Synthesis and Structure–Activity Relationships of Novel Benzofuran Derivatives with Osteoblast Differentiation-Promoting Activity

Abstract

Osteoporosis is caused by an imbalance between bone resorption and formation, which decreases bone mass and strength and increases the risk of fracture. Therefore, osteoporosis is treated with oral resorption inhibitors, such as bisphosphonates, and parenteral osteogenic drugs, including parathyroid hormone and antisclerostin antibodies. However, orally active osteogenic drugs have not yet been developed. In the present study, to find novel candidates for oral osteogenic drugs, various benzofuran derivatives were synthesized and their effects on osteoblast differentiation were examined in mouse mesenchymal stem cells (ST2 cells). Among the compounds tested, 3-{4-[2-(2-isopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23d) exhibited potent osteoblast differentiation-promoting activity, estimated as EC₂₀₀ for increasing alkaline phosphatase activity, and good oral absorption in female rats, resulting in high C_max/EC₂₀₀. Dual-energy X-ray absorptiometry scanning revealed that 23d at 10 mg/kg/d for 8 weeks increased femoral bone mineral density in ovariectomized rats with an elevation in plasma bone-type alkaline phosphatase activity, and micro-computed tomography showed that it increased bone volume, mineral contents, and strength in femoral diaphysis cortical, but not trabecular bone during the experiment period. 23d potently inhibited cyclin-dependent kinase 8 (CDK8) activity, suggesting that its osteoblastogenic activity is mediated by the suppression of CDK8, as previously reported for diphenylether derivatives. In conclusion, the structure–activity relationships of novel benzofuran derivatives were clarified and 3,5-disubstituted benzofuran was identified as a useful scaffold for orally active osteogenic compounds. Compound 23d exhibited potent osteoblastogenic activity through CDK8 inhibition and osteogenic effects in ovariectomized rats, indicating its potential as an orally active anti-osteoporotic drug.

Introduction

Osteoporosis is defined as a disease characterized by low bone mass and microarchitectural deterioration of bone tissue, leading to enhanced bone fragility and a consequent increase in fracture risk. The number of patients with osteoporosis continues to increase, affecting an estimated 200 million men and women worldwide.¹⁾

Bone structure and function are maintained through bone remodeling by osteoresorption and osteogenesis. Bone resorption exceeds bone formation in osteoporosis, resulting in decreases in bone mass and strength and increases in the risk of fracture. Bone resorption inhibitors, such as oral bisphosphonates, and bone formation stimulants, including parenteral parathyroid hormone (PTH) and anti-sclerostin antibodies, are used to treat osteoporosis. However, the long-term use of these potent bone resorption inhibitors has been associated with side effects, such as osteonecrosis of the jaw and an increased risk of atypical femur fractures.²⁾ The bone formation stimulants PTH and anti-sclerostin antibodies exhibit potent osteogenic activity; however, associated side effects include osteosarcoma and cardiovascular events.^3,4) Therefore, the development of oral osteogenic agents that are inexpensive and easily administered is desired, and a large number of small molecules have been reported to exhibit osteoblastogenic activity.

Natural derivatives, such as coumarins and flavonoids, exhibit osteoblastogenic activity.⁵⁾ Previous studies showed that osthole, fraxetin, psoralen, and sulfuretin promoted osteoblast differentiation by enhancing the bone morphogenetic protein (BMP) pathway.^6–9) Various synthesized compounds are also known to exhibit osteoblastogenic activity. Benzothiepine, benzofuran, thienopyridine, and diphenylether derivatives enhanced osteoblast differentiation by activating the estrogenic pathway, enhancing BMP signaling, activating Wnt/β-catenin, and/or inhibiting cyclin-dependent kinase 8 (CDK8).^10–14) However, none of these compounds have been successfully developed. We recently reported that diphenylether and coumarin derivatives (KY-065, KY-273, and KY-054, Fig. 1) exhibited potent blastogenic activity and exerted cortical bone-selective osteogenic effects on femoral bone in ovariectomized (OVX) rats.^14–16) These derivatives have similar substituents with a tetrahydropyranyl group. In the present study, we synthesized a series of benzofuran derivatives with various phenoxy groups, including a tetrahydropyranyl group at the 3-position (Fig. 2), and examined their promotion of osteoblast differentiation using alkaline phosphatase (ALP) activity, an osteoblast marker enzyme, in mouse mesenchymal stem cells (ST2 cells). Structure–activity relationships were clarified and 3-{4-[2-(2-isopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23d) was found to exhibit potent osteoblastogenic activity with excellent oral absorption in female rats. Compound 23d (10 mg/kg/d) was orally administered to OVX rats for 8 weeks, and changes in plasma bone-type ALP activity and femoral areal bone mineral density (aBMD) were assessed using dual-energy X-ray absorptiometry scanning (DEXA), while those in bone volume (BV) and mineral contents (BMC) were evaluated using micro-computed tomography (micro-CT).

Fig. 1. Chemical Structures of Diphenylether Derivatives and Coumarin Derivatives

Fig. 2. Chemical Structures of 3-Phenybenzufuran Derivatives

Chemistry

General approaches to the synthesis of benzofuran derivatives are outlined in Charts 1–3.

Chart 1 shows the synthesis of various 5-substituted benzofuran derivatives. Using commercially available 1a or previously reported 1d–f^17–19) as starting materials, 2a and 2d–f were synthesized by 1,2-dibromination and aromatization of the benzofuran ring. In the synthesis of 1f to 2f, an ester moiety was changed from a methyl ester to an ethyl ester in EtOH (1f: R¹ = -CO₂Me, 2f: R¹ = -CO₂Et). Bromobenzofuran 2b²⁰⁾ and 2c²¹⁾ were synthesized as previously described. Compounds 4a–f were prepared by the Suzuki coupling of each 2a–f and known pinacol boronate 3.²²⁾ Compound 4d was hydrolyzed to 5 with H₂O₂ and reduced to 6 with lithium aluminum hydride (LiAlH₄). Ester intermediate 2f was converted to 2g by hydrolysis and the Curtius reaction, which was then subjected to the Suzuki coupling reaction with 3. Compound 7 was obtained by further tert-butoxycarbonyl (Boc)-deprotection and reductive amination. Compound 8 was synthesized by the hydrolysis of compound 4f.

Chart 1. Synthesis of 5-Substituted Benzofuran Derivatives

Reagents and conditions; (i) Br₂, CH₂Cl₂ or Br₂, CH₂Cl₂-NaHCO₃ aq.; (ii) KOH, MeOH-THF or K₂CO₃, EtOH; (iii) 3, PdCl₂(dppf)·CH₂Cl₂, K₃PO₄, n-Bu₄NBr, MeCN; (iv) H₂O₂ aq., K₂CO₃, DMSO; (v) LiAlH₄, THF; (vi) HCl in IPA, AcOEt-MeOH; (vii) NaOH aq., MeOH-THF; (viii) DPPA, Et₃N, t-BuOH; (ix) HCl in IPA, HCO₂H; (x) formalin, NaBH(OAc)₃, MeCN-MeOH; (xi) NaOH aq., EtOH.

Chart 2 shows the synthesis of various 2-substituted benzofuran derivatives. 2-Methyl benzofuran 11 was synthesized by a coupling reaction with 9²³⁾ and 3 followed by hydrolysis. The bromination of methyl group 9 followed by oxidation gave carboaldehyde 12 and subsequent coupling and hydrolysis gave 14. Compound 14 was treated with Selectfluor^® to give 2-fluorobenzofuran 15. Compound 4d was converted by chlorination and the hydrolysis of nitrile to 2-chlorobenzofuran 17. An acetyl group was introduced to 2d by the Friedel-Crafts acylation to 18 and this was followed by conversion of the 3- and 5-positions to the corresponding groups with the general methods described above to 2-acetylbenzofuran derivative 20.

Chart 2. Synthesis of 2-Substituted Benzofuran Derivatives

Reagents and conditions; (i) 3, PdCl₂(dppf)·CH₂Cl₂, K₃PO₄, n-Bu₄NBr, MeCN; (ii) H₂O₂ aq., K₂CO₃, DMSO; (iii) NBS, AIBN, CCl₄; (iv) Ag₂CO₃, acetone-H₂O (v) H₂O₂ aq., NaClO₂, KH₂PO₄, DMSO-H₂O-MeCN; (vi) Selectfluor^®, CCl₄-sat. NaHCO₃ aq.; (vii) sulfuryl chloride, benzene; (viii) AcCl, AlCl₃, CHCl₃.

Chart 3 shows the synthesis of compounds with the converted benzofuran 3-position. Similar to Chart 1, the key reaction was the Suzuki coupling of 2d and pinacol boronates 21a–g, followed by hydrolysis of the nitrile group, which led to the preparation of 23a–g. The pinacol boronates 21a–g used in Suzuki coupling were either alkylated from commercial 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenol 24 in Method A or introduced into pinacol boronates after the alkylation of iodo phenol 26 in Method B. Compounds 25a and 25f were commercially available, and 25b,²⁴⁾ 27d,²⁵⁾ and 27g²⁵⁾ were synthesized as previously reported. Compounds 25c and 25e were prepared by the alkylation of 29c²⁶⁾ and 29e²⁷⁾ with 30 and this was followed by deprotection with tetrahydropyran (THP). Compound 23h was obtained via the THP-deprotection of 23g.

Chart 3. Synthesis of 3-Substituted Benzofuran Derivatives

Reagents and conditions; (i) PdCl₂(dppf)·CH₂Cl₂, K₃PO₄, n-Bu₄NBr, MeCN; (ii) H₂O₂ aq., K₂CO₃, DMSO; (iii) DIAD, PPh₃, CH₂Cl₂; (iv) K₂CO₃, KI, N,N-dimethylformamide (DMF); (v) H-Bpin, PdCl₂(dppf)·CH₂Cl₂, Et₃N, 1,4-dioxane; (vi) NaH, THF or DMF; (vii) HCl in IPA, MeOH; (viii) HCl aq., THF.

Results and Discussion

We synthesized a series of compounds to find novel osteoblastogenic compounds, and showed that coumarin and diphenylether derivatives, including KY-065, KY-273, and KY-054, exhibited potent osteoblastic activities in ST2 cells, which were assessed using ALP activity, an osteoblast marker enzyme.^14–16) They increased plasma bone-type ALP activity as well as femur cortical, but not trabecular, bone volume and mineral density in OVX rats, indicating osteogenic effects in vivo. On the other hand, various natural and synthetic derivatives, including benzofuran derivatives, are known to exhibit osteoblastogenic activity.¹²⁾ However, their osteogenic effects on cortical bone have not yet been elucidated. In the present study, we synthesized different types of benzofuran derivatives and examined their osteoblastogenic activities in ST2 cells, plasma concentrations after their oral administration at 10 mg/kg in female rats, and their effects on plasma bone-type ALP activity, the aBMD of femoral bone using DEXA, and BV, BMC, and bone strength parameters of femur diaphysis cortical bone and metaphysis trabecular bone using micro-CT. All animal experiments were conducted according to the guidelines for animal experiments of our company and the guidelines for animal experimentation approved by the Japanese Association of Laboratory Animal Science.

KY-065 and KY-273 have a tetrahydropyranyl group as a substituent (Fig. 1). We initially introduced this substituent to the 3-position of the benzofuran ring and various substituents, including the carbamoyl group employed in KY-065 and KY-273, to the 5-position (Table 1). The 5-unsubstituted derivative (4a) had no activity. The introduction of a halogen and nitrile group, an electron-withdrawing group, resulted in moderate osteoblastogenic activity (4b, 4c, 4d). However, the introduction of a trifluoromethyl group, a strong electron-withdrawing group, reduced this activity (4e). Amino and carboxyl groups all markedly reduced osteoblastogenic activity (6, 7, 8). Ionizable groups may not be suitable for interactions with target proteins. The introduction of a carbamoyl group (5) markedly increased osteoblastogenic activity over that with unsubstituted derivative 4a. Interestingly, the osteoblastogenic activity of 4a was approximately 5-fold weaker than that of KY-065 with the same substituent,¹⁴⁾ which may have been due to the different direction of the substituent fixed via the ring structure and the lack of an acetyl group. However, the plasma concentration of 4a was approximately 5-fold higher than that of KY-065 after their oral administration: benzofuran may be metabolically stable, resulting in high C_max/EC₂₀₀ of 107.5, which was similar to that of KY-065 (101.8).

Table 1. Chemical Structures and Promoting Activities on Osteoblast Differentiation (EC₂₀₀) of 5-Substituted-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)­oxy]ethoxy}phenyl)benzofuran Derivatives

a) n = 2.

We then introduced various substituents into the 2-position (Table 2). The osteoblastogenic activity of derivative 11 with 2-methyl was approximately 2-fold weaker than that of 5, while the activity of 17 with 5-chloro was approximately 1.8-fold stronger, suggesting that a bulkier group than hydrogen interfered with the interaction with a target protein; however, the electron-withdrawing group, the Cl atom, which is roughly the same size as methyl, enhanced activity (17). The C_max of 17 was markedly lower than that of 5, resulting in lower C_max/EC₂₀₀ (57.8). On the other hand, the introduction of fluorine, which is as small as hydrogen, but possesses a more potent electron-withdrawing property than Cl, markedly reduced osteoblastogenic activity. The acetyl group (20) and carboxylic acid (14) markedly reduced activity. Therefore, an electron-rich group may disturb the interaction with a target protein.

Table 2. Chemical Structures, Promoting Activities on Osteoblast Differentiation (EC₂₀₀), Maximal Plasma Concentrations (C_max) after Oral Administration at 10 mg/kg in Female Rats, and Ratios of C_max and EC₂₀₀ of 2-Substituted-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide Derivatives

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

The side chain with a tetrahydropyranyl group was changed to various groups (Table 3). In comparison with 5, the introduction of a hydroxylethyl group (23h), isopropyl group (23a), cyclopropyl group (23b), 1,4-dioxane derivative (23c), isopropyloxyethyl (23d), cyclopropyloxyethyl (23e), and ethoxyethyl (23f) reduced osteoblastogenic activity by 1.8- to 7.9-fold. The C_max of compounds 23a, 23c, 23d, 23e, and 23f were 1.5- to 3.6-fold higher than that of 5. The C_max/EC₂₀₀ of 23a was markedly lower than that of 5, while those of 23c, 23e, and 23f were similar to that of 5. Interestingly, compound 23d had a higher C_max/EC₂₀₀ than that of 5 and KY-065, indicating in vivo osteoblastogenic and osteogenic activities. Therefore, the effects of oral administration of 23d for 8 weeks (10 mg/kg/d) on plasma bone-type ALP activity, the aBMD of isolated femoral bone in DEXA scanning, and femur cortical and trabecular bone BV and BMC in in vivo micro-CT scanning were examined in OVX rats (Table 4). Ovariectomy significantly decreased uterine weights and increased body weights, whereas 23d had no effects, suggesting the lack of estrogenic effects. Compound 23d significantly increased plasma bone-type ALP activity, indicating in vivo osteoblastogenic and osteogenic effects. DEXA scanning showed that 23d increased aBMD not only in the femur distal portion, which was reduced by ovariectomy, but also in the midportion, which was not affected by ovariectomy. Changes in bone parameters during the experimental period were assessed using micro-CT scanning. Ovariectomy had no effects on changes in BV, BMC, or strength parameters, whereas 23d significantly enhanced them in diaphyseal cortical bone, which suggests that it promoted cortical bone growth. On the other hand, ovariectomy markedly reduced BV and BMC in metaphyseal trabecular bone, whereas 23d had no effect. Bisphosphonates, which are bone resorption inhibitors, have been shown to ameliorate trabecular bone loss after ovariectomy with negligible effects on cortical bone.¹⁴⁾ Therefore, the effects of 23d were considered to occur via osteogenesis rather than the inhibition of resorption, as previously reported for KY-065 and KY-273.^14,16) Furthermore, osteogenic drugs, such as PTH, increase cortical and trabecular bone.¹⁴⁾ Similar to KY-065 and KY-273, 23d may be a unique osteogenic compound with cortical bone-selective effects. Regarding the underlying molecular mechanisms, 23d inhibited CDK8 activity (IC₅₀ = 56.3 nM), suggesting osteoblastogenic activity via the suppression of CDK8.

Table 3. Chemical Structures, Promoting Activities on Osteoblast Differentiation (EC₂₀₀), Maximal Plasma Concentrations (C_max) after Oral Administration at 10 mg/kg in Female Rats, Ratios of C_max and EC₂₀₀ of 3-{4-[2-(Substituted oxy)­ethoxy]phenyl}benzofuran-5-carboxamide Derivatives

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

Table 4. Effects of the Repeated Oral Administration of 23d (10 mg/kg/d) for 8 Weeks on Plasma Bone-Type Alkaline Phosphatase (B-ALP) Activity, Areal Bone Mineral Density (aBMD) Assessed by DEXA Scanning, and Changes in the Femur Bone Structure Using in Vivo Micro-CT Scanning in Ovariectomized (OVX) Rats

	Intact	OVX
		Control	23d 10 mg/kg/d
B-ALP (IU/L)	132.3 ± 6.2	127.6 ± 4.6	157.4 ± 5.0**
DEXA aBMD (mg/cm2)
Distal portion	135.2 ± 0.9**	122.1 ± 1.1	126.1 ± 0.7*
Midportion	109.8 ± 0.6	108.9 ± 0.6	112.0 ± 0.7**
Micro-CT diaphysis cortical bone
ΔCt.BV (mm3)	2.93 ± 0.08	2.87 ± 0.17	3.57 ± 0.17**
ΔBMC (mg)	7.32 ± 0.25†	6.46 ± 0.32	7.26 ± 0.21†
ΔIaSSI (mm3)	0.31 ± 0.01	0.30 ± 0.02	0.40 ± 0.02**
Micro-CT metaphysis trabecular bone
ΔBV (cm3)	1.47 ± 0.17**	−0.42 ± 0.20	−0.03 ± 0.24
ΔBMC (mg)	0.74 ± 0.07**	−0.16 ± 0.08	0.00 ± 0.09

Mean ± standard error of the mean (S.E.M.). ^†p < 0.1, * p < 0.05, ** p < 0.01 v.s. OVX control, Dunnett’s multiple comparison test. n = 8.

In summary, we synthesized a series of benzofuran derivatives with various substituents, including the tetrahydropyranyl group in KY-065 and KY-273, which are diphenylether derivatives, and structure–activity relationships for osteoblast differentiation-promoting activity were clarified. Compound 23d exhibited lower osteoblastogenic activity and higher oral absorbability than KY-065 with the same substituents. The 3-phenoxy benzofuran skeleton was shown to be a good scaffold for orally active osteogenic compounds with excellent pharmacokinetic properties. Therefore, compound 23d has potential as an oral anti-osteoporosis agent with cortical bone-selective osteogenic effects via a new mechanism.

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 electrospray ionization (ESI)-MS spectrometer (Expression CMS-L, Advion, Ithaca, U.S.A.) and ESI-TOF/MS (micrOTOF2-kp, Bruker, Massachusetts, U.S.A.). Column chromatography was performed on silica gel (Daisogel No. 1001W; Osaka Soda Co., Ltd., Osaka, Japan). Reactions were monitored by TLC (TLC silica gel 60F254, Merck KGaA, Darmstadt, Germany). The purities of final compounds were determined by HPLC (pump, LC-8A and LC-20AT; detector, SPD-10A and SPD-20A; Shimadzu Corporation, Kyoto, Japan) using COSMOSIL 5C₁₈-AR-II column (5 µm, 4.6 × 150 mm; Nacalai Tesque, Kyoto, Japan).

2,3-Dibromo-2,3-dihydrobenzofuran (2a′)

Following the addition of Br₂ (0.87 mL, 17 mmol) to a solution of benzofuran 1a (1.00 g, 8.47 mmol) in CH₂Cl₂ (25 mL), the reaction mixture was stirred at room temperature for 30 min. The mixture was washed with 10% aqueous Na₂S₂O₃ and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give 2,3-dibromo-2,3-dihydrobenzofuran 2a′ (2.28 g, 97% yield) as a solid. ¹H-NMR (CDCl₃) δ: 5.75 (1H, s), 6.92 (1H, s), 7.02–7.08 (1H, m), 7.12–7.19 (1H, m), 7.33–7.40 (1H, m), 7.48–7.54 (1H, m).

3-Bromobenzofuran (2a)

Following the addition of 2.0 M KOH in MeOH (4.1 mL, 8.2 mmol) to a solution of 2,3-dibromo-2,3-dihydrobenzofuran 2a′ (2.28 g, 8.20 mmol) in tetrahydrofuran (THF) (30 mL), the reaction mixture was stirred at room temperature for 30 min. After the addition of water and CHCl₃, the organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure and purified by silica gel column chromatography to give 2a (1.40 g, 87% yield) as an oil. ¹H-NMR (CDCl₃) δ: 7.29–7.40 (2H, m), 7.47–7.52 (1H, m), 7.53–7.58 (1H, m), 7.65 (1H, s).

3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran (4a)

Following the addition of K₃PO₄ (585 mg, 2.76 mmol), n-Bu₄NBr (TBAB) (30 mg, 0.093 mmol), and [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride CH₂Cl₂ complex [PdCl₂(dppf)·CH₂Cl₂] (75 mg, 0.092 mmol) to a solution of 2a (200 mg, 1.02 mmol) and 3 (320 mg, 0.919 mmol) in MeCN (6 mL), the mixture was heated under reflux for 2 h under a N₂ atmosphere. After cooling, AcOEt was added, the mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography and n-hexane was added. The insoluble material was collected by filtration to give 4a (63 mg, 20% yield) as a solid. mp 72–75 °C; ¹H-NMR (CDCl₃) δ: 1.60–1.72 (2H, m), 1.89–2.01 (2H, m), 3.41–3.51 (2H, m), 3.57–3.67 (1H, m), 3.87 (2H, t, J = 5.1 Hz), 3.93–4.02 (2H, m), 4.19 (2H, t, J = 5.1 Hz), 6.99–7.07 (2H, m), 7.26–7.37 (2H, m), 7.52–7.60 (3H, m), 7.73 (1H, s), 7.77–7.83 (1H, m); ¹³C-NMR (dimethyl sulfoxide-d₆ (DMSO-d₆)) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.4 (s), 73.6 (s), 111.6 (s), 115.0 (2C, s), 120.2 (s), 120.7 (s), 123.1 (s), 123.7 (s), 124.5 (s), 125.7 (s), 128.1 (2C, s), 141.7 (s), 155.0 (s), 157.9 (s); IR (attenuated total reflectance (ATR)) cm⁻¹: 1117; high resolution (HR)-MS (ESI-TOF) Calcd for C₂₁H₂₂NaO₄ [M + Na]⁺, 361.1416. Found 361.1399; HPLC purity 90.8% (eluent: 0.01 M KH₂PO-MeCN-AcOH (30 : 70 : 0.2)).

5-Fluoro-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran (4b)

Compound 4b was prepared from 3-bromo-5-fluorobenzofuran 2b and 3 according to the procedure for the synthesis of 4a. Yield was 6.5%. mp 83–86 °C; ¹H-NMR (CDCl₃) δ: 1.60–1.71 (2H, m), 1.90–2.00 (2H, m), 3.42–3.50 (2H, m), 3.57–3.66 (1H, m), 3.83–3.90 (2H, m), 3.93–4.01 (2H, m), 4.15–4.21 (2H, m), 7.00–7.09 (3H, m), 7.41–7.47 (2H, m), 7.48–7.54 (2H, m), 7.75 (1H, s); IR (ATR) cm⁻¹: 1579; HR-MS (ESI-TOF) Calcd for C₂₁H₂₁FNaO₄ [M + Na]⁺, 379.1322. Found 379.1311; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (30 : 70 : 0.2)).

5-Chloro-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy)ethoxy)phenyl)benzofuran (4c)

Compound 4c was prepared from 3-bromo-5-chlorobenzofuran 2c and 3 according to the procedure for the synthesis of 4a. Yield was 20% as an oil. ¹H-NMR (DMSO-d₆) δ: 1.37–1.48 (2H, m), 1.84–1.92 (2H, m), 3.31–3.38 (2H, m), 3.54–3.61 (1H, m), 3.77–3.84 (4H, m), 4.13–4.17 (2H, m), 7.05–7.10 (2H, m), 7.62–7.66 (2H, m), 7.41 (1H, dd, J = 8.8, 2.0 Hz), 7.69 (1H, d, J = 8.8 Hz), 7.87 (1H, d, J = 2.0 Hz), 8.36 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.5 (s), 73.6 (s), 113.3 (s), 115.1 (2C, s), 119.6 (s), 120.5 (s), 122.9 (s), 124.5 (s), 127.3 (s), 127.7 (s), 128.2 (2C, s), 143.5 (s), 153.6 (s), 158.1 (s); IR (ATR) cm⁻¹; 1506; HR-MS (ESI-TOF) Calcd for C₂₁H₂₁ClNaO₄ [M + Na]⁺, 395.1026. Found 395.1026; HPLC purity 99.4% (eluent: 0.01 M KH₂PO₄-MeCN (20 : 80)).

3-Bromobenzofuran-5-carbonitrile (2d)

Following the addition of Br₂ (0.36 mL, 7.0 mmol) to a solution of 5-cyanobenzofuran 1d (500 mg, 3.49 mmol) in CH₂Cl₂ (10 mL), the reaction mixture was stirred at room temperature for 20 min. The mixture was washed with 1.0 M aqueous Na₂S₂O₃ and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was dissolved in THF (10 mL), and 2.0 M KOH in MeOH (1.75 mL, 3.5 mmol) was added at room temperature and stirred for 30 min. After the addition of water and CHCl₃, the organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure and i-Pr₂O was added. The insoluble material was collected by filtration to give 2d (596 mg, 77% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.55–7.65 (2H, m), 7.78 (1H, s), 7.91 (1H, d, J = 1.4 Hz).

3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carbonitrile (4d)

Compound 4d was prepared from 2d according to the procedure for the synthesis of 4a. Yield was 77% as an oil. ¹H-NMR (CDCl₃) δ: 1.62–1.72 (2H, m), 1.92–2.02 (2H, m), 3.43–3.52 (2H, m), 3.59–3.67 (1H, m), 3.85–3.91 (2H, m), 3.92–4.02 (2H, m), 4.15–4.24 (2H, m), 7.04–7.07 (2H, m), 7.49–7.57 (2H, m), 7.60–7.64 (2H, m), 7.82 (1H, s), 8.12 (1H, s); ¹³C-NMR (CDCl₃) δ: 32.4 (2C, s), 65.7 (2C, s), 66.2 (s), 67.9 (s), 74.7 (s), 106.9 (s), 113.0 (s), 115.5 (2C, s), 119.5 (s), 122.0 (s), 122.9 (s), 125.7 (s), 127.6 (s), 128.1 (s), 128.7 (2C, s), 142.6 (s), 157.3 (s), 158.9 (s); IR (ATR) cm⁻¹; 2225; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₄ [M + Na]⁺, 386.1368. Found 386.1350; HPLC purity 98.7% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (30 : 70 : 0.2)).

3-Bromo-5-(trifluoromethyl)benzofuran (2e)

Compound 2e was prepared from 1e according to the procedure for the synthesis of 2d. Yield was 85%. ¹H-NMR (CDCl₃) δ: 7.57–7.65 (2H, m), 7.76 (1H, s), 7.86 (1H, s).

3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)-5-(trifluoromethyl)benzofuran (4e)

Compound 4e was prepared from 2d according to the procedure for the synthesis of 4a. Yield was 23% as an oil. ¹H-NMR (CDCl₃) δ: 1.61–1.71 (2H, m), 1.90–2.00 (2H, m), 3.42–3.50 (2H, m), 3.58–3.66 (1H, m), 3.88 (2H, t, J = 4.9 Hz), 3.93–4.00 (2H, m), 4.20 (2H, t, J = 4.9 Hz), 7.03–7.09 (2H, m), 7.50–7.56 (2H, m), 7.57–7.64 (2H, m), 7.71 (1H, s), 8.06 (1H, s); ¹³C-NMR (CDCl₃) δ: 32.4 (2C, s), 65.7 (2C, s), 66.2 (s), 67.9 (s), 74.7 (s), 112.2 (s), 115.4 (2C, s), 118.1 (q, J = 3.9 Hz), 121.7 (q, J = 3.9 Hz), 122.3 (s), 124.7 (q, J = 272.1 Hz), 123.5 (s), 125.7 (q, J = 32.3 Hz), 126.9 (s), 128.7 (2C, s), 142.3 (s), 157.0 (s), 158.8 (s); IR (ATR) cm⁻¹; 1508; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁F₃NaO₄ [M + Na]⁺, 429.1290. Found 429.1281; HPLC purity 96.1% (eluent: 0.01 M KH₂PO-MeCN (30 : 70)).

3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide (5)

Following the addition of K₂CO₃ (68 mg, 0.50 mmol) and 30% aqueous H₂O₂ (132 mg, 1.2 mmol) in DMSO (1 mL) to a solution of 4d (300 mg, 0.826 mmol) in DMSO (2 mL), the reaction mixture was stirred at room temperature for 4 h. After water was added to the mixture, the organic phase was separated. The aqueous phase was extracted with AcOEt, and the combined organic phase 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 solvent was removed under reduced pressure and i-Pr₂O was added. The insoluble material was collected by filtration to give 5 (190 mg, 60% yield) as a solid. mp 144–145 °C; ¹H-NMR (DMSO-d₆) δ: 1.35–1.50 (2H, m), 1.82–1.92 (2H, m), 3.30–3.42 (2H, m), 3.52–3.63 (1H, m), 3.75–3.88 (4H, m), 4.12–4.20 (2H, m), 7.09–7.11 (2H, m), 7.32–7.39 (1H, br), 7.68–7.72 (3H, m), 7.91 (1H, d, J = 8.6 Hz), 8.10–8.18 (1H, br), 8.35 (1H, s), 8.40 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.5 (s), 73.6 (s), 111.3 (s), 115.1 (2C, s), 120.0 (s), 121.1 (s), 123.2 (s), 124.5 (s), 125.5 (s), 128.3 (2C, s), 129.6 (s), 142.8 (s), 156.6 (s), 158.0 (s), 167.8 (s); IR (ATR) cm⁻¹; 3363, 1643; HR-MS (ESI-TOF) Calcd for C₂₂H₂₃NNaO₅ [M + Na]⁺, 404.1474. Found 404.1463; HPLC purity 97.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

[3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-yl]methanamine Hydrochloride (6)

Following the addition of LiAlH₄ (78 mg, 2.1 mmol) to a solution of 4d (150 mg, 0.413 mmol) in THF (5 mL), the mixture was stirred at room temperature for 3 h. After the addition of water (2 drops), 15% aqueous NaOH solution (6 drops), and water (6 drops) sequentially, the mixture was stirred at room temperature for 30 min. After filtration of the insoluble material, the filtrate was evaporated under reduced pressure and purified by silica gel column chromatography. The residue obtained was dissolved in MeOH (2 mL), and 6.28 M HCl in i-PrOH (0.10 mL, 0.63 mmol) was added. After the addition of AcOEt (20 mL), the insoluble material was collected by filtration to give 6 (100 mg, 60% yield) as a solid. mp 264–266 °C; ¹H-NMR (DMSO-d₆) δ: 1.37–1.48 (2H, m), 1.85–1.93 (2H, m), 3.31–3.39 (2H, m), 3.54–3.62 (1H, m), 3.75–3.85 (4H, m), 4.12–4.20 (4H, m), 7.06–7.12 (2H, m), 7.47 (1H, dd, J = 8.5, 1.7 Hz), 7.66–7.72 (3H, m), 8.08 (1H, d, J = 1.7 Hz), 8.25–8.35 (4H, br); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 42.2 (s), 64.8 (2C, s), 65.5 (s), 67.5 (s), 73.6 (s), 111.5 (s), 115.0 (2C, s), 120.7 (s), 121.5 (s), 123.4 (s), 125.8 (s), 125.9 (s), 128.2 (2C, s), 129.0 (s), 142.6 (s), 154.9 (s), 158.0 (s); IR (ATR) cm⁻¹; 2848; HR-MS (ESI-TOF) Calcd for C₂₂H₂₅NNaO₄ [M + Na]⁺, 390.1681. Found 390.1674; HPLC purity 99.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (65 : 35 : 0.2)).

3-Bromobenzofuran-5-carboxylic Acid (2f′)

Following the addition of 5.0 M aqueous NaOH solution (3.45 mL, 17 mmol) to a solution of ethyl 3-bromobenzofuran-5-carboxylate 2f (880 mg, 3.27 mmol) in THF (10 mL) and MeOH (3 mL), the mixture was stirred at room temperature for 48 h. The reaction mixture was acidified with 5% aqueous citric acid and extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 2f′ (750 mg, 95% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.78 (1H, d, J = 8.8 Hz), 8.02 (1H, dd, J = 8.8, 1.5 Hz), 8.10 (1H, d, J = 1.5 Hz), 8.43 (1H, s).

tert-Butyl (3-Bromobenzofuran-5-yl)carbamate (2g)

Following the addition of 2f′ (600 mg, 2.49 mmol) and Et₃N (0.694 mL, 4.98 mmol) to a solution of diphenylphosphoryl azide (DPPA) (1.03 g, 3.73 mmol) in t-BuOH (10 mL), the reaction mixture was heated under reflux for 6 h. After cooling, the mixture was evaporated under reduced pressure and purified by silica gel column chromatography to give 2g (670 mg, 86% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.54 (9H, s), 6.56 (1H, s), 7.22 (1H, dd, J = 8.8, 1.7 Hz), 7.38 (1H, d, J = 8.8 Hz), 7.61 (1H, s), 7.62–7.68 (1H, m).

tert-Butyl [3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-yl]carbamate (4g)

Compound 4g was prepared from 2g according to the procedure for the synthesis of 4a. Yield was 67%. ¹H-NMR (CDCl₃) δ: 1.53 (9H, s), 1.59–1.72 (2H, m), 1.90–2.00 (2H, m), 3.42–3.50 (2H, m), 3.57–3.67 (1H, m), 3.82–3.90 (2H, m), 3.92–4.01 (2H, m), 4.15–4.20 (2H, m), 6.54 (1H, s), 6.98–7.04 (2H, m), 7.20–7.27 (1H, m), 7.42 (1H, d, J = 8.8 Hz), 7.50–7.55 (2H, m), 7.69 (1H, s), 7.80–7.84 (1H, m).

N,N-Dimethyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-amine (7)

Following the addition of 6.28 M HCl in i-PrOH (0.685 mL, 4.3 mmol) to a solution of 4g (650 mg, 1.43 mmol) in HCO₂H (3.0 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 0.5 h. After neutralization with saturated aqueous NaHCO₃, 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 obtained was dissolved in 37% formalin (540 mg, 6.70 mmol) in MeCN (10 mL). Following the addition of NaBH(OAc)₃ (1.42 g, 6.70 mmol) and MeOH (3 mL), the reaction mixture was stirred at room temperature for 0.5 h. After the addition of saturated aqueous NaHCO₃, 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 i-Pr₂O was added. The insoluble material was collected by filtration to give 7 (243 mg, 45% yield) as a solid. mp 93–94 °C; ¹H-NMR (DMSO-d₆) δ: 1.36–1.49 (2H, m), 1.83–1.92 (2H, m), 2.92 (6H, s), 3.29–3.36 (2H, m), 3.54–3.62 (1H, m), 3.75–3.85 (4H, m), 4.11–4.17 (2H, m), 6.89 (1H, dd, J = 9.0, 2.4 Hz), 7.00 (1H, d, J = 2.4 Hz), 7.04–7.10 (2H, m), 7.45 (1H, d, J = 9.0 Hz), 7.56–7.64 (2H, m), 8.10 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 41.4 (2C, s), 64.8 (2C, s), 65.5 (s), 67.4 (s), 73.6 (s), 102.2 (s), 111.5 (s), 112.1 (s), 115.1 (2C, s), 120.6 (s), 124.2 (s), 126.3 (s), 128.0 (2C, s), 141.9 (s), 147.8 (s), 148.6 (s), 157.7 (s); IR (ATR) cm⁻¹; 1612; HR-MS (ESI-TOF) Calcd for C₂₃H₂₈NO₄ [M + H]⁺, 382.2018. Found 382.2009; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN (20 : 80)).

Ethyl 3-Bromobenzofuran-5-carboxylate (2f)

Following the addition of Br₂ (1.76 mL, 34.5 mmol) and saturated aqueous NaHCO₃ (20 mL) to a solution of methyl benzofuran-5-carboxylate 1f (1.38 g, 7.83 mmol) in CH₂Cl₂ (20 mL), the reaction mixture was stirred at room temperature for 2 h. The separated organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was dissolved in EtOH (40 mL), and K₂CO₃ (4.33 g, 31.3 mmol) was added at room temperature and stirred for 43 h. After filtration of the insoluble material, the filtrate was evaporated under reduced pressure to give 2f (1.90 g, 95% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.44 (3H, t, J = 7.1 Hz), 4.42 (2H, q, J = 7.1 Hz), 7.52 (1H, d, J = 8.6 Hz), 7.71 (1H, s), 8.09 (1H, dd, J = 8.6, 2.0 Hz), 8.28 (1H, d, J = 2.0 Hz).

Ethyl 3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxylate (4f)

Compound 4f was prepared from 2a according to the procedure for the synthesis of 4a. Yield was 59%. ¹H-NMR (CDCl₃) δ: 1.42 (3H, t, J = 7.1 Hz), 1.62–1.73 (2H, m), 1.90–2.02 (2H, m), 3.43–3.55 (2H, m), 3.59–3.68 (1H, m), 3.85–3.93 (2H, m), 3.93–4.02 (2H, m), 4.17–4.28 (2H, m), 4.40 (2H, q, J = 7.1 Hz), 7.04–7.06 (2H, m), 7.54–7.58 (3H, m), 7.77 (1H, s), 8.07 (1H, dd, J = 8.8, 1.7 Hz), 8.51 (1H, d, J = 1.7 Hz).

3-(4-{2-[(Tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxylic Acid (8)

Following the addition of 5.0 M aqueous NaOH solution (0.56 mL, 2.8 mmol) to a solution of 4f (380 mg, 0.926 mmol) in EtOH (5 mL), the mixture was stirred at 40 °C for 2 h. After cooling, 5% aqueous citric acid was added to adjust to pH 2. The mixture was extracted with CHCl₃. The organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The insoluble material was collected by filtration to give 8 (244 mg, 69% yield) as a solid. mp 146–148 °C; ¹H-NMR (DMSO-d₆) δ: 1.35–1.48 (2H, m), 1.82–1.92 (2H, m), 3.30–3.40 (2H, m), 3.52–3.65 (1H, m), 3.75–3.88 (4H, m), 4.12–4.18 (2H, m), 7.11–7.14 (2H, m), 7.64–7.66 (2H, m), 7.74 (1H, d, J = 8.8 Hz), 7.98 (1H, d, J = 8.8 Hz), 8.39 (1H, s), 8.42 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.5 (s), 67.5 (s), 73.7 (s), 111.8 (s), 115.2 (2C, s), 121.2 (s), 122.0 (s), 123.0 (s), 126.0 (s), 126.0 (s), 126.1 (s), 128.3 (2C, s), 143.2 (s), 157.5 (s), 158.2 (s), 167.3 (s); IR (ATR) cm⁻¹; 1671; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁O₆ [M − H]⁻, 381.1338. Found 381.1340; HPLC purity 97.7% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (40 : 60 : 0.2)).

2-Methyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carbonitrile (10)

Compound 10 was prepared from 9 according to the procedure for the synthesis of 4a. Yield was 71%. ¹H-NMR (CDCl₃) δ: 1.60–1.70 (2H, m), 1.92–2.02 (2H, m), 2.54 (3H, s), 3.43–3.54 (2H, m), 3.60–3.70 (1H, m), 3.85–3.94 (2H, m), 3.95–4.05 (2H, m), 4.18–4.26 (2H, m), 7.05–7.07 (2H, m), 7.35–7.37 (2H, m), 7.49–7.54 (2H, m), 7.86 (1H, s).

2-Methyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide (11)

Compound 11 was prepared from 10 according to the procedure for the synthesis of 5 as a solid. Yield was 67%. mp 159–161 °C; ¹H-NMR (DMSO-d₆) δ: 1.35–1.50 (2H, m), 1.82–1.92 (2H, m), 2.54 (3H, s), 3.30–3.40 (2H, m), 3.52–3.62 (1H, m), 3.77–3.86 (4H, m), 4.15–4.20 (2H, m), 7.11–7.13 (2H, m), 7.23–7.31 (1H, br), 7.47–7.49 (2H, m), 7.48 (1H, d, J = 8.6 Hz), 7.82 (1H, d, J = 8.6 Hz), 8.00–8.05 (1H, br), 8.07 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 12.6 (s), 32.2 (2C, s), 64.8 (2C, s), 65.5 (s), 67.4 (s), 73.6 (s), 110.2 (s), 115.0 (2C, s), 116.0 (s), 118.9 (s), 123.5 (s), 127.9 (s), 129.4 (s), 129.8 (2C, s), 151.9 (s), 154.8 (s), 157.7 (s), 167.9 (s); IR (ATR) cm⁻¹; 3432, 1648; HR-MS (ESI-TOF) Calcd for C₂₃H₂₆NO₅ [M + H]⁺, 396.1811. Found 396.1810; HPLC purity 98.5% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (40 : 60 : 0.2)).

3-Bromo-2-formylbenzofuran-5-carbonitrile (12)

Following the addition of N-bromosuccinimide (NBS) (4.29 g, 24.1 mmol) and 2,2′-azobis(isobutyronitrile) (AIBN) (66 mg, 0.40 mmol) to a solution of 9 (950 mg, 4.02 mmol) in CCl₄ (30 mL), the reaction mixture was heated under reflux for 12 h. After cooling the mixture, the insoluble material was filtered off and the filtrate was concentrated under reduced pressure. Following the addition of Ag₂CO₃ (3.33 g, 12.1 mmol) and water (0.2 mL) to the solution of the residue in acetone (20 mL), the reaction mixture was stirred at room temperature for 18 h. After the addition of AcOEt and water to the mixture, the insoluble matter was filtered off and the biphasic mixture was separated. The organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 12 (721 mg, 72% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.73 (1H, dd, J = 8.8, 0.5 Hz,), 7.84 (1H, dd, J = 8.8, 1.7 Hz), 8.10 (1H, dd, J = 1.7, 0.5 Hz), 10.04 (1H, s).

2-Formyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carbonitrile (13)

Compound 13 was prepared from 12 and 3 according to the procedure for the synthesis of 4a. Yield was 83%. ¹H-NMR (CDCl₃) δ: 1.62–1.72 (2H, m), 1.91–2.00 (2H, m), 3.47 (2H, ddd, J = 12.0, 9.8, 2.7 Hz), 3.60–3.67 (1H, m), 3.88–3.92 (2H, m), 3.94–4.01 (2H, m), 4.21–4.26 (2H, m), 7.12–7.17 (2H, m), 7.52–7.57 (2H, m), 7.74 (1H, dd, J = 8.8, 0.5 Hz), 7.81 (1H, dd, J = 8.8, 1.5 Hz), 8.14 (1H, dd, J = 1.5, 0.5 Hz), 9.91 (1H, s).

5-Cyano-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-2-carboxylic Acid (14)

Following the addition of KH₂PO₄ (10 mg, 0.073 mmol) in water (0.2 mL) and NaClO₂ (40 mg, 0.35 mmol) in water (0.2 mL) to a solution of 13 (100 mg, 0.255 mmol) in DMSO (1 mL), the reaction mixture was stirred at room temperature for 14 h. MeCN (1 mL) and 30% aqueous H₂O₂ (0.04 mL, 0.4 mmol) were added to the reaction mixture and stirred for more 4 h. After the addition of water, the mixture was extracted with AcOEt. The organic layer was washed with saturated aqueous sodium bicarbonate and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give a crude residue. Compound 14 was prepared from the crude residue according to the procedure for the synthesis of 5. Yield was 24% as a solid. mp 293–296 °C; ¹H-NMR (DMSO-d₆) δ: 1.35–1.52 (2H, m), 1.82–1.97 (2H, m), 3.26–3.42 (2H, m), 3.53–3.67 (1H, m), 3.76–3.90 (4H, m), 4.14–4.25 (2H, m), 7.03–7.16 (2H, m), 7.38 (1H, s), 7.50–7.62 (2H, m), 7.78 (1H, d, J = 8.8 Hz), 8.05 (1H, d, J = 8.8 Hz), 8.12 (2H, s), 13.3–13.6 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.4 (s), 73.6 (s), 111.7 (s), 114.2 (2C, s), 121.6 (s), 121.9 (s), 127.4 (s), 127.6 (s), 127.7 (s), 130.3 (s), 131.4 (2C, s), 141.2 (s), 154.9 (s), 158.6 (s), 160.2 (s), 167.4 (s); IR (ATR) cm⁻¹; 1681, 1508; HR-MS (ESI-TOF) Calcd for C₂₃H₂₃NNaO₇ [M + Na]⁺, 448.1372. Found 448.1357; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (80 : 20 : 0.2)).

2-Fluoro-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide (15)

Following the addition of saturated aqueous NaHCO₃ (8.4 mL) and Selectfluor^® (174 mg, 0.504 mmol) to a suspension of 14 (140 mg, 0.336 mmol) in CCl₄ (2.8 mL), the mixture was stirred at room temperature for 10 h. After the addition of Selectfluor^® (174 mg, 0.504 mmol), the mixture was stirred for 14 h. The mixture was added to saturated aqueous NaHCO₃, and the resulting 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 15 (43 mg, 33% yield) as a solid. mp 124–126 °C; ¹H-NMR (DMSO-d₆) δ: 1.37–1.48 (2H, m), 1.84–1.92 (2H, m), 3.29–3.38 (2H, m), 3.54–3.62 (1H, m), 3.78–3.84 (4H, m), 4.14–4.19 (2H, m), 7.12–7.16 (2H, m), 7.39–7.45 (1H, br), 7.63–7.67 (2H, m), 7.70 (1H, d, J = 8.8 Hz), 7.91 (1H, dd, J = 8.8, 1.7 Hz), 8.12–8.18 (1H, br), 8.29 (1H, d, J = 1.7 Hz); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.5 (s), 73.6 (s), 92.9 (d, J = 8.2 Hz), 111.0 (s), 115.2 (2C, s), 119.5 (d, J = 5.3 Hz), 120.1 (d, J = 4.3 Hz), 124.1 (d, J = 3.4 Hz), 126.3 (d, J = 2.4 Hz), 128.9 (2C, d, J = 2.4 Hz), 130.6 (s), 147.9 (s), 155.7 (d, J = 281.8 Hz), 158.0 (s), 167.5 (s); IR (ATR) cm⁻¹; 3379, 3184, 1645; HR-MS (ESI-TOF) Calcd for C₂₂H₂₂FNNaO₅ [M + Na]⁺, 422.1380. Found 422.1365; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN (40 : 60)).

2-Chloro-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carbonitrile (16)

Following the addition of sulfuryl chloride (0.025 mL, 0.30 mmol) to a solution of 4d (105 mg, 0.289 mmol) in benzene (1 mL) under ice-cooling, the reaction mixture was stirred for 30 min and then at room temperature for 1.5 h. Saturated aqueous NaHCO₃ 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 16 (80 mg, 70% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.62–1.72 (2H, m), 1.92–2.02 (2H, m), 3.43–3.52 (2H, m), 3.59–3.67 (1H, m), 3.85–3.91 (2H, m), 3.92–4.02 (2H, m), 4.18–4.24 (2H, m), 7.07–7.09 (2H, m), 7.51–7.53 (2H, m), 7.55–7.61 (2H, m), 7.95 (1H, s).

2-Chloro-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide (17)

Compound 17 was prepared from 16 according to the procedure for the synthesis of 5. Yield was 66%. mp 173–175 °C; ¹H-NMR (CDCl₃) δ: 1.60–1.72 (2H, m), 1.90–2.00 (2H, m), 3.43–3.52 (2H, m), 3.58–3.67 (1H, m), 3.86–3.91 (2H, m), 3.92–4.00 (2H, m), 4.17–4.24 (2H, m), 5.45–6.25 (2H, br), 7.06–7.08 (2H, m), 7.50–7.56 (3H, m), 7.78 (1H, dd, J = 8.6, 1.7 Hz), 8.09 (1H, d, J = 1.7 Hz); ¹³C-NMR (DMSO-d₆) δ: 32.2 (2C, s), 64.8 (2C, s), 65.4 (s), 67.5 (s), 73.6 (s), 110.8 (s), 115.1 (2C, s), 115.9 (s), 119.3 (s), 120.9 (s), 124.9 (s), 126.9 (s), 129.8 (2C, s), 130.4 (s), 137.1 (s), 154.2 (s), 158.4 (s), 167.5 (s); IR (ATR) cm⁻¹; 3403, 3176, 1670; HR-MS (ESI-TOF) Calcd for C₂₂H₂₂ClNNaO₅ [M + Na]⁺, 438.1084. Found 438.1068; HPLC purity 97.0% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

2-Acetyl-3-bromobenzofuran-5-carbonitrile (18)

Following the addition of AcCl (0.19 mL, 2.7 mmol) and AlCl₃ (450 mg, 3.38 mmol) to a solution of 2d (500 mg, 2.25 mmol) in CHCl₃ (10 mL), the mixture was stirred at room temperature for 15 h. After the addition of AcCl (1.9 mL, 27 mmol) and AlCl₃ (4.50 g, 33.8 mmol), the mixture was heated under reflux for 7 h. Following the addition of water, the mixture was extracted with AcOEt. The organic layer was washed with water and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 18 (590 mg, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 2.72 (3H, s), 7.66–7.73 (1H, m), 7.77–7.86 (1H, m), 8.02–8.11 (1H, m).

2-Acetyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carbonitrile (19)

Compound 19 was prepared from 18 according to the procedure for the synthesis of 4a. Yield was 83%. ¹H-NMR (CDCl₃) δ: 1.61–1.72 (2H, m), 1.91–2.01 (2H, m), 2.53 (3H, s), 3.40–3.52 (2H, m), 3.58–3.69 (1H, m), 3.85–3.92 (2H, m), 3.93–4.02 (2H, m), 4.18–4.25 (2H, m), 7.04–7.11 (2H, m), 7.45–7.52 (2H, m), 7.65–7.72 (1H, m), 7.73–7.79 (1H, m), 7.93–7.99 (1H, m).

2-Acetyl-3-(4-{2-[(tetrahydro-2H-pyran-4-yl)oxy]ethoxy}phenyl)benzofuran-5-carboxamide (20)

Compound 20 was prepared from 19 according to the procedure for the synthesis of 5. Yield was 77%. mp 162–164 °C; ¹H-NMR (CDCl₃) δ: 1.60–1.72 (2H, m), 1.88–2.00 (2H, m), 2.50 (3H, s), 3.40–3.52 (2H, m), 3.59–3.68 (1H, m), 3.84–3.90 (2H, m), 3.90–3.98 (2H, m), 4.18–4.26 (2H, m), 5.45–6.30 (2H, br), 7.02–7.11 (2H, m), 7.45–7.54 (2H, m), 7.63 (1H, d, J = 8.7 Hz), 7.98 (1H, dd, J = 8.7, 1.7 Hz), 8.05 (1H, d, J = 1.7 Hz); ¹³C-NMR (DMSO-d₆) δ: 28.2 (s), 32.2 (2C, s), 64.8 (2C, s), 65.5 (s), 67.5 (s), 73.6 (s), 111.9 (s), 114.4 (2C, s), 121.8 (s), 122.1 (s), 126.8 (s), 127.8 (s), 128.1 (s), 130.5 (s), 131.4 (2C, s), 147.4 (s), 154.7 (s), 158.9 (s), 167.3 (s), 188.7 (s); IR (ATR) cm⁻¹; 1689, 1668, 1508; HR-MS (ESI-TOF) Calcd for C₂₄H₂₅NNaO₆ [M + Na]⁺, 446.1580. Found 446.1564; HPLC purity 99.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

2-[4-(2-Isopropoxyethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21a, Method A)

Following the addition of 25a (0.63 mL, 5.5 mmol), PPh₃ (1.43 g, 5.40 mmol), and diisopropyl azodicarboxylate (DIAD) (1.05 mL, 5.40 mmol) to a solution of 24 (1.00 g, 4.54 mmol) in CH₂Cl₂ (20 mL), the reaction mixture was stirred at room temperature for 12 h. The reaction mixture was concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give 21a (1.26 g, 91% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.19 (6H, d, J = 6.3 Hz), 1.33 (12H, s), 3.62–3.72 (1H, m), 3.78 (2H, t, J = 5.3 Hz), 4.13 (2H, t, J = 5.3 Hz), 6.89–6.91 (2H, m), 7.72–7.74 (2H, m).

3-[4-(2-Isopropoxyethoxy)phenyl]benzofuran-5-carbonitrile (22a)

Compound 22a was prepared from 21a and 2d according to the procedure for the synthesis of 4a. Yield was 44%. ¹H-NMR (DMSO-d₆) δ: 1.22 (6H, d, J = 6.1 Hz), 3.66–3.76 (1H, m), 3.82 (2H, t, J = 5.1 Hz), 4.17 (2H, t, J = 5.1 Hz), 7.04–7.07 (2H, m), 7.48–7.50 (2H, m), 7.60–7.63 (2H, m), 7.82 (1H, s), 8.11–8.13 (1H, m).

3-[4-(2-Isopropoxyethoxy)phenyl]benzofuran-5-carboxamide (23a)

Compound 23a was prepared from 22a according to the procedure for the synthesis of 5. Yield was 82%. mp 125–128 °C; ¹H-NMR (DMSO-d₆) δ: 1.20 (6H, d, J = 6.1 Hz), 3.59–3.68 (1H, m), 3.68–3.75 (2H, m), 4.10–4.16 (2H, m), 7.07–7.10 (2H, m), 7.32–7.40 (1H, br), 7.67–7.70 (3H, m), 7.90 (1H, dd, J = 8.6, 1.7 Hz), 8.10–8.17 (1H, br), 8.35–8.39 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 22.0 (2C, s), 65.9 (s), 67.6 (s), 71.1 (s), 111.4 (s), 115.1 (2C, s), 120.1 (s), 121.3 (s), 123.3 (s), 124.6 (s), 125.6 (s), 128.4 (2C, s), 129.7 (s), 142.9 (s), 156.7 (s), 158.2 (s), 168.0 (s); IR (ATR) cm⁻¹; 3361, 3178, 1644, 1606; HR-MS (ESI-TOF) Calcd for C₂₀H₂₁NNaO₄ [M + Na]⁺, 362.1368. Found 362.1358; HPLC purity 99.5% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

2-[4-(2-Cyclopropoxyethoxy)phenyl]-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21b)

Compound 21b was prepared from 24 and 25b according to Method A. Yield was 64%. ¹H-NMR (CDCl₃) δ: 0.46–0.54 (2H, m), 0.60–0.67 (2H, m), 1.33 (12H, s), 3.37–3.43 (1H, m), 3.84–3.88 (2H, m), 4.11–4.15 (2H, m), 6.88–6.93 (2H, m), 7.71–7.76 (2H, m).

3-[4-(2-Cyclopropoxyethoxy)phenyl]benzofuran-5-carbonitrile (22b)

Compound 22b was prepared from 21b and 2d according to the procedure for the synthesis of 4a. Yield was 53%. ¹H-NMR (CDCl₃) δ: 0.49–0.54 (2H, m), 0.60–0.67 (2H, m), 3.39–3.45 (1H, m), 3.88–3.92 (2H, m), 4.15–4.19 (2H, m), 7.02–7.07 (2H, m), 7.47–7.52 (2H, m), 7.61 (2H, s), 7.82 (1H, s), 8.12 (1H, s).

3-[4-(2-Cyclopropoxyethoxy)phenyl]benzofuran-5-carboxamide (23b)

Compound 23b was prepared from 22b according to the procedure for the synthesis of 5. Yield was 63%. mp 140–147 °C; ¹H-NMR (DMSO-d₆) δ: 0.42–0.55 (4H, m), 3.35–3.41 (1H, m), 3.77–3.82 (2H, m), 4.13–4.18 (2H, m), 7.07–7.12 (2H, m), 7.31–7.40 (1H, br), 7.67–7.73 (3H, m), 7.92 (1H, d, J = 8.3 Hz), 8.09–8.18 (1H, br), 8.35 (1H, s), 8.41 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 5.3 (2C, s), 52.9 (s), 67.0 (s), 68.5 (s), 111.4 (s), 115.1 (2C, s), 120.1 (s), 121.3 (s), 123.3 (s), 124.6 (s), 125.6 (s), 128.4 (2C, s), 129.7 (s), 142.9 (s), 156.7 (s), 158.1 (s), 168.0 (s); IR (ATR) cm⁻¹; 3369, 3182, 1643; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₄ [M + Na]⁺, 360.1212. Found 360.1197; HPLC purity 99.8% (eluent: 0.01 M KH₂PO₄-MeCN (40 : 60)).

2-({2-[(Tetrahydro-2H-pyran-2-yl)oxy]ethoxy}methyl)-1,4-dioxane (31c)

Following the addition of NaH (60%) (4.32 g, 0.108 mol) to a solution of 29c (11.6 g, 98.1 mmol) in THF (100 mL) under ice-cooling, the reaction mixture was stirred for 20 min. After dropping 30 (22.6 g, 0.108 mol) into THF (20 mL), the mixture was heated under reflux for 40 min. Following the addition of NaH (60%) (1.96 g, 49 mmol), the mixture was heated under reflux for 2 d. After cooling, water was added, the residue was extracted with CHCl₃, and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 31c (6.5 g, 27% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.50–1.90 (6H, m), 3.38–3.90 (15H, m), 4.60–4.65 (1H, m).

2-[(1,4-Dioxan-2-yl)methoxy]ethan-1-ol (25c)

Following the addition of 8.6 M HCl in i-PrOH (5.9 mL, 51 mmol) to a solution of 31c (4.18 g, 17.0 mmol) in MeOH (40 mL) under ice-cooling, the reaction mixture was stirred for 15 min and then at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure to give 25c (2.60 g, 95% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.90–2.10 (1H, br), 3.40–3.90 (13H, m).

2-(4-{2-[(1,4-Dioxan-2-yl)methoxy]ethoxy}phenyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21c)

Compound 21c was prepared from 24 and 25c according to Method A. Yield was 39%. ¹H-NMR (CDCl₃) δ: 1.32 (12H, s), 3.37–3.65 (4H, m), 3.66–3.89 (7H, m), 4.11–4.17 (2H, m), 6.85–6.93 (2H, m), 7.69–7.76 (2H, m).

3-(4-{2-[(1,4-Dioxan-2-yl)methoxy]ethoxy}phenyl)benzofuran-5-carbonitrile (22c)

Compound 22c was prepared from 21c and 2d according to the procedure for the synthesis of 4d. Yield was 54%. ¹H-NMR (CDCl₃) δ: 3.40–3.67 (4H, m), 3.68–3.93 (7H, m), 4.16–4.24 (2H, m), 7.01–7.09 (2H, m), 7.45–7.54 (2H, m), 7.58–7.65 (2H, m), 7.82 (1H, s), 8.12 (1H, s).

3-(4-{2-[(1,4-Dioxan-2-yl)methoxy]ethoxy}phenyl)benzofuran-5-carboxamide (23c)

Compound 23c was prepared from 22c according to the procedure for the synthesis of 5. Yield was 65%. mp 129–132 °C; ¹H-NMR (CDCl₃) δ: 3.38–3.65 (4H, m), 3.66–3.92 (7H, m), 4.16–4.23 (2H, m), 5.45–6.35 (2H, br), 6.99–7.07 (2H, m), 7.50–7.56 (2H, m), 7.56 (1H, d, J = 8.5 Hz), 7.77 (1H, s), 7.80 (1H, dd, J = 8.5, 1.7 Hz), 8.28 (1H, d, J = 1.7 Hz); ¹³C-NMR (CDCl₃) δ: 66.5 (s), 66.7 (s), 67.5 (s), 68.6 (s), 70.2 (s), 71.4 (s), 74.4 (s), 111.8 (s), 115.3 (2C, s), 120.5 (s), 122.3 (s), 123.8 (s), 124.0 (s), 127.0 (s), 128.6 (s), 128.8 (2C, s), 142.0 (s), 157.6 (s), 158.6 (s), 169.5 (s); IR (ATR) cm⁻¹;1405; HR-MS (ESI-TOF) Calcd for C₂₂H₂₃NNaO₆ [M + Na]⁺, 420.1423. Found 420.1416; HPLC purity 96.5% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (60 : 40 : 0.2)).

1-Iodo-4-[2-(2-isopropoxyethoxy)ethoxy]benzene (28d, Method B)

Following the addition of 4-iodophenol 26 (94.8 g, 0.431 mol), K₂CO₃ (89.4 g, 0.647 mol), and KI (7.15 g, 43.1 mmol) to a solution of 27d (79.0 g, 0.474 mol) in DMF (1 L), the reaction 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, 2.0 M aqueous NaOH solution, and saturated brine. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 28d (79.0 g, 52% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 3.57–3.65 (3H, m), 3.67–3.71 (2H, m), 3.83–3.87 (2H, m), 4.07–4.11 (2H, m), 6.67–6.72 (2H, m), 7.51–7.56 (2H, m).

2-{4-[2-(2-Isopropoxyethoxy)ethoxy]phenyl}-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21d)

Following the addition of 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (25.0 mL, 0.172 mol), Et₃N (85.7 mL, 0.615 mol), and PdCl₂(dppf)·CH₂Cl₂ (3.01 g, 3.69 mmol) to a solution of 28d (43.1 g, 0.123 mol) in 1,4-dioxane (430 mL), the reaction mixture was stirred at 90 °C for 4 h under a N₂ atmosphere. After cooling, AcOEt was added and the organic layer was washed with water and saturated brine. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 21d (29.2 g, 68% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 1.33 (12H, s), 3.58–3.65 (3H, m), 3.68–3.72 (2H, m), 3.85–3.89 (2H, m), 4.13–4.17 (2H, m), 6.88–6.92 (2H, m), 7.70–7.75 (2H, m).

3-{4-[2-(2-Isopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carbonitrile (22d)

Compound 22d was prepared from 21d and 2d according to the procedure for the synthesis of 4d. Yield was 77% as an oil. ¹H-NMR (CDCl₃) δ: 1.19 (6H, d, J = 6.1 Hz), 3.60–3.67 (3H, m), 3.71–3.76 (2H, m), 3.89–3.94 (2H, m), 4.18–4.23 (2H, m), 7.03–7.08 (2H, m), 7.47–7.52 (2H, m), 7.60–7.63 (2H, m), 7.82 (1H, s), 8.12–8.14 (1H, m).

3-{4-[2-(2-Isopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23d)

Compound 23d was prepared from 22d according to the procedure for the synthesis of 5. Yield was 34%. mp 108–111 °C; ¹H-NMR (DMSO-d₆) δ: 1.09 (6H, d, J = 6.1 Hz), 3.49–3.61 (5H, m), 3.76–3.81 (2H, m), 4.14–4.19 (2H, m), 7.08–7.13 (2H, m), 7.32–7.40 (1H, br), 7.68–7.74 (3H, m), 7.92 (1H, d, J = 8.8 Hz), 8.09–8.18 (1H, br), 8.35 (1H, s), 8.39–8.42 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 22.0 (2C, s), 66.8 (s), 67.2 (s), 68.9 (s), 70.3 (s), 70.9 (s), 111.4 (s), 115.1 (2C, s), 120.1 (s), 121.3 (s), 123.3 (s), 124.6 (s), 125.6 (s), 128.4 (2C, s), 129.7 (s), 142.9 (s), 156.7 (s), 158.1 (s), 168.0 (s); IR (ATR) cm⁻¹; 3359, 3182, 1645; HR-MS (ESI-TOF) Calcd for C₂₂H₂₅NNaO₅ [M + Na]⁺, 406.1630. Found 406.1626; HPLC purity 99.8% (eluent: 0.01 M KH₂PO₄-MeCN (40 : 60)).

2-[2-(2-Cyclopropoxyethoxy)ethoxy]tetrahydro-2H-pyran (31e)

Compound 31e as a crude residue was prepared from 29c and 30 according to the procedure for the synthesis of 31c.

2-(2-Cyclopropoxyethoxy)ethan-1-ol (25e)

Compound 25e was prepared from 31e according to the procedure for the synthesis of 25c. Yield was 84%. ¹H-NMR (CDCl₃) δ: 0.44–0.51 (2H, m), 0.56–0.65 (2H, m), 2.60–3.13 (1H, br), 3.31–3.37 (1H, m), 3.56–3.63 (2H, m), 3.64–3.70 (4H, m), 3.71–3.77 (2H, m).

2-{4-[2-(2-Cyclopropoxyethoxy)ethoxy]phenyl}-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21e)

Compound 21e was prepared from 24 and 25e according to Method A. Yield was 32%. ¹H-NMR (CDCl₃) δ: 0.43–0.49 (2H, m), 0.56–0.63 (2H, m), 1.32 (12H, s), 3.29–3.36 (1H, m), 3.65–3.73 (4H, m), 3.85 (2H, t, J = 4.6 Hz), 4.15 (2H, t, J = 4.6 Hz), 6.88–6.91 (2H, m), 7.71–7.34 (2H, m).

3-{4-[2-(2-Cyclopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carbonitrile (22e)

Compound 22e was prepared from 21e and 2d according to the procedure for the synthesis of 4d. Yield was quant. ¹H-NMR (CDCl₃) δ: 0.45–0.50 (2H, m), 0.58–0.64 (2H, m), 3.31–3.37 (1H, m), 3.68–3.76 (4H, m), 3.89 (2H, t, J = 4.9 Hz), 4.20 (2H, t, J = 4.9 Hz), 7.03–7.06 (2H, m), 7.48–7.51 (2H, m), 7.59–7.63 (2H, m), 7.82 (1H, s), 8.11–8.15 (1H, m).

3-{4-[2-(2-Cyclopropoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23e)

Compound 23e was prepared from 22e according to the procedure for the synthesis of 5. Yield was 51%. mp 109–111 °C; ¹H-NMR (DMSO-d₆) δ: 0.39–0.44 (2H, m), 0.45–0.49 (2H, m), 3.27–3.31 (1H, m), 3.55–3.63 (4H, m), 3.76 (2H, t, J = 4.5 Hz), 4.16 (2H, t, J = 4.5 Hz), 7.09–7.11 (2H, m), 7.34–7.40 (1H, br), 7.67–7.75 (3H, m), 7.90–7.95 (1H, m), 8.12–8.18 (1H, br), 8.36 (1H, s), 8.39–8.43 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 5.2 (2C, s), 52.7 (s), 67.2 (s), 68.8 (s), 69.2 (s), 69.6 (s), 111.3 (s), 115.0 (2C, s), 120.0 (s), 121.2 (s), 123.3 (s), 124.5 (s), 125.6 (s), 128.4 (2C, s), 129.7 (s), 142.9 (s), 156.6 (s), 158.0 (s), 167.9 (s); IR (ATR) cm⁻¹; 1648; HR-MS (ESI-TOF) Calcd for C₂₂H₂₃NNaO₅ [M + Na]⁺, 404.1474. Found 404.1470; HPLC purity 99.7% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (40 : 60 : 0.2)).

2-{4-[2-(2-Ethoxyethoxy)ethoxy]phenyl}-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (21f)

Compound 21f was prepared from 24 and 25f according to the procedure for the synthesis of 21a. Yield was 24%. ¹H-NMR (CDCl₃) δ: 1.21 (3H, t, J = 7.1 Hz), 1.33 (12H, s), 3.53 (2H, q, J = 7.1 Hz), 3.59–3.63 (2H, m), 3.70–3.74 (2H, m), 3.86 (2H, t, J = 5.1 Hz), 4.16 (2H, t, J = 5.1 Hz), 6.88–6.91 (2H, m), 7.71–7.74 (2H, m).

3-{4-[2-(2-Ethoxyethoxy)ethoxy]phenyl}benzofuran-5-carbonitrile (22f)

Compound 22f was prepared from 21f and 2d according to the procedure for the synthesis of 4d. Yield was 77%. ¹H-NMR (CDCl₃) δ: 1.23 (3H, t, J = 7.1 Hz), 3.55 (2H, q, J = 7.1 Hz), 3.63–3.66 (2H, m), 3.74–3.77 (2H, m), 3.91 (2H, t, J = 5.1 Hz), 4.21 (2H, t, J = 5.1 Hz), 7.04–7.06 (2H, m), 7.48–7.50 (2H, m), 7.60–7.62 (2H, m), 7.82 (1H, s), 8.11–8.12 (1H, m).

3-{4-[2-(2-Ethoxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23f)

Compound 23f was prepared from 22f according to the procedure for the synthesis of 5. Yield was 39%. mp 99–101 °C; ¹H-NMR (DMSO-d₆) δ: 1.11 (3H, t, J = 7.1 Hz), 3.44 (2H, q, J = 7.1 Hz), 3.49–3.52 (2H, m), 3.59–3.62 (2H, m), 3.76–3.79 (2H, m), 4.15–4.18 (2H, m), 7.09–7.11 (2H, m), 7.34–7.42 (1H, br), 7.68–7.72 (3H, m), 7.91–7.93 (1H, m), 8.11–8.17 (1H, br), 8.36 (1H, s), 8.39–8.42 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 15.0 (s), 65.5 (s), 67.2 (s), 68.9 (s), 69.2 (s), 69.9 (s), 111.3 (s), 115.0 (2C, s), 120.0 (s), 121.2 (s), 123.3 (s), 124.5 (s), 125.5 (s), 128.4 (2C, s), 129.7 (s), 142.9 (s), 156.6 (s), 158.0 (s), 167.9 (s); IR (ATR) cm⁻¹; 1646; HR-MS (ESI-TOF) Calcd for C₂₁H₂₃NNaO₅ [M + Na]⁺, 392.1474. Found 392.1459; HPLC purity 99.7% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (40 : 60 : 0.2)).

2-{2-[2-(4-Iodophenoxy)ethoxy]ethoxy}tetrahydro-2H-pyran (28g)

Compound 28g was prepared from 26 and 27g according to the procedure for the synthesis of 28d. Yield was 85%. ¹H-NMR (CDCl₃) δ: 1.47–1.89 (6H, m), 3.47–3.53 (1H, m), 3.60–3.66 (1H, m), 3.72–3.76 (2H, m), 3.83–3.92 (4H, m), 4.07–4.12 (2H, m), 4.61–4.65 (1H, m), 6.88–6.93 (2H, m), 7.71–7.76 (2H, m).

4,4,5,5-Tetramethyl-2-[4-(2-{2-[(tetrahydro-2H-pyran-2-yl)oxy]ethoxy}ethoxy)phenyl]-1,3,2-dioxaborolane (21g)

Compound 21g was prepared from 4,4,5,5-tetramethyl-1,3,2-dioxaborolane and 28g according to the procedure for the synthesis of 21d. Yield was 51%. ¹H-NMR (CDCl₃) δ: 1.33 (12H, s), 1.47–1.89 (6H, m), 3.46–3.53 (1H, m), 3.60–3.67 (1H, m), 3.73–3.77 (2H, m), 3.84–3.92 (4H, m), 4.14–4.18 (2H, m), 4.62–4.66 (1H, m), 6.88–6.93 (2H, m), 7.71–7.76 (2H, m).

3-[4-(2-{2-[(Tetrahydro-2H-pyran-2-yl)oxy]ethoxy}ethoxy)phenyl]benzofuran-5-carbonitrile (22g)

Compound 22g was prepared from 21g and 2d according to the procedure for the synthesis of 4a. Yield was 67%. ¹H-NMR (CDCl₃) δ: 1.48–1.90 (6H, m), 3.48–3.55 (1H, m), 3.63–3.69 (1H, m), 3.76–3.80 (2H, m), 3.86–3.95 (4H, m), 4.19–4.23 (2H, m), 4.64–4.67 (1H, m), 7.03–7.08 (2H, m), 7.47–7.52 (2H, m), 7.60–7.62 (2H, m), 7.82 (1H, s), 8.12–8.14 (1H, m).

3-[4-(2-{2-[(Tetrahydro-2H-pyran-2-yl)oxy]ethoxy}ethoxy)phenyl]benzofuran-5-carboxamide (23g)

Compound 23g was prepared from 22g according to the procedure for the synthesis of 5. Yield was 99%. ¹H-NMR (CDCl₃) δ: 1.47–1.66 (4H, m), 1.69–1.91 (2H, m), 3.46–3.56 (1H, m), 3.61–3.70 (1H, m), 3.74–3.81 (2H, m), 3.84–3.96 (4H, m), 4.17–4.24 (2H, m), 4.61–4.68 (1H, m), 7.01–7.07 (2H, m), 7.50–7.57 (2H, m), 7.56 (1H, d, J = 8.8 Hz), 7.78 (1H, s), 7.80 (1H, dd, J = 8.8, 1.7 Hz), 8.29 (1H, d, J = 1.7 Hz).

3-{4-[2-(2-Hydroxyethoxy)ethoxy]phenyl}benzofuran-5-carboxamide (23h)

Following the addition of 2.0 M HCl (5.0 mL, 10 mmol) to a solution of 23g (520 mg, 1.22 mmol) in THF (8 mL), the reaction mixture was stirred at room temperature for 2 h. After neutralization with saturated aqueous NaHCO₃, 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 and Et₂O (5 mL) was added. The insoluble material was collected by filtration to give 23h (248 mg, 60% yield) as a solid. mp 116–118 °C; ¹H-NMR (CDCl₃) δ: 2.02–2.28 (1H, br), 3.67–3.74 (2H, m), 3.76–3.82 (2H, m), 3.88–3.95 (2H, m), 4.17–4.25 (2H, m), 5.50–6.30 (2H, br), 7.01–7.09 (2H, m), 7.50–7.57 (2H, m), 7.56 (1H, d, J = 8.5 Hz), 7.78 (1H, s), 7.80 (1H, dd, J = 8.5, 1.4 Hz), 8.28 (1H, d, J = 1.4 Hz); ¹³C-NMR (CDCl₃) δ: 61.8 (s), 67.5 (s), 69.7 (s), 72.7 (s), 111.7 (s), 115.3 (2C, s), 120.5 (s), 122.3 (s), 123.9 (s), 124.0 (s), 126.9 (s), 128.6 (s), 128.8 (2C, s), 141.9 (s), 157.6 (s), 158.4 (s), 169.7 (s); IR (ATR) cm⁻¹; 3500–3100, 1508; HR-MS (ESI-TOF) Calcd for C₁₉H₁₉NNaO₅ [M + Na]⁺, 364.1161. Found 364.1154; HPLC purity 99.5% (eluent: 0.01 M KH₂PO₄-MeCN (60 : 40)).

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 cultured for a further 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. The concentrations at which the tested compounds increased ALP activity 2-fold were calculated (EC₂₀₀).

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 collected from the jugular vein 0.25, 0.5, 1, 3, 5, 8, and 24 h after 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 a controlled temperature, humidity, and light exposure (12-h light/dark cycle) and were provided ad libitum access to commercial standard rodent chow (CE2; CLEA Japan, Tokyo, Japan) and tap water. In the present study, 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.^15,16) 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% methyl cellulose (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.^15,16) Micro-CT data were analyzed using TRI/3D-BON software (RATOC, Tokyo, Japan).^15,16) After repeated administration, rats were deeply anesthetized with pentobarbital sodium (50 mg/kg, i.p.), fasting blood was collected from the abdominal aorta, and animals were then euthanized. Femurs were fixed with 70% ethanol and stored at 4 °C. Distal and diaphyseal femurs were scanned using DEXA (LaTheta; Aloka Co., Ltd., Tokyo, Japan) to assess aBMD.

Conflict of Interest

The authors declare no conflict of interest.

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