HETEROCYCLES

Paper | Regular issue | Vol. 83, No. 5, 2011, pp. 1067-1076
Received, 31st January, 2011, Accepted, 3rd March, 2011, Published online, 10th March, 2011.
DOI: 10.3987/COM-11-12161

■ New Antibacterial Polyacetylenes from Sunflower (Helianthus annuus L.) Seedlings

Fumie Seshimoto, Si Won Hong, Haruyuki Nakajyo, and Hideyuki Shigemori*

Graduate School of Life and Environmental Sciences, University of Tsukuba, Tsukuba, Ibaraki 305, Japan

Abstract

Three new C15 polyacetylenes 1, 2, and 3, together with two known C15 polyacetylenes 4 and 5 were isolated from the seedlings of sunflower Helianthus annuus L. cv Russia, and their structures were elucidated by spectroscopic data and chemical means. Compounds 1, 2, 4, and 5 exhibited antimicrobial activity against Staphyrococcus aureus and especially compound 1 showed strong activity.

INTRODUCTION
Polyacetylenes have been found in many families of higher plants, such as Asteraceae, Araliaceae, and Umbelliferae.1-3 It has been reported antibacterial,4,5 antifungal,5,6 and allelopathic activities.7-9 In our previous research, C17 polyacetylenes from Hedera rhombea exhibited antimicrobial activity against the Micrococcus luteus.4 We had been isolated C15 polyacetylenes, 8-(β-D-glucopyranosyloxy)-3-hydroxy-1,9, 14-pentadecatriene-4,6-diyne termed “helian”, (Z)-3,8-dihydroxy-1,9,14-pentadecatriene-4,6-diyne (4), and (Z)-8-acetoxy-3-hydroxy-1,9,14-pentadecatriene-4,6-diyne (5) and reported for its plant growth activity on rice and cress seedlings.10,11 However, to the best of our knowledge, effects on antimicrobial activity of C15 polyacetylenes have not been studied. In this paper, we describe the isolation and structure elucidation of new antibacterial polyacetylenes 1~3 from H. annuus, and the assessment of the antibacterial propaties of C15 polyacetylenes.

RESULTS AND DISCUSSION
The MeOH extract of the seedlings of H. annuus L. cv. Russia was partitioned between EtOAc and H2O. The EtOAc-soluble portion was subjected to silica gel column chromatography, C18 Sep-Pak cartridges, and reversed-phase HPLC to yield three new C15 polyacetylenes 1 (2.1×10-5%), 2 (1.4×10-4%), and 3 (6.5×10-5%), together with two known C15 polyacetylenes, (Z)-3,8-dihydroxy-1,9,14-pentadecatriene-4,6-diyne (4) and (Z)-8-acetoxy-3-hydroxy-1,9,14-pentadecatriene-4,6-diyne (5). Compounds 4 and 5 had been previously isolated from Grangea maderaspatana.12,13

(Z)-8-Acetoxy-1,2-epoxy-9,14-pentadecatriene-4,6-diyne (1), a pale yellow oil, [α]28D +68° (c 0.25, CH2Cl2) was assigned a molecular formula of C17H18O4 by HREISMS [m/z 287.1287 (M+H)+, Δ+0.4 mmu]. The UV absorptions (254, 270, and 286 nm) were in good agreement with published data of diacetylene, suggesting the presence of a conjugated diyne.14 The IR spectrum (2362, 1745, 1654, and 1637 cm-1) showed the presences of acetylene, ester carbonyl, ketone carbonyl, and olefin, respectively. The 13C NMR data (Table 1) aided by the HMQC spectrum of 1 exhibited the signals due to a ketone carbonyl carbon at δC 183.0 (C-3), an acetoxy carbon at δC 169.3 and 20.8, four olefinic carbons at δC 138.1 (C-14), 137.0 (C-10), 123.0 (C-9), and 115.1 (C-15), four acetylene quaternary carbons at δC 84.5 (C-7), 76.5 (C-5), 72.7 (C-4), and 68.1 (C-6), an acetoxy-bearing carbon at δC 59.8 (C-8), a lower-field shifted methine carbon at δC 54.0 (C-2) and methylene carbon at δC 46.7 (C-1), and three methylene carbons at δC 31.7 (C-13), 28.2 (C-12), and 27.3 (C-11).
The 1H NMR data (Table 2) showed signals for a proton attached to an acetoxy-bearing carbon at δH 6.16 (H-8), three olefinic protons at δH 5.79 (H-14), 5.72 (H-10), and 5.50 (H-9), two terminal olefinic protons at δH 5.02 (H-15a) and 4.98 (H-15b), three oxygen-bearing protons at δH 3.54 (H-2), 3.11 (H-1a), and 3.07 (H-1b), an acetoxy group at δH 2.10, and a methylene sequence at δH 2.17 (H-11), 2.07 (H-13), and 1.49(H-12). The Z-geometry of the double bond (C-9, 10) was deduced on the basis of the 1H-1H coupling constant (J9, 10 =10.5 Hz). The chemical shifts of the oxymethine proton (δH 3.54), oxymethylene proton (δH 3.11 and 3.07), and carbons (δC 54.0 and 46.7) indicated the presence of a terminal epoxide comparing with those of (Z)-8-acetoxy-1-methoxy-3-oxoheptadeca-9-ene-4,6-diyne.14

This finding was further supported by vicinal coupling constants of J1a, 2 = 2.3 Hz and J1b, 2 = 4.3 Hz. The chemical shift of H-2 (δH 3.54), which was at lower field by nearly 1 ppm compared to the value of typical epoxide protons, suggesting that the carbonyl carbon should be connected to C-2. This linkage was further confirmed by an HMBC correlation between H-1a (δH 3.11) and C-3 (δC 183.0). The higher-field shifted ketone carbonyl carbon (C-3) suggested that it was conjugated to a triple bond. This was also supported by chemical shift to lower frequency of the carbonyl peak (1654 cm-1) in the IR spectrum.14 An HMBC correlation of H-8 to OAc (δC 169.3) revealed the location of the acetoxy group at C-8. On the other hand, HMBC correlations of H-8 to C-4, C-5, C-6, and C-7 and H-9 to C-7 confirmed that the acetoxy-bearing carbon (C-8) was connected to an acetylenic carbon (C-7). Consequently, compound 1 was determined to be (Z)-8-acetoxy-1,2-epoxy-9,14- pentadecatriene-4,6-diyne.

The molecular formula of 2 was assigned as C15H20O by the HREIMS [m/z 215.1396 (M-H)+, Δ-4.0 mmu] and the 13C NMR data. In the 13C NMR data (Table 1) aided by the HMQC and HMBC of 2, signals due to four olefinic carbons at δC 139.0 (C-14), 132.9 (C-10), 122.1 (C-9), and 114.3 (C-15), four acetylene quaternary carbons at δC 85.7 (C-5), 83.4 (C-4), 79.5 (C-7), and 69.9 (C-6), an oxymethine carbon at δC 64.1 (C-3), five methylene carbons at δC 33.7 (C-13), 30.7 (C-2), 28.7 (C-12), 27.1 (C-11), and 17.7 (C-8), and a methyl carbon at δC 9.3 (C-1) were observed. The 1H NMR spectrum (Table 2) showed signals for three olefinic protons, two terminal olefinic protons, two oxygen-bearing protons, four methylene protons, and a methyl proton. The partial structures of C-1 ̶ C-3 and C-8 ̶ C-15 could be deduced from consideration of the 1H-1H COSY of 2. On the basis of this spectroscopic evidence, the structure of 2 was elucidated to be (Z)-3-hydroxy-9,14-pentadecatriene-4,6-diyne (2).

The molecular formula of 3 was assigned as C19H22O4 by the HRESIMS [m/z 337.1431 (M+Na)+, Δ+1.5 mmu] and the 13C NMR data. The 13C and 1H NMR data (Tables 1 and 2) of 3 were partially similar to those of (Z)-8-acetoxy-3-hydroxy-1,9,14-pentadecatriene-4,6-diyne (5).12,13 However, two signals of acetoxy carbons at δC 169.4, 20.8 and δC 169.4, 20.9 were observed in 3. The 13C NMR spectrum (Table 1) of 3 showed 19 carbon signals including four quaternary carbon signals (δC 76.6, 75.1, 70.7, and 69.2). Additionally, the 13C NMR spectrum (Table 2) exhibited two acetoxy-bearing carbons at δC 60.0 and 64.4. The 1H NMR spectrum of 3 showed characteristic signals for two protons at δH 6.11 and 5.90 attached to an acetoxy-bearing carbon and two acetoxy groups at δH 2.10 and 2.08. To establish the structure of 3, compound 5 was acetylated by acetic anhydride in pyridine to give compound 3 (quant.). The [α]D and 1H NMR data of the synthetic product 3 were identical with those of natural compound 3.

The structure of 1 was further confirmed by the synthesis of 1 from 5 by two steps as follows. First, compound 5 was oxidized by active MnO2 in anhydrous CH2Cl2 to afford (Z)-8-acetoxy-3-oxopentadeca- 1,9,14-triene-4,6-diyne (6) (87%). The structure of compound 6 was elucidated by spectroscopic data. Compound 6 was epoxidized by 3% H2O2 in acetone containing 1% Na2CO3 to give compound 1 (48%). Although synthetic compound 1 was a mixture of diastereomers, it was difficult to separate each isomer. Since the spectroscopic data of synthetic compound 1 was very similar to that of natural compound 1, the gross structure of 1 was also confirmed by the derivatization of 1 from 5.

The absolute configuration of 2 was determined by modified Mosher’s method15 as follows. Compound 2 was treated with (S)- and (R)-α-methoxy-α-trifluoromethyl-phenylacetyl chloride (MTPA-Cl) in pyrindine-d5 to give the (R)-MTPA ester derivative (2a) and (S)-MTPA ester derivataive (2b) of 2. In the 1H NMR spectrum of the (S)-MTPA ester (2b), proton signals assigned to H-1 and H-2 were observed at higher field than those of the (R)-MTPA ester (2a), while signals due to H-8, H-9, and H-10 in 2a were shifted to a higher field than those in 2b. Therefore, the absolute configuration at C-3 was concluded to be 3S (Figure 2).

The antibacterial activities of polyacetylenes against gram positive bacteria have been reported.4,5 Therefore, new, known, and synthetic compounds 1-6 were tested antimicrobial activities against a gram positive bacterium Staphylococcus aureus by plate diffusion assay (Table 3). Compounds 1, 2, 4, and 5 inhibited its growth, especially compound 1, possessed an epoxide ring, showed strongest activity. Additionally, the structures have some hydroxy groups exhibited activities. These results suggested that the key sites for activity of a series of polyacetylenes from H. annuus are a free hydroxy group at C-3 and an epoxide ring.

EXPERIMENTAL
General Procedures.
Optical rotations were measured with a JASCO DIP-370 polarimeter. UV spectra were recorded on a HITACHI U-2000A spectrometer. IR spectra were recorded on a JASCO FT/IR-300 spectrometer. 1H and 13C NMR spectra were measured and recorded on a Bruker Avance 500 spectrometer in CDCl3. The resonances of CDCl3 at δH 7.26 and δC 77.0 were used as internal references for the 1H and 13C NMR spectra, respectively. HRESIMS and HREIMS were recorded on Waters Xevo Q-Tof, Waters Synapt G2 mass spectrometer, and JEOL JMS-T100LC.

Plant material.
Seeds of sunflower (Helianthus annuus L. cv. Russia) were spread evenly on moist vermiculite in trays and incubated at 25 °C in the dark for 7~10 days. Some of the seedlings (hypocotyls length, ca. 12 cm) were harvested, collected, and frozen at -30 °C until use. The other were illuminated by blue light (λmax 445 nm, 1.90 μmol m-2s-1) for 1 h. The blue light-illuminated seedlings were also harvested, collected and frozen at -30 °C until use.

Extraction and Isolation of Compounds 1, 2, and 5.
The seedlings illuminated and not (2.3 kg) by blue light were homogenized in MeOH (1.5 L). The homogenate was filtered and allowed to dry in vacuo at 40 °C. The MeOH extracts were partitioned between EtOAc (100 mL×3) and H2O (100 mL). The EtOAc-soluble portion (2.14 g) was subjected to silica gel column chromatography (φ2.4×35 cm) eluting with n-hexane/acetone (20:1 to 0:1) and then CHCl3/MeOH (1:1 to 0:1) to separate into 28 fractions (EA-1~EA-28). Fraction EA-13 (18.2 mg) eluted with n-hexane/acetone (5:1) was applied to a C18 Sep-Pak cartridge (Waters, MeOH/H2O, 3:2 to 1:0) to afford 10 fractions (EA13-1~ EA13-10). Fraction EA-13-2 (6.2 mg) eluted with MeOH/H2O (3:2) was separated by reversed-phase HPLC [TSK-gel ODS-120A, TOSOH, φ7.8 mm×30 cm, flow rate 2.0 mL/min, MeCN/H2O (3:7 to 1:0)] to give 1 (0.5 mg, tR 35.1 min) and 5 (0.3 mg, tR 36.9 min), respectively. Fraction EA-8 (9.2 mg) eluted with n-hexane/acetone, 10:1 was applied to a C18 Sep-Pak cartridge eluted with MeOH/H2O (3:2 to 1:0) to give 2 (1.1 mg, EA8-4) eluted with MeOH/H2O (4:1).

Extraction and Isolation of Compounds 3 and 4.
The seedlings (1.0 kg) were freeze-dried for five days before extract. The homogenate was filtered and allowed to dry in vacuo at 40 °C. The MeOH extracts were partitioned between EtOAc (100 mL×3) and H2O (100 mL). The EtOAc-soluble portion (1.32 g) was subjected to silica gel column chromatography (φ1.0×35 cm) eluting with n-hexane/acetone (20:1 to 0:1) and then CHCl3:MeOH (1:1 to 0:1) to separate into 19 fractions (EAII-1~EAII-19). Fraction EAII-7 (20.9 mg) eluted with n-hexane/acetone, 10:1 was separated by reversed-phase HPLC [TSK-gel ODS-120A, Tosoh, Japan, φ7.8 mm×30.0 cm, flow rate 2.0 mL/min, MeCN/H2O (2:3 to 1:0)] to give 3 (0.7 mg, tR 40.6 min). The 1H and 13C NMR data of EAII-15 eluted with n-hexane/acetone (3:1) was identical to (Z)-3,8-diacetoxy-1,9,14-pentadecatriene-4,6-diyne (3, 10.9 mg).

(Z)-8-Acetoxy-1,2-epoxy-9,14-pentadecatriene-4,6-diyne (1): Pale yellow oil; [α]D28 +68 (c 0.25, CH2Cl2); IR (film) νmax 2921, 2362, 1745, 1654, 1637, and 1227 cm-1; UV (MeOH) λmax (logε) 254 (4.2), 270 (4.2), and 286 (4.1) nm; 13C and 1H NMR (Tables 1 and 2); ESIMS (positive ion) m/z 287(M+H)+; HRESIMS (positive ion) m/z 287.1287 (M+H)+, (calcd for C17H19O4, 287.1283).

(Z)-3-Hydroxy-9,14-pentadecatriene-4,6-diyne (2): Colorless oil; [α]D28 -20 (c 0.05, MeOH); IR (film) νmax 3412, 2925, 2344, and 1637 cm-1; UV (MeOH) λmax (logε) 213 (4.0), 253 (3.5), 267 (3.5), and 283 (3.5) nm; 13C and 1H NMR (Tables 1 and 2); EIMS m/z 215 (M-H)+; HREIMS m/z 215.1396 (M-H)+, (calcd for C15H19O, 215.1436).

(Z)-3,8-Diacetoxy-1,9,14-pentadecatriene-4,6-diyne (3): Pale yellow oil; [α]D28 +81 (c 0.21, CH2Cl2); IR (film) νmax 2929, 2259, 1747, 1640, and 1221 cm-1; UV (MeOH) λmax (logε) 234 (4.1), 246 (4.1), and 260 (4.0) nm; 13C and 1H NMR (Tables 1 and 2); ESIMS m/z 337(M+Na)+; HRESIMS (positive ion) m/z 337.1431 (M+Na)+, (calcd for C19H22O4Na, 337.1416).

Oxidation of 5.
Active MnO2 (33.0 mg, 3.8×10-1 mmol) was added to a solution of compound 5 (5.2 mg, 1.91×10-2 mmol) in anhydrous CH2Cl2 (1.0 mL) and the mixture was stirred at room temperature for 60 min. The reaction mixture was filtered through Celite and the residue was washed with CHCl3. The filtrate was evaporated to dryness in vacuo to give (Z)-8-acetoxy-3-oxopentadeca-1,9,14-triene-4,6-diyne (6, 4.5 mg, 87%): Pale yellow oil; 1H NMR (500 MHz, CDCl3): δH 6.56 (1H, d, J = 17.5 Hz, H-1a), 6.41 (1H, d, J =17.5 and 10.3 Hz, H-2), 6.24 (1H, d, J = 10.3 Hz, H-1b), 6.17 (1H, d, J = 8.8 Hz, H-8), 5.79 (1H, ddt, J = 17.1, 10.2, and 6.6 Hz, H-14), 5.72 (1H, dtd, J = 10.4, 7.7, and 2.7 Hz, H-10), 5.51 (1H, ddt, J = 10.3, 8.9, and 1.6 Hz, H-9), 5.02 (1H, ddt, J = 17.1, 2.0, and 1.6 Hz, H-15a), 4.98 (1H, ddt, J = 10.2, 2.0, and 1.3 Hz, H-15b), 2.18 (2H, m, H-11), 2.10 (3H, s, OAc), 2.07 (2H, m, H-13), and 1.50 (2H, m, H-12); 13C NMR (125 MHz, CDCl3): δC 177.3 (C-3), 169.4 (OAc), 138.1 (C-14), 137.6 (C-2), 136.8 (C-10), 134.7 (C-1), 123.3 (C-9), 115.1 (C-15), 83.3 (C-7), 75.2 (C-5), 74.0 (C-4), 68.3 (C-6), 59.9 (C-8), 33.1 (C-13), 28.2 (C-12), 27.3 (C-11), and 20.8 (OAc); HMBC correlations: (CDCl3, H/C) 1a/2, 1a/3, 1b/2, 1b/3, 2/1, 2/3, 2/4, 8/5, 8/6, 8/7, 8/9, 8/10, 8/CH3CO, 9/11, 10/8, 11/9, 11/10, 11/12, 11/13, 12/10, 12/11, 12/13, 13/12, 13/14, 13/15, 14/12, 14/13, 15a/13, 15b/13, and CH3CO/CH3CO.

Epoxidation of 6.
To a solution of 6 (7.3 mg, 2.7×10-2 mmol) in acetone (500 μL) at 0 °C were added 3% H2O2 (225 μL, 2.7×10-1 mmol) and 1% Na2CO3 aq. (28.7 μL, 2.7×10-3 mmol) and the mixture was stirred at 0 °C for 60 min. The reaction mixture was partitioned between EtOAc (10 mL) and H2O (10 mL) and then the EtOAc layer was dried with MgSO4, filtered, and concentrated. The residue was purified by silica gel TLC (n-hexane/acetone, 10:1) to give (Z)-8-acetoxy-1,2-epoxy-9,14-pentadecatriene-4,6-diyne (1, 3.8 mg, 48%).
Acetylation of 5.
Acetic anhydride (0.5 mL) was added to a solution of compound 5 (5.6 mg, 2.0×10-2 mmol) in pyridine (0.6 mL) and the mixture was stirred at room temperature for 60 min. After added toluene to remove pyridine it was evaporated to dryness in vacuo to give (Z)-3,8-diacetoxy-1,9,14-pentadecatriene- 4,6-diyne (3, 6.5 mg, quant).

Preparation of the (R)- and (S)-MTPA Ester Derivatives of 2.
(R)-MTPA ester of 2 (2a): (S)-MTPA chloride (2 μL) was added to a solution of compound 2 (0.4 mg) in pyridine-d5 (0.6 mL). After standing the reaction mixture at room temperature for 1day, the solution was evaporated to dryness under N2 gas stream.
1H NMR (CDCl3): 0.9984 (3H, m, H-1), 1.3653 (2H, m, H-12), 1.8757 (2H, m, H-2), 2.0294 (4H, H-11 and H-13), 3.0189 (2H, m, H-8), 4.9272 (1H, ddt, J = 10.2, 2.2, and 1.0 Hz, H-15b), 4.9851 (1H, ddt, J = 17.1, 2.2, and 1.6, H-15a), 5.3720 (1H, m, H-9), 5.4997 (1H, m, H-3), 5.5095 (1H, m, H-10), and 5.7954 (1H, ddt, J = 17.1, 10.2, and 6.8 Hz, H-14).

(S)-MTPA ester of 2 (2b): (R)-MTPA chloride (2 μL) was added to a solution of compound 2 (0.4 mg) in pyridine-d5 (0.6 mL). After standing the reaction mixture at room temperature for 1day, the solution was evaporated to dryness under N2 gas stream.
1H NM R (500 MHz, CDCl3): 0.9221 (3H, m, H-1), 1.3652 (2H, m, H-12), 1.8117 (2H, m, H-2), 2.0293 (4H, H-11 and H-13), 3.0223 (2H, m, H-8), 4.9270 (1H, ddt, J = 10.0, 2.0, and 1.0 Hz, H-15b), 4.9850 (1H, ddt, J = 17.1, 2.0, and 1.5 Hz, H-15a), 5.3724 (1H, m, H-9), 5.5331 (1H, m, H-3), 5.5120 (1H, m, H-10), and 5.7955 (1H, ddt, J = 17.1, 10.0, and 6.7 Hz, H-14).

Antimicrobial test.
Antimicrobial activity against gram positive-bacterium Staphylococcus aureus KB210 was tested by plate diffusion assay using 8 mm paper disk. Compound solutions were prepared by dissolving each compound in acetone. Each adjusted solution was added in paper disk (10 and 20 μL) and paper disk were drying. The paper disks were set on the agar plate suspended S. aureus. After cultivating microorganisms for 24 h, the strength of antimicrobial activity was estimated by measuring the diameter length of inhibition zone (mm).

ACKNOWLEDGEMENT
This work was partly supported by Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, and Culture of Japan.

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