Exploration and Biological Evaluation of Basic Heteromonocyclic Propanamide Derivatives as SARDs for the Treatment of Enzalutamide-Resistant Prostate Cancer

Article abstract of DOI:10.1021/acs.jmedchem.1c00439

A series of propanamide derivatives were designed, synthesized, and pharmacologically characterized as selective androgen receptor degraders (SARDs) and pan-antagonists that exert a broad-scope androgen receptor (AR) antagonism. Incorporating different ba

Full text of DOI:10.1021/acs.jmedchem.1c00439

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Exploration and Biological Evaluation of Basic Heteromonocyclic
Propanamide Derivatives as SARDs 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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ABSTRACT: A series of propanamide derivatives were designed, synthesized, and pharmacologically characterized as selective
androgen receptor degraders (SARDs) and pan-antagonists that exert a broad-scope androgen receptor (AR) antagonism.
Incorporating different basic heteromonocyclic B-ring structural elements in the common A-ring−linkage−B-ring nonsteroidal
antiandrogen general pharmacophore contributed to a novel scaffold of small molecules with SARD and pan-antagonist activities
even compared to our recently published AF-1 binding SARDs such as UT-69 (11), UT-155 (12), and UT-34 (13). Compound 26f
exhibited inhibitory and degradation effects in vitro in a wide array of wtAR, point mutant, and truncation mutant-driven prostate
cancers (PCs). Further, 26f inhibited tumor cell growth in a xenograft model composed of enzalutamide-resistant (EnzR) LNCaP
cells. These results demonstrate an advancement toward the development of novel SARDs and pan-antagonists with efficacy against
EnzR prostate cancers.
Although the exact mechanisms of progression to castration-
resistant PC (CRPC) are not always known, numerous
contributing factors to the emergence of CRPC have been
demonstrated and include (1) compensatory production of
intratumoral androgens (e.g., 5α-dihydrotestosterone (DHT)
synthesized from adrenal precursors);3,19,20 (2) AR gene
amplifications and overexpression;21−23 (3) AR ligand binding
domain (LBD) point mutations;22,24,25 (4) alterations in the
expression of coregulatory proteins;26,27 (5) ligand-independ-
ent activation of AR;28−32 (6) constitutively active truncated
AR splice variants (AR SVs);33 and (7) induction of intracrine
androgen synthesis.34−36 Despite resistance to AR antagonist
therapies in CRPC, AR signaling continues to be important for
tumor growth and disease progression. Correspondingly, the
INTRODUCTION
■
Prostate cancer (PC) is the most common cancer and the
second leading cause of cancer-related deaths, after lung
cancer, in American men.1,2 Since PC depends on the
activation of androgen receptor (AR) signaling for its
development, progression, growth, and survival, the AR
represents the primary therapeutic target. Blockage of the
androgen signal is very important and proven to be effective for
PC treatment.3−5 In addition to surgical or chemical castration
by gonadotropin-releasing hormone (GnRH) analogues, AR
antagonists, such as cyproterone acetate (1),6,7 flutamide (2),8
bicalutamide (3),9 nilutamide (4), enzalutamide (5),10
apalutamide (6),11,12 or darolutamide (7),13,14 or the androgen
synthesis inhibitor abiraterone acetate (8) plus prednisone
(Figure 1)15 have been used to block the androgen signal. AR
antagonists, in combination with castration, demonstrate
significant effects by blocking adrenal androgen signals as
well as suppressing transient testosterone increase induced by
GnRH analogues, with the trend toward their use earlier in the
disease to more effectively delay disease progression.16−18
Received: March 10, 2021
Published: July 16, 2021
https://doi.org/10.1021/acs.jmedchem.1c00439
© 2021 American Chemical Society
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Figure 1. Overview of known androgen receptor antagonists. Clinically approved agents include steroidal antiandrogen (1), first-generation (2−4)
second-generation (5−7) antiandrogens, as well as an indirect androgen synthesis inhibitor (8).
Figure 2. Clinical selective androgen receptor modulators (SARMs) (9 and 10) and preclinical SARDs (11−13). A series of diaryl propanamides
form the basis for a nonsteroidal general pharmacophore for AR binding that consists of A-ring−linkage−B-ring structural elements. The arrows
below indicate the evolution of propanamide AR ligands over time.
development of AR antagonists with novel mechanisms of
action to inhibit the AR-axis is urgently needed in hormone-
resistant PCs.37
In the course of exploring novel antiandrogens, our research
group has reported the discovery and characterization of UT-
69 (11) and UT-155 (12) as AR antagonists and degraders
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a
Scheme 1. Synthesis of Compounds 19a−k
a
Reagents and conditions: (a) SOCl2 in tetrahydrofuran (THF), −10 to 0 °C; (b) Et3N in THF, −10 to 0 °C, and then to 50 °C, 2−3 h; (c) 2-
butanone, K2CO3, reflux; and (d) NaH in THF, 0 °C to room temperature.
that selectively inhibit tumor growth and degrade both full-
length AR and AR splice variants (AR SVs).38 Our laboratory
also reported a series of aryl indol-1-yl propanamides and aryl
indolin-1-yl propanamides as selective androgen receptor
degraders (SARDs)39 and most recently reported an aryl
pyrazol-1-yl propanamide (13; termed UT-34 therein) as an
orally available SARD.40 Compounds 11 and 12 bind to both
the N-terminal domain (NTD) at the transcriptional activation
units (Tau)-1 and -5 (Tau-1 and Tau-5) of the activation
function-1 (AF-1) domain and competitively bind the LBD,
while compound 13 was demonstrated to bind the same Tau-1
and Tau-5 NTD sites, but it does not or very weakly binds the
LBD (Figure 2).40
and functional activity (agonist or antagonist). Different B-
rings play a major role in distinguishing how and where the
ligand binds to AR and whether the pharmacology is a selective
AR modulator (SARM) (9, 10), a traditional antiandrogen (3),
or an SARD (11−13) with broad-scope AR antagonism, AR
degradation, and/or NTD binding.39,41,42 The latter activity
seems to be elicited by basicity-located 5-bond lengths from
the A-ring. Based on this hypothesis, here we reported the
exploration of the context of the basic nitrogen within a diverse
array of monocyclic B-rings. This was accomplished through
synthesis and biological evaluation of novel SARDs and pan-
antagonists in vitro and in vivo models of CRPC and
antiandrogen resistance, including enzalutamide-resistant
(EnzR) CRPC tumors.
In the A-ring−linkage−B-ring general pharmacophore in
Figure 2, the electron-deficient aromatic A-ring substituted by
electron-withdrawing groups is required for the AR binding
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a
Scheme 2. Synthesis of Compounds 19l−n
a
Reagents and conditions: (a) Me3SiCN, ZnI2 dichloromethane (DCM); (b) HCl, reflux; (c) SOCl2 in THF, −10 to 0 °C; (d) Et3N in THF,
reflux 12 h; and (e) Cu(I)CN, microwave irradiation at 150 °C in dimethylformamide (DMF).
binding site,38,40 also function at the same site as traditional
antiandrogens. Though not prerequisite to screening in vivo,
RESULTS AND DISCUSSION
■
Chemistry. Propanamide Derivatives with Heteromono-
potent LBD binding may help achieve pan-antagonism and
cyclic B-Ring Modifications. The synthesis of compounds
thus may be an asset in the treatment of PC, where heavily
19a−k is shown in Scheme 1. Commercially available (R)-3-
pretreated patients often have a wide variety of ARs expressed
bromo-2-hydroxy-2-methylpropanoic acid 14a was treated
within tumor cells including wtAR, various point mutant ARs,
with SOCl2 to transform to its acid chloride 14b, which
and various truncated ARs, and additionally many have this
reacted with aniline 15a or 15b in the presence of
mix of ARs overexpressed. Accordingly, the broadest possible
triethylamine to afford the bromide compound 16a or 16b,
AR pan-antagonism, including mediated by the LBD, is an
respectively. Under basic conditions (e.g., K2CO3), 16a was
asset. Unfortunately, the contribution of LBD vs. NTD binding
transformed to a key oxirane intermediate 17a, while 16b was
transformed to oxirane intermediate 17b. Treatment of 17a or
17b with different nucleophiles 18a−k under basic conditions
to IC50 is not immediately clear from this in vitro panel. The
results are shown in Tables 1 and 2.
We initially explored a series of propanamides with different
(e.g., sodium hydride) generated propanamides 19a−k, as
monocyclic B-rings based on 13 as the initial lead. As shown in
shown in Scheme 1.
Scheme 2 shows the synthesis of compounds 19l−n. The
Table 1, 19a, which has a 3-fluoro-1H-pyrrol-1-yl moiety as the
B-ring, possessed binding affinity with a Ki value of 0.633 μM,
treatment of commercially available ketone 20a or 20b with
while 19b, which has a 3-cyano-1H-pyrrol-1-yl moiety as the B-
trimethylsilanecarbonitrile (Me3SiCN) in the presence of a
catalytic amount of zinc iodide (ZnI2) gave a carbonitrile
compound 21a or 21b, which was hydrolyzed under hydrogen
chloride aqueous conditions to afford the corresponding
ring, had increased binding affinity to 0.328 μM, whereas 13
demonstrated no LBD affinity (>10 μM) in this assay.
However, both 19a and 19b exhibited agonistic activity,
which cannot be tolerated in the treatment of PC and no
carboxylic acid 22a or 22b, respectively. Compound 22a or
degradation. The unsubstituted imidazol-1-yl 19c possessed
22b was treated with SOCl2 to transform to the acid chloride
weak AR inhibitory and SARD activity (IC50 = 1.856 μM; 0%
23a or 23b, which reacted with aniline 24a or 24b under basic
conditions (e.g., triethylamine) to generate the target
compound 19l or 19m, respectively. Synthesis of 19n was
achieved by reacting bromine compound 19l with Cu(I)CN
under microwave irradiation, as shown in Scheme 2.
AR FL and 30% AR SV degradation) vs 13 (IC50 = 0.199 μM;
65−100% (FL) and 60−100% (AR SV) AR degradation).
Introducing 4-bromine on the imidazole delivered 19d as a
potent partial antagonist (IC50 value of 0.14 μM) but no
degradation, while 5-bromine on the imidazole gave 19e, a
weak antagonistic activity (IC50 = 1.019 μM) but maintained
SARD activity (50%/70% AR FL/SV degradation). As such,
19e was the first pan-antagonist, albeit weak compared to 13.
Similarly, the unsubstituted 1H-1,2,4-triazol-1-yl as the B-ring,
19f, was a weak inhibitor (IC50 = 1.091 μM) lacking SARD
activity, but its 3-CF3 analogue 19g was a pan-antagonist (IC50
= 1.013 μM; 68%/100% AR FL/SV degradation) with
improved ability to degrade AR relative to 19e. 19h with a
pyrrolidine as B-ring exhibited no AR inhibitory activity and no
protein degradation activity, while 19i with a morpholine as B-
ring again showed pan-antagonism with an IC50 value of 1.018
μM and 52 and 80% AR FL and AR SV protein degradation
activities, indicating that introduction of the second heter-
oatom into the ring rescued SARD and LBD binding and
The synthesized propanamides 19a−n initially were tested
in vitro for AR LBD binding (Ki), inhibition of transactivation
(IC50), and AR degradation (% degradation) of full-length (AR
FL in LNCaP cells) and splice variant (AR SV in 22RV1 cells)
AR in prostate cancer cell lines. In vitro AR inhibition is
defined as the ability to inhibit R1881-induced wtAR
transcriptional activity as measured by luciferase assay, referred
to as in vitro AR inhibition herein. In these studies, we sought
to discover novel SARDs and AR pan-antagonists that potently
inhibit AR transactivation (IC50), optionally degrade AR FL
and AR SV, and possess in vivo efficacy in models of
antiandrogen-resistant CRPC of greater potency than 11−13.
AR LBD binding (Ki) is helpful in understanding whether
these SARDs, which are believed to function via an N-terminal
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Table 1. AR Binding and Antagonistic Activity of Propanamide Derivatives (13, 19a−n) with Monocyclic B-Ring
Modifications
a
AR binding was determined by competitive binding of 1 nM tritiated mibolerone ([3H] MIB) to recombinant LBD of wild-type AR (wtAR).
b
DHT was used in each 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 or COS-7 cells with full-length wtAR, GRE-LUC, and CMV-renilla luciferase for
transfection control. Cells were treated 24 h after transfection with a dose−response of compounds (1 pM−10 μM) in the presence of 0.1 nM
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Table 1. continued
R1881 (antagonist mode) or in the absence of R1881 (agonist mode). Luciferase assay was performed 24 h after treatment using a dual luciferase
c
(firefly and Renilla) assay kit (Promega, Madison, WI and Goldbio Luciferase kit, St. Louis, MO). SARD activity was assayed by treating LNCaP
or 22RV1 cells for determining FL AR (at 1 μM of antagonist) or AR SV (at 10 μM of antagonist) protein levels, respectively. Cells were
maintained in a 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 bands were quantified and normalized to actin bands and
d
represented as percent inhibition from vehicle-treated cells. The result was reported in the literature in the same assay as described here.40
e
f
Transcriptional activation was performed in the same assay in agonist mode. N.A. means data not available.
overall AR inhibition activity, suggesting that the oxygen atom
of the morpholine B-ring was able to serve the same purpose as
electronegative substituents of 19e and 19g.
and SARD activity (0.199 μM; 65−100 and 60−100% AR FL
and AR SV degradation), but unlike 13, 26b possessed potent
LBD binding.
Replacing the pyrrole moiety in 26b by a pyrazole gave 26c,
which showed weaker AR binding affinity (Ki = 0.612 μM) and
weaker inhibitory activity (IC50 = 0.969 μM), whereas the
tetrazole analogue 26d possessing 1,5-biaryl arrangement lost
AR inhibitory activity.
Diaryl tertiary aniline 26e with a (4-fluorophenyl)(phenyl)-
amino moiety as B-ring exhibited potent LBD binding (Ki =
0.275 μM) and inhibitory activity (IC50 = 0.172 μM) but only
low to moderate AR degradation (42%/16% FL/SV).
Compared to 11, which bears a methyl group on the basic
N-atom, there is substantial steric bulk tolerance around the
basic N-atom for LBD binding and AR inhibition, but SARD
activity is not maintained, and diphenyl B-rings were not
explored further herein.
In contrast with 13, 19j−n in some cases bound the LBD
(see 19m and 19n) but possessed no inhibitory activity (19j)
or exhibited AR agonistic activity (19k−n). Accordingly,
linking elements possessing secondary anilines (see 19j and
19k (Table 1) vs tertiary anilide 11 (Table 2)) or lacking a
basic nitrogen (19l−n) were not seen as promising features for
future structure−activity relationship (SAR) exploration for
SARD and pan-antagonist activity. Further, based on this series
and previously published SARDs, the basic N-atom seems
necessary, but not sufficient (see 19j−k), to confer activity in
our screening panel. Further, the biaryl B-ring of 11 or bicyclic
B-ring of 12 (Table 2) seems to be beneficial to SARD
activities and pan-antagonism.
Propanamide Derivatives with Biaryl or Diaryl B-Ring
Modifications. Subsequent synthetic modifications were aimed
at the replacement of the pyrazole monocyclic B-ring of 13 by
a bicyclic B-ring system such as in 11 to explore the effect of
the bicyclic system on AR inhibition and SARD activity. The
synthesis of 26a−e was performed using similar synthetic
methods as in Scheme 1, as depicted in Scheme 3. Treatment
of either bromine compound 16a or oxirane intermediate 17a
with different nucleophiles 25a−e under basic conditions (e.g.,
sodium hydride) formed propanamides 26a−e (Scheme 3).
Compounds 26f−h were synthesized as shown in Scheme 4.
Bromine compound 16a or 16c was treated with NaN3 in
DMF to afford azide 27a or 27b, respectively. 1,4-
Disubstituted-1,2,3-triazole analogues 26f and 26g were
obtained by copper(I)-catalyzed azide−alkyne cycloaddition
(CuAAC) between azide 27a or 27b and alkyne 28,
respectively. On the other hand, 1,5-disubstituted-1,2,3-triazole
26h (regioisomer of 26f) was prepared by the azide−alkyne
Huisgen cycloaddition between azide 27a and alkyne 28, as
depicted in Scheme 4. The compounds 26a−h were tested in
vitro as discussed above for AR activity (Tables 1 and 2).
Temperature. Given our difficulty in improving AR
inhibitory activity via different monocyclic B-rings, we
investigated SAR trends for biaryl (26a−d and 26f−h) and
diphenyl (26e) B-ring systems (Table 2). The 3-phenyl-1H-
pyrrol-1-yl B-ring compound 26a showed AR binding affinity
and partial inhibitory activity (Ki = 0.322 μM; IC50 = 0.178
μM). Similar to the monocyclic series (Table 1), adding an
electronegative substituent tends to improve activity in the
screening panel, e.g., introducing a para-fluorine to the 3-
phenyl-1H-pyrrol-1-yl B-ring as in 26b marginally increased
LBD binding affinity (Ki = 0.259 μM) and dramatically
increased SARD activity (100% (FL) and 60% (AR SV)
degradation) while maintaining potent AR inhibitory activity
(IC50 value of 0.226 μM). The screening profile of 26b was
comparable to that of 13 with regard to in vitro AR inhibition
Compound 26f possessed a (4-fluorophenyl)-1H-1,2,3-
triazol-1-yl B-ring and exhibited moderate inhibitory activity
(IC50 value of 0.383 μM) and moderate-to-high-efficacy
degradation (84%/74% FL/SV degradation) but no LBD
binding. 26h is a regioisomer of 26f that differs from 26f in the
1,5-biaryl arrangement of the (4-fluorophenyl) and 1,2,3-
triazol-1-yl components of the B-ring. 26h showed inhibitory
activity (IC50 = 0.317 μM) and FL AR degradation (73%
degradation), which was comparable to its regioisomer but
improved LBD binding (Ki = 0.703 μM). Contrary to tetrazole
26d, 26h suggests tolerance to the 1,5-biaryl arrangement for
AR antagonism. Replacing a carbon (CH) with a nitrogen (N)
at the 3′-position of the A-ring, i.e., 3′-pyridino derivative of
26f, delivered a more potent compound (26g) with regard to
LBD binding (Ki of 1.486 μM vs >10 μM for 26f) and AR
inhibition (IC50 value of 0.217 μM vs 0.383 μM for 26f) but
decreased AR degradation activity (12%/0% FL/SV vs 84%/
74% FL/SV for 26f).
Following our discovery of orally bioavailable pyrazole (13),
we sought to explore the SAR of other basic heteromonocycles
as templates for optimization of SARDs and pan-antagonist
activities. This survey of B-ring replacements revealed that
multiple heteromonocycles preserved FL and SV SARDs
together with inhibition of wtAR (LBD binding was optional)
fulfilling our definition of pan-antagonist and could serve as a
template for further optimization, including pyrroles (26b),
imidazoles (19e), 1,2,4-triazoles (19g), morpholino (19i),
diphenyl aniline (26e), and 1,2,3-triazoles (26f, 26g, and 26h).
Lack of an electrophilic substituent on the B-ring moiety (19c
vs 19e, 19h vs 19i, or 26a vs 26b) compromised pan-
antagonism, whereas the lack of N-substitution (19j−k) or
lack of basic nitrogen (19l−n) abrogated pan-antagonism. In
some cases, addition of a substituted 4-fluorophenyl ring to the
heteromonocycle to produce a biaryl B-ring was tolerated
(pyrazole 26c, 1,2,3-triazoles 26f−h) or improved (pyrrole
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Table 2. AR Binding and Antagonistic Activity of Propanamide Derivatives (26a−h) with Biaryl or Diaryl B-Ring
Modifications
a
b
AR binding, transactivation, and degradation assays were performed, and values are reported, as described in Table 1. The compound was
reported in the literature in the same assay as described here.40 The result was reported in the literature in the same assay as described here.38,40
c
d
e
The two values indicate SARD assays run with 1 and 10 μM of antagonist. N.A. means data not available.
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a
Scheme 3. Synthesis of Compounds 26a−e
a
Reagents and conditions: (a) NaH in THF, 0 °C to room temperature.
a
Scheme 4. Synthesis of Compounds 26f−h
a
Reagents and conditions: (a) NaN3 in DMF, 80 °C, 24 h; (b) the copper(I)-catalyzed azide−alkyne cycloaddition (CuAAC)/Cu(I)I, in
acetonitrile/H2O, room temperature, 3 d; and (c) Huisgen cycloaddition: heat, in acetonitrile/H2O.
26b vs 19a or 19b) pan-antagonist activity in our screening
panel. Though not required (see aniline 11 or heterobicycle
12), having a broad set of active heteromonocycles (in
addition to providing broad patent exclusion) affords the
advantage of being able to fine-tune biophysical properties
such as aqueous solubility and bioavailability and tune out
potential liabilities that may emerge during in vivo testing or
investigation of new drug (IND)-enabling studies.
In overview, biaryl pyrrole 26b (Ki > 0.259 μM; IC50 = 0.226
μM; 100%/60% FL/SV AR degradation) and biaryl triazole
26f (Ki > 10 μM like 13; IC50 = 0.383 μM; 84%/74% FL/SV
AR degradation) were the only compounds that exhibited
potent activity in all aspects of the screening panel. Although
26b and 26f had decreased potency LBD binding and AR
inhibition compared to 11 (Ki = 0.078 μM; IC50 = 0.048 μM;
70%/71−100% FL/SV AR degradation) and 12 (Ki > 0.267
μM; IC50 = 0.085 μM; 65−83%/60−100% FL/SV AR
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degradation), 11 and 12 suffered from a lack of in vivo activity
due to poor bioavailability. However, 26b and 26f had
comparable inhibitory potency and SARD activity relative to
13 (Ki > 10 μM; IC50 = 0.199 μM; 65−100%/60−100% FL/
SV AR degradation), which was recently demonstrated to be a
bioavailable SARD with exceptional in vivo activities in models
of EnzR PC,38,40 and 26b has the advantage of potent LBD
binding.
Biology. In Vitro Metabolic Stability in Mouse Liver
Microsomes (MLMs). Selected compounds 19c, 19e, 26a, 26b,
26e, 26f, and 26h were evaluated for in vitro metabolic
stability in mouse liver microsomes (MLMs) with cofactors for
enzymes of both phase I and phase II metabolism. The half-life
(T1/2) and intrinsic clearance (CLint) values were calculated as
a predictor of the distribution, metabolism, and pharmacoki-
netic (DMPK) properties of these compounds (Table 3). The
CRPC. Further, 26f compares favorably to other compounds
reported herein with regard to inhibitory potency and SARD
efficacy (Tables 1 and 2). Correspondingly, 26f was advanced
to testing in models of CRPC including resistance to 5 (EnzR
MR49F cells harboring F876L and T877A AR point
mutations) and 3 (bicalutamide-resistant LNCaP cells
harboring the T877A mutation).
Androgen Receptor Target Gene Expression in CRPC Cells.
To evaluate whether the observed highly potent AR
antagonism translates to inhibition of endogenous AR
function, we performed an AR target gene inhibitory
experiment to determine the effect of 26f in LNCaP cells
and compared it with enzalutamide (5) (Figure 3). 26f was
chosen as the lead compound because 26f possessed a balance
of high potency inhibition (0.383 μM), high-efficacy
degradation (84% for AR FL and 74% AR SV), and long in
vitro stability (T1/2 of 265 min) compared to other SARDs and
pan-antagonists screened. Consistent with the 0.383 μM
inhibition of wtAR transactivation, FKBP5 gene expression
induced by 0.1 nM R1881 induced in LNCaP cells was
robustly inhibited by 26f at concentrations as low as 1 μM
(Figure 3A), which was similar to 5, and 0.1 nM R1881-
induced PSA gene expression in LNCaP cells was inhibited by
26f at concentrations 10 μM as potent as 5 (Figure 3B).
Concurrently, we tested the effect of 26f on the function of
5 resistance-conferring F876L/T877A AR in the context of
LNCaP cells, i.e., MR49F cells. Similarly, dose-dependent
downregulation of FKBP5 gene expression was seen in MR49F
cells when treated with 26f but not 5, validating this as a model
of EnzR. 26f inhibited the expression of FKBP5 at
concentrations as low as 0.3 μM in EnzR LNCaP (Figure
3C), indicating that the clinically significant F876L mutant was
potently sensitive to 26f. Cumulatively, the data above support
that 26f has pan-antagonist effects in at least wtAR (IC50 value
in Table 2), T877A (AR FL degradation in Table 2), F876L/
T877A (Figure 3C; see also degradation in MR49F cells, as
reported infra), and AR SV (degradation in Table 2).
Table 3. In Vitro Metabolic Stability for Selected
Compounds in Mouse Liver Microsomes (MLMs)
a
(DMPK) MLM
compound ID
T1/2 (min)
CLint (mL/min/mg)
b
11
12
13
1.15
12.11
77.96
24.61
26.51
3.96
17.93
5.07
265
208.8
57.26
0.89
28.16
41.58
157.5
38.66
136.8
2.67
b
b
19c
19e
26a
26b
26e
26f
26h
>360
10.04 h
∼0
86.3
c
d
5 (enzalutamide)
a
Compounds were incubated together with mouse liver microsomes
(MLMs) with cofactors for phases I and II provided, as described in
b
the Experimental Section. Reported previously using the same
c
method as in the Experimental Section.38,40 T1/2 (h) after oral
d
administration in humans as previously reported in ref 43. CL (mL/
h/kg) after oral administration in humans as previously reported.43
Proliferation Studies in PC Cells. To determine whether the
potent functional inhibition and degradation of AR translate
into the inhibition of PC cell growth, the effect of 26f was
tested in an LNCaP cell model of CRPC harboring the T877A
antiandrogen resistance mutation and in an even more
refractory model of CRPC, i.e., MR49F cells. In the absence
of an AR antagonist, R1881 induced proliferation of androgen-
dependent LNCaP cells (not shown). Antiproliferative effects
were tested in the presence of a titrated dose of 26f or 5, as
shown in Figure 4A. 26f demonstrated antiproliferation at
doses as low as 10 μM and full efficacy at 30 μM, while 5
exhibited little effect on the proliferation in LNCaP cells
(Figure 4A). A modest inhibition of proliferation in the
presence of 26f, but not 5, was observed in AR-negative PC3
cells (Figure 4B) consistent with AR-dependent antiprolifera-
tion. As mentioned above, the F876L mutation confers EnzR
to MR49F cells; however, MR49F cells remain dependent on
the AR for growth. R1881-induced MR49F proliferation was
inhibited by 26f in the 10 μM and full efficacy at 30 μM
(Figure 4C) similar to the parental LNCaP (Figure 4A),
whereas 5 showed no effect of the proliferation in the MR49F
model (Figure 4C). Assuming that 26f can reach the tumors
and androgen-dependent organs in sufficient concentrations,
this potent antiproliferation of 26f suggests that 26f may
perform well in in vivo models.
CLint of these compounds 19c, 19e, 26b, 26f, and 26h was
slower than previously published SARDs 11 (T1/2 of 1.15 min)
and 12 (T1/2 of 12.11 min); especially compounds 26f and 26h
were even slower than 13, which was orally bioavailable (T1/2
of 78 min),40 producing relatively stable T1/2 values of 265 min
and >360 min in MLM, respectively. Compound 26a, which
has a 3-phenyl-1H-pyrrol-1-yl B-ring, and 26e (diphenyl
aniline) showed relatively unfavorable in vitro metabolic
stability with T1/2 values of 3.96 and 5.07 min, as well as
CLint values of 157.5 and 136.8 mL/min/mg, respectively. 26b,
in which para-fluorine is introduced on the 3-phenyl-1H-
pyrrol-1-yl B-ring, increased T1/2 by ∼4-fold to 17.93 min,
suggesting that the fluorination protected the phenyl ring from
metabolism. Compounds 26f and 26h, both of which have a
1,2,3-triazole B-ring, dramatically increased T1/2, suggesting
that the triazole moiety had a very favorable in vitro metabolic
stability. This was a significant improvement when compared
to previous SARD templates with a bicyclic B-ring such as 1.15
min for the tertiary amine 11 and 12.11 min for lead indole
12.38,39 This supports that compound 26f may be stable
enough in liver metabolism to attain optimum to high blood
levels upon oral administration and be distributed to sites of
action throughout the body, as would be necessary to treat
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Figure 3. (A) 26f antagonizes AR function in LNCaP prostate cancer cells. LNCaP cells were maintained for 2 d in a charcoal-stripped fetal bovine
serum (csFBS)-containing medium. The cells were treated with antagonists, 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 polymerase chain reaction (PCR). (B) 26f
antagonizes AR function in LNCaP prostate cancer cells. LNCaP cells were maintained in a csFBS-containing medium for 2 d. The medium was
changed and treated as indicated with 10 μM 26f in the presence of 0.1 nM R1881. Cells were harvested 24 h after treatment, and expression of
PSA was measured by real-time PCR and normalized to GAPDH. (C) Enzalutamide (5)-resistant (EnzR) LNCaP cells (MR49F) were plated in
96-well plates in a 1% csFBS-containing medium. Cells were maintained in this medium for 2 days and treated as indicated in the figure. RNA was
extracted, and real-time PCR for FKBP5 was performed and normalized to GAPDH expression.
AR FL (F876L) and AR SV (AR-V7) Degradation in Models
of CRPC. To ensure that the in vitro AR antagonism profiles of
26f were also maintained in an EnzR cell line, FL AR
degradation studies in MR49F cells were performed. Figure 5
shows that 26f, but not 5, degraded this mutant FL AR in
MR49F cells, i.e., a model of EnzR CRPC, in a dose-responsive
manner (top panel), consistent with the ability of 26f to
degrade AR in LNCaP (Table 2) and suppress AR-dependent
gene expression in LNCaP and MR49F cells (Figure 3). The
Western blots were quantified densitometrically, and the AR/
GADPH values are represented as percent change from
vehicle-treated cells.
High-efficacy SARD activity was observed with 26f at 1 μM
and complete degradation at 10 μM (Figure 5, top panel),
indicating that this mutant AR FL that confers EnzR in MR49F
LNCaP cells is susceptible to destruction by 26f.
The middle panel demonstrated that 26f did not just induce
degradation of AR FL in EnzR LNCaP (MR49F cells; Figure 5,
top panel) and LNCaP (Table 2) but can also degrade AR SVs
such as the AR-V7 (22RV1 cells; lower band of the middle
panel of Figure 5). As reported in Table 2 (see the AR SV
degradation column), 26f was able to reduce AR-V7 levels by
74% in 22RV1 cells at 10 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 resistant to approved therapies including 2−7.44 Taken
together, 26f affords a very broad scope of AR antagonistic
abilities.
In Vivo Models. Preliminary Pharmacokinetic (PK) Study
with 26f in Mice. C57BL/6 mice (n = 4/time point) were
treated with a single dose of 30 mg/kg orally of 26f. Blood was
collected 6 and 24 h after dosing, and serum 26f concentration
was measured. The average serum concentrations of 26f at 6
and 24 h after dosing were 453 and 332 nM, respectively.
These results suggested that 26f maintains serum concen-
trations above to around the AR inhibitory IC50 value of 0.383
μM for greater than 24 h after dosing. This PK behavior is
consistent with promising in vivo data such as Hershberger and
xenograft studies following oral administration, as reported
hereinbelow.
Hershberger Assays in Rats. Given the metabolic stability
in vitro (T1/2 of 265 min) and in preliminary PK observations
in vivo, we were hopeful that the in vitro SARD and pan-
antagonist activity of 26f would translate into clinically
meaningful efficacy in vivo. As expected, Hershberger assays
on 26f orally administered in intact rats demonstrated atrophy
of AR-dependent seminal vesicle tissues (Figure 6). Previously,
we demonstrated that 13 (4-F pyrazole; IC50 of 0.199 μM; T1/2
of 78 min) was able to reduce the ventral prostate weight by
∼70% at 60 mg/kg po [see Figure S4C, middle panel in
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Figure 4. (A) LNCaP cellular antiproliferation. LNCaP cells were plated and treated in a 1% charcoal-stripped serum-containing medium with the
indicated doses of the compounds in the presence of 0.1 nM R1881. Cells were treated for 6 d with medium change and retreated after 3 d. Cell-
Titer-Glo assay was performed to determine the viable cells. (B) PC3 cellular proliferation. PC3 cells were plated and treated in a 1% charcoal-
stripped serum-containing medium with the indicated doses of the compounds. Cells were treated for 6 d with medium change and retreated after 3
d. Cell-Titer-Glo assay was performed to determine the viable cells. (C) Enzalutamide-resistant LNCaP (MR49F) cellular antiproliferation.
Enzalutamide (5)-resistant (EnzR) LNCaP cells (MR49F cells) were plated and treated in a 1% charcoal-stripped serum-containing medium with
the indicated doses of the compounds in the presence of 0.1 nM R1881. Cells were treated for 6 d with medium change and retreatment after 3 d.
Cell-Titer-Glo assay was performed to determine the viable cells.
Figure 6. SARDs and pan-antagonists inhibit androgen-dependent
organs in rats. Male Sprague Dawley rats (100−120 g) were treated
orally with vehicle or 20 mg/kg 26f for 13 d. Animals were sacrificed
on the 14th day, and seminal vesicle weights were recorded. The
inhibition of the seminal vesicle growth measured as loss of organ
weight by 26f vs vehicle was statistically significant (p = 0.0031). N =
5/group.
Figure 5. 26f degrades enzalutamide (5) resistance-conferring escape
mentioned above, seminal vesicle weights were reduced by
mutant AR. EnzR LNCaP cells (MR49F cells) (top panel) or 22RV1
49% (Figure 6), demonstrating that the in vivo pharmacody-
cells (bottom panel) were maintained in a charcoal-stripped serum-
namic properties intrinsic to 26f are observable at lower doses
containing medium for 2 d and treated with 0.1 nM R1881 (agonist)
relative to 13. Compared to 13, introducing of the 4-(4-
fluorophenyl)-1H-1,2,3-triazol-1-yl moiety as the B-ring
seemed favorable for metabolic stability and in vivo
antagonism, suggesting that xenograft potencies should also
be improved. These results confirm that the oral pharmaco-
kinetics of 26f was sufficiently robust to allow 26f to be
absorbed and distributed to the site of action in AR target
and a titration of the 26f or 5, as indicated in the figure. Twenty-four
hours after treatment, cells were harvested and the protein was
extracted and blotted with an AR-N20 antibody. Blots were stripped
and reprobed with a GAPDH antibody.
Ponnusamy et al.] versus ∼40% for 30 mg/kg po of 5, both in
rats. At 20 mg/kg po of 26f, that is, one-third of the dose of 13
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organs and suggested that 26f should also distribute to tumors
in xenograft models and exert antitumor effects in sensitive
models.
series possessed moderate-to-high SARD efficacy but inhib-
itory activity of only ∼1 μM, whereas biaryls 26b, 26c, 26f, and
26h similarly possessed moderate-to-high-efficacy SARD
activity but nM level in vitro inhibition, as shown in Tables
1 and 2. 19c, 19e, 26b, 26f, and 26h improved their DMPK
properties in mouse liver microsomes (MLMs) compared to
previous SARD templates such as for the tertiary amine 11
(T1/2 of 1.15 min) and lead indole 12 (T1/2 of 12.11 min);
especially, DMPK properties in MLM of 26f (T1/2 of 265 min)
and 26h (T1/2 of >360 min), which were even better than that
of pyrazole 13 (T1/2 of 77.96 min). The lead compound 26f
effectively inhibited the expression of FKBP5 and PSA (Figure
3), as well as demonstrated a dose-responsive antiproliferation
in androgen-dependent LNCaP and EnzR LNCaP CRPC cells,
but only weakly inhibited androgen-independent PC3 PC cells
(Figure 4). Further, SARD activity was maintained in the EnzR
CRPC setting (Figure 5), all supporting testing of 26f in vivo.
Compound 26f produced AR antagonism in an intact rat
Hershberger assay, with an approximately 49% reduction in the
seminal vesicle weight compared to their intact organ weights
(Figure 6). EnzR LNCaP (MR49F) xenograft experiments
with 60 mg/kg po daily of 26f produced significant efficacy of
up to 95% TGI (Figure 7). Accordingly, biaryl SARDs such as
4-fluorophenyl-1,2,3-triazole 26f may have some advantages
over pyrazoles such as 13 and are believed to hold great
potential for overcoming enzalutamide resistance in CPRC,
which is increasingly prevalent in the clinic.
EnzR (MR49F) LNCaP Xenografts in Mice. The PC studies
described above were in vitro models that provided support
that 26f inhibited and degraded both AR FL and truncation
mutant AR (AR SV), as well as in vivo antagonism in
Hershberger assays with 49% change in seminal vesicle weights
relative to intact vehicle control. To evaluate the effect of 26f
in an enzalutamide-resistant xenograft model, EnzR LNCaP
(MR49F) cells were implanted subcutaneously in NOD SCID
γ (NSG) mice. Once tumor sizes reached 200−400 mm3, the
mice were castrated, and the tumors were allowed to regrow as
CRPC. The animals were treated with vehicle (poly(ethylene
glycol)-300/DMSO 85:15 ratio) or 60 mg/kg 26f, and the
tumor volume was measured twice weekly for 28 d. In Figure
7, 26f significantly reduced the tumor volume with an over
EXPERIMENTAL SECTION
■
Figure 7. 26f inhibits the growth of enzalutamide (5)-resistant
prostate cancer. EnzR LNCaP (MR49F) cells (5 million/mouse: 1:1
medium/Matrigel) were implanted subcutaneously in male NSG
mice. Once the tumors reach 200−400 mm3, the animals were
castrated, and the tumors were allowed to regrow as castration-
resistant prostate cancer (CRPC). Once the regrown tumors reach
400 mm3, the animals were randomized and treated orally with vehicle
or 60 mg/kg/day 26f. Tumor volume was measured twice weekly.
Animals were sacrificed 28 d after treatment, and the tumors were
processed for further analysis. * p < 0.05.
Chemistry. General Procedures, Materials, and Information. All
solvents and chemicals were used as purchased without further
purification. The progress of all reactions was monitored by the 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). All nonaqueous
reactions were performed in oven-dried glassware under an inert
atmosphere of dry nitrogen. All of the reagents and solvents were
purchased from Sigma-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
thin-layer chromatography was performed on Silica Gel GHLF 10 ×
20 cm2 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 Cartridge, silica, 50 and 100 g) was used to purify
95% tumor growth inhibition (TGI, p < 0.05; see the last four
time points), whereas 5 failed to reduce the tumor volume of
EnzR LNCaP xenografts (enzalutamide data not shown here;
see Figure 7B in Hwang et al.39).
Further, the significant levels of TGI activity indicated that
26f is orally bioavailable (unlike 11 and 12) and can reach
adequate levels in tumors to reveal the pharmacodynamic
behavior of our SARDs, albeit at high dose. The susceptibility
of these EnzR xenografts to 26f in this experiment provides
evidence that a triazole SARD, e.g., 26f, can overcome EnzR
CRPC in vivo. Moreover, no loss in body weight was observed
in animals treated with 26f for 14 or 28 d (Hershberger or
xenograft, respectively). This indicates that 26f was not toxic at
least at the efficacy doses. Extensive toxicity evaluation needs
to be conducted at a concentration that are 5−10 times higher
than the efficacy doses. In overview, these experiments provide
hope that our SARDs with their unique biological profile could
be used to overcome EnzR in CRPC patients.
1
the compounds. H NMR and 13C NMR spectra were recorded on a
Bruker Ascend 400 (400 MHz) (Billerica, MA) spectrometer.
Chemical shifts for 1H 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 (MS) were acquired using a Bruker ESQUIRE
electrospray/ion trap instrument in the positive and negative modes.
High-resolution mass spectrometer (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). High-
performance liquid chromatography (HPLC) conditions: 45%
acetonitrile at a flow rate of 1.0 mL/min using a Luna 5 μm C18
100A column (250 × 4.60 mm2) purchased from Phenomenex
(Torrance, CA) at an ambient temperature. UV detection was set at
340 or 245 nm. Purities of the compounds were established by careful
CONCLUSIONS
■
Compounds 26b, 26e, 26f, 26g, and 26h from the biaryl B-ring
series exhibited potent nM range inhibitory activity in vitro.
Compounds 19e, 19g, and 19i from the monocyclic B-ring
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integration of areas for all peaks detected and determined as ≥95% for
all compounds tested for the biological study.
evaporated to get acid 22a. The corresponding acid 22a was taken in
dry THF, cooled to 0 °C, and thionyl chloride (1.5 equiv) was added
and stirred for 90 min. To the mixture, triethylamine (5 equiv) and
corresponding aniline 24a (1 equiv) were added and refluxed
overnight. The reaction was cooled to rt and extracted with ethyl
acetate. The organic layer was dried and evaporated and subjected to
flash chromatography to afford 19l.
General Procedure A for the Synthesis of 16a−c Using (R)-3-
Bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methyl-
propanamide (16a) as an Example. (R)-3-Bromo-2-hydroxy-2-
methylpropanoic acid 14a (5.00 g, 27 mmol) was dissolved in 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, 1.30 equiv, 1 vol) was
slowly added to the reaction mixture, keeping the temperature below
12 °C. 4-Amino-2-(trifluoromethyl)benzonitrile 15a (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
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3-fluoro-1H-
pyrrol-1-yl)-2-hydroxy-2-methylpropanamide (19a). Com-
pound 19a was prepared following General Procedure C per Scheme
1. The product was purified by a silica gel column using ethyl acetate
and hexanes (1:1) as eluents to afford 0.181 g of the titled compound
1
as a white solid. Yield = 31%. H NMR (400 MHz, CDCl3) δ: 8.91
(bs, 1H, NH), 8.03 (d, J = 2.0 Hz, 1H), 7.90 (dd, J = 8.4, 2.0 Hz, 1H),
7.81 (d, J = 8.4 Hz, 1H), 6.47 (m, 1H), 6.41 (m, 1H), 5.91 (dd, J =
2.8, 2.0 Hz, 1H), 4.36 (d, J = 14.4 Hz, 1H), 3.98 (d, J = 14.4 Hz, 1H),
1.54 (s, 3H). 19F NMR (CDCl3, decoupling) δ −62.18, −164.26.
+
HRMS [C16H14F4N3O2 ]: calcd 356.1022, found 356.1021 [M + H]+.
( S ) - 3 - ( 3 - C y a n o - 1 H - p y r r o l - 1 - y l ) - N - ( 4 - c y a n o - 3 -
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
(19b). Compound 19b was prepared following General Procedure C
per Scheme 1. The product was purified by a silica gel column using
ethyl acetate and hexanes (1:1) as eluents to afford 0.26 g of the titled
concentrated to 5
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
0.5 volumes (4
0.5 wt) while maintaining
1
compound as a pinkish solid. Yield = 51.3%. H NMR (400 MHz,
volumes (4 0.5 wt), and the batch temperature was 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 16a.
DMSO-d6) δ: 10.44 (s, 1H, NH), 8.44 (s, 1H, ArH), 8.24 (d, J = 8.8
Hz, 1H, ArH), 8.10 (d, J = 8.8 Hz, 1H, ArH), 7.49 (s, 1H, pyrrole-H),
6.38 (t, J = 2.0 Hz, 1H, pyrrole-H), 6.41−6.40 (m, 2H, OH and
pyrrole-H), 4.30 (d, J = 14.0 Hz, 1H, CH), 4.14 (d, J = 14.0 Hz, 1H,
General Procedure B for the Synthesis of 17a and 17b Using (S)-
N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-methyloxirane-2-carbox-
amide (17a) as an Example. To a solution of (R)-3-bromo-N-(4-
cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
(16a) (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 an argon atmosphere. After
the end of the reaction was established by TLC, the reaction was
cooled to room temperature (rt), filtered through a pad of celite, and
the celite pad was rinsed with 15 mL of 2-butanone. The filtrate was
concentrated under vacuum and dried under 25−30 in. vacuum to
provide 17a.
+
CH), 1.34 (s, 3H, CH3). HRMS [C17H14F3N4O2 ]: calcd 363.1069,
found 363.1079 [M + H]+. Analytical HPLC showed a 97.92% purity.
(S)-N-(3-Chloro-4-cyanophenyl)-2-hydroxy-3-(1H-imidazol-
1-yl)-2-methylpropanamide (19c). Compound 19c was prepared
following General Procedure C per Scheme 1. Under an argon
atmosphere, a 2.0 M lithium diisopropylamide solution (1.25 mL, 2.5
mmol) in THF/heptane/ethylbenzene was slowly added in a
dropwise manner over 10 min to a solution of imidazole (68 mg,
1.0 mmol) in 5 mL of anhydrous THF at −78 °C and warmed to 0 °C
and stirred for 10 min and cooled again to −78 °C. To the solution
was added in a dropwise fashion a solution of bromide (315 mg, 1.0
mmol), and the reaction mixture was stirred overnight. After
quenching by the addition of sat. NH4Cl, the solution was
concentrated under reduced pressure and dispersed into excess
EtOAc and dried over Na2SO4. The product was purified by a silica
gel column using ethyl acetate and hexanes (2:1) as eluents to afford
General Procedure C for the Synthesis of 19a−k and 26a−e
Using 19b as an Example (Schemes 1 and 3). To a solution of 1H-
pyrrole-3-carbonitrile (0.10 g, 0.00108 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, 0.09 g,
0.00217 mol). After addition, the resulting mixture was stirred for 3 h.
(R)-3-Bromo-N-(4-cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methylpropanamide (16a) (0.38 g, 0.00108 mol) was added to the
above solution, and the resulting reaction mixture was allowed to stir
overnight at rt under argon. The reaction was quenched with water
and extracted with ethyl acetate. The organic layer was washed with
brine, dried with MgSO4, filtered, and concentrated under vacuum.
The product was purified by a silica gel column using ethyl acetate
and hexanes (1:1) as eluent to afford 0.26 g of 19b as a white solid.
General Procedure D for the Synthesis of 19l and 19m Using 19l
as an Example (Scheme 2). The trimethylsilanecarbonitrile
(Me3SiCN) (2 equiv) was added to a solution of ketone (20a) (1
equiv) and a catalytic amount of zinc iodide (ZnI2) in dichloro-
methane. The resulting reaction mixture was stirred at rt overnight.
After the end of reaction was established by TLC, the solvents were
evaporated under vacuum to afford crude 21a. Twenty milliliters of
concentrated HCl and 5 mL of ethyl acetate were added to the
residue of tertiary alcohol 21a, and the mixture was stirred for 1 h.
The mixture was cooled to rt, and ice-cold 1 N NaOH was added
slowly to dissolve the solid. The mixture was extracted with
dichloromethane three times. To the water layer, concentrated HCl
was added and extracted with dichloromethane, the combined
dichloromethane was dried over anhydrous sodium sulfate, and the
solvents were evaporated. The white solid was purified by redissolving
in NaOH, filtered and acidified with 1 N HCl, and extracted with
ethyl acetate. The organic layer was dried over sodium sulfate and
1
the titled compound as a white solid. Yield = 65%. H NMR (400
MHz, CDCl3) δ: 10.24 (bs, 1H, NH), 8.19 (s, 1H), 7.90 (s, 2H), 7.53
(s, 1H), 7.05 (s, 1H), 6.82 (s, 1H), 6.40 (s, 1H), 4.30 (d, J = 14.4 Hz,
1H), 4.13 (d, J = 14.4 Hz, 1H), 3.34 (bs, 1H, OH), 1.34 (s, 3H).
+
HRMS [C14H14ClN4O2 ]: calcd 305.0805, found 305.0854 [M + H]+.
Analytical HPLC showed a 99.75% purity.
(S)-3-(4-Bromo-1H-imidazol-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
(19d). Compound 19d was prepared following General Procedure C
per Scheme 1. The product was purified by a silica gel column using
DCM and methanol (19:1) as eluents to afford the titled compound
1
as a white solid. Yield = 32%. H NMR (400 MHz, CDCl3) δ: 9.48
(bs, 1H, NH), 8.15 (s, 1H), 7.97 (d, J = 8.6 Hz, 1H), 7.83 (d, J = 8.6
Hz, 1H), 7.71 (s, 1H), 6.75 (s, 1H), 4.53 (d, J = 14.4 Hz, 1H), 4.09
(d, J = 14.4 Hz, 1H), 2.84 (s, 1H, OH), 1.45 (s, 3H). 19F NMR (400
+
MHz, CDCl3) δ -62.19. HRMS [C15H13BrF3N4O2 ]: calcd 417.0174,
found 417.0174 [M + H]+. Analytical HPLC showed a 98.33% purity.
(S)-3-(5-Bromo-1H-imidazol-1-yl)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
(19e). Compound 19e was prepared following General Procedure C
per Scheme 1. The product was purified by a silica gel column using
ethyl acetate and hexanes (1:1) as eluents to afford the titled
compound as a white solid. Yield = 37%. 1H NMR (400 MHz,
acetone-d6) δ: 9.93 (bs, 1H, NH), 8.44 (d, J = 2.0 Hz, 1H), 8.26 (dd,
J = 8.6, 2.0 Hz, 1H), 8.03 (d, J = 8.6 Hz, 1H), 7.47 (s, 1H), 7.11 (s,
1H), 5.83 (s, 1H, OH), 4.50 (d, J = 14.0 Hz, 1H), 4.23 (d, J = 14.0
Hz, 1H), 1.55 (s, 3H). 19F NMR (400 MHz, acetone-d6) δ 114.69.
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+
HRMS [C15H13BrF3N4O2 ]: calcd 417.0174, found 417.0175 [M +
ArH), 6.17 (s, 1H, OH), 3.65−3.60 (m, 1H, CH), 3.38−3.34 (m, 1H,
CH), 1.43 (s, 3H, CH3). MS (ESI) m/z 457.05. [M + H]+. HRMS
H]+. Analytical HPLC showed a 95.15% purity.
+
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(1H-1,2,4-triazol-1-yl)propanamide (19f). Compound
19f was prepared following General Procedure C per Scheme 1. The
product was purified by a silica gel column using ethyl acetate and
hexanes (2:3) as eluent to afford 0.143 g of the titled compound as
[C20H15F6N4O2 ]: calcd 457.1099, found 457.1100 [M + H]+.
Analytical HPLC showed a 97.37% purity.
N-(4-Nitro-3-(trifluoromethyl)phenyl)-4-(4-fluorophenyl)-2-
hydroxy-2-methylbutanamide (19l). Compound 19l was pre-
pared following General Procedure D per Scheme 2. The product was
purified by a silica gel column using DCM and methanol (19:1) as
eluents to afford the titled compound as a brown solid. Yield = 43%.
1
white solid. Yield = 43%. H NMR (400 MHz, CDCl3) δ: 9.10 (bs,
1H, NH), 8.15 (s, 1H), 8.02 (d, J = 2.0 Hz, 1H), 7.88 (dd, J = 8.4, 2.0
Hz, 1H), 7.78 (d, J = 8.4 Hz, 1H), 5.70 (bs, 1H, OH), 4.79 (d, J =
14.0 Hz, 1H), 4.35 (d, J = 14.0 Hz, 1H), 1.53 (s, 3H). 19F NMR (400
+
HRMS [C18H19F3N3O4 ]: calcd 398.1328, found 398.1331 [M + H]+.
4-(4-Bromophenyl)-2-hydroxy-2-methyl-N-(4-cyano-3-
(trifluoromethyl)phenyl)butanamide (19m). Compound 19m
was prepared following General Procedure D per Scheme 2. The
product was purified by a silica gel column using DCM and methanol
(19:1) as eluents to afford the titled compound as a white brown
solid. Yield = 45%. 1H NMR (400 MHz, CDCl3) δ: 9.14 (s, 1H, NH),
8.09 (s, 1H, ArH), 7.91 (d, J = 8.4 Hz, 1H, ArH), 7.77 (d, J = 8.4 Hz,
1H, ArH), 7.33 (d, J = 8.4 Hz, 2H, ArH), 7.04 (d, J = 8.4 Hz, 2H,
ArH), 2.74−2.81 (m, 1H, CH), 2.52−2.59 (m, 1H, CH), 2.28−2.36
(m, 1H, CH), 1.90−1.97 (m, 1H, CH), 1.58 (s, 3H, CH3); 13C NMR
(CDCl3) δ 27.32, 29.52, 41.86, 76.65, 104.43, 115.54, 117.14, 120.75,
121.68, 130.13, 131.57, 133.92, 135.06, 139.73, 141.49, 173.88. MS
(ESI): m/z 440.8 [M − H]−.
+
MHz, CDCl3, decoupling) δ −62.22. HRMS [C14H13F3N5O2 ]: calcd
340.1021, found 340.1026 [M + H]+. Analytical HPLC showed
99.53% purity.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(3-(trifluoromethyl)-1H-1,2,4-triazol-1-yl)-
propanamide (19g). Compound 19g was prepared following
General Procedure C per Scheme 1. The product was purified by a
silica gel column using ethyl acetate and hexanes (2:3) as eluents to
afford 0.213 g of the titled compound as a white solid. Yield = 53%.
1H NMR (400 MHz, acetone-d6) δ: 9.88 (bs, 1H, NH), 9.44 (s, 1H),
8.44 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.04 (d, J = 8.4 Hz, 1H), 4.82
(d, J = 14.4 Hz, 1H), 4.61 (d, J = 14.4 Hz, 1H), 2.88 (bs, 1H, OH),
1.61 (s, 3H). 19F NMR (400 MHz, CDCl3) δ −62.26, −65.25. HRMS
N-(4-Cyano-3-(trifluoromethyl)phenyl)-4-(4-cyanophenyl)-
2-hydroxy-2-methylbutanamide (19n). Compound 19n (1
equiv) and Cu(I)CN (10 equiv) were dissolved in 2 mL of dry
dimethylformamide and subjected to microwave irradiation (80 W) at
150 °C for 1 h. After irradiation, the mixture was cooled to rt. The
mixture was extracted with ethyl acetate, the organic layer was washed
with brine and water, and dried over anhydrous sodium sulfate.
Evaporation of the solvent afforded the titled compound as a
+
[C15H12F6N5O2 ]: calcd 408.0895, found 408.0898 [M + H]+.
Analytical HPLC showed a 98.52% purity.
(S)-N-(3-Chloro-4-cyanophenyl)-2-hydroxy-2-methyl-3-(pyr-
rolidin-1-yl)propanamide (19h). Compound 19h was prepared
following General Procedure C per Scheme 1. The product was
purified by a silica gel column using ethyl acetate and hexanes (1:2) as
eluents to afford 0.277 g of the titled compound as a colorless oil.
Yield = 90%. 1H NMR (400 MHz, CDCl3) δ: 9.41 (bs, 1H, NH), 7.98
(d, J = 2.0 Hz, 1H), 7.62 (d, J = 8.8 Hz, 1H), 7.51 (dd, J = 8.8, 2.0 Hz,
1H), 3.15 (d, J = 12.4 Hz, 1H), 2.72 (d, J = 12.4 Hz, 1H), 2.62 (m,
4H), 1.76 (m, 4H), 1.52 (s, 1H, OH), 1.41 (s, 3H). HRMS
1
yellowish solid. Yield = 32%. H NMR (400 MHz, CDCl3) δ: 7.53−
7.58 (m, 3H, ArH & NH), 7.29 (d, J = 8.0 Hz, 2H, ArH), 6.95 (s, 1H,
ArH), 6.78 (d, J = 8.4 Hz, 2H, ArH), 4.46 (br. S, 1H, OH), 2.95 (t, J
= 7.6 Hz, 2H, CH2), 2.79 (t, J = 7.6 Hz, 2H, CH2), 2.14 (s, 3H, CH3);
13C NMR (400 MHz, CDCl3) δ 26.87, 29.16, 41.86, 75.92, 104.11,
+
[C15H19ClN3O2 ]: calcd 308.1166, found 308.1173 [M + H]+.
Analytical HPLC showed a 98.98% purity.
109.82, 115.60, 117.16, 119.73, 121.70, 129.24, 132.21, 133.76,
135.81, 146.28, 174.89. MS (ESI): m/z 410.1 [M + Na]+.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-morpholinopropanamide (19i). Compound 19i was
prepared following General Procedure C per Scheme 1. The product
was purified by a silica gel column using ethyl acetate and hexanes
(1:2) as eluents to afford 0.209 g of the titled compound as a white
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-2-hydroxy-2-
methyl-3-(3-phenyl-1H-pyrrol-1-yl)propanamide (26a). Com-
pound 26a was prepared following General Procedure C per Scheme
3. The product was purified by a silica gel column using ethyl acetate
and hexanes (1:2) as eluents to afford 0.90 g of the titled compound
1
solid. Yield = 88%. H NMR (400 MHz, CDCl3) δ: 9.36 (bs, 1H,
1
NH), 8.08 (d, J = 1.6 Hz, 1H), 7.94 (dd, J = 8.4, 1.6 Hz, 1H), 7.80 (d,
J = 8.4 Hz, 1H), 3.68 (m, 4H), 3.28 (d, J = 13.2 Hz, 1H), 2.55 (m,
4H), 2.42 (d, J = 13.2 Hz, 1H), 1.50 (bs, 1H, OH), 1.42 (s, 3H). 19F
as a pink solid. Yield = 62.5%. H NMR (400 MHz, DMSO-d6) δ:
10.41 (s, 1H, NH), 8.24 (d, J = 1.6 Hz, 1H, ArH), 8.17 (dd, J = 8.4
Hz, J = 2.0 Hz, 1H, ArH), 8.07 (d, J = 8.4 Hz, 1H, ArH), 7.38−7.33
(m, 4H, ArH), 7.28−7.24 (m, 1H, ArH), 6.96 (t, J = 3.0 Hz, 1H,
pyrrole-H), 6.28 (s, 1H, OH), 6.07 (t, J = 3.5 Hz, 1H, pyrrole-H),
6.03 (m, 1H, pyrrole-H), 4.30−4.22 (m, 2H, CH2), 1.01 (s, 3H,
+
NMR (400 MHz, acetone-d6) δ −62.20. HRMS [C16H19F3N3O2 ]:
calcd 358.1379, found 358.1383 [M + H]+. Analytical HPLC showed
a 97.38% purity.
+
(S)-3-((1H-1,2,4-Triazol-3-yl)amino)-N-(4-cyano-3-
(trifluoromethyl)phenyl)-2-hydroxy-2-methylpropanamide
(19j). Compound 19j was prepared following General Procedure C
per Scheme 1. The product was purified by a silica gel column using
DCM and methanol (19:1) as eluents to afford the titled compound
CH3). HRMS [C22H19F3N3O2 ]: calcd 414.1429, found 414.1432 [M
+ H]+. Analytical HPLC showed a 98.60% purity.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(3-(4-fluoro-
phenyl)-1H-pyrrol-1-yl)-2-hydroxy-2-methylpropanamide
(26b). Compound 26b was prepared following General Procedure C
per Scheme 3. The product was purified by a silica gel column using
ethyl acetate and hexanes (1:2 to 1:1) as eluents to afford 0.60 g of
the titled compound as a yellowish solid. Yield = 45%. 1H NMR (400
MHz, DMSO-d6) δ: 10.40 (s, 1H, NH), 8.42 (d, J = 2.0 Hz, 1H,
ArH), 8.24 (dd, J = 8.8 Hz, J = 2.0 Hz, 1H, ArH), 8.07 (d, J = 8.8 Hz,
1H, ArH), 7.43−7.38 (m, 2H, ArH), 7.11−7.05 (m, 3H, ArH), 6.73
(t, J = 2.0 Hz, 1H, pyrrole-H), 6.33−6.31 (m, 2H), 4.24 (d, J = 14.0
Hz, 1H, CH), 4.05 (d, J = 14.0 Hz, 1H, CH), 1.37 (s, 3H, CH3).
1
as a brown solid. Yield = 45%. H NMR (400 MHz, CDCl3) δ: 9.10
(bs, 1H, NH), 8.01 (bs, 1H, NH), 8.14 (s, 1H), 7.88−7.76 (m, 2H),
7.72 (s, 0.57H), 7.51 (bs, NH, 0.43H), 5.90 (bs, NH, 0.57H), 4.74
(bs, OH, 0.43H), 4.55 (d, J = 14.4 Hz, 0.43H), 4.54 (d, J = 13.6 Hz,
0.57H), 4.24 (bs, OH, 0.57H), 4.07 (d, J = 13.6 Hz, 0.57H), 3.97 (d, J
= 14.4 Hz, 0.43H), 1.56 (s, 1.29H), 1.48 (s, 1.71H). HRMS
+
[C14H14F3N6O2 ]: calcd 355.1130, found 355.1133 [M + H]+.
Analytical HPLC showed a 92.43% purity.
+
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-((4-cyano-3-
(trifluoromethyl)phenyl)amino)-2-hydroxy-2-methylpropana-
mide (19k). Compound 19k was prepared following General
Procedure C per Scheme 1. The product was purified by a silica gel
column using DCM and methanol (19:1) as eluents to afford the
HRMS [C22H18F4N3O2 ]: calcd 432.1335, found 432.1331 [M + H]+.
Analytical HPLC showed a 99.58% purity.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-(4-fluoro-
phenyl)-1H-pyrazol-1-yl)-2-hydroxy-2-methylpropanamide
(26c). Compound 26c was prepared following General Procedure C
per Scheme 3. The product was purified by a silica gel column using
DCM and methanol (19:1) as eluents to afford 0.33 g of the titled
compound as a white solid. Yield = 62%. 1H NMR (400 MHz,
DMSO-d6) δ: 10.29 (s, 1H, NH), 8.41 (s, 1H, ArH), 8.21 (d, J = 8.8
1
titled compound as a yellowish solid. Yield = 9%. H NMR (400
MHz, DMSO-d6) δ: 10.46 (s, 1H, NH), 8.46 (d, J = 2.0 Hz, 1H,
ArH), 8.21 (dd, J = 8.2 Hz, J = 2.0 Hz, 1H, ArH), 8.08 (d, J = 8.2 Hz,
1H, ArH), 7.19−7.15 (m, 2H), 6.98 (dd, J = 8.8 Hz, J = 2.4 Hz, 1H,
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Hz, 1H, ArH), 8.05 (d, J = 8.8 Hz, 1H, ArH), 7.68 (s, 1H, pyrazole-
H), 7.61 (t, J = 6.4 Hz, 2H, ArH), 7.08 (t, J = 8.4 Hz, 2H, ArH), 6.65
(s, 1H, pyrazole-H), 6.30 (s, 1H, OH), 4.51 (d, J = 14.0 Hz, 1H, CH),
4.31 (d, J = 14.0 Hz, 1H, CH), 1.42 (s, 3H, CH3). Mass (ESI,
filtered, and evaporated. The product was purified by a silica gel
column using ethyl acetate and hexanes (2:3) as eluents to afford
0.052 g of the titled compound as a yellow solid. Yield = 65%. H
1
NMR (400 MHz, CDCl3) δ: 9.07 (bs, 1H, NH), 7.82−7.80 (m, 1H),
7.79 (s, 1H), 7.76−7.74 (m, 2H), 7.72 (dd, J = 8.2, 2.8 Hz, 2H), 7.10
(t, J = 8.8 Hz, 2H), 5.15 (bs, 1H, OH), 4.96 (d, J = 14.0 Hz, 1H),
4.61 (d, J = 14.0 Hz, 1H), 1.62 (s, 3H). 19F NMR (CDCl3,
+
negative): 431.12 [M − H]−; HRMS [C21H17F4N4O2 ]: calcd
433.1288, found 433.1291 [M + H]+. Analytical HPLC showed a
96.01% purity.
+
decoupling) δ -62.24, -112.36. HRMS [C20H16F4N5O2 ]: calcd
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5-(4-fluoro-
phenyl)-1H-tetrazol-1-yl)-2-hydroxy-2-methylpropanamide
(26d). Compound 26d was prepared following General Procedure C
per Scheme 3. The product was purified by a silica gel column using
DCM and ethyl acetate (9:1) as eluents to afford 0.055 g of the titled
434.1240, found 434.1256 [M + H]+. Analytical HPLC showed a
99.69% purity.
(S)-N-(6-Cyano-5-(trifluoromethyl)pyridin-3-yl)-3-(4-(4-fluo-
rophenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-2-methylpropana-
mide (26g). Compound 26g was prepared in two steps per Scheme
4. Step 1. A solution of 16c (0.352 g, 1 mmol) in DMF (10 mL) was
treated with NaN3 (0.325 g, 5 mmol) under argon at 80 °C for 24 h.
The reaction mixture was cooled and extracted with CH2Cl2 (3 × 20
mL). The combined organic layers were washed with H2O (3 × 20
mL) and brine, dried, and evaporated to give a crude oil, which was
purified by silica gel chromatography (EtOAc/n-hexane = 1:2, v/v) to
afford 27b as a yellow solid. Yield = 87%. 1H NMR (400 MHz,
CDCl3) δ: 9.16 (bs, 1H, NH), 8.89 (s, 1H), 8.77 (s, 1H), 3.90 (d, J =
12.0 Hz, 1H), 3.52 (d, J = 12.0 Hz, 1H), 3.20 (bs, 1H, OH), 1.55 (s,
3H). 19F NMR (CDCl3, decoupled) δ: −62.11. MS (ESI) m/z 314.03
[M − H]−.
1
compound as a yellowish solid. Yield = 12%. H NMR (400 MHz,
DMSO-d6) δ: 10.39 (s, 1H, NH), 8.44 (s, 1H, ArH), 8.26 (d, J = 8.2
Hz, 1H, ArH), 8.10 (d, J = 8.2 Hz, 1H, ArH), 7.93−7.89 (m, 2H,
ArH), 7.30 (t, J = 8.2 Hz, 2H, ArH), 6.64 (s, 1H, OH), 5.09 (d, J =
14.0 Hz, 1H, CH), 4.92 (d, J = 14.0 Hz, 1H, CH), 1.55 (s, 3H, CH3).
+
Mass (ESI, negative): 433.17 [M − H]−; HRMS [C19H15F4N6O2 ]:
calcd 435.1193, found 435.1196 [M + H]+. Analytical HPLC showed
a 95.12% purity.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-((4-
fluorophenyl)(phenyl)amino)-2-hydroxy-2-methylpropana-
mide (26e). Compound 26e was prepared following General
Procedure C per Scheme 1. A mixture of bromide (218 mg, 0.619
mmol) and potassium carbonate (172 mg, 1.23 mmol) in 30 mL of
acetone was heated to reflux for 30 min. After complete conversion of
starting bromide to the desired intermediate epoxide as monitored by
TLC, the solvent was evaporated under reduced pressure to give a
yellowish residue, which was poured into 20 mL of anhydrous EtOAc.
The solution was filtered through a celite pad to remove the K2CO3
residue and condensed under reduced pressure to give a yellowish
solid of epoxide, which was dissolved in 5 mL of anhydrous THF to
prepare a desired solution of epoxide in THF. To the solution was
added 1 mL of lithium perchlorate and 4-fluoro-N-phenylaniline (116
mg, 0.619 mmol) in anhydrous THF (3 mL), which was cooled in an
ice−water bath under an argon atmosphere. After addition, the
resulting mixture was allowed to stir overnight at rt under argon. The
reaction was quenched by water and then extracted with ethyl acetate.
The organic layer was washed with brine, dried with MgSO4, filtered,
and concentrated under vacuum. The product was purified by a silica
gel column using ethyl acetate and hexanes (2:1) as eluents to afford
Step 2. To a suspension of copper(I) iodide (11 mg, 0.055 mmoL)
in acetonitrile (7 mL)/water (3 mL) was added 27b (57 mg, 0.182
mmol) at rt and then 28 (0.015 mL, 0.182 mmol) was added. The
resulting reaction mixture was stirred at rt for 3 d. The mixture was
evaporated under reduced pressure, poured into water/brine (1:1, v/
v), and then extracted with ethyl acetate. The combined organic
extracts were then washed with brine, dried over sodium sulfate,
filtered, and evaporated. The product was purified by a silica gel
column using ethyl acetate and hexanes (2:3) as eluents to afford
1
0.052 g of the titled compound as a yellow solid. Yield = 65%. H
NMR (400 MHz, CDCl6) δ: 10.16 (bs, 1H, NH), 9.28 (s, 1H), 8.88
(s, 1H), 8.31 (s, 1H), 7.90 (t, J = 7.8 Hz, 2H), 7.20 (t, J = 8.8 Hz,
2H), 5.73 (bs, 1H, OH), 4.94 (d, J = 14.2 Hz, 1H), 4.73 (d, J = 14.2
Hz, 1H), 1.62 (s, 3H). 19F NMR (CDCl3, decoupling) δ −61.66,
−14.58. MS (ESI) m/z 534.06 [M + H]+; 433.09 [M − H]−. HRMS
−
[C19H13F4N6O2 ]: calcd 433.1036, found 433.1039 [M − H]−.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(5-(4-fluoro-
phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-2-methylpropana-
mide (26h). Compound 26h was prepared in two steps per Scheme
4. Step 1. A solution of 16a (0.351 g, 1 mmol) in DMF (10 mL) was
treated with NaN3 (0.325 g, 5 mmol) under argon at 80 °C for 24 h.
The reaction mixture was cooled and extracted with CH2Cl2 (3 × 20
mL). The combined organic layers were washed with H2O (3 × 20
mL) and brine, dried, and evaporated to give a crude oil, which were
purified by silica gel chromatography (EtOAc/n-hexane = 1:2, v/v) to
1
the titled compound as a brown solid. Yield = 67%. H NMR (400
MHz, CDCl3) δ: 8.85 (bs, 1H, NH), 7.87 (m, 1H), 7.81−7.73 (m,
2H), 7.65 (dd, J = 8.4, 1.8 Hz, 1H), 7.20 (m, 2H), 7.05−7.00 (m,
2H), 6.94−6.89 (m, 5H), 4.54 (d, J = 15.2 Hz, 1H), 3.84 (d, J = 15.2
Hz, 1H), 3.61 (s, 1H), 1.53 (s, 3H). MS (ESI, negative) 456.1 [M −
−
H]−. HRMS [C24H18F4N3O2 ]: calcd 456.1335, found 456.1342 [M
− H]−.
(S)-N-(4-Cyano-3-(trifluoromethyl)phenyl)-3-(4-(4-fluoro-
phenyl)-1H-1,2,3-triazol-1-yl)-2-hydroxy-2-methylpropana-
mide (26f). Compound 26f was prepared in two steps per Scheme 4.
Step 1. A solution of 16a (0.351 g, 1 mmol) in DMF (10 mL) was
treated with NaN3 (0.325 g, 5 mmol) under argon at 80 °C for 24 h.
The reaction mixture was cooled and extracted with CH2Cl2 (3 × 20
mL). The combined organic layers were washed with H2O (3 × 20
mL) and brine, dried, and evaporated to give a crude oil, which were
purified by silica gel chromatography (EtOAc/n-hexane = 1:2, v/v) to
1
afford 0.224 g of 27a as a yellow solid. Yield = 72%. H NMR (400
MHz, CDCl3) δ: 9.00 (bs, 1H, NH), 8.08 (s, 1H), 7.95 (d, J = 8.4 Hz,
1H), 7.81 (d, J = 8.4 Hz, 1H), 3.92 (d, J = 12.4 Hz, 1H), 3.50 (d, J =
12.4 Hz, 1H), 2.96 (s, 1H, OH), 1.54 (s, 3H). 19F NMR (CDCl3,
decoupled) δ: −62.21. MS (ESI) m/z 314.03 [M + H]+; 312.18 [M −
H]−.
Step 2. A mixture of 27a (57 mg, 0.18 mmol), 28 (0.015 mL, 0.18
mmol), and copper iodide (11 mg, 0.055 mmol) in AcCN/H2O (1/
0.5 mL) was loaded into a vessel with a cap. The reaction vessels were
placed in a reactor block in the microwave. A programmable
microwave irradiation cycle of 30 min on 300 W at 100 °C and 25
min off (fan-cooled) was executed two times if the starting materials
were shown on TLC (total irradiation time, 60 min). The mixture was
transferred to a round-bottom flask to be concentrated under reduced
pressure and poured into EtOAc, which was washed with water, dried
over MgSO4, and concentrated. The product was purified by a silica
gel column using ethyl acetate and hexanes (2:1) as eluents to afford
1
afford 0.224 g of 27a as a yellow solid. Yield = 72%. H NMR (400
MHz, CDCl3) δ: 9.00 (bs, 1H, NH), 8.08 (s, 1H), 7.95 (d, J = 8.4 Hz,
1H), 7.81 (d, J = 8.4 Hz, 1H), 3.92 (d, J = 12.4 Hz, 1H), 3.50 (d, J =
12.4 Hz, 1H), 2.96 (s, 1H, OH), 1.54 (s, 3H). 19F NMR (CDCl3,
decoupled) δ: -62.21. MS (ESI) m/z 314.03 [M + H]+; 312.18 [M −
H]−.
Step 2. To a suspension of copper(I) iodide (11 mg, 0.055 mmoL)
in acetonitrile (7 mL)/water (3 mL) was added 27a (57 mg, 0.182
mmol) at rt and then 28 (0.015 mL, 0.182 mmol) was added. The
resulting reaction mixture was stirred at rt for 3 d. The mixture was
evaporated under reduced pressure, poured into water/brine (1:1, v/
v), and then extracted with ethyl acetate. The combined organic
extracts were then washed with brine, dried over sodium sulfate,
1
0.070 g of the titled compound as a yellow solid. Yield = 90%. H
NMR (400 MHz, acetone-d6) δ: 9.00 (bs, 1H, NH), 8.44 (s, 1H),
8.30 (s, 1H), 8.25 (d, J = 8.4 Hz, 1H), 8.02 (d, J = 8.4 Hz, 1H), 7.89
(dd, J = 8.0 Hz, 2.4 Hz, 2H), 7.20 (d, J = 8.8 Hz, 2H), 5.67 (s, 1H,
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OH), 4.92 (d, J = 14.0 Hz, 1H), 4.72 (d, J = 14.0 Hz, 1H), 1.60 (s,
3H). 19F NMR (acetone-d6, decoupling) δ 114.68, 61.64. HRMS
Tennessee 38163, United States; orcid.org/0000-0002-
6093-0985; Phone: (+1)-901-448-6026; Email: dmiller@
uthsc.edu; Fax: (+1)-901-448-3446
+
[C20H16F4N5O2 ]: calcd 434.1240, found 434.1259 [M + H]+.
Analytical HPLC showed a 98.91% purity.
Biological Methods. Competitive Ligand Binding Assay. AR
ligand binding assay was performed as described previously using the
purified AR LBD cloned from rat prostate.38,39
AR Transactivation Assay. HEK-293 cells plated in 24-well plates
at 70 000 cells/well were transfected using 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 luciferase assay
was performed 48 h after transfection. Firefly luciferase assay values
were normalized to Renilla luciferase assay numbers.
Androgen Receptor-Dependent Gene Expression in LNCaP Cells.
LNCaP cells were plated in 96-well plates in RPMI supplemented
with 1% csFBS without phenol red. Cells were maintained in this
medium for 2 d and treated with the compounds 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 probes (Life Technolo-
gies).
Authors
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
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; orcid.org/0000-0002-
1609-6524
Cellular Proliferation Assays in MR49F Cells. MR49F cells were
plated in 96-well plates in RPMI + 1% csFBS without phenol red.
Cells were treated in this medium with the compounds in the
presence of 0.1 nM R1881 for 6 d, with medium change and
retreatment after 3 d. Number of viable cells was measured using Cell-
Titer-Glo (Promega).
Ramesh Narayanan − Department of Medicine, College of
Medicine, University of Tennessee Health Science Center,
Memphis, Tennessee 38163, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00439
Western Blot. Indicated cell lines were treated for 24 h. Cells were
harvested, the protein was extracted, and Western blot for AR, AR SV,
and GAPDH was performed using the AR PG-21 rabbit polyclonal
antibody that binds to the N-terminus of the AR.38,39
In Vitro Metabolism Assays. DMPK assays were performed as
described before.38,39 Metabolism assays were performed in mouse,
rat, and human liver microsomes as described before.
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Funding
Hershberger Assay. Male Sprague Dawley rats (100−200 g) were
randomized into groups (N = 5/group) based on body weight.
Animals were orally treated with vehicle or 20 mg/kg fixed dose 26f
for 13 d. Animals were sacrificed on day 14 of treatment, and seminal
vesicle organs were removed and weighed. Organ weights were
normalized to body weight.
Xenograft Studies. Enzalutamide-resistant LNCaP cells (5
million/mouse: 1:1 medium:Matrigel) were implanted subcutane-
ously in male NSG mice. Once the tumors reach 200−400 mm3, the
animals were castrated, and the tumors were allowed to regrow as
castration-resistant prostate cancer (CRPC). Once the regrown
tumors reached 400 mm3, the animals were randomized and treated
orally with vehicle or 60 mg/kg/day 26f. Tumor volume was
measured twice weekly. Animals were sacrificed 28 d after treatment,
and the tumors were processed for further analysis. *p < 0.05.
Animal Studies. All animal studies were conducted under the
UTHSC Animal Care and Use Committee-approved protocols and in
accordance with the UTHSC guidelines.
The work in this manuscript was funded in part by a research
grant from GTx, Inc. and Oncternal Therapeutics, Inc. (R.N.
and D.D.M.), by a research grant from National Cancer
Institute (NCI) to R.N. (1R01CA229164), and by a grant
from the University of Tennessee Health Science Center
(UTHSC) for Cancer Research (R.N. and D.D.M.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors 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.
ABBREVIATIONS
■
ADT, androgen deprivation therapy; AF-1, an activation
function; AF-2, an external (solvent exposed) binding site
termed activation function-2; AR, androgen receptor; AR SV,
AR splice variant; CRPC, castration-resistant prostate cancer;
csFBS, charcoal-stripped fetal bovine serum; CSPC, castration-
sensitive PC; DBD, DNA-binding domain; DHT, 5α-
dihydrotestosterone; DMPK, distribution, metabolism, and
pharmacokinetic; EnzR, enzalutamide-resistant; ER, estrogen
receptor; FL, full length; GR, glucocorticoid receptor; GRE,
glucocorticoid response element; LBD, ligand binding domain;
MIB, milbolerone; MLM, mouse liver microsomes; NSG,
NOD SCID γ; NTD, N-terminal domain; PC, prostate cancer;
PD, pharmacodynamics; PK, pharmacokinetic; PR, progester-
one receptor; PROTACs, proteolysis-targeting chimaeras;
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00439.
■
sı
Additional information on compound characterization;
and additional biological experiments and figures (PDF)
Molecular formula strings (CSV)
AUTHOR INFORMATION
Corresponding Author
Duane D. Miller − Department of Pharmaceutical Sciences,
University of Tennessee Health Science Center, Memphis,
■
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RLUs, relative light units; rt, room temperature; SAR,
structure−activity relationship; SARDs, selective androgen
receptor degraders; SARM, selective androgen receptor
modulator; TGI, tumor growth inhibition; UPS, ubiquitin
proteasome system; VP, ventral prostate; wt, wild type
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