Facile diverted synthesis of pyrrolidinyl triazoles using organotrifluoroborate: discovery of potential mPTP blockers

Abstract

This article describes the rapid and diversified synthesis of pyrrolidinyl triazoles for the discovery of mitochondrial permeability transition pore (mPTP) blockers. The 1,3-dipolar cycloaddition of ethynyl trifluoroborate with azidopyrrolidine produced a key intermediate, triazolyl trifluoroborate 4, which subsequently underwent a Suzuki–Miyaura coupling reaction to afford a series of 1,4-disubstituted triazoles 2. Subsequent biological evaluation of these derivatives indicated 2ag and 2aj as the most potent mPTP blockers exhibiting excellent cytochrome P450 (CYP) stability when compared to the previously reported oxime analogue 1. The present work clearly demonstrates that a 1,2,3-triazole can be used as a stable oxime surrogate. Furthermore, it suggests that late-stage diversification through coupling reactions of organotrifluoroborates is suitable for the rapid discovery of biologically active molecules.

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1. Introduction

1,2,3-Triazoles are an important class of heterocycles in pharmaceutical science, chemical biology, supramolecular chemistry and materials science.^1–5 In particular, various medicinal agents contain triazoles as important pharmacophores, which can be favorable in the binding to biomolecular targets.⁶ Triazoles are often used as peptide bond isosteres because of their planarity and enhanced physiochemical properties such as increased metabolic stability and solubility.^7–9 The nitrogen atoms in the triazole rings have hydrogen bonding capabilities and are not protonated at physiological pH because of their low basicity.

The Huisgen 1,3-dipolar cycloaddition of azides and alkynes is widely used for the synthesis of 1,2,3-triazoles.^9–13 The copper(I) catalyzed version of this reaction, using azides and terminal alkynes, provides 1,4-disubstituted 1,2,3-triazoles with high regioselectivity,^14–18 making this reaction a powerful tool in medicinal chemistry for the structural modification of lead compounds.

Recently, we have identified pyrrolidinyl oxime, 1, as an mPTP (mitochondrial permeability transition pore) blocker, which is a viable therapeutic target for the treatment of Alzheimer's disease (AD).¹⁹ It effectively recovered amyloid beta-induced mitochondrial dysfunction; however, the cytochrome P450 (CYP) enzyme inhibition study revealed that it significantly reduced CYP enzymatic activities, which can cause side effects such as a drug–drug interaction. In this regard, we proposed that the stable 1,2,3-triazole could serve as an effective isostere of oxime to improve the CYP stability without reducing the mPTP inhibitory activity.²⁰ In addition, incorporation of substituents on the aromatic ring can alter the effect of CYP inhibition. Considering these structural modifications, we propose triazole derivatives 2 as new candidates for mPTP blockers (Fig. 1).

Fig. 1 Structures of pyrrolidinyl oxime 1 and triazole derivatives 2.

As described in Scheme 1, we designed an efficient, step-economical synthetic approach toward a chemical library of diverse 1,2,3-triazole derivatives. The synthesis of a triazole via a typical 1,3-cycloaddition requires the preparation of a terminal alkyne as a precursor; hence, this strategy is not effective for achieving molecular diversity. Alternatively, we envisioned that triazolyl trifluoroborate 4, derived from the annulation of ethynyl trifluoroborate 6^21,22 and azide 7, can undergo a rapid Suzuki–Miyaura coupling reaction to afford various triazole analogues 3.^23–25 A recent report showed the transformation of 6 to the corresponding triazolyl trifluoroborates, and subsequent cross-coupling reactions, to afford 1,4-disubstituted 1,2,3-triazoles.²⁶ However, this protocol was only applied to a limited number of substrates, such as primary or aromatic azides, and no further applications of the synthesized compounds were pursued.

Scheme 1 Proposed synthesis of 1-pyrrolidinyl-1,2,3-triazoles 2.

In this paper, we report an efficient and divergent synthesis of various highly functionalized 1-pyrrolidinyl 4-aryl 1,2,3-triazoles 2via triazolyl trifluoroborates for the discovery of mPTP blockers. Furthermore, we investigate the effect of structural modification of oximes into triazoles on the target efficacy and CYP enzyme stability.

2. Results and discussion

As depicted in Table 1, we performed a model study through the cycloaddition of 6, using benzyl azide 8 as a substrate. Based on the reaction conditions reported in the literature,²⁷ we employed copper(I) halides as catalysts as well as various bases and additives. Initially, the desired triazole trifluoroborate 9 was obtained in low yields when copper(I) iodide was used (entries 1–3). We explored several bases in order to improve the reaction yield, but the cycloaddition reactions did not afford 9 in any of the cases (entries 4–8). However, 9 was formed in high yield when the reaction with copper(I) bromide and cesium carbonate in the presence of N,N-dimethylethylenediamine (DMEDA) was performed at 90 °C for 30 min (entry 11).^28,29 The choice of solvent system is crucial to prevent the generation of side product 10, which is most likely formed by protodeboronation via a solvolysis mechanism.³⁰

Table 1 Optimization of the cycloaddition reaction conditions

Entry	Conditions	Temp. (°C)	Time (h)	Yield (%)
a Reaction was performed in acetone-d6. b The deboronated product 10 was formed exclusively.
1	CuI	60	17	24
2^a	CuI	60	20	40
3	CuI	90	24	28
4	CuI, TEA	50	24	—
5	CuI, K2CO3	50	24	—
6	CuI, Cs2CO3	50	24	—
7	CuI, iPr2NEt	50	24	—
8	CuI, iPr2NH	50	24	—
9^a	CuBr, Cs2CO3	60	15	—^b
10	CuBr, Cs2CO3, DMEDA	60	3	40
11	CuBr, Cs2CO3, DMEDA	90	0.5	85
12^a	CuBr, Cs2CO3, DMEDA	60	15	Trace

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After the optimal reaction conditions were selected, the scope of the cycloaddition reaction of 6 with various substrates was briefly investigated (Table 2). In general, the reactions of aromatic or aliphatic azides 11 afforded the corresponding triazoles 12 in good to excellent yields. It was found that various functional groups, such as alcohol, ester, allyl, and carbamate, were tolerated under these reaction conditions (entries 1–3, and 5). In particular, secondary pyrrolidine/piperizine azides 11d and 11e gave the desired products 12d and 12e in good yields (entries 4, 5), which could then be applied to the divergent synthesis of the proposed 1,4-disubstituted triazole library. While the sterically demanding 2,6-dimethylphenyl azide 11i was readily converted to triazole 12i within 3 h (entry 9), the use of adamantyl azide 11f did not provide the desired product 12f because of the high steric hindrance (entry 6).

Table 2 Cycloaddition reaction of 6 with azides 11 under optimized conditions

Entry	R^1	Time (h)	Product	Yield (%)
1	HOCH2CH2CH2 (11a)	0.5	12a	72
2	EtO2CCH2 (11b)	1	12b	62
3	PhCH=CHCH2 (11c)	0.5	12c	87
4		1.5	12d	71
5		1.5	12e	45
6		24	12f	No reaction
7		0.5	12g	90
8		1	12h	72
9		3	12i	69

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The Suzuki–Miyaura coupling reaction was then explored in order to produce a variety of derivatives in the late-stage functionalization, as suggested in Scheme 1. The reaction of 9 with 4-bromobenzonitrile 13 was tested, and the results are summarized in Table 3. Following a literature procedure,³¹ the initial reactions were attempted in the presence of Pd(OAc)₂ and SPhos using various bases. When K₂CO₃, Cs₂CO₃, and K₃PO₄ were used as bases, 14 was not formed at all, or was obtained in very low yield. However, the use of Na₂CO₃ as a base dramatically increased the yield to up to 99%. A variety of catalysts were also screened in order to optimize the reaction conditions. Among the catalysts tested, Pd₂(dba)₃ provided the best results, affording triazole 14 in quantitative yield. Many solvent systems were used, such as DMF/H₂O, DME, THF, and dioxane; however, all these afforded lower yields of the coupled product 14. Alternatively, ethanol gave higher yields, suggesting that it is a critical component in the reaction efficiency.

Table 3 Model study of the Suzuki–Miyaura cross-coupling reaction

Entry	Catalyst	Base	Time (h)	Yield (%)
a No ligand was used. b XPhos was used as a ligand. c Catalyst (2 mol%), ligand (4 mol%).
1	Pd(OAc)2	K2CO3	3	—
2	Pd(OAc)2	Cs2CO3	3	—
3	Pd(OAc)2	K3PO4	3	8
4	Pd(OAc)2	Na2CO3	18	99
5	PdCl2(dppf)·CH2Cl2	Na2CO3	12	78
6	Pd(PPh3)4	Na2CO3	18	76
7	Pd(dba)2	Na2CO3	7	96
8	Pd2(dba)3	Na2CO3	18	100
9^a	Pd2(dba)3	Na2CO3	24	9
10^b	Pd2(dba)3	Na2CO3	18	94
11^c	Pd2(dba)3	Na2CO3	18	88

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Given the positive results from the optimized cycloaddition reaction and the Suzuki–Miyaura coupling reaction in the model study, these reactions were applied to the synthesis of triazole derivatives 2 as mPTP blockers (Scheme 2). Starting from commercially available 3-hydroxy N-Boc-pyrrolidine 15, 3-azidopyrrolidine 7 was easily prepared by mesylation and consecutive S_N2 displacement of the azide. According to the experimental procedure previously established in Table 1, we attempted the 1,3-dipolar cycloaddition of potassium ethynyl trifluoroborate 6 with azide 7. As anticipated, the desired triazolyl trifluoroborate 4 was obtained in 70% yield. In the meantime, 2-bromo-1-benzyloxybenzenes 17, containing either a fluoride or chloride substituent at the 4-position, were easily prepared as coupling partners by benzylation of bromophenols 16. A coupling reaction of 4 and 17 could then be completed under the optimized conditions. This reaction proceeded successfully, giving 1,4-disubstituted triazoles 3 in moderate yields. Removal of the Boc protecting group on 3 using trifluoroacetic acid afforded the secondary amine 18 as a salt. This was then subjected to reductive amination, acylation, and sulfonylation to provide N-functionalized pyrrolidines 2 in good yields. Thus, we obtained a series of twenty new triazole derivatives in this concise and divergent manner using 4 as a key intermediate.

Scheme 2 Divergent synthesis of 1-pyrrolidynyl triazoles 2: (a) (i) MsCl, TEA, CH₂Cl₂, rt, 18 h, (ii) NaN₃, DMF, 80 °C, 3 h, 85%; (b) 6, CuBr, Cs₂CO₃, DMEDA, DMSO, 90 °C, 1.5 h, 70%; (c) 3,4-dichlorobenzyl chloride, K₂CO₃, KI, acetone, 60 °C, 75% (17a), and 95% (17b); (d) Pd₂(dba)₃, SPhos, Na₂CO₃, 85 °C, 18 h, 54% (3a), and 53% (3b); (e) TFA, CH₂Cl₂, rt, 0.5 h, 99% (18a), and 99% (18b); (f) ArCHO, NaBH(OAc)₃, AcOH, THF, rt, 24 h (for 2aa–e and 2ba–e), or ArCOCl, Et₃N, THF, rt, 24 h (for 2af–h and 2bf–h), or ArSO₂Cl, Et₃N, THF, rt, 24 h (for 2ai–j and 2bi–j).

The inhibitory activity of pyrrolidinyl triazoles 2 against amyloid beta-induced mPTP opening was evaluated by a cell-based JC-1 assay (Fig. 2).^32,33 In this assay, the color of the JC-1 dye changed from green to red as the mitochondrial membrane potential increased. Thus, the lower the percent increase of the green/red (g/r) ratio of the compound, the higher is the recovery of mitochondrial function in the cell. Twelve of the triazole derivatives 2 increased the inhibition of mPTP opening with less than 40% increase in the g/r ratio, indicating that the triazole is an effective structural surrogate of oxime. Further consideration of the structure–activity relationship (SAR) demonstrated the 2a series containing fluorine on the internal phenyl ring (X = F) to be more active than the chlorine-substituted 2b compounds. Smaller substituents at the R position were found to increase the inhibitory activity against mPTP opening. In particular, electron-withdrawing groups, such as carbonyl or sulfonyl, between pyrrolidine and the terminal phenyl ring significantly enhanced the inhibitory activity when compared to the initial lead compound 1.

Fig. 2 Inhibitory activity of 2 against amyloid beta-induced mPTP opening. Percent increase in the fluorescence ratio (green/red) after treatment of each compound with amyloid beta with respect to that of amyloid beta alone (100%). Cyclosporin A (CsA) was used as a control.

In order to determine the effect of triazoles on the CYP stability, the inhibitory activities of selected compounds 2 against five CYP450 isozymes in the human liver were tested. Using the CYP450 screening kits, the percent remaining activity of the CYP enzymes after treatment with 2 was obtained, as shown in Table 4. The selected compounds 2 significantly increased the percent CYP remaining activities in comparison with oxime 1. In particular, the most active triazole derivatives 2ag and 2aj, containing 4-fluorobenzoyl and 4-fluorobenzenesulfonyl groups, exhibited relatively low inhibitory activities against the tested CYP isozymes. This suggested that this triazole-based structural modification played an important role in the stability of the CYP enzymes as well as in blocking the mPTP opening.

Table 4 CYP inhibition^a,b of the selected compounds 2

Compds	% CYP isozyme remaining activity^c
	1A2	2D6	2C9	3A4	2C19
a Each value represents the mean of triplicate experiments. b Each value represents the mean of duplicated experiments. c The remaining activity of each isozyme was obtained at a concentration of 10 μM using a fluorogenic Vivid® CYP450 screening kit. d The reference compounds for each CYP isozyme assay are as follows: α-naphthoflavone (1A2), quinidine (2D6), sulfaphenazole (2C9), ketoconazole (3A4), and miconazole (2C19).
2ab	67.97	47.00	32.74	12.34	11.21
2ag	83.44	102.41	20.66	36.74	20.47
2aj	86.97	137.73	33.77	38.72	13.64
2bb	62.32	44.25	80.28	8.32	14.06
2bg	65.10	78.86	23.30	26.72	20.67
2bj	68.08	37.66	41.60	16.83	5.37
1	3.24	6.25	34.67	−0.06	—
Control^d	4.27	6.66	10.44	9.85	5.77

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3. Conclusion

In this study, we have demonstrated the synthesis of twenty pyrrolidinyl triazoles 2via cycloaddition of ethynyl trifluoroborate 6, followed by the Suzuki–Miyaura coupling reaction of the corresponding cycloadducts. These were then evaluated for their inhibitory activity against amyloid beta-induced mPTP opening. The reaction conditions that were optimized in the model system were successfully applied to the synthesis of key intermediates 4, which were transformed to a library of N-functionalized pyrrolidinyl triazoles 2 in a divergent manner with high overall yields. Thus, we believe that this methodology is useful for the rapid preparation of a large number of 1,4-disubstituted 1,2,3-triazole analogs.

Screening of the triazole analogues 2 in the JC-1 assay identified 2ag and 2aj as potent mPTP blockers. These also showed excellent CYP stability in comparison with oxime 1. These findings indicate that the 1,2,3-triazole functional group can be used as a stable isostere of oxime to improve the CYP stability while maintaining the inhibitory activity against a molecular target.

Experimental section

General

All reactions were carried out under dry nitrogen unless otherwise indicated. Commercially available reagents were used without further purification. Solvents and gases were dried according to standard procedures. Organic solvents were evaporated with reduced pressure using a rotary evaporator. Analytical thin layer chromatography (TLC) was performed using glass plates precoated with silica gel (0.25 mm). TLC plates were visualized by exposure to UV light (UV), and then were visualized with a p-anisaldehyde stain followed by brief heating on a hot plate. Flash column chromatography was performed using silica gel 60 (230–400 mesh, Merck) with the indicated solvents. ¹H, ¹¹B, ¹³C and ¹⁹F spectra were recorded on Bruker 300, Bruker 400 (376 MHz) or Varian 300 NMR spectrometers. ¹H NMR spectra are represented as follows: chemical shift, multiplicity (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet), integration, and coupling constant (J) in hertz (Hz). ¹H NMR chemical shifts are reported relative to CDCl₃ (7.26 ppm). ¹³C NMR spectra were recorded relative to the central line of CDCl₃ (77.0 ppm). ¹⁹F NMR chemical shifts were referenced to external CFCl₃ (0.0 ppm). ¹¹B NMR spectra at 128 MHz were obtained on a spectrometer equipped with the appropriate decoupling accessories. All ¹¹B NMR chemical shifts were referenced to external BF₃·OEt₂ (0.0 ppm) with a negative sign indicating an upfield shift.

Potassium ethynyltrifluoroborate (6)²². To a solution of ethynylmagnesium bromide in THF (20 mL, 10.0 mmol, 0.5 M in THF solution) was added trimethylborate (1.59 g, 15.3 mmol) at −78 °C. The solution was stirred for 1 h at this temperature, and then warmed up to −20 °C and stirred for 1 h. To the resulting white suspension, a solution of KHF₂ (4.71 g, 60.3 mmol) in distilled water (15 mL) was added at −20 °C and the solution was stirred at this temperature for 1 h and at room temperature for additional 1 h. The obtained reaction mixture was concentrated and the residue was dissolved in hot acetone. The residue was removed by filtration and the filtrate was concentrated to give the title compound 6 (1.05 g, 80%) as a white solid; mp 208.0–211.3 °C (decomp.); ¹H NMR (DMSO-d₆, 300 MHz) δ 1.90 (s, 1H). ¹³C NMR (DMSO-d₆, 75 MHz) δ 98.5, 78.9. ¹⁹F NMR (DMSO-d₆, 376 MHz) δ −132.2, −132.3, −132.4, 132.5. ¹¹B NMR (DMSO-d₆, 128 MHz) δ −1.87, −2.16, −2.44, −2.72.

Potassium (1-benzyl-1H-1,2,3-triazol-4-yl)trifluoroborate (9) (Table 1). To a solution of potassium ethynyltrifluoroborate 6 (21.0 mg, 159 μmol), CuBr (2.30 mg, 15.9 μmol), N,N-dimethylethylenediamine (3.40 μL, 31.8 μmol) and Cs₂CO₃ (51.0 mg, 159 μmol) in DMSO-d₆ (0.7 mL) in a NMR tube was added benzyl azide 8 (21.2 mg, 159 μmol) at room temperature. The reaction mixture was carried out at 90 °C for 30 min (until the ¹H NMR indicated completion of the reaction). Then, the solvent was completely removed under high vacuum. The residual product was dissolved in dry acetone (3 × 5 mL) and the insoluble salts were removed by filtration through Celite® and activated carbon. The solvent was concentrated and the crude solid was purified by dissolution in a minimal amount of dry acetone and precipitation with Et₂O to give the title compound 9 (35.8 mg, 85%) as a white solid; mp 171.9–173.8 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 7.43 (s, 1H), 7.36–7.23 (m, 5H), 5.46 (s, 2H). ¹³C NMR (DMSO-d₆, 100 MHz) δ 137.6, 128.9, 128.2, 128.1, 126.3, 52.1. ¹⁹F NMR (DMSO-d₆, 376 MHz) δ −135.6. ¹¹B NMR (DMSO-d₆, 128 MHz) δ 2.19.

4-(1-Benzyl-1H-1,2,3-triazol-4-yl)benzonitrile (14) (Table 3). A sealed tube was charged with potassium (1-benzyl-1H-1,2,3-triazol-4-yl)trifluoroborate 7 (40.0 mg, 150 μmol), 4-bromobenzonitrile (18.0 mg, 98.9 μmol), Pd₂(dba)₃ (3.70 mg, 4.04 μmol), SPhos (3.30 mg, 8.03 μmol) and Na₂CO₃ (21.0 mg, 198 μmol). The tube was sealed with a cap and purged with argon gas. Ethanol (1 mL) was added via a syringe and the reaction was carried out at 85 °C for 18 h under the protection of argon gas. Then, the reaction mixture was allowed to cool down to room temperature and filtered through a thin pad of silica gel (eluting with EtOAc) and the filtrate was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (EtOAc–n-hexane = 1 : 3) to give the title compound 14 (25.7 mg, 99.9%) as a white solid; mp 140.4–142.0 °C; ¹H NMR (CDCl₃, 400 MHz) δ 7.90 (d, J = 8.5 Hz, 2H), 7.77 (s, 1H), 7.66 (d, J = 8.5 Hz, 2H), 7.38 (m, 3H), 7.31 (m, 2H), 5.58 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 146.3, 134.9, 134.2, 132.6, 129.2, 129.0, 128.1, 126.0, 120.7, 118.7, 111.4, 54.4.

tert-Butyl 3-azidopyrrolidine-1-carboxylate (7). To a solution of t-butyl-3-(methylsulfonyloxy)pyrrolidine-1-carboxylate (0.74 g, 2.78 mmol) in DMF (7 mL) was added NaN₃ (0.45 mg, 6.95 mmol) in DMF (7 mL). The reaction mixture was stirred at 80 °C for 3 h, then cooled to room temperature and diluted with water. The aqueous phase was then extracted with ethyl acetate (3 × 20 mL). The organic layers were washed with brine, dried over anhydrous MgSO₄ and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (EtOAc–n-hexane = 15 : 1) to give the title compound 7 (0.49 mg, 85%) as a colorless oil; ¹H NMR (CDCl₃, 300 MHz) δ 4.11 (m, 1H), 3.52–3.42 (m, 4H), 2.12–1.95 (m, 2H), 1.45 (s, 9H); ¹³C NMR (CDCl₃, 100 MHz) δ 153.7, 78.8, 60.1, 59.4, 50.6, 50.3, 43.5, 43.2, 30.9, 30.1, 27.9.

Potassium 1-(1-(t-butoxycarbonyl)pyrrolidin-3-yl)-1H-1,2,3-triazole-4-yl trifluoroborate (4). Following the same procedure as that used for the synthesis of 9, the reaction of tert-butyl-3-azidopyrrolidine-1-carboxylate 7 (47.9 mg, 225 μmol), potassium ethynyltrifluoroborate 6 (27.0 mg, 207 μmol), CuBr (3.00 mg, 20.7 μmol), N,N-dimethylethylenediamine (4.40 μL, 40.8 μmol) and Cs₂CO₃ (67.0 mg, 207 μmol) in DMSO-d₆ in an NMR tube gave the title compound 4 (49.2 mg, 70%) as a white solid; mp 200.9–202.0 °C; ¹H NMR (DMSO-d₆, 400 MHz) δ 7.45 (s, 1H), 5.10 (m, 1H), 3.72 (q, J = 9.0 Hz, 1H), 3.54 (d, J = 10.7 Hz, 1H), 3.41 (m, 2H), 2.37–2.24 (m, 2H), 1.39 (s, 9H); ¹³C NMR (DMSO-d₆, 100 MHz) δ 154.0, 125.7, 79.3, 58.1, 57.4, 51.9, 51.6, 44.7, 44.5, 31.8, 31.0, 28.5; ¹⁹F NMR (DMSO-d₆, 376 MHz) δ −135.6; ¹¹B NMR (DMSO-d₆, 128 MHz) δ 2.14.

1-Bromo-4-fluoro-2-(3,4-dichlorobenzyloxy)-benzene (17a). To a solution of 2-bromo-4-fluorophenol (1.00 g, 5.26 mmol), 3,4-dichlorobenzylchloride (0.95 mL, 6.84 mmol) and K₂CO₃ (1.10 g, 7.85 mmol) in acetone (35 mL) was added KI (1.40 g, 8.38 mmol). The reaction mixture was stirred at 60 °C for 24 h, then cooled to room temperature and diluted with water. The aqueous phase was then extracted with DCM (3 × 30 mL). The organic layers were washed with brine, dried over anhydrous MgSO₄ and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (EtOAc–n-hexane = 1 : 15) to give the title compound 17a (1.38 g, 75%) as a yellow oil; ¹H NMR (CDCl₃, 300 MHz) δ 7.56 (d, J = 1.9 Hz, 1H), 7.45 (d, J = 8.2 Hz, 1H), 7.31 (td, J = 2.6, 7.8 Hz, 2H), 7.99–6.93 (m, 1H), 6.84 (q, J = 4.6 Hz, 1H), 4.99 (s, 2H).

1-Bromo-4-chloro-2-(3,4-dichlorobenzyloxy)benzene (17b). Following the same procedure as that used for the synthesis of 17a, the reaction of 2-bromo-4-chlorophenol (1.12 g, 5.41 mmol), 3,4-dichlorobenzylchloride (1.37 mL, 7.03 mmol), K₂CO₃ (1.12 g, 8.12 mmol) and KI (1.43 g, 8.66 mmol) in acetone (36 mL) gave the title compound 17b (1.89 g, 95%) as a yellowish oil; ¹H NMR (CDCl₃, 300 MHz) δ 7.56 (d, J = 2.3 Hz, 2H), 7.46 (d, J = 8.2 Hz, 1H), 7.29 (dd, J = 1.6, 8.3 Hz, 1H), 7.21 (dd, J = 2.5, 8.8 Hz, 1H), 6.80 (d, J = 8.8 Hz, 1H), 5.05 (s, 2H); ¹³C NMR (CDCl₃, 100 MHz) δ 153.4, 136.3, 133.1, 132.8, 132.2, 130.7, 128.9, 128.3, 126.9, 126.2, 114.3, 113.0.

t-Butyl-3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidine-1-carboxylate (3a). Following the same procedure as that used for the synthesis of 14, the reaction of potassium 1-(1-(t-butoxycarbonyl)pyrrolidin-3-yl)-1H-1,2,3-triazole-4-yl trifluoroborate 4 (31.4 mg, 116 μmol), 1-bromo-4-fluoro-2-(3,4-dichlorobenzyloxy)-benzene 17a (21.0 mg, 77.5 μmol), Pd₂(dba)₃ (2.20 mg, 3.09 μmol), SPhos (2.00 mg, 6.19 μmol) and Na₂CO₃ (13.0 mg, 155 μmol) in ethanol (1 mL) gave the title compound 3a (16.3 mg, 54%) as a colorless oil; ¹H NMR (CDCl₃, 400 MHz) δ 8.06 (dd, J = 3.1, 9.5 Hz, 1H), 7.93 (s, 1H), 7.53 (d, J = 1.7 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.24 (dd, J = 2.0, 8.2 Hz, 1H), 6.97 (td, J = 3.1, 8.2 Hz, 1H), 6.89 (q, J = 4.4 Hz, 1H), 5.12 (bs, 1H), 5.09 (s, 2H), 3.93 (q, J = 6.2 Hz, 1H), 3.74 (bs, 1H), 3.57 (bs, 2H), 2.46 (bs, 1H), 2.36 (bs, 1H), 1.44 (s, 9H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.6 (d, ¹J = 237.8 Hz), 154.1, 150.5, 142.3, 136.7, 132.9, 132.5, 130.9, 129.3, 126.7, 121.8, 121.2 (d, ³J = 7.9 Hz), 115.1 (d, ²J = 23.4 Hz), 114.5 (d, ²J = 25.0 Hz), 113.4 (d, ³J = 8.4 Hz), 80.0, 69.8, 59.3, 58.7, 51.4, 44.3, 43.9, 31.9, 31.0, 28.4.

t-Butyl-3-(4-(5-chloro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidine-1-carboxylate (3b). Following the same procedure as that used for the synthesis of 14, the reaction of potassium 1-(1-(t-butoxycarbonyl)pyrrolidin-3-yl)-1H-1,2,3-triazole-4-yl trifluoroborate 4 (43.6 mg, 126 μmol), 1-bromo-4-chloro-2-(3,4-dichlorobenzyloxy)benzene 17b (32.6 mg, 84.6 μmol), Pd₂(dba)₃ (3.10 mg, 3.38 μmol), SPhos (2.80 mg, 6.82 μmol) and Na₂CO₃ (18.0 mg, 169 μmol) in ethanol (1 mL) gave the title compound 3b (23.6 mg, 53%) as a white solid; mp 127.3–129.3 °C; ¹H NMR (CDCl₃, 400 MHz) δ 8.33 (d, J = 2.6 Hz, 1H), 7.91 (s, 1H), 7.53 (d, J = 1.2 Hz, 1H), 7.50 (d, J = 8.2 Hz, 1H), 7.25–7.21 (m, 2H), 6.88 (d, J = 8.8 Hz, 1H), 5.10 (bs, 3H), 3.93 (q, J = 6.2 Hz, 1H), 3.74 (m, 1H), 3.56 (bs, 2H), 2.46 (bs, 1H), 2.36 (bs, 1H), 1.44 (s, 9H); ¹³C NMR (CDCl₃, 75 MHz) δ 154.1, 152.8, 142.0, 136.5, 132.9, 132.5, 130.9, 129.3, 128.5, 127.6, 126.9, 126.7, 121.9, 121.2, 113.3, 80.0, 69.5, 59.3, 58.7, 51.4, 44.3, 44.0, 31.9, 31.0, 28.4.

3-(4-(5-Fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate (18a). To a solution of t-butyl-3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidine-1-carboxylate 17a (0.52 g, 1.03 mmol) in DCM (5 mL) was added TFA (1.90 mL, 25.7 mmol). The reaction mixture was stirred at room temperature for 0.5 h. The solvent was removed under reduced pressure to give the title compound 18a (0.57 g, 99%) as a yellowish solid; mp 157.1–159.5 °C; ¹H NMR (MeOH-d₄, 400 MHz) δ 8.38 (s, 1H), 7.81 (dd, J = 3.2, 9.6 Hz, 1H), 7.59 (d, J = 2.0 Hz, 1H), 7.50 (d, J = 8.4 Hz, 1H), 7.35 (d, J = 2.0, 8.4 Hz, 1H), 7.08–7.01 (m, 2H), 5.50 (m, 1H), 5.19 (s, 2H), 3.91 (m, 2H), 3.64 (m, 2H), 2.67 (m, 1H), 2.53 (m, 1H); ¹³C NMR (MeOH-d₄, 100 MHz) δ 157.3 (d, ¹J = 236.0 Hz), 150.7, 142.2, 137.6, 132.1, 131.5, 130.5, 129.3, 127.1, 123.9, 120.6 (d, ³J = 8.0 Hz), 115.0 (d, ²J = 24.0 Hz), 114.1 (d, ³J = 8.0 Hz), 113.3 (d, ³J = 25.0 Hz).

3-(4-(5-Chloro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate (18b). Following the same procedure as that used for the synthesis of 18a, the reaction of t-butyl-3-(4-(5-chloro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidine-1-carboxylate 17b (180 mg, 343 μmol) and TFA (0.64 mL, 8.58 mmol) in DCM (1.7 mL) gave the title compound 18b (18.5 mg, 99%) as a yellowish solid; ¹H NMR (MeOH-d₄, 400 MHz) δ 8.37 (s, 1H), 8.11 (d, J = 2.4 Hz, 1H), 7.64 (d, J = 2.0 Hz, 1H), 7.55 (d, J = 8.4 Hz, 1H), 7.39 (dd, J = 2.0, 8.0 Hz, 1H), 7.30 (dd, J = 2.6, 9.0 Hz, 1H), 7.12 (d, J = 8.8 Hz, 1H), 5.49 (m, 1H), 5.26 (s, 2H), 3.94 (dd, J = 1.6, 12.8 Hz, 1H), 3.84 (q, J = 6.5 Hz, 1H), 3.63 (m, 2H), 2.67 (m, 1H), 2.51 (m, 1H); ¹³C NMR (MeOH-d₄, 100 MHz) δ 153.2, 142.0, 137.5, 132.2, 131.6, 130.5, 129.3, 128.6, 127.1, 126.7, 126.2, 124.0, 120.7, 114.2, 69.0, 58.9, 50.1, 44.6, 31.6.

1-(1-Benzylpyrrolidin-3-yl)-4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole (2aa). To a solution of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (25.0 mg, 47.9 μmol), CH₃CO₂H (0.55 μL, 9.59 μmol) and NaBH(OAc)₃ (30.4 mg, 144 μmol) in THF (0.5 mL) was added benzaldehyde (7.33 μL, 71.9 μmol). The reaction mixture was stirred at room temperature for 24 h, and then diluted with water. The aqueous phase was then extracted with DCM (3 × 3 mL). The organic layers were washed with brine, dried over anhydrous MgSO₄ and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (EtOAc–n-hexane = 1 : 1) to give the title compound 2aa (14.8 mg, 62%) as a yellow solid; mp 148.6–149.9 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.16 (s, 1H), 8.07 (dd, J = 3.0, 9.5 Hz, 1H), 7.54 (s, 1H), 7.37 (d, J = 8.2 Hz, 1H), 7.25–7.23 (m, 6H), 7.00–6.91 (m, 2H), 5.25 (m, 1H), 5.05 (s, 2H), 3.66 (q, J = 13.1 Hz, 2H), 2.91 (m, 3H), 2.58 (m, 2H), 2.06 (m, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.7 (d, ¹J = 237.8 Hz), 150.6 (d, ⁴J = 2.0 Hz), 142.5 (d, ⁴J = 2.8 Hz), 136.7, 132.9, 132.5, 130.8, 129.5, 128.9, 128.6, 127.9, 126.8, 122.1, 121.5 (³d, J = 8.7 Hz), 115.0 (d, ²J = 23.3 Hz), 114.5 (²d, J = 25.0 Hz), 113.4 (d, ³J = 8.4 Hz), 77.2, 69.9, 59.2, 59.0, 52.6, 32.7; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₄Cl₂FN₄O 497.13057, found 497.13019.

1-(1-(4-Fluorobenzyl)pyrrolidin-3-yl)-4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole (2ab). Following the same procedure as that used for the synthesis of 2aa, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (32.6 mg, 62.5 μmol), 4-fluorobenzaldehyde (13.4 μL, 125 μmol), CH₃CO₂H (1.07 μL, 18.8 μmol) and NaBH(OAc)₃ (53.0 mg, 250 μmol) in THF (0.6 mL) gave the title compound 2ab (27.2 mg, 85%) as a yellow solid; mp 43.2–45.0 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.13 (s, 1H), 8.07 (dd, J = 3.0, 9.6 Hz, 1H), 7.55 (d, J = 1.7 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.26 (dd, J = 1.8, 8.1 Hz, 1H), 7.13 (bs, 2H), 7.01–6.88 (m, 4H), 5.23 (m, 1H), 5.08 (s, 2H), 3.58 (q, J = 12.6 Hz, 2H), 2.86 (m, 3H), 2.56 (m, 2H), 2.06 (m, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 162.3 (d, ¹J = 244.6 Hz), 157.7 (d, ¹J = 237.8 Hz), 150.6 (d, ⁴J = 1.9 Hz), 142.5 (d, ⁴J = 2.0 Hz), 136.7, 132.9, 132.5, 130.8, 130.3, 129.6, 126.9, 121.9, 121.5 (d, ³J = 8.3 Hz), 115.4 (d, ²J = 21.2 Hz), 115.0 (d, ²J = 23.3 Hz), 114.4 (d, ²J = 25.0 Hz), 113.3 (d, ³J = 8.3 Hz), 77.2, 69.9, 59.4, 59.1, 58.5, 52.5, 32.7; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₃Cl₂F₂N₄O 515.12115, found 515.12072.

1-(1-(4-Chlorobenzyl)pyrrolidin-3-yl)-4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole (2ac). Following the same procedure as that used for the synthesis of 2aa, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (33.4 mg, 64.1 μmol), 4-chlorobenzaldehyde (18.0 mg, 128 μmol), CH₃CO₂H (1.10 μL, 19.2 μmol) and NaBH(OAc)₃ (54.3 mg, 256 μmol) in THF (0.6 mL) gave the title compound 2ac (27.7 mg, 82%) as a yellow solid; mp 49.6–50.3 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.13 (s, 1H), 8.06 (dd, J = 2.9, 9.6 Hz, 1H), 7.55 (d, J = 1.8 Hz, 1H), 7.39 (d, J = 8.2 Hz, 1H), 7.25 (dd, J = 1.5, 6.6 Hz, 1H), 7.19 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.2 Hz, 2H), 6.95 (m, 2H), 5.22 (m, 1H), 5.08 (s, 2H), 3.58 (q, J = 14.1 Hz, 2H), 2.88 (m, 3H), 2.56 (m, 2H), 2.05 (m, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.7 (d, ¹J = 237.9 Hz), 150.6 (d, ⁴J = 1.9 Hz), 142.5 (d, ⁴J = 2.1 Hz), 136.7, 133.3, 132.9, 132.5, 130.8, 129.9, 129.6, 128.6, 126.9, 121.9, 121.5 (d, ³J = 8.4 Hz), 115.0 (d, ²J = 23.4 Hz), 114.4 (d, ²J = 25.1 Hz), 113.3 (d, ³J = 8.3 Hz), 77.2, 69.9, 59.5, 59.1, 58.5, 52.5, 32.7; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₃Cl₃FN₄O 531.09160, found 531.09110.

1-(1-(2,3-Dichlorobenzyl)pyrrolidin-3-yl)-4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole (2ad). Following the same procedure as that used for the synthesis of 2aa, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (34.3 mg, 65.8 μmol), 2,3-dichlorobenzaldehyde (23.0 mg, 132 μmol), CH₃CO₂H (1.13 μL, 19.7 μmol) and NaBH(OAc)₃ (55.8 mg, 263 μmol) in THF (0.6 mL) gave the title compound 2ad (28.1 mg, 76%) as a yellow solid; mp 72.4–75.4 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.20 (s, 1H), 8.06 (dd, J = 3.0, 9.6 Hz, 1H), 7.47 (dd, J = 1.4, 9.7 Hz, 1H), 7.33 (t, J = 7.2 Hz, 2H), 7.22–7.04 (m, 3H), 6.97 (td, J = 3.0, 8.2 Hz, 1H), 6.0 (q, J = 4.6 Hz, 1H), 5.28 (m, 1H), 5.09 (s, 2H), 3.74 (bs, 2H), 3.00 (bs, 2H), 2.86 (bs, 1H), 2.59 (m, 2H), 2.04 (m, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.7 (d, ¹J = 237.8 Hz), 150.6 (d, ⁴J = 1.9 Hz), 142.6 (d, ⁴J = 2.0 Hz), 136.8, 133.2, 132.8, 132.3, 132.2, 130.6, 129.4, 128.4, 127.2, 126.8, 121.8, 121.6 (d, ³J = 8.5 Hz), 114.9 (d, ²J = 23.2 Hz), 114.4 (d, ²J = 25.1 Hz), 113.3 (d, ³J = 8.3 Hz), 77.2, 69.8, 60.0, 59.3, 56.7, 52.5, 32.8; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₂Cl₄FN₄O 565.05263, found 565.05217.

1-(1-(2,4-Dichlorobenzyl)pyrrolidin-3-yl)-4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole (2ae). Following the same procedure as that used for the synthesis of 2aa, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (33.3 mg, 63.9 μmol), 2,4-dichlorobenzaldehyde (22.4 mg, 128 μmol), CH₃CO₂H (1.10 μL, 19.2 μmol) and NaBH(OAc)₃(54.2 mg, 256 μmol) in THF (0.6 mL) gave the title compound 2ae (25.3 mg, 70%) as a yellowish solid; mp 120.0–121.3 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.18 (s, 1H), 8.06 (dd, J = 2.9, 9.6 Hz, 1H), 7.50 (s, 1H), 7.36 (d, J = 8.2 Hz, 1H), 7.29 (s, 1H), 7.23–7.07 (m, 3H), 6.97 (td, J = 3.0, 8.2 Hz, 1H), 6.91 (q, J = 4.5 Hz, 1H), 5.24 (bs, 1H), 5.08 (s, 2H), 3.68 (bs, 2H), 2.98–2.83 (m, 3H), 2.57 (m, 2H), 2.04 (m, 1H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.7 (d, ¹J = 237.7 Hz), 150.6 (d, ⁴J = 2.0 Hz), 142.6 (d, ⁴J = 2.9 Hz), 142.5, 136.8, 134.6, 133.6, 132.9, 132.4, 131.1, 130.7, 129.4, 129.3, 127.1, 126.8, 121.7, 121.6 (d, ³J = 8.2 Hz), 114.9 (d, ²J = 23.3 Hz), 114.4 (d, ²J = 25.0 Hz), 113.2 (d, ³J = 8.4 Hz), 77.2, 69.8, 59.9, 59.3, 55.4, 52.5, 32.7.

(3-(4-(2-(3,4-Dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole-1-yl)pyrrolidin-1-yl)(phenyl)methanone (2af). To a solution of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (28.8 mg, 55.2 μmol) and TEA (7.78 μL, 55.8 μmol) was added benzoyl chloride (6.41 μL, 55.2 μmol). The reaction mixture was stirred at room temperature for 24 h, and then diluted with water. The aqueous phase was then extracted with DCM (3 × 3 mL). The organic layers were washed with brine, dried over anhydrous MgSO₄ and concentrated in vacuo. The resulting residue was purified by column chromatography on silica gel (EtOAc–n-hexane = 1 : 1) to give the title compound 2af (18.7 mg, 67%) as a white solid; mp 55.4–57.5 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.04 (d, J = 7.3 Hz, 1H), 7.90 (s, 1H), 7.52–7.41 (m, 7H), 7.23 (s, 1H), 6.98 (td, J = 3.0, 8.2 Hz, 1H), 6.89 (q, J = 4.4 Hz, 1H), 5.21 (bs, 1H), 5.10 (s, 2H), 4.19–3.65 (m, 4H), 2.51 (bs, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 170.1, 157.7 (d, ¹J = 238.0 Hz), 150.5, 142.4, 136.7, 135.9, 133.0, 132.6, 130.9, 130.4, 129.4, 128.4, 127.2, 126.7, 122.3, 120.9 (d, ³J = 9.2 Hz), 115.3 (d, ²J = 23.4 Hz), 114.5 (d, ²J = 25.3 Hz), 113.4 (d, ³J = 8.3 Hz), 77.2, 69.8, 65.8, 63.7, 59.3, 58.4, 54.2, 51.8, 47.7, 44.3, 32.7, 30.5; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₂Cl₂FN₄O₂ 511.10984, found 511.10888.

(3-(4-(2-(3,4-Dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole-1-yl)pyrrolidin-1-yl)(4-fluorophenyl)methanone (2ag). Following the same procedure as that used for the synthesis of 2af, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (32.7 mg, 62.7 μmol), 4-fluorobenzoyl chloride (7.41 μL, 62.7 μmol) and TEA (11.4 μL, 81.5 μmol) in THF (0.6 mL) gave the title compound 2ag (31.2 mg, 94%) as a white solid; mp 111.8–113.0 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.03 (d, J = 12.3 Hz, 1H), 7.90 (s, 1H), 7.52 (d, J = 1.4 Hz, 2H), 7.48 (d, J = 8.2 Hz, 2H), 7.22 (bs, 1H), 7.07 (t, J = 7.8 Hz, 2H), 6.97 (td, J = 3.0, 8.2 Hz, 1H), 6.89 (q, J = 4.5 Hz, 1H), 5.19 (bs, 1H), 5.09 (s, 2H), 4.16–4.01 (m, 2H), 3.85 (bs, 1H), 3.65 (bs, 1H), 2.51 (bs, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 169.0, 163.8 (d, ¹J = 249.4 Hz), 157.7 (d, ¹J = 238.2 Hz), 150.5, 142.4, 136.7, 133.0, 132.6, 132.0, 130.9, 129.6 (d, ³J = 8.5 Hz), 129.4, 126.8, 122.2, 120.9 (d, ³J = 8.4 Hz), 115.6 (d, ²J = 19.9 Hz), 115.3 (d, ²J = 22.9 Hz), 114.5 (d, ²J = 25.2 Hz), 113.3 (d, ³J = 8.4 Hz), 77.2, 69.8, 59.3, 58.4, 54.3, 52.0, 47.8, 44.5, 32.7, 30.5; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₁Cl₂F₂N₄O₂ 529.10041, found 529.09987.

(4-Chlorophenyl)(3-(4-(2-(3,4-dichlorobenzyloxy)-5-fluorophenyl)-1H-1,2,3-triazole-1-yl)pyrrolidin-1-yl)methanone (2ah). Following the same procedure as that used for the synthesis of 2af, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (33.4 mg, 64.1 μmol), 4-chlorobenzoyl chloride (8.21 μL, 64.1 μmol) and TEA (11.6 μL, 83.3 μmol) in THF (0.6 mL) gave the title compound 2ah (30.5 mg, 87%) as a white solid; mp 137.4–140.6 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.02 (d, J = 14.6 Hz, 1H), 7.90 (s, 1H), 7.52–7.37 (m, 6H), 7.26–7.22 (m, 1H), 6.97 (td, J = 3.0, 8.2 Hz, 1H), 6.89 (q, J = 4.4 Hz, 1H), 5.18 (bs, 1H), 5.08 (s, 2H), 4.14–3.99 (m, 2H), 3.85 (bs, 1H), 3.65 (bs, 1H), 2.50 (bs, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 168.9, 157.7 (d, ¹J = 238.1 Hz), 150.6 (d, ⁴J = 1.7 Hz), 142.4, 136.7, 136.5, 134.2, 133.0, 132.6, 130.9, 129.4, 128.7, 126.8, 122.3, 120.9 (d, ³J = 7.7 Hz), 115.3 (d, ²J = 23.3 Hz), 114.5 (d, ²J = 25.1 Hz), 113.3 (d, ³J = 8.3 Hz), 77.2, 69.9, 59.3, 58.3, 54.2, 52.0, 47.7, 44.4, 32.7, 30.5; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₆H₂₁Cl₃FN₄O₂ 545.07086, found 545.07018.

4-(2-(3,4-Dichlorobenzyloxy)-5-fluorophenyl)-1-(1-(phenylsulfonyl)pyrrolidin-3-yl)-1H-1,2,3-triazole (2ai). Following the same procedure as that used for the synthesis of 2af, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (25.4 mg, 48.7 μmol), benzenesulfonyl chloride (6.22 μL, 48.7 μmol) and TEA (6.68 μL, 49.2 μmol) in THF (0.5 mL) gave the title compound 2ai (10.6 mg, 40%) as a white solid; mp 136.3–137.6 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.01 (dd, J = 3.1, 9.5 Hz, 1H), 7.90 (s, 1H), 7.79 (dd, J = 1.5, 8.1 Hz, 2H), 7.56–7.47 (m, 5H), 7.28 (m, 1H), 7.00 (td, J = 2.0, 4.9 Hz, 1H), 6.90 (q, J = 4.5 Hz, 1H), 5.09 (bs, 3H), 3.72 (m, 2H), 3.47 (m, 2H), 2.41 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 157.7 (d, ¹J = 232.8 Hz), 150.5, 142.5, 136.7, 136.1, 133.1, 133.0, 132.6, 131.0, 129.5, 129.2, 127.4, 126.9, 121.7, 121.0 (d, ³J = 8.4 Hz), 115.2 (d, ²J = 23.2 Hz), 114.5 (d, ²J = 25.2 Hz), 113.3 (d, ³J = 8.3 Hz), 69.9, 58.8, 53.4, 46.2, 31.5; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₂Cl₂FN₄O₃S 547.07682, found 547.07700.

4-(2-(3,4-Dichlorobenzyloxy)-5-fluorophenyl)-1-(1-((4-fluorophenyl)sulfonyl)pyrrolidin-3-yl)-1H-1,2,3-triazole (2aj). Following the same procedure as that used for the synthesis of 2af, the reaction of 3-(4-(5-fluoro-2-(3,4-dichlorobenzyloxy)phenyl)-1H-1,2,3-triazole-1-yl)pyrrolidinium 2,2,2-trifluoroacetate 18a (32.9 mg, 63.1 μmol), 4-fluorobenzenesulfonyl chloride (12.3 mg, 63.1 μmol) and TEA (11.4 μL, 81.5 μmol) in THF (0.6 mL) gave the title compound 2aj (12.4 mg, 35%) as a yellowish solid; mp 167.0–168.5 °C; ¹H NMR (CDCl₃, 300 MHz) δ 8.03 (dd, J = 3.1, 9.5 Hz, 1H), 7.89 (s, 1H), 7.79 (m, 2H), 7.54–7.52 (m, 2H), 7.28 (m, 1H), 7.17 (t, J = 8.5 Hz, 2H), 7.00 (td, J = 3.0, 8.2 Hz, 1H), 6.91 (q, J = 4.5 Hz, 1H), 5.09 (bs, 3H), 3.72 (m, 2H), 3.48 (t, J = 7.1 Hz, 2H), 2.43 (m, 2H); ¹³C NMR (CDCl₃, 75 MHz) δ 165.4 (d, ¹J = 254.1 Hz), 157.7 (d, ¹J = 234.5 Hz), 150.5, 142.5, 136.6, 132.8 (d, ²J = 25.8 Hz), 132.3, 131.0, 130.2 (d, ³J = 9.3 Hz), 129.5, 126.9, 121.7, 120.9 (d, ³J = 8.5 Hz), 116.5 (d, ²J = 22.3 Hz), 115.3 (d, ²J = 23.1 Hz), 114.5 (d, ²J = 25.3 Hz), 113.2 (d, ³J = 8.3 Hz), 69.8, 58.8, 53.5, 46.2, 31.6; HRMS-ESI (m/z): [M + H]⁺ calcd for C₂₅H₂₀Cl₂F₂N₄O₃S 565.05263, found 565.05216.

Biological evaluation

JC-1 mitochondrial membrane potential assay

HT-22 cells (30 000 per well) were seeded into a clear 96-well plate (FALCON) at 200 μL per well one day prior to assay. 750 μM of JC-1 (Stratagene) in DMSO stock solution was dissolved in phenol red-free Opti-MEM (GIBCO) medium to make a final concentration of 7.5 μM JC-1 per well. The medium was removed from the plate, and 100 μL per well of JC-1 was added. Plates were incubated for 1 h and 15 min at 37 °C and washed twice with 100 μL per well PBS. Subsequently, cells were treated with 25 μL solution of each compound at 10 μM in Opti-MEM and incubated at 37 °C for 10 min followed by addition of 25 μL of amyloid beta (American peptide, 1–42) solution at 10 μM. Fluorescence was measured at every one hour for three hours at ex/em 530 nm/580 nm (‘red’) and ex/em 485 nm/530 nm (‘green’). The ratio of green to red fluorescence was recorded and the percent changes in the ratio for each compound were calculated and normalized using the vehicle control as 100%.

CYP inhibition assay

The test compound (40 μL of a 25 μM solution of the compound in distilled water) into a 96-well plate and then 50 μL of the Master Pre-Mix (CYP450 BACULOSOMES® Reagent and Regeneration System) were added. The plate was incubated for 20 min at 37 °C to allow the compound to interact with the CYP450. The reaction was initiated by adding Vivid® CYP450 substrate/NADP⁺ buffer (10 μL). The remaining enzyme activity was measured by reading the amount of fluorescent product using a fluorescence plate reader. The reference inhibitor for each CYP isozyme is indicated in Table 4.

Acknowledgements

This research was supported by the Korea Institute of Science and Technology (KIST-2E24670, 2E25023, 2E24510, 2V03370) and the National Research Foundation of Korea (NRF-2013R1A1A2005550 and NRF-2014M3C1A3054141) funded by the Ministry of Science, ICT and Future Planning. We thank Mr Beoung-Geon Park and Ms Min Kyung Ko for the JC-1 assay and the CYP stability experiments. We thank Dr Jungyeob Ham for helpful discussion on organotrifluoroborates.

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Footnote

† Electronic supplementary information (ESI) available. See DOI: 10.1039/c4ob01967a

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