Photochemistry, Vol. 30, No. 6, pp. 1977-1982, 1991 Printed in Great Britain.

0031-9422/91 S3.00 + 0.00 © 1991 Pergamon Press pic

ELASCLEPIAL AND OTHER TETRACYCLIC DITERPENOIDS FROM ELAEOSELINUM ASCLEPIUM M A N U E L G R A N D E , BALBINO M A N C H E Ñ O * and

M A R I A J. S A N C H E Z *

Departamento de Química Orgánica, Facultad de Ciencias Químicas, Universidad de Salamanca, Plaza de los caidos 1, E-37008 Salamanca, Spain; * División de Química Orgánica, Departamento de Ciencias Ambientales y Recursos Naturales, Universidad de Alicante, Apartado 99, E-03080 Alicante, Spain (Received in revised form 26 October 1990)

Key Word Index—Elaeoselinum asclepium; Umbelliferae; roots; beyerane, kaurane and atisane diterpene acids; elasclepic acid; diterpene aldehydes; elasclepial.

Abstract—The neutral components of the benzene extract from Elaeoselinum asclepium roots were identified as elasclepiol, erythroxylol A and four new beyerane diterpene aldehydes, enf-14/8-tigloyloxybeyer-15-en-19-al (elasclepial) and its 14j8-angeloyloxy- isomer, and eni-3j8-hydroxy-14/?-tigloyloxybeyer-15-en-19-al (3/3-hydroxyelasclepial) and its acetate. These axial aldehydes were oxidized by atmospheric oxygen to the respective carboxylic acids and decarbonylated to give hydroperoxides. Four new tetracyclic diterpene acids have been isolated from the acid fraction of the benzene extract and identified as en£-14/?-angeloyloxybeyer-15-en-19-oic acid, ent-beyer-15-en-19-oic acid, entatis-16-en-19-oic acid and ent-18-angeloyloxykaur-16-en-19-oic acid. The structures of the compounds described were established by spectroscopic methods and chemical correlations.

INTRODUCTION

Elaeoselinum asclepium (L.) Bertol subsp. asclepium, also known as Elaeoselinum asclepium (L.) Bertol var. hispanicum (Lange) Font Quer, E.hispanicum (Lánge) Pau and E. meoides (Desf) KocK ex DC. var. hispanicum [1], is native to the Comunidad Valenciana, East Spain. Although diterpenoids are not common in Umbelliferae [2, 3], some tetracyclic diterpenoids have been identified in Elaeoselinum species such as E. gummiferum [4-6], E. foetidum [7], E. tenuifolium [8], and in our previous communication [9], we established the structures of the main components of a root extract from E. asclepium as elasclepiol (6) and elasclepic acid (7). We now report the isolation and structural identification of four new natural beyerane diterpenoids (1-4), as well as erythroxylol A (5) and five tetracyclic diterpene acids with beyerane (7,15, 16), atisane (17) and kaurane (18) skeletons. Although the acids 16-18 had been detected previously as natural products [4,10,11], they were isolated and described as methyl esters after treatment of crude extracts with diazomethane. As far as we know, this is the first time that the acids 15-18 are described as natural products.

RESULTS AND DISCUSSION

The benzene extract from the roots of E. asclepium was separated into neutral and acid fractions as described previously [9]. The neutral fraction showed in TLC the presence of five predominant spots, with elasclepiol (6) as the most abundant cpmponent. The less polar neutral fractions were comprised mainly of 1, for which the name elasclepial was proposed as it showed aldehyde absorption bands in its IR and XH NMR spectra. However, our first attempts to isolate 1 were unsuccessful because it

decomposed in the chromatographic column to give two substances, compound 8, with nearly the same Rf as 1 but lacking the aldehyde function, and a more polar substance identified as elasclepic acid (7). The IR spectrum of 8 was quite similar to that of elasclepiol (6) and the *H NMR spectra of both substances showed the same signals slightly shifted, except for the AB system of the primary methoxyl group which was absent from the spectrum of 8, so the possibility of a normal dismutation of aldehyde 1 into the carboxylic acid 7 and the alcohol 6 was discounted. The molecular ion (m/z 388) observed in the mass spectrum of 8 was two units higher than that of elasclepiol. This led us to suspect that 8 is an hydroperoxide with a structure like that of elasclepiol in which the methoxyl group is replaced by an - O O H group. The presence of the hydroperoxide group in 8 was demonstrated by the oxidation of a KI soln (I 2 detected with starch), and also by the presence of fragments at m/z 371 [ M - O H ] + and 355 [ M - O O H ] + in the mass spectrum. We assigned to this oxidation product the structure 8, with the hydroperoxide group axial at C4, as deduced from deshielding effect observed on the angular methyl Me-20 («50.15) as compared with that of elasclepiol [12]. To isolate pure 1 the neutral components of a fresh benzene extract from fresh roots was subjected to flash chromatography. This allowed us to isolate pure 1 and 2, the physical constants of which were measured immediately after evaporation of the chromatography solvent in vacuo at room temperature. The IR spectrum of 1 showed absorption bands at 2710 and 1700 c m - 1 characteristic of an axial aldehyde. The stretching vibration of the carbonyl group is lowered by interaction with the methyl group Me-20. The XH NMR spectrum of 1 was quite similar to those of elasclepiol and

1977

1978

M. GRANDE et al.

hydroperoxide 8. In elasclepial the signals of a primary hydroxyl group were also absent but were replaced by a singlet at 59.78 of an axial aldehyde [13]. The axial configuration of the aldehyde explains the shielding of the

2U

—

U

1

14

1

J

>16

J is

** 18

"R2 19

R1 H H

1 2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 19

OAc OH H H H H OH

0

R3 OTgl OAng OTgl OTgl H

R2 CHO CHO CHO CHO CH2OH CH2OH COjH OOH CH2OH OCH2 COjH C02Me

CMe2

OAc OAc OH OH H H H

OTgl OTgl OTgl OTgl OTgl OTgl OTgl OTgl OTgl OAng H H

COjH C02Me C02H C0 2 H C02Me

Tgl = COCMe=CHMe

Ang = COCMe=CHMe

AngOCH 17 R = C0 2 H 20 R = C02Me

18 21

R = C0 2 H R = C02Me

methyl group Me-20 that absorbs at 50.63 (¿0.75 in 1 [9]). Also, the observed shielding effect of the aldehyde on the Me-20 carbon atom (in the isomer 2, see below) agrees with the axial configuration of the formyl group [14]. Aldehyde 2 could be isolated from the first fractions of the same chromatography that allowed the isolation of elasclepial. Substance 2 had an IR spectrum nearly identical to that of 1 and the *H and 13 C NMR showed signals of an angelate ester instead of a tiglate ester: 8 6.03 (M,qq,J = 7.3,1.6 Hz, H-3'), 1.93 (3H, dd, J = 7.3,1.6 Hz, H-4'), 1-83 (3H, quint, J = 1 . 6 H z , H-5'), 168.2 (s, C-l'), 137.4 (d, C-3'), 129.5 (s, C-2'), 20.9 (q, C-5'), 15.9 (q, C-4') [15]. The mass spectrum confirmed that 2 is the angelate isomer of 1 and both substances on reduction with LiAlH 4 gave the same diol as the reduction product from elasclepiol [9]. The spectra of aldehydes 1 and 2, their reactivity and the chemical correlations with elasclepiol and elasclepic acid let us propose for these aldehydes the structures entMjg-tigloyloxy- and ent-14/8-angeloyloxybeyer-15-en-19al respectively. The lability of the axial diterpene aldehydes had been reported previously. The autooxidation can take place through a radical mechanism as shown in Scheme 1 [12, 16, 17]. Compound 3, in common with the preceding aldehydes, showed IR absorption bands of alkene, aldehyde and conjugated ester groups, but in addition gave absorption bands for a saturated ester (acetate) at 1720 and 1240 cm" 1 . The mass spectrum confirmed the presence of the above functional groups: 442 [ M ] + (C 2 7 H 3 8 0 5 ), 355 [M-CO-HOAc]+, 343[M-TglOH]+,254[M-CO - H O A c - T g l O H ] + , 83 [Tgl] + . The signals of an acetoxy group and its geminal proton were clearly observed in the ' H N M R spectrum of 3 at <5 2.04 (3H, s) and 4.65 (1H, dd, J = 9 . 3 and 7.8 Hz) respectively. Signals of an aldehyde, a secondary tigloyloxy group, a cis-disubstituted alkene and three methyl singlets were also observed. These data suggested for the aldehyde 3 a beyerane skeleton like that of elasclepial, with a secondary acetoxyl group that could be placed equatorial on C-l, C-3, C-7 or C-1.2. The 1 3 C N M R spectrum let us assign the position of the acetoxy group: The chemical shift for the carbon atoms of the rings B, C and D of 2 and 3 are very similar, so the C-12 and C-7 can be discounted and also C-l because in this position the acetoxyl group, very close to C-10 and to C-ll, should modify the absorption of these carbon atoms. Consequently, we propose for aldehyde 3 the structure ent-Zf}acetoxy-14/J-tigloyloxybeyer-15-en-19-al, also confirmed by chemical correlations as shown below.

1

1

•-

R3C

CO

R,C

CO

" \

OO*

*• I

R «M>3C

C0 i ^3JH 1

R3C—-C02H ••

R3C*

C

O

02

R3C

OO'

Scheme 1. A radical mechanism for auto-oxidation.

R3C—OOH

(x2)

Tetracyclic diterpenoids from Elaeoselinum asclepium Compound 4 is also an aldehyde with an unsaturated ester group and a free hydroxyl group as deduced from the spectra. Both the IR and the ' H N M R spectra of 1 and 4 were nearly identical. The most important differ-' ence is the presence of an absorption band at 3480 cm" 1 and a signal at 5 3.13 (1H, m) of one proton geminal to a secondary hydroxyl group. The deshielding observed for the Me-18 singlet (51.25) as compared with that signal in 1 (¿1.06) suggest that the hydroxyl group should be placed at C-3. This assignment was in fact confirmed. Thus sodium borohydride reduction of the aldehyde gaves the diol 9, which on treatment with 2,2-dimethoxypropane afforded the isopropylidene derivative 10. The relationship of 3 and 4 was confirmed by acetylation of the latter substance which gave an acetate identical to natural 3. A substance with nearly the same Rf as 4, but esterified by angelic acid as deduced from the 1 H NMR spectra, was also detected. This substance should presumably be the angelate isomer of 4 but we could not purify it. Throughout the purification of the above aldehydes, we could detect by TLC the formation of more polar substances. These substances were isolated and identified from their spectral data as the carbóxylic acids 11 and 13, i.e. the oxidation products of the aldehydes 3 and 4 respectively. The methyl ester of 13 (diazomethane) esterified with acetic anhydride-pyridine, gaves a substance identical to the methyl ester of 11. Crude elasclepic acid (7), the main component of the acidic fraction [9], was contaminated with a minor component (15) with a similar Rf. This substance was isolated from crude 7 and purified by flash chromatography on 8% AgN0 3 -silica gel. The spectroscopic data of 15 were very similar to those of elasclepic acid: The *H and 1 3 CNMR showed characteristic signals of ah angeloyloxy group [15], and consequently we concluded that compound 15 is the angelate isomer of elasclepic acid. The acids 16 and 17, both less polar than elasclepic acid, ran very close each other on TLC. These two substances were also isolated by flash chromatography on 8% AgN0 3 -silica gel. Compound 16 showed IR absorptions bands of a carboxyl group (3600-3200, 1685 c m - 1 ) and a cis disubstituted double bond (3025, 1580, 740cm" 1 ). The ' H N M R spectrum was very similar to that of erythroxylol A (5) [18] previously isolated from the neutral fraction. However, as compared with erythroxylol A, the primary alcohol signals were absent from the spectrum of 16 and a wide signal assigned to a carboxyl group appeared at <5 10. The singlets of the methyl groups of 16 and 5 absorbed at similar field but the axial C-4 methyl of 16 [1.26 (3H, s, H-18)] was deshielded (<5 0.29) as compared with 7. These spectral data suggested for compound 16 the structure of the carboxylic acid resulting from the oxidation of erythroxylol A. This structure was confirmed by chemical correlation. Thus, treatment of 16 with diazomethane gave the methyl ester 19 [16] which was reduced with LiAlH 4 -ether to give erythroxylol A (5) [18]. The IR spectrum of 17 showed absorptions bands of a carboxylic acid (3600-3200, 1685, 1265) and a methylidene group (3060, 1650sh, 870). The ' H N M R spectrum showed signals of one exo-methylene group [54.74 (1H, q, J = 2 H z ) and 4.58 (1H, q, J = 2Hz)] and two methyl groups [51.26 (3H, s) and 0.91 (3H, s)j. The mass spectrum showed a molecular ion at m/z 302, in agreement with the molecular formula C 2 0 H 3 0 O 2 and frag;

1979

ments at 287 [ M - M e ] + , 259 [ 2 8 7 - C 2 H 4 ] + and 257 [M — C 0 2 H ] + . All these data suggested that compound 17 is a tetracyclic diterpene acid with kaurane, atisane or phyllocladane skeleton, and with an axial carboxylic acid at C-18 or C-19, which deshields the methyl groups on C-4 or C-10. The atisane skeleton was assigned to compound 17 on the basis ofthe *H NMR spectrum which showed signals for an exo-methylene group [¿4.74 (1H, q, J = 2 Hz) and 4.58 (1H, q, J = 2 Hz)] and for the H-12 proton [5 2.28] characteristic ofthe atisane skeleton. The H-13 protons in phyllocladanes and kauranes (H-12 in atisanes) absorb at ca 5 2.65 [6, 19]. The carboxyl group was assigned to position C-19 (axial) on the basis of the IR absorption bands ofthe methyl ester 20 which lacks the strong band at 1245 c m - 1 , characteristic of equatorial methoxycarbonyl groups [20]. The physical and spectral data of 20 were identical to those reported for this methyl ester previously described in the literature [4], and confirms the proposed structure for 17. Compound 18 was isolated by column chromatography from the most polar fraction of the acid components. The acid 18, C 2 5 H 3 8 0 3 ([M] + at m/z 400) must also be a tetracyclic diterpene acid with an exo-cyclic methylene group esterified by angelic acid as deduced from the IR and ' H NMR data. The presence of a signal at 5 2.64 (1H, br s) assigned to H-13, suggested for compound 18 a kaurane skeleton. The 13 C NMR signals, assigned by comparison with the spectra of known tetracyclic diterpenes [21, 22], were also in agreement with the kaurane skeleton for compound 18. Apart from the exo-cyclic methylene group and the carboxylic acid signals, the *H NMR spectrum of this compound showed one methyl singlet at 50.99 as well as an AB system (ca 5 4.77 assigned to an angeloyloxy-methylene group. According to the observed chemical shifts, the angeloyloxy group must be placed on C-18, equatorial [23], and the carboxylic acid must be axial at C-4 [20]. The methyl ester 21 (diazomethane) confirmed the proposed structure for 18. This methyl ester had been described previously [11].

EXPERIMENTAL Mps: uncorr; NMR: 1W, 200 MHz and 60 MHz; 13C, 50.3 MHz in CDC13 with TMS as int. standard; EIMS: direct inlet probe at 70 eV. Extraction and isolation. The plant material was collected at Puerto de Albaida, Alicante, Spain and the dried roots were extracted with C6H6 in a Dean-Stark apparatus. The C6H6 extract was separated into neutral and acid frs as previously described [9]. The neutral components (28.3 g, 1.8% wt of dried roots) were isolated and then chromatographed on silica gel (Merck ref. 7733) [9]. After chromatography of the enriched frs and further crystallization, pure 3 (3/?-acetoxyelasclepial, 67 mg), 4 (3^-hydroxyelasclepial, 127 mg) and 5 (erythroxylol A, 19 mg) were isolated. From the less polar frs of the main chromatography (eluent, hexane-EtOAc, 97:3), 5 frs (3.8 g) contained one predominant substance which showed 'H NMR signals of an aldehyde. However, all attempts to isolate it by conventional CC failed and only the oxidation products 7 (elasclepic acid) and 8 (0.3 g) could be purified. To isolate the natural aldehydes and to avoid their autooxidation, fresh roots of E. asclepium (590 g) were extracted with C6H6 by maceration at room temp, for 2 weeks and the organic

1980

M. GRANDE et al.

layer coned in a rotatory evaporator at room temp. (16.5 g, 442 [M] + (1), 355 (16), 343 (14), 149 (11), 148 (11), 147 (10), 133 2.8%). (17), 123 (11), 121 (19), 119{10), 109 (15), 108 (9), 105 (11), 97 (15), + + Flash chromatography of a part of the C6H6 extract (8 g) on 91 (7),'83 [Tgl] (100), 55 [Tgl-CO] (16), 43 (12), 41 (4), 29(3). silica gel (Merck ref. 7734, 300 g) under N 2 pressure, with ent-3P-Hydroxy-14fl-tigloyoxylbeyer-15-en-l9-al (4). Rf 0.24 hexane-EtOAc (19:1) as eluent, allowed us to isolate frs contain- (hexane-EtOAc, 4:1); I R v ^ c m " 1 : 3480, 3030, 2920, 2840, ing the aldehydes. Further flash chromatographies with TLC 2730,1695,1640,1440,1375,1340,1265,1135,1070,1045,1005, silica gel as stationary phase (Merck 60) and hexane-EtOAc 970, 780,730; 'H NMR (60 MHz): 5 9.82 (1H, s, H-19), 6.83 (1H, mixts as eluent, led to the isolation of pure 1 (60 mg) and 2 br q, 7 = 6 Hz, H-3'), 5.65 (1H, br d, 7 = 6 Hz, H-16), 5.46 (1H, br d, (15 mg). 7 = 6 Hz, H-15), 4.55 (1H, br s, H-14), 3.13 (1H, m, H-3), 1.82 (3H, Frs containing the aldehydes 3 and 4 also reacted with br s, H-5'), 1.75 (3H, br d, 7 = 6 Hz, H-4'), 1.25 (3H, s, H-18), 0.93 atmospheric oxygen to give the oxidation products 11 (50 mg) (3H, s, H-17), 0.70 (3H, s, H-20). and 13 (127 mg) after chromatography with C6H6-Me2CO ent-14¡l-Tigloyloxy-l9-norbeyer-l5-en-19-hydroperoxide (8). mixts. Rj 0.62 (hexane-EtOAc, 4:1); Mp 175-177° (hexane); positive 1 A part (15 g) of the acid fr. (34.8 g, 2.2% wt of dried roots) was hydroperoxide test (aq. KI-starch); IRv^Jcm" : 3335, 2940, chromatographed on silica gel (Merck ,ref. 7733) with 2840, 1670, 1640, 1445, 1370, 1340, 1270, 1140, 1080, 1040, 780, 1 hexane-EtOAc (9:1) as eluent. From the less polar frs, we 730; I R v S f ' c n T (4%): 3520, 2940, 2840, 1685, 1640, 1440, 3 1 -3 isolated compounds 7 (116 mg), 15 (25 mg), 16 (34 mg) and 17 1360, 1260, 1130, 1070, 860; IR v™? cm" (5 x 10 M): 3520, 3500-3200; 'H NMR (60 MHz): 5 7.25 (1H, br s, -OOH), 6.81 (85 mg) by CC on 8% AgN03-silica gel (Merck 60 PF 254 ) under N 2 pressure (1.5 atm). Compound 18 (60 mg) was separated from (1H, brq,J = 6 Hz, H-3'), 5.70(lH,fcrd, 7 = 6 Hz, H-16), 5.41 (1H, polar frs using CC on silica gel (Merck 60, 0.063-0.2 mm). The br d, 7 = 6 Hz, H-15), 4.52 (1H, br s, H-14), 1.83 (3H, br s, H-5'), 1.76 (3H, br d, 7 = 6 Hz, H-4'), 1.23 (3H, s, H-18), 0.91 (3H, s, Hsolid compounds were purified by crystallization in MeOH. + ent-l4P-Tigloyloxybeyer-15-en-19-al (1). Rf 0.62 (hexane-17), 0.87+ (3H, s, H-20); MS m/z+ (rel. int.): 388 [M] (2),+371 [M -1 EtOAc, 4:1); oil; [a] D +17° (CHC13; cZ2); IR v™" cm : 3040, - OH] (3), 355 [M - OOH] (10), 288 [M - TglOH] (1), 272 254 (12), 213 (6), 187 (10), 145 (17), 133 (16), 2920,2840,2710,1700,1645,1440,1370,1260,1135,1070,1050, (8), 271 (6), 255 (16), + + 2 855, 785,765, 730; 'HNMR (60 MHz): ¿9.78 (1H, s, H-19), 6.83 105 (13), 83 [Tgl] (100), 55 [Tgl-CO] ( 8)> 43 (20). (1H, br q, 7 = 6 Hz, H-3'), 5.70 (1H, br d, 7 = 6 Hz, H-16), 5.45 (1H, ent-14p-Tigloyloxybeyer-l5-en-3p,l9-dioí (9). I R v ^ c n T 1 : br d, 7=6 Hz, H-15), 4.56 (1H, br s, H-14), 1.82 (3H, br s, H-5'), 3420, 3030, 2930, 2840,1695,1640,1450,1375,1340,1260,1130, 1.76 (3H, br d, 7=6 Hz, H-4'), 0.97 (3H, s, H-18), 0.93 (3H, s, H- 1070,1050,970, 850,785,730; 'H NMR (60 MHz): S 6.83 (1H, br 17), 0.63 (3H, s, H-20). q, J• = 6 Hz, H-3'), 5.63 (1H, brd,J = 6 Hz, H-16), 5.43 (1H, br d, J e.nt-\4P-Angeloyloxybeyer-l5-en-19-al (2). Rf 0.65 (hexane-= 6 Hz, H-15), 4.52 (1H, br s, H-14), 4.20(1H, br d, J = 10.5 Hz, EtOAc, 4:1); mp 150° (hexane); [a] D +13.8° (CHC13, cl.7); H-19a), 3.30 (1H, d, J = 10.5 Hz, H-19b), 1.83 (3H, br s, H-5'), 1.76 I R v ^ c m ': 3040, 2945, 2920, 2845, 2705, 1700, 1645, 1455, (3H, br d, J = 6 Hz, H-4'), 1.20 (3H, s, H-18), 0.93 (3H, s, H-17), 1380, 1360, 1235, 1175, 1085, 1050, 860, 785, 735; 'HNMR 0.73 (3H, s, H-20). (200 MHz): ¿9.80 (1H, s, H-19), 6.04 (1H, qq, 7 = 7.2 Hz, 7 Isopropylidene derivative (10). To a soln of 9 (100 mg) in dry • = 1.5 Hz, H-3'), 5.67 (1H, br d, 7 = 6 Hz, H-16), 5.45 (1H, br d, 7 Me2CO (10 ml), 2,2-dimethoxypropane (0.3 ml) and a small =6 Hz, H-15), 4.61 (1H, br s, H-14), 1.96 (3H, dq, J = 12,1.5 Hz, crystal of p-TsOH were added. A small crystal of NaHC0 3 was H-40,1.87 (3H, quint, 7 = 1.5 Hz, H-50,1.00 (3H, s, H-18), 0.97 (3H, added after 90 min and the soln filtered and coned to give 10 s, H-17), 0.64 (3H, s, H-20); 13C NMR: 6 205.6 (d, C-19), 168.3 (s, (100 mg) which was purified by chromatography on silica gel C-l'), 137.2 (d, C-3'), 133.9 (d, C-16), 131.9 (d, C-15), 128.3 (s, C-2'),(hexane-EtOAc, 3:2); I R v ^ c m " 1 : 3060, 2940, 2860, 1710, 94.1 (d, C-14), 56.0 (d, C-5), 52.9 (s, C-8), 51.6 (d, C-9), 48.2 (s, C- 1655, 1460, 1385, 1265, 1230, 1200, 1150, 1080, 1060, 870, 840, 13), 48.2 (s, C-4), 38.9 (t, C-l), 37.6 (s, C-10), 34.2 (t, C-3), 32.3 (t, C-795, 740; XH NMR (60 MHz): 5 6.83 (1H, br q, J = 6Hz, H-3'), 12), 31.4 (t, C-7), 24.4 (q, C-18), 20.6 (q, C-5'), 19.9 (í, C-6), 19.3 (q,5.68 (1H, br d, 7 = 6 Hz, H-16), 5.45 (1H, br d, 7 = 6 Hz, H-15), C-17), 18.8* (£, C-ll), 18.4* (£, C-2), 15.8 (q, C-4'), 14.8 {q, C-20); 4.56 (1H, br s, H-14), 4.05 (1H, d, 7 = 10.5 Hz, H-19a), 3.23 (1H, d, MS m/z (rel. int.): 384 [M] + (1), 356 [ M - C O ] + (1), 301 [M 7 = 10.5 Hz, H-19b), 1.83 (3H, br s, H-5'), 1.76 (3H, br d, 7=6 Hz, -Ang] + (1), 284 [M-AngOH] + (4), 269 [284-Me] + (1), 256 H-4'), 1.43 (3H, s, H-l'), 1.36 (3H, br s, H-l"), 1.18 (3H, s, H-18), (2), 255 (2), 241 (1), 213 (1), 199 (\\ 185 (2), 173 (1), 159 (2), 145 (4), 1.00 (3H, s, H-17), 0.93 (3H, s, H-20). 131 (5), 119 (5), 105 (8), 93 (7), 83 [Ang]+ (100), 67 (5), 55 [Ang ent-3fi-Acetoxy-14f}-tigloyloxybeyer-15-en-19-oic acid (11). -CO] + (42), 41 (10), 29 (9). I R v ^ c r r T 1 : 3930, 3050, 2960, 2935, 2850, 1725, 1695, 1645, ent-3P-Acetoxy-l4p-tigloyloxybeyer-lS-en-l9al (3). Rf 0.46 1450,1375,1365,1245,1160,1130,1075,1040,970,935,855,800, (hexane-EtOAc, 4:1); mp 169-171° (hexane); I R v ^ c m - 1 : 735; 'HNMR (60MHz): ¿6.80 (1H, br q, 7=6Hz, H-3'), 5.60 3050, 2960,2840,2720,1720,1700,1695,1645,1440,1375,1265, (1H, br d, 7 = 6 Hz, H-16), 5.33 (1H, br d, 7 = 6 Hz, H-15), 4.53 1240,1130,790, 725; l H NMR (200 MHz): ¿ 10.05 (1H, s, H-19), (1H, br s, H-14), 4.46 (1H, m, H-3), 2.06 (3H, s, H-2"), 1.80 (3H, br 6.80 (1H, qq, 7 = 6,0.8 Hz, H-3'), 5.58 (1H, dd, 7 = 5.8,0.8 Hz, fi- s, H-5'), 1.73 (3H, br d, 7=6 Hz, H-4'), 1.23 (3H, s, H-18), 0.96 ló), 5.45 (lH,dd,7=5.8,1.1 Hz, H-15), 4 . 6 5 ( ^ ^ , 7 , ^ = 9 . 3 Hz, (3H, s, H-17), 0.76 (3H, s, H-20). JXB=7.8 Hz, H-3), 4.54 (1H, br s, H-14), 2.04 (3H, s, H-2"), 1.79 Methyl ester (12) (CH2N2/Et20). I R v ^ c m " 1 : 2950, 2930, (3H, br s, H-5'), 1.77 (3H, dd, 7 = 6, 0.8 Hz, H-4'), 1.06 (3H, s, H- 1725 (br), 1645, 1445, 1370, 1240, 1190, 1155, 1080, 1045, 1030, 18), 0.93 (3H, s, H-17), 0.68 (3H, s, H-20); 13C NMR (50.3 MHz): 975, 735; 'HNMR (60MHz): ¿6.82 (1H, br q, 7 = 6Hz, H-3'), 5 204.4 (d, C-19), 170.4 (s, C-l''), 168.3 (s, C-l'), 136.9 (d, C-3'), 5.63 (1H, br d, 7 = 6 Hz, H-16), 5.42 (1H, br d, 7 = 6 Hz, H-15), 134.1 (d, C-16), 131.8 (d, C-15), 128.8 (s, C-2'), 94.1 (d, C-14), 78.6 4.54 (1H, br s, H-14), 4.46 (1H, m, H-3), 3.65 (3H, s, C02Me), 2.04 (d, C-3), 56.9 (d, C-5), 52.8 (s, C-8), 51.8 (s, C-4), 51.3 (d, C-9), 48.3(3H, s, H-2"), 1.82 (3H, br s, H-5'), 1.75 (3H, br d, 7 = 6 Hz, H-4'), (s, C-13), 37.0 (s, C-10), 36.6 (t, C-l), 32.3 (t, C-12), 31.0 (í, C-7), 1.23 (3H, s, H-18), 0.93 (3H, s, H-17), 0.68 (3H, s, H-20). 24.1 ((, C-2), 21.0 (q, C-2"), 20.2 (q, C-18), 19.3 (t, C-l 1), 19.2 {q, C- ent-3fl-Hydroxy-l4fi-tigloyloxybeyer-15-en-19-oic acid (13). 17), 16.3 (q, C-20), 14.3 (q, C-4'), 12.1 (q, C-5'); MS m/z (rel. int.): 'H NMR (60 MHz): 8 6.83 (1H, br q, 7=6 Hz, H-3'), 5.70 (1H, br d, 7 = 6 Hz, H-16), 5.43 (1H, br d, 7 = 6 Hz, H-15), 4.53 (1H, br s, H-14), 3.16 (1H, m, H-3), 1.80 (3H, br s, H-5'), 1.76 (3H, br d, 7 = 6 Hz, H-4'), 1.36 (3H, s, H-18), 0.90 (3H, s, H-17), 0.70 (3H, s, H-20). * Assignments may be reversed.

Tetracyclic diterpenoids from Elaeoselinum asclepium Methyl ester (14) (CH 2 N 2 /Et 2 0). I R v ^ c n T 1 : 3520, 3330, 2940,2820,1700,1640,1450, 1370,1260,1230,1150,1005,1000, 970,850,790,730; ' H NMR (60 MHz): 5 6.83 (IH, br q, J = 6 Hz, H-3'),5.66(IH,brd,J = 6Hz,H-16), 5.43(IH,brd,J = 6Hz,H15), 4.52 (IH, br s, H-14), 3.66 (3H, s, C0 2 Me), 3.20 (IH, m, H-3), 1.83 (3H, br s, US'), 1.76 (3H, br d, J = 6 Hz, H-4'), 1.36 (3H, s, H-18), 0.93 (3H, s, H-17), 0.60 (3H, s, H-20). ent-14P-Angeloyloxybeyer-l5-en-19-oic acid (15). Rf 0.43 (hexane-EtOAc, 4:1); Mp 185° (MeOH); [a] D -18.9° (CHC1 3 ; cl.6); I R v S i c m " 1 : 3400-2200, 3015, 2920, 2830, 1680 (br), 1440, 1370, 1255, 1225, 1135, 1040, 850, 780, 725; ' H N M R (60 MHz): 56.00 (IH, br q, J = 7 Hz, H-3'), 5.70 (IH, br d, J = 6 Hz, H-16), 5.40 (IH, br d, J = 6 Hz, H-15), 4.56 (IH, br s, H14), 1.94 (3H, br d, J = 7 Hz, H-4'), 1.85 (3H, br s, H-5'), 1.20 (3H, s, H-18), 0.93 (3H, s, H-17), 0.67 (3H, s, H-20); ' H NMR (200 MHz, C 3 D 6 0): 5 6.03 (IH, qq, J = 7.3, 1.6 Hz, H-3'), 5.72 (IH, br d, J = 5.8 Hz, H-16), 5.44 (IH, br d, J = 5.8 Hz, H-15), 4.54 (IH, br s, H-14), 1.93 (3H, dd, J = 7.3, 1.6 Hz, H-4'), 1.83 (3H, quint, J = 1.6 Hz, H-5'), 1.19 (3H, s, H-18), 0.92 (3H, s, H-17^0.72 (3H, s, H-20); 13 C NMR (C 3 D 6 0): 5 179.0 (s, C-19), 168.2 (¿C-1'), 137.4 (d, C-3'), 134.2 (d, C-16), 133.1 (d, C-15), 129.5 (s, C-2'), 94.8 (d, C14), 56.9(d,C-5), 54.0(s,C-8), 53.1 (d,C-9),48.9(s,C-13),44.1 (s, C-4), 40.5 (£, C-1), 38.8 (t, C-3), 38.7 (s, C-10), 33.1 (í, C-12), 32.4 (tC-7), 29.5 (q, C-18), 21.6 (£, C-6), 20.9 fa, C-5'), 20.5 (£, C-ll), 20.1 (£, C-2), 19.7 fa, C-17), 15.9 fa, C-4'), 14.6 fa, C-20); MS m/z (rel. int.): 400 [M] + (1), 317 [ M - A n g ] + (1), 300 [ M - A n g O H ] + (8), 285 [ 3 0 0 - M e ] + (3), 272 [ 3 1 7 - C 0 2 H ] + (3), 257 (3), 243 (2), 185 (20), 173 (10), 159 (3), 145 (4), 133 (4), 121 (7), 105 (10), 83 [Ang] + (100), 67 (8), 55 [ A n g - C O ] + (81), 41 (18), 29 (27).

1981

3600-3200, 3060,2920,1685,1650,1460,1440,1360,1320,1265, 1195,1175,965,870,795,715; >H NMR (200 MHz): 5 4.74 (IH, q, J = 2 Hz, H-17a), 4.58 (IH, q, / = 2 Hz, H-17b), 2.28 (IH, m, H12), 1.26 (3H, s, H-18), 0.91 (3H, s, H-20); 13 C NMR: 5 177.7 (s, C19), 152.7 (s, C-16), 104.6 (f, C-17), 57.3 (d, C-5), 52.3 (d, C-9), 48.2 (£, C-15), 43.9 (s, C-4), 39.7 (t, C-1), 39.7 (£, C-7), 38.4 (s, C-10), 38.0 (£, C-3), 36.6 (d, C-12), 33.6 (s, C-8), 29.0 fa, C-18), 28.8* (£, C-14), 28.4* (£, C-13), 27.2 (£, C-ll), 20.3 (£, C-6), 18.8 (£,C-2), 12.1 (q, C20); MS m/z (rel. int.): 302 [M] + (34), 287 [ M - M e ] + (74), 259 [ 2 8 7 - C 2 H 4 ] + (19), 257 [ M - C 0 2 H ] + (19), 241 (27), 213 (16), 187 (10), 175 (7X159 (15), 145 (15), 131 (32), 121 (33), 105 (52), 91 (86), 79 (69), 67 (46), 55 (58), 41 (100), 29 (35). Methyl ester (20) (CH 2 N 2 -Et 2 0). Mp 125-126° (MeOH); [ a ] D -68.7° (CHC13; cl.5); I R v ^ c n T 1 : 3060, 2920, 2900, 2860, 1715,1635, 1440,1235,1190,1035, 980,970, 870, 815; *H NMR (200 MHz): 5 4.70 (IH, q, J = 2 Hz, H-17a), 4.54 (1, q, J = 2 Hz, H17b), 3.62 (3H, s, C0 2 Me), 2.28 (IH, m, H-12), 1.15 (3H, s, H-18), 0.76 (3H, s, H-20); 13 C NMR: 177.7 (s, C-19), 152.7 (s, C-16), 104.5 (£, C-17), 57.2 (d, C-5), 52.1 (d, C-9), 51.0fa, C0 2 Me), 48.2 (£, C-15), 43.8 (£, C-4), 39.7 (£, C-1), 39.7 (£, C-7), 38.2 (s, C-10), 38.2 (£, C-3), 36.6 (d, C-12), 33.4 (s, C-8), 28.7 {q, C-18), 28.7* (£, C-14), 28.3* (i, C-13), 27.2 (£, C-ll), 20.3 (£, C-6), 18.8 (£, C-2), 11.9 (q, C-2Q); MS m/z (rel. int.): 316 [ M ] + (79), 301 [ M - M e ] + (67), 288 [M - C 2 H 4 ] + (4), 273 [ 3 0 1 - C 2 H J + (21), 257 [ 3 1 6 - C 0 2 M e ] + (7), 241 (63), 213 (20), 201 (11), 187 (18), 175 (14), 159 (19), 131 (32), 121 (54), 105 (53), 91 (86), 79 (70), 67 (45), 55 (60), 41 (81), 29 (30), 15 (32).

ent-l&-Angeloyloxykaur-l6-en-19-oic acid (18). Rf 0.34 (hexane-EtOAc, 4:1); mp 154° (MeOH); [a] D -75.7° (CHC13; ent-Beyer-15-en-19-oic acid (16). Rf 0.46 (hexane-EtOAc, cl.9); I R v ^ c m " 1 : 3140, 3040, 2960, 2940, 2910, 2840, 1720, 4:1); Mp 184° (MeOH); [ a ] D + 7.0" (CHC1 3 ; c 1.8); IR v™¿ cm" 1 : 1655,1645,1440,1380,1270,1205,1170,970, 870, 850, 800, 755; 3600-3200, 3025, 2935, 2840,1685, 1580, 1445,1405,1370,1320, ' H N M R (200 MHz): 5 6.08 (IH, qq, J = 7.2, 1.5 Hz, H-3'), 4.80 1255, 1190, 970, 930, 850, 790, 750, 740; ' H N M R (200 MHz): (IH, br s, H-17a), 4.74 (IH, br s, H-17b), 4.51 (IH, d, J = 10.5 Hz, 5 5.76 (IH, d, J = 5.7Hz, H-16), 5.47 (IH, d, J = 5.7Hz, H-15), H-18a), 4.03 (IH, d, J = 10.5 Hz, H-18b), 2.64 (IH, br s, H-13), 1.26 (3H, s, H-18), 1.01 (3H, s, H-17), 0.69 (3H, s, H-20); 2.37 (IH, br d, / = 13.2 Hz, H-5), 1.97 (3H, dd, 3 = 72,1.5 Hz, H13 CNMR: 5 184.3 (s, C-19), 136.5 (d, C-16), 134.8 (d, C-15), 61.1 4'), 1.86 (3H, quint, J=1.5Hz, H-5'), 0.99 (3H, s, H-20); (£, C-14), 57.2 (d, C-5), 52.4 (d, C-9), 49.2 (s, C-8), 43.9 (s, C-4), 43.7 13 C NMR: 5 181.7 (s, C-19), 167 (s, C-1'), 155.4 (s, C-16), 138.6 (d, (s, C-13), 39.6 (£, C-1), 38.0 (t, C-3), 37.7 (t, C-7), 37.7 (s, C-10), 33.2 C-3'), 127.6 (s, C-2'), 103.2 (£, C-17), 72.0 (f, C-18), 55.3 (d, C-9), (£, C-12), 29.1 fa, C-18), 24.9 (q, C-17), 21.6 (£, C-6), 20.5 (£, C-ll), 52.4 (d, C-5), 48.9 (£, C-15), 47.8 (s, C-4), 44 (s, C-8), 43.8 (d, C-13), 19.3 (£, C-2), 13.8 fa, C-20); MS m/z (rel. int.): 302 [M] + (100), 287 40.8 (t, C-7), 40.2* (£, C-1), 39.7 (s, C-10), 39.5* (£, C-14), 33.0** (£, [ M - M e ] + (25), 259 [ 2 8 7 - C 2 H 4 ] + (9), 257 [ M - C 0 2 H ] + (7), C-12), 32.7** (£, C-3), 21.8 (£, C-6), 20.4 (q, C-5'), 18.4 (f, C-ll), 18.4 201 (6), 187 (11), 159 (17), 147 (32), 135 (85), 122 (82), 105 (76), 91 (£, C-2), 15.7 (q, C-4'), 15.6 (q, C-20); MS m/z (rel. int.): 400 [M] + (80), 79 (38), 67 (21), 55 (29), 41 (41), 29 (17). (2), 317 [ M - A n g ] + (2), 301 [ M - A n g O ] + (2), 300 [M - A n g O H ] + (23), 285 [ 3 0 0 - M e ] + (7), 272 [ 3 1 7 - C 0 2 H ] + (2), Methyl ester (19) (CH 2 N 2 -Et 2 0). Mp 118° (MeOH); [«]„ 1 257 [ 2 7 2 - M e ] + (7), 239 (4), 211 (4), 187(5), 174(10), 159 (5), 145 + 5.0° (CHC13; cl.5); IRvJSJcnT : 3040, 3020, 2940, 2860, (5), 131 (7). 119 (9), 105 (16), 83 [Ang] + (100), 55 [ A n g - C O ] + 2840, 1720, 1440, 1365, 1320, 1230, 1185, 1150, 1090, 1040, 975, 845,810,770,750,740; ' H NMR (60 MHz, CC14): 5 5.70 (IH, d, J (68), 41 (17), 29 (17). = 6 Hz,H-16), 5.40(IH,d,J = 6Hz,H-15), 3.58(3H, 5, C0 2 Me), Methyl ester (21) (CH 2 N 2 -Et 2 0). Mp 109° (MeOH); [cc]D 1.13 (3H, s, H-18), 0.98 (3H, s, H-17),0.52 (3H, s, H-20); ' H NMR -82.7° (CHC13; cl.5); I R v ^ c i r r 1 : 3060, 2980, 2940, 2880, (200 MHz): 5 5.65 (IH, d, J = 5.7 Hz, H-16), 5.38 (IH, d, J 1720, 1645, 1440, 1375, 1345, 1230, 1190, 1155, 1145, 1040, 880, = 5.7 Hz, H-15), 3.56 (3H, s, C0 2 Me), 1.10 (3H, s, H-18), 0.92 850, 755; »H NMR (200 MHz): 5 6.03 (IH, br q, J = 7 Hz, H-3'), (3H, s, H-17), 0.49 (3H, s, H-20); 1 3 CNMR: 5 177.9 (s, C-19), 4.78 (IH, br s, H-17a), 4.73 (IH, br s, H-17b), 4.46 (IH, d, J 136.4 (d, C-16), 134.7 (d, C-15), 61.1 (t, C-14), 57.1 (d, C-5), 52.3 (d, = 10.5 Hz, H-18a), 3.99 (IH, d, J = 10.5 Hz, H-18b), 3.66 (3H, s, C-9), 51.0 (q, C0 2 Me), 49.1 (s, C-8), 43.6 (s, C-4), 43.6 (s, C-13), C0 2 Me), 2.60 (IH, br s, H-13), 2.33 (IH, br d, J = 13 Hz, H-5), 39.6 (£, C-1), 38.2 (f, C-3), 37.7 (£, C-7), 37.7 (s, C-10), 33.2 (£, C-12), 1.95 (3H, br d, J = 7 Hz, H-4'), 1.85 (3H, br s, H-5'), 0.87 (3H, s, H28.9fa,C-18), 24.8 (q, C-17),21.6 (£, C-6), 20.4(£, C-ll), 19.3 (£, C20); 13 C NMR: 5 175 (s, C-19), 167 (s, C-1'), 155.5 (s, C-16), 138.4 2), 13.6 (q, C-20); MS m/z (rel. int.): 316 [ M ] + (43), 301 [M (d, C-3'), 127.6 (s, C-2'), 103.1 (t, C-17), 71.9 (£, C-18), 55.2 (d, C-9), - M e ] + (2), 273 [ 3 0 1 - C 2 H 4 ] (2), 257 [ M - C 0 2 M e ] + (11), 241 52.3 (d, C-5), 51.4 (q, C0 2 Me), 48.9 (£, C-15), 47.8 (s, C-4), 43.8, (s, (5), 229 (2), 213 (4), 194(13), 181 (13), 159(14), 148(6), 135(61), 121 C-8), 43.8 (d, C-13), 40.8 (£, C-7), 40.1* (£, C-1), 39.6* (£, C-14), 39.2 (44), 105 (68), 91 (87), 79 (54), 67 (38), 55 (61), 41 (100), 29 (54), 15 (s, C-10), 33.0** (£, C-12), 32.9** (£, C-3), 21.8 (£, C-6), 20.5 (q, C(67). 5'), 18.5 (£, C-2), 18.5 (£, C-11), 15.7 (q, C-4'), 15.4 (q, C-20); MS m/z (rel. int.): 414 [ M ] + (8), 382 [ M - M e O H ] + (2), 355 (M entv4fis-16-en-19-oic acid (17). Rf 0.50 (hexane-EtOAc, 4:1); + + + 1 C 0 (3),314 2H] (l),354[M-C02Me] (3),331[M-Ang] mp 219° (MeOH); [a] D -69.7° (CHC1 3 ; c2.0); I R v ^ í c m " : + + [ M - A n g O H ] (100), 299 [ 3 1 4 - M e ] (16), 286 (6), 271 (15), 255(19), 239(14), 227 (3),211 (13), 199(5), 187 (9), 174(31), 159 (9), 145 (7), 131 (11), 119 (11), 105 (17), 83 (73), 67 (10), 55 (53), 41 (13), 29(11). Assignments may be reversed.

1982

M.

Acknowledgements—The authors thank Prof. A. Escarré (University of Alicante) for the identification of plant material, Dr A. Fernández Rodriguez (University of Salamanca) for high field NMR measurements and Dr A. Guirado (University of Murcia) for mass spectra.

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et al. 11. Bohlmann, F. and Zdero, C. (1976) Chem Ber. 109, 1670. 12. Tanaka, U, Nihashi, S., Yanagisama, T., Nikaido and Shibata, S. (1972) Tetrahedron 28, 4523. 13. King, T. J., Yardley, J. P. (1961) J. Chem. Soc. 4308. 14. Wehrli, F. W. and Nishida, T. (1979) Fortsch. Chem. Org. Naturstoffe 36, 64. 15. Joseph-Nathan, P., Wesener, J. R. and Guenther.H. (1984) Org. Magn. Reson. 22, 190. 16. Caputo, R., Mangoni, L., Revitere, L. and Laccanino, R. (1971) Tetrahedron Letters 3731. 17. Do Khac Manh, D., Bastard, J. and Fetizon, M. (1983) J. Nat. Prod. 46, 262. 18. McCrindle, R., Martin, A. and Murray, R. D. H. (1968) J. Chem. Soc. (C), 2349. 19. Baker, M. K., Lindsay, H., Briggs, J. G., Buchanan, St. C, Cambie, R. C. Davis, B. R., Hayward, R. C, Long, G. A. S. and Rutledge, P. S. (1972) J: Chem. Soc. Perkin I 194. 20. Bory, S. and Fetizon, M. (1964) Bull. Soc. Chim. Fr. 570. 21. Yamasaki, K., Khoda, H., Kobayashi, T., Kasai, R. and Tanaka, O. (1976) Tetrahedron Letters 1005. 22. González, A. G., Fraga, B. M., Hernández, M. G. and Hanson, J. R. (1981) Phytochemistry 20, 846. 23. Gaudemer, A., Polonsky, J. and Wenkert, E. (1964) Bull. Soc. Chim. Fr. 407.