Enhanced bioactivity of silybin B methylation products

Article abstract of DOI:10.1016/j.bmc.2012.11.035

Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as partially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers). One challenge with the use of these compounds in vivo is their relatively short half-life due to conjugation, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties, particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monomethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated (5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line (Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) compared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans can increase bioactivity.

Full text of DOI:10.1016/j.bmc.2012.11.035

Bioorganic & Medicinal Chemistry 21 (2013) 742–747
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Enhanced bioactivity of silybin B methylation products
Arlene A. Sy-Cordero a, Tyler N. Graf a, Scott P. Runyon b, Mansukh C. Wani c, David J. Kroll d,
Rajesh Agarwal e, Scott J. Brantley f, Mary F. Paine f, Stephen J. Polyak g,h, Nicholas H. Oberlies a,
⇑
a Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
b Center for Organic and Medicinal Chemistry, Research Triangle Institute, Research Triangle Park, NC 27709, USA
c Natural Products Laboratory, Research Triangle Institute, Research Triangle Park, NC 27709, USA
d Department of Pharmaceutical Sciences, BRITE, North Carolina Central University, Durham, NC 27707, USA
e Department of Pharmaceutical Sciences, School of Pharmacy, University of Colorado Denver, Aurora, CO 80045, USA
f Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
g Department of Laboratory Medicine, University of Washington, Seattle, WA 98104, USA
h Department of Global Health, University of Washington, Seattle, WA 98104, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
Flavonolignans from milk thistle (Silybum marianum) have been investigated for their cellular modulatory
properties, including cancer chemoprevention and hepatoprotection, as an extract (silymarin), as par-
tially purified mixtures (silibinin and isosilibinin), and as pure compounds (a series of seven isomers).
One challenge with the use of these compounds in vivo is their relatively short half-life due to conjuga-
tion, particularly glucuronidation. In an attempt to generate analogues with improved in vivo properties,
particularly reduced metabolic liability, a semi-synthetic series was prepared in which the hydroxy
groups of silybin B were alkylated. A total of five methylated analogues of silybin B were synthesized
using standard alkylation conditions (dimethyl sulfate and potassium carbonate in acetone), purified
using preparative HPLC, and elucidated via spectroscopy and spectrometry. Of the five, one was monom-
ethylated (3), one was dimethylated (4), two were trimethylated (2 and 6), and one was tetramethylated
(5). The relative potency of all compounds was determined in a 72 h growth-inhibition assay against a
panel of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a human hepatoma cell line
(Huh7.5.1) and compared to natural silybin B. Compounds also were evaluated for inhibition of both
cytochrome P450 2C9 (CYP2C9) activity in human liver microsomes and hepatitis C virus infection in
Huh7.5.1 cells. The monomethyl and dimethyl analogues were shown to have enhanced activity in terms
of cytotoxicity, CYP2C9 inhibitory potency, and antiviral activity (up to 6-fold increased potency) com-
pared to the parent compound, silybin B. In total, these data suggested that methylation of flavonolignans
can increase bioactivity.
Received 4 September 2012
Revised 14 November 2012
Accepted 19 November 2012
Available online 5 December 2012
Keywords:
Silybin B
Milk thistle
Silybum marianum
Flavonolignans
Methylation
Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction
the natural products chemistry of these compounds led to the
gram-scale isolation of the flavonolignans,9 which consist of a ser-
Our research team has been studying the chemopreventive
properties of a series of flavonolignans isolated from an extract
(termed ‘silymarin’)1 of milk thistle [Silybum marianum (L.) Gaertn.
(Asteraceae)].2–8 In vitro and human tumor xenograft anticancer
activity of the commercial extracts, silymarin and silibinin, has
been demonstrated in models of prostate, colon, and breast cancer.
The extracts, and a subset of pure compounds derived therefrom,
have been studied most extensively in prostate carcinoma cell
lines, where they exhibit antiproliferative and pro-apoptotic
activity via inhibition of expression of cyclins and cyclin-depen-
dent kinases, induction of p21 and p27, degradation of androgen
receptor, and activation of caspase-3 and caspase-9.2–8 Studies on
ies of seven diastereo or constitutional isomers, and the isolation
and identification of two new minor analogs.10 Besides chemopre-
vention, some of these materials have been evaluated as inhibitors
of cytochrome P450 2C9 (CYP2C9)11 activity and activities associ-
ated with hepatoprotection.12–15
A challenge of working with milk thistle products has been
translating the results from in vitro studies to the clinic, likely
due to suboptimal pharmacokinetic characteristics of the com-
pounds.16 The pharmacokinetics of unconjugated and conjugated
(sulfated and glucuronidated) flavonolignans from milk thistle
were examined after oral administration of up to 700 mg/day
silymarin, and 28% and 55% of the total silymarin compounds in
plasma were found as sulfate and glucuronide conjugates, respec-
tively, after 1–3 h.17 Even with doses of 13 g/day with a silibinin
derived product in clinical studies by Flaig et al.,18 a major
⇑
Corresponding author. Tel.: +1 336 334 5474.
E-mail address: nicholas_oberlies@uncg.edu (N.H. Oberlies).
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.11.035

A. A. Sy-Cordero et al. / Bioorg. Med. Chem. 21 (2013) 742–747
743
challenge to in vivo activity was extensive conjugation at phenolic
2.2. Structure elucidation
positions. Conversely, these same phenolic moieties may present
opportunities for the semi-synthetic modification of milk thistle
compounds, if those that are or are not key for biological activity
could be identified.
The structures of the five methylated analogues of silybin B (1)
were elucidated using a suite of NMR experiments in conjunction
with HRMS data. Tables 1 and 2 summarize the 1H and 13C NMR
data, respectively, for compounds 2–6 in comparison to the well
established data for 1. The supplement includes the 1H and 13C
NMR data as stack plots (Figs. S2 and S3, respectively) and illus-
trates the ECD data (Fig. S4), which verified that all analogues re-
tained the configuration of 1.
Of the five compounds, one was monomethylated (3), one was
dimethylated (4), two were trimethylated (2 and 6), and one was
tetramethylated (5). The signals for methoxy groups in the 1H
and 13C NMR spectra (i.e. dH 3.2–3.8 and dC 55–59, respectively)
helped identify the number of methyl groups incorporated in the
structure of 1; the HRMS data served to verify the number of addi-
tional methyl groups. For example, compound 2, which was the
major product of the reaction, was 5,7,400-tri-O-methylsilybin B
(2). Key HMBC signals that established the positions of the methyl
moieties included correlations from OCH3-5 to C-5, from OCH3-7 to
C-7, and from OCH3-400 to C-400 (Fig. 2). HRESIMS data confirmed the
addition of three methyl groups in the structure (m/z 547.1579
[M+Na]+, calcd for C28H28O10Na, 547.1580). The other analogues
displayed similar HMBC correlations, thereby establishing the
structures of 7-O-methylsilybin B (3), 7,400-di-O-methylsilybin B
(4), 5,7,400,c-tetra-O-methylsilybin B (5), and 7,400,c-tri-O-methylsi-
lybin B (6). As with compound 2, these structures were supported
by HRMS data (see Section 4).
Other research groups have probed the semisynthesis of ana-
logues of silibinin19,20 (a 1:1 mixture of silybin A and silybin B)1
and/or 2,3-dehydrosilybin.21,22 The present semisynthetic studies
were designed with two central goals. The primary chemistry goal
was to determine which methylated analogues could be generated
readily using a straightforward alkylation procedure. For this,
selectivity was not critical, as it was desirable to examine the var-
ious permutations of single-, double-, triple-, and tetra-methylated
silybin B analogues. The primary pharmacology goal was to deter-
mine how these structural variations affected the antiproliferative
activity of silybin B (1). A secondary pharmacology goal was to
examine the compounds for inhibition of CYP2C9 activity and of
antiviral activity against hepatitis C virus (HCV). For the synthetic
experiments, 1 was utilized as a representative flavonolignan, since
it is a major component of both silibinin and silymarin,2 there was
an ample supply of >98% pure material on the gram scale available,
and it was the easiest of the seven flavonolignans to purify.9 Com-
pounds that were monomethylated or dimethylated were more
potent than 1, with the most potent being the dimethylated ana-
logue (compound 4; approximately 6 times more active). These
data suggested that alkylation of the phenolic moieties of the flav-
onolignans presents a strategy for their continued exploration.
2. Results and discussion
2.1. Synthesis and isolation
2.3. Bioassay results
2.3.1. Antiproliferative assays
The potency of the analogues (2–6) was determined in a 72 h
growth-inhibition assay relative to silybin B (1) and against a panel
of three prostate cancer cell lines (DU-145, PC-3, and LNCaP) and a
human hepatoma cell line (Huh7.5.1; Table 3). For inhibition of
prostate cancer cells, the most potent analogue (ꢀ6-fold increased
potency) was compound 4, corresponding to methylation of the
phenolic moieties at positions 7 and 400, yielding IC50 values that
Silybin B (1) starting material was isolated in >98% purity from
milk thistle extract (silymarin) as described in detail previously.9
Methylated analogues of 1 were prepared in a manner similar to
that of Dzubak and colleagues;22 however, those researchers used
a mixture of silybin A and silybin B (1) (often termed silibinin),1
and thus their analogues were generated as diastereomeric mix-
tures.22 Briefly, dimethyl sulfate was added to a solution of 1 and
potassium carbonate in anhydrous acetone. The mixture was
heated at reflux for 30 min, cooled to room temperature, acidified
with dilute hydrochloric acid, and extracted with ethyl acetate. The
organic extracts were combined and concentrated to dryness un-
der reduced pressure. The resulting oil was purified using RP-HPLC
to yield five major products (compounds 2–6; Fig. 1) in greater
than 95% purity as measured by analytical RP-HPLC (Fig. S1; the
numbering of the compounds corresponds to their elution order).
were 610 lM against two of the cell lines (DU-145 and PC-3).
The next most potent analogue, having a single methyl group at
the 7 position (3), was approximately a factor of two less active
than 4. However, all of the compounds were equal to or slightly
more potent (up to 6-fold increased potency) than the parent (1;
mean IC50 = 61.8 lM in DU-145 cells). Similarly, for inhibition of
hepatoma cell growth, all of the methylated compounds were
more potent than the parent. In particular, compounds 3 and 4
were over 5-fold more potent than the parent (1). In sum total,
these cytotoxicity data, regardless of cell type, suggested that mod-
ifications at any of the phenolic moieties could generate com-
pounds that enhance the antiproliferative potency of silybin B (1).
γ
O
O
CH2OR4
2.3.2. Inhibition of CYP2C9 activity
4'
3'
α
β
The inhibitory effects of four of the major compounds found in
silymarin (silybin A, silybin B (1), isosilybin A, and isosilybin B) on
the CYP2C9-mediated metabolism of (S)-warfarin were reported
recently.11 Silybin B (1) was the most potent of the four, with an
1'
R1O
O
OCH3
OR3
9
3"
7
2
3
1"
4"
4
10
OH
5
OR2
O
IC50 value of 8.2 l
M.11 Accordingly, the CYP2C9 inhibitory poten-
cies of the methylated analogues (2–5) were compared (Fig. 3);
compound 6 was not evaluated due to paucity of sample. With
the exception of 5, all compounds inhibited CYP2C9 activity in a
1
2
3
( ) R , R , R , R4 = H
1
2
1
2
3
( ) R , R , R = CH3; R4 = H
(3) R1 = CH3 ; R2, R3, R4 = H
(4) R1, R3 = CH3; R2, R4 = H
(5) R1, R2, R3, R4 = CH3
concentration-dependent manner (1 vs 10
lM). Compound 2 was
less potent than 1 at 100 M, whereas compounds 3 and 4,
l
(6) R1, R3, R4 = CH3; R2 = H
representing monomethylation at the 7-OH and dimethylation at
the 7- and 400-OH moieties, respectively, were more potent than
1, with activities below the limit of quantification when tested at
Figure 1. Structures of silybin B (1) and the methylated analogues 2–6.

744
A. A. Sy-Cordero et al. / Bioorg. Med. Chem. 21 (2013) 742–747
Table 1
1H NMR data for silybin B (1) and methylated analogues 2–6 acquired in DMSO-d6
Position
1 dH (J in Hz)
2 dH (J in Hz)
3 dH (J in Hz)
4 dH (J in Hz)
5 dH (J in Hz)
6 dH (J in Hz)
2
3
6
5.08, d (12)
4.61, d (12)
5.92, s
5.88, s
7.08, d (2)
6.98, d (9)
7.01, dd (9, 2)
4.17, ddd (8, 5, 2)
4.92, d (8)
3.54, ddd (11, 5, 2); 3.35, ddd (11, 5, 5)
7.02, d (2)
6.81 d, (8)
6.87, dd (8, 2)
—
11.90, s
—
5.05, d (11)
4.38, d (11)
6.22, d (2)
6.18, d (2)
7.08, d (2)
6.97, d (9)
7.02, dd (9, 2)
4.19, ddd (8, 4, 2)
4.97, d (8)
3.82, m; 3.74, m
7.01, d (2)
6.98, d (9)
6.99, dd (9, 2)
—
—
3.80, s
3.80, s
3.77, s
3.76, s
—
5.13, d (12)
4.66, br. d.
6.11, s
5.13, d (12)
4.66, dd (12, 7)
6.11, d (2)
6.09, d (2)
7.10, d (2)
6.97, d (9)
7.03, dd (9, 2)
4.19, ddd (8, 4, 2)
4.97, d (8)
3.55, m; 3.35, m
7.04, d (2)
6.98, d (8)
6.99, dd (8, 2)
5.88, d (7)
—
5.04, d (12)
4.66, dd (12, 7)
6.22, d (2)
6.18, d (2)
7.07, d (2)
6.99, d (9)
7.09, dd (9, 2)
4.36, m
4.94, d (8)
3.82, m; 3.74, m
7.05, d (2)
6.97, d (8)
6.98, dd (8, 2)
5.88, d (7)
—
5.13, d (12)
4.65, d (12)
6.10, s
8
6.09, s
6.08, s
20
7.09, d (2)
6.98, d (9)
7.03, dd (9, 2)
4.17, ddd (8, 5, 2)
4.91, d (8)
3.54, m; 3.35, m
7.02, d (2)
6.80, d (8)
6.86, dd (8, 2)
—
—
—
3.78, s
3.77, s
—
—
7.11, d (2)
7.01, d (9)
7.04, dd (9, 2)
4.38, ddd (8, 5, 2)
4.95, d (8)
3.81, m; 3.76, m
7.06, d (2)
6.99, d (8)
7.00, dd (8, 2)
—
—
—
3.78, s
3.78, s
3.77, s
3.22, s
50
60
a
b
c
200
500
600
3-OH
5-OH
5-OCH3
7-OCH3
300-OCH3
400-OCH3
—
3.78, s
3.77, s
3.76, s
3.80, s
3.80, s
3.78, s
3.77, s
—
3.78, s
—
—
c
-OCH3
—
3.22, s
Table 2
13C NMR data for silybin B (1) and methylated analogues 2–6 acquired in DMSO-d6
Position
1 dC, mult.
2 dC, mult.
3 dC, mult.
4 dC, mult.
5 dC, mult.
6 dC, mult.
2
3
4
5
6
7
8
9
82.5, CH
71.5, CH
197.8, C
163.3, C
96.1, CH
166.8, C
95.0, CH
162.5, C
100.5, C
130.1, C
116.7, CH
143.2, C
143.6, C
116.4, CH
121.2, CH
78.1, CH
75.9, CH
60.2, CH2
127.5, C
111.6, CH
147.6, C
147.0, C
115.3, CH
120.5, CH
—
82.1, CH
72.5, CH
190.2, C
161.6, C
92.9, CH
165.7, C
93.6, CH
163.7, C
103.5, C
130.3, C
116.5, CH
143.1, C
143.5, C
116.4, CH
121.0, CH
78.0, CH
75.7, CH
60.1, CH2
129.0, C
111.1, CH
149.1, C
148.8, C
111.5, CH
120.2, CH
55.9, CH3
55.9, CH3
55.6, CH3
55.5, CH3
—
82.6, CH
71.5, CH
198.4, C
163.0, C
94.9, CH
167.6, C
93.8, CH
162.4, C
101.4, C
129.9, C
116.6, CH
143.2, C
143.7, C
116.4, CH
121.2, CH
78.1, CH
75.8, CH
60.2, CH2
127.5, C
111.6, CH
147.6, C
147.0, C
115.3, CH
120.5, CH
—
82.9, CH
71.6, CH
198.4, C
163.0, C
94.9, CH
167.6, C
93.8, CH
162.4, C
101.4, C
130.0, C
116.6, CH
143.2, C
143.6, C
116.4, CH
121.0, CH
78.1, CH
76.0, CH
60.1, CH2
129.0, C
111.7, CH
149.1, C
148.8, C
116.2, CH
120.5, CH
—
82.1, CH
72.5, CH
190.2, C
161.6, C
92.9, CH
165.7, C
93.6, CH
163.7, C
103.5, C
130.4, C
116.6, CH
143.1, C
143.2, C
116.4, CH
121.1, CH
75.7, CH
76.4, CH
70.9, CH2
128.7, C
111.0, CH
149.1, C
148.8, C
111.5, CH
120.2, CH
56.0, CH3
55.8, CH3
55.6, CH3
55.5, CH3
58.6, CH3
82.6, CH
71.5, CH
193.3, C
162.4, C
93.8, CH
167.6, C
95.0, CH
163.6, C
101.4, C
130.2, C
116.8, CH
143.1, C
143.4, C
116.4, CH
121.3, CH
75.8, CH
76.4, CH
70.9, CH2
128.7, C
111.0, CH
149.2, C
148.8, C
111.5, CH
120.2, CH
—
10
10
20
30
40
50
60
a
b
c
100
200
300
400
500
600
5-OCH3
7-OCH3
300-OCH3
400-OCH3
—
56.0, CH3
55.7, CH3
—
56.0, CH3
55.6, CH3
55.4, CH3
—
56.0, CH3
55.6, CH3
55.5, CH3
58.6, CH3
55.7, CH3
—
—
c
-OCH3
—
γ
2.3.3. Inhibition of hepatitis C virus (HCV) infection
O
CH2OH
α
Figure 4 illustrates the antiviral profile of compounds 2–5
against HCV infection of human hepatoma Huh7.5.1 cells; com-
pound 6 was not evaluated due to paucity of sample. Previous
studies indicated that the IC50 value for inhibition of HCV infection
4'
3' O
H3CO
O
2
3
OCH3
OCH3
7
3"
β
4"
OH
5
by silybin B (1) was approximately 40 to 80 l
M.13 The tested ana-
O
O
H3C
logues of 1 inhibited HCV infection at lower concentrations, and
the dimethyl analogue 4 was the most potent. These data indicated
that methylated silybin B analogues were more active than 1 as
antivirals in the following order: dimethyl (4) > monomethyl
(3) > tetramethyl (5) > trimethyl (2) > 1. It was intriguing that both
the cytotoxicity and antiviral activities were enhanced by methyl-
ation, even though the antiviral testing was conducted at non-toxic
concentrations of the analogues.
Figure 2. Key HMBC correlations for the major product of the reaction, 5,7,400-tri-O-
methylsilybin B (2). The other analogues displayed similar data, depending on the
number of methyl groups incorporated.
100 lM. These results suggested that despite increased cytotoxic
potency, methylation of silybin B may modulate the clearance of
drugs metabolized by CYP2C9.

A. A. Sy-Cordero et al. / Bioorg. Med. Chem. 21 (2013) 742–747
745
Table 3
monomethyl (3) analogues, and irrespective of the biological assay,
the bioactivity increased relative to 1 in terms of growth inhibition
of prostate and liver cancer cells, inhibition of CYP2C9 activity, and
antiviral activity against HCV. These data suggest the feasibility of
retaining and even enhancing the bioactivity of silymarin-derived
compounds, and provides a rationale for methylating other iso-
mers to test in anticancer, antiviral, metabolic, and pharmacologic
assays. Ultimately, such studies could provide valuable structure–
activity relationships for delineating the mechanisms of action of
flavonolignans, which could aid in understanding how these com-
pounds act in many inflammatory disease states.
Antiproliferative activity of silybin B (1) and analogues 2–6 against a panel of cell
lines in culture
Compound
Mean IC50 values and standard deviations (lM)
DU-145
PC-3
LNCaP
Huh7.5.1
1
2
3
4
5
6
61.8 3.3
46.9 3.7
19.3 3.4
10.0 3.7
24.0 4.3a
16.8 3.9
60.5 4.3
36.3 3.7
16.9 3.6
8.1 3.5
14.4 4.4
21.9 4.1
69.6 3.8
40.7 3.9
20.1 3.7
11.3 4.8
30.1 3.7
19.8 4.8
56.2 2.8a
25.5 1.8
13.3 1.8
13.5 1.8
15.6 1.8
ntb
a
The IC50 values and standard deviations for these points were estimated based
on visual inspection of sigmoidal dose–response curves.
b
4. Experimental
Not tested.
4.1. General experimental methods
120
Optical rotation, UV, and IR data were acquired on a Rudolph
Research AutopolÒ III polarimeter, a Varian Cary 3 UV–Vis spectro-
photometer, and a Nicolet Avatar 360 FT-IR, respectively. Circular
dichroism spectra were obtained from an Aviv Stopped Flow Model
202 circular dichroism spectrometer. All NMR experiments were
acquired on a Varian Unity INOVA-500 instrument using a 5 mm
broad-band inverse probe with z-gradient using DMSO-d6.
LRESIMS data were acquired using an API 150EX mass spectrome-
ter. HRMS data were acquired on an Applied Biosystems 4700 TOF/
TOF instrument operating in reflectron mode using 2,5 dihydroxy-
benzoic acid as the matrix. For those data, spectra were the average
of 500–1000 laser shots, and the mass values were the mean of five
independent measurements. Some HRMS data were also acquired
on a Thermo LTQ Orbitrap XL mass spectrometer equipped with
an ESI source. Preparative HPLC was carried out on a Varian Prostar
HPLC system equipped with Prostar 210 pumps and a 335 photo-
diode array detector (PDA), with data collected and analyzed using
Galaxie Chromatography Data System software (version 1.9), using
100
*
80
60
40
20
0
*
†
#
*
*
#
#
BLQ
BLQ
Silybin B/Methylated Analogues
Figure 3. Effects of silybin B (1) and methylated analogues (2–5) on CYP2C9-
mediated (S)-warfarin 7-hydroxylation in human liver microsomes (HLM). Incuba-
tion mixtures consisted of HLM (0.1 mg/mL), (S)-warfarin (4
lM), silybin B or
methylated analogue (1, 10, or 100 M; open, solid, and hatched bars, respectively)
l
an ODS-A column (250 ꢁ 20 mm, i.d., 5
lm; YMC, Wilmington, NC)
at a flow rate of 7 mL/min. This same system and software were
and potassium phosphate buffer (100 mM, pH 7.4). Reactions were initiated by the
addition of NADPH (1 mM) and were terminated after 30 min with ice-cold MeOH
(2 volumes). Activity in the presence of vehicle control (0.75% MeOH, v/v) was
4.3 0.26 pmol/min/mg microsomal protein. Bars and error bars denote means and
SDs, respectively, of triplicate incubations. BLQ, below limit of quantification. ⁄p
used for analytical HPLC via an ODS-A column (150 ꢁ 4.6 mm,
i.d., 5 lm) at a flow rate of 1 mL/min.
<0.05, 1 versus 10
100 M (two-way ANOVA followed by Tukey’s test).
l lM; p <0.05 versus silybin B at
M; #p <0.05, 10 versus 100
l
4.2. Synthesis and purification
Silybin B (1) was isolated in >98% purity from milk thistle ex-
tract (silymarin) as described in detail previously.9 Methylated
analogs of 1 were prepared in a manner analogous to that of Dzu-
bak and colleagues.22 Dimethyl sulfate (0.20 g, 1.55 mmol) was
added to a solution of 1 (0.15 g, 0.31 mmol) and K2CO3 (0.35 g,
2.48 mmol) in anhydrous acetone (5 mL). The suspension was al-
lowed to stir at reflux for 30 min and was then cooled to room tem-
perature. The suspension was diluted with 1 N HCl (5 mL) and
extracted with EtOAc (3 ꢁ 5 mL). The organic extracts were com-
bined, dried (MgSO4), and concentrated under reduced pressure
to provide a brown oil (150 mg) as a mixture of methylated
analogs.
Figure 4. Methylated analogues (2–5) were more potent than silybin B (1) in
blocking HCV infection. Human hepatoma Huh7.5.1 cells were infected with HCV in
the presence of increasing concentrations of analogues for 72 h before cytoplasmic
proteins were extracted and HCV NS5A and core proteins detected by Western blot.
Detection of cellular actin protein served as a loading control. The concentrations
4.2.1. Purification
selected for testing were nontoxic: compound 2: 1.9, 9.4, 18.9 M; compound 3:
l
The reaction product (150 mg) was dissolved in DMSO (500 lL)
2.0, 10.1 M; compound 4: 1.9, 9.7 M; compound 5: 1.8, 9.2 lM. D refers to the
l
l
and purified via two separate rounds (ꢀ75 mg/round) of RP-HPLC
using a gradient that initiated with 60:40 MeOH:H2O and in-
creased linearly to 70:30 MeOH:H2O over 60 min, where it was
DMSO solvent control.
held isocratic for another 40 min, to yield compounds
(25.1 mg; 16.7%), (10.6 mg; 7.1%), (14.5 mg; 9.7%),
(12.0 mg; 8.0%), and 6 (2.7 mg; 1.8%).
2
5
3. Conclusion
3
4
In summary, a series of methylated analogues of silybin B (1)
were synthesized; some of these analogues have been reported
previously, albeit in the context of diastereomeric mixtures with
4.2.1.1. Silybin B (1).
The physical properties of 1 were de-
scribed in detail previously.23,24
silybin
A
analogues.22 In the case of the dimethyl (4) and

746
A. A. Sy-Cordero et al. / Bioorg. Med. Chem. 21 (2013) 742–747
4.2.1.2. 5,7,400-Tri-O-methylsilybin
B
(2).
White solid
H-2 ? C-3, 4, 9, 10, 20, 60; H-3 ? C-2, 4, 10, 10; H-6 ? C-5, 7, 8, 10;
H-8 ? C-6, 7, 9, 10; H-20 ? C-2, 10, 30, 40; H-50 ? C-10, 30, 40, 60; H-
60 ? C-2, 10, 20, 40, 50; H- , 40, 100; H-b ? C- , 30, 100, 200,
? C-b,
600; H2- , b; H-200 ? C-100, 300, 400, 600; H-500 ? C-100, 300, 400, 600;
? C-
H-600 ? C-b, 100, 200, 400, 500; OCH3-300 ? C-300; OCH3-7 ? C-7; OCH3-
400 ? C-400; OCH3- ; HRESIMS m/z 525.1740 [M+H]+ (calcd
? C-
(25.1 mg, yield 16.7% w/w); tR 7.65 min in 60:40 ? 100:0 MeOH–
H2O over 15 min with the ODS A column; ½a D25
ꢂ
ꢃ10.67 (c 0.6,
a
c
a, c
MeCN); UV (MeOH) kmax (log
e
) 283 (5.25), 205 (5.88) nm; CD
c
a
(MeOH) kext
(
D
e
) 333 (+3.3), 293 (ꢃ19.7), 233 (+21.6) nm; IR
(KBr) mmax 3438, 2934, 1672, 1606, 1571, 1508, 1422, 1257, 1214,
c
c
1106, 1021, 985, 812 cmꢃ1
;
1H and 13C NMR data, see Tables 1
for C28H29O10, 525.1755).
and 2; HMBC data, H-2 ? C-3, 4, 9, 10, 20, 60; H-3 ? C-2, 4, 10, 10;
H-6 ? C-5, 7, 8, 10; H-8 ? C-6, 7, 9, 10; H-20 ? C-2, 10, 30, 40; H-
4.3. Biological assays
50 ? C-10, 30, 40, 60; H-60 ? C-2, 10, 20, 40, 50; H-
a
? C-b,
c
, 40, 100;
, 30, 100, 200, 600; H2- , b; H-200 ? C-100, 300, 400, 600;
4.3.1. Antiproliferative assay
H-b ? C-a, c c ? C-a
H-500 ? C-100, 300, 400, 600; H-600 ? C-b, 100, 200, 400, 500; OCH3-300 ? C-
300; OCH3-5 ? C-5; OCH3-7 ? C-7; OCH3-400 ? C-400; HRESIMS m/z
547.1579 [M+Na]+ (calcd for C28H28O10Na, 547.1580).
All compounds were tested for antiproliferative/cytotoxic activ-
ity against a panel of prostate cancer cell lines in culture using
modifications of a published procedure2 that have been described
in detail.10 The prostate cancer cell lines were obtained from the
American Type Culture Collection (Manassas, VA): DU145 (HTB-
81, an androgen-independent line derived from a central nervous
system metastasis of prostate adenocarcinoma), PC-3 (CRL-1435,
an androgen-independent line derived from a bone metastasis of
prostate adenocarcinoma), and LNCaP (CRL-1740, an androgen-
dependent line derived from a lymph node metastasis of prostate
adenocarcinoma). All cell lines were cultured and maintained as
described previously,2 with the recently noted modification for
LNCaP.10 We also tested the antiproliferative/cytotoxic activity of
compounds against a liver cancer cell line, Huh7.5.1.25 Briefly,
10,000 cells per well were plated in 96-well plates, and following
overnight incubation, were incubated with increasing concentra-
tions of compounds. Seventy-two hours later, cell viability was
measured using the ATPlite kit, as described previously,13 and an
IC50 was calculated by linear regression using GraphPad Prism.
4.2.1.3. 7-O-Methylsilybin B (3).
White solid (10.6 mg, yield
7.1% w/w); tR 7.88 min in 60:40 ? 100:0 MeOH–H2O over 15 min
with the ODS A column; ½a D25
ꢂ
+2.67 (c 0.5, MeOH); UV (MeOH) kmax
(loge) 287 (5.28), 204 (5.93) nm; CD (MeOH) kext (De) 333 (+3.7),
295 (ꢃ15.5), 234 (+12.4) nm; IR (KBr) mmax 3400, 2936, 1668,
1606, 1571, 1508, 1422, 1258, 1214, 1105, 1021, 986, 813 cmꢃ1
;
1H and 13C NMR data, see Tables 1 and 2; HMBC data, H-2 ? C-3,
4, 9, 10, 20, 60; H-3 ? C-2, 4, 10, 10; H-6 ? C-5, 7, 8, 10; H-8 ? C-
6, 7, 9, 10; H-20 ? C-2, 10, 30, 40; H-50 ? C-10, 30, 40, 60; H-60 ? C-2,
10, 20, 40, 50; H-
a ? C-b, c a, c
, 40, 100; H-b ? C- , 30, 100, 200, 600; H2-
c
? C-a
, b; H-200 ? C-100, 300, 400, 600; H-500 ? C-100, 300, 400, 600; H-
600 ? C-b, 100, 200, 400, 500; OCH3-300 ? C-300; OCH3-7 ? C-7; HRESIMS
m/z 519.1271 [M+Na]+ (calcd for C26H24O10Na, 519.1267).
4.2.1.4. 7,400-Di-O-methylsilybin B (4).
White solid (14.5 mg,
yield 9.7% w/w); tR 9.17 min in 60:40 ? 100:0 MeOH–H2O over
4.3.2. Inhibition of CYP2C9 activity
15 min with the ODS A column; ½a D25
ꢂ
+8.77 (c 0.7, MeOH); UV (MeOH)
kmax (loge) 286 (5.26), 205 (5.93) nm; CD (MeOH) kext (De) 335 (+5.1),
Silybin B (1) and methylated analogues (2–5) were evaluated as
inhibitors of CYP2C9 activity using (S)-warfarin and pooled human
liver microsomes (HLM) as described previously.11 Incubation mix-
tures consisted of HLM (0.1 mg/mL microsomal protein), (S)-warfa-
294 (-21.8), 233 (+17.7) nm; IR (KBr) mmax 3432, 2937, 1670, 1606,
1572, 1508, 1422, 1258, 1214, 1105, 1021, 986, 811 cmꢃ1; 1H and 13
C
NMR data, see Tables 1 and 2; HMBC data, H-2 ? C-3, 4, 9, 10, 20, 60;
H-3 ? C-2, 4, 10, 10; H-6 ? C-5, 7, 8, 10; H-8 ? C-6, 7, 9, 10;
H-20 ? C-2, 10, 30, 40; H-50 ? C-10, 30, 40, 60; H-60 ? C-2, 10, 20, 40,
rin (4
and potassium phosphate buffer (100 mM, pH 7.4). The CYP2C9
inhibitor sulfaphenazole (1 M) was used as a positive control.
lM), silybin B or methylated analogue (1, 10, or 100 lM),
l
50; H- , 40, 100; H-b ? C- , 30, 100, 200, 600; H2-
a ? C-b, c a, c c ? C-a, b;
Control incubation mixtures contained 0.75% MeOH (v/v) in place
of silybin B/methylated analogue or sulfaphenazole. Incubation
mixtures were analyzed for 7-hydroxywarfarin by HPLC/MS–MS
as described previously.11
H-200 ? C-100, 300, 400, 600; H-500 ? C-100, 300, 400, 600; H-600 ? C-b, 100, 200, 400,
500; OCH3-300 ? C-300; OCH3-7 ? C-7; OCH3-400 ? C-400; HRESIMS m/z
533.1429 [M+Na]+ (calcd for C27H26O10Na, 533.1424).
All statistical analyses for this assay were conducted using
SigmaStat (version 3.5; Systat Software, Inc., San Jose, CA). Data
are presented as means SDs of triplicate incubations unless indi-
cated otherwise. Concentration-dependent inhibition of silybin B
and methylated analogs and comparisons between silybin B and
methylated analogs were evaluated by two-way analysis of vari-
ance (ANOVA); post hoc comparisons were made using Tukey’s test
when an overall difference resulted (p <0.05).
4.2.1.5. 5,7,400,c-Tetra-O-methylsilybin B (5).
White solid
(12.0 mg, yield 8.0% w/w); tR 9.91 min in 60:40 ? 100:0 MeOH–
H2O over 15 min with the ODS A column; ½a D25
ꢃ6.18 (c 0.1,
ꢂ
MeOH); UV (MeOH) kmax (loge) 285 (5.26), 205 (5.91) nm; CD
(MeOH) kext
(
D
e
) 334 (+3.1), 294 (ꢃ16.2), 233 (+15.5) nm; IR
(KBr) mmax 3424, 2936, 1670, 1606, 1571, 1508, 1422, 1257, 1214,
1106, 986, 812 cmꢃ1 1H and 13C NMR data, see Tables 1 and 2;
;
HMBC data, H-2 ? C-3, 4, 9, 10, 20, 60; H-3 ? C-2, 4, 10, 10; H-
6 ? C-5, 7, 8, 10; H-8 ? C-6, 7, 9, 10; H-20 ? C-2, 10, 30, 40; H-
4.3.3. Antiviral assays
50 ? C-10, 30, 40, 60; H-60 ? C-2, 10, 20, 40, 50; H-
a
? C-b,
, b; H-200 ? C-100, 300, 400, 600;
H-500 ? C-100, 300, 400, 600; H-600 ? C-b, 100, 200, 400, 500; OCH3-300 ? C-
300; OCH3-5 ? C-5; OCH3-7 ? C-7; OCH3-400 ? C-400; OCH3-
? C-
HRESIMS m/z 561.1746 [M+Na]+ (calcd for C29H30O10Na,
c
, 40, 100;
The antiviral activity of 1–5 was evaluated by choosing two
concentrations that were non-toxic to cells. For this, 150,000 cells
were plated in 12-well plates, and the next day, cells were infected
with JFH-1, a HCV that grows well in Huh7.5.1 cells, at a multiplic-
ity of infection of 0.05 focus forming units per cell. Five hours post-
infection, virus inocula were removed and replaced with fresh
medium containing compounds. Protein lysates were harvested
72 h later, and HCV proteins were detected by Western blot anal-
yses as described previously.13
H-b ? C-a, c c ? C-a
, 30, 100, 200, 600; H2-
c
c;
561.1737).
4.2.1.6. 7,400,c-Tri-O-methylsilybin B (6).
White solid (2.7 mg,
yield 1.8% w/w); tR 11.03 min in 60:40 ? 100:0 MeOH–H2O over
15 min with the ODS A column; ½a D31
ꢂ
+9.0 (c 0.05, MeOH); UV
(MeOH) kmax (loge) 286 (5.13), 205 (5.86) nm; CD (MeOH) kext
Acknowledgments
(
D
e
) 335 (+4.0), 294 (ꢃ19.7), 232 (+17.4) nm; IR (KBr) mmax 3400,
2935, 1670, 1606, 1572, 1508, 1423, 1258, 1215, 1107, 1023, 951,
815 cmꢃ1 1H and 13C NMR data, see Tables 1 and 2; HMBC data,
This research was supported by the National Institutes of
Health, with initial support from the National Cancer Institute via
;

A. A. Sy-Cordero et al. / Bioorg. Med. Chem. 21 (2013) 742–747
747
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Grant R01 CA104286 and subsequent support from the National
Institute of General Medical Sciences via Grant R01 GM077482
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and Dr. Yuka Nakanishi, then of the Natural Products Laboratory,
all at the Research Triangle Institute, for some of the high resolu-
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and Jessica Wagoner at the University of Washington for technical
assistance. We also thank Tamam El-Elimat and the Triad Mass
Spectrometry Laboratory at the University of North Carolina at
Greensboro for some of the high resolution mass spectrometry
data.
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