Influence of rhamnose substituents on the potency of SL0101, an inhibitor of the Ser/Thr kinase, RSK

Article abstract of DOI:10.1016/j.bmc.2006.05.009

We have previously reported the isolation of kaempferol 3-O-(3″,4″-di-O-acetyl-α-l-rhamnopyranoside) from Forsteronia refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem. 2006, 14, 3974-3977.]. This flavonoid glycoside, terme

Full text of DOI:10.1016/j.bmc.2006.05.009

Bioorganic & Medicinal Chemistry 14 (2006) 6034–6042
Influence of rhamnose substituents on the potency of SL0101,
an inhibitor of the Ser/Thr kinase, RSK
Jeffrey A. Smith,a,b David J. Maloney,c David E. Clark,a Yaming Xu,c
Sidney M. Hechtc and Deborah A. Lannigana,d,
*
aCenter for Cell Signaling, University of Virginia, Charlottesville, VA 22908, USA
bDepartment of Pathology, University of Virginia, Charlottesville, VA 22908, USA
cDepartment of Chemistry, University of Virginia, Charlottesville, VA 22904, USA
dDepartment of Microbiology, University of Virginia, Charlottesville, VA 22908, USA
Received 1 March 2006; revised 4 May 2006; accepted 4 May 2006
Available online 24 May 2006
Abstract—We have previously reported the isolation of kaempferol 3-O-(300,400-di-O-acetyl-a-L-rhamnopyranoside) from Forsteronia
refracta [Xu, Y.-M.; Smith, J. A.; Lannigan, D. A.; Hecht, S. M. Biorg. Med. Chem. 2006, 14, 3974–3977.]. This flavonoid glycoside,
termed SL0101, is a specific inhibitor of p90 ribosomal S6 kinase (RSK) with a dissociation constant of 1 lM. In intact cells, how-
ever, the EC50 for inhibition of RSK activity is 50 lM, which suggests that the efficacy of SL0101 could be limited by cellular uptake.
Therefore, we investigated the possibility of developing a more potent RSK inhibitor by synthesizing SL0101 analogs with increased
hydrophobic character. The total syntheses of kaempferol 3-O-(300,400-di-O-butyryl-a-L-rhamnopyranoside) (Bu-SL0101) and
kaempferol 3-O-(200,300,400-tri-O-acetyl-a-L-rhamnopyranoside) (3Ac-SL0101) were performed. The IC50 for inhibition of RSK activ-
ity in in vitro kinase assays for the analogs was similar to that obtained for SL0101. 3Ac-SL0101 demonstrated the same remarkable
specificity for inhibiting RSK activity in intact cells as SL0101; however, Bu-SL0101 was not completely specific. 3Ac-SL0101 was
ꢀ2-fold more potent at inhibiting MCF-7 cell proliferation compared to SL0101 and preferentially decreased MCF-7 cell growth, as
compared to the growth of the normal human breast line, MCF-10A. Thus the discovery of 3Ac-SL0101 as a more potent RSK-
specific inhibitor than SL0101 should facilitate the development of RSK inhibitors as anti-cancer chemotherapeutic agents.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
extract led to the isolation of an active component
kaempferol 3-O-(300,400-di-O-acetyl-a-L-rhamnopyrano-
side), also referred to as SL0101 (Fig. 1). SL0101 is
The p90-kDa ribosomal S6 kinase (RSK) family mem-
bers are downstream effectors of mitogen-activated pro-
tein kinase (MAPK). MAPK is known to be important
in proliferation and oncogenesis.1,2 However, it has only
now become possible to distinguish the function of RSK
in these processes from those of MAPK itself and of the
many other downstream MAPK effectors because of the
recent discovery of a RSK-specific inhibitor.3,4 We
reported that an extract from Forsteronia refracta, a
member of the Apocynaceae (dogbane) family found
in the South American rainforest, was able to specifically
inhibit RSK catalytic activity.4 Fractionation of the
Keywords: Kaempferol 3-O-(300,400-di-O-butyryl-a-L-rhamnopyrano-
side), Bu-SL0101; Kaempferol 3-O-(200,300,400-tri-O-acetyl-a-L-rhamno-
pyranoside), 3Ac-SL0101; RSK; Kinase inhibitor.
Figure 1. Structures of SL0101, 200,400-di-O-acetyl-SL0101, and 400-
mono-O-acetyl-SL0101.
*
Corresponding author. Tel.: +1 434 924 1144; fax: +1 434 924
1236; e-mail: dal5f@virginia.edu
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2006.05.009

J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
6035
competitive with respect to ATP with a dissociation con-
stant, Ki, of 1 lM.4 This compound is an effective and
specific inhibitor of RSK in intact cells.4 SL0101 inhibits
proliferation of the human breast cancer cell line, MCF-
7, with an efficacy paralleling its ability to inhibit RSK in
intact cells. Significantly, SL0101 does not prevent the
growth of the normal human breast line, MCF-10A,
even though it inhibits RSK activity in these cells.4 We
have found that RSK increases the transcriptional activ-
ity of estrogen receptor a (ERa) and the androgen recep-
tor (AR).5–7 Increased activity of ERa and AR is known
to be important in the etiology of some breast and pros-
tate cancers, respectively. Furthermore, we have found
that RSK levels are higher in ꢀ50% of human breast
and prostate tumors compared to normal tissue.4,7 Evi-
dence from other laboratories has demonstrated that
RSK is involved in osteosarcoma formation and in
non-small cell lung carcinoma.8,9 Taken together, these
results suggest that RSK could serve as an important
novel drug target in some types of cancers. Protein ki-
nases have been shown to be excellent targets for drug
discovery10 and thus SL0101 is a useful chemotherapeu-
tic lead compound.
side), (400-mono-O-acetyl-SL0101), differ from SL0101 in
either the position or number of acetyl groups present
on the rhamnose moiety (Fig. 1) and were obtained during
the purification of SL0101 from F. refracta. The ability of
the purified compounds to inhibit RSK activity was deter-
mined in an in vitro kinase assay using recombinant,
constitutively active RSK2.4 The data were fit using non-
linear regression analysis and the IC50 values for
400-mono-O-acetyl-SL0101 and 200,400-di-O-acetyl-SL0101
were ꢀ 140 nM and ꢀ 260 nM, respectively (Fig. 2A),
which is similar to the IC50 of ꢀ50–100 nM obtained for
SL0101. Thus all three related compounds are able to
inhibit RSK catalytic activity in vitro, which is in agree-
ment with the observations reported by Maloney and
Hecht.11 To determine whether the related compounds
The efficacy of SL0101 as a RSK inhibitor in intact
cells is ꢀ50-fold higher than its dissociation constant,
which suggests that the potency of SL0101 in cells
could be limited by cellular uptake. Therefore, we
investigated the possibility of obtaining a more effective
RSK-specific inhibitor by synthesizing analogs that
have
a higher octanol–water partition coefficient,
LogP, than SL0101. We increased the calculated LogP
by either replacing the 300- and 400-acetyl groups on the
rhamnose with butyryl groups or substituting the 200-
hydroxyl group with an acetyl group. The syntheses
of kaem-pferol 3-O-(300,400-di-O-butyryl-a-L-rhamnopyr-
anoside) (Bu-SL0101) and kaempferol 3-O-(200,300,400-tri-
O-acetyl-a-L-rhamnopyranoside) (3Ac-SL0101) were
performed. We found that 3Ac-SL0101, like synthetic
SL0101,11 is an effective and specific inhibitor of
RSK activity in intact cells. Bu-SL0101 was modestly
more potent at inhibiting MCF-7 proliferation than
SL0101 but was not completely specific for RSK inhi-
bition in intact cells. However, 3Ac-SL0101 was ꢀ 2-
fold more effective in inhibiting the growth of MCF-7
cells than SL0101. Additionally, 3Ac-SL0101 retained
the preferential ability to inhibit the growth of MCF-
7 compared to MCF-10A cells. Thus 3Ac-SL0101 is
of interest for further characterization as a potential
chemotherapeutic agent for cancer.
Figure 2. Effect of SL0101, 200,400-di-O-acetyl-SL0101, and 400-mono-O-
acetyl-SL0101 on RSK activity. (A) The potency of purified com-
pounds in inhibiting RSK catalytic activity in vitro was measured.
Kinase assays were performed using immobilized substrate. The
reactions were initiated by the addition of 10 lM ATP (final
concentration). Reactions were terminated after 30 min. All assays
measured the initial reaction velocity. The extent of phosphorylation
was determined using phosphospecific antibodies in combination with
HRP-conjugated secondary antibodies. HRP activity was measured as
described under Section 4. Maximum and minimum activity is the
relative luminescence detected in the presence of vehicle and 200 mM
EDTA, respectively. Points, mean (n = 2 in triplicate); bars = SD. (B)
The potency of purified compounds as inhibitors of MCF-7 cell
proliferation was determined. MCF-7 cells were treated with vehicle or
50 lM SL0101, 200,400-di-O-acetyl-SL0101, or 400-mono-O-acetyl-
SL0101 and cell viability was measured after 72 h of treatment. Values
given are the fold proliferation. Points, mean (n = 3 in quadruplicate);
bars = SD.
2. Results
2.1. Rhamnose acylation is critical for inhibition of MCF-
7 cell growth
To investigate the possibility of improving the potency of
SL0101, we characterized two compounds structurally
related to SL0101 for their ability to inhibit RSK activity.
These compounds, kaempferol 3-O-(200,400-di-O-acetyl-a-
L-rhamnopyranoside), (200,400-di-O-acetyl-SL0101), and
kaempferol 3-O-(400-mono-O-acetyl-a-L-rhamnopyrano-

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J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
inhibit RSK activity in intact cells, we examined their abil-
ity to inhibit the proliferation of MCF-7 cells. Previously
we have shown that SL0101 inhibits MCF-7 cell prolifera-
tion by specifically inhibiting RSK activity.4 SL0101 inhib-
ited MCF-7 cell proliferation with an EC50 of ꢀ50 lM,4
and our current results are in agreement with those obser-
vations (Fig. 2B). SL0101 and 200,400-di-O-acetyl-SL0101 at
50 lM were similarly effective at inhibiting MCF-7 cell
growth. However, 400-mono-O-acetyl-SL0101 did not
inhibit MCF-7 cell proliferation at 50 lM. These data
show that the position of the acetyl groups was not critical-
ly important in determining the in vitro affinity for RSK.
However, only the compounds with two acetyl groups
inhibitedRSKactivityinintactcells.Itislikelythattheace-
tyl groups facilitate effective uptake of the inhibitors into
the cell.
2.2. Synthesis of SL0101 analogs
The syntheses of both Bu-SL0101 (5) and 3Ac-SL0101
(8) utilized methodology and intermediates described
in the previously reported synthesis of SL0101.11
Accordingly, bis-esterification of phenyl 2-O-benzyl-1-
thio-a-L-rhamnopyranoside (1) was achieved using
butyric anhydride, NEt3, and catalytic DMAP in quan-
titative yield to afford phenylthioglycoside 2 (Scheme 1).
Bromination of the anomeric position using elemental
bromine at 0 ꢁC gave glycosyl bromide 3 in 96% yield.
Glycosylation of flavonol 4 with 3 was carried out using
heterogeneous activation conditions (Ag2O). Prepara-
tion of 4 was achieved in three steps from commercially
available 40,5,7-trihydroxyflavanone (naringenin) as
described previously.11 The crude, fully protected
Bu-SL0101 was debenzylated by hydrogenolysis
(Pd(OH)2, H2) to afford Bu-SL0101 (5) in 69% yield
for two steps. The synthesis of 3Ac-SL0101 was carried
out in a similar manner except that 1-bromo-2,3,4-tri-O-
acetyl-a-L-rhamnose (6) was used in the glycosylation
reaction with 4. Glycosyl bromide 6 was prepared using
a method reported by Morillo and co-workers in two
steps from L-rhamnose.12 Subsequent coupling of 4 with
6 gave the desired perbenzylated 3Ac-SL0101 (7) in 93%
yield, exclusively as the a-anomer as judged by the small
Based on our results we reasoned that increasing the cel-
lular uptake of SL0101 by enhancing its hydrophobic
character might afford a more potent RSK inhibitor in in-
tact cells. To test this hypothesis, kaempferol 3-O-(300,400-
di-O-butyryl-a-L-rhamnopyranoside) (Bu-SL0101) and
kaempferol 3-O-(200,300,400-tri-O-acetyl-a-L-rhamnopyr-
anoside) (3Ac-SL0101) were synthesized. The calculated
LogPs of Bu-SL0101 and 3Ac-SL0101 are 4.30 and
3.07, respectively, compared to 2.56 for SL0101.
Scheme 1.

J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
6037
J value. Complete debenzylation proceeded smoothly
using Pd(OH)2 to afford 3Ac-SL0101 (8) in 95% yield.
of the acetyl groups on the rhamnose with butyryl
groups also did not appreciably influence the in vitro
IC50.
2.3. Bu-SL0101 and 3Ac-SL0101 inhibit RSK activity
in vitro and in the intact cell
To determine whether 3Ac-SL0101 and Bu-SL0101
inhibited RSK in intact cells, we examined the phos-
phorylation of eukaryotic elongation factor 2 (eEF2)
in MCF-7 cells. eEF2 mediates the translocation step
in mRNA translation. eEF2 activity is regulated by
phosphorylation and it is inactivated by a highly specific
kinase, EF2 kinase (EF2K). RSK phosphorylates and
inactivates EF2K in response to mitogenic stimulation,
which leads to a decrease in phosphorylation of
eEF2.13 Thus, under conditions such as serum depriva-
tion, which leads to low RSK activity, eEF2 is phos-
phorylated by the active EF2K. However, stimulation
of RSK activity by mitogens results in reduced phos-
phorylation of eEF2 due to inactivation of EF2K by
RSK. Therefore, the phosphorylation state of eEF2
during mitogenic stimulation is an indicator of RSK
activity. Treatment of MCF-7 cells with the mitogen
phorbol dibutyrate (PDB) inactivated EF2K as shown
by the reduced levels of phosphorylated eEF2
(Fig. 3B, compare lanes 1 and 2). The levels of total
eEF2 were not altered by PDB treatment as shown by
the anti-eEF2 immunoblot. However, pre-incubation
of cells with SL0101 or its analogs eliminated PDB-med-
iated inactivation of eEF2K as shown by the elevated
levels of phosphorylated eEF2 (Fig. 3B, cf. lanes 3–5
with lane 2). In agreement with the literature, the levels
of eEF2 phosphorylation also remained increased in the
presence of U0126.13 We have previously demonstrated
that SL0101 does not affect the activation of MAPK, as
detected by the anti-active MAPK antibody.4 Here we
show that neither 3Ac-SL0101 nor Bu-SL0101 alters
PDB-stimulated MAPK activation (Fig. 3B). Therefore,
Bu-SL0101 and 3Ac-SL0101, like SL0101, do not inhibit
upstream kinases necessary for PDB-induced MAPK
activation, namely MEK, Raf, and protein kinase C
(PKC).
The ability of the analogs to inhibit RSK activity was
determined in an in vitro kinase assay and compared
to the results obtained with synthesized SL0101.11 The
data were fit using non-linear regression analysis and
the IC50s for Bu-SL0101 and 3Ac-SL0101 were 212
and 580 nM, respectively, as compared to 370 nM
obtained for the synthesized SL0101 (Fig. 3A). For
reasons that are not clear, we consistently obtain an
IC50 for the synthesized SL0101 that is higher than that
of the material originally isolated from F. refracta.
Nonetheless, as discussed above, kaempferol rhamno-
pyranosides with varying numbers and positions of
acetyl groups on the rhamnose moiety inhibited RSK
activity in the in vitro kinase assay with similar IC50s.
In agreement with these data, the addition of a third
acetyl group, generating 3Ac-SL0101, did not substan-
tially alter the in vitro IC50. Furthermore, replacement
2.4. 3Ac-SL0101 is a specific RSK inhibitor
To determine whether 3Ac-SL0101 and Bu-SL0101
demonstrated the same specificity as SL0101 in intact
cells, we compared the phosphorylation patterns from
MCF-7 cells pre-incubated with SL0101 or its analogs
prior to stimulation with PDB. The lysates were immu-
noblotted with multiple phospho-specific substrate anti-
bodies. We have previously shown that SL0101 does not
alter the phosphorylation patterns detected by these
antibodies, whereas changes are readily detected in cells
treated with U0126, as well as protein kinase A (PKA)
or PKC inhibitors.4 The phosphorylation patterns from
PDB-treated cells pre-incubated with vehicle, 3Ac-
SL0101 or SL0101 were similar (Fig. 4). But unlike
3Ac-SL0101 and SL0101, pre-treatment with Bu-
SL0101 decreased the PDB-induced phosphorylation
of a ꢀ25 kDa protein, as detected by the anti-phos-
pho-AKT substrate antibody. These results show that
Bu-SL0101 is not specific for inhibition of RSK activity.
However, 3Ac-SL0101 does not inhibit the phosphory-
lation of substrates associated with the kinase activity
Figure 3. Inhibition of RSK activity by Bu-SL0101 and 3Ac-SL0101.
(A) The potency of the synthesized compounds in inhibiting RSK
catalytic activity in vitro was measured as described in Figure 2A. (B)
Serum-deprived MCF-7 cells were pre-incubated with vehicle, or the
indicated concentration of inhibitor for 4 h. Cells were treated with
500 nM PDB or vehicle for 20 min prior to lysis. Protein concentration
of lysates was measured and lysates were electrophoresed, transferred,
and immunoblotted. Equal loading of lysate is demonstrated by the
anti-eEF2 and anti-ERK1/2 immunoblots.

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J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
data were fit using non-linear regression analysis.
Bu-SL0101 inhibited MCF-7 proliferation with an
EC50 of 40 lM, which was only slightly lower than that
obtained with SL0101 (Fig. 5A). Bu-SL0101 began to
significantly inhibit MCF-10A proliferation at a concen-
tration of 100 lM, which may reflect its ability to inhibit
Figure 4. Comparison of the effect of Bu-SL0101, 3Ac-SL0101 and
SL0101 on phosphorylation patterns in intact cells. MCF-7 cell lysates
normalized for protein concentration were electrophoresed, trans-
ferred, and immunoblotted with anti-phospho-PKA substrate, anti-
phospho-PKC substrate or anti-phospho-AKT substrate. Equal load-
ing of lysate is demonstrated by the anti-Ran immunoblot. The
asterisk indicates the location of a protein whose PDB-induced
phosphorylation is inhibited by pre-treatment with U0126 or
Bu-SL0101.
of other members of the AGC family, PKA, PKC, and
AKT. Likewise, 3Ac-SL0101 does not inhibit
kinases closely related to PKA, namely mitogen- and
stress-activated kinase and p70 S6 kinase (data not
shown). These data indicate that 3Ac-SL0101, like
SL0101, demonstrates specificity for RSK in intact cells.
2.5. 3Ac-SL0101 preferentially inhibits the growth of
breast cancer cells
Figure 5. 3Ac-SL0101 selectively inhibits MCF-7 cell proliferation. (A)
MCF-7 cells were treated with vehicle or the indicated concentration of
Bu-SL0101, 3Ac-SL0101, and SL0101; (B) MCF-7 and MCF-10A cells
were treated with vehicle or the indicated concentration of Bu-SL0101;
or (C) MCF-7 and MCF-10A cells were treated with vehicle or the
indicated concentration of 3Ac-SL0101. The cell number was mea-
sured after 48 h of treatment. Values given are the fold proliferation as
a percentage of that observed with vehicle-treated cells. Points, mean
(n = 2 in quadruplicate); bars = SD.
To test our hypothesis that Bu-SL0101 and 3Ac-SL0101
would be more potent inhibitors in intact cells than
SL0101, we determined their ability to inhibit the prolif-
eration of MCF-7 cells. The maximum solubility of Bu-
SL0101 and 3Ac-SL0101 in cell culture is ꢀ100 and
ꢀ50 lM, respectively, notwithstanding the higher calcu-
lated LogP of Bu-SL0101 than that of 3Ac-SL0101. The

J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
6039
kinases in addition to RSK (Fig. 5B). 3Ac-SL0101 was
ꢀ2-fold more potent than SL0101 at inhibiting the
growth of MCF-7 cells with an EC50 of 25 lM
(Fig. 5A). 3Ac-SL0101 did not substantially inhibit
MCF-10A proliferation at concentrations that effective-
ly inhibited MCF-7 cell growth by >80% (Fig. 5C). Thus
inhibition of RSK activity parallels the decrease in
MCF-7 cell proliferation;4 further, 3Ac-SL0101 demon-
strates a significantly improved inhibition of RSK
activity in intact cells relative to SL0101 without any
alteration in its specificity for RSK.
of the NTKD.4 Recently, another RSK inhibitor, a
fluoromethylketone, was identified that inhibits the
CTKD.17 Pretreatment of RSK with fluoromethylketone
in the absence of ATP produces an IC50 in an in vitro
kinase assay of 15 nM.17 In intact cells, which have an
intracellular ATP concentration of ꢀ1.4 mM18, a con-
centration of ꢀ10 lM fluoromethylketone is required
to completely inhibit the activity of ectopically expressed
RSK for phosphorylation of histone H317, a nonphysio-
logical substrate for RSK.19,20 Inhibition of the CTKD
autophosphorylation by fluoromethylketone should
inhibit mitogen-induced activation of NTKD activity.
However, the isolated NTKD is able to phosphorylate
exogenous substrates 21 and it may be that the fluorom-
ethylketone does not inhibit the basal activity of the
NTKD. Thus, by interacting with different kinase do-
mains SL0101 and the fluoromethylketone inhibit RSK
activity through different mechanisms.
3. Discussion
We have reported the identification of RSK as a novel tar-
get for anti-breast cancer and anti-prostate cancer
agents.4,7 We also described the identification and charac-
terization of SL0101, a RSK-specific inhibitor isolated
from F. refracta.4 SL0101 inhibits the growth of the
human breast cancer cell line MCF-7, and the human
prostatecancercell lines, LNCaP andPC-3, with aneffica-
cy paralleling its ability to inhibit RSK in intact cells.
However, the potency of SL0101 as a RSK inhibitor in in-
tact cells is ꢀ50-fold lower than its Ki and we postulated
that improving cellular uptake wouldincrease the potency
ofSL0101. Therefore, we synthesized the SL0101 analogs,
Bu-SL0101 and 3Ac-SL0101, which have calculated
LogP values of 4.30 and 3.07, respectively, compared to
2.56 for SL0101. 3Ac-SL0101 and SL0101 demonstrated
the same specificity for inhibition of RSK activity in intact
cells but Bu-SL0101 was not specific for RSK. Thus the
range of modifications that can be introduced on the
rhamnose while maintaining specificity for RSK inhibi-
tion may be limited. These results are in agreement with
our observations, which showed that the rhamnose moie-
ty is essential for the high affinity interaction of SL0101
withRSK.4 Despitethe>50-foldincreaseinhydrophobic-
ity the EC50 of Bu-SL0101 for inhibition of MCF-7 prolif-
eration was only slightly lower than that obtained for
SL0101. However, the EC50 of 3Ac-SL0101 for inhibiting
MCF-7 cell proliferation was >2-fold lower than that for
SL0101. Thus while increasing the hydrophobic character
of the SL0101 analogs can improve their efficacy in intact
cells, there is not a simple correlation between LogP and
EC50 for inhibition of MCF-7 cell growth.
The molecular mechanism that results in RSK playing a
dominant role in regulating proliferation in some cancer
types has not yet been elucidated. It has been proposed
that among the numerous events involved in tumorigen-
esis is an increased reliance on compromised signaling
pathways as well as the dormancy of alternative signaling
pathways.22,23 Thus it appears that some cancers
become dependent on RSK activity, rendering the prolif-
eration of these cells amenable to inhibition by a
RSK-specific inhibitor. The growth of normal cells is
presumably not dependent on RSK because intact sig-
naling pathways provide numerous mechanisms for
circumventing inhibition of a single signaling event.
The identification of RSK as a novel target and the syn-
thesis of a more potent SL0101 analog will allow the fur-
ther characterization of RSK inhibitors as potential
chemotherapeutic agents for breast and prostate cancers.
4. Experimental
4.1. Kinase assays
Glutathione-S-transferase (GST)-fusion protein (1 lg)
containing the sequence—RRRLASTNDKG (for
serine/threonine kinase assays) was adsorbed in the wells
of LumiNunc 96-well polystyrene plates (MaxiSorp
surface treatment). The wells were blocked with sterile
3% tryptone in phosphate-buffered saline. Kinase
(5 nM) in 70 lL of kinase buffer (5 mM b-glycerophos-
phate, pH 7.4, 25 mM Hepes, pH 7.4, 1.5 mM DTT,
30 mM MgCl2, and 0.15 M NaCl) was dispensed into
each well. Compound at indicated concentrations or
vehicle was added and reactions were initiated by the
addition of 30 lL ATP to a final ATP concentration
of 10 lM. Reactions were terminated after 30 min by
addition of 75 lL of 500 mM EDTA, pH 7.5. All assays
measured the initial velocity of reaction. After extensive
washing of wells, anti-p-p140 antibody, a polyclonal
phosphospecific antibody developed against the phos-
phopeptide, CGLA(pS)TND, and HRP-conjugated
anti-rabbit antibody (211-035-109, Jackson ImmunoRe-
search Laboratories, West Grove, Pennsylvania) were
used to detect serine phosphorylation of the substrate.
RSK is an unusual kinase in that it contains two non-
identical kinase domains, an N-terminal kinase domain
(NTKD) and a C-terminal kinase domain (CTKD) that
are separated by a linker region. The NTKD most closely
resembles p70 ribosomal S6 kinase and is responsible for
phosphorylating exogenous substrates.14 The CTKD
most closely resembles calmodulin-dependent kinase
and its only known substrate is RSK itself.15 In response
to mitogen treatment the CTKD is activated and auto-
phosphorylation by the CTKD enhances the activity of
the NTKD.16 The major determinant of SL0101 binding
specificity is a unique sequence present in the ATP-bind-
ing domain of the NTKD of RSK.4 Thus SL0101 acts to
directly inhibit the phosphorylation of exogenous sub-
strates via its interaction with the NTKD. SL0101 inhib-
its both the basal and mitogen-induced activity

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J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
HRP activity was measured using Western Lightning
Chemiluminescence Reagent (NEL102, PerkinElmer
Life Sciences) according to the manufacturer’s protocol.
Maximum and minimum activity is the relative lumines-
cence detected in the presence of vehicle and 200 mM
EDTA, respectively. His-tagged active RSK was ex-
pressed in Sf9 cells and purified using Ni–NTA resin
(Qiagen, Valencia, California). Baculovirus was pre-
pared using the Bac-to-Bacꢂ baculovirus expression sys-
tem (Invitrogen, Carlsbad, CA). Maximum responses
and the concentrations at half the inhibitory response
(IC50) were determined by performing a best-fit analysis
of the data (GraphPad Prism).
the residual 13C resonance of the solvent (CDCl3,
77.3 ppm; DMSO-d6, 39.5 ppm). Splitting patterns are
designated as follows: s, singlet; br, broad; d, doublet;
dd, doublet of doublets; t, triplet; q, quartet; m, multiplet.
High resolution mass spectra were obtained at the Mich-
igan State University-NIH Mass Spectrometry Facility.
LogP values were calculated using Molinspiration Prop-
erty Calculation Service (www.molinspiration.com).
4.3.1. Phenyl 3,4-di-O-butyryl-2-O-benzyl-1-thio-a-L-
rhamnopyranoside (2). A solution containing 0.87 g
(2.51 mmol) of phenyl 2-O-benzyl-1-thio-a-L-rhamno-
pyranoside (1)11, 1.19 g (1.25 mL, 7.54 mmol) of butyric
anhydride, 1.01 g (1.37 mL, 10.0 mmol) NEt3, and
0.03 g (0.25 mmol) of 4-N,N-dimethylaminopyridine in
10 mL of anhydrous CH2Cl2 was stirred at room tem-
perature under a N2 atmosphere for 2 h. The reaction
mixture was then diluted with 100 mL CH2Cl2 and
washed with two 100-mL portions of H2O. The organic
layer was separated, dried (MgSO4), and concentrated
under diminished pressure. The residue was purified
by flash chromatography on a silica gel column
(25 · 4 cm). Elution with 3:1 hexanes–ethyl acetate gave
4.2. Cell culture
For proliferation studies cells were seeded at 5000 cells
per well in 96-well tissue culture plates in the appropri-
ate medium as described by American Type Culture
Collection. After 24 h, the medium was replaced with
medium containing compound or vehicle as indicated.
Cell viability was measured at indicated time points
using CellTiter-GloTM assay reagent (Promega, Madi-
son, Wisconsin) according to manufacturer’s protocol.
Maximum responses and the concentrations of half the
effective response (EC50) were determined by performing
a best-fit analysis of the data (GraphPad Prism). For
specificity studies, cells were seeded at 3 · 105 cells/well
in 6-well plates. After 24 h, the cells were serum-starved
for 24 h then and incubated with compound or vehicle
for 4 h prior to a 20-min treatment with PDB. Cells were
lysed with boiling SDS-sample buffer without dithio-
threitol (DTT). The lysates were normalized for total
protein, and DTT was added to an aliquot, which was
electrophoresed and immunoblotted. Antibodies used
on cell lysates included anti-pan-MAPK (anti-ERK1/2)
(610124) from BD Transduction Laboratories; anti-
phospho-MAPK (V8031) from Promega; anti-eEF2
(2332), anti-phospho-eEF2 (2331), anti-phospho-Akt
Substrate (9611), anti-phospho-PKA Substrate (9621),
and anti-phospho-PKC Substrate (2261) from Cell Sig-
naling Technology. Anti-Ran was a generous gift from
Ian Macara (University of Virginia).
2 as a colorless oil: yield 1.21 g (100%); silica gel TLC Rf
21
D
0.40 (6:1 hexanes–ethyl acetate); ½aꢁ ꢂ50.1 (c 1.0,
1
CHCl3); H NMR (CDCl3) d 0.95 (q, 6H, J = 7.5 Hz),
1.24 (d, 3H, J = 6.3 Hz), 1.64 (m, 4 H), 2.26 (m, 4H),
4.11 (dd, 1H, J = 3.3 and 1.8 Hz), 4.30 (m, 1H), 4.55
(d, 1H, J= 12.3 Hz), 4.68 (d, 1H, J = 12.3 Hz), 5.20
(dd, 1H, J = 9.9 and 3.0 Hz), 5.32 (t, 1H, J = 9.9 Hz),
5.51 (d, 1H, J = 1.5 Hz), 7.31 (m, 8H) and 7.45 (m,
2H); 13C NMR (CDCl3) d 13.90, 17.72, 18.54, 18.70,
36.28, 36.41, 68.16, 71.31, 71.42, 72.80, 85.68, 127.69,
128.13, 128.64, 129.34, 131.55, 134.39, 137.67, 172.70
and 173.06; mass spectrum (FAB), m/z 487.2155 (M +
H)+ (C27H35O6S requires 487.2154).
4.3.2. 3,4-Di-O-butyryl-2-O-benzyl-a-L-rhamnopyranosyl
bromide (3). To a solution containing 0.54 g (1.12 mmol)
of 2 in 5 mL of anhydrous CH2Cl2 at 0 ꢁC under argon
was added 0.21 g (0.07 mL, 1.34 mmol) Br2. The reac-
tion mixture was stirred at 0 ꢁC for 20 min then diluted
with 20 mL CH2Cl2 and washed with 3% aq NaHSO3.
The organic layer was separated, dried (MgSO4), and
concentrated under diminished pressure. The crude
residue was then purified by flash chromatography on
a silica gel column (26 · 4 cm). Elution with 2:1
hexanes–ethyl acetate gave 3 as a colorless oil: yield
0.49 g (96%); silica gel TLC Rf 0.21 (2:1 hexanes–ethyl
acetate); 1H NMR (CDCl3) d 0.94 (q, 6H, J =
7.5 Hz), 1.25 (d, 3H, J = 6.3 Hz), 1.62 (m, 4H), 2.23 (m,
4H), 4.04 (m, 1H), 4.11 (dd, 1H, J = 3.3 and 1.8 Hz),
4.64 (q, 2H, J = 12.3 Hz), 5.30 (t, 1H, J = 9.9 Hz), 5.57
(dd, 1H, J = 10.2 and 3.3 Hz), 6.34 (d, 1H, J = 0.6 Hz)
and 7.33 (m, 5H); 13C NMR (CDCl3) d 13.71, 17.11,
18.31, 18.47, 35.99, 36.11, 69.67, 70.27, 71.26, 73.37,
79.20, 86.35, 127.96, 128.18, 128.57, 137.18, 172.31 and
172.68. Note: this compound must be used promptly;
product decomposes rather rapidly (1–2 days).
4.3. Chemistry
Reagents and solvents were of reagent grade and used
without further purification. Methylene chloride was dis-
tilled from calcium hydride, and toluene was distilled
from sodium. Anhydrous grade THF was purchased from
VWR. All reactions involving air- or moisture-sensitive
reagents or intermediates were performed under a nitro-
gen or argon atmosphere. Flash chromatography was
performed using Silicycle 40–60 mesh silica gel. Analytical
TLC was performed using 0.25 mm EM silica gel 60 F250
plates that were visualized by irradiation (254 nm) or by
staining with Hanessian’s stain (cerium molybdate). Opti-
cal rotations were obtained using a Jasco digital polarim-
1
eter. H and 13C NMR spectra were obtained using 300
and 500 MHz Varian instruments. Chemical shifts are
reported in parts per million (ppm d) referenced to the
residual H resonance of the solvent (CDCl3, 7.26 ppm;
4.3.3. Kaempferol 3-O-(300,400-di-O-butyryl-a-L-rhamno-
pyranoside) (Bu-SL0101) (5). To a stirred suspension
containing 0.13 g (0.23 mmol) of 4,11 0.11 g (0.46 mmol)
1
DMSO-d6, 2.49 ppm). 13C spectra were referenced to

J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
6041
˚
Ag2O and 4 A molecular sieves in 5 mL CH2Cl2 was
gel column (20 · 2 cm). Elution with 1:1:0.1 hexanes–
ethyl acetate–methanol gave 8 as a yellow foam: yield
0.064 g (95%); silica gel TLC Rf 0.37 (1:1:0.1 hexanes–
added 0.21 g (0.46 mmol) of 3 as a solution in 4 mL
CH2Cl2. The reaction mixture was stirred at room tem-
perature for 4 h, then diluted with 20 mL CH2Cl2 and
filtered through a Celite pad. The filtrate was concen-
trated under diminished pressure. This crude residue
was then dissolved in 5 mL of 1:1 THF–MeOH and
45 mg Pd(OH)2/C was added. The reaction vessel was
purged with H2 three times then maintained under a
H2 atmosphere for 2 h. The reaction mixture was then
filtered through Celite and the filtrate was concentrated.
The residue was purified by flash chromatography on a
silica gel column (25 · 2 cm). Elution with 1:1:0.1 hex-
anes–ethyl acetate–methanol gave 5 as a brown solid:
22
1
ethyl acetate–methanol); ½aꢁ ꢂ92.2 (c 0.6, CHCl3); H
D
NMR (CDCl3) d 0.92 (t, 3H, J = 6.3 Hz), 2.01 (s, 3H),
2.02 (s, 3H), 2.05 (s, 3H), 3.52 (m, 1H), 4.96 (t, 1H,
J = 9.9 Hz), 5.30 (s, 1H), 5.33 (dd, 1H, J = 9.9 and
3.0 Hz), 5.63 (s, 1H), 6.25 (s, 1H), 6.35 (s, 1H), 6.99
(d, 2H, J = 8.1 Hz), 7.18 (br s, 1H), 7.72 (d, 2H,
J = 8.1 Hz), 7.89 (br s, 1H) and 12.49 (s, 1H); 13C
NMR (CDCl3) d 16.97, 20.68, 20.74, 20.78, 68.33,
69.26, 70.38, 77.20, 94.18, 98.08, 99.27, 105.27, 115.66,
121.53, 130.67, 133.80, 156.78, 157.64, 159.10, 161.66,
163.08, 170.47, 170.57, 171.08 and 177.69; mass spec-
trum (FAB), m/z 559.1449 (M+H)+ (C27H27O13 requires
559.1452).
yield 0.090 g (69%); silica gel TLC Rf 0.30 (1:1:0.1 hex-
20
anes–ethyl acetate–methanol); ½aꢁ
ꢂ115.6 (c 1.2,
CHCl3); 1H NMR (DMSO-d6) D d 0.85 (d, 3H,
J = 6.3 Hz), 0.97 (m, 6H), 1.64 (m, 4H), 2.33 (m, 4H),
3.49 (m, 1H), 4.46 (s, 1H), 4.79 (br s, 1H); 5.14 (t, 1H,
J = 9.9 Hz), 5.27 (dd, 1H, J = 6.3 and 3.0 Hz), 5.66 (d,
1H, J = 1.8 Hz), 6.32 (d, 1H, J = 1.8 Hz), 6.53 (d, 1H,
J = 2.1 Hz), 7.11 (d, 2H, J = 8.7 Hz), 7.92 (d, 2H,
J = 8.4 Hz), 9.39 (br s, 2H) and 12.68 (s, 1H); mass spec-
trum (FAB), m/z 573.1970 (M+H)+ (C29H33O12 requires
573.1972).
Acknowledgments
This work was supported by Department of Defense #
DAMD17-03-1-0366 USAMRMC, by the Paul Mellon
Prostate Cancer Institute, the Prostate Cancer Founda-
tion and the Patients and Friends of the Cancer Center,
University of Virginia.
4.3.4. 5,7-Bis-(benzoxy)-2-(4-(benzoxy)phenyl)-3-[200,300,400-
tri-O-acetyl-a-L-rhamnopyranosyloxy]-4H-chromen-4-
one (7). To a stirred suspension containing 0.090 g
(0.162 mmol) of 4,11 0.075 g (0.324 mmol) Ag2O and
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4A molecular sieves in 5 mL CH2Cl2 was added
0.114 g (0.324 mmol) of 612 as a solution in 4 mL
CH2Cl2. The reaction mixture was stirred at room tem-
perature for 24 h under N2 then diluted with 20 mL
CH2Cl2 and filtered through a Celite pad. The filtrate
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22
1
½aꢁ ꢂ96.8 (c 0.5, CHCl3); H NMR (CDCl3) d 0.85 (d,
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3.39 (m, 1H), 4.94 (t, 1H, J = 9.9 Hz), 5.07 (s, 2H), 5.15
(s, 2H), 5.30 (s, 2H), 5.34 (m, 1H), 5.74 (s, 2H), 6.45(d,
1H, J = 1.8 Hz), 6.55 (d, 1H, J = 1.8 Hz), 7.16 (d, 2H,
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154.16, 158.75, 159.74, 160.49, 162.78, 169.52, 169.80,
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3-O-(200,300,400-tri-O-acetyl-a-L-
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residue was purified by flash chromatography on a silica

6042
J. A. Smith et al. / Bioorg. Med. Chem. 14 (2006) 6034–6042
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