Identification and initial SAR of silybin: An Hsp90 inhibitor

Article abstract of DOI:10.1016/j.bmcl.2010.12.088

Through Hsp90-dependent firefly luciferase refolding and Hsp90-dependent heme-regulated eIF2α kinase (HRI) activation assays, silybin was identified as a novel Hsp90 inhibitor. Subsequently, a library of silybin analogues was designed, synthesized and evaluated. Initial SAR studies identified the essential, non-essential and detrimental functionalities on silybin that contribute to Hsp90 inhibition.

Full text of DOI:10.1016/j.bmcl.2010.12.088

Bioorganic & Medicinal Chemistry Letters 21 (2011) 2659–2664
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and initial SAR of silybin: An Hsp90 inhibitor
Huiping Zhao a, Gary E. Brandt a, Lakshmi Galam b, Robert L. Matts b, Brian S. J. Blagg a,
⇑
a Department of Medicinal Chemistry, 1251 Wescoe Hall Drive, Malott 4070, The University of Kansas, Lawrence, KS 66045-7563, United States
b Department of Biochemistry and Molecular Biology, NRC 246, Oklahoma State University, Stillwater, OK 74078, United States
a r t i c l e i n f o
a b s t r a c t
Article history:
Through Hsp90-dependent firefly luciferase refolding and Hsp90-dependent heme-regulated eIF2
a
Received 1 December 2010
Revised 14 December 2010
Accepted 16 December 2010
Available online 28 December 2010
kinase (HRI) activation assays, silybin was identified as a novel Hsp90 inhibitor. Subsequently, a library
of silybin analogues was designed, synthesized and evaluated. Initial SAR studies identified the essential,
non-essential and detrimental functionalities on silybin that contribute to Hsp90 inhibition.
Keywords:
Heat shock protein 90
Hsp90 inhibitors
Silybin
Structure–activity relationships
Breast cancer
Ó 2011 Elsevier Ltd. All rights reserved.
The 90 kDa family of heat shock proteins (Hsp90) is responsible
for the conformational maturation of newly synthesized poly-
peptides and the refolding of denatured proteins into biologically
active, three-dimensional structures.1,2 To date, more than 200
Hsp90-dependent client proteins have been discovered, of which
Her2, Src family kinases, Raf, PLK, RIP, Akt, telomerase and Met
are directly associated with the six hallmarks of cancer.3–5 Conse-
quently, inhibition of the Hsp90 folding machinery provides a com-
binatorial approach towards the disruption of multiple signaling
nodes that are critical for malignant cell growth and proliferation.
Since the first-in-class drug, 17-AAG (a synthetic derivative of
geldanamycin), demonstrated therapeutic benefit at tolerable
doses, more than 30 clinical trials have commenced for the
treatment of various cancers.6,7 In contrast, recent studies have
demonstrated that Hsp90 is a potential therapeutic target for
neurodegenerative diseases, including Alzheimer’s, Parkinson’s,
Prion and Hodgkin’s diseases.8 The potential therapeutic benefits
associated with Hsp90 modulation highlight the importance of
identifying and optimizing novel Hsp90 inhibitors for the treat-
ment of these diseases.
Silymarin, a flavonolignan extract from the seed of milk thistle
(Silybum marianum), is native to the Mediterranean regions of
Europe, North Africa and the Middle East.9 It has been used since
ancient time for the treatment of liver and gallbladder disorders.
In the past three decades, silymarin has been used clinically as
an anti-hepatotoxic agent as well as a nutritional supplement to
protect the liver from diseases associated with alcohol consump-
tion and exposure to chemical and environmental toxins.10 Silybin
is a mixture of two diastereomers, A and B, in nearly 1:1 ratio, and
is the major component of silymarin complex, along with its
⇑
Corresponding author. Tel.: +1 785 864 2288; fax: +1 785 864 5326.
E-mail address: bblagg@ku.edu (B.S.J. Blagg).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.12.088

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H. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2659–2664
OH
O
E
O
O
O
OH
OMe
B
HO
HO
O
D
OMe
OH
HO
O
O
OH
A
C
OH
OH
Silybin A
Isosilybin
OH
O
OH
OH
OMe
OH
16
23
O
O 10
OH
HO
O
OH
7
19
O
O
OMe
OH
O
11
13
OH
3
OH
20
5
Silychristin
OH
O
Silybin B
OH
Figure 1. Major components of silymarin.
regio-isomers isosilybin A and B, silydianin, silychristin and isosily-
christin10 (Fig. 1).
Along with the beneficial activities exhibited by silymarin such
as hepato-, cardio-, and neuro-protective activities resulting from
the anti-oxidant and radical scavenging properties,10,11 recent
studies have demonstrated that silybin exerts cytotoxic activity
against cancer cell lines and enhances the efficacy of other chemo-
therapeutic agents.10,12 The mechanism for these activities has
been investigated at the cellular and molecular levels. For example,
in prostate cancer, both silymarin and silybin (50–100 lg/ml) have
been shown to inhibit human PC3 cell proliferation, induce cell
death, and cause G1 and G2-M cell cycle arrest in a dose-depen-
dent manner.13,14 G1 arrest is associated with a decrease in cy-
clin-dependent kinases CDK4, CDK6 and CDK2 protein levels, and
CDK2 and CDK4 kinase activity. In addition, silybin inhibits meta-
static PC3 cell mobility and adhesion,15 suggesting that it serves as
the active component in silymarin. However, it has also been sug-
gested that other silybin stereoisomers present in silymarin may
also contribute to its efficacy.10 Furthermore, silybin inhibits cell
proliferation of colon cancer cell lines by causing cell cycle arrest
due to depletion of CDK2.16 Silybin also inhibits cell growth and in-
duces apoptosis in both small cell and non-small cell human lung
carcinoma cells.17 CDK4, CDK6, and CDK2 are well-studied Hsp90-
dependent clients, suggesting that silybin may exhibit Hsp90
inhibitory activity, which may be responsible for the observed
anti-cancer activity.
To test this hypothesis, the Hsp90-dependent refolding assay
utilizing the rematuration of firefly luciferase was investigated.18
Analysis showed that silybin inhibited Hsp90-dependent refolding
of luciferase in reticulocyte lysate by 50% at a concentration of
Figure 2. (Top) Effect of geldanamycin, novobiocin and silybin on Hsp90-depen-
dent refolding of heat denatured luciferase in rabbit reticulocyte lysate. Denatured
luciferase was incubated in the presence of DMSO (vehicle control) or increasing
concentrations (mM) of geldanamycin (open circles), novobiocin (open squares) or
Figure 3. The effect of silybin on HRI maturation/activation, and the interaction of
Hsp90 and Cdc37 with HRI in rabbit reticulocyte lysate. (A) Activation of newly
synthesized [35S]-labeled His-tagged HRI matured in heme-replete (+H, lane 1) or
heme-deficient reticulocyte lysate (ÀH, lanes 2–6) in the presence of DMSO (lane 2),
silybin (open diamonds) for 30 min at 30 °C. Aliqouts (10 lL) were added to assay
buffer and relative light units produced by the refolded luciferase was measured as
previously described.18 Activity is expressed as percent of the DMSO control. Data
points correspond to one representative experiment performed in triplicate.
(Bottom) Western blot analyses of Hsp90-dependent client proteins from MCF-7
1 lg/ml geldanamycin (GA, lane 3) or the indicated concentrations of silybin (lanes
4–6) [ÀHRI, inactive HRI; ÀHRI⁄ mature/active HRI]. (B) Effect of DMSO (lane 1),
breast cancer cell lysate upon treatment with silybin. Concentrations (in
l
M) were
1 lg/ml geldanamycin (GA, lane 2) or 2 mM silybin (lane 3) on the interaction of
indicated above each line, and geldanamycin (G, 0.5
were employed as positive and negative controls.
l
M) and dimethylsulfoxide (D)
Hsp90 and Cdc37 with newly synthesized His-tagged HRI in reticulocyte lysate
assayed via anti-His-tag pull down assays and Western blotting.

H. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2659–2664
2661
250
l
M, compared to an IC50 of 2 and 350
l
M for the Hsp90
Claisen–Schmidt condensation with III, and subsequent epoxida-
tion and acid promoted cyclization.29
inhibitors, geldanamycin and novobiocin, respectively. Subsequent
evaluation in MCF-7 cells confirmed selective degradation of
Hsp90-dependent clients at a concentration that paralleled anti-
proliferative activity, linking Hsp90 inhibition to cell viability
(Fig. 2).
A late stage common intermediate is highly favored for the con-
struction of analogues to generate a diverse library of compounds.
Therefore, the latter strategy was employed to investigate substi-
tutions on the A- and D-rings, both of which originate from inter-
mediate 6. Synthesis of this key intermediate is described in
Scheme 2. Demethylation of eugenol (1) with lithium chloride in
DMF afforded catechol 2 in modest yield. Likewise, esterification
of ferulic acid (3) with methanol and thionyl chloride followed
by subsequent reduction with diisobutylaluminium hydride in tet-
rahydrofuran gave allylic alcohol, 4. Following the procedure of
Merlini,28 the hetero-Diels–Alder reaction between 2 and 4 gave
the cyclo-adduct 5, which upon isomerization and oxidative cleav-
age afforded key intermediate 6.
1,4-Benzodioxan 6 and various hydroxy-acetophenones (8–13)
were easily converted to the methoxymethyl ethers (7 and
14–18) in good yield. Claisen–Schmidt condensation of 7 and
acetophenones 14–18 were performed in ethanol in the presence
of potassium hydroxide to provide chalcones 19–23 in good yield.
Compounds produced from this reaction gave the trans products,
as established by 1H NMR. Alkaline hydrogen peroxide oxidation
of compounds 19–23 gave the corresponding epoxides, which
underwent acid promoted deprotection of the MOM-ether,
followed by in situ cyclization to afford silybin analogues 24–28
in modest yields.
To evaluate the effect of the C-3 hydroxyl group on anti-
proliferative activity, compound 34, in which the C-3 hydroxyl
group was removed, was synthesized via basic cyclization of
chalcone 29, which was obtained upon acidic deprotection of 19
(Scheme 4). Evaluation of this compound against two breast cancer
cell lines indicated that the C-3 hydroxyl group is detrimental to
anti-proliferative activity. To validate this observation, compounds
35–38, containing various phenol substitutions on the A-ring, were
prepared via the same synthetic sequence.
To assess the role of substitutions on the C- and E-rings,
Strategy 1 was applied toward the preparation of these analogues.
As described in Scheme 5, synthesis of isoeugenol (39) was accom-
plished by catalytic isomerization of eugenol (1) with platinum
dichloride. Wittig reaction of 3-methoxybenzaldehyde (40) with
(carbomethoxymethyl)-triphenylphosphonium bromide afforded
To further characterize the effect of silybin on the activity of
Hsp90 in vitro, we examined the ability of silybin to inhibit
Hsp90-dependent activation of heme-regulated eIF2
a kinase
(HRI).19–21 When newly synthesized HRI was ‘matured’ in heme-
deficient lysate, silybin inhibited the activation of HRI in a concen-
tration-dependent manner. Silybin at a concentration of 2 mM,
inhibited the Hsp90-dependent maturation and activation of HRI
to the same extent as 1 lg/mL geldanamycin (Fig. 3A, absence of
-HRI⁄). Western blotting indicated that silybin did not disrupt the
interactions between Hsp90 and Cdc37 with HRI (Fig. 3B),
suggesting that it inhibits Hsp90 similar to the Hsp90 inhibitor,
derrubone.22 Thus, silybin exhibits properties that are expected
for Hsp90-inhibitors, suggesting that other members of this family
may also exhibit Hsp90 inhibitory activity.
Although silybin was identified as an Hsp90 inhibitor, it mani-
fests poor anti-proliferative activity against the MCF-7 cell line.
Its low bioavailability and poor water solubility may contribute
to this low efficacy.10 Attempts to circumvent these issues have
been previously reported, including the esterification,23 phosphor-
ylation,24 glycosidation,25 and oxidation of the C-23 alcohol.26
Other structural modifications to the phenol and alcohol have also
been disclosed to improve efficacy, focusing primarily on the anti-
oxidant and radical-scavenging potential.11,27 Herein, we provide
an alternative approach towards improving the anti-proliferative
efficacy of silybin through the synthesis of analogues that manifest
Hsp90 inhibitory activity. Initial SAR studies focused on the role of
individual functionalities on silybin in an effort to identify essential
moieties responsible for anti-proliferative activity.
The synthesis of silybin analogues followed two strategies; (1)
the biomimetic synthesis of natural silybin through a silver oxide
promoted oxidative coupling of a taxifolin analogue (I) and a
benzylallylic alcohol (II) (Scheme 1, Strategy 1);28 however, this
oxidative coupling is not regio-selective, as nearly an equal amount
of isosilybin analogues were obtained. The other strategy was to
regio-specifically construct the substituted 1,4-benzodioxane (IV)
(Scheme 1, Strategy 2) as the key intermediate, followed by a
OMe
OH
OH
OH
a
OH
R'
2
1
O
O
+
OH
OH
OH
R
R"
b.c
OH
O
II
OMe
I
OMe
OH
Oxidative coupling
Strategy 1
OH
O
R'
10
3
4
E
O
B
O
D
O
11
R"
C
OH
2
d
R
+
A
2
5
4
OMe
OH
OH
3
O
O
5
Claisen-Schmidt condensation
Strategy 2
OH
O
O
e, f
OH
OMe
OH
O
O
R'
OHC
+
R
6
OHC
R"
O
III
Scheme 2. Synthesis of compound 6. Reagents and conditions: (a) LiCl, DMF, reflux,
48 h, 43%; (b) SOCl2, MeOH, 0 °C, 12 h, 92%; (c) DIBAL-H, THF, 0 °C, 4 h, 74%;
(d) Ag2O, benzene/acetone, 24 h, 73%; (e) PtCl2, MeOH, 12 h, 94%; (f) OsO4, NaIO4,
dioxane, 8 h, 64%.
IV
Scheme 1. Retro-synthesis of silybin analogues.

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H. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2659–2664
O
OMOM
a
6
OMe
60%
OHC
O
R1
R2
OMOM
O
O
O
OMOM
OMe
OMOM
c
7
R3
R1
R2
OH
b
R1
R2
OMOM
OMOM
55-83%
19: R1 = OMOM, R2= H, R3 = OMOM
20: R1 = OMOM, R2= H, R3 = H
21: R1 = H, R2= H, R3= OMOM
22: R1 = H, R2= H, R3= H
57-92%
R3
O
R3
O
8: R1 = OH, R2 = H, R3 = OH 14: R1 = OMOM, R2= H, R3 = OMOM
23: R1 = H, R2= OMOM, R3 = H
9: R1 = OH, R2 = H, R3 = H
11: R1 = H, R2= H, R3= OH
12: R1 = H, R2= H, R3= H
13: R1 = H, R2= OH, R3 = H
15: R1 = OMOM, R2= H, R3 = H
16: R1 = H, R2= H, R3= OMOM
17: R1 = H, R2= H, R3= H
1) d
2 steps
18-41%
2) e or f
O
E
O
18: R1 = H, R2= OMOM, R3 = H
OH
C
B
R1
R2
O
D
OMe
OH
A
OH
3
R3
O
24: R1 = OH, R2= H, R3 = OH
25: R1 = OH, R2= H, R3 = H
26: R1 = H, R2= H, R3= OH
27: R1 = H, R2= H, R3= H
28: R1 = H, R2= OH, R3 = H
Scheme 3. Synthesis of silybin analogues with a modified A ring. Reagents and conditions: (a) NaH, MOMCl, DMF; (b) NaH, MOMCl, THF; (c) KOH, EtOH; (d) 5% NaOH,
50% H2O2, MeOH; (e) concd HCl, MeOH, 55 °C; (f) TFA, DCM, 48 h.
O
O
O
O
OH
OH
R1
R2
O
O
OMe
OH
R1
R2
OH
O
OMe
OH
NaOAc, MeOH
reflux, 16-67%
c HCl, MeOH
19 23
53-82%
R3
R3
29: R1 = OH, R2 = H, R3 = OH
30: R1 = OH, R2 = H, R3 = H
31: R1 = H, R2 = H, R3 = OH
32: R1 = H, R2 = H, R3 = H
33: R1 = H, R2 = OH, R3 = H
34: R1 = OH, R2 = H, R3 = OH
35: R1 = OH, R2 = H, R3 = H
36: R1 = H, R2 = H, R3 = OH
37: R1 = H, R2 = H, R3 = H
38: R1 = H, R2 = OH, R3 = H
Scheme 4. Synthesis of silybin analogues without the C-3 hydroxy group.
the a,b-unsaturated ester 41, which was reduced with diisobutyl-
a
OMe
OH
aluminium hydride in tetrahydrofuran to give alcohol 42. Similarly,
compound 45 was obtained from (E)-3-(4-hydroxyphenyl)-acrylic
acid (43) in two steps.
1
96%
39
( )-Taxifolin 46 was synthesized in four steps following the re-
ported procedure.11 Subsequent coupling of ( )-taxifolin 46 and
coniferyl alcohol analogues (39, 42, 45, 47, 48) afforded silybin
analogues 49a–53a, along with their regio-isomers, 49b–53b, in
a 1:1 ratio. Repeated chromatography of the mixture gave 51a,
which was also independently prepared via the procedure de-
scribed in Scheme 3 to confirm its structure. Separation of other
mixtures was not pursued. To compare the anti-proliferative
activity between silybin analogues and their corresponding regio-
isomers, a mixture of silybin and isosilybin (54a and 54b) was also
synthesized, as described in Scheme 6.
Upon construction of the silybin analogues, the compounds
were evaluated for anti-proliferative activity against SKBr3 (estro-
gen receptor negative, HER2 over-expressing breast cancer cells)
and MCF-7 (estrogen receptor positive breast cancer cells) cell
lines.30,31 As shown in Table 1, silybin analogues containing modi-
fications to the A- and C-rings exhibited improved anti-prolifera-
tive activity compared to silybin. These results indicate that the
phenol on the A-ring is not necessary, since compound 27 shows
OH
OMe
O
OHC
OMe
OMe
b
c
OMe
49%
76%
40
42
41
OMe
OH
OH
O
O
d
c
OMe
OH
92%
OH
57%
OH
43
44
45
Scheme 5. Synthesis of coniferyl alcohol analogues. Reagents and conditions:
(a) PtCl2, MeOH; (b) NaH, (carbomethoxymethyl)-triphenylphosphonium bromide,
DCM; (c) DIBAL-H, THF, 0 °C; (d) SOCl2, MeOH.
comparable activity to 25, 26 and 28. However, one phenol is
tolerated on the A-ring; and the pattern of substitution is not

H. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2659–2664
2663
R'
O
O
R
OH
OH
O
O
R"
Ag2CO3
HO
O
O
R'
HO
O
O
HO
O
O
R
+
40,
43, 47
or
or
OH
R''
OH
OH
or 48
4
or , 8.6-54%
OH
OH
OH
OH
46 ( )Taxifolin
49a: R = CH3, R' = OMe, R'' = OH
49b: R = CH3, R' = OMe, R'' = OH
50b: R = CH2OH, R' = OMe, R'' = H
51b: R = CH2OH, R' = H, R'' = OH
52b: R = CH2OH, R' = R'' = H
53b: R = H, R' = OMe, R'' = OH
54a: R = CH2OH, R' = OMe, R'' = OH
OMe
OH
50a: R = CH2OH, R' = OMe, R'' = H
51a: R = CH2OH, R' = H, R'' = OH
52a: R = CH2OH, R' = R'' = H
47
48
53a: R = H, R' = OMe, R'' = OH
54a: R = CH2OH, R' = OMe, R'' = OH
Scheme 6. Synthesis of silybin analogues with modified C- and E- rings.
Table 1
Anti-proliferative activity of various silybin analogues
R5
O
O
R5
O
O
R1
R2
O
O
R6
R7
R1
OH
O
R6
R7
R4
R2
R3
R3
A
B
R1
R2
R3
R4
R5
R6
R7
SKBR3 (lM)
MCF-7 (lM)
Silybin*
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
49a + 49b
50a + 50b
51a + 51b
51a
52a + 52b
53a + 53b
54a + 54b
OH
OH
OH
H
H
H
OH
OH
H
H
H
H
H
H
H
H
OH
H
H
H
H
OH
H
H
H
H
OH
H
H
H
OH
OH
H
OH
H
H
OH
H
OH
H
H
OH
H
OH
H
OH
OH
OH
OH
OH
OH
OH
—
—
—
—
H
H
H
H
H
OH
OH
OH
OH
OH
OH
OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH3
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
OMe
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
H
197.0 45.3a
196.3 26.0
16.04 2.23
11.90 2.43
11.12 1.42
13.41 1.32
111.40 10.47
37.14 0.62
25.03 1.86
19.87 3.27
35.84 0.83
101.2 2.50
41.91 2.96
47.73 1.51
16.25 0.63
15.63 4.35
60.9 2.66
>500
222.8 3.6
196.4 28.8
11.92 0.64
17.66 6.55
13.42 2.56
17.80 7.38
109.3 6.22
16.91 4.72
22.58 2.64
12.07 0.21
12.17 0.38
104.2 5.44
41.58 3.68
50.32 2.83
13.66 2.58
15.62 0.50
69.8 7.13
140.4 14.4
151.4 26.0
138.7 33.0
>500
B
B
B
B
B
A
A
A
A
A
B
B
B
B
B
—
—
—
—
—
—
—
OH
OH
OH
H
H
H
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
OH
CH2OH
CH2OH
CH2OH
CH2OH
H
OH
OH
H
OH
OH
172.0 13.4
210.6 7.5
>500
103.3 2.62
233.5 6.15
H
H
H
H
H
H
OMe
OMe
101.3 0.92
222.7 2.76
CH2OH
*
Purchased from Sigma–Aldrich.
Values represent mean standard deviation for at least two separate experiments performed in triplicate.
a
important. Removal of the C-3 hydroxyl group resulted in an
increase in anti-proliferative activity ꢀ2-fold, compared to silybin,
suggesting that the C-3 hydroxyl group is not essential. In support
of this, removal of C-3 hydroxyl group on compounds 27 and 28 re-
sulted in similar activity (compound 37 and 38). However, removal
of the C-3 hydroxyl group on compounds 25 and 26 resulted in
decreased activity (35 and 36). Interestingly, the acyclic chalcone
intermediates 29–33 exhibited comparable anti-proliferative
activity to their cyclized counterparts. Although modifications to
the C- and E-rings resulted in two inseparable regio-isomers, eval-
uation of the mixture of 51a and 51b gave approximately the same
result as that obtained from pure 51a. This observation was also
seen in the case of silybin (24) and silybin mixtures (54a + 54b).
Consequently, it appears as though the C-23 hydroxyl group is det-
rimental to anti-proliferative activity (24 vs 49), however, removal
of the methylene group decreases activity (53 vs 49). Only one sub-
stitution on the C-ring is required for anti-proliferative activity and
the 4’-hydroxyl moiety (51) is favored over the 3’-methoxy group
(50). However, deleting both functional groups significantly dimin-
ishes activity (52).
Structure–activity relationships produced from this first gener-
ation of silybin analogues provide substantial information with re-
gards to structural features necessary for silybin and its exhibition
of anti-proliferative activity: the resorcinol structure is detrimen-
tal, and any phenol is tolerated on the A-ring. The C-3 hydroxyl
group is not essential and the C-23 hydroxyl group is detrimental.
Finally, an H-bond donor on the C-ring is more favored than a
H-bond acceptor. A summary of these observations is detailed in
Figure 4.
To confirm that anti-proliferative activities exhibited by silybin
analogues result from Hsp90 inhibition, Western blot analyses of
the MCF-7 cell lysate following administration of 27 and 38 were
performed.30,31 Figure 5 shows that the Hsp90-dependent client
proteins Her2, Raf, and Akt were degraded in a concentration-
dependent manner upon treatment with compound 27 or 38 at
concentrations that mirror its anti-proliferative activity, clearly

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H. Zhao et al. / Bioorg. Med. Chem. Lett. 21 (2011) 2659–2664
C-23 hydroxy is detrimental
O
OH
HO
O
O
OMe
OH
O
hydroxy group is likely more
potent then methoxy group
OH
one phenol is tolerated
resorcinol structure is
detremental
OH
C-3 hydroxy is not essenial
Figure 4. SAR summary of silybin analogues.
C-terminus, which validates silybin as a novel scaffold for Hsp90
inhibition and analogue development.
Acknowledgement
The authors gratefully acknowledge support of this project by
the NIH/NCI (CA120458).
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linking client protein degradation to cell viability. The non-Hsp90-
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mained constant at all concentrations tested, which is characteris-
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may bind the Hsp90 C-terminus.
In conclusion, silybin was identified as a novel inhibitor of the
Hsp90 protein folding machinery and a library of silybin analogues
was designed and synthesized to explore the structure–activity
relationships for this natural product. Upon biological evaluation,
initial SAR was produced to determine the essential, non-essential
and detrimental functionalities present on the silybin scaffold that
result in anti-proliferative activity. Western blot analyses of silybin
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