Semisynthesis, cytotoxicity, antiviral activity, and drug interaction liability of 7-O-methylated analogues of flavonolignans from milk thistle

Article abstract of DOI:10.1016/j.bmc.2013.04.017

Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, particularly for hepatoprotection. The main chemical constituents in silymarin are seven flavonolignans. Recent studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those analogues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver microsomes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced bioactivity was observed with the analogues. To further probe the biological consequences of methylation of the seven major flavonolignans, a series of 7-O-methylflavonolignans were generated. Optimization of the reaction conditions permitted selective methylation at the phenol in the 7-position in the presence of each metabolite's 4-5 other phenolic and/or alcoholic positions without the use of protecting groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal microsomes. Methylation of flavonolignans differentially modified inhibitory potency, with compounds demonstrating both increased and decreased potency depending upon the compound tested and the enzyme system investigated. In total, these data indicated that monomethylation modulates the cytotoxic, antiviral, and drug interaction potential of silymarin flavonolignans.

Full text of DOI:10.1016/j.bmc.2013.04.017

Bioorganic & Medicinal Chemistry 21 (2013) 3919–3926
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Semisynthesis, cytotoxicity, antiviral activity, and drug interaction
liability of 7-O-methylated analogues of flavonolignans from milk
thistle
Hanan S. Althagafy a, Tyler N. Graf a, Arlene A. Sy-Cordero a, Brandon T. Gufford b, Mary F. Paine b,
Jessica Wagoner c, Stephen J. Polyak c,d, Mitchell P. Croatt a, , Nicholas H. Oberlies a,
⇑
⇑
a Department of Chemistry and Biochemistry, University of North Carolina at Greensboro, Greensboro, NC 27402, USA
b Division of Pharmacotherapy and Experimental Therapeutics, UNC Eshelman School of Pharmacy, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA
c Department of Laboratory Medicine, University of Washington, Seattle, WA 98104, USA
d 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:
Silymarin, an extract of the seeds of milk thistle (Silybum marianum), is used as an herbal remedy, partic-
ularly for hepatoprotection. The main chemical constituents in silymarin are seven flavonolignans. Recent
studies explored the non-selective methylation of one flavonolignan, silybin B, and then tested those ana-
logues for cytotoxicity and inhibition of both cytochrome P450 (CYP) 2C9 activity in human liver micro-
somes and hepatitis C virus infection in a human hepatoma (Huh7.5.1) cell line. In general, enhanced
bioactivity was observed with the analogues. To further probe the biological consequences of methyla-
tion of the seven major flavonolignans, a series of 7-O-methylflavonolignans were generated. Optimiza-
tion of the reaction conditions permitted selective methylation at the phenol in the 7-position in the
presence of each metabolite’s 4–5 other phenolic and/or alcoholic positions without the use of protecting
groups. These 7-O-methylated analogues, in parallel with the corresponding parent compounds, were
evaluated for cytotoxicity against Huh7.5.1 cells; in all cases the monomethylated analogues were more
cytotoxic than the parent compounds. Moreover, parent compounds that were relatively non-toxic and
inactive or weak inhibitors of hepatitis C virus infection had enhanced cytotoxicity and anti-HCV activity
upon 7-O-methylation. Also, the compounds were tested for inhibition of major drug metabolizing
enzymes (CYP2C9, CYP3A4/5, UDP-glucuronsyltransferases) in pooled human liver or intestinal micro-
somes. Methylation of flavonolignans differentially modified inhibitory potency, with compounds dem-
onstrating both increased and decreased potency depending upon the compound tested and the
enzyme system investigated. In total, these data indicated that monomethylation modulates the cyto-
toxic, antiviral, and drug interaction potential of silymarin flavonolignans.
Received 31 January 2013
Revised 26 March 2013
Accepted 2 April 2013
Available online 16 April 2013
Keywords:
Silymarin
Milk thistle
Silybum marianum
Flavonolignans
Methylation
Hepatitis C virus
Cytochrome P450
UDP-glucuronosyl-transferase
Ó 2013 Elsevier Ltd. All rights reserved.
1. Introduction
A (5), isosilybin B (7), silychristin (9), isosilychristin (11), and
silydianin (13; Scheme 1).
The historical uses of milk thistle [Silybum marianum (L.)
Gaertn. (Asteraceae)], particularly in the areas of cancer chemopre-
vention and hepatoprotection, have been reviewed extensively.1–8
From a chemistry perspective, silymarin is a crude extract of the
seeds and affords a mixture of flavonolignans, which are diastereo-
meric and/or constitutional isomers of each other; a separate re-
view explains the nomenclature of the various constituents.9
With respect to the purified compounds, there are seven key flav-
onolignans, which are termed silybin A (1), silybin B (3), isosilybin
Recently, members of our team reported enhanced bioactivity
of methylated analogues of silybin B (3), particularly with re-
spect to assays for cytotoxic and antiviral activities.10 Therein,
a non-selective methylation method using dimethyl sulfate affor-
ded a series of five analogues of silybin B: mono-, di-, two differ-
ent tri-, and tetra-methylated analogues. Since the objective of
the research at that stage was to explore the biological activity
of a few methylated analogues, the selectivity of the reaction
was not critical. In fact, it was beneficial that those reaction con-
ditions were non-selective, as this produced multiple analogues
for testing.
⇑
Corresponding authors. Tel.: +1 336 334 3785 (M.P.C.); tel. +1 336 334 5474
(N.H.O.).
The goal of the present study was to expand upon that initial
SAR by selectively synthesizing 7-O-methyl analogues of the seven
major flavonolignans (Scheme 1). However, rather than relying
E-mail addresses: mpcroatt@uncg.edu (M.P. Croatt), nicholas_oberlies@uncg.edu
(N.H. Oberlies).
0968-0896/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2013.04.017

3920
H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
9''
5'
2'
8''
O
O
CH2OH
O
O
CH2OH
4'
3'
6'
1'
1
O
8
2''
8a
4a
2
3
RO
3'' OCH3
4''
RO
O
O
OCH3
OH
7
1''
6''
7''
6
OH
OH
OH
4
5
5''
OH
O
OH
3
R = H [silybin B ( )]
R = H [silybin A (1)]
CH3I, K2CO3
acetone
reflux, 3 h
41% yield
CH3I, K2CO3
acetone
reflux, 3.5 h
33% yield
R = CH3 [7-O-methylsilybin A (2)]
R = CH3 [7-O-methylsilybin B (4)]
5''
OH
OH
4''
6''
5'
O
4'
O
O
7''
8''
6'
3''
OCH3
OCH3
1''
1
O
2''
8
RO
O
8a
RO
2
3
7
1'
3'
O
CH2OH
CH2OH
2'
9''
6
4a
5
OH
OH
R = H [isosilybin A ( )]
4
OH
O
OH
O
5
7
R = H [isosilybin B ( )]
CH3I, K2CO3
acetone
CH3I, K2CO3
acetone
30 °C, 2.5 h
21% yield
40 °C, 2.5 h
24% yield
R = CH3 [7-O-methylisosilybin B (8)]
R = CH3 [7-O-methylisosilybin A(6)]
4'' OH
HO
OCH3
3''
4''
5''
6''
OCH3
3''
9''
1''
1''
HOH2C
2''
8''
7''
7''
O
9''
3'
5'
O
OH
HOH2C
8''
6'
4'
4'
2'
1'
1
O
8
8a
RO
RO
O
7
2
3'
1'
OH
2'
6
4a
3
OH
R = H [silychristin ( )]
OH
4
5
OH
O
OH
O
9
R = H [isosilychristin (11)]
CH3I, K2CO3
acetone
CH3I, K2CO3
acetone
40 °C, 2 h
29% yield
reflux, 2 h
40% yield
10
R = CH3 [7-O-methylsilychristin ( )]
R = CH3 [7-O-methylisosilychristin (12)]
O
OH
9''
3'
O
2' 8''
4'
5'
8
8a
4a
RO
O
O
2
3
7
1'
6'
6''
1''
2''
6
OH
5
OCH3
3''
5''
4''OH
OH
13)
R = H [silydianin (
]
CH3I, K2CO3
acetone
reflux, 2 h
33% yield
R = CH3 [7-O-methylsilydianin (14)]
Scheme 1. Structures and conditions for the synthesis of 7-O-methylated analogues of the seven major flavonolignans from silymarin.
upon protecting groups, which require two additional synthetic
steps for installation and removal, it was hypothesized that the
inherent reactivity of the molecule could be harnessed to guide
the selectivity. By judicious choice of reaction conditions, a single
position, the C-7-phenol, was methylated selectively in the pres-
ence of multiple centers with similar reactivity, including 2 or 3
other phenolic positions, a similarly acidic secondary alcohol, and
a primary alcohol. This chemoselectivity was comparable for all se-
ven flavonolignans, which enabled a site-specific analysis of the
importance of the methylated phenol for each compound’s activity
in a suite of bioassays that probed cytotoxicity, anti-hepatitis C
virus (HCV) activity, and inhibition of major drug metabolizing
enzymes.
2. Results and discussion
2.1. Chemistry
The synthesis of 7-O-methyl analogues of the natural flavono-
lignans was challenging due to the possibility of alkylation at more
than one hydroxyl group (Scheme 1). Therefore, a variety of bases
[Cs2CO3, K2CO3, and Diazabicycloundecene (DBU)], electrophilic
methylating agents (dimethyl carbonate, methyl iodide), solvents
(acetone, THF, acetonitrile), temperatures (rt to 75 °C under
standard heating or up to 130 °C using microwave heating),
and times (2 h–2 days) were examined, all with the goal of mini-
mizing the formation of polymethylated side products. Selective

H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
3921
monomethylation of the phenolic group at C-7 was possible for all
seven major flavonolignans over a temperature range of 30–60 °C
using methyl iodide in dry acetone in the presence of K2CO3. The
7-position was the most reactive for methylation, presumably
due to both acidity, being an arylogous carboxylic acid, and steric
accessibility. While the yields were moderate to low, ranging from
approximately 20–40%, the reaction conditions were optimized for
selective monomethylation rather than yield.
ments (see Tables 1 and 2 and Supplementary data). Using the data
for 7-O-methylsilybin A (2) as a representative example, two meth-
oxy singlets were apparent at dH 3.78 (3H) and 3.77 (3H), due to
the introduced methoxy group at C-7 and the natural one at C-
300, respectively. In contrast, the parent flavonolignan silybin A
(1) displayed a sharp singlet at dH 10.86 for the C-7 phenolic proton
signal. Further evidence for the methoxy group on C-7 in analogue
2 was observed in the meta-coupled protons at H-6 and H-8 (dH
6.12 and 6.10, respectively), which were shifted downfield slightly
compared to 1 (Table 1). The 13C NMR data (Table 2) of each meth-
ylated analogue displayed 26 distinct signals, which was one more
than the non-methylated starting materials, including resonances
at dC 56.0 and 55.7 due to the two methoxy moieties at C-7 and
C-300, respectively; these assignments were supported by HSQC
correlations. Moreover, data from the HMBC experiment demon-
strated the connectivity of the methoxy moieties (Fig. 1), where
correlations were observed between the protons at dH 3.78 to C-7
(dC 167.6) and dH 3.77 to C-300 (dC 147.6), respectively, verifying
that the introduced methoxy moiety was at the C-7 position in 2.
For each of the analogues, a similar set of NMR experiments
and data, coupled with comparisons to the data for the natural
flavonolignans, were utilized to establish that the methoxy moiety
2.2. Purification
Published methods were utilized to isolate the individual flav-
onolignans as starting materials for the methylation reactions.11
To purify the reaction mixtures, a PFP column was used in the re-
verse phase with a range of CH3CN/H2O (0.1% formic acid) gradi-
ents until each compound was >98% pure by analytical HPLC
(Fig. S1; Supplementary data).
2.3. Structure determination
Structures of the 7-O-methylated analogues were determined
using a suite of NMR spectroscopy and mass spectrometry experi-
Table 1
1H NMR data of flavonolignans and 7-O-methylflavonolignans in DMSO-d6 (500 MHz, 30 °C)
Position
2
1
2
3
4
5
6
7
8
9
10
11
5.17, d 5.22, d 4.86, dd 4.91, dd
(11.9) (11.4) (10.4) (10.4)
4.64, dd 4.67, dd 4.44, dd 4.48, dd
12
13
14
5.08, d
(11.5)
4.63, dd
5.14, d
(11.5)
4.69, dd
5.08, d
(11.5)
4.61, dd 4.67, dd
5.14, d
(11.5)
5.11, d
(10.9)
4.60, dd
5.16, d
(10.9)
4.66, dd
5.11, d
(11.5)
4.61, dd
5.16, d
(11.5)
4.67, dd
5.02, d
(11.4)
4.52, dd
5.05, d
(11.4)
4.58, dd
3
(11.5, 6.3) (11.5, 6.3) (11.5, 6.3) (11.5, 6.3) (10.9, 6.3) (10.9, 6.3) (11.5, 5.9) (11.5, 6.3) (11.4, 6.4) (11.4, 6.3) (11.9,
6.5)
(11.4,
6.9)
(10.4,
5.4)
(10.4,
5.4)
6
5.91, d
(1.7)
5.86, d
(1.7)
7.09, d
(1.7)
6.12, d
(1.7)
6.10, d
(1.7)
7.10, d
(1.7)
5.91, d
(2.3)
5.87, d
(2.3)
7.08, d
(1.8)
6.12, d
(2.3)
6.10, d
(2.3)
7.10, d
(2.3)
5.92, d
(2.3)
5.89, d
(2.3)
7.09, d
(2.3)
6.13, d
(2.3)
6.12, d
(2.3)
7.11, d
(2.3)
5.92, d
(2.0)
5.89, d
(2.0)
7.10, d
(1.8)
6.13, d
(2.3)
6.12, d
(2.3)
7.11, d
(1.7)
5.91, d
(2.0)
5.86, d
(2.0)
6.12, d
(2.3)
6.09, d
(2.3)
5.93, d 6.13, d 5.91, d 6.12, d
(1.9) (2.0) (2.5) (1.9)
5.88, d 6.11, d 5.88, d 6.11, d
8
(1.9)
—
(2.0)
—
(2.5)
3.44, dd 3.45, dd
(1.9)
20
6.82, d (1. 6.84, d
5)
—
(1.2)
—
(4.0,
2.0)
(4.0,
2.0)
50
6.97, d
(8.0)
6.98, d
(8.6)
6.97, d
(8.1)
6.98, d
(8.0)
6.93, d
(8.6)
6.94, d
(8.6)
6.93, d
(8.0)
6.94, d
(8.0)
6.75, d 6.74, d 3.17, dd 3.19, dd
(8.1)
(8.1)
(8.9,
3.0)
(6.9,
3.0)
60
7.00, dd
7.01, dd
7.02, dd 7.02, dd
6.98, dd
6.99, dd
6.98, dd
7.00, dd
6.87, br s 6.88, br s 6.96, d 6.97, d 5.92, d 6.01, d
(8.1) (8.1) (8.9) (6.9)
6.86, d 6.86, d 6.75, d 6.76, d
(1.5) (2.0) (2.0) (1.9)
6.70, d 6.69, d 6.63, d 6.64, d
(7.9) (8.5) (8.5) (8.4)
6.76, dd 6.76, dd 6.55, dd 6.56, dd
(8.0, 1.7) (8.6, 1.7) (8.1, 1.8) (8.0, 2.3) (8.6, 2.3) (8.6, 2.3) (8.0, 1.8) (8.0, 1.7)
7.01, d
(1.8)
6.80, d
(8.6)
6.86, dd
200
500
600
7.03, d
(1.8)
6.80, d
(8.6)
7.02, d
(1.7)
6.79, d
(8.6)
7.04, d
(1.7)
6.80, d
(8.0)
7.00, d
(1.7)
6.80, d
(8.6)
7.00, d
(1.7)
6.80, d
(8.0)
7.00, d
(2.3)
6.80, d
(8.6)
7.01, d
(1.8)
6.80, d
(8.2)
6.96, d
(2.0)
6.96, d
(1.8)
6.76, d
(8.1)
6.81, dd
(8.1, 1.8)
6.76, d
(8.4)
6.86, dd
6.86, dd 6.86, dd
6.86, dd
6.85, dd
6.86, dd
6.85, dd
6.80, dd
(8.6, 1.8) (8.6, 1.8) (8.6, 1.7) (8.0, 1.7) (8.6, 1.7) (8.0, 1.7) (8.6, 2.3) (8.2, 1.8) (8.4, 2.0)
(7.9, 1.5) (8.5,
2.0)
(8.5,
2.0)
(8.4,
1.9)
700
800
4.90, d
(7.5)
4.91, d
(7.5)
4.90, d
(7.5)
4.91, d
(7.5)
4.91, d
(8.1)
4.91, d
(7.5)
4.91, d
(7.5)
4.92, d
(7.5)
5.46, d
(7.0)
5.46, d
(6.9)
5.58, d 5.57, d 3.31, br 3.31, br
(2.5) (2.5)
s
s
4.17, ddd 4.18, ddd 4.16, ddd 4.17, ddd 4.16, ddd 4.17, ddd 4.17, ddd 4.17, ddd 3.47, ddd 3.47, ddd 3.67, m 3.67, m 2.72, br 2.73, br
(7.5, 4.6, (7.5, 4.6, (7.5, 4.1, (7.5, 4.6, (8.1, 4.6, (7.5, 4.0, (7.5, 4.6, (7.5, 4.6, (12.6, 12.0, (12.6, 12.0,
2.3) 2.3) 2.3) 2.3) 2.3) 2.3) 2.3) 1.7) 7.0) 6.9)
3.53, ddd 3.53, ddd 3.53, ddd 3.54, ddd 3.53, ddd 3.53, ddd 3.54, ddd 3.53, ddd 3.72, ddd 3.72, ddd 3.68, m 3.68, m 4.12, dd 4.13, dd
s
s
900a
(10.3, 5.2, (10.3, 5.2, (9.8, 4.6, (12.0, 5.2, (12.0, 4.6, (12.0, 4.6, (11.9, 5.2, (12.0, 5.2, (12.6, 10.9, (12.6, 10.9,
(8.0,
3.0)
(8.0,
3.0)
2.3)
2.3)
2.3)
2.3)
2.3)
2.3)
2.3)
1.7)
5.0)
5.2)
900b
3.33, m
3.34, m
3.33, m
3.33, m
3.33, m
3.33, m
3.33, m
3.33, m
3.63, ddd 3.63, ddd 3.45, m 3.44, m 3.78, d 3.78, d
(12.6, 10.9, (12.6, 10.9,
(8.0)
—
(8.0)
5.6)
5.7)
7-OCH3
—
3.78, s
3.77, s
5.90, d
(6.3)
11.86, s
—
—
3.78, s
3.77, s
5.89, d
(6.3)
11.87, s
—
—
3.79, s
3.77, s
5.91, d
(6.3)
11.87, s
—
—
3.79, s
3.77, s
5.91, d
(6.3)
11.87, s
—
—
3.78, s
3.76, s
5.81, d
(6.3)
—
3.70, s
3.80, s
3.79, s
300-OCH3 3.77, s
3.77, s
5.83, d
(6.3)
11.90, s
10.86, s
9.17, s
3.78, s
5.83, d
(6.3)
11.90, s
10.85, s
9.15, s
4.95, dd
3.77, s
5.84, d
(5.9)
11.90, s
10.86 s
9.16, s
4.96, dd
3.76, s
5.77, d
(6.4)
3.69, s 3.73, s 3.74, s
3-OH
5.83, d
(6.3)
11.90, s
10.86, s
9.18, s
4.97, dd
5.76, d 5.81, d 6.02, d 6.04, d
(6.5) (6.9) (6.3) (6.3)
11.93, s 11.90, s 11.81, s 11.77, s
5-OH
11.91, s
10.84, s
9.03, s
5.01, dd
11.87, s
—
9.02, s
5.01, dd
(5.7, 5.2)
7-OH
400-OH
900-OH
10.98, s
9.00, s
5.07, br 5.06, dd
dd (4.5, (6.5,
—
10.91, s
8.99, s 8.79, s 8.79, s
—
9.19, s
4.98, dd
9.18, s
9.18, s
4.97, dd
9.18, s
4.97, dd
4.98, dd 4.98, dd
(5.7, 5.2) (5.7, 5.2) (5.7, 4.6) (5.7, 5.2) (5.7, 4.6) (5.7, 4.6) (5.5, 5.0) (4.6, 5.2) (5.6, 5.0)
2.0)
—
4.5)
—
30-OH
—
—
—
—
—
—
—
—
—
—
7.19, s 7.17, s
Chemical shifts in d, coupling constants in Hz.

3922
H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
Table 2
13C NMR data of flavonolignans and 7-O-methylated analogues in DMSO-d6 (125 MHz, 30 °C)
Position
Type
1
2
3
4
5
6
7
8
9
10
11
12
13
14
2
3
4
4a
5
6
7
8
CH
CH
C
C
C
CH
C
CH
C
C
CH
C
82.6
71.4
82.7
71.5
82.5
71.4
82.7
71.5
82.5
71.5
82.7
71.6
82. 5
71.5
197.8
100.5
163.3
96.1
82.7
71.6
83.3
71.7
83.4
71.8
79.9
71.5
80.0
71.7
81.7
70.8
81.8
71.0
197.8
100.4
163.3
96.1
167.1
95.1
162.5
130.1
116.6
143.3
143.7
116.3
121.4
127.5
111.6
147.6
147.0
115.3
120.5
75.9
198.5
101.4
163.0
95.0
167.6
93.9
162.4
129.9
116.7
143.3
143.7
116.4
121.5
127.5
111.6
147.6
147.0
115.3
120.6
75.9
197.7
100.4
163.3
96.1
166.9
95.0
162.4
130.1
116.6
143.2
143.6
116.3
121.1
127.5
111.6
147.6
147.0
115.3
120.5
75.8
198.5
101.4
163.0
94.9
167.6
93.9
162.4
130.0
116.7
143.2
143.7
116.4
121.2
127.5
111.6
147.6
147.0
115.3
120.5
75.9
197.8
100.5
163.3
96.1
166.9
95.1
162.5
130.3
116.5
142.9
143.9
116.4
120.9
127.5
111.7
147.6
147.0
115.3
120.5
75.9
198.4
101.4
163.0
95.0
167.6
93.9
162.4
130.2
116.6
142.9
143.9
116.5
121.0
127.5
111.7
147.6
147.0
115.3
120.5
75.9
198.4
101.4
163.0
95.0
167.6
93.9
162.4
130.2
116.5
142.9
143.9
116.4
121.0
127.5
111.7
147.6
147.0
115.3
120.4
75.9
197.8
100.5
163.3
96.0
166.8
95.0
162.6
130.0
115.6
146.4
140.7
129.1
115.4
132.4
110.4
147.5
147.1
115.3
118.7
87.0
198.4
101.4
163.0
94.9
167.5
93.8
162.5
129.8
115.7
146.4
140.7
129.1
115.4
132.4
110.9
148.1
147.7
115.3
118.7
87.5
198.2
100.6
163.4
96.1
166.8
95.1
162.6
124.5
128.9
141.6
145.9
116.1
119.5
132.8
110.2
147.5
146.3
115.1
118.5
86.4
198.7
101.5
163.1
95.0
167.6
93.9
162.5
124.4
128.9
141.6
145.8
116.1
119.4
132.8
110.3
147.5
146.3
115.1
118.6
86.4
196.5
100.3
163.4
96.2
166.9
95.0
162.0
139.5
48.6
197.6
101.2
163.0
95.2
167.6
93.8
162.0
139.4
48.7
166.9
95.1
8a
162.5
130.3
116.5
142.9
143.9
116.5
120.9
127.5
111.7
147.6
147.0
115.3
120.5
75.9
10
20
30
96.7
201.9
53.4
96.7
201.9
53.3
40
C
50
CH
CH
C
CH
C
60
124.0
133.0
112.4
147.1
145.1
114.9
120.3
46.0
44.0
72.8
55.4
—
124.0
133.0
112.4
147.2
145.1
115.0
120.3
46.0
44.0
72.8
55.4
56.0
100
200
300
400
C
500
CH
CH
CH
CH
CH2
CH3
CH3
600
700
800
78.1
60.2
55.7
—
78.1
60.2
55.7
56.0
78.1
60.2
55.7
—
78.1
60.2
55.7
56.0
78.0
60.2
55.7
—
78.0
60.2
55.7
56.0
78.0
60.2
55.7
—
78.0
60.2
55.7
56.0
53.4
63.0
55.6
—
53.9
63.5
55.6
55.9
52.0
63.5
55.6
—
51.9
63.5
55.6
56.0
900
300-OCH3
7-OCH3
Chemical shifts in d. In compounds 9 and 10, position 50 is quaternary; in compounds 11 and 12, position 20 is quaternary.
Table 3
9''
5'
2'
O
O
CH2OH
4'
3'
8''
7''
6'
1'
Cytotoxic activity of flavonolignans and 7-O-methylated analogues against human
hepatoma (Huh7.5.1) cells
1
O
8
6''
1''
8a
4a
H3CO
7
2
3
5''
Compound
IC50 values (
l
M)
Fold changea,b
OH
4''OH
4
6
2''
3''
Silybin A (1)
7-O-Methylsilybin A (2)
Silybin B (3)
7-O-Methylsilybin B (4)
Isosilybin A (5)
7-O-Methylisosilybin A (6)
Isosilybin B (7)
7-O-Methylisosilybin B (8)
Silychristin (9)
7-O-Methylsilychristin (10)
Isosilychristin (11)
7-O-Methylisosilychristin (12)
Silydianin (13)
80.0
9.5
5
8.4
5.5
6.1
1.5
>2
OH
O
OCH3
68.7
12.7
80.0
13.2
27.0
18.0
>130
66.4
>130
27.6
>130
9.4
Figure 1. Key HMBC correlations of 7-O-methylsilybin A (2).
was connected at the 7 position in all of the analogues. Finally,
HRMS data supported the introduction of a single methyl moiety,
where the [MꢀH]ꢀ ion peak for all of the analogues was fourteen
mass units greater than that of the parent flavonolignans (see
experimental).
>4.7
13.8
7-O-Methylsilydianin (14)
2.4. Biological studies
Resveratrolc
10.6
a
2.4.1. Cytotoxicity and cell viability
The fold change was calculated by dividing the IC50 value for the parent flav-
onolignan by the IC50 value for the corresponding 7-O-methylated analogue.
In comparing the cytotoxicity profiles of 7-O-methylated ana-
logues versus parental counterparts (Table 3), addition of a single
methyl group increased cytotoxicity against human hepatoma cells
(Huh7.5.1). These findings were consistent with an earlier pilot
study that examined a series of mono-, di-, tri- and tetra-methylated
analogues of silybin B (3).10 Silychristin (9), isosilychristin (11),
and silydianin (13) have been reported to be relatively non-toxic
compared to the other silymarin flavonolignans.12 In the current
study, marked increases in the cytotoxicity of silydianin (13),
silybin A (1), and isosilybin A (5) were observed upon methylation
(compounds 14, 2, and 6, with 13.8-, 8.4-, and 6.1-fold, respec-
tively). However, the 7-O-methylation of isosilybin B (7) did not
produce a pronounced increase in cytotoxicity (i.e., compound 8),
which was interesting given that 7 has been shown to be the most
cytotoxic of the parent flavonolignans, particularly in assays for
prostate cancer chemoprevention.12–17
b
The average fold change for all analogues was approximately 6.
Typical average value as a positive control for the assay.
c
HCV activity [i.e. 7-O-methylsilychristin (10), 7-O-methylisosily-
christin (12)] relative to the corresponding parent compounds. In
particular, silychristin (9), isosilychristin (11), and silydianin (13)
did not show appreciable antiviral activity until they were methyl-
ated (i.e., 10, 12, and 14). On the other hand, for parent compounds
that were active in blocking HCV activity, such as silybin A (1), sily-
bin B (3), and isosilybin A (5), the activities of their methylated
counterparts were either retained (2) or reduced (4 and 6). The
most toxic parent compound, isosilybin B (7), had a slight enhance-
ment of both antiviral activity and cytotoxicity (Table 3) by meth-
ylation (compound 8). Thus, it appeared that HCV antiviral activity
tracked with cytotoxicity. That is, as compounds became more
toxic, the more likely antiviral activity was detected. However, that
is not to say that antiviral activity coincided with cytotoxicity, as
we were always able to detect anti-HCV activity that was indepen-
dent of cytotoxicity. We hypothesize that flavonolignans target
2.4.2. Antiviral activity
In evaluating the anti-HCV profiles of these compounds (Fig. 2),
some of the methylated flavonolignans displayed improved anti-

H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
3923
A
B
3
4
9
10
D
D
HCV core
Actin
HCV core
Actin
80
60
40
20
0
80
60
40
20
0
1
2
9
10
Dose 1
Dose 2
Dose 3
Dose 1
Dose 2
Dose 3
Flavonolignan Dose
Flavonolignan Dose
100
80
60
40
20
0
60
40
20
0
3
4
11
12
Dose 1
Dose 2
Dose 3
Dose 1
Dose 2
Dose 3
Flavonolignan Dose
Flavonolignan Dose
100
80
60
40
20
0
60
40
20
0
5
6
13
14
Dose 1
Dose 2
Dose 3
Dose 1
Dose 2
Dose 3
Flavonolignan Dose
Flavonolignan Dose
150
100
50
7
8
0
Dose 1
Dose 2
Dose 3
Flavonolignan Dose
Figure 2. Antiviral profile of parent and 7-O-methyl flavonolignans. Huh7.5.1 cells were infected with JFH-1 at a multiplicity of infection of 0.05. Virus inoculum was removed
after 5 h and compounds were added. Cultures were incubated for 72 h before protein lysates were analyzed for HCV core protein expression by Western blot analysis.
Detection of actin protein served as a protein loading control. Panel A depicts an example of a parent compound that lacked anti-HCV activity (9) and acquired anti-HCV
activity upon methylation (10). Panel B depicts an example of a parent compound that had anti-HCV activity (3) and decreased anti-HCV activity upon methylation (4). The
graphs below panels A and B present quantitation of HCV core pixel intensity following normalization to actin pixel intensity, expressed as percent inhibition relative to
DMSO controls. Graphs below panel A depict parent compounds that lacked anti-HCV activity (9, 11, 13) and acquired anti-HCV activity upon methylation (10, 12, 14). Graphs
below panel B depict parent compounds that had anti-HCV activity (1, 3, 5, 7), and retained (2), decreased (4, 6), or enhanced (8) anti-HCV activity upon methylation. Doses
used were derived from toxicity data, such that two non-toxic concentrations and one near the IC50 value were evaluated in the antiviral assay. Concentrations for compounds
1, 3, and 5: 6.2, 20.7, 62.1
compounds 9, 11, 12 and 13 12.4, 41.4, 124.2
l
M. Concentrations for compounds 2, 4, 6, and 14: 0.8, 2.7, 8.0
l
M. Concentrations for compounds 7 and 8: 1.6, 5.4, 16.1
M.
lM. Concentrations for
l
M. Concentrations for compound 10: 6.0, 20.1, 60.3
l
shared cellular functions that confer both cytotoxic and antiviral
activities, although this requires further experimentation to verify.
for incubations with (S)-warfarin, whereas HIM were selected for
incubations with midazolam.
All flavonolignans and 7-O-methylated analogues showed con-
centration-dependent inhibition of CYP activity (Figs. 3 and 4), ex-
cept for isosilybin A (5) against CYP2C9 activity (Fig. 3). The parent
2.4.3. Inhibition of drug metabolizing enzymes
The propensity of methylation at the 7-O position to alter drug
interaction liability was evaluated by testing all compounds as
inhibitors of major drug metabolizing enzymes using the probe
substrates (S)-warfarin (CYP2C9; Fig. 3), midazolam (CYP3A4/5;
Fig. 4), and 4-methylumbelliferone (UGT; Fig. 5) and human liver
or intestinal microsomes (HLM or HIM, respectively). Although
CYP2C9 and CYP3A4/5 are expressed in both the human intestine
and liver,18 intestinal CYP2C9 has not yet been shown to have clin-
ical impact on drug disposition in vivo. As such, HLM were selected
versus methylated analogue pairs at 100 lM differed in inhibition
potency except for silybin B (3) versus 7-O-methylsilybin B (4)
against CYP2C9 activity (Fig. 3), silydianin (13) versus 7-O-meth-
ylsilydianin (14) against CYP3A4/5 activity, and silychristin (9) ver-
sus 7-O-methylsilychristin (10) against CYP3A4/5 activity (Fig. 4).
Amongst the analogue series, compounds with an ‘iso’ configura-
tion inhibited the CYP enzymes (Figs. 3 and 4) to a greater extent
than corresponding regioisomers. For example, 7-O-methylisosily-
bin A (6), 7-O-methylisosilybin B (8), and 7-O-methylisosilychristin

3924
H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
Figure 5. Effects of selected flavonolignans and 7-O-methylated analogues on UGT-
mediated 4-methylumbelliferone (4-MU) glucuronidation in human liver micro-
Figure 3. Effects of selected flavonolignans and 7-O-methylated analogues on
CYP2C9-mediated (S)-warfarin 7-hydroxylation in human liver microsomes (HLM).
somes (HLM). Incubation mixtures consisted of HLM (0.4 mg/mL), 4-MU (100
lM),
Incubation mixtures consisted of HLM (0.1 mg/mL), (S)-warfarin (4
lM), flavono-
flavonolignan or 7-O-methylated analogue (10 or 100 M; hatched and solid bars,
l
lignan or 7-O-methylated analogue (10 or 100 M; hatched and solid bars,
l
respectively), bovine serum albumin (BSA, 0.05%), and Tris–HCl buffer (0.1 M, pH
7.5) supplemented with magnesium chloride (5 mM). Reactions were initiated by
the addition of UDPGA (4 mM) followed by fluorescence analysis of 4-MU depletion
over a period of 10 min. Activity in the presence of vehicle control (0.2% MeOH, v/v)
was 8.0 0.4 nmol/min/mg microsomal protein. Bars and error bars denote means
respectively), and potassium phosphate buffer (0.1 M, pH 7.4). Reactions were
initiated by the addition of NADPH (1 mM) and were terminated after 30 min with
ice-cold MeOH containing 4-chlorowarfarin as internal standard. Activity in the
presence of vehicle control (0.75% methanol, v/v) was 2.5 0.1 pmol/min/mg
microsomal protein. Bars and error bars denote means and SDs, respectively, of
and SDs, respectively, of triplicate incubations. ⁄p < 0.05, 10 versus 100
l
M; #p <
triplicate incubations. ⁄p < 0.05, 10 versus 100
l
M; #p < 0.05, flavonolignan versus
0.05, flavonolignan versus 7-O-methylated analogue at 100
lM (two-way ANOVA
7-O-methylated analogue at 100 lM
(two-way ANOVA with Bonferroni
with Bonferroni adjustment).
adjustment).
was 7-O-methylisosilychristin (12), which was more potent than
isosilychristin (11). Diclofenac, a non-specific inhibitor of the UGTs,
inhibited activity by 56% (data not shown).
Collectively, CYP2C9 activity was most sensitive to inhibition,
by both parent flavonolignans and corresponding methylated ana-
logues, followed by CYP3A4/5 and UGT activity (up to 91%, 72%,
and 44% inhibition, respectively). 7-O-Methylation of these
isolated flavonolignans highlights the importance of this
metabolically labile site, providing mechanistic insight into the en-
zyme–ligand interaction.
3. Conclusion
In summary, a series of 7-O-methyl flavonolignan derivatives
has been synthesized selectively, and this study is the first to
examine methylation of all seven of the major flavonolignans
in silymarin. All the derivatives showed cytotoxic effects against
human hepatoma cells. The most potent analogue was 7-O-
Figure 4. Effects of selected flavonolignans and 7-O-methylated analogues on
CYP3A-mediated midazolam 10-hydroxylation in human intestinal microsomes
(HIM). Incubation mixtures consisted of HIM (0.05 mg/ml), midazolam (4
lM),
flavonolignan or 7-O-methylated analogue (10 or 100 M; hatched and solid bars,
l
methylsilybin A, which exhibited an IC50 value of 9.5
lM com-
respectively), and potassium phosphate buffer (0.1 M, pH 7.4) supplemented with
magnesium chloride (3.3 mM). Reactions were initiated by the addition of NADPH
(1 mM) and were terminated after 4 min with ice-cold acetonitrile containing
alprazolam as internal standard. Activity in the presence of vehicle control (0.1%
DMSO, v/v) was 700 100 pmol/min/mg microsomal protein. Bars and error bars
denote means and SDs, respectively, of triplicate incubations. ⁄p <0.05, 10 versus
pared with parent silybin A with an IC50 value of 80
lM. In
terms of anti-HCV activity, parent compounds with minimal to
no antiviral activity (i.e., 9, 11, 13) showed increased antiviral
activity when converted to the 7-O-methyl derivatives. For these
compounds, acquisition of antiviral activity by 7-O-methylation
was associated with enhanced cytotoxicity. In contrast, parent
compounds with anti-HCV activity either retained (1), decreased
(3, 5), or slightly increased (7) activity upon methylation. 7-O-
Methylation differentially modified the inhibitory potency of
flavonolignans towards major drug metabolizing enzymes, with
compounds demonstrating increased, decreased, or no change
in potency depending upon the enzyme system investigated. De-
spite inconsistent patterns, the differences observed between
parent and analogue suggested the importance of the 7-position.
Cumulatively, these data suggested that silymarin-derived com-
pounds exert pleiotropic effects on cells. Ongoing efforts are
focusing on how these compounds affect cellular metabolic pro-
cesses that culminate in cytotoxicity, antiviral activity, and drug
metabolism inhibition.
100
way ANOVA with Bonferroni adjustment).
l lM (two-
M; #p < 0.05, flavonolignan versus 7-O-methylated analogue at 100
(12) were more potent against CYP activities than 7-O-methylsily-
bin A (2), 7-O-methylsilybin B (4), and 7-O-methylsilychristin (10).
Sulfaphenazole and ketoconazole, known potent inhibitors of
CYP2C9 and CYP3A4/5 activity, respectively, inhibited correspond-
ing activities by 67% and 81% (data not shown).
Compared to the CYPs, inhibition of UGTs was less extensive
(Fig. 5). However, in general, the parent compounds were more
potent than the analogues. For example, silybin A (1), silybin B
(3), and silydianin (13) at 100 lM were more potent than the
respective methylated analogues (i.e., 2, 4, and 14), suggesting
that methylation reduced activity against UGT. One exception

H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
3925
4.2.2. 7-O-Methylsilybin B (4)
4. Experimental
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 3 was reacted with 15 equiv of
CH3I at reflux for 3.5 h to yield compound 4. Yield: 10 mg, 33%;
4.1. General experimental procedures
white solid; ½a 2D0
ꢂ
+7.7 (c 0.2, MeOH); UV (MeOH) kmax (loge) 230
Optical rotation and UV data were acquired on a Rudolph Re-
search AutopolÒ III polarimeter and a Varian Cary 3 UV–vis spec-
trophotometer, respectively. All NMR experiments were
conducted in DMSO-d6 at 30 °C using a JEOL ECA-500 (operating
at 500 MHz for 1H and 125 MHz for 13C). HRESIMS data were mea-
sured using an electrospray ionization (ESI) source coupled to a Q-
TOF Premier mass spectrometer (Waters Corp., Milford, MA, USA)
in negative ionization mode via a liquid chromatographic/auto-
sampler system that consisted of an Acquity UPLC system (Waters
Corp.). HPLC was carried out on Varian Prostar HPLC systems
equipped with Prostar 210 pumps and a Prostar 335 photodiode
array detector (PDA), with data collected and analyzed using Gal-
axie Chromatography Workstation software (version 1.9.3.2). For
(4.0) , 287 (4.0) nm; CD (MeOH) kext
(
D
e) 230 (8.6), 296 (ꢀ9.2),
334 (1.3) nm; 1H and 13C NMR data, see Tables 1 and 2 and
Figs. S5 and S6; HMBC data, see Figs. S7 and S23; HRESIMS m/z
495.1290 [MꢀH]ꢀ (calcd for C26H23O10 495.1297).
4.2.3. 7-O-Methylisosilybin A (6)
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 5 was reacted with 2 equiv of CH3I
at 30 °C for 2.5 h to yield compound 6. Yield: 22 mg, 21%; white so-
lid; ½a 2D0
ꢂ
+ 46 (c 0.1, MeOH); UV (MeOH) kmax (loge) 214 (4.1), 287
(4.0) nm; CD (MeOH) kext
(D
e
) 236 (ꢀ0.9), 296 (ꢀ5.7), 331 (0.7)
nm; 1H and 13C NMR data, see Tables 1 and 2 and Figs. S8 and
S9; HMBC data, see Figs. S10 and S23; HRESIMS m/z 495.1292
[MꢀH]ꢀ (calcd for C26H23O10 495.1297).
preparative HPLC, a YMC ODS-A (5
Corp.) column was used at a 7 mL/min flow rate and a Phenomenex
PFP (pentafluorophenyl propyl; 5
m; 250 ꢁ 21 mm) column was
used at a 21.2 mL/min flow rate. For analytical HPLC, a YMC
ODS-A (5 PFP (5 m;
l
m, 250 ꢁ 20 mm; Waters
l
4.2.4. 7-O-Methylisosilybin B (8)
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 7 was reacted with 4 equiv of CH3I
at 40 °C for 2.5 h to yield compound 8. Yield: 12 mg, 24%; white so-
l
m; 150 ꢁ 4.6 mm) column and
a
l
150 ꢁ 4.6 mm) column were used, both at a 1 mL/min flow rate.
All reactions were carried out in a N2 atmosphere under anhydrous
conditions.
lid; ½a 2D0
ꢂ
ꢀ17.4 (c 0.09, MeOH); UV (MeOH) kmax (log
e) 211 (4.1),
) 229 (1.8), 294 (ꢀ4.2), 331
287 (3.8) nm; CD (MeOH) kext
(D
e
(ꢀ0.1) nm; 1H and 13C NMR data, see Tables 1 and 2 and
Figs. S11 and S12; HMBC data, see Figs. S13 and S23; HRESIMS
m/z 495.1294 [MꢀH]ꢀ (calcd for C26H23O10 495.1297).
4.2. Chemistry, synthesis and purification
The individual flavonolignans were isolated from milk thistle
extract (silymarin) in >98% purity as described in detail previ-
ously.11 To purify the reaction mixtures, two different reverse-
4.2.5. 7-O-Methylsilychristin (10)
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 9 was reacted with 12 equiv of
CH3I at reflux for 2 h to yield compound 10. Yield: 41 mg, 40%;
phase columns [ODS-A C18 (5
l
m; 250 ꢁ 20 mm) and PFP (5
lm;
250 ꢁ 21 mm)] were examined; the latter gave the best results
according to peak symmetry and resolution when using acetoni-
trile as the organic modifier (data not shown). Accordingly, each
7-O-methylated flavonolignan analogue was purified until >98%
pure, as measured by analytical HPLC (Fig. S1; Supplementary
data). 7-O-methylsilybin A (2) and 7-O-methylisosilybin A (6) were
purified using a gradient of 30:70 to 50:50 CH3CN/H2O (0.1% for-
mic acid) over 30 min. 7-O-methylsilybin B (4) was purified using
a gradient of 20:80 to 40:60 CH3CN/H2O (0.1% formic acid) over
30 min. 7-O-methylisosilybin B (8) was purified using 20:80 to
60:40 CH3CN/H2O (0.1% formic acid) over 40 min. 7-O-Methylsily-
christin (10) and 7-O-methylsilydianin (12) were purified using a
gradient of 20:80 to 42:58 CH3CN/H2O (0.1% formic acid) over
white solid; ½a 2D0
ꢂ
+56.6 (c 0.06, MeOH); UV (MeOH) kmax (loge)
215 (4.0), 288 (3.8) nm; 1H and 13C NMR data, see Tables 1 and 2
and Figs. S14 and S15; HMBC data, see Figs. S16 and S23; HRESIMS
m/z 495.1284 [MꢀH]ꢀ (calcd for C26H23O10 495.1297).
4.2.6. 7-O-Methylisosilychristin (12)
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 11 was reacted with 24 equiv of
CH3I at 40 °C for 2 h to yield compound 12. Yield: 15 mg, 29%;
white solid; ½a 2D0
ꢂ
+144.4 (c 0.05, MeOH); UV (MeOH) kmax (loge)
212 (4.1), 288 (3.7) nm; 1H and 13C NMR data, see Tables 1 and 2
and Figs. S17 and S18; HMBC data, see Figs. S19 and S23; HRESIMS
m/z 495.1284 [MꢀH]ꢀ (calcd for C26H23O10 495.1297).
45 min. 7-O-Methylisosilychristin (14) was purified using
gradient of 15:85 to 36:64 CH3CN/H2O (0.1% formic acid) over
50 min.
a
4.2.7. 7-O-Methylsilydianin (14)
4.2.1. 7-O-Methylsilybin A (2)
To a 150 mL three-neck round-bottom flask, silybin A (122 mg,
0.253 mmol) and K2CO3 (293 mg, 2.03 mmol) were dissolved in
In a manner that was otherwise consistent with the preparation
and purification of 2, compound 13 was reacted with 12 equiv of
CH3I at reflux for 2 h to yield compound 14. Yield: 20 mg, 33%;
white solid; ½a 2D0
ꢂ
+157.9 (c 0.06, MeOH); UV (MeOH) kmax (loge)
acetone (80 mL, 0.003 M). After stirring for 10 min, CH3I (244 lL,
207 (3.9), 288 (3.6) nm; 1H and 13C NMR data, see Tables 1 and 2
and Figs. S20 and S21; HMBC data, see Figs. S22 and S23; HRESIMS
m/z 495.1284 [MꢀH]ꢀ (calcd for C26H23O10 495.1297).
3.95 mmol) was added dropwise. The reaction mixture was heated
at reflux under N2 for 3 h and monitored by HPLC. After cooling to
rt, the reaction was quenched by the addition of 2% aqueous HCl
(pH 1.5) and diluted with H2O (125 mL). The mixture was ex-
tracted with EtOAc (2 ꢁ 80 mL). The organic phases were com-
bined and dried over Na2SO4, filtered, and then the solvent was
removed under reduced pressure. The compounds were purified
4.3. Biological assays
4.3.1. Antiproliferative assay
as described in Section 2.2. Yield: 51 mg, 41%; white solid; ½a D20
ꢂ
The antiproliferative/cytotoxic activities of all compounds were
examined against a human hepatoma cell line, Huh7.5.1,19 as de-
scribed previously.10 Briefly, 10,000 cells per well were plated in
96-well plates, and following overnight incubation, were incubated
with increasing concentrations of compounds. Cell viability was
measured 72 h later using the ATPlite kit, as described
+20 (c 0.1, MeOH); UV (MeOH) kmax (loge) 218 (4.2), 287 (4.1)
nm; CD (MeOH) kext
(
D
e
) 237 (ꢀ3.3), 296 (ꢀ9.2), 330 (1.7) nm;
1H and 13C NMR data, see Tables 1 and 2 and Figs. S2 and S3; HMBC
data, see Figs. 1 and S4; HRESIMS m/z 495.1281 [MꢀH]ꢀ (calcd for
C26H23O10 495.1297).

3926
H. S. Althagafy et al. / Bioorg. Med. Chem. 21 (2013) 3919–3926
previously.12 Each IC50 value was evaluated via the measurement
of three replicates over nine different concentrations, ranging from
inhibition of each flavonolignan/7-O-methylated analogue and
comparisons between flavonolignan and 7-O-methylated analogue
0 to 62.5
l
M, with the IC50 values calculated by linear regression
at 100 lM were evaluated by two-way analysis of variance (ANO-
using GraphPad Prism.
VA) with Bonferroni adjustment for multiple comparisons; p <0.05
was considered significant.
4.3.2. Antiviral assay
Acknowledgements
Cells (150,000) 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 multiplicity of infection of 0.05 focus forming units per
cell. Virus inocula were removed 5 h post-infection and replaced
with fresh medium containing test compounds. Protein lysates
were harvested 72 h later, and HCV proteins were detected by
Western blot analyses as described previously.12 Importantly, total
protein content of each sample was determined, and equal
amounts of total protein lysates were run on protein gels prior to
Western blotting.
This research was supported by the National Institutes of
Health by both the National Institute of General Medical Sciences
via Grant R01 GM077482 and the National Center for Complemen-
tary and Alternative Medicine via Grant R01 AT006842. H.S.A. was
supported by the Ministry of Higher Education of Saudi Arabia,
King Abdullah Scholarships Program.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2013.04.017.
4.3.3. Inhibition of drug metabolizing enzymes
Milk thistle flavonolignans and 7-O-methylated analogues were
evaluated as inhibitors of CYP2C9, CYP3A4/5, and UDP-glucurono-
syltransferase (UGT) activity using the probe substrates (S)-warfa-
rin, midazolam, and 4-methylumbelliferone (4-MU), respectively.
Inhibition of CYP2C9 activity. Incubation mixtures consisted of
pooled HLM (0.1 mg/mL microsomal protein), (S)-warfarin
References and notes
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M. Antivir. Ther. 2013, 18, 141.
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10. Sy-Cordero, A. A.; Graf, T. N.; Runyon, S. P.; Wani, M. C.; Kroll, D. J.; Agarwal, R.;
Brantley, S. J.; Paine, M. F.; Polyak, S. J.; Oberlies, N. H. Bioorg. Med. Chem. 2013,
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11. Graf, T. N.; Wani, M. C.; Agarwal, R.; Kroll, D. J.; Oberlies, N. H. Planta Med. 2007,
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12. Polyak, S. J.; Morishima, C.; Lohmann, V.; Pal, S.; Lee, D. Y.; Liu, Y.; Graf, T. N.;
Oberlies, N. H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 5995.
13. Deep, G.; Raina, K.; Singh, R. P.; Oberlies, N. H.; Kroll, D. J.; Agarwal, R. Int. J.
Cancer 2008, 123, 2750.
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Drug Metab. Dispos. 2006, 34, 880.
19. Zhong, J.; Gastaminza, P.; Cheng, G.; Kapadia, S.; Kato, T.; Burton, D. R.;
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(4
100
CYP2C9 inhibitor sulfaphenazole (1
l
M), flavonolignan or 7-O-methylated analogue (10 or
M), and potassium phosphate buffer (100 mM, pH 7.4). The
M) was used as a positive
l
l
control. Control incubation mixtures contained 0.75% MeOH (v/v)
in place of flavonolignan/7-O-methylated flavonolignan or sulfa-
phenazole. Incubation mixtures were analyzed for 7-hydroxywar-
farin by LC/MS–MS as described previously.20,21 Inhibition of
CYP3A4/5 activity. Incubation mixtures consisted of pooled HIM
(0.05 mg/mL microsomal protein), midazolam (4
lM), flavonolign-
ans or 7-O-methylated analogue (10 or 100 M), and potassium
l
phosphate buffer (100 mM, pH 7.4) supplemented with magne-
sium chloride (3.3 mM). The CYP3A4/5 inhibitor ketoconazole
(1 lM) was used as a positive control. Control incubation mixtures
contained 0.1% DMSO (v/v) in place of flavonolignan/7-O-methyl-
ated flavonolignan or ketoconazole. Incubation mixtures were ana-
lyzed for 10-hydroxymidazolam by LC/MS–MS as described
previously.22,23 Inhibition of UGT activity. Incubation mixtures con-
sisted of pooled HLM (0.4 mg/mL microsomal protein), 4-MU
(100
100
(0.1 M, pH 7.5) supplemented with magnesium chloride (5 mM).
The UGT inhibitor diclofenac (400 M) was used as a positive con-
l
M), flavonolignan or 7-O-methylated analogue (10 or
lM), bovine serum albumin (BSA, 0.05%), and Tris–HCl buffer
20. Brantley, S. J.; Oberlies, N. H.; Kroll, D. J.; Paine, M. F. J. Pharmacol. Exp. Ther.
2010, 1081, 332.
l
trol. Control incubation mixtures contained 0.2% MeOH (v/v) in
place of flavonolignan/7-O-methylated flavonolignan or diclofenac.
Incubation mixtures were analyzed for 4-MU depletion by fluores-
cence as described previously.24
Data were analyzed statistically using SigmaStat (version 3.5;
Systat Software, Inc., San Jose, CA) and are presented as
means SDs of triplicate incubations. Concentration-dependent
21. Ngo, N.; Brantley, S. J.; Carrizosa, D. R.; Kashuba, A. D.; Dees, E. C.; Kroll, D. J.;
Oberlies, N. H.; Paine, M. F. J. Exp. Pharmacol. 2010, 2, 83.
22. Wang, M. Z.; Wu, J. Q.; Bridges, A. S.; Zeldin, D. C.; Kornbluth, S.; Tidwell, R. R.;
Hall, J. E.; Paine, M. F. Drug Metab. Dispos. 2007, 35, 2067.
23. Ngo, N.; Yan, Z.; Graf, T. N.; Carrizosa, D. R.; Kashuba, A. D.; Dees, E. C.; Oberlies,
N. H.; Paine, M. F. Drug Metab. Dispos. 2009, 37, 514.
24. Collier, A. C.; Tingle, M. D.; Keelan, J. A.; Paxton, J. W.; Mitchell, M. D. Drug
Metab. Dispos. 2000, 28, 1184.