Pergamon Phwchtmutrv. Vol. 3’1. No. 3, PP.

855962 1994
Cop&ht 0 1994 El&in Sdena Ltd
Prmted in Chat Brilam All n&u rcsxwd
m-9422/94 s700+000

BIBENZYL CANNABINOID AND BISBIBENZYL DERIVATIVE FROM THE

LIVERWORT RADULA PERROTTETZl

MASAO TOYOTA,TOMOHIDEKINUGAWAand YOSHINOR~ ASAKAWA

Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamaahiro-cho, Tokushima 770, Japan

(Receioed 7 March 1994)

Key Word Iudex--R&h perrottetii; Bryophytes; Jungermanniales; Radulaceae; perrottetinen; biben-

zyl cannabinoid; bisbibenzyl derivatives; prenyl bibenzyls; isoperrottetin A.

Abstract-A new bibenzyl cannabinoid has been isolated as a minor component from the ether extract of the liverwort
Radula perrortetii. Its structure was established by spectral means and by comparing its spectral data with those of an
authentic compound which had been previously synthesized. A new bisbibenzyl, isoperrottetin A, has also been
isolated from the same species, along with three known bisbibenzyls, six prenyl bibenzyls, three sesquiterpenoids and 6-
tocopherol. The structures were established by spectral means and chemical evidence.

INTRODUCTION

Radula species are small stem-leafy liverworts which are

distributed worldwide and are rich sources of aromatic
compounds. About 18 species are known in Japan. The
Radulaceae are morphologically isolated from other fam-
ilies of the Jungermanniales.
Radula species elaborate several characteristic prenyl
bibenzyls and these structures have been established by
spectroscopic methods and/or synthesis [l]. Linear pre-
nyl and cyclic prenyl bibenzyls are present in this genus
C2-43. Almost all compounds of this class are distributed
in Radula species. These compounds could be useful as
chemosystematic markers for the liverworts which belong
to the Jungermanniales. Here, we wish to report on the
isolation and structure elucidation of a new bibenzyl
cannabinoid, perrottetinen (1) and a new bisbibenzyl
derivative, isoperrottetin A (15), from R. perrottetii. 4

RESULTS AND DRXUSION

The ether extract of R. perrottetii was chromato-

graphed on silica gel, Sephadex LH-20 and repurified by OH
MPLC on CN silica gel to give the new bibenzyl canna-
binoid 1 and bisbibenzyl 15, together with known ent-
sesquiterpenoids 10-12, phytol (13), bisbibenzyls 17-19, OH OH
&tocopherol (14) and prenyl bibenzyls 4-9. The spectral 6
data of the prenyl bibenzyls 4-9 were identical to those of
authentic samples [2-41.
The EI-mass spectrum of 1 showed a molecular ion
peak at m/z 348 as base peak. Its IR spectrum established
the presence of a hydroxyl group (3400 cm-‘) and an
aromatic ring (1622 and 1578 cm-‘). The ‘H and OH
“CNMR spectra (Tables 1 and 2) not only showed OH
signals due to a hydroxyl group at 64.80 (1H) which
disappeared upon addition of D20, and the aromatic 9

859
860 M. TOYOTA et al

Table 1. ‘HNMR spectral data’ for compounds 1

and 2

H 1 2

2 6.21 (br s) 6.16(brs)

3 3.57(br s) 3.53 (br s)
4 1.73(m) 1.73(m)
5 1.47(m) axial 1.50(m) axial
1.97(m) equatorial 1.95(m) equatorial
6 1.97(2H.m) I .95 (2H. m)
13 7 1.70(s) 1.67(s)
9 1.28(s) 1.27(s)
10 1.40(s) 1.39(s)
3’ 6.31 (s) 6.35(d, 1.5)
5 6.16(s) 6.26(d, 1.5)
1” 2.75 (2H. m) 2.80 (2H, m)
2” 2.86(2H, m) 2.89 (2H, m)
4”-8” 7.19(3H,m) 7.20(3H.m)
7.24(2H,m) 7.27 (2H, m)
4.80(1H,br,OH) 3.80(3H,s,OMe)

*Assignments were confirmed by ‘H-‘H COSY

spectrum, difference NOE experiments and the
HMBC spectrum. Measured in chloroform-d.

presence of one phenolic hydroxyl group. The difference

OH
ring, but also indicated the presence of a vinyl and two
tertiary methyl groups, a trisubstituted double bond and
benzylic methylenes. Methylation of 1 with methyl iodide
afforded a methyl ether (2) whose ‘HNMR spectrum
(Table 1) showed a signal at 63.80 (3H) confirming the
NOE experiments (Table 3) of 1 and 2 showed the
presence of a 1, 3,4, and Stetrasubstituted aromatic ring
in the molecule of 1. When the methoxyl group was
irradiated a NOE was observed between it and the
aromatic proton at 66.20 (H-S’). The presence of cross-
peaks in the HMBC spectrum (Tables 4 and 5) of 1 and 2
indicated that a monoterpenoid was attached to the
aromatic ring. Comparing the spectral data of 1 with
those of compound 3 which had been synthesized by
Crombie and colleagues [S], confirmed that 1 was prob-
ably an epimer of 3. Its t3C NMR spectral data (Table 2)
were very similar to those of 3, except for the chemical
shifts of the signals due to C-3,4 and 5. The stereochemis-
try of 1 was deduced by difference NOE experiments on 1
and 2. The NOES observed are summarized in Table 3.
Accordingly, the structure of perrottetinen (1) was estab-
lished as shown in formula I.
The structure of isoperrottetin A (15) was established
by comparing its spectral data with those of previously
reported bisbibenzyl derivatives which are occasionally
contained in liverworts [l, 6, 73. Compound 15, an oil,
produced a dark green colouration when treated with
F&I, solution on TLC plates. The IR and UV spectra
showed the presence of an aromatic ring and a hydroxyl
group. The ‘H (Table 6) and 13CNMR (see Experi-
mental) spectral data provided further evidence for the
presence of those functional groups. Spin-spin ‘H de-
coupling experiments indicated that a 1, 2,4,- and a 1,3-
substituted aromatic ring were present in the molecule.
The resonances of 15 in its NMR spectrum for a benzylic
proton and carbon (6,2.84,6,38.2 and 39.5) were char-
acteristic of bisbibenzyl derivatives [6]. The r3CNMR
spectrum of 15 showed 14 carbons: two sp3 methylenes,
seven sp2 methines and five sp2 quaternary carbons. Since
 Bibenzyl cannabinoid from liverwort 861

Table 2. “CNMR spectral data* for TaMe 4. The long range

compounds 1.2 and 3 ‘H-“C coupling correla-
tions of compound 1
1 2 3 c51
C H
C-l 134.6 133.8 134.3
2 122.1 122.7 123.8 8 9,lO
3 31.5 31.6 33.1 1” 3’. 5’
4 40.0 40.1 45.8 4”,8” 2”
5 20.7 20.9 25.1 3” 2”
6 29.7 29.6 29.7 4 1U
I 23.6 23.8 23.3 3’.5’ 1”
8 16.3 76.2 77.3 1 697
9 25.27 25.2t 19.3t 2 3,637
10 25.9t 25.87 27.67 7 2
1’ 109.7 111.3 109.5 6 2
2 153.8 153.5 154.9 4 2
3’ 109.9 110.1 110.0 6 5’
4 141.2 141.1 142.0 2 3
5 108.0 103.3 107.6 3 5
6 155.1 158.9 154.4 5 3
I #I 31.3 37.6 37.4
2” 37.4 38.0 37.4
3,, 142.0 142.1 141.7
4” 128.3 128.4 128.3 Table 5. The long range
5” 128.3 128.4 128.3 *H-‘%Z coupling correla-
6” 125.8 125.9 125.9 tions of compound 2
7” 128.3 128.4 128.3
8” 128.3 128.4 128.3 C H
OMe 55.3
2 6,7
*Assignments were confirmed by 5’ 1”. 3
HMBC spectrum. Measured in chloro- 3’ l”, 5
form-d. 4’ 1”,2”
tMay be interchangeable. 3” 2”. 1”
4”,8” 2”
1 6,7
Table 3. The NOE correlations of compounds 1 and 6 OMe, 5
2 2 3
2” 1I,
1 2 1” 2”, 3’, 5
8 9,lO
Irradiated Observed Irradiated Observed 4 9,lO
9 10
H-9 (6 1.40) H-4,5 H-9 (6 1.39) H-4,5 10 9
lO(61.28) 3,4 10(61.27) 3.4 6 7
7 (61.70) 2.6 7 (61.67) 2.6 1’ 3’,5
OMe (63.80) 5 .8 9.10

the EI mass spectrum of 15 showed a molecular ion peak
(m/z 426) which had the same M, as the tentatively
assigned structure, it was clarified that 15 was a sym-
metric dimer of the bibenzyl derivative. The presence of
two phenolic hydroxyl groups was confirmed by the
demonstration that methylation of 15 with methyl iodide
gave a dimethyl ether 16. The substituted position of the
methoxyl groups on the aromatic rings was established
by difference NOE experiments of the methyl ether 16.
Thus NOES were clearly observed between (i) H-6 (H-6’)
and 63.75 (OMe), (ii) H-12 (H-12’) and 63.77 (OMe) and
(iii) H-14 (H-14’) and 63.77 (OMe). Therefore, it was
confirmed that the phenolic hydroxyl groups were substi-
tuted on C-l (C-l’) and 13 (13’), respectively. Considera-
tion of these chemical and spectral data led to the
conclusion that the structure of isoperrottetin A was 15.
Marchantin-. riccardin-, and other bisbibtnzyl-types
have been isolated from liverworts in our previous studies
[l, 6-83. Compound 15 belongs to a new type of bisbi-
benzyl derivative, and was isolated as a minor component
of this species.
From the chemosystematic view point, Radulu species
are isolated from other species of the Jungermanniales in
containing the new type of bisbibenzyl. Our recent work
has resulted in the isolation of prenyl bibenzyl 5 from
both Jubula japonica and L-epidozia vitrea. It is known
862 M. TOYOTAet al.

Table 6. ‘H NMR spectral data* for isoper- CH,CI,, affording isoperrottetin A (15) (24.5 mg; 0.1 %),
rottetin A (15) and its methyl ether (16) together with perrottetin E (17) [6] (19.7 mg, O.l%),
perrottetin F (18) [63 (130.1 mg, 0.8%), perrottetin G (19)
15t W [6] (26.2 mg, 0.2%). Perrottetinen (1): oil; [a]n (22”)
- 121.3’ (CHCI,; ~0.4). IR vmsr (neat) cm-‘: 3400, 1622,
H-3 (3’) 6.87 (d, 2) 7.03 (d, 2)
1578, 1497, 1427, 1366, 1157, 1057, 752, 698; EIMS m/z
-5 (5’) 7.05 (dd, 8.2) 7.13 (dd, 8.2)
-6 (6’) 6.89 (d, 8) 6.89 (d, 8) ’
(rel. int.): 348 [M]’ (lOO), 333 [M-Me]+ (56). 305 (27),
-7,8 (7’.8’) 2.84 (4H. hr s) 2.90 (4H, s) 265 (47), 257 (36), 243 (21). 105 (16), 91 (20). Isoperrottetin
-10 (l(Y) 6.67 (br d, 8) 6.8 1(br d, 8) A (15); oil; [a&, (22”) 0” (CHCI,; c 1.0). IR v,,,
-11 (II’) 7.12 (t, 8) 7.20 (t. 8) (neat)cm-‘: 3318, 1589, 1495, 1454, 1418, 1343, 1227,
-12(12’) 6.70 (br d, 8) 6.74-6.75 (br) 1157,822,781,693; UV &,,, nm(loge): 213 (4.7), 246(4.1),
-14 (14’) 6.60 (brs) 6.14-6.75 (br) 282 (4), 294 (4); “C NMR (20% CD,OD-CDCI,); 638.2
OMe and 39.5 (CH,), 114.0, 116.6, 117.5, 121.5, 127.6, 129.7,
C-l (1’) 3.75 (3H.s) 130.2 and 133.0 (CH), 135.0, 144.9, 153.0 and 158.2 (C);
-13 (13’) 3.77 (3H, s)
EIMS m/z (rel. int.): 426 CM]’ (33), 319 (lOOk 211 (21).
199 (8), 121 (16), 107 (6), 77 (3).
*Assignments were confirmed by spin-spin
‘H decoupling and difference NOE experi-
Methylation o/ 1 and 15 with MeI. Compound 1
ments. (50 mg) and 15 (23 mg) in Me&O (3 ml) were methylated
TMeasured in 20% CD,OD-CDCI,. with Me1 (3 ml) in the presence of dry K,CO, at reflux
SMeasured in chloroform-d. temp. for 10 hr, respectively. The reaction mixt. was
filtered and the solvent of the filtrate evapd. The resulting
product was purified through a small column packed
that prenyl bibenzyl derivatives show antimicrobial, anti- with silica gel using Et,0 as solvent to give methyl ether 2
fungal and cytotoxic activity [l, 93. The structure of 1 is (45 mg) from 1 and dimethyl ether 16 (20 mg) from 15.
similar to that of A’-tetrahydrocannabinol, a known Compound 2; oil; [aIn (22’) - 157.3” (CHCI,; c 0.6). IR
hallucinogen. The pharmacological activity of 1 is under v,,,(neat) cn-‘: 1615,1574,1453,1422,1366,1229,1157,
investigation. 1100; uv A,, nm (log E):274 (3.9), 222 (4.6). Compound
16: oil; 13C NMR (CDCI,); 637.0, 38.2, 55.1, 55.9, 111.1,
111.3, 114.2, 120.9, 127.8, 128.3, 129.3, 131.5, 133.5, 143.7,
EXPERIMENTAL
155.4, 159.6.
General. TLC: silica gel precoated glass plates with n-
hexane-EtOAc (1: 1, 4: 1). Detection was with Godin Acknowledgement-We thank Dr M. Mizutani (The
reagent [lo] and 1% FeCl, aq. CC: silica gel 60 (40- Hattori Botanical Laboratory, Nichinan, Miyazaki,
63 pm) and Sephadex LH-20 (CHrCl,-MeOH, 1: 1). Japan) for identification of the liverwort.
Spectral data. NMR: 100 MHz for 13C, 400 MHz for
‘H; EIMS: 70 eV.
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(240 mg, 1.4%), 7 (12 mg, 0.1%) 8 (6.5 mg, 0.04%), 9 and Takemoto, T. (1983) Phytochemistry 22, 1413.
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(1.8 g) was rechromatographed on Sephadex LH-20 and Publications, Oxford.
silica gel, then repurified by MPLC on CN silica gel using 10. Godin, P. (1954) Nature 174, 134.