Discovery of Highly Potent and Efficient PROTAC Degraders of Androgen Receptor (AR) by Employing Weak Binding Affinity VHL E3 Ligase Ligands

Article abstract of DOI:10.1021/acs.jmedchem.9b01393

Androgen receptor (AR) is a validated therapeutic target for the treatment of metastatic castration-resistant prostate cancer (mCRPC). We report herein our design, synthesis, and biological characterization of highly potent small-molecule proteolysis targeting chimera (PROTAC) AR degraders using a potent AR antagonist and E3 ligase ligands with weak binding affinities to VHL protein. Our study resulted in the discovery of 11 (ARD-266), which effectively induces degradation of AR protein in AR-positive (AR+) LNCaP, VCaP, and 22Rv1 prostate cancer cell lines with DC₅₀ values of 0.2-1 nM. ARD-266 is capable of reducing the AR protein level by >95% in these AR+ prostate cancer cell lines and effectively reduces AR-regulated gene expression suppression. For the first time, we demonstrated that an E3 ligand with micromolar binding affinity to its E3 ligase complex can be successfully employed for the design of highly potent and efficient PROTAC degraders and this finding may have a significant implication for the field of PROTAC research.

Full text of DOI:10.1021/acs.jmedchem.9b01393

Article
pubs.acs.org/jmc
Cite This: J. Med. Chem. XXXX, XXX, XXX−XXX
Discovery of Highly Potent and Efficient PROTAC Degraders of
Androgen Receptor (AR) by Employing Weak Binding Affinity VHL
E3 Ligase Ligands
Xin Han,†,+, Lijie Zhao,†,+,⊗, Weiguo Xiang,†,+ Chong Qin,†,+ Bukeyan Miao,†,+ Tianfeng Xu,†,+
Mi Wang,†,+ Chao-Yie Yang,†,+ Krishnapriya Chinnaswamy,# Jeanne Stuckey,#
and Shaomeng Wang*,†,+,¶,§
+
¶
§
†The Rogel Cancer Center, Department of Internal Medicine, Department of Pharmacology, Department of Medicinal
#
Chemistry, and Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109, United States
⊗School of Pharmaceutical Sciences and Institute of Drug Discovery & Development, Zhengzhou University, Zhengzhou 450001,
China
S
* Supporting Information
ABSTRACT: Androgen receptor (AR) is a validated therapeutic target for the treatment of metastatic castration-resistant
prostate cancer (mCRPC). We report herein our design, synthesis, and biological characterization of highly potent small-
molecule proteolysis targeting chimera (PROTAC) AR degraders using a potent AR antagonist and E3 ligase ligands with weak
binding affinities to VHL protein. Our study resulted in the discovery of 11 (ARD-266), which effectively induces degradation
of AR protein in AR-positive (AR+) LNCaP, VCaP, and 22Rv1 prostate cancer cell lines with DC50 values of 0.2−1 nM. ARD-
266 is capable of reducing the AR protein level by >95% in these AR+ prostate cancer cell lines and effectively reduces AR-
regulated gene expression suppression. For the first time, we demonstrated that an E3 ligand with micromolar binding affinity to
its E3 ligase complex can be successfully employed for the design of highly potent and efficient PROTAC degraders and this
finding may have a significant implication for the field of PROTAC research.
the E3 ligase complex, together with an appropriate linker, are
required for the formation of a productive ternary degradation
complex to achieve efficient degradation of the target
protein.34−37 To date, the influence of the ligand affinity to
the target protein and the linker on the potency and efficiency
of PROTAC degraders have been investigated.21,26 However,
to the best of our knowledge, how the binding affinity of a
ligand for an E3 ligase complex affects potency, efficiency, and
kinetics of the resulting PROTAC degraders has not been
investigated.
We recently reported the discovery of 1 (ARD-61) as a
highly potent and efficient PROTAC AR degrader. The
structure of ARD-61 includes a potent AR antagonist, a high-
affinity ligand for the VHL/cullin2 E3 ligase complex, and an
optimized linker.26 In our present study, we have investigated
how the binding affinity of the VHL ligand portion to VHL
protein influences the potency and efficiency of the resulting
INTRODUCTION
■
In the past few years, induced targeted protein degradation
based on the proteolysis targeting chimera (PROTAC)
concept has gained momentum as a new small-molecule
therapeutic strategy.1−16 One major advantage of this new
therapeutic strategy is that, by reducing protein levels,
PROTAC small-molecule degraders can achieve more
complete target inhibition than traditional small-molecule
inhibitors and are predicted to be therapeutically more
efficacious.17−20 Indeed, our recent studies have demonstrated
that PROTAC degraders of BET proteins, MDM2 protein,
the androgen receptor (AR), and the estrogen receptor (ER)
are indeed much more potent and effective against cancer cells
than the corresponding inhibitors.21−26
A typical PROTAC degrader consists of a ligand, which
binds to the target protein of interest, and is tethered by a
chemical linker to a second ligand, which binds to and recruits
an E3 ligase complex system.27−33 In the design of a highly
potent and effective PROTAC degrader, it has generally been
assumed that high-affinity ligands for the target protein and
Received: August 22, 2019
© XXXX American Chemical Society
A
DOI: 10.1021/acs.jmedchem.9b01393
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a
Table 1. Binding Affinities of VHL Ligands to VHL Protein, from an FP-Based Binding assay
VHL ligand
VHL-a
VHL-b
VHL-c
VHL-d
VHL-e
VHL-f
VHL-g
VHL-h
>100
IC50 SD (μM)
Ki SD (μM)
0.025 0.004
0.006 0.001
0.21 0.04
0.058 0.011
1.08 0.07
0.31 0.02
0.98 0.15
0.28 0.04
2.52 0.3
0.72 0.09
10.5 1.6
3.0 0.5
9.9 0.5
2.8 0.2
a
All of the data are an average of three independent experiments.
a
Table 2. Investigation of the VHL Ligand Binding Potencies on AR Degradation in PROTAC AR Degraders
a
All of the data are an average of three independent experiments with a treatment time of 6 h.
Figure 1. Structures of VHL ligands.
PROTAC degraders. Our study demonstrates that, surpris-
ingly, highly potent and efficient PROTAC degraders can be
obtained using VHL ligands with a weak affinity (Ki = 2−3
μM) to VHL protein.
degraders based on high-affinity VHL E3 ligase ligands and
several classes of AR antagonists.26 One such highly potent
AR degrader is 1 (ARD-61) (Table 2), which achieves DC50
(concentration needed to degrade 50% of AR protein) values
of 1−10 nM in LNCaP and VCaP AR+ prostate cancer cell
lines. ARD-61 is also capable of achieving a Dmax (maximal
levels of protein degradation) of >95%. In the same study, we
found that the linker and AR antagonist portions of the AR
degraders have a major effect on the degradation potency.
RESULTS AND DISCUSSION
■
Design and Synthesis of VHL Ligands with a Wide
Range of Binding Affinities. In our previous study, we
successfully developed highly potent and efficient AR
B
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a
Table 3. Investigation of the Effect of the Linker on AR Degradation in AR Degraders
a
All of the data are an average of three independent experiments with a treatment time of 6 h.
However, the effect of the VHL portion in our previously
reported AR degraders on AR degradation has not been
investigated. We therefore decided to examine how the VHL
ligand portion affects the AR degradation in our designed AR
degraders using ARD-61 as the template molecule.
Co-crystal structures for a number of VHL ligands in a
complex with VHL protein showed that the 4-methylthiazole
group of the ligand VHL-a binds to a hydrophobic pocket in
the VHL protein.9,38−41 Accordingly, we designed and
synthesized a number of VHL ligands based on VHL-a
(Figure 1) by replacing the 4-methylthiazole group with
different groups and tested their binding affinities to VHL
protein using our optimized fluorescence polarization (FP)-
based binding assay.26
Replacement of the 4-methylthiazole group in VHL-a with
an ethyne group resulted in VHL-b, which binds to VHL
protein with an IC50 value of 210 nM (Ki = 58 nM) and is 10
times less potent than VHL-a. Replacing the 4-methylthiazole
group with a nitrile, Br, Cl, F, and H generated VHL-c, VHL-
d, VHL-e, VHL-f, and VHL-g, respectively, which bind to
VHL protein with Ki values of 0.31, 0.28, 0.72, 3.0, and 2.8
μM, respectively. Hence, VHL-c, VHL-d, VHL-e, VHL-f, and
VHL-g are 52, 47, 120, 500, and 467 times less potent than
VHL-a based on their Ki values in binding to VHL protein.
We next changed the 3-methylisoxazole group in VHL-g with
an acetyl group, yielding VHL-h, which shows no appreciable
binding to VHL protein up to 100 μM. Hence, our
modifications of VHL-a have yielded a series of VHL ligands
with a wide range of binding affinities to VHL protein.
Design and Synthesis of PROTAC AR Degraders
Using VHL Ligands with a Wide Range of Binding
Affinities. We employed 1 (ARD-61) as our template AR
degrader and synthesized a series of new PROTAC AR
degraders (Table 2) using the VHL ligands shown in Figure 1,
which have a wide range of binding affinities to VHL. We
tested these putative AR degraders at 10 nM, 100 nM, and 1
μM for their effect on the levels of AR protein in the AR+
LNCaP prostate cancer cell line, obtaining the data
summarized in Table 2.
Compound 8, which consists of a VHL ligand showing no
appreciable binding to VHL protein at 100 μM, fails to reduce
the levels of AR protein significantly at 10 nM, 100 nM, and 1
μM. Interestingly, all other putative AR degraders, consisting
of VHL ligands with a wide range of binding affinities to VHL
protein, effectively reduce the levels of AR protein at each of
the three concentrations (10 nM, 100 nM, and 1 μM).
Compounds 3, 5, and 6, all of which contain a VHL ligand
with much weaker binding affinities to VHL protein than that
in ARD-61, are as potent as ARD-61 in reducing the levels of
AR proteins. In fact, these three compounds are capable of
reducing the AR protein level by >80% at 10 nM, showing
their very high potency.
Further Optimization of the Linker in AR Degraders.
Although compound 7 is not the most potent AR degrader in
Table 2, it contains a VHL ligand with a quite weak affinity
(IC50 = 9.9 μM and Ki = 2.8 μM) to VHL protein. Our
previous study showed that optimization of the linker length
and composition of AR degraders can significantly improve
their degradation potency. Accordingly, we performed further
modifications of the linker in 7, and the results are
summarized in Table 3.
Compounds 9 and 10 were synthesized by replacing the
linker consisting of a six-six-membered ring with a four-six-
membered ring and a six-four-membered ring linker,
respectively. Our Western blotting data showed that both
compounds 9 and 10 effectively induce AR degradation in the
LNCaP AR+ cell line but are less potent than compound 7.
We removed one six-membered ring in the linker of
compound 7, yielding compound 11 (ARD-266), which
reduces the AR protein by >90% at 10 nM and is more potent
than 7. We replaced the ethyne group in the linker of ARD-
266 with an azetidine group, affording the isomeric
C
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a
Table 4. Investigation of the VHL Ligand Portion in ARD-266
a
All of the data are an average of three independent experiments with a treatment time of 6 h.
Figure 2. Western blotting analysis of AR protein in AR+ prostate cancer cell lines. (A) Immunoblots in LNCaP and VCaP cells treated with AR
degraders 11 (ARD-266) and 1 (ARD-61), with GAPDH used as the loading control at different time points; Cells were treated with 100 nM of
ARD-266 or of ARD-61 for indicated times. (B) Plots of immunoblots (SI, Figure S2) in LNCaP, VCaP, and 22Rv1 cells treated with AR
degrader ARD-266 at different concentrations for 6 hr. (C) Mechanistic investigation of AR degradation induced by ARD-266 in LNCaP cells;
cells were pretreated with AR antagonist 41, VHL-a, MLN4924, and MG132 followed by a 6 h treatment with ARD-266 at 100 nM.
compounds 12 and 13. While both 12 and 13 are very
effective in inducing near-complete AR degradation at 100 nM
and 1 μM, they reduce the levels of AR protein by only 40%
at 10 nM and are thus less potent than ARD-266. Removal of
D
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Figure 3. (A) Suppression of AR-regulated gene expression in the LNCaP cell line by the AR degrader 11 (ARD-266) and the AR antagonist 41.
LNCaP cells were treated for 24 h, and qRT-PCR was performed to determine the mRNA levels for AR and AR-regulated genes. (B) Suppression
of AR-regulated gene expression in the VCaP cell line by the AR degrader ARD-266 and the AR antagonist 41. VCaP cells were treated for 24 h,
and qRT-PCR was performed to determine the mRNA levels for AR and AR-regulated genes. (C) Cell growth inhibition in LNCaP and VCaP
cells treated with AR degrader ARD-266, 1 (ARD-61) and two AR antagonists enzalutamide and 41. LNCaP and VCaP cells were treated with
different compounds in a charcoal-stripped medium in the presence of 0.1 nM AR agonist R1881 for 7 days. Cell viability was determined by a
WST-8 assay.
the ethyne group in the linker of ARD-266 resulted in 14,
which is still a very potent AR degrader but is less potent than
ARD-266. Changing the piperidinyl group in the linker in 14
with a piperazinyl group led to 15, which is capable of
reducing the AR protein by >95% at 100 nM and 1 μM but is
less effective than 14 at 10 nM.
is capable of reducing AR protein by >90% even at 10 nM.
We performed further modifications of the VHL ligand
portion of ARD-266, with the results summarized in Table 4.
We replaced the relatively weak VHL ligand in ARD-266
with the high-affinity VHL ligand used in ARD-61, yielding
16. While 16 is very potent and effective in reducing the AR
protein in the LNCaP cell line, it is less potent than ARD-266
at 10 nM.
Examination of the VHL Ligand Portion in ARD-266.
Compound 11 (ARD-266) is a highly potent AR degrader and
E
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a
Scheme 1. Compounds 1−8
a
Reaction conditions: (a) CuI, PdCl2(PPh3)2, DMF/TEA, 100 °C; TFA, DCM, room temperature (rt); (b) K2CO3, KI, CH3CN, reflux; NaOH,
MeOH/H2O, rt; (c) NaH, DMF, rt; TFA, DCM, rt; (d) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (e) HATU, DIPEA, DMF, rt; TFA, DCM, rt;
(f) HATU, DIPEA, DMF, rt.
A F, Cl, Br, nitrile, or ethylene group was installed onto the
4-position of the phenyl ring in ARD-266, leading to
compounds 17−21, respectively. All these compounds can
reduce AR protein by >95% at concentrations of 100 nM and
1 μM. At 10 nM, 18 and 20 are still capable of reducing AR
protein by ≥80% but are less effective than ARD-266. Since a
number of these AR degraders employ a VHL ligand, which
has a greater affinity than that in ARD-266, the data showed
that employing a more potent VHL ligand does not
necessarily result in more potent AR degraders.
Finally, we synthesized compound 22 using a VHL ligand,
which does not have an appreciable binding to VHL protein
up to 100 μM. Compound 22 has no effect on the levels of
AR protein at concentrations of 10−1000 nM.
Further Evaluation of ARD-266 in AR-Positive
Prostate Cancer Cell Lines. Our data in the LNCaP cell
line demonstrated that ARD-266 is a very potent and effective
AR degrader, and next we further evaluated ARD-266 in AR-
positive prostate cancer cell lines for its activity and
mechanism of action.
We examined the kinetics of ARD-266 in the induction of
AR degradation in LNCaP and VCaP AR+ prostate cancer
cell lines, and the results are shown in Figure 2. The data
show that ARD-266 effectively reduces the AR protein level
within 3 h and achieves near-complete AR elimination with a
6 h treatment in the LNCaP cells. In VCaP cells, ARD-266
reduces the AR protein level by >90% after a 3 h treatment.
Our kinetic data thus show that the induction of AR
degradation by ARD-266 in these AR+ prostate cancer cell
lines is fairly rapid. In comparison, ARD-266 and ARD-61
degrade AR protein with a very similar kinetics (Figure 2A).
We next tested ARD-266 for its potency in LNCaP, VCaP,
and 22Rv1 AR-positive prostate cancer cell lines. Our Western
blotting data showed that ARD-266 induces AR degradation
in a dose-dependent manner in each of these three cell lines.
ARD-266 achieves DC50 values of 0.5, 1, and 0.2 nM in the
LNCaP, VCaP, and 22Rv1 cell lines, respectively. ARD-266 is
able to reduce the AR protein level by >95% at 30 nM in both
the LNCaP and VCaP cell lines and at 10 nM in the 22Rv1
cell line with a 6 h treatment time (Figure 2B and Supporting
Information).
We investigated the mechanism of action of ARD-266 in
inducing AR protein degradation in the LNCaP cell line,
obtaining the results shown in Figure 2C. Our data showed
that AR degradation induced by ARD-266 can be effectively
blocked by pretreatment with an AR antagonist (41), a VHL
ligand (VHL-a, Figure 1), a NEDD8-activating E1 enzyme
inhibitor (MLN4924) and a proteasome inhibitor (MG132)
in the LNCaP cell line. These mechanistic data clearly
demonstrate that ARD-266 is a bona fide AR degrader.
We next evaluated the ability of ARD-266 to suppress AR-
regulated gene expression in the LNCaP and VCaP cell lines,
with an AR antagonist (41) included as the control (Figure
3A,B). Our data showed that ARD-266 effectively suppresses
the expression of PSA, TMPRSS2, and FKBP5 genes in a dose-
dependent manner and is capable of reducing the mRNA
levels of PSA, TMPRSS2, and FKBP5 genes by >50% at 10
nM in the LNCaP cell line. In the VCaP cell line, ARD-266
F
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a
Scheme 2. Compounds 9−15
a
Reaction conditions: (a) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (b) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (c) HATU, DIPEA, DMF, rt.
a
Scheme 3. Compounds 16−22
a
Reaction conditions: (a) NaOH, MeOH/H2O, rt; (b) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (c) HATU, DIPEA, DMF, rt; TFA, DCM, rt; (d)
HATU, DIPEA, DMF, rt.
can also suppress the expression of TMPRSS2 and FKBP5
genes by 50% at 10 nM and ERG gene by 50% at 100 nM. In
a direct comparison, ARD-266 is therefore >10−100 times
more potent than the AR antagonist 41 in suppressing the
AR-regulated gene transcription in both the LNCaP and
VCaP cell lines.
We tested the ability of ARD-266 to inhibit cell growth,
with enzalutamide and compound 41 included as AR
antagonist controls in the LNCaP and VCaP cell lines,
obtaining the data shown in Figure 3C.
an IC50 value of 64.5 nM and is thus >30 times less potent
than ARD-266. In the VCaP cell line, ARD-266 and the AR
antagonist 41 have IC50 values of 6 and 182 nM, respectively.
Hence, ARD-266 is 30 times more potent than 41 in
inhibition of cell growth in the VCaP cell line. In both the
LNCaP and VCaP cell lines, enzalutamide is slightly less
potent than compound 41 in inhibition of cell growth (Figure
3C).
CHEMISTRY
■
Our data showed that ARD-266 potently and effectively
inhibits cell growth in the LNCaP cell line and has an IC50
value of 2 nM. In direct comparison, the AR antagonist 41 has
The synthesis of compounds 1−8 is shown in Scheme 1. The
compound 25 was synthesized from the Sonogashira coupling
G
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a
Scheme 4. Compound 41
a
Reaction conditions: (a) HATU, DIPEA, DMF, rt; (b) K2CO3, KI, CH3CN, reflux.
reaction of the compounds 23 and 24. Compound 26 was
produced through the hydrolysis and substitution reaction of
intermediate 25 with tert-butyl 4-bromopiperidine-1-carbox-
ylate. As shown in Scheme 1, the key intermediate 30 was
synthesized in two steps. Compounds 32 can be obtained
from the amidation of compound 30 with different amino
acids (31). Finally, the target compounds were obtained
through the amidation of intermediate 32 and different VHL
fragments.
Compounds 9−15 were synthesized according to the
method shown in Scheme 2. An amidation reaction of
compound 29 with different type of linkers gave the key
intermediates (33). The intermediates (34) were made by
amidation of compounds 32 with (S)-3-((tert-
butoxycarbonyl)amino)-3-phenylpropanoic acid 31a. The
target compounds 9−15 were obtained by amidation of 34
with (2S,4R)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-
5-yl)butanoyl)pyrrolidine-2-carboxylic acid.
The synthesis of compounds 16−22 is shown in Scheme 3.
Compound 35 was produced by hydrolysis of the
intermediate Boc-protected 25. Compound 36 was made by
amidation of compounds 29 and 35. As shown in Scheme 3,
the key intermediates 37 were synthesized from the amidation
of compound 34 with different amino acids (31). Finally, the
target compounds were obtained through amidation of the
intermediates 35 and various VHL fragments.
Compound 41 was synthesized according to the reported
method.42 As shown in Scheme 4, the key intermediate 39
was synthesized from the amidation of compound 29 with 34.
Then, the target compound 41 was obtained through the
substitution reaction of the intermediates 39 and 2-
bromoethanol (40).
VCaP cell lines. Our mechanistic data clearly show that ARD-
266 is a bona fide PROTAC AR degrader. Further
optimization of ARD-266 may ultimately yield a new class
of therapy for the treatment of castration-resistant prostate
cancer. While our previous potent AR degrader ARD-61 has a
molecular weight (MW) of 1095.5, ARD-266 has an MW of
915.5. We also obtained potent AR degraders 14 and 15,
which have MWs of 890.4 and 891.4, respectively. Hence,
using VHL ligands with moderate affinities, the MW of the
resulting AR degraders can be reduced, and their physi-
ochemical properties can be improved.
In summary, our present study demonstrates for the first
time that highly potent and efficient PROTAC degraders can
be successfully obtained using a high-affinity ligand to the
target protein of interest and an E3 ligase ligand with a
micromolar binding affinity to E3. Our study suggests that
even using a low-affinity ligand for an E3 ligase complex, the
resulting PROTAC degrader can induce the formation of a
productive ternary complex consisting of the target protein of
interest, the PROTAC degrader, and the E3 ligase complex in
cells, leading to efficient degradation of the target protein. Our
study may have an important implication for the design of
more druglike PROTAC degraders for the purpose of clinical
development. Furthermore, since it has been challenging to
discover high-affinity ligands for many E3 ligase complexes,
our study may open the door to employ E3 ligands with weak
binding affinities to E3 ligases for the successful design of
highly potent and efficient PROTAC degraders.
EXPERIMENTAL SECTION
■
Chemistry: General Experiment and Information. Unless
otherwise noted, all purchased reagents were used as received
1
without further purification. H NMR and 13C NMR spectra were
recorded on a Bruker Advance 400 MHz spectrometer. 1H NMR
spectra are reported in parts per million (ppm) downfield from
tetramethylsilane (TMS). All 13C NMR spectra are reported in ppm
CONCLUSIONS
■
In this study, we designed, synthesized, and evaluated a series
of putative PROTAC AR degraders using a potent AR
antagonist and VHL E3 ligase ligands with a wide range of
binding affinities to VHL protein. The study demonstrated
that highly potent and efficient AR degraders can be
successfully obtained using VHL ligands with a weak affinity
to VHL, exemplified by 1 (ARD-266). Although ARD-266
consists of a VHL ligand with a Ki value of 2.8 μM, it is in fact
more potent than a number of analogues consisting of a VHL
ligand with much higher affinities to VHL and with the same
linker and AR antagonist. ARD-266 effectively reduces the AR
protein level at concentrations as low as 1 nM in the LNCaP,
VCaP, and 22Rv1 AR-positive prostate cancer cell lines.
Furthermore, ARD-266 is capable of reducing the AR protein
level by >95% at 30 nM with a 6 h treatment time in the
LNCaP and VCaP cells. ARD-266 effectively suppresses AR-
regulated gene expression in a dose-dependent manner and is
effective at concentrations as low as 10 nM in the LNCaP and
1
and obtained with H decoupling. In the reported spectral data, the
format (δ) chemical shift (multiplicity, J values in Hz, integration)
was used with the following abbreviations: s = singlet, d = doublet, t
= triplet, q = quartet, and m = multiplet. Mass spectral (MS) analysis
was carried out with a Waters UPLC mass spectrometer. The final
compounds were all purified by a C18 reversed-phase preparative
HPLC column with solvent A (0.1% TFA in H2O) and solvent B
(0.1% TFA in CH3CN) as eluents. The purity of all the final
compounds was confirmed to be >95% by UPLC-MS or UPLC.
General Procedure for the Synthesis of Compound (11,
ARD-266). Compounds 23 (10 mmol) and 24 (1.1 equiv), CuI (0.2
equiv), and PdCl2(PPh3)2 (0.1 equiv) in DMF and TEA solvents
were placed in a 25 mL round-bottom flask under Ar. The mixture
was stirred for 4 h at 100 °C and then H2O was added into the
resulting complex, and the mixture was extracted with EtOAc three
times. The organic layer was again washed with H2O before being
dried over MgSO4, and the solvent was removed under vacuum,
leaving the crude product. The pure product Boc-protected 25 was
obtained by flash column chromatography (hexane/EtOAc = 4:1).
H
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NaOH (2 equiv) was added to a solution of Boc-protected 25 in
MeOH/H2O and stirred at rt for 2 h. Then, MeOH was removed
under reduced pressure, the pH was adjusted with 2 N HCl, and the
mixture was extracted with EtOAc. The solvent was removed to
afford the product (35), which was used without further purification.
NaH (1.2 equiv) was added at 0 °C to a solution of 28 (10 mmol)
in dry DMF. After stirring the mixture at 0 °C for 20 min, 27 was
added and the mixture was stirred at rt for 4 h. After UPLC-MS
demonstrated the full conversion of starting materials, H2O was
added and the mixture was extracted three times with EtOAc, the
combined organic layers were washed with brine, and then dried over
anhydrous Na2SO4. The solvent was removed on a rotary evaporator.
The Boc-protected intermediate was obtained by flash column
chromatography. Then, the desired intermediate 29 was obtained by
deprotection with TFA in DCM in 88% yield.
DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution
of the compound 29 (1 mmol) and 35 (1.1 equiv) in DMF (2 mL).
After 30 min at rt, the mixture was subjected to HPLC purification to
afford compound 36 in 88% yield after deprotection in TFA/DCM.
DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution
of compound 36 (0.1 mmol) and (S)-3-((tert-butoxycarbonyl)-
amino)-3-phenylpropanoic acid 31a (1.1 equiv) in DMF (2 mL).
After 30 min at rt, the mixture was subjected to HPLC purification to
afford compound 37a with 80% yield after deprotection in TFA/
DCM.
6.7 Hz, 3H), 0.91−0.84 (m, 3H). HRMS calculated for
C61H72ClN8O7S [M + H]+: 1095.4933, found 1095.4908. UPLC
retention time: 5.4 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-1-(4-ethynylphenyl)-3-oxopropyl)-4-hydroxy-1-
((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-car-
1
boxamide (2). H NMR (400 MHz, MeOD-d4) δ 7.86−7.79 (m,
2H), 7.74 (d, J = 8.7 Hz, 1H), 7.65−7.58 (m, 1H), 7.53−7.45 (m,
3H), 7.43−7.32 (m, 2H), 7.15 (s, 1H), 7.00 (d, J = 8.6 Hz, 1H),
6.25 (d, J = 3.7 Hz, 1H), 5.39 (dd, J = 17.1, 10.6 Hz, 1H), 4.68 (d, J
= 11.2 Hz, 1H), 4.53−4.42 (m, 2H), 4.31 (s, 1H), 4.15 (dd, J = 23.3,
8.9 Hz, 2H), 3.92−3.76 (m, 2H), 3.52 (ddd, J = 34.0, 24.2, 12.1 Hz,
7H), 3.27−3.22 (m, 1H), 3.17−3.03 (m, 3H), 3.02−2.75 (m, 2H),
2.63 (t, J = 12.4 Hz, 1H), 2.38 (d, J = 13.4 Hz, 2H), 2.27 (d, J = 2.7
Hz, 3H), 2.19 (s, 3H), 2.03 (dd, J = 27.5, 15.7 Hz, 4H), 1.70−1.52
(m, 1H), 1.39 (dd, J = 5.9, 3.8 Hz, 1H), 1.31 (s, 6H), 1.25 (s, 6H),
1.07 (d, J = 5.5 Hz, 3H), 0.88 (dd, J = 10.1, 4.2 Hz, 3H). UPLC−
MS calculated for C59H68ClN7O7 [M + H]+: 1021.49, found 1021.46.
UPLC: 5.5 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-1-(4-cyanophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-
3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carbox-
1
amide (3). H NMR (400 MHz, MeOD-d4) δ 7.81 (s, 2H), 7.74 (d,
J = 8.7 Hz, 3H), 7.59 (d, J = 8.0 Hz, 3H), 7.52 (d, J = 7.0 Hz, 1H),
7.15 (d, J = 2.2 Hz, 1H), 7.00 (dd, J = 8.7, 2.2 Hz, 1H), 6.31−6.21
(m, 1H), 5.49−5.42 (m, 1H), 4.67 (s, 1H), 4.47 (d, J = 7.6 Hz, 2H),
4.31 (s, 1H), 4.22 (s, 1H), 4.18 (s, 1H), 3.84 (dd, J = 23.7, 8.8 Hz,
2H), 3.58 (d, J = 19.2 Hz, 5H), 3.16 (dd, J = 18.3, 11.7 Hz, 4H),
3.02−2.86 (m, 3H), 2.65 (d, J = 12.3 Hz, 1H), 2.36 (d, J = 12.7 Hz,
2H), 2.27 (s, 3H), 2.18 (s, 4H), 2.02−1.95 (m, 2H), 1.30 (s, 6H),
1.25 (s, 6H), 1.05 (d, J = 6.1 Hz, 3H), 0.87 (d, J = 6.5 Hz, 3H).
UPLC−MS calculated for C58H67ClN8O7 [M + H]+: 1023.49, found
1023.51. UPLC: 5.0 min, purity >95%.
DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution
of the compound 37a (0.05 mmol) and (2S,4R)-4-hydroxy-1-((R)-3-
methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxylic
acid (1.1 equiv) in DMF (2 mL). After 30 min at rt, the mixture was
subject to HPLC purification to afford compound 11 (ARD-266)
with 83% yield.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-2-(3-meth-
ylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (11, ARD-
1
(2S,4R)-N-((S)-1-(4-Bromophenyl)-3-(4-((4-(((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)-
ethynyl)-[1,4′-bipiperidin]-1′-yl)-3-oxopropyl)-4-hydroxy-1-((R)-3-
methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxa-
266). H NMR (400 MHz, MeOD-d4) δ 7.81−7.77 (m, 2H), 7.74
(d, J = 8.7 Hz, 1H), 7.50 (dd, J = 8.5, 2.4 Hz, 2H), 7.41−7.32 (m,
4H), 7.30−7.24 (m, 1H), 7.15 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8,
2.4 Hz, 1H), 6.23 (dd, J = 4.4, 1.9 Hz, 1H), 5.33 (dt, J = 30.1, 7.1
Hz, 1H), 4.58−4.37 (m, 2H), 4.30 (d, J = 0.9 Hz, 1H), 4.20−4.15
(m, 1H), 4.01−3.84 (m, 2H), 3.82−3.73 (m, 2H), 3.64−3.55 (m,
1H), 3.26 (t, J = 9.8 Hz, 1H), 3.12−2.98 (m, 2H), 2.95−2.86 (m,
1H), 2.48−2.38 (m, 1H), 2.30−2.23 (m, 3H), 2.16 (t, J = 10.5 Hz,
1H), 1.99 (ddt, J = 12.9, 8.3, 4.3 Hz, 1H), 1.81 (dd, J = 36.4, 3.5 Hz,
2H), 1.50 (ddd, J = 54.1, 15.5, 8.5 Hz, 3H), 1.31 (s, 6H), 1.25 (s,
6H), 1.07 (dd, J = 6.6, 2.0 Hz, 3H), 0.94−0.84 (m, 3H). 13C NMR
(100 MHz, MeOD-d4) δ 171.73, 169.83, 169.55, 169.18, 162.97,
160.17, 159.29, 141.10, 137.56, 135.34, 133.62, 131.17, 128.25,
127.26, 126.92, 126.50, 116.59, 115.73, 114.31, 104.36, 103.08,
93.72, 84.41, 81.00, 80.84, 69.32, 59.58, 59.03, 55.81, 51.05, 50.18,
44.40, 40.23, 40.07, 39.79, 38.23, 37.44, 31.59, 30.91, 27.35, 27.23,
23.03, 22.29, 20.15, 19.59, 19.02, 9.84. HRMS calculated for
C52H60ClN6O7 [M + H]+: 915.4212, found 915.4195. UPLC
retention time: 6.6 min, purity >95%. Following the procedures
used to prepare compound ARD-266, all other compounds were
obtained using the same methods.26
1
mide (4). H NMR (400 MHz, MeOD-d4) δ 7.88−7.80 (m, 2H),
7.73 (d, J = 8.8 Hz, 1H), 7.59 (q, J = 7.4 Hz, 1H), 7.55−7.46 (m,
3H), 7.36−7.26 (m, 2H), 7.14 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.8,
2.4 Hz, 1H), 6.30−6.20 (m, 1H), 5.46−5.22 (m, 1H), 4.67 (t, J =
12.7 Hz, 1H), 4.55−4.37 (m, 2H), 4.31 (d, J = 2.6 Hz, 1H), 4.26−
4.10 (m, 2H), 3.94−3.71 (m, 2H), 3.67−3.39 (m, 5H), 3.23 (d, J =
7.3 Hz, 3H), 3.09 (dd, J = 43.6, 12.1 Hz, 3H), 2.63 (s, 1H), 2.44−
2.31 (m, 2H), 2.26 (d, J = 3.2 Hz, 3H), 2.20−2.11 (m, 3H), 2.05−
1.93 (m, 2H), 1.39 (dd, J = 6.6, 3.9 Hz, 1H), 1.31 (d, J = 2.2 Hz,
6H), 1.24 (s, 6H), 1.06 (dq, J = 13.5, 6.0, 4.7 Hz, 3H), 0.86 (ddd, J
= 9.6, 6.7, 3.7 Hz, 3H). UPLC−MS calculated for C57H68BrN7O7 [M
+ H]+: 1076.41, found 1076.42. UPLC: 5.3 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-1-(4-chlorophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-
3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carbox-
1
amide (5). H NMR (400 MHz, MeOD-d4) δ 7.86−7.77 (m, 2H),
7.74 (d, J = 8.7 Hz, 1H), 7.60 (t, J = 7.4 Hz, 1H), 7.52 (d, J = 7.8
Hz, 1H), 7.37 (d, J = 7.1 Hz, 4H), 7.15 (s, 1H), 7.00 (d, J = 8.7 Hz,
1H), 6.25 (t, J = 4.5 Hz, 1H), 5.37 (dt, J = 11.0, 6.4 Hz, 1H), 4.88−
4.77 (m, 2H), 4.68 (t, J = 12.2 Hz, 1H), 4.52−4.39 (m, 2H), 4.31 (s,
1H), 4.18 (s, 2H), 3.92−3.73 (m, 2H), 3.65−3.45 (m, 4H), 3.28−
3.09 (m, 3H), 3.07−3.00 (m, 1H), 2.98−2.83 (m, 1H), 2.64 (d, J =
9.5 Hz, 1H), 2.37 (d, J = 14.0 Hz, 2H), 2.30−2.22 (m, 3H), 2.18 (s,
4H), 2.10−1.90 (m, 3H), 1.67 (d, J = 9.3 Hz, 1H), 1.30 (s, 6H),
1.25 (s, 6H), 1.11−1.00 (m, 3H), 0.92−0.80 (m, 3H). UPLC−MS
calculated for C57H67Cl2N7O7 [M + H]+: 1032.46, found 1032.42.
UPLC: 5.2 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxo-propyl)-
4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)-
1
pyrrolidine-2-carboxamide (1). H NMR (400 MHz, MeOD-d4) δ
9.16 (t, J = 5.8 Hz, 1H), 7.81 (t, J = 8.8 Hz, 2H), 7.74 (d, J = 8.7 Hz,
1H), 7.59 (d, J = 7.5 Hz, 1H), 7.52 (dd, J = 9.1, 5.7 Hz, 5H), 7.14
(d, J = 2.1 Hz, 1H), 7.00 (dd, J = 8.7, 2.1 Hz, 1H), 6.33−6.20 (m,
1H), 5.53−5.37 (m, 1H), 4.70 (t, J = 12.9 Hz, 1H), 4.46 (t, J = 18.4
Hz, 2H), 4.31 (s, 2H), 4.17 (s, 1H), 3.90 (dd, J = 10.8, 3.8 Hz, 1H),
3.85−3.75 (m, 1H), 3.67−3.51 (m, 4H), 3.28−3.08 (m, 4H), 3.01−
2.88 (m, 1H), 2.67 (dd, J = 24.1, 11.9 Hz, 1H), 2.56−2.51 (m, 3H),
2.48−2.32 (m, 2H), 2.27 (d, J = 4.2 Hz, 3H), 2.25−2.06 (m, 6H),
1.98 (dd, J = 10.1, 5.0 Hz, 3H), 1.77 (dd, J = 29.2, 14.2 Hz, 1H),
1.62−1.42 (m, 1H), 1.30 (s, 6H), 1.25 (s, 6H), 1.07 (dd, J = 13.4,
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-1-(4-fluorophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-
3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carbox-
amide (6). 1H NMR (400 MHz, MeOD-d4) δ 7.82 (dd, J = 13.2, 8.2
I
DOI: 10.1021/acs.jmedchem.9b01393
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Hz, 2H), 7.74 (d, J = 8.6 Hz, 1H), 7.60 (t, J = 7.3 Hz, 1H), 7.52 (t, J
= 4.2 Hz, 1H), 7.48−7.35 (m, 2H), 7.15 (d, J = 2.3 Hz, 1H), 7.09
(dq, J = 14.0, 8.2, 6.1 Hz, 2H), 7.04−6.96 (m, 1H), 6.31−6.19 (m,
1H), 5.38 (dq, J = 24.1, 6.5 Hz, 1H), 4.68 (t, J = 14.4 Hz, 1H),
4.53−4.36 (m, 2H), 4.34−4.16 (m, 3H), 3.93−3.74 (m, 2H), 3.65−
3.50 (m, 4H), 3.33 (p, J = 1.7 Hz, 3H), 3.26−3.09 (m, 3H), 3.06−
2.84 (m, 2H), 2.65 (q, J = 12.6, 12.1 Hz, 1H), 2.44−2.34 (m, 2H),
2.27 (d, J = 5.8 Hz, 3H), 2.15 (t, J = 11.3 Hz, 4H), 2.05−1.93 (m,
2H), 1.79−1.64 (m, 1H), 1.52−1.40 (m, 1H), 1.33−1.23 (m, 12H),
1.05 (dt, J = 12.6, 6.5 Hz, 3H), 0.86 (ddt, J = 10.0, 6.9, 3.3 Hz, 3H).
UPLC−MS calculated for C57H68ClFN7O7 [M + H]+: 1016.49,
found 1016.52. UPLC retention time: 4.6 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)-[1,4′-bi-
piperidin]-1′-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-
2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (7).
1H NMR (400 MHz, MeOD-d4) δ 7.82 (t, J = 10.1 Hz, 2H), 7.74
(d, J = 8.7 Hz, 1H), 7.61 (t, J = 8.3 Hz, 1H), 7.52 (t, J = 6.0 Hz,
1H), 7.42−7.29 (m, 5H), 7.15 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8,
2.4 Hz, 1H), 6.24 (dd, J = 6.4, 2.7 Hz, 1H), 5.43−5.28 (m, 1H), 4.67
(d, J = 14.2 Hz, 1H), 4.47 (d, J = 13.6 Hz, 2H), 4.31 (s, 1H), 4.23−
4.08 (m, 2H), 3.93−3.75 (m, 2H), 3.64−3.47 (m, 4H), 3.23−3.02
(m, 4H), 2.98−2.77 (m, 2H), 2.64 (d, J = 12.8 Hz, 1H), 2.45−2.32
(m, 2H), 2.27 (d, J = 5.4 Hz, 3H), 2.21−1.90 (m, 8H), 1.69 (d, J =
12.8 Hz, 1H), 1.45 (d, J = 2.1 Hz, 1H), 1.31 (s, 6H), 1.25 (s, 6H),
1.07 (q, J = 6.4 Hz, 3H), 0.91−0.84 (m, 3H). UPLC−MS calculated
for C57H68ClN7O7 [M + H]+: 998.49, found 998.43. UPLC retention
time: 6.7 min, purity >95%.
1.36 (m, 1H), 1.32−1.22 (m, 12H), 1.10 (dd, J = 13.1, 6.1 Hz, 3H),
0.94−0.84 (m, 3H). UPLC−MS calculated for C55H65ClN7O7 [M +
H]+: 970.46, found 970.48. UPLC retention time: 5.0 min, purity
>95%.
(2S,4R)-N-((S)-3-(4-(1-(4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)azetidin-3-yl)-
piperidin-1-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-
2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide
1
(12). H NMR (400 MHz, MeOD-d4) δ 7.80−7.66 (m, 3H), 7.42−
7.25 (m, 5H), 7.12 (q, J = 2.1 Hz, 1H), 6.97 (dq, J = 9.0, 2.1 Hz,
1H), 6.62−6.40 (m, 2H), 6.22 (d, J = 1.8 Hz, 1H), 5.41−5.28 (m,
1H), 4.56−4.37 (m, 3H), 4.28 (d, J = 1.9 Hz, 1H), 4.15 (s, 1H),
4.00 (tt, J = 9.9, 5.4 Hz, 3H), 3.88 (dt, J = 11.3, 5.5 Hz, 1H), 3.79
(dd, J = 9.6, 7.2 Hz, 1H), 3.75−3.48 (m, 4H), 3.02 (tdt, J = 34.9,
15.2, 8.3 Hz, 3H), 2.45 (ddt, J = 37.0, 28.9, 10.9 Hz, 4H), 2.25 (d, J
= 2.0 Hz, 3H), 2.15 (q, J = 7.0 Hz, 1H), 1.99 (ttd, J = 12.9, 8.9, 8.4,
3.6 Hz, 1H), 1.67 (dd, J = 20.2, 11.6 Hz, 3H), 1.29 (s, 6H), 1.22 (d,
J = 1.9 Hz, 6H), 1.11−1.04 (m, 3H), 0.87 (d, J = 6.8 Hz, 3H).
UPLC−MS calculated for C53H65ClN7O7 [M + H]+: 946.46, found
946.48. UPLC retention time: 6.4 min, purity >95%.
(2S,4R)-N-((S)-3-(4-(3-(4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)azetidin-1-yl)-
piperidin-1-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-
2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide
1
(13). H NMR (400 MHz, MeOD-d4) δ 7.91 (dd, J = 8.2, 4.6 Hz,
2H), 7.74 (d, J = 8.7 Hz, 1H), 7.62−7.55 (m, 2H), 7.37 (tdd, J =
10.3, 7.0, 2.5 Hz, 5H), 7.29 (t, J = 6.4 Hz, 1H), 7.01 (dd, J = 8.7, 2.4
Hz, 1H), 6.38−6.21 (m, 1H), 5.57−5.29 (m, 1H), 4.75−4.37 (m,
9H), 4.32 (s, 1H), 4.19 (s, 1H), 4.02−3.55 (m, 5H), 3.28−3.19 (m,
1H), 3.17−3.03 (m, 1H), 2.95 (d, J = 53.3 Hz, 1H), 2.67 (td, J = 9.5,
8.3, 4.8 Hz, 1H), 2.45 (dt, J = 10.4, 6.5 Hz, 1H), 2.29 (d, J = 12.4
Hz, 3H), 2.19 (dd, J = 19.7, 10.1 Hz, 4H), 1.98−1.81 (m, 1H),
1.51−1.37 (m, 1H), 1.31 (d, J = 2.1 Hz, 6H), 1.25 (s, 6H), 1.13−
1.06 (m, 3H), 0.89 (t, J = 7.4 Hz, 3H). UPLC−MS calculated for
C53H65ClN7O7 [M + H]+: 946.46, found 946.49. UPLC retention
time: 5.4 min, purity >95%.
(2S,4R)-N-((S)-3-(4-(4-(((1r,3S)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)piperidin-1-yl)-3-
oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxa-
zol-5-yl)butanoyl)pyrrolidine-2-carboxamide (14). 1H NMR (400
MHz, DMSO-d6) δ 7.91 (d, J = 8.7 Hz, 1H), 7.78 (d, J = 7.8 Hz,
2H), 7.33 (t, J = 4.9 Hz, 5H), 7.27−7.18 (m, 3H), 7.01 (dd, J = 8.8,
2.4 Hz, 1H), 6.20 (dd, J = 17.7, 3.0 Hz, 1H), 5.22 (tt, J = 10.4, 8.4,
3.7 Hz, 1H), 4.53 (d, J = 12.7 Hz, 1H), 4.42−4.20 (m, 3H), 4.06 (d,
J = 3.5 Hz, 1H), 3.94 (t, J = 16.1 Hz, 1H), 3.76 (dd, J = 9.7, 4.2 Hz,
1H), 3.58 (dd, J = 11.2, 8.0 Hz, 1H), 3.12−2.74 (m, 4H), 2.62−2.52
(m, 1H), 2.33−2.22 (m, 1H), 2.20 (t, J = 4.9 Hz, 3H), 2.02−1.93
(m, 1H), 1.83−1.60 (m, 3H), 1.51−1.36 (m, 1H), 1.19 (d, J = 36.7
Hz, 12H), 0.98 (dd, J = 6.6, 2.3 Hz, 3H), 0.79 (dd, J = 14.2, 6.7 Hz,
3H). UPLC−MS calculated for C50H60ClN6O7 [M + H]+: 891.42,
found 891.40. UPLC retention time: 5.2 min, purity >95%.
(2S,4R)-N-((S)-3-(4-(4-(((1r,3S)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)piperazin-1-yl)-3-
oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-methyl-2-(3-methylisoxa-
zol-5-yl)butanoyl)pyrrolidine-2-carboxamide (15). 1H NMR (400
MHz, MeOD-d4) δ 7.81−7.76 (m, 2H), 7.72 (d, J = 8.8 Hz, 1H),
7.42−7.31 (m, 4H), 7.28−7.22 (m, 1H), 7.13 (d, J = 2.4 Hz, 1H),
7.03−6.95 (m, 3H), 6.19 (s, 1H), 5.44−5.30 (m, 1H), 4.52−4.43
(m, 2H), 4.30 (d, J = 0.9 Hz, 1H), 4.16 (d, J = 0.9 Hz, 1H), 3.87
(dd, J = 10.8, 4.3 Hz, 1H), 3.79−3.53 (m, 6H), 3.31−3.15 (m, 3H),
3.14−2.98 (m, 3H), 2.38 (dtd, J = 14.2, 8.5, 7.6, 3.1 Hz, 1H), 2.24
(d, J = 3.1 Hz, 3H), 2.16 (dddd, J = 13.2, 7.7, 3.0, 1.4 Hz, 1H),
2.07−1.93 (m, 1H), 1.26 (d, J = 26.0 Hz, 12H), 1.06 (dd, J = 11.7,
6.6 Hz, 3H), 0.88 (dd, J = 25.7, 6.7 Hz, 3H). UPLC−MS calculated
for C49H59ClN7O7 [M + H]+: 892.42, found 892.43. UPLC retention
time: 5.0 min, purity >95%.
(2S,4R)-1-(Acetyl-L-valyl)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)-
ethynyl)-[1,4′-bipiperidin]-1′-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-
1
pyrrolidine-2-carboxamide (8). H NMR (400 MHz, MeOD-d4) δ
7.86−7.78 (m, 2H), 7.74 (d, J = 8.7 Hz, 1H), 7.62 (dd, J = 8.1, 5.2
Hz, 1H), 7.51 (d, J = 8.1 Hz, 1H), 7.42−7.27 (m, 5H), 7.16−7.12
(m, 1H), 7.00 (dt, J = 8.7, 1.9 Hz, 1H), 5.33 (q, J = 6.2, 5.5 Hz, 1H),
4.65 (d, J = 13.4 Hz, 1H), 4.55 (t, J = 8.3 Hz, 1H), 4.44 (tt, J = 8.4,
4.9 Hz, 2H), 4.31 (d, J = 3.3 Hz, 1H), 4.18 (d, J = 4.9 Hz, 2H), 3.88
(d, J = 11.3 Hz, 1H), 3.77 (dq, J = 11.6, 4.1 Hz, 1H), 3.59−3.45 (m,
3H), 3.17−2.92 (m, 5H), 2.60 (q, J = 13.7 Hz, 1H), 2.39−2.32 (m,
1H), 2.23−2.06 (m, 6H), 2.04−2.01 (m, 3H), 1.95 (ddd, J = 13.2,
8.4, 4.4 Hz, 2H), 1.55 (d, J = 11.6 Hz, 1H), 1.45−1.38 (m, 1H), 1.28
(dd, J = 24.2, 2.9 Hz, 12H), 1.05−0.95 (m, 6H). UPLC−MS
calculated for C55H69ClN7O7 [M + H]+: 974.49, found 974.54.
UPLC retention time: 4.3 min, purity >95%.
(2S,4R)-N-((S)-3-(4-(3-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)azetidin-
1-yl)piperidin-1-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-
methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxa-
1
mide (9). H NMR (400 MHz, MeOD-d4) δ 7.83 (dd, J = 8.3, 2.6
Hz, 2H), 7.74 (d, J = 8.8 Hz, 1H), 7.64−7.58 (m, 2H), 7.39−7.27
(m, 5H), 7.15 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.8, 2.4 Hz, 1H),
6.37−6.21 (m, 1H), 5.62−5.13 (m, 1H), 4.74 (s, 1H), 4.57 (d, J =
9.1 Hz, 2H), 4.54−4.38 (m, 5H), 4.31 (s, 1H), 4.18 (s, 1H), 3.97−
3.80 (m, 2H), 3.72−3.53 (m, 2H), 3.30−2.99 (m, 4H), 2.69−2.61
(m, 1H), 2.46−2.37 (m, 1H), 2.32−2.24 (m, 3H), 2.15 (s, 3H),
1.97−1.85 (m, 1H), 1.50−1.37 (m, 1H), 1.31 (d, J = 1.3 Hz, 6H),
1.25 (s, 6H), 1.10−1.05 (m, 3H), 0.91−0.86 (m, 3H). UPLC−MS
calculated for C55H65ClN7O7 [M + H]+: 970.46, found 970.44.
UPLC retention time: 5.2 min, purity >95%.
(2S,4R)-N-((S)-3-(3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)azetidin-1-yl)-3-oxo-1-phenylpropyl)-4-hydroxy-1-((R)-3-meth-
yl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide
1
(10). H NMR (400 MHz, MeOD-d4) δ 7.81 (d, J = 8.0 Hz, 2H),
7.73 (dd, J = 8.7, 3.1 Hz, 1H), 7.56 (t, J = 7.9 Hz, 2H), 7.37 (dt, J =
8.5, 3.1 Hz, 4H), 7.30 (dt, J = 7.7, 3.7 Hz, 1H), 7.14 (d, J = 2.7 Hz,
1H), 7.00 (dd, J = 8.7, 2.8 Hz, 1H), 6.37−6.20 (m, 1H), 5.51−5.38
(m, 1H), 4.77−4.62 (m, 1H), 4.58−4.36 (m, 4H), 4.24 (d, J = 50.6
Hz, 4H), 3.96−3.79 (m, 2H), 3.72−3.54 (m, 2H), 2.93−2.83 (m,
1H), 2.64 (td, J = 14.7, 13.3, 8.2 Hz, 1H), 2.51−2.30 (m, 3H), 2.27
(d, J = 14.1 Hz, 3H), 2.06 (ddd, J = 57.8, 12.4, 6.7 Hz, 4H), 1.52−
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-1-(4-(4-methylthiazol-5-yl)phenyl)-3-oxopropyl)-4-hydroxy-1-
((R)-3-methyl-2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-car-
1
boxamide (16). H NMR (400 MHz, MeOD-d4) δ 9.19−9.09 (m,
1H), 7.89−7.70 (m, 4H), 7.53 (s, 3H), 7.46−7.36 (m, 2H), 7.14 (d,
J
DOI: 10.1021/acs.jmedchem.9b01393
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
J = 2.3 Hz, 1H), 7.00 (dd, J = 8.8, 2.3 Hz, 1H), 6.35−6.19 (m, 1H),
5.46−5.34 (m, 1H), 4.57−4.40 (m, 2H), 4.31 (s, 1H), 4.18 (s, 1H),
4.10−3.98 (m, 1H), 3.89 (td, J = 10.5, 4.1 Hz, 1H), 3.81 (t, J = 9.4
Hz, 2H), 3.63 (t, J = 9.4 Hz, 1H), 3.29−2.81 (m, 5H), 2.52 (d, J =
4.8 Hz, 3H), 2.47−2.39 (m, 1H), 2.26 (dd, J = 6.7, 3.0 Hz, 3H), 2.18
(dd, J = 12.1, 6.2 Hz, 1H), 2.01 (dddt, J = 17.6, 13.2, 8.6, 3.6 Hz,
1H), 1.85 (s, 2H), 1.60−1.38 (m, 2H), 1.31 (s, 6H), 1.25 (s, 6H),
1.09 (t, J = 6.1 Hz, 3H), 0.89 (td, J = 7.2, 6.6, 3.1 Hz, 3H). HRMS
calculated for C56H63ClN6O7S [M + H]+: 1012.4198, found
1012.4173. UPLC retention time: 6.2 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-1-(4-fluorophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-2-
(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (17).
1H NMR (400 MHz, MeOD-d4) δ 7.79 (dd, J = 8.4, 3.3 Hz, 2H),
7.75−7.71 (m, 1H), 7.49 (dd, J = 8.5, 3.4 Hz, 2H), 7.41 (dd, J = 9.2,
5.2 Hz, 2H), 7.15−7.06 (m, 3H), 6.99 (dt, J = 8.7, 3.0 Hz, 1H), 6.23
(d, J = 4.4 Hz, 1H), 5.35 (d, J = 6.3 Hz, 1H), 4.53−4.42 (m, 2H),
4.30 (d, J = 3.8 Hz, 1H), 4.17 (d, J = 3.5 Hz, 1H), 3.93−3.75 (m,
4H), 3.60 (d, J = 10.1 Hz, 1H), 3.39 (s, 1H), 3.25 (t, J = 7.0 Hz,
1H), 3.13−2.87 (m, 4H), 2.45−2.36 (m, 1H), 2.26 (t, J = 4.4 Hz,
3H), 2.18−2.12 (m, 1H), 1.99 (dq, J = 8.5, 4.3 Hz, 1H), 1.86 (s,
2H), 1.58 (s, 2H), 1.30 (d, J = 3.8 Hz, 6H), 1.24 (t, J = 3.0 Hz, 6H),
1.07 (d, J = 5.7 Hz, 3H), 0.87 (d, J = 6.0 Hz, 3H). UPLC−MS
calculated for C52H59ClFN6O7 [M + H]+: 933.41, found 933.42.
UPLC retention time: 6.7 min, purity >95%.
4.7 Hz, 1H), 1.86 (s, 2H), 1.56 (qd, J = 9.2, 6.2, 4.7 Hz, 1H), 1.30
(s, 6H), 1.24 (s, 6H), 1.07 (dd, J = 6.5, 1.8 Hz, 3H), 0.90−0.82 (m,
3H). HRMS calculated for C53H59ClN7O7 [M + H]+: 940.4164,
found 940.4151. UPLC retention time: 6.2 min, purity >95%.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-1-(4-ethynylphenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-
2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide
1
(21). H NMR (400 MHz, MeOD-d4) δ 7.77 (dd, J = 19.2, 8.3 Hz,
4H), 7.49 (dd, J = 17.7, 7.0 Hz, 4H), 7.38 (d, J = 8.0 Hz, 2H), 7.15
(d, J = 2.4 Hz, 1H), 7.00 (dd, J = 8.7, 2.4 Hz, 1H), 6.24 (d, J = 4.1
Hz, 1H), 5.37 (s, 1H), 4.53−4.45 (m, 2H), 4.31 (s, 1H), 4.18 (s,
1H), 3.97−3.78 (m, 5H), 3.62 (s, 1H), 3.50 (dd, J = 12.5, 7.8 Hz,
2H), 3.15−2.91 (m, 5H), 2.42 (s, 1H), 2.27 (d, J = 4.2 Hz, 3H),
2.18 (d, J = 12.4 Hz, 1H), 2.00 (d, J = 4.0 Hz, 1H), 1.85 (s, 1H),
1.58 (s, 1H), 1.31 (s, 6H), 1.25 (s, 6H), 1.08 (d, J = 6.2 Hz, 3H),
0.92−0.84 (m, 3H). HRMS calculated for C54H60ClN6O7 [M + H]+:
939.4212, found 939.4194. UPLC retention time: 6.4 min, purity
>95%.
(2S,4R)-1-(Acetyl-L-valyl)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)-
ethynyl)piperidin-1-yl)-3-oxo-1-phenylpropyl)-4-hydroxypyrroli-
1
dine-2-carboxamide (22). H NMR (400 MHz, MeOD-d4) δ 7.79
(d, J = 8.1 Hz, 2H), 7.73 (d, J = 8.7 Hz, 1H), 7.49 (dd, J = 8.4, 2.1
Hz, 2H), 7.36 (qd, J = 8.2, 1.8 Hz, 4H), 7.26 (t, J = 6.9 Hz, 1H),
7.14 (d, J = 2.4 Hz, 1H), 6.99 (dd, J = 8.8, 2.4 Hz, 1H), 5.39−5.29
(m, 1H), 4.56 (t, J = 8.2 Hz, 1H), 4.50−4.43 (m, 2H), 4.30 (s, 1H),
4.17 (s, 1H), 3.98−3.85 (m, 2H), 3.76 (dt, J = 12.7, 6.4 Hz, 2H),
3.23 (ddd, J = 12.7, 9.0, 3.2 Hz, 1H), 3.02 (dq, J = 11.4, 8.3, 7.3 Hz,
2H), 2.94−2.85 (m, 1H), 2.23−2.08 (m, 2H), 2.01 (d, J = 3.2 Hz,
3H), 1.96 (dd, J = 8.7, 4.4 Hz, 1H), 1.83 (qd, J = 10.8, 9.9, 4.8 Hz,
2H), 1.55 (dtt, J = 18.1, 9.0, 4.4 Hz, 2H), 1.43−1.35 (m, 1H), 1.27
(d, J = 23.7 Hz, 12H), 1.04−0.96 (m, 6H). UPLC−MS calculated
for C50H60ClN6O7 [M + H]+: 891.42, found 891.45. UPLC retention
time: 6.0 min, purity >95%.
General Procedure for the Synthesis of Compound (41).
DIPEA (5 equiv) and HATU (1.2 equiv) were added to a solution of
the compound 29 (1 mmol) and 38 (1.1 equiv) in DMF (2 mL).
After 30 min at rt, the mixture was subjected to HPLC purification to
afford compound 39 with 88% yield.
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-1-(4-chlorophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-
2-(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide
1
(18). H NMR (400 MHz, MeOD-d4) δ 7.79 (d, J = 7.9 Hz, 2H),
7.72 (dd, J = 7.7, 3.2 Hz, 3H), 7.58 (d, J = 8.2 Hz, 2H), 7.51 (dt, J =
8.2, 4.0 Hz, 2H), 7.13 (d, J = 2.3 Hz, 1H), 6.98 (dd, J = 8.7, 2.3 Hz,
1H), 6.25 (dd, J = 10.2, 3.1 Hz, 1H), 5.46−5.33 (m, 1H), 4.49 (dd, J
= 15.9, 7.5 Hz, 2H), 4.29 (s, 1H), 4.17 (s, 1H), 4.00−3.84 (m, 2H),
3.84−3.73 (m, 2H), 3.61 (d, J = 10.7 Hz, 1H), 3.39 (dd, J = 8.3, 4.9
Hz, 1H), 3.30−3.18 (m, 1H), 3.08 (dtt, J = 27.8, 15.5, 6.7 Hz, 2H),
2.93 (s, 1H), 2.42 (tt, J = 15.5, 7.4 Hz, 1H), 2.26 (d, J = 2.9 Hz,
3H), 2.22−2.12 (m, 1H), 1.98 (dq, J = 13.1, 4.5 Hz, 1H), 1.93−1.80
(m, 2H), 1.66−1.46 (m, 2H), 1.30 (s, 6H), 1.24 (s, 6H), 1.07 (d, J =
6.1 Hz, 3H), 0.93−0.83 (m, 3H). HRMS calculated for
C52H59Cl2N6O7 [M + H]+: 949.3822, found 949.3795. UPLC
retention time: 6.5 min, purity >95%.
(2S,4R)-N-((S)-1-(4-Bromophenyl)-3-(4-((4-(((1r,3r)-3-(3-chloro-4-
cyanophenoxy)-2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)-
ethynyl)piperidin-1-yl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-2-
(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (19).
1H NMR (400 MHz, MeOD-d4) δ 7.79 (d, J = 8.0 Hz, 2H), 7.73
(dd, J = 8.7, 2.0 Hz, 1H), 7.54−7.48 (m, 4H), 7.35−7.28 (m, 2H),
7.13 (d, J = 2.2 Hz, 1H), 6.99 (dt, J = 8.9, 2.3 Hz, 1H), 6.23 (d, J =
3.7 Hz, 1H), 5.37−5.24 (m, 1H), 4.53−4.40 (m, 2H), 4.30 (d, J =
1.9 Hz, 1H), 4.17 (s, 1H), 3.99−3.75 (m, 4H), 3.59 (d, J = 10.2 Hz,
1H), 3.44−3.35 (m, 1H), 3.31−3.20 (m, 1H), 3.09−2.87 (m, 3H),
2.41 (dq, J = 20.6, 7.3 Hz, 1H), 2.28−2.21 (m, 3H), 2.15 (t, J = 10.9
Hz, 1H), 1.98 (ddd, J = 13.6, 8.9, 4.7 Hz, 1H), 1.87 (d, J = 13.5 Hz,
2H), 1.67−1.44 (m, 2H), 1.30 (s, 6H), 1.24 (d, J = 1.7 Hz, 6H),
1.06 (d, J = 6.5 Hz, 3H), 0.88 (dd, J = 8.4, 6.3 Hz, 3H). UPLC−MS
calculated for C52H59BrClN6O7 [M + H]+: 993.33, found 993.31.
UPLC retention time: 6.6 min, purity >95%.
K2CO3 (1.2 equiv) and KI (0.2 equiv) were added to a solution of
the intermediate 39 (0.05 mmol) and 40 (1.2 equiv) in CH3CN.
After stirring the mixture overnight at 100 °C, the solvents were
evaporated under reduced pressure to afford the corresponding crude
compound 41, which was purified by flash column chromatography
(DCM/MeOH = 20:1) with 83% yield.
N-((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-2,2,4,4-tetramethylcy-
clobutyl)-1-(2-hydroxyethyl)-1H-pyrazole-4-carboxamide (41). 1H
NMR (400 MHz, MeOD-d4) δ 8.23 (d, J = 0.7 Hz, 1H), 8.02 (d, J =
0.7 Hz, 1H), 7.74 (d, J = 8.7 Hz, 1H), 7.14 (d, J = 2.4 Hz, 1H), 7.00
(dd, J = 8.7, 2.4 Hz, 1H), 4.28 (s, 2H), 4.17 (d, J = 1.0 Hz, 1H), 3.93
(t, J = 5.2 Hz, 2H), 3.33 (p, J = 1.6 Hz, 2H), 1.30 (s, 6H), 1.22 (s,
6H). UPLC−MS calculated for C21H26ClN4O3 [M + H]+: 417.17,
found 417.18. UPLC retention time: 4.4 min, purity >95%.
Cell Lines and Cell Culture. All the LNCaP, VCaP, and 22Rv1
cells used were purchased from the American Type Culture
Collection (ATCC). LNCaP and 22Rv1 cells were grown in RPMI
1640 (Invitrogen), and VCaP cells were grown in DMEM
(Dulbecco’s modified Eagle’s medium) with Glutamax (Invitrogen).
All of the cells were supplemented with 10% fetal bovine serum
(Invitrogen) at 37 °C in a humidified 5% CO2 incubator. Cell
viability was evaluated by a WST-8 assay (Dojindo) following the
manufacturer’s instructions. Western blotting analysis was performed
as previously described.43,44
(2S,4R)-N-((S)-3-(4-((4-(((1r,3r)-3-(3-Chloro-4-cyanophenoxy)-
2,2,4,4-tetramethylcyclobutyl)carbamoyl)phenyl)ethynyl)piperidin-
1-yl)-1-(4-cyanophenyl)-3-oxopropyl)-4-hydroxy-1-((R)-3-methyl-2-
(3-methylisoxazol-5-yl)butanoyl)pyrrolidine-2-carboxamide (20).
1H NMR (400 MHz, MeOD-d4) δ 7.79 (d, J = 8.2 Hz, 2H), 7.73
(d, J = 8.7 Hz, 1H), 7.50 (dd, J = 8.4, 2.4 Hz, 2H), 7.40−7.33 (m,
4H), 7.13 (d, J = 2.4 Hz, 1H), 6.98 (dd, J = 8.8, 2.4 Hz, 1H), 6.23
(d, J = 4.2 Hz, 1H), 5.39−5.29 (m, 1H), 4.55−4.42 (m, 2H), 4.29 (s,
1H), 4.17 (s, 1H), 4.01−3.86 (m, 2H), 3.83−3.74 (m, 2H), 3.60 (d,
J = 10.9 Hz, 1H), 3.43−3.35 (m, 1H), 3.30−3.18 (m, 1H), 3.13−
2.97 (m, 2H), 2.90 (dt, J = 11.0, 5.6 Hz, 1H), 2.48−2.36 (m, 1H),
2.26 (d, J = 4.1 Hz, 3H), 2.20−2.11 (m, 1H), 1.97 (ddt, J = 13.2, 9.2,
Quantitative Real-Time Polymerase Chain Reaction (qRT-
PCR). Real-time PCR was performed using a QuantStudio 7 Flex
Real-Time PCR System as described previously.23,45 RNA was
purified using the Qiagen RNase-Free DNase set, and then after
quantification, the extracted RNA was converted to cDNA using a
High Capacity RNA-to-cDNA Kit from Applied Biosystems (Thermo
Fisher Scientific). The levels of AR, TMPRSS2, FKBP5, PSA(KLK3),
K
DOI: 10.1021/acs.jmedchem.9b01393
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Journal of Medicinal Chemistry
Article
1
and GAPDH were quantified using a TaqMan Fast Advanced Master
Mix from Applied Biosystems. The level of gene expression was
evaluated using a comparative CT method, which compares the CT
value to GAPDH (ΔCT) and then to vehicle control (ΔΔCT).
Cloning and Purification of VHL-ElonginBC Complex. The
DNA sequence of VHL (coding for residues 54−213) was
constructed by PCR and inserted into a His-TEV expression vector46
using ligation-independent cloning. The DNA sequences of Elongin
B (encoding residues 1−118) and Elongin C (encoding residues 1−
96) were constructed by PCR and inserted into pCDFDuet 1 using a
Gibson assembly.47 BL21(DE3) cells were transformed simulta-
neously with both plasmids and grown in Terrific Broth at 37 °C
until an OD600 of 1.2. The cells were induced overnight with 0.4 mM
IPTG at 24 °C. Pelleted cells were freeze-thawed and then
resuspended in 20 mM Tris-HCl (pH 7.0), 200 mM NaCl, and
0.1% β-mercaptoethanol (bME) containing protease inhibitors. The
cell suspension was lysed by sonication, and the debris was removed
via centrifugation. The supernatant was incubated at 4 °C for 1 h
with Ni-NTA (Qiagen) prewashed in 20 mM Tris-HCl (pH 7.0),
200 mM NaCl, and 10 mM imidazole. The protein complex was
eluted in 20 mM Tris-HCl (pH 7.0), 200 mM NaCl, and 300 mM
imidazole, dialyzed into 20 mM Tris-HCl (pH 7.0), 150 mM NaCl,
and 0.01% bME, and incubated with TEV protease overnight at 4 °C.
The protein sample was reapplied to the Ni-NTA column to remove
the His-tag. The flow through containing the VHL complex was
diluted to 75 mM NaCl and applied to a HiTrap Q column (GE
Healthcare). The sample was eluted with a salt gradient (0.075−1 M
NaCl), concentrated, and further purified on a Superdex S75 column
(GE Healthcare) pre-equilibrated with 20 mM Bis-Tris (pH 7.0),
150 mM NaCl, and 1 mM DTT. Samples were aliquoted and stored
at −80 °C.
Binding Affinities of VHL Ligands to VHL-ElonginBC
Complex Protein. The IC50 and Ki values of compounds were
determined in competitive binding experiments. Mixtures of 5 μL of
solutions of compounds in dimethyl sulfoxide (DMSO) and 95 μL of
a preincubated protein/tracer complex solution were added into
assay plates, which were incubated at room temperature for 60 min
with gentle shaking. The final concentrations of VHL protein and
fluorescent probe were both 5 nM. Negative controls containing the
protein/probe complex only (equivalent to 0% inhibition) and
positive controls containing only free probes (equivalent to 100%
inhibition) were included in each assay plate. FP values in
millipolarization units (mP) were measured using the Infinite M-
1000 plate reader (Tecan U.S., Research Triangle Park, NC) in
Microfluor 1 96-well, black, round-bottom plates (Thermo Scientific,
Waltham, MA) at an excitation wavelength of 485 nm and an
emission wavelength of 530 nm. IC50 values were determined by
nonlinear regression fitting of the competition curves. Ki values of
competitive inhibitors were obtained directly by nonlinear regression
fitting based on the KD values of the probe and concentrations of the
protein and probe in the competitive assays. All the FP competitive
experiments were performed in duplicate in three independent
experiments.
of ARD-266 in LNCaP, VCaP, and 22Rv1 cells; H
NMR, 13C NMR, and UPLC-MS spectrum of
compound ARD-266; and chemical data for VHL-a to
VHL-h (PDF)
Molecular string files for all the final target compounds
(CSV)
AUTHOR INFORMATION
Corresponding Author
*E-mail: shaomeng@umich.edu. Phone: 1-734-615-0362.
ORCID
Shaomeng Wang: 0000-0002-8782-6950
Author Contributions
X.H. and L.Z. contributed equally to this work.
■
Notes
The authors declare the following competing financial
interest(s): The University of Michigan has filed a patent
application on these AR degraders, which has been licensed to
Oncopia Therapeutics Inc. S. Wang, X. Han, C. Wang, C. Qin,
W. Xiang, T. Xu, and C. Yang are co-inventors on these
patents. The University of Michigan has received a research
contract from Oncopia. S.W. is a co-founder of Oncopia, owns
stock in Oncopia, and is a paid consultant of Oncopia.
ACKNOWLEDGMENTS
■
This study was supported in part by funding from Oncopia
Therapeutics, LLC, and the University of Michigan
Comprehensive Cancer Center Core Grant from the National
Cancer Institute, NIH (grant P30CA046592).
ABBREVIATIONS
■
AR, androgen receptor; mCRPC, metastatic castration-
resistant prostate cancer; PROTAC, proteolysis targeting
chimera; ER, estrogen receptor; DC50, concentration needed
to degrade 50% of AR protein; Dmax, maximal levels of
degradation; FP, fluorescence polarization; ppm, parts per
million; TMS, tetramethylsilane; MS, mass spectral; ATCC,
American Type Culture Collection; qRT-PCR, quantitative
real-time polymerase chain reaction
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ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.9b01393.
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Western blotting analysis of AR proteins in LNCaP cells
treated with all AR degraders; Western blotting analysis
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