Abietane diterpenoids from Lepechinia meyeni and Lepechinia hastata

Phytochemistry, Vol. 30, No. 7, pp. 2339-2343, 1991 PrintedanGreatBritain.

ABIETANE

0031-9422/91 %3.00+0.00 Q 1991Pergamon Press plc

DITERPENOIDS FROM LEPECHINIA LEPECHINIA HASTATA

ME YENI AND

MAURIZIO BRUNO, GIUSEPPE SAVONA, FRANCO PIOZZI,* MARIA C. DE LA ToRRE,t BENJAMIN RODRIGUEZ? and MICHEL MARLIER~

Dipartimento di Chimica Organica dell’Universit8, Archirafi 20, 90123 Palermo, Italy; TInstituto de Quimica Organica, C.S.I.C., Juan de la Cierva 3, 28006 Madrid, Spain; SGroupe Spectrometrie de Masse, Facultd des Sciences Agronomiques de I’Etat, 5800 Gembloux, Belgium (Received 7 December 1990)

Key Word Index-kpechinia oids; 12-formyl-abietane

meyeni: Lepechinia hastata; Labiatae; abietane and 9(10-+20)-abeo-abietanediterpenderivative; pisiferol, rosmanol, carnosic acid, salvicanol, isosalvicanol.

Abstract-From the aerial parts of Lepechinia meyeni four previously known abietane diterpenes (pisiferol, rosmanol, carnosic acid and salvicanol) were isolated, with two unknown diterpenoids (isosalvicanol and a lZformyl-abietane derivative). The structures of the new compounds were established by spectroscopic means. Moreover, NMR spectroscopic studies allowed the assignment of a OH-1Ob configuration in salvicanol. From the aerial parts of Lepechinia hastata only the previously known carnosic acid was isolated.

INTRODUCTION

RESULTS AND DISCUSSION

In our continuing search for new diterpenoids in Labiatae species [l-3], we have studied the acetone extract of the aerial parts of Lepechinia meyeni. From this extract we have isolated the already known diterpenes pisiferol (8,11,13-abietatriene-12,20-diol) [4-6], carnosic acid (l&12-dihydroxy-8,11,13-abietatrien-20-oic acid) [7-l 11, also named salvin, rosmanol (7a,ll,l2-trihydroxy8,11,13-abietatrien-20,6jGolide [12, 133 and salvicano1 (lOfi,ll-dihydroxy-12-methoxy-9(10+20)-abeoabieta-8,11,13-triene) (1) [ 141, besides two other diterpliC compounds, namely isosalvicanol (10/&12dihydroxy-11-methoxy-9(10-+20)-abeo-abieta-8,11,13triene) (2) and 12-formyl-11-hydroxy-8,11,13abietatrien-2-oic acid methylester (3). The structures of compounds 2 and 3 were established by spectroscopic means, which also provided the configuration at C-10 of salvicanol (l), a structural feature of this diterpenoid not

Some years ago [14] salvicanol (1) was isolated as a natural constituent from the root bark of Salk canariensis. The stereochemistry at its C-10 asymmetric centre was not investigated, but a OH-1Ocr configuration was hypothesized on the basis of some theoretical considerations. From a chromatographic fraction of the acetone extract of Lepechinia meyeni, and after ethereal diazomethane treatment, we have isolated two C,,H,,O, compounds, one of which was identified as salvicanol(1) by its ‘H and 13C NMR spectra (Tables 1 and 2, respectively) and IR data, which were identical with those reported [14] for salvicanol. The other compound (2, isosalvicanol) was a regioisomer of salvicanol and their structural difference was clearly revealed by their almost identical ‘H and 13C NMR spectra (Tables 1 and 2) and by NOE experiments. Irradiation of 1 at 63.74 (12-Omethyl protons) caused NOE enhancement in the signal of H-15 (2%, at 63.20), whereas in the case of isosalvicanal (2) irradiation at 63.81 (1 l-O-methyl protons) produced NOE enhancement in one of the C-20 protons (HB20, at 63.08, 2%) and no effect was observed on H-15 (63.23).

previously

ascertained

[ 141.

As these abietane derivatives were unstable in chromatography over silica gel, and were very difficult to purify (see Experimental), the identifications of carnosic acid and rosmanol were made from their 12,20-di-Omethyl- and 12-O-methyl derivatives, respectively. These were stable compounds obtained by ethereal diazomethane treatment of the chromatographic fractions containing those compounds. Obviously, salvicanol and isosalvicanal could arise from the diazomethane methylation of the corresponding 11,12-dihydroxy-derivative; also the formyl-methyl ester 3 could be an artefact produced by the treatment with diazomethane. Investigation of the aerial parts of Lepechinia hastata indicated that the known carnosic acid [7-l l] is the only diterpenoid.

1

R’ = OH, R’ - Me H

2 R’ = OMe, R’ -

*Author to whom correspondence should he addressed.

4

2339

R’ = R’ = H

3 5

R = CHO R = OH

M. BaUNo et al.

2340

Table 1. ‘H NMR data of compounds l-3 (6 values, CDCl,, TMS as int. standard)*

Table 2. 13C NMR chemical shifts (6) of compounds (CDCl,, TMS as int. standard)*

H

1

2

3

C

18

t 2.72 m 6.54 s 3.20 ept 1.24 d 1.20 d 0.92 s 0.89 s 2.51 d 3.24 d 3 74 s .5.72 br sp

t 2.72 In 6.73 s 3.23 ept 1.25 d 1.21 d 0.93 s 0.88 s 2.56 d 3.08 d 3.81 s

3.59 dt

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 w 17g 18 19 20 CO,Me Ar-OMe CHO

‘la and 78

14 15 Me-161 Me-172 Me-18 Me-19 20A 20B Ar-OMe CO,Me CHO OH J W.4 la.lB 1#&2a l/%28 15,16(17) 20A.20B

t t t 6.9 14.1

5.61 br s§

: t 6.9 14.2

t 6.58 s 3.51 ept 1.31 d 1.28 d 0.98 s 0.78 s 3.63 s 10.25 s 13.05 SII 14.1 4.0 4.0 6.8 -

*Spectral parameters were obtained by first order approximation. All these assignments were confirmed by double resonance experiments and ‘H-‘H and ‘H-l% NMR 2D COSY spectra. ioverlapped signal. fThese carbon numbers are interchangeable. §Rapidly interchangeable with D,O. //Slowly interchangeable with D,O.

Two rearranged abietane diterpenoids with structures closely related to compounds 1 and 2 have been isolated from Ckamaecyparis pisifera (Cupressaceae), namely pisiferanol (4) [15] and pisiferadinol (the 20~-hydroxy derivative of 4) [ 151, also named pisiferdiol [16]. The OH-lo/J configuration of these compounds has been rigorously established by a combination of chemical and spectroscopic studies. Comparison of the 13C NMR spectra of 1, 2 and 4 (Table 2) clearly reveals that the lop configuration of the tertiary hydroxyl group of 4 must be also defined for 1 and 2, because the C-l to C-7 and C-15 to C-19 carbon atom resonances were almost identical in the three compounds, whereas the observed differences in the chemical shifts of the aromatic, C-IO and C-20 carbons were due to the presence (1 and 2) or absence (4) of an oxygenated substituent at C-11. In particular, the C-20 carbon appeared downfield in 4 (651.1, without substituent at C-l 1) with respect to compounds l(641.07) and 2 (642.05), in which this carbon is y with respect to the oxygen substituent at C-l 1. The well known large differences between the 13C NMR spectra of compounds possessing cis or tram fused rings [17] firmly supported to trans A/B rings junction in compounds 1 and 2, identical with that securely established for pisiferanol(4) [LSJ and pisiferadinol [ 15,161. The last diterpenoid found in the extract of L. meyeni was isolated from a chromatographic fraction previously treated with diazomethane. This substanw had a molecular formula C,,H,,O, and its structure (3) was established as follows.

1

42.15 tt 18.75 t 42.55 ta 34.40 s 58.21 d 23.92 t 36.32 t 141.41 s 119.72 s 70.64 s 147.63 s 142.42 s 139.16 s 116.76 d 26.47 d 23.57 q 23.98 q 32.25 q 21.63 q 41.07 t 61.77 q -

2 42.05 ta 18.72 t 42.38 ts 34.40 s 58.29 d 24.08 t 36.16 i 132.98 sb 125.86 s 70.80 s 146.26 s 144.46 s 135.65 sb 121.52 d 27.22 d 22.35 4 22.68 q 32.22 q 21.57 q 42.05 t

-. 61.94 q _.

3 33.43 t’ 17.82 t 41.04 t 33.68 s 54.02 d 19.85 t 33.68 ta 149.77 Sb 128.78 s 47.26 s 163.56 s 114.77 s 149.31 Sb 117.59 d 27.02 d 24.15 4 23.88 4 32.58 q 19.71 q 175.93 s 51.55 q 194.23 d

l-4

4t 41.6 t 18.7 t 42.3 t 34.4 s 58.0 d 24.4 t 35.2 t 134.9 s 133.5 s 72.3 s 118.6 d 152.3 s 132.6 s 126.6 d

26.5 d 22.6 q 22.9 q 32.3 q 21.7 4 51.1 t

-

*Assignments of carbons bearing hydrogen atoms were confirmed by ‘H-‘%Z 2D COSY spectra. iTaken from ref. [15]. SMultiplicities were determined by the DEPT pulse sequence. $These carbon numbers may be interchanged. a*bThese assignments may be interchanged.

The ‘H and 13C NMR (Tables 1 and 2) and IR spectra of 3 were in agreement with the presence of a carbomethoxyl group (vco 1720 cm- ‘; 6,175.93 s and St SS q; &, 3.63,3H, s), two C methyl groups (6,0.78,3H, s and 0.98,3H, s; 6,32.58 q and 19.71 q) and a pentasubstituted aromatic ring (five singlets and a doublet of aromatic carbons, S, 6.58, lH, s; 6, 117.59 d, Table 2) bearing a chelated phenolic function (a,, 13.05 s, slowly interchangeable with D,O) [lS], an aldehyde (vco 1630 cm-‘, chelated; S, 10.25, lH, s; 6, 194.23 d) and an isopropyl group (6, 3.51, lH, seven lines signal, J=6.8 Hz, and 1.31 and 1.28,3H each, both i&J=6.8 & 6,27.02 d, 24.15 qand23.88 q). Moreover, theC-I to C-7, C-lO,C-18,C-19 and carbomethoxyl carbon atom resonances of 3 (Table 2) were identical with those reported for carnosic acid methyl ester (5) Cl93 and related compounds [20], thus establishing that diterpenoid 3 possesses and identical structural moiety (rings A and B) to 5. The aromatic substitution pattern of ring C of 3 was established as follows. The almost identical chemical shifts of C-7 in 3 and 5 (633.68 t or 33.43 t, and 32.7 t, respectively, Table 2 and ref. [19]) was in agreement with the absence of substitution at C-14 (y-substituent with respect to C-7). In a NOE experiment, irradiation of the aldehydic proton (610.25 s) gave a clear NOE enhancement (13%) in the signal of the H-15 proton (63.51) and a minor effect on the signals of the isopropyl methyl

Diterpenoids from Lepechinia

2341

Scheme 1. Possible formation of compound 3.

groups (6 1.28 and 1.31,2% in each). Thus, the aldehyde and isopropyl substituents must be ortho. On the other hand, the UV spectra of compound 3 obtained in methanol and after addition of base (Table 3) were comparable with those report& [21] for o-hydroxybenzaldehyde, establishing an ortho relationship between the aldehyde and the phenolic function. This conclusion was also supported by the UV spectra of diterpenoid 3 obtained after addition of AlCl, and AlCl, plus HCI, which showed characteristic band shifts (Table 3) of this chromophore [22]. As the C-14 position is unsubstituted and the aldehyde function must be placed between the phenolic hydroxyl and the isopropyl substituent, it was clear that the diterpenoid possessed structure 3 of an abietane derivative. However, an alternative structure (with the isopropyl substituent at C-l 1 and the phenolic group at C-13) may also be considered for this compound, although it is less probable taking into account biogenetic considerations. Exclusion of this alternative structural possibility was firmly supported by the rather deshielded position of the PHY30:7-Q

Table 3. UV spectra of compound 3 [&,,,, nm (logs) J MeOH

+ NaOMe

+AIClf

+AICI, +HCI

230 (4.013 284 (4.07) 349 (3.61)

243 (4.04) 284 (3.96) 392 (3.74)

231 (4.01) 286 (3.98) 304sh (3.83) 358 (3.46) 392 (3.36)

231 (4.02) 288 (4.02) 358 (3.49) 39Osh (3.41)

*Recorded after 0.5 hr of addition of AK&

H-ljIprotonof3(S3.59dt, Jl,rfa= 14.1 Hx,J14,1.=J1P,2P = 4.0 Hz), which is due [23,24] to its coplanarity with the aromatic ring C and its dose proximity to the oxygen lone pairs of the C-l 1 hydroxyl group. It was not possible to undertake an X-ray analysis of 3, because it crystallixed as twin crystals from the usual solvents. From a biogenetic point of view, a 12-formyl abietane derivative such as 3 is highly surprising as a natural

2342

M.

BRUNO er al.

product, and we suppose that it could originate by treatment of the plant extract with diazomethane. However, we have not found any simple and plausible mechanism for explaining the formation of 3 as an artifact, and the possibility shown in Scheme 1 is, in our opinion, rather complicated although plausible. Indeed, the formation of 3 from carnosic acid (6) can be explained in terms ofan air-oxidation of6 into the orthoquinone 7 [9], whose tautomeric quinone methide form @) reacts with diazomethane [25] giving the epoxide 9. Cleavage of the oxirane ring ofS, probably favoured by the C-1 f hydroxyl substituent, produces 3, via its tautomeric enolate IO.

EXPERIMENTAL

Compound 3. Mp 164-166” (MeOH). [!x]i6’+ 218.3” (CHC&; c, 0.071). UV: see Table 3. IR ~2; cm-‘: 3500 (br), 3040, 2960, 2905,2870,1720,1710, 1630,1565,1470, 1455,1400,1370,1310, 1280,1225,12~5,1190,1145,1125,1040,1020,985,875,830,810, 800, ‘H NMR (300 MHz, CDCl,): see Table 1. 13C NMR (50.3 MHz, CIXY,): see Table 2. EIMS (70 eV, direct inlet) m/z jrel. int,): 358 [M] + (19)?329 (O-7),312(5), 299 (75), 298 (lOO},283 f6), 243 (8), 242 (6), 229 f28), 217 (22), 203 (29f,69 (7) (Found: C, 73.87; H, 8.49, C,,HJ,O, requires: C, 73.71; H, 8.44). Treaamen$ ofI.qerhinia husma.Driedand powdered aerial parts (40 g) were extracted as described for L. meyeni. The extract (2 g) was treated with ethereal CH,N, (1 hr, room temp.) and then subjected to CC (silica gel, 300 g); the fraction eluted with rzhexane-EtOAc 9: 1 gave 26Omg of raw material that was acetytated with Ac,O-pyridine at room temp. I48 hr). The product was identified [7] as 1I,1 2-diacetyl-20-methyl ester of carnosic acid.

Plant

material. Lepechinia meyeni (Walp.) Eplmg was collected in April 1985 in the Huarochiri-Lima region (Peru) at 3 800 m above sea level in the flowering period. Voucher specimens are deposited in the Herbarium of the Natural History Museum, San Marcos University, Lima (Peru). Lepechinia hastafa (A_ Gray) Epling, native of California, was cultivated in the Botanic Garden of the University of Palermo fltaly) and coflected in June 1983. Voucher specimens are deposited in the Herbanum of this centre. Treatment of Lepe&in&z mqelri. Dried and powdered aerial parts (110 g) were extracted (x 3) with Me&O (2 I) at room temp. for a week. The extract (3.2 g) was subjected to CC (silica gel, Merck No. 7734, deactivated with 15% H,O, 500 g) eluting with n-hexane-EtOAc mixts to yield several frs which were treated with ethereal CH,N, (4 hr, room temp.) and then rechromatographed (CC, silica gel, n-hexane-EtOAc mixts as eluent) giving the following compounds in order of their chronatographic polarities: Isosalvicanol (2, 14 mg), salvicanot (1, 9 mg) [14], pisiferolf9 mg) [4-6& compound 3 f 17 mg), carnosic acid as its 12-~-methyl-2~methyI ester derivative (50 mg) [7-l tf and the f2-U-methyl derivative of rosmanol (30 mg) [12,13], besides additional quantities (3 g) of a mixt. of ursolic and oleanolic acids as their methyl ester derivatives. The previously known compounds were identified by their physical (mp, fot&,) and spectroscopic (IR, ‘H and *3C NMR, MS) data and, m some cases, by comparison (mmp, TLC) with authentic samples. Although salvlcanol(1) is a known compound [14], we report its complete physical and spectroscopic data, as some of them have not been previously reported. Sal&anol (1). Amorphous solid. [aJi6”+ 33.3” (CHCI,; c 0.024). uv n:z)” nm (log z): 230 (3.721,274sh (3.281,281 (3.333. IR ~5:; cm -‘: 3510, 3440, 3030, 2970, 2940, 2880, 1620, 1580, 1500,1455,1425,1370,2335,1300, IlOo, 1080, f&Q 1025,1000, 975, 945, 860. ‘H NMR (200 MHz, CDCI,): see Table 1. ’ %I NMR (50.3 MHz, CDCl, ): see Table 2. EIMS (70 eV, direct infet) m/z (rel. int.): 332 CM]+ (la), 314 (XI), 299 (4), 271 (21,245 (111, 206 (IOO), 194 (23), 193 (ZO), 191 (ll), 177 (S), 55 (6). C,,H,,O, M, 332. Identical with the previously described compound [14]. Jsosalvicanol (2). Mp 129-131” (spontaneously on cooling). [c@‘+ 13.5” (CHCl,; ~0.222). UV ngg,“” nm (log&): 230 (3.69), 276931(3.29), 283 (3.32). IR vk:; cm-l: 3550, 3290, 3040, 2970, 2870,1615, 14900,1465,1455,1425, 1370,1335,1220,1080,1045, 1030, 980, 950, 945, 880, 820. ‘H NMR (300 and 200 MHz, CDCI,): see Table 1. 1‘C NMR (75.4 and 40.3 MHz, CD&): see Table 2. EIMS (70 eV, direct Inlet) m/z (rel, int.): 332 EM] + (is), 3~4~0.5),299~~=5),2~9~i),Z~~~~),~94~i4), 193(19), l91(17),69 f6h 55 (7). Found: C, 75.71; H’,9.82. C,,H,,U, requires: C, 75.86; H, 9.70%.

Acknowledgemrm~s---The authors thank Prof. 3. M. Fraga (I.P.N.U., C.S.I.C., Tenerife, Spain) for helpful discussions, This work was subsidized by the Spanish “Dire&&n General de Investigacicjn Cientifica y T&&a” (Grant PB87-U418),by “Programa Conjunta No, 3.3 C.N.R.-C.S.1.C” (~~~~~pai~~ and by the Italian “Consigk Nationale delfe Ricer&e, C.N.R.“.

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