Novel Non-carboxylate Benzoylsulfonamide-Based Protein Tyrosine Phosphatase 1B Inhibitors with Non-competitive Actions

Abstract

A novel series of benzoylsulfonamide derivatives were synthesized and biologically evaluated. Among them, 4-(biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzamide (compound 18K) was identified as a protein tyrosine phosphatase 1B (PTP1B) inhibitor with potent and selective inhibitory activity against PTP1B (IC₅₀=0.25 µM). Compound 18K functioned as a non-competitive inhibitor and bound to the allosteric site of PTP1B. It also showed high oral absorption in mice (the maximum drug concentration (C_max)=45.5 µM at 30 mg/kg), rats (C_max=53.6 µM at 30 mg/kg), and beagles (C_max=37.8 µM at 10 mg/kg), and significantly reduced plasma glucose levels at 30 mg/kg/d (per os (p.o.)) for one week with no side effects in db/db mice. In conclusion, the substituted benzoylsulfonamide was shown to be a novel scaffold of a non-competitive and allosteric PTP1B inhibitor, and compound 18K has potential as an efficacious and safe anti-diabetic drug as well as a useful tool for investigations of the physiological and pathophysiological effects of allosteric PTP1B inhibition.

In various cellular signaling pathways, protein tyrosine kinases (PTKs) mediate the phosphorylation of tyrosine residues in a number of proteins, while protein tyrosine phosphatases (PTPs) de-phosphorylate phosphorylated tyrosine.^1,2) Thus, PTPs as well as PTKs play important physiological roles and are involved in the pathogeneses of diseases such as diabetes, obesity, autoimmune disorders, cancer, and neurological disorders.^1–3) Among PTPs, PTP1B has been shown to negatively regulate insulin signals through the de-phosphorylation of insulin receptors and insulin receptor substrate (IRS), and the leptin signal by the de-phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3).^2,4) PTP1B knockout mice show obesity resistance and insulin hypersensitivity.⁵⁾ A PTP1B antisense oligonucleotide normalizes glucose levels and improves insulin resistance in diabetic mice.⁶⁾ Furthermore, PTP1B has been implicated in tumorigenesis, podocyte injury, endothelial dysfunction, and retinal light damage.^1,2,7–9) Based on this background, PTP1B has been attracting attention as a potential therapeutic target and a large number of PTP1B inhibitors have been reported as candidates for anti-diabetic and anti-obesity drugs.^1,10–12)

Classically, some types of vanadates have been shown to inhibit PTP1B and exert insulinomimetic actions.¹⁰⁾ Small molecule synthetic inhibitors started from peptidyl phosphonates, which mimic the substrate, a phosphorylated tyrosine (pTyr).¹¹⁾ Non-peptidyl compounds with two phosphonate moieties have been discovered; however, these highly charged compounds cannot permeate the cell membrane (compound I in Fig. 1).¹²⁾ Non-phosphate compounds, in which two carboxyl moieties as bioisosteres of phosphonates were introduced, have been reported to function as potent inhibitors, but were still highly charged and showed low bioavailability (compound II in Fig. 1).¹²⁾ PTP1B inhibitors with one carboxyl moiety were also discovered and shown to exhibit potent activity and efficacy (Ertiprotafib and compound III in Fig. 1).¹¹⁾ Ertiprotafib has peroxisome proliferator-activated receptor (PPAR) γ and PPARα agonist activities in addition to PTP1B inhibition, and its clinical development was discontinued.¹³⁾ We previously demonstrated that a series of 3-carboxyl tetrahydroisoquinoline derivatives exhibited PPARγ agonist activities and weak PTP1B inhibitory activities.^14–17) Carboxyl types of inhibitors appear to compete with the substrate at the catalytic site, resulting in relatively low PTP1B selectivity because the catalytic site is similar among PTPs.^11,18) Although JTT-551 with a carboxyl moiety exhibited modest activity and anti-diabetic effects in diabetic mice, clinical development was suspended possibly due to low efficacy in patients.¹⁹⁾ In order to achieve high PTP1B selectivity, allosteric inhibitors without a carboxyl moiety have been synthesized and exhibited modest activity and efficacy (Trodusquemine and compound IV in Fig. 1).^11,20) Trodusquemine is a naturally-occurring spermine derivative and an allosteric PTP1B inhibitor.^21,22) Although its parenteral administration of trodusquamine showed anti-diabetic and anti-obesity effects in mice, its clinical development was discontinued. More potent and orally bioavailable allosteric inhibitors based on a novel scaffold are strongly desired as excellent anti-diabetic and anti-obesity drugs. These inhibitors may be effective against not only diabetes and obesity, but also cancer, neuroinflammation, and retinal and renal failure.^1,2,7–9) Reported allosteric inhibitors consist of two or more aromatic rings connected via acyl, ether, or alkyl linkers, and exhibit relatively weak activity.¹¹⁾ In the present study, we synthesized a series of benzoylsulfonamide derivatives with a linker and aromatic ring. Structure–activity relationships were discussed and 4-(biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzamide (compound 18K) was selected for further evaluations. Compound 18K exhibited potent and selective non-competitive inhibitory activity, high bioavailability in mice, rats, and beagles, and anti-diabetic activity in db/db mice.

Fig. 1. Chemical Structures of PTP1B Inhibitors

Chemistry

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

Chart 1. Synthesis of Substituted Benzoylsulfonamide Derivatives with an Oxime and Amide Linker

Reagents and conditions: (i) 2A, CDI, DBU, DMF; (ii) HClO₄, 1,4-dioxane; (iii) HClO₄, 1,4-dioxane, then aqueous NaOH, MeOH; (iv) aqueous LiOH, THF, MeOH; (v) aqueous LiOH, 1,4-dioxane, then HCl in i-PrOH, solvents; (vi) HClO₄, 1,4-dioxane; (vii) 2A, CDI, DBU, DMF; (viii) HCl in i-PrOH, HCO₂H; (ix) EDC, CH₂Cl₂; (x) COCl₂, DMF, CH₂Cl₂, then i-Pr₂NEt.

Chart 2. Synthesis of Substituted Benzoylsulfonamide Derivatives with an Ether Linker

Reagents and conditions: (i) NaH, THF; (ii) Ag₂O, toluene; (iii) aqueous LiOH or aqueous NaOH, solvents; (iv) CDI, DBU, DMF, then MeONa in MeOH, MeOH; (v) aqueous LiOH, THF, MeOH; (vi) TBAF in THF, THF.

Chart 3. Synthesis of a Benzoylsulfonamide Derivative and Other Sulfonamide Derivatives with a Thioether Linker

Reagents and conditions: (i) methyl 4-(bromomethyl)benzoate 13, MeONa in MeOH, MeOH; (ii) aqueous LiOH, THF, MeOH; (iii) CDI, DBU, DMF; (iv) DPPA, Et₃N, toluene then 2K, DBU; (v) 1-bromo-4-bromomethylbenzene, MeONa in MeOH, MeOH; (vi) diethyloxalate, n-BuLi in n-hexane, THF; (vii) DPPA, Et₃N, t-BuOH, toluene; (viii) TFA, CH₂Cl₂; (ix) ethyl chloroglycoxylate, Et₃N, CH₂Cl₂; (x) aqueous LiOH, THF, MeOH; (xi) aqueous NaOH, THF, MeOH; (xii) 2K, CDI, DBU, DMF.

Chart 1 shows the general synthetic pathway for the preparation of oxime and amide linker derivatives. The starting material 1, which was prepared as described previously,²³⁾ underwent condensation with sulfonamide 2A²⁴⁾ to afford 3. Compound 3 was treated with HClO₄ in the presence of various aldehydes (4a, 4b, 4d, 4f, 4h, 4i, 4k, 4l, and 4n)^25–28) to give the corresponding oxime linker derivatives 5a, 5c, 5d, 5f, 5h, 5i, 5k, 5m, and 5o. The methyl ester of 5d was hydrolyzed with aqueous LiOH to afford 5e. Compounds 5f, 5i were converted to 5g, 5j by the deprotection of trifluoroacetyl group. Compound 3 was hydrolyzed by HClO₄, followed by acylation with 9a to prepare 10a. Separately, compound 8 was synthesized from commercially available compound 6²⁹⁾ by condensation with sulfonamide 2A and deprotection of the t-butoxycarbonyl (Boc) group. The amino group of 8 was acylated with 9a to give 11a.

The synthesis of ether linker derivatives is outlined in Chart 2. Various alcohols (12a and 12p–t)^30,31) were alkylated by methyl 4-(bromomethyl)benzoate 13 with NaH or Ag₂O, and then hydrolyzed with aqueous LiOH or NaOH to afford the carboxylic acids 14a, and 14p–t. Compounds 14a and 14p–t underwent condensation with sulfonamides 2A–D, and 2F–I^32–34) to give 15aA, 15pA–tA, 15rB, 15rC, and 15rF–rH. The methyl ester of 15rD was hydrolyzed with aqueous LiOH to afford 15rE. The tert-butyldimethylsilyl (TBS) group of 15rI was deprotected with tetrabutylammonium fluoride (TBAF) to give 15rJ.

Chart 3 shows the synthetic procedures for thioether linker derivatives. Compound 16, which was prepared as described previously,³⁵⁾ was deacetylated and alkylated with methyl 4-(bromomethyl)benzoate 13, followed by hydrolysis with aqueous LiOH to afford 17. Compound 17 was converted to 18H, 18K and 18L in a similar manner to the synthesis of 15aA, 15pA–tA, 15rB, 15rC, and 15rF–rH. Compound 17 was converted to urea 23 by the Curtius rearrangement. Separately, compound 16 was hydrolyzed and alkylated with 1-bromo-4-bromomethylbenzene in the presence of sodium methoxide, and then acylated with diethyl oxalate to give 20. Compound 20 was hydrolyzed and underwent condensation with sulfonamide 2K³⁴⁾ to give 22. Compound 19 was synthesized from compound 17 by the Curtius rearrangement, deprotection of the Boc group with trifluoroacetic acid (TFA), and acylation with ethyl chloroglyoxylate. Compound 19 was converted to 21 as described in the synthesis of 22.

Results and Discussion

Since PTP1B was discovered and shown to negatively regulate insulin and leptin signals, extensive efforts have been made to identify excellent PTP1B inhibitors that are useful for the treatment of diabetes and obesity by academia and pharmaceutical industries.^2,4) Inhibitors with two phosphonate or carboxyl substituents exhibit potent PTP1B inhibitory activity, but have low membrane permeability, resulting in poor bioavailability.¹¹⁾ Some inhibitors with a monocarboxyl moiety exhibit modest PTP1B inhibitory activity and anti-diabetic effects in mice.¹¹⁾ Most of the inhibitors with an acid moiety are considered to compete with a substrate, pTyr at the catalytic site, and, thus, may have low PTP1B selectivity and be directly influenced by the concentrations of substrates. Non-competitive inhibitors without a carboxyl or phosphonate group have recently been reported to interact with the allosteric site of PTP1B.^11,20) Among them, compound IV in Fig. 1 has a structure consisting of three aromatic rings, which line up via sulfonamide and amide linkers, and had modest activity and in vivo efficacy.^11,20) In the present study, we employed acylsulfonamide structure, which has been used in various enzyme inhibitors,³⁶⁾ to synthesize novel compounds and to find a novel allosteric PTP1B inhibitor with good bioavailability and in vivo efficacy. The chemical structures, molecular weights, calculated log P (c log P), calculated log D_7.4 (c log D_7.4), and PTP1B inhibitory activities of the compounds synthesized were shown in Tables 1–3.

Table 1. Chemical Structures, Molecular Weights, c log P, c log D_7.4, and PTP1B Inhibitory Activities of Benzoylsulfonamide Derivatives with Oxime, Amide, and Ether Linkers

a) Molecular weight as the free form. b) n=2. c) Sodium salt. d) Hydrochloride salt.

Table 2. Chemical Structures, Molecular Weights, c log P, c log D_7.4, and PTP1B Inhibitory Activities of Benzoylsulfonamide Derivatives with Ether and Thioether Linkers

a) Molecular weight as the free form. b) n=2.

Table 3. Chemical Structures, Molecular Weights, c log P, c log D_7.4, and PTP1B Inhibitory Activities of a Benzoylsulfonamide Derivative and Other Sulfonamide Derivatives

a) Molecular weight as the free form. b) n=2.

We synthesized compound 5a, the structure of which may resemble compound IV, an allosteric inhibitor (Table 1). Compound 5a has benzoylsulfonamide instead of sulfonamide in compound IV and an oxime structure instead of amide. Compound 5a exhibited weak PTP1B inhibitory activity. This activity was markedly decreased following the replacement of an oxime linker (5a) with an amide methyl linker and a hydroxamate linker (10a, 11a): the electron-rich property of carbonyl or less linear structure may not be preferable for PTP1B inhibitory activity. Changes from isopropylphenyl to indoline (5j, 5k) and indole (5m) failed to increase PTP1B inhibitory activity: benzimidazole (5o) markedly reduced activity. Phenolic, carboxyl, and anilinic moieties also markedly reduced this activity (5c, 5e, 5g). Therefore, a charged moiety introduced at R₁ is considered to disturb the interaction between an inhibitor and enzyme. Competitive inhibitors with a pTyr mimic structure, such as compounds I¹²⁾ and II,¹¹⁾ are negatively charged and bind to the phosphate-binding loop (P-loop, residues 214–221) and WPD-loop (residues 177–185) of the active site. In these competitive inhibitors, their carboxyl or phosphonate moiety also interacts with Arg24 and Arg254 of an additional non-catalytic aryl phosphate binding site (B-site), which is located close to the catalytic site. In contrast, the activities of our synthesized benzoylsulfonamide derivatives were markedly reduced by anionic and cationic substituents, suggesting that they bind to the allosteric site, but not to the catalytic and/or active site.

We then replaced the oxime with an ether linker and changed isopropyl phenyl to another substituted phenyl (Table 1). Compound 15aA with an ether linker exhibited similar activity to that of 5a with an oxime linker. Compound 15pA with 4-(4-methylpent-1-enyl)benzyl, 15rA with 4-biphenyl, and 15tA with 4-phenylethynylbenzyl exhibited more potent activity than 5a. Among them, 15pA and 15rA showed similar activities and had smaller molecular weights than the others. However, the maximum drug concentration (C_max) of 15rA after its oral administration at 10 mg/kg to male Sprague-Dawley (SD) was higher than that of 15pA; the C_max values of 15rA and 15pA were 7.0 µg/mL (13.2 µM) and 1.1 µg/mL (2.0 µM), respectively, while their area under the plasma concentration time curve (AUC) values were 81.7 µg·h/mL (155 µM·h) and 7.4 µg·h/mL (14 µM·h), respectively.

In Table 2, the dichlorophenyl moiety of 15rA was changed to another substituted phenyl and aliphatic chain in order to improve activity and bioavailability. Unfortunately, substituted phenyl, biphenyl, and fluorenyl moieties introduced at R₂ did not increase activity. Especially, benzoic acid markedly decreased activity, suggesting a disturbance in the inhibitor–enzyme interaction by the charged moiety at R₂. Compound 15rH with a octyl chain showed similar activity to that of 15rA. Compound 15rJ with pentanol showed lower activity than 15rA, and this may have been due to its weaker lipophilicity or hydrophilic moiety, and 18H, in which an ether linker of 15rH was changed to a thioether linker, exhibited markedly higher activity and bioavailability than 15rH. Compound 18K with a hexyl chain showed lower activity than 18H, but still higher activity than 15rH. Compound 18K was selected for further evaluations because of its smaller molecular weight and lower lipophilicity than 18H.

In Table 3, the benzoylsulfonamide structure was changed to other sulfonamide structures. Compound 21 had higher activity than that of 18K, but showed lower C_max and AUC values than those of 18K at 10 mg/kg in male SD rat: C_max of 1.5 µg/mL (2.9 µM) and AUC of 10.6 µg·h/mL (20.2 µM·h) in Compound 21, and C_max of 7.2 µg/mL (14.9 µM) and AUC of 63.4 µg·h/mL (132 µM·h) in Compound 18K, respectively. Among the sulfonamide derivatives, a benzoylsulfonamide derivative was considered to be the most suitable as a potent drug-like inhibitor.

In order to evaluate the inhibitory mode of compound 18K, a kinetic study using the synthetic substrate p-nitrophenylphosphate (p-NPP) was performed. Lineweaver–Burk plots demonstrated that 18K inhibited PTP1B activity in a non-competitive manner with a K_i value of 0.35 µM, suggesting that it bound to the allosteric site, but not to the catalytic site. Several X-ray crystal structure analyses and computational simulations recently revealed the binding sites of allosteric inhibitors and the inhibitory mechanisms in which a small molecule binds to allosteric sites and stabilizes the conformation associated with the inactive state of PTP1B.^37–44) Based on the molecular similarities between our compound 18K and an allosteric inhibitor bound to PTP1B (Fig. 2a), we constructed a structural model of the 18K–PTP1B complex by molecular alignment of the compounds, followed by validation and refinement of the structure obtained using a molecular dynamic (MD) simulation. Details are shown in Experimental. Our molecular overlay program, SUPERPOSE,^45,46) provided almost two patterns for the alignment between 18K and the allosteric inhibitor. The top-ranked conformations for each pattern were depicted in Fig. 2a. Of the two conformations, conformation 2 was subsequently rejected because 18K was immediately released from PTP1B during the MD simulation. The representative structure of the 18K–PTP1B complex derived from the MD simulation was depicted in Fig. 2b. In this structure, 18K formed a U-shaped conformation and bound to the allosteric site of PTP1B with the formation of a hydrogen bond and salt bridge around the benzoylsulfonamide structure. In addition, the U-shaped conformation of 18K appeared to be preferable for the formation of aromatic and van der Waals interactions around Phe196 and Phe280 of PTP1B.

Fig. 2. Structural Models of 18K–PTP1B and 18K–TCPTP Complexes

(a) SUPERPOSE alignments between the allosteric inhibitor (PDB: 1T4J; upper panel) and 18K (lower panels). The functional atoms corresponding to aromatic rings (ARs), hydrophobic atoms (HPs), hydrogen-bonding donors (HDs), and hydrogen-bonding acceptors (HAs) are colored yellow, gray, blue, and red, respectively. (b) Representative structure of the 18K–PTP1B complex derived from the MD simulation. 18K (green) and PTP1B residues within 4 Å of 18K (gray) are shown as balls and sticks, with the Connolly surface of PTP1B transparently depicted. Hydrogen bonds, salt bridges, and aromatic interactions are shown as dotted lines, and colored cyan, magenta, and orange, respectively. (c) Structural alignment between PTP1B and TCPTP. PTP1B (PDB: 1T4J) and TCPTP (PDB: 1L8K) are colored gray and magenta, respectively, and the allosteric inhibitor bound to PTP1B is colored green. The amino acids at the allosteric site are highly conserved, except for Phe280 of PTP1B. (d) Representative structure of the 18K–TCPTP complex derived from the MD simulation. 18K (green) and TCPTP residues within 4 Å of 18K (gray) are shown as balls and sticks, with the Connolly surface of TCPTP transparently depicted. Hydrogen bonds, salt bridges, and aromatic interactions are shown as dotted lines, and colored cyan, magenta, and orange, respectively.

Our structural model also explained the difference in the compound’s selectivity for PTP1B and T-cell protein tyrosine phosphatase (TCPTP), which is the most homologous phosphatase (sequence similarity of nearly 70%) to PTP1B (Fig. 2c). The structural model of the 18K–TCPTP complex based on that of the 18K–PTP1B complex was depicted in Fig. 2d. Compound 18K may not interact as strongly with TCPTP because Phe280 of PTP1B was replaced with Cys278 in TCPTP, causing the disappearance of aromatic and van der Waals interactions with 18K. It is important to note that the hexyl chain of 18K was less ordered during the MD simulation in the 18K–TCPTP complex, and this may have been due to the loss of interactions with Phe280. The IC₅₀ values of compound 18K were 0.25 µM for PTP1B, 1.24 µM for TCPTP, >100 µM for CD45, and >100 µM for leukocyte common antigen-related protein tyrosine phosphatase (LAR).

The C_max value of 18K reached 21.9 µg/mL (45.5 µM) in male ICR mice at 30 mg/kg, 25.8 µg/mL (53.6 µM) in male SD rats at 30 mg/kg, and 18.2 µg/mL (37.8 µM) in male beagles at 10 mg/kg, all of which exceeded its IC₅₀ value for PTP1B inhibition. Furthermore, compound 18K lowered plasma glucose, triglyceride (TG), and homeostasis model assessment (HOMA) levels at 30 mg/kg/d (per os (p.o.)) for one week in db/db mice (Table 4).

Table 4. Effects of the Repeated Administration of 18K at 30 mg/kg/d for One Week in Male db/db Mice

Compound	Glucose (mg/dL)	TG (mg/dL)	HOMA value
Control	280.0±38.9	151.7±8.9	2613.7±685.3
18K	182.8±7.2*	115.8±4.8**	894.1±117.2*

Mean±S.E. (n=6). * p<0.05, ** p<0.01 vs. Control, the Student’s t-test.

In conclusion, a series of benzoylsulfonamide derivatives were synthesized and a substituted benzoylsulfonamide structure was shown to be a novel scaffold for a non-competitive allosteric PTP1B inhibitor. Among the derivatives synthesized, compound 18K is a potential candidate for anti-diabetic drug as well as a useful tool for investigations of the physiological and pathophysiological effects of allosteric PTP1B inhibition.

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 nuclear magnetic resonance spectrometer at 400 MHz (JNM-AL400; JEOL Ltd., Tokyo, Japan) using tetramethylsilane (TMS) as an internal standard. IR spectra were recorded with an infrared spectrometer (FT-IR8200PC; Shimadzu Corporation, Kyoto, Japan). Mass spectra were obtained on a QTRAP LC/MS/MS system (API2000; Thermo Fisher Scientific Inc., Foster, 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).

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(1-ethoxyethylideneaminooxy)benzamide (3)

1,1′-Carbonylbis-1H-imidazole (CDI) (3.38 g, 19.7 mmol) was added to a solution of 1²³⁾ (4.00 g, 17.9 mmol) in N,N-dimethylformamide (DMF) (40 mL), and the mixture was stirred at room temperature for 1 h. 2,4-Dichlorobenzenesulfonamide 2A²⁴⁾ (4.46 g, 19.7 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) (2.95 mL, 19.7 mmol) were added to the reaction mixture, and stirred at room temperature for 5.5 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt and the organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was recrystallized from AcOEt–n-hexane to give 3 (6.51 g, 84% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.36 (3H, t, J=7.1 Hz), 2.11 (3H, s), 4.19 (2H, q, J=7.1 Hz), 7.15–7.20 (2H, m), 7.45–7.52 (2H, m), 7.72–7.79 (2H, m), 8.30 (1H, d, J=8.5 Hz).

Compound 4i was prepared from 5-(tert-butyldimethylsilanyloxymethyl)indole²⁶⁾ via compounds 24, 25, and 26 as follows:

5-(tert-Butyldimethylsilanyloxymethyl)-2,3-dihydro-1H-indole (24)

NaBH₃CN (5.34 g, 84.9 mmol) was added to a solution of 5-(tert-butyldimethylsilanyloxymethyl)indole²⁶⁾ (4.44 g, 17.0 mmol) in AcOH (31.1 mL) under ice-cooling, and the mixture was stirred at room temperature for 2.5 h. The reaction mixture was diluted with water, neutralized with K₂CO₃, 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 obtained was purified by silica gel column chromatography to give 24 (1.58 g, 35% yield) as an oil. ¹H-NMR (CDCl₃) δ: 0.08 (6H, s), 0.93 (9H, s), 3.01 (2H, t, J=8.3 Hz), 3.54 (2H, t, J=8.3 Hz), 4.61 (2H, s), 6.59 (1H, d, J=7.8 Hz), 6.95 (1H, d, J=7.8 Hz), 7.06–7.11 (1H, m).

1-[5-(tert-Butyldimethylsilanyloxymethyl)]-2,2,2-trifluoroacetyl-2,3-dihydro-1H-indole (25)

Triethylamine (Et₃N) (7.09 mL, 7.80 mmol) and trifluoroacetic anhydride (TFAA) (0.92 mL, 6.6 mmol) were added to a solution of 24 (1.58 g, 6.00 mmol) in CH₂Cl₂ (16 mL), and the mixture was stirred at room temperature for 30 min. The reaction mixture was diluted with water and neutralized with K₂CO₃, which was extracted twice with AcOEt. The organic layer was washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 25 (1.94 g, 90% yield) as an oil. ¹H-NMR (CDCl₃) δ: 0.10 (6H, s), 0.94 (9H, s), 3.25 (2H, t, J=8.1 Hz), 4.28 (2H, t, J=8.1 Hz), 4.71 (2H, s), 7.19 (1H, d, J=8.3 Hz), 7.25 (1H, s), 8.14 (1H, d, J=8.3 Hz).

1-(2,2,2-Trifluoroacetyl)-5-hydroxymethyl-2,3-dihydro-1H-indole (26)

One molar TBAF in tetrahydrofuran (THF) (5.8 mL, 5.8 mmol) was added to a solution of 25 (1.75 g, 4.87 mmol) in THF (35 mL), and the mixture was stirred at room temperature for 75 min. After the addition of water, the mixture was extracted twice with AcOEt, and the organic layer was washed with saturated brine and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 26 (670 mg, 56% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.65–1.78 (1H, br), 3.26 (2H, t, J=8.0 Hz), 4.30 (2H, t, J=8.0 Hz), 4.65–4.72 (2H, m), 7.20–7.32 (2H, m), 8.17 (1H, d, J=8.3 Hz).

1-(2,2,2-Trifluoroacetyl)-2,3-dihydro-1H-indole-5-carbaldehyde (4i)

Dimethyl sulfoxide (DMSO) (0.39 mL, 5.5 mmol), Et₃N (0.76 mL, 5.5 mmol), and SO₃·pyridine (869 mg, 5.46 mmol) were added to a stirred solution of 26 (670 mg, 2.73 mmol) in CH₂Cl₂ (6.7 mL), followed by stirring at room temperature for 2 h. After the addition of 10% aqueous citric acid solution, the mixture was extracted twice with AcOEt, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 4i (340 mg, 51% yield) as a solid. ¹H-NMR (CDCl₃) δ: 3.34 (2H, t, J=8.3 Hz), 4.38 (2H, t, J=8.3 Hz), 7.77–7.84 (2H, m), 8.35 (1H, d, J=8.3 Hz), 9.95 (1H, s).

Compound 4k was prepared from 2,3-dihydro-1H-indole via compound 27 as follows:

1-Ethyl-2,3-dihydro-1H-indole (27)

EtI (2.42 mL, 30.2 mmol) and i-Pr₂NEt (5.29 mL, 37.8 mmol) were added to a solution of 2,3-dihydro-1H-indole (3.00 g, 25.2 mmol) in DMF (2.4 mL), which was stirred at 80°C for 14.5 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 27 (3.32 g, 90% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.20 (3H, t, J=7.4 Hz), 2.94 (2H, t, J=8.0 Hz), 3.15 (2H, q, J=7.4 Hz), 3.32 (2H, t, J=8.0 Hz), 6.45–6.52 (1H, m), 6.60–6.68 (1H, m), 7.00–7.09 (2H, m).

1-Ethyl-2,3-dihydro-1H-indole-5-carbaldehyde (4k)

Vilsmeier reagent was prepared by adding POCl₃ (6.29 mL, 67.4 mmol) dropwise to DMF (8.5 mL, 112 mmol) under ice-cooling, and the mixture was stirred at the same temperature for 15 min. Vilsmeier reagent was added to a solution of 27 (3.31 g, 22.5 mmol) in DMF (112 mL), followed by stirring at 40°C for 45 min. The reaction mixture was poured into cold water and basified with K₂CO₃, which was extracted twice with AcOEt. The organic layer was dried over Na₂SO₄ and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give 4k (2.32 g, 59% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.21 (3H, t, J=7.3 Hz), 3.03 (2H, t, J=8.6 Hz), 3.28 (2H, q, J=7.3 Hz), 3.59 (2H, t, J=8.6 Hz), 6.34–6.40 (1H, m), 7.50–7.57 (2H, m), 9.64 (1H, s).

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(4-isopropylbenzylideneaminooxy)benzamide (5a)

Compound 4a (172 mg, 1.16 mmol) and 60% aqueous HClO₄ solution (0.19 mL, 1.7 mmol) were added to a solution of 3 (500 mg, 1.16 mmol) in 1,4-dioxane (17 mL) under ice-cooling, and the mixture was stirred at room temperature for 14 h. AcOEt was added to the reaction mixture, which was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of Et₂O (10 mL) and n-hexane (20 mL), the mixture was stirred at room temperature for 3 h, and the precipitate was then collected by filtration to give 5a (502 mg, 88% yield) as a white solid, mp 90–91°C. ¹H-NMR (CDCl₃) δ: 1.27 (6H, d, J=6.8 Hz), 2.90–3.03 (1H, m), 7.30 (2H, d, J=8.0 Hz), 7.27–7.35 (2H, m), 7.49 (1H, dd, J=8.6, 2.0 Hz), 7.52 (1H, d, J=2.0 Hz), 7.64 (2H, d, J=8.0 Hz), 7.78–7.86 (2H, m), 8.32 (1H, d, J=8.6 Hz), 8.40 (1H, s), 8.99–9.42 (1H, br). IR (attenuated total reflectance (ATR)) cm⁻¹: 1678. MS m/z: 513 [M+Na]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(4-hydroxybenzylideneaminooxy)benzamide (5c)

Sixty percent aqueous HClO₄ solution (5.8 mL, 53 mmol) was added to a solution of 3 (500 mg, 1.16 mmol) and 4-acetoxybenzaldehyde 4b (190 mg, 1.16 mmol) in 1,4-dioxane (10 mL), and the mixture was stirred at room temperature for 14 h. After the addition of 5.0 M aqueous NaOH solution (1.2 mL, 6.0 mmol), the reaction mixture was stirred at room temperature for 2 h. The mixture was acidified by 5% citric acid solution and extracted twice with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 5c (296 mg, 55% yield) as a pale brown solid, mp 154–157°C. ¹H-NMR (DMSO-d₆) δ: 6.84–6.88 (2H, m), 7.29–7.32 (2H, m), 7.63–7.66 (2H, m), 7.72 (1H, dd, J=8.8, 2.2 Hz), 7.89 (1H, d, J=2.0 Hz), 7.94–7.97 (2H, m), 8.16 (1H, d, J=8.6 Hz), 8.63 (1H, s), 10.11 (1H, s), 12.60–13.20 (1H, br). IR (ATR) cm⁻¹: 1708. MS m/z: 487 [M+Na]⁺.

Methyl (E)-4-{[4-(2,4-Dichlorobenzenesulfonylaminocarbonyl)phenoxyimino]methyl}benzoate (5d)

Compound 5d was synthesized from 3 and 4d according to the procedure for the synthesis of 5a. Yield 93%.¹H-NMR (CDCl₃) δ: 3.95 (3H, s), 7.30–7.37 (2H, m), 7.46–7.54 (2H, m), 7.75–7.85 (4H, m), 8.07–8.14 (2H, m), 8.32 (1H, d, J=8.6 Hz), 8.47 (1H, s), 8.90–9.30 (1H, br).

(E)-4-{[4-(2,4-Dichlorobenzenesulfonylaminocarbon‑yl)phenoxyimino]methyl}benzoic Acid (5e)

One molar aqueous LiOH solution (3.3 mL, 3.3 mmol) was added to a solution of 5d (550 mg, 1.08 mmol) in MeOH (3.0 mL) and THF (9.0 mL), and the mixture was stirred at room temperature for 14 h. The reaction mixture was neutralized with 2.0 M aqueous HCl solution and extracted twice with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of Et₂O, the precipitate was collected by filtration to give 5e (440 mg, 89% yield) as a white solid, mp 208–211°C. ¹H-NMR (DMSO-d₆) δ: 7.35–7.40 (2H, m), 7.72 (1H, dd, J=8.6, 1.7 Hz), 7.88 (1H, d, J=1.7 Hz), 7.92–8.02 (4H, m), 8.04 (1H, d, J=8.6 Hz), 8.15–8.19 (2H, m), 8.87 (1H, s), 12.70–13.50 (2H, br). IR (ATR) cm⁻¹: 1687. MS m/z: 491 [M−H]⁻.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-[4-(N′-2,2,2-trifluoroacetylamino)benzylideneaminooxy]benzamide (5f)

Compound 5f was synthesized from 3 and 4f²⁵⁾ according to the procedure for the synthesis of 5a. Yield quant. ¹H-NMR (CDCl₃) δ: 7.30–7.35 (2H, m), 7.49 (1H, dd, J=8.6, 2.0 Hz), 7.52 (1H, d, J=2.0 Hz), 7.67–7.73 (2H, m), 7.74–7.84 (4H, m), 8.07–8.17 (1H, br), 8.31 (1H, d, J=8.6 Hz), 8.42 (1H, s), 8.95–9.12 (1H, br).

(E)-N-[4-(4-Aminobenzylideneaminooxy)benzoyl]-2,4-dichlorobenzamide Hydrochloride Salt (5g)

One molar aqueous LiOH solution (6.8 mL, 6.8 mmol) was added to a solution of 5f (1.27 g, 2.27 mmol) in 1,4-dioxane (13 mL), and the mixture was stirred at room temperature for 2.5 h and at 40°C for 25 min. After the addition of water, the mixture was extracted three times with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

A total of 8.6 M HCl in i-PrOH (0.40 mL, 3.4 mmol) and Et₂O (20 mL) were added to a solution of the residue obtained in MeOH (2 mL) under ice-cooling, and the reaction mixture was stirred at the same temperature for 1 h. After the addition of MeOH (8 mL) and Et₂O (70 mL), the mixture was further stirred under ice-cooling for 1 h. The precipitate was collected by filtration to give 5g (435 mg, 38% yield) as a brown solid, mp 145–150°C. ¹H-NMR (DMSO-d₆) δ: 4.35–6.01 (3H, br), 7.05–7.13 (2H, m), 7.29–7.37 (1H, m), 7.65–7.80 (3H, m), 7.87–7.93 (1H, m), 7.97 (2H, m), 8.17 (1H, d, J=8.3 Hz), 8.66 (1H, s). IR (ATR) cm⁻¹: 1676. MS m/z: 464 [M+H]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(4-chlorobenzylideneaminooxy)benzamide (5h)

Compound 5h was synthesized from 3 and 4h according to the procedure for the synthesis of 5a. Yield 71%. A white solid, mp 177–179°C. ¹H-NMR (CDCl₃) δ: 7.28–7.37 (2H, m), 7.38–7.48 (2H, m), 7.49 (1H, dd, J=8.5, 2.0 Hz), 7.52 (1H, d, J=2.0 Hz), 7.61–7.72 (2H, m), 7.76–7.87 (2H, m), 8.32 (1H, d, J=8.5 Hz), 8.40 (1H, s), 8.80–9.18 (1H, br). IR (ATR) cm⁻¹: 1699. MS m/z: 505 [M+Na]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-[1-(2,2,2-trifluoroacetyl)-2,3-dihydro-1H-indole-5-ylmethyleneaminooxy]benzamide (5i)

Compound 5i was synthesized from 3 and 4i according to the procedure for the synthesis of 5a. Yield 78%. ¹H-NMR (CDCl₃) δ: 3.33 (2H, t, J=8.3 Hz), 4.36 (2H, t, J=8.3 Hz), 7.29–7.36 (2H, m), 7.45–7.59 (3H, m), 7.72 (1H, s), 7.76–7.84 (2H, m), 8.27 (1H, d, J=8.3 Hz), 8.33 (1H, d, J=8.5 Hz), 8.41 (1H, s), 8.81–9.08 (1H, br).

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(indolin-5-ylmethyleneaminooxy)benzamide Hydrochloride Salt (5j)

One molar aqueous LiOH solution (1.4 mL, 1.4 mmol) was added to a solution of 5i (540 mg, 0.899 mmol) in 1,4-dioxane (5.4 mL), which was stirred at room temperature for 45 min. One molar aqueous LiOH solution (1.4 mL, 1.4 mmol) was then added to the reaction mixture, which was further stirred at the same temperature for 50 min. After water was added, the mixture was extracted twice with AcOEt. The organic layer was washed with saturated brine and dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue was purified by column chromatography to give the free form of 5j.

A total of 8.6 M HCl in i-PrOH (0.07 mL, 0.6 mmol) was added to a solution of the residue obtained in CHCl₃ (10 mL) and MeOH (3 mL) under ice-cooling, and the mixture was stirred at the same temperature for 10 min. After the addition of Et₂O (40 mL), the mixture was stirred under ice-cooling for 1 h. The precipitate was collected by filtration to give 5j (14 mg, 14% yield) as a brown solid, mp 145–150°C. ¹H-NMR (DMSO-d₆) δ: 3.09 (2H, t, J=8.3 Hz), 3.62 (2H, t, J=8.3 Hz), 3.95–5.40 (3H, m), 6.92 (1H, d, J=7.8 Hz), 7.29–7.35 (2H, m), 7.51 (1H, d, J=7.8 Hz), 7.67 (1H, s), 7.73 (1H, dd, J=8.5 Hz, 1.7 Hz), 7.90 (1H, d, J=1.7 Hz), 7.93–7.99 (2H, m), 8.17 (1H, d, J=8.5 Hz), 8.64 (1H, s). IR (ATR) cm⁻¹: 1687. MS m/z: 490 [M−H]⁻.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(1-ethyl-2,3-dihydro-1H-indol-5-ylmethyleneaminooxy)benzamide (5k)

Compound 5k was synthesized from 3 and 4k according to the procedure for the synthesis of 5a. Yield 65%. A white solid, mp 135–137°C. ¹H-NMR (DMSO-d₆) δ: 1.19 (3H, t, J=7.3 Hz), 3.02 (2H, t, J=8.3 Hz), 3.22 (2H, q, J=7.3 Hz), 3.49 (2H, t, J=8.3 Hz), 6.38 (1H, d, J=8.1 Hz), 7.25–7.30 (3H, m), 7.45–7.53 (3H, m), 7.75–7.80 (2H, m), 8.25–8.33 (2H, m), 8.95–9.17 (1H, br). IR (ATR) cm⁻¹: 1693. MS m/z: 518 [M+H]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(1H-indol-5-ylmethyleneaminooxy)benzamide (5m)

Compound 5m was synthesized from 3 and 4l²⁷⁾ according to the procedure for the synthesis of 5a. Yield 68%. A white solid, mp 172–173°C. ¹H-NMR (DMSO-d₆) δ: 6.52–6.57 (1H, m), 7.32–7.38 (2H, m), 7.41–7.47 (1H, m), 7.49 (1H, d, J=8.6 Hz), 7.63 (1H, dd, J=8.6, 1.4 Hz), 7.73 (1H, dd, J=8.8, 2.2 Hz), 7.90 (1H, d, J=2.2 Hz), 7.95–8.01 (3H, m), 8.17 (1H, d, J=8.8 Hz), 8.79 (1H, s), 11.39–11.47 (1H, br), 12.65–13.25 (1H, br). IR (ATR) cm⁻¹: 1693. MS m/z: 510 [M+Na]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(benzoimidazol-5-ylmethyleneaminooxy)benzamide (5o)

Compound 5o was synthesized from 3 and 4n²⁸⁾ according to the procedure for the synthesis of 5a. Yield 56%. A pale brown solid, mp 150–155°C. ¹H-NMR (DMSO-d₆) δ: 7.31–7.38 (2H, m), 7.67–7.74 (2H, m), 7.75–7.82 (1H, m), 7.85 (1H, d, J=2.0 Hz), 7.94–8.00 (2H, m), 8.05 (1H, s), 8.15 (1H, d, J=8.6 Hz), 8.49–8.53 (1H, s), 8.89 (1H, s). IR (ATR) cm⁻¹: 1598. MS m/z: 489 [M+H]⁺.

N-(4-Aminooxybenzoyl)-2,4-dichlorobenzenesulfonamide (7)

Sixty percent aqueous HClO₄ solution (0.39 mL, 3.6 mmol) was added to a solution of 3 (1.00 g, 2.32 mmol) in 1,4-dioxane (10 mL), which was stirred at room temperature for 14 h. After the addition of 5.0 M aqueous NaOH solution (0.46 mL, 2.3 mmol), the reaction mixture was extracted twice with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of i-Pr₂O, the precipitate that formed was collected by filtration to give 7 (890 mg, quant.) as a solid. ¹H-NMR (CDCl₃) δ: 4.05–4.14 (2H, m), 7.50–7.59 (2H, m), 7.69 (1H, dd, J=8.5, 2.2 Hz), 7.84 (1H, d, J=2.2 Hz), 7.90–7.94 (2H, m), 8.13 (1H, d, J=8.5 Hz), 8.20–8.40 (3H, br).

N-(2,4-Dichlorobenzenesulfonyl)-4-aminomethylbenzamide Hydrochloride Salt (8)

CDI (948 mg, 5.84 mmol) was added to a solution of 6²⁹⁾ (1.22 g, 4.87 mmol) in DMF (10 mL), and the mixture was stirred at room temperature for 1 h. After the addition of 2,4-dichlorobenzenesulfonamide 2A (1.10 g, 4.87 mmol) and DBU (0.87 mL, 5.8 mmol), the reaction mixture was stirred at room temperature for 16 h. The reaction mixture was acidified with 5% aqueous citric acid solution, and extracted twice 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 obtained was dissolved in HCO₂H (5.0 mL), to which 8.6 M HCl in i-PrOH (1.3 mL, 11 mmol) was added under ice-cooling, and this was followed by stirring at the same temperature for 10 min. The precipitate was collected by filtration to give 8 (880 mg, quant.) as a solid. ¹H-NMR (DMSO-d₆) δ: 4.05–4.12 (2H, m), 7.51–7.58 (2H, m), 7.64–7.73 (1H, m), 7.81–7.86 (1H, m), 7.89–7.95 (2H, m), 8.12–8.15 (1H, m), 8.20–8.40 (3H, m).

N-[4-(2,4-Dichlorobenzenesulfonylaminocarbonyl)phenoxy]-4-isopropylbenzamide (10a)

Compound 9a (175 mg, 1.07 mmol) and 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) (241 mg, 1.26 mmol) were added to a solution of 7 in CH₂Cl₂ (2.5 mL), and the mixture was stirred at room temperature for 14 h. After the addition of 5% aqueous citric acid solution, the reaction mixture was extracted twice with AcOEt, washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was dissolved in AcOEt and n-hexane was added to the solution. The precipitate was collected by filtration to give 10a (182 mg, 37%) as a white solid, mp 208–210°C. ¹H-NMR (DMSO-d₆) δ: 1.21 (6H, d, J=6.8 Hz), 2.94 (1H, quintet, J=7.1 Hz), 4.51 (2H, d, J=6.1 Hz), 7.32–7.35 (2H, m), 7.37–7.42 (2H, m), 7.71 (1H, dd, J=8.8, 2.2 Hz), 7.79–7.83 (2H, m), 7.83–7.88 (3H, m), 8.15 (1H, d, J=8.8 Hz), 9.02 (1H, t, J=6.1 Hz). IR (ATR) cm⁻¹: 1697. MS m/z: 529 [M+Na]⁺.

N-[4-(2,4-Dichlorobenzenesulfonylaminocarbonyl)benzyl]-4-isopropylbenzamide (11a)

(COCl)₂ (80 µL, 0.93 mmol) and 1 drop of DMF were added to a solution of 9a (151 mg, 0.917 mmol) in CH₂Cl₂ (5.0 mL), and the mixture was stirred at room temperature for 15 min. After the addition of 8 (330 mg, 0.834 mmol) and i-Pr₂NEt (0.71 mL, 4.2 mmol), the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was acidified with 5% aqueous citric acid solution, which was extracted twice 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 obtained was dissolved in AcOEt, to which n-hexane was added, and the precipitate was collected by filtration to give 11a (240 mg, 57%) as a white solid, mp 225–227°C. ¹H-NMR (DMSO-d₆) δ: 1.23 (6H, d, J=7.1 Hz), 2.97 (1H, quintet, J=7.1 Hz), 7.15–7.22 (2H, m), 7.36–7.44 (2H, m), 7.72 (1H, dd, J=8.6, 2.0 Hz), 7.78–7.85 (2H, m), 7.89 (1H, d, J=2.0 Hz), 7.90–7.96 (2H, m), 8.16 (1H, d, J=8.6 Hz), 12.57 (1H, s), 12.70–13.20 (1H, br). IR (ATR) cm⁻¹: 1658. MS m/z: 527 [M+Na]⁺.

4-(4-Isopropylbenzyloxymethyl)benzoic Acid (14a)

A 60% suspension of NaH in mineral oil (800 mg, 20 mmol) was added portionwise to a stirred solution of 12a (3.00 g, 20.0 mmol) in THF (20 mL), which was stirred at 50°C for 30 min. After the addition of 13 (2.29 g, 10.0 mmol) in THF (10 mL), the reaction mixture was stirred at room temperature for 1.5 h, and 5.0 M aqueous NaOH solution (10 mL, 50 mmol) and MeOH (10 mL) were then added to the mixture, which was stirred at 50°C for 30 min. After cooling, water and Et₂O were added to the reaction mixture, the aqueous layer was then acidified with concentrated hydrochloric acid, followed by extraction 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 obtained was purified by silica gel column chromatography to give 14a (1.19 g, 42% yield for 2 steps) as a solid. ¹H-NMR (CDCl₃) δ: 1.25 (6H, d, J=6.8 Hz), 2.91 (1H, septet, J=6.8 Hz), 4.56 (2H, s), 4.62 (2H, s), 7.21–7.32 (4H, m), 7.45–7.52 (2H, m), 8.07–8.13 (2H, m).

Compound 14p was prepared from ethyl 4-iodobenzoate via compound 28 as follows:

Ethyl 4-(4-Methylpent-1-enyl)benzoate (28)

A mixture of ethyl 4-iodobenzoate (7.35 g, 26.6 mmol), 4-methyl-1-pentene (3.36 g, 39.9 mmol), Pd(OAc)₂ (119 mg, 0.532 mmol), tri(o-tolyl)phosphine (405 mg, 1.33 mmol), and Et₃N (13.0 mL, 93.3 mmol) in toluene (43 mL) was stirred at 80°C for 16 h under a nitrogen atmosphere. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 28 (6.69 g, quant.) as an oil. ¹H-NMR (CDCl₃) δ: 0.95 (6H, d, J=6.6 Hz), 1.26 (3H, t, J=7.1 Hz), 1.69–1.82 (1H, m), 2.12 (2H, t, J=7.1 Hz), 4.12 (2H, q, J=7.1 Hz), 6.35–6.43 (2H, m), 7.36–7.41 (2H, m), 7.92–7.98 (2H, m).

[4-(4-Methylpent-1-enyl)benzyloxymethyl]benzoic Acid (14p)

Compound 28 (6.69 g, 28.8 mmol) in THF (20 mL) was added dropwise to the suspension of lithium aluminium hydride (LiAlH₄) (1.31 g, 34.6 mmol) in THF (134 mL), which was stirred at room temperature for 15 min. After the addition of water under ice-cooling, the precipitate was removed by filtration and washed with AcOEt. The filtrate was dried over Na₂SO₄ and then evaporated under reduced pressure to give 12p (5.61 g, crude).

A 60% suspension of NaH in mineral oil (1.91 g, 48 mmol) was added portionwise to the crude product (3.64 g) and compound 13 (2.19 g, 9.55 mmol) in THF (44 mL) under ice-cooling, and the mixture was stirred at room temperature for 18.5 h. After the addition of 2.0 M aqueous NaOH solution (23.9 mL, 48 mmol), the reaction mixture was stirred at 50°C for 2.5 h, and water and Et₂O were then added. The aqueous layer was acidified with 10% aqueous citric acid solution, followed by extraction with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was recrystallized from AcOEt and n-hexane to give 14p (1.61 g, 52% yield for 3 steps) as a solid. ¹H-NMR (CDCl₃) δ: 0.94 (6H, d, J=6.6 Hz), 1.66–1.80 (1H, m), 2.10 (2H, t, J=7.3 Hz), 4.57 (2H, s), 4.61 (2H, s), 6.23 (1H, dt, J=15.8, 7.3 Hz), 6.36 (1H, d, J=15.8 Hz), 7.26–7.37 (4H, m), 7.43–7.49 (2H, m), 8.05–8.12 (2H, m).

Compound 14q was prepared from ethyl 4-iodobenzoate via compound 29 as follows:

Ethyl 4-(5-Methyl-1-hexenyl)benzoate (29)

Compound 29 was synthesized according to the procedure for the synthesis of 28. Yield quant. ¹H-NMR (CDCl₃) δ: 0.92 (6H, d, J=6.6 Hz), 1.34–1.42 (5H, m), 1.56–1.66 (1H, m), 2.19–2.28 (2H, m), 4.36 (2H, q, J=7.1 Hz), 6.30–6.40 (2H, m), 7.34–7.41 (2H, m), 7.91–7.98 (2H, m).

4-[4-(5-Methyl-1-hexenyl)benzyloxymethyl]benzoic Acid (14q)

Compound 14q was synthesized from 29 according to the procedure for the synthesis of 14p. Yield 13% for 3 steps. ¹H-NMR (CDCl₃) δ: 0.91 (6H, d, J=6.6 Hz), 1.31–1.40 (2H, m), 1.56–1.67 (1H, m), 2.16–2.26 (2H, m), 4.56 (2H, s), 4.60 (2H, s), 6.17–6.28 (1H, m), 6.38 (1H, d, J=15.8 Hz), 7.26–7.37 (4H, m), 7.43–7.49 (2H, m), 8.05–8.12 (2H, m).

4-(Biphenyl-4-ylmethoxymethyl)benzoic Acid (14r)

Compound 14r was synthesized from 12r and 13 according to the procedure for the synthesis of 14a. Yield 61% for 2 steps. ¹H-NMR (CDCl₃) δ: 4.64 (2H, s), 4.67 (2H, s), 7.31–7.39 (1H, m), 7.40–7.63 (10H, m), 8.08–8.15 (2H, m).

4-(4-Styrylbenzyloxymethyl)benzoic Acid (14s)

Compound 14s was synthesized from 12s³⁰⁾ and 13 according to the procedure for the synthesis of 14a. Yield 61% for 2 steps. ¹H-NMR (DMSO-d₆) δ: 4.57 (2H, s), 4.62 (2H, s), 7.24–7.30 (3H, m), 7.34–7.41 (4H, m), 7.45–7.52 (2H, m), 7.57–7.64 (4H, m), 7.91–7.96 (2H, m), 12.60–13.15 (1H, br).

Compound 14t was prepared from methyl 4-iodobenzoate via compound 30 as follows:

Methyl 4-(4-Phenylethynylbenzyloxymethyl)benzoate (30)

Ag₂O (4.00 g, 17.3 mmol) was added to a solution of 12t³¹⁾ (1.20 g, 5.76 mmol) and 13 (1.32 g, 5.76 mmol) in toluene (20 mL), and the mixture was stirred at 60°C for 2 h under an argon atmosphere. After the removal of Ag₂O by filtration, the filtrate was concentrated under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 30 (250 mg, 12%) as a solid. ¹H-NMR (CDCl₃) δ: 3.91 (3H, s), 4.58 (2H, s), 4.61 (2H, s), 7.32–7.38 (5H, m), 7.40–7.45 (2H, m), 7.50–7.56 (4H, m), 8.00–8.05 (2H, m).

4-(4-Phenylethynylbenzyloxymethyl)benzoic Acid (14t)

One molar aqueous LiOH solution (3.4 mL, 3.4 mmol) was added to a solution of 30 (250 mg, 0.701 mmol) in MeOH (2.0 mL) and THF (2.0 mL), and the mixture was stirred at 60°C for 2 h. The reaction mixture was neutralized with 1.0 M aqueous HCl solution, which was concentrated under reduced pressure. After the addition of water, the precipitate was collected by filtration to give 14t (200 mg, 83% yield) as a solid. ¹H-NMR (CDCl₃) δ: 4.59 (2H, s), 4.63 (2H, s), 7.32–7.38 (5H, m), 7.42–7.47 (2H, m), 7.50–7.56 (4H, m), 8.01–8.06 (2H, m).

9H-Fluorene-2-sulfonamide (2G)

Concentrated hydrochloric acid (27 mL) was added to a solution of 9H-fluoreneyl-2-amine (3.00 g, 16.6 mmol) in MeCN (38 mL) at −5°C, and the mixture was stirred at the same temperature for 10 min. NaNO₂ (1.37 g, 19.9 mmol) was dissolved in water (3.8 mL), which was added dropwise at −5°C to the above reaction mixture. After stirring for 5 min, 30% SO₂ in AcOH (42.5 mL) and 2.2 M aqueous CuCl₂ solution (3.8 mL, 8.4 mmol) were added dropwise at −5°C to the reaction mixture, followed by stirring at room temperature for 45 h. After the addition of water, the mixture was extracted twice 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 obtained was dissolved in THF (40 mL), to which 28% aqueous NH₃ solution (7.1 mL, 83 mmol) was added, and the mixture was stirred at room temperature for 10 min. AcOEt was added to the reaction mixture, which was washed with 10% aqueous citric acid solution, water, and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue was rinsed with Et₂O to give 2G (2.47 g, 61% yield for 2 steps) as a solid. ¹H-NMR (DMSO-d₆) δ: 4.04 (2H, s), 7.32–7.36 (2H, br), 7.37–7.46 (2H, m), 7.63–7.65 (1H, m), 7.84–7.88 (1H, m), 7.98–8.01 (1H, m), 8.02–8.03 (1H, m), 8.06–8.09 (1H, m).

Compound 2I was prepared from 5-benzyloxypentylbromide via compounds 31, 32, 33, and 2J as follows:

S-(5-Benzyloxypentyl)thioacetate (31)

AcSK (2.23 g, 19.5 mmol) was added to a stirred solution of 5-benzyloxypentylbromide (2.51 g, 9.76 mmol) in acetone (25 mL), and the reaction mixture was stirred at room temperature for 19 h. After the addition of water, the mixture was extracted twice with AcOEt, washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 31 (2.40 g, 97% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.38–1.50 (2H, m), 1.55–1.68 (4H, m), 2.32 (3H, s), 2.87 (2H, t, J=7.3 Hz), 3.46 (2H, t, J=6.6 Hz), 4.49 (2H, s), 7.26–7.37 (5H, m).

5-(2-Chlorobenzyloxy)pentane-1-sulfonamide (32) and 5-Benzyloxypentane-1-sulfonamide (33)

AcONa (7.80 g, 95.1 mmol) was added to a stirred solution of 31 (2.40 g, 9.51 mmol) in AcOH (35 mL) and water (7 mL), and the mixture was bubbled with Cl₂ gas under ice-cooling for 30 min. The reaction mixture was degassed by purging with argon, which was diluted with water and extracted with CHCl₃. The organic layer was washed with 10% aqueous Na₂S₂O₃ solution and saturated aqueous NaHCO₃ solution, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

The residue obtained was dissolved in THF (60 mL), to which 28% NH₃ solution (3.3 mL, 54 mmol) was added, followed by stirring at room temperature for 14 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt and the organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 32 (370 mg, 12% yield) as an oil and 33 (270 mg, 9.7% yield) as an oil.

Compound 32: ¹H-NMR (CDCl₃) δ: 1.52–1.63 (2H, m), 1.65–1.75 (2H, m), 1.85–1.95 (2H, m), 3.08–3.17 (2H, m), 3.56 (2H, t, J=5.9 Hz), 4.48–4.58 (2H, br), 4.58 (2H, s), 7.17–7.38 (3H, m), 7.42–7.49 (1H, m).

Compound 33: ¹H-NMR (CDCl₃) δ: 1.49–1.60 (2H, m), 1.61–1.72 (2H, m), 1.83–1.95 (2H, m), 3.06–3.18 (2H, m), 3.47 (2H, t, J=6.1 Hz), 4.45 (2H, s), 4.51–4.58 (2H, br), 7.21–7.38 (5H, m).

5-(tert-Butyldimethylsilanyloxy)pentane-1-sulfonamide (2I) and 5-Hydroxypentane-1-sulfonamide (2J)

A solution of 32 (370 mg, 1.27 mmol) in MeOH (7.5 mL) was hydrogenated at 0.4 MPa in the presence of Pd–C (40 mg) at room temperature for 22 h. After removal of the catalyst by filtration, the filtrate was evaporated under reduced pressure to give 5-hydroxypentane-1-sulfonamide 2J (150 mg) as an oil.

Compound 33 (270 mg, 1.05 mmol) was hydrogenated as described above to give 5-hydroxypentane-1-sulfonamide 2J (220 mg) as an oil.

Imidazole (196 mg, 2.87 mmol) and tert-butylchlorodimethylsilane (TBSCl) (350 mg, 2.32 mmol) were added to a solution of 5-hydroxypentane-1-sulfonamide (370 mg, 2.21 mmol) in CH₂Cl₂ (4.0 mL), and the mixture was stirred at room temperature for 3 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt and the organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography to give 2I (490 mg, 75% yield for 2 steps) as an oil. ¹H-NMR (CDCl₃) δ: 0.05 (6H, s), 0.89 (9H, s), 1.45–1.65 (4H, m), 1.83–1.97 (2H, m), 3.09–3.22 (2H, m), 3.62 (2H, t, J=5.9 Hz), 4.47–4.66 (2H, br).

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-(4-isopropylbenzyloxymethyl)benzamide Sodium Salt (15aA)

CDI (116 mg, 0.715 mmol) was added to a solution of 14a (170 mg, 0.570 mmol) in DMF (2.0 mL), and the mixture was stirred at room temperature for 1 h. After the addition of 2,4-dichlorobenzenesulfonamide 2A (162 mg, 0.716 mmol) and DBU (0.11 mL, 0.72 mmol), the reaction mixture was stirred at room temperature for 14 h. The mixture was acidified with 10% aqueous citric acid solution, followed by extraction with AcOEt and the organic layer was washed with saturated aqueous NaHCO₃ solution and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of Et₂O, the precipitate was collected by filtration to give 15aA (125 mg, 43% yield) as a white solid, mp 185–190°C. ¹H-NMR (DMSO-d₆) δ: 1.18 (6H, d, J=6.8 Hz), 2.82–2.92 (1H, m), 4.46 (2H, s), 4.51 (2H, s), 7.18–7.30 (6H, m), 7.82–7.88 (2H, m), 7.45 (1H, dd, J=8.6, 2.0 Hz), 7.50 (1H, d, J=2.0 Hz), 7.98 (1H, d, J=8.6 Hz). IR (ATR) cm⁻¹: 1593. MS m/z: 514 [M+Na]⁺.

(E)-N-(2,4-Dichlorobenzenesulfonyl)-4-[4-(4-methylpent-1-enyl)benzyloxymethyl]benzamide Sodium Salt (15 pA)

CDI (275 mg, 1.69 mmol) was added to a solution of 14p (500 mg, 1.54 mmol) in DMF (5.0 mL), and the mixture was stirred at room temperature for 1 h. 2,4-Dichlorobenzenesulfonamide 2A (382 mg, 1.69 mmol) and DBU (0.25 mL, 1.7 mmol) were added to the reaction mixture, followed by stirring at room temperature for 13.5 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt and the organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give the free form of 15pA.

Five molar MeONa in MeOH (0.31 mL, 0.15 mmol) was added to a solution of the residue obtained in THF (10 mL) under ice-cooling, and the mixture was stirred at the same temperature for 5 min. Et₂O (50 mL) and n-hexane (20 mL) were added to the mixture and the precipitate was collected by filtration to give 15 pA (450 mg, 53% yield for 2 steps) as a white solid, mp 190–191°C. ¹H-NMR (DMSO-d₆) δ: 0.91 (6H, d, J=6.6 Hz), 1.62–1.79 (1H, m), 2.04–2.11 (2H, m), 4.49 (2H, s), 4.52 (2H, s), 6.27 (1H, dt, J=15.8, 6.8 Hz), 6.38 (1H, d, J=15.8 Hz), 7.25–7.31 (4H, m), 7.35–7.40 (2H, m), 7.46 (1H, dd, J=8.5, 2.2 Hz), 7.51 (1H, d, J=2.2 Hz), 7.83–7.88 (2H, m), 7.99 (1H, d, J=8.5 Hz). IR (ATR) cm⁻¹: 1593, 1554, 1332. MS m/z: 554 [M+Na]⁺.

N-(2,4-Dichlorobenzenesulfonyl)-4-[4-(5-methyl-1-hexenyl)benzyloxymethyl]benzamide Sodium Salt (15qA)

Compound 15qA was synthesized from 14q and 2A according to the procedure for the synthesis of 15aA. Yield 87% for 2 steps. A white solid, mp 165–175°C. ¹H-NMR (DMSO-d₆) δ: 0.85 (6H, d, J=6.6 Hz), 1.25–1.32 (2H, m), 1.48–1.58 (1H, m), 2.10–2.19 (2H, m), 4.44 (2H, s), 4.82 (2H, s), 6.23 (1H, dt, J=16.1, 6.8 Hz), 6.35 (1H, d, J=16.1 Hz), 7.21–7.28 (4H, m), 7.30–7.35 (2H, m), 7.44 (1H, dd, J=8.6, 2.2 Hz), 7.49 (1H, d, J=2.2 Hz), 7.80–7.86 (2H, m), 7.96 (1H, d, J=8.6 Hz). IR (ATR) cm⁻¹: 1655. MS m/z: 568 [M+Na]⁺.

N-(2,4-Dichlorobenzesulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rA)

Compound 15rA was synthesized from 14r and 2A according to the procedure for the synthesis of 15aA. Yield 69%. A white solid, mp 114–115°C. ¹H-NMR (CDCl₃) δ: 4.56–4.65 (4H, m), 7.31–7.55 (9H, m), 7.55–7.63 (4H, m), 7.76–7.83 (2H, m), 8.31 (1H, d, J=8.6 Hz), 8.80–9.50 (1H, br). IR (ATR) cm⁻¹: 1693, 1610. MS m/z: 548 [M+Na]⁺. Anal. Calcd for C₂₇H₂₁Cl₂NO₄S: C, 61.60; H, 4.02; N, 2.66. Found: C, 61.53; H, 4.06; N, 2.49.

N-(2,4-Dichlorobenzenesulfonyl)-4-(4-styrylbenzyloxymethyl)benzamide Sodium Salt (15sA)

Compound 15sA was synthesized from 14s and 2A according to the procedure for the synthesis of 15 pA. Yield 65% for 2 steps. A white solid, mp 171–173°C. ¹H-NMR (DMSO-d₆) δ: 4.53 (2H, s), 4.55 (2H, s), 7.24–7.32 (3H, m), 7.33–7.40 (5H, m), 7.43–7.52 (2H, m), 7.56–7.64 (4H, m), 7.85–7.92 (3H, m), 7.97–8.01 (1H, m). IR (ATR) cm⁻¹: 1670. MS m/z: 574 [M+Na]⁺.

N-(2,4-Dichlorobenzenesulfonyl)-4-(4-phenylethynylbenzyloxymethyl)benzamide Sodium Salt (15tA)

Compound 15tA was synthesized from 14t and 2A according to the procedure for the synthesis of 15 pA. Yield 41% for 2 steps. A white solid, mp 160–170°C. ¹H-NMR (DMSO-d₆) δ: 4.55 (4H, s), 7.27–7.32 (2H, m), 7.38–7.44 (5H, m), 7.46 (1H, dd, J=8.3, 2.2 Hz), 7.51 (1H, d, J=2.2 Hz), 7.52–7.58 (4H, m), 7.85–7.90 (2H, m), 7.98 (1H, d, J=8.3 Hz). IR (ATR) cm⁻¹: 1591. MS m/z: 572 [M+Na]⁺.

N-(2,6-Dichlorobenzenesulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rB)

Compound 15rB was synthesized from 14r and 2B³²⁾ according to the procedure for the synthesis of 15aA. Yield 24%. A white solid, mp 127–128°C. ¹H-NMR (CDCl₃) δ: 4.60–4.66 (4H, m), 7.32–7.63 (14H, m), 7.77–7.85 (2H, m), 9.02–9.33 (1H, br). IR (ATR) cm⁻¹: 1668, 1610. MS m/z: 548 [M+Na]⁺.

N-(2-Cyanobenzenesulfony)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rC)

Compound 15rC was synthesized from 14r and 2C according to the procedure for the synthesis of 15aA. Yield 47%. A white solid, mp 184–186°C. ¹H-NMR (DMSO-d₆) δ: 4.64 (2H, s), 4.70 (2H, s), 7.33–7.39 (1H, m), 7.44–7.52 (4H, m), 7.57–7.64 (2H, m), 7.65–7.72 (4H, m), 7.86–7.96 (2H, m), 7.98–8.04 (2H, m), 8.12–8.18 (1H, m), 8.48–8.56 (1H, m), 11.64–11.75 (1H, br). IR (ATR) cm⁻¹: 1698. MS m/z: 505 [M+Na]⁺.

Methyl 2-[4-(Biphenyl-4-ylmethoxymethyl)benzoylsulfamoyl]benzoate (15rD)

Compound 15rD was synthesized from 14r and 2D according to the procedure for the synthesis of 15aA. Yield 50%. ¹H-NMR (CDCl₃) δ: 3.99 (3H, s), 4.61 (2H, s), 4.62 (2H, s), 7.31–7.52 (7H, m), 7.55–7.63 (4H, m), 7.64–7.75 (2H, m), 7.77–7.89 (3H, m), 8.41–8.48 (1H, m), 9.30–9.45 (1H, br).

2-[4-(Biphenyl-4-ylmethoxymethyl)benzoylsulfamoyl]benzoic Acid (15rE)

Five molar aqueous LiOH solution (1.24 mL, 6.29 mmol) was added to a solution of 15rD (640 mg, 1.24 mmol) in MeOH (5.0 mL) and THF (10 mL), and the mixture was stirred at 50°C for 4 h. The reaction mixture was neutralized with 6.0 M aqueous HCl solution and extracted twice with AcOEt. The organic layer was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of Et₂O, the precipitate was collected by filtration to give 15rE (183 mg, 29% yield) as a white solid, mp 97–99°C. ¹H-NMR (DMSO-d₆) δ: 4.60 (2H, s), 4.63 (2H, s), 7.33–7.40 (1H, m), 7.42–7.52 (6H, m), 7.63–7.80 (7H, m), 7.88–7.95 (2H, m), 8.10–8.17 (1H, m), 11.50–14.50 (2H, br). IR (ATR) cm⁻¹: 1724, 1687. MS m/z: 524 [M+Na]⁺.

N-(Biphenyl-4-sulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rF)

Compound 15rF was synthesized from 14r and 2F³³⁾ according to the procedure for the synthesis of 15aA. Yield 41%. A white solid, mp 181–183°C. ¹H-NMR (CDCl₃) δ: 4.60 (2H, s), 4.63 (2H, s), 7.32–7.38 (1H, m), 7.41–7.54 (9H, m), 7.63–7.67 (4H, m), 7.73–7.76 (2H, m), 7.87–7.90 (2H, m), 7.90–7.95 (2H, m), 8.00–8.09 (2H, m), 12.53–12.67 (1H, br). IR (ATR) cm⁻¹: 1697. MS m/z: 556 [M+Na]⁺.

N-(9H-Fluorene-2-sulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rG)

Compound 15rG was synthesized from 14r and 2G according to the procedure for the synthesis of 15aA. Yield 34%. A white solid, mp 180–182°C. ¹H-NMR (DMSO-d₆) δ: 4.08 (2H, s), 4.59 (2H, s), 4.62 (2H, s), 7.33–7.38 (1H, m), 7.40–7.48 (8H, m), 7.63–7.66 (5H, m), 7.86–7.88 (2H, m), 8.02–8.04 (2H, m), 8.14–8.16 (1H, m), 8.19–8.20 (1H, m), 12.52–12.60 (1H, br). IR (ATR) cm⁻¹: 1698. MS m/z: 568 [M+Na]⁺.

N-(Octane-1-sulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rH)

Compound 15rH was synthesized from 14r and 2H according to the procedure for the synthesis of 15aA. Yield 37%. A white solid, mp 107–109°C. ¹H-NMR (DMSO-d₆) δ: 0.80–0.86 (3H, m), 1.15–1.31 (8H, m), 1.34–1.45 (2H, m), 1.65–1.75 (2H, m), 3.46–3.55 (2H, m), 4.62 (2H, s), 4.65 (2H, s), 7.33–7.39 (1H, m), 7.43–7.55 (6H, m), 7.65–7.70 (4H, m), 7.91–7.97 (2H, m), 12.05 (1H, s). IR (ATR) cm⁻¹: 1675. MS m/z: 516 [M+Na]⁺.

N-[5-(tert-Butyldimethylsilanyloxy)pentane-1-sulfonyl]-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rI)

Compound 15rI was synthesized from 14r and 2I according to the procedure for the synthesis of 15aA. Yeild quant. ¹H-NMR (CDCl₃) δ: 0.03 (6H, s), 0.87 (9H, s), 1.48–1.62 (4H, m), 1.85–1.98 (2H, m), 3.55–3.68 (4H, m), 4.64 (2H, s), 4.66 (2H, s), 7.26–7.65 (11H, m), 7.78–7.85 (2H, m), 8.17–8.23 (1H, br).

N-(5-Hydroxypentane-1-sulfonyl)-4-(biphenyl-4-ylmethoxymethyl)benzamide (15rJ)

One molar TBAF in THF (1.9 mL, 1.9 mmol) was added to a solution of 15rI (560 mg, 0.962 mmol) in THF (8.4 mL) under ice-cooling, and the mixture was stirred at the same temperature for 16 h. After the addition of water, the reaction mixture was extracted twice with AcOEt, washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. Et₂O (5.0 mL) was added to the residue obtained and the precipitate that formed was collected by filtration to give 15rJ (135 mg, 31% yield for 2 steps) as a white solid, mp 113–114°C. ¹H-NMR (CDCl₃) δ: 1.48–1.75 (4H, m), 1.80–2.00 (2H, m), 3.55–3.68 (4H, m), 4.64 (2H, s), 4.64 (2H, s), 7.30–7.38 (1H, m), 7.39–7.47 (4H, m), 7.47–7.54 (2H, m), 7.56–7.65 (4H, m), 7.80–7.89 (2H, m), 8.45–9.10 (1H, br). IR (ATR) cm⁻¹: 1678, 1612. MS m/z: 590 [M+Na]⁺.

Compound 17 was prepared from compound 16³⁵⁾ via compound 34 as follows:

Methyl 4-(Biphenyl-4-ylmethylsulfanylmethyl)benzoate (34)

Five molar MeONa in MeOH (5.5 mL, 28 mmol) was added to a stirred solution of 16 (2.42 g, 10.0 mmol) in MeOH (30 mL), and the mixture was stirred at room temperature for 30 min. Methyl 4-(bromomethyl)benzoate 13 (2.29 g, 10.0 mmol) was added to the reaction mixture, and this was followed by stirring at room temperature for 30 min. After the addition of AcOEt and water to the reaction mixture, the organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure, and the residue obtained was purified by silica gel column chromatography and recrystallized from AcOEt and n-hexane to give 34 (3.41 g, 98% yield) as a solid. ¹H-NMR (CDCl₃) δ: 3.63 (2H, s), 3.66 (2H, s), 3.91 (3H, s), 7.30–7.40 (5H, m), 7.40–7.47 (2H, m), 7.51–7.61 (4H, m), 7.96–8.02 (2H, m).

4-(Biphenyl-4-ylmethylsulfanylmethyl)benzoic Acid (17)

One molar aqueous LiOH solution (5.2 mL, 5.2 mmol) was added to a solution of 34 (600 mg, 1.72 mmol) in MeOH (3.0 mL) and THF (9.0 mL), and the mixture was stirred at room temperature for 1 h. The reaction mixture was acidified by 10% citric acid solution and extracted with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. After the addition of n-hexane, the precipitate was collected by filtration to give 17 (410 mg, 71% yield) as a solid. ¹H-NMR (DMSO-d₆) δ: 3.71 (2H, s), 3.76 (2H, s), 7.33–7.49 (7H, m), 7.58–7.68 (4H, m), 7.87–7.92 (2H, m), 12.70–13.10 (1H, br).

4-(Biphenyl-4-ylmethylsulfanylmethyl)-N-(octane-1-sulfonyl)benzamide (18H)

Compound 18H was synthesized from 2H³⁴⁾ and 17 according to the procedure for the synthesis of 15aA. Yield 30%. A white solid, mp 138–140°C. ¹H-NMR (DMSO-d₆) δ: 0.83 (3H, t, J=6.6 Hz), 1.15–1.30 (8H, m), 1.34–1.44 (2H, m), 1.65–1.75 (2H, m), 3.47–3.54 (2H, m), 3.71 (2H, s), 3.77 (2H, s), 7.32–7.39 (3H, m), 7.42–7.48 (4H, m), 7.59–7.68 (4H, m), 7.87–7.92 (2H, m), 12.01 (1H, s). IR (ATR) cm⁻¹: 1678, 1441, 1336. MS m/z: 532 [M+Na]⁺. Anal. Calcd for C₂₉H₃₅NO₃S₂: C, 68.33; H, 6.92; N, 2.75. Found: C, 68.30; H, 6.92; N, 2.65.

4-(Biphenyl-4-ylmethylsulfanylmethyl)-N-(hexane-1-sulfonyl)benzamide (18K)

Compound 18K was synthesized from 2K³⁴⁾ and 17 according to the procedure for the synthesis of 15aA. Yield 36%. A white solid, mp 134–136°C. ¹H-NMR (DMSO-d₆) δ: 0.80–0.88 (3H, m), 1.20–1.30 (4H, m), 1.34–1.45 (2H, m), 1.69 (2H, quintet, J=7.6 Hz), 3.51 (2H, t, J=7.6 Hz), 3.71 (2H, s), 3.77 (2H, s), 7.32–7.39 (3H, m), 7.42–7.50 (4H, m), 7.58–7.68 (4H, m), 7.86–7.93 (2H, m), 11.97–12.07 (1H, br). IR (ATR) cm⁻¹: 1681. MS m/z: 504 [M+Na]⁺. Anal. Calcd for C₂₇H₃₁NO₃S₂: C, 67.33; H, 6.49; N, 2.91. Found: C, 67.31; H, 6.52; N, 2.85.

4-(Biphenyl-4-ylsulfanylmethyl)-N′-(hexane-1-aminosulfonyl)benzamide (18L)

Compound 18L was synthesized from 2L⁴⁷⁾ and 17 according to the procedure for the synthesis of 15aA. Yield 60%. A white solid, mp 136–137°C. ¹H-NMR (DMSO-d₆) δ: 0.77–0.84 (3H, m), 1.12–1.29 (6H, m), 1.39–1.49 (2H, m), 2.87–2.95 (2H, m), 3.71 (2H, s), 3.76 (2H, s), 7.32–7.40 (3H, m), 7.40–7.49 (4H, m), 7.58–7.68 (4H, m), 7.74–7.80 (1H, m), 7.85–7.91 (2H, m), 11.72 (1H, s). IR (ATR) cm⁻¹: 1687, 1604. MS m/z: 547 [M+H]⁺.

Compound 19 was prepared from compound 17 via compound 35 as follows:

tert-Butyl N-[4-(Biphenyl-4-ylmethylsulfanylmethyl)phenyl]carbamate (35)

Diphenylphosphoryl azide (DPPA) (3.9 mL, 18 mmol), Et₃N (2.7 mL, 19 mmol), and t-BuOH (33 mL), which were stirred at 100°C for 3 h, were added to a stirred solution of 17 (5.01 g, 15.0 mmol) in toluene (33 mL). After cooling, water was added to the reaction mixture, followed by extraction with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by silica gel column chromatography to give 35 (3.66 g, 60% yield) as a solid. ¹H-NMR (CDCl₃) δ: 1.52 (9H, s), 3.59 (2H, s), 3.61 (2H, s), 6.40–6.48 (1H, br), 7.14–7.45 (9H, m), 7.50–7.56 (2H, m), 7.57–7.62 (2H, m).

Ethyl N-[4-(Biphenyl-4-ylmethylsulfanylmethyl)phenyl]oxalamate (19)

TFA (10 mL, 59 mmol) was added to a solution of 35 (4.20 g, 10.4 mmol) in CH₂Cl₂ (10 mL) under ice-cooling, and the mixture was stirred at the same temperature for 50 min. The reaction mixture was basified by saturated aqueous NaHCO₃ solution, followed by extraction with CH₂Cl₂. The organic layer was washed with saturated brine, and dried over Na₂SO₄. The solvent was removed under reduced pressure.

Et₃N (1.1 mL, 7.3 mmol) and ethyl chloroglyoxylate (0.69 mL, 6.2 mmol) were added to a solution of the residue obtained (1.72 g, 5.63 mmol) in CH₂Cl₂ (15 mL) under ice-cooling, and the mixture was stirred at the same temperature for 40 min. The reaction mixture was washed with water and saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. Et₂O (2.0 mL) and n-hexane (4.0 mL) were added to the mixture and the precipitate was collected by filtration to give 19 (2.30 g, 55% yields for 2 steps) as a solid. ¹H-NMR (CDCl₃) δ: 1.44 (3H, t, J=7.1 Hz), 3.62 (2H, s), 3.63 (2H, s), 4.42 (2H, q, J=7.1 Hz), 7.28–7.37 (5H, m), 7.40–7.46 (2H, m), 7.52–7.62 (6H, m), 8.82–8.91 (1H, br).

Compound 20 was prepared from 16 via compound 36 as follows:

4-(4-Bromobenzylsulfanylmethyl)biphenyl (36)

Compound 36 was synthesized from 16 and 1-bromo-4-bromomethylbenzene according to the procedure for the synthesis of 34. Yield 87% for 2 steps. ¹H-NMR (CDCl₃) δ: 3.57 (2H, s), 3.63 (2H, s), 7.14–7.19 (2H, m), 7.30–7.37 (3H, m), 7.40–7.47 (4H, m), 7.51–7.62 (4H, m).

Ethyl [4-(Biphenyl-4-ylmethylsulfanylmethyl)phenyl]oxoacetate (20)

A total of 1.6 M n-BuLi in n-hexane (3.3 mL, 5.3 mmol) was added dropwise at −78°C to a solution of 36 (1.62 g, 4.39 mmol) in THF (10 mL), and the mixture was stirred at the same temperature for 30 min. Diethyl oxalate (0.71 mL, 5.3 mmol) was added to the reaction mixture, followed by stirring at room temperature for 2 h. After the addition of 10% aqueous citric acid solution, the reaction mixture was extracted with AcOEt and the organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure. The residue obtained was purified by column chromatography to give 20 (500 mg, 29% yield) as an oil. ¹H-NMR (CDCl₃) δ: 1.43 (3H, t, J=7.1 Hz), 3.64 (2H, s), 3.67 (2H, s), 4.45 (2H, q, J=7.1 Hz), 7.30–7.37 (3H, m), 7.41–7.47 (4H, m), 7.52–7.62 (4H, m), 7.96–8.00 (2H, m).

N-[4-(Biphenyl-4-ylmethylsulfanylmethyl)phenyl]-2-(hexane-1-sulfonylamino)-2-oxoacetamide (21)

One molar aqueous LiOH solution (14 mL, 14 mmol) was added to a solution of 19 (2.30 g, 5.67 mmol) in MeOH (14 mL) and THF (28 mL), and the mixture was stirred at room temperature for 30 min. The reaction mixture was acidified with 2.0 M aqueous HCl solution, which was extracted with AcOEt. The organic layer was washed with saturated brine, and then dried over Na₂SO₄. The solvent was removed under reduced pressure.

CDI (1.83 g, 11.3 mmol) was added to a solution of the residue obtained in DMF (15 mL), and the reaction mixture was stirred at room temperature for 30 min. After the addition of 2K³⁴⁾ (930 mg, 5.63 mmol) and DBU (2.5 mL, 17 mmol), the mixuture was stirred at 50°C for 14 h. The reaction mixture was acidified with 2.0 M aqueous HCl solution, and the precipitate was then collected by filtration. CHCl₃ (2.0 mL) and MeOH (1.0 mL) were added to the solid obtained, and the precipitate was then collected by filtration to give 21 (250 mg, 8.5% yield for 2 steps) as a white solid, mp 199–201°C. ¹H-NMR (DMSO-d₆) δ: 0.82–0.88 (3H, m), 1.22–1.30 (4H, m), 1.36–1.44 (2H, m), 1.64–1.74 (2H, m), 3.40–3.50 (2H, m), 3.67 (2H, s), 3.70 (2H, s), 7.26–7.33 (2H, m), 7.37–7.40 (3H, m), 7.41–7.48 (2H, m), 7.58–7.67 (5H, m), 7.68–7.74 (2H, m), 10.87–10.90 (1H, br). IR (ATR) cm⁻¹: 1676. MS m/z: 519 [M+Na]⁺.

{2-[4-(Biphenyl-4-ylmethylsulfanylmethyl)phenyl]-N-(hexane-1-sulfonyl)-2-oxoacetamide (22)

Compound 22 was synthesized from 20 and 2K³⁴⁾ according to the procedure for the synthesis of 21. Yield 12% for 2 steps. A white solid, mp 135–137°C. ¹H-NMR (DMSO-d₆) δ: 0.85–0.92 (3H, m), 1.26–1.34 (4H, m), 1.39–1.48 (2H, m), 1.68–1.74 (2H, m), 3.07–3.17 (2H, m), 3.73 (2H, s), 3.80 (2H, s), 7.20–7.32 (3H, m), 7.20–7.32 (2H, m), 7.52–7.58 (2H, m), 7.20–7.32 (4H, m), 7.84–7.89 (2H, m). IR (ATR) cm⁻¹: 1670. MS m/z: 519 [M+Na]⁺.

N-[4-(Biphenyl-4-ylmethylsulfanylmethyl)]-N′-(hexane-1-sulfonyl)urea (23)

DPPA (0.45 mL, 2.1 mmol) and Et₃N (0.31 mL, 4.2 mmol) were added to a stirred suspention of 17 (670 mg, 2.00 mmol) in toluene (6.7 mL), and the mixture was stirred at 100°C for 25 min. Compound 2K³⁴⁾ (500 mg, 3.03 mmol) and DBU (0.45 mL, 3.0 mmol) were added to the reaction mixture, followed by stirring at 100°C for 30 min. After cooling, 1.0 M aqueous HCl solution and Et₂O were added to the reaction mixture, and the precipitate was then collected by filtration and washed with water, MeOH, and AcOEt. The solid obtained was dissolved in DMF (2.0 mL), to which AcOEt (20 mL) was added, and the mixture was stirred at room temperature for 1 h. The precipitate was removed by filtration and washed with AcOEt and Et₂O. The filtrate was evaporated under reduced pressure, and the residue obtained was washed with Et₂O to give 23 (310 mg, 50% yield for 2 steps) as a white solid, mp 140–141°C. ¹H-NMR (DMSO-d₆) δ: 0.77–0.84 (3H, m), 1.15–1.30 (6H, m), 1.40–1.50 (2H, m), 2.88–2.94 (2H, m), 3.71 (2H, s), 3.76 (2H, s), 7.32–7.50 (7H, m), 7.58–7.68 (4H, m), 7.75–7.81 (1H, m), 7.85–7.91 (2H, m), 11.72 (1H, s). IR (ATR) cm⁻¹: 1687, 1604, 1546. MS m/z: 532 [M+Na]⁺.

PTP1B Inhibitory Activity and Enzyme Selectivity

Inhibitory activity against recombinant human PTP1B (Enzo Life Sciences, Inc., Farmingdale, NY, U.S.A.), recombinant human T-cell PTP (TCPTP) (CALBIOCHEM, EMD Biosciences, Darmstadt, Germany), recombinant human CD45 PTP (CD45) (Enzo Life Sciences, Inc.) and recombinant human leucocyte common antigen-related (LAR) PTP (Enzo Life Sciences, Inc.) was measured using p-NPP as a substrate. Briefly, PTP1B inhibitory activities were assessed in the absence and presence of the test compounds in 100 mM 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) buffer (pH 7.2) containing the enzyme, 1 mM dithiothreitol (DTT), 1 mM ethylenediaminetetraacetic acid (EDTA), and 0.0005% Triton-X. The reaction was started by the addition of p-NPP and stopped by the addition of 1 M NaOH after a 30-min incubation at 37°C. The absorbance of p-nitrophenol produced was measured at 405 nm.

PTP1B Inhibitory Mode

The inhibitory mode was examined using recombinant human PTP1B (Enzo Life Sciences, Inc.) and p-NPP as the substrate. In order to clarify the inhibitory mode, phosphatase activities were measured at a fixed PTP1B concentration, while concentrations of the substrate and inhibitor varied.

The enzyme (23.3 ng/mL final) solved in 100 mM HEPES buffer (pH 7.2) containing 1 mM DTT, 1 mM EDTA 0.0005% Triton-X, and various concentrations of 18K was added to the wells of a 96-well plate. The reaction was started by the addition of p-NPP (0.15, 0.25, 0.5, 1, 3, 10, 30 µM) and stopped by the addition of 1 M NaOH after a 30-min incubation at 37°C, and the absorbance of p-nitrophenol produced was then measured at 405 nm.

The 1/s and 1/v values, which were obtained from substrate concentration (s) and enzyme activity (v) at 0, 0.1, 0.2, and 0.5 µM of 18K, were plotted on the x-axis and y-axis, respectively. The inhibitory mode was evaluated from the intersection characteristics of the approximation straight lines obtained.

Plasma Concentrations after Oral Administration to Male ICR Mice, Male SD Rats, and Male Beagles

Male ICR mice (8 weeks old; Japan SLC, Inc.), male SD rats (7 weeks old; Japan SLC, Inc.), and male beagles (45 months old; Narc Corporation, Chiba, Japan) were used. All animal experiments in the present study were conducted according to the guidelines for animal experiments of our institute and the guidelines for animal experimentation approved by the Japanese Association of Laboratory Animal Science. Test compounds suspended in 0.5% methylcellulose solution were orally administered at 30 mg/kg to mice and rats, and at 10 mg/kg to beagles. Blood samples were taken from the tail vein in mice, and from the jugular vein in rats and beagles 0.25, 0.5, 1, 3, 5, and 8 h after administration. Plasma concentrations of the test compounds were assessed using a COSMOSIL 5C18-MS-II column (4.6×150 mm; Nacalai Tesque) and Hitachi Elite LaChrom HPLC system (Hitachi High Technologies, Tokyo, Japan) consisting of a pump (L-2130), autosampler (L-2200), column oven (L-2300), and UV detector (L-2400) in mice and rats, and a COSMOSIL ODS-MS-II column (4.6 mm×150 mm; Nacalai Tesque) and HPLC system consisting of a pump (LC-10ATvp, Shimadzu Techno-Research, Kyoto, Japan), autosampler (SIL-HTc, Shimadzu Techno-Research), column oven (CTO-10Avp, Shimadzu Techno-Research), degasser (DGU-14A, Shimadzu Techno-research), and UV detector (SPD-10Avp, Shimadzu Techno-Research) in beagles.

Anti-diabetic Effects in Male db/db Mice

Male db/db diabetic mice (9 weeks old; Institute for Animal Reproduction, Japan) were allocated to vehicle-treated and 18K-treated groups (n=6). Compound 18K suspended in 0.5% methylcellulose solution was orally administered at 30 mg/kg/d to mice for one week. On day 7, blood samples were taken from the tail vein in fasted mice 50 min after the final administration and plasma glucose and triglyceride levels were measured. In order to estimate insulin resistance, the HOMA value was calculated.⁴⁸⁾

Protein Structure Preparation for in Silico Analyses

The crystal structures of the allosteric inhibitor–PTP1B complex (residue 1–298, but no coordinates from 283 to the last; PDB: 1T4J)³⁷⁾ and TCPTP (residue 1–314, but no coordinates from 278 to the last; PDB: 1L8K)⁴⁹⁾ were used. All crystallographic water molecules were removed. The missing amino acids of TCPTP (Cys278, Ile279, and Lys280, corresponding to Phe280, Ile281, and Met282 of PTP1B) were compensated for using the Prime program from Schrödinger Suite 2012. Structures were minimized using force-field OPLS_2005.

Structural Model of the 18K–PTP1B Complex

The initial superposition between compound 18K and the allosteric inhibitor bound to PTP1B was performed using our molecular overlay program SUPERPOSE.^45,46) This program was developed based on the premise that compounds binding to the same site of a protein possess common 3D physicochemical features. Two molecules are superposed based on their respective physicochemical properties using a pseudo-molecule consisting of functional atoms instead of a real molecule. The properties of the functional atoms are divided into five types, comprising aromatic rings (ARs), hydrophobic atoms (HPs), hydrogen-bonding donors (HDs), hydrogen-bonding acceptors (HAs), and hydrogen-bonding donors/acceptors (DAs), each of which is represented as a sphere with a predefined radius (1.0 Å). After molecular superposition, overlaps of the spheres are scored using the scoring matrix (Table 5). In this study, 220 conformers of compound 18K, generated by the CAMDAS program,⁵⁰⁾ were superposed on the conformation of the allosteric inhibitor bound to PTP1B, and the top-ranked conformations were then validated and refined by the MD simulation described below.

Table 5. Scoring Matrix for the SUPERPOSE Analysis

	HP	AR	HD	HA	DA
HP	+3	+3	−2	−2	−2
AR	+3	+4a)	−2	−2	−2
HD	−2	−2	+2	−2	+1
HA	−2	−2	−2	+2	+1
DA	−2	−2	+1	+1	+1

a) If the planes of two rings cannot be superposed upon each other, a score of +3 is given.

The MD simulation presented here used the force-field OPLS_2005 with an explicit solvent, and was run using the default parameters in the Desmond program from Schrödinger Suite 2012. The SPC water model was used. The systems were neutralized with Na⁺ or Cl⁻ ions, and 0.15 M NaCl was also added. Periodic boundary conditions and a 9.0-Å cut-off for non-bonding interactions were used, with electrostatic interactions treated using the particle mesh Ewald method with a tolerance of 10⁻⁹. A Desmond default relaxation protocol was employed prior to the MD simulation. After relaxation, a 9.0-ns simulation in a normal pressure and temperature ensemble [temperature=300 K, thermostat relaxation time=1.0 ps, pressure=1 atm, and barostat relaxation time=2.0 ps] was performed for each system using a Nose–Hoover thermostat and Martyna–Tobias–Klein barostat. Trajectory atomic coordinate data were recorded every 20 ps. Based on the MD trajectories obtained for the ligand-binding residues (within 4 Å of each ligand), conformations between 6.0 and 9.0 ns were clustered using the distance matrix of the Maestro clustering program from Schrödinger Suite 2012, and a representative structure was then selected as the closest to the average root-mean-square value in the cluster area.

Structural Model of the 18K–TCPTP Complex

The protein backbone heavy atoms of the prepared TCPTP structure and representative structure of the 18K–PTP1B complex were aligned using the Maestro protein structure alignment tool from Schrödinger Suite 2012. The PTP1B protein structure was removed, and the remaining structure of the 18K–TCPTP complex was then used as the initial structure for the MD simulation. The MD simulation and selection of a representative structure were performed as described above.

Conflict of Interest

The authors declare no conflict of interest.

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