New halogenated sesquiterpenes from the red alga Laurencia caespitosa MANUEL NORTE,'RAFAEL GONZALEZ,AGUST~N PADILLA, J O SJ.~ FERNANDEZ, A N D JES~TS T. VAZQUEZ Centro de Productos Naturales Organicos "Antonio Gonzdlez", Instituto Universitario de Bio-Orgdnica, Universidad de La Laguna, Carretera de La Esperanza, 2 , 38206 La Laguna, Tenerife, Spain Received August 22, 1990 Can. J. Chem. Downloaded from www.nrcresearchpress.com by 3.236.55.199 on 06/14/20 For personal use only. MANUEL NORTE, RAFAEL GONZALEZ, AGUST~N PADILLA. J O S J~. FERNANDEZ, and J ~ s d T. s VAZQUEZ. Can. J. Chem. 69, 518 (1991). Three new brominated sesquiterpenes have been isolated from the red alga Laurencia caespitosa. The structures of these metabolites have been elucidated by spectral analysis and chemical correlations. Their absolute configurations were established by CD methods. Key words: red alga, Rhodomelaceae, halogenated sesquiterpenes, snyderane. MANUEL NORTE,RAFAEL GONZALEZ, AGUST~N PADILLA, J O S ~J. FERNANDEZ et J ~ s d T. s VAZQUEZ. Can. J. Chem. 69, 518 (1991). On a isolt trois nouveaux sesquiterpknes bromts j. partir de l'algue rouge Laur-encia caespitosa. On a tlucidt les structures de ces mttabolites en se basant sur des analyses spectrales et sur des corrtlations chimiques. On a ttabli leurs configurations absolues j. l'aide de mtthodes de DC. Mots c l b : algue rouge, ~hodomelaceae,sesquiterpenes halogtnts, snyderane. [Traduit par la rtdaction] The red alga Laurencia caespitosa collected at La Graciosa (Canary Islands) was found to be a rich source of regular and irregular halogenated sesquiterpenes (1). We recently published the isolation of three new irregular rearranged sesquiterpenoids, the laucapyranoids A , 1; B, 2, and C, 3 (2), which are related to caespitol4 and isocaespitol 5, metabolites previously isolated from the same alga (3). We report here the isolation and structure elucidation of the monocyclofarnesane derivatives 6-8, one of them with an unprecedented y-snyderol system. In contrast with other kinds of sesquiterpenoid skeletons, the number of halogenated sesquiterpenes with a snyderane skeleton isolated from Laurencia is small (4). These compounds were isolated by chromatography on silica gel followed by gel filtration chromatography on Sephadex LH-20, the final purification being carried out by HPLC. The major metabolite of this series, compound 6, was isolated as an oil, [a],+8.7 ( c 0.172, CHC13). Its molecular formula was established as C,5H2502Bron the basis of its HRMS. The 'H NMR spectrum showed the presence of two methines a to heteroatoms (6 4.28 and 4.50), an exocyclic double bond (6 4.77 and 5. lo), and the characteristic chemical shifts due to the double bond of the last isoprene unit for the monocyclofarnesane derivatives (6 5.07,5.2 1, and 5.9 1). This was confirmed by the 13cNMR spectrum as well as by the loss of the fragment (C5H90) observed in the mass spectrum. The cyclic proton assignments begin with the direct coupling of H-8 (6 4.28) to H-9 (6 2.35 and 2.17) and, further, H-9 to H-10 (6 4.50), and the direct coupling of protons finishes with the presence of quaternary centers at C-7 and C-11. The relative positions were confirmed by the long range coupling of H-8 to H-14 (6 5.10). The nature of the heteroatoms at C-8 and C-10 was easily established as oxygen and bromine, respectively, by the correlation of the assigned proton signals to carbons in the 'H-13C COSY(HETC0R) spectrum: H-8 (6 74.5) and H-10 (6 63.5). The relative configurations of the chiral centers at C-6, C-8, and C-10 were established as follows. The coupling constant values observed for H-8 and H-10 (Table 1) showed that the hydroxyl group must be axial and the bromine atom 'Author to whom correspondence may be addressed. Printed in Canada i Imprim6 au Canada equatorial, which implies that they possess opposite orientations. On the other hand, the NOEDIFF experiment showed nOe enhancement for the H-10 signal when the H-6 signal at 6 2.3 was irradiated. These spectroscopic data agree with the structure of 8-hydroxy-P-snyderol for compound 6. Compound 7,oil, [a], 16.8 (c 0.136, CHC13). Its HRMS + NORTE ET AL. 519 TABLE1. NMR spectral data for 6 , 7 , and 8 I3c Carbon 6" 7b 'H< 8 8 7 6 5.21 dd, 17.4; 1.2 5.07 dd, 10.7; 1.2 5.91 dd, 17.4; 10.7 5.23 dd, 17.3; 1.3 5.10 dd, 10.7; 1.3 5.92 dd, 17.3; 10.7 5.18 dd, 17.3, 1.2 5.04 dd, 10.7; 1.2 5.87 dd, 17.3; 10.7 - Can. J. Chem. Downloaded from www.nrcresearchpress.com by 3.236.55.199 on 06/14/20 For personal use only. 1.58 m 4.28 t, 3.3 2.35 ddd, 13.8; 4.5; 3.3 2.17 ddd, 13.8; 12.4; 3.3 4.50 dd, 12.4; 4.5 3.9 t, 3.4 2.38 ddd, 13.8; 4.5; 3.4 2.29 ddd, 13.8; 11.4;3.4 4.43 dd, 1 1.4; 4.5 1.07 s 1.19 s 1.73 s 6.65 dd, 10.2, 1.4 "Assignments made by 'H-13C heteronuclear COSY 'Assignments made by comparison with 6. 'Assignments made by homonuclear COSY. showed it to be an isomer of 6 and the comparison of their NMR spectra showed that they are structurally related. Thus, the main differences observed, with respect to 6, in the 'H NMR spectrum of compound 7 were the absence of signals for the protons H-6 and H-14 together with the presence of a vinylic methyl group at 6 1.73. This was confirmed in the I3C NMR spectrum by the presence of a Z tetrasubstituted double bond (6 128.28, 140.92, and 22.56). On the basis of these data we assigned the structure of 7 as 8-hydroxy-y-snyderol, which is the first example of a y-snyderol derivative isolated from marine sources. Compound 8, oil, [ a ] , +13.6 ( c 0.77, CHC13), has a molecular formula of C15H2202.The presence in the molecule of an a,P-unsaturated carbonyl group was evidenced by the strong absorption at 1660 cm-' in the IR spectrum and by a band in the UV spectrum at 274 nm. It was confirmed by the NMR spectral data, which allowed us to place the carbonyl group at the carbon C-8 and the double bond between the carbons C-9 and C- 10. Moreover, comparison of these data with those observed for 6 and 7 showed that this compound possesses the common side chain and that it belongs to the P-snyderol series. The final confirmation of the structure was obtained through the chemical correlation between 6 and 8. Treatment of 6 with M n 0 2 at room temperature for 2 h gave a compound that showed physical and spectroscopical data identical with those observed for compound 8. The circular dichroic exciton chirality method has proved to be an excellent tool for the determination of absolute configurations (5). We have applied the CD allylic benzoate method (6-8) to compounds 6 and 7, which possess allylic alcohols. Their bromobenzoylation with 4-bromobenzoyl chloride in pyridine with DMAP as catalyst yielded the allylic benzoates 9 and 10 (CH3CN, 243 nm, E = 21 300), respectively. Their CD spectra exhibited a negative Cotton effect at 241.5 nm (AE = - 1.5) for 9 and a positive Cotton effect at 241.0 nm (AE = 13.9) for 10. Since the sign of the CD Cotton effect represents the chirality of the double bond - benzoate system, the observed negative exciton chirality for 9 and the positive one for 10 led to the absolute configuration shown for these compounds. + Experimental part NMR spectra were recorded on a Bruker model WP 200 SY spectrometer (200 MHz), chemical shifts are reported relative to Me4% (6, O), and coupling constants are given in hertz. The 2D-NMR spectra were obtained using Bruker microprograms. Samples for nOe experiments were degassed by bubbling Ar through the solution. IR and UV spectra were recorded on Perkin-Elmer model 257 and Perkin-Elmer model 550D spectrophotometers. CD spectra were recorded on a Jasco 5-600 spectropolarimeter. Optical rotations were determined for solution in CHCI3 with a Perkin-Elmer model 241 polarimeter. Low and high resolution mass spectra were obtained from a VG Micromass ZAB-2F spectrometer. HPLC was carried out on a semipreparative silica gel p-Porasil column attached to a Waters pump model 6000 and monitored with a differential diffractometer detector model R-401. Sephadex LH-20 obtained from Pharmacia was used for gel filtration chromatographies. Silica gel column chromatography, TLC, and PLC were performed on silica gel 60 G. The TLC plates were developed by spraying with 6 N sulphuric acid and heating. All solvents were purified by standard techniques. Anhydrous magnesium sulphate was used for drying solutions. Collection, extraction, and chromatographic separation Laurencia caespitosa was collected in shallow water at low tide at the island of La Graciosa in September 1986. The air-dried alga (5 kg) was extracted with acetone and diethyl ether. The solvents were evaporated in vacuo to afford 62 g of crude extract. This was chromatographed on a silica gel column eluted with a mixture of n-hexanelethyl acetate of increasing polarity, and 50 fractions of 1 L each were collected. The fractions eluted with n-hexanelethyl acetate 60:40 afforded after solvent evaporation the products 6 , 7 , and 8 . 520 CAN. J . CHEM. VOL. 69, 1991 These were chromatographed on Sephadex LH-20 (using as eluent CHC13/methanol/n-hexane 1:1:2) and, after silica gel column chromatography, gave pure 8 (25 mg) and a mixture of 6 and 7 (168 mg). This mixture was chromatographed on HPLC using n-hexanelethyl acetate 70:30, affording the pure products 6 (136 mg) and 7 (30 mg). Can. J. Chem. Downloaded from www.nrcresearchpress.com by 3.236.55.199 on 06/14/20 For personal use only. Compound 6 (8-hydroxy- P-snyderol) Oil, [ a ] ,+8.7 (c 0.172, CHC13). IR (CHC13) v,,,: 3590, 3000, 2970, 1640, and 1370 cm-'. 'H and I3C NMR (200 MHz, CDC13), see Table 1. HRMS mlz: c ~ ~ 285.0069 H ~ (calcd. ~ 285.0677). ~ ~ ~ MS (EI), mlz (relative intensity): 318, 316 (M', not obs.), 285, 283 (1, 1); 233, 231 (7, 7); 219 (19); 201 (10); 173 (6); 149 (14); and 133 (29). Compound 7 (8-hydroq- y-snyderol) Oil, [ a ] ,+ 16.2 ( c 0.136, CHCI,). IR (CHC13)v,,,: 3590, 3000, 2970, 1600, 1410, and 1360 cm-I. 'H and I3cNMR (200 MHz, CDC13), see Table 1. HRMS mlz: C14H2008'Br285.0658 (calcd. 285.0677). MS (EI), m/z (relative intensity): 318,316 (M+, not obs.); 285, 283 (1, 1); 233, 231 (41, 41); 219 (46); 201 (16); 161 (59); and 123 (41). Compound 8 (8-keto-10-dehydrobrominated-P-snyderol) Oil, [ a ] ,+13.6 ( c 0.77, CHC13). IR (CHC13) v,,,: 3570, 3010, 274 nm 3000, 2940, 1660, 1590, and 1420 cm-I. UV (EtOH) A,,: ( E 8900). 'H and I3C NMR (200 MHz, CDC13), see Table 1. HRMS mlz: Cl5H2*O2234.1599 (calcd. 234.1620). MS (EI), mlz (relative intensity): 234 (M+, 2), 219 (4), 201 (9), 175 (7), 161 (lo), 149 (56), 135 (31), and 121 (40). Compound 9 (8-(p-bromobenzoy1)-P-snyderol) To compound 6 (16 mg, 0.05 rnrnol) dissolved in dry pyridine (2 mL) was added p-bromobenzoyl chloride (23 mg, 0.1 mmol) and the mixture was stirred for 5 h at room temperature under Ar. The usual work-up gave an oily residue, which was chromatographed on HPLC to furnish 14 mg of 9; oil, [ a ] , +0.9 ( c 0.22, CHCl,). 'H NMR (CDC13)6: 7.86 (2H, d, J = 8.6 Hz), 7.60 (2H, d, J = 8.6 Hz), 5.87 J=2.8Hz),5.34(1H,s), ( l H , d d , J = 10.6and17.1Hz),5.53(1H,t, 5.19 (lH, dd, J = 1.2 and 17.1 Hz), 5.04 (lH, dd, J = 1.2 and 10.6Hz),4.40(1H,dd, J = 4 . 5 a n d 12.5Hz),2.59(1H,ddd,J = 3.9, 4 and 14.4 Hz), 1.26 (3H, s), 1.23 (3H, s), and 0.85 (3H, s). MS (EI), mlz (relative intensity): 416, 414, 412 (8, 16, 8); 334, 332 (2, 2); 267, 265 (5, 6); 254, 252 (8, 7); and 185, 183 (100, 99). Compound I 0 (8-(p-bromobenzoy1)-y-snyderol) Compound 7 (16 mg, 0.05 mmol) was converted into 10 under the same conditions employed to transform 6 into 9. Purification afforded 13 mg of 10: oil [ a ] ,f89.5 ( c 0.19, CHC13). 'H NMR (CDC13) 6: 7.89(2H,d,J=8.6Hz),7.60(2H,d,J=8.6Hz),5.95(1H,dd,J= 10.7and17.2Hz),5.35 ( l H , d d , J = 2.5and2.8Hz),5.26(1H,dd, J = 1.2 and 17.2 Hz), 5.15 (lH, dd, J = 1.2 and 10.7 Hz), 4.39 (lH, dd, J = 4.4 and 11.9 Hz), 1.65 (3H, s), 1.33 (3H, s), 1.26 (3H, s), and 1.13 (3H, s). MS (EI), mlz (relative intensity): 416, 414, 412 (3,6,3); 334, 332 (20, 19) and 235, 233 (96, 100); and219 (10). B ~ Transformation of 6 into 8 To a solution of 6 (16 mg, 0.05 mmol) in CH2CI2(3 mL) was added Mn02 (10 mg, 0.12 mmol) and the mixture was stirred for 2 h at room temperature, filtered off, the solvent evaporated, and the residue chromatographed by HPLC. The physical and spectral properties of the ketone obtained were identical with those observed for compound 8. Acknowledgments This research was supported by a grant from the Plan Nacional d e Investigacibn ref. FAR88-0500. R . G . and A .P. thank the Ministry of Education for a F.P.I. fellowship. Nat. Prod. Rep. 6 , 613 (1988), and references 1. D. J. FAULKNER. therein. 2. M. CHANG, J. T. VAZQUEZ,K . NAKANISHI, F. CATALDO, D. M. ESTRADA, J. J. FERNANDEZ, A. GALLARDO, J . D. M A R T ~ N , M . NORTE,R. P ~ R E Zand , M. L. RODRIGUEZ. Phytochemistry, 28, 1417 (1989). 3. A. G. GONZALEZ,J. DARIAS,J . D. MARTIN,and C. P ~ R E Z . Tetrahedron Lett. 1249 (1974). 4. (a) B. M. HOWARDand W. FENICAL.Tetrahedron Lett. 41 (1976); (b) J. D. M A R T ~and N J. DARIAS.In Marine natural products. Vol. I. Edited by P. J. Scheuer. Academic Press, New In Marine natural York. 1978. p. 125; (c) K. L. ERICKSON. products. Vol. V. Edited by P. J. Scheuer. Academic Press, New York. 1983. p. 132. Circular dichroic spectroscopy5. N. HARADA and K. NAKANISHI. Exciton coupling in organic stereochemistry. University Science Books, Mill Valley, CA. 1983. J. IWABUCHI, Y. YOKOTA,H. UDA, and K. 6. N. HARADA, NAKANISHI. J. Am. Chem. Soc. 103,5590 (1981). 7. N. C. GONELLA, K. NAKANISHI, V. S. M A R T ~ N and , K. B. SHARPLESS. J. Am. Chem. Soc. 104, 3775 (1982). J. IWABUCHI, H. UDA,and M. OCHI. 8. N. HARADA, Y. YOKOTA, J. Chem. Soc. Chem. Commun. 1220 (1984).