Exploration of Alternative Scaffolds for P2Y14Receptor Antagonists Containing a Biaryl Core

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Exploration of Alternative Scaﬀolds for P2Y₁₄Receptor Antagonists

Containing a Biaryl Core

Young-Hwan Jung,^⊥Jinha Yu,^⊥Zhiwei Wen, Veronica Salmaso, Tadeusz P. Karcz, Ngan B. Phung,

Zhoumou Chen, Sierra Duca, John M. Bennett, Steven Dudas, Daniela Salvemini, Zhan-Guo Gao,

Donald N. Cook, and Kenneth A. Jacobson *

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ABSTRACT: Various heteroaryl and bicyclo-aliphatic analogues

of zwitterionic biaryl P2Y₁₄receptor (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.

on P2Y₁₄R antagonists.¹⁴⁻¹⁶Examined across the entire P2YR

INTRODUCTION

■

family of eight G protein-coupled receptors (GPCRs), 1 was

The P2Y₁₄receptor (P2Y₁₄R, previously termed GPR105) is

characterized as a highly potent (K_i< 1 nM) and selective

activated by uridine-5′-diphosphoglucose (UDPG) and is

P2Y₁₄R antagonist in functional assays and in reduction of

expressed in human immune cells, including mast cells and

UDPG-induced chemotaxis of human neutrophils.^17,18How-

neutrophils.¹As with other P2 receptors that sense

ever, 1 showed poor aqueous solubility and bioavailability,

extracellular nucleotides as danger signals,²UDPG released

suggesting that a prodrug approach is needed.¹⁶

by stressed cells serves as a damage-associated molecular

Our approach to exploring P2Y₁₄R antagonist SAR is based

pattern (DAMP) to activate the innate immune system via the

on insights gained from modeling of the receptor binding.

P2Y₁₄R. Thus, modulation by ligands and regulation of the

Although lacking an X-ray structure, the P2Y₁₄R can be

P2Y₁₄R are considered a target for the treatment of

modeled on the closely related human (h) P2Y₁₂R structure

inﬂammation, diabetes, cardiac progenitor cell therapy, pain,

(45% sequence identity), within the Gi-coupled P2YR

and pulmonary diseases, such as cystic ﬁbrosis and asthma.¹⁻¹²

subfamily.^19,20We initially probed the piperidine moiety

In renal intercalated cells, P2Y₁₄R activation by endogenous

(yellow region of 1, Chart 1) in this series of naphthalenes

UDPG leads to neutrophil recruitment and elevated

and found it to have ﬂexibility of substitution of the secondary

inﬂammation, which is reduced by a P2Y₁₄R antagonist.^4,5

amine, which we proposed is directed toward the extracellular

Furthermore, in the eye, the P2Y₁₄R gene is expressed at the

space when receptor-bound. This secondary amine was

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

Received: May 3, 2020

The original non-nucleotide lead, 1 (4-[4-(4-piperidinyl)-

phenyl]-7-[4-(triﬂuoromethyl)phenyl]-2-naphthalenecarbox-

ylic acid, PPTN, Chart 1), in our structure−activity relation-

ship (SAR) studies of P2Y₁₄R antagonists appeared in a patent

and later publications by Black and co-workers and Belley et al.

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A

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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.

Chart 1. Structures of Two Keys, Previously Reported

P2Y₁₄R Antagonists Comparing Analogous Regions That

Were Structurally Modiﬁed in This Study

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.²⁵Simple alkyl or

acyl extensions of the piperidine nitrogen in the form of

tertiary amines or amides were reported by Kiselev et al.¹⁹with

signiﬁcant retention of P2Y₁₄R aﬃnity, and their SAR is

expanded here. Synthetic procedures for simple acyl derivatives

8 and 9 were recently reported.¹²Several of the alkyl-extended

analogues in Table 1A were tested previously using a

functional assay¹⁹and are re-assayed here using our standard

whole cell ﬂuorescence binding assay.

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 con-

strained conformation may be the receptor-preferred one. By

analogy, we previously applied the approach of conformation-

ally constraining a ring moiety, i.e., ribose, to increase the

potency and selectivity of purinergic nucleoside and nucleotide

ligands.²⁶The 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 solubility²⁷while 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 replace-

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,²¹we previously focused on replacing this

moiety of 1 with a bioisostere to improve the physicochemical

properties of the series.²²Thus, we introduced the biaryl

(phenyl-triazole) ring system (2, blue region) as a bioisosteric

substitute for naphthalene. In a subsequent P2Y₁₄R SAR

study,²³we 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.^22,23From 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.²³Thus, 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

B

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C

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Table 1B. hP2Y₁₄R Inhibitory Potency of Alternative Scaﬀolds Containing a Biaryl Core

a

IC₅₀values were determined by ﬂow cytometry of hP2Y₁₄R-CHO cells using ﬂuorescent antagonist tracer 36 and expressed as mean SEM (n =

3−5). IC₅₀values were from Junker et al.,²²Yu et al.,²³and Mufti et al.¹²No inhibition by the compound discerned at the highest concentration;

b

c

therefore, IC₅₀> 100 μM. cLogP calculated using ALOGPS 2.1 program (www.vcclab.org/lab/alogps/).²⁴

d

ment of the 4-triﬂuoromethyl-phenyl ring was introduced in

28, based on a published report that cubane can serve as a

benzene ring bioisostere.^28,29We 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.

D

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a

Scheme 1. (A−D) Synthesis of Piperidine-Modiﬁed Analogues of 1 and 2

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%.

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., 37.²³This 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.

The bromoaryl intermediates 96−101 for the synthesis of

bridged piperidine products 17−21 were prepared as described

in the Supporting Information (Scheme S1). Phenylquinucli-

dine 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

46,²³as 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-

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a

Scheme 2. Synthesis of Reverse Triazole Analogues of 2

a

Reagents 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-

BrPhCONH₂or 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).

(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, respec-

tively, 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.

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 carbox-

amide 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-BrPhCONH₂or 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 69,³⁰followed by a

Suzuki reaction with 4-aminocarbonylphenylboronic acid

pinacol ester 70, which was hydrolyzed to yield 29.

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-dihy-

dropyridine-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 synthe-

size 91 followed by deprotection to aﬀord 35.

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-

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Scheme 3. (A−D) Synthesis of Analogues of 2 in Which the Triazole Moiety Has Been Replaced with an Amide or Fused with

a

the Terminal 4-Triﬂuoromethylaryl Ring

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%.

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.

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.,²³by

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.^12,23Here, 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 IC₅₀value 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

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a

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

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%.

of the piperidine nitrogen were tolerated. The (PEG)₆amide-

linked conjugates 11−14 were intermediate in binding aﬃnity

(IC₅₀≈ 1−3 μM), although at least 160-fold weaker than the

parent compound 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 IC₅₀values > 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 R¹substituent. Nevertheless, the aﬃnities

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 IC₅₀value 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, respec-

tively, 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.

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a

Scheme 5. Synthesis of [³H]1

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

a

compound

mP2Y₁₄R, IC₅₀(nM)

hP2Y₁₄R, IC₅₀(nM)

1

2

4

7a

8

12

17

18

19

21

22

23

25

21.6 7.0

142 58

499 57

130 30

29.7 9.3

487 130

21.4 7.9

845 150

1160 330

56.3 19.0

902 344

384 88

246 63

6.0 0.1

31.7 8.0

233 26

76.3 24.4

27.6 4.3

963 417

20.0 4.4

105 10

359 69

38.3 2.2

269 121

169 42

644 175

a

IC₅₀values 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.

and organic phases. There was no correlation between the

cLogP and IC₅₀values (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.³²The

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.

a

3

Reagents and conditions: (a) Pd/C, H₂, EtOAc, rt, 100 psi; (b)

TFA/THF = 2:1, rt, 0.5 h; (c) KOH, MeOH, H₂O, 50 °C, 5 h.

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 IC₅₀value 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.¹²The 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.¹²The

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 IC₅₀values 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.

The pH dependence of hP2Y₁₄R aﬃnity was examined for

two potent derivatives, 1 and 17. These antagonists showed

only a minor increase in IC₅₀values 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

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

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.^8,17Oﬀ-target non-P2YR

interactions of selected antagonists (4, 6b, 7a, 8, 12, 17−19,

and 22−26) were determined at 46 receptors, transporters,

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Table 3. pH Dependence of IC₅₀of Two Antagonists in

Inhibition of Fluorescent Antagonist 36 Binding at

hP2Y₁₄R-Expressing CHO Cells (n = 3, Measured in

Triplicate)

a

compound

pH

IC₅₀SEM (nM)

1

7.4

7.0

6.5

6.0

7.4

7.0

6.5

6.0

6.01 0.87

9.75 3.29

14.5 5.8

19.0 2.8

30.8 2.2

38.1 2.1

57.0 8.7

84.5 1.1

17

a

The 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.

Table 4. Solubility and Relative Lipophilicity Determined

for Selected P2Y₁₄R Antagonists

a

solubility (μg/mL)

lipophilicity (Δk), relative to

b

compound

pH 4.0

pH 7.4

1

8

12

17

18

21

1.10 0.07

0.09 0.02

8.02 1.01

1.50 0.04

2.87 0.10

2.24 0.19

0.36 0.03

2.32 0.18

7.16 1.16

0.55 0.02

1.10 0.09

1.72 0.07

0

−0.48 0.01

0.71 0.03

1.24 0.78

−0.09 0.13

0.24 0.03

a

Determined using the pION method at the pH indicated

b

(Supporting Information). Determined from the RP-HPLC

retention time (C18 column), with a 20 min gradient of 10 to

100% acetonitrile in 10 mM triethylammonium acetate.

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),³⁴was the lack of appreciable binding at other sites

in the PDSP screen.

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 IC₅₀values 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.

ADME-toxicity properties were predicted for compounds

7a, 8, and 25 using online resources.^35,36None 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_1/2values

of compounds 17 and 18 were 5.1 and 4.6 h, respectively (1

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_ivalues of

6.79 and 2.75 μM, respectively, and compounds 2, 6b, and 7a

had no detected oﬀ-target interactions.^8,9Several 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

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

0.54 (5HT_1DR), and 0.51 (TSPO); 17,

(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

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a

Table 5. In Vitro and In Vivo ADME-Tox Data for Two Representative P2Y₁₄R Antagonists

b

test

22

17

18

simulated intestinal ﬂuid (% remaining at 120 min)

simulated gastric ﬂuid (% remaining at 120 min)

CYP1A2 (IC₅₀, μM)

CYP2C9 (IC₅₀, μM)

CYP2C19 (IC₅₀, μM)

100

>30

94−100

145 (m), 108 (r), 145 (h)

0.166

0.20 (iv)

138

ND^f

99.7

74.2

8.69

>30

3.29

102

106

14.1

>30

9.94

CYP2D6 (IC₅₀, μM)

CYP3A4 (IC₅₀, μM)

plasma stability (three species) (% remaining at 120 min)

d

100 (h); 79.9 (r); 100 (m)

67.2 (m), 80.0 (r), >240 (h)

16.4

5.1 (1 mg/kg, ip); 3.46 (iv)

0.44 0.06

86.8 (h); 89.4 (r); 79.5 (m)

311 (m), 108 (r), 192 (h)

16.8

4.6 (1 mg/kg, ip); 0.72 (iv)

<0.1

microsomal stability(t_1/2, min)

hERG, IC₅₀(μM)

e

t_1/2(h), administration mode

c

aqueous solubility (pH 7.4, μg/mL)

aqueous solubility (pH 4.0, μg/mL)

4

c

14.3 0.5

14.4 0.3

a

b

c

d

Procedure is in the Supporting Information. Data determined by Yu et al.²³Mean SD, pION method. Species tested for plasma stability were

e

f

human, rat, and mouse; species as indicated for microsomal stability. By iv administration: 0.5 mg/kg dose. ND, not determined.

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).

mP2Y₁₄R aﬃnity in vitro, the diﬀerences in in vivo activity

probably arose from varying pharmacokinetic properties.

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.¹²Compound 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.

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.²²The

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,^22,23so it was

applicable to the current study.

Table 6. Plasma Protein Binding at 10 μM in Three Species,

a

Expressed as Unbound Percent

b

compound

human, % free

mouse, % free

rat, % free

12

21

23

25

0.48

1.13

0.00

1.25

0.29

0.94

0.83

1.33

0.00

a

Procedure is in the Supporting Information. Verapamil and warfarin

b

served as positive controls. Mean 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.³⁷The 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.¹¹A 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

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 conformation-

ally 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 ana-

logues.^22,23The carboxylate engaged in electrostatic and H-

bond interactions with the side chains of Lys77^2.60(using

Ballesteros−Weinstein convention for residue numbering³⁸)

and Lys277^7.35and H-bonding with the Tyr102^3.33phenolic

group. Compound 17 made interactions with Lys77^2.60and

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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.

Lys277^7.35, but an ideal H-bond with Tyr102^3.33was 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.

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 Lys77^2.60and/or

Lys277^7.35were maintained during much of the simulation

time, with the addition of the previously observed H-bond with

Tyr102^3.33for >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

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 Arg253^6.55and 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,²²the piperidine of compound 1, simulated in the more

stable chair conformation, made a H-bond with Gly80^2.63and

deviated to interact with Ile170^EL2during MD simulation,

while the reverse behavior was observed for compound 2. The

involvement in binding: Asn90^3.21, Cys94^3.25, Ala98^3.29

,

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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^EL1and 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^EL2in 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.

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.¹²The 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.

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 Lys277^7.35was largely maintained, while the

Lys77^2.60H-bond was more persistent in replicate 3 and the

Tyr102^3.33H-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

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-

Figure 4. Docking pose of compound 17 at the hP2Y₁₄R putative binding site. The P2Y₁₂R crystal structure (PDB ID: 4PXZ)²⁰was 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.

M

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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 (triﬂuor-

omethyl 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 benzimida-

zole 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 phenyl-

piperidine 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.

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 IC₅₀value

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.³⁹Furthermore, amides and

1,2,3-triazole groups have been identiﬁed as bioisosteres, and

amides may have better water-solubilizing properties than the

triazoles.⁵⁸The “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.

DISCUSSION

■

In previous SAR studies of P2Y₁₄R antagonists, the aﬃnity of 1

was seldom surpassed.^19,22,23Attempts 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.²³In the SAR progression in our previous three

studies,^19,22,23there was a tendency toward lower aﬃnity at

the hP2Y₁₄R (IC₅₀value), 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 micro-

graphs show the presence of speciﬁc mP2Y₁₄R binding in the

HEK293 cells,¹²which were used to demonstrate that the

species diﬀerences in antagonist aﬃnity were relatively minor,

compared to species diﬀerences in other GPCRs, such as the

A₃adenosine receptor.¹³Furthermore, 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 Informa-

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 Tyr102^3.33and 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.

tion). The cAMP assay values were lower by 18

6-fold,

indicating that the ﬂuorescence binding consistently under-

estimates the intrinsic aﬃnity of these antagonists.

CONCLUSIONS

We have previously implicated the naphthalene moiety as a

source of the unfavorable physicochemical properties of the

potent, amphiphilic antagonist 1.⁸Nevertheless, naphthalene is

a component of many useful drug substances.²¹Therefore, 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

■

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 modiﬁca-

tions, such as reversal of a triazole moiety maintained only

N

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(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]⁺.

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.

4′-(Piperazin-1-yl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-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,

1

95% (R_t= 12.49 min); H 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]⁺.

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 CDCl₃and 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) measure-

ments 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.³⁶Simulated

pharmacokinetic parameters were calculated as reported.³

4-(4-(1-(Prop-2-yn-1-yl)piperidin-4-yl)phenyl)-7-(4-

(triﬂuoromethyl)phenyl)-2-naphthoic Acid (6a). Method A: yield,

1

52%; HPLC purity, 99% (R_t= 13.64 min); H 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)-

1

phenyl)-2-naphthoic Acid (6b). Method A: yield, 56%; H 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,

1

48%; HPLC purity, 100% (R_t= 13.71 min); H 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). Syn-

thetic procedures followed, and conﬁrmation of structural identity and

purity were recently reported.¹²

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 (dichloro-

methane/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,

1

88%; HPLC purity, 95% (R_t= 14.76 min); H NMR (400 MHz,

4-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-7-(4-

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

(triﬂuoromethyl)phenyl)-2-naphthoic Acid (10). Method A: yield,

1

71%; HPLC purity, 95% (R_t= 16.26 min); H NMR (400 MHz,

CD₃OD): δ 8.74 (s, 1H), 8.43 (s, 1H), 8.05−7.99 (m, 4H), 7.93 (d, J

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= 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). Meth-

od 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

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]⁺.

+

(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

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=

1

Acid (12). Method B: yield, 91%; H NMR (400 MHz, CD₃OD): δ

1

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]⁺.

11.78 min); H 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-Methylquinuclidin-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-tria-

zol-4-yl)-[1,1′-biphenyl]-3-carboxylic Acid (25). Synthetic proce-

dures followed, and conﬁrmation of structural identity and purity were

recently reported.¹²

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-naph-

thoic 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 (dichloro-

methane/methanol = 20:1) to aﬀord compound 13 (3.7 mg, 59%) as

1

a white solid; HPLC purity, 99% (R_t= 13.38 min); H 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-aza-

hexacosan-26-oyl)piperidin-4-yl)phenyl)-7-(4-(triﬂuoromethyl)-

phenyl)-2-naphthoic Acid (14). Method A: yield, 79%; HPLC purity,

3-(5-((3-Aminopropyl)carbamoyl)thiophen-2-yl)-5-(1-(4-

1

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

97% (R_t= 14.17 min); H NMR (400 MHz, CD₃OD): δ 8.71 (s,

1

Method B: yield, 45%; HPLC purity, 98% (R_t= 10.23 min); H

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):

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 C₂₄H₂₁N₅O₃F₃³²S, 516.1317; found, 516.1316 [M +

1]⁺.

+

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′-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′-(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),

P

https://dx.doi.org/10.1021/acs.jmedchem.0c00745

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

4′-Carbamoyl-5-((4-(triﬂuoromethyl)phenyl)carbamoyl)-[1,1′-bi-

phenyl]-3-carboxylic Acid (29). Method A: yield, 72%; HPLC purity,

99% (R_t= 11.30 min); H 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-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)-3,6-dihydro-

pyridine-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

1

4′-Carbamoyl-5-(5-(triﬂuoromethyl)-1H-indol-2-yl)-[1,1′-biphen-

yl]-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

1

aﬀord compound 38 (35 mg, 48%) as a white solid; H 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,

1

solid; HPLC purity, 96% (R_t= 11.75 min); (96%); H 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]⁺.

1

90%; H NMR (400 MHz, CDCl₃): δ 8.69 (s, 1H), 8.24 (s, 1H),

4′-Carbamoyl-5-(6-(triﬂuoromethyl)-1H-indol-2-yl)-[1,1′-biphen-

yl]-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]⁺.

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

1

compound 43a (5 mg, 55%) as a white solid; H 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]⁺.

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-car-

boxylic Acid (33). Method A: yield, 30%; HPLC purity, 99% (R_t=

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 N₂atmosphere.

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

10.35 min); H 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:

1

yield, 72%; HPLC purity, 99% (R_t= 10.37 min); H 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]⁺.

1

solid; H 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

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)-car-

boxylate (38). A mixture of compound 37 (60 mg, 0.121 mmol,

synthesized according to literature procedures reported), tert-butyl 4-

Q

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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; H 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]⁺.

1]⁺; ESI-HRMS calcd m/z for C₃₄H₃₃NO₃F₃, 560.2413; found,

560.2419 [M + 1]⁺.

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 chromatog-

raphy (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-(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%)

1

as a white solid; H 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-(2,5,8,11,14,17-Hexaoxaicosan-20-oyl)piperidin-4-

yl)phenyl)-7-(4-(triﬂuoromethyl)phenyl)-2-naphthoate (44b). Com-

pound 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

1

compound 44a; H 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]⁺.

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

1

43e (26 mg, 49%) as a white solid; H 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 +

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

1

solid; H 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−

R

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2.43 (m, 6H), 1.47 (t, J = 7.12 Hz, 3H); MS (ESI, m/z): 544.3 [M +

1]⁺.

1H), 1.11−1.06 (m, 1H), 0.91−0.86 (m, 1H); MS (ESI, m/z): 549.2

[M + 1-tert-butyl]⁺.

tert-Butyl 4-(3′-(Methoxycarbonyl)-5′-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-yl)piperazine-1-car-

boxylate (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

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

[1,1′-biphenyl]-3-carboxylic Acid (49d). Method A: yield, 94%; H

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-Tet-

ramethyl-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-tria-

zol-4-yl)-[1,1′-biphenyl]-4-yl)piperidine-1-carboxylate (59b). Syn-

thesis 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 dimethoxy-

ethane (2 mL) and 2 M Na₂CO₃aqueous 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

1

a similar procedure used in the preparation of compound 48a; H

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]⁺.

1

solid; H NMR (400 MHz, CDCl₃): δ 8.53−8.52 (m, 2H), 8.45 (s,

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

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]⁺.

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

procedure used in the preparation of compound 48a; H 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]⁺.

1

preparation of compound 59a; H 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-

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

1

a similar procedure used in the preparation of compound 48a; H

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]⁺.

1

carboxylic Acid (60b). Method A: yield, 60%; H 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).

Methyl 4′-(Piperazin-1-yl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

3-(5-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-

thiophen-2-yl)-5-(1-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-4-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (49a). Method B:

1

yield, 76%; H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.46 (s,

1

yl)benzoic Acid (60c). Method A: yield, 99%; H NMR (400 MHz,

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′-biphen-

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-amino-

benzoic 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.

1

yl]-3-carboxylic Acid (49c). Method A: yield, 76%; H 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,

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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%)

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

1

procedure used in the preparation of compound 64a; H NMR (400

as a yellow solid; H NMR (400 MHz, DMSO-d₆): δ 7.16 (dd, J =

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′-(piperi-

din-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]⁺.

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]⁺.

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

1

compound 67 (287 mg, 54%) as a white solid; H 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 NaHCO₃aqueous 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

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

1

63a; H 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 CHCl₃peak, 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]⁺.

1

aﬀord compound 68 (25 mg, 78%) as a white solid; H 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 4′-Carbamoyl-5-(4-(triﬂuoromethyl)benzamido)-[1,1′-bi-

phenyl]-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%)

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 triethyl-

amine (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 dichloro-

methane. 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

1

as a white solid; H 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]⁺.

1

aﬀord compound 69 (14 mg, 45%) as a white solid; H NMR (400

MHz, CDCl₃): δ 8.42 (s, 1H), 8.38 (s, 1H), 8.29 (s, 1H), 8.03 (s,

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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-(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-phenylenedi-

amine (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-((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

1

aﬀord compound 70 (9 mg, 63%) as a white solid; H 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-bro-

mobenzoate (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 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

1

compound 69 at 80 °C for 15 h; H 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]⁺.

Methyl 3-((2-Amino-4-(triﬂuoromethyl)phenyl)ethynyl)-5-bro-

mobenzoate (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

1

procedure used in the preparation of compound 71a; H NMR (400

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

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

1

compound 72a (13 mg, 65%) as a white solid; H 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

1

procedure used in the preparation of compound 72a; H 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]⁺.

1

beige solid; H 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

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[M + 1]⁺; ESI-HRMS calcd m/z for C₁₃H₁₄N₂O₂⁷⁹Br, 309.0239;

found, 309.0240 [M + 1]⁺.

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; H

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).

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)-car-

boxylate (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

1

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 =

1

30:1) to aﬀord compound 85 (66 mg, 93%) as a white solid; H

= 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]⁺.

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

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

1

aﬀord compound 80 (16 mg, 75%) as a beige solid; H 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]⁺.

= 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

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-dihydro-

pyridine-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

1

solid; H NMR (400 MHz, CDCl₃): δ 8.35 (s, 1H), 8.28 (s, 1H),

(16 mg, 77%) as a beige solid; H NMR (400 MHz, CDCl₃): δ 9.32

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]⁺.

(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]⁺.

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]⁺.

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-car-

boxylate (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

Methyl 4-Bromo-2-(4-(triﬂuoromethyl)phenyl)-1H-benzo[d]-

imidazole-6-carboxylate (90). To a solution of compound 89 (200

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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,

ﬂow cytometry.⁷The 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,⁷the 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 ﬂuores-

cence 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.⁹For 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. IC₅₀values were determined from the gathered

data using Prism 8.0 (GraphPad, San Diego, CA).

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:

1

yield, 82%; H 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]⁺.

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. IC₅₀values 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

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.³⁹The 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

W

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J. Med. Chem. XXXX, XXX, XXX−XXX

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Article

protease model of murine asthma.^36,38C57BL/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.

and 7.5 Å. The particle mesh Ewald (PME)⁵⁵method (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 in-

house Tcl script employing VMD 1.9.3.⁴⁵The 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.⁵⁶The data were plotted

using the Gnuplot (version 5.0) software.⁵⁷

Behavioral testing of mechano-allodynia was measured as

described¹²in the CCI model using Hsd:ICR (CD-1) outbred

adult male (20−30 g, Envigo, Indianapolis, IN).¹²All 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.

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c00745.

■

sı

Synthesis of additional compounds (4-bromophenyl

intermediates and carboxylate substitutions on the

central phenyl ring of 2); oﬀ-target binding inhibition

curves; pharmacological data; molecular modeling data,

including docking poses of selected compounds, number

of contacts between the receptor residues and the ligand

in each MD replicate, RMSD and ligand−receptor

interaction pattern during MD replicate 3, RMSD and

ligand-receptor interaction pattern during MD replicate

4, and analysis of MD replicates; predicted pharmaco-

kinetic parameters; predicted log D and log S; and

methods to measure solubility and lipohilicity (PDF)

NMR and mass spectra of ﬁnal compounds (PDF)

3D coordinates of hP2Y₁₄R in complex with 17,

obtained through molecular docking (PDB)

MD trajectory (replicate 3) of the complex between

compound 17 and hP2Y₁₄R, after alignment of the

receptor Cα atoms to the initial frame (MP4)

MD trajectory (replicate 4) of the complex between

compound 17 and hP2Y₁₄R, after alignment of the

receptor Cα atoms to the initial frame (MP4)

Computational Methods. Molecular Modeling. Protein Prep-

aration. We used a previously published⁸hP2Y₁₄R 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 tool⁴⁰of the

Schrodinger suite (Maestro 2019-1)⁴¹was used to assign the histidine

̈

protonation and tautomeric states, with His184, His217, and His280

protonated at N^δnitrogen (named HSD according to the CHARMM

nomenclature) and with His264 doubly protonated (HSP).

Molecular Docking. The compounds were drawn using the

41

̈

Schrodinger suite (Maestro 2019-1) and minimized using the

OPLS3 force ﬁeld.⁴²The molecules were docked to the hP2Y₁₄R

homology model with Glide SP⁴³on 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

HTMD⁴⁴module. A 90 Å × 90 Å 1-palmitoyl-2-oleoyl-sn-glycero-3-

phosphocholine (POPC) lipid bilayer was generated through the

VMD membrane plugin,⁴⁵and the system was inserted into the

membrane according to the orientation provided by the Positioning of

Proteins in Membrane (PPM)⁴⁶web server. TIP3P⁴⁷water 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.

Molecular formula strings of P2Y₁₄R antagonists (PDF)

AUTHOR INFORMATION

Corresponding Author

■

Kenneth A. Jacobson − Molecular Recognition Section,

Laboratory of Bioorganic Chemistry, National Institute of

Diabetes and Digestive and Kidney Diseases, National Institutes

of Health, Bethesda, Maryland 20892, United States;

orcid.org/0000-0001-8104-1493; Email: kennethj@

niddk.nih.gov

The simulations were run employing ACEMD⁴⁸as a molecular

dynamics engine and CHARMM36^49,50force ﬁeld for protein, lipids,

water, and ions. The CGenFF^51,52force ﬁeld was used for the ligand,

assigning the missing parameters by analogy through the Para-

mChem⁵³web service.

Authors

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⁻¹Å⁻²for ligand atoms, 0.85 kcal mol⁻¹

Å⁻²for Cα carbon atoms, and 0.4 kcal mol⁻¹Å⁻²for 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-SHAKE⁵⁴algorithm was used

to constrain bonds containing hydrogen atoms. The nonbonded

interaction cutoﬀ and the switching distance were set respectively to 9

Young-Hwan Jung − Molecular Recognition Section, Laboratory

of Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Jinha Yu − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Zhiwei Wen − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States; orcid.org/0000-

0002-0821-1369

X

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Veronica Salmaso − Molecular Recognition Section, Laboratory

of Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

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-dimethylfor-

mamide; 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 hexaﬂuorophos-

phate; IE, interaction energy; MD, molecular dynamics;

PDSP, Psychoactive Drug Screening Program; PEG, poly-

ethylene 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 ammo-

nium ﬂuoride; TEA, triethylamine; TFA, triﬂuoroacetic acid;

THF, tetrahydrofuran; TM, transmembrane helix; TMS,

trimethylsilyl

Tadeusz P. Karcz − Immunity, Inﬂammation, and Disease

Laboratory, National Institute of Environmental Health

Sciences, National Institutes of Health, Durham, North

Carolina 27709, United States; Jagiellonian University, Krakow

31-007, Poland

́

Ngan B. Phung − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Zhoumou Chen − Department of Pharmacology and Physiology

and the Henry and Amelia Nasrallah Center for Neuroscience,

Saint Louis University School of Medicine, Saint Louis, Missouri

63104, United States

Sierra Duca − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

John M. Bennett − Molecular Recognition Section, Laboratory

of Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Steven Dudas − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Daniela Salvemini − Department of Pharmacology and

Physiology and the Henry and Amelia Nasrallah Center for

Neuroscience, Saint Louis University School of Medicine, Saint

Louis, Missouri 63104, United States

Zhan-Guo Gao − Molecular Recognition Section, Laboratory of

Bioorganic Chemistry, National Institute of Diabetes and

Digestive and Kidney Diseases, National Institutes of Health,

Bethesda, Maryland 20892, United States

Donald N. Cook − Immunity, Inﬂammation, and Disease

Laboratory, National Institute of Environmental Health

Sciences, National Institutes of Health, Durham, North

Carolina 27709, United States

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ACKNOWLEDGMENTS

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We acknowledge support from the NIDDK (ZIA DK031116)

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Exploration of Alternative Scaffolds for P2Y14Receptor Antagonists Containing a Biaryl Core

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