Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one Derivatives as Orally eIF4A3-Selective Inhibitors

Article abstract of DOI:10.1021/acsmedchemlett.7b00283

Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed antitumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of compounds represents promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.

Full text of DOI:10.1021/acsmedchemlett.7b00283

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Letter
Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-
one Derivatives as Orally eIF4A3-selective Inhibitors
Ryo Mizojiri, Daisuke Nakata, Yoshihiko Sato, Daisuke Morishita, Sachio Shibata, Misa Iwatani-
Yoshihara, Yohei Kosugi, Mai Kosaka, Junpei Takeda, Shigekazu Sasaki, Kazuaki Takami, Koichiro
Fukuda, Masahiro Kamaura, Shinobu Sasaki, Ryosuke Arai, Douglas R Cary, and Yasuhiro Imaeda
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.7b00283 • Publication Date (Web): 08 Sep 2017
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Discovery of Novel 5-(Piperazine-1-carbonyl)pyridin-2(1H)-one De-
rivatives as Orally eIF4A3-selective Inhibitors
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Ryo Mizojiri, Daisuke Nakata, Yoshihiko Satoh, Daisuke Morishita, Sachio Shibata, Misa Iwatani-
Yoshihara, Yohei Kosugi, Mai Kosaka, Junpei Takeda, Shigekazu Sasaki, Kazuaki Takami, Koichiro
Fukuda, Masahiro Kamaura, Shinobu Sasaki, Ryosuke Arai, Douglas R. Cary, and Yasuhiro Imaeda
Research, Takeda Pharmaceutical Company Ltd., Fujisawa, Kanagawa 251-8555, Japan
KEYWORDS. Eukaryotic initiation factor 4A3 (eIF4A3) inhibitor, RNA helicase, solubility, metabolic stability, P-gp sub-
strate, pyridin-2(1H)-one derivative
ABSTRACT: Starting from our previous eIF4A3-selective inhibitor 1a, a novel series of (piperazine-1-carbonyl)pyridin-2(1H)-
one derivatives was designed, synthesized, and evaluated for identification of orally bioavailable probe molecules. Compounds 1o
and 1q showed improved physicochemical and ADMET profiles, while maintaining potent and subtype-selective eIF4A3 inhibitory
potency. In accord with their promising PK profiles and results from initial in vivo PD studies, compounds 1o and 1q showed anti-
tumor efficacy with T/C values of 54% and 29%, respectively, without severe body weight loss. Thus, our novel series of com-
pounds represent promising probe molecules for the in vivo pharmacological study of selective eIF4A3 inhibition.
The eukaryotic initiation factor 4A(eIF4A) family is a member
of the ATP-dependent RNA helicase protein family and con-
stability, and pharmacokinetics were insufficient for conduct-
ing further biological and pharmacological studies. To identi-
fy improved probe molecules for the study of eIF4A3 function,
we continued our chemistry efforts on allosteric inhibitors
using the novel 3-(4-chlorophenyl)-1,4-diacylpiperazine de-
rivative 1a as a lead compound.
1–4
sists of three isoforms, eIF4A1, eIF4A2 and eIF4A3.
eIF4A1 and eIF4A2 are required for translation initiation,
While
5–7
eIF4A3 controls RNA metabolism including nonsense-
mediated mRNA decay (NMD) and RNA localization as a
8–15
core component of the exon junction complex (EJC).
NMD is a surveillance pathway to eliminate mRNAs that con-
tain premature termination codons (PTCs), preventing the
generation of detrimental gain-of-function or dominant-
negative proteins. Cancer cells often harbor a wide varie-
ty of mutations introducing PTCs, and cancer cells are consid-
Table 1. Biological and physicochemical profile of lead
compound 1a
16–18
ered to be highly dependent on the NMD pathway to avoid the
19–22
accumulation of aberrant proteins.
Therefore, targeting
eIF4A3 inhibition could potentially represent a promising
approach for anticancer therapy, however, the detailed physio-
logical functions of eIF4A3 are not clear. While some re-
searchers have reported on the biological function of pan-
eIF4A inhibition utilizing a known natural product, hippuri-
d
Mouse cassette PK
eIF4A3
Solubility
b
(mg/mL)
Metabolic stability
c
(µL/min/mg)
AUCpo
F
2
3–26
27
a
IC50 (µM)
stanol
and Pateamine A, selective eIF4A3 inhibition
e
f
(ng h/mL)
(%)
would be required to enable more definitive biological and
pharmacological evaluation both in vitro and in vivo.
0
.11
1
a
0.46
227
9.0
5.7
(
0.09–0.13)
Recently, we discovered several classes of both ATP-
competitive and allosteric eIF4A3 inhibitors which showed
a
IC50 is presented as the mean of duplicate experiments along with
b
5% confidence intervals (95%CI) in parentheses. Solubility in
2
8–30
9
pH 6.8. Metabolic stability in mice microsomes. Male ICR
potent and selective eIF4A3 inhibition
from hit com-
c
d
pounds through HTS of an in-house compound library.
Among these, ATP-competitive inhibitors were characterized
e
mice (n = 3). Dose: i.v. at 0.1 mg/kg; p.o. at 1 mg/kg. Area under
f
the curve from 0 to 8 hours. Bioavailability.
2
using ITC and SPR analyses. On the other hand, the alloster-
8
ic eIF4A3 inhibitors exhibited significant NMD inhibition in a
29
cellular reporter assay along with eIF4A3 inhibitory potency
and were examined for understanding the function of eIF4A3
According to our previous research, we found that the (S)-3-
phenyldiacylpiperazine scaffold of 1a was essential for potent
30
in RNA homeostasis.
Although the allosteric inhibitors
29
eIF4A3 inhibition. Substitution or modification of the piper-
showed potent enzymatic activity as well as cellular potency,
the physicochemical properties such as solubility, metabolic
azine ring, including investigations of the corresponding (R)-
enantiomer demonstrated substantially reduced biological
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activity. In addition, SAR around the pendant 4-chlorophenyl
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moiety was also found to be narrow, with most modifications
resulting in equal or reduced activity, as well. Therefore, we
fixed the Core Scaffold as shown in Figure 1 and thought to
modify the two amide moieties, the 4-bromobenzoyl moiety in
the East Region and the pyrazolo[1,5-a]pyridin-3-yl moiety in
the West Region, to optimize the biological and physicochem-
ical properties of 1a.
1
1
a
CH (0.09–
0.46
3.8
227
207
9.0
5.7
0
.13)
.28
0
b
N
(0.17–
0.46)
55.9
11.4
2
.7
1
1
1
c
d
e
CH (1.7–
3.2
5.1
11
143
202
16
129.1
1.5
19.7
0.4
4
1
.1)
.6
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0
CH (1.0–
2
.5)
0
.29
CH (0.17–
.50)
97.3
9.6
0
a
IC50 is presented as the mean of duplicate experiments along with
b
95% confidence intervals (95%CI) in parentheses. Solubility in
c
pH 6.8. Metabolic stability in mice microsomes. Male ICR
d
Figure 1. Synthetic strategies for improvement of physicochemi-
cal properties and PK profile
e
mice (n = 3). Dose: i.v. at 0.1 mg/kg; p.o. at 1 mg/kg. Area under
f
the curve from 0 to 8 h. Bioavailability.
During the chemical modification of the East Region, it was
found that replacement of the 4-bromobenzoyl moiety with a
As the start of compound optimization for 1e, we examined
the replacement of bicyclic ring of 1f, the racemate of 1e, into
biaryl moiety in order to facilitate synthetic accessibility to
new analogues during our synthesis campaign aimed at effi-
cient SAR data collection. The 3-phenylpyridin-2(1H)-one
derivative 1g thus synthesized showed potent eIF4A3 inhibi-
5
-bromo-2-picolinoyl moiety generally improved solubility
while maintaining eIF4A3 inhibitory potency (1b eIF4A3
IC : 0.28 µM). Although broad investigation of other substit-
50
2
9
uents in the East Region, acyl and otherwise, generally
showed substantially reduced biological activity. On the other
hand, an initial SAR campaign suggested that modification of
the West Region was tolerated for wide variety of substituents
to modulate both potency and physicochemical properties.
Thus, we focused on the chemical modification of this region
for further investigation. During derivative synthesis around
this amide moiety, quinoline derivative 1c and isoquinoline
derivative 1d were identified as promising bicyclic analogues.
Further investigation of bicyclic 6-6 fused ring systems led us
to find isoquinolin-1(2H)-one derivative 1e as a potent lead
compound with improved solubility and metabolic stability,
while still maintaining eIF4A3 inhibitory potency. Addition-
ally, 1e showed a slightly improved PK profile compared to 1a,
along with improved physicochemical properties as shown in
Table 2.
tion (1g eIF4A3 IC : 0.41 µM). Therefore, we chose 1g as a
50
new lead compound and conducted further optimization of
related 3-arylpyridin-2(1H)-one derivatives. In order to col-
lect SAR information rapidly for these new pyridin-2(1H)-one
derivatives, we examined substituent effects on the phenyl
ring of pyridin-2(1H)-one utilizing parallel synthesis. Assay
results for representative compounds are listed in Table 3.
Table 3. Representative compounds in parallel synthesis
for 3-phenylpyridin-2(1H)-one derivatives
Table 2. Initial SAR of diacyl moieties – all compounds
are (S)-enantiomer
eIF4A3
IC50 (µM)
0.42
eIF4A3
IC50 (µM)
0.19
R
R
a
a
1
h
2-Cl
3-Cl
4-Cl
1k
1l
3-CN
(0.33–0.53)
(0.12–0.29)
0.29
0.7
1
i
3-CF
3
(0.25–0.34)
(0.5–0.9)
1.1
0.49
1
j
1m
2
3-SO Me
(0.8–1.5)
(0.35–0.69)
Mouse cassette
d
PK
eIF4A3
Metabolic
stability
a
Solubility
b
(mg/mL)
IC50 is presented as the mean of duplicate experiments along with
95% confidence intervals (95%CI) in parentheses.
2
R
X
IC50
AUCpo
ng
h/mL)
F
a
c
(µM)
(µL/min/mg)
(
f
e
(%)
Investigation of substituents on the benzene ring (compound
1h–1j) suggested that substitution at the C3 position was fa-
vorable for obtaining potent eIF4A3 inhibitory activity (1i
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eIF4A3 IC : 0.29 µM). Therefore, we focused our examina-
substituents into the pyridin-2(1H)-one moiety, using the cal-
culated total polar surface area (tPSA) value for reference (1o
50
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
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tion of substituent effects at the benzene C3-position. Re-
placement of the chloro group with a cyano group slightly
improved potency (1k eIF4A3 IC : 0.19 µM) compared to 1i.
2
32
has a high tPSA value of 99.3 Ǻ ). Replacement of the 3-
cyanophenyl group with a trifluoromethyl group reduced the
5
0
2
We postulated that an electron-withdrawing group (EWG)
could be effective at enhancing eIF4A3 inhibition. However,
further investigation of EWGs such as a trifluoromethyl group
tPSA value to 75.5 Ǻ . Since eIF4A3 inhibitory potency was
decreased by this modification (1p eIF4A3 IC50: 0.75 µM), we
hypothesized that the biaryl scaffold was important for obtain-
ing high potency. Based on this hypothesis, we investigated
the introduction of an aryl substituent into the pyridin-2(1H)-
one moiety while maintaining a tPSA value comparable to 1p.
Accordingly, the introduction of an N-phenyl group instead of
an N-methyl group into the pyridin-2(1H)-one moiety im-
proved eIF4A3 inhibitory potency (1q IC50: 0.14 µM) compa-
rable to 1o (1o IC : 0.10 µM) while maintaining a low tPSA
(1l) or a methanesulfonyl group (1m) at the C3-position did
not yield improvements relative to the 3-cyano group. As a
result, we concluded the 3-cyanophenyl group to be the most
potent substituent for this series. Next, we synthesized the
optically active (S)-pyridin-2(1H)-one derivative with a 3-
cyanophenyl group possessing a 5-bromo-2-picolinoyl moiety
in the East Region. As expected, compound 1n showed highly
potent eIF4A3 inhibitory activity (1n eIF4A3 IC : 0.05 µM)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
2
5
0
value of 75.5 Ǻ . During this modification, 1q showed lower
P-gp efflux (efflux ratio of 0.8) compared to 1o (efflux ratio of
25.0). The PK profile of pyridin-2(1H)-one derivatives 1o and
1q are shown in Table 5. Expectedly, their PK profiles were
dramatically improved relative to 1a.
with acceptable solubility (2.4 mg/mL at pH 6.8), however,
exhibited poor metabolic stability in mouse microsomes
(
113µL/min/mg). We hypothesized that its instability in
mouse microsomes originated from the acidity of the pyridin-
(1H)-one moiety. Based on this hypothesis, we designed and
2
synthesized N-methyl compound 1o to reduce the acidity of
the compound, which showed dramatically improved solubili-
ty and metabolic stability while maintaining eIF4A3 inhibitory
potency, as expected. However, 1o was found to act as a P-gp
substrate, with a high efflux ratio of 25.0. It is well known
that P-gp mediated efflux can have a negative impact a com-
a
Table 5. PK profile of 1o and 1q in mouse cassette
b
c
AUCpo (ng h/mL)
F (%)
5.7
1a
1o
1q
9.0
146.6
315.4
16.1
23.7
3
1
pound's PK profile. Thus, we conducted further modifica-
tion of these pyridin-2(1H)-one derivatives to address this
issue.
a
Male ICR mice (n = 3). Dose: i.v. at 0.1 mg/kg; p.o. at 1 mg/kg.
c
Area under the curve from 0 to 8 h. Bioavailability.
b
Table 4. Compound optimization of pyridin-2(1H)-one
derivatives – all compounds are (S)-enantiomer
Given the discovery of potent eIF4A3 inhibitors 1o and 1q,
which showed acceptable PK profiles, we examined their bio-
chemical selectivity. As shown in Table 6, compounds 1o and
1
q were both found to show highly selective eIF4A3 inhibito-
ry activity over not only other eIF4A family proteins (eIF4A1
and eIF4A2) but also against other ATP-dependent RNA hel-
icases such as BRR2 and DHX29, which is consistent with the
activity found for compound 1a. Accordingly, we advanced
these potent and selective eIF4A3 inhibitors into further in
vitro and in vivo biological evaluation.
eIF4A3
Metabolic
stability
Solubility
b
(mg/mL)
tPSA
2 e
1
2
Efflux
d
ratio
R
R
IC50
a
(Ǻ )
c
(
µM)
.05
(0.04–
(
µL/min/mg)
Table 6. Biochemical selectivity of 1o and 1q.
0
3-
CNPh
1
n
o
H
2.4
12
113
6
35.0
25.0
7.4
110.2
99.3
75.5
75.5
eIF4A1
eIF4A2
eIF4A3
IC50 (µM)
0.11
BRR2
DHX29
0
.05)
a
IC50 (µM) IC50 (µM)
IC50 (µM) IC50 (µM)
0
.1
3-
CNPh
1
Me
Me
Ph
(0.06–
0.15)
1a
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
(0.09–0.13)
0.1
0
.75
1
1
o
1p
CF
3
3
(0.52–
1.1)
32
0
(
0.06–0.15)
.14
0.09–0.22)
0
q
0.14
(
1
q
CF
(0.09–
0
5.9
0
0.8
a
.22)
IC50 is presented as the mean of duplicate experiments along with
5% confidence intervals (95%CI) in parentheses.
9
a
IC50 is presented as the mean of duplicate experiments along with
b
95% confidence intervals (95%CI) in parentheses. Solubility in
c
pH 6.8. Metabolic stability in mice microsomes. Efflux ratio in
e
vitro MDR1 overexpressing cell line. Calculated by Daylight
software.
d
In a separate report, we developed a luciferase-based cellular
NMD reporter assay to evaluate the cellular potency of our
28
eIF4A3 inhibitors. However, this assay method could not be
To reduce the potential for P-gp recognition, our medicinal
chemistry strategy focused on the introduction of less polar
applied to in vivo pharmacodynamics (PD) evaluation. On the
other hand, serine/arginine-rich splicing factor 2 (SC35) 1.6
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33
and 1.7 kb have been known as substrates for NMD and are
measurable in vivo. Thus, their mRNA expression levels
could be a potential PD marker for NMD inhibition in vivo.
To evaluate cellular potency, we chose SC35 1.6 kb as a PD
marker in consideration of its applicability in vivo studies. Six
hour treatment of 1o and 1q upregulated SC35 1.6 kb mRNA
expression by ca. 8-fold at 3 µM and ca. 20-fold at 10 µM in
colorectal carcinoma HCT-116 cells, whereas 1a showed 2.59-
fold upregulation at 3 µM and 12.88-fold at 10 µM at seven
hours, respectively. Consistent with these results, both 1o and
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Figure 2. Effects of compounds 1o and 1q on expression of SC35
.6kb in HCT-116 xenograft tumors. . *P < 0.05 versus vehicle
group by Dunnett's test.
1
q exhibited more potent growth inhibitory activity in HCT-
16 cancer cells compared to 1a, as shown in Table 7. Ac-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
1
cording to these results, ca. 2-fold upregulation of SC35 1.6 kb
mRNA expression appeared to correlate with a meaningful
change in GI value.
50
The in vivo anti-tumor activity of 1o and 1q was examined in
an HCT-116 xenograft mouse model. As shown in Figure 3,
oral administration of both compounds in our solubilizing
formulation (100 mg/kg, bid) for 14 days exhibited anti-tumor
efficacy with T/C values of 54% and 29%, respectively. The
compounds were tolerated during the course of the study,
showing less than 5% decrease in body weight. These data
suggested that a long-acting profile in PD studies might be
favorable for anti-tumor efficacy. Accordingly, compound 1q
is an orally bioavailable eIF4A3-selective inhibitor with in
vivo anti-tumor efficacy, representing a useful new tool for
assessing the pharmaceutical potential of eIF4A3 inhibition in
cancer.
a
Table 7. Cellular potency of eIF4A3 inhibitors
b
SC35 1.6kb (fold of change)
HCT-116
c
1
µM
3 µ M
10 µ M
GI50 (µM)
d
d
c
1
a
0.9
2.59
12.88
3.06
1.34
1.18
1o
1q
2.17
1.79
8.41
8.31
19.08
21.76
a
b
Data are representative of three independent experiments. Fold
change relative to control for mRNA expression of SC35 1.6 kb
c
after 6 h treatment. Intra-cellular ATP amount measured 72 h
after compound treatment. GI50 calculated using GraphPad
d
Prism5.
after 7 h treatment.
Fold change relative to control of mRNA expression
After confirming cellular potency of 1o and 1q in HCT-116
cells, we designed in vivo studies for further assessment of our
eIF4A3 inhibitors. In order to improve the exposure of 1o and
1
q effectively for in vivo evaluation, we examined solubilized
formulations for both compounds. As a result of our investi-
gations, the compounds were dissolved in 10% DMSO/10%
Cremophor EL/30% PEG400/20% propylene glycol (PG)/30%
0.4 mol/L citrate solution. Next, we conducted in vivo PD
studies in an HCT-116 xenograft model. The in vivo PD pro-
files of 1o and 1q are shown in Figure 2. Expression of SC35
Figure 3. Anti-tumor efficacy of compounds 1o and 1q in HCT-
116 xenograft model mice. **P < 0.001 versus vehicle group by
Dunnett's test.
1
.6 kb in tumors was measured at 4 h, 8 h and 24 h after oral
administration of 1o and 1q at 100 mg/kg in the above solubil-
izing formulation in HCT-116 xenograft model mice. As a
result, PD responses were confirmed by single oral administra-
tion of 1o and 1q. Remarkably, 1o showed 4-fold upregula-
tion of SC35 1.6 kb mRNA expression after 4 h as a rapid-
acting profile, while 1q displayed ca. 2-fold upregulation of
SC35 1.6 kb mRNA expression after 24 h as a long-acting
profile. Thus, we examined both compounds for anti-tumor
efficacy study to assess the differing profiles of these eIF4A3
inhibitors.
We have described the identification of a novel series of orally
bioavailable eIF4A3-selective inhibitors as a chemical probes
for oncology research and potential use in the development of
anti-cancer agents. Our initial lead 1a was optimized to im-
prove its physicochemical properties and PK profile and con-
verted to 3-(3-cyanophenyl)pyridin-2(1H)-one derivative 1o.
Compound 1o showed improved solubility and metabolic sta-
bility relative to compound 1a, while maintaining eIF4A3
inhibitory potency, however further testing suggested the need
to reduce P-gp efflux.
To address this need, a 3-
trifluoropyridin-2(1H)-one derivative was prepared using the
tPSA value as a guide for reducing polarity, which lead us to
the discovery of N-phenyl-3-trifluoropyridin-2(1H)-one deriv-
ative 1q. Compounds 1o and 1q showed improved physico-
chemical and ADMET profiles, while maintaining potent and
subtype-selective eIF4A3 inhibitory potency. Based on their
promising PK profiles and results from initial in vivo PD stud-
ies, both compounds demonstrated anti-tumor efficacy in xen-
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ograft model. As a result, compounds 1o and 1q showed anti- dimethylformamide, DMF; dimethylsulfoxide, DMSO; tet-
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tumor efficacy with T/C values of 54% and 29%, respectively,
without severe body weight loss. Accordingly, compound 1q
was judged to be a useful in vivo probe molecule to further
study the fundamental biology and therapeutic potential of
selective eIF4A3 inhibition in cancer.
rahydrofuran, THF; trifluoroacetic acid, TFA; trimethylsilyl
chloride, TMSCl.
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