Identification of an Orally Bioavailable Chromene-Based Selective Estrogen Receptor Degrader (SERD) That Demonstrates Robust Activity in a Model of Tamoxifen-Resistant Breast Cancer

Article abstract of DOI:10.1021/acs.jmedchem.8b00921

About 75% of breast cancers are estrogen receptor alpha (ER-α) positive, and women typically initially respond well to antihormonal therapies such as tamoxifen and aromatase inhibitors, but resistance often emerges. Fulvestrant is a steroid-based, selective estrogen receptor degrader (SERD) that both antagonizes and degrades ER-α and shows some activity in patients who have progressed on antihormonal agents. However, fulvestrant must be administered by intramuscular injections that limit its efficacy. We describe the optimization of ER-α degradation efficacy of a chromene series of ER modulators resulting in highly potent and efficacious SERDs such as 14n. When examined in a xenograft model of tamoxifen-resistant breast cancer, 14n (ER-α degradation efficacy = 91%) demonstrated robust activity, while, despite superior oral exposure, 15g (ER-α degradation efficacy = 82%) was essentially inactive. This result suggests that optimizing ER-α degradation efficacy in the MCF-7 cell line leads to compounds with robust effects in models of tamoxifen-resistant breast cancer derived from an MCF-7 background.

Full text of DOI:10.1021/acs.jmedchem.8b00921

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Article
Identification of an orally bioavailable chromene-based Selective
Estrogen Receptor Degrader (SERD) that demonstrates
robust activity in a model of tamoxifen-resistant breast cancer
Johnny Nagasawa, Steven Govek, Mehmet Kahraman, Andiliy Lai, Celine Bonnefous,
Karensa Douglas, John Sensintaffar, Nhin Lu, Kyoungjin Lee, Anna Aparicio, Josh Kaufman,
Jing Qian, Gang Shao, Rene Prudente, James D Joseph, Beatrice Darimond, Daniel
Brigham, Kate Maheu, Richard Heyman, Peter Rix, Jeffrey H Hager, and Nicholas D. Smith
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00921 • Publication Date (Web): 07 Aug 2018
Downloaded from http://pubs.acs.org on August 8, 2018
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Identification of an orally bioavailable chromeneꢀ
based Selective Estrogen Receptor Degrader
(SERD) that demonstrates robust activity in a model
of tamoxifenꢀresistant breast cancer
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Johnny Nagasawa, a Steven Govek,a Mehmet Kahraman, a Andiliy Lai,a Celine Bonnefous,a
Karensa Douglas,a John Sensintaffar, b Nhin Lu,b KyoungJin Lee,c Anna Aparicio,c Josh
Kaufman,c Jing Qian,b Gang Shao,b Rene Prudente,b James D. Joseph,b Beatrice Darimont,b
Daniel Brigham,b Kate Maheu,b Richard Heyman, b Peter J. Rix,c Jeffrey H. Hager,b Nicholas D.
Smith a*
Seragon Pharmaceuticals, 12780 El Camino Real, Suite 302, San Diego, California 92130.
a
b
c
Department of Chemistry, Department of Biology, Department of Drug Safety and
Disposition
ABSTRACT About seventyꢀfive percent of breast cancers are estrogen receptor alpha (ERꢀα)
positive, and women typically initially respond well to antiꢀhormonal therapies such as
tamoxifen and aromatase inhibitors, resistance often emerges. Fulvestrant is a steroidꢀbased,
selective estrogen receptor degrader (SERD) that both antagonizes and degrades ERꢀα, and
shows some activity in patients who have progressed on antiꢀhormonal agents. However
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fulvestrant must be administered by intraꢀmuscular injections that limits its efficacy. We describe
the optimization of ERꢀα degradation efficacy of a chromene series of ER modulators resulting
in highly potent and efficacious SERDs such as 14n. When examined in a xenograft model of
tamoxifenꢀresistant breast cancer, 14n (ERꢀα degradation efficacy = 91%) demonstrated robust
activity, while despite superior oral exposure, 15g (ERꢀα degradation efficacy = 82%) was
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essentially inactive. This result suggests that optimizing ERꢀα degradation efficacy in the MCFꢀ7
cell line leads to compounds with robust effects in models of tamoxifenꢀresistant breast cancer
derived from an MCFꢀ7 backꢀground.
KEYWORDS: Estrogen receptor, estrogen receptor degrader, SERD, antagonist, tamoxifenꢀ
resistant, breast cancer, chromene.
INTRODUCTION
The estrogen receptor ERα is a ligandꢀregulated transcription factor, and mediates the activity of
estrogens in a number of physiological processes including reproduction, the cardiovascular
system and bone density/remodeling.1 The estrogen receptor (ER) is a well validated therapeutic
target for a number of indications, and there are several ER ligands that are approved drugs for
the treatment of cancer (tamoxifen, fulvestrant) and osteoporosis (raloxifene, bazodoxifene).2
Tamoxifen (1) and its active metabolite, 4ꢀhydroxytamoxifen (2) are selective estrogen receptor
modulators (SERMs), in that they function as an antagonist or agonist depending on the tissue
context.1 Unfortunately, despite many women with breast cancer initially responding to
tamoxifen, resistance often emerges.3 ER has been shown to still play an active role in this
resistant state,4 and fulvestrant (3), a selective estrogen receptor degrader (SERD) that both
antagonizes and induces degradation of ERꢀα, is of some clinical benefit in this patient
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population.5 However fulvestrant has poor pharmaceutical properties requiring delivery through
an intraꢀmuscular injection that limits the amount of drug that can be administered, and its
maximal clinical response. We thus set out to identify SERD’s where ERꢀα degradation efficacy
was optimized prospectively, and which were orally bioavailable allowing robust, therapeutic
levels of exposure to be achieved rapidly.
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Figure 1: ER ligands
There is a wealth of information in the literature which provides an understanding of the
structural elements required for high affinity ER ligand binding, but little has been described as
to what is required for maximal ERꢀα degradation while maintaining antagonist activity (i.e.
SERD activity). In order to prospectively optimize ERꢀα degradation, we utilized an inꢀcell
western assay that monitored the levels of ERꢀα in MCFꢀ7 breast cancer cells. This assay was
key to defining the SAR of the program, including the identification of the clinical compound
ARNꢀ810/GDCꢀ0810 (4) which was previously described (Figure 1).6 This paper describes our
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preliminary optimization of the chromene scaffold found in EMꢀ652 (5, Figure 1),7 ultimately
leading to 14n, a highly efficacious, orally bioavailable ERꢀα degrader with robust activity in a
model of tamoxifenꢀresistant breast cancer.
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Similar to the native ligand estradiol and many potent ER ligands in the literature, EMꢀ652 has
two free hydroxyls on its core structure, and these form key interactions in the ligand binding
domain (LBD) of ERꢀα.8 Appended to this core is a sideꢀchain that is proposed to modulate the
position of helix 12 of ER, and hence is the primary determinant of the antagonist/agonist
properties of the ligand. Due to the presence of one or more phenols that can be readily
glucuronidated in vivo, ER ligands typically suffer from low oral bioavailability and high
clearance due to gutꢀwall and liver glucuronidation.9 This is indeed the case with EMꢀ652 which
in our hands had low exposure following oral dosing in mice (AUC=0.09 ꢀg·h/mL; 10 mg/kg
peroral). However more recent work on related scaffolds had shown that the position of the
phenol on the bicyclic core can have an unexpectedly dramatic effect on the PK properties. As
shown in Figure 2 for dihydrobenzoxathiin10 and chromane11 scaffolds, the 6ꢀOH phenol isomers
have significantly higher rat oral bioavailability than the corresponding 7ꢀOH phenol isomers
(%F = 31 and 45, compared to %F = 0 and 2). In an attempt to maximize oral exposure, we
applied this finding to the chromene scaffold and from the outset focused our SAR studies at
maximizing ERꢀα degradation on a chromene core with a 6ꢀOH.
Figure 2: Effect of phenol position on inꢀvivo clearance and bioavailability of
dihydrobenzoxathiin and chromane scaffolds
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OH
O
OH
O
6
R1
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R1
7
S
O
O
N
N
9
Dihydrobenzoxathiin scaffold:
Chromane scaffold:
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R1 = 6-OH: CL = 5 mL/min/Kg; %F = 31
R1 = 7-OH: CL = 154 mL/min/Kg; %F = 0
R1 = 6-OH: CL = 7.7 mL/min/Kg; %F = 45
R1 = 7-OH: CL = 9.5 mL/min/Kg; %F = 2
CHEMISTRY
The general strategy for the synthesis of new ER ligands was the coupling of an aminoꢀalcohol
sideꢀchain (Scheme 1) and a central chromene core (Scheme 2) to produce the desired
compounds (Schemes 2ꢀ4). Unsubstituted aminoꢀalcohol sideꢀchains 7bꢀd were purchased from
commercial sources, while 7a, 7e and 7h were prepared by alkylation of the appropriate cyclic
amine with 2ꢀbromoethanol (Scheme 1). The rest of the sideꢀchains, all of which contained a
stereoꢀcenter, were prepared by alkylation of an aminoꢀalcohol with a bisꢀelectrophile, so
forming the cyclic amine structure. Thus 7f and 7g were prepared by reacting 2ꢀaminoethanol
with (R)ꢀ or (S)ꢀ2ꢀmethylbutaneꢀ1,4ꢀdiylꢀdimethanesulfonate to give the pyrrolidine with the
desired stereocenter on the ring. Compounds 7i, kꢀm, with an (S)ꢀmethyl substituent on the chain
were in turn prepared by alkylation of (S)ꢀ2ꢀaminopropanꢀ1ꢀol ((S)ꢀ8) with a diꢀhalo alkane as
shown. Similarly, alkylation of (S)ꢀ8 or (R)ꢀ8 with a bisꢀelectrophile 9 containing a methyl
substituent with either (S)ꢀ or (R)ꢀ stereochemistry, produced 7nꢀo and 7j, 7p, 7q respectively.
Scheme 1
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Analogs 14aꢀq were prepared from a common chromene intermediate 13a, which was prepared
in an eight step linear sequence as shown in Scheme 2. Starting material 2,5ꢀdimethoxybenzoic
acid (10) was converted to the Weinreb amide, and then treated with (3ꢀ
methoxybenzyl)magnesium chloride to afford a triꢀmethoxy ketone intermediate. Demethylation
with BBr3 afforded 11 in 58% yield over the three steps. The 5 and 3’ phenols of 11 were
selectively protected with a THP group, and then cyclization with 4ꢀiodobenzaldehyde gave
THPꢀprotected chromanone 12. To install the double bond of the chromene, 12 was treated with
methylmagnesium chloride to give the tertiary alcohol, followed by elimination to the alkene
under acidic conditions using AcOH/H2O. Under these elimination conditions deprotection of the
THP groups also occurred, requiring reꢀprotection to afford chromene 13a in 56% over three
steps. Finally, Ullmann coupling between 13a and aminoꢀalcohols 7aꢀq in the presence of copper
catalyst, followed by THPꢀdeprotection using acetic acid, gave chromene analogs (14aꢀq) in 6ꢀ
96% yield over 2 steps. The key Ullmann reaction was carried out in neat sideꢀchain or at high
concentration using butyronitrile or mꢀxylenes as a solvent, and in some cases, 2,2’bipyridine
was used as a ligand.
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Scheme 2
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As shown in Scheme 3, chromene phenol isomers 15bꢀd were prepared by Ullman coupling of
sideꢀchain 7n with iodochromene intermediates 13bꢀd (which were prepared in an analogous
way to that shown for 13a in scheme 2), followed by acetic acid/water deprotection of the THP
groups. In a similar way the monophenol analogues 15eꢀg were prepared.
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Scheme 3
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Other derivatives were prepared by palladiumꢀcatalyzed coupling with aryl halide intermediates
as shown in scheme 4. Thus nitrile derivatives 15hꢀi were prepared from the bromo or
chlorophenyl intermediates 16hꢀi using Zn(CN)2 and [1,1'ꢀbinaphthalen]ꢀ2ꢀyldiꢀtertꢀ
butylphosphine12 followed by THP deprotection. In turn, 15jꢀk were prepared from 16hꢀi (R =
Br) using sodium methanesulfinate and catalytic CuI/proline to install the sulfone group,13
followed by THP deprotection.
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RESULTS AND DISCUSSION
Figure 3 shows the degree of ERꢀα degradation by 4ꢀhydroxytamoxifen, fulvestrant and EMꢀ652
as determined by the key inꢀcell western assay in MCFꢀ7 cells. Fulvestrant was used as an
internal control in each assay run, and we report degradation efficacy of compounds as a percent
of this fulvestrant control. It should be noted that although potent, both 4ꢀhydroxytamoxifen and
EMꢀ652 do not have the same level of ERꢀα degradation efficacy as fulvestrant. Despite being a
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robust degrader, a small percentage of the ERꢀα still remains after fulvestrant treatment. This
may reflect an inaccessible ERꢀα population resistant to degradation such as a nuclear insoluble
fraction that is revealed upon cell permeabilization and antibody interrogation.14
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Figure 3: MCFꢀ7 ERꢀα degradation assaya
a: ERꢀα inꢀcell western in MCFꢀ7 breast cancer cells in phenolꢀred free RPMI medium containing 5% charcoal dextran treated FBS; readings
taken after 4 hours incubation using a SPꢀ1 antiꢀER rabbit monoclonal antibody
We initially examined the effect of ring size and alkyl substitution of the sideꢀchain, in particular
seeking to maximize ERꢀα degradation efficacy as monitored by the MCFꢀ7 inꢀcell western
assay. As shown in Table 1, all derivatives were potent ERꢀα degraders (IC50 < 1.0 nM) and had
similar potency as antagonists of cell proliferation in the MCFꢀ7 breast cancer cell line (IC50 <
0.5 nM). However the ERꢀα degradation efficacy varied depending on the ring size and
substitution pattern of the sideꢀchain. Of the unsubstituted nitrogenꢀcontaining heteroalkyl rings
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of sizes from 4 to 7 (14aꢀd), piperidine 14c had the highest degradation at 83% of the fulvestrant
control. Interestingly, with methyl substitution on the ring (14eꢀh), an increase in degradation
efficacy was obtained for azetidine 14e (86%) and pyrrolidine 14f (89%), while the piperidine
derivative 14h had a significant reduction in degradation efficacy (64%). Of note, the
stereochemistry of the pyrrolidine methyl was important with the (S)-configuration isomer 14g
being an inferior degrader (77%) compared to the (R)-configuration isomer 14f (89%). The
importance of stereochemistry was also observed when substitution was examined on the linker,
with (S)-methyl derivative 14i being a good ERꢀα degrader (86%), while 14j, with the opposite
configuration was a poor degrader (58%). Whereas this (S)ꢀconfiguration of the methyl on the
linker increased degradation efficacy for 14i over the parent pyrrolidine 14b (86% versus 75%),
installation of this methyl on the linker of other ring sizes, giving azetidine 14k, piperidine 14l or
homopiperidine 14m, did not lead to an increase in degradation efficacy over the unsubstituted
parent compounds 14a, 14c or 14d.
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Table 1: Ring size and stereochemistry of methyl substitution of sideꢀchain modulates
degradation efficacy
MCFꢀ7
ER Degradationa
Proliferationb
Compound
R1
IC50 (nM)
Efficacyc
IC50 (nM)
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14b
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0.2
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14l
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14m
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14n
14o
14p
14q
a: ERꢀα inꢀcell western in MCFꢀ7 breast cancer cells in phenolꢀred free RPMI medium containing 5% charcoal dextran treated FBS; readings
taken after 4 hours incubation using a SPꢀ1 antiꢀER rabbit monoclonal antibody (n ≥ 4) b: MCFꢀ7 proliferation assay in RPMI medium
containing 10% FBS; 5 day incubation (n ≥ 3) c: Efficacy recorded as percent of efficacy of fulvestrant control
Having identified substituted pyrrolidines 14f and 14i as compounds with increased ERꢀα
degradation efficacy over the unsubstituted pyrrolidine, we combined both pieces of SAR (14n)
and gained a further increase in ERꢀα degradation efficacy to 91% of the fulvestrant control. The
importance of stereochemistry of the methyl substitution is again apparent as the stereoisomers
14oꢀq are less effective degraders. Of further note is that for the pyrrolidine system, the linker
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methyl is ‘dominant’ in driving ER degradation efficacy, as 14o (87%) is more efficacious than
14p (76%).
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To maximize efficiency during our SAR studies, the compounds were prepared and tested in
biological assays as a 1:1 mixture of stereosiomers at the chromene core. For key compounds in
the program, we confirmed that ERꢀα binding and SERD activity lay in one isomer by separation
and characterization of the stereoisomers. For example, the lead compound 14n was separated by
chiral supercritical fluid chromatography into two stereoisomers, 14na and 14nb. The active
diastereoisomer 14na had an ERꢀα binding IC50 = 2.8 nM, ERꢀα degradation IC50 = 0.1 nM and
MCFꢀ7 antiꢀproliferation IC50 = 0.1 nM. In contrast, diastereoisomer 14nb was 50ꢀ100x less
active in these assays, with this remaining activity likely due to residual 14na in the sample (~
1% remaining following separation of 14n). Further, using a 3xERE transcription assay, we
confirmed that 14nb had no ERꢀα agonist properties, indicating that working with enantiomeric
or diastereoisomeric mixtures was acceptable during SAR studies (that the ER activity resided in
one chromene stereoisomer was confirmed for a number of pairs of compounds on the program).
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Table 2: SERD activity resides in one chromene stereoisomer
MCFꢀ7
ERꢀα Degradationa
ERꢀα Binding
Proliferationb
Cpd
14n
Structure
IC50 (nM)
IC50 (nM) Efficacyc
IC50 (nM)
5.1
0.2
91
0.2
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2.8
0.1
9.7
91
90
0.1
59
14na
14nb
136
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a: ERꢀα inꢀcell western in MCFꢀ7 breast cancer cells in phenolꢀred free RPMI medium containing 5% charcoal dextran treated FBS; readings
taken after 4 hours incubation using a SPꢀ1 antiꢀER rabbit monoclonal antibody (n ≥ 4) b: MCFꢀ7 proliferation assay in RPMI medium
containing 10% FBS; 5 day incubation (n ≥ 3) c: Efficacy recorded as percent of efficacy of fulvestrant control
An (S)-stereochemistry at the chromene core of 14na was unambiguously assigned by a singleꢀ
crystal Xꢀray diffraction pattern of the (R)ꢀmandelate salt (Figure 3).
Figure 3: Singleꢀcrystal Xꢀray structure of 14na.mandalate salt confirms (S)ꢀconfiguration at the
chromene core
HO
OH
OH
O
.
O-
N+
H
O
O
With the optimized chiral sideꢀchain of 14n, we examined the effect of the position of the two
phenols of 14n by preparing 3 regioisomers 15b, 15c and 15d (Table 3). The in vitro profile of
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these regioisomers were similar to that of 14n, with all compounds demonstrating high potency
and efficacy in the ERꢀα degradation assay (IC50 < 0.5 nM, efficacy >90%), and MCFꢀ7
proliferation assay (IC50 < 0.3 nM). 6ꢀOH Phenol 14n was determined to be the preferred phenol
configuration in terms of pharmacokinetics as it had 10ꢀfold higher exposure following oral
dosing in mice than its 7ꢀOH phenol isomer 15b. A similar trend was also observed when
comparing the exposure of 15c to 15d.
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Table 3:
MCFꢀ7
Mouse po
Plasma AUC15
(ꢀg·h/mL)
0.60
ER Degradationa
Proliferationb
Cpd
Phenol
R1
IC50 (nM) Efficacyc
IC50 (nM)
0.2
6ꢀOH
7ꢀOH
6ꢀOH
7ꢀOH
6ꢀOH
6ꢀOH
6ꢀOH
3’ꢀOH
3’ꢀOH
4’ꢀOH
4’ꢀOH
H
0.2
0.2
0.1
0.3
0.4
0.5
0.4
91
92
92
92
84
88
82
14n
15b
15c
15d
15e
15f
0.3
0.05
0.1
0.46
0.2
0.12
0.4
1.0
3’ꢀF
1.0
0.35
4’ꢀF
0.4
2.0
15g
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6ꢀOH
6ꢀOH
3’ꢀCN
4’ꢀCN
0.7
1.5
1.4
5.7
83
84
73
77
2.6
10
ND
2.0
15h
15i
6ꢀOH 3’ꢀSO2Me
6ꢀOH 4’ꢀSO2Me
4.3
27
ND
ND
15j
15k
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a: ERꢀα inꢀcell western in MCFꢀ7 breast cancer cells in phenolꢀred free RPMI medium containing 5% charcoal dextran treated FBS; readings
taken after 4 hours incubation using a SPꢀ1 antiꢀER rabbit monoclonal antibody (n ≥ 4) b: MCFꢀ7 proliferation assay in RPMI medium containing
10% FBS; 5 day incubation (n ≥ 3) c: Efficacy recorded as percent of efficacy of fulvestrant control
The major route of metabolism of 14n is glucuronidation of one or both of the phenols, and so in
an attempt to increase the oral exposure of the series, we examined replacement or removal of
these metabolic liabilities. Indeed, removal of the 3’ꢀOH of 15e, led to an almost doubling in
exposure over 14n (15e mouse po AUC (10 mpk) = 1.0 ꢀg·h/mL), and also retained ERꢀα
degradation and MCFꢀ7 proliferation potency. However, a pronounced reduction in ERꢀα
degradation efficacy of 15e compared to 14n was observed (84% versus 91% respectively).
Examination of a fluoro, cyano or methylsulfone group at the 3’ꢀ or 4’ꢀpositions of this ring (15fꢀ
k), did not lead to compounds with equivalent ERꢀα degradation to 14n, but did indicate that
lipophilic groups were preferred for potency in both the degradation and MCFꢀ7 proliferation
assays (compare: 15f vs 15h vs 15j and 15g vs 15i vs 15k). Of note, monoꢀphenols 15g and 15i
displayed a further 3ꢀfold improvement in oral exposure over 14n. However 15i was
significantly less potent in the degradation and MCFꢀ7 proliferation assays when compared to
both 14n and 15g.
To rapidly assess ERꢀbased pharmacodynamics in an ER responsive tissue, we utilized a 4ꢀday
immature rat uterine wet weight assay. The assay was run in two modalities, either in antagonist
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mode where the test compound competes against ethynyl estradiol, or in agonist mode where no
ethynyl estradiol was administered (revealing any inherent estrogenic activity of the ligand).
Table 4 shows the reduction in ethynyl estradiol stimulated uterine weight (antagonist mode) for
select compounds at either the SAR screening dose of 1 mg/kg or at a higher 10 mg/kg dose (the
table also includes exposure from separate mouse PK studies). The 10 mg/kg dose was used to
assess the maximum efficacy of the compound in this assay.
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Table 4: Immature rat uterine wet weight assay (antagonist mode) and mouse pharmacokinetics
Rat Uterine Wet
Mouse PKc
Weight Assaya
ER
ER
Reduction in
uterine weightb
(dose)
po AUC
Degradation Degradation
Cpd
(ꢀg·h/mL)
efficacy (%)
IC50 (nM)
78%
(60 mg/kg)
110%
0.1d
0.1
51d
ND
ND
Tamoxifen (1)
Fulvestrant (3)
100
(200 mg/kg)
84%
0.3
0.2
58
91
ND
14j
(10 mg/kg)
111%e
0.59
14n
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(10 mg/kg)
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113%
(1 mg/kg)
111%
0.1
0.4
92
82
0.46
2.0
15c
15g
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(1 mg/kg)
a: Compounds administered orally for 3 days (1 mg/kg po) followed by an oral dose of 0.1 mg/kg ethynyl estradiol. On the
fourth day, 24 hours after dose, the animals were euthanized and the uterus was removed and weighed. Plasma was taken at the
time point indicated. b: 100 x [(VehicleEE – Cpd)/(VehicleEE – Vehicle)], where ‘VehicleEE’ is the uterine wet weight of the
vehicle animals dosed with ethynyl estradiol (0.1 mg/kg); ‘Cpd’ is the uterine wet weight of the of the compound treated animals
dosed with ethynyl estradiol (0.1 mg/kg); ‘Vehicle’ is the uterine wet weight of the of the vehicle animals. c: 10 mg/kg po. d:
Values for 4ꢀhydroxytamoxifen. e: At 1 mg/kg, reduction in uterine weight was 99%.
In this assay, 14n, 15c and 15g were confirmed as compounds of interest as they had a robust
reduction of >110% in uterine weight compared to the ethynyl estradiol treated vehicle animals,
indicating they were inverse agonists in this ERꢀresponsive tissue.16 In contrast, tamoxifen (1)
and 14j did not reduce the uterine weight to that of ethynyl estradiol treated vehicle animals in
this assay (78% and 84% reduction), and when run in agonist mode in the immature rat uterine
wet weight assay, were shown to be partial agonists (figure 4). This partial agonist activity
observed in the uterus (SERM activity) is well known for tamoxifen and is manifested both in
preclinical models and in patients.1 Of note, 14j is a partial agonist, while the close structural
analogue 14n is an inverse agonist, reducing uterine wet weight below that of vehicle control.
This divergent activity was also seen in the efficacy difference in the ERꢀα degradation assay
(58% efficacy versus 91% respectively). High efficacy ERꢀα degraders were found to be robust
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inverse agonists in the UWW assay throughout the program, although there is clearly a threshold
as 15g, which had subꢀoptimal ERꢀα degradation efficacy (82%), was a full antagonist in the
uterine assay. Due to this and its improved exposure in mice (AUC = 2 ꢀg.h/mL following a 10
mg/kg po dose), 15g was selected for further profiling along with 14n.
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Figure 4: Tamoxifen (1) and 14j are partial agonists in the immature rat uterine wet weight
assaya
a: Compounds administered orally for 3 days (14n and 14j at 10 mg/kg po; tamoxifen at 60 mg/kg). On the fourth day, 24 hours
after dose, the animals were euthanized and the uterus was removed and weighed. Uterine wet weight is normalized to total body
weight.
The full rat and mouse pharmacokinetic profiles for 14n and 15g are shown in Table 6. As would
be expected monoꢀphenol 15g has a lower clearance (CL = 21ꢀ30 mL/min/kg versus 33ꢀ38
mL/min/kg) and longer halfꢀlife (t1/2 = 5ꢀ7 h versus 2ꢀ3 h) compared to biꢀphenol 14n, as well as
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higher bioavailability (%F = 35ꢀ54 versus 13ꢀ18) and oral exposure (AUC = 2.0 – 4.5 versus
5
6
0.59 – 0.93 ꢀg.h/mL).
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Table 6: Rodent PK of 14n and 15g
Compounda
CL
Vss
t1/2
(h)
Cmax
AUC0ꢀinf
Oral
%F
(Species)
(mL/min/kg)
(L/kg)
(Oral)
(µg/mL)
0.1
(Oral)
(µg·h/mL)
0.59
38
33
30
21
6.2
8.0
11
3.6
2.9
5.2
7.4
13
18
35
54
14n
(Mouse)
14n
0.14
0.17
0.28
0.93
2.0
(Rat)
15g
(Mouse)
15g
12
4.5
(Rat)
a: Dosed intravenously at 3 mg/kg and per orally at 10 mg/kg in PEG400 / PVP / TW80 / 0.5% CMC in water, 9:0.5:0.5:90
14n and 15g were profiled in MCFꢀ7 based xenograft models of breast cancer using subꢀ
cutaneous estradiol pellets to deliver sufficient levels of estradiol to drive tumor growth. This
supplemental estradiol (E2) is used to drive xenograft tumor growth in vivo, and leads to high E2
concentrations (300ꢀ400 pg/mL), thus requiring more ER ligand to outꢀcompete the native ligand
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(E2) and so mediate an inhibitory effect. This is of relevance as plasma E2 levels in the target
patient population of postꢀmenopausal women (~5 pg/mL) are substantially lower than those of
the various preꢀclinical efficacy models.17 Thus efficacy models run in the context of high E2
plasma concentrations potentially overestimate ER ligand dose and plasma levels necessary to
drive efficacy in a postꢀmenopausal setting. When examined in a MCFꢀ7 tamoxifenꢀsensitive
xenograft model, 14n and 15g (both dosed at 6mg/kg per orally QD), as well as tamoxifen (1)
(60 mg/kg per orally QD) showed robust activity even in this high estradiol setting (Figure 5).
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Figure 5: 14n and 15g show robust activity in a tamoxifenꢀsensitive MCFꢀ7 xenograft model
As the target patient population for SERDs is women who have progressed on endocrine therapy,
including tamoxifen, we developed models of endocrine resistance by continuous dosing of
tamoxifen to mice bearing MCFꢀ7 xenograft tumors until reꢀgrowth occurred. These individual
tumors that were resistant to tamoxifen were then propagated under chronic tamoxifen dosing
and used as the source material for tamoxifenꢀresistant xenograft studies. The activity of 14n and
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15g in one such model is shown in Figure 6, and an interesting divergence of activities was
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observed.
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Figure 6: Optimized ERꢀα degrader 14n demonstrated tumor stasis in an MCFꢀ7 tamoxifenꢀ
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resistant xenograft model
Encouragingly, 14n was active in this tamoxifenꢀresistant model giving tumor stasis at 60
mg/Kg. In contrast to the tamoxifenꢀsensitive xenograft (figure 5), 15g had minimal activity,
effectively growing like vehicle control. The inferior profile of 15g compared to 14n is not due
to potency against the estrogen receptor (ERꢀα degradation IC50: 15g = 0.4 nM, versus 14n = 0.2
nM; MCFꢀ7 proliferation IC50: 15g = 0.4 nM versus 14n = 0.2 nM) or pharmacokinetics (day 28
xenograft AUC: 14n = 4.6 ꢀg·h/mL; 15g = 36 ꢀg·h/mL). Of note however, is 15g is a less
efficacious ERꢀα degrader than 14n in vitro (ERꢀα degradation efficacy = 82% versus 91%).
This is consistent with our previous conclusion that compounds with lower ERꢀα degradation
efficacy did not perform as well in a tamoxifenꢀresistant xenograft setting.6
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In summary, we have optimized ERꢀα degradation efficacy on a chromene series of estrogen
receptor modulators resulting in highly potent and efficacious SERDs. Sideꢀchain ring size and
stereochemical elements were central in driving degradation efficacy, leading to the
identification of 14n. SAR studies revealed that potency could be maintained and
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pharmacokinetic parameters improved when one of the two phenol moieties was removed, but
this resulted in a significant erosion in degradation efficacy. In a xenograft model of tamoxifenꢀ
resistant breast cancer, 14n (ERꢀα degradation efficacy = 91%) demonstrated robust activity,
while despite superior oral exposure, monoꢀphenol 15g (ERꢀα degradation efficacy = 82%) was
essentially inactive. This result suggests that optimizing ERꢀα degradation efficacy in the MCFꢀ7
cell line leads to compounds with robust effects in models of tamoxifenꢀresistant breast cancer
derived from an MCFꢀ7 backꢀground.
EXPERIMENTAL SECTION
Experimental Procedures (Chemistry)
All air and moisture sensitive reactions were carried out under an inert atmosphere of nitrogen.
All reactive liquid reagents were transferred by syringe or cannula and were added into the flask
through a rubber septum. All other solvents and reagents were obtained from commercial
sources and used as received unless otherwise stated. Both 1H and 13C spectra were obtained on a
Bruker 400 spectrometer at 400 MHz and 100 MHz, respectively. Chemical shifts are reported
in parts per million (δ) from an internal standard of residual DMSO (2.50 ppm or 39.5 ppm),
methanol (3.31 ppm or 49.0 ppm), or chloroform (7.26 ppm or 77.2 ppm). Proton chemical shift
data are reported as follows: chemical shift, multiplicity (s=singlet, d=doublet, t = triplet, q =
quartet, m = multiplet, b = broad), coupling constant (J) reported in hertz, integration. Analytical
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thinꢀlayer chromatography (TLC) was performed on commercial silica plates (Merck 60ꢀF 254,
0.25 mm thickness); compounds were visualized by UV light (254 nm). All yields reported are
not optimized. Normal phase purifications were performed on silica gel columns using a Biotage
(SP1 or SP4), an ISCO Combiflash Companion XL or ISCO Combiflash Torrent using preꢀ
packed silica gel columns. Reversed phase semipreparative HPLC purifications were carried out
using a Shimadzu Discovery VP system with a SPDꢀ20A prominence UV/vis detector (190ꢀ700
nm range). The columns used were a Waters SunFire C18 (19 mm x 150 mm) or a YMCꢀPack
Pro ODSꢀA (20 mm x 150 mm) or a Phenomenex, Luna 5 ꢀ C18 semipreparative (250 x 10 mm)
column with an acetonitrileꢀwater solvent mixture in the presence of 0.1% TFA. The purity of
target compounds was determined by 1H NMR and by analytical HPLC and shown to be > 95%
pure prior to biological testing. Lowꢀresolution mass spectra (LRMS) were recorded on a
WatersMicromass ZQ using electrospray positive ionization. Highꢀresolution mass spectra
(HRMS) were obtained on an Agilent ESIꢀTOF mass spectrometer using electrospray positive or
negative ionization.
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For full experimental details of all compounds, see Supplementary Information
(R)ꢀ2ꢀMethylbutaneꢀ1,4ꢀdiyl dimethanesulfonate. Triethylamine (100 mL, 0.72 mol) was
added to a solution of (R)ꢀ2ꢀmethylbutaneꢀ1,4ꢀdiol (30 g, 0.29 mol) in DCM (600 mL) at room
temperature. The solution was cooled to ꢀ20 °C, and methanesulfonyl chloride (49 mL, 0.63 mol)
was added dropwise over 30 min with vigorous stirring. The resulting mixture was stirred for
additional 1 h while the temperature was maintained between ꢀ20 and ꢀ15 °C. The mixture was
allowed to warm to 0 °C and then poured into cold 1N HCl solution (100 mL). The organic layer
was separated, and the aqueous phase was extracted with DCM (100 mL). The combined organic
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extracts were washed (saturated NaHCO3 and then brine), dried (MgSO4), and concentrated in
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1
vacuo. The resulting product (75.9 g, quant) was used directly for the next step. H NMR (400
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9
MHz, DMSOꢀd6): δ 4.32ꢀ4.22 (m, 2H), 4.13ꢀ4.03 (m, 2H), 3.18 (s, 2 x 3H), 2.02ꢀ1.92 (m, 1H),
1.87ꢀ1.77 (m, 1H), 1.61ꢀ1.50 (m, 1H), 1.07 (d, J = 6.8 Hz, 3H).
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(S)ꢀ2ꢀ((R)ꢀ3ꢀMethylpyrrolidinꢀ1ꢀyl)propanꢀ1ꢀol.
(R)ꢀ2ꢀMethylbutaneꢀ1,4ꢀdiyl
dimethanesulfonate (37.5 g, 0.144 mol,) was added to neat (S)ꢀ2ꢀaminopropanꢀ1ꢀol (54.8 g,
0.730 mol) at room temperature. The mixture was stirred in a room temperature water bath to
minimize the exotherm. After 24 h, the reaction was diluted with DCM (150 mL), saturated
K2CO3 solution (150 mL), and just enough water (60 mL) to dissolve the resulting precipitate.
The layers were separated, and the aqueous layer was extracted with DCM (150 mL). The
organic layers were combined, dried (Na2SO4), concentrated, and purified by silica gel
chromatography (10: 7; ethyl acetate: hexanes → 10: 7: 2: 1; ethyl acetate: hexanes: methanol:
triethylamine) to give the title compound (17.9 g, 87%) as a pale yellow oil. 1H NMR (400 MHz,
DMSOꢀd6): δ 4.33 (t, J = 5.4 Hz, 1H), 3.51ꢀ3.43 (m, 1H), 3.21ꢀ3.13 (m, 1H), 2.79 (dd, J = 8.6,
7.6 Hz, 1H), 2.62ꢀ2.54 (m, 1H), 2.51ꢀ2.43 (m, 1H), 2.30ꢀ2.20 (m, 1H), 2.15ꢀ2.03 (m, 1H), 2.01
(dd, J = 8.6, 6.8 Hz, 1H), 1.91ꢀ1.81 (m, 1H), 1.25ꢀ1.15 (m, 1H), 0.98 (d, J = 6.4 Hz, 3H), 0.96 (d,
J = 6.5 Hz, 3H); 13C NMR (100 MHz, DMSOꢀd6): δ 64.5, 60.1, 58.8, 50.4, 32.0, 31.1, 20.3, 12.2;
LCMS: 144.3 [M+H]+.
2ꢀ(4ꢀIodophenyl)ꢀ4ꢀmethylꢀ6ꢀ((tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)ꢀ3ꢀ(3ꢀ((tetrahydroꢀ2Hꢀpyranꢀ
2ꢀyl)oxy)phenyl)ꢀ2Hꢀchromene (13a)
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Journal of Medicinal Chemistry
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Step 1: N,2,5ꢀTrimethoxyꢀNꢀmethylbenzamide. Oxalyl chloride (3.6 mL, 41.3 mmol) was
added to a solution of 2,5ꢀdimethoxybenzoic acid (10) (6.00 g, 33.0 mmol) in DCM (100 mL) at
room temperature. Then, DMF (0.2 mL) was added to the solution. The resulting solution was
stirred at room temperature for 2 h, and the solvent was removed on a rotary evaporator. The
crude material was placed under vacuum for 30 minutes (to remove the residual oxalyl chloride),
dissolved in DCM (100 mL), and cooled to 0 °C. To this solution, N,Oꢀdimethylhydroxylamine
hydrochloride (4.03 g, 41.32 mmol) and triethylamine (6.8 mL, 48.78 mmol) were added. The
resulting mixture was stirred at 0 °C for 30 min and then at room temperature for additional 30
min. The reaction was diluted with DCM (50 mL), washed (2×100 mL water and then 100 mL
brine), dried (Na2SO4), and concentrated under reduced pressure. The crude material was
purified by silica gel chromatography to yield the title compound (7.32 g, 99%) as clear oil
which solidified over time. 1H NMR (400 MHz, CDCl3): δ 6.95ꢀ6.84 (m, 3H), 3.82 (s, 3H), 3.79
(s, 3H), 3.58 (br, 3H), 3.32 (br, 3H).
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Step 2: 1ꢀ(2,5ꢀDimethoxyphenyl)ꢀ2ꢀ(3ꢀmethoxyphenyl)ethanone. A 5 mL portion of 3ꢀ
methoxybenzyl chloride (12.8 mL, 88.1 mmol) in THF (60 mL) was added to a mixture of
magnesium (2.88 g, 118 mmol) and iodine (1 crystal) in THF (30 mL) at room temperature. The
reaction mixture was stirred until the color disappeared and the remaining solution of 3ꢀ
methoxybenzyl chloride was added dropwise over 45 min. The mixture was heated at 60 °C for 1
h and then cooled to 0 °C. A solution of N,2,5ꢀtrimethoxyꢀNꢀmethylbenzamide (6.65 g, 29.6
mmol) in THF (70 mL) was added to this mixture over 30 min at 0 °C. The reaction was stirred
for 30 min at 0 °C, quenched with brine (50 mL), and then extracted with ethyl acetate (3×100
mL). The combined organic extracts were washed (50 mL brine), dried (Na2SO4), and
concentrated under reduced pressure to give the title compound (7.99 g, 95%) as a white solid.
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Page 29 of 45
Journal of Medicinal Chemistry
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1H NMR (400 MHz, CDCl3): δ 7.27ꢀ7.20 (m, 2H), 7.01 (dd, J = 9.0, 3.2 Hz, 1H), 6.92 (d, J = 9.0
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Hz, 1H), 6.86ꢀ6.76 (m, 3H), 4.30 (s, 2H), 3.90 (s, 3H), 3.82 (s, 3H), 3.79 (s, 3H).
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Step 3: 1ꢀ(2,5ꢀDihydroxyphenyl)ꢀ2ꢀ(3ꢀhydroxyphenyl)ethanone. Boron tribromide (1M in
DCM, 48.0 mL, 48.0 mmol) was added dropwise to a solution of 1ꢀ(2,5ꢀdimethoxyphenyl)ꢀ2ꢀ(3ꢀ
methoxyphenyl)ethanone (3.35 g, 11.7 mmol) in DCM (50 mL) at ꢀ78 °C. The reaction mixture
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was warmed to 0 °C, stirred for 30 min, reꢀcooled to ꢀ78 °C, and then quenched with methanol
(15 mL). The reaction mixture was warmed to room temperature, concentrated under reduced
pressure, and purified by silica gel chromatography to give the title compound (1.78 g, 62%) as a
yellow solid. 1H NMR (400 MHz, DMSOꢀd6): δ 11.24 (s, 1H), 9.34 (s, 1H), 9.20 (s, 1H), 7.26 (d,
J = 3.0 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 6.98 (dd, J = 8.9, 3.0 Hz, 1H), 6.83 (d, J = 8.9 Hz, 1H),
6.70ꢀ6.58 (m, 3H), 4.24 (s, 2H); LCMS: 243.0 [MꢀH]ꢀ.
Step 4: 1ꢀ(2ꢀHydroxyꢀ5ꢀ((tetrahydroꢀ2Hꢀpyranꢀ2ꢀyl)oxy)phenyl)ꢀ2ꢀ(3ꢀ((tetrahydroꢀ2Hꢀ
pyranꢀ2ꢀyl)oxy)phenyl)ethanone. A solution of 3,4ꢀdihydroꢀ2Hꢀpyran (2.65 g, 30.8 mmol) in
DCM (6 mL) was added to a mixture of 11 (1.50 g, 6.15 mmol) and pyridinium pꢀ
toluenesulfonate (320 mg, 1.27 mmol) in DCM (40 mL) at room temperature. The reaction
mixture was stirred for 1 h and then diluted with DCM (100 mL). The solution was washed
(2×50 mL saturated NaHCO3 and then 50 mL brine), dried (Na2SO4), and concentrated under
reduced pressure. The crude material was purified by silica gel chromatography to give the title
compound (2.42 g, 96%) as yellow oil which solidified over time. 1H NMR (400 MHz, CDCl3):
δ 11.88 (s, 1H), 7.63ꢀ7.56 (m, 1H), 7.31ꢀ7.20 (m, 2H), 7.03ꢀ6.96 (m, 2H), 6.94ꢀ6.92 (m, 2H),
5.46ꢀ5.40 (m, 1H), 5.31ꢀ5.25 (m, 1H), 4.25 (s, 2H), 4.00ꢀ3.86 (m, 2H), 3.70ꢀ3.52 (m, 2H), 2.07ꢀ
1.55 (m, 12H); LCMS: 413.2 [M+H]+.
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