Discovery of Novel Chromenopyridine Derivatives as Readthrough-Inducing Drugs

Abstract

Hurler syndrome, a type of Mucopolysaccharidosis type I, is an inherited disorder caused by the accumulation of glycosaminoglycans (GAG) due to a deficiency in lysosomal α-L-iduronidase (IDUA), resulting in multiorgan dysfunction. In many patients with Hurler syndrome, IDUA proteins are not produced due to nonsense mutations in their genes; therefore, readthrough-inducing compounds, such as gentamycin, are expected to restore IDUA proteins by skipping the premature termination codon. In the present study, we synthesized a series of chromenopyridine derivatives to identify novel readthrough-inducing compounds. The readthrough-inducing activities of synthesized compounds were examined by measuring cellular IDUA activities and GAG concentrations in Hurler syndrome patient-derived cells. Compounds with a difluorophenyl group at the 2-position of chromenopyridine, a cyclobutyl group at the 3-position, and a basic side chain or basic fused ring exhibited excellent readthrough-inducing activities. KY-640, a chromenopyridine derivative with a tetrahydroisoquinoline sub-structure, increased the cellular IDUA activities of patient-derived cells by 3.2-fold at 0.3 µM and significantly reduced GAG concentrations, and also significantly increased enzyme activity in mouse models, suggesting its therapeutic potential in patients with Hurler syndrome.

Introduction

Mucopolysaccharidosis type I is a rare disease characterized by systemic organ dysfunction caused by the accumulation of glycosaminoglycans (GAG) in lysosomes due to a congenital deficiency in or reduced activity of α-L-iduronidase (IDUA), a lysosomal enzyme. It is classified into three subtypes according to the time of onset and severity of the disease, with Hurler syndrome being the most severe type. The residual IDUA enzyme activity of patients with Hurler syndrome is less than 1% that of normal individuals,¹⁾ and they present with a number of systemic symptoms, such as characteristic facial features, developmental delays, skeletal abnormalities, and corneal clouding, which become more severe with age.²⁾ Enzyme replacement therapy and hematopoietic stem cell transplantation are currently available as root-cause therapy. However, enzyme replacement therapy is not effective against neurological symptoms and bone deformities, and hematopoietic stem cell transplantation has severe side effects, such as graft failure and graft-versus-host disease, limiting its use; therefore, the development of more effective treatment options is desired.

Two common nonsense mutations, W402X and Q70X, have been reported in analyses of the IDUA gene in patients with mucopolysaccharidosis type I, and these two nonsense mutations account for approximately 70% of mutated alleles.³⁾ Furthermore, the percentage of nonsense mutations was shown to be higher in patients with the severe form of the disease than in those with the mild form, suggesting a correlation between genotype and phenotype.⁴⁾ Therefore, nonsense mutations in the IDUA gene play a major role in the onset and severity of mucopolysaccharidosis type I, and treatment that restores enzyme activity that is defective due to nonsense mutations is necessary. The readthrough mechanism, which restores the biosynthesis of full-length proteins by misreading a premature termination codon and allowing translation to continue, has recently been actively investigated as a treatment for nonsense mutation-related diseases, such as Hurler syndrome.⁵⁾ Ataluren is the only readthrough-inducing compound approved for Duchenne muscular dystrophy,⁶⁾ and various small molecule readthrough-inducing compounds, such as aminoglycosides,⁷⁾ negamycin derivatives,⁸⁾ RTC-13,⁹⁾ and Novartis’ compounds¹⁰⁾ have been reported (Fig. 1). We previously identified a compound (KY-516) with more potent readthrough-inducing activity than ataluren by structural optimization based on ataluren.¹¹⁾

Fig. 1. Chemical Structures of Readthrough-Inducing Compounds

In the present study, we synthesized various derivatives based on the chromenopyridine skeleton, which we discovered in our skeletal discovery research, and evaluated their readthrough-inducing activities. Structure–activity relationships were discussed and a representative compound 30 with a basic fused ring structure was named KY-640 and selected for further evaluations. KY-640 exhibited strong readthrough-inducing activity, increased IDUA activity, and inhibited GAG accumulation in Hurler patient-derived fibroblasts, and also significantly increased enzyme activity in mouse models.

Chemistry

Chart 1 shows the synthetic route of 2-substituted chromenopyridine derivatives. 6-Cyclobutyl-4-hydroxy-2H-pyran-2-one 1 synthesized by a previously reported method¹²⁾ was acylated with 2-fluorobenzoyl chloride to give ester 2, which was treated with KCN, Et₃N, and 18-crown-6, and the rearrangement and aromatic nucleophilic substitution proceeded sequentially to give pyrano[4,3-b]chromen-1,10-dione 3. Compound 3 was reacted with amines 4a–4f in the presence of acetic acid (AcOH) to give corresponding 2-substituted chromenopyridine derivatives 5a–5f.

Chart 1. Synthesis of 2-Substituted Chromenopyridine Derivatives

Reagents and conditions; (i) 2-fluorobenzoyl chloride, Et₃N, CH₂Cl₂; (ii) KCN, Et₃N, 18-crown-6, toluene; (iii) amine 4a–4f, Et₃N (in the case of HCl salt), AcOH, CF₃CH₂OH.

The preparation of 2,3-substituted chromenopyridine derivatives is shown in Chart 2. 2-Substituted-3-cyclobutyl derivatives 17a, 18a, and 19a were synthesized from compound 1 via Route A in Chart 2. Compound 1 was reacted with the corresponding aniline in the presence of AcOH to give 6-cyclobutyl-4-hydroxy-pyridine-2(1H)-ones 6a, 7a, and 8a, which were acylated with 2-fluorobenzoyl chloride, and cyclized by the same method as before to give 2-substituted 3-cyclobutyl chromenopyridine derivatives 17a, 18a, and 19a.

Chart 2. Synthesis of 2- and 3-Substituted Chromenopyridine Derivatives

Reagents and conditions; (i) amine, AcOH, H₂O; (ii) 2,3-difluoroaniline, CHCl₃; (iii) NaH, phenol; (iv) POCl₃; (v) amine, i-Pr₂NEt, CH₂Cl₂; (vi) Boc₂O, DMAP, MeCN; (vii) aqueous NaOH, 1,4-dioxane; (viii) trimethyloxonium tetrafluoroborate, i-Pr₂NEt, CH₂Cl₂; (ix) 2-fluorobenzoyl chloride, Et₃N, CH₂Cl₂; (x) KCN, Et₃N, 18-crown-6, toluene; (xi) TFA, CH₂Cl₂; (xii) HBr in AcOH.

Regarding the synthesis of 3-substituted chromenopyridine derivatives 17b–17d, 6-substituted 4-hydroxy-2H-pyran-2-ones 1b–1d were reacted with aniline to give 4-hydroxy-1-phenylpyridine-2(1H)-ones 6b–6d via Route A in Chart 2, which were derivatized to the final compounds in the same manner as described above. Starting material 1b was purchased, and 1c and 1d were prepared according to the synthesis of compound 1.

On the other hand, derivatives with a substituent containing a heteroatom at 3-position was synthesized as shown in Route B in Chart 2. Compound 9, prepared according to the literature,¹³⁾ was reacted with 2,3-difluoroaniline, followed by treatment with NaH to give 7-hydroxydioxinopyridinedione 11. The hydroxy group of compound 11 was chlorinated with POCl₃, reacted with the corresponding amine, and treated with aqueous NaOH and tert-butoxycarbonyl protection in the case of the ethylamino group to give compounds 8e and 8f. Compounds 8e and 8f were derivatized to chromenopyridine derivatives 19e and 19f by the same method as before, and compound 19f was subjected to tert-butoxycarbonyl deprotection to give compound 19g. Regarding the synthesis of derivatives with methoxy or hydroxyl groups, compound 11 was methylated with trimethyloxonium tetrafluoroborate and then treated with aqueous NaOH to give compound 8h with methoxy groups. 8h was then derivatized to chromenopyridine derivative 19h, which was demethylated by a treatment with HBr in AcOH solution to obtain compound 19i.

The general synthetic scheme for skeletal transformation compounds and methyl group-introduced compounds with a phenyl group at the 2-position is shown in Chart 3. Carboxylic acids 20a,¹⁴⁾ 20b–20e, and 20f, which were purchased or prepared according to the literature, were converted to acyl chlorides with (COCl)₂ or SOCl₂, followed by condensation with compound 6a to give intermediate esters 21a–20f. Compounds 21a–21f were transformed to compounds with a tricyclic skeleton 22a–22f in the same manner as before.

Chart 3. General Synthetic Scheme for Skeletal Transformation Compounds and Methyl Group Substituted Compounds

Chart 4 shows the synthetic scheme for skeletal transformation compounds with a 2,3-difluorophenyl group at the 2-position. Purchased or synthesized carboxylic acids 20g–20m were condensed with compound 8a using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) and 4-dimethylaminopyridine (DMAP) to give esters 21g–21m, which were subjected to rearrangement and cyclization reactions under the same conditions as above. Deprotection of the tert-butoxycarbonyl group gave compounds 24, 25, 27, 29, and 30, while demethylation of the amino group of compounds 25 and 27 gave compounds 26 and 28, respectively.

Chart 4. General Synthetic Scheme for Skeletal Transformation Compounds

Reagents and conditions; (i) KCN, Et₃N, 18-crown-6, toluene; (ii) BBr₃, CH₂Cl₂; (iii) HCl in 1,4-dioxane; (iv) HCl in i-PrOH, AcOEt; (v) formalin, Et₃N, NaBH(OAc)₃, MeOH.

Carboxylic acids 20h,¹⁵⁾ 20i,¹⁶⁾ and 20m¹⁷⁾ were prepared according to the previously reported method. Chart 5 shows the synthetic scheme for carboxylic acids 20g, 20j, 20j, and 20l. 3-Fluorofuran-2-carboxylic acid 20g was synthesized from 3-bromofuran-2-carboaldehyde 31. 3-Bromofuran-2-carboaldehyde 31 was acetal-protected to give 32, and the bromine in compound 32 was converted to fluorine. After the deprotection of the acetal, the oxidation of the aldehyde to carboxylic acid gave compound 20g. Compound 20j was synthesized from methyl 2-bromo-3-fluorobenzoate 33. The Heck reaction of methyl 2-bromo-3-fluorobenzoate 33 and tert-butyl acrylate gave compound 34, which was hydrogenated to give compound 35. Compound 35 was treated with trifluoroacetic acid (TFA) to give carboxylic acid 36. The Curtius rearrangement of carboxylic acid 36 gave tert-butoxycarbonyl (Boc)-protected amine 37, and hydrolysis of the methyl ester gave compound 20j. Similarly, compound 20k was prepared from 3-(3-fluoro-4-methoxycarbonylphenyl)propionic acid 38, which was prepared according to the literature,¹⁸⁾ by the Curtius rearrangement followed by ester hydrolysis. Tetrahydroisoquinoline 20l was synthesized from tert-butyl 8-fluoro-7-hydroxy-1,2,3,4-tetrahydroisoquinoline-2-carboxylate 40, which was prepared according to the literature,¹⁹⁾ by hydroxyl group triflation followed by Pd-catalyzed carboxylation.

Chart 5. Synthesis of Carboxylic Acids 20g, 20j, 20k, and 20l

Reagents and conditions; (i) p-TsOH·H₂O, propylene glycol; (ii) n-BuLi in n-hexane, N-fluorobenzenesulfonimide, THF; (iii) p-TsOH·H₂O, 1,4-dioxane, H₂O; (iv) NaH₂PO₄, NaClO₂, 2-methyl-2-butene, t-BuOH, H₂O, THF; (v) tert-butyl acrylate, Pd(OAc)₂, P(o-tolyl)₃, Et₃N, DMF; (vi) H₂, Pd-C, MeOH; (vii) TFA, CH₂Cl₂; (viii) DPPA, Et₃N, t-BuOH; (ix) aqueous NaOH, MeOH; (x) Tf₂O, Et₃N, CH₂Cl₂; (xi) HCO₂Li·H₂O, xantphos, Pd₂(dba)₃, LiCl, i-Pr₂NEt, Ac₂O, DMF.

Results and Discussion

The prevalence of mucopolysaccharidosis type I was previously reported to be as high as 1 in 7300,²⁰⁾ and although there is an urgent need for the development of therapeutic agents, few drugs have so far been developed for mucopolysaccharidosis type I. A phase 1 study on ataluren for nonsense mutation-related mucopolysaccharidosis type I was initiated in 2015; however, patient enrollment was halted in 2017 due to the difficulties associated with identifying qualified patients for the study. Novartis has also patented a series of pyrimido[4,5-b]quinoline-4,5 (3H,10H)-dione derivatives, such as Ex. 1.5 in Fig. 1, with readthrough-inducing activity against IDUA nonsense mutations.¹⁰⁾ We synthesized a number of compounds based on this structure, transforming the skeleton, and found original lead compound 17a. Compound 17a was effective in Hurler patient-derived cells from 3 µM, retaining activity. Therefore, we conducted an optimization study to further enhance the activity of compound 17a. Hurler patient-derived fibroblasts carrying the IDUA W402X mutation, which accounts for a large percentage of Hurler syndrome,³⁾ were used to evaluate the synthesized compounds. IDUA enzyme activity restored by readthrough was evaluated using a fluorescent substrate as previously described.¹¹⁾ The oral absorption of the synthesized compounds was evaluated by measuring the plasma concentrations of the compounds in male C57BL/6J mice after their oral administration at 30 mg/kg. All animal experiments in the present study were conducted according to the guidelines for animal experiments of our company and the guidelines for animal experimentation approved by the Japanese Association of Laboratory Animal Science.

IDUA enzyme activity was 1.6- and 7.4-fold higher in Hurler patient-derived cells treated with 1 and 3 µM of compound 17a than in control cells. To enhance the readthrough-inducing activity of compound 17a and improve oral absorption, we converted the R¹ side chain of compound 17a (Table 1). We initially attempted to convert the phenyl group of compound 17a to an aliphatic ring. Enzyme activity was up to 4.4-fold higher in cells treated with 1 µM of compound 5b with a cyclopentyl group than in control cells. On the other hand, compound 5a with a cyclopropyl group, compound 5c with a tetrahydrofuran ring, and compound 5d with a tetrahydropyran ring by introducing an oxygen atom lost activity. Since the introduction of a heteroatom into the aliphatic ring resulted in a loss of activity, we attempted to introduce a heteroatom into the aromatic ring; however, compound 5e with a pyridine ring and compound 5f with a methoxyphenyl group both lost activity. To improve pharmacokinetics profile while maintaining activity, the introduction of fluorine was examined, and compound 18a with an ortho-fluorophenyl group retained its activity. The introduction of fluorine at both the ortho and meta positions further enhanced activity, increasing enzyme activity by 6.7-fold at 1 µM and by 10.2-fold at 3 µM from that in control cells (compound 19a).

Table 1. Chemical Structures, Molecular Weights, c log D_7.0 Values, and Readthrough Activities of 2-Substituted Chromenopyridine Derivatives

a) Cell-based readthrough-inducing activity ratio relative to control in patient-derived cells, compounds are evaluated at the indicated concentrations.

The R² side chain was then converted based on compounds 17a and 19a (Table 2). The cyclobutyl group was converted to the alkyl chain group; however, the activities of compounds 17b, 17c, and 17d disappeared. The introduction of a heteroatom was then examined. The activity of compound 19e, in which a cyclobutane ring was converted to an azetidine ring, decreased. Conversions to the ethylamino (19g), methoxy (19h), and hydroxyl groups (19i) also resulted in a loss of activity, indicating that heteroatoms in both the R¹ and R² side chains were not acceptable for activity.

Table 2. Chemical Structures, Molecular Weights, c log D_7.0 Values, and Readthrough Activities of 2,3-Substituted Chromenopyridine Derivatives

a) Cell-based readthrough-inducing activity ratio relative to control in patient-derived cells, compounds are evaluated at the indicated concentrations.

The benzene ring portion of the chromenopyridine skeleton was then converted (Table 3). Compound 22a with an aliphatic ring and compounds 22b and 22c with a pyridine ring by introducing a nitrogen atom all lost activity. Aromatic 5-membered rings were also examined; however, the activity of the furan derivative 22g disappeared. Therefore, we fixed the compounds to the benzene ring and investigated the introduction of substituents. All compounds in which methyl groups were introduced at each position exhibited reduced activity (22d–22f). Methoxy group (22h) and hydroxyl group (23) were introduced as substituents containing heteroatoms, but also exhibited reduced activity. Various types of substituents were examined, and compound 24 with an amino group retained activity (6.4-fold higher than the control at 1 µM); therefore, the introduction of a basic side chain was investigated. Compound 25 with an aminoethyl group was more active (10.0-fold higher than the control at 1 µM), but was poorly absorbed orally (C_max below the limit of detection). Based on this result, we dimethylated the amino group of compound 25 to improve membrane permeability, which further increased enzyme activity to 14.1-fold that of the control at 1 µM and improved oral absorption (C_max = 4.97 µM) (compound 26). Activity decreased in the case of a dimethylaminomethyl or dimethylaminopropyl group, and two carbon chain lengths were optimal (data not shown). Compound 28, in which the dimethylaminoethyl group was moved to the 6-position, also retained activity.

Table 3. Chemical Structures, Molecular Weights, c log D_7.0 Values, and Readthrough Activities of Skeletal Transformation Derivatives (FibroLife S2)

a) Cell-based readthrough-inducing activity ratio relative to control in patient-derived cells, compounds are evaluated at the indicated concentrations.

In our experiments, the evaluation of the readthrough inducing activity of the synthesized compounds was conducted by culturing Hurler patient-derived fibroblasts in low serum liquid medium for human fibroblast growth (FibroLife S2; LifeLine Cell Technology, Frederick, MD, U.S.A.). In the FibroLife S2 medium, patient-derived fibroblasts, which are more susceptible to cell death than healthy donor fibroblasts, showed rapid proliferation, enabling stable compound screening. At this point, to evaluate the compounds under conditions more similar to actual living organisms, the medium used was changed to minimal essential medium (MEM) containing 10% fetal bovine serum (FBS), in which cell proliferation was slow (Table 4). Compounds with a dimethylaminoethyl group at the 5- or 6-position (26, 28) exhibited good activities, suggesting that the placement of a nitrogen atom at an appropriate position from the 5- or 6-position of the chromenopyridine ring is important for activity. Therefore, the tetrahydroisoquinoline derivatives 29 and 30 were synthesized by cyclization at the 5- and 6- positions. Both compounds exhibited good activities, which were approximately 3-fold higher from 0.3 µM from that in control cells. Compound 30 also showed better oral absorption (C_max = 8.82 µM) than compound 29 (C_max = 2.95 µM), was named KY-640, and was subjected to further evaluations.

Table 4. Chemical Structures, Molecular Weights, c log D_7.0 Values, and Readthrough Activities of Chromenopyridine Derivatives with a Basic Side Chain (MEM)

a) Cell-based readthrough-inducing activity ratio relative to control in patient-derived cells, compounds are evaluated at the indicated concentrations. b) not detected.

We examined the exposure time dependency of the effects of KY-640 on Hurler patient-derived fibroblasts (Fig. 2). A 2-d KY-640 treatment significantly increased IDUA activity in Hurler patient-derived fibroblasts in a concentration-dependent manner at 0.3 and 1 µM, whereas ataluren did not, even at 100 µM. To establish whether this increase in IDUA activity was sufficient to reduce cellular GAG levels, we quantified GAG levels using GAG-binding Blyscan™ dye. Cellular GAG levels were significantly higher in Hurler patient-derived fibroblasts than in normal human fibroblasts. A 2-d KY-640 treatment at 0.1–1 µM did not affect GAG levels, while a 6-d treatment significantly reduced GAG levels in a concentration-dependent manner. After enzyme expression by the readthrough-inducing activity of KY-640, GAG may have been slowly metabolized by IDUA.

Fig. 2. A) IDUA Activity and B) Cellular GAG Levels in Hurler Patient-Derived Fibroblasts Treated with KY-640 for 2 and 6 d

Hurler patient derived-fibroblasts carrying the IDUA W402X nonsense mutation were treated with increasing concentrations of KY-640 for 2 or 6 d. Data shown are expressed as the mean ± S.E. of representative experiments (n = 3–6). Exact p values were calculated using the unpaired, two-tailed t-test comparing values in treated cells to vehicle-treated cells. ** p < 0.01, ^##p < 0.01.

We previously reported that the novel triaryl derivative KY-516 exhibited more potent readthrough-inducing activity than ataluren, which is approved for Duchenne muscular dystrophy. KY-516 was effective against the Q70X nonsense mutation, but less effective against the W402X nonsense mutation.¹¹⁾ In addition, KY-516 was ineffective even at 30 µM in the Hurler patient-derived fibroblasts carrying the W402X mutation used in this study. Therefore, we investigated the sequence specificity of the readthrough-inducing activity of KY-640 using the previously reported luciferase assay system.¹¹⁾ The results obtained showed that the readthrough-inducing activity of KY-640 was stronger against the W402X mutation than the Q70X mutation (data not shown), in contrast to that of KY-516. The reason for the difference in sequence specificities between KY-516 and KY-640 remains unknown; however, each of the previously reported readthrough-inducing compounds was previously shown to have different sequence specificities, which may also be affected by the peripheral sequence of a nonsense mutation.²¹⁾

We then investigated whether KY-640 suppressed nonsense mutations in vivo. We generated a knock-in mouse carrying the IDUA-W392X (TGG→TAG) mutation, which is analogous to the human IDUA-W402X mutation. The homozygous IDUA-W392X mutation resulted in a severe IDUA deficiency in all organs, and the accumulation of GAG was observed in the liver, heart, kidney, and lungs. KY-640 (30 and 100 mg/10 mL/kg) was orally administered twice a day (BID) for 8 d. KY-640 at 100 mg/kg (BID), but not at 30 mg/kg, significantly increased IDUA activity in the liver, spleen, and brain (Fig. 3).

Fig. 3. IDUA Activity in Liver, Spleen, and Brain Homogenates of Female IDUA-W392X Knock-in Mice Treated with KY-640 at 30 or 100 mg/kg (BID) Orally for 8 d

Female IDUA-W392X knock-in mice were established using the CRISPR/Cas9 system and used at 5–7 weeks old. KY-640 (30 and 100 mg/10 mL/kg) was suspended in 0.5% MC and orally administered twice a day (BID) for 8 d. Data shown are expressed as the mean ± S.E. of representative experiments (n = 7). Exact p values were calculated using the unpaired, two-tailed t-test comparing values in treated cells to vehicle-treated cells. * p < 0.05.

In conclusion, we herein demonstrated that 1,10-dioxochromenopyridines with substituents at the 2- and 3-positions and fused to the tetrahydroisoquinoline are useful as a scaffold for novel readthrough inducers. Among the synthesized compounds, KY-640 potently increased cellular IDUA activities and reduced GAG concentrations in Hurler patient-derived cells, showed high oral absorption, and also increased IDUA activity in the liver, spleen, and brain in mouse models, suggesting its therapeutic potential for patients with Hurler syndrome.

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

6-Cyclobutyl-2-oxo-2H-pyran-4-yl 2-Fluorobenzoate (2)

Following the addition of Et₃N (4.18 mL, 30.0 mmol) to a suspension of 1 (2.49 g, 15.0 mmol) in toluene (270 mL), 2-fluorobenzoyl chloride (2.68 mL, 22.5 mmol) was added dropwise at room temperature and stirred at the same temperature for 30 min. After the addition of saturated aqueous NH₄Cl, the mixture was extracted with AcOEt. The organic layer was washed with saturated aqueous NH₄Cl 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 2 (4.8 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.84–2.12 (2H, m), 2.22–2.40 (4H, m), 3.32–3.45 (1H, m), 6.04–6.09 (1H, m), 6.14–6.18 (1H, m), 7.16–7.36 (2H, m), 7.59–7.69 (1H, m), 7.97–8.08 (1H, m).

3-Cyclobutylpyrano[4,3-b]chromene-1,10-dione (3)

Following the addition of KCN (1.47 g, 22.5 mmol), Et₃N (3.14 mL, 22.5 mmol), and 18-crown-6 (0.40 g, 1.5 mmol) to a solution of 2 (4.80 g, 15.0 mmol) in toluene (375 mL), the mixture was stirred at room temperature for 10 min, at 60 °C for 30 min, and at 100 °C for 45 min. After cooling, water was added to the reaction mixture, which was then 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 3 (1.08 g, 27% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.85–2.19 (2H, m), 2.27–2.46 (4H, m), 3.36–3.50 (1H, m), 6.23 (1H, s), 7.41–7.49 (2H, m), 7.66–7.73 (1H, m), 8.25–8.34 (1H, m).

3-Cyclobutyl-2-(tetrahydrofuran-3-yl)-2H-10-oxa-2-azaanthracene-1,9-dione (5d)

Following the addition of Et₃N 1.1 mL (8.0 mmol), 3 (270 mg, 1.00 mmol), and AcOH (1.4 mL) to a suspension of tetrahydropyran-3-ylamine hydrochloride (920 mg, 6.69 mmol) in CF₃CH₂OH (2.8 mL), the reaction mixture was stirred at 110 °C for 14 h. After cooling, water was added to the reaction mixture, which was then extracted with AcOEt. 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 (3 mL) was added. The insoluble material was collected by filtration to give 5d (57 mg, 16% yield) as a solid. mp 194–196 °C; ¹H-NMR (dimethyl sulfoxide (DMSO)-d₆) δ: 1.62–2.00 (4H, m), 2.06–2.36 (3H, m), 2.38–2.52 (2H, m), 3.10–3.26 (1H, m), 3.40–3.64 (2H, m), 3.68–3.78 (1H, m), 3.86–3.97 (1H, m), 4.00–4.12 (1H, m), 4.79 (1H, t, J = 10.2 Hz), 6.13 (1H, s), 7.31–7.40 (2H, m), 7.58–7.77 (1H, m), 8.26–8.33 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 17.1 (s), 25.1 (s), 26.3 (s), 27.55 (s), 27.60 (s), 37.7 (s), 56.6 (s), 67.0 (s), 67.2 (s), 95.6 (s), 107.1 (s), 117.5 (s), 123.6 (s), 125.2 (s), 125.6 (s), 134.3 (s), 153.9 (s), 159.6 (s), 159.8 (s), 165.5 (s), 172.8 (s); IR (attenuated total reflectance (ATR)) cm⁻¹: 1693. High resolution (HR)-MS (ESI-TOF) Calcd for C₂₁H₂₁NNaO₄ (M + Na)⁺ 374.1368. Found 374.1394.

3-Cyclobutyl-2-cyclopropyl-2H-10-oxa-2-azaanthracene-1,9-dione (5a)

Compound 5a was prepared from 3 according to the procedure for the synthesis of 5d. Yield was 27%. mp 276–281 °C; ¹H-NMR (DMSO-d₆) δ: 0.72–0.81 (2H, m), 1.12–1.22 (2H, m), 1.73–1.85 (1H, m), 1.95–2.11 (1H, m), 2.15–2.27 (2H, m), 2.35–2.45 (2H, m), 2.69–2.78 (1H, m), 3.95–4.08 (1H, m), 6.24 (1H, s), 7.41–7.49 (1H, m), 7.55 (1H, d, J = 8.3 Hz), 7.72–7.83 (1H, m), 8.05 (1H, d, J = 7.8 Hz); ¹³C-NMR (DMSO-d₆) δ: 9.9 (s, 2C), 17.2 (s), 27.3 (s), 27.8 (s, 2C), 37.3 (s), 94.9 (s), 106.7 (s), 117.5 (s), 123.5 (s), 125.2 (s), 125.5 (s), 134.2 (s), 154.0 (s), 160.0 (s), 162.3 (s), 165.4 (s), 172.8 (s); IR (ATR) cm⁻¹: 1685. HR-MS (ESI-TOF) Calcd for C₁₉H₁₇NNaO₃ (M + Na)⁺ 330.1106. Found 330.1128.

3-Cyclobutyl-2-cyclopentyl-2H-10-oxa-2-azaanthracene-1,9-dione (5b)

Compound 5b was prepared from 3 according to the procedure for the synthesis of 5d. Yield was 32%. mp 230–233 °C; ¹H-NMR (DMSO-d₆) δ: 1.47–1.64 (2H, m), 1.66–1.88 (3H, m), 1.90–2.08 (3H, m), 2.10–2.29 (4H, m), 2.30–2.43 (2H, m), 3.72–3.86 (1H, m), 4.43–4.60 (1H, m), 6.22 (1H, s), 7.37–7.49 (1H, m), 7.51–7.59 (1H, m), 7.72–7.83 (1H, m), 8.05 (1H, d, J = 7.8 Hz); ¹³C-NMR (DMSO-d₆) δ: 17.1 (s), 25.7 (s, 2C), 27.7 (s, 2C), 28.1 (s, 2C), 37.7 (s), 58.4 (s), 94.9 (s), 107.0 (s), 117.5 (s), 123.6 (s), 125.1 (s), 125.5 (s), 134.2 (s), 153.9 (s), 158.8 (s), 159.8 (s), 165.3 (s), 173.0 (s); IR (ATR) cm⁻¹: 1678. HR-MS (ESI-TOF) Calcd for C₂₁H₂₁NNaO₃ (M + Na)⁺ 358.1419. Found 358.1428.

3-Cyclobutyl-2-(tetrahydrofuran-3-yl)-2H-10-oxa-2-azaanthracene-1,9-dione (5c)

Compound 5c was prepared from 3 according to the procedure for the synthesis of 5d. Yield was 24%. mp 205–206 °C; ¹H-NMR (DMSO-d₆) δ: 1.70–2.45 (8H, m), 3.80–3.94 (4H, m), 3.99–4.07 (1H, m), 4.70–4.90 (1H, m), 6.29 (1H, s), 7.43–7.50 (1H, m), 7.53–7.61 (1H, m), 7.73–7.84 (1H, m), 8.06 (1H, d, J = 7.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 17.1 (s), 27.56 (s), 27.62 (s), 28.7 (s), 37.4 (s), 56.7 (s), 66.9 (s), 68.1 (s), 95.5 (s), 107.1 (s), 117.5 (s), 123.6 (s), 125.2 (s), 125.5 (s), 134.3 (s), 153.9 (s), 158.9 (s), 159.9 (s), 165.4 (s), 172.9 (s); IR (ATR) cm⁻¹: 1682. HR-MS (ESI-TOF) Calcd for C₂₀H₂₀NO₄ (M + H)⁺ 338.1392. Found 338.1372.

3-Cyclobutyl-2-pyridin-3-yl-2H-10-oxa-2-azaanthracene-1,9-dione (5e)

Compound 5e was prepared from 3 according to the procedure for the synthesis of 5d. Yield was 32%. mp 219–220 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.78 (4H, m), 2.00–2.20 (2H, m), 3.12–3.30 (1H, m), 6.48 (1H, s), 7.47–7.55 (1H, m), 7.57–7.67 (2H, m), 7.78–7.90 (2H, m), 8.04–8.13 (1H, m), 8.52 (1H, d, J = 2.0 Hz), 8.69 (1H, d, J = 4.4 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.7 (s), 26.9 (s), 27.0 (s), 37.7 (s), 95.4 (s), 106.9 (s), 117.7 (s), 123.5 (s), 124.1 (s), 125.4 (s), 125.6 (s), 134.2 (s), 134.5 (s), 136.8 (s), 149.5 (s), 149.6 (s), 154.0 (s), 159.3 (s), 159.4 (s), 166.4 (s), 172.7 (s); IR (ATR) cm⁻¹: 1689. HR-MS (ESI-TOF) Calcd for C₂₁H₁₆N₂NaO₃ (M + Na)⁺ 367.1059. Found 367.1069.

3-Cyclobutyl-2-(2-methoxyphenyl)-2H-10-oxa-2-azaanthracene-1,9-dione (5f)

Compound 5f was prepared from 3 according to the procedure for the synthesis of 5d. Yield was 29%. mp 248–258 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.81 (4H, m), 1.95–2.22 (2H, m), 3.02–3.15 (1H, m), 3.72 (3H, s), 6.43 (1H, s), 7.07 (1H, t, J = 7.7 Hz), 7.16–7.27 (2H, m), 7.42–7.53 (2H, m), 7.60 (1H, d, J = 8.3 Hz), 7.75–7.86 (1H, m), 8.06 (1H, d, J = 7.4 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.6 (s), 27.2 (s), 37.6 (s), 55.5 (s), 94.8 (s), 106.8 (s), 112.1 (s), 117.6 (s), 120.5 (s), 123.6 (s), 125.3 (s), 125.5 (s), 125.7 (s), 129.8 (s), 130.5 (s), 134.4 (s), 154.0 (s), 154.5 (s), 158.6 (s), 160.0 (s), 166.4 (s), 172.8 (s); IR (ATR) cm⁻¹: 1697. HR-MS (ESI-TOF) Calcd for C₂₃H₁₉NNaO₄ (M + Na)⁺ 396.1212. Found 396.1213.

6-Cyclobutyl-4-hydroxy-1-phenylpyridine-2(1H)-one (6a)

1 (1.86 g, 11.2 mmol) was suspended in AcOH (40 mL) and water (80 mL). Following the addition of aniline (1.03 mL, 11.2 mmol) to the reaction mixture, it was stirred at 85 °C for 18 h. After cooling, the reaction mixture was concentrated under reduced pressure. Following the addition of toluene (10 mL), the mixture was stirred at 50 °C for 10 min. The precipitate was collected by filtration. The resulting powder was washed with toluene and Et₂O to give 6a (1.11 g, 41% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.45–1.65 (4H, m), 1.81–2.00 (2H, m), 2.94–3.09 (1H, m), 5.54 (1H, s), 5.91 (1H, s), 7.11–7.23 (2H, m), 7.34–7.54 (3H, m), 10.45–10.80 (1H, br).

2-(6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridine-4-yl) 2-Fluorobenzoate (14a)

Et₃N (0.47 mL, 3.4 mmol) and 2-fluorobenzoyl chloride (0.30 mL, 2.5 mmol) were added to a suspension of 6a (400 mg, 1.66 mmol) in toluene (40 mL), and the mixture was stirred at room temperature for 40 min. Saturated aqueous NH₄Cl was added to the reaction mixture, which was then extracted with AcOEt, and the organic layer was washed with saturated aqueous NH₄Cl, water, and saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by silica gel column chromatography to give 14a (651 mg, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 1.71–1.76 (4H, m), 1.93–2.04 (2H, m), 3.10–3.20 (1H, m), 6.16 (1H, d, J = 2.1 Hz), 6.44 (1H, d, J = 2.1 Hz), 7.11–7.24 (3H, m), 7.27–7.33 (1H, m), 7.42–7.54 (3H, m), 7.60–7.67 (1H, m), 8.06–8.15 (1H, m).

3-Cyclobutyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (17a)

Following the addition of KCN (162 mg, 2.49 mmol), Et₃N (0.35 mL, 2.5 mmol), and 18-crown-6 (44 mg, 0.17 mmol) to a solution of 14a (651 mg, 1.66 mmol) in toluene (40 mL), the mixture was stirred at room temperature for 16 h. After the addition of water, the mixture was extracted with AcOEt. 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 and t-BuOMe (10 mL) was added. The insoluble material was collected by filtration to give 17a (312 mg, 55% yield) as a solid. mp: 240–243 °C; ¹H-NMR (DMSO-d₆) δ: 1.56–1.73 (4H, m), 2.02–2.17 (2H, m), 3.11–3.23 (1H, m), 6.44 (1H, s), 7.25–7.33 (2H, m), 7.45–7.57 (4H, m), 7.61 (1H, d, J = 8.3 Hz), 7.77–7.84 (1H, m), 8.07 (1H, d, J = 7.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.9 (s), 27.3 (s, 2C), 38.0 (s), 94.9 (s), 107.0 (s), 117.7 (s), 123.6 (s), 125.4 (s), 125.6 (s), 128.7 (s), 128.9 (s, 2C), 129.2 (s, 2C), 134.5 (s), 137.4 (s), 154.1 (s), 159.3 (s), 159.6 (s), 166.3 (s), 172.8 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₂H₁₇NNaO₃ (M + Na)⁺ 366.1106. Found 366.1122.

6-Cyclobutyl-1-(2-fluorophenyl)-4-hydroxy-1H-pyridin-2-one (7a)

Compound 7a was prepared from 1 according to the procedure for the synthesis of 6a. Yield was 28%. ¹H-NMR (CDCl₃) δ: 1.61–1.81 (4H, m), 1.94–2.08 (2H, m), 3.01–3.13 (1H, m), 5.95–6.02 (2H, m), 7.14–7.26 (3H, m), 7.40–7.48 (1H, m).

2-[6-Cyclobutyl-1-(2-fluorophenyl)-2-oxo-1,2-dihydropyridine-4-yl] 2-Fluorobenzoate (15a)

Compound 15a was prepared from 7a according to the procedure for the synthesis of 14a. Yield quant. ¹H-NMR (CDCl₃) δ: 1.62–1.85 (4H, m), 1.97–2.12 (2H, m), 3.09–3.21 (1H, m), 6.14–6.19 (1H, m), 6.43–6.47 (1H, m), 7.18–7.34 (5H, m), 7.42–7.50 (1H, m), 7.60–7.69 (1H, m), 8.05–8.12 (1H, m).

3-Cyclobutyl-2-(2-fluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione (18a)

Compound 18a was prepared from 15a according to the procedure for the synthesis of 17a. Yield was 24%. mp 220–249 °C; ¹H-NMR (DMSO-d₆) δ: 1.47–1.89 (4H, m), 1.95–2.09 (1H, m), 2.15–2.29 (1H, m), 3.11–3.25 (1H, m), 6.52 (1H, s), 7.33–7.54 (6H, m), 7.78–7.89 (1H, m), 8.06–8.12 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.81 (s), 26.84 (s), 37.6 (s), 95.6 (s), 106.8 (s), 116.2 (d), 117.7 (s), 123.6 (s), 124.8 (d), 125.2 (d), 125.5 (s), 125.6 (s), 130.8 (s), 131.4 (d), 134.5 (s), 154.0 (s), 157.4 (d), 158.5 (s), 159.3 (s), 166.6 (s), 172.7 (s); IR (ATR) cm⁻¹: 1693; HR-MS (ESI-TOF) Calcd for C₂₂H₁₆FNNaO₃ (M + Na)⁺ 384.1012. Found 384.1024.

6-Cyclobutyl-1-(2,3-difluorophenyl)-4-hydroxy-1H-pyridin-2-one (8a)

Compound 8a was prepared from 1 according to the procedure for the synthesis of 6a. Yield was 27%. ¹H-NMR (DMSO-d₆) δ: 1.40–1.52 (1H, m), 1.53–2.10 (5H, m), 3.07 (1H, quintet, J = 8.6 Hz), 5.56 (1H, s), 5.88 (1H, s), 7.10–7.37 (2H, m), 7.50–7.60 (1H, m).

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (16a)

Compound 16a was prepared from 8a according to the procedure for the synthesis of 14a. Yield quant. ¹H-NMR (CDCl₃) δ: 1.65–1.88 (4H, m), 1.99–2.15 (2H, m), 3.07–3.23 (1H, m), 6.18 (1H, d, J = 2.2 Hz), 6.46 (1H, d, J = 2.2 Hz), 6.98–7.07 (1H, m), 7.19–7.36 (4H, m), 7.60–7.70 (1H, m), 8.06–8.12 (1H, m).

3-Cyclobutyl-2-(2,3-difluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione (19a)

Compound 19a was prepared from 16a according to the procedure for the synthesis of 17a. Yield was 45%. mp 230–233 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.95 (4H, m), 1.97–2.10 (1H, m), 2.18–2.30 (1H, m), 3.18–3.30 (1H, m), 6.55 (1H, s), 7.31–7.46 (2H, m), 7.47–7.55 (1H, m), 7.60–7.75 (2H, m), 7.78–7.89 (1H, m), 8.08 (1H, d, J = 7.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.8 (s), 26.9 (s), 37.4 (s), 96.0 (s), 106.8 (s), 117.7 (s), 118.7 (d), 123.5 (s), 125.0 (dd), 125.5 (s), 125.6 (s), 126.2 (d), 126.5 (d), 134.6 (s), 146.0 (dd), 149.9 (dd), 154.0 (s), 158.4 (s), 159.0 (s), 166.7 (s), 172.7 (s); IR (ATR) cm⁻¹: 1697; HR-MS (ESI-TOF) Calcd for C₂₂H₁₅F₂NNaO₃ (M + Na)⁺ 402.0918. Found 402.0927.

4-Hydroxy-6-methyl-1-phenyl-1H-pyridin-2-one (6b)

Compound 6b was prepared from 1b according to the procedure for the synthesis of 6a. Yield was 60%. ¹H-NMR (DMSO-d₆) δ: 1.82 (3H, s), 5.54 (1H, d, J = 3.0 Hz), 5.86–5.89 (1H, m), 7.16–7.21 (2H, m), 7.39–7.51 (3H, m), 10.51–10.64 (1H, br).

6-Methyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (14b)

Compound 14b was prepared from 6b according to the procedure for the synthesis of 14a. Yield was 98%. ¹H-NMR (CDCl₃) δ: 1.99 (3H, s), 6.15–6.18 (1H, m), 6.45 (1H, d, J = 2.2 Hz), 7.19–7.32 (4H, m), 7.44–7.56 (3H, m), 7.60–7.67 (1H, m), 8.07 (1H, td, J = 7.8, 1.7 Hz).

3-Methyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (17b)

Compound 17b was prepared from 14b according to the procedure for the synthesis of 17a. Yield was 6.4%. mp 255–259 °C; ¹H-NMR (DMSO-d₆) δ: 2.02 (3H, s), 6.57 (1H, s), 7.30–7.35 (2H, m), 7.45–7.63 (5H, m), 7.77–7.83 (1H, m), 8.07 (1H, d, J = 7.8 Hz); ¹³C-NMR (DMSO-d₆) δ: 21.8 (s), 97.6 (s), 107.0 (s), 117.7 (s), 123.6 (s), 125.4 (s), 125.6 (s), 128.4 (s, 2C), 128.8 (s), 129.5 (s, 2C), 134.4 (s), 137.9 (s), 153.98 (s), 154.01 (s), 159.3 (s), 166.0 (s), 172.9 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) Calcd for C₁₉H₁₃NNaO₃ (M + Na)⁺ 326.0793. Found 326.0794.

6-Butyl-4-hydroxy-1-phenyl-1H-pyridin-2-one (6c)

Compound 6c was prepared from 1c according to the procedure for the synthesis of 6a. Yield was 33%. ¹H-NMR (DMSO-d₆) δ: 0.67 (3H, t, J = 7.3 Hz), 1.01–1.15 (2H, m), 1.21–1.34 (2H, m), 2.23 (2H, t, J = 7.6 Hz), 5.53 (1H, d, J = 2.4 Hz), 5.83 (1H, d, J = 2.4 Hz), 7.13–7.22 (2H, m), 7.37–7.53 (3H, m), 10.58 (1H, s).

6-Butyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (14c)

Compound 14c was prepared from 6c according to the procedure for the synthesis of 14a. Yield quant. ¹H-NMR (DMSO-d₆) δ: 0.77 (3H, t, J = 7.4 Hz), 1.13–1.30 (2H, m), 1.37–1.49 (2H, m), 2.23 (2H, t, J = 7.6 Hz), 6.15 (1H, d, J = 2.4 Hz), 6.44 (1H, d, J = 2.4 Hz), 7.18–7.34 (4H, m), 7.43–7.58 (3H, m), 7.60–7.69 (1H, m), 8.05–8.15 (1H, m).

3-Butyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (17c)

Compound 17c was prepared from 14c according to the procedure for the synthesis of 17a. Yield was 17%. mp 244–247 °C; ¹H-NMR (DMSO-d₆) δ: 0.70 (3H, t, J = 7.3 Hz), 1.07–1.20 (2H, m), 1.35–1.47 (2H, m), 2.27 (2H, t, J = 8.6 Hz), 6.50 (1H, s), 7.30–7.38 (2H, m), 7.44–7.66 (5H, m), 7.76–7.85 (1H, m), 8.06 (1H, d, J = 8.0 Hz); ¹³C-NMR (DMSO-d₆) δ: 13.3 (s), 21.5 (s), 29.1 (s), 33.2 (s), 96.7 (s), 107.0 (s), 117.7 (s), 123.6 (s), 125.4 (s), 125.6 (s), 128.80 (s, 2C), 128.83 (s), 129.4 (s, 2C), 134.5 (s), 137.5 (s), 154.1 (s), 157.5 (s), 159.4 (s), 166.1 (s), 172.9 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) Calcd for C₂₂H₁₉NNaO₃ (M + Na)⁺ 368.1263. Found 368.1272.

4-Hydroxy-6-isopropyl-1-phenyl-1H-pyridin-2-one (6d)

Compound 6d was prepared from 1d according to the procedure for the synthesis of 6a. Yield was 32%. ¹H-NMR (CDCl₃) δ: 1.04 (6H, d, J = 6.8 Hz), 2.46 (1H, septet, J = 6.8 Hz), 5.97–6.03 (2H, m), 7.61–7.22 (2H, m), 7.41–7.54 (3H, m).

6-Isopropyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (14d)

Compound 14d was prepared from 6d according to the procedure for the synthesis of 14a. Yield was 95%. ¹H-NMR (DMSO-d₆) δ: 1.11 (6H, d, J = 6.7 Hz), 2.57 (1H, septet, J = 6.7 Hz), 6.17 (1H, d, J = 2.3 Hz), 6.43 (1H, d, J = 2.3 Hz), 7.19–7.33 (3H, m), 7.43–7.57 (4H, m), 7.60–7.68 (1H, m), 8.05–8.13 (1H, m).

3-Isopropyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (17d)

Compound 17d was prepared from 14d according to the procedure for the synthesis of 17a. Yield was 5.1%. mp 209–211 °C; ¹H-NMR (DMSO-d₆) δ: 1.13 (6H, d, J = 6.6 Hz), 2.43 (1H, septet, J = 6.6 Hz), 6.55 (1H, s), 7.32–7.39 (2H, m), 7.45–7.64 (5H, m), 7.76–7.85 (1H, m), 8.07 (1H, d, J = 7.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 21.5 (s, 2C), 31.1 (s), 94.0 (s), 106.8 (s), 117.6 (s), 123.5 (s), 125.3 (s), 125.6 (s), 128.7 (s, 2C), 128.8 (s), 129.4 (s, 2C), 134.4 (s), 137.5 (s), 154.0 (s), 159.3 (s), 163.6 (s), 166.4 (s), 172.7 (s); IR (ATR) cm⁻¹: 1693; HR-MS (ESI-TOF) Calcd for C₂₁H₁₇NNaO₃ (M + Na)⁺ 354.1106. Found 354.1118.

7-(2,3-Difluorophenylamino)-2,2-dimethylpyrano-[4,3-d][1,3]dioxine-4,5-dione (10)

Following the addition of 2,3-difluoroaniline (2.9 mL, 29 mmol) to a solution of 9 (3.00 g, 13.0 mmol) in CHCl₃ (30 mL), the reaction mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure, and water (40 mL) and t-BuOMe (40 mL) were added. The insoluble material was collected by filtration to give 10 (3.47 g, 83% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 1.67 (6H, s), 5.36 (1H, s), 7.24–7.49 (3H, m), 10.93–11.07 (1H, br).

6-(2,3-Difluorophenyl)-7-hydroxy-2,2-dimethyl-6H-[1,3]dioxino[5,4-c]pyridine-4,5-dione (11)

Following the addition of NaH (p = 60%) (1.72 g, 43 mmol) to phenol (20 mL) under ice-cooling, the mixture was stirred at room temperature for 10 min. After the addition of 10 (3.47 g, 10.8 mmol) in phenol (5 mL) to the mixture, the reaction mixture was stirred at 110 °C for 15 min. After cooling, water (120 mL) was added and the reaction mixture was washed with Et₂O. The separated aqueous layer was acidified by adding 6.0 M aqueous HCl solution (20 mL), and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure and AcOEt (10 mL) and t-BuOMe (30 mL) was added. The insoluble material was collected by filtration to give 11 (1.13 g, 33% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 1.63 (6H, s), 5.09 (1H, s), 7.07–7.18 (1H, m), 7.20–7.30 (1H, m), 7.40–7.58 (1H, m).

6-(2,3-Difluorophenyl)-7-ethylamino-2,2-dimethyl-6H-[1,3]dioxino[5,4-c]pyridine-4,5-dione (12g)

A suspension of 11 (2.04 g, 6.31 mmol) in POCl₃ (17 mL) was stirred at 100 °C for 15 min. After cooling, the reaction mixture was concentrated under reduced pressure and saturated aqueous NaHCO₃ was added. The mixture was extracted with AcOEt. The organic layer was washed with saturated aqueous NaHCO₃ and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give an intermediate (1.54 g).

The solid obtained was dissolved in CH₂Cl₂ (15 mL), and i-Pr₂NEt (1.09 mL, 6.31 mmol) and 2.0 M EtNH₂ in tetrahydrofuran (THF) (2.5 mL, 5.0 mmol) were added under ice-cooling. The reaction mixture was stirred at room temperature for 2.5 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. The residue was purified by silica gel column chromatography to give 12g (1.02 g, 46% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.15–1.23 (3H, m), 1.72–1.77 (6H, m), 3.14–3.26 (2H, m), 4.23–4.34 (1H, br), 5.16 (1H, s), 7.00–7.11 (1H, m), 7.26–7.42 (2H, m).

tert-Butyl [6-(2,3-Difluorophenyl)-2,2-dimethyl-4,5-dioxo-5,6-dihydro-4H-[1,3]dioxino[5,4-c]pyridin-7-yl]ethylcarbamate (12f)

Following the addition of DMAP (52 mg, 0.43 mmol) and Boc₂O (93 mg, 0.43 mmol) to a solution of 12g (100 mg, 0.285 mmol) in MeCN (1 mL), the reaction mixture was stirred at room temperature for 16 h. After the addition of Boc₂O (93 mg, 0.43 mmol), the reaction mixture was stirred at 40 °C for 2 h and then at 60 °C for 3 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 12f (55 mg, 43% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.12–1.20 (3H, m), 1.37 (9H, s), 1.77–1.82 (6H, m), 2.80–3.10 (1H, br), 3.30–3.50 (1H, br), 5.92 (1H, s), 7.00–7.19 (2H, m), 7.26–7.34 (1H, m).

tert-Butyl [1-(2,3-Difluorophenyl)-4-hydroxy-6-oxo-1,6-dihydropyridin-2-yl]ethylcarbamate (8f)

Following the addition of 4.0 M aqueous NaOH (0.40 mL, 1.6 mmol) to a solution of 12f (50 mg, 0.11 mmol) in 1,4-dioxane (1 mL), the reaction mixture was heated to reflux for 15 h. After cooling, 0.1 M aqueous HCl solution (30 mL) was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated NaCl and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 8f (32 mg, 79% yield) as a solid.

¹H-NMR (CDCl₃) δ: 1.09 (3H, t, J = 7.1 Hz), 1.38 (9H, s), 2.70–3.00 (1H, br), 3.25–3.45 (1H, br), 6.01 (1H, s), 6.08 (1H, s), 7.03–7.17 (2H, m), 7.24–7.33 (1H, m).

6-(tert-Butoxycarbonylethylamino)-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (16f)

Compound 16f was prepared from 8f according to the procedure for the synthesis of 14a. Yield was 83%. ¹H-NMR (CDCl₃) δ: 1.13 (3H, t, J = 7.1 Hz), 1.40 (9H, s), 2.70–3.06 (1H, br), 3.20–3.52 (1H, br), 6.17–6.30 (1H, m), 6.55–6.65 (1H, m), 7.00–7.32 (5H, m), 7.60–7.69 (1H, m), 8.02–8.10 (1H, m).

tert-Butyl [2-(2,3-Difluorophenyl)-1,9-dioxo-2,9-dihydro-1H-10-oxa-2-azaanthracen-3-yl]ethylcarbamate (19f)

Compound 19f was prepared from 16f according to the procedure for the synthesis of 17a. Yield was 30%. ¹H-NMR (DMSO-d₆) δ: 1.00–1.45 (12H, m), 3.20–3.60 (2H, m), 6.75–6.90 (1H, m), 7.27–7.46 (2H, m), 7.49–7.56 (1H, m), 7.57–7.75 (2H, m), 7.81–7.89 (1H, m), 8.07–8.13 (1H, m).

2-(2,3-Difluorophenyl)-3-ethylamino-2H-10-oxa-2-azaanthracene-1,9-dione (19g)

After the addition of TFA (0.5 mL) to a solution of 19f (82 mg, 0.18 mmol) in CH₂Cl₂ (0.5 mL), the reaction mixture was stirred at room temperature for 1 h. Saturated aqueous NaHCO₃ was added and the mixture was extracted with CHCl₃. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography and t-BuOMe (5 mL) was added. The insoluble material was collected by filtration to give 19g (47 mg, 73% yield) as a solid. mp 305–310 °C; ¹H-NMR (DMSO-d₆) δ: 1.09 (3H, t, J = 7.1 Hz), 3.20–3.30 (2H, m), 5.71 (1H, s), 6.80–6.88 (1H, m), 7.21–7.30 (1H, m), 7.34–7.48 (3H, m), 7.61–7.75 (2H, m), 7.99 (1H, dd, J = 7.6, 1.5 Hz); ¹³C-NMR (DMSO-d₆) δ: 13.8 (s), 37.2 (s), 75.2 (s), 98.8 (s), 116.9 (s), 119.0 (d), 123.3 (s), 123.9 (d), 124.6 (s), 125.5 (s), 125.7 (dd), 126.9 (d), 133.6 (s), 147.0 (dd), 150.7 (dd), 153.5 (s), 154.1 (s), 158.2 (s), 167.7 (s), 171.6 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) Calcd for C₂₀H₁₄F₂N₂NaO₃ (M + Na)⁺ 391.0870. Found 391.0900.

7-Azetidin-1-yl-6-(2,3-difluorophenyl)-2,2-dimethyl-6H-[1,3]dioxino[5,4-c]pyridine-4,5-dione (12e)

Compound 12e was prepared from 11 according to the procedure for the synthesis of 12g. Yield was 42%. ¹H-NMR (CDCl₃) δ: 1.70–1.75 (6H, m), 2.20 (2H, quintet, J = 7.8 Hz), 3.55–3.75 (4H, m), 4.88 (1H, s), 7.02–7.12 (1H, m), 7.14–7.35 (2H, m).

6-Azetidin-1-yl-1-(2,3-difluoro-phenyl)-4-hydroxy-1H-pyridin-2-one (8e)

Compound 8e was prepared from 12e according to the procedure for the synthesis of 8f. Yield was 70%. ¹H-NMR (DMSO-d₆) δ: 1.99 (2H, quintet, J = 7.6 Hz), 3.34–3.44 (4H, m), 4.96 (1H, d, J = 2.2 Hz), 5.14 (1H, d, J = 2.2 Hz), 7.14–7.22 (1H, m), 7.26–7.36 (1H, m), 7.50–7.60 (1H, m), 10.45 (1H, s).

6-Azetidin-1-yl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 2-Fluorobenzate (16e)

Compound 16e was prepared from 8e according to the procedure for the synthesis of 14a. Yield was 81%. ¹H-NMR (CDCl₃) δ: 2.12 (2H, quintet, J = 7.6 Hz), 3.47–3.63 (4H, m), 5.26 (1H, d, J = 2.2 Hz), 5.94 (1H, d, J = 2.2 Hz), 7.08–7.15 (1H, m), 7.17–7.34 (4H, m), 7.58–7.66 (1H, m), 8.01–8.11 (1H, m).

3-Azetidin-1-yl-2-(2,3-difluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione (19e)

Compound 19e was prepared from 16e according to the procedure for the synthesis of 17a. Yield was 3.8%. ¹H-NMR (DMSO-d₆) δ: 2.02–2.18 (2H, m), 3.54–3.73 (4H, m), 5.43 (1H, s), 7.32–7.50 (4H, m), 7.56–7.75 (2H, m), 7.94–8.04 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 15.0 (s), 53.1 (s, 2C), 76.8 (s), 98.7 (s), 116.9 (s), 119.0 (d), 123.2 (s), 124.6 (s), 124.8 (dd), 125.2 (d), 125.5 (s), 127.4 (d), 133.7 (s), 147.1 (dd), 149.9 (dd), 154.0 (s), 154.2 (s), 157.9 (s), 166.8 (s), 171.5 (s); IR (ATR) cm⁻¹: 1682; HR-MS (ESI-TOF) Calcd for C₂₁H₁₄F₂N₂NaO₃ (M + Na)⁺ 403.0870. Found 403.0905.

6-(2,3-Difluorophenyl)-7-methoxy-2,2-dimethyl-6H-[1,3]dioxino[5,4-c]pyridine-4,5-dione (13)

Following the addition of i-Pr₂NEt (0.770 mL, 4.49 mmol) and trimethyloxonium tetrafluoroborate (697 mg, 4.71 mmol) to a suspension of 11 (1.45 g, 4.49 mmol) in CH₂Cl₂ (15 mL), the reaction mixture was stirred at room temperature for 15 h under a N₂ atmosphere. After the addition of i-Pr₂NEt (0.390 mL, 2.28 mmol) and trimethyloxonium tetrafluoroborate (350 mg, 2.28 mmol), the reaction mixture was stirred for 5 h. Following the addition of 1.0 M aqueous HCl solution, 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 13 (860 mg, 57% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.78 (6H, s), 3.88 (3H, s), 6.21 (1H, s), 6.98–7.07 (1H, m), 7.12–7.20 (1H, m), 7.22–7.32 (1H, m).

1-(2,3-Difluorophenyl)-4-hydroxy-6-methoxy-1H-pyridin-2-one (8h)

Compound 8h was prepared from 13 according to the procedure for the synthesis of 8f. Yield was 8.4%. ¹H-NMR (CDCl₃) δ: 3.73 (3H, s) 5.37 (1H, s), 5.47 (1H, s), 7.10–7.22 (1H, m), 7.23–7.35 (1H, m), 7.45–7.58 (1H, m), 10.88 (1H, s).

1-(2,3-Difluorophenyl)-6-methoxy-2-oxo-1,2-dihydropyridin-4-yl 2-Fluorobenzoate (16h)

Compound 16h was prepared from 8h according to the procedure for the synthesis of 14a. Yield was 83%. ¹H-NMR (CDCl₃) δ: 3.81 (3H, s), 5.65 (1H, d, J = 2.2 Hz), 6.23 (1H, d, J = 2.2 Hz), 7.03–7.10 (1H, m), 7.15–7.34 (4H, m), 7.60–7.68 (1H, m), 8.03–8.11 (1H, m).

2-(2,3-Difluorophenyl)-3-methoxy-2H-10-oxa-2-azaanthracene-1,9-dione (19h)

Compound 19h was prepared from 16h according to the procedure for the synthesis of 17a. Yield was 18%. mp: 246–254 °C; ¹H-NMR (DMSO-d₆) δ: 3.97 (3H, s), 7.30–7.43 (2H, m), 7.54–7.53 (1H, m), 7.54–7.67 (2H, m), 7.75–7.84 (1H, m), 8.02–8.11 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 58.5 (s), 78.3 (s), 103.4 (s), 117.5 (s), 118.4 (d), 123.3 (s), 124.0 (d), 124.9 (dd), 125.5 (s), 125.6 (s), 126.5 (d), 134.4 (s), 145.9 (dd), 150.0 (dd), 154.1 (s), 157.3 (s), 160.4 (s), 168.7 (s), 172.2 (s); IR (ATR) cm⁻¹: HR-MS (ESI-TOF) Calcd for C₁₉H₁₁F₂NNaO₄ (M + Na)⁺ 378.0554. Found 378.0581.

2-(2,3-Difluorophenyl)-3-hydroxy-2H-10-oxa-2-azaanthracene-1,9-dione (19i)

After the addition of 5.1 M HBr in AcOH (1.3 mL, 6.6 mmol) to 19h (50 mg, 0.141 mmol), the reaction mixture was stirred at 80 °C for 1 h. After cooling, saturated aqueous NaHCO₃ and NaCl were added and the mixture was extracted with AcOEt. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography and t-BuOMe was added. The insoluble material was collected by filtration to give 19i (24 mg, 50% yield) as a solid. mp: 336–341 °C; ¹H-NMR (DMSO-d₆) δ: 5.10–5.51 (1H, m), 6.60–7.98 (8H, m); ¹³C-NMR (DMSO-d₆) δ: 85.6 (s), 116.7 (s), 120.6 (d), 123.5 (s), 124.1 (s), 125.2 (s), 126.0 (dd), 128.8 (d), 131.5 (d), 132.1 (s), 134.6 (s), 147.9 (dd), 149.9 (dd), 154.6 (s), 162.1 (s), 164.2 (s), 165.0 (s), 174.0 (s). HR-MS (ESI-TOF) Calcd for C₁₈H₈F₂NO₄ (M − H)⁻ 340.0421. Found 340.0425.

6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Chlorocyclohexenecarboxylate (21a)

Following the addition of (COCl)₂ (0.25 mL, 2.9 mmol) and N,N-dimethylformamide (DMF) 1 drop to a solution of 20a (300 mg, 1.87 mmol) under ice-cooling, the reaction mixture was stirred at room temperature for 30 min. The reaction mixture was concentrated under reduced pressure to give an intermediate (0.40 g) as an oil.

Following the addition of Et₃N (0.35 mL, 2.5 mmol) and the intermediate (0.40 g) to 6a (300 mg, 1.24 mmol) in CH₂Cl₂ (5 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 1 h. After the removal of the solvent under reduced pressure, 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 21a (398 mg, 84% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.61–1.84 (8H, m), 1.95–2.03 (2H, m), 2.50–2.57 (4H, m), 3.06–3.17 (1H, m), 6.07 (1H, d, J = 2.4 Hz), 6.34 (1H, d, J = 2.4 Hz), 7.13–7.18 (2H, m), 7.40–7.51 (3H, m).

3-Cyclobutyl-2-phenyl-5,6,7,8-tetrahydro-2H-10-oxa-2-azaanthracene-1,9-dione (22a)

Compound 22a was prepared from 21a according to the procedure for the synthesis of 17a. Yield was 4.8%. mp: 185–188 °C; ¹H-NMR (DMSO-d₆) δ: 1.54–1.69 (6H, m), 1.72–1.82 (2H, m), 1.97–2.10 (2H, m), 2.26–2.34 (2H, m), 2.57–2.63 (2H, m), 3.04–3.16 (1H, m), 6.32 (1H, s), 7.20–7.26 (2H, m), 7.44–7.54 (3H, m); ¹³C-NMR (DMSO-d₆) δ: 16.9 (s), 20.6 (s), 20.9 (s), 21.2 (s), 26.6 (s), 27.3 (s, 2C), 37.8 (s), 94.6 (s), 108.4 (s), 121.2 (s), 128.6 (s), 128.9 (s, 2C), 129.1 (s, 2C), 137.6 (s), 157.5 (s), 159.3 (s), 160.8 (s), 165.6 (s), 174.0 (s); IR (ATR) cm⁻¹: 1682; HR-MS (ESI-TOF) Calcd for C₂₂H₂₁NNaO₃ (M + Na)⁺ 370.1419. Found 370.1425.

3-(6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridine-4-yl) 4-Chloronicotininate (21b)

Compound 21b was prepared from 20b according to the procedure for the synthesis of 21a. Yield was 79%. ¹H-NMR (CDCl₃) δ: 1.64–1.78 (4H, m), 1.93–2.04 (2H, m), 3.09–3.21 (1H, m), 6.16 (1H, d, J = 2.2 Hz), 6.47 (1H, d, J = 2.2 Hz), 7.16–7.22 (2H, m), 7.43–7.55 (4H, m), 8.69 (1H, d, J = 5.4 Hz), 9.24 (1H, s).

3-Cyclobutyl-2-phenyl-2H-10-oxa-2,7-diazaanthracene-1,9-dione (22b)

Compound 22b was prepared from 21b according to the procedure for the synthesis of 17a. Yield was 25%. mp: 267–272 °C; ¹H-NMR (DMSO-d₆) δ: 1.57–1.75 (4H, m), 2.03–2.18 (2H, m), 3.13–3.24 (1H, m), 6.50 (1H, s), 7.26–7.32 (2H, m), 7.47–7.58 (3H, m), 7.63 (1H, d, J = 5.6 Hz), 8.84 (1H, d, J = 5.6 Hz), 9.17 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 16.9 (s), 27.4 (s, 2C), 38.1 (s), 94.9 (s), 108.3 (s), 112.5 (s), 119.3 (s), 128.7 (s, 2C), 128.9 (s), 129.3 (s, 2C), 137.2 (s), 148.8 (s), 153.9 (s), 158.9 (s), 159.5 (s), 160.7 (s), 166.6 (s), 172.2 (s); IR (ATR) cm⁻¹: 1701; HR-MS (ESI-TOF) Calcd for C₂₁H₁₆N₂NaO₃ (M + Na)⁺ 367.1059. Found 367.1077.

6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Chloronicotinate (21c)

Compound 21c was prepared from 20c according to the procedure for the synthesis of 21a. Yield was 89%. ¹H-NMR (CDCl₃) δ: 1.62–1.78 (4H, m), 1.92–2.09 (2H, m), 3.09–3.23 (1H, m), 6.19 (1H, d, J = 2.2 Hz), 6.50 (1H, d, J = 2.2 Hz), 7.16–7.22 (2H, m), 7.40–7.54 (4H, m), 8.43–8.40 (1H, m), 8.60–8.66 (1H, m).

7-Cyclobutyl-6-phenyl-6H-9-oxa-1,6-diazaanthracene-5,10-dione (22c)

Compound 22c was prepared from 21c according to the procedure for the synthesis of 17a. Yield was 28%. mp: 215–216 °C; ¹H-NMR (DMSO-d₆) δ: 1.56–1.75 (4H, m), 2.05–2.21 (2H, m), 3.12–3.24 (1H, m), 6.57 (1H, s), 7.25–7.33 (2H, m), 7.46–7.58 (3H, m), 7.61 (1H, dd, J = 7.6, 4.6 Hz), 8.51 (1H, dd, J = 7.6, 1.8 Hz), 8.74 (1H, dd, J = 4.6, 1.8 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 27.3 (s, 2C), 38.0 (s), 95.1 (s), 106.9 (s), 118.5 (s), 122.6 (s), 128.7 (s, 2C), 128.8 (s), 129.2 (s, 2C), 136.4 (s), 137.2 (s), 153.1 (s), 158.7 (s), 159.1 (s), 160.2 (s), 166.4 (s), 173.4 (s); IR (ATR) cm⁻¹: 1693; HR-MS (ESI-TOF) Calcd for C₂₁H₁₆N₂NaO₃ (M + Na)⁺ 367.1059. Found 367.1062.

(6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl) 2-Fluoro-5-methylbenzoate (21d)

Compound 21d was prepared from 20d according to the procedure for the synthesis of 21a. Yield was 97%. ¹H-NMR (CDCl₃) δ: 1.63–1.77 (4H, m), 1.92–2.05 (2H, m), 2.41 (3H, s), 3.09–3.21 (1H, m), 6.15 (1H, d, J = 2.2 Hz), 6.43 (1H, d, J = 2.2 Hz), 7.07–7.15 (1H, m), 7.15–7.22 (2H, m), 7.38–7.54 (4H, m), 7.83–7.89 (1H, m).

3-Cyclobutyl-7-methyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (22d)

Compound 22d was prepared from 13d according to the procedure for the synthesis of 21a. Yield was 8.5%. mp: 247–251 °C; ¹H-NMR (DMSO-d₆) δ: 1.53–1.73 (4H, m), 2.00–2.17 (2H, m), 2.43 (3H, s), 3.10–3.23 (1H, m), 6.43 (1H, s), 7.23–7.34 (2H, m), 7.44–7.57 (4H, m), 7.62 (1H, d, J = 8.0 Hz), 7.86 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 20.3 (s), 27.2 (s, 2C), 37.9 (s), 94.8 (s), 106.9 (s), 117.4 (s), 123.2 (s), 125.0 (s), 128.6 (s), 128.8 (s, 2C), 129.1 (s, 2C), 134.8 (s), 135.3 (s), 137.4 (s), 152.2 (s), 159.28 (s), 159.33 (s), 166.2 (s), 172.8 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₃H₁₉NNaO₃ (M + Na)⁺ 380.1263. Found 380.1274.

6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl 2-Fluoro-4-methylbenzoate (21e)

Compound 21e was prepared from 20e according to the procedure for the synthesis of 21a. Yield was 44%. ¹H-NMR (CDCl₃) δ: 1.62–1.75 (4H, m), 1.95–2.05 (2H, m), 2.45 (3H, s), 3.09–3.19 (1H, m), 6.16 (1H, d, J = 2.2 Hz), 6.43 (1H, d, J = 2.2 Hz), 7.04 (1H, d, J = 11.7 Hz), 7.09 (1H, d, J = 7.8 Hz), 7.16–7.21 (2H, m), 7.42–7.53 (3H, m), 7.97 (1H, t, J = 7.8 Hz).

3-Cyclobutyl-6-methyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (22e)

Compound 22e was prepared from 21e according to the procedure for the synthesis of 17a. Yield was 13%. mp: 239–244 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.72 (4H, m), 2.02–2.16 (2H, m), 2.48 (3H, s), 3.11–3.22 (1H, m), 6.41 (1H, s), 7.26–7.32 (3H, m), 7.42 (1H, s), 7.47–7.57 (3H, m), 7.95 (1H, d, J = 8.1 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 21.1 (s), 27.3 (s, 2C), 37.9 (s), 94.8 (s), 106.9 (s), 117.3 (s), 121.3 (s), 125.4 (s), 126.6 (s), 128.6 (s), 128.8 (s, 2C), 128.9 (s), 129.1 (s, 2C), 137.4 (s), 145.4 (s), 154.1 (s), 159.3 (s), 166.2 (s), 172.6 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₃H₁₉NNaO₃ (M + Na)⁺ 380.1263. Found 380.1273.

(6-Cyclobutyl-2-oxo-1-phenyl-1,2-dihydropyridin-4-yl) 2-Fluoro-3-methylbenzoate (21f)

Compound 21f was prepared from 20f according to the procedure for the synthesis of 21a. Yield was 80%. ¹H-NMR (CDCl₃) δ: 1.63–1.76 (4H, m), 1.92–2.06 (2H, m), 2.37 (3H, d, J = 2.0 Hz), 3.09–3.22 (1H, m), 6.16 (1H, d, J = 2.2 Hz), 6.43 (1H, d, J = 2.2 Hz), 7.12–7.21 (3H, m), 7.42–7.54 (4H, m), 7.86–7.96 (1H, m).

3-Cyclobutyl-5-methyl-2-phenyl-2H-10-oxa-2-azaanthracene-1,9-dione (22f)

Compound 22f was prepared from 21f according to the procedure for the synthesis of 17a. Yield was 21%. mp: 239–242 °C; ¹H-NMR (DMSO-d₆) δ: 1.55–1.74 (4H, m), 2.05–2.19 (2H, m), 2.52 (3H, s), 3.12–3.23 (1H, m), 6.49 (1H, s), 7.24–7.32 (2H, m), 7.33–7.39 (1H, m), 7.45–7.58 (3H, m), 7.63–7.69 (1H, m), 7.85–7.93 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 14.9 (s), 16.8 (s), 27.3 (s, 2C), 38.0 (s), 95.0 (s), 106.7 (s), 123.1 (s), 123.5 (s), 124.7 (s), 126.7 (s), 128.7 (s), 128.8 (s, 2C), 129.1 (s, 2C), 135.1 (s), 137.4 (s), 152.4 (s), 159.29 (s), 159.31 (s), 166.0 (s), 173.0 (s); IR (ATR) cm⁻¹: 1693; HR-MS (ESI-TOF) Calcd for C₂₃H₁₉NNaO₃ (M + Na)⁺ 380.1263. Found 380.1268.

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 2-Fluoro-3-methoxy-benzoate (21h)

Following the addition of EDC·HCl (114 mg, 0.595 mmol) and DMAP (7 mg, 0.05 mmol) to a suspension of 20h (92 mg, 0.54 mmol) and 8a (150 mg, 0.540 mmol) in CH₂Cl₂ (10 mL) and DMF (1 mL), the reaction mixture was stirred at room temperature for 1.5 h. After the addition of 20h (28 mg, 0.16 mmol) and EDC·HCl (34 mg, 0.18 mmol), the mixture was stirred for 0.5 h. Water was added and the mixture was extracted with AcOEt. The organic layer was washed with 1.0 M aqueous HCl solution, saturated aqueous NaHCO₃, and saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give 21h (0.18 g, 77% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.65–1.89 (4H, m), 1.94–2.14 (2H, m), 3.08–3.20 (1H, m), 3.95 (3H, s), 6.15–6.21 (1H, m), 6.45–6.50 (1H, m), 6.98–7.05 (1H, m), 7.17–7.35 (4H, m), 7.56–7.65 (1H, m).

3-Cyclobutyl-2-(2,3-difluorophenyl)-5-methoxy-2H-10-oxa-2-azaanthracene-1,9-dione (22h)

Compound 22h was prepared from 21h according to the procedure for the synthesis of 17a. Yield was 14%. mp: 217–218 °C; ¹H-NMR (DMSO-d6) δ: 1.49–1.95 (4H, m), 1.96–2.12 (1H, m), 2.17–2.31 (1H, m), 3.17–3.28 (1H, m), 3.99 (3H, s), 6.53 (1H, s), 7.31–7.56 (4H, m), 7.57–7.74 (2H, m); ¹³C-NMR (DMSO-d₆) δ: 16.9 (s), 26.9 (s), 27.0 (s), 37.5 (s), 56.3 (s), 96.2 (s), 106.8 (s), 116.0 (s), 116.1 (s), 118.7 (d), 124.6 (s), 125.1 (dd), 125.4 (s), 126.3 (d), 126.7 (d), 144.1 (s), 146.1 (dd), 148.1 (s), 150.0 (dd), 158.5 (s), 159.0 (s), 166.4 (s), 172.7 (s); IR (ATR) cm⁻¹; 1697; HR-MS (ESI-TOF) Calcd for C₂₃H₁₇F₂NNaO₄ (M + Na)⁺ 432.1023. Found 432.1050.

3-Cyclobutyl-2-(2,3-difluorophenyl)-5-hydroxy-2H-10-oxa-2-azaanthracene-1,9-dione (23)

Following the addition of 1.0 M BBr₃ in CH₂Cl₂ (27 mL, 27 mmol) to a solution of 22h (1.1 g, 2.7 mmol) in CH₂Cl₂ (20 mL) under ice-cooling, the reaction mixture was stirred at room temperature for 2 h. Water (100 mL) was added dropwise under ice-cooling, followed by AcOEt (100 mL). After filtration of the insoluble material, the filtrate was dissolved in MeOH and dried over Na₂SO₄. The solvent was removed under reduced pressure and t-BuOMe was added. The insoluble material was collected by filtration to give 23 (346 mg, 33% yield) as a solid. mp: 298–301 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.83 (4H, m), 1.84–1.94 (1H, m), 1.96–2.09 (1H, m), 3.19–3.32 (1H, m), 6.49 (1H, s), 7.24–7.30 (2H, m), 7.33–7.51 (3H, m), 7.61–7.71 (1H, m), 10.55 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.8 (s), 26.9 (s), 37.3 (s), 96.0 (s), 106.5 (s), 114.8 (s), 118.6 (d), 119.7 (s), 124.8 (s), 125.0 (dd), 125.3 (s), 126.2 (d), 126.6 (d), 143.4 (s), 146.0 (dd), 146.3 (s), 149.9 (dd), 158.5 (s), 158.7 (s), 166.2 (s), 172.9 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) Calcd for C₂₂H₁₅F₂NNaO₄ (M + Na)⁺ 418.0867. Found 418.0905.

2-(3-Bromofuran-2-yl)-[1,3]dioxane (32)

Following the addition of p-TsOH·H₂O (1.66 g, 8.71 mmol) to a solution of 31 (3.81 g, 21.8 mmol) in propylene glycol (20 mL, 0.27 mol), the reaction mixture was stirred at room temperature for 2 h. After neutralization with 10% aqueous K₂CO₃, AcOEt was added. The organic layer was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 32 (4.70 g, 93% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.40–1.49 (1H, m), 2.21–2.38 (1H, m), 3.92–4.05 (2H, m), 4.18–4.35 (2H, m), 5.65 (1H, s), 6.41 (1H, d, J = 2.0 Hz), 7.37 (1H, d, J = 2.0 Hz).

3-Fluorofuran-2-carboxylic Acid (20g)

After the addition of 1.6 M n-BuLi in n-hexane (13.2 mL, 21 mmol) to a solution of 32 (4.70 g, 20.2 mmol) in THF (20 mL) at −78 °C over 30 min, the reaction mixture was stirred at the same temperature for 15 min. Following the addition of N-fluorobenzenesulfonimide (6.69 g, 21.2 mmol) in THF (20 mL) over 45 min, the mixture was stirred at room temperature for 1 h. After the addition of water (18 mL) and Et₃N (1.8 mL) under ice-cooling, Et₂O was added. The mixture was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give an intermediate (1.64 g) as an oil.

Following the addition of p-TsOH·H₂O (0.72 g, 3.8 mmol) to a solution of the intermediate obtained (1.64 g) in 1,4-dioxane (8 mL) and water (4 mL), the reaction mixture was stirred at room temperature for 1 h. After neutralization with 10% aqueous K₂CO₃ solution, AcOEt was added. The mixture was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give an intermediate (420 mg) as an oil.

Following the addition of 2-methyl-2-butene (3.90 mL, 36.8 mmol) to a solution of the intermediate obtained (420 mg) in t-BuOH (12 mL) and THF (6 mL), a solution of NaH₂PO₄ (3.53 g, 29.4 mmol) and NaClO₂ (520 mg, 4.60 mmol) in water (4 mL) was added dropwise under ice-cooling and stirred at room temperature for 1.5 h. The mixture was extracted with AcOEt and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 20g (157 mg, 4.9% yield for 3 steps) as a solid. ¹H-NMR (CDCl₃) δ: 6.41–6.50 (1H, m), 7.41–7.48 (1H, m).

6-Cyclobutyl-7-(2,3-difluorophenyl)-7H-1,4-dioxa-7-azacyclopenta[b]naphthalene-8,9-dione (22g)

Compound 22g was prepared from 20g according to the procedure for the synthesis of 22h. Yield was 2.3% for 2 steps. mp: 247–264 °C; ¹H-NMR (DMSO-d₆) δ: 1.47–1.95 (4H, m), 1.95–2.09 (1H, m), 2.15–2.29 (1H, m), 3.15–3.28 (1H, m), 6.60 (1H, s), 7.14 (1H, d, J = 2.0 Hz), 7.28–7.46 (2H, m), 7.58–7.72 (1H, m), 8.21 (1H, d, J = 2.0 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.7 (s), 26.7 (s), 26.8 (s), 37.1 (s), 96.1 (s), 103.0 (s), 109.7 (s), 118.7 (d), 125.0 (dd), 126.2 (d), 126.5 (d), 138.0 (s), 148.7 (dd), 148.8 (s), 149.9 (dd), 150.9 (s), 157.0 (s), 158.9 (s), 163.3 (s), 166.5 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₀H₁₃F₂NNaO₄ (M + Na)⁺ 392.0710. Found 390.0740.

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 3-tert-Butoxycarbonylamino-2-fluorobenzoate (21i)

Compound 21i was prepared from 20i according to the procedure for the synthesis of 21h. Yield was 82%. ¹H-NMR (CDCl₃) δ: 1.45–1.63 (9H, m), 1.64–1.88 (4H, m), 1.98–2.13 (2H, m), 3.08–3.21 (1H, m), 6.14–6.20 (1H, m), 6.46 (1H, d, J = 2.2 Hz), 6.80–6.90 (1H, m), 6.98–7.04 (1H, m), 7.19–7.35 (3H, m), 7.65–7.71 (1H, m), 8.38–8.48 (1H, m).

tert-Butyl [3-Cyclobutyl-2-(2,3-difluorophenyl)-1,9-dioxo-2,9-dihydro-1H-10-oxa-2-azaanthracen-5-yl]carbamate (22i)

Compound 22i was prepared from 21i according to the procedure for the synthesis of 17a. Yield was 32%. ¹H-NMR (CDCl₃) δ: 1.60 (9H, s), 1.70–1.94 (4H, m), 2.09–2.26 (2H, m), 3.14–3.28 (1H, m), 6.38 (1H, s), 6.97–7.04 (1H, m), 7.05–7.13 (1H, m), 7.18–7.38 (3H, m), 7.93 (1H, dd, J = 7.8, 1.2 Hz), 8.34–8.46 (1H, m).

5-Amino-3-cyclobutyl-2-(2,3-difluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione Hydrochloride (24)

After the addition of 4.0 M HCl in 1,4-dioxane (0.89 mL, 3.6 mmol) to 22i (117 mg, 0.237 mmol) under ice-cooling, the reaction mixture was stirred at the same temperature for 50 min and at room temperature for 1 h. Following the addition of 1,4-dioxane (2 mL) and AcOEt (1 mL), the insoluble material was collected by filtration to give 24 (84.8 mg, 83% yield) as a solid. mp: >152 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 1.53–2.25 (6H, m), 3.20–3.32 (1H, m), 6.60 (1H, s), 7.01–7.06 (1H, m), 7.11–7.21 (2H, m), 7.32–7.46 (2H, m), 7.62–7.72 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.8 (s), 26.9 (s), 37.3 (s), 96.1 (s), 106.3 (s), 111.6 (s), 117.8 (s), 118.6 (d), 123.9 (s), 125.0 (dd), 125.4 (s), 126.3 (d), 126.7 (d), 137.0 (s), 142.2 (s), 146.0 (dd), 149.9 (dd), 158.2 (s), 158.6 (s), 166.0 (s), 173.2 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₂H₁₆F₂N₂NaO₃ (M + Na)⁺ 417.1027. Found 417.1068.

Methyl 4-(2-tert-Butoxycarbonylaminoethyl)-2-fluorobenzoate (39)

Following the addition of Et₃N (9.7 mL, 70 mmol) and diphenylphosphoryl azide (DPPA) (15 mL, 70 mmol) to a suspension of 38 (11.4 g, 46.6 mmol) in t-BuOH (94 mL), the reaction mixture was heated to 100 °C over 30 min and then stirred at the same temperature for 3.5 h. After cooling, water was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 39 (6.61 g, 48% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.43 (9H, s), 2.55 (2H, t, J = 7.8 Hz), 3.30–3.46 (2H, m), 3.92 (3H, s), 4.41–4.60 (1H, br), 6.92–7.06 (2H, m), 7.82–7.90 (1H, m).

4-(2-tert-Butoxycarbonylaminoethyl)-2-fluorobenzoic Acid (20k)

Following the addition of 1.0 M aqueous NaOH solution (67 mL, 67 mmol) to a solution of 39 (6.61 g, 22.2 mmol) in MeOH (130 mL), the reaction mixture was stirred at 40 °C for 1 h. After cooling, 1.0 M aqueous HCl solution (80 mL, 80 mmol) was added and the mixture was extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 20k (6.28 g, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 1.34 (9H, s), 2.72–2.82 (2H, m), 3.12–3.23 (2H, m), 6.78–6.94 (1H, m), 7.03–7.23 (2H, m), 7.70–7.83 (1H, m), 12.84–13.19 (1H, br).

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 4-(2-tert-Butoxycarbonyl-aminoethyl)-2-fluorobenzoate (21k)

Compound 21k was prepared from 20k according to the procedure for the synthesis of 21h. Yield was 99%. ¹H-NMR (CDCl₃) δ: 1.45 (9H, s), 1.64–1.93 (4H, m), 1.96–2.18 (2H, m), 2.81–2.98 (2H, m), 3.08–3.23 (1H, m), 3.35–3.52 (2H, m), 4.47–4.64 (1H, m), 6.15–6.20 (1H, m), 6.41–6.47 (1H, m), 6.97–7.17 (5H, m), 7.70–7.83 (1H, m).

tert-Butyl{2-[3-Cyclobutyl-2-(2,3-difluorophenyl)-1,9-dioxo-2,9-dihydro-1H-10-oxa-2-azaanthracen-6-yl]ethyl}carbamate (22k)

Compound 22k was prepared from 21k according to the procedure for the synthesis of 17a. mp: 228–231 °C; ¹H-NMR (DMSO-d₆) δ: 1.11 (9H, s), 1.50–1.94 (4H, m), 1.97–2.10 (1H, m), 2.17–2.30 (1H, m), 2.87 (2H, t, J = 6.4 Hz), 3.18–3.30 (3H, m), 6.51 (1H, s), 6.90–6.99 (1H, br), 7.30–7.47 (4H, m), 7.60–7.72 (1H, m), 7.98 (1H, d, J = 8.1 Hz).

6-(2-Aminoethyl)-3-cyclobutyl-2-(2,3-difluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione (27)

Compound 27 was prepared from 20k according to the procedure for the synthesis of 24. ¹H-NMR (DMSO-d₆) δ: 1.72–1.93 (4H, m), 2.07–2.23 (2H, m), 2.89 (2H, t, J = 6.7 Hz), 3.07 (2H, t, J = 6.7 Hz), 3.13–3.26 (1H, m), 6.28 (1H, s), 6.98–7.07 (1H, m), 7.18–7.38 (4H, m), 8.23 (1H, d, J = 7.8 Hz).

3-Cyclobutyl-2-(2,3-difluorophenyl)-6-(2-dimethylaminoethyl)-2H-10-oxa-2-azaanthracene-1,9-dione (28)

Following the addition of Et₃N (0.030 mL, 0.22 mmol), formalin (p = 37%) (0.080 mL, 1.1 mmol), and NaBH(OAc)₃ (228 mg, 1.08 mmol) to a solution of 27 (91 mg, 0.22 mmol) in MeOH (10 mL), the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was concentrated under reduced pressure and saturated aqueous NaHCO₃ was added. 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 AcOEt (1 mL) was added. The insoluble material was collected by filtration to give 28 (40 mg, 41% yield) as a solid. mp: 174–177 °C; ¹H-NMR (DMSO-d₆) δ: 1.51–1.94 (4H, m), 1.97–2.09 (1H, m), 2.17–2.30 (1H, m), 2.34 (6H, s), 2.75 (2H, t, J = 7.0 Hz), 2.95 (2H, t, J = 7.0 Hz), 6.50 (1H, s), 7.31–7.47 (3H, m), 7.51 (1H, s), 7.60–7.71 (1H, m), 7.98 (1H, d, J = 8.1 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.8 (s), 26.9 (s), 32.8 (s), 37.3 (s), 44.9 (s, 2C), 59.6 (s), 96.0 (s), 106.7 (s), 117.2 (s), 118.6 (d), 121.6 (s), 125.0 (dd), 125.3 (s), 126.2 (d), 126.4 (s), 126.6 (d), 146.0 (dd), 148.6 (s), 149.9 (dd), 154.0 (s), 158.4 (s), 158.8 (s), 166.6 (s), 172.5 (s); IR (ATR) cm⁻¹; HR-MS (ESI-TOF) Calcd for C₂₆H₂₄F₂N₂NaO₃ (M + Na)⁺ 473.1653. Found 473.1683.

Methyl 3-(2-tert-Butoxycarbonylvinyl)-2-fluorobenzoate (34)

After the addition of Pd(OAc)₂ (449 mg, 2.00 mmol) and P(o-tolyl)₃ (1.22 g, 4.00 mmol) to DMF (20 mL), the reaction mixture was stirred at room temperature for 5 min. Following the addition of 33 (4.66 g, 20.0 mmol), tert-butyl acrylate (5.8 mL, 40 mmol), and Et₃N (5.8 mL, 40 mmol), the reaction mixture was stirred at 100 °C for 16 h and at 120 °C for 2 h. After cooling, AcOEt was added and the mixture was extracted with AcOEt. The organic layer was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 34 (4.2 g, 68% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.54 (9H, s), 3.94 (3H, s), 6.47 (1H, d, J = 16.1 Hz), 7.21 (1H, t, J = 7.8 Hz), 7.64–7.80 (2H, m), 7.86–7.96 (1H, m).

Methyl 3-(2-tert-Butoxycarbonylethyl)-2-fluorobenzoate (35)

A solution of 34 (4.24 g, 13.7 mmol) in MeOH (75 mL) was hydrogenated at 0.4 MPa in the presence of 10% Pd-C (770 mg) at room temperature for 15 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 35 (3.80 g, 98% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.40 (9H, s), 2.55 (2H, t, J = 7.6 Hz), 2.98 (2H, t, J = 7.6 Hz), 3.92 (3H, s), 7.07–7.14 (1H, m), 7.36–7.44 (1H, m), 7.72–7.80 (1H, m).

3-(2-Fluoro-3-methoxycarbonylphenyl)propionic Acid (36)

After the addition of 35 (3.80 g, 13.5 mmol) to CH₂Cl₂ (13.5 mL) and TFA (13.5 mL), the reaction mixture was stirred at room temperature for 2.5 h. The reaction mixture was concentrated under reduced pressure and AcOEt was then added. The mixture was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 36 (3.67 g, 87% yield) as an oil. ¹H-NMR (CDCl₃) δ: 2.55 (2H, t, J = 7.6 Hz), 2.89 (2H, t, J = 7.6 Hz), 3.85 (3H, s), 7.19–7.28 (1H, m), 7.54–7.62 (1H, m), 7.64–7.78 (1H, m).

Methyl 3-(2-tert-Butoxycarbonylaminoethyl)-2-fluorobenzoate (37)

Following the addition of Et₃N (2.43 mL, 17.5 mmol) and DPPA (3.77 mL, 17.5 mmol) to a suspension of 36 (3.67 g, 11.7 mmol) in t-BuOH (25 mL), the reaction mixture was heated to 100 °C over 23 min, and the mixture was stirred at the same temperature for 1.5 h. After cooling, water was added and the mixture was extracted with AcOEt. 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 37 (1.41 g, 41% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.42 (9H, s), 2.82–2.97 (2H, m), 3.29–3.45 (2H, m), 3.92 (3H, s), 4.48–4.64 (1H, br), 7.13 (1H, t, J = 7.6 Hz), 7.32–7.44 (1H, m), 7.74–7.83 (1H, m).

3-(2-tert-Butoxycarbonylaminoethyl)-2-fluorobenzoic Acid (20j)

Following the addition of 1.0 M aqueous NaOH (14.2 mL, 14 mmol) to a solution of 37 (1.41 g, 4.74 mmol) in MeOH (36 mL), the reaction mixture was stirred at 40 °C for 40 min. After cooling, 1.0 M aqueous HCl solution was added to the reaction solution to make it weakly acidic. The mixture was extracted with AcOEt and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 20j (1.07 g, 80% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 1.34 (9H, s), 2.70–2.83 (2H, m), 3.05–3.20 (2H, m), 6.81–6.99 (1H, m), 7.19 (1H, t, J = 7.6 Hz), 7.35–7.53 (1H, m), 7.56–7.78 (1H, m).

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 3-(2-tert-Butoxycarbonylaminoethyl)-2-fluorobenzoate (21j)

Compound 21j was prepared from 20j according to the procedure for the synthesis of 21h. Yield quant. ¹H-NMR (CDCl₃) δ: 1.43 (9H, s), 1.67–1.90 (4H, m), 1.97–2.16 (2H, m), 2.83–3.03 (2H, m), 3.05–3.26 (1H, m), 3.34–3.48 (2H, m), 4.52–4.67 (1H, m), 6.17 (1H, s), 6.46 (1H, s), 6.97–7.06 (1H, m), 7.15–7.36 (3H, m), 7.47–7.57 (1H, m), 7.91–7.99 (1H, m).

tert-Butyl{2-[3-Cyclobutyl-2-(2,3-difluorophenyl)-1,9-dioxo-2,9-dihydro-1H-10-oxa-2-azaanthracen-5-yl]ethyl}carbamate (22j)

Compound 22j was prepared from 21j according to the procedure for the synthesis of 17a. mp: 264–265 °C; ¹H-NMR (CDCl₃) δ: 1.29 (9H, s), 1.51–2.11 (5H, m), 2.17–2.32 (1H, m), 2.94–3.09 (2H, m), 3.18–3.42 (3H, m), 6.70 (1H, s), 6.85–6.98 (1H, m), 7.30–7.49 (3H, m), 7.56–7.73 (2H, m), 7.93 (1H, d, J = 7.3 Hz) ; IR (ATR) cm⁻¹: 1685.

5-(2-Aminoethyl)-3-cyclobutyl-2-(2,3-difluorophenyl)-2H-10-oxa-2-azaanthracene-1,9-dione Hydrochloride (25)

Following the addition of 9.1 M HCl in i-PrOH (0.28 mL, 2.6 mmol) to a suspension of 22j (134 mg, 0.256 mmol) in AcOEt (0.57 mL) under ice-cooling, the reaction mixture was stirred at the same temperature for 2 h. After the addition of 9.1 M HCl in i-PrOH (0.28 mL, 2.6 mmol) under ice-cooling, the mixture was stirred at the same temperature for 1 h. The insoluble material was collected by filtration to give 25 (51 mg, 47% yield) as a solid. mp: >242 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 1.52–1.98 (4H, m), 1.98–2.13 (1H, m), 2.19–2.34 (1H, m), 3.14–3.29 (5H, m), 6.81 (1H, s), 7.27–7.52 (3H, m), 7.60–7.79 (2H, m), 7.97 (1H, d, J = 7.6 Hz), 8.02–8.19 (3H, br); ¹³C-NMR (DMSO-d₆) δ: 16.9 (s), 26.9 (s), 27.1 (s), 27.2 (s, 2C), 37.5 (s), 96.8 (s), 106.6 (s), 118.8 (d), 123.7 (s), 124.4 (s), 125.1 (s) 125.2 (d), 126.3 (d), 126.6 (s), 126.7 (s), 135.6 (s), 146.0 (dd), 150.0 (dd), 152.7 (s), 158.6 (s), 158.9 (s), 166.6 (s), 173.1 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₄H₂₀F₂N₂NaO₃ (M + Na)⁺ 445.1340. Found 445.1368.

3-Cyclobutyl-2-(2,3-difluorophenyl)-5-(2-dimethylaminoethyl)-2H-10-oxa-2-azaanthracene-1,9-dione (26)

Compound 26 was prepared from 25 according to the procedure for the synthesis of 28. mp: 191–194 °C; ¹H-NMR (DMSO-d₆) δ: 1.51–2.14 (5H, m), 2.16–2.31 (1H, m), 2.43 (6H, s), 2.74–2.93 (2H, m), 3.05–3.50 (3H, m), 6.58 (1H, s), 7.24–7.55 (3H, m), 7.57–7.82 (2H, m), 7.94 (1H, d, J = 7.6 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 26.5 (s), 26.9 (s), 27.0 (s), 37.4 (s), 45.1 (s, 2C), 59.1 (s), 96.2 (s), 106.5 (s), 118.7 (d), 123.4 (s), 123.6 (s), 125.0 (s), 125.1 (d), 126.3 (d), 126.7 (d), 129.8 (s), 135.0 (s), 146.1 (dd), 150.0 (dd), 152.3 (s), 158.5 (s), 159.0 (s), 166.5 (s), 173.0 (s); IR (ATR) cm⁻¹: 1685; HR-MS (ESI-TOF) Calcd for C₂₆H₂₄F₂N₂NaO₃ (M + Na)⁺ 473.1653. Found 473.1697.

2-tert-Butoxycarbonyl-8-fluoro-1,2,3,4-tetrahydroisoquinoline-7-carboxylic Acid (20l)

Following the addition of pyridine (6.7 mL, 83 mmol) to a solution of 40 (4.44 mL, 16.6 mmol) in CH₂Cl₂ (60 mL), Tf₂O (4.08 mL, 24.9 mmol) was added dropwise under ice-cooling, and the reaction mixture was stirred at room temperature for 1 h. Water was added and the mixture was extracted with CHCl₃. The organic layer was washed with 1.0 M aqueous HCl solution and dried over Na₂SO₄. The solvent was removed under reduced pressure.

The residue obtained was dissolved in DMF (70 mL), and HCO₂Li·H₂O (3.50 g, 50.0 mmol), 9,9-dimethyl-9H-xanthene-4,5-diyl)bis(diphenylphosphane) (577 mg, 0.997 mmol), tris(dibenzylideneacetone)dipalladium (912 mg, 0.996 mmol), LiCl (2.53 g, 59.7 mmol), i-Pr₂NEt (6.8 mL, 40 mmol), and Ac₂O (3.8 mL, 40 mmol) were added. The reaction mixture was stirred at 80 °C for 14 h under a N₂ atmosphere. After cooling, water was added to the mixture, and the insoluble material was filtered off. One molar aqueous HCl solution was added to the filtrate to acidify it, and the mixture was extracted with AcOEt. The organic layer was washed with water and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 20l (2.61 g, 83% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 2.82–2.93 (2H, m), 3.57–3.76 (2H, m), 4.60–4.73 (2H, br), 7.02 (1H, d, J = 8.0 Hz), 7.78–7.86 (1H, m).

6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl 2-tert-Butoxycarbonyl-8-fluoro-1,2,3,4-tetrahydroisoquinoline-7-carboxylate (21l)

Compound 21l was prepared from 20l according to the procedure for the synthesis of 21f. Yield was 71%. ¹H-NMR (CDCl₃) δ: 1.51 (9H, m), 1.64–1.89 (4H, m), 1.96–2.14 (2H, m), 2.84–2.98 (2H, m), 3.07–3.22 (1H, m), 3.61–3.76 (2H, m), 4.58–4.73 (2H, br), 6.14–6.20 (1H, m), 6.41–6.47 (1H, m), 6.95–7.35 (4H, m), 7.81–7.92 (1H, m).

tert-Butyl 10-Cyclobutyl-9-(2,3-difluorophenyl)-7,8-dioxo-4,7,8,9-tetrahydro-1H, 3H-12-oxa-2,9-diazabenzo[a]anthracene-2-carboxylate (22l)

Compound 22l was prepared from 21l according to the procedure for the synthesis of 17a. Yield was 37%. ¹H-NMR (CDCl₃) δ: 1.47–1.68 (9H, m), 1.72–1.96 (4H, m), 2.07–2.26 (2H, m), 2.89–3.02 (2H, m), 3.14–3.29 (1H, m), 3.62–3.83 (2H, m), 4.71–4.95 (2H, m), 6.32 (1H, s), 6.97–7.06 (1H, m), 7.16–7.36 (3H, m), 8.11 (1H, d, J = 8.3 Hz).

10-Cyclobutyl-9-(2,3-difluorophenyl)-1,2,3,4-tetrahydro-9H-12-oxa-2,9-diazabenzo[a]anthracene-7,8-dione Hydrochloride (29)

Compound 29 was prepared from 22l according to the procedure for the synthesis of 24. Yield was 85%. mp: 235–243 °C; ¹H-NMR (DMSO-d₆) δ: 1.51–2.12 (5H, m), 2.16–2.31 (1H, m), 3.13–3.20 (2H, m), 3.21–3.31 (1H, m), 3.43–3.53 (2H, m), 4.43–4.60 (2H, m), 6.68 (1H, s), 7.25–7.48 (3H, m), 7.58–7.74 (3H, m), 7.96 (1H, d, J = 8.0 Hz), 9.51–9.88 (2H, br); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 18.5 (s), 25.0 (s), 26.9 (s), 27.0 (s), 37.5 (s), 56.0 (s), 96.3 (s), 106.9 (s), 118.4 (s), 118.8 (d), 121.7 (s), 124.0 (s), 125.1 (dd), 125.9 (s), 126.3 (d), 126.6 (d), 139.5 (s), 146.0 (dd), 150.0 (dd), 150.8 (s), 158.5 (s), 159.2 (s), 166.4 (s), 172.4 (s); IR (ATR) cm⁻¹: 1689; HR-MS (ESI-TOF) Calcd for C₂₅H₂₀F₂N₂NaO₃ (M + Na)⁺ 457.1340. Found 457.1391.

6-[6-Cyclobutyl-1-(2,3-difluorophenyl)-2-oxo-1,2-dihydropyridin-4-yl] 2-tert-Butoxycarbonyl-5-fluoro-3,4-dihydro-1H-isoquinoline-6-carboxylate (21 m)

Compound 21 m was prepared from 20 m according to the procedure for the synthesis of 21h. Yield was 63%. ¹H-NMR (CDCl₃) δ: 1.50 (9H, s), 1.65–1.88 (4H, m), 2.00–2.14 (2H, m), 2.85–2.95 (2H, m), 3.08–3.22 (1H, m), 3.65–3.75 (2H, m), 4.61–4.70 (2H, m), 6.15–6.21 (1H, m), 6.44–6.50 (1H, m), 6.97–7.10 (2H, m), 7.18–7.37 (2H, m), 7.82–7.94 (1H, m).

tert-Butyl 10-Cyclobutyl-9-(2,3-difluorophenyl)-7,8-dioxo-1,2,4,7,8,9-hexahydro-12-oxa-3,9-diazabenzo[a]anthracene-3-carboxylate (22 m)

Compound 22 m was prepared from 21 m according to the procedure for the synthesis of 17a. Yield was 18%. ¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 1.70–1.95 (4H, m), 2.08–2.22 (2H, m), 3.02–3.11 (2H, m), 3.15–3.28 (1H, m), 3.70–3.84 (2H, m), 4.65–4.75 (2H, m), 6.31 (1H, s), 6.95–7.38 (4H, m), 8.13 (1H, d, J = 8.1 Hz).

10-Cyclobutyl-9-(2,3-difluorophenyl)-1,2,3,4-tetrahydro-9H-12-oxa-3,9-diazabenzo[a]anthracene-7,8-dione (30)

Compound 30 was prepared from 22 m according to the procedure for the synthesis of 24. Yield was 69%. mp: 228–232 °C; ¹H-NMR (DMSO-d₆) δ: 1.50–1.94 (4H, m), 1.96–2.10 (1H, m), 2.17–2.30 (1H, m), 2.90–3.30 (5H, m), 4.01–4.10 (2H, m), 6.56 (1H, s), 7.20 (1H, d, J = 8.1 Hz), 7.32–7.48 (2H, m), 7.60–7.72 (1H, m), 7.83 (1H, d, J = 8.1 Hz); ¹³C-NMR (DMSO-d₆) δ: 16.8 (s), 22.5 (s), 26.8 (s), 26.9 (s), 37.3 (s), 42.1 (s), 47.8 (s), 96.2 (s), 106.6 (s), 118.6 (d), 121.2 (s), 121.9 (s), 123.4 (s), 124.3 (s), 125.0 (dd), 126.2 (d), 126.6 (d), 143.9 (s), 146.0 (dd), 149.9 (dd), 152.1 (s), 158.5 (s), 158.6 (s), 166.3 (s), 172.8 (s); IR (ATR) cm⁻¹: 1682; HR-MS (ESI-TOF) Calcd for C₂₅H₂₀F₂N₂NaO₃ (M + Na)⁺ 457.1340. Found 457.1388; Anal. Calcd for C₂₅H₂₀F₂N₂O₃: C, 69.12; H, 4.64; N, 6.45. Found: C, 68.92; H, 4.68; N, 6.41; HPLC purity 99.4% (eluent: 10 mM NaH₂PO₄-Na₂HPO₄ (pH 7.0)/MeCN = 35/65).

Evaluation of IDUA Activity and Cellular GAG Levels in Hurler Patient-Derived Fibroblasts

1. 1.   Cell culture
   • Healthy human neonatal dermal fibroblasts (healthy donor fibroblasts) were purchased from Zen-Bio, Inc. (Research Triangle Park, NC, U.S.A.). Hurler patient-derived fibroblasts (GM00798; IDUA-W402X) were obtained from the Coriell Institute for Medical Research (Camden, NJ, U.S.A.). The Coriell Institute for Medical Research guarantees that skin samples were collected under IRB approval and with patient informed consent. The use of human samples was approved by the ethical committee of Kyoto Pharmaceutical Industries, Ltd.

2. 2.   Treatment
   • Fibroblasts in MEM (Thermo Fisher Scientific, Waltham, MA, U.S.A.) supplemented with 10% FBS (MEM/10% FBS) or in FibroLife S2 Comp kit medium (LifeLine Cell Technology, Frederick, MD, U.S.A.) were seeded at 1.25 × 10⁵ cells/well (500 µL/well) in 24-well plates and incubated under 5% CO₂ at 37 °C for 24 h. Five hundred microliters of the compound dissolved in medium was added to cells, followed by an incubation under 5% CO₂ at 37 °C. Cells were treated with the compound for 2 or 6 d.

3. 3.   IDUA activity
   • Cells were lysed in 100 µL of Mammalian Cell PE LB (G-Bioscience, St. Louis, MO, U.S.A.). Protein concentrations were measured using the Protein Assay Bicinchoninate Kit (Nacalai Tesque). Twenty microliters of the cell lysate was mixed with 20 µL of 50 µM 4-methylumbelliferyl-α-Iduronide substrate (Glycosynth, Cheshire, U.K.) in 0.1 M sodium formate buffer (pH 3.0) in black half-area 96-well plates (Greiner, Frickenhausen, Germany). The mixture was incubated at 37 °C for 2 h. The reaction was stopped by 50 µL of 0.2 M sodium glycine buffer (pH 10.5). Before and after the incubation, the fluorescence of 4-methylumbelliferone produced by IDUA (Ex 360 nm, Em 460 nm) was measured using a microplate reader (Power Scan HT, BioTek, Winooski, VT, U.S.A.).

4. 4.   GAG levels
   • Cells were washed with D-phosphate buffered saline (PBS) and then sonicated and digested by 125 µg/mL papain in 0.2 M sodium phosphate buffer (pH 6.4) at 65 °C for 24 h. After digestion, cell lysates were centrifuged at 10000 × g at 4 °C for 10 min and GAG levels in the supernatants were quantified using the Blyscan Glycosaminoglycan Assay (Biocolor, County Antrim, U.K.). Briefly, cell lysates and Blyscan dye reagent were mixed for 30 min, followed by centrifugation at 12000 rpm for 10 min to precipitate the GAG-dye complex. The precipitate obtained was resuspended in Blyscan dye dissociation reagent. Absorbance at 656 nm was measured using a microplate reader. DNA contents were measured using the DNA Quantity Kit (Cosmo Bio, Tokyo, Japan). Briefly, 40 µL of the lysate, 160 µL of dilution buffer, and 10 µL of color development reagent were mixed in black half-area 96-well plates and fluorescence (Ex. 360 nm, Em. 460 nm) was measured using a microplate reader (Power Scan HT; BioTek, Winooski, VT, U.S.A.).

Effects of the Repeated Administration of KY-640 on Hurler Model Mice

1. 1.   Animals
   • Female IDUA-W392X (TGG→TAG) knock-in mice (C57BL/6J background) were established using the CRISPR/Cas9 system in the Laboratory Animal Resource Center, the University of Tsukuba, and used at 5–7 weeks old. All procedures were conducted in accordance with “Regulation/Procedure on Animal Experimentation at Kyoto Pharmaceutical Industries, Ltd.” and approved by the animal ethical committee of Kyoto Pharmaceutical Industries, Ltd.

2. 2.   Administration of KY-640
   • KY-640 (30 and 100 mg/10 mL/kg) was suspended in 0.5% methyl cellulose #400 and orally administered twice a day (BID) for 8 d. During the administration period, body weight and food consumption were monitored and clinical signs were observed daily.

3. 3.   IDUA activity
   • Three hours after the last administration on day 8 in the morning, mice were euthanized by exsanguination from the heart under deep anesthesia by the intraperitoneal administration of ketamine (37.5 mg/kg) and xylazine (7.5 mg/kg).
   • Livers, spleens, brains, and kidneys were excised and their wet weights were measured. Relative organ weights were calculated as percentages of body weight [(organ weight/body weight) × 100]. Tissue samples were homogenized in 0.01 M phosphate buffer (pH 7.4) containing 0.1% Triton X-100 (MP Biomedicals, Santa Ana, CA, U.S.A.) and Protease Inhibitor Cocktail (100 mg tissue/mL) (Nacalai Tesque), followed by centrifugation at 20000 × g at 4 °C for 20 min. Protein concentrations in the supernatants were measured using Protein Assay Bicinchoninate Kit. Forty microliters of diluted tissue lysates (100-fold for the liver, 30-fold for the spleen, and 10-fold for the brain) were mixed with 40 µL of 200 µM 4-methylumbelliferyl-α-iduronide substrate in 0.1 M sodium formate buffer (pH 3.5) in 96-well plates. The mixture was incubated at 37 °C for 2, 6, and 24 h. The reaction was stopped by 100 µL of 0.5 M sodium glycine buffer (pH 10.5). The fluorescence of 4-methylumbelliferone produced by IDUA (Ex. 360 nm, Em. 460 nm) was measured using a microplate reader.

Conflict of Interest

The authors declare no conflict of interest.

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