INFORMATION FOR AUTHORS Full details of how to submit a manuscript for publication in Natural Product Communications are given in Information for Authors on our Web site http://www.naturalproduct.us. Authors may reproduce/republish portions of their published contribution without seeking permission from NPC, provided that any such republication is accompanied by an acknowledgment (original citation)-Reproduced by permission of Natural Product Communications. Any unauthorized reproduction, transmission or storage may result in either civil or criminal liability. The publication of each of the articles contained herein is protected by copyright. Except as allowed under national “fair use” laws, copying is not permitted by any means or for any purpose, such as for distribution to any third party (whether by sale, loan, gift, or otherwise); as agent (express or implied) of any third party; for purposes of advertising or promotion; or to create collective or derivative works. Such permission requests, or other inquiries, should be addressed to the Natural Product Inc. (NPI). A photocopy license is available from the NPI for institutional subscribers that need to make multiple copies of single articles for internal study or research purposes. To Subscribe: Natural Product Communications is a journal published monthly. 2013 subscription price: US$2,395 (Print, ISSN# 1934-578X); US$2,395 (Web edition, ISSN# 1555-9475); US$2,795 (Print + single site online); US$595 (Personal online). Orders should be addressed to Subscription Department, Natural Product Communications, Natural Product Inc., 7963 Anderson Park Lane, Westerville, Ohio 43081, USA. Subscriptions are renewed on an annual basis. Claims for nonreceipt of issues will be honored if made within three months of publication of the issue. All issues are dispatched by airmail throughout the world, excluding the USA and Canada.

NPC Natural Product Communications

EDITOR-IN-CHIEF

DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA agrawal@naturalproduct.us EDITORS

PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain afbarre@ugr.es

PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy braca@farm.unipi.it

PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China gda5958@163.com

PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan mimakiy@ps.toyaku.ac.jp

PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia spyne@uow.edu.au

PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA m.reinecke@tcu.edu

PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA wsetzer@chemistry.uah.edu

PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan tezuka@inm.u-toyama.ac.jp

PROFESSOR DAVID E. THURSTON Department of Pharmaceutical and Biological Chemistry, The School of Pharmacy, University of London, 29-39 Brunswick Square, London WC1N 1AX, UK david.thurston@pharmacy.ac.uk

ADVISORY BOARD Prof. Berhanu M. Abegaz Gaborone, Botswana

Prof. Viqar Uddin Ahmad Karachi, Pakistan

Prof. Øyvind M. Andersen Bergen, Norway

Prof. Giovanni Appendino Novara, Italy

Prof. Yoshinori Asakawa Tokushima, Japan

Prof. Lee Banting Portsmouth, U.K.

Prof. Julie Banerji Kolkata, India

Prof. Anna R. Bilia Florence, Italy

Prof. Maurizio Bruno Palermo, Italy

Prof. César A. N. Catalán Tucumán, Argentina

Prof. Josep Coll Barcelona, Spain

Prof. Geoffrey Cordell Chicago, IL, USA

Prof. Ana Cristina Figueiredo Lisbon, Portugal

Prof. Cristina Gracia-Viguera Murcia, Spain

Prof. Duvvuru Gunasekar Tirupati, India

Prof. Kurt Hostettmann Lausanne, Switzerland

Prof. Martin A. Iglesias Arteaga Mexico, D. F, Mexico

Prof. Leopold Jirovetz Vienna, Austria

Prof. Vladimir I Kalinin Vladivostok, Russia

Prof. Niel A. Koorbanally Durban, South Africa

Prof. Karsten Krohn Paderborn, Germany

Prof. Chiaki Kuroda Tokyo, Japan

Prof. Hartmut Laatsch Gottingen, Germany

Prof. Marie Lacaille-Dubois Dijon, France

Prof. Shoei-Sheng Lee Taipei, Taiwan

Prof. Francisco Macias Cadiz, Spain

Prof. Imre Mathe Szeged, Hungary

Prof. Ermino Murano Trieste, Italy

Prof. M. Soledade C. Pedras Saskatoon, Canada

Prof. Luc Pieters Antwerp, Belgium

Prof. Peter Proksch Düsseldorf, Germany

Prof. Phila Raharivelomanana Tahiti, French Polynesia

Prof. Luca Rastrelli Fisciano, Italy

Prof. Monique Simmonds Richmond, UK

Dr. Bikram Singh Palampur, India

Prof. John L. Sorensen Manitoba, Canada

Prof. Valentin Stonik Vladivostok, Russia

Prof. Winston F. Tinto Barbados, West Indies

Prof. Sylvia Urban Melbourne, Australia

Prof. Karen Valant-Vetschera Vienna, Austria

HONORARY EDITOR

PROFESSOR GERALD BLUNDEN The School of Pharmacy & Biomedical Sciences,

University of Portsmouth, Portsmouth, PO1 2DT U.K.

axuf64@dsl.pipex.com

Cyclic Peroxides from a Two-Sponge Association of Plakortis communis-Agelas mauritiana Pinus Jumaryatnoa,b, Lynette K. Lambertc, John N. A. Hooperd, Joanne T. Blanchfielda and Mary J. Garsona,* aSchool of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia

bPharmacy Department, Faculty of Mathematic and Natural Sciences, Universitas Islam Indonesia, Kampus Terpadu UII, Jalan Kaliurang KM 14.5, Yogyakarta 55284, Indonesia

cCentre for Advanced Imaging, The University of Queensland, Brisbane QLD 4072, Australia

dQueensland Museum, PO Box 3300, South Brisbane QLD 4101, Australia m.garson@uq.edu.au Received: October 25th, 2012; Accepted: November 20th, 2012

A cyclic peroxide 1 with an unusual phenethenyl side chain, together with the known peroxide 2 with a C4-sidechain have been isolated from a two-sponge association of Plakortis communis – Agelas mauritiana (Carter, 1883) collected near Mooloolaba, South-East Queensland, Australia. Metabolite purification was complicated by the presence of the free carboxylic acid groups in 1 and 2; therefore, diazomethane treatment was undertaken to afford methyl ester 3. Following RP-HPLC purification, the ring-opened analogues 4 and 5 were also obtained. The structures of the new compounds were elucidated on the basis of their 1D and 2D NMR and MS data, and by comparison with literature data. The relative configuration of the isolated peroxides was determined by the interpretation of JH-H values and comparison of the 13C chemical shift data with literature data for related compounds. The bromopyrrole alkaloid longamide (6) was also isolated. Keywords: Cyclic peroxides, Plakortis, Relative configuration, JH-H values. Marine sponges of the family Plakinidae are known to be a prolific source of secondary metabolites with a wide-ranging structural diversity. Among them, cyclic peroxides and structurally related metabolites are the prominent family of compounds which are frequently isolated from marine sponges of the genera Plakortis and Plakinastrella [1]. Most of the cyclic peroxide metabolites from these sponges are assumed to derive from small carboxylic acids via the polyketide pathway [2]. The majority of their carbon skeletons possess a 1,2-dioxane ring with an acetate residue at position C-3 and alkyl substituents at positions C-4 and C-6, while a growing number of cyclic peroxides with the rare 1,2-dioxolane ring system have also been reported. In continuation of our current studies on marine peroxy metabolites [3,4], we now describe the isolation and structure elucidation of cyclic peroxides from a two-sponge association Plakortis communis-Agelas mauritiana. The sponge sample was extracted with CH2Cl2/MeOH followed by partition between H2O and EtOAc. Fractionation by NP and by RP flash chromatography gave two fractions of interest, one of which contained a mixture containing cyclic peroxy acids 1 and 2 and was poorly soluble in CDCl3. Berrué et al. [5] have previously noted that cyclic peroxides containing carboxylic acid substituents cannot easily be separated by RP-HPLC. Hence, treatment with diazomethane in Et2O was conducted to convert the mixture of peroxy acids to their methyl ester derivatives in order to optimize their separation. A second benefit was that the mixture of methyl ester products was now CDCl3-soluble. The methylation product was subjected to RP-HPLC using MeOH/H2O as a solvent, and the resulting peroxide/diol products 3-5 individually identified by 1H NMR and MS. The other fraction of interest provided the known bromopyrrole alkaloid longamide (6) [6,7] by RP-HPLC. For peroxyacid 1, a molecular formula of C18H24O4 was established from the HRESIMS sodiated parent ion at m/z 327.1572. The 1H

Figure 1: Structures of peroxy compounds, their associated diols, and of the alkaloid

longamide isolated from Plakortis communis-Agelas mauritiana NMR (MeOH-d4) and HSQC data revealed signals for an aromatic ring (įH 7.21, 7.31 and 7.40; įC 128.3, 129.2 and 127.2), a disubstituted olefin (įH 6.50 and 6.30; įC 131.5 and 133.2), an oxygenated methine (įH 4.20; įC 83.8), a geminal methylene (įH 2.68 and 2.17; įC 37.6) and two methyl groups (įH 0.89 and 0.97; įC 7.6 and 10.7). There was also a carbonyl group (įC 174.8). The molecular formula of compound 1 implied seven degrees of unsaturation. Since one carbonyl bond, one olefinic bond and one aromatic ring accounted for six degrees of unsaturation, one other ring must be present. These MS data together with HSQC, HMBC and DQFCOSY secured the peroxide-containing ring functionality. In the DQFCOSY spectrum, the methine proton at įH 4.20 (H-3) correlated to the non-equivalent methylene protons at įH 2.68 and 2.17 (H2-2), and to another methine proton at įH 1.66 (H-4) that was further coupled to methylene protons at įH 2.25 and 1.42 (H2-5). HMBC data showed correlations from H-3 to the signal at įC 174.8 (C-1) and to C-2 at įC 37.6. H-5 correlated to an oxygenated quaternary signal at įC 85.5 (C-6). The methyl signals at įH 0.89 and at įH 0.97 showed DQFCOSY correlations to a methylene

OO

COOR

5

16 18

2

9

1 R = H3 R = Me

OHHO

COOMe

5

16 18

2

9

4

OO

COOH7

5

2

10

12 14

2

OHHO

COOMe

5

2

10

12 14

5

HNN

Br

Br

O

OH

6

NPC Natural Product Communications 2013 Vol. 8 No. 6

725 - 728

726 Natural Product Communications Vol. 8 (6) 2013 Jumaryatno et al.

Figure 2: Key DQFCOSY and HMBC correlations for peroxide 1

Table 1: NMR spectroscopic data for peroxide 1.

Position į1H (J)a,b į13C HMBCc,d DQFCOSY 1 - 174.8 - - 2 2.17 m 37.6 C-1, C-3, C-4 H-3 2.68 dd (3.2, 15.9) 3 4.20 dt (3.2, 9.6, 9.6) 83.8 C-1, C-2 H2, H-4 4 1.66 m 38.0 - H-3, H-5 5 1.42 t (13.1) 37.7 C-6, C-7, C-15, C-17 H-4 2.25 dd (4.2, 13.1) 6 - 85.5 - - 7 6.30 d (16.7) 133.2 C-5, C-6, C-9, C-15 H-8 8 6.50 d (16.7) 131.5 C-6, C-9, C-10, C-14 H-7, H-10, H-14 9 - 138.4 - - 10,14 7.40 d (7.3) 127.2 C-8, C-12 H-8, H-11, H-13 11,13 7.31 t (7.6) 129.2 C-9 H-10, H-12, H-14 12 7.21 t (7.3) 128.3 C-10, C-14 H-11, H-13 15 1.59 q (7.6) 33.8 C-5, C-6, C-7, Me-16 Me-16 16 0.89 t (7.6) 7.6 C-6, C-15 H-15 17 1.15 m 24.7 - Me-18 1.54 m 18 0.97 t (7.2) 10.7 C-4, C-17 H-17

a500 MHz, MeOH-d4 referenced to 1H at į 3.30 ppm; bcoupling constant in Hz; cHMBC connectivity from H to C; dcorrelations observed for long range JC-H of 8 Hz signal at įH 1.59 (2H), and at įH 1.15 and 1.54, respectively. In addition, there were HMBC correlations from įH 0.89 (Me-16) and 0.97 (Me-18) to įC 33.8 (C-15) and 24.7 (C-17), respectively. Hence two ethyl moieties were present in the structure. Me-16 showed an HMBC correlation to the signal at įC 85.5 for C-6, while Me-18 had an HMBC correlation to a methine carbon at įC 38.0 for C-4. The olefinic protons at įH 6.50 (H-8) and įH 6.30 (H-7) showed HMBC correlations to C-6 and C-5, respectively, while H-7 also showed an HMBC correlation to C-15. The aromatic ring was placed next to the olefinic group by observation of HMBC correlations from H-7 at įH 6.30 to C-9 at įC 138.4 and from H-8 at įH 6.50 to C-10 at įC 127.2. The 16.7 Hz coupling between H-7 and H-8 established the trans configuration of the phenethenyl sidechain. The full NMR assignments of compound 1 are given in Table 1. The relative stereochemistry of 1 was deduced by interpretation of the proton coupling constants and the value of carbon chemical shifts compared with literature values [2,5,8-10]. The proton chemical shifts for H-2 [įH 2.68 (dd, J = 3.2, 15.9 Hz) and 2.17 (m)] and H-3 [įH 4.20 (dt, J = 3.2, 9.6, 9.6 Hz)] matched the corresponding signals in epiplakortin (H-2 [įH 2.66 (dd, J = 3.5, 15.5 Hz) and 2.38 (dd, J = 9.0, 15.5 Hz)]; H-3 [įH 4.16 (m, J = 3.5, 9.0, 9.0 Hz)]) [2] rather than the signals for plakortin (H-2 [įH 3.05 (dd, J = 9.5, 16.0 Hz) and 2.35 (dd, J = 3.5, 16.0 Hz)]; H-3 [įH 4.49 (m, J = 3.5, 6.0, 9.0 Hz)]) [8]. These data suggested that 1 has an epiplakortin configuration. In particular, the 9.6 Hz value of the vicinal H-3/H-4 coupling constant supported a trans diaxial relationship of H-3 and H-4, and so the acetic acid substituent at C-3 was placed in an equatorial position. An empirical rule determines the relative configuration of a 1,2-dioxane ring using carbon chemical shift data. The acetic acid substituent at C-3 occupies an equatorial or axial position when C-2 resonates above įC 36 or below įC 32, respectively [5,10]. The chemical shift value of C-2 at įC 37.6 was fully in agreement with the proposed equatorial orientation. In a similar way, an equatorial or axial position is assigned for the ethyl group at C-6 of a cyclic peroxy compound when the methylene carbon of the ethyl group resonates above įC 30 or below įC 27, respectively. The ethyl substituent at C-6 in 1 was assigned as equatorial due to the chemical shift of C-15 at įC 33.8. Moreover, the ethyl substituent at C-4 must be equatorial since H-4 was axial. This configuration was also supported by the chemical

shift of C-17 at įC 24.7; the ethyl substituent at C-4 is equatorial if the associated methylene carbon resonates near įC 25 [5,9,10]. For peroxyacid 2, the HRESIMS indicated a [M+Na]+ ion at 281.1716 corresponding to the molecular formula C14H26O4, implying two degrees of unsaturation. In the 1H NMR spectrum (MeOH-d4), a methine proton signal (H-3) at įH 4.07 (dt, J = 3.2, 9.6, 9.6 Hz) displayed COSY correlations to a mutually coupled pair of signals at įH 2.25 and 2.63 (H2-2), and to another proton at įH 1.68 (H-4). The latter proton was further coupled to signals at įH 1.20 and 1.85 (H2-5). Inspection of 1H NMR and HSQC revealed the presence of three alkyl functionalities in compound 2 due to the methyl triplets at įH 0.86 (įC 7.2), įH 0.92 (įC 10.6) and įH 0.93 (įC 14.2). Each of these methyl signals was coupled to methylene groups from the COSY data (įH 0.86 to įH 1.38 and 1.48; įH 0.92 to įH 1.15 and 1.55; įH 0.93 to įH 1.35). Analysis of HMBC spectra showed that the methyl protons at įH 0.86 and 0.92 had long-range correlations to signals at įC 83.9 and 37.6 placing ethyl groups at positions C-6 and C-4, respectively. These NMR data matched those of the 1,2-dioxane ring of compound 1. The remaining methyl group was part of a chain with three or more methylenes. The triplet at įH 0.93 linked to an upfield carbon at įC 14.2 by HSQC, showed a COSY correlation to methylene protons at įH 1.35 and HMBC correlations to methylene carbons at įC 23.8 and 26.2. An HMBC correlation was observed from įH 1.20 (H-5) to the signal at įC 32.5 (C-7) linked by an HSQC correlation to a methylene signal at įH 1.58. These details connected a 4C aliphatic side chain to the 1,2-dioxane ring in agreement with the molecular formula, and established the planar structure of 2. Peroxide 2 has an epiplakortin configuration since the proton chemical shifts at H-2 [įH 2.63 (dd, J = 3.2, 15.9 Hz) and 2.25 (m)] and H-3 [įH 4.07 (dt, J = 3.2, 9.6, 9.6 Hz)] were similar to the corresponding signals in epiplakortin and different to those for plakortin. The large value of the vicinal coupling constant J3-H/4-H = 9.6 Hz was in agreement with the trans diaxial relationship between H-3 and H-4. The equatorial position was established for the acetic acid substituent at C-3 since C-2 resonated at įC 37.4. The ethyl groups at C-4 and C-6 were both equatorial from the chemical shift values of C-13 (įC 24.7) and C-11 (įC 30.3). Moreover, C-7 resonated at įC 32.5, as is observed in plakortide F [9] and ethyl plakortide Z [10]; therefore, a cis relationship was deduced between the C-4 and C-6 substituents. A peroxy compound with the same structure as 2 was isolated by Toth and Schmitz from Callyspongia communis, but with no report of the configuration at C-6 [11]. The 13C NMR data of 2 closely matched those (CDCl3/C6D6) reported by Toth and Schmitz. The ethyl ester of 2 has been reported by Harrison and Crews from Plakortis lita [10]. Methylation of the fraction containing 1-2 followed by RP-HPLC yielded ester 3 and two diol products 4 and 5 that resulted from peroxide ring opening on exposure to MeOH during HPLC purification. The methyl ester derivative 3 was isolated as a colorless film with a HRESIMS sodiated ion at m/z 341.1735 corresponding to a molecular formula of C19H26O4. The 1H NMR spectrum was acquired in CDCl3 using a Shigemi tube and revealed signals for a methine proton at įH 4.27 (dt, J = 3.3, 9.5, 9.5 Hz) and two methylene protons at įH 2.30 (m) and 2.62 (dd, J = 3.2, 15.7 Hz). These proton signals were distinctive for H-3 and H-2, respectively, of an epiplakortin analogue [2]. The 1H NMR data also contained one disubstituted olefin group [įH 6.45 (d, J = 16.7 Hz) and 6.30 (d, J = 16.7 Hz)], a methoxy group [įH 3.68 (s)] and two methyl groups [įH 0.89 (t, J = 7.5 Hz) and 0.94 (t, J = 7.5 Hz)]. In addition, aromatic proton signals were observed at įH 7.41, 7.34 and 7.23. These data revealed that the methyl ester derivative 3 has an

Cyclic peroxides from a two-sponge association Natural Product Communications Vol. 8 (6) 2013 727

Figure 3: Important HMBC correlations for compound 4.

Table 2: NMR spectroscopic data for diol methyl ester 4

Position į1H (J)a,b į13Cc HMBCd,e 1 - 174.3f - 2 2.35 dd (10.0, 16.7) 39.0 - 2.63 dd (2.3, 16.7) 3 3.80 dt (2.3, 10.0, 10.0) 71.1 - 4 1.56 mg 39.5 - 5 1.62 mg 42.0 - 1.92 dd (6.1, 15.0) 6 - 74.8f - 7 6.06 d (15.9) 135.7 C-9 8 6.66 d (15.9) 128.5 C-6, C-10, C-14 9 - 137.5f - 10,14 7.37 d (7.5) 126.2 C-11, C-12, C-13 11,13 7.31 t (7.5) 128.4 C-9 12 7.20 t (7.5) 126.9 C-10, C-14 15 1.62 mg 35.8 - 16 0.93 t (7.5) 7.8 C-6, C-15 17 1.17 m 25.3 - 1.35 m 18 0.85 t (7.4) 10.2 C-4, C-17 -OMe 3.71 s 51.6 C-1

a500 MHz, CDCl3 referenced to 1H at į 7.26 ppm; bcoupling constant in Hz; ctaken from HSQC at 900 MHz, CDCl3 referenced to 13C at į 77.0 ppm; d500 MHz, HMBC connectivity from H to C; ecorrelations observed for long range JC-H of 6 Hz; fchemical shifts taken from HMBC experiment at 500 MHz; gsignal multiplicity unresolved due to overlapping signals.

epiplakortin ring system, a trans-disubstituted olefin and an aromatic ring. However, owing to the very small quantity of isolated compound, 2D NMR experiments of 3 could not be achieved. Diol 4 was isolated as a colorless film and HRESIMS indicated a [M+Na]+ ion at m/z 343.1877 corresponding to the molecular formula C19H28O4 and implying an additional two hydrogen atoms compared with the molecular formula of 3. Inspection of the NMR spectra (Table 2) established the disubstituted olefin (įH 6.66 and 6.06; įC 128.5 and 135.7) from 1H NMR and HSQC data. Furthermore, a methine proton at įH 3.80, methylene protons at įH 2.63 and 2.35, two methyl triplets at įH 0.85 and 0.93, and a methoxy signal at įH 3.71 were observed, as well as aromatic signals at įH 7.20, 7.31 and 7.37. The methoxy signal at įH 3.71 showed an HMBC correlation to a carbonyl at įC 174.3 confirming the methyl ester functionality. The presence of two oxygenated carbons was inferred from a correlation in the HMBC spectrum between the methyl signal at įH 0.93 and a carbon at įC 74.8, and by an HSQC correlation from a methine proton at įH 3.80 to a signal at įC 71.1. According to the molecular formula, there were six degrees of unsaturation (one double bond, one ester carbonyl and the phenyl group), and consequently, the two oxygenated carbons at įC 71.1 and 74.8 did not form a peroxide ring, and so had hydroxy substituents. Therefore, compound 4 was the ring-opened form of peroxide 3. In the HMBC spectrum of 4, there were correlations from įH 0.93 (Me-16) to C-15 at įC 35.8 and C-6 at įC 74.8 as well as from įH 0.85 (Me-18) to C-17 at įC 25.3 and C-4 at įC 39.5. TOCSY data of 4 showed a correlation from Me-16 to H-15 and from Me-18 to H-17 and H-4. These allowed the placement of ethyl groups at C-4 and C-6. In addition, 1D TOCSY spectra of 4 also revealed that the signal at įH 2.63 assigned as H-2 had correlations to H-3 at įH 3.80 and H-4 at įH 1.56. The remaining signals of compound 4 resembled those attributed to the side chains of compounds 1 and 3. HMBC analysis showed that both protons at įH 7.31 (H-11 and H-13) and 6.06 (H-7) correlated to a quaternary aromatic carbon at įC 137.5 (C-9) while H-8 at įH 6.66 showed long-range correlations to an aromatic signal at įC 126.2 (C-10 and C-14) and to C-6 at įC 74.8.

Diol 4 was likely derived from ester 3 and so should have the same relative stereochemistry at C-3, C-4 and C-6. In the proton spectrum, the H-4 multiplet of 4 shared a 10.0 Hz coupling with H-3 indicating an anti arrangement. Moreover, the chemical shift values for C-2 and C-15 of compound 4 were comparable with those reported for a semisynthetic sample of ethyl seco-plakortide Z [10]. The trans geometry between H-7 and H-8 was assigned on the basis of the large coupling constant (J7-H/8-H = 15.9 Hz). The optical rotation measurement of 4 was -73 (c 0.006, CHCl3); that of ethyl seco-plakortide Z isolated by Harrison and Crews [10] was reported as -24.2 (c 1.1, CH2Cl2). The negative sign suggested that diol 4 was likely to have the same absolute configuration as ethyl seco-plakortide Z and so was tentatively assigned 3S, 4R and 6R configuration. The HRESIMS of 5 gave a sodiated ion at m/z 297.2036 corresponding to a molecular formula of C15H30O4Na suggesting two hydrogen atoms more than for a cycloperoxy methyl ester product. Owing to the limited sample quantity, 1H NMR data was obtained using a Shigemi NMR tube at 750 MHz, with H-2 and H-3 giving resonances at įH 2.42 and 2.65, and įH 3.85, respectively. The proton signal at įH 3.85 was of comparable chemical shift to that of H-3 in diol 4 and in analogous published diol structures [10,12]. The amounts of individually purified metabolites were insufficient for specific rotation measurements, and so the absolute configuration shown for 1-3 and 5 is selected by comparison with 4. Examples of cyclic peroxy metabolites containing a Ph(CH2)n- sidechain (n = 8-10 or 12) are known from sponges of the family Plakinidae and these frequently possess a plakortolide skeleton [1,3]; we are not aware of any examples among the plakortin/epiplakortin suite of cyclic peroxy metabolites that have a phenyl group in the side chain. The Plakortis communis investigated in this study thus uses both phenylacetate and acetate as the chain starter unit in peroxyacid biosynthesis. Also isolated was longamide (6), identified by comparison of its 1H and 13C NMR data with a synthetic sample [7]. The specific rotation was -6 (c 0.039, MeOH) compared with a value of +86.9 (c 0.001, MeOH) for a sample isolated from the sponge Agelas longissima [6], and suggested that the present sample was close to racemic. It is known that longamide undergoes facile racemisation through inter-conversion of carbinolamine and aldehyde-pyrrole tautomers [13]. Experimental

General: 1D and 2D NMR, Bruker Avance 500 MHz spectrometer; HRESIMS, MicroTof Q instrument; Reverse phase HPLC was carried out on an Agilent 1100 series instrument fitted with either a Phenomenex Gemini C18 (250 x 10 mm i.d., 5 P) column or a C18 analytical column. UV detection was at 254 nm. Silica gel 60 G and precoated silica TLC plates F254 were purchased from Merck. Animal material: The sponge was collected using SCUBA at a depth of 10-12 m at Mudjimba Island, Mooloolaba, South-East Queensland, Australia, on 1 December 2004. A voucher specimen (QM G331184) is lodged at the Queensland Museum and was identified as a two sponge association of Agelas mauritiana (Carter, 1883) and an unidentified Plakortis communis The combined samples were stored at -20°C until extraction. Extraction and isolation of metabolites: The frozen sponge (189 g wet weight) was diced finely and extracted with DCM/MeOH (400 mL, 1:1) 4 times, followed by partition between H2O (50 mL) and EtOAc (3 x 150 mL). The organic layers were combined and evaporated in vacuo to give a crude extract (417 mg). This was

728 Natural Product Communications Vol. 8 (6) 2013 Jumaryatno et al.

dissolved in CHCl3 and adsorbed onto flash silica gel (845 mg) before loading onto a silica column pre-equilibrated with hexanes, then subjected to silica flash chromatography using a solvent gradient (hexaneso DCMoEtOAcoMeOH) to give 8 fractions (F1-F8) by NP-TLC analysis. The sixth fraction (F6) was further fractionated by NP flash chromatography using a solvent gradient (DCMoEtOAcoMeOH) to give 5 fractions (F6A-F6E) by NP TLC. Fraction F6B (40.8 mg) was dissolved in MeOH and adsorbed onto C18 silica gel (103 mg) before loading onto a C18 column pre-equilibrated with MeOH/H2O (30:70), then subjected to flash chromatography using a solvent gradient (MeOH/H2O 30:70o100% MeOH) to yield 5 fractions (RPF1-RPF5) based on RP-TLC analysis. Longamide (6) was isolated from fraction RPF1 using a gradient of MeOH/H2O 20:80o100% MeOH over 40 min. A portion of RPF4 (0.7 mg) was treated with diazomethane in diethyl ether prior to additional HPLC separation. Methylation of a mixture of cyclic peroxides: The cyclic peroxide mixture (0.7 mg) was dissolved in MeOH (1 mL) and CH2N2 in Et2O was added drop wise until a stable yellow color was obtained. The resulting mixture was stirred for 3-5 min at room temperature, then excess reagent and solvent were removed under a stream of N2 followed by high vacuum treatment overnight. The product mixture was purified by RP-HPLC using MeOH/H2O (70:30) for 60 min, followed by 100% MeOH for 20 min to obtain compound 4 (~ 0.1 mg). The HPLC fraction eluting from 100% MeOH was re-chromatographed by RP-HPLC using MeOH/H2O (70:30) for 20 min, followed by a gradient of MeOH/H2O (70:30) to 100% MeOH over 40 min to afford, in order of elution, compounds 4 (~ 0.1 mg), 3 (<0.01 mg) and 5 (< 0.01mg). Peroxide 1 Colorless film 1H and 13C NMR (MeOH-d4): Table 1. HRESIMS: m/z [M+Na]+ calcd for C18H24O4Na: 327.1567; found: 327.1572. Peroxide 2 Colorless film 1H NMR (500 MHz, MeOH-d4): į 4.07 (1H, dt, J = 3.2, 9.6, 9.6 Hz, H-3), 2.63 (1H, dd, J = 3.2, 15.9 Hz, H-2), 2.25 (1H, m , H-2), 1.85

(1H, dd, J = 4.4, 13.3 Hz , H-5), 1.68 (1H, m, H-4), 1.58 (2H, m, H-7), 1.55 (1H, m, H-13), 1.48 (1H, m, H-11), 1.38 (1H, m, H-11), 1.35 (2H, m, H-9), 1.34 (1H, m, H-8), 1.20 (1H, t, J = 13.1 Hz, H-5), 1.19 (1H, m, H-8), 1.15 (1H, m, H-13), 0.93 (3H, t, J = 7.1 Hz, Me-10), 0.92 (3H, t, J = 7.7 Hz, Me-14 ), 0.86 (3H, t, J = 7.8 Hz, Me-12). 13C NMR (500 MHz, MeOH-d4): 174.8 (C-1), 83.9 (C-6), 83.4 (C-3), 37.6 (C-4), 37.4 (C-2), 37.0 (C-5), 32.5 (C-7), 30.3 (C-11), 26.2 (C-8), 24.7 (C-13), 23.8 (C-9), 14.2 (C-10), 10.6 (C-14), 7.2 (C-12). HRESIMS: m/z [M+Na]+ calcd for C14H26O4Na: 281.1723; found: 281.1716. Peroxide methyl ester 3 Colorless film 1H NMR (500 MHz, CDCl3): į 7.41 (2H, d, J = 7.3 Hz, H-10 & H-14), 7.34 (2H, overlap with CDCl3, H-11 & H-13), 7.23 (1H, overlap with CDCl3, H-12), 6.45 (1H, d, J = 16.7 Hz, H-8), 6.30 (1H, d, J =16.7 Hz, H-7), 4.27 (1H, dt, J = 3.3, 9.5 Hz, H-3), 3.68 (3H, s, -OMe), 2.62 (1H, dd, J = 3.2, 15.7 Hz, H-2), 2.30 (1H, m, H-2), 2.16 (1H, dd, J = 4.1, 13.5 Hz, H-5a), 0.94 (3H, t, J = 7.5 Hz, Me-18), 0.89 (3H, t, J = 7.5 Hz, Me-16); Signals for H-4, H-5b, H-15, H-17 were obscured. HRESIMS: m/z [M+Na]+ calcd for C19H26O4Na: 341.1723; found: 341.1735. Diol methyl ester 4 Colorless film [D]D: -73 (c 0.006, CDCl3). 1H and 13C NMR (CDCl3): Table 2. HRESIMS: m/z [M+Na]+ calcd for C19H28O4Na: 343.1880; found: 343.1877. Acknowledgments - We thank the Indonesian Directorate of Higher Education (DIKTI) for a PhD Scholarship, the Australia Research Council and the University of Queensland for funding, and Mr Graham McFarlane for assistance with high resolution mass spectrometric analysis, respectively. Sponge collection was undertaken under permit from the Queensland government and was facilitated by ScubaWorld Mooloolaba. Mr Michael Somerville and Dr Ernesto Mollo assisted with the collection.

References

[1] Rahm F, Hayes PY, Kitching W. (2004) Metabolites from marine sponges of the genus Plakortis. Heterocycles, 64, 523-575. [2] Stierle DB, Faulkner DJ. (1980) Metabolites of three marine sponges of the genus Plakortis. Journal of Organic Chemistry, 45, 3396-3401. [3] Yong KWL, De Voss JJ, Hooper JNA, Garson MJ. (2011) Configurational assignment of cyclic peroxy metabolites provides an insight into their

biosynthesis: isolation of plakortolides, seco-plakortolides, and plakortones from the Australian marine sponge Plakinastrella clathrata. Journal of Natural Products, 74, 194-207.

[4] Yong KWL, Lambert LK, Hayes PY, De Voss JJ, Garson MJ. (2012) Oxidative processes in the Australian marine sponge Plakinastrella clathrata: isolation of plakortolides with oxidatively modified side chains. Journal of Natural Products, 75, 351-360.

[5] Berrué F, Thomas OP, Funel-Le Bon C, Reyes F, Amade P. (2005) New bioactive cyclic peroxide from the Caribbean marine sponge Plakortis zyggompha. Tetrahedron, 61, 11843-11849.

[6] Cafieri F, Fattorusso E, Mangoni A, Taglialatela-Scafati O. (1995) Longamide and 3,7-dimethylisoguanine, two novel alkaloids from the marine sponge Agelas longissima. Tetrahedron Letters, 36, 7893-7896.

[7] Banwell MG, Bray AM, Willis AC, Wong DJ. (1999) First syntheses of the pyrroloketopiperazine marine natural products (±)-longamide, (±)-longamide B, (±)-longamide B methyl ester and (±)-hanishin. New Journal of Chemistry, 23, 687-690.

[8] Higgs MD, Faulkner DJ. (1978) Plakortin, an antibiotic from Plakortis halichondrioides. Journal of Organic Chemistry, 43, 3454-3457. [9] Patil AD, Freyer AJ, Carte B, Johnson RK, Lahouratate P. (1996) Plakortides, novel cyclic peroxides from the sponge Plakortis halichondrioides:

activators of cardiac SR-Ca2+ -pumping ATPase. Journal of Natural Products, 59, 219-223. [10] Harrison B, Crews P. (1998) Cyclic polyketide peroxides and acyclic diol analogues from the sponge Plakortis lita. Journal of Natural Products,

61, 1033-1037. [11] Toth SI, Schmitz FJ. (1994) Two new cytotoxic peroxide-containing acids from a New Guinea sponge, Callyspongia sp. Journal of Natural

Products, 57, 123-127. [12] Fattorusso E, Taglialatela-Scafati O, Di Rosa M, Ianaro A. (2000) Metabolites from the sponge Plakortis simplex. Part 3: Isolation and

stereostructure of novel bioactive cycloperoxides and diol analogues. Tetrahedron, 56, 7959-7967. [13] Marchais S, Al Mourabit A, Ahond A, Poupat C, Potier P. (1999) Synthesis of the marine carbinolamine (±) longamide: control of N-1 and C-3

bromopyrrole nucleophilicity. Tetrahedron Letters, 40, 5519-5522.

Natural Product Communications Vol. 8 (6) 2013 Published online (www.naturalproduct.us)

Synthesis and Mass Spectral Fragmentation Patterns of Brassinolide Early Biosynthetic Precursors Labeled at C-26 Vladimir A. Khripach, Danuše Tarkowská, Vladimir N. Zhabinskii, Olga V. Gulyakevich, Yurii V. Ermolovich, Pavel Drašar and Miroslav Strnad 771

Severibuxine, Isolated from Severinia buxifolia, Induces Apoptosis in HL-60 Leukemia Cells Chihiro Ito, Tomiyasu Murata, Midori Kato, Natsu Suzuki, Tian-Shung Wu, Norio Kaneda, Hiroshi Furukawa and Masataka Itoigawa 775

Two New Morphinandienone Alkaloids from Croton micradenus Armando L. Payo-Hill, Daysi Sandoval-López, Hermán T. Vélez-Castro, IraidaSpengler-Salabarría and Luca Rastrelli 779

Isolation and Cholinesterase Inhibitory Activity of Narcissus Extracts and Amaryllidaceae Alkaloid Lucie Cahlíková, Miroslav Loþárek, Nina Benešová, Radim Kuþera, Jakub Chlebek, ZdenČk Novák and Lubomír Opletal 781

Antioxidant Activity and Mechanism of Tetrahydroamentoflavone in vitro Xican Li, Weijuan Han, Wenqiong Mai and Li Wang 787

Blue Mood for Superfood Kari Taulavuori, Riitta Julkunen-Tiitto, Valtteri Hyöky and Erja Taulavuori 791

Acetylcholinesterase Inhibitors from Croton sylvaticus Ethyl Acetate Leaf Extract and their Mutagenic Effects Mutalib A. Aderogba, Ashwell R. Ndhlala and Johannes Van Staden 795

Coumarin Precursor from Micromelum integerrimum Leaves Wong Phakhodee, Preetiya Pongparn, Angkana Saovapakhiran and Surat Laphookhieo 799

Antifilarial Activity of Constituents of Calophyllum inophyllum and their Derivatives Jankiprasad, Gunaganti Naresh, Jyoti Gupta, Shailja M. Bhattacharya, Siron M. Rajendran, Shailendra K. Awasthi and Tadigoppula Narender 803

Two New Lignans from the Wood of Cunninghamia konishii Chi-I Chang, Yen-Cheng Li, Ching-Chuan Kuo, Che-Yi Chao, Hsun-Shuo Chang, Jyh-Horng Wu, Sheng-Yang Wang and Yueh-Hsiung Kuo 805

Biological Studies of Turmeric Oil, Part 1: Selective in vitro Anticancer Activity of Turmeric Oil (TO) and TO-Paclitaxel Combination James N. Jacob and Masoud Toloue 807

Biological Studies of Turmeric Oil, Part 2: Isolation and Characterization of Turmeric Oil Components; Cytotoxic Activity Against Pancreatic Cancer Cells Wudan Yan, Wayne D. Bowen, Russell Hopson, Abraham E. Mathew and James N. Jacob 811

Ushikulide C, a New Immunosuppressant from Streptomyces sp. IUK-102 Kosaku Takahashi, Eri Fukushi, Jun Kawabata, Hideyuki Matsuura and Kazuhiko Kurosawa 815

Influence of the Bioclimatic Area on the Polyphenolic Composition, and Antioxidant and Bacteriostatic Activities of Rosmarinus officinalis Maria J. Jordán, Vanesa Lax, Maria C. Rota, Susana Lorán and José A. Sotomayor 817

Antibacterial and Antioxidant Activities of Various Medicinal Plants Used in Oriental Medicine Junho Seo, Jiyeon Kim, Geon Go, Jung-Suk Sung and Kwang-Geun Lee 823

Aconitamide, a Novel Alkaloid from the Roots of Aconitum carmichaeli Tzong-Huei Lee, Yu-Chang Chen, Ching-Kuo Lee, Hsun-Shuo Chang, Ching-Chuan Kuo, Che-Yi Chao, Jing-Jer Lin, Lee-Chiang Lo and Yueh-Hsiung Kuo 827

The Chemical Composition and Antibacterial Activities of the Essential Oils from Three Aframomum Species from Cameroon, and Their Potential as Sources of (E)-(R)-Nerolidol Sylvie Kwanga Nguikwie, Maximilienne A. Nyegue, Florentine Ndoye-Foe Belinga, Rosalie A. Ngono Ngane, Bernard Romestand, Achraf Kouzayha, Hervé Casabianca, Paul H. Amvam Zollo and Chantal Menut 829

Chemical Composition and Antimicrobial Activity of Daucus aureus Essential Oils from Algeria Nawel Meliani, Mohammed El Amine Dib, Nassim Djabou, Jean Costa, Hocine Allali, Boufeldja Tabti and Alain Muselli 835

Chemical Composition of the Essential Oils of Three Species of Apiaceae Growing Wild in Sicily: Bonannia graeca, Eryngium maritimum and Opopanax chironium Antonella Maggio, Maurizio Bruno, Carmen Formisano, Daniela Rigano and Felice Senatore 841

Natural Product Communications 2013

Volume 8, Number 6

Contents

Australasian Natural Product Studies (Guest Editor: Sylvia Urban)

Original Paper Page

Epimanool and a New 2,6-Dideoxy-hexopyran-3-ulose Derivative from Celmisia viscosa Catherine E. Sansom, Lesley Larsen, Alison C. Evans and Nigel B. Perry 689

7-Bromo-1-ethyl-E-carboline, an Alkaloid from the New Zealand Marine Bryozoan Pterocella vesiculosa Michèle R. Prinsep and Mérine Dumté 693

Alkaloids from the Roots and Leaves of Stichoneuron halabalensis and their Acetylcholinesterase Inhibitory Activities Rosdayati Alino Ramli, Wilford Lie and Stephen G. Pyne 695

Establishment of a Phenotypic-based Sand Dollar Fellaster zelandiae Embryo Development Assay and its Application in Defining the Structure-Activity Relationship of Discorhabdin Alkaloids Tanja Grkovic and Brent R. Copp 699

Sharing of Pyrazine Semiochemicals Between Genera of Sexually Deceptive Orchids Bjorn Bohman, Ryan D. Phillips, Gavin Flematti, Rod Peakall and Russell A. Barrow 701

Constituents of the Indonesian Epiphytic Medicinal Plant Drynaria rigidula Ari S. Nugraha, Rachada Haritakun and Paul A. Keller 703

Variability of the Polyphenolic Content and Antioxidant Capacity of Methanolic Extracts of Pomegranate Peel Antony Kam, Kong M Li, Valentina Razmovski-Naumovski, Srinivas Nammi, Kelvin Chan and George Q Li 707

Combination Effects of Curcumin and Aqueous Extract of Lignosus rhinocerotis Mycelium on Neurite Outgrowth Stimulation Activity in PC-12 Cells Priscilla A. John, Kah - Hui Wong, Murali Naidu, Vikineswary Sabaratnam and Pamela David 711

HPLC-NMR Chemical Profiling and Dereplication Studies of the Marine Brown Alga, Cystophora torulosa Sylvia Urban and Michael Timmers 715

Two Polyketides from a Co-culture of Two Marine-derived Fungal Strains Miriam H. Kossuga, Antonio G. Ferreira, Lara D. Sette and Roberto G. S. Berlinck 721

Cyclic Peroxides from a Two-Sponge Association of Plakortis communis-Agelas mauritiana Pinus Jumaryatno, Lynette K. Lambert, John N. A. Hooper, Joanne T. Blanchfield and Mary J. Garson 725

Relative Configuration of the Marine Natural Product Elatenyne using NMR Spectroscopic and Chemical Derivatization Methodologies Robert Brkljaþa and Sylvia Urban 729

Antimicrobial, Antimalarial and Cytoxicity Activities of Constituents of a Bhutanese Variety of Ajania nubigena Phurpa Wangchuk, Paul A. Keller, Stephen G. Pyne, John Korth, Samten, Malai Taweechotipatr, Roonglawan Rattanajak and Sumalee Kamchonwongpaisan 733

Dihydrotagetone, an Unusual Fruity Ketone, is Found in Enantiopure and Enantioenriched Forms in Additional Australian Native Taxa of Phebalium (Rutaceae: Boronieae) Nicholas J. Sadgrove, Ian R. H. Telford, Ben W. Greatrex, Ashley Dowell and Graham L. Jones 737

Antimicrobial Activity of Essential Oils and Solvent Extracts from Zieria species (Rutaceae) Nicholas J. Sadgrove and Graham L. Jones 741

Characterization and Bioactivity of Essential Oils from Geijera parviflora (Rutaceae): A Native Bush Medicine from Australia Nicholas J. Sadgrove and Graham L. Jones 747

-------------------- Synthesis and Insecticidal Activities of Novel Derivatives of 1ȕ,4Į,6Į,9Į-Tetrahydroxy-2ȕ,12-epoxymethano-ȕ-dihydroagarofuran Jiwen Zhang, Zhan Hu, Shengkun Li, Shuding Yang and Wenjun Wu 753

Novel�D-Glucosidase Activator from Pulicaria undulata Nasir Rasool, Muhammad Abid Rashid, Saleha Suleman Khan, Zulfiqar Ali, Muhammd Zubair, Viqar Uddin Ahmad, Shamsun Nahar Khan, M. Iqbal Choudhary and Rasool Bakhsh Tareen 757

Phytochemical, Antimicrobial and Antiplasmodial Investigations of Terminalia brownii Francis Machumi, Jacob O. Midiwo, Melissa R. Jacob, Shabana I. Khan, Babu L. Tekwani, Jin Zhang, Larry A. Walker and Ilias Muhammad 761

A New Sulfated Triterpene Saponin from Gypsophila trichotoma Ilina Krasteva, Maya Yotova, Kristina Jenett-Siems, Petranka Zdraveva and Stefan Nikolov 765

Bioactive Triterpene Saponins from the Leaves of Schefflera elegantissima Amira S. Wanas, Mostafa A. Fouad, Mohamed S, Kamel, Katsuyoshi Matsunami and Hideaki Otsuka 767

Continued Inside backcover