Triterpenes from Cedrela odorata

003 1 9422/91 $3.00 + 0.00 ic;: 1991 Pergamon Press plc

Phytochemisrry, Vol. 30, No. 4. pp 1225 1229. 1991 Printed m Great Bntm.

TRITERPENES ANGELA

M.

CAMPOS,

FROM

CEDRELA

ODORATA*

FRANCISCO S. OLIVEIRA, MARIA IRACEMA L. MACHADO, RAIMUNDO BRAZ-FILHOt and FRANCKO J. A. MATOS

Departamento de Quimica Orglnica e Inorglnica, Centro de Ciencias, Universidade Federal do CearL, C.P. 12.200, 60.150Fortaleza, Ceari, Brasil; TDepartamento de Quimica, Instituto de Ciincias Exatas, Universidade Federal Rural do Rio de Janeiro, C.P. 74.541, 23851, Seropedica, Rio de Janeiro, RJ, Brasil

(Receioed 15 June 1990) Key Word Index----Cedrela o&rata; Meliaceae;

one; essential

bark; wood; triterpenes;

three-23,24,25-trihydroxytirucall-7-en-3-

oil.

Abstract-From the ethanolic extract of the heartwood of Cedrela odorata were isolated gedunin, 3,%0-/?-Dglucopyranosyl-24-methyllenecholesterol, oleanonic acid, sitosterol, n-octacosanol and a new triterpene threo23,24,25-trihydroxytirucall-7-en-3-one. Structural determinations of these compounds were made by spectrometric data. CC-MS analysis of the essential oil identified the sesquiterpenoids a-copaene, b-elemene, a-muurolene, calamenene, guayazulene, dihydroguayazulene, torreyol, y-muurolene and a-cubebene.

INTRODUCTION

Cedrela odorata Veil., is a tree distributed throughout Brazil and other countries in South America. It is reputed in folk medicine for its use as an astringent, emetic, leucorrheal and anti-ulcer agent [l]. The characteristic constituents of this genus are the triterpenoids of the types pentaol (1) odoratone (2), odoratol @a), iso-doratol (2b) and the limonoids gedunin (3) and desoxycedrelone (4). which have been isolated from C. odorata, C. yluzior+i, C. mexicana and C. toona [2]. We report on the isolation of a new triterpene (5) and other known compounds: 3/I0-/I-D-glucopyranosyl-24-methylenecholesterol (6), oleanonic acid (7) sitosterol, stearic, oleic and linoleic acids and the presence of n-octacosanol. Analysis by GC-MS of the essential oil showed the presence of the sesquiterpenes acopaene, /I-elemene, a-muurolene, calamenene, guayazulene, dihydroguayazulene, torreyol, y-muurolene and cubebene. RESULTS AND DISCUSSION

The known compounds were identified mainly by comparison of their spectral properties with literature data (see Experimental). The identification of compounds 3 and 7 was confirmed by a comparative study of the 13CNMR spectra of these compounds and the corresponding data reported in the literature [2, 33. The presence of a methyfene group at C-24 of the acetate derivative 6a was revealed by chemical shifts of C-24 (6 156.5, s) and C-28 (6 105.9, t). These chemical shifts are almost identical with those of the corresponding carbons

*This paper is based on the M.Sc. thesis submitted by A. M. to Universidade Federal do Ceari (1983); for a preliminary communication see (1982) Ci&rc. Cult. (So Paulo) 34

Campos

(Suplemento), 490. tAuthor to whom correspondence

should

be addressed.

in cycloeucalenol [4]. The i3CNMR spectrum also showed the presence of an additional double bond between C-5 (6140.1, s) and C-6 (6 121.9, d) and the moiety 3j-O-/.?-D-tetra-O-acetylglucopyranosyl: 99.5 (d, C-l’), 71.6 (d, C-2’), 71.4 (d, C-3’). 68.5 (d, C-4’), 72.9 (d, C-5’) and 62.0 (t, C-6). The localization of this moiety at C-3 was deduced from chemical shifts of C-2 (629.2, t), C-3 (679.9, d) and C-4 (639.7, t), from the downfield shift of C-3 @-effect, +8.3 ppm) and upfield shifts of C-2 (‘i-effect, - 2.4 ppm) and C-4 (y-effect, - 2.5 ppm). The 13C NMR assignments for all carbon atoms of 6a were determined by comparison with those made previously for known related compounds [S-7]. The molecular formula C,,H,,O, for 5 was deduced from its mass spectrum (CM]” at m!.z 474) in combinaspectra and its IR tion with the ‘H and “CNMR spectrum (vcEo 1700 and vOH3450 cm- ‘). It possesses one secondary (6 1.02, d, J = 6 Hz) and seven tertiary (6 1.34, 1.34, 1.14, 1.08, 1.06, 1.06 and 0.85) methyl groups, a carbonyl(6,216.8), two secondary hydroxyls [S, 4.14 dd, J = 9 and 4.5 Hz, H-23) and 3.20 (br s, H-24); Sc 69.7 (d, C23) and 74.2 (d, C-24)], one tertiary hydroxyl(6c75.0, s, C25) and one trisubstituted double bond [S, 5.36 (m. H-7); &145.6 (s, C-8) and 117.8 (d, C-7)]. Comparison of the ‘H and “C NMR spectral data of 5 and its acetyl derivative 5a with those of model compounds 8 [8], 9 [9], 10 and 1Oa [lo] indicated that all these compounds have the same tetracyclic system (Table 1). Additional support for this proposal was obtained from the important fragment in the mass spectrum of 5 at m/z 369 (base peak) (11) [lo] and this was chemically confirmed by periodate oxidation, which gave a tetra-noraldehyde 12 [S, 9.78 (br s, H-23); [Ml” at m/z 384, C,,H,,Oz]. The ’ 3C NMR spectral data of the natural compounds 5, 8 and 10 (Table 1) differed significantly only in the chemical shifts of the side-chain carbons at C-17, suggesting that they are stereoisomers. The only significant difference in the spectra of 5 and 8 [8] was observed in the absorption of a methyl group [6c_2t 24.2 or 24.7 (8) and 18.9 (5)]. The 1225

1226

A. M.CAMNS

2 2a 2b

6 6a

eral.

R'= O.R'= p-OH R'= a-OH.H.R'= P-OH R' = a-OH.H.R'= o-OH

R=H R = AC

10 IOa

R = H R =

AC

11 R = I2

assignments were based on the application of the usual shift parameters, comparison with literature data [&lo] and observed multiplicities of signals (Tables I). In our correlations of the chemical shifts of the carbon atoms of 8 we also used the comparison with those of reported derivatives [S]. Thus, the tetracyclic triterpenoids 5 [mp 199.-202” (petrol): [r];” - 23.4” (MeOH; c l.O)], 8 (amorphous) and piscidinol A [mp 195’ (MeOH); [a];’ -90” CHCI,; c l.O)], isolated from Cedrela odorata (this paper), Turraea nitolica [8], Trichiris hispida [lo] and

@,m/z36Y(lOo%)

R = Me

Walsura piscidiu [ 111, respectively, differ in the side-chain at C-17. The formation of an acetonide was consistent with either a 1,2- or 1,3-diol grouping and a decision in favour of the former (5b) was reached when a singlet was observed at 6108.1 corresponding to acetonide C-2’ (Table 1) in the “C NMR spectra (broadband decoupled and APT). The chemical shifts of acetonidc carbon in the hexacyclic 1,3-dioxane system appear at ca 698.0 (e.g. 13 [12]). Furthermore, the chemical shifts of C-22 (642.0), C-23 (677.3). C-24 (687.6) and C-25 (669.7) are consistent

Triterpenes from Cedrela odorata

1227

Table 1. ‘%NMR spectral data for compounds 5, SI and 91 (25.2MH2, CDCI,) and comparison with models 8 ]a],9 (CDCI,) 191,10 (pyridine-d,) and 1Oa(CDCI,) Ilo( [S (ppm) relative to TMS] C

S

Sa

1

38.5 34.9 216.8 47.8 52.2 24.5 117.8 145.6 48.4 34.9 18.3 33.9 43.5 51.1 33.7 28.5 53.8 12.7 22.0 33.7 18.9 40.4 69.7 74.2 75.0 26.2 27.3 24.3 21.6 27.3

38.4 34.9 216.8 47.8 52.1 24.5 117.7 145.6 48.4 34.9 18.1 33.9 43.5 51.1 33.8 27.8 53.6 12.8 21.9 32.8 18.4 37.9 69 6 73.9 82.5 22.3 22.6 24.3 21.6 27.4 169.9 169.9 169.8 21.5 21.4 20.7

2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 2.4 25 26 27 28 29 30 OAc

2 Me-2 Me-2

_..

8

9

38.4 34.9 217.0 47.8 52.2 24.4 117.8 145.7 48.8 34.8 18.8 33.9 43.4 51.0 33.7 28.3 53.7 12.7 21.9 33.6 24.2/18.8$ 40.3 69.6 74.2 74.9 26.1 27.2 24.7 21.5 27.2

38.4 34.9 216.6 47.8 52.3 24.4 117.7 146.0 48.2 34.9 27.3’ 34.0 43.5 51.1 33.6 28.2 52.9 12.7 18.3. 35.8 18.3 36.1

5h 38.4 34.9 216.3 47.7 52.2 24.3 117.7 145.6 48.4 34.8 18.3 34.0 43.4 51.1 33.5 28.5 53.2 12.8 21.8 36.2 19.7 42.0 77.3 87.6 69.7 27.2 27.4 24.5 21.6 27.4

_. 108.1 24.8 27.7

-. -

24.4 21.5 21.5, --

10 37.6 28.6 78.3 39.5 51.2 24.4 118.5 146.1 49.3 35.2 18.4 34.3 43.8 51.4 34.3 28.6 54.4 13.5 22.1 34.3 19.6 42.3 69.5 76.8 73.8 27.2 27.8 28.3 15.5 27.4

llh 36.8 24.2 81.2 37.9 50.8 23.8 117.8 145.7 48.8 34.8 18.1 33.9 43.6 51.1 33.9 27.9 53.6 13.2 21.9 33.9 21.47 38.0 70 4 76.8 72.5 26.3 27.2 27.2’ 15.9 27.6 171.0 170.7 170.5 21.4 20.9 18.47

*These comparative analyses revealed the assignments corresponding for these carbons: 6 18.3 (C-l I), 21.5 (C-19) and 27.3 (C-30). tit is necessary to interchange these values: 621.4 or 21.9 (MeCOJ and 18.4 (C-21). $Probable superposition with the signal of C-l 1 (I). ‘These values are interchangeable.

with the relative stereochemistry shown in 5b. This deduction was based on the comparison of the difference between the chemical shifts of C-24 and C-23 (664.5 ppm) of natural compound 5 and that observed in the acetonide 91 (A610.6 ppm). This major difference (A6 10.6/4.5 > 2.3 ppm), which is the minor downfield shift of C-23 (A67.6 ppm) when compared with that of C-24 (Ah 13.4 ppm), is consistent with y-effects at the C-23 attributed to hydroxyl and two methyl groups at C-25 since the /?-effect of C-2’ and yeffect of methyl group at C2’ might be expected to be the same in C-23 and C-24 (Table 1). Thus, the relative three-configuration of the

23,24diol moiety of the natural product 5 isolated from Cedrelu odorata was proposed. The erythro-configuration forms a cis-acetonide derivative. Finally, the configuration (S) at C-20 is assumed on biogenetic grounds since tirucallane derivatives occur widely in the Meliaceae while euphanes (20 R) in this family are restricted to Melia species [13]. EXPERIMENTAL Mps: uncorr; ‘H (100 MHz) and “CNMR (25.2 MHz): CDCI,, TMS as int. standard; MS: direct inlet with 70 eV ion-

1228

A. M.

Table

2. Constituents

of the essential

CAMPOSet al. oils from Cedrela

odorata

and C. odorata

C. odorata C. odorata

x-Copaene t(-Elemene x-Muurolene Calamenene Guayazulene Dihydroguayazulene Torreyol 7-Muurolene Nerolidol cr-Cubcbene

17.49 1.57 3.71 4.05 0.12 2.77

A

(%)

16.64 0.84 IO.87 26.60 3.91 4.82 4.45 3.52

1.15 -

ization; CC: silica gel (Merck 0.05-0.20 mm) or florisil (Merck 0.154.25 mm); TLC: silica gel H or G (Merck) and the spots were visuahzed by UV (254 nm), exposure to iodine vapour and Ce,SO,-H,SO,. Plant materiul. Cedrela odorata Veil. was collected in September 1979. from the National Park of Ubajara-Ceara-Brazil and identified by Professor Afrlnio Fernandes (Federal University of Ceara, Fortaleza). A voucher specimen (No. 6897) is deposited at the Herbarium Prisco Bezerra of the Biology Department, University of Ceara, Brazil. Extraction and isolation of the bark constiruents. The powdered bark (9.6 kg) was extracted with hexane at room temp. for 48 hr. Solvent was removed under vacuum to yield 47 g of residue. Steam distillation of this residue furnished a volatile fraction (2.5 ml) that was separated into A (1 ml) and B (1 ml) by chromatography on a silica gel column using hexane as eluent. The remaining residue was treated with KOH in H,O-EtOH under reflux for 30 min. After addition of H,O, the ppt. was filtered (32 g) and submitted to CC (silica gel, 500 g). Elution with &Hi+ C,H,.-CHCI, (7:3), CHCI, and CHCI,-EtOAc (9: 1) gave, respectively, oil A (1.2 ml), sitosterol (500 mg), n-octacosanol(500 mg) and 7 (600 mg). The soln was extracted with Et,O. The Et,0 soln was dried and evapd under vacuum giving a mixture of stearic, oleic and linoleic acids (3 g). This mixture was methylated with BF,-MeOH and its methyl ester was identified by CC-MS through comparison with literature data [14, IS]. Extraction and isolation of the sapwood constituents. The powdered sapwood (3.5 kg) was extracted with C,H, and EtOH, successively, at room temp. Solvent was removed in uacuo to yield 38 and 83 g of residues, respectively. The C,H, extract was submitted to steam distillation to give a volatile fraction C(1 ml). The EtOH extract (72 g) was chromatographed on a silica gel column and eluted with C,H,,, CHCI,, Et,O, Me&O and MeOH. yieldmg frs I (10 g), II (17 g), III (24 g), IV (3 g) and V (6 g). respectively. Fr. I yield a volatile oil D (3 ml, steam distillation), sitosterol (130 mg) and a mixture of stearic, oleic and linoleic acids (1.3 g). Fr. II was rechromatographed on a silica gel (600 g) column and the fraction eluted with CHCI,-Me,CO (9: 1) furnished compound 3 (4OBmg) after crystallization from MeOH, mp 200-202. An additional quantity of 3 (80 mg) was isolated from fr. III. Extracrion and isolation of the heartwood consrituents. The powdered hearwood (2.5 kg) was extd with EtOH at room temp. The residue. obtained (180 g) after distillation of the solvent, was chromatographed (160 g) on a silica gel column. Elution with petrol. CHCI,. Me&O and MeOH afforded the frs Ia (15 g). IIa

0.13 5.31 4.45 1.66 1.84

(%)

c

D

4.03 0.22 2.92 11.10 3.37 2.29 2.40 1.18

3.26 0.21 1.99 6.69 1.66 1.21 0.67

B

[ 173

E 0.98 2.64 2.25 28.96 2.04 1.72

1.03

-

0.94

(10 g), IIIa (14 g) and IVa (30 g). respectively. Fr. Ia was rechromatographed on a silica gel column to yield a volatile oil E (0.3 ml), 5 (600 mg) and 6a after acetylation with Ac,O--pyridine (IOOmg), eluted with petrol. CHCI,-Me,CO (9: I) and Me&O-MeOH (4: I), respectively. An additional quantity of 5 (3 g) was isolated from fr. IIa. Analysis of the essential oils A-E. The identification of the essential oils constituents were made by a computer library search program which involved CC-MS data [16]. Comparative analysis with the essential oil from Cedrrla odoraro [ 171, furnished by Dierbcrger Essential Oils S.A.. was used for conlirmation (Table 2). Gedunin (3). Mp 2W202 (MeOH); [aIF + 54’ (MeOH; c 1) (ref. [18] mp 196-198” (EtOAc-petrol), [x]n +W (MeOH; c 1.2). Spectral data, mainly MS, ‘H (100 MHz) and 13C (25.2 MHz) NMR (CDCI,) [19. 201. are in agreement with literature values. 3-Oxo-thrco-23.24,25-Trihydroxytirucall-7-enc (5). Mp 199.. 202” (C6H,,); [a]; - 23.4’ (MeOH; c I); IR v$‘: cm- ‘: 3450, 1700. 1680; ‘H NMR: 65.36 (m, H-7), 4.41 (dd, J =9 and 4.5 HI H-23). 3.20 [brs, 4H, (OH), and H-241, 1.34 (s, Me-26 and Me27). 1.14 (s. Me-30). 1.06 (s, Me-19 and Me-29). 1.02 (d, J = 6 Hz Me-21). 0.85 (s, Me-18): “C NMR: see Table 1; MS m/z (rel. int.): 474 ([Ml”. IO), 441 (16). 423 (6). 385 (7). 384 (7) 383 (14). 369 (100). 351 (23). 341 (5) 325 (54). 313 (5). 271 (7). 161 (23). Acetylation of compound 5. The acetate Sa was prepared by treatment of 5 (200 mg) with Ac,O (5 ml) and pyridine (1 ml). The usual work-up followed by CC on silica gel (CHCI,) of the crude product yielded 5~ (150 mg) as an amorphous solid, mp 141-144’. [z]:“ -40” (MeOH; c I). ‘H NMR: 65.4532 (m. H-7, H-23 and H-24), 2.17 (s. OAc), 2.06 (s, OAc), 1.94 (s. OAc), 1.56, 1.44 (s, Ha-26 and H,-27). 1.10 (s. H,-30). 1.04 (d, J = 6 Hz. H,-21). 0.98 (s, H,-19, H,-28 and H,-29) 0.80 (s, H,-18); “CNMR: see Table 1. Periodate oxidalion of compound 5. A soln of 5 (50 mg) in EtOH (60 ml) was treated with aq. soln (9 ml) of NaIO, (0.058 M) and stirred at room temp. for 12 hr. The soln was filtered and extracted with CHCI,.

The crude product, obtained

after evapn of the solvent, was chromatographed Elution

with CHCI,

gave the aldehyde

over silica gel.

12 (30 mg) as an amor-

phous solid, mp 119-122’. MS m/z (rel. int): 384 ([Ml +, 4). 369 (20). 356 (7) 355 (8), 351 (73), 342 (12). 341 (38), 327 (6), 325 (13). 43(lOO);‘HNMR:S9.78(brs,H-23),5.3(m.H-7). 1.12(s,HJ-28) 1.04 (d, J=6 Hz, H,-21). 1.01 (s, H,-19 and H,-29). 0.98 (s, H,30). 0.83 (s, H,-18). Preparation

ofaceronide

5b. The natural product 5 (200 mg) in

Triterpenes

from Cedrela odorato

dry Me&O (10 ml) and HISO, (two drops) was stirred at room temp. for 2 hr. Filtration and removal of solvent gave the acetonide 5h (190mg). 13C NMR: sex Table I. 3P-0-8-rrTetra-0-ocetylylucopyranosyl-24_cerol (I%). Mp 159-160”; ‘HNMR: S5.26-4.93 (m, H-2’. H-3’. H-4’ and H-28a), 4.7 (ms, H-28b), 4.60 (d, J =8 Hz, H-l’), 4.34.0 (m, H-6’a and H&b), 3.9-3.4 (m, H-3 and H-5’). 2.1 (s, OAc), 2.06 (s. OAc), 2.03 (s, 20Ac). 1.03 (d, J=6 HI H,-21). 1.0 (s, H,-19). 0.83 (d, J=6.5 Hz, H,-26 and H,-27). 0.70 (s, H,-18); ‘“C NMR: 6 170.3 (OAc), 170.0 (OAc), 169.1 (OAc), 169.0 (OAc), 156.5 (C-24), 140.1 (C-5), 121.9(C-6), 105.9(C-28), 99.5 (C-l’), 79.9 (C-3). 72.9 (C-S), 71.6 (C-2’), 71.4 (C-3’). 68.5 (C-4’), 62.0 (C-6’). 56.7 (C-14). 56.O(C-17), 50.1 (C-9). 42.3 (C-13). 39.7 (C-4). 38.9 (C12), 37.1 (C-l), 36.7 (C-lo), 36.1 (C-20). 35.0 (C-22). 33.9 (C-25), 31.8 (C-7 and C-8). 29.4(C-23). 29.2 (C-2), 28.1 (C-16), 24.3 (C-15), 21.8 (C-l l), 21.O(C-26 and C-27), 20.6 (OAc), 19.3 (C-19). 18.8 (C21). 11.8 (C-18). Oleanonic acid (7). Mp 192-194” (petrol); [a]io +42.3” (MeOH; c 1); Methyl oleanonate, mp. 18G182.5”. [z];“ +68” (MeOH, c 1) (lit. [21], mp 181-182”; [LY],, +76”). Spectral data, mainly MS, ‘H and ‘%NMR [3], were in agreement with literature values. Acknowledgements-This work was supported by CNPq research fellowships and by grants from Conselho National de Desenvolvimento Cientilico e Tecnol6gico (CNPq), Financiadora de Estudos e Projectos (FINEP) and CoordenaCHo de Aperfeiqoamento de Pessoal de Ensino Superior (CAPES). The authors are grateful to Professor A. J. R. da Silva, NPPN, Universidade Federal do Rio de Janeiro, for acquisition of the mass, ‘H and ‘%NMR spectra.

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