Development of Benziodarone Analogues with Enhanced Potency for Selective Binding to Transthyretin in Human Plasma

Abstract

Transthyretin amyloidosis is a fatal disorder caused by transthyretin amyloid aggregation. Stabilizing the native structure of transthyretin is an effective approach to inhibit amyloid aggregation. To develop kinetic stabilizers of transthyretin, it is crucial to explore compounds that selectively bind to transthyretin in plasma. Our recent findings demonstrated that the uricosuric agent benziodarone selectively binds to transthyretin in plasma. Here, we report the development of benziodarone analogues with enhanced potency for selective binding to transthyretin in plasma compared to benziodarone. These analogues featured substituents of chlorine, bromine, iodine, a methyl group, or a trifluoromethyl group, at the 4-position of the benzofuran ring. X-ray crystal structure analysis revealed that CH···O hydrogen bonds and a halogen bond are important for the binding of the compounds to the thyroxine-binding sites. The bioavailability of benziodarone analogues with 4-Br, 4-Cl, or 4-CH₃ was comparable to that of tafamidis, a current therapeutic agent for transthyretin amyloidosis.

Introduction

Transthyretin (TTR) amyloidosis is a group of diseases caused by the deposition of amyloid fibrils formed by TTR in various organs, such as the nerves, heart, eyes, and brain. TTR amyloidosis encompasses familial amyloid polyneuropathy, familial amyloid cardiomyopathy, ocular amyloidosis, and leptomeningeal amyloidosis/cerebral amyloid angiopathy.¹⁻⁹ TTR is a tetrameric protein that serves as a transport protein for thyroxine in plasma and the cerebrospinal fluid. The native ligand thyroxine binds to two hydrophobic sites located in the dimer–dimer interface of the tetramer.¹⁰ Although TTR is a stable protein, it has propensity to aggregate into amyloid fibrils both in vivo and in vitro. The process of amyloid aggregation begins with the dissociation of the native TTR tetramer followed by partial unfolding into an aggregation-prone intermediate, which subsequently self-assembles into amyloid fibrils.¹¹⁻¹⁸ To hinder the molecular cascade leading to TTR amyloid aggregation, the stabilization of the tetramer through kinetic stabilizers is effective in preventing the conversion of the native structure into the aggregation-prone intermediate.¹⁹⁻²¹ Tafamidis (1), which is currently used for the treatment of wild-type and variant TTR amyloidosis, strongly binds to the thyroxine-binding sites, stabilizes the native tetramer, and impedes TTR amyloid aggregation (Figure 1).²¹ In addition to tafamidis, the kinetic stabilizers tolcapone and AG10 have been shown to stabilize the native tetramer of TTR and are currently undergoing clinical trials.²²⁻²⁵

Figure 1.

Chemical structures of tafamidis (1), benzbromarone (2), benziodarone (3), and benziodarone analogues (4–20).

Treatment with 1 is well-tolerated and has been shown to slow the progression of polyneuropathy and cardiomyopathy symptoms in TTR amyloidosis.^21,26−28 While the development of 1 is a great advancement for the treatment of TTR amyloidosis, approximately 30% of patients in a retrospective cohort study of 210 patients with hereditary TTR amyloidosis experienced continued disease progression despite the treatment with 1.²⁹ Therefore, there is still a need for the development of alternative drugs that offer better clinical outcomes.^29,30

One important strategy for developing a kinetic stabilizer of TTR is to explore compounds that can selectively bind to TTR in human plasma.^21,31−34 This is crucial because the kinetic stabilizer needs to specifically bind to TTR in human plasma, where numerous proteins, including albumin and TTR, exist.^21,31−34 The currently used drug 1 is known to bind to TTR in human plasma, thereby inhibiting amyloid aggregation by stabilizing the native tetramer.²¹ However, serum albumin has been shown to hinder the binding of 1 to TTR, which may reduce the effectiveness of 1 in terms of binding potency and selectivity in human plasma.³³ Cotrina et al. recently suggested that the uricosuric drug benzbromarone (2) could be a kinetic stabilizer of TTR for repositioning purposes, based on their findings that 2 bound to wild-type TTR with nM affinity and inhibited TTR amyloid aggregation in vitro (Figure 1).³⁵ In addition, we have recently demonstrated that benziodarone (3) and its possible metabolite 6-hydroxybenziodarone are potent for selective binding to TTR in human plasma (Figure 1).³⁶ The compound 3 with a hydroxy diiodophenyl ring was more potent in binding to TTR in plasma than 2 with a hydroxy dibromophenyl ring.³⁶ Therefore, we considered that 3 was a promising lead compound for the development of kinetic stabilizers with high potency for selective binding to TTR in plasma. Here, we report the development of benziodarone analogues with enhanced potency and selectivity for binding to TTR in human plasma compared to 1 and 3. These analogues featured substituents, such as iodine, bromine, or chlorine at the 4-position of the benzofuran ring (4, 5, 6, and 7). Other analogues featured a methyl group or a trifluoromethyl group at the same position (8 and 9) (Figure 1). We further characterized the amyloid inhibitory activity of compounds 4, 5, 6, 7, and 8 using thioflavin-T fluorescence. The X-ray crystal structure analysis of 4 and 7 revealed that the benzofuran ring was located within the inner binding site of TTR, while the hydroxy diiodophenyl group was positioned at the entrance of the binding site. In the analysis of thioflavin-T fluorescence and X-ray crystallography, V30M-TTR was used since it is the most frequent variant in familial amyloid polyneuropathy.^1,2 In vivo pharmacokinetic studies demonstrated that the bioavailabilities of 5, 6, and 8 were comparable to that of 1, with a mean bioavailability of 64% in rats (Figure 1).

Results and Discussion

Exploring the Benziodarone Analogues with Enhanced Potency for Selective Binding to TTR in Human Plasma

Since human plasma contains numerous proteins, it is important to develop kinetic stabilizers that selectively bind to TTR in human plasma. Nonselective binding to plasma components other than TTR compromises the efficacy of kinetic stabilizers.^21,31−34 We have previously demonstrated that 3 is a potent and selective kinetic stabilizer of TTR.³⁶ In this study, we report the development of benziodarone analogues that are more potent in selective binding to plasma TTR than 3. First, we synthesized benziodarone analogues and investigated their binding to TTR in human plasma.

The synthesis of the benzofuran derivatives 4–20 is the same as our previous synthetic method.³⁶ The appropriate salicylaldehydes 21a–m were converted to the corresponding ketones 22a–m, which were then subjected to Wolff–Kishner reduction to afford the ethyl benzofurans 23a–m, as shown in Scheme 1.

Scheme 1. Synthesis of the Benzofurans 23a–m.

To investigate the effect of the ethyl group at the 2-position of the benzofuran skeleton on the biological activity, a Wittig reaction was performed on 22e followed by hydrogenation of the resulting olefin 23e’ to obtain the isopropyl derivative 23e”. To further clarify the importance of the benzofuran moiety, benzothiophene derivative 26a and indole derivative 26b were also prepared (Scheme 2).

Scheme 2. Synthesis of the Isopropyl Derivatives 23e”, Benzothiophene 26a, and Indole 26b.

After Friedel–Crafts acylation of 23a–m, 23e”, and 26a–b, the methoxy group of the resulting acylated compounds 27a–e and 27g–p was deprotected using BBr₃ to synthesize the corresponding phenol derivatives 28a–d, 16, and 28g–p. Finally, the desired benzofuran derivatives 4–8, 10–15, and 17–20 were synthesized by iodination, as shown in Scheme 3.

Scheme 3. Synthesis of the Desired Benzofuran Derivatives 4–8, 10–15, and 17–20.

Friedel–Crafts acylation of 23f did not proceed at all due to the strong electron-withdrawing of the CF₃ group, and acylated compound 27f was not obtained. In order to synthesize the desired benzofuran derivative 9, trifluoromethylation of 28a was investigated. However, trifluoromethylation of 28a also did not proceed at all, resulting in the recovery of the starting material. We expected the reaction to proceed by protecting the phenolic hydroxyl group of 28a with a MOM group. As a result, the desired trifluoromethylated product 30 was successfully obtained by reacting TMSCF₃ with the prepared MOM ether 29 in the presence of catalytic amounts of copper iodide and 1,10-phenanthroline in dimethyl sulfoxide (DMSO).³⁷ After removal of the MOM group in 30, iodination was performed on the resulting phenol derivative 28f to afford the desired benzofuran derivative 9 (Scheme 4).

Scheme 4. Synthesis of Benzofuran Derivative 9.

To assess the selective binding to TTR in plasma, we performed ex vivo competitive binding experiments with a fluorogenic probe, (E)-S-phenyl 3-(4-hydroxy-3,5-dimethylstyryl)benzothioate, developed by Choi and Kelly.^31,33 This probe binds to the thyroxine-binding site of TTR, forming a covalently linked conjugate with the side chain of Lys15, which is located at the entrance of the thyroxine-binding sites.

The time-dependent increase in fluorescence intensity indicates the formation of a fluorescent conjugate between the probe and the side chain of Lys15 (Figure 2). This formation is inhibited by a kinetic stabilizer that occupies the thyroxine-binding site of TTR in human plasma. The lower the fluorescence of the TTR-probe conjugate in this assay, the higher the binding potency and selectivity of the candidate kinetic stabilizer to TTR in plasma.³¹ We used a commercially available human plasma containing 5 μM wild-type TTR.³³ This is crucial to maintain uniform TTR concentrations across all compounds, ensuring consistency in our experimental conditions. In the presence of 4 at 20 μM, the fluorescence intensity of the probe is reduced to approximately 10% of that in the control (DMSO), suggesting competitive occupation of the thyroxine-binding sites of TTR by 4 in human plasma (Figure 2). In contrast, in the presence of 1 at 20 μM, the fluorescence intensity is approximately 50% of that in the control. This is due to the presence of a high concentration of human serum albumin: the concentration of albumin in plasma is about 600 μM, which is much higher than that of TTR in plasma (about 5 μM). The presence of albumin, which exists at extremely high concentrations in plasma, has been shown to prevent the binding of 1 to TTR in human plasma.³³ The binding of 3 was intermediate between 4 and 1: the fluorescence intensity was approximately 30% of the intensity in the control (Figure 2). These results indicate that 4 is more potent for selective binding to TTR in plasma than 1 and 3. It is worth noting that compounds 1, 3, and 4 interact with serum albumin in human plasma. Our data show that 4 tightly binds to 5 μM TTR in human plasma, even in the presence of 600 μM serum albumin.

Figure 2.

Time-dependent fluorescence change of the fluorogenic probe, (E)-S-phenyl 3-(4-hydroxy-3,5-dimethylstyryl)benzothioate, caused by the formation of the fluorescent conjugate with Lys15 of TTR in human plasma. Circles indicate the fluorescence intensity in the absence of a compound (control DMSO). Diamonds, triangles, and squares indicate the fluorescence intensity in the presence of tafamidis (1), benziodarone (3), and 4, respectively. The concentration of compounds was set to be 20 μM. Each experiment was independently conducted three times. Average and standard deviation values are shown.

In addition to 4, compounds 5, 6, 7, 8, and 9 were highly potent for selective binding to TTR in human plasma (Figures 1 and 3). These compounds were characterized by the presence of iodine, bromine, chlorine, a methyl group, or a trifluoromethyl group at the 4-position of the benzofuran B-ring (Figure 1). Therefore, these substituents at the 4-position contribute to the selective binding of these benziodarone analogues. The importance of the 4-Cl on the benzofuran B-ring was also revealed through a comparison of 6 (4-Cl), 10 (5-Cl), 11 (6-Cl), and 12 (7-Cl): the binding of 6 with 4-Cl to TTR in plasma was more potent than that of 10 (5-Cl), 11 (6-Cl), or 12 (7-Cl) (Figures 1 and 3). Additionally, the binding of 4 (4-I), 5 (4-Br), and 6 (4-Cl) was slightly more potent than that of 13, which has a fluorine atom at the 4-position of the benzofuran B-ring (Figures 1 and 3).

Figure 3.

Fluorescence intensity of the fluorogenic probe, (E)-S-phenyl 3-(4-hydroxy-3,5-dimethylstyryl)benzothioate, in human plasma in the presence of the compounds at 6 h of incubation. The fluorescence intensity in the absence of the compound (DMSO) is designated as C. Each experiment was independently conducted three times. Average and standard deviation values are shown.

Compound 14, which has a benzo[b]thiophene ring instead of the benzofuran ring, exhibited weaker binding compared to 3 (Figures 1 and 3). However, the binding of 15, which contains the 1H-indole ring, was similar to that of 3. These findings suggest that the 1H-indole ring can be used as an alternative to the benzofuran ring in the development of benziodarone analogues as kinetic stabilizers of TTR (Figures 1 and 3).

Figure 4 shows the percent TTR occupancy by 4, 5, 6, 7, and 8 in human plasma.³⁶ The percent TTR occupancy by 4, 5, 6, 7, and 8 was higher than that of 1 and 3 across the concentration range of 2.5 to 20 μM (Figures 1 and 4). Compounds 1 and 3 showed less than 50% TTR occupancy at a concentration of 10 μM, while 4, 5, 6, 7, and 8 exhibited over 80% TTR occupancy at the same concentration (Figure 4). An 80% TTR occupancy indicates a stoichiometry of 1:1.6 between the TTR tetramer and the compounds at a concentration of 10 μM. At a concentration of 5 μM, 4, 5, 6, 7, and 8 showed approximately 50–70% TTR occupancy, corresponding to a stoichiometry of at least 1:1 between the TTR tetramer and the compounds (Figure 4). At the same concentration, 1 exhibited 15% TTR occupancy, while 3 exhibited 30% TTR occupancy. The concentration of TTR in human plasma has been reported to be around 5.5 μM (tetramer concentration).³⁸ These studies show that the binding of 4, 5, 6, 7, and 8 to TTR in plasma is more potent and selective than that of 1 and 3. Half-maximal effective concentration (EC₅₀) values were calculated to identify potential potency differences among compounds 4, 5, 6, 7, and 8. Notably, compounds 6 and 8 exhibited slightly greater potency than 4 and 7 in selectively binding to TTR in human plasma (Table 1).

Figure 4.

Percent TTR occupancy by compounds in the presence of the fluorogenic probe measured after 6 h of incubation in human plasma. The percent TTR occupancy was calculated as 1 – (I_compound – I₀)/(I_DMSO – I₀), where I_DMSO and I_compound represent the fluorescence intensity of the probe in the absence and presence of the compound, respectively, and I₀ represents the intensity without the probe. Tafamidis (1), yellow; benziodarone (3), gray; 4, cyan; 5, green; 6, red; 7, orange; 8, blue. Each experiment was independently conducted three times. Average and standard deviation values are shown.

Table 1. Half-Maximal Effective Concentration (EC₅₀) Values for Selective Binding of Compounds 4–8 to Wild-Type TTR in Human Plasma^a.

compound	EC50 (μM)
4	3.5 ± 0.2
5	3.0 ± 0.1
6	2.5 ± 0.1
7	3.9 ± 0.2
8	2.7 ± 0.1
1	15.6 ± 1.2
3	8.9 ± 0.4

^aThe TTR concentration is 5 μM (tetramer concentration).

We have synthesized additional compounds with a benzofuran skeleton, which did not exhibit superior efficacy compared to compounds 4, 5, 6, 7, and 8 (Figures S2–S4). Details on these synthesized compounds and their structure–activity relationships are provided in the Supporting Information file.

Amyloid Inhibitory Activity of Benziodarone Analogues

The inhibitory activity of benziodarone analogues against V30M-TTR aggregation was analyzed using thioflavin-T fluorescence (Figure 5). V30M-TTR was selected since it is the most frequent variant in familial amyloid polyneuropathy.^1,2 Amyloid aggregation of V30M-TTR was induced by a pH jump from pH 7.0 to 4.7 in the absence or presence of benziodarone analogues.^11,12 The time course of aggregation was monitored by measuring the thioflavin-T fluorescence intensity of V30M-TTR in the presence of 20 μM compounds (Figure 5A). The concentration of TTR was 10 μM (tetramer concentration), i.e., the molar ratio of TTR and compounds was 1:2. In the aggregation assay, we focused on 4, 5, 6, 7, and 8, which showed high potency for selective binding to TTR in human plasma (Figures 1, 3, and 4). These benziodarone analogues clearly inhibited the amyloid aggregation of V30M-TTR (Figure 5A). The amyloid aggregation of V30M-TTR was also analyzed at various compound concentrations ranging from 0 to 50 μM (Figure 5B).³⁶ The values of the half-maximal inhibitory concentration (IC₅₀) of the benziodarone analogues are summarized in Table 2. The IC₅₀ values of 4, 5, 6, 7, and 8 were similar to that of 1 (Table 2). It is crucial to note that the best achievable IC₅₀ value under our experimental conditions is 5 μM. This value represents the maximum inhibition attainable in our aggregation assays, given that the tetramer is present at 10 μM, and the stoichiometry of the compound and the tetramer is 1:1 at a 10 μM compound concentration (i.e., one of the two binding sites occupied). The IC₅₀ values for compounds 1–8 were approximately 5 μM. The dissociation constants (K_d) between V30M-TTR and the compounds were analyzed by isothermal titration calorimetry (ITC). The compounds 4, 5, 6, 7, 8, and 3 bound to V30M-TTR with nM affinity (Table 3).

Figure 5.

Amyloid inhibitory activity of benziodarone analogues monitored by thioflavin-T fluorescence. Each experiment was independently conducted three times. Average and standard deviation values are shown. (A) Time course of aggregation of V30M-TTR at pH 4.7 in the presence of 20 μM compounds. Tafamidis (1), yellow; benziodarone (3), gray; 4, cyan; 5, green; 6, red; 7, orange; 8, blue. Aggregation of V30M-TTR in the absence of a compound (control DMSO) is represented by black circles. (B) Inhibition ratio of benziodarone analogues against amyloid aggregation of V30M-TTR. The solutions at pH 4.7 containing V30M-TTR (10 μM tetramer concentration) and 0–50 μM compounds were incubated for 7 days at 37 °C. Tafamidis (1), yellow; benziodarone (3), gray; 4, cyan; 5, green; 6, red; 7, orange; 8, blue.

Table 2. Inhibitory Activities of 4, 5, 6, 7, and 8 against Aggregation of V30M-TTR^b.

compound	IC50 (μM)
4	6.5 ± 0.4
5	6.1 ± 0.5
6	5.0 ± 0.2
7	5.4 ± 0.3
8	5.2 ± 0.5
1a	5.5 ± 0.5
3a	4.5 ± 0.2

^aThe data are from our previous study.³⁶

^bThe inhibitory activity is represented by the half-maximal inhibitory concentrations (IC₅₀) against amyloid aggregation of V30M-TTR. The TTR concentration is 10 μM (tetramer concentration). The IC₅₀ values of 1 and 3 are also indicated for comparison. Each experiment was independently conducted three times. All results are presented as average values with their corresponding standard deviations.

Table 3. Dissociation Constants (K_d) of 4, 5, 6, 7, and 8 Determined by ITC Measurements^a.

compound	Kd (nM)
4	120 ± 30
5	40 ± 19
6	66 ± 3.0
7	53 ± 16
8	42 ± 9.0
1	138 ± 13
3	78 ± 16

^aThe K_d values of 1 and 3 are indicated for comparison. Each experiment was independently conducted four to six times. All results are presented as average values with their corresponding standard deviations.

Binding Modes of Benziodarone Analogues

To clarify the binding modes of the benziodarone analogues, we analyzed the X-ray crystal structure of V30M-TTR in complex with 4, 7, or 20 (Table 4). The electron densities of the compounds bound to V30M-TTR were clearly observed in the complex (Figure 6). In all complexes, the hydroxy diiodophenyl group was located at the entrance of the thyroxine-binding site, and the benzofuran ring was located in the inner binding site. The hydroxy group and two iodine atoms of the hydroxy diiodophenyl group were located near Lys15 (Figure 7). The importance of the two iodine atoms was demonstrated in ex vivo competitive binding assays of 8 and 16: removal of iodine atoms from the diiodophenyl group markedly reduced the binding to TTR in plasma (Figures 1 and 3).

Table 4. X-ray Crystallographic Data and Refinement Data Statistics for V30M-TTR in Complex with Selected Compounds.

	4	7	20
Crystal data
beamline	PF-17A	PF-AR-NE3A	SLS-X06SA
wavelength (Å)	1.70	1.00	1.00
resolution range (Å)	42.97–1.80 (1.87–1.80)	42.64–1.70 (1.76–1.70)	38.58–1.51 (1.56–1.51)
space group	P21212	P21212	P21212
unit-cell parameters a, b, and c (Å)	43.3, 85.9, 63.9	43.5, 85.3, 63.8	43.2, 85.5, 63.7
observed reflections	188,835 (17,083)	200,826 (16,814)	345,560 (33,840)
unique reflections	22,569 (2123)	26,271 (2445)	37,996 (3698)
completeness (%)	99.15 (95.46)	97.27 (92.58)	99.80 (99.09)
Rmeas (%)	8.4 (61)	11.6 (55.8)	5.4 (98.6)
Rpim (%)	2.8 (20.9)	4 (21.3)	1.8 (32.3)
redundancy	8.37 (8.05)	7.64 (6.88)	9.09 (9.15)
I/sigma(I)	16.2 (3.7)	13.7 (5.5)	20.8 (2.1)
CC1/2	0.998 (0.939)	0.995 (0.896)	0.999 (0.798)
Refinement data
R, Rfree (%)	18.7, 21.4	20.3, 23.1	18.9, 21.3
RMSD bonds and angles (Å and °)	0.009, 1.16	0.008, 1.06	0.008, 0.99
average B-factor protein, water, and inhibitor (Å2)	32.4, 40.2, 32.2	21.5, 30.4, 15.9	26.3, 36, 29.1
Ramachandran plot favored, allowed, and disallowed (%)	97.8, 2.2	98.7, 1.3	98.2, 1.8
PDB code	8WGS	8WGT	8WGU

Figure 6.

Crystal structure of V30M-TTR in complex with 4 (PDB code 8WGS). (A) Tetramer of V30M-TTR. Subunits A, B, C, and D are colored in green, cyan, magenta, and yellow, respectively. (B) Visibility of 4 in the crystal structure. The omitted difference Fourier map (polder map) is contoured at 3.3 σ (cyan mesh). The anomalous difference Fourier map is contoured at 7 σ (magenta mesh). The oxygen and iodine atoms are colored in red and black, respectively. The structure models were prepared using PyMOL (Schrödinger, New York, NY).

Figure 7.

Close-up view of 4, 7, and 20 in the thyroxine-binding site of V30M-TTR. Compounds 4, 7, and 20 are colored gray and are represented by a stick model. The oxygen, nitrogen, and fluorine atoms are colored red, blue, and cyan, respectively. The iodine and chlorine atoms are colored black. The distances of CH···O hydrogen bonds and halogen bonds were indicated in Å. The panels show (A) 4 in binding mode 1, (B) 4 in binding mode 2, (C) 7, and (D) 20. The PDB ID codes are 8WGS, 8WGT, and 8WGU for V30M-TTR in complex with 4, 7, and 20, respectively.

Compound 4 adopted two binding modes in the thyroxine-binding site of V30M-TTR (Figures 6 and 7). In both binding modes, the hydroxy diiodophenyl group of 4 overlapped with that of 3 (Figure 8A,B).³⁶ In addition, two iodine atoms of 4 were located at the same position as the iodine atoms of thyroxine in complex with wild-type TTR (Figure 8C,D).¹⁰ The iodine atoms of thyroxine are important for its interaction with the halogen-binding pockets 1–3, which leads to stabilization of the tetramer.^10,19,39 In binding mode 1, the carbonyl oxygen of 4 formed CH···O hydrogen bonds with the side chain C^γ of Thr119 and the side chain C^δ of Leu17 (Figure 7A).⁴⁰ The distances were 3.0 and 3.2 Å, respectively. The binding energy of a CH···O hydrogen bond is approximately one-third that of a conventional hydrogen bond, as reported by Jiang and Lai.⁴⁰ While the conventional hydrogen bond exhibits a binding energy of −5.5 kJ/mol, the CH···O hydrogen bond has a binding energy of −1.9 kJ/mol. Our X-ray structures of V30M-TTR in complex with benziodarone analogues revealed the presence of multiple CH···O hydrogen bonds, emphasizing their contribution to the binding of the benziodarone analogues. The iodine atom of the benzofuran ring was surrounded by Leu17, Ala108, and Ala109, and the iodine atom formed a halogen bond with the main chain carbonyl oxygen of Ala109 (Figure 7A).⁴¹⁻⁴⁴ The distance between I and O was 3.2 Å. The binding mode 1 of 4 was similar to that of 3 in complex with V30M-TTR (8II1; Figure 8A).³⁶ The only difference between 3 and 4 was the iodine atom at the 4-position in the B-ring (Figure 1).

Figure 8.

Overlay of 4 with 3 (A,B; PDB codes 8WGS and 8II1) and 4 with thyroxine (C,D; PDB code 8WGS and 2ROX). Compounds 4, 3, and thyroxine are represented by a stick model. 4 is colored in gray. 3 and thyroxine are colored in yellow. The oxygen, nitrogen, and iodine atoms are colored red, blue, and black, respectively. The panels show (A) 4 in binding mode 1 and 3, (B) 4 in binding mode 2 and 3, (C) 4 in binding mode 1 and thyroxine, and (D) 4 in binding mode 2 and thyroxine. (C,D) Overlapped iodine atoms are highlighted by circles.

In binding mode 2, the CH···O hydrogen bonds were also important for the interaction between 4 and V30M-TTR (Figure 7B). The ethyl group of 4 formed CH···O hydrogen bonds with the backbone carbonyl oxygens of Lys15 and Ala109. The distances were 3.0 and 2.9 Å, respectively. The importance of the ethyl group in the benzofuran B-ring was also indicated by the ex vivo competitive binding assay of 8 and 17: the substitution of the ethyl group of the benzofuran ring with an isopropyl group greatly reduced the binding potency to TTR in plasma (Figures 1 and 3). In addition, in binding mode 2, the carbonyl oxygen of 4 formed CH···O hydrogen bonds with the C^β atom of Ala108 and the C^γ atom of Thr119, at distances of 3.3 and 3.0 Å, respectively (Figure 7B).

We also investigated the binding mode of 7 by analyzing the crystal structure of TTR-7 (Figure 7C). The binding mode of 7 was similar to binding mode 2 of 4 (Figure 7B,C). The ethyl group of 7 formed CH···O hydrogen bonds with the main chain carbonyl oxygens of Lys15 and Ala109. The carbonyl oxygen of 7 formed CH···O hydrogen bonds with the C^β atom of Ala108 and the C^γ atom of Thr119 (Figure 7C). Additionally, the chlorine atom at position 7 of compound 7 formed a halogen bond with the main chain carbonyl oxygen of Ser117, with a distance of 3.2 Å between the 7-Cl and the carbonyl oxygen (Figure 7C).⁴¹⁻⁴⁴ The importance of the 4-Cl and 7-Cl in the B-ring of 7 was further demonstrated by the ex vivo competitive binding assay (Figure 3). The binding potency of 18 and 19, which had chlorine atoms located at different positions in the benzofuran ring, was remarkably reduced compared to that of 7 (Figures 1 and 3). This indicates that the 5-Cl and 6-Cl cannot be accommodated in the benziodarone analogues as a TTR kinetic stabilizer (Figures 1 and 7).

The ex vivo competitive binding assay showed that the binding of 7 to plasma TTR was stronger than that of 20 (Figures 1 and 3): the substitution of two chlorine atoms for fluorine atoms in the B-ring reduced the binding potency to TTR in plasma. This difference in binding was due to the variation in halogen atoms at positions 4 and 7 (Figure 1). We further analyzed the crystal structure of V30M-TTR in complex with 20, in which the chlorine atoms in the benzofuran ring of 7 were substituted with fluorine atoms (Figures 1 and 7D). We chose compound 20 in addition to 7 for X-ray crystallographic analysis because the ex vivo competitive binding assay demonstrated that the potency of 20 is weaker than that of 7 (Figure 3). The binding mode of 20 was similar to that of 4 in binding mode 1 (Figure 7A,D). In the complex with 20, the fluorine atom at position 4 made contact with the C^δ atom of Leu17, with a distance of 3.0 Å (Figure 7D). The carbonyl oxygen of 20 formed CH···O hydrogen bonds with the C^β atom of Ala108, the C^γ atom of Thr119, and the C^δ atom of Leu17. Furthermore, the ethyl group in the benzofuran ring of 20 formed a CH···O hydrogen bond with the main chain carbonyl oxygen atom of Ser117, with a distance of 3.3 Å (Figure 7D).

In Vitro ADME Assay and In Vivo Pharmacokinetic Study

We performed an in vitro absorption, distribution, metabolism and excretion (ADME) assay and an in vivo pharmacokinetic study.^45,46Table 5 summarizes the solubility, membrane permeability, hepatic microsomal metabolic stability in rats, and plasma protein binding in rats of the compounds. At pH 6.8, the solubility of 4, 5, 6, and 8 was high and comparable to that of 1. On the other hand, the solubility of 7 at pH 6.8 was slightly lower (Table 5). At pH 1.2, the solubility of 4, 6, 7, and 8 was lower than that of 1, but the solubility of 5 was similar to that of 1. The solubility of benziodarone analogues significantly varied between acidic (pH 1.2) and neutral (pH 6.8) conditions. The observed large difference in solubility can be attributed to the phenol ring with two iodine atoms, responsible for high solubility at pH 6.8 and low solubility at pH 1.2. The results of the parallel artificial membrane permeability assay (PAMPA) showed that all compounds were absorbed through membranes, suggesting their absorption from the gastrointestinal tract (Table 5). Computational predictions using pkCSM indicated high Caco-2 permeability and intestinal absorption for benziodarone analogues (Table S1), aligning with our PAMPA assay results.⁴⁷ Liver microsomal metabolic stability assays indicated that 4, 5, 6, 7, and 8 were less metabolically stable than 1 (Table 5). To determine the unbound fraction in rat plasma, rat plasma was mixed with the test compound (1 μM). Due to the compound’s low concentration and the strong affinity between the compound and TTR, the unbound fraction was substantially low (Table 5). The unbound fractions of 4, 5, 6, 7, and 8 to plasma proteins were lower than that of 1, implying strong binding of these compounds to plasma proteins. The ex vivo competitive binding assay demonstrated that compounds 4, 5, 6, 7, and 8 selectively bound to TTR in plasma (Figures 3 and 4). Consequently, the higher plasma binding observed for these compounds is primarily attributed to their interaction with TTR in the plasma (Table 5).

Table 5. Summary of In Vitro ADME Properties of 4, 5, 6, 7, 8, and 1.

	aqueous solubility (μM)
compound	pH 1.2	pH 6.8	PAMPA permeability (×10–6 cm/s)	hepatic microsome stability in rats (mL/min/kg)	plasma protein binding in rats (% unbound)
4	<0.02	>100	>50	171	<0.01
5	0.054	>100	>50	237	<0.01
6	<0.01	>100	>50	275	<0.01
7	<0.02	89.5	>50	123	<0.01
8	<0.01	>100	>50	296	<0.01
1a	0.088	>100	>50	38.7	0.51

^aThe data are from our previous studies.^45,46

In an in vivo pharmacokinetic study, compound solutions were intravenously administered (iv) to two male SD rats and given orally (po) to two other male SD rats using cassette dosing. The dose was set to 0.1 mg/kg for each compound.^45,46 The selection of a low dose (0.1 mg/kg) was based on two considerations. First, a low dose enables the assessment of the pharmacokinetic profile without potential saturation effects. Second, it helps avoid adverse reactions that might occur at higher doses. Blood samples were collected from the caudal vein at each time point, and the concentrations of the compounds in plasma were determined using liquid chromatography-tandem mass spectrometry (LC-MS/MS) (Figure S1). The pharmacokinetic parameters are summarized in Table 6. The values of total clearance (CLtot) for 4, 5, 6, 7, and 8 were 21–40 mL/h/kg, which were greater than that of 1. This observation aligns with our in vitro ADME assays, which consistently demonstrated that 4, 5, 6, 7, and 8 were less metabolically stable than 1 (Table 5). The Vdss values of 4, 5, 6, and 8 were significantly lower than that of 1. A low Vdss value suggests reduced distribution of a compound into tissues and organs, with the compound primarily staying in the bloodstream. Although compounds 4, 5, 6, and 8 showed higher clearance than tafamidis, their Vdss were smaller than those of tafamidis, resulting in higher C_max values for compounds 4, 5, 6, and 8 than for tafamidis. The concentrations of 5, 6, and 8 in plasma after oral dosing were significantly higher than those of 4 and 7 (Figure S1). The bioavailabilities (BA) of 5, 6, and 8 were 67, 68, and 62%, respectively, which were similar to that of 1. The BA values of 4 and 7 were lower than those of 5, 6, and 8. These results suggest that the first-pass effect was high for compounds 4 and 7 but comparable to 1 for compounds 5, 6, and 8. We were unable to calculate the half-life after oral administration because all of the compounds evaluated in this study had long half-lives after oral administration, with a time to maximum concentration (T_max) of 4 or 8 h (Figure S1). Consequently, we assessed the first-pass effect of the compounds based on BA, which could be derived from the AUC. Taking into account the ex vivo competitive binding experiments that demonstrated the highly potent and selective binding of 5, 6, and 8 to TTR in human plasma (Figures 3 and 4), compounds 5, 6, and 8 have the potential to be effective therapeutic agents for TTR amyloidosis.

Table 6. Pharmacokinetic Parameters of 4, 5, 6, 7, 8, and 1 from an In Vivo Pharmacokinetic Study.

compound	route	dose (mg/kg)	C0 or Cmax (ng/mL)	CLtot (mL/h/kg)	Vdss (mL/kg)	AUC0–8h (ng/mL·hr)	BA (%)
4	iv	0.1	1543	24.3	153	2961
	po	0.1	240			1489	50.3
5	iv	0.1	1743	20.6	113	3708
	po	0.1	390			2476	66.8
6	iv	0.1	1718	20.6	112	3726
	po	0.1	395			2524	67.7
7	iv	0.1	1337	39.7	269	1746
	po	0.1	77			350	20.1
8	iv	0.1	1645	26.4	123	3102
	po	0.1	303			1918	61.8
1	iv	0.1	519	5.96	311	2363
	po	0.1	235			1500	63.5

Conclusions

The ex vivo competitive binding study demonstrated that compounds 4, 5, 6, 7, and 8 exhibited superior binding potency to TTR in human plasma compared to the currently used therapeutic agent (1). This is crucial because the kinetic stabilizer needs to specifically bind to TTR in human plasma, where numerous proteins, including albumin and TTR, exist.^21,31−34 Serum albumin has been shown to hinder the binding of 1 to TTR, which may reduce the effectiveness of 1 in terms of binding potency and selectivity in human plasma.³³ Notably, this study highlighted the critical role of iodine, bromine, chlorine, a methyl group, or a trifluoromethyl group at the 4-position of the benzofuran B-ring for selective binding to TTR in plasma. Additionally, the presence of iodine atoms in the hydroxy diiodophenyl ring, the carbonyl oxygen, and the ethyl group in the benzofuran ring proved crucial for their selective binding to TTR in plasma. The structure–activity relationships are summarized in Figure S3. Structural insights gained from the X-ray crystal structures of V30M-TTR in complex with compounds 4, 7, and 20 revealed the significance of CH···O hydrogen bonds and a halogen bond in mediating the interaction between the benziodarone analogues and residues in the thyroxine-binding sites of V30M-TTR. The in vivo pharmacokinetic study further revealed that compounds 5, 6, and 8 exhibited bioavailabilities comparable to the currently used therapeutic agent (1). Collectively, our findings contribute to the understanding of the structure–activity relationship and pharmacokinetic profiles of benziodarone analogues, paving the way for their potential development as effective therapeutic agents for TTR amyloidosis.

Experimental Section

Chemistry

General Methods of Chemistry

Chemicals were purchased from Sigma-Aldrich, Merck (Darmstadt, Germany), FUJIFILM Wako Chemicals (Osaka, Japan), Nacalai Tesque, Tokyo Chemical Industry (Tokyo), and Kanto Chemical (Tokyo) and used without further purification. Column chromatography was done on Cica silica gel 60N (spherical, neutral; particle size, 63-210 nm; Kanto Chemical), while thin-layer chromatography was performed using Merck silica gel 60F₂₅₄ plates. Melting points were taken on a Yanaco micromelting point apparatus and are uncorrected. The nuclear magnetic resonance (NMR) spectra were acquired in the specified solvent in a JEOL JNM-A400 (400 and 100 MHz for ¹H and ¹³C, respectively) or a JEOL JNM-ECX500 (500 and 125 MHz for ¹H and ¹³C, respectively). The chemical shifts (δ) are reported in ppm downfield from tetramethylsilane, and coupling constants (J) are expressed in hertz. Infrared spectroscopy (IR) spectra were measured with a JASCO FT/IR-460 Plus spectrophotometer (JASCO Corp., Tokyo). The high-resolution mass spectra were obtained with a Shimadzu LCMS-9090 equipped with dual ion source probes, which continuously performed both electrospray ionization (ESI) and atmospheric-pressure chemical ionization (APCI). All synthesized benzofuran derivatives 4–20 were >95% pure by high-performance liquid chromatography (HPLC) analysis. Analytical HPLC was performed on a Hitachi Chromaster 5110 pump with a Hitachi Chromaster 5410 UV detector using an InertSustain C18 column (5 μL, 4.6 mm × 250 mm) and MeCN and formic acid (100:1) as the mobile phase.

General Procedure for the Synthesis of 23a–m

To a stirred solution of 21a–m (5.53 mmol) in acetone (15 mL) were added chloroacetone (0.67 mL, 8.30 mmol) and K₂CO₃ (915 mg, 6.64 mmol) at room temperature, and the resulting mixture was refluxed for 24 h. After cooling, the solvent was evaporated to give the residue, which was directly used in the next step. To a stirred solution of the above oil in ethylene glycol (15 mL) was added hydrazine monohydrate (1.08 mL, 22.12 mmol) at 0 °C, and the reaction mixture was stirred at 120 °C for 30 min. Then, KOH (930 mg, 16.59 mmol) was added to the reaction mixture at room temperature, and the resulting mixture was stirred at 160 °C for 5 h. After cooling, the reaction was quenched with water, and the aqueous mixture was extracted with Et₂O (3 mL × 3). The organic extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (20 g, n-hexane/EtOAc = 50/1–20/1) to give the title compounds.

2-Ethyl-4-iodobenzofuran (23a)

Yield: 56% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.54 (1H, d, J = 8.0 Hz), 7.37 (1H, d, J = 8.0 Hz), 6.95 (1H, t, J = 8.0 Hz), 6.31 (1H, s), 2.80 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 161.59, 153.07, 134.58, 131.75, 124.64, 110.70, 104.41, 85.08, 21.99, 11.89; IR (neat): 2974, 2935, 2878, 1595, 1570, 1420, 1256, 1163, 897, 766 cm^–1; high-resolution mass spectrometry (HRMS) (ESI): calcd for C₁₀H₉IO, 272.9771 ([M + H]⁺); found, 272.9763.

4-Bromo-2-ethylbenzofuran (23b)

Yield: 77% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.35 (1H, d, J = 8.0 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.07 (1H, t, J = 8.0 Hz), 6.43 (1H, d, J = 0.8 Hz), 2.80 (2H, qd, J = 7.6, 0.8 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (125 MHz, CDCl₃) δ: 161.86, 154.45, 130.56, 125.47, 124.15, 113.26, 109.88, 101.41, 21.94, 11.83; IR (neat): 2976, 2935, 1595, 1578, 1472, 1423, 1258, 1161, 806, 910 cm^–1; HRMS (ESI): calcd for C₁₀H₉BrO, 224.9910 ([M + H]⁺); found, 224.9913.

4-Chloro-2-ethylbenzofuran (23c)

Yield: 54% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.31 (1H, dt, J = 8.0, 0.8 Hz), 7.18 (1H, dd, J = 8.0, 0.8 Hz), 7.13 (1H, t, J = 8.0 Hz), 6.49 (1H, d, J = 0.8 Hz), 2.81 (2H, qd, J = 7.6, 0.8 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 161.93, 155.01, 128.51, 125.23, 123.79, 122.46, 109.41, 99.86, 21.95, 11.88; IR (neat): 2982, 2941, 1609, 1589, 1454, 1427, 1258, 1167, 935, 773 cm^–1; HRMS (ESI): calcd for C₁₀H₉OCl, 179.0269 ([M-H]⁻); found, 179.0269.

4,7-Dichloro-2-ethylbenzofuran (23d)

Yield: 54% in 2 steps (pale-yellow solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.13 (1H, d, J = 8.0 Hz), 7.10 (1H, d, J = 8.0 Hz), 6.51 (1H, t, J = 1.2 Hz), 2.85 (2H, qd, J = 8.0, 1.2 Hz), 1.37 (3H, t, J = 8.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 163.01, 150.44, 129.71, 123.88, 123.78, 123.24, 114.96, 100.75, 21.92, 11.77; IR (KBr): 3126, 2978, 2916, 1475, 1391 cm^–1; melting point (mp): 31–32 °C; HRMS (ESI): calcd for C₁₀H₈Cl₂O, 215.0025 ([M + H]⁺); found, 215.0034.

2-Ethyl-4-methylbenzofuran (23e)

Yield: 58% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.25 (1H, d, J = 7.6 Hz), 7.11 (1H, t, J = 7.6 Hz), 6.98 (1H, d, J = 7.6 Hz), 6.39 (1H, d, J = 0.8 Hz), 2.81 (2H, qd, J = 7.6, 0.8 Hz), 2.49 (3H, s), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 160.39, 154.36, 130.07, 128.65, 122.98, 122.65, 108.09, 99.59, 21.81, 18.58, 11.95; IR (neat): 2922, 2852, 1558, 1533, 1496, 1458, 1259, 1244, 920, 796 cm^–1; HRMS (ESI): calcd for C₁₁H₁₂O, 161.0961 ([M + H]⁺); found, 161.0949.

2-Ethyl-4-(trifluoromethyl)benzofuran (23f)

Yield: 47% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.57 (1H, d, J = 8.4 Hz), 7.45 (1H, d, J = 8.4 Hz), 7.27 (1H, t, J = 8.4 Hz), 6.58 (1H, s), 2.84 (2H, qd, J = 7.6, 0.8 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (125 MHz, CDCl₃) δ: 163.04, 154.90, 126.29, 124.40 (q, J = 269.5 Hz), 122.63, 121.89 (q, J = 32.3 Hz), 119.62 (q, J = 4.8 Hz), 114.09, 100.05, 21.81, 11.66; IR (neat): 3020, 2982, 1541, 1508, 1474, 1456, 1338, 1217, 688, 669 cm^–1; HRMS (ESI): calcd for C₁₁H₉F₃O, 215.0678 ([M + H]⁺); found, 215.0689.

5-Chloro-2-ethylbenzofuran (23g)

Yield: 81% in 2 steps; ¹H NMR (400 MHz, CDCl₃) δ: 7.44 (1H, d, J = 1.8 Hz), 7.31 (1H, d, J = 8.4 Hz), 7.16 (1H, dd, J = 8.4, 1.8 Hz), 6.33 (1H, q, J = 1.0 Hz), 2.79 (2H, qd, J = 7.6, 1.0 Hz), 1.33 (3H, t, J = 7.6 Hz).⁴⁸

6-Chloro-2-ethylbenzofuran (23h)

Yield: 62% in 2 steps; ¹H NMR (400 MHz, CDCl₃) δ: 7.41 (1H, s), 7.37 (1H, d, J = 8.0 Hz), 7.16 (1H, dd, J = 8.0, 1.8 Hz), 6.35 (1H, q, J = 1.0 Hz), 2.78 (2H, qd, J = 7.6, 1.0 Hz), 1.33 (3H, t, J = 7.6 Hz).⁴⁹

7-Chloro-2-ethylbenzofuran (23i)

Yield: 61% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.37 (1H, dd, J = 8.0, 1.0 Hz), 7.21 (1H, dd, J = 8.0, 1.0 Hz), 7.11 (1H, t, J = 8.0 Hz), 6.42 (1H, t, J = 1.0 Hz), 2.85 (2H, qd, J = 7.6, 1.0 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 162.24, 150.49, 130.77, 123.45, 123.43, 118.88, 116.25, 101.86, 21.89, 11.93; IR (neat): 2976, 2941, 1601, 1475, 1427, 1171, 1144, 920, 810, 735 cm^–1; HRMS (ESI): calcd for C₁₀H₉ClO, 181.0415 ([M + H]⁺); found, 181.0411.

2-Ethyl-4-fluorobenzofuran (23j)

Yield: 53% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.22 (1H, d, J = 8.4 Hz), 7.13 (1H, ddd, J = 8.4, 7.6, 5.2 Hz), 6.87 (1H, dd, J = 9.6, 8.4 Hz), 6.47 (1H, d, J = 0.8 Hz), 2.80 (2H, qd, J = 7.6, 0.8 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 161.14, 156.76 (d, J = 10.5 Hz), 155.55 (d, J = 246.9 Hz), 123.60 (d, J = 6.7 Hz), 118.02 (d, J = 21.0 Hz), 108.12 (d, J = 19.1 Hz), 107.05 (d, J = 3.8 Hz), 97.34, 21.84, 11.90; IR (neat): 2978, 2941, 1591, 1495, 1439, 1267, 1242, 1204, 1022, 770 cm^–1; HRMS (ESI): calcd for C₁₀H₉FO, 165.0710 ([M + H]⁺); found, 165.0710.

4,5-Dichloro-2-ethylbenzofuran (23k)

Yield: 50% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.27 (1H, d, J = 8.6 Hz), 7.24 (1H, dd, J = 8.6, 0.8 Hz), 6.47 (1H, s), 2.80 (2H, qd, J = 7.6, 1.2 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 163.29, 152.98, 130.05, 126.23, 124.51, 123.18, 110.18, 100.51, 21.97, 11.77; IR (neat): 2978, 2939, 1593, 1447, 1427, 1265, 1159, 1146, 949, 797 cm^–1; HRMS (ESI): calcd for C₁₀H₈Cl₂O, 215.0025 ([M + H]⁺); found, 215.0020.

6,7-Dichloro-2-ethylbenzofuran (23l)

Yield: 49% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.29 (1H, d, J = 8.4 Hz), 7.26 (1H, d, J = 8.4 Hz), 6.39 (1H, q, J = 0.8 Hz), 2.83 (2H, qd, J = 7.4, 0.8 Hz), 1.35 (3H, t, J = 7.4 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 163.00, 151.31, 128.98, 127.36, 124.49, 118.67, 115.45, 101.86, 21.88, 11.81; IR (neat): 2978, 2939, 1601, 1454, 1421, 1285, 1155, 966, 920, 818 cm^–1; HRMS (ESI): calcd for C₁₀H₈Cl₂O, 215.0025 ([M + H]⁺); found, 215.0017.

2-Ethyl-4,7-difluorobenzofuran (23m)

Yield: 41% in 2 steps (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 6.86 (1H, ddd, J = 10.0, 8.8, 3.2 Hz), 6.77 (1H, td, J = 8.8, 3.2 Hz), 6.49 (1H, td, J = 2.4, 1.2 Hz), 2.83 (2H, qd, J = 7.6, 1.2 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 162.35, 151.14 (dd, J = 242.7, 2.4 Hz), 144.42 (dd, J = 242.2, 3.8 Hz), 142.81 (dd, J = 13.3, 9.5 Hz), 120.77 (dd, J = 23.9, 3.8 Hz), 109.41 (dd, J = 19.1, 8.6 Hz), 107.95 (dd, J = 21.9, 6.7 Hz), 98.16, 21.79, 11.82; IR (neat): 2980, 2943, 1599, 1512, 1418, 1236, 1184, 1015, 924, 741 cm^–1; HRMS (ESI): calcd for C₁₀H₈F₂O, 183.0616 ([M + H]⁺); found, 183.0623.

General Procedure for the Synthesis of 23e’ and 25a–b

To a stirred solution of MeP⁺Ph₃Br^– (690 mg, 1.93 mmol) in THF (5 mL) was added n-BuLi (1.6 M in n-hexane, 1.06 mL, 1.69 mmol) at 0 °C, and the reaction mixture was stirred at 0 °C for 5 min. To the solution was added a solution of 23e’ or 25a–b (0.97 mmol) in THF (3 mL) at 0 °C, and the resulting mixture was stirred at room temperature for 15 h. The reaction was quenched with sat. NH₄Cl (aq.), and the organic layer was separated. The aqueous layer was extracted with CH₂Cl₂ (3 mL × 3). The organic layer and extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (10 g, n-hexane/EtOAc = 50/1–30/1) to give the title compounds.

4-Methyl-2-(1-methylethenyl)benzofuran (23e’)

Yield: 92% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.28 (1H, d, J = 8.8 Hz), 7.17 (1H, dd, J = 8.8, 7.2 Hz), 6.99 (1H, d, J = 7.2 Hz), 6.65 (1H, s), 5.78 (1H, s), 5.17 (1H, t, J = 1.0 Hz), 2.51 (3H, s), 2.15 (3H, d, J = 1.0 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 156.42, 154.71, 132.97, 131.01, 128.90, 124.60, 123.06, 113.02, 108.51, 101.61, 19.51, 18.68; IR (neat): 3057, 2953, 2855, 1614, 1551, 1491, 1454, 1421, 1246, 1175 cm^–1; HRMS (ESI): calcd for C₁₂H₁₂O, 173.0961 ([M + H]⁺); found, 173.0955.

2-Vinylbenzo[b]thiophene (25a)

Yield: 85%; ¹H NMR (400 MHz, CDCl₃) δ: 7.81–7.75 (1H, m), 7.70–7.67 (1H, m), 7.36–7.28 (2H, m), 6.93 (1H, dd, J = 17.2, 10.4 Hz), 5.68 (1H, d, J = 17.4 Hz), 5.32 (1H, d, J = 10.4 Hz).⁵⁰

2-Vinyl-1H-indole (25b)

Yield: 93%; ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (1H, br), 7.57 (1H, d, J = 7.6 Hz), 7.33 (1H, dd, J = 7.6, 0.8 Hz), 7.19 (1H, td, J = 7.6, 0.8 Hz), 7.09 (1H, td, J = 7.6, 0.8 Hz), 6.75 (1H, dd, J = 17.6, 11.2 Hz), 6.51 (1H, s), 5.54 (1H, d, J = 17.6 Hz), 5.27 (1H, d, J = 11.2 Hz).⁵¹

Synthesis of 4-Methyl-2-(1-methylethyl)benzofuran (23e”)

To a stirred solution of 23e’ (150 mg, 0.87 mmol) in EtOAc (3 mL) was added 10% Pd/C (3 mg), and the resulting mixture was hydrogenated at 1 atm for 20 h. The catalyst was removed through a Celite pad and washed with EtOAc (2 mL × 3). The filtrate and washings were combined and evaporated to give 23e” (127 mg, 84%, 0.73 mmol) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ: 7.26 (1H, d, J = 7.6 Hz), 7.11 (1H, t, J = 7.6 Hz), 6.98 (1H, d, J = 7.6 Hz), 6.38 (1H, s), 3.08 (1H, sep, J = 6.8 Hz), 2.49 (3H, s), 1.36 (6H, d, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 164.45, 154.48, 130.30, 128.68, 123.16, 122.76, 108.31, 98.44, 28.39, 21.14, 18.71; IR (neat): 2966, 2924, 2855, 1585, 1493, 1458, 1423, 1248, 770 cm^–1; HRMS (ESI): calcd for C₁₂H₁₄O, 175.1117 ([M + H]⁺); found, 175.1124.

Synthesis of 2-Ethylbenzo[b]thiophene (26a)

A solution of 25a (85 mg, 0.53 mmol), NiCl₂ (35 mg, 0.27 mmol), and Zn (260 mg, 3.98 mmol) in H₂O (2.7 mL) and THF (0.3 mL) was placed in a vessel suited for microwave irradiation and irradiated at 90 °C for 5 h. After cooling to room temperature, nickel and zinc were removed through a Celite pad and washed with EtOAc (2 mL × 3). The filtrate and washings were combined to give the residue, which was chromatographed on SiO₂ (10 g, n-hexane/EtOAc = 30/1) to give 26a (45 mg, 52%, 0.28 mmol). ¹H NMR (400 MHz, CDCl₃) δ: 7.78 (1H, d, J = 7.6 Hz), 7.68 (1H, d, J = 7.6 Hz), 7.32 (1H, td, J = 7.6, 1.2 Hz), 7.26 (1H, td, J = 7.6, 1.2 Hz), 7.03 (1H, d, J = 0.8 Hz), 2.96 (2H, qd, J = 7.6, 0.8 Hz), 1.40 (3H, t, J = 7.6 Hz).⁵²

Synthesis of 2-Ethyl-1H-indole (26b)

To a stirred solution of 25b (195 mg, 1.36 mmol) in EtOAc (3 mL) was added 10% Pd/C (5 mg), and the resulting mixture was hydrogenated at 1 atm for 18 h. The catalyst was removed through a Celite pad and washed with EtOAc (2 mL × 3). The filtrate and washings were combined and evaporated to give 26b (188 mg, 95%, 1.29 mmol). ¹H NMR (400 MHz, CDCl₃) δ: 7.87 (1H, br), 7.54 (1H, d, J = 7.8 Hz), 7.30 (1H, dd, J = 7.8, 1.2 Hz), 7.12 (1H, td, J = 7.8, 1.2 Hz), 7.07 (1H, td, J = 7.8, 1.2 Hz), 6.25 (1H, t, J = 0.8 Hz), 2.80 (2H, qd, J = 7.6, 0.8 Hz), 1.35 (3H, t, J = 7.6 Hz).⁵³

General Procedure for the Synthesis of 27a–e and 27g–p

To a stirred solution of 23a–m, 23e”, or 26a–b (3.13 mmol) in CH₂Cl₂ (10 mL for 23a–d, 23g–j, 23k–m,and 26b) or CS₂ (10 mL for 23e, 23e”, and 26a) were added 4-methoxybenzoyl chloride (690 mg, 4.04 mmol) and AlCl₃ (540 mg, 4.04 mmol for 23a–d, 23g–j, 23k–m, and 26b) or SnCl₄ (4.04 mL, 1 M in CH₂Cl₂, 4.04 mmol for 23e, 23e”, and 26a) at 0 °C, and the resulting mixture was stirred at room temperature for 12 h. The reaction was quenched with sat. NaHCO₃ (aq.), and the layers were separated. The aqueous layer was extracted with CH₂Cl₂ (3 mL × 3). The organic layer and extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (20 g, n-hexane/EtOAc = 20/1–10/1) to give the title compounds.

(2-Ethyl-4-iodobenzofuran-3-yl)(4-methoxyphenyl)methanone (27a)

Yield: 98% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.86 (2H, d, J = 8.8 Hz), 7.62 (1H, d, J = 8.0 Hz), 7.48 (1H, d, J = 8.0 Hz), 7.00 (1H, t, J = 8.0 Hz), 6.93 (2H, d, J = 8.8 Hz), 3.87 (3H, s), 2.70 (2H, q, J = 7.6 Hz), 1.25 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.51, 164.11, 160.67, 153.22, 134.23, 132.91, 132.23, 131.24, 125.57, 117.75, 114.01, 111.04, 84.12, 55.60, 21.15, 12.48; IR (neat): 3067, 2976, 2935, 2837, 1651, 1599, 1574, 1464, 1259, 1161 cm^–1; HRMS (ESI): calcd for C₁₈H₁₅O₃I, 407.0139 ([M + H]⁺); found, 407.0124.

(4-Bromo-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27b)

Yield: 94% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.87 (2H, d, J = 8.8 Hz), 7.45 (1H, dd, J = 8.0, 0.8 Hz), 7.36 (1H, dd, J = 8.0, 0.8 Hz), 7.15 (1H, t, J = 8.0 Hz), 6.93 (2H, d, J = 8.8 Hz), 3.88 (3H, s), 2.73 (2H, q, J = 7.6 Hz), 1.27 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.60, 164.06, 161.04, 154.06, 132.26, 132.14, 127.93, 127.43, 125.22, 116.50, 113.93, 113.30, 110.26, 55.58, 21.12, 12.45; IR (neat): 2976, 2839, 1651, 1601, 1463, 1411, 1248, 1161, 1028, 914, 841 cm^–1; HRMS (ESI): calcd for C₁₈H₁₅O₃Br, 359.0277 ([M + H]⁺); found, 359.0267.

(4-Chloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27c)

Yield: 95% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.86 (2H, d, J = 8.8 Hz), 7.40 (1H, dd, J = 8.0, 1.2 Hz), 7.23 (1H, t, J = 8.0 Hz), 7.18 (1H, dd, J = 8.0, 1.2 Hz), 6.93 (2H, d, J = 8.8 Hz), 3.87 (3H, s), 2.75 (2H, q, J = 7.6 Hz), 1.28 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.58, 164.07, 161.54, 154.39, 132.14, 132.06, 126.16, 125.81, 124.97, 124.27, 115.85, 113.92, 109.78, 55.63, 21.17, 12.57; IR (neat): 2978, 2937, 1740, 1653, 1576, 1462, 1426, 1261, 1165, 939 cm^–1; HRMS (ESI): calcd for C₁₈H₁₅O₃Cl, 315.0783 ([M + H]⁺); found, 315.0774.

(4,7-Dichloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27d)

Yield: 82% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.85 (2H, d, J = 9.2 Hz), 7.22 (1H, d, J = 8.4 Hz), 7.12 (1H, d, J = 8.4 Hz), 6.93 (2H, d, J = 9.2 Hz), 3.88 (3H, s), 2.78 (2H, q, J = 7.6 Hz), 1.31 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.63, 164.18, 162.11, 149.90, 132.05, 131.64, 127.25, 124.95, 124.87, 124.23, 116.46, 115.41, 113.96, 55.59, 21.14, 12.53; IR (neat): 3075, 2974, 2939, 2839, 1659, 1601, 1574, 1472, 1242, 1161 cm^–1; HRMS (ESI): calcd for C₁₈H₁₄O₃Cl₂, 349.0393 ([M + H]⁺); found, 349.0381.

(2-Ethyl-4-methylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27e)

Yield: 44% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.88 (2H, d, J = 8.8 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.19 (1H, t, J = 8.0 Hz), 6.98 (1H, d, J = 8.0 Hz), 6.94 (2H, d, J = 8.8 Hz), 3.88 (3H, s), 2.69 (2H, q, J = 7.6 Hz), 2.16 (3H, s), 1.26 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.99, 164.01, 160.60, 153.95, 132.13, 132.06, 131.44, 126.62, 124.83, 124.34, 116.73, 113.95, 108.58, 55.57, 21.34, 20.15, 12.58; IR (neat): 2974, 2935, 2841, 1651, 1599, 1508, 1452, 1259, 1169, 1028 cm^–1; HRMS (ESI): calcd for C₁₉H₁₈O₃, 295.1329 ([M + H]⁺); found, 295.1326.

(5-Chloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27g)

Yield: 80% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.82 (2H, d, J = 8.8 Hz), 7.40 (1H, d, J = 2.4 Hz), 7.39 (1H, d, J = 8.4 Hz), 7.24 (1H, dd, J = 8.4, 2.4 Hz,), 6.98 (2H, d, J = 8.8 Hz), 3.91 (3H, s), 2.89 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.01, 166.71, 163.80, 152.15, 131.79, 131.63, 129.26, 128.74, 124.64, 121.05, 116.07, 113.97, 112.07, 55.68, 21.97, 12.39; IR (KBr): 3090, 2944, 1643, 1598, 1503, 1459, 1255, 930 cm^–1; mp: 77–79 °C; HRMS (ESI): calcd for C₁₈H₁₅O₃Cl, 315.0783 ([M + H]⁺); found, 315.0783.

2(6-Chloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27h)

Yield: 94% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.82 (2H, d, J = 8.8 Hz), 7.49 (1H, d, J = 2.0 Hz), 7.31 (1H, d, J = 8.0 Hz), 7.18 (1H, dd, J = 8.0, 2.0 Hz), 6.96 (2H, d, J = 8.8 Hz), 3.90 (3H, s), 2.89 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.15, 166.07, 163.74, 153.78, 131.80, 131.75, 130.22, 126.07, 124.21, 121.96, 116.19, 113.90, 111.68, 55.67, 21.90, 12.44; IR (KBr): 3069, 2840, 1648, 1605, 1508, 1308, 1231, 1167 cm^–1; mp: 73–75 °C; HRMS (ESI): calcd for C₁₈H₁₅O₃Cl, 315.0783 ([M + H]⁺); found, 315.0771.

(7-Chloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27i)

Yield: 91% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.83 (2H, d, J = 9.0 Hz), 7.30 (1H, dd, J = 7.6, 1.2 Hz), 7.28 (1H, d, J = 7.6 Hz), 7.13 (1H, t, J = 7.6 Hz), 6.97 (2H, d, J = 9.0 Hz), 3.90 (3H, s), 2.93 (2H, q, J = 7.6 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.09, 166.00, 163.78, 149.57, 131.86, 131.62, 129.03, 124.55, 124.39, 119.90, 116.88, 116.64, 113.91, 55.66, 21.90, 12.58; IR (neat): 3078, 2976, 2841, 1645, 1600, 1510, 1472, 1427, 1312, 1238, 1169, 1030 cm^–1; HRMS (ESI): calcd for C₁₈H₁₅O₃Cl, 315.0783 ([M + H]⁺); found, 315.0773.

(2-Ethyl-4-fluorobenzofuran-3-yl)(4-methoxyphenyl)methanone (27j)

Yield: 95% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.87 (2H, d, J = 8.8 Hz), 7.30 (1H, d, J = 8.0 Hz), 7.23 (1H, td, J = 8.0, 4.8 Hz), 6.94 (2H, d, J = 8.8 Hz), 6.89 (1H, ddd, J = 10.0, 8.0, 0.8 Hz), 3.88 (3H, s), 2.85 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.74, 163.83, 163.53, 155.45 (d, J = 8.6 Hz), 155.43 (d, J = 250.8 Hz), 131.91, 131.61 (d, J = 2.0 Hz), 124.99 (d, J = 7.7 Hz), 116.26 (d, J = 19.1 Hz), 114.55 (d, J = 3.8 Hz), 113.73, 109.81 (d, J = 20.0 Hz), 107.38 (d, J = 4.8 Hz), 55.58, 21.34, 12.59; IR (KBr): 2976, 2935, 2839, 1651, 1599, 1495, 1437, 1261, 1171, 1030 cm^–1; mp: 57–59 °C; HRMS (ESI): calcd for C₁₈H₁₅O₃F, 299.1078 ([M + H]⁺); found, 299.1066.

(2-Ethylbenzo[b]thien-3-yl)(4-methoxyphenyl)methanone (27k)

Yield: 64%; ¹H NMR (400 MHz, CDCl₃) δ: 7.84 (2H, d, J = 9.0 Hz), 7.80 (1H, dt, J = 7.6, 1.4 Hz), 7.45 (1H, dd, J = 7.6, 1.4 Hz), 7.29 (1H, td, J = 7.6, 1.4 Hz), 7.26 (1H, td, J = 7.6, 1.4 Hz), 6.93 (2H, d, J = 9.0 Hz), 3.88 (3H, s), 2.89 (2H, q, J = 7.6 Hz), 1.30 (3H, t, J = 7.6 Hz).⁵⁴

(2-Ethyl-1H-indol-3-yl)(4-methoxyphenyl)methanone (27l)

Yield: 30% (pale-yellow oil); ¹H NMR (400 MHz, DMSO-d₆) δ: 11.84 (1H, s), 7.65 (2H, d, J = 8.4 Hz), 7.40 (1H, d, J = 8.0 Hz), 7.23 (1H, d, J = 8.0 Hz), 7.10 (1H, td, J = 8.0, 1.2 Hz), 7.03 (2H, d, J = 8.4 Hz), 6.99 (1H, td, J = 8.0, 1.2 Hz), 3.85 (3H, s), 2.83 (2H, q, J = 7.6 Hz), 1.23 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.56, 161.96, 148.74, 134.99, 133.61, 130.77, 127.17, 121.58, 119.96, 113.52, 111.85, 111.36, 55.40, 20.69, 13.94; IR (KBr): 3390, 3078, 2941, 1609, 1457, 1277 cm^–1; HRMS (ESI): calcd for C₁₈H₁₇O₂N, 278.1187 ([M-H]⁻); found, 278.1190.

(4-Methyl-2-(1-methylethyl)benzofuran-3-yl)(4-methoxyphenyl)methanone (27m)

Yield: 60% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.89 (2H, d, J = 9.0 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.18 (1H, t, J = 8.0 Hz), 6.97 (1H, d, J = 8.0 Hz), 6.94 (2H, d, J = 9.0 Hz), 3.88 (3H, s), 3.03 (1H, sep, J = 6.8 Hz), 2.14 (3H, s), 1.29 (6H, d, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 192.37, 164.04, 163.23, 153.79, 132.14, 131.40, 126.64, 124.70, 124.28, 115.53, 113.95, 108.67, 55.61, 27.74, 21.23, 20.07; IR (neat): 2974, 2936, 1651, 1597, 1508, 1456, 1352, 1256, 1169, 1030 cm^–1; HRMS (ESI): calcd for C₂₀H₂₀O₃, 309.1485 ([M + H]⁺); found, 309.1488.

(4,5-Dichloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27n)

Yield: 81% (colorless oil); ¹H NMR (500 MHz, CDCl₃) δ: 7.84 (2H, d, J = 9.0 Hz), 7.37 (1H, d, J = 8.8 Hz), 7.35 (1H, d, J = 8.8 Hz), 6.94 (2H, d, J = 9.0 Hz), 3.88 (3H, s), 2.74 (2H, q, J = 7.5 Hz), 1.27 (3H, t, J = 7.5 Hz); ¹³C NMR (125 MHz, CDCl₃) δ: 190.14, 164.20, 162.45, 152.39, 132.09, 131.83, 127.86, 127.62, 125.78, 123.83, 116.17, 114.03, 110.55, 55.65, 21.21, 12.45; IR (neat): 3083, 2980, 2935, 2843, 1651, 1601, 1508, 1425, 1256, 1157, 901 cm^–1; HRMS (ESI): calcd for C₁₈H₁₄O₃Cl₂, 349.0393 ([M + H]⁺); found, 349.0380.

(6,7-Dichloro-2-ethylbenzofuran-3-yl)(4-methoxyphenyl)methanone (27o)

Yield: 83% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.81 (2H, d, J = 9.2 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.23 (1H, d, J = 8.0 Hz), 6.97 (2H, d, J = 9.2 Hz), 3.90 (3H, s), 2.92 (2H, q, J = 7.6 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.63, 166.49, 163.90, 150.40, 131.82, 131.43, 128.74, 127.33, 125.51, 119.76, 116.86, 115.92, 113.98, 55.69, 21.94, 12.49; IR (KBr): 3026, 2937, 1645, 1601, 1555, 1512, 1456, 1265, 897 cm^–1; mp: 78–80 °C; HRMS (ESI): calcd for C₁₈H₁₄O₃Cl₂, 349.0393 ([M + H]⁺); found, 349.0377.

(2-Ethyl-4,7-difluorobenzofuran-3-yl)(4-methoxyphenyl)methanone (27p)

Yield: 75% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.86 (2H, d, J = 8.8 Hz), 6.97 (1H, ddd, J = 9.6, 8.8, 3.6 Hz), 6.94 (2H, d, J = 8.8 Hz), 6.80 (1H, td, J = 8.8, 3.6 Hz), 3.88 (3H, s), 2.86 (2H, q, J = 7.6 Hz), 1.34 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.00, 164.17, 164.04, 151.00 (dd, J = 246.5, 2.4 Hz), 144.37 (dd, J = 244.1, 3.8 Hz), 141.83 (dd, J = 13.3, 9.5 Hz), 131.93, 131.26 (d, J = 1.9 Hz), 118.86 (dd, J = 21.5, 2.4 Hz), 115.06 (d, J = 2.9 Hz), 113.83, 110.74 (dd, J = 18.5, 8.1 Hz), 109.62 (dd, J = 22.4, 6.2 Hz), 55.60, 21.35, 12.52; IR (KBr): 2970, 2934, 1647, 1607, 1578, 1510, 1248, 1171, 1052, 903 cm^–1; mp: 62–63 °C; HRMS (ESI): calcd for C₁₈H₁₄O₃F₂, 317.0984 ([M + H]⁺); found, 317.0972.

General Procedure for the Synthesis of 28a–d, 16, and 28g–p

To a stirred solution of 27a–e or 27g–p (0.81 mmol) in CH₂Cl₂ (3 mL) was added BBr₃ (2.43 mL, 1 M in CH₂Cl₂, 2.43 mmol) at 0 °C, and the resulting mixture was stirred at room temperature for 24 h. The reaction was quenched with water, and the layers were separated. The aqueous layer was extracted with EtOAc (1 mL × 3). The organic layer and extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (10 g, n-hexane/EtOAc = 10/1–5/1) to give the title compounds.

(2-Ethyl-4-iodobenzofuran-3-yl)(4-hydroxyphenyl)methanone (28a)

Yield: 98% (white solid); ¹H NMR (500 MHz, CDCl₃) δ: 7.80 (2H, d, J = 9.0 Hz), 7.62 (1H, dd, J = 8.0, 0.7 Hz), 7.47 (1H, dd, J = 8.0, 0.7 Hz), 7.00 (1H, t, J = 8.0 Hz), 6.88 (2H, d, J = 9.0 Hz), 2.70 (2H, q, J = 7.5 Hz), 1.25 (3H, t, J = 7.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.61, 161.68, 160.96, 153.30, 134.40, 132.85, 132.55, 131.22, 125.72, 117.63, 115.92, 111.14, 84.19, 21.25, 12.47; IR (KBr): 3279, 2978, 2866, 1637, 1595, 1577, 1572, 1418, 1232, 1159 cm^–1; mp: 53–54 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃I, 392.9982 ([M + H]⁺); found, 392.9969.

(4-Bromo-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28b)

Yield: 95% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.79 (2H, d, J = 8.8 Hz), 7.44 (1H, d, J = 8.0 Hz), 7.35 (1H, d, J = 8.0 Hz), 7.14 (1H, t, J = 8.0 Hz), 6.88 (2H, d, J = 8.8 Hz), 6.84 (1H, br), 2.71 (2H, q, J = 7.6 Hz), 1.26 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.99, 161.97, 161.27, 154.10, 132.79, 131.60, 127.85, 127.57, 125.40, 116.28, 115.85, 113.31, 110.34, 21.19, 12.43; IR (KBr): 3250, 2982, 2841, 1639, 1578, 1597, 1421, 1361, 1232, 1159 cm^–1; mp: 57–58 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃Br, 345.0121 ([M + H]⁺); found, 345.0108.

(4-Choro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28c)

Yield: 69% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.82 (2H, d, J = 8.8 Hz), 7.40 (1H, dd, J = 7.6, 1.2 Hz), 7.22 (1H, t, J = 7.6 Hz), 7.18 (1H, dd, J = 7.6 1.2 Hz), 6.87 (2H, d, J = 8.8 Hz), 5.66 (1H, br), 2.75 (2H, q, J = 7.6 Hz), 1.28 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.56, 161.72, 161.52, 154.39, 132.67, 131.56, 126.03, 125.77, 125.09, 124.36, 115.73, 115.64, 109.83, 21.21, 12.49; IR (KBr): 3215, 2829, 1637, 1583, 1564, 1477, 1361, 1288, 1242, 1165 cm^–1; mp: 126–127 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃Cl, 301.0626 ([M + H]⁺); found, 301.0617.

(4,7-Dichoro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28d)

Yield: 75% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.81 (2H, d, J = 8.8 Hz), 7.23 (1H, d, J = 8.4 Hz), 7.12 (1H, d, J = 8.4 Hz), 6.88 (2H, d, J = 8.8 Hz), 5.71 (1H, br), 2.78 (2H, q, J = 7.6 Hz), 1.30 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.79, 162.38, 161.74, 149.99, 132.70, 131.26, 127.17, 125.14, 125.04, 124.26, 116.27, 115.83, 115.56, 21.26, 12.54; IR (KBr): 3281, 2974, 2920, 1637, 1578, 1474, 1394, 1238, 1159, 901 cm^–1; mp: 55–56 °C; HRMS (ESI): calcd for C₁₇H₁₂O₃Cl₂, 335.0236 ([M + H]⁺); found, 335.0224.

(2-Ethyl-4-methylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (16)

Yield: 53% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.81 (2H, d, J = 8.8 Hz), 7.32 (1H, d, J = 8.0 Hz), 7.18 (1H, t, J = 8.0 Hz), 6.98 (1H, d, J = 8.0 Hz), 6.89 (2H, d, J = 8.8 Hz), 2.69 (2H, q, J = 7.6 Hz), 2.15 (3H, s), 1.25 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 193.22, 161.64, 161.01, 154.00, 132.74, 131.58, 131.43, 126.56, 125.01, 124.52, 116.61, 115.80, 108.69, 21.39, 20.24, 12.59; IR (KBr): 3169, 2978, 2908, 1583, 1574, 1564, 1510, 1360, 1288, 1238 cm^–1; mp: 100–101 °C; HRMS (ESI): calcd for C₁₈H₁₆O₃, 281.1172 ([M + H]⁺); found, 281.1164.

(5-Choro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28g)

Yield: 61% (pale-yellow solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.78 (2H, d, J = 8.8 Hz), 7.39 (1H, d, J = 8.4 Hz), 7.39 (1H, d, J = 2.4 Hz), 7.24 (1H, dd, J = 8.4, 2.4 Hz), 6.93 (2H, d, J = 8.8 Hz), 6.19 (1H, br), 2.89 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.74, 165.64, 162.38, 151.59, 131.72, 129.45, 128.54, 128.06, 124.49, 120.08, 115.62, 115.41, 112.79, 21.37, 11.97; IR (KBr): 3566, 3070, 2940, 1591, 1520, 1349, 1260, 1133 cm^–1; mp: 154–156 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃Cl, 301.0630 ([M + H]⁺); found, 301.0633.

(6-Chloro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28h)

Yield: 100% (pale-yellow solid); ¹H NMR (400 MHz, DMSO-d₆) δ: 10.49 (1H, s), 7.85 (1H, d, J = 1.6 Hz), 7.69 (2H, d, J = 8.8 Hz), 7.37 (1H, d, J = 8.4 Hz), 7.32 (1H, dd, J = 8.4, 1.6 Hz), 6.89 (2H, d, J = 8.8 Hz), 2.80 (2H, q, J = 7.6 Hz), 1.23 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.79, 164.87, 162.40, 153.12, 131.73, 129.48, 129.05, 125.93, 124.08, 121.78, 115.60, 115.40, 111.56, 21.24, 12.04; IR (KBr): 3412, 2848, 1591, 1540, 1399, 1311, 1260, 943 cm^–1; mp: 150–152 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃Cl, 301.0630 ([M + H]⁺); found, 301.0630.

(7-Choro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28i)

Yield: 93% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.78 (2H, d, J = 8.8 Hz), 7.31 (1H, dd, J = 8.0, 1.2 Hz), 7.28 (1H, dd, J = 8.0, 1.2 Hz), 7.13 (1H, t, J = 8.0 Hz), 6.93 (2H, d, J = 8.8 Hz), 2.93 (2H, q, J = 7.6 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.85, 166.32, 160.99, 149.59, 132.30, 131.31, 128.91, 124.68, 124.50, 119.85, 116.81, 116.71, 115.65, 21.96, 12.55; IR (KBr): 3223, 2939, 1583, 1556, 1425, 1354, 1265, 1165, 1148, 899 cm^–1; mp: 152–153 °C; HRMS (ESI): calcd for C₁₇H₁₃O₃Cl, 301.0630 ([M + H]⁺); found, 301.0622.

(2-Ethyl-4-fluorobenzofuran-3-yl)(4-hydroxyphenyl)methanone (28j)

Yield: 70% (colorless oil); ¹H NMR (400 MHz, CDCl₃) δ: 7.89 (2H, d, J = 8.8 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.22 (1H, td, J = 8.4, 5.2 Hz), 6.91 (2H, d, J = 8.8 Hz), 6.88 (1H, dd, J = 8.4, 8.0 Hz), 2.81 (2H, q, J = 7.6 Hz), 1.30 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.03, 163.72, 161.74, 155.48 (d, J = 9.6 Hz), 155.40 (d, J = 250.8 Hz), 132.49, 130.85, 125.18 (d, J = 7.6 Hz), 116.13 (d, J = 20.1 Hz), 115.62, 114.39, 109.92 (d, J = 19.1 Hz), 107.41 (d, J = 3.8 Hz), 21.43, 12.43; IR (neat): 3325, 2982, 2939, 1634, 1591, 1495, 1362, 1242, 1167, 1032 cm^–1; HRMS (ESI): calcd for C₁₇H₁₃O₃F, 285.0922 ([M + H]⁺); found, 285.0918.

(2-Ethylbenzo[b]thiophen-3-yl)(4-hydroxyphenyl)methanone (28k)

Removal of the methyl group of 27k using BBr₃ gave a complex mixture containing 28k. Since 28k could not be purified by column chromatography, the mixture containing 28k was subjected directly to the next iodination reaction.

(2-Ethyl-1H-indol-3-yl)(4-hydroxyphenyl)methanone (28l)

Yield: 86% (yellow oil); ¹H NMR (400 MHz, DMSO-d₆) δ: 11.78 (1H, s), 10.14 (1H, s), 7.55 (2H, d, J = 8.4 Hz), 7.38 (1H, d, J = 8.0 Hz), 7.25 (1H, d, J = 8.0 Hz), 7.09 (1H, t, J = 8.0 Hz), 6.99 (1H, t, J = 8.0 Hz), 6.84 (2H, d, J = 8.4 Hz), 2.82 (2H, q, J = 7.6 Hz), 1.23 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 190.60, 160.77, 148.29, 134.97, 132.08, 131.08, 127.24, 121.50, 120.46, 120.00, 114.86, 111.99, 111.30, 20.64, 13.97; IR (neat): 3402, 3006, 2980, 2939, 1601, 1561, 1497, 1247 cm^–1; HRMS (ESI): calcd for C₁₇H₁₅O₂N, 266.1176 ([M + H]⁺); found, 266.1174.

(4-Hydroxyphenyl)(4-methyl-2-(1-methylethyl)benzofuran-3-yl)methanone (28m)

Yield: 71% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.83 (2H, d, J = 9.0 Hz), 7.33 (1H, d, J = 8.0 Hz), 7.18 (1H, t, J = 8.0 Hz), 7.97 (1H, d, J = 8.0 Hz), 6.88 (2H, d, J = 9.0 Hz), 6.25 (1H, br), 3.02 (1H, sep, J = 6.8 Hz), 2.13 (3H, s), 1.28 (6H, d, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 193.94, 163.59, 162.01, 153.78, 132.79, 131.37, 131.30, 126.52, 124.85, 124.46, 115.83, 115.30, 108.76, 27.77, 21.21, 20.09; IR (KBr): 3163, 3084, 2822, 1560, 1541, 1516, 1358, 1288, 1238, 1171, 910 cm^–1; mp: 184–185 °C; HRMS (ESI): calcd for C₁₉H₁₈O₃, 295.1329 ([M + H]⁺); found, 295.1322.

(4,5-Dichoro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28n)

Yield: 84% (white solid); ¹H NMR (500 MHz, CDCl₃) δ: 7.80 (2H, d, J = 9.0 Hz), 7.37 (1H, d, J = 9.0 Hz), 7.34 (1H, d, J = 9.0 Hz), 6.88 (2H, d, J = 9.0 Hz), 2.73 (2H, q, J = 7.5 Hz), 1.27 (3H, t, J = 7.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.78, 162.63, 161.25, 152.42, 132.57, 131.72, 127.96, 127.54, 125.90, 123.83, 116.02, 115.77, 110.9, 21.26, 12.44; IR (KBr): 3360, 3119, 1641, 1605, 1572, 1427, 1356, 1231, 1157, 901 cm^–1; mp: 152–153 °C; HRMS (ESI): calcd for C₁₇H₁₂O₃Cl₂, 335.0236 ([M + H]⁺); found, 335.0224.

(6,7-Dichoro-2-ethylbenzofuran-3-yl)(4-hydroxyphenyl)methanone (28o)

Yield: 74% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.77 (2H, d, J = 8.4 Hz), 7.29 (1H, d, J = 8.0 Hz), 7.23 (1H, d, J = 8.0 Hz), 6.91 (2H, d, J = 8.4 Hz), 5.82 (1H, s), 2.92 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 188.37, 165.51, 162.61, 149.45, 131.92, 129.20, 127.46, 127.34, 125.65, 120.14, 116.43, 115.49, 114.42, 21.32, 12.01; IR (KBr): 3221, 2970, 1622, 1580, 1543, 1516, 1456, 1367, 1271, 1161 cm^–1; mp: 175–176 °C; HRMS (ESI): calcd for C₁₇H₁₂O₃Cl₂, 335.0236 ([M + H]⁺); found, 335.0223.

(2-Ethyl-4,7-difluorobenzofuran-3-yl)(4-hydroxyphenyl)methanone (28p)

Yield: 75% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 7.81 (2H, d, J = 8.8 Hz), 6.97 (1H, td, J = 9.2, 3.6 Hz), 6.92 (2H, d, J = 8.8 Hz), 6.80 (1H, td, J = 9.2, 3.2 Hz), 2.84 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.46, 164.41, 161.92, 150.96 (dd, J = 246.5 Hz), 144.53 (dd, J = 245.0, 3.8 Hz), 141.88 (dd, J = 13.4, 9.6 Hz), 132.59, 130.55, 118.69 (dd, J = 21.9, 1.9 Hz), 115.77, 114.88 (d, J = 3.8 Hz), 110.99 (dd, J = 18.6, 8.1 Hz), 109.80 (dd, J = 22.4, 6.2 Hz), 21.47, 12.38; IR (KBr): 3126, 2949, 2878, 1602, 1622, 1549, 1512, 1250, 1169, 905 cm^–1; mp: 119–120 °C; HRMS (ESI): calcd for C₁₇H₁₂O₃F₂, 303.0827 ([M + H]⁺); found, 303.0818.

General Procedure for the Synthesis of 4–8, 10–15, and 17–20

To a stirred solution of 28a–d, 16, or 28g–p (0.11 mmol) in EtOH (3 mL) were added I₂ (108 mg, 0.42 mmol) and K₂CO₃ (44 mg, 0.32 mmol) at room temperature, and the resulting mixture was refluxed for 12 h. The reaction was quenched with 10% HCl (aq.). The aqueous mixture was extracted with EtOAc (3 mL × 3). The organic extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (10 g, n-hexane/EtOAc = 10/1–5/1) to give the title compounds.

(2-Ethyl-4-iodobenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (4)

Yield: 93% (white solid); ¹H NMR (500 MHz, CDCl₃) δ: 8.23 (2H, s), 7.67 (1H, d, J = 8.0 Hz), 7.51 (1H, d, J = 8.0 Hz), 7.05 (1H, t, J = 8.0 Hz), 2.69 (2H, q, J = 7.5 Hz), 1.28 (3H, t, J = 7.5 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.72, 161.69, 157.90, 153.42, 141.27, 136.00, 134.70, 130.83, 126.03, 116.75, 111.30, 84.04, 82.56, 21.43, 12.52; IR (KBr): 3440, 2966, 2928, 1647, 1570, 1533, 1454, 1418, 1350, 1227 cm^–1; mp: 107–108 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃I₃, 644.7915 ([M + H]⁺); found, 644.7911.

(4-Bromo-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (5)

Yield: 84% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.21 (2H, s), 7.48 (1H, dd, J = 8.0, 0.8 Hz), 7.40 (1H, d, J = 8.0 Hz), 7.19 (1H, t, J = 8.0 Hz), 2.72 (2H, q, J = 7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.74, 162.14, 157.79, 154.19, 141.19, 135.37, 127.88, 127.45, 125.70, 115.50, 113.21, 110.52, 82.41, 21.37, 12.54; IR (KBr): 3346, 2935, 2878, 1632, 1569, 1543, 1303, 1221, 1169, 1111 cm^–1; mp: 148–150 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃BrI₂, 596.8054 ([M + H]⁺); found, 596.8032.

(4-Chloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (6)

Yield: 97% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.21 (2H, s), 7.43, (1H, dd, J = 7.6, 1.6 Hz), 7.25 (1H, t, J = 7.6 Hz), 7.22 (1H, dd, J = 7.6, 1.6 Hz), 6.24 (1H, br), 2.75 (2H, q, J = 7.6 Hz), 1.31 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.61, 162.62, 157.78, 154.42, 141.12, 135.08, 125.61, 125.54, 125.38, 124.64, 114.81, 110.00, 82.35, 21.35, 12.54; IR (KBr): 3386, 2922, 1639, 1572, 1456, 1393, 1283, 1132, 947, 766 cm^–1; mp: 143–145 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃ClI₂, 552.8559 ([M + H]⁺); found, 552.8536.

(4,7-Dichloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (7)

Yield: 93% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.19 (2H, s), 7.27 (1H, d, J = 8.4 Hz), 7.17 (1H, d, J = 8.4 Hz), 6.24 (1H, br), 2.78 (2H, q, J = 7.6 Hz), 1.33 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 186.86, 163.21, 157.98, 150.07, 141.11, 134.80, 126.75, 126.46, 125.46, 125.33, 124.14, 115.77, 115.49, 82.42, 21.41, 12.58; IR (KBr): 3254, 2939, 2885, 1649, 1570, 1456, 1288, 1227, 1123, 925 cm^–1; mp: 58–60 °C; HRMS (ESI): calcd for C₁₇H₁₀O₃Cl₂I₂, 586.8169 ([M + H]⁺); found, 586.8164.

(2-Ethyl-4-methylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (8)

Yield: 85% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.25 (2H, s), 7.35 (1H, d, J = 8.0 Hz), 7.22 (1H, t, J = 8.0 Hz), 7.03 (1H, d, J = 8.0 Hz), 6.23 (1H, br), 2.65 (2H, q, J = 7.6 Hz), 2.23 (3H, s), 1.28 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 188.92, 161.68, 157.78, 154.17, 141.26, 135.18, 131.56, 126.06, 125.34, 124.83, 115.82, 108.79, 82.43, 21.75, 20.52, 12.53; IR (KBr): 3449, 2932, 1649, 1578, 1429, 1360, 1308, 1229, 1163, 1124 cm^–1; mp: 103–104 °C; HRMS (ESI): calcd for C₁₈H₁₄O₃I₂, 532.9105 ([M + H]⁺); found, 532.9096.

(5-Chloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (10)

Yield: 78% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (2H, s), 7.48 (1H, d, J = 2.0 Hz), 7.41 (1H, d, J = 8.8 Hz), 7.27 (1H, dd, J = 8.8, 2.0 Hz), 6.23 (1H, s), 2.85 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 186.98, 167.42, 157.50, 152.25, 140.77, 134.92, 129.74, 128.19, 125.15, 120.95, 115.29, 112.23, 82.27, 22.30, 12.26; IR (KBr): 3204, 2935, 1620, 1570, 1448, 1390, 1288, 1231, 1128, 995 cm^–1; mp: 152–153 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃ClI₂, 552.8559 ([M + H]⁺); found, 552.8549.

(6-Chloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (11)

Yield: 78% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (2H, s), 7.51 (1H, d, J = 1.6 Hz), 7.36 (1H, d, J = 8.4 Hz), 7.23 (1H, dd, J = 8.4, 1.6 Hz), 6.20 (1H, s), 2.87 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.08, 166.96, 157.45, 153.86, 140.78, 135.01, 130.69, 125.48, 124.63, 121.74, 115.40, 111.89, 82.22, 22.16, 12.32; IR (KBr): 3325, 2935, 1639, 1578, 1558, 1528, 1305, 1267, 1126, 810 cm^–1; mp: 200–201 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃ClI₂, 552.8559 ([M + H]⁺); found, 552.8544.

(7-Chloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (12)

Yield: 93% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.17 (2H, s), 7.34 (1H, dd, J = 8.0, 1.2 Hz), 7.31 (1H, d, J = 8.0 Hz), 7.18 (1H, t, J = 8.0 Hz), 2.91 (2H, q, J = 7.6 Hz), 1.39 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.08, 166.86, 157.53, 149.66, 140.85, 134.84, 128.42, 125.01, 124.82, 119.67, 116.87, 116.06, 82.27, 22.17, 12.49; IR (KBr): 3287, 2947, 2910, 1638, 1574, 1522, 1456, 1425, 1306, 1219, 1119 cm^–1; mp: 138–140 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃ClI₂, 552.8559 ([M + H]⁺); found, 552.8552.

(2-Ethyl-4-fluorobenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (13)

Yield: 91% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.20 (2H, s), 7.33 (1H, dd, J = 8.4, 0.8 Hz), 7.27 (1H, td, J = 8.4, 5.2 Hz), 6.94 (1H, ddd, J = 10.0, 8.4, 0.8 Hz), 6.20 (1H, br), 2.86 (2H, q, J = 7.6 Hz), 1.35 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 186.75, 164.82, 157.48, 155.43 (d, J = 8.6 Hz), 155.26 (d, J = 256.7 Hz), 140.95, 134.81 (d, J = 1.9 Hz), 125.48 (d, J = 8.5 Hz), 115.60 (d, J = 19.1 Hz), 113.65 (d, J = 3.8 Hz), 110.17 (d, J = 20.1 Hz), 107.60 (d, J = 3.8 Hz), 82.04, 21.55, 12.50; IR (KBr): 3354, 3092, 2936, 1637, 1578, 1529, 1495, 1308, 1238, 1121 cm^–1; mp: 146–148 °C; HRMS (ESI): calcd for C₁₇H₁₁O₃FI₂, 536.8854 ([M + H]⁺); found, 536.8845.

(2-Ethylbenzo[b]thien-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (14)

Yield: 21% in 2 steps from 27k (yellow solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.18 (2H, s), 7.83–7.81 (1H, m), 7.51–7.47 (1H, m), 7.34–7.31 (2H, m), 6.22 (1H, br), 2.87 (2H, q, J = 7.6 Hz), 1.32 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.41, 157.70, 153.48, 141.26, 138.70, 138.05, 134.81, 130.50, 125.08, 124.68, 123.14, 122.23, 82.39, 23.75, 16.33; IR (KBr): 3355, 3075, 2908, 1621, 1549, 1311, 1232 cm^–1; mp: 131–132 °C; HRMS (ESI): calcd for C₁₇H₁₂O₂SI₂, 532.8675 ([M-H]⁻); found, 532.8555.

(2-Ethyl-1H-indol-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (15)

Yield: 52% (pale-yellow solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.50 (1H, br), 8.14 (2H, s), 7.39 (1H. d, J = 6.8 Hz), 7.37 (1H, d, J = 6.8 Hz), 7.21 (1H, ddd, J = 8.0, 6.8, 0.8 Hz), 7.13 (1H, ddd, J = 8.0, 6.8, 0.8 Hz), 6.10 (1H, s), 3.00 (2H, q, J = 7.6 Hz), 1.36 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, DMSO-d₆) δ: 187.50, 158.30, 149.52, 139.73, 136.50, 135.09, 126.94, 121.92, 120.92, 119.87, 111.54, 111.17, 86.09, 20.78, 13.78; IR (KBr): 3417, 3299, 3044, 2912, 1606, 1550 cm^–1; mp: 148–149 °C; HRMS (ESI): calcd for C₁₇H₁₃O₂NO₂I₂, 515.8963 ([M-H]⁻); found, 515.8947.

(4-Methyl-2-(1-methylethyl)benzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (17)

Yield: 70% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.27 (2H, s), 7.35 (1H, d, J = 7.6 Hz), 7.22 (1H, t, J = 7.6 Hz), 7.02 (1H, d, J = 7.6 Hz), 6.23 (1H, br), 2.92 (1H, sep, J = 6.8 Hz), 2.23 (3H, s), 1.30 (6H, d, J = 6.8 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 189.15, 164.37, 157.84, 154.05, 141.25, 135.22, 131.55, 126.05, 125.20, 124.76, 114.53, 108.86, 82.44, 28.11, 21.24, 20.41; IR (KBr): 3369, 2963, 2932, 1643, 1570, 1531, 1456, 1356, 1209 cm^–1; mp: 68–70 °C; HRMS (ESI): calcd for C₁₉H₁₆O₃I₂, 544.9116 ([M-H]⁻); found, 544.9104.

(4,5-Dichloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (18)

Yield: 91% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.20 (2H, s), 7.41 (1H, d, J = 8.8 Hz), 7.37 (1H, d, J = 8.8 Hz), 6.24 (1H, br), 2.72 (2H, q, J = 7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 187.29, 163.26, 157.98, 152.46, 141.07, 134.85, 128.25, 127.07, 126.23, 123.70, 115.07, 110.76, 82.49, 21.43, 12.43; IR (KBr): 3342, 2995, 2932, 1653, 1570, 1425, 1389, 1219, 1190, 1115 cm^–1; mp: 147–149 °C; HRMS (ESI): calcd for C₁₇H₁₀O₃Cl₂I₂, 586.8169 ([M + H]⁺); found, 586.8164.

(6,7-Dichloro-2-ethylbenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (19)

Yield: 85% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.16 (2H, s), 7.34 (1H, d, J = 8.6 Hz), 7.28 (1H, d, J = 8.6 Hz), 6.22 (1H, br), 2.90 (2H, q, J = 7.6 Hz), 1.39 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 186.66, 167.5, 157.62, 150.51, 140.80, 134.69, 129.26, 126.74, 125.92, 119.52, 116.21, 116.07, 82.28, 22.21, 12.39; IR (KBr): 3258, 3074, 2924, 1639, 1572, 1528, 1456, 1306, 1229, 1121 cm^–1; mp: 201–202 °C; HRMS (ESI): calcd for C₁₇H₁₀O₃Cl₂I₂, 586.8169 ([M + H]⁺); found, 586.8162.

(2-Ethyl-4,7-difluorobenzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (20)

Yield: 96% (white solid); ¹H NMR (400 MHz, CDCl₃) δ: 8.19 (2H, s), 7.01 (1H, td, J = 9.2, 4.0 Hz), 6.85 (1H, td, J = 9.2, 3.2 Hz), 6.23 (1H, br), 2.87 (2H, q, J = 7.6 Hz), 1.37 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 186.11, 165.42, 157.72, 150.82 (dd, J = 247.0, 2.9 Hz), 144.42 (dd, J = 245.0, 3.8 Hz), 141.88 (dd, J = 13.4, 9.5 Hz), 140.95, 134.42, 118.18 (dd, J = 21.5, 2.4 Hz), 114.15, 111.26 (dd, J = 18.6, 8.1 Hz), 110.03 (dd, J = 22.9, 6.6 Hz), 82.13, 21.56, 12.45; IR (KBr): 3366, 2935, 2874, 1636, 1508, 1462, 1304, 1246, 1204, 1124 cm^–1; mp: 152–153 °C; HRMS (ESI): calcd for C₁₇H₁₀O₃F₂I₂, 554.8760 ([M + H]⁺); found, 554.8749.

Synthesis of (2-Ethyl-4-iodobenzofuran-3-yl)(4-(methoxymethoxy)phenyl)methanone (29)

To a stirred solution of 28a (50 mg, 0.13 mmol) in CH₂Cl₂ (3 mL) were added DIPEA (0.03 mL, 0.16 mmol) and MOMCl (0.01 mL, 0.16 mmol) at room temperature, and the resulting mixture was refluxed for 1 h. The solvent was evaporated to give the residue, which was chromatographed on SiO₂ (5 g, n-hexane/EtOAc = 15/1) to give 29 (49 mg, 88%, 0.11 mmol) as a colorless oil. ¹H NMR (400 MHz, CDCl₃) δ: 7.86 (2H, d, J = 8.8 Hz), 7.62 (1H, dd, J = 8.0, 0.8 Hz), 7.48 (1H, dd, J = 8.0, 0.8 Hz), 7.07 (2H, d, J = 8.8 Hz), 7.00 (1H, t, J = 8.0 Hz), 5.24 (2H, s), 3.49 (3H, s), 2.70 (2H, q, J = 7.2 Hz), 1.26 (3H, t, J = 7.2 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 190.74, 161.82, 160.83, 153.32, 134.34, 133.83, 132.21, 131.34, 125.66, 117.78, 116.02, 111.11, 94.26, 84.20, 56.52, 21.25, 12.54; IR (neat): 2978, 2935, 1655, 1599, 1576, 1418, 1238, 1153, 1082 cm^–1; HRMS (ESI): calcd for C₁₉H₁₇O₄I, 437.0244 ([M + H]⁺); found, 437.0241.

Synthesis of (2-Ethyl-4-(trifluoromethyl)benzofuran-3-yl)(4-(methoxymethoxy)phenyl)methanone (30)

To a stirred solution of 29 (49 mg, 0.11 mmol) in DMSO (2 mL) were added CuI (4 mg, 0.02 mmol), 1,10-phenanthroline (4 mg, 0.02 mmol), KF (33 mg, 0.55 mmol), B(OMe)₃ (0.07 mL, 0.55 mmol), and TMSCF₃ (0.08 mL, 0.55 mmol) at room temperature, and the resulting mixture was heated at 50 °C for 1 h. The reaction was quenched with water. The aqueous mixture was extracted with CH₂Cl₂ (1 mL × 3). The organic extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (5 g, n-hexane/EtOAc = 7/1) to give 30 (21 mg, 50%, 0.06 mmol) as a pale-yellow oil. ¹H NMR (400 MHz, CDCl₃) δ: 7.86 (2H, d, J = 8.8 Hz), 7.68 (1H, d, J = 8.0 Hz), 7.54 (1H, d, J = 8.0 Hz), 7.38 (1H, t, J = 8.0 Hz), 7.06 (2H, d, J = 8.8 Hz), 5.24 (2H, s), 3.49 (3H, s), 2.68 (2H, q, J = 7.6 Hz), 1.26 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.05, 161.88, 161.29, 154.55, 132.08, 132.02, 124.06, 123.93, 123.79 (q, J = 270.8 Hz), 122.57 (q, J = 33.4 Hz), 121.46 (q, J = 4.8 Hz), 115.92, 115.54, 114.89, 94.25, 56.50, 21.33, 12.33; IR (neat): 2980, 2939, 1663, 1601, 1578, 1431, 1366, 1240, 1126, 1082 cm^–1; HRMS (ESI): calcd for C₂₀H₁₇O₄F₃, 379.1152 ([M + H]⁺); found, 379.1149.

Synthesis of (2-Ethyl-4-(trifluoromethyl)benzofuran-3-yl)(4-hydroxyphenyl)methanone (28f)

To a stirred solution of 30 (17 mg, 0.04 mmol) in MeOH (3 mL) was added conc. HCl (1 drop) at room temperature, and the resulting mixture was refluxed for 1 h. The solvent was evaporated to give the residue, which was chromatographed on SiO₂ (5 g, n-hexane/EtOAc = 5/1) to give 28f (15 mg, 100%, 0.04 mmol) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ: 7.82 (2H, d, J = 8.8 Hz), 7.67 (1H, d, J = 8.0 Hz), 7.54 (1H, d, J = 8.0 Hz), 7.38 (1H, t, J = 8.0 Hz), 6.87 (2H, d, J = 8.8 Hz), 6.26 (1H, br), 2.67 (2H, q, J = 7.6 Hz), 1.25 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 191.70, 161.45, 161.41, 154.53, 132.65, 130.93, 123.98, 123.78 (q, J = 270.8 Hz), 122.53 (q, J = 33.4 Hz), 121.50 (q, J = 4.8 Hz), 115.70, 115.44, 114.92, 21.31, 12.27; IR (KBr): 3306, 2953, 2858, 1637, 1601, 1564, 1431, 2390, 1161, 1109 cm^–1; mp: 144–145 °C; HRMS (ESI): calcd for C₁₈H₁₃O₃F₃, 335.0890 ([M + H]⁺); found, 335.0885.

Synthesis of (2-Ethyl-4-(trifluoromethyl)benzofuran-3-yl)(4-hydroxy-3,5-diiodophenyl)methanone (9)

To a stirred solution of 28f (15 mg, 0.04 mmol) in EtOH (2 mL) were added I₂ (46 mg, 0.18 mmol) and K₂CO₃ (19 mg, 0.13 mmol) at room temperature, and the resulting mixture was refluxed for 1 h. The reaction was quenched with 10% HCl (aq.). The aqueous mixture was extracted with EtOAc (1 mL × 3). The organic extracts were combined, dried over Na₂SO₄, and evaporated to give the residue, which was chromatographed on SiO₂ (5 g, n-hexane/EtOAc = 5/1) to give 9 (23 mg, 87%, 0.04 mmol) as a white solid. ¹H NMR (400 MHz, CDCl₃) δ: 8.23 (2H, s), 7.71 (1H, d, J = 8.0 Hz), 7.58 (1H, d, J = 8.0 Hz), 7.42 (1H, t, J = 8.0 Hz), 6.24 (1H, s), 2.67 (2H, q, J = 7.6 Hz), 1.29 (3H, t, J = 7.6 Hz); ¹³C NMR (100 MHz, CDCl₃) δ: 188.11, 161.96, 157.99, 154.59, 141.17, 134.19, 124.33, 123.74 (q, J = 270.8 Hz), 122.53 (q, J = 33.4 Hz), 121.76 (q, J = 4.8 Hz), 115.11, 114.54, 77.16, 21.50, 12.32; IR (KBr): 3431, 3103, 1649, 1427, 1360, 1231, 1161, 1111 cm^–1; mp: 143–144 °C; HRMS (ESI): calcd for C₁₈H₁₁O₃F₃I₂, 584.8677 ([M-H]⁻); found, 584.8635.

Protein Expression and Purification

The protein was expressed in the Escherichia coli (E. coli) M-15 strain, which was transformed with the expression vector pQE30.⁵⁵ The expression vector for V30M-TTR was constructed using a QuikChange site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA). After inducing protein expression with isopropyl β-d-thiogalactopyranoside, E. coli cells were cultivated at 37 °C for 3–4 h followed by centrifugation at 9000g for 15 min at 4 °C. The cells were then resuspended in buffer A containing 20 mM Tris-HCl (pH 8.0 at 20 °C) and sonicated on ice. Proteins were subsequently purified using a nickel chelation affinity column. Protein purity was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Ex Vivo Competitive Binding Assay Using a Fluorogenic Probe

The fluorogenic probe (E)-S-phenyl 3-(4-hydroxy-3,5-dimethylstyryl)benzothioate was employed for an ex vivo competitive binding assay.^31,33 A 2 mM stock solution of compounds in DMSO was prepared. Subsequently, 5 μL of the DMSO stock solution was added to 490 μL of human plasma (human male AB plasma of USA origin, sterile-filtered; containing 5 μM TTR concentration; Sigma: catalog no. H4522). Next, 5 μL of the probe solution (0.36 mM stock solution in DMSO) was introduced into the human plasma with the compound. The fluorescence intensity was measured at room temperature, using an excitation wavelength of 328 nm and an emission wavelength of 384 nm.

Acid-Induced Aggregation Experiments

A solution with a pH of 7.0 containing both TTR and the compound was incubated for 30 min at room temperature. The aggregation of TTR was initiated by mixing the protein solution with a 100 mM acetate buffer at pH 4.7. Solutions with a pH of 4.7 containing TTR (at a tetramer concentration of 10 μM) and compounds ranging from 0 to 50 μM were incubated at 37 °C for 7 days. Following incubation, the pH 4.7 solutions were mixed with a 5-fold volume of a pH 8.0 solution consisting of 200 mM Tris-HCl and 20 μM thioflavin-T. Fluorescence measurements were conducted using a Hitachi F-4500 fluorescence spectrophotometer, with an excitation wavelength of 440 nm at 25 °C. The fluorescence intensity at 484 nm was employed to monitor the aggregation of TTR. The IC₅₀ values of the compounds were calculated using the four-parameter logistic model.⁵⁶

ITC Measurements

A compound solution comprising 30 μM of the compound, 50 mM sodium phosphate (pH 7.5), 50 mM NaCl, and 0.3% DMSO was prepared. The protein solution was dialyzed against a solution of 50 mM sodium phosphate and 50 mM NaCl at pH 7.5. Following dialysis, DMSO was added to achieve a final concentration of 0.3%. Prior to the ITC experiment, all solutions were degassed through centrifugation (20,652g, 10 min). In order to decrease the concentration of DMSO in both the protein solution and the compound solution, we conducted a reverse titration experiment.^45,46 In this experiment, the compound solution was placed in the cell, while the protein solution was loaded into the syringe. The compound solution (30 μM) was titrated using small injections of the protein solution (200 μM). The ITC measurements were carried out with a MicroCal iTC200 microcalorimeter (Malvern Instruments, Malvern, UK). Titrations were performed with 18 injections, each separated by a 180 s delay, while maintaining a stirring rate of 750 rpm at 25 °C. Before performing the curve fitting, the contribution of heat of dilution was subtracted from the titration data. The resulting data were analyzed and plotted using SEDPHAT, GUSSI, and NITPIC (University of Texas Southwestern Medical Center, Dallas, TX). The ITC curves were fitted under the assumption that the tetramer possesses two identical and independent ligand-binding sites.^32,33,45,46

Protein Crystallography

Crystals of V30M-TTR-4 and V30M-TTR-7 were obtained by a soaking technique. Parent crystals in an apo form were grown with a crystallization buffer containing 20 mg/mL V30M-TTR, 29% polyethylene glycol 400 (PEG400), 0.4 M calcium chloride, and 0.1 M sodium acetate pH 5.6. The crystals were soaked in a buffer containing 1 mM 4 or 7, 30% PEG400, 0.4 M calcium chloride, and 0.1 M sodium acetate pH 5.6 for a week at 293 K. Crystals of V30M-TTR-20 were obtained by the cocrystallization method with the buffer containing 20 mg/mL V30M-TTR, 1 mM 20, 0.4 M calcium chloride, and 0.1 M sodium acetate pH 5.6. The crystals were cryo-protected using a cryo-buffer containing 32% PEG400, 0.4 M calcium chloride, 0.1 M sodium acetate pH 5.6, and 5% DMSO and then directly frozen in liquid nitrogen until data collection. X-ray diffraction experiments were conducted on beamline BL-17A at the Photon Factory (Japan), on beamline NE3A at the Photon Factory Advanced ring, or on beamline X06SA at the Swiss Light Source (Switzerland). The obtained diffraction data were processed with XDS software.⁵⁷ The crystal complex belonged to the space group P2₁2₁2 and showed isomorphism with previously obtained crystals.^58,59 Consequently, structure refinements were directly performed without molecular replacement phasing, utilizing the apo V30M-TTR (PDB ID 4pwe) as an initial model.^58,59 Manual implementation of crystal structure refinement from diffraction data was carried out using PHENIX.REFINE and COOT.^60,61 The 3D structures and compound library data were prepared using PRODRG {Schuttelkopf, 2004 #1122}. The final coordinates and structure factors of the V30M-TTR complexed with 4, 7, and 20 have been deposited in the Protein Data Bank (PDB) under the accession codes 8WGS, 8WGT, and 8WGU, respectively.

In Vitro ADME Assay

We used the Japanese Pharmacopoeia (JP) first test fluid (pH 1.2) and JP second test fluid (pH 6.8) for the solubility test. Solutions of the compounds were prepared by diluting a 10 mM DMSO stock solution (2 μL) with either the JP first or second fluid (165 μL) followed by mixing at 37 °C for 4 h with rotation at 1000 rpm. The resulting mixed solution was loaded into 96-well MultiScreen HTS-PCF filter plates for solubility assays (product number MSSLBPC10; Millipore, Bedford, MA), and filtration was performed by centrifugation. The obtained filtrates were subjected to analysis via HPLC with UV detection at 254 nm or liquid chromatography-tandem mass spectrometry (LC-MS/MS). Solubility was determined by comparing the peak area of the filtrate with that of a 100 μM standard solution. When the peak area of the filtrate exceeded that of the standard solution, the solubility was designated as >100 μM. Each test was performed in duplicate.

To determine passive membrane diffusion rates, we conducted the PAMPA assay using a Corning Gentest precoated PAMPA plate system. The acceptor plate was prepared by adding 200 μL of a 0.1 M phosphate buffer (pH 7.4) supplemented with 5% DMSO to each well. Subsequently, 300 μL of a solution containing 80 (7) or 100 μM compounds in a 0.1 M phosphate buffer (pH 6.4) with 5% DMSO was added to the donor wells. The acceptor plate was then placed on top of the donor plate and incubated at 37 °C for 4 h without agitation. After incubation, the plates were separated, and the solutions from each well of both the acceptor plate and the donor plate were transferred to 96-well plates and mixed with acetonitrile. The final concentrations of compounds in both the donor and acceptor wells, as well as the concentrations of the initial donor solutions, were analyzed using LC-MS/MS. We calculated the compound permeability as described in a previous study.⁶² The recovery rate for 7 was 42%, while those of the others exceeded 60%. We used antipyrine (100 μM), metoprolol (500 μM), and sulfasalazine (500 μM) as reference compounds. The permeabilities of antipyrine, metoprolol, and sulfasalazine were 20, 1.8, and 0.038 × 10^–6 cm/s, respectively. Two technical replicates were performed.

To assess hepatic microsomal stability, we analyzed the reduction of the parent compound over time using LC-MS/MS analysis. The initial compound amount served as the reference point. Following a 5 min preincubation, we introduced 1 mM NADPH (final concentration, as per the following steps) into a mixture containing 1 μM of the compound, 0.2 mg/mL of rat liver microsomes (Sekisui XenoTech LLC, Kansas City, KS), 1 mM EDTA, and a 0.1 M phosphate buffer (pH 7.4). This mixture was then incubated at 37 °C for 30 min with rotation at 60 rpm. Subsequently, a 50 μL aliquot of the incubation mixture was combined with 250 μL of chilled acetonitrile along with an internal standard (IS; methyltestosterone). After centrifugation at 3150g for 15 min at 4 °C, we subjected the supernatants to LC-MS/MS analysis. We calculated hepatic microsomal stability (mL/min/kg, CLint) as outlined in a previous report, using scaling factors of 44.8 mg microsomal protein/g liver and 40.0 g liver/kg body weight.⁶³ Briefly, the half-life was calculated from the parent compound reduction rate in 30 min, assuming that the compound was reduced by the primary reaction, and the clearance was further calculated using the microsomal concentration. This process was carried out with two technical replicates.

To determine the unbound fraction in rat plasma, we employed an equilibrium dialysis apparatus. We used an HTDialysis complete unit (HTD96b) and dialysis membrane strips MWCO 12–14 kDa (HTDialysis, Galesferry, CT). Rat plasma was mixed with the test compound (1 μM), and 150 μL aliquots were loaded into the apparatus and then dialyzed against 150 μL of a 0.1 M phosphate buffer (pH 7.4) at 37 °C for 6 h with rotation at 80 rpm. We mixed a 100 μL aliquot of the buffer from the receiver side and 10 μL of rat blank plasma. Additionally, we mixed a 10 μL aliquot of plasma from the donor side and 100 μL of the blank buffer. These mixtures were combined with 400 μL of chilled acetonitrile/IS. After centrifuging at 3150g for 15 min at 4 °C, we analyzed the supernatants using LC-MS/MS. The unbound fraction (f_u) was calculated as the ratio of the concentration on the receiver side (C_buffer) to that on the donor side (C_plasma). Plasma protein binding was calculated as a percentage according to the following formula: plasma protein binding (% unbound) = f_u × 100 = (C_buffer/C_plasma) × 100. Plasma protein binding (% bound) could be calculated as 100 – % unbound. This process was performed twice for technical accuracy.

Animals

Seven-weeks-old male Sprague–Dawley rats were acquired from SLC, Inc. (Hamamatsu, Japan). All the rats were housed in air-conditioned rooms maintained at a temperature of 20 ± 2 °C, a relative humidity of 50 ± 10%, and a 12 h alternating light–dark cycle. The rats had unrestricted access to water and food (MF; Oriental Yeast Co.). The studies were carried out in accordance with the guidelines of the Animal Care and Use Committee of Osaka University.

In Vivo Pharmacokinetic Assay

A test compound solution in 10% DMSO/0.1 M phosphate buffer (pH 8.5) was administered intravenously (iv) or orally (po) to male SD rats. The dosage of the test compounds was 0.1 mg/kg in a 5 mL/kg solution. Blood samples were collected from the caudal vein at 5 min (iv), 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h. Plasma was obtained by centrifuging the blood samples and stored at −80 °C. These plasma samples were precipitated with 4 volumes of acetonitrile/IS and then centrifuged at 15,000g at 4 °C for 10 min. The resulting supernatants were analyzed using LC-MS/MS. Standard noncompartmental analysis was conducted to determine various pharmacokinetic parameters: AUC from time zero to 8 h (AUC_0–8h), total clearance (CL_tot), and the volume of distribution at the steady state (V_dss). Additionally, the absolute bioavailability (BA) of the oral dose was calculated as AUC_0–8h(po)/AUC_0–8h(iv). For each sample, at least two biological replicates were generated.

LC-MS/MS Quantification Methods

We utilized an LC-MS8060 instrument equipped with a Shimadzu Nexera series LC system (Shimadzu, Kyoto, Japan) for our analysis. All compounds were analyzed in the multireaction monitoring mode under electrospray ionization conditions. The analytical column employed was a CAPCELLPAK C18 MGIII (3 μm × 2.0 mm ID × 35 mm; OSAKA SODA, Osaka, Japan) maintained at 50 °C. The gradient mobile phase consisted of 0.1% formic acid in water (mobile phase A) and 0.1% formic acid in acetonitrile (mobile phase B) at a total flow rate of 1 mL/min. Initially, the mobile phase composition was 10% B and was held constant for 0.5 min. It was then linearly increased to 90% B over 1 min followed by a constant hold for 0.8 min. The mobile phase was then returned to the initial condition of 10% B over 0.01 min and re-equilibrated for 1 min. The transitions (precursor ion > product ion) of 1, 4, 5, 6, 7, 8, and IS (methyltestosterone) were 339.0 > 187.1, 642.8 > 127.0, 594.8 > 127.0, 550.8 > 127.0, 584.8 > 345.0, 530.9 > 127.0 (negative), and 303.1 > 109.1 (positive), respectively.

Glossary

ABBREVIATIONS

ADME

absorption, distribution, metabolism, and excretion

DMSO

dimethyl sulfoxide

EC₅₀

half-maximal effective concentration

HPLC

high-performance liquid chromatography

HRMS

high-resolution mass spectrometry

IC₅₀

half-maximal inhibitory concentration

IR

infrared spectroscopy

JP

Japanese Pharmacopoeia

K_d

dissociation constant

LC-MS/MS

liquid chromatography-tandem mass spectrometry

mp

melting point

MS

mass spectrometry

NMR

nuclear magnetic resonance

PAMPA

parallel artificial membrane permeability assay

PDB

Protein Data Bank

TTR

transthyretin

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02286.

• Table S1, Figure S1, ¹H and ¹³C NMR spectra, HRMS spectra, HPLC data, and structure–activity relationship study (PDF)

• Molecular formula strings (CSV)

Accession Codes

The final structure coordinates and structure factors of V30M-TTR in complex with 4, 7, and 20 have been deposited in PDB under the accession codes 8WGS, 8WGT, and 8WGU, respectively. The authors will release the atomic coordinates upon article publication.

Author Contributions

N.T. and M.M. designed and initiated the project. Y.N., T.O., K.T., and N.N.T.L. synthesized the compounds. K.F., Y.N., S.Y., M.U., and Y.A. contributed to the ex vivo competitive binding study. K.F. performed fluorescence and ITC measurements. T.Y. performed X-ray crystallography. K.K. and S.N. performed the in vitro ADME and in vivo pharmacokinetic studies. M.M. prepared the manuscript with contributions from the other authors.

The authors declare no competing financial interest.