Pyrazol-1-yl-propanamides as SARD and Pan-Antagonists for the Treatment of Enzalutamide-Resistant Prostate Cancer

Article abstract of DOI:10.1021/acs.jmedchem.0c00943

We report herein the design, synthesis, and pharmacological characterization of a library of novel aryl pyrazol-1-yl-propanamides as selective androgen receptor degraders (SARDs) and pan-Antagonists that exert broad-scope AR antagonism. Pharmacological ev

Full text of DOI:10.1021/acs.jmedchem.0c00943

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Pyrazol-1-yl-propanamides as SARD and Pan-Antagonists for the
Treatment of Enzalutamide-Resistant Prostate Cancer
Yali He, Dong-Jin Hwang, Suriyan Ponnusamy, Thirumagal Thiyagarajan, Michael L. Mohler,
Ramesh Narayanan, and Duane D. Miller*
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sı Supporting Information
ABSTRACT: We report herein the design, synthesis, and pharmacological characterization of a library of novel aryl pyrazol-1-yl-
propanamides as selective androgen receptor degraders (SARDs) and pan-antagonists that exert broad-scope AR antagonism.
Pharmacological evaluation demonstrated that introducing a pyrazole moiety as the B-ring structural element in the common A-
ring−linkage−B-ring nonsteroidal antiandrogens’ general pharmacophore allowed the development of a new scaffold of small
molecules with unique SARD and pan-antagonist activities even compared to our recently published AF-1 binding SARDs such as
UT-155 (9) and UT-34 (10). Novel B-ring pyrazoles exhibited potent AR antagonist activities, including promising distribution,
metabolism, and pharmacokinetic properties, and broad-spectrum AR antagonist properties, including potent in vivo antitumor
activity. 26a was able to induce an 80% tumor growth inhibition of xenografts derived from the enzalutamide-resistant (Enz-R)
VCaP cell line. These results represent an advancement toward the development of novel AR antagonists for the treatment of Enz-R
prostate cancer.
1
. INTRODUCTION
synthesis inhibition such as abiraterone acetate (7) plus
15
prednisone (Figure 1). Secondary hormonal suppression,
that is, added to ADT, has been approved to treat castration-
Prostate cancer (PC) is the second leading cause of cancer-
related death, after lung cancer, in American men. Globally, both
the number of PC cases and mortality have increased
significantly. Longer life expectancy and increasing geriatric
male population are some of the contributors to increasing PC
incidence. PC depends on the activation of androgen receptor
sensitive PC (CSPC) or castration-resistant prostate cancer
16−18
3
,4
(CRPC),
with the approval trend toward their use earlier in
the natural history of the disease in order to more effectively
19
delay disease progression.
ADT is initially effective for advanced PCs; however,
sustained ADT treatment, in combination with antiandrogens,
often only stabilizes the disease for 2−3 years before PC
becomes refractory, resulting in a more aggressive CRPC tumor
phenotype where tumors become resistant to (ongoing ADT
(
AR) signaling for its development, progression, growth, and
5
−7
survival.
Approximately 20−40% of PC patients treated with radiation
4
and radical prostatectomy will experience tumor recurrence.
Once the tumor recurs, androgen ablation therapy or androgen
deprivation therapy (ADT) is the standard of care for most
patients. ADT is achieved through surgical castration (orchi-
ectomy) or chemical castration (injection of gonadotropin-
releasing hormone agonist or antagonist), both of which cause a
reduction in testosterone biosynthesis by testes. In addition to
ADT, secondary hormonal suppression is provided by direct
competitive ligand binding domain (LBD)-directed AR
20
and) secondary hormonal therapies. Resistance to any one of
2
, 4, 5, or 7 can emerge just months after initiation and studies
suggest that 6 (darolutamide) may behave similarly in the CPRC
Received: June 2, 2020
8
antagonists termed as antiandrogens such as flutamide (1),
bicalutamide (2), nilutamide (3), enzalutamide (4),
apalutamide (5),
9
10
1
1,12
13,14
or darolutamide (6)
or androgen
©
XXXX American Chemical Society
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A
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Figure 1. Overview of direct antiandrogens (1−6), an indirect LBD antagonist (7), and our preclinical SARDs (8−10). Clinically approved agents
include first-generation (1−3) and second-generation (4−6) antiandrogens and an indirect androgen synthesis inhibitor (7). Also shown are our
SARDs (8−10), which are pan-antagonists in preclinical development for the treatment of antiandrogen-resistant PC.
1
4
population (6 approved for mCSPC). Despite resistance to
secondary hormonal therapies in CRPC whether direct (1−6)
or indirect (7), AR signaling continues to be fundamental for
tumor growth and disease progression. Correspondingly, novel
mechanisms to inhibit the AR axis are needed in hormone-
states with sufficient potency to maintain suppression of the AR
axis as PC becomes progressively more refractory to treatment.
Binding to a non-LBD site and degradation of the AR protein
are promising preclinical approaches to rationally target CRPC;
42−46
however, a clinical proof of concept is still needed.
2
1
Degradation of AR can be achieved by genetic knockdown
technologies, such as antisense oligonucleotides, RNA interfer-
ence, and DNA editing. Despite genetic approaches having great
therapeutic potential, it remains clinically challenging because of
technical difficulties in delivering oligonucleotides (polyanionic
macromolecules) to the prostate and metastatic tumors.
Furthermore, oligonucleotide uptake into the tumor cells is
resistant PCs.
Although the exact mechanisms of CRPC progression are not
always known clinically nor are they mutually exclusive,
preclinical and clinical research has demonstrated numerous
contributing factors to the emergence of CRPC that include (1)
compensatory production of intratumoral androgens (e.g., DHT
5
,22,23
synthesized from adrenal precursors),
(2) AR gene
(3) AR LBD point
47,48
2
4−26
poor.
Alternatively, targeted destruction of the AR by
amplifications and overexpression,
2
5,27,28
protein knockdown technologies which degrade AR via the
ubiquitin proteasome system (UPS) remains a promising group
of options yet to be tested definitively in the clinical
mutations,
tory proteins,
(4) alterations in the expression of coregula-
2
9,30
(5) ligand-independent activation of AR,31−35
(
6) constitutively active truncated AR splice variants (AR
SVs), and (7) induction of intracrine androgen metabolic
enzymes. Direct and indirect antiandrogen therapies all
target AR at the LBD and eventually fail because of the resistance
2,44,49,50
3
6
setting.
2
0,37,38
In recent antitumor studies, our laboratory has reported the
discovery and characterization of UT-69 (8) and UT-155 (9) as
first-in-class AR antagonists that selectively inhibit tumor
39
mechanisms mentioned above. The development of AR
antagonists for CRPC with novel mechanisms of action that
are capable of durably treating patients with resistance to 2, 4, 5,
and 7 (cross-resistance of 7 to 4 and 5 is common; 1 and 3 are
rarely used) or darolutamide (6) (approved in 2019; patterns of
resistance to 6 are still emerging) is an urgent need.
To provide clinical benefit for CRPC or to circumvent the
emergence of CRPC, the next generation of AR-targeted
therapeutics ideally should be able to bind to novel and/or
multiple domains of the AR and inhibit a broad scope of AR
functions across the broad scope of AR sequences present and
growth and degrade the AR (full-length) and AR SVs
1
(
truncated) within these tumors. We also reported a novel
series of aryl indol-1-yl propanamides and aryl indolin-1-yl
propanamides as selective androgen receptor degraders
51
(
SARDs). These SARD activities were mediated through the
1,2
UPS as determined by UPS inhibitor studies. Among these
SARDs, 8 and 9 bind both to the N-terminal domain (NTD) at
the transcriptional activation units (Tau)-1 and -5 (Tau-1 and
Tau-5) of the AF-1 domain, which has not been targeted for
degradation previously, and additionally, these SARDs com-
petitively bind the LBD. Recently, an aryl pyrazol-1-yl
propanamide (10; termed UT-34 therein) was demonstrated
to bind the same Tau-1 and Tau-5 NTD sites but have improved
pharmacokinetic (PK) properties and was characterized to have
4
0,41
emerging in the heavily pretreated CPRC population.
novel antagonists ideally will maintain activity in wild-type (wt),
point mutant, AR SVs, and/or AR overexpressing pathogenic
Such
B
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Table 1. Structures of Pyrazol-1-yl-propanamide AR Antagonists
a
Scheme 1. Synthesis of Pyrazol-1-yl-propanamides 16a−16x
a
Reagents and conditions: (a) 1. SOCl in THF, −10 to 0 °C. 2. Et N in THF, −10 to 0 °C, and then to 50 °C, 2−3 h; (b) 2-butanone, K CO ,
2
3
2
3
reflux; and (c) NaH in THF, 0 °C to rt.
unprecedented xenograft efficacy in the models of enzalutamide
exhibited in vitro inhibitory potency in screening assays (e.g.,
LBD binding, transcriptional inhibition, AR degradation, and
antiproliferative assays) and greater in vivo efficacy (Hershberger
assay and various AR-dependent CPRC xenografts) than the
2
(4) resistance (Enz-R).
All our preclinical SARDs reported so far have degraded AR
and inhibited AR function, and our leads from each scaffold
C
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Table 2. In Vitro AR Activity of 16a−16x (Series I) and Approved Antiandrogens
binding (K )/transactivation (IC )
i
50
(μM)
SARD activity (% degradation)
a
b
c
c
compound ID (R-group)
Ki (DHT = 1 nM)
IC50
full length (LNCaP) at 1 μM
splice variant (22RV1) at 10 μM
F.L. DC50 (μM)
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
1
2
4
5
6
6a (4-H)
0 (4-F)
6b (3-F)
6c (4-Cl)
6d (4-Br)
6e (4-I)
6f (4-COCH3)
6g (4-CF3)
6h (3-CF3)
6i (4-CN)
6j (4-NO2)
6k (4-OCH3)
6l (4-CH3)
6m (4-phenyl)
6n (3-phenyl)
6o [4-(4-fluorophenyl)]
6p [3-(4-fluorophenyl)]
6q (4-ethynyl)
6r [4-(4-OH-but-1-yn-1-yl)]
6s (4-carbamoyl)
6t (4-NH2)
7.398
>10
0.821
>10
1.442
0.199
0.220
0.136
0.427
2.038
0.758
0.071
0.205
0.045
0.036
no effect
8.087
1.152
4.770
0.969
0
0
d
100
82
71
42
100
73
34
0
0.74
0.47
0.97
>10
N.A.
e
e
e
N.A.
N.A.
1.056
0.898
0.512
1.499
2.225
N.A.
1.552
>10
3.660
0.612
>10
N.A.
>10
N.A.
0.223
1.382
>10
>10
>10
30
80
67
90
20
0
N.A.
0
0
72
54
7, 28
51, 80
N.A.
N.A.
100
54
100
N.A.
0
N.A.
0
0
0.29
0.73
0.32
0
81
0.86
0.99
1.069
0.276
f
N.A.
N.A.
N.A.
N.A.
0
N.A.
N.A.
N.A.
f
no effect
no effect
agonist
1.153
no effect
no effect
0.827
0.97
6u (4-NHCOOtBu)
6v (4-NHCOCH3)
6w (4-NHCOCH Cl)
6x (4-NHCOOCH )
(R-bicalutamide)
(enzalutamide)
(apalutamide)
(darolutamide)
20
N.A.
N.A.
2
3
g
0.509
3.641
1.452
0.248
0.216
g
g
0.160
0.065
h
h
0.011
N.A.
N.A.
a
3
AR binding was determined by competitive binding of 1 nM [ H] MIB to recombinant LBD of wild-type AR (wtAR). DHT was used in each
b
experiment as a standard agent and the values are normalized to DHT, with the IC50 of DHT taken as 1 nM. Inhibition of transactivation was
determined by transfecting HEK-293 cells with full-length wtAR, GRE-LUC, and CMV-renilla luciferase for transfection control. Cells were treated
2
4 h after transfection with a dose response of compounds (1 pM to 10 μM) in the presence of 0.1 nM R1881 (antagonist mode) or in the absence
of R1881 (agonist mode). Luciferase assay was performed 24 h after treatment using a dual-luciferase (firefly and Renilla) assay kit (Promega,
Madison, WI). SARD activity was assayed by treating LNCaP or 22RV1 cells for determining FL AR (at 1 μM of antagonist) or SV AR (at 10 μM
c
of antagonist) protein levels, respectively. Cells were maintained in charcoal-stripped, serum-containing medium for 48 h and treated with the
indicated doses of antagonist for 24 h in the presence of 0.1 nM R1881 (agonist). Cells were harvested and Western blot for AR was performed
using AR-N20 or PG-21 antibody that is directed toward the NTD of AR and actin (internal control for protein loading). The AR FL and AR SV
d
bands were quantified and normalized to actin bands and represented as percent inhibition from vehicle-treated cells. Result was reported in the
2
e
f
literature in the same assay as described here. N.A. indicates data not available. The two values indicate SARD assays run with 1 and 10 μM of
g
56 h
antagonist. Transcriptional activation was performed in the same assay in antagonist mode and the IC values are reported.
and wtAR inhibition of transactivation were reported in the literature for the mixture of diastereomers of darolutamide.
Binding affinity
5
0
57
approved AR antagonists. To discover preclinical leads to
advance to clinical testing, herein, we explored the structure−
activity relationships (SARs) within the pyrazol-1-yl series with
the goal of improving upon the unprecedented activities of 10.
the pyrazole B-ring (Series III), or modifications of the linkage
moiety (Series IV), as shown in Table 1.
2
.1.1. Series I: Monosubstitutions of the Pyrazole Moiety
B-Ring). The syntheses of 16a−16x were performed according
to Scheme 1. Commercially available (R)-3-bromo-2-hydroxy-
-methylpropanoic acid (11) was treated with SOCl2 to
(
2
2
. RESULTS AND DISCUSSION
.1. Chemistry. We designed and synthesized a series of
transform acid 11 to acid chloride (R)-3-bromo-2-hydroxy-2-
methylpropanoyl chloride (not shown), which reacted with
aniline (12) to afford the bromide compound (13).
Intermediate 13 has also been synthesized previously by Tucker
(PMID: 3625091 and 3361581) and our lab (PMID: 8867996).
2
pyrazol-1-yl-propanamide compounds similar to 10 with varying
2
mono-substituents of the pyrazole B-ring (Series I), variations
of the aromatic A-ring (Series II), varying the disubstituents of
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Table 3. In Vitro AR Activity of 21a−21j (Series II)
a
b
AR binding, transactivation, and degradation assays were performed and values are reported as described in Table 2. N.A. indicates data not
available.
Under basic conditions (e.g., K CO ), 13 was transformed to a
μM) > 16d (4-Br, 0.427 μM) > 16a (4-H, 1.442 μM) > 16e (4-I,
2.038 μM). Compounds with 4-substitution exhibited more
potent AR inhibitory activity than that of their 3-substitution
counterparts. For example, compare 10 (4-F) to 16b (3-F), 16g
(4-CF ) to 16h (3-CF ), 16m (4-phenyl) to 16n (3-phenyl),
2
3
key oxirane intermediate (14). Alkylation of commercially
available pyrazoles (15) by their reaction with 14 afforded the
pyrazol-1-yl-propanamides 16a−16x, as shown in Scheme 1.
Series I and all other compounds tested herein were screened in
3
3
vitro for AR LBD binding (K ), inhibition of transactivation
and 16o [4-(4-fluorophenyl)] to 16p [3-(4-fluorophenyl)],
i
(
IC ), AR degradation (% degradation) of full-length (AR FL in
respectively.
50
LNCaP cells) and SV (AR SV in 22RV1 cells) ARs in PC cell
lines, and degradation potency (DC50 values) in LNCaP cells
In general, the stronger the electron-withdrawing group
(EWGs) is on the pyrazole ring, the more potent is the AR
inhibitory activity, with the potency order of 16j (4-NO 0.036
(Table 2). Optimal SARDs and pan-antagonists are compounds
2
,
that potently inhibit AR transactivation (IC ) and optionally
μM) > 16i (4-CN, 0.045 μM) > 16g (4-CF 0.071 μM) > 16h
50
3,
degrade AR FL or AR SV and possess in vivo efficacy in models of
antiandrogen-resistant CRPC of greater potency than 10.
Compound 16a, which has no substitution on the pyrazole
ring, possessed weak AR inhibitory activity with an IC value of
(3-CF 0.205 μM) > 16q (4-ethynyl, 0.276 μM) > 16f (4-
3
,
COCH 0.758 μM). Compounds bearing an electron-donating
3
,
group on the pyrazole ring showed low-potency AR inhibitory
activity (16l, 16u, and 16x), no AR inhibitory activity (16k, 16s,
16v, and 16w), or even AR agonist activity (16t which is 4-
50
1
.442 μM. AR inhibition in vitro is defined as the ability to inhibit
R1881-induced wtAR transcriptional activity as measured by the
NH ). Interestingly, 16r bearing [4-(4-OH-but-1-yn-1-yl)] on
2
luciferase assay [see values in the transactivation (IC ) column
the pyrazole ring exhibited no AR inhibitory activity but showed
51% AR full-length protein degradation activity.
With regard to SARD activity, substitution of the pyrazole ring
seems to be necessary (16a; 0%/0% in % degradation) but some
50
of Table 2], referred to as in vitro AR inhibition herein.
Introducing a halogen on the pyrazole significantly increased the
AR inhibitory activity, except for the 4-iodo compound 16e. The
order of AR inhibitory potency with halogen substitution was
electron-donating groups such as 4-OCH of 16k and 4-phenyl
3
1
6c (4-Cl, 0.136 μM) > 10 (4-F, 0.199 μM) > 16b (3-F, 0.220
or 3-phenyl in 16m and 16n are inactive. Like AR inhibitory
E
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Table 4. In Vitro AR Activity of 26a−26h (Series III)
binding (K )/transactivation
i
(
IC ) (μM)
SARD activity (% degradation)
5
0
a
splice variant (22RV1) at
F.L. DC50
(μM)
a
a
a
ID
X
R2
R3
Ki (DHT = 1 nM)
IC50
full length (LNCaP) at 1 μM, 10 μM
10 μM
2
6a
6b
6c
6d
6e
6f
CH
CH
CH
CH
CH
N
F
F
Br
Cl
Br
F
Br
phenyl
Br
0.607
0.601
0.202
1.345
4.935
0.567
0.084
0.285
0.181
0.332
0.138
0.035
5.481
0.579
70
N.A.
41, 23
41, 83
N.A.
8, 15
40, 80
9, 55
80
toxic
32
N.A.
N.A.
N.A.
N.A.
N.A.
0.86
c
2
2
2
2
2
2
2
4-F-phenyl
CN
CH3
Cl
Br
CN
b
b
c
c
c
b
c
c
b
c
6g
6h
N
N
N.A.
N.A.
c
b
c
CN
N.A.
a
b
AR binding, transactivation, and degradation assays were performed and values are reported as described in Table 2. The two values indicate the
c
SARD assays run with 1 and 10 μM of antagonist. N.A. indicates data not available.
Table 5. In Vitro AR Activity of 29a−29f (Series IV)
a
b
AR binding, transactivation, and degradation assays were performed and values are reported as described in Table 2. N.A. indicates data not
c
available. The two values indicate SARD assays run with 1 and 10 μM of antagonist.
potency (discussed above), the strength of the EWGs and 4-
substitution seem to contribute favorably as seen in 10 (4-F;
its 4-position isomer 16o (4-(4-fluorophenyl)) with 54%/81%
versus 72%/0% degradation efficacies.
51
1
1
00%/100% for AR FL and AR SV efficacies), 16g (4-CF ; 80%/
00% efficacy), and 16i (4-CN; 90%/100% efficacy), whereas 3-
As seen previously, inhibitory potency (IC ) does not
3
50
always correlate with % degradation. For example, the most
substituted EWGs possessed slightly lower SARD activity as can
potent inhibitor 16j (4-NO ; 0.036 μM) was a poor degrader,
2
be seen in 16b (3-F; 82%/73% efficacy) and 16h (3-CF ; 67%/
also the most potent Series I halogen 16c (4-Cl; 0.136 μM)
3
5
4% efficacy). However, 16p (3-(4-fluorophenyl)) is superior to
demonstrated only moderate SARD activity (71%/34%). Also
F
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a
Scheme 2. Synthesis of Pyrazol-1-yl-propanamides 21a−21j
a
Reagents and conditions: (a) 1. SOCl in THF, −10 to 0 °C. 2. Et N in THF, −10 to 0 °C and then heat to 50 °C, 2−3 h; (b) 2-butanone,
2
3
K CO , reflux; and (c) NaH in THF, 0 °C to rt.
2
3
a
Scheme 3. Synthesis of Pyrazol-1-yl-propanamides 26a−26h
a
Reagents and conditions: (a) 1. SOCl in THF, −10 °C to 0 °C. 2. Et N in THF, −10 to 0 °C and then heated to 50 °C, 2−3 h; (b) 2-butanone,
2
3
K CO , and reflux; and (c) NaH in THF, 0 °C to rt.
2
3
5
1
as seen previously, LBD binding (K ) does not correlate with
possessing only 20% AR FL efficacy (N.A. for SV), but very
potent inhibition (0.036 μM) compared to LBD binding (2.225
μM), and 26f, which possessed only 8, 15% AR FL efficacy at 1
and 10 μM but extremely potent inhibition (0.035 μM), which is
>10-fold more potent than LBD binding (0.567 μM).
Therefore, we have reconsidered placing primary emphasis of
these molecules as AR degraders (i.e., SARDs) instead of placing
emphasis of their ability to inhibit, and in most cases degrade, all
AR forms tested to date. In an effort to determine the
contribution of AR SARD activity to the AR antagonism
observed, the degradation potency values (DC50 values) in
LNCaP cells have been reported here for the first time. These
values were approximately 4- to 10-fold greater than IC values
i
AR inhibitory potency (IC ) or SARD activity. For example,
nonbinders 10 and 16c (K values >10 μM) inhibited and
50
i
degraded, whereas nonbinder 16r degraded but was not an
inhibitor. At this point, it is not clear how to differentiate the
SARs of the AR inhibition and the SARD activity seen with these
noncanonical ligands. Further complicating the SAR of these
compounds is that the structural information for the AF-1 region
has not been elucidated, and AR antagonism observed may be
NTD-mediated for some compounds, while other compounds
may have contributions from both NTD and LBD. We are in the
process of learning about how our molecules interact with AR
and the importance of each binding site.
5
0
The SARs of % efficacy of SARD activity (considering AR FL
SARD and AR SV SARD activity in aggregate because of their
limitation as semiquantitative values) seems to correlate with the
(Tables 2−5), suggesting that SARD activity alone may not
explain the potent AR pan-antagonism of these compounds.
Hence, these broad-scope and potent noncanonical AR
antagonists are discussed as SARDs and pan-antagonists herein.
2.1.2. Series II: Modifications of Aromatic A-Ring.
Subsequent synthetic modifications were aimed at the
replacement of the A-ring of 10 to explore the effect of different
A-rings or A-ring substitutions on AR inhibition and SARD
activity. 21a−21j were prepared by the route shown in Scheme
AR inhibitory potency to some degree and LBD K to a [much]
i
51
lesser degree. As discussed previously, the screening profile is
intended to allow us to maximize FL and SV SARD efficacies and
AR inhibitory potency to provide the most potent and broad-
scope antagonists for testing in the models of antiandrogen-
resistant CRPC. In some cases, such as 16g, 16i, and 26a, high-
efficacy SARDs (>70% for both AR FL and AR SV) are potent
AR inhibitors (<0.100 μM IC ); and moderate efficacy SARDs
2. Treatment of acid 11 with SOCl provided the acid chloride
2
(R)-3-bromo-2-hydroxy-2-methylpropanoyl chloride (not
shown), which was reacted with various amines (17) under
50
were moderate potency inhibitors such as 16b, 16c, 16h, and
6c. However, as discussed herein, % SARD efficacy does not
2
basic Et N conditions to furnish bromoamides 18 with different
3
always correlate well with in vitro inhibitory potency or LBD
binding. As mentioned above, 16j was a poor degrader,
A-rings. Basic conditions (e.g., K CO ) transformed bromoa-
mides 18 to the oxirane intermediates 19, followed by coupling
2
3
G
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Article
a
Scheme 4. Synthesis of Pyrazol-1-yl-propanamides 29a−29f
a
Reagents and conditions: (a) 1. SOCl in THF, −10 to 0 °C. 2. Et N in THF, −10 to 0 °C and then heat to 50 °C, 2−3 h, and (b) NaH in THF,
2
3
0
°C to rt.
with various pyrazoles 20 under the sodium hydride basic
conditions to produce the target compounds 21a−21j, as shown
in Scheme 2. The compounds were tested in vitro as discussed
above for AR activity (Table 3).
monosubstituted analogues and retained or improved upon
degradation properties, supportive of further exploration of
disubstitution. Replacing a carbon (CH) with a nitrogen (N) in
the A-ring of 26a delivered the 3′-pyridino 26f, which was a very
potent AR inhibitor with an IC value of 0.035 μM but poor
For the 4-F pyrazole, replacing a carbon (CH) with a nitrogen
5
0
(
N) at the 3′-position of the A-ring, that is, 3′-pyridino
SARD activity (8, 15%/N.A. for FL/SV) (Table 4). Compounds
derivative of 10, delivered a more potent compound (21a) with
an AR inhibitory IC50 value of 0.062 μM compared to its
counterpart 10 (IC = 0.199 μM). However, in other instances,
26b−26e with disubstituents on pyrazole showed inhibitory
activity comparable to 10 (0.199 μM) with AR inhibitory IC
50
values in the order of 26e (3-Br, 4-Cl; 0.138 μM) > 26c (3-Br, 4-
CN; 0.202 μM) > 26b (3-Br, 4-(4-fluorophenyl); 0.285 μM) >
26d (3-Cl, 4-methyl); 0.332 μM). Compounds 26e (0.138 μM)
and 26c (0.202 μM) did not improve upon their monosub-
stituted analogues 16c (4-Cl; 0.136 μM) and 16i (4-CN; 0.045
μM). However, addition of halogens to 4-EDG pyrazoles at least
partially rescued the activity, for example, compare to 26b [3-Br,
4-(4-fluorophenyl); 0.285 μM] and 16o [4-(4-fluorophenyl);
0.969 μM] and 26d [(3-Cl, 4-methyl); 0.332 μM] and 16l (4-
methyl; 8.087 μM). Again, these results suggest that the EWG
strength of the pyrazole substituents contribute favorably to
inhibitory activity. Interestingly, the 3′-pyridino A-ring version
of 26c afforded a >10-fold less potent inhibitor 26g with an AR
inhibitory IC value of 5.481 μM despite 80% SARD efficacy in
5
0
3
′-pyridino derivatives were equipotent or less potent than their
phenyl A-ring analogues. The 3′-pyridino 21c (4-CN; 0.059
μM) showed almost equally potent AR inhibitory activity
compared to its A-ring phenyl analogue 16i (IC = 0.045 μM).
However, 3′-pyridino compounds 21b (4-CF ) and 21d (4-
NHCOOtBu) showed lower activity (IC values of 0.208 and
6
1
50
3
50
.108 μM, respectively) than their phenyl A-ring counterparts
6g and 16u. Other A-ring modifications of 10 decreased the AR
inhibitory activity and % degradation when compared to 10
0.199 μM; 100%/100%), including replacing the 3′-CF with a
(
3
3
′-Cl (21e; 0.427 μM; 42%/0% degradation), replacing the 4′-
CN with a 4′-NO (21f; partial agonist; N.A. % degradation),
2
and other modifications as in 21g−21j. Interestingly, unlike
5
0
other pyrazole propanamides, which show low or no AR LBD
AR FL (but no efficacy in AR SV). Introducing an extra bromo
(26g) or a phenyl (26h) group on the 3-position of the pyrazole
greatly decreased the inhibitory activity to 5.481 or 0.579 μM,
respectively, compared to 21c (4-CN; 0.059 μM). We found
that 3-Br and 4-CN substituents on pyrazole promoted a tighter
binding affinity (K ), we found that the combination of 4-CN
substituent in pyrazole and 3′-pyridino A-ring promoted the
tight LBD binding seen for 21c (K = 0.089 μM) but relatively
i
i
52
poor SARD activity (15%/N.A.).
2.1.3. Series III: Disubstitution of the Pyrazole B-Ring.
LBD binding (K = 0.202 μM) for 26c, and 3-F and 4-Br
i
Disubstitutions on pyrazole ring system might enhance
inhibitory activity or improve metabolic stability and provide a
better understanding of the SAR of the pyrazole ring. Series III
compounds 26a−26h were designed and synthesized utilizing
similar synthetic methods as in Schemes 1 and 2, as depicted in
Scheme 3, and tested for AR activity (Table 4).
substituents on pyrazole (26a) delivered a potent inhibitor (IC50
= 0.084 μM) while retaining the SARD activity in AR FL and AR
SV (70%/80%). Correspondingly, 26a and 26c were selected for
further study.
2.1.4. Series IV: Modification of the Linkage Moiety.
Subsequent modifications were aimed at introducing different
linkage/spacer groups between the aromatic A-ring and pyrazole
B-ring to explore the importance of the structure of the linking
element to improve the inhibitory potency and SARD activity.
Series IV compounds 29a−29f were designed and synthesized
utilizing similar synthetic methods as in Schemes 1−3, as
depicted in Scheme 4, and 29a−29f were tested for their AR
activity (Table 5 and Table 1).
Given our difficulty in improving the activity via A-ring
modification [see Series II (Table 3); all changes except for 21a
and 21c were not favorable], we expanded our attempts to
optimize the pyrazole ring system for inhibitory activity by 3,4-
disubstitution. 26a possessed two EWGs (3-F and 4-Br) on the
pyrazole ring and exhibited potent inhibitory activity (IC value
5
0
of 0.084 μM) and moderate- to high-efficacy AR FL and AR SV
degradation (70−80% degradation). Compound 26a improved
the AR inhibitory potency by 3−4-fold over the 3-F (16b; 0.220
μM; 82%/73%) and 4-Br (16d; 0.427 μM; 42%/0%)
1
Similar to the previously reported R-isomer of 9, switching
the chirality of 10 (the S-isomer) afforded the almost equipotent
29a (the R-isomer) with an AR inhibitory IC50 value of 0.192
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μM and slightly decreased to 84% degradation compared to 10
Table 6. In Vitro Metabolic Stability for Selected Compounds
in MLMs
(
2
100%). Removal of the 2-hydroxyl moiety from the 2-hydroxy-
-methyl propanamide linker of 10 produced 29b with reduced
a
MLM
AR inhibitory activity (0.462 μM) and 60%/70% FL/SV SARD
activity compared to 10 (0.199 μM; 100%/100%). Removal of
compound ID
T1/2 (min)
CLint (mL/min/mg)
b
2
-methyl and 2-hydroxy moieties from linkage of 10 to produce
10 (4-F)
77.96
64.07
>360
0.89
1.02
0
0
the linear propanamide 29c further decreased the AR inhibitory
and SARD activities. As an oxazolidin-2,4-dione linker variant of
16b (3-F)
16g (4-CF
16h (3-CF
)
3
1
0, 29d possessed groups similar to the amide and hydroxyl (as
)
3
330
the oxygen in the carbamate) groups of the linker. 29d still
showed activity but with substantially decreased AR inhibitory
16i (4-CN)
16m (4-phenyl)
21a (4-F)
>360
48.45
>360
0
14.31
0
(
1
1.131 μM) and SARD (18, 50%/N.A.) activities compared to
0 (0.199 μM; 100%/100%). Acylation of the 2-hydroxy AR
26a (3-F, 4-Br)
29b (4-F)
8 /9
4 (enzalutamide)
>360
>360
1.15/12.11
10.04 h
0
0
agonist 16t (4-NH ) produced 29e, which recovered some
2
b
b
antagonist activity with an AR inhibitory IC value of 0.901 μM,
208.8/57.26
86.3
50
c
d
whereas introducing a second amide into the linker and varying
the pyrazole attachment position as in 29f (see Table 1 for
structure) produced an agonist. Although the linker element was
not optimized in this initial SAR of Series IV, tolerance to chiral
center inversion was again observed (unlike structurally similar
propanamide SARMs), and it was established that there is no
absolute requirement for the 2-hydroxy-2-methylpropamide
linker for inhibitory and SARD activities.
a
Compounds were incubated together with MLMs with cofactors for
phases I and II provided, as described in the Experimental Section.
b
Reported previously in using the same method as in the
1,2 c
Experimental Section. T1/2 (h) after oral administration in humans
as previously reported in ref. 58 CL (mL/h/kg) after oral
administration in humans as previously reported.
d
58
The AR LBD affinity (for some compounds) and in vitro
antagonist properties of Series I−III ranged from comparable to
favorable relative to the known standard AR antagonists
currently employed clinically for the treatment of PC. For
example, 2, 4, 5, and 6 had LBD binding affinities of 0.509,
indoline B-ring compounds previously published in the same in
51
vitro assay and an improvement over 10 (T1/2 of 77.96 min).
The likely metabolic liability in aryl bicycles such as indoles
and indolines may be aryl hydroxylation of the B-ring. The A-
ring and propanamide portions have been incorporated into
many bioavailable compounds such as 2 (N-[4-cyano-3-
3
.641, 1.452, and 0.011 μM (values for 2, 4, and 5 are internally
determined vs for 6 is from the literature), and in vitro inhibition
of 0.248, 0.216, 0.160, and 0.065 μM (values for 2, 4, and 5 are
internally determined vs for 6 is from the literature); compared
to 10 binding of >10 μM and antagonism of 0.199 μM. We
found that compounds 16b, 16c, 16g, 16h, 16i, and 16m from
Series I; 21a from Series II; 26a and 26c from Series III; and 29a
from Series IV exhibited relatively potent AR inhibitory IC50
values in the range from 0.041 to 0.220 μM but, unlike 2, 4−6,
were SARDs with degradation activity values in the range from
(
trifluoromethyl)phenyl]-3-(4-fluorophenyl)sulfonyl-2-hy-
droxy-2-methylpropanamide) and enobosarm ((2S)-3-(4-cya-
nophenoxy)-N-[4-cyano-3-(trifluoromethyl)phenyl]-2-hy-
droxy-2-methylpropanamide), leaving the B-ring as the likely
metabolically labile site. A possible rationale for improved PK
properties with pyrazoles is the elimination of some of the
possible aryl hydroxylation sites on the B-ring. Also, it is possible
that the increased positive charged on the 2-position nitrogen
atom of the pyrazole makes the compounds poor substrates for
metabolic enzymes and/or improves biological partitioning.
Further, the design principle of 3,4-dihalogenated pyrazoles like
26a was an attempt to more effectively block the metabolism of
the B-ring, thereby decreasing hepatic CL.
Four further compounds were also characterized in rat liver
microsomes (RLM) and human liver microsomes (HLM) as
these readouts are relevant to suggest the stability of compounds
for in vivo testing in PD models such as the rat Hershberger assay
and xenograft in SRG rats (see infra) and ultimately in the clinic
(Table 7). Series I compounds 16c and 16g were stable in RLM
(T1/2 of >120 min) but 16c was less stable in HLM (T1/2 of 102
min). 21a (3′-pyridino, 4-F) and 26a (3-F, 4-Br) were stable
(T of >120 min) in both RLM and HLM, which was similar to
previously published data for 10 in RLM (181 min) and HLM
(274 min) (cite ref. 2). The stability in RLM and HLM is
consistent with the possibility of oral bioavailability of these
pyrazoles, as previously seen with 10. However, 21a and 26a
have improved in vitro efficacy relative to 16c and 10.
Correspondingly, if sufficiently high blood levels of 21a and
26a are attained and the compounds are distributed to the site of
action, that is, the tumor(s) throughout the body, it may be
possible to improve the efficacy to treat antiandrogen-resistant
CRPC compared to 10. Correspondingly, a few pyrazoles (16i,
21a, and 26a) with a variety of activity profiles were advanced to
testing in the models of CRPC including resistance to 4
1
1
00 to 45%. Because these compounds with the exception of
6m were comparable to improved inhibitors relative to known
LBD-targeted antiandrogens but possessed novel pan-antago-
nism and SARD activities, they were selected for further in vitro
and in vivo study to help optimize PK properties and explore the
pharmacodynamic (PD) potential of these NTD binding
noncanonical antagonists. A particular emphasis was placed on
improving upon pyrazole 10, which was the early lead in this
chemical class of compounds.
2.2. In Vitro Metabolic Stability in Mouse, Rat, and
Human Liver Microsomes. Compounds with potent
inhibitory activity of each series were selected to be further
evaluated for in vitro metabolic stability in mouse liver
microsomes (MLM) with cofactors for enzymes of both phase
I and phase II metabolism. The half-life (T1/2) and intrinsic
1
/2
clearance (CL ) values were calculated as a predictor of the
int
metabolism and PK properties of these compounds (Table 6).
In overview, the CLint of these compounds was slower than
previous generations of SARDs, producing relatively stable T1/2
values that range from 48.45 to >360 min for these pyrazol-1-yl-
propanamides (16b, 16g, 16h, 16i, 16m, 21a, 26a, and 29a)
with six of the nine tested pyrazoles being stable for >330 min in
MLM. This is a vast improvement when compared to previous
SARD templates such as 1.15 min for the tertiary amine 8, 12.11
1
min for lead indole 9, and 9−36 min for a variety of indole and
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Table 7. In Vitro Metabolic Stability for Selective Compounds
mutant AR with IC values of 0.043, 0.063, 0.084, and 0.219 μM
50
in RLM and HLM
(Figure 2) that are comparable to wtAR IC50 values of 0.045,
.062, 0.084, and 0.199 μM (Tables 2-4). The ability to
0
a
b
RLM
HLM
equipotently inhibit F876L and wtAR indicates that these
SARDs exhibit pan-antagonism in a model of Enz-R. Further,
this pan-antagonism cannot be explained by the AR LBD Ki
values of 1.499, >10, 0.607, and >10 μM for 16i, 21a, 26a, and
10. Moreover, the increased potency of 16i, 21a, and 26a
relative to 10 in wtAR inhibitory potency translated into this
model of Enz-R.
T
CL
T
CL
int
(mL/min/mg)
1
/2
int
1/2
(min)
compound ID
(min)
(mL/min/mg)
1
1
2
2
6c (4-Cl)
>120
>120
>120
>120
0
0
0
0
102
6.78
6g (4-CF3)
>120
>120
>120
0
0
0
1a (4-F)
6a
(
3-F, 4-Br)
Similar to our previously reported propanamide SARDs, these
molecules also inhibited wtPR activity with IC values of 3.540,
a
Compounds were incubated together with RLM with cofactors for
5
0
phases I and II provided, as described in the Experimental Section.
b
0.235, 1.101, and 0.403 μM for 16i, 21a, 26a, and 10 (Figure 3)
Compounds were incubated together with HLM with cofactors for
phases I and II provided, as described in the Experimental Section.
(
(
MR49F cells harboring F876L AR point mutant) and 1
LNCaP cells harboring the T877A).
2.3. In Vitro PD in Models of Castration-Resistant PC.
As discussed above, compounds were screened in vitro in a
competitive LBD binding assay (K ), inhibitory AR trans-
i
activation assay (IC ), and AR FL (in LNCaP cells) and AR SV
50
(
2
in 22RV1 cells) degradation assays (% degradation) (Tables
−5 above). Once strong in vitro screening profiles were
accomplished for single molecule(s), in vitro metabolic stability
criteria were also considered in the selection of compounds to be
tested further in vitro (Tables 6 and 7 above). In order to
improve the efficacy in in vivo testing, compounds were sought
with superior in vitro screening profiles compared to 10 and
tested further for transactivation selectivity between AR and PR,
AR target gene expression in LNCaP cells, and proliferation
studies in Enz-R PC cells (MR49F LNCaP cells).
Figure 3. Antagonism of wtPR transactivation. COS cells were
transfected with wtPR and a transactivation study was performed as
indicated in the legend for Figure 2. IC50 values were calculated and are
provided in the figure.
2.3.1. Mutant AR and wtPR Antagonist Effects. The selected
compounds 16i (4-CN), 21a (3′-pyridino, 4-F), 26a (3-F, 4-
Br), and 10 (4-F) were tested for their ability to antagonize a
LBD point mutant AR, which confers an enzalutamide (4)-
resistant (Enz-R) phenotype to PC cells. This F876L-mutant AR
or wild-type PR (wtPR) was transfected into COS cells, a non-
PC cell line, and quantified by luciferase assay (Figure 2). The
compounds 16i, 21a, 26a, and 10 robustly inhibited the F876L-
versus 0.045, 0.062, 0.084, and 0.199 μM for wtAR inhibition
(
Tables 2−5). Though wtPR inhibition was conserved,
selectivity ratios ([PR IC ]/[AR IC ]) varied with values of
50
50
7
9-, 3.8-, 13.1-, and 2.0-fold for the compounds selected for
testing, suggesting that AR selectivity could also be optimized
with further testing. Importantly, none of these molecules had
any effect on GR, MR, or ER transactivation (data not shown).
2.3.2. AR Target Gene Expression in CRPC Cells. As 10 was
reported to be effective in inhibiting the expression of FKBP5 in
2
MR49F cells, we performed an AR target gene inhibitory
experiment to determine the effect of lead pyrazole 26a on
R1881-induced AR target gene expression in LNCaP cells
(
Figure 4). The LNCaP cell line is a very well-characterized
model of CRPC that expresses the T877A point mutation of AR
that confers resistance to 1. Compound 26a was chosen as the
lead pyrazole as 26a possessed a balance of high-potency
inhibition (0.084 μM) and high-efficacy degradation (70−80%
for both AR FL and AR SV) with 3,4-disubstitution that blocked
metabolism relative to 10 [T1/2 > 360 min vs 77.96 min in MLM
(
Table 6)] and 26a is also stable in RLM and HLM (>120 min).
Consistent with the nM inhibition of wtAR (0.084 μM; Table 4)
and F876L AR (0.084 μM; Figure 2) transactivation, FKBP5
gene expression in LNCaP cells was robustly inhibited by 26a at
concentrations as low as 0.1 μM, indicating that the
antiandrogenic effects include inhibition of endogenous gene
expression (Figure 4) in another model of antiandrogen-
resistant CRPC without loss of potency. As expected, the
antiandrogen 4 also inhibited expression of the FKBP5 but at a
slightly lower potency. The same results were observed with
other AR target genes such as PSA and TMPRSS2 (data not
Figure 2. Antagonism of F876L-mutant AR transactivation. AR with
phenylalanine 876 mutated to leucine (F876L), GRE-LUC, and CMV-
renilla LUC were transfected in COS cells. Cells were treated 24 h after
transfection with 0.1 nM R1881 (agonist) and a dose response of
antagonists. Luciferase assay was performed 48 h after transfection. The
effect of each compound was conducted in antagonistic mode (in the
presence of 0.1 nM R1881). IC50 values were calculated and are
provided in the figure.
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The Enz-R of the MR49F model was demonstrated as the
antiproliferation of 4 was ∼100-fold less potent. For example 10
μM of 4 produced effects comparable to 0.1 μM of 26a, which
was weak at ∼20% efficacy and not significantly different from
vehicle. Assuming that 26a can reach the tumors, this potent
antiproliferation suggests that 26a may perform well in in vivo
models of Enz-R CRPC.
2.3.4. AR FL (F876L) and AR SV (AR-V7) Degradation in
Models of CRPC. FL AR degradation studies in MR49F cells
were performed in order to confirm that the robust in vitro AR
antagonism profiles of 16i (0.045 μM, 90, and 100% in wtAR
inhibition, AR FL and AR SV degradation assays) and 26a
(
0.084 μM, 70, 80%) predicted SARD activity in this model of
highly refractory CRPC. Compound 26a possessed the ability to
suppress AR-dependent gene expression in LNCaP cells and
suppress proliferation in MR49F cells as described above and
was able to also degrade the FL AR (Figure 6 upper panel) in the
Figure 4. SARDs antagonize AR function in PC cell, LNCaP. LNCaP
cells were maintained for 2 d in charcoal-stripped, serum-containing
medium. The cells were treated with antagonist as indicated in the
figure for 20−24 h, RNA was isolated, and expression of AR target gene,
FKBP5, was measured and normalized to GAPDH using real-time PCR.
shown). Cumulatively, the above data support that 26a has pan-
antagonist effects in at least wtAR (Table 4), F876L (Figure 2),
T877A (Figure 4), and AR SV (Table 4).
2
.3.3. Proliferation Studies in Enz-R LNCaP Cells. Pro-
liferation studies were conducted with 26a to confirm that
potent inhibition of AR-dependent gene expression in a model
of CRPC harboring the T877A antiandrogen-resistant mutation
Figure 6. SARDs degrade enzalutamide resistance conferring escape
mutant AR. Enzalutamide (4)-resistant (Enz-R) LNCaP cells
(i.e., LNCaP cells) translated into antiproliferation in an even
more refractory model of CRPC, that is, MR49F LNCaP cells
harboring F876L and T877A point mutations of AR. As
mentioned above, the F876L mutation confers enzalutamide (4)
resistance (Enz-R) to MR49F cells; however, MR49F cells
remain dependent on the AR for growth. MR49F cells were
tested in the presence of a titrated dose of 26a or 4 as shown in
Figure 5. Compound 26a demonstrated dose-responsive
antiproliferation that showed potent, but partial, efficacy
(
MR49F) (top panel) or 22RV1 cells (bottom panel) were maintained
in charcoal-stripped, serum containing medium for 2 d and treated with
.1 nM R1881 (agonist) and a titration of the SARD or 4 as indicated in
0
the figure. Twenty-four hours after treatment, the cells were harvested
and protein-extracted, and the proteins were blotted with AR-N20
antibody. Blots were stripped and reprobed with a GAPDH antibody.
The ratio of AR to GAPDH or each lane is given under each blot.
(∼50−60% reduction from vehicle) at doses as low as 0.1 μM.
Enz-R CRPC setting. Western blotting is not a quantitative
method and it can be difficult to compare the AR levels between
compounds based on relative band densities. Accordingly,
GAPDH was also included as a protein loading control in each
lane. The levels of AR are normalized to the level of GADPH in
that lane. The western blots were quantified densitometrically
and the AR/GADPH values are represented as fold change
(
under blots in Figure 6) or percent change from vehicle-treated
cells (Tables 2−5).
High-efficacy SARD activity was observed with 26a at 3 μM
and complete degradation at 10 μM (Figure 6, top panel),
indicating that this mutant AR FL that confers Enz-R in MR49F
LNCaP cells is susceptible to destruction by 26a. 16i also
demonstrated SARD activity but not full efficacy, whereas 4
produced no AR degradation in MR49F cells.
The lower panel demonstrates that the SARD activity is not
just present for T877A (LNCaP; Tables 2−5) and F876L/
T877A (MR49F LNCaP cells; Figure 6 upper panel) AR FL
with point mutations in the LBD but also can degrade AR SVs
such as the AR-V7 that lack the expression of the LBD (22RV1
cells; lower panel of Figure 6). As reported in Tables 4 and 2 (see
AR SV degradation column), 26a and 16i were able to reduce
Figure 5. Enzalutamide-resistant LNCaP (MR49F) cellular antiprolif-
eration. Enzalutamide (4)-resistant (Enz-R) LNCaP (MR49F) cells
were plated in 1% charcoal-stripped, serum-containing medium and
treated with 0.1 nM R1881 and a titration of antagonist as indicated in
the figure. Cells were retreated 3 d after the first treatment and the
number of viable cells was measured by the CellTiter-Glo assay
(
Promega, Madison, WI). N = 3.
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Figure 7. Concentration−time plots in rats for 26a. Twelve week old male Sprague Dawley rats were dosed in five groups of five animals each (N = 5) at
the doses shown. Blood samples were drawn at the shown time points and analyte concentrations were determined by MS/MS. The concentration−
time plots for all dose groups are shown for day 1 (left) and day 7 (right).
a
Table 8. Summary of 26a PK Parameters in Rat
dose
group
dose Level
(mg/kg/day)
C
C
DN C
AUC
DN AUC
0−24
(h*ng/mL)/(mg/kg/day)
0
max
(ng/mL)
max
0−24
(ng/mL)
(ng/mL)/(mg/kg/day)
Tmax (h)
(h*ng/mL)
1
2
3
4
5
5
10
20
30
NA
NA
NA
NA
4200
2570
2680
3420
3650
3940
515
268
171
122
394
3.00
3.00
12.0
24.0
0.083
26,800
44,600
64,100
71,500
45,500
5350
4460
3200
2380
4550
10 (iv)
a
C back-extrapolated concentration at time 0 (group 5 only). C maximum observed concentration. DN C dose normalized Cmax,
0
max
max
calculated as C /dose level. T time of maximum observed concentration. AUC area under the concentration−time curve from time 0 to
max
max
0−24
2
4 h, estimated by linear trapezoidal rule. DN AUC dose normalized AUC , calculated as AUC /dose level.
0−24
0−24
0−24
AR-V7 levels in 22RV1 cells at 10 μM. Figure 6 confirmed the
AR-V7 SARD activity at 3 and 10 μM, but % degradation was
not complete for either SARD in this particular experiment.
Lower % degradation for AR SV than AR FL is consistent with
earlier reports and Tables 2−5, which revealed that AR SV
degradation can be complete but generally at higher treatment
concentrations (screened at 10 μM) than for AR FL (screening
remain sensitive to our SARDs and pan-antagonists. Further, our
SARDs and pan-antagonists performed well in models of AR
overexpression and/or AR gene recombination such as present
in VCaP cells, suggesting that these PCs will not be able to resist
this treatment either. In view of the fact that SARD activity may
not be necessary for these activities, we have added discussion of
our agents as AR pan-antagonists in this publication. Compound
26a was tested as a lead SARD and pan-antagonist in vitro
(supra) and subjected to a series of in vivo tests to describe its PK
(Section 2.4) and PD (Section 2.5) profiles in healthy rats and
models of antiandrogen-resistant PC in rats.
5
1
at 1 μM). PCs expressing AR SVs possess no binding site for
traditional (or canonical) antiandrogens to bind AR, are
associated with poor prognosis, and are believed to be pan-
53
resistant to approved therapies including 1−7. Accordingly,
1
,2
the pyrazole SARDs and pan-antagonists such as 10, 21a, and
6a (discussed infra) that possess PK properties compatible
2.4. In Vivo Rat PK. The overall goal of this initial pyrazole
SAR study was to improve upon 10 as a pyrazole lead. Assuming
comparable oral bioavailability, Sections 2.1 and 2.3 demon-
strate improved potency compared to 10 and a broad spectrum
of in vitro activities for 26a, suggesting the possibility of
improved in vivo AR-dependent tumor growth inhibition (TGI)
over 10 including antiandrogen-resistant and/or CRPC
2
with oral administration at low dose afford a very broad scope of
AR antagonistic abilities in at least:
(
(
1) wtAR (IC values in Tables 2−5),
5
0
2) T877A (LNCaP AR FL degradation in Tables 2−5 and
inhibition of AR-dependent gene expression in Figure 4),
3) F876L (inhibition in COS cells in Figure 2),
4) F876L/T877A comutant (proliferation in MR49F cells in
Figure 5),
2
tumors. Rat PK studies were conducted to confirm that
(
(
2
pyrazole 26a, like 10, possessed improved PK properties
compared to previous generations of our SARDs such as tertiary
amine 8 and indole 9. Further, these studies allow more
informed comparisons between the pyrazoles 26a and 10 in view
of their PK criteria. Optimized PK properties within the pyrazole
template provide the best chance to reveal optimized in vivo PD
profiles for our molecules with their unique AR mechanism of
(
5) AR-V7 (degradation of AR SV in 22RV1 cells in Tables
2
−5 and Figure 6), and
(
6) AR amplification/overexpression (see VCaP data re-
ported infra).
2
The broad-scope AR antagonism across various resistance-
conferring AR mutants helps to ensure that treated tumors that
are evolving to contain these and/or other AR mutations will
action in in vivo models of advanced PCs.
Male Sprague Dawley rats were given a single oral (po) daily
dose on seven consecutive days or a single intravenous (iv) dose
L
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2
on day 1, and blood was sampled periodically at 0.083, 0.25, 0.5,
resistance, similar to 10. In summary, the more potent in
vitro profile of 26a (Sections 2.1 and 2.3) relative to 10 and the
more stable μM range plasma concentrations of 26a with daily
dosing (Figure 7) relative to 10 or 21a suggest that a robust in
vivo PD profile will be observed with 26a in models of CRPC
and antiandrogen resistance.
1
, 3, 6, 12, and 24 h post dose. The doses of 5, 10, 20, and 30 mg/
kg po (groups 1−4) and 10 mg/kg iv (group 5) were selected
based on in vivo efficacies seen in a series of pilot experiments,
which were similar to the Hershberger study discussed in detail
in this section. Concentration−time curves were plotted from
this data for 26a (Figure 7), and the PK parameters were
calculated for 26a from this data (Table 8).
Like 10, 26a demonstrated a robust PK profile in rats
characterized by micromolar blood levels and a long terminal
elimination half-life (t1/2) (Table 8) consistent with daily oral
2.5. In Vivo AR Antagonist Activity. 2.5.1. Hershberger
Assays. In order to prove that these compounds with robust PK
properties have clinically meaningful SARD and pan-antagonist
activity in vivo, we performed Hershberger assays in intact rats
for 21a and 26a which, as shown above, demonstrated oral
bioavailability in rats (Figures S1 and 7). The Hershberger assay
has been used to demonstrate anabolic selectivity of androgens
dosing. An advantage of 26a over 10 is its relatively long t1/2
,
which is in excess of 24 versus 2.6 h (calculated based on the 7
2
54
day rat PK data reported in Ponnusamy’s paper for 10). The
for decades. Rat ventral prostate (VP), seminal vesicle (SV),
exact t1/2 value of 26a could not be calculated as the t1/2 was
longer than the 24 h dosing interval (Figure 7). 26a had
decreasing oral bioavailability at higher doses as revealed by the
decreasing dose-normalized area under the concentration−time
and levator ani (LA) muscle are AR-dependent tissues whose
size (reflected by their weight) responds rapidly to castration.
Upon castration, these organs atrophy within 3−7 days to organ
weights that are approximately 85% (VP), 90% (SV), and 50%
(LA) reduced compared to their intact organ weights.
Traditionally, agonists are dosed to prevent (whereby agonist
is given upon castration) or restore (agonist is given after tissue
atrophies) anabolic tissue weights [LA or other skeletal muscles
and bone (the latter takes months not days to atrophy and
restore)] to intact levels or greater, without increasing
androgenic tissue (SV or VP) weights back to intact levels. As
employed herein, that is, antagonist mode, young intact animals
were used wherein the endogenous androgen milieu provided
AR-mediated support for the VP, SV, and LA weights, as
reflected by the 0% change for the vehicle columns in Figure 8.
Exogenous antagonists 21a and 26a, with potent in vitro
inhibition [0.062 and 0.084 μM (Tables 3 and 4)], were dosed
to observe their AR antagonism in vivo. Previously, we
demonstrated that 10 (4-F pyrazole; 0.199 μM inhibition in
vitro) was able to reduce the VP weight by ∼70−80% at 60 mg/
kg po [Figure S4C, middle panel in Ponnusamy’s paper] versus
curve from 0 to 24 h (DN AUC0 ) values and increasing time
−24
of maximum concentration (Tmax) values for groups 1−4 with
increasing 26a dose (Table 8). The calculated oral bioavail-
abilities for 5, 10, 20, and 30 mg/kg doses of 26a were 1.18,
0
.982, 0.705, and 0.524. Nonetheless, the longer t1/2 of 26a
relative to 10 at least partially offset the decreasing oral
bioavailability at high doses and 26a attained marginally
increased absolute exposures compared to 10. For example,
the AUC0−24 values for 30 mg/kg po 26a and 10 were 71,500
and 62,000 h*ng/mL, respectively. The latter value, again, is
calculated from the 7 day rat PK data presented in Ponnusamy’s
2
paper.
Correspondingly, 26a exhibits a PK profile sufficiently robust
to maintain high blood levels in vivo via oral daily dosing in rats.
Also shown is preliminary rat PK data for 30 mg po 21a (Figure
S1). The concentration versus time plot demonstrated reduced
in vivo stability, with the vast majority of 21a eliminated by 24 h,
which is in sharp contrast to 30 mg po 26a where blood levels at
2
∼40% for 30 mg/kg po of 4, both in rats. Improved potency of
2
4 h were barely reduced from their Cmax (Figure 7).
in vivo AR antagonism was seen for derivatives of 10 with (1) the
addition of the 3′-pyridino N to 10 as in 21a or (2) an additional
halogen on the pyrazole such as 3-F or 4-Br in 26a. At 20 mg/kg
of 21a and 26a, that is, one-third of the dose of 10 mentioned
above, VP weights were reduced by approximately 35 and 30%
(Figure 8, top panel), demonstrating that the in vivo PD
properties intrinsic to 21a and 26a are observable at lower doses
relative to 10. In SV at this dose, approximately 45−50%
reductions were provided by 21a and 26a. Addition of the 3′-
pyridino (21a) or dihalogenation of pyrazole (26a) of 10
seemed favorable for in vivo antagonism, suggesting that
xenograft potencies should also be improved. Consistent with
Section 2.4, these results confirm that orally administered 26a
was absorbed and distributed to the site of action in AR target
organs and suggest that these compounds should also distribute
to tumors in xenograft models and exert antitumor effects in
sensitive models.
Nonetheless, 21a at 30 mg po demonstrated sufficiently low
CL to allow observation of its PD character in rats.
[Unfortunately, despite an interesting and potent in vitro panel
of activities, 16i demonstrated lethality at 5 mg/kg in vivo and
was not evaluated further]. Correspondingly, 21a and 26a were
studied in rat Hershberger assays, but 26a was chosen as the lead
for the xenograft studies reported infra.
The micromolar Cmax blood levels and long t1/2 observed for
6a suggested PK properties in rats consistent with revealing any
2
high efficacy AR antagonism of 26a in vivo that was engendered
by the data in Sections 2.1 and 2.3. Sections 2.1 and 2.3
demonstrated that 26a inhibited a broad spectrum of
antiandrogen activities in vitro with increased potency compared
to 10, including in the models of antiandrogen-resistant CRPC.
Oral daily dosing in rats with 26a should be able to maintain
blood levels above the IC value of AR antagonism (Table 4)
50
and inhibitory effects on AR-dependent transcription (Figure 4)
and proliferation (Figure 5), as would be necessary to suppress
the AR axis in AR-dependent xenografts. Further, the low
micromolar drug levels seen for 21a (Figure S1), 26a (Figure 7
2.5.2. Enz-R (MDVR) VCaP Xenografts in Rats. The VCaP
cell line is derived from a vertebral bone metastasis from a
patient with hormone refractory PC (https://atcc.org/
55
Products/ all/CRL-2876.aspx; accessed January 20, 2020).
2
and Table 8), and 10 were in excess of DC values for 21a (880
VCaP is commonly used as a model of CRPC, which expresses
both AR SV (AR-V7) and overexpression of AR FL (TMPRSS2-
ERG gene fusion). VCaP, the parental cell line for the MDVR
VCaP used in the experiments below, is a model of highly
advanced PC where multiple mechanisms of hormone resistance
have emerged in response to androgen ablation in a single AR
50
nM), 26a (860 nM), and 10 (740 nM) (see Tables 2−4),
suggesting that SARD activity may contribute to in vivo AR
antagonism, as seen previously with 10 where intratumoral
2
degradation was observed. Correspondingly, we expected to
observe TGI in models of CRPC and/or antiandrogen
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Figure 9. SARDs and pan-antagonists inhibit growth of Enz-R PC. Enz-
6
R MDVR cells (10 × 10 cells/rat) were implanted subcutaneously in
male SRG (Sprague Dawley-Rag2: IL2rg KO) rats. When the tumors
Figure 8. SARDs and pan-antagonists inhibit androgen-dependent
organs in rats. The top and bottom panels show the reduction of VP and
SV weights following the treatment of intact rats with 20 mg/kg (mpk)
po daily of antagonist or vehicle for 14 days (n = 5/group). Rats were
sacrificed at the end of the treatment period and weights of prostate and
SVs were measured and normalized to body weight.
3
reached 1000−3000 mm , the animals were randomized and treated
3
(
intact). Once the tumors attain 2000−3000 mm , the animals were
treated orally with vehicle (DMSO/PEG-300 15:85) or 10 mg/kg/day
of 26a. Tumor volume (T.V.) was measured twice weekly and
represented as a percent change (upper panel) or weight at sacrifice
(lower panel).
axis-driven cell line. The parental VCaP cells are nonetheless
sensitive to enzalutamide (4); however, MDVR VCaP cells
possess acquired Enz-R in addition to the resistance mechanisms
in the parental cell line. Previously, we demonstrated that VCaP
are partially sensitive to 4, whereas MDVR VCaP are not
versus 10 required 20−30 mg/kg to achieve similar results [see
Figure 6E, left panel of ref. 2], whereas 4 failed to durably
achieve any effect (not shown; previously published) [see Figure
6, panel C]. Tumor weights measured at the end of the study
also demonstrated a significant inhibition (lower panel of Figure
9).
Consistent with the observed high-potency antitumor activity,
26a was observed in this study at an average concentration
within the tumors of 881 nM, which is 10-fold higher than its
IC50 value in wtAR or F876L (both 84 nM). Further,
intratumoral levels were only slightly reduced from the 1319
nM average concentration of 26a in the blood of these animals
(Table 9). This supports efficient distribution of 26a into
tumors, in addition to VP and SV, and supports its use in
advanced PC.
2
2
sensitive. Furthermore, 60 mg/kg po of 10 regressed MDVR
2
VCaP xenografts and degraded AR and AR-V7 intratumorally,
whereas 10 mg/kg po of 10 only produced about 50% TGI
[
Figure 6E left panel of ref. 2].
Following the demonstration with 26a of an in vitro screening
panel that was superior to 10, in vitro activity in MR49F (an Enz-
R LNCaP cell line) and in vivo antagonism in Hershberger
assays, there was confidence in our ability to demonstrate
activity in Enz-R MDVR VCaP xenografts. In order to allow
direct comparison of 26a to 10, the MDVR VCaP xenografts
2
were performed as previously published for 10. For 10,
The in vitro DC values (concentration of the 50th percentile
5
0
castration was not necessary to demonstrate the efficacy in this
model (unlike all previous AR antagonists to our knowledge);
however, 10 was not stable in mice; therefore, intact SRG rats
were used as the host for MDVR VCaP xenograft experiments.
Treatment of intact SRG rats (studies performed at HERA
Biolabs, Lexington KY) with 10 mg/kg po daily of 26a produced
comparable efficacy of up to 83% TGI (Figure 9, top panel)
of degradation efficacy) in LNCaP cells for 21a (880 nM) and
26a (860 nM) reported in Tables 3 and 4 were comparable to
the intratumoral levels attained in the MDVR VCaP xenografts.
Despite the different cell types between in vitro and in vivo
studies, the data suggest the possibility of suboptimal exposures
for full-efficacy SARD activity in the tumors of this experiment.
Nonetheless, this presumed half-efficacy intratumoral SARD
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Table 9. Serum and Tumor Drug Concentration of 26a
vitro (Tables 2−4). Compared to previous SARD templates
such as 1.15 min for the tertiary amine 8 and 12.11 min for lead
indole 9, these pyrazol-1-yl-propanamides, such as 16g, 16i, 21a,
and 26a, significantly improved their stability in vitro in MLM
(Table 6), and 21a and 26a were stable in RLM and HLM
(Table7). Compounds 16i, 21a, and 26a robustly inhibited the
F876L-mutant AR with IC50 values of 0.043, 0.063, and 0.084
μM (Figure 2), as well as inhibited wt PR activity with IC50
values of 3.540, 0.235, and 1.101 μM (Figure 3). Compound 26a
effectively inhibited the expression of FKBP5 in LNCaP cells at
concentrations as low as 0.1 μM, indicating that the
antiandrogenic effects include inhibition of endogenous gene
expression (Figure 4), as well as demonstrated dose-responsive
antiproliferation at doses as low as 0.1 μM (Figure 5).
Compound 26a also produced superior in vivo rat PK and PD
properties compared to 10 and 21a, with relatively long t1/2
values that were well in excess of 24 h (Figure 7) and AR
antagonism in rat Hershberger assay, with approximately 30%
(VP), and 50% (SV) reduced compared to their intact organ
weights (Figure 8), which was comparable to 21a.
2
6a (3-F, 4-Br)
a
a
serum (nM)
tumor (nM)
animal 1
animal 2
animal 3
animal 4
average
S. E.
1611.663
1360.036
1143.556
1160.002
1318.814
109.3146
962.6859
913.4912
666.0278
983.6887
881.4484
73.29447
a
Twenty to 24 h after the last dose (day 28), the animals were
sacrificed, and blood and tumors were collected for further analysis.
The serum was separated from blood, and drug concentration in
serum and tumor was measured using the LC−MS/MS method (n =
4
).
activity may contribute to the TGI. In overview, it may be
possible to improve the antitumor activity with increased
intratumoral levels, that is, at increased dose of 26a, or with
improved degradation potency analogues.
The results clearly indicate that 26a was stable in rats (like 10)
and was very potent and highly efficacious in this AR
overexpressing and AR-V7 expressing model of Enz-R CRPC.
The results further suggest that the improved PK and PD of 26a
translated into more potent in vivo efficacies compared to 10,
providing a dose-sparing SARD and pan-antagonist if, as of yet
to be unobserved, toxicities become dose limiting. Further, the
improved PK may translate into improved penetration
throughout the cancer patient allowing better suppression of
distant metastatic growth. All the above increase our chances of
observing clinically significant reduction in disease burden when
trialed in a human population (as supported by HLM studies in
Table 7) expressing a broad spectrum of CRPC-resistant
mechanisms. This population would still be sensitive even if
expressing AR SVs (like AR-V7), AR gene amplications to
overexpress AR (like TMPRSS2-ERG), or LBD-directed
antiandrogen resistance (like Enz-R and/or darolutamide
resistance observed in MR49F or MDV VCaP cells) or
combinations thereof as in MDVR VCaP.
Enz-R (MDVR) VCaP xenograft experiments with 10 mg/kg
po daily of 26a in an intact rat model demonstrated high drug
levels intratumorally (881 nM) and producing an efficacy of 83%
TGI (Figure 9, top panel), which was comparable to 10 at 20−
30 mg/kg po. The results clearly indicate that 26a was very
potent and highly efficacious in this AR overexpressing and AR-
V7 expressing model of Enz-R CRPC and collectively satisfied
all the criteria for a next-generation AR antagonist for Enz-R PC.
Compound 26a is believed to hold great potential for
overcoming multiple mechanisms of CPRC present in the clinic
and is one of the several compounds being evaluated for their
potential to advance to IND enabling studies.
4. EXPERIMENTAL SECTION
4
.1. Chemistry. 4.1.1. General Procedures, Materials, and
Information. All solvents and chemicals were used as purchased
without further purification. The progress of all reactions was
monitored by thin-layer chromatography (TLC) analysis on silica gel
60 F254 plates (Merck). Column chromatography was performed with
a silica gel column (Merck Kieselgel 60, 70−230 mesh, Merck).
General methods: All nonaqueous reactions were performed in oven-
dried glassware under an inert atmosphere of dry nitrogen. All the
reagents and solvents were purchased from Aldrich (St. Louis, MO),
Alfa-Aesar (Ward Hill, MA), Combi-Blocks (San Diego, CA), and Ark
Pharm (Libertyville, IL) and used without further purification.
Analytical TLC was performed on Silica Gel GHLF 10 × 20 cm
Analtech TLC Uniplates (Analtech, Newark, DE) and was visualized by
fluorescence quenching under UV light. The Biotage SP1 Flash
Chromatography Purification System (Charlotte, NC) (Biotage SNAP
These results confirm that for 26a, our in vitro screening
paradigm was successful in selecting an improved lead
compound from our library of SARDs and pan-antagonists
that was highly efficacious in an in vivo model of CRPC. Though
full-efficacy in vitro SARD activity such as published for 10 is
unique and should be beneficial in AR-dependent disease, it may
not be necessary for efficacy in the clinic. This is supported by
the more potent and comparable efficacy antitumor activity in
vivo for 26a, which was not a full-efficacy SARD in vitro (70%/
1
Cartridge, silica, 50 & 100 g) was used to purify the compounds. H
8
0%; Table 4), unlike 10 (100%/100%; Table 2). Although
1
3
NMR and C NMR spectra were recorded on a Bruker Ascend 400
exact and incontrovertible mechanistic explanations of the high
efficacy of 26a are not possible, its potent in vivo efficacy is also
incontrovertible. The pyrazole template represents the optimal
B-ring template presented to date, and 26a is an optimized lead
from this template. 10 or 26a is believed to hold great potential
for overcoming multiple mechanisms of CPRC present in the
clinic.
1
(
400 MHz) (Billerica, MA) spectrometer. Chemical shifts for H NMR
were reported in parts per million (ppm) downfield from
tetramethylsilane (δ) as the internal standard in deuterated solvent
and coupling constants (J) are in hertz (Hz). The following
abbreviations are used for spin multiplicity: s = singlet, d = doublet, t
= triplet, q = quartet, quin = quintet, dd = doublet of doublets, dt =
doublet of triplets, qd = quartet of doublets, dquin = doublet of quintets,
m = multiplet, and br s = broad singlet. Low-resolution mass spectra
were acquired using a Brucker ESQUIRE electrospray/ion trap
instrument in the positive and negative modes. High-resolution mass
spectrometry (HRMS) data were acquired on a Waters Xevo G2-S
QTOF (Milford, MA) system equipped with an Acquity I-class UPLC
system. The purity of the final compounds was analyzed by an Agilent
1100 HPLC system (Santa Clara, CA). HPLC conditions: 45%
acetonitrile at flow rate of 1.0 mL/min using a LUNA 5 μ C18 100A
3
. CONCLUSIONS
Compounds 16c, 16g, 16i, and 16j from Series I; 21a and 21c
from Series II; and 26a, 26c, 26e, and 26f from Series III
exhibited potent inhibitory activity in vitro, while compounds
1
2
6b, 16c, 16g, and 16i from Series I; only 21a from Series II; and
6a and 26g from Series III possessed potent SARD activity in
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column (250 × 4.60 mm) purchased from Phenomenex (Torrance,
CA) at ambient temperature. UV detection was set at 340 or 245 nm.
Purities of the compounds were established by careful integration of
areas for all peaks detected and determined as ≥95% for all compounds
tested for biological study.
NMR (400 MHz, DMSO-d ): δ 10.39 (s, 1H, NH), 8.48 (d, J = 2.0 Hz,
6
1H, ArH), 8.22 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, ArH), 8.08 (d, J = 8.2 Hz,
1H, ArH), 7.66−7.65 (m, 1H, pyrazole-H), 7.39−7.38 (m, 1H,
pyrazole-H), 6.28 (s, 1H, OH), 6.25−6.23 (m, 1H, pyrazole-H), 4.50
(d, J = 13.6 Hz, 1H, CH), 4.29 (d, J = 13.6 Hz, 1H, CH), 1.35 (s, 3H,
+
2
4.1.2. Synthesis of 13, 18, 23, and 28 Using 13 as an Example.
CH ). HRMS [C H F N O ]: calcd, 339.1099; found, 339.1105.
3
15 14
3
4
(
R)-3-Bromo-2-hydroxy-2-methylpropanoic acid 11 (5.00 g, 27 mmol)
4.1.4.2. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-
1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (10 (UT-34)).
10 was prepared following general procedure A as per Scheme 1. The
crude product was purified by a silica gel column using ethyl acetate and
was dissolved in tetrahydrofuran (THF) (27 mL, 5.4 vol) in an
EasyMax 100 mL reactor. Agitation was set to 400 rpm and the solution
was cooled to 2.5 °C. Thionyl chloride (2.39 mL, 1.20 equiv, 0.48 vol)
was slowly added to the reaction mixture over 30 min while maintaining
the reaction temperature below 12 °C. The reaction mixture was stirred
for 1.5 h. The reaction was cooled to −5 °C. Triethylamine (5.0 mL,
hexanes (1:1) as eluents to afford 0.13 g of the titled compound as a
1
white solid. Yield = 32%. H NMR (400 MHz, DMSO-d
): δ 10.39 (s,
6
1H, NH), 8.47 (d, J = 1.6 Hz, 1H, ArH), 8.24 (dd, J = 8.4 Hz, J = 2.0 Hz,
1H, ArH), 8.10 (d, J = 8.4 Hz, 1H, ArH), 7.73 (d, J = 4.4 Hz, 1H,
pyrazole-H), 7.41 (d, J = 4.4 Hz, 1H, pyrazole-H), 6.31 (s, 1H, OH),
4.38 (d, J = 14.0 Hz, 1H, CH), 4.21 (d, J = 14.0 Hz, 1H, CH), 1.34 (s,
1
.30 equiv, 1 vol) was slowly added to the reaction mixture, keeping the
temperature below 12 °C. 4-Amino-2-(trifluoromethyl)benzonitrile 12
4.85 g, 0.95 equiv, 0.97 wt) and THF (3.37 mL, 0.67 vol) were then
(
+
charged to the batch. The batch was then heated to 50 ± 5 °C and
agitated for 2 h. The batch was then cooled to 20 ± 5 °C, followed by
the addition of water (14.7 mL, 2.9 vol) and toluene (20.2 mL, 4.0 vol).
After brief agitation, the layers were separated. The organic layer was
then washed with water (14.7 mL, 2.9 vol). The batch was then
concentrated to 5 ± 0.5 vol (4 ± 0.5 wt) while maintaining the batch
temperature below 50 °C, followed by the addition of toluene (30 mL, 6
vol). The batch was then distilled to 5 ± 0.5 vol (4 ± 0.5 wt) and the
batch temperature reduced to 2.5 ± 2.5 °C. The batch was then filtered
and the filter cake was washed with toluene twice (8.5 mL each, 1.7 vol
each). The batch was then dried under 25−30 in. vacuum to provide
3H, CH ). HRMS [C H F N O ]: calcd, 357.0975; found,
3
15 13
4
4
2
357.0966.
4.1.4.3. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3-fluoro-
1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (16b). 16b was
prepared following general procedure A as per Scheme 1. The crude
product was purified by a silica gel column using ethyl acetate and
hexanes (2:1) as eluents to afford 0.36 g of the titled compound as white
1
needles. Yield = 44%. H NMR (400 MHz, DMSO-d
): δ 10.39 (s, 1H,
6
NH), 8.47 (d, J = 2.0 Hz, 1H, ArH), 8.24 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H,
ArH), 8.11 (d, J = 8.8 Hz, 1H, ArH), 7.55 (t, J = 3.0 Hz, 1H, yrazole-H),
6.29 (s, 1H, OH), 5.93−5.91 (m, 1H, pyrazole-H), 4.34 (d, J = 13.6 Hz,
(
R)-3-bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
1H, CH), 4.15 (d, J = 13.6 Hz, 1H, CH), 1.36 (s, 3H, CH ). HRMS
3
1
+
methylpropanamide 13. Yield = 85%. H NMR (400 MHz, DMSO-d ):
[C15H F N O ]: calcd, 357.0975; found, 357.0985.
13 4 4 2
6
δ 10.55 (s, 1H, NH), 8.55 (s, 1H, ArH), 8.32 (d, J = 8.8 Hz, 1H, ArH),
4.1.4.4. (S)-3-(4-Chloro-1H-pyrazol-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16c).
16c was prepared following general procedure A as per Scheme 1.
The crude product was purified by a silica gel column using
dichloromethane (DCM) and ethyl acetate (19:1) as eluents to afford
8
1
.12 (d, J = 8.8 Hz, 1H, ArH), 6.43 (s, 1H, OH), 3.84 (d, J = 10.4 Hz,
H, CH), 3.59 (d, J = 10.4 Hz, 1H, CH), 1.48 (s, 3H, CH ). HRMS
3
+
[
C H BrF N O ]: calcd, 350.9956; found, 350.9961.
12
11
3
2
2
4
.1.3. Synthesis of 14, 19, and 24 Using 14 as an Example. To a
1
solution of (R)-3-bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
hydroxy-2-methylpropanamide 13 (5.00 g, 0.018504 mol) in 25 mL
of 2-butanone was added potassium carbonate (3.836 g, 0.027756
mol). The resulting reaction mixture was heated at reflux for 2 h under
argon atmosphere. After ending the reaction by establishing TLC, the
reaction was cooled to room temperature (rt), filtered through a pad of
Celite, and rinsed the pad of Celite with 15 mL of 2-butanone. The
filtrate was concentrated under vacuum and dried under 25−30 in.
vacuum to provide (S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
0.30 g of the titled compound as a white solid. Yield = 55%. H NMR
(400 MHz, DMSO-d ): δ 10.38 (s, 1H, NH), 8.46 (s, 1H, ArH), 8.23
6
(d, J = 8.6 Hz, J = 1.2 Hz, 1H, ArH), 8.10 (d, J = 8.6 Hz, 1H, ArH), 7.83
(s, 1H, pyrazole-H), 7.47 (s, 1H, pyrazole-H), 6.34 (s, 1H, OH), 4.45
(d, J = 14.0 Hz, 1H, CH), 4.27 (d, J = 14.0 Hz, 1H, CH), 1.36 (s, 3H,
+
2
CH
Purity: 97.69% (HPLC).
). HRMS [C15
H
13ClF
N
4
O
]: calcd, 373.0679; found, 373.0678.
3
3
4.1.4.5. (S)-3-(4-Bromo-1H-pyrazol-1-yl)-N-(4-cyano-3-
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16d).
(
1
1
6d was prepared following general procedure A as per Scheme 1.
methyloxirane-2-carboxamide 14. Yield = 99%. H NMR (400 MHz,
DMSO-d ): δ 10.22 (s, 1H, NH), 8.41 (s, 1H, ArH), 8.22 (d, J = 8.8 Hz,
The crude product was purified by a silica gel column using DCM and
6
1
3
H, ArH), 8.09 (d, J = 8.8 Hz, 1H, ArH), 3.09 (d, J = 4.8 Hz, 1H, CH),
ethyl acetate (19:1) as eluents to afford 0.47 g of the titled compound as
1
.02 (d, J = 4.8 Hz, 1H, CH), 1.55 (s, 3H, CH ). HRMS
a white form. Yield = 79.6%. H NMR (400 MHz, CDCl
): δ 9.08 (s,
3
3
+
[
C H F N O ]: calcd, 271.0694; found, 271.0696.
1H, NH), 8.00 (d, J = 2.0 Hz, 1H, ArH), 7.87 (dd, J = 8.4 Hz, J = 2.0 Hz,
1H, ArH), 7.79 (d, J = 8.4 Hz, 1H, ArH), 7.49 (s, 1H, pyrazole-H), 7.47
(s, 1H, pyrazole-H), 5.92 (s, 1H, OH), 4.64 (d, J = 14.0 Hz, 1H, CH),
12
10
3
2
2
4
.1.4. General Procedure A for the Synthesis of 16(a−y), 21(a−j),
2
6(a−h), and 29(a−p) Using 10 (UT-34) as an Example. To a
solution of 4-fluoro-pyrazole (0.10 g, 0.00116 mol), or general pyrazole
4.24 (d, J = 14.0 Hz, 1H, CH), 1.47 (s, 3H, CH ). HRMS
3
+
1
5, in anhydrous THF (10 mL), which was cooled in an ice−water bath
[C H BrF N O ]: calcd, 417.0174; found, 417.0167. Purity:
1
5
13
3
4
2
under an argon atmosphere, was added sodium hydride (60%
dispersion in oil, 0.12 g, 0.00291 mol). After addition, the resulting
mixture was stirred for 3 h. (R)-3-Bromo-N-(4-cyano-3-
99.53% (HPLC).
4.1.4.6. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-
(4-iodo-1H-pyrazol-1-yl)-2-methylpropanamide (16e). 16e was
prepared following general procedure A as per Scheme 1. The crude
product was purified by a silica gel column using DCM and ethyl acetate
(
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide 13 (0.41
g, 0.00116 mol) or (S)-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-
methyloxirane-2-carboxamide 14 (0.313 g, 0.00116 mol) was added
to the above solution, and the resulting reaction mixture was allowed to
stir overnight at rt under argon atmosphere. The reaction was quenched
with water and extracted with ethyl acetate. The organic layer was
(19:1) as eluents to afford 0.25 g of the titled compound as an off-white
1
solid. Yield = 52%. H NMR (400 MHz, DMSO-d
): δ 10.36 (s, 1H,
6
NH), 8.45 (s, 1H, ArH), 8.23 (d, J = 8.8 Hz, J = 1.2 Hz, 1H, ArH), 8.10
(d, J = 8.8 Hz, 1H, ArH), 7.78 (s, 1H, pyrazole-H), 7.46 (s, 1H,
washed with brine, dried with MgSO , filtered, and concentrated under
pyrazole-H), 6.31 (s, 1H, OH), 4.48 (d, J = 14.0 Hz, 1H, CH), 4.31 (d, J
4
+
2
vacuum. The product was purified by a silica gel column using ethyl
acetate and hexanes (1:1) as eluents to afford 0.13 g of 10 as a white
solid.
= 14.0 Hz, 1H, CH), 1.35 (s, 3H, CH
calcd, 465.0035; found, 465.0045.
3
). HRMS [C15
H
F
13
IN
3
O
4
]:
4.1.4.7. (S)-3-(4-Acetyl-1H-pyrazol-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16f).
16f was prepared following general procedure A as per Scheme 1.
The product was purified by a silica gel column using DCM and ethyl
4.1.4.1. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(1H-pyrazol-1-yl)propanamide (16a). 16a was prepared
following general procedure A. The crude product was purified by a
silica gel column using ethyl acetate and hexanes (2:1) as eluents to
acetate (19:1) as eluents to afford 70 mg of the titled compound as a
1
1
afford 0.52 g of the titled compound as a white solid. Yield = 52%. H
yellowish solid. Yield = 20%. H NMR (400 MHz, DMSO-d ): δ 10.37
6
P
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
8
7
s, 1H, NH), 8.45 (d, J = 1.2 Hz, 1H, ArH), 8.25 (s, 1H, pyrazole-H),
.23 (d, J = 8.2 Hz, J = 1.2 Hz, 1H, ArH), 8.10 (d, J = 8.2 Hz, 1H, ArH),
.86 (s, 1H, pyrazole-H), 6.37 (s, 1H, OH), 4.50 (d, J = 14.0 Hz, 1H,
J = 8.8 Hz, 1H, ArH), 7.41 (s, 1H, pyrazole-H), 7.17 (s, 1H, pyrazole-
H), 6.24 (s, 1H, OH), 4.40 (d, J = 14.0 Hz, 1H, CH), 4.22 (d, J = 14.0
Hz, 1H, CH), 1.97 (s, 3H, CH ), 1.36 (s, 3H, CH ). HRMS
3
3
+
CH), 4.33 (d, J = 14.0 Hz, 1H, CH), 2.34 (s, 3H, CH ), 1.39 (s, 3H,
CH ). HRMS [C H F N O ]: calcd, 381.1175; found, 381.1178.
[C H F N O ]: calcd, 353.1225; found, 353.1232. Purity: 99.75%
3
16 16 3 4 2
+
(HPLC).
3
17 16
3
4
3
Purity: 95.66% (HPLC).
.1.4.8. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(4-(trifluoromethyl)-1H-pyrazol-1-yl)propanamide (16g).
6g was prepared following general procedure A as per Scheme 1. The
product was purified by a silica gel column using DCM and ethyl acetate
19:1) as eluents to afford 0.30 g of the titled compound as a white
4.1.4.14. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(4-phenyl-1H-pyrazol-1-yl)propanamide (16m). 16m was
prepared following general procedure A as per Scheme 1. The product
was purified by a silica gel column using ethyl acetate and hexanes (1:2)
4
1
as eluents to afford 0.90 g of the titled compound as white needles. Yield
1
(
= 68.5%. H NMR (400 MHz, DMSO-d
6
): δ 10.40 (s, 1H, NH), 8.46
1
foam. Yield = 50%. H NMR (400 MHz, DMSO-d ): δ 10.38 (s, 1H,
(d, J = 2.0 Hz, 1H, ArH), 8.24 (dd, J = 8.4 Hz, J = 2.0 Hz, 1H, ArH), 8.09
(d, J = 8.4 Hz, 1H, ArH), 8.05 (s, 1H, pyrazole-H), 7.82 (s, 1H,
pyrazole-H), 7.52−7.45 (m, 2H, ArH), 7.35−7.31 (m, 2H, ArH),
7.20−7.16 (m, 1H, ArH), 6.33 (s, 1H, OH), 4.50 (d, J = 14.0 Hz, 1H,
6
NH), 8.45 (d, J = 2.0 Hz, 1H, ArH), 8.25−8.22 (m, 2H, ArH &
pyrazole-H), 8.11 (d, J = 8.2 Hz, 1H, ArH), 7.82 (s, 1H, pyrazole-H),
6
1
4
.39 (s, 1H, OH), 4.55 (d, J = 14.0 Hz, 1H, CH), 4.37 (d, J = 14.0 Hz,
+
CH), 4.30 (d, J = 14.0 Hz, 1H, CH), 1.40 (s, 3H, CH ). HRMS
H, CH), 1.40 (s, 3H, CH ). HRMS [C H F N O ]: calcd,
3
3
16 13
6
4
2
+
[
C H F N O ], calcd, 415.1382; found, 415.1391.
07.0943; found, 407.0945.
.1.4.9. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(3-(trifluoromethyl)-1H-pyrazol-1-yl)propanamide (16h).
6h was prepared following general procedure A as per Scheme 1. The
21 18 3 4 2
4
.1.4.15. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
4
methyl-3-(3-phenyl-1H-pyrazol-1-yl)propanamide (16n). 16n was
prepared following general procedure A as per Scheme 1. The product
was purified by a silica gel column using ethyl acetate and hexanes (1:3
1
product was purified by a silica gel column using ethyl acetate and
to 1:2) as eluents to afford 0.60 g of the titled compound as white
hexanes (2:1) as eluents to afford 0.31 g of the titled compound as a
1
1
needles. Yield = 41.7%. H NMR (400 MHz, DMSO-d ): δ 10.33 (s,
white solid. Yield = 50%. H NMR (400 MHz, DMSO-d ): δ 10.31 (s,
6
6
1
1
7
1
H, NH), 8.48 (d, J = 2.0 Hz, 1H, ArH), 8.22 (dd, J = 8.2 Hz, J = 2.0 Hz,
H, ArH), 8.05 (d, J = 8.2 Hz, 1H, ArH), 7.69 (d, J = 2.0 Hz, 1H, ArH),
.60−7.57 (m, 2H, ArH), 7.28−7.21 (m, 3H, ArH), 6.66 (d, J = 3.0 Hz,
1
H, NH), 8.42 (d, J = 2.0 Hz, 1H, ArH), 8.18 (dd, J = 8.8 Hz, J = 2.0 Hz,
H, ArH), 8.09 (d, J = 8.8 Hz, 1H, ArH), 7.84−7.83 (m, 1H, pyrazole-
1
H), 6.67 (d, J = 2.4 Hz, 1H, pyrazole-H), 6.41 (s, 1H, OH), 4.56 (d, J =
H, ArH), 6.31 (s, 1H, OH), 4.52 (d, J = 14.6 Hz, 1H, CH), 4.32 (d, J =
1
4.0 Hz, 1H, CH), 4.38 (d, J = 14.0 Hz, 1H, CH), 1.40 (s, 3H, CH ).
3
+
2
+
14.6 Hz, 1H, CH), 1.43 (s, 3H, CH ). HRMS [C H F N O ]: calcd,
HRMS [C H F N O ]: calcd, 407.0943; found, 407.0945.
3
21 18
3
4
16
13
6
4
2
4
15.1382; found, 514.1423.
.1.4.16. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-(4-fluo-
rophenyl)-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide
16o). 16o was prepared following general procedure A as per Scheme
. The product was purified by a silica gel column using DCM and ethyl
4
.1.4.10. (S)-3-(4-Cyano-1H-pyrazol-1-yl)-N-(4-cyano-3-
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16i).
6i was prepared following general procedure A as per Scheme 1.
4
(
1
(
1
The product was purified by a silica gel column using hexanes and ethyl
acetate (1:1−1:2) as eluents to afford 0.18 g of the titled compound as a
1
acetate (19:1) as eluents to afford 0.33 g of the titled compound as a
white solid. Yield = 46%. H NMR (400 MHz, DMSO-d ): δ 10.35 (s,
6
1
white solid. Yield = 62%. H NMR (400 MHz, DMSO-d ): δ 10.29 (s,
6
1
H, NH), 8.45 (d, J = 1.2 Hz, 1H, ArH), 8.43 (s, 1H, pyrazole-H), 8.22
1
8
H, NH), 8.41 (s, 1H, ArH), 8.21 (d, J = 8.8 Hz, 1H, ArH), 8.05 (d, J =
.8 Hz, 1H, ArH), 7.68 (s, 1H, pyrazole-H), 7.61 (t, J = 6.4 Hz, 2H,
(d, J = 8.8 Hz, J = 1.2 Hz, 1H, ArH), 8.10 (d, J = 8.8 Hz, 1H, ArH), 7.98
s, 1H, pyrazole-H), 6.41 (s, 1H, OH), 4.45 (d, J = 14.0 Hz, 1H, CH),
(
ArH), 7.08 (t, J = 8.4 Hz, 2H, ArH), 6.65 (s, 1H, pyrazole-H), 6.30 (s,
4
.36 (d, J = 14.0 Hz, 1H, CH), 1.38 (s, 3H, CH ). HRMS
16 13 3 5 2
3
+
1H, OH), 4.51 (d, J = 14.0 Hz, 1H, CH), 4.31 (d, J = 14.0 Hz, 1H, CH),
[
C H F N O ]: calcd, 364.1021; found, 364.1016. Purity: 98.48%
+
2
1
.42 (s, 3H, CH ). HRMS [C H F N O ]: calcd, 433.1288; found,
3
21 17
4
4
(
HPLC).
.1.4.11. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
4
33.1291. Purity: 96.01% (HPLC).
.1.4.17. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3-(4-fluo-
rophenyl)-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide
16p). 16p was prepared following general procedure A as per Scheme
. The product was purified by a silica gel column using DCM and ethyl
4
4
methyl-3-(4-nitro-1H-pyrazol-1-yl)propanamide (16j). 16j was pre-
pared following general procedure A as per Scheme 1. The product was
purified by a silica gel column using hexanes and ethyl acetate (1:1) as
(
1
eluents to afford 0.15 g of the titled compound as an off-white solid.
acetate (19:1) as eluents to afford 0.27 g of the titled compound as a
1
Yield = 44%. H NMR (400 MHz, DMSO-d ): δ 10.36 (s, 1H, NH),
1
6
white solid. Yield = 43%. H NMR (400 MHz, DMSO-d ): δ 10.29 (s,
6
8
.69 (s, 1H, pyrazole-H), 8.45 (d, J = 1.2 Hz, 1H, ArH), 8.23 (d, J = 8.8
1
8
H, NH), 8.41 (s, 1H, ArH), 8.21 (d, J = 8.8 Hz, 1H, ArH), 8.05 (d, J =
.8 Hz, 1H, ArH), 7.69 (s, 1H, pyrazole-H), 7.61 (t, J = 6.4 Hz, 2H,
Hz, J = 1.2 Hz, 1H, ArH), 8.19 (s, 1H, pyrazole-H), 8.11 (d, J = 8.8 Hz,
1
1
3
H, ArH), 6.47 (s, 1H, OH), 4.56 (d, J = 14.0 Hz, 1H, CH), 4.38 (d, J =
ArH), 7.08 (t, J = 8.4 Hz, 2H, ArH), 6.65 (s, 1H, pyrazole-H), 6.30 (s,
+
4.0 Hz, 1H, CH), 1.41 (s, 3H, CH ). HRMS [C H F N O ]: calcd,
3
15 13
3
5
4
1H, OH), 4.51 (d, J = 14.0 Hz, 1H, CH), 4.31 (d, J = 14.0 Hz, 1H, CH),
84.0920; found, 384.0932. Purity: 99.58% (HPLC).
.1.4.12. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-
4-methoxy-1H-pyrazol-1-yl)-2-methylpropanamide (16k). 16k was
+
2
1
.42 (s, 3H, CH ). HRMS [C H F N O ]: calcd, 433.1288; found,
3
21 17
4
4
4
4
33.1290.
.1.4.18. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-ethynyl-
H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (16q). 16q was
(
4
prepared following general procedure A as per Scheme 1. The product
was purified by a silica gel column using DCM and ethyl acetate (9:1) as
1
prepared following general procedure A as per Scheme 1. The product
was purified by a silica gel column using DCM and ethyl acetate (95:5)
eluents to afford 0.30 g of the titled compound as a white solid. Yield =
1
6
0%. H NMR (400 MHz, DMSO-d ): δ 10.38 (s, 1H, NH), 8.46 (d, J
6
as eluents to afford 0.37 g of the titled compound as a white foam. Yield
=
=
2.0 Hz, 1H, ArH), 8.24 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, ArH), 8.10 (d, J
8.2 Hz, 1H, ArH), 7.35 (d, J = 0.8 Hz, 1H, pyrazole-H), 7.15 (d, J =
1
=
62.7%. H NMR (400 MHz, DMSO-d ): δ 10.40 (s, 1H, NH), 8.47
6
(
s, 1H, ArH), 8.24 (d, J = 8.8 Hz, 1H, ArH), 8.11 (d, J = 8.8 Hz, 1H,
0
.8 Hz, 1H, pyrazole-H), 6.25 (s, 1H, OH), 4.35 (d, J = 14.0 Hz, 1H,
ArH), 7.91 (s 1H, pyrazole-H), 7.57 (s 1H, pyrazole-H), 6.35 (s, 1H,
CH), 4.18 (d, J = 14.0 Hz, 1H, CH), 3.61 (s, 3H, CH ), 1.36 (s, 3H,
3
OH), 4.46 (d, J = 14.4 Hz, 1H, CH), 4.29 (d, J = 14.4 Hz, 1H, CH), 4.00
+
+
2
CH ). HRMS [C H F N O ]: calcd, 369.1175; found, 369.1182.
3
16 16
3
4
3
(s, 1H, CH), 1.35 (s, 3H, CH ). HRMS [C H F N O ]: calcd,
3
17 14
3
4
Purity: 99.28% (HPLC).
3
63.1069; found, 363.1026. Purity: 99.55% (HPLC).
4.1.4.13. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
4
.1.4.19. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-3-
methyl-3-(4-methyl-1H-pyrazol-1-yl)propanamide (16l). 16l was
prepared following general procedure A as per Scheme 1. The product
was purified by a silica gel column using DCM and ethyl acetate (19:1)
(4-(4-hydroxybut-1-yn-1-yl)-1H-pyrazol-1-yl)-2-methylpropana-
mide (16r). 16r was prepared following general procedure A as per
Scheme 1. The product was purified by a silica gel column using DCM
as eluents to afford 0.28 g of the titled compound as a white solid. Yield
and methanol (95:5) as eluents to afford 0.477 g of the titled compound
1
1
=
66%. H NMR (400 MHz, DMSO-d ): δ 10.38 (s, 1H, NH), 8.46 (d,
as a yellowish solid. Yield = 20%. H NMR (400 MHz, DMSO-d ): δ
6
6
J = 2.0 Hz, 1H, ArH), 8.23 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H, ArH), 8.10 (d,
12.99 (br s, 1H), 10.47 (s, 1H, NH), 8.55 (s, 1H, ArH), 8.29 (d, J = 8.8
Q
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
Hz, 1H, ArH), 8.08 (d, J = 8.8 Hz, 1H, ArH), 7.87 (s 1H, pyrazole-H),
1H, OH), 4.50 (d, J = 14.0 Hz, 1H), 4.13 (d, J = 14.0 Hz, 1H), 2.04 (s,
1
9
7
.49 (s 1H, pyrazole-H), 6.00 (s, 1H, OH), 3.64 (d, J = 8.2 Hz, 1H,
3H), 1.39 (s, 3H). F NMR (CDCl , decoupled): δ −62.20. MS (ESI)
3
+ −
CH), 4.50 (d, J = 9.6 Hz, 1H, CH), 3.60−3.56 (m, 2H, CH ), 2.59−
m/z: 356.11 [M + H] ; 354.06 [M − H] .
2
+
2
.55 (m, 2H, CH ), 1.31 (s, 3H, CH ). HRMS [C H F N O ]:
4.1.4.24. (S)-3-(4-(2-Chloroacetamido)-1H-pyrazol-1-yl)-N-(4-
cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropana-
mide (16w). Under argon atmosphere, to a solution of 16t (0.17 g, 0.48
mmol) and triethylamine (0.16 mL, 1.15 mmol) in 10 mL of anhydrous
DCM was added 2-chloroacetyl chloride (0.04 mL, 0.58 mmol) in an
ice−water bath. After stirring for 30 min, the temperature raised to rt
and the mixture was stirred for 2 h. The reaction mixture was condensed
under vacuum and then dispersed into 10 mL of ethyl acetate, washed
2
3
19 18
3
4
3
calcd, 407.1331; found, 407.1267.
.1.4.20. (S)-1-(3-((4-Cyano-3-(trifluoromethyl)phenyl)amino)-2-
hydroxy-2-methyl-3-oxopropyl)-1H-pyrazole-4-carboxamide (16s).
6s was prepared in two steps. In the first step, (S)-tert-butyl (1-(3-((4-
4
1
cyano-3-(trifluoromethyl)phenyl)amino)-2-hydroxy-2-methyl-3-oxo-
propyl)-1H-pyrazol-4-yl)carbamate 16u (an intermediate compound
for 16s) was synthesized following general procedure A as per Scheme 1
as described in detail infra. The compound was purified by a silica gel
with water, evaporated, dried over anhydrous MgSO , and evaporated
4
to dryness. The mixture was purified with flash column chromatography
column using hexanes and ethyl acetate (2:1) as eluents to afford the
1
using hexanes and ethyl acetate as eluents (2/1, v/v) to produce the
compound as a white solid. Yield = 69%. H NMR (400 MHz, CDCl ):
3
1
titled compound as yellow solids. Yield = 68%. H NMR (400 MHz,
δ 9.13 (br s, 1H, NH), 8.18 (d, J = 10.8 Hz, 1H), 8.02 (d, J = 1.2 Hz,
CDCl ): δ 9.12 (br s, 1H, NH), 8.12 (br s, 1H, NH), 7.99 (d, J = 1.6 Hz,
1
7
1
H), 7.94 (s, 1H), 7.89 (d, J = 8.4 Hz, 1H), 7.77 (d, J = 8.4 Hz, 1H),
3
1H), 7.92 (br s, 1H), 7.88 (dd, J = 8.6, 1.6 Hz, 1H), 7.78 (d, J = 8.6 Hz,
.66 (br s, C(O)NHC(O)), 5.79 (br s, 1H, OH), 4.70 (d, J = 13.8 Hz,
1
9
1H), 7.61 (br s, 1H), 6.11 (br s, 1H, OH), 4.60 (d, J = 13.8 Hz, 1H),
H), 4.32 (d, J = 13.8 Hz, 1H), 1.52 (s, 3H), 1.50 (s, 9H). F NMR
1
9
−
4.23 (d, J = 13.8 Hz, 1H), 4.17 (s, 2H), 1.47 (s, 3H); F NMR (CDCl ,
(
400 MHz, CDCl ): δ −62.20. MS (ESI) m/z: 480.23 [M − H] .
The second step: To a solution of 16u (0.721 g, 2.05 mmol) in EtOH
10 mL) was added dropwise acetyl chloride (3 mL) at 0 °C and further
3
3
+
decoupled): δ −62.19. MS (ESI) m/z: 452.01 [M + Na] ; 428.00 [M −
−
H] .
(
4
.1.4.25. (S)-Methyl (1-(3-((4-cyano-3-(trifluoromethyl)phenyl)-
stirred at rt for 3 h. After removing the solvent under reduced pressure,
amino)-2-hydroxy-2-methyl-3-oxopropyl)-1H-pyrazol-4-yl)-
carbamate (16x). Under argon atmosphere, to a solution of 16t (0.17
g, 0.48 mmol) and triethylamine (0.16 mL, 1.15 mmol) in 10 mL of
anhydrous DCM was added methyl carbonochloridate (0.04 mL, 0.58
mmol) in an ice−water bath. After stirring for 30 min, the temperature
raised to rt and the mixture was stirred for 2 h. The reaction mixture was
condensed under vacuum and then dispersed into 10 mL of ethyl
the mixture was treated with ethyl acetate and hexane (2:1) to afford the
desired compound as a yellowish solid. Yield = 95%. UV max 194.45,
1
2
8
70.45. H NMR (400 MHz, DMSO-d ): δ 10.39 (br s, 1H, NHC(O)),
6
.46 (d, J = 1.6 Hz, 1H), 8.20 (dd, J = 8.6, 1.6 Hz, 1H), 8.08 (d, J = 8.6
Hz, 1H), 8.05 (s, 1H), 7.75 (s, 1H), 7.55 (br s, 2H, C(O)NH ), 6.99 (br
2
s, 1H, OH), 4.45 (d, J = 13.8 Hz, 1H), 4.28 (d, J = 13.8 Hz, 1H), 1.34 (s,
1
9
3
[
H). F NMR (DMSO-d , decoupled): δ −61.13. HRMS
6
acetate, washed with water, evaporated, dried over anhydrous MgSO4,
and evaporated to dryness. The mixture was purified with flash column
+
C H F N O ]: calcd, 382.1127; found, 382.1282. HPLC purity:
16 15 3 5 3
9
8.75%.
.1.4.21. (S)-3-(4-Amino-1H-pyrazol-1-yl)-N-(4-cyano-3-
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16t).
chromatography using hexanes and ethyl acetate as eluents (2/1, v/v)
4
1
to produce the titled compound as a white solid. Yield = 71%. H NMR
(
(
400 MHz, CDCl ): δ 9.07 (br s, 1H,C(O)NH), 7.91 (s, 1H, ArH),
3
To a solution of 16u (see below) (0.815 g, 0.0018 mol) in absolute
EtOH (10 mL) was added acetyl chloride (0.4 mL, 5.4 mmol) at 0 °C
and further stirred at rt for 3 h. After removing the solvent under
vacuum, the resulting mixture was purified by flash column
chromatography using hexanes and ethyl acetate (1:1, v/v) to afford
7
.79 (d, J = 7.2 Hz, 1H, ArH), 7.69 (d, J = 7.2 Hz, 1H, ArH), 7.57 (s, 1H,
ArH), 7.40 (s, 1H, ArH), 6.33 (br s, 1H, NH), 6.08 (br s, 1H, OH), 4.50
d, J = 13.6 Hz, 1H, CH ), 4.12 (d, J = 13.6 Hz, 1H, CH ), 3.67 (s, 3H,
(
2
2
19
NH(CO)OCH ), 1.39 (s, 3H, CH ); F NMR (CDCl decoupled): δ
−
3
3
3,
‑
+
62.21. MS (ESI) m/z: 410.30 [M-H] ; 413.21 [M + H] .
.1.4.26. (S)-N-(6-Cyano-5-(trifluoromethyl)pyridin-3-yl)-3-(4-flu-
1
the desired compound as a brown solid. Yield = 91%. H NMR (400
4
MHz, DMSO-d ): δ 10.31 (br s, 1H, NH), 10.21 (br s, 2H, NH ), 8.20
6
2
oro-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (21a). 21a
was prepared following general procedure A as per Scheme 2, where 17
was 5-amino-3-(trifluoromethyl)picolinonitrile. The product was
purified by a silica gel column using hexanes and ethyl acetate (1:1)
(
7
=
1
s, 1H), 7.98 (d, J = 7.6 Hz, 1H), 7.77−7.73 (m, 2H), 7.62 (br s, 1H),
.21 (br s, 1H), 6.28 (br s, 1H, OH), 4.23 (d, J = 14.0 Hz, 1H), 4.04 (d, J
1
9
14.0 Hz, 1H), 1.04 (s, 3H); F NMR (acetone-d , decoupled): δ
6
+
−
14.77. MS (ESI) m/z: 354.08 [M + H] ; 351.98 [M − H] .
as eluents to afford 0.50 g of the titled compound as a white solid. Yield
4
.1.4.22. (S)-tert-Butyl (1-(3-((4-cyano-3-(trifluoromethyl)-
1
=
60.2%. H NMR (400 MHz, DMSO-d ): δ 10.64 (s, 1H, NH), 9.32
6
phenyl)amino)-2-hydroxy-2-methyl-3-oxopropyl)-1H-pyrazol-4-yl)-
carbamate (16u). 16u was prepared following general procedure A as
per Scheme 1. The product was purified by a silica gel column using
(d, J = 2.0 Hz, 1H, ArH), 8.82 (d, J = 2.0 Hz, 1H, ArH), 7.75 (d, J = 4.8
Hz, 1H, pyrazole-H), 7.40 (d, J = 4.0 Hz, 1H, pyrazole-H), 6.41 (s, 1H,
OH), 4.39 (d, J = 14.0 Hz, 1H, CH), 4.22 (d, J = 14.0 Hz, 1H, CH), 1.36
hexanes and ethyl acetate (2:1) as eluents to afford the desired
+
(
s, 3H, CH ). HRMS [C H F N O ]: calcd, 358.0927; found,
1
3
14 12
4
5
2
compound as a brown solid. H NMR (400 MHz, CDCl ): δ 9.12 (br s,
3
3
58.0932.
H, NH), 8.01 (d, J = 1.6 Hz, 1H), 7.85 (dd, J = 8.4, 1.6 Hz, 1H), 7.76
4
.1.4.27. (S)-N-(6-Cyano-5-(trifluoromethyl)pyridin-3-yl)-2-hy-
droxy-2-methyl-3-(4-(trifluoromethyl)-1H-pyrazol-1-yl)-
propanamide (21b). 21b was prepared following general procedure A
as per Scheme 2, where 17 was 5-amino-3-(trifluoromethyl)-
picolinonitrile. The product was purified by a silica gel column using
DCM and ethyl acetate (19:1) as eluents to afford 0.18 g of the titled
6
.17 (br s, 1H, OH), 4.54 (d, J = 14.0 Hz, 1H), 4.17 (d, J = 14.0 Hz,
1
9
1
H), 1.47 (s, 9H), 1.45 (s, 3H); F NMR (CDCl , decoupled): δ
3
‑
+
−
+
62.21. MS (ESI) m/z: 452.11 [M − H] ; 454.11 [M + H] ; 476.12 [M
+
Na] .
4
.1.4.23. (S)-3-(4-Acetamido-1H-pyrazol-1-yl)-N-(4-cyano-3-
1
compound as a white solid. Yield = 60%. H NMR (400 MHz, DMSO-
(
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (16v).
d6): δ 10.63 (s, 1H, NH), 9.31 (s, 1H, ArH), 8.80 (s, 1H, ArH), 8.32 (s,
Under argon atmosphere, to a solution of 16t (0.17 g, 0.48 mmol)
and triethylamine (0.16 mL, 1.15 mmol) in 10 mL of anhydrous DCM
was added acetyl chloride (0.04 mL, 0.58 mmol) in an ice−water bath.
After stirring for 30 min, the temperature raised to rt and the mixture
was stirred for 2 h. The reaction mixture was condensed under reduced
pressure and then dispersed into 10 mL of ethyl acetate, washed with
1
H, pyrazole-H), 7.81 (s, 1H, pyrazole-H), 6.48 (s, 1H, OH), 4.55 (d, J
=
14.0 Hz, 1H, CH), 4.37 (d, J = 14.0 Hz, 1H, CH), 1.42 (s, 3H, CH ).
3
+
HRMS [C H F N O ]: calcd, 408.0892; found, 408.0890. Purity:
1
5
12
6
5
2
9
6.81% (HPLC).
.1.4.28. (S)-3-(4-Cyano-1H-pyrazol-1-yl)-N-(6-cyano-5-
trifluoromethyl)pyridin-3-yl)-2-hydroxy-2-methylpropanamide
4
(
water, evaporated, dried over anhydrous MgSO , and evaporated to
(21c). 21c was prepared following general procedure A as per Scheme 2,
where 17 was 5-amino-3-(trifluoromethyl)picolinonitrile. The product
was purified by a silica gel column using hexanes and ethyl acetate (2:1)
as eluents to the titled compound as an off-white solid. Yield = 52%. mp
4
dryness. The mixture was purified with flash column chromatography
using hexanes and ethyl acetate as eluents (2/1, v/v) to produce the
1
desired compound as a yellow solid. Yield = 92%. H NMR (400 MHz,
1
CDCl ): δ 9.08 (br s, 1H, NH), 7.92 (br s, 1H, NH), 7.82−7.80 (m,
169.7−169.9 °C; UV max 195.45, 274.45 ; H NMR (400 MHz,
3
2
H), 7.69 (d, J = 8.4 Hz, 1H), 7.44 (br s, 1H), 7.15 (br s, 1H), 6.10 (br s,
CDCl ): δ 9.17 (br s, 1H, NH), 8.83 (s, 1H), 8.67 (d, J = 1.6 Hz, 1H),
3
R
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

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pubs.acs.org/jmc
Article
7
.92 (s, 1H), 7.85 (s, 1H), 5.58 (s, OH), 4.73 (d, J = 14.0 Hz, 1H), 4.34
allowed to stir overnight at rt under argon atmosphere. The reaction
was quenched by water and extracted with ethyl acetate. The organic
1
9
(
d, J = 14.0 Hz, 1H), 1.53 (s, 3H); F NMR (CDCl , decoupled): δ
3
+
2
−
62.11. HRMS [C H F N O ]: calcd, 365.0974; found, 365.0931.
layer was washed with brine, dried with MgSO , filtered, and
15
12
3
6
4
4
.1.4.29. (S)-tert-Butyl (1-(3-((6-cyano-5-(trifluoromethyl)pyridin-
concentrated under vacuum. The product was purified by a silica gel
column using DCM and methanol (19:1) as eluents to afford 50 mg of
3
-yl)amino)-2-hydroxy-2-methyl-3-oxopropyl)-1H-pyrazol-4-yl)-
1
carbamate (21d). 21d was prepared following general procedure A as
per Scheme 2, where 17 was 5-amino-3-(trifluoromethyl)-
picolinonitrile. The product was purified by a silica gel column using
hexanes and ethyl acetate (2:1) as eluents to afford the desired
the titled compound as a yellow solid. Yield = 13.7%. H NMR (400
MHz, DMSO-d ): δ 10.10 (s, 1H, NH), 9.54 (s, 1H, ArH), 9.21 (s, 1H,
6
ArH), 8.64 (d, J = 2.4 Hz, 1H, ArH), 8.22 (dd, J = 8.6 Hz, J = 2.4 Hz, 1H,
ArH), 7.97 (d, J = 8.6 Hz, 1H, ArH), 7.75 (d, J = 4.8 Hz, 1H, pyrazole-
H), 7.43 (d, J = 4.0 Hz, 1H, pyrazole-H), 6.26 (s, 1H, OH), 4.42 (d, J =
1
compound as a brown solid. Yield = 60%. H NMR (400 MHz,
CDCl ): δ 9.28 (br s, 1H, NH), 8.80 (s, 1H), 7.63 (br s, 1H), 7.43 (br s,
3
14.0 Hz, 1H, CH), 4.25 (d, J = 14.0 Hz, 1H, CH), 1.36 (s, 3H, CH ).
3
−
1
1
H), 6.29 (br s, 1H, NH), 6.21 (br s, 1H, OH), 4.55 (d, J = 14.0 Hz,
H), 4.17 (d, J = 14.0 Hz, 1H), 1.47 (s, 3H); F NMR (CDCl ,
MS (ESI) m/z: 314.05 [M − H] .
1
9
3
4.1.4.34. (S)-N-(2-Chloropyridin-4-yl)-3-(4-fluoro-1H-pyrazol-1-
yl)-2-hydroxy-2-methylpropanamide (21i). 21i was prepared in two
steps. In the first step, (R)-3-bromo-N-(2-chloropyridin-4-yl)-2-
hydroxy-2-methylpropanamide as an intermediate compound was
synthesized following Scheme 2, where 17 was 2-chloropyridin-4-
amine. Thionyl chloride (11.2 mL, 0.154 mol) was added dropwise to a
cooled solution (less than 4 °C) of 11 (18.3 g, 0.100 mol) in 100 mL of
THF under an argon atmosphere. The resulting mixture was stirred for
‑
decoupled): δ −62.11. MS (ESI) m/z: 453.16 [M − H] ; 477.16 [M +
Na] .
+
4
.1.4.30. (S)-N-(3-Chloro-4-cyanophenyl)-3-(4-fluoro-1H-pyrazol-
1
-yl)-2-hydroxy-2-methylpropanamide (21e). 21e was prepared
following general procedure A as per Scheme 2, where 17 was 4-
amino-2-chlorobenzonitrile. The product was purified by a silica gel
column using hexanes and ethyl acetate (2:1) as eluents to afford the
1
desired compound as a white solid. Yield = 71%. H NMR (400 MHz,
CDCl ): δ 8.97 (br s, 1H, NH), 7.87 (d, J = 2.0 Hz, 1H), 7.60 (d, J = 8.4
Hz, 1H), 7.45 (dd, J = 8.4, 2.0 Hz, 1H), 7.36 (dd, J = 8.8, J = 4.0 Hz,
3 h under the same condition. To this was added Et N (25.7 mL, 0.185
3
3
mol) and stirred for 20 min under the same condition. After 20 min, 2-
chloropyridin-4-amine (17; 9.89 g, 0.077 mol) and 100 mL of THF
were added and then the mixture was allowed to stir overnight at rt. The
solvent was removed under reduced pressure to afford a solid, which
1
H), 5.86 (br s, 1H, OH), 4.55 (d, J = 14.0 Hz, 1H), 4.16 (d, J = 14.0
1
9
Hz, 1H), 1.46 (s, 3H); F NMR (CDCl3, decoupled): δ −176.47.
HRMS [C H FN O ]: calcd, 323.0711; found, 323.0710.
+
14
13
4
2
was treated with 100 mL of H O, extracted with ethyl acetate (2 × 50
2
4
.1.4.31. (S)-3-(4-Fluoro-1H-pyrazol-1-yl)-2-hydroxy-2-methyl-N-
mL). The combined organic extracts were washed with saturated
(
4-nitro-3-(trifluoromethyl)phenyl)propanamide (21f). 21f was
NaHCO solution (2 × 100 mL) and brine (100 mL). The organic layer
3
prepared following general procedure A as per Scheme 2, where 17
was 4-nitro-3-(trifluoromethyl)aniline. The product was purified by a
silica gel column using hexanes and ethyl acetate (2:1) as eluents to
was dried over MgSO and concentrated under reduced pressure to
4
afford a solid, which was purified from column chromatography using
ethyl acetate and DCM (80:20) to afford a solid. This solid was
recrystallized from DCM and hexane to afford 12.6 g of the
1
afford the desired compound as a yellowish solid. Yield = 67%. H NMR
(
2
400 MHz, CDCl ): δ 9.14 (br s, 1H, NH), 8.01 (s, 1H), 7.97−7.91 (m,
1
3
intermediate compound as a light-yellow solid. Yield = 43%.
H
H), 7.38 (d, J = 3.6 Hz, 1H), 7.35 (d, J = 4.4 Hz, 1H), 5.95 (s, 1H,
NMR (400 MHz, CDCl ): δ 9.06 (br s, 1H, NH), 8.31 (d, J = 5.6 Hz,
3
OH), 4.56 (d, J = 14.0 Hz, 1H), 4.17 (d, J = 14.0 Hz, 1H), 1.48 (s, 3H);
1H), 7.77 (d, J = 0.8 Hz, 1H), 7.45 (dd, J = 5.6, 0.8 Hz, 1H), 4.81 (br s,
19
F NMR (CDCl decoupled): δ −60.13, −176.47. MS (ESI) m/z:
3,
−
1H, OH), 3.97 (d, J = 10.6 Hz, 1H), 3.60 (d, J = 10.6 Hz, 1H), 1.64 (s,
+
+
+
3
75.08 [M − H] ; 377.22 [M + H] ; 399.04 [M + Na] .
.1.4.32. (S)-5-(3-(4-Fluoro-1H-pyrazol-1-yl)-2-hydroxy-2-
3H). MS (ESI) m/z: 295.28 [M + H] .
4
In the second step, 21i was prepared following general procedure A
as per Scheme 2, where 18 was (R)-3-bromo-N-(2-chloropyridin-4-yl)-
methylpropanamido)picolinamide (21g). 21g was prepared follow-
ing general procedure A as per Scheme 2, where 17 of step a was 5-
cyano-6-(trifluoromethyl)picolinamide. In step c, to a solution of 4-
fluoro-pyrazole (20; 0.20 g, 0.0023237 mol) in anhydrous THF (5 mL)
which was cooled in an ice−water bath under an argon atmosphere was
added sodium hydride (60% dispersion in oil, 0.28 g, 0.0069711 mol).
After addition, the resulting mixture was stirred for 3 h. (R)-3-Bromo-
N-(6-cyanopyridin-3-yl)-2-hydroxy-2-methylpropanamide 18 (0.66 g,
2-hydroxy-2-methylpropanamide. The product was purified by a silica
gel column using hexanes and ethyl acetate (2:1) as eluents to afford the
1
desired compound as a white solid. Yield = 55%. H NMR (400 MHz,
CDCl ): δ 8.90 (br s, 1H, NH), 8.26 (d, J = 5.6 Hz, 1H), 7.63 (s, 1H),
3
7.75 (d, J = 4.2 Hz, 1H), 7.33 (d, J = 4.2 Hz, 1H), 7.31 (dd, J = 5.6, 1.2
Hz, 1H), 5.88 (s, 1H, OH), 4.53 (d, J = 13.6 Hz, 1H), 4.14 (d, J = 13.6
1
9
Hz, 1H), 1.45 (s, 3H); F NMR (CDCl decoupled): δ −176.47. MS
3
,
+
−
0
.0023237 mol) was added to the above solution, and the resulting
(
ESI) m/z: 298.98 [M + H] ; 296.96 [M − H] .
.1.4.35. (S)-N-(4-Cyano-2-iodo-5-(trifluoromethyl)phenyl)-3-(4-
reaction mixture was allowed to stir overnight at rt under argon
atmosphere. The reaction was quenched by water and extracted with
ethyl acetate. The organic layer was washed with brine, dried with
4
fluoro-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (21j).
21j was prepared following general procedure A as per Scheme 2,
where 17 of step (a) was 4-cyano-2-iodoaniline. In step (c), 20 was 4-
fluoro-1H-pyrazole (0.09 g, 0.001048 mol). The product was purified
by a silica gel column using hexanes and ethyl acetate (2:1 to 1:1) as
MgSO , filtered, and concentrated under vacuum. The product was
purified by a silica gel column using DCM and methanol (9:1) as
eluents to afford 0.10 g of the titled compound as a white solid. Yield =
4
1
1
4.1%. H NMR (400 MHz, DMSO-d ): δ 10.08 (s, 1H, NH), 8.89 (d, J
eluents to afford 0.32 g of the titled compound as a white solid. Yield =
6
1
=
2.4 Hz, 1H, ArH), 8.30 (dd, J = 8.2 Hz, J = 2.4 Hz, 1H, ArH), 8.01 (s,
H, NH), 7.98 (d, J = 8.2 Hz, 1H, ArH), 7.73 (d, J = 4.4 Hz, 1H,
pyrazole-H), 7.51 (s, 1H, NH), 7.42 (d, J = 4.0 Hz, 1H, pyrazole-H),
64%. H NMR (400 MHz, CDCl ): δ 9.60 (s, 1H, NH), 8.76 (s, 1H,
3
1
ArH), 8.69 (s, 1H, ArH), 7.76 (d, J = 4.8 Hz, 1H, pyrazole-H), 7.36 (d, J
= 4.4 Hz, 1H, pyrazole-H), 6.85 (s, 1H, OH), 4.39 (d, J = 14.0 Hz, 1H,
6
1
3
.24 (s, 1H, OH), 4.38 (d, J = 14.0 Hz, 1H, CH), 4.42 (d, J = 14.0 Hz,
CH), 4.20 (d, J = 14.0 Hz, 1H, CH), 1.41 (s, 3H, CH ). Mass (ESI) m/
3
+
−
H, CH), 1.34 (s, 3H, CH ). HRMS [C H FN O ]: calcd,
z: 481.00 [M − H] .
3
13 15
5
3
08.1159; found, 308.1177.
.1.4.33. (S)-3-(4-Fluoro-1H-pyrazol-1-yl)-2-hydroxy-2-methyl-N-
quinazolin-6-yl) propanamide (21h). 21h was prepared following
4.1.4.36. (S)-3-(4-Bromo-3-fluoro-1H-pyrazol-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (26a).
26a was prepared following general procedure A as per Scheme 3. To
a solution of 4-bromo-3-fluoro-pyrazole (25; 0.30 g, 0.001819 mol) in
anhydrous THF (10 mL), which was cooled in an ice−water bath under
an argon atmosphere, was added sodium hydride (60% dispersion in oil,
0.26 g, 0.006365 mol). After addition, the resulting mixture was stirred
for 3 h. 23 (where X is CH; 0.64 g, 0.001819 mol) was added to the
above solution, and the resulting reaction mixture was allowed to stir
overnight at rt under argon atmosphere. The reaction was quenched
with water and extracted with ethyl acetate. The organic layer was
4
(
general procedure A as per Scheme 2, where 17 of step (a) was
quinazolin-6-amine. In step (c), a solution of 4-fluoro-pyrazole (20;
0
.20 g, 0.0023237 mol) in anhydrous THF (5 mL), which was cooled in
an ice−water bath under an argon atmosphere, was added sodium
hydride (60% dispersion in oil, 0.28 g, 0.0069711 mol). After addition,
the resulting mixture was stirred for 3 h. (R)-3-Bromo-2-hydroxy-2-
methyl-N-(quinazolin-6-yl) propanamide (18; 0.72 g, 0.0023237 mol)
was added to the above solution, and the resulting reaction mixture was
S
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
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Article
washed with brine, dried with MgSO , filtered, and concentrated under
d6): δ 10.67 (s, 1H, NH), 9.32 (d, J = 2.0 Hz, 1H, ArH), 8.82 (d, J = 2.0
Hz, 1H, ArH), 7.85 (d, J = 2.0 Hz 1H, pyrazole-H), 6.47 (s, 1H, OH),
4.35 (d, J = 14.0 Hz, 1H, CH), 4.17 (d, J = 14.0 Hz, 1H, CH), 1.39 (s,
4
vacuum. The product was purified by a silica gel column using ethyl
acetate and hexanes (2:1) as eluents to afford 0.34 g of the titled
1
+
compound as a pinkish solid. Yield = 34%. mp 110−112 °C. H NMR
3H, CH ). HRMS [C H BrClF N O ]: calcd, 434.9954; found,
3
15 12
3
4
2
(
1
1
400 MHz, DMSO-d ): δ 10.38 (s, 1H, NH), 8.45 (d, J = 2.0−1.6 Hz,
435.9997. Purity: 93.41% (HPLC).
6
H, ArH), 8.23 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, ArH), 8.11 (d, J = 8.2 Hz,
H, ArH), 7.82 (d, J = 2.0 Hz, 1H, pyrazole-H), 6.35 (s, 1H, OH), 4.35
4.1.4.42. (S)-3-(3-Bromo-4-cyano-1H-pyrazol-1-yl)-N-(6-cyano-5-
(trifluoromethyl)pyridin-3-yl)-2-hydroxy-2-methylpropanamide
(26g). 26g was prepared following general procedure A as per Scheme
3, where X of 22 (step a) is N and 25 of step c is 3-bromo-4-cyano-
pyrazole. The product was purified by a silica gel column using hexanes
and ethyl acetate (2:1) as eluents to afford the titled compound as a
(
d, J = 14.0 Hz, 1H, CH), 4.04 (d, J = 14.0 Hz, 1H, CH), 1.37 (s, 3H,
+
CH ). HRMS [C H BrF N O ]: calcd, 435.0080; found, 435.0080.
3
15 12
4
4
2
Purity: 96.98% (HPLC).
.1.4.37. (S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3-fluoro-
-(4-fluorophenyl)-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropana-
mide (26b). 26b was prepared by Suzuki reaction mixing 26a (0.20 g,
.4596 mmol), 4-fluoro boronic acid (77 mg, 0.5515 mmol),
Pd(II)(OAc) (2−3 mg, 0.009192 mmol), PPh (7−8 mg, 0.02758
4
1
4
white solid. Yield = 81%. mp 172.5−173.6 °C; H NMR (400 MHz,
DMSO-d ): δ 10.60 (br s, 1H, NH), 9.29 (s, 1H), 8.79 (s, 1H), 8.53 (s,
6
0
1H), 6.59 (s, OH), 4.50 (d, J = 14.0 Hz, 1H), 4.32 (d, J = 14.0 Hz, 1H),
1
9
2
3
1.43 (s, 3H); F NMR (CDCl , decoupled): δ −61.25. HRMS
3
+
mmol), and K CO (0.13 g, 0.965 mmol) into ACN (4−5 mL) and
2
3
[C H BrF N O ]: calcd, 443.0079; found, 443.0083.
1
5
11
3
6
2
H O (2−3 mL). The mixture was degassed and refilled with argon three
2
4.1.4.43. (S)-3-(4-Cyano-3-phenyl-1H-pyrazol-1-yl)-N-(6-cyano-
5-(trifluoromethyl)pyridin-3-yl)-2-hydroxy-2-methylpropanamide
(26h). A flask equipped with a reflux condenser, a septum inlet, and a
magnetic stirring bar charged with 26g (0.053 g, 0.23 mmol),
tetrakis(triphenylphosphine) palladium (0) (9 mg, 0.07 mmol), and
phenyl boronic acid (35 mg, 0.28 mmol) in THF/MeOH (5 mL/1 mL)
with sodium carbonate (50 mg, 0.48 mmol) in deoxygenated water (1
mL) was stirred and heated to reflux for 2 h until the starting material
was not detectable on TLC. The mixture was cooled to rt and the
solvent was removed in vacuo, then poured into ethyl acetate (10 mL),
and extracted with ethyl acetate. The combined organic layers were
washed with sat. NH Cl and water and dried over MgSO . The solvent
times. The resulting reacting mixture was heated at reflux for 3 h under
argon atmosphere. The product was purified by a silica gel column using
hexanes and ethyl acetate (2:1−1:1) as eluents to afford 51 mg of the
1
titled compound as a yellowish solid. Yield = 25%. H NMR (400 MHz,
CDCl ): δ 9.12 (s, 1H, NH), 8.06 (d, J = 1.6 Hz, 1H, ArH), 7.85 (dd, J =
6
8
3
.2 Hz, J = 1.6 Hz, 1H, ArH), 7.77 (d, J = 8.2 Hz, 1H, ArH), 7.51 (d, J =
.0 Hz, 1H, pyrazole-H), 7.43−7.40 (m, 2H, ArH), 7.08−7.04 (m, 2H,
ArH), 4.57 (d, J = 10.5 Hz, 1H, CH), 4.17 (d, J = 10.5 Hz, 1H, CH),
+
1
.26 (s, 3H, CH ). HRMS [C H F N O ]: calcd, 451.1193; found,
3 21 16 5 4 2
4
51.1196.
.1.4.38. (S)-3-(3-Bromo-4-cyano-1H-pyrazol-1-yl)-N-(4-cyano-3-
trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide (26c).
6c was prepared following general procedure A as per Scheme 3,
4
4
4
(
2
was removed in vacuo and then purified by flash column
chromatography on silica gel using hexanes and ethyl acetate (1:1) as
where X of 22 (step a) is CH and 25 of step c is 3-bromo-4-cyano-
pyrazole. The product was purified by a silica gel column using ethyl
acetate and hexanes (2:1) as eluents to afford 0.10 g of the titled
eluents to afford 36 mg of the targeted compound as a yellowish solid.
1
Yield = 69%. mp 112.3−124.4 °C; H NMR (400 MHz, CDCl ): δ 9.17
3
(
br s, 1H, NH), 8.76 (s, 1H), 8.60 (s, 1H), 7.77 (s, 1H), 7.57−7.52 (m,
H), 7.18 (d, J = 8.8 Hz, 2H), 5.32 (s, OH), 4.60 (d, J = 14.0 Hz, 1H),
1
compound as an off-white solid. Yield = 20%. H NMR (400 MHz,
3
DMSO-d ): δ 10.32 (s, 1H, NH), 8.50 (s 1H, pyrazole-H), 8.41 (s, 1H,
19
6
4.23 (d, J = 14.0 Hz, 1H), 1.47 (s, 3H). F NMR (CDCl , decoupled):
3
ArH), 8.20 (d, J = 8.4 Hz, 1H, ArH), 8.11 (d, J = 8.4 Hz, 1H, ArH), 6.47
+
δ −62.09. HRMS [C H F N O ]: calcd, 441.1287; found, 441.1291.
2
1
16
3
6
2
(
s, 1H, OH), 4.52 (d, J = 13.6 Hz, 1H, CH), 4.33 (d, J = 13.6 Hz, 1H,
4
.1.4.44. (R)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-
+
CH), 1.41 (s, 3H, CH ). HRMS [C H BrF N O ]: calcd, 442.0126;
3
16 12
3
5
2
1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide (29a). 29a was
prepared following general procedure A as per Scheme 1 as exemplified
above for 10, except that instead of 11, the opposite isomer was used
found, 442.0109. Purity: 98.84% (HPLC).
.1.4.39. (S)-3-(3-Chloro-4-methyl-1H-pyrazol-1-yl)-N-(4-cyano-
-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
26d). 26d was prepared following general procedure A as per Scheme
, where X of 22 (step a) is CH and 25 of step c is 3-chloro-4-methyl-
4
3
(
[(S)-11 or (S)-3-bromo-2-hydroxy-2-methylpropanoic acid]. The
product was purified by a silica gel column using hexanes and ethyl
acetate (1:1) as eluents to afford the titled compound as a yellowish
3
pyrazole. The product was purified by a silica gel column using DCM
and ethyl acetate (98:2 to 95:5) as eluents to afford 0.27 g of the titled
24
1
solid. Yield = 64%. [α]D +126.7° (c 1.0, MeOH); H NMR (400 MHz,
CDCl ): δ 9.07 (br s, 1H, NH), 8.01 (d, J = 2.0 Hz, 1H), 7.95 (dd, J =
.4, J = 2.0 Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 7.38 (d, J = 4.0 Hz, 1H),
.34 (d, J = 4.4 Hz, 1H), 5.92 (s, OH), 4.54 (d, J = 14.0 Hz, 1H), 4.16
1
3
compound as a white solid. Yield = 54%. H NMR (400 MHz, DMSO-
8
7
d6): δ 10.33 (s, 1H, NH), 8.42 (d, J = 0.8 Hz, 1H, ArH), 8.21 (dd, J = 8.4
Hz, J = 0.8 Hz, 1H, ArH), 8.10 (d, J = 8.2 Hz, 1H, ArH), 7.50 (s 1H,
pyrazole-H), 6.29 (s, 1H, OH), 4.36 (d, J = 14.4 Hz, 1H, CH), 4.18 (d, J
1
9
(d, J = 14.4 Hz, 1H), 1.47 (s, 3H); F NMR (CDCl , decoupled): δ
3
+
2
−
62.23, −176.47. HRMS [C H F N O ]: calcd, 357.0975; found,
1
5
13
4
4
=
14.4 Hz, 1H, CH), 1.91 (s, 3H, CH ), 1.35 (s, 3H, CH ). HRMS
16 15 3 4 2
3
3
+
357.0984.
[
C H ClF N O ]: calcd, 387.0836; found, 387.0839. Purity: 97.07%
4
.1.4.45. N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-1H-
(
pyrazol-1-yl)-2-methylpropanamide (29b). 29b was prepared follow-
ing general procedure A as per Scheme 4, where Y in 27 is a tertiary
carbon [CH(CH )] instead of quaternary [C(OH)(CH )] like all
3
2
3
3
previous compounds. To a solution of 4-fluoro-pyrazole (20; 0.20 g,
.0023237 mol) in anhydrous THF (5 mL), which was cooled in an
where X of 22 (step a) is CH and 25 of step c is 3-bromo-4-chloro-
pyrazole. The product was purified by a silica gel column using DCM
and ethyl acetate (95:5) as eluents to afford 0.25 g of the titled
0
ice−water bath under an argon atmosphere, was added sodium hydride
(60% dispersion in oil, 0.28 g, 0.0069711 mol). After addition, the
resulting mixture was stirred for 3 h. 3-Bromo-N-(4-cyano-3-
1
compound as a white solid. Yield = 50%. H NMR (400 MHz, DMSO-
d6): δ 10.34 (s, 1H, NH), 8.41 (s, 1H, ArH), 8.20 (d, J = 8.8 Hz, 1H,
ArH), 8.11 (d, J = 8.8 Hz, 1H, ArH), 7.93 (s 1H, pyrazole-H), 6.39 (s,
(
0
trifluoromethyl)phenyl)-2-methylpropanamide (28; 0.78 g,
.0023237 mol) was added to the above solution, and the resulting
1
1
H, OH), 4.43 (d, J = 14.0 Hz, 1H, CH), 4.25 (d, J = 14.0 Hz, 1H, CH),
+
reaction mixture was allowed to stir overnight at rt under argon. The
reaction was quenched with water and extracted with ethyl acetate. The
.38 (s, 3H, CH ). HRMS [C H BrClF N O ]: calcd, 450.9784;
3
15 12
3
4
2
found, 450.9807. Purity: 96.55% (HPLC).
organic layer was washed with brine, dried with MgSO , filtered, and
4
.1.4.41. (S)-3-(4-Bromo-3-fluoro-1H-pyrazol-1-yl)-N-(6-cyano-5-
4
concentrated under vacuum. The product was purified by a silica gel
column using hexanes and ethyl acetate (1:1) as eluents to afford 0.050
(
(
trifluoromethyl)pyridin-3-yl)-2-hydroxy-2-methylpropanamide
26f). 26f was prepared following general procedure A as per Scheme 3,
1
where X of 22 (step a) is N and 25 of step c is 4-bromo-3-fluoro-
pyrazole. The product was purified by a silica gel column using hexanes
and ethyl acetate (2:1 to 1:1) as eluents to afford 0.28 g of the titled
g of the titled compound as a yellowish solid. Yield = 6.3%. H NMR
(400 MHz, DMSO-d ): δ 10.77 (s, 1H, NH), 8.25 (s, 1H, ArH), 8.10
6
(d, J = 8.2 Hz, 1H, ArH), 7.96 (d, J = 8.2 Hz, 1H, ArH), 7.85 (d, J = 4.4
Hz, 1H, pyrazole-H), 7.47 (d, J = 4.4 Hz, 1H, pyrazole-H), 4.35−4.30
1
compound as a white solid. Yield = 54%. H NMR (400 MHz, DMSO-
T
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
(
m, 1H, CH), 4.12−4.07 (m, 1H, CH), 3.12−3.10 (m, 1H, CH), 1.22
4.2. Biological Methods. 4.2.1. Competitive Ligand-Binding
Assay. AR ligand-binding assay was performed as described previously
using purified AR-LBD cloned from rat prostate.
+
d, J = 6.8 Hz, 3H, CH ). MS (ESI) m/z: 341.14 [M + H] .
3
1
,51
4
.1.4.46. N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-fluoro-1H-
pyrazol-1-yl)propanamide (29c). 29c was prepared following general
procedure A as per Scheme 4, where Y in 27 is secondary carbon
4.2.2. AR Transactivation Assay. HEK-293 cells plated in 24-well
plates at 70,000 cells/well were transfected using the lipofectamine
transfection reagent (Life Technologies, Carlsbad, CA). Cells were
transfected with 0.25 μg of GRE-LUC, 25 ng of CMV-hAR, and 10 ng
of CMV-LUC. Cells were treated 24 h after transfection and the
luciferase assay performed 48 h after transfection. Firefly luciferase
assay values were normalized to Renilla luciferase assay numbers.
4.2.3. Mutant AR (F876L) and wt PR Transactivation Assay. COS
cells were plated at 70,000 cells/well of a 24-well plate in DME plus 5%
csFBS without phenol red. Cells were transfected with 0.25 μg of GRE-
LUC, 10 ng of CMV-renilla LUC, and 50 ng of pCR3.1-hPR(wt) or
F876L AR using the lipofectamine transfection reagent in an optiMEM
medium. Medium was changed 24 h after transfection to DME +5%
csFBS without phenol red and treated with a dose response of various
drugs (1 pM to 10 μM) in the presence or absence of 0.1 nM
progesterone (PR) or R1881 (F876L AR). The luciferase assay was
performed 24 h after treatment on a Biotek synergy 4 plate reader.
Firefly luciferase values were normalized to Renilla luciferase values.
4.2.4. AR-Dependent Gene Expression in LNCaP Cells (Figure 4).
LNCaP cells were plated in 96-well plates in RPMI plus 1% csFBS
without phenol red. Cells were maintained in this medium for 2 days
and treated in the presence of 0.1 nM R1881. Twenty-four hours after
treatment, the cells were harvested, RNA was isolated, and cDNA was
prepared using Cells-to-CT kit (Life Technologies). Expression of
genes was measured using real-time PCR using TaqMan primers and
probe (Life Technologies).
[
CH ]] instead of quarternary [C(OH)(CH )]. To a solution of 4-
2
3
fluoro-pyrazole (20; 0.20 g, 0.0023237 mol) in anhydrous THF (5
mL), which was cooled in an ice−water bath under an argon
atmosphere, was added sodium hydride (60% dispersion in oil, 0.28
g, 0.0069711 mol). After addition, the resulting mixture was stirred for 3
h. 3-Bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)propanamide (28;
0
.75 g, 0.0023237 mol) was added to the above solution, and the
resulting reaction mixture was allowed to stir overnight at rt under
argon atmosphere. The reaction was quenched with water and extracted
with ethyl acetate. The organic layer was washed with brine, dried with
MgSO , filtered, and concentrated under vacuum. The product was
4
purified by a silica gel column using DCM and methanol (19:1) as
eluents to afford 0.75 mg of the titled compound as a white solid. Yield =
1
1
0%. H NMR (400 MHz, DMSO-d ): δ 10.81 (s, 1H, NH), 8.25 (d, J
6
=
=
2.4 Hz, 1H, ArH), 8.10 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H, ArH), 7.95 (d, J
8.8 Hz, 1H, ArH), 7.88 (s, 1H, pyrazole-H), 7.46 (s, 1H, pyrazole-H),
4
.35 (t, J = 6.0 Hz, 2H, CH ), 2.79 (t, J = 6.0 Hz, 2H, CH ). MS (ESI)
2
2
−
m/z: 325.03 [M − H] .
.1.4.47. (S)-4-(5-((4-Fluoro-1H-pyrazol-1-yl)methyl)-5-methyl-
,4-dioxooxazolidin-3-yl)-2-(trifluoromethyl)benzonitrile (29d).
4
2
Preparation of 29d proceeded by cyclization of the 2-methyl-2-
hydroxy-propanamide linker of 10 form an oxazolidinedione ring
system. To a solution of 10 (0.234 g, 0.0006568 mol) in anhydrous
pyridine (8 mL) was added 1,1′-carbonyldiimidazole (CDI) (0.16 g,
0
.0009825 mol). After addition, the resulting mixture was allowed to
4.2.5. Cellular Proliferation Assays in MR49F LNCaP Cells (Figure
5). MR49F cells were plated in 96-well plates in RPMI plus 1% csFBS
without phenol red. Cells were treated in this medium in the presence of
0.1 nM R1881 for 6 days with a medium change and retreatment after 3
days. The number of viable cells was measured using CellTiter-Glo
(Promega).
stir overnight at rt under argon atmosphere. The reaction was quenched
with water and extracted with ethyl acetate. The organic layer was
washed with brine, dried with MgSO , filtered, and concentrated under
4
vacuum. The product was purified by a silica gel column using hexanes
and ethyl acetate (2:1) as eluents to afford 0.134 g of the titled
1
4
.2.6. Western Blot. Indicated cell lines were treated for 24 h. Cells
compound as a white foam. Yield = 42%. H NMR (400 MHz, DMSO-
were harvested and protein-extracted, and Western blot for AR, AR-SV,
and GAPDH was performed using an AR PG-21 rabbit polyclonal
d6): δ 8.41 (d, J = 8.0 Hz, 1H, ArH), 7.98 (s, 1H, ArH), 7.94 (d, J = 4.0
Hz, 1H, pyrazole-H), 7.85 (d, J = 8.2 Hz, 1H, ArH), 7.58 (d, J = 4.4 Hz,
1
,51
antibody that binds to the N-terminus of the AR.
.2.7. In Vitro Metabolism Assays. In vitro metabolism assays were
1
1
3
H, pyrazole-H), 4.78 (d, J = 14.8 Hz, 1H, CH), 4.69 (d, J = 14.8 Hz,
+
4
H, CH), 1.71 (s, 3H, CH ). HRMS [C H F N O ]: calcd,
3
16 11
4
4
3
1
,51
performed as described before. Metabolism assays were performed
83.0767; found, 383.0726. Purity: 97.64% (HPLC).
.1.4.48. (S)-3-(4-Amino-1H-pyrazol-1-yl)-1-((4-cyano-3-
trifluoromethyl)phenyl)amino)-2-methyl-1-oxopropan-2-yl 2-
chloroacetate (29e). Under argon atmosphere, to a solution of 16t
0.17 g, 0.48 mmol) and triethylamine (0.16 mL, 1.15 mmol) in 10 mL
in MLM, RLM, and HLM as described before.
4.2.8. In Vivo PK in Rats. PK studies were conducted at Covance
using standard methods as briefly discussed below.
4
(
(
4.2.9. Animal Husbandry and Experimental Design. Male Sprague
Dawley rats from Envigo RMS, Inc. were acclimated to study conditions
for 5 days prior to initial dose administration. At initial dosing, the
animals were 12 weeks of age. The animals were group-housed (up to
three animals/cage/group) in polycarbonate cages with hardwood chip
bedding. Certified Rodent Diet #2016C (Envigo RMS, Inc.) was
provided ad libitum. Water was provided fresh daily, ad libitum.
Environmental controls for the animal room were set to maintain a
temperature of 20−26 °C, a relative humidity of 50 ± 20%, and a 12 h
light/12 h dark cycle. As necessary, the 12-h dark cycle was interrupted
to accommodate study procedures. The test article was prepared in 15%
dimethyl sulfoxide (DMSO)/85% polyethylene glycol (PEG) 300 by
Covance. Individual doses were calculated based on body weights
recorded on day 1 and day 7 of dose administration. A single oral daily
dose was administered via a gavage needle on seven consecutive days,
and blood was sampled as described below. A single iv dose was
administered via a tail vein and blood sample on day 1.
Additional detailed information for the 26a experiment, including
the groups, number of animals per group, dose (oral 5, 10, 20, and 30
mg/kg per day for 7 days; iv 10 mg/kg on day 1), and route, is given in
the Experimental Design section. Animals were observed for mortality
and signs of pain and distress twice daily (a.m. and p.m.), and cage side
observations for general health and appearance were done once daily.
Animals were weighed at the time of animal selection and on day 1 and
day 7 of dose administration.
of anhydrous DCM was added 2-chloroacetyl chloride (0.04 mL, 0.58
mmol) in an ice−water bath. After stirring for 30 min, the temperature
was raised to rt and the mixture was stirred for 2 h. The reaction mixture
was condensed under vacuum and then dispersed into 10 mL of EtOAc,
washed with water, evaporated, dried over anhydrous MgSO , and
4
evaporated to dryness. The mixture was purified with flash column
chromatography using hexanes and ethyl acetate as eluents (2/1, v/v)
1
to produce the titled compound as yellow solids. Yield = 19%. H NMR
(
(
(
400 MHz, CDCl ): δ 9.22 (br s, 1H, NH), 8.10 (br s, 2H, NH ), 7.93
3
2
d, J = 1.6 Hz, 1H), 7.89−7.86 (m, 2H), 7.78 (d, J = 8.4 Hz, 1H), 7.53
br s, 1H), 5.16 (d, J = 14.8 Hz, 1H), 4.61 (d, J = 14.8 Hz, 1H), 4.15 (s,
9
2
H), 1.78 (s, 3H); 1 F NMR (CDCl , decoupled): δ −62.19. MS (ESI)
3
+
−
m/z: 452.01 [M + Na] ; 428.03 [M − H] .
4
.1.4.49. (S)-N-(3-((4-Cyano-3-(trifluoromethyl)phenyl)amino)-2-
hydroxy-2-methyl-3-oxopropyl)-1H-pyrazole-4-carboxamide (29f).
9f was prepared following general procedure A as per Scheme 4. The
product was purified by a silica gel column using DCM and methanol
19:1) as an eluent to afford the titled compound as a brown solid. Yield
2
(
=
1
43%. H NMR (400 MHz, acetone-d ): δ 9.92 (br s, 1H, NHCO),
6
8
8
.44 (d, J = 1.8 Hz, 1H), 8.24 (dd, J = 8.8, J = 1.8 Hz, 1H), 8.12 (s, 1H),
.03 (d, J = 1.8 Hz, 1H), 7.84 (s, 1H), 7.11 (br s, 1H, NHCO), 6.38 (br
s, 1H, NH), 5.74 (s, OH), 4.67 (d, J = 14.0 Hz, 1H), 4.39 (d, J = 14.0
1
9
Hz, 1H), 1.50 (s, 3H). F NMR (acetone-d , decoupled): δ 114.69.
6
+
HRMS [C H F N O ]: calcd, 382.1127; found, 382.1051.
16
15
3
5
3
U
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
4
.2.10. Sample Collection. Blood (approximately 0.5 mL) was
Suriyan Ponnusamy − Department of Medicine, College of
Medicine, University of Tennessee Health Science Center,
Memphis, Tennessee 38163, United States
Thirumagal Thiyagarajan − Department of Medicine, College of
Medicine, University of Tennessee Health Science Center,
Memphis, Tennessee 38163, United States
Michael L. Mohler − Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States
Ramesh Narayanan − Department of Medicine, College of
Medicine, University of Tennessee Health Science Center,
Memphis, Tennessee 38163, United States
collected via the jugular vein via a syringe and a needle and transferred
into tubes containing K EDTA on days 1 and 7 from three animals/
group predose (day 7 only) and at approximately 0.083, 0.25, 0.5, 1, 3,
3
6
, 12, and 24 h postdose. For iv group, blood (approximately 0.5 mL)
was collected via the jugular vein at approximately 0.083, 0.25, 0.5, 1, 3,
, 12, and 24 h postdose. Blood was maintained in chilled cryoracks
prior to centrifugation to obtain plasma. Centrifugation began within 1
h of collection. Plasma was placed into 96-well tubes with barcode
labels. Plasma was maintained on dry ice prior to storage at
approximately −70 °C. Drug concentrations were measured by
established chromatography/mass spectrometry (LC−MS/MS) meth-
ods.
6
4
.2.11. Hershberger Assay. Male rats (6−8 weeks old) were
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c00943
randomized into groups based on body weight. Animals were treated
with drugs by oral administration as indicated in the figures for 14 days.
Animals were sacrificed, prostate and SVs were weighed, and organ
weights were normalized to body weight. Male rats (n = 5/group) were
left intact for 13 days. Intact rats were treated with the indicated
compounds at the indicated dose by mouth daily for 13 days. Rats were
sacrificed on day 14 of treatment and prostate and SVs organs were
removed and weighed. Organ weights were normalized to body weight.
This 20 mg/kg fixed dose screening Hershberger, which was performed
for 10, 21a, 16i (toxic so no data), and 26a. The goal of the experiments
was to find compounds with in vivo antiandrogen efficacies greater than
Author Contributions
This manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
This work was funded in part by a research grant from GTx, Inc.,
and Oncternal Therapeutics, Inc. (RN and DDM), and by a
research grant from National Cancer Institute (NCI) to RN
1
0.
4
.2.12. Xenograft Studies. Xenograft studies were performed at
(
1R01CA229164).
Hera Biolabs (Lexington, KY). Enz-R VCaP (MDVR VCaP; licensed
from Dr. Donald McDonnell, Duke University, Durham, NC) cells
were implanted subcutaneously in SRG rats (n = 5−7/group) (Hera
Notes
The authors declare no competing financial interest.
3
Biolabs). Once the tumors grow to 1000−2000 mm , the animals were
randomized and treated with the indicated drugs. Tumor volume was
measured thrice weekly. Thirty days after treatment, the animals were
sacrificed, tumors were weighed, and stored for further analysis.
ACKNOWLEDGMENTS
■
We thank GTx, Inc., and Oncternal Therapeutics, Inc., for
supporting this project and Dr. Dejian Ma of UTHSC, College
of Pharmacy, for assistance with HPLC purity and HRMS
experiments.
4
.2.13. Animal Studies. All animal studies were conducted under
UTHSC Animal Care and Use Committee approved protocols and in
accordance to the UTHSC guidelines. Xenograft studies were
conducted at HERA biolabs under ACUC protocols approved by the
University of Kentucky ACUC committee.
ABBREVIATIONS
■
AR, androgen receptor; PC, prostate cancer; CRPC, castration-
resistant prostate cancer; SARDs, selective androgen receptor
degraders; DMPK, distribution, metabolism, and pharmacoki-
netic; Enz-R, enzalutamide-resistant; SV, splice variant; NTD,
N-terminal domain; DBD, DNA binding domain; LBD, ligand
binding domain; AF-1, an activation function; AF-2, an external
ASSOCIATED CONTENT
■
*
sı Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00943.
Additional information on compound characterization,
additional biological experiments, and figures (PDF)
(
solvent exposed) binding site termed activation function-2;
DHT, 5α-dihydrotestosterone; SAR, structure−activity rela-
tionship; PD, pharmacodynamics; PK, pharmacokinetic; ADT,
androgen deprivation therapy; CSPC, castration sensitive PC;
wt, wild-type; UPS, ubiquitin proteasome system; SV, seminal
vesicles; VP, ventral prostate; FL, full length; EWGs, with-
drawing groups; SARM, selective androgen receptor modulator;
GR, glucocorticoid receptor; ER, estrogen receptor; PR,
progesterone receptor; MLM, mouse liver microsomes; HLM,
human liver microsomes; RLM, rat liver microsomes; RLU,
relative light units; MIB, mibolerone; PROTACs, proteolysis-
targeting chimaeras; csFBS, charcoal-stripped fetal bovine
serum; GRE, glucocorticoid response element; NSG, NOD
SCID gamma; TGI, tumor growth inhibition
Molecular formula strings (CSV)
PK method in rats for 21a (PDF)
AUTHOR INFORMATION
■
Corresponding Author
Duane D. Miller − Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States; orcid.org/0000-0002-
6
093-0985; Phone: (+1)-901-448-6026; Email: dmiller@
uthsc.edu; Fax: (+1)-901-448-3446
Authors
REFERENCES
■
Yali He − Department of Pharmaceutical Sciences, University of
Tennessee Health Science Center, Memphis, Tennessee 38163,
United States
Dong-Jin Hwang − Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis,
Tennessee 38163, United States
(
1) Ponnusamy, S.; Coss, C. C.; Thiyagarajan, T.; Watts, K.; Hwang,
D.-J.; He, Y.; Selth, L. A.; McEwan, I. J.; Duke, C. B.; Pagadala, J.; Singh,
G.; Wake, R. W.; Ledbetter, C.; Tilley, W. D.; Moldoveanu, T.; Dalton,
J. T.; Miller, D. D.; Narayanan, R. Novel selective agents for the
degradation of androgen receptor variants to treat castration-resistant
prostate cancer. Cancer Res. 2017, 77, 6282−6298.
V
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
2) Ponnusamy, S.; He, Y.; Hwang, D.-J.; Thiyagarajan, T.; Houtman,
(20) Scher, H. I.; Sawyers, C. L. Biology of progressive, castration-
resistant prostate cancer: directed therapies targeting the androgen-
receptor signaling axis. J. Clin. Oncol. 2005, 23, 8253−8261.
(21) Ito, Y.; Sadar, M. D. Enzalutamide and blocking androgen
receptor in advanced prostate cancer: lessons learnt from the history of
drug development of antiandrogens. Res. Rep. Urol. 2018, 10, 23−32.
(22) Harris, W. P.; Mostaghel, E. A.; Nelson, P. S.; Montgomery, B.
Androgen deprivation therapy: progress in understanding mechanisms
of resistance and optimizing androgen depletion. Nat. Clin. Pract. Urol.
2009, 6, 76.
R.; Bocharova, V.; Sumpter, B. G.; Fernandez, E.; Johnson, D.; Du, Z.;
Pfeffer, L. M.; Getzenberg, R. H.; McEwan, I. J.; Miller, D. D.;
Narayanan, R. Orally bioavailable androgen receptor degrader,
potential next-generation therapeutic for enzalutamide-resistant
prostate cancer. Clin. Cancer Res. 2019, 25, 6764−6780.
(
3) Aragon-Ching, J. B. The evolution of prostate cancer therapy:
targeting the androgen receptor. Front. Oncol. 2014, 4, 295.
4) Siegel, R. L.; Miller, K. D.; Jemal, A. Cancer statistics, 2015. Ca-
Cancer J. Clin. 2015, 65, 5−29.
5) Chen, C. D.; Welsbie, D. S.; Tran, C.; Baek, S. H.; Chen, R.;
(
(
(23) Tan, M. E.; Li, J.; Xu, H. E.; Melcher, K.; Yong, E.-l. Androgen
receptor: structure, role in prostate cancer and drug discovery. Acta
Pharmacol. Sin. 2014, 36, 3.
Vessella, R.; Rosenfeld, M. G.; Sawyers, C. L. Molecular determinants of
resistance to antiandrogen therapy. Nat. Med. 2003, 10, 33.
(
6) Torre, L. A.; Bray, F.; Siegel, R. L.; Ferlay, J.; Lortet-tieulent, J.;
Jemal, A. Global cancer statistics, 2012. Ca-Cancer J. Clin. 2015, 65, 87−
08.
7) Tian, X.; He, Y.; Zhou, J. Progress in antiandrogen design targeting
(24) Visakorpi, T.; Hyytinen, E.; Koivisto, P.; Tanner, M.; Keinanen,
̈
R.; Palmberg, C.; Palotie, A.; Tammela, T.; Isola, J.; Kallioniemi, O.-P.
In vivo amplification of the androgen receptor gene and progression of
human prostate cancer. Nat. Genet. 1995, 9, 401.
1
(
hormone binding pocket to circumvent mutation based resistance.
Front. Pharmacol. 2015, 6, 57.
(25) Linja, M. J.; Savinainen, K. J.; Saramaki, O. R.; Tammela, T. L. J.;
̈
Vessella, R. L.; Visakorpi, T. Amplification and overexpression of
androgen receptor gene in hormone-refractory prostate cancer. Cancer
Res. 2001, 61, 3550−3555.
(
8) Delaere, K. P.; Van Thillo, E. L. Flutamide monotherapy as
primary treatment in advanced prostatic carcinoma. Semin. Oncol. 1991,
8, 13−18.
9) Kolvenbag, G. J. C. M.; Blackledge, G. R.; Gotting-Smith, K.
1
(
(26) Yamaoka, M.; Hara, T.; Kusaka, M. Overcoming persistent
dependency on androgen signaling after progression to castration-
resistant prostate cancer. Clin. Cancer Res. 2010, 16, 4319−4324.
(27) Veldscholte, J.; Ris-Stalpers, C.; Kuiper, G. G. J. M.; Jenster, G.;
Berrevoets, C.; Claassen, E.; van Rooij, H. C. J.; Trapman, J.;
Brinkmann, A. O.; Mulder, E. A mutation in the ligand binding domain
of the androgen receptor of human INCaP cells affects steroid binding
characteristics and response to anti-androgens. Biochem. Biophys. Res.
Commun. 1990, 173, 534−540.
Bicalutamide (casodex) in the treatment of prostate cancer: history of
clinical development. Prostate 1998, 34, 61−72.
(
10) Scher, H. I.; Beer, T. M.; Higano, C. S.; Anand, A.; Taplin, M.-E.;
Efstathiou, E.; Rathkopf, D.; Shelkey, J.; Yu, E. Y.; Alumkal, J.; Hung,
D.; Hirmand, M.; Seely, L.; Morris, M. J.; Danila, D. C.; Humm, J.;
Larson, S.; Fleisher, M.; Sawyers, C. L. Antitumour activity of
MDV3100 in castration-resistant prostate cancer: a phase 1-2 study.
Lancet 2010, 375, 1437−1446.
(28) Grasso, C. S.; Wu, Y.-M.; Robinson, D. R.; Cao, X.;
Dhanasekaran, S. M.; Khan, A. P.; Quist, M. J.; Jing, X.; Lonigro, R.
J.; Brenner, J. C.; Asangani, I. A.; Ateeq, B.; Chun, S. Y.; Siddiqui, J.;
Sam, L.; Anstett, M.; Mehra, R.; Prensner, J. R.; Palanisamy, N.; Ryslik,
G. A.; Vandin, F.; Raphael, B. J.; Kunju, L. P.; Rhodes, D. R.; Pienta, K.
J.; Chinnaiyan, A. M.; Tomlins, S. A. The mutational landscape of lethal
castration-resistant prostate cancer. Nature 2012, 487, 239.
(29) Scher, H. I.; Sayers, C. L. Biology of progressive, castration-
resistant prostate cancer: directed therapies targeting the androgen-
receptor signaling axis. J. Clin. Oncol. 2005, 23, 8253.
(30) Gregory, C. W.; He, B.; Johnson, R. T.; Ford, O. H.; Mohler, J. L.;
French, F. S.; Wilson, E. M. A Mechanism for androgen receptor-
mediated prostate cancer recurrence after androgen deprivation
therapy. Cancer Res. 2001, 61, 4315−4319.
(
11) Chong, J.; Oh, W.; Liaw, B. Profile of apalutamide in the
treatment of metastatic castration-resistant prostate cancer: evidence to
date. OncoTargets Ther. 2018, 11, 2141−2147.
(
12) Dellis, A. E.; Papatsoris, A. G. Apalutamide: The established and
emerging roles in the treatment of advanced prostate cancer. Expet
Opin. Invest. Drugs 2018, 27, 553−559.
(
13) Sugawara, T.; Baumgart, S. J.; Nevedomskaya, E.; Reichert, K.;
Steuber, H.; Lejeune, P.; Mumberg, D.; Haendler, B. Darolutamide is a
potent androgen receptor antagonist with strong efficacy in prostate
cancer models. Int. J. Cancer 2019, 145, 1382−1394.
(
14) Bastos, D. A.; Antonarakis, E. S. Darolutamide for castration-
resistant prostate cancer. OncoTargets Ther. 2019, 12, 8769−8777.
(
15) Attard, G.; Reid, A. H. M.; Auchus, R. J.; Hughes, B. A.; Cassidy,
A. M.; Thompson, E.; Oommen, N. B.; Folkerd, E.; Dowsett, M.; Arlt,
W.; de Bono, J. S. Clinical and biochemical consequences of CYP17A1
inhibition with abiraterone given with and without exogenous
glucocorticoids in castrate men with advanced prostate cancer. J. Clin.
Endocrinol. Metab. 2012, 97, 507−516.
(31) Wen, Y.; Hu, M. C.; Makino, K.; Spohn, B.; Bartholomeusz, G.;
Yan, D. H.; Hung, M. C. HER-2/neu promotes androgen-independent
survival and growth of prostate cancer cells through the Akt pathway.
Cancer Res. 2000, 60, 6841.
(32) Majumder, P. K.; Sellers, W. R. Akt-regulated pathways in
prostate cancer. Oncogene 2005, 24, 7465−7474.
(33) Ueda, T.; Sadar, M. D.; Suzuki, H.; Akakura, K.; Sakamoto, S.;
Shimbo, M.; Suyama, T.; Imamoto, T.; Komiya, A.; Yukio, N.;
Ichikawa, T. Interleukin-4 in patients with prostate cancer. Anticancer
Res. 2005, 25, 4595−4598.
(34) Ueda, T.; Mawji, N. R.; Bruchovsky, N.; Sadar, M. D. Ligand-
independent activation of the androgen receptor by interleukin-6 and
the role of steroid receptor coactivator-1 in prostate cancer cells. J. Biol.
Chem. 2002, 277, 38087−38094.
(35) Culig, Z.; Hobisch, A.; Cronauer, M. V.; Radmayr, C.; Trapman,
J.; Hittmair, A.; Bartsch, G.; Klocke, R. H. Androgen receptor activation
in prostatic tumor cell lines by insulin-like growth factor-I, keratinocyte
growth factor, and epidermal growth factor. Cancer Res. 1994, 54,
5447−5448.
(
16) Chi, K. N.; Agarwal, N.; Bjartell, A.; Chung, B. H.; Pereira de
́
Santana Gomes, A. J.; Given, R.; Juar
́
ez Soto, A.; Merseburger, A. S.;
̈
Ozgu
̈
rog
̆
lu, M.; Uemura, H.; Ye, D.; Deprince, K.; Naini, V.; Li, J.;
Cheng, S.; Yu, M. K.; Zhang, K.; Larsen, J. S.; McCarthy, S.;
Chowdhury, S.; TITAN Investigators. Apalutamide for metastatic,
castration-sensitive prostate cancer. N. Engl. J. Med. 2019, 381, 13−24.
(
17) Heidegger, I.; Brandt, M. P.; Heck, M. M. Treatment of non-
metastatic castration resistant prostate cancer in 2020: What is the best?
Urol. Oncol.: Semin. Orig. Invest. 2020, 38, 129−136.
(
18) Nuhn, P.; De Bono, J. S.; Fizazi, K.; Freedland, S. J.; Grilli, M.;
Kantoff, P. W.; Sonpavde, G.; Sternberg, C. N.; Yegnasubramanian, S.;
Antonarakis, E. S. Update on systemic prostate cancer therapies:
management of metastatic castration-resistant prostate cancer in the era
of precision oncology. Eur. Urol. 2019, 75, 88−99.
(
19) Kwan, E. M.; Thangasamy, I. A.; Teh, J.; Alghazo, O.;
(36) Antonarakis, E. S.; Chandhasin, C.; Osbourne, E.; Luo, J.; Sadar,
M. D.; Perabo, F. Targeting the N-Terminal Domain of the Androgen
Receptor: A New Approach for the Treatment of Advanced Prostate
Cancer. Oncologist 2016, 21, 1427−1435.
Sathianathen, N. J.; Lawrentschuk, N.; Azad, A. A. Navigating systemic
̈
therapy for metastatic castration-naive prostate cancer. World J. Urol.
020, 38. DOI: 10.1007/s00345-019-03060-7.
2
W
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
(
37) Dutt, S. S.; Gao, A. C. Molecular mechanisms of castration-
resistant prostate cancer progression. Future Oncol. 2009, 5, 1403−
413.
38) De Maeseneer, D. J.; Van Praet, C.; Lumen, N.; Rottey, S.
(54) Hershberger, L. G.; Shipley, E. G.; Meyer, R. K. Myotrophic
activity of 19-nortestosterone and other steroids determined by
modified levator ani muscle method. Proc. Soc. Exp. Biol. Med. 1953,
83, 175−180.
1
(
Battling resistance mechanisms in antihormonal prostate cancer
treatment: novel agents and combinations. Urol. Oncol.: Semin. Orig.
Invest. 2015, 33, 310−321.
(55) Youson, J. H. Absorption and transport of ferritin and exogenous
horseradish peroxidase in the opisthonephric kidney of the sea lamprey.
I. The renal corpuscle. Can. J. Zool. 1975, 53, 571−581.
(56) Clegg, N. J.; Wongvipat, J.; Joseph, J. D.; Tran, C.; Ouk, S.;
(
39) Taplin, M. E.; Bubley, G. J.; Ko, Y.-J.; Small, E. J.; Upton, M.;
Dilhas, A.; Chen, Y.; Grillot, K.; Bischoff, E. D.; Cai, L.; Aparicio, A.;
Dorow, S.; Arora, V.; Shao, G.; Qian, J.; Zhao, H.; Yang, G.; Cao, C.;
Sensintaffar, J.; Wasielewska, T.; Herbert, M. R.; Bonnefous, C.;
Darimont, B.; Scher, H. I.; Smith-Jones, P.; Klang, M.; Smith, N. D.; De
Stanchina, E.; Wu, N.; Ouerfelli, O.; Rix, P. J.; Heyman, R. A.; Jung, M.
E.; Sawyers, C. L.; Hager, J. H. ARN-509: a novel antiandrogen for
prostate cancer treatment. Cancer Res. 2012, 72, 1494.
Rajeshkumar, B.; Balk, S. P. Selection for androgen receptor mutations
in prostate cancers treated with androgen antagonist. Cancer Res. 1999,
5
(
9, 2511−2515.
40) Borgmann, H.; Lallous, N.; Ozistanbullu, D.; Beraldi, E.; Paul, N.;
Dalal, K.; Fazli, L.; Haferkamp, A.; Lejeune, P.; Cherkasov, A.; Gleave,
M. E. Moving towards precision urologic oncology: targeting
enzalutamide-resistant prostate cancer and mutated forms of the
androgen receptor using the novel inhibitor darolutamide (ODM-201).
Eur. Urol. 2018, 73, 4−8.
(57) Moilanen, A.-M.; Riikonen, R.; Oksala, R.; Ravanti, L.; Aho, E.;
Wohlfahrt, G.; Nykanen, P. S.; Tormakangas, O. P.; Palvimo, J. J.;
Kallio, P. J. Discovery of ODM-201, a new-generation androgen
receptor inhibitor targeting resistance mechanisms to androgen
signaling-directed prostate cancer therapies. Sci. Rep. 2015, 5, 12007.
(
41) Li, Y.; Yang, R.; Henzler, C. M.; Ho, Y.; Passow, C.; Auch, B.;
Carreira, S.; Nava Rodrigues, D.; Bertan, C.; Hwang, T. H.; Quigley, D.
A.; Dang, H. X.; Morrissey, C.; Fraser, M.; Plymate, S. R.; Maher, C. A.;
Feng, F. Y.; de Bono, J. S.; Dehm, S. M. Diverse AR gene
rearrangements mediate resistance to androgen receptor inhibitors in
metastatic prostate cancer. Clin. Cancer Res. 2020, 26, 1965−1976.
(58) Clegg, N. J.; Wongvipat, J.; Joseph, J. D.; Tran, C.; Ouk, S.;
Dilhas, A.; Chen, Y.; Grillot, K.; Bischoff, E. D.; Cai, L.; Aparicio, A.;
Dorow, S.; Arora, V.; Shao, G.; Qian, J.; Zhao, H.; Yang, G.; Cao, C.;
Sensintaffar, J.; Wasielewska, T.; Herbert, M. R.; Bonnefous, C.;
Darimont, B.; Scher, H. I.; Smith-Jones, P.; Klang, M.; Smith, N. D.; De
Stanchina, E.; Wu, N.; Ouerfelli, O.; Rix, P. J.; Heyman, R. A.; Jung, M.
E.; Sawyers, C. L.; Hager, J. H. ARN-509: a novel antiandrogen for
prostate cancer treatment. Cancer Res. 2012, 72, 1494−1503.
(
42) Sadar, M. D. Small molecule inhibitors targeting the ‘Achilles’
heel of androgen receptor activity. Cancer Res. 2011, 71, 1208−1213.
43) Crews, C. M. Inducing Protein Degradation as a Therapeutic
Strategy. J. Med. Chem. 2018, 61, 403−404.
44) Han, X.; Wang, C.; Qin, C.; Xiang, W.; Fernandez-Salas, E.; Yang,
(
(
C.-Y.; Wang, M.; Zhao, L.; Xu, T.; Chinnaswamy, K.; Delproposto, J.;
Stuckey, J.; Wang, S. Discovery of ARD-69 as a highly potent
proteolysis targeting chimera (PROTAC) degrader of androgen
receptor (AR) for the treatment of prostate cancer. J. Med. Chem.
2
(
019, 62, 941−964.
45) Churcher, I. Protac-induced protein degradation in drug
discovery: breaking the rules or just making new ones? J. Med. Chem.
018, 61, 444−452.
46) Shibata, N.; Nagai, K.; Morita, Y.; Ujikawa, O.; Ohoka, N.;
2
(
Hattori, T.; Koyama, R.; Sano, O.; Imaeda, Y.; Nara, H.; Cho, N.; Naito,
M. Development of protein degradation inducers of androgen receptor
by conjugation of androgen receptor ligands and inhibitor of apoptosis
protein ligands. J. Med. Chem. 2018, 61, 543−575.
(
47) Kim, D. H.; Rossi, J. J. Strategies for silencing human disease
using RNA interference. Nat. Rev. Genet. 2007, 8, 173.
48) Davidson, B. L.; McCray, P. B., Jr. Current prospects for RNA
interference-based therapies. Nat. Rev. Genet. 2011, 12, 329.
49) Salami, J.; Alabi, S.; Willard, R. R.; Vitale, N. J.; Wang, J.; Dong,
(
(
H.; Jin, M.; McDonnell, D. P.; Crew, A. P.; Neklesa, T. K.; Crews, C. M.
Androgen receptor degradation by the proteolysis-targeting chimera
ARCC-4 outperforms enzalutamide in cellular models of prostate
cancer drug resistance. Commun. Biol. 2018, 1, 100.
(
2
(
50) Stone, L. UT-34: a promising new AR degrader. Nat. Rev. Urol.
019, 16, 640.
51) Hwang, D.-J.; He, Y.; Ponnusamy, S.; Mohler, M. L.;
Thiyagarajan, T.; McEwan, I. J.; Narayanan, R.; Miller, D. D. New
generation of selective androgen receptor degraders: our initial design,
synthesis, and biological evaluation of new compounds with
enzalutamide-resistant prostate cancer activity. J. Med. Chem. 2019,
6
(
2, 491−511.
52) de Jesus Cortez, F.; Nguyen, P.; Truillet, C.; Tian, B.;
Kuchenbecker, K. M.; Evans, M. J.; Webb, P.; Jacobson, M. P.;
Fletterick, R. J.; England, P. M. Development of 5N-bicalutamide, a
high-affinity reversible covalent antiandrogen. ACS Chem. Biol. 2017,
1
(
2, 2934−2939.
53) Okegawa, T.; Ninomiya, N.; Masuda, K.; Nakamura, Y.; Tambo,
M.; Nutahara, K. AR-V7 in circulating tumor cells cluster as a predictive
biomarker of abiraterone acetate and enzalutamide treatment in
castration-resistant prostate cancer patients. Prostate 2018, 78, 576−
5
82.
X
https://dx.doi.org/10.1021/acs.jmedchem.0c00943
J. Med. Chem. XXXX, XXX, XXX−XXX