Cyclic Peroxides from a Two-Sponge Association of Plakortis communis-Agelas mauritiana

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Natural Product Communications

EDITOR-IN-CHIEF DR. PAWAN K AGRAWAL Natural Product Inc. 7963, Anderson Park Lane, Westerville, Ohio 43081, USA

[email protected] EDITORS PROFESSOR ALEJANDRO F. BARRERO Department of Organic Chemistry, University of Granada, Campus de Fuente Nueva, s/n, 18071, Granada, Spain [email protected] PROFESSOR ALESSANDRA BRACA Dipartimento di Chimica Bioorganicae Biofarmacia, Universita di Pisa, via Bonanno 33, 56126 Pisa, Italy [email protected] PROFESSOR DEAN GUO State Key Laboratory of Natural and Biomimetic Drugs, School of Pharmaceutical Sciences, Peking University, Beijing 100083, China [email protected] PROFESSOR YOSHIHIRO MIMAKI School of Pharmacy, Tokyo University of Pharmacy and Life Sciences, Horinouchi 1432-1, Hachioji, Tokyo 192-0392, Japan [email protected] PROFESSOR STEPHEN G. PYNE Department of Chemistry University of Wollongong Wollongong, New South Wales, 2522, Australia [email protected] PROFESSOR MANFRED G. REINECKE Department of Chemistry, Texas Christian University, Forts Worth, TX 76129, USA [email protected] PROFESSOR WILLIAM N. SETZER Department of Chemistry The University of Alabama in Huntsville Huntsville, AL 35809, USA [email protected] PROFESSOR YASUHIRO TEZUKA Institute of Natural Medicine Institute of Natural Medicine, University of Toyama, 2630-Sugitani, Toyama 930-0194, Japan [email protected] 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 [email protected]

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2013 Vol. 8 No. 6 725 - 728

Natural Product Communications

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,* a

School of Chemistry and Molecular Biosciences, The University of Queensland, Brisbane QLD 4072, Australia Pharmacy Department, Faculty of Mathematic and Natural Sciences, Universitas Islam Indonesia, Kampus Terpadu UII, Jalan Kaliurang KM 14.5, Yogyakarta 55284, Indonesia c Centre for Advanced Imaging, The University of Queensland, Brisbane QLD 4072, Australia d Queensland Museum, PO Box 3300, South Brisbane QLD 4101, Australia b

[email protected] 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

12

18

16

14 5

5 10 9

O

O

7

COOR

O

2

O

COOH 2

O

2

1R=H 3 R = Me

HN

Br

N OH 18

16

12

14

5

Br

6

5 10

9

OH HO 4

COOMe 2

OH HO

COOMe 2

5

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 ( H 7.21, 7.31 and 7.40; C 128.3, 129.2 and 127.2), a disubstituted olefin ( H 6.50 and 6.30; C 131.5 and 133.2), an oxygenated methine ( H 4.20; C 83.8), a geminal methylene ( H 2.68 and 2.17; C 37.6) and two methyl groups ( H 0.89 and 0.97; C 7.6 and 10.7). There was also a carbonyl group ( C 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 H 4.20 (H-3) correlated to the non-equivalent methylene protons at H 2.68 and 2.17 (H2-2), and to another methine proton at H 1.66 (H-4) that was further coupled to methylene protons at H 2.25 and 1.42 (H2-5). HMBC data showed correlations from H-3 to the signal at C 174.8 (C-1) and to C-2 at C 37.6. H-5 correlated to an oxygenated quaternary signal at C 85.5 (C-6). The methyl signals at H 0.89 and at H 0.97 showed DQFCOSY correlations to a methylene

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Jumaryatno et al.

shift of C-17 at C 24.7; the ethyl substituent at C-4 is equatorial if the associated methylene carbon resonates near C 25 [5,9,10].

Figure 2: Key DQFCOSY and HMBC correlations for peroxide 1 Table 1: NMR spectroscopic data for peroxide 1. Position 1 2 3 4 5 6 7 8 9 10,14 11,13 12 15 16 17 18

1 H (J)a,b 2.17 m 2.68 dd (3.2, 15.9) 4.20 dt (3.2, 9.6, 9.6) 1.66 m 1.42 t (13.1) 2.25 dd (4.2, 13.1) 6.30 d (16.7) 6.50 d (16.7) 7.40 d (7.3) 7.31 t (7.6) 7.21 t (7.3) 1.59 q (7.6) 0.89 t (7.6) 1.15 m 1.54 m 0.97 t (7.2)

13 C 174.8 37.6

HMBCc,d C-1, C-3, C-4

DQFCOSY H-3

83.8 38.0 37.7

C-1, C-2 C-6, C-7, C-15, C-17

H2, H-4 H-3, H-5 H-4

85.5 133.2 131.5 138.4 127.2 129.2 128.3 33.8 7.6 24.7

C-5, C-6, C-9, C-15 C-6, C-9, C-10, C-14 C-8, C-12 C-9 C-10, C-14 C-5, C-6, C-7, Me-16 C-6, C-15 -

H-8 H-7, H-10, H-14 H-8, H-11, H-13 H-10, H-12, H-14 H-11, H-13 Me-16 H-15 Me-18

10.7

C-4, C-17

H-17

a

500 MHz, MeOH-d4 referenced to 1H at 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 H 1.59 (2H), and at H 1.15 and 1.54, respectively. In addition, there were HMBC correlations from H 0.89 (Me-16) and 0.97 (Me-18) to C 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 C 85.5 for C-6, while Me-18 had an HMBC correlation to a methine carbon at C 38.0 for C-4. The olefinic protons at H 6.50 (H-8) and H 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 H 6.30 to C-9 at C 138.4 and from H-8 at H 6.50 to C-10 at C 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 [ H 2.68 (dd, J = 3.2, 15.9 Hz) and 2.17 (m)] and H-3 [ H 4.20 (dt, J = 3.2, 9.6, 9.6 Hz)] matched the corresponding signals in epiplakortin (H-2 [ H 2.66 (dd, J = 3.5, 15.5 Hz) and 2.38 (dd, J = 9.0, 15.5 Hz)]; H-3 [ H 4.16 (m, J = 3.5, 9.0, 9.0 Hz)]) [2] rather than the signals for plakortin (H-2 [ H 3.05 (dd, J = 9.5, 16.0 Hz) and 2.35 (dd, J = 3.5, 16.0 Hz)]; H-3 [ H 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 C3 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 C 36 or below C 32, respectively [5,10]. The chemical shift value of C-2 at C 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 C 30 or below C 27, respectively. The ethyl substituent at C-6 in 1 was assigned as equatorial due to the chemical shift of C-15 at C 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

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 H 4.07 (dt, J = 3.2, 9.6, 9.6 Hz) displayed COSY correlations to a mutually coupled pair of signals at H 2.25 and 2.63 (H2-2), and to another proton at H 1.68 (H-4). The latter proton was further coupled to signals at 1 H 1.20 and 1.85 (H2-5). Inspection of H NMR and HSQC revealed the presence of three alkyl functionalities in compound 2 due to the methyl triplets at H 0.86 ( C 7.2), H 0.92 ( C 10.6) and H 0.93 ( C 14.2). Each of these methyl signals was coupled to methylene groups from the COSY data ( H 0.86 to H 1.38 and 1.48; H 0.92 to H 1.15 and 1.55; H 0.93 to H 1.35). Analysis of HMBC spectra showed that the methyl protons at H 0.86 and 0.92 had long-range correlations to signals at C 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 H 0.93 linked to an upfield carbon at C 14.2 by HSQC, showed a COSY correlation to methylene protons at H 1.35 and HMBC correlations to methylene carbons at C 23.8 and 26.2. An HMBC correlation was observed from H 1.20 (H-5) to the signal at C 32.5 (C-7) linked by an HSQC correlation to a methylene signal at H 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 [ H 2.63 (dd, J = 3.2, 15.9 Hz) and 2.25 (m)] and H-3 [ H 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 C 37.4. The ethyl groups at C-4 and C-6 were both equatorial from the chemical shift values of C-13 ( C 24.7) and C-11 ( C 30.3). Moreover, C-7 resonated at C 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 13 C 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 H 4.27 (dt, J = 3.3, 9.5, 9.5 Hz) and two methylene protons at H 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 [ H 6.45 (d, J = 16.7 Hz) and 6.30 (d, J = 16.7 Hz)], a methoxy group [ H 3.68 (s)] and two methyl groups [ H 0.89 (t, J = 7.5 Hz) and 0.94 (t, J = 7.5 Hz)]. In addition, aromatic proton signals were observed at H 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

Figure 3: Important HMBC correlations for compound 4. Table 2: NMR spectroscopic data for diol methyl ester 4 Position 1 2 3 4 5 6 7 8 9 10,14 11,13 12 15 16 17 18 -OMe a

1 H (J)a,b 2.35 dd (10.0, 16.7) 2.63 dd (2.3, 16.7) 3.80 dt (2.3, 10.0, 10.0) 1.56 mg 1.62 mg 1.92 dd (6.1, 15.0) 6.06 d (15.9) 6.66 d (15.9) 7.37 d (7.5) 7.31 t (7.5) 7.20 t (7.5) 1.62 mg 0.93 t (7.5) 1.17 m 1.35 m 0.85 t (7.4) 3.71 s 1

13 c

HMBCd,e -

C 174.3f 39.0 71.1 39.5 42.0

-

74.8f 135.7 128.5 137.5f 126.2 128.4 126.9 35.8 7.8 25.3

C-9 C-6, C-10, C-14 C-11, C-12, C-13 C-9 C-10, C-14 C-6, C-15 -

10.2 51.6

C-4, C-17 C-1 b

500 MHz, CDCl3 referenced to H at 7.26 ppm; coupling constant in Hz; ctaken from HSQC at 900 MHz, CDCl3 referenced to 13C at 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 ( H 6.66 and 6.06; C 128.5 and 135.7) from 1H NMR and HSQC data. Furthermore, a methine proton at H 3.80, methylene protons at H 2.63 and 2.35, two methyl triplets at H 0.85 and 0.93, and a methoxy signal at H 3.71 were observed, as well as aromatic signals at H 7.20, 7.31 and 7.37. The methoxy signal at H 3.71 showed an HMBC correlation to a carbonyl at C 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 H 0.93 and a carbon at C 74.8, and by an HSQC correlation from a methine proton at H 3.80 to a signal at C 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 C 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 H 0.93 (Me-16) to C-15 at C 35.8 and C-6 at C 74.8 as well as from H 0.85 (Me-18) to C-17 at C 25.3 and C-4 at C 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 H 2.63 assigned as H-2 had correlations to H-3 at H 3.80 and H-4 at H 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 H 7.31 (H-11 and H-13) and 6.06 (H-7) correlated to a quaternary aromatic carbon at C 137.5 (C-9) while H-8 at H 6.66 showed long-range correlations to an aromatic signal at C 126.2 (C-10 and C-14) and to C-6 at C 74.8.

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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 secoplakortide 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 H 2.42 and 2.65, and H 3.85, respectively. The proton signal at H 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)nsidechain (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 interconversion 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 ) 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

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 (hexanes DCM EtOAc MeOH) 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 (DCM EtOAc MeOH) 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 30:70 100% 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 20:80 100% 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 rechromatographed 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 (