Lignans from polar extracts ofJuniperus thurifera

Phytochemistry, Vol. 31, No. 1, pp. 267 270, 1992 Pnnted in Great Britain.

LIGNANS FROM POLAR EXTRACTS ARTURO

003 l-9422/92 $5.00 + 00 1991 PergamonPressplc

0

SAN FELICIANO,* JOSE M. MIGUEL DEL

CORRAL,

OF JUNIPERUS

THURZFERA

JOSE L. LOPEZ and BEATRIZ DE

PASCUAL-TERESA

Departamento de Quimica OrgBnica, Facultad de Farmacia, 37007 Salamanca, Spain (Receioed 28 May 1991)

Key Word Index---Juniperus thurifera;Cupressaceae; methyl deoxypodophyllotoxinate; 7B-hydroxydihydrosesamin;

podophyllotoxinic

acid;

lignans.

Abstract-Three new natural lignans, methyl deoxypodophyllotoxinate, podophyllotoxinic acid and 7/%hydroxydihydrosesamin, 11 known lignans and two cinnamyl alcohols were isolated and identified from a chloroform extract of Juniper-us thurifera leaves. Assignment of the 13C NMR spectrum of b-methyl peltatin B was also performed through direct and long-range heteronuclear 2D NMR analysis, amending previous assignments.

INTRODUCTION

deoxypodophyllotoxin although important differences were apparent. Thus, its IR showed a band at 1730 cm- ’ instead of a y-lactone band at 1780 cm-‘. Its 13C NMR, also close to that of deoxypodophyllotoxin, showed an additional signal at 651.38 corresponding to a methoxy group. These data, together with the signal in the ‘H NMR spectrum at 63.60 assignable to the cited methoxy group, indicated the presence of a methyl ester in the molecule instead of the usual lactone group, thereby prompting us to propose structure 1 for this compound. The trans configuration of substituents at C-8/C-8 was deduced from the value of JHs, “,,, = 11.4 Hz [4]; indirect confirmation of the structure was achieved through synthesis of its %epimer 4, which showed a value of J H8,Hsg= 3.7 Hz. Attempts to synthesize 1 from podophyllotoxin were unsuccessful due to the tendency of the latter to epimerize at C-8. Compound 4, on standing, progressively lactonized to deoxypicropodophyllotoxin, whereas 1 was stable. Additionally, reduction of 1 with LiAlH, yielded the diol 5, whose spectroscopic data (Tables 1 and 2) were consistent with those of the reduction product of podophyllotoxin, once again confirming the trans stereochemistry of the new compound

In previous studies on the chemical composition of the leaves of Juniperus thurifera we described the isolation

and characterization of a number of lignans from the nhexane extract [l]. In the present work, we describe the lignans isolated from a chloroform extract obtained after depletion with n-hexane. The presence of lignans, substances with hitherto poorly understood biological activities in the different extracts of this plant led us to develop some activity assays with either extracts and fractions or purified substances. The hexane and chloroform extracts have both shown significant cytotoxic and cytostatic effects against KB cells together with inhibition of tubulin polymerization (San Feliciano et al. unpublished data).

RESULTS AND DISCUSSION

Dried and triturated leaves of Juniperus thurijera were extracted first with hexane and then with chloroform in a Soxhlet apparatus. From the neutral part, obtained after partitioning with basic aqueous solutions, by repeated chromatography 15 lignans were isolated and identified. Most of these were already known, viz. yatein, podorhiwl, nemerosin, deoxypodophyllotoxin, deoxypicropodophyllotoxin, podophyllotoxin, picropodophyllotoxin, epipicropodophyllotoxin, p-methylpeltatin B, dihydrosesamin [ 1, 21 and (2R,3R)-2,3-bis-(3,4_dimethoxybenzyl)-1,4-butanodiol [3], which was isolated as its diacetate identical to ariensin. Two new lignans, methyl deoxypodophyllotoxinate (1) and 7’/3-hydroxy-dihydrosesamin (2) were also isolated as their corresponding diacetates, together with 3’,4’-dimethoxy- and 3’,4’,5’trimethoxycinnamyl alcohols. The only lignan found in the acidic part of the extract, mainly composed of diterpene acids, was podophyllotoxinic acid, which was isolated as its diacetate methyl ester 3. Compound 1 was an oil, with a [M]’ at m/z 430 corresponding to the molecular formula C,,H,,Os. Its ‘H and “C NMR (Table 1) data suggested a cyclolignan structure, with methylenedioxy and 3,4,5-trimethoxyphenyl groups presenting signals similar to those of

at C-8/C-8’. Compound 1 cannot be an artefact, because it is well known that deoxypodophyllotoxin in methanol epimerizes to deoxypicropodophyllotoxin even in the absence of acid or base.

Compound 2 isolated after acetylation was also an oil with a [M]’ at m/z 456 corresponding to the molecular formula CZ4HZb09. Its IR spectrum showed bands for acetate (1740 cm- I), aromatic rings (1620, 1505 cm- ‘) and methylenedioxy groups (2865, 935 cm-‘). These data, together with the study of its NMR spectra (Tables 1 and 2), led us to propose a monoepoxylignan structure for this compound. Both spectra displayed signals very similar to those shown by dihydrosesamin acetate (9). However, 2 showed an additional secondary acetate, which was deduced by the presence in the ‘H spectrum of a methyl singlet at 6 1.99 and a one proton doublet at 65.67 as well as signals for two acetate groups in the 13C NMR spectrum. The location of the second acetate group at the benzyl position 7’ became obvious and the configuration

267

9’a 9’b llya 1Wb 2’-OMe 9-OAc 7’-OAc 9’-OAc CO,Me

6 7 8 9a 9b 10 11 12 2' s 6' 7'a 1% 8

2 5

H

-.

3.60 s

_.

5.82 s -_

2.44 m 3.68 dd (6.4, 4.5)

3.51 s

-

5.85 s _

4.18 dd (5.4, 10.8)

2.98 dd (5.8, 16.0) 2.55 M 4.09 dd (3.0, 10.8)

3.08 d (5.8)

s s s s s

2.96 dd (5.8, 10.9)

6.09 s 4.33 d (5.8)

6.09 s -

18

3.76 s 3.78 s 3.76 s 6.58 s 6.36 s 2.66 dd (10.6, 16.0)

3.75 3.80 3.75 6.63 6.40 -

6.13 s 6.13 s 4.38 d (5.7) 3.00 dd (1 X.4, 5.7)

1

5.95 s -. 2.05 s 1.99 s -.. -

3.70 dd (16.8, 7.2)

5.67 d (9.3) 2.60 m 3.18 dd (9.7, 5.3)

6.74-6.87 m

5.96 s -

4.52 d (8.0) 2.34 m 4.26dd (11.2, 4.6) 4.10 dd (11.2, 6.1)

I 6.74-6.87 m

2.03 s 2.16 s 3.60 s

5.92 s -

6.27 s 2.61 m 4.14 dd (11.6, 2.3) 4.26 dd (11.6, 3.6)

3.71 s 3.80 s 3.77 s 6.73 s 6.43 s

-

-..

3.19 dd (13.2, 5.2)

6.34 s 4.31 d (5.1)

6.34 s

-

-

3.63 s

-. -

5.86 s -

3.64 m -.

3.70 m -5.85 d (1.4) 5.88 d (1.4) __.

2.80 m .2.11 m

3.75 s 3.80 s 3.75 s 6.61 s 6.36 s -

3.49 m -

4.09 d (5.1) 2.ltm

6.24 s

6.24 s

5

274 dd (5.4, 36.7) 2.94 dd (7.6, 16.7) 2.41 m

__ 3.77 s 3.82 s 3.77 s 6.58 s 6.36 s

3.01 dd (6.4, 3.7)

6.26 s 4.36 d (6.4)

6.26 s

-

Table 1. “H NMR spectral data for compounds l-7 (2aOMHz) 2 3 4

3.78 s 3.82 s 3.78 s 6.33 s 2.78 dd (16.0, 6.3)

s s s s

3.79 3.84 3.79 6.63

6.56 s 4.38 d (3.4)

-2.65 m 3.70 m -. 5.92 d (l-4) 5.90 d (1.4) _.”

3.29 dd (9.3, 3.0)

2.65 dd (16.0, 5.8) 2.98 m 3.96 dd (9.2, 3.0) 4.43 dd (9.2, 7.0) 5.88 d (1.5) 5.91 d (1.5) 4.00 s

6.34 s 4.34 d (3.0)

3.48 m -

6.34 s

7

6.36 s 6.36 s 4.67 d [SS) 2.80 m

6

269

;

4 5

Me0

OMe OMe

6

of the new chiral centre was deduced from the value of the coupling constant J,,,, nsp=9.3 Hz. This implied a predominantly anti conformation for these two protons. After detailed study of Dreidiug models for both epimers at 7, it was found that for the 7’R epimer the most stable conformation corresponded to values for the coupling constant of JH,,, Hs,= S-10 Hz and for the 7’5 the conformation corresponds to values of JH,,, t++,= 4-6 Hz; this led us to assign structure 2 for this compound. From the acidic part extracted with aqueous sodium bicarbonate, after acetylation and methylation with diazomethane, only lignan 3 was isolated, whose spectroscopic data indicated a cyclolignan structure. Its EI mass spectrum with CM]’ at m/z 530 corresponded to the mol~ular formula C$,H,,O,,. Its IR, ‘HNMR and 13CNMR (Tables 1 and 2) spectra were very similar to those of methyl deoxypodophyllotoxinate acetate, isolated from the neutral fraction. Compared to la, compound 3 showed more intense acetate bands in its IR spectrum and the mass spectrum displayed the loss of two acetic acid molecules, indicating the presence of two acetate groups. This was confirmed by the presence in the ‘HNMR spectrum of two singlets at ca 2.1 ppm. The location of the second acetate group at 7’ was deduced fram the presence of a methine signal at ca 671 in the 13C NMR spectrum and the absence of the signal corresponding to the methylene carbon in the 13CNMR spectrum (632.2), as well as those of its protons in the ‘H NMR spectrum (62.16 and 2.03). The configuration of C-7’ was established considering there was no coupling in the signal corresponding to H-7’; this observation can only be explained if this proton is rruns to H-g’. The structure of methyl di~tylp~ophyllotoxinate was thus established for compound 3 and confirmed by hydride reduction of 3 and podophyllotoxin, both of which gave the same trio1 6 (Tables 1 and 2). P~ophyllotoxini~ acid, the corresponding natural product, has not been described before as a component of plants, but its natural

R=OAc R=H

R’=H, R*=OH, R3=COOMe, R4=H R’=H, R2=oAc, R3--cooMe, R4=H RkAc, R*=OAc, R3=CUOMe, R4=H R’=H, R*=OH, R3= H, R4=COOMe R’=H, R*=OH, R3=CH20H, R4=H R’=OH, R*=OH, R3=CH20H, R4=H

character is certain because it cannot be obtained under acidic or basic conditions from podophyllotoxin. Complementary to the isolation of the above products, the unambiguous assignment of the i3C NMR spectra of some compounds was made by direct H-C and longrange H/C 2D NMR correlations. These particular experiments allowed us to assign spectra (Tables 1 and 2) for ~-me~yl~ltatin B (7), amen~g the previously published [S] 13CNMR data. In addition, we were able to ascertain that in the cis-lactone series, the chemical shifts for carbon atoms at positions 1 and 4 are, respectively, 6 139.0 and 137.9, contrary to the ~si~rnen~ performed by us for compounds of the truns-lactone series.

EXPERIMENTAL

General. Mps arc uncorr. Optical rotations were measured in

CHQ,. UV spectra were recorded in EtQH, IR spectra in CHCl,, ‘H NMR (200 MHz) and i3C NMR (SOMHz) spectra were measured in CDCl, with TMS as int. std; 6 values are expressed in ppm. EKMS were obtained at ‘70eV. Flash CC was run on silica gel. Plant material, extraction and isolation. Juniperus thwtfera was collected in November at PrHdena (Segovia, Spain). Voucher

specimens are deposited at the Botany Departmen& Faculty of Pharmacy, Salamanca (register number SALAP No. 15980). Leaves (4.6 kg) were extracted in a Soxhlet first with hexane and then with CHCl,. The CHCI, extract was partition& between a 4% aq. soln of NaOH obtaining an acidic part (76.8 g) and a neutral part (46.1 g), which were successively defatted with M&H and a satd soln of urea in MeOH. Once defatted (15.3 g, 12.4%), the neutral part was c~omato~ph~ over silica gel with hexane-EtOAc mixts of increasing polarity yiehling yatein (0.6 g), podorhixol (0.2 g), nemerosin (0.1 g), deoxypodophyllotoxin (0.2 g), deoxypi~~ophyllotoxin (Ll g), pi~o~opbyilotoxin (3.0 g), epipicropodophyllotoxin (0.2 g), @uthyIpeltatin B (20 mg), dihydrosesamin (0.2 g) and (2R,3R)-2,3-bis-(3,4-

270

A. SAN FELICIANOet al. Table C

2. 13CNMR

spectral

data for compounds

l-7

1

la

2

3

4

5

6

7

137.88 107.41 152.96 137.88 152.96 107.41 48.64 47.91 173.72 56.34 60.84 56.34 128.58 108.00 146.80 146.37 109.92 130.27 32.23 33.19 65.98 100.86

137.63 107.20 152.91 148 31 152.9 1 107.20 47.67 47.67 172.52 56.28 60.80 56.28 128.01 107.87 146.37 146.81 109.27 129.97 31.87 30.18 66.69 10086

130.30 106.93 148.18 148.18 108.19 120.13 85.33 50.41 64.49 101.32 -133.33 108.33 155.69 145.69 107.42 121.20 77.05 47.93 69.58 101.32

135.91 107.12 153.03 137.66 153.03 107.12 46.43 47.84 171.40 56.20 60.84 56.20 127.91 107.34 147.98 147.56 108.66 131.67 71.02 36.04 63.06 101.34

137.23 106.78 153.25 140.93 153.25 106.78 49.68 46.07 174.44 56.37 60.82 56.37 128.28 108.36 146.30 146.48 109.77 129.82 30.97 35.97 63.90 100.80

137.20 107.59 152.88 138.60 152.88 107.69 43.55 48.66 65.36 56.33 60.85 56.33 129.01 109.32 146.10 146.41 107.97 131.85 33.00 34.98 6494 loo.70

136.87 107.04 153.22 138.87 153.22 107.04 47.49 48.17 68.92 56.36 60.83 56.36 129.96 109.93 147.67 146.25 108.00 132.26 77.03 43.25 60.21 100.99

181.31 20.24 170.77 20.72 -

_ 171.02 21.29 170.61

‘__

,_ __

139.0 108.1 154.0 137.9 154.0 108.1 45.9 46.8 178.8 56.8 61.6 56.8 120.7 141 5 136.0 148.5 104.8 132.0 24.8 33.0 73.6 101.4 60.2 .

._-

-

--

.-

--

_

1 2 3 4 5 6 7 8 9 10 11 12 1’ 2’ 3’ 4 5’ 6 7 8’ 9 10 2’-OMe 9-CO-Me 9-CO-Me 7’-CO-Me 7’“CO-Me 9’-CO-Me

.---

9’-CO-Me CO,Me

-51.38

-.-.

170.98 20.81 51.33

-

20.75 51.55

dimethoxybenzyl)-1,Cbutanodiol diacetate (2 mg), 3’,4’-dimethoxycinamyl alcohol (60 mg), 3’,4’,5’-trimethoxycinamyl alcohol (70 mg), 1 (90 mg) and 2 (5 mg). Methyl podopkyllotoxinate (1). Oil. eluted with CH,C12EtOAc. [a]23(n) -72.5” (589); -75.0” (578); -90.0” (546); - 106.25” (436) (EtOH; cO.l%). EIMS m/z (rel. int.): 430 (87) 429(51),414(19),398(16),397(11),353(16~352(13),340(13),339 (59), 338 (49), 324 (1 l), 283 (33), 282 (39). UV&,,, (8): 215 (20215), 289 (3750). IR v cm-l: 3620,3460, 173?, 1600, 1510, 1490, 1240, 1130, 1050, 1010, 945. ‘H NMR (Table 1); 13CNMR (Table 2). Acetate (la). Oil. IR: 1780, 1740, 1600, 1505, 1485, 1240, 1130, 1045,1005,945 cm- ’ , ‘HNMR (Table 1); 13CNMR (Table 2). Compound 9 (30 mg) in 5 ml of 0.25% KOH in MeOH was stirred for 10 mm at room temp. After neutralization and extraction with EtOAc the reaction mixt. was methylated to afford 25 mg of Me deoxypicropodophyllotoxinate (4). Oil. IR v cm-‘: 3320, 1730, 1600, 1510. 1240, 1135, 1050, 1010, 9.50. ‘HNMR (Table 1); i3CNMR (Table 2). LiAlH, reduction of compound 1. To an Et,0 soln of 1(25 mg), LiAlH, (30 mg) was added and the mixt. stirred for 4 hr. Usual work-up of the reaction mixt. afforded 24 mg of 5, identical to the reduction product obtained from deoxypodophyllotoxin. IRvcn-‘: 3600, 1600, 1510, 1470, 1240, 1130, 1040, 1010, 940. ‘H NMR (Table 1); i3C NMR (Table 2). 7jhHydroxydikydrosesamin diacetate (2). Oil, eluted with CH,Cl,-EtOAc. UV L,,, (a): 284 (15300), 235 (238003, 205 (83650). EIMS m/z (rel. int.): 456 (1 l), 336 (5), 202 (lo), 187 (1 l), 161 (31), 151 (42), 150 (20), 149 (84), 134 (30). IRv cm-‘: 2865, 1740,1620,1505,1455,1390,1260,1060,935. ‘H NMR (Table 1); 13C NMR (Table 2).

-

51.77

---

From the acidic part, after acetylation and methylation, by standard methods with Ac,O-pyridine and CH*N,-Et,O, respectively, repeated CC yielded 25 mg of 3. Methyl podopkyllotoxinate diacetate (3). Oil eluted with n-164,O” (589); -156.0” (578); hexane-EtOAc. [~]‘~(i), -133,0”(546); -131”(436)(cO,8%). UVi,,,,(~):288(3000),207 (34600). EIMS m/z (rel. int.): 530 (6), 470 (l), 410 (4), 364 (6), 351 (5), 350 (18). IRvcm-‘: 1735, 1590, 1505. 1240, 1130, 1045. 935 cm-i. lH NMR (Table 1); i3CNMR (Table 2). LiAlH, reduction ofcompound 3. To an Et,0 soln of 3 (25 mg), LiA1H4 (30 mg) was added and the mixt. stirred for 3 hr. Usual work-up afforded 24 mg of 6, identical to the reduction product ofpodophyllotoxin. IR: 3620,1600,1510,1240. 1130,1045,1010, 995 cm-‘. ‘H NMR (Tabte 1); i3CNMR (Table 2). Acknowledgement-Financial tained from DGICYT, grant

support for this work PB 89-0394.

was ob-

1

REFERENCES

1. San Feliciano, A., Medarde, M., Lopez, J. L., Puebla, P., Miguel del Corral, J. M. and Barrero, A. F. (1989) Pkytockemistry 28, 2863. 2. San Feliciano, A., Miguel de1 Corral, J. M., Lopeg J. L. and Pascual-Teresa, I3 de. (1990) An. Quim. 86, 561. 3. Hernandez, J. D., Roman, L. U., Espifieira, J. and JosephNathan, J. (1983) Planta Med. 47, 215. 4. Hartwell, J. L. and Schrecker, A. W. (1953) J. Am. Ckem. Sot. 75, 5916. 5. San Feliciano, A., Miguel de1 Corral, J. M., Gordaliza. M. and Castro, M. (1990) Pkytockemistry 29, 1335.