A-C Estrogens as Potent and Selective Estrogen Receptor-Beta Agonists (SERBAs) to Enhance Memory Consolidation under Low-Estrogen Conditions

Article abstract of DOI:10.1021/acs.jmedchem.7b01601

Estrogen receptor-beta (ERβ) is a drug target for memory consolidation in postmenopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs) with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The

Full text of DOI:10.1021/acs.jmedchem.7b01601

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Article
A-C Estrogens as Potent and Selective Estrogen Receptor-beta Agonists
SERBAs) to Enhance Memory Consolidation under Low-estrogen Conditions
(
Alicia M Hanson, K.L. Iresha Sampathi Perera, Jaekyoon Kim, Rajesh K. Pandey,
Noreena Sweeney, Xingyun Lu, Andrea Imhoff, Alexander Craig Mackinnon, Adam J
Wargolet, Rochelle M. Van Hart, Karyn M Frick, William A. Donaldson, and Daniel S. Sem
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01601 • Publication Date (Web): 09 May 2018
Downloaded from http://pubs.acs.org on May 9, 2018
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A-C Estrogens as Potent and Selective Estrogen
Receptor-beta Agonists (SERBAs) to Enhance
Memory Consolidation under Low-estrogen
Conditions
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a
b
c
b
Alicia M. Hanson , K. L. Iresha Sampathi Perera , Jaekyoon Kim , Rajesh K. Pandey , Noreena
a
a
a
d
e
Sweeney , Xingyun Lu , Andrea Imhoff , Alexander Craig Mackinnon , Adam J. Wargolet ,
e
*‡,c
*‡,b
*‡,a
Rochelle M. Van Hart , Karyn M. Frick , William A. Donaldson , and Daniel S. Sem
a
Department of Pharmaceutical Sciences, Center for Structure-based Drug Design and Development, Concordia University Wisconsin, Mequon,
WI 53097, United States
b
Department of Chemistry, Marquette University, P. O. Box 1881, Milwaukee, WI 53201-1881, United States
Department of Psychology, University of Wisconsin-Milwaukee, 2441 E. Hartford Ave., Milwaukee, WI 53211, United States
Department of Pathology, Medical College of Wisconsin, 9200 W. Wisconsin Ave., Milwaukee, WI 53226, United States
Department of Natural Science, Concordia University Wisconsin, Mequon, WI 53097, United States
c
d
e
KEYWORDS. Memory consolidation, cognition, estrogen receptor, agonist, drug, phenol.
ABSTRACT. Estrogen receptorꢀbeta (ERβ) is a drug target for memory consolidation in postꢀ
menopausal women. Herein is reported a series of potent and selective ERβ agonists (SERBAs)
with in vivo efficacy that are A-C estrogens, lacking the B and D estrogen rings. The most potent
and selective A-C estrogen is selective for activating ER relative to seven other nuclear hormone
receptors, with a surprising 750ꢀfold selectivity for the beta over alpha isoform, and with EC ’s
5
0
of 20ꢀ30 nM in cellꢀbased and direct binding assays. Comparison of potency in different assays
suggests that the ER isoform selectivity is related to the compound’s ability to drive the
productive conformational change needed to activate transcription. The compound also shows in
vivo efficacy after microinfusion into the dorsal hippocampus, and after intraperitoneal injection
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(
0.5 mg/kg) or oral gavage delivery (5 mg/kg). This simple yet novel A-C estrogen is selective,
brain penetrant, and facilitates memory consolidation.
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Introduction
1
ERβ agonists have a number of promising clinical applications . Current ERβ agonist
drug lead molecules possess a phenolic ring, with varying substituted aromatic ring systems on
the other half of the molecule, typically comprising another phenolic or indoleꢀlike ring
systems (Fig. 1a). One of these, WAYꢀ200070 (benzoxazole), has shown efficacy as an
1
ꢀ3
anxiolytic/antidepressant and has 68ꢀfold selectivity for ERβ over ERα. Some ERβ agonists
have progressed into human clinical trials for different disease indications, ranging from
schizophrenia (Eli Lilly; NCT01874756), to FragileꢀX syndrome (Parc de Salut Mar;
NCT01855971), to memory loss and hot flashes (National Institutes on Aging;
4
NCT01723917). Studies presented herein focus on one of the more promising new clinical
applications of ERβ agonists, for treating neuronal symptoms caused by estrogen deficiency in
5
menopause, as illustrated in animal model studies using diarylpropionitrile (DPN).
1
7βꢀestradiol (E ) is a critical modulator of hippocampal synaptic plasticity and
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hippocampalꢀdependent memory formation in male and female rodents. E levels decrease in
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both sexes as people age, but drop much more precipitously in women during the menopausal
transition. ERβ is the predominant ER isoform in the hippocampus and plays an important role in
mediating estradiol’s effects on neural plasticity and neuroprotection, which could be pivotal
during aging and in Alzheimer’s disease (AD). For example, overexpression of ERβ in a rat
model of AD significantly reduced hippocampal AD pathology and improved learning and
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memory. Moreover, specific alleles of the gene for ERβ (Esr2), but not ERα, are associated
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with decreased AD risk in men and women, supporting ERβ as a putative drug target for AD.
APOE4 is the most well established genetic risk factor for Alzheimer’s disease (AD).
Women with the APOE4 genotype are 2ꢀ4 times more likely to develop AD than women without
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ꢀ11
APOE4 or than men of any other APOE genotype.
APOE4 carriers are also much more likely
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to show symptoms of anxiety and depression. A major contributor to these risks in women is
menopausal estrogen loss, as estrogens are neuroprotective for brain regions like the
13
hippocampus and cortex that mediate cognitive function and deteriorate in AD. As such, drugs
that facilitate estrogenꢀmediated effects on cognition, like the selective ERβ agonists (SERBAs)
being developed herein, may reverse memory loss and alleviate anxiety and depression in aging
females. But, estrogenꢀbased hormone replacement therapy is associated with increased risk of
various diseases (thought to be associated with ERα agonist activity), including breast cancer
14ꢀ18
(
esp. lobular) as well as stroke, gallbladder disease and venous thromboembolism
.
Accordingly, any ERβ agonist therapeutic should be selective for ERβ over ERα agonist
activity.
Recently, we reported a novel ERβ agonist that was more selective for ERβ versus
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ERα activation than previously reported clinical candidates . This ERβ agonist was in a
unique structural class, comprised of a phenol ring tethered to a 4ꢀhydroxymethylꢀcycloheptane
ring system. However, the presence of the 4ꢀsubstituted cycloheptane ring presents synthetic
and stereochemistry challenges, making it less desirable as a drug lead. Herein is reported the
optimization and characterization of a related class of molecules, comprised of a 4ꢀ
hydroxymethylꢀcyclohexane ring tethered to a phenol ring, making it an A-C estrogen that
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closely resembles the naturally occurring estrogen molecule, but lacks the B and D rings (Fig.s
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b-d). Whereas A-CD estrogens have been widely studied and reported to have up to 15ꢀfold
16−23
selectivity for ERβ,
the simpler A-C estrogens reported herein show even higher selectivity
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for ERβ over ERα. These A-C estrogens represent a surprisingly simple yet novel class of
isoform selective ERβ agonists, with potential for treating ageꢀrelated memory decline in postꢀ
menopausal women.
Results
Compound synthesis. Commercially available 4ꢀ(4ꢀhydroxyphenyl)cyclohexanone 1
o
o
was transformed into 2 or 3 alcohols 2 or 3 by reaction with NaBH or excess methyl lithium
4
respectively, or into oxime 4 by condensation with hydroxylamine (Scheme 1). The
o
stereochemistries of 2 and 3 were assigned based on their NMR spectral data. In particular, for 2
alcohol 2, the alcohol methane proton appears as a triplet of triplets at δ 2.38 (J = 11.8, 3.4 Hz);
the large couplings are consistent with an axialꢀaxial disposition of this proton, and thus the
1
3
hydroxyl group is equatorial. Signals at δ 69.5 and 31.1 ppm in the C NMR spectrum of 3,
o
assigned to the 3 alcohol and methyl carbons, are in good agreement with cisꢀ1,4ꢀalcohols of
24
this type . The tꢀbutyldimethylsilyl ether 5 underwent olefination with the ylide generated from
methyltriphenylphosphonium bromide to give 6. Cleavage of the silyl ether using TBAF gave 7;
catalytic hydrogenation of 7 gave 8 as a mixture of stereoisomers. Reaction of 7 with excess
paraformaldehyde, MgCl and NEt gave the substituted salicaldehyde 9, which upon reaction
2
3
with hydroxylamine afforded the oxime 10. Dihydroxylation of 6, followed by cleavage of the
silyl ether gave 12, as a single stereoisomer after chromatographic purification. The
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stereochemistry of 12 was assigned as indicated, based on the known stereochemistry of
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5
osmiumꢀcatalyzed dihydroxylation of 4ꢀtꢀbutylmethylenecyclohexane.
Hydroboration–
oxidation of 6 using BH ꢀTHF, produced an inseparable mixture of stereoisomeric primary
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alcohols cisꢀ13 and transꢀ14, in a 2:1 ratio as determined by integration of the H NMR signals
for the hydroxymethylene protons for each (δ 3.60 and 3.39 ppm respectively). The
stereochemistry of the isomers was tentatively assigned on the basis of the relative chemical shift
of these two signals; the signal for an axial hydroxymethylene (i.e. cis-isomer) appears
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downfield compared to that for an equatorial hydroxymethylene. Alternatively, hydroborationꢀ
oxidation using 9ꢀBBN afforded a mixture in which transꢀ14 was in greater proportion compared
to cisꢀ13 (2:3, cis:trans). The use of these two borane reagents to tune the cis:trans outcome for
26, 27
4
ꢀsubstituted methylenecyclohexanes has previously been reported.
Cleavage of the silyl
ether using TBAF gave a mixture of stereoisomeric 4ꢀ(4ꢀhydroxymethylcyclohexyl)phenols cisꢀ
15/transꢀ16. Treatment of a mixture of the stereoisomers 15/16 (2:3, cis:trans) with DDQ (0.5
equiv.) led to a separable mixture of a bicyclic ether 17 and transꢀ16. The tentative structural
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assignment for trans-16 was corroborated by single crystal Xꢀray diffraction analysis (Fig. 7a).
Isolation of the unreacted transꢀ16 is rationalized on the basis of the faster rate of oxidation of
cisꢀ15. Since oxidation of either cisꢀ or transꢀ4ꢀ(4ꢀhydroxymethylcyclohexyl)phenol proceeds
via the same benzylic carbocation intermediate (i.e. 18, Scheme 2), the activation energy for the
formation of this intermediate will be lower for the less stable cisꢀ15 in comparison to transꢀ16,
and thus oxidative cyclization of the cisꢀisomer will be faster. Reaction of 17 with MgCl and
2
trimethylamine led to an intramolecular elimination reaction to afford the cyclohexene (±)ꢀ19
(Scheme 1). The structure of 19 was assigned on the basis of its NMR spectral data; in
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particular, the signal for the olefinic proton appears as a narrow multiplet at ca. δ 5.95 ppm. This
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signal is characteristic of other 1ꢀ(4ꢀhydroxyphenyl)cyclohexenes.
Silyl ether 20 (prepared from 1) underwent HornerꢀEmmons olefination with triethyl
phosphonoacetate to afford the unsaturated ester (±)ꢀ21; desilylation with TBAF gave the phenol
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(±)ꢀ22 (Scheme 3). Reduction of 21 with DIBAL, followed by deprotection of the silyl ether
yielded the allylic alcohol (±)ꢀ24. Catalytic hydrogenation of 24 gave a separable mixture of
alcohol 25 and the over reduced ethyl cyclohexane derivative 26.
TR-FRET and Cell-based Transcriptional Assays. Initial screening of compounds was
performed in a TRꢀFRET displacement assay, which detects binding to the ERβ LBD (see Fig. 2
for doseꢀresponse curves for selected compounds and Fig. S1 for doseꢀresponse curves for all
compounds). In this assay EC s were measured for all compounds that were synthesized, with
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EC values summarized in Table 1. The most potent compounds were 16 (hereafter referred to
50
as ISP358-2) (hydroxymethyl substitution), 25 (hydroxyethyl substitution), and 8 (methyl
substitution) which all had EC s < 30 nM for ERβ. ISP358-2 is the pure trans isomer, and was
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found to bind with higher affinity to ERβ than the mixture of cis- and trans-stereoisomers
(15/16). While having a methylene (ISP358-2) or ethylene (25) linker to the hydroxyl group
leads to potency, the direct substitution of the hydroxyl on the cyclohexane ring yields a
significant decrease in affinity (EC of 7,250 nM for 2). While introduction of unsaturation into
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the alkyl linker (24) also led to a decrease in affinity (676 nM), introduction of unsaturation into
the cyclohexane ring (18) only decreased affinity modestly (49 nM). ISP358-2 was also tested
for binding to ERα, and bound with only 12ꢀfold higher affinity to ERβ (EC of 24 nM; Fig.
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2
b) in this TRꢀFRET assay, which measures direct binding to the isolated LBD.
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ISP358-2 was further screened in a nuclear hormone receptor functional assay (Fig. 3),
where transcriptional activation was measured due to binding and activation of a chimeric
receptor comprised of the LBD of a hormone receptor of interest (e.g. ERβ) tethered to the DNA
binding domain (DBD) of GAL4. This assay was done to assess selectivity for activating
estrogen receptor, versus other nuclear hormone receptors. No significant agonist activity was
observed for compound ISP358-2 for any of the nuclear hormone receptors tested (except for the
estrogen receptor) at concentrations ranging from 0.25 to 25 ꢀM (Fig. 3a). Thus, ISP358-2 is not
an agonist for the following receptors: Androgen Receptor (AR), Glucocorticoid Receptor (GR),
Mineralocorticoid Receptor (MR), Peroxisome ProliferatorꢀActivated Receptor (PPARδ),
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Progesterone Receptor (PR), Thyroid Hormone Receptor (TRβ), and Vitamin D Receptor (VDR)
(see Table S1 for control compound EC s for each nuclear hormone tested). In a followꢀup 10ꢀ
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point titration in this same assay, ISP358-2 was found to be 2.6ꢀfold selective for binding and
activating the fullꢀlength chimeric ERβ (357 + 26 nM) relative to fullꢀlength chimeric ERα (930
+ 69 nM). This assay (Fig. 3) measures activation of transcription, rather than simply binding of
agonist to the ER LBD (as in Fig. 2). But, it uses an unnatural chimeric protein (ER LBD fused
to a GAL4 DBD) that may not accurately reflect the actual agonistꢀinduced activation that occurs
under native conditions.
When the coactivator form of the TRꢀFRET LBD binding assay was performed (Fig. 4a),
the ISP358-2 compound (Fig. 4b) was found to be 15ꢀfold selective (Fig. 4c) for binding to ER
and recruiting the PPARγ coactivator peptide to ERβ (161 + 15 nM) relative to ERα (2,940 +
3
90 nM). This assay measures activation of the ER LBD, in that it measures binding and agonistꢀ
induced recruitment of the coactivator peptide, rather than simply binding of agonist to the
receptor.
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Finally, a cellꢀbased transcriptional activation assay, which employs a full length and
native ER (comprised of an ER LBD and an ER DBD), was performed. Unlike the previous
assays, this assay is cellꢀbased, so best mimics the in vivo situation. The most potent and
selective compound tested in this assay is ISP358-2, which has an ERβ agonist potency of 31 + 7
nM (Fig. 5a and Fig S2a; the assay was performed in duplicate, with values of 31 nM and 23 nM
obtained, for an average of 27 nM), and an ERα agonist potency of 20,419 + 859 nM (Fig. 5b).
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This makes the ERβ/ERα selectivity ratio ~ 750 in this more physiologically relevant assay.
ISP358-2 showed no antagonist activity for ERβ (Fig. 5c) or ERα (Fig. 5d) at concentrations up
to 10 ꢀM.
In Vitro Druggability – CYP450 Binding, hERG and Nephelometry. ISP358-2 shows
no inhibition of CYP1A2 and CYP2D6, and only weak inhibition of CYP2C9 (EC = 34 + 4.7
50
ꢀ
M) and CYP3A4 (EC = 89 + 18 ꢀM) (Fig. 6). ISP358-2 does not bind to hERG, showing
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only 14% activity at 100 ꢀM; and, nephelometry (done for ISP171, 15/16, the mixture of
isomers) shows no significant aggregation, indicating good solubility at concentrations up to 300
ꢀ
M (Fig. S3).
Docking Studies. ISP358-2 (Fig. 7a) was docked into the binding site of agonistꢀ
conformation ERα in two conformations with similar docking energy. In one binding mode
Figs. 6d and 6e), the phenolic hydroxyl interacts with the Arg394/Glu353 (energy = ꢀ7.6
(
kcal/mol), and in another mode the ISP358-2 molecule is flipped 180 degrees (energy = ꢀ7.8
kcal/mol) with the aliphatic hydroxyl interacting with Arg394/Glu353. ISP358-2 binds in the
agonistꢀconformation ERβ pocket (Figs. 7f and 7g) with the phenolic hydroxyl interacting with
the Arg394/Glu353 in the lowest energy docking pose (energy = ꢀ8.0 kcal/mol). In both cases,
there are significant hydrophobic interactions between binding site residues and the bound
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ISP358-2, although the ERβ pocket is smaller and makes for a tighter fit. In both cases, the
hydroxymethyl group is proximal enough (3.0 Å) to the His524 to participate in the hydrogen
bonding interaction that is typically seen for ER agonists, although in ERβ the hydrogen bond to
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the aliphatic alcohol may be with the backbone carbonyl of Gly472. In ERβ there are more
hydrophobic interactions that constrain the hydroxymethylꢀcyclohexyl ring (Ring C) to be nearly
planar with the phenolic ring (Ring A) (Fig. 7c), as it is in the native estrogen molecule. This is
in contrast to the binding pose in ERα, where the two rings are nearly orthogonal (Fig. 7b). The
ERβ hydrophobic interactions are with Phe356, Met340, Phe355, Leu298, near the phenol ring,
and with Leu476, Ile373 near the hydroxymethyl group and with Ala302, Leu298 near the
cyclohexane ring (Fig. 7f,g). A control docking study of E reproduced the expected binding
2
orientation, based on the crystal structure (Fig. S4).
Assessment of Memory Consolidation. As described in detail in the Experimental
Section, memory consolidation was assessed in all studies using acute treatments administered
immediately after training. That is, for each task, mice completed a single training trial and then
were immediately treated with control vehicle or experimental compounds. Memory
consolidation was measured 24 or 48 h later. Briefly, in the object recognition task, mice
explored two identical objects during training, immediately after which they were treated with
the compounds described below. Memory for the training objects was tested 48 h later by
allowing mice to explore one training object and one novel object. In the object placement task,
mice again explored two identical objects and received immediate postꢀtraining treatments.
Memory was tested 24 h later by allowing mice to explore a training object in its original
location and a training object in a new location. More time than chance, or than the control
group, spent with the novel or moved object indicated intact consolidation of memory for the
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training objects. Each mouse completed both tasks, the order of which was counterbalanced
within each group. Two weeks separated bouts of testing to allow any acute effects of the drugs
to dissipate before the next treatment.
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Dorsal hippocampal infusion.
We first investigated the effects of direct
intrahippocampal infusion of ISP358-2 on object recognition and spatial memory consolidation
in ovariectomized mice (Fig. 8a). Five groups of mice were tested (Fig. 8b,c): vehicle (negative
control), DPN (positive control), and three doses of ISP358-2 (10 pg/hemisphere, 100
pg/hemisphere, and 1 ng/hemisphere). For object placement (Fig. 8b), oneꢀsample tꢀtests
indicated that mice receiving vehicle or 10 pg ISP358-2 did not spend significantly more time
than chance with the moved object (ts(7) = 0.44 and 1.19, respectively, p > 0.05; n = 8),
indicating that these groups did not exhibit a memory for the training object location. In
contrast, mice receiving DPN, 100 pg ISP358-2, or 1 ng ISP358-2 spent significantly more time
than chance with the moved object (ts(6) = 4.5, 10.3, and 3.4, respectively, p < 0.05; n = 7),
indicating robust memories for the training object location. In addition, a oneꢀway ANOVA
conducted on the time spent with the moved object indicated a significant main effect of
treatment (F(4,32) = 2.97, p = 0.034). Fisher’s LSD posthoc tests indicated that the DPN, 100 pg,
and 1 ng groups spent significantly more time with the moved object than the vehicle group,
whereas the vehicle and 10 pg groups did not differ from each other. Together, these data
suggest that dorsal hippocampal infusion of 100 pg or 1 ng ISP358-2 enhanced object placement
memory consolidation.
Results for object recognition (Fig. 8c) were nearly identical. Neither the vehicle nor 10
pg ISP358-2 groups showed a preference for the novel object (ts(9ꢀ10) = 1.08 and 0.88,
respectively, p > 0.05; n = 10ꢀ11). However, the DPN, 100 pg ISP358-2, and 1 ng ISP358-2
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groups all spent significantly more time than chance with the novel object (ts(9ꢀ10) = 2.35, 3.16,
and 1.08, respectively, p < 0.05; n = 10ꢀ11). Moreover, the main effect of treatment was
significant (F(4,48) = 3.69, p = 0.011), and posthoc tests confirmed that the DPN, 100 pg ISP358-
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, and 1 ng ISP358-2 groups, but not the 10 pg ISP358-2 group, differed significantly from
vehicle. As with object placement, these data indicate that dorsal hippocampal infusion of 100
pg or 1 ng ISP358-2, but not 10 pg ISP358-2, enhanced object recognition memory
consolidation.
Intraperitoneal injection. We next used a new set of mice to investigate whether
systemic administration of ISP358-2 also provide similar memory enhancing effects as
intrahippocampal infusion (Fig. 8d,e). Intraperitoneal (IP) injection is a common, reliable, and
convenient systemic treatment in which the injected drug is absorbed into the blood vessels
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through the peritoneum. Because the doses of the drugs for intrahippocampal infusion are much
smaller than that needed to cross the blood brain barrier, we examined a range of IP doses based
on the cellꢀbased assay and DH infusion results above and previous work showing that IP
31
injections of 0.05 mg/kg DPN enhanced object recognition memory. In our cellꢀbased assays,
the EC50 of ISP358-2 was approximately 10 times higher than that of DPN. Moreover, our
behavioral tests showed that ISP358-2 enhances hippocampal memory at a concentration 10
times higher than DPN. Therefore, our IP doses of ISP358-2 were at least 10 times higher than
DPN (0.5 mg/kg and 5 mg/kg). We thus tested four groups of mice as follows: vehicle (negative
control), DPN (positive control), and two doses of ISP358-2 (0.5 mg/kg and 5 mg/kg).
For object placement (Fig. 8d), oneꢀsample tꢀtests indicated that mice receiving vehicle
did not show a preference for the moved object (t = 0.68, p > 0.05; n = 9). However, the DPN,
(
8)
0.5 mg/kg ISP358-2, and 5 mg/kg ISP358-2 groups all spent significantly more time than chance
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with the moved object (ts(9ꢀ11) = 3.20, 3.93, and 2.78, respectively, p < 0.05; n = 10ꢀ12),
suggesting that systemic administration of ISP358-2 enhanced object placement memory
consolidation. Moreover, the main effect of treatment was significant (F(3,38) = 3.63, p = 0.021),
and posthoc tests showed that the 0.5 mg/kg ISP358-2 group differed significantly from vehicle.
These data demonstrate that IP injection of ISP358-2 enhances spatial memory consolidation in
a manner similar to dorsal hippocampal infusion.
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Similar results were observed for object recognition (Fig. 8e). Oneꢀsample tꢀtest results
showed that mice receiving vehicle did not spend significantly more time than chance with the
novel object (t = 1.40, p > 0.05; n = 10). In contrast, the DPN and 0.5 mg/kg ISP358ꢀ2 groups
(9)
exhibited a significant preference for the novel object relative to chance (ts(8 and11) = 3.52 and
.17, respectively, p < 0.01; n = 12 and 9). There was also somewhat of a trend for the 5 mg/kg
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ISP358-2 to prefer the novel object (t(12) = 1.65, p = 0.125; n = 13). In addition, the main effect
of treatment was significant (F(3,40) = 5.05, p = 0.005), and posthoc tests verified that the DPN,
0
.5 mg/kg ISP358-2, and 5 mg/kg ISP358ꢀ2 groups differed significantly from the vehicle group.
Together, the object placement and object recognition data suggest that IP administration of
ISP358-2, particularly the 0.5 mg/kg dose, enhances object recognition and spatial memory
consolidation similar to dorsal hippocampal infusion. Importantly, these data also demonstrate
brain penetrance and behavioral efficacy in ovariectomized mice.
Oral gavage. Given the mnemonic effectiveness of IP injection, we next assessed
whether oral administration of ISP358-2 could enhance memory consolidation (Fig. 8f,g). Oral
gavage is a common procedure in scientific experiments delivering the drug directly into the
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stomach by means of a syringe. Although it is highly effective and more accurate than other
oral administration methods such as administration through delivery in food and/or water, it is
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more invasive and stressful. Because we observed that IP injection of ISP358-2 enhanced
hippocampal memory consolidation, we used the same doses for oral gavage as for IP injections
(vehicle, 0.5 or 5 mg/kg ISP358-2 and 0.05 mg/kg DPN).
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Similar results were observed for oral administration as were observed for IP injection
ISP358-2. For object placement (Fig. 8f), oneꢀsample tꢀtests results showed that mice receiving
vehicle did not spend significantly more time than chance with the moved object (t = 0.54, p >
(8)
0.05; n = 9). However, the DPN, 0.5 mg/kg ISP358-2, and 5 mg/kg ISP358-2 groups all
exhibited a significant preference for the moved object relative to chance (ts(8ꢀ9) = 2.76, 3.65,
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.06, respectively, p < 0.05; n = 9ꢀ10), suggesting that oral administration of ISP358-2 enhanced
spatial memory consolidation. Also, oneꢀway ANOVA showed that the main effect of treatment
was significant (F(3,33) = 5.04, p = 0.006), and posthoc tests verified that the DPN, 0.5 mg/kg
ISP358-2, and 5 mg/kg ISP358-2 groups differed significantly from the vehicle group. Likewise,
for object recognition (Fig. 8g), oneꢀsample tꢀtests indicated that mice receiving vehicle did not
show a preference for the novel object (t(8) = 0.25, p > 0.05; n = 9). In contrast, the DPN, 0.5
mg/kg ISP358-2, and 5 mg/kg ISP358-2 groups all spent significantly more time with the novel
object relative to chance (ts(7ꢀ9) = 3.89, 5.37, 2.36, respectively, p < 0.05; n = 8ꢀ10). Moreover,
the main effect of treatment was significant (F(3,32) = 3.02, p = 0.044), and posthoc tests showed
that the DPN, 0.5 mg/kg ISP358-2, and 5 mg/kg ISP358-2 groups differed significantly from
vehicle. Together, the object placement and object recognition behavior results demonstrate that
oral administration of ISP358-2 enhances object recognition and spatial memory consolidation
similar to dorsal hippocampal infusion or IP injection. These data also suggest the oral
bioavailability of ISP358-2 in ovariectomized mice.
Finally, we collected preliminary data to assess the effectiveness of orallyꢀgavaged
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ISP358-2 on spatial memory consolidation in mice who experienced longꢀterm estrogen
deprivation. Mice that received i.p. injections of vehicle, DPN, or ISP358-2 above remained in
our colony for 4 months after ovariectomy. They were then trained in the object placement task
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(using new objects) and then immediately given vehicle, DPN, or ISP358-2 via oral gavage in
the same doses described above (n = 9ꢀ12/group). Unlike mice gavaged within 1 month of
ovariectomy (Fig. 8f), DPN or ISP358-2 did not enhance spatial memory consolidation in mice
treated within 4 months of ovariectomy (Fig. S5a). We then measured ERα and ERβ levels in
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the DH using Western blotting as per our previous work (ERα, 1:200, Santa Cruz
Biotechnology; ERβ, 1:200, Santa Cruz Biotechnology); tissue was collected approximately 2
and 5 months after ovariectomy. Levels of ERβ were significantly reduced 5 months after
ovariectomy relative to 2 months after ovariectomy (Fig. S5b; t = 2.46, p < 0.05). In contrast,
9
levels of ERα did not change (Fig. S5c). These data suggest that DPN or ISP358-2 did not
enhance memory after longꢀterm ovariectomy, because of the reduced levels of ERβ. By
extension, these data also support in vivo selectivity of ISP358-2 for ERβ; because, if ISP358-2
enhanced memory via binding to ERα, then it should have been able to enhance memory
consolidation after longꢀterm ovariectomy because ERα levels were not reduced. However, the
fact that ERβ levels were reduced at a time at which ISP358-2 did not enhance memory supports
our hypothesis that that it regulates memory via ERβ and not ERα.
Assessment of Peripheral Pathology or Cell Proliferation Due to ISP358-2
Treatment. In general, all the tissues from the twenty different specimens appeared similar.
Heart: the cardiac tissues were all unremarkable. The ventricular walls were intact and normal
thickness. The atrial walls were intact with normal thickness. There was no evidence of
congenital defects such as myofiber disarray or ischemic heart disease or ischemic injury. There
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was no evidence of inflammation or myocarditis. Kidney: The kidneys were all unremarkable.
The glomeruli were intact. The tubules appeared normal. There was no evidence of inflammation
involving any of the structures of the kidneys. Liver: The general architecture of the liver was
intact and normal appearing with large portalꢀtypes veins running together with hepatic ducts and
hepatic arteries. The central veins were present and normal appearing. A generalized appearance
of low grade/mild ischemic injury was present in all samples. This seemed nonꢀspecific, was
appreciated in all specimens, and could be secondary to early ischemic damage or autolysis that
occurred post mortem. In several animals, small foci of cellular necrosis were observed likely
secondary to ischemia. Two animals showed areas with mild, low grade inflammation that was
small and focal. One animal (R15ꢀIPꢀ24 V) had multifocal areas of an organized inflammatory
infiltrate composed primarily of mononuclear lymphocytes. Overall there was no evidence of
acute inflammation composed of neutrophils or damage to structures in the liver, such as hepatic
ducts. Bloodwork chemistry and hematology data (Fig. S7) for treated animals do not show
significant deviations from expected reference ranges, relative to vehicle, except for modest
effects due to hemolysis that was likely due to sample collection via cardia puncture. Finally,
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while E caused statistically significant proliferation of MCFꢀ7 breast cancer cells at doses of 10,
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00 and 1,000 ꢀM, neither ISP358-2 nor DPN showed any significant proliferation, relative to
untreated control cells.
Discussion
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ERβ has previously been pursued as a drug target for a wide range of conditions,
including anxiety, depression, schizophrenia, and Alzheimer’s disease, with representative ERβ
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drug lead agonist compounds shown in Fig. 1a. Compounds presented herein (Table 1) differ
from these previously reported compounds in that they are more selective for ERβ over ERα (~
750 fold), and in that they are A-C estrogens that resemble the native 17βꢀestradiol molecule,
lacking only the B and D rings.
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Structure Activity Relationship for the A-C Estrogens. The binding affinity of 4ꢀ(4ꢀ
substituted cyclohexyl)phenols were assessed in a TRꢀFRET ERβ binding assay (Table 1 and
Fig. 2). In particular, compounds bearing a hydroxymethyl functionality attached to the
cyclohexyl core showed higher affinities, in the range 20ꢀ200 nM. Of the two components in the
2:1 mixture of cis- and trans-stereoisomers 15/16 (EC = 184 nM), the transꢀisomer was found
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to be more potent (ISP358-2, EC = 24 nM) than the mixture. Introduction of unsaturation
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within the sixꢀmembered ring (18, EC = 49 nM) did not greatly reduce the binding affinity
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compared to ISP358-2; however, conformational restriction, such as is present in the exocyclic
allylic alcohol, reduced affinity (24, EC = 676 nM). The presence of a third hydroxyl group led
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to greatly reduced binding affinity (12, EC = 2,700 nM). Finally, varying the distance between
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the phenolic OH and the aliphatic alcohol group gave a larger range of binding affinities (2, EC50
7250 nM, 25, EC = 11 nM). Those analogs having EC values < 200 nM were further tested
=
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in a cellꢀbased transcriptional activation assay to evaluate their ERβ selectivity both in terms of
binding affinity and of efficacy in activating transcription, in a biologically relevant system. The
transꢀstereoisomer ISP358-2, 8, 18, and 25 exhibited similar ERβ agonist potencies (EC ~ 30ꢀ
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5 nM) in the TRꢀFRET assay; but, ISP358-2 stands out as being the most potent and selective
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in this more biologically relevant cellꢀbased functional assay. Interestingly, the hydroxyethyl
analog was less potent in the cellꢀbased functional assay (25, EC = 75 nM vs. 11 nM).
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The potency differences observed in the assays may be due to the nature of what the
assays measure. The TRꢀFRET assay in Fig. 2 measures only displacement of a fluorescently
labelled estradiol ligand from the ligand binding domain (LBD), which reflects binding affinity
for the ligand that competitively displaces the fluorescent probe. In contrast, the cellꢀbased assay
is more complicated and measures the entire sequence of molecular events leading to
transcriptional activation. This sequence of events involves a series of conformational changes
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(e.g. a rotation of Helixꢀ12 of the ligand binding domain) that are triggered by the initial
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hormone binding. The protein conformational change in the estrogen receptor induced by
agonist binding results in recruitment of a coactivator protein and also triggers protein
dimerization, ultimately resulting in DNA binding and transcriptional activation. Additional
interactions between the aliphatic hydroxyl group and the His475 residue of ERβ plays a role in
the conformational change involving nearby Helixꢀ12, which would be reflected in EC values
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in the cellꢀbased functional assay (Fig. 5); but, not in the TRꢀFRET binding assay (Fig. 2).
All compounds tested showed no significant ERβ or ERα antagonist activity (EC50
>
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0,000 nM), thus also demonstrating their selectivity as agonist vs. antagonist activity (Table 1
and Fig. 5c,d). Of the ERβ agonists, ISP358-2 was the most selective, with ~ 750ꢀfold agonist
selectivity for ERβ over ERα in the cellꢀbased functional assay. But, it had only modest
selectivity in the TRꢀFRET binding assay (Fig. 2b).
Assay Differences Suggest Mechanism for Isoform Selectivity. As mentioned above,
the cellꢀbased assay for ISP358-2 indicates that it is ~ 750 fold selective for ERβ agonist activity
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over ERα agonist activity (Fig. 5a,b); whereas, the TRꢀFRET binding assay shows more modest
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2ꢀfold selectivity for ERβ (Fig. 2b). This could be because the TRꢀFRET binding assay simply
measures binding affinity (to an isolated LBD), whereas the cellꢀbased assay measures
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transcription that is induced by agonist binding to a fullꢀlength and native ERβ, which causes a
productive conformational change in ERβ that includes rotation of Helixꢀ12 (leading to
recruitment of coactivators, dimerization, DNA binding and then transcriptional activation). To
test the hypothesis that assay differences are due to these downstream activation events, two
other assays were performed. In a first assay, transcriptional activation was measured in a
different cellꢀbased assay, now using an unnatural chimeric receptor (ER LBD fused to a GLA4
DBD). In this assay (Fig. 3b), there was a modest 2.6ꢀfold selectivity for ERβ. In a second assay,
ability of agonist binding to recruit binding of a coactivator peptide to the ER LBD was
measured (Fig. 4a). In this assay, 15ꢀfold selectivity for ERβ was observed (Fig. 4c). Thus, ERβ
versus ERα selectivity for ISP358-2 varies significantly, based on how well the assay
incorporates native downstream activation events that occur subsequent to binding to the ERβ
binding pocket, as a result of hormoneꢀinduced conformational changes. It therefore appears that
the significant ERβ versus ERα selectivity shown by ISP358-2 is not just a function of binding
affinity for the ERβ receptor (as measured in the TRꢀFRET assay in Fig. 2b); but, rather is a
function of the ability to induce the productive conformational change that leads to downstream
activation of transcription (Fig. 5a).
Consistent with the above hypothesis that ISP358-2 potency and selectivity is related to
its ability to drive a productive conformational change, docking studies show that ISP358-2
docks into the ERβ active site in a conformation that differs significantly from that for the ERα
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binding site. Key differences occur where the estrogen C ring is normally located (Fig. 7c, f, g),
thereby affecting positioning of the aliphatic hydroxyl group that interacts with the His524
and/or Gly472 (backbone carbonyl) residues (Fig. 7f-g) in the region known to be important for
driving the Helixꢀ12 conformational change that permits binding of coactivator. The ERβ
hydrophobic interactions include PiꢀPi stacking between Phe356 and the ISP358-2 phenol ring,
along with Ala302, Leu298, Leu476 and Ile373 that constrain the cyclohexyl ring and its
attached hydroxymethyl methyl group in the “C ring” region (Fig. 1d). These unique
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o
hydrophobic interactions in the ERβ active site may be what drives the 90 rotation of the C
(cyclohexane) ring of ISP358-2 relative to the phenol ring, in the ERβ pocket relative to the ERα
pocket (Fig. 7f, g); and, in this way they could affect the adjacent coactivator pocket. This region
of the 17βꢀestradiol binding pocket is known to affect the accessibility and structure of the
coactivator binding pocket, so could be the reason for the large differences in agonist activity
that were observed for ISP358-2. Future structural characterization studies are being planned to
address this question.
Druggability and Preliminary Safety Toxicity. While ISP358-2 binds to ER and
activates transcription, it shows no significant offꢀtarget activity with seven other nuclear
hormone receptors (Fig. 3a). ISP358-2 also showed no significant activity against the heart
potassium ion channel hERG (Supplementary Fig. S3b) and no significant inhibition of the
major drug metabolizing cytochrome P450 enzymes, CYP2D6, CYP3A4, CYP2C9 and CYP1A2
(
Fig. 6). ISP358-2 was also reasonably soluble, showing no aggregation in nephelometry assays
(Supplementary Fig. S3a).
To assess the potential of ISP358-2 to stimulate breast cancer cell growth, MTT assays
with MCFꢀ7 human breast cancer cells were performed (Supplementary Fig. S8). No significant
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changes in the growth of MCFꢀ7 cells were observed following treatment at any concentration of
the ERβ agonists ISP358-2 or DPN compared to untreated controls (Supplementary Fig.
S8b,c). However, the proliferation of MCFꢀ7 cells treated with 1, 0.1 or 0.01 µM E was
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significantly increased (n=3; p<0.02, 0.05, 0.00, respectively) compared to untreated controls
(Supplementary Fig. S8a). Furthermore, cell proliferation was significantly lower (n=3; p≤0.04
for both compounds) in comparison to positive control MCFꢀ7 cells treated with 0.01 µM E2.
To assess potential peripheral pathology due to ISP358-2 treatment, a histological
analysis of tissue slices of treated animals was performed (Supplementary Fig. S6). Overall,
tissue changes due to treatment were unremarkable. Mild, global ischemic changes were noted in
the livers of all animals. It is difficult to assign a specific pattern or significance to this finding.
These changes likely represent hypoꢀperfusion and subsequent mild ischemic changes in the post
mortem period. One animal showed organized lymphoid hyperplasia in the liver. None of the
animals demonstrated any significant pathological changes in the heart or kidney.
In vivo Efficacy. A single postꢀtraining systemic injection or intracranial infusion of E2
or agonists of ERα or ERβ, including DPN, enhances memory consolidation in the object
recognition and object placement via rapid nonꢀclassical (aka, nonꢀgenomic)
6
,34,38,39,40,42,62
effects.
These compounds activate numerous cell signaling pathways in the dorsal
hippocampus within 5 minutes of dorsal hippocampal infusion, and pharmacological inhibition
6
,34,59
of this activation prevents E , DPN, and the ERα agonist DPN from enhancing memory.
2
The nonꢀclassical nature of these effects have been demonstrated in studies showing that the
require interactions among ERα, ERβ, and metabotropic glutamate receptors at the plasma
3
4
membrane. In the present study, in vivo behavioral assays measuring object placement or object
recognition (Fig. 8a), showed efficacy for all three routes of administration: microinfusion into
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the dorsal hippocampus, intraperitoneal injection, or oral gavage (Fig. 8). Thus, ISP358-2 can
enhance object recognition and spatial memory consolidation in ovariectomized female mice.
Intrahippocampal infusion of 100 pg and 1 ng ISP358-2 enhanced memory consolidation in the
object recognition and object placement tasks as effectively as the ERβ agonists DPN (Fig.
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b,c). In systemic administration experiments, 0.5 mg/kg ISP358-2 most effectively enhanced
consolidation in both tasks when delivered intraperitoneally (Fig. 8d,e), whereas 5 mg/kg
ISP358-2 was most effective via oral gavage (Fig. 8f,g). These data are consistent with previous
findings showing that intrahippocampal or systemic administration of the ERβ agonists DPN or
WAY200070 enhance hippocampalꢀdependent memory in ovariectomized rats and mice in tasks
3, 34, 39ꢀ42
including object recognition, object placement, and the radial arm maze.
As such,
ISP358-2 mimics the memoryꢀenhancing effects of other ERβ agonists with different chemical
structures and could potentially be used to reduce memory dysfunction in numerous
neuropsychiatric conditions for which women are at increased risk, including AD, depression,
4
3
43
and schizophrenia. Moreover, women are at greater risk of anxiety disorders than men, and
DPN decreases anxietyꢀrelated behaviors among rodents tested in the open field and elevated
4
4, 45
plus maze tasks.
Thus, ISP358-2 has the potential to not only facilitate memory
consolidation but also to reduce anxiety. Although promising, numerous issues remain to be
addressed in future studies, including the extent to which the beneficial effects of ISP358-2
generalize to males, older subjects, rodent models of AD and other disorders, and other forms of
memory.
Finally, while it was observed that ISP358-2 is highly selective for ERβ over ERα in the
more biologically relevant cellꢀbased assay, it is not known if it has this same selectivity for ERβ
in vivo. However, our preliminary studies have shown a correlation between behavioral results
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and levels of ERβ in the brain, consistent with the effect being related to ERβ agonist activity
(Supplementary Fig. S5). While the observed behavioral effects are consistent with the
predicted blood brain barrier (BBB) penetration (Supplementary Fig. S10) and subsequent
activation of ERβ, future studies will be directed to determining the pharmacological mechanism
of ISP358-2 in vivo, including in vivo studies of isoform selectivity, effects on signaling
cascades and neural morphology changes in the brain, as well as pharmacokinetics and
pharmacodynamics.
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Conclusion
The results of the current study demonstrate that our lead compound, ISP358-2, is
selective for ERβ, and shows no obvious signs of peripheral toxicity. Importantly, ISP358-2 also
enhances multiple types of memory dependent on the hippocampus, a brain region involved in
4
3, 46
numerous disorders including AD, depression, and schizophrenia.
ISP358-2 is distinct from
previously reported ERβ agonists in that it has higher selectivity for ERβ over ERα, and in that it
more closely resembles that naturally occurring 17βꢀestradiol molecule (Fig. 1d), as an A-C
estrogen. Our studies also demonstrated biological efficacy in behavioral assays that were
performed via three routes of administration: direct dorsal hippocampal infusion, intraperitoneal
injection, and oral gavage, the latter two of which illustrate brain penetrance of the effective
doses (Fig. 8). Overall, these findings suggest that the novel ERβ agonist ISP358-2 could be a
promising drug candidate for enhancing memory in a variety of disorders characterized by
memory dysfunction that occurs under low estrogen conditions, such as menopause.
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Experimental Section
Compound Synthesis. All the chemicals were purchased from SigmaꢀAldrich, Matrix
Scientific, or Alfa Aesar and used as received. Reactions with moistureꢀ or airꢀsensitive reagents
were conducted under an inert atmosphere of nitrogen in ovenꢀdried glassware with anhydrous
solvents. Reactions were followed by TLC on precoated silica plates (60 Å, F , EMD
1
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54
Chemicals Inc) and were visualized by UV lamp (UVGLꢀ25, 254/365 nm). Flash column
chromatography was performed by using flash silica gel (32–63 ꢁ). NMR spectra were recorded
on Varian UnityInova 400 MHz instrument. CDCl , d –acetone, and CD OD were purchased
3
6
3
1
from Cambridge Isotope Laboratories. H NMR spectra were calibrated to δ = 7.26 ppm for
13
residual CHCl , δ = 2.05 ppm for d ꢀacetone and δ = 3.30 ppm for residual d ꢀCD OD.
C
3
5
3
3
NMR spectra were calibrated from the central peak at δ = 77.23 ppm for CDCl δ = 29.92 ppm
3,
for d ꢀacetone and δ = 49.00 ppm for CD OD. Purity of all compounds was >95%, determined
6
3
with chromatography and NMR.
2
trans-4-(4-Hydroxycyclohexyl)phenol (2). To a solution of 1 (0.200 g, 5.30 mmol) in
anhydrous methanol (15 mL) at room temperature was added solid NaBH (0.400 g, 10.6 mmol).
4
The mixture was stirred for 3 h and then extracted with several times with ethyl acetate. The
combined extracts were concentrated to give 2 (0.181 g, 90%) as a colorless solid. mp 196ꢀ208
1
°
C. H NMR (400 MHz, CD OD) δ 7.00 and 6.67 (AA’XX’, J = 8.7 Hz, 4H), 3.61ꢀ3.53 (m,
3
AX
1H), 2.38 (tt, J = 11.8, 3.4 Hz, 1H), 2.05ꢀ1.98 (m, 2H), 1.87ꢀ1.78 (m, 2H), 1.56ꢀ1.30 (m, 4H)
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13
ppm. C NMR (100 MHz, CD OD) δ 156.6, 139.2, 128.7, 116.1, 71.4, 49.3, 44.3, 36.9, 34.2
3
–
2
+
ppm. HRMS m/z 191.1077 [calcd for C H O (M–H ) 191.1077].
1
2
15
0
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(3)
4
-(4-Hydroxy-4-methylcyclohexyl)phenol (3). To a solution of 1 (0.100 g, 0.526 mmol) in dry
ether (20 mL) at –78 °C under N , was slowly added a solution of methyllithiumꢀlithium
2
bromide complex (1.5 M in ether, 0.78 mL, 1.2 mmol). The mixture was stirred at –78 °C for 30
min, warmed to room temperature and stirred for another 1 h. The mixture was cooled to 0 °C
and quenched with water. The mixture was extracted several times with ether, and the combined
extracts dried (Na SO ) and concentrated. The residue was purified by column chromatography
2
4
(
SiO , hexanes–ethyl acetate = 4:1) to give 3 (0.040 g, 37%) as a colorless solid. mp 126ꢀ131
2
1
°
C; H NMR (400 MHz, CD OD) δ 7.03 and 6.67 (AA’XX’, J = 8.3 Hz, 4H), 2.35 (tt, J =
3
AX
1
3
12.4, 3.6 Hz, 1H), 1.87ꢀ1.69 (m, 4H), 1.61ꢀ1.44 (m, 4H) 1.21 (s, 3H); C NMR (100 MHz,
CD OD) δ 156.5, 140.0, 128.8, 116.1, 69.5, 44.5, 40.0, 31.9, 31.1 ppm. HRMS m/z 205.1234
3
–
+
[
calcd for C H O (M–H ) 205.1234]. Anal. calcd. for C H O : C, 75.69; H 8.79. Found: C,
13 17 2 13 18 2
75.33; H, 8.83.
4
4-(4-Hydroxyphenyl)cyclohexanone oxime (4). To a solution of 1 (0.050 g, 0.26 mmol) in
ethanol (10 mL), were added Amberlyst (0.060 g) and hydroxylamine hydrochloride (0.039 g,
0
.560 mmol). The mixture was stirred at room temperature for 2 h and then filtered. The filtrate
was concentrated and extracted several times with ethyl acetate. The combined organic extracts
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were washed with water, dried (MgSO ), and concentrated to give 4 as a colorless solid (0.037 g,
4
o
1
7
0%). mp 171ꢀ174 C; H NMR (400 MHz, CD OD) δ 7.00 and 6.69 (AA’XX’, J = 8.2 Hz,
3 AX
4H), 3.39 (broad d, J = 13.5 Hz, 1H), 2.67 (t, J = 12.8 Hz, 1H), 2.41 (broad d, J = 14.0 Hz, 1H),
1
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6
.20 (td, J = 14.6, 5.4 Hz, 1H), 1.93 (broad t, J = 15.8 Hz, 2H), 1.81 (td, J = 14.0, 5.2 Hz, 1H),
13
.61ꢀ1.42 (m, 2H) ppm. C NMR (100 MHz, CD OD) δ 160.8, 156.7, 138.3, 128.6, 116.2, 44.1,
3
5.8, 34.6, 32.8, 25.1 ppm. Anal. calcd. for C H NO : C, 70.22; H 7.36; N, 6.83. Found: C,
12 15
2
9.93; H, 7.36; N, 6.63.
5
4-(4-t-Butyldimethylsilyloxyphenyl)cyclohexan-1-one (5). To a solution of 1 (0.500 g, 2.62
mmol) in anhydrous CH Cl (30 mL) at 0 ˚C under N , was added imidazole (0.357 g, 5.24
2
2
2
mmol). After 30 min tꢀbutyldimethylsilyl chloride (0.594 g, 3.94 mmol) was added and the
mixture was gradually warmed to room temperature overnight. The resulting mixture was
diluted with brine (25 mL) and extracted several times with CH Cl . The combined organic
2
2
extracts were dried (Na SO ) and concentrated. The residue was purified by column
2
4
chromatography (SiO , hexanesꢀethyl acetate = 9:1) to give 5 (0.664, 83%) as a colorless solid.
2
1
mp 39ꢀ42 °C. H NMR (400 MHz, CDCl ) δ 7.08 and 6.78 (AA’XX’, J = 8.4 Hz, 4H), 2.96
3
AX
(t, J = 12.3 Hz, 1H), 2.56ꢀ2.40 (m, 4H), 2.25ꢀ2.14, (m, 2H), 1.97ꢀ1.82 (m, 2 H), 0.98 (s, 9H),
1
3
0
.19 (s, 6H) ppm. C NMR (100 MHz, CDCl ) δ 211.6, 154.3, 137.7, 127.7, 120.1, 42.2, 41.6,
3
34.6, 25.9, 18.4, ꢀ4.2 ppm.
6
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t-Butyldimethyl(4-(4-methylenecyclohexyl)phenoxy)silane (6). To
a
solution of
methyltriphenylphosphonium bromide (0.836 g, 2.34 mmol) in dry THF (20 mL) at –10 °C
under N , was slowly added a solution of nꢀbutyllithium (1.6 M in hexane, 1.50 mL, 2.4 mmol).
2
0
1
2
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After 30 min, a solution of 5 (0.502 g, 1.17 mmol) in dry THF (8 mL) was added dropwise. The
reaction mixture was slowly warmed to room temperature and stirred overnight. After this time,
the mixture was diluted with water (20 mL), extracted several times with ethyl acetate, and the
combined extracts were dried (Na SO ) and concentrated. Purification of the crude residue by
2
4
column chromatography (SiO , hexanesꢀethyl acetate = 9:1) gave 6 (1.678 g, 84%) as a colorless
2
1
oil. H NMR (400 MHz, CDCl ) δ 7.06 and 6.77 (AA’XX’, J = 8.3 Hz, 4H), 4.68 (s, 2H),
3
AX
2.62 (tt, J = 12.1, 3.4 Hz, 1H), 2.42 (broad d, J = 13.5 Hz, 2H), 2.18 (broad t, J = 13.2, 2H), 2.00ꢀ
13
1
1
.93 (m, 2H), 1.57ꢀ1.45 (m, 2H), 0.99 (s, 9H), 0.20 (s, 6H) ppm. C NMR (100 MHz, CDCl ) δ
3
53.9, 149.2, 139.8, 127.8, 119.9, 107.4, 43.5, 35.9, 35.4, 25.9, 18.4, ꢀ4.2 ppm. Anal. calcd. for
C H O Si: C, 75.43; H, 9.99. Found: C, 75.71; H, 10.02.
19
30
2
(7)
4-(4-Hydroxyphenyl)methylenecyclohexane (7). To a solution of 6 (0.739 g, 0.244 mmol) in
anhydrous THF (20 mL) was added a solution of TBAF (1 M in THF, 9.8 mL, 9.8 mmol). The
mixture was heated at reflux for 5 h. After cooling, the solution was partitioned between ethyl
acetate and water, and the aqueous layer was extracted several times with ethyl acetate. The
combined organic layers were washed with brine, dried (Na SO ) and concentrated. Purification
2
4
of the residue by column chromatography (SiO , hexanes–ethyl acetate = 4:1) gave 7 (0.379 g,
2
1
8
3%) as a colorless solid. mp 82ꢀ84 °C; H NMR (400 MHz, CD OD) δ 6.99 and 6.67
3
(AA’XX’, JAB = 8.5 Hz, 4H), 4.63 (t, J = 1.7 Hz, 2H), 2.57 (tt, J = 12.3, 4.3 Hz, 1H), 2.41ꢀ2.33
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Page 27 of 71
Journal of Medicinal Chemistry
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9
13
(
m, 2H), 2.22ꢀ2.11 (m, 2H), 1.94ꢀ1.85 (m, 2H), 1.45 (qd, J = 12.3, 4.3 Hz, 2H); C NMR (100
MHz, CD OD) δ 156.6, 150.3, 139.3, 128.8, 116.2, 107.8, 44.8, 37.3, 36.4 ppm. HRMS m/z
3
–
+
187.1128 [calcd for C H O (M–H ) 187.1128]. Anal. calcd. for C H O : C, 82.93; H 8.57.
1
3
15
13 16
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0
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Found: C, 82.71; H, 8.58.
(8)
4-(4-Methylcyclohexyl)phenol (8). To a solution of 7 (0.150 g, 0.797 mmol) in methanol (10
mL) was added 10% Pd/C (85 mg, 10 mol %). The mixtures was stirred under a balloon filled
with H , at room temperature, for 12 h. The reaction mixture was filtered through a pad of celite,
2
dried (Na SO ) and concentrated. The residue was purified by column chromatography (SiO ,
2
4
2
hexanes–ethyl acetate = 4:1) to give 8 (0.121 g, 80%) as a colorless solid. This was determined
1
1
to be a mixture of cisꢀ and transꢀ stereoisomers by H NMR spectroscopy. mp 93ꢀ99 °C; H
NMR (400 MHz, CD OD) δ 7.05ꢀ6.96 (m, 2H), 6.70ꢀ6.64 (m, 2H), 2.48ꢀ2.28 (m, 1H), 1.83ꢀ1.34
3
1
3
(
m, 8H), 1.13ꢀ1.04 (m, 1H), 1.03 (d, J = 7.2 Hz, 1H), 0.92 (d, J = 6.6 Hz, 2H); C NMR (100
MHz, CD OD) d 156.3, 140.0, 128.6, 116.0, 44.7, 36.9, 35.9, 33.7, 33.1, 30.1, 23.1 ppm.
3
(9)
2
-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde (9). To a solution of 7 (0.100 g, 0.531
mmol) in dry CH CN (20 mL) were sequentially added MgCl (0.076g, 0.797), triethylamine
3
2
(
0.28 mL, 2.0 mmol), followed by paraformaldehyde (0.108 g, 3.59 mmol). The mixture was
heated at reflux for 6 h. The mixture was cooled to room temperature and quenched with 10%
HCl (10 mL) and extracted several times with ethyl acetate. The combined extracts were washed
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Journal of Medicinal Chemistry
Page 28 of 71
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with brine, dried (Na SO ) and concentrated. Purification of the residue by column
2
4
1
chromatography (SiO , hexanes–diethyl ether = 4:1) gave 9 (0.046 g, 40%) as a colorless oil. H
2
NMR (400 MHz, CD OD) δ 9.96 (s, 1H), 7.50 (s, 1H), 7.39 (d, J = 8.5 Hz, 1H), 6.85 (d, J = 8.5
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0
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0
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Hz, 1H), 4.65 (t, J =1.7 Hz, 2H), 2.68 (tt, J = 12.0, 3.4 Hz, 1H), 2.44ꢀ2.34 (m, 2H), 2.24ꢀ2.13 (m,
13
2
1
2
H), 1.97ꢀ1.89 (m, 2H), 1.49 (qd, J =13.0, 4.0 Hz, 2H); C NMR (100 MHz, CD OD) δ 197.5,
3
61.0, 149.8, 139.9, 137.0, 131.6, 122.5, 118.2, 108.2, 44.3, 36.9, 36.2 ppm. HRMS m/z
–
+
31.1027 [calcd for C H O (M–H ) 231.1027].
1
4
15
3
(10)
2
-Hydroxy-5-(4-methylenecyclohexyl)benzaldehyde oxime (10). To a solution of 9 (0.050 g,
0
.232 mmol) in pure ethanol (10 mL), were added sodium bicarbonate (0.024 g, 0.278 mmol)
and hydroxylamine hydrochloride (0.025 g, 0.348 mmol). The reaction was heated at 80°C for 5
h and the mixture was extracted several times with ethyl acetate. The combined organic extracts
were dried (MgSO ) and concentrated. Purification of the residue by column chromatography
4
(SiO , hexanes–ethyl acetate = 13:7) gave 10 (0.037 g, 69%) as a colorless solid. mp 120ꢀ125
2
1
°
C; H NMR (400 MHz, CD OD) δ 8.20 (s, 1H), 7.09–7.06 (m, 1H), 7.05 (d, J = 2.4 Hz, 1.8H),
3
6
.99 (d, J = 7.9 Hz, 0.2H), 6.78 (d, J = 8.1 Hz, 0.8H), 6.68 (d, J = 8.6 Hz, 0.2H), 4.63 (t, J = 1.6
Hz, 2H), 2.60 (tt, J =12.2, 3.3 Hz, 1H), 2.42ꢀ2.33 (m, 2H), 2.22ꢀ2.10 (m, 2H), 1.94ꢀ1.85 (m, 2H),
1
3
1
1
.46 (qd, J =12.5, 4.0 Hz, 2H); C NMR (100 MHz, CD OD) δ 156.6, 152.4, 150.1, 139.4,
3
30.2, 129.2, 128.7, 118.5, 117.2, 116.2, 108.0, 107.7, 44.5, 37.3, 37.1, 36.4, 36.3 ppm. HRMS
–
3
+
m/z 230.1187 [calcd for C H NO (M–H ) 230.1186].
14 16
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Page 29 of 71
Journal of Medicinal Chemistry
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4
-(4-((t-Butyldimethylsilyl)oxy)phenyl)-1-(hydroxymethyl)cyclohexan-1-ol (11).
To a
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solution of 6 (0.280 g, 0.926 mmol) and NꢀmethylmorpholineꢀNꢀoxide (0.13 mL, 1.3 mmol) in
acetone (6 mL) and distilled water (0.3 mL) was added a solution of OsO in tertꢀbutanol (2.5%,
4
90 µL). The mixture was stirred overnight and saturated aqueous NaHSO (10 mL) was added to
3
quench the reaction. The mixture was diluted with ether and washed several times with water.
The organic layer was dried (MgSO ), concentrated and the residue purified by column
4
chromatography (SiO , hexanes–ethyl acetate = 1:4) to give 11 (0.267 g, 86%) as a colorless
2
1
solid. mp 80ꢀ86 °C; H NMR (400 MHz, CDCl ) δ 7.04 and 6.75 (AA’XX’, J = 8.5 Hz, 4H),
3
AX
3
.69 (s, 2H), 2.52 (tt, J = 11.4, 3.6 Hz, 1H), 2.04ꢀ1.72 (m, 4H, solvent peak overlap), 1.61ꢀ1.37
1
3
(m, 4H), 0.97 (s, 9H), 0.18 (s, 6H); C NMR (100 MHz, CDCl ) δ 154.0, 138.9, 127.7, 120.0,
3
7
2.4, 66.2, 42.8, 35.4, 31.3, 25.9, 18.4, ꢀ4.2 ppm.
(12)
4
-(4-Hydroxy-4-(hydroxymethyl)cyclohexyl)phenol (12). To a solution of 11 (0.230 g, 0.683
mmol) in anhydrous THF (10 mL) was added a solution of TBAF (1M in THF, 2.8 mL, 2.8
mmol). The mixture was heated at reflux for 6 h and cooled to room temperature. The solution
was partitioned between ethyl acetate and water. The combined organic layers were washed
with brine, dried (Na SO ) and concentrated. Purification of the residue by column
2
4
chromatography (SiO , ethyl acetate–methanol = 9:1) gave 12 (0.118 g, 78%) as a colorless
2
1
solid. mp 182ꢀ188 °C; H NMR (400 MHz, CD OD) δ 7.02 and 6.68 (AA’XX’, J = 8.5 Hz,
3
AX
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