Pergamon 0031-9422(95)00848-9 Phytochemistty, Vol. 42, No. 1. pp.

185-188, 1996
Copyright (~) 1996 Published by Elsevier Science Ltd
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MORE PHLOROGLUCINOLS FROM HYPERICUM BRASILIENSE*

LEANDROROCHA, ANDREWMARSTON,OLIVIERPOJq'ERAT,M. A. C. KAPLAN'~and KURT HOSTETTMANN~

Institut de Pharmacognosie et Phytochimie, Universit6 de Lausanne, BEE CH-1015 Lausanne-Dorigny, Switzerland: +NPPN,
Universidade Federal do Rio de Janeiro, Rio de Janeiro, 21941, Brazil

(Received 26 September 1995)

Key Wordlndex--Hypericum brasiliense; Guttiferae; phloroglucinols; antibacterial activity;

Bacillus subtilis; hyperbrasilols B and C; isohyperbrasilol B.

Abstract--Three new phloroglucinols (hyperbrasilols B and C, and isohyperbrasilol B) have been isolated from a
petrol extract of the leaves and flowers of Hypericum brasiliense. Their structures were established by
spectroscopic methods including two-dimensional-NMR heteronuclear correlations. EI and D/Cl-mass spectral,
NMR, IR and UV data are reported. All three phloroglucinols were antibacterial against Bacillus subtilis in a TLC
bioautographic assay.

INTRODUCTION graphy (CPC) provided compounds 1-3, which ap-

peared as red spots on TLC plates when sprayed with
As part of our phytochemical and biological inves-
Godin reagent [5]. Their IR spectra showed broad
tigations on plants of the genus Hypericum, we recently
absorption in the 3500-3000cm ~ region, together
reported on the bioactive constituents of H. brasiliense,
with intense peaks from 1600 to 1650cm ~. This
a species growing in south and south-east Brazil.
suggested compounds 1-3 to be phloroglucinol deriva-
Xanthones and a new y-pyrone derivative isolated from
tives containing an enolic 1,3-diketo system or a 2-
the stems and roots showed antifungal properties [2]. In
hydroxyaryl ketone [6].
addition, they were found to be inhibitors of mono-
The UV spectrum of 1 exhibited maxima at 357, 287
amine oxidases (MAO), enzymes which are involved in
and 226nm. A [ M ] + was detected in the EI-mass
the regulation of some physiological amines and are
spectrum at m/z 552. The M r was further confirmed by
thought to contribute to the management of depression.
a [ M + H ] + pseudomolecular ion at m/z 553 in the
Phloroglucinols, including the new compound, hyper-
D/C1 mass spectrum. The molecular formula was
brasilol A, have been isolated from the petrol extract of
deduced to be C32H4oO 8. The ~H and ~3C NMR data of
the leaves and flowers [3]. These compounds exhibited
1 (Tables 1 and 2) indicated a close structural similarity
strong antibacterial activity against Bacillus subtilis in a
to isouliginosin B (4), a compound that has been
TLC bioautographic assay. Further studies on the petrol
previously isolated from H. brasiliense [3]. Compared
extract of the leaves and flowers of H. brasiliense have
with that of 4, however, the NMR spectra of 1
now led to the isolation of three additional antibiotic
exhibited additional signals corresponding to a prenyl
compounds, hyperbrasilol B (1), isohyperbrasilol B (2)
side-chain, while only one methyl group (6, 1.51; 6c
and hyperbrasilol C (3).
22.8) was found to be present at C-4. In the ~~C NMR
spectrum of 1, the resonance of C-4 (6 49.8) was
shifted ca 5 ppm downfield from that in isouliginosin
RESULTS AND DISCUSSION
B. Hence, the prenyl side-chain was placed at C-4,
Leaves and flowers of H. brasiliense were extracted together with one methyl group. Such a deshielding
with petrol. The resulting extract showed several inhibi- effect on C-4 when a methyl group is replaced by a
tion zones against B. subtilis in a TLC bioautographic prenyl chain has been observed in drummondins [7]. In
assay [4]. After evaporation to dryness, treatment of the the t H NMR spectrum of 1, a pair of doublets (J =
petrol extract with acetone provided an acetone-soluble 10 Hz) at 6 5.59 and 6 6.66, respectively, revealed the
fraction and an insoluble fatty residue. Fractionation of presence of a 2,2-dimethyl chromene moiety. The
the bioactive acetone-soluble fraction by a combination fusion of the pyran ring in 1 was inferred by direct
of silica gel column chromatography, gel filtration on comparison of the hydroxyl resonances, in particular
Sephadex LH-20 and centrifugal partition chromato- HO-7' (6 16.35) and HO-5' (6 11.40), with those
observed in the isomers drummondin C and isodrum-
*Part of the PhD Thesis of L. R. [l]. mondin C [7]. Recently, selective INEPT experiments
~Author to whom correspondence should be addressed. on the last compounds have confirmed the reliability of

185
186 L. ROCHA et al.

Table 1. ~H NMR spectral data of compounds 1-3 (J values

in Hz)*
H 1 2t 3

11' ~ .,,,"J1" 4-Me 1.51 (s) 1.26 (s):~ -

6-Me - - l. 17 (s)
7 3.55 (s) 3.55 (brs) 3.41 (s)
9 4.19(sepJ=7) 4.20(sepJ=7) -
9-Me 1.16(dJ=7) 1.17(d J = 7 ) a -
1.17(d J = 7 ) -
2'-Me 1.53 (s) 1.52 (unres.) 1.57 (s)
1.53 (s) 1.52 (unres.) 1.72 (s)
3' 5.59(d J = 10) 5.48 (d J = 10) 5.22(brtJ=7)
4' 6.66 (d J = 10) 6.74(d J = 10) 3.20(brdJ=7)
12' 4.00(sep J= 7) 4.11(sep J= 7) 4.19(sep J = 7)
12'-Me 1.20(d J = 7 ) 1.18(dJ=7) ~ 1.11 (d J = 7 )
1.21 (d J = 7)
1" 2.8-2.5 (m) 2.8-2.5 (m) 2.41 (br d J = 7)
2" 4.60 (br t) 4.8-4.4 (m) 4.81 (br t J = 7)
11' 3"-Me 1.31 (s) 1.49 (unres.) 1.39 (br s)

~OHHo~OH 2"
2"-Me
-
-
1.34 (s)
-
-
1.59 (unres.) 1.44 (br s)
3.98 (sep J = 7)
1.05 (d J = 7)
1.07 (d J = 7)
3-OH 9.90 (s) 9.00 (s) n.o.
5-OH 18.80 (s) 18.64 (s) 19.40 (s)
5'-0H 11.40(s) - n.o.
T-OH 16.35(s) 11.64 (s) 14.00 (s)
9'-OH - 14.13 (s) n.o.
2
*In acetone-d6 (1 and 3) or CDCI 3 (2): attributions in 1 and 3 are
supported by COSY and HETCOR data.
tQuatemary methyl groups were better resolved in acetone-d6:
6 1.55 (s, 4-Me), 1.46 (s, 2'-Me2), 1.23 and 1.31 (2s, 3"-Me2);
hydroxyl resonances in acetone-d6:8 18.66, 11.71, 14.02 and 9.15 (br).
~tTentative assignment.
n.o., Not observed.
11'

HO '~ OH Ha .

and '3C N M R data of 2 are listed in Tables 1 and 2,

respectively.
C o m p o u n d 3 exhibited U V absorption m a x i m a at
348, 301 and 224 nm. The El-mass spectrum exhibited
O a [M] + at m / z 554. In the D / C l - m a s s spectrum, a
pseudomolecular ion was observed at m / z 555 ([M +
H] ÷). The difference of 2 amu compared with the M r of
1 and 2 suggested that 3 contained two uncyclized
hydroxyl 1H N M R signals for determination of the prenyl chains. N M R data (Tables 1 and 2) confirmed
orientation of the cyclization [8]. C o m p o u n d 1 has been the molecular formula to be C32H420 8. The phloro-
n a m e d hyperbrasilol B. glucinol moiety was found to be identical to that found
The U V spectrum of 2 resembled that of 1 with in uliginosin A, bearing a prenyl group at C-10' and an
absorption m a x i m a at 357, 279 and 216 nm. The isobutyryl substituent at C-8' [3]. The location of both
molecular formula was deduced to be C32H4oO8 from remaining carbon chains at C-4 was established from
EI ( m / z 552), D/C1 ( m / z 553, [M + H I - ) mass spec- the chemical shift of C-4 (6 57.5) which appeared
trometry and N M R data. Hence, c o m p o u n d 2 is an 8 p p m more downfield than in 1 and 2 and ca 13 p p m
isomer of 1. N M R data of 1 and 2 were very similar. more deshielded than in c o m p o u n d s bearing a gem-
The only significant difference concerned the signals dimethyl group. The chemical shift of C-4 was in full
corresponding to hydroxyl groups. These were detected agreement with the value (8 57.4) observed for the
in the 1H N M R spectrum of 2 at 8 9.00 (HO-3), 11.64 corresponding carbon in kolanone (5), a polyiso-
(HO-7'), 14.13 ( H O - 9 ' ) and 18.64 (HO-5). Thus, the prenylated b e n z o p h e n o n e which has been isolated from
cyclization pattern must involve 0 - 5 ' instead of 0 - 9 ' . fruits of Garcinia kola [9]. Thus, an isobutyryl sub-
Hydroxyl resonances in 2 are in full agreement with the stituent was located together with a prenyl chain at C-4
values reported for i s o d r u m m o n d i n C [7]. We assigned and the remaining methyl group was placed at C-6. The
the n a m e isohyperbrasilol B to c o m p o u n d 2. The ' H position of attachment of the prenyl chain was con-
 Phloroglucinols from Hypericum brasiliense 187

Table 2. k3C NMR spectral data of compounds 1-3 (multip-

C 1
licities in brackets)*
2 3
L,x//o
1 199.2 (s) 199.1 (s) 195.5 (s)
2 109.8 (s)a 109.8 (s)a 109.0 (s)a
3 170.7 (s) 170.2 (s) 183.8 (s)
4 49.8 (s) 49.7 (s) 57.5 (s)
4-Me 22.8 (q) 22.9 (q)
5 188.5 (s) 188.7 (s) 199.6 (s)
6 114.2 (s) a 114.1 (s) 105.0 (s)"
6-Me - - 23.8 (q)
7 16.9 (t) 17.1 (t) 18.9 (t)
8 211.9 (s)" 212.3 (s)" - 4
9 37.2 (d) 37.4 (d) -
9-Me 19.4 (q) 19.5 (q) -
19.5 (q) 19.5 (q) -
2' 79.2 (s) 81.5 (s) 129.6 (s)
2'-Me 27.8 (q) 27.6 (q) 17.8 (q)
27.9 (q) 28.0 (q) 25.9 (q)
3' 126.1 (d) 125.8 (d) 125.2 (d)
4' 117.5 (d) 117.2 (d) 22.6 (t)
5' 160.0 (s)~ 160.9 (s)c 159.0 (s) b
6' 107.2 (s)" 106.3 (s)" 107.5 (s) a
7' 162.7 (s)~ 161.0 (s)" 162.5 (s) h
8' 104.3 (s) a 106.2 (s)" 104.9 (s)"
9' 156.1 (s)~ 155.9 (s)c 163.1 (s) b
10' 104.2 (s) ~ 102.8 (s)~ 107.7 (s)"
l 1' 210.8 (s)" 211.0 (s) h 211.1 (s)
12' 39.7 (d) 40.1 (d) 39.2 (d)
12'-Me 18.9 (q) 18.7 (q) 19.8 (q)?
19.8 (q) 19.6 (q) 19.8 (q)?
1" 39.3 (t) 39.1 (t) 39.5 (t)
2" 118.2 (d) 118.1 (d) 121.2 (d)
3" 136.5 (s) 136.7 (s) 133.0 (s)
3"-Me 17.5 (q) 17.6 (q) 17.8 (q)
25.6 (q) 25.6 (q) 25.9 (q)
1'" - - 197.3 (s)
5
2'" - - 33.5 (d)
2"-Me - - 19.8 (q)?
19.8 (q)t
*In acetone-d6; assignments are supported by HETCOR
o f 1 - 3 were comparable to those o f japonicine A,
data in 1 and 3 and further by COLOC and pulsed-field-
uliginosin A, isouliginosin B and hyperbrasilol A, the
gradient HMBC experiments in 3.
?Broad signal, unresolved. antibacterial phloroglucinol derivatives previously iso-
~Values with the same superscripts in each column are lated from H. brasiliense [2].
interchangeable.

EXPERIMENTAL

firmed by a long-range coupling between H-I" and C-4 General. M p s uncorr. ~H and 13C NMR: 200 and
in the C O L O C spectrum. Conversely, no correlations 50 MHz, respectively. H E T C O R and COLOC: 400 and
could be detected from the methine protons o f the 100 MHz, respectively. The pulsed-field-gradient
isobutyryl residue to any C-atom o f the filicinic acid H M B C spectrum o f 3 was recorded at 5 0 0 M H z .
ring, neither in C O L O C nor in pulsed-field-gradient Distinction o f carbon multiplicities by the use o f DEPT
H M B C [10] experiments. C o m p o u n d 3 has been named sequences. ~H chemical shifts relative to TMS; ~3C
hyperbrasilol C. chemical shifts referenced to solvent peak. TLC: silica
All three isolated compounds were inhibitory to B. gel precoated A1 sheets and RP-18 H P T L C glass plates
subtilis in a bioautography assay [4]. The m i n i m u m (Merck). CPC: Pharma-Tech CCC-1000, 2.6 m m i.d.
quantities o f 1 - 3 required to inhibit the growth o f the Teflon coils, capacity 660 ml (Pharma-Tech, U.S.A.).
bacterium on the TLC plate were 0.6, 0.2 and 0 . 1 6 / z g , MS: triple-stage quadrupole spectrometer; El: 70eV;
respectively. In the same test, the crude petrol extract D/CI: positive ion mode, NH 3.
was active at 2 / z g , while the reference antibiotics Plant material. Hypericum brasiliense Choisy was
ampicillin and chloramphenicol inhibited the growth of collected in Nova Friburgo, Brazil, in March 1991. A
B. subtilis at 0.01 and 0.001 /zg, respectively. Activities herbarium specimen (no. R F A 23.257) is deposited at
188 L. ROCHAet al.

the Botany Department, Institute of Biology, Federal (13), 275 (93), 259 (39), 247 (34), 167 (28), 69 (74).
University of Rio de Janeiro, Brazil. D/C1MS m/z: 553 ([M + H]+), 263.
Extraction and isolation. Ground, air-dried leaves Hyperbrasilol C (3). Pale yellow needles from
and flowers (1.31 kg) were extracted at room temp. MeOH, mp 152-153 °. [ce]D - 5 ° (MeOH; c 0.25). TLC
with petrol. After evapn to dryness, the petrol extract (silica gel, CHC13): Rf 0.10. HPTLC (RP-18, MeCN):
was treated with Me2CO to afford an Me2CO-sol. fr. RI 0.32. UVA M°°H nm (log e): 348 (4.01), 301 (4.17),
(32 g) and an insol, fatty residue (11.8 g). A portion 224 (4.27). IR v KBr cm ~: 3474 (br), 2974, 2929, 2874,
( 17.5 g) of the Me2CO-sol. ft. was sepd by gel filtration 1619, 1501, 1426, 1383, 1286, 1235, 1207, 1137, I101,
over Sephadex LH-20 with CHC13-MeOH (1:1) to 987, 906, 802. ~H and ~3C NMR: Tables 1 and 2. ElMS
give 3 frs (A-C). Fr. B, which exhibited antibacterial m/z (rel. int.): 554 ([M] +, 3), 467 (3), 277 (20), 264
properties, was further fractionated by CC on silica gel (56), 221 (87), 209 (24), 165 (100), 69 (36). D/C1MS
with CHC13-MeOH mixts of increasing polarity m/z: 555 ([M + HI+), 335, 279, 265, 148.
(CHCI3-MeOH 1:0---~4: 1) to yield seven frs (I-VII). Antibacterial testing. Tests were carried out against
Sepn of fr. II (80 mg) by CPC with n-hexane-EtOAc- B. subtilis ATCC 6633 using the bioautographic meth-
M e O H - H 2 0 ( 10 : 5 : 5 : 1, upper phase as mobile phase) odology [4] on silica gel glass-backed plates.
yielded 1 (31 mg). Compound 2 (80 mg) was purified
from fr. III (792 mg) by CPC with n-hexane-MeCN- Acknowledgements--This work was supported by the
MeOH (8:5:2, upper phase as mobile phase) followed Swiss National Science Foundation. Scholarships were
by CC on silica gel with n - h e x a n e - E t O A c - M e O H - awarded to L. R. by the Commission F6d6rale des
H20 ( 1 0 : 5 : 5 : 1 , upper phase). Fr. V was further Bourses pour Etudiants Etrangers of the Swiss Govern-
fractionated using CPC with n-hexane-EtOAc- ment and the CNPq from Brazil.
M e O H - H 2 0 ( 10:5 : 5 : 1, upper phase as mobile phase).
Subsequent sepn by CC on silica gel with n-hexane-
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