BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus ON Selenastrum capricornutum

Journal of Chemical Ecology, Vol. 30, No. 4, April 2004 ( C 2004) BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus ON Selenastrum capricornutum MARINA DELLAGRECA, 1 MARINA ISIDORI, 2 MARGHERITA LAVORGNA, 2 PIETRO MONACO, 2 LUCIO PREVITERA, 1 and ARMANDO ZARRELLI 1, 1 Dipartimento di Chimica Organica e Biochimica, Università Federico II Complesso Universitario Monte S. Angelo, via Cinthia I-80126 Napoli, Italy 2 Dipartimento di Scienze della Vita, Seconda Università di Napoli via Vivaldi 43, 81100 Caserta, Italy (Received April 22, 2003; accepted December 15, 2003) Abstract Twenty-five 9,10-dihydrophenanthrenes, four phenanthrenes, a dihydrodibenzoxepin, and a pyrene, isolated from the wetland plant Juncus acutus, were tested to detect their effects on the green alga Selenastrum capricornutum. Nine of the compounds were isolated and identified for the first time. Most of the compounds caused inhibition of algal growth. The 9,10-dihydrophenanthrenes 1, 5, 21, and 22 were the most active. Key Words Juncus acutus, dihydrophenanthrenes, phenanthrenes, dihydrodibenzoxepin, pyrene, algiecides, Selenastrum capricornutum. INTRODUCTION As part of our effort to discover natural products with potential use as algicides, we have investigated the aerial part of Juncus effusus and reported that many of the 25 dihydrophenanthrenes isolated from the plant possess significant antialgal activity (DellaGreca et al., 1996, 1997). A toxicity evaluation of these compounds on aquatic species from various taxonomic groups and their possible selective toxicity in aquatic ecosystems has been also reported (DellaGreca et al., 2001a). In continuing our studies, we have considered another Juncaceae, Juncus acutus, a wetland plant widely distributed in the Mediterranean area. From samples collected in Sardinia, nine 9,10-dihydrophenanthrenes, three phenanthrenes, To whom correspondence should be addressed. E-mail: zarrelli@unina.it 867 0098-0331/04/0400-0867/0 C 2004 Plenum Publishing Corporation

868 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI and a pyrene were isolated and described in a recent paper (DellaGreca et al., 2002). Many of them have inhibitory activity on Selenastrum capricornutum, the algal species selected for standardized studies in aquatic environments (ISO, 1989; OECD, 1994; ASTM, 1998). The ability of some natural products to inhibit microalgae may be of ecotoxicological significance because algae are a key functional group in freshwater, and specific toxic effects may be expressed in algae at low concentrations. Furthermore, alterations of the phytoplankton community due to a toxic stress may affect the structure and functioning of the whole ecosystem (Nyholm and Källqvist, 1989). In this paper, we report a study from samples collected in Sabaudia, near Rome. Eight new 9,10-dihydrophenanthrenes and a phenanthrene were isolated in addition to already reported compounds that were identified on the basis of their spectroscopic features. METHODS AND MATERIALS Plant Material. Juncus acutus plants were collected on land 300 m from the sea, in Sabaudia (near Rome) during summer (July). They were identified by Professor Antonino Pollio of the Dipartimento di Biologia Vegetale of University of Naples Federico II. A voucher specimen (HERBNAPY250) was deposited at the Dipartimento di Biologia Vegetale of University of Naples Federico II. General Experimental Procedures. NMR spectra were recorded at 500 MHz for 1 H and 125 Hz for 13 C on a Varian Unity Inova spectrometer in CDCl 3 or CD 3 OD solutions, at 25 C. Proton-detected heteronuclear correlations were measured using HMQC (optimized for 1 J HC = 160 Hz) and HMBC (optimized for 1 J HC = 7 Hz). IR spectra were determined in CHCl 3 solutions on a FT-IR Perkin- Elmer 1740 spectrometer. UV spectra were obtained on a Perkin-Elmer Lambda 7 spectrophotometer in CHCl 3 solutions. MS spectra were obtained with an HP 6890 spectrometer equipped with a MS 5973 N detector. The HPLC was performed on an Agilent 1100 series apparatus by using a UV detector. Preparative HPLC was performed by using RP-8 (Luna 10 µm, 250 10 mm ID, Phenomenex), SiO 2 (Maxsil 10 µm, 250 10 mm ID, Phenomenex), or RP-18 (Kromasil 10 µm, 250 10 mm ID, Phenomenex) columns. Analytical TLC was performed on Merck Kieselgel 60 F 254 or RP-18 F 254 plates with 0.2 mm layer thickness. Spots were visualized by UV light or by spraying with H 2 SO 4 AcOH H 2 O (1:20:4). The plates were then heated for 5 min at 110 C. Preparative TLC was performed on Merck Kieselgel 60 F 254 plates, with 0.5 or 1 mm film thickness. Flash column chromatography (FCC) was performed on Merck Kieselgel 60 (230 400 mesh) at medium pressure. Column chromatography (CC) was performed on Merck Kieselgel 60 (70 240 mesh). Extraction and Isolation of 9,10-Dihydrophenanthrenes and Phenanthrenes from J. acutus. Air-dried plants (6 kg) were sequentially and exhaustively extracted

BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus 869 with light petrol (20 l), ethyl acetate (EtOAc, 18 l), and methanol (MeOH, 18 l) at room temperature for 10 d. The light petrol extract (130 g) was dissolved in ethyl ether (900 ml) and shaken with 2 M NaOH (2 400 ml). The organic layer was washed until neutral and dried on Na 2 SO 4 to give, after evaporation of solvent in vacuo, 65 g of neutral material. The alkaline solutions were reacidified with 2 M HCl and extracted with EtOAc. The organic layer was washed until neutral and dried on Na 2 SO 4 to give, after evaporation of solvent in vacuo, 40 g of acidic material. Neutral Fraction Separation. The neutral fraction (65 g) was chromatographed on silica gel (400 g). The fractions eluted with hexane were separated by silica gel flash column chromatography (9:1 hexane Et 2 O) to give pure 1 (21 mg), 4 (24 mg), and 17 (30 mg). Fractions eluted with hexane CHCl 3 (4:1) were purified by preparative TLC (3:1 hexane Et 2 O) to give pure 25 (8 mg) and 26 (10 mg). Fractions eluted with hexane CHCl 3 (3:1) were purified on an HPLC silica gel column (9:1 hexane acetone) to give pure 6 (25 mg) and 8 (7 mg). Fractions eluted with CHCl 3 (100%) were separated by flash column chromatography (9:1 CHCl 3 acetone) and successively purified by preparative TLC (9:1 CHCl 3 EtOAc) to give pure 5 (16 mg), 15 (18 mg), 20 (12 mg), and 21 (15 mg). The EtOAc extract (200 g) was separated into an acidic (60 g) and a neutral fraction (80 g), as previously described for the light petrol extract. Neutral Fraction Separation. The neutral fraction was chromatographed on silica gel (300 g) by using a hexane EtOAc gradient, and the fractions eluted with hexane EtOAc (9:1) were further separated by flash column chromatography (9:1 hexane EtOAc) to give 10 fractions. Subfractions 2 5 were purified by HPLC silica gel column (4:1 hexane EtOAc) to give pure 22 (7 mg), 28 (23 mg), and 29 (15 mg). Subfractions 6 10 were purified by HPLC silica gel column (9:1 hexane EtOAc) to give pure 2 (5 mg) and 3 (8 mg). The acidic fraction was separated by flash column chromatography by using a CHCl 3 EtOAc gradient. Fractions eluted with CHCl 3 EtOAc (6:1) were further purified by HPLC C-18 column (2:2:1 MeOH H 2 O MeCN) to give pure 27 (45 mg) and 31 (18 mg). Fractions eluted with CHCl 3 EtOAc (1:1) were rechromatographed on silica gel (24:1 CHCl 3 MeOH) to give eight fractions. The fractions 1 and 2 were purified by HPLC C-18 column (5:4:1 MeOH H 2 O MeCN) to give pure 7 (15 mg), 16 (18 mg), and 19 (4 mg). Fraction 3 was purified by preparative TLC (3:1 hexane EtOAc) to give pure 18 (9 mg). Fractions 4 6 were purified by HPLC NH 2 column (49:1 CHCl 3 MeCN) to give 10 (6 mg), 11 (4 mg), and 30 (9 mg). Fractions 7 and 8 were purified by HPLC C-18 column (5:3:2 MeOH H 2 O MeCN) to give pure 12 (5 mg), 13 (6 mg), and 14 (4 mg). Fractions eluted with EtOAc were rechromatographed on silica gel (17:3 CHCl 3 acetone 3) and purified by HPLC C-18 column (6:3:1 MeOH H 2 O MeCN) to give pure 9 (10 mg), 23 (8 mg), and 24 (11 mg). Toxicity Testing. The bioactivity of all the compounds was tested on the freshwater green alga Selenastrum capricornutum. The 9,10-dihydrophenanthrenes

870 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI (1, 4 7, 10 13, 15 17, 20 22) were previously assayed using the algal growth inhibition method in a 4-d static test with laboratory cultures of algae (DellaGreca et al., 1996, 1997). The algal growth inhibition tests for the compounds isolated only from J. acutus were run in 72 hr according to the International Standard Organization procedure 8692 (ISO, 1989). The algal inoculum was taken from an exponentially growing preculture and added to 25 ml of test solution to obtain an initial cell density on the order of 10 4 cells/ml. Each compound was tested in five concentrations, with three replicates per concentration and a negative control. All chemicals were dissolved in dimethyl sulfoxide (DMSO), and in the final test solutions DMSO concentration was kept constant at 0.01% (v/v). A solvent only control was included in each experiment to detect the possible effect of the DMSO. Flasks were placed in a growth chamber at 25 C under continuous illumination (8000 lux). Cell density was measured at 0 time and every 24 hr for 3dbyan electronic particle dual threshold counter (Coulter Counter Z2, 100 µm capillary, Instrumentation Laboratory, Miami, FL, USA), and from these data the algal growth inhibition was calculated by integrating the mean values from t 0 to t 72 hr (area under the curve). Inhibition (percentage) values were reported against logtransformed data of concentrations (in µm) and processed by a regression analysis technique to obtain the respective IC 50 value (the test concentration corresponding to 50% reduction in growth relative to the control). IC 50 values were also calculated from the results of previous studies (DellaGreca et al., 1996, 1997) to allow a comparison of algal growth inhibition for all compounds isolated from J. acutus. RESULTS AND DISCUSSION The whole plants (aerial and rhizome) were extracted with solvents of increasing polarity. Preliminary assays on S. capricornutum showed an algal growth inhibition of petrol (50%, 1.0 mg/l) and ethyl acetate extracts (60%, 1.0 mg/l), while the MeOH extract was inactive. The examination of the active extracts of J. acutus revealed 9,10-dihydrophenanthrenes, phenanthrenes, a dihydrodibenzoxepin, and a pyrene (Figures 1 and 2). Some of them had been previously isolated from another Juncaceae, J. effusus, and were identified by comparison of NMR data for the compounds 1, 4 7, 10 13, 15 17, 19 22 with NMR data from the literature (DellaGreca et al., 1993a,b, 1996, 1997). The examination of petrol and ethyl acetate extracts of J. acutus collected in Sabaudia revealed known 9,10-dihydrophenanthrenes 25 and 26, phenanthrenes 27 29, and pyrene 31 (DellaGreca et al., 2002) together with the novel compounds 2, 3, 8, 9, 14, 18, 23, 24, and 30. Compound 2 had in the EIMS spectrum a molecular ion peak at m/z 266 and showed 18 carbon signals in the 13 C NMR spectrum (Table 2), consistent with a molecular formula of C 18 H 18 O 2. A DEPT experiment defined the carbons as two methyls, three methylenes, five methines, and eight quaternary carbons.

BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus 871 FIG. 1. Structures of 9, 10-dihydrophenanthrenes.

872 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI FIG. 1. CONTINUED. The 1 H NMR spectrum (Table 1) showed four aromatic protons, three vinyl protons, a methoxyl, two methylenes, and a methyl. These data resembled those of effusol (7), except for the presence of a methoxyl group. In a NOE experiment, the methoxyl had relation with the doublet at δ 6.78 and was assigned to the H-3 proton. The EIMS spectrum of compound 3 had a molecular ion peak at m/z 264 and showed 19 carbon signals in the 13 C NMR spectrum (Table 2), consistent with a molecular formula of C 19 H 20 O. A DEPT experiment defined the carbons as three methyls, three methylenes, five methines, and eight quaternary carbons. The 1 H NMR spectrum (Table 1) showed four aromatic protons, three vinyl protons, a methoxyl, two methylenes, and two methyl groups. These data resembled those of juncunol (4), except for the presence of a methoxyl group. In a NOE experiment, the methoxyl had relation to the doublet at δ 6.73 and was assigned to the H-3 proton. Compound 8 had molecular formula C 18 H 18 O according to the molecular ion at m/z 250 in the EIMS spectrum. Four aromatic ortho-coupled protons, three double doublets of a vinyl group, two methylene multiplets, and two methyl singlets were present in the 1 H NMR spectrum (Table 1). Thirteen carbon signals were present in the 13 C NMR spectrum (Table 2), identified by DEPT experiment as

BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus 873 FIG. 2. Structures of phenanthrenes and pyrene. two methyls, three methylenes, five methines, and eight quaternary carbons. The protons were assigned to the corresponding carbons by an HMQC experiment, and in combination with an HMBC experiment (Figure 3), allowed assignment of the structure 2-hydroxy-1,6-dimethyl-5-vinyl-9,10-dihydrophenathrene to compound 8. In the HMBC spectrum, correlations between the H-12 and H-14 protons with the C-5 and C-6 carbons were observed. Furthermore, the H-7 and H-8 protons were correlated to the C-6 and C-8a carbons. The assignment of the methyl at C-6 was also confirmed by NOE between that methyl and the H-7 and H-12 protons. The molecular ion at m/z 282 in the EIMS spectrum and the presence of 18 signals in the 13 C NMR spectrum defined a molecular formula C 18 H 18 O 3 for compound 9. A DEPT experiment defined the carbons as a methyl, four methylenes, four methines, and nine quaternary carbons. The 1 H NMR spectrum (Table 1) showed the H-3 and H-4 doublets, the H-8 singlet, the H-9 and H-10 broad singlet, the H-12 and H-13 double doublets, and the H-11 and H-14 singlets. The protons were assigned to the corresponding carbons by an HMQC experiment that, in combination with an HMBC experiment, allowed the assignment of the structure 2,7-dihydroxy-6-hydroxymethyl-1-methyl-5-vinyl-9,10-dihydrophenathrene to the compound. In the HMBC spectrum, correlations between the H-12 and hydroxymethyl protons with the C-5 and C-6 carbons were observed. Furthermore, the H-8 proton was correlated to the C-6, C-7, and C-8a carbons. In addition, a NOE correlation between the hydroxymethyl protons and the H-12 proton was

874 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI TABLE 1. 1 H NMR DATA FOR 2, 3, 8, 9, 14, 18, 23, 24, 30 (CDCl 3) a Position 2 3 8 9 4 b 18 23 b 24 b 30 b 3 6.78 d 6.78 d 6.73 d 6.77 d 6.85 d 6.68 d 6.67 d 7.20 d (8.2) (8.6) (8.4) (8.4) (8.4) (8.0) (8.0) (9.0) 4 7.40 d 7.48 d 7.47 d 7.65 d 7.54 d 7.44 s 7.33 d 7.28 d 8.45 d (8.2) (8.6) (8.4) (8.4) (8.4) 7.44 s (8.0) (8.0) (9.0) 6 6.88 d 7.25 s 6.90 d 6.90 s (2.5) (2.5) 7 7.12 d 7.65 d 6.68 s 8.56 d (7.8) (8.3) (8.5) 8 6.69 d 7.02 s 7.49 d 6.97 s 7.76 d 6.64 d 7.72 d (2.5) (7.8) (8.3) (2.5) (8.5) 9 2.72 m 2.74 m 2.89 m 2.66 brs 2.88 m 2.71 m 2.62 brs 2.63 brs 8.04 d (9.5) 10 2.72 m 2.74 m 2.78 m 2.66 brs 2.74 m 2.64 m 2.62 brs 2.63 brs 7.89 d (9.5) 11 2.24 s 2.26 s 2.24 s 2.29 s 2.19 s 2.32 s 2.19 s 2.18 s 2.54 s 12 6.93 dd 6.99 dd 6.77 dd 6.70 dd 7.11 dd 6.86 dd 4.66 s 4.64 s 7.18 dd (17.4, 11.2) (18.0, 10.5) (17.8, 11.6) (18.4, 11.4) (18.0, 11.2) (17.6, 11.8) 4.66 s 4.64 s (17.5, 11.5) 13 5.68 dd 5.71 dd 5.60 dd 5.77 dd 5.46 dd 5.46 dd 5.84 dd (17.4, 1.8), (18.0, 1.5), (11.6, 2.0), (11.4, 1.8), (11.2, 1.8), (11.8, 2.1), (11.5, 2.0), 5.26 dd 5.25 dd 5.22 dd 5.70 dd 5.19 dd 5.14 dd 5.45 dd (11.2, 1.8) (10.5, 1.5) (17.8, 2.0) (18.4, 1.8) (18.0, 1.8) (17.6, 2.1) (17.5, 2.0) 14 2.38 s 2.32 s 5.01 s 2.23 s 2.21 s 4.90 s OMe 3.86 s 3.87 s a J values (in Hz) in parentheses. b Recorded in CD 3OD.

BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus 875 TABLE 2. 13 C NMR DATA FOR 2, 3, 8, 9, 14, 18, 23, 24, 30 (CDCl 3 ) Position 2 3 8 9 14 a 18 23 a 24 a 30 a 1 120.3 122.8 120.7 122.4 123.6 122.0 122.4 121.9 116.7 2 155.8 156.6 153.7 155.6 153.1 150.3 155.5 155.2 152.4 3 107.4 107.0 113.8 113.1 110.3 140.2 113.1 112.9 115.7 4 128.0 127.5 128.8 127.6 124.5 113.2 127.7 127.6 121.7 5 136.2 135.0 135.0 142.1 134.4 137.9 139.6 136.1 134.5 6 112.5 127.1 135.6 127.6 129.3 137.5 115.1 115.1 134.1 7 154.6 135.7 123.0 156.8 119.3 129.2 156.5 154.8 125.5 8 114.0 127.7 123.0 115.1 120.3 152.4 116.7 121.6 123.2 9 30.0 29.9 26.8 28.3 23.5 30.3 32.3 27.7 120.4 10 26.1 25.6 26.0 27.1 23.5 25.9 27.2 26.9 120.5 1a 139.2 139.4 135.6 154.9 135.7 129.2 153.8 140.2 131.3 4a 127.0 126.6 129.0 119.2 129.3 127.7 111.3 129.1 123.4 5a 127.4 131.3 134.7 123.2 131.8 137.9 127.0 128.1 127.4 8a 139.3 138.8 129.0 136.7 135.3 120.8 129.6 140.2 129.7 11 11.8 11.7 12.1 12.0 7.8 12.4 12.3 12.1 9.8 12 138.0 139.0 128.8 139.7 133.2 137.9 64.6 64.5 132.9 13 113.5 113.6 113.8 116.7 115.6 119.8 120.7 14 21.1 21.3 62.7 168.0 13.2 61.8 OMe 55.5 55.8 a Recorded in CD 3 OD. observed. The molecular ion at m/z 280 in the EIMS spectrum and the presence of 18 signals in the 13 C NMR spectrum (Table 2) defined a molecular formula of C 18 H 16 O 3 for compound 14. Its IR spectrum showed the presence of hydroxyl and carboxyl functions with absorptions at 3340, 3200, and 1682 cm 1. A DEPT experiment defined the carbons as a methyl, three methylenes, five methines, and nine quaternary carbons. Four aromatic ortho-coupled protons, three double doublets of a vinyl group, two methylene multiplets, and a methyl singlet were present in the 1 H NMR spectrum (Table 1). These data were similar to those of compound 12 previously isolated from J. effusus (DellaGreca et al., 1993b). However, the different correlations observed in the HMBC spectrum defined the isomeric structure with the carboxyl group at C-6. The H-12 vinyl and H-7 protons gave correlations with the C-5 and C-6 quaternary carbons. Confirmation of this was given by the NOE interaction observed between the methoxyl, of the methylated 14, and the H-12 and H-7 protons. The structure of 2,3,8-trihydroxy-1,6-dimethyl-5-vinyl-9,10-dihydrophenanhrene was attributed to compound 18. It had the molecular formula C 18 H 18 O 3 with the molecular ion at m/z 282 in the EIMS spectrum. The 1 H NMR spectrum (Table 1) showed the H-4 and H-7 singlets, the H-9 and H-10 multiplets, the H-12 and H-13 double doublets, the H-11 and H-14 singlets. In the HMBC spectrum, the H-4 proton was correlated to the C-2, C-3, C-1a, C-4a, and C-5a carbons. The

876 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI FIG. 3. Selected HMBC correlations for 9,10-dihydrophenanthrene 8. H-7, H-12 vinyl, and H-14 methyl gave cross peaks with the C-5, C-6 carbons and the first proton was also correlated to C-8 and C-8a. As expected from the structure, a NOE interaction between the H-14 methyl and H-7 proton was seen. Compound 23 had the molecular formula C 16 H 16 O 3 with the molecular ion at m/z 256 in the EIMS spectrum. Accordingly, 16 carbon signals were present in the 13 C NMR spectrum (Table 2), identified by a DEPT experiment as a methyl, three methylenes, four methines, and eight quaternary carbons. Four aromatic protons (two ortho- and two meta-coupled), three methylenes (one singlet and two multiplets), and a methyl singlet were present in the 1 H NMR spectrum (Table 1). The protons were assigned to the corresponding carbons by an HMQC experiment and, in combination with an HMBC experiment, the structure 2,7-dihydroxy-5- hydroxymethyl-1-methyl-9,10-dihydrophenathrene was assigned to compound 23. In the HMBC spectrum, the H-6 and H-12 protons showed correlations with the C-5 and C-5a carbons. The first proton was also correlated to the C-7 and C-8 carbons. The assignment of the hydroxymethyl at C-5 was also confirmed by the NOE between these protons and the H-6 proton. The molecular ion at m/z 270 in the EIMS spectrum and the presence of 17 signals in the 13 C NMR spectrum defined a molecular formula C 17 H 18 O 3 for compound 24. A DEPT experiment defined the carbons as two methyls, three methylenes, three methines, and nine quaternary carbons. The 1 H NMR spectrum (Table 1) showed the H-3 and H-4 doublets, the H-6 singlet, the H-9 and H-10 broad singlet, the H-12 singlet, and the H-11 and H-14 methyl singlets. The protons were assigned to the corresponding carbons by an HMQC experiment that, in combination with an HMBC experiment, allowed a structure of 2,7-dihydroxy-5-hydroxymethyl-1,8-dimethyl-9,10-dihydrophenathrene to be assigned to compound 24. In the HMBC spectrum, correlations of the H-12 and H-6 protons with the C-5 and C-5a carbons were observed. Furthermore, the H-6

BIOACTIVITY OF PHENANTHRENES FROM Juncus acutus 877 proton was correlated to the C-7 and C-8 carbons. A NOE correlation between the hydroxymethyl and the H-6 protons was observed. Structure 2-hydroxy-6-hydroxymethyl-1-methyl-5-vinyl-phenanthrene was attributed to compound 30. The molecular peak at m/z 264 in the EIMS spectrum defined the molecular formula C 18 H 16 O 2. The 1 H NMR (Table 1) exhibited six aromatic protons ortho-coupled, three vinyl protons as double doublets, and a methylene and a methyl singlet. In the 13 C NMR spectrum (Table 2), 18 carbon signals were present, which were defined by a DEPT experiment as a methyl, two methylenes, seven methines, and eight quaternary carbons. The HMBC spectrum showed cross peaks of the H-11 methyl protons with the C-1, C-2, and C-1a carbons. The H-3 proton was heterocorrelated with the C-1, C-2, and C-4a carbons. According to the 1 H- 1 H COSY, the proton at δ 8.45 was attributed to the H-4. It gave interactions with the C-2, C-1a, and C-5a carbons. The interaction of the H-12 vinyl proton to the C-5a carbon located the vinyl chain at C-5 and, accordingly, this carbon was correlated with the H-13 protons. In the same experiment, both the H-7, at δ 8.56, and the H-14 methylene protons were correlated to the C-5 carbon according to the location of the hydroxymethyl at the C-6 position. Finally, the H-9 proton gave cross peaks with the C-1a and C-5a, and the H-10 proton gave cross peaks with the C-1 and C-4a carbons. A NOE correlation between the H-14 hydroxymethyl and the H-7 proton was observed. The phytoxicity of all the compounds was tested on the freshwater green alga Selenastrum capricornutum and the respective median inhibition concentrations (IC 50 ) are reported in Table 3. Among the substances tested, the most active was compound 1 (IC 50 = 11.1 µm) with an activity of 9, and 11 times that of compounds 3 (103.4 µm) and 31 (126.8 µm), respectively, that showed the least toxic values in IC 50. However, compounds 7, 8, 18, 19, 23, and 30 did not reach an IC 50 value, showing only slight inhibition at the highest concentrations assayed. Two compounds, 26 and 29, were found to biostimulate the algal growth even at higher concentrations (90% at concentration of 410.9 µm for 29). The presence of one hydroxyl on the molecule seems important for the activity. Compound 4 is twice as active as compound 3, which does not have a hydroxyl group. Compounds 1, 4, and 17, which have a single hydroxyl group, are six, three, and two times, respectively, more toxic than 15, 7, and 16 that have two hydroxyl groups. For compound 18, the presence of three hydroxyl groups cause a further reduction of the activity (35% inhibition at 82.1 µm). The isomeric compounds 5 and 6, 10 and 11, 12, 13, and 14 exhibit comparable responses, indicating that the relative position of the groups is not very important. The presence of a vinyl, a hydroxyethyl, or a hydroxymethyl at C-5 does not cause significant differences in the antialgal activity; compare the isomeric 6, 7, and 15 with 21, 22, 23, and 24. The presence of a phytoxyl group in compound 26 causes a biostimulation of algal growth. Phenanthrenes 27 and 30 are less active than the corresponding

878 DELLAGRECA, ISIDORI, LAVORGNA, MONACO,PREVITERA, AND ZARRELLI TABLE 3. MEDIAN INHIBITION CONCENTRATIONS (IC 50 ) FOR Selenastrum capricornutum Compound IC 50 (µm) 95% confidence interval 1 11.1 8.6 15.3 2 23.8 21.2 26.7 3 103.4 92.2 116.1 4 45.6 37.6 58.0 5 19.9 12.8 35.7 6 26.2 20.3 33.7 7 15% inhibition at 100.0 µm 8 36% inhibition at 120.0 µm 9 65.0 55.7 75.5 10 77.5 59.6 100.8 11 66.4 35.2 125.3 12 26.0 21.8 31.0 13 27.0 22.4 32.6 14 54.1 47.6 62.6 15 69.0 43.6 185.6 16 68.0 61.4 76.0 17 28.0 24.5 32.4 18 35% inhibition at 82.1 µm 19 19% inhibition at 106.4 µm 20 35.6 29.9 41.6 21 16.8 7.5 25.5 22 16.2 13.9 18.9 23 7% inhibition at 89.8 µm 24 75.3 56.4 100.4 25 49.7 43.2 58.3 26 30% biostimulation at 43.4 µm 27 72.9 67.9 78.4 28 83.1 63.9 119.1 29 90% biostimulation at 410.9 µm 30 27% inhibition at 125.0 µm 31 126.8 114.2 139.5 9,10-dihydrophenanthrenes 5 and 11. On the contrary, phenanthrene 28 is more active than 9,10-dihydrophenanthrene 8. Structure activity studies carried out on synthetic phenanthrenes and dihydrophenanthrenes also indicated that the first compunds were less active than the second (DellaGreca et al., 2000, 2001a,b). The phytotoxicity of phenanthrenes components might justify the reported chemical interaction toward dominance of Juncus over species from Cyperaceae found growing sympatric with, but subordinate to, Juncus and the autotoxicity response in these species (Ervin and Wetzel, 2000).

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