O-Aminoalkyl-O-trimethyl-2,3-dehydrosilybins: Synthesis and in vitro effects towards prostate cancer cells

Article abstract of DOI:10.3390/molecules23123142

As part of our ongoing silybin project, this study aims to introduce a basic nitrogen-containing group to 7-OH of 3,5,20-O-trimethyl-2,3-dehydrosilybin or 3-OH of 5,7,20-O-trimethyl- 2,3-dehydrosilybin via an appropriate linker for in vitro evaluation as

Full text of DOI:10.3390/molecules23123142

molecules
Article
O-Aminoalkyl-O-Trimethyl-2,3-Dehydrosilybins:
Synthesis and In Vitro Effects Towards Prostate
Cancer Cells
Bao Vue 1, Sheng Zhang 1, Andre Vignau 1, Guanglin Chen 1, Xiaojie Zhang 1, William Diaz 1,
Qiang Zhang 2, Shilong Zheng 2, Guangdi Wang 2 and Qiao-Hong Chen 1,
*
1
Department of Chemistry, California State University, Fresno, 2555 E. San Ramon Avenues, M/S SB70,
Fresno, CA 93740, USA; baov@csufresno.edu (B.V.); shengzhang2014@mail.fresnostate.edu (S.Z.);
andre1357@mail.fresnostate.edu (A.V.); chen.bmc@gmail.com (G.C.); zhangxiaojie87@gmail.com (X.Z.);
diazwill101@mail.fresnostate.edu (W.D.)
Department of Chemistry and RCMI Cancer Research Center, Xavier University of Louisiana, 1 Drexel Drive,
New Orleans, LA 70125, USA; qzhang1@xula.edu (Q.Z.); szheng@xula.edu (S.Z.); gwang@xula.edu (G.W.)
Correspondence: qchen@csufresno.edu; Tel.: +1-559-278-2394
2
*
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
Received: 31 October 2018; Accepted: 29 November 2018; Published: 29 November 2018
ꢀꢁꢂꢃꢄꢅꢆ
Abstract: As part of our ongoing silybin project, this study aims to introduce a basic
nitrogen-containing group to 7-OH of 3,5,20-O-trimethyl-2,3-dehydrosilybin or 3-OH of
5,7,20-O-trimethyl- 2,3-dehydrosilybin via an appropriate linker for in vitro evaluation as potential
anti-prostate cancer agents. The synthetic approaches to 7-O-substituted-3,5,20-O-trimethyl-
2,3-dehydrosilybins through a five-step procedure and to 3-O-substituted-5,7,20-O-trimethyl-2,3-
dehydrosilybins via a four-step transformation have been developed. Thirty-two nitrogen-containing
derivatives of silybin have been achieved through these synthetic methods for the evaluation of
their antiproliferative activities towards both androgen-sensitive (LNCaP) and androgen-insensitive
prostate cancer cell lines (PC-3 and DU145) using the WST-1 cell proliferation assay. These derivatives
exhibited greater in vitro antiproliferative potency than silibinin. Among them, 11
were identified as five optimal derivatives with IC50 values in the range of 1.40–3.06
a 17- to 52-fold improvement in potency compared to silibinin. All these five optimal derivatives can
,
29
, 31, 37, and 40
µM, representing
arrest the PC-3 cell cycle in the G0/G1 phase and promote PC-3 cell apoptosis. Derivatives 11
, 37,
and 40 are more effective than 29 and 31 in activating PC-3 cell apoptosis.
Keywords: silybin; prostate cancer; 2,3-dehydrosilybin; cell proliferation; cell apoptosis
1. Introduction
Silybin ((
mixture of two diastereomers of silybin A and silybin B with opposite configurations at C-10 and
C-11 [ ]. They are hard to separate by conventional purification methods, but can be separated by the
HPLC method [ ]. Silybin A and silybin B biogenetically originate from a taxifolin moiety (flavonoid)
and a coniferyl alcohol unit (lignan), which was presumed to follow a non-stereoselective radical
coupling reaction [ ]. The mixture of diastereoisomeric silybin A and silybin B was originally thought
to be a pure compound and named silybin [ ]. Later, it was re-termed as silibinin to emphasize that
it is a mixture [ ]. In this article, silibinin was thus used to represent the mixture of silybin A and
silybin B. Silibinin ( ) is the first and well-studied member of flavonolignans [ ] and the key chemical
and medicinal component of milk thistle (Silybum marianum L. Gaertner, Asteraceae) [ ]. The earliest
record on the medicinal merit of Milk thistle for preventing and treating various hepatotoxicity
1), Figure 1), also known as silibinin, exists in nature as an approximately equimolar
1
2
3
4
5
1
4
6
is Hieronymus Bock’s book published in 1539 [
7,
8]. Milk thistle and silibinin are now attractive
Molecules 2018, 23, 3142; doi:10.3390/molecules23123142
www.mdpi.com/journal/molecules

Molecules 2018, 23, 3142
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to scientists, not only for their well-known chemotherapeutic use for hepatotoxicity in Europe [
9],
but also for their potential in treating and preventing cancers [ 10 12]. Silymarin (crude extract of
5, –
milk thistle), silibinin (diastereomeric mixture), silybin A (optically pure), and silybin B (optically pure)
have been demonstrated by various in vitro cell-based and in vivo animal-based assays to possess
therapeutic potential in treating prostate cancer [12–14]. The detailed mechanism of silibinin and
structurally-related flavonolignans as anti-prostate cancer agents has previously been summarized by
us in a review article [15]. Briefly, this group of natural products can arrest the prostate cancer cell cycle
at G1 phase and induce cancer cell apoptosis. Also, they have been demonstrated to inhibit the secretion
of prostate cancer specific antigen (PSA) and androgen receptor (AR). They have been revealed to
possess anti-angiogenesis properties by decreasing the density of prostate tumor microvessels and
VEGF immunoreactivity. Additionally, silibinin and structurally-related compounds can suppress
prostate cancer cell migration and invasion through down-regulating the vimentin protein and MMP-2
mRNA, up-regulating E-cadherin expression, and reversing the epithelial-to-mesenchymal transition
(EMT). Additionally, the non-toxic profiles of silibinin have been confirmed by its long-term use
as a dietary supplement and a Phase I clinical trial of silybin-phytosome, a formula of silibinin, at
a dose of 13 g/day [16]. However, the development of silibinin as an anti-prostate cancer drug
is hindered, at least partly, by its moderate potency, with its IC50 values of 40–106
µM in prostate
cancer cell models [12 13 15 17] and by its poor pharmacokinetic profiles [16]. Structural modification
,
,
,
of silibinin can serve as a viable strategy to enhance its potency. Methylated silybins have been
reported to be capable of increasing antiproliferative activities towards prostate cancer cells [18].
Additionally, 2,3-dehydrosilybin has been shown to be a significantly better anticancer agent than
silibinin [19]. Structural manipulations on the phenolic and alcoholic hydroxyl groups of silibinin and
2,3-dehydrosilybin have been applied to overcome their pharmacokinetic limitations [20–22].
Figure 1. Structures of silibinin and derivatives.
Our previous studies on the structure-activity relationships of silibinin revealed that the
antiproliferative potency of 2,3-dehydrosilybin in three prostate cancer cell models could be
further improved through introducing a suitable alkyl group on 7-OH and 3-OH, as exemplified
by 7-O-ethyl-2,3-dehydrosilybin (3) and 3-O-propyl-2,3-dehydrosilybin (4) (Figure 1) [23,24].
This encouraged us to further investigate the effects of nitrogen-containing groups on 7-OH
and 3-OH of 2,3-dehydrosilybin on the biological profiles in prostate cancer cell models.
Consequently, this study started with the development of general synthetic approaches to
7-O-substituted-3,5,20-O-trimethyl-2,3-dehydrosilybins and 3-O-substituted-5,7,20-O-trimethyl-2,3-
dehydrosilybins, followed by the synthesis of thirty-two new derivatives of silibinin,
including six 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybins and 26 3-O-aminoalkyl-5,7,20-O-
trimethyl-2,3-dehydrosilybins. Additionally, the phenolic hydroxyl groups in all synthetic derivatives
were converted to methoxyl groups. This conversion was expected to overcome, to some degree,
pharmacokinetic limitations caused by the phenolic hydroxyl groups and to pave an avenue to the
selective incorporation of a basic nitrogen-containing group into the phenolic hydroxyl group at either
C-7 or C-3. The in vitro anticancer activities of these derivatives have been evaluated in three prostate

Molecules 2018, 23, 3142
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cancer cell models. The design, synthesis, antiproliferative activity, and structure-activity relationships
of these silibinin derivatives are presented in this paper. The cell apoptosis induction and cell cycle
regulation by five representative derivatives are also reported.
2. Results and Discussion
2.1. Chemistry
2.1.1. Synthesis of 7-O-Aminoalkyl-3,5,20-O-Trimethyl-2,3-Dehydrosilybins (9–14)
Some oxidative conversions of silibinin to 2,3-dehydrosilybin have been reported [25
As illustrated in Scheme 1, the synthesis of 7-O-substituted-2,3-dehydrosilybins ( 14) started
with selective benzylation (81%) of the C-7 phenolic hydroxyl group of silibinin, according to the
procedure reported by Kren et al. and us [23 28]. It is worth noting that anaerobic conditions are
essential to achieving high yields for the selective benzylation. This is because the simultaneous
presence of a base and air led to the aerobic oxidation of silibinin to 2,3-dehydrosilybin [ 24
and the 3-OH in 2,3-dehydrosilybin is readily benzylated or alkylated [ 24 28], which has been
rationalized by the electrochemistry measurements and bond dissociation energy calculations [29].
7-O-Benzyl-3,5,20-O-trimethyl-2,3-dehydrosilybin ( ) was then achieved by the one-pot reaction
of the base-mediated oxidation of 7-O-benzylsilybin ( ), followed by trimethylation of the
corresponding 7-O-benzyl-2,3-dehydrosilybin. 3,5,20-O-Trimethyl-2,3-dehydrosiliybin ( ) was
obtained by debenzylation of aryl benzyl ether using ammonium formate as the hydrogen
source, catalyzed by palladium on carbon. 7-O-bromopropyl-3,5,20-O-trimethyl-2,3-dehydrosilybin
) was prepared by O-alkylating with 1,3-dibromopropane mediated by potassium carbonate.
–27].
9–
,
8, ]
8
, ,
6
5
7
6
(8
7
7-O-Aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybins (
bromoalkyl side chain of 8 with the appropriate amine.
9–14) were achieved by N-alkylation of the
Scheme 1. Synthesis of 7-O-aminopropyl-3,5,20-O-trimethyl-2,3-dehydrosilybins (9–14). Reagents and
conditions: i. K2CO3 (4 equiv.), BnBr (1 equiv.), acetone (0.1 M), reflux overnight, 80%; ii. (a) K2CO3
(3 equiv.), DMF (0.5 M), rt, 3 h; (b) MeI (6 equiv.), rt, overnight, 48%; iii. HCOONH4 (10 equiv.), Pd/C
(10%, w/w), MeOH (0.2 M), reflux, overnight, 67%; iv. 1,3-dibromopropane (4 equiv.), K2CO3 (4 equiv.),
◦
DMF (1 M), 60 C, overnight; v. amine (3 equiv.), K2CO3 (3 equiv.), acetone (0.1 M), reflux, overnight, 40%.

Molecules 2018, 23, 3142
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2.1.2. Synthesis of 3-O-Aminoalkyl-5,7,20-O-Trimethyl-2,3-Dehydrosilybins (20–45)
3-O-Substituted-2,3-dehydrosilybins (20
as shown in Scheme 2. Specifically, 5,7,20-O-trimethylsilybin (15) was achieved by treating
silibinin ( ) with dimethylsulfate in the presence of potassium carbonate under strictly anaerobic
–45) were synthesized following a four-step procedure,
1
conditions. Note that a small amount of 3,5,7,20-O-tetramethyl-2,3-dehydrosilybin can be
formed if anaerobic conditions are not well-controlled, which would complicate the purification
process and decrease the yield. Even though 5,7,20-O-tribenzylsilybin was much easier to
aerobically oxidize than that in silibinin [30], the oxidation of 5,7,20-O-trimethylsilybin (15
)
under the same conditions led to a mixture of products instead of the desired oxidation product.
After several trials with different oxidation conditions, 5,7,20-O-trimethyl-2,3-dehydrosilybin (16
)
was eventually obtained by the oxidation of 15 with sodium hydroxide and hydrogen peroxide.
A 10–14 h reaction time serves as a critical factor for the optimal yield (40–55%) of this oxidation
reaction. We also found that this oxidation cannot be quenched with hydrochloric acid because
it selectively demethylated the 5-OMe of the product. 5,7,20-O-Trimethyl-2,3-dehydrosilybin
(16) was then converted to 3-O-bromoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybins (17
–19)
via O-alkylation with the appropriate dibromoalkanes, using potassium carbonate
as the base and DMF as the aprotic solvent.
3-O-bromoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybins (17
The subsequent N-alkylation of the
19) with the corresponding amine
–
furnished the respective 3-O-aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybin (20–45).
Scheme 2. Synthesis of 3-O-aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybins (20–45). Reagents and
conditions: i. dimethyl sulfate (8 equiv.), K2CO3 (8 equiv.), acetone, argon, reflux 4 h; ii. H2O2 (30%),
NaOH (16%), methanol/THF, rt overnight; iii. Dibromoalkane (10 equiv.), K2CO3 (6 equiv.), DMF,
rt 24–48 h; iv. Amine (16 equiv.), K2CO3 (6 equiv.), acetone, rt 48 h.
2.1.3. Structure Determination
The 1H-NMR and 13C-NMR data of
5 are consistent with those reported in the literature [28].
The structure of
6 was characterized by the presence of three single signals at 3.94, 3.924, and 3.916
1
in its H-NMR spectrum and at 60.1, 56.6, and 56.2 in its 13C-NMR spectrum for three additional
1
methoxyl groups when compared with
H-3 of . The structure of
5
and the absence of the H-NMR signal at 4.52 for the
1
5
7
was established based on the absence of the H-NMR signals at

Molecules 2018, 23, 3142
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7.48–7.34 (m, 5H) and 5.12 (s, 2H) for the benzyl group of
6
. The signals in the 1H-NMR spectrum
[4.17 (t, J = 5.7 Hz, 2H), 3.61 (t, J = 6.3 Hz, 2H), 2.36 (quin, J = 6.0 Hz, 2H)] of compound
confirmed the addition of a 3-bromopropyl group when compared with . The structures of the
six 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybins ( 14) were characterized by interpreting
8
7
9–
their NMR, HRMS, and FTIR data (for details see Experimental Section and Supporting Information).
Specifically, the presence of the 1H-NMR and 13C-NMR signals for each alkylamine group suggests
the incorporation of the corresponding alkylamine group, which was corroborated by the HRMS data
1
for each compound. H-NMR and 13C-NMR data for
9–14 were assigned (Experimental Section and
Supporting Information) by comparing them with the NMR data of 7-O-propyl-2,3-dehydrosilybin
because derivatives 14 and 7-O-propyl-2,3-dehydrosilybin share an identical core structure and
9
–
all NMR signals of 7-O-propyl-2,3-dehydrosilybin have been fully assigned by us, according to the
interpretation of its COSY, HMQC, and HMBC data [23].
The structure of 15 was determined by the existence of three single signals for three additional
1
methoxyl groups at 3.91, 3.90, and 3.90 in its H-NMR spectrum when compared with silibinin.
1
The structure of 16 was confirmed by the absence of the H-NMR signal at 4.42 (1H) for H-3
of 15
.
The signals in the 1H-NMR spectra [4.14 (t, 2H), 3.60 (t, 2H), 2.31–2.26 (m, 2H) of
17; 4.14 (t, 2H), 3.46 (t, 2H), 2.09–2.00 (m, 2H), 1.90–1.81 (m, 2H) of 18; 4.02 (t, 2H), 3.34
(t, 2H), 1.85 (quin, 2H), 1.74 (quin, 2H), 1.59–1.49 (m, 2H) of 19] confirmed the addition of an
appropriate bromoalkyl group to each of 17
3-O-aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybins (20
–
19 when compared with 16. The structures of the 26
45) were characterized by interpreting
–
their NMR, HRMS, and FTIR data (for details, see Experimental Section and Supporting Information).
Specifically, the presence of the 1H-NMR and 13C-NMR signals for each alkylamine group suggests the
incorporation of the corresponding alkylamine group, which was further supported by the HRMS data
1
for each compound. H-NMR and 13C-NMR data for 20
–
45 were assigned (Experimental Section and
),
possess an identical core structure and all NMR
have been fully assigned by us, according to the interpretation of its COSY,
Supporting Information) by comparing them with the NMR data of 3-O-propyl-2,3-dehydrosilybin (
due to the fact that derivatives 20 45 and compound
signals of compound
4
–
4
4
HMQC, and HMBC data [24].
2.2. Antiproliferative Activity towards Prostate Cancer Cell Lines and Structure-Activity Relationships
The in vitro antiproliferative activities of six 7-O-substituted and 26 3-O-substituted
silybin derivatives were evaluated using the WST-1 cell proliferation assay, according
to the procedure as described in the Experimental Section in both androgen-sensitive
(LNCaP) and androgen-insensitive (PC-3 and DU145) human prostate cancer cell lines.
Silibinin and docetaxel were used as a positive control for comparison in the parallel
experiments and the IC50 values calculated from the dose-response curves are summarized
in Table 1. Clearly, 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybins and 3-O-aminoalkyl-
5,7,20-O-trimethyl-2,3-dehydrosilybins are more potent in suppressing both androgen-sensitive and
androgen-insensitive prostate cancer cell proliferation than silibinin. This conclusion is supported
by the following data: i) the optimal 7-O-substituted derivative (11) is 52-, 51-, and 24-fold more
potent than silibinin toward PC-3, DU145, and LNCaP prostate cancer cell lines, respectively; and
ii) the optimal 3-O-substituted derivatives (29
more potent than silibinin against PC-3, DU145, and LNCaP prostate cancer cell lines, respectively.
Furthermore, the most potent derivatives 11 29 31 37, and 40 are more active than 2,3-dehydrosilybin
), 7-O-ethyl-2,3-dehydrosilybin ( ), and 3-O-propyl-2,3-dehydrosilybin ( ) in suppressing DU145
prostate cancer cell proliferation. Additionally, the dibutylamino group in derivatives 11 29 and 31
the morpholino moiety in 37; and the piperidino unit in 40, are the favorable nitrogen-containing
, 31, 37, and 40) are 26–27, 31–37 and 17–22 times
,
,
,
(2
3
4
,
;
groups for the greater potency. A three-carbon linker in 11 and 29, as well as a five-carbon linker in 31
37 and 40, are beneficial to the potency.
,

Molecules 2018, 23, 3142
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Table 1. Anti-proliferative activity of dialkylaminoalkyl-2,3-dehydrosilybins.
IC50 (µM) a
DU145 c
IC50 (Silibinin)/IC50(Derivative)
Compound
Docetaxel
DU145 c
LNCaP d
PC-3 b
LNCaP d
PC-3b
0.0019 ± 0.0006 0.0012 ± 0.0003 0.0002 ± 0.0001
-
-
-
Silibinin
2 [23]
3 [24]
4 [24]
8
72.65 ± 3.15
9.45 ± 0.56
3.25 ± 0.31
1.71 ± 0.45
26.09 ± 3.58
42.64 ± 6.61
9.92 ± 0.43
1.40 ± 0.17
25.05 ± 1.00
26.47 ± 1.00
25.65 ± 3.39
5.76 ± 1.36
9.09 ± 1.48
5.15 ± 2.13
3.47 ± 2.23
5.16 ± 0.94
5.76 ± 1.36
6.41 ± 0.40
2.03 ± 0.69
3.77 ± 0.41
3.30 ± 0.86
2.73 ± 0.12
3.77 ± 0.41
2.84 ± 0.10
2.86 ± 0.79
7.49 ± 1.98
2.95 ± 0.76
2.37 ± 0.70
24.09 ± 10.55
2.71 ± 0.23
2.94 ± 0.13
3.49 ± 0.24
2.72 ± 0.08
9.98 ± 4.94
5.30 ± 0.66
7.87 ± 1.47
8.45 ± 2.89
4.72 ± 0.88
93.34 ± 13.67
11.48 ± 1.42
7.59 ± 0.66
11.04 ± 0.68
11.47 ± 2.38
39.64 ± 9.49
8.62 ± 0.32
1.84 ± 0.14
19.59 ± 0.47
45.10 ± 11.53
19.42 ± 0.88
8.13 ± 0.42
32.71 ± 5.32
9.97 ± 3.34
6.73 ± 0.45
9.21 ± 0.37
8.18 ± 0.42
6.64 ± 0.54
8.39 ± 1.38
5.39 ± 0.53
5.78 ± 1.36
2.51 ± 0.04
3.07 ± 0.51
2.85 ± 0.23
14.12 ± 2.79
19.84 ± 2.38
5.90 ± 1.30
7.26 ± 1.12
66.96 ± 13.65
2.69 ± 0.03
10.60 ± 0.63
6.36 ± 0.37
3.06 ± 0.13
>50
43.73 ± 10.90
3.09 ± 1.30
2.58 ± 0.07
2.07 ± 0.18
5.71 ± 2.13
12.08 ± 1.81
7.49 ± 0.16
1.82 ± 0.14
11.00 ± 0.99
12.72 ± 6.28
16.35 ± 3.47
5.19 ± 2.08
20.69 ± 5.34
5.13 ± 0.89
5.07 ± 0.52
3.09 ± 0.15
5.19 ± 2.08
6.33 ± 0.34
3.89 ± 0.54
4.02 ± 1.55
2.70 ± 0.47
2.21 ± 0.17
3.84 ± 0.51
2.51 ± 0.31
5.89 ± 0.30
8.06 ± 1.44
3.92 ± 1.67
2.28 ± 0.42
27.30 ± 5.42
1.99 ± 0.23
1.74 ± 0.97
2.84 ± 0.22
2.23 ± 0.18
11.41 ± 5.91
4.58 ± 1.77
14.39 ± 7.49
12.80 ± 7.80
5.66 ± 0.44
-
8
22
42
3
2
7
52
3
3
-
8
12
8
8
2
11
51
5
2
5
11
3
9
14
10
8
14
11
17
16
37
30
33
7
-
14
17
21
8
4
6
24
4
3
3
8
9
10
11
12
13
14
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
3
13
8
2
9
9
14
21
14
11
11
36
19
22
27
19
26
25
10
25
31
3
27
25
21
27
7
14
9
9
14
13
7
11
11
16
20
11
17
7
5
5
16
13
1
35
9
15
31
<2
14
2
11
19
2
22
25
15
20
4
6.85 ± 0.77
48.12 ± 16.27
18.57 ± 7.67
8.59 ± 2.23
10
3
3
5
11
45
15
8
a
IC50 value is the compound concentration effective at inhibiting 50% of the cell viability measured by the
WST-1 cell proliferation assay after three days of exposure. The data are presented as the mean
±
SD from
b
n = 3. Human androgen-insensitive prostate cancer cell line derived from bone metastasis of a prostate tumor.
c
d
Human androgen-insensitive prostate cancer cell line derived from brain metastasis of a prostate tumor. Human
androgen-sensitive prostate cancer cell line derived from lymph node metastasis of a prostate tumor.
2.3. Antiproliferative Activity towards MCF 10A and PWR-1E Non-Neoplastic Human Epithelial Cell Lines
Silibinin and five potent derivatives (11, 29, 31, 37, and 40) were selected for further
evaluation against the MCF 10A non-neoplastic human mammary epithelial cell line and PWR-1E
non-neoplastic human prostate epithelial cells. The five derivatives were chosen based on the
following grounds: 11 is the most potent 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybin
considering its overall potency towards three prostate cancer cell models; 29, 31, 37, and 40
are the most promising 3-O-aminoalkyl-5,7,20-O-trimethyl-2,3-dehydrosilybins. As shown in
Table 2, silibinin demonstrates a significantly higher capability of suppressing non-neoplastic cell
(MCF 10A and PWR-1E) proliferation than prostate cancer cell proliferation. Four 3-O-aminoalkyl-

Molecules 2018, 23, 3142
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5,7,20-O-trimethyl-2,3-dehydrosilybins (29
,
31
,
37, and 40) did not exhibit significantly different
responses to prostate cancer cells and to non-neoplastic MCF 10A and PWR-1E cells. However,
the 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybin 11 does not demonstrate apparent
antiproliferative activity towards PWR-1E and MCF 10A non-neoplastic epithelial cells up to a 50 µM
concentration. Consequently, 7-O-aminoalkyl-3,5,20-O-trimethyl-2,3-dehydrosilybin 11 illustrates a
good selectivity of inhibiting prostate cancer cell proliferation over non-neoplastic MCF 10A and
PWR-1E human epithelial cell proliferation (Table 2), which suggests that modification at 7-OH of
3,5,20-O-trimethyl-2,3-dehydrosilybin only improves the antiproliferative potency towards prostate
cancer cells, and not non-neoplastic epithelial cells.
Table 2. Antiproliferative activity of selected derivatives against MCF 10A and PWR-1E cells.
IC50 (µM) a
Compound
PC-3
DU145
LNCaP
MCF 10A
PWR-1E
Silibinin
72.65 ± 3.15
1.40 ± 0.17
2.73 ± 0.12
2.84 ± 0.10
2.71 ± 0.23
2.72 ± 0.08
93.34 ± 13.67
1.84 ± 0.14
2.51 ± 0.04
2.85 ± 0.23
2.69 ± 0.03
3.06 ± 0.13
43.73 ± 10.90
1.82 ± 0.14
2.21 ± 0.17
2.51 ± 0.31
1.99 ± 0.23
2.23 ± 0.18
23.84 ± 0.96
20.45 ± 4.09
11
29
31
37
40
>50
<5
<5
<5
<5
>50
<5
<5
<5
<5
a
IC50 is the drug concentration effective at inhibiting 50% of the cell viability measured by the WST-1 cell
proliferation assay after three days of exposure. The data are presented as the mean ± SD from n = 3.
2.4. Cell Cycle Regulation and Cell Apoptosis
Silibinin can arrest the rat (H-7 and I-8) and human prostate cancer cell (LNCaP) cycle at the
G1 phase [31
,32], and cause G1 and G2-M PC-3 prostate cancer cell cycle arrest [33]. Five optimal
derivatives, consisting of 11
,
29 31 37, and 40, were selected for flow cytometry evaluation of their
,
,
effect on PC-3 cell cycle regulation because they exhibited optimal cell proliferation inhibition on
both androgen-dependent LNCaP and androgen-independent PC-3 prostate cancer cell models, with
≤
3.0 µM IC50 values. At 20 µM, all these five derivatives can cause PC-3 cell accumulation in a G0/G1
phase by increasing the cell population in this phase at 16 h from 55.7% (control) to 66.3% (treated with
11), from 36.2% (control) to 50.3% (treated with 29), from 33.1% (control) to 35.9% (treated with 31),
from 33.1% (control) to 34.6% (treated with 37), and from 33.1% (control) to 43.4% (treated with 40).
Silibinin was revealed by Agarwal and co-workers to activate cell apoptosis in PC-3 tumor
xenografts [34]. An F2N12S and SYTOX AADvanced double staining flow cytometry-based assay was
used to discriminate PC-3 cells dying from apoptosis from those dying from necrosis in response to
various concentrations of derivatives 11
compound for 16 h. As shown in Figure 2, derivatives 11
apoptotic cell death in the androgen-insensitive PC-3 prostate cancer cell line in a dose-responsive
manner after 16 h of treatment. Specifically, 5 M of derivatives 11 37, and 40 could induce a
substantial early phase of apoptosis (26–59%) in PC-3 cells compared with control cells: treatment
with 10 M of these three optimal derivatives led to 56–76% early apoptotic cells and 6–40% late
apoptotic/necrotic cells; 20 M of derivatives 11 37, and 40 also activated notable apoptosis, with
54–75% early apoptotic cells and 16–44% late apoptotic/necrotic cells. The apoptotic cell population
reached its maximum when PC-3 cancer cells were exposed to derivative 11 37, and 40 at 5 M, 10 M,
and 30 M, respectively. In contrast, derivatives 29 and 31 did not induce significant levels of apoptotic
cell death (less than 10%) up to a 10 M concentration. Only 50 M of derivatives 29 and 31 resulted
in the maximum apoptotic cell population (71% and 95%, respectively).
,
29
,
31,
37, and 40. PC-3 cells were incubated with the test
,
37, and 40 induced appreciable levels of
µ
,
µ
µ
,
,
µ
µ
µ
µ
µ

Molecules 2018, 23, 3142
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Figure 2. Apoptosis in PC-3 cells treated with 11
, 29, 31, 37, and 40 at 5 µM, 10 µM, and 20 µM (by
F2N12S and SYTOX AADvanced double staining).

Molecules 2018, 23, 3142
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3. Materials and Methods
3.1. General Procedures
HRMS were obtained on an Orbitrap mass spectrometer with electrospray ionization (ESI)
(Thermo Fisher Scientific, Waltham, MA, USA). NMR spectra were obtained on a Bruker Fourier
300 spectrometer (Billerica, MA, USA) in CDCl3, or DMSO-d6. The chemical shifts are given in ppm
referenced to the respective solvent peak, and coupling constants are reported in Hz. Anhydrous THF
and dichloromethane were purified by the PureSolv MD 7 Solvent Purification System from Innovative
Technologies (MB-SPS-800) (Herndon, VA, USA). All other reagents and solvents were purchased from
commercial sources and were used without further purification. Silica gel column chromatography
was performed using silica gel (32–63 µm) (SiliCycle Inc, Quebect, QC, Canada). Preparative thin-layer
chromatography (PTLC) separations were carried out on thin layer chromatography plates loaded
with silica gel 60 GF254 (EMD Millipore Corporation, MA, USA). Silibinin (>98.0%) was purchased
from Fisher Scientific (TCI America, Portland, OR, USA, Cat # 50-014-46874).
3.2. Synthesis of 7-O-Benzylsilybin (5)
Following the procedure described in the literature [23,28], 7-O-benzylsilybin (5) was prepared
from silibinin in an 80% yield as a light yellow solid. m.p. 93–95 ◦C. IR (film) νmax: 3432, 2937, 1634,
1
1571, 1507 cm−1; H-NMR (300 MHz, CDCl3):
δ 11.25 (11.24) (s, 1H), 7.43–7.34 (m, 5H), 7.19 (dd,
J = 4.2, 1.5 Hz, 1H), 7.10–7.01 (m, 2H), 6.97–6.88 (overlapped, 3H), 6.21 (d, J = 1.8 Hz, 1H), 6.13 (6.12)
(d, J = 2.4 Hz, 1H), 5.96 (br.s, 1H), 5.07 (s, 2H), 4.99 (d, J = 11.7 Hz, 1H), 4.93 (d, J = 8.4 Hz, 1H), 4.52 (dd,
J = 11.7, 3.3 Hz, 1H), 4.09–3.99 (m, 1H), 3.89 (s, 3H), 3.80 (dd, J = 12.3, 2.1 Hz, 1H), 3.55 (dd, J = 12.3,
3.6 Hz, 1H); 13C-NMR (75 MHz, CDCl3):
δ 196.2, 167.8, 163.6, 162.8, 147.0, 146.4, 144.1, 143.9, 135.6,
129.5, 128.8, 128.4, 127.9, 127.5, 121.2 (121.1), 120.8, 117.3 (117.2), 116.6, 114.9, 109.8, 101.1, 96.4, 95.4,
83.0, 78.4, 76.3, 72.4, 70.5, 61.6, 56.1; HRMS-ESI m/z [M + H]+ calcd for C32H29O10: 573.1761, found:
573.1769.
3.3. Synthesis of 7-O-Benzyl-3,5,20-O-Trimethyl-2,3-Dehydrosilybin (6)
Potassium carbonate (3 eq.) was added to a solution of benzylsilybin (1 eq.) in DMF (0.5 M) and
the reaction mixture was exposed to air with stirring at room temperature (or 60 ◦C) for 3 h. When
most of the 7-O-benzylsilybin was oxidized to 7-O-benzyl-2,3-dehydrobenzylsilybin, as monitored
by TLC, the reaction mixture was cooled down to room temperature. Potassium carbonate (3 eq.),
followed by methyl iodide (6 eq.), were added to the reaction mixture and the reaction was allowed
to proceed at room temperature overnight prior to being quenched with HCl (1 M). The subsequent
mixture was diluted with water and extracted with ethyl acetate. The combined organic extracts
were rinsed with brine and dried over anhydrous sodium sulfate. After filtration, the volatile
components were evaporated under vacuum to give the crude product, which was purified by column
chromatography over silica gel or PTLC eluting with 5% methanol in dichloromethane to generate
7-O-benzyl-3,5,20-O-trimethyl-2,3-dehydrosilybin (
6
): 48% yield, yellow solid, m.p. 118–119 ◦C. IR
7.76 (d, J = 2.1 Hz, 1H),
(film) νmax: 3401, 2932, 1601, 1504, 1440 cm−1; 1H-NMR (300 MHz, CDCl3):
δ
7.73 (dd, J = 8.7, 2.1 Hz, 1H), 7.48–7.34 (m, 5H), 7.06 (d, J = 8.7 Hz,1H), 7.04 (dd, J = 8.4, 2.1Hz, 1H),
6.98 (d, J = 1.8 Hz, 1H), 6.93 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 2.1 Hz, 1H), 6.41 (d, J = 2.4 Hz, 1H), 5.12 (s,
2H), 5.02 (d, J = 8.4 Hz,1H), 4.17–4.10 (m,1H), 3.94 (s, 3H), 3.924 (s, 3H), 3.916 (s, 3H), 3.89 (s, 3H), 3.85
(dd, J = 12.3, 2.1 Hz, 1H), 3.58 (dd, J = 12.6, 3.9 Hz, 1H); 13C-NMR (75 MHz, CDCl3):
δ 174.2, 163.1,
161.2, 158.8, 152.1, 150.0, 149.6, 145.3, 143.8, 141.6, 135.8, 128.9, 128.6, 128.4, 127.7, 124.5, 122.4, 120.3,
117.3, 117.2, 111.5, 110.3, 109.8, 96.5, 93.4, 78.8, 76.5, 70.6, 61.8, 60.1, 56.6, 56.2, 56.1; HRMS-ESI m/z
[M + H]+ calcd for C35H33O10: 613.2074, found: 613.2071.

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3.4. Synthesis of 3,5,20-O-Trimethyl-2,3-Dehydrosilybin (7)
To the solution of 7-O-benzyl-3,5,20-O-trimethyl-2,3-dehydrosilybin (
6
, 1 eq.) in methanol (0.2 M),
Pd-C (50% wet, 10% w/w) and ammonium formate (10 eq.) were sequentially added. The reaction
mixture was refluxed overnight under argon. After being cooled to room temperature, the reaction
mixture was filtered through silica gel pad eluting with THF. The filtrate was concentrated under
vacuum to afford 3,5,20-O-trimethyl-2,3-dehydrosilybin (
235–236 ◦C. IR (film) νmax: 3545, 2955, 2924, 2853, 2177, 2159, 2028, 1992, 1978, 1968, 1728, 1593, 1557,
1508 cm−1; 1H-NMR (300 MHz, DMSO-d6):
7.74–7.64 (m, 1H), 7.60–7.55 (m, 2H), 7.13–6.98 (m, 6H),
7) in 67% yield as a light yellow solid. M.p.
δ
6.42 (s, 1H), 6.33 (s, 1H), 5.02 (d, J = 7.8 Hz, 1H), 4.36–4.28 (m, 1H), 3.78 (s, 12H), 3.76 (s, 3H), 3.57 (dd,
J = 12.9, 2.1 Hz, 1H), 3.35 (dd, J = 12.9, 4.8 Hz, 1H). HRMS-ESI m/z [M + H]+ calcd for C28H27O10:
523.1604, found: 523.1598.
3.5. Synthesis of 7-O-(30-Bromo)Propyl-3,5,20-O-Trimethyl-2,3-Dehydrosilybin (8)
To a solution of 3,5,20-O-trimethyl-2,3-dehydrosilybin in DMF (1 M), potassium carbonate (4 eq.)
◦
followed by 1,3-dibromopropane (4 eq.) were added. The reaction mixture was stirred at 60 C
overnight prior to being quenched by the addition of 1 M HCl. The subsequent reaction mixture was
diluted with water and extracted with ethyl acetate. The combined organic extracts were rinsed with
brine, dried over anhydrous sodium sulfate, and concentrated under vacuum to yield a crude product,
which was subjected to column chromatography or PTLC purification over silica gel eluting with 5%
methanol in dichloromethane to afford 7-O-(30-bromo)propyl-3,5,20-O-trimethyl-2,3-dehydrosilybin
(8
) as a yellow solid. m.p. 115–116 ◦C. IR (film) νmax: 2932, 1604, 1506, 1463, 1441, 1345, 1264 cm−1
1H-NMR (300 MHz, CDCl3):
7.76 (d, J = 2.1 Hz, 1H), 7.73 (dd, J = 8.7, 2.4 Hz, 1H), 7.07–6.97 (m, 3H),
;
δ
6.92 (d, J = 8.4 Hz, 1H), 6.48 (d, J = 2.1 Hz, 1H), 6.32 (d, J = 2.1 Hz, 1H), 5.02 (d, J = 8.4 Hz, 1H), 4.20–4.11
(m, 1H), 4.17 (t, J = 5.7 Hz, 2H), 3.95 (s, 3H), 3.92 (s, 3H), 3.91 (s, 3H), 3.89 (s, 3H), 3.85 (dd, J = 12.6,
2.7 Hz, 1H), 3.66-3.56 (m, 1H), 3.61 (t, J = 6.3 Hz, 2H), 2.36 (quin, J = 6.0 Hz, 2H); 13C-NMR (75 MHz,
CDCl3):
δ 174.2, 163.0, 161.2, 158.8, 152.1, 149.9, 149.6, 145.3, 143.8, 141.5, 128.4, 124.4, 122.3, 120.3, 117.3,
117.2, 111.4, 110.2, 109.7, 96.1, 93.0, 78.8, 76.4, 66.0, 61.8, 60.1, 56.5, 56.2, 56.1, 32.1, 29.6; HRMS-ESI m/z
[M + H]+ calcd for C31H32BrO10: 643.1179, 645.1158, found: 643.1173, 645.1151.
3.6. General Procedure for the Synthesis of
7-O-(N,N-Dialkylamino)Propyl-3,5,20-O-Trimethyl-2,3-Dehydrosilybins
Potassium carbonate (3 eq.) and the appropriate amine (3 eq.) were added to a solution of
7-O-(30-bromo)propyl-3,5,20-O-trimethyl-2,3-dehydrosilybin (
8) in dry acetone (0.1 M). The reaction
mixture was refluxed overnight before the removal of acetone under vacuum. The residue was diluted
with water and extracted with ethyl acetate. The combined organic extracts were rinsed with brine,
dried over anhydrous sodium sulfate, and concentrated in vacuo to generate the crude products,
which were subjected to PTLC purification eluting with 10% methanol in dichloromethane to yield the
respective 7-O-(30-amino)propyl-3,5,20-O-trimethyl-2,3-dehydrosilybin.
7-O-(N-Methylaminopropyl)-3,5,20-O-trimethyl-2,3-dehydrosilybin (9). 40% yield, light yellow solid, m.p.
◦
1
125–127 C. IR (film) νmax: 3401, 2928, 1625, 1606, 1507 cm−1; H-NMR (300 MHz, CDCl3):
δ 7.59
(d, J = 2.1 Hz, 1H, H-13), 7.53 (dd, J = 8.7, 2.1 Hz, 1H, H-15), 7.04 (dd, J = 8.1, 1.2 Hz, 1H, H-16), 6.97
(d, J = 1.5 Hz, 1H, H-18), 6.93 (dd, J = 8.7, 2.7 Hz, 2H, H-21 & H-22), 6.22 (d, J = 1.8 Hz, 1H, H-8), 6.18
(d, J = 1.8 Hz, 1H, H-6), 4.99 (d, J = 8.4 Hz, 1H, H-11), 4.17–4.06 (overlapped, 3H, H-10 & 7-OCH2),
3.92 (s, 3H, OCH3), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.80 (s, 3H, OCH3), 3.85–3.80 (overlapped,
1H, H-23), 3.57 (dd, J = 12.9, 3.9 Hz, 1H, H-23), 3.25 (t, J = 6.3 Hz, 2H, 7-O-CH2CH2CH2-), 2.81 (s,
3H, NHCH3), 2.34–2.30 (m, 2H, 7-O-CH2CH2CH2-), 2.01 (d, J = 1.2 Hz, 1H, 23-OH); 13C-NMR (75
MHz, CDCl3):
δ 174.4 (C-4), 162.9 (C-7), 160.4 (C-5), 158.1 (C-8a), 152.6 (C-19), 150.0 (C-20), 149.6
(C-16a), 145.6 (C-2), 143.7 (C-12a), 140.9 (C-3), 128.3 (C-17), 123.5 (C-14), 122.0 (C-15), 120.3 (C-22),
117.2 (C-16), 117.1 (C-13), 111.5 (C-21), 110.4 (C-18), 108.7 (C-4a), 96.3 (C-6), 92.9 (C-8), 78.9 (C-10),

Molecules 2018, 23, 3142
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76.3 (C-11), 65.2 (7-OCH2), 61.6 (C-23), 59.9 (OCH3), 56.8 (OCH3), 56.2 (OCH3), 56.1 (OCH3), 47.5
(7-O-CH2CH2CH2-), 34.2 (NHCH3), 25.7 (7-O-CH2CH2CH2-); HRMS-ESI m/z [M + H]+ calcd for
C32H36NO10: 594.2339, found: 594.2334. The yields and spectral data for compounds 10–14 are
included in Supporting Information.
3.7. Synthesis of 5,7,20-O-Trimethylsilybin (15)
A three-neck round bottom flask was charged with silibinin (2.01 g, 4.2 mmol) and potassium
carbonate (3.43 g, 25.1 mmol), which was vacuumed three times under argon prior to the addition
of acetone (30.0 mL). The reaction mixture was refluxed for 15 min before dimethylsulfate (3.13 mL,
33.1 mmol) was added through a needle. The reaction was continued with refluxing for an additional
4 h when the reaction was completed, as monitored by TLC. After cooling down to room temperature,
saturated ammonium chloride was added to quench the reaction, and the subsequent mixture was
extracted with ethyl acetatethree times. The organic layers were combined, washed with brine
twice, and dried over anhydrous sodium sulfate. Purification of the crude product through column
chromatography, eluting with ethyl acetate/hexane (50/50 to 70/30, v/v), gave the product (15) as a
1
white crystal in 80% yield. H-NMR (300 MHz, CDCl3):
δ 7.21 (dd, J = 9.9, 1.8 Hz, 1H), 7.08(dd, J = 8.4,
2.1 Hz, 1H), 7.04 (d, J = 3.3 Hz, 1H), 7.00 (dd, J = 8.7, 2.7 Hz, 1H), 6.96 (s, 1H), 6.90 (d, J = 8.4 Hz, 1H),
6.12 (d, J = 2.1 Hz, 1H), 6.11 (d, J = 1.5 Hz, 1H), 4.98 (d, J = 9.0 Hz, 1H), 4.94 (d, J = 12.3 Hz, 1H), 4.42
(dd, J = 12.3, 5.1 Hz, 1H), 4.06–4.03 (m, 1H), 3.91 (s, 3H), 3.90 (s, 3H), 3.90 (s, 3H), 3.81 (d, J = 1.8 Hz,
3H), 3.81 (dd, J = 12.0, 2.4 Hz, 1H), 3.55 (dd, J = 12.3, 3.9 Hz).
3.8. Synthesis of 5,7,20-O-Trimethyl-2,3-Dehydrosilybin (16)
A 10 mL round flask was charged with 5,7,20-O-trimethylsiybin (150.0 mg, 0.23 mmol) in methanol
(2.0 mL) and tetrahydrofuran (2.0 mL). The solution was stirred for 10 min at room temperature prior
to being added to hydrogen peroxide (0.85 mL, 30%) and sodium hydroxide aqueous solution (0.65 mL,
◦
16%) at 0 C. The reaction mixture was slowly warmed to room temperature and then stirred overnight,
before being quenched with saturated ammonium chloride. The subsequent mixture was extracted
with dichloromethane three times, and the combined extracts were dried over sodium sulfate and
concentrated under vacuum. The crude product was obtained in 49% yield, which is pure enough
1
for the next step of the reaction without purification. H-NMR (300 MHz, CDCl3):
δ 7.89 (s, 1H), 7.86
(d, J = 8.4 Hz, 1H), 7.14 (d, J = 8.7 Hz, 1H), 7.04 (d, J = 8.4 Hz, 1H), 6.98 (s, 1H), 6.93 (d, J = 8.1 Hz, 1H),
6.53 (d, J = 1.8 Hz, 1H), 6.35 (d, J = 2.1 Hz, 1H), 5.03 (d, J = 8.1 Hz, 1H), 4.17–4.11 (m, 1H), 3.98 (s, 3H),
3.93 (s, 3H), 3.92 (s, 3H), 3.90 (s, 3H), 3.85 (d, J = 13.8 Hz, 1H), 3.59 (d, J = 10.5 Hz, 1H).
3.9. General Procedure for the Synthesis of 3-O-Bromoalkyl-5,7,20-O-Trimethyl-2,3-Dehydrosilybins (17–19)
A round bottom flask (10 mL) was charged with 5,7,20-O-trimethyl-2,3-dehydrosilybin
16, 83.2 mg, 0.16 mmol), potassium carbonate (352.0 mg, 2.55 mmol), and DMF (5.0 mL). The mixture
(
was stirred for 10 min prior to being added to 1,3-, 1,4-, or 1,5-dibromalkane (2.56 mmol, 16 equiv.).
The reaction was continued with stirring at room temperature for 24–48 h, before the reaction
was quenched with water. The subsequent mixture was extracted with ethyl acetate three
times, and the combined extracts were dried over anhydrous sodium sulfate and concentrated
in vacuo. The crude products were subjected to PTLC purification eluting with DCM/methanol
(100/5, v/v) to yield the respective 3-O-bromoalkyl-5,7,10-O-trimethyl-2,3-dehydrosilybin.
3-O-(30-Bromo)propyl-5,7,20-O-trimethyl-2,3-dehydrosilybin (17). 81% yield, 1H-NMR (300 MHz, CDCl3):
δ
7.72 (s, 1H), 7.70 (d, J = 9.6 Hz, 1H), 7.07 (d, J = 7.8 Hz, 1H), 7.06 (dd, J = 8.1, 1.8 Hz, 1H), 7.00 (d, J = 1.8
Hz, 1H), 6.94 (d, J = 8.4 Hz, 1H), 6.49 (d, J = 2.1 Hz, 1H), 6.35 (d, J = 2.4 Hz, 1H), 5.05 (d, J = 8.4 Hz, 1H),
4.14 (t, J = 6.0 Hz, 2H), 4.16–4.12 (overlapped, 1H), 3.96 (s, 3H), 3.94 (s, 3H), 3.93 (s, 3H), 3.89 (s, 3H),
3.86 (dd, J = 12.6, 2.7 Hz, 1H), 3.60 (t, J = 6.9 Hz, 2H), 3.63–3.58 (overlapped, 1H), 2.31–2.26 (m, 2H).
The yields and spectral data for compounds 18–19 are included in Supporting Information.

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3.10. General Procedure for the Synthesis of 3-O-(Alkylamino)Alkyl-5,7,20-O-Trimethyl-2,3-Dehydrosilybins
A round bottom reaction flask (10 mL) was charged with 3-O-bromoalkyl-5,7,10-O-trimethyl-
2,3-dehydrosilybin (1 eq.) and potassium carbonate (10 eq.) in acetone (2.0 mL, 0.029 M).
The solution was stirred for 10 min prior to being added the appropriate amine (16 eq.).
The reaction was allowed to proceed with stirring at room temperature for 24–48 h, before being
quenched with water. The subsequent mixture was extracted with ethyl acetate three times,
and the combined extracts were dried over anhydrous sodium sulfate and concentrated in vacuo.
The crude product was subjected to PTLC purification eluting with DCM/methanol (100:5, v/v).
Each desired nitrogen-containing compound was retrieved from PTLC silica gel by washing with
dichloromethane/methanol/ammonium hydroxide (100:10:5, v/v/v).
3-O-(N,N-Dimethylamino)propyl-5,7,20-O-trimethyl-2,3-dehydrosilybin (20). 97% yield, white solid, white
1
wax. IR (film) νmax: 3364, 2940, 2837, 1625, 1604, 1517, 1505, 1492, 1462 cm−1; H-NMR (300 MHz,
CDCl3):
δ 7.64 (d, J = 6.2 Hz, 1H, H-15), 7.63 (s, 1H, H-13), 7.10 (d, J = 10.1 Hz, 1H, H-16), 7.03 (d, J = 8.4
Hz, 1H, H-22), 6.98 (s, 1H, H-18), 6.91 (d, J = 8.1 Hz, 1H, H-21), 6.47 (s, 1H, H-8), 6.33 (s, 1H, H-6),
5.05 (d, J = 7.7 Hz, 1H, H-11), 4.19–4.16 (m, 1H, H-10), 4.00–3.97 (overlapped, 2H, 3-OCH2), 3.94 (s,
3H, OCH3), 3.91 (s, 3H, OCH3), 3.90 (s, 3H, OCH3), 3.86 (s, 3H, OCH3), 3.86–3.83 (overlapped, 1H,
H-23), 3.57 (dd, J = 12.2, 2.9 Hz, 1H, H-23), 3.52–3.42 (m, 2H, NCH2), 2.88 (s, 6H, 2
×
NCH3), 2.33–2.22
(m, 2H, 3-OCH2CH2); 13C-NMR (75 MHz, CDCl3):
δ
174.4 (C-4), 164.5 (C-7), 160.8 (C-5), 158.9 (C-8a),
153.4 (C-16a), 149.8 (C-19), 149.4 (C-20), 145.8 (C-2), 143.9 (C-12a), 139.6 C-3), 128.2 (C-17), 123.3 (C-14),
122.2 (C-15), 120.2 (C-22), 117.5 (C-13), 117.0 (C-16), 111.4 (C-21), 110.3 (C-18), 108.9 (C-4a), 96.2 (C-6),
92.5 (C-8), 78.7 (C-10), 76.3 (C-11), 69.3 (3-OCH2), 61.4 (C-23), 56.5 (OCH3), 56.1 (OCH3), 56.0 (OCH3),
55.9 (OCH3), 43.3 (NCH2), 42.3 (NCH3), 25.4 (3-O-CH2CH2CH2-); HRMS-ESI m/z [M + H]+ calcd
for C33H38NO10: 608.2496, found: 608.2490. The yields and spectral data for compounds 21–45 are
included in Supporting Information.
3.11. Cell Culture
All cell lines were initially purchased from American Type Culture Collection (ATCCTM
)
(Manassas, VA, USA). The PC-3 and LNCaP prostate cancer cell lines were routinely cultured in
RPIM-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin. The DU145 prostate
cancer cells were routinely cultured in Eagle’s Minimum Essential Medium (EMEM) supplemented
with 10% FBS and 1% penicillin/streptomycin. Cultures were maintained in a high humidity
environment supplemented with 5% carbon dioxide at a temperature of 37 ◦C.
3.12. WST-1 Cell Proliferation Assay
PC-3, DU145, or LNCaP cells were plated in 96-well plates at a density of 3,200 per well in 200 µL
of culture medium. The cells were then separately treated with silibinin, or synthesized silibinin
derivatives, at five different doses for three days, while equal treatment volumes of DMSO (0.25%)
were used as the vehicle control. The cells were cultured in a CO2 incubator at 37 ◦C for three days.
A total of 10 µL of the premixed WST-1 cell proliferation reagent (Clontech) was added to each well.
After mixing gently for one min on an orbital shaker, the cells were incubated for an additional 3 h at
◦
37 C. To ensure a homogeneous distribution of color, it is important to mix gently on an orbital shaker
for one min. The absorbance of each well was measured using a microplate reader (Synergy HT, BioTek,
Winooski, VT, USA) at a wavelength of 430 nm. The IC50 value is the concentration of each compound
that inhibits cell proliferation by 50% under the experimental conditions and is the average of at least
triplicate determinations so is reproducible and statistically significant. For calculating the IC50 values,
a linear proliferative inhibition was made based on at least five dosages for each compound.

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3.13. Cell Cycle Analysis
PC-3 cells were plated in 24-well plates at a density of 200,000 per well in 400
µL of culture
medium. After 3 h of cell attachment, the cells were then treated with compound 30 at 5 µM for 15 h,
while equal treatment vol◦umes of DMSO were used as the vehicle control. The cells were cultured
in a CO2 incubator at 37 C for 22 h and 31 h, respectively. Both attached and floating cells were
collected in a centrifuge tube by centrifugation at an rcf value of 450 g for 5 min. After discarding
the supernatant, the collected cells were re-suspended with 500
µL 80% cold ethanol to fix them for
30 min in 4 ◦C. The fixed cells could be stored at 20 ◦C for one week. After fixation, the ethanol was
−
removed after centrifuging and the cells were washed with PBS. The cells were then re-suspend with
◦
100
µ
L of 100 mg/mL ribonuclease and were cultured at 37 C for 30 min to degrade all RNA. The cells
◦
were stained with 200
µL of 50
µg/mLpropidium iodide stock solution for 30 min at
−
20 C, and then
the fluorescence intensity of PI was detected in individual PC-3 cells using an Attune flow cytometer
(Life Technologies, Carlsbad, CA, USA) within 0.5 to 1 h after staining.
3.14. F2N12S and SYTOX AADvanced Double Staining Assay
PC-3 cells were plated in 24-well plates at a density of 200,000 per well in 400 µL of culture
medium. After 3 h of cell attachment, the cells were then treated with each test compound at different
concentrations for 15 h, while equal treatment volumes of DMSO were used as the vehicle control.
The cells were cultured in a CO2 incubator at 37 ◦C for 15 h. Both attached and floating cells were
collected in a centrifuge tube by centrifugation at an rcf value of 450 g for 5 to 6 min. The collected
cells were re-suspended with 500
were centrifuged again. After discarding the supernatant, the collected cells were re-suspended with
300 L HBSS and stained with 0.3 L of F2N12S for 3–5 min, followed by 0.3 L SytoxAAdvanced for
µL HBSS to remove proteins which may affect the flow signal and
µ
µ
µ
an additional 5 min. The fluorescence intensity of the two probes was further measured in individual
PC-3 cells using an Attune flow cytometer (Life Technologies) 0.5 to 1 h after staining.
3.15. Statistical Analysis:
All data are represented as the mean
indicated. Other differences between treated and control groups were analyzed using the Student’s
±
standard deviation (SD) for the number of experiments
t-test. A p-value < 0.05 was considered statistically significant.
4. Conclusions
In summary, six 7-O-aminoalkyl-3,5-20-O-trimethyl-2,3-dehydrosilybins and 26 3-O-aminoalkyl-
5,7,20-O-trimethyl-2,3-dehydrosilybins have been successfully synthesized through a five-step
and four-step sequence, respectively. The synthetic methods can be used for the general
synthesis of 7-O-substituted-3,5,20-O-trimethyl-2,3-dehydrosilybins and 3-O-substituted-5,7,20-O-
trimethyl-2,3-dehydrosilybins. The antiproliferative activities of the 32 derivatives against three
prostate cancer cell lines have been evaluated using the WST-1 cell proliferation assay. All of them
showed better prostate cancer cell proliferation inhibition than silybin. Derivatives 11
and 40 were identified as the optimal derivatives, with IC50 values in the range of 1.40–3.06
,
µ
29
, 31, 37,
M toward
these three prostate cancer cell lines, representing a 17- to 52-fold improvement in potency compared
to silibinin. All these five optimal derivatives can cause PC-3 cell accumulation in a G0/G1 phase by
increasing the cell population in this phase at 16 h. Derivatives 11
than derivatives 29 and 31 in activating PC-3 cell apoptosis by inducing appreciable levels of apoptotic
cell death at a 5 M concentration after 16 h of treatment. In contrast, derivatives 29 and 31 did not
, 37, and 40 show a stronger ability
µ
induce significant levels of apoptotic cell death (less than 10%) up to a 10 µM concentration.
Supplementary Materials: The following are available online at http://www.mdpi.com/1420-3049/23/12/3142/
s1, The yields and spectral data for compounds 10–14, 18–19, and 21–
45; NMR-spectra (1H and 13C) of the new
silybin derivatives.

Molecules 2018, 23, 3142
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Author Contributions: Conceptualization and design: Q.C., B.V., and G.W.; performed the experiment and
analyzed data: B.V., S.Z., A.V., G.C., X.Z., W.D., Q.Z., S.Z., and Q.C.; drafted and proofread the paper: Q.C., B.V.,
S.Z., and G.W.; all authors finally reviewed and approved the manuscript.
Funding: This work was financially supported by California State University (CSU)-Fresno. HRMS were
supported by the NIH RCMI program at Xavier University of Louisiana through Grant [2G12MD007595] (G. Wang).
We are also grateful to (i) the ASI at CSU-Fresno for a Graduate Research Grant (to S. Zhang), (ii) the Graduate Net
Initiative at CSU-Fresno for 2015–2016 Graduate Research Fellowships (to S. Zhang and X. Zhang), and (iii) the
Undergraduate Research Grant program at CSU-Fresno for the funding (to A. Vignau and W. Diaz). We are grateful
to the Henry Madden Library Open Access mini-grant program (CSU-Fresno) for an Open Access Mini-Grant.
Acknowledgments: We thank the Department of Chemistry and College of Science and Mathematics at CSU-Fresno
for all administrative support. We also thank Mr. Douglas Kliewer (CSU-Fresno) and Mr. Alan Preston (CSU-Fresno)
for technical help.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds 9–14 and 20–45 are available from the authors.
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2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).