A Novel Series of Coumarin Derivatives That Exert Osteoblastogenic Effects in Mesenchymal Stem Cells and Osteogenic Effects in Ovariectomized Female Rats

Abstract

Osteoporosis is treated with oral and parenteral resorption inhibitors and parenteral osteogenic drugs. However, orally active small-molecule osteogenic drugs are not clinically available. Natural coumarin derivatives, such as osthole, exert osteoblastogenic effects. In the present study, novel 4,6-substituted coumarin derivatives were synthesized, and their osteoblastogenic effects were assessed in a bone mesenchymal stem cell line (ST2 cell), and structure–activity relationships were discussed. Among the derivatives tested, the osteoblastogenic effects of 2-oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxamide (11m) and 2-oxo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]-2H-chromene-6-carboxamide (29v) were potent: EC₂₀₀ for increasing alkaline phosphatase (ALP) activity were 34 and 24 nM, respectively. The maximal plasma concentrations (C_max) of 11m and 29v (10 mg/kg, per os (p.o.)) in female rats were 3637 and 975 nM, respectively, resulting in high C_max/EC₂₀₀ ratios of 105.9 and 40.8, respectively, indicating possible osteoblastogenic effects in vivo. Compound 11m (10 mg/kg, p.o., 8 weeks) was previously reported to increase plasma bone-type ALP activity as well as femoral metaphyseal and diaphyseal cortical bone volumes and mineral contents in micro-computed tomography analyses of ovariectomized female rats (OVX rats). Compound 29v at the same dose also exerted osteoblastogenic and osteogenic effects in OVX rats; however, these effects were weaker than those of 11m. Furthermore, 11m and 29v inhibited cyclin-dependent kinase 8 (CDK8) activity, suggesting that their osteoblastogenic effects involved the suppression of CDK8. In conclusion, a synthetic 4,6-substituted coumarin structure is a useful scaffold for osteoblastogenic and osteogenic compounds via the inhibition of CDK8, and 11m and 29v have potential as anti-osteoporotic drugs that exert osteogenic effects on cortical bone.

Introduction

Bones are continuously remodeled by osteogenesis via osteoblasts and osteoresorption via osteoclasts. The excessive activation of osteoresorption causes osteoporosis, which is treated with oral resorption inhibitors, such as bisphosphonates, and parenteral osteogenic drugs, including parathyroid hormone (PTH) and anti-sclerostin antibody.^1,2) Therefore, the efficacy of treatments for osteoporosis is increasing as more drugs become available. Orally active small-molecule osteogenic drugs have long been desired and various natural and synthetic compounds have been reported to exert osteoblastogenic and osteogenic effects; however, none have been successful, which may be attributed to poor oral absorption, low efficacy, or toxicity. Therefore, new chemotypes of small-molecule osteogenic compounds are needed.

Among natural compounds, the 7,8-substituted coumarin derivative, osthole and 6,7-substituted coumarin derivative, scopolin were found to exert osteoblastogenic effects by enhancing bone morphogenetic protein (BMP) signaling^3–5) (Fig. 1). Among synthetic compounds, benzothiepine, benzofuran, and thienopyridine derivatives promoted osteoblast differentiation by activating the estrogenic pathway, enhancing BMP signaling, activating wnt/β-catenin, and/or inhibiting cyclin-dependent kinase 8 (CDK8).^6–10) We also synthesized diphenylether derivatives, such as KY-065 and KY-273, which exerted osteoblastogenic effects by inhibiting CDK8 as well as osteogenic effects in ovariectomized female rats (OVX rats)^11,12) (Fig. 1). To discover novel coumarin derivatives with higher efficacy and safety than currently known natural coumarin derivatives, we synthesized a novel series of 4,6-substituted coumarin derivatives using the para-substituted phenyl group employed in the diphenylether derivatives KY-065 and KY-273 as substituents at the 4-position (Fig. 2). We then identified 2-oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxamide (11m, KY-054) as a potent osteoblastogenic agent, which exerted osteogenic effects in OVX rats; it increased femur metaphyseal and diaphyseal cortical, but not metaphyseal trabecular, bone volumes (BV) and mineral contents (BMC).¹³⁾ The mechanisms by which 11m exerts its cortical bone-selective osteogenic effects may differ from those of natural coumarin derivatives because osthole and scopolin were previously reported to increase BV and bone mineral density in trabecular bone rather than in cortical bone.^3,5)

Fig. 1. Chemical Structures of Natural Coumarins and Diphenylether Derivatives

Fig. 2. Chemical Structures of 4,6-Substituted Coumarin Derivatives Synthesized

Among the compounds synthesized, the effects of 2-oxo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]-2H-chromene-6-carboxamide (29v) on femurs in OVX rats were investigated using dual-energy X-ray absorptiometry (DEXA) and in vivo micro-computed tomography (micro-CT). The results obtained on 29v were compared with previous findings on 11m.¹³⁾ The mechanisms by which both compounds exert their osteoblastogenic effects remain unclear; their impact on CDK8 activity was assessed because both have a substituent similar to KY-065 and KY-273, diphenylether derivatives, which exert osteoblastogenic effects by inhibiting CDK8.^11,12)

Chemistry

General approaches to the synthesis of 4-phenyl-coumarin derivatives are outlined in Charts 1–5.

Chart 1. Synthesis of 4′-Alkyl or Alcohol Derivatives

(i) PdCl₂(dppf)·CH₂Cl₂, K₃PO₄, n-Bu₄NBr, MeCN; (ii) Zn(CN)₂, Pd(PPh₃)₄, DMF; (iii) H₂O₂ aq. NaOH aq.; (iv) HCl aq.; (v) bis(pinacolato)diboron, PdCl₂(PPh₃)₂, AcOK, 1,4-dioxane.

Chart 2. Synthesis of 4-Benzyl Ether Derivatives

(i) PdCl₂(dppf)·CH₂Cl_2, K₃PO_4,n-Bu₄NBr, MeCN; (ii) Zn(CN)₂, Pd(PPh₃)₄, DMF; (iii) H₂O₂ aq., NaOH aq., DMSO; (iv) NaBO₃·4H₂O, H₂O, MeOH, THF; (v) HCl aq.; (vi) NaH, DMF; (vii) bis(pinacolato)diboron, PdCl₂(PPh₃)₂, AcOK, 1,4-dioxane; (viii) TBSCl, imidazole, DMF; (ix) 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, PdCl₂(dppf)·CH₂Cl₂, Et₃N, 1,4-dioxane.

Chart 3. Synthesis of 6-Substituted Derivatives

(i) PdCl₂(dppf)·CH₂Cl₂, K₃PO₄, n-Bu₄NBr, MeCN; (ii) trimethylsilylacetylene, PdCl₂(PPh₃)₂, CuI, Et₃N; (iii) H₂O, HCO₂H; (iv) H₂O, HCO₂H; (v) Raney Nickel 2800, NaH₂PO₂·H₂O, AcOH, H₂O, pyridine; (vi) H₂O₂ aq., NaClO₂, KH₂PO₄, DMSO, H₂O, MeCN; (vii) DAST, CH₂Cl₂.

Chart 4. Synthesis of 4-Phenyl Ether Derivatives

(i) PdCl₂(dppf)·CH₂Cl_2, K₃PO_4,n-Bu₄NBr, MeCN; (ii) Zn(CN)₂, Pd(PPh₃)₄, DMF; (iii) H₂O₂ aq., NaOH aq., DMSO; (iv) NaBO₃·4H₂O, H₂O, MeOH, THF; (v) HCl aq.; (vi) HCl in IPA, HCO₂H (vii) formalin, NaBH(OAc)₃, MeCN, MeOH; (viii) KI, Cs₂CO₃, DMF; (ix) DIAD, PPh₃, CH₂Cl₂; (x) MsCl, Et₃N, CH₂Cl₂; (xi) K₂CO₃, DMF; (xii) 4-iodophenol, KI, Cs₂CO₃, DMF; (xiii) 4,4,5,5-tetramethyl-1,3,2-dioxaborolane, PdCl₂(dppf)·CH₂Cl_2, Et₃N, 1,4-dioxane.

Chart 5. Synthesis of Skeletal Transformation Derivatives

(i) 2-Propyn-1-ol, Pd(PPh₃)₄, CuI, pyrrolidine, H₂O; (ii) 4-cyanophenol or ethyl 4-hydroxybenzoate, DIAD, PPh₃, CH₂Cl₂; (iii) PtCl₄·5H₂O, 1,4-dioxane; (iv) H₂O₂ aq., K₂CO₃, DMSO; (v) H₂, Pd-C, MeOH; (vi) NaOH aq., MeOH; (vii) i-BuOCOCl, NMM, THF; (viii) NH₃ aq.

Chart 1 shows the synthesis of compounds with various substituents (R³) on a 4-phenyl-coumarin skeleton (6a–h). Previously reported 1¹⁴⁾ was coupled with boronic acids 2a–d or boronic esters 3e–h to afford 4a–h, the bromine group was converted to a cyano group to give 5a–h, and the cyano group was hydrolyzed to give 6a–h. Compounds 3g–h were obtained from 7g–h by Miyaura–Ishiyama borylation.

Chart 2 shows the synthesis of compounds with various substituents (R⁴) on a 4-phenyl-coumarin skeleton (11i–q). Similar to chart 1, 1¹⁴⁾ was coupled with boronic esters 8i–q to afford 9i–q, followed by cyanation to give 10i–q and hydrolysis to give 11i–q. Compound 12 was reacted with alcohols 13i–n and 13q to afford ethers 14i–n and 14q, and the bromine group was converted to boronic esters to give 8i–n and 8q. Compound 8o was obtained from compounds 15 and 13o: they reacted to 16o by Williamson ether synthesis, the remaining hydroxy group was protected by tert-butyldimethylsilyl to give compound 17o, and the iodine group was then converted to boronic ester by Miyaura–Ishiyama borylation to give 8o. Compound 8p was obtained by the esterification of 15 with 13p followed by borylation of the iodine group in 16p.

Chart 3 shows the synthesis of compounds with 6-substituted 4-phenyl-coumarin derivatives. Previously reported 18¹⁴⁾ and 8m were converted to 19 by Suzuki–Miyaura coupling. Compound 9m was converted to 20 by Sonogashira coupling, followed by desilylation to give 21. The acetylene group of 21 was hydrolyzed to afford compound 22. Compound 10m was converted to 23 by the reduction of the cyano group, followed by the oxidation of aldehyde to give 24. Compound 23 was converted to 25 by fluorination of the carbonyl group.

Chart 4 shows the synthesis of compounds with various substituents (R⁵) on a 4-phenyl-coumarin skeleton. Similar to chart 1, 1¹⁴⁾ and boronic esters 26r–y were converted to 27r–y by Suzuki-Miyaura coupling reaction. The bromine group was converted to a cyano group to give 28r–y, and converted by hydrolysis to afford 29r–y. The tert-butoxycarbonyl (Boc) group of 29w was removed, and this was followed by methylation to give 30w. Compound 26u was obtained from 31 and 32u by Williamson ether synthesis. Compound 26t was obtained from 31 and 33t, and 26v from 31 and 33v by Mitsunobu reaction. The hydroxyl group of 33w was mesylated, and this was followed by etherification with 31 to give 26w. Compound 26y was obtained by the etherification of 34 with 32y followed by Miyaura–Ishiyama borylation.

Chart 5 shows the synthesis of compounds with various chromene derivatives. Compound 14m was converted to 35 by Sonogashira coupling. The hydroxy group of 35 was alkylated to give 36z and 36α, followed by cyclization to afford 37z and 37α. The cyano group of 37z was hydrolyzed to a carbamoyl group to give 38, followed by hydrogenation to afford 39. Compound 37α was converted to 40 by hydrolysis and amidation.

Results and Discussion

In the present study, novel 4,6-substituted coumarin derivatives were synthesized, and their osteoblastogenic activities in parenchymal stem cells, oral absorption in female rats, and osteogenic effects in OVX rats were evaluated.

We initially synthesized 6-carbamoyl-4-phenylcoumarin derivatives with various alkyl and hydroxy alkyl substituents at the 4′-position of the 4-phenyl ring (Table 1). These derivatives exerted weak osteoblastogenic effects with EC₂₀₀ values of 111–325 nM. Among them, 6f and 6h were the most potent compounds with EC₂₀₀ of 139 and 111 nM, respectively. The maximal plasma concentrations (C_max) of 6f and 6h after their oral administration at 10 mg/kg were 2193 and <0.3 nM, respectively, in female rats: C_max/EC₂₀₀ calculated as an index of in vivo osteoblastogenic activity were 15.8 and <0.003, respectively. The effects of compound 6f on femoral bone were examined in OVX rats, and the results obtained revealed its negligible effects on plasma bone-type alkaline phosphatase (ALP) activity and the structure of femoral bone (data not shown). Therefore, stronger osteoblastogenic activity and/or C_max/EC₂₀₀ >15.8 may be required for osteoblastogenic and osteogenic effects to be exerted in vivo.

Table 1. Chemical Structures, Promoting Activities on Osteoblast Differentiation (EC₂₀₀), Maximal Plasma Concentration (C_max) after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of C_max and EC₂₀₀ (C_max/EC₂₀₀) of 4-(4-Substituted)-2-oxo-2H-chromene-6-carboxamide Derivatives

a) n = 2, b) Mean ± standard error (S.E.) (n = 3).

Various benzyloxy derivatives were then synthesized (Table 2). Compounds 11i, 11j, 11l, 11m, and 11q exerted stronger osteoblastogenic effects than 6f, with EC₂₀₀ values <100 nM. However, the osteoblastogenic effects of 11k and 11o with a hydroxyl group and 11n with an oxetane ring were weaker with EC₂₀₀ >100 nM. An oxygen atom in the ether structure may play an important role in the interaction with a target protein, while a hydroxy group may disturb it. Compound 11p with a carboxyl group did not exhibit any activity. An ionizable moiety may completely disturb the interaction with a target protein. The most potent osteoblastogenic compound 11m showed the highest ratio of 105.9, suggesting strong osteoblastogenic effects in vivo. 11m was previously reported to enhance plasma bone-type ALP activity and increased femur metaphysis and diaphysis cortical BV and BMC.¹³⁾

Table 2. Chemical Structures, Promoting Activities on Osteoblast Differentiation (EC₂₀₀), Maximal Plasma Concentration (C_max) after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of C_max and EC₂₀₀ (C_max/EC₂₀₀) of 4-(4-Substituted)-2-oxo-2H-chromene-6-carboxamide Derivatives

a) n = 2, b) Mean ± S.E. (n = 3). ND: not determined.

The effects of 6-substituents on osteoblast differentiation were subsequently examined (Table 3). In comparison with a carbamoyl group (11m), an acetyl group (22) markedly reduced osteoblastogenic activity, and chlorine (19), CHF₂ (25), and a carboxyl group (24) almost abolished it, indicating that a 6-carbamoyl coumarin structure is the most suitable for osteoblastogenic effects. In the coumarin ring, the deletion of 2-oxo moderately, while that of 2-oxo and the 3,4-double bond markedly reduced osteoblastogenic activity, indicating that a coumarin ring is essential for osteoblastogenic effects (Table 4).

Table 3. Chemical Structures and Promoting Activities on Osteoblast Differentiation (EC₂₀₀) of 6-Substituted-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chromen-2-one Derivatives

n = 2.

Table 4. Chemical Structures and Promoting Activities on Osteoblast Differentiation (EC₂₀₀) of 4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl-6-carbamoyl-chromene Derivatives

n = 2.

Derivatives with various phenyloxy substituents, which were employed in diphenylamine and diphenylether derivatives,^11,12) were synthesized and their osteoblastogenic effects were examined (Table 5). Compound 29r with hydroxyethyl, 29s with cyclopropylmethyl, and 29u with hydroxyethoxyethyl exhibited weaker activity and lower C_max than 11m. Compound 29t with cyclopropyloxyethyl, 29v with tetrahydropyranylmethyl, and 30w with N-methyl piperidinyl methyl exhibited slightly higher activity than 11m; however, their plasma concentrations were lower. Compound 29x with tetrahydropyranyloxyethyl and 29y with isopropyloxyethoxyethyl exhibited markedly higher activity and C_max than 11m, resulting in higher C_max/EC₂₀₀. Compound 29x exhibited higher activity and C_max than 29v, suggesting the importance of the length or ether structure of substituents for osteoblastogenic activity and plasma concentrations. The isopropyloxy structure in 29y appeared to be preferable for the interaction with a target protein, and was more resistant to metabolism than the hydroxy structure in 29u. Compound 29v is structurally similar to 11m and exhibited slightly higher activity than 11m; however, its plasma concentration was lower and, thus, C_max/EC₂₀₀ was lower. The effects of 29v on OVX rats were examined and compared with those of 11m previously reported. Compound 29v had no effects on uterine weight reduced by ovariectomy (Intact 388.3 ± 16.4**, OVX-Control 87.9 ± 4.1, OVX-29v 114.7 ± 15.3 mg, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test), indicating no estrogenic effects, and significantly increased plasma bone-type ALP activity (Intact 104.6 ± 4.4*, OVX control 116.5 ± 1.6, OVX-29v 134.6 ± 3.3** IU/L, * p < 0.05, ** p < 0.01 vs. OVX control, Dunnett’s multiple comparison test), suggesting osteoblastogenic effects in vivo. Compounds 11m and 29v increased areal bone mineral density (aBMD) in the femoral bone midportion, which was not affected by ovariectomy in DEXA analyses, and slightly increased femur diaphyseal cortical BV and BMC, which was not affected by ovariectomy, in micro-CT analyses; however, it had no effects on trabecular bone (Table 6). These cortical bone-selective osteogenic effects of 29v were weaker than 11m, possibly due to its lower C_max/EC₂₀₀.

Table 5. Chemical Structures, Promoting Activities on Osteoblast Differentiation (EC₂₀₀), Maximal Plasma Concentration (C_max) after Oral Administration at 10 mg/kg in Female Rats, and the Ratio of C_max and EC₂₀₀ (C_max/EC₂₀₀) of 4-(4-Substituted)-2-oxo-2H-chromene-6-carboxamide Derivatives

a) n = 2, b) Mean ± S.E. (n = 3).

Table 6. Effects of the Repeated Administration of 11m and 29v at 10 mg/kg/d for 8 Weeks on Areal Bone Mineral Density (aBMD) Assessed by DEXA, and Femur Bone Structure on in Vivo Micro-CT in Ovariectomized Rats

	Compound 11ma)			Compound 29vb)
	Intact	OVX		Intact	OVX
		Control	10 mg/kg/d		Control	10 mg/kg/d
DEXA aBMD (mg/cm2)
Distal portion	135.2 ± 0.9**	122.1 ± 1.1	127.7 ± 1.3**	139.9 ± 2.0**	125.4 ± 1.5	125.8 ± 1.3
Midportion	109.8 ± 0.6	108.9 ± 0.6	112.2 ± 1.1*	110.4 ± 0.5	109.9 ± 0.7	113.3 ± 0.8**
µCT diaphysis cortical bone
BV (mm3)	20.36 ± 0.16	20.34 ± 0.25	21.16 ± 0.31†	20.69 ± 0.24	20.60 ± 0.21	21.38 ± 0.28†
BMC (mg)	28.14 ± 0.29†	27.38 ± 0.28	28.51 ± 0.34*	23.32 ± 0.31	22.99 ± 0.23	23.89 ± 0.29†
µCT metaphysis cortical bone
BV (mm3)	8.83 ± 0.12	8.57 ± 0.10	9.03 ± 0.14*	8.80 ± 0.12	8.63 ± 0.09	8.77 ± 0.11
BMC (mg)	10.69 ± 0.14*	10.16 ± 0.13	10.77 ± 0.14*	8.76 ± 0.11*	8.42 ± 0.06	8.63 ± 0.10
µCT metaphysis trabecular bone
BV (mm3)	2.79 ± 0.17**	1.18 ± 0.11	1.31 ± 0.07	3.40 ± 0.17**	1.51 ± 0.13	1.31 ± 0.20
BMC (mg)	1.20 ± 0.07**	0.41 ± 0.04	0.47 ± 0.03	1.44 ± 0.08**	0.55 ± 0.05	0.45 ± 0.07

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.

aBMD: areal bone mineral density, µCT: micro-CT, BV: bone volume, BMC: bone mineral contents, a) n = 8, cited from Reference 13. b) n = 7.

The molecular mechanisms by which these coumarin derivatives exert their osteoblastogenic effects have yet to be elucidated in detail. The natural coumarin derivatives, osthole and scopolin also exert osteoblastogenic effects, which are mediated by enhancements in BMP signaling.^3–5) However, their first target protein remains unknown. On the other hand, the osteoblastogenic effects of thienopyridine derivatives correlated with their suppression of CDK8 activity, suggesting the involvement of the inhibition of CDK8 in these effects.^9,10) We previously reported that diphenylether derivatives exerted osteoblastogenic effects by inhibiting CDK8.^11,12) Coumarin derivatives in the present study and biphenyl ether derivatives have similar substituents: various benzyloxy and phenoxy groups, suggesting that the present coumarin derivatives also exert their effects by inhibiting CDK8. Therefore, the effects of 11m and 29v on CDK8 activity were examined and compared with those of the natural coumarin derivative, osthole. Compounds 11m and 29v potently inhibited CDK8 activity with IC₅₀ of 17.1 and 8.3 nM, respectively, while osthole had no effects; therefore, unlike a natural coumarin derivative, 11m and 29v increased femur metaphysis and diaphysis cortical BV and BMC as well as plasma bone-type ALP activity by osteoblastogenesis via the inhibition of CDK8. The first target protein of natural coumarin derivatives is unknown and it is also unclear whether their osteoblastogenic effects contribute to their in vivo effects. They have been shown to restore destructured trabecular bone, but not cortical bone, in OVX rats and exert more potent osteoclastogenic inhibitory effects than osteoblastogenic effects, suggesting the involvement of the suppression of bone resorption.^3,5)

The structure–activity relationships of 4,6-substituted coumarin derivatives for osteoblastogenic activity are summarized in Fig. 3. The results obtained herein showed that the 6-carbamoyl-4-phenylcoumarin structure is an excellent scaffold for osteoblastogenic drugs. Furthermore, the insertion of ether structures increased the osteoblastogenic activities of both phenoxy and benzyl type substituents. Among the derivatives synthesized, 11m and 29v exerted osteogenic effects in OVX rats. Further studies are needed to clarify their potential as orally active anti-osteoporotic drugs.

Fig. 3. Summary of Structure–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; Osaka Soda Co., Ltd., Osaka, Japan). Reactions were monitored by TLC (TLC silica gel 60 F₂₅₄, Merck KGaA, Darmstadt, Germany). The purity of the final compounds was assessed by HPLC (pump, LC-8A and LC-20AT; detector, SPD-10A and SPD-20A; Shimadzu Corporation, Kyoto, Japan) using the COSMOSIL 5C₁₈-AR-II column (5 µm, 4.6 × 150 mm; Nacalai Tesque, Kyoto, Japan).

6-Bromo-4-(4-isobutylphenyl)chromen-2-one (4a)

Following the addition of K₃PO₄ (3.58 g, 16.9 mmol), n-Bu₄NBr (TBAB) (181 mg, 0.562 mmol), and the [1,1′-bis(diphenylphosphino)ferrocene]palladium dichloride CH₂Cl₂ complex [PdCl₂(dppf)·CH₂Cl₂] (459 mg, 0.562 mmol) to a solution of 6-bromo-4-chloro-chromen-2-one (1)¹⁴⁾ (1.46 g, 5.62 mmol) and 2a (1.00 g, 5.62 mmol) in MeCN (30 mL), the mixture was heated to reflux for 2 h under a N₂ atmosphere. After cooling, AcOEt was added, and 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 to give 4a (2.00 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 0.97 (6H, d, J = 6.8 Hz), 1.89–2.01 (1H, m), 2.57 (2H, d, J = 7.3 Hz), 6.39 (1H, s), 7.27–7.40 (5H, m), 7.60–7.70 (2H, m).

4-(4-Isobutylphenyl)-2-oxo-2H-chromene-6-carbonitrile (5a)

Following the addition of Zn(CN)₂ (1.31 g, 11.2 mmol) and Pd(PPh₃)₄ (0.97 g, 0.84 mmol) to a solution of 4a (2.00 g, 5.60 mmol) in N,N-dimethylformamide (DMF) (40 mL), the reaction mixture was stirred at 90 °C for 3 h under a N₂ atmosphere. After cooling, AcOEt was added and the insoluble material was filtrated. The filtrate 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 5a (530 mg, 31% yield) as a solid. ¹H-NMR (CDCl₃) δ: 0.98 (6H, d, J = 6.6 Hz), 1.88–2.02 (1H, m), 2.59 (2H, d, J = 7.1 Hz), 6.47 (1H, s), 7.29–7.39 (4H, m), 7.49 (1H, d, J = 8.5 Hz), 7.80 (1H, dd, J = 8.6, 2.0 Hz), 7.88 (1H, d, J = 2.0 Hz).

4-(4-Isobutylphenyl)-2-oxo-2H-chromene-6-carboxamide (6a)

Following the addition of 5.0 M aqueous NaOH (1.8 mL, 8.7 mmol) and 30% aqueous H₂O₂ (0.89 mL, 8.7 mmol) to a solution of 5a (530 mg, 1.75 mmol) in dimethyl sulfoxide (DMSO) (10 mL), the mixture was stirred at room temperature for 1 h. After the addition of 30% aqueous H₂O₂ (0.89 mL, 8.7 mmol), the mixture was stirred for 1 h, 6.0 M HCl (3.5 mL, 21 mmol) and MeCN (10 mL) were added, and the mixture was then stirred for 1 h. 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 and the powder obtained was recrystallized with AcOEt (12 mL) to give 6a (250 mg, 45% yield) as a solid. mp 201–204 °C; ¹H-NMR (DMSO-d₆) δ: 0.93 (6H, d, J = 6.6 Hz), 1.86–2.00 (1H, m), 2.57 (2H, d, J = 7.1 Hz), 6.47 (1H, s), 7.35–7.42 (2H, m), 7.44–7.52 (3H, m), 7.56 (1H, d, J = 8.5 Hz), 8.02 (1H, d, J = 2.0 Hz), 8.08–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 22.1 (s), 29.4 (s), 44.2 (s), 115.0 (s), 116.8 (s), 118.1 (s), 126.8 (s), 128.4 (s), 129.4 (s), 130.3 (s), 130.6 (s), 131.8 (s), 143.2 (s), 154.7 (s), 155.2 (s), 159.2 (s), 166.5 (s); IR (attenuated total reflectance (ATR)) cm⁻¹: 3154, 1722, 1662; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₃ 344.1263. Found 344.1244; HPLC purity 99.6% (eluent: 0.01 M KH₂PO₄-MeCN (30 : 70)).

6-Bromo-4-(4-tert-butylphenyl)chromen-2-one (4b)

Compound 4b was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 2b according to the procedure for the synthesis of 4a. Yield was quant. ¹H-NMR (CDCl₃) δ: 1.40 (9H, s), 6.39 (1H, s), 7.29 (1H, d, J = 8.6 Hz), 7.36–7.40 (2H, m), 7.54–7.58 (2H, m), 7.63 (1H, dd, J = 8.6, 2.2 Hz), 7.67 (1H, d, J = 2.2 Hz).

4-(4-tert-Butylphenyl)-2-oxo-2H-chromene-6-carbonitrile (5b)

Compound 5b was prepared from 4b according to the procedure for the synthesis of 5a. Yield was 61%. ¹H-NMR (CDCl₃) δ: 1.41 (9H, s), 6.47 (1H, s), 7.34–7.38 (2H, m), 7.49 (1H, d, J = 8.6 Hz), 7.57–7.61 (2H, m), 7.79 (1H, dd, J = 8.6, 1.9 Hz), 7.89 (1H, d, J = 1.9 Hz).

4-(4-tert-Butylphenyl)-2-oxo-2H-chromene-6-carboxamide (6b)

Compound 6b was prepared from 5b according to the procedure for the synthesis of 6a. Yield was 63%. mp 230–232 °C; ¹H-NMR (DMSO-d₆) δ: 1.36 (9H, s), 6.48 (1H, s), 7.47–7.55 (3H, m), 7.56 (1H, d, J = 8.8 Hz), 7.61–7.68 (2H, m), 8.04 (1H, d, J = 2.2 Hz), 8.11–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 30.9 (s), 34.5 (s), 115.1 (s), 116.8 (s), 118.1 (s), 125.7 (s), 126.8 (s), 128.4 (s), 130.4 (s), 130.6 (s), 131.6 (s), 152.5 (s), 154.6 (s), 155.3 (s), 159.2 (s), 166.6 (s); IR (ATR) cm⁻¹: 3161, 1716, 1674; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₃ 344.1263. Found 344.1241; HPLC purity 99.4% (eluent: 0.01 M KH₂PO₄-MeCN (30 : 70)).

6-Bromo-4-(4-trifluoromethylphenyl)chromen-2-one (4c)

Compound 4c was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 2c according to the procedure for the synthesis of 4a. Yield was 51%. ¹H-NMR (CDCl₃) δ: 6.41 (1H, s), 7.32 (1H, d, J = 8.8 Hz), 7.47 (1H, d, J = 2.2 Hz), 7.53–7.61 (2H, m), 7.67 (1H, dd, J = 8.8, 2.2 Hz), 7.80–7.88 (2H, m).

2-Oxo-4-(4-trifluoromethylphenyl)-2H-chromene-6-carbonitrile (5c)

Compound 5c was prepared from 4c according to the procedure for the synthesis of 5a. Yield was 98%. ¹H-NMR (CDCl₃) δ: 6.49 (1H, s), 7.53 (1H, d, J = 8.5 Hz), 7.54–7.62 (2H, m), 7.70 (1H, d, J = 2.0 Hz), 7.83–7.92 (3H, m).

2-Oxo-4-(4-trifluoromethylphenyl)-2H-chromene-6-carboxamide (6c)

Compound 6c was prepared from 5c according to the procedure for the synthesis of 6a. Yield was 48%. mp 218–220 °C; ¹H-NMR (DMSO-d₆) δ: 6.60 (1H, s), 7.50 (1H, brs), 7.59 (1H, d, J = 8.6 Hz), 7.78–7.85 (2H, m), 7.88 (1H, d, J = 1.7 Hz), 7.94–8.05 (2H, m), 8.10–8.18 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 116.2 (s), 117.0 (s), 117.8 (s), 124.02 (q), 125.8 (q), 126.3 (s), 129.6 (s), 130.1 (q), 130.5 (s), 131.1 (s), 138.5 (s), 153.4 (s), 155.3 (s), 159.1 (s), 166.4 (s); IR (ATR) cm⁻¹: 1710, 1614; HR-MS (ESI-TOF) Calcd for C₁₇H₁₀F₃NNaO₃ 356.0510. Found 356.0499; HPLC purity 99.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

6-Bromo-4-[4-(1-cyclohexyl)phenyl]chromen-2-one (4d)

Compound 4d was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 2d according to the procedure for the synthesis of 4a. Yield was 82%. ¹H-NMR (CDCl₃) δ: 1.23–1.36 (1H, m), 1.37–1.56 (4H, m), 1.76–1.83 (1H, m), 1.84–2.00 (4H, m), 2.56–2.65 (1H, m), 6.38 (1H, s), 7.28 (1H, d, J = 8.8 Hz), 7.34–7.40 (4H, m), 7.63 (1H, dd, J = 8.8, 2.2 Hz), 7.66 (1H, d, J = 2.2 Hz).

4-(4-Cyclohexylphenyl)-2-oxo-2H-chromene-6-carbonitrile (5d)

Compound 5d was prepared from 4d according to the procedure for the synthesis of 5a. Yield was 42%. ¹H-NMR (CDCl₃) δ: 1.24–1.37 (1H, m), 1.38–1.53 (4H, m), 1.76–1.84 (1H, m), 1.85–2.00 (4H, m), 2.57–2.67 (1H, m), 6.46 (1H, s), 7.33–7.42 (4H, m), 7.49 (1H, d, J = 8.8 Hz), 7.79 (1H, dd, J = 8.8, 2.0 Hz), 7.88 (1H, d, J = 2.0 Hz).

4-(4-Cyclohexylphenyl)-2-oxo-2H-chromene-6-carboxamide (6d)

Compound 6d was prepared from 5d according to the procedure for the synthesis of 6a. Yield was 13%. mp 247–250 °C; ¹H-NMR (DMSO-d₆) δ: 1.19–1.31 (1H, m), 1.35–1.58 (4H, m), 1.73 (1H, d, J = 12.5 Hz), 1.78–1.96 (4H, m), 2.62 (1H, t, J = 11.2 Hz), 6.47 (1H, s), 7.42–7.54 (5H, m), 7.56 (1H, d, J = 8.6 Hz), 8.04 (1H, d, J = 2.0 Hz), 8.13 (2H, dd, J = 8.4, 2.0 Hz); ¹³C-NMR (DMSO-d₆) δ: 25.6 (s), 26.3 (s), 33.7 (s), 43.6 (s), 115.1 (s), 116.9 (s), 118.2 (s), 126.9 (s), 127.3 (s), 128.7 (s), 130.4 (s), 130.7 (s), 132.0 (s), 149.5 (s), 154.8 (s), 155.4 (s), 159.3 (s), 166.7 (s); IR (ATR) cm⁻¹: 1714, 1614; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₃ 370.1419. Found 370.1412; HPLC purity 96.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (30 : 70 : 0.2)).

6-Bromo-4-[4-(1-hydroxy-1-methylethyl)phenyl]chromen-2-one (4e)

Compound 4e was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 3e according to the procedure for the synthesis of 4a. Yield was 61%. ¹H-NMR (CDCl₃) δ: 1.67 (6H, s), 1.70–1.90 (1H, m), 6.39 (1H, s), 7.28–7.34 (1H, m), 7.40–7.45 (2H, m), 7.63 (1H, s), 7.64–7.70 (3H, m).

4-[4-(1-Hydroxy-1-methylethyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (5e)

Compound 5e was prepared from 4e according to the procedure for the synthesis of 5a. Yield was 84%. ¹H-NMR (CDCl₃) δ: 1.69 (6H, s), 1.86 (1H, brs), 6.49 (1H, s), 7.40–7.45 (2H, m), 7.51 (1H, d, J = 8.5 Hz), 7.70–7.75 (2H, m), 7.82 (1H, dd, J = 8.5, 2.0 Hz), 7.87 (1H, d, J = 2.0 Hz).

4-[4-(1-Hydroxy-1-methylethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (6e)

Compound 6e was prepared from 5e according to the procedure for the synthesis of 6a. Yield was 69%. mp 225–228 °C; ¹H-NMR (DMSO-d₆) δ: 1.52 (6H, s), 5.21 (1H, s), 6.49 (1H, s), 7.48–7.62 (4H, m), 7.66–7.77 (2H, m), 8.02–8.10 (1H, m), 8.10–8.20 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 31.7 (s), 70.6 (s), 115.1 (s), 116.8 (s), 118.1 (s), 125.1 (s), 126.9 (s), 128.1 (s), 130.4 (s), 130.6 (s), 132.1 (s), 152.4 (s), 154.7 (s), 155.3 (s), 159.2 (s), 166.6 (s); IR (ATR) cm⁻¹: 1726, 1660; HR-MS (ESI-TOF) Calcd for C₁₉H₁₇NNaO₄ 346.1055. Found 346.1041; HPLC purity 96.0% (eluent: 0.01 M KH₂PO₄-MeCN (40 : 60)).

6-Bromo-4-[4-(2-hydroxy-1,1-dimethylethyl)phenyl]chromen-2-one (4f)

Compound 4f was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 3f¹⁵⁾ according to the procedure for the synthesis of 4a. Yield was 80%. ¹H-NMR (CDCl₃) δ: 1.24 (6H, s), 1.48 (1H, t, J = 5.9 Hz), 3.72 (2H, d, J = 5.8 Hz), 6.39 (1H, s), 7.29 (1H, d, J = 8.6 Hz), 7.39–7.46 (2H, m), 7.54–7.61 (2H, m), 7.61–7.70 (2H, m).

4-[4-(2-Hydroxy-1,1-dimethylethyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (5f)

Compound 5f was prepared from 4f according to the procedure for the synthesis of 5a. Yield was 71%. ¹H-NMR (CDCl₃) δ: 1.43 (6H, s), 1.51–1.68 (1H, m), 3.73 (2H, s), 6.46 (1H, s), 7.37–7.45 (2H, m), 7.50 (1H, d, J = 8.5 Hz), 7.56–7.65 (2H, m), 7.80 (1H, dd, J = 8.5, 1.4 Hz), 7.88 (1H, d, J = 1.4 Hz).

4-[4-(2-Hydroxy-1,1-dimethylethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (6f)

Compound 6f was prepared from 5f according to the procedure for the synthesis of 6a. Yield was 52%. mp 214–215 °C; ¹H-NMR (DMSO-d₆) δ: 1.31 (6H, s), 3.52 (2H, d, J = 5.4 Hz), 4.80 (1H, t, J = 5.4 Hz), 6.47 (1H, s), 7.48–7.85 (3H, m), 7.57 (1H, d, J = 8.6 Hz), 7.60–7.73 (2H, m), 8.08 (1H, d, J = 1.8 Hz), 8.13–8.23 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 25.5 (s), 39.9 (s), 70.8 (s), 115.1 (s), 116.9 (s), 118.2 (s), 126.89 (s), 126.94 (s), 128.2 (s), 130.5 (s), 130.7 (s), 131.7 (s), 150.1 (s), 154.8 (s), 155.4 (s), 159.3 (s), 166.7 (s); IR (ATR) cm⁻¹: 3209, 1725, 1654; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₄ 360.1212. Found 360.1200; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN (60 : 40)).

2-Methyl-1-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)phenyl]propan-2-ol (3g)

Following the addition of bis(pinacolato)diboron (1.50 g, 5.91 mmol), PdCl₂(PPh₃)₂ (0.19 g, 0.27 mmol), and AcOK (1.84 g, 18.7 mmol) to a solution of 7g¹⁶⁾ (1.23 g, 5.37 mmol) in 1,4-dioxane (30 mL), the reaction mixture was stirred at 80 °C for 8 h under a N₂ atmosphere. After cooling, the reaction mixture was concentrated under reduced pressure. Water and AcOEt were added and the insoluble material was filtrated. The filtrate was separated, and 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 3g (1.46 g, 98% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.22 (6H, s), 1.27 (1H, s), 1.34 (12H, s), 2.78 (2H, s), 7.21–7.26 (2H, m), 7.74–7.79 (2H, m).

6-Bromo-4-[4-(2-hydroxy-2-methylpropyl)phenyl]chromen-2-one (4g)

Compound 4g was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 3g according to the procedure for the synthesis of 4a. Yield was 89%. ¹H-NMR (CDCl₃) δ: 1.31 (6H, s), 1.37 (1H, s), 2.87 (2H, s), 6.39 (1H, s), 7.26–7.32 (1H, m), 7.36–7.43 (4H, m), 7.61–7.66 (2H, m)

4-[4-(2-Hydroxy-2-methylpropyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (5g)

Compound 5g was prepared from 4g according to the procedure for the synthesis of 5a. Yield was 38%. ¹H-NMR (CDCl₃) δ: 1.32 (6H, s), 1.38 (1H, brs), 2.89 (2H, s), 6.47 (1H, s), 7.36–7.40 (2H, m), 7.42–7.47 (2H, m), 7.50 (1H, d, J = 8.6 Hz), 7.80 (1H, dd, J = 8.6, 2.0 Hz), 7.87 (1H, d, J = 2.0 Hz).

4-[4-(2-Hydroxy-2-methylpropyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (6g)

Compound 6g was prepared from 5g according to the procedure for the synthesis of 6a. Yield was 55%. mp 173–175 °C; ¹H-NMR (DMSO-d₆) δ: 1.14 (6H, s), 2.78 (2H, s), 4.45 (1H, s), 6.47 (1H, s), 7.42–7.53 (5H, m), 7.57 (1H, d, J = 8.6 Hz), 8.03–8.07 (1H, m), 8.14 (2H, dd, J = 8.6, 2.0 Hz); ¹³C-NMR (DMSO-d₆) δ: 29.3 (s), 49.0 (s), 69.4 (s), 115.0 (s), 116.9 (s), 118.2 (s), 127.0 (s), 127.9 (s), 130.4 (s), 130.7 (s), 131.0 (s), 131.9 (s), 141.1 (s), 154.9 (s), 155.4 (s), 159.3 (s), 166.7 (s); IR (ATR) cm⁻¹: 1724, 1670; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₄ 360.1212. Found 360.1200; HPLC purity 97.7% (eluent: 0.01 M KH₂PO₄-MeCN (40 : 60)).

2-Methyl-4-[4-(4,4,5,5-tetramethyl-[1,3,2]dioxaborolan-2-yl)phenyl]butan-2-ol (3h)

Compound 3h was prepared from 7h¹⁷⁾ according to the procedure for the synthesis of 3g. The crude product was used in the next reaction without further purification.

6-Bromo-4-[4-(3-hydroxy-3-methylbutyl)phenyl]chromen-2-one (4h)

Compound 4h was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 3h according to the procedure for the synthesis of 4a. Yield was 28% (2 steps). ¹H-NMR (CDCl₃) δ: 1.34 (6H, s), 1.83–1.90 (2H, m), 2.79–2.86 (2H, m), 6.38 (1H, s), 7.29 (1H, d, J = 9.6 Hz), 7.33–7.44 (4H, m), 7.61–7.66 (2H, m).

4-[4-(3-Hydroxy-3-methylbutyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (5h)

Compound 5h was prepared from 4h according to the procedure for the synthesis of 5a. Yield was 42%. ¹H-NMR (CDCl₃) δ: 1.34 (6H, s), 1.83–1.92 (2H, m), 2.80–2.89 (2H, m), 6.46 (1H, s), 7.33–7.38 (2H, m), 7.39–7.44 (2H, m), 7.49 (1H, d, J = 8.5 Hz), 7.80 (1H, dd, J = 8.6, 1.9 Hz), 7.86 (1H, d, J = 1.9 Hz).

4-[4-(3-Hydroxy-3-methylbutyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (6h)

Compound 6h was prepared from 5h according to the procedure for the synthesis of 6a. Yield was 78%. mp 234–235 °C; ¹H-NMR (DMSO-d₆) δ: 1.19 (6H, s), 1.68–1.78 (2H, m), 2.68–2.81 (2H, m), 4.31 (1H, s), 6.47 (1H, s), 7.39–7.44 (2H, m), 7.46–7.52 (3H, m), 7.56 (1H, d, J = 8.7 Hz), 8.04 (1H, d, J = 2.0 Hz), 8.10–8.20 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 29.2 (s), 30.0 (s), 45.3 (s), 68.6 (s), 114.9 (s), 116.8 (s), 118.1 (s), 126.8 (s), 128.6 (s), 128.7 (s), 130.3 (s), 130.7 (s), 131.6 (s), 145.1 (s), 154.8 (s), 155.3 (s), 159.2 (s), 166.5 (s); IR (ATR) cm⁻¹: 3398, 1712, 1677; HR-MS (ESI-TOF) Calcd for C₂₁H₂₁NNaO₄ 374.1368. Found 374.1363; HPLC purity 98.8% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

1-Bromo-4-tert-butoxymethylbenzene (14i)

Following the addition of t-BuOK (20.0 g, 178 mmol) to a solution of 12 (10.0 g, 40.0 mmol) in t-BuOH (100 mL), the reaction mixture was stirred at 70 °C for 12 h. Water was added and 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 14i (9.41 g, 97% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.27 (9H, s), 4.39 (2H, s), 7.17–7.24 (2H, m), 7.40–7.46 (2H, m).

2-(4-tert-Butoxymethylphenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (8i)

Compound 8i was prepared from 14i according to the procedure for the synthesis of 3g. Yield was 85%. ¹H-NMR (CDCl₃) δ: 1.28 (9H, s), 1.34 (12H, s), 4.46 (2H, s), 7.30–7.39 (2H, m), 7.72–7.82 (2H, m).

6-Bromo-4-(4-tert-Butoxymethylphenyl)chromen-2-one (9i)

Compound 9i was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8i according to the procedure for the synthesis of 4a. The crude product was used in the next reaction without further purification.

4-(4-tert-Butoxymethylphenyl)-2-oxo-2H-chromene-6-carbonitrile (10i)

Compound 10i was prepared from 9i according to the procedure for the synthesis of 5a. Yield was 12% (2 steps). ¹H-NMR (CDCl₃) δ: 1.35 (9H, s), 4.56 (2H, s), 6.46 (1H, s), 7.36–7.43 (2H, m), 7.49 (1H, d, J = 8.5 Hz), 7.53–7.60 (2H, m), 7.79 (1H, dd, J = 8.5, 2.0 Hz), 7.84 (1H, d, J = 2.0 Hz).

4-(4-tert-Butoxymethylphenyl)-2-oxo-2H-chromene-6-carboxamide (11i)

Compound 11i was prepared from 10i according to the procedure for the synthesis of 6a. Yield was 21%. mp 182–185 °C; ¹H-NMR (DMSO-d₆) δ: 1.27 (9H, s), 4.53 (2H,s), 6.48 (1H, s), 7.47 (1H, brs), 7.50–7.63 (5H, m), 8.01 (1H, d, J = 2.0 Hz), 8.06–8.18 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 27.4 (s), 62.9 (s), 73.1 (s), 115.2 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.6 (s), 128.4 (s), 130.4 (s), 130.8 (s), 133.0 (s), 141.9 (s), 154.8 (s), 155.3 (s), 159.3 (s), 166.6 (s); IR (ATR) cm⁻¹: 1735, 1654; HR-MS (ESI-TOF) Calcd for C₂₁H₂₁NNaO₄ 374.1368. Found 374.1360; HPLC purity 96.3% (eluent: 0.01 M KH₂PO₄-MeCN (30 : 70)).

1-Bromo-4-isopropoxymethylbenzene (14j)

Following the addition of NaH (60% oil dispersion, 0.32 g, 8.0 mmol) to a solution of i-PrOH (0.51 mL, 6.7 mmol) in DMF (10 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 30 min. After the addition of 12 (2.00 g, 8.00 mmol) in DMF (10 mL), the mixture was stirred at room temperature for 1 h. Water was added and 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 14j (0.65 g, 43% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.21 (6H, d, J = 6.1 Hz), 3.66 (1H, septet, J = 6.1 Hz), 4.45 (2H, s), 7.17–7.24 (2H, m), 7.41–7.50 (2H, m).

2-(4-Isopropoxymethylphenyl)-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (8j)

Compound 8j was prepared from 14j according to the procedure for the synthesis of 3g. Yield was 81%. ¹H-NMR (CDCl₃) δ: 1.20 (6H, d, J = 6.1 Hz), 1.34 (12H, s), 3.66 (1H, septet, J = 6.1 Hz), 4.53 (2H, s), 7.31–7.40 (2H, m), 7.74–7.83 (2H, m).

6-Bromo-4-(4-isopropoxymethylphenyl)chromen-2-one (9j)

Compound 9j was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8j according to the procedure for the synthesis of 4a. Yield was 57%. ¹H-NMR (CDCl₃) δ: 1.27–1.31 (6H, m), 3.72–3.83 (1H, m), 4.61 (2H, s), 6.38 (1H, s), 7.27–7.32 (1H, m), 7.37–7.45 (2H, m), 7.50–7.58 (2H, m), 7.59–7.68 (2H, m).

4-(4-Isopropoxymethylphenyl)-2-oxo-2H-chromene-6-carbonitrile (10j)

Compound 10j was prepared from 9j according to the procedure for the synthesis of 5a. Yield was 77%. ¹H-NMR (CDCl₃) δ: 1.28 (6H, d, J = 6.1 Hz), 3.77 (1H, septet, J = 6.1 Hz), 4.62 (2H, s), 6.46 (1H, s), 7.36–7.44 (2H, m), 7.49 (1H, d, J = 8.5 Hz), 7.53–7.60 (2H, m), 7.76–7.87 (2H, m).

4-(4-Isopropoxymethylphenyl)-2-oxo-2H-chromene-6-carboxamide (11j)

Compound 11j was prepared from 10j according to the procedure for the synthesis of 6a. Yield was 19%. mp 170–173 °C; ¹H-NMR (CDCl₃) δ: 1.20 (6H, d, J = 6.1 Hz), 3.72 (1H, septet, J = 6.1 Hz), 4.59 (2H, s), 6.49 (1H, s), 7.47 (1H, brs), 7.52–7.61 (5H, m), 8.00 (1H, d, J = 1.8 Hz), 8.08–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 22.0 (s), 68.6 (s), 70.7 (s), 115.2 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.6 (s), 128.5 (s), 130.4 (s), 130.8 (s), 133.2 (s), 141.2 (s), 154.7 (s), 155.3 (s), 159.3 (s), 166.5 (s); IR (ATR) cm⁻¹: 1714, 1660; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₄ 360.1212. Found 360.1202; HPLC purity 98.8% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

2-[2-(4-Bromobenzyloxy)ethoxy]tetrahydro-2H-pyran (14k)

Compound 14k was prepared from 12 and 13k according to the procedure for the synthesis of 14j. Yield was 79%. ¹H-NMR (CDCl₃) δ: 1.50–1.90 (6H, m), 3.47–3.54 (1H, m), 3.60–3.69 (3H, m), 3.83–3.93 (2H, m), 4.49–4.58 (2H, m), 4.64 (1H, t, J = 3.6 Hz), 7.20–7.25 (2H, m), 7.44–7.48 (2H, m).

2-{2-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)benzyloxy]ethoxy}-tetrahydro-2H-pyran (8k)

Compound 8k was prepared from 14k according to the procedure for the synthesis of 3g. Yield was 79%. ¹H-NMR (CDCl₃) δ: 1.34 (12H, s), 1.49–1.89 (6H, m), 3.44–3.53 (1H, m), 3.60–3.68 (3H, m), 3.82–3.94 (2H, m), 4.57–4.68 (3H, m), 7.33–7.38 (2H, m), 7.76–7.81 (2H, m).

6-Bromo-4-{4-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxymethyl]phenyl}chromen-2-one (9k)

Compound 9k was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8k according to the procedure for the synthesis of 4a. Yield was 74%. ¹H-NMR (CDCl₃) δ: 1.50–1.90 (6H, m), 3.49–3.58 (1H, m), 3.67–3.79 (3H, m), 3.86–4.00 (2H, m), 4.65–4.73 (3H, m), 6.39 (1H, s), 7.29 (1H, d, J = 8.5 Hz), 7.39–7.44 (2H, m), 7.53–7.58 (2H, m), 7.60 (1H, d, J = 2.2 Hz), 7.64 (1H, dd, J = 8.5, 2.2 Hz).

2-Oxo-4-{4-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxymethyl]phenyl}-2H-chromene-6-carbonitrile (10k)

Compound 10k was prepared from 9k according to the procedure for the synthesis of 5a. Yield was 67%. ¹H-NMR (CDCl₃) δ: 1.50–1.91 (6H, m), 3.50–3.58 (1H, m), 3.66–3.79 (3H, m), 3.86–4.00 (2H, m), 4.66–4.74 (3H, m), 6.47 (1H, s), 7.39–7.44 (2H, m), 7.50 (1H, d, J = 8.5 Hz), 7.55–7.60 (2H, m), 7.80 (1H, dd, J = 8.5, 2.0 Hz), 7.83 (1H, d, J = 2.0 Hz).

4-[4-(2-Hydroxyethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (11k)

Compound 11k was prepared from 10k according to the procedure for the synthesis of 6a. Yield was 39%. mp 186–189 °C; ¹H-NMR (DMSO-d₆) δ: 3.55–3.69 (4H, m), 4.63 (2H, s), 4.71 (1H, t, J = 5.2 Hz), 6.49 (1H, s), 7.49 (1H, s), 7.53–7.64 (5H, m), 7.98–8.02 (1H, m), 8.13–8.24 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 60.3 (s), 71.6 (s), 72.1 (s), 115.2 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.8 (s), 128.5 (s), 130.4 (s), 130.8 (s), 133.4 (s), 140.6 (s), 154.7 (s), 155.3 (s), 159.3 (s), 166.6 (s); IR (ATR) cm⁻¹: 1732, 1672; HR-MS (ESI-TOF) Calcd for C₁₉H₁₇NNaO₅ 362.1004. Found 362.1004; HPLC purity 98.2% (eluent: 0.01 M KH₂PO₄-MeCN (60 : 40)).

1-Bromo-4-(2-fluoroethoxymethyl)benzene (14l)

Compound 14l was prepared from 12 and 13l according to the procedure for the synthesis of 14j. Yield was 88%. ¹H-NMR (CDCl₃) δ: 3.65–3.77 (2H, m), 4.51–4.67 (4H, m), 7.20–7.25 (2H, m), 7.45–7.49 (2H, m).

2-[4-(2-Fluoroethoxymethyl)phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (8l)

Compound 8l was prepared from 14l according to the procedure for the synthesis of 3g. Yield was 97%. ¹H-NMR (CDCl₃) δ: 1.35 (12H, s), 3.65–3.77 (2H, m), 4.51–4.67 (4H, m), 7.33–7.38 (2H, m), 7.78–7.82 (2H, m).

6-Bromo-4-[4-(2-fluoroethoxymethyl)phenyl]chromen-2-one (9l)

Compound 9l was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8l according to the procedure for the synthesis of 4a. Yield was 37%. ¹H-NMR (CDCl₃) δ: 3.83 (2H, ddd, J = 29.8, 4.4, 3.6 Hz), 4.58–4.74 (4H, m), 6.39 (1H, s), 7.29 (1H, d, J = 8.8 Hz), 7.41–7.45 (2H, m), 7.54–7.58 (2H, m), 7.59 (1H, d, J = 2.2 Hz), 7.64 (1H, dd, J = 8.8, 2.2 Hz).

4-[4-(2-Fluoroethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (10l)

Compound 10l was prepared from 9l according to the procedure for the synthesis of 5a. Yield was 88%. ¹H-NMR (CDCl₃) δ: 3.78–3.89 (2H, m), 4.58–4.73 (4H, m), 6.39 (1H, s), 7.30 (1H, d, J = 8.8 Hz), 7.41–7.45 (2H, m), 7.53–7.57 (2H, m), 7.59 (1H, d, J = 2.4 Hz), 7.64 (1H, dd, J = 8.8, 2.4 Hz).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]-2H-chromene-6-carboxamide (11l)

Compound 11l was prepared from 10l according to the procedure for the synthesis of 6a. Yield was 55%. mp 161–162 °C; ¹H-NMR (DMSO-d₆) δ: 3.73–3.83 (2H, m), 4.56–4.73 (4H, m), 6.50 (1H, s), 7.49 (1H, brs), 7.55–7.65 (5H, m), 7.97–8.05 (1H, m), 8.13–8.20 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 69.3 (d), 71.5 (s), 83.0 (d), 115.3 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.8 (s), 128.6 (s), 130.5 (s), 130.8 (s), 133.5 (s), 140.2 (s), 154.6 (s), 155.3 (s), 159.3 (s), 166.6 (s); IR (ATR) cm⁻¹: 1720, 1697; HR-MS (ESI-TOF) Calcd for C₁₉H₁₆FNNaO₄ 364.0961. Found 364.0947; HPLC purity 97.2% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

4-(4-Bromobenzyloxy)tetrahydro-2H-pyran (14m)

Compound 14m was prepared from 12 and 13m according to the procedure for the synthesis of 14j. Yield was 47%. ¹H-NMR (CDCl₃) δ: 1.59–1.71 (2H, m), 1.88–1.97 (2H, m), 3.40–3.48 (2H, m), 3.53–3.62 (1H, m), 3.92–4.00 (2H, m), 4.51 (2H, s), 7.20–7.25 (2H, m), 7.44–7.49 (2H, m).

4-[4-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)benzyloxy]tetrahydro-2H-pyran (8m)

Compound 8m was prepared from 14m according to the procedure for the synthesis of 3g. Yield was 97%. ¹H-NMR (CDCl₃) δ: 1.34 (12H, s), 1.59–1.72 (2H, m), 1.87–1.97 (2H, m), 3.38–3.47 (2H, m), 3.52–3.61 (1H, m), 3.91–3.99 (2H, m), 4.58 (2H, s), 7.31–7.36 (2H, m), 7.76–7.81 (2H, m)

6-Bromo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chromen-2-one (9m)

Compound 9m was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8m according to the procedure for the synthesis of 4a. Yield was 57%. ¹H-NMR (CDCl₃) δ: 1.66–1.77 (2H, m), 1.97–2.07 (2H, m), 3.45–3.55 (2H, m), 3.64–3.73 (1H, m), 3.97–4.05 (2H, m), 4.66 (2H, s), 6.39 (1H, s), 7.29 (1H, d, J = 8.5 Hz), 7.39–7.45 (2H, m), 7.52–7.57 (2H, m), 7.60 (1H, d, J = 2.4 Hz), 7.64 (1H, d, J = 8.5, 2.4 Hz).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carbonitrile (10m)

Compound 10m was prepared from 9m according to the procedure for the synthesis of 5a. Yield was 89%. ¹H-NMR (CDCl₃) δ: 1.66–1.78 (2H, m), 1.96–2.05 (2H, m), 3.45–3.55 (2H, m), 3.65–3.73 (1H, m), 3.97–4.04 (2H, m), 4.67 (2H, s), 6.46 (1H, s), 7.39–7.43 (2H, m), 7.49 (1H, d, J = 8.6 Hz), 7.54–7.59 (2H, m), 7.80 (1H, d, J = 8.6, 2.0 Hz), 7.83 (1H, d, J = 2.0 Hz).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxamide (11m)

Compound 11m was prepared from 10m according to the procedure for the synthesis of 6a. Yield was 35%. mp 197–199 °C; ¹H-NMR (DMSO-d₆) δ: 1.44–1.55 (2H, m), 1.88–1.98 (2H, m), 3.30–3.39 (2H, m), 3.60–3.67 (1H, m), 3.83 (2H, dt, J = 11.6, 4.3 Hz), 4.63 (2H, s), 6.47 (1H, s), 7.47 (1H, s), 7.52–7.56 (5H, m), 7.99 (1H, d, J = 2.0 Hz), 8.08–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 32.2 (s), 64.8 (s), 68.1 (s), 73.3 (s), 115.2 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.6 (s), 128.6 (s), 130.4 (s), 130.8 (s), 133.3 (s), 141.0 (s), 154.7 (s), 155.3 (s), 159.3 (s), 166.6 (s); IR (ATR) cm⁻¹: 1724, 1671; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₅ 402.1317. Found 402.1308; HPLC purity 98.9% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

3-(4-Bromobenzyloxymethyl)-3-methyloxetane (14n)

Compound 14n was prepared from 12 and 13n according to the procedure for the synthesis of 14j. Yield was 81%. ¹H-NMR (CDCl₃) δ: 1.33 (3H, s), 3.51 (2H, s), 4.36 (2H, d, J = 5.6 Hz), 4.50–4.53 (4H, m), 7.19–7.23 (2H, m), 7.45–7.49 (2H, m).

4,4,5,5-Tetramethyl-2-[4-(3-methyloxetan-3-ylmethoxymethyl)phenyl][1,3,2]dioxaborolane (8n)

Compound 8n was prepared from 14n according to the procedure for the synthesis of 3g. Yield was 93%. ¹H-NMR (CDCl₃) δ: 1.32 (3H, s), 1.34 (12H, s), 3.50 (2H, s), 4.35 (2H, d, J = 5.6 Hz), 4.51 (2H, d, J = 5.6 Hz), 4.59 (2H, s), 7.32–7.36 (2H, m), 7.77–7.82 (2H, m).

6-Bromo-4-[4-(3-methyloxetan-3-ylmethoxymethyl)phenyl]chromen-2-one (9n)

Compound 9n was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8n according to the procedure for the synthesis of 4a. Yield was 56%. ¹H-NMR (CDCl₃) δ: 1.39 (3H, s), 3.64 (2H, s), 4.42 (2H, d, J = 5.6 Hz), 4.58 (2H, d, J = 5.6 Hz), 4.68 (2H, s), 6.40 (1H, s), 7.30 (1H, d, J = 8.8 Hz), 7.41–7.45 (2H, m), 7.51–7.56 (2H, m), 7.61 (1H, d, J = 2.0 Hz), 7.64 (1H, dd, J = 8.8, 2.0 Hz).

4-[4-(3-Methyloxetan-3-ylmethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (10n)

Compound 10n was prepared from 9n according to the procedure for the synthesis of 5a. Yield was 86%. ¹H-NMR (CDCl₃) δ: 1.39 (3H, s), 3.64 (2H, s), 4.42 (2H, d, J = 5.6 Hz), 4.58 (2H, d, J = 5.6 Hz), 4.69 (2H, s), 6.48 (1H, s), 7.40–7.45 (2H, m), 7.50 (1H, d, J = 8.5 Hz), 7.53–7.58 (2H, m), 7.79–7.82 (1H, m), 7.83 (1H, d, J = 1.5 Hz).

4-[4-(3-Methyloxetan-3-ylmethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (11n)

Compound 11n was prepared from 10n according to the procedure for the synthesis of 6a. Yield was 61%. mp 165–169 °C; ¹H-NMR (DMSO-d₆) δ: 1.36 (3H, s), 3.64 (2H, s), 4.30 (2H, d, J = 5.6 Hz), 4.50 (2H, d, J = 5.6 Hz), 4.72 (2H, s), 6.55 (1H, s), 7.54 (1H, s), 7.59–7.82 (5H, m), 8.07 (1H, s), 8.16–8.23 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 21.2 (s), 39.4 (s), 71.9 (s), 75.2 (s), 78.8 (s), 115.3 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.7 (s), 128.6 (s), 130.5 (s), 130.8 (s), 133.5 (s), 140.4 (s), 154.6 (s), 155.3 (s), 159.3 (s), 166.6 (s); IR (ATR) cm⁻¹: 3365, 3120, 1716, 1662; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₅ 402.1317. Found 402.1314; HPLC purity 97.3% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (60 : 40 : 0.2)).

4-(4-Iodobenzyloxy)tetrahydrofuran-3-ol (16o)

Compound 16o was prepared from 15 and 13o according to the procedure for the synthesis of 14j. Yield was 66%. ¹H-NMR (CDCl₃) δ: 2.58–2.72 (1H, m), 3.75 (1H, dd, J = 9.5, 4.2 Hz), 3.78 (1H, dd, J = 9.5, 5.4 Hz), 3.90 (2H, dd, J = 9.5, 5.6 Hz), 4.02–4.07 (1H, m), 4.22–4.31 (1H, m), 4.56 (2H, s), 7.06–7.12 (2H, m), 7.67–7.73 (2H, m).

tert-Butyl-[4-(4-iodobenzyloxy)tetrahydrofuran-3-yloxy]dimethylsilane (17o)

Following the addition of imidazole (600 mg, 8.81 mmol) and tert-butyldimethylchlorosilane (TBSCl) (730 mg, 4.84 mmol) to a solution of 16o (1.41 g, 4.40 mmol) in DMF (15 mL), the reaction mixture was stirred at room temperature for 2 h. AcOEt was added, and 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 to give 17o (1.74 g, 91% yield) as an oil. ¹H-NMR (CDCl₃) δ: 0.09 (3H, s), 0.10 (3H, s), 0.91 (9H, s), 3.67 (1H, dd, J = 8.6, 6.4 Hz), 3.81 (1H, dd, J = 9.0, 4.2 Hz), 3.86–3.92 (2H, m), 3.95 (1H, dd, J = 9.0, 5.2 Hz), 4.27–4.34 (1H, m), 4.50 (1H, d, J = 12.4 Hz), 4.71 (1H, d, J = 12.4 Hz), 7.07–7.12 (2H, m), 7.63–7.68 (2H, m).

2-{4-[4-(tert-Butyldimethylsilyloxy)tetrahydrofuran-3-yloxymethyl]phenyl}-4,4,5,5-tetra-methyl[1,3,2]dioxaborolane (8o)

Following the addition of triethylamine (Et₃N) (2.77 mL, 19.9 mmol), 4,4,5,5-tetramethyl-1,3,2-dioxaborolane (0.87 mL, 5.9 mmol), and PdCl₂(dppf)·CH₂Cl₂ (100 mg, 0.122 mmol) to a solution of 17o (1.73 g, 3.98 mmol) in 1,4-dioxane (20 mL), the reaction mixture was stirred at 100 °C for 3 h under a N₂ atmosphere. After cooling, AcOEt was added, and the reaction 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 to give 8o (980 mg, crude) as an oil. The crude product was used in the next reaction without further purification.

6-Bromo-4-{4-[4-(tert-butyldimethylsilyloxy)tetrahydrofuran-3-yloxymethyl]phenyl}-chromen-2-one (9o)

Compound 9o was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8o according to the procedure for the synthesis of 4a. Yield was 25% (2 steps). ¹H-NMR (CDCl₃) δ: 0.12 (3H, s), 0.13 (3H, s), 0.93 (9H, s), 3.72 (1H, dd, J = 8.6, 6.6 Hz), 3.90 (1H, dd, J = 8.8, 3.7 Hz), 3.94 (1H, dd, J = 8.6, 5.9 Hz), 3.97–4.06 (2H, m), 4.33–4.40 (1H, m), 4.68 (1H, d, J = 12.2 Hz), 4.90 (1H, d, J = 12.2 Hz), 6.39 (1H, s), 7.29 (1H, d, J = 8.8 Hz), 7.38–7.44 (2H, m), 7.52–7.57 (2H, m), 7.59 (1H, d, J = 2.2 Hz), 7.64 (1H, dd, J = 8.8, 2.2 Hz).

4-{4-[4-(tert-Butyldimethylsilyloxy)tetrahydrofuran-3-yloxymethyl]phenyl}-2-oxo-2H-chromene-6-carbonitrile (10o)

Compound 10o was prepared from 9o according to the procedure for the synthesis of 5a. Yield was 98%. ¹H-NMR (CDCl₃) δ: 0.12 (3H, s), 0.14 (3H, s), 0.93 (9H, s), 3.72 (1H, dd, J = 8.3, 6.6 Hz), 3.90 (1H, dd, J = 8.8, 3.6 Hz), 3.94 (1H, dd, J = 8.3, 5.9 Hz), 3.97–4.07 (2H, m), 4.33–4.40 (1H, m), 4.69 (1H, d, J = 12.4 Hz), 4.91 (1H, d, J = 12.4 Hz), 6.47 (1H, s), 7.38–7.43 (2H, m), 7.49 (1H, d, J = 8.8 Hz), 7.54–7.60 (2H, m), 7.77–7.83 (2H, m).

4-[4-(4-Hydroxytetrahydrofuran-3-yloxymethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (11o)

Compound 11o was prepared from 10o according to the procedure for the synthesis of 6a. Yield was 21%. mp 156–159 °C; ¹H-NMR (CDCl₃) δ: 3.56 (1H, dd, J = 8.7, 4.9 Hz), 3.68 (1H, dd, J = 8.9, 5.5 Hz), 3.80–3.90 (2H, m), 4.00–4.06 (1H, m), 4.22–4.30 (1H, m), 4.65 (1H, d, J = 12.4 Hz), 4.81 (1H, d, J = 12.4 Hz), 4.93 (1H, d, J = 5.7 Hz), 6.49 (1H, s), 7.48 (1H, s), 7.54–7.63 (5H, m), 8.00 (1H, d, J = 2.0 Hz), 8.10–8.20 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 69.3 (s), 69.6 (s), 70.5 (s), 72.1 (s), 78.7 (s), 115.2 (s), 116.8 (s), 118.1 (s), 126.7 (s), 127.8 (s), 128.4 (s), 130.4 (s), 130.7 (s), 133.3 (s), 140.5 (s), 154.6 (s), 155.2 (s), 159.2 (s), 166.5 (s); IR (ATR) cm⁻¹: 1714, 1695; HR-MS (ESI-TOF) Calcd for C₂₁H₁₉NNaO₆ 404.1110. Found 404.1103; HPLC purity 99.9% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (70 : 30 : 0.2)).

Methyl 2-(4-Iodobenzyloxy)-2-methylpropionate (16p)

Compound 16p was prepared from 15 and 13p according to the procedure for the synthesis of 14j. Yield was 51%. ¹H-NMR (CDCl₃) δ: 1.50 (6H, s), 3.75 (3H, s), 4.41 (2H, s), 7.11–7.16 (2H, m), 7.63–7.68 (2H, m).

Methyl 2-Methyl-2-[4-(4,4,5,5-tetramethyl[1,3,2]dioxaborolan-2-yl)benzyloxy]propionate (8p)

Compound 8p was prepared from 16p according to the procedure for the synthesis of 8o. Yield was 48%. ¹H-NMR (CDCl₃) δ: 1.34 (12H, s), 1.51 (6H, s), 3.75 (3H, s), 4.49 (2H, s), 7.35–7.39 (2H, m), 7.75–7.79 (2H, m).

Methyl 2-[4-(6-Bromo-2-oxo-2H-chromen-4-yl)benzyloxy]-2-methylpropionate (9p)

Compound 9p was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8p according to the procedure for the synthesis of 4a. Yield was 56% ¹H-NMR (CDCl₃) δ: 1.57 (6H, s), 3.80 (3H, s), 4.58 (2H, s), 6.38 (1H, s), 7.29 (1H, d, J = 8.6 Hz), 7.39–7.44 (2H, m), 7.57–7.66 (4H, m).

Methyl 2-[4-(6-Cyano-2-oxo-2H-chromen-4-yl)benzyloxy]-2-methylpropionate (10p)

Compound 10p was prepared from 9p according to the procedure for the synthesis of 5a. Yield was 80%. ¹H-NMR (CDCl₃) δ: 1.57 (6H, s), 3.80 (3H, s), 4.59 (2H, s), 6.46 (1H, s), 7.39–7.43 (2H, m), 7.49 (1H, d, J = 8.6 Hz), 7.60–7.64 (2H, m), 7.80 (1H, dd, J = 8.6, 2.0 Hz), 7.84 (1H, d, J = 2.0 Hz).

2-[4-(6-Carbamoyl-2-oxo-2H-chromen-4-yl)benzyloxy]-2-methylpropionic Acid (11p)

Compound 11p was prepared from 10p according to the procedure for the synthesis of 6a. Yield was 66%. mp 223–227 °C; ¹H-NMR (DMSO-d₆) δ: 1.46 (6H, s), 4.57 (2H, s), 6.49 (1H, s), 7.48 (1H, s), 7.50–7.80 (5H, m), 8.02 (1H, s), 8.10–8.25 (2H, m), 12.76 (1H, brs); ¹³C-NMR (DMSO-d₆) δ: 24.6 (s), 65.6 (s), 77.2 (s), 115.2 (s), 116.9 (s), 118.2 (s), 126.8 (s), 127.9 (s), 128.4 (s), 130.5 (s), 130.8 (s), 133.3 (s), 140.9 (s), 154.8 (s), 155.4 (s), 159.3 (s), 166.6 (s), 175.6 (s); IR (ATR) cm⁻¹: 3398, 3199, 1714, 1693; HR-MS (ESI-TOF) Calcd for C₂₁H₁₉NNaO₆ 404.1110. Found 404.1108; HPLC purity 96.6% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (60 : 40 : 0.2)).

1-Bromo-4-(2-isopropoxyethoxymethyl)benzene (14q)

Compound 14q was prepared from 12 and 13q according to the procedure for the synthesis of 14j. Yield was 81%. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 3.57–3.66 (5H, m), 4.52 (2H, s), 7.20–7.25 (2H, m), 7.43–7.48 (2H, m).

2-[4-(2-Isopropoxyethoxymethyl)phenyl]-4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (8q)

Compound 8q was prepared from 14q according to the procedure for the synthesis of 3g. Yield was 93%. ¹H-NMR (CDCl₃) δ: 1.17 (6H, d, J = 6.1 Hz), 1.34 (12H, s), 3.57–3.66 (5H, m), 4.60 (2H, s), 7.33–7.38 (2H, m), 7.76–7.81 (2H, m).

6-Bromo-4-[4-(2-isopropoxyethoxymethyl)phenyl]chromen-2-one (9q)

Compound 9q was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 8q according to the procedure for the synthesis of 4a. Yield was 65%. ¹H-NMR (CDCl₃) δ: 1.20 (6H, d, J = 6.1 Hz), 3.62–3.74 (5H, m), 4.68 (2H, s), 6.39 (1H, s), 7.29 (1H, d, J = 8.8 Hz), 7.39–7.44 (2H, m), 7.52–7.57 (2H, m), 7.60 (1H, d, J = 2.2 Hz), 7.63 (1H, dd, J = 8.8, 2.2 Hz).

4-[4-(2-Isopropoxyethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carbonitrile (10q)

Compound 10q was prepared from 9q according to the procedure for the synthesis of 5a. Yield was 79%. ¹H-NMR (CDCl₃) δ: 1.21 (6H, d, J = 6.1 Hz), 3.62–3.74 (5H, m), 4.70 (2H, s), 6.47 (1H, s), 7.38–7.43 (2H, m), 7.50 (1H, d, J = 8.5 Hz), 7.55–7.60 (2H, m), 7.80 (1H, dd, J = 8.5, 2.0 Hz), 7.83 (1H, d, J = 2.0 Hz).

4-[4-(2-Isopropoxyethoxymethyl)phenyl]-2-oxo-2H-chromene-6-carboxamide (11q)

Compound 11q was prepared from 10q according to the procedure for the synthesis of 6a. Yield was 37%. mp 89–91 °C; ¹H-NMR (DMSO-d₆) δ: 1.11 (6H, d, J = 6.1 Hz), 3.56–3.73 (5H, m), 4.63 (2H, s), 6.49 (1H, s), 7.48 (1H, s), 7.50–7.71 (5H, m), 8.02 (1H, s), 8.08–8.22 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 22.0 (s), 66.7 (s), 69.8 (s), 70.9 (s), 71.5 (s), 115.2 (s), 116.9 (s), 118.1 (s), 126.8 (s), 127.7 (s), 128.5 (s), 130.5 (s), 130.8 (s), 133.4 (s), 140.6 (s), 154.7 (s), 155.3 (s), 159.2 (s), 166.6 (s); IR (ATR) cm⁻¹: 1724, 1664; HR-MS (ESI-TOF) Calcd for C₂₂H₂₃NNaO₅ 404.1474. Found 404.1466; HPLC purity 98.8% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

6-Chloro-4-(4-{[(tetrahydro-2H-pyran-4-yl)oxy]methyl}phenyl)-2H-chromen-2-one (19)

Compound 19 was prepared from 18¹⁴⁾ and 8m according to the procedure for the synthesis of 4a. Yield was 41%. mp 99–101 °C; ¹H-NMR (CDCl₃) δ: 1.47–1.56 (2H, m), 1.90–1.98 (2H, m), 3.33–3.42 (2H, m), 3.61–3.70 (1H, m), 3.85 (2H, dt, J = 11.6, 4.3 Hz), 4.64 (2H, s), 6.51 (1H, s), 7.37 (1H, d, J = 2.4 Hz), 7.50–7.60 (5H, m), 7.72 (1H, dd, J = 8.9, 2.4 Hz); ¹³C-NMR (DMSO-d₆) δ: 32.2 (s), 64.8 (s), 68.0 (s), 73.2 (s), 115.8 (s), 119.1 (s), 119.9 (s), 125.6 (s), 127.7 (s), 128.3 (s), 128.4 (s), 131.8 (s), 132.9 (s), 141.1 (s), 152.2 (s), 153.5 (s), 159.1 (s); IR (ATR) cm⁻¹: 1718; HR-MS (ESI-TOF) Calcd for C₂₁H₁₉ClNaO₄ 393.0870. Found 393.0885; HPLC purity 99.1% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (60 : 40 : 0.2)).

4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-6-trimethylsilylethynylchromen-2-one (20)

Following the addition of trimethylsilylacetylene (0.90 mL, 6.5 mmol), PdCl₂(PPh₃)₂ (18 mg, 0.026 mmol), CuI (5 mg, 0.03 mmol), and Et₃N (9 mL) to 9m (527 mg, 1.3 mmol), the reaction mixture was heated to reflux for 2 h under a N₂ atmosphere. After cooling, the mixture was concentrated under reduced pressure. Water and AcOEt were added and the insoluble material was filtrated. The filtrate was separated, and 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 20 (377 mg, 67% yield) as a solid. ¹H-NMR (CDCl₃) δ: 0.23 (9H, s), 1.67–1.78 (2H, m), 1.97–2.06 (2H, m), 3.45–3.54 (2H, m), 3.65–3.72 (1H, m), 3.97–4.04 (2H, m), 4.67 (2H, s), 6.37 (1H, s), 7.33 (1H, d, J = 8.5 Hz), 7.41–7.46 (2H, m), 7.52–7.57 (3H, m), 7.62 (1H, dd, J = 8.5, 1.7 Hz).

6-Ethynyl-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chromen-2-one (21)

Following the addition of water (0.25 mL) and HCO₂H (5 mL) to 20 (377 mg, 0.872 mmol), the reaction mixture was stirred at 40 °C for 4 h. Water was added and the mixture was neutralized with saturated aqueous NaHCO₃. The mixture was extracted with AcOEt, and the organic layer was washed with saturated aqueous NaHCO₃ 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 Et₂O was added. The insoluble material was collected by filtration to give 21 (230 mg, 70% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.67–1.78 (2H, m), 1.97–2.06 (2H, m), 3.05 (1H, s), 3.45–3.54 (2H, m), 3.64–3.72 (1H, m), 3.97–4.04 (2H, m), 4.66 (2H, s), 6.39 (1H, s), 7.36 (1H, d, J = 8.3 Hz), 7.41–7.46 (2H, m), 7.52–7.57 (2H, m), 7.60–7.67 (2H, m).

6-Acetyl-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chromen-2-one (22)

Following the addition of water (0.4 mL) and HCO₂H (4 mL) to 21 (210 mg, 0.583 mmol), the reaction mixture was stirred at 90 °C for 5 h. After cooling, water was added and the mixture was neutralized with saturated aqueous NaHCO₃. The mixture was extracted with AcOEt, and the organic layer was washed with saturated aqueous NaHCO₃ 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 t-BuOMe was added. The insoluble material was collected by filtration to give 22 (121 mg, 55% yield) as a solid. mp 123–125 °C; ¹H-NMR (CDCl₃) δ: 1.46–1.58 (2H, m), 1.91–1.99 (2H, m), 2.55 (3H, s), 3.37–3.42 (2H, m), 3.62–3.71 (1H, m), 3.85 (2H, dt, J = 11.6, 4.3 Hz), 4.66 (2H, s), 6.52 (1H, s), 7.55–7.62 (5H, m), 8.01 (1H, d, J = 2.0 Hz), 8.24 (1H, dd, J = 8.6, 2.0 Hz); ¹³C-NMR (DMSO-d₆) δ: 26.6 (s), 32.2 (s), 64.8 (s), 68.1 (s), 73.3 (s), 115.4 (s), 117.4 (s), 118.3 (s), 126.8 (s), 127.7 (s), 128.5 (s), 132.1 (s), 132.8 (s), 133.1 (s), 141.2 (s), 154.4 (s), 156.4 (s), 159.0 (s), 196.3 (s); IR (ATR) cm⁻¹: 1736, 1676; HR-MS (ESI-TOF) Calcd for C₂₃H₂₂NaO₅ 401.1365. Found 401.1353; HPLC purity 98.1% (eluent: 0.01 M KH₂PO₄-MeCN (30 : 70)).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carbaldehyde (23)

Following the addition of pyridine (5 mL), NaH₂PO₂·H₂O (1.17 g, 11.0 mmol) in H₂O (5 mL), and Raney Ni 2800 (1.0 g) to a solution of 10m (495 mg, 1.37 mmol) in AcOH (5 mL), the reaction mixture was stirred at 40 °C for 3 h. After cooling, AcOEt was added and the insoluble material was filtrated. The filtrate was washed with water, 2.0 M HCl, saturated aqueous NaHCO₃, 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 23 (90 mg, 18% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.67–1.78 (2H, m), 1.97–2.06 (2H, m), 3.45–3.55 (2H, m), 3.66–3.74 (1H, m), 3.98–4.05 (2H, m), 4.68 (2H, s), 6.46 (1H, s), 7.45–7.50 (2H, m), 7.53–7.59 (3H, m), 8.04 (1H, d, J = 1.7 Hz), 8.08 (1H, dd, J = 8.8, 1.7 Hz), 9.95 (1H, s).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxylic Acid (24)

Following the addition of 80% NaClO₂ (32 mg, 0.28 mmol) in H₂O (0.5 mL), KH₂PO₄ (8 mg, 0.06 mmol), and 30% aqueous H₂O₂ (0.027 mL, 0.22 mmol) to a solution of 23 (72 mg, 0.20 mmol) in DMSO-MeCN (1 : 1) (1 mL) at room temperature, the reaction mixture was stirred for 2 h. Water and Et₂O were added and the insoluble material was collected by filtration to give 24 (38 mg, 50% yield) as a solid. mp 159–168 °C; ¹H-NMR (DMSO-d₆) δ: 1.42–1.58 (2H, m), 1.88–2.02 (2H, m), 3.60–3.72 (1H, m), 3.78–3.90 (2H, m), 4.65 (2H, s), 6.50–6.58 (1H, m), 7.52–7.62 (5H, m), 8.04 (1H, s), 8.16 (1H, d, J = 8.4 Hz); ¹³C-NMR (DMSO-d₆) δ: 32.1 (s), 64.7 (s), 68.0 (s), 73.2 (s), 115.3 (s), 117.4 (s), 118.4 (s), 126.8 (s), 127.6 (s), 128.1 (s), 128.4 (s), 132.6 (s), 133.1 (s), 141.0 (s), 154.3 (s), 156.3 (s), 159.1 (s), 166.0 (s); IR (ATR) cm⁻¹: 1716; HR-MS (ESI-TOF) Calcd for C₂₂H₂₀NaO₆ 403.1158. Found 403.1153; HPLC purity 93.0% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (60 : 40 : 0.2)).

6-Difluoromethyl-4-[4-(tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chromen-2-one (25)

Following the addition of diethylaminosulfur trifluoride (DAST) (40 mg, 0.25 mmol) to a solution of 23 (90 mg, 0.25 mmol) in CH₂Cl₂ (2 mL), the reaction mixture was stirred at room temperature for 13 h. After the addition of DAST (200 mg, 1.24 mmol), the mixture was heated to reflux for 2 h. After cooling, 10% aqueous citric acid was added, the mixture 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, and n-hexane (10 mL) and t-BuOMe (1 mL) were then added. The insoluble material was collected by filtration to give 25 (54 mg, 57% yield) as a solid. mp 113–115 °C; ¹H-NMR (DMSO-d₆) δ: 1.44–1.58 (2H, m), 1.88–2.02 (2H, m), 3.30–3.48 (2H, m), 3.61–3.72 (1H, m), 3.85 (2H, dt, J = 11.3, 4.1 Hz), 4.65 (2H, s), 6.53 (1H, s), 7.11 (1H, t, J = 55.6 Hz), 7.52–7.60 (4H, m), 7.62–7.70 (2H, m), 7.87 (1H, d, J = 8.7 Hz) ; ¹³C-NMR (DMSO-d₆) δ: 32.1 (s), 64.7 (s), 68.0 (s), 73.2 (s), 114.1 (t), 115.6 (s), 117.9 (s), 118.5 (s), 124.4 (t), 127.6 (s), 128.4 (s),129.1 (t), 130.1 (t), 133.0 (s), 141.0 (s), 154.1 (s), 154.9 (s), 159.1 (s); IR (ATR) cm⁻¹: 1720; HR-MS (ESI-TOF) Calcd for C₂₂H₂₀F₂NaO₄ 409.1227. Found 409.1224; HPLC purity 97.9% (eluent: 0.01 M KH₂PO₄-MeCN (30 : 70)).

6-Bromo-4-{4-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]phenyl}chromen-2-one (27r)

Compound 27r was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26r¹⁸⁾ according to the procedure for the synthesis of 4a. Yield was 69%. ¹H-NMR (CDCl₃) δ: 1.50–1.92 (6H, m), 3.52–3.60 (1H, m), 3.82–3.96 (2H, m), 4.07–4.15 (1H, m), 4.22–4.28 (2H, m), 4.71–4.75 (1H, m), 6.37 (1H, s), 7.07–7.13 (2H, m), 7.28 (1H, d, J = 8.6 Hz), 7.35–7.41 (2H, m), 7.63 (1H, dd, J = 8.6, 2.2 Hz), 7.65 (1H, d, J = 2.2 Hz).

2-Oxo-4-{4-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]phenyl}chromene-6-carbonitrile (28r)

Compound 28r was prepared from 27r according to the procedure for the synthesis of 5a. Yield was 86%. ¹H-NMR (CDCl₃) δ: 1.50–1.90 (6H, m), 3.53–3.60 (1H, m), 3.84–3.96 (2H, m), 4.09–4.15 (1H, m), 4.24–4.30 (2H, m), 4.71–4.75 (1H, m), 6.44 (1H, s), 7.09–7.15 (2H, m), 7.35–7.41 (2H, m), 7.49 (1H, d, J = 8.6 Hz), 7.80 (1H, dd, J = 8.6, 2.0 Hz), 7.89 (1H, d, J = 2.0 Hz).

4-[4-(2-Hydroxyethoxy)phenyl]-2-oxochromene-6-carboxamide (29r)

Compound 29r was prepared from 28r according to the procedure for the synthesis of 6a. Yield was 2.1%. ¹H-NMR (DMSO-d₆) δ: 3.76 (2H, td, J = 5.3, 4.8 Hz), 4.10 (2H, t, J = 4.8 Hz), 4.93 (1H, t, J = 5.3 Hz), 6.45 (1H, s), 7.12–7.19 (2H, m), 7.44–7.58 (4H, m), 8.06 (1H, d, J = 2.0 Hz), 8.08–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 59.4 (s), 69.6 (s), 114.5 (s), 114.8 (s), 116.8 (s), 118.2 (s), 126.4 (s), 126.8 (s), 130.1 (s), 130.3 (s), 130.6 (s), 154.5 (s), 155.3 (s), 159.3 (s), 159.9 (s), 166.5 (s); IR (ATR) cm⁻¹: 1712, 1608; HR-MS (ESI-TOF) Calcd for C₁₈H₁₅NNaO₅ 348.0848. Found 348.0842; HPLC purity 95.6% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (70 : 30 : 0.2)).

6-Bromo-4-(4-cyclopropylmethoxyphenyl)chromen-2-one (27s)

Compound 27s was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26s according to the procedure for the synthesis of 4a. Yield was 51%. ¹H-NMR (CDCl₃) δ: 0.36–0.43 (2H, m), 0.67–0.73 (2H, m), 1.27–1.38 (1H, m), 3.89 (2H, d, J = 7.1 Hz), 6.36 (1H, s), 7.03–7.08 (2H, m), 7.28 (1H, d, J = 8.8 Hz), 7.34–7.39 (2H, m), 7.62 (1H, dd, J = 8.8, 2.4 Hz), 7.66 (1H, d, J = 2.4 Hz).

4-(4-Cyclopropylmethoxyphenyl)-2-oxochromene-6-carbonitrile (28s)

Compound 28s was prepared from 27s according to the procedure for the synthesis of 5a. Yield was 80%. ¹H-NMR (CDCl₃) δ: 0.37–0.44 (2H, m), 0.67–0.74 (2H, m), 1.27–1.39 (1H, m), 3.90 (2H, d, J = 7.1 Hz), 6.44 (1H, s), 7.05–7.11 (2H, m), 7.33–7.39 (2H, m), 7.48 (1H, d, J = 8.8 Hz), 7.79 (1H, dd, J = 8.8, 2.0 Hz), 7.90 (1H, d, J = 2.0 Hz).

4-(4-Cyclopropylmethoxyphenyl)-2-oxochromene-6-carboxamide (29s)

Compound 29s was prepared from 28s according to the procedure for the synthesis of 6a. Yield was 45%. mp 182–186 °C; ¹H-NMR (DMSO-d₆) δ: 0.30–0.41 (2H, m), 0.52–0.68 (2H, m), 1.21–1.31 (1H, m), 3.91 (2H, d, J = 7.0 Hz), 6.41 (1H, s), 7.07–7.18 (2H, m), 7.40–7.56 (4H, m), 8.05 (1H, d, J = 1.8 Hz), 8.07–8.15 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 3.1 (s), 10.1 (s), 72.2 (s), 114.5 (s), 114.8 (s), 116.8 (s), 118.3 (s), 126.4 (s), 126.9 (s), 130.1 (s), 130.4 (s), 130.7 (s), 154.6 (s), 155.3 (s), 159.3 (s), 159.9 (s), 166.6 (s); IR (ATR) cm⁻¹: 1711, 1663; HR-MS (ESI-TOF) Calcd for C₂₀H₁₇NNaO₄ 358.1055. Found 358.1045; HPLC purity 98.5% (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 (26t)

Following the addition of PPh₃ (17.9 g, 68.2 mmol) and diisopropyl azodicarboxylate (DIAD) (13.2 mL, 68.2 mmol) to a solution of 31 (10.0 g, 45.4 mmol) and 33t¹⁹⁾ (6.96 g, 68.2 mmol) in CH₂Cl₂ (200 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 48 h. The mixture was concentrated under reduced pressure, and the residue was purified by silica gel column chromatography to give 26t (10.0 g, 72% yield) as an oil. ¹H-NMR (CDCl₃) δ: 0.46–0.54 (2H, m), 0.60–0.67 (2H, m), 1.33 (12H, s), 3.36–3.43 (1H, m), 3.84–3.88 (2H, m), 4.10–4.16 (2H, m), 6.88–6.93 (2H, m), 7.71–7.76 (2H, m).

6-Bromo-4-[4-(2-cyclopropoxyethoxy)phenyl]chromen-2-one (27t)

Compound 27t was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26t according to the procedure for the synthesis of 4a. Yield was 66%. ¹H-NMR (CDCl₃) δ: 0.48–0.56 (2H, m), 0.61–0.69 (2H, m), 3.39–3.46 (1H, m), 3.87–3.95 (2H, m), 4.16–4.23 (2H, m), 6.36 (1H, s), 7.03–7.11 (2H, m), 7.28 (1H, d, J = 8.8 Hz), 7.33–7.41 (2H, m), 7.59–7.67 (2H, m).

4-[4-(2-Cyclopropoxyethoxy)phenyl]-2-oxo-2H-chromene-6-carbonitrile (28t)

Compound 28t was prepared from 27t according to the procedure for the synthesis of 5a. Yield was 79%. ¹H-NMR (CDCl₃) δ: 0.50–0.57 (2H, m), 0.63–0.69 (2H, m), 3.40–3.46 (1H, m), 3.89–3.95 (2H, m), 4.17–4.23 (2H, m), 6.44 (1H, s), 7.06–7.13 (2H, m), 7.33–7.40 (2H, m), 7.48 (1H, d, J = 8.6 Hz), 7.79 (1H, dd, J = 8.6, 2.0 Hz), 7.88 (1H, d, J = 2.0 Hz).

4-[4-(2-Cyclopropoxyethoxy)phenyl]-2-oxo-2H-chromene-6-carboxamide (29t)

Compound 29t was prepared from 28t according to the procedure for the synthesis of 6a. Yield was 50%. mp 176–180 °C; ¹H-NMR (DMSO-d₆) δ: 0.41–0.65 (4H, m), 3.36–3.42 (1H, m), 3.81 (2H, t, J = 4.3 Hz), 4.20 (2H, t, J = 4.3 Hz), 6.43 (1H, s), 7.08–7.22 (2H, m), 7.42–7.60 (4H, m), 8.07 (1H, s), 8.09–8.22 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 5.3 (s), 52.9 (s), 67.1 (s), 68.4 (s), 114.6 (s), 114.9 (s), 116.9 (s), 118.3 (s), 126.7 (s), 126.9 (s), 130.2 (s), 130.4 (s), 130.7 (s), 154.5 (s), 155.4 (s), 159.4 (s), 159.8 (s), 166.7 (s); IR (ATR) cm⁻¹: 1714, 1656, 1604; HR-MS (ESI-TOF) Calcd for C₂₁H₁₉NNaO₅ 388.1161. Found 388.1152; HPLC purity 99.5% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50)).

2-(2-{2-[4-(4,4,5,5-Tetramethyl[1,3,2]dioxaborolan-2-yl)phenoxy]ethoxy}ethoxy)tetrahydro-2H-pyran (26u)

Following the addition of KI (378 mg, 2.28 mmol) and Cs₂CO₃ (5.57 g, 17.1 mmol) to a solution of 31 (2.50 g, 11.4 mmol) and 32u²⁰⁾ (2.84 g, 13.6 mmol) in DMF (30 mL), the reaction mixture was stirred at 70 °C for 11 h. Water was added and 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 26u (1.25 g, 28% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.33 (12H, s), 1.47–1.87 (6H, m), 3.46–3.53 (1H, m), 3.60–3.66 (1H, m), 3.73–3.77 (2H, m), 3.84–3.92 (4H, m), 4.14–4.18 (2H, m), 4.62–4.65 (1H, m), 6.88–6.92 (2H, m), 7.71–7.75 (2H, m).

6-Bromo-4-(4-{2-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]ethoxy}phenyl)chromen-2-one (27u)

Compound 27u was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26u according to the procedure for the synthesis of 4a. Yield was 47%. ¹H-NMR (CDCl₃) δ: 1.47–1.87 (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), 4.21–4.25 (2H, m), 4.64–4.67 (1H, m), 6.36 (1H, s), 7.06–7.10 (2H, m), 7.28 (1H, dd, J = 8.3, 0.7 Hz), 7.35–7.39 (2H, m), 7.61–7.66 (2H, m).

2-Oxo-4-(4-{2-[2-(tetrahydro-2H-pyran-2-yloxy)ethoxy]ethoxy}phenyl)-2H-chromene-6-carbonitrile (28u)

Compound 28u was prepared from 27u according to the procedure for the synthesis of 5a. Yield was 83%. ¹H-NMR (CDCl₃) δ: 1.47–1.87 (6H, m), 3.48–3.55 (1H, m), 3.63–3.70 (1H, m), 3.77–3.81 (2H, m), 3.86–3.96 (4H, m), 4.22–4.26 (2H, m), 4.64–4.67 (1H, m), 6.44 (1H, s), 7.08–7.13 (2H, m), 7.34–7.39 (2H, m), 7.48 (1H, d, J = 8.5 Hz), 7.79 (1H, dd, J = 8.5, 2.0 Hz), 7.89 (1H, d, J = 2.0 Hz).

4-{4-[2-(2-Hydroxyethoxy)ethoxy]phenyl}-2-oxo-2H-chromene-6-carboxamide (29u)

Compound 29u was prepared from 28u according to the procedure for the synthesis of 6a. Yield was 33%. mp 165–167 °C; ¹H-NMR (DMSO-d₆) δ: 3.47–3.58 (4H, m), 3.80 (2H, t, J = 4.6 Hz), 4.22 (2H, t, J = 4.6 Hz), 4.64 (1H, s), 6.44 (1H, s), 7.14–7.22 (2H, m), 7.47 (1H, s), 7.50–7.62 (3H, m), 8.06 (1H, d, J = 2.0 Hz), 8.09–8.21 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 60.1 (s), 67.3 (s), 68.7 (s), 72.4 (s), 114.5 (s), 114.8 (s), 116.8 (s), 118.2 (s), 126.5 (s), 126.8 (s), 130.1 (s), 130.3 (s), 130.6 (s), 154.4 (s), 155.3 (s), 159.3 (s), 159.7 (s), 166.5 (s); IR (ATR) cm⁻¹: 3413, 3167, 1714, 1693; HR-MS (ESI-TOF) Calcd for C₂₀H₁₉NNaO₆ 392.1110. Found 392.1106; HPLC purity 96.5% (eluent: 0.01 M KH₂PO₄-MeCN (70 : 30)).

4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)phenoxymethyl]tetrahydro-2H-pyran (26v)

Compound 26v was prepared from 33v according to the procedure for the synthesis of 26t. Yield was 57%. ¹H-NMR (CDCl₃) δ: 1.33 (12H, s), 1.38–1.53 (2H, m), 1.70–1.81 (2H, m), 2.01–2.12 (1H, m), 3.37–3.50 (2H, m), 3.83 (2H, d, J = 6.4 Hz), 3.95–4.06 (2H, m), 6.83–6.92 (2H, m), 7.70–7.78 (2H, m).

6-Bromo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]chromen-2-one (27v)

Compound 27v was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26v according to the procedure for the synthesis of 4a. Yield was 74%. ¹H-NMR (CDCl₃) δ: 1.43–1.57 (2H, m), 1.74–1.86 (2H, m), 2.07–2.19 (1H, m), 3.42–3.54 (2H, m), 3.90 (2H, d, J = 6.3 Hz), 4.00–4.09 (2H, m), 6.36 (1H, s), 7.01–7.09 (2H, m), 7.28 (1H, d, J = 8.8 Hz), 7.35–7.41 (2H, m), 7.63 (1H, dd, J = 8.8, 2.2 Hz), 7.66 (1H, d, J = 2.2 Hz).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]-2H-chromene-6-carbonitrile (28v)

Compound 28v was prepared from 27v according to the procedure for the synthesis of 5a. Yield was 86%. ¹H-NMR (CDCl₃) δ: 1.45–1.54 (2H, m), 1.75–1.85 (2H, m), 2.07–2.20 (1H, m), 3.42–3.54 (2H, m), 3.91 (2H, d, J = 6.3 Hz), 4.01–4.09 (2H, m), 6.43 (1H, s), 7.03–7.11 (2H, m), 7.35–7.41 (2H, m), 7.48 (1H, d, J = 8.5 Hz), 7.79 (1H, dd, J = 8.5, 1.9 Hz), 7.88 (1H, d, J = 1.9 Hz).

2-Oxo-4-[4-(tetrahydro-2H-pyran-4-ylmethoxy)phenyl]-2H-chromene-6-carboxamide (29v)

Compound 29v was prepared from 28v according to the procedure for the synthesis of 6a. Yield was 46%. mp 206–209 °C; ¹H-NMR (CDCl₃) δ: 1.29–1.43 (2H, m), 1.66–1.80 (2H, m), 2.00–2.10 (1H, m), 3.29–3.42 (2H, m), 3.84–3.98 (4H, m), 6.43 (1H, s), 7.10–7.20 (2H, m), 7.46–7.58 (4H, m), 8.08 (1H, d, J = 1.8 Hz), 8.11–8.20 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 29.2 (s), 34.4 (s), 66.6 (s), 72.2 (s), 114.6 (s), 114.9 (s), 116.9 (s), 118.3 (s), 126.5 (s), 126.9 (s), 130.2 (s), 130.4 (s), 130.7 (s), 154.6 (s), 155.4 (s), 159.4 (s), 160.0 (s), 166.6 (s); IR (ATR) cm⁻¹: 1712, 1691; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₅ 402.1317. Found 402.1310; HPLC purity 99.4% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (50 : 50 : 0.2)).

tert-Butyl 4-[4-(4,4,5,5-Tetramethyl-[1,3,2]dioxaborolan-2-yl)phenoxymethyl] Piperidine-1-carboxylate (26w)

Following the addition of methanesulfonyl chloride (MsCl) (1.62 mL, 20.9 mmol) and Et₃N (3.87 mL, 27.8 mmol) to a solution of 33w (3.00 g, 13.9 mmol) in CH₂Cl₂ (30 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 2 h. Water was added and the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure to give an intermediate.

Following the addition of the intermediate in DMF (30 mL) to a solution of 31 (3.06 g, 13.9 mmol) in DMF (30 mL), the reaction mixture was stirred at 60 °C for 18 h. After the addition of KI (460 mg, 2.78 mmol), the mixture was stirred at 90 °C for 4 h. 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 26w (1.51 g, 26% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.22–1.32 (2H, m), 1.33 (12H, s), 1.46 (9H, s), 1.76–1.87 (2H, m), 1.90–2.02 (1H, m), 2.66–2.83 (2H, m), 3.82 (2H, d, J = 6.4 Hz), 4.07–4.28 (2H, m), 6.83–6.92 (2H, m), 7.70–7.78 (2H, m).

tert-Butyl 4-[4-(6-Bromo-2-oxo-2H-chromen-4-yl)phenoxymethyl]piperidine-1-carboxylate (27w)

Compound 27w was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26w according to the procedure for the synthesis of 4a. Yield was 68%. ¹H-NMR (CDCl₃) δ: 1.27–1.39 (2H, m), 1.48 (9H, s), 1.81–1.91 (2H, m), 1.95–2.08 (1H, m), 2.70–2.86 (2H, m), 3.89 (2H, d, J = 6.4 Hz), 4.10–4.28 (2H, m), 6.36 (1H, s), 7.00–7.07 (2H, m), 7.28 (1H, d, J = 8.5 Hz), 7.35–7.42 (2H, m), 7.63 (1H, dd, J = 8.5, 2.4 Hz), 7.65 (1H, d, J = 2.4 Hz).

tert-Butyl 4-[4-(6-Cyano-2-oxo-2H-chromen-4-yl)phenoxymethyl]piperidine-1-carboxylate (28w)

Compound 28w was prepared from 27w according to the procedure for the synthesis of 5a. Yield was 82%. ¹H-NMR (CDCl₃) δ: 1.29–1.39 (2H, m), 1.48 (9H, s), 1.82–1.91 (2H, m), 1.95–2.08 (1H, m), 2.70–2.86 (2H, m), 3.90 (2H, d, J = 6.4 Hz), 4.14–4.26 (2H, m), 6.44 (1H, s), 7.02–7.10 (2H, m), 7.33–7.41 (2H, m), 7.48 (1H, d, J = 8.8 Hz), 7.79 (1H, dd, J = 8.8, 2.0 Hz), 7.88 (1H, d, J = 2.0 Hz).

tert-Butyl 4-[4-(6-Carbamoyl-2-oxo-2H-chromen-4-yl)phenoxymethyl]piperidine-1-carboxylate (29w)

Following the addition of water (2 mL) and NaBO₃·4H₂O (1.16 g, 7.52 mmol) to a solution of 28w (900 mg, 1.88 mmol) in MeOH-tetrahydrofuran (THF) (2 : 1) (6 mL), the reaction mixture was stirred at 50 °C for 4 h. After the addition of 1.0 M HCl, the mixture was stirred for 30 min. 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 to give 29w (397 mg, 44% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.28–1.39 (2H, m), 1.48 (9H, s), 1.81–1.92 (2H, m), 1.96–2.07 (1H, m), 2.71–2.87 (2H, m), 3.89 (2H, d, J = 6.3 Hz), 4.11–4.28 (2H, m), 5.50–6.10 (2H, m), 6.40 (1H, s), 7.01–7.09 (2H, m), 7.36–7.43 (2H, m), 7.45 (1H, d, J = 8.5 Hz), 7.94 (1H, dd, J = 8.5, 2.2 Hz), 8.08 (1H, d, J = 2.2 Hz).

4-[4-(1-Methylpiperidin-4-ylmethoxy)phenyl]-2-oxo-2H-chromene-6-carboxamide (30w)

Following the addition of 6.3 M HCl in i-PrOH (0.20 mL, 1.3 mmol) to a solution of 29w (397 mg, 0.83 mmol) in HCO₂H (4 mL) under ice-cooling, the reaction mixture was stirred at the same temperature for 30 min. After neutralization with 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 obtained was dissolved in MeCN-MeOH (1 : 1) (4 mL). After the addition of 37% formalin (0.19 mL, 2.5 mmol) and NaBH(OAc)₃ (880 mg, 4.15 mmol) under ice-cooling, the reaction mixture was stirred at the same temperature for 1 h. The mixture was concentrated under reduced pressure and AcOEt was added. The mixture was washed with saturated aqueous NaHCO₃ 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 Et₂O was added. The insoluble material was collected by filtration to give 30w (33 mg, 10% yield) as a solid. mp 191–194 °C; ¹H-NMR (DMSO-d₆) δ: 1.25–1.40 (2H, m), 1.64–1.80 (3H, m), 1.82–1.92 (2H, m), 2.16 (3H, s), 2.73–2.85 (2H, m), 3.93 (2H, d, J = 5.9 Hz), 6.44 (1H, s), 7.11–7.18 (2H, m), 7.46–7.59 (4H, m), 8.06 (1H, d, J = 2.0 Hz), 8.08–8.14 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 28.4 (s), 34.7 (s), 46.1 (s), 54.8 (s), 72.2 (s), 114.5 (s), 114.8 (s), 116.8 (s), 118.2 (s), 126.4 (s), 126.8 (s), 130.1 (s), 130.3 (s), 130.6 (s), 154.5 (s), 155.3 (s), 159.3 (s), 160.0 (s), 166.5 (s); IR (ATR) cm⁻¹: 1720, 1664; HR-MS (ESI-TOF) Calcd for C₂₃H₂₅N₂O₄ 393.1814. Found 393.1798; HPLC purity 97.1% (eluent: 0.01 M KH₂PO₄-MeCN-AcOH (75 : 25 : 0.2)).

6-Bromo-4-{4-[2-(tetrahydro-2H-pyran-4-yloxy)ethoxy]phenyl}chromen-2-one (27x)

Compound 27x was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26x¹⁸⁾ according to the procedure for the synthesis of 4a. Yield was 74%. ¹H-NMR (CDCl₃) δ: 1.55–1.70 (2H, m), 1.92–1.98 (2H, m), 3.43–3.50 (2H, m), 3.59–3.68 (1H, m), 3.87–3.91 (2H, m), 3.93–4.02 (2H, m), 4.20–4.24 (2H, m), 6.37 (1H, s), 7.06–7.11 (2H, m), 7.29 (1H, d, J = 8.5 Hz), 7.35–7.40 (2H, m), 7.60–7.65 (2H, m).

6-Bromo-4-{4-[2-(tetrahydro-2H-pyran-4-yloxy)ethoxy]phenyl}chromen-2-one (28x)

Compound 28x was prepared from 27x according to the procedure for the synthesis of 5a. Yield was 84%. ¹H-NMR (CDCl₃) δ: 1.60–1.73 (2H, m), 1.92–2.01 (2H, m), 3.43–3.53 (2H, m), 3.59–3.68 (1H, m), 3.88–3.91 (2H, m), 3.94–4.00 (2H, m), 4.21–4.24 (2H, m), 6.44 (1H, s), 7.08–7.12 (2H, m), 7.35–7.39 (2H, m), 7.49 (1H, d, J = 8.6 Hz), 7.79 (1H, dd, J = 8.6, 2.0 Hz), 7.88 (1H, d, J = 2.0 Hz).

2-Oxo-4-{4-[2-(tetrahydro-2H-pyran-4-yloxy)ethoxy]phenyl}-2H-chromene-6-carboxamide (29x)

Compound 29x was prepared from 28x according to the procedure for the synthesis of 6a. Yield was 10%. mp 142–144 °C; ¹H-NMR (DMSO-d₆) δ: 1.38–1.50 (2H, m), 1.85–1.94 (2H, m), 3.30–3.40 (2H, m), 3.55–3.64 (1H, m), 3.78–3.86 (4H, m), 4.21 (2H, t, J = 4.7 Hz), 6.44 (1H, s), 7.12–7.22 (2H, m), 7.46–7.58 (4H, m), 8.07 (1H, d, J = 1.8 Hz), 8.11–8.22 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 32.3 (s), 64.9 (s), 65.5 (s), 67.7 (s), 73.7 (s), 114.6 (s), 115.0 (s), 116.9 (s), 118.3 (s), 126.6 (s), 126.9 (s), 130.2 (s), 130.4 (s), 130.7 (s), 154.5 (s), 155.4 (s), 159.4 (s), 159.9 (s), 166.6 (s); IR (ATR) cm⁻¹: 3450, 1733; HR-MS (ESI-TOF) Calcd for C₂₃H₂₃NNaO₆ 432.1423. Found 432.1422; HPLC purity 96.1% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

2-{4-[2-(2-Isopropoxyethoxy)ethoxy]phenyl}-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (26y)

Following the addition of 34 (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 32y²¹⁾ (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, and then dried over Na₂SO₄. The solvent was removed under reduced pressure and the residue was purified by silica gel column chromatography to give an intermediate (79.0 g) as an oil.

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 the intermediate (43.1 g) 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 26y (29.2 g, 35% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.16 (6H, d, J = 6.1 Hz), 1.33 (12H, s), 3.57–3.65 (3H, m), 3.67–3.72 (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)

6-Bromo-4-{4-[2-(2-isopropoxyethoxy)ethoxy]phenyl}-chromen-2-one (27y)

Compound 27y was prepared from 6-bromo-4-chloro-chromen-2-one (1) and 26y according to the procedure for the synthesis of 4a. Yield was 55%. ¹H-NMR (CDCl₃) δ: 1.18 (6H, d, J = 6.1 Hz), 3.58–3.67 (3H, m), 3.69–3.76 (2H, m), 3.90–3.95 (2H, m), 4.20–4.25 (2H, m), 6.36 (1H, s), 7.04–7.12 (2H, m), 7.28 (1H, d, J = 8.5 Hz), 7.33–7.40 (2H, m), 7.59–7.67 (2H, m).

2-Oxo-4-{4-[2-(2-isopropoxyethoxy)ethoxy]phenyl}-2H-chromene-6-carbonitrile (28y)

Compound 28y was prepared from 27y according to the procedure for the synthesis of 5a. Crude product was used in the next reaction without further purification.

4-{4-[2-(2-Isopropoxyethoxy)ethoxy]phenyl}-2-oxo-2H-chromene-6-carboxamide (29y)

Compound 29y was prepared from 28y according to the procedure for the synthesis of 5a and 6a. Yield was 27% (2 steps). mp 102–105 °C; ¹H-NMR (DMSO-d₆) δ: 1.09 (6H, d, J = 6.1 Hz), 3.47–3.63 (5H, m), 3.76–3.84 (2H, m), 4.18–4.25 (2H, m), 6.43 (1H, s), 7.11–7.21 (2H, m), 7.46 (1H, s), 7.50–7.58 (3H, m), 8.06 (1H, d, J = 2.0 Hz), 8.07–8.16 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 22.0 (s), 66.8 (s), 67.4 (s), 68.9 (s), 70.2 (s), 70.8 (s), 114.6 (s), 114.9 (s), 116.8 (s), 118.3 (s), 126.6 (s), 126.8 (s), 130.2 (s), 130.4 (s), 130.7 (s), 154.5 (s), 155.3 (s), 159.3 (s), 159.8 (s), 166.6 (s); IR (ATR) cm⁻¹: 1731, 1660; HR-MS (ESI-TOF) Calcd for C₂₃H₂₅NNaO₆ 434.1580. Found 434.1585; HPLC purity 96.6% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

3-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]prop-2-yn-1-ol (35)

Following the addition of 2-propyn-1-ol (0.32 mL, 5.2 mmol), pyrrolidine (0.46 mL, 5.6 mmol), CuI (8 mg, 0.04 mmol), Pd(PPh₃)₄ (214 mg, 0.185 mmol), and H₂O (18 mL) to 14m (1.00 g, 3.69 mmol), the reaction mixture was stirred at 80 °C for 10 h under a N₂ atmosphere. After cooling, water (50 mL) was added, the mixture was extracted with AcOEt, and 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 35 (0.72 g, 79% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.61–1.71 (3H, m), 1.90–2.00 (2H, m), 3.44 (2H, ddd, J = 12.0, 9.8, 2.7 Hz), 3.54–3.64 (1H, m), 3.96 (2H, dt, J = 12.0, 4.4 Hz), 4.50 (2H, d, J = 6.1 Hz), 4.55 (2H, s), 7.27–7.33 (2H, m), 7.40–7.47 (2H, m).

4-{3-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]prop-2-ynyloxy}benzonitrile (36z)

Following the addition of 4-cyanophenol (170 mg, 1.43 mmol), PPh₃ (409 mg, 1.56 mmol), and DIAD (0.30 mL, 1.6 mmol) to a solution of 35 (0.32 g, 1.3 mmol) in CH₂Cl₂ (6 mL) under ice-cooling, the reaction mixture was stirred for 1.5 h. The mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give 36z (0.54 g, crude) as a solid. The crude product was used in the next reaction without further purification.

4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carbonitrile (37z)

Following the addition of PtCl₄·5H₂O (30 mg, 0.070 mmol) to a solution of 36z (0.54 g) in 1,4-dioxane (5 mL), the reaction mixture was stirred at 90 °C for 5 h. After cooling, water (10 mL) 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 37z (173 mg, crude) as a solid. The crude product was used in the next reaction without further purification.

4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxamide (38)

Following the addition of K₂CO₃ (205 mg, 1.48 mmol) and 30% aqueous H₂O₂ (0.15 mL, 1.5 mmol) to a solution of 37z (172 mg) in DMSO (5 mL), the mixture was stirred at room temperature for 10 min. After the addition of 30% aqueous H₂O₂ (0.15 mL, 1.5 mmol), the mixture was stirred at room temperature for 10 min and at 60 °C for 20 min. Water was added and 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 and Et₂O was added. The insoluble material was collected by filtration to give 38 (77 mg, crude) as a solid. The crude product was used in the next reaction without further purification.

4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]chroman-6-carboxamide (39)

A solution of 38 (35 mg) in MeOH (3 mL) was hydrogenated at 0.3 MPa in the presence of 10% Pd-C (14 mg) at room temperature for 1 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure. The residue was purified by silica gel column chromatography and Et₂O was added. The insoluble material was collected by filtration to give 39 (16 mg, 7.5%, 4 steps) as a solid. mp 144–147 °C; ¹H-NMR (CDCl₃) δ: 1.61–1.71 (2H, m), 1.91–2.02 (2H, m), 2.03–2.12 (1H, m), 2.28–2.37 (1H, m), 3.42–3.52 (2H, m), 3.56–3.66 (1H, m), 3.97 (2H, dt, J = 11.7, 4.4 Hz), 4.17–4.27 (3H, m), 4.54 (2H, s), 5.20–6.00 (2H, m), 6.91 (1H, d, J = 8.5 Hz), 7.06–7.12 (2H, m), 7.27–7.34 (2H, m), 7.36 (1H, d, J = 2.2 Hz), 7.60 (1H, dd, J = 8.5, 2.4 Hz); ¹³C-NMR (DMSO-d₆) δ: 30.6 (s), 32.1 (s), 63.7 (s), 64.7 (s), 68.2 (s), 72.8 (s), 79.1 (s), 115.9 (s), 124.3 (s), 125.9 (s), 127.0 (s), 127.5 (s), 128.2 (s), 130.3 (s), 137.1 (s), 144.0 (s), 157.1 (s), 167.2 (s); IR (ATR) cm⁻¹: 1653; HR-MS (ESI-TOF) Calcd for C₂₂H₂₅NNaO₄ 390.1681. Found 390.168; HPLC purity 96.7% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

Ethyl 4-{3-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]prop-2-ynyloxy}benzoate (36α)

Compound 36α was prepared from 35 according to the procedure for the synthesis of 36z. Crude 36α was used in the next reaction without further purification.

Ethyl 4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxylate (37α)

Compound 37α was prepared from 36α according to the procedure for the synthesis of 37z.Yield was 27% (2 steps). ¹H-NMR (CDCl₃) δ: 1.31 (3H, t, J = 7.1 Hz), 1.65–1.75 (2H, m), 1.94–2.03 (2H, m), 3.47 (2H, ddd, J = 11.4, 9.8, 2.7 Hz), 3.61–3.69 (1H, m), 3.99 (2H, dt, J = 11.4, 4.4 Hz), 4.28 (2H, q, J = 7.1 Hz), 4.61 (2H, s), 4.93 (2H, d, J = 3.9 Hz), 5.79 (1H, t, J = 3.9 Hz), 6.89 (1H, d, J = 8.6 Hz), 7.29–7.34 (2H, m), 7.37–7.43 (2H, m), 7.72 (1H, d, J = 1.9 Hz), 7.85 (1H, dd, J = 8.6, 1.9 Hz).

4-[4-(Tetrahydro-2H-pyran-4-yloxymethyl)phenyl]-2H-chromene-6-carboxamide (40)

A solution of 5.0 M aqueous NaOH (1.4 mL, 7.0 mmol) was added to a solution of compound 37α (0.54 g, 1.4 mmol) in MeOH (14 mL), and the reaction mixture was stirred at 40 °C for 75 min and then at 60 °C for 75 min. MeOH was distilled off under reduced pressure, the residue was acidified with 3.0 M HCl 5.0 mL and appropriately diluted with water, and then extraction with AcOEt was performed. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was dissolved in 5 mL THF, N-methylmorphiline (NMM) (0.20 mL, 0.18 mmol) and i-BuOCOCl (0.20 mL, 1.5 mmol) were added slowly under ice-cooling, and the reaction mixture was stirred for 30 min. Then, 28% NH₃ aq. (0.46 mL, 6.8 mmol) was added to the reaction mixture under ice-cooling and stirred for 1 h. Water was added to the reaction and extracted with AcOEt. The organic layer was washed with saturated NaHCO₃ 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 40 (122 mg, 24% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 1.39–1.58 (2H, m), 1.88–1.99 (2H, m), 3.30–3.42 (2H, m), 3.56–3.71 (1H, m), 3.83 (2H, dt, J = 11.6, 4.1 Hz), 4.57 (2H, s), 4.89 (2H, d, J = 3.8 Hz), 5.93 (1H, t, J = 3.8 Hz), 6.92 (1H, d, J = 8.4 Hz), 7.16 (1H, brs), 7.28–7.34 (2H, m), 7.36–7.44 (2H, m), 7.51 (1H, d, J = 2.0 Hz), 7.73 (1H, dd, J = 8.4, 2.0 Hz), 7.80 (1H, brs); ¹³C-NMR (DMSO-d₆) δ: 32.2 (s), 64.8 (s), 65.1 (s), 68.3 (s), 73.1 (s), 115.6 (s), 121.4 (s), 122.5 (s), 125.2 (s), 127.3 (s), 127.6 (s), 128.1 (s), 128.6 (s), 135.1 (s), 136.3 (s), 138.9 (s), 156.8 (s), 167.4 (s) ; IR (ATR) cm⁻¹: 1711, 1678; HR-MS (ESI-TOF) Calcd for C₂₂H₂₃NNaO₄ 388.1525. Found 388.1515; HPLC purity 98.3% (eluent: 0.01 M KH₂PO₄-MeCN (50 : 50)).

CDK8 Kinase Assay

CDK8 activity was measured using the QSS Assist CDK8 enzyme-linked immunosorbent assay (ELISA) Kit (Carna Bioscience, Kobe, Japan) according to the manufacturer’s protocol as previously reported.¹²⁾

In Vitro Osteoblast Differentiation

Osteoblast differentiation was investigated using ST2 cells derived from mouse bone marrow mesenchymal stem cells (Riken BioResource Research Center, Tsukuba, Japan) as previously reported.¹³⁾ Briefly, ST2 cells were cultured for 24 h in α-MEM, osteoblast differentiation medium, compounds were added, and cells were then 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 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-week-old, Japan SLC, Inc. Hamamatsu, Japan) were orally administered the compounds (10 mg/kg) suspended in 0.5% methyl cellulose (MC) solution. Blood was taken 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 Corporation) and autoinjector (Nexera X2, SIL-30AC, Shimadzu Corporation). 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, and with all procedures approved by the Animal Ethical Committee of Kyoto Pharmaceutical Industries, Ltd.

OVX Rats

Twelve-week-old female F344/NSlc rats were used. Ovariectomy was performed as previously reported.¹³⁾ 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 groups. Rats were orally administered vehicle (0.5% MC) or 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.¹³⁾ Micro-CT data were analyzed using TRI/3D-BON software (RATOC, Tokyo, Japan).^22,23) After repeated administration, rats were deeply anaesthetized 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.

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) Chen J., Liao X., Gan J., Front. Pharmacol., 14, 1236893 (2023).
• 4) 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).
• 5) Park E., Kim J., Jin H.-S., Choi C. W., Choi T. H., Choi S., Huh D., Jeong S.-Y., Nutrients, 12, 3565 (2020).
• 6) Rosa A. L., Beloti M. M., J. Cell. Biochem., 91, 749–755 (2004).
• 7) Kushwaha P., Tripathi A. K., Gupta S., Kothari P., Upadhyay A., Ahmad N., Sharma T., Siddiqi M. I., Trivedi R., Sashidhara K. V., Eur. J. Med. Chem., 156, 103–117 (2018).
• 8) Tripathi A. K., Rai D., Kothari P., Kushwaha P., Sashidhara K. V., Trivedi R., Apoptosis, 27, 90–111 (2022).
• 9) 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).
• 10) Saito K., Shinozuka T., Nakao A., Kunikata T., Nakai D., Nagai Y., Naito S., Bioorg. Med. Chem. Lett., 54, 128440 (2021).
• 11) Kitao T., Ito Y., Fukui M., Yamamoto M., Shoji Y., Takeda S., Shirahase H., Yakugaku Zasshi, 139, 19–25 (2019).
• 12) 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).
• 13) Yamamoto M., Ito Y., Fukui M., Otake K., Shoji Y., Kitao T., Shirahase H., Hinoi E., Biol. Pharm. Bull., 46, 1435–1443 (2023).
• 14) Rao M. L. N., Kumar A., Tetrahedron, 70, 6995–7005 (2014).
• 15) Chen L., Zhao J., Patent CN109896991 (2017).
• 16) Hirayama T., Fujimoto J., Cary D. R., Okaniwa M., Hirata Y., Patent U.S.2017/0044132 (2017).
• 17) Saito T., Nishimoto Y., Yasuda M., Baba A., J. Org. Chem., 72, 8588–8590 (2007).
• 18) Kanno O., Nakajima K., Aoki K., Tanaka R., Hirano S., Oizumi K., Naito S., Patent WO2010116915 (2010).
• 19) Huang J.-W., Chen C.-D., Leung M.-K., Tetrahedron Lett., 40, 8647–8650 (1999).
• 20) Gibson H. W., Lee S. H., Engen P. T., Lecavalier P., Sze J., Shen Y. X., Bheda M., J. Org. Chem., 58, 3748–3756 (1993).
• 21) Cheng Y.-X., Tomaszewski M., Patent WO2007/142584 (2007).
• 22) Nango N., Kubota S., Horiguchi Y., Osteoporotic Fracture and Systemic Skeletal Disorders, 15, 129–160 (2021).
• 23) Ferretti J. L., Capozza R. F., Zanchetta J. R., Bone, 18, 97–102 (1996).