(+)-Echinobetaine B: isolation, structure elucidation, synthesis and preliminary SAR studies on a new nematocidal betaine from a southern Australian marine sponge, Echinodictyum sp.

Abstract

The principle nematocidal agent present in a southern Australian marine sponge of the genus Echinodictyum has been isolated and identified as the novel betaine (+)-echinobetaine B (6), and the structure assigned by spectroscopic analysis has been confirmed by total synthesis. Preliminary SAR conclusions are drawn from analysis of synthetic intermediates and the known marine metabolites zooanemonin (12) and norzooanemonin (13), and the new sponge metabolite norzooanemonin methyl ester (14). The latter compound is reported for the first time from a selection of Australian sponges, including an Axinyssa sp., a Niphates sp., an Axinella sp. and a Ptilocaulis sp.

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Introduction

During earlier investigations into new agrochemical agents from the Australian marine sponge Echinodictyum sp. we reported a series of novel antibacterial metabolites, echinosulfonic acids A–C (1–3) and echinosulfone (4),¹ along with the nematocidal betaine (−)-echinobetaine A (5).² Although initial interest in this Echinodictyum sp. was prompted by promising nematocidal bioassay data against the endo parasite Haemonchus contortus, it is noteworthy that 1–4 displayed no nematocidal activity while the nematocidal properties of (−)-echinobetaine A (5) were insufficient (LD₉₉ 83 µg mL⁻¹) to account for the level of activity observed in the crude EtOH extract (LD₉₉ 22 µg mL⁻¹). The identity of the more potent Echinodictyum sp. nematocidal agent can now be revealed as the novel betaine (+)-echinobetaine B (6) (LD₉₉ 8.3 µg mL⁻¹).

Results and discussion

The EtOH extract of the Echinodictyum specimen was decanted, concentrated in vacuo and triturated with DCM after which the residual solid was partitioned between n-BuOH and H₂O. Whereas the n-BuOH solubles displayed antibacterial activity and ultimately led to the isolation and identification of the echinosulfonic acids A–C (1–3) and echinosulfone (4),¹ the crude H₂O solubles displayed significant nematocidal activity. The H₂O solubles were initially further fractionated by elution through Sephadex G-10 (H₂O) followed by isocratic C₁₈ HPLC (0.1% TFA in H₂O) to yield nematocide (−)-echinobetaine A (5).² More extensive HPLC studies on nematocidal active fractions have since yielded the principle nematocidal agent as (+)-echinobetaine B (6).

High resolution ESI(+)MS analysis of 6 revealed a highest mass ion at m/z 185 consistent with a molecular formula (C₈H₁₂N₂O₃, Δ 0.5 mmu) requiring four double bond equivalents. Examination of the NMR data for 6 (see Table 1) revealed resonances consistent with an OMe (¹H: δ 3.22 (s); ¹³C: 57.1 ppm), two NMe's (¹H: δ 3.78 and 3.63; ¹³C: 35.7 and 33.9 ppm), two sp² methines (¹H: δ 8.52 and 7.31; ¹³C: 138.1 and 123.6 ppm), one deshielded sp³ oxymethine (¹H: δ 4.78; ¹³C: 73.3 ppm), one carboxylic acid or amide carbonyl (¹³C: δ 172.5 ppm), and a quaternary sp² hybridized carbon (¹³C: 130.7 ppm). The presence of the carbonyl functionality was further supported by a characteristic IR absorbance (1689 cm⁻¹), while a measurable [α]_D (+30) confirmed the presence of a chiral centre. With a total of four sp² carbons, one attributed to a carboxylic acid moiety, 6 was determined to contain an N-heterocycle. Analysis of the gHMBC NMR data (Table 1) confirmed that the chiral tertiary carbon (C-2) was substituted by both OMe and carbonyl moieties. Further gHMBC NMR correlations from N5-Me to C-4 and C-6, from N7-Me to C-3 and C-6, and from H-2 to C-3 and C-4, suggested that (+)-echinobetaine B was the substituted imidazole betaine as shown. To confirm the assigned structure a total synthesis of (±)-echinobetaine B (11) was undertaken following the synthetic pathway outlined in Scheme 1.

Table 1 NMR (400 MHz; D₂O) data for (+)-echinobetaine B (6)

Number	^13C (ppm)	^1H δ (multiplicity)	gHMBC (^1H–^13C)
1	172.5
2	73.3	4.78 (s)	C-1, C-3, C-4, 2-OMe
2-OMe	57.1	3.22 (s)	C-2
3	130.7
4	123.6	7.31 (s)	N5–Me, C-3, C-6,
N5-Me	35.7	3.78 (s)	C-6, C-4
6	138.1	8.52 (s)	C-3, C-4
N7-Me	33.9	3.63 (s)	C-6, C-3

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Scheme 1 Synthesis of (±)-echinobetaine B (11). Reagents and conditions: (i) NaH, MeI, DMF, N₂, 0 °C to RT, 3.5 h; (ii) 10% HCl_(aq), RT; (iii) p-TsOH, MeOH, N₂, 64 °C, 12 h; (iv) TMSCN, SnCl₂, CH₃CN, N₂, RT, 96 h; (v) MeI, acetone, N₂, 56 °C, 15 h; (vi) 10 M HCl_(aq), 100 °C, 36 h; (vii) 10 M HCl_(aq), 100 °C, 15 h; (viii) MeI, DMF, N₂, 70 °C, 14 h.

Commercially available imidazole carboxaldehyde (7) was viewed as a suitable starting material for the synthesis of (±)-echinobetaine B (11). As initially conceived, treatment of 7 with cyanide might be expected to return the cyanohydrin, which on per-methylation about oxygen (2-OMe) and nitrogen (N5-Me and N7-Me) could yield a suitable organonitrile precursor. This precursor would in turn be a potential substrate for commercially available stereoselective nitrilases^3,4 leading to an asymmetric synthetic outcome. In practice, the proposed cyanohydrin derived from treatment of 7 with either cyanide salts or HCN proved unstable and difficult to handle, necessitating a modification (see Scheme 1) to the strategy as detailed above. Monomethylation of 7 yielded the mixture 8a and 8b, which was readily converted in good yield to the mixed acetals 9a and 9b. Subsequent treatment with trimethylsilylcyanide and the Lewis acid SnCl₂^5,6 successfully converted the acetals in high yield to the unstable organonitrile mixture 10a and 10b. The noted instability of cyanohydrin and organonitrile precursors precluded stereoselective enzymatic hydrolysis, and necessitated a more direct chemical approach. Acid hydrolysis of the organonitrile moiety followed by N-methylation proceeded in low yield (27%), with the alternative strategy of N-methylation followed by acid hydrolysis returning a slightly improved yield (39%). The product obtained by either route, (±)-echinobetaine B (11), was identical by NMR, MS, UV and HPLC retention time comparison to the natural product (+)-echinobetaine B (6), differing only in the lack of a measurable optical rotation, as befits a racemate. Attempts to resolve the racemate by chiral HPLC proved unsuccessful.

Of particular note, the nematocidal properties for (±)-echinobetaine B (11) (LD₉₉ 65 µg mL⁻¹) were considerably lower than those for the natural product (+)-echinobetaine B (6) (LD₉₉ 8.3 µg mL⁻¹), implicating stereochemistry as a critical factor in the echinobetaine B pharmacophore. A similar observation had been made regarding the pharmacophore of the nematocidal co-metabolite (−)-echinobetaine A (5).²

(+)-Echinobetaine B (6) is a new example of a rare class of naturally occurring imidazole betaine. The first example of this structure class was zooanemonin (12) reported by Ackermann and Menssen in 1960 from the “horse sponge”, Hippospongia equina.⁷ Although the structure of 12 was revised shortly thereafter (following communication on its synthesis by Woodward and List),⁸ few subsequent references have appeared in the scientific literature. The most recent reference to 12 was in 2001 by Hattori et al., in which 12 was assessed as an antifouling constituent of the sponge Protophilaspongia aga.⁹ As with zooanemonin (12), the closely related homologue norzooanemonin (13) is known but remains equally elusive in the scientific literature. First reported in 1973 by Weinheimer et al. from the gorgonian Pseudopterogorgia americana,¹⁰13 was also described in 1977 by Gupta et al. from the hydroid Tubularia larynx.¹¹

During our earlier investigation into Australian marine sponges we isolated norzooanemonin (13) from an Axinyssa and a Niphates sp. The latter sponge, together with an Axinella sp. and a Ptilocaulis sp., also yielded the previously undescribed norzooanemonin methyl ester 14. The molecular formula assigned to 14 by high resolution mass spectrometry (C₇H₁₁N₂O₂, Δmmu −0.3) was consistent with a salt possessing 4 double bond equivalents. Analysis of the ¹H NMR (400 MHz, D₂O) data for 14 revealed resonances diagnostic for a deshielded sp² methine (H_δ 8.27, H-5) that proved to be D₂O exchangeable (see below), as well as an isolated deshielded sp² methine (H_δ 7.07, H-3), a CO₂Me (H_δ 3.46) and two quaternary ammonium methyl moieties (H_δ 3.56 and 3.66, N6-Me and N4-Me respectively). The data presented above supported a doubly N-methylated imidazole heterocycle substituted by a methyl ester moiety (as shown). Both ¹³C and 2D NMR correlations supported this determination and facilitated assignment of the NMR data. In this way 14 was confirmed to be the methyl ester of norzooanemonin as indicated. As all sponge samples were extracted with EtOH, and were stored for several years in EtOH prior to investigation, we are confident that the methyl ester 14 is not an esterification artifact. Curiously, during acquisition of ¹H NMR data in neutral deuterated protic solvents (CD₃OD or D₂O) the imidazole methine H-5 in both 13 and 14 underwent slow but complete deuterium exchange. While such exchange processes have been described for a range of heterocycles, they are typically pH or metal-ion activated.^12,13 Such an exchange was not observed for 6 or 12 suggesting that the conjugated carbonyl present in 13 and 14 may play a pivotal role in this process, and revealing a measure of specificity not previously recorded.

It is worthwhile noting that the nematocidal activity displayed against Haemonchus contortus by the natural product (+)-echinobetaine B (6) (LD₉₉ 8.3 µg mL⁻¹) in our assay system is comparable to that of the two commercial synthetic anthelmintics closantel and levamisole (LD₉₉ 5–10 µg mL⁻¹). By contrast, none of 12–14 displayed nematocidal activity, and the synthetic racemate 11 was considerably less active (LD₉₉ 65 µg mL⁻¹). Thus it would appear that the 2-OMe and stereochemistry are critical components in the echinobetaine B nematocidal pharmacophore. Ongoing studies are directed at confirming enantiopurity and assignment of absolute stereochemistry for 6, and towards further SAR investigations.

Experimental

General procedures

Solvents were dried according to standard techniques. Anhydrous SnCl₂ was prepared by heating commercial SnCl₂ dihydrate to 200 °C under an atmosphere of N₂ for 1 hour. Column chromatography (flash) was performed using Merck silica gel 60. Thin layer chromatography (TLC) was performed on Merck Silica 60 F₂₅₄ sheets. Compounds were visualised with 254 nm UV light and/or heating of the TLC plate that had been dipped in p-anisaldehyde, phosphomolybdic acid or potassium permanganate reagent solutions. High performance liquid chromatography (HPLC) was performed using either a Waters 2790 Separations Module equipped with a Waters 996 Photodiode Array Detector, Alltech 500 Evaporative Light Scattering Detector and a Waters Fraction Collector II, running Waters Millennium software or an Agilent 1100 Series Separations Module equipped with a six column switching capability, Agilent 1100 Series Diode Array and/or Multiple Wavelength Detectors, Polymer Laboratories PL-ELS1000 Evaporative Light Scattering Detector (ELSD) and an Agilent 1100 Series Fraction Collector and running ChemStation Rev.9.03A and Purify version A.1.2 software. Unless otherwise specified, a constant level of 0.01% TFA was used in all HPLC separations. Chiroptical measurements ([α]_D), given in 10⁻¹ deg cm² g⁻¹, were obtained on either a Jasco Dip-1000 digital polarimeter or a Jasco P-1010 Intelligent Remote Module type polarimeter, at ambient temperature, in a 100 × 3 mm cell. Ultraviolet (UV) absorption spectra were obtained using either a Shimadzu UV-1650PC or a CARY3 UV-visible Spectrophotometer. Infrared (IR) spectra were acquired using either a Shimadzu FTIR-8400 or a Jasco FT/IR-460Plus Spectrometer, with samples examined as films on NaCl discs. ¹H and ¹³C NMR spectra were acquired on a Varian Inova 400, a Varian Unity 400 plus, a Bruker Avance 500 or a Bruker Avance 600 Spectrometer, in the solvents indicated and referenced to residual ¹H and ¹³C signals in the deuterated solvents. Electrospray Ionisation Mass Spectra (ESI-MS) and Atmospheric Pressure Chemical Ionisation Mass Spectra (APCI-MS) were acquired using either a Waters 2790 Separations Module equipped with a Micromass ZMD mass detector or an Agilent 1100 Series Separations Module equipped with an Agilent 1100 Series LC/MSD mass detector. High resolution (HR) ESI-MS measurements were obtained on either a Bruker BioApex 47E FT mass spectrometer at a cone voltage of 100 kV or a Finnigan MAT 900 XL-Trap instrument with a Finnigan API III source, while EI-MS measurements were obtained on a Kratos MS25RFA mass spectrometer at 70 eV.

Parasitic nematode larval development assay. Nematode eggs were applied in duplicate to the surface of an agar matrix containing the “test solution” (extract, chromatographic fraction and/or pure compound at a concentration range of ∼100 to 0.5 ppm). The eggs were allowed to hatch and develop through to the L3 infective stage (6 days). The extent of larval development was then determined quantitatively by counting the number of eggs, L1s, L2s and L3 larvae to establish the proportion of undeveloped larvae (eggs, L1s and L2s) at each concentration. The resulting data were fitted to a log-concentration–logit model to determine the LD₉₉ value.

Collection, extraction and isolation

The marine sponge Echinodictyum sp. (Museum of Victoria Registry Number MVF88741) was collected by beam trawl from the Great Australian Bight at a depth of 85 m at position 32°58′92″S : 128°00′84″E. A taxonomic description has been reported.¹ The sponge was diced, steeped in EtOH and kept at −20 °C prior to extraction. The EtOH extract was decanted and concentrated in vacuo to yield a bright orange solid (4.08 g). Trituration with DCM returned insoluble soluble material that was partitioned between n-BuOH and H₂O, with the nematocidal activity of the crude extract concentrated into the H₂O solubles (2.96 g, 73%). The n-BuOH solubles yielded (in prior studies) echinosulfonic acids A-C (1–3) and echinosulfone (4).¹ This nematocidal H₂O soluble material was concentrated in vacuo and fractionated by Sephadex G-10 (H₂O) chromatography, followed by isocratic C₁₈ HPLC (2 mL min⁻¹ H₂O + 0.1% TFA, Zorbax C₁₈ 10 µm 250 × 10 mm column), to yield the nematocidal agent (−)-echinobetaine A (5) (as documented in prior studies).² Further bioassay directed HPLC fractionation, with careful peak shaving, has yielded the dominant nematocidal agent as (+)-echinobetaine B (6) (25 mg, 0.044%) (H. contortus, LD₉₉ 8.3 µg mL⁻¹) as the trifluoroacetate salt. Note that percentage yields of natural products are calculated against the total dry weight mass of the crude EtOH extract.

The marine sponge Axinyssa sp. (Museum of Victoria Registry Number MVF83453) was collected by hand (SCUBA) off Beware Reef, Cape Conran, Victoria, 38°12′S : 148°47′E. The sponge was diced, steeped in EtOH and kept at −20 °C prior to extraction. The crude EtOH extract was processed as described above for the Echinodictyum sp. to return an H₂O soluble fraction that was fractionated by Sephadex G-10 (H₂O) chromatography, followed by isocratic C₁₈ HPLC (2 mL min⁻¹ H₂O + 0.1% TFA, Zorbax C₁₈ 10 µm 250 × 10 mm column), to yield trigonelline¹⁴ (0.4 mg, 0.037%) together with norzooanemonin (13) (1.1 mg, 0.10%). Norzooanemonin possessed MS and ¹H NMR data consistent with those reported in the literature.¹⁵

The marine sponge Axinella sp. (Northern Territory Museum Registry Number NTMZ3725) was collected by trawling in the Great Australian Bight. The sponge was diced, steeped in EtOH and kept at −20 °C prior to extraction. The crude EtOH extract was processed as described above for the Axinyssa sp. to return an H₂O soluble fraction that was fractionated by Sephadex G-10 (H₂O) chromatography, followed by isocratic C₁₈ HPLC (1 mL min⁻¹ H₂O + 0.1% TFA, Phenomenex Aqua C₁₈ 5 µm 250 × 10 mm column), to yield norzooanemonin methyl ester (14) (1.1 mg, 1.45%). λ_max (MeOH)/nm 264; δ_H (400 MHz; D₂O) 8.27 (1 H, s), 7.07 (1 H, s), 3.66 (3 H, s), 3.56 (3 H, s) and 3.46 (3 H, s); δ_C (100 MHz; D₂O) 175.0 (C-1), 131.0 (C-2), 35 (N4-Me), 33.0 (N6-Me) and 32.0 (CO₂Me); ESI(+)MS m/z 177 (M − H + Na), 155 (M); HRESI(+)MS m/z 177.0639 (C₇H₁₀N₂O₂Na requires 177.0640) and 155.0817 (C₇H₁₁N₂O₂ requires 155.0820).

The marine sponge Niphates sp. (Museum of Victoria Registry Number MVF83541) was collected by trawling off the east coast of Tasmania. The sponge was diced, steeped in EtOH and kept at −20 °C prior to extraction. The crude EtOH extract was processed as described above for the Axinyssa sp. to return norzooanemonin (13) (1.6 mg, 0.36%) and the methyl ester 14 (27.0 mg, 6.14%).

The marine sponge Ptilocaulis sp. (Museum of Victoria Registry Number MVF83529) was collected by scientific trawling in the Great Australian Bight, 38°02′S : 128°28′E. The sponge was diced, steeped in EtOH and kept at −20 °C prior to extraction. The crude EtOH extract was processed as described above for the Axinyssa sp. to return norzooanemonin methyl ester (14) (1.7 mg, 0.7%).

(+)-Echinobetaine B (6). [α]_D +30.0 (c 0.59 in MeOH); λ_max (MeOH)/nm (ε/dm³ mol⁻¹ cm⁻¹) 214 (2344); ν_max (film)/cm⁻¹ 1689; ¹H and ¹³C NMR data (400 MHz; D₂O) see Table 1; HRESI(+)MS m/z 185.0921 (M + H, C₈H₁₃N₂O₃ requires 185.0926).

1-Methyl-4-imidazolecarboxaldehyde¹⁶ (8a) and 1-methyl-5-imidazolecarboxaldehyde¹⁶ (8b). A solution of 4(5)-imidazolecarboxaldehyde (7) (1.05 g, 10.90 mmol) in anhydrous DMF (20 mL) was added dropwise to a suspension of NaH (95%) (0.27 g, 11.25 mmol) in anhydrous DMF (15 mL) at 0 °C under an atmosphere of N₂. The effervescent mixture was stirred for 30 min at 0 °C after which MeI (0.80 mL, 12.85 mmol) was slowly added and the reaction mixture stirred for a further 3 h at RT. At this point the reaction product was concentrated in vacuo, and the residue dissolved in DCM (90 mL), washed with water (3 × 50 mL), and the combined aqueous fractions extracted with DCM (2 × 70 mL). The combined organic extracts were dried (MgSO₄), filtered, concentrated in vacuo and the residue purified by silica gel column chromatography (EtOAc : hexane (1 : 1) to EtOAc then from MeOH : DCM (1:49) to MeOH : DCM (1 : 1), via a stepwise gradient elution) to afford a mixture of mono-N-methylated imidazoles¹⁶8a and 8b (0.44 g, 36%) as a colourless oil which solidified to white crystals on cooling to −30 °C. The above combined aqueous fractions were concentrated in vacuo, MeOH added and the mixture filtered through a pad of celite. The filtrate was concentrated in vacuo and a solution of the residue in H₂O (5 mL) eluted through an Alltech C₁₈ Solid Phase Extraction (SPE) cartridge (2 g) with water (15 mL). The eluent was concentrated in vacuo (0.44 g) and subjected to silica gel column chromatography under identical conditions as above to provide a further yield of recovered pure mono-N-methylated imidazoles8a and 8b (0.26 g, 22%) as a colourless oil: ν_max (film)/cm⁻¹ 3111, 2956, 2833 and 1673; δ_H (8a) (500 MHz; CDCl₃) 3.70 (3 H, s, N1-Me), 7.45 (1 H, s, 5-H), 7.53 (1 H, s, 2-H) and 9.78 (1 H, s, CHO); δ_C (8a) (125 MHz; CDCl₃) 34.0 (q, N1-Me); 125.5 (d, C-5), 139.5 (d, C-2), 142.7 (s, C-4) and 186.1 (s, CHO); δ_H (8b) (500 MHz; CDCl₃) 3.86 (3 H, s, N1-Me), 7.55 (1 H, s, 4-H), 7.70 (1 H, s, 2-H) and 9.69 (1 H, s, CHO); δ_C (8b) (125 MHz; CDCl₃) 34.1 (q, N1-Me), 131.7 (d, C-4), 143.3 (d, C-2), 144.2 (s, C-5) and 179.5 (s, CHO); ESI(+)MS (30V) m/z 111.1 (M + H⁺).

1-Methyl-4-(dimethoxymethyl)-imidazole (9a) and 1-methyl-5-(dimethoxymethyl)-imidazole (9b). 10% HCl_aq (10 mL) was added to a mixture of imidazoles 8a and 8b (0.41 g, 3.70 mmol) and the solution concentrated in vacuo. A solution of the resultant oily residue and p-toluenesulfonic acid monohydrate (0.07 g, 0.37 mmol) in anhydrous methanol (20 mL) was refluxed for 12 h under an atmosphere of N₂, after which it was cooled to RT and quenched with saturated aqueous NaHCO₃ (15 mL) and the organic solvent removed in vacuo. The resulting aqueous solution was extracted with ethyl acetate (3 × 20 mL) and the combined organic fractions dried (MgSO₄), filtered and concentrated in vacuo to provide a quantitative yield of a 7 : 3 mixture of dimethoxy acetals9a and 9b (0.51 g, 87%) as a colourless oil. The ratio of 7 : 3 for the mixture 9a and 9b was determined by integration of the two singlets at δ_H 3.31 (9a) and 3.27 (9b), respectively, in the ¹H NMR spectrum: ν_max (film)/cm⁻¹ 3126, 2939, 1507 and 1102; δ_H (9a) (500 MHz; CDCl₃) 3.31 (6 H, s, 2 × OMe), 3.60 (3 H, s, N1-Me), 5.37 (1 H, s, 6-H), 6.87 (1 H, s, 5-H) and 7.33 (1 H, s, 2-H); δ_H (9b) (500 MHz; CDCl₃) 3.27 (6 H, s, 2 × OMe), 3.59 (3H, s, N1-Me), 5.32 (1 H, s, 6-H), 7.02 (1 H, s, 4-H) and 7.35 (1 H, s, 2-H); δ_C (9a and 9b) (125 MHz; CDCl₃) 32.3, 33.5, 53.0, 53.3, 98.3, 100.4, 118.5, 128.2, 129.8, 137.7, 139.5 and 140.5; HR(EI)MS m/z 156.0897 (M, C₇H₁₂N₂O₂ requires 156.0899).

1-Methyl-4-[cyano(methoxy)methyl]imidazole (10a) and 1-methyl-5-[cyano(methoxy)methyl]imidazole (10b). Trimethylsilyl cyanide (0.40 mL, 3.00 mmol) and anhydrous SnCl₂ (0.19 g, 1.00 mmol) were successively added to a solution of acetals 9a and 9b (0.16 g, 1.00 mmol) in dry MeCN (7 mL), under an atmosphere of N₂, and the mixture stirred at RT for 96 h. The reaction mixture was quenched with H₂O (30 mL), extracted with DCM (3 × 40 ml) and the combined organic fractions washed with H₂O (20 mL), dried (MgSO₄), filtered and concentrated in vacuo. Purification of the resultant residue by silica gel column chromatography (a stepwise gradient elution from MeOH : DCM (1 : 49) to MeOH : DCM (1 : 1)) afforded a mixture of methylated cyanohydrins10a and 10b (0.14 g, 93%) as a colourless oil: ν_max (film)/cm⁻¹ 3066, 2954, 1504 and 1081; δ_H (10a) (500 MHz; CDCl₃) 3.47 (3 H, s, OMe), 3.65 (3 H, s, N1-Me), 5.17 (1 H, s, 6-H), 7.07 (1 H, s, 5-H) and 7.39 (1 H, s, 2-H); δ_H (10b) (500 MHz; CDCl₃) 3.41 (3 H, s, OMe), 3.68 (3H, s, N1-Me), 5.26 (1 H, s, 6-H), 7.19 (1 H, s, 4-H) and 7.61 (1 H, s, 2-H); δ_C (10a and 10b) (125 MHz; CDCl₃) 32.2, 33.8, 56.5, 57.1, 63.2, 67.0, 115.3, 116.9, 119.8, 123.7, 131.5, 135.7, 138.6 and 141.1; HR(EI)MS m/z 151.0745 (M, C₇H₉N₃O requires 151.0746).

(±)-Echinobetaine B (11)—methylation followed by hydrolysis. A solution of methylated cyanohydrins 10a and 10b (0.06 g, 0.37 mmol), and MeI (0.23 mL, 3.70 mmol) in anhydrous acetone (8 mL) was refluxed for 15 h under an atmosphere of N₂ after which the solution was concentrated in vacuo to an orange solid (0.12 g) tentatively identified by NMR as the desired methylated product. δ_H (500 MHz, CD₃OD) 3.58 (3-H, s, CO₂CH₃), 3.92 (6-H, s, N5-Me and N7-Me), 6.01 (1-H, s, 2-H), 7.93 (1-H, s, 4-H) and 9.08 (1-H, s, 6-H). This intermediate product (0.12 g) was refluxed in 10 M HCl_aq (10 mL) for 36 h, after which the reaction mixture was cooled to RT and the solvent removed in vacuo to give a yellow oil (0.10 g) which was purified by C₁₈ HPLC (4.2 mL min⁻¹ H₂O + 0.1% TFA, Agilent Zorbax SB-Aq 5 µm 150 × 9.4 mm column) to yield (±)-echinobetaine B (11) (27%) as a colourless oil with ESI(+)MS, and ¹H and ¹³C NMR data in agreement with data for the natural product (+)-echinobetaine B (6).

(±)-Echinobetaine B (11)—hydrolysis followed by methylation. A solution of methylated cyanohydrins 10a and 10b (0.10 g, 0.66 mmol) was refluxed in 10 M HCl_aq (20 mL) for 15 h, after which the reaction mixture was cooled to RT and the solvent removed in vacuo to give a white solid (0.218 g), which was added to H₂O and the pH adjusted to 8.0 prior to concentration in vacuo. The resultant residue was dissolved in anhydrous DMF (4 mL) and MeI (0.30 mL, 4.80 mmol) and heated at 70 °C under N₂ for 14 h before cooling to RT and concentrating to dryness in vacuo. Purification by C₁₈ HPLC (4.2 mL min⁻¹ H₂O + 0.1% TFA, Agilent Zorbax SB-Aq 5 µm 150 × 9.4 mm column) to yield (±)-echinobetaine B (11) (39%) as a colourless oil with ESI(+)MS, and ¹H and ¹³C NMR data in agreement with data for the natural product (+)-echinobetaine B (6).

Acknowledgements

We acknowledge the assistance of L. Goudie in identifying sponge material, D. Howse for database support, and T. Friedel and K. Heiland for facilitating access to bioassays. This research was partially funded by the Australian Research Council, together with Novartis Animal Health Australasia Pty Ltd.

References

1. S. P. B. Ovenden and R. J. Capon, J. Nat. Prod., 1999, 62(9), 1246–1249.
2. R. J. Capon, D. Vuong, E. Lacey and J. H. Gill, J. Nat. Prod., 2004 (accepted).
3. C. O'Reilly and P. D. Turner, J. Appl. Microbiol., 2003, 95(6), 1161–1174.
4. L. Martinkova and V. Mylerova, Curr. Org. Chem., 2003, 7(13), 1279–1295.
5. K. Utimoto, Y. Wakabayashi, Y. Shishiyama, M. Inoue and H. Nozaki, Tetrahedron Lett., 1981, 22(43), 4279–4280.
6. K. Utimoto, Y. Wakabayashi, T. Horiie, M. Inoue, Y. Shishiyama, M. Obayashi and H. Nozaki, Tetrahedron, 1983, 39(6), 967–973.
7. D. Ackermann and H. G. Menssen, Hoppe-Seyler’s Z. Physiol. Chem., 1960, 322, 198–207.
8. D. Ackermann and P. H. List, Hoppe-Seyler’s Z. Physiol. Chem., 1960, 318(3/6), 281.
9. T. Hattori, S. Matsuo, K. Adachi and Y. Shizuri, Fish. Sci., 2001, 67(4), 690–693.
10. A. J. Weinheimer, E. K. Metzner and M. L. Mole Jr., Tetrahedron, 1973, 29(20), 3125–3126.
11. K. C. Gupta, R. L. Miller, J. R. Williams and J. F. Blount, Experientia, 1977, 33(12), 1556.
12. E. Buncel, O. Clement and I. Onyido, Acc. Chem. Res., 2000, 33(10), 672–678.
13. E. Buncel and I. Onyido, J. Labelled Compd. Radiopharm., 2002, 45(4), 291–306.
14. E. Schulze and G. Trier, Hoppe-Seyler’s Z. Physiol. Chem., 1911, 67, 46–58.
15. T. Jahn, G. M. Konig, A. D. Wright, G. Worheide and J. Reitner, Tetrahedron Lett., 1997, 38(22), 3883–3884.
16. M. Kodera, N. Terasako, T. Kita, Y. Tachi, K. Kano, M. Yamazaki, M. Koikawa and T. Tokii, Inorg. Chem., 1997, 36(18), 3861–3868.

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