Discovery of [cis -3-({(5 R)-5-[(7-Fluoro-1,1-dimethyl-2,3-dihydro-1 H -inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-naphthyridin-6(5 H)-yl}carbonyl)cyclobutyl]acetic Acid (TAK-828F) as a Potent, selective, and orally available novel retinoic acid rece

Article abstract of DOI:10.1021/acs.jmedchem.8b00061

A series of tetrahydronaphthyridine derivatives as novel RORγt inverse agonists were designed and synthesized. We reduced the lipophilicity of tetrahydroisoquinoline compound 1 by replacement of the trimethylsilyl group and SBDD-guided scaffold exchange,

Full text of DOI:10.1021/acs.jmedchem.8b00061

Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE
Discovery of [cis-3-({(5R)-5-[(7-fluoro-1,1-dimethyl-2,3-
dihydro-1H-inden-5-yl)carbamoyl]-2-methoxy-7,8-dihydro-1,6-
naphthyridin-6(5H)-yl}carbonyl)cyclobutyl]acetic acid (TAK-828F)
as a Potent, Selective, and Orally Available Novel Retinoic
Acid Receptor-Related Orphan Receptor #t Inverse Agonist
Mitsunori Kono, Atsuko Ochida, Tsuneo Oda, Takashi Imada, Yoshihiro Banno, Naohiro
Taya, Shinichi Masada, Tetsuji Kawamoto, Kazuko Yonemori, Yoshi Nara, Yoshiyuki
Fukase, Tomoya Yukawa, Hidekazu Tokuhara, Robert Skene, Bi-Ching Sang, Isaac D.
Hoffman, Gyorgy P. Snell, Keiko Uga, Akira Shibata, Keiko Igaki, Yoshiki Nakamura, Hideyuki
Nakagawa, Noboru Tsuchimori, Masashi Yamasaki, Junya Shirai, and Satoshi Yamamoto
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 06 Mar 2018
Downloaded from http://pubs.acs.org on March 6, 2018
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Discovery of [cisꢀ3ꢀ({(5R)ꢀ5ꢀ[(7ꢀfluoroꢀ1,1ꢀ
dimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl]ꢀ2ꢀ
methoxyꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀ
yl}carbonyl)cyclobutyl]acetic acid (TAKꢀ828F) as a
Potent, Selective, and Orally Available Novel
Retinoic Acid ReceptorꢀRelated Orphan Receptor γt
Inverse Agonist
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Mitsunori Kono,* Atsuko Ochida, Tsuneo Oda, Takashi Imada, Yoshihiro Banno, Naohiro
†
,§
†
†,§
†
†,§
Taya, Shinichi Masada, Tetsuji Kawamoto, Kazuko Yonemori, Yoshi Nara, Yoshiyuki
†
,
∥
†
†,§
‡
‡
Fukase, Tomoya Yukawa, Hidekazu Tokuhara, Robert Skene, Bi-Ching Sang, Isaac D.
‡
‡
†,§
†
†,§
†
Hoffman, Gyorgy P. Snell, Keiko Uga, Akira Shibata, Keiko Igaki, Yoshiki Nakamura,
†
†
†,§
†,
⊥
Hideyuki Nakagawa, Noboru Tsuchimori, Masashi Yamasaki, Junya Shirai, and Satoshi
†
Yamamoto*
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†
Pharmaceutical Research Division, Takeda Pharmaceutical Company Limited, 26ꢀ1 Muraokaꢀ
Higashi 2ꢀchome, Fujisawa, Kanagawa 251ꢀ8555, Japan
‡
Takeda California, 10410 Science Center Drive, San Diego, CA 92121, USA
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ABSTRACT
A series of tetrahydronaphthyridine derivatives as novel RORγt inverse agonists were designed
and synthesized. We reduced the lipophilicity of tetrahydroisoquinoline compound 1 by
replacement of the trimethylsilyl group and SBDDꢀguided scaffold exchange, which successfully
afforded compound 7 with a lower logD value and tolerable in vitro activity. Consideration of
LLE values in the subsequent optimization of the carboxylate tether led to the discovery of [cisꢀ
3
ꢀ({(5R)ꢀ5ꢀ[(7ꢀfluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl]ꢀ2ꢀmethoxyꢀ7,8ꢀ
dihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl}carbonyl)cyclobutyl]acetic acid, TAKꢀ828F (10), which
showed potent RORγt inverse agonistic activity, excellent selectivity against other ROR isoforms
and nuclear receptors, and a good pharmacokinetic profile. In animal studies, oral administration
of compound 10 exhibited robust and doseꢀdependent inhibition of ILꢀ17A cytokine expression
in a mouse IL23ꢀinduced gene expression assay. Furthermore, development of clinical symptoms
in a mouse experimental autoimmune encephalomyelitis model was significantly reduced.
Compound 10 was selected as a clinical compound for the treatment of Th17ꢀdriven autoimmune
diseases.
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Introduction
The retinoic acidꢀrelated orphan receptor (ROR) is a subclass of orphan nuclear receptors, and
1
there are three subtypes known as RORα, RORβ and RORγ. In particular, RORγt (the second
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isoform of γ which is highly restricted to expression in the thymus) is a master regulator for
differentiation of human Th17 cells, which is a subset of CD4+T cells that are essential in the
2
,3
immune response. The Th17 cells, and the inflammatory cytokines they produce (ILꢀ17A, ILꢀ
7F etc.) play a central role in the pathogenesis of various autoimmune diseases such as
inflammatory bowel disease, psoriasis, rheumatoid arthritis, multiple sclerosis, chronic
1
4
ꢀ8
obstructive pulmonary disease, and asthma. The biologics targeting ILꢀ17A and the ILꢀ17A
9ꢀ11
receptor have demonstrated clinical efficacy in psoriasis, rheumatoid arthritis, and uveitis.
Therefore, Th17 cells have emerged as an attractive target for therapeutic intervention in these
immuneꢀrelated diseases. RORγt contributes to the production of not only ILꢀ17A, but also other
proꢀinflammatory cytokines such as ILꢀ17F, ILꢀ22, ILꢀ23 receptor, and granulocyte macrophage
colonyꢀstimulating factor, from various immune cells such as Th17, ILꢀ17/IFNꢀγ coꢀproducing
1
2,13
cells (Th1/17 cells), innate lymphoid cells, and γδ T cells.
The deletion of RORγt results in
impaired Th17 differentiation in murine and human cell and RORγt knockout mice are resistant
to various autoimmune disease models, such as colitis and experimental autoimmune
encephalomyelitis (EAE). Hence, the inhibition of RORγt transcription could be promising for a
new potential therapeutic option for autoimmune diseases, by acting as a replacement for
existing immunosuppressive agents.
Consequently, a large number of RORγt inhibitors have been reported from many research
1
4ꢀ17
groups.
Among them, some compounds such as VTPꢀ43742, JNJꢀ3534, ARNꢀ6039, AZDꢀ
1
8–21
0
284, and JTEꢀ451 are under clinical trials.
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Previously, we reported a series of novel tetrahydroisoquinoline derivatives as potent and
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orally available RORγt inverse agonists. We identified compound 1, which has potent in vitro
RORγt inverse agonistic activity and favorable pharmacokinetic (PK) profile. Compound 1
showed robust and doseꢀdependent inhibition of ILꢀ17A cytokine expression after oral
administration in a mouse ILꢀ23ꢀinduced cytokine expression pharmacodynamic (PD) model
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(
ED50 = 2.8 mg/kg). Despite the attractive biological profiles for target potency and ADME
properties, compound 1 has a lipophilicity (logD: 3.96) outside the typical optimal range for drug
23
discovery (logD: 1–3). It is known that nuclear receptor ligands will often require high
lipophilicity to achieve the required potency and the lipophilicity is generally higher compared
2
3
with other target classes (approximate logD average: 2.5–3.5). Even when this is accounted for
the lipophilicity of compound 1 is a little too high; a reduction in lipophilicity is usually
important to achieve favorable drugꢀlike properties. Hence, the balance between potency and
lipophilicity is important for the discovery of clinical candidates, and potency by lipophilicity
(
i.e., LLE) may be informative for the optimization and assessment of synthesized compounds.
Therefore, to generate new candidates, we included a consideration of the LLE values.
Initially, we divided the compound 1 into three components (AꢀC). The structureꢀactivity
2
2
relationships (SAR) study described in our previous report indicated that it would be difficult to
reduce lipophilicity by the modification of the carboxylic acid tether linker (C). Therefore, we
investigated lipophilicity reduction by the modification of the anilide moiety having the highly
lipophilic trimethylsilyl group (A) and the central core scaffold (B) (Figure 1). Then, for the
compound possessing the optimized moieties of (A) and (B), we conducted optimization of the
linker (C), which focused on the improvement of in vitro activity to identify a clinical candidate.
In this study, we have reported the design, synthesis, and biological evaluation of novel RORγt
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inverse agonists and the discovery of [cisꢀ3ꢀ({(5R)ꢀ5ꢀ[(7ꢀfluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀ
indenꢀ5ꢀyl)carbamoyl]ꢀ2ꢀmethoxyꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀ
yl}carbonyl)cyclobutyl]acetic acid (compound 10) as a potent, selective and orally available
clinical candidate.
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Figure 1. Modification strategy of 1,2,3,4ꢀtetrahydroisoquinoline derivative 1
Result and Discussion
The synthesized compounds were initially evaluated using a RORγt binding inhibitory assay
and a RORγt reporter gene assay. In the binding assay (cellꢀfree system), our original ligand with
BODIPY label was used to evaluate the affinity of the test compounds to human RORγt by timeꢀ
resolved fluorescence resonance energy transfer (TRꢀFRET) technology. In the reporter gene
assay (cellꢀbased system), human Jurkat cells were used to evaluate the functional ability of the
test compounds to affect the transcriptional activity of RORγt by measurement of luciferase
activity. All compounds were also evaluated for lipophilicity (logD at pH 7.4) and LLE (=
reporter pIC – logD). The PK profiles of the compounds were analyzed by mouse cassetteꢀ
5
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dosing tests. The selectivity against ROR isoforms (α and β) was determined by reporter gene
assay in human Jurkat cells, and specificity against other subclass NRs was confirmed by
®
Invitrogen SelectScreen Cellꢀbased Nuclear Receptor Profiling in 20 kinds of receptors with
1
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agonist and antagonist modes. The in vivo PD efficacy of the selected compound was evaluated
by ILꢀ23ꢀinduced cytokine expression in mice. The in vivo pharmacological efficacy in an
animal disease model was evaluated in mouse EAE model.
Design Strategy and Validation of Lipophilicity Reduction. In order to reduce the
lipophilicity of compound 1, we conducted clogPꢀguided compound design and optimization by
focusing on the experimental logD and the reporter activityꢀbased LLE value. First, we
considered the modification of the anilide moiety which has a highly lipophilic trimethylsilyl
group. As we described in our previous report on the phenylglycinamide series, the quaternary
structure such as the trimethylsilyl group or tertꢀbutyl group was very important for functional
2
4
activity in the reporter gene assay. Therefore, we implemented three approaches to lower the
lipophilicity of the quaternary structure (Figure 2). First was the replacement of the
trimethylsilyl substituent by the conversion of silicon to carbon atom. As for the second
approach, conversion to a cyclized dimethylindan group which was promising in a previously
2
4
examined phenylglycinamide series. Third was the introduction of polar substituents such as
alkoxy group into the terminal substituent.
The results of anilide moiety modification are summarized in Table 1. Replacement of
trimethylsilyl group with a tertꢀbutyl group slightly decreased the reporter activity (Compound 3).
Although the clogP value was reduced by 0.75 by the replacement of silicon with carbon, the
experimental logD value remained slightly decreased and LLE was decreased. Conversion to a
dimethylindan group by the cyclization of tertꢀbutylphenyl group resulted in being still potent in
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vitro activity (compound 4). Interestingly, the compound had a significantly lowered logD value
and increased LLE value, whereas the clogP values between compound 3 and 4 were unchanged.
The introduction of an alkoxy group afforded the methoxy methyl analog 5, which showed an
additional decrease in the lipophilicity and a high LLE value. However, the compound led to the
diminishment of plasma exposure compared to the compound 1.
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In addition to modification of the anilide moiety, we attempted to change the core scaffold and
evaluated tetrahydronaphthyridine derivatives. We deemed that core scaffold exchange was
another strategy for lipophilicity reduction, and that replacement of the benzene ring in the center
core with a heterocycle, such as pyridine, was an attractive approach. In the course of studying
the tetrahydroisoquinoline series, we identified an active compound 2 (binding IC = 1.9 nM,
5
0
reporter gene IC = 20 nM) and succeeded in acquiring the crystal structure of the RORγt protein
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in complex with the compound (Figure 3). Using the crystal structure information, we
investigated changing the phenyl ring to a pyridine. In the ring change, there are three possible
positions to which a nitrogen atom can be introduced: the 5ꢀ, 7ꢀ, and 8ꢀpositions of the
tetrahydroisoquinoline ring. Among them, the 5ꢀ and 8ꢀpositions have a chance to interact with
the nearby amino acid residue Gln286 and the backbone carbonyl of the Phe377, respectively.
However, the backbone carbonyl of the Phe377 has already formed a hydrogen bond with the
NH of the anilide moiety. Thus we attempted to introduce nitrogen atom into the 5ꢀposition of
the tetrahydroisoquinoline ring, as we expected a hydrogen bonding interaction with Gln286
residue, and designed the tetrahydronaphthyridine scaffold as a novel bicyclic core.
As expected, the replacement of the tetrahydroisoquinoline core with tetrahydronaphthyridine
was tolerable. Compound 6 showed equipotent in vitro binding and reporter activity to the
corresponding tetrahydroisoquinoline analog 1, although the naphthyridine scaffold did not
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affect the lipophilicity despite the lower clogP value (1 vs 6). Interestingly, the combination of
the dimethylindan group as region (A) and tetrahydronaphthyridine core as region (B) improved
reporter activity and led to an improved LLE value (4 vs 7). High plasma exposure following
oral administration was also observed for compound 7. These results indicated that we
successfully lowered the lipophilicity of compound 1 without a significant reduction of in vitro
activity and plasma exposure by the modification of central core scaffold and anilide moiety. In
next step, we evaluated in vivo PD efficacy of compound 4 and 7 in a mouse IL23ꢀinduced gene
expression assay. As the result, both compounds showed potent and doseꢀdependent inhibition of
ILꢀ17A cytokine expression after oral administration. ED value of compound 4 and 7 is 5.4
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mg/kg and 2.0 mg/kg, respectively. Therefore, we selected compound 7 and conducted a final
optimization of the carboxylate tether moiety.
Figure 2. Modification strategy of anilide moiety
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Figure 3. SBDDꢀguided scaffold exchange and crystal structure RORγt protein in complex with
compound 2
Table 1. In vitro RORγt binding and reporter activities of derivatives modifying the anilide
moiety
Bindinga
IC50 (nM)
Reporter geneb
mouse PKg
d
e
f
Compound
R
X
clogP
logD
LLE
IC50 (nM)
AUC0-8h
F
95%CI]c
[95%CI]c
[
(ꢁg·h/mL)
(%)
3
.0
23
[19–29]
1
3
4
CH
CH
CH
5.80
5.05
5.04
3.96
3.77
3.37
3.68
3.54
3.76
6894
94
62
91
[2.4–3.8]
4
.0
49
[41–59]
2838
3442
[
3.4–4.7]
4.8
[4.2–5.5]
74
[52–110]
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2.5
2.1–2.9]
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[54–78]
5
6
7
CH
N
3.93
5.21
4.44
3.16
4.03
3.73
3.96
389
2884
2625
78
51
58
[
[
[
3
.2
17
[11–26]
4.04
3.39
2.5–4.0]
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
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
3.2
2.6–3.9]
45
[33–62]
N
a
b
Displacement assay of BODIPYꢀlabeled ligand with apo RORγt Inhibition of RORγt
c
transcriptional activity in human Jurkat cells 95% confidence intervals (CI) in duplicate (n = 2)
d
e
f
clogP value determined using Daylight software Measured at pH 7.4 LLE = reporter gene
g
pIC – logD The values shown as means of three determinations (1 mg/kg, po)
5
0
Linker Optimization in the Carboxylate Tether and Candidate Selection. We previously
revealed that the carboxylic acid was an essential functional group for plasma exposure and oral
2
2
bioavailability. Thus, we optimized the linker between the core and the carboxylate in
compound 7. We hypothesized that changing the flexible linker to a rigid structure would lead to
an improvement of in vitro potency through the reduction of the entropy loss of the carboxylic
acid group, which occurs through interaction with amino acid residues in the binding site. We
carefully designed rigid linker structures by calculating the clogP, and conducted the tether
optimization that accounted for the LLE values. The results are summarized in Table 2. First, we
introduced a cyclobutyl ring, and the both cisꢀ and transꢀisomers (8 and 9) showed slightly
increased reporter activity. In this transformation, LLE of cisꢀisomer 8 was improved since the
logD value did not change from the alkyl compound 7. Next, we adjusted the linker length and
synthesized compounds with an increase of one carbon atom. Comparable, or more potent,
reporter activities were observed for all compounds (10ꢀ13 vs 7). In particular, the cisꢀisomer of
a cyclobutane acetic acid derivative 10 demonstrated potent, nanomolarꢀlevel, reporter activity
with slightly increased lipophilicity, which resulted in a significant increase in the LLE value
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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
compared with compound 7. Moreover, compound 10 showed the highest plasma exposure and
oral bioavailability in the series at a dose of 1 mg/kg, po in mice (Cmax: 1562 ng/mL, Tmax: 2.3 h,
AUC0ꢀ8h: 10076 ꢁg·h/mL, MRT: 4.1 h, F: 96%, the values shown as means of three
determinations). In addition to cyclobutane, we investigated a heterocyclic azetidine ring as a
rigid linker without a stereocenter in the carboxylate tether. Although the urea derivative 14
retained moderate reporter activity, the LLE value decreased (7 vs 14). The basic azetidine
compound 15 resulted in a significant loss of in vitro activity. From these results, we selected
compound 10 as one of the most promising compounds in our chemical series for further in vivo
evaluation.
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
Table 2. In vitro RORγt binding and reporter activities of derivatives modifying the carboxylate
tether
Bindinga
IC50 (nM)
Reporter geneb
d
e
f
Compound
R
clogP
logD
LLE
IC50 (nM)
[95%CI]c
[
95%CI]c
3
.2
45
[33–62]
7
8
9
4.44
4.90
4.90
3.39
3.39
3.46
3.96
4.12
4.01
[
2.6–3.9]
2
.0
31
[24–39]
[
1.5–2.6]
2
.6
34
[28–42]
[2.0–3.4]
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1.9
1.6–2.2]
6.1
[3.7–10]
1
0
1
3.88
3.88
3.88
3.88
5.24
3.63
3.53
3.54
3.51
3.49
3.42
3.39
4.68
4.31
4.03
4.08
3.69
2.29
[
[
[
[
[
[
3
.1
14
[12–18]
1
2.6–3.6]
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
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
O
O
3.3
3.0–3.7]
29
[17–50]
OH
OH
1
1
1
1
2
3
4
5
3
.0
27
[14–53]
2.8–3.3]
4
.4
78
[66–93]
3.6–5.3]
6
.4
2100
[1500–3000]
4.5–9.3]
a
b
Displacement assay of BODIPYꢀlabeled ligand with apo RORγt Inhibition of RORγt
c
transcriptional activity in human Jurkat cells 95% confidence intervals (CI) in duplicate (n = 2)
d
e
f
clogP value determined using Daylight software Measured at pH 7.4 LLE = reporter gene
pIC – logD
5
0
Nuclear Receptor Selectivity and In Vivo Evaluation of Compound 10. To investigate the
selectivity against other ROR isoforms, we performed the reporter assay with human RORα and
RORβ. Compound 10 showed excellent selectivity (IC = >30000 nM, >5000ꢀfold) for these
5
0
ROR isoforms. We also tested the selectivity against other nuclear receptors in a panel of cellular
reporter assays (Invitrogen) and found the compound 10 was a selective RORγt inverse agonist
with >800ꢀfold selectivity over the other 20 nuclear receptors in both agonist and antagonist
25
mode.
In order to determine if our RORγt inverse agonists could modulate the cytokine production
through the suppression of differentiation and activation of Th17 and Th1/17 cells, we evaluated
the in vivo PD efficacy in the mouse ILꢀ23ꢀinduced cytokine expression model. Compound 10
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1
1
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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
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4
4
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5
5
5
5
5
5
5
5
5
6
showed robust and doseꢀdependent inhibition of ILꢀ17A expression after oral administration at
2
6
0
.3, 1 and 3 mg/kg bid. (ED = 0.5 mg/kg bid.). Since the ED80 value of the
80
tetrahydroisoquinoline derivative 1 in this model was 7.6 mg/kg bid., compound 10 showed more
potent efficacy than compound 1 owing to its stronger in vitro activity and better PK profile. In
addition to ILꢀ17A expression, compound 10 also demonstrated doseꢀdependent inhibition of
RORγt regulated gene expression such as ILꢀ17F, ILꢀ22, and IL23 receptor, but no effect against
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
2
7
IFNꢀγ expression which is not regulated by RORγt. Next, we evaluated the in vivo therapeutic
potential of compound 10 in a mouse EAE model which is one of the most commonly used
experimental models for the human inflammatory demyelinating disease, multiple sclerosis. The
oral administration of compound 10 at 1 and 3 mg/kg, bid. significantly reduced the development
of clinical symptoms with inhibition rates of 50% and 44%, respectively (Figure 4). These
results indicated that the oral administration of compound 10 protected against the development
of clinical symptoms caused by immunization with myelin oligodendrocyte glycoprotein (MOG)
peptide with maximum efficacy doses at 1 and 3 mg/kg. Furthermore, compound 10 showed in
vivo efficacy in activated T cell transfer mouse colitis model and ILꢀ23 induces mouse psoriasis
2
8
model by oral administration. From these results, we selected compound 10 as a clinical
candidate for the treatment of Th17ꢀdriven autoimmune diseases, and conducted clinical trials.
The clinical pharmacological data of compound 10 in healthy subjects will be published.
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0
1
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9
0
1
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 4. Effect of compound 10 on MOGꢀinduced EAE model
Compound 10 (0.3, 1 and 3 mg/kg, bid) or vehicle (0.5% methylcellulose) was orally
administered to mice for 28 days. (A) EAE clinical score and (B) cumulative EAE clinical score,
from day 0 to day 28. Data are presented as the mean ± S.E. of 4 (healthy control [normal]) or 12
*
(MOGꢀimmunized mice). p<0.025 (oneꢀtailed ShirleyꢀWilliams' test) vs vehicleꢀtreated MOGꢀ
immunized control group
Crystal Structure of RORγt in Complex with Compound 10. We succeeded in acquiring the
crystal structure of RORγt protein in complex with compound 10. An Xꢀray crystal structure of
1
0 bound to RORγt revealed the expected binding mode (Figure 5). The NH of the anilide
moiety and the carbonyl group of the carboxylate tether formed a hydrogen bond with the
backbone carbonyl of Phe377 and NH of Glu379, respectively. The methoxy group on the
central scaffold engaged in waterꢀmediated interactions with Arg364. As expected, the pyridine
nitrogen atom on the tetrahydronaphthyridine made an additional waterꢀmediated interaction
with Gln286. The fluoroꢀdimethylindanyl group packed hydrophobic pocket was surrounded by
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5
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5
5
6
lipophilic amino acid residues, such as Phe388. The cyclobutyl acetic acid portion extended out
towards the solvent exposed region, curling back towards the backbone amide of Gly380.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
9
0
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0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Figure 5. Crystal structure of RORγt protein in complex with compound 10
Chemistry.
The synthesis of the tetrahydroisoquinoline derivatives is described in Scheme 1. Preparation
of the anilines 17aꢀc was detailed in the Supporting Information. Amination of the key
29
intermediate 16 was achieved by the use of propylphosphonic acid anhydride cyclic trimer
(T3P) in the presence of stoichiometric amount of DMAP to obtain the racemic amides 18aꢀc.
Optically pure analogues 19aꢀc were prepared by chiral HPLC resolution of the corresponding
racemates. The target compounds (3ꢀ5) were synthesized from compounds 19aꢀc by deprotection
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of amino group and the subsequent reaction with glutaric anhydride. We previously reported that
2
2
the eutomer in the tetrahydroisoquionoline series was the (R)ꢀisomer. We determined the
absolute configuration of compounds 3ꢀ5 by confirming the absolute configuration of the key
intermediates 21aꢀc. The compounds 21aꢀc were prepared from the acylation of compounds 19aꢀ
c with pꢀbromobenzoic acid. The absolute stereochemistry of compounds 21aꢀc were determined
as the (R)ꢀconfiguration by single Xꢀray crystallographic analysis (see Supporting Information).
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
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
a
Scheme 1. Synthesis of Compounds 3–5
a
i
Reagents and conditions: (a) RꢀNH (17aꢀc), T3P, DMAP, Pr NEt, EtOAc, 80 °C, 39–86%; (b)
2
2
chiral HPLC, hexane/EtOH, 47–49% for 19aꢀc; (c) 4 N HClꢀEtOAc, rt, 85%–quant. for 20aꢀc;
i
(
d) glutaric anhydride, Et N, THF, 41–86% for 3ꢀ5 (e) pꢀbromobenzoic acid, HATU, Pr NEt,
3
2
DMF, 50–54%
The synthesis of the tetrahydronaphthyridine scaffold is shown in Scheme 2. Starting from 2ꢀ
methoxyꢀ6ꢀmethylpyridine 22, the hydroxymethylation of the benzylic position with
3
0
paraformaldehyde and subsequent Gabriel reaction afforded primary amine 25. The
tetrahydronaphthyridine scaffold was then constructed by the reaction of 25 with ethyl
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2
2
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2
2
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3
3
3
3
3
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3
3
3
3
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5
5
5
5
5
5
5
5
5
5
6
glyoxylate in acidic conditions and simultaneous deꢀmethylation, followed by Boc protection,
which yielded compound 27. OꢀSelective methylation was successfully conducted by the
reaction with methyl iodide in the presence of silver carbonate, followed by the hydrolysis of
ethyl ester 28 to give carboxylic acid 29.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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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
a
Scheme 2. Synthesis of Compound 29
a
Reagents and conditions: (a) nꢀBuLi, (HCHO) , THF, ꢀ78 °C–rt, 37%; (b) phthalimide, ADDP,
n
tributylphosphine, THF, rt, 69%; (c) hydrazine monohydrate, EtOH, reflux, 92%; (d) ethyl
glyoxylate, 4 N HClꢀCPME, EtOH, reflux, 82%; (e) (Boc) O, Et N, THF, H O, rt, 67%; (f) MeI,
2
3
2
Ag CO , THF, rt–50 °C, 89%; (g) 2 N LiOH, EtOH, THF, rt, quant.
2
3
Optically pure tetrahydronaphthyridine derivatives were synthesized as shown in Scheme 3.
The amidation of key intermediate 29 with aminodimethylindan 17b was performed by T3P to
obtain the racemic intermediate 30. The optical resolution of 30 was conducted by chiral HPLC
to afford two enantiomers (31 and 33). The target carboxylic acid compounds 10 and 38 were
prepared by acylation with half ester 35 and the subsequent deprotection of the tertꢀbutyl group;
The preparation of half ester 35 was detailed in the Supporting Information. The binding assay of
the compound 10 showed more potent RORγt binding activity than compound 38, which
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indicated that 10 was the eutomer. We confirmed the absolute configuration of compound 10 by
Xꢀray crystallographic analysis (Figure 6). A single crystal of compound 10 was successfully
obtained as a sulfate salt. Thus, the absolute configuration of the eutomer 10 was proved to be
the (R)ꢀconfiguration, and we also confirmed the cis geometry of the cyclobutyl carboxylic tether
moiety.
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
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
a
Scheme 3. Synthesis of Compound 10 and 38
a
i
Reagents and conditions: (a) 17b, T3P, DMAP, Pr NEt, EtOAc, 65 °C, 81%; (b) chiral HPLC,
2
hexane/EtOH, 46% for 31 and 44% for 33; (c) TFA, rt, 97% for 32 and quant. for 34; (d) 35,
i
HATU, Pr NEt, DMF, 95% for 36 and 83% for 37; (e) TFA, rt, 92% for 10 and 68% for 38
2
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0
1
2
3
4
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9
0
1
2
3
4
5
6
7
8
9
0
Figure 6. ORTEP of sulfate of compound 10, thermal ellipsoids are drawn at 20% probability.
(Sulfuric acid molecule and water were omitted.)
The synthesis of the tetrahydronaphthyridine derivative that possessed a trimethylsilylanilide
moiety is described in Scheme 4. The optically pure isomer 40 was synthesized by amidation
and optical resolution by chiral HPLC in the same manner as shown in Scheme 3. Deprotection
of the amino group was conducted carefully over a short time (3 minutes) owing to the instability
of the trimethylsilyl group in the acidic condition, and the chiral amine 41 was obtained. The
target compound 6 was synthesized from compounds 41 by the reaction with glutaric anhydride.
The absolute configuration of 41 was determined as the (R)ꢀconfiguration by Xꢀray
crystallographic analysis of the compound 41 tartrate (see Supporting Information). Therefore,
compound 6 was proven to be the (R)ꢀconfiguration.
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a
Scheme 4. Synthesis of Compound 6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
3
3
3
3
3
3
4
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5
5
5
6
0
1
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6
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9
0
1
2
3
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a
i
Reagents and conditions: (a) 3,5ꢀdifluoroꢀ4ꢀ(trimethylsilyl)aniline, T3P, DMAP, Pr NEt,
EtOAc, 65 °C, 80%; (b) chiral HPLC, hexane/EtOH, 46%; (c) TFA, rt, 95%; (d) glutaric
2
anhydride, Et N, THF, 46%
3
The synthetic route for the naphthyridine derivatives with a variety of carboxylate tether
moieties is described in Scheme 5. Preparation of the carboxylic acid tether units was detailed in
the Supporting Information. From the key chiral intermeditate 32, the reaction with glutaric
anhydride afforded the target compound 7. Acylation with carboxylic acids and the subsequent
deprotection gave the desired compounds (8, 9, 11ꢀ13, 15, 42ꢀ44, and 46). The urea derivative 14
was synthesized via urea formation using triphosgene and the deprotection of the benzyl group.
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a
Scheme 5. Synthesis of Compounds 7–9, 11, and 12–15
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a
Reagents and conditions: (a) glutaric anhydride, Et N, THF, rt, 38% for 7; (b) carboxylic acids,
HATU, Pr NEt, DMF, rt, 18ꢀ92% for 8, 9, 42ꢀ44, and 46; (c) triphosgene, pyridine, Pr NEt,
benzyl 2ꢀ(azetidinꢀ3ꢀyl)acetate, THF, 0 °C, 54% for 45; (d) TFA, rt, 47ꢀ96%; (e) Pd/C (10%), H2,
MeOH, rt, 56% for 12 and 85% for 14
3
i
i
2
2
Conclusion
In the course of the exploration of novel RORγt inverse agonists that are therapeutically
effective against various autoimmune diseases, we designed, synthesized and evaluated a series
of tetrahydronaphthyridine derivatives. In order to lower the lipophilicity of the
tetrahydroisoquinoline derivative 1, we conducted the modification of anilide moiety having the
trimethylsilyl group and the central core scaffold. Replacement of the trimethylsilylaniline unit
with the fluorineꢀsubstituted dimethylindan moiety was tolerable. SBDDꢀguided scaffold
exchange successfully identified a new core scaffold, tetrahydronaphthyridine. The combination
of the dimethylindan anilide moiety and tetrahydronaphthyridine core significantly lowered the
value of logD and increased the value of LLE, with a favorable PK profile. Subsequent
optimization of the carboxylic acid tether with consideration of the LLE values led to the
discovery of [cisꢀ3ꢀ({(5R)ꢀ5ꢀ[(7ꢀfluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl]ꢀ2ꢀ
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methoxyꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl}carbonyl)cyclobutyl]acetic acid TAKꢀ828F
(
10). The compound showed potent RORγt inverse agonistic activity (reporter IC = 6.1 nM)
50
and excellent ROR isoforms selectivity (>5000ꢀfold selectivity against human RORα and
1
1
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0
RORβ) without exhibiting significant activity against other nuclear receptors (>800ꢀfold
selectivity for RORγt over the other 20 nuclear receptors in both agonist and antagonist mode).
In animal studies, compound 10 exhibited robust and doseꢀdependent inhibition of ILꢀ17A
cytokine level in a mouse IL23ꢀinduced gene expression assay by oral treatment (ED80 = 0.5
mg/kg, bid.). Furthermore, oral administration of compound 10 significantly reduced the
development of clinical symptoms in mouse EAE model. On the basis of the potent in vivo
efficacy and favorable preclinical PK profile, compound 10 was selected as a clinical compound
for the treatment of Th17ꢀdriven autoimmune diseases.
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Experimental section
Chemistry
Reagents and solvents were obtained from commercial sources and used without further
purification. Reaction progress was determined by thin layer chromatography (TLC) analysis on
Merck Kieselgel 60 F254 plates or Fuji Silysia NH plates. Silica gel column chromatography
was performed on PurifꢀPack (SI or NH, SHOKO SCIENTIFIC). Proton nuclear magnetic
resonance (1H NMR) spectra were recorded on Bruker Ultra Shieldꢀ300 (300 MHz) instruments.
Chemical shifts are given in parts per million (ppm) with tetramethylsilane as an internal
standard. Abbreviations are used as follows: s = singlet, d = doublet, t = triplet, q = quartet, m =
multiplet, dd = doublets of doublet, br = broad. Coupling constants (J values) are given in hertz
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(
Hz). LC–MS analysis was performed on a Shimadzu LCꢀ20AD separations module or Agilent
200 series, operating in ESI (+ or ꢀ) ionization mode. Analytes were eluted using a linear
gradient of 0.05% TFA containing H O/CH CN or 0.01% TFA containing H O/CH CN or 5 mM
1
2
3
2
3
ammonium acetate containing H O/CH CN or 10 mM NH HCO containing H O/CH CN or
2
3
4
3
2
3
0
.01% heptafluorobutyric acid /1.0% isopropyl alcohol containing H O/CH CN mobile phase.
2 3
Preparative HPLC was performed on a Waters 2525 separations module (Lꢀcolumn2 ODS (20
x150 mm I.D., CERI, Japan); MS spectra were recorded using a Waters ZQ2000 with
electrospray ionization or on a GILSON system, equipped with a Lꢀcolumn2 ODS (20 x150 mm
I.D., CERI, Japan) or on Waters Deltaprep 300 system or on Shimadzu 10A VP system. Samples
were eluted using a linear gradient of 0.1% TFA in H O/CH CN or 10 mM NH HCO in H O
2
3
4
3
2
/
CH CN, or H O/CH CN. Purity data were collected by a HPLC with Corona CAD (Charged
3 2 3
Aerosol Detector), Nano quantity analyte detector (NQAD), or photo diode array detector. The
column was a Capcell Pak C18AQ (50 mm x 3.0 mm I.D., Shiseido, Japan) or Lꢀcolumn 2 ODS
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(
30 mm x 2.0 mm I.D., CERI, Japan) with a temperature of 50 °C and a flow rate of 0.5 mL/min.
Mobile phase A and B under a neutral condition were a mixture of 50 mM ammonium acetate,
H O and CH CN (1:8:1, v/v/v) and a mixture of 50 mM ammonium acetate and CH CN (1:9,
2
3
3
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0
v/v), respectively. The ratio of mobile phase B was increased linearly from 5% to 95% over 3
min, 95% over the next 1 min. Mobile phase A and B under an acidic condition were a mixture
of 0.2% formic acid in 10 mM ammonium formate and 0.2% formic acid in CH CN,
3
respectively. The ratio of mobile phase B was increased linearly from 14% to 86% over 3 min,
8
6% over the next 1 min. The purity of all test compounds was assessed by analytical HPLC
>95%).
5ꢀ((1R)ꢀ1ꢀ((4ꢀtertꢀButylꢀ3,5ꢀdifluorophenyl)carbamoyl)ꢀ6ꢀmethoxyꢀ3,4ꢀdihydroisoquinolinꢀ
(
2
(1H)ꢀyl)ꢀ5ꢀoxopentanoic acid (3) A mixture of 20a (100 mg, 0.27 mmol), dihydroꢀ2Hꢀpyranꢀ
,6(3H)ꢀdione (45.7 mg, 0.40 mmol), TEA (0.093 ml, 0.67 mmol), and THF (2.0 mL) was
2
stirrred at room temperature for 3 h. The mixture was concentrated in vacuo. The residue was
purified by column chromatography (silica gel, eluted with 50%–100% EtOAc in hexane) to give
1
3
(52.8 mg, 0.108 mmol, 41%) as a colorless amorphous solid. H NMR (300 MHz, DMSOꢀd )
6
δ 0.30 (9H, s), 1.63 – 1.82 (2H, m), 2.16 – 2.32 (3H, m), 2.35 – 2.48 (1H, m), 2.51 – 2.63 (1H,
m), 2.70 – 2.88 (1H, m), 3.04 – 3.20 (1H, m), 3.42 – 3.58 (1H, m), 3.73 (3H, s), 5.58 – 5.65 (1H,
m), 6.76 – 6.87 (2H, m), 7.13 – 7.26 (2H, m), 7.46 (1H, d, J = 9.4 Hz), 10.76 (1H, s), 12.03 (1H,
25
brs). LC/MS m/z 489.2 (M + H). Optical rotation: [α] –5.7 (c 0.252, MeOH). HPLC purity
D
9
7.8%
5
ꢀ((1R)ꢀ1ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ2(1H)ꢀyl)ꢀ5ꢀoxopentanoic acid (4) Compound 4 was prepared in a manner
similar to that described for 3, using compound 20b in 86% yield as a colorless amorphous solid.
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1
H NMR (300 MHz, DMSOꢀd ) δ 1.28 (6H, d, J = 1.5 Hz), 1.63 – 1.81 (2H, m), 1.86 (2H, t, J =
6
7
.4 Hz), 2.20 – 2.32 (2H, m), 2.35 – 2.62 (2H, m), 2.69 – 2.90 (3H, m), 3.04 – 3.19 (1H, m), 3.45
– 3.60 (1H, m), 3.72 (3H, s), 3.92 – 4.08 (1H, m), 5.53 – 5.69 (1H, m), 6.75 – 6.86 (2H, m), 7.12
7.27 (2H, m), 7.41 – 7.52 (1H, m), 10.35 – 10.48 (1H, m), 12.05 (1H, brs). LC/MS m/z 481.2
0
1
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3
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5
6
7
8
9
0
1
2
3
4
5
6
7
8
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9
0
–
25
(M – H). Optical rotation: [α] +2.7 (c 0.254, MeOH). HPLC purity 98.4%
D
5ꢀ((1R)ꢀ1ꢀ((3,5ꢀDifluoroꢀ4ꢀ(1ꢀmethoxyꢀ2ꢀmethylpropanꢀ2ꢀyl)phenyl)carbamoyl)ꢀ6ꢀmethoxyꢀ
,4ꢀdihydroisoquinolinꢀ2(1H)ꢀyl)ꢀ5ꢀoxopentanoic acid (5) Compound 5 was prepared in a
3
manner similar to that described for 3, using compound 20c in 86% yield as a colorless
1
amorphous solid. H NMR (300 MHz, DMSOꢀd ) δ 1.28 (6H, d, J = 1.5 Hz), 1.63 – 1.81 (2H,
6
m), 1.86 (2H, t, J = 7.4 Hz), 2.20 – 2.32 (2H, m), 2.35 – 2.62 (2H, m), 2.69 – 2.90 (3H, m), 3.04
–
–
3.19 (1H, m), 3.45 – 3.60 (1H, m), 3.72 (3H, s), 3.92 – 4.08 (1H, m), 5.53 – 5.69 (1H, m), 6.75
6.86 (2H, m), 7.12 – 7.27 (2H, m), 7.41 – 7.52 (1H, m), 10.35 – 10.48 (1H, m), 12.05 (1H,
2
5
brs). LC/MS m/z 517.3 (M – H). Optical rotation: [α] –5.7 (c 0.253, MeOH). HPLC purity
D
9
6.8%
5
ꢀ((1R)ꢀ1ꢀ((3,5ꢀDifluoroꢀ4ꢀ(trimethylsilyl)phenyl)carbamoyl)ꢀ6ꢀmethoxyꢀ3,4ꢀ
dihydroisoquinolinꢀ2(1H)ꢀyl)ꢀ5ꢀoxopentanoic acid (6) Compound 6 was prepared in a manner
similar to that described for 3, using compound 41 in 46% yield as a colorless amorphous solid.
1
H NMR (300 MHz, CDCl ) δ 0.27 (9H, s), 2.06 – 2.20 (2H, m), 2.43 (2H, t), 2.55 (1H, ddd, J =
3
14.5, 9.1, 5.5 Hz), 2.93 – 3.10 (2H, m), 3.22 – 3.34 (1H, m), 3.92 (3H, s), 3.94 – 4.00 (1H, m),
4
.07 – 4.17 (1H, m), 5.83 (1H, s), 6.63 (1H, d, J = 8.3 Hz), 6.73 – 6.81 (2H, m), 7.74 (1H, d, J =
25
8
.7 Hz), 9.94 (1H, s). LC/MS m/z 506.4 (M + H). Optical rotation: [α]
+93.2 (c 0.253,
D
MeOH). HPLC purity 96.5%
5
ꢀ((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀmethoxyꢀ7,8ꢀ
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dihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)ꢀ5ꢀoxopentanoic acid (7) Compound 7 was prepared in a
manner similar to that described for 3, using compound 32 in 38% yield as a colorless
1
amorphous solid. H NMR (300 MHz, CDCl ) δ 1.28 (6H, d, J = 1.9 Hz), 1.85 (2H, t, J = 7.4
3
1
1
1
1
1
1
1
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Hz), 2.02 – 2.16 (3H, m), 2.45 (2H, t, J = 6.4 Hz), 2.57 (1H, dt, J = 15.0, 7.4 Hz), 2.73 – 2.91
(3H, m), 2.96 – 3.20 (2H, m), 3.91 (3H, s), 3.94 – 4.00 (2H, m), 5.95 (1H, s), 6.63 (1H, d, J = 8.3
Hz), 6.90 (1H, s), 7.03 (1H, d, J = 12.1 Hz), 7.61 (1H, d, J = 8.7 Hz), 9.41 (1H, s). LC/MS m/z
2
5
4
84.3 (M + H). [α] +102.5 (c 0.253, MeOH). HPLC purity 100%
D
cisꢀ3ꢀ(((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀmethoxyꢀ
7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)carbonyl)cyclobutanecarboxylic acid (8) To a stirred
solution of 32 (100 mg, 0.27 mmol), N,N'ꢀdiisopropylethylamine (0.093 mL, 0.54 mmol),
cyclobutaneꢀ1,3ꢀdicarboxylic acid (58.5 mg, 0.41 mmol) in DMF (2.0 mL) was added HATU
(124 mg, 0.32 mmol) at room temperature. The mixture was stirred at room temperature for 5 h.
The mixture was quenched with water and extracted with EtOAc. The organic layer was
separated, washed with water and brine, dried over MgSO and concentrated in vacuo. The
4
residue was purified by column chromatography (silica gel, eluted with 20%–90% EtOAc in
1
hexane) and prep. HPLC to give 8 (44.5 mg, 33%) as colorless amorphous solid. H NMR (300
MHz, DMSOꢀd ) δ 1.29 (6 H, s), 1.87 (2 H, t, J = 7.4 Hz), 2.23 – 2.45 (4 H, m), 2.71 – 2.91 (3
6
H, m), 2.92 – 3.09 (2 H, m), 3.37 – 3.51 (1 H, m), 3.69 – 3.80 (1 H, m), 3.82 (3 H, s), 3.89 – 4.02
(1 H, m), 5.70 (1 H, s), 6.72 (1 H, d, J = 8.3 Hz), 7.11 – 7.31 (2 H, m), 7.78 (1 H, d, J = 8.7 Hz),
25
1
0.53 (1 H, s), 12.18 (1 H, brs). LC/MS m/z 494.2 (M – H). LC/MS m/z 484.3 (M + H). [α]
D
+114.7 (c 0.250, MeOH). HPLC purity 98.7%
transꢀ3ꢀ(((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀmethoxyꢀ
7
,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)carbonyl)cyclobutanecarboxylic acid (9) Compound
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6
9
was prepared in a manner similar to that described for 8, using transꢀcyclobutaneꢀ1,3ꢀ
1
dicarboxylic acid in 18% yield as a colorless amorphous solid. H NMR (300 MHz, DMSOꢀd )
6
δ 1.29 (6 H, s), 1.81 – 1.93 (2 H, m), 2.28 – 2.47 (4 H, m), 2.77 – 3.03 (5 H, m), 3.44 – 3.60 (1
0
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H, m), 3.60 – 3.74 (1 H, m), 3.82 (3 H, s), 3.86 – 3.99 (1 H, m), 5.72 (1 H, s), 6.72 (1 H, d, J =
8
.7 Hz), 7.10 – 7.29 (2 H, m), 7.79 (1 H, d, J = 8.7 Hz), 10.54 (1 H, s), 12.26 (1 H, brs). LC/MS
25
m/z 494.2 (M – H). [α] +110.1 (c 0.255, MeOH). HPLC purity 99.2%
D
cisꢀ3ꢀ({(5R)ꢀ5ꢀ[(7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl]ꢀ2ꢀmethoxyꢀ
7
,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl}carbonyl)cyclobutyl]acetic acid (10) Trifluoroacetic
acid (5 mL) was added to 36 (288 mg, 0.51 mmol) at room temperature. After being stirred at
room temperature for 30 min, the reaction mixture was concentrated in vacuo. The evaporation
was repeated after the addition of toluene. The residual oil was purified by column
chromatography (silica gel, eluted with 30%–100% EtOAc in hexane) to give 10 (238 mg, 92%)
1
as a colorless amorphous solid. H NMR (300 MHz, CDCl ) δ 1.31 (6H, s), 1.88 (2H, t, J = 7.4
3
Hz), 2.05 – 2.18 (2H, m), 2.44 – 2.56 (4H, m), 2.62 – 2.77 (1H, m), 2.84 (2H, t, J = 7.4 Hz), 2.88
–
3.05 (2H, m), 3.31 (1H, quin, J = 8.9 Hz), 3.67 – 3.78 (1H, m), 3.80 – 3.88 (1H, m), 3.90 (3H,
s), 5.97 (1H, s), 6.63 (1H, d, J = 8.3 Hz), 7.04 (1H, s), 7.10 (1H, d, J = 11.7 Hz), 7.46 (1H, d, J =
25
8
.7 Hz), 9.21 (1H, s). LC/MS m/z 510.2 (M + H). Optical rotation: [α]
+113.5 (c 0.251,
D
MeOH). HPLC purity 100%
(transꢀ3ꢀ(((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀ
methoxyꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)carbonyl)cyclobutyl)acetic acid
(11)
Compound 11 was prepared in a manner similar to that described for 10 in 96% yield as a
1
colorless amorphous solid. H NMR (300 MHz, DMSOꢀd ) δ 1.32 (6H, s), 1.89 (2H, t, J = 7.4
6
Hz), 2.08 – 2.19 (2H, m), 2.50 – 2.69 (4H, m), 2.70 – 2.89 (3H, m), 2.96 (2H, d, J = 4.2 Hz),
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Page 29 of 54
Journal of Medicinal Chemistry
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9
3
.34 – 3.53 (1H, m), 3.60 – 3.84 (2H, m), 3.91 (3H, s), 6.01 (1H, s), 6.64 (1H, d, J = 8.7 Hz),
.04 (1H, s), 7.11 (1H, d, J = 11.7 Hz), 7.46 (1H, d, J = 8.3 Hz), 9.19 (1H, s). LC/MS m/z 508.2
7
25
(M – H). [α] +109.0 (c 0.252, MeOH). HPLC purity 99.0%
D
1
1
1
1
1
1
1
1
1
1
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5
6
0
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0
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0
cisꢀ3ꢀ(2ꢀ((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀmethoxyꢀ
7
,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)ꢀ2ꢀoxoethyl)cyclobutanecarboxylic acid (12)
A
mixture of 43 (108 mg, 0.18 mmol) and 10% palladium on carbon (50% wet, 20 mg) in THF (2.0
mL) was hydrogenated under balloon pressure at room temperature for 3 h. The catalyst was
removed by filtration, washed with THF and the filtrate was concentrated in vacuo. The residue
was purified by column chromatography (diolꢀsilica gel, eluted with 20%–90% EtOAc in
1
hexane) to give 12 (51.0 mg, 56%) as a colorless amorphous solid. H NMR (300 MHz, DMSOꢀ
d6) δ 1.29 (6H, s), 1.70 – 1.92 (4H, m), 2.17 – 2.38 (3H, m), 2.52 – 2.68 (1H, m), 2.78 – 3.00
(4H, m), 3.20 – 3.41 (2H, m), 3.82 (4H, s), 3.90 – 4.04 (1H, m), 5.71 (1H, s), 6.71 (1H, d, J = 8.3
Hz), 7.11 – 7.30 (2H, m), 7.72 – 7.84 (1H, m), 10.39 – 10.53 (1H, m), 11.99 (1H, brs). LC/MS
25
m/z 508.1 (M – H). [α] +105.8 (c 0.252, MeOH). HPLC purity 98.1%
D
transꢀ3ꢀ(2ꢀ((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀ
methoxyꢀ7,8ꢀdihydroꢀ1,6ꢀnaphthyridinꢀ6(5H)ꢀyl)ꢀ2ꢀoxoethyl)cyclobutanecarboxylic
acid
(13) Compound 13 was prepared in a manner similar to that described for 10, using compound
1
4
4 in 78% yield as a colorless amorphous solid. H NMR (300 MHz, DMSOꢀd ) δ 1.28 (6H, s),
6
1.83 – 1.96 (4H, m), 2.21 – 2.32 (3H, m), 2.60 – 2.79 (2H, m), 2.81 – 2.96 (3H, m), 2.97 – 3.11
2H, m), 3.78 – 3.91 (4H, m), 3.97 – 4.11 (1H, m), 5.71 (1H, s), 6.72 (1H, d, J = 8.3 Hz), 7.12 –
.28 (2H, m), 7.72 – 7.82 (1H, m), 10.44 (1H, s), 12.01 (1H, s). LC/MS m/z 508.1 (M – H).
(
7
25
[α] +91.2 (c 0.250, MeOH). HPLC purity 96.8%
D
(1ꢀ(((5R)ꢀ5ꢀ((7ꢀFluoroꢀ1,1ꢀdimethylꢀ2,3ꢀdihydroꢀ1Hꢀindenꢀ5ꢀyl)carbamoyl)ꢀ2ꢀmethoxyꢀ7,8ꢀ
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