Synthesis and biological evaluation of novel 4-hydroxytamoxifen analogs as estrogen-related receptor gamma inverse agonists

Article abstract of DOI:10.1016/j.ejmech.2016.04.076

Estrogen-related receptor gamma (ERRγ) has recently been recognized as an attractive target for treating inflammation, cancer, and metabolic disorders. Herein, we discovered and demonstrated the in vitro pharmacology as well as the absorption, distribution, metabolism, excretion, and toxicity (ADMET) properties of chemical entities that could act as highly selective inverse agonists for ERRγ. The results were comparable to those for GSK5182 (4), a leading ERRγ inverse agonist ligand. Briefly, the half-maximal inhibitory concentration (IC₅₀) range of the synthesized compounds for ERRγ was 0.1-10 μM. Impressively, compound 24e exhibited potency comparable to 4 but was more selective for ERRγ over three other subtypes: ERRα, ERRβ, and estrogen receptor α. Furthermore, compound 24e exhibited a superior in vitro ADMET profile compared to the other compounds. Thus, the newly synthesized class of ERRγ inverse agonists could be lead candidates for developing clinical therapies for ERRγ-related disorders.

Full text of DOI:10.1016/j.ejmech.2016.04.076

Accepted Manuscript
Synthesis and biological evaluation of novel 4-hydroxytamoxifen analogs as estrogen-
related receptor gamma inverse agonists
Jina Kim, Jungwook Chin, Chun Young Im, Eun Kyung Yoo, Seoyeon Woo, Hee Jong
Hwang, Joong-heui Cho, Kyung-ah Seo, Jaeyoung Song, Hayoung Hwang, Kyung-
Hee Kim, Nam Doo Kim, Suk Kyoon Yoon, Jae-Han Jeon, Seung-Yun Yoon, Yong
Hyun Jeon, Hueng-Sik Choi, In-Kyu Lee, Seong Heon Kim, Sung Jin Cho
PII:
S0223-5234(16)30393-2
10.1016/j.ejmech.2016.04.076
EJMECH 8605
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 February 2016
Revised Date: 27 April 2016
Accepted Date: 28 April 2016
Please cite this article as: J. Kim, J. Chin, C.Y. Im, E.K. Yoo, S. Woo, H.J. Hwang, J.-h. Cho, K.-a. Seo,
J. Song, H. Hwang, K.-H. Kim, N.D. Kim, S.K. Yoon, J.-H. Jeon, S.-Y. Yoon, Y.H. Jeon, H.-S. Choi, I.-
K. Lee, S.H. Kim, S.J. Cho, Synthesis and biological evaluation of novel 4-hydroxytamoxifen analogs as
estrogen-related receptor gamma inverse agonists, European Journal of Medicinal Chemistry (2016),
doi: 10.1016/j.ejmech.2016.04.076.
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ACCEPTED MANUSCRIPT
Graphical Abstract
ꢀ

ACCEPTED MANUSCRIPT
Synthesis and Biological Evaluation of Novel 4-Hydroxytamoxifen Analogs as Estrogen-
related Receptor Gamma Inverse Agonists
Jina Kima,†, Jungwook China,†, Chun Young Ima, Eun Kyung Yoob, Seoyeon Wooa, Hee Jong
Hwanga, Joong-heui Choa, Kyung-ah Seoa, Jaeyoung Songa, Hayoung Hwanga, Kyung-Hee
Kima, Nam Doo Kima, Suk Kyoon Yoona, Jae-Han Jeonb,c, Seung-Yun Yoond, Yong Hyun
Jeond, Hueng-Sik Choie, In-Kyu Leeb,c, Seong Heon Kima,§, *and Sung Jin Choa,b,*
a New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea
b
Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease,
Kyungpook National University Hospital, Daegu 41404, Korea
c Department of Internal Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea
d Department of Nuclear Medicine, School of Medicine, Kyungpook National University, Daegu 41944, Korea
e
National Creative Research Initiatives Center for Nuclear Receptor Signals and Hormone Research Center,
School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Republic of
Korea
†
These two authors contributed equally to this work.
§
Current address: Boryung R&D Center, Chemistry Research Unit, Gyeonggi-do 15425, Korea
* Corresponding Authors:
Director/Ph.D. Seong Heon Kim
Boryung R&D Center, Chemistry Research Unit, Gyeonggi-do 15425, Korea
rose1998@boryung.co.kr; Tel.: +82-31-490-2271 (ex 520)
Prof. Sung Jin Cho
New Drug Development Center, Daegu-Gyeongbuk Medical Innovation Foundation, Daegu 41061, Korea;
Leading-edge Research Center for Drug Discovery and Development for Diabetes and Metabolic Disease,
Kyungpook National University Hospital, Daegu 41404, Korea;
sjcho@dgmif.re.kr; Tel.: +82-53-790-5226
Abbreviations:
4-OHT, 4-hydroxytamoxifen; ADMET, absorption, distribution, metabolism, excretion, and
toxicity; CYP, cytochrome P450; DCM, dichloromethane; DES, diethylstilbestrol; ER,
estrogen receptor; ERR, estrogen-related receptor; NIS, sodium iodide symporter; ATC,
anaplastic thyroid cancer; IC50, half-maximal inhibitory concentration; MeOH, methanol;
PAMPA, parallel artificial membrane permeability assay; SAR, structure-activity relationship
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Abstract
Estrogen-related receptor gamma (ERRγ) has recently been recognized as an attractive target
for treating inflammation, cancer, and metabolic disorders. Herein, we discovered and
demonstrated the in vitro pharmacology as well as the absorption, distribution, metabolism,
excretion, and toxicity (ADMET) properties of chemical entities that could act as highly
selective inverse agonists for ERRγ. The results were comparable to those for GSK5182 (4), a
leading ERRγ inverse agonist ligand. Briefly, the half-maximal inhibitory concentration
(IC50) range of the synthesized compounds for ERRγ was 0.1–10 ꢀM. Impressively,
compound 24e exhibited potency comparable to 4 but was more selective for ERRγ over
three other subtypes: ERRα, ERRβ, and estrogen receptor α. Furthermore, compound 24e
exhibited a superior in vitro ADMET profile compared to the other compounds. Thus, the
newly synthesized class of ERRγ inverse agonists could be lead candidates for developing
clinical therapies for ERRγ-related disorders.
Keywords: ERRγ inverse agonist, Nuclear receptor, ADMET, GSK5182
1. Introduction
The orphan estrogen-related receptors (ERRs), comprising ERRα, ERRβ, and ERRγ, are
constitutively active nuclear receptors that have a high degree of sequence homology with the
estrogen receptors (ERs) ERα [1,2] and ERβ [3]. ERRs are primarily expressed in the heart,
brain, kidney, pancreas, placenta, and liver [4] and are involved in the regulation of genes
relevant to cellular metabolism, energy homeostasis, and cancer [5]. Therefore, they are
regarded as potential therapeutic targets for the treatment of associated diseases [6]. In
particular, the ERRγ subtype is involved in metabolic diseases such as type 2 diabetes
mellitus, alcohol-induced oxidative stress, liver injury, microbial infections caused by
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impaired hepatic gluconeogenesis [7,8], damaged hepatic insulin signaling [9], and impaired
iron metabolism [10]. Furthermore, ERRγ is thought to play an important role in the
metabolic transcriptional pathway depending on the cellular environment of cancer cells [11]
and in regulating metabolic reprogramming in primarily hormone-dependent, breast, and
prostate cancers by modulating a Warburg-like effect [12,13].
Based on recent findings, ERRγ has emerged as a promising target for treating certain
metabolic disorders [14] and cancers [15]. Despite its biological versatility, few studies have
investigated ERRγ modulators over the past two decades. Consequently, there are currently
few synthetic compounds targeting ERRγ with useful pharmacological activity (Figure 1).
The initial synthetic compounds, 4-hydroxytamoxifen (4-OHT, 1) [16] and diethylstilbestrol
(DES, 2) [17], were identified as nonselective ERRγ inverse agonists. In addition, the acyl
hydrazine compound, GSK4716 (3), was reported as a more selective ERRγ agonist with
mixed ERRγ/ERRβ functional activities [18]. This mandated development of a more selective
inverse agonist for ERRγ; therefore, GSK5182 (4), which showed better selectivity for ERRγ
than the structurally related nuclear ERα, was developed as a 4-OHT (1) analog [19]. In our
previous study, we structurally optimized 4 to synthesize moderately active analogs that were
selective ERRγ inverse agonists with improved absorption, distribution, metabolism,
excretion, and toxicity (ADMET) profiles [20].
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Figure 1. Synthetic estrogen-related receptor gamma (ERRγ) ligands. 4-Hydroxytamoxifen (4-OHT, 1), an
estrogen receptor α (ERα) antagonist/ERRγ inverse agonist; diethylstilbestrol (DES, 2), an ERα agonist/ERRγ
inverse agonist; GSK4716 (3), a selective ERRγ agonist; and GSK5182 (4), a selective ERRγ inverse agonist.
The ERRγ inverse agonist 4 mitigates diabetes by inhibiting hepatic gluconeogenesis
through peroxisome proliferator-activated receptor-gamma coactivator 1α [8] and exhibits
antimicrobial effects by reducing the expression of ERRγ-mediated hepcidin mRNA [10].
Recently, it was reported that 4 facilitates the responsiveness to radioiodine therapy by
modulating sodium iodide symporter (NIS) function in anaplastic thyroid cancer (ATC) cells
through the ERRγ and MAP kinase signaling pathway [21]. Nonetheless, the discovery of
new small ERRγ ligands with superior pharmacological and ADMET profiles is needed to
facilitate the development of novel therapeutic agents.
Therefore, the aim of this study was to identify new ERRγ inverse agonists that possess a
tetrasubstituted scaffold similar to that of 4 with increased in vitro potency and selectivity,
including an in vitro ADMET profile better than that of currently available agents.
Considering the requirement for more potent and selective ERRγ inverse agonists with
validated therapeutic potential for various diseases associated with ERRγ, we conducted a
new series of lead optimization processes on this promising scaffold.
Based on our continuing efforts in this field, we focused the lead optimization efforts on
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modifying the B-ring phenyl group with various substituted aryl or heterocyclic substituents
and made minor alterations to the A-ring as well to adequately improve the in vitro ADMET
profile for subsequent in vivo assessments. Here, we report the chemical synthesis and
structure-activity relationship (SAR) results obtained for the new compounds as well as the
molecular modeling study, and in vitro ADMET profile.
2. Results and Discussion
2.1. Chemistry
As shown in Scheme 1, starting from acyl chloride containing various substituents,
Friedel-Crafts acylation was performed with methoxybenzene to obtain the corresponding
ketone derivatives 6a–d. Lewis acid (either with aluminum chloride (AlCl3) or boron
tribromide (BBr3))-mediated deprotection of methyl ether generated the corresponding
phenol, which was subsequently coupled with methyl 3-benzoylpropionate under typical
cross McMurry conditions to yield mostly (ca. 80%) E-olefins 8a–d. Thereafter, compounds
9a–d were prepared using either the Mitsunobu reaction or Williamson ether synthesis. The
final reduction of the ester with lithium aluminum hydride (LiAlH4) yielded the chemical
entities 10a–d; 10c further underwent reductive debromination under the same conditions,
ultimately leading to the formation of 10e. The HCl salts of 10a–e were formed by treatment
with 1 M aqueous HCl mixed with dichloromethane (CH2Cl2, DCM)/methanol (MeOH)
solution.
The commercially available 11 (Scheme 2) was protected with pivaloyl chloride, and then
methanolysis was carefully performed to produce 13, which was subsequently coupled with
methyl 3-benzoylpropionate under cross McMurry conditions to yield E-olefin 14 (ca. 80%).
The various amino alcohols were attached by Mitsunobu reactions to obtain compounds 15a–
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c, which were then deprotected and reduced with LiAlH4 to their corresponding pivaloyl and
ester functionalities, yielding the chemical entities 4 and 16b–c, respectively [19]. Then,
compounds 4 and 16b–c were treated with 1 M aqueous HCl mixed with DCM/MeOH
solution to obtain their HCl salts.
Scheme 1. Synthesis of compounds 10a–e. (a) Aluminum chloride (AlCl3), dichloromethane (CH2Cl2, DCM),
room temperature (rt), 99%; (b) AlCl3, 1,2-dichloroethane, 70 °C, 99%; (c) AlCl3, toluene, 100 °C, 85–91%; (d)
Boron tribromide (BBr3), DCM, 30 °C, 77%; (e) Methyl 3-benzoylpropionate, titanium tetrachloride (TiCl4), Zn,
60 °C, 23–73%; (f) 2-Chloro-N,N-dimethylethanamine, potassium carbonate (K2CO3), acetone, 48–72%; (g) 2-
(dimethylamino)ethanol, diisopropyl azodicarboxylate (DIAD), triphenylphosphine (PPh3), DCM, 99%; (h)
Lithium aluminum hydride (LiAlH4), tetrahydrofuran (THF), 0 °C, 5–71%; (i) 1 M Hydrochloric acid (HCl) in
water (H2O), methanol (MeOH):DCM (1:1), 0 °C.
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Scheme 2. Synthesis of compounds 4 and 16b–c. (a) Pivaloyl chloride, triethylamine (TEA), dichloromethane
(CH2Cl2, DCM)/tetrahydrofuran (THF), room temperature (rt), 91%; (b) Potassium carbonate (K2CO3),
methanol (MeOH)/DCM, rt, 58%; (c) Methyl 3-benzoylpropionate, titanium tetrachloride (TiCl4), Zn, THF,
60 °C, 70%; (d) R-OH, diisopropyl azodicarboxylate (DIAD), triphenylphosphine (PPh3), DCM, 92–99%; (e)
Lithium aluminum hydride (LiAlH4), THF, 0 °C, 22–42%; (f) 1 M Hydrochloric acid (HCl) in water (H2O),
MeOH:DCM (1:1), 0 °C.
Compound 17 was produced by a Mitsunobu reaction between 14 and a Boc-protected
amino alcohol (Scheme 3). LiAlH4 was used to both deprotect the pivaloyl group and reduce
its ester functionality to give the primary alcohol 18, which was treated with 4 M HCl
solution in dioxane to deprotect the Boc-group and form the corresponding HCl salt,
compound 19.
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Scheme 3. Synthesis of compound 19. (a) tert-Butyl(2-hydroxyethyl)(methyl)carbamate, diisopropyl
azodicarboxylate (DIAD), triphenylphosphine (PPh3), dichloromethane (CH2Cl2), 40%; (b) Lithium aluminum
hydride (LiAlH4), tetrahydrofuran (THF), 0 °C, 88%; (c) 4 M Hydrochloric acid (HCl) in dioxane, 0 °C, 2%.
Scheme 4. Synthesis of compounds 24a–o. (a) Bis(benzonitrile)palladium chloride (PdCl2[PhCN]2), potassium
carbonate (K2CO3), dimethylformamide (DMF)/water (H2O), 45 °C, 3–59%; (b) Lithium aluminum hydride
(LiAlH4), 0 °C, 6–79%; (c) Lithium borohydride (LiBH4), tetrahydrofuran (THF), 0 °C, 11%; (d)
Diisobutylaluminium hydride (DIBAL-H), THF, 0 °C, 46–79%; (e) 1 M Hydrochloric acid (HCl) in H2O,
methanol (MeOH):H2O (1:1), 0 °C.
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Scheme 5. Synthesis of compound 25. (a) Dimethylaminosulfur trifluoride, dichloromethane (CH2Cl2, DCM),
room temperature (rt), 8%; (b) 1 M Hydrochloric acid (HCl) in water (H2O), methanol (MeOH):DCM (1:1),
0 °C.
Scheme 6. Synthesis of compounds 27–29. (a) Dess-Martin periodinane, dichloromethane (CH2Cl2, DCM),
room temperature (rt), 89%; (b) Dimethylaminosulfur trifluoride, DCM, rt, 6%; (c)
Tris(dibenzylideneacetone)dipalladium(0) (Pd2(dba)3, 2-di-tert-butylphosphino-2',4',6'-triisopropylbiphenyl (t-
BuXPhos), potassium hydroxide (KOH), 1,4-dioxane:water (H2O, 1:1), 80 °C, 6%; (d) 1 M Hydrochloric acid
(HCl) in H2O, methanol (MeOH):DCM (1:1), 0 °C.
Palladium-catalyzed three-component reactions of iodobenzene (A-ring), internal alkynes
(B-ring) with various substituents on the phenyl ring (refer to Table 2), and arylborates (22)
in DMF/water with a base, such as potassium carbonate, yielded tetrasubstituted olefins [22].
The ester compounds 23a–o were reduced using various reagents and conditions with lithium
borohydride, LiAlH4, or diisobutylaluminium hydride (DIBAL-H) to primary alcohols, which
were subsequently treated with 1 M HCl solution in a mixture of MeOH and water to form
the HCl salts 24a–o (Scheme 4). Compound 25 was prepared by treating 4 with DAST and 1
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M HCl solution in MeOH/DCM (Scheme 5). Compound 10c was converted to 29 by
treatment with DAST and 1 M HCl solution in MeOH/DCM or to the aldehyde 26 by
oxidation under Dess-Martin conditions (Scheme 6). Compound 26 was further treated with
DAST to obtain the geminal difluorides, and subsequent treatment with 1 M HCl solution in a
mixture of MeOH and DCM generated 27. Finally, palladium-catalyzed O-arylation with 1 M
HCl solution in a mixture of MeOH and DCM ultimately gave 28 [23].
2.2. SAR
Here, we report the rational synthesis and development of novel ERRγ inverse agonists
exhibiting drug-like profiles comparable to that of 4 based on in vitro evaluations and the
ADMET index. Using the structural motifs of 4 as a starting point, we performed targeted
structural modifications designed to improve the therapeutic index based on molecular
modeling studies while adhering to accessible synthetic procedures.
Table 1. Estrogen-related receptor gamma (ERRγ) binding and functional assays.
In vitro assay
Functional assay at 10 ꢀM
Entry
Compound
Binding assay, IC50 (ꢀM)
(% of control)
3.90
0.52
0.11
6.70
1.50
0.18
0.09
0.19
0.11
10.2
1
2
3
4
5
6
7
8
9
10a
1.90
NDa
10b
10c
39.1
10d
2.80
10e
16b
ND
3.72
16c
7.75
19
5.10
4 (GSK5182)
aND, not determined; IC50, half-maximal inhibitory concentration.
Molecular docking analyses indicated that the newly synthesized inverse agonists bound
to the active site via hydrogen bonding with the Asp 273, Glu 275, and Asn 346 residues of
ERRγ [20]. This observation prompted us to focus on the structural flexibility at the ligand
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binding domain of the ERRγ active site, which was not explored fully even in characterizing
the current leading ERRγ inverse agonists. Therefore, this provided sufficient groundwork to
develop more potent and selective ERRγ inverse agonists and perform SAR studies, which
commenced following the simple structural modifications. We first explored the role of the
B-ring of compound 4 by incorporating various halogens at the para and ortho positions.
Furthermore, we systematically modified the length of the carbon chain on the C-ring
(Schemes 2 and 3). These modifications produced compound 10c, which displayed an
excellent potency against hERRγ (IC50, 0.11; Table 1), but the functional assay data are not
shown because of cell death-related problems with the analysis. The initial SAR studies
indicated that the presence of p-bromo or chloro substituents on the B-ring produced
compounds (10b–c) with better activities than those of the fluoro-substituted compounds
(10a and 10d); the non-substituted compound 10e exhibited moderate activity.
Table 2. Structure, estrogen-related receptor gamma (ERRγ) binding, and functional screening of compounds
24a–o.
N
O
HCl
A
(
-ring)
B
(
-ring)
OH
Binding
assay, IC50
(ꢀM)
Functional assay at 10
ꢀM (% of control)
Compounds
A-ring
B-ring
0.11
1.3
6.6
4 (GSK5182)
15.8
24a
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>10
>10
>10
108.4
13.7
24b
24c
24d
108.6
0.27
1.0
4.6
5.9
24e
24f
a
>10
0.5
24g
24h
ND
4.9
1.1
38.8
24i
>10
>10
>10
87.2
85.9
24j
24k
24l
116.6
>10
5.6
5.3
44.3
24m
24n
24o
a
ND
a
ND
aND, not detected; IC50, half-maximal inhibitory concentration.
The effects of different alkyl chain lengths were inconclusive. Specifically, compound
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16c, which possesses an extra methylene unit, was more potent than 4 but showed less
selectivity for other ERR subtypes (Table 4). Therefore, the length of alkyl chains on the C-
ring was arbitrarily fixed to that in 4 because two other compounds with mono- and diethyl
amines on the C-ring (16b and 19) showed relatively good pharmacological activities. At this
juncture, we expanded our modification simultaneously to the A- and B-rings in a
synthetically pleasing way and subsequently applied a palladium-catalyzed three-component
reaction of a borate (22, C-ring), internal alkyne (20, B-ring), and iodobenzene (21, A-ring).
This method enabled nearly exclusive generation of the desired regioisomer of a
tetrasubstituted alkene without requiring difficult and tedious chemical transformations.
Impressively, 24e and 24h (para-substituted benzenes) exhibited considerable potency (Table
2); in particular, 24e exhibited improved functional activity relative to 4.
Other functional groups such as trifluoromethyl, methoxy, nitrile, and bromo (24a, 24f,
24g, and 24i, respectively) exhibited moderate binding and functional activities. When the B-
ring was changed to a pyridine (24c–d), we observed no binding activity. Compounds 24j–m
showed >100-fold decreased binding affinity relative to compound 4. The remaining N-
methyl imidazole compounds, 24n–o, exhibited only modest binding affinities.
Table 3. Estrogen-related receptor gamma (ERRγ) binding and functional screening of compounds 25–29.
In vitro assay
Entry
Compound
Binding assay, IC50 (ꢀM)
Functional assay at 10
ꢀM (% of Control)
1
2
3
4
5
25
0.02
0.89
0.59
0.48
0.11
1.7
1.9
4.0
4.2
6.6
27
28
29
4 (GSK5182
)
We next examined the change in binding affinity following the conversion of aliphatic
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alcohols to fluoride and geminal difluorides (Table 3). Interestingly, compound 25 showed
more potent binding and functional activity than compound 4 despite the relatively minor
change. The other branched series, 27–29, showed a slight improvement in functional
potency against hERRγ (Table 3). These results suggest that incorporation of a para-
substituted phenyl group (24e) at the B-ring of the main scaffold enhanced the affinity for
hERRγ and imparted selectivity over other subtypes (ERRα, ERRβ, and ERα) by fitting
more tightly into the binding pocket.
Next, the properties of compounds that exhibited promising activities in the in vitro
screening tests were determined using a binding selectivity test, cytochrome P450 (CYP)
screening, liver microsomal metabolic stability test, and parallel artificial membrane
permeability assay (PAMPA) (Table 4). The binding selectivity profile of 24e to ERRγ was
comparable to that of 4 with no observable affinity for ERRα, ERRβ, and ERα at the
maximum concentration tested (10 ꢀM). Furthermore, 10 µM 24e exhibited 40% inhibition of
CYP2C9 but did not measurably inhibit the other CYP subtypes (2C19, 2D6, and 3A4). In
contrast, compound 4 broadly inhibited the CYP enzymes by 15–27%. We used PAMPA to
investigate the permeability of the newly synthesized compounds compared to that of
compound 4, and compound 24e had a much higher permeability than the other compounds
tested. In addition to the binding assay, assessing functional activity is a key step in the
screening and selection of potent therapeutic candidates for subsequent in vivo testing.
Among the selected compounds, 16b, 16c, and 25 showed promisingly potent functional
activities comparable to that of 4 but had comparatively poor selectivity for the ERR subtypes.
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Table 4. In vitro binding and functional analysis of in vitro absorption, distribution, metabolism, excretion,
and toxicity (ADMET) profiles.
ERRγ
functional
assay, IC50
(ꢀM)
CYP inhibition (% of
control)
Binding assay, IC50 (ꢀM)
ERRγ ERRα ERRβ ERα
MS (% of remaining) PAMPA
(10-6
Compounds
cm/s)
1A2 2C9 2C19 2D6 3A4 Human Dog Rat Mouse
0.110 >10 >10
2
0.077
0.719
0.079
0.058
0.187
0.669
0.048
0.111
84 73 78 82 83
>100 86 61 99 88
82 90 70 98 93
96 90 64 93 79
91 44 76 89 85
>100 60 >100 >100 >100
>100 99 72 >100 93
14 60 11 12 11
43
42
18
72
83
59
72
77
9.6 26 6.8
0.43
6.52
1.02
0.36
0.03
9.94
2.12
0.54
4 (GSK5182)
0.117 >10 6.95 >10
0.180 >10 >10 0.875
0.090 >10 >10 0.99
0.190 >10 >10 3.34
0.270 >10 >10 >10
0.020 8.01 0.722 0.02
0.480 8.1 >10 1.7
5.8 30
22 32
19 19
76 79
13 25
21 35
73 66
35
10
13
20
16
24
91
10c
16b
16c
19
24e
25
28
ERR, estrogen-related receptor; ER, estrogen receptor; IC50, half-maximal inhibitory concentration; CYP,
cytochrome P450; PAMPA, parallel artificial membrane permeability assay
Leu 440
Leu 440
Ile 438
Ile 438
(a)
(b)
Asp 273
Glu 275
Asp 273
Glu 275
His 434
His 434
Tyr 326
Asn 346
Asn 346
Tyr 326
Figure 2. Binding model prediction of ERRγ inverse agonists. Green dashed lines denote hydrogen bonding
interactions. (a) Binding model prediction of compound 24e and ERRγ. (b) Overlay of the predicted binding
model of compound 24e and X-ray crystal complex structure of compound 4 with ERRγ.
Next, we performed a docking simulation analysis to construct a molecular model for the
binding of compound 24e to ERRγ. The docking study revealed that compound 24e might
bind to the active site via hydrogen bonding with Asp 273, Glu 275, and Asn 346 of ERRγ.
The docking study results suggest that compound 24e overlaps the active site of ERRγ in a
similar manner as 4 (Figure 2).
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2.3. Validation of effect of 24e on NIS protein expression in ATC cells
Previously, we demonstrated that 4 increased NIS protein expression by decreasing
endogenous ERRγ protein and activating MAP kinase in ATC cells [21]. Based on our
previous findings, we examined whether the newly synthesized 24e also increased NIS
protein levels in the ATC cell line CAL62 via regulation of ERRγ protein and MAP kinase.
As shown in Figure 3, treatment with 24e significantly reduced ERRγ protein levels in
CAL62 cells by 1.6-fold relative to controls (Figure 3a and b). Significant increases were
observed in phosphorylated MAP kinase levels, such as p44 and p42 extracellular signal-
regulated kinase, in 24e-treated CAL62 cells, leading to 2.0- and 1.2-fold increases in p44
and p42 MAP kinase, respectively (Figure 3a and c). Importantly, there was a marked 2.1-
fold increase of NIS protein in 24e-treated CAL62 cells (Figure 3a and d).
Figure 3. Compound 24e induced NIS protein expression via downregulation of endogenous ERRγ and
activation of MAP kinase in CAL62 cells. (a) CAL62 cells were treated with either vehicle or 6 µM 24e for 24 h,
and then proteins were subjected to immunoblotting. (b–d) Quantification (mean ± SD) of band intensities in (a)
using ImageJ software. *, P<0.05 when compared with vehicle.
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3. Conclusion
In conclusion, compound 24e represents a new structural class of promising analogs of
compound 4 that are fully subtype-selective inverse agonists of ERRγ. In addition, this novel
compound exhibited a significantly improved in vitro ADMET profile. Our study suggests
that the newly synthesized 24e has advantageous pharmacological and ADMET properties
and the potential to be a lead compound for the development of therapeutic agents to treat
various ERRγ-related disorders. Particularly, is a promising enhancer for restoring NIS
protein expression in ATC cells as a key protein for radioiodine therapy. Additional studies
analyzing compound 24e in relevant animal disease models are currently underway.
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4. Experimental
All NMR experiments were carried out using an Avance III 400 MHz NMR spectrometer
equipped with a 5-mm broadband-observed probe head (Bruker, Billerica, MA, USA). The
NMR spectrum optimization was conducted using the Bruker Topspin 3.1 software, and all
parameters were set in it. The compounds were dissolved in CDCl3 or CD3OD and the spectra
were acquired at 25 °C. Mass spectra were measured in positive electrospray ionization (ESI)
mode on LCMS-2020 system (Shimadzu, Tokyo, Japan). Column chromatography was
performed using a CombiFlash® Rf system with RediSep® Rf (Teledyne Isco, Lincoln, NE,
USA). For the final compounds, further purification was performed by preparative HPLC on
Kinetex® 5 ꢀm Biphenyl 100 Å (GX-281 HPLC system, Gilson, Middleton, WI, USA;
column tube: 250 mm × 21.2 mm ID) with ACN/H2O as eluent. The purity of the target
compounds was determined to be >95% by analytical HPLC using dual different wavelength
UV detector. Starting materials were obtained from Aldrich (St. Louis, MO, USA), or Alfa
Aesar (Ward Hill, MA, USA). Solvents were obtained from Fisher Scientific (Hampton, NH,
USA) or Aldrich and were used without further purification unless noted otherwise.
4.1. Chemistry
4.1.1. Synthetic Procedure for 6a–c
To a solution of aluminum chloride (1.1 eq.) and the acid chloride (1.1 eq.) in CH2Cl2 90 mL
was added anisole (1 eq.) in CH2Cl2 (10 mL). The resulting reaction mixture was stirred at rt
for 3 h and then poured onto 1N HCl 50 mL at 0 °C. The quenched reaction mixture was
extracted with EtOAc and washed with brine, dried over Na2SO4, and filtered. The solvent
was concentrated under reduced pressure which was used for the next step without further
purification.
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(4-Fluorophenyl)(4-methoxyphenyl)methanone (6a) The title compound was synthesized
from aluminum chloride (6.8 g, 50.9 mmol), 4-fluorobenzoyl chloride (8.1 g, 50.9 mmol) and
anisole (5 g, 46.2 mmol) in CH2Cl2 (100 mL) according to general procedure for 6a–c (10 g,
99% yield).
(4-chlorophenyl)(4-methoxyphenyl)methanone (6b) The title compound was synthesized
from aluminum chloride (6.8 g, 50.9 mmol), 4-chlorobenzoyl chloride (8.9 g, 50.9 mmol) and
anisole (5 g, 46.2 mmol) in CH2Cl2 (100 mL) according to general procedure for 6a–c (11 g,
99% yield).
(4-bromophenyl)(4-methoxyphenyl)methanone (6c) The title compound was synthesized
from aluminum chloride (6.1 g, 50.9 mmol), 4-bromobenzoyl chloride (8.2 g, 50.9 mmol) and
anisole (5 g, 46.2 mmol) in CH2Cl2 (100 mL) according to general procedure for 6a–c (11 g,
99% yield).
(2,4-difluorophenyl)(4-methoxyphenyl)methanone (6d) In a 100 mL round-bottomed flask
was placed alumium chloride (3.0 g, 22.2 mmol) in 1,2-dichloroethane (15 mL) at 0 °C. 2,4-
difluorobenzoyl chloride (2.6 mL, 18.5 mmol) was added, and anisole (2.0 g, 18.5 mmol) was
added dropwise in 1,2-dichloroethane (2 mL). The reaction mixture was heated at 70 °C.
After 3 h, 6N HCl (3 mL) was added at 0 °C. The combined aqueous layer was extracted with
CH2Cl2. The combined organic layer was washed with aq. NaHCO3, brine and dried over
Na2SO4, filtered and concentrated under reduced pressure (4.6 g, 99% yield).
4.1.2. Synthetic Procedure for 7a–c
To a solution of 6a–c (1 eq.) in toluene was added aluminum chloride (1.2–2.0 eq.) at 0 °C.
The resulting reaction mixture was refluxed for 4 h. The reaction mixture was allowed to cool
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to rt and then poured into 1N HCl. The aqueous phase was extracted with EtOAc. The
combined organic layer was washed with aq. NaHCO3 and brine, dried over Na2SO4 and
filtered. The filtrate was concentrated under reduced pressure. This crude compound was
crystallized from hexane.
(4-fluorophenyl)(4-hydroxyphenyl)methanone (7a) The title compound was synthesized from
6a (5 g, 21.7 mmol), aluminum chloride (7.2 g, 54.3 mmol) in toluene (40 mL) according to
general procedure for 7a–c (4.3 g, 91% yield).
(4-chlorophenyl)(4-hydroxyphenyl)methanone (7b) The title compound was synthesized
from 6b (5 g, 20.3 mmol), aluminum chloride (6.8 g, 50.7 mmol) in toluene (40 mL)
according to general procedure for 7a–c (4.3 g, 90% yield).
(4-bromophenyl)(4-hydroxyphenyl)methanone (7c) The title compound was synthesized
from 6c (10 g, 34.3 mmol), aluminum chloride (11.5 g, 86 mmol) in toluene (80 mL)
according to general procedure for 7a–c (8.1 g, 85% yield).
(2,4-difluorophenyl)(4-hydroxyphenyl)methanone (7d) A solution of 6d (1 g, 4.0 mmol) in
CH2Cl2 (20 mL) was cooled to 0 °C. Boron tribromide in CH2Cl2 (10.1 mL, 10.1 mmol) was
added dropwise. After 15 min the reaction mixture was allowed to come to room temperature.
The reaction mixture was refluxed at 30 °C overnight. The reaction mixture was quenched
with water. The resulting mixture was treated with EtOAc, aq. NaHCO3 and then the aqueous
layer was extracted with EtOAc several times. The organic layer was washed with brine. The
organic layer was dried Na2SO4, filtered and concentrated under reduced pressure (0.7 g, 77%
yield).
4.1.3. Synthetic Procedure for 8a–d
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To a solution of zinc (8 eq.) in THF was added TiCl4 (4 eq.) was added at 0 °C. The
resulting reaction mixture was refluxed at 60 °C for 2 h. A solution of 7a–d (1 eq.) and
methyl 3-benzoylpropionate (1.5 eq.) were added. The resulting mixture was refluxed at
50 °C for 1 h. The mixture was poured onto a mixture of 10% K2CO3 solution and EtOAc.
The resulting black suspension was stirred for 30 min and filtered through a short pad of
Celite®. The combined aqueous layers and extracted with EtOAc. The combined organic
layers was washed with brine, dried over Na2SO4, filtered and concentrated under reduced
pressure. This crude compound was purified by column chromatography.
(E)-methyl 5-(4-fluorophenyl)-5-(4-hydroxyphenyl)-4-phenylpent-4-enoate (8a) The title
compound was synthesized from zinc (1.7 g, 25.9 mmol), TiCl4 (1.4 mL, 13.0 mmol), 7a (0.7
g, 3.2 mmol) and methyl 3-benzoylpropionate (0.9 g, 4.7 mmol) in THF (5 mL) according to
general procedure for 8a–d (0.7 g, 59% yield).
(E)-methyl 5-(4-chlorophenyl)-5-(4-hydroxyphenyl)-4-phenylpent-4-enoate (8b) The title
compound was synthesized from zinc (1.6 g, 24.1 mmol), TiCl4 (1.3 mL, 12.0 mmol), 7b (0.7
g, 3.0 mmol) and methyl 3-benzoylpropionate (0.9 g, 4.5 mmol) in THF (5 mL) according to
general procedure for 8a–d (0.9 g, 73% yield).
(E)-methyl 5-(4-bromophenyl)-5-(4-hydroxyphenyl)-4-phenylpent-4-enoate (8c) The title
compound was synthesized from zinc (1.9 g, 28.9 mmol), TiCl4 (1.6 mL, 14.4 mmol), 7b (1 g,
3.6 mmol) and methyl 3-benzoylpropionate (1.0 g, 5.4 mmol) in THF (10 mL) according to
general procedure for 8a–d (0.61 g, 39% yield).
(E)-methyl 5-(2,4-difluorophenyl)-5-(4-hydroxyphenyl)-4-phenylpent-4-enoate (8d) The title
compound was synthesized from zinc (0.3 g, 4.8 mmol), TiCl4 (0.27 mL, 2.4 mmol), 7c (0.14
g, 0.60 mmol) and methyl 3-benzoylpropionate (0.17 g, 0.91 mmol) in THF (4 mL) according
21

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to general procedure for 8a–d (54 mg, 23% yield).
4.1.4. Synthetic Procedure for 9a–c
To a solution of 8a–c (1 eq.) and 2-chloro-N,N-dimethylethanamine (3.5 eq.) in acetone was
added K2CO3 (3.5 eq.). The resulting reaction mixture was refluxed at 60 °C overnight and
then concentrated under reduced pressure. The crude mixture was purified by column
chromatography.
(E)-methyl
5-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(4-fluorophenyl)-4-phenylpent-4-
enoate (9a) The title compound was synthesized from 8a (0.26 g, 0.68 mmol), 2-chloro-N,N-
dimethylethanamine (0.26 g, 2.4 mmol) and K2CO3 (0.3 g, 2.4 mmol) in acetone (5 mL)
according to general procedure for 9a–c (0.2 g, 49% yield).
(E)-methyl
5-(4-chlorophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-
enoate (9b) The title compound was synthesized from 8b (0.26 g, 0.65 mmol), 2-chloro-N,N-
dimethylethanamine (0.33 g, 2.3 mmol) and K2CO3 (0.3 g, 2.3 mmol) in acetone (5 mL)
according to general procedure for 9a–c (0.2 g, 48% yield).
methyl
(E)-5-(4-bromophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-
enoate (9c) The title compound was synthesized from 8c (0.2 g, 0.46 mmol), 2-chloro-N,N-
dimethylethanamine (0.15 g, 1.38 mmol) and K2CO3 (0.19 g, 1.38 mmol) in acetone (5 mL)
according to general procedure for 9a–c (0.2 g, 72% yield).
methyl (E)-5-(2,4-difluorophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-
enoate (9d) A solution of 8d (54 mg, 0.13 mmol), 2-(dimethylamino)ethanol (41 ꢀL, 0.41
mmol) and triphenylphosphine (0.11 g, 0.41 mmol) in CH2Cl2 (2 mL) was cooled to 0 °C.
DIAD (81 ꢀL, 0.41 mmol) was added dropwise. After 15 min the reaction mixture was
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allowed to come to room temperature. The reaction mixture was stirred at rt for 1 h. The
reaction mixture was added with water. The aqueous layer was extracted with EtOAc several
times. The organic layer was washed with brine. The organic layer was dried Na2SO4, filtered
and concentrated under reduced pressure (64 mg, 99% yield).
4.1.5. Synthetic Procedure for 10a–e
To a solution of 9a–e was dissolved in THF and added 1 M LiAlH4 in THF (1.5 eq.)
dropwise at 0 °C. The solution was stirred at rt for 1 h and was quenched by adding water.
The resulting solution was extracted with EtOAc, washed with brine, dried over Na2SO4,
filtered and concentrated under reduced pressure. The crude compound was purified by
preparative HPLC using eluent A (0.1% TFA H2O) and eluent B (0.1% TFA ACN) (A/B =
70/30). To a solution of this compound in MeOH/CH2Cl2 (1:1) was added a 1M solution of
hydrochloric acid (1.2 eq.) in water at 0 °C. After stirring for 10 min, the volatiles were
removed in vacuum to yield the desired compound.
(E)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-5-(4-fluorophenyl)-4-phenylpent-4-en-1-ol
hydrochloride salt (10a) The title compound was synthesized from 9a (0.15 g, 0.34 mmol),
1 M LiAlH4 in THF (0.5 mL, 0.50 mmol) in THF (2 mL) according to general procedure for
10a–e (7 mg, 5% yield). 1H NMR (400 MHz, CD3OD) : δ 7.27-7.08 (m, 7H), 6.86 (d, J = 8.8
Hz, 2H), 6.71 (d, J = 8.8 Hz, 2H), 4.23 (t, J = 4.8 Hz, 2H), 3.53 (t, J = 4.8 Hz, 2H), 3.42 (t, J
= 6.7 Hz, 2H), 2.95 (s, 6H), 2.53-2.49 (m, 2H), 1.59-1.51 (m, 2H). MS (ESI, m/z) : calcd for
C27H31ClFNO2 [M+H] + 420.54, found 420.23.
(E)-5-(4-chlorophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-en-1-ol
hydrochloride salt (10b) The title compound was synthesized from 9b (0.15 g, 0.32 mmol),
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1 M LiAlH4 in THF (0.5 mL, 0.47 mmol) in THF (2 mL) according to general procedure for
10a–e (15 mg, 10% yield). 1H NMR (400 MHz, CD3OD) : δ 7.38 (d, J = 8.4 Hz, 2H), 7.24-
7.12 (m, 7H), 6.86 (d, J = 8.8 Hz, 2H), 6.72 (d, J = 8.8 Hz, 2H), 4.23 (t, J = 4.8 Hz, 2H), 3.54
(t, J = 4.8 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 2.97 (s, 6H), 2.53-2.49 (m, 2H), 1.59-1.52 (m,
2H). MS (ESI, m/z) : calcd for C27H31Cl2NO2 [M+H] + 436.99, found 436.20.
(E)-5-(4-bromophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-en-1-ol
hydrochloride salt (10c) The title compound was synthesized from 9c (0.48 g, 0.93 mmol), 1
M LiAlH4 in THF (1.4 mL, 1.4 mmol) in THF (10 mL) according to general procedure for
10a–e (0.32 g, 71% yield). 1H NMR (400 MHz, CD3OD) : δ 7.47 (d, J = 8.4 Hz, 2H), 7.18-
7.09 (m, 7H), 6.73 (d, J = 8.8 Hz, 2H), 6.56 (d, J = 9.0 Hz, 2H), 3.93 (t, J = 5.8 Hz, 2H), 3.51
(t, J = 6.6 Hz, 2H), 2.65 (t, J = 5.7 Hz, 2H), 2.50-2.46 (m, 2H), 2.29 (s, 6H), 2.59-2.55 (m,
2H), 1.62-1.54 (m, 2H). 13C NMR (100 MHz, CD3OD) : δ 159.95, 142.63, 142.13, 140.22,
137.89, 135.11, 131.52, 131.05, 130.98, 129.32, 127.62, 125.98, 120.16, 113.17, 64.89, 61.44,
57.61, 44.31, 39.00, 31.95, 31.50. MS (ESI, m/z) : calcd for C27H31BrClNO2 [M+H] + 481.45,
found 480.15.
(E)-5-(2,4-difluorophenyl)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenylpent-4-en-1-ol
hydrochloride salt (10d) The title compound was synthesized from 9d (64 mg, 0.14 mmol), 1
M LiAlH4 in THF (0.17 mL, 0.17 mmol) in THF (2 mL) according to general procedure for
10a–e (31 mg, 53% yield). 1H NMR (400 MHz, CDCl3) : δ 7.22-7.11 (m, 7H), 6.90-6.83 (m,
2H), 6.78(d, J = 6.8 Hz, 2H), 6.57 (d, J = 6.8 Hz, 2H), 3.92 (t, J = 4.6 Hz, 2H), 3.49 (t, J =
5.2 Hz, 2H), 2.64 (t, J = 4.6 Hz, 2H), 2.41 (t, J = 6.3 Hz, 2H), 2.28 (s, 6H), 1.65-1.53 (m, 2H).
MS (ESI, m/z) : calcd for C27H30ClF2NO2 [M+H] + 438.53, found 438.22.
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(Z)-5-(4-(2-(dimethylamino)ethoxy)phenyl)-4,5-diphenylpent-4-en-1-ol hydrochloride salt
(10e) The title compound was synthesized from 9c (0.48 g, 0.93 mmol), 1 M LiAlH4 in THF
(1.4 mL, 1.4 mmol) in THF (10 mL) according to general procedure for 10a–e (6.8 mg, 2%
yield). 1H NMR (400 MHz, CDCl3) : δ 7.36-7.10 (m, 10H), 6.81 (d, J = 7.7 Hz, 2H), 6.53 (d,
J = 7.9 Hz, 2H), 4.31 (br, 2H), 3.48 (t, J = 6.5 Hz, 2H), 3.38 (br, 2H), 2.84 (s, 6H), 2.48 (t, J =
7.6 Hz, 2H), 1.61-1.54 (m, 2H). MS (ESI, m/z) : calcd for C27H32ClNO2 [M+H] + 402.55,
found 402.24.
4.1.6. Synthesis of [4-[4-(2,2-dimethylpropanoyloxy)benzoyl]phenyl] 2,2-dimethylpropanoate
(12)
To the solution of pivaloyl chloride (19.7 g, 186 mmol) in CH2Cl2 (140 mL) and THF (40
mL) was added 4,4-hydroxybenzophenone (10 g, 46.6 mmol). Triethylamine (26 mL, 186
mmol) was added slowly to the solution. The reaction mixture was turned to suspension. The
resulting mixture was stirred at room temperature overnight. The reaction was quenched by
adding water. The mixture was transferred to a separatory funnel and CH2Cl2 was added. The
layer was separated. The organic layer was washed with water, aq. NaHCO3, and brine. The
organic layer was dried over anhydrous Na2SO4, filtered and concentrated to give white solid.
This crude compound was crystallized from ethyl acetate to give white solid product (16g,
91% yield).
4.1.7. Synthesis of compounds 13–19
[4-(4-hydroxybenzoyl)phenyl] 2,2-dimethylpropanoate (13) Compound 12 (12.4 g, 32.3
mmol) and K2CO3 (2.2 g, 16.2 mmol) were dissolved in MeOH (360 mL) and CH2Cl2 (60
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mL). The resulting mixture was stirred at room temperature overnight. The mixture was
concentrated to remove some solvent and acidified with 1.0 M citric acid solution (16.2 mL,
16.2 mmol), and then extracted with ethyl acetate three times. The combined ethyl acetate
layer were washed with brine, dried over anhydrous Na2SO4, and then concentrated. This
crude compound was purified by column chromatography (5.6 g, 58% yield).
(E)-5-[4-(2,2-dimethylpropanoyloxy)phenyl]-5-(4-hydroxyphenyl)-4-phenyl-pent-4-enoate
(14) The title compound was synthesized from zinc (8.8 g, 134 mmol), TiCl4 (7.35 mL, 67.0
mmol), 13 (5 g, 16.8 mmol) and methyl 3-benzoylpropionate (4.8 g, 25.1 mmol) in THF (130
mL) according to general procedure for 8a–d (5.4 g, 70% yield).
(E)-methyl 5-(4-(2-(dimethylamino)ethoxy)phenyl)-4-phenyl-5-(4-(pivaloyloxy)phenyl)pent-
4-enoate (15a) The title compound was synthesized from 14 (2 g, 4.4 mmol), 2-
(dimethylamino)ethanol (1.3 mL, 13.1 mmol), DIAD (2.5 mL, 13.1 mmol) and PPh3 (3.4 g,
13.1 mmol) in CH2Cl2 (40 mL) according to general procedure for 9d (2.2 g, 99% yield).
(E)-methyl 5-(4-(2-(diethylamino)ethoxy)phenyl)-4-phenyl-5-(4-(pivaloyloxy)phenyl)pent-4-
enoate (15b) The title compound was synthesized from 14 (0.1 g, 0.22 mmol), 2-
(diethylamino)ethanol (87 ꢀL, 0.65 mmol), DIAD (0.13 mL, 0.65 mmol) and PPh3 (0.172 g,
0.65 mmol) in CH2Cl2 (3 mL) according to general procedure for 9d (0.1 g, 92% yield).
(E)-methyl
5-(4-(3-(dimethylamino)propoxy)phenyl)-4-phenyl-5-(4-
(pivaloyloxy)phenyl)pent-4-enoate (15c) The title compound was synthesized from 14 (0.1 g,
0.22 mmol), 3-dimethylamino-1-propanol (77 ꢀL, 0.65 mmol), DIAD (0.13 mL, 0.65 mmol)
and PPh3 (0.172 g, 0.65 mmol) in CH2Cl2 (3 mL) according to general procedure for 9d (0.1
g, 99% yield).
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4-[(Z)-1-[4-(2-dimethylaminoethyloxy)phenyl]-5-hydroxy-2-phenyl-pent-1-enyl]phenol
hydrochloride salt (4) The title compound was synthesized from 15a (2.2 g, 4.3 mmol), 1 M
LiAlH4 in THF (6.5 mL, 6.5 mmol) in THF (100 mL) according to general procedure for
10a–e (0.56 g, 31% yield). 1H NMR (400 MHz, CD3OD) : δ 7.17-7.05 (m, 7H), 6.85 (d, J =
8.6 Hz, 2H), 6.79 (d, J = 8.4 Hz, 2H), 6.71 (d, J = 8.6 Hz, 2H), 4.23 (t, J = 4.5 Hz, 2H), 3.53
(t, J = 4.6 Hz, 2H), 3.43 (t, J = 6.6 Hz, 2H), 2.94 (s, 6H), 2.54 (t, J = 7.9 Hz, 2H), 1.56 (m, J
= 7.7 Hz, 2H). MS (ESI, m/z) : calcd for C27H32ClNO3 [M+H] + 418.55, found 418.23.
(Z)-4-(1-(4-(2-(diethylamino)ethoxy)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol
hydrochloride salt (16b) The title compound was synthesized from 15b (0.1 g, 0.20 mmol),
1 M LiAlH4 in THF (0.3 mL, 0.30 mmol) in THF (2 mL) according to general procedure for
10a–e (20 mg, 22% yield). 1H NMR (400 MHz, CD3OD) : δ 7.18-7.09 (m, 5H), 7.05 (d, J =
8.6 Hz, 2H), 6.80-6.77 (m, 4H), 6.59 (d, J = 8.8 Hz, 2H), 4.01 (t, J = 5.6 Hz, 2H), 3.43 (t, J =
6.8 Hz, 4H), 2.94-2.93 (m, 2H), 2.74-2.70 (m, 2H), 2.55-2.51 (m, 2H), 1.60-1.52 (m, 2H),
1.11 (t, J = 7.2 Hz, 6H). 13C NMR (100 MHz, CD3OD) : δ 156.02, 155.58, 142.72, 139.36,
138.94, 137.24, 134.55, 131.78, 130.20, 129.45, 127.55, 125.69, 114.54, 113.10, 61.67, 50.86,
46.95, 39.01, 32.02, 31.66, 7.70. MS (ESI, m/z) : calcd for C29H36ClNO3 [M+H] + 446.60,
found 446.26.
(Z)-4-(1-(4-(3-(dimethylamino)propoxy)phenyl)-5-hydroxy-2-phenylpent-1-en-1-yl)phenol
hydrochloride salt (16c) The title compound was synthesized from 15c (0.1 g, 0.19 mmol),
1 M LiAlH4 in THF (0.29 mL, 0.29 mmol) in THF (2 mL) according to general procedure for
10a–e (35 mg, 42% yield). 1H NMR (400 MHz, CD3OD) : δ 7.18-7.08 (m, 5H), 7.04 (d, J =
8.6 Hz, 2H), 6.82-6.77 (m, 4H), 6.61 (d, J = 8.8 Hz, 2H), 4.00 (t, J = 5.6 Hz, 2H), 3.43 (t, J =
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ACCEPTED MANUSCRIPT
6.8 Hz, 2H), 3.91 (s, 6H), 2.55-2.51 (m, 2H), 2.19-2.12 (m, 2H), 1.59-1.52 (m, 2H). 13C NMR
(100 MHz, CD3OD) : δ 156.22, 155.97, 142.79, 138.87, 136.77, 134.67, 131.65, 130.34,
129.45, 127.54, 125.69, 114.49, 113.77, 112.93, 64.54, 61.62, 55.53, 42.31, 32.01, 31.69,
24.42. MS (ESI, m/z) : calcd for C28H34ClNO3 [M+H] + 432.58, found 432.25.
(E)-methyl
5-(4-(2-((tert-butoxycarbonyl)(methyl)amino)ethoxy)phenyl)-4-phenyl-5-(4-
(pivaloyloxy)phenyl)pent-4-enoate (17) The title compound was synthesized from 14 (1.78 g,
3.9 mmol), tert-butyl (2-hydroxyethyl)(methyl)carbamate (2.0 g, 11.7 mmol), DIAD (2.3 mL,
11.7 mmol) and PPh3 (3.1 g, 11.7 mmol) in CH2Cl2 (40 mL) according to general procedure
for 9d (0.96 g, 40% yield).
(Z)-tert-butyl
(2-(4-(5-hydroxy-1-(4-hydroxyphenyl)-2-phenylpent-1-en-1-
yl)phenoxy)ethyl)(methyl)carbamate (18) To a solution of 17 (0.96 g, 1.6 mmol) was
dissolved in THF (10 mL) and added 1 M LiAlH4 in THF (2.3 mL, 2.3 mmol) dropwise at
0 °C. The solution was stirred at rt for 1 h and was quenched by adding water. The resulting
solution was extracted with EtOAc, washed with brine, dried over Na2SO4, filtered and
concentrated under reduced pressure. This crude compound was used for next step without
further purification (0.69 g, 88% yield).
(Z)-4-(5-hydroxy-1-(4-(2-(methylamino)ethoxy)phenyl)-2-phenylpent-1-en-1-yl)phenol
hydrochloride salt (19) Compound 17 (0.96 g, 1.6 mmol) was added 4 M HCl in dioxane (1.4
mL, 5.5 mmol) dropwise at 0 °C. The solution was stirred at rt for 4 h and was concentrated
under reduced pressure. The crude compound was purified by preparative HPLC using eluent
A (H2O) and eluent B (ACN) (A/B = 70/30) (12 mg, 2% yield). 1H NMR (400 MHz,
CD3OD) : δ 8.53(s, 1H), 7.18-6.99 (m, 7H), 6.81 (d, J = 6.8 Hz, 2H), 6.76 (d, J = 6.6 Hz, 2H),
6.54 (d, J = 6.8 Hz, 2H), 4.11 (t, J = 3.8 Hz, 2H), 3.40 (t, J = 5.4 Hz, 2H), 2.70 (s, 3H), 2.53-
28