Discovery of a potent and selective Axl inhibitor in preclinical model

Bioorg. Med. Chem. 39 (2021) 116137

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Bioorganic & Medicinal Chemistry

journal homepage: www.elsevier.com/locate/bmc

Discovery of a potent and selective Axl inhibitor in preclinical model

,

Satoshi Inoue^{a *}, Yoshinobu Yamane^a, Shuntaro Tsukamoto^b, Hiroshi Azuma^a, Satoshi Nagao^a,

Norio Murai^a, Kyoko Nishibata^b, Sayo Fukushima^b, Kenji Ichikawa^b, Takayuki Nakagawa^b,

Naoko Hata Sugi^b, Daisuke Ito^b, Yu Kato^b, Aya Goto^c, Dai Kakiuchi^c, Takashi Ueno^d,

Junji Matsui^b, Tomohiro Matsushima^a

^aMedicinal Chemistry, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

^bBiopharmacology, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

^cDrug Safety, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

^dDrug Metabolism and Pharmacokinetics, Tsukuba Research Laboratories, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan

A R T I C L E I N F O

A B S T R A C T

Keywords:

Axl and Mer are a members of the TAM (Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when

activated, can promote tumor cell survival, proliferation, migration, invasion, angiogenesis, and tumor-host

interactions. Chronic inhibition of Mer leads to retinal toxicity in mice. Therefore, successful development of

an Axl targeting agent requires ensuring that it is safe for prolonged treatment. Here, to clarify whether enzyme

inhibition of Mer by a small molecule leads to retinal toxicity in mice, we designed and synthesized Axl/Mer

inhibitors and Axl-selective inhibitors. We identiﬁed an Axl/Mer dual inhibitor 28a, which showed retinal

toxicity at a dose of 100 mg/kg in mice. Subsequent derivatization of a pyridine derivative led to the discovery of

a pyrimidine derivative, 33g, which selectively inhibited the activity of Axl over Mer without retinal toxicity at a

dose of 100 mg/kg in mice. Additionally, the compound displayed in vivo anti-tumor effects without inﬂuencing

body weight in a Ba/F3-Axl isogenic subcutaneous model.

Axl

Mer

Retinal toxicity

Structure–activity relationships

Cancer

Inhibitor

Small molecule

1. Introduction

therapies.^4–9Inhibition of Axl signaling suppresses such resistance in a

variety of cancer types.¹⁰Therefore, we consider Axl to be a crucial drug

Drug resistance to conventional, targeted therapy and immune

checkpoint inhibitors is a major cause of failure of anticancer treatment.

Understanding of resistance mechanisms and identiﬁcation of the mol-

ecules driving resistance will allow development of targeted agents that

can destroy the drug resistant cancer. Axl is a member of the TAM

(Tyro3-Axl-Mer) family of receptor tyrosine kinases, which, when acti-

vated, can promote tumor cell survival, proliferation, migration and

invasion, angiogenesis, and tumor host interactions.¹Axl is often highly

expressed in resistant tumor cells that show mesenchymal

phenotypes.^2–3Furthermore, Axl is involved in acquired drug resistance

to multiple chemotherapies and molecular targeted-, radio- and immuno

target to eradicate therapy-resistant cancer cells to improve treatment

outcomes.

Several small molecular inhibitors showing Axl inhbitory activity¹¹

have already been developed, e.g. gilteritinib (ASP2215),¹²TP-0903,¹³

foretinib (XL-880),¹⁴and S49076.¹⁵However, these agents are multi-

kinase inhibitors with high potency against not only Axl but also other

relevant kinases, including FLT3, c-Met (also known as MET), ALK, and

VEGFR2 kinase (also known as KDR). Recently, some selective inhibitors

have been developed: BGB324¹⁶and DS-1205b¹⁷were reported as se-

lective Axl inhibitors; ONO-7475¹⁸and RXDX-106¹⁹were reported as a

selective Axl/Mer inhibitor (Fig. 1).

Abbreviations: ALK, anaplastic lymphorma kinase; Boc, tert-butoxycarbonyl; Cbz, benzyloxycarbonyl; DCM, dichloromethane; DDR1, discoidin domain receptor 1;

DMF, N,N-dimethylformamide; DMSO, dimethyl sulfoxide; EtOH, ethanol; Et₃N, triethylamine; EtOAc, ethyl acetate; EtONa, sodium ethoxide; FLT3, fms-like tyrosine

kinase 3; FLT4, fms-related tyrosine kinase 4; HATU, 1-[Bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-Oxide Hexaﬂuorophosphate; iPr₂NEt,

N,N-Diisopropylethylamine; K₂CO₃, potassium carbonate; KDR, kinase insert domain receptor; MeOH, methanol; MgSO₄, magnesium sulfate; i-PrOH, 2-propanol;

NaHCO₃, sodium hydrogen carbonate; NH₄Cl, ammonium chloride; NMP, N-methylpyrrolidone; RT, room temperature; TAM, Tyro3-Axl-Mer; TBME, tert-butyl

methyl ether; TFA, triﬂuoroacetic acid; THF, tetrahydrofuran; TLR, toll-like receptor; VEGFR2, vascular endothelial growth factor receptor 2.

* Corresponding author at: Satoshi Inoue, Eisai Co., Ltd., 5-1-3 Tokodai, Tsukuba-shi, Ibaraki 300-2635, Japan.

E-mail address: s4-inoue@hhc.eisai.co.jp (S. Inoue).

https://doi.org/10.1016/j.bmc.2021.116137

Received 3 January 2021; Received in revised form 18 March 2021; Accepted 22 March 2021

Available online 21 April 2021

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

The main function of TAM family members in normal tissues is

synthesize aniline derivatives 19d-e, 14a-b were synthesized from 4-

chloropyridine-2-carboxamide (8) by S_NAr in the presence of amino-

phenol derivatives 9a-b. Protection of the aniline moiety of 10a-b by

benzyloxycarbonyl (Cbz), followed by Hofmann rearrangement, affor-

ded 12a-b, respectively. Condensation of 12a-b with 1-Boc-piperidine-

4-ylacetic acid, followed by deprotection of the Boc group and reduc-

tive amination with formaldehyde generated 19d-e, respectively.

Condensation of 19d with 7b produced 20c.

clearance of apoptotic cells and dampening of Toll-like receptor (TLR)-

dependent inﬂammatory responses and natural killer cell activity.^20–21

Chronic inhibition of Mer leads to retinal toxicity in a mouse model.²²In

Mer knock-out mice, retinal pigment epithelial cells cannot clear

apoptotic-like outersegment debris, resulting in retinal degeneration.

The retinal toxicity of the multi kinase inhibitors as mentioned above

and the four selective Axl inhibitors shown in Fig. 1 has not been pub-

lished, except in patents where the retinal toxicity of DS-1205b has been

mentioned. To clarify whether enzyme inhibition of Mer by small mol-

ecules leads to retinal toxicity in mice, we focused on the selective Axl/

Mer inhibitors RXDX-106 and ONO-7475. We speculated that the Axl

selectivity of these compounds might be increased if the right end part

were convert to the uracil or hexahydroquinoline-2,5-dione moiety,

because some multi kinase inhibitors consist of a smaller moiety like

pyridone or cyclopropane dicarboxamide. In the present study, we

replaced a pyridine and a pyrimidine in the quinoline moiety of RXDX-

106 and ONO-7475 in an attempt to produce an original Axl inhibitor

with drug-like properties.

Pyridine derivatives containing hexahydroquinoline-2,5-dione were

synthesized as shown in Scheme 3. Carboxylic acid (24) was synthesized

by cyclization of ethyl (ethoxymethylene)cyanoacetate (21), followed

by amidation and hydroxylation. Compounds 25a-d were synthesized

by condensation of 19b-e with 24.

With the aim of improving the potency and the microsomal stability

of 20a-e and 25a-d, ureidopyridine derivatives 28a-b were synthesized

from 10b by condensation with 7b and 24, followed by Hofmann

rearrangement and ureidation via phenyl carbamate as shown in

Scheme 4.

To investigate Axl selectivity over Mer in the cell based assay using

Ba/F3 driven with activity of each kinase, pyrimidine derivatives 33a-j

were synthesized as shown in Scheme 5. Compound 31 was synthesized

by S_NAr from aminochloropyrimidine (29) in the presence of amino-

phenol (30). Condensation of 31 with 7a-d afforded 32a-d, respectively.

Amidation of 32a with cyclopropanecarbonyl chloride generated 33a.

Ureidation of 32a-d with the corresponding cyclic amine via phenyl

carbamate produced 33b-j, respectively.

We report the synthesis of a series of pyridine and pyrimidine com-

pounds and the selection of the optimal compound for Axl inhibition by

analysis of structure–activity relationships (SARs), optimization of drug-

likeness parameters, and pathological evaluation for in vivo retinal

toxicity.

2. Chemistry

The primary purpose of the chemistry research was to develop a

novel Axl inhibitor. RXDX-106 and ONO-7475 each contain a quinoline

moiety. To investigate the effect of uracil substituents on the pyridine

framework, we synthesized pyridine derivatives of RXDX-106 as shown

in Schemes 1 and 2. To produce uracil-5-carboxylic acid derivatives 7a-

c, 3 was synthesized from 1 by amination using ammonia, and 4 was

produced from 3 by ureidation with 4-ﬂuorophenyl isocyanate. Cycli-

zation of 4 in the presence of sodium ethoxide generated 5. Alkylation of

5 with iodomethane or 2-iodopropane or cyclopentyl bromide, followed

by hydroxylation, generated the derivatives 7a-c, respectively. A

different synthetic route was used to produce uracil-5-carboxylic acid

derivative 7d. Compound 1 was aminated with cyclopropylamine to

produce 2, and then cyclization with 4-ﬂuorophenyl isocyanate in the

presence of K₂CO₃generated 6d. Hydroxylation of 6d under acidic

condition generated 7d.

3. Results and discussion

To investigate intrinsic inhibitory activity against target kinases, the

pyridine and pyrimidine derivatives were ﬁrstly evaluated with two

assays, a cell-free Axl kinase assay and a cell-free Mer kinase assay.

Additionally, some compounds were evaluated with a cell proliferation

assay against Axl-dependent Ba/F3 cells (Ba/F3-Axl) and Mer-

dependent Ba/F3 cells (Ba/F3-Mer). A solubility assay and microsomal

stability assay in mouse liver microsomes were also conducted for drug-

like properties.

Table 1 lists the results of the inhibitory activity, solubility, and

microsomal stability assays for the pyridine derivatives containing a

uracil moiety. Regarding the substituents on R³, 20b showed more

potent inhibitory activity than 20a in the cell-free Axl and Mer assays.

Regarding the substituents on R⁴, 20e showed more potent inhibitory

activity than 20d in the cell-free Axl and Mer assays. No clear effect of

ﬂuorine substituents on R¹and R²was seen. The Axl inhibitory activity

of 20b and 20e was more than 10-fold higher that the Mer inhibitory

activity in the cell-free assays, indicating Axl selectivity; however only a

small difference was observed in the cell-based assay.

Various aniline derivatives, 19a-e, were created to investigate the

substituent effect of ﬂuorine. The synthetic route differed depending on

the position of the ﬂuorine. To produce aniline derivatives 19a-c, 17a-b

were synthesized from 2-amino-4-chloropyridine (15) by nucleophilic

aromatic substitution (S_NAr) in the presence of nitrophenol derivatives

16a-b. Amidation of 17a with N,N-dimethylglycine, and amidation of

17a-b with 1-Boc-piperidine-4-ylacetic acid generated 18a-c. Depro-

tection of the Boc group of 18b-c, followed by reductive amination with

formaldehyde, afforded 18d-e, respectively. Reduction of the nitro

group of 18a and 18d-e with Pd/C afforded 19a-c, respectively.

Condensation of 19a-b and 19d with 7b generated 20a-c, respectively.

Condensation of 19c with 7d and 7c generated 20d-e, respectively. To

Table 2 lists the result for the equivalent analyses of pyridine de-

rivatives containing hexahydroquinoline-2,5-dione, 25a-d. In general, it

was found that the hexahydroquinoline-2,5-dione derivatives showed

moderate potency, but weaker potency than the uracil derivatives in the

cell-free assay. Regarding the effect of ﬂuorine substituents on R¹and

R², 25d which had ﬂuorine substituents on both R¹and R², showed the

highest Axl selectivity in the cell-free assay (Table 2), but this selectivity

Fig. 1. Chemical structure of previously reported Axl selective inhibitors.

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Bioorganic & Medicinal Chemistry 39 (2021) 116137

Scheme 1. Reagents and conditions: (a) cyclopropylamide, EtOH, RT; (b) 4-ﬂuorophenyl isocyanate, K₂CO₃, DMF, 60 ^◦C; (c) ammonia in MeOH, 0 ^◦C to RT; (d) 4-

ﬂuorophenyl isocyanate, iPr₂NEt, 1,2-dichloroethane, reﬂux; (e) EtONa, EtOH, 0 ^◦C to RT; (f) alkyl halide, K₂CO₃, DMF, 70 ^◦C; (g) HCl in 1,4-dioxane/H₂O, 70–80 ^◦C.

Scheme 2. Reagents and conditions: (a) t-BuOK, DMSO, 80 ^◦C or 90 ^◦C; (b) benzyl chloroformate, NaHCO₃, acetone, H₂O, RT; (c) [bis(triﬂuoroacetoxy)iodo]

benzene, pyridine, DMF, H₂O, RT; (d) 1) benzotriazole, thionyl chloride, 1-Boc-piperidine-4-ylacetic acid, DCM, RT, 2) Et₃N, THF, 0 ^◦C to RT; (e) TFA, DCM, RT, then

formaldehyde, NaBH(OAc)₃, DCM, RT; (f) Palladium on carbon, hydrogen, MeOH, RT; (g) iPr₂NEt, NMP, 150 ^◦C; (h) N,N-dimethylglycine, HATU, iPr₂NEt, DMF,

80 ^◦C; (i) HATU, iPr₂NEt, DMF, RT to 70 ^◦C.

was not as high as that of the uracil derivatives (Table 1).

assay model, but no selectivity was observed in the cell-based assay.

Of these two compounds, 28a showed higher solubility and micro-

somal stability with potency against Axl and Mer like RXDX-106 and

ONO-7475 (Table S2). We therefore, performed an in vivo test using 28a

to evaluate its retinal toxicity. Compound 28a was administered once

daily at 100 mg/kg for 14 days to BALB/c mice. There was no signiﬁcant

We then generated ureidopyridine derivatives in an attempt to in-

crease solubility and microsomal stability while keeping potency

(Table 3). Similar to the trend described above for pyridine derivatives

containing a uracil moiety, 28a-b respectively showed more than 47-

fold and 19-fold greater selectivity for Axl than for Mer in the cell-free

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S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

Scheme 3. Reagents and conditions: (a) tBuOK, DMF, RT; (b) aniline, EtOH, RT; (c) HATU, iPr₂NEt, DMF, RT or 80 ^◦C.

Scheme 4. Reagents and conditions: (a) 7b or 24, HATU, iPr₂NEt, RT to 75 ^◦C; (b) [bis(triﬂuoroacetoxy)iodo]benzene, pyridine, DMF, H₂O, RT;(c) 1) phenyl

chloroformate, iPr₂NEt, THF, 0 ^◦C to RT, 2) 1-methyl-4-(piperidin-4-yl)-piperazine, iPr₂NEt, DMF, RT.

Scheme 5. Reagents and conditions: (a) t-BuOK, DMSO, 100 ^◦C; (b) 7a-d, HATU, iPr₂NEt, DMF, RT or 70 ^◦C; (c) cyclopropanecarbonyl chloride, iPr₂NEt, THF, RT or

1) phenyl chloroformate, iPr₂NEt, THF, RT, 2) amine, iPr₂NEt, DMF, RT.

body weight loss during the dosing period (Fig. S1). The eyes were

collected and submitted to the histopathologic examination. Similar to

chronic inhibition of Mer,²²retinal toxicity, characterized by degener-

ation or necrosis of photoreceptor cells in the outer nuclear layer

throughout the retina, was observed in all treated mice (Fig. 2). We then

performed cell-free kinase assays on a panel of 52 kinases to clarify the

kinase selectivity of 28a (Table S1). Compound 28a showed inhibitory

activity not only against Axl and Mer, but also against Tyro3, c-Met, and

LCK. Although there are no reports of retinal toxicity based on inhibition

of Tyro3, c-Met or LCK, we hypothesized that retinal toxicity led to Mer

inhibition and could be avoided by increasing the inhibitor’s selectivity

against Axl over Mer.

cell-based assay (33j > 33g > 33i > 33h).

Because 33g showed good solubility and microsomal stability for

oral administration with 4-fold Axl selectivity in the cell-based assay

(Table 4), we explored this compound further. First, we evaluated

pharmacokinetic proﬁles of 33g after single oral administration of 10,

30 or 100 mg/kg in female Balb/c mice (Table 5). Plasma concentration

of 33g peaked at 2 h postdose, and C_maxand AUC_(0-24h)of 33g increased

dose-dependently in the dose range of 10–100 mg/kg. To evaluate the

retinal toxicity, histopathologic examination was conducted in the eyes

of BALB/c mice that were administered 33g at 100 mg/kg once daily for

14 days. There was no signiﬁcant body weight loss (Fig. S1) and no

histological change in the retina (Fig. 2). We conclude that 33g did not

induce retinal toxicity. Next, we evaluated the antitumor effects of 33g

in the Ba/F3-Axl isogenic subcutaneous model (Fig. 3). Oral adminis-

tration of 33g at 1, 3 and 10 mg/kg once daily for 4 days inhibited tumor

growth in this model without signiﬁcant body weight loss. We then

conducted kinome assay (as described above for 28a) to clarify the ki-

nase selectivity of 33g (Table S1). Unlike 28a, compound 33g showed

highly selective Axl inhibition. These results suggest that ureidopyr-

imidine derivatives may be highly selective Axl inhibitors. Further

studies are needed to clarify the precise mechanism of ureidopyrimidine

action.

In an attempt to improve the Axl selective of the inhibitor, we

designed and synthesized pyrimidine derivatives containing a uracil

moiety. Table 4 lists the result of the inhibitory activity, solubility, and

microsomal stability assays for the resultant derivatives, 33a-j. All of

these compounds showed good potency in the cell-free Axl except for

33h. The amide derivative 33a did not show selectivity for Axl over Mer

in the cell-based assay, but the ureidopyrimidine derivatives 33b-g

showed 3- to 6-fold more selective for Axl than for Mer. Compound 33c

showed good potency and selectivity in the cell-based assay, but solu-

bility was low. Though 33e and 33f were unstable in mouse liver

microsome, other compounds showed moderate to high microsomal

stability. As for the effect of substituents on R¹, compounds with bulky

moieties showed more potency and selectivity in the cell-free assay and

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Bioorganic & Medicinal Chemistry 39 (2021) 116137

Table 1

Effect of substitution of R¹-R⁴on in vitro biological activity, solubility and microsomal stability of pyridine derivatives containing uracil moiety.

^*1Solubility in Dulbecco’s phosphate-buffered saline.

^*2Microsomal stability in mouse liver microsomes. The ratio of peak area responses relative to the internal standard was determined, and the residual ratio of the test

articles in the presence of NADPH relative to that in the absence of NADPH was calculated.

Table 2

Effect of substitution of R¹-R²on in vitro biological activity, solubility and microsomal stability of pyridine derivatives containing hexahydroquinoline-2,5-dione

moiety.

4. Conclusions

pyrimidine derivative 33g, which at a dose of 100 mg/kg selectively

inhibited the activity of Axl over Mer without retinal toxicity in mouse.

Additionally, 33g displayed in vivo anti-tumor effects without inﬂu-

encing body weight in the Ba/F3-Axl isogenic subcutaneous model.

We designed a novel series of pyridine derivatives as Axl/Mer in-

hibitors. Compound 28a at a dose of 100 mg/kg showed retinal toxicity

in mouse. However, subsequent derivatization led to the discovery of

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Bioorganic & Medicinal Chemistry 39 (2021) 116137

Table 3

In vitro proﬁle of biological activity, solubility and microsomal stability of ureidopyridine derivatives.

Fig. 2. Histological assessment of the retina. (A)

Normal retina in control (untreated) mice, (B) Retina

in mice treated with 28a at 100 mg/kg. Degeneration/

necrosis of photoreceptor cells in the outer granular

layer (arrows), resulting in thinning of the retina, was

observed, (C) Retina in mice treated with 33g at 100

mg/kg. No histopathologic changes were detected. All

slides were stained with hematoxylin-eosin (HE)

stained slides. Scale bars, 100

μm. Images are repre-

sentative of sections from 3 or 5 mice.

5. Experimental sections

3.7 Hz, 1H), 4.19 (dq, J = 18.5, 7.3 Hz, 4H), 8.09 (d, J = 14.1 Hz, 1H),

9.21 (br d, J = 12.2 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 6.6, 14.4,

14.5, 29.6, 59.6, 59.9, 90.5, 160.3, 166.0, 169.2; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₁H₁₈NO⁺₄: 228.1230, found: 228.1226.

5.1. Chemistry

5.1.1. General methods

¹H and ¹³C nuclear magnetic resonance (NMR) spectra were recor-

ded on a JEOL spectrometer (operating at 500 MHz for ¹H and 126 MHz

for ¹³C or 400 MHz for ¹H and 101 MHz for ¹³C). Chemical shifts were

expressed in ppm (δ) from the residual CHCl₃signal at δ 7.26 ppm and

δ_C77.0 ppm in CDCl₃or the residual CHD₂SOCD₃signal^Hat δ_H2.50 ppm

and δ_C39.5 ppm in CD₃SOCD₃(s = singlet, d = doublet, t = triplet, q =

qualtet, quin = quintet, sep = septet, m = multiplet, and br = broad).

Coupling constants (J) are given in Hertz (Hz). High-resolution mass

spectra (HRMS) were recorded on a Thermo Fisher LTQ Orbitrap XL

spectrometer using electrospray ionization (ESI) and a Waters GCT

Premier using electron ionization (EI). Column chromatography was

5.1.3. Diethyl 2-((3-(4-ﬂuorophenyl)ureido)methylene)malonate (4)

To a solution of 1,3-diethyl 2-(aminomethylidene)propanedioate²³

(3) (11.3 g, 60.3 mmol) in 1,2-dichloroethane (50 mL) were added 4-ﬂu-

orophenyl isocyanate (6.86 mL, 60.3 mmol) and N,N-diisopropylethyl-

amine (11.1 mL, 63.3 mmol) at room temperature. The reaction mixture

was reﬂuxed for 21 h. The mixture was cooled to room temperature, and

the precipitate was collected by ﬁltration, rinsed with TBME. The

collected precipitate was puriﬁed with column chromatography on silica

gel (n-heptane/ethyl acetate (EtOAc) = 1/1 ~ EtOAc) to afford the title

compound 4 as a white solid (8.30 g, 25.6 mmol, 42.4%).

¹H NMR (500 MHz, DMSO‑d₆): δ = 1.10–1.33 (m, 6H), 4.11 (q, J =

7.3 Hz, 2H), 4.20 (q, J = 6.7 Hz, 2H), 7.15 (t, J = 8.9 Hz, 2H), 7.47 (dd, J

= 8.6, 4.9 Hz, 2H), 8.33–8.53 (m, 1H), 10.26–10.44 (m, 1H),

10.46–10.72 (m, 1H); ¹³C NMR (126 MHz, DMSO‑d₆): δ = 14.6, 14.7,

60.5, 60.8, 98.4, 116.2 (d, J_CF= 22.7 Hz), 121.1 (d, J_CF= 7.6 Hz), 135.1,

148.3, 150.5, 158.7 (d, J_CF= 239.4 Hz), 164.8, 166.9; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₅H₁₈FN₂O⁺₅: 325.1194, found: 325.1185.

carried out using silica gel 60 (spherical) (40–50 μm, Kanto Chemical

Co., Inc.), Chromotorex (200–350 mesh, Fuji Silysia Chemical Ltd.),

Isolera™ One (silica gel and NH-silica gel, Biotage) and a Hi-Flash™

column (silica gel and NH-silica gel, Yamazen Corporation). Chemicals

and solvents used in the study were commercially available.

5.1.2. Diethyl 2-((cyclopropylamino)methylene)malonate (2)

To a solution of diethyl ethoxymethylenemalonate (1) (1.00 g, 4.63

mmol) in EtOH (20 mL) was added cyclopropylamine (290 mg, 5.09

mmol) in EtOH (2 mL) at room temperature. The reaction mixture was

stirred at room temperature for 30 min. The reaction mixture was

concentrated and the residue was puriﬁed with column chromatography

on NH silica gel (n-heptane/EtOAc = 9/1 ~ 1/1) to afford the title

compound 2 as a colorless oil (1.03 g, 4.53 mmol, 98%).

5.1.4. Ethyl 3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

carboxylate (5)

To a solution of diethyl 2-((3-(4-ﬂuorophenyl)ureido)methylene)

malonate (4) (8.27 g, 25.5 mmol) in EtOH (100 mL) was added 20%

◦

NaOEt in EtOH solution (19.7 mL, 51.0 mmol) at 0 C and stirred at

room temperature for 2 h. The reaction mixture was concentrated. The

residue was poured into saturated aqueous NH₄Cl and extracted with

DCM/i-PrOH = 10/1 solution, 5 times. The organic extract was dried

over MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed

¹H NMR (500 MHz, CDCl₃): δ = 0.71–0.72 (m, 2H), 0.79–0.81 (m,

2H), 1.28 (t, J = 7.0 Hz, 3H), 1.32 (t, J = 7.0 Hz, 3H), 2.84 (qd, J = 6.7,

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S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

Table 4

Effect of substitution of R¹-R²on in vitro biological activity, solubility and microsomal stability pyrimidine derivatives containing uracil moiety.

with column chromatography on silica gel (n-heptane/EtOAc = 1/1 ~

EtOAc ~ EtOAc/MeOH = 9/1) to afford the title compound 5 as a white

solid (6.15 g, 22.1 mmol, 87%).

5.1.5. Ethyl 3-(4-ﬂuorophenyl)-1-methyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylate (6a)

To a solution of ethyl 3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahy-

dropyrimidine-5-carboxylate (5) (300 mg, 1.08 mmol) in DMF (6 mL)

¹H NMR (400 MHz, DMSO‑d₆): δ = 1.19 (t, J = 7.1 Hz, 3H), 4.14 (q, J

= 6.9 Hz, 2H), 7.13–7.36 (m, 4H), 8.21 (s, 1H), 11.76–12.15 (m, 1H);

were added iodomethane (134 μL, 2.16 mmol) and K₂CO₃(298 mg, 2.16

¹³C NMR (126 MHz, DMSO‑d₆): δ = 14.7, 60.6, 103.7, 116.2 (d, J_CF

=

mmol) at room temperature. The reaction mixture was stirred at 70 ^◦C

overnight, then cooled to room temperature. The reaction mixture was

poured into water and extracted with EtOAc. The organic extract was

washed with water and brine, dried over MgSO₄, ﬁltered and then

concentrated. The residue was puriﬁed with column chromatography on

silica gel (n-heptane/EtOAc = 7/3 ~ EtOAc) to afford the title

22.7 Hz), 131.5 (d, J_CF= 8.8 Hz), 132.0, 149.0, 151.3, 159.8, 162.1 (d,

J_CF

=

244.4 Hz), 163.2; HRMS–ESI m/z [M+H]⁺calcd. for

C

₁₃H₁₂FN₂O⁺₄: 279.0776, found: 279.0768.

7

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

(2) (976 mg, 4.30 mmol) in DMF (20 mL) were added 4-ﬂuorophenyl

Table 5

Pharmacokinetic parameters* of 33g in mice.

isocyanate (928

μL, 8.16 mmol) and K₂CO₃(1.13 g, 8.16 mmol) at

room temperature. The reaction mixture was stirred at 60 ^◦C overnight

under nitrogen, then cooled to room temperature. The reaction mixture

was poured into water and extracted with EtOAc. The organic extract

was washed with water and brine, dried over MgSO₄, ﬁltered and then

concentrated. The residue was puriﬁed with column chromatography on

silica gel (n-heptane/EtOAc = 7/3 ~ EtOAc) to afford the title com-

pound 6d as a white solid (879 mg, 2.76 mmol, 64.3%).

Parameter

Dose (mg/kg)

10

30

100

C_max

(ng/mL)

(h)

1580

2

3790

2

13,500

2

t_max

AUC_(0–24h)

(ng⋅h/mL)

7290

18,100

72,700

*

After a single oral administration of 33g at 10, 30 and 100 mg/kg to female

mice, blood was collected from retinal vein at 0.5, 1, 2, 8, and 24 h postdose and

centrifuged to obtain plasma samples. Plasma concentrations of compound 33g

were measured using liquid chromatography-tandem mass spectrometry.

¹H NMR (500 MHz, CDCl₃): δ = 0.96–0.98 (m, 2H), 1.11–1.18 (m,

2H), 1.34 (t, J = 7.0 Hz, 3H), 3.24 (tt, J = 7.3, 3.7 Hz, 1H), 4.33 (q, J =

6.9 Hz, 2H), 7.14–7.16 (m, 4H), 8.33 (s, 1H); ¹³C NMR (126 MHz,

CDCl₃): δ = 7.2, 14.3, 33.0, 61.6, 105.0, 116.5 (d, J_CF= 25.2 Hz), 130.2

(d, J_CF= 10.1 Hz), 130.5, 150.6, 151.5, 158.9, 162.6 (d, J_CF= 249.5 Hz),

163.3; HRMS–ESI m/z [M+H]⁺calcd. for C₁₆H₁₆FN₂O⁺₄: 319.1089,

found: 319.1079.

compound 6a as a white solid (298 mg, 1.02 mmol, 95%).

¹H NMR (500 MHz, CDCl₃): δ = 1.31–1.37 (m, 3H), 3.53 (s, 3H), 4.33

(q, J = 6.9 Hz, 2H), 7.16 (d, J = 6.1 Hz, 4H), 8.30 (s, 1H); ¹³C NMR (126

MHz, CDCl₃): δ = 13.9, 37.5, 61.1, 104.5, 116.1 (d, J_CF= 22.7 Hz), 129.8

(d, J_CF= 7.6 Hz), 130.2, 150.7, 150.9, 158.8, 162.2 (d, J_CF= 249.5 Hz),

162.8; HRMS–ESI m/z [M+H]⁺calcd. for C₁₄H₁₄FN₂O⁺₄: 293.0932,

found: 293.0925.

5.1.9. 3-(4-ﬂuorophenyl)-1-methyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylic acid (7a)

4 M HCl in 1,4-dioxane (4.00 mL, 16.0 mmol) and water (2.00 mL)

were added to ethyl 3-(4-ﬂuorophenyl)-1-methyl-2,4-dioxo-1,2,3,4-tet-

5.1.6. Ethyl 3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylate (6b)

rahydropyrimidine-5-carboxylate (6a) (290 mg, 992

μmol), and the

reaction mixture was stirred at 80 ^◦C for 29 h. The reaction mixture was

cooled to room temperature, then poured into water. The reaction

mixture was extracted with DCM, the organic extract was dried over

MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed with

column chromatography on silica gel (n-heptane/EtOAc = 1/1 ~ EtOAc

~ EtOAc/MeOH = 9/1) to afford the title compound 7a as a white solid

(245 mg, 0.927 mmol, 93%).

The title compound was prepared from 5 using a method analogous

to that described for 6a in 87% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.35 (td, J = 7.2, 1.5 Hz, 3H), 1.43

(dd, J = 6.7, 1.2 Hz, 6H), 4.34 (qd, J = 7.1, 1.2 Hz, 2H), 4.84–4.97 (m,

1H), 7.10–7.21 (m, 4H), 8.34 (d, J = 1.2 Hz, 1H); ¹³C NMR (126 MHz,

CDCl₃): δ = 14.3, 21.6, 49.9, 61.6, 105.4, 116.5 (d, J_CF= 23.9 Hz), 130.2

(d, J_CF= 8.8 Hz), 130.9, 146.8, 150.9, 158.7, 162.6 (d, J_CF= 248.2 Hz),

163.7; HRMS–ESI m/z [M+H]⁺calcd. for C₁₆H₁₈FN₂O⁺₄: 321.1245,

found: 321.1235.

¹H NMR (500 MHz, CDCl₃): δ = 3.61 (s, 3H), 7.16–7.25 (m, 4H), 8.53

(s, 1H), 12.26 (br s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 38.6, 102.0,

117.0 (d, J_CF= 22.7 Hz), 129.0 (d, J_CF= 3.8 Hz), 129.8 (d, J_CF= 8.8 Hz),

150.2, 152.3, 163.0 (d, J_CF= 250.7 Hz), 163.0, 165.4; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₂H₁₀FN₂O⁺₄: 265.0619, found: 265.0614.

5.1.7. Ethyl 1-cyclopentyl-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahy-

dropyrimidine-5-carboxylate (6c)

The title compound was prepared from 5 using a method analogous

to that described for 6a in 74.2% as a white solid.

5.1.10. 3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylic acid (7b)

¹H NMR (500 MHz, CDCl₃): δ = 1.34 (td, J = 7.0, 2.5 Hz, 3H), 1.54

(d, J = 2.5 Hz, 2H), 1.74 (br s, 2H), 1.89 (br s, 2H), 2.19 (br s, 2H), 4.33

(qd, J = 7.0, 2.1 Hz, 2H), 4.85–4.99 (m, 1H), 7.11–7.21 (m, 4H), 8.35 (d,

J = 2.5 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 14.3, 24.1, 31.8, 59.4,

61.6, 105.3, 116.5 (d, J_CF= 23.9 Hz), 130.2 (d, J_CF= 8.8 Hz), 130.9 (d,

J_CF= 2.5 Hz), 147.7, 151.2, 158.8, 162.6 (d, J_CF= 248.2 Hz), 163.7;

HRMS–ESI m/z [M+H]⁺calcd. for C₁₈H₂₀FN₂O⁺₄: 347.1402, found:

347.1391.

4 M HCl in 1,4-dioxane (30 mL, 120 mmol) and water (10 mL) were

added to ethyl 3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetra-

hydropyrimidine-5-carboxylate (6b) (4.97 g, 15.5 mmol), and the re-

action mixture was stirred at 70 ^◦C for 45 h. The reaction mixture was

cooled to room temperature, then concentrated. The residue was poured

into water and the precipitate was collected by ﬁltration, rinsed with

water and dried to afford the title compound 7b as a white solid (3.81 g,

13.0 mmol, 84%).

¹H NMR (500 MHz, CDCl₃): δ = 1.47 (d, J = 6.7 Hz, 6H), 4.94 (spt, J

5.1.8. Ethyl 1-cyclopropyl-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahy-

dropyrimidine-5-carboxylate (6d)

= 6.8 Hz, 1H), 7.22 (d, J = 6.7 Hz, 4H), 8.57 (s, 1H), 12.34 (br s, 1H); ¹³

C

NMR (126 MHz, CDCl₃): δ = 21.6, 51.2, 102.1, 116.9 (d, J_CF= 23.9 Hz),

129.4, 129.9 (d, J_CF= 10.1 Hz), 148.1, 150.0, 163.0 (d, J_CF= 250.7 Hz),

To a solution of diethyl 2-((cyclopropylamino)methylene)malonate

Ba/F3-Axl

1.3

800

Control

1.2

1 mg/kg

600

1.1

1.0

0.9

0.8

0.7

3 mg/kg

10 mg/kg

400

*

***

200

0

1

2

3

4

5

0

1

2

3

4

5

Days

Fig. 3. Anti-tumor effect of 33g in the Ba/F3-Axl isograft model. Mice were orally administered 33g at the indicated dose once daily for 4 days. Data are means ± SD

(n = 5). Tumor volumes were compared between the control (non-treated) and 33g-treated group at the last time point. *P < 0.05; ***P < 0.001 (Dunnett’s Multiple

Comparison vs a Control).

8

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

163.3, 165.0; HRMS–ESI m/z [M+H]⁺calcd. for C₁₄H₁₄FN₂O₄⁺:

over MgSO₄, ﬁltered and then concentrated. To the residue was added

TBME/EtOAc = 10/1 solution, and the precipitate was collected by

ﬁltration, rinsed with TBME to afford the title compound 11a as a white

solid (1.01 g, 2.65 mmol, 78%).

293.0932, found: 293.0924.

5.1.11. 1-cyclopentyl-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylic acid (7c)

¹H NMR (500 MHz, DMSO‑d₆): δ = 5.14 (s, 2H), 7.03 (dd, J = 8.6,

1.8 Hz, 1H), 7.16 (dd, J = 5.5, 2.5 Hz, 1H), 7.23–7.28 (m, 1H),

7.29–7.33 (m, 1H), 7.34–7.42 (m, 5H), 7.62–7.77 (m, 2H), 8.08–8.09

(m, 1H), 8.49 (d, J = 5.5 Hz, 1H), 9.53 (br s, 1H); ¹³C NMR (126 MHz,

DMSO‑d₆): δ = 66.7, 109.7, 109.8 (d, J_CF= 21.4 Hz), 115.0, 117.5 (d,

J_CF= 3.8 Hz), 124.4, 124.5, 126.2, 128.6 (d, J_CF= 7.6 Hz), 129.0, 137.0,

150.3, 151.1, 153.3, 154.5, 155.1 (d, J_CF= 247.0 Hz), 165.8, 165.9;

The title compound was prepared from 6c using a method analogous

to that described for 7a in 80% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.69–1.83 (m, 4H), 1.89–1.93 (m,

2H), 2.19–2.24 (m, 2H), 4.94 (quin, J = 8.0 Hz, 1H), 7.22 (d, J = 6.1 Hz,

4H), 8.56 (s, 1H), 12.35 (br s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 24.0,

31.9, 60.3, 102.0, 116.9 (d, J_CF= 22.7 Hz), 129.4, 129.9, 148.8, 150.3,

163.0 (d, J_CF= 250.7 Hz), 163.3, 165.0; HRMS–ESI m/z [M+H]⁺calcd.

for C₁₆H₁₆FN₂O⁺₄: 319.1089, found: 319.1080.

HRMS–ESI m/z [M+H]⁺calcd. for C₂₀

H

FN₃O⁺₄: 382.1198, found:

17

382.1183.

5.1.12. 1-cyclopropyl-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxylic acid (7d)

5.1.16. Benzyl (4-((2-carbamoylpyridin-4-yl)oxy)-2,5-diﬂuorophenyl)

carbamate (11b)

The title compound was prepared from 6d using a method analogous

to that described for 7b in 79% as a white solid.

To a solution of 4-(4-amino-2,5-diﬂuorophenoxy)picolinamide (583

mg, 2.20 mmol) in acetone (20 mL) and H₂O (5 mL) was added benzyl

¹H NMR (500 MHz, CDCl₃): δ = 1.00–1.03 (m, 2H), 1.18–1.21 (m,

2H), 3.27–3.36 (m, 1H), 7.20–7.23 (m, 4H), 8.57 (s, 1H), 12.29 (br s,

chloroformate (471 μL, 3.30 mmol) at room temperature, and stirred at

room temperature for 31 h. The reaction mixture was poured into brine

and extracted with EtOAc. The organic extract was washed with brine,

dried over MgSO₄, ﬁltered and then concentrated. The residue was pu-

riﬁed with column chromatography on silica gel (n-heptane/EtOAc = 4/

1 ~ 1/4) to afford the title compound 11b as a white solid (684 mg, 1.71

mmol, 78%).

1H); ¹³C NMR (126 MHz, CDCl₃): δ = 7.2, 33.7, 101.7, 116.9 (d, J_CF

22.7 Hz), 129.1, 129.9 (d, J_CF= 10.1 Hz), 150.6, 151.7, 163.0 (d, J_CF

=

249.5 Hz), 163.0, 165.2; HRMS–ESI m/z [M+H]⁺calcd. for

C

₁₄H₁₂FN₂O⁺₄: 291.0776, found: 291.0769.

5.1.13. 4-(4-amino-3-ﬂuorophenoxy)picolinamide (10a)

¹H NMR (500 MHz, CDCl₃): δ = 5.23 (s, 2H), 5.62 (br s, 1H),

6.93–6.99 (m, 2H), 7.00 (dd, J = 5.5, 2.5 Hz, 1H), 7.35–7.42 (m, 5H),

7.64 (d, J = 2.5 Hz, 1H), 7.81 (br s, 1H), 8.15 (br s, 1H), 8.43 (d, J = 5.5

Hz, 1H); ¹³C NMR (126 MHz, DMSO‑d₆): δ = 66.9, 108.8, 111.8, 112.0

(d, J_CF= 23.9 Hz), 114.1, 125.9 (dd, J_CF= 13.9, 8.8 Hz), 128.58, 128.64,

129.0, 135.6 (dd, J_CF= 15.1, 10.8 Hz), 136.8, 150.0 (d, J_CF= 247.0 Hz),

150.2 (d, J_CF= 245.7 Hz), 151.3, 153.5, 154.2, 165.3, 165.8; HRMS–ESI

m/z [M+H]⁺calcd. for C₂₀H₁₆F₂N₃O⁺₄: 400.1103, found: 400.1088.

To a solution of 4-amino-3-ﬂuorophenol (2.27 g, 17.9 mmol) in

DMSO (20 mL) was added t-BuOK (2.15 g, 19.2 mmol) at room tem-

perature, followed by stirring for 15 min. To the reaction mixture was

added 4-chloropicolylamide (8) (2.00 g, 12.8 mmol) at room tempera-

◦

ture, and stirred at 80 C for 50 min. Then the reaction mixture was

cooled to room temperature, poured into 1 N NaOH and extracted with

EtOAc. The organic extract was washed with water and brine, dried over

MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed with

column chromatography on silica gel (n-heptane/EtOAc = 1/1 ~

EtOAc) and then NH silica gel (n-heptane/EtOAc = 1/1 ~ EtOAc ~

EtOAc/MeOH = 9/1) to afford the title compound 10a as a white solid

(1.87 g, 7.56 mmol, 59.1%).

5.1.17. Benzyl (4-((2-aminopyridin-4-yl)oxy)-2-ﬂuorophenyl)carbamate

(12a)

To a solution of benzyl (4-((2-carbamoylpyridin-4-yl)oxy)-2-ﬂuo-

rophenyl)carbamate (11a) (1.01 g, 2.65 mmol) in DMF (20 mL), pyri-

¹H NMR (500 MHz, DMSO‑d₆): δ = 5.18 (s, 2H), 6.73–6.76 (m, 1H),

6.79–6.86 (m, 1H), 6.94–7.01 (m, 1H), 7.07 (dd, J = 5.5, 2.5 Hz, 1H),

7.33 (d, J = 3.1 Hz, 1H), 7.64–7.66 (m, 1H), 8.06 (br s, 1H), 8.43 (d, J =

dine (859 μL, 10.6 mmol) and H₂O (239 μL, 13.3 mmol) was added [bis

(triﬂuoroacetoxy)iodo]benzene (2.28 g, 5.31 mmol) at room tempera-

ture. The reaction mixture was stirred at room temperature for 6 h. The

reaction mixture was poured into 1 N NaOH and H₂O and extracted with

EtOAc. The organic extract was washed with H₂O and brine, dried over

MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed with

column chromatography on silica gel (n-heptane/EtOAc = 1/1 ~

EtOAc) to afford the title compound 12a as a white solid (557 mg, 1.58

mmol, 59.4%).

5.5 Hz, 1H); ¹³C NMR (126 MHz, DMSO‑d₆): δ = 109.1, 109.6 (d, J_CF

=

21.4 Hz), 114.4, 117.0 (d, J_CF= 5.0 Hz), 117.9 (d, J_CF= 2.5 Hz), 135.3

(d, J_CF= 11.3 Hz), 142.7 (d, J_CF= 7.6 Hz), 150.7 (d, J_CF= 239.4 Hz),

150.8, 153.1, 166.0, 166.9; HRMS–ESI m/z [M+H]⁺calcd. for

C₁₂H₁₁FN₃O⁺₂: 248.0830, found: 248.0823.

5.1.14. 4-(4-amino-2,5-diﬂuorophenoxy)picolinamide (10b)

¹H NMR (500 MHz, CDCl₃): δ = 4.40 (br s, 2H), 5.22 (s, 2H), 5.93 (s,

1H), 6.26 (dd, J = 6.1, 1.8 Hz, 1H), 6.77–6.94 (m, 3H), 7.30–7.48 (m,

5H), 7.93 (d, J = 5.5 Hz, 1H), 8.10 (br s, 1H); ¹³C NMR (126 MHz,

The title compound was prepared from 8 and 9b using a method

analogous to that described for 10a in 52.5% as a white solid.

¹H NMR (500 MHz, DMSO‑d₆): δ = 5.53 (s, 2H), 6.72 (dd, J = 12.2,

8.6 Hz, 1H), 7.13 (dd, J = 5.5, 2.5 Hz, 1H), 7.21 (dd, J = 11.0, 7.3 Hz,

1H), 7.33 (d, J = 3.1 Hz, 1H), 7.68 (br s, 1H), 8.08 (br s, 1H), 8.47 (d, J

= 5.5 Hz, 1H); ¹³C NMR (126 MHz, DMSO‑d₆): δ = 103.4 (dd, J_CF= 23.5,

5.4 Hz), 108.4, 111.3 (d, J_CF= 22.7 Hz), 113.9, 127.8 (dd, J_CF= 14.5,

10.9 Hz), 136.7 (dd, J_CF= 15.7, 10.9 Hz), 146.3 (d, J_CF= 238.1 Hz),

151.02, 151.1 (d, J_CF= 240.7 Hz), 153.3, 165.9, 166.3; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₂H₁₀F₂N₃O⁺₂: 266.0736, found: 266.0726.

CDCl₃): δ = 67.6, 95.5, 104.2, 108.6 (d, J_CF= 21.4 Hz), 117.0 (d, J_CF

=

3.8 Hz), 121.5, 123.5, 123.6, 128.5, 128.6, 128.8, 135.8, 149.9, 152.6

(d, J_CF= 245.7 Hz), 153.3, 160.4, 166.1; HRMS–ESI m/z [M+H]⁺calcd.

for C₁₉H₁₇FN₃O⁺₃: 354.1248, found: 354.1234.

5.1.18. Benzyl (4-((2-aminopyridin-4-yl)oxy)-2,5-diﬂuorophenyl)

carbamate (12b)

The title compound was prepared from 11b using a method analo-

gous to that described for 12a in 85% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 4.45 (br s, 2H), 5.22 (s, 2H), 5.91 (s,

1H), 6.24 (d, J = 5.5 Hz, 1H), 6.92 (dd, J = 10.4, 6.7 Hz, 1H), 6.98 (br s,

5.1.15. Benzyl (4-((2-carbamoylpyridin-4-yl)oxy)-2-ﬂuorophenyl)

carbamate (11a)

To a solution of 4-(4-amino-3-ﬂuorophenoxy)picolinamide (10a)

(846 mg, 3.42 mmol) in acetone (20 mL) and H₂O (10 mL) was added

1H), 7.31–7.47 (m, 5H), 7.93 (d, J = 6.1 Hz, 1H), 8.09 (br s, 1H); ¹³

C

NMR (126 MHz, CDCl₃): δ = 67.8, 94.5, 103.2, 108.8, 109.0, 110.3 (d,

J_CF= 23.9 Hz), 124.6 (dd, J_CF= 22.7, 11.3 Hz), 128.6, 128.8, 128.8,

135.5, 147.5 (d, J_CF= 241.9 Hz), 149.9, 150.9 (d, J_CF= 248.2 Hz),

152.9, 160.3, 165.8; HRMS–ESI m/z [M+H]⁺calcd. for C₁₉H₁₆F₂N₃O₃⁺:

benzyl chloroformate (733 μ

L, 5.13 mmol) at 0 ^◦C, and stirred at room

temperature for 20 h. The reaction mixture was poured into brine and

extracted with EtOAc. The organic extract was washed with brine, dried

9

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

372.1154, found: 372.1138.

2.24–2.26 (m, 5H), 2.81 (d, J = 11.6 Hz, 2H), 5.22 (s, 2H), 6.57 (dd, J =

5.8, 2.1 Hz, 1H), 6.84–6.90 (m, 3H), 7.31–7.45 (m, 5H), 7.80 (d, J = 1.8

Hz, 1H), 7.85 (br s, 1H), 8.09 (d, J = 5.5 Hz, 1H), 8.14 (br s, 1H); ¹³C

NMR (126 MHz, CDCl₃): δ = 32.1, 32.7, 44.4, 46.4, 55.6, 67.4, 102.6,

108.5, 108.6, 116.8, 121.9, 123.9, 124.0, 128.4, 128.5, 128.7, 135.9,

148.9, 149.5, 152.7 (d, J_CF= 215.5 Hz), 153.8, 166.4, 171.3; HRMS–ESI

m/z [M+H]⁺calcd. for C₂₇H₃₀FN₄O⁺₄: 493.2246, found: 493.2230.

5.1.19. tert-butyl 4-(2-((4-(4-(((benzyloxy)carbonyl)amino)-3-

ﬂuorophenoxy)pyridin-2-yl)amino)-2-oxoethyl)piperidine-1-carboxylate

(13a)

To a solution of benzotriazole (927 mg, 7.78 mmol) in DCM (10 mL)

was added thionyl chloride (565 μL, 7.78 mmol) at room temperature.

The reaction mixture was stirred at room temperature for 5 min. To the

reaction mixture was added 1-Boc-piperidin-4-ylacetic acid (1.33 g,

5.45 mmol) at room temperature, and stirred at room temperature for

30 min. The precipitate was ﬁltered off through Celite and washed with

small amount of DCM. The ﬁltrate was added dropwise to a solution of

benzyl (4-((2-aminopyridin-4-yl)oxy)-2-ﬂuorophenyl)carbamate (12a)

(550 mg, 1.56 mmol) in THF (10 mL) at 0 ^◦C, then iPr₂NEt (4.08 mL,

23.3 mmol) was at 0 ^◦C. The reaction mixture was stirred at room

temperature for 24 h. The reaction mixture was poured into H₂O and

extracted with EtOAc. The organic extract was washed with brine, dried

over MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed

with column chromatography on NH silica gel (n-heptane/EtOAc = 1/1

~ EtOAc) to afford the title compound 13a as a white solid (824 mg,

1.42 mmol, 91%).

5.1.22. Benzyl (2,5-diﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)

pyridin-4-yl)oxy)phenyl)carbamate (14b)

The title compound was prepared from 13b using a method analo-

gous to that described for 14a in 74.4% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.27–1.39 (m, 2H), 1.71–1.75 (m,

2H), 1.78–1.88 (m, 1H) 1.92 (td,

J = 11.8, 2.1 Hz, 2H), 2.23–2.26 (m, 5H), 2.81 (d, J = 11.6 Hz, 2H),

5.23 (s, 2H), 6.58 (dd, J = 5.5, 2.5 Hz, 1H), 6.90–7.00 (m, 2H),

7.32–7.44 (m, 5H), 7.79 (d, J = 1.8 Hz, 1H), 8.01 (s, 1H), 8.10 (d, J =

6.1 Hz, 1H), 8.13 (br s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 32.2, 32.7,

44.6, 46.4, 55.6, 67.7, 101.3, 107.9, 109.1 (d, J_CF= 25.2 Hz), 110.3 (d,

J_CF= 23.9 Hz), 125.01 (d, J_CF= 10.1 Hz), 128.5, 128.7, 128.8, 135.2,

135.6, 147.6 (d, J_CF= 241.9 Hz), 149.0, 150.7 (d, J_CF= 245.7 Hz),

153.0, 153.62, 166.0, 171.2; HRMS–ESI m/z [M+H]⁺calcd. for

C₂₇H₂₉F₂N₄O⁺₄: 511.2151, found: 511.2133.

¹H NMR (500 MHz, CDCl₃): δ = 1.11–1.22 (m, 2H), 1.44 (s, 9H), 1.71

(d, J = 12.8 Hz, 2H), 2.02 (ttt, J = 11.2, 7.4, 3.7 Hz, 1H), 2.26 (d, J = 6.7

Hz, 2H), 2.70 (br s, 2H), 3.94–4.21 (m, 2H), 5.22 (s, 2H), 6.58 (dd, J =

5.8, 2.1 Hz, 1H), 6.84–6.91 (m, 3H), 7.31–7.44 (m, 5H), 7.80 (s, 1H),

7.94 (s, 1H), 8.09 (d, J = 6.1 Hz, 1H), 8.14 (br s, 1H); ¹³C NMR (126

MHz, CDCl₃): δ = 28.5, 31.9, 33.3, 44.0, 44.4, 67.5, 79.5, 102.4, 108.5

(d, J_CF= 21.4 Hz), 108.7, 116.9, 121.6, 123.9, 124.0, 128.4, 128.6,

128.7, 135.8, 149.0, 149.5, 152.5 (d, J_CF= 211.7 Hz), 153.5, 154.9,

166.4, 170.7; HRMS–ESI m/z [M+H]⁺calcd. for C₃₁H₃₆FN₄O₆⁺:

579.2613, found: 579.2589.

5.1.23. 4-(4-nitrophenoxy)pyridin-2-amine (17a)

To a solution of 2-amino-4-chloropyridine (15) (4.00 g, 31.1 mmol)

in NMP (40.0 mL) were added p-nitrophenol (16a) (8.66 g, 62.2 mmol)

and iPr₂NEt (21.7 mL, 124 mmol) at room temperature. The reaction

mixture was stirred at 150 ^◦C for 137 h under nigrogen, then cooled to

room temperature. The reaction mixture was poured into water and

extracted with EtOAc. The organic extract was washed with water and

brine, dried over MgSO₄, ﬁltered and then concentrated. The residue

was puriﬁed with column chromatography on silica gel (n-heptane/

EtOAc = 7/3 ~ EtOAc) and then NH silica gel (n-heptane/EtOAc = 7/3

~ EtOAc) to afford the title compound 17a as a slightly yellow solid

(3.50 g, 15.2 mmol, 48.7%).

5.1.20. tert-butyl 4-(2-((4-(4-(((benzyloxy)carbonyl)amino)-2,5-

diﬂuorophenoxy)pyridin-2-yl)amino)-2-oxoethyl)piperidine-1-carboxylate

(13b)

The title compound was prepared from 12b using a method analo-

gous to that described for 13a in 56.7% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 4.51 (br s, 2H), 6.08–6.09 (m, 1H),

6.33 (dd, J = 6.4, 1.5 Hz, 1H), 7.15 (d, J = 9.2 Hz, 2H), 8.03 (d, J = 5.5

Hz, 1H), 8.25 (d, J = 9.2 Hz, 2H); ¹³C NMR (126 MHz, CDCl₃): δ = 97.5,

105.3, 119.6, 126.1, 144.1, 150.6, 160.5, 160.6, 164.2; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₁H₁₀N₃O⁺₃: 232.0717, found: 232.0711.

¹H NMR (500 MHz, CDCl₃): δ = 1.17 (dd, J = 11.9, 4.0 Hz, 2H), 1.44

(s, 9H), 1.71 (d, J = 12.8 Hz, 2H), 1.95–2.08 (m, 1H), 2.25 (d, J = 7.3 Hz,

2H), 2.70 (br s, 2H), 4.07 (br s, 2H), 5.23 (s, 2H), 6.59 (dd, J = 5.8, 2.1

Hz, 1H), 6.92–6.96 (m, 2H), 7.35–7.42 (m, 5H), 7.78 (s, 1H), 7.85 (s,

1H), 8.07–8.20 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ = 28.5, 31.9,

33.3, 43.8, 44.3, 67.7, 79.4, 101.4, 107.9, 109.1, 110.3 (d, J_CF= 23.9

Hz), 125.1 (dd, J_CF= 20.2, 10.1 Hz), 128.4, 128.6, 128.7, 135.1, 135.6,

147.7 (d, J_CF= 244.4 Hz), 149.0, 150.7 (d, J_CF= 245.7 Hz), 153.0,

153.6, 154.9, 166.0, 170.9; HRMS–ESI m/z [M+H]⁺calcd. for

5.1.24. 4-(2-ﬂuoro-4-nitrophenoxy)pyridin-2-amine (17b)

The title compound was prepared from 15 and 16b using a method

analogous to that described for 17a in 50.5% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 4.51 (br s, 2H), 6.04 (d, J = 2.5 Hz,

1H), 6.30 (dd, J = 5.8, 2.1 Hz, 1H), 7.19–7.30 (m, 1H), 8.02 (d, J = 6.11

Hz, 1H), 8.05–8.14 (m, 2H); ¹³C NMR (126 MHz, CDCl₃): δ = 96.1,

104.0, 113.6 (d, J_CF= 23.9 Hz), 120.7, 122.0, 144.5, 148.0, 150.5, 153.4

(d, J_CF= 257.0 Hz), 160.6, 164.2; HRMS–ESI m/z [M+H]⁺calcd. for

C

₃₁H₃₅F₂N₄O⁺₆: 597.2519, found: 597.2496.

5.1.21. Benzyl (2-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)

pyridin-4-yl)oxy)phenyl)carbamate (14a)

To a solution of tert-butyl 4-(2-((4-(4-(((benzyloxy)carbonyl)amino)-

3-ﬂuorophenoxy)pyridin-2-yl)amino)-2-oxoethyl)piperidine-1-carbox-

ylate (13a) (803 mg, 1.39 mmol) in DCM (5 mL) was added TFA (5 mL)

at room temperature. The reaction mixture was stirred at room tem-

perature for 20 min and then concentrated. To a solution of the residue

C

₁₁H₉FN₃O⁺₃: 250.0622, found: 250.0616.

5.1.25. 2-(dimethylamino)-N-(4-(4-nitrophenoxy)pyridin-2-yl)acetamide

(18a)

To a solution of 4-(4-nitrophenoxy)pyridin-2-amine (17a) (500 mg,

2.16 mmol) in DMF (10 mL) was added N, N-dimethylglycine (335 mg,

3.24 mmol), HATU (1.23 g, 3.24 mmol) and iPr₂NEt (1.13 mL, 6.49

mmol) at room temperature. The reaction mixture was stirred at 80 ^◦C

for 18 h, then cooled to room temperature. The reaction mixture was

poured into water and extracted with EtOAc. The organic extract was

washed with water and brine, dried over MgSO4, ﬁltered and then

concentrated. The residue was puriﬁed with column chromatography on

silica gel (n-heptane/EtOAc = 9/1 ~ 2/8) to afford the title compound

18a as a white solid (403 mg, 1.27 mmol, 58.9%).

in THF (10 mL) were added 37% formaldehyde (205 μL, 2.78 mmol) and

sodium triacetoxyborohydride (441 mg, 2.08 mmol) at room tempera-

ture. The reaction mixture was stirred at room temperature for 40 min,

poured into saturated aqueous NaHCO₃and extracted with EtOAc. The

organic extract was washed with brine, dried over MgSO₄, ﬁltered and

then concentrated. The residue was puriﬁed with column chromatog-

raphy on NH silica gel (n-heptane/EtOAc = 3/7 ~ EtOAc ~ EtOAc/

MeOH = 9/1) to afford the title compound 14a as a white solid (560 mg,

1.14 mmol, 82%).

¹H NMR (500 MHz, CDCl₃): δ = 1.33 (qd, J = 12.2, 3.7 Hz, 2H), 1.74

(d, J = 12.8 Hz, 2H), 1.79–1.89 (m, 1H), 1.93 (td, J = 11.8, 2.1 Hz, 2H),

¹H NMR (500 MHz, CDCl₃): δ = 2.36 (s, 6H), 3.07 (s, 2H), 6.71 (dd, J

= 5.2, 2.1 Hz, 1H), 7.19 (d, J = 8.6 Hz, 2H), 7.96 (d, J = 2.5 Hz, 1H),

10

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

8.25 (d, J = 6.1 Hz, 1H), 8.28 (d, J = 8.6 Hz, 2H), 9.75 (br s, 1H); ¹³

C

(balloon) atmosphere for 1.5 h. The reaction mixture was ﬁltered

through Celite. The ﬁltrate was concentrated. The residue was puriﬁed

with column chromatography on NH silica gel (n-heptane/EtOAc = 1/1

~ EtOAc) to afford the title compound 19a as a white solid (255 mg,

NMR (126 MHz, CDCl₃): δ = 46.0, 63.6, 103.4, 110.2, 119.9, 126.2,

144.4, 145.0, 153.3, 160.1, 164.4, 170.0; HRMS–ESI m/z [M+H]⁺calcd.

for C₁₅H₁₇N₄O⁺₄: 317.1244, found: 317.1236.

891 μmol, 91%).

5.1.26. tert-butyl 4-(2-((4-(4-nitrophenoxy)pyridin-2-yl)amino)-2-

oxoethyl)piperidine-1-carboxylate (18b)

¹H NMR (500 MHz, CDCl₃): δ = 2.34 (s, 6H), 3.03 (s, 2H), 3.64 (br s,

2H), 6.53–6.55 (m, 1H), 6.69 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.0 Hz,

2H), 7.79 (s, 1H), 8.08 (d, J = 6.1 Hz, 1H), 9.60 (br s, 1H); ¹³C NMR

(126 MHz, CDCl₃): δ = 46.0, 63.6, 101.3, 108.3, 116.3, 122.0, 144.2,

145.9, 149.1, 152.7, 167.6, 169.7; HRMS–ESI m/z [M+H]⁺calcd. for

C₁₅H₁₉N₄O⁺₂: 287.1503, found: 287.1495.

The title compound was prepared from 17a using a method analo-

gous to that described for 13a in 81% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.15–1.26 (m, 2H), 1.44 (s, 9H), 1.72

(d, J = 12.8 Hz, 2H), 1.94–2.11 (m, 1H), 2.28 (d, J = 6.7 Hz, 2H), 2.70

(br s, 2H), 4.08 (br s, 2H), 6.71 (dd, J = 5.8, 2.1 Hz, 1H), 7.19 (d, J = 9.2

Hz, 2H), 7.89–7.92 (m, 2H), 8.21 (d, J = 5.5 Hz, 1H), 8.28 (d, J = 9.2 Hz,

2H); ¹³C NMR (126 MHz, CDCl₃): δ = 28.5, 31.9, 33.3, 43.8, 44.4, 79.5,

103.7, 110.2, 120.0, 126.2, 144.4, 149.6, 153.7, 154.9, 159.8, 164.5,

170.8; HRMS–ESI m/z [M+H]⁺calcd. for C₂₃H₂₉N₄O⁺₆: 457.2082,

found: 457.2068.

5.1.31. N-(4-(4-aminophenoxy)pyridin-2-yl)-2-(1-methylpiperidin-4-yl)

acetamide (19b)

The title compound was prepared from 18d using a method analo-

gous to that described for 19a in 94% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.27–1.39 (m, 2H), 1.72–1.76 (m,

2H), 1.78–1.88 (m, 1H), 1.92 (td, J = 11.9, 2.5 Hz, 2H), 2.23–2.25 (m,

5H), 2.81 (d, J = 11.6 Hz, 2H), 3.64 (s, 2H), 6.52 (s, 1H), 6.68–6.70 (m,

2H), 6.87–6.90 (m, 2H), 7.74–7.78 (m, 1H), 7.81 (br s, 1H), 8.03 (d, J =

5.5 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 32.2, 32.8, 44.7, 46.5,

55.6, 101.8, 108.3, 116.4, 122.0, 144.2, 145.9, 148.7, 153.3, 167.7,

170.9; HRMS–ESI m/z [M+H]⁺calcd. for C₁₉H₂₅N₄O⁺₂: 341.1972,

found: 341.1966.

5.1.27. tert-butyl 4-(2-((4-(2-ﬂuoro-4-nitrophenoxy)pyridin-2-yl)amino)-

2-oxoethyl)piperidine-1-carboxylate (18c)

The title compound was prepared from 17b using a method analo-

gous to that described for 13a in 55.3% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.17 (qd, J = 12.4, 4.3 Hz, 2H), 1.43

(s, 9H), 1.71 (d, J = 12.8 Hz, 2H), 1.95–2.08 (m, 1H), 2.27 (d, J = 7.3 Hz,

2H), 2.70 (br s, 2H), 3.95–4.21 (m, 2H), 6.69 (dd, J = 6.1, 2.5 Hz, 1H),

7.28–7.34 (m, 1H), 7.86 (d, J = 2.5 Hz, 1H), 7.90 (s, 1H), 8.07–8.16 (m,

2H), 8.20 (d, J = 6.1 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 28.5,

31.9, 33.3, 43.7, 44.3, 79.4, 102.3, 108.9, 113.8 (d, J_CF= 22.7 Hz),

120.9 (d, J_CF= 2.5 Hz), 122.6, 145.0, 147.1 (d, J_CF= 11.3 Hz), 149.4,

153.4 (d, J_CF= 255.8 Hz), 153.8, 154.8, 164.5, 171.0; HRMS–ESI m/z

[M+H]⁺calcd. for C₂₃H₂₈FN₄O⁺₆: 475.1987, found: 475.1974.

5.1.32. N-(4-(4-amino-2-ﬂuorophenoxy)pyridin-2-yl)-2-(1-

methylpiperidin-4-yl)acetamide (19c)

The title compound was prepared from 18e using a method analo-

gous to that described for 19a in 89% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.33 (qd, J = 12.2, 3.7 Hz, 2H), 1.74

(d, J = 12.8 Hz, 2H), 1.78–1.88 (m, 1H), 1.92 (t, J = 11.6 Hz, 2H),

2.23–2.25 (m, 5H), 2.81 (d, J = 11.0 Hz, 2H), 3.67–3.78 (m, 2H),

6.43–6.45 (m, 1H), 6.50 (d, J = 12.2 Hz, 1H), 6.56–6.58 (m, 1H), 6.94 (t,

J = 8.6 Hz, 1H), 7.77 (s, 1H), 7.84 (br s, 1H), 8.06 (d, J = 5.5 Hz, 1H);

¹³C NMR (126 MHz, CDCl₃):δ = 32.2, 32.7, 44.6, 46.4, 55.6, 101.3,

103.6 (d, J_CF= 21.4 Hz), 107.8, 111.0 (d, J_CF= 2.5 Hz), 124.1, 132.0 (d,

5.1.28. 2-(1-methylpiperidin-4-yl)-N-(4-(4-nitrophenoxy)pyridin-2-yl)

acetamide (18d)

The title compound was prepared from 18b using a method analo-

gous to that described for 14a in 83% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.34 (qd, J = 12.2, 3.7 Hz, 2H),

1.69–1.77 (m, 2H), 1.79–1.88 (m, 1H), 1.93 (td, J = 11.9, 2.5 Hz, 2H),

2.24 (s, 3H), 2.27 (d, J = 6.7 Hz, 2H), 2.81 (d, J = 11.6 Hz, 2H), 6.70 (dd,

J = 5.5, 2.5 Hz, 1H), 7.16–7.21 (m, 2H), 7.92 (d, J = 2.5 Hz, 1H), 7.96 (s,

1H), 8.21 (d, J = 5.5 Hz, 1H), 8.26–8.31 (m, 2H); ¹³C NMR (126 MHz,

CDCl₃):δ = 32.2, 32.7, 44.5, 46.4, 55.6, 103.8, 110.1, 112.0, 126.2,

144.4, 149.5, 153.9, 159.8, 164.5, 171.3; HRMS–ESI m/z [M+H]⁺calcd.

for C₁₉H₂₃N₄O⁺₄: 371.1714, found: 371.1708.

J_CF= 13.9 Hz), 145.9 (d, J_CF= 10.1 Hz), 148.7, 153.5, 155.0 (d, J_CF=

249.5 Hz), 167.1, 171.2; HRMS–ESI m/z [M+H]⁺calcd. for

C

₁₉H₂₄FN₄O⁺₂: 359.1878, found: 359.1868.

5.1.33. N-(4-(4-amino-3-ﬂuorophenoxy)pyridin-2-yl)-2-(1-

methylpiperidin-4-yl)acetamide (19d)

The title compound was prepared from 14a using a method analo-

gous to that described for 19a in 90% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.27–1.39 (m, 2H), 1.73 (d, J = 12.8

Hz, 2H), 1.83 (ttt, J = 11.3, 7.4, 3.6 Hz, 1H), 1.92 (td, J = 11.8, 2.1 Hz,

2H), 2.23–2.25 (m, 5H), 2.81 (d, J = 11.6 Hz, 2H), 3.62–3.75 (m, 2H),

6.55 (dd, J = 5.8, 2.1 Hz, 1H), 6.70–6.73 (m, 1H), 6.76–6.80 (m, 2H),

7.77 (br s, 1H), 7.96 (br s, 1H), 8.06 (d, J = 6.1 Hz, 1H); ¹³C NMR (126

MHz, CDCl₃): δ = 32.2, 32.7, 44.5, 46.4, 55.6, 102.0, 108.3, 109.1 (d,

J_CF= 20.2 Hz), 117.1 (d, J_CF= 3.8 Hz), 117.3 (d, J_CF= 5.0 Hz), 132.5 (d,

J_CF= 13.9 Hz), 145.2, 148.7, 151.4 (d, J_CF= 241.9 Hz), 153.6, 167.2,

171.2; HRMS–ESI m/z [M+H]⁺calcd. for C₁₉H₂₄FN₄O⁺₂: 359.1878,

found: 359.1868.

5.1.29. N-(4-(2-ﬂuoro-4-nitrophenoxy)pyridin-2-yl)-2-(1-methylpiperidin-

4-yl)acetamide (18e)

The title compound was prepared from 18c using a method analo-

gous to that described for 14a in 86% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.33 (qd, J = 12.2, 3.7 Hz, 2H),

1.72–1.75 (m, 2H), 1.78–1.88 (m, 1H), 1.93 (td, J = 11.8, 2.1 Hz, 2H),

2.24 (s, 3H), 2.27 (d, J = 6.7 Hz, 2H), 2.81 (d, J = 11.6 Hz, 2H), 6.69 (dd,

J = 5.8, 2.1 Hz, 1H), 7.27–7.34 (m, 1H), 7.87 (d, J = 1.8 Hz, 1H), 8.04 (s,

1H), 8.08–8.15 (m, 2H), 8.19 (d, J = 5.5 Hz, 1H); ¹³C NMR (126 MHz,

CDCl₃): δ = 32.2, 32.7, 44.7, 46.4, 55.6, 102.1, 109.0, 113.8 (d, J_CF

=

22.7 Hz), 120.9 (d, J_CF= 3.8 Hz), 122.5, 145.0 (d, J_CF= 7.6 Hz), 147.2

(d, J_CF= 11.3 Hz), 149.6, 153.5 (d, J_CF= 255.8 Hz), 153.6, 164.5, 171.1;

HRMS–ESI m/z [M+H]⁺calcd. for C₁₉H₂₂FN₄O⁺₄: 389.1620, found:

389.1606.

5.1.34. N-(4-(4-amino-2,5-diﬂuorophenoxy)pyridin-2-yl)-2-(1-

methylpiperidin-4-yl)acetamide (19e)

The title compound was prepared from 14b using a method analo-

gous to that described for 19a in 81% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.33 (qd, J = 12.1, 3.4 Hz, 2H), 1.73

(d, J = 12.8 Hz, 2H), 1.78–1.88 (m, 1H), 1.92 (t, J = 11.6 Hz, 2H),

2.23–2.26 (m, 5H), 2.81 (d, J = 11.0 Hz, 2H), 3.79 (s, 2H), 6.53–6.67 (m,

2H), 6.85 (dd, J = 10.4, 7.3 Hz, 1H), 7.78 (s, 1H), 7.96 (br s, 1H), 8.08

(d, J = 6.1 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 32.2, 32.7, 44.6,

5.1.30. N-(4-(4-aminophenoxy)pyridin-2-yl)-2-(dimethylamino)

acetamide (19a)

To a solution of 2-(dimethylamino)-N-(4-(4-nitrophenoxy)pyridin-2-

yl)acetamide (310 mg, 980

μmol) in MeOH (6 mL) was added 5%

palladium on carbon (104 mg, 0.0490 mmol) at room temperature, and

the reaction mixture was stirred at room temperature under a hydrogen

46.4, 55.6, 101.14, 104.5 (dd, J_CF= 23.5, 4.2 Hz), 107.8, 110.7 (d, J_CF=

22.7 Hz), 130.7 (dd, J_CF= 14.5, 9.7 Hz), 133.6 (dd, J_CF= 14.5, 9.7 Hz),

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S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

146.7 (d, J_CF= 238.1 Hz), 148.8, 151.1 (d, J_CF= 247.0 Hz), 153.5,

166.7, 171.2; HRMS–ESI m/z [M+H]⁺calcd. for C₁₉H₂₃F₂N₄O₂⁺:

377.1784, found: 377.1772.

22.7 Hz), 116.3, 116.9 (d, J_CF= 22.7 Hz), 123.6, 130.0 (d, J_CF= 8.8 Hz),

136.6 (d, J_CF= 10.1 Hz), 136.9, 137.0, 149.0, 150.3, 151.1, 153.2, 154.3

(d, J_CF= 250.7 Hz), 159.8, 162.9 (d, J_CF= 250.7 Hz), 163.2, 166.3,

170.8; HRMS–ESI m/z [M+H]⁺calcd. for C₃₃H₃₃F₂N₆O⁺₅: 631.2475,

found: 631.2454.

5.1.35. N-(4-((2-(2-(dimethylamino)acetamido)pyridin-4-yl)oxy)

phenyl)-3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (20a)

5.1.39. 1-cyclopentyl-N-(3-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)

acetamido)pyridin-4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (20e)

The title compound was prepared from 19a and 7b using a method

analogous to that described for 18a in 89% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.49 (d, J = 6.7 Hz, 6H), 2.35 (s, 6H),

3.04 (s, 2H), 4.89–5.04 (m, 1H), 6.57 (dd, J = 6.1, 1.8 Hz, 1H), 7.06 (d, J

= 8.6 Hz, 2H), 7.24 (s, 4H), 7.67 (d, J = 9.2 Hz, 2H), 7.85 (d, J = 1.8 Hz,

1H), 8.11 (d, J = 6.1 Hz, 1H), 8.67 (s, 1H), 9.63 (s, 1H), 10.80 (s, 1H);

¹³C NMR (126 MHz, CDCl₃): δ = 21.6, 46.0, 50.5, 63.6, 101.9, 105.8,

108.6, 116.9 (d, J_CF= 23.9 Hz), 121.4, 121.8, 130.0 (d, J_CF= 7.6 Hz),

130.4 (d, J_CF= 2.5 Hz), 135.4, 146.2, 149.3, 150.4, 150.5, 152.8, 159.9,

162.8 (d, J_CF= 248.2 Hz), 163.0, 166.7, 169.8; HRMS–ESI m/z [M+H]⁺

calcd. for C₂₉H₃₀FN₆O⁺₅: 561.2256, found: 561.2241.

The title compound was prepared from 19c and 7c using a method

analogous to that described for 18a in 80% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.32 (qd, J = 12.2, 3.7 Hz, 2H),

1.66–1.86 (m, 9H), 1.88–1.98 (m, 4H), 2.21–2.25 (m, 5H), 2.80 (d, J =

11.6 Hz, 2H), 4.98 (quin, J = 7.8 Hz, 1H), 6.57 (dd, J = 5.8, 2.1 Hz, 1H),

7.06–7.14 (m, 1H), 7.20–7.25 (m, 5H), 7.80 (d, J = 1.8 Hz, 1H), 7.83

(dd, J = 12.2, 2.5 Hz, 1H), 8.06 (s, 1H), 8.08 (d, J = 6.1 Hz, 1H), 8.65 (s,

1H), 10.89 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 24.2, 31.9, 32.2,

32.7, 44.8, 46.4, 55.6, 59.8, 101.2, 105.4, 107.9, 109.7 (d, J_CF= 22.7

Hz), 116.3, 116.9 (d, J_CF= 22.7 Hz), 123.6, 130.0 (d, J_CF= 8.8 Hz),

130.3, 136.7 (d, J_CF= 10.1 Hz), 136.9 (d, J_CF= 11.3 Hz), 147.3, 149.0,

150.7, 153.2, 154.3 (d, J_CF= 249.5 Hz), 160.1, 162.8 (d, J_CF= 250.7

Hz), 163.0, 166.4, 170.8; HRMS–ESI m/z [M+H]⁺calcd. for

C₃₅H₃₇F₂N₆O⁺₅: 659.2788, found: 659.2766.

5.1.36. 3-(4-ﬂuorophenyl)-1-isopropyl-N-(4-((2-(2-(1-methylpiperidin-4-

yl)acetamido)pyridin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (20b)

The title compound was prepared from 19b and 7b using a method

analogous to that described for 18a in 81% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.32 (qd, J = 12.1, 3.4 Hz, 2H), 1.48

(d, J = 6.1 Hz, 6H), 1.73 (d, J = 12.8 Hz, 2H), 1.78–1.88 (m, 1H), 1.92 (t,

J = 11.6 Hz, 2H), 2.23–2.25 (m, 5H), 2.81 (d, J = 11.0 Hz, 2H),

4.88–5.04 (m, 1H), 6.50–6.60 (m, 1H), 7.05 (dd, J = 9.2, 1.2 Hz, 2H),

7.23–7.25 (m, 4H), 7.67 (dd, J = 9.2, 1.2 Hz, 2H), 7.81 (s, 1H), 7.92 (s,

5.1.40. Ethyl 2,5-dioxo-5,6,7,8-tetrahydro-2H-chromene-3-carboxylate

(23)

To a solution of 1,3-cyclohexanedione (22) (1.00 g, 8.92 mmol) and

ethyl (ethoxymethylene)cyanoacetate (21) (1.51 g, 8.92 mmol) in DMF

(15 mL) was added potassium tert-butoxide (1.00 g, 8.92 mmol) at room

temperature. The reaction mixture was stirred at room temperature for

2 h. The reaction mixture was poured into 1 N HCl (10.7 mL) and water

(40 mL), then extracted with EtOAc. The organic extract was washed

with water and brine, dried over Na₂SO₄, ﬁltered and then concentrated.

The residue was puriﬁed with column chromatography on silica gel (n-

heptane/EtOAc = 4/1 ~ 3/7) to afford the title compound 23 as a red oil

(1.08 g, 4.58 mmol, 51.4%).

1H), 8.06 (dd, J = 6.1, 1.2 Hz, 1H), 8.67–8.68 (m, 1H), 10.81 (s, 1H); ¹³

C

NMR (126 MHz, CDCl₃): δ = 21.7, 32.2, 32.7, 44.8, 46.4, 50.5, 55.6,

102.2, 105.8, 108.7, 116.9 (d, J_CF= 22.7 Hz), 121.4, 121.9, 130.0 (d,

J_CF= 7.6 Hz), 130.3 (d, J_CF= 3.8 Hz), 135.4, 146.2, 149.0, 150.3, 150.5,

153.2, 159.9, 162.8 (d, J_CF= 249.5 Hz), 163.0, 166.8, 170.8; HRMS–ESI

m/z [M+H]⁺calcd. for C₃₃H₃₆FN₆O⁺₅: 615.2726, found: 615.2713.

5.1.37. N-(2-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)pyridin-

4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (20c)

¹H NMR (400 MHz, CDCl₃): δ = 1.36 (t, J = 7.1 Hz, 3H), 2.09–2.26

(m, 2H), 2.50–2.61 (m, 2H), 2.81–2.96 (m, 2H), 4.26–4.44 (m, 2H), 8.62

(s, 1H); ¹³C NMR (101 MHz, CDCl₃): δ = 14.2, 20.0, 28.5, 36.5, 61.9,

114.2, 115.4, 144.9, 155.9, 162.3, 178.4, 193.1; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₂H₁₃O⁺₅: 237.0757, found: 237.0748.

The title compound was prepared from 19d and 7b using a method

analogous to that described for 18a in 71.2% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.33 (qd, J = 12.2, 3.7 Hz, 2H), 1.49

(d, J = 6.7 Hz, 6H), 1.74 (d, J = 12.8 Hz, 2H), 1.79–1.89 (m, 1H),

1.89–1.97 (m, 2H), 2.24–2.26 (m, 5H), 2.81 (d, J = 11.6 Hz, 2H), 4.95

(spt, J = 6.8 Hz, 1H), 6.59 (dd, J = 6.1, 2.5 Hz, 1H), 6.84–6.94 (m, 2H),

7.21–7.25 (m, 4H), 7.83 (d, J = 2.5 Hz, 1H), 7.90 (s, 1H), 8.10 (d, J =

5.1.41. 2,5-dioxo-1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-carboxylic

acid (24)

To a solution of ethyl 2,5-dioxo-5,6,7,8-tetrahydro-2H-chromene-3-

carboxylate (23) (917 mg, 3.88 mmol) in EtOH (15 mL) was added

aniline (362 mg, 3.88 mmol) at room temperature. The reaction mixture

was stirred at room temperature for 16 h. The precipitate was collected

by ﬁltration, then rinsed with EtOH to afford the title compound 24 as a

white solid (585 mg, 2.06 mmol, 53.2%).

5.5 Hz, 1H), 8.41–8.49 (m, 1H), 8.66 (s, 1H), 10.98–10.99 (m, 1H); ¹³

C

NMR (126 MHz, CDCl₃): δ = 21.7, 32.2, 32.7, 44.9, 46.4, 50.5, 55.6,

102.3, 105.8, 108.5 (d, J_CF= 21.4 Hz), 108.9, 116.5 (d, J_CF= 2.5 Hz),

116.9 (d, J_CF= 22.7 Hz), 123.1, 124.0 (d, J_CF= 8.8 Hz), 130.1 (d, J_CF

10.1 Hz), 130.26, 146.3, 149.2, 150.3, 150.6, 153.2, 153.3 (d, J_CF

=

¹H NMR (500 MHz, CDCl₃): δ = 2.05–2.15 (m, 2H), 2.53–2.65 (m,

4H), 7.23 (s, 1H), 7.48–7.74 (m, 4H), 9.19 (s, 1H), 12.98–13.50 (m, 1H);

¹³C NMR (126 MHz, CDCl₃): δ = 21.0, 29.4, 36.5, 116.4, 116.7, 127.2,

130.6, 130.7, 135.9, 143.8, 160.9, 164.1, 165.4, 192.5; HRMS–ESI m/z

[M+H]⁺calcd. for C₁₆H₁₄NO⁺₄: 284.0917, found: 284.0909.

250.7 Hz), 160.2, 161.8, 162.8, 166.2, 170.7; HRMS–ESI m/z [M+H]⁺

calcd. for C₃₃H₃₆F₂N₆O⁺₅: 633.2632, found: 633.2611.

5.1.38. 1-cyclopropyl-N-(3-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)

acetamido)pyridin-4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (20d)

5.1.42. N-(4-((2-(2-(1-methylpiperidin-4-yl)acetamido)pyridin-4-yl)oxy)

phenyl)-2,5-dioxo-1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-

carboxamide (25a)

The title compound was prepared from 19c and 7d using a method

analogous to that described for 18a in 81% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 0.99–1.06 (m, 2H), 1.16–1.23 (m,

2H), 1.32 (qd, J = 12.2, 3.7 Hz, 2H), 1.70–1.74 (m, 2H), 1.77–1.87 (m,

1H), 1.88–1.97 (m, 2H), 2.23–2.25 (m, 5H), 2.80 (d, J = 11.6 Hz, 2H),

3.27–3.35 (m, 1H), 6.57 (dd, J = 5.8, 2.1 Hz, 1H), 7.06–7.14 (m, 1H),

7.20–7.24 (m, 5H), 7.76–7.87 (m, 2H), 7.99 (s, 1H), 8.08 (d, J = 5.5 Hz,

1H), 8.64 (s, 1H), 10.84 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 7.2,

The title compound was prepared from 19b and 24 using a method

analogous to that described for 18a in 73.6% as a white solid.

¹H NMR (400 MHz, CDCl₃): δ = 1.25–1.40 (m, 2H), 1.69–1.77 (m,

2H), 1.78–1.87 (m, 1H), 1.88–1.97 (m, 2H), 2.04–2.14 (m, 2H),

2.18–2.30 (m, 5H), 2.48–2.65 (m, 4H), 2.80 (d, J = 11.9 Hz, 2H), 6.53

(dd, J = 5.5, 2.3 Hz, 1H), 7.03 (d, J = 7.7 Hz, 2H), 7.24–7.30 (m, 2H),

7.55–7.66 (m, 3H), 7.74 (d, J = 7.7 Hz, 2H), 7.78–7.89 (m, 2H), 8.05 (d,

32.2, 32.7, 33.4, 44.9, 46.4, 55.6, 101.1, 105.1, 107.9, 109.4 (d, J_CF

=

12

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

J = 5.5 Hz, 1H), 9.31 (s, 1H), 11/39 (s, 1H); ¹³C NMR (101 MHz, CDCl₃):

δ = 21.2, 29.4, 32.2, 32.7, 36.6, 44.8, 46.4, 55.6, 102.3, 108.5, 115.7,

120.0, 121.3, 122.0, 127.4, 130.2, 130.6, 135.7, 137.0, 142.0, 149.0,

150.1, 153.2, 159.6, 160.7, 163.5, 166.9, 170.8, 193.0; HRMS–ESI m/z

[M+H]⁺calcd. for C₃₅H₃₆N₅O⁺₅: 606.2711, found: 606.2696.

= 6.8 Hz, 1H), 6.06–6.09 (m, 1H), 6.96 (dd, J = 10.1, 7.0 Hz, 1H), 7.00

(dd, J = 5.5, 2.5 Hz, 1H), 7.19–7.24 (m, 4H), 7.64 (d, J = 2.5 Hz, 1H),

7.82 (d, J = 4.3 Hz, 1H), 8.41 (d, J = 5.5 Hz, 1H), 8.47 (dd, J = 12.2, 7.3

Hz, 1H), 8.66 (s, 1H) 11.14 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ =

21.6, 50.7, 105.4, 109.6, 110.3 (d, J_CF= 22.7 Hz), 110.6 (d, J_CF= 25.2,

Hz), 114.0, 116.9 (d, J_CF= 22.7 Hz), 125.3 (dd, J_CF= 22.7, 12.6 Hz),

130.1 (d, J_CF= 8.8 Hz), 130.2 (d, J_CF= 3.8 Hz), 135.5 (dd, J_CF= 14.5,

9.7 Hz), 146.7, 148.5 (d, J_CF= 245.7 Hz), 150.2, 150.3 (d, J_CF= 243.2

Hz), 150.5, 152.1, 160.4, 162.8 (d, J_CF= 250.7 Hz), 162.8, 165.5, 166.2;

HRMS–ESI m/z [M+H]⁺calcd. for C₂₆H₂₁F₃N₅O⁺₅: 540.1489, found:

540.1468.

5.1.43. N-(3-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)pyridin-

4-yl)oxy)phenyl)-2,5-dioxo-1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-

carboxamide (25b)

The title compound was prepared from 19c and 24 using a method

analogous to that described for 18a in 65.3% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.27–1.39 (m, 2H), 1.68–1.76 (m,

2H), 1.78–1.88 (m, 1H), 1.89–1.96 (m, 2H), 2.06–2.14 (m, 2H),

2.20–2.27 (m, 5H), 2.51–2.65 (m, 4H), 2.80 (d, J = 11.6 Hz, 2H), 6.55

(dd, J = 5.5, 2.5 Hz, 1H), 7.06–7.12 (m, 1H), 7.25–7.28 (m, 2H),

7.29–7.34 (m, 1H), 7.57–7.70 (m, 3H), 7.78–7.92 (m, 3H), 8.07 (d, J =

6.1 Hz, 1H), 9.31 (s, 1H), 11.49 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ =

21.2, 29.4, 32.2, 32.7, 36.6, 44.8, 46.4, 55.6, 101.3, 107.7, 109.7 (d, J_CF

= 22.7 Hz), 115.7, 116.4 (d, J_CF= 2.5 Hz), 119.7, 123.5, 127.4, 130.3,

5.1.47. N-(4-((2-carbamoylpyridin-4-yl)oxy)-2,5-diﬂuorophenyl)-2,5-

dioxo-1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-carboxamide (26b)

The title compound was prepared from 10b and 24 using a method

analogous to that described for 18a in 82% as a white solid.

¹H NMR (400 MHz, CDCl₃): δ = 2.09 (quin, J = 6.4 Hz, 2H), 2.54 (t, J

= 6.2 Hz, 2H), 2.58–2.62 (m, 2H), 5.50 (d, J = 3.2 Hz, 1H), 6.93–7.00

(m, 2H), 7.26–7.28 (m, 1H), 7.52–7.67 (m, 4H), 7.69 (d, J = 2.3 Hz, 1H),

7.80 (d, J = 3.2 Hz, 1H), 8.43 (d, J = 5.5 Hz, 1H), 8.61 (dd, J = 12.4, 6.9

Hz, 1H), 9.31 (s, 1H), 11.71 (s, 1H); ¹³C NMR (101 MHz, DMSO‑d₆): δ =

21.0, 29.6, 36.5, 108.9, 110.0 (d, J_CF= 26.3 Hz), 111.8 (d, J_CF= 23.2

Hz), 114.1, 115.3, 117.9, 125.7 (dd, J_CF= 11.6, 11.6 Hz), 128.3, 130.2,

130.6, 136.8 (d, J_CF= 22.7 Hz), 136.9 (d, J_CF= 10.1 Hz), 137.0 (d, J_CF

=

10.1 Hz), 142.2, 149.0, 153.2, 154.6 (d, J_CF= 249.5 Hz), 159.9, 160.9,

163.5, 166.4, 170.8, 192.9; HRMS–ESI m/z [M+H]⁺calcd. for

C₃₅H₃₅FN₅O⁺₅: 624.2617, found: 624.2602.

130.4, 135.2 (dd, J_CF= 13.0, 13.0 Hz), 137.5, 141.1, 148.6 (d, J_CF

=

5.1.44. N-(2-ﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)pyridin-

4-yl)oxy)phenyl)-2,5-dioxo-1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-

carboxamide (25c)

242.4 Hz), 150.2 (d, J_CF= 243.4 Hz), 151.3, 153.4, 161.7, 163.2, 163.5,

165.3, 165.8, 194.1; HRMS–ESI m/z [M+H]⁺calcd. for C₂₈H₂₁F₂N₄O₅⁺:

531.1475, found: 531.1461.

The title compound was prepared from 19d and 24 using a method

analogous to that described for 18a in 66.1% as a white solid.

¹H NMR (400 MHz, CDCl₃): δ = 1.26–1.39 (m, 2H), 1.68–1.77 (m,

2H), 1.79–1.89 (m, 1H), 1.89–1.99 (m, 2H), 2.04–2.16 (m, 2H),

2.20–2.29 (m, 5H), 2.45–2.55 (m, 2H), 2.57–2.64 (m, 2H), 2.81 (d, J =

11.9 Hz, 2H), 6.56 (dd, J = 6.0, 2.3 Hz, 1H), 6.80–6.95 (m, 2H),

7.20–7.30(m, 2H), 7.54–7.70 (m, 3H), 7.83 (d, J = 2.3 Hz, 2H), 8.08 (d,

J = 6.0 Hz, 1H), 8.52–8.63 (m, 1H), 9.30 (s, 1H), 11.55 (s, 1H); ¹³C NMR

(101 MHz, CDCl₃): δ = 21.2, 29.4, 32.2, 32.7, 36.6, 44.8, 46.4, 55.6,

102.4, 108.4 (d, J_CF= 22.2 Hz), 108,7, 115.5, 116.6, 119.9, 123.3, 124.3

(d, J_CF= 11.1 Hz), 127.5, 130.1, 130.6, 136.9, 142.1, 149.1, 150.1 (d,

J_CF= 11.1 Hz), 153.2, 153.2 (d, J_CF= 249.5 Hz), 160.0, 161.0, 163.4,

166.3, 170.8, 193.0; HRMS–ESI m/z [M+H]⁺calcd. for C₃₅H₃₅FN₅O₅⁺:

624.2617, found: 624.2598.

5.1.48. N-(4-((2-aminopyridin-4-yl)oxy)-2,5-diﬂuorophenyl)-3-(4-

ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

carboxamide (27a)

The title compound was prepared from 26a using a method analo-

gous to that described for 12a in 61.1% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 6.7 Hz, 6H), 4.46 (br s,

2H), 4.95 (spt, J = 6.8 Hz, 1H), 5.94 (d, J = 1.8 Hz, 1H), 6.26 (dd, J =

5.8, 2.1 Hz, 1H), 6.93 (dd, J = 10.4, 6.7 Hz, 1H), 7.21–7.25 (m, 4H),

7.93 (d, J = 5.5 Hz, 1H), 8.41 (dd, J = 11.9, 7.0 Hz, 1H), 8.65 (s, 1H),

11.08 (br s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 21.6, 50.6, 94.6,

103.3, 105.4, 110.2 (d, J_CF= 22.7 Hz), 110.5 (d, J_CF= 26.5 Hz), 116.9

(d, J_CF= 22.7 Hz), 124.5 (dd, J_CF= 22.7, 11.3 Hz), 130.0 (d, J_CF= 8.8

Hz), 130.2 (d, J_CF= 3.8 Hz), 136.4 (dd, J_CF= 14.5, 9.7 Hz), 146.6, 148.5

(d, J_CF= 247.0 Hz), 149.9, 150.4, 150.4 (d, J_CF= 249.5 Hz), 160.3,

160.4, 162.8, 162.8 (d, J_CF= 250.7 Hz), 165.7; HRMS–ESI m/z [M+H]⁺

calcd. for C₂₅H₂₁F₃N₅O⁺₄: 512.1540, found: 512.1521.

5.1.45. N-(2,5-diﬂuoro-4-((2-(2-(1-methylpiperidin-4-yl)acetamido)

pyridin-4-yl)oxy)phenyl)-2,5-dioxo-1-phenyl-1,2,5,6,7,8-

hexahydroquinoline-3-carboxamide (25d)

The title compound was prepared from 19e and 24 using a method

analogous to that described for 18a in 34.8% as a pink-colored solid.

¹H NMR (400 MHz, CDCl₃): δ = 1.26–1.40 (m, 2H), 1.68–1.78 (m,

2H), 1.78–1.88 (m, 1H), 1.88–1.98 (m, 2H), 2.04–2.15 (m, 2H),

2.19–2.30 (m, 5H), 2.48–2.67 (m, 4H), 2.81 (d, J = 11.4 Hz, 2H), 6.57

(dd, J = 5.7, 2.5 Hz, 1H), 6.88–6.98 (m, 1H), 7.24–7.27 (m, 2H),

7.50–7.68 (m, 3H), 7.77–7.96 (m, 2H), 8.09 (d, J = 5.5 Hz, 1H),

8.53–8.67 (m, 1H), 9.29 (s, 1H), 11.68 (s, 1H); ¹³C NMR (101 MHz,

CDCl₃): δ = 21.2, 29.5, 32.2, 32.7, 36.6, 44.9, 46.4, 55.6, 101.3, 107.8,

110.0 (d, J_CF= 23.2 Hz), 110.7 (d, J_CF= 27.3 Hz), 115.6, 119.6, 125.2

(dd, J_CF= 22.2, 11.1 Hz), 127.4, 130.2, 130.6, 135.8 (dd, J_CF= 14.5,

5.1.49. N-(4-((2-aminopyridin-4-yl)oxy)-2,5-diﬂuorophenyl)-2,5-dioxo-

1-phenyl-1,2,5,6,7,8-hexahydroquinoline-3-carboxamide (27b)

The title compound was prepared from 26b using a method analo-

gous to that described for 12a in 85% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 2.07–2.10 (m, 2H), 2.53 (t, J = 6.1

Hz, 2H), 2.57–2.63 (m, 2H), 4.39 (s, 2H), 5.93 (d, J = 2.5 Hz, 1H),

6.26–6.28 (m, 1H), 6.91 (dd, J = 10.7, 7.0 Hz, 1H), 7.24–7.26 (m, 2H),

7.55–7.66 (m, 3H), 7.93 (d, J = 6.1 Hz, 1H), 8.53 (dd, J = 11.9, 7.0 Hz,

1H), 9.29 (s, 1H), 11.65 (s, 1H); ¹³C NMR (126 MHz, DMSO‑d₆): δ =

21.0, 29.6, 36.5, 93.1, 101.5, 109.8 (d, J_CF= 26.5 Hz), 111.8 (d, J_CF

=

22.7 Hz), 115.3, 117.9, 125.0 (dd, J_CF= 11.3, 11.3 Hz), 128.3, 130.2,

10.6 Hz), 136.8, 142.4, 148.5 (d, J_CF= 244.4 Hz), 149.2, 150.4 (d, J_CF

=

130.5, 136.0 (dd, J_CF= 15.1, 10.1 Hz), 137.4, 141.1, 148.5 (d, J_CF=

240.4 Hz), 153.1, 160.2, 161.1, 163.3, 165.9, 170.7, 192.9; HRMS–ESI

m/z [M+H]⁺calcd. for C₃₅H₃₄F₂N₅O⁺₅: 642.2523, found: 642.2505.

241.9 Hz), 150.2, 150.4 (d, J_CF= 240.7 Hz), 161.7, 162.2, 163.1, 163.4,

165.2, 194.1; HRMS–ESI m/z [M+H]⁺calcd. for C₂₇H₂₁F₂N₄O₄⁺:

503.1525, found: 503.1513.

5.1.46. N-(4-((2-carbamoylpyridin-4-yl)oxy)-2,5-diﬂuorophenyl)-3-(4-

ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

carboxamide (26a)

5.1.50. N-(2,5-diﬂuoro-4-((2-(4-(4-methylpiperazin-1-yl)piperidine-1-

carboxamido)pyridin-4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-1-isopropyl-2,4-

dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (28a)

The title compound was prepared from 7b and 10b using a method

analogous to that described for 18a in 79% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.47 (d, J = 7.3 Hz, 6H), 4.94 (spt, J

To

a

solution of N-(4-((2-aminopyridin-4-yl)oxy)-2,5-diﬂuor-

ophenyl)-3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

13

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

tetrahydropyrimidine-5-carboxamide (27a) (34.0 mg, 66

μ

mol) in THF

477.1681, found: 477.1669.

(2 mL) were added iPr₂NEt (35 μL, 199 μmol) and phenyl chloroformate

(25.0 mg, 160

μ

mol) at 0 ^◦C. The reaction mixture was stirred for 30 min

5.1.54. N-(4-((6-aminopyrimidin-4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-1-

methyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (32b)

The title compound was prepared from 31 and 7a using a method

analogous to that described for 18a in 92% as a white solid.

at room temperature. The reaction mixture was poured into water and

extracted with EtOAc. The organic extract was washed with brine, dried

over MgSO₄, ﬁltered and then concentrated. To a solution of the residue

in DMF (2 mL) were added iPr₂NEt (35

μ

L, 199

μ

mol) and 1-methyl-4-

¹H NMR (500 MHz, DMSO‑d₆): δ = 3.49 (s, 3H), 5.67 (s, 1H), 6.79 (s,

2H), 7.08 (d, J = 8.6 Hz, 2H), 7.28–7.44 (m, 4H), 7.66 (d, J = 8.6 Hz,

2H), 8.02 (s, 1H), 8.81 (s, 1H), 10.81 (s, 1H); ¹³C NMR (126 MHz,

DMSO‑d₆): δ = 37.9, 86.8, 104.5, 116.5 (d, J_CF= 23.9 Hz), 121.6, 122.6,

131.3 (d, J_CF= 8.8 Hz), 131.9, 135.7, 149.1, 151.1, 152.4, 158.7, 160.6,

162.3 (d, J_CF= 245.7 Hz), 163.9, 166.5, 167.0; HRMS–ESI m/z [M+H]⁺

calcd. for C₂₂H₁₈FN₆O⁺₄: 449.1368, found: 449.1355.

(piperidine-4-yl)-piperazine (24.4 mg, 133

μ

mol) at room tempera-

ture. The reaction mixture was stirred for 17 h at room temperature. The

reaction mixture was poured into water and extracted with EtOAc. The

organic extract was washed with water and brine, dried over MgSO₄,

ﬁltered and then concentrated. The residue was puriﬁed with column

chromatography on NH silica gel (EtOAc ~ EtOAc/MeOH = 4/1) to

afford the title compound 28a as a white solid (33 mg, 46 μmol, 69%).

¹H NMR (500 MHz, CDCl₃): δ = 1.49 (d, J = 7.3 Hz, 8H), 1.87 (d, J =

11.0 Hz, 2H), 2.25–2.30 (m, 4H), 2.38–2.50 (m, 4H), 2.50–2.65 (m, 4H),

2.80–2.93(m, 2H), 4.08 (d, J = 13.5 Hz, 2H), 4.89–5.02 (m, 1H), 6.52

(dd, J = 6.1, 2.5 Hz, 1H), 6.95 (dd, J = 10.4, 7.3 Hz, 1H), 7.19–7.25 (m,

5H), 7.62 (d, J = 2.5 Hz, 1H), 8.04 (d, J = 5.5 Hz, 1H), 8.45 (dd, J =

11.9, 7.0 Hz, 1H), 8.65 (s, 1H), 11.09 (s, 1H); ¹³C NMR (126 MHz,

CDCl₃): δ = 21.7, 28.3, 43.7, 46.1, 49.1, 50.6, 55.5, 61.5, 100.1, 105.5,

5.1.55. N-(4-((6-aminopyrimidin-4-yl)oxy)phenyl)-1-cyclopropyl-3-(4-

ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide

(32c)

The title compound was prepared from 31 and 7d using a method

analogous to that described for 18a in 91% as a white solid.

¹H NMR (500 MHz, DMSO‑d₆): δ = 0.94–1.02 (m, 4H), 3.20–3.26 (m,

1H), 5.67 (s, 1H), 6.79 (s, 2H), 7.07 (d, J = 9.2 Hz, 2H), 7.29–7.40 (m,

4H), 7.66 (d, J = 9.2 Hz, 2H), 8.02 (s, 1H), 8.45 (s, 1H), 10.77 (s, 1H);

¹³C NMR (126 MHz, DMSO‑d₆): δ = 7.0, 33.5, 86.9, 104.5, 116.5 (d, J_CF

= 23.9 Hz), 121.6, 122.6, 131.4 (d, J_CF= 8.8 Hz), 131.9, 135.7, 149.2,

151.0, 151.3, 158.7, 160.4, 162.3 (d, J_CF= 245.7 Hz), 163.5, 166.5,

169.9; HRMS–ESI m/z [M+H]⁺calcd. for C₂₄H₂₀FN₆O⁺₄: 475.1525,

found: 475.1512.

107.0, 110.1 (d, J_CF= 22.7 Hz), 110.5 (d, J_CF= 26.5 Hz), 116.9 (d, J_CF

=

22.7 Hz), 124.7, 130.1 (d, J_CF= 7.6 Hz), 130.2, 136.3, 146.5, 148.4 (d,

J_CF= 242.6 Hz), 149.0, 150.4 (d, J_CF= 242.6 Hz), 150.5, 153.5, 154.8,

160.2, 162.8, 162.8 (d, J_CF= 249.5 Hz), 165.7; HRMS–ESI m/z [M+H]⁺

calcd. for C₃₆H₄₀F₃N₈O⁺₅: 721.3068, found: 721.3041.

5.1.51. N-(2,5-diﬂuoro-4-((2-(4-(4-methylpiperazin-1-yl)piperidine-1-

carboxamido)pyridin-4-yl)oxy)phenyl)-2,5-dioxo-1-phenyl-1,2,5,6,7,8-

hexahydroquinoline-3-carboxamide (28b)

5.1.56. N-(4-((6-aminopyrimidin-4-yl)oxy)phenyl)-1-cyclopentyl-3-(4-

ﬂuorophenyl)-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide

(32d)

The title compound was prepared from 27b using a method analo-

gous to that described for 28a in 59.3% as a white solid.

The title compound was prepared from 31 and 7c using a method

analogous to that described for 18a in 94% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.70–1.86 (m, 4H), 1.88–2.01 (m,

2H), 2.17–2.27 (m, 2H), 4.80 (s, 2H), 4.98 (quin, J = 7.8 Hz, 1H), 5.81

(s, 1H), 7.08 (d, J = 8.6 Hz, 2H), 7.23 (s, 4H), 7.67 (d, J = 9.2 Hz, 2H),

8.27 (s, 1H), 8.66 (s, 1H), 10.79 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ =

24.2, 31.9, 59.7, 87.8, 105.7, 117.0 (d, J_CF= 23.9 Hz), 121.6, 122.2,

130.0 (d, J_CF= 7.6 Hz), 130.4, 135.4, 147.1, 149.1, 150.8, 158.7, 159.9,

162.8 (d, J_CF= 250.7 Hz), 163.1, 165.1, 170.5; HRMS–ESI m/z [M+H]⁺

calcd. for C₂₆H₂₄FN₆O⁺₄: 503.1838, found: 503.1824.

¹H NMR (500 MHz, CDCl₃): δ = 1.43–1.53 (m, 2H), 1.87 (d, J = 11.6

Hz, 2H), 2.09 (quin, J = 6.4 Hz, 2H), 2.27 (s, 3H), 2.35–2.48 (m, 5H),

2.50–2.61 (m, 8H), 2.82–2.92 (m, 2H), 4.08 (d, J = 13.5 Hz, 2H), 6.49

(dd, J = 6.1, 2.5 Hz, 1H), 6.93 (dd, J = 10.4, 7.3 Hz, 1H), 7.19 (s, 1H),

7.24–7.30 (m, 2H), 7.58–7.60 (m, 1H), 7.61–7.65 (m, 3H), 8.03 (d, J =

5.5 Hz, 1H), 8.57 (dd, J = 11.9, 7.0 Hz, 1H), 9.29 (s, 1H), 11.66 (s, 1H);

¹³C NMR (126 MHz, CDCl₃): δ = 21.2, 28.3, 29.5, 36.6, 43.7, 46.1, 49.1,

55.5, 61.6, 100.2, 106.8, 110.0 (d, J_CF= 22.7 Hz), 110.7 (d, J_CF= 26.5

Hz), 115.6, 119.6, 125.0 (dd, J_CF= 11.5, 11.5 Hz), 127.4, 130.2, 130.6,

136.1 (d, J_CF= 13.9, 10.1 Hz), 136.9, 142.4, 148.5 (d, J_CF= 241.9 Hz),

148.9, 150.4 (d, J_CF= 244.4 Hz), 153.5, 154.8, 160.1, 161.1, 163.3,

165.7, 192.9; HRMS–ESI m/z [M+H]⁺calcd. for C₃₈H₄₀F₂N₇O₅⁺:

712.3054, found: 712.3040.

5.1.57. N-(4-((6-(cyclopropanecarboxamido)pyrimidin-4-yl)oxy)phenyl)-

3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

carboxamide (33a)

To a solution of N-(4-((6-aminopyrimidin-4-yl)oxy)phenyl)-3-(4-

ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

5.1.52. 6-(4-aminophenoxy)pyrimidin-4-amine (31)

The title compound was prepared from 29 and 30 using a method

analogous to that described for 10a in 67.7% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 3.64 (br s, 2H), 4.75 (br s, 2H), 5.74

(s, 1H), 6.68–6.70 (m, 2H), 6.89–6.92 (m, 2H), 8.28 (s, 1H); ¹³C NMR

(126 MHz, methanol‑d₄): δ = 86.0, 116.1, 121.7, 144.3, 145.6, 157.8,

166.2, 171.0; HRMS–ESI m/z [M+H]⁺calcd. for C₁₀H₁₁N₄O⁺:

203.0927, found: 203.0921.

carboxamide (32a) (40 mg, 84

(73

μmol) in THF (2 mL) was added iPr₂NEt

μ

L, 0.42 mmol) and cyclopropanecarbonyl chloride (31 μL, 0.34

mmol) at room temperature. The reaction mixture was stirred at room

temperature for 3 days. The reaction mixture was poured into water and

extracted with EtOAc. The organic extract was washed with brine, dried

over MgSO₄, ﬁltered and then concentrated. The residue was puriﬁed

with column chromatography on NH silica gel (heptane/EtOAc = 1/1 ~

EtOAc) to afford the title compound 33a as a white solid (35 mg, 64

5.1.53. N-(4-((6-aminopyrimidin-4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-1-

isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-carboxamide (32a)

The title compound was prepared from 31 and 7b using a method

analogous to that described for 18a in 93% as a white solid.

μmol, 77%).

¹H NMR (500 MHz, CDCl₃): δ = 0.90–0.96 (m, 2H), 1.08–1.14 (m,

2H), 1.48 (d, J = 6.7 Hz, 6H), 1.53–1.55 (m, 1H), 4.95 (spt, J = 6.8 Hz,

1H), 7.08–7.10 (m, 2H), 7.23–7.25 (m, 4H), 7.65 (d, J = 1.2 Hz, 1H),

7.67–7.72 (m, 2H), 8.18 (s, 1H), 8.43 (s, 1H), 8.67 (s, 1H), 10.81 (s, 1H);

¹³C NMR (126 MHz, CDCl₃): δ = 9.14, 16.0, 21.6, 50.5, 95.5, 105.9,

116.9 (d, J_CF= 22.7 Hz), 121.5, 122.1, 130.0 (d, J_CF= 7.6 Hz), 130.4 (d,

J_CF= 3.8 Hz), 135.7, 146.3, 148.8, 150.5, 157.8, 158.9, 159.9, 162.8 (d,

J_CF= 250.7 Hz), 163.0, 171.3, 173.3; HRMS–ESI m/z [M+H]⁺calcd. for

¹H NMR (500 MHz, CDCl₃): δ = 1.47 (d, J = 6.7 Hz, 6H), 4.92–4.97

(m, 3H), 5.79 (s, 1H), 7.07 (d, J = 9.2 Hz, 2H), 7.20–7.28 (m, 4H), 7.65

(d, J = 8.6 Hz, 2H), 8.25 (s, 1H), 8.66 (s, 1H), 10.79 (s, 1H); ¹³C NMR

(126 MHz, CDCl₃): δ = 21.6, 50.5, 87.8, 105.8, 116.9 (d, J_CF= 22.7 Hz),

121.7, 122.2, 130.0 (d, J_CF= 8.8 Hz), 130.3 (d, J_CF= 3.8 Hz), 135.4,

146.3, 149.1, 150.5, 158.8, 159.9, 162.8 (d, J_CF= 250.7 Hz), 163.0,

165.1, 170.5; HRMS–ESI m/z [M+H]⁺calcd. for C₂₄H₂₂FN₆O₄⁺:

C

₂₈H₂₆FN₆O⁺₅: 545.1943, found: 545.1929.

14

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

5.1.58. N-(4-((6-(azetidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-

3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-tetrahydropyrimidine-5-

carboxamide (33b)

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 7.3 Hz, 6H), 1.77–1.87

(m, 4H), 2.48–2.51 (m, 4H), 3.36 (tt, J = 7.0, 4.9 Hz, 1H), 3.98 (dd, J =

8.6, 4.9 Hz, 2H), 4.07–4.15 (m, 2H), 4.88–5.02 (m, 1H), 6.89 (s, 1H),

7.06–7.14 (m, 2H), 7.22–7.28 (m, 4H), 7.53 (s, 1H), 7.64–7.72 (m, 2H),

8.35 (s, 1H), 8.67 (s, 1H), 10.79 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ =

21.6, 23.6, 50.5, 50.7, 52.3, 53.7, 93.8, 105.9, 116.9 (d, J_CF= 22.7 Hz),

121.5, 122.1, 130.0 (d, J_CF= 7.6 Hz), 130.4 (d, J_CF= 2.5 Hz), 135.6,

146.2, 148.9, 150.5, 154.2, 157.7, 159.8 (2C), 162.8 (d, J_CF= 249.5 Hz),

163.0, 170.9; HRMS–ESI m/z [M+H]⁺calcd. for C₃₂H₃₄FN₈O₅⁺:

629.2631, found: 629.2614.

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 89% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 6.7 Hz, 6H), 2.33 (quin,

J = 7.5 Hz, 2H), 4.10 (t, J = 7.6 Hz, 4H), 4.95 (spt, J = 6.8 Hz, 1H), 6.85

(s, 1H), 7.07–7.13 (m, 2H), 7.20–7.28 (m, 4H), 7.53–7.54 (m, 1H),

7.66–7.71 (m, 2H), 8.35 (s, 1H), 8.67 (s, 1H), 10.79 (s, 1H); ¹³C NMR

(126 MHz, CDCl₃): δ = 15.2, 21.6, 49.2, 50.5, 93.7, 105.9, 116.9 (d, J_CF

= 22.7 Hz), 121.5, 122.1, 130.0 (d, J_CF= 7.6 Hz), 130.4 (d, J_CF= 3.8

Hz), 135.6, 146.2, 148.9, 150.5, 154.1, 157.7, 159.8 (2C), 162.8 (d, J_CF

= 249.5 Hz), 163.0, 171.0; HRMS–ESI m/z [M+H]⁺calcd. for

5.1.63. 3-(4-ﬂuorophenyl)-1-isopropyl-N-(4-((6-(4-(4-methylpiperazin-1-

yl)piperidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-2,4-dioxo-

1,2,3,4-tetrahydropyrimidine-5-carboxamide (33 g)

C

₂₈H₂₇FN₇O⁺₅: 560.2052, found: 560.2035.

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 80% as a white solid.

5.1.59. 3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-N-(4-((6-(pyrrolidine-1-

carboxamido)pyrimidin-4-yl)oxy)phenyl)-1,2,3,4-tetrahydropyrimidine-5-

carboxamide (33c)

¹H NMR (500 MHz, CDCl₃): δ = 1.45–2.03 (m, 2H), 1.48 (d, J = 6.7

Hz, 6H), 1.90 (d, J = 11.6 Hz, 2H), 2.27 (s, 3H), 2.32–2.52 (m, 5H), 2.58

(br s, 4H), 2.92 (t, J = 11.6 Hz, 2H), 4.09 (d, J = 12.8 Hz, 2H), 4.89–5.01

(m, 1H), 7.09 (d, J = 9.2 Hz, 2H), 7.20–7.28 (m, 4H), 7.35 (s, 1H), 7.49

(s, 1H), 7.68 (d, J = 8.6 Hz, 2H), 8.35 (s, 1H), 8.67 (s, 1H), 10.79 (s, 1H);

¹³C NMR (126 MHz, CDCl₃): δ = 21.6, 28.3, 43.8, 46.1, 49.1, 50.5, 55.4,

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 89% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 6.7 Hz, 6H), 1.97 (br s,

4H), 3.46 (br s, 4H), 4.88–5.02 (m, 1H), 7.09–7.11 (m, 3H), 7.20–7.28

(m, 4H), 7.58–7.59 (m, 1H), 7.67–7.69 (m, 2H), 8.36 (s, 1H), 8.67 (s,

1H), 10.79 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 21.6, 25.6, 46.0,

61.4, 94.2, 105.9, 116.9 (d, J_CF= 22.7 Hz), 121.5, 122.1, 130.0 (d, J_CF

=

7.6 Hz), 130.4 (d, J_CF= 3.8 Hz), 135.6, 146.2, 148.9, 150.5, 152.8,

157.6, 159.8, 160.5, 162.8 (d, J_CF= 248.2 Hz), 163.0, 170.9; HRMS–ESI

m/z [M+H]⁺calcd. for C₃₅H₄₁FN₉O⁺₅: 686.3209, found: 686.3193.

50.5, 93.9, 105.9, 116.9 (d, J_CF= 22.7 Hz), 121.5, 122.1, 130.0 (d, J_CF

=

7.6 Hz), 130.4 (d, J_CF= 2.5 Hz), 135.6, 146.2, 149.0, 150.5, 152.2,

157.7, 159.8, 160.2, 162.8 (d, J_CF= 248.2 Hz), 163.0, 171.0; HRMS–ESI

m/z [M+H]⁺calcd. for C₂₉H₂₉FN₇O⁺₅: 574.2209, found: 574.2192.

5.1.64. 3-(4-ﬂuorophenyl)-1-methyl-N-(4-((6-(4-(4-methylpiperazin-1-yl)

piperidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (33 h)

5.1.60. 3-(4-ﬂuorophenyl)-N-(4-((6-(3-hydroxyazetidine-1-carboxamido)

pyrimidin-4-yl)oxy)phenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

The title compound was prepared from 32b using a method analo-

gous to that described for 28a in 76% as a white solid.

tetrahydropyrimidine-5-carboxamide (33d)

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 85% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.42–1.57 (m, 2H), 1.90 (d, J = 12.2

Hz, 2H), 2.24–2.32 (m, 4H), 2.44 (t, J = 10.7 Hz, 4H), 2.58 (br s, 4H),

2.91 (t, J = 12.5 Hz, 2H), 3.60 (s, 3H), 4.09 (d, J = 12.8 Hz, 2H), 7.09 (d,

J = 7.3 Hz, 2H), 7.20–7.30 (m, 4H), 7.36 (s, 1H), 7.49 (s, 1H), 7.67 (d, J

= 7.3 Hz, 2H), 8.35 (s, 1H), 8.60 (s, 1H), 10.73 (s, 1H); ¹³C NMR (126

MHz, CDCl₃): δ = 28.3, 38.2, 43.8, 46.1, 49.1, 55.4, 61.4, 94.1, 105.7,

117.0 (d, J_CF= 22.7 Hz), 121.6, 122.2, 130.0 (d, J_CF= 8.8 Hz), 130.1 (d,

J_CF= 3.8 Hz), 135.5, 149.0, 150.6, 150.8, 152.8, 157.6, 159.6, 160.5,

162.9 (d, J_CF= 249.5 Hz), 163.5, 170.9; HRMS–ESI m/z [M+H]⁺calcd.

for C₃₃H₃₇FN₉O⁺₅: 658.2896, found: 658.2883.

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 6.7 Hz, 6H), 2.25 (d, J =

6.1 Hz, 1H), 3.96 (dd, J = 10.4, 4.3 Hz, 2H), 4.31 (dd, J = 9.4, 7.0 Hz,

2H), 4.67–4.75 (m, 1H), 4.90–5.00 (m, 1H), 6.90 (s, 1H), 7.06–7.14 (m,

2H), 7.24 (d, J = 2.5 Hz, 4H), 7.53 (d, J = 1.2 Hz, 1H), 7.65–7.74 (m,

2H), 8.36–8.36 (m, 1H), 8.67 (s, 1H), 10.80 (s, 1H); ¹³C NMR (126 MHz,

DMSO‑d₆): δ = 21.2, 51.0, 60.2, 79.7, 93.0, 105.2, 116.4 (d, J_CF= 22.9

Hz), 121.5, 122.7, 131.4 (d, J_CF= 8.8 Hz), 132.1, 136.0, 147.5, 148.8,

150.6, 155.8, 158.1, 160.6, 161.6, 162.3 (d, J_CF= 247.0 Hz), 163.3,

170.6; HRMS–ESI m/z [M+H]⁺calcd. for C₂₈H₂₇FN₇O⁺₆: 576.2001,

found: 576.1983.

5.1.65. 1-cyclopropyl-3-(4-ﬂuorophenyl)-N-(4-((6-(4-(4-methylpiperazin-

1-yl)piperidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-2,4-dioxo-

1,2,3,4-tetrahydropyrimidine-5-carboxamide (33i)

5.1.61. N-(4-((6-(3-(dimethylamino)azetidine-1-carboxamido)pyrimidin-

4-yl)oxy)phenyl)-3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (33e)

The title compound was prepared from 32c using a method analo-

gous to that described for 28a in 76% as a white solid.

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 89% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 0.99–1.05 (m, 2H), 1.15–1.22 (m,

2H), 1.50 (qd, J = 12.1, 4.0 Hz, 2H), 1.90 (d, J = 11.0 Hz, 2H), 2.27 (s,

3H), 2.38–2.48 (m, 5H), 2.58 (br s, 4H), 2.85–2.98 (m, 2H), 3.30 (tt, J =

7.3, 3.7 Hz, 1H), 4.09 (d, J = 13.5 Hz, 2H), 7.08–7.10 (m, 2H),

7.20–7.27 (m, 4H), 7.33 (s, 1H), 7.49 (s, 1H), 7.66–7.69 (m, 2H), 8.35

(d, J = 1.2 Hz, 1H), 8.64 (s, 1H), 10.74 (s, 1H); ¹³C NMR (126 MHz,

CDCl₃): δ = 7.2, 28.3, 33.3, 43.8, 46.1, 49.1, 55.4, 61.4, 94.2, 105.5,

116.9 (d, J_CF= 22.7 Hz), 121.5, 122.1, 129.3, 130.0 (d, J_CF= 8.8 Hz),

¹H NMR (500 MHz, CDCl₃): δ = 1.48 (d, J = 6.7 Hz, 6H), 2.18 (d, J =

1.2 Hz, 6H), 3.10–3.18 (m, 1H), 3.88–3.95 (m, 2H), 4.02–4.09 (m, 2H),

4.95 (spt, J = 6.6 Hz, 1H), 6.91 (s, 1H), 7.09 (d, J = 7.3 Hz, 2H),

7.20–7.27 (m, 4H), 7.53 (s, 1H), 7.68 (d, J = 8.0 Hz, 2H), 8.35 (s, 1H),

8.67 (d, J = 1.2 Hz, 1H), 10.79 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ =

21.6, 41.8, 50.5, 53.4, 54.9, 93.8, 105.9, 116.9 (d, J_CF= 22.7 Hz), 121.5,

122.1, 130.0 (d, J_CF= 7.6 Hz), 130.4 (d, J_CF= 3.8 Hz), 135.6, 146.2,

148.9, 150.5, 154.2, 157.7, 159.8 (2C), 162.8 (d, J_CF= 249.5 Hz), 163.0,

171.0; HRMS–ESI m/z [M+H]⁺calcd. for C₃₀H₃₂FN₈O⁺₅: 603.2474,

found: 603.2456.

135.5, 148.9, 150.0, 151.2, 152.8, 157.6, 159.6, 160.5, 162.8 (d, J_CF

=

249.5 Hz), 163.2, 170.9; HRMS–ESI m/z [M+H]⁺calcd. for

C

₃₅H₃₉FN₉O⁺₅: 684.3053, found: 684.3037.

5.1.66. 1-cyclopentyl-3-(4-ﬂuorophenyl)-N-(4-((6-(4-(4-methylpiperazin-

1-yl)piperidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-2,4-dioxo-

1,2,3,4-tetrahydropyrimidine-5-carboxamide (33j)

5.1.62. 3-(4-ﬂuorophenyl)-1-isopropyl-2,4-dioxo-N-(4-((6-(3-(pyrrolidin-

1-yl)azetidine-1-carboxamido)pyrimidin-4-yl)oxy)phenyl)-1,2,3,4-

tetrahydropyrimidine-5-carboxamide (33f)

The title compound was prepared from 32d using a method analo-

gous to that described for 28a in 70.6% as a white solid.

The title compound was prepared from 32a using a method analo-

gous to that described for 28a in 87% as a white solid.

¹H NMR (500 MHz, CDCl₃): δ = 1.50 (qd, J = 12.1, 4.0 Hz, 2H),

15

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

1.74–1.83 (m, 4H), 1.88–1.95 (m, 4H), 2.19–2.23 (m, 2H), 2.27 (s, 3H),

2.36–2.52 (m, 5H), 2.58 (br s, 4H), 2.86–2.97 (m, 2H), 4.05–4.14 (m,

2H), 4.97 (quin, J = 7.8 Hz, 1H), 7.08–7.10 (m, 2H), 7.23 (d, J = 2.5 Hz,

4H), 7.34 (s, 1H), 7.49 (d, J = 1.2 Hz, 1H), 7.65–7.71 (m, 2H), 8.35 (s,

1H), 8.66 (s, 1H), 10.79 (s, 1H); ¹³C NMR (126 MHz, CDCl₃): δ = 24.2,

28.3, 31.9, 43.8, 46.1, 49.1, 55.4, 59.7, 61.4, 94.2, 105.8, 116.9 (d, J_CF

= 22.7 Hz), 121.5, 122.1, 130.0 (d, J_CF= 7.6 Hz), 130.4 (d, J_CF= 3.8

Hz), 135.6, 147.0, 148.9, 150.8, 152.8, 157.6, 159.8, 160.5, 162.8 (d,

J_CF= 249.5 Hz), 163.0, 170.9; HRMS–ESI m/z [M+H]⁺calcd. for

the resulting supernatant was subjected to an LC-MS/MS analysis. The

ratio of peak area responses relative to the internal standard was

determined, and the residual ratio of the test articles in the presence of

NADPH relative to that in the absence of NADPH was calculated.

5.2.6. Evaluation of pharmacokinetic proﬁle in mice

Compound 33g was formulated for oral administrations at 10, 30,

and 100 mg/kg in DMSO/Tween80/5% glucose (7:13:80, v/v/v) and

administered to female CAnN.Cg-Foxn1nu/CrlCrlj mice. After adminis-

tration, blood was collected from retinal vein at 0.5, 1, 2, 8, and 24 h

postdose and centrifuged to obtain plasma samples. Plasma concentra-

tions of compound 33g were measured using liquid chromatography-

tandem mass spectrometry (LC-MS/MS). Plasma samples were precipi-

tated with 20 volumes of acetonitrile/methanol (7:3, v/v) containing

internal standards (10 ng/mL propranolol and 500 ng/mL niﬂumic

acid). Following vortex mixing and centrifugation, the supernatant was

ﬁltered, and the resulting ﬁltrate was injected into an LC-MS/MS. For

samples above the upper limit of quantiﬁcation, the resulting ﬁltrate

was 100-fold diluted with 50% acetonitrile and subjected to an LC-MS/

MS analysis. Detection was accomplished by multiple reaction moni-

toring in positive ionization mode. The ratio of the peak area responses

relative to the internal standard were used to construct a standard curve

using linear least squares regression with a 1/x²weighting. PK param-

eters, maximum drug concentration (C_max), area under the concen-

tration–time curve (AUC_(0-24h)) from zero to 24 h postdose, and time to

reach maximum (peak) concentration following drug administration

were determined based on the mean plasma concentration.

C

₃₇H₄₃FN₉O⁺₅: 712.3366, found: 712.3349.

5.2. Biology

5.2.1. Cells and compounds

Recombinat human Axl-expressing or Mer-expressing mouse Ba/F3

cells were used for cell-based Axl inhibition assay (Carna Biosciences,

Inc.), and cells were cultured in RPMI1640 (WAKO) containing 10% (v/

v) FBS (Sigma-Aldrich), penicillin–streptomycin (WAKO). Cells were

cultured at 37 ^◦C under a 5% CO₂atmosphere.

5.2.2. Cell free Axl/Mer assay

GST fused human Axl or Mer cytoplasmic domain was incubated

with serial dilution of compound Km concentration of ATP, and

respective substrates in appropriate buffer for reaction at room tem-

perature for 1 h in <0.1% DMSO solution. For Axl kinase assay, after

reaction with ADP-Glo™ kinase assay kit, activities of kinase were

measured using luminometer. For Mer kinase assay, phosphorylation of

biotinylated substrates was measured by TR-FRET system using Eu-

labeled anti-phosphotyrosine antibody and APC-labeled streptavidin.

All experiments were performed in duplicate wells for each condition (n

= 2), and conducted once or twice independently. IC₅₀values were

calculated from plots of concentration versus percent inhibition by using

Microsoft Excel.

5.2.7. Ba/F3-Axl isogenic subcutaneous model

Mice were maintained under speciﬁc pathogen-free conditions and

housed in barrier facilities on a 12-hour light/dark cycle, with food and

water ad libitum. Animal experiments were conducted in accordance

with the Institutional Animal Care and Use Committee guidelines of

Eisai Co., Ltd.

Cultured Ba/F3-Axl cells were prepared in Hank’s balanced salt so-

lution (Thermo Fisher Scientiﬁc Inc.) to yield a suspension of 2.0 × 10⁶

cells/mL. A 0.1 mL aliquot of the cell suspension was transplanted

subcutaneously into the right ﬂank region of female C3H mice (7 weeks

old, CLEA Japan or Japan SLC). When tumor volumes reached around

200 mm³, mice were selected on the basis of their tumor volumes and

general condition, and were randomly divided into groups according to

their tumor volumes (n = 5 per group). The oral administration was

started on day 0, and the administration continued once daily for 4 days.

The differences in tumor volume between the non-treated and 33g-

treated groups were analyzed by one-way ANOVA followed by the

Dunnett’s multiple comparison test. A value of P < 0.05 (two-sided) was

considered statistically signiﬁcant. Statistical analyses were performed

using GraphPad Prism.

5.2.3. Cell based Ba/F3 Axl/Mer assay

Transformation of Ba/F3 cells required the kinase activity of the TEL-

Axl fusion or TEL-Mer fusionprotein. If a compound inhibits the activity

of the activity of the recombinant kinase, the proliferation of trans-

formed cells will suppress. Ba/F3-Axl or -Mer cells (500 cells) were

seeded in a 384-well cell culture plate in RPMI1640 (WAKO) containing

10% (v/v) FBS (Sigma-Aldrich), 1% penicillin–streptomycin (WAKO).

And, various concentrations of compounds were added, and the cells

were incubated at 37 ^◦C for 2 days in culture medium containing 0.1%

DMSO. Then, Cell Titer-Glo-2.0 (Promega) was added to each well,

viable cell number was measured with a EnVision or ARVO (Perkin

Elmer) microplate reader. All experiments were performed in quadru-

plicate wells for each condition (n = 4), and conducted once or twice

independently. IC₅₀values were calculated from plots of concentration

versus percent inhibition by using Microsoft Excel.

5.2.8. In vivo study in mice to detect the retinal toxicity

28a and 33g were orally administrated once a daily for 14 days to

female BALB/cAJcl mice (n = 3 or 5/group) at 30 and 100 mg/kg or 100

mg/kg, respectively. Control animals were not treated during the dosing

period. All animals were sacriﬁced according to the guideline for animal

experiment of Eisai Co., Ltd. and necropsied after a 14-day dosing

period. Eyes collected from all animals were ﬁxed in a mixture of 3%

glutaraldehyde and 2.5% formalin. Tissues were embedded in parafﬁn,

sectioned and stained with hematoxylin-eosin (HE) slide and were

submitted for histopathologic examination.

5.2.4. Solubility assay

Kinetic solubility was determined in Dulbecco’s phosphate-buffered

saline (pH 7, Invitrogen Corporation) containing 1% (v/v) DMSO.

Firstly, DMSO stock solution was prepared at the concentration of 10

mM. The stock solution was diluted by 100 times with test ﬂuid and then

stirred for 15 min at room temperature. After vacuum suction ﬁltration,

the concentration of the ﬁltrate was measured by using HPLC-UV

method (wavelength: 254 nm).

5.2.5. Microsomal stability screening in liver microsomes

5.2.9. Statistical analysis

Test articles were incubated with mouse liver microsomes (0.5 mg/

mL) for 30 min at 37 ^◦C in the reaction mixture containing 0.1 M po-

tassium phosphate (pH 7.4), 0.1 mM EDTA, 0.33 mM β-NADP⁺, 8 mM

glucose-6-phosphate, 0.1 unit/mL glucose-6-phosphate dehydrogenase,

and 6 mM MgCl₂. After adding acetonitrile/methanol (7:3, v/v) mixture

containing propranolol as the internal standard to the reaction mixture,

All data are presented as means ± SD. The differences between the

means of groups were analyzed by one-way analysis of variance

(ANOVA) followed by Dunnett’s multiple comparison test. P values less

than 0.05 were considered statistically signiﬁcant. Statistical analyses

were performed using GraphPad Prism version 8.3.1.

16

S. Inoue et al.

Bioorganic & Medicinal Chemistry 39 (2021) 116137

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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.

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Acknowledgements

The authors would like to thank the colleagues who helped to

generate all of the data reported in this manuscript. In particular, special

thanks are owed to Nao Shibuguchi for HRMS analyses, Ikuo Kushida

and Takako Yoshiba for solubility analyses.

13 Mollard Alexis, Warner Steven L, Call Lee T, et al. Design, synthesis and biological

evaluation of a series of novel Axl kinase inhibitors. ACS Med Chem Lett. 2011;2(12):

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Appendix A. Supplementary material

Supplementary data to this article can be found online at https://doi.

org/10.1016/j.bmc.2021.116137.

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inhibitor of MET, AXL, and FGFR with strong preclinical activity alone and in

association with bevacizumab. Mol Cancer Therap. 2013;12:1749–1762. https://doi.

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17

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Discovery of a potent and selective Axl inhibitor in preclinical model

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