear or cyclized polyketides derived from caffeyl-CoA (1). While 7 appears to be a shunt product resulting from a premature release from the polyketide synthase, 4, 5, 9 and 11 are the result of two rounds of elongation. The structurally unusual 11 could be the product of a decarboxylation-radical ring closure sequence via the known metabolite hispolon 10.12 A related sequence could be involved in the formation of the tri- and tetrahydroxyaurone aglycones of sulfurein and cernuosides.13,14
All compounds were evaluated for their inhibitory activities in hydroxysteroid dehydrogenase (3α-HSD), COX-1, COX-2 and XO enzyme assays according to previously documented procedures. Their inhibitory potencies, expressed as IC50 values, are shown in Table 1 and are compared with those of the references, indomethacin and allopurinol. The results in the present study demonstrated that the phenolic compounds exhibit strong COX inhibitory effects with a prevalence for COX-2 in the case of the compounds 4, 7, 9 and 11. It should be highlighted that hispidin (4) and the novel inotilone (11) selectively inhibit COX-2 at concentrations as low as those of the marketed selective inhibitors meloxicam and nimesulide.3 In all cases, except for compound 11, strong 3α-HSD inhibitory effects were noted, as well as moderate inhibitory effects toward XO, except hispidin (4), which exhibited an inhibitory activity at a level comparable with that of the standard allopurinol. As far as the tautomeric compounds 4 and 5 are concerned, it seems that the α-pyrone is more active than the γ-pyrone.
Compound | IC50/µM | ||||
---|---|---|---|---|---|
3α-HSD | COX-1 | COX-2 | COX-2/COX-1 | XO | |
4 | 8.1 | 0.01 | 8 × 10−4 | 0.08 | 4.4 |
5 | 12.1 | 0.05 | 0.13 | 2.6 | 13.8 |
7 | 8.9 | 0.03 | 0.01 | 0.3 | 10.1 |
9 | 16.1 | 0.46 | 0.21 | 0.4 | 7.1 |
11 | 50.4 | 0.36 | 0.03 | 0.08 | 9.1 |
Indomethacin | 15.4 | 0.10 | 6.00 | 60 | n.a. |
Allopurinol | n.a. | n.a. | n.a. | n.a. | 4.4 |
In summary, we have isolated and characterized three new phenylpropanoid polyketides with potent COX and XO inhibitory activities from the mushroom Inonotus sp. Apart from their potent anti-arthritic activities, these metabolites represent new members of caffeyl derived polyketides, out of which the structure of inotilone is most notable.
N° | 5 | 9 | 11 | |||
---|---|---|---|---|---|---|
δ1H (J/Hz) | δ13C | δ1H (J/Hz) | δ13C | δ1H (J/Hz) | δ13C | |
a Recorded in DMSO-d6. | ||||||
1 | 167.9 | |||||
2 | 165.4 | 3.55 s | 45.6 | 180.4 | ||
3 | 4.42 d (1.2) | 86.5 | 191.8 | 5.82 q | 105.5 | |
4 | 179.1 | 5.91 s | 100.3 | 186.6 | ||
5 | 5.59 d (1.2) | 109.0 | 178.3 | 144.3 | ||
6 | 156.1 | 6.50 d (15.8) | 118.6 | 6.49 s | 111.9 | |
7 | 6.12 d (15.8) | 118.5 | 7.45 d (15.8) | 141.0 | 122.9 | |
8 | 6.87 d (15.8) | 130.8 | 126.2 | 7.35 d (2.0) | 117.9 | |
9 | 127.4 | 7.07 d (1.8) | 114.7 | 145.4 | ||
10 | 6.94 d (1.5) | 113.5 | 145.6 | 148.1 | ||
11 | 145.6 | 148.4 | 6.80 d (8.2) | 115.9 | ||
12 | 146.5 | 6.77 d (8.1) | 115.7 | 7.17 dd (8.2, 2.0) | 124.7 | |
13 | 6.70 d (8.1) | 115.7 | 7.02 dd (8.1,1.8) | 121.5 | 2.39 s | 15.67 |
14 | 6.82 dd (8.1,1.5) | 119.2 | ||||
1′ | 3.65 s | 51.8 |
The peroxidative activity of cyclooxygenases I and II was measured using luminol as a specific chemiluminescent substrate according to the method described by Forghani et al.16 The inhibitory activities of the test compounds are given in terms of IC50. Indomethacin was used as reference.
The xanthine oxidase activity was measured using lucigenin as the chemiluminescence substrate, and conducted according to the method described by Pierce et al.17 The inhibitory activities of the test compounds are indicated in terms of IC50. Allopurinol was used as the reference.
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