Isolation and Structure Elucidation of a Novel Symmetrical Macrocyclic Phthalate Hexaester

: A novel symmetrical macrocyclic phthalate hexaester ( 1 ) and a known macrocyclic phthalate tetraester ( 2 ) were isolated during a natural product-exploring program on the cyanobacterium Moorea producens . Their structures were elucidated based on spectroscopic data, including nuclear magnetic resonance and high-resolution mass spectra. In the antibacterial activity test, compounds 1 and 2 showed no bioactivity at the concentrations tested.

The cyanobacterium Moorea producens (formerly Lyngbya majuscula) is a species known for producing many bioactive compounds [21]. Some of these compounds have been recognized as potential pharmaceutical compounds [22,23]. M. producens has been involved in causing contact dermatitis, also known as "swimmer's itch", in many Pacific areas [24]. The causative agents of this contact dermatitis have been reported to be aplysiatoxins and lyngbyatoxins produced by M. producens [25][26][27][28]. In addition, it has been reported that food tainted with aplysiatoxins that led to food poisoning was contaminated with M. producens [29]. Recently, we isolated and reported new toxic constituents from M. producens [30][31][32][33][34]. During the exploration of aplysiatoxin-related compounds from the cyanobacterium M. producens, we isolated compounds with interesting symmetrical structures: a novel symmetrical macrocyclic hexaester 1 ( Figure 1) and a known macrocyclic tetraester 2 ( Figure 1). The true origin of the phthalate esters is controversial, as we mentioned above; however, the record of finding of new phthalates is valuable. Thus, here, we report the isolation, structure elucidation and bioactivities of these cyclic phthalate compounds 1 and 2. The cyanobacterium Moorea producens (formerly Lyngbya majuscula) is a species known for producing many bioactive compounds [21]. Some of these compounds have been recognized as potential pharmaceutical compounds [22,23]. M. producens has been involved in causing contact dermatitis, also known as "swimmer's itch", in many Pacific areas [24]. The causative agents of this contact dermatitis have been reported to be aplysiatoxins and lyngbyatoxins produced by M. producens [25][26][27][28]. In addition, it has been reported that food tainted with aplysiatoxins that led to food poisoning was contaminated with M. producens [29]. Recently, we isolated and reported new toxic constituents from M. producens [30][31][32][33][34]. During the exploration of aplysiatoxin-related compounds from the cyanobacterium M. producens, we isolated compounds with interesting symmetrical structures: a novel symmetrical macrocyclic hexaester 1 ( Figure 1) and a known macrocyclic tetraester 2 ( Figure 1). The true origin of the phthalate esters is controversial, as we mentioned above; however, the record of finding of new phthalates is valuable. Thus, here, we report the isolation, structure elucidation and bioactivities of these cyclic phthalate compounds 1 and 2.

General Experimental Procedures
Reversed-phase high-performance liquid chromatography (RP-HPLC) was carried out using an HPLC system equipped with a UV-975 Intelligent Ultraviolet-visible (UV/VIS) Detector (JASCO Co., Tokyo, Japan). A HPLC Senshu Scientific SSC-1310 Recycle Unit (Senshu Scientific Co., Tokyo, Japan) was also used, which consisted of a LC-10AD VP pump (SHIMADZU Co., Kyoto, Japan) and a SPD-6AV detector. HR-ESI-MS spectra data were obtained using a Bruker MicrOTOF QII (Bruker Co., Billerica, MA, USA) mass spectrometer. NMR spectra were recorded in CD3OD at 800 MHz (or 600 MHz) on a Bruker AVANCE III 800 MHz (or 600 MHz, Bruker Co., Billerica, MA, USA) spectrometer. The chemical shifts were reported in δ units (ppm) using CD3OD solvent (δH at 3.31 ppm and δC at 49.0 ppm) as the internal standard signals. The UV spectra were measured on a HITACHI U-3000 (Hitachi High-Tech Fielding Co., Tokyo, Japan) spectrometer.

The Cyanobacterium
The cyanobacterium M. producens was collected from Kuba Beach, Nakagusuku, Okinawa, Japan, on 13 July 2010. The collected samples were immediately stored in a freezer (−30 °C) without lyophilization. The Okinawan collection was identified as M. producens.

General Experimental Procedures
Reversed-phase high-performance liquid chromatography (RP-HPLC) was carried out using an HPLC system equipped with a UV-975 Intelligent Ultraviolet-visible (UV/VIS) Detector (JASCO Co., Tokyo, Japan). A HPLC Senshu Scientific SSC-1310 Recycle Unit (Senshu Scientific Co., Tokyo, Japan) was also used, which consisted of a LC-10AD VP pump (SHIMADZU Co., Kyoto, Japan) and a SPD-6AV detector. HR-ESI-MS spectra data were obtained using a Bruker MicrOTOF QII (Bruker Co., Billerica, MA, USA) mass spectrometer. NMR spectra were recorded in CD 3 OD at 800 MHz (or 600 MHz) on a Bruker AVANCE III 800 MHz (or 600 MHz, Bruker Co., Billerica, MA, USA) spectrometer. The chemical shifts were reported in δ units (ppm) using CD 3 OD solvent (δH at 3.31 ppm and δC at 49.0 ppm) as the internal standard signals. The UV spectra were measured on a HITACHI U-3000 (Hitachi High-Tech Fielding Co., Tokyo, Japan) spectrometer.

The Cyanobacterium
The cyanobacterium M. producens was collected from Kuba Beach, Nakagusuku, Okinawa, Japan, on 13 July 2010. The collected samples were immediately stored in a freezer (−30 • C) without lyophilization. The Okinawan collection was identified as M. producens. A voucher specimen (#20100713-a) was also retained.

Bioactive Assays
The antibacterial assays of compounds 1 and 2 were carried out using Escherichia coli JCM No. 20135 and Pyricularia oryzae Ina 86-137. E. coli was cultured at 25 • C for 3 days in B-1 medium, consisting of 5.0 g/L Bactom TM Peptone, 3.0 g/L beef extract, 3.0 g/L NaCl, and 15.0 g/L agar powder (Kanto Chemical Co. Inc., Tokyo, Japan). P. oryzae was cultured at RT in Ottaviani and Agosti (OA) medium containing 50 g/L of oatmeal, 5 g/L sucrose, and 30 g/L agar powder. Both media were prepared using distilled water. Compounds 1 and 2 were dissolved in MeOH and then absorbed on paper discs (8 mm in diameter). After placing the discs on to the assay plates were incubated at 27 • C for 18 h (E. coli) and at RT for 13 days (P. oryzae).

Results and Discussion
Compound 1 (0.10 mg) and compound 2 (0.16 mg) were isolated during exploring natural products from the Okinawan cyanobacterium M. producens (10.1 kg wet wt.  Figure S1) and the [M + Na] + ion peak at m/z 767.3042 (calcd. for C 42 H 48 O 12 Na, 767.3038). The UV spectral data of compound 1 suggested the existence of a conjugated ring system (UV λ max (ethanol) nm (ε) 225 (27,788), 274 (5686)), the structure of which was predominantly determined by 1D and 2D nuclear magnetic resonance (NMR) spectral analyses. The 1 H-NMR spectrum revealed the existence of an ethylene group at H-5 (δH 4.30, dd, J = 6.6 Hz, 6.6 Hz) connected to the oxygen of the carboxyl group. The proton signals with chemical shifts of δH 7.60 (H-1, dd, J = 3.3 Hz, 5.7 Hz) and δH 7.71 (H-2, dd, J = 3.3 Hz, 5.7 Hz) indicated a benzene ring. Furthermore, the existence of two ethylene groups was revealed by the proton signals of δH 1.77 (H-6, m) and δH 1.49 (H-7, m). From the 13 C-NMR spectrum, the signals of C-1 (δC 132.3), C-2 (δC 129.9), and C-3 (δC 133.6) confirmed the existence of a benzene ring and identified three methylene groups at C-5 (δC 66.9), C-6 (δC 29.6), and C-7 (δC 26.9). 1 H-1 H COSY spectrum analysis ( Figure S4) revealed the correlation between H-1 and H-2, which further confirmed the existence of a benzene ring. The correlations of H-5/H-6 and H-6/H-7 were also detected. Moreover, the correlations of H-5/C-4, C-6, and C-7; and H-6/C-5 and C-7, were detected from the 1 H-13 C HMBC spectrum ( Figure S6), indicating the partial structure of compound 1 (Figure 2). Furthermore, the molecular weight of compound 1 was detected to be 744 Da, which corresponds to exactly three times of the molecular weight of the partial structure (MW 248 Da, C 14 H 16 O 4 ) ( Figure 2). Meanwhile, the correlation of H-7/C-7 was detected from the HMBC spectrum (Figure 3), indicating that C-7 was connected to a carbon with the same situation as C-7. Thus, compound 1 was revealed to be a novel macrocyclic hexaester ( Figure 1). NMR data are summarized in Table 1. Compounds 1 and 2 showed no antibacterial activity against E. coli JCM No 20135 and P. oryzae Ina 86-137 at the concentrations tested (1, 12 µg/disc; 2, 10 µg/disc; methanol was tested as the control).
A novel symmetrical macrocyclic hexaester (1) and a known macrocyclic tetraester (2) were isolated during a natural product-exploring program on the cyanobacterium Moorea producens. These molecules could be produced by the cyanobacterium. Further study is needed to determine the true origin of these symmetrical compounds.  and P. oryzae Ina 86-137 at the concentrations tested (1, 12 µg/disc; 2, 10 µg/disc; methanol was tested as the control). A novel symmetrical macrocyclic hexaester (1) and a known macrocyclic tetraester (2) were isolated during a natural product-exploring program on the cyanobacterium Moorea producens. These molecules could be produced by the cyanobacterium. Further study is needed to determine the true origin of these symmetrical compounds.    1 and 2, the proton signals were found to be identical, revealing that compound 1 and 2 had the same partial structure. Furthermore, the molecular weight of compound 2 was measured to be 496 Da, which was exactly twice that of the partial structure (MW 248 Da) of compound 1 (Figure 2), indicating that compound 2 is a macrocyclic tetraester (See Figure 1). Compound 2 was recently reported as a natural compound [16]. Therefore, compounds 1 and 2 are possibly natural products.
Compounds 1 and 2 showed no antibacterial activity against E. coli JCM No 20135 and P. oryzae Ina 86-137 at the concentrations tested (1, 12 µg/disc; 2, 10 µg/disc; methanol was tested as the control). A novel symmetrical macrocyclic hexaester (1) and a known macrocyclic tetraester (2) were isolated during a natural product-exploring program on the cyanobacterium Moorea producens. These molecules could be produced by the cyanobacterium. Further study is needed to determine the true origin of these symmetrical compounds.