Four New Cryptoporic Acid Derivatives from the Fruiting Bodies ofCryptoporus sinensis, and Their Inhibitory Effects on Nitric Oxide Production

by Wen Wua)b), Feng Zhaoc), Rong Dingb), Li Baoa), Hao Gaod), Jin-Cai Lu*b), Xin-Sheng Yaod),Xiao-Qing Zhanga), and Hong-Wei Liu*a)

a) State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, No. 8Beiertiao, Zhongguancun, Haidian District, Beijing 100090, P. R. China

(phone: þ86-10-62566577; fax: þ86-10-62566511; e-mail: liuhw@im.ac.cn; lujincai99@yahoo.com.cn)b) College of Traditional Chinese Materia Medica, Shenyang Pharmaceutical University, No. 103

Wenhua-Road, Shenyang 110016, P. R. China (e-mail: lujincai99@yahoo.com.cn)c) School of Pharmacy, Yantai University, No. 32 QingQuan Road, Laishan District, Yantai 264005, P. R.

Chinad) Institute of Traditional Chinese Medicine and Natural Products, College of Pharmacy, Jinan

University, Guangzhou 510632, P. R. China

Four new drimane-type sesquiterpenoid ethers of isocitric acid, named cryptoporic acids J –M, wereisolated from the fruiting bodies of Cryptoporus sinensis, together with six known cryptoporic acids. Theirstructures were elucidated on the basis of extensive spectroscopic analyses. Among them, cryptoporicacid D showed strong inhibition against nitric oxide (NO) production in macrophages with an IC50 of45.8�3.6 mm, comparable to the positive control of hydrocortisone (IC50 of 40.6�2.5 mm).

Introduction. – Inedible mushrooms have been demonstrated to be a rich source ofbiologically active substances. Various secondary metabolites with characteristicpharmacological activities have been isolated from inedible mushrooms around theworld [1]. In the course of our chemical investigations on inedible mushrooms in China,fruiting bodies from Cryptoporus sinensis were collected from the Yunnan Province ofChina. The fungus C. sinensis was first described as new fungus and separated from C.volvatus in 2000 [2], and, to date, only two species, C. volvatus and C. sinensis, havebeen described in the genus Cryptoporus. The fruiting bodies of C. sinensis have beenused as herbal medicine for the treatment of asthma and bronchitis in southwest China.Previous chemical investigations of the metabolites from C. volvatus have resulted inthe identification of bitter drimane sesquiterpenoids, cryptoporic acids A– G, whichshowed inhibitory activities on superoxide anion-radical release, colon and skin cancerdevelopment in mouse, and activity against TNF-a release [3 – 9]. The secondarymetabolites of C. sinensis have rarely been studied.

To discover novel bioactive secondary metabolites from C. sinensis, a comprehen-sive chromatographic separation was conducted on its EtOH extract. As a result, tendifferent cryptoporic acids, including four new compounds, 1, 2, 5, and 6, were obtained(Fig. 1). Their structures were determined on the basis of 1D- and 2D-NMR, and MSdata along with comparison with literature. The inhibitory activity of these metabolitesagainst NO release from macrophages was also investigated. In this article, we reportthe isolation and structure elucidation of four new compounds, and their NO inhibitoryactivity.

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� 2011 Verlag Helvetica Chimica Acta AG, Z�rich

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Fig. 1. Structures of cryptoporic acids J, K, B, L, M, E, G, D (1–3, 5–8, and 10, resp.) , methyl cryptoporicacid B (4) , and demethyl cryptoporic acid D (9)

Results and Discussion. – Structure Elucidation. Cryptoporic acid J (1) was isolatedas a colorless oil. The molecular formula, C23H36O8, was determined by the positive-ion-mode HR-TOF-MS. The 1H-NMR spectrum of 1 showed three Me signals at d(H)0.75, 0.76, and 0.98, two MeO signals at 3.63 and 3.74, two broad singlets at d(H) 4.76and 4.83, characteristic of geminal olefinic H-atoms, as well as signals due to six CH2

and three CH groups (Table 1). The 13C-NMR spectrum revealed the presence of aC¼C bond (d(C) 108.6 and 148.1), three O-bearing C-atoms (d(C) 69.3, 79.4, and 79.8),and three COO C-atoms (d(C) 172.7, 173.0, and 174.8). 1H,1H-COSY Spectrumallowed the identification of the partial structures �CH2�CH2�CH�O�, �CH�CH2�CH2�C(¼CH2), �CH�CH2�O�, and �O�CH�CH�CH2�. The structure of 1was finally established by HMBC spectrum (Fig. 2). A drimenol substructure wasconfirmed by HMBC correlations from H�C(14) and H�C(15) to C(3), C(4), andC(5), from H�C(13) to C(1), C(5), C(9), and C(10), and from H�C(12) to C(7), C(8),and C(9). The HMBC correlations between H�C(1’), and C(4’) and C(5’); H�C(2’),and C(4’), C(5’), and C(6’); H�C(3’), and C(5’) and C(6’) indicated the presence of anisocitric moiety. The connection between the isocitric moiety and drimenol wasconfirmed by HMBC from H�C(11) to C(1’), and from H�C(1’) to C(11). Thepositions of the two MeO groups at C(5’) and C(4’) were determined by the HMBCcorrelations from the MeO group at d(H) 3.63, H�C(1’), and H�C(3’) to C(5’), and thecorrelations from the MeO group at d(H) 3.74 and H�C(1’) to C(4’). The relativeconfiguration of 1 was deduced to be the same as cryptoporic acid I by NOESYspectrum and comparison with literature [10]. Full structural assignments wereachieved by interpretation of the 1H,1H-COSY, HMQC, HMBC, and NOESY NMRspectral data (Table 1). The absolute configurations of C(1’) and C(2’) in the isocitricmoiety was proposed to be either (1’S,2’R) or (1’R,2’S) by comparing signals of H�C(1’)(d(H) 4.13) and H�C(2’) (d(H) 3.33) with those of the four diastereoisomers ofcryptoporic acid A methyl ester recently synthesized [9]. Further comparison of theoptical rotation value of 1 ([a]25

D ¼ þ16.2) and cryptoporic acid I ([a]D¼ þ17.0)confirmed the (1’R,2’S)-configuration of 1 [10]. Thus, the structure for compound 1 wasidentified as 4’,5’-dimethoxylcryptoporic acid I and named cryptoporic acid J.

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Fig. 1 (cont.)

The molecular formula of 2, C23H38O9, was determined by the positive-ion-modeHR-TOF-MS. The 1H- and 13C-NMR spectra of 2 were similar to those of 1, except forthe loss of the olefinic signals. Three Me signals at d(H) 0.72, 0.88, at 1.24, two MeOsignals at d(H) 3.64 and 3.76, two O-bearing CH2 signals at d(H) 2.99 (d, J¼11.2), 3.37(overlapped), 3.64 (dd, J¼9.2, 3.4), and 3.93 (dd, J¼9.2, 6.8), and a cluster of signalsdue to the isocitric moiety at d(H) 4.16 (d, J¼4.2), 3.35– 3.38 (m), 2.57 (dd, J¼17.2,5.4), and 2.79 (dd, J¼17.2, 9.2) were observed. The 13C-NMR spectrum exhibitedsignals of four O-bearing C-atoms at d(C) 70.8, 72.0, 74.1, and 80.2, and of three COOgroups at d(C) 172.7, 172.9, and 174.8. The HMBC correlations observed from H�C(12)to C(7), C(8), and C(9), from H�C(13) to C(1), C(5), C(9), and C(10), and fromH�C(14) to C(3), C(4), C(5), and C(15) in combination with 1H,1H-COSY spectralanalyses confirmed the presence of 8,15-dihydroxyldrimenol as the sesquiterpenoidportion of 2. The HMBC from H�C(11) to C(1’) and from H�C(1’) to C(11) connectedthe isocitrate part to C(11) via an ether linkage. In the HMBC NMR spectrum, thecorrelations from the MeO group at d(H) 3.63, H�C(1’), and H�C(3’) to C(5’), and thecorrelations from the MeO group at d(H) 3.74 and H�C(1’) to C(4’) confirmed the

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Table 1. 1H- and 13C-NMR Data (at 400 and 100 MHz, resp., in CD3OD) of Compounds 1 and 2. d inppm, J in Hz.

Position 1 2

d(H) d(C) d(H) d(C)

1 1.30–1.33 (m), 1.72 –1.75 (m) 38.6 1.07 (dd, J¼2.7, 12.8),1.75–1.77 (m)

40.9

2 1.61–1.63 (m) 28.5 1.49–1.51 (m), 1.67–1.70 (m) 18.93 3.20 (dd, J¼10.0, 5.5) 79.4 1.52–1.54 (m), 1.22–1.24 (m) 36.24 40.2 38.65 1.13 (dd, J¼12.5, 2.5) 55.8 1.28 (dd, J¼12.2, 2.9) 49.96 1.40–1.43 (m), 1.73 –1.75 (m) 24.7 1.32–1.35 (m), 1.55–1.57 (m) 20.77 2.02–2.05 (m), 2.37–2.40 (m) 38.4 1.54–1.56 (m), 1.75–1.78 (m) 43.98 148.1 74.19 1.90–1.93 (m) 56.7 1.57–1.60 (m) 61.2

10 39.5 38.611 3.53 (dd, J¼9.8, 3.8),

3.89 (dd, J¼9.8, 7.9)69.3 3.64 (dd, J¼9.2, 3.4),

3.93 (dd, J¼9.2, 6.8)70.8

12 4.76 (br. s), 4.83 (br. s) 108.6 1.24 (s) 24.913 0.75 (s) 15.8 0.88 (s) 17.114 0.76 (s) 16.2 0.72 (s) 17.915 0.98 (s) 28.9 2.99 (d, J¼11.2),

3.37 (overlapped)72.0

1’ 4.13 (d, J¼4.6) 79.8 4.16 (d, J¼4.2) 80.22’ 3.31–3.34 (m) 45.9 3.35–3.38 (m) 45.83’ 2.53 (dd, J¼17.2, 5.4),

2.72 (dd, J¼17.2, 9.0)32.8 2.57 (dd, J¼17.2, 5.4),

2.79 (dd, J¼17.2, 9.2)33.3

4’ 172.7 172.75’ 173.0 172.96’ 174.8 174.84’-MeO 3.74 (s) 52.5 3.76 (s) 52.65’-MeO 3.63 (s) 52.6 3.64 (s) 52.7

attachment of two MeO groups at C(5’) and C(4’), respectively. The relativeconfiguration of 2 was deduced from the NOESY NMR spectrum (Fig. 2). Theabsolute configurations of C(1’) and C(2’) in the isocitric moiety of 2 was assumed to bethe same as those in 1 by the NMR data comparison between 1 and 2, and considerationof the same biosynthetic origin. Full signal assignments were achieved by the analysesmentioned above, and the structure of compound 2, named cryptoporic acid K, wasdetermined.

Compound 5 was obtained as a colorless oil. The molecular formula C44H66O15 of 5was determined by the positive-ion-mode HR-TOF-MS. The 1H- and 13C-NMR spectraof 5 showed signals corresponding to four Me groups and two MeO groups, four olefinicH-atoms, four O-bearing CH2, two ester C¼O, and three COO groups. Its 1H- and13C-NMR spectra were quite similar to those of cryptoporic acid E [5], except for theloss of a MeO group. Compound 5 was deduced to be a dimer, like cryptoporic acid E,from the analyses above, which was supported by 1H,1H-COSY, HMQC, and HMBCanalyses (Table 2). The ester connection between the OH group at C(15’’) and theCOOH group at C(2’) was confirmed by HMBC correlations from H�C(15’’), H�C(1’),and H�C(3’) to C(5’). The positions of two MeO groups were deduced from HMBCcorrelations from the MeO group at d(H) 3.65, H�C(1’’’), and H�C(3’’’) to C(5’’’), andfrom the MeO group at d(H) 3.77 and H�C(1’) to C(4’). The relative configuration of 3was resolved by NOESY analyses (Fig. 2). To determine the absolute configuration in

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Fig. 2. Selected HMBC (H!C) and NOESY (H$H) correlations of compounds 1, 2, 5, and 6

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Table 2. 1H- and 13C-NMR (at 400 and 100 MHz, resp.) Data of Compounds 5a) and 6b). d in ppm, J in Hz.

Position 5c) 6d)

d(H) d(C) d(H) d(C)

1 1.20 –1.23 (m), 1.71 –1.73 (m) 39.7 1.68 –1.70 (m) 38.12 1.55 –1.57 (m), 1.61 –1.63 (m) 19.5 1.56 –1.58 (m) 18.23 1.29 –1.31 (m), 1.52 –1.54 (m) 36.5 1.43 –1.46 (m) 35.04 39.0 36.65 1.48 –1.51 (m) 49.2 1.52 –1.54 (m) 47.56 1.33 –1.34 (m), 1.55 –1.57 (m) 24.7 1.62 –1.64 (m) 23.37 2.07–2.10 (m), 2.32–2.35 (m) 38.4 2.03–2.06 (m), 2.35–2.37 (m) 37.68 148.2 146.59 2.06–2.08 (m) 56.6 2.00–2.03 (m) 54.8

10 39.6 38.311 3.57–3.59 (m), 3.92–3.94 (m) 69.4 3.62–3.65 (m),

3.86 (dd, J¼8.9, 8.9)67.8

12 4.76 (br. s), 4.83 (br. s) 108.6 4.69 (br. s), 4.85 (br. s) 106.613 0.79 (s) 16.2 0.73 (s) 15.414 0.73 (s) 18.1 0.73 (s) 17.415 2.98 (d, J¼11.1),

3.34 (d, J¼11.1)72.0 3.04 (d, J¼11.2),

3.42 (overlapped)71.1

1’ 4.12 (d, J¼4.0) 79.9 4.08 (d, J¼3.8) 78.32’ 3.39–3.41 (m) 46.2 3.41–3.43 (m) 43.93’ 2.57 (t, J¼4.4),

2.78 (dd, J¼17.2, 9.8)33.7 2.59 (dd, J¼17.6, 5.4),

2.81 (dd, J¼17.6, 9.1)31.7

4’ 172.6 172.75’ 172.5 171.26’ 175.1 174.11’’ 1.24 –1.27 (m), 1.70 –1.73 (m) 40.1 1.68 –1.70 (m) 38.52’’ 1.55 –1.57 (m), 1.61 –1.63 (m) 19.7 1.56 –1.58 (m) 18.33’’ 1.29 –1.31 (m), 1.52 –1.54 (m) 36.8 1.43 –1.46 (m) 35.24’’ 38.0 36.95’’ 1.48 –1.51 (m) 49.6 1.52 –1.54 (m) 47.66’’ 1.33 –1.35 (m), 1.62 –1.64 (m) 24.8 1.62 –1.64 (m) 23.37’’ 2.07–2.10 (m), 2.32–2.35 (m) 38.5 2.03–2.06 (m),

2.35–2.37 (m)37.6

8’’ 148.3 146.69’’ 2.00–2.03 (m) 57.1 2.00–2.03 (m) 55.3

10’’ 39.6 38.311’’ 3.57–3.59 (m), 3.92–3.94 (m) 69.0 3.62–3.65 (m),

3.98 (dd, J¼9.9, 9.9)68.1

12’’ 4.76 (br. s), 4.83 (br. s) 108.6 4.76 (br. s), 4.85 (br. s) 107.613’’ 0.79 (s) 16.4 0.76 (s) 15.514’’ 0.81 (s) 18.2 0.78 (s) 17.415’’ 3.58 (overlapped),

3.86 (d, J¼10.9)74.1 3.57 (d, J¼11.0),

3.93 (d, J¼11.0)72.7

1’’’ 4.08 (d, J¼4.8) 80.0 4.18 (d, J¼4.5) 78.02’’’ 3.31–3.34 (m) 45.8 3.50–3.52 (m) 44.23’’’ 2.53 (t, J¼4.4),

2.73 (dd, J¼17.2, 9.9)33.2 2.63 (dd, J¼17.6, 5.4),

2.86 (dd, J¼17.6, 9.1)32.0

4’’’ 174.1 172.85’’’ 173.3 171.9

the isocitric moieties, compound 5 was first hydrolyzed in 10% aqueous KOH, and thenmethylated with MeI to give a permethylated ester derivative. Its NMR data andoptical rotation was in accordance with those of methyl cryptoporic acid B, indicatingthe absolute configurations in the isocitric moieties as (1’R,1’’R,2’S,2’’S) [5]. On thebasis of the above data, the structure of 5, named cryptoporic acid L, was determined.

Compound 6 was obtained as a colorless oil. Its molecular formula C43H64O15 wasdetermined by the positive-ion-mode HR-TOF-MS. Analysis of its 1H- and 13C-NMRdata revealed structural features similar to those found in 5, except that the signals forthe MeO group at d(H) 3.65 were absent. The ester linkage between the OH group atC(15’’) and the COOH group at C(2’), and the position of the MeO group with thesignal at d(H) 3.69 was further confirmed by HMBC correlations (Fig. 2). 1H,1H-COSY, HMQC, and HMBC spectral analyses established the gross structure of 6 andenabled full structural assignment (Table 2). The relative configuration of 6 wasassigned on the basis of the NOESY correlations (Fig. 2). The same permethylatedester derivative, methylcryptoporic acid B, derived from compound 5, was alsoobtained from 6 when treated in the same manner, indicating the absoluteconfiguration of (1’R,1’’R,2’S,2’’S) in the isocitric moieties. Compound 6 was namedcryptoporic acid M.

Six known cryptoporic acids, but new to this species, were also isolated. Theirstructures were identified as cryptoporic acid B (3), methylcryptoporic acid B (4),cryptoporic acid E (7), cryptoporic acid G (8), demethylcryptoporic acid D (9), andcryptoporic acid D (10) by 1H- and 13C-NMR, and MS analysis, and comparison withliterature data [5] [7] [10] [11].

Biological Study. NO, a free radical, readily interacts with various substances, suchas aqueous oxygen, superoxide, transition metals, and iron or zinc�sulfur clusters, toaffect the functions of diverse types of cells. Overproduction of NO has been implicatedin the pathogenesis of many disorders, including inflammatory autoimmune diseasesand cancer [12] [13]. Therefore, inhibition of NO production in cells could controlinflammatory diseases and cancer. Compounds 1 –10 were evaluated for theirinhibitory activities of NO production from macrophage RAW 264.7. Compounds 3,5, 8, 9, and 10 inhibited the NO production with IC50 values less than 100 mm (Table 3).Compound 10 showed the strongest activity with an IC50 value of 45.8�3.6 mm, which iscomparable to the positive control of hydrocortisone (IC50 40.6�2.5 mm).

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Table 2 (cont.)

Position 5c) 6d)

d(H) d(C) d(H) d(C)

6’’’ 175.3 174.64’-MeO 3.77 (s) 52.5 52.05’’’-MeO 3.65 (s) 52.6 3.69 (s)

a) Recorded in CD3OD. b) Recorded in CDCl3/CD3OD 1 : 1. c) Signals for C(1–3), C(5–9) and C(12 –14) are interchangeable. d) Signals for C(1–3), C(5–8), and C(12–14) are interchangeable.

Conclusions. Chemical investigation of the fruiting bodies of C. sinensis led to theisolation of ten sesquiterpenoid derivatives, including cryptoporic acids J – M. Chemi-cally, cryptoporic acids have an unusual structure with drimane-type sesquiterpeneslinked to isocitric acid moiety by an ether bond. So far, this kind of natural products hasonly been found in the metabolites of polyporus fungi, i.e., C. volvatus [5], Polyporusarcularius, and P. ciliatus [11], and Ganoderma neo-japonicum [10]. In this study,compounds 1 – 10 were confirmed to inhibit NO production from macrophage, which,in part, contributes to the anti-inflammatory effects of C. sinensis as herbal medicine.

This work was supported by the National Key Basic Research Project of China (2009CB522300), theMOST (2007DFB31620) and grants from the National Natural Science Foundation of China (U0633008).

Experimental Part

General. TLC: Silica gel 60 F254 (SiO2); visualization by spraying with 10% H2SO4 and heating.Column chromatography (CC): LH-20 (Amersham Biosciences) and ODS (Lobar, 40–63 mm, Merck).Prep. HPLC: Agilent 1200 HPLC system with an ODS column (RP-18, 250�10 mm, Shimadzu Pak,5 mm; detector: UV) with a flow rate of 3.0 ml/min. Optical rotations: P-1020 digital polarimeter(JASCO). UV Spectra: Shimadzu UV2401PC UV/VIS spectrophotometer, in MeOH; lmax (log e) in nm.1H- and 13C-, and 2D-NMR spectra: Bruker AV-400 (400 MHz for 1H, 100 MHz for 13C) NMRspectrometer; d in ppm rel. to Me4Si as internal standard, J in Hz. ESI-MS: Bruker esquire 2000 massspectrometer. HR-TOF-MS: Bruker microTOF-Q instrument with the pos.-ion source.

Plant Material. The fruiting bodies of Cryptoporus sinensis were collected from the Yunnan Provinceof China in September 2009, and identified by X.-Q. Zhang, Institute of Microbiology, Chinese Academyof Sciences. A voucher specimen (LHW-2009–01) was deposited with the Institute of Microbiology,Chinese Academy of Sciences.

Extraction and Isolation. Dried fruiting bodies of C. sinensis (2.0 kg) were percolated with EtOH(3�10 l) at r.t. The combined filtrates were concentrated under reduced pressure to give 420 g of residue.The residue was dissolved in H2O (1 l) and partitioned with CHCl3 to yield CHCl3-soluble extract (CS-C ;40.6 g). A portion of CS-C (20 g) was subjected to CC (SiO2; CH2Cl2/MeOH 1 : 0!0 : 1) to afford tensubfractions, CS-C1–C10. The main Fr. CS-C2 (5.6 g) was selected for further separation. Fr. CS-C2 wasapplied to an ODS column eluted with a gradient of MeOH/H2O 40, 50, 60, 70, 80, 90, and 100% to giveeight subfractions, CS-C21–CS-C28. The Subfr. C23 (0.8 g) was first subjected to CC (SiO2; with CHCl3/acetone 10 : 1!0 : 1) to give eleven subfractions, C23-1–C23-11. Cryptoporic acid J (1; 6.0 mg; tR

12.8 min), 3 (10.6 mg; tR 14.8 min), and 4 (9.6 mg; tR 27.9 min) were isolated from Fr. C23-6 by areversed-phase (RP) HPLC column using 55% MeCN in 0.4‰ CF3COOH (TFA)/H2O as the mobilephase. Purification of the Fr. C23-11 by RP-HPLC (50% MeCN in 0.4‰ TFA/H2O) afforded cryptoporicacid K (2 ; 9.9 mg; tR 9.4 min). Fr. CS-C24 (1.5 g) was subjected to CC (SiO2; CHCl3/acetone 1 :0–0 : 1) togive 13 subfractions, CS-C24-1–CS-C24-13. Subfr. CS-C24-12 was subjected to a RP-HPLC (78% MeOHin 0.4‰ TFA/H2O) to give cryptoporic acid L (5 ; 16.8 mg; tR 17.6 min) and 8 (6.0 mg; tR 19.9 min). Subfr.

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Table 3. Effects of Compounds 1–10 on the NO Production Induced by LPS in Macrophages

Compound IC50 [mm] Compound IC50 [mm]

1 >100 7 >1002 >100 8 69.7�4.23 92.0�4.5 9 >1004 >100 10 45.8�3.65 86.4�3.8 Hydrocortisone 40.6�2.56 >100

CS-C24-13 was first isolated from an LH-20 column eluted with MeOH, and further purified by a RP-HPLC (85% MeOH in 0.4‰ TFA/H2O) to yield cryptoporic acid M (6 ; 6.0 mg; tR 19.8 min) andcompound 9 (6.0 mg; tR 13.0 min). Compounds 7 (15.6 mg, tR 30.9 min) and 10 (7.5 mg; tR 29.2 min) wereobtained from Fr. CS-C-24-5 by a RP-HPLC (80% MeOH in 0.4‰ TFA/H2O).

(3S,4R)-4-{[(1S,4aR,6S,8aS)-Decahydro-6-hydroxy-5,5,8a-trimethyl-2-methylidenenaphthalen-1-yl]-methoxy}-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic Acid (¼Cryptoporic Acid J ; 1) . Colorless oil.[a]25

D ¼ þ16.2 (c¼0.85, MeOH). UV (MeOH): 206 (3.80), 220 (sh, 2.06). 1H- and 13C-NMR: Table 1.ESI-MS (pos.): 919 ([2MþK]þ ). ESI-MS (neg.): 879 ([2M�H]� ). HR-TOF-MS (pos.): 463.2315([MþNa]þ , C23H36NaOþ

8 ; calc. 463.2302).(3S,4R)-4-{[(1S,2R,4aR,5R,8aS)-Decahydro-2-hydroxy-5-(hydroxymethyl)-2,5,8a-trimethylnaph-

thalen-1-yl]methoxy}-5-methoxy-3-(methoxycarbonyl)-5-oxopentanoic Acid (¼Cryptoporic Acid K ; 2) .Colorless oil. [a]25

D ¼ þ12.8 (c¼0.58, MeOH). UV (MeOH): 206 (3.95). 1H- and 13C-NMR: Table 1.ESI-MS (pos.): 955 ([2MþK]þ ). ESI-MS (neg.): 915 ([2M�H]� ). HR-TOF-MS (pos.): 481.2416([MþNa]þ , C23H38NaOþ

9 ; calc. 481.2408).(2R,3S)-2-{[(1S,4aR,5R,8aS)-5-({[(2S,3R)-2-(Carboxymethyl)-3-{[(1S,4aR,5R,8aS)-decahydro-5-

(hydroxymethyl)-5,8a-dimethyl-2-methylidenenaphthalen-1-yl]methoxy}-4-methoxy-4-oxobutanoyl]oxy}-methyl-decahydro)-5,8a-dimethyl-2-methylidenenaphthalen-1-yl]methoxy}-3-(methoxycarbonyl)pentane-dioic Acid (¼Cryptoporic Acid L ; 5). Colorless oil. [a]25

D ¼ þ38.5 (c¼0.76, MeOH). UV (MeOH): 206(4.25), 220 (sh, 2.45). 1H- and 13C-NMR: Table 2. ESI-MS (pos.): 858 ([MþNa]þ ). ESI-MS (neg.): 834([M�H]� ). HR-TOF-MS (pos.): 857.4298 ([MþNa]þ , C44H66NaOþ

15 ; calc. 857.4294).(2R,3S)-2-{[(1S,4aR,5R,8aS)-5-({[(2S,3R)-3-Carboxy-2-(carboxymethyl)-3-{[(1S,4aR,5R,8aS)-dec-

ahydro-5-(hydroxymethyl)-5,8a-dimethyl-2-methylidenenaphthalen-1-yl]methoxy}propanoyl]oxy}meth-yl)-decahydro-5,8a-dimethyl-2-methylidenenaphthalen-1-yl]methoxy}-3-(methoxycarbonyl)pentanedioicAcid (¼Cryptoporic Acid M ; 6) . Colorless oil. [a]25

D ¼ þ48.6 (c¼0.68, MeOH). UV (MeOH): 206(3.65), 220 (sh, 2.62). 1H- and 13C-NMR: Table 2. ESI-MS (pos.): 843 ([MþNa]þ ). ESI-MS (neg.): 819([M�H]� ). HR-TOF-MS (pos.): 843.4128 ([MþNa]þ , C43H64NaOþ

15 ; calc. 843.4137).Alkaline Hydrolysis and Permethylation of 5 and 6. Solns. of 5 and 6 (each, 5 mg) in 10% aq. KOH/

50% aq. dioxane 1 :1 (4 ml) were stirred at 508 for 1 h. After cooling in the air, the pH value was adjustedto 3 by addition of 10% HCl. The residue obtained after removal of solvent under N2 was dissolved inH2O (5 ml) and extracted with AcOEt/BuOH 1 : 1 (5 ml). After drying (anh. Na2SO4), the org. layer wasevaporated in vacuum to give a residue, which was further reacted with MeI (1 ml) and anh. K2CO3

(500 mg) in dry acetone (10 ml) under reflux for 12 h. The residue, after evaporation, was further purifiedby CC (SiO2; CHCl3/acetone 20 : 1) to give the corresponding methyl ether (methylcryptoporic acid B(4)). Oil. [a]25

D ¼ þ40.5 (c¼0.56, MeOH). ESI-MS (pos.): 477.6 ([MþNa]þ ). 1H-NMR (CD3OD): 0.73(s, H�C(13)); 0.78 (s, H�C(14)); 1.97 (H�C(9)); 2.08 (dt, J¼5.4, 12.9, H�C(7)); 2.36 (dq, J¼2.3, 13.1,H�C(7)); 2.97 (d, J¼11.1, H�C(15)); 3.35 (H�C(15)); 3.35 (H�C(2’)); 3.56 (dd, J¼9.9, 3.4, H�C(11));3.89 (dd, J¼9.8, 8.0, H�C(11)); 4.13 (d, J¼4.5, H�C(1’)); 4.75 (br. s, H�C(12)); 4.82 (br. s, H�C(12)).13C-NMR (CD3OD): 16.3 (C(13)); 18.2 (C(14)); 19.6 (C(2)); 24.7 (C(6)); 33.0 (C(3’)); 36.5 (C(3)); 38.4(C(7)); 39.6 (C(10)); 40.0 (C(1)); 45.8 (C(2’)); 49.0 (C(5)); 52.5 (MeO); 52.7 (MeO); 52.8 (MeO); 56.9(C(9)); 69.3 (C(11)); 72.0 (C(15)); 79.6 (C(1’)); 108.5 (C(12)); 148.3 (C(8)); 172.7 (CO); 173.8 (CO);172.8 (CO).

NO Inhibition Assay. Mouse monocyte-macrophages RAW 264.7 (ATCC TIB-71) were purchasedfrom the Chinese Academy of Sciences. RPMI 1640 Medium, penicillin, streptomycin, and fetal bovineserum (FBS) were purchased from Invitrogen (N.Y., USA). Lipopolysaccharide (LPS), DMSO, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT), and hydrocortisone were obtainedfrom Sigma Co. RAW 264.7 Cells were maintained in RPMI 1640 medium supplemented with penicillin(100 U/ml), streptomycin (100 mg/ml), and 10% heat-inactivated FBS at 378 in a humidified incubatorwith 5% CO2 and 95% air. The medium was routinely changed every 2 d. RAW264.7 Cells were passagedby trypsinization until they attained confluence, and were used for assays during the exponential growthphase. Compounds 1–10 were dissolved in DMSO and further diluted with the culture medium to give afinal DMSO concentration of 0.2% in the assay. This concentration of DMSO had no significant effect onthe growth of the cell line tested. Cell concentration was adjusted to 5�105 cells/ml, and 200 ml wereseeded in every well of a 96-well plate. After 1 h incubation, the cells were treated with 1 mg/ml of LPS

CHEMISTRY & BIODIVERSITY – Vol. 8 (2011) 1537

and various concentrations of test compounds for 24 h. Control groups received an equal amount ofDMSO. As a parameter of NO release, the nitrite concentration was measured in the supernatant ofRAW 264.7 cells by the Griess reaction. Briefly, 100 ml of culture medium in each well was transferred toanother plate, and the level of NO was assessed by measuring the accumulation of nitrite (NO�

2 ) using100 ml of Griess agent (mixture of 0.1% N-(naphthalen-1-yl)ethylenediamine in 5% H3PO4 and 1%sulfanilamide). The concentration of NO�

2 was calculated by a standard curve from 0, 1, 2, 5, 10, 20, 50,and 100 mm NaNO2 solns. The inhibitory rate of the compounds on NO production induced by LPS wascalculated by the NO�

2 levels [14]. Every experiment was performed in triplicate; data are expressed asmean�S.D. of three independent experiments.

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Received June 25, 2010

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