F~yr~c~e~~s~~~,Vol. 34, No. 3, pp. 813-818, 1993 0031~9422/93 $6.00+0.

00
Printed in Great Britain. Pefgamon Press Ltd

TWO CAROTENES AND A PRENYLATED BENZOIC ACID DERIVATIVE

FROM ~~~~~ ~~~~CU~

JIMMYORIALA,C. A. J. ERDELMEIER+*
ANTHONYD. WRIGHT, TOPUL RALII and 0~0 STICHER

apartment of Pharmacy, Swiss Federal fnstitute of T~hnol~gy (ETH) Zurich, CH-8092 Z%ich Switzerland; ~~par~ent of
Chemistry, University of Papua New Guinea, Port Moresby, Papua New Guinea

(Received18 January 1993)

Key Word Index-Piper a&cum; Piperaceae; leaves; chromene derivatives; prenylated benzoic acids;
antimicrobial; moliu~cidai.

Abstract-In addition to sti~asterol, piper&on, methyl Z,Z~~ethyl-2~~hromene-6-~r~xylate, methyl 3-(2-

hydroxy-3-methyl-3-butenyl)~-hydroxy-~~oate and methyl (6~-2-tru~-6-hydroxy-2,6-dimethyl-~7-~~dienoate,
three new natural products were isolated from Piper aunt and chara~te~d as methyl S-hydroxy-2,2~~ethyl-
2%chromene-bcarboxylate, 2,2dimethyl-8-(3-methyl-2-bu~~yl~2H~hromene-6~rboxy~ic acid and methyl 3-(6-
hydroxy-3,7-dimethyl-~7-octadienyi~-~methoxy-~nzoate. The structures of all isolates were elucidated by spectro-
scopic methods, mainly 1D and 2D NMR spectroscopy. The antibacterial, antifungal and molluscicidal activities of the
isolates were also investigated.

INTRODUCTION presence of ester (1703 cm-“), hydroxyl(3380 cm-l) and

aromatic (159Q1480 cm-‘) moieties, while the tJV spec-
Piper ~~~~~ L. is a small tree commonly found in
trum showed three absorption maxima at 254,277 and
Papua New Guinea (P.N.G.). Villagers from the coastal
326 nm (log E4.38,3.76 and 3.22) indicating the aromatic
areas of the moron Province of P.N.G. use this plant to
character of I.
heal wounds [l]. In earlier investigations of P. ~u~~~
The ‘H NMR spe&rum of 1 revealed a set of two meta-
phenylpropanoids, like myristicin and dillapiol, benzoic
coupled protons (67.48 d and 7.32 d, J = 1.8 Hz), which
acid derivatives, flavonoids and terpenes were reported
implied the presence of a 1,3,4,5+mbstituted benzene ring,
[2-41. Some of these metabolites were also found to
two olefinic protons (an AB system at 65.66 and 6.35 with
exhibit antibacterial activities f5]. In our study on biolo-
J A,e==9.9Hz), a gem-dimethyl group attached to an
gically active metabol~~s derived from plants which are
oxy~n-~a~ng carbon (6H, 61.49 s), a sharp methyl
employed in the traditional medicine of P.N.G., we are
singlet at 63.88 cloning to a methyl ester and an
currently inv~~gating the leaves of P. educe.
exchangeable resonance (65.46, s), which implied a phea-
The crude petrol extract of the leaves from P. ud~~
olic hydroxyi.
showed, in in vitro biological screening, significant anti-
Methylation of I gave the monomethoxyl derivate lb,
bacterial activity against ~~~~~~#s~bt~lis, ~icr~Q~~
which lacked hydroxyl abso~tions in the ‘H NMR spec-
luteus and ~~cbe~icbiu colt as well as antifungal activity
trum, but gave an additional methoxyl resonance (63.90,
against Pe~iciZlJu~ oxoJic~~. Mollu~cidal potential
s), hence con~~ing the nature of the hydroxyl function.
against Biomphalaria glabrata was also detected.
The five delineated molecular fragments were then
We report on the isolation and s,tructural elucidation of
associated from the results of a 2D NOESY measurement
six bioactive molecules from P. ~a~~~~, i.e. two new
made with 1 and lb. Thus the NOE observed in 1 between
chromene derivatives (I, 2) and a new benzoic acid
l&-9 and H-3, as well as between H,-10 and H-3 implied
derivative (3) as well as the known compounds 4-6,
the ~e~~imethyl function to be allylic. Further, the C-4
together with an as~ssment of their antimicrobial and
to C-10 fragment could be positioned at C-3 on the basis
molluscicidai potential.
of the NOE observed between H-4 and H-5. The final
diagnostic NOE was between the methoxy~ group and H-
7 in lb, thus fixing the methoxyl function in Ib at C-8 and
~n~uen~y also the hydroxyl group in 1. The ester
Compound I showed a molecular ion in the EI mass
group must then reside at C-6 and an ether bridge exists
spectrum at m/z 234, corresponding to the molecular
between C-2 and C-9, as consistent with the 13CNMR
formula of CI$II,Qa. The IR spectrum showed the
data.
To conclusively prove these deductions, I was syn-
*Present address: Rr Willmar Schwabe Arznaimittel, Re- thesized as outlined in the Experimental. The inter-
search and ~ei~prne~t, ~W~7~ Karlsruhe, F.R.C. mediates lc and Id in thii synthesis were both fully

813
814 J. ORJALA et al.

Rl R2

1 CH, OH

lb CH3 OCH,

4’

,CMQ
2 H CH2-CH =C,,
1’ 2’ 3 CH3
5’

OH

COOCH3
I

characterized by ‘II, 13CNMR and mass spectrometry. The key NOE being from Hz-l’ to H-7, thus 2 is 2,2-
Compound 1 is methyl S-hydroxy-2,2-dimethyl-2H- dimethy1-8-(3-methyl-2-buteny1)-2H-chromene-6-car~-
chromene&carboxylate. xylic acid.
Compound 2 had the molecular formula C,,H,,O, by The molecular formula of 3, Ci9Hz604, was estab-
mass spectrometry. The IR spectrum of 2 showed, as the lished by means of EI-mass spectrometry and ’ 3CNMR
only major difference to 1, the presence of an aromatic spectroscopy. In its IR spectrum, absorptions for hy-
acid carbonyl group (1680 cm- ‘). droxyl(3400 cm-‘), ester (1710crn-if and aromatic ring
The ‘H and r3CNMR spectra of 2 (Tables 1 and 2) (1600, 1490 cm- ‘) functions were present, while the IJV
exhibited enough characteristic features of 1 to suggest it spectrum showed an absorption maximum at 255 nm (log
was a C-8 substituted carboxylic acid derivative of I. The E 3.89) confuming the aromatic character of 3.
nature of the substituent at C-8 was deduced from a single The ‘HNMR spectrum of 3 contained a set of three
lH-‘H double resonance experiment. Thus, the signal at coupled aromatic resonances (66.85, d, 1H; 7.83, d, 1H;
63.28 (Hz-l’) collapsed to a singlet upon irradiation of the 7.88, dd, 1H; Jortb=8.5 Hz, J,,= 1.8 Hz), for a 1,3,4-
proton absorbing at 6 5.27 (H-2’), the signals at 6 1.73 (Hs- substituted aryl ring, and two signals at 63.88 (3H) and
4’ and H,-5’) thereby losing line-broadening. 3.89 (3H) for an aryl methoxyl group and an aryl methyl
A 2D NOESY measurement ma& with 2 confirmed ester function, which were also evident in the 13C NMR
the position of the y,y-dimethyl ally1 side chain at C-8. spectrum (55.5 and 51.8). The ‘HNMR spectrum of 3
 Constituents of Piper uduncum 815

Table 1. ‘H NMR (300 MHz, CDCI,) data of compounds 1,2, lb, le and Id

Proton (s) at
carbon 1 2 lb lc Id

3 5.66 d (9.9) 5.63 d (9.8) 5.65 d (9.9) 1.82 I (6.7) 1.79 t (6.9)
4 6.35 d (9.9) 6.33 d (9.8) 6.33 d (9.9) 2.78 t (6.7) 2.75 t (6.9)
5 7.33 d (1.8) 7.59 6r s 7.31 d (1.9) 7.38’ nt 7.34 s
7 7.48 d (1.8) 7.74 br s 7.45 d (1.9) 7.408 m -
9+10 1.49 s 1.44 s 1.50 s 1.37 s 1.36 s
1’ - 3.28 d (7.3) - 3.08 t (6.9)
2’ _- 5.27 t (7.3) --- - 1.77 t (6.9)
4’+5 - 1.73 s - - 1.34 s
COOMe 3.88 s - 3.88 s 3.85 s 3.83 s
OMe - 3.90 s -
OH 5.46 s - 5.68 -

~A~ignments interchangeable.

Table 2. r3C NMR data (75.5 MHz, CDCL,) of compounds 1, 2, Ib, le and Id

C 1 2 lb le Id

2 78.5 s 77.2 s 77.6 s 76.6 s 75.0” s

3 130.8 d 130.7 d 131.0 d 21.9 t 21.8 t
4 122.8 d 122.1p d 121.9 d 32.6 c 32Sb t
4a 12W s 120.4b s 121.2s s 120.6b s 1 19.4d s
5 119.9 d 126.6 d 121.0 d 122.7 d 123.6 d
6 122.8b s 121.6b s 122.Obs 121.5b s 121.4” s
7 116.3 d 131.7 d 113.1 d 113.1 d 118.36 s
8 143.48 s 129.3 s 148.ff q 144.8’ s 147.6’ s
8a 144.1a s 155.4 s 146.3’ s 145.28 s 143.7” s
9+10 28.3 q x 2 28.3 ij x 2 28.2 g x 2 26.8 q x 2 26.7 9 x 2
1’ - 28.2 t - - 22.3 t
2 -- 122.0” d - 32.1b t
3 - 132.5 s - 73.5” s
4 -^ 25.8 q - - 26.5 q
5 - 17.9 q - - 26.5 q
COOR 166.8 s 171.2 s 166.9 s 167.1 s 167.8 s
COOMe 51.9 q - 51.9 q 51.8 q 51.4 q
OMe -- 56.3 q - -

“-dAssignments interchangeable.

contained a further set of coupled spins for a hy- - 1391’ (M - C,H t $0) in the EI mass spectrum of 3. The
droxylated monoterpene side chain, whose structure stereochemistry of the A2’*3’double bond was established
could be determined from the ‘H-‘H and’H-t3C! (one as (E) on the basis of the 13CNMR shifts of the vinyl
bond, J= 136 Hz) COSY spectra. Thus, the methylene methyl group (C-9’) [6].
protons at C-l’ (63.33) coupled to the olefinic proton at With the basic fragments of 3 established, the connec-
C-2’ (65.35) which in turn showed an ally& coupling to tivity between them required solution. From the results of
the protons of the C-4’ methylene group (62.11) and to the a 2D-NOESY experiment, it was evident that the 6-
C-9’ methyl group (61.72). The two protons of the C-4’ hydroxy-3,7-dimethyl-2,7octadienyl, methoxyl and
methylene group further coupled to the methylene pro- methyl ester functions had the regiochemical relationship
tons at C-S (S 1.6’7),which in turn coupled to the oxygen- as shown in 3. The diagnostic NOEs being from Hz-l’ to
bearing methine proton at C-6’ (64.06). The latter proton H-2 and the methoxyl group, and from the methoxyl
displayed allylic coupling to one of the C-g’ exo-methyl- group to H-5, clearly indicating the methoxyl group to be
ene protons (64.93). Both C-8’ exo-methylene protons adjacent to H-5 and the 6-hydroxy-3,7-dimethyl-2,7-
(64.82,4.93) showed allylic coupling to the protons of the octadienyl side chain. The methyl ester function must
C-10’ methyl group (61.72), thus establishing the side therefore be at C-l. Confirmation of the above structural
chain to be 6-hydroxy-3,7-dimethyl-2,7-~dienyl. deductions came from comparison of the ‘II and
The presence of a 6-hydroxy-3,7-~~ethyl-2,7-~ta~ 13C NMR data with those of compound 5 and with those
dienyl chain was also supported by the major ion at [M published for methyl 3~3,7-dimethyl-2,6-octadienyl~
816 J. ORJALA et al.

4-methoxy-benzoate [7]. Compound 3 is methyl Plant material. The plant material was collected near
3-(6-hydroxy-3,7-dimethyl-2,7-octadienyl)-4-methoxy- Gawam village, Morobe Province of P.N.G., during
benzoate. September, 1988 [12]. Herbarium specimens are depos-
Together with the new compounds the three known ited at Herbarium ZT, ETH, Zurich, Switzerland, as well
metabolites 4, methyl 2,2-dimethyl-2H-chromene-6- as at UPNG Herbarium, Port Moresby, P.N.G. and at
carboxylate [S], 5, methyl 3-(2-hydroxy-3-methyl-3- the National Herbarium in Lae. P.N.G.
butenyl)-4-hydroxy-benzoate [8] and 6, (6S)-2-trans-6- Extraction and isolation. Air-dried and powdered
hydroxy-2,6-dimethyl-2,7-octadienoate [9, lo] were also leaves (I .55 kg) were percolated with petrol at room temp.
isolated. Compounds 4 and 5 were reported from Piper Removal of the solvent under red. pres. furnished a
hostmannianum (Piperaceae) [S], while 6 was reported resinous mass (91.0 g, 5.9%). The extract was divided into
from Gymnoclndus chinensis (Leguminosae) and Artemi- 5 parts and each part was subjected to VLC (RP-18
sia santolinifolia (Compositae) [9, lo]. This is, however, material, 40 g) using a step gradient of MeOH-H,O (3:2,
the first report of compound 6 from Piperaceae. 4:1, lO:O) to give three frs, A (1.5 g), B (48.3 g), and C
The isolates 1-6 were tested for their biological activi,ty (15.2 g). Frs A and B were worked up as follows: Fr. A was
against the bacteria Bacillus suhtilis, Micrococcus luteus subjected to VLC (silica gel, 50 g) using a hexane-EtOAc
and Escherichin coli, and the fungus Penicillium oxalicum step gradient to afford 4 frs (Al-A4). From fr. A3, 5
using a TLC bioassay [I 11. Further the molluscicidal (7.2mg) was isolated by RP 18 HPLC (MeOH-H,O,
effect against Biomphalaria glabrata was evaluated. Mini- 3:2). Fr. B was subjected to VLC (silica gel, I50 g) using a
mum growth inhibition concentrations on TLC as well as hexane-EtOAc step gradient to yield 5 frs (Bl-B5). Fr.
the lethal concentration are given in Table 3. B2 was further fractionated by MPLC (silica gel, column
The biological activities reported here suggest that the A) giving 8 frs (B2.1-B2.8), the mobile phase being
topical application of P. aduncum leaves will have a EtOAc hexane (1:9). Fr. B2.2 was further purified by
beneficial effect on infected wounds and the antimicrobial HPLC on RP18 material using a mixt. of MeOH-H,O
activity of these metabolites also support the traditional (9:l) as eluent, to give 4 (8.5 mg). Fr. 82.4 yielded pipe-
use as a remedy for wounds. riton. Fr. B3 was further fractionated into 10 frs
(83.1. B3.10) by MPLC (silica gel, column B), the mobile
phase was Me,CO-hexane (1:9). Fr. B3.3 was further
EXPERIMENTAL
purified by HPLC on RP18 material using MeOH-H,O
General. Mps: uncorr; UV: MeOH; IR: KBr or film; (4: I) as eluent, to give 1 (20.4 mg). Fr. 3.4 gave stigma-
optical rotations: MeOH; EIMS 70 eV, ‘HNMR sterol upon addition of Me&O. Fr. B3.7 was further
(300 MHz) and 13CNMR (75.5 MHz): CDCI, using purified by HPLC on RPl8 material using a mixt. of
TMS or solvent (67.26 resp. 77.0) as int. standard. MeOH-H,O (7:3) as eluent, to afford 2 (10.7 mg). Fr. B4
Separation. All solvents were of analyt. quality. Silica was further fractionated by MPLC (silica gel, column B)
gel (Merck) and RP-l&material (Baker) for VLC had a yielding 9 frs (B4.1-B4.9). The mobile phase being
particle size of 40--63 pm resp. average particle size of Me&O-hexane (1:9). Fr. B4.3 gave 6 (15.3 mg) and fr.
40 pm. MPLC sepn was carried out using Biichi MPLC B4.5 yielded 3 (6.3 mg), both using prep. TLC with
columns 80 cm x 4.9 cm (column A), 46 x 3.6 cm (column toluene-EtOAc (9:l) as an eluent.
B), and 46 cm x 2.6 cm (column C). The columns were Bioassay procedures. The bioautographic assays were
drypacked with TLC silica gel HF 254 (Merck), particle carried out as previously described [ 111. Test organisms
size 1.5pm. HPLC sepns were performed on a Spherisorb were B. subtilis (ATCC 6633), M. luteus (ATCC 9341), E.
S5 ODS II, 5 pm, 250 x 16 mm column with UV detection coli (ATCC 25922) and P. oxalicum (Table 3). The screen-
at 254 nm and Lichrosorb Si 60, 5 pm, 250 x 8 mm with ing for molluscicidal potential was carried out as pre-
UV detection at 340 nm. viously described [13], with the modification that the

Table 3. Biological activity of isolates l-6

Compound

Organism 1 2 3 4 5 6 CA M.NO,

E. coli* 8.5 - 0.15 NT

M. lute& 8.5 3.2 10.8 --- 6.6 0.10 NT
B. subtilis* 8.5 2.0 10.8 --- 13.0 _~ 0.10 NT
P. oxalicum* 17.0 15.5 - -- NT 1.66
B. glabratat 30 30 NT NT $ $ NT NT

*Minimum growth inhibition concentration in nmol on TLC.

tlOO% lethal concentration in ppm.
$ = No activity at 35 ppm.
CA = Chloramphenicol; M.NO, = miconazole NO,; - = no inhibition at 20 nmol; NT=not tested.
 Constituents of Piper aduncum 817

samples were first dissolved in 100 ~1 of EtOH and then (14), 257 [M- 151’ (lOO),227 (2), 197 (3), 128 (4), 115 (4);
diluted to 100 ml with distilled H,O. The test organism ‘HNMR: Table 1; ‘jCNMR: Table 2.
was Biomphalaria glabrata. Methyl 3-(6-hydroxy-3,7-dimethyl-2,7-octadienyl)-4-
Methyl 8-hydroxy-2,2-dimethyl-2H-chromene-6- methoxy-benzoate (3) (6.3 mg, 0.0004%). Clear oil; [alLo:
carboxylate (1) (20.4 mg, 0.0013%). Amorphous solid; mp - 10.0” (MeOH; c 0.23); UV 1zc” nm (log E):285 nm sh
94”; uv n%:” nm (log E):254 nm (4.38), 277 nm sh (3.76), (3.37), 257 nm (3.89); IR $A!$cm-‘: 3400, 2920, 1710,
326 nm (3.22); IR YE:: cm - ‘: 3380,1708,1590,1480,1438, 1600, 1490, 1435, 1295, 1265, 1250, 1120; EI-MS m/z
1322, 1204, EIMS m/z (rel.int.): 234 [M]’ (14), 219 (100x (rel.int.): 318 [Ml+ (3), 300(6), 286(21), 232(16), 179 (lOO),
174 (8), 160 (lP), 129 (17), 115 (7), 103 (P), 91 (lo), 77 (15); 161 (17), 149 (15), 121 (24); ‘HNMR (CDCl,, 300 MHz):
‘HNMR: Table 1, 13CNMR: Table 2. 6 1.67 (2H, m, H-S), 1.72 (6H, s, H-P’ and H-10’), 2.11(2H,
Methylation of 1 (4.2 mg) with CH,N, afforded lb t,J=6.9 Hz,H-4’), 3.33 (2H,d,J=7.3 Hz, H-l’), 3.87(3H,
(4.3 mg, 97%). s, COO&&$, 3.88 (3H, s, OMe), 4.06 (lH, t, J=6.2 Hz, H-
Methyl 8-methoxy-2,2-dimethyl-2H-chromene-6- 6’), 4.82 (lH, s, Ha-8’), 4.93 (lH, s, Hb-8’), 5.35 (lH, t, J
carboxylate (lb). Clear oil; UV eiyH nm (log E): 290 nm =7.4Hz,H-2’),6.85(1H,d,J=8.5Hz,H-5),7.83(1H,d,J
(3.48), 249 nm (4.18), 243 nm (4.16); IR ~2; cm-‘: 2940, =1.8 Hz, H-2), 7.88 (lH, dd, 5=1.8, 8.5 Hz, H-6);
1710, 1365, 1305, 1200, 1090; EI-MS m/z (rel.int.): 248 ’ 3C NMR (CDCI,, 75.5 MHz): 6 16.0* (q, C-P’), 17.7* (q,
[M]’ (29), 233 [M-Me]+ (lOO), 218 (7), 174 (6), 129 (8), C-10’), 28.3 (t, C-l’), 33.0 (t, C-5’), 35.8 (t, C-4’), 51.8 (q,
77 (8); ‘HNMR: Table 1; 13CNMR: Table 2. COOMe), 55.5(q, Om, 75.5 (d, C-6’), 109.5 (d, C-5), 110.9
Total synthesis of 1. Methyl 3,4_dihydroxybenzoate was (t, C-8’), 122.2 (s, C-l), 122.3 (d, C-2’), 129.4 (d, C-6), 129.9
obtained by dissolving 4 g 3,4-dihydroxybenzoic acid (s, C-3), 130.8 (d, C-5), 136.3 (s, C-3’), 147.6 (s, C-7’), 161.1
(Fluka, purum) in 10 ml MeOH (Fluka p.a), adding cont. (t, C-4), 167.2 (s, COOMe). *Assignments may be inter-
H,SO, (1 ml) and heating the resultant soln under reflux changed.
for 5 hr. Aq. work-up followed by VLC (silica gel, Methyl 2,2-dimethyl-2H-chromene-6-carboxylate (4)
EtOAc-hexane step-gradient), gave 3.5 g of the methyl (8.5 mg, 0.0005%). Spectroscopic and chemical data are
ester. The ester (1.5 g) was dissolved in toluene and identical with those previously reported [8].
condensed with isoprene (Fluka purum) as described Methyl 3-(2-hydroxy-3-methyl-3-butenyl)-4-hydroxy-
elsewhere [14]. The condensation with isoprene gave a benzoate (5) (7.2 mg, 0.0005%). [a]$‘: +4.8” (MeOH; c
mixt. of 2 products in a ratio of 2: 1, which were sepd by 0.29); spectroscopic and chemical data are identical with
MPLC (silica gel, column C) using a EtOAc-hexane those previously reported [S].
gradient. The more polar product lc (600 mg), methyl 8- Methyl (6S)-2-trans-6-hydroxy-2,6-dimethyl-2,7-octa-
hydroxy-2,2dimethylchroman-6-carboxylate, showed dienoate (6) (15.3 mg, 0.0010%). [a];‘: + 1.4” (MeOH;
the introduction and condensation of one isoprene c 2.01); Spectroscopic and chemical data are identical
moiety, while the least polar compound Id (350 mg), with those previously reported [P, lo].
methyl 2,2,9,9-tetramethyltetrahydropyrano[3,2-h]
chroman-6-carboxylate, showed the introduction of two Acknowledgements-This research was supported by the
isoprene units. Compound lc (100 mg) was de- Swiss National Science Foundation. We are indebted to
hydrogenated with DDQ in CsH6, as described [S]. The Dr M. Baltisberger (ETH Zurich) for the preparation of
resulting product (15 mg) was purified by HPLC (silica the herbarium specimens and for his assistance with
gel) using Me,CO-hexane (1:4) as eluent. The synthetic collection of plant materials. Our thanks are also due to
product showed identical chemical and spectroscopic Mr P. Katik (National Herbarium, Lae) for his valuable
data as the isolated compound 1. help concerning botanical matters. We are also grateful to
Methyl 8-hydroxy-2,2-dimethylchroman-B-earboxylate Mr R. Htiiger and Mr 0. Greter (ETH Zurich) for
(lc). Crystalline solid; mp 95”; UV 1zz” nm (log s): recording the mass spectra and to Dr E. Zass (ETH
298 nm (3.77), 268 nm (4.05); IR vkti cm-‘: 3400 br, 2970, Zurich) for performing literature searches.
1700, 1590, 1490, 1370, 1330, 1200, 1120; EI-MS m/z
(rel.int.): 236 [M]’ (14), 221 (7), 205 (24), 189 (ll), 181
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