Synthesis and Evaluation of Novel Triaryl Derivatives with Readthrough-Inducing Activity

Abstract

The readthrough mechanism, which skips the premature termination codon and restores the biosynthesis of the defective enzyme, is an emerging therapeutic tactic for nonsense mutation-related diseases, such as Hurler syndrome, a type of mucopolysaccharidosis. In the present study, novel triaryl derivatives were synthesized and their readthrough-inducing activities were evaluated by a luciferase reporter assay with a partial α-L-iduronidase (IDUA) DNA sequence containing the Q70X nonsense mutation found in Hurler syndrome and by measuring the enzyme activity of IDUA knockout cells transfected with the mutant IDUA gene. KY-516, a representative compound in which the meta position carboxyl group of the left ring of the clinically used ataluren was converted to the para position sulfamoylamino group, the central ring to triazole, and the right ring to cyanobenzene, exhibited the most potent readthrough-inducing activity in the Q70X/luciferase reporter assay. In Q70X mutant IDUA transgenic cells, KY-516 significantly increased enzyme activity at 0.1 µM. After the oral administration of KY-516 (10 mg/kg), the highest plasma concentration of KY-516 was above 5 µM in rats. These results indicate that KY-516, a novel triaryl derivative, exhibits potent readthrough-inducing activity and has potential as a therapeutic agent for Hurler syndrome.

Introduction

A nonsense mutation is a single point mutation in a DNA sequence that causes a change from a codon encoding an amino acid to a stop codon. When the mutated gene is translated into a protein, protein translation terminates upstream of the normal stop codon, resulting in a truncated, incomplete protein that is generally non-functional and susceptible to degradation, which leads to the onset of various diseases.¹⁾ More than 10% of genetic disorders are estimated to be caused by a nonsense mutation, such as some type of Duchenne muscular dystrophy (DMD),²⁾ cystic fibrosis³⁾ and mucopolysaccharidosis,⁴⁾ bringing the total number of these diseases to approximately 1800.⁵⁾

In recent years, a new therapeutic strategy, called nonsense suppression or premature termination codon (PTC) readthrough, which promotes the insertion of an amino acid at the PTC, resulting in the production of the full-length, functional proteins, has attracted attention for genetic diseases caused by a nonsense mutation.^6–9) Several small molecules with readthrough-inducing activity have been reported (Fig. 1). Aminoglycoside antibiotics, such as gentamicin¹⁰⁾ and G418,¹¹⁾ are the most traditional and well-known readthrough agents. They have been reported to suppress PTCs and restore the expression of defective proteins in various genetic disease models and clinical trials. However, the long-term administration of aminoglycoside antibiotics as readthrough drugs is considered to be difficult due to their ototoxicity and nephrotoxicity.^12,13) Therefore, to develop safer and more effective agents, the search for compounds with non-aminoglycoside structures, such as negamycin derivatives and RTC13, is ongoing.^14–17)

Fig. 1. Chemical Structures of Readthrough Compounds

Only ataluren is currently approved for the treatment of DMD. In 2007, a 4-week study on the effects of ataluren in mdx mice reported the restoration of dystrophin expression levels in the tibialis anterior muscle, diaphragm, and cardiac muscle.¹⁸⁾ Although clinical trials were subsequently initiated for DMD, ataluren did not exhibit sufficient efficacy in a late phase 2 study.¹⁹⁾ Based on the findings of a retrospective analysis of this trial, ataluren was conditionally approved by the European Medicines Agency (EMA) in 2014; however, its application was rejected by the U.S. Food and Drug Administration (FDA). It was also being developed for the treatment of other diseases, such as cystic fibrosis,^20,21) hemophilia, and mucopolysaccharidosis type I, but was discontinued due to the lack of efficacy or difficulties with patient inclusion in clinical trials.

The selection of indications for readthrough therapy is also an important factor. Mucopolysaccharidosis type I is an autosomal recessive disorder caused by a congenital deficiency of α-L-iduronidase (IDUA), a lysosomal enzyme that is necessary for the degradation of glycosaminoglycans, and is mainly caused by the Q70X and W402X nonsense mutations in the IDUA gene. Hurler syndrome is the most severe type of mucopolysaccharidosis type I, with enzyme activity that is less than 1% that in normal individuals.²²⁾ Since it is an enzyme-deficient disease, the restoration of even a small amount of protein expression is expected to improve the course of the disease.

In the present study, we synthesized and evaluated novel triaryl derivatives based on the structure of ataluren for the discovery of novel readthrough agents that are more potent than ataluren. Various transformations of the three rings of the triaryl structure were performed to reveal structure–activity relationships and N-{4-[2-(2-cyanophenyl)[1,2,3]triazol-4-yl]phenyl}sulfamide (KY-516) was selected for further evaluations. The readthrough activity of KY-516 was approximately 460-fold more potent than that of ataluren and it also exhibited markedly increased IDUA enzyme activity and favorable oral absorption in rats.

Chemistry

Charts 1 and 2 show the synthetic routes of meta- and para-substituted oxadiazole derivatives. Ataluren (1) synthesized by the previously reported method¹⁸⁾ was amidated with SOCl₂ and ammonium hydroxide, followed by dehydration with trifluoroacetic anhydride (TFAA) to afford benzonitrile 2. The tetrazole cyclization of compound 2 gave 3. Similarly, ataluren was converted to acetyl compound 4 via weinreb amide, followed by reduction with NaBH₄ to give benzyl alcohol 5. Methanesulfonamide derivative 7 and sulfamoylamine derivative 8 were obtained from aniline compound 6 synthesized from ataluren by the Curtius rearrangement.

Chart 1. Synthesis of meta-Substituted Oxadiazole Derivatives

Reagents and conditions; (i) SOCl₂; (ii) NH₃ aq., toluene; (iii) TFAA, Et₃N, CH₂Cl₂; (iv) NaN₃, NH₄Cl, DMF; (v) N,O-dimethylhydroxylamine hydrochloride, EDC·HCl, CH₂Cl₂; (vi) MeMgBr in THF, THF; (vii) NaBH₄, MeOH; (viii) DPPA, Et₃N, toluene, HCl aq.; (ix) MsCl, pyridine, CH₂Cl₂; (x) BocNHSO₂Cl, Et₃N, CH₂Cl₂; (xi) TFA.

Chart 2. Synthesis of meta- and para-Substituted Oxadiazole Derivatives

Reagents and conditions; (i) NH₂OH, 2-fluorobenzoyl chloride, Et₃N, t-BuOH for 10a–c; NH₂OH, 2-fluorobenzoyl chloride, Et₃N, t-BuOH, then tetrabutylammonium hydroxide, THF for 10d; (ii) LiOH aq., MeOH, THF; (iii) methyl bromoacetate, K₂CO₃, DMF; (iv) t-BuOK, DMF; (v) SnCl₂·2H₂O, EtOH; (vi) BocNHSO₂Cl, Et₃N, CH₂Cl₂; (vii) TFA.

The preparation of oxadiazole derivatives 10b, 10c, 11, and 12 is shown in Chart 2. Commercially available benzonitriles with various substituents were treated with hydroxylamine to give the intermediate benzamidoxime, followed by O-alkylation and cyclization to give 10a, 10b, 10c, and 10d. The methyl ester of compound 10a was hydrolyzed with aqueous LiOH, followed by alkylation with methyl bromoacetate and cyclization to give 11. The reduction of compound 10d and its subsequent sufamoylation gave sulfamoylamine derivative 12.

The general scheme for derivatives with various types of central rings is shown in Chart 3, and synthetic conditions for the construction of the central ring are indicated in the table. Benzohydrazide was acylated with p-nitrobenzoyl chloride, followed by cyclization with SOCl₂ to construct the 1,3,4-oxadiazole ring. The reduction of the nitro group gave 13a and subsequent sulfamoylation gave 14a. Aniline compounds 13b–i synthesized by various methods described below were also derivatized to the final compounds 14b–i by the same method. 1H-1,2,3-Triazole derivative 13b was prepared by constructing triazole rings via the Huisgen reaction. 1,2,4-Triazole derivative 13c was synthesized by bromination of the hydrazone prepared from commercially available hydrazine and benzaldehyde and then treating it with tetrazole to construct a 1,2,4-triazole ring, followed by the reduction of the nitro group. 2H-1,2,3-Triazole derivative 13d was synthesized by the Pd-catalyzed coupling reaction of the 2H-1,2,3-triazole component prepared by the previously reported method and 2-bromofluorobenzene, followed by the reduction of the nitro group. Bromoacetophenone was mixed with 2-fluorobenzylamine in the presence of AgOTf, followed by the reduction of the nitro group to give oxazole derivative 13e. The coupling reaction of 4-bromonitrobenzene and the 5-aryl-1,3-oxazole component prepared by the previously reported method and subsequent reduction of the nitro group gave oxazole derivative 13f, the other isomer of 13e. In addition, 2-bromoacetophenone was aminated and acylated with 2-fluorobenzoic acid, and the product was then cyclized with POCl₃ followed by the reduction of the nitro group to give another oxazole derivative 13g. Oxazole derivatives with a substituent at the 4-position (13h, 13i) were synthesized by the following method. 2-Bromopropiophenone was reacted with 2-fluorobenzylamine in the presence of I₂ and K₂CO₃ followed by the reduction of the nitro group to give 4-methyloxazole 13h. In the case of 4-cyanooxazole derivative 13i, simultaneous halogenation and cyclization with 2-fluorobenzylamine afforded the ester group-substituted oxazole derivative. This ester was hydrolyzed with aqueous NaOH and amidated with aqueous NH₃, followed by dehydration with TFAA to give 4-cyanooxazole 13i.

Chart 3. Synthesis of Various Central Ring Derivatives

Reagents and conditions; (i) Na₂CO₃, 1,4-dioxane; (ii) SOCl₂, pyridine; (iii) CuSO₄·5H₂O, sodium L-ascorbate, t-BuOH-H₂O; (iv) EtOH, H₂SO₄ aq.; (v) NBS, dimethylsulfide, CH₂Cl₂; (vi) tetrazole, Et₃N, EtOH; (vii) Pd₂(dba)₃, tetramethyl t-BuXPhos, K₃PO₄, toluene; (viii) AgOTf, AcOEt; (ix) Pd(PPh₃)₄, t-BuOLi, 1,4-dioxane; (x) EDC·HCl, HOBt, Et₃N, CH₂Cl₂; (xi) POCl₃; (xii) I₂, K₂CO₃, DMF; (xiii) TBHP in n-decane, I₂, Cu(OAc)₂, DMF; (xiv) NaOH aq., MeOH, THF; (xv) NH₃ aq., EDC·HCl, HOBt, DMF; (xvi) TFAA, pyridine, 1,4-dioxane; (xvii) SnCl₂·2H₂O, THF; (xviii) BocNHSO₂Cl, Et₃N, CH₂Cl₂; (xix) TFA, CH₂Cl₂.

The synthesis of 2,5-aryl-1,3-oxazole derivatives is shown in Chart 4. α-Amino-4-nitroacetophenone 15 was acylated with various aryl carbonyl chlorides 16a–c, followed by cyclization with POCl₃ to give 19a–c. Compounds 19a–c were converted to 21a–c in the same manner as described before. Alternatively, in the case of pyrazine derivative 21d and cyanobenzene derivative 21e, compounds 19d and 19e were obtained by the coupling reaction of oxazole 17 and aryl iodides 18d and 18e.

Chart 4. Synthesis of Oxazole Derivatives

Reagents and conditions; (i) Et₃N, CH₂Cl₂; (ii) POCl₃; (iii) Pd(PPh₃)₄, t-BuOLi, 1,4-dioxane; (iv) SnCl₂·2H₂O, EtOH; (v) ClSO₂NCO, t-BuOH, Et₃N, CH₂Cl₂; (vi) TFA, CH₂Cl₂.

The synthesis of triazole derivatives is shown in Chart 5. Compounds 23a, 23c, and 23f were synthesized by the aromatic nucleophilic substitution reaction of various aryl fluorides with triazole 22. Compounds 23a, 23c, and 23f were converted to 24a, 24c, and 24f by the same method as before. Acetyl derivative 24a was reduced with NaBH₄ to give 24b. In the case of hydroxymethyl derivative 23d, the coupling reaction of compound 22 with (3-bromophenyl)methanol gave compound 23d. The hydroxylmethyl group of compound 23d was converted to a dimethylaminomethyl group with MsCl and Me₂NH to give compound 23e. The conversion of compound 23e to 24e was performed as described above.

Chart 5. Synthesis of Triazole Derivatives

Reagents and conditions; (i) R³F, K₂CO₃, DMSO, or DMF for 23a, 23c, and 23f; (ii) R³Br, Pd₂(dba)₃, tetramethyl t-BuXPhos, K₃PO₄, toluene for 23d; (iii) SnCl₂·2H₂O, EtOH; (iv) ClSO₂NCO, t-BuOH, Et₃N, CH₂Cl₂; (v) TFA, CH₂Cl₂; (vi) NaBH₄, MeOH; (vii) MsCl, Et₃N, THF; (viii) Me₂NH in THF.

Results and Discussion

Only ataluren is in clinical use as a readthrough inducer; however, as discussed above, it was approved by the EMA, not the FDA for the treatment of DMD. In addition, its development for cystic fibrosis and mucopolysaccharidosis has been discontinued, raising the need for research on more effective agents. Therefore, we aimed to develop a more potent compound based on ataluren by acquiring a new lead compound and optimizing the lead from it. A luciferase assay system was constructed and used for a readthrough activity evaluation of synthesized compounds. In clinical cases of mucopolysaccharidosis type I Hurler syndrome, two common nonsense mutations (Q70X and W402X) account for approximately 70% of mutant alleles, and both of these PTC are TAG.⁴⁾ Therefore, a vector containing the luciferase gene downstream of the PTC sequence (Q70X) was generated and stably expressed in HeLa cells. In the presence of a compound with readthrough-inducing activity, PTC is skipped, translation proceeds, and downstream luciferase is also expressed. We evaluated readthrough-inducing activity using the activity of the expressed luciferase as an indicator. The oral absorption of the synthesized compounds was evaluated by measuring the plasma concentrations of the compounds in Sprague-Dawley (SD) rats after the oral administration of 10 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.

As shown in Fig. 1, the compounds exhibiting readthrough-inducing activity reported to date have a polar group at the hydrophobic moiety end as a structural feature. Compounds without acidic groups, such as NB54²³⁾ or Novartis’s compound (Ex. 1.37),²⁴⁾ have also been reported to exhibit readthrough-inducing activity, which suggests that the carboxyl group is not essential for activity. Therefore, we synthesized derivatives of ataluren by converting the carboxyl group to various substituents (Table 1). The activities of all synthesized compounds with an acidic group were markedly reduced, while compound 5, which has a neutral hydroxyl group, exhibited increased activity (EC₂₀₀ = 10.8 µM), approximately twice that of ataluren (EC₂₀₀ = 22.9 µM). A further search for neutral to weakly acidic substituents revealed that benzenesulfonamide derivative 10b did not enhance activity, while methanesulfonamide derivative 7 and sulfamoylamine derivative 8 exhibited increased activity (EC₂₀₀ = 4.2 and 6.1 µM, respectively). When the substitution position of the methanesulfonamide group was examined, activity disappeared at the ortho position (data not shown), but increased 2-fold at the para position (EC₂₀₀ = 2.1 µM). The sulfamoylamino group, which also exhibited good activity, was similarly moved to the para position, and a marked increase in activity was observed (Compound 12, EC₂₀₀ = 0.39 µM). The maximum blood concentration (C_max) of compound 12 in SD rats after the oral administration of 10 mg/kg was 4.16 µM, which was lower than that of ataluren (C_max = 21.4 µM); however, it was absorbed well orally. Furthermore, the EC₂₀₀ ratio of compound 12 was approximately 59-fold higher than that of ataluren. Further optimization was conducted using compound 12 as the lead compound.

Table 1. Chemical Structures, Molecular Weights, c Log P Values, and Readthrough Activities of Oxadiazole Derivatives

The oxadiazole ring of compound 12 was converted to various other aromatic heterocycles (Table 2). Conversion to 1,3,4-oxadiazole (14a), 1H-1,2,3-triazole (14b), and 1,2,4-triazole (14c) did not improve activity, whereas that to 2H-1,2,3-triazole (14d) and oxazole (14f and 14g) resulted in 3- to 8-fold increases in activity. Differences in activity were observed depending on the position of the oxadiazole and triazole heteroatoms, suggesting that a nitrogen atom in the appropriate position may function as a hydrogen bond acceptor. The introduction of various substituents into the oxazole ring of compound 14g was also investigated. The introduction of a methyl group retained activity, whereas that of a cyano group or carboxyl group (data not shown) abolished this activity, indicating that the introduction of a polar group is not suitable and the size of substituents also affects activity. Oxazoles 14f and 14g exhibited significantly higher activity than compound 12; however, their oral absorption was not good (14f, C_max = 0.18 µM, area under the curve (AUC) = 0.72 µM·h; 14g, C_max = 0.33 µM, AUC = 1.33 µM·h). Various 5-membered aromatic rings were examined, and triazole 14d (EC₂₀₀ = 0.15 µM) and oxazoles 14f (EC₂₀₀ = 0.08 µM) and 14g (EC₂₀₀ = 0.05 µM) exhibited good activity. Further optimization study was performed with two lead compounds, triazole 14d and oxazole 14g. Oxazole 14g was selected because of its better pharmacokinetics profile compared to 14f, although it was still insufficient.

Table 2. Chemical Structures, Molecular Weights, c Log P Values, and Readthrough Activities of Various Central Ring Derivatives

In the course of the optimization study of oxazole derivative 14g, fluorobenzene ring was converted (Table 3). The fluorine atom in the para position (21b) decreased activity, whereas the meta position (21a) retained activity. Although the introduction of a nitrogen atom was attempted to improve pharmacokinetics, the conversion to pyridine (21c) and pyrazine (21d) rings decreased activity. These results indicated that hydrophobic substituents were preferred for activity. When a cyano group, which is also known as a fluorine bioisostere,²⁵⁾ was introduced in place of the o-fluorine in compound 14g, activity improved (EC₂₀₀ = 0.02 µM), whereas oral absorption deteriorated (C_max below the limit of detection).

Table 3. Chemical Structures, Molecular Weights, c Log P Values, and Readthrough Activities of Oxazole Derivatives

An optimization study of triazole derivative 14d was then conducted, and right-hand ring transformation was also performed (Table 4). Decreased activity was observed with the introduction of acetyl (24a) or trifluoromethyl (24c) groups, which have the same electron-withdrawing properties as fluorine, as well as with neutral or basic substituents, such as hydroxyethyl (24b) or dimethylaminomethyl (24e) groups. We investigated the substitution of cyano groups, found that activity increased when the central ring was an oxazole ring, and obtained compound 24f, which exhibited increased activity (EC₂₀₀ = 0.05 µM) and good oral absorption (C_max = 5.44 µM, AUC = 26.1 µM·h). Compared to 21e with an oxazole central ring and cyanophenyl group, compound 24f with a triazole central ring showed a 2–3 fold decrease in activity, however it had a significantly improved pharmacokinetics profile. Therefore, compound 24f was named KY-516, and subjected to further biological evaluations.

Table 4. Chemical Structures, Molecular Weights, c Log P Values, and Readthrough Activities of Triazole Derivatives

KY-516 exhibited dose-dependent readthrough-inducing activity in the Q70X/luciferase assay, which was approximately 460-fold stronger than that of ataluren (Fig. 2A). KY-516 was also evaluated for the W402X nonsense mutation, which is a common mutation as well as Q70X. In contrast to the Q70X mutation, KY-516 exhibited moderate activity against the W402X mutant sequence, which was still more potent than ataluren (Fig. 2B). We generated IDUA knockout cells expressing the full-length mutant IDUA enzyme and evaluated the readthrough-inducing activity of KY-516. The activity of the IDUA enzyme generated by readthrough induction was evaluated using 4-methylumbelliferyl-α-iduronide as a fluorescent substrate. The results obtained showed that ataluren significantly increased IDUA enzyme activity at 100 µM, while KY-516 significantly increased IDUA enzyme activity from 0.1 µM in IDUA knockout cells transfected with Q70X mutant cDNA (Fig. 3A). In contrast, KY-516 failed to increase IDUA activity in the case of the W402X mutation (Fig. 3B). Although the Q70X and W402X mutations had the same TAG stop codon, the difference in KY-516 activity between the two mutations in the two assays indicated sequence specificity in the readthrough-inducing effects of KY-516. The sequence around PTC is known to have a significant effect on readthrough efficiency,²⁶⁾ and KY-516 may be effective for diseases with specific nonsense mutations.

Fig. 2. Readthrough-Inducing Activities of KY-516 and Ataluren Evaluated by Luciferase Reporter Assays

HeLa cells stably transfected with the plasmid carrying IDUA DNA fragments containing A) Q70X or B) W402X nonsense mutation were treated with increasing concentrations of KY-516 or ataluren, and assayed for luciferase activity. Data shown are expressed as the mean ± S.E. of representative experiments (n = 4–7).

Fig. 3. IDUA Activity in IDUA Knockout Cells Transfected with Full-Length Mutant IDUA

IDUA knockout HeLa cells transfected with full-length IDUA cDNA with A) Q70X or B) W402X mutation were treated with the indicated concentrations of KY-516 or ataluren. The IDUA activity of cell lysates was measured using a fluorescent substrate. Each bar represents the mean ± S.E. of representative experiments (n = 4–8). Exact p values were calculated using the unpaired, two-tailed t-test comparing values in treated cells to vehicle-treated cells. ** indicates p < 0.01.

In conclusion, KY-516, which has a sulfamoylamino group in the para position of the left ring, a triazole ring in the central ring, and a cyano group in the ortho position of the right ring, showed approximately 460-fold stronger readthrough activity than ataluren and significantly increased IDUA enzyme activity.²⁷⁾ KY-516 potentially has efficacy as a treatment for Hurler syndrome with the IDUA Q70X mutation and may also be a valuable tool for elucidating the mechanisms underlying readthrough.

Experimental

General

Melting points were measured on a melting point apparatus (YAMATO MP-21; Yamato Scientific Co., Ltd., Tokyo, Japan) and were uncorrected. ¹H-NMR spectra were obtained on a NMR 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 IR spectrometer (HORIBA FT-720, HORIBA, Kyoto, Japan). Mass spectra were obtained on an electrospray ionization (ESI)-MS spectrometer (Expression CMS-L, Advion, Ithaca, U.S.A.) and ESI-TOF/MS (micrOTOF2-kp, Bruker, Massachusetts, U.S.A.). Column chromatography was performed on silica gel (Daisogel No.1001W; 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 determined 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).

3-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzamide (25)

3-[5-(2-Fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid (1) (470 mg, 1.65 mmol) was suspended in SOCl₂ (5.0 mL, 69 mmol) and heated to reflux for 1 h. The reaction mixture was concentrated under reduced pressure. Following the addition of 28% ammonium hydroxide (1.0 mL, 15 mmol) under ice-cooling, the mixture was stirred at the same temperature for 15 min. The precipitate was collected by filtration to give 25 (420 mg, 90% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.48–7.60 (3H, m), 7.70 (1H, t, J = 7.8 Hz), 7.78–7.85 (1H, m), 8.09–8.14 (1H, m), 8.20–8.30 (3H, m), 8.57–8.62 (1H, m).

3-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]benzonitrile (2)

Et₃N (0.41 mL, 3.0 mmol) and TFAA (0.41 mL, 2.9 mmol) were added to a suspension of 25 (420 mg, 1.48 mmol) in CH₂Cl₂ (2 mL) under ice-cooling, and the mixture was stirred at room temperature for 1 h. After the addition of saturated aqueous NaHCO₃, the mixture was extracted with CH₂Cl₂ and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure to give 2 (420 mg, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 7.48–7.60 (2H, m), 7.77–7.87 (2H, m), 8.08–8.14 (1H, m), 8.20–8.30 (1H, m), 8.37–8.43 (1H, m), 8.52–8.56 (1H, m).

5-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}-1H-tetrazole (3)

NH₄Cl (52 mg, 0.98 mmol) and NaN₃ (64 mg, 0.98 mmol) were added to a solution of 2 (173 mg, 0.652 mmol) in N,N-dimethylformamide (DMF) (2 mL), and the mixture was stirred at 120 °C for 14 h. After cooling, 2.0 M aqueous HCl solution (10 mL) was added to the reaction mixture and the insoluble material was collected by filtration. The residue was washed with water and t-BuOMe and purified by octadecyl silica (ODS) column chromatography. The target fractions were collected and 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 dissolved in t-BuOMe (2 mL) and n-hexane (5 mL) was added to the solution. The precipitate was collected by filtration to give 3 (25 mg, 12% yield) as a solid. mp 227–230 °C; ¹H-NMR (dimethyl sulfoxide (DMSO)-d₆) δ: 7.45–7.65 (2H, m), 7.77–7.92 (2H, m), 8.20–8.35 (3H, m), 8.75–8.82 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 111.6 (d), 117.3 (d), 125.3 (s), 125.5 (s), 125.5 (s), 127.0 (s), 129.4 (s), 129.9 (s), 130.6 (s), 130.9 (s), 135.7 (s), 135.8 (s), 159.9 (d), 167.3 (s), 172.7 (d); IR (attenuated total reflectance (ATR)) cm⁻¹: 1620; high resolution (HR)-MS ESI-TOF Calcd for C₁₅H₉FN₆NaO [M + Na]⁺ 331.0720. Found 331.0721.

3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]-N-methoxy-N-methylbenzamide (26)

Et₃N (0.30 mL, 2.2 mmol), compound 1 (500 mg, 1.76 mmol), and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC·HCl) (405 mg, 2.11 mmol) were added to a solution of N,O-dimethylhydroxylamine hydrochloride (190 mg, 1.95 mmol) in CH₂Cl₂ (5 mL), and the mixture was stirred at room temperature for 1 h. After the removal of the solvent under reduced pressure, the residue was dissolved in AcOEt, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure to give 26 (629 mg, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 3.40 (3H, s), 3.60 (3H, s), 7.26–7.37 (2H, m), 7.54–7.65 (2H, m), 7.84 (1H, d, J = 7.6 Hz), 8.14–8.32 (2H, m), 8.50 (1H, s).

1-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}ethanone (4)

Following the addition of 1.0 M MeMgBr solution in tetrahydrofuran (THF) (2.1 mL, 2.1 mmol) to a solution of 26 (620 mg, 1.76 mmol) in THF (10 mL) under ice-cooling, the mixture was stirred at the same temperature for 30 min. The same MeMgBr solution in THF (2.1 mL, 2.1 mmol) was added again to the mixture, which was then stirred at the same temperature for another 30 min. After the addition of saturated aqueous NH₄Cl, 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 t-BuOMe (5 mL) was added. The insoluble material was collected by filtration to give 4 (341 mg, 69% yield) as a solid. mp 145–147 °C; ¹H-NMR (DMSO-d₆) δ: 2.68 (3H, s), 7.47–7.59 (2H, m), 7.75–7.84 (2H, m), 8.21 (1H, d, J = 7.8 Hz), 8.23–8.29 (1H, m), 8.33 (1H, d, J = 7.8 Hz), 8.58 (1H, s); IR (ATR) cm⁻¹; 1680.

1-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}ethanol (5)

NaBH₄ (14 mg, 0.37 mmol) was added to a solution of 4 (100 mg, 0.354 mmol) in MeOH (1.0 mL), and the mixture was stirred at room temperature for 30 min. 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. t-BuOMe:n-hexane (1 : 3) (4 mL) was added to the solid, and the insoluble material was collected by filtration to give 5 (58 mg, 58% yield) as a solid. mp 99–100 °C; ¹H-NMR (DMSO-d₆) δ: 1.39 (3H, d, J = 6.4 Hz), 4.81–4.90 (1H, m), 5.37 (1H, d, J = 4.2 Hz), 7.47–7.60 (4H, m), 7.77–7.84 (1H, m), 7.93–7.98 (1H, m), 8.11 (1H, s), 8.22–8.28 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 25.8 (s), 67.6 (s), 111.7 (d), 117.2 (d), 123.9 (s), 125.3 (s), 125.4 (d), 125.6 (s), 128.7 (s), 128.9 (s), 130.8 (s), 135.6 (d), 148.6 (s), 159.9 (d), 168.1 (s), 172.4 (d); IR (ATR) cm⁻¹; 1618; HR-MS (ESI-TOF) Calcd for C₁₆H₁₃FN₂NaO₂ [M + Na]⁺ 307.0859. Found 307.0830.

3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenylamine (6)

Et₃N (1.90 mL, 13.7 mmol) and diphenylphosphory azide (DPPA) (2.72 mL, 12.7 mmol) were added to a suspension of 1 (3.00 g, 10.6 mmol) in toluene (30 mL), and the mixture was stirred at 100 °C for 1 h. Following the addition of 6.0 M aqueous HCl solution (20 mL) to the mixture, it was stirred at the same temperature for 1 h. After cooling, the reaction mixture was neutralized with saturated aqueous NaHCO₃ and the organic solvent was removed under reduced pressure. The insoluble material was collected by filtration, washed with water, and then dissolved in Et₂O. The solution 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 6 (450 mg, 17% yield) as a solid. ¹H-NMR (CDCl₃) δ: 5.45 (2H, s), 6.72–6.81 (1H, m), 7.18–7.25 (2H, m), 7.33 (1H, s), 7.45–7.58 (2H, m), 7.74–7.85 (1H, m), 8.16–8.24 (1H, m).

N-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}methanesulfonamide (7)

Pyridine (0.05 mL, 0.6 mmol) and MsCl (0.03 mL, 0.4 mmol) were added to a suspension of 6 (100 mg, 0.392 mmol) in CH₂Cl₂ (0.5 mL) under ice-cooling, and the mixture was stirred at room temperature for 1 h. After 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. t-BuOMe (5 mL) was added to the solid, and the insoluble material was collected by filtration to give 7 (56.8 mg, 43% yield) as a solid. mp 157–158 °C; ¹H-NMR (DMSO-d₆) δ: 3.06 (3H, s), 7.42–7.62 (4H, m), 7.74–7.87 (2H, m), 7.98 (1H, s), 8.16–8.26 (1H, m), 10.05 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 39.4 (s), 111.7 (d), 117.3 (d), 117.7 (s), 122.3 (s), 122.4 (s), 125.4 (d), 126.9 (s), 130.4 (s), 130.8 (s), 135.7 (d), 139.3 (s), 159.9 (d), 167.7 (s), 172.5 (d); IR (ATR) cm⁻¹; 1619; HR-MS (ESI-TOF) Calcd for C₁₅H₁₂FN₃NaO₃S [M + Na]⁺ 356.0481. Found 356.0479.

N-{3-[5-(2-Fluorophenyl)-[1,2,4]oxadiazol-3-yl]phenyl}sulfamoylamine (8)

Et₃N (0.21 mL, 1.5 mmol) and 1.0 M BocNHSO₂Cl solution in CH₂Cl₂ (0.76 mL, 0.76 mmol) were added to a solution of 6 (130 mg, 0.509 mmol) in CH₂Cl₂ (3 mL) under ice-cooling, and the mixture was stirred at room temperature for 16 h. After the addition of 1.0 M aqueous HCl solution, 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 obtained residue was dissolved in trifluoracetic acid (TFA) (1 mL), and the mixture was stirred at room temperature for 30 min. After the addition of saturated aqueous NaHCO₃, 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. CHCl₃ (0.5 mL) was added to the solid, and the insoluble material was collected by filtration to give 8 (23.7 mg, 14% yield) as a solid. mp 160–161 °C; ¹H-NMR (DMSO-d₆) δ: 7.22 (2H, s), 7.41 (1H, d, J = 8.4 Hz), 7.46–7.59 (3H, m), 7.71 (1H, d, J = 7.8 Hz), 7.75–7.84 (1H, m), 7.92 (1H, s), 8.17–8.25 (1H, m), 9.80 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 111.7 (d), 116.4 (s), 117.2 (d), 120.6 (s), 120.9 (s), 125.4 (d), 126.5 (s), 129.8 (s), 130.8 (s), 135.6 (d), 140.3 (s), 159.9 (d), 167.9 (s), 172.3 (d); IR (ATR) cm⁻¹; 1623; HR-MS (ESI-TOF) Calcd for C₁₄H₁₁FN₄NaO₃S [M + Na]⁺ 357.0434. Found 357.0408.

Methyl 3-{5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl}phenylacetate (10a)

Following the addition of 50% aqueous NH₂OH (1.67 g, 25 mmol) to a solution of 9a (4.03 g, 23.0 mmol), the mixture was stirred at 60 °C for 4 h. After cooling to room temperature, Et₃N (3.6 mL, 26 mmol) and 2-fluorobenzoyl chloride (3.0 mL, 25 mmol) were added to the reaction mixture, stirred at room temperature for 30 min, heated to 80 °C, and then stirred for 14 h. After cooling and the addition of water, 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 10a (4.97 g, 69% yield) as a solid. ¹H-NMR (CDCl₃) δ: 3.72 (3H, s), 3.74 (2H, s), 7.26–7.37 (2H, m), 7.43–7.52 (2H, m), 7.56–7.64 (1H, m), 8.08–8.12 (2H, m), 8.19–8.25 (1H, m).

3-{5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl}phenylacetic Acid (27)

Following the addition of 1.0 M aqueous LiOH solution (15 mL, 15 mmol) to a solution of 10a (2.38 g, 7.62 mmol) in MeOH (20 mL) and THF (20 mL), the mixture was stirred at room temperature for 30 min. After the addition of 6.0 M aqueous HCl solution (3.0 mL, 18 mmol), the mixture was concentrated to approximately 20 mL under reduced pressure and extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure to give 27 (1.96 g, 86% yield) as a solid. ¹H-NMR (CDCl₃) δ: 3.76 (2H, s), 7.25–7.36 (2H, m), 7.43–7.52 (2H, m), 7.56–7.63 (1H, m), 8.09–8.13 (2H, m), 8.18–8.24 (1H, m).

Methoxycarbonylmethyl{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}acetate (28)

K₂CO₃ (350 mg, 2.53 mmol) and methyl bromoacetate (0.20 mL, 2.2 mmol) were added to a solution of 27 (583 mg, 1.95 mmol) in DMF (5 mL), and the mixture was stirred at room temperature for 1 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 resulting oily residue was left in a freezer (− 19 °C) overnight to solidify. n-Hexane:i-Pr₂O (5 : 1) (6 mL) was added to the solid and the insoluble material was collected by filtration to give 28 (614 mg, 85% yield) as a solid. ¹H-NMR (CDCl₃) δ: 3.75 (3H, s), 3.85 (2H, s), 4.67 (2H, s), 7.25–7.37 (2H, m), 7.47–7.52 (2H, m), 7.56–7.64 (1H, m), 8.08–8.14 (2H, m), 8.19–8.25 (1H, m).

3-{3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}-4-hydroxy-5H-furan-2-one (11)

t-BuOK (277 mg, 2.47 mmol) was added to a solution of 28 (610 mg, 1.65 mmol) in THF (10 mL) under ice-cooling, and the mixture was stirred at room temperature for 2 h. After the addition of 0.5 M aqueous HCl solution (20 mL, 10 mmol), 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, the solvent of the target fractions were removed under reduced pressure, and the residue was purified again by ODS column chromatography. The target fractions were collected and extracted with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure. t-BuOMe (1 mL) was added to the residue and the insoluble material was collected by filtration to give 11 (10.3 mg, 1.8% yield) as a solid. mp 240–246 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 4.78 (2H, s), 7.48–7.63 (3H, m), 7.76–7.84 (1H, m), 7.91–7.96 (1H, m), 8.17–8.26 (2H, m), 8.75 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 66.3 (s), 111.8 (d), 117.2 (d), 124.4 (s), 124.6 (s), 125.4 (d), 125.7 (s), 128.9 (s), 129.0 (s), 130.8 (s), 132.0 (s), 135.5 (s), 135.6 (s), 159.9 (d), 168.1 (s), 172.4 (s), 172.9 (s), 177.0 (s); IR (ATR) cm⁻¹; 1697; HR-MS (ESI-TOF) Calcd for C₁₈H₁₂FN₂O₄ [M + H]⁺ 339.0781. Found 339.0739.

3-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]benzenesulfonamide (10b)

Following the addition of 50% aqueous NH₂OH (170 mg, 2.60 mmol) to a suspension of 9b (425 mg, 2.33 mmol) in t-BuOH (5 mL), the mixture was stirred at 40 °C for 14 h. After cooling, Et₃N (0.36 mL, 2.6 mmol) and 2-fluorobenzoyl chloride (0.29 mL, 2.4 mmol) were added to the mixture, which was stirred at room temperature for 1.5 h and then at 80 °C for another 22h. After cooling, 20 mL of water was added to the reaction mixture and the precipitate was collected by filtration. The resulting powder was washed with water and t-BuOH to give 10b (592 mg, 80% yield) as a solid. mp 210–212 °C; ¹H-NMR (DMSO-d₆) δ: 7.48–7.62 (4H, m), 7.78–7.86 (2H, m), 8.06 (1H, d, J = 7.8 Hz), 8.22–8.28 (1H, m), 8.31 (1H, d, J = 7.8 Hz), 8.55 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 111.6 (d), 117.3 (d), 124.2 (s), 125.5 (d), 126.6 (s), 128.6 (s), 130.1 (s), 130.3 (s), 130.8 (s), 135.8 (d), 145.1 (s), 159.9 (d), 167.1 (s), 172.8 (d); IR (ATR) cm⁻¹; 3329, 3234; HR-MS (ESI-TOF) Calcd for C₁₄H₁₁FN₃O₃S [M + H]⁺ 320.0505. Found 320.0472.

N-{4-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}methanesulfonamide (10c)

Compound 10c was prepared according to the procedure for the synthesis of 10b. Yield was 83%. mp 202–205 °C; ¹H-NMR (DMSO-d₆) δ: 3.12 (3H, s), 7.38–7.43 (2H, m), 7.47–7.59 (2H, m), 7.77–7.84 (1H, m), 8.04–8.09 (2H, m), 8.19–8.25 (1H, m), 10.27 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 111.8 (d), 117.2 (d), 118.7 (s, 2C), 120.6 (s), 125.4 (d), 128.4 (s, 2C), 130.8 (s), 135.5 (s), 135.6 (s), 141.6 (s), 159.9 (d), 167.5 (s), 172.2 (d); IR (ATR) cm⁻¹; 3207; HR-MS (ESI-TOF) Calcd for C₁₅H₁₃FN₃O₃S [M + H]⁺ 334.0662. Found 334.0624; Anal. Calcd for C₁₅H₁₂FN₃O₃S: C, 54.05; H, 3.63; N, 12.61. Found: C, 54.06; H, 3.42; N, 12.60.

(Z)-O-(2-Fluorobenzoyl)-4-nitrobenzamidoxime (29)

Following the addition of 50% aqueous NH₂OH (2.45 g, 37.0 mmol) to a suspension of 9d (5.00 g, 33.8 mmol) in t-BuOH (70 mL), the mixture was heated to reflux for 3 h. After cooling and 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. t-BuOMe (30 mL) was added to the residue, and the insoluble material was collected by filtration. Et₃N (1.44 mL, 12.1 mmol) and 2-fluorobenzoyl chloride (1.68 mL, 12.1 mmol) were added to a solution of the collected powder (2.00 g, 11.0 mmol), and the mixture was stirred at room temperature for 0.5 h. Water and AcOEt were added to the reaction mixture, and the insoluble material was collected by filtration to give 29 (1.79 g, 31% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.10–7.25 (2H, br), 7.31–7.45 (2H, m), 7.66–7.77 (1H, m), 8.01–8.15 (3H, m), 8.30–8.43 (2H, m).

5-(2-Fluorophenyl)-3-(4-nitrophenyl)[1,2,4]oxadiazole (10d)

Following the addition of 1.0 M methanol solution of tetrabutylammonium hydroxide (3.3 mL, 3.3 mmol) to a suspension of 29 (1.00 g, 3.3 mmol) in THF (7 mL), the mixture was stirred at room temperature for 15 min and 1.0 M aqueous HCl solution was then added. The precipitate was collected by filtration to give 10d (790 mg, 84% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.28–7.42 (2H, m), 7.60–7.70 (1H, m), 8.20–8.27 (1H, m), 8.36–8.42 (4H, m).

4-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenylamine (30)

SnCl₂·2H₂O (2.07 g, 9.17 mmol) was added to a suspension of 10d (778 mg, 2.73 mmol) in EtOH (15 mL), and the mixture was heated to reflux for 0.5 h. After cooling, water was added to the reaction mixture 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 30 (700 mg, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 3.80–4.20 (2H, br), 6.73–6.80 (2H, m), 7.26–7.36 (2H, m), 7.54–7.62 (1H, m), 7.95–8.02 (2H, m), 8.15–8.25 (1H, m).

N-{4-[5-(2-Fluorophenyl)[1,2,4]oxadiazol-3-yl]phenyl}sulfamide (12)

Compound 12 was prepared from 30 according to the procedure for the synthesis of 8. Yield was 15% for 2 steps. mp 190–193 °C; ¹H-NMR (DMSO-d₆) δ: 7.30–7.40 (4H, m), 7.44–7.63 (2H, m), 7.74–7.85 (1H, m), 7.95–8.05 (2H, m), 8.18–8.30 (1H, m), 10.00–10.15 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 111.8 (d), 117.1 (s), 117.3 (s, 2C), 118.8 (s), 125.4 (d), 128.0 (s, 2C), 130.8 (s), 135.5 (d), 142.6 (s), 159.9 (d), 167.7 (s), 172.1 (d); IR (ATR) cm⁻¹; 3273; HR-MS (ESI-TOF) Calcd for C₁₄H₁₂FN₄O₃S [M + H]⁺ 335.0614. Found 335.0579; Anal. Calcd for C₁₄H₁₁FN₄O₃S: C, 50.30; H, 3.32; N, 16.76. Found: C, 50.25; H, 3.25; N, 16.74.

N'-(2-Fluorobenzoyl)-4-nitrobenzohydrazide (31)

Na₂CO₃ (365 mg, 3.44 mmol) and 4-nitrobenzoyl chloride (1.28 g, 6.88 mmol) were added to a suspension of 2-fluorobenzohydrazide (1.06 g, 6.88 mmol) in 1,4-dioxane (28 mL), and the mixture was stirred at room temperature for 1 h. The solvent was removed under reduced pressure and the residue was added to water (20 mL). The insoluble material was collected by filtration to give 31 (1.31 g, 63% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.31–7.38 (2H, m), 7.55–7.63 (1H, m), 7.69–7.75 (1H, m), 8.12–8.17 (2H, m), 8.31–8.36 (2H, m), 10.40–11.00 (2H, br).

2-(2-Fluorophenyl)-5-(4-nitrophenyl)[1,3,4]oxadiazole (32)

Pyridine (2.1 mL) and SOCl₂ (1.23 mL, 17.0 mmol) were added to compound 31 (1.31 g, 4.32 mmol), and the mixture was stirred at 80 °C for 0.5 h. After cooling, MeOH (1 mL) and water (20 mL) were added to the reaction mixture, which was extracted with AcOEt. The organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. AcOEt (5 mL) was added to the residue, and the insoluble material was collected by filtration to give 32 (501 mg, 41% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.46–7.58 (2H, m), 7.71–7.78 (1H, m), 8.19–8.25 (1H, m), 8.34–8.39 (2H, m), 8.44–8.49 (2H, m).

N-{4-[5-(2-Fluorophenyl)[1,3,4]oxadiazol-2-yl]phenyl}sulfamide (14a)

Compound 14a was prepared from 32 according to the procedure for the synthesis of 12. Yield was 16% for 3 steps. mp 226–230 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.32–7.37 (2H, m), 7.37–7.43 (2H, m), 7.43–7.55 (2H, m), 7.67–7.75 (1H, m), 7.98–8.04 (2H, m), 8.12–8.18 (1H, m); ¹³C-NMR (DMSO-d₆) δ: 111.8 (d), 116.0 (s), 117.1 (d), 117.3 (s, 2C), 125.3 (d), 127.8 (s, 2C), 129.7 (s), 134.1 (d), 143.1 (s), 159.2 (d), 160.2 (d), 164.2 (s); IR (ATR) cm⁻¹; 3294, 1614; HR-MS (ESI-TOF) Calcd for C₁₄H₁₂FN₄O₃S [M + H]⁺ 335.0614. Found 335.0571.

4-[1-(2-Fluorophenyl)-1H-[1,2,3]triazol-4-yl]phenylamine (33)

Water (10 mL), 4-ethynylaniline (924 mg, 7.89 mmol), sodium L-ascorbate (156 mg, 0.789 mmol), and CuSO₄·5H₂O (20 mg, 0.079 mmol) were added to a solution of 1-azido-2-fluorobenzene (1.08 g, 7.89 mmol) in t-BuOH (10 mL), and the mixture was stirred at 60 °C for 27 h. After cooling, water (20 mL) was added to the reaction mixture and the insoluble material was collected by filtration to give 33 (1.52 g, 76% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 5.25–5.37 (2H, br), 6.63–6.68 (2H, m), 7.42–7.48 (1H, m), 7.55–7.65 (3H, m), 7.84–7.89 (1H, m), 8.75 (1H, d, J = 2.0 Hz).

N-{4-[1-(2-Fluorophenyl)[1,2,3]triazol-4-yl]phenyl}sulfamide (14b)

Compound 14b was prepared from 33 according to the procedure for the synthesis of 12. Yield was 6.0% for 2 steps. mp 213–220 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.15–7.22 (2H, br), 7.23–7.28 (2H, m), 7.44–7.50 (1H, m), 7.56–7.67 (2H, m), 7.82–7.92 (3H, m), 8.75 (1H, d, J = 2.0 Hz), 9.65–9.72 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 117.1 (d), 117.9 (s, 2C), 121.9 (d), 123.5 (s), 124.7 (d), 125.5 (d), 125.9 (s), 126.0 (s, 2C), 131.2 (d), 139.5 (s), 146.8 (s), 153.7 (d); IR (ATR) cm⁻¹; 3305, 1618; HR-MS (ESI-TOF) Calcd for C₁₄H₁₃FN₅O₂S [M + H]⁺ 334.0774. Found 334.0729.

3-(2-Fluorophenyl)-1-(4-nitrophenyl)-1H-[1,2,4]triazole (34)

2-Fluorobenzaldehyde (3.58 mL, 34.3 mmol) and EtOH (50 mL) were added to a suspension of (4-nitrophenyl)hydrazine (5.00 g, 33.0 mmol) in 2.0 M sulfuric acid solution (33 mL) and EtOH (20 mL), and the mixture was stirred at room temperature for 4.5 h. The insoluble material was collected by filtration to give an intermediate (11.50 g). N-Bromosuccinimide (NBS) (1.2 g, 6.8 mmol) was dissolved in CH₂Cl₂ (25 mL), dimethylsulfide (0.92 mL, 12.4 mol) was added under ice-cooling, and a solution of the intermediate (1.4 g, 4.0 mmol) in CH₂Cl₂ (25 mL) was added dropwise at −20 °C and stirred at room temperature for 3 h. CH₂Cl₂ (200 mL) was added to the reaction mixture, washed with water and saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. The resulting brown powder (1.18 g) was suspended in EtOH (35 mL), Et₃N (0.33 mL, 2.4 mmol) and tetrazole (0.17 g, 2.4 mmol) were added, and the mixture was heated to reflux for 4.25 h. After cooling, water was added to the reaction mixture and the precipitate was collected by filtration. The solid obtained was purified by silica gel column chromatography to give 34 (325 mg, 29% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.15–7.36 (2H, m), 7.42–7.52 (1H, m), 7.93–8.09 (2H, m), 8.11–8.19 (1H, m), 8.35–8.47 (2H, m), 8.76 (1H, s).

N-[4-[3-(2-Fluorophenyl)[1,2,4]triazol-1-yl]phenyl]sulfamide (14c)

Compound 14c was prepared from 34 according to the procedure for the synthesis of 12. Yield was 52% for 3 steps. mp 191–195 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.24 (2H, s), 7.29–7.40 (4H, m), 7.47–7.57 (1H, m), 7.75–7.86 (2H, m), 8.07 (1H, td, J = 7.6, 1.5 Hz), 9.27 (1H, s), 9.79 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 116.6 (d), 118.4 (s), 118.4 (s, 2C), 120.4 (s, 2C), 124.6 (s), 124.6 (s), 129.9 (s), 131.1 (s), 131. 3 (d), 139.2 (s), 142.7 (s), 158.0 (d), 159.4 (d); IR (ATR) cm⁻¹; 1520; HR-MS (ESI-TOF) Calcd for C₁₄H₁₂FN₅NaO₂S [M + Na]⁺ 356.0593. Found 356.0612.

4-(4-Nitrophenyl)-2H-[1,2,3]triazole (22)

Formalin (p = 37%) (44 mL, 0.59 mol) and AcOH (5.1 mL, 89 mmol) were dissolved in THF (60 mL) and stirred at room temperature for 15 min. 1-Ethynyl-4-nitrobenzene (8.63 g, 58.7 mmol) and NaN₃ (5.72 g, 88.0 mmol) were added to the mixture and stirred at room temperature for 10 min. A solution of sodium-L-ascorbate (2.33 g, 11.8 mmol) and CuSO₄·5H₂O (733 mg, 2.94 mmol) in water (5 mL) was added, and the mixture was stirred at room temperature for 15 h. Water was added to the reaction mixture and extracted with AcOEt, and the organic layers were combined, washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by column chromatography. CH₂Cl₂ was added to the obtained solid, and the insoluble material was collected by filtration to give 22 (9.57 g, 86% yield) as a solid. ¹H-NMR (DMSO-d)₆ δ: 8.12–8.19 (2H, m), 8.30–8.36 (2H, m), 8.52 (0.8H, s), 8.89 (0.2H, s).

2-(2-Fluorophenyl)-4-(4-nitrophenyl)-2H-[1,2,3]triazole (35)

1-Bromo-2-fluorobenzene (0.21 mL, 1.9 mmol), di-tert-butyl[3,4,5,6-tetrmethyl-2′,4′,6′-tris(propan-2-yl)-[1,1′-biphenyl]-2-yl]phosphane (76 mg, 0.16 mmol), Pd₂(dba)₃ (72 mg, 0.079 mmol), and K₃PO₄ (671 mg, 3.16 mmol) were added to a suspension of 22 (300 mg, 1.58 mmol) in toluene (5 mL), and the mixture was heated to reflux for 19 h. After cooling, the reaction mixture was diluted with AcOEt, washed with saturated aqueous NH₄Cl, water, 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 35 (132 mg, 29% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.30–7.37 (2H, m), 7.42–7.49 (1H, m), 7.86–7.93 (1H, m), 8.04–8.09 (2H, m), 8.22 (1H, s), 8.31–8.36 (2H, m).

N-{4-[2-(2-Fluorophenyl)-2H-[1,2,3]triazol-4-yl]phenyl}sulfamide (14d)

Compound 14d was prepared from 35 according to the procedure for the synthesis of 12. Yield was 45% for 3 steps. mp 186–189 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.22–7.31 (4H, m), 7.40–7.46 (1H, m), 7.50–7.60 (2H, m), 7.83–7.93 (3H, m), 8.57 (1H, s), 9.78 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 117.4 (d) 117.8 (s, 2C), 122.5 (s), 125.2 (d), 125.3 (s), 126.6 (s, 2C), 127.6 (s), 130.3 (d), 133.4 (s), 140.3 (s), 148.6 (s), 153.8 (d); IR (ATR) cm⁻¹; 3257; HR-MS (ESI-TOF) Calcd for C₁₄H₁₂FN₅NaO₂S [M + Na]⁺ 356.0593. Found 356.0600; Anal. Calcd for C₁₄H₁₂FN₅O₂S·0.125AcOEt: C, 50.59; H, 3.83; N, 20.25. Found: C, 50.30; H, 3.80; N, 20.07.

2-(2-Fluorophenyl)-4-(4-nitrophenyl)oxazole (36)

2-Bromo-1-(4-nitrophenyl)ethan-1-one (1.00 g, 4.10 mmol) and 2-fluorobenzamide (713 mg, 5.12 mmol) were suspended in AcOEt (6 mL), AgOTf (1.32 g, 5.12 mmol) was added, and the mixture was stirred at 60 °C for 1 h. AgOTf (421 mg, 1.64 mmol) was added and stirred at the same temperature for another 1.5 h. AcOEt and saturated brine were added to the reaction mixture, and after stirring at room temperature for 10 min, the insoluble material was filtered off and the filtrate was divided. The organic layer was washed with water, saturated aqueous NaHCO₃, 1.0 M HCl, water, and saturated brine, dried over Na₂SO₄, and the solvent was removed under reduced pressure. t-BuOMe (10 mL) was added to the residue, and the insoluble material was collected by filtration to give 36 (458 mg, 39% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.20–7.34 (2H, m), 7.45–7.56 (1H, m), 7.95–8.07 (2H, m), 8.10–8.20 (2H, m), 8.27–8.35 (2H, m).

N-{4-[2-(2-Fluorophenyl)oxazol-4-yl]phenyl}sulfamide (14e)

Compound 14e was prepared from 36 according to the procedure for the synthesis of 12. Yield was 20% for 3 steps. mp 174–176 °C; ¹H-NMR (DMSO-d₆) δ: 7.15–7.30 (4H, m), 7.35–7.50 (2H, m), 7.54–7.66 (1H, m), 7.72–7.80 (2H, m), 8.05–8.17 (1H, m), 8.67 (1H, s), 9.68 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 114.8 (d), 116.9 (d), 117.9 (s, 2C), 124.1 (s), 125.0 (d), 125.9 (s, 2C), 129.5 (s), 132.7 (d), 134.8 (s), 139.5 (s), 140.8 (s), 157.3 (d), 159.1 (d); IR (ATR) cm⁻¹; 3275; HR-MS (ESI-TOF) Calcd for C₁₅H₁₂FN₃NaO₃S [M + Na]⁺ 356.0481. Found 356.0475.

5-(2-Fluorophenyl)oxazole (37)

1-Isocyanomethanesulfonyl-4-methylbenzene (4.28 g, 21.9 mmol) and K₂CO₃ (3.58 g, 25.9 mmol) were added to a solution of 2-fluorobenzaldehyde (2.08 mL, 19.9 mmol) in MeOH (50 mL), and the mixture was stirred at room temperature for 2 h. The reaction mixture was concentrated under reduced pressure and the residue was purified by silica gel column chromatography to give 37 (0.67 g, 21% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.11–7.39 (3H, m), 7.51 (1H, d, J = 3.9 Hz), 7.77 (1H, dt, J = 5.4, 1.7 Hz), 7.95 (1H, s).

5-(2-Fluorophenyl)-2-(4-nitrophenyl)oxazole (38)

1-Bromo-4-nitrobenzene (531 mg, 2.63 mmol), Pd(PPh₃)₄ (0.10 g, 0.088 mmol) and t-BuOLi (0.21 g, 2.6 mmol) were added to a solution of compound 37 (286 mg, 1.75 mmol) in 1,4-dioxane (10 mL), and the reaction mixture was stirred at 120 °C for 200 min. Pd(PPh₃)₄ (0.07 g, 0.06 mmol) and t-BuOLi (0.20 g, 2.5 mmol) were added, and the mixture was stirred at 120 °C for 1 h. After cooling, the reaction mixture was diluted with AcOEt, washed with saturated aqueous NH₄Cl 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 38 (0.18 g, 36% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.18–7.41 (2H, m), 7.50–7.58 (1H, m), 7.68 (1H, d, J = 3.9 Hz), 7.89 (1H, dt, J = 7.6, 1.3 Hz), 8.25–8.32 (2H, m), 8.33–8.38 (2H, m).

N-[4-{5-(2-Fluorophenyl)oxazol-2-yl}phenyl]sulfamide (14f)

Compound 14f was prepared from 38 according to the procedure for the synthesis of 12. Yield was 53% for 3 steps. mp 194–195 °C; ¹H-NMR (DMSO-d₆) δ: 7.18–7.49 (7H, m), 7.52–7.68 (1H, m), 7.88–8.12 (3H, m), 9.90–10.08 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 115.6 (d), 116.1 (d), 117.3 (s, 2C), 119.7 (s), 125.1 (d), 126.0 (s), 127.1 (s, 2C), 127.3 (d), 129.9 (d), 142.0 (s), 144.5 (d), 157.9 (d), 160.4 (s); IR (ATR) cm⁻¹; 1490; HR-MS (ESI-TOF) Calcd for C₁₅H₁₃FN₃O₃S [M + H]⁺ 334.0662. Found 334.0594.

2-(2-Fluorophenyl)-5-(4-nitrophenyl)oxazole (39)

2-Amino-1-(4-nitrophenyl)ethan-1-one hydrochloride (3.96 g, 18.3 mmol) and 2-fluorobenzoic acid (2.56 g, 18.3 mmol) were suspended in CH₂Cl₂ (50 mL), EDC·HCl (4.21 g, 21.9 mmol), 1-hydroxybenzotriazole (HOBt) (2.96 g, 21.9 mmol), and Et₃N (3.8 mL, 27 mmol) were added, and the reaction mixture was stirred at room temperature for 13 h. Water and CHCl₃ were added to the reaction mixture, and the insoluble material was filtered off. The reaction mixture was extracted with CHCl₃, and the organic layer was washed with 5% citric acid water, saturated aqueous NaHCO₃, and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, AcOEt was added to the residue, and the insoluble material was collected by filtration to give 1.31 g of crude powder. The resulting powder (300 mg) was suspended in POCl₃ (5 mL) and heated to reflux for 4 h. After cooling, water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with water, saturated aqueous NaHCO₃, 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 39 (101 mg, 8.5% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.26–7.34 (2H, br), 7.45–7.56 (1H, m), 7.70 (1H, s), 7.86–7.92 (2H, m), 8.10–8.17 (1H, m), 8.29–8.36 (2H, m).

N-[4-{2-(2-Fluorophenyl)oxazole-5-yl}phenyl]sulfamide (14g)

Compound 14g was prepared from 39 according to the procedure for the synthesis of 12. Yield was 31% for 3 steps. mp 187–192 °C; ¹H-NMR (DMSO-d₆) δ: 7.19–7.32 (4H, m), 7.35–7.48 (2H, m), 7.55–7.65 (1H, m), 7.67–7.82 (3H, m), 8.05–8.18 (1H, m), 9.81 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 114.9 (d), 116.9 (d), 117.8 (s, 2C), 120.6 (s), 122.6 (s), 124.9 (s, 2C), 125.0 (d), 129.1 (s), 132.4 (d), 140.0 (s), 151.1 (s), 155.9 (d), 158.9 (d); IR (ATR) cm⁻¹; 1616; HR-MS (ESI-TOF) Calcd for C₁₅H₁₃FN₃O₃S [M + H]⁺ 334.0662. Found 334.0608.

2-(2-Fluorophenyl)-4-methyl-5-(4-nitrophenyl)oxazole (40)

2-Fluorobenzylamine (1.23 mL, 10.7 mmol), K₂CO₃ (4.95 g, 35.8 mmol), and I₂ (5.00 g, 19.7 mmol) were added to a solution of 2-bromo-1-(4-nitrophenyl)propan-1-one (2.31 g, 8.95 mmol) in DMF (36 mL), and the mixture was stirred at 80 °C for 5 h. After cooling, AcOEt was added to the reaction mixture, and the mixture was washed with 10% Na₂S₂O₃ water, 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 40 (2.34 g, 88% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 2.55 (3H, s), 7.38–7.49 (2H, m), 7.59–7.66 (1H, m), 7.95–8.00 (2H, m), 8.11–8.17 (1H, m), 8.34–8.39 (2H, m).

N-{4-[2-(2-Fluorophenyl)-4-methyloxazol-5-yl]phenyl}sulfamide (14h)

Compound 14h was prepared from 40 according to the procedure for the synthesis of 12. Yield was 11% for 3 steps. mp 193–196 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 2.42 (3H, s), 7.19–7.25 (2H, m), 7.27–7.32 (2H, m), 7.34–7.45 (2H, m), 7.53–7.65 (3H, m), 8.04–8.10 (1H, m), 9.77 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 12.9 (s), 114.8 (d), 116.9 (d), 117.9 (s, 2C), 121.6 (s), 124.9 (d), 125.9 (s, 2C), 129.1 (s), 131.8 (s), 132.3 (d), 139.4 (s), 145.3 (s), 154.3 (d), 159.0 (d); IR (ATR) cm⁻¹; 3323, 3260; HR-MS (ESI-TOF) Calcd for C₁₆H₁₅FN₃O₃S [M + H]⁺ 348.0818. Found 348.0756.

Ethyl 2-(2-Fluorophenyl)-5-(4-nitrophenyl)oxazole-4-carboxylate (41)

Ethyl 3-(4-nitrophenyl)-3-oxopropanoate (10.0 g, 42.2 mmol), I₂ (12.9 g, 50.8 mmol), Cu(OAc)₂ (766 mg, 4.22 mmol) and 5.5 M decane solution of tert-butyl hydroperoxide (15 mL, 84 mmol) were added to a solution of 2-fluorobenzylamine (7.2 mL, 64 mmol) in DMF (100 mL), and the mixture was stirred at room temperature for 1 h. 2-Fluorobenzylamine (2.4 mL, 21 mmol) was added and stirred at the same temperature for 1 h. Water was added to the reaction mixture, and the mixture was extracted with AcOEt. The organic layer was washed with 10% aqueous Na₂S₂O₃ solution, 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 41 (6.06 g, 41% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.46 (3H, t, J = 7.1 Hz), 4.50 (2H, q, J = 7.1 Hz), 7.22–7.23 (2H, m), 7.49–7.57 (1H, m), 8.16–8.22 (1H, m), 8.32–8.37 (2H, m), 8.38–8.44 (2H, m).

2-(2-Fluorophenyl)-5-(4-nitrophenyl)oxazole-4-carboxamide (42)

Following the addition of 5.0 M aqueous NaOH (3.0 mL, 15 mmol) to a suspension of compound 41 (2.66 g, 7.47 mmol) in MeOH (30 mL) and THF (30 mL), the mixture was stirred at 40 °C for 0.5 h and 6.0 M HCl (5.0 mL, 30 mmol) was then added under ice-cooling. The solvent was removed under reduced pressure. Water (50 mL) was added to the residue, and the insoluble material was collected by filtration. The resulting powder was washed with 50 mL of t-BuOMe-n-hexane (1 : 1), dissolved in AcOEt-THF (2 : 1), and dried over Na₂SO₄. The solvent was removed under reduced pressure.

The obtained residue was dissolved in DMF (10 mL), following the addition of HOBt (617 mg, 4.57 mmol), EDC·HCl (876 mg, 4.57 mmol), and 28% ammonium hydroxide (0.31 mL, 4.6 mmol), and the mixture was stirred at room temperature for 8 h. Water and AcOEt were added to the reaction mixture, and after stirring at room temperature for 0.5 h, the precipitate was collected by filtration to give 42 (679 mg, 68% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.42–7.53 (2H, m), 7.65–7.73 (1H, m), 7.86–7.92 (1H, br), 7.92–7.98 (1H, br), 8.19–8.25 (1H, m), 8.36–8.41 (2H, m), 8.57–8.62 (2H, m).

2-(2-Fluorophenyl)-5-(4-nitrophenyl)oxazole-4-carbonitrile (43)

Pyridine (0.20 mL, 2.5 mmol) and TFAA (0.25 mL, 1.8 mmol) were added to a suspension of compound 42 (400 mg, 1.22 mmol) in 1,4-dioxane (10 mL), and the mixture was stirred at room temperature for 1 h. The reaction mixture was diluted with AcOEt, washed with water, 1.0 M HCl, and saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. t-BuOMe (10 mL) was added to the residue, and the insoluble material was collected by filtration to give 43 (314 mg, 83% yield) as a solid. ¹H-NMR (CDCl₃) δ: 7.26–7.37 (2H, m), 7.55–7.62 (1H, m), 8.11–8.17 (1H, m), 8.20–8.25 (2H, m), 8.39–8.44 (2H, m).

2-(2-Fluorophenyl)-5-(4-sulfamoylaminophenyl)oxazole-4-carbonitrile (14i)

Compound 14i was prepared from 43 according to the procedure for the synthesis of 12. Yield was 23% for 3 steps. mp 199–204 °C; ¹H-NMR (DMSO-d₆) δ: 7.35–7.52 (6H, m), 7.65–7.73 (1H, m), 7.88–7.93 (2H, m), 8.12–8.18 (1H, m), 10.19 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 106.7 (s), 113.3 (d), 114.0 (s), 117.1 (d), 117.3 (s), 117.4 (s, 2C), 125.2 (s), 126.7 (s, 2C), 129.6 (s), 133.9 (d), 142.6 (s), 156.0 (d), 157.8 (s), 159.3 (d); IR (ATR) cm⁻¹; 3263, 2239; HR-MS (ESI-TOF) Calcd for C₁₆H₁₁FN₄NaO₃S [M + Na]⁺ 381.0434. Found 381.0440.

N-[4-{2-(3-Fluorophenyl)oxazole-5-yl}phenyl]sulfamide (21a)

Compound 21a was prepared from 15 according to the procedure for the synthesis of 14g. Yield was 6.9% for 5 steps. mp >195 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.16–7.33 (4H, m), 7.34–7.43 (1H, m), 7.55–7.69 (1H, m), 7.73 (1H, s), 7.75–7.81 (2H, m), 7.81–7.89 (1H, d, J = 9.6 Hz), 7.92 (1H, d, J = 7.8 Hz), 9.80 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 112.4 (d), 117.2 (d), 117.8 (s, 2C), 120.7 (s), 121.9 (d), 122.8 (s), 125.0 (s, 2C), 128.9 (d), 131.4 (d), 140.0 (s), 151.3 (s), 158.4 (d), 162.3 (d); IR (ATR) cm⁻¹; 1487; HR-MS (ESI-TOF) Calcd for C₁₅H₁₃FN₃O₃S [M + H]⁺ 334.0662. Found 334.0612.

N-{4-[2-(4-Fluorophenyl)oxazol-5-yl]phenyl}sulfamide (21b)

Compound 21b was prepared from 15 according to the procedure for the synthesis of 14g. Yield was 10% for 5 steps. mp >270 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.21–7.30 (4H, m), 7.36–7.45 (2H, m), 7.69 (1H, s,), 7.72–7.79 (2H, m), 8.08–8.17 (2H, m), 9.79 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 116.2 (d), 117.8 (s, 2C), 120.9 (s), 122.7 (s), 123.5 (s), 123.6 (s), 124.8 (s, 2C), 128.2 (s), 128.2 (s), 139.9 (s), 150.9 (s), 158.8 (s), 163.2 (d); IR (ATR) cm⁻¹; 1619, 1506; HR-MS (ESI-TOF) Calcd for C₁₅H₁₃FN₃O₃S [M + H]⁺ 334.0662. Found 334.0597.

N-[4-(2-Pyridin-2-yloxazol-5-yl)phenyl]sulfamide (21c)

Compound 21c was prepared from 15 according to the procedure for the synthesis of 14g. Yield was 3.5% for 5 steps. mp 205–209 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.21–7.32 (4H, m), 7.48–7.58 (1H, m), 7.69–7.82 (3H, m), 7.93–8.05 (1H, m), 8.15 (1H, d, J = 7.8 Hz), 8.73 (1H, d, J = 4.4 Hz), 9.78–9.91 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 117.8 (s, 2C), 120.6 (s), 121.8 (s), 123.0 (s), 124.8 (s), 125.0 (s, 2C), 137.4 (s), 140.1 (s), 145.3 (s), 149.8 (s), 151.8 (s), 158.9 (s); IR (ATR) cm⁻¹; 1590; HR-MS (ESI-TOF) Calcd for C₁₄H₁₂N₄NaO₃S [M + Na]⁺ 339.0528. Found 339.0546.

5-(4-Nitrophenyl)oxazole (17)

1-Isocyanomethanesulfonyl-4-methylbenzene (9.24 g, 47.3 mmol) and K₂CO₃ (7.73 g, 55.9 mmol) were added to a solution of 4-nitrobenzaldehyde (6.50 g, 43.0 mmol) in MeOH (108 mL), and the mixture was stirred at room temperature for 15.5 h and heated to reflux for 6 h. After cooling, water was added to the reaction mixture and the mixture was extracted with AcOEt. The organic layers were combined, dried over Na₂SO₄, and the solvent was removed under reduced pressure. t-BuOMe (70 mL) was added to the residue, and insoluble material was collected by filtration. The solid was purified by silica gel column chromatography to give 17 (3.46 g, 42% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 7.94–8.05 (3H, m), 8.27–8.37 (2H, m), 8.59 (1H, s).

5-(4-Nitrophenyl)-2-(pyrazin-2-yl)oxazole (19d)

Compound 17 (500 mg, 2.63 mmol), t-BuOLi (421 mg, 5.26 mmol), and Pd(PPh₃)₄ (303 mg, 0.263 mmol) were added to a suspension of 2-iodopyrazine (0.52 mL, 5.3 mmol) in 1,4-dioxane (13 mL), and heated to reflux for 15 h. After cooling, saturated aqueous NH₄Cl was added to the reaction mixture and extracted with AcOEt. The organic layer was washed with saturated brine, dried over Na₂SO₄, and the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 19d (730 mg, quant.) as a solid. ¹H-NMR (DMSO-d₆) δ: 8.08–8.20 (2H, m), 8.31 (1H, s), 8.34–8.45 (2H, m), 8.76–8.92 (2H, m), 9.42 (1H, d, J = 1.2 Hz).

N-{4-(2-Pyrazin-2-yloxazol-5-yl)phenyl}sulfamide (21d)

Compound 21d was prepared from 19d according to the procedure for the synthesis of 12. Yield was 64% for 3 steps. mp 218–221 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.23–7.33 (4H, m), 7.73–7.81 (2H, m), 7.87 (1H, s), 8.75–8.83 (2H, m), 9.35 (1H, d, J = 1.2 Hz), 9.86 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 117.7 (s, 2C) 120.3 (s), 123.3 (s), 125.2 (s), 140.4 (s), 141.2 (s), 142.8 (s, 2C), 144.6 (s), 145.3 (s), 152.5 (s), 156.9 (s); IR (ATR) cm⁻¹; 3134, 1616; HR-MS (ESI-TOF) Calcd for C₁₃H₁₁N₅NaO₃S [M + Na]⁺ 340.0480. Found 340.0472.

N-{4-[2-(2-Cyanophenyl)oxazol-5-yl]phenyl}sulfamide (21e)

Compound 21e was prepared from 17 according to the procedure for the synthesis of 21d. Yield was 4.6% for 4 steps. mp 202–206 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.24–7.31 (4H, m), 7.71 (1H, t, J = 7.6 Hz), 7.76–7.82 (2H, m), 7.84–7.91 (2H, m), 8.04 (1H, d, J = 7.6 Hz), 8.30 (1H, d, J = 7.6 Hz), 9.85 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 107.9 (s) 117.6 (s, 2C), 117.9 (s), 120.3 (s), 123.0 (s), 125.1 (s, 2C), 128.1 (s), 128.2 (s), 130.6 (s), 133.6 (s), 135.1 (s), 140.3 (s), 151.8 (s), 156.6 (s); IR (ATR) cm⁻¹; 3219; HR-MS (ESI-TOF) Calcd for C₁₆H₁₂N₄NaO₃S [M + Na]⁺ 363.0528. Found 363.0505.

1-{2-[4-(4-Nitrophenyl)[1,2,3]triazol-2-yl]phenyl}ethanone (23a)

1-(2-Fluorophenyl)ethan-1-one (0.70 mL, 5.8 mmol) and K₂CO₃ (872 mg, 6.31 mmol) were added to a solution of compound 22 (1.00 g, 5.26 mmol) in DMF (10 mL), and the mixture was stirred at 120 °C for 2.5 h. 1-(2-Fluorophenyl)ethan-1-one (0.30 mL, 2.5 mmol) was added to the reaction mixture and stirred at the same temperature for another 9 h. After cooling, the reaction mixture was diluted with AcOEt, washed with saturated aqueous NH₄Cl, 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 23a (270 mg, 17% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.33 (3H, s), 7.49–7.57 (2H, m), 7.59–7.65 (1H, m), 7.96 (1H, d, J = 7.8 Hz), 8.00–8.05 (2H, m), 8.19 (1H, s), 8.31–8.35 (2H, m).

N-{4-[2-(2-Acetylphenyl)-2H-[1,2,3]triazol-4-yl]phenyl}sulfamide (24a)

Compound 24a was prepared from 23a according to the procedure for the synthesis of 12. Yield was 25% for 3 steps. mp 138–140 °C; ¹H-NMR (DMSO-d₆) δ: 2.28 (3H, s), 7.21 (2H, s), 7.25–7.30 (2H, m), 7.54–7.64 (2H, m), 7.68–7.74 (1H, m), 7.80–7.84 (2H, m), 7.92 (1H, d, J = 7.8 Hz), 8.55 (1H, s), 9.76 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 30.1 (s) 117.8 (s, 2C), 122.4 (s), 122.5 (s), 126.5 (s, 2C), 127.8 (s), 128.4 (s), 131.3 (s), 133.5 (s), 134.7 (s), 135.9 (s), 140.3 (s), 148.8 (s), 200.7 (s); IR (ATR) cm⁻¹; 3348, 1680; HR-MS (ESI-TOF) Calcd for C₁₆H₁₅N₅NaO₃S [M + Na]⁺ 380.0793. Found 380.0794.

N-(4-{2-[2-(1-Hydroxyethyl)phenyl]-2H-[1,2,3]triazol-4-yl}phenyl)sulfamide (24b)

NaBH₄ (10 mg, 0.26 mmol) was added to the solution of compound 24a (78 mg, 0.22 mmol) in MeOH (2 mL), and the mixture was stirred at room temperature for 0.5 h. After the addition of water and 6.0 M HCl, the reaction mixture was extracted with CH₂Cl₂, and the organic layer was dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography to give 24b (50 mg, 63% yield) as a solid. mp 62–67 °C; ¹H-NMR (DMSO-d₆) δ: 1.27 (3H, d, J = 6.4 Hz), 4.94–5.02 (1H, m), 5.25 (1H, d, J = 4.2 Hz), 7.15–7.30 (4H, m), 7.41–7.47 (1H, m), 7.51–7.58 (2H, m), 7.78–7.86 (3H, m), 8.50 (1H, s), 9.65–9.84 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 25.3 (s), 63.3 (s), 117.9 (s, 2C), 122.9 (s), 124.8 (s), 126.4 (s, 2C), 126.8 (s), 127.3 (s), 129.2 (s), 132.4 (s), 136.7 (s), 140.1 (s), 142.0 (s), 147.9 (s); IR (ATR) cm⁻¹; 3301; HR-MS (ESI-TOF) Calcd for C₁₆H₁₇N₅NaO₃S [M + Na]⁺ 382.0950. Found 382.0945.

N-{4-[2-(2-Trifluoromethylphenyl)-2H-[1,2,3]triazol-4-yl]phenyl}sulfamide (24c)

Compound 24c was prepared from 22 according to the procedure for the synthesis of 24a. Yield was 3.1% for 4 steps. mp 178–180 °C (decomp.); ¹H-NMR (DMSO-d₆) δ: 7.19–7.29 (4H, m), 7.78–7.96 (5H, m), 8.02 (1H, d, J = 7.6 Hz), 8.57 (1H, s), 9.74–9.80 (1H, br); ¹³C-NMR (DMSO-d₆) δ: 117.8 (s, 2C), 121.5 (s), 122.5 (s), 123.6 (q) 126.5 (s, 2C), 127.7 (q), 127.9 (s), 130.1 (s), 133.3 (s), 133.8 (s), 137.2 (s), 140.3 (s), 148.6 (s); IR (ATR) cm⁻¹; 3275; HR-MS (ESI-TOF) Calcd for C₁₅H₁₂F₃N₅NaO₂S [M + Na]⁺ 406.0561. Found 406.0563.

2-(3-Hydroxymethylphenyl)-4-(4-nitrophenyl)-2H-[1,2,3]triazole (23d)

Compound 23d was prepared from 22 according to the procedure for the synthesis of 23a. Yield was 43%. ¹H-NMR (CDCl₃) δ: 1.81 (1H, t, J = 5.8 Hz), 4.84 (2H, d, J = 5.8 Hz), 7.41 (1H, d, J = 7.6 Hz), 7.52 (1H, t, J = 7.6 Hz), 8.04–8.24 (5H, m), 8.28–8.41 (2H, m).

2-(3-Dimethylaminomethylphenyl)-4-(4-nitrophenyl)-2H-[1,2,3]triazole (23e)

Et₃N (0.19 mL, 1.4 mmol) and MsCl (0.083 mL, 1.1 mmol) were added to a solution of compound 23d (203 mg, 0.685 mmol) in THF (5 mL) under ice-cooling, and the mixture was stirred at the same temperature for 55 min. Following the addition of 2.0 M THF solution of dimethylamine (1.71 mL, 3.4 mmol) to the mixture, it was stirred at room temperature for 250 min. After the addition of water, 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 23e (148 mg, 67% yield) as a solid. ¹H-NMR (CDCl₃) δ: 2.30 (6H, s), 3.54 (2H, s), 7.37 (1H, d, J = 7.6 Hz), 7.47 (1H, t, J = 7.6 Hz), 8.02–8.19 (5H, m), 8.29–8.37 (2H, m).

N-{4-[2-(3-dimethylaminomethylphenyl)[1,2,3]triazol-4-yl]phenyl}sulfamide (24e)

Compound 24e was prepared from 23e according to the procedure for the synthesis of 12. Yield was 27% for 3 steps. mp 146–148 °C; ¹H-NMR (DMSO-d₆) δ: 2.20 (6H, s), 3.50 (2H, s), 7.16–7.40 (5H, m), 7.52 (1H, t, J = 7.8 Hz), 7.94–8.09 (4H, m), 8.52 (1H, s), 9.76 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 44.9 (s, 2C), 62.8 (s), 116.8 (s), 117.8 (s, 2C), 118.1 (s), 122.7 (s), 126.6 (s, 2C), 127.7 (s), 129.4 (s), 133.1 (s), 139.1 (s), 140.2 (s), 140.8 (s), 148.3 (s); IR (ATR) cm⁻¹; 1485; HR-MS (ESI-TOF) Calcd for C₁₇H₂₁N₆O₂S [M + H]⁺ 373.1447. Found 373.1458.

N-{4-[2-(2-Cyanophenyl)[1,2,3]triazol-4-yl]phenyl}sulfamide (24f)

Compound 24f was prepared from 22 according to the procedure for the synthesis of 24a. Yield was 15% for 4 steps. mp 186–189 °C; ¹H-NMR (DMSO-d₆) δ: 7.23–7.27 (2H, br), 7.27–7.32 (2H, m), 7.61–7.67 (1H, m), 7.88–7.96 (3H, m), 8.05–8.10 (1H, m), 8.14–8.18 (1H, m), 8.69 (1H, s), 9.82 (1H, s); ¹³C-NMR (DMSO-d₆) δ: 103.3 (s), 116.7 (s), 117.7 (s, 2C), 122.1 (s), 122.2 (s), 126.8 (s, 2C), 128.4 (s), 134.3 (s), 134.6 (s), 135.4 (s), 139.5 (s), 140.6 (s), 149.2 (s); IR (ATR) cm⁻¹; 3251, 2229; HR-MS (ESI-TOF) Calcd for C₁₅H₁₂N₆NaO₂S [M + Na]⁺ 363.0640. Found 363.0631; Anal. Calcd for C₁₅H₁₂N₆O₂S·0.05AcOEt: C, 52.95; H, 3.63; N, 24.36. Found: C, 52.70; H, 3.60; N, 24.31; HPLC purity 98.8% (eluent: 20 mM NaH₂PO₄-Na₂HPO₄ (pH 7.0)/MeCN = 50/50).

Vector Construction

1. Construction of the pNLF-C-IDUA/Q70X Vector (IDUA/Q70X-Luc)

The vector IDUA/Q70X-Luc was generated by GenScript (Piscataway, NJ). A DNA fragment (47 bp) derived from IDUA cDNA (NM_000203), including the Q70X (c.208C > T) nonsense mutation and 5 upstream and 7 downstream flanking codons, tagged with the Kozak sequence (GCCACC) and the start codon (ATG) at the 5′-end (i.e., GCCACCatgGTCCTCAGCTGGGACtagCAGCTCAACCTCGCCTATGT) was created. The fragment was inserted in-frame at the EcoRV restriction site of the pNLF1-C [CMV/Hygro] Vector (Promega, Madison, WI, U.S.A.).

2. Construction of the pcDNA6/myc-His/LacZ–IDUA/W402X-NLuc Vector (IDUA/ W402X-Luc)

The vector IDUA/W402X-Luc was generated by GenScript. A DNA fragment (39 bp) derived from IDUA cDNA, including the W402X (c.1205G > A) nonsense mutation and 6 upstream and downstream flanking codons (i.e., ctggatgaggagcagctcTAGgccgaa gtgtcgcaggcc), tagged with the NLuc luciferase sequence at the 3′-end was created. The fragment was inserted into the pcDNA6/myc-His/LacZ vector (Carlsbad, CA, U.S.A.).

Luciferase Assay

HeLa cells were maintained in MEM medium (Nacalai Tesque) containing 10% fetal bovine serum (FBS, Life Technologies, Carlsbad, CA, U.S.A.) at 37 °C under 5% CO₂. Cells were seeded in 10 cm dishes at 5 × 10⁵ cells/dish. The day after seeding, cells were stably transfected with a vector using FuGENE^® HD Transfection Reagent (Promega, Madison, WI, U.S.A.). Cells stably transfected with IDUA/Q70X-Luc or IDUA/W402X-Luc were seeded at 1 × 10⁴ cells/well (50 µL/well) in white half-area 96-well plates (Greiner, Frickenhausen, Germany) and incubated at 37 °C under 5% CO₂ for 24 h. Then, 50 µL of the compound dissolved in MEM/10% FBS was added to the cells, followed by incubation under 5% CO₂ at 37 °C for 24 h. Luciferase activity was measured using the Nano-Glo Luciferase Assay System (Promega). Briefly, culture medium was removed and 20 µL of the Nano-Glo Luciferase Assay Reagent was added to the cells. Luminescence was measured using a microplate reader (Power Scan HT, BioTek, Winooski, VT, U.S.A.). The luciferase activity was expressed as a relative value (%): the activity in the cells untreated with the compound was taken as 100%.

Construction of the pcDNA6-IDUA Mutant Vector

The wild-type IDUA gene with the Kozak and EcoRI sequences at the 5′ end and the NotI sequence at the 3′ end was synthesized and introduced into the pcDNA3.1 (+) vector (Thermo Fisher Scientific Inc., Foster, U.S.A.). The Q70X or W402X mutation was introduced using the wild-type IDUA vector as a template. The synthesized pcDXA3.1 (+)-IDUA Q70X or W402X vector was PCR amplified with gccaccATG~TGA as 5′:EcoRI, 3′:XhoI and cloned into the EcoRI-XhoI site of the pcDNA6 myc–His C vector (Thermo Fischer Scientific, Waltham, MA, U.S.A.).

Evaluation of Readthrough-Inducing Activity Using IDUA Knockout HeLa Cells Transiently Transfected with IDUA cDNA with a Nonsense Mutation

IDUA gene knockout HeLa cells were generated using the CRISPR/Cas9 system from TaKaRa Bio (Otsu, Japan), and maintained in MEM medium containing 10% FBS at 37 °C under 5% CO₂. Cells were suspended in medium at 2.0 × 10⁵ cells/mL, seeded on 24-well plates at 500 µL/well, and incubated at 37 °C under 5% CO₂. The day after seeding, cells were transiently transfected with the pcDNA6-IDUA Q70X or W402X vector using FuGENE^® HD Transfection Reagent. After incubation under 5% CO₂ at 37 °C for 24 h, compound was added to the cells, followed by incubation under 5% CO₂ at 37 °C for 24 h. Cells were then lysed in 100 µL of Mammalian Cell PE LB (G-Bioscience, St. Louis, MO, U.S.A.). The protein concentration was measured by Protein Assay Bicinchoninate Kit (Nacalai Tesque). Twenty microliters of 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 incubation, 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.

References

• 1) Mort M., Ivanov D., Cooper D. N., Chuzhanova N. A., Hum. Mutat., 29, 1037–1047 (2008).
• 2) Korinthenberg R., Dev. Med. Child Neurol., 61, 292–297 (2019).
• 3) Pranke I., Golec A., Hinzpeter A., Edelman A., Gaudelus I. S., Front. Pharmacol., 10, 121–141 (2019).
• 4) Bunge S., Kleijer W. J., Steglich C., Beck M., Zuther C., Morris C. P., Schwinger E., Hopwood J. J., Scott H. S., Gal A., Hum. Mol. Genet., 3, 861–866 (1994).
• 5) Kellermayer R., Eur. J. Med. Genet., 49, 445–450 (2006).
• 6) Dabrowski M., Bieryllo Z. B., Zietkiewicz E., Mol. Med., 24, 25–39 (2018).
• 7) Dias P. M., Romão L., Cell. Mol. Life Sci., 78, 4677–4701 (2021).
• 8) Spelier S., van Doorn E. P. M., van der Ent C. K., Beekman J. M., Koppens M. A. J., Trends Mol. Med., 29, 297–314 (2023).
• 9) Campofelice A., Lentini L., Leonardo A. D., Melfi R., Tutone M., Pace A., Pibiri I., Int. J. Mol. Sci., 20, 3329–3346 (2019).
• 10) Keeling K. M., Bedwell D. M., Current Pharmacogenomics, 3, 259–269 (2005).
• 11) Burke J. F., Mogg A. E., Nucleic Acids Res., 13, 6265–6272 (1985).
• 12) Huth M. E., Ricci A. J., Cheng A. G., Int. J. Otolaryngol., 2011, 937861–937879 (2011).
• 13) Mingeot-Leclercq M. P., Tulkens P. M., Antimicrob. Agents Chemother., 43, 1003–1012 (1999).
• 14) Taguchi A., Hamada K., Kotake M., Shiozuka M., Nakaminami H., Pillaiyar T., Takayama K., Yakushiji F., Noguchi N., Usui T., Matsuda R., Hayashi Y., ChemMedChem, 9, 2233–2237 (2014).
• 15) Taguchi A., Hamada K., Shiozuka M., Kobayashi M., Murakami S., Takayama K., Taniguchi A., Usui T., Matsuda R., Hayashi Y., ACS Med. Chem. Lett., 8, 1060–1065 (2017).
• 16) Hamada K., Omura N., Taguchi A., Heravi A. B., Kotake M., Arai M., Takayama K., Taniguchi A., Roberge M., Hayashi Y., ACS Med. Chem. Lett., 10, 1450–1456 (2019).
• 17) Du L., Damoiseaux R., Nahas S., Gao K., Hu H., Pollard J. M., Goldstine J., Jung M. E., Henning S. M., Bertoni C., Gatti R. A., J. Exp. Med., 206, 2285–2297 (2009).
• 18) Welch E. M., Barton E. R., Zhuo J., et al., Nature (London), 447, 87–91 (2007).
• 19) Bushby K., Finkel R., Wong B., et al., Muscle Nerve, 50, 477–487 (2014).
• 20) Kerem E., Hirawat S., Armoni S., Yaakov Y., Shoseyov D., Cohen M., Nissim-Rafinia M., Blau H., Rivlin J., Aviram M., Elfring G. L., Northcutt V. J., Miller L. L., Kerem B., Wilschanski M., Lancet, 372, 719–727 (2008).
• 21) Kerem E., Konstan M. W., De Boeck K., et al., Lancet Respir. Med., 2, 539–547 (2014).
• 22) Oussoren E., Keulemans J., van Diggelen O. P., Oemardien L. F., Timmermans R. G., van der Ploeg A. T., Ruijter G. J., Mol. Genet. Metab., 109, 377–381 (2013).
• 23) Nudelman I., Sabbah A. R., Cherniavsky M., Belakhov V., Hainrichson M., Chen F., Schacht J., Pilch D. S., Yosef T. B., Baasov T., J. Med. Chem., 52, 2836–2845 (2009).
• 24) Jacoby E., Reinhardt J., Schmiedeberg N., Spanka C., WO2014/091446 (2014).
• 25) Meanwell N. A., J. Med. Chem., 61, 5822–5880 (2018).
• 26) Howard M. T., Shirts B. H., Petros L. M., Flanigan K. M., Gesteland R. F., Atkins J. F., Ann. Neurol., 48, 164–169 (2000).
• 27) Takeda S., Shirahase H., Takashima S., Kitao T., WO2018/186365 (2018).