Balancing potency and basicity by incorporating fluoropyridine moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitor that affords robust and sustained central Aβ reduction

Article abstract of DOI:10.1016/j.ejmech.2021.113270

β-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the treatment of Alzheimer's disease (AD). In this report, we describe the discovery of BACE1 inhibitors with a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine rings, which showed increased biochemical and cellular potency, along with reduced basicity on the amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained cerebrospinal fluid (CSF) Aβ reduction in dog. The crystal structure of compound 11 bound to BACE1 confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.

Full text of DOI:10.1016/j.ejmech.2021.113270

European Journal of Medicinal Chemistry 216 (2021) 113270
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Balancing potency and basicity by incorporating fluoropyridine
moieties: Discovery of a 1-amino-3,4-dihydro-2,6-naphthyridine
BACE1 inhibitor that affords robust and sustained central Ab reduction
,
*
,
,
Kenji Nakahara a 1, Yasunori Mitsuoka a 1, Satoshi Kasuya a, Takahiko Yamamoto a,
Shiho Yamamoto a, Hisanori Ito a, Yasuto Kido b, Ken-ichi Kusakabe a
,
**
a Discovery Research Laboratory for Core Therapeutic Areas, Shionogi Pharmaceutical Research Center, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka, 561-
0825, Japan
b Research Laboratory for Development, Shionogi Pharmaceutical Research Center, 1-1 Futaba-cho 3-chome, Toyonaka, Osaka, 561-0825, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
b
-Site amyloid precursor protein cleaving enzyme 1 (BACE1) has been pursued as a prime target for the
Received 18 October 2020
Received in revised form
22 January 2021
Accepted 1 February 2021
Available online 7 February 2021
treatment of Alzheimer’s disease (AD). In this report, we describe the discovery of BACE1 inhibitors with
a 1-amino-3,4-dihydro-2,6-naphthyridine scaffold. Leveraging known inhibitors 2a and 2b, we designed
the naphthyridine-based compounds by removing a structurally labile moiety and incorporating pyridine
rings, which showed increased biochemical and cellular potency, along with reduced basicity on the
amidine moiety. Introduction of a fluorine atom on the pyridine culminated in compound 11 which had
improved cellular activity as well as further reduced basicity and demonstrated a robust and sustained
cerebrospinal fluid (CSF) Ab reduction in dog. The crystal structure of compound 11 bound to BACE1
confirmed van der Waals interactions between the fluorine on the pyridine and Tyr71 in the flap.
© 2021 Elsevier Masson SAS. All rights reserved.
Keywords:
BACE1
b
-secretase
Inhibitor
Naphthyridine
Basicity
X-ray analysis
1. Introduction
effects, thus leaving an urgent unmet need to develop disease-
modifying therapies [1].
Alzheimer’s disease (AD) is a progressive neurodegenerative
disease and the most common cause of dementia. The clinical
symptoms of AD are progressive memory loss, learning impair-
ment, and behavioral and psychiatric disturbances. Currently
available treatments using acetylcholinesterase inhibitors and the
The pathological hallmarks of AD patients are extracellular
amyloid- (A ) plaques and intraneuronal neurofibrillary tangles
consisting of aggregates of phosphorylated tau protein [1]. As A
levels become abnormal before the accumulation of tau proteins
and A 42 oligomers, a toxic component of A , inducing tau
hyperphosphorylation, A is thought to be a causative factor in the
development of AD [2]. Amyloid precursor protein (APP) is pro-
cessed sequentially by -site amyloid precursor protein cleaving
enzyme 1 (BACE1, also known as -secretase) and -secretase,
leading to the formation of A peptides [3]. Because the clinical use
of -secretase inhibitors was abandoned in the clinic due to the
mechanism-based toxicities arising from inhibition of Notch pro-
cessing, the inhibition of BACE1 remained as a promising thera-
peutic approach for the development of disease-modifying
therapies for AD [4].
The large size of the catalytic site in BACE1 has hampered the
search for BACE1 inhibitors. Initial investigations identified pepti-
domimetic inhibitors with high molecular weight and large polar
surface area, which prevented both cellular and brain penetration
b
b
b
b
b
N-methyl-D-aspartate receptor antagonist do not halt or even delay
b
the disease progression and only provide temporary symptomatic
b
b
g
b
Abbreviations: Alzheimer’s disease, AD; amyloid-
tein, APP; area under the concentration-time curve, AUC;
b
, A
b
; amyloid precursor pro-
-site amyloid precursor
g
b
protein cleaving enzyme 1, BACE1; cerebrospinal fluid, CSF; N,N-dimethylforma-
mide, DMF; efflux ratio, ER; 1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo
[4,5-b]pyridinium 3-oxid hexafluorophosphate, HATU; homogeneous time-
resolved fluorescence, HTRF.
* Corresponding author.
** Corresponding author.
E-mail addresses: kenji.nakahara@shionogi.co.jp (K. Nakahara), ken-ichi.
kusakabe@shionogi.co.jp (K.-i. Kusakabe).
1
K$N and Y.M contributed equally to this work.
https://doi.org/10.1016/j.ejmech.2021.113270
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
[4]. Identification of amidine-based inhibitors mitigated these is-
sues and consequently led to the discovery of several clinical
compounds, such as verubecestat (MK-8931) [5], elenbecestat
(Eꢀ2609) [6], and atabecestat (JNJ-54861911) [7]. During the course
of the research, controlling the basicity of the amidine moiety
together with retaining the potency was key to the development of
centrally active inhibitors, as they tend to show high basicity being
associated with poor brain penetration due to recognition of the P-
gp transporter [8]. Recently, we identified dihydro-1,3-oxazine 1
Having identified promising pyridine-fused amidine scaffolds
(1-amino-3,4-dihydro-2,6-naphthyridines), such as 5 and 6, that
showed cellular IC50 values of <2 nM, we optimized the scaffolds to
reduce basicity. To this end, we examined incorporation of a fluo-
rine atom on the pyridine ring. Compound 10 with a fluorine at the
1-positon decreased both enzymatic and cellular potency, whereas
introduction of a fluorine at the 4-position yielded compounds with
improved cellular potency (8, 9, and 11). As expected, further
reduction of pKa was realized by the incorporation of a fluorine on
the pyridine; compounds 8 and 11 with a fluorine at the 4-position
showed reduced pKa values of 7.6 and 8.2, relative to those in non-
substituted 5 and 6 (pKa ¼ 9.5 and 9.0, respectively), although we
observed a difference in the extent to which the fluorine atom at
the 4-position reduced basicity (5 vs 8; 6 vs 11). Although incor-
poration of a fluorine at the 1-position achieved the lowest pKa of
7.3, it was accompanied by loss in potency. As expected, the less
basic profile translated into reduced P-gp efflux. Indeed, non-
fluorine substituted 6 with a pKa of 9.0 had a high P-gp efflux ra-
tio (ER) of 37 in LLC-PK cells expressing the human MDR1 gene.
Conversely, the corresponding fluorine analogue 11 with a pKa of
8.2 displayed a reduced, but still significant P-gp ER of 13; com-
pound 8 with a further reduced pKa of 7.3 also exhibited the same P-
gp ER value of 13. Unfortunately, all the less basic compounds of 8,
10, and 11 were found to be strong hERG inhibitors with percent
which led to significant Ab reduction in vivo at a low dose through
mitigation of the P-gp efflux resulting from moderate basicity
(pKa ¼ 7.2). The fluorine at the 5-position in 1 reduced the basicity
along with increasing the potency via stabilization of an active
conformation (Fig. 1) [9]. Herein we report an alternative approach
to controlling pKa along with increasing potency, starting with
fused inhibitors 2a and 2b [10a], which led to the identification of
novel 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitors
[11]. Optimization efforts culminated in an orally efficacious com-
pound 11, which showed robust and sustained Ab reduction in dog.
2. Results and discussion
We have reported benzene-fused amidines 2a and 2b with
moderate to slightly high pKa values of 7.7 and 7.2 in the patent
literature [10a], which had comparable potency relative to non-
substituted dihydro-1,3-oxazine 3 [9]. Because diversification of
dihydro-1,3-oxazine head groups, as in 1 and 3, was considered in
our optimization strategy, compounds 2a and 2b served as prom-
ising scaffolds for the design of structurally differentiated in-
hibitors. The potential concern of racemization for 2 did occur
when a racemic close analogue was subjected to chiral chroma-
tography, and the enantiomers racemized again. Therefore, our
effort started with removal of the labile N,O-acetal substructure and
replacement of the phenyl moiety on the amidine with a variety of
pyridine rings, in order to control basicity (Fig. 2).
Table 1 summarizes the key SAR for the newly designed com-
pounds. Clearly, incorporation of pyridine rings impacted potency.
Compound 4 with a nitrogen at the 1-position, with the atom
shown in Table 1, exhibited modest biochemical and cellular po-
tency, whereas compounds with nitrogen at other positions, such
as compounds 5, 6, and 7, were found to increase potency relative to
4 and the lead 2a and 2b. Of these, compounds 5 and 6 with ni-
trogen atoms at the 2- and 3-positions respectively, significantly
improved BACE1 activity associated with excellent cellular potency.
Introduction of the pyridine rings successfully reduced basicity for
compounds 4e7 (pKa ¼ 9.0 to 9.8) when compared with the cor-
responding benzene analogue (pKa ¼ 10.8) reported by scientists at
Roche [10b], although the pKa values were still too high to gain
brain penetration due to the potential of being P-gp substrates,
according to our previous research and other reports [8,9].
inhibitions at 5 mM of 72%, 92%, and 88% in an automated patch
clamp assay, which were comparable to those of non-substituted 5
and 6 (83% and 85%, respectively). As shown in a previous paper
[12a], a pKa reduction of less than 7 seems to be required to achieve
preferable profiles for P-gp efflux and hERG inhibition, along with
modifications at the amide substituent of the cyanopyridine group.
Nevertheless, a representative compound of 11 was evaluated
in vivo for the ability to reduce A
b in the brain. Table 2.
Before measuring A reduction in vivo, we profiled the phar-
b
macokinetics properties for 11 in rat and dog, together with
measuring metabolic stability. In rat, clearance was high (45 mL/
min/kg) due to poor microsomal stability (22% remaining after
30 min). Combined with high tissue distribution (14 L/kg), this
translated into a low maximum concentration (Cmax) and area
under the concentration-time curve (AUC). Reflecting high P-gp
efflux, 11 exhibited a low total brain-to-plasma ratio (Kp) of 0.28. In
dog, improved microsomal stability (93% remaining after 30 min)
led to good PK profiles with a relatively high Cmax and AUC value
following oral administration (1 mg/kg), which prompted us to
advance to PK/PD study. As shown in Table 3, compound 11
demonstrated robust and sustained reduction of total Ab in the
cerebrospinal fluid (CSF) by 49% and 47% at the 10 and 24 h time
points, respectively, despite the low CSF-to-unbound plasma ratios
of 0.070 and 0.083. The significant and sustained Ab reduction was
rationalized by the drug concentrations in the CSF at 10 and 24 h,
where 11 exceeded the cellular IC50 value by 3.3- and 3.0-fold.
Fig. 1. Dihydro-1,3-oxazine BACE1 inhibitors 1 and 3 and lead compounds 2a and 2b
2

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Fig. 2. Design of 1-amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitors.
Table 1
and the nitrile substituent projects into the S3 pocket. Interestingly,
the fluorine on the pyridine in 11 engages in van der Waals in-
teractions with Tyr71 located in the flap. The corresponding
hydrogen in 6 is likely to be involved in the same interaction, giving
comparable activities to 11. In contrast, the nitrogen atom at the
same position on the pyridine in 7 may disrupt this favorable
interaction, explaining why 7 showed reduced potency relative to
the corresponding hydrogen or fluorine analogues, such as 6 and 11.
We also reasoned that the nitrogen and fluorine atoms at the 1-
position (shown in Table 1) in 4 and 10, respectively, would be
proximal to the catalytic site and consequently cause electrostati-
cally unfavorable interactions, resulting in reduced potency [13][
[15]]. [10],
Exploration of pyridine-fused amidines.
IC50 (nM)a
BACE1b
Cellular Abc
pKa
9.8
d
compd
4
394
26
3. Chemistry
5
8.1
1.3
9.5
Synthesis of compound 4 started with commercially available
acetophenone 12 (Scheme 1). The ketone moiety in 12 was con-
verted to the corresponding boronate 14 via Wittig reaction and
subsequent Miyaura borylation reaction [14]. Cross-coupling of 14
with 3-bromo-2-cyanopyridine gave compound 15, which was
then heated with ammonium chloride in the presence of trimethyl
aluminum to afford 16. Cyclization of the amidine 16 furnished a 1-
amino-3,4-dihydro-2,6-naphthyridine scaffold followed by nitra-
tion of the fluorobenzene gave 17. The amide moiety in 18 was
successfully introduced through amidation with 5-cyanopicolinic
acid, following Boc protection of 17 and subsequent reduction of
the nitro group. Finally, deprotection of the Boc group in 18 under
acidic condition furnished compound 4.
The synthesis of compound 5 is shown in Scheme 2. Homolo-
gation of 20-fluoroacetophenone 12 was achieved by nucleophilic
addition of 3-cyano-4-methylpyridine and subsequent elimination
of water to afford amide 19, which was then dehydrated with tri-
fluoroacetic anhydride to yield nitrile 20. According to the methods
described in Scheme 1, compound 5 was prepared from interme-
diate 20 as shown in Scheme 2. The synthesis of compounds 6 and 7
also followed the procedures described in Scheme 1 with some
modifications to Suzuki cross-coupling conditions of boronate 14
(Schemes 3 and 4). The synthesis of compounds 8e11 started with
Suzuki cross-coupling reaction of commercially available reagents
33, 39, and 45 with boronate 14 (Schemes 5e7). Intermediates 34,
40, and 46 were converted to the final compounds 8e11, according
to the procedures described in Scheme 1. Chiral separation of
compound 9 using supercritical fluid chromatography (SFC) affor-
ded the desired enantiomer 11.
6
7
8
21
54
12
1.5
9.0
9.4
7.6
4.1
0.25
9
14
0.36
11
NT
7.3
10
118
11 (chiral, S)
9.1
0.41
8.2
a
Values represent the mean values of at least two determinations.
Biochemical homogeneous time-resolved fluorescence (HTRF)-based assay.
IC50 determined by measuring the levels of secreted A 40 in human APP-
b
c
b
transfected human neuroblastoma (SH-SY5Y) cells via an HTRF-based assay.
d
pKa determined by capillary electrophoresis. NT ¼ not tested.
The X-ray cocrystal structure of 11 bound to BACE1 was solved at
2.3 Å resolution (PDB code 7D36 Fig. 3). The structure confirmed a
binding mode similar to those seen for amidine-based BACE1 in-
hibitors [4,5a,12]. The amidine moiety displays hydrogen bond in-
teractions with the catalytic aspartate dyad (Asp32 and Asp228).
The amide NeH forms a hydrogen bond with the backbone
carbonyl oxygen of Gly230. The quaternary center positions the
fluorophenyl ring in an axial orientation to occupy the S1 pocket,
4. Conclusions
1-Amino-3,4-dihydro-2,6-naphthyridine BACE1 inhibitors were
identified through incorporation of pyridine moieties and removal
3

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Table 2
Pharmacokinetic profiles of 11 in rat and dog.
LM (%)a
Serum fu
iv, 0.5 mg/kg, n ¼ 2
po, 1 mg/kg, n ¼ 2 (rat), 3 (dog)
b
species
CL (mL/min/kg)c
Vdss (L/kg)d
B/Pe
AUC (ng･h/mL)f
Cmax (ng/mL)g
Tmax (h)h
F (%)i
rat
beagle dog
22
93
0.15
0.14
45
NT
14
NT
0.28
NT
122
3890
9.0
78
4.0
4.3
33
NC
a
% remaining in rat and dog liver microsomes after 30 min incubation.
Fraction unbound in rat and dog serum.
Total clearance.
Volume of distribution at steady state.
Total brain-to-plasma ratio (Kp).
Area under the concentration-time curve.
Maximum plasma concentration.
Time at maximum concentration.
b
c
d
e
f
g
h
i
Oral bioavailability. NT ¼ not tested. NC ¼ not calculated.
Table 3
automated purification system using Yamazen or Fuji Silysia pre-
packed silica gel columns. 1H NMR spectra were recorded on a
Bruker Avance 400 MHz Analytical LC/MS was carried out on a
Pharmacokinetic and pharmacodynamic profiles of 11 in dog.
time after administration / po, 1 mg/kg, n ¼ 3a
Shimadzu Shim-pack XR-ODS (C18, 2.2
m
m, 3.0 ꢁ 50 mm, a linear
3 h
7 h
10 h
24 h
gradient from 10% to 100% B over 3 min and then 100% B for 1 min
(A ¼ water þ 0.1% formic acid, B ¼ MeCN þ 0.1% formic acid), flow
rate 1.6 mL/min) using a Shimadzu UFLC system equipped with an
LCMS-2020 mass spectrometer, LC-20AD binary gradient module,
SPD-M20A photodiode array detector (detection at 254 nm), and
SIL-20AC sample manager.
Cp (ng/ml)b
72
NT
NT
NT
65
NT
NT
NT
64
47
CCSF (ng/ml)c
0.56
0.070
49
0.52
0.083
47
d
CCSF/Cp,u
CSF total A
(%)
b reduction
CCSF /Cell IC50
NT
NT
3.3
3.0
a
Dosed to beagle dogs as a suspension in 0.5% MC.
Plasma concentration.
CSF concentration.
CSF-to-unbound plasma ratio. NT ¼ not tested.
1-(1-Bromoprop-1-en-2-yl)-2-fluorobenzene (13)
b
c
To a mixture of (bromomethyl)triphenylphosphonium bromide
(7.58 g, 17.4 mmol) in THF (20 mL) was added dropwise tert-BuOK
(1 M in THF; 17.4 mL, 17.4 mmol) at 0 ꢂC. After the mixture was
stirred at the same temperature for 10 min, 1-(2-fluorophenyl)
ethanone (12, 1.79 mL, 14.5 mmol) was added dropwise to the
mixture at 0 ꢂC. The mixture was stirred at the same temperature
for 90 min and then quenched with saturated aqueous NH4Cl so-
lution. The aqueous layer was separated and extracted with Et2O.
The combined organic layers were washed with brine, dried over
MgSO4, filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel; hexane) to give a 3:2 diaste-
reomeric mixture of 13 (1.57 g, 50%) as a colorless oil. 1H NMR
d
(400 MHz, CDCl3)
d
2.10 (d, J ¼ 1.5 Hz, 3H, major isomer) and 2.19 (t,
J ¼ 1.4 Hz, 3H, minor isomer), 6.32 (q, J ¼ 1.5 Hz, 1H, major isomer)
and 6.39 (q, J ¼ 1.4 Hz, 1H, minor isomer), 7.01e7.34 (4H, m).
2-(2-(2-Fluorophenyl)prop-1-en-1-yl)-4,4,5,5-tetramethyl-
1,3,2-dioxaborolane (14)
A mixture of 13 (2.24 g, 10.4 mmol), bis(4,4,5,5-tetramethyl-
[1,3]dioxolan-2-yl)borane (3.97 g, 15.6 mmol), KOAc (3.07 g,
31.2 mmol), and PdCl2(dppf)ꢃCH2Cl2 (0.425 g, 0.521 mmol) in 1,4-
dioxane (45 mL) was stirred at 65 ꢂC for 18 h under N2. The
mixture was filtered through a Celite pad, and the filtrate was
evaporated. The residue was purified by flash column chromatog-
raphy (silica gel; EtOAc/hexane, gradient: 0e10% EtOAc) to give a
3:2 diastereomeric mixture of 14 (2.15 g, 79%) as a yellow oil. 1H
Fig. 3. Crystal structure of compound 11 complexed with BACE1.
of the N,O-acetal group in initial leads 2a and 2b. Further optimi-
zation by introducing a fluorine atom on the pyridine ring balanced
basicity and cellular potency, leading to the discovery of compound
NMR (400 MHz, CDCl3)
d 1.09 (s, 12H, major isomer), 1.31 (s, 12H,
11 which showed a robust and sustained CSF Ab reduction in dog,
minor isomer), 2.18 (br s, 3H, major isomer), 2.37 (t, J ¼ 1.2 Hz, 3H,
minor isomer), 5.53 (s, 1H, minor isomer), 5.61 (q, J ¼ 1.2 Hz, 1H,
major isomer), 6.95e7.09 (2H, m), 7.14e7.30 (2H, m). MS-ESI (m/z):
263 [M þ H]þ.
along with reduced P-gp efflux. The X-ray structure of compound
11 bound to BACE1 revealed the molecular interactions around the
fluoropyridine moiety, which provide opportunities for further
optimization of this analogue.
3-(2-(2-Fluorophenyl)prop-1-en-1-yl)picolinonitrile (15)
A
mixture of 14 (223 mg, 0.852 mmol), 3-bromo-2-
cyanopyridine (187 mg, 1.02 mmol), K2CO3 (2 M aqueous solu-
tion; 1.28 mL, 2.56 mmol), and PdCl2(dppf)ꢃCH2Cl2 (34.8 mg,
0.043 mmol) in THF (7 mL) was stirred at 70 ꢂC for 9 h under N2. The
mixture was filtered through a Celite pad, and then the aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with H2O and brine, dried over Na2SO4,
5. Experimental section
5.1. General chemistry
All commercial reagents and solvents were used without further
purification. Flash column chromatography was conducted on an
4

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Scheme 1. Synthesis of Compound 4. Reagents and conditions: (a) t-BuOK, bromomethyltriphenylphosphonium bromide, THF, 0 ꢂC, 50%, dr ¼ 3:2; (b) bis(4,4,5,5-tetramethyl-[1,3]-
dioxolan-2-yl)borane, KOAc, PdCl2(dppf)ꢃCH2Cl2, 1,4-dioxane, 100 ꢂC, 79%, dr ¼ 3:2; (c) 3-bromo-2-cyanopyridine, K2CO3, PdCl2(dppf)ꢃCH2Cl2, THF-H2O, 70 ꢂC; (d) trimethylalu-
minum, NH4Cl, toluene, 100 ꢂC, 31% for 2 steps, dr ¼ 3:2; (e) H2SO4, rt; then HNO3, 0 ꢂC, 75%; (f) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC; (iii) 5-cyanopicolinic acid
hydrate, HATU, Et3N, THF, rt, 71% for 3 steps; (g) formic acid, rt, 59%.
Scheme 2. Synthesis of Compound 5. Reagents and conditions: (a) t-BuONa, 3-cyano-4-methylpyridine, DMF, rt, 66%, dr ¼ 1:1; (b) trifluoroacetic anhydride, pyridine, 1,4-dioxane,
rt; (c) trimethylaluminum, NH4Cl, toluene, 100 ꢂC, 74% for 2 steps, dr ¼ 3:2; (d) (i) H2SO4, rt; then HNO3, 0 ꢂC; (ii) Boc2O, THF, rt, 70% for 2 steps; (e) (i) Fe, NH4Cl, THF-MeOH-H2O,
70 ꢂC; (ii) 5-cyano-2-pyridinecarboxylic acid, diphenyl phosphorochloridate, Et3N, EtOAc, 0 ꢂC, 77% for 2 steps; (f) TFA, CH2Cl2, rt, 67%.
filtered, and evaporated. The residue was purified by flash column
chromatography (silica gel; EtOAc/hexane, gradient: 0e10% EtOAc)
to give a 1:1 diastereomeric mixture of 15 (207 mg, 3-bromo-2-
cyanopyridine was contained) as a colorless amorphous. 1H NMR
residue was purified by flash column chromatography (silica gel;
MeOH/CHCl3, gradient: 0e10% MeOH) to give a 3:2 diastereomeric
mixture of 16 (65.3 mg, 31% for 2 steps) as a brown gum. 1H NMR
(400 MHz, CDCl3)
d
2.22 (t, J ¼ 1.3 Hz, 3H, minor isomer), 2.26 (d,
(400 MHz, CDCl3)
d
2.28 (t, J ¼ 1.2 Hz, 3H, major isomer) and 2.44 (d,
J ¼ 1.5 Hz, 3H, major isomer), 6.65e6.66 (1H, m), 6.87e7.40 (5H, m),
8.01 (s, 1H, major isomer), 8.71 (s, 1H, minor isomer), 8.35 (d,
J ¼ 5.0 Hz, 1H, major isomer) and 8.58 (d, J ¼ 4.9 Hz, 1H, minor
isomer). MS-ESI (m/z): 256 [M þ H]þ.
J ¼ 1.5 Hz, 3H, minor isomer), 6.75 (1H, br s), 6.97e7.56 (5H, m), 8.17
(s, major isomer) and 8.88 (s, minor isomer), 8.55 (d, J ¼ 5.0 Hz, 1H,
major isomer), 8.78 (d, J ¼ 5.0 Hz, 1H, minor isomer). MS-ESI (m/z):
239 [M þ H]þ.
6-(2-Fluoro-5-nitrophenyl)-6-methyl-5,6-dihydro-1,7-
naphthyridin-8-amine (17)
3-(2-(2-Fluorophenyl)prop-1-en-1-yl)picolinimidamide (16)
To a suspension of NH4Cl (1.56 g, 29.2 mmol) in toluene (2 mL)
was added dropwise trimethyl aluminum (14.6 mL, 29.2 mmol) at
0 ꢂC under N2. After the mixture was stirred at rt for 1 h, 15 (199 mg,
0.833 mmol) in toluene (4 mL) was added. The mixture was stirred
at 100 ꢂC for 24 h. The reaction was quenched with potassium so-
dium tartrate and NaOH (2 M aqueous solution) at 0 ꢂC. The
mixture was stirred at rt for 1 h. The aqueous layer was separated
and extracted with CHCl3. The combined organic layers were
washed with brine, dried over Na2SO4, filtered, and evaporated. The
After a mixture of 16 (61.3 mg, 0.240 mmol) and conc. H2SO4
(0.50 mL, 9.00 mmol) was stirred at rt for 16 h, HNO3 (0.021 mL,
0.480 mmol) was added at 0 ꢂC, and the mixture was then stirred at
0
ꢂC for 1 h. The reaction was quenched with NaOH (2 M aqueous
solution) and basified with saturated aqueous NaHCO3 solution.
The aqueous layer was separated and extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na2SO4, filtered, and evaporated to give 17 (53.7 mg, 75%) as a
brown solid. 1H NMR (400 MHz, CDCl3)
d 1.53 (3H, s), 3.18 (1H, d,
5

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Scheme 3. Synthesis of Compound 6. Reagents and conditions: (a) 3-bromo-4-cyanopyridine, K2CO3, PdCl2(dppf)ꢃCH2Cl2, THF-H2O, 70 ꢂC, 84%, dr ¼ 3:2; (b) trimethylaluminum,
NH4Cl, toluene, 100 ꢂC, 97%; (c) conc. H2SO4, rt; then HNO3, 0 ꢂC, 86%; (c) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC; (d) 5-cyanopicolinic acid hydrate, HATU, Et3N, THF,
rt, 97% for 3 steps; (e) formic acid, rt, 52%.
Scheme 4. Synthesis of Compound 7. Reagents and conditions: (a) 2-chloro-3-cyanopyridine, K2CO3, PdCl2(dppf)ꢃCH2Cl2, 1,4-dioxane-H2O, 100 ꢂC, 84%, dr ¼ 3:2; (b) trimethy-
laluminum, NH4Cl, toluene, 100 ꢂC, 97%, dr ¼ 3:2; (c) conc. H2SO4, rt; then HNO3, 0 ꢂC, 81% for 2 steps; (d) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC, 70% for 2 steps; (e)
5-cyanopicolinic acid hydrate, HATU, Et3N, THF, rt, 96%; (f) formic acid, rt, 84%.
J ¼ 15.9 Hz), 3.30 (1H, d, J ¼ 15.9 Hz), 7.15 (1H, dd, J ¼ 10.9, 8.9 Hz),
7.31 (1H, d, J ¼ 5.0 Hz), 8.10 (1H, ddd, J ¼ 8.9, 4.0, 3.0 Hz), 8.55 (1H,
s), 8.63 (1H, d, J ¼ 5.0 Hz), 8.68 (1H, dd, J ¼ 7.0, 3.0 Hz). MS-ESI (m/
z): 301 [M þ H]þ.
H2O and saturated aqueous NaHCO3 solution. The aqueous layer
was separated and extracted with EtOAc. The combined organic
layers were washed with H2O and brine, dried over Na2SO4, filtered,
and evaporated to give the residue as a brown amorphous
(64.5 mg). To the residue (64.5 mg) in THF (1.3 mL) were added 5-
cyanopicolinic acid hydrate (35.0 mg, 0.211 mmol), HATU (87.0 mg,
0.228 mmol), and Et3N (0.063 mL, 0.456 mmol) at 0 ꢂC. The mixture
was stirred at rt for 90 min. The reaction was quenched with
saturated aqueous NaHCO3 solution. The aqueous layer was sepa-
rated and extracted with EtOAc. The combined organic layers were
washed with H2O and brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column
tert-Butyl (6-(5-(5-cyanopicolinamido)-2-fluorophenyl)-6-
methyl-5,6-dihydro-1,7-naphthyridin-8-yl)carbamate (18)
After a mixture of 17 (54.9 mg, 0.183 mmol) and Boc2O
(0.042 mL, 0.183 mmol) in THF (0.83 mL) was stirred at rt for 15 h,
MeOH (0.6 mL), H2O (0.2 mL), Fe (57.2 mg, 2.90 mmol), and NH4Cl
(44.0 mg, 0.823 mmol) were added. The mixture was stirred at
70 ꢂC for 90 min, and cooled to rt, and filtered through a Celite pad,
and then the filtrate was evaporated. The residue was diluted with
6

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Scheme 5. Synthesis of Compound 8. Reagents and conditions: (a) 14, K2CO3, PdCl2(dppf)ꢃCH2Cl2, THF-H2O, 70 ꢂC, 81%, dr ¼ 3:2; (d) trimethylaluminum, NH4Cl, toluene, 100 ꢂC,
99%, dr ¼ 3:2; (e) conc. H2SO4, rt; then HNO3, 0 ꢂC, 81%; (f) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC, 92% for 2 steps; (g) 5-cyano-2-pyridinecarboxylic acid, HATU,
Et3N, THF, rt, 96%; (h) formic acid, rt, 78%.
Scheme 6. Synthesis of Compounds 9 and 11. Reagents and conditions: (a) 14, K2CO3, PdCl2(dppf)ꢃCH2Cl2, 1,4-dioxane-H2O, 100 ꢂC, 32%, dr ¼ 1:1; (b) trimethylaluminum, NH4Cl,
toluene, 100 ꢂC, 39%; (c) conc. H2SO4, rt; then HNO3, 0 ꢂC, 81% for 2 steps; (d) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC, 70% for 2 steps; (e) 5-cyano-2-
pyridinecarboxylic acid, HATU, Et3N, THF, rt, 80%; (f) formic acid, rt or HCl in EtOAc, rt. (g) chiral separation by SFC, 29% for 2 steps.
chromatography (silica gel; EtOAc/hexane, gradient: 50e75%
EtOAc) to give 18 (62.5 mg, 71% for 3 steps) as a white solid. 1H NMR
was separated and extracted with EtOAc. The combined organic
layers were washed with H2O and brine, dried over Na2SO4, filtered,
and evaporated. The residue was purified by flash column chro-
matography (silica gel; MeOH/CHCl3, gradient: 0e5% MeOH). The
crude product was recrystallized from hexane/EtOAc to give 4
(400 MHz, CDCl3)
d
1.63 (9H, s), 1.89 (3H, s), 3.27 (1H, d, J ¼ 15.9 Hz),
3.77 (1H, d, J ¼ 15.9 Hz), 7.07 (1H, dd, J ¼ 11.6, 8.9 Hz), 7.39 (1H, dd,
J ¼ 7.2, 2.5 Hz), 7.73e7.79 (1H, m), 8.02 (1H, d, J ¼ 5.0 Hz), 8.19 (1H,
dd, J ¼ 8.2, 2.0 Hz), 8.37 (1H, dd, J ¼ 8.2, 0.8 Hz), 8.50 (1H, s), 8.54
(1H, d, J ¼ 5.0 Hz), 8.88 (1H, dd, J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72
(1H, s). MS-ESI (m/z): 501 [M þ H]þ.
(20.9 mg, 59%) as a white solid. 1H NMR (400 MHz, CD3OD)
d 1.75
(3H, s), 3.27 (1H, d, J ¼ 15.7 Hz), 3.63 (1H, d, J ¼ 15.7 Hz), 7.08 (1H,
dd, J ¼ 12.2, 8.6 Hz), 7.39 (1H, dd, J ¼ 7.6, 5.1 Hz), 7.67 (1H, d,
J ¼ 7.6 Hz), 7.72e7.78 (2H, m), 8.34 (1H, d, J ¼ 8.1 Hz), 8.41e8.47 (2H,
m), 9.04e9.05 (1H, m). MS-ESI (m/z): 401 [M þ H]þ.
N-(3-(8-Amino-6-methyl-5,6-dihydro-1,7-naphthyridin-6-
yl)-4-fluorophenyl)-5-cyanopicolinamide (4)
A mixture of 18 (44.5 mg, 0.089 mmol) and formic acid (0.50 mL,
13.0 mmol) was stirred at rt for 14 h. The reaction was quenched
with saturated aqueous NaHCO3 solution at 0 ꢂC. The aqueous layer
4-(2-(2-Fluorophenyl)prop-1-en-1-yl)nicotinamide (19)
To a mixture of tert-BuONa (4.49 g, 46.7 mmol) in DMF (18 mL)
was added dropwise 3-cyano-4-methylpyridine (3.68 g, 31.2 mmol)
7

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Scheme 7. Synthesis of Compound 10. Reagents and conditions: (a) 14, K2CO3, PdCl2(dppf)ꢃCH2Cl2, THF-H2O, 70 ꢂC, 80%, dr ¼ 3:2; (b) trimethylaluminum, NH4Cl, toluene, 100 ꢂC,
82%; (c) conc. H2SO4, rt; then HNO3, 0 ꢂC, 86%; (d) (i) Boc2O, THF, rt; (ii) Fe, NH4Cl, THF-MeOH-H2O, 70 ꢂC, 88% for 2 steps; (g) (i) 5-cyano-2-pyridinecarboxylic acid, HATU, Et3N, THF,
rt; (ii) formic acid, rt, 62% for 2 steps.
at 0 ꢂC. After the mixture was stirred at 0 ꢂC for 1 h, 1-(2-
fluorophenyl)ethanone (4.03 mL, 32.7 mmol) was added drop-
wise at 0 ꢂC. The resulting mixture was stirred at 0 ꢂC for 7 h and
then rt for 15 h. The reaction was quenched with sat NH4Cl at 0 ꢂC.
The aqueous layer was separated and extracted with EtOAc. The
combined organic layers were washed with H2O and brine, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
flash column chromatography (silica gel; MeOH/CHCl3, gradient:
0e5% MeOH). The crude product was recrystallized from EtOAc/
hexane to give a 1:1 diastereomeric mixture of 19 (5.27 g, 66%) as a
4-(2-(2-Fluorophenyl)prop-1-en-1-yl)nicotinonitrile (22)
After a mixture of 21 (25.5 mg, 0.100 mmol) and conc. H2SO4
(0.50 mL, 9.00 mmol) was stirred at rt for 19 h, HNO3 (8.93
0.20 mmol) was added at 0 ꢂC, and the mixture was then stirred at
ꢂC for 1 h. The reaction was quenched with NaOH (2 M aqueous
solution) and basified with saturated aqueous NaHCO3 solution.
The aqueous layer was separated and extracted with EtOAc. The
combined organic layers were washed with H2O and brine, dried
over Na2SO4, filtered, and evaporated to give the residue as a white
mL,
0
solid (21.5 mg). After
a mixture of the residue (21.3 mg,
brown gum. 1H NMR (400 MHz, CDCl3)
d
2.19 (3H, t, J ¼ 2.2 Hz,
0.071 mmol) and Boc2O (0.016 mL, 0.071 mmol) in THF (0.4 mL) was
stirred at rt for 17 h, the mixture was evaporated. The residue was
purified by flash column chromatography (silica gel; MeOH/CHCl3,
gradient: 0e5% MeOH) to give 22 (28.2 mg, 70% for 2 steps) as a
major isomer), 2.29 (3H, d, J ¼ 1.5 Hz, minor isomer), 7.07e7.41 (6H,
m), 8.27 (1H, d, J ¼ 5.2 Hz, minor isomer), 8.70 (1H, d, J ¼ 5.0 Hz,
major isomer), 8.81 (1H, s, minor isomer), 9.04 (1H, s, major isomer).
MS-ESI (m/z): 257 [M þ H]þ.
white solid. 1H NMR (400 MHz, CDCl3)
d
2.20 (t, J ¼ 1.5 Hz, 3H, minor
4-(2-(2-Fluorophenyl)prop-1-en-1-yl)nicotinimidamide (21)
To a solution of 19 (105 mg, 0.411 mmol) in 1,4-dioxane (1 mL)
were added pyridine (0.073 mL, 0.904 mmol) and trifluoroacetic
anhydride (0.064 mL, 0.452 mmol) at 0 ꢂC. The mixture was stirred
at rt for 3.5 h. The reaction was quenched with H2O. The aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with 10% citric acid, H2O and brine,
dried over Na2SO4, filtered, and evaporated to give 20 as a brown oil
(103 mg). To a mixture of NH4Cl (279 mg, 5.21 mmol) in toluene
(0.35 mL) was added dropwise trimethylaluminum (2.61 mL,
5.21 mmol) at 0 ꢂC under N2. After the mixture was stirred at rt for
1 h, 20 (35.5 mg, 0.149 mmol) in toluene (0.7 mL) was added. The
mixture was stirred at 100 ꢂC for 27 h. The reaction was quenched
with potassium sodium tartrate and NaOH (2 M aqueous solution)
at 0 ꢂC. The mixture was stirred at rt for 30 min. The aqueous layer
was separated and extracted with CHCl3. The combined organic
layers were washed with brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column chromatog-
raphy (silica gel; MeOH/CHCl3, gradient: 0e10% MeOH) to give a 3:2
diastereomeric mixture of 21 (28.1 mg, 74% for 2 steps) as a brown
isomer), 2.45 (d, J ¼ 1.5 Hz, 3H, major isomer), 6.23e6.26 (1H, m),
6.87e7.40 (5H, m), 8.15 (s, 1H, major isomer), 8.88 (s, 1H, minor
isomer), 8.35 (d, J ¼ 5.0 Hz, 1H, major isomer), 8.58 (d, J ¼ 5.0 Hz, 1H,
minor isomer). MS-ESI (m/z): 401 [M þ H]þ.
tert-Butyl (3-(5-(5-cyanopicolinamido)-2-fluorophenyl)-3-
methyl-3,4-dihydro-2,7-naphthyridin-1-yl)carbamate (23)
A
mixture of 22 (34.0 mg, 0.085 mmol), Fe (26.6 mg,
0.476 mmol), and NH4Cl (20.4 mg, 0.382 mmol) in THF (0.51 mL),
MeOH (0.34 mL), and H2O (0.14 mL) was stirred at 70 ꢂC for 2 h.
After cooling, the mixture was filtered through a Celite pad, and the
filtrate was evaporated. The residue was diluted with H2O and
aqueous saturated NaHCO3. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated to
give a white solid (30.9 mg). To the residue in EtOAc (0.6 mL) were
added 5-cyanopicolinic acid hydrate (15.0 mg, 0.090 mmol),
diphenyl phosphorochloridate (0.020 mL, 0.098 mmol), and Et3N
(0.057 mL, 0.410 mmol) at 0 ꢂC. The mixture was stirred at the same
temperature for 90 min. The reaction was quenched with saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (silica
gel; EtOAc/hexane, gradient: 50e75% EtOAc) to give 23 (31.5 mg,
gum. 1H NMR (400 MHz, CDCl3)
d
2.22 (t, J ¼ 1.3 Hz, 3H, minor
isomer), 2.26 (d, J ¼ 1.5 Hz, 3H, major isomer), 6.65e6.66 (1H, m),
6.87e7.40 (5H, m), 8.01 (s, 1H, major isomer), 8.71 (s, 1H, minor
isomer), 8.35 (d, J ¼ 5.0 Hz, 1H, major isomer), 8.58 (d, J ¼ 4.9 Hz, 1H,
minor isomer). MS-ESI (m/z): 256 [M þ H]þ.
77% for 2 steps) as a white solid. 1H NMR (400 MHz, CDCl3)
d 1.63
8

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
(9H, s),1.89 (3H, s), 3.27 (1H, d, J ¼ 15.9 Hz), 3.77 (1H, d, J ¼ 15.9 Hz),
7.07 (1H, dd, J ¼ 11.6, 8.9 Hz), 7.39 (1H, dd, J ¼ 7.2, 2.5 Hz), 7.73e7.79
(1H, m), 8.02 (1H, d, J ¼ 5.0 Hz), 8.19 (1H, dd, J ¼ 8.2, 2.0 Hz), 8.37
(1H, dd, J ¼ 8.2, 0.8 Hz), 8.50 (1H, s), 8.54 (1H, d, J ¼ 5.0 Hz), 8.88
(1H, dd, J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72 (1H, s). MS-ESI (m/z): 501
[M þ H]þ.
added HNO3 (0.062 mL, 1.40 mmol) at 0 ꢂC, which was then stirred
at the same temperature for 1 h. The reaction was quenched with
NaOH (2 M aqueous solution) and basified with saturated aqueous
NaHCO3 solution. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and evaporated to give 26 (181 mg, 86%)
N-(3-(1-Amino-3-methyl-3,4-dihydro-2,7-naphthyridin-3-
yl)-4-fluorophenyl)-5-cyanopicolinamide (5)
as a yellow solid. 1H NMR (400 MHz, CDCl3)
d 1.53 (3H, s), 3.18 (1H,
d, J ¼ 15.9 Hz), 3.30 (1H, d, J ¼ 15.9 Hz), 7.15 (1H, dd, J ¼ 10.9, 8.9 Hz),
7.31 (1H, d, J ¼ 5.0 Hz), 8.10 (1H, ddd, J ¼ 8.9, 4.0, 3.0 Hz), 8.55 (1H,
s), 8.63 (1H, d, J ¼ 5.0 Hz), 8.68 (1H, dd, J ¼ 7.0, 3.0 Hz). MS-ESI (m/
z): 301 [M þ H]þ.
A mixture of 23 (31.2 mg, 0.062 mmol) and TFA (0.144 mL,
1.870 mmol) in CH2Cl2 (0.6 mL) was stirred at rt for 50 min. The
reaction was quenched with saturated aqueous NaHCO3 solution at
0
ꢂC. The aqueous layer was separated and extracted with EtOAc.
tert-Butyl (3-(5-(5-cyanopicolinamido)-2-fluorophenyl)-3-
methyl-3,4-dihydro-2,6-naphthyridin-1-yl)carbamate (27)
After a mixture of 26 (156 mg, 0.519 mmol) and Boc2O (0.120 mL,
0.519 mmol) in THF (2.3 mL) was stirred at rt for 15 h, MeOH
(1.5 mL), H2O (0.6 mL), Fe (162 mg, 2.90 mmol), and NH4Cl (125 mg,
2.33 mmol) were added at rt. The mixture was stirred at 70 ꢂC for
90 min and then filtered through a Celite pad, and the filtrate was
evaporated. The residue was diluted with H2O and saturated
aqueous NaHCO3. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, filtered, and evaporated to give a residue as a
yellow solid (228 mg). To the residue (68.8 mg) in THF (1.4 mL)
were added 5-cyanopicolinic acid hydrate (37.0 mg, 0.223 mmol),
HATU (92.0 mg, 0.241 mmol), and Et3N (0.067 mL, 0.483 mmol) at
The combined organic layers were washed with H2O and brine,
dried over Na2SO4, filtered, and evaporated. The residue was puri-
fied by flash column chromatography (silica gel; MeOH/CHCl3,
gradient: 0e5% MeOH). The crude product was recrystallized from
hexane/EtOAc/MeOH to give 5 (16.7 mg, 67%) as a white solid. 1H
NMR (400 MHz, DMSO‑d6)
d
1.44 (3H, s), 3.09 (1H, d, J ¼ 15.2 Hz),
3.18 (1H, d, J ¼ 15.2 Hz), 6.36 (2H, s), 7.11 (1H, dd, J ¼ 11.9, 8.9 Hz),
7.29 (1H, d, J ¼ 4.6 Hz), 7.68e7.71 (1H, m), 7.98e7.99 (1H, m), 8.26
(1H, d, J ¼ 8.1 Hz), 8.47 (1H, d, J ¼ 4.6 Hz), 8.57 (1H, dd, J ¼ 8.1.
2.0 Hz), 8.82 (1H, s), 9.19 (1H, d, J ¼ 1.5 Hz), 10.69 (1H, s). MS-ESI (m/
z): 401 [M þ H]þ.
3-(2-(2-Fluorophenyl)prop-1-en-1-yl)isonicotinonitrile (24)
A mixture of 14 (267 mg, 1.02 mmol), 3-bromo-4-cyanopyridine
(205 mg, 1.12 mmol), K2CO3 (2 M aqueous solution; 1.53 mL,
3.06 mmol), and PdCl2(dppf)ꢃCH2Cl2 (41.6 mg, 0.051 mmol) in THF
(8 mL) was stirred at 70 ꢂC for 14 h under N2. The resulting mixture
was filtered through a Celite pad, and then the aqueous layer was
separated and extracted with EtOAc. The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and evapo-
rated. The residue was purified by flash column chromatography
(silica gel; EtOAc/hexane, gradient: 10e20% EtOAc) to give a 3:2
diastereomeric mixture of 24 (203 mg, 84%) as a colorless oil. 1H
0
ꢂC. The mixture was stirred at rt for 90 min and quenched with
saturated aqueous NaHCO3 solution. The aqueous layer was sepa-
rated and extracted with EtOAc. The combined organic layers were
washed with H2O and brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column chromatog-
raphy (silica gel; EtOAc/hexane, gradient: 50e75% EtOAc) to give 27
(90.3 mg, 97% for 3 steps) as a white solid. 1H NMR (400 MHz,
CDCl3)
d
1.55 (9H, s), 1.99 (3H, s), 3.26 (1H, d, J ¼ 152 Hz), 3.78(1H, d,
J ¼ 15.2 Hz), 7.11 (1H, dd, J ¼ 11.6, 8.9 Hz), 7.50 (1H, dd, J ¼ 7.2,
2.5 Hz), 7.73e7.79 (1H, m), 8.02 (1H, d, J ¼ 5.0 Hz), 8.33 (1H, dd,
J ¼ 8.2, 2.0 Hz), 8.37 (1H, dd, J ¼ 8.5, 1.0 Hz), 8.50 (1H, s), 8.54 (1H, d,
J ¼ 5.0 Hz), 8.88 (1H, dd, J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72 (1H, s).
MS-ESI (m/z): 501 [M þ H]þ.
NMR (400 MHz, CDCl3)
d
2.26 (t, J ¼ 1.2 Hz, 3H, major isomer) and
2.32 (d, J ¼ 1.5 Hz, 3H, minor isomer), 6.75 (1H, br s), 6.97e7.56 (5H,
m), 8.17 (s, major isomer) and 8.88 (s, minor isomer), 8.42 (d,
J ¼ 5.0 Hz, 1H, major isomer), 8.66 (d, J ¼ 5.0 Hz, 1H, minor isomer).
MS-ESI (m/z): 239 [M þ H]þ.
N-(3-(1-Amino-3-methyl-3,4-dihydro-2,6-naphthyridin-3-
yl)-4-fluorophenyl)-5-cyanopicolinamide (6)
3-(2-(2-Fluorophenyl)prop-1-en-1-yl)isonicotinimidamide
(25)
A mixture of 27 (64.3 mg, 0.128 mmol) and formic acid (0.50 mL,
13.0 mmol) was stirred at rt for 14 h. The reaction was quenched
with NaOH (2 M aqueous solution) and saturated aqueous NaHCO3
solution at 0 ꢂC. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with H2O
and brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; MeOH/
CHCl3, gradient: 0e5% MeOH). The crude product was recrystal-
lized from hexane/EtOAc/MeOH to give 6 (26.5 mg, 52%) as a white
solid. 1H NMR (400 MHz, CD3OD) d 1.75 (3H, s), 3.13 (1H, d,
J ¼ 16.2 Hz), 3.62 (1H, d, J ¼ 16.2 Hz), 7.08 (1H, dd, J ¼ 11.9, 8.9 Hz),
7.64e7.70 (2H, m), 7.77 (1H, dd, J ¼ 7.4, 2.8 Hz), 8.34 (1H, dd, J ¼ 8.1,
1.0 Hz), 8.42 (1H, dd, J ¼ 8.1, 2.0 Hz), 8.46 (1H, s), 8.50 (1H, d,
J ¼ 5.1 Hz), 9.04 (1H, d, J ¼ 1.5 Hz). MS-ESI (m/z): 401 [M þ H]þ.
2-(2-(2-Fluorophenyl)prop-1-en-1-yl)nicotinonitrile (28)
To a mixture of NH4Cl (1.44 g, 27.0 mmol) in toluene (2 mL) was
added dropwise trimethylaluminum (13.5 mL, 27.0 mmol) at 0 ꢂC
under N2. After the mixture was stirred at rt for 1 h, 24 (184 mg,
0.771 mmol) in toluene (4 mL) was added. The mixture was stirred
at 100 ꢂC for 26 h. The reaction was quenched with potassium so-
dium tartrate and NaOH (2 M aqueous solution) at 0 ꢂC, which was
then stirred at rt for 10 min. The aqueous layer was separated and
extracted with CHCl3. The combined organic layers were washed
with brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; MeOH/
CHCl3, gradient: 0e5% MeOH) to give a 3:2 diastereomeric mixture
of 25 (190 mg, 97%) as a brown gum. 1H NMR (400 MHz, CDCl3)
d
2.22 (t, J ¼ 1.3 Hz, 3H, minor isomer), 2.26 (d, J ¼ 1.5 Hz, 3H, major
isomer), 6.65e6.66 (1H, m), 6.87e7.40 (5H, m), 7.99 (s, 1H, major
isomer), 8.33 (s, 1H, minor isomer), 8.45 (d, J ¼ 5.0 Hz, 1H, major
isomer), 8.99 (d, J ¼ 5.0 Hz, 1H, minor isomer). MS-ESI (m/z): 256
[M þ H]þ.
A
mixture of 14 (172 mg, 0.656 mmol), 2-chloro-3-
cyanopyridine (109 mg, 0.787 mmol), K2CO3 (2 M aqueous solu-
tion; 0.984 mL, 1.97 mmol), and PdCl2(dppf)ꢃCH2Cl2 (26.8 mg,
0.033 mmol) in 1,4-dioxane (5 mL) was stirred at 100 ꢂC for 4.5 h
under N2. The resulting mixture was filtered through a Celite pad.
The aqueous layer was separated and extracted with EtOAc. The
combined organic layers were washed with brine, dried over
3-(2-Fluoro-5-nitrophenyl)-3-methyl-3,4-dihydro-2,6-
naphthyridin-1-amine (26)
After a mixture of 25 (179 mg, 0.699 mmol) and conc. H2SO4
(1.00 mL, 18.0 mmol) was stirred at rt for 15 h, to this mixture was
9

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
Na2SO4, filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel; EtOAc/hexane, gradient: 0e10%
EtOAc) to give a 3:2 diastereomeric mixture of 28 (203 mg, 84%) as a
(26.6 mg, 0.070 mmol), and Et3N (0.019 mL, 0.140 mmol) at 0 ꢂC.
The mixture was stirred at rt for 1.5 h and then quenched with
saturated aqueous NaHCO3 solution. The aqueous layer was sepa-
rated and extracted with EtOAc. The combined organic layers were
washed with H2O and brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column chromatog-
raphy (silica gel; EtOAc/hexane, gradient: 50e75% EtOAc) to give 32
colorless oil. 1H NMR (400 MHz, CDCl3)
d
2.26 (t, J ¼ 1.2 Hz, 3H,
major isomer) and 2.32 (d, J ¼ 1.5 Hz, 3H, minor isomer), 6.75 (1H, br
s), 6.97e7.56 (5H, m), 8.17 (s, major isomer) and 8.88 (s, minor iso-
mer), 8.42 (d, J ¼ 5.0 Hz, 1H, major isomer), 8.66 (d, J ¼ 5.0 Hz, 1H,
minor isomer). MS-ESI (m/z): 239 [M þ H]þ.
(21.4 mg, 80%) as a white solid. 1H NMR (400 MHz, CDCl3)
d 1.63
2-(2-(2-Fluorophenyl)prop-1-en-1-yl)nicotinimidamide (29)
To a suspension of NH4Cl (411 mg, 7.68 mmol) in toluene
(0.5 mL) was added dropwise trimethylaluminum (3.84 mL,
7.68 mmol) at 0 ꢂC under N2. After the mixture was stirred at rt for
1 h, 28 (52.3 mg, 0.220 mmol) in toluene (1 mL) was added. The
mixture was stirred at 100 ꢂC for 48 h. The reaction was quenched
with potassium sodium tartrate and NaOH (2 M aqueous solution)
at 0 ꢂC. The mixture was stirred at rt for 1 h, and the aqueous layer
was then separated and extracted with CHCl3. The combined
organic layers were washed with brine, dried over Na2SO4, filtered,
and evaporated. The residue was purified by flash column chro-
matography (silica gel; MeOH/CHCl3, gradient: 0e10% MeOH) to
give a 3:2 diastereomeric mixture of 29 (21.7 mg, 39%) as a brown
(9H, s), 1.89 (3H, s), 3.27 (1H, d, J ¼ 15.9 Hz), 3.77 (1H, d, J ¼ 15.9 Hz),
7.07 (1H, dd, J ¼ 11.6, 8.9 Hz), 7.39 (1H, dd, J ¼ 7.2, 2.5 Hz), 7.73e7.79
(1H, m), 8.02 (1H, d, J ¼ 5.0 Hz), 8.19 (1H, dd, J ¼ 8.2, 2.0 Hz), 8.37
(1H, dd, J ¼ 8.2, 0.8 Hz), 8.50 (1H, s), 8.54 (1H, d, J ¼ 5.0 Hz), 8.88
(1H, dd, J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72 (1H, s). MS-ESI (m/z): 501
[M þ H]þ.
N-(3-(5-Amino-7-methyl-7,8-dihydro-1,6-naphthyridin-7-
yl)-4-fluorophenyl)-5-cyanopicolinamide (7)
A mixture of 32 (21.1 mg, 0.042 mmol) and formic acid (0.50 mL,
13.0 mmol) was stirred at rt for 14 h. The reaction was quenched
with NaOH (2 M aqueous solution) and saturated aqueous NaHCO3
solution at 0 ꢂC. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with H2O
and brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; MeOH/
CHCl3, gradient: 0e5% MeOH). The crude product was recrystal-
lized from hexane/CH2Cl2 to give 7 (12.3 mg, 73%) as a white solid.
gum. 1H NMR (400 MHz, CDCl3)
d
2.22 (t, J ¼ 1.3 Hz, 3H, minor
isomer), 2.26 (d, J ¼ 1.5 Hz, 3H, major isomer), 6.65e6.66 (1H, m),
6.87e7.40 (5H, m), 8.01 (s, 1H, major isomer), 8.71 (s, 1H, minor
isomer), 8.35 (d, J ¼ 5.0 Hz, 1H, major isomer) and 8.58 (d, J ¼ 4.9 Hz,
1H, minor isomer). MS-ESI (m/z): 256 [M þ H]þ.
1H NMR (400 MHz, DMSO‑d6)
d
1.47 (3H, s), 3.22 (1H, d, J ¼ 15.2 Hz),
7-(2-Fluoro-5-nitrophenyl)-7-methyl-7,8-dihydro-1,6-
naphthyridin-5-amine (30)
6.33 (2H, brs), 7.12 (1H, dd, J ¼ 11.9, 8.9 Hz), 7.31 (1H, dd, J ¼ 7.6,
5.1 Hz), 7.67e7.71 (1H, m), 7.97e8.05 (2H, br m), 8.26 (1H, d,
J ¼ 8.1 Hz), 8.45 (1H, dd, J ¼ 4.6, 1.5 Hz), 8.57 (1H, dd, J ¼ 8.1, 2.0 Hz),
9.18e9.19 (1H, m), 10.69 (1H, s). MS-ESI (m/z): 401 [M þ H]þ.
5-Fluoro-4-(2-(2-fluorophenyl)prop-1-en-1-yl)nicotinoni-
trile (34)
After a mixture of 29 (179 mg, 0.699 mmol) and conc. H2SO4
(0.50 mL, 9.00 mmol) was stirred at rt for 14 h, HNO3 (7.5
mL,
0.168 mmol) was added at 0 ꢂC. The mixture was stirred at 0 ꢂC for
1 h. The reaction was quenched with NaOH (2 M solution) and
basified with saturated aqueous NaHCO3 solution. The aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, filtered,
and evaporated to give 30 (181 mg) as a yellow solid. 1H NMR
A mixture of 14 (223 mg, 0.852 mmol), 4-chloro-3-cyano-5-
fluoropyridine (33, 209 mg, 1.33 mmol), K2CO3 (2 M aqueous so-
lution; 2.00 mL, 4.00 mmol), and PdCl2(dppf)ꢃCH2Cl2 (54.4 mg,
0.067 mmol) in THF (11 mL) was stirred at 70 ꢂC for 16 h under N2.
The resulting mixture was filtered through a Celite pad. The
aqueous layer was separated and extracted with EtOAc. The com-
bined organic layers were washed with H2O and brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel; EtOAc/hexane, gradient: 0e10%
EtOAc) to give a 3:2 diastereomeric mixture of 34 (277 mg, 81%) as a
(400 MHz, CDCl3)
d
1.63 (9H, s), 1.89 (3H, s), 3.27 (1H, d, J ¼ 15.9 Hz),
3.77 (1H, d, J ¼ 15.9 Hz), 7.07 (1H, dd, J ¼ 11.6, 8.9 Hz), 7.39 (1H, dd,
J ¼ 7.2, 2.5 Hz), 7.73e7.79 (1H, m), 8.02 (1H, d, J ¼ 5.0 Hz), 8.19 (1H,
dd, J ¼ 8.2, 2.0 Hz), 8.37 (1H, dd, J ¼ 8.2, 0.8 Hz), 8.50 (1H, s), 8.54
(1H, d, J ¼ 5.0 Hz), 8.88 (1H, dd, J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72
(1H, s). MS-ESI (m/z): 301 [M þ H]þ.
tert-Butyl
(7-(5-amino-2-fluorophenyl)-7-methyl-7,8-
colorless amorphous. 1H NMR (400 MHz, CDCl3)
d
2.26 (t, J ¼ 1.2 Hz,
dihydro-1,6-naphthyridin-5-yl)carbamate (31)
3H, major isomer) and 2.32 (d, J ¼ 1.5 Hz, 3H, minor isomer), 6.75
(1H, br s), 6.97e7.56 (5H, m), 8.17 (s, major isomer) and 8.88 (s,
minor isomer), 8.42 (d, J ¼ 5.0 Hz, 1H, major isomer), 8.66 (d,
J ¼ 5.0 Hz, 1H, minor isomer). MS-ESI (m/z): 257 [M þ H]þ.
5-Fluoro-4-(2-(2-fluorophenyl)prop-1-en-1-yl)nic-
After a mixture of 30 (20.5 mg, 0.068 mmol) and Boc2O
(0.016 mL, 0.068 mmol) in THF (0.6 mL) was stirred at rt for 13 h,
MeOH (0.4 mL), H2O (0.16 mL), Fe (21.4 mg, 0.382 mmol), and NH4Cl
(16.4 mg, 0.307 mmol) were added at rt. The mixture was stirred at
70 ꢂC for 2 h and filtered through a Celite pad, and the filtrate was
evaporated. The residue was diluted with H2O and saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; MeOH/
CHCl3, gradient: 0e5% MeOH) to give a 3:2 diastereomeric mixture
of 31 (21.7 mg, 39%) as a brown gum. 1H NMR (400 MHz, CDCl3)
otinimidamide (35)
To a suspension of NH4Cl (1.88 g, 35.1 mmol) in toluene (2.5 mL)
was added dropwise trimethylaluminum (15.5 mL, 35.1 mmol) at
0 ꢂC under N2. After the mixture was stirred at rt for 1 h, 34 (257 mg,
1.00 mmol) in toluene (5 mL) was added. The mixture was stirred at
100 ꢂC for 21 h and then quenched with potassium sodium tartrate
and NaOH (2 M solution) at 0 ꢂC. The mixture was stirred at rt for
10 min. The aqueous layer was separated and extracted with CHCl3.
The combined organic layers were washed with brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel; MeOH/CHCl3, gradient: 0e10%
MeOH) to give a 3:2 diastereomeric mixture of 35 (270 mg, 99%) as
d
2.22 (t, J ¼ 1.3 Hz, 3H, minor isomer), 2.26 (d, J ¼ 1.5 Hz, 3H, major
isomer), 6.65e6.66 (1H, m), 6.87e7.40 (5H, m), 8.01 (s, 1H, major
isomer), 8.71 (s, 1H, minor isomer), 8.35 (d, J ¼ 5.0 Hz, 1H, major
isomer) and 8.58 (d, J ¼ 4.9 Hz, 1H, minor isomer). MS-ESI (m/z): 371
[M þ H]þ.
a brown gum. 1H NMR (400 MHz, CDCl3)
d
2.22 (t, J ¼ 1.3 Hz, 3H,
tert-Butyl (7-(5-(5-cyanopicolinamido)-2-fluorophenyl)-7-
methyl-7,8-dihydro-1,6-naphthyridin-5-yl)carbamate (32)
To a solution of 31 (19.9 mg) in THF (0.6 mL) were added 5-
cyanopicolinic acid hydrate (10.7 mg, 0.064 mmol), HATU
minor isomer), 2.26 (d, J ¼ 1.5 Hz, 3H, major isomer), 6.65e6.66 (1H,
m), 6.87e7.40 (5H, m), 8.01 (s, 1H, major isomer), 8.71 (s, 1H, minor
isomer), 8.35 (d, J ¼ 5.0 Hz, 1H, major isomer) and 8.58 (d, J ¼ 4.9 Hz,
1H, minor isomer). MS-ESI (m/z): 274 [M þ H]þ.
10

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
5-Fluoro-3-(2-fluoro-5-nitrophenyl)-3-methyl-3,4-dihydro-
2,7-naphthyridin-1-amine (36)
(1H, d, J ¼ 16.2 Hz), 7.08 (1H, dd, J ¼ 11.7, 8.6 Hz), 7.64e7.68 (1H, m),
7.78 (1H, dd, J ¼ 7.4, 2.8 Hz), 8.33 (1H, dd, J ¼ 8.1, 1.0 Hz), 8.41 (1H,
dd, J ¼ 8.1, 2.0 Hz), 8.45 (1H, s), 9.03e9.04 (1H, m). MS-ESI (m/z):
419 [M þ H]þ.
After a mixture of 35 (234 mg, 0.857 mmol) and conc. H2SO4
(1.00 mL, 18.0 mmol) was stirred at rt for 13 h, HNO3 (0.077 mL,
1.71 mmol) was added at 0 ꢂC. The mixture was stirred at 0 ꢂC for
1.5 h, then quenched with NaOH (2 M aqueous solution), and
basified with saturated aqueous NaHCO3 solution. The aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with H2O and brine, dried over Na2SO4,
filtered, and evaporated. The residue was purified by flash column
chromatography (silica gel; MeOH/CHCl3, gradient: 0e10% MeOH)
to give 36 (220 mg, 81%) as a brown gum. 1H NMR (400 MHz, CDCl3)
d 1.53 (3H, s), 3.18 (1H, d, J ¼ 15.9 Hz), 3.30 (1H, d, J ¼ 15.9 Hz), 7.15
(1H, dd, J ¼ 10.9, 8.9 Hz), 7.31 (1H, d, J ¼ 5.0 Hz), 8.10 (1H, ddd,
J ¼ 8.9, 4.0, 3.0 Hz), 8.55 (1H, s), 8.63 (1H, d, J ¼ 5.0 Hz), 8.68 (1H, dd,
J ¼ 7.0, 3.0 Hz). MS-ESI (m/z): 319 [M þ H]þ.
2-Fluoro-3-(2-(2-fluorophenyl)prop-1-en-1-yl)iso-
nicotinonitrile (40)
A
mixture of 14 (365 mg, 1.39 mmol), 2-chloro-3-
fluorobenzonitrile (218 mg, 1.39 mmol), K2CO3 (2 M in H2O;
2.09 mL, 4.18 mmol), and PdCl2(dppf)ꢃCH2Cl2 (56.9 mg,
0.070 mmol) in 1,4-dioxane (11 mL) was stirred at 100 ꢂC for 18 h
under N2. The resulting mixture was filtered through a Celite pad,
and the aqueous layer was then separated and extracted with
EtOAc. The combined organic layers were washed with brine, dried
over Na2SO4, filtered, and evaporated. The residue was purified by
flash column chromatography (silica gel; EtOAc/hexane, gradient:
0e10% EtOAc) to give a 1:1 diastereomeric mixture of 40 (174 mg,
tert-Butyl (3-(5-amino-2-fluorophenyl)-5-fluoro-3-methyl-
3,4-dihydro-2,7-naphthyridin-1-yl)carbamate (37)
49%) as a yellow oil. 1H NMR (400 MHz, CDCl3)
d
2.12 (d J ¼ 1.3 Hz,
3H, one isomer) and 2.36 (d, J ¼ 1.5 Hz, 3H, the other isomer), 6.52
(1H, s, one isomer), 6.56 (1H, s, the other isomer), 6.91e7.54 (5H, m),
8.12 (d, J ¼ 4.9 Hz, 1H, one isomer), 8.28 (d, J ¼ 5.0 Hz, 1H, one iso-
mer), 8.31 (d, J ¼ 5.0 Hz, 1H, the other isomer). MS-ESI (m/z): 257
[M þ H]þ.
After a mixture of 36 (174 mg, 0.549 mmol) and Boc2O (0.127 mL,
0.549 mmol) in THF (2.6 mL) was stirred at rt for 16 h, MeOH
(1.8 mL), H2O (0.7 mL), Fe (172 mg, 3.07 mmol), and NH4Cl (132 mg,
2.47 mmol) were added at rt. The mixture was stirred at 70 ꢂC for
1.5 h and then filtered through a Celite pad, and the filtrate was
evaporated. The residue was diluted with H2O and saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (silica
gel; EtOAc/hexane, gradient: 33e50% MeOH) to give 37 (196 mg,
2-Fluoro-3-(2-(2-fluorophenyl)prop-1-en-1-yl)iso-
nicotinimidamide (41)
To a suspension of NH4Cl (1.16 g, 21.7 mmol) in toluene (1.6 mL)
was added dropwise trimethylaluminum (10.8 mL, 21.7 mmol, 2 M
in toluene) at 0 ꢂC under N2. After the mixture was stirred at rt for
1 h, 40 (159 mg, 0.621 mmol) in toluene (3.2 mL) was added. The
mixture was stirred at 100 ꢂC for 23 h and then quenched with
potassium sodium tartrate and 2 M aqueous NaOH solution at 0 ꢂC.
The mixture was stirred at rt for 50 min. The aqueous layer was
separated and extracted with CHCl3. The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and evapo-
rated. The residue was purified by flash column chromatography
(silica gel; MeOH/CHCl3, gradient: 0e5% MeOH) to give a 3:2 dia-
stereomeric mixture of 41 (98.3 mg, 58%) as a colorless gum. 1H
92%) as a colorless amorphous. 1H NMR (400 MHz, CDCl3)
d 1.53
(3H, s), 3.18 (1H, d, J ¼ 15.9 Hz), 3.30 (1H, d, J ¼ 15.9 Hz), 7.15 (1H,
dd, J ¼ 10.9, 8.9 Hz), 7.31 (1H, d, J ¼ 5.0 Hz), 8.10 (1H, ddd, J ¼ 8.9,
4.0, 3.0 Hz), 8.55 (1H, s), 8.63 (1H, d, J ¼ 5.0 Hz), 8.68 (1H, dd, J ¼ 7.0,
3.0 Hz). MS-ESI (m/z): 389 [M þ H]þ.
tert-Butyl (3-(5-(5-cyanopicolinamido)-2-fluorophenyl)-5-
fluoro-3-methyl-3,4-dihydro-2,7-naphthyridin-1-yl)carbamate
(38)
NMR (400 MHz, CDCl3)
d
2.06 (t, J ¼ 1.4 Hz, 3H, minor isomer), 2.26
To a solution of 37 (80.9 mg, 0.208 mmol) in THF (1.6 mL) were
added 5-cyanopicolinic acid hydrate (41.5 mg, 0.250 mmol), HATU
(103 mg, 0.271 mmol), and Et3N (0.075 mL, 0.542 mmol) at 0 ꢂC. The
mixture was stirred at rt for 2 h and then quenched with saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (silica
gel; EtOAc/hexane, gradient: 25e40% EtOAc) to give 38 (104 mg,
(d, J ¼ 0.9 Hz, 3H, major isomer), 6.47 (1H, s), 6.86e7.45 (5H, m), 8.03
(d, J ¼ 5.0 Hz, 1H, major isomer), 8.23 (d, J ¼ 5.0 Hz, 1H, minor iso-
mer). MS-ESI (m/z): 274 [M þ H]þ.
5-Fluoro-3-(2-fluoro-5-nitrophenyl)-3-methyl-3,4-dihydro-
2,6-naphthyridin-1-amine (42)
After a mixture of 41 (82.9 mg, 0.303 mmol) and conc. H2SO4
(0.50 mL, 9.00 mmol) was stirred at rt for 15 h, HNO3 (0.027 mL,
0.607 mmol) was added at 0 ꢂC. The mixture was stirred at 0 ꢂC for
1 h and then quenched with 2 M aqueous NaOH solution and
basified with saturated aqueous NaHCO3 solution. The aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with H2O and brine, dried over Na2SO4,
filtered, and evaporated. The residue was purified by flash column
chromatography (silica gel; MeOH/CHCl3, gradient: 0e5% MeOH) to
give 42 (71.6 mg, 74%) as a white solid. 1H NMR (400 MHz, CDCl3)
96%) as a white solid. 1H NMR (400 MHz, CDCl3)
d 1.63 (9H, s), 1.89
(3H, s), 3.27 (1H, d, J ¼ 15.9 Hz), 3.77 (1H, d, J ¼ 15.9 Hz), 7.07 (1H,
dd, J ¼ 11.6, 8.9 Hz), 7.39 (1H, dd, J ¼ 7.2, 2.5 Hz), 7.73e7.79 (1H, m),
8.02 (1H, d, J ¼ 5.0 Hz), 8.19 (1H, dd, J ¼ 8.2, 2.0 Hz), 8.37 (1H, dd,
J ¼ 8.2, 0.8 Hz), 8.50 (1H, s), 8.54 (1H, d, J ¼ 5.0 Hz), 8.88 (1H, dd,
J ¼ 2.0, 0.8 Hz), 9.70 (1H, s), 10.72 (1H, s). MS-ESI (m/z): 519 [M þ
H]þ.
d
1.54 (3H, s), 3.11 (1H, d, J ¼ 16.5 Hz), 3.37 (1H, d, J ¼ 16.5 Hz), 4.93
N-(3-(1-Amino-5-fluoro-3-methyl-3,4-dihydro-2,7-
(1H, brs), 7.18 (1H, dd, J ¼ 10.8, 9.0 Hz), 8.13 (1H, dt, J ¼ 8.8, 3.2 Hz),
8.23 (1H, d, J ¼ 5.0 Hz), 8.70 (1H, d, J ¼ 4.3 Hz). MS-ESI (m/z): 319
[M þ H]þ.
naphthyridin-3-yl)-4-fluorophenyl)-5-cyanopicolinamide (8)
A mixture of 38 (65.0 mg, 0.125 mmol) and formic acid (0.65 mL,
17.0 mmol) was stirred at rt for 2.5 h. The reaction was quenched
with saturated aqueous NaHCO3 solution at 0 ꢂC. The aqueous layer
was separated and extracted with EtOAc and then CHCl3/MeOH.
The combined organic layers were washed with H2O and brine,
dried over Na2SO4, filtered, and evaporated. The residue was puri-
fied by flash column chromatography (silica gel; MeOH/CHCl3,
gradient: 0e3% MeOH). The crude product was recrystallized from
hexane/EtOAc/MeOH to give 8 (43.8 mg, 84%) as a white solid. 1H
tert-Butyl (3-(5-amino-2-fluorophenyl)-5-fluoro-3-methyl-
3,4-dihydro-2,6-naphthyridin-1-yl)carbamate (43)
After a mixture of 42 (58.6 mg, 0.184 mmol) and Boc2O
(0.043 mL, 0.184 mmol) in THF (0.9 mL) was stirred at rt for 16 h,
MeOH (0.6 mL), H2O (0.2 mL), Fe (57.6 mg, 1.03 mmol), and NH4Cl
(44.3 mg, 0.829 mmol) were added at rt. The mixture was stirred at
70 ꢂC for 1.5 h and filtered through a Celite pad, and the filtrate was
evaporated. The residue was diluted with H2O and saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
NMR (400 MHz, CD3OD)
d
1.75 (3H, s), 3.02 (1H, d, J ¼ 16.2 Hz), 3.78
11

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (silica
gel; EtOAc/hexane, gradient: 10e33% EtOAc) to give 43 (68.5 mg,
EtOAc) to give a 3:2 diastereomeric mixture of 46 (208 mg, 80%) as a
colorless oil. 1H NMR (400 MHz, CDCl3)
d
2.28 (t, J ¼ 1.5 Hz, 3H,
minor isomer), 2.34 (d, J ¼ 1.5 Hz, 3H, major isomer), 6.71 (1H, s, 1H,
major), 6.72 (1H, s, 1H, minor), 7.01e7.43 (5H, m), 7.98 (s, major
isomer), 8.34 (s, minor isomer), 8.56 (s, minor isomer), 8.71 (s, 1H,
minor isomer). MS-ESI (m/z): 257 [M þ H]þ.
96%) as a colorless amorphous. 1H NMR (400 MHz, CDCl3)
d 1.54
(3H, s), 1.84 (3H, s), 3.04 (1H, d, J ¼ 16.7 Hz), 3.50 (1H, brs), 3.84 (1H,
d, J ¼ 16.7 Hz), 6.32 (1H, dd, J ¼ 6.8, 2.8 Hz), 6.46 (1H, td, J ¼ 5.8,
3.0 Hz), 6.82 (1H, dd, J ¼ 11.7, 8.6 Hz), 7.94 (2H, d, J ¼ 5.6 Hz), 8.12
(2H, d, J ¼ 5.6 Hz), 10.57 (1H, s). MS-ESI (m/z): 389 [M þ H]þ.
tert-Butyl (3-(5-(5-cyanopicolinamido)-2-fluorophenyl)-5-
fluoro-3-methyl-3,4-dihydro-2,6-naphthyridin-1-yl)carbamate
(44)
3-Fluoro-5-(2-(2-fluorophenyl)prop-1-en-1-yl)iso-
nicotinimidamide (47)
To a suspension of NH4Cl (1.50 g, 28.0 mmol) in toluene (2.0 mL)
was added dropwise trimethylaluminum (14.0 mL, 28.0 mmol, 2 M
in toluene) at 0 ꢂC under N2. After the mixture was stirred at rt for
1 h, 46 (205 mg, 0.800 mmol) in toluene (4.0 mL) was added. The
mixture was stirred at 100 ꢂC for 25 h and then quenched with
potassium sodium tartrate and 2 M aqueous NaOH solution at 0 ꢂC.
The mixture was stirred at rt for 50 min. The aqueous layer was
separated and extracted with CHCl3. The combined organic layers
were washed with brine, dried over Na2SO4, filtered, and evapo-
rated. The residue was purified by flash column chromatography
(silica gel; MeOH/CHCl3, gradient: 0e5% MeOH) to give a 2:1 dia-
stereomeric mixture of 47 (180 mg, 82%) as a white solid. 1H NMR
To a solution of 43 (49.2 mg, 0.127 mmol) in THF (1.0 mL) at 0 ꢂC
were added 5-cyanopicolinic acid hydrate (25.3 mg, 0.152 mmol),
HATU (62.6 mg, 0.165 mmol), and Et3N (0.046 mL, 0.329 mmol). The
mixture was stirred at rt for 1.5 h and then quenched with saturated
aqueous NaHCO3 solution. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by flash column chromatography (silica
gel; EtOAc/hexane, gradient: 25e40% EtOAc) to give 44 (65.7 mg,
(400 MHz, CDCl3)
d 2.25e2.26 (s, 3H), 6.59 (1H, s), 6.90e7.39 (5H,
100%) as a white solid. 1H NMR (400 MHz, CDCl3)
d
1.64 (9H, s), 1.91
m), 7.82 (s, 1H, major isomer), 8.23 (s, 1H, major isomer), 8.46 (s, 1H,
minor isomer), 8.57 (s, 1H, minor isomer). MS-ESI (m/z): 274 [M þ
H]þ.
tert-Butyl (3-(5-amino-2-fluorophenyl)-8-fluoro-3-methyl-
3,4-dihydro-2,6-naphthyridin-1-yl)carbamate (48)
After a mixture of 47 (155 mg, 0.568 mmol) and conc. H2SO4
(1.0 mL, 18.000 mmol) was stirred at rt for 13 h, HNO3 (0.051 mL,
1.14 mmol) was added at 0 ꢂC. The mixture was stirred at 0 ꢂC for
2.5 h and then quenched with 2 M aqueous NaOH solution and
basified with saturated aqueous NaHCO3 solution. The aqueous
layer was separated and extracted with EtOAc. The combined
organic layers were washed with H2O and brine, dried over Na2SO4,
filtered, and evaporated to give 48 (155 mg, 86%) as a brown solid.
(3H, s), 3.13 (1H, d, J ¼ 16.8 Hz), 3.93 (1H, d, J ¼ 16.8 Hz), 7.09 (1H,
dd, J ¼ 11.5, 8.9 Hz), 7.42 (1H, dd, J ¼ 7.2, 2.6 Hz), 7.72e7.78 (1H, m),
7.96 (1H, d, J ¼ 5.2 Hz), 8.12 (1H, d, J ¼ 5.2 Hz), 8.19 (1H, dd, J ¼ 8.2,
2.1 Hz), 8.36 (1H, d, J ¼ 8.1 Hz), 8.88 (1H, d, J ¼ 1.7 Hz), 9.71 (1H, s),
10.71 (1H, s). MS-ESI (m/z): 519 [M þ H]þ.
N-(3-(1-Amino-5-fluoro-3-methyl-3,4-dihydro-2,6-
naphthyridin-3-yl)-4-fluorophenyl)-5-cyanopicolinamide (9)
A mixture of 44 (51.2 mg, 0.099 mmol) and formic acid (0.50 mL,
13.0 mmol) was stirred at rt for 14 h. The reaction was quenched
with NaOH (2 M aqueous solution) and saturated aqueous NaHCO3
solution at 0 ꢂC. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with H2O
and brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; MeOH/
CHCl3, gradient: 0e3% MeOH). The crude product was recrystal-
lized from hexane/EtOAc to give 9 (21.3 mg, 52%) as a white solid. 1H
NMR (400 MHz, CD3OD) d 1.74 (3H, s), 3.01 (1H, d, J ¼ 16.2 Hz), 3.66
(1H, d, J ¼ 16.2 Hz), 7.09 (1H, dd, J ¼ 11.9, 8.9 Hz), 7.64e7.67 (1H, m),
7.78 (1H, dd, J ¼ 7.4, 2.8 Hz), 8.13 (1H, d, J ¼ 5.1 Hz), 8.41 (1H, dd,
J ¼ 8.1, 2.0 Hz), 9.03e9.04 (1H, m). MS-ESI (m/z): 419 [M þ H]þ.
(S)eN-(3-(1-Amino-8-fluoro-3-methyl-3,4-dihydro-2,6-
1H NMR (400 MHz, CDCl3)
d
1.51 (3H, s), 3.12 (1H, d, J ¼ 15.8 Hz),
3.33 (1H, d, J ¼ 15.8 Hz), 7.16 (1H, dd, J ¼ 10.9, 8.9 Hz), 8.12 (1H, ddd,
J ¼ 8.9, 4.0, 3.0 Hz), 8.38 (1H, s), 8.48 (1H, d, J ¼ 2.2 Hz), 8.74 (1H, dd,
J ¼ 6.9, 3.0 Hz). MS-ESI (m/z): 319 [M þ H]þ.
tert-Butyl (3-(5-amino-2-fluorophenyl)-8-fluoro-3-methyl-
3,4-dihydro-2,6-naphthyridin-1-yl)carbamate (49)
After a mixture of 48 (153 mg, 0.480 mmol) and Boc2O (0.111 mL,
0.480 mmol) in THF (2.3 mL) was stirred at rt for 13 h, MeOH
(1.5 mL), H2O (0.6 mL), Fe (150 mg, 2.69 mmol), and NH4Cl (115 mg,
2.16 mmol) were added at rt. The mixture was stirred at 70 ꢂC for
1.5 h and filtered through a Celite pad, and the filtrate was evapo-
rated. The residue was diluted with H2O and saturated aqueous
NaHCO3 solution. The aqueous layer was separated and extracted
with EtOAc. The combined organic layers were washed with H2O
and brine, dried over Na2SO4, filtered, and evaporated. The residue
was purified by flash column chromatography (silica gel; EtOAc/
hexane, gradient: 10e33% EtOAc) to give 49 (164 mg, 88%) as a
naphthyridin-3-yl)-4-fluorophenyl)-5-cyanopicolinamide (11)
A mixture of 44 (2.00 g, 3.85 mmol) and HCl (EtOAc solution,
50 mL) was stirred at overnight. The reaction was quenched with
saturated aqueous NaHCO3. The aqueous layer was separated and
extracted with EtOAc. The combined organic layers were washed
with H2O and brine, dried over Na2SO4, filtered, and evaporated.
The residue was purified by SFC to give 9 (445 mg, 29%) as a white
solid. 1H NMR (400 MHz, CD3OD)
d 1.72 (3H, s), 3.10 (1H, d,
J ¼ 16.1 Hz), 3.60 (1H, d, J ¼ 16.1 Hz), 7.08 (1H, dd, J ¼ 11.8, 8.8 Hz),
7.66e7.71 (1H, m), 7.80 (1H, dd, J ¼ 7.4, 2.7 Hz), 8.33e8.36 (2H, m),
8.41e8.44 (2H, m), 9.04e9.05 (1H, m). MS-ESI (m/z): 419 [M þ H]þ.
3-Fluoro-5-(2-(2-fluorophenyl)prop-1-en-1-yl)iso-
nicotinonitrile (46)
yellow solid. 1H NMR (400 MHz, CDCl3)
d 1.59 (9H, s), 1.81 (3H, s),
3.17 (1H, d, J ¼ 15.7 Hz), 3.52 (2H, s), 3.74 (1H, d, J ¼ 15.7 Hz),
6.41e6.54 (1H, m), 6.87 (1H, dd, J ¼ 11.7, 8.6 Hz), 8.22 (1H, s), 8.39
(1H, d, J ¼ 2.5 Hz), 10.68 (1H, s). MS-ESI (m/z): 389 [M þ H]þ.
N-(3-(1-Amino-8-fluoro-3-methyl-3,4-dihydro-2,6-
A
mixture of 14 (267 mg, 1.02 mmol), 3-bromo-5-
naphthyridin-3-yl)-4-fluorophenyl)-5-cyanopicolinamide (10)
To a solution of 49 (80.3 mg, 0.207 mmol) in THF (2.4 mL) at 0 ꢂC
were added 5-cyanopicolinic acid hydrate (41.2 mg, 0.248 mmol),
HATU (102 mg, 0.269 mmol), and Et3N (0.075 mL, 0.538 mmol). The
mixture was stirred at rt for 2.5 h and then quenched with satu-
rated aqueous NaHCO3 solution. The aqueous layer was separated
and extracted with EtOAc. The combined organic layers were
washed with H2O and brine, dried over Na2SO4, filtered, and
evaporated. The residue was purified by flash column
fluoroisonicotinonitrile (45, 205 mg, 1.02 mmol), K2CO3 (2 M in
H2O; 1.53 mL, 3.06 mmol), and PdCl2(dppf)ꢃCH2Cl2 (41.7 mg,
0.051 mmol) in THF (8 mL) was stirred at 70 ꢂC for 24 h under N2.
The resulting mixture was filtered through a Celite pad, and the
aqueous layer was then separated and extracted with EtOAc. The
combined organic layers were washed with brine, dried over
Na2SO4, filtered, and evaporated. The residue was purified by flash
column chromatography (silica gel; EtOAc/hexane, gradient: 0e15%
12

K. Nakahara, Y. Mitsuoka, S. Kasuya et al.
European Journal of Medicinal Chemistry 216 (2021) 113270
chromatography (silica gel; EtOAc/hexane, gradient: 33e67%
EtOAc) to give a white solid (133 mg, impure).
PDB code
7D36
A mixture of the solid (97.1 mg) and formic acid (0.50 mL,
13.0 mmol) was stirred at rt for 18 h. The reaction was quenched
with NaOH (2 M aqueous solution) and saturated aqueous NaHCO3
at 0 ꢂC. The aqueous layer was separated and extracted with EtOAc.
The combined organic layers were washed with H2O and brine,
dried over Na2SO4, filtered, and evaporated. The residue was puri-
fied by flash column chromatography (silica gel; EtOAc/hexane,
gradient: 50e80% EtOAc). The crude product was recrystallized
from hexane/EtOAc/MeOH to give 10 (48.6 mg, 62%) as a white
Wavelength (Å)
Resolution range (Å)
Space group
1.5418
50.0e2.30 (2.34e2.30)
P 6122
102.531 102.531 170.115 90.0 90.0120.0
24 100
24 100
99.5 (99.3)
8.3 (8.4)
24.5 (3.7)
46.2
Unit cell
Total reflections
Unique reflections
Completeness (%)
Redundancy
Mean I/sigma(I)
Wilson B-factor
R-merge
Reflections used in the refinement
R-work
R-free
solid. 1H NMR (400 MHz, CD3OD)
d 1.72 (3H, s), 3.10 (1H, d,
9.5 (52.0)
21 623
0.213
0.265
J ¼ 16.1 Hz), 3.60 (1H, d, J ¼ 16.1 Hz), 7.08 (1H, dd, J ¼ 11.8, 8.8 Hz),
7.66e7.71 (1H, m), 7.80 (1H, dd, J ¼ 7.4, 2.7 Hz), 8.33e8.36 (2H, m),
8.41e8.44 (2H, m), 9.04e9.05 (1H, m). MS-ESI (m/z): 419 [M þ H]þ.
Number of non-hydrogen atoms
macromolecules
ligands
2902
32
5.2. Biochemical BACE1 Assay
RMS (bonds)
RMS (angles)
0.011
1.363
97.6
2.40
0.00
0
Ramachandran favored (%)
Ramachandran allowed (%)
Ramachandran outliers (%)
Rotamer outliers (%)
Average B-factor
macromolecules
Ligands
The biochemical BACE1 IC50 values were measured by HTRF
assay using an APP-derived peptide as reported previously.
5.3. Cellular A
b
Assay
IC50 values were determined by measuring Ab40
45.2
56.1
45.3
The cellular A
b
Solvent
using HTRF assay in SH-SY5Y cells expressing human APP as re-
ported previously.
Accession codes
5.4. In Vitro ADMET Assays
The PDB accession code for the X-ray structure of 11 bound to
Metabolic stability in microsomes, P-gp efflux ratio, solubility,
protein binding, and hERG inhibitory activity were determined
according to procedures reported previously.
human BACE1 is 7D36.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
5.5. pKa assays
pKa values were determined using capillary electrophoresis
method according to procedures reported previously.9,12a
Acknowledgements
5.6. In vivo experiments
We thank Maki Hattori, Masanori Nakae, and Maito Douma for
the biological assays and Eriko Matsuoka for the X-ray analysis. We
gratefully thank the members of the Janssen-Shionogi research
collaboration for the support of this program. We gratefully thank
Dr. Harrie J.M. Gijsen for his help with reviewing the manuscript.
We are grateful to Judy Noguchi for proofreading the manuscript.
All the procedures for the animal studies were in accordance
with regulations and established guidelines and were approved by
the Shionogi Animal Care and Use Committee.
5.6.1. In vivo pharmacokinetics study
The test compound was dissolved in 20% HPBCD, and the details
were reported previously.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113270.
5.6.2. Dog PK/PD study
The test compound was suspended in 0.5% MC and was orally
administered to beagle dogs (n ¼ 3) at 1 mg/kg dose. The total A
b
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European Journal of Medicinal Chemistry 216 (2021) 113270
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