1. PhytochemistryV, ol. 29, No. 2, pp. 459463, 1990.
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0031-9422/90$ 3.00+0.00
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METHOXY FURAN AURANOLS WITH FUNGISTATIC ACTIVITY FROM
LONCHOCARPUSCASTILLOI*
FEDERICO G~MEZ-GARIBAY, RICARDO REYES CHILPA, LEOVIGILDO QUIJANO, Josh S. CALDER~N PARDO and TIRSO
Rios CASTILLO
Instituto de Quimica, Universidad National Autbnoma de Mbxico, Circuit0 Exterior, Ciudad Universitaria, Coyoacan 04510,
Mtxico, D.F.
(Received in reoisedform 6 June 1989)
Key Word Index-Lonchocarpus castifloi; Leguminosae; heartwood; auranols; 2-O- and 2-O-, 3-O-methyl auranols.
Abstract-Chemical analysis of heartwood of Lonchocarpus castilloi afforded, in addition to /3-sitosterol, tour methyl
furan auranols and one a-hydroxydihydrofuran chalcone, named castillenes A-E. Their structures were established by
chemical and spectral methods. The compounds exhibited fungistatic activity against Len&es trabea.
INTRODUCTION
In continuation of our biochemical systematic work on
the chemistry of the Leguminosae, we have investigated
flavonoids and rotenoids with special emphasis on two
related genera, Tephrosia and Lonchocarpus. We have
previously reported the isolation of a wide range of
flavonoids and rotenoids from T. madrensis [ 11, T. nitens
[2], T. watsoniana [3], T. uiridijlora [4] and T. abbottiae
[5]. Similarly, the genus Lonchocarpus is specially inter-esting
because of the wide range of flavonoids which it has
been shown to produce, viz. chalcones and flavanones
[6-81, isoflavones [9-l 1],4-hydroxy-3-phenylcoumarins
[lo, 1 l] and rotenoids [12].
We now wish to report on the flavonoids present in the
heartwood of L. castilloi, a large tree which grows in the
northern part of the state of Chiapas, the southern part of
the State of Tabasco and the Yucatan Peninsula.
RESULTSAND DISCUSSION
The residue from the petrol extract of the heartwood of
L. castilloi separated by column chromatography on
silica gel and prep. TLC on silica gel (see Experimental)
afforded five new flavonoids named castillene A (l), B (Z),
C (3), D (4) and E (5), besides the ubiquitous /I-sitosterol.
Castillene A(l), C,,H,,O, ([Ml’ m/z 294), [~l]n
= 32.12”, was a yellowish oil whose IR spectrum showed
the presence of a carbonyl group in a five-membered ring
(1715 cm-‘) and a benzofuran group (3130, 3050, 1025
and 870 cm- ‘). The UV spectrum ;Imax nm (E): 277 (6481),
332 (3470), indicated the presence of an unconjugated
aromatic system. Structure 1 followed from the typical
‘H NMR signals together with mass spectral peaks which
indicated the presence of a 2-methoxy-2-benzyl furan
coumaranone. The ‘H NMR spectrum (Table 1) showed
chemical shifts for one methoxyl group at 63.30, a
benzylic methylene group as two AB doublets at 63.1 and
*Contribution No. 977 from Instituto de Quimica de la
UNAM (MCxico).
3.39, and two doublets corresponding to protons a, B of a
furan ring at 68.62 (5=2 Hz) and 6.94 (5=2 Hz). These
signals are indicative of a 2-benzyl-2-methoxy furan
coumaranone.
The mass spectrum of 1 exhibited peaks which correl-ated
with anticipated fragments (Ia) and (Ib) derived from
the characteristic fragmentation of a 2-benzyl-2-
methoxy-coumaranone [ 131.
Confirmation of structure 1 was achieved by treatment
with acetic sulphuric acid which furnished the known
aurone 7. All the spectroscopic data of the obtained
product were identical to those of the aurone 7 [143. The
nature of structure 1 followed from typical ‘HNMR
signals together with mass spectral peaks which indicated
the presence of a 2-methoxy-2-benzyl furan coumaran-one.
Castillene B (2), Ci9H1s04 (CM]’ m/z 310), [u]n=
+ 36.8 was a yellowish oil whose IR spectrum exhibited
the presence of a benzofuran (3100,3040,885 cm-‘) with
no carbonyl groups. The UV spectrum I,,, nm (E): 281
(4696) and 293 (4678) indicated the presence of an un-conjugated
aromatic system. The structure 2 was deduced
from the ‘HNMR spectrum (Table 1) which was very
similar to that of 1. Castillene B (2) differs from 1 by the
presence of an extra methoxyl group at the C-3 position,
as the ‘HNMR spectrum of 2 showed a new sharp
methoxy singlet at 63.53 and a one proton singlet at
64.78. The mass spectrum of 2 supported the proposed
structure as it showed a [Ml+ at m/z 310 and other
significant peaks at m/z 219 and 91, corresponding with
fragments IIa and Ib, respectively.
Castillene C (3) was isolated as a yellowish oil, [cr]n=
+ 72.6 and analysed for C,,,H,,O, (CM]’ m/z 354). Its IR
spectrum showed absorptions at 3020, 1605, 1504 and
685 cm-‘, suggesting a furan-methoxyl auranol. The UV
spectrum i,,, nm (E): 284 (4078), 293 (3463), indicated the
presence of a flavonoid with an unconjugated aromatic
system. Structure 3 was deduced from the ‘HNMR
spectrum (Table 1) which was in part close to that of 2,
differing only in the substitution pattern of the B ring.
Castillene C (3) showed two AB doublets (1H each) at
459

2. 460 F. G~MEZ-GARIBAY et al.
O?qTJOJt q%-JJ
OMe
6 w-, ;/ O- -
5 R’ = H, R’ = 0
% R’ = AC, RZ = 0
Sb R’ = H. Ra = OH
0
7
o%oMe @
bhle
11a m/z 219 ( 100%) Ib m/z ‘)I (37’~)
63.02 and 63.23 (J= 14 Hz) due to the benzylic protons,
and two doublets (1H each) centred at 67.55 and 66.85 (J
= 2 Hz) due to protons tl and p of the furan ring. While 1
and 2 have an unsubstituted B-ring, 3 has a meth-ylenedioxy
group attached at C-3’, C-4’ as indicated by
the sharp singlet (2H) at 65.9, and the three proton signals
of the B-ring which were observed as a non-first order
pattern at 6.86 and 6.7. As in case of 2, 3 has a di-substituted
C-ring. The ‘H NMR spectrum also showed
signals for two methoxyl groups (3H each) at 63.41 and
63.52 and one singlet (1H) at 64.75 due to H-3. The mass
spectrum showed a [M]’ at m/z 354 and other significant
peaks at m/z 219 (C,,H, ,O,) and I35 (C,H,O,), which
were consistent with the proposed structure for 3.
Castillene D (4) C,9H,4d6 ([MI’ n~jz 338), [cl&=
+25.7 was another furan-methoxvl auranol which was
also similar in structure to 1. It differed from 1 by the
presence of an extra methylenedioxy group at C-3’, C-4
as indicated in the ‘H NMR by the sharp singlet (2H) at
65.81. The mass spectrum of 4 supported the proposed
structure 4, because the [M]’ was observed at m/z 338
together with other peaks at m/z 203 (C, ,H,O,) and 135
(C,H,O,) derived from the characteristic fragmentation
of 2-benzyl-2-methoxy-furanauranols [ 131.
Castillene E (5) C,,H,,O,, ([Ml’ m/z 282) was iso-lated
as a yellowish oil which lacked optical activity. The
UV spectrum indicated that it could be a dihydrochal-cone,
since it showed bands at 277 and 333 nm typical of a
dihydrochalcone [ 151. The presence of an a-hydro-xyketone
group was suggested by the positive test with

3. Auranols from Lonchocarpus castilloi 461
Table 1. ‘H NMR data* of castillenes l-5, Sa, 5b and 6
1 2 3 4 5 5a 5b 6
H-3 - 4.78 s
H-4 1.4 d 7.11 d
(8) (8)
H-5 7.01 d 7.05 d
(8) (8)
H-6 -
H-7 _ -
H-U 6.94 dd 6.85 d
(132) (2)
H-B 8.62 d 7.55 d
(2) (2)
-dJ 7.1 m 7.1-7.3 WI
3.1 d 3.12 d
+CH,- 3.39 d 3.4 d
(14) (14)
0
‘CH, -
d
-0Me
3.3 s
-OH -
-0Ac -
H-a’ -
H-B’ -
H-5’ -
H-6 -
CH=O -
3.53 s
3.34 s
-
-
-
-
-
-
-
4.75 s
7.15 d
(8)
7.03 d
(8)
6.85t
7.55 d
(2)
6.866.7 m
-
7.45 d
(8)
7.1 dd
(8, 1.5)
-
6.96 dd
(192)
1.65 d
(2)
6.76.55
3.02 d
3.23 d
(14)
5.9 s
3.41 s
3.52 s
3.1 d
3.3 d
(14)
5.81 s
3.3 s
in
6.98 d 6.1 dd
(2) (2)
7.57 d 7.62 d
(2) (2)
7.2 m 1.2 br d
- -
10.5 s
2.07 s
2.4 s
1.35 dd 6.02 dd
(436) (476)
2.91 dd
3.26 dd 3.05 m
(4 and 14,
6 and 14)
7.05 d 7.71 d
(8) (8)
7.60 d 7.4 dd
(8) (8.1.5)
- -
- 7.40 d
(8)
7.10d
(8)
6.92 d 6.97 dd
(2) (2,4)
7.5 d 1.51 d
(2) (2)
1.24 br s
- -
-
- -
2.03 s 11.9 s
- -
5.0 m -
2.75 m -
6.85 d
(8)
7.05 d
(8)
-
-
-
9.85 s
*Run at 80 MHz in CDCl, with TMS as internal standard.
Values are in ppm (6). Values in parentheses are coupling constants in Hz.
tobscured by other signals.
periodic acid and by the IR spectrum since it showed
absorption bands for hydroxy (3500 cm- ‘) and carbonyl
groups (1689 cm-‘). The nature of the hydroxyl group
was confirmed by acetylation of 2, which afforded the
diacetate 5s (IR 1766, 1741 cm-‘; ‘H NMR 62.4 s, 3H,
and 2.07 s, 3H). The ‘HNMR spectrum (Table 1)
confirmed that 5 must be an a-hydroxy-furan dihydro-chalcone,
as it showed three proton signals as an ABX
system pattern at 6 7.35 (.I = 4,10 Hz), 2.97 and 3.26 (J = 4,
14 Hz) due to protons H-a’ and H-p. As in case of the
flavonoids 1 and 2, castillene E (5) has an unsubstituted B-ring
and a benzofuran A-ring because its ‘H NMR spec-trum
showed two doublets (1H each) at 67.57 and 6.98 (J
= 2 Hz) due to protons 0: and jI of the furan ring, and two
doublets (1H each) at 6 7.60 and 7.05 (J = 8 Hz) assigned
to H-6’ and H-S of the benzofuran A-ring.
The mass spectrum of 5 was in accordance with the
proposed structure. It showed a [M]’ at m/z 282
[C,,H,,OJ and other significant peaks at m/z 264 [M
-HzO]+, 191 [M-C,H,]+ and 161 [CgH50J+, de-rived
from the expected fragmentation for this com-pound.
Confirmation of structure 5 was achieved by
reduction with sodium borohydride which furnished 5b.
All the spectroscopic data of the obtained product were in
accordance with the reduction product 5b. Oxidation
of 5b with periodic acid afforded a product which was
spectroscopically identified as 4-hydroxy-Sformyl-ben-zofuran
(6). Compound 6 must therefore be derived from

4. 462 F. GbMEZ-GARIBAY et d.
Table 2. Effect of castillens* and /&sitosterol* on the mycel-ial
growth of the wood-rotting fungus Lrnzitrs trubeu
Compound Growtht (cm) % Inhibition
Control (ether)
/&sitosterol
Control (acetone)
Castillen A
Castillen B
Castillen C
Castillen D
Castillen E
4.01 & 0.05 0
4.00 _+ 0.50 0.25
3.87 & 0.06 0
3.04 + 0.09 21.45
3.48 &O.Ol 10.08
3.09 * 0.09 20.16
2.56 _+ 0.27 33.86
1.23_+0.09 6X.22
*Concentration =0.25 mg,‘ml growth medium.
t Mean of three replicates.
the A-ring. The isolation of 5 is of particular interest as
it is probably the biogenetic precursor of the other
castillens.
Lonchocarpus castilloi heartwood has been classified as
highly resistant against several wood-rotting fungi, in-cluding
Lenaites trabea [16]. Because wood decay resist-ance
is attributed to the presence of inhibitory com-pounds
[17]. the antifungal activity of castillens and b-sitosterol
was examined against L. trabeu. It was found
that all castillens partially inhibited the growth of the
fungus (Table 2). Castillen E was the most active com-pound,
whereas fl-sitosterol did not show any effect. The
biological properties of the six previously known benzyi
coumaranones. all of them isolated from the heartwood
of trees belonging to the Anacardiaceae, Leguminosae
and Rhamnaceae families [I 8, 191, have not been investi-gated
yet. On the other hand, j?-sitosterol in a similar
bioassay and at the same concentration used here
(0.025%), has been reported as inhibitory to the growth of
the wood-rotting fungus Fomes annosus [20].
EXPERIMENTAL
Mps: uncorr. Analysis was done by Miss Gail R. Hutchens
(Galbraith Laboratories, Inc., USA). Lonchocarpus castilloi
Standley was collected in the Lacandon Woods, Chiapas,
Mtxico, April 1977. A voucher is deposited at the Herbarium of
Institute National de Investigaciones sobre Recursos Bibticos,
Xalapa, Ver.
Extraction and separation. Drillings (670 g) from the heart-wood
of L. castilloi were extracted for I day with boiling petrol
(10 1) and the solvent removed in cacuo. The petrol extract (5.9 g)
was percolated on a column packed with 60 g of Tonsil optimum
extra and eluted with petrol, CH,CI,, EtOAc and MeOH. From
frs 3-5 eluted with petrol, /&sitosterol was obtained. Frs 1 and 2
eluted with petrol were combined (2 g) and chromatographed on
a column packed with 20 g of Tonsil and eluted with petrol and
petrol-CH,CI, (9: 1). (3: 1) and (1: I) mixts. Fr. 1 (60 mg), eluted
with petrol, was purified by prep. TLC (CH,Cl,) to give 20 mg of
castillene B (2). Frs 3--6 eluted with petrol-CH,CI, (9: I) were
combined (325 mg) and purified by prep. TLC (CH,CI,) to give
10 mg of castillene A (I), 12 mg of castillene B (2) and 15 mg of
castillene C (3). In the same way frs 10 and 11 (100 mg) eluted
with petrol-CH,CI, (3: 1) were combined and purified by prep.
TLC (CH,Cl,) to give 22 mg of castillene A (1) and 7 mg of
castillene D (4). Finally, frs 7--9 eluted with petrol~CHzC1, (I : 1)
were combined (95 mg) and purltied by prep. TLC (C’H,C12 x 2)
to give 12 mg of castlllene D (4) and 50 mg of ca&llene E (5).
Castlllene .4 (1). C,,H,,O, yellowish oil; [r),+32.17-
(CHCI,3: (’ 1.6). UV ;.::p” nm (c): 705 (21 602). 236 (32 595), 227
(6481), 332 (3470). IR ,Ly,‘,‘:“c’m -‘: 3040. 1715, 1630. 1600 EIMS
(probe) 70eV. rn,r (rel. mt.1: 294 [M]’ (14). 203 [C,,H,O,]’
(100). 175 [C,,H-O,]’ (8). 161 [C,H,O,]‘ (IO). 160
[C,H,03] + (22). 116 [C,H,] + (15). 91 [C-H,] + (37). (Found: C.
73.45; H. 4.90; 0. 21.90 C,,H,.O, requires: C. 73.46; H. 4.80:
0, 21.75%).
C~~stiiirr~r B(2). C,,H,,O,, yellowish 011, Lx&,+ 36.8 (CHCI,;
(’ 1.25). uvi;;;l’n nm (I.): 2 I 1 (47 533). 244 (16 282). 252 (I 6 I88),
281 (4696). 293 (4678). IR vL,‘,‘,““cm- ‘: 3110. 3040. 1600. 1465.
EIMS (probe) 70eV ,?Z 2 (ml. Int.1: 310 [MJ’ (26). 27X [M
-MeOH]* (22), 719 [C,ZH,,O,]’ (100). 201 [M-MeOH
-C,H,]+ (35). 188 [C,,H,O,] r (181, 91 [C;H,] L (74).
Caslillme C (3). C2,,H,,0, yellowish oil. LrlD+ 72.6 (CHCI,;
(’ 1.9). uv i,“y nm 0:): 204 (24 636). 212 (24636) shoulder. 238
(6530) shoulder. 244 (7629). 257 (6713). 2X4 (4078). 293 (3463~.
1R~C,‘~“cm~‘: 3130, 3045. 1715, 1630. 1599. EIMS (probe)
70 eV m,: (rel. Int.): 354 LMJ’ (1X). 32 [M--M&H]* (5), 219
[C,,H,,O,]’ (1001. 18X [C,,H,O,] (13), 135 [C,H&]’ (39).
Castlllenr D (4). C,,H,,O,. yellowish oil. [r],,+25.7
(CHCI,: c’ 1.71 j. UV ;.;:;“’ nm(c): 203 (15 916). 235(15271),2X6
(4143), 329 (1561). IR I,:‘,:” cm -I. 1045, 1715, 1630. 3599. EIMS
(probe) 70eV m,‘: (rel. Int.): 33X [Ml- (50). 203 LC, ,H.O,]’
(55), 187 [C’,,O:O,]+ (371, 135 LCBHi02]- (100).
Castillene E (5). C,:H,.O,, yellowish oil. [IX]~ 0 (CHCI,;
c 1.8). uv;“,“::” nm (I:): 205 (18 441). 236 (2X 651). 277 (8301), 333
(3140). IRv~~~‘~crn-‘: 3500. 3065, 1689, 1496. EIMS (probe)
70eV rn:~ (rel. int.): 282 [M]+ (10). 264 [M-H,O]’ (4), 191
[C,,H,O,]’ (13, 162 [C,H,O,]’ (15). 161 [C,H,O,]+ (lOO),
133 CC,H,O,]- (ll), [C,H,O]‘ (3). 105 [C,H,O]’ (13). 91
[C,H,]+ (37). 77 [C,H,] + (35).
Bioassays. Solns (0.3 mg,‘ml) of each compound were prepd
(castillens in Me,CO, p-sitosterol m Et,O). A portion (0.5 ml) of
a given test soln was poured into a petri dish (60 x 15 mm) and
immediately 6 ml of hot sterilized growth medium (malt-agar
2%) was added. Control plates were treated uith solvent (0.5 ml)
only. The plates were left to stand overnight inside a sterile hood
to remove solvent. Each plate was inoculated with a plug
(6 mm 4) of Lenzites trahra mycelium, taken from the edge of a
12 day culture. Three replicates per treatment were run simultan-eously.
After incubation at 25 for 7 days, inhibition was
determined by measurement of the linear growth of the fungus.
1.
2.
3.
4.
5.
6.
7.
8.
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