Hydnocarpin-Type Flavonolignans: Semisynthesis and Inhibitory Effects on Staphylococcus aureus Biofilm Formation

Article abstract of DOI:10.1021/acs.jnatprod.5b00430

A new, efficient, and general semisynthesis of hydnocarpin-type flavonolignans was developed and optimized, enabling gram-scale production of hydnocarpin D (2). Moreover, the syntheses of optically pure hydnocarpin isomers [(10R,11R)-hydnocarpin (1a), (10R,11R)-hydnocarpin D (2a), and (10S,11S)-hydnocarpin D (2b)], as well as the synthesis of isohydnocarpin (8), were achieved for the first time utilizing this new method. The synthesis is based on the two-step transformation of the readily available flavonolignans from milk thistle (Silybum marianum), accessible by isolation from the commercial extract silymarin. The first step relies on the regioselective formylation of the C-3 hydroxy group of the dihydroflavonol-type precursor using the Vilsmeier-Haack reagent, followed by formic acid elimination by triethylamine in the second step. The synthesized compounds were effective inhibitors of Staphylococcus aureus biofilm formation, with (10S,11S)-hydnocarpin D (2b) being the most potent inhibitor. Furthermore, the effect of glucose on biofilm formation was tested, and glucose decreased the biofilm inhibitory activity of 2b. Moreover, 2b increased the susceptibility of Staph. aureus to enrofloxacin. (Chemical Equation Presented).

Full text of DOI:10.1021/acs.jnatprod.5b00430

Article
pubs.acs.org/jnp
Hydnocarpin-Type Flavonolignans: Semisynthesis and Inhibitory
Effects on Staphylococcus aureus Biofilm Formation
§
†,‡
Vladimir Vimberg,† Marek Kuzma,† Eva Stodulkova,† Petr Novak,†,‡ Lucie Bednarova, Miroslav Sulc,
̌
̊
́
́
́
́
and Radek Gazak*,†
̌
́
†Institute of Microbiology of the Academy of Sciences of the Czech Republic, v.v.i., Víden
Czech Republic
‡Department of Biochemistry, Faculty of Science, Charles University in Prague, Hlavova 8, Prague 2, CZ 128 40, Czech Republic
§Institute of Organic Chemistry and Biochemistry of the AS CR, v.v.i., Flemingovo nam
estí 2, Prague 6, CZ 166 10, Czech Republic
̌ ́
ska 1083, Prague 4, CZ 142 20,
́
̌
S
* Supporting Information
ABSTRACT: A new, efficient, and general semisynthesis
of hydnocarpin-type flavonolignans was developed and
optimized, enabling gram-scale production of hydnocarpin
D (2). Moreover, the syntheses of optically pure hydnocarpin
isomers [(10R,11R)-hydnocarpin (1a), (10R,11R)-hydnocarpin
D (2a), and (10S,11S)-hydnocarpin D (2b)], as well as the
synthesis of isohydnocarpin (8), were achieved for the first
time utilizing this new method. The synthesis is based on the
two-step transformation of the readily available flavonolignans
from milk thistle (Silybum marianum), accessible by isolation
from the commercial extract silymarin. The first step relies on
the regioselective formylation of the C-3 hydroxy group of the
dihydroflavonol-type precursor using the Vilsmeier−Haack reagent, followed by formic acid elimination by triethylamine in the
second step. The synthesized compounds were effective inhibitors of Staphylococcus aureus biofilm formation, with (10S,11S)-
hydnocarpin D (2b) being the most potent inhibitor. Furthermore, the effect of glucose on biofilm formation was tested, and
glucose decreased the biofilm inhibitory activity of 2b. Moreover, 2b increased the susceptibility of Staph. aureus to enrofloxacin.
3-Deoxy-2,3-dehydroflavonolignans, also called hydnocarpin-
type flavonolignans (henceforth referred to as hydnocarpins), are
a relatively small group of natural flavonoids named according to
the first member, hydnocarpin (1), from Hydnocarpus wightiana,1
which is structurally related to the flavonolignan isosilybin
(11) contained in Silybum marianum extract (Figure 1). Cur-
rently, nine representatives of this group of flavonolignans have
been isolated (Figure 1): hydnocarpin (1),1,2 its regioisomer
hydnocarpin D (2; D in the name indicates “D-ring down”),3
sinaiticin (3),4 5′-methoxyhydnocarpin D (4),5 5″-methoxyhydno-
carpin D (5),6 palstatin (6),3 hydnowightin (7),7 isohydno-
carpin (8),8 and neohydnocarpin (9).7 Moreover, several
regioisomers of these natural compounds were obtained by
chemical synthesis, e.g., regioisomers of 3 and 4.9
In contrast to the related flavonolignans from S. marianum,
specifically silybin (10), isosilybin (11), and silychristin (12)
(Figure 1), knowledge of the biological activity of hydnocarpins
is rather limited due to their poor availability. These compounds
are obtained either by complicated isolation from a few tropical
plants (usually in minute amounts) or by inefficient synthesis
yielding mostly regioisomeric mixtures.9
numerous cancer cell lines (ED50 < 10 μM).4,7 In another study,
(−)-hydnocarpin (1b) (Figure 2) was inactive (ED50 > 10 μM)
against the MCF-7 human breast cancer cell line, but it po-
tentiated the cytotoxic effects of bruceantin and bruceine A by
7- and 10-fold, respectively.11 Moreover, 2 was the most potent
inhibitor of Staphylococcus aureus multidrug resistance efflux
protein Nor A in the presence of subinhibitory quantities of the
alkaloid berberine, whereas 4 and 1 exhibited 10 and 30 times
lower activity, respectively.12
Previous studies on the antimicrobial activities of hydnocar-
pins reported moderate or low effects; however, these studies
did not focus on microbial biofilms. Bacterial biofilms and the
emergence of multiple drug resistance have become a major
threat for the current medical treatment of nosocomial infec-
tions. In fact, approximately 65−80% of microbial infections in
the developed countries are associated with biofilms.13 The treat-
ment of microbial biofilms is still a challenge, as even the most
potent antibiotics have little effect on the viability of micro-
organisms forming biofilms, especially during late-stage chronic
infections.14,15 Staphylococcus aureus, one of the most important
human pathogens that causes a wide range of infections and is
Although biological studies of the hydnocarpins are sporadic
(ca. 12 papers published), they show that hydnocarpins are not
only effective antioxidants10 but also promising anticancer com-
pounds. The anticancer efficiency of 1 has been reported in
Received: May 14, 2015
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
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Figure 1. Naturally occurring hydnocarpins (1−9) and related flavonolignans from S. marianum (10−13).
Figure 2. Synthesized enantiomers of hydnocarpin (1) and hydnocarpin D (2) (except 1b).
B
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Figure 3. Preparation of flavones from corresponding 3-hydroxyflavanones.
responsible for thousands of deaths, is regularly found in biofilms
from catheters, implanted devices, and wounds, and its virulence
is frequently associated with biofilm formation.16−19 Com-
pounds that inhibit biofilm formation may facilitate treatment
and prevention of staphylococci-associated infections.20
This approach afforded a single regioisomer (in the form of the
racemic mixture); however, it represents multistep synthesis with
moderate overall yield. Moreover, starting aldehyde 15 is not
commercially available, which further complicates synthesis of
the target compound.
The objective of this work is to develop a new, efficient, and
general method for the semisynthesis of hydnocarpins from
related and readily available flavonolignans present in
S. marianum, specifically 10, 11, and 12. Additionally, the evalua-
tion of the inhibitory effect of the synthesized hydnocarpins on
biofilm formation of Staph. aureus is presented and compared
to the antibiofilm effect of the starting compounds and the
structurally related flavonoid 2,3-dehydrosilybin (13) (Figure 1).
To overcome the drawbacks of the previous syntheses of these
rare natural compounds, we used their “hydrated” analogues, 10,
11, and 12 (Figure 1), which are readily available flavonolignans
from the extract of S. marianum seeds, for transformation into the
corresponding hydnocarpins. To achieve this aim, we exploited
a reaction that we developed during our attempts to introduce
a formyl group into 10 using the Vilsmeier−Haack reaction.
Reacting 10 with (COCl)2 in DMF resulted in a complex mixture
of products that was difficult to separate. However, using 23-O-
acetylsilybin (18)21 in the Vilsmeier−Haack reaction resulted in
a single product, 19 (Scheme 2), in excellent yield (88%). The
use of POCl3 instead of (COCl)2 for generation of the
Vilsmeier−Haack reagent was assessed as well, but the yield of
19 was substantially lower in this case.
The elimination of formic acid from 19 was assessed using
three different bases (Table 1), where Et3N was the most effec-
tive. Because the use of anhydrous conditions did not lead to
simultaneous deacetylation, 23-O-acetylhydnocarpin D (20) was
ultimately treated with K2CO3 in MeOH/H2O (9:1), which
resulted in the formation of 2 in ca. 60% overall yield (Scheme 2,
Figure 3).
RESULTS AND DISCUSSION
■
Chemistry. The first syntheses of hydnocarpin (1) were
accomplished by an oxidative coupling reaction of the flavonoid
luteolin (24) (Figure 3) with coniferyl alcohol catalyzed by either
horseradish peroxidase or Ag2CO3.9 Both coupling reactions
afforded regioisomeric mixtures of 1 and 2 (1:2 ratio of 3:2 and 1:9,
respectively). These reactions were also used for the preparation of
hydnocarpins 3 and 5 (Figure 1) with similar regioselectivities and
yields.12 Aside from the low regioselectivity of both coupling
reactions, drawbacks of this type of synthesis are the relatively low
yield (less than 40% of the mixture of 1 and 2) and the requisite use
of a rather expensive starting material, luteolin (24). A second
approach to the synthesis of flavonolignan 4 but also applicable to
the synthesis of 1 and 2 was based on the aldol condensation of
MOM-protected acetophenone 14 with aldehyde 15 (Scheme 1).9
The same procedure was applied for the semisyntheses of
optically pure isomers of hydnocarpins from corresponding
stereochemically pure flavonolignans from S. marianum, which
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Scheme 1. Reported9 Synthesis of Hydnocarpin 4
Scheme 2. Synthesis of Hydnocarpin D (2)
Table 1. Bases Tested for Elimination of HCO2H from 19
The silymarin dihydroflavonol (3-hydroxyflavanone), (2R,3R)-
taxifolin (23) [(+)-taxifolin], was subjected to formylation and
subsequent elimination to verify the applicability of this pro-
cedure for structurally related flavonoids as well. As a result, the
flavone luteolin (24) was obtained in 29% yield (Figure 3).
Notably, the new procedure did not require separation after each
individual step, and only the final compound had to be purified
by silica gel column chromatography, then typically recrystallized
from MeOH or MeOH/H2O.
yield
of 20 [%]
a
entry base
note
1
2
3
Et3N
74
isolated yield
DIPEA
DBU
20
low conversion (ca. 20%)
n.d.
elimination is accompanied by decomposition
a
Procedure: compound 19, base (10 equiv), CH2Cl2, 48 h; n.d., not
determined.
However, the regioselective acetylation of the 23-OH function
prior to formylation was problematic with isosilybin A (11a) and
especially silychristin (12), as enzymatic transesterification either
proceeded with moderate conversion for 11a or did not proceed at
all for 12. Therefore, an alternative acetylation method had to be
developed for 12 (Ac2O/BF3·OEt2, THF), which resulted in the
formation of acetate 22 in moderate yield only. To overcome the
problem of the selective protection of the primary 23-OH function
in the corresponding flavonolignans, we also applied a simpler
procedure that consisted of nonselective per-acylation of compound
10 and subsequent elimination by the aforementioned method
(Et3N, CH2Cl2). However, these attempts failed to give elimination
products at C-2 and C-3, and only degradation was observed.
S. marianum extract is produced in large quantities for its
application in the pharmaceutical industry, and the separation
were obtained by using recently developed enzymatic separation
methods.21−23 Utilizing this procedure, (10R,11R)-hydnocarpin
(1a) from 11a, (10R,11R)-hydnocarpin D (2a) from 10a, and
(10S,11S)-hydnocarpin D (2b)from10b were prepared (Figure 2).
Because the description of optically pure compounds according
to their specific optical rotations [α]TD is rather impractical for
comparison of their structures and may be rather confusing ([α]DT
of 1a and 2a are opposite, in spite of having the same absolute
configuration), we strictly use the R/S notation for these isomers.
Determination of their absolute configurations was based on ECD,
NMR, and optical rotation measurements as well as comparison of
these data with those of closely related enantiomers of 13.24
In addition to the enantiomers of 1 and 2, isohydnocarpin (8)
was synthesized for the first time (Figure 3), starting from 12.
D
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of 10 and other constituents is well described, affording gram
quantities of each starting compound (from 200 g of the dried
S. marianum extract, ca. 80 g of 10, 3 g of 11a, 30 g of 12, and 2 g
of 23 can be obtained22), including stereochemically pure
isomers of silybin 10a and 10b.21,23 Therefore, the method
described above for the semisyntheses of hydnocarpins and
luteolin represents a feasible way to synthesize sufficient amounts
of these rare compounds for more detailed biological testing and
for the synthesis of their derivatives.
Effect of Flavonolignans on Staph. aureus Biofilm
Formation. The effect of flavonolignans on Staph. aureus 8325-4
growth and biofilm formation in a static microtiter plate
was investigated. Although there was little or no effect of the
compounds on bacterial growth, all flavonolignans inhibited
biofilm formation. The efficiency of inhibition strongly depended
on the structure of the compound (Figure 4A). Isosilybin
configurations at C-9 and C-10 are important for influencing
inhibition of biofilm formation. (10S,11S)-Hydnocarpin D (2b)
was the most active compound from our series of flavonolignans,
while its enantiomer 2a (10R,11R) possessed significantly lower
activity. The racemic mixture of both compounds, hydnocarpin
D (2), exhibited the approximate median activity of the two
enantiomers; that is, it was a better inhibitor than 2a but worse
than 2b. In contrast, the regiochemistry at C-10 and C-11 seems
to be less important, as the activities of (10R,11R)-hydnocarpin
(1a) and (10R,11R)-hydnocarpin D (2a) were practically
identical.
In addition to the effects of the tested compounds on biofilm
inhibition, their ability to disrupt preformed biofilms was also
evaluated. However, we did not observe any significant effects of
the tested compounds in this case.
Glucose Affects the (10S,11S)-Hydnocarpin D Inhibi-
tion of Staph. aureus Biofilm Formation. The effects of the
most potent inhibitor from the series, (10S,11S)-hydnocarpin
D (2b), on biofilm formation in the presence of 1% glucose,
which is frequently added to growth media as a biofilm formation
enhancer, were assessed. Glucose positively regulates the two
main mechanisms of biofilm formation in Staph. aureus:
icaADBC-dependent and icaADBC-independent. Although the
icaADBC operon, required for the synthesis of polymeric
N-acetylglucosamine, is considered the main factor for biofilm
formation,25 glucose predominantly influences the icaADBC-
independent mechanism of biofilm formation.26,27 When glucose
was added to the growth medium, biofilm formation was inhib-
ited by 80% at a 8 mM concentration of (10S,11S)-hydnocarpin
D (2b), while complete inhibition of biofilm formation occurred
only at 128 mM of 2b (Figure 4B). Two-level inhibition
may suggest that 2b efficiently inhibits the icaADBC-dependent
biofilm formation pathway but has a moderate effect on the
icaADBC-independent mechanism of biofilm formation, which is
induced after the addition of the glucose.
Staph. aureus Susceptibility to Enrofloxacin Is In-
creased by (10S,11S)-Hydnocarpin D. The antibiofilm
activity of the flavonolignans may be associated with inhibition
of the cell transport system, since hydnocarpin D (2) has been
shown to inhibit NorA transporter,12 responsible for Staph.
aureus quinolone resistance and important for bacterial intra-
cellular invasion.28 Indeed, addition of 2b increased susceptibility
to enrofloxacin of the Staph. aureus 8325−4 strain, as well as two
other tested Staph. aureus strains: ATCC29213, a control strain
for MIC measurements, and N315, a methicillin-resistant
strain (Figure 4C). Although this result suggests that the NorA
transporter may be affected by (10S,11S)-hydnocarpin D (2b),
further investigation is required to prove inhibition of the NorA
transporter.
In conclusion, the chemoselective O-formylation of the 3-OH
group of the flavonolignans from S. marianum, i.e., isosilybin
A (11a), silybin (10), and silychristin (12), by the Vilsmeier−
Haack reaction followed by the elimination of formic acid using
Et3N represents a straightforward way to prepare three members
of the hydnocarpin-type flavonolignans, compounds 1a, 2, and 8.
This convenient procedure enables biological studies of these
rare and neglected natural compounds, including their optically
pure isomers, which are not accessible by isolation or by using
reported synthetic methods.
An SAR study of the series of flavonolignans resulted in deter-
mination of the basic structural features important for effective
inhibition of Staph. aureus biofilm formation: the presence of a
2,3-double bond together with a dihydrobenzodioxine moiety
Figure 4. Inhibitory effect of flavonolignans on biofilm formation by
the Staph. aureus strain 8325-4. (A) EC50 and minimal antibiofilm
concentration of flavonoids required for antibiofilm activity in the
absence of glucose. (B) Effect of glucose on inhibition of biofilm
formation by 2b. The graphic demonstrates the mean values obtained
from three independent experiments with their statistical deviations.
(C) Minimal inhibitory concentration of enrofloxacin in the presence of
2b for Staph. aureus strains ATCC29213, 8325-4, and N315.
A (11a), isohydnocarpin (8), and silybin (10) were the least
efficient inhibitors. (10R,11R)-Hydnocarpin (1a), 2,3-dehydro-
silybin (13), and (10R,11R)-hydnocarpin D (2a) showed
moderate inhibition of biofilm formation. The most effective
compound was (10S,11S)-hydnocarpin D (2b), followed by
racemic hydnocarpin D (2). On the basis of the effects of the
tested compounds on the inhibition of Staph. aureus biofilm
formation, it is possible to propose important structural motifs
that should be present in the structure to achieve significant
inhibitory activity. The most important feature is the presence of
a 2,3-double bond (cf. compounds 1a and 2 vs their saturated
analogues 11a and 10, respectively, or compounds 13 and 10).
However, the presence of a dihydrobenzofuran instead of
a dihydrobenzodioxine moiety (cf. 8 vs 1a, 2, and 13) leads to
an almost complete loss of inhibitory activity despite the pres-
ence of the 2,3-double bond. 2,3-Dehydrosilybin (13) is the
3-OH analogue of hydnocarpin D (2) and demonstrates that the
presence of a 3-OH group of flavonolignans decreases the biofilm
inhibitory activity of the compound. Comparing the activities of
racemic 2 and its enantiomers 2a and 2b demonstrated that the
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Table 2. NMR Spectroscopic Data (700.13 MHz, DMSO-d6) for Compounds 19 and 21
19
21
position
δC, type
δH (J in Hz)
5.551, d (11.8)
δC, type
δH (J in Hz)
5.102, d (11.2)
2
3
79.64, CH; 79.58, CH
71.69, CH; 71.64, CH
82.52, CH
71.52, CH
6.106, dd (0.8, 11.8);
6.094, dd (0.8, 11.8)
4.608, dd (6.2, 11.2)
4
190.77, C; 190.76, C
100.57, C
197.72, C
100.51, C
163.35, C
96.13, CH
166.89, C
95.10, CH
4a
5
163.24, C
6
96.57, CH
5.974, d (2.1)
5.923, d (2.1)
5.884, d (2.1)
7
167.46, C
8
95.50, CH
5.935, d (2.1);
5.933, d (2.1)
8a
10
162.37, C; 162.35, C
75.07, CH; 75.06, CH
162.49, C
75.92, CH
4.555, ddd (2.7, 5.1, 9.3);
4.535, ddd (2.7, 5.2, 9.3)
4.951, d (8.0); 4.941, d (8.0)
4.940, d (8.0)
11
75.86, CH; 75.84, CH
143.54, C; 143.50, C
116.82, CH
74.97, CH
142.39, C
4.498, ddd (2.6, 5.2, 8.0)
7.142, d (2.0)
12a
13
7.203, d (2.0); 7.193, d (2.0)
116.55, CH
130.67, C
14
128.55, C
15
121.61, CH;
7.079, dd (2.0, 8.6);
7.074, dd (2.1, 8.6)
7.027, d (8.3)
121.38, CH
7.018, dd (2.0, 8.3)
6.964, d (8.3)
121.52, CH
16
116.82, CH
116.62, CH
143.90, C
16a
17
143.70, C; 143.69, C
126.51, C; 126.50, C
111.77, CH
126.60, C
18
7.030, d (1.8);
7.025, d (1.6)
111.79, CH
7.019, d (2.0)
19
20
21
147.80, C
147.77, C
147.35, C
115.52, CH
147.37, C; 147.36, C
115.49, CH
6.810, d(8.0);
6.816, d (8.0)
6.809, d (8.1)
22
23
120.68, CH;
120.64, CH
62.58, CH2
6.870, dd (2.0, 8.4);
6.868, dd (2.0, 8.4)
4.087, dd (2.6, 12.4)
3.929, dd (5.1, 12.4);
3.925, dd (5.0, 12.4)
3.777, s; 3.773, s
120.59, CH
62.64, CH2
6.867, dd (2.0, 8.0)
4.085, dd (2.6, 12.4)
3.932, dd (5.2, 12.4)
19-OMe
55.74, CH3;
55.72, CH3
160.71, CH;
160.70, CH
170.05, C
55.74, CH3
3.777, s
3-OC(O)H
8.325, d (0.8);
8.321, d (0.8)
23-CO
23-OAc
3-OH
170.09, C
20.456, CH3
2.026, s; 2.025, s
20.48, CH3
2.030, s
5.796, d (6.3)
11.884, s
10.827, s
9.185, s
5-OH
11.353, s
11.005, s
9.179, s
7-OH
20-OH
rotations were measured in MeOH (UV/vis purity) using an Autopol IV
polarimeter (Rudolph Research Analytical, NJ, USA). The ECD spectra
were recorded on a Jasco-815 spectrometer (Japan) in MeOH.
NMR spectra were recorded on a Bruker Avance III 400 MHz
spectrometer (400.00 MHz for 1H and 100.58 MHz for 13C), a Bruker
and the absence of a 3-OH group. (10S,11S)-Hydnocarpin D
(2b) was the most effective inhibitor of Staph. aureus biofilm
formation from this series. This compound was also an effective
chemosensitizer of Staph. aureus to quinolones. Additionally,
testing the biofilm inhibitory activity of 2b in the presence and/or
absence of glucose suggested that2b most likely effectively inhibits
the icaADBC-dependent biofilm formation pathway but has only a
moderate effect on the icaADBC-independent mechanism.
This is the first report regarding the effect of flavonolignans on
biofilm formation. Moreover, the preparation and biological
studies of both enantiomers of 2 are quite unique.
1
Avance III 600 MHz spectrometer (600.23 MHz for H and 150.93
MHz for 13C), and a Bruker Avance III 700 MHz spectrometer (700.13
MHz for 1H and 176.05 MHz for 13C) in DMSO-d6 (99.8 atom % D,
VWR Chemicals, Leuven, Belgium) at 30 °C. Residual solvent signals
were used as an internal standard (δH 2.500, δC 39.60). The 1H and 13
C
NMR, J-resolved, COSY, HSQC, HSQC-TOCSY, HMBC, band-
selective HMBC, and 1D TOCSY experiments were performed using
the manufacturer’s software. 1H and 13C NMR spectra were zero filled to
4-fold data points and multiplied by a window function before Fourier
transformation. The two-parameter double-exponential Lorentz−Gauss
function was applied for 1H to improve resolution, and line broadening
(1 Hz) was applied to get better 13C signal-to-noise ratio. Chemical shift
EXPERIMENTAL SECTION
■
General Experimental Procedures. Melting points (mp) were
recorded on a Kofler hot-stage microscope and are uncorrected. Optical
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Table 3. 1H NMR Spectroscopic Data (700.13 MHz,
DMSO-d6) for Hydnocarpins 1a, 2a, 2b, 2, and 20
values are presented in the δ-scale with digital resolution justifying the
reported values to three (δH) or two (δC) decimal places.
Mass spectra were measured on a MALDI-TOF/TOF ultraFLEX III
mass spectrometer (Bruker-Daltonics, Bremen, Germany). Positive mass
spectra were externally calibrated using the monoisotopic [M + H]+/
[M − H]− ions of the PepMixII calibrant (Bruker-Daltonics, Bremen).
HR-MS were measured on a commercial 12T solariX FTMS
instrument (Bruker Daltonics, Billerica MA, USA) equipped with an
ESI/MALDI ion source and ParaCell. The analyses were performed
using electrospray ionization (ESI), and the spectra were acquired in
positive ion mode. The instrument was externally calibrated using singly
charged NaTFA clusters, resulting in sub-ppm accuracy. The spectra were
apodized using sine apodization with one zero filling. Data were processed
with the software Data Analysis 4.2, and the possible elemental
compositions were calculated using Smart Formula calculations.
The HPLC system consisted of a pump equipped with a 600E system
controller, a 717 autosampler, and 2487 dual UV detector, and the data
were processed using Empower 2 software (Waters, Milford, MA, USA).
Water-containing mobile phases were filtered through a 0.22 μM GS
filter (Millipore, Billerica, MA, USA) and degassed in an ultrasonic bath
for 10 min before use. A Gemini 5 μm C18 column (250 × 4.6 mm;
Phenomenex, CA, USA) with a guard column was used for analysis.
The mobile phase consisted of 5% MeOH in H2O with TFA (1%) (A) and
MeOH with 1% TFA (B). Gradient elution started at 30% B (0 min),
increasing linearly to 100% B over 20 min at a flow rate of 1.0 mL/min.
UV detection was performed at 340 nm. The purity of the tested com-
pounds that was determined under HPLC conditions and instrumenta-
tion mentioned above exceeded 95% in all cases.
1a
2a
2b
2
20
position δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz) δH (J in Hz)
3
6.862, s
6.868, s
6.853, s
6.844, s
6.857, s
6
6.204, d (2.0) 6.193, d (2.1) 6.193, d (2.1) 6.190, d (1.9) 6.192, d (2.1)
6.517, d (2.0) 6.505, d (2.1) 6.500, d (2.1) 6.495, d (1.9) 6.497, d (2.1)
8
10
5.020, d (7.8) 4.972, d (8.0) 4.972, d (8.0) 4.300, ddd
4.632, ddd
(2.8, 4.5, 8.0) (2.6, 5.1, 8.0)
4.967, d (8.0) 5.000, d (8.0)
11
4.265, ddd
(2.5, 4.6, 7.8) (2.5, 4.5, 8.0) (2.6, 4.6, 8.0)
7.656, d (2.1) 7.666, d (2.2) 7.655, d (2.3) 7.647, d (2.0) 7.680, d (2.2)
4.312, ddd
4.306, ddd
13
15
7.592, dd
(2.1, 8.5)
7.629, dd
(2.2, 8.6)
7.620, dd
(2.3, 8.6)
7.613, dd
(2.0, 8.5)
7.629, dd
(2.2, 8.6)
16
18
21
22
7.078, d (8.5) 7.120, d (8.6) 7.114, d (8.6) 7.108, d (8.5) 7.139, d (8.6)
7.039, d (1.7) 7.048, d (2.0) 7.048, d (2.0) 7.047, d (1.3) 7.057, d (2.0)
6.815, d (8.1) 6.819, d (8.0) 6.821, d (8.0) 6.822, d (8.0) 6.829, d (8.0)
6.883, dd
(1.7, 8.1)
3.588, ddd
6.889, dd
(2.0, 8.0)
3.575, dm
(12.2)
6.891, dd
(2.0, 8.0)
3.579, ddd
6.891, dd
(1.3, 8.0)
3.579, ddd
6.891, dd
(2.0, 8.0)
4.114, dd
(2.6, 12.5)
23
(2.5, 4.9,
12.3)
(2.6, 4.8,
12.4)
(2.8, 3.5,
12.2)
3.384, ddd
3.367, dm
(12.2)
3.373, ddd
3.373, ddd
3.967, dd
(4.6, 5.6,
12.3)
(4.6, 6.1,
12.4)
(4.5, 5.7,
12.2)
(5.1, 12.5)
5-OH 12.903, s
7-OH 10.835, s
12.902, s
10.890, s
3.787, s
12.895, s
10.827, s
3.790, s
12.893, s
10.828, s
3.791, s
12.878, s
10.829, s
3.787, s
The reactions were monitored by TLC on F254 silica gel (Merck,
Germany), and the spots were visualized with UV light and by charring
with an ethanolic solution of H2SO4 (5% v/v).
19-
OMe
3.786, s
Materials. Silymarin (denoted as “milk thistle extract 60% silymarin
(HPLC)”) was purchased from Naturalin International Co. Ltd.,
China. Silybin (10), isosilybin A (11a), silychristin (12), and taxifolin
(23) were isolated by the previously reported method.22 Silybin stereo-
isomers 10a and 10b were separated by lipase-catalyzed discrimi-
nation.21,23 23-O-Acetylsilybin (18), 23-O-acetylsilybin A (18a), and
23-O-acetylsilybin B (18b) were prepared by enzymatic transacetylation
as described previously.21,23 All other reagents and chemicals were of
analytical grade from Sigma-Aldrich and were used without further
purification.
20-OH 9.148, s
23-OH 4.959, dd
(4.9, 5.6)
9.176, s
9.151, s
9.154, s
9.215, s
5.000, br s
4.982, dd
(4.8, 6.1)
4.984, dd
(3.8, 5.2)
23-Ac
2.037, s
(1H, dd, J = 6.2, 11.3 Hz, H-3), 5.031 (1H, d, J = 11.3 Hz, H-2), 5.441
(1H, d, J = 7.7 Hz, H-10), 5.760 (1H, d, J = 6.2 Hz, 3-OH), 5.870 (1H, d,
J = 2.1 Hz, H-8), 5.915 (1H, d, J = 2.1 Hz, H-6), 6.781 (1H, d, J = 8.0 Hz,
H-20), 6.823 (1H, dd, J = 2.0, 8.0 Hz, H-21), 6.867 (1H, d, J = 1.7 Hz,
H-14), 6.894 (1H, dd, J = 0.5, 1.7 Hz, H-12), 6.985 (1H, d, J = 2.0 Hz,
H-17), 9.039 (1H, s, 19-OH), 9.406 (1H, s, 15-OH), 10.799 (1H, s, 7-OH),
11.888 (1H, s, 12-OH); 13C NMR (DMSO-d6, 176.05 MHz) δ 20.62
(CH3, 22-COCH3), 49.56 (CH, C-11), 55.72 (CH3, 18-OMe), 65.11
(CH2, C-22), 71.74 (CH, C-3), 83.14 (C, C-2), 87.49 (CH, C-10),
95.03 (CH, C-8), 96.07 (CH, C-6), 100.51 (C, C-4a), 110.62 (CH,
C-17), 115.10 (CH, C-12), 115.37 (CH, C-20), 116.25 (CH, C-14),
119.07 (CH, C-21), 127.76 (C, C-11a), 130.42 (C, C-13), 131.45 (C,
C-16), 140.94 (C, C-15), 146.69 (C, C-19), 147.07 (C, C-15a), 147.64
(C, C-18), 162.55 (C, C-8a), 163.34 (C, C-5), 166.82 (C, C-7), 170.38
(C, 22-CO), 197.76 (C, C-4); ESIMS m/z 547 [M + Na]+.
Staph. aureus 8325-4 was kindly provided by Dorte Free. Staph. aureus
N315 was a gift from Dr. Malcolm Horsburgh.
23-O-Acetylisosilybin A (21). Isosilybin A (11a) (1 g, 2.1 mmol) was
dissolved in a mixture of acetone/vinyl acetate (150 mL, 9:1, v/v),
Novozym 435 (1 g, ≥10 000 U/g, 100% w/w) was added, and the
mixture was shaken at 45 °C and 650 rpm for 48 h. Immobilized enzyme
was removed by filtration and washed with acetone, and the combined
filtrates were evaporated. The crude evaporated residue was purified by
column chromatography (CHCl3/acetone/HCO2H, 90:10:1), yielding
21 (0.76 g, 70% yield). For 1H and 13C NMR data, see Table 2; ESIMS
m/z 547 [M + Na]+.
23-O-Acetylsilychristin (22). To a cooled solution (0 °C) of
silychristin (12; 1.5 g, 3.1 mmol) in dry THF (75 mL) was added
Ac2O (0.5 mL, 5.6 mmol) and then BF3·OEt2 (1.5 mL, 6.1 mmol, 50%
solution in Et2O), and the reaction mixture was stirred at 0 °C for 1 h.
The reaction mixture was diluted with an ice-cold saturated solution of
NaHCO3 (100 mL), extracted with EtOAc (2 × 75 mL), dried over
anhydrous Na2SO4, and evaporated to give a viscous oil. The oil was
dissolved in acetone (10 mL), and n-hexane (100 mL) was added.
The solution was briefly shaken and carefully decanted. The remaining
oily solid was washed once again with n-hexane (100 mL), the solvent
was decanted, and the remaining precipitate was dissolved in acetone
and evaporated under reduced pressure to afford a solid residue. This
residue provided, after chromatography (first CHCl3/acetone/HCO2H,
95:5:1−90:10:1; second EtOAc/hexanes with 1% HCO2H 1:2−1:1),
title compound 22 (0.798 g, 49%) as a white, amorphous solid: 1H NMR
(DMSO-d6, 700.13 MHz) δ 1.975 (3H, s, 22-Ac), 3.764 (1H, dddd,
J = 0.5, 5.8, 7.4, 7.7 Hz, H-11), 3.769 (3H, s, 18-OMe), 4.250 (1H, dd,
J = 7.4, 11.0 Hz, H-22u), 4.375 (1H, dd, J = 5.8, 11.0 Hz, H-22d), 4.529
General Procedure A. Regioselective Formylation. Dry DMF
(20 mL) was cooled to 0 °C by ice, and then (COCl)2 (4 mL, 8 mmol,
2 M solution in CH2Cl2) was added dropwise (caution: gas evolution
occurs violently−the release of gas has to be ensured), to form a white
precipitate of the Vilsmeier−Haack reagent. The resulting mixture was
stirred at 0 °C for 10 min; then a solution of corresponding
3-hydroxyflavonoid (3.8 mmol) in dry DMF (15 mL) was added
dropwise, and the mixture was briefly stirred at 0 °C. The reaction
mixture was warmed to 50 °C and maintained at this temperature for 2 h
(the color of the solution typically changed from slightly yellow to
purple-red within ca. 30 min of heating). The reaction mixture was
cooled to room temperature, diluted in ice-cold H2O, and extracted with
EtOAc (2 × 75 mL). The combined organic phases were washed with a
saturated NaCl solution and H2O, dried over anhydrous Na2SO4, and
evaporated to dryness to afford the crude 3-O-formyl derivative, which
was used in the next synthetic step without further purification.
General Procedure B. Elimination of Formic Acid. To a solution
of the corresponding 3-formate obtained following general procedure
G
DOI: 10.1021/acs.jnatprod.5b00430
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Table 4. 13C NMR Spectroscopic Data (176.05 MHz, DMSO-d6) for Hydnocarpins 1a, 2a, 2b, 2, and 20
1a
2a
2b
2
20
position
δC
type
δC
type
δC
type
δC
type
δC
type
2
162.98
103.99
181.82
103.87
161.49
98.97
C
162.99
103.93
181.84
103.83
161.48
98.98
C
162.98
103.92
181.81
103.84
161.47
98.95
C
162.97
103.92
181.81
103.85
161.48
98.95
C
162.83
104.10
181.84
103.88
161.50
98.99
C
3
CH
C
CH
C
CH
C
CH
C
CH
C
4
4a
C
C
C
C
C
5
C
C
C
C
C
6
CH
C
CH
C
CH
C
CH
C
CH
C
7
164.31
94.14
164.37
94.15
164.28
94.11
164.29
94.12
164.35
94.15
8
CH
C
CH
C
CH
C
CH
C
CH
C
8a
157.40
76.43
157.41
75.97
157.38
75.95
157.38
78.63
157.41
75.51
10
CH
CH
C
CH
CH
C
CH
CH
C
CH
CH
C
CH
CH
C
11
78.08
78.62
78.62
75.96
75.96
12a
13
143.71
114.87
123.76
119.95
117.56
147.15
127.03
111.90
147.72
147.24
115.43
120.66
60.14
144.05
115.12
123.51
120.20
117.43
146.94
127.09
111.85
147.72
147.22
115.40
120.74
60.09
144.03
115.09
123.49
120.17
117.40
146.92
127.09
111.88
147.71
147.21
115.40
120.72
60.08
144.03
115.08
123.49
120.17
117.40
146.92
127.10
111.89
147.72
147.22
115.41
120.72
60.09
144.02
115.26
123.96
120.33
117.54
146.24
126.26
111.85
147.87
147.51
115.55
120.79
62.51
CH
C
CH
C
CH
C
CH
C
CH
C
14
15
CH
CH
C
CH
CH
C
CH
CH
C
CH
CH
C
CH
CH
C
16
16a
17
C
C
C
C
C
18
CH
C
CH
C
CH
C
CH
C
CH
C
19
20
C
C
C
C
C
21
CH
CH
CH2
CH3
CH
CH
CH2
CH3
CH
CH
CH2
CH3
CH
CH
CH2
CH3
CH
CH
CH2
CH3
C
22
23
19-OMe
23-CO
23-Ac
55.81
55.78
55.79
55.80
55.78
170.10
20.48
CH3
A in CH2Cl2 (70 mL) was added Et3N (5 mL), and the solution was stirred
at room temperature for 2 days. The solvents were evaporated to dryness by
coevaporation with toluene, affording crude 3-deoxy-2,3-dehydroflavonoid,
which was subjected to basic hydrolysis without purification.
23-O-Acetyl-3-O-formylsilybin (19). The title compound was
prepared according to general procedure A, starting from 18 (2 g,
3.8 mmol). Column chromatography (hexanes/EtOAc, 2:1−1:1, v/v)
of the crude reaction mixture afforded pure 19 (1.849 g, 88%) as a white,
amorphous solid. For 1H and 13C NMR data, see Table 2; ESIMS m/z
575 [M + Na]+.
23-O-Acetylhydnocarpin D (20). The title compound was prepared
according to general procedure B, starting from 19 (1.82 g, 3.3 mmol).
Column chromatography (CHCl3/acetone/HCO2H, 90:10:1) of
the crude reaction mixture afforded 20 (1.23 g, 74%) as a yellowish,
amorphous solid. For 1H and 13C NMR data, see Tables 3 and 4; ESIMS
m/z 507 [M + H]+.
Hydnocarpin D (2). Compound 20 (1.2 g, 2.4 mmol) was dissolved
in a mixture of MeOH/H2O (22 mL, 10:1 v/v), K2CO3 (1.2 g) was
added, and the mixture was stirred at room temperature for 4 h. The
solution was diluted with ice-cold H2O and acidified with HCl, and
the precipitate was filtered off, washed with H2O, and dried. The crude
product was purified by silica gel column chromatography (CHCl3/
acetone/HCO2H, 80:20:1), affording nearly pure title compound. This
compound was recrystallized from MeOH/H2O (9:1 v/v) to yield pure
2 (1.064 g, 97%) as a slightly yellowish, microcrystalline solid: mp 186−
188 °C. For 1H and 13C NMR data, see Tables 3 and 4; HRESIMS m/z
465.1187 [M + H]+ (calcd for C25H21O9, 465.1180).
(10S,11S)-Hydnocarpin D (2b). Compound 2b (0.514 g, 58%) was
prepared from 18b (1 g, 1.9 mmol) by the procedure described for 2.
The final recrystallization of 2b was performed in MeOH/H2O (9:1): mp
182−184 °C; [α]2D5 +46 (c 0.2, MeOH); ECD spectrum, see Supporting
Information (Figure S22); for 1H and 13C NMR data, see Tables 3 and 4;
HRESIMS m/z 465.1188 [M + H]+ (calcd for C25H21O9, 465.1180).
(10R,11R)-Hydnocarpin (1a). The solid residue obtained from
general procedures A and B, from acetate 21 (0.76 g, 1.449 mmol), was
dissolved in a mixture of MeOH/H2O (11 mL, 10:1 v/v), K2CO3
(0.76 g) was added, and the mixture was stirred at room temperature
for 4 h. The solution was diluted with ice-cold H2O and acidified with
HCl, and the precipitate was filtered off, washed with H2O, and dried.
The crude product was purified by silica gel column chromatography
(CHCl3/acetone/HCO2H, 80:20:1), affording nearly pure title com-
pound, which was recrystallized from MeOH/H2O (9:1 v/v) to provide
pure 1a (0.39 g, 58%) as a slightly yellowish, microcrystalline solid: mp
256−258 °C; [α]2D5 +39 (c 0.1, MeOH); ECD spectrum, see Supporting
Information (Figure S21); for 1H and 13C NMR data, see Tables 3 and 4;
HRESIMS m/z 465.1186 [M + H]+ (calcd for C25H21O9, 465.1180).
Isohydnocarpin (8). Compound 8 was prepared analogously to
compound 2, starting from acetate 22 (1.7 g, 3.2 mmol). Recrystalliza-
tion (MeOH) yielded pure title compound (0.63 g, 42%) as a yellowish,
1
microcrystalline solid: mp 237−239 °C; H NMR (DMSO-d6, 700.13
MHz) δ 3.552 (1H, ddd, J = 5.0, 6.4, 6.7 Hz, H-11), 3.718 (1H, dd, J =
6.4, 10.5 Hz, H-22u), 3.757 (3H, s, 18-OMe), 3.786 (1H, dd, J = 5.0,
10.5 Hz, H-22d), 5.066 (1H, br s, 22-OH), 5.597 (1H, d, J = 6.7 Hz,
H-10), 6.194 (1H, d, J = 2.0 Hz, H-6), 6.447 (1H, d, J = 2.0 Hz, H-8),
6.703 (1H, s, H-3), 6.775 (1H, d, J = 8.1 Hz, H-20), 6.813 (1H, dd, J =
2.0, 8.1 Hz, H-21), 6.965 (1H, d, J = 2.0 Hz, H-17), 7.362 (1H, d, J = 1.8 Hz,
H-14), 7.510 (1H, m, H-12), 9.045 (1H, s, 19-OH), 9.728 (1H, s, 15-OH),
10.837 (1H, s, 7-OH), 12.952 (1H, s, 12-OH); 13C NMR (DMSO-d6,
176.05 MHz) δ 52.75 (CH, C-11), 55.75 (CH3, 18-OMe), 62.66 (CH2,
C-22), 88.02 (CH, C-10), 93.92 (CH, C-8), 98.93 (CH, C-6), 103.31
(CH, C-3), 103.76 (C, C-4a), 110.60 (CH, C-17), 114.26 (CH, C-14),
(10R,11R)-Hydnocarpin D (2a). Compound 2a (0.531 g, 60%) was
prepared analogously to 2, starting from 18a (1 g, 1.907 mmol).
The final recrystallization of 2a was performed in MeOH/H2O (9:1): mp
190−192 °C; [α]2D5 −48 (c 0.2, MeOH); ECD spectrum, see Supporting
1
Information (Figures S21 and S22); for H and 13C NMR data, see
Tables 3 and 4; HRESIMS m/z 465.1188 [M + H]+ (calcd for C25H21O9,
465.1180).
H
DOI: 10.1021/acs.jnatprod.5b00430
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
114.71 (CH, C-12), 115.45 (CH, C-20), 118.88 (CH, C-21), 123.50 (C,
C-13), 130.67 (C, C-11a), 131.91 (C, C-16), 141.56 (C, C-15), 146.67
(C, C-19), 147.67 (C, C-18), 150.63 (C, C-15a), 157.38 (C, C-8a),
161.54 (C, C-5), 163.93 (C, C-2), 164.31 (C, C-7), 181.69 (C, C-4);
HRESIMS m/z 465.1183 [M + H]+ (calcd for C25H21O9, 465.1183).
Luteolin (24). The solid residue obtained according to general pro-
cedures A and B, starting from compound 23 (0.2 g, 0.7 mmol), was
purified by silica gel column chromatography (CHCl3/acetone/
HCO2H, 80:20:1), affording nearly pure title compound, which was
recrystallized from MeOH/H2O (9:1) to give pure 24 (55 mg, 29%) as a
yellow, microcrystalline solid: mp 326−328 °C (reported29 mp 326 °C);
1H NMR (DMSO-d6, 700.13 MHz) δ 6.187 (1H, d, J = 2.1 Hz, H-8),
6.441 (1H, d, J = 2.1 Hz, H-6), 6.664 (1H, s, H-3), 6.887 (1H, d, J =
8.3 Hz, H-13), 7.392 (1H, d, J = 2.3 Hz, H-10), 7.412 (1H, dd, J = 2.3,
8.3 Hz, H-14), 9.902, 9.391 (2H, br s, 11-OH, 12-OH), 10.814 (1H, br s,
7-OH), 12.969 (1H, s, 5-OH); 13C NMR (DMSO-d6, 176.05 MHz) δ
93.90 (CH, C-8), 98.89 (CH, C-6), 102.94 (CH, C-3), 103.77 (C,
C-4a), 113.44 (CH, C-10), 116.08 (CH, C-13), 119.06 (CH, C-14),
121.58 (C, C-9), 145.80 (C, C-11), 149.76 (C, C-12), 157.36 (C, C-8a),
161.55 (C, C-5), 163.96 (C, C-2), 164.19 (C, C-7), 181.72 (C, C-4);
HRESIMS m/z 287.0553 [M + H]+ (calcd for C15H11O6, 287.0550).
Preparation of Monosodium Salts of Compounds. To improve
water solubility of the tested compounds, their monosodium salts were
prepared. The corresponding flavonolignan (1 equiv) was dissolved in a 1 M
methanolic solution of NaOH (1 equiv, MeOH/H2O, 95:5 v/v), evaporated
to dryness, redissolved in MeOH, and evaporated to dryness again by
coevaporation with toluene, affording the monosodium salt of the compound.
Biofilm Assay. Biofilm formation was determined under static
conditions by a microtiter plate assay;30 however, instead of a 24-well
plate, a 96-well plate was used. Staph. aureus 8325-4, pregrown overnight
on a brain heart infusion (BHI) agar plate, was resuspended in 0.9%
NaCl to McF = 0.5, and 5 μL of resuspended cells was inoculated into
each well of the 96-well plate containing 100 μL of tryptic soy (TS)
medium supplemented with the tested compounds (their monosodium
salts) at concentrations of 0−124 mM. In the experiment with glucose, 1%
glucose was added to the TS medium. The plate was incubated for
24 h at 37 °C, and the wells were then gently rinsed three times with water
to remove nonadherent cells. The cells that remained attached to the wells
were stained with 50 μL of 1% crystal violet for 15 min, then rinsed gently
with water five times and air-dried. Crystal violet was recovered from the
stained cells by adding 100 μL of a 96% ethanol solution. The optical
density of the plate was measured at A630 nm using a BioTek Synergy HT
spectrophotometer. Every experiment was performed at least in triplicate.
Minimum Inhibitory Concentration (MIC). The MIC values were
determined by the standard microbroth dilution method31 in Mueller-
Hinton broth. Experiments were performed with two biological
replicates, and each biological replicate was performed in triplicate.
from the Ministry of Education, Youth and Sports of the Czech
Republic and by BIOCEV - Biotechnology and Biomedicine
Centre of the Academy of Sciences, and Charles University No.
CZ.1.05/1.1.00/02.0109 from the European Regional Develop-
ment Fund in the Czech Republic.
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* Supporting Information
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2a, and 2b (PDF)
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AUTHOR INFORMATION
Corresponding Author
3480−3490.
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*Phone: (+420) 296 442 350. Fax: (+420) 296 442 374. E-mail:
gazak@biomed.cas.cz.
Notes
The authors declare no competing financial interest.
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(31) ISO 20776-1 (2006).
ACKNOWLEDGMENTS
This work was supported by project UNCE204025/2012 from the
Charles University in Prague, project CZ.1.07/2.3.00/30.0003
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DOI: 10.1021/acs.jnatprod.5b00430
J. Nat. Prod. XXXX, XXX, XXX−XXX