Exploration of Alternative Scaffolds for P2Y₁₄ Receptor Antagonists Containing a Biaryl Core

Abstract

Various heteroaryl and bicyclo-aliphatic analogues of zwitterionic biaryl P2Y₁₄ receptor (P2Y₁₄R) antagonists were synthesized, and affinity was measured in P2Y₁₄R-expressing Chinese hamster ovary cells by flow cytometry. Given this series’ low water solubility, various polyethylene glycol derivatives of the distally binding piperidin-4-yl moiety of moderate affinity were synthesized. Rotation of previously identified 1,2,3-triazole attached to the central m-benzoic acid core (25) provided moderate affinity but not indole and benzimidazole substitution of the aryl-triazole. The corresponding P2Y₁₄R region is predicted by homology modeling as a deep, sterically limited hydrophobic pocket, with the outward pointing piperidine moiety being the most flexible. Bicyclic-substituted piperidine ring derivatives of naphthalene antagonist 1, e.g., quinuclidine 17 (MRS4608, IC₅₀ ≈ 20 nM at hP2Y₁₄R/mP2Y₁₄R), or of triazole 2, preserved affinity. Potent antagonists 1, 7a, 17, and 23 (10 mg/kg) protected in an ovalbumin/Aspergillus mouse asthma model, and PEG conjugate 12 reduced chronic pain. Thus, we expanded P2Y₁₄R antagonist structure–activity relationship, introducing diverse physical–chemical properties.

Graphical Abstract

INTRODUCTION

The P2Y₁₄ receptor (P2Y₁₄R, previously termed GPR105) is activated by uridine-5′-diphosphoglucose (UDPG) and is expressed in human immune cells, including mast cells and neutrophils.¹ As with other P2 receptors that sense extracellular nucleotides as danger signals,² UDPG released by stressed cells serves as a damage-associated molecular pattern (DAMP) to activate the innate immune system via the P2Y₁₄R. Thus, modulation by ligands and regulation of the P2Y₁₄R are considered a target for the treatment of inflammation, diabetes, cardiac progenitor cell therapy, pain, and pulmonary diseases, such as cystic fibrosis and asthma.^1–12 In renal intercalated cells, P2Y₁₄R activation by endogenous UDPG leads to neutrophil recruitment and elevated inflammation, which is reduced by a P2Y₁₄R antagonist.^4,5 Furthermore, in the eye, the P2Y₁₄R gene is expressed at the highest levels in the trabecular meshwork, suggesting that this receptor might have a role in the regulation of outflow resistance in the context of glaucoma treatment.¹³

The original non-nucleotide lead, 1 (4-[4-(4-piperidinyl)-phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthalenecarboxylic acid, PPTN, Chart 1), in our structure–activity relationship (SAR) studies of P2Y₁₄R antagonists appeared in a patent and later publications by Black and co-workers and Belley et al. on P2Y₁₄R antagonists.^14–16 Examined across the entire P2YR family of eight G protein-coupled receptors (GPCRs), 1 was characterized as a highly potent (K_i < 1 nM) and selective P2Y₁₄R antagonist in functional assays and in reduction of UDPG-induced chemotaxis of human neutrophils.^17,18 However, 1 showed poor aqueous solubility and bioavailability, suggesting that a prodrug approach is needed.¹⁶

Chart 1.

Structures of Two Keys, Previously Reported P2Y₁₄R Antagonists Comparing Analogous Regions That Were Structurally Modified in This Study

Our approach to exploring P2Y₁₄R antagonist SAR is based on insights gained from modeling of the receptor binding. Although lacking an X-ray structure, the P2Y₁₄R can be modeled on the closely related human (h) P2Y₁₂R structure (45% sequence identity), within the Gi-coupled P2YR subfamily.^19,20 We initially probed the piperidine moiety (yellow region of 1, Chart 1) in this series of naphthalenes and found it to have flexibility of substitution of the secondary amine, which we proposed is directed toward the extracellular space when receptor-bound. This secondary amine was suitable for chain extension, including creating a site for fluorophore attachment through a click reaction of an azide-functionalized fluorophore with an extended alkyne.¹⁹ A resulting fluorescent antagonist of high affinity (MRS4174 36, K_i = 0.08 nM in a hP2Y₁₄R functional assay, structure in the Supporting Information) containing an Alexa Fluor 488 (AF488) fluorophore is now our standard tool for drug screening at this receptor using flow cytometry of whole cells expressing the receptor.

Although a naphthalene moiety is found in many important bioactive molecules,²¹ we previously focused on replacing this moiety of 1 with a bioisostere to improve the physicochemical properties of the series.²² Thus, we introduced the biaryl (phenyl-triazole) ring system (2, blue region) as a bioisosteric substitute for naphthalene. In a subsequent P2Y₁₄R SAR study,²³ we preserved the red (with hydrophobic substituents favored) and blue regions and varied the green and yellow regions with other aryl groups containing hydrophilic substituents, including furan and thiophene substitution of the phenyl ring. The selection of aryl and extended groups was guided by molecular modeling, using both docking in a P2Y₁₂R-based homology model and molecular dynamics (MD) simulation.^22,23 From that study, a 2,5-disubstituted thienyl group emerged as a favored bioisosteric replacement for this p-substituted phenyl ring (green region). However, the substitution with nitrogen heteroatoms within either of the p-substituted phenyl moieties (green and red regions) greatly reduced binding affinity. Moreover, the purple region of 1 and 2 was found empirically to be best kept as a carboxylate, consistent with a predicted interaction of this anionic group with three amino acid residues in the receptor. An attempt to substitute this carboxylate with the bioisostere tetrazole²⁴ resulted in a 7- to 30-fold affinity reduction of potent P2Y₁₄R antagonists.²³ Thus, the reported biaryl scaffolds in P2Y₁₄R antagonists in 1 and 2 appeared to be of limited ability to be further modified to enhance P2Y₁₄R affinity.

In this study, we have re-examined each of the regions of 1 and 2, with the intention of identifying alternative scaffolds for P2Y₁₄R antagonists. Both empirical SAR probing of alternative scaffolds and the computational modeling of the new antagonists with both the human (h) and mouse (m) P2Y₁₄R are included here. One important objective is to identify new P2Y₁₄R antagonists that could be useful for in vivo studies. We have quantified modest improvements in aqueous solubility, and we demonstrate the efficacy of selected analogues in mouse models of asthma and chronic neuropathic pain.

RESULTS

Selection of Target Structures.

We have re-examined substitution in all regions of the previously reported key naphthalene 1 and triazole 2 derivatives as P2Y₁₄R antagonist scaffolds. The new target structures selected are shown in Tables 1A and 1B. Initially, we probed minor modifications (3 and 4), and terminal N-alkyl and acyl extensions (5–14) of the piperidine moiety of 1, including amide-linked polyethylene glycol (PEG) chains (11–14), were introduced to enhance water solubility (Table 1A). In general, covalently bound PEG is known to reduce aggregation in aqueous solutions of hydrophobic or highly H-bonding drug substances and biologics with a low liability of toxicity.²⁵ Simple alkyl or acyl extensions of the piperidine nitrogen in the form of tertiary amines or amides were reported by Kiselev et al.¹⁹ with significant retention of P2Y₁₄R affinity, and their SAR is expanded here. Synthetic procedures for simple acyl derivatives 8 and 9 were recently reported.¹² Several of the alkyl-extended analogues in Table 1A were tested previously using a functional assay¹⁹ and are re-assayed here using our standard whole cell fluorescence binding assay.

Table 1A.

hP2Y₁₄R Inhibitory Potency of Piperidine Variations of Naphthalene and Phenyl-Triazole Derivatives^e

Compound	R1 =, other changes	cLogPd	IC50 (μM)a
1b		6.18	0.0060±0.0001
2b		4.64	0.0317±0.0080
3		6.09	0.018±0.002
4		3.95	0.233±0.026
5		7.00	0.195±0.120
6a		6.55	0.139±0.019
6b		7.96	0.133±0.111
7a		7.54	0.0763±0.0244
7b		8.80	0.131±0.011
8b		6.47	0.0276±0.0043
9b		6.74	0.0418±0.0018
10		7.41	2.44±1.54
11		5.52	1.41±0.56
12		2.46	0.963±0.417
13		5.27	0.979±0.331
14		5.84	2.83±1.15
15		4.39	c
16		5.34	42.1±8.4
17		6.32	0.0200±0.0044
18		4.96	0.105±0.010
19		4.58	0.359±0.069
20		4.57	0.639±0.136
21		2.72	0.0383±0.0022

^aIC₅₀ values were determined by flow cytometry of hP2Y₁₄R-CHO cells using fluorescent antagonist tracer 36 and expressed as mean ± SEM (n = 3–5).

^bIC₅₀ values were from Junker et al.,²² Yu et al.,²³ and Mufti et al.¹²

^cNo inhibition by the compound discerned at the highest concentration; therefore, IC₅₀ > 100 μM.

^dcLogP calculated using ALOGPS 2.1 program (www.vcclab.org/lab/alogps/).²⁴

^eR² = CO₂H, unless noted.

Table 1B.

hP2Y₁₄R Inhibitory Potency of Alternative Scaffolds Containing a Biaryl Core

Compound	R3 =	R1 =	cLogPd	IC50 (μM)a
22b		CONH2	3.63	0.269±0.121
23b		CONH(CH2)3NH2	0.84	0.169±0.042
24		CONH2	3.65	1.68±0.38
25b			4.64	0.644±0.175
26		CONH(CH2)3NH2	0.85	2.60±0.56
27		CONH2	3.50	3.05±0.21
28			1.14	c
29		CONH2	3.52	6.04±0.81
30		CONH2	4.31	2.44±0.43
31		CONH2	4.30	2.03±0.34
32		CONH2	3.77	24.4±3.3
33			3.92	c
34			4.66	11.1±1.6
35			4.82	c

^aIC₅₀ values were determined by flow cytometry of hP2Y₁₄R-CHO cells using fluorescent antagonist tracer 36 and expressed as mean ± SEM (n = 3–5).

^bIC₅₀ values were from Junker et al.,²² Yu et al.,²³ and Mufti et al.¹²

^cNo inhibition by the compound discerned at the highest concentration; therefore, IC₅₀ > 100 μM.

^dcLogP calculated using ALOGPS 2.1 program (www.vcclab.org/lab/alogps/).²⁴

Bicyclic analogues of the piperidine moiety of either 1 or 2 were included (17–21), with the aim to conformationally constrain the compounds and empirically test if the constrained conformation may be the receptor-preferred one. By analogy, we previously applied the approach of conformationally constraining a ring moiety, i.e., ribose, to increase the potency and selectivity of purinergic nucleoside and nucleotide ligands.²⁶ The absence of an experimental P2Y₁₄R structure is limiting the predictive power of modeling, so binding affinity of conformationally constrained ligands could indirectly provide structural information on the preferred binding mode of the compounds. Adding carbon bridges to the piperidine ring of 2 was also intended to reduce hydrophobicity, paradoxically, and increase solubility²⁷ while retaining P2Y₁₄R affinity. The compact 3D-shaped conformation of a bridged piperidine might improve the solubility by increasing the fraction of the solvent-exposed polar surface area. Also, this region in the receptor binding site is predicted to be less sterically crowded than deeper in the site, so three-dimensionality, e.g., in the form of a bicyclic quinuclidine, might be tolerated. The alkene derivative 3 was desired both as a precursor for introduction of a fused cyclopropane ring and for radiolabeling by catalytic reduction. We also re-examined substitution of the carboxylic acids of 1 and 2, which appeared to be already optimized based on the earlier studies. Thus, two uncharged substitutions of this carboxylate of 2 were included in this study (15 and 16).

We probed the effect on P2Y₁₄R affinity of other scaffold modifications and compared them to known reference compounds 2, 22, and 23 (Table 1B). Modifications included a rotation of the 1,2,3-triazole ring of the blue region of 2 by a single position to form 24–26 and its replacement with an amide group (27–29). Synthetic procedures for rotated triazole 25 and its P2Y₁₄R affinity were recently reported.¹² Rotation of the triazole was predicted by modeling to maintain a geometry of the polycyclic scaffold of 2. Among the amide derivatives synthesized, a three-dimensional cubane replacement of the 4-trifluoromethyl-phenyl ring was introduced in 28, based on a published report that cubane can serve as a benzene ring bioisostere.^28,29 We also fused two aryl rings of 2 to form various heterocycles (30–32), e.g., to form benzimidazoles and an indole from the fusion of the 4-trifluoromethyl-phenyl ring (red, Chart 1) with the 1,2,3-triazole. The SAR of the central phenyl ring of 2 was not previously explored; here, we introduce various heterocyclic substitutions, i.e., a pyrrole (33), and methylation of the phenyl ring (34). Benzimidazole derivative 35 was an additional variation of ring fusion, by combining ring equivalents of the central 1,3,5-trisubstituted phenyl ring and the 1,2,3-triazole.

Chemical Synthesis.

The synthesis of the novel heteroaryl, piperidinyl, and bicyclo-aliphatic analogues (3, 4, 6, 7b, 10–21, 24, and 26–35) is shown in Schemes 1–4. The synthesis of the piperidin-4-yl derivatives of the potent naphthalene antagonist 1 in which the secondary amine was alkylated or acylated with small groups (5–10) or acylated with water-solubilizing PEG groups (11–14) is shown in Scheme 1A,B. An amide linkage, rather than an alkylated piperidine group, was chosen for the PEG conjugates due to the relatively high P2Y₁₄R affinity of simple acyl derivatives 8 and 9. First, a key intermediate, e.g., aryl dioxaborolane 40, was subjected to a Pd-catalyzed Suzuki coupling with either a bromoaryl or aryl triflate precursor, e.g., 37.²³ This affixed the p-substituted phenyl ring to the central naphthalene or phenyl ring of reference antagonist 1 or 2, respectively. For most of the analogues, removal of the Boc-piperidine protection, e.g., of 41, and saponification of the ethyl ester, e.g., of 42, were performed sequentially. However, when N-acylation with a PEG group was desired, this group was installed first on secondary amine 42 before saponification. The (PEG)₆ conjugates 11–14 were more water soluble than parent 1, as expected. For example, PEG derivative 12 was found to be completely soluble in water at a concentration of 150 mM.

Scheme 1. (A–D) Synthesis of Piperidine-Modified Analogues of 1 and 2^a.

^a(A) Reagents and conditions: (a) B₂pin₂, PdCl₂(dppf), KOAc, dioxane, 85 °C, 4 h, 61%; (b) tert-butyl 4-(4-bromophenyl)piperidine-1-carboxylate, Pd(PPh₃)₄, K₂CO₃, DMF, 80 °C, 5 h, 59%; (c) TFA/THF = 1:1, rt, 1 h, 90–93%; (d) CH₃I or HC≡CCH₂Br or CH₃(CH₂)₂I, K₂CO₃, CH₃CN, rt or 50 °C, 15 h, 55% (43a) or 65% (43b) or 68% (43c); 6-bromohexyne-1, K₂CO₃, DMF, rt, 15 h, 70% for 43d; Ac₂O, Et₃N, CH₃CN, rt, 0.5 h, 49% for 43e; propionic acid, HATU, DIPEA, DMF, rt, 2 h, 54% for 43f; (e) KOH, MeOH, H₂O, 50 °C, 48–88%; (f) H₂, Rh/C, MeOH/EtOAc (1:1), 100 psi, 92%; (g) tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate, PdCl₂(PPh₃)₂, Na₂CO₃, 1,4-dioxane/water (10:1), 80 °C, 12 h, 48%. (B) Reagents and conditions: (a) Boc-NH-PEG₆-CH₂CH₂COOH or mPEG₅-CH₂CH₂COOH, HATU, DIPEA, DMF, rt, 1 h, 94% (44a) or 93% (44b); (b) KOH, MeOH, H₂O, 50 °C, 15 h, 65% (11) or 79% (14); (c) TFA/THF = 1:1, rt, 1 h, 91%; (d) Ac₂O, pyr, rt, 1 h, 59%. (C) Reagents and conditions: (a) 4-(4-bromophenyl)quinuclidine 96, Pd(PPh₃)₄, K₂CO₃, DMF, 80 °C, 3 h, 88%; (b) CH₃I, K₂CO₃, CH₃CN/MeOH (1:1), 37 °C, 1 h, 55%; (c) KOH, MeOH, H₂O, 50 °C, 12 h, 53% (17) or 71% (21). (D) Reagents and conditions: (a) B₂pin₂, PdCl₂(dppf), KOAc, dioxane, 70 °C, 15 h, 87%; (b) tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate, 96, 99, or 101, Pd(PPh₃)₄, K₂CO₃, DMF, 80 °C, 3 h, 32–41%; (c) KOH, MeOH, H₂O, 50 °C, 59–94%; (d) TFA/THF = 2:1, rt, 0.5 h, 35–79%.

Scheme 4. (A–C) Synthesis of Analogues of 2 in Which the Central Phenyl Ring Has Been Modified^a.

^a(A) Reagents and conditions: (a) Ts₂O, TEA, DCM, rt, 3 h; (b) NaOEt, diethyl aminomalonate hydrochloride, EtOH, THF, rt, 0.5 h, 40%; (c) tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate, PdCl₂(dppf), NaOH, DMF, rt, 1 h, 78%; (d) NaNO₂, NaN₃, 4 M HCl(aq), 0 °C to rt, 0.5 h, 75%; (e) 4-ethynyl-α,α,α-trifluorotoluene, sodium ascorbate, CuSO₄·5H₂O, dimethyl sulfoxide/water = 9:1, rt, 1 h, 77%; (f) TFA/THF = 2:1, rt, 0.5 h, 60%; (g) KOH, MeOH, H₂O, 50 °C, 5 h, 30%. (B) Reagents and conditions: (a) TMS-acetylene, PdCl₂(PPh₃)₂, CuI, Et₃N, DMF, rt, 5 h, 99%; (b) TBAF, THF, rt, 0.5 h, 93%; (c) 1-azido-4-(trifluoromethyl)benzene, CuSO₄·5H₂O, Na ascorbate, THF/H₂O, rt, 1 h, 66%; (d) tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate, Pd(PPh)₄, K₂CO₃, DMF, 85 °C, 12 h, 70%; (e) TFA/THF = 2:1, rt, 0.5 h, 79%; (f) KOH, MeOH, H₂O, 50 °C, 5 h, 72%. (C) Reagents and conditions: (a) 4-(trifluoromethyl)benzaldehyde, Na₂S₂O₅, DMF, 130 °C, 12 h, 65%; (b) tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-piperidine-1-carboxylate, Pd(PPh₃)₄, Na₂CO₃, 1,4-dioxane/water (10:1), 80 °C, 12 h, 43%; (c) TFA/THF = 2:1, rt, 0.5 h, 82%; (d) KOH, MeOH, H₂O, 50 °C, 5 h, 63%.

The bromoaryl intermediates 96–101 for the synthesis of bridged piperidine products 17–21 were prepared as described in the Supporting Information (Scheme S1). Phenylquinuclidine derivative 17 related to 1 was prepared by a Suzuki coupling, as shown in Scheme 1C from intermediate 40, protected as an ethyl ester. Other bridged piperidine derivatives of 2 were prepared from bromo intermediate 46,²³ as shown in Scheme 1D. The attempted substitution of the carboxylic acid of the central phenyl ring with closely related uncharged groups was performed, as described in Scheme S2.

Rotation of the triazole ring of reference compound 2 and thienyl analogue 23 by one position was accomplished, as shown in Scheme 2, to provide 25 and 26, respectively. The carboxylic group of commercial 1,3,5-trisubstituted benzene derivative 53 was first protected as the methyl ester 54 followed by substitution of iodo of the central phenyl ring with TMS-acetylene to yield 55. Compound 55 was reacted with TBAF for deprotection of TMS to afford 56 having an alkyne group. Subsequently, 56 was reacted with 1-azido-4-(trifluoromethyl)benzene in the presence of copper sulfate to afford rotated triazole 57, which was then reacted with bis(pinacolato)diboron (B₂pin₂) to yield 58. Suzuki reactions of 58 with 4-BrPhCONH₂, tert-butyl 4-(4-bromophenyl)-piperidine-1-carboxylate, or tert-butyl (3-(5-bromothiophene-2-carboxamido)propyl) carbamate yielded 59a–59c, respectively, and were followed by hydrolysis of the methyl ester to provide 24 and 60b and 60c, respectively. Removal of the N-Boc protecting groups of 60b and 60c afforded rotated triazole analogues 25 and 26, respectively.

Scheme 2. Synthesis of Reverse Triazole Analogues of 2^a.

^aReagents and conditions: (a) SOCl₂, MeOH, rt, 15 h, 96%; (b) TMS-acetylene, PdCl₂(PPh₃)₂, CuI, Et₃N, DMF, rt, 5 h, 92%; (c) TBAF, THF, rt, 0.5 h, 94%; (d) 1-azido-4-(trifluoromethyl)benzene, CuSO₄·5H₂O, Na ascorbate, THF/H₂O, rt, 1 h, 46%; (e) B₂(pin)₂, KOAc, PdCl₂(dppf), dioxane, 70 °C, 15 h, 76%; (f) tert-butyl 4-(4-bromophenyl)piperidine-1-carboxylate, Pd(PPh)₄, K₂CO₃, DMF, 85 °C, 2 h for 59b (39%) or 4-BrPhCONH₂ or tert-butyl (3-(5-bromothiophene-2-carboxamido)propyl) carbamate, PdCl₂(dppf)₂, NaCO₃, DME, 50 °C, 46% (59a) or 52% (59c); (g) KOH, MeOH, H₂O, 50 °C, 15 h, 60–99%; (h) TFA/THF = 1:1, rt, 1 h, 61% (25) or 45% (26).

Amides in place of the triazole ring were prepared, as shown in Scheme 3A. A Suzuki reaction established the biaryl group of 63 prior to the amide formation using HATU. Amide analogues of reference compound 2 and a primary carboxamide 22, i.e., 27 and 28, respectively, were included. A cubane amide was introduced in compound 28. The direction of the amide linking group in primary carboxamide analogue 29 was reversed for comparison to 27 (Scheme 3B). The carboxylic group of commercial precursor 61a was protected as the methyl ester 61b, which was coupled with dioxaborolane group 62, followed by Suzuki reactions with 4-BrPhCONH₂ or tert-butyl 4-(4-bromophenyl)piperidine-1-carboxylate to afford 63a and 63b, respectively. Subsequently, 63a and 63b were coupled with p-CF₃PhCOOH or 4-(HOCH₂)cubane-1-COOH to yield 64a and 64b, respectively, which were then deprotected by one of the two methods to yield 27 and 28, respectively. For the synthesis of 29 in which the direction of the amide linking group was reversed compared with 27, esterification of 66 was conducted to afford 67, which was coupled with p-trifluoromethylaniline 69,³⁰ followed by a Suzuki reaction with 4-aminocarbonylphenylboronic acid pinacol ester 70, which was hydrolyzed to yield 29.

Scheme 3. (A–D) Synthesis of Analogues of 2 in Which the Triazole Moiety Has Been Replaced with an Amide or Fused with the Terminal 4-Trifluoromethylaryl Ring^a.

^a(A) Reagents and conditions: (a) SOCl₂, MeOH, rt, 15 h, 98%; (b) B₂(pin)₂, KOAc, PdCl₂(dppf), dioxane, 95 °C, 15 h, 68%; (c) 4-BrPhCONH₂ or tert-butyl 4-(4-bromophenyl)piperidine-1-carboxylate, Pd(PPh)₄, K₂CO₃, DMF, 80 °C, 15 h, 63% (63a) or 41% (63b); (d) p-CF₃PhCOOH or 4-(HOCH₂)cubane-1-COOH, HATU, DIPEA, DMF, rt, 15 h, 99% (64a) or 69% (64b); (e) KOH, MeOH, H₂O, 50 °C, 15 h, 70% for 27; (f) (i) 1 N HCl, dioxane, rt, 15 h, 67%; (ii) KOH, MeOH, H₂O, 50 °C, 15 h, 39% for 28. (B) Reagents and conditions: (a) H₂SO₄, CH₃OH, 60 °C, 15 h, 54%; (b) oxone, DMF, rt, 15 h, 78%; (c) (i) SOCl₂, Et₃N, DCM, 0 °C, 1 h; (ii) p-trifluoromethylaniline, Et₃N, DCM, rt, 15 h, 45%; (d) 4-aminocarbonylphenylboronic acid pinacol ester, PdCl₂(PPh₃)₂, Na₂CO₃, dioxane, H₂O, 80 °C, 2 h, 63%; (e) KOH, MeOH, H₂O, 50 °C, 15 h, 72%. (C) Reagents and conditions: (a) iodo-(trifluoromethyl)aniline, PdCl₂(PPh)₂, CuI, Et₃N, rt, 1 h, 82% (71a) or 87% (71b); (b) PdCl₂, DMF, 110 °C, 10 min, μW, 65% (72a) or 62% (72b); (c) 4-aminocarbonylphenylboronic acid pinacol ester, PdCl₂(PPh)₂, Na₂CO₃, dioxane, H₂O, 80 °C, 15 h, 55% (73a) or 72% (73b); (d) KOH, MeOH, H₂O, 70 °C, 3 h, 59% (30) or 67% (31). (D) Reagents and conditions: (a) Na₂S₂O₅, 4-trifluoromethyloxy-phenylenediamine, DMF, 130 °C, 15 h, 97%; (b) 4-aminocarbonyl-phenylboronic acid pinacol ester, PdCl₂(PPh₃)₂, Na₂CO₃, dioxane, H₂O, 80 °C, 15 h, 38%; (c) KOH, MeOH, H₂O, 70 °C, 3 h, 99%.

Fused rings derived from the p-trifluoromethylphenyl group were prepared in 30–32, as shown in Scheme 3C,D. The Sonogashira reactions of 56 with trifluoromethyl-substituted 2-iodo-anilines were performed to prepare 71a and 71b, which were cyclized to indoles 72a and 72b, respectively. Compounds 72a and 72b were subjected to a Suzuki reaction to afford 73a and 73b, respectively, which were hydrolyzed to yield 30 and 31, respectively. To synthesize 32, intermediate 66b was first cyclized to a benzimidazole 74. Compound 74 was coupled with 4-aminocarbonylphenylboronic acid pinacol ester through a Suzuki reaction, and the product 75 was then hydrolyzed to afford 32.

Modification of the central phenyl ring of 2 was accomplished, as shown in Scheme 4A (pyrrole analogue 33, which was also substituted with an alkene in the piperidine ring), Scheme 4B (aryl-methyl analogue 34), and Scheme 4C (benzimidazole 35). For preparation of 33, the pyrrole scaffold of 78 was formed from 77, which was synthesized with commercial 76 and p-toluenesulfonic anhydride (Ts₂O). Subsequently, a Suzuki reaction was conducted with tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate to afford 79 followed by conversion of the amine functionality into an azide 80. The azide was subjected to a click reaction with 4-ethynyl-α,α,α-trifluorotoluene to afford triazole 81, which was then deprotected to yield 33. For the synthesis of 34, the iodo group of 83 was substituted with TMS-acetylene to afford 84, which was reacted with TBAF for deprotection of TMS to yield 85. This was followed by the click reaction with 1-azido-4-(trifluoromethyl)benzene to afford 86. A Suzuki reaction of 86 with tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate yielded 87, which was then deprotected at the N-Boc group 88 and hydrolyzed to afford 34. To prepare 35, compound 89 was cyclized to benzimidazole 90, which was coupled with tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-piperidine-1-carboxylate through a Suzuki reaction to synthesize 91 followed by deprotection to afford 35.

In search of an alternative assay to the fluorescence binding assay using 36, we prepared [³H]1 as a potential radioligand, as shown in Scheme 5. The piperidine alkene intermediate 39, containing both Boc and ester protecting groups, was catalytically hydrogenated with tritium gas in ethyl acetate as the solvent to yield 93. The protecting groups were used, rather than one-step catalytic reduction of 3, because having no easily ionizable groups present improved the solubility of both the hydrogenation substrate and the product. Facile, sequential removal of the protecting groups provided the desired tritiated compound [³H]1 in high purity following preparative HPLC (Supporting Information). The catalytic reduction sequence shown in Scheme 5 was first performed using nonradioactive hydrogen gas.

Scheme 5. Synthesis of [³H]1^a.

^aReagents and conditions: (a) Pd/C, ³H₂, EtOAc, rt, 100 psi; (b) TFA/THF = 2:1, rt, 0.5 h; (c) KOH, MeOH, H₂O, 50 °C, 5 h.

In Vitro Pharmacological Assays.

Feasibility testing of [³H]1 as a radioligand in cell membranes was disappointing as the compound demonstrated low solubility in the aqueous medium and adsorbed to plastic surfaces at higher μM concentrations. In hP2Y₁₄R-expressing cell membranes, a signal for specific receptor binding was lacking due to the high nonspecific binding. Therefore, the affinity measurement of the new antagonist analogues was performed using the previously reported fluorescence binding assay, using as tracer the AF488 conjugate 36 prepared according to Yu et al.,²³ by flow cytometry of whole Chinese hamster ovary (CHO) cells stably expressing the hP2Y₁₄R in a 96-well format.

Various small N-alkyl and N-acyl derivatives of the piperidine ring of 1 were previously shown to have moderate hP2Y₁₄R antagonist potency.^12,23 Here, as shown in Table 1A, the loss of affinity in the fluorescence assay of small N-alkyl derivatives of 1 was in the range of 13- to 33-fold (compounds 5–7). Notably, an N-acetyl derivative 8, which is no longer a zwitterion, showed only 3-fold lower affinity than 1. Derivatives with other simple modifications of the piperidine rings of 1 and 2, i.e., alkene 3 and piperazine 4 derivatives, respectively, bound relatively well to the hP2Y₁₄R with affinity reduction of only 3- to 7-fold. The IC₅₀ value of 3 in inhibition of binding of 36 was 18 nM, i.e., it ranked among the most potent analogues in this study. However, the Boc derivative 10 showed relatively weak affinity. Therefore, not all modifications of the piperidine nitrogen were tolerated. The (PEG)₆ amide-linked conjugates 11–14 were intermediate in binding affinity (IC₅₀ ≈ 1–3 μM), although at least 160-fold weaker than the parent compound 1.

The approach of modifying the piperidine ring as bicyclic ring systems was especially fruitful. Phenylquinuclidine derivative 17 proved to have high affinity at the hP2Y₁₄R with an IC₅₀ value of 20 nM, i.e., ∼3-fold lower than its reference naphthalene 1. Similarly, the corresponding triazole derivative 18 showed a P2Y₁₄R affinity ∼3-fold lower than its reference 2. Bridged cyclopropyl derivatives 19 and 20 (both racemic) showed 11-fold and 20-fold lower affinity, respectively, than the reference triazole 2. The quaternized N-methylquinuclidinium derivative 21 had an improved cLogP and showed only ∼6-fold lower hP2Y₁₄R affinity than parent zwitterion 1.

Compounds with modifications elsewhere on the scaffold (Table 1B) were generally of much lower P2Y₁₄R affinity than the reference compounds. Cubane 28, pyrrole 33, and benzimidazole 35 derivatives had affinities that were too low to be measured (estimated >100 μM) using the current method and given the solubility limitations of the compounds. Benzimidazole 32 and aryl-methyl 34 derivatives only weakly inhibited binding, with IC₅₀ values > 10 μM. More effective preservation of P2Y₁₄R affinity was achieved with amides 27 and 29 and trifluoromethyl-indoles 30 and 31, which had IC₅₀ values between 1 and 10 μM. Amide 27 with NH on the central phenyl ring had 11-fold lower affinity than its reference triazole derivative 22, and it bound to the P2Y₁₄R with twice the affinity of the corresponding reversed amide 29. Among the analogues in Table 1B, the best retention of P2Y₁₄R affinity was observed with the rotated triazole derivatives 24–26, which differed in the R¹ substituent. Nevertheless, the affinities were moderately reduced (fold) compared to respective reference compound: 24 (6.2-fold vs 22), 25 (20-fold vs 2), and 26 (15-fold vs 23). The IC₅₀ value of primary carboxamide derivative 25 was 0.644 μM, i.e., 20-fold weaker than reference triazole 2.

To examine the species dependence of affinity, the mP2Y₁₄R was stably transfected in human embryonic kidney (HEK) 293 cells.¹² The intact cell binding of 36 in mP2Y₁₄R-expressing HEK293 cells was almost completely inhibited in the presence of 1 μM 1, as shown in fluorescence micrographs.¹² The primary screen of binding affinity was P2Y₁₄R fluorescence binding quantified using flow cytometry. The affinity of selected compounds at the mP2Y₁₄R was determined (Table 2). Representative fluorescence binding inhibition curves at the two species homologues are shown for key antagonists (Figure 1). Compound 17 showed the same affinity at the two P2Y₁₄R species homologues and also the same affinity as 1 at the mP2Y₁₄R. PEG derivative 12 and rotated triazole 25 showed 2-fold greater affinity at the mouse homologue than at the hP2Y₁₄R, while other compounds had higher IC₅₀ values at mP2Y₁₄R than at hP2Y₁₄R: 1 (3.6-fold), 2 (4.5-fold), 4 (2.1-fold), 8 (1.7-fold), 18 (8.0-fold), 19 (3.3-fold), 22 (3.4-fold), and 23 (2.3-fold). Thus, in the quinuclidine series, the naphthalene moiety of 17 better preserved mP2Y₁₄R affinity than the triazole core structure of 18.

Table 2.

Inhibitory Potency of Antagonists at the mP2Y₁₄R Expressed in HEK293 Cells, Using the Fluorescence Binding Method, Compared to the hP2Y₁₄R Expressed in CHO Cells

Compound	mP2Y14R, IC50 (nM)a	hP2Y14R, IC50 (nM)a
1	21.6 ± 7.0	6.0 ± 0.1
2	142 ± 58	31.7 ± 8.0
4	499 ± 57	233 ± 26
7a	130 ± 30	76.3 ± 24.4
8	29.7 ± 9.3	27.6 ± 4.3
12	487 ± 130	963 ± 417
17	21.4 ± 7.9	20.0 ± 4.4
18	845 ± 150	105 ± 10
19	1160 ± 330	359 ± 69
21	56.3 ± 19.0	38.3 ± 2.2
22	902 ± 344	269 ± 121
23	384 ± 88	169 ± 42
25	246 ± 63	644 ± 175

^aIC₅₀ values were determined by flow cytometry of mP2Y₁₄R-HEK293 cells using fluorescent antagonist tracer 36 and expressed as mean ± SEM (n = 3–5). hP2Y₁₄R values were from Tables 1A and 1B.

Figure 1.

(A, B) Fluorescence inhibition assay by flow cytometry in (A) hP2Y₁₄R-expressing CHO cells and (B) mP2Y₁₄R-expressing HEK293 cells with the AF488-labeled tracer 36. The IC₅₀ values are given in Tables 1A and 1B. In (C), the pH was varied between 7.4 and 6.0, and binding was measured using hP2Y₁₄R-expressing CHO cells.

The pH dependence of hP2Y₁₄R affinity was examined for two potent derivatives, 1 and 17. These antagonists showed only a minor increase in IC₅₀ values when the pH was lowered from 7.4 to 6.0 (Figure 1C and Table 3). Thus, the antagonists can be expected to be effective under slightly acidic conditions as might occur during inflammation, cancer, and ischemia.³¹

Table 3.

pH Dependence of IC₅₀ of Two Antagonists in Inhibition of Fluorescent Antagonist 36 Binding at hP2Y₁₄R-Expressing CHO Cells (n = 3, Measured in Triplicate)

Compound	pHa	IC50 ± SEM (nM)
1	7.4	6.01 ± 0.87
	7.0	9.75 ± 3.29
	6.5	14.5 ± 5.8
	6.0	19.0 ± 2.8
17	7.4	30.8 ± 2.2
	7.0	38.1 ± 2.1
	6.5	57.0 ± 8.7
	6.0	84.5 ± 1.1

^aThe levels of total fluorescence binding were comparable at the different pH values (arbitrary fluorescence units): pH 7.4, 71.37 ± 3.82; pH 7.0, 76.98 ± 4.39; pH 6.5, 78.80 ± 7.83; and pH 6.0, 79.29 ± 6.49.

One objective was to overcome the low aqueous solubility of 1. The cLogP values (Tables 1A and 1B) were calculated for the antagonists as an indication of partition between aqueous and organic phases. There was no correlation between the cLogP and IC₅₀ values (Supporting Information), indicating that the binding affinity was not a function of hydrophobicity as one would expect if association with a lipid membrane was required, for example with a P2Y₁R allosteric antagonist.³² The five antagonists of highest affinity, 1, 2, 3, 17, and 18, were relatively nonpolar, with cLogP values of 5–6, but the cLogP was consistently lower in the triazole compared to the naphthalene series. Compound 23 showed a balance between moderately high affinity and hydrophilicity, with a cLogP of 0.84.²³

The solubility of six analogues was determined using the pION method (Table 4), and lipophilicity was determined based on the HPLC retention time relative to 1 (methods in the Supporting Information). The mean aqueous solubility at pH 7.4 varied in the order 12 > 8, 21 > 18 > 17, 1. Thus, modest improvements in the solubility at physiological pH were observed, especially for the PEG conjugate 12 (7.16 μg/mL) compared to parent reference compound 1 (0.36 μg/mL). At pH 4.0, the solubility was greater than at pH 7.4, except for the nonzwitterionic carboxylic acid 8, but the same compound showed reduced lipophilicity relative to 1. For in vivo administration, aqueous DMSO was used as a solubilizing vehicle. Quinuclidine derivative 17 was readily soluble at 1 mM in DMSO as a zwitterion, while the parent 1 as a zwitterion failed to dissolve in DMSO at 1 mM, unless it was treated with aqueous NaOH or HCl. Compound 17 dissolved readily in DMSO without basification and was not subjected to precipitation upon dilution; it was completely soluble at 1 mg/mL in 10% DMSO in water.

Table 4.

Solubility and Relative Lipophilicity Determined for Selected P2Y₁₄R Antagonists

	solubility (μg/mL)a
compound	pH 4.0	pH 7.4	lipophilicity (Δk), relative to 1b
1	1.10 ± 0.07	0.36 ± 0.03	0
8	0.09 ± 0.02	2.32 ± 0.18	−0.48 ± 0.01
12	8.02 ± 1.01	7.16 ± 1.16	0.71 ± 0.03
17	1.50 ± 0.04	0.55 ± 0.02	1.24 ± 0.78
18	2.87 ± 0.10	1.10 ± 0.09	−0.09 ± 0.13
21	2.24 ± 0.19	1.72 ± 0.07	0.24 ± 0.03

^aDetermined using the pION method at the pH indicated (Supporting Information).

^bDetermined from the RP-HPLC retention time (C18 column), with a 20 min gradient of 10 to 100% acetonitrile in 10 mM triethylammonium acetate.

OFF-TARGET AND ADME-TOXICITY TESTING

Although the novel antagonists in this study were not evaluated for P2Y₁₄R selectivity within the P2Y family, there is precedent for exclusive interaction with P2Y₁₄R as compounds 1 and 2 and other members of the triazole series were inactive at other P2YRs.^8,17 Off-target non-P2YR interactions of selected antagonists (4, 6b, 7a, 8, 12, 17–19, and 22–26) were determined at 46 receptors, transporters, and ion channels (comprehensive screen) by the Psychoactive Drug Screening Program (PDSP, Supporting Information).³³ Compound 1 was previously reported to bind to the D₃ dopamine and δ-opioid (DOR) receptors with K_i values of 6.79 and 2.75 μM, respectively, and compounds 2, 6b, and 7a had no detected off-target interactions.^8,9 Several off-target interactions were noted for the new analogues (K_i, μM, receptor): 4, 6.7 ± 0.5 (σ₂R); 8, 4.31 ± 0.11 (D₃R), 4.38 ± 0.62 (D₅R), 1.75 ± 0.54 (5HT_1DR), and 0.51 (TSPO); 17, 2.29 ± 0.81 (σ₂R); 18, 1.37 ± 0.04 (σ₂R); 19, 2.63 ± 0.13 (DOR) and 0.30 ± 0.13 (σ₂R); 21, 0.42 (σ₂R); 22, 6.89 (5HT₆R); 23, 3.26 ± 0.64 (DOR) and 4.34 (5HT_1DR); 24, 2.48 (DOR), 0.50 (σ₁R), and 2.91 (σ₂R); 25, 3.79 (DOR), 0.96 (α_2AR), 5.33 (α_2BR), 2.71 ± 0.21 (α_2CR), 1.48 (σ₁R), and 2.09 (σ₂R); and 26, 2.96 (DOR) and 1.11 (σ₁R). Furthermore, compounds 1 and 25 were inactive (EC₅₀, IC₅₀ > 10 μM) as functional agonist or antagonist of CB1 and CB2 cannabinoid receptors, as determined by PDSP (Supporting Information). An indication that the representative tested compounds were not promiscuous, i.e., pan-assay interference compounds (PAINS),³⁴ was the lack of appreciable binding at other sites in the PDSP screen.

ADME-toxicity properties were predicted for compounds 7a, 8, and 25 using online resources.^35,36 None of the three were predicted to inhibit hERG-I. The only CYP interactions predicted were as follows: all three as CYP3A4 substrates and 8 additionally as the CYP2C9 inhibitor. Furthermore, compounds 17 and 18 were tested for bioavailability when administered ip (1, 3, and 10 mg/kg compared to 0.5 mg/kg iv) in the rat and in other in vitro tests predictive of ADME-toxicity properties (Table 5). Several unusual pharmacokinetic features were observed (Supporting Information), the first of which was >100% F for ip administration of both 17 and 18. The bioavailability was dose-correlated such that the highest dose of 10 mg/kg showed a faster clearance than at 1 and 3 mg/kg. The 10 mg/kg dose also had a higher volume of distribution. The maximal plasma concentrations of 17 and 18 (∼900 ng/mL at the highest dose of 10 mg/kg) were reached at 3–4 h (Figure S6, Supporting Information). The highest dose still maintained a concentration of ∼200–400 ng/kg at the 12 h time point. At the 1 mg/kg dose, the plasma concentrations of compounds 17 and 18 were 165 and 206 nM (2 h) and 292 and 163 nM (4 h), respectively. The t_1/2 values of compounds 17 and 18 were 5.1 and 4.6 h, respectively (1 mg/kg, ip). Thus, the in vivo clearance of both compounds was low, but there was a lag observed in the entry into circulation with ip but not iv administration. Caco-2 cell permeability values of 17 and 18 (P_app, apical to basal) were 0.00 and 0.09, respectively, and they were not cytotoxic to Hep-G2 cells (IC₅₀ > 20 μM, Table S2). Compounds 17 and 18 were stable in plasma and simulated body fluids and showed an acceptable hERG inhibition, which was greatly improved over the earlier compound 22. In addition, the plasma protein binding of compounds 12, 17, 18, 21, 23, and 25 tested in three species was high, with 21 and 25 having a more favorable free fraction in human and mouse but not rat (Table 6).

Table 5.

In Vitro and In Vivo ADME-Tox Data for Two Representative P2Y₁₄R Antagonists^a

Test	22b	17	18
simulated intestinal fluid (% remaining at 120 min)	100	99.7	102
simulated gastric fluid (% remaining at 120 min)	100	74.2	106
CYP1A2 (IC50, μM)	>30	8.69	14.1
CYP2C9 (IC50, μM)	>30	>30	>30
CYP2C19 (IC50, μM)	>30	>30	>30
CYP2D6 (IC50, μM)	>30	>30	>30
CYP3A4 (IC50, μM)	>30	3.29	9.94
plasma stability (three species)d(% remaining at 120 min)	94–100	100 (h); 79.9 (r); 100 (m)	86.8 (h); 89.4 (r); 79.5 (m)
microsomal stability(t1/2, min)	145 (m), 108 (r), 145 (h)	67.2 (m), 80.0 (r), >240 (h)	311 (m), 108 (r), 192 (h)
hERG, IC50 (μM)	0.166	16.4	16.8
t1/2 (h), administration modee	0.20 (iv)	5.1 (1 mg/kg, ip); 3.46 (iv)	4.6 (1 mg/kg, ip); 0.72 (iv)
aqueous solubilityc (pH 7.4, μg/mL)	138 ± 4	0.44 ± 0.06	<0.1
aqueous solubilityc (pH 4.0, μg/mL)	NDf	14.3 ± 0.5	14.4 ± 0.3

^aProcedure is in the Supporting Information.

^bData determined by Yu et al.²³

^cMean ± SD, pION method.

^dSpecies tested for plasma stability were human, rat, and mouse; species as indicated for microsomal stability.

^eBy iv administration: 0.5 mg/kg dose.

^fND, not determined.

Table 6.

Plasma Protein Binding at 10 μM in Three Species, Expressed as Unbound Percent^a

compound	human, % freeb	mouse, % freeb	rat, % free
12	0.48	0.29	0.00
21	1.13	0.94	0.00
23	0.00	0.83	0.00
25	1.25	1.33	0.00

^aProcedure is in the Supporting Information. Verapamil and warfarin served as positive controls.

^bMean ± SD.

Evaluation of Selected Analogues in an In Vivo Assay of Antiasthmatic Activity.

The ability of selected analogues to reduce allergic lung inflammation was studied in an Aspergillus protease-mediated mouse model of allergic asthma.³⁷ The test compounds were injected intraperitoneally at 10 mg/kg 30 min prior to allergen challenge, and airway inflammation was assessed 2 days post-challenge (Figure 2)¹¹ by cell differential counts in the bronchoalveolar lavage fluid (BALF) of mice. As shown previously, mP2Y₁₄R blockade by reference antagonist 1 strongly attenuated eosinophilic airway inflammation.¹¹ A comparison of activity for other derivatives revealed that the highest potency of reducing eosinophil infiltration was associated with the previously reported N-hexynyl 7a (10 mg/kg, ip) and that in vivo effects of quinuclidine 17 and primary amino 23 derivatives were similar to 1. N-Acetyl 8 and rotated triazole 25 derivatives had a less pronounced but still significant anti-inflammatory activity. By contrast, a PEG derivative 12 and reference triazole 2 at the same dose were inactive when applied in this rodent model of asthma. Although many of the tested compounds had similar mP2Y₁₄R affinity in vitro, the differences in in vivo activity probably arose from varying pharmacokinetic properties.

Figure 2.

P2Y₁₄R blockade during allergen challenge reduces eosinophilic airway inflammation in the protease model of asthma. (A) Timeline for ASP/OVA sensitization, P2Y₁₄R antagonist treatment, and OVA challenge. (B) Effect on BALF leukocytes of ip injection of the P2Y₁₄R antagonists 2 days post-challenge of ASP/OVA-sensitized mice. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001 vs vehicle by one-way ANOVA followed by Dunnett’s test.

Evaluation of 12 in Chronic Constriction Injury Mouse Model of Neuropathic Pain.

Since the PEG conjugate 12 showed ∼20-fold greater aqueous solubility at pH 7.4 than the reference compound 1, we measured its in vivo efficacy in a mouse model of chronic neuropathic pain (7 days post-chronic constriction injury, CCI, of the sciatic nerve in adult male mice), as we recently reported for various P2Y₁₄R antagonists in the work of Mufti et al.¹² Compound 12 was fully efficacious in reversing established, CCI-induced mechano-allodynia (Figure 3) at a dose that was already shown to be effective for other more potent P2Y₁₄R antagonists such as 1 and 8 (10 μmol/kg, ip). Peak protection was achieved at 1–2 h post-injection. No effect of the drug was seen on the hind paw response contralateral to the injury.

Figure 3.

In vivo efficacy of compound 12 in a mouse model of unilateral chronic neuropathic pain (7 days post-chronic constriction injury, CCI, of the sciatic nerve in adult male ICR mice), as described by Mufti et al.¹² The dose was 10 μmol/kg, ip, injected in a volume of 0.2 mL in a vehicle of 30% DMSO in phosphate-buffered saline. Mechano-allodynia in the ipsilateral and contralateral hind paws was measured manually using von Frey filaments. Data (mean ± SD) were analyzed by two-way ANOVA, *P < 0.05 versus day 0 and ^†P < 0.05 versus day 7, n = 3.

Molecular Modeling.

We used our previously published homology model of hP2Y₁₄R based on a high-resolution X-ray structure of the closely related hP2Y₁₂R as a template.²² The agonist-bound P2Y₁₂R structure served as the template (PDB ID: 4PXZ²⁰), instead of a non-nucleotide antagonist-bound structure, because the extracellular loops (ELs) are defined. This model was used effectively in our previous docking and MD simulation of hP2Y₁₄R antagonist binding,^22,23 so it was applicable to the current study.

This study has two principal aims: investigating piperidine substitution of lead compound 1 with bicyclic analogues and the replacement of the naphthalene or phenyl-triazole moieties of lead compounds 1 and 2 with alternative scaffolds. Focusing on the first aim, piperidine substitution with a conformationally constrained quinuclidine was proposed, and molecular modeling tested the hP2Y₁₄R binding site fit of the potent protonated quinuclidinium form of analogue 17. The initial docking pose of 17 (Figure 4) indicates a binding mode resembling that of 1 and 2 (Figure S7A). The carboxylate group of the central naphthalene/phenyl ring of 1 and 2 interacted with three residues in the transmembrane domains (TMs) 2, 3, and 7, which was used as an essential predictor of P2Y₁₄R affinity among newly synthesized antagonist analogues.^22,23 The carboxylate engaged in electrostatic and H-bond interactions with the side chains of Lys77^2.60 (using Ballesteros–Weinstein convention for residue numbering³⁸) and Lys277^7.35 and H-bonding with the Tyr102^3.33 phenolic group. Compound 17 made interactions with Lys77^2.60 and Lys277^7.35, but an ideal H-bond with Tyr102^3.33 was not observed, even if the conformation of the compound was in proximity to allow this kind of interaction. To further test the requirement of the carboxylate, the carboxamide-substituted 15, analogue of 2, was docked to hP2Y₁₄R, and the pose lacked the predicted important carboxylate interactions with the three amino acid residues (Figure S7B), consistent with the loss of affinity.

Figure 4.

Docking pose of compound 17 at the hP2Y₁₄R putative binding site. The P2Y₁₂R crystal structure (PDB ID: 4PXZ)²⁰ was used as a starting point for molecular modeling. The ligand is represented by lime sticks, and the receptor by light gray ribbon. Residues within 3 Å from the ligand, in addition to EL1 and EL2 residues interacting with the ligand during MD simulations, are depicted by gray sticks, and their surface is colored according to the Coulombic potential.

Compound 17 occupied the binding pocket spanning from the transmembrane helical domain 2 (TM2), TM3, and TM7 regions, i.e., the carboxylate interface, to trifluoromethyl moiety insertion at TM4 and TM5. The 7-phenyl moiety of the naphthalene ring was stabilized by a charge–π and a π–π interaction with Arg253^6.55 and His184^5.36, respectively, while the 4-phenyl ring made a charge–π interaction with Arg274^7.32. The protonated phenyl-quinuclidinium form of 17 pointed toward the P2Y₁₄R extracellular region, surrounded by TM4, TM5, extracellular loop 1 (EL1), and EL2. In the previous study,²² the piperidine of compound 1, simulated in the more stable chair conformation, made a H-bond with Gly80^2.63 and deviated to interact with Ile170^EL2 during MD simulation, while the reverse behavior was observed for compound 2. The direct or water-mediated interaction mainly involved an axial piperidine N proton. The quinuclidine may be considered as a constrained boat form of the piperidine, and the single hydrogen atom bound to the protonated nitrogen has a geometry different than the piperidine axial hydrogen. In fact, in the predicted docking pose of 17, the quinuclidine protonated nitrogen did not interact with any residue. Given similar binding affinity of compounds 17 and 1, we explored if some quinuclidine interactions could be observed by increasing the flexibility level of the model. For this reason, we subjected the complex obtained through docking to MD simulations, and five 50 ns replicates were simulated. In all the replicates, the ligand deviated only slightly from its initial geometry, with an average RMSD over the five replicates of 3.05 Å (Table S4). Except for replicate 2, H-bonds with Lys77^2.60 and/or Lys277^7.35 were maintained during much of the simulation time, with the addition of the previously observed H-bond with Tyr102^3.33 for >50% of the simulation in replicate 4. Eleven residues were in contact (≤4 Å) with the ligand for >50% of the time in all the replicates (Figure S8), suggesting their involvement in binding: Asn90^3.21, Cys94^3.25, Ala98^3.29, Val99^3.30, Tyr102^3.33, Cys172^EL2, His184^5.36, Asn188^5.40, Phe191^5.43, Arg253^6.55, and Lys277^7.35.

The geometrical rearrangement of the complex enabled ligand interactions with residues of EL1 and EL2 (Figure S8). We focused on the two replicates with an intrareplicate ligand RMSD below the interreplicate average, i.e., replicates 3 and 4 (with an intrareplicate average RMSD values of 2.22 and 2.97 Å, respectively, as reported in Table S4). In both replicates, the H-bond with Lys277^7.35 was largely maintained, while the Lys77^2.60 H-bond was more persistent in replicate 3 and the Tyr102^3.33 H-bond was more persistent during replicate 4 (Table S4 and Figures S9 and S10C). The ligand electrostatic interactions of those residues, in addition to Arg253^6.55, were intensely negative during the simulations (Figures S9 and S10D). Moreover, additional electrostatic interactions and transient hydrogen bonds were observed between the ligand and residues in EL1 and EL2, according to the replicate. In the case of replicate 3, negative electrostatic interactions were observed between the protonated quinuclidinium form of 17 and residues Gly80^2.63, Leu84^EL1, and Gly85^2.63 (Figure S9D), with water-mediated H-bonds with Leu84^EL1 and Gly85^2.63 appearing (Video S1). In replicate 4, the quinuclidinium moiety showed negative electrostatic interactions with Gly80^2.63, Gly83^2.66, Thr168^EL2, Gln169^EL2, and Ile170^EL2 (Figure S10D), with the last three residues observed to be in proximity of the ligand during most of replicates 1, 2, and 5 as well as reported in Figure S8. Transient direct H-bonds were observed with Thr168^EL2, but for most of the simulation, a water molecule mediated H-bonds between the ligand and Thr168^EL2, Gln169^EL2, and Leu84^EL1 (Video S2).

The simulations showed a possible involvement of EL1 and EL2 in stabilizing the ligand-bound state. Electrostatic interactions are a molecular mechanics approximation of either ionic or dipolar interactions; in our simulations, no ionic interaction was observed between the cationic quinuclidinium ion and anionic residues, instead suggesting charge–dipole interactions. However, limitations to our observations of other possible relevant interactions arise from the use of a homology model and from the conformational uncertainty of the sequence variable and highly flexible EL regions. However, potentially interacting proximal residues include the negatively charged E166^EL2 in EL2. Moreover, the high affinity of N-methyl-quaternized 21, similar to 17, suggests that the electrostatic interaction within the hP2Y₁₄R binding site is more important than H-bonding of the quinuclidine nitrogen atom.

The second aim of this work was the investigation of possible substitution of the naphthalene or phenyl-triazole scaffolds of lead compounds 1 and 2. We investigated the amide analogues 27 and 29, indoles (30 and 31), and benzimidazole (32) obtained by the fusion of the 4-trifluoromethyl-phenyl and triazole rings of 2, and the pyrrole analogue of the central trisubstituted phenyl ring of 2 (33). Unlike compound 1, where the trifluoromethyl-carboxylate distance (around 11.5 Å) was conformation independent, in all these compounds, the distance between those two (trifluoromethyl and carboxylate) moieties varied with the ligand conformation. However, the docking poses of these ligands resembled the bound conformations of 1 and 2 (Figure S7C,D). In fact, even with greatly reduced affinities, these ligands were able to occupy the putative binding site (with the exception of 33, which lacked affinity and showed a slightly modified docking pose and slightly less deep in the binding pocket, as reported in Figure S7E). To test the relevance of compound geometry for the SAR, we designed a benzimidazole analogue 35, by fusing the central trisubstituted phenyl and triazole moieties of compound 2. Compound 35 had a conformation-independent fixed trifluoromethyl-carboxylate distance similar to compound 1 (Figure S7F), but an alignment of those moieties to receptor-bound 1 reflected a different geometry of the compound, forcing the phenylpiperidine to point in a region virtually occupied by EL2. In fact, no docking pose was obtained for this compound, and the experimental lack of affinity of 35 for hP2Y₁₄R was consistent with our model’s topology.

DISCUSSION

In previous SAR studies of P2Y₁₄R antagonists, the affinity of 1 was seldom surpassed.^19,22,23 Attempts to enhance the drug-likeness in this chemical series often resulted in much lower affinity and in at least one case, i.e., 22, rapid metabolism in vivo.²³ In the SAR progression in our previous three studies,^19,22,23 there was a tendency toward lower affinity at the hP2Y₁₄R (IC₅₀ value), i.e., in the progression of 1 (6.0 nM) to 2 (31.7 nM) to 23 (169 nM). Therefore, we re-examined the entire scaffold of both 1 and 2 to identify alternative scaffold variations with greater flexibility of substitution and retention of receptor affinity.

In an effort to improve the assay method, we synthesized tritiated 1 and found that it was not only of limited solubility but also tended to adsorb to plastic surfaces and was therefore not suitable as a radiolabeled tracer for drug screening. Thus, we applied the fluorescent tracer 36 using our previous method to determine the binding affinity of the newly synthesized analogues at both the hP2Y₁₄R (expressed in CHO cells) and mP2Y₁₄R (expressed in HEK293 cells). Fluorescence micrographs show the presence of specific mP2Y₁₄R binding in the HEK293 cells,¹² which were used to demonstrate that the species differences in antagonist affinity were relatively minor, compared to species differences in other GPCRs, such as the A₃ adenosine receptor.¹³ Furthermore, we compared data obtained for three compounds in the fluorescence (whole cell binding) assay with our previously published forskolin-stimulated cAMP inhibition (Table S1, Supporting Information). The cAMP assay values were lower by 18 ± 6-fold, indicating that the fluorescence binding consistently underestimates the intrinsic affinity of these antagonists.

We have previously implicated the naphthalene moiety as a source of the unfavorable physicochemical properties of the potent, amphiphilic antagonist 1.⁸ Nevertheless, naphthalene is a component of many useful drug substances.²¹ Therefore, we now focus on modification of other scaffold regions, particularly the piperidine ring as a means of enhancing the drug-like properties of this antagonist series. We found that steric constraint of this piperidine ring is especially suitable for retaining high P2Y₁₄R affinity. Specifically, bicyclic substitution of the piperidine ring to decrease the conformational flexibility led to the most successful analogues, i.e., quinuclidine analogue 17, which was only 3-fold less potent than 1 with an IC₅₀ value of 20 nM. Similarly, 18 was 3-fold less potent than its piperidine equivalent 2. Also, a single alkene group in 3 preserved affinity, and substitution with a piperazine in 4 only moderately reduced affinity. The previously reported amino derivative 23, which had a more favorable cLogP (0.84 compared to 4.64 for 2), was active in vivo in reducing the eosinophil content in the BALF.

Previously, rotating a bridging 1,2,3-triazole ring by one position (equivalent to its inversion) in other GPCR pharmacophores was shown to preserve biological activity, e.g., in adenosine receptor agonists.³⁹ Furthermore, amides and 1,2,3-triazole groups have been identified as bioisosteres, and amides may have better water-solubilizing properties than the triazoles.⁵⁸ The “reversed” triazole derivative 25 maintained moderate affinity and in vivo activity. However, extension of the piperidine nitrogen in the form of a water-solubilizing PEG group in 12 resulted in inactivity. The most effective antagonist in lowering the eosinophil content in the BALF was the hexynyl derivative 7a.

Modification of the central phenyl ring and its carboxylate group, e.g., nitrile 16, pyrrole 33, and C-methyl derivative 34, or fusion of two aryl groups, e.g., benzimidazoles 32 and 35, in general, dramatically reduced the affinity. This indicated that the “upper” part of the scaffold is conformationally constrained in its deeper P2Y₁₄R binding pocket. Also, planarity of this part of the scaffold seems to be required as an attempt to introduce three-dimensionality in cubane derivative 28 failed. Thus, the overall placement and orientation of this ligand series in the orthosteric P2Y₁₄R binding site are consistent with the piperidine moiety of 1 and 2 facing the extracellular medium. This region is less sterically constrained than the deeper binding pocket that is mainly hydrophobic, except for its small hydrophilic region for electrostatic interaction of the essential carboxylate. Replacement of this carboxylate group of 2 with a polar and H bonding but uncharged carboxamide group of 15 eliminated its binding ability.

Bicyclic substitution of the piperidine ring, e.g., quinuclidine 17 (IC₅₀ = 20 nM) in the naphthalene series and 18 (IC₅₀ ≈ 100 nM) in the triazole series, preserved P2Y₁₄R affinity. Molecular modeling of the interaction of quinuclidine derivative 17 with the hP2Y₁₄R showed a binding mode similar to the parent compound 1. MD simulations showed the conservation of electrostatic interactions with Lys77^2.60, Lys277^7.35, and Tyr102^3.33 and additional interactions with residues of EL1 and EL2. N-Methyl quinuclidinium analogue 21 showed nearly as high affinity as the parent quinuclidine 17, suggesting that a positively charged, non-H-bonding N atom is as suitable for high hP2Y₁₄R affinity as a neutral, H-bonding N atom in 8. Thus, multiple modes of interaction between the piperidine/quinuclidine ring at the receptor are possible.

CONCLUSIONS

P2Y₁₄R antagonism is one of the potential therapeutic strategies for pain and renal and pulmonary conditions. Exploration of alternative scaffolds for P2Y₁₄R antagonists containing a biaryl core has yielded high affinity derivatives in which the piperidine moiety is constrained. Other modifications, such as reversal of a triazole moiety maintained only moderate affinity. Thus, other regions of the antagonist scaffold are highly sensitive to structural changes. Quinuclidine 17 showed high affinity receptor binding by flow cytometric analysis at hP2Y₁₄R (IC₅₀ = 20 nM) and mP2Y₁₄R (IC₅₀ = 21 nM) and in vivo efficacy. Modest improvement in aqueous solubility was achieved for several analogues, including a PEG conjugate 12 with 20-fold higher solubility than potent antagonist 1. Moreover, in vivo efficacy in models of asthma and chronic neuropathic pain was demonstrated. Future studies potentially will be able to identify leads for clinically useful drug candidates for kidney inflammation, chronic neuropathic pain, pulmonary diseases, and other conditions.

EXPERIMENTAL SECTION

Reagents and Instrumentation.

All reagents and solvents were purchased from Sigma-Aldrich (St. Louis, MO) or Ark Pharm, Inc. (Libertyville, IL; compound 83).¹H NMR spectra were obtained using a Bruker 400 spectrometer with CDCl₃, CD₃OD, and DMSO-d₆ as solvents. Chemical shifts are expressed in δ values (ppm) with tetramethylsilane (δ 0.00) for CDCl₃ and water (δ 3.30) for CD₃OD. NMR spectra were collected using a Bruker AV spectrometer equipped with a z-gradient [¹H, ¹³C, ¹⁵N]-cryoprobe. TLC analysis was carried out on glass sheets precoated with silica gel F₂₅₄ (0.2 mm) from Sigma-Aldrich. The purity of final compounds was checked using a Hewlett–Packard 1100 HPLC equipped with a Zorbax SB-Aq 5 μm analytical column (50 × 4.6 mm, Agilent Technologies Inc., Palo Alto, CA). Mobile phase: linear gradient solvent system, 5 mM tetrabutylammonium dihydrogen phosphate–CH₃CN from 100:0 to 0:100 in 15 min; the flow rate was 0.5 mL/min. Peaks were detected by UV absorption with a diode array detector at 210, 254, and 280 nm. All derivatives tested for biological activity showed >95% purity by HPLC analysis (detection at 254 nm). Low-resolution mass spectrometry was performed using a JEOL SX102 spectrometer with 6 kV Xe atoms following desorption from a glycerol matrix or on an Agilent LC/MS 1100 MSD, with a Waters (Milford, MA) Atlantis C18 column. High-resolution mass spectroscopy (HRMS) measurements were performed on a proteomics optimized Q-TOF-2 (MicromassWaters) using external calibration with polyalanine, unless noted. Observed mass accuracies are those expected based on known instrument performance as well as trends in masses of standard compounds observed at intervals during the series of measurements. Reported masses are observed masses uncorrected for this time-dependent drift in mass accuracy. The total polar surface area (tPSA) was calculated using ChemDraw Professional (PerkinElmer, Boston, MA, v. 16.0). cLogP was calculated as reported.³⁶ Simulated pharmacokinetic parameters were calculated as reported.³

General Procedure: Deprotection Reaction.

Method A.

A mixture of compound containing an ester (1 equiv) and potassium hydroxide (5 equiv) in methanol/water (2:1) was stirred at 50 °C. This mixture was neutralized with 1 N HCl until pH was 5–6. The slightly acidic mixture was evaporated under reduced pressure and purified by silica gel column chromatography (dichloromethane/methanol/acetic acid = 95:5:0.1) or semipreparative HPLC (10 mM triethylammonium acetate buffer/acetonitrile = 80:20 to 20:80 in 40 min) to afford the compound as a white solid.

Method B.

A solution of compound in trifluoroacetic acid/tetrahydrofuran (1:1 or 2:1) was stirred at room temperature. The solvent was evaporated with toluene under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 95:5) or semipreparative HPLC (10 mM triethylammonium acetate buffer/acetonitrile = 80:20 to 20:80 in 40 min) to afford the compound as a white solid.

4-(4-(1,2,3,6-Tetrahydropyridin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (3).

Method A: yield, 88%; HPLC purity, 95% (R_t = 14.76 min); ¹H NMR (400 MHz, CD₃OD): δ 8.58 (s, 1H), 8.36 (s, 1H), 8.06–7.98 (m, 4H), 7.86–7.78 (m, 3H), 7.63 (d, J = 8.00 Hz, 2H), 7.55 (d, J = 8.00 Hz, 2H), 6.31 (broad s, 1H), 3.66 (broad s, 2H), 3.25 (broad s, 2H), 2.71 (broad s, 2H); MS (ESI, m/z): 474.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₉H₂₃NO₂F₃, 474.1681; found, 474.1683 [M + 1]⁺.

4′-(Piperazin-1-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (4).

Method A: yield, 59%; HPLC purity, 95% (R_t = 6.41 min); ¹H NMR (400 MHz, CD₃OD): δ 9.17 (s, 1H), 8.45 (s, 1H), 8.37 (s, 1H), 8.23 (s, 1H), 8.16 (m, 2H), 7.81–7.75 (m, 4H), 7.20–7.14 (m, 2H), 3.51 (broad s, 4H), 3.40 (broad s, 4H); MS (ESI, m/z): 494.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₆H₂₃N₅O₂F₃, 494.1804; found, 494.1807 [M + 1]⁺.

4-(4-(1-Methylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (5).

Method A: yield, 65%; HPLC purity, 95% (R_t = 12.49 min); ¹H NMR (400 MHz, DMSO-d₆): δ 8.59 (s, 1H), 8.52 (s, 1H), 8.06 (d, J = 8.28 Hz, 2H), 7.96–7.86 (m, 5H), 7.46 (d, J = 8.48 Hz, 2H), 7.43 (d, J = 8.52 Hz, 2H), 2.93 (d, J = 11.56 Hz, 2H), 2.23 (s, 3H), 2.06–2.00 (m, 2H), 1.91–1.77 (m, 4H); MS (ESI, m/z): 490.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₀H₂₇NO₂F₃, 490.1994; found, 490.1988 [M + 1]⁺.

4-(4-(1-(Prop-2-yn-1-yl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (6a).

Method A: yield, 52%; HPLC purity, 99% (R_t = 13.64 min); ¹H NMR (400 MHz, CD₃OD): δ 8.72 (s, 1H), 8.41 (s, 1H), 8.06–7.98 (m, 4H), 7.91–7.89 (m, 1H), 7.80 (d, J = 8.08 Hz, 2H), 7.50–7.45 (m, 4H), 3.74 (broad s, 2H), 3.45–3.42 (m, 2H), 3.05 (m, 1H), 2.88–2.80 (m, 3H), 2.15–1.94 (m, 4H); MS (ESI, m/z): 514.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₂₇NO₂F₃, 514.1994; found, 514.2001 [M + 1]⁺.

4-(4-(1-Propylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (6b).

Method A: yield, 56%; ¹H NMR (400 MHz, CD₃OD): δ 8.63 (s, 1H), 8.35 (s, 1H), 8.05 (s, 1H), 7.98–7.94 (m, 3H), 7.81–7.78 (m, 3H), 7.46 (d, J = 7.64 Hz, 2H), 7.41 (d, J = 7.84 Hz 2H), 3.65 (d, J = 11.80 Hz, 2H), 3.19–3.09 (m, 4H), 2.19–2.03 (m, 3H), 1.97 (s, 2H), 1.88–1.82 (m, 2H), 1.08 (t, J = 7.32 Hz, 3H); MS (ESI, m/z): 518.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₃₁NO₂F₃, 518.2307; found, 518.2301 [M + 1]⁺.

4-(4-(1-(Hex-5-yn-1-yl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (7a).

Method A: yield, 48%; HPLC purity, 100% (R_t = 13.71 min); ¹H NMR (400 MHz, CD₃OD): δ 8.74 (s, 1H), 8.41 (d, J = 1.76 Hz, 1H), 8.04–7.95 (m, 4H), 7.91 (dd, J = 1.88, 8.91 Hz, 1H), 7.80 (d, J = 8.28 Hz, 2H), 7.50 (q, J = 8.28 Hz, 4H), 3.76 (d, J = 12.05 Hz, 2H), 3.26–3.14 (m, 4H), 3.10–3.00 (m, 1H), 2.38–2.30 (m, 3H), 2.26 (d, J = 14.05 Hz, 2H), 2.17–2.06 (m, 2H), 2.01–1.90 (m, 2H), 1.65 (quin, J = 7.15 Hz, 2H); negative ion ESI-MS m/z (rel intensity): 554 [M − H⁺, 100]; ESI-HRMS [M − H⁺] calcd m/z for C₃₅H₃₁F₃NO₂, 554.2307; found, 554.2288.

4-(4-(1-Hexylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (7b).

To a solution of compound 7a (4 mg, 0.007 mmol) in methanol (0.5 mL) and ethyl acetate (0.5 mL) was added the Rh/C catalyst. The resulting reaction mixture was stirred at room temperature in a hydrogen atmosphere (100 psi) for 14 h. The mixture was filtered through a cake of Celite, and the filtrate was evaporated under reduced pressure. The residue was purified by semipreparative HPLC (10 mM triethylammonium acetate buffer/acetonitrile = 80:20 to 20:80 in 40 min) to afford the compound 9 (3.7 mg, 92%) as a white solid; HPLC purity, 95% (R_t = 13.98 min); ¹H NMR (400 MHz, CD₃OD): δ 8.58 (s, 1H), 8.36 (s, 1H), 8.02 (s, 1H), 7.98–7.92 (m, 3H), 7.81–7.77 (m, 3H), 7.46 (d, J = 8.20 Hz, 2H), 7.40 (d, J = 8.16 Hz, 2H), 3.60 (d, J = 11.56 Hz, 2H), 3.04–2.89 (m, 4H), 2.15–2.01 (m, 3H), 1.77–1.74 (m, 1H), 1.45–1.34 (m, 8H), 0.95 (t, J = 6.80 Hz, 3H), 0.91–0.87 (m, 1H); MS (ESI, m/z): 560.3 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₅H₃₇NO₂F₃, 560.2776; found, 560.2782 [M + 1]⁺.

4-(4-(1-(Acetyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (8) and 4-(4-(1-(Propionyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (9).

Synthetic procedures followed, and confirmation of structural identity and purity were recently reported.¹²

4-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (10).

Method A: yield, 71%; HPLC purity, 95% (R_t = 16.26 min); ¹H NMR (400 MHz, CD₃OD): δ 8.74 (s, 1H), 8.43 (s, 1H), 8.05–7.99 (m, 4H), 7.93 (d, J = 8.72 Hz, 1H), 7.82 (d, J = 8.20 Hz, 2H), 7.48 (d, J = 8.08 Hz, 2H), 7.44 (d, J = 8.12 Hz, 2H), 4.28 (d, J = 12.6 Hz, 2H), 2.95–2.85 (m, 3H), 1.96 (d, J = 12.40 Hz, 2H), 1.77–1.685 (m, 2H), 1.52 (s, 9H); MS (ESI, m/z): 520.1 [M + 1-tert-butyl]⁺, 476.2 [M + 1-Boc]⁺.

4-(4-(1-(2,5,8,11,14,17-Hexaoxaicosan-20-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (11).

Method A: yield, 65%; HPLC purity, 97% (R_t = 14.01 min); ¹H NMR (400 MHz, CD₃OD): δ 8.71 (s, 1H), 8.41 (s, 1H), 8.04–7.99 (m, 4H), 7.90 (d, J = 8.92 Hz, 1H), 7.81 (d, J = 8.20 Hz, 2H), 7.48 (d, J = 8.16 Hz, 2H), 7.44 (d, J = 8.20 Hz, 2H), 4.77 (d, J = 13.22 Hz, 1H), 4.23 (d, J = 13.6 Hz, 1H), 3.87–3.77 (m, 2H), 3.66–3.59 (m, 21H), 3.53–3.50 (m, 2H), 3.29–3.26 (m, 1H), 3.01–2.95 (m, 1H), 2.88–2.77 (m, 2H), 2.72–2.65 (m, 1H), 2.02 (t, J = 11.88 Hz, 2H), 1.87–1.67 (m, 2H); MS (ESI, m/z): 782.4 [M + 1]⁺, 799.4 [M + NH₄⁺]⁺; ESI-HRMS calcd m/z for C₄₃H₅₁NO₉F₃, 782.3516; found, 782.33530 [M + 1]⁺.

4-(4-(1-(1-Amino-3,6,9,12,15,18-hexaoxahenicosan-21-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (12).

Method B: yield, 91%; ¹H NMR (400 MHz, CD₃OD): δ 8.74 (s, 1H), 8.42 (s, 1H), 8.04–7.98 (m, 4H), 7.91 (d, J = 8.84 Hz, 1H), 7.81 (d, J = 8.20 Hz, 2H), 7.48 (d, J = 8.24 Hz, 2H), 7.45 (d, J = 8.32 Hz, 2H), 4.79 (d, J = 12.6 Hz, 1H), 4.19 (d, J = 13.4 Hz, 1H), 3.83 (t, J = 6.06 Hz, 2H), 3.79 (t, J = 8.08 Hz, 2H), 3.73–3.67 (m, 21H), 3.16 (t, J = 4.86 Hz, 2H), 3.02–2.96 (m, 1H), 2.88–2.71 (m, 3H), 2.07–2.01 (m, 2H), 1.86–1.67 (m, 2H); MS (ESI, m/z): 811.4 [M + 1]⁺; ESI-HRMS calcd m/z for C₄₄H₅₄N₂O₉F₃, 811.3781; found, 811.3793 [M + 1]⁺.

4-(4-(1-(2-Oxo-6,9,12,15,18,21-hexaoxa-3-azatetracosan-24-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (13).

To a solution of compound 12 (6.3 mg, 7.77 μmol) in pyridine (0.5 mL) was added acetic anhydride (8 μL, 84 μmol), and then this reaction mixture was stirred at room temperature for 1 h. After all volatiles were evaporated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol = 20:1) to afford compound 13 (3.7 mg, 59%) as a white solid; HPLC purity, 99% (R_t = 13.38 min); ¹H NMR (400 MHz, CD₃OD): δ 8.73 (s, 1H), 8.37 (s, 1H), 7.98–7.80 (m, 7H), 7.43 (s, 4H), 4.77 (d, J = 9.04 Hz, 1H), 4.22 (d, J = 11.04 Hz, 1H), 3.82 (d, J = 5.24 Hz, 2H), 3.66–3.60 (m, 20H), 3.52–3.50 (m, 2H), 3.29–3.25 (m, 1H), 2.96–2.70 (m, 4H), 2.00 (m, 2H), 1.94 (s, 3H), 1.83–1.68 (m, 2H); MS (ESI, m/z): 853.4 [M + 1]⁺, 870.5 [M + NH₄⁺]⁺; ESI-HRMS calcd m/z for C₄₆H₅₆N₂O₁₀F₃, 853.3887; found, 853.3893 [M + 1]⁺.

4-(4-(1-(2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23-heptaoxa-5-azahexacosan-26-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (14).

Method A: yield, 79%; HPLC purity, 97% (R_t = 14.17 min); ¹H NMR (400 MHz, CD₃OD): δ 8.71 (s, 1H), 8.40 (s, 1H), 8.02–7.98 (m, 4H), 7.88 (d, J = 8.92 Hz, 1H), 7.80 (d, J = 8.20 Hz, 2H), 7.46 (d, J = 8.20 Hz, 2H), 7.43 (d, J = 8.16 Hz, 2H), 4.76 (d, J = 12.6 Hz, 1H), 4.22 (d, J = 12.7 Hz, 1H), 3.86–3.77 (m, 2H), 3.66–3.57 (m, 20H), 3.49 (t, J = 9.52 Hz, 2H), 3.27–3.25 (m, 1H), 3.21 (t, J = 5.52 Hz, 2H), 2.99–2.93 (m, 1H), 2.87–2.76 (m, 2H), 2.71–2.65 (m, 1H), 2.01 (t, J = 11.82 Hz, 2H), 1.86–1.80 (m, 1H), 1.75–1.68 (m, 1H), 1.43 (s, 9H); MS (ESI, m/z): 811.4 [M + 1-Boc]⁺, 911.4 [M + 1]⁺, 928.4 [M + NH₄⁺]⁺; ESI-HRMS calcd m/z for C₄₉H₆₂N₂O₁₁F₃, 911.4306; found, 911.4300 [M + 1]⁺.

4-(4-(Quinuclidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic Acid (17).

Method A: yield, 53%; HPLC purity, 98% (R_t = 10.13 min); melting point, 203–205 °C; ¹H NMR (400 MHz, CD₃OD): δ 8.76 (s, 1H), 8.43 (s, 1H), 8.01–7.92 (m, 5H), 7.82–7.80 (m, 2H), 7.65–7.54 (m, 4H), 3.58–3.54 (m, 6H), 2.37–2.33 (m, 6H); MS (ESI, m/z): 502.2 [M + 1]⁺; ¹³C NMR (100 MHz, DMSO-d₆): δ 167.2, 145.2, 143.2, 139.2, 137.3, 136.5, 133.1, 132.2, 130.5, 129.7, 128.7, 128.0, 127.7, 127.4, 126.7, 126.4, 126.1, 125.8, 125.6, 124.7, 45.9, 31.3, 28.4; ESI-HRMS calcd m/z for C₃₁H₂₇NO₂F₃, 502.1994; found, 502.1993 [M + 1]⁺.

4′-(Quinuclidin-4-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (18).

Method A: yield, 66%; HPLC purity, 99% (R_t = 8.70 min); melting point, 308–310 °C; ¹H NMR (400 MHz, CD₃OD): δ 9.24 (s, 1H), 8.51 (broad s, 1H), 8.39 (broad s, 1H), 8.37 (broad s, 1H), 8.17 (d, J = 8.00 Hz, 2H), 7.83–7.75 (m, 4H), 7.58 (d, J = 8.00 Hz, 2H), 3.58–3.49 (m, 6H), 2.31–2.24 (m, 6H); MS (ESI, m/z): 519.2 [M + 1]⁺; ¹³C NMR (100 MHz, DMSO-d₆): δ 166.2, 146.7, 146.1, 146.0, 141.7, 137.2, 136.2, 134.0, 129.3, 128.2, 127.1, 126.1, 125.9, 125.8, 125.5, 124.2, 121.5, 121.2, 45.7, 31.3, 28.3; ESI-HRMS calcd m/z for C₂₉H₂₆N₄O₂F₃, 519.2008; found, 519.2013 [M + 1]⁺.

4′-(3-Azabicyclo[4.1.0]heptan-6-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (19).

Method B: yield, 79%; HPLC purity, 99% (R_t = 24.09 min); ¹H NMR (400 MHz, CD₃OD): δ 9.09 (s, 1H), 8.45 (s, 1H), 8.34 (s, 1H), 8.17 (s, 1H), 8.13–8.04 (m, 2H), 7.75–7.63 (m, 4H), 7.50–7.40 (m, 2H), 3.83–3.77 (m, 1H), 3.30–3.22 (m, 2H), 2.94–2.87 (m, 1H), 2.35 (broad s, 2H), 1.60–1.56 (m, 1H), 1.31–1.23 (m, 1H), 1.12–1.10 (m, 1H); MS (ESI, m/z): 505.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₈H₂₄N₄O₂F₃, 505.1851; found, 505.1848 [M + 1]⁺.

4′-(7,7-Difluoro-3-azabicyclo[4.1.0]heptan-6-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (20).

Method B: yield, 35%; HPLC purity, 99% (R_t = 11.78 min); ¹H NMR (400 MHz, CD₃OD): δ 9.19 (s, 1H), 8.50 (s, 1H), 8.38–8.30 (m, 2H), 8.17–8.11 (m, 2H), 7.83–7.72 (m, 4H), 7.59–7.54 (m, 2H), 3.98–3.86 (m, 1H), 3.73 (broad s, 1H), 3.43–3.37 (m, 1H), 3.04–2.96 (m, 1H), 2.66–2.46 (m, 2H), 2.33–2.22 (m, 1H); MS (ESI, m/z): 541.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₈H₂₂N₄O₂F₅, 541.1663; found, 541.1668 [M + 1]⁺.

4-(4-(1-Methylquinucl i din-1-ium-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (21).

Method A: yield, 71%; HPLC purity, 97% (R_t = 9.57 min); ¹H NMR (400 MHz, CD₃OD): δ 8.58 (s, 1H), 8.36 (s, 1H), 8.02–7.93 (m, 4H), 7.79–7.77 (m, 3H), 7.54–7.52 (m, 3H), 3.69–3.65 (m, 6H), 3.09 (s, 3H), 2.40–2.37 (m, 6H); MS (ESI, m/z): 516.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₂₉NO₂F₃, 516.2150; found, 516.2159 [M + 1]⁺.

4′-Carbamoyl-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (24).

Method A: yield, 88%; HPLC purity, 99% (R_t = 11.77 min); ¹H NMR (400 MHz, CD₃OD): δ 9.28 (s, 1H), 8.64 (s, 1H), 8.51 (s, 1H), 8.35 (s, 1H), 8.21 (d, J = 8.40 Hz, 2H), 8.04 (d, J = 8.28 Hz, 2H), 7.96 (d, J = 8.52 Hz, 2H), 7.88 (d, J = 8.24 Hz, 2H); MS (ESI, m/z): 453.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₆N₄O₃F₃, 453.1175; found, 453.1169 [M + 1]⁺.

4′-(Piperidin-4-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (25).

Synthetic procedures followed, and confirmation of structural identity and purity were recently reported.¹²

3-(5-((3-Aminopropyl)carbamoyl)thiophen-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic Acid (26).

Method B: yield, 45%; HPLC purity, 98% (R_t = 10.23 min); ¹H NMR (400 MHz, DMSO-d₆): δ 9.75 (s, 1H), 8.82 (broad s, 1H; NH), 8.53 (s, 1H), 8.46 (s, 1H), 8.25 (d, J = 8.44 Hz, 2H), 8.23 (s, 1H), 8.06 (d, J = 8.52 Hz, 2H), 7.89 (s, 1H), 7.75 (d, J = 3.36 Hz, 1H), 3.35 (merged with water peak), 2.90 (t, J = 7.24 Hz, 2H), 1.84 (t, J = 7.02 Hz, 2H); MS (ESI, m/z): 516.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₄H₂₁N₅O₃F_{3 32}S, 516.1317; found, 516.1316 [M + 1]⁺.

4′-Carbamoyl-5-(4-(trifluoromethyl)benzamido)-[1,1′-biphenyl]-3-carboxylic Acid (27).

Method A: yield, 70%; HPLC purity, 96% (R_t = 11.11 min); ¹H NMR (400 MHz, CD₃OD): δ 8.42 (s, 1H), 8.37 (s, 1H), 8.17 (d, J = 7.96 Hz, 2H), 8.14 (s, 1H), 8.02 (d, J = 7.76 Hz, 2H), 7.86 (d, J = 7.96 Hz, 2H), 7.81 (d, J = 7.80 Hz, 2H); MS (ESI, m/z): 429.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₂H₁₆N₂O₄F₃, 429.1062; found, 429.1069 [M + 1]⁺.

5-(4-(Hydroxymethyl)cubane-1-carboxamido)-4′-(piperidin-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (28).

Method A: yield, 39%; HPLC purity, 96% (R_t = 7.39 min); ¹H NMR (400 MHz, DMSO-d₆): δ 9.69 (s, 1H), 8.13 (s, 2H), 7.90 (s, 1H), 7.57 (d, J = 7.88 Hz, 2H), 7.35 (d, J = 7.96 Hz, 2H), 4.15 (t, J = 4.68 Hz, 3H), 3.80 (t, J = 4.50 Hz, 3H), 3.55 (s, 2H), 3.20 (d, J = 11.76 Hz, 2H), 2.79–2.75 (m, 2H), 1.80–1.76 (m, 3H); MS (ESI, m/z): 457.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₈H₂₉N₂O₄, 457.2127; found, 457.2129 [M + 1]⁺.

4′-Carbamoyl-5-((4-(trifluoromethyl)phenyl)carbamoyl)-[1,1′-biphenyl]-3-carboxylic Acid (29).

Method A: yield, 72%; HPLC purity, 99% (R_t = 11.30 min); ¹H NMR (400 MHz, CD₃OD): δ 8.61 (s, 1H), 8.53 (s, 1H), 8.44 (s, 1H), 8.03 (d, J = 8.12 Hz, 2H), 7.99 (d, J = 8.48 Hz, 2H), 7.87 (d, J = 8.12 Hz, 2H), 7.68 (d, J = 8.48 Hz, 2H); MS (ESI, m/z): 429.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₂H₁₆N₂O₄F₃, 429.1062; found, 429.1065 [M + 1]⁺.

4′-Carbamoyl-5-(5-(trifluoromethyl)-1H-indol-2-yl)-[1,1′-biphenyl]-3-carboxylic Acid (30).

The suspension of compound 73a (8 mg, 18.2 μmol) and potassium hydroxide (5.2 mg, 91.2 μmol) in methanol (1 mL) and water (0.5 mL) was stirred at 70 °C for 3 h. The reaction mixture was acidified with acetic acid, and the solvent was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol/acetic acid = 10:1:0.01) to afford compound 30 (4.6 mg, 59%) as a white solid; HPLC purity, 96% (R_t = 11.75 min); (96%); ¹H NMR (400 MHz, CD₃OD): δ 8.62 (s, 1H), 8.34 (s, 1H), 8.29 (s, 1H), 8.05 (d, J = 7.84 Hz, 2H), 7.93 (s, 1H), 7.89 (d, J = 7.92 Hz, 2H), 7.58 (d, J = 8.48 Hz, 1H), 7.40 (d, J = 8.44 Hz, 1H), 7.14 (s, 1H); MS (ESI, m/z): 425.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₆N₂O₃F₃, 425.1113; found, 425.1112 [M + 1]⁺.

4′-Carbamoyl-5-(6-(trifluoromethyl)-1H-indol-2-yl)-[1,1′-biphenyl]-3-carboxylic Acid (31).

Compound 73b (20 mg, 50.2 μmol) was converted to compound 31 (13 mg, 67%) as a white solid, using a similar procedure used in the preparation of compound 30; HPLC purity, 97% (R_t = 11.94 min); ¹H NMR (400 MHz, CD₃OD): δ 8.54 (s, 1H), 8.38 (s, 1H), 8.31 (s, 1H), 8.06 (d, J = 8.24 Hz, 2H), 7.90 (d, J = 8.28 Hz, 2H), 7.75–7.74 (m, 2H), 7.30 (d, J = 8.64 Hz, 1H), 7.12 (s, 1H); MS (ESI, m/z): 425.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₆N₂O₃F₃, 425.1113; found, 425.1108 [M + 1]⁺.

4′-Carbamoyl-5-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)-[1,1′-biphenyl]-3-carboxylic Acid (32).

Compound 75 (5 mg, 11.4 μmol) was converted to compound 32 (5 mg, 99%) as a white solid, using a similar procedure used in the preparation of compound 30; HPLC purity, 99% (R_t = 10.64 min); ¹H NMR (400 MHz, CD₃OD): δ 8.77 (s, 1H), 8.62 (s, 1H), 8.49 (s, 1H), 8.04 (d, J = 7.48 Hz, 2H), 7.96 (s, 1H), 7.91 (d, J = 7.52 Hz, 2H), 7.79 (d, J = 7.92 Hz, 1H), 7.58 (d, J = 8.48 Hz, 1H); MS (ESI, m/z): 426.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₂H₁₅N₃O₃F₃, 426.1066; found, 426.1063 [M + 1]⁺.

5-(4-(1,2,3,6-Tetrahydropyridin-4-yl)phenyl)-3-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrole-2-carboxylic Acid (33).

Method A: yield, 30%; HPLC purity, 99% (R_t = 10.35 min); ¹H NMR (400 MHz, CD₃OD): δ 9.23 (s, 1H), 8.13–8.05 (m, 2H), 7.80–7.68 (m, 3H), 7.58–49 (m, 2H), 7.37–7.31 (m, 1H), 7.01–6.92 (m, 1H), 6.22 (broad s, 1H), 3.83–3.76 (m, 2H), 3.48–3.37 (m, 2H), 2.84–2.74 (m, 2H); MS (ESI, m/z): 480.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₅H₂₁N₅O₂F₃, 480.1647; found, 480.1649 [M + 1]⁺.

2-Methyl-4′-(piperidin-4-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (34).

Method A: yield, 72%; HPLC purity, 99% (R_t = 10.37 min); ¹H NMR (400 MHz, DMSO-d₆): δ 8.31 (s, 1H), 8.22 (d, J = 8.40 Hz, 2H), 8.04 (d, J = 8.40 Hz, 2H), 7.88 (s, 1H), 7.39–7.34 (m, 3H), 7.01 (d, J = 8.40 Hz, 1H), 6.72 (d, J = 8.000 Hz, 1H), 3.42–3.35 (m, 2H), 3.05–2.91 (m, 3H), 2.38 (s, 3H), 2.01–1.83 (m, 4H); MS (ESI, m/z): 507.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₈H₂₆N₄O₂F₃, 507.2008; found, 507.2009 [M + 1]⁺.

4-(4-(Piperidin-4-yl)phenyl)-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-6-carboxylic Acid (35).

Method B: yield, 63%; HPLC purity, 99% (R_t = 5.59 min); ¹H NMR (400 MHz, CD₃OD): δ 8.37 (d, J = 8.00 Hz, 2H), 8.28 (broad s, 1H), 8.12–8.02 (m, 3H), 7.89 (d, J = 8.40 Hz, 2H), 7.53–7.45 (m, 2H), 3.58 (d, J = 12.80 Hz, 2H), 3.25 (t, J = 13.20 Hz, 2H), 3.08–3.00 (m, 1H), 2.23–2.15 (m, 2H), 2.06–1.95 (m, 2H); MS (ESI, m/z): 466.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₆H₂₃N₃O₂F₃, 466.1742; found, 466.1747 [M + 1]⁺.

tert-Butyl 4-(4-(3-(Ethoxycarbonyl)-6-(4-(trifluoromethyl)-phenyl)naphthalen-1-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (38).

A mixture of compound 37 (60 mg, 0.121 mmol, synthesized according to literature procedures reported), tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (47 mg, 0.121 mmol), PdCl₂(PPh₃)₂ (8 mg, 0.012 mmol), and Na₂CO₃ (47 mg, 0.240 mmol) in 1,4-dioxane/water (10:1, 5 mL) was purged with nitrogen gas for 15 min and then stirred at 80 °C for 12 h under a nitrogen atmosphere. After cooling at room temperature, the mixture was partitioned with ethyl acetate (20 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3:1) to afford compound 38 (35 mg, 48%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.68 (s, 1H), 8.23 (s, 1H), 8.06–8.03 (m, 2H), 7.83–7.74 (m, 5H), 7.55–7.49 (m, 4H), 6.17 (broad s, 1H), 4.46 (q, J = 7.12 Hz, 2H), 4.14 (broad s, 2H), 3.71–3.68 (m, 2H), 2.63 (broad s, 2H), 1.51 (s, 9H), 1.45 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 546.2 [M + 1-tert-butyl]⁺; ESI-HRMS calcd m/z for C₃₂H₂₇NO₄F₃, 546.1892; found, 546.1902 [M + 1-tert-butyl]⁺.

Ethyl 4-(4-(1,2,3,6-Tetrahydropyridin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (39).

Method B: yield, 90%; ¹H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1H), 8.24 (s, 1H), 8.05–8.00 (m, 2H), 7.83–7.74 (m, 5H), 7.58–7.52 (m, 4H), 6.16 (broad s, 1H), 4.46 (q, J = 7.12 Hz, 2H), 3.95 (broad s, 2H), 3.56–3.48 (m, 2H), 2.93 (broad s, 2H), 1.45 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 502.3 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₁H₂₇NO₂F₃, 502.1994; found, 502.1996 [M + 1]⁺.

Ethyl 4-(4-(1-Methylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43a).

To a solution of compound 42 (10 mg, 16.2 μmol; synthesized according to literature procedures reported) in acetonitrile (1 mL) were added potassium carbonate (6.7 mg, 48.6 μmol) and iodomethane (36 μL, 17.8 μmol, 0.5 M solution in acetonitrile), and then this reaction mixture was stirred at room temperature for 15 h. The reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 10:1) to afford compound 43a (5 mg, 55%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.74 (s, 1H), 8.44 (s, 1H), 8.04–7.93 (m, 5H), 7.82 (d, J = 7.96 Hz, 2H), 7.52–7.47 (m, 4H), 4.50–4.45 (dd, J = 7.03 Hz, 2H), 3.28 (m, 2H), 2.86–2.80 (m, 1H), 2.64–2.60 (m, 5H), 2.08 (d, J = 12.00 Hz, 2H), 2.03–1.94 (m, 2H), 1.47 (t, J = 7.00 Hz, 3H); MS (ESI, m/z): 518.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₃₁NO₂F₃, 518.2307; found, 518.2297 [M + 1]⁺.

Ethyl 4-(4-(1-(Prop-2-yn-1-yl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43b).

To a solution of compound 42 (24 mg, 0.04 mmol), which was synthesized according to literature procedures reported, in acetonitrile (2 mL) was added potassium carbonate (17.0 mg, 0.12 mmol), and then propargyl bromide (4 μL, 0.047 mmol, 1 M solution in acetonitrile) was added to the reaction mixture by dropwise addition under a N₂ atmosphere. This reaction mixture was stirred at 50 °C temperature for 15 h. This mixture was partitioned with ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3:1) to afford compound 43b (14 mg, 65%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.67 (s, 1H), 8.22 (s, 1H), 8.08–8.04 (m, 2H), 7.82 (d, J = 8.24 Hz, 2H), 7.79–7.73 (m, 3H), 7.48 (d, J = 8.04 Hz, 2H), 7.40 (d, J = 8.04 Hz, 2H), 4.45 (q, J = 7.12 Hz, 2H), 3.42 (broad s, 2H), 3.11–3.07 (m, 2H), 2.64–2.60 (m, 1H), 2.48–2.38 (m, 1H), 2.05–1.92 (m, 4H), 1.64–1.54 (m, 2H), 1.44 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 542.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₁NO₂F₃, 542.2307; found, 542.2305 [M + 1]⁺.

Ethyl 4-(4-(1-Propylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43c).

To a solution of compound 42 (5 mg, 8.10 μmol), which was synthesized according to literature procedures reported, in acetonitrile (1 mL) were added potassium carbonate (6.7 mg, 48.6 μmol) and 1-iodopropane (9 μL, 8.91 μmol, 1 M solution in acetonitrile), and then this reaction mixture was stirred at room temperature for 15 h. The reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 20:1) to afford compound 43c (3 mg, 68%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.70 (s, 1H), 8.25 (s, 1H), 8.06–8.04 (m, 2H), 7.86–7.77 (m, 5H), 7.51 (d, J = 8.08 Hz, 2H), 7.45 (d, J = 8.08 Hz, 2H), 4.48 (q, J = 7.12 Hz, 2H), 3.59 (d, J = 6.52 Hz, 2H), 2.91–2.85 (m, 2H), 2.71–2.60 (m, 3H), 2.13 (d, J = 12.88 Hz, 2H), 1.97–1.92 (m, 2H), 1.47 (t, J = 7.12 Hz, 3H), 1.05 (t, J = 7.32 Hz, 3H); MS (ESI, m/z): 546.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₅NO₂F₃, 546.2620; found, 546.2627 [M + 1]⁺.

Ethyl 4-(4-(1-(Hex-5-yn-1-yl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43d).

To a solution of compound 42 (50 mg, 0.081 mmol), which was synthesized according to literature procedures reported, in DMF (N,N-dimethylformamide, 3 mL) were added potassium carbonate (34 mg, 0.024 mmol) and 6-bromohex-1-yne (65 mg, 0.405 mmol, 1 M solution in DMF), and then this reaction mixture was stirred at room temperature for 15 h. This mixture was partitioned with ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3:1) to afford compound 43d (50 mg, 95%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.70 (s, 1H), 8.24 (d, J = 1.80 Hz, 1H), 8.05 (d, J = 1.60 Hz, 1H), 8.02 (d, J = 8.84 Hz, 1H), 7.84 (d, J = 8.16 Hz, 2H), 7.81–7.76 (m, 3H), 7.52 (d, J = 8.20 Hz, 2H), 7.47 (d, J = 8.20 Hz, 2H), 4.48 (q, J = 7.08 Hz, 2H), 3.77 (d, J = 10.36 Hz, 2H), 3.10–3.04 (m, 2H), 2.92–2.79 (m, 4H), 2.36–2.32 (m, 2H), 2.20–2.14 (m, 4H), 2.03 (t, J = 2.62 Hz, 1H), 1.72–1.65 (m, 2H), 1.47 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 584.3 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₇H₃₇NO₂F₃, 584.2776; found, 584.2783 [M + 1]⁺.

Ethyl 4-(4-(1-Acetylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43e).

To a solution of compound 42 (30.0 mg, 0.060 mmol) in acetonitrile (2.0 mL) was added triethylamine (12 μL, 0.120 mmol) by dropwise addition at room temperature. After this reaction mixture was stirred for 5 min, acetic anhydride (18 μL, 0.180 mmol) was added to the reaction mixture. Then, this reaction mixture was stirred at room temperature for 30 min. This mixture was partitioned with ethyl acetate (5 mL) and brine (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL × 2), and then the combined organic layer was dried with MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) to afford 43e (26 mg, 49%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.68 (s, 1H), 8.23 (s, 1H), 8.05–8.03 (m, 2H), 7.83–7.74 (m, 5H), 7.49 (d, J = 7.96 Hz, 2H), 7.37 (d, J = 7.88 Hz, 2H), 4.86–4.83 (m, 1H), 4.47 (q, J = 7.08 Hz, 2H), 4.01–3.98 (m, 1H), 3.27–3.20 (m, 1H), 2.89–2.83 (m, 1H), 2.72–2.66 (m, 1H), 2.17 (s, 3H), 2.04–1.98 (m, 2H), 1.75–1.70 (m, 2H), 1.45 (t, J = 7.08 Hz, 3H); MS (ESI, m/z): 546.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₃H₃₁NO₃F₃, 546.2256; found, 546.2250 [M + 1]⁺.

Ethyl 4-(4-(1-Propionylpiperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (43f).

To a solution of compound 42 (33 mg, 0.065 mmol) in DMF (2.0 mL) were added propionic acid (5 μL, 0.072 mmol), HATU (27.0 mg, 0.072 mmol), and N,N-diisopropylethylamine (15 μL, 0.085 mmol), and then this reaction mixture was stirred at room temperature for 2 h. The reaction mixture was partitioned with ethyl acetate (5 mL) and water (5 mL), and the aqueous layer was extracted with ethyl acetate (5 mL × 2). The combined organic layer was washed with brine (3 mL), dried with MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 3:1) to afford compound 43f (20 mg, 54%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.60 (s, 1H), 8.28 (s, 1H), 7.92–7.87 (m, 4H), 7.79–7.73 (m, 3H), 7.35 (s, 4H), 4.74–4.70 (m, 1H), 4.42 (q, J = 7.12 Hz, 2H), 4.12–4.07 (m, 1H), 3.26–3.19 (m, 1H), 2.94–2.86 (m, 1H), 2.77–2.70 (m, 1H), 2.48 (q, J = 7.48 Hz, 2H), 2.02–1.91 (m, 2H), 1.76–1.58 (m, 2H), 1.42 (t, J = 7.12 Hz, 3H), 1.16 (t, J = 7.48 Hz, 3H); MS (ESI, m/z): 560.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₃NO₃F₃, 560.2413; found, 560.2419 [M + 1]⁺.

Ethyl 4-(4-(1-(2,2-Dimethyl-4-oxo-3,8,11,14,17,20,23-heptaoxa-5-azahexacosan-26-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (44a).

To a solution of compound 42 (5 mg, 8.10 μmol) in DMF (0.5 mL) were added Boc-NH-PEG₆-CH₂-CH₂-COOH (7 mg, 15.4 μmol), HATU (3.4 mg, 8.94 μmol), and N,N-diisopropylethylamine (4 μL, 24.3 μmol), and then this reaction mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned with ethyl acetate (5 mL) and water (5 mL), and the aqueous layer was extracted with ethyl acetate (5 mL × 2). The combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/methanol = 50:1) to afford compound 44a (7 mg, 94%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.70 (s, 1H), 8.25 (s, 1H), 8.08–8.06 (m, 2H), 7.84 (d, J = 8.16 Hz, 2H), 7.82–7.77 (m, 3H), 7.50 (d, J = 7.92 Hz, 2H), 7.38 (d, J = 8.00 Hz, 2H), 5.09 (s, 1H), 4.86 (d, J = 13.24 Hz, 1H), 4.48 (q, J = 7.12 Hz, 2H), 4.09 (d, J = 13.32 Hz, 1H), 3.86 (t, J = 6.58 Hz, 2H), 3.71–3.65 (m, 20H), 3.56 (t, J = 5.08 Hz, 2H), 3.33 (d, J = 4.88 Hz, 2H), 3.25–3.19 (m, 1H), 2.92–2.86 (m, 1H), 2.76 (t, J = 6.68 Hz, 2H), 2.07–2.00 (m, 2H), 1.80–1.70 (m, 2H), 1.49–1.46 (m, 12H); MS (ESI, m/z): 939.5 [M + 1]⁺, 956.4 [M + NH₄]⁺; ESI-HRMS calcd m/z for C₅₁H₆₆N₂O₁₁F₃, 939.4619; found, 939.4625 [M + 1]⁺.

Ethyl 4-(4-(1-(2,5,8,11,14,17-Hexaoxaicosan-20-oyl)piperidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (44b).

Compound 42 (11 mg, 14.9 μmol) with mPEG₅-CH₂-CH₂-COOH (7.4 mg, 22.8 μmol) was converted to compound 44b (11 mg, 93%) as a white foam, using a similar procedure used in the preparation of compound 44a; ¹H NMR (400 MHz, CDCl₃): δ 8.70 (s, 1H), 8.25 (s, 1H), 8.07 (d, J = 7.88 Hz, 2H), 7.86–7.77 (m, 5H), 7.50 (d, J = 7.88 Hz, 2H), 7.38 (d, J = 7.88 Hz, 2H), 4.84 (d, J = 13.8 Hz, 1H), 4.48 (q, J = 7.10 Hz, 2H), 4.10 (d, J = 13.6 Hz, 1H), 3.89–3.82 (m, 2H), 3.70–3.65 (m, 18H), 3.59–3.57 (m, 2H), 3.51 (s, 1H), 3.40 (s, 3H), 3.24 (t, J = 12.8 Hz, 1H), 2.90 (t, J = 12.1 Hz, 1H), 2.76 (t, J = 12.7 Hz, 2H), 2.04 (t, J = 13.5 Hz, 2H), 1.81–1.71 (m, 2H), 1.47 (t, J = 7.15 Hz, 3H); MS (ESI, m/z): 810.4 [M + 1]⁺; ESI-HRMS calcd m/z for C₄₅H₅₅NO₉F₃, 810.3829; found, 810.3831 [M + 1]⁺.

Ethyl 4-(4-(Quinuclidin-4-yl)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (45a).

The mixture of compound 40 (10 mg, 0.021 mmol), Pd(PPh₃)₄ (2 mg, 1.73 μmol), and potassium carbonate (8 mg, 0.057 mmol) in DMF (2 mL) was purged with nitrogen gas for 15 min, and then 4-(4-bromophenyl)quinuclidine (96, 7 mg, 0.025 mmol) was added to the mixture. The mixture was stirred at 80 °C for 3 h and then allowed to be cooled to room temperature. This mixture was partitioned with ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 45a (10 mg, 88%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.75 (s, 1H), 8.44 (s, 1H), 8.01–7.92 (m, 5H), 7.82 (d, J = 8.12 Hz, 2H), 7.62 (d, J = 8.16 Hz, 2H), 7.57 (d, J = 8.24 Hz, 2H), 4.49 (q, J = 7.12 Hz, 2H), 3.58–3.54 (m, 6H), 2.37–2.33 (m, 6H), 1.47 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 530.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₃H₃₁NO₂F₃, 530.2307; found, 530.2302 [M + 1]⁺.

4-(4-(3-(Ethoxycarbonyl)-6-(4-(trifluoromethyl)phenyl)-naphthalen-1-yl)phenyl)-1-methylquinuclidin-1-ium (45b).

To a solution of compound 45a (10.0 mg, 0.018 mmol) in acetonitrile/methanol (1:1, 1 mL) were added potassium carbonate (10.0 mg, 0.075 mmol) and iodomethane (6.0 μL, 0.094 mmol), and then this reaction mixture was stirred at room temperature for 1 h. The reaction mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (chloroform/methanol = 10:1) to afford compound 45b (5.6 mg, 55%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.77 (s, 1H), 8.45 (s, 1H), 8.03–7.96 (m, 5H), 7.84 (d, J = 8.00 Hz, 2H), 7.66–7.55 (m, 4H), 4.48 (q, J = 7.12 Hz, 2H), 3.79–3.71 (m, 6H), 3.17 (s, 3H), 2.47–2.43 (m, 6H), 1.47 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 544.3 [M + 1]⁺.

tert-Butyl 4-(3′-(Methoxycarbonyl)-5′-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-yl)piperazine-1-carboxylate (48a).

The mixture of compound 47 (30 mg, 0.063 mmol), Pd(PPh₃)₄ (5.0 mg, 4.32 μmol), and potassium carbonate (25 mg, 0.180 mmol) in DMF (1.5 mL) was purged with nitrogen gas for 15 min, and then tert-butyl 4-(4-bromophenyl)piperazine-1-carboxylate (26 mg, 0.076 mmol) was added to the mixture. The mixture was stirred at 80 °C for 3 h and then allowed to be cooled to room temperature. This mixture was partitioned with ethyl acetate (5 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 48a (14 mg, 36%) as a colorless oil; ¹H NMR (400 MHz, CDCl₃): δ 8.39 (s, 1H), 8.34 (s, 1H), 8.29 (s, 1H), 8.25 (s, 1H), 8.07 (d, J = 7.96 Hz, 2H), 7.77 (d, J = 7.96 Hz, 2H), 7.65 (d, J = 8.60 Hz, 2H), 7.04 (d, J = 8.60 Hz, 2H), 4.01 (s, 3H), 3.62 (broad s, 4H), 3.24 (broad s, 4H), 1.49 (s, 9H); MS (ESI, m/z): 608.3 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₃₃N₅O₄F₃, 608.2485; found, 608.2483 [M + 1]⁺.

Methyl 4′-(Quinuclidin-4-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (48b).

Compound 47 (43 mg, 0.091 mmol) and compound 96 (29 mg, 0.109 mmol) were coupled to compound 48b (20 mg, 41%) as a white solid, using a similar procedure used in the preparation of compound 48a; ¹H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.50 (s, 1H), 8.42 (s, 1H), 8.35 (s, 1H), 8.15 (d, J = 8.00 Hz, 2H), 7.83–7.75 (m, 4H), 7.59–7.55 (m, 2H), 4.00 (s, 3H), 3.55–3.46 (m, 6H), 2.30–2.23 (m, 6H); MS (ESI, m/z): 533.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₀H₂₈N₄O₂F₃, 533.2164; found, 533.2170 [M + 1]⁺.

tert-Butyl 6-(3′-(Methoxycarbonyl)-5′-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-yl)-3-azabicyclo-[4.1.0]heptane-3-carboxylate (48c).

Compound 47 (36 mg, 0.076 mmol) and compound 99 (32 mg, 0.091 mmol) were coupled to compound 48c (15 mg, 32%) as a white solid, using a similar procedure used in the preparation of compound 48a; ¹H NMR (400 MHz, CD₃OD): δ 9.18 (s, 1H), 8.40 (s, 1H), 8.31 (s, 1H), 8.26 (s, 1H), 8.11 (d, J = 8.04 Hz, 2H), 7.77 (d, J = 8.08 Hz, 2H), 7.65 (d, J = 8.04 Hz, 2H), 7.39 (d, J = 8.08 Hz, 2H), 3.97 (s, 3H), 3.78 (broad s, 2H), 3.41–3.36 (m, 2H), 2.20–2.11 (m, 2H), 1.48 (s, 9H), 1.29–1.24 (m, 1H), 1.08–1.04 (m, 1H), 0.89–0.86 (m, 1H); MS (ESI, m/z): 619.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₄N₄O₄F₃, 619.2532; found, 619.2524 [M + 1]⁺.

tert-Butyl 7,7-Difluoro-6-(3′-(methoxycarbonyl)-5′-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-yl)-3-azabicyclo[4.1.0]heptane-3-carboxylate (48d).

Compound 47 (53 mg, 0.112 mmol) and compound 101 (36 mg, 0.093 mmol) were coupled to compound 48d (25 mg, 41%) as a white solid, using a similar procedure used in the preparation of compound 48a; ¹H NMR (400 MHz, CD₃OD): δ 9.27 (s, 1H), 8.53 (s, 1H), 8.43 (s, 1H), 8.37 (s, 1H), 8.17 (d, J = 8.00 Hz, 2H), 7.82–7.76 (m, 4H), 7.50 (d, J = 8.40 Hz, 2H), 4.00 (s, 3H), 3.93 (broad s, 1H), 3.79 (broad s, 1H), 3.53 (broad s, 1H), 3.20–3.09 (m, 1H), 2.38–2.15 (m, 2H), 1.99–1.93 (m, 1H), 1.50 (s, 9H); MS (ESI, m/z): 655.2 [M + 1]⁺.

Methyl 4′-(Piperazin-1-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (49a).

Method B: yield, 76%; ¹H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.46 (s, 1H), 8.40 (s, 1H), 8.35 (s, 1H), 8.16 (d, J = 8.12 Hz, 2H), 7.81–7.75 (m, 4H), 7.20 (d, J = 8.60 Hz, 2H), 4.01 (s, 3H), 3.54 (broad s, 4H), 3.42 (broad s, 4H); MS (ESI, m/z): 508.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₇H₂₅N₅O₂F₃, 508.1960; found, 508.1964 [M + 1]⁺.

4′-(3-(tert-Butoxycarbonyl)-3-azabicyclo[4.1.0]heptan-6-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (49c).

Method A: yield, 76%; ¹H NMR (400 MHz, CD₃OD): δ 9.24 (s, 1H), 8.49 (s, 1H), 8.38 (d, J = 6.6 Hz, 2H), 8.16 (d, J = 8.04 Hz, 2H), 7.79 (d, J = 8.12 Hz, 2H), 7.72 (d, J = 8.04 Hz, 2H), 7.45 (d, J = 7.96 Hz, 2H), 3.79 (broad s, 2H), 3.41–3.36 (m, 2H), 2.21–2.11 (m, 2H), 1.48 (s, 9H), 1.25 (d, J = 6.40 Hz, 1H), 1.11–1.06 (m, 1H), 0.91–0.86 (m, 1H); MS (ESI, m/z): 549.2 [M + 1-tert-butyl]⁺.

4′-(3-(tert-Butoxycarbonyl)-7,7-difluoro-3-azabicyclo[4.1.0]-heptan-6-yl)-5-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (49d).

Method A: yield, 94%; ¹H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.54 (s, 1H), 8.41 (d, J = 7.20 Hz, 2H), 8.17 (d, J = 8.00 Hz, 2H), 7.83–7.76 (m, 4H), 7.51 (d, J = 8.00 Hz, 2H), 3.98–3.73 (m, 2H), 3.53 (broad s, 1H), 3.19–3.12 (m, 1H), 2.38–2.15 (m, 2H), 2.00–1.93 (m, 1H), 1.50 (s, 9H); MS (ESI, m/z): 641.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₃H₃₀N₄O₄F₅, 641.2187; found, 641.2179 [M + 1]⁺.

Synthesis of Methyl 3-Bromo-5-iodobenzoate (54), Methyl 3-Bromo-5-((trimethylsilyl)ethynyl)benzoate (55), Methyl 3-Bromo-5-ethynylbenzoate (56), Methyl 3-Bromo-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoate (57), Methyl 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoate (58), and tert-Butyl 4-(3′-(Methoxycarbonyl)-5′-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-4-yl)piperidine-1-carboxylate (59b).

Synthesis of compounds 54–59 is reported by Mufti et al.¹²

Methyl 4′-Carbamoyl-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylate (59a).

A mixture of compound 58 (51 mg, 0.106 mmol), 4-bromobenzamide (26 mg, 0.127 mmol), and PdCl₂(dppf) (9 mg, 10.6 μmol) in dimethoxyethane (2 mL) and 2 M Na₂CO₃ aqueous solution (0.2 mL) was stirred at 50 °C for 3 h. After cooling at room temperature, the mixture was partitioned with diethyl ether (5 mL) and water (10 mL). The aqueous layer was extracted with diethyl ether (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) to afford compound 59a (24 mg, 46%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.53–8.52 (m, 2H), 8.45 (s, 1H), 8.34 (s, 1H), 8.02 (d, J = 8.40 Hz, 2H), 7.97 (d, J = 8.16 Hz, 2H), 7.88 (d, J = 8.48 Hz, 2H), 7.83 (d, J = 8.16 Hz, 2H), 4.03 (s, 3H); MS (ESI, m/z): 467.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₄H₁₈N₄O₃F₃, 467.1331; found, 467.1325 [M + 1]⁺.

Methyl 3-(5-((3-((tert-Butoxycarbonyl)amino)propyl)-carbamoyl)thiophen-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoate (59c).

Compound 58 (45 mg, 0.095 mmol) and tert-butyl (3-(5-bromothiophene-2-carboxamido)propyl) carbamate (38 mg, 0.105 mmol) were converted to compound 59c (31 mg, 52%) as a white solid, using a similar procedure used in the preparation of compound 59a; ¹H NMR (400 MHz, CDCl₃): δ 8.47 (s, 1H), 8.45–8.44 (m, 2H), 8.31 (s, 1H), 8.02 (d, J = 8.44 Hz, 2H), 7.87 (d, J = 8.56 Hz, 2H), 7.61 (d, J = 3.64 Hz, 1H), 7.47 (d, J = 3.88 Hz, 1H), 7.36 (broad s, 1H), 4.92 (broad s, 1H), 4.01 (s, 3H), 3.53 (q, J = 6.03 Hz, 2H), 3.30 (q, J = 5.97 Hz, 2H), 1.79–1.73 (m, 2H), 1.50 (s, 9H); MS (ESI, m/z): 530.1 [M + 1-Boc]⁺; ESI-HRMS calcd m/z for C₂₅H₂₃N₅O₃F₃³²S, 530.1474; found, 530.1476 [M + 1-Boc]⁺.

4′-(1-(tert-Butoxycarbonyl)piperidin-4-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (60b).

Method A: yield, 60%; ¹H NMR (400 MHz, CD₃OD): δ 9.20 (s, 1H), 8.53 (s, 1H), 8.39 (s, 1H), 8.25 (s, 1H), 8.18 (d, J = 7.92 Hz, 2H), 7.93 (d, J = 8.20 Hz, 2H), 7.68 (d, J = 7.76 Hz, 2H), 7.37 (d, J = 7.84 Hz, 2H), 4.25 (d, J = 13.12 Hz, 2H), 2.91 (broad s, 2H), 2.80 (t, J = 12.02 Hz, 1H), 1.88 (d, J = 12.68 Hz, 2H), 1.70–1.60 (m, 2H), 1.51 (s, 9H).

3-(5-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)thiophen-2-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoic Acid (60c).

Method A: yield, 99%; ¹H NMR (400 MHz, CD₃OD): δ 9.13 (s, 1H), 8.48 (s, 1H), 8.34 (s, 1H), 8.23 (s, 1H), 8.13 (s, 2H), 7.90 (s, 2H), 7.65 (s, 1H), 7.50 (s, 1H), 3.41 (s, 2H), 3.16 (s, 2H), 1.78 (s, 2H), 1.46 (s, 9H); MS (ESI, m/z): 516.1 [M + 1-Boc]⁺; ESI-HRMS calcd m/z for C₂₄H₂₁N₅O₃F₃³²S, 516.1317; found, 516.1316 [M + 1-Boc]⁺.

Methyl 3-Amino-5-bromobenzoate (61b).

3-Bromo-5-aminobenzoic acid (61a, 1.01 g, 4.62 mmol) was stirred in methanol (15 mL) with ice cooling, and the yellow solution was treated with thionyl chloride (4.00 mL, 55.0 mmol) dropwise over 20 min. The resulting mixture was warmed up to room temperature and left stirring for 15 h. The reaction mixture was quenched with aqueous saturated NaHCO₃ solution at 0 °C. The solvent was then removed under vacuum, and the residue was suspended in ethyl acetate (200 mL). The organic phase was washed with brine (100 mL), dried over Na₂SO₄, and concentrated in vacuo to afford the title compound 61b (1.08 g, 98%) as a yellow solid; ¹H NMR (400 MHz, DMSO-d₆): δ 7.16 (dd, J = 1.48, 2.12 Hz, 1H), 7.13 (t, J = 1.64 Hz, 1H), 6.96 (t, J = 2.00 Hz, 1H), 5.74 (s, 2H), 3.81 (s, 3H); MS (ESI, m/z): 231 [M + 1]⁺; ESI-HRMS calcd m/z for C₈H₈BrNO₂, 229.9817; found, 229.9818 [M + 1]⁺.

Methyl 3-Amino-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzoate (62).

To a solution of methyl 3-amino-5-bromobenzoate (61b, 219 mg, 0.950 mmol) in 1,4-dioxane (20 mL) were added bis(pinacolato)diboron (290 mg, 1.14 mmol), PdCl₂(dppf) (23 mg, 28.5 μmol), and potassium acetate (279 mg, 2.85 mmol), and then this reaction mixture was stirred at 95 °C for 15 h. The reaction mixture was partitioned with ethyl acetate (20 mL) and water (10 mL), and the aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 62 (180 mg, 68%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 7.88 (s, 1H), 7.46 (s, 1H), 7.31 (d, J = 2.4 Hz, 1H), 3.91 (s, 3H), 1.36 (s, 12H); MS (ESI, m/z): 278.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₄H₂₁NO₄¹¹B, 278.1564; found, 278.1565 [M + 1]⁺.

Methyl 5-Amino-4′-carbamoyl-[1,1′-biphenyl]-3-carboxylate (63a).

To a solution of compound 62 (90 mg, 0.325 mmol) in 1,2-dimethoxyethane (4 mL) were added compound 4-bromobenzamide (71 mg, 0.357 mmol), Pd(PPh₃)₄ (7.5 mg, 6.5 μmol), and potassium carbonate (90 mg, 0.650 mmol), and then this reaction mixture was purged with nitrogen for 30 min and stirred at 80 °C for 15 h. The reaction mixture was partitioned with ethyl acetate (20 mL) and water (10 mL), and the aqueous layer was extracted with ethyl acetate (20 mL × 2). The combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) to afford compound 63a (55 mg, 63%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 7.90 (d, J = 8.20 Hz, 2H), 7.70–7.68 (m, 3H), 7.40 (s, 1H), 7.11 (s, 1H), 3.95 (s, 3H), 3.92 (broad s, 1.5H; NH₂); MS (ESI, m/z): 271.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₅H₁₅N₂O₃, 271.1083; found, 271.1080 [M + 1]⁺.

tert-Butyl 4-(3′-Amino-5′-(methoxycarbonyl)-[1,1′-biphenyl]-4-yl)piperidine-1-carboxylate (63b).

Compound 62 (90 mg, 0.325 mmol) was converted to compound 63b (54 mg, 41%) as a white solid, using a similar procedure used in the preparation of compound 63a; ¹H NMR (400 MHz, CDCl₃): δ 7.68 (s, 1H), 7.56 (d, J = 8.16 Hz, 2H), 7.34 (s, 1H), 7.30 (merged with CHCl₃ peak, 2H), 7.09–7.08 (m, 1H), 4.03 (broad s, 2H), 3.93 (s, 3H), 2.84 (t, J = 12.06 Hz, 2H), 2.75–2.67 (m, 1H), 1.88 (d, J = 13.6 Hz, 2H), 1.73–1.62 (m, 2H), 1.51 (s, 9H); MS (ESI, m/z): 355.1 [M + 1-tert-butyl]⁺, 323.1 [M + 1-Boc]⁺.

Methyl 4′-Carbamoyl-5-(4-(trifluoromethyl)benzamido)-[1,1′-biphenyl]-3-carboxylate (64a).

To a solution of compound 63a (20 mg, 0.074 mmol) in DMF (2 mL) were added 4-(trifluoromethyl)-benzoic acid (21 mg, 0.111 mmol), HATU (31 mg, 0.081 mmol), and N,N-diisopropylethylamine (39 μL, 0.222 mmol), and then this reaction mixture was stirred at room temperature for 15 h. The reaction mixture was partitioned with ethyl acetate (10 mL) and water (10 mL), and the aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 1:1) to afford compound 64a (33 mg, 99%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.42 (s, 1H), 8.15 (d, J = 9.60 Hz, 2H), 8.07 (s, 1H), 8.05 (s, 1H), 7.94 (d, J = 7.80 Hz, 2H), 7.83 (d, J = 8.12 Hz, 2H), 7.78 (d, J = 8.16 Hz, 2H), 4.00 (s, 3H); MS (ESI, m/z): 443.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₈N₂O₄F₃, 443.1219; found, 443.1227 [M + 1]⁺.

tert-Butyl 4-(3′-(4-(Hydroxymethyl)cubane-1-carboxamido)-5′-(methoxycarbonyl)-[1,1′-biphenyl]-4-yl)piperidine-1-carboxylate (64b).

Compound 63b (28 mg, 68.2 μmol) and 4-(hydroxymethyl)-cubane-1-carboxylic acid (13 mg, 75.0 μmol) were converted to compound 64b (27 mg, 69%) as a white solid, using a similar procedure used in the preparation of compound 64a; ¹H NMR (400 MHz, CDCl₃): δ 8.30 (s, 1H), 8.04 (s, 1H), 7.61 (d, J = 7.96 Hz, 2H), 7.34 (s, 1H), 7.30 (d, J = 8.28 Hz, 2H), 4.27 (s, 5H), 3.98 (s, 3H), 3.96 (s, 3H), 3.85 (s, 2H), 2.91–2.82 (m, 2H), 2.75–2.69 (m, 1H), 1.87 (d, J = 12.24 Hz, 2H), 1.73–1.65 (m, 2H), 1.51 (s, 9H).

Methyl 5-(4-(Hydroxymethyl)cubane-1-carboxamido)-4′-(piperidin-4-yl)-[1,1′-biphenyl]-3-carboxylate (65).

The reaction mixture of compound 64b (20 mg, 35.0 μmol) in 1 N HCl/dioxane solution (1 mL) was stirred at room temperature for 15 h. After all volatiles was evaporated under reduced pressure, the residue was purified by silica gel column chromatography (dichloromethane/methanol = 3:1) to afford a methyl ester compound (65, 11 mg, 67%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.27 (s, 1H), 8.25 (s, 1H), 8.00 (t, J = 1.50 Hz, 1H), 7.65 (d, J = 8.28 Hz, 2H), 7.41 (d, J = 8.24 Hz, 2H), 4.25 (t, J = 4.90 Hz, 3H), 3.96 (s, 3H), 3.93 (t, J = 4.94 Hz, 3H), 3.73 (s, 2H), 3.49 (d, J = 12.52 Hz, 2H), 3.18–3.11 (m, 2H), 3.00–2.94 (m, 1H), 2.11 (d, J = 12.84 Hz, 2H), 2.03–1.93 (m, 2H); MS (ESI, m/z): 471.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₉H₃₁N₂O₄, 471.2284; found, 471.2282 [M + 1]⁺.

Methyl 3-Bromo-5-formylbenzoate (67).

To a solution of 3-bromo-5-formylbenzoic acid (66, 500 mg, 2.18 mmol) in methanol (25 mL) was added concentrated H₂SO₄ (1.16 mL, 21.8 mmol) at room temperature, and this reaction mixture was stirred at 60 °C for 15 h. After the solvent was evaporated under reduced pressure, the residue was partitioned with ethyl acetate (20 mL) and saturated sodium bicarbonate solution (20 mL), and the aqueous phase was extracted with ethyl acetate (20 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 50:1) to afford compound 67 (287 mg, 54%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 10.04 (s, 1H), 8.47 (s, 1H), 8.43 (s, 1H), 8.22 (s, 1H), 4.00 (s, 3H); MS (ESI, m/z): 243.0, 245.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₉H₈O₃⁷⁹Br, 242.9657; found, 242.9656 [M + 1]⁺.

3-Bromo-5-(methoxycarbonyl)benzoic Acid (68).

To a solution of compound 67 (30 mg, 0.123 mmol) in DMF (1 mL) was added oxone (38 mg, 0.123 mmol), and this reaction mixture was stirred at room temperature for 15 h. The reaction mixture was partitioned with ethyl acetate (5 mL) and saturated NaHCO₃ aqueous solution (5 mL), and the organic layer was extracted with saturated NaHCO₃ aqueous solution (5 mL × 2). The basic aqueous layer was acidified with 4 N aqueous HCl and extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure to afford compound 68 (25 mg, 78%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1H), 8.43 (s, 2H), 3.99 (s, 3H); MS (ESI, m/z): 259.0, 261.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₉H₈O₄⁷⁹Br, 258.9606; found, 258.9609 [M + 1]⁺.

Methyl 3-Bromo-5-((4-(trifluoromethyl)phenyl)carbamoyl)-benzoate (69).

To a solution of compound 68 (20 mg, 0.0778 mmol) in dichloromethane (3 mL) were added thionyl chloride (86 μL, 0.0856 mmol; 1 M solution in dichloromethane) and triethylamine (16 μL, 0.117 mmol) at 0 °C, and this reaction mixture was stirred at the same temperature for 1 h. After removal of the solvent under reduced pressure, the residue was dissolved in dichloromethane. p-(Trifluoromethyl)aniline (30 μL, 0.234 mmol) and triethylamine (16 μL, 0.117 mmol) were added, and the reaction mixture was stirred at room temperature for 15 h. The reaction mixture was partitioned with dichloromethane (10 mL) and water (5 mL), and the aqueous phase was extracted with dichloromethane (10 mL × 2). The combined organic layer was dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 6:1) to afford compound 69 (14 mg, 45%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.42 (s, 1H), 8.38 (s, 1H), 8.29 (s, 1H), 8.03 (s, 1H), 7.81 (d, J = 8.44 Hz, 2H), 7.67 (d, J = 8.52 Hz, 2H), 4.00 (s, 3H); MS (ESI, m/z): 402.0, 404.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₆H₁₂NO₃F₃⁷⁹Br, 401.9953; found, 401.9950 [M + 1]⁺.

Methyl 4′-Carbamoyl-5-((4-(trifluoromethyl)phenyl)carbamoyl)-[1,1′-biphenyl]-3-carboxylate (70).

To a solution of compound 69 (13 mg, 32.3 μmol) in 1,4-dioxane (2 mL) and water (0.2 mL) were added 4-aminocarbonylphenylboronic acid pinacol ester⁹ (16 mg, 64.6 μmol), PdCl₂(PPh₃)₂ (2.3 mg, 3.23 μmol), and sodium carbonate (6.5 mg, 64.6 μmol), and then this reaction mixture was stirred at 80 °C for 2 h. The reaction mixture was partitioned with ethyl acetate (10 mL) and water (5 mL), and the aqueous layer was extracted with ethyl acetate (5 mL × 2). The combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (dichloromethane/ethyl acetate = 1:1) to afford compound 70 (9 mg, 63%) as a white solid; ¹H NMR (400 MHz, CD₃OD): δ 8.64 (s, 1H), 8.54 (s, 1H), 8.53 (s, 1H), 8.05 (d, J = 8.20 Hz, 2H), 8.00 (d, J = 8.36 Hz, 2H), 7.89 (d, J = 8.28 Hz, 2H), 7.70 (d, J = 8.72 Hz, 2H), 4.02 (s, 3H); MS (ESI, m/z): 443.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₈N₂O₄F₃, 443.1219; found, 443.1217 [M + 1]⁺.

Methyl 3-((2-Amino-5-(trifluoromethyl)phenyl)ethynyl)-5-bromobenzoate (71a).

To a solution of 2-iodo-4-(trifluoromethyl)aniline (119 mg, 0.417 mmol), PdCl₂(PPh₃)₂ (2.4 mg, 3.47 μmol), and copper iodide (0.7 mg, 3.47 μmol) in triethylamine (6 mL) was added dropwise a solution of 56 (83 mg, 0.347 mmol) in triethylamine (4 mL), and then the reaction mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned with ethyl acetate (20 mL) and water (10 mL), and the aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 71a (113 mg, 82%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.17 (s, 1H), 8.13 (s, 1H), 7.86 (s, 1H), 7.64 (s, 1H), 7.40 (d, J = 8.52 Hz, 1H), 6.78 (d, J = 8.56 Hz, 1H), 4.61(broad s, 2H), 3.97 (s, 3H); MS (ESI, m/z): 398.0, 400.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₇H₁₂NO₂F₃⁷⁹Br, 398.0003; found, 398.0007 [M + 1]⁺.

Methyl 3-((2-Amino-4-(trifluoromethyl)phenyl)ethynyl)-5-bromobenzoate (71b).

Compound 70 (60 mg, 0.251 mmol) and 2-iodo-5-(trifluoromethyl)aniline (97 mg, 0.301 mmol) were converted to compound 71b (87 mg, 87%) as a white solid, using a similar procedure used in the preparation of compound 71a; ¹H NMR (400 MHz, CDCl₃): δ 8.18 (s, 1H), 8.14 (s, 1H), 7.87 (s, 1H), 7.46 (d, J = 8.52 Hz, 1H), 6.99–6.97 (m, 2H), 4.49 (broad s, 2H), 3.98 (s, 3H); MS (ESI, m/z): 398.0, 400.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₇H₁₂NO₂F₃⁷⁹Br, 398.0003; found, 398.0009 [M + 1]⁺.

Methyl 3-Bromo-5-(5-(trifluoromethyl)-1H-indol-2-yl)benzoate (72a).

The mixture of compound 71a (20 mg, 50.2 μmol) and PdCl₂ (1 mg, 5.02 μmol) in DMF (2 mL) was stirred at 110 °C for 10 min in microwave. After microwave irradiation, the solvent was removed under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 6:1) to afford compound 72a (13 mg, 65%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.68 (s, 1H), 8.27 (s, 1H), 8.16 (s, 1H), 8.03 (s, 1H), 7.96 (s, 1H), 7.51 (d, J = 8.48 Hz, 1H), 7.48 (d, J = 8.56 Hz, 1H), 7.01 (s, 1H), 4.00 (s, 3H); MS (ESI, m/z): 398.0, 400.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₇H₁₂NO₂F₃⁷⁹Br, 398.0003; found, 398.0000 [M + 1]⁺.

Methyl 3-Bromo-5-(6-(trifluoromethyl)-1H-indol-2-yl)benzoate (72b).

Compound 71b (76 mg, 0.191 mmol) was converted to compound 72b (47 mg, 62%) as a white solid, using a similar procedure used in the preparation of compound 72a; ¹H NMR (400 MHz, CDCl₃): δ 8.65 (broad s, 1H), 8.28 (s, 1H), 8.17 (s, 1H), 8.04 (s, 1H), 7.76–7.72 (m, 2H), 7.40 (d, J = 8.44 Hz, 1H), 7.00 (s, 1H), 4.01 (s, 3H); MS (ESI, m/z): 398.0, 400.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₇H₁₂NO₂F₃⁷⁹Br, 398.0003; found, 398.0002 [M + 1]⁺.

Methyl 4′-Carbamoyl-5-(5-(trifluoromethyl)-1H-indol-2-yl)-[1,1′-biphenyl]-3-carboxylate (73a).

Compound 72a (13 mg, 32.6 μmol) was converted to compound 73a (8 mg, 55%) as a white solid, using a similar procedure used in the preparation of compound 69 at 80 °C for 15 h; ¹H NMR (400 MHz, CDCl₃): δ 8.54 (s, 1H), 8.40 (s, 1H), 8.29 (s, 1H), 8.06 (d, J = 7.24 Hz, 2H), 7.95 (s, 1H), 7.90 (d, J = 7.25 Hz, 2H), 7.59 (d, J = 8.48 Hz, 1H), 7.41 (d, J = 8.20 Hz, 1H), 7.18 (s, 1H), 4.03 (s, 3H); MS (ESI, m/z): 439.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₄H₁₈N₂O₃F₃, 439.1270; found, 439.1272 [M + 1]⁺.

Methyl 4′-Carbamoyl-5-(6-(trifluoromethyl)-1H-indol-2-yl)-[1,1′-biphenyl]-3-carboxylate (73b).

Compound 72b (25 mg, 62.8 μmol) was converted to compound 73b (20 mg, 72%) as a white solid, using a similar procedure used in the preparation of compound 69 at 80 °C for 15 h; ¹H NMR (400 MHz, CD₃OD): δ 8.52 (s, 1H), 8.38 (s, 1H), 8.28 (s, 1H), 8.05 (d, J = 8.16 Hz, 2H), 7.88 (d, J = 8.24 Hz, 2H), 7.76–7.74 (m, 2H), 7.30 (d, J = 8.60 Hz, 1H), 7.13 (s, 1H), 4.01 (s, 3H); MS (ESI, m/z): 439.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₄H₁₈N₂O₃F₃, 439.1270; found, 439.1272 [M + 1]⁺.

Methyl 3-Bromo-5-(5-(trifluoromethyl)-1H-benzo[d]imidazol-2-yl)benzoate (74).

To a solution of compound 66b (20 mg, 0.083 mmol) in DMF (3 mL) was added 4-trifluoromethyl-O-phenylenediamine (29 mg, 0.166 mmol) and sodium metabisulfite (32 mg, 0.166 mmol) at room temperature, and this reaction mixture was stirred at 130 °C for 15 h. After cooling, the reaction mixture was partitioned with ethyl acetate (20 mL) and water (20 mL), and the aqueous phase was extracted with ethyl acetate (20 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 6:1) to afford compound 74 (32 mg, 97%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.57–8.56 (m, 2H), 8.32 (s, 1H), 8.01 (broad s, 1H), 7.76 (broad s, 1H), 7.60 (d, J = 8.24 Hz, 1H), 4.01 (s, 3H); MS (ESI, m/z): 399.0, 401.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₆H₁₁N₂O₂F₃⁷⁹Br, 398.9956; found, 398.9953 [M + 1]⁺.

Methyl 4′-Carbamoyl-5-(5-(trifluoromethyl)-1H-benzo[d]-imidazol-2-yl)-[1,1′-biphenyl]-3-carboxylate (75).

Compound 74 (12 mg, 30.1 μmol) was converted to compound 75 (5 mg, 38%) as a white solid, using a similar procedure used in the preparation of compound 69 at 80 °C for 15 h; ¹H NMR (400 MHz, CD₃OD): δ 8.83 (s, 1H), 8.73 (s, 1H), 8.48 (s, 1H), 8.07 (d, J = 8.04 Hz, 2H), 7.98 (broad s, 1H), 7.92 (d, J = 8.16 Hz, 2H), 7.80 (broad s, 1H), 7.60 (d, J = 8.08 Hz, 1H), 4.04 (s, 3H); MS (ESI, m/z): 440.1 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₃H₁₇N₃O₃F₃, 440.1222; found, 440.1223 [M + 1]⁺.

Ethyl 3-Amino-5-(4-bromophenyl)-1H-pyrrole-2-carboxylate (78).

To a mixture of (4-bromobenzoyl)acetonitrile (76, 287 mg, 1.28 mmol) in dichloromethane (1 mL) were added p-toluenesulfonic anhydride (502 mg, 1.54 mmol) and triethylamine (194 mg, 0.27 mL, 1.92 mmol), and the reaction mixture was stirred at room temperature for 3 h. The reaction mixture was partitioned with dichloromethane (10 mL) and water (10 mL), and the aqueous phase was extracted with dichloromethane (10 mL × 2). The combined organic layer was dried over MgSO₄, filtered, and evaporated under reduced pressure to give a beige solid (526 mg, >100%). To a solution of sodium ethoxide (262 mg, 1.25 mL, 3.85 mmol, 21 wt % ethanol solution) in ethanol (4 mL) was added a solution of the obtained beige solid (426 mg, 1.28 mmol) and diethyl aminomalonate hydrochloride (281 mg, 1.33 mmol) in ethanol (6 mL) and tetrahydrofuran (3 mL) dropwise over 10 min. This reaction mixture was stirred at room temperature for 30 min, and all solvent was removed under reduced pressure. The residue was partitioned with ethyl acetate (10 mL) and water (10 mL), and the aqueous phase was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 5:1) to afford compound 78 (127 mg, 40% from 76) as a beige solid; ¹H NMR (400 MHz, CDCl₃): δ 7.53 (d, J = 8.40 Hz, 2H), 7.38 (d, J = 8.40 Hz, 2H), 6.03 (d, J = 2.84 Hz, 1H), 4.37 (q, J = 7.06 Hz, 2H), 1.40 (t, J = 7.10 Hz, 3H); MS (ESI, m/z): 309.0, 311.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₃H₁₄N₂O₂⁷⁹Br, 309.0239; found, 309.0240 [M + 1]⁺.

tert-Butyl 4-(4-(4-Amino-5-(ethoxycarbonyl)-1H-pyrrol-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (79).

To a mixture of compound 78 (23 mg, 74.4 μmol), tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H)-carboxylate (28 mg, 89.2 μmol), and PdCl₂(dppf) (6 mg, 7.44 μmol) in DMF (1 mL) was added 2 M NaOH (75 μL, 0.148 mmol), and this reaction mixture was stirred at room temperature for 1 h. The mixture was partitioned with ethyl acetate (10 mL) and water (10 mL), and the aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 2:1) to afford compound 79 (24 mg, 78%) as a beige solid; ¹H NMR (400 MHz, CDCl₃): δ 7.49 (d, J = 8.20 Hz, 2H), 7.42 (d, J = 8.28 Hz, 2H), 6.11 (broad s, 1H), 6.05 (d, J = 2.84 Hz, 1H), 4.37 (q, J = 7.01 Hz, 2H), 4.12 (s, 2H), 3.67 (t, J = 5.66 Hz, 2H), 2.56 (s, 2H), 1.52 (s, 9H), 1.41 (t, J = 7.10 Hz, 3H); MS (ESI, m/z): 412.2 [M + 1]⁺.

tert-Butyl 4-(4-(4-Azido-5-(ethoxycarbonyl)-1H-pyrrol-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (80).

To a mixture of 79 (20 mg, 48.6 μmol) in DMF (1.4 mL) and water (0.6 mL) was added 4 N aqueous HCl (24 μL, 97.2 μmol). After 5 min, sodium nitrite (7 mg, 0.101 mmol) was added to the above reaction mixture at 0 °C, and then sodium azide was added after 30 min. The mixture was stirred at room temperature for 30 min and partitioned with ethyl acetate (10 mL) and water (15 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 80 (16 mg, 75%) as a beige solid; ¹H NMR (400 MHz, CDCl₃): δ 8.94 (s, 1H), 7.51 (d, J = 8.40 Hz, 2H), 7.45 (d, J = 8.44 Hz, 2H), 6.40 (d, J = 3.08 Hz, 1H), 6.13 (s, 1H), 4.41 (q, J = 7.10 Hz, 2H), 4.12 (s, 2H), 3.68 (t, J = 5.32 Hz, 2H), 2.56 (s, 2H), 1.52 (s, 9H), 1.43 (t, J = 7.06 Hz, 3H); MS (ESI, m/z): 410.2 [M + 1-N₂]⁺; ESI-HRMS calcd m/z for C₂₃H₂₈N₃O₄, 410.2080; found, 410.2086 [M + 1-N₂]⁺.

tert-Butyl 4-(4-(5-(Ethoxycarbonyl)-4-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrol-2-yl)phenyl)-3,6-dihydropyridine-1(2H)-carboxylate (81).

To a mixture of compound 80 (15 mg, 34.3 μmol) and 4-ethynyl-α,α,α-trifluorotoluene (9 μL, 9.39 mg, 55.2 μmol) in dimethyl sulfoxide/water (9:1, 1 mL) were added sodium ascorbate (10 mg, 51.4 μmol) and CuSO₄·5H₂O (4 mg, 17.1 μmol) sequentially. The reaction mixture was stirred at room temperature for 1 h and partitioned between ethyl acetate (10 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 2:1) to afford compound 81 (16 mg, 77%) as a beige solid; ¹H NMR (400 MHz, CDCl₃): δ 9.32 (s, 1H), 8.79 (s, 1H), 8.07 (d, J = 8.16 Hz, 2H), 7.73 (d, J = 8.00 Hz, 2H), 7.61 (d, J = 8.08 Hz, 2H), 7.51 (d, J = 7.92 Hz, 2H), 7.11 (s, 1H), 6.17 (s, 1H), 4.39 (q, J = 7.00 Hz, 2H), 4.14 (s, 2H), 3.69 (t, J = 5.40 Hz, 2H), 2.58 (s, 2H), 1.53 (s, 9H), 1.36 (t, J = 6.98 Hz, 3H); MS (ESI, m/z): 608.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₃₃N₅O₄F₃, 608.2485; found, 608.2491 [M + 1]⁺.

Ethyl 5-(4-(1,2,3,6-Tetrahydropyridin-4-yl)phenyl)-3-(4-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-1H-pyrrole-2-carboxylate (82).

Method B: yield, 60%; ¹H NMR (400 MHz, CD₃OD): δ 8.90 (s, 1H), 8.13 (d, J = 8.04 Hz, 2H), 7.86 (d, J = 8.36 Hz, 2H), 7.80 (d, J = 8.16 Hz, 2H), 7.62 (d, J = 8.44 Hz, 2H), 7.01 (s, 1H), 6.28 (broad s, 1H), 4.29 (q, J = 7.12 Hz, 2H), 3.91–3.88 (m, 2H), 3.50 (t, J = 6.12 Hz, 2H), 2.87–2.84 (m, 2H), 0.91 (t, J = 6.12 Hz, 3H); MS (ESI, m/z): 508.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₇H₂₅N₅O₂F₃, 508.1960; found, 508.1960 [M + 1]⁺.

Methyl 3-Bromo-2-methyl-5-((trimethylsilyl)ethynyl)benzoate (84).

To a solution of compound 83 (100 mg, 0.281 mmol) in DMF (2 mL) were added PdCl₂(PPh₃)₂ (40 mg, 0.056 mmol), copper iodide (6 mg, 0.030 mmol), triethylamine (0.120 mL, 0.843 mmol), and TMS-acetylene (0.043 mL, 0.309 mmol), and then this reaction mixture was stirred at room temperature for 5 h. After the solvent was evaporated under reduced pressure, the residue was purified by silica gel column chromatography (hexane/ethyl acetate = 50:1) to afford compound 84 (91 mg, 99%) as a colorless syrup; ¹H NMR (400 MHz, CDCl₃): δ 7.82 (s, 1H), 7.79 (s, 1H), 3.89 (s, 3H), 2.61 (s, 3H), 0.24 (s, 9H).

Methyl 3-Bromo-5-ethynyl-2-methylbenzoate (85).

To a solution of compound 84 (91 mg, 0.279 mmol) in tetrahydrofuran (10 mL) was added tetrabutylammonium fluoride (0.028 mL, 1 M solution in tetrahydrofuran), and then this reaction mixture was stirred at room temperature for 0.5 h. After being neutralized with acetic acid, the mixture was evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 30:1) to afford compound 85 (66 mg, 93%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 7.85 (s, 1H), 7.81 (s, 1H), 3.90 (s, 3H), 3.10 (s, 1H), 2.62 (s, 3H).

Methyl 3-Bromo-2-methyl-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)benzoate (86).

To a solution of compound 85 (66 mg, 0.149 mmol) and 1-azido-4-(trifluoromethyl)benzene (42 mg, 0.224 mmol, synthesized according to literature procedures reported) in tetrahydrofuran/water (2 mL, 1:1) were added CuSO₄· 5H₂O (19 mg, 0.076 mmol) and sodium ascorbate (43 mg, 0.217 mmol, freshly prepared 1 M aqueous solution), and then this reaction mixture was stirred at room temperature for 1 h. The reaction mixture was partitioned with diethyl ether (10 mL) and water (5 mL), and the aqueous layer was extracted with diethyl ether (10 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 9:1) to afford compound 86 (76 mg, 66%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.30 (s, 2H), 8.27 (s, 1H), 7.97 (d, J = 8.36 Hz, 2H), 7.85 (d, J = 8.40 Hz, 2H), 3.95 (s, 3H), 2.68 (s, 3H); MS (ESI, m/z): 440.0, 442.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₈H₁₄N₃O₂F₃⁷⁹Br, 440.0221; found, 440.0227 [M + 1]⁺.

tert-Butyl 4-(3′-(Methoxycarbonyl)-2′-methyl-5′-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-4-yl)piperidine-1-carboxylate (87).

The mixture of compound 86 (40 mg, 0.090 mmol), Pd(PPh₃)₄ (6 mg, 5.19 μmol), and potassium carbonate (37 mg, 0.267 mmol) in DMF (3 mL) was purged with nitrogen gas for 15 min, and then tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate (53 mg, 0.136 mmol) was added to the mixture. The mixture was stirred at 85 °C for 12 h and then allowed to be cooled to room temperature. This mixture was partitioned with diethyl ether (5 mL) and water (10 mL). The aqueous layer was extracted with diethyl ether (5 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 4:1) to afford compound 87 (40 mg, 70%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1H), 8.28 (s, 1H), 7.96–7.92 (m, 3H), 7.84 (d, J = 8.44 Hz, 2H), 7.07 (d, J = 8.52 Hz, 2H), 6.78 (d, J = 8.56 Hz, 2H), 4.30–4.19 (m, 2H), 3.96 (s, 3H), 2.95–2.72 (m, 3H), 2.47 (s, 3H), 1.90 (d, J = 13.12 Hz, 2H), 1.80 (d, J = 13.12 Hz, 2H), 1.49 (s, 9H); MS (ESI, m/z): 621.3 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₆N₄O₄F₃, 621.2689; found 621.2690 [M + 1]⁺.

Methyl 2-Methyl-4′-(piperidin-4-yl)-5-(1-(4-(trifluoromethyl)phenyl)-1H-1,2,3-triazol-4-yl)-[1,1′-biphenyl]-3-carboxylate (88).

Method B: yield, 79%; ¹H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1H), 8.29 (s, 1H), 7.96–7.92 (m, 3H), 7.84 (d, J = 8.52 Hz, 2H), 7.34–7.29 (m, 2H), 7.09 (d, J = 8.40 Hz, 1H), 6.81 (d, J = 8.40 Hz, 1H), 3.96 (s, 3H), 3.62–3.52 (m, 2H), 2.90–2.83 (m, 2H), 2.74–2.67 (m, 1H), 2.46 (s, 3H), 2.17–2.11 (m, 2H), 2.06–1.97 (m, 2H); MS (ESI, m/z): 521.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₉H₂₈N₄O₂F₃, 521.2164; found, 521.2173 [M + 1]⁺.

Methyl 4-Bromo-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]-imidazole-6-carboxylate (90).

To a solution of compound 89 (200 mg, 0.816 mmol) in DMF (10 mL) was added 4-(trifluoromethyl)-benzaldehyde (0.222 mL, 1.632 mmol) and sodium metabisulfite (310 mg, 1.632 mmol) at room temperature, and this reaction mixture was stirred at 130 °C for 12 h. After cooling, the reaction mixture was partitioned with ethyl acetate (20 mL) and water (20 mL), and the aqueous phase was extracted with ethyl acetate (20 mL × 2). The combined organic layer was washed with brine (5 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 6:1) to afford compound 90 (212 mg, 65%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.38 (broad s, 1H), 8.25–8.18 (m, 3H), 7.82 (d, J = 8.16 Hz, 2H), 3.97 (s, 3H); MS (ESI, m/z): 399.0, 401.0 [M + 1]⁺; ESI-HRMS calcd m/z for C₁₆H₁₁N₂O₂F₃⁷⁹Br, 398.9956; found, 398.9950 [M + 1]⁺.

Methyl 4-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-6-carboxylate (91).

Compound 90 (30 mg, 0.075 mmol) and tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate (34 mg, 0.090 mmol) were dissolved in degassed 2 M Na₂CO₃ aqueous solution (15 mg, 0.141 mmol) and 1,4-dioxane (3 mL), and then Pd(PPh₃)₄ (5 mg, 4.32 μmol) was added to the reaction mixture. The mixture was stirred at 80 °C for 12 h under a nitrogen atmosphere. After cooling at room temperature, the mixture was partitioned with ethyl acetate (20 mL) and water (10 mL). The aqueous layer was extracted with ethyl acetate (10 mL × 2), and then the combined organic layer was washed with brine (3 mL), dried over MgSO₄, filtered, and evaporated under reduced pressure. The residue was purified by silica gel column chromatography (hexane/ethyl acetate = 5:1) to afford compound 91 (19 mg, 43%) as a white solid; ¹H NMR (400 MHz, CDCl₃): δ 8.42 (broad s, 1H), 8.23–8.17 (m, 3H), 7.79 (d, J = 8.04 Hz, 2H), 7.40 (d, J = 7.84 Hz, 2H), 7.27–7.23 (m, 2H), 3.97 (s, 3H), 2.90–2.70 (m, 3H), 1.93–1.86 (m, 2H), 1.74–1.65 (m, 2H), 1.50 (s, 9H), 1.28–1.24 (m, 2H); MS (ESI, m/z): 580.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₃₂H₃₃N₃O₄F₃, 580.2423; found, 580.2434 [M + 1]⁺.

Methyl 4-(4-(Piperidin-4-yl)phenyl)-2-(4-(trifluoromethyl)phenyl)-1H-benzo[d]imidazole-6-carboxylate (92).

Method B: yield, 82%; ¹H NMR (400 MHz, CD₃OD): δ 8.36 (d, J = 8.00 Hz, 2H), 8.28 (s, 1H), 8.06 (s, 1H), 7.97 (d, J = 8.00 Hz, 2H), 7.88 (d, J = 8.40 Hz, 2H), 7.48 (d, J = 8.00 Hz, 2H), 3.98 (s, 3H), 3.57 (d, J = 13.12 Hz, 2H), 3.25–3.17 (m, 2H), 3.08–2.98 (m, 1H), 2.23–2.15 (m, 2H), 2.06–1.95 (m, 2H); MS (ESI, m/z): 480.2 [M + 1]⁺; ESI-HRMS calcd m/z for C₂₇H₂₅N₃O₂F₃, 480.1899; found, 480.1902 [M + 1]⁺.

tert-Butyl 4-(4-(3-(Ethoxycarbonyl)-6-(4-(trifluoromethyl)phenyl)naphthalen-1-yl)phenyl)piperidine-1-carboxylate-3,4-t₂ (93).

To a solution of compound 39 (6 mg, 0.010 mmol) in ethyl acetate (0.2 mL) was added 10% Pd/C (5 mg). The reaction mixture was stirred under a tritium atmosphere for 24 h at room temperature. The reaction mixture was filtered and evaporated with methanol three times.

Ethyl 4-(4-(Piperidin-4-yl-3,4-t₂)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoate (94).

To a solution of compound 93 in THF (1 mL) was added dropwise TFA (1 mL) and stirred for 4 h at room temperature. The reaction mixture was evaporated to dryness.

4-(4-(Piperidin-4-yl-3,4-t₂)phenyl)-7-(4-(trifluoromethyl)phenyl)-2-naphthoic acid ([³H]1).

HPLC purity, 97% (R_t = 13.67 min). To a solution of compound 94 in water/methanol (1 mL, 1:1) was added dropwise 1 N KOH (0.1 mL) and stirred for 18 h at room temperature. The reaction mixture was evaporated to dryness. The residue was purified by HPLC (0.1% TFA/acetonitrile = 100:0 to 0:100).

Pharmacological Assays.

Mammalian Cells Expressing the P2Y₁₄R.

We recently described the establishment and validation using fluorescent antagonist 36 of an HEK293 cell line stably expressing the mP2Y₁₄R.³⁹ The cloning vector contained an N-terminal human influenza hemagglutinin (HA)-TAG, which allowed the initial selection by flow cytometry detection of cell colonies highly expressing the receptor protein. A secondary selection process identified cell lines having high levels of 36 binding, as indicated by flow cytometry.⁷ The growth medium consisted of Eagle’s minimal essential medium (EMEM) supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and G418 sulfate (0.555 mg/mL). Cells were maintained in a humidified atmosphere and sterile incubating conditions held at 37 °C and 5% CO₂(g). Fluorescence assays were performed using a BD FACSCalibur flow cytometer (BD Biosciences, San Jose, CA) in conjunction with the software packages BD Bioscience PlateManager and CellQuest. Fluorescence microscopy of the HEK293-mP2Y₁₄R cell line, after incubation of the cells with a fixed concentration of 36 (200 nM) for 45 min, in the presence and absence of competing nonfluorescent antagonist 1, validated the presence of functional receptors.³⁹

As in our previous studies,⁷ the growth medium for Chinese hamster ovary (CHO) cells stably expressing hP2Y₁₄R (CHO-hP2Y₁₄R) consisted of Dulbecco’s modified Eagle’s medium/Ham’s F12 (DMEM/F12) 1:1 supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and G418 sulfate (0.500 mg/mL). Cells were maintained in a humidified atmosphere and sterile incubating conditions held at 37 °C and 5% CO₂(g).

Competitive Binding Assay of P2Y₁₄R.

All competitive fluorescence assays were performed using a BD FACSCalibur flow cytometer in conjunction with the software packages BD Bioscience PlateManager and CellQuest. The assay of antagonist binding to the hP2Y₁₄R expressed in CHO cells using flat-bottom 96-well plates was performed as described.⁹ For the binding assay at the mouse homologue, HEK293-mP2Y₁₄R cells were grown in a 24-well plate and used when 80–90% confluency was reached. Unlabeled ligands are stored as 5 mM stock solutions in dimethyl sulfoxide (DMSO). A serial dilution of each compound was prepared in EMEM supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and G418 sulfate (0.555 mg/mL) to generate solutions with different concentrations of unlabeled antagonist ranging from 1 nM to 100 μM. Cells were initially incubated with unlabeled antagonists (prepared as described) for 30 min at 37 °C and 5% CO₂(g). Cells were then incubated with 36 for 30 min at a final concentration of 20 nM. At the end of the incubation, cells were washed three times with 500 μL of sterile 1× DPBS minus Ca²⁺/Mg²⁺. HEK293-mP2Y₁₄R cells were then detached from the plate using Corning Cellstripper. Cells were resuspended in sterile 1× DPBS minus Ca²⁺/Mg²⁺ and transferred to a 96-well plate for flow cytometry. IC₅₀ values were determined from the gathered data using Prism 8.0 (GraphPad, San Diego, CA).

Competitive Binding Assay of CHO-hP2Y₁₄R Cells at Different pH Levels.

CHO-hP2Y₁₄R cells were grown to approximately 80–90% confluency prior to the assay in a flat-bottom 96-well plate in DMEM/F12 1:1 supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and G418 sulfate (0.500 mg/mL). HCl solution (1 N) was added dropwise to DMEM/F12 1:1 supplemented with 10% fetal bovine serum, penicillin (100 units/mL), streptomycin (100 μg/mL), and G418 sulfate (0.500 mg/mL) to generate cell media with pH values of 7.0, 6.5, and 6.0. These cell media with different pH levels were then used to perform serial dilution and prepare solutions with different concentrations of the tested antagonist ranging from 100 μM to 1 nM. CHO-hP2Y₁₄R cells were initially incubated with tested antagonists (prepared in different pH level cell media as described) for 30 min at 37 °C and 5% CO₂(g). Cells were then incubated with 36 for 30 min at a final concentration of 20 nM. The pH level of sterile 1× DPBS minus Ca²⁺/Mg²⁺ was also adjusted by adding 1 N HCl dropwise to prepare three different appropriate washing buffers for the cells with pH levels of 7.0, 6.5, and 6.0. After the incubation period, cells were washed three times with appropriate 1× DPBS minus Ca²⁺/Mg²⁺. Cells were then detached from the plate using Corning Cellstripper and were resuspended in 1× DPBS minus Ca²⁺/Mg²⁺ with an appropriate pH level for flow cytometry. IC₅₀ values were determined from the gathered data using Prism 8.0 (GraphPad, San Diego, CA).

Animal Model of Asthma and Chronic Neuropathic Pain.

Selected antagonists were examined in the ovalbumin/Aspergillus protease model of murine asthma.^36,38 C57BL/6J wild-type mice from the Jackson Laboratory (Bar Harbor, ME) were sensitized twice, week apart, by oropharyngeal instillation (op) of 50 μg of LPS-free OVA (Worthington Biomedical, CA) and 20 μg of protease from Aspergillus oryzae (Sigma-Aldrich, St. Louis, MO) in a total volume of 50 μL of PBS. On day 14, mice were injected intraperitoneally with tested compounds at 10 mg/kg or with 10 mL/kg vehicle (10% DMSO, 30% PEG-400, 60% water); 30 min later, animals were subjected to inhalation of 1% OVA aerosol in PBS for 1 h. BALF cells were collected and counted 48 h after OVA challenge. These animal studies were reviewed and approved by the Institutional Animal Care and Use Committee at the National Institute of Environmental Health Sciences, Research Triangle Park, NC.

Behavioral testing of mechano-allodynia was measured as described¹² in the CCI model using Hsd:ICR (CD-1) outbred adult male (20–30 g, Envigo, Indianapolis, IN).¹² All procedures were conducted in accordance with the International Association for the Study of Pain, the National Institutes of Health guidelines on laboratory animal welfare guidelines, and the approval of Saint Louis University Institutional Animal Care and Use Committee.

Computational Methods.

Molecular Modeling. Protein Preparation.

We used a previously published⁸ hP2Y₁₄R homology model, which was built using an agonist-bound X-ray structure (PDB ID: 4PXZ¹⁷) of hP2Y₁₂R as the template and subsequently refined through MD simulation. The Protein Preparation Wizard tool⁴⁰ of the Schrödinger suite (Maestro 2019–1)⁴¹ was used to assign the histidine protonation and tautomeric states, with His184, His217, and His280 protonated at N^δ nitrogen (named HSD according to the CHARMM nomenclature) and with His264 doubly protonated (HSP).

Molecular Docking.

The compounds were drawn using the Schrödinger suite (Maestro 2019–1)⁴¹ and minimized using the OPLS3 force field.⁴² The molecules were docked to the hP2Y₁₄R homology model with Glide SP⁴³ on a grid centered on the previously published pose of 1, with an inner box of 10 Å and an outer box of 30 Å. Maximum 20 poses were generated for each compound. In the case of compound 17, a post-docking minimization of the ligand was included in the docking protocol. Successively, a pose was selected for each compound by visual inspection: the capability to maintain interactions with Lys77^2.60, Tyr102^3.33, and Lys277^7.35, in analogy with the published 1 pose, was taken into consideration.

Molecular Dynamics.

The complex between hP2Y₁₄R and compound 17 was prepared for MD simulations using the HTMD⁴⁴ module. A 90 Å × 90 Å 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) lipid bilayer was generated through the VMD membrane plugin,⁴⁵ and the system was inserted into the membrane according to the orientation provided by the Positioning of Proteins in Membrane (PPM)⁴⁶ web server. TIP3P⁴⁷ water molecules (with a positive and negative padding of 15 Å on the z axis) and Na⁺/Cl⁻ counterions (0.154 M) were added to the system, reaching charge neutrality.

The simulations were run employing ACEMD⁴⁸ as a molecular dynamics engine and CHARMM36^49,50 force field for protein, lipids, water, and ions. The CGenFF^51,52 force field was used for the ligand, assigning the missing parameters by analogy through the ParamChem⁵³ web service.

The system was submitted to 5000 conjugate-gradient steps of minimization and then equilibrated for 40 ns of MD simulation in the NPT ensemble, applying positional harmonic restraints to ligand and protein atoms (0.8 kcal mol⁻¹ Å⁻² for ligand atoms, 0.85 kcal mol⁻¹ Å⁻² for Cα carbon atoms, and 0.4 kcal mol⁻¹ Å⁻² for the other protein atoms) that were linearly reduced after 20 ns. Five 50 ns replicates of MD simulations were run in the NVT ensemble, starting from the equilibrated system. A Berendsen barostat (relaxation time, 800 fs) was employed to maintain the pressure at around 1 atm during the equilibration, and a Langevin thermostat was used to keep the temperature at around 310 K (damping constants of 1 and 0.1 ps⁻¹ for equilibration and production, respectively). The timestep was set to 2 fs in all the simulations, and the M-SHAKE⁵⁴ algorithm was used to constrain bonds containing hydrogen atoms. The nonbonded interaction cutoff and the switching distance were set respectively to 9 and 7.5 Å. The particle mesh Ewald (PME)⁵⁵ method (1 Å grid spacing) was used to compute the long-range electrostatic interactions beyond the cutoff. All the simulations were run on NVIDIA Tesla P100 GPUs of the NIH HPC Biowulf cluster.

Trajectory Analysis.

The MD trajectories were analyzed with an inhouse Tcl script employing VMD 1.9.3.⁴⁵ The system was aligned to the initial conformation by superposing the protein Cα carbon atoms. The VMD HBonds plugin was used to estimate hydrogen bonds, using a donor–acceptor distance of 3.5 Å and a donor–H–acceptor angle of 30°. Ligand–protein electrostatic and van der Waals interactions were computed with NAMD.⁵⁶ The data were plotted using the Gnuplot (version 5.0) software.⁵⁷

ABBREVIATIONS

ADME

absorption, distribution, metabolism, and excretion

AF488

Alexa Fluor 488

CHO

Chinese hamster ovary

DCM

dichloromethane

DIPEA

diisopropylethylamine

DMEM

Dulbecco’s modified Eagle’s medium

DMF

N,N-dimethylformamide

DPPF

1,1′-bis(diphenylphosphino)ferrocene

EL

extracellular loop

HATU

1-[bis(dimethylamino)methylene]-1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxid hexafluorophosphate

IE

interaction energy

MD

molecular dynamics

PDSP

Psychoactive Drug Screening Program

PEG

polyethylene glycol

PPTN

4-(4-(piperidin-4-yl)-phenyl)-7-(4-(trifluoromethyl)-phenyl)-2-naphthoic acid

RMSD

root-mean-square deviation

SAR

structure–activity relationship

UDPG

uridine-5′-diphosphoglucose

TBAF

tetrabutyl ammonium fluoride

TEA

triethylamine

TFA

trifluoroacetic acid

THF

tetrahydrofuran

TM

transmembrane helix

TMS

trimethylsilyl