Aryltetralols from Holostylis reniformis and syntheses of lignan analogous

Article abstract of DOI:10.1016/j.phytol.2015.06.001

Abstract Two new lignans, an aryltetralol and its methyl ether analogous, were isolated from Holostylis reniformis (Aristolochiaceae) together with futokadsurin C and (-)-8′-epi-aristoligone. The latter was also obtained as an enantiomeric mixture by synt

Full text of DOI:10.1016/j.phytol.2015.06.001

Phytochemistry Letters 13 (2015) 200–205
Contents lists available at ScienceDirect
Phytochemistry Letters
journal homepage: www.elsevier.com/locate/phytol
Aryltetralols from Holostylis reniformis and syntheses of lignan
analogous
Marcos D.P. Pereiraa, Matheus R. Ferreiraa, Gisele B. Messianoa,1, Isabela Penna Cerávolob
b,**
, Lucia M.X. Lopesa, , Antoniana U. Krettli
*
a
Institute of Chemistry, São Paulo State University, UNESP, C.P. 355, 14801-970, Araraquara, SP, Brazil
Centro de Pesquisas René Rachou/FIOCRUZ, Av. Augusto de Lima 1715, 30190-002, Belo Horizonte, MG, Brazil
b
A R T I C L E I N F O
A B S T R A C T
Article history:
Two new lignans, an aryltetralol and its methyl ether analogous, were isolated from Holostylis reniformis
(Aristolochiaceae) together with futokadsurin C and (ꢀ)-80-epi-aristoligone. The latter was also obtained
as an enantiomeric mixture by synthesis and was transformed into aryltetralols and aryltetralenes that
were subjected to chiral-HPLC separations. The compound structures were determined by spectroscopic
methods. Several of these lignans had their antiplasmodial activity (against Plasmodium falciparum, W2
clone, anti-HRPII) and toxicity to mammalian kidney cells (MDL50) evaluated. (ꢀ)-Cyclogalgravin and
Received 18 March 2015
Received in revised form 26 May 2015
Accepted 4 June 2015
Available online xxx
Keywords:
(ꢀ)-aristoligol exhibited activity (IC50 ꢁ10.8 and 8.4
mM, respectively), the latter exhibited lower toxicity.
Holostylis reniformis
Aristolochiaceae
Lignans
ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.
Aryltetralol
Arytetralene
Antiplasmodial activity
1. Introduction
and aryltetralene lignans obtained by regioselective syntheses.
These lignans belong to 2,70-cyclolignan subclass of lignans or
Holostylis reniformis Duch. (Aristolochiaceae) synthesizes a
variety of seco compounds, including lignans and sesquiterpenes
(da Silva and Lopes, 2004, 2006; Lopes et al., 2012; Martins et al.,
2014; Pereira et al., 2012). More than 25 aryltetralone and furan
lignans without oxygenation at C-9,90 have been isolated from
extracts of this species (da Silva and Lopes, 2004, 2006; Lopes et al.,
2012), and isoeugenol is shown to be their biosynthetic
intermediate (da Silva and Lopes, 2004, 2006; Messiano et al.,
2008, 2009). The anti-Plasmodium falciparum activities and
toxicities of lignans and extracts obtained from H. reniformis have
been demonstrated (da Silva et al., 2004; da Silva and Lopes, 2004;
de Andrade-Neto et al., 2007). As part of our continuing studies on
the chemical constituents of H. reniformis, in this paper we report
the results of the in vitro antiplasmodial and toxicity evaluation of
two new natural lignans (1 and 2) and of aryltetralone, aryltetralol,
arylnaphthalene type of lignans. Lignans of this subclass, without
oxygenation at C-9,90, have already been obtained by syntheses
using commercially available compounds (Kuo and Lin, 1993;
Takeya et al., 1983; Yvon et al., 2001), via transformations of
7,70-epoxylignans (furan lignans) (Blears and Haworth, 1958;
Crossley and Djerassi, 1962; Purushothaman et al., 1984), and by
biotransformations from fungi (Messiano et al., 2010). However,
stereoselective syntheses of 2,70-cyclolignans normally involve
more than 10 steps (Peng et al., 2013; Rye and Barker, 2011), and
those regioselective carried out in fewer steps showed poor yields
(Barba et al., 1990; Iguchi et al., 1978). Aiming to obtain lignan
analogous to advances in pharmacological studies of these lignans,
this report describes a sequence for the syntheses of aryltetralone,
aryltetralol, and aryltetralene lignans in four, five, and six steps,
successively by improving a route for obtaining aryltetralone
lignans proposed by Müller and Vajda (1952).
2. Results and discussion
* Corresponding author. Fax: +55 16 3301 9692.
** Corresponding author. Fax: +55 31 3295 3115.
E-mail addresses: mpelicon@bol.com.br (M.D.P. Pereira),
matheusrf_sud@hotmail.com (M.R. Ferreira), gbaraldi@ifsp.edu.br (G.B. Messiano)
, ceravolo@cpqrr.fiocruz.br (I.P. Cerávolo), lopesxl@iq.unesp.br (L.M.X. Lopes),
akrettli@cpqrr.fiocruz.br (A.U. Krettli).
Present address: Instituto Federal de São Paulo, R. Stéfano D’Avassi 625, 15991-
502 Matão, SP, Brazil.
The acetone extract of H. reniformis roots was subjected to
column chromatography followed by HPLC to yield two minor new
lignans (1 and 2, Fig. 1) together with the known compounds
(ꢀ)-futokadsurin C (3) and (ꢀ)-80-epi-aristoligone (4a). They were
analyzed by spectroscopic methods, and the known compounds
1
http://dx.doi.org/10.1016/j.phytol.2015.06.001
1874-3900/ã 2015 Phytochemical Society of Europe. Published by Elsevier B.V. All rights reserved.

M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205
201
OR
7
O
9
1
H3CO
R1O
H3CO
7'
1'
H3CO
9'
3
OCH3
O
O
3'
OCH3
O
OCH3
OCH3
OCH3
OR2
R1 R2
3
4a
R
1
2
5
6
7
H
H
H
CH3
CH3
CH3
H
CH3 CH3
CH3 CH3 CH3
CH3 CH3
H
Fig. 1. Chemical structures of compounds 1-3, 4a, and 5–7.
were identified by comparison with data reported in the literature
(da Silva and Lopes, 2004, 2006; Konishi et al., 2005; Kuo et al.,
1989) (Fig. 1).
(1: dC 74.1; dH 4.38; 2: dC 83.4; dH 3.92) and CH-8 (1: dC 39.4;
dH 1.99; 2: dC 34.6; dH 2.19), suggested that these compounds differ
by the presence of a methoxyl group at C-7 in 2 instead of a
hydroxyl group in 1. Furthermore, gHMBC experiments supported
correlations between C-7 and OCH3-7, H-6, and 3H-9. The
magnitude of the coupling constants of the methine hydrogens
in the B ring evidenced trans diaxial, cis axial–equatorial, and trans
diequatorial positions for H-70,80 (J = 10.0 ꢂ 0.5 Hz), H-80,8
(J = 3.5 Hz), and H-7,8 (J = 3.7 ꢂ 0.3 Hz), respectively. Moreover,
gNOESY experiments showed spatial interactions between H-7
and 3H-9, as well as between H-70 and H-3, H-20, H-60, 3H-9, and
3H-90 for both compounds. Furthermore, spatial interactions of
The 1H and 13C NMR, IR, and UV spectroscopic data for
compounds 1 and 2 were very similar to those reported for the
aryltetralol derivatives, previously isolated from H. reniformis and
obtained by microbial transformation of the aryltetralin lignans
5-7 (da Silva and Lopes, 2006; Messiano et al., 2008, 2010).
Compounds 1 and 2 were also suggested to be aryltetralol lignans
based on the HRMS spectra. They displayed quasi-molecular ions at
m/z 381.1663 [M + Na]+ and at m/z 371.1445 [M ꢀ H]ꢀ, respectively,
which were consistent with the molecular formula C21H26O5 for 1
and C22H28O5 for 2 (14
m
higher than 1). The IR spectra of both
H-6 (
observed in gNOESY experiments of 2. Both compounds showed
very similar CD curves with a positive Cotton effect at
ꢁ 290 nm,
consistent with a 70R configuration (da Silva et al., 2004). Thus,
d 6.68) with H-7, OCH3-7 (d 3.40), and OCH3-5 (d 3.84) were
compounds showed characteristic absorptions of hydroxyl groups
at ꢁ3440 cmꢀ1. The 1H and 13C NMR spectra of both compounds
(Tables 1 and 2, Figs. S1–S4) suggested the presence of a veratryl
group (C ring), and one methoxyl group linked to 1,2,4,5-
tetrasubstituted aromatic ring (A). These spectra also showed
signals for two methyl groups and four methine carbons, of
which one is oxygenated. In addition, signals for hydroxyl groups
l
the absolute configuration of
1 and 2 was determined as
(7R,70R,8S,80S), which was identical to that previously determined
for 5–7 (da Silva and Lopes, 2006; Messiano et al., 2010).
The synthesis of aryltetralone (4 and 11), aryltetralol (12), and
aryltetralene (13) lignans was achieved from four to six steps,
successively, starting from stable and easily available materials.
Improving the scheme proposed by Müller and Vajda (1952), via
at dH ꢁ 5.4 were observed. Compound
2 showed additional
resonances reminiscent of an aliphatic methoxyl group (dC 56.0;
dH 3.40). The differences between the chemical shifts of carbons
and hydrogens on the B ring of 1 and 2, particularly of CH-7
Reformatsky, by using veratrylacetone and
a-bromopropianate,
Table 1
1H NMR spectroscopic data for compounds 1 and 2 (CDCl3, 11.7 T).
Position
H
1
2
dH (J in Hz)a
gNOESY
dH (J in Hz)a
gNOESY
3
6
7
8
6.28, d (1.0)
6.78, s
70
6.29, d (0.5)
6.68, s
70
7, OCH3-5
6, 8, 9
7, OCH3-5, OCH3-7
6, 8, 9, OCH3-7
7, 9, 80, OCH3-7
7, 8, 70, 90
70, 80, OCH3-30
OCH3-40
4.38, d (4.0)
1.99, ddq (4.0, 3.5, 7.5)
0.83, d (7.5)
6.55, d (2.0)
6.70, d (8.0)
6.56, dd (8.0, 2.0)
3.36, br d (9.5)
2.32, ddq (9.5, 3.5, 7.0)
0.81, d (7.0)
3.73, s
3.92, d (3.5)
2.19, ddq (3.5, 3.5, 7.0)
0.79, d (7.0)
6.55, d (2.0)
6.71, d (8.0)
6.63, dd (8.0, 2.0)
3.30, br d (10.5)
2.35, ddq, (10.5, 3.5, 7.0)
0.81, d (7.0)
3.73, s
7, 9, 80, 90
7, 8, 70, 90
70, 80, OCH3-30
OCH3-40
50, 70, 80
3, 9, 20, 60, 90
8, 20, 60, 90
8, 9, 80
20
9
20
50
60
70
70
3, 9, 20, 60, 90
8, 20, 90
80
90
80
OCH3-30
OCH3-40
OCH3-5
OCH3-7
OH-4
3.78, s
3.81, s
50
6
3.80, s
3.84, s
3.40, s
5.37, br s
7, 8
5.49, br s
a
Multiplicities were determined with the assistance of 1H–1H COSY spectra and simulation using ACD/C + 1H NMR predictors (ACD, 2010).

202
M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205
The similarity of the 1H and 13C NMR data and optical activities
of 13a ([ 290 ꢀ10330) with those from
]D = ꢀ96 (CHCl3; c 0.21), [
the natural product ([
(da Silva and Lopes, 2006) confirmed the assigned relative and
absolute configurations for 13a.
Table 2
13C NMR spectroscopic data for compounds 1 and 2 (CDCl3, 11.7 T).
a
u]
a
]D = ꢀ106 (CHCl3; c 1.07), [ 290 ꢀ1254)
u
]
Position
H
1
2
dC, typea
gHMBCb
dC, typea
gHMBCb
1
2
3
4
5
6
7
8
128.6, C
133.0, C
H-3, 7, 70
H-6, 7, 70
126.0, C
134.0, C
115.5, CH
H-3
H-6
The compound 12a and 13a exhibited the highest activity
115.6, CH H-70
(IC50 8.4 and 10.8
moderate activity (IC50 30
1 and 5 also showed moderate activity (IC50 33.6 and 33.1
m
M, respectively), whereas 13b showed
M) (Table S1). The natural aryltetralols
M,
145.3, C
145.4, C
H-3 and/or H-6
H-3 and/or H-6, OCH3-5 144.9, C
145.3, C
H-6
H-3, OCH3-5
m
m
111.4, CH H-7
112.2, CH H-7
respectively), while the C-7 OCH3 analogous 2 and 6 were inactive
(Table S1). These results suggest that steric effects also have
influence on the activity. Among the tested lignans, 12a and 13a
exhibited good selectivity index (SI > 28).
74.1, CH
39.4, CH
11.7, CH3 H-7
138.1, C
112.4, CH H-60, 70
148.9, C
147.5, C
111.0, CH
121.7, CH H-20, 50, 70
48.7, CH
35.1, CH
16.8, CH3 H-70
H-9
H-9
83.4, CH
34.6, CH
11.1, CH3
138.4, C
H-6, 9, OCH3-7
H-70, 90
9
10
20
30
40
50
60
70
80
90
H-50, 70
H-50
112.2, CH H-60, H-700
H-50, OCH3-30
H-20, 60, OCH3-40
148.9, C
147.4, C
110.8, CH
121.8, CH H-20
48.8, CH
35.3, CH
17.2, CH3 H-70
56.0, CH3
55.9, CH3
55.9, CH3
56.0, CH3
H-50, OCH3-30
3. Experimental
H-20, 60, OCH3-40
3.1. General experimental procedures
H-3, 20, 60, 90
H-7, 9, 70
H-3, 20, 90
One-dimensional (1H, 13C, DEPT, HOMODEC, TOCSY, and
gNOESY) and two-dimensional (1H–1H gCOSY, gNOESY, gHMQC,
gHSQC, and gHMBC) NMR experiments were performed on a
Varian INOVA 500 spectrometer (11.7 T) at 500 MHz (1H), and
126 MHz (13C), using deuterated solvents (CDCl3 and DMSO-d6) (P
99.9% D) as an internal standards for 13C NMR chemical shifts and
OCH3-30 55.9, CH3
OCH3-40 55.9, CH3
OCH3-5 55.9, CH3
OCH3-7
a
Chemical shifts and multiplicities were determined with the assistance of DEPT
and gHMQC experiments.
residual solvent as an internal standard for 1H NMR.
d values are
b
gHMBC correlations, optimized for 8 Hz, are from carbon stated to the indicated
reported relative toTMS. Mass spectra (ESI-MS) were obtained on a
LCQ Fleet—Thermo Scientific, and flow injection into the electro-
spray source was used for LC-ESI-MS. High-resolution mass spectra
(HRMS) were obtained on a Bruker Daltonics ultrOTOFq (ESI-
TOFMS). IR spectra were obtained on a PerkinElmer 1600 FT-IR
spectrometer using KBr discs. Optical rotations were measured on
a PerkinElmer 341-LC polarimeter. Ultraviolet (UV) absorptions
were measured on a PerkinElmer UV–vis Lambda 14P diode array
spectrophotometer. Circular dichroism (CD) spectra were recorded
on a JASCO J-815 spectrometer, using 0.2 mm cell. HPLC analyses
were performed using a Shimadzu liquid chromatograph (SPD-10
Avp), equipped with UV–vis and 341-LC polarimeter detectors, and
using a Jasco LC-NetII/ADC, equipped with photodiode array (MD-
2018 Plus) and CD (2095 Plus) detectors, and chromatograms were
acquired at 270 nm and 254 nm. The columns RP-18 (C18, Varian),
hydrogen(s).
two pairs of aryltetralone enantiomers (4 + 11) were obtained,
after HPLC separation, in four steps with good yield (87.5%). The
major bottleneck in 4 + 11 synthesis was the lactonization after
Reformatsky reaction. By extracting the intermediate lactone (9)
with CHCl3 instead of distilling it (Müller and Vajda, 1952), the
yield of this step increased from 28% to 99.8%.
After separation by HPLC, each pair of enantiomers (4 and 11)
was evaluated in vitro for their antiplasmodial activity. The
immunoenzymatic test with monoclonal antibodies to the parasite
protein histidine- and alanine-rich (HRPII), as shown in SI, was
used to assess the inhibition of growth of P. falciparum in the
presence of these compounds (Krettli et al., 2009; Noedl et al.,
2002). Enantiomers 11 and enantiomers 4 showed to be inactive
Chiralpak1 IC (DAICEL), Lux 5
m
Cellulose-1 (Phenomenex1), and
m, were
(IC50 ꢃ 82
mM, Table S1), suggesting that the stereochemistry is
b
-CD BR Chiralpak1 (YMC) having a particle size of 5
m
very important for the antiplasmodial activity of this type of
lignans. Aiming to obtain pure enantiomers to advance in the
biological tests and chemical transformations, to evaluate the
importance of C-7 carbonyl group for the activity, samples of 4
were analyzed by chiral-HPLC by using several conditions in
analytical scales (columns, solvents and flows). Unfortunately, no
suitable condition to separate the enantiomers 4 in a semi-
preparative scale was achieved. Thus, samples of 4 + 11 and the
natural aryltetralone 4a [(ꢀ)-80-epi-aristoligone] were individually
subjected to reduction with NaBH4 in methanol to give 12 and 12a,
respectively. The structure of 12a was confirmed by comparison of
used for analytical analysis (250 ꢄ 4.6 mm) and for semi-
preparative analysis (250 ꢄ10 mm). All reactions were monitored
by TLC using 0.25 mm E. Merk silica gel plates (60 PF254) with UV, I2
vapor, or 10% H2SO4—heat as developing agent. All reactions were
carried out under N2 atmosphere with freshly distilled solvents
under anhydrous conditions unless otherwise noted. Reagents
were purchased from Sigma–Aldrich Co., and used without
purification, except where noted. Solvents employed were HPLC
grade from Mallinckrodt. All yields refer to chromatographically
and spectroscopically (1H and 13C NMR, aD) homogenous material
unless otherwise stated. Ultrapure water was obtained from
Milli-Q Gradient A10 from Millipore.
its 1D and 2D NMR spectra and optical activity ([
a
]D = ꢀ27 (CHCl3,
c 0.10)) with those of an authentic sample of (ꢀ)-aristoligol (12a)
[
a
]D = ꢀ20 (CHCl3, c 0.10) (da Silva and Lopes, 2006). Interestingly,
3.2. Plant materials
the stereochemistry of C-7 of 12a is different from that of the
natural aryltetralol (1). Finally, subjecting 12 and 12a to dehydra-
tion with p-toluenesulfonic acid, the products 13 and 13a were
obtained in 83.3% and 85% yield, respectively (Fig. 2).
The plant materials were collected in Ituiutaba, MG, Brazil, in
February 2010, and identified as Holostylis reniformis Duch. by
Dr. Vinícius C. Souza and Dr. Lindolpho Cappellari Jr. A voucher
specimen (ESA 110,744/2010) was deposited at the herbarium of
the Escola Superior de Agricultura, Luiz de Queiroz (ESALQ),
Piracicaba, SP, Brazil. Authorization CGEN/MMA number 10,586/
2012-1. The material was separated according to the plant parts
and dried (ꢁ45 ꢅC).
A
better chromatographic condition, with lower tR, was
achieved for the separation of the enantiomers 13 than for the
aryltetralones 4 using chiral columns (Fig. S5). Thus, 13 was
subjected to semi-preparative chiral-HPLC to give (ꢀ)-cyclo-
galgravin 13a (tR = 14.3 min, 50.9%) and (+)-cyclogalgravin 13b
(tR = 13.4 min, 49.1%).

M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205
203
Fig. 2. Total syntheses of aryltetralenes 13a and 13b.

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M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205
3.3. Extraction and isolation of the chemical constituents
solution was heated until the reflux began, this being maintained
for 4 h. Then, the mixture was cooled and the ester (4.24 g,
16.89 mmol, 98.2%) extracted with CHCl3 (3 ꢄ 50 mL).
The roots (3.7 kg) were ground and exhaustively extracted
successively at room temperature with hexanes, acetone, and
ethanol [4 ꢄ (ꢁ200 mL, 2 days and shaken manually every 12 h for
2 min) each solvent] (da Silva and Lopes, 2006; Messiano et al.,
2009). The residues were extracted with ethanol in a Soxhlet
apparatus and the extracts were individually concentrated.
The crude acetone extract (6.6 g) was subjected to CC
(40.0 ꢄ 5.0 cm, silica gel 60H, 203.5 g, n-hexanes/EtOAc gradient,
19:1 to 100% EtOAc) to give 31 fractions (ca. 120 mL each), as
previously described (da Silva et al., 2004). After semi-preparative
HPLC (C18, MeOH/H2O, 7:3, flow rate: 8 mL/min) fractions 16 and
19 gave 3 (8.2 mg) and 4a (1140.0 mg), respectively. Fraction 23
(280.0 mg) gave 1 (43.5 mg) and 2 (8.9 mg) after semi-preparative
HPLC (C18, MeOH/H2O, 3:2, flow rate: 8 mL/min).
3.4.2.
a,b-Dimethyl-g-(3,4-dimethoxyphenyl) butyrolactone (9)
The ester (3.92 g, 15.6 mmol) was dissolved in CH3CO2H (13 mL)
and then ice-cooling. To this cooling and stirring solution sulfuric
acid (3 mL) was added by drops over 1 h. After 4 h on steam-bath,
the organic portion was successively extracted with CHCl3
(3 ꢄ 50 mL), neutralized with NaHCO3, washed with water and
dried (CaCl2), and concentrated at reduced pressure to give the
lactone 9 (3.43 g, 15.6 mmol, 99.8%).
3.4.3. 4,4-Bis(3,4-dimethoxyphenyl)-2,3-dimethyl-butyric acid (10)
A solution of AlCl3 (2.23 g, 16.8 mmol) in veratrol (1 mL,
7.97 mmol) was slowly dropwise to veratrol (1 mL, 7.97 mmol,
15 min) and the lactone 9 (2.2 g, 8.78 mmol). The resulting mixture
was then stirred at 80 ꢅC for 3 h, and then the complex was
decomposed by dropwise addition of EtOH (5 mL) at room
temperature, followed by addition of 10% HCl (10 mL). After
disappearance of solid particles, the mixture was extracted with
CHCl3 (3 ꢄ 50 mL), washed with 8% NaHCO3 (3 ꢄ 50 mL) and then
with 10% HCl until a white solution was obtained. The removal of
solvents under reduced pressure yielded the intermediary acid 10
(3.20 g, 8.24 mmol, 93.8 %).
3.3.1. (ꢀ)-(7R,70R,8S,80S)-4,7-Dihydroxy-30,40,5-trimethoxy-2,70-
cyclolignan [(ꢀ)-4-O-demethyl-7-hydroxyisogalbulin, 1]
Yellow oil (CHCl3); ½a D25
ꢆ
ꢀ38 (c 0.7, CHCl3); UV lMmaexOH see Fig. S6;
IR (KBr) nmax 3434, 1590, 1513, 1463 cmꢀ1; 1H and 13C NMR (CDCl3)
see Tables 1 and 2; ESIMS 20 eV, positive mode, m/z (rel. int.): 359
[M + H]+ (100); HRESIMS (probe) 4.5 eV, positive mode, m/z
(rel. int.): 381.1663 [M + Na]+ (100) (calcd for C21H26O5Na,
381.1672); CD (CHCl3, c 0.50): [
u
]
ꢀ5626, [
u
]
296
+22895,
307
[
u
]
279
ꢀ44311, [ ꢀ14739, [ ꢀ141569.
u
]
u]
241
260
3.4.4. 80-epi-Aristoligone and aristoligone (4 + 11)
The acid 10 (3.02 g, 8.1 mmol) was dissolved in dry benzene
(6 mL), then PCl5 (1.98 g, 9.5 mmol) was added to this solution,
which was kept stirring in ice-bath for 30 min. Then, the stirring
mixture was warmed up to 40 ꢅC and occasionally shacked until
the reactants dissolved. After that, the mixture was chilled and a
solution of SnCl4 (2.84 g, 10.9 mmol) in dry benzene (4 mL) was
added to it. In a few minutes, a red precipitate was observed,
which was dissolved with concentrate HCl. A mixture comprising
the enantiomer pairs 4 + 11 (2.85 g, 7.7 mmol, 95.1%) was
obtained by extraction with CHCl3, followed by removal of the
solvents.
3.3.2. (7R,70R,8S,80S)-4-Hydroxy-30,40,5,7-tetramethoxy-2,70-
cyclolignan [(ꢀ)-4-O-demethyl-7-methoxyisogalbulin, 2]
Yellow oil (CHCl3); ½a D25
ꢆ
ꢀ26 (c 0.6, CHCl3); UV lMmaexOH see Fig. S6;
IR (KBr) nmax 3446, 1590, 1520, 1430 cmꢀ1; 1H and 13C NMR (CDCl3)
see Tables 1 and 2; HRESIMS (probe) 4.5 eV, negative mode, m/z
(rel. int.): 371.1445 [M ꢀ H]ꢀ (100) (calcd for C22H27O5, 371.1853);
CD (CHCl3, c 0.57): [
u
]
+5494, [
u
]
ꢀ18353, [
u]
245
ꢀ113950.
297
278
3.3.3. (7R,70R,8S,80S)-7-Hydroxy-30,40,4,5-tetramethoxy-2,70-
cyclolignan [(–)-holostylol, 12a]
Yellow oil (CHCl3); ½a D25
ꢀ28 (c 0.10, CHCl3) lit. (da Silva et al.,
ꢆ
2006) ½a 2D5
ꢆ
ꢀ20 (c 1.6, CHCl3). 1H NMR (CDCl3, 500 MHz):
d 6.16
3.4.5. Aryltetralol 12
(1H, s, H-3), 7.08 (1H, s, H-6), 4.93 (1H, d, J = 4.5 Hz, H-7), 2.16 (1H,
ddq, J = 3.0, 4.5, 7.0 Hz, H-8), 0.86 (3H, d, J = 7.0 Hz, H-9), 6.48 (1H, d,
J = 2.0 Hz, H-20), 6.72 (1H, d, J = 8.0 Hz, H-50), 6.58 (1H, dd, J = 8.0,
2.0 Hz, H-60), 3.48 (1H, d, J = 10.0 Hz, H-70), 2.01 (1H, ddq, J = 3.0,
10.0, 7.0 Hz, H-80), 0.84 (3H, d, J = 7.0 Hz, H-90), 3.54 (s, OCH3-4), 3.84
(s, OCH3-5), 3.74 (s, OCH3-30), 3.81 (s, OCH3-40).
To a stirring solution of 4 + 11 (2.00 g, 5.40 mmol) in MeOH
(20 mL) a methanol solution of NaBH4 (204.2 mg, 5.40 mmol,
20 mL) was added. The mixture was stirred in an ice-bath for 4 h.
Following reduction, the excess of NaBH4 was quenched by
dropwise addition of MeOH and water. The organic phase resulting
from EtOAc (3 ꢄ 30 mL) extraction was washed with water,
dried (MgSO4), and concentrated to yield 12 (2.01 g, 5.40 mmol,
100.0%).
3.3.4. (70R,80S)-30,40,4,5-Tetramethoxy-2,70-cyclolignan-7-eno
[(ꢀ)-cyclogalgravin, 13a]
3.4.6. (ꢀ)-(70R,80S)- and (+)-(70S,80R)-30,40,4,5-Tetramethoxy-2,70-
cyclolignan-7-ene [(ꢀ)-cyclogalgravin, 13a and (+)-cyclogalgravin,
13b]
Yellow oil (CHCl3); ½a D25
ꢀ 103 (c 0.10, CHCl3), lit. (da Silva et al.,
ꢆ
2006) ½a 2D5
ꢆ
ꢀ106 (c 1.07, CHCl3). 1H NMR (CDCl3, 300 MHz):
d 6.49
(1H, s, H-3), 6.56 (1H, s, H-6), 6.08 (1H, br s, H-7), 1.73 (3H, d,
J = 1,2 Hz, H-9), 6.60 (1H, d, J = 2.1 Hz, H-20), 6.65 (1H, d, J = 8.4 Hz, H-
50), 6.49 (1H, dd, J = 8.4, 2.1 Hz, H-60), 3.62 (1H, d, J = 3.3 Hz, H-70),
2.33 (1H, dq, J = 7.2, 3.3 Hz, H-80),1.02 (3H, d, J = 7.2 Hz, H-90), 3.76 (s,
OCH3-4), 3.82 (s, OCH3-5); 3.72 (s, OCH3-30), 3.72 (s, OCH3-40).
To a stirring solution of the alcohol 12 (1.93 g, 5.20 mmol) in dry
benzene (15 mL) TsOH (0.72 g, 0.40 mmol) was slowly added. After
stirring under reflux, this solution was cooled, extracted with
CHCl3 and the solvent removed under reduced pressure to give
13 (1.53 g, 4.33 mmol, 83.3%). This product was subjected to
chiral-HPLC analysis [cellulose tris-(3,5-dichlorophenylcarbamate
column) to give (ꢀ)-13a (0.78 g, 2.20 mmol, ee > 99.9%), and
(+)-13b (0.75 g, 2.13 mmol, ee > 99.9%).
3.4. Syntheses and chemical transformations
3.4.1. Benzenebutanoic acid,
-dimethyl-, ethyl ester (8):
-Bromopropionate (2.89 g, 17.4 mmol) was dropwise added to
b-hydroxy-3,4-dimethoxy-
(ꢀ)-13a: yellow oil (CHCl3); ½a D25
ꢀ96 (c 0.21, MeOH); (+)-13b:
ꢆ
a,b
Yellow oil (CHCl3); ½a D25
ꢆ
+106 (c 0.20, MeOH), lit. (Messiano et al.,
296 +24783,
+543, [
a
2010)] ½a 2D5
ꢆ
ꢀ106 (c 1.07, CHCl3); CD (CHCl3, c 0.3): [
u]
a stirred solution of veratrylacetone (3.08 g, 17.2 mmol) in 9 mL of
dry benzene (under N2) and zinc (previously washed with acetone
and activated at 100 ꢅC for 8 h (Chavan, 2004)). The stirring
[
u
]
+10406, [
u
]
+71184, [
u
]
ꢀ15366, [
u
]
u]
226
290
274
249
240
+71706, [
u
]
220–52760, [
u
]
210
ꢀ153856, [
u
]
+113946.
201

M.D.P. Pereira et al. / Phytochemistry Letters 13 (2015) 200–205
205
3.4.7. HPLC separation of 4 + 11
Aliquots (30 mg) of 4 + 11 were subjected to semi-preparative
HPLC (20 times) using RP-C18 ODS Chrompack column, UV
da Silva, T., Krettli, A.U., de Andrade-Neto, V.F., Lopes, L.M.X., 2004. Set of lignans and
lignan extracts comprises constituents of malaria prevention compositions.
BR200404986-A. Patent PI0404986-1, 2004. Lignanas, lignanas ariltetralônicas,
extratos, processos de obtenção de lignanas, processo de obtenção de extratos,
uso de lignanas, uso de extratos e composição farmacêutica para prevenir e
tratar malária. Rev. Prop. Ind. 1774.
(l 254 nm) detector, flow rate of 8 mL/min, and MeOH/H2O (7:3,
v/v) as mobile phases.
da Silva, T., Lopes, L.M.X., 2004. Aryltetralone lignans and 7,8-seco-lignans from
Holostylis reniformis. Phytochemistry 65, 751–759.
3.4.8. Chiral-HPLC analysis of 4 (4a + 4b) and 13 (13a + 13b)
Aryltetralone (4, 4 mg) and aryltetralene (13, 4 mg) lignans were
subjected to HPLC (44 times each sample) using Chiralpak1 IC
da Silva, T., Lopes, L.M.X., 2006. Aryltetralol and aryltetralone lignans from Holostylis
reniformis. Phytochemistry 67, 929–937.
de Andrade-Neto, V.F., da Silva, T., Xavier Lopes, L.M., do Rosario, V.E., de Pilla
Varotti, F., Krettli, A.U., 2007. Antiplasmodial activity of aryltetralone lignans fro
Holostylis reniformis. Antimicrob. Agents Chemother. 51, 2346–2350.
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of E-isoeugenol and Z-isoeugenol. Chem. Lett. 1015–1018.
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column, UV (l 270 nm) and polarimeter (l 365 nm) detectors, flow
rate of 0.5 mL/min and 0.7 mL/min for 4 and 13, respectively, and
n-hexanes/EtOAc (17:3, v/v for 4 and 87:13, v/v for 13) as mobile
phases (Fig. S5).
Konishi, T., Konoshima, T., Daikonya, A., Kitanaka, S., 2005. Neolignans from Piper
futokadsura and their inhibition of nitric oxide production. Chem. Pharm. Bull.
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4. Conclusions
Two new lignans were isolated from H. reniformis. Aryltetr-
alone, aryltetralol, and aryltetralene lignans were synthesized by a
regioselective route in four, five, and six steps, successively, with
good overall yields (81.0%, 80.9%, and 67.6%). (ꢀ)-Cyclogalgravin
(13a) and (ꢀ)-aristoligol (12a) exhibited activity (IC50 ꢁ10.8 and
Kuo, Y.H., Lin, S.T., 1993. Studies on chromium trioxide-based oxidative coupling
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Holostylis reniformis. Pharm. Microbiol. 50, 643–644.
Martins, G.F., Pereira, M.D.P., Lopes, L.M.X., da Silva, T., Vieira e Rosa, P. de T., Barbosa,
F.P., Messiano, G.B., Krettli, A.U., 2014. Intraspecific variability of Holostylis
reniformis: concentration of lignans, as determined by maceration and
supercritical fluid extraction (SFE-CO2), as a function of plant provenance and
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8.4 mM, respectively), the latter exhibited lower toxicity.
Acknowledgments
Messiano, G.B., da Silva, T., Nascimento, I.R., Lopes, L.M.X., 2008. Biosynthesis of
antimalarial aryltetralone lignans fro Holostylis reniformis. Planta Med. 74,
924–924.
The authors thank Vinicius C. Souza and Lindolpho Capellari Jr.
for plant identification, and the Fundação de Amparo à Pesquisa do
Estado de São Paulo (FAPESP), Coordenação de Aperfeiçoamento de
Pessoal de Nível Superior (CAPES), and Conselho Nacional de
Desenvolvimento Científico e Tecnológico (CNPq/MCT/MS/PRO-
NEX, Brazil) for financial support and fellowships.
Messiano, G.B., da Silva, T., Nascimento, I.R., Lopes, L.M.X., 2009. Biosynthesis of
antimalarial lignans from Holostylis reniformis. Phytochemistry 70, 590–596.
Messiano, G.B., Wijeratne, E.M.K., Lopes, L.M.X., Gunatilaka, A.A.L., 2010. Microbial
transformations of aryltetralone and aryltetralin lignans by Cunninghamella
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Appendix A. Supplementary data
Supplementary data associated with this article can be
found, in the online version, at http://dx.doi.org/10.1016/j.
phytol.2015.06.001.
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