Bicyclo[2.2.2]octanes: Close structural mimics of the nuclear receptor-binding motif of steroid receptor coactivators

Article abstract of DOI:10.1016/j.bmcl.2007.05.058

Nuclear hormone receptor (NR) function relies on association of agonist-bound receptors with steroid receptor coactivator (SRC) proteins through a small pentapeptide motif (LXXLL) of the SRC that binds to a hydrophobic groove on the NR. We have synthesize

Full text of DOI:10.1016/j.bmcl.2007.05.058

Bioorganic & Medicinal Chemistry Letters 17 (2007) 4118–4122
Bicyclo[2.2.2]octanes: Close structural mimics of the nuclear
receptor-binding motif of steroid receptor coactivators
Hai-Bing Zhou, Margaret L. Collins, Jillian R. Gunther,
John S. Comninos and John A. Katzenellenbogen*
Department of Chemistry, University of Illinois, 600 South Mathews Avenue, Urbana, IL 61801, USA
Received 20 April 2007; revised 14 May 2007; accepted 17 May 2007
Available online 23 May 2007
Abstract—Nuclear hormone receptor (NR) function relies on association of agonist-bound receptors with steroid receptor coacti-
vator (SRC) proteins through a small pentapeptide motif (LXXLL) of the SRC that binds to a hydrophobic groove on the NR. We
have synthesized a series of bicyclo[2.2.2]octanes that are close structural mimics of the two key leucine residues of this SRC
sequence as bound in the hydrophobic groove of the estrogen receptor. These bicyclic systems block the NR–SRC interaction with
modest potency.
Ó 2007 Elsevier Ltd. All rights reserved.
Nuclear receptors (NRs) are transcription factors that
control many physiological and pathological processes
by directly regulating the expression of select target
genes.1 Members of the NR gene superfamily have mul-
ti-domain structures, with a conserved DNA-binding
domain and a ligand-binding domain (LBD). The tran-
scriptional activity of NRs is initiated by hormone bind-
ing that stabilizes the conformation of the LBD. When
an agonist ligand binds, conformational stabilization
rigidifies surface features that function as docking sites
for coactivator protein complexes that are recruited to
modify chromatin structure and activate RNA polymer-
ase II. The most notable of these are the p160 class of
steroid receptor coactivators (SRCs).2,3
places the first and third leucine residues (L690 and
L694) in a deep, solvent-exposed hydrophobic groove
made up of residues from various helices of the LBD;
histidine and arginine residues of the peptide (H691
and R692, removed for clarity) are solvent exposed
and appear unnecessary for the interaction.5 The intrin-
sic dipole moment of the coactivator a-helix is aligned
with charged residues on the surface of the ERa-LBD
(E542 interacts with the peptide N-terminus and K362
with its C-terminus), together forming a ‘charge clamp’.5
The traditional strategy to block agonist signaling of
NRs involves hormone antagonists (antihormones) that
bind to the LBD, displacing the agonist and stabilizing
alternative conformations that cannot interact with the
coactivators.4–8 The effectiveness of NR antagonists
can be compromised by cellular adaptations that enable
coactivators to bind to NR–antihormone complexes,
such as increased coactivator expression10 or covalent
modifications of the interacting components.11,12 This
is exemplified by the development of resistance to the
antiestrogen tamoxifen in ER-based endocrine therapies
for breast cancer, a phenomenon that is a serious limita-
tion on such therapies.11,12
X-ray crystallography has revealed details of these NR–
SRC interactions,4–9 which are largely mediated by a
short, conserved, pentapeptide LXXLL (L = leucine
and X = amino acid) motif of the SRC, termed the
NR box. The interaction between the estrogen receptor
a (ERa) LBD and a peptide corresponding to NR box 2
of SRC1 (RHKILHRLLQE) is shown in Figure 1.5
Selected residues of the peptide interact with the LBD
through a two-turn amphipathic a-helical motif that
An alternative approach to interrupting NR signaling is
the use of compounds capable of blocking the step
following NR-ligand binding, namely, the interaction
between the agonist-liganded ER and coactivators. Such
compounds could be termed coactivator-binding
Keywords: Steroid receptor coactivators; Coactivator-binding inhibi-
tors; Bicyclo[2.2.2]octanes; Estrogen receptor.
*
Corresponding author. Tel.: +1 217 333 6310; fax: +1 217 333
7325; e-mail: jkatzene@uiuc.edu
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.05.058

H.-B. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4118–4122
4119
Figure 1. Schematic of a crystal structure of an SRC NR2 peptide
bound to ERa LBD. Residues in yellow are from the LBD, white from
the peptide.
Figure 2. Cyclohexane connector (purple) creates a bicyclo[2.2.2]-
octane CBI core from L690 and L694 of an SRC NR2 box peptide.
inhibitors (CBIs). Biochemical studies using peptides
and peptide mimics of the LXXLL motifs of the SRCs
have demonstrated that this alternative approach is fea-
sible.13–16 If disruption of this helix-groove protein–pro-
tein interaction between the ERs and SRCs could be
effected with small molecules, as is possible in other
helix-groove interactions,17–23 such molecules would be
intriguing molecular probes for studying ER signaling.
They might also afford an alternative strategy for block-
ing estrogen action in breast cancer less likely to be com-
promised by the cellular adaptations responsible for
tamoxifen resistance.11,12
adorned with functionality mimicking the N-terminal
isoleucine (I689) and the second leucine (L693); appro-
priately charged groups could also be added to mimic
the helix dipole of the NR box sequence.
Retrosynthetic analysis shows that this bicyclo[2.2.2]-oc-
tane system, for example, compound 1, can be assem-
bled in two key steps: addition of a side arm and a
Diels–Alder reaction of a 5-substituted 1,3-cyclohexadi-
ene with methacrolein to form the bicyclo[2.2.2]octane
core (Scheme 1). The synthesis, therefore, was divided
into three parts: synthesis of the 5-substituted 1,3-cyclo-
hexadiene, the Diels–Alder reaction, and addition of the
side arms.
Recently, we reported that certain small molecules, par-
ticularly 2,4,6-trisubstituted pyrimidines, are inhibitors
of coactivator binding to ERa24; since then, examples
of related compounds from other functional group clas-
ses have appeared.25 These reports provide a proof-of-
principle that small molecule CBIs can be developed,24
and as part of our interest in ER CBIs, we are pursuing
refinements to improve their potency.
Preparation of the desired diene (Scheme 2) began with
the cyclohexadienyl cation salt 3, prepared by abstrac-
tion of a hydride from 1,3-cyclohexadiene iron tricar-
bonyl 2 with triphenylcarbenium tetrafluoroborate.
Nucleophilic addition of isobutyraldehyde provided
aldehyde 4, in which the two methyl groups necessary
to mimic L690 of the coactivator peptide have been
introduced. To install one of the side arms, a Henry
reaction of aldehyde 4 with nitromethane gave nitroalk-
ene 5, which was reduced to amine 6 with lithium boro-
hydride and Me3SiCl and converted to amide 7 with
isoamyl acid chloride. We chose to make the leucine
mimic instead of the isoleucine (I689) to reduce the
number of potential stereoisomeric products. Trimethyl-
amine N-oxide decomplexation of the iron tricarbonyl
gave the iron-free diene 8.
Previously, we took an ‘outside-in’ design, in which the
heterocycle (pyrimidine) functioned as a peptide scaffold
mimic onto which we appended groups mimicking the
key leucine residues.24 Here, we have taken an alterna-
tive ‘inside-out’ approach, beginning with a structural
surrogate of the two leucine residues in the ILHRLL
sequence (L690 and L694, bolded) that extend most dee-
ply into the hydrophobic pocket (Fig. 1) and attaching
to it structural elements that mimic other residues and
functional elements of the NR box helix.
The key step in preparing bicyclo[2.2.2]octane aldehyde
10 is a Diels–Alder reaction between diene 8 and meth-
acrolein 9 (Scheme 3). Lewis acids are typically used in
catalytic quantities to accelerate Diels–Alder reactions,
and they enhance stereo- and regioselectivity. However,
because of the amide function in diene 8, stoichiometric
amounts of Lewis acids were needed. Ytterbium trichlo-
ride was an effective catalyst.26
Figure 2 illustrates how this can be done using a bicy-
clo[2.2.2]octane as the core structural mimic of the first
and third leucine residues (L690 and L694) in the NR
box motif. The alignment of the two isopropyl groups
of these two leucine residues can be replicated almost
exactly by interposing a boat cyclohexane ring (Fig. 2,
purple). Two atoms of one of the isopropyl groups make
a 1,4-bridge on the cyclohexane, creating the bicy-
clo[2.2.2]octane system; the other isopropyl group is
attached to the cyclohexane by its tertiary carbon. This
‘inside’ portion of a potential CBI can then be further
Because of secondary orbital overlap, Diels–Alder reac-
tions, when run under conditions of kinetic control, par-
ticularly with Lewis acid catalysis, favor products with

4120
H.-B. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4118–4122
Me
Me
Me
Me
Me
Me
HN
O
HN
CO2R
CO2R
HN
O
O
+
NHCOR
CO2H
+
Me
Me
Me
Me
Me
CHO
Me
Me
CHO
Me
Me
1
Scheme 1. Retrosynthetic analysis of bicyclo[2.2.2]octane CBI 1.
Me
O
Me
NO2
+
CH3NO2,
NH4OAc
Ph3CBF4
CH2Cl2
H
O
H
Me
Me
Me
Me
Fe(CO)3
Fe(CO)3
Me
Fe(CO)3
EtOH
-
Fe(CO)3
BF4
5
2
3
4
Me
Me
Me
Me
Me
NH2
LiBH4,
TMSCl
THF
HN
O
HN
O
Cl
O
(CH3)3NO
acetone, 90%
Me
Me
Et3N,
Fe(CO)3
Me
Me
Me
Me
CH2Cl2
Fe(CO)3
6
7
8
Scheme 2. Synthesis of 5-substituted 1,3-cyclohexadiene.
Me
Me
Me
Me
Me
Me
Me
Me
1.3 eq. YbCl3
CH2Cl2, 36h
HN
HN
HN
O
O
O
HN
O
+
Me
CHO
Me
Me
Me
Me
CHO
Me
Me
Me
Me
Me
CHO
1,4-exo (42%) 1,3-regioisomer (8%)
[34.56 Kcal/mol] [34.54 Kcal/mol] [35.58 Kcal/mol]
CHO
Me
1,4-endo (50%)
Me
8
9
10a
10b
10c
chemical shift
of C
(ppm)
HO
9.34
9.56
9.29
a Reported data in
refrences 32 and 33
STD 1
endo
STD2
CHO
Me
9.31a
9.54a
exo
Me
CHO
Scheme 3. Diels–Alder reaction products, relative yields, steric energies, and aldehyde 1H NMR data.
endo stereochemistry.27,28 In the reaction of diene 8 with
methacrolein 9, three distinct aldehyde peaks were ob-
served between 9.25 and 9.65 ppm, indicating that three
isomeric products were formed (Scheme 3). We expected
that the 5-alkyl substituent on the 1,3-cyclohexadiene
would control facial selectivity, directing dienophile ap-
proach from the less hindered side to give the desired
stereoisomer, the one having the new dimethylene bridge
anti to the C-5 group. Because alkyl substitution of the
diene is at a remote position (carbon 5), there were no
groups on the diene itself to strongly direct the regiose-
lectivity of the reaction. Nevertheless, steric factors
would still be expected to favor formation of regioisom-
ers (10a and 10b), in which the carboxyaldehyde and
tertiary alkyl substituents were in a 1,4-rather than a
1,3-relationship (10c) with respect to one another.
The endo and exo isomers could readily be distinguished
by H NMR spectral comparison to reported analogs
1
(Scheme 3), because when the double bond is syn to
the aldehyde, its anisotropic effect shields the aldehyde
proton, shifting its signal upfield.28,29 The structure of
a minor isomer was tentatively assigned as regioisomer
10c, based on steric energy calculations (MacroModel),
which predicted it to be the third most stable isomer
(The steric energy values for the three isomers are given
in brackets). Further synthesis was done on the major,
desired isomer, 10a.
To add a second short side chain containing a carboxylic
acid for interaction with the lysine charge clamp residue
(K362), aldehyde 10a was converted to the unsaturated
methyl ester 12 by a Horner–Wadsworth–Emmons

H.-B. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4118–4122
4121
reaction (Scheme 4). Hydrogenation gave the saturated
methyl ester 14, and both esters were saponified, giving
acids 13 and 15.
these bicyclic compounds to compete with the nuclear
receptor domain of SRC3 (627-829, encompassing
all three NR boxes) for agonist-liganded ER (The
development of this assay will be described elsewhere).
Biotin-labeled ERa LBD, tagged with terbium-labeled
streptavidin, was incubated with estradiol and fluores-
cein-labeled SRC3 NRD, labeled non-specifically
through cysteine residues with iodoacetamide-fluores-
cein. The Fl-SRC3 binds to the estradiol-liganded
Tb-ER complex, and CBI activity is assayed by the
ability of increasing concentrations of compound to
compete for ER-SRC binding, reducing the FRET
signal. Unlabeled SRC1-NR Box2 peptide (LTERH-
Part of our original design of the bicyclo[2.2.2]octane core
CBI involved addition of a more extended side chain bear-
ing another amide group to mimic the third leucine resi-
due, but introduction of this second amide proved
surprisingly difficult. Condensations of methyl iso-
cyanoacetate, N-(benzyloxycarbonyl)-phosphono-gly-
cine trimethyl ester or methyl nitroacetate with aldehyde
10 proved to be unsuccessful in our hands. Aldehyde 10
did react with dimethyl malonate 16 and TiCl4 to give
unsaturated diester 17 (Scheme 5). Hydrogenation gave
the saturated ester 18, and saponification, the saturated
monocarboxylic acid 19. Curtius rearrangement via the
acyl azide gave an isocyanate, which was trapped with
benzyl alcohol to give the Cbz-protected amino ester 20.
Curiously, attempts to deprotect the carbobenzyloxy
group of amino ester 20 under various conditions were
only marginally successful; so only a trace amount of
the desired amino ester 21 was obtained.
KILHRLLQEGSPSD) and
a pyrimidine prepared
previously24 are used as CBI positive controls. Vehicle
(DMF) and abV peptide (that binds to tamoxifen-li-
ganded ER) were used as negative controls.13,14 IC50
values were converted to Ki values. Representative
competition-binding curves for the bicyclo[2.2.2]-oc-
tanes are shown in Figure 3; additional ones are listed.
While the control peptide and a pyridimine prepared
previously show full competition curves with good po-
tency, the bicyclo-octane CBIs have curves that are right
shifted and show less complete competition. Ki values
We used a time-resolved fluorescence resonance energy
transfer (TR-FRET) assay to evaluate the potential of
Me
Me
+
Me
Me
O
Me
Me
O
NaH
2N NaOH
THF, 80%
EtOH, 90%
O
O
HN
HN
HN
O
O
O
P
MeO
OMe
OMe
OH
OMe
Me
Me
Me
Me
Me
Me
CHO
(mixture of
isomers)
Me
Me
Me
10
11
12
H2, 5% Pd/C
13
Me
Me
O
Me
Me
O
2N NaOH
EtOH
HN
HN
O
O
OMe
OH
Me
Me
Me
Me
Me
14
15
Me
Scheme 4. Synthesis of carboxylic acid side arm.
Me
Me
Me
Me
Me
Me
O
O
MeO
OMe
(6 eq.)
aq KOH
HN
HN
Pd/C, H2
Quant.
16
O
O
HN
O
+
CO2Me
10 eq TiCl4
20 eq PyH
THF, 3 days
CO2Me
CO2Me
Me
Me
Me
Me
Me
Me
CO2Me
CHO
Me
Me
10
Me
17 Me
18
Me
Me
Me
Me
Me
(1) Cyanuric Chloride
NMM
Pd/C, MeOH
2 days
(2) NaN3
(3) PhCH2OH, reflux
HN
HN
HN
O
O
O
CO2H
CO2Me
NHCO2CH2Ph
NH2
Me
Me
Me
Me
Me
Me
CO2Me
CO2Me
Me
Me
Me
21,trace
19
20, 25%
Scheme 5. Installation of the amide side arm.

4122
H.-B. Zhou et al. / Bioorg. Med. Chem. Lett. 17 (2007) 4118–4122
CBI
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3000
src box II peptide
αβV peptide
pyrimidine
DMF
0.19
1100
0.91
n/a
7.1
12
2000
1000
0
18
19
21
32
7.9
17
20
38
-8
-7
-6
-5
-4
-3
-2
log M [CBI]
Figure 3. TR-FRET assay of bicyclo[2.2.2]octane CBI and SRC1
NR2, ab V peptide and pyrimidine control compounds and vehicle
activity on ERa-SRC3. Pyrimidine control is compound 12a from Ref.
24.
are in the range of 7–40 lM, indicating lower potency
than the peptide and pyrimidine control compounds.
While we are not certain how to interpret the incomplete
competition curves, they could represent stabilization by
these compounds of an ER complex that does not result
in full displacement of the Fl-peptide but binds it with
lower affinity or altered geometry, reducing but not
eliminating the FRET signal. Despite the structural
mimicry of the LXXLL peptide motif by these bicyclo-
octanes, there is also no obvious relationship between
their structures and Ki values.
Compounds that block the interaction of agonist-li-
ganded ER with CoAs could provide unique pharmaco-
logical tools for interrupting the signal transduction of
this receptor and might also allow the estrogen receptor
to circumvent certain modes of cellular resistance to con-
ventional antagonists. In this paper, we describe the
structure-inspired de novo design, synthesis, and evalua-
tion of such small molecule coactivator-binding inhibi-
tors (CBIs), based on a bicyclo[2.2.2]octane skeleton.
Despite their accurate structural mimicry of the key res-
idues involved in the ER-coactivator interaction, their
potency and effectiveness is limited. Nevertheless, this
work contributes to the proof-of-principle that effective
small molecule CBIs for the ER can be developed.
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Acknowledgments
The present work was supported by grants from the NIH
(PHS 5R37 DK15556). Funding for NMR and MS
instrumentation is from the Keck Foundation, NIH,
and NSF. We are grateful to Dr. Sung Hoon Kim and
Terry Moore for helpful comments, and Jaimin Amin
for conducting the coactivator-binding assays.
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