2013 Volume 61 Issue 7 Pages 695-699
Several novel sulfides, called garlicnins B2 (1), B3 (2), B4 (3), C2 (4), and C3 (5), were isolated from acetone extracts of garlic, Allium sativum L. and characterized. These garlicnins are capable of suppressing M2 macrophage activation and they have a novel skeleton of cyclic sulfoxide. The structures of the former 3 and latter of 2 were deduced to be 2-(sulfenic acid)-5-(allyl)-3,4-dimethyltetrahydrothiophene-S-oxides and 2-(allyldithiine)-5-(propenylsulfoxide)-3,4-dimethyltetrahydrothiophene-S-oxides, respectively. The mechanism of the proposed production of these compounds is discussed. The identification of these novel sulfoxides from garlic accumulates a great deal of new chemistry in the Allium sulfide field, and future pharmacological investigations of these compounds will aid the development of natural, healthy foods and anti-cancer agents that may prevent or combat disease.
Garlic, Allium sativum L. (Liliaceae), is at the top of the National Cancer Institute list of designer foods that prevent cancer.1) The various biological activities of garlic are generally classified into 2 categories: cardiovascular disease prevention and cancer prevention.2–5) Activities of the former include inhibition of cholesterol synthesis, platelet aggregation, and arterial smooth muscle cell proliferation as well as anti-inflammatory, antioxidant, and hydrogen sulfide-mediated vasodilatory effects. Effects on carcinogen metabolism (enhanced cellular glutathione synthesis induces cell cycle arrest and apoptosis) and prevention of Helicobacter pylori infection, gastric cancer, and colorectal cancer are effects of the latter category.2–5) The chemistry of garlic sulfides is summarized in a textbook edited by Block.4) In previous work, we isolated a novel stable sulfide, 5-(1-propenyl)-3,4-dimethyl-tetrahydrothiophene-2-sulfoxide-S-oxide, from the acetone extract of Allium cepa L. and found that it inhibits macrophage activation. We named this compound onionin A.6)
Similarly, the sulfur-containing substances, garlicnins A,7) B1, C1, and D8) from an acetone-soluble extract of A. sativum L. have been isolated and characterized to aid the development of natural, healthy foods that may prevent and combat disease, particularly tumors. Successively, the cyclic sulfoxides correlated with garlicnins A, B1, and C1—garlicnins B2, B3, B4, C2, and C3—have been isolated and characterized. This paper discusses their chemical structure characterization.
Chinese garlic (1061 g) was roughly chopped and blended with acetone in a mixer. Subsequently, the mixture was soaked in additional acetone for 3 d at room temperature. The filtrate was evaporated at 40°C in vacuo to obtain a residue (26.1 g), which was then subjected to polystyrene gel column chromatography (Diaion HP-20). It was first eluted with water, then with methanol (residue after evaporation, 5.90 g), and finally with acetone (residue after evaporation, 0.32 g). Part of the methanolic eluate (3.0 g) was partitioned between ethyl acetate and water. The respective residues (Fr. 1: ethyl acetate layer, 0.8 g; Fr. 2: aqueous layer, 2.1 g) were examined for their capability to control macrophage activation.
Macrophages that infiltrate cancer tissues are referred to as tumor-associated macrophages (TAMs) and are closely involved in the development of the tumor microenvironment.9–11) TAMs are alternatively activated macrophages (M2) because of their anti-inflammatory functions.12,13) In certain tumors, the presence of TAMs is associated with poor prognosis.11,14,15) Inhibition of M2-macrophage polarization suppresses tumor cell proliferation. Incubation of human monocyte-derived macrophages with interleukin 10 for 2 d increased the expression of the M2 macrophage marker CD163. Under the same conditions, the effects of Fr. 1 and Fr. 2 on interleukin 10-induced CD163 expression were measured in this work. We found that Fr. 1 and Fr. 2 inhibited CD163 expression, suggesting that they are capable of suppressing M2 macrophages.
The remaining methanolic residue (5.90 g) was partitioned between ethyl acetate and water. Fraction 1 (ethyl acetate layer) was more active than Fr. 2, including saccharides; therefore, the constituents of Fr. 1 were examined. Fraction 1 was evaporated to produce a residue (2.14 g), which was repeatedly chromatographed on silica gel with CHCl3–methanol (200 : 1) to obtain 17 compounds, of which 5 have been previously reported as (E)-ajoene (79.7 mg)16) and garlicnin A (48.2 mg),7) B1 (52.0 mg), C1 (16.4 mg), and D (42.0 mg).8) Five of the other compounds were identified as new substances and called garlicnins B2 (1: 47.2 mg), B3 (2: 9.8 mg), B4 (3: 9.3 mg), C2 (4, 13.4 mg), C3 (5, 14.6 mg). The results of a qualitative analysis using a sodium nitroprusside test confirmed the presence of these compounds.
Garlicnin B2 (1) was obtained as a resinous syrup showing [α]D −10.2°(CHCl3). The IR spectrum of 1 shows absorption bands at 1025 cm−1 corresponding to a sulfoxide group. Positive high-resolution fast atom bombardment mass spectrometry (HR-FAB-MS) of garlicnin B1 (1) showed a peak corresponding to [M+H]+ at m/z 221.0672 (Calcd for C9H17O2S2 to be 221.0670). The 1H-NMR spectrum of compound 1 showed signals assigned to 2 methyl groups at δ 1.11 (3H, d, J=6.8 Hz) and 1.35 (3H, d, J=7.5 Hz); 2 methylene protons at δ 3.43 (1H, dd, J=8.3, 12.4 Hz) and 3.59 (1H, dd, J=6.3, 12.4 Hz); 4 methine protons at δ 1.92 (1H, m), 2.35 (1H, m), 4.06 (1H, d, J=6.3 Hz), and 5.07 (1H, d, J=3.4 Hz); and 3 olefinic protons at δ 5.35 (1H, d, J=6.9 Hz), 5.37 (1H, s), and 5.68 (1H, m). The 13C-NMR spectrum exhibited signals for 2 methyl groups at δ 15.7, and 17.9; 1 methylene carbon at δ 54.4; 2 methine carbons at δ 50.5, 53.5; 2 methine carbons attached to an electron-withdrawing atom at δ 73.5, 90.2; and 2 olefinic carbons at δ 124.3 and 125.8. The 1H–1H correlation spectroscopy (COSY) spectrum showed the presence of the following 4 sequential correlations: first from the methine proton at δ 5.07→to the methine proton at δ 1.92→to the methine proton at δ 2.35→to the methine proton at δ 4.06; second from the methine proton at δ 1.92→to the methyl protons at δ 1.11; third from the methine proton at δ 2.35→to the methyl protons at δ 1.35; and fourth from the methylene protons at δ 3.43 and 3.59→to the olefinic proton at δ 5.68→to the olefinic methylene protons at δ 5.35 and 5.37. The heteronuclear multiple bond correlation (HMBC) spectrum exhibited the following correlations: from the methine proton at δ 5.07 to the methine carbon at δ 73.5; from the methyl protons at δ 1.11 to the carbons at δ 50.5, 53.5, and 90.2; from the methyl protons at δ 1.35 to the carbons at δ 50.5, 53.5, and 73.5; from the methine proton at δ 4.06 to the carbon at δ 90.2; and from the methylene protons at δ 3.43 and 3.59 to the methine carbon at δ 73.5 and the olefinic carbons at δ 124.3 and 125.8, as shown in Fig. 1. Thus, plane-structured garlicnin B2 (1) was constructed as 2-sulfenic acid-5-allyl-3,4-dimethyltetrahydrothiophene-S-oxide. Furthermore, the nuclear Overhauser effect spectroscopy (NOESY) spectrum showed the following 1H–1H correlations: H-2 and H-3; H-3 and CH3 at C-4; CH3 at C-3 and H-4; and H-4 and H-5. To determine the relative configuration of substitutions, we used the aromatic solvent-induced NMR shift,17,18) as in the case of onionin A. The thermodynamic conformational preference of the sulfoxide is the axial form19–21) (lower α-side; Fig. 1). When the 1H-NMR spectrum of 1 is measured in C6D6, the molecule forms a collision complex18,22) against to the sulfoxide—that is, C6D6 orients to the opposite side (upper β-side) against the sulfoxide and affects chemical shifts. The comparison of the 1H-NMR spectrum of compound 1 in CDCl3 with that in C6D6 showed that most of the signals in the latter were shifted upfield: H-2 (+0.07); H-3 (−0.05); H-4 (−0.33); CH3 (−0.30) at C-3; CH3 (−0.18) at C-4; and H-5 (−0.66). In summary, the signals assigned to CH3 at C-3, H-4, and H-5 shifted significantly toward the higher field and the signals that remained relatively unchanged were those of H-2, H-3, and CH3 at C-4 remained relatively unchanged. Therefore, a proposed relative structure of 1 is shown in Fig. 1 and is a stereo-isomer of garlicnin B1.
Similar to that of garlicnin B2 (1), the structures of garlicnins B3 (2) and B4 (3) were examined using HR-FAB-MS, 1H-NMR, 13C-NMR (in Experimental), and various 2D-NMR techniques (1H–1H COSY, HMQC, HMBC, and NOESY; Fig. 1) and shown to be stereo-isomers of 1 (see Fig. 1). The methyl group at C-3 in 1 and 3 appeared slightly lower field compared with that of 2 owing to the cis orientation of the sulfenic acid group at C-2. These compounds are speculated to have strong capabilities for suppressing M2 macrophage activation because their structures are analogous to that of onionin A.6) The framework of cyclic sulfoxide with a tetrahydrothiophene is relatively stable and might be crucial for exhibiting various bioactivities. Pharmacological test with mice for anticancer activity and X-ray analysis to determine the absolute configuration are planned.
Garlicnin C2 (4) was obtained as a resinous syrup showing [α]D −7.8°(CHCl3). The IR spectrum of 4 showed absorption bands at 1025 cm−1 corresponding to a sulfoxide group. Positive HR-FAB-MS of 4 showed a peak corresponding to [M+H]+ at m/z 325.0422 (Calcd for C12H21O2S4 to be 325.0424). The 1H-NMR spectrum of compound 4 displayed signals assigned to 3 methyl groups at δ 1.22 (3H, d, J=6.9 Hz), 1.25 (3H, d, J=6.9 Hz), and 1.97 (3H, d, J=6.9 Hz); 2 methylene protons at δ 3.43 (1H, d, J=6.9 Hz) and 3.46 (1H, br s); 4 methine protons at δ 2.26 (1H, m), 2.52 (1H, m), 3.55 (1H, d, J=12.7 Hz), and 3.60 (1H, d, J=6.9 Hz); and 5 olefinic protons at δ 5.19 (1H, d, J=4.6 Hz), 5.26 (1H, d, J=14.3 Hz), 5.82 (1H, m), 6.19 (1H, d, J=14.9 Hz), and 6.49 (1H, m). The 13C-NMR spectrum exhibited signals for 3 methyl groups at δ 15.6, 17.9, and 20.7; 1 methylene carbon at δ 42.8; 2 methine carbons signals at δ 38.4 and 44.5; 2 methine carbons attached to an electron-withdrawing atom at δ 80.6 and 89.5; and 4 olefinic carbon signals at δ 119.7, 130.8, 132.5 and 138.4. The 1H–1H COSY spectrum revealed the presence of an allyl group at δ 3.43, 3.46, 5.82, 5.19, and 5.26; a propenyl group at δ 1.97, 6.49, and 6.19; a sequence correlation of a methine proton at δ 3.60→to a methine proton at δ 2.26→to a methine proton at δ 2.52→to a methine proton at δ 3.55; and 2 vicinal correlations from the methine proton at δ 2.26 to the methyl protons at δ 1.25 and from the methine proton at δ 2.52 to the methyl protons at δ 1.22. The key HMBC spectrum exhibited the following correlations: from the methine proton at δ 3.60 to the methine carbon at δ 80.6; from the methine proton at δ 3.55 to the carbons at δ 89.5; and from the methine proton at δ 3.55 to the carbons at δ 130.8. Taking into consideration the molecular formula and mechanism of production for cyclic sulfoxides such as garlicnins A,7) B1, and C1,8) we used the HMBC spectrum to characterize the structure as 2-(allyldithiine)-5-(propenylsulfoxide)-3,4-dimethyltetrahydrothiophene-S-oxide. Furthermore, the NOESY spectrum showed the following 1H–1H correlations: H-2 and CH3 at C-3; CH3 at C-3 and CH3 at C-4; and H-4 and H-5. Comparison of the 1H-NMR spectrum of compound 4 in CDCl3 with that in C6D6 showed that most of the signals in the latter were shifted upfield: H-2 (−0.34); H-3 (−0.07); H-4 (−0.10); CH3 (−0.39) at C-3; CH3 (−0.25) at C-4; and H-5 (−0.05). In summary, the signals assigned to H-2, CH3 at C-3, and CH3 at C-4, significantly shifted higher field, and the signals of H-3, H-4, and H-5 remained relatively unchanged. The proposed relative structure of 4 is shown in Fig. 2.
Similar to that of garlicnin C2 (4), the structures of garlicnin C3 (5) was examined using HR-FAB-MS, 1H-NMR, 13C-NMR (in Experimental), and various 2D-NMR techniques (1H–1H COSY, HMQC, HMBC, and NOESY; Fig. 2) and shown to be the stereo-isomer of 4 (see Fig. 2). Compounds of 4 and 5 also reportedly have activities that suppress M2 macrophage activation because they have the fundamental tetrahydrothiophene skeleton.
The proposed mechanism of the proposed production of these compounds is shown in Chart 1. Allyl sulfenic acid derived from (+)-S-allyl L-cysteine sulfoxide (alliin), present in garlic, is proposed to yield diallyl thiosulfinate (allicin). Allicin is then transformed to 1-propenyl-1-propene-thiosulfinate and then become converted to 2,3-dimethylbutanedithial-1-oxide via [3,3]-sigmatropic rearrangement.23) Next, this compound may be ring-closed to a dimethylthiophene-sulfenic acid adduct, which is further attached by 1-propenesulfenic acid (a) derived from allicin to produce garlicnins B2–B4. By contrast, the mechanism of garlicnin C production is deduced to be the further addition of 1-propenesulfenic acid (a) and allyl thiosulfenic acid (b) to the intermediate dimethylthiophene-sulfenic acid adduct, mentioned above in the formation of garlicnin B (see Chart 1).
Finding of these novel sulfoxides from onion and garlic extracts accumulates a great deal of new chemistry in the Allium sulfide field. Pharmacological investigations will aid the development of natural, healthy foods and anti-cancer agents that may prevent or combat disease.
Optical rotation was measured using a JASCO P-1020 (l=0.5) automatic digital polarimeter. The IR spectrum was measured using a Fourier Transform FT/IR-4200 spectrometer (JASCO). The 1H- and 13C-NMR spectra were measured in CDCl3 and C6D6 using a JEOL alpha 500 spectrometer at 500 and 125 MHz, respectively, and the chemical shifts were found to be on the δ (ppm) scale. The HR-FAB-MS were measured using a JEOL JMS-DX303HF mass spectrometer and taken in a glycerol matrix containing NaI. Column chromatography was carried out on Diaion HP-20 (Mitsubishi Chemical Industries), and silica gel 60 (230–400 mesh, Merck). Thin layer chromatography (TLC) was performed on silica gel plates (Kieselgel 60 F254; Merck). TLC spots were visualized under UV light (254/366 nm), sprayed with 10% H2SO4 and anisaldehyde, and then heated.
Plant MaterialThe garlic bulbs (A. sativum L. family Liliaceae) imported from China were identified by Prof. Kohtaro Murakami. A voucher specimen (SBGH 11-07-16-215) was deposited in the Herbarium of the Botanical Garden at Sojo University, Kumamoto, Japan.
Extraction and IsolationPeeled Chinese garlic bulbs (1061 g) of garlics were roughly chopped and homogenized in a mixer along with acetone. The mixture was subsequently soaked in acetone for 3 d at room temperature. The filtrate was concentrated at 40°C in vacuo to obtain a syrup residue (26.1 g), which was then subjected to polystyrene gel column chromatography (Diaion HP-20). It was first eluted with water, then with methanol (residue after evaporation: 5.90 g), and finally with acetone (the residue after evaporation: 0.32 g). Part of the methanolic eluate 3.00 g) was partitioned between ethyl acetate and water. The respective residues (Fr. 1: ethyl acetate residue 0.8 g; Fr. 2: aquous residue 2.1 g) were examined for the ability to control macrophage activation.
The remaining methanolic residue (5.90 g) was partitioned between ethyl acetate and water. The ethyl acetate layer was taken and evaporated to produce a residue (2.14 g), which was repeatedly chromatographed on silica gel with CHCl3–methanol=200 : 1 to obtain eleven compounds, two of which were identified as previously reported garlicnin A (48.2 mg),7) garlicnin B1 (52.0 mg), garlicnin C1 (16.4 mg), garlicnin D (42.0 mg),8) and (E)-ajoene (79.7 mg).16) The other compounds were recognized to be new substances, thus named as garlicnins B2 (1: 47.2 mg), B3 (2: 9.8 mg), B4 (3: 9.3 mg), C2 (4, 13.4 mg), and C3 (5: 14.6 mg). The results of a qualitative analysis using a sodium nitroprusside test confirmed the presence of these compounds.
Determination of the Inhibitory Effect of Fractions on CD163 ExpressionHuman monocyte-derived macrophages (5×104 cells per well of a 96-well plate) were incubated with fractions (100 µg/mL) for 24 h after treatment with IL-10 (20 nM) for 2 d, followed by the determination of CD163 expression by cell enzyme-linked immunosorbent assay (cell-ELISA).
Cell-ELISAExpression of CD163 on human monocyte-derived macrophages was evaluated using a cell-ELISA procedure, as described previously.24) Briefly, each well of a 96-well plate was blocked with Block Ace, and washed 3 times with phosphate buffered saline containing 0.05% Tween 20 (washing buffer). The wells were incubated with anti-CD163 antibody AM3K (2 µg/mL) and dissolved in washing buffer for 1 h. The wells were then washed with washing buffer 3 times and reacted with HRP-conjugated anti-mouse immunoglobulin G (IgG) antibody followed by reaction with Ultrasensitive TMB (Moss, Inc., Pasadena, MD, U.S.A.). The reaction was terminated by the addition of 1 M sulfuric acid, and the absorbance at 450 nm was then read on a micro-ELISA plate reader.
StatisticsAll data are representative 2 or 3 independent experiments. Data are expressed as means (S.D.). Mann–Whitney’s U-test was used for 2-group comparison. A value of p<0.05 was considered statistically significant.
Garlicnin B2 (1): Colorless resinous syrup, [α]D24 −10.2° (c=0.5, CHCl3); IR νmax (KBr) 1025 (sulfoxide) cm−1; positive HR-FAB-MS (m/z): 221.0672 [M+H]+ (Calcd for C9H17O2S2, 221.0670). 1H-NMR (CDCl3, 500 MHz) δ: 1.11 (3H, d, J=6.8 Hz, CH3 at C-3), 1.35 (3H, d, J=7.5 Hz, CH3 at C-4), 1.92 (1H, m, H-3), 2.35 (1H, m, H-4), 3.43 (1H, dd, J=8.3, 12.4 Hz, Ha-1′), 3.59 (1H, dd, J=6.3, 12.4 Hz, Hb-1′), 4.06 (1H, d, J=6.3 Hz, H-5), 5.07 (1H, d, J=3.4 Hz, H-2), 5.35 (1H, d, J=6.9 Hz, Hb-3′), 5.37 (1H, s, Ha-3′), and 5.68 (1H, m, H-2′). 1H-NMR (C6D6, 500 MHz) δ: 0.81 (3H, d, J=7.0 Hz, CH3 at C-3), 1.17 (3H, d, J=7.0 Hz, CH3 at C-4), 1.87 (1H, m, H-3), 2.02 (1H, m, H-4), 3.40 (1H, d, J=5.8 Hz, H-5), 5.14 (1H, d, J=3.5 Hz, H-2). 13C-NMR (CDCl3, 125 MHz) δ: 15.7 (CH3 at C-4), 17.9 (CH3 at C-3), 50.5 (C-4), 53.5 (C-3), 54.4 (C-1′), 73.5 (C-5), 90.2 (C-2), 124.3 (C-3′), and 125.8 (C-2′).
Garlicnin B3 (2): Colorless resinous syrup, [α]D24 −16.0° (c=0.5, CHCl3); IR νmax (KBr) 1026 (sulfoxide) cm−1; Positive HR-FAB-MS (m/z): 221.0671 [M+H]+ (Calcd for C9H17O2S2, 221.0670). 1H-NMR (CDCl3, 500 MHz) δ: 0.95 (3H, d, J=7.4 Hz, CH3 at C-3), 1.19 (3H, d, J=6.8 Hz, CH3 at C-4), 2.46 (1H, m, H-3), 2.84 (1H, m, H-4), 3.30 (1H, dd, J=8.6, 12.6 Hz, Ha-1′), 3.63 (1H, t-like, J=12.6 Hz, Hb-1′), 3.94 (1H, d, J=6.9 Hz, H-5), 4.98 (1H, s, H-2), 5.37 (1H, d, J=6.3 Hz, Hb-3′), 5.39 (1H, s, Ha-3′), and 5.69 (1H, m, H-2′). 1H-NMR (C6D6, 500 MHz) δ: 0.69 (3H, d, J=7.2 Hz, CH3 at C-3), 0.82 (3H, d, J=7.1 Hz, CH3 at C-4), 2.22 (1H, m, H-3), 2.78 (1H, m, H-4), 3.84 (1H, d, J=6.2 Hz, H-5), 4.66 (1H, s, H-2). 13C-NMR (CDCl3, 125 MHz) δ: 13.7 (CH3 at C-3), 14.7 (CH3 at C-4), 39.8 (C-3), 51.7 (C-4), 56.2 (C-1′), 73.9 (C-5), 88.0 (C-2), 124.4 (C-2′), and 125.3 (C-3′).
Garlicnin B4 (3): Colorless resinous syrup, [α]D24 −14.5° (c=0.5, CHCl3); IR νmax (KBr) 1025 (sulfoxide) cm−1; Positive HR-FAB-MS (m/z): 221.0671 [M+H]+ (Calcd for C9H17O2S2, 221.0670). 1H-NMR (CDCl3, 500 MHz) δ: 1.10 (3H, d, J=6.9 Hz, CH3 at C-3), 1.30 (3H, d, J=6.8 Hz, CH3 at C-4), 2.04 (1H, m, H-3), 2.51 (1H, m, H-4), 3.36 (2H, d, J=7.4 Hz, H2-1′), 4.08 (1H, d, J=5.7 Hz, H-5), 5.14 (1H, d, J=4.0 Hz, H-2), 5.41 (1H, d, J=18.3 Hz, Ha-3′), 5.44 (1H, d, J=10.3 Hz, Hb-3′), and 5.86 (1H, m, H-2′). 1H-NMR (C6D6, 500 MHz) δ: 0.84 (3H, d, J=7.0 Hz, CH3 at C-3), 0.97 (3H, d, J=6.9 Hz, CH3 at C-4), 1.99 (1H, m, H-3), 2.47 (1H, m, H-4), 3.63 (1H, d, J=5.8 Hz, H-5), 4.98 (1H, d, J=3.5 Hz, H-2). 13C-NMR (CDCl3, 125 MHz) δ: 13.6 (CH3 at C-3), 19.8 (CH3 at C-4), 39.2 (C-4), 53.4 (C-1′), 55.1 (C-3), 71.3 (C-5), 85.6 (C-2), 124.1 (C-2′), and 125.7 (C-3′).
Garlicnin C2 (4): Colorless resinous syrup, [α]D24 −7.8° (c=0.5, CHCl3); IR νmax (KBr) 1025 (sulfoxide) cm−1; Positive HR-FAB-MS (m/z): 325.0422 [M+H]+ (Calcd for C12H21O2S4, 325.0424). 1H-NMR (CDCl3, 500 MHz) δ: 1.22 (3H, d, J=6.9 Hz, CH3 at C-4), 1.25 (3H, d, J=6.9 Hz, CH3 at C-3), 1.97 (3H, d, J=6.9 Hz, CH3 at C-8′), 2.26 (1H, m, H-3), 2.52 (1H, m, H-4), 3.43 (1H, d, J=6.9 Hz, Ha-3′), 3.46 (1H, br s, Hb-3′), 3.55 (1H, d, J=12.7 Hz, H-5), 3.60 (1H, d, J=6.9 Hz, H-2), 5.19 (1H, d, J=4.6 Hz, Ha-5′), 5.26 (1H, d, J=14.3 Hz, Hb-5′), 5.82 (1H, m, H-4′), 6.19 (1H, d, J=14.9 Hz, H-7′), and 6.49 (1H, m, H-8′). 1H-NMR (C6D6, 500 MHz) δ: 0.86 (3H, d, J=6.9 Hz, CH3 at C-3), 0.97 (3H, d, J=6.9 Hz, CH3 at C-4), 2.19 (1H, m, H-3), 2.62 (1H, m, H-4), 3.26 (1H, d, J=6.9 Hz, H-2), 3.60 (1H, d, J=7.0 Hz, H-5). 13C-NMR (CDCl3, 125 MHz) δ: 15.6 (CH3 at C-4), 20.7 (CH3 at C-3), 17.9 (CH3 at C-8′), 38.4 (C-4), 42.8 (C-3′), 44.5 (C-3), 80.6 (C-5), 89.5 (C-2), 119.7 (C-5′), 130.8 (C-7′), 132.5 (C-4′), and 138.4 (C-8′).
Garlicnin C3 (5): Colorless resinous syrup, [α]D24 −12.8° (c=0.5, CHCl3); IR νmax (KBr) 1025 (sulfoxide) cm−1; Positive HR-FAB-MS (m/z): 325.0422 [M+H]+ (Calcd for C12H21O2S4, 325.0424). 1H-NMR (CDCl3, 500 MHz) δ: 1.24 (3H, d, J=6.9 Hz, CH3 at C-4), 1.26 (3H, d, J=6.3 Hz, CH3 at C-3), 1.71 (1H, m, H-3), 1.97 (3H, d, J=5.1 Hz, CH3 at C-8′), 2.79 (1H, m, H-4), 3.43 (1H, d, J=6.9 Hz, Ha-3′), 3.45 (1H, d, J=7.5 Hz, Hb-3′), 3.60 (1H, d, J=9.1 Hz, H-5), 3.92 (1H, d, J=10.3 Hz, H-2), 5.19 (1H, d, J=9.8 Hz, Ha-5′), 5.23 (1H, d, J=16.9 Hz, Hb-5′), 5.81 (1H, m, H-4′), and 6.58 (2H, m, H-7′, H-8′). 1H-NMR (C6D6, 500 MHz) δ: 1.12 (3H, d, J=6.8 Hz, CH3 at C-3), 1.16 (3H, d, J=7.0 Hz, CH3 at C-4), 1.40 (1H, m, H-3), 2.45 (1H, m, H-4), 3.22 (1H, d, J=8.4 Hz, H-5), 3.60 (1H, d, J=9.8 Hz, H-2). 13C-NMR (CDCl3, 125 MHz) δ: 16.5 (CH3 at C-3), 18.3 (CH3 at C-4), 18.1 (CH3-9′), 39.6 (C-4), 43.3 (C-3′), 44.0 (C-3), 81.5 (C-5), 81.6 (C-2), 119.9 (C-5′), 131.0 (C-8′), 132.3 (C-4′), and 139.0 (C-7′).
