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SUPPLEMENTARY DATABioactive Constituents of Clausena lansium and

a Method for Discrimination of Aldose Enantiomers

De-Yang Shen a, Chih-Hua Chao b, Hsiu-Hui Chan b, Guan-Jhong Huang c, Tsong-

Long Hwang d, Chin-Yu Lai e, Kuo-Hsiung Lee b,e, Tran Dinh Thang f,

and Tian-Shung Wu a, b,g ,*

a Department of Chemistry, National Cheng Kung University, Tainan 70101, Taiwanb Chinese Medicine Research and Development Center, China Medical University and Hospital, Taichung 40402, Taiwan

c Institute of Chinese Pharmaceutical Science and Chinese Medicine Resources, China Medical University, Taichung 40402, Taiwan

d Graduate Institute of Natural Products, College of Medicine, Chang Gung University, Taoyuan 333, Taiwan

e Natural Products Research Laboratories, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina 27599-7568,USA

f Department of Chemistry, Vinh University, Vinh City, Vietnamg Department of Pharmacy, China Medical University, Taichung 40402, Taiwan

* Corresponding author. Tel.: 886-6-2757575 ext 65333. Fax: 886-6-2740552.

E-mail address: [email protected] (T. S. Wu)

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Supporting Information Content:

S1. Extraction and isolation of all new and known compounds.S2. Identities and reference material for the 50 isolated known compounds.S3. Table 5. Cell viability and effect of compounds 737 on LPS-induced NO

production in macrophages.S4. Table 6. Effect of compounds 7, 2427, 31, and 3637 on LPS-induced TNF-

production in macrophages.S5. The 1H NMR spectrum of 1 (acetone-d6, 500 MHz)S6. The 13C NMR spectrum of 1 (acetone-d6, 125 MHz)S7. The HR-ESI spectrum of 1S8. The 1H NMR spectrum of 2 (acetone-d6, 500 MHz)S9. The 13C NMR spectrum of 2 (acetone-d6, 125 MHz)S10. The HR-ESI spectrum of 2S11. The 1H NMR spectrum of 3 (CDCl3, 300 MHz)S12. The 13C NMR spectrum of 3 (CDCl3, 75 MHz)S13. The HR-ESI spectrum of 3S14. The 1H NMR spectrum of 4 (CDCl3, 400 MHz)S15. The 13C NMR spectrum of 4 (CDCl3, 100 MHz)S16. The HR-ESI spectrum of 4S17. The CD spectrum of 4S18. The 1H NMR spectrum of 5 (acetone-d6, 400 MHz)S19. The 13C NMR spectrum of 5 (acetone-d6, 100 MHz)S20. The HR-ESI spectrum of 5S21. The CD spectrum of 5S22. The 1H NMR spectrum of 7a (CDCl3, 400 MHz)S23. The 13C NMR spectrum of 7a (CDCl3, 100 MHz)S24. The HR-ESI spectrum of 7aS25. The 1H NMR spectrum of 7b (CDCl3, 400 MHz)S26. The 13C NMR spectrum of 7b (CDCl3, 100 MHz)S27. The HR-ESI spectrum of 7bS28. Separation of the o-toylthiocarbamate derivatives derived from seven aldoses

using the elution system reported in the literature.S29. Separation of the o-toylthiocarbamate derivatives derived from ten aldoses using

the modified elution system.

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S1. Extraction and Isolation of New and Known Compounds

Air-dried and powdered stem of C. lansium (5 kg) was extracted with MeOH (6 × 20 L) under reflux for 8 h and concentrated to give brown syrup (420 g). The extract was suspended in H2O and then partitioned successively with CHCl3 (3 × 2 L). The CHCl3

extract (127 g) was subjected to column chromatography, eluted with n-hexane–acetone (5:1), to afford six fractions. Fraction 3 was chromatographed on silica gel with n-hexane–EtOAc (9:1) to give five subfractions (Subfr. 3.1–3.5). Subfr. 3.4 was chromatographed on silica gel with n-hexane–diisopropyl ether (3:1) to obtain mellein (2.1 mg). Subfr. 3.5 was chromatographed on silica gel with n-hexane–EtOAc (5:1) to give isoimperatorin (9) (1.2 mg), osthol (24) (3.1 mg), claulamine A (4) (2.1 mg), and imperatorin (8) (2 g). Fraction 4 was chromatographed on silica gel using n-hexane–EtOAc (5:1) to yield seven subfractions (Subfr. 4.1–4.7). Subfr. 4.3 was chromatographed on silica gel with n-hexane–diisopropyl ether (2:1) to give (+)-(E)--santalen-12-oic acid (10) (3.1 g), 6-methoxyheptaphylline (26) (0.3 mg), indizoline (7) (12 mg), and alloisoimperatorin (34) (1.0 mg). Subfr. 4.4 was chromatographed on silica gel with n-hexane–diisopropyl ether (2:1) to yield methyl carbazole-3-carboxylate (29) (3.4 mg), lansiumarin A (28) (4.1 mg) and 8-geranyloxypsolaren (11) (3.1 mg). Subfr. 4.5 was chromatographed on silica gel with n-hexane–acetone (1:1) to afford 3-formyl-9H-carbazole (12) (2.5 mg), N-phenethylcinnaamide (13) (1.4 mg), lansiumarin C (1.3 mg), 3-formyl-2-methoxy-9H-carbazole (27) (0.9 mg), dictamnine (30) (1.6 mg), xanthotoxol (20) (3.2 mg), N-phenethylbenzamide (14) (0.7 mg), clausenaline A (3) (1.2 mg), (E)-8-(6-hydroperoxy-3,7,-dimethylocta-2,7-dienyloxy)psoralen (1.2 mg), and mafaicheenamine A (6) (14.7 mg). Fraction 5 was subjected to column chromatography on silica gel using n-hexane–acetone (3:1) to give five subfractions (Subfr. 5.1–5.5). Subfr. 5.4 was chromatographed on silica gel with chloroform–MeOH (49:1) to give five subfractions (Subfr. 5.4.1–5.4.5). Subfr. 5.4.2 was chromatographed on silica gel with n-hexane–EtOAc (2:1) to give wampetin (35) (2.1 mg). Subfr. 5.4.3 was chromatographed on silica gel with n-hexane–EtOAc (2:1) to give dihydroalatamide (19) (1.4 mg), isoheraclenin (22) (0.8 mg), 2′,3′-epoxyanisolactone (0.4 mg), -fagarine (1.1 mg), (E,E)-8-(7-hydroxy-3,7-dimethyocta-2,5-dienyloxy)psoralen (1.7 mg), 3-formyl-6-methoxycarbazole (25) (1.9 mg), isogospherol (23) (1.3 mg), and 8-(3-chloro-2-hydroxy-3-methylbutoxy)psoralen (2.4 mg). Subfr. 5.4.5 was chromatographed on silica gel with n-hexane–acetone (2:1) to give marmesin (1.2 mg), N-(2-hydroxy-2-phenylethyl)-3-phenyl-2-propenamide (1.4 mg), tembamide (18) (2.0 mg), t-O-methylheraclenol (1.9 mg), and claulamine B (5) (3.7 mg). Subfr. 5.5 was chromatographed on silica gel with diisopropyl ether–MeOH (9:1) to give clausine D (31) (1.3 mg). Fraction 6 was

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subjected to column chromatography on silica gel using CHCl3–MeOH (14:1) to yield 4-methoxyquinolin-2-one (21) (2.2 mg). The H2O-soluble layer (208 g) was directly subjected to Diaion HP-20 column chromatography using H2O containing increasing proportions of MeOH to give seven fractions. Fraction 2 was chromatographed on silica gel using CHCl3–MeOH–H2O (3:1:0.05) to obtain uracil (0.9 mg). Fraction 3 was chromatographed on silica gel using CHCl3–MeOH–H2O (60:20:1) to afford isotachioside (15) (1.1 mg), nicotinamide (1.4 mg), and adenosine (0.8 mg). Fraction 4 was chromatographed on silica gel using CHCl3–MeOH–H2O (80:20:1) to afford scopolin (1.9 mg), haploperoside A (0.6 mg), and 4-hydroxybenzoic acid (0.3 mg). Fraction 5 was chromatographed on RP-18 using MeOH–H2O (3:1) to give clausenoside A (1) (2.4 mg), clausenoside B (2) (1.6 mg), vanillic acid (33) (1.2 mg), isorutarin (1.3 mg), methyl salicylate D-glucoside (16) (0.8 mg), and xanthotoxol 8-O--D-glucopyranoside (17) (1.6 mg). Fraction 6 was chromatographed on RP-18 using MeOH–H2O (1:1) to give 4-hydroxycinnamic acid (32) (2.1 mg) and marmesin 4′-O--D-apiofuranosyl-(1→6)--D-glucopyranoside (1.4 mg). Fraction 7 was chromatographed on silica gel using CHCl3–MeOH–H2O (5:1:0.05) to obtain nodakenin (1.9 mg).

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S2. Identities and Reference Material for the 50 Isolated Known Compounds.

Mafaicheenamine A (6): Maneerat, W., Laphookhieo, S., 2010. Antitumoral alkaloids from Clausea lansium. Heterocycles 81, 12611269.

Indizoline (7): Li, W.S., McChesney, J.D., El-Feraly, F.S., 1991. Carbazole alkaloids from Clausena lansium. Phytochemistry 30, 343–346.

Imperatorin (8): Ngadjui, B.T., Ayafor, J.F., Sondengam, B.L., Connolly, J.D., 1989. Coumarins from Clausena anisata. Phytochemistry 28, 585–589.

Isoimperatorin (9): Bergendorff, O., Dekermendjian, K., Nielsen, M., Shan, R., Witt, R., Ai, J., Sterner, O., 1997. Furanocoumarins with affinity to brain benzodiazepine receptors in vitro. Phytochemistry 44, 1121–1124.

(＋)-(E)--Santalen-12-oic acid (10): Coates, R.M., Denissen, J.F., Juvik, J.A., Babka, B.A., 1988. Identification of -santalenoic and endo--bergamotenoic acids as moth oviposition stimulants from wild tomato leaves. J. Org. Chem. 53, 2186–2192.

8-Geranyloxypsolaren (11): Kitajima, J., Okamura, C., Ishikawa, T., Tanaka, Y., 1998b. New glycosides and furocoumarin from the Glehnia littoralis root and rhizoma. Chem. Pharm. Bull. 46, 1939–1940.

3-Formyl-9H-carbazole (12): Li, W.S., McChesney, J.D., El-Feraly, F.S., 1991. Carbazole alkaloids from Clausena lansium. Phytochemistry 30, 343–346.

N-Phenethylcinnaamide (13): Riemer, B., Hofer, O., Greger, H., 1997. Tryptamine derived amides from Clausena indica. Phytochemistry. 45, 337–341.

N-(Phenethyl)benzamide (14): Kita, Y., Akai, S., Ajimura, N., Yoshigi, M., Tsugoshi, T., Yasuda, H., Tamura, Y., 1986. Facile and efficient syntheses of carboxylic anhydrides and amides using (trimethylsilyl)ethoxyacetylene. J. Org. Chem. 51, 4150–4158.

Isotachioside (15): Inoshiri, S., Sasaki, M., Kohda, H., Otsuka, H., Yamasaki, K., 1987. Aromatic glycosides from Berchemia racemosa. Phytochemistry 26, 2811–2814.

Methyl salicylate D-glucoside (16): Ushiyama, M., Furuya, T., 1989. Glycosylation of phenolic compounds by root culture of Panax ginseng. Phytochemistry 28, 3009–3013.

Xanthotoxol 8-O--D-glucopyranoside (17): Kitajima, J., Aoki, Y., Ishikawa, T., Tanaka, Y., 1999. Monoterpenoid glucosides of Cnidium monnieri fruit. Chem. Pharm. Bull. 47, 639–641.

Tembamide (18): Marcano, D.D.C., Hasegawa, M., Castaldi, A., 1972. Neutral compounds and alkaloids of Zanthoxylum ocumarense. Phytochemistry 11, 1531–1532.

Dihydroalatamide (19): Minor, D.L., Wyrick, S.D., Charifson, P.S., Watts, V.J., Nichols, D.E., Mailman, R.B., 1994. Synthesis and molecular modeling of 1-phenyl-1,2,3,4-tetrahydroisoquinolines and related 5,6,8,9-tetrahydro-13bH-dibenzo[a,h]quinolizines as D1 dopamine antagonists. J. Med. Chem. 37, 4317–4328.

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Xanthotoxol (20): Harkar, S., Razdan, T.K., Waight, E.S., 1984. Steroids, chromone and coumarins from Angelica officinalis. Phytochemistry 23, 419–426.

4-Methoxyquinolin-2-one (21): Bessonova, I.A., 2000. Components of Haplophyllum bucharicum. Chem. Nat. Compd. 36, 323–324.

Isoheraclenin (22): Razdan, T.K., Kachroo, V., Harkar, S., Koul, G.L., 1982. Furanocoumarins from Heracleum canescens. Phytochemistry 21, 923–927.

Isogospherol (23): Razdan, T.K., Kachroo, V., Harkar, S., Koul, G.L., 1982. Furanocoumarins from Heracleum canescens. Phytochemistry 21, 923–927.

Osthol (24): Harkar, S., Razdan, T.K., Waight, E.S., 1984. Steroids, chromone and coumarins from Angelica officinalis. Phytochemistry 23, 419–426.

3-Formyl-6-methoxycarbazole (25): Li, W.S., McChesney, J.D., El-Feraly, F.S., 1991. Carbazole alkaloids from Clausena lansium. Phytochemistry 30, 343–346.

6-Methoxyheptaphylline (26): Joshi, B.S., Gawa, D.H., Kamat, V.N., 1972. 6-Methoxyheptaphylline, a new carbazole alkaloid from Clausena-indica Oliv. Indian J. Chem. 10, 1123–1124.

3-Formyl-2-methoxy-9H-carbazole (27): Jash, S.S., Biswas, G.K., Bhattacharyya, S.K., Bhattacharyya, P., Chakraborty, A., Chowdhury, B.K., 1992. Carbazole alkaloids from Glycosmis pentaphylla. Phytochemistry 31, 2503–2505.

Lansiumarin A (28): Ito, C., Katsuno, S., Furukawa, H., 1998a. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium. Chem. Pharm. Bull. 46, 341–343.

Methyl carbazole-3-carboxylate (29): Li, W.S., McChesney, J.D., El-Feraly, F.S., 1991. Carbazole alkaloids from Clausena lansium. Phytochemistry 30, 343–346.

Dictamnine (30): Bhoga, U., Mali, R.S., Adapa, S.R., 2004. New synthesis of linear furoquinoline alkaloids. Tetrahedron Lett. 45, 9483–9485.

Clausine D (31): Wu, T.S., Huang, S.C., Wu, P.L., Teng, C.M., 1996. Carbazole alkaloids from Clausena excavata and their biological activity. Phytochemistry 43, 133–140.

4-Hydroxycinnamic acid (32): Teresa, J.P., Moran, J.R., Hernandez, J.M., Grande, M., 1985. Tovarol and other germacrane derivatives from Thapsia villosa. Chem. Pharm. Bull. 24, 1779–1783.

Vanillic acid (33): Kuo, P.C., Kuo, T.H., Su, C.R., Liou, M.J., Wu, T.S., 2008. Cytotoxic principles and α-pyrone ring-opening derivatives of bufadienolides from Kalanchoe hybrid. Tetrahedron 64, 3392–3396.

Alloisoimperatorin (34): Razdan, T.K., Qadri, B., Harkar, S., Waight, E.S., 1987. Chromones and coumarins from Skimmia laureola. Phytochemistry 26, 2063–2069.

Wampetin (35): Khan, N.U., Naqvi, S.W.I., Ishratullah, K., 1983. Wampetin, a furocoumarin from Clausena wampi. Phytochemistry 22, 2624–2625.

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(E,E)-8-(7-Hydroxy-3,7-dimethylocta-2,5-dienyloxy)psoralen (36): Ito, C., Katsuno, S., Furukawa, H., 1998a. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium. Chem. Pharm. Bull. 46, 341–343.

Lansiumarin C (37): Ito, C., Katsuno, S., Furukawa, H., 1998a. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium. Chem. Pharm. Bull. 46, 341–343.

(E)-8-(6-Hydroperoxy-3,7-dimethylocta-2,7-dienyloxy)psoralen: Ito, C., Katsuno, S., Furukawa, H., 1998a. Structures of lansiumarin-A, -B, -C, three new furocoumarins from Clausena lansium. Chem. Pharm. Bull. 46, 341–343.

2′,3′-Epoxyanisolactone: Lakshmi, V., Prakash, D., Raj, K., Kapil, R.S., Popli, S.P., 1984. Monoterpenoid furanocoumarin lactones from Clausena anisata. Phytochemistry 23, 2629–2631.

-Fagarine: Couillerot, E., Caron, C., Comoe, L., Audran, J.C., Molinatti, P., Zeches, M., Men-Olivier, L.L., Jardillier, J.C., Chenieux, J.C., 1994. Benzophenanthridine and furoquinoline accumulation in cell suspension cultures of Fagara zanthoxyloides. Phytochemistry 37, 425–428.

Marmesin: Hagemeier, J., Batz, O., Schmidt, J., Wray, V., Hahlbrock, K., Strack, D., 1999. Accumulation of phthalides in elicitor-treated cell suspension cultures of Petroselinum crispum. Phytochemistry 51, 629–635.

N-(2-Hydroxy-2-phenylethyl)-3-phenyl-2-propenamide: Kojima, K., Purevsuren, S., Narantuya, S., Tsetsegmaa, S., Jamyansan, Y., Isaka, K., Ogihara, Y., 2001. Alkaloids from Oxytropis myriophylla (Pall) DC. Sci. Pharm. 69, 383–388.

t-O-Methylheraclenol: Bandopadyay, M., Seshadri, T.R., 1970. Components of Heracleum candicans: Part I. Indian J. Chem. 8, 855856.

8-(3-Chloro-2-hydroxy-3-methylbutoxy)psoralen: Xiao, Y.Q., Liu, X.H., Taniguchi, M., Baba, K., 1997. Bicoumarins from Pleurospermum rivulorum. Phytochemistry 45, 1275–1277.

Uracil: McCarthy, O., Gilbert, I. H., Musso-Buendia, A., Ruiz-Perez, L. M., Pacanowska, D.G., Kaiser, M., Brun, R., Johansson, N.G., 2009. Design, synthesis and evaluation of novel uracil acetamide derivatives as potential inhibitors of Plasmodium falciparum dUTP nucleotidohydrolase. Eur. J. Med. Chem. 44, 678688.

Nicotinamide: Singha, N.C., Anand, J., Sathyanarayana, D.N., 1998. Proton magnetic resonance study of the molecular conformation of N-(3-pyridinyl)3-pyridinecarboxamide, N-(3-pyridinyl)acetamide and 3-pyridinecarboxamide. J. Mol. Struct. 443, 1–8.

Adenosine: Hruska, F.E., Wood, D.J., McCaig, T.N., Smith, A.A., Holy, A., 1974. A nuclear magnetic resonance study of nucleoside conformation in solution. The effect of

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structure and conformation on the magnetic nonequivalence of the 5′-methylene hydrogens. Can. J. Chem. 52, 497–508.

Scopolin: Li, W.W., Barz, W., 2006. Structure and accumulation of phenolics in elicited Echinacea purpurea cell cultures. Planta Med. 72, 248–254.

Haploperoside A: Yuldashev, M.P., Batirov, E.K., Bdovin, A.D., Malikov, V.M., Yagudaev, M.R., 1985. Coumarin glycosides of Haplophyllum performatum. structures of haploperosides C, D, and E. Chem. Nat. Compd. 21, 25–32.

4-Hydroxybenzoic acid: Machida, K., Kikuchi, M., 1996. Norisoprenoids from Viburnum dilatatum. Phytochemistry 41, 1333–1336.

Isorutarin: Bertrand, C., Fabre, N., Moulis, C., 2004. A new coumarin glucoside, coumarins and alkaloids from Ruta corsica roots. Fitoterapia 75, 242–244.

Marmesin 4′-O--D-apiofuranosyl-(1→6)--D-glucopyranoside: Kitajima, J., Okamura, C., Ishikawa, T. Tanaka, Y., 1998a. Coumarin glycosides of Glehnia littoralis root and rhizome. Chem. Pharm. Bull. 46, 1404–1407.

Nodakenin: Ma, Z., Xu, W., Liu-Chen, L.Y., Lee, D.Y.W., 2008. Novel coumarin glycoside and phenethyl vanillate from Notopterygium forbesii and their binding affinities for opioid and dopamine receptors. Bioorg. Med. Chem. 16, 3218–3223.

Mullein: Pietrusiewicz, K.M., Salamonczyk, I., 1988. Useful route to partially saturated isocoumarins. Biomimetic syntheses of mellein, ramulosin, and epiramulosin. J. Org. Chem. 53, 2837–2840.

-Sitosterol: Pant, P., Rastogi, R.P., 1977. Castanopsone and castanopsol two new triterpenoids from Castanopsis indica. Phytochemistry 16, 1787–1789.

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S3. Table 5

Cell viability and effect of compounds 737 on LPS-induced NO production in macrophages a

Dose (g/mL)

Cell viability(% of control)

NO level(M)

NO inhibition(% of control)

control (-) 99.0 ± 0.6 0.1 ± 0.1 (-)LPS (+) 98.2 ± 0.5 39.8 ± 0.6### (-)

Indomethacin 25 95.8 ± 1.2 27.5 ± 0.7* 30.9 ± 0.550 94.3 ± 1.6 20.4 ± 0.4** 48.7 ± 0.6

7 3.1256.2512.5

103.2 ± 1.799.5 ± 1.078.3 ± 1.7

20.5 ± 0.1**

14.5 ± 0.3***

10.6 ± 0.2***

48.6 ± 0.763.5 ± 2.673.3 ± 2.3

8 12.52550

96.0 ± 0.587.1 ± 4.266.2 ± 2.2

29.8 ± 0.2*

13.8 ± 0.3**

2.8 ± 0.2***

25.1 ± 0.865.4 ± 1.993.1 ± 5.5

9 12.52550

89.6 ± 0.385.1 ± 1.976.1 ± 0.9

34.5 ± 0.429.4 ± 0.3*

27.9 ± 0.4*

13.4 ± 1.026.1 ± 1.129.9 ± 1.3

10 12.52550

101.2 ± 1.1100.2 ± 1.694.5 ± 0.8

32.5 ± 0.435.7 ± 0.430.5 ± 0.4*

19.3 ± 1.410.2 ± 1.124.4 ± 1.4

11 3.1256.2512.5

101.6 ± 2.0101.1 ± 0.889.9 ± 0.7

25.4 ± 0.3*

20.3 ± 0.8**

16.4 ± 0.2***

37.4 ± 0.471.4 ± 0.698.4 ± 0.9

12 12.52550

96.1 ± 3.791.5 ± 2.756.4 ± 3.2

29.4 ± 0.4*

20.5 ± 0.1**

3.2 ± 0.1***

26.1 ± 1.349.9 ± 0.492.1 ± 4.5

13 12.52550

100.3 ± 2.890.9 ± 4.782.5 ± 4.2

30.9 ± 0.4*

20.6 ± 0.1**

16.8 ± 0.1***

22.4 ± 0.848.3 ± 1.357.8 ± 2.1

14 12.52550

94.1 ± 1.385.1 ± 0.971.5 ± 1.5

45.7 ± 0.540.2 ± 0.3

(-)

-14.7 ± 1.0-1.0 ± 0.8

(-)15 12.5

2550

99.3 ± 1.198.6 ± 1.498.1 ± 0.9

40.9 ± 0.542.0 ± 1.238.7 ± 0.7

-2.6 ± 1.1-5.5 ± 2.92.7 ± 1.8

16 12.5 99.0 ± 0.5 42.0 ± 0.4 -5.6 ± 0.9

S9

2550

98.6 ± 0.397.2 ± 0.4

40.2 ± 0.239.6 ± 0.3*

-1.0 ± 0.50.5 ± 0.7

17 12.52550

99.3 ± 0.999.0 ± 0.698.5 ± 1.2

44.4 ± 1.442.3 ± 0.240.6 ± 0.2

-11.5 ± 3.1-6.3 ± 0.5-1.9 ± 0.4

18 12.52550

99.0 ± 0.797.8 ± 0.696.5 ± 1.8

38.9 ± 0.437.6 ± 0.536.7 ± 0.6

2.2 ± 0.95.6 ± 1.37.8 ± 1.6

19 12.52550

95.9 ± 0.995.9 ± 1.377.3 ± 1.8

39.4 ± 0.632.4 ± 0.527.5 ± 1.0*

1.1 ± 1.518.7 ± 1.631.0 ± 3.5

20 12.52550

90.9 ± 1.787.7 ± 2.394.2 ± 0.6

29.5 ± 0.2*

21.3 ± 0.7**

9.6 ± 0.4***

25.9 ± 0.646.4 ± 3.375.9 ± 3.8

21 12.52550

95.5 ± 1.694.7 ± 1.593.3 ± 1.3

30.4 ± 0.5*

20.8 ± 0.1**

7.8 ± 0.3***

23.7 ± 1.747.8 ± 0.680.5 ± 3.5

22 12.52550

92.4 ± 0.889.5 ± 0.678.2 ± 0.4

36.2 ± 0.327.5 ± 0.2*

20.1 ± 0.2**

9.1 ± 0.830.6 ± 0.719.4 ± 1.2

23 12.52550

92.1 ± 1.792.9 ± 1.491.7 ± 1.4

43.7 ± 0.233.0 ± 0.2

20.7 ± 0.3**

-9.7 ± 1.917.1 ± 1.148.0 ± 0.9

24 12.52550

97.4 ± 2.294.3 ± 2.064.4 ± 3.1

21.3 ± 0.9**

10.0 ± 0.3***

(-)

46.1 ± 0.674.8 ± 0.3

(-)25 12.5

2550

97.0 ± 0.693.9 ± 1.138.9 ± 0.6

31.1 ± 0.418.0 ± 0.3

(-)

21.9 ± 1.354.7 ± 1.5

(-)26 3.125

6.2512.5

105.5 ± 1.195.2 ± 0.688.6 ± 1.8

26.2 ± 0.4*

21.0 ± 0.4**

12.4 ± 0.2***

34.2 ± 1.647.2 ± 1.768.8 ± 1.7

27 12.52550

103.6 ± 3.898.8 ± 1.246.8 ± 1.1

12.8 ± 0.4***

4.4 ± 0.2***

(-)

67.9 ± 2.989.1 ± 5.2

(-)28 3.125

6.2512.5

104.9 ± 1.197.4 ± 0.414.0 ± 1.4

3.6 ± 0.1***

0.6 ± 0.8***

(-)

90.9 ± 3.298.4 ± 1.6

(-)

S10

29 12.52550

92.3 ± 2.883.0 ± 1.746.0 ± 1.9

23.0 ± 0.18.6 ± 0.1***

(-)

42.2 ± 0.278.5 ± 1.2

(-)30 12.5

2550

94.7 ± 0.990.5 ± 1.171.2 ± 1.6

34.4 ± 0.529.7 ± 0.1*

(-)

13.6 ± 1.325.3 ± 0.3

(-)31 3.125

6.2512.5

79.8 ± 1.771.5 ± 0.870.4 ± 1.4

20.8 ± 0.1(-)(-)

47.7 ± 0.4(-)(-)

32 12.52550

98.2 ± 1.095.1 ± 1.694.0 ± 1.0

33.0 ± 0.2**

31.9 ± 0.2***

31.8 ± 0.3***

17.2 ± 0.619.9 ± 0.820.2 ± 1.0

33 12.52550

95.4 ± 0.495.2 ± 0.293.4 ± 0.8

31.2 ± 0.130.7 ± 0.7*

29.2 ± 0.4*

21.56 ± 0.222.8 ± 2.326.6 ± 1.4

34 12.52550

93.4 ± 2.287.4 ± 3.455.3 ± 2.2

27.0 ± 0.4**

13.9 ± 0.2***

(-)

32.2 ± 1.565.0 ± 1.3

(-)35 12.5

2550

103.7 ± 0.979.4 ± 2.525.5 ± 0.7

4.9 ± 0.3***

0.4 ± 0.2***

(-)

87.7 ± 2.498.9 ± 0.6

(-)36 12.5

2550

103.3 ± 0.497.4 ± 3.386.1 ± 2.9

12.9 ± 0.4**

3.2 ± 0.2***

1.5 ± 0.2***

87.7 ± 1.692.0 ± 3.196.3 ± 3.2

37 12.52550

91.8 ± 2.770.9 ± 1.028.3 ± 0.6

9.1 ± 0.4***

(-)(-)

77.1 ± 4.5(-)(-)

CHCl3 layer extract

62.51252505001000

101.7 ± 1.3102.6 ± 0.8103.3 ± 1.2103.8 ± 0.899.5 ± 0.3

31.6 ± 0.128.4 ± 0.1*

22.1 ± 0.4**

13.4 ± 0.6***

7.6 ± 0.9***

20.7 ± 1.028.6 ± 0.644.5 ± 0.366.5 ± 0.980.9 ± 2.0

a The data are presented as mean ± S.D. for three different experiments performed in triplicate. ###compared with sample of control group. *p < 0.05, **p < 0.01, and ***p < 0.001 were compared with LPS-alone group.

S11

S4. Table 6

Effect of 7, 2427, 31, and 3637 on LPS-induced TNF- production in macrophages

Dose (μg/mL) TNF- level (pg/mL)control 1.4 ± 0.2

LPS 103.0 ± 0.2###

Indomethacin 25 74.7 ± 0.9*

50 58.4 ± 1.2**

7 1.56 82.8 ± 2.2*

3.125 72.7 ± 1.2**

6.25 74.4 ± 3.3**

24 6.25 25.3 ± 0.6***

12.5 22.5 ± 0.3***

25 19.6 ± 0.3***

25 6.25 62.1 ± 0.4**

12.5 48.7 ± 1.4***

25 32.9 ± 0.6***

26 0.78 101.8 ± 0.31.56 79.8 ± 1.2**

3.13 60.5 ± 0.6**

27 6.25 54.8 ± 4.2**

12.5 55.2 ± 4.3**

25 36.1 ± 0.8***

31 1 74.4 ± 1.3**

2 79.4 ± 2.6**

4 64.2 ± 0.3**

35 3.12 53.2 ± 0.2**

6.25 52.2 ± 1.6**

12.5 47.1 ± 2.0***

36 12.5 54.3 ± 1.6**

25 54.1 ± 3.7**

50 48.3 ± 1.8***

37 3.12 51.0 ± 1.0**

6.25 48.5 ± 0.9***

12.5 26.5 ± 2.2***

The data are presented as mean ± S.D. for three different experiments performed in triplicate. ###compared with sample of control group. *p < 0.05, **p < 0.01, and ***p < 0.001 were compared with LPS-alone group.

S12

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

1.0792

1.0914

1.1258

1.1382

2.0496

3.2506

3.2560

3.2617

3.2691

3.2753

3.2806

3.2860

3.3110

3.3288

3.3475

3.3628

3.3803

3.3966

3.5389

3.5569

3.5747

3.6203

3.6314

3.6436

3.6547

3.7911

3.7964

3.8179

3.8369

3.9308

3.9433

3.9556

3.9680

3.9805

4.0340

4.2439

4.2629

4.3388

4.3614

4.4001

4.4158

4.4198

4.4425

5.4947

6.9167

6.9340

7.9256

7.9430

1.44

1.49

0.53

0.53

0.63

0.55

0.63

1.13

0.63

0.52

0.52

1.59

0.50

1.02

1.01

CLSW 53412 Acetone-d6 2008/2/22 AV500

S14

S5. The 1H NMR spectrum of 1 (acetone-d6, 500 MHz)

200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

21.91

23.97

62.88

68.43

70.99

72.08

75.29

77.04

77.32

77.95

78.75

100.48

109.58

116.00

122.22

132.62

162.82

166.51

CLSW 53412 C13 Acetone-d6 2008/2/22 AV500

S15

S6. The 13C NMR spectrum of 1 (acetone-d6, 125 MHz)

S16

S7. The HR-ESI spectrum of 1

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

1.0907

1.1028

1.1334

1.1455

3.2769

3.3084

3.3267

3.3455

3.3632

3.3796

3.3969

3.5367

3.5551

3.5728

3.5892

3.6205

3.6434

3.6533

3.7946

3.8196

3.8387

3.9062

3.9620

3.9741

4.0345

4.2479

4.2669

4.3479

4.3703

4.4013

4.4168

4.4473

4.4699

5.4962

6.9086

6.9248

7.5754

7.6105

7.6269

1.90

3.11

1.42

3.26

2.18

2.64

2.85

3.65

1.37

1.41

1.38

4.28

0.99

1.13

0.94

1.03

CLSW 53411 Acetone-d6 2008/2/22 AV500

S17


200 190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

21.91

23.98

56.37

62.94

68.68

70.96

72.15

75.29

77.07

77.27

78.01

78.83

100.47

109.57

113.39

115.57

122.48

124.81

148.10

152.30

166.51

CLSW 53411 C13 Acetone-d6 2008/2/22 AV500

S18


S19


S20

S11. The 1H NMR spectrum of 3 (CDCl3, 300 MHz)

S21

S12. The 13C NMR spectrum of 3 (CDCl3, 75 MHz)

S22


9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 ppm

1.3236

1.5549

1.9417

2.1709

3.1408

3.1520

3.1805

3.3468

3.3534

3.3868

3.3939

3.4910

3.9550

3.9799

4.5123

4.9374

4.9614

4.9653

5.0680

5.2099

7.2595

7.2955

7.3094

7.4927

8.0804

8.0999

8.4107

8.7039

5.43

1.96

2.05

3.25

6.71

1.64

1.84

1.85

2.82

3.66

1.39

1.43

1.00

S23

S14. The 1H NMR spectrum of 4 (CDCl3, 400 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

18.45

18.51

26.87

29.68

30.91

61.21

79.43

80.94

111.14

113.94

117.26

120.04

120.89

120.97

123.79

124.41

126.98

127.77

136.18

139.72

140.61

142.11

166.41

S24

S15. The 13C NMR spectrum of 4 (CDCl3, 100 MHz)

S25


S26

S17. The CD spectrum of 4

S27


S28


S29


S30

S21. The CD spectrum of 5

9.5 9.0 8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

1.95

39

2.17

26

3.18

373.

2215

3.23

913.

3473

3.37

073.

3851

4.06

79

5.04

215.

1428

5.23

635.

2477

7.21

557.

2329

7.23

497.

2611

7.39

297.

4112

7.42

777.

4475

7.85

548.

0091

8.02

838.

2268

cls-rxn step 1 CDCl3 2010/9/15 AVIII 400

S31

S22. The 1H NMR spectrum of 7a (CDCl3, 400 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

23.05

29.69

30.74

30.92

53.45

60.16

74.92

110.82

111.81

112.93

119.48

120.29

123.87

124.60

125.60

129.66

132.84

137.93

139.55

141.10

143.94


S32

S23. The 1H NMR spectrum of 7a (CDCl3, 100 MHz)

S33

S24. The HR-ESI spectrum of 7a

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0 0.5 0.0 ppm

1.2574

1.7425

2.0568

2.4957

3.2368

3.2462

3.2786

3.2878

3.5116

3.5209

3.5323

3.5416

3.5874

3.6084

3.6289

3.6498

3.8710

4.1449

4.9811

5.0004

5.0040

5.0077

7.2780

7.2897

7.3008

7.3096

7.4598

7.4669

7.4694

7.4789

7.4803

8.0703

8.0899

8.3005

8.3160

8.4096

1.31

1.32

0.76

0.65

0.80

5.57

2.63

1.91

4.01

1.98

1.81

1.24


S34

S25. The 1H NMR spectrum of 7b (CDCl3, 400 MHz)

190 180 170 160 150 140 130 120 110 100 90 80 70 60 50 40 30 20 ppm

19.65

29.87

55.74

60.15

111.15

112.40

114.25

120.62

121.01

123.95

125.98

126.96

130.67

137.59

138.51

140.14

141.03

142.90

191.81


S35

S26. The 1H NMR spectrum of 7b (CDCl3, 100 MHz)

S36

S27. The HR-ESI spectrum of 7b

Minutes

2 4 6 8 10 12 14 16 18 20 22 24 26 28 30 32 34 36 38 40

Volts

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

Volts

-0.04

-0.02

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

aAnalytical HPLC was performed on a 250×4.6 mm i.d. Cosmosil 5C18-AR II column (Nacalai Tesque Inc.) at 35 °C with isocratic elution of 25% CH3CN in 50mM H3PO4 for 40 min a flow rate 0.8 mL/min.

S37

S28. Separation of the o-toylthiocarbamate derivatives derived from seven aldoses using the elution system reported in the literaturea

Minutes26 28 30 32 34 36 38 40 42 44 46 48

mAU

0

50

100

150

200

mAU

0

50

100

150

200

aHPLC profile of o-toylthiocarbamate derivatives derived from seven aldoses. Column: Purospher STAR RP-8e column; 250 × 4.6 mm; i.d. 5 m. Mobile phase A (0.05 M, pH 5.0 acetate buffer). Mobile phase B (CH3CN). Flow rate: 0.8 mL/min. A two-step gradient was used: 0 to 20 min (phase A from 85 to 80%) and 20-50 min (phase A from 80 to 70%).

S38

S29. Separation of the o-toylthiocarbamate derivatives derived from ten aldoses using the modified elution system.a

311

12

7,19

18

5,2,6

1,4

16

1315

14,209

108,17

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