J. CHEM. SOC. PERKIN TRANS. I 1983 Bark Acids of Seven Calophylhm Species (Guttiferae) Upasiri Samaraweera, Subramaniam Sotheeswaran,. and M. Uvais S. Sultanbawa Department of Chemistry, University of Peradeniya, Sri Lanka Sinnathamby Balasubramaniam Department of Botany, University of Peradeniya, Sri Lanka Caloph yllum bracteaturn, C. calaba (var. calaba) ,C. moonii (=C. soulattri), C. th waitesii, C. traperifolium, C. walkeri, and C. zeylanicurn (=C. lankaensis) have been examined for their bark acids. A new acid, calozeylanic acid, has been isolated from three species and has been identified as (2R,3R) -2,3-dimethyl-5- hydroxy-6- (3-methyl butyl) -6- (3,7-dimethylocta-3,6-dienyl) -8-(2-carboxy-1 -phenylet hyl) -2,3,6,7- tetrahydroben~opyran-4~7-dione.Four species were found to be different, having the enantiomer of apetalic acid, isoapetalic acid and a minor acid identified as the structural isomer of the latter.Some 15 acids have been isolated 1-6 from the various parts of several Calophyllumspecies. Most of these acids possess either a phloroglucinol ring system or a cyclohexadienone (a potential phloroglucinol) system fused to a 2,3-dimethyl-chromanone ring (1) and (2). A majority of the acids possess the system (1). We have found in the chemical investigation of the bark acids of seven Sri Lankan endemic Calophyllum species that three of them namely: C. thwaitesii Planch and Trian., C. walkeri Wight; and C. zeylanicum Kosterm. (=C.lankaensis Kosterm) were similar and contained a new acid designated calozeylanic acid (3) 'containing system (2) whereas the four species: C.bracteaturn Thw., C. calaba var. calaba; C. rnoonii (=C.soulatri) and C.frapezifoliurnThw. gave a dif-ferent set of acids identified as the enantiomer of apetalic acid (4a), isoapetalic acid (5a), and a minor acid isomeric with (5a) which has been assigned structure (6a). Both the former acids possess the ring system (1). Several new xanthones have been reported previously from these Calophyllum species. Culozeylanic Acid (3).-The acid was isolated by prepar- ative t.1.c. separation of the Na2C03 fraction of the bark light-petroleum extractives of C. thwaitesii, C. walkeri, and C. zeylanicum. A molecular formula of C35H4606 (M562.3309)has been assigned to calozeylanic acid from an accurate mass measurement of the molecular ion.From the i.r. spectrum, the presence of the following groups were inferred: monosub- stituted phenyl (vmax-753 and 699 cm-I), conjugated carbonyl (1 665) and hydroxy-group (3 500 and 3 200). Me2S04 methylation gave a compound which had a COzMe (6 3.45) and an OMe (6 3.73) group as shown by its 'H n.m.r. spectrum. The 'H n.m.r. spectrum of the acid showed the presence of 5 aromatic protons (6 7-7.4), 13 allylic protons (6 1.4-2.0), 2 olefinic protons and a 1, one-proton low-field signal (6 4.12) which was identified as due to a benzylic proton. Reaction of calozeylanic acid with toluene-p-sulphonic acid gave a lactone(vmax,1 775 cm-') which was identified as a new lactone, calozeylanolactone (7), C25H2R05 (M+ 408.1923), m.p.125-126 "C, .ID + 123.7". The 'H n.m.r. spectrum of this lactone showed the presence of 5 aromatic protons (6 7.2), a triplet at 6 4.5 for a low-field benzylic proton and a doublet at 6 2.98 for 2 protons of the type CH,CO. It also showed the presence of a chelated OH group. The lactone readily lost a C4H9 chain during mass fragmentation (see Scheme) and the mass spec- trum showed the base peak at rn/z 35I. The high resolution of this ion showed it to have the formula CZ1HI9O5.Methylation of the lactone with Me2S04 gave a monomethyl ether (6 3.76 for OMe). The i.r. spectrum of the lactone also indicated the presence of a mono-substituted phenyl ring (760 and 699 cm-I), R')$amp; HO (2) OH 0R2w 0 (41 a;R= H b; R = Me OH 0 (51 (6) a; R=H a;R=H b; R = Me b; R = Me a lactone carbonyl group (I 775 cm ') and a chromanone carbonyl absorption (I 665 cm-I).These data showed that calozeylanolactone has the structure (7). The evidence for the 2,3-.dimethylchromanone ring can be obtained from the chemical shifts of the 2-H and 3-H. They appeared as multiplets at 6 3.73 and 2.1 respectively. The 3-Me protons appeared as a doublet (J 6 Hz) centred at 6 1.10, whereas the C-2 Me protons appeared centred at 6 1.25 as a doublet (J 6 Hz). Due to the position of the phenyl substituent, 2-H and 3-H were found shifted to higher fields in calozeylanolactone (and in calo-zeylanic acid) than in the enantiomer of apetalic acid (4a).The 2-H,3-H coupling constant was found to be 12 Hz indicating a trans-diaxial orientation as in isoapetalic acid 704 J. CHEM. SOC. PERKIN TRANS. I 1983 Table 1. 6 Values of (4a) and (5a) OH 0 2-H 3-H 6-H 7-H 11-H (4a) 4.52 (m,J1.*3.2 Hz) 2.4 (m) 6.56 5.38 (d, J 10 Hz) (d, J 10 Hz) 3.64 (m) (5a) 4.13 (m,J,,212 Hz) 2.5 6.60 5.44 (m) (d, J 10 Hz) (d,J 10 Hz) 3.75 (m) OH 0 OH 0 Ph (71 (6) C5Hll = CH2CH2CHMe2 CwH17 = CH2CH2CMe=CHCH2CH=CMe2 (5a). The nature of the C5Hl, chain in (7) remains to be elucidated. The 'H n.m.r. spectrum of (7) showed the pre- sence of a two-proton triplet for benzylic protons at 6 2.60 and another two-proton triplet at 6 1.73. The signals for Me groups of the side chain appeared along with the chromanone ring Me groups.These and the ready elimination of the C4H9 chain giving rise to the base peak at m/z 351 in its mass spectrum showed that calozeylanolactone (7) has an iso-pentanyl side chain. Calozeylanolactone (7), calozeylanic acid (3), and tetra-hydrocalozeylanic acid, all gave the same base peak at m/z 351 in their mass spectra. The structure of this fragment, (8), and the molecular formula for the acid show clearly that the acid belongs to type (2). The base peak arises from the mole- cular ion by the loss of the chains C4H9 and CIoHl7 and a concomitant lactonisation in the mass spectrometer's probe. The fragmentation patterns are given in the Scheme. The formation of a tetrahydro-derivative when the acid (3) is hydrogenated and the presence of 13 allylic protons in the acid as shown by its 'H n.m.r.spectrum pointed to the assign- ment of calozeylanic acid as (2R,3R)-2,3-dimethyl-5-hydroxy-6-( 3-methylbutyl)-6-( 3,7-dimethylocta-3,6-dienyl)-8-(2-carboxyl-1-phenylethyl)-2,3,6,7-tetrahydrobenzopyran-4,7-dione (3). Calozeylanic acid (3) isolated from the three Calophyllum species were found to be identical ('H n.m.r., co-t.l.c., and i.r.). Etiatitiotiier of Apetalic Acid (4a).-The Na,C03 fraction of the bark light petroleum extractives of C. bracteatum, C. calaba var. calaba, C. moonii, and C. trapezifolium gave a mixture of acids. The mixture was esterified with CH2N2, in each case, and the methyl esters were separated by preparative t.1.c.The more polar ester was found to be a stereoisomer of apetalic acid methyl ester (4b), C23H3006 (M+, 402.2048, re- quires 402.2042). The 'H n.m.r. chemical shift assignments for the protons are given in Table 1. These and other (i.r. and u.v.) data were identical with those of the methyl ester of apetalic 12-H C02CH3 2-Me 3-Me 8-Me 5-OH 2.61 3.52 1.35 1.15 1.45 12.3 (dd) (s) (d,J6.5 Hz) (d,J7 Hz) (s) (s) 2.70 3.57 I .48 1.19 1.43 12.5 (dd) (s) (J6.5 Hz) (J7 Hz) (s) (s) Table 2 a;inkg b;ing c; d C. zeylanicum 3.1 56 0.42 Madugoda sub-montane forest C. walker; 4.15 44 0.45 Ambawela montane forest C. thwaitesii 0.5 10 0.60 Kanneliyalowland forest C. trapezifalium 12.0 64 0.18 Uduwela sub-montane forest C.moonii 6.0 110 0.36 Kanneliyalowland rain forest C. calaba 1.9 15 0.26 Pallai coastal lowland scrub C. bracteaturn 0.6 12 0.58 Kanneliyalowland rain forest acid. However, the specific rotation for the methyl ester isolated was -58 "C and hence it should be the enantiomer of the methyl ester of apetalic acid which has an alD+ 13.4".1deg; Isoapetalic Acid (Sa).-The less polar methyl ester isolated from the above mixture had the formula C23H3006 (Found: M+, 402.2048, requires 402.2042) and was found to be identical with the methyl ester of isoapetalic acid (5b).'O 'H N.m.r. chemical shifts are given in Table 1. A contaminant, (6a), of isoapetalic acid (5a) which was difficult to separate from the latter even after methylation with CH2N2 was found in all the four Calophyllum species: C.bracteatum, C. calaba, C. moonii, and C. trapezifolium. The methyl ester of (6a) had a 'Hn.m.r. spectrum very similar to that of (5b). The main differences being: the C-6 olefinic proton which appeared at 6 6.60 for (5b) appeared at 6 6.45; the chelated OH which appeared at 6 12.5 for (5b) appeared at 6 12.65. These data indicate that (6a) is a structural isomer of isoapetalic acid. Experimental The plant materials were collected in different parts of Sri Lanka. For other details see ref. 3. Table 2 gives the weight of dry plant material (a), weight of the total light petroleum extract (b), percent weight of acid (c), and locality/vegetative type (d).Acid refers to the Na2C03-soluble fraction of the light petroleum extracts. The Na2C03-soluble fractions were puri- fied on a silica gel column and subsequently by preparative J. CHEM. SOC. PERKIN TRANS. I 1983 OH 0 Calozeylanic (3 ) b acid m/z 562 HO2C4 Ph bsol; m/z 351 AH19 Ph tm/z 478 // Tetrahydroca lozey lanic acid m/z 566 Scheme. Mass fragmentations of Calozeylanic acid and its derivatives t.1.c. (p.t.1.c.). The acids were obtained as non-crystallisable gums. Isolation of Calozeylanic Acid (3).-The pure acid (gum) had aJD+12.6 (CHC13) (Found M+,562.3309 C35H460.5 I'eqUireS 562.3294), m/z 562 (7), 494 (25), 476 (20), 450 (14), 439 (21), 413 (13), 407 (23), 354 (24), 353 (loo), 351 (25), and 345 (12); vmas.(CHCIj) 3 540-3 250, I 720, I 710, 1 665, 1 590, 753, and 699 ern-.'; h,,,,.282 (E 12 980), 362 (3 304); 6 (CCI,, 60 MHz), 7.00-7.40 (5 H, m, aromatic), 5.15 (1 H, olefinic), 4.73 (I H, olefinic), 4.12 (1 H, CHPh), 3.60 (1 H, CHO), 2.60 (2 H, m), 2.50 (2 H, m), 1.97 (2 H, m), 1.70 (9 H, CH,C=), and 0.8-1.4 (CH3 and CH2). Calozeylanic acid (3) was isolated from the bark extractives of C. thwaitesii, C. walkeri, and C. reylanicum. Methylation of Calozeylanic Acid.-Calozeylanic acid (250 mg) was dissolved in diethyl ether and was treated with CH2N2. P.t.1.c. separation of the reaction mixture gave the major product as a gum (180 mg), v,,,,. I 735, 1 660, 1 590, 1 460, 1 300, 1 170, 1 020, 850, 760, 780, and 690 cm-I; h,,,,.275 (E 25 290) and 360 (2 810); 6 (CCI,) 7.2 (5 H, aromatic), 5.15 (I H, olefinic), 4.77 (I H, olefinic), 4.07 (1 H, CHPh) 3.81 (3 H,s, OCH3), 3.78 (1 H, m, CH-0), 3.45 (3 H,s, C02Me), 2.8 (3 H, m), 2.5 (4 H, m), 1.3-2.0 (13 H, allylic protons), 1.2 (6 H, CH3), 0.97 (3 H, d, J 5 Hz), and 0.78 (3 H, d, J 5 Hz). Tetrahydrocalozeylanic Acid.-Calozeylanic acid (500 mg) was dissolved in EtOH (10 ml) and was hydrogenated using Pd-C catalyst. The product was purified by p.t.1.c. and the pure product was isolated as a gum, aID +17" (CHCIJ (Found: M+, 566.3606. C35H5006requires 566.3606) ; rn/z 566 (5479, 551 (22), 548 (33), 530 (22), 520 (13), 495 (59, 478 (39,410 (64),409 (85), 353 (91), 351 (lOO), 309 (26), and 107 (38); vmax.(KBr) 3400-3600, 3000-3 100, 1720, 1 600-1 650, 1 330, 1 200,920,760, and 699 cm-'; 6 (CDC13) 7-8.3 (5 H, aromatic), 4.18 (1 H, m, CHPh), 3.80 (1 H, m, CHO), 2.3-3.0 (low field CHI and CH), and 0.7-2.2 (CH, and CH3). Dimethyl Sulphate Methylation of Calozeylanic Acid.-Calozeylanic acid (150 mg) was dissolved in acetone (5 ml). Anhydrous K2C03 (50 mg) and dimethyl sulphate (0.5 ml) were added and the mixture was heated in an oil-bath (80 "C, 6 h). The methylated product was separated by p.t.1.c. to give the pure product (102 mg) as a gum; 6 (CDCIJ 7.0-7.2 (5 H, aromatic), 5.15 (1 H, olefinic), 4.73 (1 H, olefinic), 4.13 (1 H, CHPh2),3.73 (3 H, s, OCH3), 3.78 (1 H, m, CHO), 3.45 (3 H, s, C02CH3), 2.3-2.5 (4 H, m), 2.1-3.0 (13 H, allylic protons), 1.2 (8 H, 2 x CH3, 2 H, aliphatic), 1.0 (3 H, d, J 5 Hz), and 0.82 (3 H, d, J 5 Hz).Calozeylanolactone (7).-Calozeylanic acid (200 mg) was dissolved in benzene (10 ml) and toluene-p-sulphonic acid 706 (25 mg) was added. The reaction mixture was cooled and solvent was evaporated. The residue was dissolved in ether and was shaken with water to remove excess toluene- p-sulphonic acid. The ether layer was dried and solvent was evaporated. The residue was purified by p.t.1.c. to give calozeylanolactone (7) (125 mg). Pure lactone was ob- tained by recrystallisation (light petroleum-MeOH) to give white needles, m.p. 125-126 "C, oilD +123.7 (CHC13) (Found: M+, 408.1923. C25H2805requires 408.2395), m/t 408 (48), 394 (12), 352 (43), 351 (loo), 331 (12), 317 (98), 295 (70), 261 (40), 253 (54), and 149 (98); vmas, (KBr) 3 240, 2 900-3 000, I 775, 1 665, 1 440, 1420, 1 363, 1 325, 1 230, 1 188, 1 157, 1 142, 1 110, 1 030,970, 880,760, and 699 cm-'; Amax, 238 (E 5 406), 290 (4 386), 324 (1 122), and 392 nm (306); 6 (CDCI3)14.3 (1 H, s, OH), 7.20(5 H, m,aromatic),4.50(1 H, t, J 4.5 Hz), 3.73 (1 H, t, J 6 Hz), 2.98 (2 H, d, J 4.5 Hz), 2.6 (2 H, t, J6 Hz), 2.1 (1 H, m), 1.73 (2 H, t, J6 Hz), 1.25 (3 H, d, J 6 Hz), 1.10 (3 H, d, J 6 Hz), and 0.9-1.2 (CH2 and CH,).M~~noiiietltylcalozeylanolactone.-Calozeylanolactone (70 ing) was dissolved in acetone (10 ml) and heated (12 h) after the addition of dimethyl sulphate (0.5 ml) and KzCOJ (100 mg).An hydrous conditions were maintained throughout. Acetone was evaporated and dimethyl sulphate was destroyed by adding ammonia. The product was partitioned between ct her-water.Evaporation of ether gave monomethylcalozeyl- anolactone as a gum, 6 (CDCI,) 7.2 (5 H, s, aromatic), 4.48 (1 H, t, J 5 Hz, COCH, CHPh2), 3.76 (3 H, s, OMe), 2.91 (2 H, d, J 5 Hz, COCH,CHPh2), 2.63 (2 H, m, side chain protons), 2.1 (I H, m), 1.22 (3 H, d, J 6 Hz, CH3), 1.10 (3 H, d, J 6 Hz, CHJ, and 1.1 (6 H, 2 CH,). lwlntioii of the Enantionier of Apetalic Acid (4a).-The Na,C03-soluble fractions of the bark extractives of C. hr.crc.tcntiriii, C. calaba, C. nioonii, and C. trapezifolium gave a mixture of acids which were separated as the methyl esters obtained by esterifying the acid mixture with CH2N2.P.t.1.c. separation gave the enantiomer of apetalic acid methyl ester as a gum, dD -58" (Found: M+, 402.2048. C23H3006 requires M, 402.2042), iii/z 402 (19), 388 (30), 387 (loo), 375 (8), 365 (8), 359 (6), 329 (lo), 327 (9), 289 (9), 285 (6), and 271 (6); vrnns.(CHC13) 3 200-3 500, 1 730, 1 650, 1 580, 1 460, 1440, 1400, 1380, 1360, 1340, 1 280, 1200, 1 160, 1120, I 020, 920, 890, 750, and 660 cm-l; h,,;,,. (EtOH) 228 (log E 3.59), 267 (4.62), 312 (4.02), and 363 nm (3.32); 6 (CCI,) 12.3 (chelated OH), 6.56 (1 H, d, J 10 Hz), 5.38 (1 H, d, J 10 Hz), 4.52 (I H, m, J 3.2 Hz), 3.64 (1 H, m), 3.52 (3 H, s, J. CHEM. SOC. PERKIN TRANS. 1 1983 CO2CH3),2.61 (2H, dd), 2.4 (1 H, m), 1.45 (6 H, s, 2CH3), 1.35 (3 H, d, J6.54 Hz), and 1.15 (3 H, d, J7 Hz).Isolation of Isoapetalic Acid (Sa).-P.t.l.c. separation of the CH2N2 reaction product of the above acid mixture gave methyl ester of isoapetalic acid as a gum, aID -91' (lit.,lo -68.3') (Found: M+,402.2048. C23HSOOs requires 402.2042), nz/z 402 (2179, 388 (25), 287 (loo), 359 (3), 329 (I), 313 (7), 285 (15), 271 (12), 257 (ll), 243 (9), and 229 (15); vmx. (CHCI,) 2 900,l 740, 1 660-1 640, 1 593, 1 475, 1 465, 1 415, 1400, 1369, 1307, 1280, 1 176, 1 160, 1 140, 1097, 1070, I 025, and 907 cm-'; Lmx. (EtOH) 228 (log 4.01), 268 (4.42), 274 (4.63), 301 (4.02), 315 (4.01), and 371 nm (3.62); 6 (CCI,) 12.50 (1 H, s, chelated OH), 6.60 (1 H, d, J 10 Hz),5.44 (1 H, d, J 10 Hz), 4.13 (1 H, m, J 12 Hz), 3.75 (1 H, m), 3.57 (3 H, s, C0,CH3), 2.70 (2 H, dd), 2.5 (1 H, m), 1.48 (3 H, d, J6.5 Hz), 1.43 (6 H, s, 2CH3), 1.19 (3 H, d, J 7 Hz), and 0.88 (3 H).The identity of the methyl ester of isoapetalic acid was confirmed by comparison with an authentic sample and by preparation of its acetate. Acknowledgements We thank the World Health Organisation for financial support and Dr. P. Bladon for high-resolution mass spectral data. References 1 U. Samaraweera, M.Phi1. Thesis, 1982, University of Peradeniya,Sri Lanka. 2 G. H. Stout, M. M. Krahn, and G. D. Breck, Tetrahedron Lett., 1968,3285. 3 G. H. Stout and G. D. Breck, Tetrahedron Lett., 1970, 1999. 4 J. Gantier, G. Kunesch, and J. Polonsky, Tetrahedron Lett., 1972,2715. 5 G. H. Stout and K. D. Sears,J. Org. Chem., 1968,33,4185. 6 G. H. Stout, G. K. Hickernell, and K. D. Sears, J. Org. Chern., 1968,33,4191. 7 U. Samaraweera, S. Sotheeswaran, and M. U. S. Sultanbawa, Tetrahedron Lett., 1981, 5083. 8 S. P. Gunasekera, S. Sotheeswaran, and M.U. S. Sultanbawa,J. Chem. Soc., Perkin Trans. I, 1981, 1831. 9 M. U. S. Sultanbawa, Tetrahedron, 1980, 36, 1465. 10 E. Guerreiro, G. Kunesch, and J. Polonsky, Phytochemistry, 1971, 10, 2139. Received 2nd June 1982; Paper 2/890
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