Full text of DOI:10.1016/S0031-9422(00)89891-X

1 9 7 1 , Vol. 1 0 , pp. 2 4 3 3 t o 2 4 5 0 .
Prin t ed in En glan d.
THE FLAVONES OF APULEIA
R. B R AZ
and 0. R.
da Produtos Naturais de
de Amparo Pesquisa do Estado de
Paulo, Brasil
Paulo, Universidade
de
(Received 9 July 1970)
Abstract-The wood of
yielded (+)-pinitol,
oxyayanin-A
(Vog.) Macbr. (Leguminosae, subfamily
and ten flavones. Three of these flavones are the known compounds ayanin
and oxyayanin-B (IX). The constitutions of the seven new flavones,
apuleisin
apuleitrin
apuleirin
and
oxyayanin-A
were established. A structural proposal is also advancedfor leiocarpin (XVI), a new
isolated from the bark. The unusual oxygenation pattern of the flavones is discussed.
INTRODUCTION
leiocurpa
Macbr. (= A.
Mart.) (Leguminosae-Caesalpinioideae) is
a tree which may attain considerable proportions. The fine-textured durable yellow timber
is highly appreciated for heavy construction, flooring, door frames, wheel-wright work, shafts
of vehicles and fence posts.’ The species occurs over a huge territory from Northern Argentine.
cites also A.
Spr. ex Bth, which grows in the Amazon valley.2 More recently,
however, the opinion prevails that the genus Apuleia Mart. is monotypic and that A.
leiocarpa is the binomial to be retained. We have compared, by TLC, extracts of different
parts of specimens of A.
collected near
State, and classified by the
botanist J. Pires, and of A.
collected near Brasilia, D. F., and classified by
the botanist E. P. Heringer, and were unable to detect any difference in chemical
composition.
The trunk of the last mentioned specimen was separated into heartwood,
bark, which were extracted separately. By a combination of fractional crystallization and
column chromatography (+)-pinitol, and nine flavones were isolated from the
heartwood extract. A tenth flavone was shown to be present by TLC. The same flavones and
were also detected in the extract. The composition of the bark extract,
and
however, was found to be different. Instead of the flavones, a new pterocarpan and
sitosterol were present.
The derivation from a
skeleton of the group of nine heartwood constituents was
immediately apparent upon inspection of their UV spectra. In order to classify the substances
*
Part XXIX in the series “The Chemistry of Brazilian Leguminosae”. For Part XVIII see
42, Supplement (1970).
This paper is based on the D.S. Thesis submitted by R. Braz Filho to the Universidade Federal Rural
do Rio de Janeiro (1970); for preliminary communications see
42,
Supplement 55 (1970); 43, (1971) in press.
On leave of absence from
de Quimica, Universidade Federal do
Fortaleza.
Pesquisador-Conferencista, Conselho
de
Brasil.
W. B.
and C. T.
Useful Plants of Brazil, p. 124, Holden-Day, San Francisco (1966).
A. DUCKE
p. 112,
C. T.
,
a Flora
do
II. As Leguminosas da Amazonia
2nd edition,
do Norte, No. 18,
do Rio de Janeiro, personal communication (1969); see also S. J.
and P. W. HESS
,
Timbers of the New World, p. 233, Yale University Press, New Haven (1943).
2433

2434
R.
FILHO and 0. R.
TABLE 1. NMR SPECTRA OF Apukia
2’-OH OH OH
4.55
Compound
MHz
8-H
3.26
5 ’ - H
6-H
100
100
100
60
60
60
2.13
Ib
Ic
-2.28
4.67
3.37
3.18
3.08
3.36
3.15
3.16
3.42
2.69
2.57
Id
Ie
Va
3.26
d
-2.24
4.56
d
Vb
vc
Vd
Ve
Vf
3.33
3.20
3.42
3.23
2.86
60
60
60
60
60
d
d
2.98
d
2.47
d
4.52
3.28
d
-2.53
3.19
d
2.79
d
3.20
d
2.82
d
3.18
2.64
- 2 4 3
3.45
3.08
264
2.32
d
60
60
d
J2.3
2.55
2.61
d
3.15
3.25
2.55
2.33
d
60
60
v u
3.27
d
-2.46
-2.35
1.95
2.14
3.62
d
3.54
d
J2.3
3.25
d
J2.3
3.56
d
60
60
d
3.35
d
2.96
d
2.42
d
4.72
3.41
3.13
3.16
2.73
2.58
2.65
100
60
3.63
d
J2
3.56
d
3.63
d
3.26
d
3.41
d
60
J2.6
3.68
d
J2.6
3.56
d
X
-2.86
60
2.32
d
J2
d
J 9
dd
J2
J2
m, includes the
of the two phenyl groups.
Unless otherwise indicated, the chemical shift values
refer to singlets. d
doublet; dd
double
doublet. Identical values for two or more signals indicate overlap of the corresponding spectral lines. When
localization of groups is not specified, individualization of signals was either doubtful, or omitted to allow
inclusion of values for differently located groups under the same heading.

2435
The flavones of Apuleia Ieiocarpa
CONSTITUENTS AND OF THEIR
6.05
6.07
6.12
6.15
6.31
6.12
6.13
6.34
7.78
7.75
7.65
6.10
6.08
6.11
6.12
6.37
6.09
7.68
7.65
6.03
6.08
7.47
7.86
6.06
6.09
6.06
6.35
6.06
6.06
6.06
6.23
6.09
7.76
7.53
7.65
768
6.05
6.05
6.05
605
6.05
6.05
6.12
6.20
6.28
5.99
6.07
6.05
6.07
6.12
6.18
607
7.63
5.93
6.07
5.97
6.07
5.97
6.10
6.02
6.10
6.08
6.13
7.65
7.80
602
6.09
6.10
6.04
6.13
6.35
6.17
6.12
6.17
6.10
613
6.12
7.70
7.66
629
7.78
6.39
6.12
6.02

2436
R. BRAZ FILHO and 0. R.
into groups with identical oxygenation patterns, all were exhaustively methylated with
sulphate and Counting the number of the methoxyl groups of the de-
rivatives by NMR spectroscopy revealed that five compounds were heptaoxygenated, while
three were hexaoxygenated and one was pentaoxygenated.
Heptaoxygenated Flavones
Two of the heptaoxygenated flavones yielded an identical methyl ether. A hydroxyl-
methoxyl count revealed that one of the parent compounds, for which we propose the name
apulein, is a dihydroxypentamethoxyflavone (Table
is a trihydroxytetramethoxyflavone (Table 1, Compound Ib). The hydroxylproton signals
in the NMR spectra of apulein (2.00 and and of the trihydroxyflavone (-2.28,
and indicated not only that a pair of OH groups may be situated in identical environ-
ments in both compounds, but also that the additional hydroxyl in the latter compound
must be strongly chelated. The structure of had thus to be considered
Compound
and the other one
for this compound. Aq. HCI promotes selective demethylation of 5-methoxyl groups in
and the use of this reagent to convert apulein to the trihydroxy derivative confirmed
their structural relationship.
The two remaining hydroxyls must be located in different environments, the one
represented by a proton signal at about
(Table I, Compounds
and b) being involved in
a weak intramolecular hydrogen bond. Since a hydroxyl at position 3 gives rise to a proton
signal at about
the OH group in question was placed at
where it would be
TABLE 2. MA S S SPECTRA OF Apuleia CONSTITUENTS AND OF THEIR METHYL ETHERS:
DUE TO CLEAVAGE
OF THE HETEROCYCLE
X
Y
Z
V
W
T
Ion
Com p ou n d m /e
%
%
167
167 31
195
167
195
331
181
181
167
167
19.5 13
181 15
167 51
153 15
167 13
211
197
211
7
4
7
194
194 14
6
179 21
210
1
1
1
3
2
179
207
179
207
343
193
193
179
179
32
1
8
3
5
3
3
3
12
Ib
If
V a
Vb
Vd
196
210
182
210
182
182
196
166
166
180
180
166
222
194
222
358
208
208
194
194
194
222
164
1
9
1
16
2
1
2
7
7
1
195
167
195
6
5
8
139
167
139
139
153
123
123
3
12
1
2
7
7
10
183 13
211
183
183
197
167
167
181 25
181 12
167 13
10
5
5
4
14
26
167 17
1
6
167
181
151
151
165
165
151
2
7
2
7
5
2
7
5
2
8
17
3
2
137 13
167 13
195
151
179 18
137
3
1
207
163
1
2
X
2
123
4
1
Key to Table-2.
-M e’
- M e ’-
v
T
Y
F.
and A.
9,143 (1960).
T. J.
and R. J. HIG H ET, Australian Chem. 17,428 (1964).

The
of Apuleia leiocarpa
2437
TABLE 3. MASS SP ECTRA
Apuleia CONSTITUENTS
OF
METHYL
P EAKS DUE TO CLEAVAGE
OF THE SUBSTITUENTS ON C-2’
3
I o n
M
N(M-1)
O(M-15)
P (M-17)
R(M-31)
S(M-43)
C o m p o u n d
%
mle
%
mle
%
mle
%
%
mle
404 98
390 100
432 46
376 100
432 42
540 100
390 100
404 100
360 100
360 100
374 82
402 53
344 100
403 31
389 21
431
389 100
375 34
417 100
361 14
417 100
387 56
373 25
374 16
360 25
402 16
373 59
359 49
401 65
345 16
361
347
389
333
389
497
347 10
361
317 20
317 33
331 12
5
I b
If
Va
Vb
Vd
6
8
9
2
7
375
359 36
346
402
510
3
13
1
431 13
539 22
389 33
403 43
359 25
359 27
373 40
401 15
343 79
401
509
58
3
525
2
523
373
2
6
375 33
389 71
345 10
359 15
373 10
329 32
329 88
343 100
371 100
360 18
387 10
343 52
343 58
357 87
374
330
8
7
4
345
359
7
7
330 21
344 24
372 24
387 54
329
359
301 45
2
X
9
327
7
314
3
313
5
R
X (Table
subjected to the polarizing influence of the heterocyclic oxygen. This seemed reasonable, if
considered in conjunction with evidence pointing to the presence of the other hydroxyl at
the
position: The UV spectrum of apulein is not altered upon addition of
or but revealed that decomposition of the substance takes place upon
addition of
A preliminary indication of the localization of the four methoxy groups in the partial
structures of apulein and was obtained by mass
The well known fragmentation mode, through compensated displacement of electrons
in the heterocycle, led to ions (Table 2, Compounds and Ib) whose mass revealed the
presence of two of these methoxyls in ring A, while the third one must be located either on
the heterocycle or, together with the fourth one, on ring B. These assignments, however,
L.
in The Chemistry of Flavonoid Compounds (ed it ed by T. A. G E ISSMAN ), p . 107,
Oxfor d (1962).
C. S.
A. P E LT
a n d J . L.
, P .
Australian J. Ch em . 17,975 (1964).
a n d M. BARBER , J. Heterocyclic Chem. 2,262 (1965).
E
R

2438
R . BR AZ F I L H O a n d 0. R .
are somewhat ambiguous. Not only are the pertinent ions of low relative abundance, but, as
Table 2 also shows, fragments of identical mass may be formulated as arising either from
ring A (ions V) or ring B (ions X). Confirmation was secured through the more reliable
information contained in Table 3. Loss of 43 mass units from the molecular ion, as featured
in the reactions
the presence of a hydroxyl at position 2’ had already been ascertained, the highly favoured
losses of 17 (M P) and of 31 mass units (M R) were to be and constitute
additional evidence for the presence of a methoxyl group at C-3.
Mass spectral analysis thus authorized the expansion of the partial formula
respectively to and b. The proton signals in the aromatic region of the NMR spectra of
0
S and M
S, is typical of
Since at this stage
and b
all apulein derivatives (Table
Compounds
were represented by singlets, indicating
the absence of ortho or
related aromatic hydrogens. This fact assigned the methoxyl of
ring B to position 4’. The NMR signals at
with the protons at C-6’ and C-3’. The resonance
and
had, consequently. to be correlated
frequency of the remaining aromatic
proton, however, was compatible with its presence either at C-6 or at C-8, and thus was not
immediately helpful to decide unequivocally between two structural alternatives.
Definite proposals concerning the structures of apulein and of 5-0-demethylapulein could
be advanced only after degradation experiments. Alkaline hydrolysis of apulein led to a
m i x t u r e o f
and of
trimethoxybenzoic acid. These fragments must correspond to ring A, since under the
conditions ring B must have been destroyed. The substitution pattern of ring B could be
defined after alkaline degradation of di-0-ethylapulein which led, in addition to the previous
fragments, to
to the reaction, yielded,
acid. Di-0-methylapulein, when submitted
acid. All these degradation products were
characterized by physical and spectrometric means and by direct comparison with authentic
samples. Clearly, they not only defined the localization of all oxygen functions, but also
corroborated the assumption that the compounds are flavones. Accordingly, apulein must be
and 5-0-demethylapulein must be
(Ib).
Once the structures of the apuleins had been established, it became necessary to re-
interpret an experimental fact which did not fit the
pattern for the
apuleins. Nitric acid is known to effect oxidative demethylations leading to
and treatment of apulein and of 5-0-demethylapulein with this reagent led to a single red
quinone. This was reduced to a quinol which was acetylated without purification. NMR
spectrometry revealed the product to be the trimethoxytetraacetoxyflavone of structure Ie,
since a signal at relatively low field places one of the four acetyl groups at C-5 of the flavone
Treatment of apulein and of
the quinone IV and it must be concluded that nitric acid produces not only
with nitric acid thus afforded
but also
ortho-quinones. A reaction of this type, involving chromic oxide-acetic acid as the reagent,
has been reported
The oxygenation pattern of apuleisin, a third heptaoxygenated flavone of
leiocarpa, differs from the pattern of the apuleins only with respect to ring B. Indeed, the
fully 0-methylated derivative of this tetrahydroxytrimethoxyflavone yielded, upon
J. H.
a n d D. W. CAM E R O N ,
(1966).
A. P ELTER a n d P . ST AN T O N ,
M. KR I S H N AMU R T I , T. R.
Chem.
(C) 1933 (1967).
a n d P . R. S H AN KAR AN , Tetrahedron
J .
J .-P .
a n d S.
Bull.
Chim. 2712 (1963).
3767 (1966).
D. KU MAR I , S. K. M UKE R J E E a n d T. R. S E S H AD R I ,

The flavones of Apuleia Ieiocarpa
2439
ment with alkali,
acid, besides the
methoxyacetophenone which had also been obtained upon degradation of the apuleins.
The ortho relationship of the two ring B protons which was thus indicated, could also be
appreciated through examination of the NMR spectrum of apuleisin (Table 1, Compound
Va) which shows two ortho split doublets at
As in two of the free hydroxyls in apuleisin must occupy positions
5 and since their protons also gave rise to NMR signals respectively at and
and
Both these hydroxyls form part of ortho-dihydroxy systems. This was ascertained by heating
apuleisin with dichlorodiphenylmethane. As indicated by mass spectrometry (Tables 2, and 3,
Compound Vd), a diphenylmethylenedioxydihydroxy trimethoxyflavone resulted. The two
hydroxyls of this derivative form an ortho-quinol function involving the hydroxyl at C-5,
since the compound is labile in alkali and originates a PMR signal at
Compound Vd). On this evidence, the structure
flavone (Va) is proposed for apuleisin.
(Table 1,
Apuleitrin and apuleirin, the fourth and fifth heptaoxygenated flavones of Apuleia
Zeiocarpa, again yielded an identical methyl ether. Apuleitrin differs structurally from
apuleisin
Compound
only with respect to ring B. Indeed, while its mass spectrum (Tables 2 and 3,
reproduces fully all peaks originating through fragmentation of ring A of
apuleisin, alkaline degradation of tri-O-methylapuleitrin afforded
acid. The structure of this methyl ether must consequently be represented by
firmation was obtained by direct comparison with
prepared by methylation of
Con-
kindly
supplied by Prof. P. R.
The mass spectral indication of the substitution of ring A in apuleitrin was confirmed
by NMR and UV spectrometry. The existence of a chelated hydroxyl (-2.43 Table 1,
Compound
borate) was easily demonstrated. At this point only the relative site of one hydroxyl and
two methoxyls at positions 4’ and 5’ remained to be established. The protons at C-2’ and
6’ of apuleitrin gave rise to a single NMR band at (Table 1, Compound In spite
and of an ortho-dihydroxy-system (positive shift in
spectrum with
of this fact, ring B must be asymmetrically substituted, because the corresponding signals
in the spectrum of tri-O-acetylapuleitrin were characterized by two distinct chemical shift
values (Table 1, Compound
Only
can thus represent the structure of apuleitrin.
Apuleirin is a dihydroxypentamethoxy derivative (Table Compound
Since the
structure of its methyl ether
of the substituents remained to be established. NMR (Table 1, Compound
data (no shift with
had already been determined, only the relative positions
and UV
showed the absence of hydroxyls from both the and 5-positions.
The NMR spectrum contains a two-proton singlet at
which can only be attributed to
the hydrogens at C-2’ and 6’. The magnetic equivalence of these protons does not persist in
apuleirin acetate, since they are represented by two doublets in the spectrum of
this derivative (Table 1, Compound Asymmetric substitution of ring B requires the
presence of a hydroxyl at C-3’. The only alternative asymmetric substitution pattern,
namely a grouping, could be ruled out, because apuleirin is
stable in alkali and no alteration of its W spectrum was observed upon addition of
Absence of a paramagnetic shift of the H-8 NMR signal upon acetylation, and a
bathochromic shift of the UV band II upon addition of
can only be rationalized if
P. R.
J . R. KNOX and E. J. MIDDLETON , Australian
15,532 (1962).

R . BR AZ
a n d 0. R.
2440
the remaining OH group is at C-6. The mass spectral data (Tables 2 and 3) are in accord
with the structure of
which was
consequently assigned to apuleirin.
Hexaoxygenated Flavones
The distribution of hydroxyls and methoxyls on the skeleton of apuleidin, one of the
hexaoxygenated flavones, was determined by mass spectrometry (Table 3, Compound
The masses of ions of types T, and V (Table 2, Compound
a hydroxy-methoxylated A-ring, while the masses of the ions Y and
with a dihydroxy-methoxylated B-ring. The UV spectrum revealed the vicinal relationship
indicated the existence of
were compatible
of the two hydroxyls in ring B (spectral shifts with
Consequently, when the characteristic bands at -2.46 and
and decomposition in
were found in the NMR
spectrum of apuleidin (Table 1, Compound
5 and had to be considered. The same spectrum indicated also the presence of four
aromatic protons in and ortho related pairs. The chemical shifts of the corresponding
signals suggested their situation respectively on ring A and ring B. All these data lead to
structure for apuleidin.
the location of the hydroxyls at positions
The fully 0-methylated derivatives of the two other hexaoxygenated flavones isolated
from Apuleia leiocarpa were identical. Mass spectra characterized one of the parent com-
pounds (Table 3, Compound
(Table 3, Compound VIIIa) as a trihydroxytrimethoxyflavone. The NMR spectra indicated,
through a proton signal at -2.35 the presence of a group only in the
flavone (Table Compound VIIIa). This suggested again a relationship and,
as a dihydroxy tetramethoxyflavone and the other one
indeed, the tetramethoxyflavone was easily converted into the trimethoxyflavone upon
heating with aqueous
UV, NMR (Table 1, Compound VIIIa) and mass spectral (Tables 2 and 3, Compound
measurements, as well as degradation by alkali (see Experimental), indicated that the
5-hydroxy-compound was
methoxy-compound
and the
is the
structure of oxyayanin-A, a constituent of Distemonanthus
Jain et
synthesized
and found it
to be different, by IR spectroscopy, from a sample of oxyayanin-A supplied by Prof. F. E.
King, although they recorded close agreement in
This, in addition to the fact that the
reactions, UV spectra and
procedure for the isolation of oxyayanin-A
from Distemonanthus
the Indian workers to consider the structural proposal VIIIa advanced for the natural
compound as
disubstituted B-ring might be present in
included alkaline treatment which destroys VIIIa, forced
and stimulated Dreyer and Bertelli to suggest that a
Such a hypothesis would, of
course, never have been formulated, had the authors been able to examine the NMR
spectrum. This shows (Table 1, Compound VIIIa) two doublets whose chemical shifts and
coupling constant allow correlation only with protons at C-6 and C-8, and two singlets
revealing a pair of
consequently be substituted at positions
related protons. These can only be placed on ring B, which must
and 5’.
F . E . KING, T. J . KING a n d P . J .
A. C. J AIN, S. K. MATHUR a n d
S
TOKES
,
4587 (1954).
R. SE SH ADR I, Indian J. Chem. 4,365 (1966).
Zndian Chem. 7,110 (1969).
, (1967).
S. C.
V. V. S.
a n d T. R. SE S H AD R I
,
D.
D
R E YE R a n d D. J . BE R TE LLI Tetrahedron

The
of Apuleia leiocarpa
2441
We were able to identify the
of Apuleia leiocarpa with oxyayanin-A
by direct comparison (IR and UV spectra, TLC and mixed m.p.) with an authentic sample
from Dr. T. J. King. Consequently, when Prof. T. R. Seshadri kindly informed us that he
found our oxyayanin-A ex A, leiocarpa identical with his synthetic
the structural
proposal originally advanced for oxyayanin-A was established as correct, and the remaining
question concerned only the nature of the sample which had been sent from Nottingham to
Delhi.
This problem was solved when it was found that oxyayanin-A, crystallized from ethyl
acetate-light petroleum, ethyl acetate-benzene or methanol, gave an
spectrum (in
identical with that published by Seshadri et al.
for synthetic VIIIa. When oxyayanin-A,
however, was dissolved in chloroform and the solvent removed by evaporation, the residue,
although indistinguishable from oxyayanin-A by UV all other methods, gave rise to an IR
spectrum (in
identical to that published by Seshadri et
for the natural
A received from England.
Distemonanthus benthamianus Baill. is an African timber closely akin to Apuleia
Interestingly, both species belong to monotypic genera. The finding of oxyayanin-A
in A. leiocarpa stimulated the search for other constituents of D. benthamianus in the Brazil-
ian species. Indeed, the presence in the corresponding extract of still another
ted flavone, namely oxyayanin-B
could be demonstrated by TLC.
Pentaoxygenated Flavone
The sole pentaoxygenated flavone isolated from A. leiocarpa was, at this stage, identified
easily through spectral measurements (cf. Tables l-3, Compound X; Experimental) with yet
another constituent of D. benthamianus, namely ayanin
Mass Spectra
Table 4 summarizes some of the mass spectral data contained in Table 2. Clearly, the
substitution at C-2’ and 6’ of
can be recognized upon examination of the
relative abundances of their M-l, M-17 and M-31 ions. Absence of substitution confers
high stability to the molecular ion, favouring only the expulsion of a hydrogen atom.
Substitution by hydroxyl favours the expulsion of OH and
by methoxyl favours expulsion only of
radicals, while substitution
TABLE 4. ABUNDANCE OF IONS
MASS SPECTRA OF THE
AND R IN THE
FLAVONES LISTED IN TABLE
2
G = H
G = O H
Ion
82-100
21-40
42-53
M-l
9-15
6-10
5-15
25-87
M-31
16-100
58-100
A. ENGLER and K.
Die
Vol. 3, Part 3, p. 156, Engelmann, Leipzig
92 (1952).
(1894).
F. E. KING, T. J. KING and K. SELLARS,

2442
R . BRAZ FILHO and 0. R .
Leiocarpin
The aromatic constituent of A. leiocarpa bark was classified as a pterocarpan by means
of its very typical and spectra. The mass spectrum suggested, through its
molecular ion, substitution by a methylenedioxy- and a unit. The
prominent M-15 peak was attributed to the benzopyrilium ion, formed by loss of a methyl
radical from the chromene portion of the molecule. Direct assignment of the substituents to
rings A or B of the pterocarpan skeleton through mass spectrometry was not feasable for,
as shown previously,
the major fragments can be reasonably formulated as arising from
either ring (e.g. XI, XII). As expected, however, the interpretation of the mass spectrum of
the isoflavan, derived from leiocarpin by catalytic hydrogenation, was straightforward. The
retro-Diels Alder fragmentation mode yielded the ions XIII and XIV in high relative
abundance. The
can thus be located only on ring A, while the
methylenedioxy-group must occupy positions of ring B.
The aromatic region of the NMR spectrum of the isoflavan showed four bands, each
representing one proton two doublets centered at
at and Clearly, the chemical shift of the low field signal reveals the existence of a
proton which keeps neither an ortho- nor apara relationship to an a condition
and
Hz) and two singlets
which can be met only by a H at C-5 of a C-7-oxygenated A-ring. Since the proton signal
under scrutiny is characterized by an ortho-coupling constant, a second H must occur at C-6.
The ring B protons gave rise to two singlets and must be
consequently to be assigned to the tetrahydrogenated derivative of leiocarpin.
The for the isoflavan XV was deduced upon inspection of its ORD
curve. This showed a negative Cotton effect in the 260-300 nm region, in analogy with the
curves of known
It was now possible to formulate structure and configuration of leiocarpin (XVI) on the
reasonable assumption that the heterocyclic rings are cis Confirmation of the
structure was provided by all features of the NMR spectrum. Observed and
expected chemical shift values for the singlets due to the C-7 and C-10
related. Structure XV had
which do not bear an additional substituent at
protons are in agreement. Confirmation of the absolute configuration was obtained by
inspection of the ORD curve. This followed, between 3.50 and 220 nm, precisely the course
expected for
D I S C U S S I O N
Methylation of the
natural Two additional conspicuous examples are eupatin
not only on account of their close structural relationship with oxyayanin-B
in preference to the 3’-hydroxyl is relatively rare in
and
(IX), but also because they show moderate cytotoxicity against human carcinoma of the
D. R. P E R R IN , Tetrahedron Letters 29 (1964).
K. G. P ACHLER a n d W. G. E. U N D E R WO O D, Tetrahedron
(1967).
K.
M. NAKAYAMA a n d T. H AR ANO , Bull. Chem.
Japan 42,233 (1969).
B. L.
J. Org. Chem.
J. Chem.
Tetrahedron
(1961).
A.
P ELTER a n d P .
(C) 887 (1969).
J . A. BALLANTINE a n d C. T.
(1967).
K K U R O S A W A W. D.
B.
I. 0. S U T H E R L A N D , 0. R .
a n d H .
Chem.
H. SUGINOME ,
H.
1265
Japan 39,409 (1966).
a n d T. I WAD AR E , Experientia 18,163 (1962).
a n d P . R . J EF F ERIES , Australian J. Chem. 17,934 (1964).
C. A.
M. K U P C H AN , C. W. SIGEL, J. R. KNOX a n d M. S. U D AYAM U R T H Y, J. Org. Chem. 34, 1460 (1969).

2443
The
of Apuleia leiocarpa
R
Me
H
Me Me
AC Me
A C A C
Me Me
Me Me Et
R = M e
b R = H
Me
Me
H
apulein
H
b
AC
AC
e
f
A
C
Me
H
H
R - M e
R - H
IV
b
OR
0
OR
H
0
H
H
Me
Me Me Me Me Me
Me Me
AC A C Me
Me Me
Me Me AC A C Me
H
Me
apuleisin
apuleitrin
Va
b
c
H
H
b
c
d
e
f
Me Me Me
H
H
H
AC
A C A C
H
AC Me
Me
H
H
Me Me
Me
Et
H
AC Me
Et
Et

R. BRAZ FILHO and 0. R.
OR
2444
OR
0
R
oxyayanin-A
apuleidin
H
H
H
H
AC
b
e
Me
b
Me Me
Me AC
A C A C
OH
0
IX oxyayanin-B
X ayanin
XIV m/e 164
XII m/ e 213
XIII m/ e 190
XI m/e 213
3
XVI leiocarpin
OR
0
R = H
eupatin
R = Me eupatoretin
b

The
of Apuleia leiocarpa
oxyoyonin
ayanin
apulein
oxyayanln-A
- A
O H
OH
L
-
OH
OH
apuleisin
OH
apuleitrin
opuleirin
CHART 1. POSTULATED DERIVATION OF B-RING OXYGENATION IN
ISOLATED FROM A.
leiocarpa.
The postulated derivation of trioxygenated B-rings of Apuleia constituents from
genated precursors is presented in Chart 2. It seems obvious that the hydroxyl, and not the
methoxyl, controls the orientation of electrophyllic attack by
positions. Effects of this nature were attributed to the ability of the hydroxyl groups to
establish a hydrogen bond with the neighbouring methoxy As a result of such
intramolecular hydrogen bonding, and inductive electron release toward
or positions would be expected to be enhanced for the hydroxyl, and repressed for the
to its
or
methoxyl. This may explain why the usual
diarylpropanoids leads to the usual
Distemonanthus and Apuleia species, the unusual
substituion in natural
derivatives, while, at least in
substitution leads
to the unusual
and
patterns.
pattern is indeed usual among flavonoids was again
and apuleirin
Identically substituted derivatives have been located in such unrelated species as
That the
brought out in the present work with respect to apuleitrin
and
(L.) Jack,
F. Muell.
In contradistinction, while
isoflavonoids of the
this fact
B-rings are reasonably widespread among the
and a proposal has been advanced which rationalizes
oxyayanin-A was until now the only
M.
D. L.
H.
W D.
B
ARROETA
,
G.
and J.
Z
ABICKY
,
Org.
(1 9 6 6 ).
Chem.
(1969).
G
RISEBACH andOWrg..D.
Experientia
(1961).
(edited by T. J.
in Recent Advances in
Vol. 1, p. 348, Appleton-Century-Crofts, New York (1968).
M
ABRY, R. E.
and V. C.

R.
and 0. R.
2446
reported in the family. Nevertheless, recent
mention 27 additional repre-
sentatives of this class from members of the Acanthaceae, Datiscaceae, Labiatae, Meliaceae,
Moraceae and Rutaceae. Their biosynthesis, however, does not necessarily follow the
course which is suggested for the Apuleia constituents, and at least some of these com-
pounds may be derived by some departure from the usual acetate-shikimate
Finally, it has been reported that ayan wood (Distemonanthus
may cause
dermatitis due to the presence of oxyayanin-A and
Clearly, this is another
indication designed to stimulate an investigation into the physiological activity of the con-
stituents of garapa wood (Apuleia leiocarpa).
EXPERIMENTAL
NMR spectra were determined in
using a Kofler hot stage microscope and are uncorrected. Separations by column chromatography were
carried out usine Merck mm. Merck’s G was used for TLC. During isolation
using TMS as an internal standard.
were determined
processes the appropriate combination of fractions was determined by examination of their i.r. spectra and
TLC behaviour. TLC plates were examined under UV illumination and after exposure to iodine vapour.
The majority of NMR and mass spectral figures are given in Tables 1-3.
of Apuleia Constituents
Benzene extraction of the heartwood of A. leiocarpa. Isolation of
and
The powdered
heartwood kg) was continuously extracted with hot benzene. Upon partial evaporation of the solvent
separated an oil which solidified by standing (Hi). Further concentration of the benzene solution produced a
crystalline precipitate which was separated into an acetone insoluble
The acetone soluble portion was chromatographed on silica giving fractions
and an acetone soluble portion.
in this order, by elution
and
with benzene-AcOEt(l:l). Two more successive crops of crystals
of the original benzene
solution were collected. The remaining benzene solution was divided into two parts,
and
Hi was washed with a small quantity of benzene, and freed from an insoluble portion by treatment with
The
was evaporated. The residue, purified by passage of its methanol solution
through a column of Sephadex LH-20 gelified with methanol, and
afforded apuleisin (Va, 530
from the same solvent,
was recrystallized from
was recrystallized from acetone yielding apulein
g).
yielding oxyayanin-A
160 mg).
was recrystallized from benzene yielding
apulein (Ib, 180 mg).
was recrystallized from acetone yielding apulein
140 mg).
900 mg).
was recrystallized from acetone,
and recrystallization from methanol,
was recrystallized
from acetone yielding
purified by chromatography on silica, elution with benzene-AcOEt(1
yielding ayanin (X, 9 mg).
was recrystallized from ethanol yielding apuleisin (Va, 40
was
evaporated. The ethanol soluble part of the residue was chromatographed on a polyamide column.
eluted a product in which the presence of oxyayanin-B (IX) was detected by TLC (silica
was partially evaporated. An oil precipitated and was separated. Treatment of this oil with
produced a crystalline mass which was shown by TLC to be composed of
(Ib), apuleisin
(Va) and oxyayanin-A (VIIIa). The filtered
solution was concentrated to a small volume and
tographed through a column of Sephadex LH-20 gelified with
In order of elution were collected
(30 mg), a fraction which after recrystallization from AcOEt-light petroleum yielded apuleitrin
30 mg), and a fraction which after recrystallization from
Further evaporation of benzene from the original
yielded ayanin (X, 25 mg).
afforded a second crop of oily precipitate
which was again treated with
The crystals were collected and again shown by TLC to be composed
of
(Ib), apuleisin (Va), and oxyayanin-A (VIIIa). The filtered
solution was
evaporated and the residue dissolved in
This solution was extracted several times with
aqueous borax. The aqueous layers were united, washed with
acidified with
and extracted
with
The
was washed with water, dried, evaporated and the residue (1.9 g)
chromatographed on silica (60 g). In order of elution with benzene-AcOEt were collected a fraction which
P C
P. V.
B. R.
S. S.
and K.
J. Chem. 7,
T. R.
M.
and P. S.
J. Chem. 7, 306 (1969).
D. L. DREYER, J. Org. Chem.
(1968).
J. W. W. MORGAN and J. THOMSON, Brit. J. Ind. Med. 24,156 (1967).

2441
The
of
leiocarpa
after recrystallization from
from acetone-light petroleum yielded apuleitrin
from yielded apuleirin
yielded apuleidin
129 mg), a fraction which after recrystallization
109 mg), and a fraction which after recrystallization
22 mg). In the mother-liquor, which remained after the
filtration of apuleitrin, the presence of oxyayanin-B (IX) was demonstrated by TLC.
Ethanol extraction of the heartwood of A. leiocarpa. Isolation of
extraction with hot benzene, see above, was continuously extracted with
solvent produced a crystalline deposit which through recrystallization from
The ground wood, after
Partial evaporation of the
yielded
Benzene extraction of the
extracted with hot benzene. Upon removal of the solvent a residue (100
revealed the presenceof
apuleisin (Va), apuleitrin
Benzene extraction of the bark of A. leiocarpa. Isolation of
of A. leiocarpa. The powdered
kg) was continuously
remained. Column or TLC
oxyayanin-A(VIIIa),
ayanin (X) and
and Ieiocarpin (XVI). The powdered
bark (1.2 kg) was continuously extracted with hot benzene.’ Evaporation of the solvent yielded a residue
Elution with
afforded
(XVI, 130 mg).
(16 g), part of which (3 g) was chromatographed on silica (90 g).
mg) and a fraction which after recrystallization from
yielded
Apulein and Derivatives
Apulein Pale yellow rectangular plates, m.p. 211-213”.
ll,lOO), 244, 287,
Gibbs test,
(Ib). Orange needles,
255,298,
(cm-‘): 3458, 1658,
250, 315, 335 infl. 21,450, 12,400,
238, 290, unchanged on addition of NaOAc,
NaOAc or
NaOAc,
450, 690 nm.
(cm-‘):
1510.
260, 305, 350
270, 360,
+ NaOAc.
267, 314, 357, unchanged in presence of
1493.
Apulein diacetate (Zc). White needles from hexane-benzene, m.p. 158-160”.
(cm-‘): 1768, 1640, 1613, 1565, 1517. M.S.: 488
431 430 429 416 415 404
385 383 371 369
341 202
168 150
230, 254, 308 nm
474
473
388
355
181
136
446
387
445
386
343
179
123
403
359
194
139
389 (8).
357 (11),
188
375
327
165
105 (5).
374
315
153
373
211
151
345
180
329
167
195
149
135
122
109
107
triacetate (Id). White rectangular plates from
m.p.
(cm-‘):
nm
Apulein
ether (Zf). White rectangular plates from methanol (463 mg), m.p.
(cm-‘): 1637, 1613, 1570, 1515. M.S.:
ether.
247, 320 nm 21,700,
245, 320 nm 23,200,
Tables
no alteration with
also afforded apulein
White needles from benzene, m.p. 129-131”.
of
Apulein
ether
(cm-‘): 1628,
Apuleisin and Derivatives
Apuleisin (Va). Yellow needles,
193-195”.
237, 272, 328
25,600, 21,600, 19,750)
280,385,
Gibbs test:
460,590 nm.
(cm-‘):
Apuleisin
ether
White needles from
235, 255, 308 nm
230, 250, 308 nm
(cm-‘):
Apuleisin tetraacetate (Vc). White crystals from
19,800).
(Vd). A mixture of apuleisin (35 mg) and
for 20 min. After cooling to room temp., benzene was added. The
(200 mg) was maintained at
solid which precipitated (11 mg) was filtered. Upon addition of light petroleum to the filtrate an additional
quantity of solid material (6 mg) was obtained. The united precipitates were crystallized from
yielding yellowish
plates, m.p. 232-234”.
223, 255, 360,
215, 260,
285, 325,
1555,
240, 295,
alteration with NaOAc.
(cm-‘):
497 464
149 109
broad,
Mass spectra:
m/e
165
539
151
463
357
343
340
167
166
105 (40).

R.
FILHO and O.R.
2448
By treatment of apuleisin with
white crystals from
17,650, 11,500,
White crystals from hexane-benzene, m.p.
nm
330
234,261, 312,
(cm-‘):
272, 295, 338,
broad,
272,295
(open
1635, 1620, 1570, 1495.
1750,
233, 250
306
18,550, 13,400, 13,600).
(cm-r):
tetraethyl ether
White crystals from light petroleum-benzene,
12”.
235, 252, 306 nm 20,050, 15,300, 13,400).
(cm-‘): 1639, 1605, 1493.
Apuleitrin, Apuleirin and Derivatives
238,282,
13,600, 16,050,
Apuleitrin
Yellow rectangular plates,
300, 365,
256 298 374
175-177”.
235, 275, 314,
240, 288, 335,
240
240, 256, 298, 365 nm, no alteration with NaOAc.
(cm-‘): 3333, 1653, 1575, 1550, 1504.
Apuleitrin trimethyl ether
White rectangular plates from light
m.p. 152-154”
(cm-r): 1639, 1600, 1577, 1504.
(lit.‘”
237, 260, 325 nm 17,550, 13,050, 21,100).
Methylation of apuleirin with
Apuleitrin triacetate
also afforded apuleitrin trimethyl ether.
White rectangular plates from
m.p. 215-218”.
230, 245, 320,
(cm-‘):
337 nm
18,850, 14,950, 14,550, 14,050).
236, 270, 330 11,550, 8750,
Apuleirin
Pale yellow crystals,
253, 293, 340,
235, 270, 330,
218-220”.
236, 267, 330,
235, 270, 330,
230, 240, 295, 332,
235, 270, 330 nm.
(cm-‘): 3425, 1620,
Apuleirin diacetate
1515.
White crystals from light petroleum-benzene,
cm-r.
174-175”.
1761,
Apuleidin and Derivatives
Apuleidin
11,250, 5300,
Pale yellow rectangular plates,
265, 360,
230, 260, 300, 333
10,650,
255, 290, 320,
238, 255, 300,
335,
233, 260, 295, 343,
268, 300, 360, 400,
270,
300, 334, 38Snm.
(cm-‘)
Apuleidin triacetate
White rectangular plates from
(cm-‘):
198-200”.
225, 253,
305 nm
Oxyayanin-A and Derivatives
Oxyayanin-A
14,000,
with NaOAc,
S-0-Methyloxyayanin-A
250,
Orange needles, m.p.
268, 358,
(lit.
254,294,
229-230).
257, 291, 350 43,250
270, 314,400, no alteration
+NaOAc.
(cm-‘):
White crystals, m.p. 258-260”.
248,295 nm, no alteration with NaOAc,
253, 345 43,000,
1515, 1499.
Oxyuyanin-A trimethyl ether
White crystals from benzene-light petroleum and methanol,
(lit.”
245, 295, 324 nm 20,600, 7800, 10,050).
(cm-‘): 1634, 1626, 1514.
also afforded oxyayanin-A trimethyl ether.
White crystals from light petroleum-benzene,
Methylation of S-0-methyloxyayanin-A with
S-0-Methylaxyayanin-A diacetate
Oxyayanin-A triacetate
183-184”).
White crystals from light petroleum-benzene,
cm-r.
(lit.15

2449
The
of Apuleia Ieiocarpa
Other Apuleia Constituents
White needles, m.p.
172-174”).
255, 265, 295, 353,
265, 275, 295, 353,
2.76
270, 290, 380,
393 nm, no alteration with NaOAc,
(XVI). White rectangular plates,
no alteration with
H-l), 3.31 (s, H-7), 3.34 (d, 9.4 Hz, H-4’), 3.48 (d,
+ NaOAc.
98-100”.
(cm-‘): 1639, 1613, 1587, 1504. NMR spectrum (CCL,
Hz, H-2), 3.58 (s,
(cm-‘):
nm
402 (s,
4.48
(s,
(d,
HZ, H-3’),
(d,
Hz, H-l la),
m/e
(c
(m,
185
Mass spectra:
336
335 321 317
nm):
173
168
167 (6). ORD
White crystals, m.p.
-37”.
White crystals, m.p. 185-187”
+ 60” (c 0.89,
+ 65”).
Selective Demethylation of Apuleia Constituents
Formation of
A mixture of apulein (100 mg) and 20% aq.
ml), was
heated on a steam with occasional agitation for 30 min, cooled to room temp., diluted with water and
extracted with
The
solution was washed with water, dried and evaporated. The residue was
separated by thick layer chromatography (silica, benzene-EtOAc
apulein.
into apuiein and
Formation of oxyayanin-A
5-0-Methyloxyayanin-A (20 mg) was treated with
by the pro-
cedure described above (1 hr heating on the steam bath), yielding, besides starting material, oxyayanin-A.
Oxidative Demethylation of
Formation of the Quinone
A mixture of apulein (344 mg) and
occasional warming on the steam bath, for 30 min, left for an additional 30 min in an ice bath, poured into
water, and extracted with The solution was dried and evaporated, yielding a deep orange
solid (210 mg) 120-l 30”. 1696, 1660,
Formation of
(1 and anhydrous
diluted with water and extracted with
(d
ml) was shaken, with
A mixture of the quinone IV (200 mg),
(4 ml) was heated on the steam bath for 3 min, cooled to room temp.,
The solution was dried and evaporated, yielding a
yellowish solid,
250-260”.
Formation of
The quinol Ih was
without
: benzene-AcOEt
230, 250, 306 nm
purification, the crude product being purified by passage through a silica column
when it crystallized from benzene as white needles (41 mg), m.p. 213-215”.
15,650,
m/e
(cm-‘):
1582, 1515, 1492. Mass spectrum:
417 401 388 387 377
194 179 167 153 151
107 106 105 104 103 (6).
544
376
149
502
359
461
345
460
231
115
459
230
111
418
195
109
375
137 (7). 135 (IO), 129
122
Alkaline Hydrolysis of Apuleia Constituents and of Their Derivatives
Formation of
acid and of
aq. KOH 5 ml), and
A
mixture of apulein
ether (If, 150
(10 ml) was heated
atm)
on a steam bath for 8 hr. The ethanol was removed by distillation and simultaneously substituted with water.
The aqueous solution was acidified
and extracted with ether. The ether solution was extracted
and with aq. (3 The solution was acidified (2N
solution was evaporated, and the residue purified by filtration
and crystallization from petroleum yielding white needles of
acid (14 mg), m.p. and mixed m.p. with an authentic sample
(cm-‘): 3400, 3225, 2500 broad, 1719, 1613, 1590, 1518. The solution was acidified
and extracted with The solution was evaporated, and the residue purified by
filtration through silica (eluant: and crystallization from light petroleum yielding white needles of
successively with aq.
and extracted with
The
through silica (eluant:
143-144”);
(5 mg),
and mixed m.p. with an authentic sample
70-71”);
(nm):
(cm-i):
196 195
216, 234, 290, 305 sh.
Mass spectrum:
167 153
256
149 (5).
m/e
212
W.
(1958).
211
210
184
169
168
K
ARRER,
Konstitution und Vorkommen der Organischen
p. 117, Birkhluser Verlag, Base1

2450
Formation of
R.
FILHO and 0. R.
acid and
A mixture of apulein diethyl ether (Ig, 40 mg) and
KOH in
(10 ml) was heated
atm) on a
steam bath for hr. Treatment as described above, afforded an acidic and a phenolic fraction. The acidic
fraction yielded, upon sublimation,
with an authentic sample
acid (2 mg), m.p. and mixed
(cm-‘): 2500 broad,
196 195
139,
109
155-156”);
212 211
151
111
Mass spectrum:
168 167
240
166
119
m/e
194
184
183
165
144
110
138
108
137
107
135
105
was demonstrated by
128
12.5
123
122
115
103 (6). the
phenolic fraction, the presence of
TLC (silica, benzene-AcOEt 1
Formation of
acid and of
phenone. A mixture of apulein
20 mg), 20%
(in
1
(1.5 ml)) was heated under
reflux on a steam bath during 4 hr. Treatment as described above, afforded an acidic and a phenolic fraction.
The acidic fraction yielded, upon sublimation,
acid (2 mg), m.p. 113-
115”.
210
(cm-‘): 3323, 3168, 1679, 1613, 1586, 1493. Mass spectrum:
228
111
solution was acidified and extracted with
m/e
105
211
83
196
55
195
184
169
168
167
141
139
69
66
53 (18). The
In the phenolic
fraction, the presence of
was demonstrated by TLC.
Formation of
acid
of
A
mixture of apuleisin tetramethyl ether (Vb, 80 mg), KOH (1.5
under reflux until TLC indicated complete absence of starting material in the reaction mixture. Treatment
as described above, afforded an acidic and a phenolic fraction. The acidic fraction was crystallized from
(11 ml) and water (3 ml) was heated
light petroleum benzene, yielding
(cm-‘): 3021, 2600 broad, 1681, 1590, 1493. NMR spectrum
acid (10 mg), m.p. 99-100” (lit.“’ 100”).
6.28
(s,
(d,
Hz, H-5), 2.38 (d,
Hz, H-6). The phenolic fraction was crystallized from
and mixed m.p. with an
light petroleum, yielding
authentic sample 67-69” (lit.“’ 70-71”).
of Leiocarpin
Formation of
(XV). A
(15 ml) was saturated with hydrogen at room temp. and pressure.
(5 ml) was added and hydrogenation continued to the dis-
suspension of 10
(250 mg) in
A solution of leiocarpin (100 mg) in
appearance (TLC) of starting material. The mixture was filtered, evaporated and the residue crystallized
from
yielding white crystals (72 mg), m.p. 184-186”.
230, 288, 300 12,750,
6550);
227, 280, 317 nm. Gibbs test: negative.
(cm-‘): 3420,
1510, 1490. NMR
Hz, H-6), 4.12
spectrum in
3.16 (d,
Hz, H-5), 3.31 (s, H-6’), 3.49 (s, H-3’),
(d,
177
136
8650.
Mass spectrum:
354
150
m/e
216
149
192
147
4280,
191
137
176
135
175
165
ORD in
164
152
0.18):
151
149.5
133 (15).
0,
0,
Acknowledgements-We thank Dr. E. P.
and Dr. J.
Pires, Universidade de Brasilia, for the
collection and identification of the plant material used in this investigation; Prof. P. R. Jefferies, University
of Western Australia, Nedlands, for a sample of
and
Prof. B. R. Presidency College, Madras, for a sample of
acid.
Holzwirt B. M. Haussen, Holzchemie, Hamburg,
attention to ref. 39. We
thank Professor W. D. Ollis, The University, Sheffield, who arranged for the determination of the mass and
the 100 MHz spectra.
K. R.
A.
and A. ROBERTSON, J.
Chem. 8,273 (1958).