Synthesis and Evaluation of a Novel Series of Diphenylamine and Diphenylether Derivatives with Osteoblastogenic and Osteogenic Effects via CDK8 Inhibition

Abstract

Osteoporosis is induced by an imbalance between osteogenesis and bone resorption, and is treated with osteogenic drugs and/or resorption inhibitors. Resorption inhibitors, such as bisphosphonates, are orally used; however, orally active small molecules with osteogenic activity are not clinically available. We synthesized various types of small molecules and identified a series of diphenylamine and diphenylether derivatives that promoted osteoblast differentiation. Among them, diphenylether derivatives 13a, 13g, and 13h potently promoted osteoblast differentiation (EC₂₀₀ for increasing alkaline phosphatase activity = 11.3, 31.1, and 12.3 nM, respectively) and inhibited cyclin-dependent kinase 8 (CDK8) activity (IC₅₀ = 2.5, 7.8, and 3.9 nM, respectively), suggesting that their osteoblastgenic effects are mediated by the inhibition of CDK8. The ratio of the maximal plasma concentration after oral administration at 10 mg/kg in female rats and EC₂₀₀ for osteoblastogenesis was 148.1 for compound 13a, 53.4 for 13g, and 101.8 for 13h, indicating possible in vivo osteoblastogenic and osteogenic effects. In ovariectomized female rats, 13g and 13h at 10 mg/kg/d for 8 weeks increased plasma bone-type alkaline phosphatase activity, indicating enhanced in vivo osteoblastogenesis. Furthermore, micro-computed tomography (micro-CT) showed that both compounds increased femoral cortical bone volume and mineral contents, which were unaffected by ovariectomy, while having negligible effects on trabecular bone volume and mineral contents, which were markedly reduced by ovariectomy. In conclusion, diphenylamine and diphenylether structures are novel scaffolds for osteoblastogenesis enhancers via the inhibition of CDK8. Among them, 13g and 13h are candidates for anti-osteoporotic drugs with cortical bone-selective osteogenic effects.

Introduction

Bones are structurally and functionally maintained through remodeling by bone formation via osteoblasts and resorption via osteoclasts; therefore, an imbalance between bone formation and resorption causes osteoporosis in the elderly men and postmenopausal women. Osteoporosis has been treated with bone resorption inhibitors, such as oral and parenteral bisphosphonates, and osteogenic agents, including parenteral parathyroid hormone (PTH) and anti-sclerostin antibodies.^1,2) A large number of small molecules have been reported to accelerate osteoblast differentiation and are candidates as orally active osteogenic drugs. Among them, benzothiepine, benzofuran, thienopyridine, and coumarin derivatives enhanced osteoblast differentiation and exerted osteogenic effects in rodent bone defect models and postmenopausal osteoporosis models.^3–13) These derivatives promoted osteoblast differentiation by activating the estrogenic pathway, enhancing bone morphogenetic protein signaling, activating Wnt/β-catenin, and/or inhibiting cyclin-dependent kinase 8 (CDK8).^5,6,10–13) However, small molecules that exert osteogenic effects via osteoblastogenesis have not been successfully developed as anti-osteoporotic drugs. Therefore, new chemotypes of small molecules that enhance osteoblastogenesis have been desired for a long time. We synthesized various types of small molecules and examined their effects on osteoblast differentiation in bone marrow parenchymal cells.^14–16) Among them, 4-acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-065), a diphenylether derivative, exhibited potent osteoblastogenic activity in bone parenchymal cells and osteogenic effects in ovariectomized female rats (OVX rats).¹⁴⁾ It also potently inhibited CDK8 activity, suggesting that its osteoblastogenic activity is mediated by the inhibition of CDK8. We briefly presented some of our findings on the effects of KY-065 at a symposium in the annual meeting of the Pharmaceutical Society of Japan (2018).¹⁴⁾ We recently showed that (R)-4-(1-hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (KY-273), a diphenylether derivative, exerted osteoblastogenic effects, possibly via the inhibition of CDK8, and osteogenic effects on femur diaphyseal and metaphyseal cortical bone, but not trabecular bone, via osteoblastogenesis in OVX rats.¹⁶⁾

In the present study, we described the synthesis and evaluation of a series of diphenylamine and diphenylether derivatives, including KY-065 (Fig. 1). Osteoblastogenic effects were assessed by measuring alkaline phosphatase activity in a mouse bone marrow-derived cell line (ST2 cells), and structure–activity relationships were discussed. The effects of selected osteogenic compounds on CDK8 activity were then examined because KY-065 and KY-273 are known to inhibit CDK8 activity. Furthermore, the plasma concentrations of selected compounds after their oral administration were assessed in female rats, and the ratio of the maximal plasma concentration and EC₂₀₀ for osteoblastogenesis (C_max/EC₂₀₀) was calculated to estimate the potentiality of in vivo osteoblastogenic activity. The effects of 13g and 13h with high C_max/EC₂₀₀ on femoral bone were investigated in OVX rats using an in vivo micro-computed tomography (micro-CT) device.

Fig. 1. Structures of Diphenylamine and Diphenylether Derivatives

Chemistry

General approaches to the synthesis of diphenylamine and diphenylether derivatives are outlined in Charts 1–5. Chart 1 shows the synthesis of diphenylamine derivatives 7a–7g. Previously reported 1¹⁷⁾ and 2¹⁸⁾ were converted to the corresponding acetals 3 and 4, respectively, followed by the Buchwald–Hartwig coupling reaction with aniline to give 5a, 5b, and 5e–5g. The esters 5a, 5b, and 5e–5g were hydrolyzed and then amidated to give 6a, 6b, and 6e–6g. Compounds 7a and 7e–7g were obtained by the deprotection of the acetal. Compounds 7b and 7c were synthesized by the deprotection of the acetal and tert-butoxycarbonyl (Boc) of 6b followed by alkylation of the side chain piperidine. Compound 7d was synthesized by the Buchwald–Hartwig coupling, hydrolysis, and amidation from compound 4 as in the method described above, followed by reduction of the double bond in the side chain at the end.

Chart 1. Synthesis of 4-Acetyl-3-[(4-substituted)phenylamino]benzamide Derivatives 7a–7g

(i) 1,2-Bis(trimethylsilyloxy)ethane, TMSOTf, CH₂Cl₂; (ii) various anilines, Pd(OAc)₂, BINAP, Cs₂CO₃, 1,4-dioxane; (iii) LiOH aq., MeOH, THF; (iv) EDC·HCl, HOBt, NH₃ aq., DMF; (v) HCl aq., THF; (vi) HCl in IPA, HCO₂H, then glycolaldehyde dimer, NaBH₃CN, MeOH (for 7b); (vii) HCl in IPA, HCO₂H, then formalin, NaBH₃CN, MeOH (for 7c); (viii) HCl aq., THF then H₂, Pd-C, MeOH.

Chart 2 shows the synthesis of diphenylamine derivatives 7h and 7i. Compound 8, which was prepared from 4-acetyl-3-hydroxybenzonitrile,¹⁹⁾ was converted to 9h and 9i by the Buchwald–Hartwig coupling with aniline, and the cyano group of 9h and 9i was hydrolyzed with aqueous hydrogen peroxide solution to give 10h and 10i. Compound 7h was synthesized by the deprotection of the acetal of 10h, while 7i was synthesized by the acetal deprotection of 10i followed by hydrolysis.

Chart 2. Synthesis of 4-Acetyl-3-[(4-substituted)phenylamino]benzamide Derivatives 7h and 7i

(i) Various anilines, Pd(OAc)₂, BINAP, Cs₂CO₃, 1,4-dioxane; (ii) H₂O₂ aq., K₂CO₃, DMSO; (iii) HCl aq., THF; (iv) HCl aq., THF then LiOH aq., MeOH, THF.

Chart 3 shows the synthesis of diphenylether derivatives. Compounds 12a, 12g, 12h, 12j, 12k, 12m, and 12o were prepared from previously reported 11²⁰⁾ by a nucleophilic aromatic substitution reaction. Compounds 13g, 13h, 13j, 13k, 13m, and 13o were synthesized by hydrolyzing the cyano groups of 12g, 12h, 12j, 12k, 12m, and 12o, respectively, and 13a was synthesized by the further deprotection of the tetrahydropyranyl (THP) group. Compound 13n was obtained by a reduction of the double bond in the side chain of 13o. Separately, 13l was synthesized by the hydrolysis and amidation of 15l, which was obtained by an aromatic nucleophilic substitution reaction from previously reported compound 14.²¹⁾

Chart 3. Synthesis of 4-Acetyl-3-[(4-substituted)phenoxy]benzamide Derivatives 13a, 13g, 13h, 13j–13o

(i) Various phenols, K₂CO₃, DMF; (ii) H₂O₂ aq., K₂CO₃, DMSO then HCl aq., THF (for 13a); (iii) H₂O₂ aq., K₂CO₃, DMSO; (iv) H₂, Pd-C, MeOH; (v) 4-[(2-isopropoxyethoxy)methyl]phenol, K₂CO₃, DMF; (vi) LiOH aq., MeOH; (vii) ClCO₂i-Bu, NMM, THF then NH₃ aq.

Chart 4 shows the synthesis of compounds with various conversions of the R² substituent of diphenylether derivatives. Compounds 21h and 22h were synthesized from 16²²⁾ and 17, which were prepared from 3-fluoro-4-formylbenzonitrile,²³⁾ by an aromatic nucleophilic substitution reaction followed by a hydrolysis. Compound 20h was obtained by fluorination of the carbonyl of compound 12h using bis(2-methoxyethyl)aminosulfur trifluoride (Deoxo-Fluor^® reagent), and the cyano group of 20h was then hydrolyzed to obtain compound 24h. The carbonyl of 13h was reduced to synthesize compound 25h, and the hydroxyl group of 25h was further fluorinated using N,N-diethylaminosulfur trifluoride (DAST) to obtain compound 23h.

Chart 4. Synthesis of 4-Substituted-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide Derivatives 21h–25h

(i) Phenol 62, K₂CO₃, NMP; (ii) Deoxofluor^®, CHCl₃; (iii) H₂O₂ aq., K₂CO₃, DMSO; (iv) NaBH₄, MeOH; (v) DAST, CHCl₃.

Chart 5 shows the synthesis of derivatives with methylene, thioether, and sulfonyl linkages. Compound 27 was synthesized by the oxidation of compound 26²⁴⁾ to give an aldehyde, followed by condensation with phenol in the presence of sulfuric acid. The hydroxyl group of 27 was alkylated by the Mitsunobu reaction to obtain 28h. Compound 28h was converted to carboxylic acid by ionic hydrogenation using triethylsilane and titanium tetrachloride, and condensed with N,O-dimethylhydroxylamine to form the Weinreb amide 29h. Compound 29h was converted to the ketone 30h using methyl lithium, followed by cyanation and hydrolysis to give compound 32h. Compound 34h was synthesized by the addition of 4-hydroxybenzenethiol to compound 14 followed by alkylation of the hydroxyl group, hydrolysis, and amidation. Compound 34h was oxidized with m-chloroperoxybenzoic acid (m-CPBA) to obtain 35h.

Chart 5. Synthesis of Derivatives 32h, 34h, and 35h with the Converted Linker Structure

(i) SO₃·pyridine, i-Pr₂NEt, DMSO, CH₂Cl₂; (ii) phenol, H₂SO₄ aq.; (iii) 2-[(tetrahydropyran-4-yl)oxy]ethanol, DIAD, PPh₃, CH₂Cl₂; (iv) Et₃SiH, TiCl₄, CH₂Cl₂; (v) N,O-Dimethylhydroxylamine hydrochloride, EDC·HCl, Et₃N, CH₂Cl₂; (vi) MeLi in Et₂O, THF; (vii) CuCN, CuI, DMF; (viii) H₂O₂ aq., NaOH aq., DMSO; (ix) 4-hydroxybenzenethiol, Et₃N, DMF; (x) 2-[(tetrahydropyran-4-yl)oxy]ethyl methanesulfonate, K₂CO₃, DMF; (xi) LiOH aq., MeOH, THF; (xii) ClCO₂i-Bu, NMM, THF then NH₃ aq.; (xiii) m-CPBA, CH₂Cl₂.

Results and Discussion

Osteoporosis is treated with osteogenic drugs, such as parenteral PTH and anti-sclerostin antibodies, and bone resorption inhibitors, including oral and parenteral bisphosphonates.^1,2) In spite of many years of efforts by medicinal chemists and pharmaceutical companies, no oral osteogenic drug has been successfully developed and clinically used.

In the present study, we synthesized a series of diphenylamine and diphenylether derivatives and screened them to find a new osteoblastogenic compound, which is expected to promote osteogenesis. We synthesized a series of diphenylamine derivatives with various substituents at the 4-position of the phenyl ring (Table 1). Among these, 7a with 3-(2-hydroxyethoxy)azetidine as R¹ exerted potent osteoblastogenic effects (EC₂₀₀ = 81.2 nM), which was similar to the previously reported thieopyridine derivative.⁸⁾ The activities of 7b, 7c, and 7d with piperidine and morpholine groups, basic substituents, and 7i with a carboxy group, an acidic substituent, were markedly lower than that of 7a with an almost neutral substituent. In 7e, the removal of the azetidine ring reduced its activity: the length or direction of the substituent may not be appropriate. The activity of compound 7f with an ethoxy group instead of the azetidine in 7a was reduced by approximately 4-fold: the length of substituents was similar in both compounds, whereas the direction may not be appropriate in 7f. Compounds 7g and 7h with tetrahydrofuran and tetrahydropyran structures, respectively, at the end of the R¹ substituent exhibited activity that was 2-fold stronger than that of 7f with a hydroxy group; their bulky ring structures may be beneficial for interactions with target proteins. Therefore, neutral substituents are preferred for interactions with target proteins, and a hydroxyl group may not be essential for activity. The plasma C_max of 7a, 7g, and 7h after their oral administration at 10 mg/kg in female rats were 3.8, 2.9, and 4.3 µM, respectively: therefore, C_max/EC₂₀₀ were 46.7, 17.4, and 32.5, respectively.

Table 1. Chemical Structures, Promotion of Osteoblast Differentiation, Maximal Plasma Concentrations after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of the Maximal Plasma Concentration and EC₂₀₀ for Osteoblastogenesis in ST2 Cells of 4-Acetyl-3-[(4-substituted)phenylamino]benzamide Derivatives

a) n = 2. b) n = 3. Mean ± standard error (S.E.). ND: Not determined.

We then synthesized and evaluated a series of diphenylether derivatives (Table 2). The osteoblastogenic activities of diphenylether derivatives 13a, 13g, and 13h were 7.2-, 5.3-, and 10.7-fold stronger, respectively, than those of the corresponding diphenylamine derivatives 7a, 7g, and 7h: the diphenylether structure was more suitable as a skeleton for an osteoblastogenic compound than the diphenylamine structure. In diphenylamine derivatives, the proton of an amino group may disturb the interaction with a target protein and/or the direction of a substituted phenyl group may not be appropriate. Compound 13j, in which the azetidine structure of 13a was changed to a piperidine structure, exhibited 6-fold weaker activity, suggesting its inappropriate conformation. Compound 13g with tetrahydrofuran was slightly weaker than 13h with tetrahydropyran. However, 13g is a racemate: if either enantiomer had no activity, the other enantiomer may exhibit similar activity to 13h. Compound 13k with a linear isopropoxyethyl group was slightly less active than 13g and 13h. This result suggests that a cyclic structure or bulky substituent is preferred for interactions with target proteins. The activities of the four compounds 13l, 13m, 13n, and 13o with an alkyl or an alkenyl linkage in the R¹ group were markedly lower, by approximately 4- to 12-fold, than that of 13h with an ether linkage. The activities of the four compounds 13l, 13m, 13n, and 13o with a methylene linkage in the R¹ group were markedly lower, by approximately 10-fold, than that of 13h with an ether linkage. In these compounds, the electron density of the benzene ring in the side chain was slightly lower than that in 13h, which may be related to their weaker activity. The orientation or electron density of the substituent may be suitable in 13a, 13g, and 13h for interactions with proteins due to a nitrogen atom or an oxygen atom. The C_max of 13a, 13g, and 13h were 1.7, 1.7, and 1.3 µM, respectively, and C_max/EC₂₀₀ were 148.1, 53.4, and 101.8, respectively.

Table 2. Chemical Structures, Promotion of Osteoblast Differentiation, Maximal Plasma Concentrations after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of the Maximal Plasma Concentration and EC₂₀₀ for Osteoblastogenesis in ST2 Cells of 4-Acetyl-3-[(4-substituted)phenoxy]benzamide Derivatives

a) n = 2. b) n = 3. Mean ± S.E. ND: Not determined.

In Table 3, the acetyl group in the para-position of the carbamoyl group was replaced by various groups, and the oxygen at the diphenylether linkage was converted to other atoms. Compounds with alkyl groups having 1–3 fluorine atoms (21h, 22h, 23h, 24h) and hydroxyethyl 25h exhibited markedly weaker activity than 13h with an acetyl group. The carbonyl group may play an important role as a hydrogen bond acceptor in interactions with target proteins. Compounds 23h and 25h were racemates; therefore, either enantiomer may exhibit stronger activity. The R form enantiomer of 25h was recently reported to exhibit moderately potent osteoblastogenic activity and cortical bone-selective osteogenic effects on femoral bone in OVX rats.¹⁶⁾ The replacement of the oxygen of the diphenylether by CH₂, S, and SO₂ markedly reduced activity: the activity order was O > NH >S > CH₂ > SO₂. The orientation of the side chains appeared to be the most suitable in the oxygen linkage for interactions with proteins.

Table 3. Chemical Structures, Promotion of Osteoblast Differentiation, Maximal Plasma Concentrations after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of the Maximal Plasma Concentration and EC₂₀₀ for Osteoblastogenesis in ST2 Cells of 3,4-Substituted-benzamide Derivatives

a) n = 2. b) n = 3. Mean ± S.E. ND: Not determined.

Compounds 13a, 13g, and 13h with high C_max/EC₂₀₀ were speculated to exert in vivo osteogenic effects. Among these, 13g and 13h (KY-065) were selected for further evaluations in OVX rats (Table 4). Ovariectomy markedly reduced uterine weights: both compounds at 10 mg/kg/d for 8 weeks did not affect uterine weights, indicating no estrogenic effects. Ovariectomy markedly reduced bone volume (BV) and bone mineral contents (BMC) in metaphyseal trabecular bone in the femur and slightly reduced these parameters in metaphyseal cortical bone, but had no effects on BV or BMC in diaphyseal cortical bone. Micro-CT scanning showed that compound 13h significantly increased plasma bone-type alkaline phosphatase (ALP) activity, suggesting in vivo osteogenesis, and BV and BMC in femur diaphyseal and metaphyseal cortical bone, but not metaphyseal trabecular bone. Compound 13g slightly increased bone-type ALP activity and significantly increased BV and BMC in femur metaphyseal cortical bone; its effects were weaker than those of 13h, which may be related to smaller C_max/EC₂₀₀. These effects were attributed to enhanced osteogenesis rather than the inhibition of resorption because alendronate, a resorption inhibitor, markedly restored reductions in femur trabecular bone following ovariectomy without affecting non-damaged diaphyseal cortical bone.¹⁶⁾

Table 4. Effects of the Repeated Administration of 13g and 13h (10 mg/kg/d) for 8 Weeks on Plasma Bone-Type Alkaline Phosphatase and the Femur Bone Structure in in Vivo Micro-CT Scanning in Ovariectomized Rats

	Experiment 1a)			Experiment 2b)
	Intact	OVX		Intact	OVX
		Control	13g (10 mg/kg/d)		Control	13h (10 mg/kg/d)
BALP (IU/L)	102.3 ± 1.8	117.8 ± 9.0	133.3 ± 10.6	103.8 ± 5.6	98.8 ± 3.8	136.0 ± 7.8**
Diaphysis cortical bone
BV (mm3)	19.5 ± 0.29	19.4 ± 0.32	20.5 ± 0.42	19.8 ± 0.17	19.7 ± 0.32	21.3 ± 0.29**
BMC (mg)	22.0 ± 0.28	21.5 ± 0.37	22.7 ± 0.37	22.1 ± 0.18	21.5 ± 0.34	23.2 ± 0.29**
Metaphysis cortical bone
BV (mm3)	8.93 ± 0.22**	8.09 ± 0.13	8.93 ± 0.18**	8.63 ± 0.12	8.30 ± 0.11	9.20 ± 0.13**
BMC (mg)	8.92 ± 0.19**	8.02 ± 0.14	8.89 ± 0.13**	8.38 ± 0.12	8.03 ± 0.15	9.03 ± 0.15**
Metaphysis trabecular bone
BV (mm3)	3.95 ± 0.19**	1.75 ± 0.21	1.69 ± 0.14	4.17 ± 0.18**	1.58 ± 0.15	1.78 ± 0.12
BMC (mg)	1.72 ± 0.09**	0.62 ± 0.08	0.58 ± 0.06	1.75 ± 0.10**	0.57 ± 0.06	0.61 ± 0.04

a) n = 7. b) n = 8. Mean ± S.E. * p < 0.05, ** p < 0.01 vs. OVX Control, Dunnett’s method. BALP: plasma bone-type alkaline phosphatase activity. BV: bone volume. BMC: bone mineral contents.

Regarding the molecular mechanisms responsible for osteoblastogenic effects, KY-065 and KY-273, both diphenylether derivatives, have been reported to inhibit CDK8 activity.^14,16) Therefore, the effects of six potent osteoblastogenic compounds, including KY-065, on CDK8 activity were examined. Compounds 7a, 13a, 13k, 13g, 13h (KY-065), and 13l exhibited potent CDK8 inhibitory activity: their ratios of IC₅₀ for CDK8 inhibition and EC₂₀₀ were similar, suggesting that osteoblastogenesis was mediated by the inhibition of CDK8 (Fig. 2).

Fig. 2. Promotion (-logEC₂₀₀) of Osteoblast Differentiation and Inhibition (-logIC₅₀) of the CDK8 Enzyme by Diphenylamine and Diphenylether Derivatives (n = 2)

In conclusion, a series of diphenylamine and diphenylether derivatives were synthesized and their structure-osteoblastogenic activity relationships were clarified (Fig. 3). A diphenylether structure was shown to be a better scaffold than a diphenylamine structure for an osteoblastogenic compound. Among the derivatives examined, 13g and 13h exhibited potent osteoblastogenic activity, had high C_max/EC₂₀₀, and exerted osteogenic effects on femoral cortical bone, possibly by osteoblastogenesis via the inhibition of CDK8 in OVX rats.

Fig. 3. Summary of Structure-Osteoblastogenic Activity Relationships

Experimental

General

Melting points were measured on a melting point apparatus (MP-500P; Yanaco Technical Science Co., Ltd., Tokyo, Japan) and were uncorrected. ¹H-NMR and ¹³C-NMR spectra were obtained on a nuclear magnetic resonance spectrometer at 400 MHz (JNM-AL400 and JNM-ECZL400S; JEOL Ltd., Tokyo, Japan) using tetramethylsilane as an internal standard. IR spectra were recorded with an infrared spectrometer (HORIBA FT-720, HORIBA, Kyoto, Japan). Mass spectra were obtained on an ESI-MS spectrometer (Expression CMS-L, Advion, Ithaca, NY, U.S.A.) and ESI-TOF/MS (micrOTOF2-kp, Bruker, MA, U.S.A.). Column chromatography was performed on silica gel (Daisogel No.1001W; Daiso Co., Ltd., Osaka, Japan). Reactions were monitored by TLC (TLC silica gel 60F254, Merck KGaA, Darmstadt, Germany).

Ethyl 4-(2-Methyl-[1,3]dioxolan-2-yl)-3-trifluoromethanesul-fonyloxybenzoate (3)

Following the addition of 1,2-bis(trime-thylsilyloxy)ethane (0.43 mL, 1.8 mmol) and trimethylsilyl trifluoromethanesulfonate (TMSOTf) (0.05 mL, 0.3 mmol) to a solution of 1 (500 mg, 1.47 mmol) in CH₂Cl₂ (10 mL) under ice cooling, the reaction mixture was stirred at room temperature for 18 h. The mixture was washed with saturated aqueous NaHCO₃ solution and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 3 (490 mg, 87% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.40 (3H, t, J = 7.1 Hz), 1.74 (3H, s), 3.78–3.87 (2H, m), 4.05–4.15 (2H, m), 4.40 (2H, q, J = 7.1 Hz), 7.74 (1H, d, J = 8.1 Hz), 7.89 (1H, d, J = 1.4 Hz), 8.01 (1H, dd, J = 8.1, 1.4 Hz).

Methyl 3-Bromo-4-(2-methyl-[1,3]dioxolan-2-yl)benzoate (4)

Following the addition of 1,2-bis(trimethylsilyloxy)ethane (1.43 mL, 5.83 mmol) and TMSOTf (0.14 mL, 0.78 mmol) to a solution of 2 (1.00 g, 3.89 mmol) in CH₂Cl₂ (10 mL) under ice cooling, the reaction mixture was stirred at room temperature for 16 h. The mixture was washed with saturated aqueous NaHCO₃ solution and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 4 (1.13 g, 97% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.81 (3H, s), 3.70–3.80 (2H, m), 3.93 (3H, s), 4.05–4.15 (2H, m), 7.73 (1H, d, J = 8.0 Hz), 7.93 (1H, dd, J = 8.0, 1.7 Hz), 8.25 (1H, d, J = 1.7 Hz).

Compound 5a was synthesized from 3 and aniline 38, which was obtained via 36 and 37 from 1-(diphenylmethyl)-3-hydroxyazetidine as the starting material.

1-Benzhydryl-3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidine (36)

Following the addition of NaH (60%) (1.26 g, 31 mmol) to a solution of 1-(diphenylmethyl)-3-hydroxyazetidine (5.00 g, 20.9 mmol) in N,N-dimethylformamide (DMF) (50 mL) under ice-cooling, the reaction mixture was stirred at the same temperature for 45 min. After the addition of 2-(2-bromoethoxy)tetrahydropyran (4.46 mL, 31.4 mmol), the mixture was stirred at 70 °C for 10 h. Water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 36 (5.60 g, 73% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.45–1.86 (6H, m), 2.90–2.98 (2H, m), 3.45–3.60 (6H, m), 3.78–3.89 (2H, m), 4.15–4.24 (1H, m), 4.36 (1H, s), 4.57–4.62 (1H, m), 7.15–7.42 (10H, m).

1-(4-Nitrophenyl)-3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidine (37)

A solution of 36 (19 g, 52 mmol) in MeOH (200 mL) was hydrogenated at 0.4 MPa in the presence of 20% Pd(OH)₂-C (950 mg) at room temperature for 25 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give an intermediate (19.5 g).

Following the addition of i-Pr₂NEt (13.3 mL, 78.2 mmol) and p-fluoronitrobenzene (8.75 g, 62.0 mmol) to a solution of the intermediate (19.5 g) in DMF (200 mL), the reaction mixture was stirred at 80 °C for 20 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 37 (15.0 g, 90% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.48–1.65 (4H, m), 1.69–1.88 (2H, m), 3.48–3.56 (1H, m), 3.59–3.70 (3H, m), 3.83–3.98 (4H, m), 4.20–4.28 (2H, m), 4.53–4.59 (1H, m), 4.60–4.65 (1H, m), 6.28–6.34 (2H, m), 8.07–8.13 (2H, m).

(2-Methoxy-5-nitropyridin-4-yl)-(4-{3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidin-1-yl}phenyl)-amine (38)

A solution of 37 (15.0 g, 46.5 mmol) in MeOH (150 mL) was hydrogenated at 0.4 MPa in the presence of 10% Pd-C (0.75 g) at room temperature for 1.5 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 38 (13.1 g) as a crude product. The crude product was used in the next reaction without further purification.

Ethyl 4-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-{3-[2-(tetrahy-dropyran-2-yloxy)ethoxy]azetidin-1-yl}phenylamino)-benzoate (5a)

Following the addition of 3 (470 mg, 1.22 mmol), Pd(OAc)₂ (55 mg, 0.24 mmol), 2,2′-bis(diphenylphosphino)-1,1′-binaphthalene (BINAP) (304 mg, 0.488 mmol), and Cs₂CO₃ (795 mg, 2.44 mmol) to a solution of 38 (357 mg, 1.22 mmol) in 1,4-dioxane (10 mL), the reaction mixture was stirred at 80 °C for 1 h under a N₂ atmosphere. After cooling, AcOEt was added, and the mixture was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 5a (158 mg) as a crude product.

tert-Butyl 4-{4-[5-Methoxycarbonyl-2-(2-methyl[1,3]dioxolan-2-yl)phenylamino]phenyl}piperidine-1-carboxylate (5b)

Compound 5b was prepared from 4 and tert-butyl 4-(4-aminophenyl)piperidin-1-carboxylate²⁵⁾ according to the procedure for the synthesis of 5a. Yield was 93%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.55–1.67 (2H, m), 1.70 (3H, s), 1.80–1.87 (2H, m), 2.56–2.65 (1H, m), 2.74–2.85 (2H, m), 3.84–3.88 (5H, m), 4.05–4.15 (2H, m), 4.17–4.31 (2H, m), 7.03–7.08 (2H, m), 7.10–7.15 (2H, m), 7.41–7.44 (1H, s), 7.48 (1H, dd, J = 8.1, 1.4 Hz), 7.51 (1H, d, J = 8.1 Hz), 7.95 (1H, d, J = 1.4 Hz).

Compound 5d was synthesized from 4 and aniline 40, which was obtained via 39 from (E)-4-nitrocinnamyl alcohol²⁶⁾ as the starting material.

(E)-4-[3-(4-Nitrophenyl)allyl]morpholine (39)

Following the addition of SOCl₂ (0.92 mL, 13 mmol) to a solution of (E)-4-nitrocinnamyl alcohol²⁶⁾ (1.50 g, 8.37 mmol) and pyridine (5.1 mL, 63 mmol) in CH₂Cl₂ (15 mL) under ice cooling, the reaction mixture was stirred at room temperature for 30 min. The mixture was washed with water, 1.0 M aqueous HCl solution, and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. Morpholine (10 mL, 0.12 mol) was added to the residue, and the mixture was stirred at room temperature for 30 min. Et₂O was added, and the mixture was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 39 (870 mg, 42% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.48–2.55 (4H, m), 3.20 (2H, dd, J = 6.6, 1.3 Hz), 3.71–3.78 (4H, m), 6.45 (1H, dt, J = 15.8, 6.6 Hz), 6.58–6.65 (1H, m), 7.46–7.52 (2H, m), 8.14–8.20 (2H, m).

(E)-4-(3-Morpholin-4-ylpropenyl)phenylamine (40)

Following the addition of SnCl₂·2H₂O (3.50 g, 15.5 mmol) to a solution of 39 (880 mg, 3.10 mmol) in EtOH (40 mL), the reaction mixture was stirred at 80 °C for 1 h under a N₂ atmosphere. After cooling, AcOEt was added and neutralized with saturated aqueous NaHCO₃ solution. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 40 (467 mg, 69% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.44–2.53 (4H, m), 3.11 (2H, dd, J = 6.8, 1.2 Hz), 3.60–3.76 (6H, m), 6.04 (1H, dt, J = 15.9, 6.8 Hz), 6.37–6.45 (1H, m), 6.59–6.65 (2H, m), 7.15–7.21 (2H, m).

Methyl (E)-4-(2-Methyl-[1,3]dioxolan-2-yl)-3-[4-(3-morpholin-4-ylpropenyl)phenylamino]benzoate (5d)

Compound 5d was prepared from 4 and 40 according to the procedure for the synthesis of 5a. Yield was 40%. ¹H-NMR (CDCl₃) δ: 1.68 (3H, s), 2.45–2.55 (4H, m), 3.15 (2H, d, J = 6.8 Hz), 3.72–3.77 (4H, m), 3.84–3.89 (3H, m), 4.07–4.12 (2H, m), 6.13 (1H, dt, J = 15.8, 6.8 Hz), 6.52 (1H, d, J = 15.8 Hz), 7.01–7.07 (2H, m), 7.28–7.34 (2H, m), 7.46–7.55 (3H, m), 7.98 (1H, s).

Methyl 4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydropyran-2-yloxy)ethoxy]phenylamino}benzoate (5e)

Compound 5e was prepared from 4 and 4-{[2-(tetrahydropyran-2-yloxy)ethyl]oxy}aniline²⁷⁾ according to the procedure for the synthesis of 5a. Yield was quant. ¹H-NMR (CDCl₃) δ: 1.50–1.69 (4H, m), 1.70–1.90 (2H, m), 1.72 (3H, s), 3.50–3.59 (1H, m), 3.78–3.95 (4H, m), 3.84 (3H, s), 4.02–4.20 (5H, m), 4.70–4.75 (1H, m), 6.88–6.95 (2H, m), 7.02–7.09 (2H, m), 7.27 (1H, s), 7.41 (1H, dd, J = 8.0, 1.4 Hz), 7.48 (1H, d, J = 8.0 Hz), 7.71 (1H, d, J = 1.4 Hz).

Compound 5f was synthesized from 4 and aniline 42, which was obtained via 41 from 4-nitrophenol as the starting material.

2-{2-[2-(4-Nitrophenoxy)ethoxy]ethoxy}tetrahydropyran (41)

Following the addition of K₂CO₃ (7.24 g, 52.4 mmol) to a solution of 2-[2-(2-chloroethoxy)ethoxy]tetrahydropyran²⁸⁾ (8.0 g, 38 mmol) and 4-nitrophenol (4.85 g, 34.9 mmol) in DMF (80 mL), the reaction mixture was stirred at 80 °C for 24 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 41 (10.1 g, 93% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.46–1.65 (4H, m), 1.68–1.90 (2H, m), 3.46–3.56 (1H, m), 3.59–3.68 (1H, m), 3.72–3.80 (2H, m), 3.83–3.96 (4H, m), 4.20–4.29 (2H, m), 4.60–4.68 (1H, m), 6.94–7.03 (2H, m), 8.15–8.24 (2H, m).

4-{2-[2-(Tetrahydropyran-2-yloxy)ethoxy]ethoxy}phenylamine (42)

A solution of 41 (10.1 g, 32.3 mmol) in MeOH (200 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd-C (2.01 g) at room temperature for 2 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 42 (9.09 g, quant.). ¹H-NMR (CDCl₃) δ: 1.45–1.65 (4H, m), 1.67–1.91 (2H, m), 3.36–3.46 (2H, br), 3.47–3.56 (1H, m), 3.59–3.68 (1H, m), 3.70–3.78 (2H, m), 3.80–3.93 (4H, m), 4.01–4.09 (2H, m), 4.60–4.68 (1H, m), 6.58–6.66 (2H, m), 6.72–6.80 (2H, m).

Methyl 4-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-{2-[2-(tetrahydropyran-2-yloxy)ethoxy]ethoxy}phenylamino)benzoate (5f)

Compound 5f was prepared from 4 and 42 according to the procedure for the synthesis of 5a. Yield was 87%. ¹H-NMR (CDCl₃) δ: 1.47–1.63 (4H, m), 1.69–1.78 (4H, m), 1.79–1.90 (1H, m), 3.46–3.56 (1H, m), 3.61–3.70 (1H, m), 3.74–3.80 (2H, m), 3.82–3.95 (9H, m), 4.07–4.17 (4H, m), 4.62–4.68 (1H, m), 6.86–6.94 (2H, m), 7.01–7.08 (2H, m), 7.27 (1H, s), 7.41 (1H, dd, J = 8.0, 1.4 Hz), 7.48 (1H, d, J = 8.0 Hz), 7.71 (1H, d, J = 1.4 Hz).

Compound 5g was synthesized from 4 and aniline 44, which was obtained via 43 from 2-(tetrahydrofuran-3-yloxy)ethanol²⁹⁾ as the starting material.

3-[2-(4-Nitrophenoxy)ethoxy]tetrahydrofuran (43)

Following the addition of triethylamine (Et₃N) (2.37 mL, 17.0 mmol) and methanesulfonyl chloride (MsCl) (1.14 mL, 14.8 mmol) to a solution of 2-(tetrahydrofuran-3-yloxy)ethanol²⁹⁾ (1.5 g, 11 mmol) in CH₂Cl₂ (30 mL) under ice cooling, the reaction mixture was stirred at room temperature for 30 min. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give an intermediate (2.38 g) as an oil.

Following the addition of 4-nitrophenol (1.89 g, 13.6 mmol) and K₂CO₃ (3.12 g, 22.6 mmol) to a solution of the intermediate (2.38 g) in DMF (50 mL), the reaction mixture was stirred at 80 °C for 15 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 43 (2.7 g, 95% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.99–2.07 (2H, m), 3.76–3.96 (6H, m), 4.18–4.26 (3H, m), 6.93–7.04 (2H, m), 8.15–8.25 (2H, m).

4-[2-(Tetrahydrofuran-3-yloxy)ethoxy]phenylamine (44)

Compound 44 was prepared from 43 according to the procedure for the synthesis of 42. Yield was 95%. ¹H-NMR (CDCl₃) δ: 1.95–2.07 (2H, m), 3.38–3.49 (2H, br), 3.69–3.95 (6H, m), 4.03 (2H, t, J = 4.8 Hz), 4.19–4.25 (1H, m), 6.60–6.66 (2H, m), 6.70–6.79 (2H, m).

Methyl 4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydrofuran-3-yloxy)ethoxy]phenylamino}benzoate (5g)

Compound 5g was prepared from 4 and 44 according to the procedure for the synthesis of 5a. Yield was 86%. ¹H-NMR (CDCl₃) δ: 1.72 (3H, s), 1.98–2.09 (2H, m), 3.75–3.96 (11H, m), 4.07–4.15 (4H, m), 4.22–4.28 (1H, m), 6.86–6.92 (2H, m), 7.02–7.08 (2H, m), 7.28 (1H, s), 7.42 (1H, dd, J = 8.0, 1.7 Hz), 7.49 (1H, d, J = 8.0 Hz), 7.72 (1H, d, J = 1.7 Hz).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-{3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidin-1-yl}phenylamino)benzamide (6a)

Following the addition of MeOH (1 mL) and 1.0 M aqueous LiOH solution (0.86 mL, 0.86 mmol) to a solution of 5a (158 mg, crude) in tetrahydrofuran (THF) (3 mL), the reaction mixture was stirred at room temperature for 1 h and at 40 °C for 4 h. Water and toluene were added, and the mixture was neutralized with 2.0 M aqueous HCl solution. The mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give carboxylic acid (70 mg) as an intermediate.

Following the addition of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl) (54 mg, 0.28 mmol) and 1-hydroxybenzotriazole (HOBt) (0.38 mg, 0.28 mmol) to a solution of carboxylic acid (70 mg) in DMF (1 mL), the reaction mixture was stirred at room temperature for 10 min. After the addition of 14.8 M aqueous ammonia solution (0.05 mL, 0.7 mmol), the mixture was stirred at the same temperature for 3 h. After the addition of EDC·HCl (54 mg, 0.28 mmol), HOBt (0.38 mg, 0.28 mmol), and 14.8 M aqueous ammonia (0.05 mL, 0.7 mmol), the mixture was stirred for 16 h. AcOEt was added, and the mixture was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 6a (52 mg, 8.6% yield for 3 steps) as a solid. ¹H-NMR (CDCl₃) δ: 1.47–1.92 (9H, m), 3.46–3.66 (4H, m), 3.69–3.78 (2H, m), 3.84–3.94 (4H, m), 4.06–4.16 (4H, m), 4.46–4.54 (1H, m), 4.61–4.66 (1H, m), 5.27–5.58 (1H, br), 5.70–6.02 (1H, br), 6.44–6.50 (2H, m), 6.98–7.06 (2H, m), 7.12 (1H, dd, J = 8.0, 1.7 Hz), 7.22 (1H, s), 7.36 (1H, d, J = 1.7 Hz), 7.46 (1H, d, J = 8.0 Hz).

tert-Butyl 4-{4-[5-Carbamoyl-2-(2-methyl[1,3]dioxolan-2-yl)phenylamino]phenyl}piperidine-1-carboxylate (6b)

Compound 6b was prepared from 5b according to the procedure for the synthesis of 6a. Yield was 77%. ¹H-NMR (CDCl₃) δ: 1.49 (9H, s), 1.55–1.67 (2H, m), 1.70 (3H, s), 1.79–1.86 (2H, m), 2.56–2.65 (1H, m), 2.74–2.86 (2H, m), 3.82–3.91 (2H, m), 4.05–4.15 (2H, m), 4.17–4.31 (2H, m), 5.38–5.68 (1H, br), 5.80–6.09 (1H, br), 7.04–7.09 (2H, m), 7.10–7.15 (2H, m), 7.19 (1H, dd, J = 8.1, 1.7 Hz), 7.42–7.45 (1H, s), 7.52 (1H, d, J = 8.1 Hz), 7.72 (1H, d, J = 1.7 Hz).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-[4-(3-morpholin-4-ylpropenyl)phenylamino]benzamide (6d)

Compound 6d was prepared from 5d according to the procedure for the synthesis of 6a. Yield was 66%. ¹H-NMR (CDCl₃) δ: 1.69 (3H, s), 2.46–2.55 (4H, m), 3.14 (2H, d, J = 6.8 Hz), 3.72–3.77 (4H, m), 3.83–3.89 (2H, m), 4.07–4.13 (2H, m), 5.40–5.70 (1H, br), 5.75–6.10 (1H, br), 6.13 (1H, dt, J = 15.9, 6.8 Hz), 6.48 (1H, d, J = 15.9 Hz), 7.02–7.07 (2H, m), 7.24 (1H, dd, J = 8.0, 1.7 Hz), 7.27–7.33 (2H, m), 7.53 (1H, d, J = 8.0 Hz), 7.76 (1H, d, J = 1.7 Hz), 8.01 (1H, s).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydropyran-2-yloxy)ethoxy]phenylamino}benzamide (6e)

Compound 6e was prepared from 5e according to the procedure for the synthesis of 6a. Yield was 86%. ¹H-NMR (CDCl₃) δ: 1.48–1.70 (4H, m), 1.73 (3H, s), 1.74–1.90 (2H, m), 3.49–3.60 (1H, m), 3.78–3.97 (4H, m), 4.00–4.20 (5H, m), 4.69–4.75 (1H, m), 5.40–5.60 (1H, br), 5.80–6.00 (1H, br), 6.87–6.94 (2H, m), 7.01–7.09 (2H, m), 7.15 (1H, dd, J = 7.8, 1.2 Hz), 7.29 (1H, s), 7.46 (1H, d, J = 1.2 Hz), 7.48 (1H, d, J = 7.8 Hz).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-(4-{2-[2-(tetrahydropyran-2-yloxy)ethoxy]ethoxy}phenylamino)benzamide (6f)

Compound 6f was prepared from 5f according to the procedure for the synthesis of 6a. Yield was quant. ¹H-NMR (CDCl₃) δ: 1.47–1.68 (4H, m), 1.69–1.89 (5H, m), 3.47–3.56 (1H, m), 3.61–3.70 (1H, m), 3.74–3.80 (2H, m), 3.84–3.95 (6H, m), 4.08–4.17 (4H, m), 4.62–4.68 (1H, m), 5.42–5.66 (1H, br), 5.84–6.06 (1H, br), 6.86–6.93 (2H, m), 7.01–7.09 (2H, m), 7.15 (1H, dd, J = 8.0, 1.7 Hz), 7.29 (1H, s), 7.46 (1H, d, J = 1.7 Hz), 7.48 (1H, d, J = 8.0 Hz).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydrofuran-3-yloxy)ethoxy]phenylamino}benzamide (6g)

Compound 6g was prepared from 5g according to the procedure for the synthesis of 6a. Yield was 50%. ¹H-NMR (CDCl₃) δ: 1.73 (3H, s), 1.98–2.09 (2H, m), 3.73–3.95 (8H, m), 4.05–4.16 (4H, m), 4.22–4.27 (1H, m), 5.50–6.20 (2H, br), 6.86–6.91 (2H, m), 7.03–7.08 (2H, m), 7.15 (1H, dd, J = 8.0, 1.7 Hz), 7.30 (1H, s), 7.48 (1H, d, J = 1.7 Hz), 7.48 (1H, d, J = 8.0 Hz).

4-Acetyl-3-{4-[3-(2-hydroxyethoxy)azetidin-1-yl]phenylamino}benzamide (7a)

Following the addition of 2.0 M aqueous HCl solution (1.0 mL, 2.0 mmol) to a solution of 6a (52 mg, 0.11 mmol) in THF (1.0 mL), the reaction mixture was stirred at room temperature for 3.5 h. After the addition of saturated aqueous NaHCO₃ solution, the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure and Et₂O was added. The insoluble material was collected by filtration to give 7a (25 mg, 64% yield) as a solid. mp 175–178 °C; ¹H-NMR (CDCl₃) δ: 1.96–2.07 (1H, br), 2.65 (3H, s), 3.57 (2H, t, J = 4.4 Hz), 3.72–3.82 (4H, m), 4.11–4.17 (2H, m), 4.45–4.52 (1H, m), 5.45–5.75 (1H, br), 5.75–6.10 (1H, br), 6.45–6.51 (2H, m), 6.97 (1H, dd, J = 8.3, 1.4 Hz), 7.05–7.12 (2H, m), 7.30 (1H, d, J = 1.4 Hz), 7.82 (1H, d, J = 8.3 Hz), 10.33 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.2 (s), 59.1(2C, s), 60.1 (s), 68.1 (s), 70.0, 112.3 (2C, s), 112.5 (s), 113.8 (s), 118.9 (s), 125.4 (2C, s), 128.9 (s), 133.0 (s), 139.6 (s), 148.6 (s), 149.2 (s), 167.4 (s), 201.1 (s); IR (ATR) cm⁻¹: 1677, 1637. HR-MS (ESI-TOF) m/z: 392.1582 [M + Na]⁺ (Calcd for C₂₀H₂₃N₃NaO₄, 392.1586).

4-Acetyl-3-{4-[1-(2-hydroxyethyl)piperidin-4-yl]phenylamino}benzamide (7b)

Following the addition of 6.3 M HCl in i-PrOH (0.53 mL, 3.3 mmol) to a solution of 6b (400 mg, 0.831 mmol) in HCO₂H under ice cooling, the reaction mixture was stirred at the same temperature for 0.5 h. After the addition of i-Pr₂O (50 mL), the supernatant was decanted. The residue was concentrated under reduced pressure and dissolved in MeOH (5 mL). After the addition of a glycolaldehyde dimer (500 mg, 4.16 mmol) and NaBH₃CN (63 mg, 1.0 mmol) under ice cooling, the reaction mixture was stirred at the same temperature for 0.5 h. Saturated aqueous NaHCO₃ solution was added and MeOH was removed under reduced pressure. The mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography and t-BuOMe was added. The insoluble material was collected by filtration to give 7b (94 mg, 30% yield) as a solid. mp 160–164 °C; ¹H-NMR (dimethyl sulfoxide-d₆ (DMSO-d₆)) δ: 1.59–1.79 (4H, m), 2.03–2.13 (2H, m), 2.40–2.54 (3H, m), 2.64 (3H, s), 2.95–3.03 (2H, m), 3.49–3.55 (2H, m), 4.36–4.45 (1H, br), 7.17–7.22 (3H, m), 7.24–7.29 (2H, m), 7.46–7.52 (1H, br), 7.63 (1H, s), 7.97–8.07 (2H, m), 10.35 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.4 (s), 33.0 (2C, s), 41.1 (s), 54.1 (2C, s), 58.5 (s), 60.5 (s), 112.9 (s), 115.0 (s), 120.1 (s), 122.3 (2C, s), 127.7 (2C, s), 133.0 (s), 137.5 (s), 139.5 (s), 142.0 (s), 146.6 (s), 167.2 (s), 201.4 (s); IR (ATR) cm⁻¹: 3375, 3155, 1637; HR-MS (ESI-TOF) m/z: 382.2125 [M + H]⁺ (Calcd for C₂₂H₂₈N₃O₃, 382.2131).

4-Acetyl-3-[4-(1-methylpiperidin-4-yl)phenylamino]benzamide (7c)

Following the addition of 6.3 M HCl in i-PrOH (0.53 mL, 3.3 mmol) to a solution of 6b (400 mg, 0.831 mmol) in HCO₂H under ice cooling, the reaction mixture was stirred at the same temperature for 0.5 h. After the addition of i-Pr₂O (50 mL), the supernatant was decanted. The residue was concentrated under reduced pressure and dissolved in MeOH (5 mL). After the addition of formalin (37%) (0.31 mL, 4.1 mmol) and NaBH₃CN (63 mg, 1.0 mmol) under ice cooling, the reaction mixture was stirred at the same temperature for 10 min. Saturated aqueous NaHCO₃ solution was added, and MeOH was removed under reduced pressure. The mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography and t-BuOMe was added. The insoluble material was collected by filtration to give 7c (59 mg, 20% yield) as a solid. mp 174–176 °C; ¹H-NMR (DMSO-d₆) δ: 1.60–1.79 (4H, m), 1.92–2.01 (2H, m), 2.19 (3H, s), 2.40–2.49 (1H, m), 2.74–2.86 (2H, m), 2.64 (3H, s), 2.83–2.91 (2H, m), 7.17–7.22 (3H, m), 7.23–7.28 (2H, m), 7.45–7.52 (1H, br), 7.63 (1H, s), 7.97–8.06 (2H, m), 10.36 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 28.4 (s), 32.9 (s), 40.6 (s), 46.1 (s), 55.7 (s), 112.9 (s), 115.0 (s), 120.1 (s), 122.3 (2C, s), 127.7 (2C, s), 133.0 (s), 137.5 (s), 139.5 (s), 141.9 (s), 146.6 (s), 167.3 (s), 201.4 (s); IR (ATR) cm⁻¹: 3234, 3113, 1697, 1630. HR-MS (ESI-TOF) m/z: 352.2016 [M + H]⁺ (Calcd for C₂₁H₂₆N₃O₂, 352.2025).

Compound 7d was synthesized via 45d obtained from 6d.

4-Acetyl-3-[4-(3-morpholin-4-ylpropenyl)phenylamino]-benzamide (45d)

Compound 45d was prepared from 6d according to the procedure for the synthesis of 7a. Yield was 48%. ¹H-NMR (DMSO-d₆) δ: 2.34–2.43 (4H, m), 2.65 (3H, s), 3.08 (2H, d, J = 6.4 Hz), 3.54–3.62 (4H, m), 6.22 (1H, dt, J = 15.8, 6.4 Hz), 6.52 (1H, d, J = 15.8 Hz), 7.18–7.27 (3H, m), 7.42–7.53 (3H, m), 7.69 (1H, d, J = 1.2 Hz), 8.00 (1H, d, J = 8.3 Hz), 8.02–8.09 (1H, br), 10.35 (1H, s).

4-Acetyl-3-[4-(3-morpholin-4-ylpropyl)phenylamino]-benzamide (7d)

A suspension of 45d (69 mg, 0.18 mmol) in MeOH (3 mL) was hydrogenated at 0.2 MPa in the presence of 10% Pd-C (10 mg) at room temperature for 8 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 7d (32 mg, 46% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.77–1.88 (2H, m), 2.33–2.42 (2H, m), 2.42–2.50 (4H, m), 2.62–2.68 (5H, m), 3.72 (4H, t, J = 4.6 Hz), 5.40–5.70 (1H, br), 5.80–6.10 (1H, br), 7.02 (1H, dd, J = 8.3, 1.7 Hz), 7.12–7.21 (4H, m), 7.57 (1H, d, J = 1.7 Hz), 7.86 (1H, d, J = 8.3 Hz), 10.50 (1H, s); ¹³C-NMR (CDCl₃) δ: 27.7 (s), 28.4 (s), 32.2 (s), 53.2 (2C, s), 57.5 (s), 66.1 (2C, s), 112.9 (s), 115.0 (s), 120.1 (s), 122.4 (2C, s), 129.3 (2C, s), 133.0 (s), 137.2 (s), 137.8 (s), 139.5 (s), 146.7 (s), 167.2 (s), 201.4 (s); IR (ATR) cm⁻¹: 1668, 1641. HR-MS (ESI-TOF) m/z: 404.1950 [M + Na]⁺ (Calcd for C₂₂H₂₇N₃NaO₃, 404.1950).

4-Acetyl-3-[4-(2-hydroxyethoxy)phenylamino]benzamide (7e)

Compound 7e was prepared from 6e according to the procedure for the synthesis of 7a. Yield was 40%. mp 158–161 °C; ¹H-NMR (CDCl₃) δ: 2.00–2.10 (1H, br), 2.66 (3H, s), 3.94–4.04 (2H, m), 4.07–4.15 (2H, m), 5.44–5.66 (1H, br), 5.84–6.02 (1H, br), 6.91–6.98 (2H, m), 6.99 (1H, dd, J = 8.3, 1.2 Hz), 7.13–7.22 (2H, m), 7.38 (1H, d, J = 1.4 Hz), 7.85 (1H, d, J = 8.3 Hz), 10.39 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.3 (s), 59.5 (s), 69.6 (s), 112.5 (s), 114.3 (s), 115.3 (2C, s), 119.3 (s), 125.4 (2C, s), 132.0 (s), 133.0 (s), 139.6 (s), 148.0 (s), 155.8 (s), 167.3 (s), 201.2 (s); IR (ATR) cm⁻¹: 3500–3100, 1592, 1509. HR-MS (ESI-TOF) m/z: 337.1153 [M + Na]⁺ (Calcd for C₁₇H₁₈N₂NaO₄, 337.1164).

4-Acetyl-3-{4-[2-(2-hydroxyethoxy)ethoxy]phenylamino}-benzamide (7f)

Compound 7f was prepared from 6f according to the procedure for the synthesis of 7a. Yield was 66%. mp 121–123 °C; ¹H-NMR (CDCl₃) δ: 2.06–2.30 (1H, br), 2.66 (3H, s), 3.64–3.71 (2H, m), 3.72–3.78 (2H, m), 3.85–3.94 (2H, m), 4.16–4.24 (2H, m), 5.60–5.80 (1H, br), 6.04–6.28 (1H, br), 6.92–7.00 (2H, m), 7.03 (1H, dd, J = 8.3, 1.4 Hz), 7.12–7.21 (2H, m), 7.33 (1H, d, J = 1.4 Hz), 7.84 (1H, d, J = 8.3 Hz), 10.37 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.3 (s), 60.2 (s), 67.3 (s), 68.8 (s), 72.4 (s), 112.5 (s), 114.4 (s), 115.3 (2C, s), 119.4 (s), 125.3 (2C, s), 132.2 (s), 133.0 (s), 139.6 (s), 147.9 (s), 155.6 (s), 167.3 (s), 201.2 (s); IR (ATR) cm⁻¹: 3500–3100, 1563, 1509. HR-MS (ESI-TOF) m/z: 381.1421 [M + Na]⁺ (Calcd for C₁₉H₂₂N₂NaO₅, 381.1426).

4-Acetyl-3-{4-[2-(tetrahydrofuran-3-yloxy)ethoxy]phenylamino}benzamide (7g)

Compound 7g was prepared from 6g according to the procedure for the synthesis of 7a. Yield was 87%. mp 104–106 °C; ¹H-NMR (CDCl₃) δ: 1.98–2.08 (2H, m), 2.66 (3H, s), 3.75–3.95 (6H, m), 4.13 (2H, t, J = 4.9 Hz), 4.21–4.27 (1H, m), 5.50–6.20 (2H, br), 6.89–6.96 (2H, m), 7.00 (1H, dd, J = 8.0, 1.7 Hz), 7.12–7.18 (2H, m), 7.36 (1H, d, J = 1.7 Hz), 7.84 (1H, d, J = 8.0 Hz), 10.38 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.3 (s), 32.0 (s), 66.1 (s), 66.9 (s), 67.3 (s), 72.0 (s), 79.2 (s), 112.5 (s), 114.4 (s), 115.4 (2C, s), 119.4 (s), 125.3 (2C, s), 132.2 (s), 133.0 (s), 139.6 (s), 147.9 (s), 155.6 (s), 167.3 (s), 201.2 (s); IR (ATR) cm⁻¹: 1658, 1626; HR-MS (ESI-TOF) m/z: 407.1576 [M + Na]⁺ (Calcd for C₂₁H₂₄N₂NaO₅, 407.1583).

2-Acetyl-5-cyanophenyl Trifluoromethanesulfonate (46)

Following the addition of pyridine (1.90 mL, 21.4 mmol) and trifluoromethanesulfonic anhydride (1.93 mL, 11.7 mmol) to a solution of 4-acetyl-3-hydroxybenzonitrile¹⁹⁾ (1.72 g, 10.7 mmol) in CH₂Cl₂ (20 mL) under ice cooling, the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was washed with 1.0 M aqueous HCl solution and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 46 (2.93 g, 93% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.67 (3H, s), 7.64 (1H, d, J = 1.2 Hz), 7.79 (1H, dd, J = 8.0, 1.2 Hz), 7.89 (1H, d, J = 8.0 Hz).

5-Cyano-2-(2-methyl-[1,3]dioxolan-2-yl)phenyl Trifluoromethanesulfonate (8)

Following the addition of ethylenedioxybis(trimethylsilane) (3.67 mL, 15.0 mmol) and trimethylsilyl trifluoromethanesulfonate (0.36 mL, 2.0 mmol) to a solution of 46 (2.93 g, 9.99 mmol) in CH₂Cl₂ (30 mL) under ice cooling, the reaction mixture was stirred at room temperature for 19 h The reaction mixture was washed with saturated aqueous NaHCO₃ solution and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 8 (2.58 g, 77% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.72 (3H, s), 3.77–3.88 (2H, m), 4.06–4.17 (2H, m), 7.53 (1H, d, J = 1.4 Hz), 7.66 (1H, dd, J = 8.0, 1.4 Hz), 7.81 (1H, d, J = 8.0 Hz).

Compound 9h was synthesized from 8 and aniline 48, which was obtained via 47 from 2-[(tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ as the starting material.

4-[2-(4-Nitrophenoxy)ethoxy]tetrahydropyran (47)

Compound 47 was prepared from 2-[(tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ according to the procedure for the synthesis of 43. Yield was 70%. ¹H-NMR (CDCl₃) δ: 1.57–1.69 (2H, m), 1.88–1.98 (2H, m), 3.40–3.50 (2H, m), 3.54–3.65 (1H, m), 3.83–3.90 (2H, m), 3.91–4.00 (2H, m), 4.20–4.25 (2H, m), 6.95–7.02 (2H, m), 8.16–8.24 (2H, m).

4-[2-(Tetrahydropyran-4-yloxy)ethoxy]phenylamine (48)

Compound 48 was prepared from 47 according to the procedure for the synthesis of 42. Yield was 95%. ¹H-NMR (CDCl₃) δ: 1.55–1.72 (2H, m), 1.85–1.98 (2H, m), 3.37–3.50 (4H, m), 3.53–3.64 (1H, m), 3.75–3.83 (2H, m), 3.90–3.99 (2H, m), 4.00–4.08 (2H, m), 6.60–6.67 (2H, m), 6.72–6.80 (2H, m).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenylamino}benzonitrile (9h)

Following the addition of 8 (3.39 g, 10.1 mmol), Pd(OAc)₂ (451 mg, 2.01 mmol), BINAP (2.50 g, 4.02 mmol), and Cs₂CO₃ (9.83 g, 30.2 mmol) to a solution of 48 (2.17 g, 9.14 mmol) 1,4-dioxane (100 mL), the reaction mixture was stirred at 50 °C for 18 h under an N₂ atmosphere. After cooling, AcOEt was added and the insoluble material was filtered. The filtrate was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 9h (3.30 g, 85% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.73 (3H, s), 1.92–1.99 (2H, m), 3.42–3.50 (2H, m), 3.58–3.66 (1H, m), 3.82–3.89 (4H, m), 3.97 (2H, dt, J = 12.0, 4.4 Hz), 4.08–4.15 (4H, m), 6.90–6.95 (2H, m), 7.00 (1H, dd, J = 7.8, 1.5 Hz), 7.01–7.06 (2H, m), 7.19 (1H, d, J = 1.5 Hz), 7.36 (1H, s), 7.48 (1H, d, J = 7.8 Hz).

Compound 9i was synthesized from 8 and aniline 52, which was obtained via 49, 50, and 51 from 4-nitrophenol as the starting material.

2-[2-(4-Nitrophenoxy)ethoxy]ethanol (49)

Compound 49 was prepared from 4-nitrophenol according to the procedure for the synthesis of 41. Yield was 60%. ¹H-NMR (CDCl₃) δ: 1.81–2.15 (1H, br), 3.67–3.71 (2H, m), 3.75–3.80 (2H, m), 3.89–3.94 (2H, m), 4.12–4.27 (2H, m), 6.96–7.01 (2H, m), 8.11–8.22 (2H, m).

Diethyl 2-{2-[2-(4-Nitrophenoxy)ethoxy]ethyl}malonate (50)

Following the addition of Et₃N (8.41 mL, 60.3 mmol) and MsCl (4.00 mL, 51.7 mmol) to a solution of 49 (9.79 g, 43.1 mmol) in CH₂Cl₂ (100 mL) under ice cooling, the reaction mixture was stirred at the same temperature for 30 min. Water was added, the mixture was extracted with CHCl₃, and the organic layer was washed with saturated aqueous NaHCO₃ solution and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure.

Following the addition of NaH (60%) (2.59 g, 65 mmol) to a solution of diethyl malonate (9.77 mL, 64.7 mmol) in DMF (120 mL) under ice cooling, the reaction mixture was stirred at the same temperature for 30 min. After the addition of the residue obtained in DMF (30 mL), the mixture was stirred at 80 °C for 18 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 50 (7.06 g, 44% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.25 (6H, t, J = 7.1 Hz), 2.17–2.26 (2H, m), 3.54 (1H, t, J = 7.5 Hz), 3.58–3.63 (2H, m), 3.78–3.84 (2H, m), 4.10–4.24 (6H, m), 6.95–7.00 (2H, m), 8.17–8.21 (2H, m).

Ethyl 4-[2-(4-Nitrophenoxy)ethoxy]butyrate (51)

Following the addition of water (0.69 mL, 38 mmol) and NaCl (2.23 g, 38.2 mmol) to a solution of 50 (7.06 g, 19.1 mmol) in DMSO (35 mL), the reaction mixture was heated to reflux for 5 h. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 51 (4.40 g, 77% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz), 1.89–1.97 (2H, m), 2.40 (2H, t, J = 7.3 Hz), 3.55–3.61 (2H, m), 3.79–3.84 (2H, m), 4.12 (2H, q, J = 7.1 Hz), 4.17–4.23 (2H, m), 6.95–7.02 (2H, m), 8.17–8.23 (2H, m).

Ethyl 4-[2-(4-Aminophenoxy)ethoxy]butyrate (52)

Compound 52 was prepared from 51 according to the procedure for the synthesis of 42. Yield was 99%. ¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz), 1.89–1.97 (2H, m), 2.41 (2H, t, J = 7.3 Hz), 3.35–3.48 (2H, br), 3.54–3.59 (2H, m), 3.71–3.77 (2H, m), 4.00–4.06 (2H, m), 4.12 (2H, q, J = 7.1 Hz), 6.60–6.67 (2H, m), 6.72–6.78 (2H, m).

Ethyl 4-(2-{4-[5-Cyano-2-(2-methyl-[1,3]dioxolan-2-yl)-phenylamino]phenoxy}ethoxy)butyrate (9i)

Compound 9i was prepared from 8 and 52 according to the procedure for the synthesis of 9h. Yield was 39%. ¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz), 1.72 (3H, s), 1.90–2.00 (2H, m), 2.42 (2H, t, J = 7.3 Hz), 3.59 (2H, t, J = 6.1 Hz), 3.77–3.82 (2H, m), 3.82–3.88 (2H, m), 4.08–4.17 (6H, m), 6.90–6.95 (2H, m), 6.99 (1H, dd, J = 7.8, 1.4 Hz), 7.01–7.07 (2H, m), 7.18 (1H, d, J = 1.4 Hz), 7.35 (1H, s), 7.48 (1H, d, J = 7.8 Hz).

4-(2-Methyl-[1,3]dioxolan-2-yl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]-phenylamino}benzamide (10h)

Following the addition of K₂CO₃ (1.07 g, 7.77 mmol) and 9.79 M aqueous H₂O₂ solution (1.59 mL, 15.5 mmol) to a solution of 9h (3.30 g, 7.77 mmol) in DMSO (10 mL), the reaction mixture was stirred at room temperature for 1.5 h. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 10h (3.14 g, 91% yield) as an oil. ¹H-NMR (DMSO-d₆) δ: 1.55–1.70 (2H, m), 1.73 (3H, s), 1.90–1.98 (2H, m), 3.42–3.49 (2H, m), 3.57–3.66 (1H, m), 3.82–3.88 (4H, m), 3.92–4.00 (2H, m), 4.09–4.20 (4H, m), 5.40–5.60 (1H, br), 5.80–6.05 (1H, br), 6.89–6.91 (2H, m), 7.05–7.07 (2H, m), 7.14 (1H, dd, J = 8.1, 1.7 Hz), 7.31 (1H, s), 7.48–7.50 (2H, m).

Ethyl 4-(2-{4-[5-Carbamoyl-2-(2-methyl-[1,3]dioxolan-2-yl)phenylamino]phenoxy}ethoxy)butyrate (10i)

Compound 10i was prepared from 9i according to the procedure for the synthesis of 10h. Yield was 58%. ¹H-NMR (CDCl₃) δ: 1.25 (3H, t, J = 7.1 Hz), 1.73 (3H, s), 1.89–1.99 (2H, m), 2.41 (2H, t, J = 7.3 Hz), 3.58 (2H, t, J = 6.1 Hz), 3.74–3.81 (2H, m), 3.83–3.90 (2H, m), 4.07–4.16 (6H, m), 5.40–5.60 (1H, br), 5.80–6.00 (1H, br), 6.86–6.93 (2H, m), 7.01–7.08 (2H, m), 7.15 (1H, dd, J = 8.0, 1.7 Hz), 7.29 (1H, s), 7.46 (1H, d, J = 1.7 Hz), 7.48 (1H, d, J = 8.0 Hz).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenylamino}benzamide (7h)

Compound 7h was prepared from 10h according to the procedure for the synthesis of 7a. Yield was 63%. mp 126–130 °C; ¹H-NMR (DMSO-d₆) δ: 1.60–1.70 (2H, m), 1.90–1.98 (2H, m), 2.66 (3H, s), 3.42–3.52 (2H, m), 3.57–3.66 (1H, m), 3.82–3.88 (2H, m), 3.92–4.02 (2H, m), 4.10–4.16 (2H, m), 5.40–5.60 (1H, br), 5.80–6.00 (1H, br), 6.93–6.95 (2H, m), 7.00 (1H, dd, J = 8.3, 1.7 Hz), 7.15–7.17 (2H, m), 7.36 (1H, d, J = 1.7 Hz), 7.84 (1H, d, J = 8.3 Hz), 10.39 (1H, s); ¹³C-NMR (CDCl₃) δ: 28.2 (s), 59.1 (2C, s), 60.1 (s), 68.1 (s), 70.0, 112.3 (2C, s), 112.5 (s), 113.8 (s), 118.9 (s), 125.4 (2C, s), 128.9 (s), 133.0 (s), 139.6 (s), 148.6 (s), 149.2 (s), 167.4 (s), 201.1 (s); IR (ATR) cm⁻¹: 3374, 3166, 1654, 1635. HR-MS (ESI-TOF) m/z: 421.1726 [M + Na]⁺ (Calcd for C₂₂H₂₆N₂NaO₅, 421.1739).

Compound 7i was synthesized via 53i obtained from 10i.

Ethyl 4-{2-[4-(2-Acetyl-5-carbamoylphenylamino)phenoxy]ethoxy}butyrate (53i)

Compound 53i was prepared from 10i according to the procedure for the synthesis of 7a. Yield was 81%. ¹H-NMR (CDCl₃) δ: 1.24 (3H, t, J = 7.1 Hz), 1.90–1.99 (2H, m), 2.41 (2H, t, J = 7.3 Hz), 2.66 (3H, s), 3.58 (2H, t, J = 6.1 Hz), 3.76–3.84 (2H, m), 4.08–4.17 (4H, m), 5.45–5.65 (1H, br), 5.85–6.05 (1H, br), 6.90–6.98 (2H, m), 7.01 (1H, dd, J = 8.6, 1.7 Hz), 7.12–7.20 (2H, m), 7.35 (1H, d, J = 1.7 Hz), 7.84 (1H, d, J = 8.6 Hz), 10.38 (1H, s).

4-{2-[4-(2-Acetyl-5-carbamoylphenylamino)phenoxy]-ethoxy}butyric Acid (7i)

Following the addition of 1.0 M aqueous LiOH solution (3.3 mL, 3.3 mmol) to a solution of 53i (470 mg, 1.10 mmol) in THF (10 mL) and MeOH (3.3 mL), the reaction mixture was stirred at room temperature for 1 h. The mixture was washed with Et₂O and the water layer was acidified with 10% aqueous citric acid solution. The mixture was extracted with AcOEt, the organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was recrystallized with EtOH (15 mL) to give 7i (222 mg, 50% yield) as a solid. mp 126–130 °C; ¹H-NMR (CDCl₃) δ: 1.87–2.00 (2H, m), 2.46 (2H, t, J = 7.1 Hz), 2.65 (3H, s), 3.60 (2H, t, J = 6.1 Hz), 3.76–3.87 (2H, m), 4.10–4.20 (2H, m), 6.06–6.22 (1H, br), 6.40–6.56 (1H, br), 6.92–7.02 (3H, m), 7.10–7.18 (2H, m), 7.31–7.37 (1H, m), 7.82 (1H, d, J = 8.0 Hz), 10.35 (1H, s); ¹³C-NMR (CDCl₃) δ: 24.6 (s), 28.3 (s), 30.2 (s), 67.3 (s), 68.5 (s), 69.4 (s), 112.5 (s), 114.4 (s), 115.4 (2C, s), 119.4 (s), 125.3 (2C, s), 132.2 (s), 133.0 (s), 139.6 (s), 147.9 (s), 155.6 (s), 167.4 (s), 174.3 (s), 201.3 (s); IR (ATR) cm⁻¹: 1704, 1635, 1562; HR-MS (ESI-TOF) m/z: 423.1522 [M + Na]⁺ (Calcd for C₂₁H₂₄N₂NaO₆, 423.1532).

Compound 12a was synthesized from 11 and phenol 57, which was obtained via 54, 55, and 56 from 36 as the starting material.

1-Phenyl-3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidine (54)

A solution of 36 (17.7 g, 48.2 mmol) in MeOH (200 mL) was hydrogenated at 0.3 MPa in the presence of 20% Pd(OH)₂-C (0.90 g) at room temperature for 46 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give an intermediate.

Following the addition of PhI (7.0 mL, 63 mmol), CuI (918 mg, 4.82 mmol), L-proline (1.11 g, 9.64 mmol), K₂CO₃ (13.3 g, 96.2 mmol), and DMSO (24 mL) to the intermediate, the reaction mixture was stirred at 70 °C for 19 h under a N₂ atmosphere. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 54 (7.8 g, 55% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.40–1.90 (6H, m), 3.47–3.67 (4H, m), 3.69–3.78 (2H, m), 3.82–3.92 (2H, m), 4.08–4.16 (2H, m), 4.47–4.55 (1H, m), 4.62–4.68 (1H, m), 6.44–6.52 (2H, m), 6.70–6.78 (1H, m), 7.16–7.26 (2H, m).

4-Iodo-1-phenyl-3-[2-(tetrahydropyran-2-yloxy)ethoxy]-azetidine (55)

Following the addition of NaHCO₃ (3.5 g, 42 mmol) and water (40 mL) to a solution of 54 (7.8 g, 28 mmol) in CH₂Cl₂ (40 mL), iodine (7.46 g, 29.4 mmol) was added in portions over 1 h, and the mixture was stirred for 1.5 h. After the addition of 5% aqueous Na₂SO₃ solution (40 mL), the mixture was stirred for 30 min. The mixture was concentrated under reduced pressure and then extracted with AcOEt. The organic layer was washed with 5% aqueous Na₂SO₃ solution and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 55 (10.34 g, 92% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.40–1.90 (6H, m), 3.47–3.65 (4H, m), 3.68–3.75 (2H, m), 3.83–3.91 (2H, m), 4.07 (2H, t, J = 7.1 Hz), 4.44–4.52 (1H, m), 4.60–4.65 (1H, m), 6.20–6.25 (2H, m), 7.43–7.47 (2H, m).

1-[4-(4,4,5,5-Teramethyl-1,3,2-dioxaborolan-2-yl)-phenyl]-3-[2-(tetrahydropyran-2-yloxy)ethoxy]azetidine (56)

Following the addition of [1,1′-bis(diphenylphosphino)ferrocene]palladium (II) dichloride dichloromethane adduct (PdCl₂(dppf)₂・CH₂Cl₂) (101 mg, 0.124 mmol), 4,4,5,5–tetramethyl-1,3,2-dioxaborolane (1.35 mL, 9.30 mmol), and Et₃N (4.32 mL, 31.0 mmol) to a solution of 55 (2.50 g, 6.20 mmol) in 1,4-dioxane (10 mL), the reaction mixture was stirred at 80 °C for 40 min under a N₂ atmosphere. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 56 (1.83 g, 73% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.29–1.78 (18H, m), 3.46–3.54 (1H, m), 3.55–3.66 (3H, m), 3.73–3.81 (2H, m), 3.82–3.91 (2H, m), 4.08–4.17 (2H, m), 4.46–4.54 (1H, m), 4.59–4.65 (1H, m), 6.37–6.45 (2H, m).

4-{3-[2-(Tetrahydropyran-2-yloxy)ethoxy]azetidin-1-yl}-phenol (57)

Following the addition of 9.79 M aqueous H₂O₂ solution (1.52 mL, 14.9 mmol) and 5.0 M aqueous NaOH solution (3.0 mL, 15 mmol) to a solution of 56 (2.00 g, 4.96 mmol) in THF (25 mL), the mixture was stirred at room temperature for 9 h. After the addition of 6.0 M aqueous HCl solution (2.5 mL, 15 mmol), the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 57 (490 mg, 34% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.48–1.68 (4H, m), 1.70–1.90 (2H, m), 3.47–3.55 (1H, m), 3.57–3.69 (3H, m), 3.75–3.82 (2H, m), 3.84–3.93 (2H, m), 4.10–4.17 (2H, m), 4.48–4.58 (1H, m), 4.60–4.65 (1H, m), 6.36–6.42 (2H, m), 6.68–6.74 (2H, m).

4-Acetyl-3-(4-{3-[2-(tetrahydropyran-2-yloxy)ethoxy]-azetidin-1-yl}phenoxy)benzonitrile (12a)

Following the addition of K₂CO₃ (463 mg, 3.35 mmol) to a solution of 57 (490 mg, 1.67 mmol) and 11 (273 mg, 1.67 mmol) in DMF (10 mL), the reaction mixture was stirred at 100 °C for 11 h. After cooling, AcOEt was added, and the mixture was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 12a (236 mg, 32% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.47–1.66 (4H, m), 1.69–1.89 (2H, m), 2.72 (3H, s), 3.46–3.56 (1H, m), 3.59–3.68 (1H, m), 3.66 (2H, t, J = 6.8 Hz), 3.75–3.81 (2H, m), 3.85–3.93 (2H, m), 4.15 (2H, t, J = 6.8 Hz), 4.49–4.56 (1H, m), 4.61–4.66 (1H, m), 6.47–6.53 (2H, m), 6.90–6.96 (2H, m), 7.00 (1H, d, J = 1.2 Hz), 7.31 (1H, dd, J = 8.1, 1.2 Hz), 7.82 (1H, d, J = 8.1 Hz).

Compound 12g was synthesized from 11 and phenol 59, which was obtained via 58 from 2-(tetrahydrofuran-3-yloxy)ethanol²⁹⁾ as the starting material.

3-[2-(4-Benzyloxyphenoxy)ethoxy]tetrahydrofuran (58)

Following the addition of Et₃N (2.79 mL, 20.0 mmol) and MsCl (1.03 mL, 13.3 mmol) to a solution of 2-(tetrahydrofuran-3-yloxy)ethanol²⁹⁾ (1.76 g, 13.3 mmol) in CH₂Cl₂ (40 mL) under ice cooling, the reaction mixture was stirred at room temperature for 40 min. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give an intermediate (2.51 g) as an oil.

Following the addition of K₂CO₃ (7.24 g, 52.4 mmol) to a solution of the intermediate (2.51 g, 11.9 mmol) and 4-benzyloxyphenol (2.40 g, 12.0 mmol) in DMF (40 mL), the reaction mixture was stirred at 80 °C for 13 h. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 58 (2.40 g, 57% yield for 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 1.99–2.04 (2H, m), 3.73–3.94 (6H, m), 4.04–4.08 (2H, m), 4.20–4.25 (1H, m), 5.00 (2H, s), 6.82–6.85 (2H, m), 6.88–6.92 (2H, m), 7.28–7.44 (5H, m).

4-[2-(Tetrahydrofuran-3-yloxy)ethoxy]phenol (59)

A solution of 58 (2.4 g, 7.6 mmol) in MeOH (75 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd-C (480 mg) at room temperature for 1.5 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 59 (1.56 g, 92% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.99–2.04 (2H, m), 3.71–3.96 (6H, m), 4.03–4.05 (2H, m), 4.20–4.25 (1H, m), 6.72–6.75 (2H, m), 6.77–6.79 (2H, m).

4-Acetyl-3-{4-[2-(tetrahydrofuran-3-yloxy)ethoxy]phenoxy}benzonitrile (12g)

Compound 12g was prepared from 11 and 59 according to the procedure for the synthesis of 12a. Yield was 89%. ¹H-NMR (CDCl₃) δ: 2.02–2.07 (2H, m), 2.71 (3H, s), 3.77–3.96 (6H, m), 4.13–4.16 (2H, m), 4.23–4.27 (1H, m), 6.73–6.83 (4H, m), 7.01 (1H, d, J = 1.0 Hz), 7.34 (1H, dd, J = 7.9, 1.0 Hz), 7.83 (1H, d, J = 7.9 Hz).

Compound 12h was synthesized from 11 and phenol 62, which was obtained via 60 and 61 from 2-[(tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ as the starting material.

4-(2-Bromoethoxy)tetrahydropyran (60)

Following the addition of PPh₃ (49.3 g, 188 mmol) to a solution of 2-[(tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ (25.0 g, 171 mmol) and CBr₄ (68.1 g, 205 mmol) in CH₂Cl₂ (170 mL) under ice cooling, the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure. After the addition of i-Pr₂O to the residue, the insoluble material was removed by filtration and the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 60 (30.3 g, 85% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.55–1.77 (2H, m), 1.86–1.96 (2H, m), 3.41–3.50 (4H, m), 3.52–3.62 (1H, m), 3.79 (2H, t, J = 6.1 Hz), 3.91–4.00 (2H, m).

4-[2-(4-Benzyloxyphenoxy)ethoxy]tetrahydropyran (61)

Following the addition of 4-benzyloxyphenol (15.2 g, 76.0 mmol), K₂CO₃ (4.53 g, 32.8 mmol), and KI (1.33 g, 8.00 mmol) to a solution of 60 (16.7 g, 80.0 mmol) in DMF (80 mL), the reaction mixture was stirred at 80 °C for 14 h. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 61 (23.3 g, 95% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.77–1.92 (2H, m), 2.09–2.20 (2H, m), 2.46–2.58 (2H, m), 2.76–2.89 (2H, m), 3.38–3.48 (1H, m), 3.80–3.88 (2H, m), 4.18–4.25 (2H, m), 6.94–7.02 (2H, m), 8.16–8.24 (2H, m).

4-[2-(Tetrahydropyran-4-yloxy)ethoxy]phenol (62)

A solution of 61 (23.7 g, 72.2 mmol) in MeOH (200 mL) was hydrogenated at 0.4 MPa in the presence of 10% Pd-C (2.50 g) at room temperature for 2 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. After the addition of i-Pr₂O to the residue, the insoluble material was collected by filtration to give 62 (16.8 g, 98% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.95–2.07 (2H, m), 3.38–3.49 (2H, br), 3.69–3.95 (6H, m), 4.03 (2H, t, J = 4.8 Hz), 4.19–4.25 (1H, m), 6.60–6.66 (2H, m), 6.70–6.79 (2H, m).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzonitrile (12h)

Compound 12h was prepared from 11 and 62 according to the procedure for the synthesis of 12a. Yield was 80%. ¹H-NMR (CDCl₃) δ: 1.24 (3H, t, J = 7.1 Hz), 1.90–1.99 (2H, m), 2.41 (2H, t, J = 7.3 Hz), 2.66 (3H, s), 3.58 (2H, t, J = 6.1 Hz), 3.76–3.84 (2H, m), 4.08–4.17 (4H, m), 5.45–5.65 (1H, br), 5.85–6.05 (1H, br), 6.90–6.98 (2H, m), 7.01 (1H, dd, J = 8.6, 1.7 Hz), 7.12–7.20 (2H, m), 7.35 (1H, d, J = 1.7 Hz), 7.84 (1H, d, J = 8.6 Hz), 10.38 (1H, s).

Compound 12j was synthesized from 11 and phenol 65, which was obtained via 63 and 64 from tert-butyl 4-(2-hydroxyethoxy)piperidine-1-carboxylate³¹⁾ as the starting material.

4-[2-(tert-Butyldimethylsilanyloxy)ethoxy]piperidine (63)

Trifluoroacetic acid (TFA) was added to tert-butyl 4-(2-hydroxyethoxy)piperidine-1-carboxylate³¹⁾ (3.01 g, 12.3 mmol), and the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure.

Following the addition of Et₃N (14 mL, 98 mmol) and tert-butyldimethylchlorosilane (3.71 g, 24.6 mmol) to a solution of the residue in THF (14 mL), the reaction mixture was stirred at room temperature for 50 min. The solvent was removed under reduced pressure. AcOEt was added, the mixture was washed with saturated aqueous NaHCO₃ solution and saturated brine, and was then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 63 (3.46 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 0.07 (6H, s), 0.90 (9H, s), 1.46–1.56 (2H, m), 1.88–1.98 (2H, m), 2.20–2.45 (1H, br), 2.64–2.72 (2H, m), 3.12 (2H, dt, J = 13.2, 4.6 Hz), 3.41–3.48 (1H, m), 3.53 (2H, t, J = 5.6 Hz), 3.75 (2H, t, J = 5.6 Hz).

1-(4-Benzyloxyphenyl)-4-[2-(tert-butyldimethylsilanyloxy)ethoxy]piperidine (64)

Following the addition of 1-(benzyloxy)-4-iodobenzene (3.47 g, 11.2 mmol), Pd(OAc)₂ (126 mg, 0.561 mmol), (2-biphenyl)dicyclohexylphosphine (393 mg, 1.12 mmol), and tert-BuONa (1.61 g, 16.8 mmol) to a solution of 63 (3.46 g, 12.3 mmol) in 1,4-dioxane (35 mL), the reaction mixture was stirred at 80 °C for 10 h under a N₂ atmosphere. After cooling, AcOEt was added and the insoluble material was filtered. The filtrate was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 64 (560 mg, 25% yield) as an oil. ¹H-NMR (CDCl₃) δ: 0.07 (6H, s), 0.90 (9H, s), 1.68–1.80 (2H, m), 1.95–2.06 (2H, m), 2.76–2.85 (2H, m), 3.33–3.41 (2H, m), 3.43–3.51 (1H, m), 3.55 (2H, t, J = 5.6 Hz), 3.76 (2H, t, J = 5.6 Hz), 5.01 (2H, s), 6.87–6.91 (4H, m), 7.27–7.45 (5H, m).

4-{4-[2-(tert-Butyldimethylsilanyloxy)ethoxy]piperidin-1-yl}phenol (65)

A solution of 64 (550 mg, 1.25 mmol) in AcOEt (10 mL) was hydrogenated at 0.4 MPa in the presence of 10% Pd-C (55 mg) at room temperature for 1 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 65 (9.09 g) as a crude product.

4-Acetyl-3-{4-[4-(2-hydroxyethoxy)piperidin-1-yl]phenoxy}benzonitrile (12j)

Compound 12j was prepared from 11 and 65 according to the procedure for the synthesis of 12a. Yield was 46%. ¹H-NMR (CDCl₃) δ: 1.73–1.83 (2H, m), 1.97–2.10 (3H, m), 2.71 (3H, s), 2.94–3.03 (2H, m), 3.47–3.58 (3H, m), 3.60–3.65 (2H, m), 3.73–3.78 (2H, m), 6.94–7.01 (4H, m), 7.03 (1H, d, J = 1.5 Hz), 7.32 (1H, dd, J = 7.8, 1.5 Hz), 7.83 (1H, d, J = 7.8 Hz).

Compound 12k was synthesized from 11 and phenol 67, which was obtained via 66 from 2-[2-(2-chloroethoxy)ethoxy]propane³²⁾ as the starting material.

4-Benzyloxy-[2-(3-isopropoxyethoxy)ethoxy]benzene (66)

Following the addition of 2-[2-(2-chloroethoxy)ethoxy]propane³²⁾ (2.74 g, 16.4 mmol) and K₂CO₃ (4.53 g, 32.8 mmol) to a solution of 4-benzyloxyphenol (3.85 g, 19.2 mmol) in DMF (80 mL), the reaction mixture was stirred at 80 °C for 12 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 66 (1.95 g, 36% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.16 (6H, d, J = 6.1 Hz), 3.56–3.63 (3H, m), 3.66–3.71 (2H, m), 3.80–3.86 (2H, m), 4.05–4.10 (2H, m), 5.00 (2H, s), 6.81–6.92 (4H, m), 7.27–7.45 (5H, m).

4-[2-(2-Isopropoxyethoxy)ethoxy]phenol (67)

A solution of 66 (1.95 g, 5.90 mmol) in MeOH (40 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd-C (390 mg) at room temperature for 1.5 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 67 (1.36 g, 96% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 3.59–3.65 (3H, m), 3.67–3.72 (2H, m), 3.79–3.85 (2H, m), 4.00–4.06 (2H, m), 6.70–6.80 (4H, m).

4-Acetyl-3-{4-[2-(2-isopropoxyethoxy)ethoxy]phenoxy}-benzonitrile (12k)

Compound 12k was prepared from 11 and 67 according to the procedure for the synthesis of 12a. Yield was 84%. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 2.70 (3H, s), 3.58–3.66 (3H, m), 3.69–3.75 (2H, m), 3.86–3.93 (2H, m), 4.13–4.19 (2H, m), 6.98 (4H, m), 7.01 (1H, d, J = 1.2 Hz), 7.34 (1H, dd, J = 7.8, 1.2 Hz), 7.83 (1H, d, J = 7.8 Hz).

Compound 12m was synthesized from 11 and phenol 68, which was obtained from 4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)benzyloxy]tetrahydropyran³³⁾ as the starting material.

4-(Tetrahydropyran-4-yloxymethyl)phenol (68)

Following the addition of 9.79 M aqueous H₂O₂ solution (4.8 mL, 47 mmol) and 5.0 M aqueous NaOH solution (9.4 mL, 47 mmol) to a solution of 4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)benzyloxy]tetrahydropyran³³⁾ (5.00 g, 15.7 mmol) in THF (50 mL), the mixture was stirred at room temperature for 0.5 h. Following the addition of 6.0 M aqueous HCl solution, the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 68 (4.28 g). The crude product was used in the next reaction without further purification.

4-Acetyl-3-[4-(tetrahydropyran-4-yloxymethyl)phenoxy]-benzonitrile (12m)

Compound 12m was prepared from 11 and 68 according to the procedure for the synthesis of 12a. Yield was 71%. ¹H-NMR (CDCl₃) δ: 1.62–1.75 (2H, m), 1.93–2.02 (2H, m), 2.69 (3H, s), 3.42–3.52 (2H, m), 3.59–3.69 (1H, m), 3.94–4.04 (2H, m), 4.58 (2H, s), 7.01–7.06 (2H, m), 7.10 (1H, d, J = 1.2 Hz), 7.39 (1H, dd, J = 1.2, 8.0 Hz), 7.40–7.46 (2H, m), 7.86 (1H, d, J = 8.0 Hz).

Compound 12o was synthesized from 11 and phenol 71, which was obtained via 69 and 70 from tetrahydropyran-4-ol as the starting material.

4-Allyloxytetrahydropyran (69)

Following the addition of NaH (60%) (940 mg, 24 mmol) to a solution of tetrahydropyran-4-ol (2.00 g, 19.6 mmol) in DMF (40 mL) in portions at room temperature, the reaction mixture was stirred at the same temperature for 30 min. After the addition of allyl bromide (1.66 mL, 19.6 mmol), the mixture was stirred at the same temperature for 3 h. AcOEt was added, and the mixture was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 69 (1.26 g, 45% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.56–1.66 (2H, m), 1.85–1.95 (2H, m), 3.39–3.48 (2H, m), 3.49–3.57 (1H, m), 3.91–3.99 (2H, m), 4.00–4.06 (2H, m), 5.14–5.21 (1H, m), 5.25–5.33 (1H, m), 5.87–5.99 (1H, m).

4-[3-(Tetrahydropyran-4-yloxy)propenyl]phenyl Acetate (70)

Following the addition of Pd(OAc)₂ (247 mg, 1.10 mmol), PPh₃ (577 mg, 2.20 mmol), and AcOAg (3.67 g, 22.0 mmol) to a solution of 4-iodophenyl acetate (1.92 g, 7.33 mmol) and 69 (1.04 g, 7.31 mmol) in DMF (30 mL), the reaction mixture was stirred at 70 °C for 23 h under a N₂ atmosphere. After cooling, AcOEt was added and the insoluble material was filtered. The filtrate was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 70 (0.46 g, 23% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.58–1.68 (2H, m), 1.88–1.98 (2H, m), 2.29 (3H, s), 3.40–3.49 (2H, m), 3.54–3.63 (1H, m), 3.92–4.00 (2H, m), 4.16–4.21 (2H, m), 6.25 (1H, dt, J = 15.9, 5.8 Hz), 6.59 (1H, d, J = 15.9 Hz), 7.01–7.07 (2H, m), 7.35–7.42 (2H, m).

4-[3-(Tetrahydropyran-4-yloxy)propenyl]phenol (71)

Following the addition of 1.0 M aqueous LiOH solution (5.0 mL, 5.0 mmol) to a solution of 70 (0.46 g, 1.7 mmol) in MeOH (15 mL), the reaction mixture was stirred at room temperature for 30 min. Following the addition of 6.0 M aqueous HCl solution to adjust acidic conditions, the mixture was extracted with AcOEt, and the organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 71 (0.29 g, 74% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.90–1.98 (2H, m), 3.42–3.51 (2H, m), 3.57–3.65 (1H, m), 3.95–4.02 (2H, m), 4.15–4.19 (2H, m), 6.14 (1H, dt, J = 15.6, 6.3 Hz), 6.53 (1H, d, J = 15.6 Hz), 6.74–6.79 (2H, m), 7.23–7.28 (2H, m).

4-Acetyl-3-{4-[3-(tetrahydropyran-4-yloxy)propenyl]phenoxy}benzonitrile (12o)

Compound 12o was prepared from 11 and 71 according to the procedure for the synthesis of 12a. Yield was quant. ¹H-NMR (CDCl₃) δ: 1.60–1.71 (2H, m), 1.90–1.99 (2H, m), 2.68 (3H, s), 3.42–3.50 (2H, m), 3.56–3.65 (1H, m), 3.94–4.02 (2H, m), 4.19–4.24 (2H, m), 6.29 (1H, dt, J = 15.9, 5.8 Hz), 6.63 (1H, d, J = 15.9 Hz), 6.97–7.03 (2H, m), 7.11 (1H, d, J = 1.4 Hz), 7.41 (1H, dd, J = 8.0, 1.4 Hz), 7.43–7.48 (2H, m), 7.86 (1H, d, J = 8.0 Hz).

Compound 13a was synthesized via 72a obtained from 12a.

4-Acetyl-3-(4-{3-[2-(tetrahydropyran-2-yloxy)ethoxy]-azetidin-1-yl}phenoxy)benzamide (72a)

Following the addition of K₂CO₃ (87 mg, 0.63 mmol) and 9.79 M aqueous H₂O₂ solution (0.19 mL, 1.9 mmol) to a solution of 12a (230 mg, 0.526 mmol) in DMSO (5 mL), the reaction mixture was stirred at room temperature for 3 h. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 72a (215 mg, 90% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.48–1.68 (4H, m), 1.69–1.91 (2H, m), 2.70 (3H, s), 3.47–3.55 (1H, m), 3.58–3.69 (3H, m), 3.73–3.79 (2H, m), 3.84–3.92 (2H, m), 4.13 (2H, t, J = 6.8 Hz), 4.47–4.55 (1H, m), 4.61–4.66 (1H, m), 6.45–6.51 (2H, m), 6.90–6.96 (2H, m), 7.23 (1H, d, J = 1.7 Hz), 7.41 (1H, dd, J = 8.0, 1.7 Hz), 7.82 (1H, d, J = 8.0 Hz).

4-Acetyl-3-{4-[3-(2-hydroxyethoxy)azetidin-1-yl]phenoxy}benzamide (13a)

Compound 13a was prepared from 72a according to the procedure for the synthesis of 7a. Yield was 71%. mp 58–62 °C; ¹H-NMR (CDCl₃) δ: 2.06–2.17 (1H, br), 2.70 (3H, s), 3.54–3.61 (2H, m), 3.70–3.83 (4H, m), 4.13 (2H, t, J = 6.9 Hz), 4.44–4.52 (1H, m), 5.60–6.30 (2H, br), 6.45–6.52 (2H, m), 6.90–6.97 (2H, m), 7.23–7.27 (1H, m), 7.37–7.43 (1H, m), 7.82 (1H, d, J = 8.1 Hz); ¹³C-NMR (CDCl₃) δ: 31.0 (s), 31.0 (s), 31.1 (s), 32.2 (s), 64.8 (s), 65.8 (s), 73.1 (s), 118.0 (s), 118.8 (2C, s), 121.9 (s), 129.8 (s), 130.0 (2C, s), 132.1 (s), 137.6 (s), 138.8 (s), 153.7 (s), 155.5 (s), 166.2 (s), 198.2 (s); IR (ATR) cm⁻¹: 1709, 1682. HR-MS (ESI-TOF) m/z: 393.1421 [M + Na]⁺ (Calcd for C₂₀H₂₂N₂NaO₅, 393.1426).

4-Acetyl-3-{4-[2-(tetrahydrofuran-3-yloxy)ethoxy]phenoxy}benzamide (13g)

Compound 13g was prepared from 12g according to the procedure for the synthesis of 72a. Yield was 52%. mp 91–92 °C; ¹H-NMR (DMSO-d₆) δ: 1.85–2.01 (2H, m), 2.60 (3H, s), 3.64–3.77 (6H, m), 4.07–4.12 (2H, m), 4.18–4.23 (1H, m), 7.00–7.03 (2H, m), 7.06–7.08 (2H, m), 7.29 (1H, d, J = 1.2 Hz), 7.50–7.58 (1H, br), 7.63 (1H, d, J = 8.0, 1.2 Hz), 7.73 (1H, d, J = 8.0 Hz), 8.05–8.12 (1H, br); ¹³C-NMR (CDCl₃) δ: 31.1 (s), 32.0 (s), 66.1 (s), 66.9 (s), 67.5 (s), 72.0 (s), 79.2 (s), 115.9 (2C, s), 116.9 (s), 120.7 (2C, s), 121.3 (s), 129.7 (s), 131.5 (s), 138.8 (s), 148.7 (s), 155.2 (s), 156.5 (s), 166.3 (s), 198.3 (s); IR (ATR) cm⁻¹: 1652. HR-MS (ESI-TOF) m/z: 408.1407 [M + Na]⁺ (Calcd for C₂₁H₂₃NNaO₆, 408.1423).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (13h)

Compound 13h was prepared from 12h according to the procedure for the synthesis of 72a. Yield was 87%. mp 99–100 °C; ¹H-NMR (CDCl₃) δ: 1.57–1.68 (2H, m), 1.91–1.99 (2H, m), 2.70 (3H, s), 3.42–3.50 (2H, m), 3.57–3.65 (1H, m), 3.85 (2H, t, J = 5.1 Hz), 3.91–3.98 (2H, m), 4.14 (2H, t, J = 5.1 Hz), 5.40–5.70 (1H, br), 5.85–6.10 (1H, br), 6.93–7.01 (2H, m), 7.28 (1H, d, J = 1.0 Hz), 7.43 (1H, dd, J = 8.1, 1.0 Hz), 7.85 (1H, d, J = 8.1 Hz); ¹³C-NMR (CDCl₃) δ: 31.1 (s), 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.8 (s), 73.6 (s), 115.9 (2C, s), 116.8 (s), 120.7 (2C, s), 121.3 (s), 129.7 (s), 131.5 (s), 138.8 (s), 148.7 (s), 155.3 (s), 156.5 (s), 166.3 (s), 198.3 (s); IR (ATR) cm⁻¹: 1666; HR-MS (ESI-TOF) m/z: 422.1574 [M + Na]⁺ (Calcd for C₂₂H₂₅NNaO₆, 422.1580).

4-Acetyl-3-{4-[4-(2-hydroxyethoxy)piperidin-1-yl]phenoxy}benzamide (13j)

Compound 13j was prepared from 12j according to the procedure for the synthesis of 72a. Yield was 72%. mp 116–117 °C; ¹H-NMR (CDCl₃) δ: 1.70–1.84 (2H, m), 1.97–2.03 (3H, m), 2.88–3.02 (2H, m), 2.69 (3H, s), 2.88–3.00 (2H, m), 3.43–3.57 (3H, m), 3.62 (2H, t, J = 4.4 Hz), 3.73–3.79 (2H, m), 6.92–7.00 (4H, m), 7.30 (1H, d, J = 1.2 Hz), 7.42 (1H, dd, J = 8.0, 1.2 Hz), 7.83 (1H, d, J = 8.0 Hz); IR (ATR) cm⁻¹: 1709, 1682. HR-MS (ESI-TOF) m/z: 421.1742 [M + Na]⁺ (Calcd for C₂₂H₂₆N₂NaO₅, 421.1739).

4-Acetyl-3-{4-[2-(2-isopropoxyethoxy)ethoxy]phenoxy}-benzamide (13k)

Compound 13k was prepared from 12k according to the procedure for the synthesis of 72a. Yield was 80%. mp 77–79 °C; ¹H-NMR (DMSO-d₆) δ: 1.06 (6H, d, J = 6.1 Hz), 2.58 (3H, s), 3.45–3.60 (5H, m), 3.70–3.78 (2H, m), 4.05–4.13 (2H, m), 6.96–7.10 (4H, m), 7.28 (1H, d, J = 1.5 Hz), 7.45–7.55 (1H, br), 7.62 (1H, dd, J = 8.0, 1.5 Hz), 7.71 (1H, d, J = 8.0 Hz), 8.00–8.10 (1H, br); IR (ATR) cm⁻¹: 1664.

4-Acetyl-3-[4-(tetrahydropyran-4-yloxymethyl)phenoxy]-benzamide (13m)

Compound 13m was prepared from 12m according to the procedure for the synthesis of 72a. Yield was 88%. mp 139–141 °C; ¹H-NMR (CDCl₃) δ: 1.42–1.55 (2H, m), 1.85–1.95 (2H, m), 2.57 (3H, s), 3.30–3.38 (2H, m), 3.54–3.64 (1H, m), 3.77–3.87 (2H, m), 4.52 (2H, s), 7.03–7.09 (2H, m), 7.35–7.42 (3H, m), 7.55 (1H, s), 7.67–7.74 (1H, m), 7.77 (1H, d, J = 8.0 Hz), 8.10 (1H, s); ¹³C-NMR (CDCl₃) δ: 31.0 (s), 32.2 (2C, s), 64.8 (2C, s), 67.9 (s), 72.9 (s), 118.2 (s), 118.6 (2C, s), 122.2 (s), 129.3 (2C, s), 129.9 (s), 132.2 (s), 134.8 (s), 138.9 (s), 155.0 (s), 155.2 (s), 166.2 (s), 198.1 (s); IR (ATR) cm⁻¹: 1666. HR-MS (ESI-TOF) m/z: 392.1464 [M + Na]⁺ (Calcd for C₂₁H₂₃NNaO₅, 392.1474).

4-Acetyl-3-{4-[3-(tetrahydropyran-4-yloxy)propenyl]phenoxy}benzamide (13o)

Compound 13o was prepared from 12o according to the procedure for the synthesis of 72a. Yield was 73%. mp 101–104 °C; ¹H-NMR (CDCl₃) δ: 1.62–1.71 (2H, m), 1.90–1.99 (2H, m), 2.66 (3H, s), 3.42–3.51 (2H, m), 3.56–3.65 (1H, m), 3.94–4.02 (2H, m), 4.18–4.23 (2H, m), 5.60–6.20 (2H, br), 6.25 (1H, dt, J = 16.1, 5.8 Hz), 6.63 (1H, d, J = 16.1 Hz), 6.95–7.01 (2H, m), 7.38 (1H, d, J = 1.5 Hz), 7.38–7.43 (2H, m), 7.49 (1H, dd, J = 8.0, 1.5 Hz), 7.87 (1H, d, J = 8.0 Hz); ¹³C-NMR (CDCl₃) δ: 30.9 (s), 32.3 (2C, s), 64.8 (2C, s), 67.2 (s), 72.7 (s), 118.5 (s), 118.8 (2C, s), 122.4 (s), 126.8 (s), 128.1 (2C, s), 129.8 (s), 129.9 (s), 132.3 (s), 132.5 (s), 138.9 (s), 155.0 (s), 155.3 (s), 166.1 (s), 198.1 (s); IR (ATR) cm⁻¹: 1676, 1664. HR-MS (ESI-TOF) m/z: 418.1622 [M + Na]⁺ (Calcd for C₂₃H₂₅NNaO₅, 418.1630).

4-Acetyl-3-{4-[3-(tetrahydropyran-4-yloxy)propyl]phenoxy}benzamide (13n)

Compound 13n was prepared from 13o according to the procedure for the synthesis of 7d. Yield was 49%. mp 53–55 °C; ¹H-NMR (CDCl₃) δ: 1.52–1.69 (2H, m), 1.85–1.95 (4H, m), 2.67 (3H, s), 2.71 (2H, t, J = 7.6 Hz), 3.39–3.52 (5H, m), 3.90–3.98 (2H, m), 5.60–6.30 (2H, br), 6.92–6.98 (2H, m), 7.18–7.23 (2H, m), 7.37 (1H, d, J = 1.5 Hz), 7.46 (1H, dd, J = 8.1, 1.5 Hz), 7.85 (1H, d, J = 8.1 Hz); ¹³C-NMR (CDCl₃) δ: 31.0 (s), 31.0 (s), 31.1 (s), 32.2 (s), 64.8 (s), 65.8 (s), 73.1 (s), 118.0 (s), 118.8 (2C, s), 121.9 (s), 129.8 (s), 130.0 (2C, s), 132.1 (s), 137.6 (s), 138.8 (s), 153.7 (s), 155.5 (s), 166.2 (s), 198.2 (s); IR (ATR) cm⁻¹: 1676, 1662. HR-MS (ESI-TOF) m/z: 420.1775 [M + Na]⁺ (Calcd for C₂₃H₂₇NNaO₅, 420.1787).

Methyl 4-Acetyl-3-[4-(2-isopropoxyethoxymethyl)phenoxy]benzoate (15l)

Following the addition of 14 (630 mg, 3.21 mmol) and K₂CO₃ (666 mg, 4.82 mmol) to a solution of 4-[(2-isopropoxyethoxy)methyl]phenol³⁴⁾ (1.16 g, 3.85 mmol) in DMF (10 mL), the reaction mixture was stirred at 100 °C for 15 h. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 15l (810 mg, 65% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.18 (6H, d, J = 6.1 Hz), 2.66 (3H, s), 3.56–3.75 (5H, m), 3.87 (3H, s), 4.58 (2H, s), 6.99–7.01 (2H, m), 7.36–7.39 (2H, m), 7.56 (1H, d, J = 8.5 Hz), 7.78–7.89 (2H, m).

Compound 13l was synthesized via 73l obtained from 15l.

4-Acetyl-3-[4-(2-isopropoxyethoxymethyl)phenoxy]benzoic Acid (73l)

Following the addition of 1.0 M aqueous LiOH solution (6.3 mL, 6.3 mmol) to a solution of 15l (810 mg, 2.10 mmol) in MeOH (10 mL), the reaction mixture was stirred at 50 °C for 1 h. Following the addition of 5% aqueous citric acid solution to adjust pH to 2, the mixture was extracted with CHCl₃, and the organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 73l (730 mg, 93% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.18 (6H, d, J = 6.1 Hz), 2.67 (3H, s), 3.58–3.72 (5H, m), 4.58 (2H, s), 6.99–7.01 (2H, m), 7.37–7.39 (2H, m), 7.58 (1H, s), 7.82–7.87 (2H, m).

4-Acetyl-3-[4-(2-isopropoxyethoxymethyl)phenoxy]benzamide (13l)

Following the addition of N-methylmorpholine (NMM) (0.28 mL, 2.6 mmol) and ClCO₂i-Bu (0.28 mL, 2.2 mmol) to a solution of 73l (730 mg, 1.96 mmol) under ice cooling, the reaction mixture was stirred at the same temperature for 30 min. After the addition of 28% aqueous ammonia solution (0.66 mL, 9.8 mmol), the mixture was stirred for 1 h. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure and i-Pr₂O was added. The insoluble material was collected by filtration to give 13l (370 mg, 51% yield) as a solid. mp 73–75 °C; ¹H-NMR (CDCl₃) δ: 1.18 (6H, d, J = 6.1 Hz), 2.67 (3H, s), 3.60–3.72 (5H, m), 4.57 (2H, s), 5.45–6.25 (2H, br), 6.99–7.01 (2H, m), 7.37–7.39 (3H, m), 7.50 (1H, d, J = 8.0 Hz), 7.87 (1H, d, J = 8.0 Hz); ¹³C-NMR (CDCl₃) δ: 21.9 (2C, s), 30.9 (s), 66.6 (s), 69.4 (s), 70.8 (s), 71.3 (s), 118.3 (s), 118.5 (2C, s), 122.2 (s), 129.4 (2C, s), 129.8 (s), 132.3 (s), 134.3 (s), 138.9 (s), 155.1 (s), 155.1 (s), 166.1 (s), 198.1 (s); IR (ATR) cm⁻¹: 3411, 3195, 1654. HR-MS (ESI-TOF) m/z: 394.1633 [M + Na]⁺ (Calcd for C₂₁H₂₅NNaO₅, 394.1630).

3-{4-[2-(Tetrahydropyran-4-yloxy)ethoxy]phenoxy}-4-trifluoromethylbenzonitrile (18h)

Following the addition of 16 (778 mg, 4.11 mmol) and K₂CO₃ (610 mg, 4.41 mmol) to a solution of 62 (700 mg, 2.94 mmol) in N-methyl-2-pyrrolidone (NMP) (10 mL), the reaction mixture was stirred at 120 °C for 16 h. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 18h (791 mg, 66% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.91–1.99 (2H, m), 3.46 (2H, ddd, J = 12.0, 9.8. 2.7 Hz), 3.58–3.65 (1H, m), 3.84–3.88 (2H, m), 3.97 (2H, dt, J = 12.0, 4.2 Hz), 4.13–4.17 (2H, m), 6.96–7.04 (5H, m), 7.37 (1H, dd, J = 8.1 Hz), 7.74 (1H, d, J = 8.1 Hz).

4-Difluoromethyl-3-fluorobenzonitrile (17)

Following the addition of N,N-diethylsulfur trifluoride (1.82 g, 11.3 mmol) to a solution of 3-fluoro-4-formylbenzonitrile (1.53 g, 10.3 mmol) in CH₂Cl₂ (20 mL) under ice cooling, the reaction mixture was stirred at room temperature for 21 h. The reaction mixture was washed with saturated aqueous NaHCO₃ solution and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 17 (1.40 g, 80% yield) as a solid. ¹H-NMR (CDCl₃) δ: 6.91 (1H, t, J = 54.4 Hz), 7.46 (1H, dd, J = 9.3, 1.2 Hz), 7.58 (1H, d, J = 8.0 Hz), 7.72–7.77 (1H, m).

4-Difluoromethyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzonitrile (19h)

Compound 19h was prepared from 17 and 62 according to the procedure for the synthesis of 18h. Yield was 88%. ¹H-NMR (CDCl₃) δ: 1.58–2.04 (2H, m), 1.92–1.99 (2H, m), 3.42–3.49 (2H, m), 3.57–3.64 (1H, m), 3.83–3.87 (2H, m), 3.93–4.00 (2H, m), 4.10–4.17 (2H, m), 6.92–7.22 (6H, m), 7.38–7.42 (1H, m), 7.72–7.76 (1H, m).

4-(1,1-Difluoroethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)-ethoxy]phenoxy}benzonitrile (20h)

Following the addition of Deoxo-Fluor^® (8.3 mL, 45 mmol) to a solution of 12h (2.15 g, 5.63 mmol) in CHCl₃ (20 mL), the reaction mixture was stirred at 70 °C for 14 h. After cooling, the mixture was poured into saturated aqueous NaHCO₃ solution. The mixture was extracted with AcOEt, and the organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 20h (2.21 g, 97% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.90–1.99 (2H, m), 2.09 (3H, t, J = 18.6 Hz), 3.42–3.50 (2H, m), 3.57–3.66 (1H, m), 3.85 (2H, t, J = 5.1 Hz), 3.93–4.00 (2H, m), 4.14 (2H, t, J = 5.1 Hz), 6.94–7.01 (5H, m), 7.34–7.36 (1H, m), 7.67–7.70 (1H, m).

3-{4-[2-(Tetrahydropyran-4-yloxy)ethoxy]phenoxy}-4-trifluoromethylbenzamide (21h)

Compound 21h was prepared from 18h according to the procedure for the synthesis of 72a. Yield was 69%. mp 122–124 °C; ¹H-NMR (DMSO-d₆) δ: 1.35–1.46 (2H, m), 1.82–1.90 (2H, m), 3.29–3.37 (2H, m), 3.52–3.60 (1H, m), 3.74–3.84 (4H, m), 4.08–4.12 (2H, m), 7.00–7.07 (4H, m), 7.33 (1H, s), 7.58–7.64 (1H, br), 7.70 (1H, d, J = 8.0 Hz), 7.84 (1H, d, J = 8.0 Hz), 8.13–8.19 (1H, br); ¹³C-NMR (CDCl₃) δ: 2.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.7 (s), 73.6 (s), 115.9 (2C, s), 116.6 (s), 120.8 (1C, q, J = 31.3 Hz), 121.0 (2C, s), 121.1 (s), 123.2 (1C, q, J = 272.6 Hz), 127.2 (1C, q, J = 4.8 Hz), 139.8 (s), 148.3 (s), 155.5 (s), 155.8 (s), 165.9 (s); IR (ATR) cm⁻¹: 3392, 3207, 1666. HR-MS (ESI-TOF) m/z: 448.1345 [M + Na]⁺ (Calcd for C₂₁H₂₂F₃NNaO₅, 448.1348).

4-Difluoromethyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (22h)

Compound 22h was prepared from 19h according to the procedure for the synthesis of 72a. Yield was 86%. mp 128–129 °C; ¹H-NMR (DMSO-d₆) δ: 1.34–1.45 (2H, m), 1.81–1.90 (2H, m), 2.99–3.36 (2H, m), 3.52–3.60 (1H, m), 3.72–3.84 (4H, m), 4.07–4.13 (2H, m), 6.98–7.07 (4H, m), 7.11–7.41 (2H, m), 7.48–7.56 (1H, br), 7.64–7.72 (2H, m), 8.03–8.12 (1H, br); ¹³C-NMR (CDCl₃) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.7 (s), 73.6 (s), 111.8 (1C, t, J = 236.5 Hz), 115.6 (s), 115.8 (2C, s), 121.2 (2C, s), 121.3 (s), 125.8 (1C, t, J = 22.2 Hz), 126.5 (1C, t, J = 5.3 Hz), 138.2 (s), 148.5 (s), 155.4 (s), 156.0 (1C, t, J = 5.3 Hz), 166.4 (s); IR (ATR) cm⁻¹: 1685. HR-MS (ESI-TOF) m/z: 430.1434 [M + Na]⁺ (Calcd for C₂₁H₂₃F₂NNaO₅, 430.1442).

4-(1,1-Difluoroethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)-ethoxy]phenoxy}benzamide (24h)

Compound 24h was prepared from 20h according to the procedure for the synthesis of 72a. Yield was 20%. mp 128–129 °C; ¹H-NMR (DMSO-d₆) δ: 1.34–1.47 (2H, m), 1.82–1.90 (2H, m), 2.06 (3H, t, J = 19.0 Hz), 3.30–3.37 (2H, m), 3.52–3.61 (1H, m), 3.76 (2H, t, J = 4.4 Hz), 3.77–3.84 (2H, m), 4.09 (2H, t, J = 4.4 Hz), 6.98–7.03 (4H, m), 7.27–7.31 (1H, m), 7.45–7.53 (1H, br), 7.61–7.68 (2H, m), 8.02–8.11 (1H, br); ¹³C-NMR (CDCl₃) δ: 24.4 (1C, t, J = 28.0 Hz), 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.7 (s), 73.6 (s), 115.8 (2C, s), 117.0 (s), 120.6 (2C, s), 121.1 (1C, t, J = 239.9 Hz), 121.3 (s), 126.1 (1C, t, J = 8.2 Hz), 129.3 (1C, t, J = 26.0 Hz), 137.5 (s), 149.1 (s), 155.0 (1C, t, J = 3.9 Hz), 155.2 (s), 166.3 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) m/z: 444.1592 [M + Na]⁺ (Calcd for C₂₂H₂₅F₂NNaO₅, 444.1598).

4-(1-Hydroxyethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (25h)

Following the addition of NaBH₄ (6 mg, 0.2 mmol) to a solution of 13h (50 mg, 0.13 mmol) in MeOH (1 mL), the reaction mixture was stirred at room temperature for 30 min. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography. The residue was recrystallized with AcOEt (3 mL) and t-BuOMe (1 mL) to give 25h (30 mg, 60% yield) as a solid. mp 138–140 °C; ¹H-NMR (DMSO-d₆) δ: 1.32 (3H, d, J = 6.4 Hz), 1.35–1.46 (2H, m), 1.81–1.90 (2H, m), 3.31–3.37 (2H, m), 3.51–3.60 (1H, m), 3.72–3.85 (4H, m), 4.03–4.11 (2H, m), 5.01–5.10 (1H, m), 5.25 (1H, d, J = 4.4 Hz), 6.90–7.01 (4H, m), 7.16–7.21 (1H, m), 7.27–7.35 (1H, br), 7.57–7.64 (2H, m), 7.87–7.94 (1H, br); ¹³C-NMR (CDCl₃) δ: 24.6 (s), 32.2 (s), 62.5 (s), 64.8 (s), 65.5 (s), 67.7 (s), 73.6 (s), 115.7 (2C, s), 116.1 (s), 119.9 (2C, s), 121.8 (s), 126.0 (s), 133.9 (s), 140.8 (s), 149.8 (s), 153.4 (s), 154.6 (s), 167.1 (s); IR (ATR) cm⁻¹: 1675; HR-MS (ESI-TOF) m/z: 424.1731 [M + Na]⁺ (Calcd for C₂₂H₂₇NNaO₆, 424.1736).

4-(1-Fluoroethyl)-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenoxy}benzamide (23h)

Following the addition of DAST (0.89 mL, 6.7 mmol) to a solution of 25h (900 mg, 2.24 mmol) in CHCl₃ (9 mL) under ice cooling, the reaction mixture was stirred at the same temperature for 30 min. The mixture was washed with saturated aqueous NaHCO₃ solution and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography. The residue (610 mg) was recrystallized with AcOEt (1 mL) and t-BuOMe (3 mL) to give 23h (378 mg, 42% yield) as a solid. mp 101–103 °C; ¹H-NMR (DMSO-d₆) δ: 1.34–1.46 (2H, m), 1.62 (3H, dd, J = 24, 6.3 Hz), 1.82–1.91 (2H, m), 3.30–3.37 (2H, m), 3.52–3.60 (1H, m), 3.75 (2H, t, J = 4.4 Hz), 3.77–3.84 (2H, m), 4.08 (2H, t, J = 4.4 Hz), 5.92–6.11 (1H, m), 6.98–7.02 (4H, m), 7.19–7.23 (1H, m), 7.36–7.45 (1H, br), 7.52–7.57 (1H, m), 7.62–7.68 (1H, m), 7.94–8.02 (1H, br); ¹³C-NMR (CDCl₃) δ: 21.6 (1C, d, J = 25.1 Hz), 32.2 (s), 64.8 (2C, s), 65.4 (s), 67.7 (s), 73.6 (s), 85.9 (1C, d, J = 166.2 Hz), 115.8 (3C, s), 120.5 (2C, s), 121.7 (s), 125.7 (s), 134.0 (1C, d, J = 20.2 Hz), 135.5 (1C, d, J = 1.4 Hz), 149.1 (s), 154.0 (1C, d, J = 5.3 Hz), 155.0 (s), 166.7 (s); IR (ATR) cm⁻¹: 1654. HR-MS (ESI-TOF) m/z: 426.1687 [M + Na]⁺ (Calcd for C₂₂H₂₆FNNaO₅, 426.1693).

5-Bromo-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]-phenyl}-3H-isobenzofuran-1-one (28h)

DMSO (7.7 mL, 0.11 mmol), i-Pr₂NEt (9.4 mL, 55 mmol), and sulfur trioxide pyridine complex (8.66 g, 54.4 mmol) were added to a solution of compound 26²⁵⁾ (4.19 g, 18.1 mmol) in CH₂Cl₂ (80 mL) under ice cooling, and the mixture was stirred at the same temperature for 0.5 h. The reaction mixture was evaporated under reduced pressure. After the addition of 1.0 M aqueous HCl solution, the mixture was extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure.

Phenol (1.71 g, 18.2 mmol) was added to a suspension of the residue obtained in 60% aqueous sulfuric acid solution (40 mL), and the mixture was stirred at room temperature for 14 h. The mixture was poured into water, and the precipitate that formed was collected by filtration. After the addition of AcOEt to the precipitate obtained, the mixture was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. t-BuOMe was added to the residue, the mixture was stirred at room temperature for 1 h, and the precipitate was filtered to give compound 27 (3.57 g, crude) as a solid.

2-[(Tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ (287 mg, 1.96 mmol), triphenylphosphine (PPh₃) (620 mg, 2.36 mmol), and diisopropyl azodicarboxylate (DIAD) (0.46 mL, 2.4 mmol) were added to a solution of compound 27 (1.00 g) in CH₂Cl₂ (15 mL), and the mixture was stirred at room temperature for 2 h. 2-[(Tetrahydropyran-4-yl)oxy]ethanol³⁰⁾ (144 mg, 0.985 mmol), PPh₃ (310 mg, 1.18 mmol), and DIAD (0.23 mL, 1.2 mmol) were added to the reaction mixture, which was stirred at the same temperature for 1 h. The mixture was concentrated under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 28h (730 mg, yield 57% for 3 steps) as a solid. ¹H-NMR (DMSO-d₆) δ: 1.34–1.45 (2H, m), 1.81–1.89 (2H, m), 3.28–3.35 (2H, m), 3.50–3.58 (1H, m), 3.73–3.82 (4H, m), 4.07–4.12 (2H, m), 6.66 (1H, s), 6.95–7.00 (2H, m), 7.19–7.24 (2H, m), 7.65–7.68 (1H, m), 7.80–7.87 (2H, m).

4-Bromo-N-methoxy-N-methyl-2-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]benzyl}benzamide (29h)

Triethylsilane (0.80 mL, 5.0 mmol) and titanium tetrachloride (0.21 mL, 1.9 mmol) were added to a solution of compound 28h (0.73 g, 1.7 mmol) in CH₂Cl₂ (15 mL) under ice cooling, and the mixture was stirred at the same temperature for 1 h. Titanium tetrachloride (0.10 mL, 0.91 mmol) was added to the reaction mixture under ice cooling, which was stirred at the same temperature for 1 h. After the addition of water, the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. n-Hexane was added to the residue and the mixture was stirred at room temperature for 1 h. The precipitate that formed was obtained after repeated decantation with n-hexane.

N,O-Dimethylhydroxylamine hydrochloride (229 mg, 2.35 mmol), Et₃N (0.35 mL, 2.5 mmol), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (419 mg, 2.19 mmol) were added to a solution of the precipitate obtained in CH₂Cl₂ (15 mL), and the mixture was stirred at room temperature for 14 h. After the addition of water, the mixture was extracted with AcOEt. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 29h (580 mg, yield 72% for 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 1.56–1.68 (2H, m), 1.89–1.96 (2H, m), 3.05–3.40 (6H, br), 3.40–3.48 (2H, m), 3.55–3.62 (1H, m), 3.79–3.84 (2H, m), 3.91–3.98 (4H, m), 4.06–4.11 (2H, m), 6.81–6.85 (2H, m), 7.05–7.10 (2H, m), 7.11–7.19 (1H, br), 7.26–7.32 (1H, br), 7.35 (1H, dd, J = 8.3, 1.7 Hz).

1-(4-Bromo-2-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]-benzyl}phenyl)ethanone (30h)

To a solution of compound 29h (693 mg, 1.45 mmol) in THF (10 mL) was added 1.0 M methyllithium in Et₂O (2.9 mL, 2.9 mmol) under ice cooling, and the mixture was stirred at the same temperature for 0.5 h. After the addition of saturated aqueous NH₄Cl solution, the mixture was extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 30h (434 mg, yield 69%) as an oil. ¹H-NMR (CDCl₃) δ: 1.56–1.68 (2H, m), 1.89–1.96 (2H, m), 2.43 (3H, s), 3.44 (2H, ddd, J = 12.0, 9.8, 2.7 Hz), 3.55–3.62 (1H, m), 3.79–3.83 (2H, m), 3.95 (2H, dt, J = 12.0, 4.4 Hz), 4.07–4.11 (2H, m), 4.16 (2H, s), 6.80–6.85 (2H, m), 7.00–7.05 (2H, m), 7.34 (1H, d, J = 2.0 Hz), 7.42 (1H, dd, J = 8.3, 2.0 Hz), 7.49 (1H, d, J = 8.3 Hz).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]-benzyl}benzonitrile (31h)

Copper(I) cyanide (175 mg, 1.95 mmol) and copper(I) iodide (594 mg, 3.12 mmol) were added to a solution of compound 30h (338 mg, 0.780 mmol) in DMF (10 mL), and the mixture was refluxed for 4 h. After the addition of AcOEt, the mixture was washed with 10% aqueous sodium cyanide solution, water, and saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 31h (161 mg, yield 54%) as an oil. ¹H-NMR (CDCl₃) δ: 1.56–1.68 (2H, m), 1.90–1.97 (2H, m), 2.42 (3H, s), 3.40–3.48 (2H, m), 3.56–3.63 (1H, m), 3.80–3.85 (2H, m), 3.92–3.99 (2H, m), 4.08–4.12 (2H, m), 4.14 (2H, s), 6.82–6.87 (2H, m), 6.97–7.02 (2H, m), 7.47 (1H, d, J = 1.5 Hz), 7.58 (1H, dd, J = 8.0, 1.5 Hz), 7.60 (1H, d, J = 8.0 Hz).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]benzyl}benzamide (32h)

Compound 32h was prepared according to the procedure for the synthesis of 71a. Yield was 64%. A white solid, mp 103–105 °C. ¹H-NMR (DMSO-d₆) δ: 1.33–1.43 (2H, m), 1.80–1.88 (2H, m), 2.45 (3H, s), 3.27–3.35 (2H, m), 3.49–3.56 (1H, m), 3.70–3.74 (2H, m), 3.75–3.82 (2H, m), 3.99–4.04 (2H, m), 4.09 (2H, s), 6.80–6.85 (2H, m), 6.99–7.04 (2H, m), 7.47–7.52 (1H, br), 7.76–7.81 (3H, m), 8.04–8.09 (1H, br); ¹³C-NMR (CDCl₃) δ: 30.1 (s), 32.2 (2C, s), 37.0 (s), 64.8 (s), 65.4 (s), 67.2 (s), 73.6 (s), 114.2 (2C, s), 125.0 (s), 128.6 (s), 129.7 (2C, s), 130.3 (s), 132.5 (s), 136.1 (s), 139.9 (s), 140.6 (s), 156.7 (s), 167.0 (s), 202.4 (s); IR (ATR) cm⁻¹: 3348, 3163, 1682. HR-MS (ESI-TOF) m/z: 420.1786 [M + Na]⁺ (Calcd for C₂₃H₂₇NNaO₅, 420.1787).

Methyl 4-Acetyl-3-(4-hydroxyphenylsulfanyl)benzoate (33)

Following the addition of 4-hydroybenzenethiol (901 mg, 7.14 mmol) and Et₃N (1.3 mL, 9.4 mmol) to a solution of 14 (467 mg, 2.38 mmol) in DMF (5 mL), the reaction mixture was stirred at 100 °C for 16 h and then at 120 °C for 11 h. The reaction mixture was added to 4-hydroybenzenethiol (450 mg, 3.57 mmol), which was stirred at 120 °C for 16 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 33 (243 mg, 34% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.69 (3H, s), 3.82 (3H, s), 5.20 (1H, s), 6.88–6.94 (2H, m), 7.39–7.45 (2H, m), 7.54 (1H, d, J = 1.4 Hz), 7.75 (1H, dd, J = 8.3, 1.4 Hz), 7.84 (1H, d, J = 8.3 Hz).

Compound 34h was synthesized via 74h obtained from 33.

Methyl 4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenylsulfanyl}benzoate (74h)

Following the addition of K₂CO₃ (210 mg, 1.52 mmol) to a solution of 33 (230 mg, 0.761 mmol) and 2-(tetrahydropyran-4-yloxy)ethyl methanesulfonate³⁵⁾ (170 mg, 0.761 mmol) in DMF (5 mL), the reaction mixture was stirred at 80 °C for 16 h. After cooling, water was added, and the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 74h (300 mg, 92% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.92–2.00 (2H, m), 2.68 (3H, s), 3.42–3.50 (2H, m), 3.57–3.66 (1H, m), 3.82 (3H, s), 3.84–3.89 (2H, m), 3.94–4.01 (2H, m), 4.15–4.19 (2H, m), 6.95–7.01 (2H, m), 7.42–7.49 (2H, m), 7.55 (1H, d, J = 1.5 Hz), 7.75 (1H, dd, J = 8.0, 1.5 Hz), 7.83 (1H, d, J = 8.0 Hz).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]phenylamino}benzamide (34h)

Following the addition of 1.0 M aqueous LiOH solution (2.1 mL, 2.1 mmol) to a solution of 74h (300 mg, 0.697 mmol) in MeOH (2 mL) and THF (4 mL), the reaction mixture was stirred at 50 °C for 2 h. After the addition of 1.0 M aqueous HCl solution, the mixture was extracted with CHCl₃, and the organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

Following the addition of NMM (0.076 mL, 0.69 mmol) and ClCO₂i-Bu (0.090 mL, 0.69 mmol) to a solution of the residue obtained in THF (3 mL), the reaction mixture was stirred at room temperature for 0.5 h. Water was added and the mixture was extracted with CHCl₃. The organic layer was washed with water and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography. After the addition of AcOEt to the residue, the insoluble material was collected by filtration to give 34h (170 mg, 59% yield for 2 steps) as a solid, mp 152–154 °C. ¹H-NMR (DMSO-d₆) δ: 1.36–1.48 (2H, m), 1.83–1.93 (2H, m), 2.64 (3H, s), 3.30–3.38 (2H, m), 3.52–3.63 (1H, m), 3.76–3.85 (4H, m), 4.12–4.20 (2H, m), 7.03–7.10 (2H, m), 7.31 (1H, d, J = 1.4 Hz), 7.39–7.47 (3H, m), 7.64 (1H, dd, J = 8.0, 1.4 Hz), 7.99 (1H, s), 8.03 (1H, d, J = 8.0 Hz); ¹³C-NMR (CDCl₃) δ: 28.4 (s), 32.2 (2C, s), 64.8 (2C, s), 65.3 (s), 67.5 (s), 73.6 (s), 116.1 (2C, s), 122.1 (s), 122.7 (s), 126.3 (s), 131.0 (s), 135.3 (s), 136.8 (2C, s), 137.2 (s), 142.0 (s), 159.4 (s), 166.7 (s), 198.8 (s); IR (ATR) cm⁻¹: 1675, 1654, 1590. HR-MS (ESI-TOF) m/z: 470.1248 [M + Na]⁺ (Calcd for C₂₂H₂₅NNaO₇S, 470.1249).

4-Acetyl-3-{4-[2-(tetrahydropyran-4-yloxy)ethoxy]benzenesulfonyl}benzamide (35h)

m-CPBA (75%) (154 mg, 0.67 mmol) was added to a solution of compound 34h (185 mg, 0.445 mmol) in CH₂Cl₂ (4 mL), and the mixture was stirred at room temperature for 2 h. m-CPBA (75%) (154 mg, 0.67 mmol) was added to the reaction mixture, which was stirred at room temperature for 2 h. After the addition of 5% aqueous sodium thiosulfate solution and 5% aqueous K₂CO₃ solution, the mixture was extracted with CHCl₃. The organic layer was washed with saturated brine, dried over Na₂SO₄, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography. After the addition of Et₂O, the precipitate that formed was collected by filtration to give compound 35h (160 mg, 80% yield) as a solid, mp 145–147 °C. ¹H-NMR (DMSO-d₆) δ: 1.30–1.44 (2H, m), 1.78–1.88 (2H, m), 2.60 (3H, s), 3.24–3.35 (2H, m), 3.49–3.58 (1H, m), 3.73–3.82 (4H, m), 4.15–4.20 (2H, m), 7.14–7.20 (2H, m), 7.66–7.75 (2H, m), 7.80–7.87 (2H, m), 8.17 (1H, dd, J = 7.8, 1.4 Hz), 8.32 (1H, s), 8.44 (1H, d, J = 1.4 Hz); ¹³C-NMR (CDCl₃) δ: 31.6, (s) 32.1 (2C, s), 64.7 (2C, s), 65.1 (s), 68.0 (s), 73.6 (s), 115.2 (2C, s), 126.8 (s), 128.2 (s), 130.1 (2C, s), 131.8 (s), 132.3 (s), 135.7 (s), 138.1 (s), 143.5 (s), 162.6 (s), 165.4 (s), 202.4 (s); IR (ATR) cm⁻¹: 1708, 1637, 1590. HR-MS (ESI-TOF) m/z: 470.1248 [M + Na]⁺ (Calcd for C₂₂H₂₅NNaO₇S, 470.1249).

CDK8 Kinase Assay

CDK8 activity was measured using the QSS Assist CDK8 enzyme-linked immunosorbent assay (ELISA) kit (Carna Bioscience, Kobe, Japan) following the manufacturer’s protocol, as previously reported.¹⁶⁾

In Vitro Osteoblast Differentiation in ST2 Cells

Osteoblast differentiation was investigated using ST2 cells derived from mouse bone marrow mesenchymal stem cells (Riken BioResource Research Center, Tsukuba, Japan) as previously reported.¹⁵⁾ Briefly, ST2 cells were cultured for 24 h in α-MEM, osteoblast differentiation medium, compounds were added, and cells were further cultured for 4 d. Cells were washed with phosphate-buffered saline (pH 7.4) and lysed in 1% Triton X-100 solution, an ALP-mediated reaction was initiated by the addition of p-nitrophenyl phosphate (pNPP), and after a 30-min incubation at 37 °C, ALP activity was assessed by measuring absorbance at 405 nm.

Plasma Concentrations in Female Rats

Female rats (F344/NSlc, 12 weeks old, Japan SLC, Inc., Hamamatsu, Japan) were orally administered compounds (10 mg/kg) suspended in 0.5% MC solution. Blood was taken from the jugular vein 0.25, 0.5, 1, 3, 5, 8, and 24 h after their administration. Plasma concentrations were assessed using LC/MS/MS (QTRAP5500, AB Sciex, Framingham, MA, U.S.A) with a pump (Nexera X2, LC-30AD, Shimadzu, Kyoto, Japan) and autoinjector (Nexera X2, SIL-30AC, Shimadzu). Animals were housed under conditions with controlled temperature, humidity, and light exposure (12-h light–dark cycle) and provided ad libitum access to commercial standard rodent chow (CE2; CLEA Japan, Tokyo, Japan) and tap water. Animals were handled in accordance with the “Guidelines for Animal Experimentation” approved by The Japanese Pharmacological Society with all procedures approved by the Animal Ethical Committee of Kyoto Pharmaceutical Industries, Ltd.

Bone Structures in OVX Rats

Twelve-week-old female F344/NSlc rats were used. Ovariectomy was performed as previously reported.¹⁵⁾ Rats were anesthetized using ketamine (37.5 mg/kg, intraperitoneally (i.p.)) and xylazine (7.5 mg/kg, i.p.), and underwent a sham operation in the ovaries-intact control group and were bilaterally ovariectomized in the OVX-control and test compound-treated group. Rats were orally administered vehicle (0.5% MC) and the test compound suspended in 0.5% MC for 8 weeks. The right femur was scanned, under anesthesia with isoflurane, using micro-CT (R_μCT; Rigaku, Tokyo, Japan) 1 d before initiating the administration protocol and on the final day of administration.¹⁵⁾ Micro-CT data were analyzed using TRI/3D-BON software (RATOC, Tokyo, Japan).^36,37) After repeated administration, rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.), fasting blood was collected from the abdominal aorta, and rats were euthanized.

Conflict of Interest

The authors declare no conflict of interest.

References

• 1) Ukon Y., Makino T., Kodama J., Tsukazaki H., Tateiwa D., Yoshikawa H., Kaito T., Int. J. Mol. Sci., 20, 2557 (2019).
• 2) Noh J. Y., Yang Y., Jung H., Int. J. Mol. Sci., 21, 7623 (2020).
• 3) Notoya K., Nagai H., Oda T., Gotoh M., Hoshino T., Muranishi H., Taketomi S., Sohda T., Makino H., J. Pharmacol. Exp. Ther., 290, 1054–1064 (1999).
• 4) Rosa A. L., Beloti M. M., J. Cell. Biochem., 89, 1148–1153 (2003).
• 5) Rosa A. L., Beloti M. M., J. Cell. Biochem., 91, 749–755 (2004).
• 6) Tripathi A. K., Rai D., Kothari P., Kushwaha P., Sashidhara K. V., Trivedi R., Apoptosis, 27, 90–111 (2022).
• 7) Saito K., Nakao A., Shinozuka T., Shimada K., Matsui S., Oizumi K., Yano K., Ohata K., Nakai D., Nagai Y., Naito S., Bioorg. Med. Chem., 21, 1628–1642 (2013).
• 8) Saito K., Shinozuka T., Nakao A., Kunikata T., Nakai D., Nagai Y., Naito S., Bioorg. Med. Chem. Lett., 54, 128440 (2021).
• 9) Na W., Kang M. K., Park S. H., Kim D. Y., Oh S. Y., Oh M. S., Park S., Kang I. J., Kang Y. H., Int. J. Mol. Sci., 22, 12391 (2021).
• 10) Tang D. Z., Hou W., Zhou Q., Zhang M., Holz J., Sheu T. J., Li T. F., Cheng S. D., Shi Q., Harris S. E., Chen D., Wang Y. J., J. Bone Miner. Res., 25, 1234–1245 (2010).
• 11) Tang D. Z., Yang F., Yang Z., Huang J., Shi Q., Chen D., Wang Y. J., Biochem. Biophys. Res. Commun., 405, 256–261 (2011).
• 12) Wang X., Tian Y., Liang X., Yin C., Huai Y., Zhao Y., Huang Q., Chu X., Wang W., Qian A., Food Funct., 13, 2913–2924 (2022).
• 13) Xiao J. J., Zhao W. J., Zhang X. T., Zhao W. L., Wang X. X., Yin S. H., Jiang F., Zhao Y. X., Chen F. N., Li S. L., Mol. Cell. Biochem., 409, 113–122 (2015).
• 14) Kitao T., Ito Y., Fukui M., Yamamoto M., Shoji Y., Takeda S., Shirahase H., Yakugaku Zasshi, 139, 19–25 (2019).
• 15) Yamamoto M., Ito Y., Fukui M., Otake K., Shoji Y., Kitao T., Shirahase H., Hinoi E., Biol. Pharm. Bull., 46, 1435–1443 (2023).
• 16) Yamamoto M., Shibata Y., Ito Y., Fukui M., Kioka H., Shoji Y., Kitao T., Shirahase H., Hinoi E., Biol. Pharm. Bull., 47, 669–679 (2024).
• 17) Devgen, WO2007/042321 (2007).
• 18) Schulte B., Froehlich R., Studer A., Tetrahedron, 64, 11852–11859 (2008).
• 19) Pfizer Products, WO2008/065508 (2008).
• 20) Constellation Pharmaceuticals, WO2021/021893 (2021).
• 21) Mitsubishi Tanabe Pharma, WO2012/124825 (2012).
• 22) Anbarasan P., Neumann H., Beller M., Chem. Eur. J., 17, 4217–4222 (2011).
• 23) Olsen J., Seiler P., Wagner B., Fischer H., Tschopp T., Obst-Sander U., Banner D. W., Kansy M., Müller K., Diederich F., Org. Biomol. Chem., 2, 1339–1352 (2004).
• 24) Moya-Garzón M. D., Martín Higueras C., Peñalver P., Romera M., Fernandes M. X., Franco-Montalbán F., Gómez-Vidal J. A., Salido E., Díaz-Gavilán M., J. Med. Chem., 61, 7144–7167 (2018).
• 25) Merck Sharp Dohme, WO2018/068297 (2018).
• 26) Adolfsson D. E., Tyagi M., Singh P., Deuschmann A., Ådén J., Gharibyan A. L., Jayaweera S. W., Lindgren A. E. G., Olofsson A., Almqvist F., J. Org. Chem., 85, 14174–14189 (2020).
• 27) Elhalem E., Bailey B. N., Docampo R., Ujváry I., Szajnman S. H., Rodriguez J. B., J. Med. Chem., 45, 3984–3999 (2002).
• 28) Hydra Biosciences, WO2014/143799 (2014).
• 29) Abbvie, US10213433 (2019).
• 30) Daiichi Sankyo Company Limited, EP2380878 (2011).
• 31) Dong Y., Wang X., Kamaraj S., et al., J. Med. Chem., 60, 2654–2668 (2017).
• 32) AstraZeneca, WO2007/142584 (2007).
• 33) Incyte, WO2022/006457 (2022).
• 34) Kang J., More K. N., Pyo A., Jung Y., Kim D. Y., Chang D. J., Bioorg. Med. Chem. Lett., 36, 127789 (2021).
• 35) FUJIFILM, US2015/322063 (2015).
• 36) Nango N., Kubota S., Horiguchi Y., Osteoporotic Fracture and Systemic Skeletal Disorders, 15, 129–160 (2021).
• 37) Ferretti J. L., Capozza R. F., Zanchetta J. R., Bone, 18, 97–102 (1996).