Discovery of JNJ-63576253: A Clinical Stage Androgen Receptor Antagonist for F877L Mutant and Wild-Type Castration-Resistant Prostate Cancer (mCRPC)

Article abstract of DOI:10.1021/acs.jmedchem.0c01563

Persistent androgen receptor (AR) activation drives therapeutic resistance to second-generation AR pathway inhibitors and contributes to the progression of advanced prostate cancer. One resistance mechanism is point mutations in the ligand binding domain

Full text of DOI:10.1021/acs.jmedchem.0c01563

pubs.acs.org/jmc
Drug Annotation
Discovery of JNJ-63576253: A Clinical Stage Androgen Receptor
Antagonist for F877L Mutant and Wild-Type Castration-Resistant
Prostate Cancer (mCRPC)
Zhuming Zhang,* Peter J. Connolly, Heng Keang Lim, Vineet Pande, Lieven Meerpoel,
Christopher Teleha, Jonathan R. Branch, Janine Ondrus, Ian Hickson, Tammy Bush, Leopoldo Luistro,
Kathryn Packman, James R. Bischoff, Salam Ibrahim, Christopher Parrett, Yolanda Chong,
Marco M. Gottardis, and Gilles Bignan*
Cite This: J. Med. Chem. 2021, 64, 909−924
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ABSTRACT: Persistent androgen receptor (AR) activation drives therapeutic resistance to second-generation AR pathway
inhibitors and contributes to the progression of advanced prostate cancer. One resistance mechanism is point mutations in the ligand
binding domain of AR that can transform antagonists into agonists. The AR F877L mutation, identified in patients treated with
enzalutamide or apalutamide, confers resistance to both enzalutamide and apalutamide. Compound 4 (JNJ-pan-AR) was identified
as a pan-AR antagonist with potent activity against wild-type and clinically relevant AR mutations including F877L. Metabolite
identification studies revealed a latent bioactivation pathway associated with 4. Subsequent lead optimization of 4 led to amelioration
of this pathway and nomination of 5 (JNJ-63576253) as a clinical stage, next-generation AR antagonist for the treatment of
castration-resistant prostate cancer (CRPC).
advanced prostate cancers, these second-generation AR
antagonists show improved metastasis-free survival (MFS)
for both castration-sensitive and castration-resistant prostate
cancers.5,4 However, as with other molecularly targeted cancer
therapies in precision medicine, their efficacy is short-lived in
many patients, and acquired resistance almost invariably
emerges, resulting in disease progression to metastatic
castration-resistant prostate cancer (mCRPC).6 Genomic
studies have established numerous resistance mechanisms
such as gene alteration, amplification, and overexpression,7,8
splice variant isoform expression (AR-V7),9 ligand binding
INTRODUCTION
■
Oncogenic activation of the androgen receptor (AR) is crucial
for all stages of progressive prostate cancer.1 Androgen
deprivation therapy (ADT) such as abiraterone acetate
effectively suppresses biosynthesis of endogenous androgens
for AR activation.2 AR-targeted antiandrogens such as
enzalutamide (1, Figure 1) and apalutamide (3, ARN-509,
Figure 2) block the binding of androgens to the androgen
receptor.3,4 Now approved by FDA as the standard of care for
Received: September 7, 2020
Published: January 20, 2021
Figure 1. Chemical structures of enzalutamide, 1, and darolutamide,
2.
https://dx.doi.org/10.1021/acs.jmedchem.0c01563
© 2021 American Chemical Society
J. Med. Chem. 2021, 64, 909−924
909

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Drug Annotation
work on full characterization of 4 and additional studies
leading to the selection of compound 5, known as JNJ-
63576253 (TRC-253) (Figure 2), highlighting data supporting
its suitability for clinical development.
RESULTS AND DISCUSSION
In wild-type androgen receptor competitive radioligand
binding assays (Table 1), 4 showed 2-fold better binding
■
a
Table 1. Radioligand Binding of Compounds 4 and 1
4
1
IC50 (nM)
Ki (nM)
IC50 (nM)
Ki (nM)
Figure 2. Chemical structures of 2 (Apalutamide ARN-509), 4 (JNJ-
pan-AR), and 5 (JNJ-63576253). Ring “A” and Ring “B” (and its
periphery) were the focus of our lead optimization efforts.
AR
GR
19
20000
8.4
9900
38
29000
17
14000
a
Radioligand binding inhibition and affinity calculations were
domain (LBD) point mutations,10,11 and glucocorticoid
receptor bypass.12 Aberrant activation and persistent androgen
receptor transcriptional signaling is now understood as the
central core of resistant mechanisms in CRPC.13 Continued
reliance on AR provides a powerful rationale for retargeting the
androgen/AR axis with next-generation AR pathway inhib-
itors.14 Therefore, reactivation of AR signaling represents a
major therapeutic opportunity as well as a challenge for
treating mCRPC.15
Androgen receptor is a ligand-inducible transcription factor
consisting of the N-terminal domain (NTD), DNA binding
domain (DBD), and C-terminal ligand binding domain (LBD).
To control the expression of AR regulated genes, the highly
conserved DBD directly binds to DNA and thus allows
constitutively active activation function 1 (AF1) in the NTD
and ligand-dependent activation function 2 (AF2) in the LBD
to stimulate transcriptional activities.16
Point mutations in the LBD of AR accounts for 10−20% of
resistance and are characterized by receptor activation rather
than inhibition upon ligand binding.17 For example, four
clinically relevant resistant mutations L702H, T878A, W742C,
and W875L (formerly known as L701H, T877A, W741C, and
W874L respectively) were identified for first-generation anti-
androgens flutamide or bicalutamide.18−21 Second-generation
antiandrogens 1 and 3 were in part developed to overcome
these resistance mechanisms.3,4 In 2013, the mutation of
phenylalanine to leucine at AR amino acid 877 (F877L,
formerly known as F876L) was reported to confer resistance to
both 1 and 3, leading to antagonist-to-agonist switch in
response to preclinical treatment in vitro.10,11 In the clinic, AR
F877L was detected in the plasma ctDNA for 3 of 29
progressing patients enrolled in the ARN-509 Phase I trial.17
Recently, darolutamide (ODM-201, 2, Figure 1) was reported
to target resistance mechanisms including F877L mutation as a
next-generation AR antagonist.22
In parallel to the development of 3, we collaborated with
Aragon Pharmaceuticals and screened a library of compounds
to evaluate activity against wild-type AR as well as AR F877L
and other clinically relevant mutants.23 From this work,
compound 4 (JNJ-pan-AR, Figure 2) was identified as a
potential lead with favorable in vitro ADME and in vivo
pharmacological properties. However, unacceptable toxicity
findings hampered its further developability. We subsequently
discovered an in vitro bioactivation pathway that could
potentially explain the observed in vivo toxicity. Our hypothesis
of blocking the bioactivation pathway served as the basis to
initiate SAR studies for lead optimization. Here, we report our
determined using [3H]methyltrienolone and [3H]dexamethasone for
AR and GR, respectively.
affinity (IC50 = 19 nM, Ki = 8.4 nM) than enzalutamide (1,
IC50 = 38 nM, Ki = 17 nM) using labeled synthetic androgen
R1881 (methyltrienolone) as the radioligand. Both com-
pounds were highly selective over glucocorticoid receptor, GR
(Table 1), and neither compound showed appreciable affinity
for estrogen receptor, ER.
Having determined that 4 is a potent, competitive binder of
AR, it was necessary to establish whether 4 acts as an
antagonist or agonist.10,24 The VP16 AR (WT and F877L)
reporter system was chosen due to its highly sensitive response
to ligand-dependent agonism (Supplementary Section S-2).25
In these transcriptional reporter assays, we defined antagonism
as the ability of a test compound to inhibit the response
stimulated by low concentration of the synthetic androgen R-
1881. Conversely, agonism was defined as the ability of a test
compound to cause a response in the absence of exogenous
androgen stimulation. In our hands, 4 completely inhibited
signaling in HepG2 cells transiently transfected with VP16-AR
F877L (IC50 = 127 nM) in the presence of R1881 (90 pM,
antagonist mode), whereas in the same assay 1 only reached a
maximal inhibition of 50% at any concentrations up to 30 μM
(Table 6, Supplementary Figure S1a). When the VP16-AR
F877L reporter assay was run in agonist mode (Supplementary
Figure S1b), 1 elicited activation of the androgen receptor in
the absence of R1881 in a dose-dependent manner starting at 1
nM, reaching 40% activity at 3 μM (Table 6, Supplementary
Figure S1b). In contrast, 4 did not significantly activate AR and
elicited only 10% maximal agonism at 300 nM in the absence
of R1881. Because VP16-AR is constitutively nuclear and
drives transcription through androgen response elements
(AREs) in the absence of coactivator protein recruitment,
this assay provides a direct assessment of ligand-induced DNA
binding. We therefore conclude that 4 is effective in blocking
ligand-induced DNA binding by AR.
To explore the activity of 4 in a native AR setting, ARE-
luciferase reporter constructs were introduced into LNCaP
prostate cancer cells, a model of prostate adenocarcinoma, that
stably express either wild-type AR (LNCaP AR cs) or F877L
mutant AR (LNCaP F877L).26 Compound 4 was a full
antagonist of the androgen-dependent reporter gene activity of
both AR F877L and AR WT in the presence of R1881. As
expected, enzalutamide (1) was an antagonist of AR WT.
However, 1 was an incomplete antagonist in the presence of
R1881 and behaved as an agonist in AR F877L cells at 10 μM
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Drug Annotation
in the absence of R1881 (Table 2). To demonstrate that AR
antagonism has an antiproliferative effect in androgen-depend-
Table 2. Transcriptional Reporter Assay Activity of 1−18 in
LNCaP F877L (Mutant) and LNCaP AR cs (WT) Cells
LNCaP F877L
LNCaP WT
b
a
b
a
compound
IC50 (nM)
agonism
IC50 (nM)
agonism
c
1
4
5
7
8
9
10
11
12
13
14
15
16
6
not fit
yes
no
no
no
no
no
no
no
no
no
no
no
no
no
no
117 66
191 120
54 53
115 89
240 580
no
no
no
no
no
no
no
no
no
no
no
no
no
no
no
98 63
37 47
85 68
145 200
427 190
417 64
d
e
309 92
501
210
214 92
102 110
81 43
47 27
37 16
204 150
182 45
1000 380
302 250
162 210
120 78
50 36
76 47
355 170
204 130
2239 700
17
a
LNCaP F877L and LNCaP WT reporter assays were repeated ≥3
times. Agonism is defined as complete, indicating activity was equal to
that of R1881 stimulated reporter output. Max inhibition >90% for
all compounds except specifically mentioned. Complete agonist at 10
μM. Max inhibition 67%. Max inhibition 85%.
b
c
d
e
ent tumor cell lines, 4 and 1 were evaluated in the growth
inhibition of VCaP cells (WT AR). Compound 4 inhibited the
growth of VCaP cells with an IC50 of 92 nM, and the IC50 for 1
was 149 nM (Table 3 and Supplementary Figure S3). We thus
conclude that 4 is equipotent to 1 in wild-type AR in vitro.
Additionally, 4 was capable of effectively inhibiting trans-
activation of other clinically relevant AR ligand binding domain
mutations, L702H, T878A, W742C, and W875L, in transiently
transfected HepG2 cells (Supplementary Table S1).
Androgen receptor nuclear translocation occurs following
ligand binding and intramolecular dimerization.27 In an
immunofluorescent imaging assay for AR protein localization
(Supplementary Section S-5, Figure S2), 4 potently blocked
nuclear translocation of AR F877L in both the presence and
absence of R1881 in LNCaP F877L cells. Enzalutamide (1)
partially inhibited nuclear translocation in the presence of
R1881 and induced nuclear translocation of AR F877L to
∼50% of R1881-stimulated levels in the absence of R1881.25 In
contrast, both 4 and 1 inhibited androgen-stimulated nuclear
translocation in wild-type LNCaP cells to a similar extent
(Supplementary Figure S2). In this assay, 4 also inhibited the
expression of PSA (KLK3), a protein whose expression is
transcriptionally regulated by AR, in the presence of R1881 for
both AR F877L and AR WT (data not shown).
The effect of 4 on androgen-dependent signaling was also
assessed using the Hershberger assay as an in vivo PD model.
In this assay, changes in weight of five androgen sensitive
organs (ASOs) under stimulation by testosterone propionate
(TP) were measured with or without AR antagonist treatment.
Compound 4 was found to be a potent antagonist of ASO
development in vivo, with activity equivalent to that of 1 when
dosed at 30 mg/kg QD for 10 days (Supplementary Figure
S4a).
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To demonstrate in vivo activity in a prostate cancer model
driven by AR F877L that is resistant to 1, LNCaP F877L
tumors were established in castrated SHO mice, and the
antitumor activity of 4 was determined at 10 and 30 mg/kg
orally once daily. At the end of study, tumor growth inhibition
(TGI) was calculated using initial and final tumor volume
measurements. TGIs of 80.2% and 100.8% (complete stasis)
were obtained for the 10 mg/kg and 30 mg/kg cohorts
respectively (Table 8 and Supplementary Figure S5a).
Treatment with 4 led to complete stasis of tumor growth
inhibition in a F877L-driven xenograft, whereas in a separate
experiment no antitumor activity was observed in animals
treated with 1 at 30 mg/kg orally once daily (Supplementary
Section S-8).
To better understand the potential structural mechanism
and binding mode of 1 in wild-type AR and AR F877L, we
built a homology model representing a putative antagonistic
AR conformation. It should be noted that to date no
experimental structures of AR LBD in antagonist mode have
been reported.33 Our homology model of AR LBD was built
using an antagonist-bound structure of glucocorticoid receptor
as a template and used for various ligand docking calculations
(Supplementary Section S-13).29 The mechanism for antago-
nist-to-agonist switch could be rationalized by analogy,
comparing the open and closed conformations, respectively,
of Helix 12 located in LBD (Figure 3).30,31 Enzalutamide (1)
conformation and should favor the antagonist conformation,
resulting in functional antagonism of AR F877L and
inactivation of transcription (Figure 3). This structural
hypothesis would need additional experimental validation to
broadly establish the idea that such a conformational switch is
responsible for the observed pharmacological properties.
The attractive in vitro and in vivo pharmacological properties
of 4 supported its further progression into single and repeat
oral dose toleration 14-day studies in rats and dogs.
Unfortunately, 4 displayed unacceptable hepatotoxicity in
dogs, including liver necrosis, bile degeneration, and liver
enzyme elevation (ALT, ALP, and GGT) at all doses (data not
shown). Three major in vivo metabolites were detected in rat
and dog plasma (respectively) by high resolution LC-MS on
day 13: N-oxide M2 (2%, 20%), hydantoin M3 (2%, 0.2%),
and N-des-methylpiperidine M4 (19%, 31%), consistent with
findings from in vitro metabolite identification studies in
human, rat, and dog hepatocytes. Significantly, a small amount
of phenolic metabolite 6 (M1) was also observed in vivo, even
though the percentage could not be estimated due to different
ionization method utilized for identification by LC-MS (Figure
4).
Figure 4. Proposed metabolic pathway of 4 in rat and dog plasma
samples and potential bioactivation via M4 in vivo.
The detection of 6 (M1) in vivo raised the possibility of
CYP450-mediated bioactivation of 4. Phenolic structures are
known to have safety liabilities; for example, it was reported
that substituted phenols could act as uncouplers in
mitochondrial oxidative phosphorylation.28 In a GSH trapping
experiment, an authentic sample of 6 formed glutathione
adducts with a composition of M1+O+GSH-2H (2.1%) during
incubation in dog liver microsomes in the presence of GSH
and NADPH (Table 7). However, no GSH conjugate was
detected from incubation of parent 4 in dog liver microsomes
under the same conditions, suggesting the formation of 6 (M1)
is a slow process due to low turnover of 4 in vitro. A putative
bioactivation pathway was thus proposed involving the initial
oxidative formation of o-quinone (M1+O) (Figure 4).
To determine whether reactive metabolites from phenol M4
were indeed responsible for observed hepatotoxicity (Figure
4), we began SAR studies to evade this bioactivation pathway
and block the formation of a phenolic metabolite while
simultaneously balancing the desirable in vitro and in vivo
pharmacological parameters of 4. Our systematic SAR analysis
of the thiohydantoin chemotype suggested that keeping the
basic piperidine nitrogen was favorable for full F877L
Figure 3. A manual docking model of 4 (orange balls and sticks with
gray surface) with a potentially antagonistic conformation (orange
tubes; homology model of AR built using GR PDB ID: 1NHZ) of AR
LBD. Superposition of a crystal structure of AR in agonistic
conformation (cyan tubes; PDB ID: 1T5Z) is shown as a reference
to demonstrate the potential role of Helix 12 in modulating the
pharmacology of AR.
was predicted to bind to the AR WT pocket in close proximity
to Helix 12 and keep Helix 12 in an open, extended
conformation described as the antagonist conformation.32
Utilizing our molecular docking models, we hypothesized that
the AR F877L mutation reduced steric hindrance in the ligand
binding pocket and subsequently allowed 1 to freely rotate and
point toward Helix 11 and the loop between Helices 11 and
12. The model suggested that this change potentially allowed
Helix 12 to close, resulting in an agonistic conformation for 1.
Conversely, compound 4 was predicted to bind in close
proximity to Helix 12 in an open conformation. Because of the
presence of the bulky and charged piperidine group, 4 would
have severe clashes with the protein in a putative agonistic
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Drug Annotation
antagonism. Additionally, prior studies indicated that the
middle thiohydantoin core, including the spiro-cyclobutyl
feature, has been optimized.23,34 Therefore, our next round of
SAR iteration was focused on rational design of substituents on
ring “A”, ring “B”, and its periphery by preserving the structural
features of the key pharmacophore such as the critical cyano
group (ring “A”) that interacts with Arg752 via a hydrogen
bond (Figure 3) and the pyridine nitrogen moiety (ring “A”)
for optimal potency and better plasma protein binding free
fraction.
Scheme 2. Synthesis of Thiohydantoin Analogues 4, 5, 7, 8,
13, 14, and 15
a
More than 200 analogues of 4 were synthesized with
modifications on the ring “A” or “B” region.35 Selected
representatives are highlighted in Figure 5, including those
a
Reagents and conditions: (a) DIAD or DEAD, PPh3, THF, 60 °C;
(b) HCl, 1,4-dioxane; (c) TFA; (d) HCHO, NaBH(OAc)3.
provided corresponding analogues 4, 11, and 12 as HCl salts.
Similarly, the Mitsunobu reaction of 6, 17, 22c−e with
commercially available 24, followed by deprotection reaction
and treatment with HCl, provided analogues 8, 5, 13−15,
respectively. Reductive amination of 5 with formaldehyde and
sodium triacetoxyborohydride led to the formation of 7.
Compounds 9 and 10 were prepared according to Scheme 3.
Cyano derivative 19c was prepared via a Strecker reaction by
Figure 5. Chemical structures for close analogues of 4 designed to
address bioactivation issues. The pyridone metabolite 17 of 5 is also
shown.
with a metabolic soft spot remote from the bioactivation site
(ring “A”, 11, 12, and 13), electronic alteration (ring “B”, 5, 7,
9, 10, 12, 13, 14), or a carbon-linker in place of oxygen (ring
“B”, 16). Doubly modified compound 15 was chosen as a
comparator to both 8 and 14. Efforts to block or attenuate
bioactivation by steric hindrance from additional substituents
on ring “B” were also attempted but proved unsuccessful (data
not shown).
a
Scheme 3. Synthesis of Thiohydantoin Analogues 9 and 10
The general syntheses of analogues and metabolites are
outlined in Schemes 1−4.35 In Scheme 1, cyano derivatives
Scheme 1. Synthesis of Thiohydantoin Analogues
a
Metabolites or Intermediates 6, 17, and 22a−e
a
Reagents and Cconditions: (a) NaCN, AcOH; (b) DMA, 60 °C; (c)
HCl, 1,4-dioxane; (d) HCHO, NaBH(OAc)3.
heating commercially available amine 18c in acetic acid with
cyclobutanone in the presence of sodium cyanide. 19c was
then cyclized to 22f by heating with freshly prepared
isothiocyanate 21a, in dimethylacetamide. Treatment with
anhydrous HCl in 1,4-dioxane solution provided 10 as HCl
salt. Reductive amination of 10 with formaldehyde and sodium
triacetoxyborohydride, followed by treatment of anhydrous
HCl, afforded 9. A variation of synthetic route Scheme 3,
replacing 18c with commercially available 18d, afforded
analogue 16 as shown in Scheme 4.
The synthesized analogues and selected metabolites
(intermediates 6 and 17) were evaluated in LNCaP AR
F877L and LNCaP AR WT cellular reporter assays for
luciferase transcriptional activities. The results are summarized
in Table 2. Analogues 5, 7−16, and phenolic metabolites 6 and
17 all acted as full antagonists and inhibited luciferase
transcription with IC50 values ranging from 30 to >2000 nM
(Table 2). No agonism of either AR F877L or AR WT was
observed for these compounds under the assay conditions.
a
Reagents and conditions: (a) TMSCN, ZnI2 (cat), MeOH, rt or
heating; (b) CHCl3/H2O/DMA, rt; (c) DMA, 60 °C.
19a−b were prepared via a Strecker reaction by heating
commercially available or synthetically accessible amines 18a−
b in methanol or neat with cyclobutanone in the presence of
trimethylsilyl cyanide and a catalytic amount of zinc iodide.
Cyano derivatives 19a−b were then cyclized to 6, 17, and
22a−e in a one-pot reaction in dimethylacetamide by heating
with isothiocyanates 21a−d, freshly prepared from the
corresponding anilines 20a−d, followed by treatment with
aqueous HCl solution.
In Scheme 2, the Mitsunobu reaction of 6, 22a−b with
commercially available 23, followed by treatment with HCl
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Drug Annotation
a
ranging from 250 to >2700 nM (Table 3). Despite comparable
or better potency in LNCaP reporter assays, demethylated 8
(VCaP IC50 = 306 nM) was less potent compared to
corresponding 4 (VCaP IC50 = 92 nM). Pyridone metabolite
16 is the least potent compound (VCaP IC50 = 1692 nM).
In mouse single-dose PK studies, 8, the major active
metabolite M4 of 4 (Figure 2) displayed comparable AUC
(10.4 μg·h/mL) and Cmax (1.17 μM) to that of 4 (AUC 9.4 μg·
h/mL, Cmax 0.98 μM) after oral dosing, with a shorter but still
reasonable half-life (T1/210.5 h) compared to that of 4
(T1/246.4 h) in IV dosing (Table 4). The same trend was
observed by comparing 5 and 7 in terms of exposure and half-
life, indicating demethylated analogues still maintained the
desirable PK characteristics of their parents. Furthermore, both
8 (HLM T1/2 > 180 min) and 5 (HLM T1/2 > 180 min) are
more stable in human liver microsomes compared to 4 (HLM
Scheme 4. Synthesis of Thiohydantoin Analogue 16
a
Reagents and conditions: (a) NaCN, AcOH; (b) DMA, 60 °C; (c)
TFA; (d) HCl, 1,4-dioxane.
Interestingly, analogue 8 (F877L IC50 = 145 nM; WT IC50
=
240 nM) was equally potent to N-methyl parent 4 (F877L IC50
= 98 nM; WT IC50 = 191 nM, Table 2). The potency of des-
methyl 5 (F877L IC50 = 37 nM; WT IC50 = 54 nM) was 2-fold
T
1/2 = 93.1 min) and 7 (HLM T1/2 = 173 min). All analogues
displayed favorable PK parameters with oral bioavailabilities
ranging from 45% to >100% (Table 4)
better compared to that of 7 (F877L IC50 = 85 nM; WT IC50
=
All analogues except for 7 were tested in the Hershberger
assay for their inhibitory effect on the five androgen sensitive
organs (ASOs) under stimulation by testosterone propionate
(TP). The pharmacodynamic (PD) results are summarized in
Table 5. Because in vivo metabolite studies indicated that 5 was
115 nM), confirming that piperidine without methyl
substitution still maintained potency. Furthermore, both 5
and 7 were comparable or slightly more potent than 4 and 8,
indicating a pyridinyl “B” ring is well tolerated. In contrast, the
pyrimidinyl “B” ring in 9 (F877L IC50 = 427 nM; WT IC50
=
417 nM) and 10 (F877L IC50 = 309 nM; WT IC50 = 501 nM)
was less tolerated, evidenced by the loss of more than 3-fold
Table 5. PD Effect of 5, 8, 11−16 in Hershberger Assay on
ASO (Seminal Vesicles and Prostate): QD × 10 Days
Dosing
potency. Methoxy (ring “A”) modification (11, F877L IC50
=
214 nM; WT IC50 = 302 nM) lost 2-fold in potency, but
potency was restored in combination with a pyridinyl “B” ring
(12, F877L IC50 = 102 nM; WT IC50 = 162 nM). Similarly,
methyl or chloro-modification in ring “A” in combination with
pyridinyl (13, 14) maintained good potency, similar to
counterpart 15. C-linker modification (16) reduced potency
in both AR F877L mutant and AR WT (F877L IC50 = 204
nM; WT IC50 = 355 nM) compared to 4 or 8. In contrast to
phenol 6, pyridone metabolite 17 lost 5- or 10-fold potency
(F877L IC50 = 1000 nM; WT IC50 = 2239 nM) in relation to
its corresponding parent 5 (or 7).
To correlate AR antagonism with the antiproliferative effect
in androgen-dependent tumor cell lines, 5, 7, 8, and 11−16
were also evaluated in the growth inhibition of AR WT-
dependent VCaP cells. Inhibition of luciferase transcription
appeared to translate into antiproliferative activities in VCaP
cells, tracking with LNCaP WT potency with IC50 values
compound
PO dose (mg/kg) 30
ASO inhibition yes
5
8
11
12
13
14
15
16
10
yes
30
yes
30
yes
30
no
30
yes
30
yes
30
no
the major circulating, stable metabolite of 7 in mouse plasma,
accumulating significantly in repeat-dosing PK studies,
compound 5 was selected to be included for PD studies in
the Hershberger assay. Compounds 5, 8, 11, 12, 14, and 15
showed significant efficacy with 30 mg/kg once daily oral
dosing (Table 5), comparable to that of control group
flutamide with 10 mg/kg once daily oral dosing.
Compounds 13 and 16 displayed no statically significant
inhibitory effect in the Hershberger assay. Introduction of a
metabolic soft spot in 13 and weaker in vitro potency in 16
(LNCaP WT IC50 350 nM; VCaP IC50 2779 nM) may
contribute to the loss of in vivo efficacy. Nevertheless, the lack
Table 4. Human Liver Microsomal T1/2, Permeability, and Mean Single Dose PK Parameters of Compound 4, 5, 7, 8, 11−16 in
CD-1 Male Mice by PO and IV Dosing
compound
4
5
7
8
11
12
13
14
15
16
a
HLM_T1/2 (min)
93.1
23.5
10
9.4
0.98
2
4.12
46.4
12.0
56
>180
5.62
10
4.9
0.66
2
15.0
5.99
6.11
45
173
16.2
10
10.9
1.3
>180
6.8
10
10.4
1.17
2
114
−
10
20.1
2.8
2
6.9
7.8
3.7
87
135
22.1
10
6.1
1.32
2
8.8
4.2
2.93
116
>180
3.38
10
7.8
1.88
2
19.4
3
4.35
92
>180
11.5
10
6.0
1.88
2
25.4
4.1
6.92
118
>180
12.2
10
4.6
0.78
2
26.7
3.7
8.47
50
>180
6.31
10
8.7
0.84
2
18.2
12.1
16.8
97
c
Papp A > B (+inh.) (cm/s × 10−6
PO dose (mg/kg)
AUClast (μg·h/mL)
Cmax (μM)
IV dose (mg/kg)
CL (mL/min/kg)
T1/2 (h)
)
2
8.14
12.5
7.69
56
8.3
10.5
5.56
49
Vdss (L/kg)
b
F (%)
a
b
c
For HLM T1/2: High stability >180 min; 33 min < medium stability <180 min; low stability <33 min. Oral bioavailability. Passive permeability
was measured from the apical (A) to the basolateral side (B) of the MDCK-MDR1 cells in the presence of a P-gp inhibitor with mass balance
>60%.
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Drug Annotation
Table 6. Summary of Maximal Inhibition (Antagonist Activity), EC50 and Maximal Stimulation (Agonist Activity) for 1, 4, 5, 8,
a
11−15 in VP16-AR F877L Mutant and VP16-AR WT HepG2 Reporter Assays
VP16-AR F877L
VP16-AR WT
b
b
compound
% Inhmax
% Emax
EC50 (nM)
% Inhmax
% Emax
EC50 (nM)
1
4
5
8
11
12
13
14
15
86
40
10
10
10
35
40
<5
31
34
61.8 65.1
NA
NA
100
100
100
100
100
>90
100
100
100
<5
<5
<5
<5
<5
<5
<5
<5
<5
NA
NA
NA
NA
NA
NA
NA
NA
NA
100
100
100
100
86
100
100
>90
NA
649 310
838 527
NA
NA
45 20
a
Agonist (EC50) values for each of the AR cDNA used in the reporter assays are summarized. VP16-AR F877L and VP16-AR WT reporter assays
were repeated ≥3 times, and similar results were obtained. Each compound was tested in concentrations ranging from 1 nM to 30 μM with a
b
dilution factor 12. All values are calculated relative to the activity of R1881-induced androgen receptor activity (n ≥ 3) for indicated maximal
inhibition or stimulation of induction of androgen dependent signaling (%).
a
Table 7. Incubation of 8, 6, 5, 17 in Dog Liver Microsomes with GSH in the Absence or Presence of NADPH
compound
8
6
5
17
b
NADPH
UD
UD+O
−
100%
−
−
+
−
100%
−
−
+
−
100%
−
−
+
−
100%
−
−
+
c
99.8%
0.01%
−
91.9%
6.1%
2.1%
NA
98.8%
0.6%
−
100%
0.02%
−
UD+O+GSH-2
phenolic (6 or 17)
d
−
0.2%
NA
−
0.6%
NA
NA
a
Final concentrations of the substrates (UD), dog liver microsomes, glutathione (GSH), and NADPH were 10 μM, 1 mg/mL, 5 mM, and 1 mM,
respectively. The percent compositions of parent drug and its metabolites were based on peak areas from 5 ppm accurate mass measurements and
b
c
with assumptions of equal positive electrospray ionizations. ∓ means absence or in the presence of NADPH. UD is defined as incubated substrate
d
unchanged. Phenolic refers to either metabolite 6 or 17.
of in vivo activity for 13 was unexpected given its in vitro
potency in VCaP (Table 3) and in vivo PK parameters (Table
4), which are comparable to those of 5, 11, and 14.
motifs on AF2 induced after antagonist or agonist binding in
LBD. Nevertheless, taken together, our results suggest that the
CF3 moiety on ring “A” has been optimized.
In a follow-up studies, 11, 12, 14, and 15 showed signs of
partial agonism ranging from 30% to 43% maximal stimulation
in the absence of R1881 in HepG2 cells transiently transfected
with VP16-AR F877L, while 5, 8, and 13 exhibited no signs of
agonism (<10%) in both AR F877L and AR WT cells (Table
6). The results were not in total agreement with those from
natively stable AR LNCaP reporter assays. In particular, the
phenotype of 11 and 12 strikingly resembles 1 (Table 6).
Compound 12 is an incomplete antagonist and partial agonist
in the VP16-AR F877L reporter assay. As noted previously, the
VP16-AR reporter system is a highly sensitive assay for
determining antagonism and agonism of the androgen
receptor. This fusion protein elicits as much as a 20-fold
stronger transcriptional activity than the natural AR but still
reflects the intrinsic propensity of antagonism or agonism.
These data were surprising because, compared to the CF3 or
Me group in 4, 5, 8, and 13, the MeO or Cl moiety in ring “A”
of 11, 12, or 14 was considered to represent a minor change in
terms of binding to the hydrophobic area of the LBD pocket in
our homology model. The results indicate that there is still a
lack of molecular understanding as to how exactly Helix 12
regulates the antagonist−agonist switchfor example, how
Helix 12 affects coactivator binding to the leucine rich LxxLL
The chemical structures of 5 and 8 are similar except for a
critical bioisosteric N/CH exchange in ring “B” (Figure 5).
Both compounds share the identical ring “A” and hydantoin
core, as well as the same peripheral piperidin-4-yloxy
decoration. Their overall in vitro and in vivo profiles also
track well and mirror each other (Tables 2−6). In fact, 5 and 8
were both first identified from metabolite studies of 4 and 7
respectively. The difference lies in the reactivity of key
metabolites. Specifically, phenol 6, the common metabolite
from both 4 and 8, can be bioactivated as indicated by the
formation of a GSH adduct (2.1% UD+O+GSH-2H, Table 7),
whereas pyridone 17, the corresponding metabolite from 5, is
negative in the same GSH trapping experiments, as
summarized in Table 7. Like 6, pyridone 17 was also produced
in only trace amounts (0.6%) presumably due to the low
turnover of 5 under the in vitro assay conditions.
To demonstrate that improved safety did not come at the
expense of in vivo efficacy, 5 was further evaluated in the
prostate LNCaP SRα F877L tumor xenograft model driven by
AR F877L in castrated SHO mice. In this model, 5 showed
statistically significant tumor growth inhibition at 30 mg/kg,
while 1 was not effective at 30 mg/kg QD compared to vehicle
control (Figure 6). The antitumor activity of 5 was determined
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Drug Annotation
AR-V7 splicing variant is a useful biomarker but also represents
a validated therapeutic target for novel drug discovery
approaches.9 Personalized treatment demands diversified
cancer therapeutics in an era of precision medicine. The
development of AR F877L antagonists such as 5 would provide
an additional opportunity for the treatment of prostate
cancer.42
EXPERIMENTAL SECTION
■
Chemistry. All commercial reagents and anhydrous solvents were
purchased and used without further purification, unless otherwise
specified. Mass spectra (MS) were obtained on a SHIMADZU
LCMS-2020 MSD or Agilent 1200\G6110A MSD using electrospray
ionization (ESI) in positive mode unless otherwise indicated.
Calculated (calcd.) mass corresponds to the exact mass. Nuclear
magnetic resonance (NMR) spectra were obtained on Bruker model
AVIII 400 spectrometers. Definitions for multiplicity are as follows: s
= singlet, d = doublet, t = triplet, q = quartet, dd = doublet of
doublets, ddd = doublet of doublet of doublets, td = triplet of
doublets, dt = doublet of triplets, spt = septet, quin = quintet, m =
multiplet, br = broad. 1H NMR chemical shifts are expressed in parts
per million (δ) downfield from tetramethylsilane as a standard.
Normal-phase silica gel chromatography (FCC) was performed on
silica gel (SiO2) using prepacked cartridges. Enzalutamide (1 (MDV-
3100)) was obtained from commercially available source. All
compounds sent for biological tests were confirmed with purity
>95% in quantitative HPLC analysis [method: Gilson GX-281-RP-
HPLC with Phenomenex Gemini C18 (10 μm, 150 × 25 mm), or
Waters XBridge C18 column (5 μm, 150 × 30 mm), mobile phase of
5−99% ACN in water (10 mM NH4HCO3) over 10 min and then
held at 100% ACN for 2 min, at a flow rate of 25 mL/min] or
Figure 6. Oral in vivo efficacy profile of 5 (JNJ-63756253) in castrated
male SHO mice implanted with LNCaP F877L tumors cells. Tumors
were measured twice weekly, and the results presented as the average
tumor volume, expressed in mm3
SEM of each group. *Tumor
growth inhibition (TGI) was statistically significant (p < 0.05) using a
one-way ANOVA with multiple comparisons Dunnett’s multiple
comparisons post-test using Graph Pad Prism software (version 6).
at 30 mg/kg QD comparable to that of 4 as shown in Table 8.
Taken together, our results support the continued develop-
ment and advancement of 5 into human clinical trials.35,36
Table 8. In vivo Efficacy Data for 5 compared to 4 Based on
the LNCaP F877L Tumor Xenograft Model
compound
dose (mg/kg)
4
5
1
30
30
elemental analysis in addition to LCMS and H NMR.
Preparation of 5-(5-(4-Hydroxyphenyl)-8-oxo-6-thioxo-5,7-
diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile
(6). To a solution of l-((4-hydroxyphenyl)amino)cyclobutane-l-
carbonitrile (19a, Supplementary Section S-9) (6.93 g, 29 mmol) in
DMA (60 mL) was added solution of freshly prepared 5-
isothiocyanato-3-(trifluoromethyl)picolinonitrile (21a, Supplementa-
ry Section S-9) (6.67 g, 29 mmol) in DMA (60 mL). The mixture was
heated at 60 °C for overnight and then allowed to cool to room
temperature. To the mixture was added with MeOH (58 mL) and
aqueous HCl solution (1 M, 58 mL). The resulting reaction mixture
was stirred at room temperature for 1 h. The mixture was diluted with
ethyl acetate, washed with water, saturated aqueous NaHCO3
solution, and brine. The organic layer was separated, and the aqueous
layer was extracted with ethyl acetate. The combined organic extract
was dried over MgSO4, filtered, and concentrated. The residue was
purified by chromatography (SiO2, 0−65% EtOAc in heptane) to give
the desired product, which was further purified by crystallization in
acetonitrile to give the title compound as a white solid (7.9 g, 63%).
mass calcd. for C19H13F3N4O2S, 418.1; m/z found, 419.0 [M + H]+;
1H NMR (300 MHz, DMSO-d6) δ 9.92 (s, 1H), 9.22 (d, J = 2.0 Hz,
tumor growth inhib. (%)
plasma exposure (ng/mL)
100.8
748
87
1750
Both 8 and 5 were advanced into single and repeat dose 14-
day toleration studies. Compound 8 displayed unacceptable
hepatotoxicity similar to that of 4 in dogs. In contrast, no
hepatotoxicity or other major safety issues were observed in
dogs for 5 (data not shown). Our studies indicate that a subtle
and simple N/C exchange of phenyl with bioisosteric pyridinyl
(“B” ring) can lead to a dramatically improved safety window
in vivo. Our results also support the hypothesis that the
bioactivation pathway of phenol 6 from 4 and 8 contributes to
initiating a cascade of events leading toward the hepatotoxicity
observed uniquely in dogs.
CONCLUSION
■
In summary, we described our detailed studies of AR pan-
antagonist 4, including impressive antitumor efficacy in an
enzalutamide-resistant AR F877L LNCaP mouse xenograft
model. Despite its attractive in vitro and in vivo pharmaco-
logical properties, idiopathic hepatotoxicity findings in dogs
rendered 4 undevelopable. Metabolite identification studies
revealed a latent bioactivation pathway associated with 4. We
hypothesized the link between in vitro bioactivation and in vivo
toxicity by profiling close analogues of 4. A simple bioisosteric
C/N exchange from phenyl to pyridyl (“B” ring) was able to
abolish the bioactivation. Our SAR studies led to the discovery
of 5, a clinical stage AR antagonist with improved safety
margins and more balanced pharmacological profile.
1H), 8.76 (d, J = 2.0 Hz, 1H), 7.18 (d, J = 8.7 Hz, 2H), 6.95 (d, J =
8.7 Hz, 2H), 2.54−2.71 (m, 2H), 2.35−2.49 (m, 2H), 1.87−2.07 (m,
1H), 1.45−1.66 (m, 1H) ppm.
Preparation of 5-(5-(6-Hydroxypyridin-3-yl)-8-oxo-6-thio-
xo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)-
picolinonitrile (17). To a solution of 1-((6-hydroxypyridin-3-
yl)amino)cyclobutanecarbonitrile (19b, Supplementary Section S-
9S-9) (10.4 g, 54.5 mmol) in DMA (60 mL) was added a solution of
5-isothiocyanato-3-(trifluoromethyl)picolinonitrile (21a, Supplemen-
tary Section S-9) (13 g, 45.4 mmol) in DMA (60 mL). The mixture
was heated at 60 °C for 2 h and then allowed to cool to room
temperature. The mixture was treated with MeOH (100 mL) and
HCl (2 M, 100 mL). The resulting suspension was stirred at 60 °C for
1 h. The mixture was filtered, and the filter cake was washed with
water, MeOH, and then dried in vacuo to give the title compound as a
gray solid (16.7 g, 86%). MS: mass calcd. for C18H12F3N5O2S, 419.1;
With improved understanding of AR signaling in prostate
cancer, there has been renewed interest in AR as a
transcriptional factor.37−41 For example, the discovery of the
1
m/z found, 420.0 [M + H]+; H NMR (400 MHz, DMSO-d6) δ
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Drug Annotation
12.01 (br s., 1H), 9.15 (s, 1H), 8.67 (s, 1H), 7.58 (br s, 1H), 7.40
(dd, J = 9.66, 2.32 Hz, 1H), 6.48 (d, J = 9.54 Hz, 1H), 2.53−2.60 (m,
2H), 2.36−2.44 (m, 2H), 1.88−2.01 (m, 1H), 1.60−1.71 (m, 1H)
ppm.
57.3 mmol) in DMA (50 mL). The mixture was heated at 60 °C for 2
h and then allowed to cool to room temperature. The mixture was
treated with MeOH (100 mL) and HCl (2 M, 100 mL). The resulting
suspension was stirred at 60 °C for 2 h. The mixture was cooled to
room temperature, and water (100 mL) was added. The precipitate
was collected by filtration, washed with water and methanol, and dried
in vacuo to give the title compound as a white solid (15 g, 66%). MS:
mass calcd. for C17H12ClN5O2S, 385.0; m/z found, 386.0 [M + H]+;
1H NMR (400 MHz, DMSO-d6) δ 11.96 (br s, 1H), 8.83 (d, J = 1.96
Hz, 1H), 8.47 (d, J = 1.96 Hz, 1H), 7.58 (d, J = 2.45 Hz, 1H), 7.40
(dd, J = 9.66, 2.81 Hz, 1H), 6.48 (d, J = 9.78 Hz, 1H), 2.50−2.62 (m,
2H), 2.36−2.45 (m, 2H), 1.88−2.03 (m, 1H), 1.57−1.73 (m, 1H)
ppm.
Preparation of 5-(5-(4-hydroxyphenyl)-8-oxo-6-thioxo-5,7-
diazaspiro[3.4]octan-7-yl)-3-methoxypicolinonitrile (22a). To
a solution of l-((4-hydroxyphenyl)amino)cyclobutane-l-carbonitrile
(19a, Supplementary Section S-9) (8.4 g, 44.7 mmol) in DMA (100
mL) was added a solution of 5-isothiocyanato-3-methoxypicolinoni-
trile (21b, Supplementary Section S-9) (10.3 g, 54 mmol) in DMA
(100 mL). The mixture was heated at 60 °C for 1 h and then allowed
to cool to room temperature. The mixture was treated with MeOH
(45 mL) and HCl (1 M, 45 mL). The resulting suspension stirred at
room temperature for overnight. Water (50 mL) was added, and the
mixture was filtered. The precipitate was collected and dissolved into
ethyl acetate (200 mL). The solution was washed with aqueous
Na2CO3 solution, dried over MgSO4, filtered, and concentrated. The
product was dried in vacuo to give the title compound as a white solid
(15 g, 88%). MS: mass calcd. for C19H16N4O3S, 380.1; m/z found,
381.1 [M + H]+; 1H NMR (400 MHz, CDCl3) δ 8.43 (d, J = 1.98 Hz,
1H), 7.56 (d, J = 1.76 Hz, 1H), 7.14−7.22 (m, 2H), 6.97−7.06 (m,
2H), 5.13 (s, 1H), 4.00 (s, 3H), 2.61−2.71 (m, 2H), 2.50−2.60 (m,
2H), 2.17−2.29 (m, 1H), 1.63−1.73 (m, 1H) ppm.
Preparation of 3-Chloro-5-(5-(4-hydroxyphenyl)-8-oxo-6-
thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile (22e). To
a solution of l-((4-hydroxyphenyl)amino)cyclobutane-l-carbonitrile
(19a, Supplementary Section S-9) (11 g, 58.6 mmol) in DMA (50
mL) was added a solution of 3-chloro-5-isothiocyanatopicolinonitrile
(21d, Supplementary Section S-9) (11.9 g, 48.8 mmol) in DMA (50
mL). The mixture was heated at 60 °C for 2 h and then allowed to
cool to room temperature. The mixture was treated with MeOH (100
mL) and HCl (2 M, 100 mL). The resulting suspension was stirred at
60 °C for 2 h. The mixture was cooled to room temperature, and
water (100 mL) was added. The precipitate was collected by filtration,
washed with water and methanol, and dried in vacuo to give the title
compound as a white solid (17 g, 90%). MS: mass calcd. for
Preparation of 5-(5-(6-Hydroxypyridin-3-yl)-8-oxo-6-thio-
xo-5,7-diazaspiro[3.4]octan-7-yl)-3-methoxypicolinonitrile
(22b). To a solution of 1-((6-hydroxypyridin-3-yl)amino)-
cyclobutanecarbonitrile (19b, Supplementary Section S-9) (7.3 g,
38.4 mmol) in DMA (50 mL) was added a solution of 5-
isothiocyanato-3-methoxypicolinonitrile (21b, Supplementary Section
S-9) (7.6 g, 32 mmol) in DMA (50 mL). The mixture was heated at
60 °C for 2 h and then allowed to cool to room temperature. The
mixture was treated with MeOH (50 mL) and HCl (2 M, 50 mL).
The resulting suspension was stirred at 60 °C for 2 h. Water (50 mL)
was added, and the mixture was filtered. The precipitate was collected,
washed with water and methanol, and dried in vacuo to give the title
compound as a white solid (9 g, 74%). MS: mass calcd. for
1
C18H13ClN4O2S, 384.0; m/z found, 385.0 [M + H]+; H NMR (400
MHz, DMSO-d6) δ 9.93 (s, 1H), 8.90 (d, J = 2.01 Hz, 1H), 8.54 (d, J
= 2.01 Hz, 1H), 7.18 (d, J = 8.78 Hz, 2H), 6.94 (d, J = 8.78 Hz, 2H),
2.54−2.65 (m, 2H), 2.38−2.46 (m, 2H), 1.89−2.01 (m, 1H), 1.48−
1.60 (m, 1H) ppm.
Preparation of 5-(5-(4-((1-Methylpiperidin-4-yl)oxy)-
phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (4). To a mixture of 5-(5-(4-
hydroxyphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-me-
thoxypicolinonitrile (6) (6.3 g, 15 mmol), 1-methylpiperidin-4-ol (2.1
g, 18 mmol), and triphenylphosphine (7.9 g, 30 mmol) in anhydrous
THF (150 mL) was added diethyl azodicarboxylate (DEAD, 5.2 g, 30
mmol) dropwise over a period of 15−20 min under nitrogen. Upon
completion of the addition, the reaction mixture was stirred at room
temperature for 5 h. The mixture was concentrated. The crude residue
was purified by chromatography (SiO2, 0−10% MeOH in EtOAc) to
give the desired product. The solid was dissolved into 1,4-dioxane (40
mL), and a 1,4-dioxane (4 M) solution of HCl (2.8 mL, 11.2 mmol)
was added. The mixture was stirred at room temperature for 2 h and
then concentrated. The residue was triturated in diethyl ether (200
mL) for overnight. The precipitate was collected by filtration, washed
with diethyl ether, and dried in vacuo to give the title compound as a
HCl salt: a white solid (5.83 g, 70%). LCMS (API): mass calcd. for
1
C18H15N5O3S, 381.1; m/z found, 382.1 [M + H]+; H NMR (300
MHz, DMSO-d6) δ 12.02 (br s, 1H), 8.42 (d, J = 1.65 Hz, 1H), 8.00
(d, J = 1.65 Hz, 1H), 7.63 (d, J = 2.75 Hz, 1H), 7.45 (dd, J = 2.75,
9.90 Hz, 1H), 6.51 (d, J = 9.90 Hz, 1H), 4.00 (s, 3H), 2.27−2.73 (m,
4H), 1.85−2.13 (m, 1H), 1.59−1.80 (m, 1H) ppm.
Preparation of 5-(5-(6-Hydroxypyridin-3-yl)-8-oxo-6-thio-
xo-5,7-diazaspiro[3.4]octan-7-yl)-3-methylpicolinonitrile
(22c). To a solution of 1-((6-hydroxypyridin-3-yl)amino)-
cyclobutanecarbonitrile (19b, Supplementary Section S-9) (8.2 g,
43.3 mmol) in DMA (90 mL) was added a solution of 5-
isothiocyanato-3-methylpicolinonitrile (21c, Supplementary Section
S-9) (11.4 g, 65 mmol) in DMA (90 mL). The mixture was heated at
60 °C for 4 h and then allowed to cool to room temperature. The
mixture was treated with MeOH (90 mL) and HCl (1 M, 87 mL).
The resulting suspension was stirred at room temperature for 1 h. The
mixture was diluted with ethyl acetate, washed with water, saturated
aqueous NaHCO3 solution, and brine. The organic layer was
separated, and the aqueous layer was extracted with ethyl acetate.
The combined organic extract was dried over MgSO4, filtered, and
concentrated. The residue was purified by chromatography (SiO2, 0−
65% EtOAc in heptane) to give the desired product, which was
further purified by crystallization in acetonitrile to give the title
compound as a brown solid (12.8 g, 69%). MS: mass calcd. for
1
C25H24F3N5O2S·HCl, 515.2; m/z found, 516.1 [M + H]+; H NMR
(300 MHz, DMSO-d6) δ 9.23 (d, J = 1.10 Hz, 1H), 8.77 (d, J = 1.65
Hz, 1H), 7.31 (d, J = 8.80 Hz, 2H), 7.16 (d, J = 8.80 Hz, 2H), 4.47
(td, J = 4.26, 7.97 Hz, 1H), 2.54−2.77 (m, 5H), 2.38−2.50 (m, 2H),
2.28 (br s, 1H), 2.20−2.32 (m, 1H), 2.24 (s, 3H), 1.92−2.06 (m,
3H), 1.63−1.80 (m, 2H), 1.49−1.64 (m, 1H) ppm. Anal. Calcd for
C25H24F3N5O2S: C, 58.24; H, 4.69; N, 13.58. Found: C, 57.99; H,
4.47; N, 13.33.
Preparation of 3-Methoxy-5-(5-(4-((1-methylpiperidin-4-yl)-
oxy)phenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-
picolinonitrile (11). To a mixture of 5-(5-(4-hydroxyphenyl)-8-oxo-
6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-methoxypicolinonitrile
(22a) (9 g, 23.7 mmol), 1-methylpiperidin-4-ol (5.4 g, 47.3 mmol),
and triphenylphosphine (12.4 g, 47.3 mmol) in anhydrous THF (150
mL) was added diethyl azodicarboxylate (DEAD, 8.2 g, 47.3 mmol)
dropwise over a period of 15−20 min under nitrogen. Upon
completion of the addition, the reaction mixture was stirred at 30
°C for overnight. The mixture was concentrated. The crude residue
was purified by chromatography (SiO2, 0−20% MeOH in EtOAc) to
give the desired product as a yellow solid. The solid was dissolved into
1,4-dioxane (60 mL), and a 1,4-dioxane (4 M) solution of HCl (4.2
1
C18H15N5O2S, 365.1; m/z found, 366.1 [M + H]+; H NMR (400
MHz, CDCl3) δ 13.20 (br s, 1H), 8.66 (d, J = 1.98 Hz, 1H), 7.82 (d, J
= 1.76 Hz, 1H), 7.48 (d, J = 2.65 Hz, 1H), 7.42 (dd, J = 9.70, 2.65 Hz,
1H), 6.75 (d, J = 9.70 Hz, 1H), 2.61−2.75 (m, 5H), 2.43−2.56 (m,
2H), 2.25−2.37 (m, 1H), 1.77−1.89 (m, 1H) ppm.
Preparation of 3-Chloro-5-(5-(6-hydroxypyridin-3-yl)-8-
oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile
(22d). To a solution of 1-((6-hydroxypyridin-3-yl)amino)-
cyclobutanecarbonitrile (19b, Supplementary Section S-9) (10.9 g,
57.3 mmol) in DMA (50 mL) was added a solution of 3-chloro-5-
isothiocyanatopicolinonitrile (21d, Supplementary Section S-9) (14 g,
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Drug Annotation
mL, 16.8 mmol) was added. The mixture was stirred at room
temperature for 2 h and then concentrated. The residue was triturated
in diethyl ether (250 mL) for overnight. The precipitate was collected
by filtration, washed with diethyl ether, and dried in vacuo to give the
title compound as a HCl salt: a white solid (6.86 g, 56%). LCMS
(API): mass calcd. for C25H27N5O3S·HCl, 477.2; m/z found, 478.0
[M + H]+; 1H NMR (300 MHz, DMSO-d6) δ 10.17 (br s, 1H), 8.45
(d, J = 1.7 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H), 7.35 (d, J = 8.4 Hz, 2H),
7.21 (d, J = 8.4 Hz, 2H), 4.57−4.90 (m, 1H), 3.99 (s, 3H), 3.43−3.61
(m, 1H), 3.29−3.36 (m, 1H), 3.00−3.27 (m, 2H), 2.79 (br s, 3H),
2.56−2.68 (m, 2H), 2.36−2.46 (m, 2H), 2.23−2.36 (m, 1H), 2.04−
2.19 (m, 2H), 1.77−2.04 (m, 2H), 1.44−1.63 (m, 1H) ppm. Anal.
Calcd for C25H24F3N5O2S·HCl·H2O: C, 56.44; H, 5.68; N, 13.16.
Found: C, 56.40; H, 5.70; N, 12.72.
Preparation of 3-Methoxy-5-(5-(6-((1-methylpiperidin-4-yl)-
oxy)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-
yl)picolinonitrile (12). To a mixture of 5-(5-(6-hydroxypyridin-3-
yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-methoxypicolino-
nitrile (22b) (6.87 g, 18 mmol), 1-methylpiperidin-4-ol (4.1 g, 36
mmol), and triphenylphosphine (9.4 g, 36 mmol) in anhydrous THF
(150 mL) was added diethyl azodicarboxylate (DEAD, 6.27 g, 36
mmol) dropwise over a period of 15−20 min under nitrogen. Upon
completion of the addition, the reaction mixture was stirred at 30 °C
for overnight. The mixture was concentrated. The crude residue was
purified by chromatography (SiO2, 0−20% MeOH in EtOAc) to give
the desired product as a yellow solid. The solid was dissolved into 1,4-
dioxane (40 mL), and a 1,4-dioxane (4 M) solution of HCl (3 mL, 12
mmol) was added. The mixture was stirred at room temperature for 2
h and then concentrated. The residue was triturated in diethyl ether
(200 mL) for overnight. The precipitate was collected by filtration,
washed with diethyl ether, and dried in vacuo to give the title
compound as a HCl salt: a beige solid (5.5 g, 62%). LCMS (API):
mass calcd. for C24H26N6O3S·HCl, 478.2; m/z found, 479.1 [M +
H]+; 1H NMR (300 MHz, DMSO-d6) δ 10.62 (br s, 1H), 8.45 (d, J =
1.8 Hz, 1H), 8.24 (dd, J = 4.5, 2.5 Hz, 1H), 8.04 (d, J = 1.8 Hz, 1H),
7.82 (ddd, J = 9.0, 6.6, 2.5 Hz, 1H), 7.07 (dd, J = 8.8, 4.1 Hz, 1H),
5.16−5.42 (m, 1H), 3.99 (s, 3H), 3.42−3.54 (m, 1H), 3.30−3.42 (m,
1H), 3.05−3.28 (m, 2H), 2.78 (dd, J = 11.6, 4.6 Hz, 3H), 2.57−2.71
(m, 2H), 2.37−2.46 (m, 2H), 2.22−2.37 (m, 1H), 2.11−2.24 (m,
2H), 1.86−2.10 (m, 2H), 1.47−1.70 (m, 1H) ppm. Anal. Calcd for
C24H26N6O3S·HCl·H2O: C, 54.08; H, 5.48; N, 15.77. Found: C,
53.71; H, 5.26; N, 15.32.
Preparation of 5-(8-Oxo-5-(4-(piperidin-4-yloxy)phenyl)-6-
thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)-
picolinonitrile (8). Step A. To a mixture of 5-(5-(4-hydroxyphenyl)-
8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)-
picolinonitrile (6) (25 g, 59.7 mmol), tert-butyl 4-hydroxypiperidine-
1-carboxylate (18.6 g, 89.7 mmol), and triphenylphosphine (31.4 g,
120 mmol) in anhydrous THF (400 mL) was added a solution of
diisopropyl azodicarboxylate (DIAD, 23.5 mL, 120 mmol) in
anhydrous THF (250 mL) dropwise over a period of 30 min under
nitrogen. Upon completion of the addition, the reaction mixture was
stirred at room temperature for 0.5 h. The mixture was concentrated.
The crude residue was purified by chromatography (SiO2, 0−60%
EtOAc in heptane) to give the crude product, which was further
purified by reverse phase chromatography [C18 colomn, Start (90%
H2O-10% ACN-MeOH)-End (54% H2O-46% ACN-MeOH-0.1%
HCOOH)]. The product was neutralized with Na2CO3 solution and
then extracted with DCM. The organic extract was separated, dried
over MgSO4, filtered, and concentrated. The residue was triturated in
diethyl ether to give tert-butyl 4-(4-(7-(6-cyano-5-(trifluoromethyl)-
pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)-
piperidine-1-carboxylate as a white foam (18 g, 49%). LCMS (API):
mass calcd. for C29H30F3N5O4S, 601.2; m/z found, 602.1 [M + H]+;
1H NMR (400 MHz, CDCl3) δ 9.11 (d, J = 2.26 Hz, 1H), 8.37 (d, J =
oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)phenoxy)piperidine-1-car-
boxylate (8 g, 13 mmol) in DCM (130 mL) was added TFA (14 mL).
The reaction mixture was stirred at room temperature for 2 h. The
mixture concentrated, and toluene was added to the residue. The
solvent was evaporated, and the remaining mixture was poured into
aqueous saturated NaHCO3 and extracted with EtOAc. The organic
layer was washed with water, brine, dried over MgSO4, filtered, and
concentrated. The crude material was purified by chromatography
(SiO2, 0−10% MeOH in DCM) to provide the desired product (6.86
g). The product was dissolved into 1,4-dioxane (50 mL), and a 1,4-
dioxane solution (4 M) of HCl (3.3 mL, 13.2 mmol) was added. The
mixture was stirred at room temperature for 1 h and then
concentrated. The residue was triturated in diethyl ether, and the
precipitate was collected, washed with diethyl ether, and dried in
vacuo to give the title compound as a HCl salt: white solid (6.08 g,
86%). LCMS (API): mass calcd. for C24H22F3N5O2S·HCl, 501.1; m/z
1
found, 502.1 [M + H]+; H NMR (400 MHz, CDCl3) δ 9.65 (br s,
2H), 9.09 (s, 1H), 8.35 (s, 1H), 7.21−7.26 (m, 2H), 7.08 (d, J = 7.58
Hz, 2H), 4.76 (br s, 1H), 3.40 (br s, 4H), 2.67 (d, J = 8.80 Hz, 2H),
2.49−2.62 (m, 2H), 2.38 (br s, 2H), 2.24 (d, J = 9.05 Hz, 3H), 1.69
(d, J = 10.03 Hz, 1H) ppm.
Preparation of 5-(8-Oxo-5-(6-(piperidin-4-yloxy)pyridin-3-
yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)-
picolinonitrile (5). Step A. To a mixture of 5-(5-(6-hydroxypyridin-
3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (17) (16.6 g, 39.6 mmol), tert-butyl 4-
hydroxypiperidine-1-carboxylate (8.94 g, 43.5 mmol), and triphenyl-
phosphine (22.8 g, 87.1 mmol) in anhydrous THF (150 mL) was
added a solution of diisopropyl azodicarboxylate (DIAD, 15.6 mL,
79.1 mmol) in anhydrous THF (50 mL) dropwise over a period of 10
min under nitrogen. Upon completion of the addition, the reaction
mixture was stirred at 50 °C for 3 h. The mixture was cooled to room
temperature and concentrated. The crude residue was purified by
chromatography (SiO2, 0−30% EtOAc in heptane) to give crude tert-
butyl 4-((5-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thi-
oxo-5,7-diazaspiro[3.4]octan-5-yl)pyridin-2-yl)oxy)piperidine-1-car-
boxylate as an amorphous foam (43.4 g), which was used directly to
next step without further purification. MS: mass calcd. for
1
C28H29F3N6O4S, 602.2; m/z found, 547.1 [M + H-tBu]+; H NMR
(300 MHz, CDCl3) δ 9.09 (d, J = 2.2 Hz, 1H), 8.36 (d, J = 2.2 Hz,
1H), 8.09 (d, J = 2.6 Hz, 1H), 7.52 (dd, J = 8.8, 2.6 Hz, 1H), 6.91 (d,
J = 8.8 Hz, 1H), 5.21−5.35 (m, 1H), 3.70−3.90 (m, 2H), 3.22−3.40
(m, 2H), 2.63−2.80 (m, 2H), 2.44−2.62 (m, 2H), 2.15−2.35 (m,
1H), 1.94−2.10 (m, 2H), 1.66−1.86 (m, 3H), 1.47 (s, 9H) ppm.
Step B. To a solution of crude tert-butyl 4-((5-(7-(6-cyano-5-
(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]-
octan-5-yl)pyridin-2-yl)oxy)piperidine-1-carboxylate (43.4 g) in
DCM (300 mL) was added TFA (60 mL). The reaction mixture
was stirred at room temperature for overnight. The mixture
concentrated, and the residue was taken in toluene (150 mL) and
again concentrated (three times). The crude residue was then purified
by column chromatography on silica gel (gradient of MeOH in DCM
from 0 to 10%) to afford a yellowish amorphous solid (19.8 g). Final
purification was performed by preparative LC (gradient of a mixture
ACN/MeOH (1/1, v/v) in 0.1% aqueous formic acid from 10 to
54%). The pure fractions were collected, and the “pH” was brought to
8−9 by addition of solid Na2CO3. The product was extracted with
ethyl acetate (3 × 400 mL). The combined organic layers were
washed with brine (300 mL), dried over MgSO4, filtered, and
concentrated to give 5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-
thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)-
picolinonitrile (5) as a white foam (9.57 g, 47% for two steps). MS:
mass calcd. for C23H21F3N6O2S, 502.1; m/z found, 503.1 [M + H]+;
1H NMR (300 MHz, CDCl3) δ 8.91 (d, J = 2.2 Hz, 1H), 8.24 (d, J =
2.2 Hz, 1H), 7.90 (d, J = 2.6 Hz, 1H), 7.39 (dd, J = 8.7, 2.6 Hz, 1H),
6.74 (d, J = 8.7 Hz, 1H), 5.15−5.27 (m, 1H), 3.10−3.30 (m, 2H),
2.94−3.09 (m, 2H), 2.42−2.60 (m, 2H), 2.20−2.41 (m, 2H), 1.87−
2.15 (m, 5H), 1.45−1.57 (m, 1H) ppm.
2.26 Hz, 1H), 7.22 (d, J = 8.78 Hz, 2H), 7.08 (d, J = 8.78 Hz, 2H),
4.56 (d, J = 3.01 Hz, 1H), 3.67−3.79 (m, 2H), 3.34−3.44 (m, 2H),
2.51−2.73 (m, 4H), 2.19−2.30 (m, 1H), 1.97 (br s, 2H), 1.83 (d, J =
3.01 Hz, 2H), 1.70 (d, J = 10.79 Hz, 1H), 1.49 (s, 9H) ppm. Step B.
To tert-butyl 4-(4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-
Step C. 5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-
5,7-diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile (5)
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Drug Annotation
(9.57 g, 19.0 mmol) was dissolved into 1,4-dioxane (54 mL), and a
1,4-dioxane solution (4 M) of HCl (5.24 mL, 20.9 mmol) was added.
The mixture was stirred at room temperature for 1 h and then
concentrated. The residue was triturated in diethyl ether (50 mL) for
overnight. The precipitate was collected by filtration through a
sintered funnel, washed with diethyl ether (2 × 15 mL), and dried in
vacuo to give the title compound as a HCl salt: a white solid (9.85 g,
93%). LCMS (API): mass calcd. for C23H21F3N6O2S·HCl, 502.1; m/z
found, 503.1 [M + H]+; 1H NMR (300 MHz, DMSO-d6) δ 9.21 (d, J
= 2.0 Hz, 1H), 8.92 (br s, 2H), 8.75 (d, J = 2.0 Hz, 1H), 8.22 (d, J =
2.6 Hz, 1H), 7.80 (dd, J = 8.8, 2.6 Hz, 1H), 7.09 (d, J = 8.8 Hz, 1H),
5.26−5.40 (m, Hz, 1H), 3.21−3.35 (m, 2H), 3.04−3.20 (m, 2H),
2.58−2.73 (m, 2H), 2.34−2.48 (m, 2H), 2.12−2.29 (m, 2H), 1.87−
2.04 (m, 3H), 1.48−1.69 (m, 1H) ppm; Anal. Calcd for
C23H21F3N6O2S·HCl: C, 51.26; H, 4.11; N, 15.59; Found: C,
51.02; H, 4.01; N, 15.57.
was stirred at room temperature for 4 h and then concentrated. The
yellow solid was triturated in diethyl ether (100 mL) for overnight.
The precipitate was collected by filtration through a sintered funnel,
washed with diethyl ether, and dried in vacuo to give the title
compound as a HCl salt: a white solid (8.62 g, 91%). LCMS (API):
mass calcd. for C23H24N6O2S·HCl, 448.2; m/z found, 449.1 [M +
H]+; 1H NMR (300 MHz, DMSO-d6) δ 8.88−9.16 (m, 2H), 8.72 (d,
J = 1.65 Hz, 1H), 8.25 (d, J = 2.20 Hz, 1H), 8.14 (d, J = 1.65 Hz, 1H),
7.83 (dd, J = 2.20, 8.80 Hz, 1H), 7.08 (d, J = 8.80 Hz, 1H), 5.21−5.43
(m, 1H), 3.27 (br s, 2H), 3.14 (br d, J = 3.85 Hz, 2H), 2.60−2.70 (m,
2H), 2.59 (s, 3H), 2.35−2.49 (m, 2H), 2.12−2.29 (m, 2H), 1.86−
2.08 (m, 3H), 1.47−1.71 (m, 1H) ppm.
Preparation of 3-Chloro-5-(8-oxo-5-(6-(piperidin-4-yloxy)-
pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-
picolinonitrile (14). Step A. To a mixture of 3-chloro-5-(5-(6-
hydroxypyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-
picolinonitrile (22d) (5 g, 12.9 mmol), tert-butyl 4-hydroxypiper-
idine-1-carboxylate (3.9 g, 19 mmol), and triphenylphosphine (6.1 g,
23 mmol) in anhydrous THF (25 mL) was added a solution of
diisopropyl azodicarboxylate (DIAD, 4.6 mL, 23 mmol) in anhydrous
THF (25 mL) dropwise over a period of 10 min under nitrogen.
Upon completion of the addition, the reaction mixture was stirred at
50 °C for 2 h. The mixture was cooled to room temperature and
concentrated. The residue was triturated in a mixture of ethyl acetate
and heptane. The precipitate of triphenylphosphine oxide was filtered,
and the filtrate was concentrated. The crude residue was purified by
chromatography (SiO2, 0−30% EtOAc in heptane) to give tert-butyl
4-((5-(7-(5-chloro-6-cyanopyridin-3-yl)-8-oxo-6-thioxo-5,7-
diazaspiro[3.4]octan-5-yl)pyridin-2-yl)oxy)piperidine-1-carboxylate
as a yellow foam (7.6 g, 100%). MS: mass calcd. for C27H29ClN6O4S,
568.2; m/z found, 513.0 [M + H-tBu]+; 1H NMR (300 MHz, CDCl3)
δ 8.76−8.82 (m, 1H), 8.09 (dd, J = 9.66, 2.32 Hz, 2H), 7.50 (dd, J =
8.68, 2.81 Hz, 1H), 6.90 (d, J = 8.80 Hz, 1H), 5.28 (tt, J = 7.89, 3.97
Hz, 1H), 3.74−3.85 (m, 2H), 3.30 (ddd, J = 13.27, 8.99, 3.67 Hz,
2H), 2.62−2.75 (m, 2H), 2.43−2.57 (m, 2H), 2.19−2.33 (m, 1H),
2.02 (d, J = 9.05 Hz, 2H), 1.70−1.83 (m, 3H), 1.47 (s, 9H) ppm.
Step B. To a solution of tert-butyl 4-((5-(7-(5-chloro-6-
cyanopyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-
pyridin-2-yl)oxy)piperidine-1-carboxylate (7.4 g, 13 mmol) in DCM
(65 mL) was added TFA (14 mL). The reaction mixture was stirred
at room temperature for 2 h. The mixture concentrated and toluene
was added to the residue. The solvent was evaporated, and the
remaining mixture was poured into aqueous saturated NaHCO3 and
extracted with EtOAc. The organic layer was washed with water,
brine, dried over MgSO4, filtered, and concentrated. The crude
material was purified by chromatography (SiO2, 0−10% MeOH in
DCM) to provide the desired product. The product was dissolved
into 1,4-dioxane (50 mL), and a 1,4-dioxane solution (4 M) of HCl
(10 mL, 40 mmol) was added. The mixture was stirred at room
temperature for 1 h and then concentrated. The residue was triturated
in diethyl ether, and the precipitate was collected, washed with diethyl
ether, and dried in vacuo to give the title compound as a HCl salt: a
white solid (4.95 g, 78%). LCMS (API): mass calcd. for
Preparation of 3-Methyl-5-(8-oxo-5-(6-(piperidin-4-yloxy)-
pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-
picolinonitrile (13). Step A. To a mixture of 5-(5-(6-hydroxypyr-
idin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (17) (12.8 g, 35 mmol), tert-butyl 4-
hydroxypiperidine-1-carboxylate (8.46 g, 32 mmol), and triphenyl-
phosphine (16.5 g, 63 mmol) in anhydrous THF (125 mL) was
added a solution of diisopropyl azodicarboxylate (DIAD, 12.4 mL, 63
mmol) in anhydrous THF (250 mL) dropwise over a period of 30
min under nitrogen. Upon completion of the addition, the reaction
mixture was stirred at 50 °C for 2 h. The mixture was cooled to room
temperature and concentrated. The residue was triturated in a mixture
of ethyl acetate and heptane. The precipitate of triphenylphosphine
oxide was filtered, and the filtrate was concentrated. The crude
residue was purified by chromatography (SiO2, 0−45% EtOAc in
heptane) to give tert-butyl 4-((5-(7-(6-cyano-5-methylpyridin-3-yl)-8-
oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)pyridin-2-yl)oxy)-
piperidine-1-carboxylate as a yellow foam (14.9 g, 77%). MS: mass
1
calcd. for C28H32N6O4S, 548.2; m/z found, 493.1 [M + H-tBu]+; H
NMR (300 MHz, DMSO-d6) δ 8.66−8.77 (m, 1H), 8.24 (d, J = 2.20
Hz, 1H), 8.15 (s, 1H), 7.79 (dd, J = 2.75, 8.80 Hz, 1H), 7.04 (d, J =
8.80 Hz, 1H), 5.17−5.34 (m, 1H), 3.67−3.85 (m, 2H), 3.08−3.27
(m, 2H), 2.62−2.72 (m, 1H), 2.35−2.54 (m, 5H), 1.90−2.12 (m,
3H), 1.51−1.72 (m, 3H), 1.43 (s, 9H), 1.19−1.32 (m, 1H) ppm.
Step B. To a solution of tert-butyl 4-((5-(7-(6-cyano-5-methylpyr-
idin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)pyridin-2-yl)-
oxy)piperidine-1-carboxylate (14.9 g, 27 mmol) in DCM (110 mL)
was added TFA (54 mL). The reaction mixture was stirred at room
temperature for 1 h. The mixture concentrated, and the residue was
taken in toluene (150 mL) and again concentrated (3 × 50 mL). The
crude residue was diluted with DCM (500 mL) and washed with
aqueous Na2CO3 solution (1 M, 250 mL). The organic layer was
separated, and aqueous layer was extracted with DCM (500 mL). The
combined organic extract was dried over MgSO4, filtered, and
concentrated. The residue was purified by chromatography (SiO2,
MeOH in DCM from 0 to 10%) to afford a yellowish amorphous
solid. Final purification was performed by preparative LC ([Start
(90% H2O-10% ACN)-End (54% H2O-46% ACN]-[25 mM
NH4HCO3]). The pure fractions were collected, and brine was
added (200 mL). The mixture was extracted with DCM (200 mL).
The organic layer was separated, dried over MgSO4, filtered, and
concentrated to give 3-methyl-5-(8-oxo-5-(6-(piperidin-4-yloxy)-
pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile
(13) as a yellow solid (8.56 g, 69% for two steps). MS: mass calcd. for
1
C22H21ClN6O2S·HCl, 468.1; m/z found, 468.9 [M + H]+; H NMR
(300 MHz, DMSO-d6) δ 9.23 (s, 2H), 8.91 (d, J = 2.0 Hz, 1H), 8.55
(d, J = 2.1 Hz, 1H), 8.24 (d, J = 2.6 Hz, 1H), 7.81 (dd, J = 8.8, 2.6 Hz,
1H), 7.09 (d, J = 8.8 Hz, 1H), 5.28−5.37 (m, 1H), 3.07−3.31 (m,
4H), 2.59−2.71 (m, 2H), 2.39−2.47 (m, 2H), 2.17−2.31 (m, 2H),
1.93−2.05 (m, 3H), 1.59 (d, J = 10.7 Hz, 1H) ppm.
Preparation of 3-Chloro-5-(8-oxo-5-(4-(piperidin-4-yloxy)-
phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile
(15). Step A. To a mixture of 3-chloro-5-(5-(4-hydroxyphenyl)-8-oxo-
6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile (22e) (4.15 g,
10.1 mmol), tert-butyl 4-hydroxypiperidine-1-carboxylate (3.16 g, 15.2
mmol), and triphenylphosphine (5.3 g, 20.2 mmol) in anhydrous
toluene (35 mL) and DMF (5 mL) was added a solution of
diisopropyl azodicarboxylate (DIAD, 4.4 mL, 22 mmol) in anhydrous
toluene (15 mL) dropwise over a period of 10 min under nitrogen.
Upon completion of the addition, the reaction mixture was stirred at
50 °C for 1 h. The mixture was cooled to room temperature and
1
C23H24N6O2S, 448.2; m/z found, 449.1 [M + H]+; H NMR (300
MHz, DMSO-d6) δ 8.99 (br s, 2H), 8.71 (d, J = 2.1 Hz, 1H), 8.23 (d,
J = 2.6 Hz, 1H), 8.13 (d, J = 2.2 Hz, 1H), 7.82 (dd, J = 8.8, 2.6 Hz,
1H), 7.07 (d, J = 8.8 Hz, 1H), 5.20−5.40 (m, 1H), 3.20−3.36 (m,
2H), 3.02−3.20 (m, 2H), 2.55−2.70 (m, 2H), 2.29−2.47 (m, 2H),
2.11−2.29 (m, 2H), 1.84−2.06 (m, 3H), 1.49−1.69 (m, 1H) ppm.
Step C. 3-methyl-5-(8-oxo-5-(6-(piperidin-4-yloxy)pyridin-3-yl)-6-
thioxo-5,7-diazaspiro[3.4]octan-7-yl)picolinonitrile (13) (8.56 g, 19.0
mmol) was dissolved into 1,4-dioxane (75 mL), and a 1,4-dioxane
solution (4 M) of HCl (5.25 mL, 21 mmol) was added. The mixture
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Drug Annotation
concentrated. The residue was triturated in a mixture of ethyl acetate
and heptane. The precipitate of triphenylphosphine oxide was filtered,
and the filtrate was concentrated. The crude residue was purified by
chromatography (SiO2, 0−65% EtOAc in heptane) to give crude tert-
butyl 4-(4-(7-(5-chloro-6-cyanopyridin-3-yl)-8-oxo-6-thioxo-5,7-
diazaspiro[3.4]octan-5-yl)phenoxy)piperidine-1-carboxylate as a
white foam (5.7 g, 100%). MS: mass calcd. for C28H30ClN5O4S,
567.2; m/z found, 512.1 [M+H-tBu]+; 1H NMR (400 MHz, DMSO-
d6) δ 8.88 (d, J = 1.47 Hz, 1H), 8.52 (d, J = 1.47 Hz, 1H), 7.29 (d, J =
8.56 Hz, 2H), 7.16 (d, J = 8.80 Hz, 2H), 4.62 (br s, 1H), 3.63−3.78
(m, 2H), 3.18 (br s, 2H), 2.54−2.66 (m, 2H), 2.33−2.45 (m, 2H),
1.88−2.04 (m, 3H), 1.48−1.65 (m, 3H), 1.40 (s, 9H), ppm.
Step B. To a solution of tert-butyl tert-butyl 4-(4-(7-(5-chloro-6-
cyanopyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-
phenoxy)piperidine-1-carboxylate (5.7 g, 10 mmol) in DCM (100
mL) was added TFA (10 mL). The reaction mixture was stirred at
room temperature for 2 h. The mixture concentrated and toluene was
added to the residue. The solvent was evaporated, and the remaining
mixture was poured into aqueous saturated NaHCO3 and extracted
with DCM. The organic layer was washed with water, brine, dried
over MgSO4, filtered, and concentrated. The crude material was
purified by chromatography (SiO2, 0−10% MeOH in DCM) to
provide the desired product. The product was dissolved into 1,4-
dioxane (8 mL), and a 1,4-dioxane solution (4 M) of HCl (4 mL, 16
mmol) was added. The mixture was stirred at room temperature for 1
h and then concentrated. The residue was triturated in diethyl ether,
and the precipitate was collected, washed with diethyl ether, and dried
in vacuo to give the title compound as a HCl salt: a white solid (4.4 g,
86%). LCMS (API): mass calcd. for C23H22ClN5O2S·HCl, 467.1; m/z
found, 468.1 [M + H]+; 1H NMR (300 MHz, DMSO-d6) δ 9.03 (br s,
2H), 8.89 (d, J = 1.9 Hz, 1H), 8.53 (d, J = 2.0 Hz, 1H), 7.33 (d, J =
8.6 Hz, 2H), 7.21 (d, J = 8.5 Hz, 2H), 4.63−4.84 (m, 1H), 3.20−3.29
(m, 2H), 3.02−3.17 (m, 2H), 2.54−2.67 (m, 2H), 2.34−2.46 (m,
2H), 2.09−2.25 (m, 2H), 1.82−2.05 (m, 3H), 1.45−1.63 (m, 1H)
ppm.
sodium cyanide (0.90 g, 18.5 mmol). The reaction mixture was stirred
at room temperature for overnight. The mixture was then
concentrated under reduced pressure in a fume hood. The residue
was taken up in ethyl acetate (50 mL) and washed with 1 M Na2CO3
(100 mL) and brine (25 mL) successively. The organic layer was
separated, dried over MgSO4, filtered, and concentrated. The crude
oily residue was purified by chromatography (SiO2, 1−60% EA in
heptane) to give tert-butyl 4-((5-((1-cyanocyclobutyl)amino)-
pyrimidin-2-yl)oxy)piperidine-1-carboxylate (19c) as a colorless oil
(2.47 g, 71%). MS: mass calcd. for C19H27N5O3, 373.2; m/z found,
1
318.0 [M + H-tBu]+; H NMR (300 MHz, CDCl3) δ 8.05 (s, 2H),
5.00−5.16 (m, 1H), 3.70−3.91 (m, 3H), 3.17−3.37 (m, 2H), 2.68−
2.88 (m, 2H), 2.31−2.45 (m, 2H), 2.12−2.31 (m, 2H), 1.91−2.07
(m, 2H), 1.72−1.86 (m, 2H), 1.47 (s, 9H) ppm.
Step B. To a solution of tert-butyl 4-((5-((1-cyanocyclobutyl)-
amino)pyrimidin-2-yl)oxy)piperidine-1-carboxylate (19c) (2.47 g,
6.61 mmol) in DMA (35 mL) was added freshly prepared 5-
isothiocyanato-3-trifluoromethyl-pyridine-2-carbonitrile (21a) (2.73
g, 11.9 mmol). The reaction mixture was heated at 60 °C for 4 h. The
mixture was cooled to room temperature, and MeOH (7 mL) and
HCl (1 M, 7 mL) were added. The mixture was stirred at room
temperature for overnight. Ethyl acetate (50 mL) was added, and the
mixture washed with water (100 mL), aqueous saturated Na2CO3
(1M, 30 mL), and brine (50 mL) successively. The organic layer was
separated, dried over MgSO4, filtered, and concentrated. The residue
was purified by chromatography (SiO2, 0−50% EtOAc in heptane) to
yield tert-butyl 4-((5-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-
oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)pyrimidin-2-yl)oxy)-
piperidine-1-carboxylate (22f) as an amorphous solid (3.40 g, 85%).
MS: mass calcd. for C27H28F3N7O4S, 603.2; m/z found, 547.9 [M+H-
tBu]+; 1H NMR (300 MHz, CDCl3) δ 9.08 (d, J = 2.2 Hz, 1H), 8.50
(s, 2H), 8.34 (d, J = 2.2 Hz, 1H), 5.21−5.40 (m, 1H), 3.76−3.96 (m,
2H), 3.25−3.44 (m, 2H), 2.69−2.87 (m, 2H), 2.42−2.60 (m, 2H),
2.23−2.40 (m, 1H), 2.00−2.18 (m, 2H), 1.73−1.98 (m, 3H), 1.48 (s,
9H) ppm.
Preparation of 5-(5-(6-((1-methylpiperidin-4-yl)oxy)pyridin-
3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (7). To a mixture of 5-(8-oxo-5-
(6-(piperidin-4-yloxy)pyridin-3-yl)-6-thioxo-5,7-diazaspiro[3.4]octan-
7-yl)-3-(trifluoromethyl)picolinonitrile TFA salt, 5 (17 g, 27.8 mmol)
in THF (150 mL) was added an aqueous solution (37%) of
formaldehyde (4.2 mL, 55.6 mmol), followed by the addition of
sodium triacetoxyborohydride (9.3 g, 41.7 mmol). The reaction
mixture was stirred at room temperature for overnight. The mixture
was partitioned between ethyl acetate and aqueous Na2CO3 solution
(1 M). The organic layer was separated, and the aqueous layer was
extracted with ethyl acetate. The combined organic extract was
concentrated. The crude residue was purified by chromatography
(SiO2, 0−10% MeOH in DCM) to give the crude product, which was
triturated in diethyl ether to give pure salt free product (16.19 g). The
product was dissolved into 1,4-dioxane (150 mL), and a 1,4-dioxane
solution (4 M) of HCl (8.6 mL, 34 mmol) was added. The mixture
was stirred at room temperature for 1 h and then concentrated. The
residue was triturated in diethyl ether, and the precipitate was
collected, washed with diethyl ether, and dried in vacuo to give the
title compound as a HCl salt: a white solid (13.6 g, 89%). LCMS
(API): mass calcd. for C24H23F3N6O2S·HCl, 516.2; m/z found, 517.1
[M + H]+; 1H NMR (300 MHz, DMSO-d6) δ 10.72 (s, 1H), 9.22 (d,
J = 2.1 Hz, 1H), 8.75 (d, J = 2.1 Hz, 1H), 8.23 (d, J = 2.6 Hz, 1H),
7.81 (dd, J = 8.7, 2.6 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 5.10−5.51
(m, 1H), 3.32−3.54 (m, 2H), 3.04−3.26 (m, 2H), 2.77 (br s, 3H),
2.57−2.71 (m, 2H), 2.37−2.51 (m, 2H), 1.87−2.36 (m, 5H), 1.50−
1.70 (m, 1H) ppm; Anal. Calcd for C24H23F3N6O2S·HCl·H2O: C,
50.48; H, 4.59; N, 14.72; Found: C, 51.31; H, 4.45; N, 14.46.
Preparation of 5-(8-oxo-5-(2-(piperidin-4-yloxy)pyrimidin-
5-yl)-6-thioxo-5, 7-diazaspiro[3. 4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (10). Step A. To a solution of tert-
butyl 4-((5-aminopyrimidin-2-yl)oxy)piperidine-1-carboxylate (18c)
(Matrix, 2.71 g, 9.23 mmol) in acetic acid (45 mL) was added
cyclobutanone (1.38 mL, 18.5 mmol), followed by the addition of
Step C. To a solution of tert-butyl 4-((5-(7-(6-cyano-5-
(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]-
octan-5-yl)pyrimidin-2-yl)oxy)piperidine-1-carboxylate (22f) (3.40 g,
5.63 mmol) in dioxane (25 mL) was added an anhydrous 1,4-dioxane
solution (4 N) of HCl (14.0 mL, 56.0 mmol). The reaction mixture
was stirred at room temperature for overnight. The mixture was
diluted with diethyl ether (150 mL) and triturated for 2 h. The
precipitate was collected by filtration and dried in vacuo to give the
title compound as a HCl salt: a white solid (2.86 g, 94%). MS: mass
calcd. for C22H20F3N7O2S·HCl, 503.2; m/z found, 504.0 [M + H]+;
1H NMR (300 MHz, DMSO-d6) δ 9.21 (d, J = 2.0 Hz, 1H), 9.08 (br
s, 2H), 8.75 (d, J = 2.0 Hz, 1H), 8.74 (s, 2H), 5.17-5.47 (m, 1H),
3.21-3.35 (m, 2H), 3.05-3.21 (m, 2H), 2.58-2.73 (m, 2H), 2.42-2.59
(m, 2H), 2.17-2.32 (m, 2H), 1.92-2.11 (m, 3H), 1.52-1.75 (m, 1H)
ppm.
Preparation of 5-(5-(2-((1-methylpiperidin-4-yl)oxy)-
pyrimidin-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-
3-(trifluoromethyl)picolinonitrile (9). Step A. To a solution of 5-
(8-oxo-5-(2-(piperidin-4-yloxy)pyrimidin-5-yl)-6-thioxo-5,7-
diazaspiro[3.4]octan-7-yl)-3-(trifluoromethyl)picolinonitrile, 10 (2.88
g, 5.31 mmol) and sodium acetate (0.436 g, 5.31 mmol) in DCE (15
mL) was added formaldehyde (37 wt % in water, 2.8 mL, 37.2 mmol).
The mixture was stirred at room temperature for 40 min, and then
sodium triacetoxyborohydride (1.78 g, 7.96 mmol) was added in three
portions during a period of 45 min. The reaction mixture was stirred
for 2 h and then diluted with DCM (125 mL). The mixture was
washed successively with aqueous Na2CO3 (1 M, 100 mL) and water
(50 mL). The organic layer was separated, dried over MgSO4, filtered,
and concentrated. The residue was purified by chromatography (SiO2,
0−15% MeOH in DCM to give the title compound as a white foam
(2.27 g, 82%). MS: mass calcd. for C23H22F3N7O2S, 517.2; m/z
found, 518.1 [M + H]+; 1H NMR (300 MHz, CDCl3) δ 9.09 (d, J =
2.2 Hz, 1H), 8.50 (s, 2H), 8.35 (d, J = 2.2 Hz, 1H), 5.00-5.27 (m,
1H), 2.69-2.94 (m, 4H), 2.42-2.59 (m, 2H), 2.35 (s, 3H), 2.25-2.42
920
https://dx.doi.org/10.1021/acs.jmedchem.0c01563
J. Med. Chem. 2021, 64, 909−924

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
(m, 3H), 2.09-2.23 (m, 2H), 1.94-2.09 (m, 2H), 1.69-1.90 (m, 1H)
ppm.
was purified by reverse phase preparative LC start (70% H2O-30%
MeCN-MeOH)-end (27%H2O-73%MeCN-MeOH)]-[25 mM
NH4HCO3]). The salt-free desired product (4.1 g) was dissolved in
dioxane (8 mL), and an anhydrous 1,4-dioxane solution (4 N) of HCl
(2.25 mL, 9 mmol) was added. The mixture was stirred at room
temperature for 1 h. The mixture was diluted with diethyl ether and
triturated for overnight. The precipitate was collected by filtration and
dried in vacuo to give the title compound as a HCl salt: a white solid
(4.2 g, 52%). MS: mass calcd. for C24H23F3N6OS·HCl, 500.2; m/z
Step B. To the solution of 5-(5-(2-((1-methylpiperidin-4-yl)oxy)-
pyrimidin-5-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile, 9 (2.27 g, 4.38 mmol) in 1,4-dioxane
(15 mL) was added a 1,4-dioxane solution (4 N) of HCl (1.26 mL,
5.04 mmol). The mixture was stirred at room temperature for 1.5 h,
and then diethyl ether (50 mL) was added. The resulting suspension
was stirred for another 30 min. The precipitate was collected by
filtration through a sintered funnel, washed with diethyl ether (2 × 15
mL), and dried under in vacuo to yield the title compound as a HCl
salt: a white solid (2.24 g, 89%). MS: mass calcd. for C23H22F3N7O2S·
HCl, 517.2; m/z found, 518.1 [M + H]+; 1H NMR (300 MHz,
DMSO-d6) δ 10.76 (br s, 1H), 9.22 (s, 1H), 8.75 (s, 3H), 5.38 (br s,
1H), 3.45−3.58 (m, 1H), 3.31−3.43 (m, 2H), 3.08−3.29 (m, 2H),
2.79 (br dd, J = 3.30, 13.75 Hz, 3H), 2.60−2.72 (m, 2H), 2.43−2.58
(m, 1H), 2.36 (br d, J = 12.65 Hz, 1H), 2.23 (br s, 2H), 1.87−2.15
(m, 2H), 1.54−1.73 (m, 1H) ppm.
1
found, 500.9 [M + H]+; H NMR (300 MHz, DMSO-d6) δ 9.24 (s,
1H), 8.95 (br s, 1H), 8.78 (d, J = 1.10 Hz, 1H), 7.58 (br d, J = 7.70
Hz, 2H), 7.41 (br d, J = 8.25 Hz, 2H), 3.91−4.26 (m, 1H), 3.68 (br s,
2H), 3.58 (s, 1H), 3.26−3.45 (m, 3H), 3.02−3.21 (m, 4H), 2.66 (br
s, 2H), 2.45 (br d, J = 10.45 Hz, 2H), 1.84−2.14 (m, 1H), 1.43−1.66
(m, 1H) ppm.
Identification of In Vitro and In Vivo Metabolites. The in vitro
metabolism was conducted by incubation of 10 μM 4 (JNJ-pan-AR)
in 24-well HepatoPac, containing rat, dog, and human hepatocytes
cocultured with stroma cells, according to the vendor’s protocol at 37
°C for 0 and 7 days. The final organic concentration was 0.1%. Each
well was sonicated and scraped prior to protein precipitation with 6
vol of ice-cold 1:1 ratio of acetonitrile to isopropyl alcohol containing
0.1% formic acid. The content from each well was mixed and then
transferred to test tube. The precipitated proteins were then sonicated
and pelleted by centrifugation at 3000g for 10 min. The supernatants
were transferred to clean test tubes and evaporated to dryness under a
gentle stream of nitrogen at room temperature. The residue in each
test tube was reconstituted in 300 μL of a 1:2:1 ratio of acetonitrile/
water/isopropyl alcohol fortified with 0.1% formic acid, sonicated for
5 min, and the filtrate from centrifugal filtration, using 0.45 μm nylon
filter at 20800g for 10 min at 8 °C, was used for metabolite profiling
by LC-MS.
The in vivo metabolism was investigated using plasma samples from
14-day repeat dose investigation of oral toxicity study of 4 (JNJ-pan-
AR) at 50 mg/kg/day and 45 mg/kg/day in rats and dogs,
respectively. The rat and dog plasma samples were pooled by equal
volumes to create pooled samples corresponding to day 0 and 13
samples. The pooled plasma samples were protein precipitated with
two volumes of a 1:1 ratio of acetonitrile and isopropyl alcohol
containing 0.1% formic acid. The resulting samples were sonicated
and centrifuged at 3000 g for 10 min. The supernatants were diluted
with equal volume of 0.1% formic acid, and then filtered through 0.45
μm nylon filters by centrifugation at 20800g for 10 min at 8 °C prior
to analysis by LC-MS.
All LC-MS analysis was conducted on an Accela UHPLC
connected to an LTQ Orbitrap XL (Thermo Scientific, Bremen,
Germany) operated in positive ionization mode unless indicated. The
chromatographic separation was achieved on a Phenyl-Hexyl LC
column (150 × 1 mm ID, 5 μm; Thermo Scientific, Bellefonte, Pa)
using a nonlinear gradient consisting of 0.2% formic acid (mobile
phase A) and acetonitrile containing 0.2% formic acid (mobile phase
B). The column was flow at 0.1 mL/min and kept at 45 °C
throughout the analysis. All mass spectrometric analysis was carried
out using high resolution accurate mass data-dependent multiple-stage
mass analysis. The mass resolution was set at 30,000 and 7,500 for full
scan and data-dependent multiple stage mass analysis, respectively.
The drug and its metabolites were detected using MsCompare
(MsMetrix, The Netherlands).
4 (JNJ-pan-AR) was metabolized in vitro to 4 metabolites, M1−4.
Structure elucidation of metabolites M1, M2, M3, and M4 was based
on the chemical formulas of C19H14F3N4O2S (13.5 RDB, 0.006 ppm),
C25H25F3N5O3S (14.5 RDB, −0.003 ppm), C25H25F3N5O3 (14.5
RDB, −0.001 ppm), and C24H23F3N5O2S (14.5 RDB, 0.006 ppm)
determined from the protonated molecules at m/z 419.0784,
532.1625, 500.1904 and 502.1519, respectively. Therefore, M1, M2,
M3, and M4 was postulated from metabolism of 4 (JNJ-pan-AR) via
O-dealkylation, N-oxidation, oxidative desulfuration and N-demethy-
lation, respectively. The individual structure of M1, M2, M3, or M4
metabolite was confirmed using respective synthetic standard.
Metabolites M1−M4 were also identified in pooled day 0 and 13
Preparation of 5-(8-oxo-5-(4-(piperazin-1-ylmethyl)-
phenyl)-6-thioxo-5,7-diazaspiro[3.4]octan-7-yl)-3-
(trifluoromethyl)picolinonitrile (16). Step A. Step A. To a
solution of tert-butyl 4-(4-aminobenzyl)piperazine-1-carboxylate
(18d) (Combi-Blocks, 8.53 g, 29.3 mmol) in acetic acid (120 mL)
was added cyclobutanone (4.4 mL, 58.5 mmol), followed by the
addition of sodium cyanide (2.96 g, 58.5 mmol). The reaction mixture
was stirred at room temperature for 3 h. The mixture was then
concentrated under reduced pressure in a fume hood. The residue was
taken up in ethyl acetate (200 mL) and washed with 1 M Na2CO3
(150 mL) and brine (100 mL) successively. The organic layer was
separated, dried over MgSO4, filtered, and concentrated. The crude
oily residue was purified by chromatography (SiO2, 10−100% EA in
heptane) to give tert-butyl 4-(4-((1-cyanocyclobutyl)amino)benzyl)-
piperazine-1-carboxylate (19d) as a colorless oil (8.1 g, 67%). MS:
1
mass calcd. for C21H30N4O2, 370.2; m/z found, 371.2 [M + H]+; H
NMR (400 MHz, CDCl3) δ 7.17 (d, J = 8.28 Hz, 2H), 6.60 (d, J =
8.53 Hz, 2H), 4.03 (s, 1H), 3.37−3.45 (m, 6H), 2.75−2.86 (m, 2H),
2.32−2.42 (m, 6H), 2.11−2.30 (m, 2H), 1.45 (s, 9H) ppm.
Step B. To a solution of tert-butyl 4-(4-((1-cyanocyclobutyl)-
amino)benzyl)piperazine-1-carboxylate (19d) (8.1 g, 21.9 mmol) in
DMA (30 mL) was added freshly prepared 5-isothiocyanato-3-
trifluoromethyl-pyridine-2-carbonitrile (21a) (7.5 g, 32.9 mmol). The
reaction mixture was heated at 70 °C for 1.5 h. The mixture was
cooled to room temperature and MeOH (30 mL) and HCl (1 M,
32.9 mL) were added. The mixture was stirred at room temperature
for overnight. Ethyl acetate (50 mL) was added, and the mixture
washed with water (100 mL), aqueous saturated Na2CO3 (1 M, 30
mL), and brine (50 mL) successively. The organic layer was
separated, dried over MgSO4, filtered, and concentrated. The residue
was purified by chromatography (SiO2, 10−65% EtOAc in heptane).
The product was further purified by reverse phase chromatography
[C18, start (39% H2O-61%MeCN-MeOH)-end (11% H2O-89%
MeCN-MeOH)]-(25 mM NH4HCO3)] to yield tert-butyl 4-(4-(7-
(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-
diazaspiro[3.4]octan-5-yl)benzyl)piperazine-1-carboxylate (22g) as a
white foam (8.7 g, 66%). MS: mass calcd. for C29H31F3N6O3S, 600.2;
m/z found, 601.0 [M + H]+; 1H NMR (300 MHz, CDCl3) δ 9.11 (d,
J = 2.3 Hz, 1H), 8.37 (d, J = 2.3 Hz, 1H), 7.57 (d, J = 7.9 Hz, 2H),
7.27 (d, J = 7.1 Hz, 2H), 3.61 (s, 2H), 3.38−3.56 (m, 4H), 2.52−2.81
(m, 4H), 2.40−2.51 (m, 4H), 2.16−2.35 (m, 1H), 1.59−1.80 (m,
1H), 1.46 (s, 9H) ppm.
Step C. To a solution of tert-butyl 4-(4-(7-(6-cyano-5-
(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]-
octan-5-yl)benzyl)piperazine-1-carboxylate (22g) (8.7 g, 14.5 mmol)
in DCM (70 mL) was added TFA (15 mL). The reaction mixture was
stirred at room temperature for 2 h. The mixture concentrated, and
the residue was taken in toluene (40 mL) and again concentrated (2
× 40 mL). The crude residue was diluted with DCM and washed with
aqueous Na2CO3 solution (1 M). The organic layer was separated,
and aqueous layer was extracted with DCM. The combined organic
extract was dried over MgSO4, filtered, and concentrated. The residue
921
https://dx.doi.org/10.1021/acs.jmedchem.0c01563
J. Med. Chem. 2021, 64, 909−924

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
plasma samples from repeated dose investigation of oral toxicity of 4
(JNJ-pan-AR) in rats and dogs.
Heng Keang Lim − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
Vineet Pande − Janssen Research and Development, B-2340
Beerse, Belgium
Lieven Meerpoel − Janssen Research and Development, B-
2340 Beerse, Belgium
Christopher Teleha − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
Jonathan R. Branch − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States;
orcid.org/0000-0003-4169-5753
Janine Ondrus − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Ian Hickson − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States; orcid.org/
0000-0002-9167-0514
Tammy Bush − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
Leopoldo Luistro − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
Kathryn Packman − Janssen Research and Development,
Cambridge, Massachusetts 02142, United States
James R. Bischoff − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
Salam Ibrahim − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States
GSH Screen for Phenolic Metabolites of Androgen
Receptor Antagonists. The bioactivation of potential phenolic
metabolites of androgen receptor antagonists was evaluated by
modification of trapping procedure previously reported by Lim et al.,
2008. Briefly, incubation of each potential phenolic metabolite was
conducted in a final volume 1 mL in a test tune, containing 10 μM
test compound, 1 mg/mL dog liver microsomes, 5 mM glutathione
mixture (GSH: [13C2, 15N-glycine]GSH 2:1), and 1 mM NADPH in
0.1 M phosphate buffer. The control sample contained all except the
NADPH cofactor. The samples were incubated for 60 min. Once the
incubations were completed, the reaction was quenched with 5.0 mL
of a 1:1 ratio of acetonitrile and isopropyl alcohol containing 0.1%
formic acid. The resulting mixture was vortex mixed and sonicated,
followed by centrifugation at 3000g for 10 min at 5 °C. The
supernatants were transferred into clean tubes and dried under
nitrogen at room temperature to dryness. The residue was dissolved
in 300 μL of a 4:1 ratio of 0.1% formic acid and acetonitrile,
sonicated, and filtered through 0.45 μm Nylon filters prior to LC-MS
analysis.
The glutathione conjugate was detected by isotopic pattern
triggered data-dependent multiple-stage high resolution accurate
mass analysis. The mining for glutathione conjugates was by high
resolution accurate mass isotopic pattern filtering (MsMetrix, The
Netherlands) of full scan MS data. The product ion mass spectra were
used to elucidate the site of bioactivation.
The identification of glutathione conjugate of phenol 6 at 24.2 min
was from the correct chemical formula of C29H29O9F3S2 (17.5 RDB,
1.76 ppm), which upon collision-induced dissociation (CID) resulted
in cleavage of the C−S bond linking the sulfur atom to the carbon
atom of the tripeptide to give m/z 465.0316 (C19H12O3N4F3S2, 14.5
RDB, 3.97 ppm). Such bond cleavage is diagnostic of an aryl
thioether. Further isolation of m/z 465 for further CID produced the
base peak at m/z 208.0422 (6.5 RDB, −1.90 ppm), which localized
the site of hydroxylation and addition of glutathione molecule to the
phenolic moiety. The identity of the glutathione conjugate is
consistent with bioactivation of phenol 6 to an o-quinone prior to
addition of the glutathione molecule.
Christopher Parrett − VWR Avantor, Spring House,
Pennsylvania 19477, United States
Yolanda Chong − Janssen Research and Development, B-2340
Beerse, Belgium
Marco M. Gottardis − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01563
All experimental procedures were conducted in accordance with
Janssen’s Institutional Animal Care and Use Committee and U.S.
Department of Agriculture regulations.
Author Contributions
The manuscript was written through contributions of all
authors. Contributions by C.T., I.H., J.R.B., and Y.C. were
performed while employed at Janssen Research and Develop-
ment.
ASSOCIATED CONTENT
■
sı
* Supporting Information
Notes
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01563.
The authors declare no competing financial interest.
Homology model coordinates (PDB)
ACKNOWLEDGMENTS
■
Molecular formula strings and some data (CSV)
Assay and protocols: radioligand binding, transcriptional
reporter, high-content imaging, proliferation, Hershberg-
er, tumor xenograft efficacy, chemistry, 1H NMR/
LCMS/HPLC traces of compounds 4, 5, 8, and
molecular modeling (PDF)
The authors would like to thank Nick Smith and Mark Herbert
(Aragon Pharmaceuticals, San Diego, CA) for their valuable
insights into early SAR of the thiohydantoin series. We are also
́
grateful to Luis Trabalon Escolar and Christian Rocaboy
(Eurofins Villapharma, Spain) and Bing Huang (Wuxi AppTec,
Shanghai, China) for their contributions to synthesis of
compounds described herein.
AUTHOR INFORMATION
■
Corresponding Authors
ABBREVIATIONS
■
Zhuming Zhang − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States; orcid.org/
0000-0002-5638-5320; Email: zzhang85@its.jnj.com
Gilles Bignan − Janssen Research and Development, Spring
House, Pennsylvania 19477, United States;
AR, androgen receptor; ADT, androgen deprivation therapy;
CRPC, castration resistant prostate cancer; LBD, ligand
binding domain; GSH, glutathione; NADPH, nicotinamide
adenine dinucleotide phosphate hydrogen; MFS, metastasis-
free survival; NTD, N-terminal domain; DBD, DNA-binding
domain; AF1, activation function 1; AF2, activation function 2;
ctDNA, circulating tumor DNA; GR, glucocorticoid receptor;
ER, estrogen receptor; PSA, prostate-specific antigen; ARE,
androgen response element; KLK3, kallikrein-3, also known as
Email: gbignan@its.jnj.com
Authors
Peter J. Connolly − Janssen Research and Development,
Spring House, Pennsylvania 19477, United States
922
https://dx.doi.org/10.1021/acs.jmedchem.0c01563
J. Med. Chem. 2021, 64, 909−924

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Drug Annotation
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PSA; ASO, androgen sensitive organ; TP, testosterone
propionate; TGI, tumor growth inhibition; ALP, alkaline
phosphatase; ALT, alanine aminotransferase; GGT, gamma-
glutamyl transferase; PD, pharmacodynamics; PK, pharmaco-
kinetics; HLM, human liver microsomes; ESI, electrospray
ionization; API, atmospheric pressure chemical ionization;
DMA, dimethylacetamide; EtOAc, ethyl acetate
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