Chemical constituents from Piper marginatum Jacq. (Piperaceae)-antifungal activities and kinetic resolution of (RS)-marginatumol by Candida antarctica lipase (Novozym 435)

Article abstract of DOI:10.1016/j.tetasy.2007.05.006

The leaves of Piper marginatum contain the antifungal compounds 3,4-methylenedioxypropiophenone 1, 2-methoxy-4,5-methylenedioxypropiophenone 2, 1-(3,4-methylenedioxyphenyl)propan-1-ol 3 (marginatumol), 5,4′-dihydroxy-7-methoxyflavanone 4 and 5,7-dihydroxy-4′-methoxyflavanone 5. The absolute configuration of natural marginatumol was determined as (+)-(R)-3 (ee 48%) by comparison of its optical properties with the chiral forms obtained by kinetic resolution of racemic 3 using Candida antarctica lipase (Novozym 435).

Full text of DOI:10.1016/j.tetasy.2007.05.006

Tetrahedron: Asymmetry 18 (2007) 1054–1058
Chemical constituents from Piper marginatum Jacq.
(Piperaceae)—antifungal activities and kinetic resolution of
(RS)-marginatumol by Candida antarctica lipase (Novozym 435)
Juliana B. Reigada,a Celize. M. Tcacenco,a Leandro H. Andrade,a Massuo J. Kato,a
b
c,
*
´
Andre L. M. Porto and Joao Henrique G. Lago
˜
aInstituto de Quı´mica, Universidade de Sa˜o Paulo, CEP 05508-900, Sa˜o Paulo, SP, Brazil
bInstituto de Quı´mica de Sa˜o Carlos, Universidade de Sa˜o Paulo, CEP 13560-970, Sa˜o Carlos, SP, Brazil
ˆ
Centro de Ciencias e Humanidades, Universidade Presbiteriana Mackenzie, CEP 01302-907, Sa˜o Paulo, SP, Brazil
c
Received 23 February 2007; accepted 3 May 2007
Available online 1 June 2007
Abstract—The leaves of Piper marginatum contain the antifungal compounds 3,4-methylenedioxypropiophenone 1, 2-methoxy-4,5-
methylenedioxypropiophenone 2, 1-(3,4-methylenedioxyphenyl)propan-1-ol 3 (marginatumol), 5,40-dihydroxy-7-methoxyflavanone 4
and 5,7-dihydroxy-40-methoxyflavanone 5. The absolute configuration of natural marginatumol was determined as (+)-(R)-3 (ee 48%)
by comparison of its optical properties with the chiral forms obtained by kinetic resolution of racemic 3 using Candida antarctica lipase
(Novozym 435).
ꢀ 2007 Elsevier Ltd. All rights reserved.
1. Introduction
bioactivity-guided phytochemical investigation, due to its
potent activity observed against Cladosporium cladosporio-
ides and Cladosporium sphaerospermum. Thus, the major
bioactive compounds isolated were 3,4-methylenedioxy-
propiophenone 1, 2-methoxy-4,5-methylenedioxypropio-
phenone 2, 1-(3,4-methylenedioxyphenyl)propan-1-ol 3
(marginatumol), 5,40-dihydroxy-7-methoxyflavanone 4 and
5,7-dihydroxy-40-methoxyflavanone 5.
The Piperaceae family comprises 14 genera and ca. 1950
species,1 and among these the genus Piper is the most abun-
dant with approximately 700 species.2 Phytochemical anal-
ysis from the Piper species showed the occurrence of several
secondary metabolites, many of which exhibit a variety of
biological activities, mainly as antifungal activity.3
Piper marginatum Jacq. (Piperaceae) is a common shrub of
Most natural products are frequently accumulated in plant
species as one major stereoisomer.13 Such an aspect has
recently been investigated by means of chiral resolution
of enantiomers using specific stationary phases. Surpris-
ingly, chiral analysis has shown that many natural products
can be found as enantiomeric mixtures of variable propor-
tions. The chiral synthesis involved in the biocatalytic pro-
cess has been proven as an effective tool in the preparation
of enantiomerically pure compounds. Herein we report the
resolution of the racemic (RS)-marginatumol 3, prepared
by a Grignard reaction using piperonal and ethylbromide,
in order to determine the configuration of the natural mar-
ginatumol isolated from P. marginatum. The asymmetric
synthesis of the natural products was carried out through
a chemoenzymatic resolution with lipase of Candida ant-
arctica (Novozym 435).
4
´
the Amazon region, popularly known as ‘malvaısco’. The
extract of its leaves has been used in popular medicine to
treat liver and vesicle diseases and also as a tonic with car-
minative and antispasmodic action.5 Previous chemical
studies carried out on P. marginatum have described the
occurrence of propiophenones,6–8 amides,9 flavonoids,10
phenylalkanoids11 and aristolactams.12
Over the course of our search aimed at unraveling new
antifungal agents from Piper species, the crude MeOH
extract from leaves of P. marginatum was selected for
*
Corresponding author. Tel.: +55 11 3091 3813; e-mail: joaolago@
iq.usp.br
0957-4166/$ - see front matter ꢀ 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2007.05.006

J. B. Reigada et al. / Tetrahedron: Asymmetry 18 (2007) 1054–1058
1055
2. Results and discussion
typical broad band at 3383 cmꢀ1. Finally, in the LREIMS
of 3 was observed the molecular ion peak at m/z 180 Da, in
agreement with the molecular formula C10H12O3. There-
fore, the structure of 3 was elucidated as 1-(3,4-methylene-
dioxyphenyl)propan-1-ol.
2.1. Characterization of P. marginatum constituents
The MeOH extract from the leaves of P. marginatum was
submitted to bioactivity-guided fractionation using column
chromatography on silica gel and Sephadex LH-20 followed
by prep. TLC to yield compounds 1 (3,4-methylenedioxy-
propiophenone), 2 (2-methoxy-4,5-methylenedioxypropio-
phenone), 3 [1-(3,4-methylenedioxyphenyl)propan-1-ol], 4
The natural product 3 was submitted to chiral GC analysis
using a b-cyclodextrin column, which indicated the pres-
ence of two compounds with tR = 49.3 and 49.8 min
(48% excess of the compound with tR = 49.3 min). At-
tempts to determine the configuration of natural product
3, further NMR analysis and chiral GC analysis were pre-
ceded by its fast intermolecular dehydration. Therefore, a
study involving the preparation of racemic 3 on a large
scale by Grignard synthesis (Section 3.5) followed by enzy-
matic resolution was carried out. The comparison of the
retention times of the resolved synthetic enantiomers and
the natural 3 by GC analysis allowed the determination
of its configuration as (R), and the enantiomeric excess in
comparison to the proportion of the (S)-derivative.
(5,40-dihydroxy-7-methoxyflavanone) and
5 (5,7-dihy-
droxy-40-methoxyflavanone), as shown in Figure 1. Com-
pounds 1, 2, 4 and 5 were identified by comparison of
their spectral data with those previously reported.3b,6
O
O
OH
6
3
2
2
5
3
4
1
2
9
1
5
4
3
4
O
O
1
9
O
O
7
7
9
O
O
7
8
8
8
6
6
OMe
HO
5
2
1
3
2.2. Enzymatic resolution
OH
OMe
The evaluation of the chemoenzymatic esterification of 3
was performed with a lipase from Candida antarctica (Nov-
ozym 435, CALB) and vinyl acetate as the acetate donor,
using previous conditions as described by our group.14,15
As can be seen in Table 2, the small scale (50 mg) resolu-
tion was very efficient and yielded the (S)-alcohol 3 (ee
92%) and (R)-acetate 3a in high enantiomeric excess
(>99%) and good conversion (c 50%) at 24 h (Table 2,
entry 6). The kinetic resolution of 3 by CALB on a
preparative scale (500 mg) yielded the (S)-alcohol 3 and
(R)-acetate 3a with high enantiomeric excess (ee >99%)
and with good isolated yields after 48 h (alcohol 3: 43%;
acetate 3a: 45%; Table 2, entry 7).
MeO
O
O
O
OH
OH
O
4
5
Figure 1. Compounds 1–5 isolated from the leaves of P. marginatum.
1
The H NMR spectrum of 3 indicated the presence of a
1,3,4-trisubstituted aromatic ring since three hydrogen res-
onances were observed at d 6.55 (dd, J = 8.8 and 1.8 Hz),
6.82 (d, J = 1.8 Hz) and 6.63 (d, J = 8.8 Hz). In addition,
the spectrum showed the presence of an ethyl group due
to the signals at d 0.79 (t, J = 7.5 Hz, 3H) and d 1.55 (m,
2H) and a methylenedioxy group due to the singlet at d
5.30 (2H), which was positioned at C-3/C-4 similarly to
compound 1. The signal at d 4.15 (t, J = 6.6 Hz, H-7) indi-
cated an oxybenzyl hydrogen, which was confirmed by the
presence of an oxymethine carbon at d 75.9 in the 13C
NMR spectra (Table 1). The presence of the hydroxyl
group in 3 was confirmed by IR spectroscopy due to the
2.3. Determination of the enantiomeric excesses and absolute
configuration
The enantiomeric excesses of alcohol 3 and acetate 3a were
calculated from the peak areas observed in the chiral GC
chromatograms and by comparison with those observed
in the racemic samples.16 (R)-Acetate 3a was hydrolyzed
to the (R)-alcohol 3 (tR = 49.2 min), which showed
Table 1. 1H and 13C NMR (300 and 75 MHz, d ppm, CDCl3) spectral data for compounds 1–3 isolated from P. marginatum
Position
1
2
3
dH (m, J/Hz)a
dC
a
dH (m, J/Hz)
dC
dH (m, J/Hz)
dC
1
2
3
4
5
6
7
8
—
131.8
107.7
148.1
151.5
107.8
124.0
198.8
31.5
—
—
120.6
156.6
94.2
152.0
141.6
109.1
200.6
36.8
—
138.7
106.4
146.9
147.8
108.0
119.4
75.9
31.8
10.1
100.9
—
7.51 (d, 1.5)
—
—
6.92 (d, 8.5)
7.65 (dd, 8.5, 1.5)
—
3.00 (q, 7.6)
1.30 (t, 7.6)
6.10 (s)
6.82 (d, 1.8)
—
—
6.46 (d, 1.2)
—
—
7.24 (d, 1.2)
—
2.87 (q, 7.5)
1.46 (t, 7.5)
6.10 (s)
3.78 (s)
6.63 (d, 8.8)
6.55 (dd, 8.8, 1.8)
4.15 (t, 6.6)
1.55 (m)
0.79 (t, 7.5)
5.30 (s)
9
8.4
101.7
—
8.5
101.8
56.2
OCH2O
OCH3
—
—
a Spectra recorded in C6D6.

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J. B. Reigada et al. / Tetrahedron: Asymmetry 18 (2007) 1054–1058
Table 2. Kinetic resolution of (RS)-3 catalyzed by Novozym 435a
OH
OH
OAc
O
O
O
O
O
O
Novozym 435
Hexane
+
(RS)-3
(R)-3a
(S)-3
ee >99%
ee >99%
Entry
t
c 3a
ee (AC) 3a
ee (AC) 3
E
1
2
3
4
5
6
7b
8
1
3
5
7
9
24
48
—
Nc
Nc
22
30
35
48
50
—
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
>99 (R)
—
9 (S)
16 (S)
28 (S)
42 (S)
53 (S)
92 (S)
—
—
—
—
—
>200
>200
—
>99 (S)
48c (R)
t: time (h); c (%): conversion, calculated from the ee’s of the substrate (ees)
and the product (eep); c: ees/(eep + ees)16; ee (%): enantiomeric excess; AC:
absolute configuration; E: enantiomeric ratio; Nc: not calculated.
a The small scale reaction was carried out at 32 ꢁC (160 rpm) using alcohol
3 (50 mg), vinyl acetate (1 mL), hexane (10 mL) and Novozyme 435
(100 mg).
b Preparative scale enzymatic reaction (see Section 3.6.2: 500 mg of 3).
c Natural product isolated from P. marginatum.
Figure 3. (a) GC chiral chromatogram of (RS)-3; (b) GC chiral
chromatogram of natural product 3 isolated from P. marginatum.
½aꢁD ¼ þ31:6 (c 3.20, CHCl3). The absolute configuration
of the (S)-alcohol 3 and (R)-acetate 3a were suggested by
stereochemical preference by CALB based on Kazlauskas
rule.17 In addition, the chemoenzymatic resolution of the
(RS)-1-phenylpropanol 6 produced (S)-alcohol 6 and (R)-
1-phenylpropane acetate 6a in accordance with Kazlauskas
rule (Fig. 2).18
Table 3. Minimum amount of compounds 1–5 isolated from P. margin-
atum required for the inhibition of fungal growth on thin-layer chro-
matographic plates (TLC)
Compounds
Antifungal activity (lg)
C. cladosporioides
C. sphaerospermum
1
5.0
5.0
5.0
5.0
2
3
10.0
1.0
10.0
1.0
4
OAc
5
1.0
1.0
1.0
1.0
1.0
1.0
O
Nystatin
Miconazole
O
(R)-3a
H
OH
enantiomer resolved by CALB
OAc
be seen by the detection limit of these compounds (Table
3), all the substances described displayed antifungal activ-
ity when submitted to bioautographic assays. Flavanones
4 and 5 were the most potent with activities comparable
to the controls, and compounds 1 and 2 showed higher
activity than 3. Therefore, the carbonyl group appeared
to be important to the antifungal activity, since compounds
1 and 2 have their potential increased by the presence of
this group. There has been no previous report describing
the antifungal activity of compounds 1–3.
L
M
M = medium sized group
L = large group
(R)-6a
enantiomer resolved by CALB
Figure 2. Kazlauskas rule for the esterifications of 3 and phenyl-propanol
6 using CALB (Novozyme 435).17,18
Finally, the natural compound 3 isolated from P. margina-
tum was determined as having an (R)-configuration (Fig. 3)
based on the well established configurations and retention
times of the (R)-3 and (S)-3 derivatives and an enantio-
meric excess of 48% (Table 2, entry 8 and Fig. 2).
3. Experimental
3.1. General
Silica gel (Merck 230–400 mesh) and Sephadex LH-20
(Pharmacia) were used for column chromatography sepa-
rations and silica gel 60 PF254 (Merck) for preparative
TLC purifications (1.0 mm). 1H NMR (300 MHz) and
13C NMR (75 MHz) spectra were measured in CDCl3
and C6D6 on Bruker DPX-300 instrument with 1% of
2.4. Antifungal activity
The antifungal activity of compounds 1–5 was determined
by means of a direct bioautography on TLC plate.19 As can

J. B. Reigada et al. / Tetrahedron: Asymmetry 18 (2007) 1054–1058
1057
TMS as internal standard; LREIMS were measured at
70 eV on a HP 5990/5988A spectrometer; IR spectra were
obtained on an FT-IR 510 Nicolet spectrometer and UV
spectra were recorded on a UV/Visible Shimadzu 1650-
PC spectrophotometer.
three sub-fractions. Bioactive sub-fraction 2 (100 mg) was
composed of pure 1.
Fraction 2 (100 mg) was submitted to prep. TLC (hexane–
CH2Cl2 8:2) to afford three sub-fractions. Compound 2
(39 mg) was isolated in pure form from bioactive sub-frac-
tion 2.
The conversions and enantiomeric excesses of the enzyme-
catalyzed reactions were determined using a Shimadzu
GC-17A gas chromatograph equipped with a chiral
capillary column Chirasil-Dex CB b-cyclodextrin (25 m ·
0.25 mm · 0.25 lm). The carrier gas was hydrogen with a
pressure of 100 kPa. Optical rotation values were measured
in a Jasco DIP-378 polarimeter and the reported data refer
to the Na-line value using a 1 dm cuvette. Novozym 435
immobilized lipase from Candida antarctica was obtained
Fraction 3 (59 mg) was chromatographed on a Sephadex
LH-20 column eluted with hexane–CH2Cl2 (1:4) followed
by CH2Cl2–Me2CO 3:2 and CH2Cl2–Me2CO 1:4 to yield
ten sub-fractions. Compound 3 (5 mg) was isolated from
sub-fraction 6, and compounds 4 (4 mg) and 5 (4 mg) were
isolated from sub-fractions 9 and 10, respectively.
´
as a gift from Novozymes Latin America Ltda (Parana-
3.4.1. 1-(3,4-Methylenedioxyphenyl)propan-1-ol 3 [(+)-(R)-
marginatumol]. Yellow oil. IR (film) (cmꢀ1): 3383, 2965,
2877, 1503, 1487, 1441, 1248, 1040, 930, 811; UV kmax
(CHCl3) nm (loge): 286 (3.55), 240 (3.56); LREIMS m/z
(relative intensity): 180 [M+] (25), 151 (89), 93 (100), 77
Brazil).20
3.2. Plant material
1
(76); H NMR and 13C NMR (Table 1).
Leaves of P. marginatum Jacq. were collected at Manaus
(Amazonia State, Brazil) in May 2002 and identified by
Dr. Elsie F. Guimaraes. A voucher specimen (Kato-0223)
˜
was deposited in the Herbarium of Instituto de Botaˆnica,
Sao Paulo, SP, Brazil.
˜
3.5. Synthesis of (RS)-1-(3,4-methylenedioxyphenyl)propan-
1-ol 3 [(RS)-marginatumol]
To a 125 mL three-necked flask equipped with an addi-
tional funnel, one-neck stoppered and one fitted with a
condenser, were added 486 mg (20 mmoles) of dry magne-
sium turnings and the whole system was dried by heating.
Then 30 mL of anhydrous ether with a crystal of iodine
was added to the flask. Next, a solution of 2.21 g (20 mmo-
les) of dry ethylbromine in 10 mL of anhydrous ether was
added using a separatory funnel. After preparation, the
Grignard reagent was cooled (0 ꢁC) and had the piperonal
(3 g, 20 mmoles) slowly added. The mixture was stirred
until all of the piperonal had been consumed after which
the mixture was kept stirring at room temperature for
60 min. Then, the mixture was extracted between aqueous
NH4Cl (20 mL) and EtOAc (3 · 50 mL). The organic
phases were combined, dried over MgSO4, concentrated
under reduced pressure and the product chromatographed
over silica gel eluted with hexane–EtOAc (9:1) to give 3 as a
yellow oil (2.98 g, 17 mmoles, 84% yield).
3.3. Antifungal assay
The microorganisms used in the antifungal assays C. clado-
sporioides (Fresen) de Vries SPC 140 and C. sphaerosper-
mum (Perzig) SPC 491 have been maintained at the
Instituto de Botaˆnica, Sao Paulo, SP, Brazil. For the anti-
˜
fungal assays, 10.0 lL of the solutions of the crude extract,
fractions and pure compounds were prepared, under differ-
ent concentrations, corresponding to 100.0 lg of crude
extract and 10.0, 5.0 and 1.0 lg of fractions and pure com-
pounds. The samples were applied to TLC plates, which
were eluted with CHCl3–MeOH (9:1) and dried for com-
plete removal of solvents. The chromatograms were
sprayed with a spore suspension of C. cladosporioides or
C. sphaerospermum in a nutritive medium19 and incubated
for 48 h at 25 ꢁC. After incubation, clear inhibition zones
appeared against a dark background chromatogram. Nys-
tatin and miconazole were used as positive controls
whereas ampicillin and chloramphenicol were used as neg-
ative controls.
3.6. Enzymatic reaction
3.6.1. Small scale enzymatic reaction. To a 50 mL Erlen-
meyer flask containing 10 mL of hexane (HPLC grade),
1 mL of vinyl acetate and 100 mg Novozym 435 was added
3 (50 mg). The reaction mixture was stirred on a rotatory
shaker (32 ꢁC, 160 rpm) and analyzed until the starting
material had been totally consumed (Table 2).
3.4. Extraction and isolation of constituents
The dried and powdered leaves of P. marginatum (82.2 g)
were exhaustively extracted with MeOH at room tempera-
ture. The resulting MeOH extract was filtered and concen-
trated in vacuum to yield 3.43 g of crude extract, which was
dissolved in MeOH–H2O (1:1) and extracted with EtOAc.
The bioactive EtOAc phase (1.22 g) was subjected to col-
umn chromatography over silica gel (gradient of hexane
to EtOAc) to yield nine fractions, in which bioactivity
was detected in three of them (fractions 1–3).
3.6.2. Preparative scale enzymatic reaction. To a 250 mL
Erlenmeyer flask containing 100 mL of hexane (HPLC
grade), 1 mL of vinyl acetate and 1 g Novozym 435 was
added 500 mg of 3. The reaction mixture was stirred on a
rotatory shaker (32 ꢁC, 160 rpm) and analyzed for con-
sumption of the starting material (48 h, Table 2). The mix-
ture was then filtered and the solvent evaporated. The
residue was purified by silica gel column chromatography
Fraction 1 (200 mg) was applied to a silica gel column and
eluted with gradient mixtures of EtOAc in hexane giving

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J. B. Reigada et al. / Tetrahedron: Asymmetry 18 (2007) 1054–1058
using hexane–EtOAc (9:1) as eluent to afford (S)-3 (43%)
and (R)-3a (45%).
Acknowledgements
´
The authors acknowledge Novo Nordisk (Curitiba-Parana-
Brazil) for providing CALB. M. J. Kato, C. M. Tcacenco,
J. H. G. Lago, L. H. Andrade and A. L. M. Porto thank
FAPESP for financial support. J. B. Reigada, M. J. Kato
and J. H. G. Lago acknowledge CNPq for providing a
scholarship.
3.6.3. Control of the chemoenzymatic resolution of 3 by
Novozym 435. The reaction progress was monitored by
collecting 0.1 mL samples every 1 h until 48 h (Table 2).
These samples were previously analyzed by GC/FID in a
chiral capillary column. Alcohol 3 and acetate 3a were
compared with the racemic mixtures previously analyzed.
References
General GC conditions: Injector: 200 ꢁC; Detector: 220 ꢁC.
Pressure: 100 kPa. Oven 100–150 ꢁC; rate 1 ꢁC; 150–
180 ꢁC; rate 50 ꢁC; retention time for alcohol 3 [(R)-iso-
mer = 49.2 min; (S)-isomer = 49.7 min] and acetate 3a
[(S)-isomer = 30.2 min; (R)-isomer = 31.2 min].
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6a have already been described in the literature.18
25
(ꢀ)-(S)-1-(3,4-Methylenedioxyphenyl)propan-1-ol 3: ½aꢁD
¼
ꢀ34:1 (c 3.26, CHCl3), ee 99%.
25
(+)-(R)-1-(3,4-Methylenedioxyphenyl)propan-1-ol 3: ½aꢁD
¼
þ31:6 (c 3.20, CHCl3), ee 99% alcohol obtained by hydro-
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14. Raminelli, C.; Comasseto, J. V.; Andrade, L. H.; Porto, A. L.
M. Tetrahedron: Asymmetry 2004, 15, 3117.
15. Vieira, T. O.; Ferraz, H. M. C.; Andrade, L. H.; Porto, A. L.
M. Tetrahedron: Asymmetry 2006, 17, 1990.
16. Faber, K.. Biotransformation in Organic Chemistry, 4th ed.;
Springer: Berlin, 2000.
(+)-(R)-1-(3,4-Methylenedioxyphenyl)propyl acetate 3a:
½aꢁD ¼ þ101:3 (c 3.10, CHCl3), ee 99%.
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(ꢀ)-(S)-1-Phenylpropan-1-ol 6: ½aꢁD ¼ ꢀ50:7 (c 3.73,
CHCl3), ee 99%.
17. Kazlauskas, R. J.; Weissfloch, A. N. E.; Rappaport, A. T.;
Cuccia, L. A. J. Org. Chem. 1991, 56, 2656.
18. Raminelli, C.; Kagohara, E.; Comasseto, J. V.; Pellizari, V.;
Andrade, L. H.; Porto, A. L. M. Enzyme Microb. Technol.
2007, 40, 362.
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(ꢀ)-(S)-1-Phenylpropan-1-ol 6: ½aꢁD ¼ ꢀ28:0 (c 1.0,
MeOH).17
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(+)-(R)-1-Phenylpropyl acetate 6a: ½aꢁD ¼ þ80:4 (c 3.77,
19. Homans, A. L.; Fuchs, A. J. Chromatogr. 1970, 51, 327.
20. Novozymes Latin America Ltda—Rua Professor Francisco
CHCl3), ee 99%.
´
´
Ribeiro, 683, CEP 83707-660, Araucaria–Parana-Brazil. Tel.:
+55 41 641 1000, fax: +55 41 643 1443,
www.novozymes.com.
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(+)-(R)-1-Phenylpropyl acetate 6a: ½aꢁD ¼ þ100:0 (c 1.0,
CHCl3).18