Acyclic Sulfides, Garlicnins L-1–L-4, E, and F, from Allium sativum

Abstract

Six novel acyclic sulfides, named garlicnins L-1–L-4 (1–4), E (5), and F (6), were isolated from the acetone extracts, with the ability to suppress M2 macrophage activation, of the bulbs of garlic (Allium sativum L.), and their chemical structures were characterized.

Garlic, Allium sativum L. (Liliaceae) is at the top of the National Cancer Institute’s list of designer foods that prevent cancer.¹⁾ Various biological activities of garlic are generally classified into two categories: (i) cardiovascular disease prevention and (ii) cancer prevention.^2–5) The activities of the former include the inhibition of cholesterol synthesis, platelet aggregation, and arterial smooth muscle cell proliferation as well as anti-inflammatory, antioxidant, and hydrogen sulfide-mediated vasodilatory effects. The activities of the latter include the effects on carcinogen metabolism (enhanced cellular glutathione synthesis induced cell cycle arrest and apoptosis) and the 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 text book edited by Block.⁴⁾ In a previous work, we isolated a novel, stable sulfide, named as onionin A,⁶⁾ 5-(1-propenyl)-3,4-dimethyltetrahydrothiophene-2-sulfoxide-S-oxide, from the acetone extract of Allium cepa L. and found that it inhibits macrophage activation. Similarly, the sulfur-containing substances, garlicnins A,⁷⁾ B₁,⁸⁾ B₂, B₃, B₄,⁹⁾ C₁,⁸⁾ C₂, C₃,⁹⁾ and D⁸⁾ from the acetone-soluble extract of A. sativum L. were isolated along with E-azoene¹⁰⁾ and characterized to help in the development of natural, healthy foods that may prevent and combat disease, particularly tumors. Successively, the related acyclic sulfides garlicnins L-1–L-4, E, and F, were isolated and characterized. This paper discusses the characterization of the chemical structure of these compounds.

Chinese garlic (1061 g) was roughly chopped and blended in a mixer along with acetone. 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 (the residue after evaporation: 5.90 g), and finally with acetone (the residue after evaporation: 0.32 g). A part of the methanolic eluate (3.0 g) was partitioned between ethyl acetate (EtOAc) and water. The respective residues (Fr. 1: EtOAc layer 0.8 g; Fr. 2: aqueous layer 2.1 g) were examined for their capability to control macrophage activation.

The macrophages that infiltrate cancer tissues are referred to as tumor-associated macrophages (TAMs) and are closely involved in the development of the tumor microenvironment.^11–13) TAMs are considered to belong to alternatively activated macrophages (M2) because of their anti-inflammatory functions.^14,15) In the case of certain tumor types, the presence of TAMs is associated with a poor prognosis of patients.^13,16,17) The inhibition of M2-macrophage polarization suppresses tumor cell proliferation. The 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 Frs. 1 and 2 on the interleukin 10-induced CD163 expression were measured in this study. Fractions 1 and 2 inhibited the CD163 expression, indicating that they are capable of suppressing M2 macrophages.

The methanolic residue (5.90 g) freshly prepared in the same manner as the above method was partitioned between EtOAc and water. Fraction 1 (EtOAc layer) was more active than that of Fr. 2, including saccharides, therefore, the constituents of Fr. 1 was investigated. Fraction 1 was evaporated to afford a residue (2.14 g), which was repeatedly chromatographed on silica gel using CHCl₃–methanol (200 : 1) and n-hexane–acetone (4 : 1) to afford sixteen compounds; ten of them have been previously reported as garlicnin A (48.2 mg),⁷⁾ E-ajoene (79.7 mg),¹⁰⁾ garlicnins B₁ (52.0 mg), B₂ (47.2 mg), B₃ (9.8 mg), B₄ (9.3 mg), garlicnins C₁ (16.4 mg), C₂ (13.4 mg), C₃ (14.6 mg), and D (42.0 mg).^8,9) Six of the other compounds were recognized as new substances and named as garlicnins L-1 (1: 47.2 mg), L-2 (2: 9.8 mg), L-3 (3: 9.3 mg), L-4 (4, 13.4 mg), E (5, 14.6 mg), and F (6, 15.2 mg). The results of the qualitative analysis using the sodium nitroprusside test confirmed the presence of these compounds.

Garlicnin L-1 (1) was obtained as syrup with [α]_D −11.8° (CHCl₃). The IR spectrum of 1 shows absorption bands at 1715, 1025 cm⁻¹ corresponding to a carbonyl and a sulfoxide groups, respectively. The positive high-resolution fast-atom-bombardment mass spectrometry (HR-FAB-MS) of 1 showed the [M+H]⁺ peak at m/z 175.0790 (Calcd for C₈H₁₅O₂S: 175.0793). The ¹H-NMR spectrum of 1 showed three methyl groups at δ 1.29 (3H, d, J=6.9 Hz), 1.96 (3H, d, J=6.9 Hz), and 2.18 (3H, s); two methylene protons at δ 2.55 (1H, dd, J=9.3, 18.6 Hz) and 3.14 (1H, dd, J=3.5, 18.6 Hz); one methine proton at δ 3.46 (1H, m); and two olefinic protons at δ 6.21 (1H, dd, J=1.5, 15.4 Hz), and 6.89 (1H, dq, J=6.9, 15.4 Hz). The ¹³C-NMR spectrum showed three methyl carbons at δ 14.1, 17.7, and 30.5; one methylene carbon at δ 46.6; one methine carbon at δ 54.1; two olefinic carbons at δ 127.2, and 146.6; and one carbonyl carbon signal at δ 204.2. The ¹H–¹H correlation spectroscopy (COSY) spectrum showed the presence of the following two sequential correlations: (i) from the methylene protons at δ 2.55, and 3.14 to the methine proton at δ 3.46 to the methyl protons at δ 1.29; and (ii) from the olefinic proton at δ 6.21 to the olefinic proton at δ 6.89 to the methyl protons at δ 1.96. The heteronuclear multiple-bond correlation (HMBC) spectrum exhibited the following correlations: (i) from the methyl protons at δ 2.18 to the carbonyl carbon carbon at δ 204.2 and the methylene carbon at δ 46.6; (ii) from the methyl protons at δ 1.29 to the methylene carbon at δ 46.6 and the methine carbon at δ 54.1; and (iii) from the methyl protons at δ 1.96 to the olefinic carbons at δ 127.2 and 146.6. The chemical shift of the methine carbon at δ 54.1 indicates it to be adjacent to the sulfoxide group and the double bond lies in the E-configuration as shown by the coupling constant (J=15.4 Hz) of the olefinic protons. Thus, the structure of 1 was deduced as E-5-thiaocta- 6-ene 4-methyl-2,5-dioxide.

Garlicnin L-2 (2) was obtained as syrup with [α]_D −2.7° (CHCl₃). The positive HR-FAB-MS of 2 showed the [M+H]⁺ peak at m/z 209.0132 (Calcd for C₇H₁₃OS₃: 209.0129). The ¹H–¹H COSY spectrum of 2 showed the presence of one methyl group at δ 2.51 (3H, s); one allyl group at δ 3.31 (2H, d, J=7.3 Hz), 5.13 (1H, d, J=9.7 Hz), 5.15 (1H, d, J=17.1 Hz), and 5.78 (1H, ddd, J=7.3, 9.7, 17.1 Hz); and one propenyl group at δ 3.45 (1H, dd, J=8.0, 13.2 Hz), 3.48 (1H, dd, J=8.0, 13.2 Hz), 5.88 (1H, dd, J=8.0, 14.9 Hz), and 6.34 (1H, d, J=14.9 Hz). The ¹³C-NMR spectrum exhibited one methyl carbon at δ 37.4; two methylene carbons at δ 41.4 and 56.6; and four olefinic carbons at δ 116.6, 119.4, 132.6, and 134.9. The HMBC spectrum exhibited the following key correlations: from the methyl protons at δ 2.51 to the methylene carbon at δ 56.6. Thus, the structure of 2 was deduced as E-2,6,7-trithiadeca-4,9-diene 2-oxide. This compound was identical to the product obtained by the decomposition of allyl methanethiosulfinate.¹⁸⁾ The isomers of 2, Z-2,6,7-trithiadeca-4,9-diene 2-oxide¹⁹⁾ and E-2,6,7-trithiadeca-4,8-diene 2-oxide²⁰⁾ were also obtained from the oil-macerated garlic extract.

Garlicnin L-3 (3) was obtained as syrup. The positive HR-FAB-MS of 3 showed the [M+H]⁺ peak at m/z 251.0059 (Calcd for C₉H₁₅S_4: 251.0057). The ¹H–¹H COSY spectrum of 3 showed the presence of two allyl groups at δ 3.34 (4H, m), 5.84 (2H, m), and 5.14 (4H, m); one propenyl group at δ 3.34 (2H, m), 5.84 (1H, m), and 6.16 (1H, d, J=13.7 Hz). The ¹³C-NMR spectrum showed three methylene carbons at δ 41.0, 41.3 and 42.3; and six olefinic carbons at 118.8, 119.1, 126.2, 129.4, 132.8, and 133.4. The two-dimensional NMR spectroscopy, heteronuclear multiple-quantum coherence (HMQC), and HMBC, indicated that the structure of 3 can be represented as a sequence of an allyl—a dithiine—a propenyl—a dithiine—an allyl group. Based on the coupling constant of the olefinic proton, the geometry of the inner double bond was regarded as Z. This was supported by the nuclear Overhauser effect (NOE) experiment. Thus, the structure of 3 was deduced as Z-4,5,9,10-tetrathiatrideca-1,7,12-triene.

Garlicnin L-4 (4) was obtained as a syrup with [α]_D +4.9° (CHCl₃). The IR spectrum of 4 shows an absorption band at 1725 cm⁻¹ corresponding to a carbonyl group. The positive HR-FAB-MS of 4 showed the [M+H]⁺ peak at m/z 203.0568 (Calcd for C₉H₁₅OS₂: 203.0564). The ¹H–¹H COSY spectrum of 4 showed one allyl group at δ 3.32 (2H, d, J=8.0 Hz), 5.14 (1H, d, J=11.1 Hz), 5.21 (1H, d, J=17.0 Hz), and 5.83 (1H, m); one methyl group at δ 1.76 (3H, d, J=1.1 Hz); two methylene groups at δ 2.74 (2H, triplet-like, J=6.3 Hz), and 2.80 (2H, d, J=6.3 Hz); one olefinic proton at δ 6.48 (1H, dd, J=1.1, 6.3 Hz); and one aldehyde proton at δ 9.41 (1H, s). The ¹³C-NMR spectrum showed four olefinic carbons at δ 118.8, 133.5, 140.7, and 151.3; one methyl carbon at δ 9.5; one aldehyde carbonyl carbon at δ 195.0; and three methylene carbons at δ 28.5, 36.8, and 42.3. The HMBC spectrum showed the following correlations: (i) from the methyl protons at δ 1.76 to the two olefinic carbons at δ 140.7, 151.3 and the carbonyl carbon at δ 195.0; (ii) from the methylene protons at δ 2.74 to the carbons at δ 36.8, 140.7, and 151,3; (iii) from the methylene protons at δ 2.80 to the carbons at δ 28.5 and 151.3; and (iv) from the methylene protons at δ 3.32 to the carbons at δ 118.8 and 133.5. The NOE was observed between the methyl protons at δ 1.76 and the proton at δ 2.74. Therefore, the structure of 4 was deduced as E-6,7-dithiadeca-2,9-diene 2-methyl-1-oxide.

Garlicnin E (5) was obtained as syrup with [α]_D +11.5° (CHCl₃). The positive HR-FAB-MS of 5 showed the [M+H]⁺ peak at m/z 372.9921 (Calcd for C₁₂H₂₁OS₆: 372.9917). The ¹H‒¹H COSY spectrum showed the presence of two 1-propenyl groups at δ 1.83 (3H, d, J=6.9 Hz), 1.88 (3H, d, J=6.1 Hz), 6.26–6.32 (2H, m), 6.33 (1H, d, J=15.3 Hz), and 6.39 (1H, d, J=15.1 Hz); one proton at δ 5.44 (1H, overlapped) attached to the electron-withdrawing group; and two propenyl groups at δ 3.76 (4H, m), 5.44 (2H, d, J=15.2 Hz), and 5.91 (2H, m). Based on the molecular formula, two dithiines and one thiosulfenic acid groups form 5 along with two 1-propenyl and two propenyl groups. The HMBC spectrum showed the following correlations: (i) from the methyl protons at δ 1.83 to the carbons at δ 116.8 and 137.5; and (ii) from the methyl protons at δ 1.88 to the carbons at δ 113.6 and 144.3. The olefinic proton signal at δ 6.33 coupled to the proton attached to the thiosulfenic acid group at δ 5.44. Therefore, the structure of 5 was deduced as shown in Fig. 1.

Fig. 1. Structures of Garlicnins L-1–L-4 (1–4), E (5) and F (6)

Garlicnin F (6) was obtained as syrup with [α]_D +12.1° (CHCl₃). The IR spectrum of 6 shows absorption bands at 1728 and 1025 cm⁻¹ corresponding to a carbonyl and a sulfoxide groups, respectively. The positive HR-FAB-MS of 6 showed the [M+H]⁺ at m/z 517.0166 (Calcd for C₁₈H₂₉O₃S₇ to be 517.0162). The ¹H–¹H COSY spectrum indicated the presence of the following sequential groups: (i) one aldehyde proton at δ 9.68 (1H, s), one methine proton at δ 2.77 (1H, m), one methine proton at δ 2.77 (1H, m), one methine proton adjacent to the oxygen-containing group at δ 4.23 (1H, d, J=4.6 Hz); (ii) one methine proton at δ 2.77 (1H, m), one methyl group at δ 1.03 (3H, d, J=6.9 Hz); (iii) one methine proton at δ 2.77 (1H, m), one methyl group at δ 1.12 (3H, d, J=6.9 Hz); (iv) three propenyl groups at δ 5.20 (1H, d, J=15.4 Hz), 5.84 (1H, m), and 3.23–3.50 (2H, m); at δ 5.21 (1H, d, J=15.2 Hz), 5.84 (1H, m), and 3.23–3.50 (2H, m); and at δ 5.25 (1H, d, J=15.3 Hz), 5.84 (1H, m), and 3.23–3.50 (2H, m); (v) one allyl group at δ 5.37 (1H, d, J=16.6 Hz), 6.28 (1H, d, J=10.3 Hz), 5.84 (1H, m), and 3.23–3.50 (2H, m). The ¹³C-NMR spectrum showed one aldehyde carbonyl at δ 203.7; two methyl carbons at δ 10.5 and 12.9; two methine carbons at δ 36.5 and 48.5; one methine carbon adjacent to the oxygen-containing group at δ 69.9; three propenyl groups at δ 134.2, 118.6, and 41.9, at δ 119.7, 132.5, and 42.6, and at δ 119.4, 133.0 and 42.6; and one allyl group at δ 122.1, 117.3, and 45.2. The NOE showed a correlation between the methine proton at δ 4.23 and an olefinic proton at δ 5.20; however, the inner two propenyl groups are not defined in terms of their alignment and direction. Four methylene carbon signals appeared at δ 41.9, 45.2, and 2×42.6, which are adjacent to the dithiine groups in terms of their chemical shifts. Therefore, the structure of 6 was deduced as shown in Fig. 1; however, the stereo configurations at C-2, C-3, and C-4 positions, and the geometry of the double bonds remain to be solved. The sulfides, so far obtained from the acetone-soluble extracts of the Chinese bulbs of Allium sativum are summarized in Table 1.

Table 1. Three Types of Sulfides Obtained from Chinese Garlic

Experimental

General Experimental Procedures

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 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 Material

The garlic bulbs (A. sativum L. family Liliaceae) imported from China were identified by Prof. Kotaro 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 Isolation

Peeled 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: aqueous residue 2.1 g) were examined for the ability to control macrophage activation.

The methanolic residue (5.90 g) freshly prepared in the same manner as the above method 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) and n-hexane–acetone (4 : 1) to obtain eleven compounds, two of which were identified as previously reported garlicnin A (48.2 mg),⁷⁾ garlicnins B₁ (52.0 mg), B₂ (1: 47.2 mg), B₃ (2: 9.8 mg), B₄ (3: 9.3 mg), garlicnins C₁ (16.4 mg), C₂ (4, 13.4 mg), and C₃ (5: 14.6 mg), garlicnin D (42.0 mg),^8,9) and (E)-ajoene (79.7 mg).¹⁰⁾ The other compounds were recognized to be new substances, thus named as garlicnins L-1 (1: 47.2 mg), L-2 (2: 9.8 mg), L-3 (3: 9.3 mg), L-4 (4, 13.4 mg), E (5, 14.6 mg), and F (6, 15.2 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 Expression

Human 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-ELISA

Expression of CD163 on human monocyte-derived macrophages was evaluated using a cell-ELISA procedure, as described previously.²¹⁾ 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 horseradish peroxidase (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.

Statistics

All 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 L-1 (1): Syrup, [α]_D²⁴ −11.8° (c=0.5, CHCl₃); IR ν_max (KBr) 1715 (carbonyl), 1025 (sulfoxide) cm⁻¹; positive HR-FAB-MS (m/z): 175.0790 [M+H]⁺ (Calcd for C₈H₁₅O₂S, 175.0793). ¹H-NMR (CDCl₃) δ 1.29 (3H, d, J=6.9 Hz, H₃-9), 1.96 (3H, d, J=6.9 Hz, H₃-8), 2.18 (3H, s, H₃-1), 2.55 (1H, dd, J=9.3, 18.6 Hz, Ha-3), 3.14 (1H, dd, J=3.5, 18.6 Hz, Hb-3), 3.46 (1H, m, H-4), 6.21 (1H, d, J=1.5, 15.4 Hz, H-6), 6.89 (1H, dq, J=6.9, 15.4 Hz, H-7). ¹³C-NMR (CDCl₃) δ 14.1 (C-9), 17.7 (C-8), 30.5 (C-1), 46.6 (C-3), 54.1 (C-4), 127.2 (C-6), 146.6 (C-7), 204.2 (C-2).

Garlicnin L-2 (2): Syrup, [α]_D²⁴ −2.7° (c=0.5, CHCl₃); IR ν_max (KBr) 1026 (sulfoxide) cm⁻¹; positive HR-FAB-MS (m/z): 209.0132 [M+H]⁺ (Calcd for C₇H₁₃OS₃, 209.0129). ¹H-NMR (CDCl₃) δ 2.51 (3H, s, H₃-1), 3.31 (2H, d, J=7.3 Hz, H₂-8), 3.45 (1H, dd, J=8.0, 13.2 Hz, Ha-3), 3.48 (1H, dd, J=8.0, 13.2 Hz, Hb-3), 5.13 (1H, d, J=9.7 Hz, Ha-10), 5.15 (1H, d, J=17.1 Hz, Hb-10), 5.78 (1H, ddd, J=7.3, 9.7, 17.1 Hz, H-9), 5.88 (1H, dd, J=8.0, 14.9 Hz, H-4), 6.34 (1H, d, J=14.9 Hz, H-5). ¹³C-NMR (CDCl₃) δ 37.4 (C-1), 41.4 (C-8), 56.6 (C-3), 116.6 (C-4), 119.4 (C-10), 132.6 (C-9), 134.9 (C-5).

Garlicnin L-3 (3): Syrup, positive HR-FAB-MS (m/z): 251.0059 [M+H]⁺ (Calcd for C₉H₁₅S₄, 251.0057). ¹H-NMR (CDCl₃) δ 3.34 (6H, m, H₂-3, 6, 11), 5.14 (4H, m, H₂-1, 13), 5.84 (3H, m, H₂-2, 7, 12), 6.16 (1H, d, J=13.7 Hz, H-8). ¹³C-NMR (CDCl₃) δ 41.0 (C-3), 41.3 (C-11), 42.3 (C-6), 118.8 (C-1), 119.1 (C-13), 126.2 (C-12), 129.4 (C-2), 132.8 (C-7), 133.4 (C-8).

Garlicnin L-4 (4): Syrup, [α]_D²⁴ +4.9° (c=0.5, CHCl₃); IR ν_max (KBr) 1725 (carbonyl) cm⁻¹; positive HR-FAB-MS (m/z): 203.0568 [M+H]⁺ (Calcd for C₉H₁₅OS₂, 203.0564). ¹H-NMR (CDCl₃) δ 1.76 (3H, d, J=1.1 Hz, H₃-11), 2.74 (2H, t-like, J=6.3 Hz, H₂-4), 2.80 (2H, d, J=6.3 Hz, H₂-5), 3.32 (2H, d, J=8.0 Hz, H₂-8), 5.14 (1H, d, J=11.1 Hz, Ha-10), 5.21 (1H, d, J=17.0 Hz, Hb-10), 5.83 (1H, m, H-9), 6.48 (1H, dd, J=1.1, 6.3 Hz, H-3), 9.41 (1H, s, H-1). ¹³C-NMR (CDCl₃) δ 9.5 (C-11), 28.5 (C-4), 36.8 (C-5), 42.3 (C-8), 118.8 (C-10), 133.5 (C-9), 140.7 (C-2), 151.3 (C-3), 195.0 (C-1).

Garlicnin E (5): Syrup, [α]_D²⁴ +11.5° (c=0.5, CHCl₃); IR ν_max (KBr) 1026 (sulfoxide) cm⁻¹; positive HR-FAB-MS (m/z): 372.9921 [M+H]⁺ (Calcd for C₁₂H₂₁OS₆, 372.9917). ¹H-NMR (CDCl₃) δ 1.83 (3H, d, J=6.9 Hz, H₃-1), 1.88 (3H, d, J=6.1 Hz, H₃-18), 3.76 (4H, m, H₂-8, 11), 5.44 (2H, d, J=15.2 Hz, H-6, 13), 5.44 (1H, m, H-15), 5.91 (2H, m, H-7, 12), 6.26–6.32 (2H, m, H-2, 17), 6.33 (1H, d, J=15.3 Hz, H-16), 6.39 (1H, d, J=15.1 Hz, H-3). ¹³C-NMR (CDCl₃) δ 15.3 (C-1), 116.8 (C-2), 137.5 (C-3), 125.8 (C-6), 124.1 (C-7), 59.7 (C-8), 60.2 (C-11), 124.3 (C-12), 125.9 (C-13), 144.3 (C-16), 113.6 (C-17), 19.2 (C-18).

Garlicnin F (6): Syrup, [α]_D²⁴ +12.1° (c=0.5, CHCl₃); IR ν_max (KBr) 1728 (carbonyl), 1025 (sulfoxide) cm⁻¹; positive HR-FAB-MS (m/z): 517.0166 [M+H]⁺ (Calcd for C₁₈H₂₉O₃S₇, 517.0162). ¹H-NMR (CDCl₃, 500 MHz) δ 1.03 (3H, d, J=6.9 Hz, H₃ at C-3), 1.12 (3H, d, J=6.9 Hz, H₃ at C-2), 2.77 (2H, m, H-2, 3), 3.23–3.50 (8H, m, H₂-8, 13, 16, 21), 4.23 (1H, d, J=4.6 Hz, H-4), 5.20 (1H, d, J=15.4 Hz, H-6), 5.21 (1H, d, J=15.2 Hz, H-11), 5.25 (1H, d, J=15.3 Hz, H-18), 5.37 (1H, d, J=16.6 Hz, Ha-23), 5.84 (4H, m, H-7, 12, 17, 22), 6.28 (1H, d, J=10.3 Hz, Hb-23), 9.68 (1H, s, H-1). ¹³C-NMR (CDCl₃) δ 203.7 (C-1), 48.5 (C-2), 10.5 (CH₃ at C-2), 36.5 (C-3), 12.0 (CH₃ at C-3), 69.9 (C-4), 134.2 (C-6), 118.6 (C-7), 41.9 (C-8), 119.7 (C-11), 132.5 (C-12), 42.6 (C-13), 42.6 (C-16), 133.0 (C-17), 119.4 (C-18), 45.2 (C-21), 117.3 (C-22), 122.1 (C-23).

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