《有机化学》课程教学资源（参考书籍）Nature pruduct commucations，An International Journal for Communications and Reviews Covering all Aspects of Natural Products Research，Pawan K. Agrawal.pdf_P11-P15

1076 Natural Product Communications Vol.1(12)2006 Piacente etal. prior to or at the time Furthe exessure to a syrupy)The crude ration corr o its EC 20%6)After monitoring by TLC [Si gel 4 inhibited the interaction between gpl20 and CD4 CHCl-MeOH(9:1)],the fractions were combined to by55%. give A (350 mg),B(150 mg),C (200 mg)and D (280 mg).Fractions A-D were submitted to HPLC on The activity exerted by betulin esters (9-11)is very a H-Bondapack C-18 column (30 cm x 7.8 mm i.d. ver t 8A-nD( 11 lower than that ted for salaspermic acid 10] 0 12 m4 min)fr =20 further a-OH group at C-2 (8 mg Rt=19 min)from C.1(7 mg Rt=8 min)2 (5 mg,Rt 12 min),3 (12 mg.Rt 10 min),4 (10 Experimental mg.Rt 15 min).5 (7 mg.Rt=11 min),6 (9 mg.Rt General 16.5 min).and 13(18 mg.Rt=7 min)from D rotations me Th spectra R on 61 of anti-Hctivty Bruker IFS-48 spectrometer.Melting with HIV-I d in RPMI determined using a Bausch Lomb apparatus 1640 with 10%fetal calf serum.Forty-tho sand c Accurate molecular weights were measured by a per microtiter plate well were mixed with 5-fold Voyager DE mass spectrometer Samples were dilutions of compounds prior to addition of 10 CCIDso units of virus and incubated for 5-6 days analyzed by natrix assisted laser ionization(MALDI)mass spectrometry. A mixture o Format ed from 2 days f solution -cyano- bition or acid (Si plat by estima ions from -infected cells and infected cell and Angiotensin III at 931.5154 Da as interna controls using the XTT-formazan method standards.ESI-MS analyses were performed using a ThermoFinnigan LCQ Deca XP Max ion trap mas Virus infectiviry assay:The total progeny virus was spectrometer equipped with Xcalibur softwar titrated in microtiter plates using double dilutions of er DRX-600 freshly upernatants and C8166 cells.The experiments spec T-f CD tite (CCID.)is ev sed as the re cal of the fo DOF-COSY. HMBC and ROESY TLC was performed or the silica gel F254 (Merek)plates,and reagent grade effects of compounds on virus infectivity HIV- chemicals(Carlo Erba)were used throughout. (10-10CCIDso)was incubated with test compound at 37C for Ih,the virus was serially diluted,and the Plant material infectivity end-point determined Maytenus macrocarpa was collected in the Ucayali Gp120-sCD4 interaction Department h CD4 al Sciences lerno.Italy bound to microtiter plate e wells at a concentration of 005 well.Various dilutions of com unds were Extraction and isolation:The dried and powdered bark of M.macrocarpa (310 g)was defatted with mixed with equal volumes of recombinant gpl20 (004 ug/ml)and added to CD4 coated wells Afte light petroleum and then extracted by maceration at incubation at 37C for 3-5 h,the binding of gp120 was detected using human anti-HIV serum and anti- human Ig conjugated to horseradish peroxidase

1076 Natural Product Communications Vol. 1 (12) 2006 Piacente et al. prior to or at the time of virus infection. Further experiments confirmed that it inhibited the binding of gp120 to sCD4 in a dose dependent manner. At a concentration corresponding to its EC50, compound 4 inhibited the interaction between gp120 and CD4 by 55%. The activity exerted by betulin esters (9-11) is very much lower than that reported for betulinic acid and its derivatives by Hashimoto et al. [8]. Also orthosphenic acid (13) showed an anti-HIV activity lower than that reported for salaspermic acid [10], from which it differs only by the occurrence of a further α-OH group at C-2. Experimental General procedures: Optical rotations were measured on a Jasco DIP 1000 polarimeter. UV spectra were obtained on a Beckman DU 670 spectrometer. IR measurements were obtained on a Bruker IFS-48 spectrometer. Melting points were determined using a Bausch & Lomb apparatus. Accurate molecular weights were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix assisted laser desorption ionization (MALDI) mass spectrometry. A mixture of analyte solution and α-cyano-4-hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions from ACTH (fragment 18-39) at 2465.1989 Da and Angiotensin III at 931.5154 Da as internal standards. ESI-MS analyses were performed using a ThermoFinnigan LCQ Deca XP Max ion trap mass spectrometer equipped with Xcalibur software. NMR experiments were performed on a Bruker DRX-600 spectrometer at 300 K. All the 2D-NMR spectra were acquired in CD3OD. Standard pulse sequence and phase cycling were used for DQF-COSY, HSQC, HMBC and ROESY spectra. TLC was performed on silica gel F254 (Merck) plates, and reagent grade chemicals (Carlo Erba) were used throughout. Plant material: Maytenus macrocarpa was collected in the Ucayali Region (Pucallpa), Peru. A voucher specimen is deposited at the Department of Pharmaceutical Sciences, Salerno, Italy. Extraction and isolation: The dried and powdered bark of M. macrocarpa (310 g) was defatted with light petroleum and then extracted by maceration at room temperature with CHCl3 until exhaustion. The CHCl3 extract was concentrated under reduced pressure to a syrupy consistency (2.5 g). The crude extract was chromatographed on a silica gel column using CHCl3 and increasing amounts of MeOH (up to 20%). After monitoring by TLC [Si gel plates, CHCl3-MeOH (9:1)], the fractions were combined to give A (350 mg), B (150 mg), C (200 mg) and D (280 mg). Fractions A-D were submitted to HPLC on a μ-Bondapack C-18 column (30 cm x 7.8 mm i.d., flow rate 2.0 mL/min) using MeOH: H2O in the ratio 85:15 for A-C and 3:1 for D (isocratic conditions). Pure 11 (12 mg, Rt = 12 min), 9 (8 mg, Rt = 15 min) and 10 (6 mg, Rt = 19 min) were obtained from A; 12 (9 mg, Rt = 24 min) from B; 7 (5 mg, Rt = 20 min), 8 (8 mg, Rt = 19 min) from C; 1 (7 mg, Rt = 8 min), 2 (5 mg, Rt = 12 min), 3 (12 mg, Rt = 10 min), 4 (10 mg, Rt = 15 min), 5 (7 mg, Rt = 11 min), 6 (9 mg, Rt = 16.5 min), and 13 (18 mg, Rt = 7 min) from D. Antiviral assays: The anti-HIV activity and toxicity of compounds 1-13 were assessed in C8166 cells infected with HIV-1MN. Cells were cultured in RPMI 1640 with 10% fetal calf serum. Forty-thousand cells per microtiter plate well were mixed with 5-fold dilutions of compounds prior to addition of 10 CCID50 units of virus and incubated for 5-6 days. Formation of syncytia was examined from 2 days post-infection. The inhibition of HIV-infection was determined by examining syncytia, by estimating antigen gp120 by ELISA, and by measuring cell viability for virus-infected cells and uninfected cell controls using the XTT-formazan method. Virus infectivity assay: The total progeny virus was titrated in microtiter plates using double dilutions of freshly collected supernatants and C8166 cells. The end point was determined by examining syncytia formation and by the XTT-formazan method. Virus titer (CCID50) is expressed as the reciprocal of the dilution that gave a 50% end point. To measure the effects of compounds on virus infectivity, HIV-1MN (104 -105 CCID50) was incubated with test compound at 37°C for 1h, the virus was serially diluted, and the infectivity end-point determined. Gp120-sCD4 interaction assay: Gp120-sCD4 interaction was measured by ELISA; sCD4 was bound to microtiter plate wells at a concentration of 0.05 μg/well. Various dilutions of compounds were mixed with equal volumes of recombinant gp120 (0.04 μg/mL) and added to CD4 coated wells. After incubation at 37°C for 3-5 h, the binding of gp120 was detected using human anti-HIV serum and antihuman Ig conjugated to horseradish peroxidase

Triterpenes from Maytenus macrocarpa Natural Product Communications Vol. 1 (12) 2006 1077 Using WIACALC (Pharmacia LKB), the percent inhibition was calculated from linear logarithmic plots using three concentrations of gp120 alone as standard. Macrocarpoic acid A (1) MP: 272-274ºC. [α]D: +48.2º (c 0.1, CHCl3). Rf : 0.6 (CHCl3-MeOH, 9:1). IR (KBr): 3480, 2970-2880, 1690, 1450, 1360, 1230, 1070 cm-1. 1 H NMR (600 MHz, MeOH): 0.82 (3H, s, Me-23), 0.94 (3H, s, Me-28), 1.00 (3H, s, Me-25), 1.03 (3H, s, Me-24), 1.05 (3H, s, Me-26), 1.26 (3H, s, Me-29), 1.33 (3H, s, Me-27), 3.19 (1H, dd, J = 3.5 and 11.5 Hz), 3.62 (1H, dd, J = 3.0 and 11.0 Hz), 5.24 (1H, t, J = 3.5). 13C NMR (150 MHz MeOH): 15.7 (CH3, C-25), 16.1 (CH3, C-24), 17.3 (CH3, C-26), 19.0 (CH2, C-6), 24.4 (CH2, C-11), 25.8 (CH3, C-28), 26.3 (CH3, C-27), 26.5 (CH2, C-15), 28.2 (CH2, C-2), 28.3 (CH3, C-23), 29.0 (CH2, C-16), 33.4 (CH2, C-7), 34.1 (CH3, C-29), 37.8 (C, C-10), 38.9 (CH2, C-1), 39.5 (C, C-17), 40.1 (C, C-4), 40.7 (CH2, C-21), 41.2 (C, C-8), 43.5 (C, C-14), 44.3 (CH2, C-19), 46.2 (C, C-20), 48.9 (CH, C-9), 49.9 (CH, C-18), 56.5 (CH, C-5), 78.8 (CH, C-22), 79.6 (CH, C-3), 123.5 (CH, C-12), 144.5 (C, C-13), 181.1 (C, C-30). ESI-MS: 473 [M + H+ ] , 495 [M + Na+ ] . HRMS-MALDI: m/z [M + Na+ ] calcd for C30H48NaO4 495.3450, found 495.3458. Macrocarpoic acid B (2) MP: 264-266ºC. [α]D: +76.2º (c 0.1, CHCl3). Rf : 0.7 (CHCl3-MeOH, 9:1). IR (KBr): 3450, 2980-2840, 1730, 1710, 1450, 1380, 1230, 1120 cm-1. 1 H NMR (600 MHz, MeOH): 0.95 (3H, s, Me-28), 1.09 (3H, s, Me-24), 1.11 (6H, s, Me-23, Me-26), 1.13 (3H, s, Me-25), 1.26 (3H, s, Me-29)1.33 (3H, s, Me-27), 3.63 (1H, dd, J = 3.0 and 11.0 Hz), 5.25 (1H, t, J = 3.5). 13C NMR (150 MHz MeOH): 15.5 (CH3, C-25), 17.2 (CH3, C-26), 20.3 (CH2, C-6), 21.7 (CH3, C-24), 24.0 (CH2, C-11), 25.8 (CH3, C-28), 26.2 (CH3, C-27), 26.3 (CH2, C-15), 26.6 (CH3, C-23), 26.7 (CH2, C-7), 29.0 (CH2, C-16), 34.3 (CH3, C-29), 35.0 (CH2, C-2), 37.8 (C, C-10), 39.5 (C, C-17), 40.1 (CH2, C-1), 40.6 (CH2, C-21), 40.5 (C, C-8), 43.4 (C, C-14), 44.2 (CH2, C-19), 46.0 (C, C-20), 48.0 (C, C-4), 47.5 (CH, C-9), 49.8 (CH, C-18), 56.0 (CH, C-5), 78.7 (CH, C-22), 123.4 (CH, C-12), 143.5 (C, C-13), 181.0 (C, C-30), 219.0 (C, C-3). ESI-MS: 471 [M + H+ ] , 493 [M + Na+ ] . HRMS-MALDI: m/z [M + Na+ ] calcd for C30H46NaO4 493.3294, found 493.3301. Macrocarpol A (7) MP: 310-312ºC. [α]D: +42.0º (c 0.1, CHCl3). Rf : 0.7 (CHCl3-MeOH, 9:1). IR (KBr): 3440, 2930, 1680, 1600, 1188 cm-1. 1 H NMR (600 MHz, MeOH): 0.88 (3H, s, Me-29), 0.96 (3H, s, Me-23), 0.97 (3H, s, Me-30), 1.01 (3H, s, Me-24), 1.08 (3H, s, Me-25), 1.09 (3H, s, Me-26), 1.17 (3H, s, Me-27), 3.08 (1H, d, J = 11.0 Hz, H-28a), 3.59 (1H, d, J = 11.0 Hz, H-28b), 4.58 (1H, dd, J = 3.5, 12.0 Hz), 5.21 (1H, t, J = 3.5, H-12), 6.32 (1H, d, J = 15.9), 6.83 (1H, d, J = 8.0, H-3’, H-5’), 7.50 (1H, d, J = 8.0, H-2’, H-6’), 7.64 (1H, d, J = 15.9), 13C NMR (150 MHz MeOH): 16.3 (CH3, C-25), 17.3 (CH3, C-26), 17.5 (CH3, C-24), 17.8 (CH3, C-29), 18.8 (CH2, C-6), 21.6 (C, C-30), 23.9 (CH3, C-27), 24.1 (CH2, C-16), 24.2 (CH2, C-11), 25.9 (CH2, C-2), 26.7 (CH2, C-15), 28.3 (CH3, C-23), 32.2 (CH2, C-21), 34.3 (CH2, C-7), 36.5 (CH, C-22), 37.7 (C, C-17), 38.0 (C, C-10), 38.7 (C, C-4), 39.8 (CH2, C-1), 40.7 (CH, C-20), 41.3 (CH, C-8), 41.4 (CH, C-19), 43.3 (C, C-14), 49.4 (CH, C-9), 55.3 (CH, C-18), 56.7 (CH, C-5), 70.5 (CH3, C-28), 115.8 (CH, C-3’, C-5’), 116.4 (CH, C-8’), 125.1 (CH, C-12), 127.3 (C, C-1’), 129.5 (CH, C-2’, C-6’), 140.8 (C, C-13), 143.8 (CH, C-7’), 157.4 (C, C-4’) 167.2 (C, C-9’). ESI-MS: 589 [M + H+ ] , 611 [M + Na+ ] . HRMS-MALDI: m/z [M + Na+ ] calcd for C39H56NaO4 611.4076, found 611.4082. Acknowledgments - The authors thanks Dr Juan de Dioz Zuniga Quiroz of Agro Selva Zuniga, Parque Caceres Dorregaray 86-C Pueblo Libre, Lima 21, Peru, for providing the plant material. References [1] Piacente S, De Tommasi N, Pizza C. (1999) Laevisines A and B: two new sesquiterpene-pyridine alkaloids from Maytenus laevis. Journal of Natural Products, 62, 161-163

1078 Natural Product Communications Vol. 1 (12) 2006 Piacente et al. [2] Muhammad I, El Sayed KA, Mossa JS, Al-Said MS, El-Feraly FS, Clark AM, Hufford CD, Oh S, Mayer AMS. (2000) Bioactive 12-oleanene triterpene and secotriterpene acids from Maytenus undata. Journal of Natural Products, 63, 605-610. [3] Chavez H, Estevez-Braun AE, Ravelo AG, Gonzalez AG. (1997) First examples of dammarane triterpenes isolated from Celastraceae. Tetrahedron, 53, 6465-6472. [4] Chavez H, Estevez-Braun AE, Ravelo AG, Gonzalez AG. (1998) Friedelane triterpenoids from Maytenus macrocarpa. Journal of Natural Products, 61, 82-85. [5] Chavez H, Callo N, Estevez-Braun AE, Ravelo AG, Gonzalez AG. (1999) Sesquiterpene polyolesters from the leaves of Maytenus macrocarpa. Journal of Natural Products, 62, 1576-1577. [6] Chavez H, Rodriguez G, Estevez-Braun AE, Ravelo AG, Estevez-Reyes R, Gonzalez AG, Fdez-Puente JL, Garcia-Gravalos D. (2000) Macrocarpins A-D, new cytotoxic nor-triterpenes from the leaves of Maytenus macrocarpa. Biorganic & Medicinal Chemistry Letters, 10, 759-762. [7] Perez-Victoria JM, Tincusi BM, Jimenez IA, Bazzocchi IL, Gupta MP, Castanys S, Gamarro F, Ravelo AG. (1999) New natural sesquiterpenes as modulators of daunomycin resistance in a multidrug-resistant Leishmania tropica line. Maytenus macrocarpa. Journal of Medicinal Chemistry, 42, 4388-4393. [8] Hashimoto F, Kashiwada Y, Cosentino LM, Chen CH, Garrett PE, Lee KH. (1997) Anti-AIDS agents-XXVII. Synthesis and antiHIV activity of betulinic acid and dihydrobetulinic acid derivatives. Biorganic & Medicinal Chemistry, 5, 2133-2143. [9] Sun IC, Chen CH, Kashiwada Y, Wu JH, Wang HK, Lee KH. (2002) Anti-AIDS agents 49. Synthesis, anti-HIV, and anti-fusion activities of IC9564 analogues based on betulinic acid. Journal of Medicinal Chemistry, 45, 4271-4275. [10] Chen K, Shi Q, Kashiwada Y, Zhand DC, Hu CQ, Jin JQ, Nozaki H, Kilkuskie R, Tramontano E, Cheng YC, McPhail DR, McPhail AT, Lee KH. (1992) Anti-Aids agents, 6. Salaspermic acid, an anti-HIV principle from Tripterygium wilfordii, and the structureactivity correlation with its related compounds. Journal of Natural Products, 55, 340-346. [11] Nozaki H, Suzuki H, Hirayama T, Kasai R, Wu RY, Lee KH. (1986) Antitumour triterpenes of Maytenus diversifolia. Phytochemistry, 28, 479-485. [12] Mahato SB, Kundu AP. (1994) 13C NMR spectra of pentacyclic triterpenoids – A compilation and some salient features. Phytochemistry, 37, 1517-1575. [13] Kaneda N, Pezzuto JM, Kinghorn AD, Farnsworth NR. (1992) Plant anticancer agents, L. Cytotoxic triterpenes from Sandoricum koetjape stems. Journal of Natural Products, 55, 654-659. [14] Nakagawa H, Takaishi Y, Fujimoto Y, Duque C, Garzon C, Sato M, Okamoto M, Oshikawa T, Ahmed SU. (2004) Chemical constituents from the Colombian medicinal plant Maytenus laevis. Journal of Natural Products, 67, 1919-1924. [15] Kutney JP, Hewitt GM, Lee G, Piotrowska K, Roberts M, Rettig SJ. (1992) Studies with tissue cultures of the Chinese herbal plant Tripterygium wilfordii. Isolation of metabolites of interest in rheumatoid arthritis, immunosuppression, and male contraceptive activity. Canadian Journal of Chemistry, 70, 1455-1480. [16] Silva GDF, Duarte LP, Vieira Filho SA, Doriguetto AC, Mascarenhas YP, Ellena J, Castellano EE, Cota AB. (2002) Epikatonic acid from Austroplenckia populnea: structure elucidation by 2D NMR spectroscopy and X-ray crystallography. Magnetic Resonance in Chemistry, 40, 366-370. [17] Rashid MA, Gray AI, Waterman PG. (1992) Coumarins from Phebalium tuberculosum ssp. megaphyllum and Phebalium filifolium. Journal of Natural Products, 55, 851-858. [18] Fuchino H, Satho T, Tanaka N. (1995) Chemical evaluation of Betula species in Japan. I. Constituents of Betula ermanii Chemical and Pharmaceutical Bulletin, 43, 1937-1942. [19] Gao H, Wu L, Kuroyanagi M, Harada K, Kawahara N, Nakane T, Umehara K, Hirasawa A, Nakamura Y. (2003) Antitumorpromoting constituents from Chaenomeles sinensis Koehne and their activities in JB6 mouse epidermal cells. Chemical and Pharmaceutical Bulletin, 51, 1318-1321. [20] Es Saady D, Delage C, Simon A, Chulia AJ. (1995) Antiproliferative effects of uvaol. Fitoterapia, 66, 366-369

New Oxidized 4-Oxo Fatty Acids from Hygrophorus discoxanthus Gianluca Gilardoni, Marco Clericuzio, Alberto Marchetti, Paola Vita Finzi, Giuseppe Zanoni and Giovanni Vidari* Dipartimento di Chimica Organica, University of Pavia, Via Taramelli 10, 27100 Pavia, Italy vidari@unipv.it. Received: July 24th, 2006; Accepted: August 28th, 2006 Dedicated to the memory of Professor Ivano Morelli. The results are reported from the first investigation of the secondary metabolites of the basidiomycete Hygrophorus discoxanthus (Fr.) Rea. Five new oxidized 4-oxo fatty acids (C16, C18) were isolated from the fruiting bodies and their structures established on the basis of their spectroscopic data and an ozonolysis experiment. Preliminary data indicate a moderate fungicidal activity, suggesting a possible function of these acids as chemical deterrents against mushroom parasites and predators. Keywords: Hygrophorus discoxanthus, Basidiomycetes, 4-oxo-fatty acids, fungicidal activities. In a search for new prototype (bioactive) agents from higher mushrooms (Basidiomycetes) [1], we were attracted by the species Hygrophorus discoxanthus (Fr.) Rea (fam. Hygrophoraceae) [2]. This is a mycorrhizal fungal species, growing solitary, scattered to gregarious in hardwood forests, particularly in the presence of Fagus trees, and fruiting in the fall. It is easily recognized by a whitish, viscid cap, with an ochreous-brown border, hence the name, and by the widely spaced, cream colored decurrent gills, turning rust-colored on rubbing. Our own field observations revealed that the fruiting bodies of H. discoxanthus are hardly ever attacked by either insects or parasitic fungi. Fungicidal 4-oxo-2-alkenoic fatty acids were recently isolated from H. eburneus (Bull.: Fr.) Fr. [3], and related cyclopentenone and cyclopentenedione derivatives were found in the extracts of various Hygrophorus species [4,5]. In addition to the common fungal sterol ergosterol and derivatives, the aroma components of various Hygrophorus species were investigated by GC-MS [6]; a ceramide was reported from a Chinese Hygrophorus species [7], malodorous indole derivatives were isolated from H. paupertinus A. H. Smith & Hesler [8], while muscaflavine and hygrophoric acid were identified as pigments of some Hygrophorus fruiting bodies [9]. No investigation of the secondary metabolites of H. discoxanthus has yet appeared in the literature. Along with the ecological observations, this prompted a study of the chemical constituents of this mushroom. To prevent undesired enzymatic reactions, the fresh fruiting bodies were frozen after collection and extracted with EtOAc at –20°C. The crude extract was subsequently partitioned between n-hexane and MeCN, and the residue from the more polar layer was separated by chromatography on multiple reverse-phase C-18 columns to give acids 1-5. Remarkably, these compounds exhibit brownochreous spots on C-18 TLC-plates sprayed with a sulfovanillin solution, followed by heating, and are thus well differentiable from the fungal ubiquitous oleic and linoleic acids, and methyl linoleate, of similar chromatographic polarity, which are detected as purple spots with the same reagent. In addition, TLC-spots of compounds 1 and 2 respond to UV light (fluorescence quenching at 254 nm). NPC Natural Product Communications 2006 Vol. 1 No. 12 1079 - 1084

1080 Natural Product Communications Vol.1(12)2006 Gilardonieral. Acids 1-5(Cor be divided between those （1-2 3.51 a mutua system (Figure 1).Additionally vicina indic ve some compe nd possess either an internal Z-configured double bond z-conngure d tha or a terminal one.Compounds 3-5 are optically npound 1 corresponds.from C-9 to C-18.to that active.Acid 1 was obtained as a whitish sticky solid of oleic acid.The remaining eight carbons were The UV spectrum showed an intense absorption band assembled as a .c-unsaturated y-oxocrotonate unit. at Ama 234 nm (Log s 4.34)attributable to a attached to C-9 by a C2 linker,on the basis of the π→r transition of a conjugated keto group.which abcu9ee6oa following NMR information.The proton doublets at in the IR pe a0. 06.75 15.7 Hz),which an IR bro 0280 ò129 and 19. em along with a strong band at 1693 cm the presence of an unsaturated carboxylic acid.These d double attributions were firmly confirmed by the signals at positioned bet the carboxylic and the 6 170.2 and 6 1881 in the'C NMR spectrum of 1 grouD. The carbon signal of the ketone displayed belonging to an unsaturated carboxylic group and an additional HMBC cross peaks with two other olefinic unsa group, respe methine resonances at 66.39 and 67.06 (IH each, trum sho an ion vicinal coupling JAB =15.9 Hz)constituting an the NMR E-configured double bond,which was joined to C-9 与 through molecular formula CHO and quarte hC6andC.9 (Figure2） Figure Selected HMBC The spectral data of compound 2 were closely related to 1,the most significant difference being the lack of evidence for an internal non-conjugated double bond In fact,the UV absorption band at =235nm along with the IR peaks at 1690 and 1 Figure Acids 1-5 isolated from Hygrophorus discox 664c2 o ear The upfield portion of the 'H NMR spectrum of te as co nd 1 compound I was typical of a long chain unsaturated From the mass spectral data,the length of the fatty acid chain in compound 2 could be determined as methy group oad signa .105 Ci6,while the terminal double bond was identified by to th 02 the signals from the three spin system at 65.83(1H icalofprot ddtJ=17.0,10.3,6.7Hz),64.95(1H,did,J=10.3 and a qu (C-ID COSY 1.8,1.5Hz)and65.02(1H,did,J=17.0,1.8,1.5 d HMBC (F lati Hz). oup was linked to a 1.2-disubstituted double bo whos caon signals were found at 6 127.1 and 311 und 3 was 131.6,respectively. The corresponding protons negative

1080 Natural Product Communications Vol. 1 (12) 2006 Gilardoni et al. Acids 1-5 (C16 or C18) can be divided between those (1-2) presenting an δ,ε-unsaturated γ-oxocrotonate partial structure and those (3-5) containing a chetol system (Figure 1). Additionally, some compounds possess either an internal Z-configured double bond or a terminal one. Compounds 3-5 are optically active. Acid 1 was obtained as a whitish sticky solid. The UV spectrum showed an intense absorption band at λmax = 234 nm (Log ε = 4.34) attributable to a π→π* transition of a conjugated keto group, which was corroborated by an intense absorption peak at about 1666 cm-1 in the IR spectrum. On the other hand, an IR broad band extending from 3600 to 2800 cm-1, along with a strong band at 1693 cm-1 revealed the presence of an unsaturated carboxylic acid. These attributions were firmly confirmed by the signals at δ 170.2 and δ 188.1 in the 13C NMR spectrum of 1, belonging to an unsaturated carboxylic group and an unsaturated carbonyl group, respectively. The negative ion ESI mass spectrum showed an ion at m/z 291 [M-H]- which, in accordance with data obtained from the NMR spectra, corresponded to the molecular formula C18H28O3. Figure 1: Acids 1-5 isolated from Hygrophorus discoxanthus. The upfield portion of the 1 H NMR spectrum of compound 1 was typical of a long chain unsaturated fatty acid, as indicated by the distorted triplet at δ 0.88, integrating for 3H, attributable to the ω1 methyl group, a broad signal at δ 1.10−1.45, integrating for 12H, assignable to the ω2−ω7 methylene protons, and a distorted quartet at δ 2.05 typical of an allylic methylene group (C-11). COSY and HMBC (Figure 2) correlations proved that this group was linked to a 1,2-disubstituted double bond, whose carbon signals were found at δ 127.1 and 131.6, respectively. The corresponding protons resonated as well separated doublets of triplets at δ 5.30 and δ 5.42, respectively, and showed a mutual vicinal coupling constant of 10.3 Hz, indicative of a Z-configured double bond. Comparison of these data with the literature [10] showed that the structure of compound 1 corresponds, from C-9 to C-18, to that of oleic acid. The remaining eight carbons were assembled as a δ,ε-unsaturated γ-oxocrotonate unit, attached to C-9 by a C2 linker, on the basis of the following NMR information. The proton doublets at δ 6.75 and δ 7.48 (1H each, JAB = 15.7 Hz), which showed HSQC correlations with the carbon signals at δ 129.8 and δ 139.7, respectively, and HMBC correlations (Figure 2) with the signals at δ 170.2 and δ 188.1, indicated an E-configured double bond positioned between the carboxylic and the carbonyl group. The carbon signal of the ketone displayed additional HMBC cross peaks with two other olefinic methine resonances at δ 6.39 and δ 7.06 (1H each, vicinal coupling JAB = 15.9 Hz) constituting an E-configured double bond, which was joined to C-9 through a CH2CH2 group. These two methylenes gave rise to two, well-resolved distorted quartets at δ 2.27 (H2-8) and δ 2.38 (H2-7), respectively, which showed two and three bond HMBC correlations with both C-6 and C-9 (Figure 2). OH O O Figure 2: Selected HMBC correlations of compound 1. The spectral data of compound 2 were closely related to 1, the most significant difference being the lack of evidence for an internal non-conjugated double bond. In fact, the UV absorption band at λmax = 235 nm, along with the IR peaks at 1690 and 1664 cm-1, and the almost superimposable patterns of the 1 H- and 13C NMR signals for the C(1)-C(6) moiety clearly proved that acid 2 contains the same E,E-configured δ,ε-unsaturated γ-oxocrotonate unit as compound 1. From the mass spectral data, the length of the fatty acid chain in compound 2 could be determined as C16, while the terminal double bond was identified by the signals from the three spin system at δ 5.83 (1H, ddt, J = 17.0, 10.3, 6.7 Hz), δ 4.95 (1H, dtd, J = 10.3, 1.8, 1.5 Hz), and δ 5.02 (1H, dtd, J = 17.0, 1.8, 1.5 Hz). The molecular formula C18H32O4 of compound 3 was deduced from the ion at m/z 311 [M–H]– in the negative ion ESI spectrum, combined with the O OH O O OH O O OH O OH O OH O OH O OH O OH 9 4 1 4 1 1 2 4 1 3 4 1 4 4 1 5 11