Structural optimization and biological screening of a steroidal scaffold possessing cucurbitacin-like functionalities as B-Raf inhibitors

Article abstract of DOI:10.1002/cmdc.201300523

Inhibition of the mitogen-activated protein kinase (MAPK) pathway by targeting the commonly occurring mutated B-Raf in melanoma has become a practical method for the development of drugs and drug candidates. In order to expand upon the currently reported structural scaffolds used to target the MAPK pathway, molecular docking studies led to the installation an α,β-unsaturated ketone side chain, related to the cucurbitacin class of natural products, on to an estrone core via an aldol condensation reaction, along with installation of the Δ^9,11 olefin to assemble what has been defined as a pseudo-cis configuration at the B/C ring juncture. Combination of these cucurbitacin-like features resulted in a compound with an enhanced biological profile against the A-375 mutant B-Raf cell line, in regards to their cytotoxicity and inhibitory activity toward phosphorylated extracellular-signal-regulated kinase (ERK). A hybrid scaffold: Molecular docking studies resulted in the design of hybrid compounds containing the α,β-unsaturated ketone of cucurbitacin D, a triterpene natural product, and an estrone core, along with a Δ^9,11 olefin. When evaluated in A-375 mutant B-Raf cells, compounds with this combination of cucurbitacin-like and steroidal features exhibited potent inhibition of phosphorylated extracellular-signal-regulated kinase (ERK).

Full text of DOI:10.1002/cmdc.201300523

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DOI: 10.1002/cmdc.201300523
Structural Optimization and Biological Screening of
a Steroidal Scaffold Possessing Cucurbitacin-Like
Functionalities as B-Raf Inhibitors
Mahmoud S. Ahmed, Lucas C. Kopel, and Fathi T. Halaweish*[a]
Inhibition of the mitogen-activated protein kinase (MAPK)
pathway by targeting the commonly occurring mutated B-Raf
in melanoma has become a practical method for the develop-
ment of drugs and drug candidates. In order to expand upon
the currently reported structural scaffolds used to target the
MAPK pathway, molecular docking studies led to the installa-
tion an a,b-unsaturated ketone side chain, related to the cu-
curbitacin class of natural products, on to an estrone core via
an aldol condensation reaction, along with installation of the
D9,11 olefin to assemble what has been defined as a pseudo-cis
configuration at the B/C ring juncture. Combination of these
cucurbitacin-like features resulted in a compound with an en-
hanced biological profile against the A-375 mutant B-Raf cell
line, in regards to their cytotoxicity and inhibitory activity
toward phosphorylated extracellular-signal-regulated kinase
(ERK).
tion loop.[6] This mutation keeps the kinase pathway constitu-
tively active, leading to aberrant growth of melanoma cells.
Therefore, inhibition of the MAPK signaling cascade at the
level of B-Raf provides an approach for the treatment of mela-
noma.
Cucurbitacins exhibit a broad spectrum of biological activi-
ties because of their ability to target multiple biological path-
ways, sometimes undesirably.[7] However, this does not elimi-
nate them as a potential source for drug development. Struc-
tural modifications to natural products and their derivatives
continue to allow for increased selectivity in regards to a de-
sired biological response, while at the same time decreasing
adverse effects.[8] Instrumental to this process is the identifica-
tion of important structural features that will remain and
which to modify or replace.
Cucurbitacin analogues resulting from synthetic transforma-
tions on the isolated natural products have already been
shown by Guo et al., Lang et al., and Bartalis et al. to have sig-
nificant effects on the cytotoxicity of these compounds to-
wards various cancer cell lines.[9] The structure–activity relation-
ships of cucurbitacins suggests the importance of the 23,24-
olefin of the a,b-unsaturated ketone side chain.[10] Therefore,
the side chain containing an enone was identified as an impor-
tant pharmacophore to maintain during analogue develop-
ment. This leaves the tetracyclic core for further modification.
Upon first glance at the chemical structure of cucurbitiacins,
known as [19(10!9b)-abeo-10a-lanost-5-ene],[2d] one recog-
nizes the similarities they possess with steroids (Figure 1). How-
ever, the cucurbitane ring system differs from that of steroids
by the presence of a C-19 methyl group at C-9 instead of the
usual C-10 position and the relative orientation at the B/C ring
juncture—cis for cucurbitacins and trans for steroids. Due to
their similarities and the ability to install new functionality,
a steroid core was identified as a potential replacement for the
highly functionalized tetracyclic core of the cucurbitacins.[11] In
particular, the estrone skeleton was identified for derivatization
due to the synthetic ability to install various functionalities
while affecting the geometry of the B/C ring juncture, a key
structural element of the cucurbitacin scaffold. Support for this
decision comes from the ability of estrogen analogues to
affect other biological processes without producing the nega-
tive side effects associated with estrogen treatment.[12]
Natural products have been an important source for drug dis-
covery over the years. They are commonly used directly, or in-
directly via semi-synthetic modifications to their structures in
order to potentiate their activity, in an attempt to solve various
clinical problems. Natural products continue to show potential
as sources for drug candidates targeting a wide range of appli-
cations: antihypertensive, antidiabetics, antibacterial, anti-
fungal, antimicrobial, and anticancer agents.[1]
Cucurbitacins are highly oxygenated triterpene natural prod-
ucts, found mainly in the Cucurbitaceae family of plants, that
possess an array of biological activities: anti-inflammatory, hep-
atoprotective and antiproliferative activities against various
cancer cell lines.[2] However, only a limited number of studies
have been reported describing the biological effect of cucurbi-
tacins towards melanoma cell lines,[3] as well as their ability to
target the mitogen-activated protein kinase (MAPK) pathway,
a target for the treatment of melanoma.[4] The Ras/Raf/MEK/
ERK signaling pathway is a kinase receptor pathway that has
been heavily studied for the treatment of melanoma due to its
major role in multiple cellular processes, such as proliferation,
differentiation, survival, and apoptosis.[5] B-Raf mutations show
high incidence, about 66%, in melanoma. The most common
mutation (~90%) is the change of Val600 to Glu in the activa-
[a] M. S. Ahmed, Dr. L. C. Kopel, Prof. Dr. F. T. Halaweish
Department of Chemistry & Biochemistry, South Dakota State University
Box 2202, Brookings, SD 57007 (USA)
In an effort to maximize analogue development for targeting
the MAPK pathway, molecular modeling was used to investi-
gate the installation of key structural features by systematically
integrating cucurbitacin functionality with the steroidal skele-
ton and docking these virtual compounds against the mutant
E-mail: fathi.halaweish@sdstate.edu
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300523.
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aliphatic- and aromatic-substituted a,b-unsaturated ketones on
the side chain, when compared with the known B-Raf inhibitor
PLX-4032. The binding modes of alkyl- and aromatic-substitut-
ed enone systems installed on the estrone skeleton, such as
MSA-1, MSA-2 and MSA-3, showed hydrophobic–hydrophobic
interactions. Upon installation of the cucurbitacin D side chain
to the steroidal skeleton to form MSA-4, the binding affinity
was improved via hydrophobic–hydrophobic interactions and
a hydrogen-bonding interaction between the hydroxy group
at C-25 and Asp594:A in the Asp-Phe-Gly (DFG) motif. Mean-
while, installation of the D9,11 olefin at the B/C ring juncture of
MSA-4 to obtain MSA-5 showed a greater predicted binding
affinity via the hydrogen-bonding interaction between the car-
bonyl of the a,b-unsaturated ketone and Asp594:A in the DFG
motif, as shown in Figure 3.
Previously reported synthetic efforts towards assembling the
a,b-unsaturated ketone side chain onto the steroidal scaffold
involved taking advantage of the aldol coupling reaction, by
Figure 1. Structures of relevant cucurbitacins and estrone starting materials.
B-Raf receptor.[13] The two structural regions of the estrone
scaffold explored were 1) incorporation of various substituted
enone side chains at the C-17 position, and 2) modification of
the B/C ring juncture, as shown in Figure 2.
The molecular docking studies were conducted using Open-
Eye Scientific Software: Fast Rigid Exhaustive Docking (FRED)
Receptor, Omega,[14] FRED[15] and VIDA. The molecular docking
results revealed significant binding affinity towards the crystal
structure of mutant B-Raf for the estrone derivatives containing
Figure 3. Visual Representation of A) MSA-4 and B) MSA-5 in the ATP bind-
ing site of B-Raf V600E, where the arrow shows hydrogen bond with
Asp594:A.
Figure 2. Modifications to the steroidal skeleton identified via molecular
docking.
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Scheme 1. A) Synthesis of reagent 4 and B) synthesis of analogue MSA-4 and installation of a D9,11 olefin at the B/C ring juncture for the synthesis of MSA-5.
Reagents and conditions: a) TBSCl, imidazole, DMF, 70%; b) DIBAL-H, hexane, ꢀ788C, 80%; c) TPAP, NMO, CH2Cl2, 4 ꢁ, 51%; d) LDA, THF, ꢀ788C, 58%; e) TBAF,
THF, 60%; f) DDQ, MeOH, CH2Cl2, 80%; g) LDA, THF, ꢀ788C, 60%; h) TBAF, THF, 70%.
reacting the desired a-hydroxy ketone (5) with various com-
mercially available aldehydes, followed by in situ elimination
to form the enone.[16] The aldol reaction here allows for the in-
stallation of a wide range of substituents onto the a,b-unsatu-
rated ketone, simply by changing the aldehyde fragment. The
new analogue (MSA-3) was synthesized via the previously de-
scribed method.[16] Unfortunately, the aldehyde fragment
needed for installation of the cucurbitacin D side chain re-
quired synthesis.
sure of hydroxy ketone 5 to 2,3-dichloro-5,6-dicyano-1,4-ben-
zoquinone (DDQ) in dichloromethane provided the newly
formed alkene at C-9 and C-11 in 80% yield.[19] The resulting
ketone was then treated with identical aldol and silyl deprotec-
tion conditions as before to afford enone 8 and MSA-5 in 60%
and 70% yields, respectively, completing this initial series of
synthesized analogues (Scheme 1B).
The synthesized analogues were consequently subjected to
in vitro biological screening to validate their ability to target
the MAPK pathway by initially employing an MTT cell viability
assay. Analogues MSA-1, MSA-2 and MSA-3 exhibited modest
cytotoxicity with IC50 values ranging from 20–30 mm towards
the mutant B-Raf A-375 cell line, while MSA-4 and MSA-5 dis-
played improved inhibitory activity towards the same cell line.
The higher IC50 values for MSA-1, MSA-2, and MSA-3 can po-
tentially be attributed to their higher logP values.[20] Their high
lipophilicity can lead to a greater plasma-protein binding affini-
ty, which can lead to higher IC50 values. The lipophilicity pro-
files for MSA-4 and MSA-5 appear to be improved compared
with the other analogues and are hypothesized to have
a direct impact on the activity, as shown in Table 1.
The three-step synthetic sequence to aldehyde 4 began
with protection of methyl 2-hydroxyisobutyrate (1) with tert-
butyldimethylsilyl chloride (TBSCl) (Scheme 1A).[17] The result-
ing ester was reduced with diisobutylaluminum hydride
(DIBAL-H) to afford alcohol 3 in 80% yield. This was followed
by a controlled oxidation using tetrapropylammonium per-
ruthenate (TPAP) and N-methylmorpholine N-oxide (NMO) to
arrive at desired volatile aldehyde 4 in 51% yield.
Attempts to react a-hydroxy ketone 5 with protected alde-
hyde 4 via an aldol condensation reaction using lithium hy-
droxide as the base, in a refluxing mixture of THF/water, result-
ed in the recovery of starting ketone 5 and the disappearance
of aldehyde 4. Therefore, an alternative procedure was used in-
volving preforming the enolate with lithium diisopropylamine
(LDA) at ꢀ788C, followed by addition of TBS-protected alde-
hyde 4 and warming the reaction mixture to room tempera-
ture to yield enone 6 in 58% yield, along with some unreacted
ketone 5. Final treatment of enone 6 with tetrabutylammoni-
um fluoride (TBAF) provided MSA-4 in 60% yield
(Scheme 1B).[18]
Cucurbitacins were previously shown to possess inhibitory
activity towards phosphorylated extracellular-signal-regulated
kinase (ERK), as well as total ERK levels.[4a] This was attributed
to the ability of cucurbitacins to target multiple biological tar-
gets without any specificity. However, when cucurbitacin ana-
logues MSA-4 and MSA-5 were tested employing a cell-based
enzyme-linked immunosorbent assay (ELISA) in A-375 melano-
ma cells, the synthesized analogues showed the ability to only
inhibit the phosphorylated ERK levels induced by epidermal
growth factor (EGF), as shown in Figure 4.
Upon completing the installation of the cucurbitacin D side
chain onto the estrone core, attention was directed toward the
installation of the D9,11 olefin at the B/C ring juncture of the
steroidal skeleton to attain the pseudo-cis configuration. Expo-
In conclusion, steroidal analogues were designed using mo-
lecular modeling in order to mimic the cucurbitacin skeleton
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using ChemOffice Ultra 2004 (CambridgeSoft Corp.). The energies
of the 3D structures were minimized using semi-empirical PM3 cal-
culations. The energy-minimized structures were then converted
into .pdb files, maintaining all heavy atoms.
Table 1. Cell viability data against the A-375 cell line, molecular weight
(MW), and calculated LogP values of MSA analogues.
Compd
IC50 [mm][a]
MW [gmolꢀ1
]
cLogP[b]
LogP calculations: cLogP values were calculated using ALOGPS
2.1 software (Virtual Computational Chemistry Laboratory) using
the energy-minimized structures.[21]
MSA-1
MSA-2
MSA-3
MSA-4
MSA-5
PLX-4032
26.80ꢁ0.44
30.74ꢁ1.90
27.59ꢁ0.12
19.90ꢁ0.21
12.20ꢁ0.12
3.01ꢁ0.30
474.63
424.61
462.59
426.78
424.57
413.82
6.30ꢁ0.98
6.23ꢁ0.80
6.51ꢁ0.86
4.86ꢁ0.74
4.58ꢁ0.92
3.42ꢁ0.49
Generation of conformers: The energy-minimized structures were
combined into a single continuous pdb file for use as an input for
Omega.[22] The Omega utility uses the MMFF94 force field to gen-
erate multiple conformations for each input ligand in the library in
order to induce ligand flexibility in an otherwise rigid model. Modi-
fications applied to the default settings of OMEGA were: 1) inde-
pendent of conformers with an energy difference to the global
minimum of >5.0 kcalmolꢀ1 (GP_ENERGY_WINDOW), 2) maximum
number of output conformers 400 (GP NUM OUTPUT CONFS),
and 3) low-energy selection of conformers from the final ensemble
(GP_SELECT_RANDOM false), with the root-mean square deviation
(RMSD) cut-off of 0.8 ꢁ (GP_RMS_CUTOFF). In addition, the maxi-
mum number of rotatable bonds in the molecule was raised to 30
(GP_MAX_ROTORS) in order to generate conformers for all ligands
of our data set.[23]
[a] Data represent the mean ꢁSEM of n=3 independent experiments
performed in triplicate. [b] Calculated LogP values were determined
using ALOGPS 2.1, and data represent the correlation between the IC50,
the molecular weight, and the lipophilicity profile of the tested com-
pounds.
Receptor preparation: The B-Raf V600E receptor structure was taken
from the RCSB Protein Data Bank (PDB ID: 3OG7[15]) and prepared
for modeling using Fast Rigid Exhaustive Docking (FRED).[24] The
application allows for the creation a grid box by the mode selec-
tion pane and adjustment of its size using the mode controls. The
box size should never exceed 50000–60000 ꢁ. Once the grid box
size has been adjusted, the receptor is ready for use in the docking
calculations.[15]
Figure 4. Graphical representation for the inhibitory effect of the MSA ana-
logues over the total (&) and phosphorylated (&) ERK expression induced
by epidermal growth factor (EGF).
Docking: Multiple scoring functions were employed in order to
obtain a consensus structure and score in the final output.[25] The
scoring functions include Shapegauss, Chemgauss3, Oechemscore,
Screen score, and PLP. For further details of each scoring function
and consensus score, see the OpenEye Scientific Software FRED
manual.[24,25] Snapshots and visualization of the chemical interac-
tions between the analogues and receptor were obtained using
the VIDA application.[26]
and their ability to bind to the mutant B-Raf receptor, while at-
tempting to increase activity and selectivity. A variety of side
chains containing substituted a,b-unsaturated ketones were
assembled at the C-17 position of estrone via aldol condensa-
tion reactions using different aldehydes, such as para-fluoro-
benzaldehyde, para-methoxybenzaldehyde, pivaldehyde, and
TBS-protected 2-hydroxy isobutyraldehyde (4). The cytotoxicity
data exhibited the following increasing order of activity with
respect to substitution on the enone: tert-butyl (MSA-2) <
para-fluorophenyl (MSA-3) < para-methoxyphenyl (MSA-1) <
propan-2-ol (MSA-4). Added modification of the estrone core
of MSA-4 by formation of the D9,11 olefin at the B/C ring junc-
ture provided MSA-5 with what has been classified as
a pseudo-cis configuration. This change in the structure led to
an improvement in the activity of MSA-5 compared with MSA-
4, exhibiting greater cytotoxicity and inhibitory activity toward
phosphorylated ERK by up to 33%.
Chemistry
General methods: 1H and 13C NMR spectra were acquired on
a Bruker AVANCE-400 MHz NMR spectrometer, in CDCl3 using tetra-
1
methylsilane (d=0 ppm) as the internal standard for H NMR and
the residual solvent peak (d=77.16 ppm) for 13C NMR. Chemical
shifts (d) are reported in parts per million (ppm), with coupling
constants (J) in Hertz (Hz) and the signal multiplicities as singlet (s),
doublet (d), triplet (t), quartet (q), doublet of doublets (dd), dou-
blet of triplets (dt), multiplet (m), or broad (br). High-resolution
mass spectrometry (HRMS) data were obtained using either elec-
tron ionization (EI) on a ThermoFinnigan MAT 95 XL mass spec-
trometer or electrospray ionization (ESI) on a ThermoFinnigan LCQ
Advantage IonTrap liquid chromatography–mass spectrometer (LC/
MS). Melting points (mp) were determined on a Vernier Melt Sta-
tion melting point apparatus and are uncorrected. All reagents and
solvents were obtained from commercial suppliers and used as re-
ceived. All reactions requiring anhydrous conditions were per-
formed in oven-dried glassware under an atmosphere of nitrogen.
Experimental Section
Molecular modeling
2D and 3D Structures: A virtual library of 100 structurally modified
C-17 estrone analogues containing an enone side chain, standard
cucurbitacins, and known mutant B-Raf inhibitors were prepared
(20R,22E)-24-(p-fluorophenyl)-20-hydroxy-3-methoxy-19-norcho-
lan-1,3,5(10),23-tetraen-22-one (MSA-3):
A stirred solution of
(iPr)2NH (0.57 mL, 3.70 mmol) in THF (11.6 mL) was cooled to
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ꢀ788C, treated dropwise with nBuLi (2.5m in hexanes, 1.52 mL,
3.70 mmol), and stirred at ꢀ788C for 1 h. A solution of a-hydroxy
ketone 5 (0.40 g, 1.09 mmol) in THF (2.18 mL) was then added, and
the reaction was stirred for an additional 1 h at ꢀ788C. A solution
of para-fluorobenzaldehyde (0.29 g, 2.18 mmol) in THF (14.5 mL)
was then added at ꢀ788C, and the solution was allowed to slowly
warm to RT and stirred for 20 h. The reaction was quenched by the
addition of saturated aq NH4Cl (20 mL), followed by extraction of
the aqueous layer with EtOAc (3ꢂ50 mL). The combined organic
phases were dried over Na2SO4, filtered and concentrated in vacuo.
The crude product was purified by silica gel column chromatogra-
phy (hexane/EtOAc, 95:5) to yield MSA-3 as a white solid (0.32 g,
60%); mp: 95–978C; 1H NMR (400 MHz, CDCl3): d=7.80 (d, J=
15.6 Hz, 1H), 7.60 (dd, J=5.2, 8.8 Hz, 2H), 7.20 (d, J=8.5 Hz, 1H),
7.10 (dd, J=5.2, 8.8 Hz, 2H), 6.99 (d, J=15.6 Hz, 1H), 6.72 (dd, J=
8.5, 2.7 Hz, 1H), 6.62 (d, J=2.7 Hz, 1H), 4.18 (s, 1H), 3.76 (s, 3H),
2.92–2.76 (m, 2H), 2.39–2.11 (m, 3H), 1.97–1.16 (m, 11H), 1.56 (s,
3H), 0.96 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=201.8, 165.5,
163.0, 157.4, 144.4, 138.0, 132.7, 130.7, 130.7, 130.6, 126.2, 118.2,
116.3, 116.1, 113.8, 111.5, 79.2, 55.7, 55.2, 55.1, 50.8, 44.3, 43.8, 43.0,
38.1, 29.8, 27.6, 26.7, 24.3, 23.7, 22.1,13.7 ppm; HRMS (EI): m/z [M]+
calcd for C30H35FO3: 462.2569, found: 462.2565.
1.17 (s, 6H), 0.79 (s, 9H), 0.08 ppm (s, 6H); 13C NMR (100 MHz,
CDCl3): d=206.4, 80.3, 28.0, 27.2, 20.4, 0.3 ppm. Note: The product
is unstable at RT and should be stored at ꢀ208C; the aldehyde is
stable for two weeks under argon at ꢀ208C.
(20R,22E)-25-tert-butyldimethylsilyloxy-20-hydroxy-3-methoxy-
19-norcholesta-1,3,5(10),23-tetraene-22-one (6): A stirred solution
of (iPr)2NH (0.57 mL, 3.70 mmol) in THF (11.6 mL) was cooled to
ꢀ788C, treated dropwise with nBuLi (2.5m in hexanes, 1.52 mL,
3.70 mmol), and stirred at ꢀ788C for 1 h. A solution of a-hydroxy
ketone 5 (0.40 g, 1.09 mmol) in THF (2.18 mL) was then added, and
the reaction was stirred for an additional 1 h at ꢀ788C. A solution
of 4 (0.44 g, 2.18 mmol) in THF (14.5 mL) was added at ꢀ788C, and
the solution was slowly warmed to RT and stirred for 20 h. The re-
action was quenched by addition of saturated aq NH4Cl (20 mL),
followed by extraction of the aqueous layer with EtOAc (3ꢂ50 mL).
The combined organic phases were dried over Na2SO4, filtered, and
concentrated in vacuo. The crude product was purified by silica gel
column chromatography (hexane/EtOAc, 4:1) to yield 6 as an off-
1
white solid (0.35 g, 58%); H NMR (400 MHz, CDCl3): d=7.10 (d, J=
8.5 Hz, 1H), 6.95 (d, J=15.1 Hz), 6.60 (d, J=14.9 Hz, 1H), 6.60 (dd,
J=8.8, 2.7 Hz, 1H), 6.51 (d, J=2.52 Hz, 1H), 4.01 (s, 1H), 3.66 (s,
3H), 2.96–2.76 (m, 2H), 2.40–2.16 (m, 3H), 1.92–1.09 (m, 11H), 1.48
(s, 6H), 1.12 (s, 3H), 0.92 (s, 3H), 0.75 (s, 9H), 0.08 ppm (s, 6H);
13C NMR (100 MHz, CDCl3): d=204.5, 173.2, 159.4, 140.0, 134.7,
128.3, 120.3, 115.8, 113.5, 81.1, 75.6, 62.4, 57.9, 57.2, 56.9, 46.3, 45.9,
42.7, 40.2, 32.0, 29.6, 28.3, 27.9, 27.8, 27.4, 26.2, 25.7, 23.1, 20.3,
20.2, 16.2, 15.6, 0.0 ppm; HRMS (ESI): m/z [M+Na]+ calcd for
C33H52O4Si: 563.3535, found: 563.3527.
Methyl 2-tert-butyldimethylsilyloxy-2-methyl propanoate (2):
Methyl 2-hydroxy isobutyrate 1 (5.00 g, 42.3 mmol) was dissolved
in DMF (12.5 mL), and the solution was treated with TBSCl (7.65 g,
50.75 mmol) and imidazole (7.45 g, 110.0 mmol). The reaction was
allowed to stir at RT for 2 d. Saturated aq NaHCO3 (30 mL) was
added to the reaction, followed by the addition of hexane/EtOAc
(96:4, 10 mL). The aqueous layer was extracted using hexane/
EtOAc (96:4, 3ꢂ50 mL), and the combined organic layers were
dried over Na2SO4, filtered and concentrated in vacuo. The crude
material was purified using silica gel column chromatography
(hexane/EtOAc, 9:1) to yield 2 as a pale yellow oil (7.00 g, 70%);
1H NMR (400 MHz, CDCl3): d=3.60 (s, 3H), 1.34 (s, 6H), 0.80 (s, 9H),
0.08 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=175.8, 74.5, 51.4,
28.4, 25.5, 17.9, ꢀ3.2 ppm.
(20R,22E)-20,25-dihydroxy-3-methoxy-19-norcholesta-1,3,5,(10),
23-tetraen-22-one (MSA-4): TBAF (1m in THF, 4.0 mL, 4.00 mmol)
was added to a stirred solution of 6 (0.70 g, 1.29 mmol) in THF
(20.0 mL), and the reaction was stirred for 6 h at RT. The reaction
mixture was quenched by addition of saturated aq NH4Cl (20 mL).
The aqueous layer was extracted with EtOAc (3ꢂ50 mL), and the
combined organic layers were dried over Na2SO4, filtered and con-
centrated in vacuo. The crude material was purified using silica gel
column chromatography (hexane/EtOAc, 4:1!7:3!3:2) to yield
MSA-4 as a white solid (0.34 g, 60%); mp: 80–838C; 1H NMR
(400 MHz, CDCl3): d=7.21 (d, J=8.5 Hz, 1H), 7.15 (d, J=15.3 Hz,
1H), 6.71 (dd, J=8.5, 2.7 Hz, 1H), 6.69 (d, J=15.3 Hz, 1H), 6.63 (d,
J=2.8 Hz, 1H), 4.11 (s, 1H), 3.77 (s, 3H), 2.96–2.76 (m, 2H), 2.40–
2.16 (m, 3H), 1.92–1.09 (m, 11H), 1.49 (s, 3H), 1.40 (s, 6H),
0.92 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=202.3, 157.4, 155.9,
137.9, 132.7, 126.2, 118.1, 113.7, 111.4, 79.2, 71.3, 55.6, 55.2, 54.6,
44.3, 43.8, 40.6, 38.1, 29.8, 29.5, 27.6, 26.6, 24.1, 23.6, 21.9, 14.2,
13.6 ppm; HRMS (ESI): m/z [M+Na]+ calcd for C27H38O4: 449.2669,
found: 449.2662.
2-tert-Butyldimethylsilyloxy-2-methyl propanol (3): DIBAL-H
(1.2m in hexane, 10.0 mL, 12.0 mmol) was added to a stirred solu-
tion of ester 2 (1.27 g, 5.46 mmol) in hexane (15.6 mL) at ꢀ788C
over 10 min, and then the solution was allowed to stir for 20 min.
The reaction was warmed to RT and stirred for 20 min until TLC
confirmed the disappearance of the starting material. The reaction
was quenched by addition of a saturated aq solution of potassium
sodium tartrate (20 mL) at ꢀ788C, and the mixture was allowed to
warm to RT and vigorously stirred overnight. The reaction mixture
was extracted with EtOAc (3ꢂ50 mL), and the combined organic
layers were dried over Na2SO4, filtered and concentrated in vacuo
1
to provide 3 as colorless clear oil (1.05 g, 90%); H NMR (400 MHz,
(20R)-3-Methoxy-20-hydroxy-19,24-dinorcholan-1,3,5(10),9(11)-
tetraen-22-one (7): A solution of DDQ (0.41 g, 1.80 mmol) in
MeOH (15.0 mL) was added to a stirred solution of 5 (0.50 g,
1.40 mmol) in CH2Cl2 (7.0 mL) via syringe at 08C, and the reaction
was allowed to warm to RT. The color of the reaction mixture
turned brown upon addition of DDQ and then faded over time.
The reaction was stirred for 1 h until TLC confirmed the disappear-
ance of all starting material. The reaction mixture was concentrated
in vacuo, and the crude material was purified using silica gel
column chromatography (hexane/EtOAc, 9:1) to yield 7 as a white
CDCl3): d=3.19 (s, 2H), 2.31 (s, 1H), 1.11 (s, 6H), 0.76 (s, 9H),
0.08 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=76.3, 74.4, 28.5,
28.0, 20.3, 0.3 ppm. The crude product was used in the next reac-
tion without further purification.
2-tert-Butyldimethylsilyloxy-2-methyl propanal (4): TBS-protected
alcohol 3 (0.600 g, 2.93 mmol) was dissolved in CH2Cl2 (29.3 mL),
and 4 ꢁ molecular sieves (0.68 g) were added. NMO (0.680 g,
5.86 mmol) was added to the stirred solution followed by the addi-
tion of TPAP (0.102 g, 0.290 mmol) at 08C. The reaction was al-
lowed to stir at 08C for 2 h, warmed to RT, and stirred for 24 h. The
reaction mixture was filtered through a pad of silica gel using Et2O
as the eluting solvent. The crude product was concentrated in
vacuo at low temperature using an ice bath to yield 4 as a colorless
1
solid (0.40 g, 80%); mp: 125–1288C; H NMR (400 MHz, CDCl3): d=
7.53 (d, J=8.8 Hz, 1H), 6.72 (dd, J=8.8, 2.7 Hz, 1H), 6.59 (d, J=
2.8 Hz, 1H), 6.09 (m, 1H), 4.00 (s, 1H), 3.79 (s, 3H), 2.95–2.75 (m,
2H), 2.36–2.11 (m, 2H), 2.23 (s, 2H), 1.94–1.15 (m, 10H), 1.48 (s,
3H), 0.94 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=211.8, 158.3,
1
clear oil (0.300 g, 50%); H NMR (400 MHz, CDCl3): d=9.44 (s, 1H),
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137.5, 134.9, 127.3, 125.1, 117.5, 113.2, 112.6, 80.0, 55.2, 52.7, 43.1,
42.3, 38.1, 30.0, 28.6, 24.7, 24.3, 23.3, 22.2, 13.6 ppm; HRMS (EI):
m/z [M]+ calcd for C23H30O3: 354.2189, found: 354.2196. Note: the
Rf values of the product and starting material are so close that
they require multiple runs on the TLC plate using hexane/EtOAc
(9:1) for separation.
a Biotech plate reader at 570 nm 4 h after lysing. The IC50 value
was determined from the exponential curve of viability versus con-
centration. The viability was calculated using Equation (1), where
Adrug is the absorbance at 570 nm for the compound, ANC is the ab-
sorbance for the negative control (no cells), and APC is the absorb-
ance of positive control (cells with no drug). The experiments were
run at least in triplicates, along with negative and positive controls.
(20R,22E)-25-tert-Butyldimethylsilyloxy-20-hydroxy-3-methoxy-
19-norcholesta-1,3,5(10),9(11),23-pentaen-22-one (8):
A stirred
ðAdrugꢀANC
Þ
ð1Þ
Viability ð%Þ ¼ 100 ꢂ
solution of (iPr)2NH (0.57 mL, 3.70 mmol) in THF (11.6 mL) was
cooled to ꢀ788C, treated dropwise with nBuLi (2.5m in hexanes,
1.52 mL, 3.70 mmol), and stirred at ꢀ788C for 1 h. A solution of 7
(0.40 g, 1.09 mmol) in THF (2.18 mL) was then added, and the reac-
tion was stirred for an additional 1 h at ꢀ788C. A solution of 4
(0.44 g, 2.18 mmol) in THF (14.5 mL) was then added at ꢀ788C,
and the solution was slowly warmed to RT and stirred for 20 h. The
reaction was quenched by the addition of saturated aq NH4Cl
(20 mL), followed by extraction of the aqueous layer with EtOAc
(3ꢂ50 mL). The combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo. The crude product was purified
by silica gel column chromatography (hexane/EtOAc, 4:1) to yield
ðAPCꢀANC
Þ
Enzyme-linked immunosorbent assay (ELISA): Thermo Scientific
Pierce ERK1/2 colorimetric in-cell ELISA kit, including buffers,
target-specific primary antibodies (anti-ERK1/2 (Thr202/Tyr204)
and anti-ERK1/2 antibodies), and horseradish peroxidase (HRP)-con-
jugated detection reagent, was used. A-375 cells were seeded in
96-well plates at 20000 cells per well in the corresponding
medium, and incubated at 378C for 24 h. Cells were treated with
test compounds and PLX-4032 for different time intervals, then
stimulated with EGF (100 ngmLꢀ1) prior to fixation using 4% form-
aldehyde. Permeabilization buffer, quenching solution, and block-
ing buffer were added to the fixed cells, which were then treated
with primary antibody and incubated overnight. Then, HRP conju-
gate was added followed by 3,3’,5,5’-tetramethylbenzidine (TMB)
substrate and TMB stop solution. The absorbance was measured
using an ELISA plate reader at 450 nm, and the collected data was
normalized by staining the whole cell using Janus green, followed
by measuring absorbance again at 615 nm. The experiments were
run at least in triplicate.
1
8 as a white solid (0.36 g, 60%); H NMR (400 MHz, CDCl3): d=7.42
(d, J=8.8 Hz, 1H), 6.97 (d, J=14.9 Hz, 1H), 6.61 (d, J=14.9 Hz, 1H),
6.60 (dd, J=8.8, 2.7 Hz, 1H), 6.48 (d, J=2.5 Hz, 1H), 5.99 (m, 1H),
4.06 (s, 1H), 3.67 (s, 3H), 2.83–2.65 (m, 2H), 2.47–2.14 (m, 2H),
1.92–1.09 (m, 10H), 1.48, (s, 6H), 1.12 (s, 2H), 0.92 (s, 3H), 0.81 (s,
9H), 0.08 ppm (s, 6H); 13C NMR (100 MHz, CDCl3): d=204.5, 160.4,
159.4, 139.6, 136.9, 129.4, 127.4, 120.2, 119.7, 115.3, 114.7, 81.0,
75.6, 57.3, 57.0, 54.8, 45.1, 44.3, 40.2, 32.3, 32.1, 31.9, 30.6, 27.9,
26.8, 25.9, 24.1, 20.3, 15.7, 0.0 ppm; HRMS (ESI): m/z [M+Na]+
calcd for C33H50O4Si: 561.3371, found: 561.3376.
(20R,22E)-20,25-Dihydroxy-3-methoxy-19-norcholesta-
Acknowledgements
1,3,5(10),9(11),23-pentaen-22-one (MSA-5): TBAF (1m in THF,
4.0 mL, 4.00 mmol) was added to a stirred solution of 8 (0.70 g,
1.29 mmol) in THF (20.0 mL), and the reaction was stirred for 6 h at
RT. The reaction mixture was quenched by addition of a saturated
aq NH4Cl (20 mL). The aqueous layer was extracted with EtOAc (3ꢂ
50 mL), and the combined organic layers were dried over Na2SO4,
filtered and concentrated in vacuo. The crude material was purified
using silica gel column chromatography (hexane/EtOAc, 4:1!
7:3!3:2) to yield MSA-5 as a white solid (0.38 g, 70%); mp: 88–
90; 1H NMR (400 MHz, CDCl3): d=7.52 (d, J=9.1 Hz, 1H), 7.15 (d,
J=15.1 Hz, 1H), 6.71 (dd, J=8.8, 2.7 Hz, 1H), 6.68 (d, J=15.3 Hz,
1H), 6.59 (d, J=2.5 Hz, 1H), 6.08 (m, 1H), 4.15 (s, 1H), 3.78 (s, 3H),
2.96–2.76 (m, 2H), 2.40–2.16 (m, 2H), 1.92–1.09 (m, 10H), 1.50 (s,
3H), 1.39 (s, 6H), 0.95 ppm (s, 3H); 13C NMR (100 MHz, CDCl3): d=
202.4, 158.3, 156.2, 137.5, 134.8, 127.4, 125.1, 118.0, 117.6, 113.3,
112.6, 79.1, 71.2, 55.2, 54.7, 52.6, 42.9, 42.3, 38.1, 30.1, 29.5, 29.4,
28.6, 24.7, 23.8, 22.1, 13.8 ppm; HRMS (EI): m/z [M]+ calcd for
C27H36O4: 424.2614, found: 424.2608.
The authors would like to acknowledge South Dakota Board of
Regents-2010 Biological Control and Analysis by Applied Photon-
ics (BCAAP) Center for financial support, OpenEye Scientific Soft-
ware Inc. (Santa Fe, USA) for providing an academic license for
the molecular modeling software, and Dr. Alice Bergmann (Uni-
versity of Buffalo, USA) for performing high-resolution mass spec-
trometry; the mass spectrometers used by Dr. Bergmann were
obtained through the Chemistry Research Instrumentation and
Facilities (CRIF) Program of the National Science Foundation
(award CHE0091977).
Keywords: anticancer agents · B-Raf · cucurbitacins · kinases ·
molecular modeling · natural products
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Received: December 12, 2013
Published online on March 28, 2014
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ChemMedChem 2014, 9, 1361 – 1367 1367