Structure-Activity Relationship of Heterocyclic P2Y14Receptor Antagonists: Removal of the Zwitterionic Character with Piperidine Bioisosteres

pubs.acs.org/jmc

Article

Structure−Activity Relationship of Heterocyclic P2Y₁₄Receptor

Antagonists: Removal of the Zwitterionic Character with Piperidine

Bioisosteres

Young-Hwan Jung, Veronica Salmaso, Zhiwei Wen, John M. Bennett, Ngan B. Phung,

David I. Lieberman, Varun Gopinatth, John C. R. Randle, Zhoumou Chen, Daniela Salvemini,

Tadeusz P. Karcz, Donald N. Cook, and Kenneth A. Jacobson *

Cite This: J. Med. Chem. 2021, 64, 5099 −5122

Read Online

sı

*

Metrics & More

Article Recommendations

Supporting Information

ACCESS

ABSTRACT: A known zwitterionic, heterocyclic P2Y₁₄R antago-

nist 3a was substituted with diverse groups on the central phenyl

and terminal piperidine moieties, following a computational

selection process. The most potent analogues contained an

uncharged piperidine bioisostere, prescreened in silico, while an

aza-scan (central phenyl ring) reduced P2Y₁₄R aﬃnity. Piperidine

amide 11, 3-aminopropynyl 19, and 5-(hydroxymethyl)isoxazol-3-

yl) 29 congeners in the triazole series maintained moderate

receptor aﬃnity. Adaption of 5-(hydroxymethyl)isoxazol-3-yl gave

the most potent naphthalene-containing (32; MRS4654; IC₅₀, 15

nM) and less active phenylamide-containing (33) scaﬀolds. Thus,

a zwitterion was nonessential for receptor binding, and molecular

docking and dynamics probed the hydroxymethylisoxazole

interaction with extracellular loops. Also, amidomethyl ester prodrugs were explored to reversibly block the conserved carboxylate

group to provide neutral analogues, which were cleavable by liver esterase, and in vivo eﬃcacy demonstrated. We have, in stages,

converted zwitterionic antagonists into neutral molecules designed to produce potent P2Y₁₄R antagonists for in vivo application.

(MD) simulation to the design of new antagonist ligands.^1,2,14

INTRODUCTION

■

The nucleotide agonist-bound P2Y₁₂R structure¹⁵serves as a

The P2Y₁₄receptor (P2Y₁₄R) is a target for the treatment of

inﬂammatory diseases, including pulmonary and renal

conditions, and selective P2Y₁₄R antagonists have demon-

suitable protein template for P2Y₁₄R homology, with 44%

sequence identity between P2Y₁₂R and P2Y₁₄R.^1,2,14Previous

studies have analyzed the structure−activity relationship

strated eﬃcacy in animal models of asthma, pain, diabetes, and

(SAR) of high aﬃnity antagonist 4-[4-(4-piperidinyl)phenyl]-

acute kidney injury.¹⁻⁸Its endogenous agonists are uridine-5′-

7-[4-(triﬂuoromethyl)phenyl]-2-naphthalene-carboxylic acid

diphosphoglucose (UDPG) and other UDP-sugars, and UDP

itself acts as a partial agonist. This G_iprotein-coupled receptor

(PPTN, 1a, Chart 1) and its analogues, e.g., potent N-acetyl

derivative 1b and triazole derivatives 3a, 4, and 5.^{1,2,7,11,14,16}

is one of three P2Y₁₂R-like receptors in purinergic signaling

Compound 1a is a highly selective antagonist for the P2Y₁₄R

pathways.⁹UDPG acts as damage-associated molecular pattern

among the family of eight P2YRs; this chemical series has no

(DAMP), and thus, competitive P2Y₁₄R antagonists have anti-

evidence of an interaction with other P2YRs or of residual

inﬂammatory eﬀects.³P2Y₁₄R antagonists are considered for

receptor agonism.^16,17Therefore, we consider this to be a

the development of new treatments of asthma and peripheral

viable, high aﬃnity scaﬀold for the design of new P2Y₁₄R

inﬂammation, although antagonist eﬀects in the CNS are not

antagonists. As a screening tool, we previously introduced a

well characterized and might be detrimental. For example,

high aﬃnity ﬂuorescent antagonist 2, which shows a functional

tonic P2Y₁₄R activation helps to maintain astrocytes by

preventing the secretion of matrix metalloproteinase-9.¹⁰

Received: January 28, 2021

Published: March 31, 2021

Various P2Y₁₄R antagonists have been reported, and the

most well explored class is derived from 7-phenyl-2-

naphthalenecarboxylic acid.^1,2,11−13CHANGE REFS. 11-13

to 11-14. Although there is no report yet of a P2Y₁₄R X-ray

crystallographic structure, we have applied a structure-guided

approach using homology modeling and molecular dynamics

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

5099

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Chart 1. Structure of Reported P2Y₁₄R Antagonist Ligands, Including High Aﬃnity Fluorescent Conjugate 2 Used to

a

Determine Receptor Aﬃnity in a Whole Cell Flow Cytometry Assay

a

One of the compounds shown, 3b, was synthesized and characterized in the present study.

K_iof 80 pM and serves as a versatile screening tool in a ﬂow

cytometry assay using Chinese hamster ovary (CHO) cells

overexpressing the human (h) P2Y₁₄R and human embryonic

kidney (HEK)293 cells overexpressing the mouse (m)

P2Y₁₄R.^2,7,16Consistently reproducible IC₅₀values can be

obtained for newly synthesized antagonists, although these

inhibitory constants underestimate the compounds’ aﬃnity by

at least one order of magnitude.²

prodrug derivatives showed eﬃcacy in a mouse asthma model

that surpassed the eﬃcacy of the parent drugs.

Another structural modiﬁcation of 1a found to lower its

hydrophobicity was the bioisosteric replacement of the

hydrophobic naphthalene moiety of 1a in the form of a

triazolyl-phenyl group, which was the starting point of our SAR

study.¹⁴This biaryl moiety is predicted to maintain a similar

conformation to the naphthalene when receptor-bound, as we

showed previously for moderately potent P2Y₁₄R antagonists

3a, 4, and 5. A further impediment to attaining drug-like

properties in this chemical series is the secondary amine in the

piperidine moiety. Finding an alternative terminal group in

place of piperidine, especially one that lacks a positively

charged group, would be desirable and has been attempted in

previous SAR reports.^1,2For example, compound 5 provides

moderate aﬃnity and enhanced aqueous solubility with a

simple 4-carboxamido group in place of piperidine. However,

this compound shows rapid clearance in vivo and unacceptable

hERG inhibition.²In this study, we have modiﬁed the

piperidine moiety and the central phenyl ring of the triazole

derivatives to search for alternatives that might be more drug-

like than reference zwitterionic compounds 1a and 3a.

Piperidine bioisosteres were screened by modeling the

proposed analogues’ interaction with a P2Y₁₄R model, and

the most aﬃnity enhancing group (5-(hydroxymethyl)isoxazol-

3-yl) was predicted as a high scoring hit. Subsequently, we

combined this neutral bioisostere of piperidine with the

naphthalene core. Thus, both structure-based and empirical

approaches were followed to probe the P2Y₁₄R SAR.

However, the prototypical antagonist 1a is not optimal for in

vivo administration as it shows a high cLogP and low oral

bioavailability (5%, mouse).¹⁸Its hydrophobicity precluded

the use of [³H]1a as a radioligand for P2Y₁₄R assays due to

high nonspeciﬁc binding.²Furthermore, 1a is a zwitterionic

ligand, which aﬀects assessment of its drug-likeness, lip-

ophilicity and absorption in vivo, although various zwitterionic

drugs are used therapeutically.¹⁹Previously, we introduced

alternative scaﬀolds to replace the phenyl-naphthalene moiety

and substituted the phenyl-piperidine moiety with extended

polar chains that reduced the cLogP but did not increase the

receptor aﬃnity of 1a and in some cases introduced other

liabilities, such as human ether-a-go-go-related gene (hERG)

inhibition.^1,2,14One possible solution to overcome the

deﬁciencies of 1a is described in a report on its prodrug

derivative in which the essential benzoic acid group was

protected as a neutral ester moiety.¹⁸Here, we applied the

same prodrug approach that was reported for the prodrug of

1a, by introducing a N,N-dimethylamidomethyl ester at the

essential aryl carboxylate group in three high aﬃnity P2Y₁₄R

antagonists. We demonstrated that the parent, active drug in

each case could be recovered upon treatment of the prodrug

form with puriﬁed liver esterase in vitro. Furthermore, several

5100

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Scheme 1. (A) Synthesis of Triazole-Containing Derivatives with Acyl or Acylamino Replacement of the Piperidine Ring of

a

3a

a

Reagents and conditions: (a) tert-butyl (4-bromophenyl)carbamate, Pd(PPh₃)₄, K₂CO₃, DMF, 85 °C, 5 h, and 40−68%; (b) TFA/THF = 2:1, rt,

0.5 h, and 57−87%; (c) KOH, MeOH/H₂O = 2:1, 50 °C, and 50−82%; (d) acetic anhydride, DCM, rt, 2 h, and 76%; (e) benzoyl chloride, TEA,

DCM, rt, 18 h, and 63%; and (f) boc-isonipecotic acid (Boc-Inp-OH), HATU, DIPEA, DMF, rt, 2 h, and 94%. (B) Synthesis of N-acyl

naphthalene-containing derivatives. reagents and conditions: 1c: (a) Acetic formic anhydride (from Ac₂O/formic acid = 2:1, v/v), 0 °C for 3 h →

room temperature for 12 h, and 71%. 1d: (b) Triﬂuoroacetic Anhydride and 92%.

were found to be maintained during MD simulation of

receptor−antagonist complexes.^1,14

RESULTS

■

Computational Selection of Substituent Groups and

Following our earlier SAR studies of heterocyclic P2Y₁₄R

antagonists,^1,2,14we turned our attention to the replacement of

the piperidine moiety of 3a initially and subsequently of 1a,

with diverse groups, including cyclic and acyclic and charged

and uncharged moieties. The selection of groups was based

both on the commercial availability of functionalized p-

substituted phenyl starting materials for the Suzuki coupling

(Schemes 1−3) and on computational predictions. We

Initial Chemical Synthesis. A previously reported hP2Y₁₄R

homology model was used to guide scaﬀold and functional

group modiﬁcation in this antagonist series, featuring a

conserved aryl carboxylate on the central ring of the scaﬀold

interacting with three polar side chains in TMs (trans-

membrane helices) 2, 3, and 7, speciﬁcally Lys77^2.60

Tyr102^3.33, and Lys277^7.35, respectively.^1,14These interactions

,

5101

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 2. Synthesis of Triazole-Containing Derivatives with Acyclic Replacement of the Piperidine Ring of 3a

a

Synthesis of intermediate 54 is in Scheme S1 (Supporting Information). Reagents and conditions: (a) Pd(PPh₃)₄, K₂CO₃, DMF, 85 °C, 5 h, and

27−55%; (b) PdCl₂(dppf), dimethoxyethane/Na₂CO₃(aq, 2 M) = 10:1, 60 °C, 4 h, and 73%; (c) 3-(4-bromophenyl)propanenitrile, Pd(PPh₃)₄,

K₂CO₃, DMF/H₂O = 1:1, 90 °C, 12 h, and 64%; (d) KOH, MeOH/H₂O = 2:1, 50 °C, and 39−88%; and (e) TFA/THF = 2:1, rt, 0.5 h, and 62−

98%.

initiated this study with a computational selection of favored p-

substituted phenyl derivatives to enable high aﬃnity receptor

binding, by varying group “R” in the triazole series related to

3a (Scheme 1). A hP2Y₁₄R homology model,¹⁴based on a

nucleotide-bound hP2Y₁₂R¹⁵structure (sequence identity,

44%) and reﬁned with induced ﬁt docking of high aﬃnity

antagonist 1a followed by MD simulations, was used for the

computational work. A computational screening based on

docking and MD simulation of analogues containing the

proposed R groups was employed to prioritize the candidate

synthesis.

The R groups were selected from a manually assembled list

of 20 commercially available bromo-aryl precursors for the

Suzuki reaction with intermediate 38 (Scheme 1), together

with ﬁve compounds that could be easily synthesized using

commercial precursors (compounds 8, 9, 11, 14, and 19). The

list of 24 screened compounds is reported in Figure S1

(Supporting Information). The azido R group in 93 was

discarded and not included in computation and subsequent

synthesis because of lack of MD parameters for this

substituent. The R groups were incorporated on the antagonist

scaﬀold computationally, the resulting compounds were

docked to the receptor structure, and the stability of the

receptor-bound complexes was assessed using MD. Three 30

ns MD replicates were run for each complex, and the average

results over the three replicates were compared to reference

compounds 1a and 3a. With the objective being to account for

both enduring geometric and energetic stabilities of the

complexes, we employed the weighted dynamic scoring

function²⁰(wDSF) method to evaluate the outcome of the

simulations. This method computes the cumulative ligand−

receptor energy during the MD simulation (here the sum of

electrostatic and van der Waals contributions), which is frame-

by-frame weighted by the RMSD of the ligand heavy atoms (eq

1)

The slope of the line ﬁtting the wDSF values versus the

number of frames was used to compare the complexes. This

scoring system evaluates the enduring strength of the

interaction, as previously reported,²⁰and a lower slope value

(higher absolute value) identiﬁes a more stable ligand pose.

We used the average slope of 3a over the three MD replicates

as cutoﬀ to prioritize the synthesis of our dataset of

compounds. In the case of compound 31, whose tetrazole

moiety might exist as two possible tautomers, both tautomers

were simulated, and the 1H-tetrazole was selected as the one

minimizing the wDSF/time slope. Thus, compounds 8, 11, 14,

18, 19, 22, 23, 25, 27, 29, and 31, showing a lower wDSF/time

slope as compared to 3a (Table S1, Supporting Information),

were selected as best candidates of the series and consequently

prioritized for synthesis and testing, followed then by all the

other compounds of the series (Table 1). Compound 6 with a

piperazine substituent, already synthesized and whose binding

aﬃnity was reported to be 233 nM,²showed a wDSF/time

slope lower than 3a. Two compounds reported in Figure S1

have not been synthesized: the synthesis of the hydroxy-

oxadiazole-bearing compound 91 was not successful, while

compound 92 with the 4-hydroxy-piperidine substituent,

selected for its low wDSF/time slope value, was not

synthesized because this moiety combined with the naphthoic

acid scaﬀold was previously reported to provide high aﬃnity.⁵⁸

The synthetic routes for R group substitution, either

according to modeling predictions for bioisosteres or with

other substitution, are shown in Schemes 1−Scheme 5 Among

the synthesized analogues in the triazole series, the terminal

group contained an aryl−CO or aryl−NH linker (7−14), a

halogen (15), a carbon-linked acyclic moiety (16−21), or a

carbon-linked cyclic moiety (22−31). Scheme 1 shows the

synthetic route to triazole analogues containing an aryl−CO or

aryl−NH linker (7−14). The Suzuki reaction of 38 with N-

(tert-butoxycarbonyl)-4-bromoaniline, 4-bromobenzoic acid,

or tert-butyl (3-(4-bromobenzamido)propyl)carbamate yielded

39, 44, and 45, respectively, followed by deprotection reactions

(removal of methyl ester and N-Boc protecting groups) to

n

∑

_t=0IE_L−R

wDSF =

(1)

RMSD

5102

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Table 1. Inhibition of Fluorescent Antagonist (2) Binding in hP2Y₁₄R-Expressing CHO Cells (X, Y, and Z = CH, Unless

Noted)

a

p-Phenyl substituted, unless noted. The structure of 3b is shown in Chart 1. IC₅₀values are mean

SEM for three to four separate

determinations, unless noted. Reported by Yu et al.,¹Jung et al.,²and Mufti et al.⁷cLogP calculated using the ALOGPS 2.1 program

b

c

(www.vcclab.org/lab/alogps/).⁵⁷

provide 7, 10, 13, and 14. The primary amine of compound 40

was N-acetylated (41), N-benzoylated (42), or coupled with

the piperidinyl group (43), and the products were then

deprotected to yield 8, 9, 11, and 12. Since the N-acetyl

derivative 1b was particularly potent in binding and in vivo

eﬃcacy,^2,7we prepared the corresponding N-formyl derivative

1c using reported formylation conditions using acetic formic

anhydride²¹and N-triﬂuoroacetyl derivative 1d.

5103

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 3. Synthesis of Triazole-Containing Derivatives with Cyclic Replacement of the Piperidine Ring of 3a

a

Reagents and conditions: (a) Pd(PPh₃)₄, K₂CO₃, DMF, 85 °C, 5 h, and 46−91%; (b) Pd(PPh₃)₄, K₂CO₃, DMF, μW, 100 °C, 0.5 h, and 80−91%;

(c) Pd(PPh₃)₄, K₂CO₃, DMF/H₂O = 1:1, 90 °C, 12 h, and 30−70%; (d) KOH, MeOH/H₂O = 2:1, 50 °C, and 43−74%; and (e) TFA/THF =

2:1, rt, 0.5 h, and 52−90%.

The triazole series containing a halogen (15), a carbon-

linked acyclic moiety (16−21), or a carbon-linked cyclic

moiety (22−31) was synthesized, as shown in Schemes 2 and

3, to provide 15−31. The Suzuki reaction of 38 with various

aryl groups containing a halogen (47), a carbon-linked acyclic

moiety (17 and 48−51), or a carbon-linked cyclic moiety

(55−63) followed by deprotection reactions provided 15, 16,

18−21, and 22−31.

contact with 15 residues for more than 95% of the time in all

three replicates (Figure S2C). In addition to Lys77^2.60

,

Lys277^7.35, and Tyr102^3.33, the central scaﬀold was surrounded

by Asp81^2.64, Val93^3.24, Ala98^3.29, Val99^3.30, Cys172^EL2, and

Phe191^5.43. More externally, Gly80^2.63, Gly83^2.66, Leu84^EL1

,

Gly85^EL1, Asn90^3.21, and Ile170^EL2surrounded the 5-

(hydroxymethyl)isoxazol-3-yl substituent of 29. This particular

R group appeared involved in a H bond with Asn90^3.21in the

docking pose, which was transient during the MD simulations.

During the three MD replicates, the 5-(hydroxymethyl)-

isoxazol-3-yl group transiently interacted with residues in

EL1 and EL2 or on the extracellular tip of TMs, making H

bonds especially with Gly85^EL1, Asn90^3.21, and Ile170^EL2

(Figure S4A,C,E for the three replicates and Video S1,

Supporting Information). The capability of the hydroxymethyl

moiety to stabilize the ligand through H bonds supports the

binding aﬃnity improvement of compound 29 (5-

(hydroxymethyl)isoxazole) as compared to compound 28

(plain isoxazole), which was not included in the list of

prioritized compounds (wDSF/time slope lower than com-

pound 3a, Table S1).

Combination of Favored Groups: Extended SAR and

Modeling. The main success of the computational screening

and of the consequent synthetic eﬀort in the triazole series was

the identiﬁcation of a piperidine bioisostere in compound 29,

where the removal of the zwitterionic character was compatible

with relatively high P2Y₁₄R aﬃnity (IC₅₀, 296 nM). For this

reason, we focused on this compound and we report here its

hypothetical binding mode. The docking pose of compound

29 (Figure 1) resembles the one previously reported for its

piperidine-bearing analogue 3a.^1,14The carboxylate moiety is

involved in electrostatic and H bond interactions with

Lys77^2.60, Lys277^7.35, and Tyr102^3.33. Arg274^7.32, Arg253^6.55

,

and His184^5.36(using Ballesteros−Weinstein notation²²) are

compatible with π−charge and π−π interactions with the p-

CF₃-phenyl, triazolyl, and peripheral aryl substituents of the

central benzoic acid (Figure 1A). During 30 ns MD simulation,

the ligand appeared stable (Figure S2A, Supporting Informa-

tion), with an inter-replicate average RMSD of 2.23 Å (Table

S2, Supporting Information). The interaction energy ﬂuctuated

slightly around a plateau during the simulations (Figure S2B),

with an inter-replicate average value of −207 kcal mol⁻¹. The

H bond interactions between the ligand carboxylate moiety

and Lys77^2.60, Tyr102^3.33, and Lys277^7.35were endured during

the simulations, with an inter-replicate average persistence of

about 93, 88, and 77%, respectively. Compound 29 was in

The piperidine ring substitution with a 5-(hydroxymethyl)-

isoxazol-3-yl moiety was thus able to contribute to ligand

stabilization with the aforementioned interaction pattern.

Given the success of this biososteric group in the initial

triazole series, its receptor interactions were analyzed in the

context of naphthalene-containing (32) and phenyl-amide-

containing (33) scaﬀolds.^2,11The hypothetical binding mode

of compound 32 at the hP2Y₁₄R model (Figure 1B) resembles

the pose previously reported for 1a.¹⁴As previously discussed

for compound 29, the carboxylate of compound 32 makes H

bonds with Lys77^2.60, Lys277^7.35, and Tyr102^3.33that are

maintained on average during the three MD replicates for 77,

5104

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 4. (A) Synthesis of Naphthalene-Containing Isoxazole Derivative 32

a

Reagents and conditions: (a) (3-(4-bromophenyl)isoxazol-5-yl)methanol, Pd(PPh₃)₄, K₂CO₃, DMF, μW, 100 °C, 1.5 h, and 50% and (b) KOH,

MeOH/H₂O = 2:1, 50 °C, and 98%. (B) Synthesis of amide-containing isoxazole derivative 33. Reagents and conditions: (a) SOCl₂, MeOH, rt, 15

h, and 98%; (b) 4-(triﬂuoromethyl)benzoic acid, HATU, DIPEA, DMF, rt, 5 h, and 70%; (c) B₂pin₂, PdCl₂(dppf), KOAc, dioxane, 70 °C, 6 h, and

65%; (d) (3-(4-bromophenyl)isoxazol-5-yl)methanol, Pd(PPh₃)₄, K₂CO₃, DMF, 85 °C, 5 h, and 45%; and (e) KOH, MeOH, H₂O, 50 °C, 3 h, and

62%.

81, and 62% of the simulation time, respectively (Table S2 and

Figure S4B,D,F, Supporting Information). As compared to 29,

compound 32 appeared more geometrically stable during the

simulations, with small RMSD deviations from a plateau: the

inter-replicate average RMSD value was 2.07 Å, with

ﬂuctuations within a 1−3 Å range (Table S2 and Figure

S3A, Supporting Information). With an inter-replicate average

interaction energy of −211 kcal mol⁻¹and a wDSF/time slope

of −100.70 (Table S2, Supporting Information), compound 32

seemed encouraging, and in fact, its aﬃnity for hP2Y₁₄R turned

out to be 15 nM, as will be discussed below. In addition to

Lys77^2.60, Lys277^7.35, and Tyr102^3.33, compound 32 was

especially in replicates 1 and 2 (Figure S4B,D,F, Supporting

Information). The isoxazole oxygen and nitrogen atoms

transiently interacted with Cys172^EL2, bridging interactions

between EL1 and EL2 (Video S2, Supporting Information).

The same stability was not reached in the case of compound

33, where the 5-(hydroxymethyl)isoxazol-3-yl substituent was

combined to the phenyl-amide-containing scaﬀold. An average

inter-replicate RMSD of 4.53 Å was experienced by the ligand,

with huge deviations from the initial state especially in replicate

2 (Table S2 and Figure S5A,E, Supporting Information). The

H bond with Lys277^7.35was not conserved during the three

replicates, and the interaction with Lys77^2.60was lost in

replicate 2. The average ligand−receptor interaction energy

(−148 kcal mol⁻¹) signiﬁcantly worsened as compared to

compounds 29 and 32, with the 5-(hydroxymethyl)isoxazol-3-

yl substituent not making stable contacts with speciﬁc residues.

The amido group replacing half of the naphthalene scaﬀold

introduced higher ﬂexibility to the molecule, which rotated to

surrounded by residues Gly80^2.63, Asp81^2.64, Gly85^EL1

Asn90^3.21, Val93^3.24, Cys94^3.25, Ala98^3.29, Val99^3.30, Asn156^4.60

,

Cys172^EL2, His184^5.36, Ser187^5.39, Asn188^5.40, Phe191^5.43, and

Arg253^6.55for more than 95% of the simulation time. A stable

H bond was maintained between Gly85^EL1and the hydroxyl

group of the 5-(hydroxymethyl)isoxazol-3-yl substituent,

5105

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Scheme 5. Synthesis of Triazole-Containing Derivatives 34−36 for the Aza-Scan of the Central Phenyl Ring

a

Reagents and Conditions: (a) SOCl₂, MeOH, rt, 15 h, and 40%; (b) tert-butyl nitrite, azidotrimethylsilane, acetonitrile, rt, 1 h, and 37−65%; (c)

4-ethynyl-α,α,α-triﬂuorotoluene, CuSO₄·5H₂O, Na ascorbate, TBTA, tert-BuOH/H₂O = 1:1, rt, 45 min, and 36−69%; (d) tert-butyl 4-(4-(4,4,5,5-

tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl)piperidine-1-carboxylate, Pd(PPh₃)₄, K₂CO₃, DMF, 85 °C, 4 h, and 35−91%; (e) KOH, MeOH/H₂O

= 2:1, 50 °C, 5 h, and 68−91%; and (f) TFA/THF = 2:1, rt, 0.5 h, and 60−65%.

Figure 1. Docking pose of compound 29 (A), in green, and compound 32 (B), in orange, at the P2Y₁₄R structure (gray), obtained by MD-reﬁned

homology modeling using an agonist-bound P2Y₁₂R structure (PDB ID: 4PXZ³⁴) as the template.

interact mainly with Arg253^6.55in replicates 1 and 3, and with

His184^5.36in replicate 2, with a general deviation from the

initial state and the binding mode of compounds 29 and 32.

Scheme 4A shows the combination of the 5-

(hydroxymethyl)isoxazol-3-yl group of potent derivative 29

with the naphthalene ring of 1a to give compound 32. The

Suzuki reaction product of 64 with (3-(4-bromophenyl)-

isoxazol-5-yl)methanol was hydrolyzed to yield 32. A 5-

(hydroxymethyl)isoxazol-3-yl analogue 33 with an amide

group replacing the triazole ring was synthesized, as shown

in Scheme 4B. This follows our previous report and that of

Zhang et al. on amide incorporation.^2,11For synthesis of amide

analogue 33, the carboxylic acid of 65 was converted into a

methyl ester in 66, which then coupled with 4-

(triﬂuoromethyl)benzoic acid. Compound 67 reacted with

bis(pinacolato)diboron (B₂pin₂, 68) followed by the Suzuki

reaction to yield 69. Subsequently, 69 was hydrolyzed to aﬀord

33.

Subsequently, with the aim to further explore modiﬁcations

of the central scaﬀold in this antagonist series, we empirically

modiﬁed the central phenyl ring with nitrogen substitution

(“aza-scan”), leading to triazole derivatives 34−36 (Scheme 5),

which could be compared to 3a. Adding a ring nitrogen or

shifting its position within an aromatic ring is a known

approach to modulate the activity of bioactive molecules.^23,24

5106

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

The aza-scan of the central phenyl ring of 3a was

accomplished, as shown in Scheme 5. The carboxylic groups

of 69 and 70 were protected as methyl esters in 72 and 73.

The amine functionality of 71−73 was converted into an azide

in 74−76 followed by the click reaction with 4-ethynyl-α,α,α-

triﬂuorotoluene to yield 77−79. Subsequently, a Suzuki

reaction with tert-butyl 4-(4-(4,4,5,5-tetramethyl-1,3,2-dioxa-

borolan-2-yl)phenyl)piperidine-1-carboxylate yielded 80−82,

which then were deprotected to aﬀord 34−36, respectively.

Since many P2YR ligands contain anionic groups, we wanted

to test experimentally if a phosphate monoester could serve the

same role as the essential carboxylate of 3a.^2,16Therefore, we

synthesized phosphate ester 3b (Scheme S1, Supporting

Information) and determined its hP2Y₁₄R aﬃnity. The

synthetic route to phosphate derivative 3b began with LAH

reduction of the methyl ester of compound 88 to a primary

alcohol 89, which was phosphorylated with di-tert-butyl N,N-

diethylphosphoramidite to aﬀord 90. Subsequently, a depro-

tection reaction yielded 3b.

Pharmacological Characterization. The inhibition by

the newly synthesized analogues of binding of ﬂuorescent

tracer 2 was measured in hP2Y₁₄R-expressing CHO cells

(Table 1).^1,2The computational screening procedure adopted

enabled the discrimination of submicromolar ligands, with

some false negative and false positive exceptions. In particular,

13 out of 24 compounds showed a wDSF/time slope lower

than compound 3a (−70.55), in addition to 1b, compounds 8,

11, 14, 18, 19, 22, 23, 25, 27, 29, and 31 and the piperazine 6

and 4-hydroxy-piperidine 92 compounds (Table S1, Support-

ing Information). A threshold of 1 μM was used to deﬁne

positive compounds, and according to this metric, we were

able to identify 8 true positives out of the 13 selected

compounds: ligands 8, 11, 14, 18, 19, 29, and 31 and the

piperazine compound 6²had submicromolar aﬃnity. The

compound bearing the 4-hydroxy-piperidine substituent 92

was not part of this study, aimed to explore chemical groups

diﬀerent than piperidine. However, this modiﬁcation in the

naphthoic acid series was associated with high aﬃnity for the

chimpanzee P2Y₁₄R.⁵⁸Compounds 22, 23, 25, and 27 were

false positives (aﬃnity higher than 1 μM), while ligands 13 and

30, whose IC₅₀turned out to be <1 μM, were missed (false

negatives). This screening was not meant to accurately predict

a precise ranking of binding aﬃnities. Given the limitations of a

structure obtained by homology modeling, we did not expect a

perfect correlation between computational and experimental

results, but we exploited modeling to drive the synthesis of new

compounds, and we were able to identify seven new

submicromolar hP2Y₁₄R ligands.

derivative 4.¹Neutral functional group substitutions of the

phenyl ring, 15−17, showed only weak to intermediate aﬃnity.

Tetrazole, a common carboxylate bioisostere,¹in compound

31 (IC₅₀≈ 0.9 μM) maintained the intermediate aﬃnity of the

corresponding carboxylate 13.

Homologues of potent 18 and 29 (unsaturated chain in

equipotent 19 and isoxazole derivatives 28 and 30,

respectively) were compared. Hydroxybutyl derivative 21 and

small cycloalkyl-amino derivatives, 22−26, either Boc-

protected or unprotected, and oxetane 27 all bound weakly.

The hydroxymethylisoxazole derivative 29 showed higher

aﬃnity than its hydroxyethyl homologue 30, and the

unsubstituted isoxazole 28 was 7.5-fold weaker than 29. The

5-(hydroxymethyl)isoxazol-3-yl group was incorporated into

naphthalene-containing 32 and phenyl-amide-containing 33

scaﬀolds. Compound 32 proved to be antagonist of the highest

hP2Y₁₄R aﬃnity in the present set of compounds with an IC₅₀

of 15 nM. Thus, the restoration of the naphthalene scaﬀold in

32 increased the aﬃnity by 20-fold, but the phenyl-amide

scaﬀold reduced aﬃnity 25-fold compared to the triazole

series.

We turned our attention to the central phenyl ring’s SAR in

the triazole series. An aza-scan of this ring indicated reduced

P2Y₁₄R aﬃnity of compounds 34−36 in relation to parent 3a.

4-Phenylpicolinic acid derivative 34 had slightly greater aﬃnity

than 2-phenylisonicotinic 35 and 6-phenylpicolinic 36 acid

derivatives, but the aﬃnities were nevertheless in the μM

range. Thus, an aza-scan at any of the three CH positions failed

to enhance the aﬃnity (≥53-fold weaker) compared to 3a.

Eﬀorts to replace the important carboxylate group of 3a with a

diﬀerent anionic group, i.e., phosphate 3b, greatly reduced

aﬃnity. The measured aﬃnity of 3b was 1900-fold weaker

(IC₅₀, 60.5 6.9 μM) than 3a. Thus, this carboxylate group

that we propose to be buried deeply in the binding site

remained an essential requirement for high P2Y₁₄R aﬃnity.

It is important to maintain receptor aﬃnity across species, in

order to perform in vivo preclinical testing in small animals,

with the mouse being the most commonly used species in

initial evaluation.³⁻⁷Therefore, several of the new compounds

(11, 19, 29, and 32) were compared in aﬃnity at hP2Y₁₄R and

mP2Y₁₄R (Figure 2 and Table 2) using our reported

ﬂuorescence binding procedures.²Compound 29 had slightly

enhanced aﬃnity (IC₅₀, 184 nM) at the mP2Y₁₄R versus the

human homologue, while 11, 19, and 32 had nearly the same

aﬃnity in the two species.

The solubility (using the pION method)^2,25,26and lip-

ophilicity (based on the HPLC retention time)^2,27were

determined for the 5-(hydroxymethyl)isoxazol-3-yl com-

Compounds 1a, 3a, and 4−6 (Table 1) are included as

archival compounds for comparison. The N-triﬂuoroacetyl

derivative 1d in the naphthalene series had 8.7-fold lower

hP2Y₁₄R aﬃnity than the corresponding N-acetyl 1b, which

was similar to the corresponding N-formyl analogue 1c (IC₅₀≈

30 nM). Four aniline and simple acylated aniline derivatives in

the triazole series 7−10 had relatively weak aﬃnity (1−3 μM).

Two zwitterionic molecules, i.e., piperidine amide 11 and 3-

aminopropyl 18 derivatives, and anionic isoxazole 29 were

found to maintain moderate receptor aﬃnity with IC₅₀values

≤300 nM. The N-Boc protected forms of 11 and 18, i.e., 12

and 20, were >100-fold weaker in binding than the parent

amines. p-Carboxyphenyl derivative 13 and its amino-

propylamide 14 were equipotent (IC₅₀≈ 0.6 μM), with 14

having 3.5-fold lower aﬃnity than the corresponding thienyl

pounds 29 and 32. The aqueous solubility (mean

SEM)

of 29 was 3.72 0.87 μg mL⁻¹(7.34 μM) at pH 7.4 and 0.60

0.09 μg mL⁻¹at pH 4.0, and the compound showed a

hydrophilicity gain of −1.06 cLogP units compared to 1a. The

aqueous solubility values of 32 were 2.43

0.13 μg mL⁻¹

(4.96 μM, pH 7.4) and 0.14 0.02 μg mL⁻¹(pH 4.0). Potent

antagonist 32 showed a hydrophilicity gain of −0.80 cLogP

units compared to 1a. It is worth noting that the three

prodrugs 37a−37c (Table 3) were readily soluble in DMSO at

20 mg mL⁻¹as this solvent has been required for the in vivo

testing of this chemical series. Reference compound 1a could

not dissolve at a concentration of 20 mg mL⁻¹in DMSO and

required the addition of 1 N NaOH (1 equiv) in order to

dissolve in DMSO at a near maximal concentration of 5 mM

(2.38 mg mL⁻¹).

5107

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Oﬀ-target interactions of selected analogues (11, 13, 18, 16,

18, 19, 29−32, 37a, and 37c) were analyzed by radioligand

binding at 46 receptors and channels by the Psychoactive Drug

Screening Program (PDSP, Figure S6, Supporting Informa-

tion), performed as reported previously.²⁸Several compounds

in this chemical series previously were found to have oﬀ-target

activity at DOR (δ-opioid receptor, 2.75 μM, 1a).¹Weak oﬀ-

target interactions were detected, but not extensively (K_i, μM,

receptor): 11, 2.33 0.15 (σ₁R), 2.0 0.4 (DOR); 16, 0.931

0.388 (σ₂R), 2.81 0.18 (TSPO); 29, 2.65 0.74 (H₁R),

0.751 0.101 (TSPO), 6.9 1.9 (DOR); 31, 2.26 1.28

(σ₂R), 3.30 1.76 (DOR); and 32, 2.68 0.21 (σ₁R), 4.78

1.00 (σ₂R), 4.63 0.87 (TSPO). Compounds 6, 13, 18, and

19 did not show any oﬀ-target interactions in the standard

PDSP screen. N-Formyl 1c (% inhibition at 10 μM or K_i, μM:

5HT_1D, 81%; α_1B, 2.65; α_2A, 3.97; α_2B, 82%; D₁, 0.52; D₅, 1.44;

σ₁, 0.759; σ₂, 84%; H₄, 1.70) and previously reported⁷N-acetyl

1b (5HT_1D, 1.75 0.54; 5HT_1E, 3.91 0.35; 5HT_5A, 2.34

1.25; D₁, 1.39 0.12; α_1A, 3.14 0.68; α_2C, 4.31 0.82; β₃,

0.86 0.40; TSPO, 0.508 0.044) analogues showed some

oﬀ-target interactions. Prodrug 37a was promiscuous in oﬀ-

target binding, with 18 interactions found in the μM range,

while prodrug 37c only inhibited binding at σ₂R (0.59 μM).

Absorption, distribution, metabolism, excretion, and toxicol-

ogy (ADMET) properties were determined for compound 32

(Tables 4 and 5), by the same methods as in Jung et al.²

Moderate inhibition by 32 of CYP2C9 was observed (IC₅₀, 10

μM), but there was no inhibition of four other CYPs. The

compound was stable in simulated gastric and intestinal ﬂuids,

in plasma of three species, and in human and mouse liver

Figure 2. Inhibition of binding of ﬂuorescent antagonist 2 at hP2Y₁₄R

(A) and at mP2Y₁₄R (B) by four novel P2Y₁₄R antagonist analogues

(mean

triplicate).

SD, n = 3−4 independent determinations, performed in

Table 2. Species Dependence of Binding of Various P2Y₁₄R

Antagonists, as Determined in a Fluorescent Antagonist

c

Binding Assay in Whole Cells (n = 3−4)

a

compound

mP2Y₁₄R, IC₅₀(nM)

hP2Y₁₄R, IC₅₀(nM)

b

Table 4. In Vitro and In Vivo ADMET Data for Compound

32 Compared to Known P2Y₁₄R Antagonist 5

1b

3a

29.7 9.3

142 58

384 88

902 344

499 57

155 79

375 175

184 24

18.6 8.4

27.6 4.3

31.7 8.0

169 42

269 121

233 26

197 29

308 63

296 13

15.0 4.4

a

b

4

b

test

5

32

b

5

simulated intestinal ﬂuid

(t_1/2, min)

simulated gastric ﬂuid (t_1/2

min)

>240

161

b

6

11

19

29

32

,

CYP1A2 (IC₅₀, μM)

CYP2C9 (IC₅₀, μM)

CYP2C19 (IC₅₀, μM)

CYP2D6 (IC₅₀, μM)

CYP3A4 (IC₅₀, μM)

>30

10.4

>30

a

Fluorescence binding assays using 2 (20 nM, for a 45 min

incubation) as a tracer. The hP2Y₁₄R was stably expressed in CHO

b

cells, and the mP2Y₁₄R was stably expressed in HEK293 cells. Data

from Jung et al.²and Mufti et al.⁷See Table 1 for structures.

c

plasma stability (species)

(t_1/2, min)

>240 (h), >240 (r),

>240 (m)

>240 (r), >240 (m)

microsomal stability (t_1/2

,

145 (m), 108 (r), 145 >240 (h), 47.7 (r),

Table 3. Properties of Amidomethyl Ester Prodrugs 37a−

min)

(h)

>240 (m)

>30 (patch clamp)

8.47

a

37c

d

hERG, IC₅₀(μM)

0.166 (ﬂuorescent)

>30

compound

(molecular

charge, pH 7)

t_1/2(min,

HepG2 cell toxicity, IC₅₀

MW, TPSA

(Å²)

parent

drug

mean SEM) for

(μM)

b

c

e

cLogP

esterase cleavage

t_1/2(r)

0.20 (0.5 mg kg⁻¹

i.v.)

,

7.23 (1.0 mg kg⁻¹

i.p.)

,

37a (+1)

37b (uncharged) 574.6, 88.4

37c (uncharged) 602.7, 66.9

577.2, 86.6

4.59

5.65

6.27

3a

32

1b

20.2 1.9

>72 (32.6 0.9%)

>72 (27.2 0.3%)

f

d

aqueous solubility (pH 7.4, 138

4

7.63 1.93

μg mL⁻¹

aqueous solubility (pH 4.0, ND

μg mL⁻¹

)

d

f

g

0.14 0.02

a

b

Structures are shown in Scheme 6. cLogP calculated using the

)

ALOGPS 2.1 program (www.vcclab.org/lab/alogps/).⁵⁷At pH 7.3 in

the aqueous medium in the presence of PLE. See Experimental

Procedures and Supporting Information (Figures S7 −S9) for

conditions and graphical representation of results. The prodrugs are

stable under the same incubation conditions for 24 h in the absence of

c

a

Procedures are in the Supporting Information and in Yu et al.¹

Dosing formulation: for i.v., DMSO/20% 2-hydroxypropyl-β-cyclo-

detrin (Sigma-Aldrich, 10:90); for i.p., DMSO/Kolliphor EL (Sigma-

Aldrich)/PBS (15:15:70). Data determined by Yu et al.¹Species

b

c

d

PLE. Percent cleavage at 72 h in parentheses.

tested for plasma stability were human, rat, and mouse; species as

d

e

indicated for microsomal stability. Method noted in parentheses. By

administration in rat (plasma half-life, h). See Table 5 for complete

f

g

results for 32. Mean SD, pION method. ND, not determined.

5108

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

a

Table 5. Pharmacokinetic Parameters for Compound 32 Administered by Intravenous and Intraperitoneal Routes

dose

C_max

T_max

(h)

AUC_0‑last

AUC_0‑∞

MRT_last

(h)

V_d

K_el

F

(%)

Cl

(mg kg⁻¹

)

(ng mL⁻¹

)

(h ng mL⁻¹

)

(h ng mL⁻¹

)

t_1/2(h)

(mL kg⁻¹

)

(1 h⁻¹

)

(mL h⁻¹kg⁻¹

)

0.5, i.v.

1.0, i.p.

3.0, i.p.

10, i.p.

359

88.4

315

786

0.083

2.0

234

650

2060

4900

238

700

2120

4920

0.689

7.23

4.68

2.98

0.703

6.24

5.68

5.02

2090

14,900

9570

1.01

100

139

147

105

2110

1430

1420

2030

0.096

0.148

0.233

4.0

8730

a

In healthy adult male Wistar rats; with 3 per group. Dosing formulation: for i.v., DMSO/20% 2-hydroxypropyl-β-cyclodetrin (Sigma-Aldrich,

10:90); for i.p., DMSO/Kolliphor EL (Sigma-Aldrich)/PBS (15:15:70). Abbreviations: C_max, maximal concentration; T_max, time at which maximal

concentration observed; AUC₀-_last, area under the plasma drug concentration−time curve up to the last quantiﬁable time point; AUC₀-_∞, area

under the plasma drug concentration−time curve to inﬁnite time; T_1/2, terminal half-life; MRT_last, mean residence time; Vd, volume of distribution;

Kel, elimination rate constant; F, biovailability; and Cl, total body clearance.

a

Scheme 6. Synthesis of Prodrug Derivatives 37a−37c

a

Reagents and conditions: (a) 2-chloro-N,N-dimethylacetamide, K₂CO₃, DMF, 45 °C, 1 h, and 70−72%; (b) TFA/THF = 2:1, rt, 0.5 h, and 78%;

and (c) 2-chloro-N,N-dimethylacetamide, Cs₂CO₃, DMF, 45 °C, 1 h, and 68−72%.

microsomes. However, the half-life in rat microsomes was 48

min. The rat and mouse plasma protein binding of 10 μM 32

was measured as ∼100%. High plasma protein binding has

been a recurrent disadvantage of this series of P2Y₁₄R

antagonists.^1,223In vivo pharmacokinetics in the rat (Table 5

and Figure S10, Supporting Information) indicated a high i.p.

bioavailabilty, with a much longer half-life than the reference

carboxamide 5, and linear systemic exposure with increasing

dose. We did not determine oral biovailability of 32 because of

the reported low %F of 1a and 5 in this chemical series.^1,18The

peak concentration of 32 (10 mg kg⁻¹, i.p., 4 h post-injection)

was 786 ng mL⁻¹(1.61 μM). The clinical signs for all rat

groups at all time points (up to 24 h) were normal.

Carboxamide analogue 5 was previously shown to potently

inhibit hERG (Table 4). hERG inhibition was determined by

two methods (ﬂuorescence and electrophysiological)^29,30as

have been done for other P2Y₁₄R antagonists.²The

ﬂuorescence assay utilized a commercial hERG binding

ﬂuorescence polarization assay with a tetramethyl-6-carbox-

yrhodamine (TAMRA) ﬂuorophore.³⁰This ﬂuorescence

method indicated an IC₅₀of >30 μM for the novel antagonist

32. Also, compounds 1a, 1b, and 32 were measured using a

patch clamp assay with CHO cells expressing the full length

hERG protein, by measuring the block of hERG outward

current amplitude (Supporting Information).²⁹By this electro-

physiological method, compounds 1a and 32 did not inhibit

hERG (IC₅₀or EC₅₀, >30 μM), while compound 1b showed a

slight activation at high concentrations (EC₅₀, 16.0 μM). Thus,

an improvement in the hERG liability was demonstrated for

the bioisostere-containing compound 32.

Prodrug Synthesis and Cleavage by Liver Esterases.

Following the lead of Robichaud et al.¹⁸with the report of a

N,N-dimethylamidomethyl ester prodrug of potent zwitter-

ionic antagonist 1a, prodrug derivatives of novel, potent

P2Y₁₄R antagonists were synthesized. Fully uncharged prodrug

derivative of the 5-(hydroxymethyl)isoxazol-3-yl derivative 32,

the potent bioisostere of the piperidine moiety, was

synthesized in the naphthalene series (37b). Also, the prodrug

37c derived similarly from N-acetyl derivative 1b is fully

uncharged. For comparison, we also applied the same prodrug

derivatization to reference triazole derivative 3a to provide the

cationic molecule 37a. Scheme 6 shows the preparation of

prodrugs (37a− 37c) having a N,N-dimethylamidomethyl

ester as a promoiety. The Boc-protected piperidine 86, 5-

(hydroxymethyl)isoxazol-3-yl derivative 32, and N-acetyl

derivative 1b were utilized for esterifying the free carboxylic

5109

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

acid using 2-chloro-N,N-dimethylacetamide in the presence of

K₂CO₃or Cs₂CO₃as the base to provide 87, 37b, and 37c,

respectively. Subsequently, the deprotection reaction of the

Boc group of 87 yielded 37a.

reduction. Other immune cells showed no signiﬁcant eﬀect,

except the lymphocyte count following administration of

prodrug 37a trended higher than in a vehicle control.

DISCUSSION

As an in vitro model system for studying the relative

cleavage rates of the ester prodrugs, we used porcine liver

esterase (PLE).³¹The prodrug derivatives 37a−37c were

shown to regenerate the corresponding parent drugs, 3a, 32,

and 1b, respectively, upon incubation with PLE (Table 3 and

Figures S7 −S9, Supporting Information). The triazole-

containing prodrug 37a was the most rapidly cleaved, while

the naphthalene-containing prodrugs 37b and 37c did not

reach 50% cleavage after 72 h at 37 °C. All of the prodrugs

were stable for 24 h upon aqueous incubation in the absence of

PLE.

In Vivo Eﬃcacy of Novel P2Y₁₄R Antagonist 32. We

ﬁrst tested the potent 5-(hydroxymethyl)isoxazol-3-yl deriva-

tive 32 in a well-established mouse model of neuropathic pain

caused by chronic constriction of the sciatic nerve (CCI)³²at a

dose of 10 μmol kg⁻¹(4.9 mg kg⁻¹, i.p., Figure 3). Previously,

■

This study has been guided by insights from P2Y₁₄R molecular

modeling. The potent analogues 29 and 32 contained an

uncharged piperidine bioisostere, which was among the hits

selected using in silico prescreening. The hypothetical receptor

interactions of the favored 5-(hydroxymethyl)isoxazol-3-yl

moiety were analyzed by subsequent docking and MD

simulations. Although the piperidine-amide derivative 11 in

the triazole series showed comparable aﬃnity to 29, it was

poorly soluble in aqueous medium, and we did not prepare the

corresponding naphthalene derivative.

We have sought to increase the druglikeness³⁴of this

chemical series of potent P2Y₁₄R antagonists. Through SAR

exploration, we have, in stages, removed the zwitterionic

character of the widely used antagonist 1a. We have identiﬁed

the uncharged 5-(hydroxymethyl)isoxazol-3-yl substituent in

compounds 29 and 32 as a piperidine bioisostere in the

P2Y₁₄R binding site. The discovery of potent antagonist 32

having the preferred naphthalene scaﬀold and active in vivo in

a chronic pain model greatly improved the unacceptable hERG

activity of a previously reported non-amino containing

carboxamide 5, but it showed high plasma protein binding.

Another concern was that isoxazole rings have been reported

to be susceptible to ring opening biotransformations, although

many experimental drugs, even some approved drugs

(sulﬁsoxazole, risperidone, and leﬂunomide), contain this

moiety.^35,36For example, an isoxazole moiety was found to

be beneﬁcial in a series of antinociceptive σ₁R antagonists.³⁷

The high microsomal stability and in vivo rat pharmacokinetics

of compound 32 indicated that it is not rapidly metabolized.

Nevertheless, it will be useful to further study the in vivo

metabolism of 29 and 32.

Aﬃnity diﬀerences between two species, human and mouse,

are not a major concern for this compound class.²In the more

potent naphthalene series, compound 32 showed IC₅₀values

of 15 and 19 nM at the hP2Y₁₄R and mP2Y₁₄R, respectively,

which are similar to the aﬃnity of compound 1a. This

chemically neutral substituent allowed us to prepare a

completely neutral prodrug derivative 37b, which was shown

to be cleaved with liver esterase to regenerate the active drug

32. Although we determined the relative parent drug recovery

in all three cases after ester cleavage catalyzed by liver esterase,

this model system cannot be used to predict the cleavage

kinetics in vivo for a given route of administration. There are

multiple esterases present in the plasma, stomach, intestines,

and other organs, which would also be expected to catalyze this

cleavage. Interspecies diﬀerences would also apply to general-

izing the in vitro results to the in vivo biotransformation.³⁸

The activity of 32 in a mouse model of chronic neuropathic

pain establishes the ability of this bioisosteric approach to

discover P2Y₁₄R antagonists with substantial in vivo eﬃcacy.

However, it is to be noted that at the dose of 10 μmol kg⁻¹, the

reversal of mechano-allodynia was less prolonged or eﬃcacious

than with 1a or 1b.⁷

Figure 3. Reversal of CCI-induced mechano-allodynia in the mouse

induced by compound 32 (10 μmol kg⁻¹, i.p.). Mean SD is shown,

n = 3; ANOVA, *P < 0.05 vs day 0 and ^†P < 0.05 vs day 7 post-injury.

32 was dissolved in 5% DMSO in water as a vehicle, and a volume of

0.2 mL was injected.

we demonstrated that 1a, 1b, and 3a and other P2Y₁₄R

antagonists of this series were eﬀective in reducing chronic

pain in this model and reached full reversal in some cases

(Table S3, Supporting Information).^2,7A maximal 71.0

17.4% reversal of CCI-induced mechano-allodynia was

observed 1 h post-injection of 32, and the degree of protection

declined during the following 2 h and was not statistically

signiﬁcant at 3 or 5 h. No side eﬀects were evident at this dose.

Selected compounds were examined for their ability to

reduce eosinophils in the bronchoalveolar lavage ﬂuid (BALF)

in an ovalbumin/Aspergillus mouse asthma model.^2,17,33

Compounds 1a, 1b, 3a, and 32 (10 mg kg⁻¹) were

administered prior to allergen challenge at a dose of 20

μmol kg⁻¹, i.p. Airway inﬂammation was determined 2 days

post-challenge (Figure 4). Also, the corresponding prodrug

derivatives 37a−37c of triazole 3a, 5-(hydroxymethyl)-

isoxazole 32, and N-acetyl-piperidine 1b derivatives, respec-

tively, were administered at the same dose. Eosinophil counts

in the BALF were reduced following administration of

reference antagonist 1a or several of the prodrugs. In fact,

37b and 37c signiﬁcantly reduced eosinophils to a greater

extent than the parent antagonists, which showed no signiﬁcant

The lack of in vivo activity of the prodrug 37a in the

protease model of murine asthma might be a result of its

relatively rapid enzymatic cleavage to 3a, which itself was

inactive in the same in vivo assay. Thus, the prodrug 37a might

be largely cleaved prior to reaching the site of action. The

5110

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Figure 4. Eﬀects of P2Y₁₄R antagonists and their prodrugs, compared to reference antagonist 1a, during allergen challenge reduce eosinophilic

airway inﬂammation in the protease model of asthma. Eﬀect on BALF leukocytes of i.p. 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.

other prodrugs 37b and 37c were highly eﬃcacious in the

asthma assay and were more slowly cleaved to the active drugs

in the model esterase system. The correlation of slower ester

cleavage with the naphthalene scaﬀold suggests that these

esters might be more sterically hindered than the triazole 37a

in the esterase active site to attenuate hydrolysis. The higher in

vivo eﬃcacy might also be related to 37b and 37c both being

neutral molecules as the lack of a formal charge could promote

their distribution/diﬀusion to the site of action followed by

cleavage to the active parent drug. However, additional

pharmacokinetic studies of the prodrugs are needed in order

to point to a mechanism for the diﬀerential activities.

Thus, a zwitterion was nonessential for receptor binding,

which was probed through docking and MD simulation. Also, a

reported N,N-dimethylamidomethyl ester prodrug moiety was

used to reversibly block the conserved carboxylate group to

provide neutral analogues, which were proven cleavable by

liver esterase. We have, in stages, converted zwitterionic

antagonists into neutral molecules that are designed to produce

potent P2Y₁₄R antagonists for in vivo application. Among the

analogues derivatized as prodrugs are the novel and potent

compound 32 containing a neutral bioisostere of the piperidine

group present in reference antagonist 1a. The resulting

prodrug 37b and N-acetyl derivative 37c showed substantial

eﬃcacy in a protease mouse model of allergic asthma, which

surpassed the parent drugs’ eﬃcacy. Thus, by searching for

piperidine bioisosteres and applying a prodrug strategy, we

have, in stages, arrived at novel, tractable P2Y₁₄R antagonists

for in vivo studies. Thus, by using insights into the drug−

receptor interaction, we have expanded this heterocyclic class

of potent P2Y₁₄R antagonists, which hold promise for treating

a range of inﬂammatory diseases.

CONCLUSIONS

■

In a structure-guided approach, we systematically replaced the

piperidine and central phenyl groups of P2Y₁₄R antagonist 3a

with diverse groups. A computational selection process

provided a priority list for the initial synthetic foray. A suitable

bioisostere for the piperidine moiety was identiﬁed as the 5-

(hydroxymethyl)isoxazol-3-yl group, which was transferred

from the triazole-containing scaﬀold of 3a to the naphthalene

series to provide 32 with high aﬃnity at both hP2Y₁₄R and

mP2Y₁₄R. However, an empirical aza-scan of the central phenyl

ring of 3a was unsuccessful in maintaining aﬃnity.

EXPERIMENTAL PROCEDURES

■

Molecular Modeling. Molecular Docking. A previously pub-

lished model of hP2Y₁₄R¹⁴was employed in this study. The structure

5111

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

was obtained by homology modeling using an agonist-bound hP2Y₁₂R

CDCl₃and water (δ 3.30) for CD₃OD. NMR spectra were collected

with 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 an Agilent 1260 Inﬁnity

HPLC equipped with an Agilent Eclipse 5 μm XDB-C18 analytical

column (250 × 4.6 mm; Agilent Technologies Inc.; Palo Alto, CA).

Mobile phase: linear gradient solvent system, 10 mM triethylammo-

nium acetate/CH₃CN from 95:5 to 5:95 in 20 min or CH₃CN/0.1%

formic acid and 5% methanol in water from 30:70 to 55:45 in 20 min;

the ﬂow rate was 1.0 mL min⁻¹. 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 with 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 spectroscopic (HRMS) measurements were per-

formed 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. tPSA (total polar surface area) was calculated using

ChemDraw Professional (PerkinElmer, Boston, MA, v. 16.0). cLogP

was calculated as reported.⁵⁷The synthesis of 1b was recently

described.⁷

General Procedures: Deprotection Reactions. Method A. A

mixture of compound (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 (2:1) was stirred at room temperature. The solvent

was evaporated with toluene under reduced pressure. The residue was

puriﬁed by silica gel column chromatography (dichloromethane/

methanol = 95:5) or semipreparative HPLC (10 mM triethylammo-

nium acetate buﬀer/acetonitrile = 80:20 to 20:80 in 40 min) to aﬀord

the compound as a white solid.

General Procedures: Suzuki Reaction. Method C. The mixture of

compound 38 (1 equiv), Pd(PPh₃)₄(0.06 equiv), and potassium

carbonate (3 equiv) in N,N-dimethylformamide was purged with

nitrogen gas for 15 min, and then various aryl halides (1.2 equiv) were

added to the mixture. The mixture was stirred at 85 °C for 5 h and

then allowed to be cooled to room temperature. This mixture was

partitioned between 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

(MgSO₄or Na₂SO₄), ﬁltered, and evaporated under reduced pressure.

The residue was puriﬁed by silica gel column chromatography to

aﬀord the compound as a white solid.

Method D. The mixture of compound 38 (1 equiv) and aryl halide

(1.2 equiv) in dimethoxyethane/2 M Na₂CO₃aqueous solution

(10:1) was purged with nitrogen gas for 30 min, and then

PdCl₂(dppf) (0.1 equiv) was added to the mixture. The mixture

was stirred at 60 °C for 4 h. After cooling at room temperature, this

mixture was partitioned between 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 (MgSO₄or Na₂SO₄), ﬁltered, and evaporated under reduced

pressure. The residue was puriﬁed by silica gel column chromatog-

raphy to aﬀord the compound as a white solid.

X-ray structure (PDB ID: 4PXZ¹⁵) as a template and then reﬁned

through induced ﬁt docking of the antagonist 1a and MD simulations

39

̈

of the complex. The Schrodinger suite (Maestro 2020-1) was used

for general modeling operations, like protein preparation through the

Protein Preparation Wizard⁴⁰tool and drawing and minimization of

the compounds using the OPLS3 force ﬁeld.⁴¹All His residues were

protonated at the N^δnitrogen (designated HSD in the CHARMM

nomenclature).

Docking was performed with Glide SP,⁴²employing a grid centered

on the previously published pose of the antagonist 1a, with an inner

box of 10 Å and an outer box of 30 Å. Maximum 20 poses were

generated for each compound, and a post-docking minimization was

included. One pose was selected for each compound by visual

inspection, considering the presence of interactions with Lys77^2.60

,

Y102^3.33, and K277^7.35, similarly to the previously published 1a

binding mode.

Molecular Dynamics. The complexes between hP2Y₁₄R and each

compound were prepared for MD simulations using the HTMD⁴³

module and then inserted in a 90 Å × 90 Å 1-palmitoyl-2-oleoyl-sn-

glycero-3-phosphocholine (POPC) lipid bilayer generated through

the VMD Membrane Plugin,⁴⁴according to the orientation provided

by the Positioning of Proteins in Membrane (PPM)⁴⁵web server.

TIP3P⁴⁶water molecules were added to the systems (with a positive

and negative padding of 15 Å on the z axis), which were then

neutralized by Na⁺/Cl⁻counterions at a concentration of 0.154 M.

The MD simulations were carried out with ACEMD⁴⁷as a

molecular dynamics engine. The CHARMM36^48,49force ﬁeld was

employed for proteins, lipids, water, and ions and the CGenFF^50,51

force ﬁeld for the ligands, with assignment of missing parameters by

analogy through the ParamChem⁵²web service.

The systems were minimized (5000 conjugate gradient steps) and

then submitted to an equilibration stage of 40 ns 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⁻¹Å⁻²for Cα carbon atoms, and 0.4 kcal mol⁻¹Å⁻²for the other

protein atoms) that were linearly reduced after 20 ns. Three 30 ns

replicates of MD simulations were run in the NVT ensemble, starting

from the equilibrated system. A pressure around 1 atm was

maintained by a Berendsen barostat (relaxation time, 800 fs) during

the equilibration, and a temperature of about 310 K was kept by a

Langevin thermostat (damping constants of 1 and 0.1 ps⁻¹for

equilibration and production, respectively). The time step was set to 2

fs, and the hydrogen-containing bonds were constrained by the M-

SHAKE⁵³algorithm. A 9 Å nonbonded interaction cutoﬀ and a 7.5 Å

switching distance were employed, and long-range electrostatic

interactions beyond the cutoﬀ were computed with the particle

mesh Ewald (PME)⁵⁴method (1 Å grid spacing). All the simulations

were run on NVIDIA Tesla P100 GPUs of the NIH HPC Biowulf

cluster.

Molecular Dynamics Analysis. The MD trajectories were analyzed

with an in-house script written in Tcl, employing VMD 1.9.3.⁴⁴The

complexes were aligned to their initial conformation by superposition

of the protein Cα carbon atoms. H bonds were estimated using the

VMD Hbonds plugin, 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.⁵⁵The data

were plotted using gnuplot⁵⁶(version 5.0) software. The weighted

dynamic scoring function (wDSF) was computed with a python3

script, employing Pandas and SciPy for the linear ﬁtting of the points

and slope computation. Frames every 100 ps were taken into account

for the wDSF analysis.

Chemical Synthesis. Reagents and Instrumentation. All

reagents and solvents were purchased from Sigma-Aldrich (St.

Louis, MO), Enamine, Ltd. (Monmouth Jct., NJ; compound 69),

Alchem Pharmtech, Inc. (Monmouth Jct., NJ; compound 70), or

ChemScene, LLC (Monmouth Jct., NJ; compound 71) unless noted.

¹H NMR spectra were obtained with a Bruker 400 spectrometer using

CDCl₃, CD₃OD, and DMSO-d₆as solvents. Chemical shifts are

expressed in δ values (ppm) with tetramethylsilane (δ 0.00) for

Method E. A mixture of compound 38 (1 equiv) and aryl bromide

(1.2 equiv) in DMF/H₂O (10:1; 20 mM) was purged with nitrogen

gas for 30 min, and then Pd(PPh₃)₄(0.05 equiv) and Na₂CO₃(3

5112

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

equiv) were added to the mixture, which was stirred for 1 h at 40−120

°C. The aqueous layer was extracted with ethyl acetate twice, and

then the combined organic layer was washed with brine, dried

(MgSO₄or Na₂SO₄), ﬁltered, and evaporated under reduced pressure.

The residue was puriﬁed by silica gel column chromatography to

aﬀord the compound as white solids.

Method F. The mixture of compound 38 (1 equiv), Pd(PPh₃)₄

(0.06 equiv), potassium carbonate (3 equiv), and various aryl halides

(1.2 equiv) in N,N-dimethylformamide was purged with nitrogen gas

for 15 min. The mixture was stirred at 100 °C for 30−90 min in

microwave. After microwave irradiation, the mixture was allowed to

be cooled at room temperature. This mixture was partitioned between

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 (Na₂SO₄), ﬁltered,

and evaporated under reduced pressure. The residue was puriﬁed by

silica gel column chromatography to aﬀord the compound as a white

solid.

4-(4-(1-Formylpiperidin-4-yl)phenyl)-7-(4-(triﬂuoromethyl)-

phenyl)-2-naphthoic Acid (1c). To a solution of acetic formic

anhydride (1 mL from Ac₂O and formic acid = 2:1, v/v) was slowly

added compound 1a (10 mg, 0.019 mmol) at 0 °C. After complete

addition, cooling was continued for 3 h, and the mixture was then

stirred at rt for 12 h. The reaction mixture was evaporated under

reduced pressure. The residue was puriﬁed by silica gel column

chromatography (dichloromethane/methanol = 50:1) to aﬀord the

compound 1c (7.0 mg, 71%) as a white solid. HPLC purity: 98% (R_t

= 14.62 min). ¹H NMR (400 MHz, DMSO-d₆): δ 8.73 (s, 1H), 8.64

(s, 1H), 8.08−7.96 (m, 5H), 7.90−7.88 (m, 3H), 7.50−7.44 (m, 4H),

4.37−4.34 (m, 1H), 3.85−3.81 (m, 1H), 3.22−3.16 (m, 1H), 2.97−

2.90 (m, 1H), 2.77−2.70 (m, 1H), 1.96−1.89 (m, 2H), 1.68−1.47

(m, 2H). MS (ESI, m/z): 504.2 [M + 1]⁺. ESI-HRMS (m/z): calcd

[M + 1]⁺for C₃₀H₂₅NO₃F₃, 503.1787; found, 504.1792.

(ESI, m/z): 467.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₄H₁₈N₄O₃F₃, 467.1331; found, 467.1330.

4′-Benzamido-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-

1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (9). Method A. Yield: 73%.

HPLC purity: 99% (R_t= 9.48 min). ¹H NMR (400 MHz, DMSO-d₆):

δ 9.70 (s, 1H), 8.41 (s, 1H), 8.29 (broad s, 2H), 8.21 (d, J = 7.60 Hz,

2H), 8.00−7.97 (m, 4H), 7.90−7.83 (m, 4H), 7.62−7.54 (m, 3H).

MS (ESI, m/z): 529.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₂₉H₂₀N₄O₃F₃, 529.1488; found, 529.1489.

4′-((tert-Butoxycarbonyl)amino)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(10). Method A. Yield: 50%. HPLC purity: 99% (R_t= 10.36 min).

¹H NMR (400 MHz, CD₃OD): δ 9.25 (s, 1H), 8.48 (s, 1H), 8.37−

8.35 (m, 2H), 8.16 (d, J = 8.40 Hz, 2H), 7.78 (d, J = 8.00 Hz, 2H),

7.71 (d, J = 8.40 Hz, 2H), 7.58 (d, J = 8.40 Hz, 2H), 1.54 (s, 9H). MS

(ESI, m/z): 525.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₇H₂₄N₄O₄F₃, 525.1750; found, 525.1755.

4′-(Piperidine-4-carboxamido)-5-(4-(4-(triﬂuoromethyl)phenyl)-

1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (11). Meth-

od B. Yield: 87%. HPLC purity: 99% (R_t= 21.20 min). ¹H NMR (400

MHz, DMSO-d₆): δ 8.03−8.01 (m, 4H), 7.83 (s, 1H), 7.68 (d, J =

8.40 Hz, 2H), 7.30−7.25 (m, 5H), 2.70−2.68 (m, 2H), 2.23−2.20

(m, 2H), 2.00−1.93 (m, 1H), 1.44−1.27 (m, 4H). MS (ESI, m/z):

536.2 [M + Na]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₈H₂₅N₅O₃F₃, 536.1909; found, 536.1913.

4′-(1-(tert-Butoxycarbonyl)piperidine-4-carboxamido)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylic Acid (12). Method A. Yield: 82%. HPLC purity: 98% (R_t=

24.10 min). ¹H NMR (400 MHz, DMSO-d₆): δ 9.73 (s, 1H), 8.43 (s,

1H), 8.38 (s, 1H), 8.27 (s, 1H), 8.20 (d, J = 8.00 Hz, 2H), 7.90 (d, J =

8.40 Hz, 2H), 7.91−7.77 (m, 4H), 4.05−3.97 (m, 2H), 2.92−2.75

(m, 3H), 1.85−1.76 (m, 2H), 1.56−1.45 (m, 2H), 1.41 (s, 9H). MS

(ESI, m/z): 658.2 [M + Na]⁺. ESI-HRMS (m/z): [M + Na]⁺calcd

for C₃₃H₃₂N₅O₅F₃Na, 658.2253; found, 658.2256.

5-(4-(4-(Triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-bi-

4-(4-(1-(2,2,2-Triﬂuoroacetyl)piperidin-4-yl)phenyl)-7-(4-

(triﬂuoromethyl)phenyl)-2-naphthoic Acid (1d). To triﬂuoroacetic

anhydride (100 μL) was slowly added compound 1a (10 mg, 0.019

mmol) at 0 °C. After complete addition, cooling was continued for 1

h, and the mixture was then stirred at rt for 2 h. The reaction mixture

was evaporated under reduced pressure. The residue was puriﬁed by

silica gel column chromatography (dichloromethane/methanol =

50:1) to aﬀord compound 1d (10.0 mg, 92%) as a white solid. HPLC

phenyl]-3,4′-dicarboxylic Acid (13). Method A. Yield: 68%. HPLC

1

purity: 99% (R_t= 13.46 min). H NMR (400 MHz, DMSO-d₆): δ

9.77 (s, 1H), 8.56−8.53 (m, 2H), 8.36 (broad s, 1H), 8.20 (d, J =

8.00 Hz, 2H), 8.11 (d, J = 8.40 Hz, 2H), 8.00 (d, J = 8.40 Hz, 2H),

7.91 (d, J = 8.40 Hz, 2H). MS (ESI, m/z): 454.1 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₂₃H₁₅N₃O₄F₃, 454.1015; found,

454.1017.

4′-((3-Aminopropyl)carbamoyl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(14). Method B. Yield: 63%. HPLC purity: 97% (R_t= 12.68 min).

¹H NMR (400 MHz, DMSO-d₆): δ 9.73 (s, 1H), 8.55 (s, 1H), 8.51

(s, 1H), 8.37 (s, 1H), 8.23−8.20 (m, 4H), 8.00 (d, J = 8.40 Hz, 2H),

7.90 (d, J = 8.00 Hz, 2H), 3.51−3.46 (m, 2H), 3.00−2.99 (m, 2H),

1.93−1.90 (m, 2H). MS (ESI, m/z): 510.2 [M + 1]⁺. ESI-HRMS (m/

z): [M + 1]⁺calcd for C₂₆H₂₃N₅O₃F₃, 510.1753; found, 510.1754.

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

[1,1′-biphenyl]-3-carboxylic Acid (15). Method A. Yield: 77%. HPLC

purity: 99% (R_t= 12.93 min). ¹H NMR (400 MHz, CD₃OD): δ 9.17

(s, 1H), 8.43 (s, 1H), 8.35 (s, 1H), 8.25 (s, 1H), 8.16 (d, J = 8.00 Hz,

2H), 7.79 (d, J = 8.00 Hz, 2H), 7.72 (d, J = 8.00 Hz, 2H), 7.66 (d, J =

8.40 Hz, 2H). MS (ESI, m/z): 488.0, 490.0 [M + 1]⁺. ESI-HRMS

(m/z): [M + 1]⁺calcd for C₂₂H₁₄N₃O₂F₃Br, 488.0221; found,

488.0218.

4′-(2-Amino-2-oxoethyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (16). Method A.

Yield: 64%. HPLC purity: 99% (R_t= 8.55 min). ¹H NMR (400 MHz,

CD₃OD): δ 9.24 (s, 1H), 8.50 (s, 1H), 8.38 (s, 2H), 8.16 (d, J = 8.00

Hz, 2H), 7.79−7.74 (m, 4H), 7.48 (d, J = 7.60 Hz, 2H), 3.60 (s, 2H).

MS (ESI, m/z): 467.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₂₄H₁₈N₄O₃F₃, 467.1331; found, 467.1332.

1

purity: 99% (R_t= 16.92 min). H NMR (400 MHz, CDCl₃): δ 8.80

(s, 1H), 8.27 (s, 1H), 8.12−8.07 (m, 2H), 7.86−7.77 (m, 5H), 7.53

(d, J = 8.00 Hz, 2H), 7.39 (d, J = 8.00 Hz, 2H), 4.81−4.78 (m, 1H),

4.25−4.22 (m, 1H), 3.37−3.30 (m, 1H), 3.01−2.92 (m, 2H), 2.15−

2.11 (m, 2H), 1.91−1.81 (m, 2H). MS (ESI, m/z): 570.1 [M − 1]⁻.

ESI-HRMS (m/z): [M − 1]⁻calcd for C₃₁H₂₂NO₃F₆, 570.1504;

found, 570.1493.

(4′-(Piperidin-4-yl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tri-

azol-1-yl)-[1,1′-biphenyl]-3-yl)methyl Dihydrogen Phosphate (3b).

Method B. Yield: 69%. HPLC purity: 96% (R_t= 8.58 min). ¹H NMR

(400 MHz, DMSO-d₆): δ 9.20−9.19 (m, 1H), 8.05−8.03 (m, 2H),

7.96 (s, 1H), 7.85 (s, 1H), 7.79 (d, J = 8.00 Hz, 2H), 7.74 (s, 1H),

7.67 (d, J = 8.40 Hz, 2H), 7.34 (d, J = 7.60 Hz, 2H), 4.82−4.80 (m,

2H), 2.96−2.92 (m, 2H), 2.66−2.58 (m, 3H), 1.71−1.67 (m, 2H),

1.56−1.49 (m, 2H). MS (ESI, m/z): 559.2 [M + 1]⁺. ESI-HRMS (m/

z): [M + 1]⁺calcd for C₂₇H₂₇N₄O₄F₃P, 559.1722; found, 559.1716.

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

[1,1′-biphenyl]-3-carboxylic Acid (7). Method A. Yield: 62%. HPLC

1

purity: 97% (R_t= 8.96 min). H NMR (400 MHz, CD₃OD): δ 9.25

(s, 1H), 8.48 (s, 1H), 8.37 (s, 2H), 8.16 (d, J = 8.00 Hz, 2H), 7.79 (d,

J = 8.00 Hz, 2H), 7.73 (d, J = 8.40 Hz, 2H), 7.62 (d, J = 8.40 Hz, 2H).

MS (ESI, m/z): 425.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₂₂H₁₆N₄O₂F₃, 425.1225; found, 425.1221.

4′-Acetamido-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-

1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (8). Method A. Yield: 76%.

HPLC purity: 99% (R_t= 7.95 min). ¹H NMR (400 MHz, CD₃OD): δ

9.21 (s, 1H), 8.46 (s, 1H), 8.34 (d, J = 7.20 Hz, 2H), 8.13 (d, J = 8.00

Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H), 7.72 (s, 4H), 2.15 (s, 3H). MS

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

zol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (17). The mixture of

compound 38 (30 mg, 0.063 mmol), Pd(PPh₃)₄(4 mg, 0.003

mmol), and 2 M K₂CO₃aqueous solution (90 μL, 0.180 mmol) in

N,N-dimethylformamide/water (1:1, 3 mL) was purged with nitrogen

gas for 15 min, and then 3-(4-bromophenyl)propionitrile (16 mg,

5113

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

0.076 mmol) was added to the mixture. The mixture was stirred at 90

°C for 12 h and then allowed to be cooled at room temperature. This

mixture was partitioned between 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 (MgSO₄), ﬁltered, and evaporated under reduced pressure. The

residue was puriﬁed by silica gel column chromatography (chloro-

form/methanol = 10:1) to aﬀord the compound 17 (18 mg, 61%) as a

white solid. HPLC purity: 99% (R_t= 7.63 min). ¹H NMR (400 MHz,

CD₃OD): δ 9.27 (s, 1H), 8.53 (s, 1H), 8.40 (m, 2H), 8.17 (d, J =

8.00 Hz, 2H), 7.80−7.77 (m, 4H), 7.48 (d, J = 8.00 Hz, 2H), 3.03 (t,

J = 7.20 Hz, 2H), 2.81 (t, J = 7.20 Hz, 2H). MS (ESI, m/z): 463.1 [M

+ 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for C₂₅H₁₈N₄O₂F₃,

463.1382; found, 463.1381.

4′-(3-Aminopropyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tri-

azol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (18). Method B. Yield:

98%. HPLC purity: 98% (R_t= 8.14 min). ¹H NMR (400 MHz,

CD₃OD): δ 9.27 (s, 1H), 8.52 (s, 1H), 8.42 (s, 1H), 8.38 (s, 1H),

8.17 (d, J = 7.60 Hz, 2H), 7.80−7.75 (m, 4H), 7.43 (d, J = 8.00 Hz,

2H), 2.99 (t, J = 7.60 Hz, 2H), 2.82 (t, J = 7.60 Hz, 2H), 2.05−2.02

(m, 2H). MS (ESI, m/z): 467.2 [M + 1]⁺. ESI-HRMS (m/z): [M +

1]⁺calcd for C₂₅H₂₂N₄O₂F₃, 467.1695; found, 467.1689.

[M

+

Na]⁺. ESI-HRMS (m/z): [M

+

Na]⁺calcd for

C₃₁H₂₉N₄O₄F₃Na, 601.2039; found, 601.2040.

4′-(1-Aminocyclobutyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (25). Method B.

Yield: 65%. HPLC purity: 99% (R_t= 8.69 min). ¹H NMR (400 MHz,

CD₃OD): δ 9.27 (s, 1H), 8.55 (s, 1H), 8.45 (s, 1H), 8.41 (s, 1H),

8.16 (d, J = 8.00 Hz, 2H), 7.93 (d, J = 8.40 Hz, 2H), 7.79 (d, J = 8.40

Hz, 2H), 7.69 (d, J = 8.40 Hz, 2H), 2.89−2.82 (m, 2H), 2.70−2.63

(m, 2H), 2.33−2.24 (m, 1H), 2.07−1.97 (m, 1H). MS (ESI, m/z):

479.3 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₆H₂₂N₄O₂F₃, 479.1695; found, 479.1696.

4′-(1-((tert-Butoxycarbonyl)amino)cyclobutyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylic Acid (26). Method A. Yield: 47%. HPLC purity: 99% (R_t=

10.76 min). H NMR (400 MHz, CD₃OD): δ 9.18 (s, 1H), 8.40−

8.39 (m, 2H), 8.26 (s, 1H), 8.17 (d, J = 8.00 Hz, 2H), 7.80−7.76 (m,

4H), 7.59 (d, J = 8.40 Hz, 2H), 2.60−2.46 (m, 4H), 2.16−2.07 (m,

1H), 1.99−1.88 (m, 1H), 1.40−1.25 (m, 9H). MS (ESI, m/z): 579.2

[M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for C₃₁H₃₀N₄O₄F₃,

579.2219; found, 579.2219.

4′-(3-(Hydroxymethyl)oxetan-3-yl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(27). Method A. Yield: 47%. HPLC purity: 99% (R_t= 8.71 min).

¹H NMR (400 MHz, CD₃OD): δ 9.25 (s, 1H), 8.51 (s, 1H), 8.41 (s,

1

4′-(3-Aminoprop-1-yn-1-yl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (19). Method B.

1H), 8.39 (s, 1H), 8.15 (d, J = 8.40 Hz, 2H), 7.80−7.77 (m, 4H),

7.32 (d, J = 8.00 Hz, 2H), 4.97 (d, J = 5.60 Hz, 2H), 4.89 (d, J = 6.00

Hz, 2H), 3.94 (s, 2H). MS (ESI, m/z): 496.2 [M + 1]⁺. ESI-HRMS

(m/z): [M + 1]⁺calcd for C₂₆H₂₁N₃O₄F₃, 496.1484; found, 496.1488.

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

zol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (28). Method A. Yield:

1

Yield: 62%. HPLC purity: 97% (R_t= 10.49 min). H NMR (400

MHz, DMSO-d₆): δ 9.67 (s, 1H), 8.42 (s, 1H), 8.26 (s, 1H), 8.21 (d,

J = 8.00 Hz, 2H), 8.16 (s, 1H), 7.88 (d, J = 8.40 Hz, 2H), 7.79 (d, J =

8.00 Hz, 2H), 7.53 (d, J = 8.00 Hz, 2H), 3.53 (s, 2H). MS (ESI, m/

z): 463.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₅H₁₈N₄O₂F₃, 463.1382; found, 463.1380.

1

74%. HPLC purity: 98% (R_t= 11.86 min). H NMR (400 MHz,

4′-(3-((tert-Butoxycarbonyl)amino)propyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylic Acid (20). Method A. Yield: 66%. HPLC purity: 99% (R_t=

CD₃OD): δ 9.28 (s, 1H), 8.75 (s, 1H), 8.57 (s, 1H), 8.46 (s, 2H),

8.17 (d, J = 7.60 Hz, 2H), 8.05 (d, J = 8.00 Hz, 2H), 7.94 (d, J = 8.40

Hz, 2H), 7.79 (d, J = 8.00 Hz, 2H), 7.00 (s, 1H). MS (ESI, m/z):

477.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₅H₁₆N₄O₃F₃, 477.1175; found, 477.1177.

4′-(5-(Hydroxymethyl)isoxazol-3-yl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(29). Method A. Yield: 43%. HPLC purity: 98% (R_t= 20.10 min).

¹H NMR (400 MHz, DMSO-d₆): δ 9.77 (s, 1H), 8.57 (s, 1H), 8.54

1

10.56 min). H NMR (400 MHz, CD₃OD): δ 9.24 (s, 1H), 8.49 (s,

1H), 8.38−8.36 (m, 2H), 8.16 (d, J = 8.00 Hz, 2H), 7.78 (d, J = 8.00

Hz, 2H), 7.70 (d, J = 8.40 Hz, 2H), 7.37 (d, J = 8.00 Hz, 2H), 3.10 (t,

J = 7.20 Hz, 2H), 2.71 (t, J = 7.20 Hz, 2H), 1.87−1.78 (m, 2H), 1.45

(s, 9H). MS (ESI, m/z): 589.2 [M + Na]⁺. ESI-HRMS (m/z): [M +

1]⁺calcd for C₃₀H₂₉N₄O₄F₃Na, 589.2039; found, 589.2043.

4′-(4-Hydroxybutyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-

triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (21). Method A.

Yield: 39%. HPLC purity: 99% (R_t= 7.40 min). ¹H NMR (400

MHz, CD₃OD): δ 9.25 (s, 1H), 8.50 (s, 1H), 8.38 (s, 2H), 8.16 (d, J

= 8.00 Hz, 2H), 7.79 (d, J = 8.40 Hz, 2H), 7.70 (d, J = 8.00 Hz, 2H),

7.37 (d, J = 8.00 Hz, 2H), 3.59 (t, J = 6.40 Hz, 2H), 2.72 (t, J = 7.60

Hz, 2H), 1.78−1.71 (m, 2H), 1.64−1.57 (m, 2H). MS (ESI, m/z):

482.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₆H₂₃N₃O₃F₃, 482.1692; found, 482.1694.

4′-(1-Aminocyclopropyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (22). Method A.

Yield: 69%. HPLC purity: 98% (R_t= 6.80 min). ¹H NMR (400 MHz,

CD₃OD): δ 9.27 (s, 1H), 8.55−8.39 (m, 3H), 8.15 (broad s, 2H),

7.88 (broad s, 2H), 7.78 (broad s, 2H), 7.63 (broad s, 2H), 1.43−1.38

(m, 4H). MS (ESI, m/z): 465.2 [M + 1]⁺. ESI-HRMS (m/z): [M +

1]⁺calcd for C₂₅H₂₀N₄O₂F₃, 465.1538; found, 465.1539.

4′-(1-(Aminomethyl)cyclopropyl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(23). Method B. Yield: 52%. HPLC purity: 97% (R_t= 10.85 min).

¹H NMR (400 MHz, CD₃OD): δ 9.08 (s, 1H), 8.38 (s, 1H), 8.30 (s,

1H), 8.16 (s, 1H), 8.09 (d, J = 8.00 Hz, 2H), 7.77−7.73 (m, 4H),

7.55 (d, J = 8.00 Hz, 2H), 3.22 (s, 2H), 1.09−1.06 (m, 4H). MS (ESI,

m/z): 479.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₆H₂₂N₄O₂F₃, 479.1695; found, 479.1693.

(s, 1H), 8.36 (s, 1H), 8.20 (d, J = 8.00 Hz, 2H), 8.08−8.01 (m, 4H),

7.91 (d, J = 8.00 Hz, 2H), 7.05 (s, 1H), 5.75 (t, J = 6.00 Hz, 1H), 4.64

(d, J = 6.00 Hz, 2H). MS (ESI, m/z): 507.1 [M + 1]⁺. ESI-HRMS

(m/z): [M + 1]⁺calcd for C₂₆H₁₈N₄O₄F₃, 507.1280; found, 507.1271.

4′-(5-(2-Hydroxyethyl)isoxazol-3-yl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid

(30). Method A. Yield: 55%. HPLC purity: 95% (R_t= 10.69 min).

¹H NMR (400 MHz, DMSO-d₆): δ 9.74 (s, 1H), 8.51 (s, 1H), 8.46

(s, 1H), 8.35 (s, 1H), 8.20 (d, J = 8.00 Hz, 2H), 8.03−7.98 (m, 4H),

7.90 (d, J = 8.00 Hz, 2H), 6.93 (s, 1H), 3.77 (t, J = 6.40 Hz, 2H), 2.97

(d, J = 6.40 Hz, 2H). MS (ESI, m/z): 521.1 [M + 1]⁺. ESI-HRMS

(m/z): [M + 1]⁺calcd for C₂₇H₂₀N₄O₄F₃, 521.1437; found, 521.1435.

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

triazol-1-yl)-[1,1′-biphenyl]-3-carboxylic Acid (31). Method A.

1

Yield: 43%. HPLC purity: 98% (R_t= 15.95 min). H NMR (400

MHz, CD₃OD): δ 9.24 (s, 1H), 8.49 (s, 1H), 8.45 (s, 1H), 8.35 (s,

1H), 8.22−8.17 (m, 4H), 7.92 (d, J = 8.40 Hz, 2H), 7.80 (d, J = 8.00

Hz, 2H). MS (ESI, m/z): 478.1 [M + 1]⁺. ESI-HRMS (m/z): [M +

1]⁺calcd for C₂₃H₁₅N₇O₂F₃, 478.1239; found, 478.1242.

4-(4-(5-(Hydroxymethyl)isoxazol-3-yl)phenyl)-7-(4-

(triﬂuoromethyl)phenyl)-2-naphthoic Acid (32). Method A. Yield:

1

93%. HPLC purity: 98% (R_t= 12.07 min). H NMR (400 MHz,

DMSO-d₆): δ 8.79 (s, 1H), 8.68 (s, 1H), 8.09−8.05 (m, 5H), 8.01−

7.98 (m, 1H), 7.95 (m, 1H), 7.91−7.89 (m, 2H), 7.70 (d, J = 8.40 Hz,

2H), 7.04 (s, 1H), 5.80−5.73 (m, 1H), 4.66−4.63 (m, 2H). MS (ESI,

m/z): 490.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₈H₁₉NO₄F₃, 490.1266; found, 490.1269.

4′-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclopropyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylic Acid (24). Method A. Yield: 51%. HPLC purity: 99% (R_t=

1

21.57 min). H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.51 (s,

4′-(5-(Hydroxymethyl)isoxazol-3-yl)-5-(4-(triﬂuoromethyl)-

1H), 8.40−8.38 (m, 2H), 8.17 (d, J = 8.00 Hz, 2H), 7.80 (d, J = 8.00

Hz, 2H), 7.73 (d, J = 8.00 Hz, 2H), 7.51 (d, J = 8.00 Hz, 2H), 3.34 (s,

2H), 1.39−1.28 (m, 9H), 0.94−0.86 (m, 4H). MS (ESI, m/z): 601.2

benzamido)-[1,1′-biphenyl]-3-carboxylic Acid (33). Method A.

1

Yield: 62%. HPLC purity: 99% (R_t= 10.29 min). H NMR (400

MHz, CD₃OD): δ 8.35 (broad s, 2H), 8.17−8.13 (m, 3H), 7.97 (d, J

5114

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

= 8.40 Hz, 2H), 7.86−7.81 (m, 4H), 6.82 (s, 1H), 4.74 (s, 2H). MS

(ESI, m/z): 483.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₅H₁₈N₂O₅F₃, 483.1168; found, 483.1170.

4-(4-(Piperidin-4-yl)phenyl)-6-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)picolinic Acid (34). Method B. Yield: 60%. HPLC

purity: 99% (R_t= 7.87 min). ¹H NMR (400 MHz, DMSO-d₆): δ 9.55

(s, 1H), 8.31−8.26 (m, 4H), 7.91−7.85 (m, 4H), 7.47 (d, J = 8.00

Hz, 2H), 3.68−3.65 (m, 1H), 3.02−2.88 (m, 4H), 1.95−1.83 (m,

4H). MS (ESI, m/z): 494.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₂₆H₂₃N₅O₂F₃, 494.1804; found, 494.1805.

8.00 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H), 7.64 (d, J = 8.40 Hz, 2H),

7.52 (d, J = 8.40 Hz, 2H), 4.01 (s, 3H), 1.54 (s, 9H). MS (ESI, m/z):

539.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₈H₂₆N₄O₄F₃, 539.1906; found, 539.1916.

Methyl 4′-Amino-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-1-yl)-[1,1′-biphenyl]-3-carboxylate (40). Method B. Yield: 57%.

¹H NMR (400 MHz, CDCl₃): δ 8.38 (s, 1H), 8.32 (s, 1H), 8.26−8.24

(m, 2H), 8.06 (d, J = 8.00 Hz, 2H), 7.74 (d, J = 8.00 Hz, 2H), 7.54

(d, J = 8.40 Hz, 2H), 6.81 (d, J = 8.40 Hz, 2H), 4.00 (s, 3H). MS

(ESI, m/z): 439.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₃H₁₈N₄O₂F₃, 439.1382; found, 439.1378.

2-(4-(Piperidin-4-yl)phenyl)-6-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)isonicotinic Acid (35). Method B. Yield: 65%.

Methyl 4′-Acetamido-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-

triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (41). 40 (12 mg, 0.027

mmol) was dissolved in dichloromethane (1 mL) and degassed for 30

min with nitrogen gas. Acetic anhydride (3.2 μL, 0.033 mmol) was

added to the reaction mixture and was stirred for 2 h at room

temperature. The reaction mixture was partitioned between dichloro-

methane (10 mL) and saturated aqueous Na₂CO₃solution (10 mL),

and the aqueous phase was extracted with dichloromethane (10 mL ×

2). The combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated under pressure. The residue was puriﬁed by silica gel

chromatography (hexane/ethyl acetate = 2:1) to aﬀord compound 41

1

HPLC purity: 95% (R_t= 10.72 min). H NMR (400 MHz, DMSO-

d₆): δ 9.79 (s, 1H), 8.42 (s, 2H), 8.34−8.33 (m, 4H), 7.89 (d, J = 8.00

Hz, 2H), 7.44 (d, J = 8.00 Hz, 2H), 3.62−3.59 (m, 1H), 3.07−2.92

(m, 4H), 2.03−1.89 (m, 4H). MS (ESI, m/z): 494.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₂₆H₂₃N₅O₂F₃, 494.1804; found,

494.1805.

6-(4-(Piperidin-4-yl)phenyl)-4-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)picolinic Acid (36). Method B. Yield: 65%. HPLC

1

purity: 99% (R_t= 11.24 min). H NMR (400 MHz, DMSO-d₆): δ

9.94 (s, 1H), 8.71 (s, 1H), 8.56 (s, 1H), 8.28 (d, J = 7.20 Hz, 2H),

8.19 (d, J = 7.60 Hz, 2H), 7.93 (d, J = 7.20 Hz, 2H), 7.46 (d, J = 7.20

Hz, 2H), 3.43−3.40 (m, 2H), 3.07−2.92 (m, 3H), 2.03−2.00 (m,

2H), 1.91−1.84 (m, 2H). MS (ESI, m/z): 494.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₂₆H₂₃N₅O₂F₃, 494.1804; found,

494.1808.

1

(10 mg, 76%) as a white solid. H NMR (400 MHz, CDCl₃): δ 8.40

(s, 1H), 8.34 (s, 1H), 8.32−8.30 (m, 2H), 8.06 (d, J = 8.00 Hz, 2H),

7.74 (d, J = 8.40 Hz, 2H), 7.67 (s, 4H), 4.01 (s, 3H), 2.23 (s, 3H).

MS (ESI, m/z): 481.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₂₅H₂₀N₄O₃F₃, 481.1488; found, 481.1487.

2-(Dimethylamino)-2-oxoethyl 4′-(Piperidin-4-yl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (37a). Method B. Yield: 78%. HPLC purity: 97% (R_t=

Methyl 4′-Benzamido-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-

triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (42). 40 (12 mg, 0.027

mmol) was dissolved in dichloromethane (1 mL) at 0 °C.

Triethylamine (4.2 μL, 0.030 mmol) and benzoyl chloride (3.2 μL,

0.027 mmol) were added, and the mixture was stirred for 18 h at

room temperature. The reaction mixture was partitioned betweeen

dichloromethane (10 mL) and water (10 mL), and the aqueous phase

was extracted with dichloromethane (10 mL × 2). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated

under pressure. The residue was puriﬁed by silica gel chromatography

(hexane/ethyl acetate = 3:1) to aﬀord compound 42 (9 mg, 63%) as a

1

20.45 min). H NMR (400 MHz, CD₃OD): δ 9.29 (s, 1H), 8.59 (s,

1H), 8.47 (s, 1H), 8.45 (s, 1H), 8.17 (d, J = 8.00 Hz, 2H), 7.82−7.79

(m, 4H), 7.47 (d, J = 8.00 Hz, 2H), 5.18 (s, 2H), 3.56−3.53 (m, 2H),

3.23−3.16 (s, 2H), 3.13 (s, 3H), 3.05−2.99 (m, 1H), 3.02 (s, 3H),

2.18−2.14 (m, 2H), 2.03−1.92 (m, 2H). MS (ESI, m/z): 578.3 [M +

1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for C₃₁H₃₁N₅O₃F₃, 578.2379;

found, 578.2390.

2-(Dimethylamino)-2-oxoethyl 4-(4-(5-(Hydroxymethyl)isoxazol-

3-yl)phenyl)-7-(4-(triﬂuoromethyl)phenyl)-2-naphthoate (37b). To

a solution of 32 (3.5 mg, 0.007 mmol) in DMF (1 mL) was added

Cs₂CO₃(3.0 mg, 0.010 mmol). 2-Chloro-N,N-dimethylacetamide

(1.0 mg, 0.007 mmol) was added to the reaction mixture under a N₂

atmosphere, and the reaction mixture was stirred at 45 °C for 1 h

under a N₂atmosphere. The reaction mixture was partitioned

between ethyl acetate (5 mL) and water (5 mL), and the aqueous

phase was extracted with ethyl acetate (2 × 5 mL). The combined

organic layers were dried over Na₂SO₄, ﬁltered, and concentrated

under pressure. The residue was puriﬁed by silica gel chromatography

(chloroform/methanol = 30:1) to aﬀord compound 37b (2.7 mg,

68%) as a white solid. HPLC purity: 99% (R_t= 17.73 min). ¹H NMR

(400 MHz, CD₃OD): δ 8.85−8.84 (m, 1H), 8.45−8.44 (m, 1H),

8.08−7.97 (m, 7H), 7.81 (d, J = 8.40 Hz, 2H), 7.66 (d, J = 8.00 Hz,

2H), 6.88 (s, 1H), 5.14 (s, 2H), 4.76 (s, 2H), 3.12 (s, 3H), 3.00 (s,

3H). MS (ESI, m/z): 575.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₃₂H₂₆N₂O₅F₃, 575.1794; found, 575.1786.

1

white solid. H NMR (400 MHz, CDCl₃): δ 8.41 (s, 1H), 8.37 (s,

1H), 8.33 (broad s, 2H), 8.10−8.05 (m, 2H), 7.94−7.90 (m, 3H),

7.82 (d, J = 8.40 Hz, 2H), 7.75−7.72 (m, 3H), 7.61−7.45 (m, 3H),

4.02 (s, 3H). MS (ESI, m/z): 543.2 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₃₀H₂₁N₄O₃F₃, 543.1644; found, 543.1647.

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

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

piperidine-1-carboxylate (43). To a solution of compound 40 (27

mg, 0.061 mmol) in N,N-dimethylformamide (2 mL) were added

Boc-Inp-OH (15 mg, 0.067 mmol), HATU (25 mg, 0.067 mmol),

and N,N-diisopropylethylamine (13 μL, 0.080 mmol), and then this

reaction mixture was stirred at room temperature for 2 h. The

reaction mixture was partitioned between 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 (hexane/ethyl acetate = 3:1) to aﬀord compound 43 (37 mg,

2-(Dimethylamino)-2-oxoethyl 4-(4-(1-Acetylpiperidin-4-yl)-

phenyl)-7-(4-(triﬂuoromethyl)phenyl)-2-naphthoate (37c). Com-

pound 1b (24 mg, 0.046 mmol) was converted to compound 37c

(20 mg, 72%) as a white solid, using a similar procedure to that used

in the preparation of compound 87. HPLC purity: 99% (R_t= 16.43

1

94%) as a white solid. H NMR (400 MHz, CDCl₃): δ 8.40 (s, 1H),

8.34−8.33 (m, 1H), 8.32−8.30 (m, 2H), 8.06 (d, J = 8.40 Hz, 2H),

7.74 (d, J = 8.00 Hz, 2H), 7.68 (s, 4H), 4.25−4.18 (m, 2H), 4.01 (s,

3H), 2.84−2.78 (m, 2H), 2.46−2.40 (m, 1H), 1.95−1.92 (m, 2H),

1.78−1.76 (m, 2H), 1.47 (s, 9H). MS (ESI, m/z): 672.2 [M + Na]⁺.

ESI-HRMS (m/z): [M + Na]⁺calcd for C₃₄H₃₄N₅O₅F₃Na, 672.2410;

found, 672.2413.

1

min). H NMR (400 MHz, CD₃OD): δ 8.80 (s, 1H), 8.42 (s, 1H),

8.03−7.98 (m, 4H), 7.93−7.91 (m, 1H), 7.80 (d, J = 7.60 Hz, 2H),

7.47−7.43 (m, 4H), 5.14 (s, 2H), 4.75−4.72 (m, 1H), 4.12−4.08 (m,

1H), 3.27−3.24 (m, 1H), 3.12 (s, 3H), 3.00 (s, 3H), 2.95−2.92 (m,

1H), 2.81−2.75 (m, 1H), 2.18 (s, 3H), 2.04−1.97 (m, 2H), 1.84−

1.68 (m, 2H). MS (ESI, m/z): 603.2 [M + 1]⁺. ESI-HRMS (m/z):

[M + 1]⁺calcd for C₃₅H₃₄N₂O₄F₃, 603.2471; found, 603.2479.

Methyl 4′-((tert-Butoxycarbonyl)amino)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (39). Method C. Yield: 40%. ¹H NMR (400 MHz,

CDCl₃): δ 8.40 (s, 1H), 8.33 (s, 1H), 8.29 (broad s, 2H), 8.06 (d, J =

3′-(Methoxycarbonyl)-5′-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-carboxylic Acid (44). Method C.

1

Yield: 45%. H NMR (400 MHz, DMSO-d₆): δ 9.78 (s, 1H), 8.59−

8.56 (m, 2H), 8.36 (s, 1H), 8.19 (d, J = 8.00 Hz, 2H), 8.10 (d, J =

8.40 Hz, 2H), 8.00 (d, J = 8.40 Hz, 2H), 7.91 (d, J = 8.40 Hz, 2H),

3.98 (s, 3H). MS (ESI, m/z): 468.1 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₂₄H₁₇N₃O₄F₃, 468.1171; found, 468.1169.

5115

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Methyl 4′-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-

5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphen-

Hz, 2H), 7.57 (d, J = 8.40 Hz, 2H), 3.51−3.46 (m, 2H), 3.26−3.22

(m, 2H), 1.71−1.68 (m, 2H), 1.44 (s, 9H).

1

yl]-3-carboxylate (45). Method C. Yield: 68%. H NMR (400 MHz,

tert-Butyl (3-(4-Bromophenyl)prop-2-yn-1-yl)carbamate (54b).

To a mixture of 1-bromo-4-iodobenzene 53b (Scheme S2, Supporting

Information, 300 mg, 1.06 mmol), PdCl₂(PPh₃)₂(37 mg, 0.053

mmol), and copper iodide (20 mg, 0.106 mmol) in THF were added

triethylamine (295 μL, 2.12 mmol) and N-Boc-propargylamine (247

mg, 1.59 mmol) at room temperature under a N₂atmosphere, and

then this reaction mixture was stirred at room temperature for 12 h

under a N₂atmosphere. The reaction mixture was partitioned with

ethyl acetate (100 mL) and aqueous saturated NH₄Cl solution (50

mL), and then the aqueous layer was extracted with ethyl acetate (100

mL × 2). The combined organic layer was dried (Na₂SO₄), ﬁltered,

and evaporated under reduced pressure. The residue was puriﬁed by

silica gel column chromatography (hexane/ethyl acetate = 5:1) to

CD₃OD): δ 9.15 (s, 1H), 8.41 (s, 1H), 8.32 (s, 1H), 8.25 (s, 1H),

8.04 (d, J = 8.00 Hz, 2H), 7.93 (d, J = 8.40 Hz, 2H), 7.78 (d, J = 8.40

Hz, 2H), 7.71 (d, J = 8.00 Hz, 2H), 3.95 (s, 3H), 3.44 (t, J = 6.80 Hz,

2H), 3.17−3.14 (m, 2H), 1.80−1.76 (m, 2H). MS (ESI, m/z): 646.2

[M

C₃₂H₃₂N₅O₅F₃Na, 646.2253; found, 646.2255.

+ +

Na]⁺. ESI-HRMS (m/z): [M Na]⁺calcd for

4′-((3-((tert-Butoxycarbonyl)amino)propyl)carbamoyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

1

carboxylic Acid (46). Method A. Yield: 77%. H NMR (400 MHz,

CD₃OD): δ 9.27 (s, 1H), 8.57 (s, 1H), 8.46 (s, 1H), 8.41 (s, 1H),

8.15 (d, J = 8.00 Hz, 2H), 7.99 (d, J = 7.60 Hz, 2H), 7.89 (d, J = 8.00

Hz, 2H), 7.78 (d, J = 8.00 Hz, 2H), 3.47−3.43 (m, 2H), 3.17−3.14

(m, 2H), 1.80−1.77 (m, 2H). MS (ESI, m/z): 610.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₃₁H₃₁N₅O₅F₃, 610.2277; found,

610.2270.

Methyl 4′-Bromo-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-1-yl)-[1,1′-biphenyl]-3-carboxylate (47). Method C. Yield: 53%.

¹H NMR (400 MHz, CDCl₃): δ 8.40 (s, 1H), 8.35−8.32 (m, 3H),

8.06 (d, J = 8.00 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H), 7.65 (d, J = 8.40

Hz, 2H), 7.57 (d, J = 8.40 Hz, 2H), 4.02 (s, 3H). MS (ESI, m/z):

502.0, 504.0 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₃H₁₆N₃O₂F₃Br, 502.0378; found, 502.0382.

1

aﬀord the compound 54b (310 mg, 94%) as a white solid. H NMR

(400 MHz, CDCl₃): δ 7.45 (d, J = 8.40 Hz, 2H), 7.28 (d, J = 8.40 Hz,

2H), 4.16−4.15 (m, 2H), 1.48 (s, 9H).

Methyl 4′-(1-((tert-Butoxycarbonyl)amino)cyclopropyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (55). Method E. Yield: 30%. ¹H NMR (400 MHz,

CDCl₃): δ 8.39 (s, 1H), 8.35−8.30 (m, 3H), 8.06 (d, J = 8.00 Hz,

2H), 7.74 (d, J = 8.00 Hz, 2H), 7.63 (d, J = 8.40 Hz, 2H), 7.34 (d, J =

8.00 Hz, 2H), 4.01 (s, 3H), 1.46 (s, 9H), 1.35−1.30 (m, 4H). MS

(ESI, m/z): 579.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₃₁H₃₀N₄O₄F₃, 579.2219; found, 579.2220.

Methyl 4′-(2-Amino-2-oxoethyl)-5-(4-(4-(triﬂuoromethyl)-

Methyl 4′-(1-Aminocyclopropyl)-5-(4-(4-(triﬂuoromethyl)-

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (48).

1

phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (56).

Method D. Yield: 73%. H NMR (400 MHz, CDCl₃): δ 8.41 (s,

1

Method B. Yield: 90%. H NMR (400 MHz, CD₃OD): δ 9.29 (s,

1H), 8.36−8.33 (m, 3H), 8.06 (d, J = 8.00 Hz, 2H), 7.75−7.69 (m,

2H), 7.50−7.44 (m, 2H), 7.16 (d, J = 8.40 Hz, 2H), 4.02 (s, 3H),

3.67 (s, 2H). MS (ESI, m/z): 481.2 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₂₅H₂₀N₄O₃F₃, 481.1488; found, 481.1492.

Methyl 4′-(3-((tert-Butoxycarbonyl)amino)propyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (49). Method E. Yield: 55%. ¹H NMR (400 MHz,

CDCl₃): δ 8.40 (s, 1H), 8.35 (s, 1H), 8.33−8.31 (m, 2H), 8.06 (d, J =

8.00 Hz, 2H), 7.74 (d, J = 8.40 Hz, 2H), 7.62 (d, J = 8.40 Hz, 2H),

7.33 (d, J = 8.00 Hz, 2H), 4.01 (s, 3H), 3.20−3.18 (m, 2H), 2.72 (t, J

= 7.60 Hz, 2H), 1.90−1.82 (m, 2H), 1.45 (s, 9H). MS (ESI, m/z):

525.2 [M + 1-tert-butyl]⁺.

Methyl 4′-(3-((tert-Butoxycarbonyl)amino)prop-1-yn-1-yl)-5-(4-

(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (50). Method C. Yield: 35%. ¹H NMR (400 MHz,

CD₃OD): δ 9.24 (s, 1H), 8.51 (s, 1H), 8.40 (s, 1H), 8.34 (s, 1H),

8.13 (d, J = 8.00 Hz, 2H), 7.78−7.74 (m, 4H), 7.56 (d, J = 8.40 Hz,

2H), 4.10−4.08 (m, 2H), 4.00 (s, 3H), 1.48 (s, 9H). MS (ESI, m/z):

577.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₃₁H₂₈N₄O₄F₃, 577.2063; found, 577.2057.

1H), 8.56 (s, 1H), 8.49 (s, 1H), 8.41 (s, 1H), 8.17 (d, J = 8.00 Hz,

2H), 7.90 (d, J = 8.40 Hz, 2H), 7.80 (d, J = 8.00 Hz, 2H), 7.64 (d, J =

8.00 Hz, 2H), 4.02 (s, 3H), 1.43−1.40 (m, 4H). MS (ESI, m/z):

479.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₆H₂₂N₄O₂F₃, 479.1695; found, 479.1699.

Methyl 4′-(1-(((tert-Butoxycarbonyl)amino)methyl)cyclopropyl)-

5-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphen-

1

yl]-3-carboxylate (57). Method E. Yield: 30%. H NMR (400 MHz,

CDCl₃): δ 8.40 (s, 1H), 8.35−8.30 (m, 3H), 8.06 (d, J = 8.40 Hz,

2H), 7.74 (d, J = 8.40 Hz, 2H), 7.64 (d, J = 8.00 Hz, 2H), 7.44 (d, J =

8.00 Hz, 2H), 4.01 (s, 3H), 3.39−3.35 (m, 2H), 1.40 (s, 9H), 0.93−

0.79 (m, 4H). MS (ESI, m/z): 615.2 [M + Na]⁺. ESI-HRMS (m/z):

[M + Na]⁺calcd for C₃₂H₃₁N₄O₄F₃Na, 615.2195; found, 615.2199.

Methyl 4′-(1-((tert-Butoxycarbonyl)amino)cyclobutyl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (58). Method E. Yield: 70%. ¹H NMR (400 MHz,

CDCl₃): δ 8.40 (s, 1H), 8.38 (s, 1H), 8.33 (s, 2H), 8.06 (d, J = 8.00

Hz, 2H), 7.74 (d, J = 8.00 Hz, 2H), 7.68 (d, J = 8.00 Hz, 2H), 7.57

(d, J = 7.60 Hz, 2H), 4.01 (s, 3H), 2.63−2.44 (m, 4H), 2.20−2.09

(m, 1H), 1.97−1.85 (m, 1H), 1.47−1.29 (m, 9H). MS (ESI, m/z):

593.3 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₃₂H₃₂N₄O₄F₃, 593.2376; found, 593.2383.

Methyl 4′-(3-(Hydroxymethyl)oxetan-3-yl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (59). Method E. Yield: 44%. ¹H NMR (400 MHz,

CDCl₃): δ 8.41 (s, 1H), 8.36 (s, 1H), 8.34 (s, 2H), 8.06 (d, J = 8.00

Hz, 2H), 7.75−7.64 (m, 4H), 7.54 (d, J = 8.00 Hz, 2H), 5.01 (d, J =

5.60 Hz, 2H), 4.83 (d, J = 5.60 Hz, 2H), 4.11 (d, J = 5.60 Hz, 2H),

4.02 (s, 3H). MS (ESI, m/z): 510.2 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₂₇H₂₃N₃O₄F₃, 510.1641; found, 510.1640.

Methyl 4′-(4-Hydroxybutyl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (51). Method E.

1

Yield: 27%. H NMR (400 MHz, CDCl₃): δ 8.48 (s, 1H), 8.40 (s,

1H), 8.32 (s, 2H), 8.06 (d, J = 8.00 Hz, 2H), 7.74 (d, J = 8.00 Hz,

2H), 7.62 (d, J = 8.00 Hz, 2H), 7.33 (d, J = 8.00 Hz, 2H), 4.01 (s,

3H), 3.72−3.68 (m, 2H), 2.73 (t, J = 7.60 Hz, 2H), 1.80−1.72 (m,

2H), 1.68−1.58 (m, 2H). MS (ESI, m/z): 496.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₂₇H₂₅N₃O₃F₃, 496.1848; found,

496.1855.

4′-(3-((tert-Butoxycarbonyl)amino)prop-1-yn-1-yl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

Methyl 4′-(Isoxazol-3-yl)-5-(4-(4-(triﬂuoromethyl)phenyl)-1H-

1

carboxylic Acid (52). Method A. Yield: 88%. H NMR (400 MHz,

1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (60). Method F.

1

DMSO-d₆): δ 9.76 (s, 1H), 8.50 (d, J = 6.40 Hz, 2H), 8.30 (s, 1H),

8.19 (d, J = 8.00 Hz, 2H), 7.91−7.88 (m, 4H), 7.58 (d, J = 8.40 Hz,

2H), 4.02 (s, 2H), 1.41 (s, 9H). MS (ESI, m/z): 563.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₃₀H₂₆N₄O₄F₃, 563.1906; found,

563.1897.

Yield: 91%. H NMR (400 MHz, acetone-d₆): δ 9.48 (s, 1H), 8.88

(d, J = 1.60 Hz, 1H), 8.62−8.61 (m, 2H), 8.46−8.45 (m, 1H), 8.26

(d, J = 8.00 Hz, 2H), 8.14 (d, J = 8.40 Hz, 2H), 8.04 (d, J = 8.40 Hz,

2H), 7.88 (d, J = 8.00 Hz, 2H), 7.13 (d, J = 1.60 Hz, 1H), 4.03 (s,

3H). MS (ESI, m/z): 491.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₂₆H₁₈N₄O₃F₃, 491.1331; found, 491.1328.

Methyl 4′-(5-(Hydroxymethyl)isoxazol-3-yl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (61). Method E. Yield: 46%. ¹H NMR (400 MHz,

CDCl₃): δ 8.40 (s, 1H), 8.38 (s, 1H), 8.36−8.35 (m, 2H), 8.04 (d, J =

tert-Butyl (3-(4-Bromobenzamido)propyl)carbamate (54a). 4-

Bromobenzoic acid 53a (Scheme S2, Supporting Information, 100

mg, 0.497 mmol) was converted to compound 54a (54 mg, 31%) as a

white solid, using a similar procedure to that used in the preparation

1

of compound 43. H NMR (400 MHz, CDCl₃): δ 7.73 (d, J = 8.00

5116

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

8.40 Hz, 2H), 7.93 (d, J = 8.00 Hz, 2H), 7.78 (d, J = 8.40 Hz, 2H),

7.72 (d, J = 8.00 Hz, 2H), 6.62 (s, 1H), 4.84 (s, 2H), 4.00 (s, 3H).

MS (ESI, m/z): 521.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₂₇H₂₀N₄O₄F₃, 521.1437; found, 521.1430.

raphy (hexane/ethyl acetate = 2:1) to aﬀord compound 68 (29.0 mg,

65%) as a white solid. H NMR (400 MHz, CDCl₃): δ 8.50 (s, 1H),

8.29 (s, 1H), 8.15 (s, 1H), 7.99 (d, J = 8.00 Hz, 2H), 7.78 (d, J = 8.40

Hz, 2H), 3.93 (s, 3H), 1.35 (s, 12H). MS (ESI, m/z): 450.2 [M + 1]⁺.

ESI-HRMS (m/z): [M + 1]⁺calcd for C₂₂H₂₄NO₅F₃B, 450.1700;

found, 450.1701.

1

Methyl 4′-(5-(2-Hydroxyethyl)isoxazol-3-yl)-5-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-

carboxylate (62). Method F. Yield: 80%. ¹H NMR (400 MHz,

DMSO-d₆): δ 9.76 (s, 1H), 8.58 (s, 1H), 8.54 (s, 1H), 8.35 (s, 1H),

8.18 (d, J = 8.40 Hz, 2H), 8.04−8.00 (m, 4H), 7.90 (d, J = 8.40 Hz,

2H), 6.94 (s, 1H), 3.97 (s, 3H), 3.77 (q, J = 6.40 Hz, 2H), 2.97 (d, J =

6.40 Hz, 2H). MS (ESI, m/z): 535.2 [M + 1]⁺. ESI-HRMS (m/z):

[M + 1]⁺calcd for C₂₈H₂₂N₄O₄F₃, 535.1593; found, 535.1592.

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

1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-3-carboxylate (63). Method

Methyl 4′-(5-(Hydroxymethyl)isoxazol-3-yl)-5-(4-

(triﬂuoromethyl)benzamido)-[1,1′-biphenyl]-3-carboxylate (69).

1

Method C. Yield: 45%. H NMR (400 MHz, CD₃OD): δ 8.44 (m,

1H), 8.36−8.35 (m, 1H), 8.17−8.13 (m, 3H), 7.98 (d, J = 8.40 Hz,

2H), 7.87−7.82 (m, 4H), 6.83 (s, 1H), 4.74 (s, 2H), 3.97 (s, 3H). MS

(ESI, m/z): 497.1 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₂₆H₂₀N₂O₅F₃, 497.1324; found, 497.1323.

Methyl 2-Amino-6-bromoisonicotinate (72). Compound 69 (200

mg, 0.922 mmol) was converted to compound 72 (83 mg, 40%) as a

white solid, using a similar procedure to that used in the preparation

1

C. Yield: 53%. H NMR (400 MHz, CD₃OD): δ 9.26 (s, 1H), 8.52

(s, 1H), 8.44 (s, 1H), 8.39 (s, 1H), 8.18 (d, J = 8.40 Hz, 2H), 8.12 (d,

J = 8.40 Hz, 2H), 7.90 (d, J = 8.40 Hz, 2H), 7.76 (d, J = 8.40 Hz, 2H),

4.00 (s, 3H). MS (ESI, m/z): 492.1 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₂₄H₁₇N₇O₂F₃, 492.1396; found, 492.1399.

Ethyl 4-(4-(5-(Hydroxymethyl)isoxazol-3-yl)phenyl)-7-(4-

(triﬂuoromethyl)phenyl)-2-naphthoate (65). Method F. Yield:

50%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.81 (s, 1H), 8.71 (s,

1H), 8.09−8.05 (m, 5H), 7.99−7.97 (m, 1H), 7.94−7.93 (m, 1H),

7.89 (d, J = 8.00 Hz, 2H), 7.68 (d, J = 8.00 Hz, 2H), 7.04 (s, 1H),

5.77 (t, J = 6.00 Hz, 1H), 4.66 (d, J = 6.00 Hz, 2H), 4.41 (q, J = 7.20

Hz, 2H), 1.38 (t, J = 7.20 Hz, 3H). MS (ESI, m/z): 518.2 [M + 1]⁺.

ESI-HRMS (m/z): [M + 1]⁺calcd for C₃₀H₂₃NO₄F₃, 518.1579;

found, 518.1582.

1

of compound 66. H NMR (400 MHz, CD₃OD): δ 7.13 (d, 1H),

7.00 (d, 1H), 3.89 (s, 3H). MS (ESI, m/z): 231.0 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₇H₈BrN₂O₂, 230.9769; found,

230.9769.

Methyl 4-Amino-6-bromopicolinate (73). Compound 70 (320

mg, 1.47 mmol) was converted to compound 73 (136 mg, 40%) as a

white solid, using a similar procedure to that used in the preparation

1

of compound 66. H NMR (400 MHz, DMSO-d₆): δ 7.23 (d, 1H),

6.79 (d, 1H), 3.81 (s, 3H). MS (ESI, m/z): 231.0 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₇H₈BrN₂O₂, 230.9769; found,

230.9765.

Methyl 6-Azido-4-bromopicolinate (74). Compound 71 (121 mg,

0.523 mmol) was dissolved in acetonitrile (4 mL) and cooled to 0 °C

in an ice bath. tert-Butyl nitrite (124 μL, 1.047 mmol) was added to

the reaction mixture and stirred at 0 °C for 30 min. After that,

azidotrimethylsilane (206 μL, 1.57 mmol) was added to the reaction

mixture at 0 °C dropwise. The resulting solution was stirred at room

temperature for 12 h. The reaction mixture was concentrated under

reduced pressure. The residue was puriﬁed by silica gel column

chromatography (hexane/ethyl acetate = 3:1) to aﬀord compound 74

Methyl 3-Amino-5-bromobenzoate (66). 3-Bromo-5-aminoben-

zoic acid (65, 1.01 g, 4.62 mmol) was stirred in methanol (15 mL)

with ice cooling, and the yellow solution was treated with thionyl

chloride (4.00 mL, 55.0 mmol) dropwise over 20 min. The resulting

mixture was warmed up to room temperature and left stirring for 15 h.

The reaction mixture was quenched with aqueous saturated NaHCO₃

solution at 0 °C. The solvent was then removed under vacuum, and

the residue was suspended in ethyl acetate (200 mL). The organic

phase was washed with brine (100 mL), dried (Na₂SO₄), and

concentrated in vacuo to aﬀord the title compound 66 (1.08 g, 98%)

1

(50 mg, 37%) as a white solid. H NMR (400 MHz, CDCl₃): δ 7.98

(s, 1H), 7.26 (s, 1H), 3.99 (s, 3H).

1

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

Methyl 2-Azido-6-bromoisonicotinate (75). Compound 72 (27

mg, 0.107 mmol) was converted to compound 75 (30 mg, 65%) as a

white solid, using a similar procedure to that used in the preparation

of compound 74. ¹H NMR (400 MHz, CDCl₃): δ 7.76 (d, 1H), 7.29

(d, 1H), 3.94 (s, 3H).

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 (m/z): [M + 1]⁺calcd for C₈H₈BrNO₂, 229.9817; found,

229.9818.

Methyl 3-Bromo-5-(4-(triﬂuoromethyl)benzamido)benzoate

(67). To a solution of compound 66 (70 mg, 0.304 mmol) in N,N-

dimethylformamide (6 mL) were added 4-(triﬂuoromethyl)benzoic

acid (136 mg, 0.717 mmol), HATU (218 mg, 0.573 mmol), and N,N-

diisopropylethylamine (250 μL, 1.43 mmol), and then this reaction

mixture was stirred at room temperature for 5 h. The reaction mixture

was partitioned between 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 (10 mL), dried

over Na₂SO₄, ﬁltered, and evaporated under reduced pressure. The

residue was puriﬁed by silica gel column chromatography (hexane/

ethyl acetate = 7:1) to aﬀord compound 67 (86 mg, 70%) as a white

solid. ¹H NMR (400 MHz, CDCl₃): δ 8.32−8.31 (m, 1H), 8.05−8.04

(m, 1H), 7.99−7.98 (m, 3H), 7.79 (d, J = 8.40 Hz, 2H), 3.93 (s, 3H).

MS (ESI, m/z): 402.0 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd

for C₁₆H₁₂NO₃F₃Br, 401.9953; found, 401.9948.

Methyl 3-(4,4,5,5-Tetramethyl-1,3,2-dioxaborolan-2-yl)-5-(4-

(triﬂuoromethyl)benzamido)benzoate (68). To a solution of

compound 67 (40 mg, 0.099 mmol) in 1,4-dioxane (3 mL) were

added bis(pinacolato)diboron (63 mg, 0.248 mmol), PdCl₂(dppf)

(2.0 mg, 0.002 μmol), and potassium acetate (30 mg, 0.298 mmol),

and then this reaction mixture was stirred at 70 °C for 6 h. The

reaction mixture was partitioned between 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 Na₂SO₄, ﬁltered, and evaporated under reduced

pressure. The residue was puriﬁed by silica gel column chromatog-

Methyl 4-Azido-6-bromopicolinate (76). Compound 73 (21 mg,

0.090 mmol) was converted to compound 76 (12 mg, 52%) as a white

solid, using a similar procedure to that used in the preparation of

compound 74. H NMR (400 MHz, CDCl₃): δ 7.75 (broad s, 1H),

7.31 (broad s, 1H), 4.02 (s, 3H).

1

Methyl 4-Bromo-6-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-1-yl)picolinate (77). To a solution of compound 74 (50 mg,

0.195 mmol) in tert-BuOH/water (5 mL, 1:1) were added 4-ethynyl-

α,α,α-triﬂuorotoluene (50 μL, 0.292 mmol) and tris[(1-benzyl-1H-

1,2,3-triazol-4-yl)methyl]amine (TBTA, 7 mg, 0.014 mmol). Sub-

sequently, sodium ascorbate (58 mg, 0.292 mmol, freshly prepared 1

M aqueous solution) and CuSO₄·5H₂O (37 mg, 0.146 mmol, freshly

prepared 7.5% aqueous solution) were added into the reaction

mixture, and then this reaction mixture was stirred at room

temperature for 1.5 h. The solvent of the reaction mixture was

evaporated under reduced pressure. The residue was puriﬁed by silica

gel column chromatography (hexane/ethyl acetate = 3:1) to aﬀord

1

compound 77 (30 mg, 36%) as a white solid. H NMR (400 MHz,

CDCl₃): δ 8.98 (s, 1H), 8.65 (d, 1H), 8.31 (d, 1H), 8.08 (d, J = 8.00

Hz, 2H), 7.74 (d, J = 8.00 Hz, 2H), 4.06 (s, 3H). MS (ESI, m/z):

427.0 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₁₆H₁₁BrN₄O₂F₃, 427.0017; found, 427.0013.

Methyl 2-Bromo-6-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-1-yl)isonicotinate (78). Compound 75 (27 mg, 0.107 mmol)

was converted to compound 78 (30 mg, 65%) as a white solid, using a

similar procedure to that used in the preparation of compound 77. ¹H

NMR (400 MHz, CD₃OD): δ 9.28 (s, 1H), 8.65 (d, 1H), 8.20−8.18

5117

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(m, 3H), 7.79 (d, J = 8.00 Hz, 2H), 4.03 (s, 3H). MS (ESI, m/z):

427.0 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₁₆H₁₁BrN₄O₂F₃, 427.0017; found, 427.0018.

pressure. The residue was puriﬁed by silica gel chromatography

(hexane/ethyl acetate = 3:1) to aﬀord compound 87 (205 mg, 70%)

as a white solid. ¹H NMR (400 MHz, CDCl₃): δ 8.43−8.41 (m, 2H),

8.38−8.36 (m, 2H), 8.05 (d, J = 8.00 Hz, 2H), 7.73 (d, J = 8.40 Hz,

2H), 7.65 (d, J = 8.40 Hz, 2H), 7.34 (d, J = 8.40 Hz, 2H), 5.05 (s,

2H), 3.76−3.72 (m, 2H), 3.07 (s, 3H), 3.02 (s, 3H), 2.87−2.77 (m,

2H), 2.76−2.67 (m, 1H), 1.89−1.83 (m, 2H), 1.72−1.62 (m, 2H),

1.49 (s, 9H). MS (ESI, m/z): 622.2 [M + 1-tert-butyl]⁺.

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

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

boxylate (89). To a solution of 88 (35 mg, 0.058 mmol), which was

synthesized according to literature procedures reported,¹in THF at 0

°C, 2 M lithium aluminum hydride solution in THF (2.6 mg, 0.070

mmol) was added dropwise over a period of 0.5 h under a N₂

atmosphere. After the addition was complete, the reaction mixture

was stirred at 0 °C under a N₂atmosphere for 1 h. The unreacted

LAH was quenched with saturated aqueous NH₄Cl, and the reaction

mixture was extracted with dichloromethane three times. The

combined organic extracts were dried with anhydrous Na₂SO₄, and

the mixture was ﬁltered through a Celite pad. The ﬁltered mixture was

concentrated in vacuum and chromatographed on silica gel with

hexanes/ethyl acetate = 3:1 to aﬀord 89 (15 mg, 45%) as a white

solid. ¹H NMR (400 MHz, CD₃OD): δ 9.16 (s, 1H), 8.15 (d, J = 8.00

Hz, 2H), 8.05 (s, 1H), 7.90 (s, 1H), 7.80−7.76 (m, 3H), 7.70 (d, J =

8.40 Hz, 2H), 7.38 (d, J = 8.40 Hz, 2H), 4.80 (s, 2H), 4.25−4.22 (m,

2H), 2.96−2.77 (m, 3H), 1.88−1.85 (m, 2H), 1.68−1.59 (m, 2H),

1.49 (s, 9H). MS (ESI, m/z): 579.2 [M + 1]⁺. ESI-HRMS (m/z): [M

+ 1]⁺calcd for C₃₂H₃₄N₄O₃F₃, 579.2583; found, 579.2588.

Methyl 6-Bromo-4-(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-tria-

zol-1-yl)picolinate (79). Compound 76 (7.0 mg, 0.027 mmol) was

converted to compound 79 (8.0 mg, 69%) as a white solid, using a

similar procedure to that used in the preparation of compound 77. ¹H

NMR (400 MHz, CD₃OD): δ 9.24 (s, 1H), 8.68 (s, 1H), 8.42 (s,

1H), 8.08 (d, J = 8.00 Hz, 2H), 7.73 (d, J = 8.00 Hz, 2H), 4.04 (s,

3H). MS (ESI, m/z): 427.0 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₁₆H₁₁BrN₄O₂F₃, 427.0017; found, 427.0022.

Methyl 4-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-6-(4-

(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)picolinate (80).

1

Method C. Yield: 35%. H NMR (400 MHz, CDCl₃): δ 9.05 (s,

1H), 8.67 (d, 1H), 8.41 (d, 1H), 8.10 (d, J = 8.00 Hz, 2H), 7.78−7.73

(m, 4H), 7.41 (d, J = 8.40 Hz, 2H), 4.35−4.21 (m, 2H), 2.90−2.79

(m, 3H), 1.93−1.89 (m, 2H), 1.75−1.63 (m, 2H), 1.49 (s, 9H). MS

(ESI, m/z): 608.3 [M + 1]⁺.

Methyl 2-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-6-(4-

(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)isonicotinate (81).

1

Method C. Yield: 63%. H NMR (400 MHz, CD₃OD): δ 9.36 (s,

1H), 8.49 (s, 1H), 8.38 (s, 1H), 8.20−8.16 (m, 4H), 7.78 (d, J = 8.40

Hz, 2H), 7.41 (d, J = 8.40 Hz, 2H), 4.26−4.16 (m, 2H), 4.04 (s, 3H),

2.95−2.77 (m, 3H), 1.88−1.85 (m, 2H), 1.69−1.59 (m, 2H), 1.49 (s,

9H). MS (ESI, m/z): 608.3 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₃₂H₃₃N₅O₄F₃, 608.2485; found, 608.2492.

Methyl 6-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-4-(4-

(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)picolinate (82).

Method C. Yield: 91%. ¹H NMR (400 MHz, CD₃OD): δ 9.44−

9.36 (m, 1H), 8.57−8.46 (m, 2H), 8.15−8.10 (m, 4H), 7.81−7.76

(m, 2H), 7.40−7.35 (m, 2H), 4.24−4.21 (m, 2H), 4.04 (s, 3H),

2.88−2.77 (m, 3H), 1.86−1.84 (m, 2H), 1.65−1.62 (m, 2H), 1.49 (s,

9H). MS (ESI, m/z): 608.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺

calcd for C₃₂H₃₃N₅O₄F₃, 608.2485; found, 608.2474.

tert-Butyl 4-(3′-(((Di-tert-butoxyphosphoryl)oxy)methyl)-5′-(4-

(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphenyl]-4-

yl)piperidine-1-carboxylate (90). A stirred solution of 89 (12 mg,

0.020 mmol) in dry THF (1.0 mL) was treated successively with

tetrazole (17 mg, 0.242 mmol) and di-tert-butyl N,N-diethylphos-

phoramidite (115 μL, 0.414 mmol) at room temperature under a N₂

atmosphere. The resulting solution was stirred at room temperature

for 1 h under a N₂atmosphere, and the reaction mixture was cooled

to −78 °C. 3-Chloroperbenzoic acid (MCPBA, contains 50%, 78 mg)

was added, and the reaction mixture was warmed to 0 °C and stirred

for 0.5 h. The reaction mixture was partitioned between ethyl acetate

(10 mL) and aqueous saturated NaHCO₃solution (10 mL), and the

aqueous phase was extracted with ethyl acetate (2 × 10 mL). The

combined organic layers were dried over Na₂SO₄, ﬁltered, and

concentrated under pressure. The residue was puriﬁed by silica gel

chromatography (chloroform/methanol = 30:1) to aﬀord compound

4-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-6-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)picolinic Acid (83).

1

Method A. Yield: 68%. H NMR (400 MHz, DMSO-d₆): δ 9.64 (s,

1H), 8.57 (s, 1H), 8.41 (s, 1H), 8.30 (d, J = 8.00 Hz, 2H), 7.94 (d, J =

8.40 Hz, 2H), 7.87 (d, J = 8.00 Hz, 2H), 7.48 (d, J = 8.40 Hz, 2H),

4.11−4.05 (m, 2H), 2.90−2.73 (m, 3H), 1.82−1.79 (m, 2H), 1.60−

1.50 (m, 2H), 1.42 (s, 9H). MS (ESI, m/z): 594.2 [M + 1]⁺. ESI-

HRMS (m/z): [M + 1]⁺calcd for C₃₁H₃₁N₅O₄F₃, 594.2328; found,

594.2320.

2-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-6-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)isonicotinic Acid

1

90 (13 mg, 84%) as a white solid. H NMR (400 MHz, CD₃OD): δ

1

(84). Method A. Yield: 91%. H NMR (400 MHz, CD₃OD): δ 9.38

9.22 (s, 1H), 8.18−8.16 (m, 3H), 8.01 (s, 1H), 7.83−7.79 (m, 3H),

7.73 (d, J = 8.40 Hz, 2H), 7.41 (d, J = 8.40 Hz, 2H), 5.23−5.22 (m,

2H), 4.27−4.23 (m, 2H), 2.96−2.77 (m, 3H), 1.91−1.87 (m, 2H),

1.71−1.59 (m, 2H), 1.52−1.50 (m, 27H). MS (ESI, m/z): 793.3 [M

+ Na]⁺. ESI-HRMS (m/z): [M + Na]⁺calcd for C₄₀H₅₀N₄O₆F₃PNa,

793.3318; found, 793.3303.

(s, 1H), 8.53 (s, 1H), 8.42 (s, 1H), 8.19 (t, J = 8.40 Hz, 4H), 7.79 (d,

J = 8.40 Hz, 2H), 7.42 (d, J = 8.00 Hz, 2H), 4.26−4.15 (m, 2H),

2.96−2.73 (m, 3H), 1.89−1.76 (m, 2H), 1.70−1.55 (m, 2H), 1.49 (s,

9H). MS (ESI, m/z): 616.3 [M + Na]⁺. ESI-HRMS (m/z): [M +

Na]⁺calcd for C₃₁H₃₀N₅O₄F₃Na, 616.2148; found, 616.2158.

6-(4-(1-(tert-Butoxycarbonyl)piperidin-4-yl)phenyl)-4-(4-(4-

(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)picolinic Acid (85).

Solubility and Lipophilicity Measurements. Solubility was

measured using a modiﬁed pION method.^2,25,26Lipophilicity was

determined based on the HPLC retention time as preported.^2,27

Competitive Binding Assay Using CHO Cells Expressing

hP2Y₁₄R (CHO-hP2Y₁₄R). The ﬂuorescence assay procedures used

are similar to previous reports.^2,7Twenty-four hours prior to the

assay, CHO-hP2Y₁₄R cells were plated in a ﬂat bottom 96-well plate

at a density of 1 × 10⁴cells well⁻¹and grown at 37 °C and 5%

CO₂(g) in Gibco Dulbecco’s modiﬁed Eagle’s medium/nutrient

mixture F-12 (DMEM/F12, 1:1) supplemented with 10% fetal bovine

serum, 100 units mL⁻¹penicillin, 100 μg mL⁻¹streptomycin, and

0.500 mg mL⁻¹selective antibiotic G418 sulfate. Cells were used for

binding assays when conﬂuency reached 80−90%. Unlabeled P2Y₁₄R

antagonists (test compounds) were stored frozen as 5 mM stock

solutions in DMSO. A 10-fold serial dilution of each ligand was

prepared in the appropriate complete cell medium to generate

solutions of unlabeled ligands with concentrations ranging from 100

μM to 100 pM. CHO-hP2Y₁₄R cells were ﬁrst incubated with

unlabeled ligand solutions for 30 min in an incubator at 37 °C and 5%

1

Method A. Yield: 77%. H NMR (400 MHz, CD₃OD): δ 9.27 (s,

1H), 8.58−8.52 (m, 2H), 8.10 (m, 4H), 7.74 (d, J = 7.60 Hz, 2H),

7.39 (broad s, 2H), 4.24−4.21 (m, 2H), 2.89−2.73 (m, 3H), 1.89−

1.86 (m, 2H), 1.70−1.60 (m, 2H), 1.47 (s, 9H). MS (ESI, m/z):

594.2 [M + 1]⁺. ESI-HRMS (m/z): [M + 1]⁺calcd for

C₃₁H₃₁N₅O₄F₃, 594.2328; found, 594.2318.

tert-Butyl 4-(3′-((2-(Dimethylamino)-2-oxoethoxy)carbonyl)-5′-

(4-(4-(triﬂuoromethyl)phenyl)-1H-1,2,3-triazol-1-yl)-[1,1′-biphen-

yl]-4-yl)piperidine-1-carboxylate (87). To a solution of 86 (166 mg,

0.280 mmol), which was synthesized according to literature

procedures reported,¹in DMF (10 mL) was added K₂CO₃(116

mg, 0.840 mmol). 2-Chloro-N,N-dimethylacetamide (68 mg, 0.560

mmol) was added to the reaction mixture under a N₂atmosphere, and

the reaction mixture was stirred at 45 °C for 1 h under a N₂

atmosphere. The reaction mixture was partitioned between ethyl

acetate (10 mL) and water (10 mL), and the aqueous phase was

extracted with ethyl acetate (2 × 10 mL). The combined organic

layers were dried over Na₂SO₄, ﬁltered, and concentrated under

5118

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

CO₂(g) without shaking followed by another 30 min of incubation

with 20 nM ﬂuorescent antagonist 2 under the same condition. After

the incubation periods, the cell medium was removed, and the cells

were washed three times with sterile 1× Dulbecco’s phosphate-

buﬀered saline (DPBS) minus Ca²⁺/Mg²⁺. Cells were then detached

from the plate using Corning Cellstripper and resuspended in sterile

1× DPBS minus Ca²⁺/Mg²⁺for ﬂow cytometry analysis. All

competitive binding assays were performed on a BD FACSCalibur

ﬂow cytometer in conjunction with the software programs BD

Bioscience PlateManager and CellQuest. The gathered data were

analyzed to obtain IC₅₀values using GraphPad Prism 8.0 (GraphPad,

San Diego, CA).

Competitive Binding Assay Using HEK293 Cells Expressing

mP2Y₁₄R (HEK293-mP2Y₁₄R). HEK293-mP2Y₁₄R cells were plated

in a ﬂat bottom 96-well plate at a density of 2 × 10⁴cells well⁻¹48 h

prior to the assay and were grown at 37 °C and 5% CO₂(g) in Eagle’s

minimum essential medium (EMEM) supplemented with 10% fetal

bovine serum, 100 units mL⁻¹penicillin, 100 μg mL⁻¹streptomycin,

and 0.555 mg mL⁻¹G418 sulfate. Cells were used for binding assays

when conﬂuency reached 80−90%. A 10-fold serial dilution of each

test compounds (5 mM stock solution, DMSO) was prepared in the

appropriate complete cell medium to generate solutions of unlabeled

ligands with concentrations ranging from 100 μM to 1 nM. HEK293-

mP2Y₁₄R cells were ﬁrst incubated with unlabeled ligand solutions for

30 min in an incubator at 37 °C and 5% CO₂(g) without shaking

followed by another 30 min incubation with 20 nM ﬂuorescent

antagonist 2 under the same condition. After the incubation periods,

the cell medium was removed, and the cells were washed three times

with sterile 1× DPBS minus Ca²⁺/Mg²⁺. Cells were then detached

from the plate using Corning Cellstripper and resuspended in sterile

1× DPBS minus Ca²⁺/Mg²⁺for ﬂow cytometry analysis. All

competitive binding assays were performed on a BD FACSCalibur

ﬂow cytometer in conjunction with the software programs BD

Bioscience PlateManager and CellQuest. The gathered data were

analyzed to obtain IC₅₀values using GraphPad Prism 8.0.

Esterase Cleavage of Prodrugs 37a−37c. HPLC Analysis. The

retention times of compounds were determined by Agilent 1100 series

HPLC equipped with an Agilent Eclipse 5 μm XDB-C18 analytical

column (250 × 4.6 mm, Agilent Technologies Inc., Palo Alto, CA).

Separations were achieved with a ﬂow rate of 1 mL min⁻¹and the

following mobile phase: (1) Method A for 37a: linear gradient solvent

system, CH₃CN/0.1% formic acid and 5% methanol in water from

30:70 to 55:45 in 20 min, and then to 100% CH₃CN for 5 min and

(2) Method B for 37b and 37c: linear gradient solvent system,

CH₃CN/0.1% formic acid in water from 50:50 to 95:5 in 20 min, and

then to 100% CH₃CN for 5 min. Peaks were detected by UV

absorption with a diode array detector at 210, 254, and 280 nm.

Analysis of the Enzymatic Hydrolysis. A solution of prodrug was

prepared in HEPES buﬀer (50 mM, pH 7.3) with 10% DMSO and

10% Kolliphor EL at a concentration of 1.5 mM. Three aliquots (200

μL) from the 1.5 mM stock solution were each treated with a 100 μL

solution of porcine liver esterase (PLE, 40 U in 400 μL HEPES buﬀer,

50 mM, pH 7.3). The samples were incubated (37a for 5, 35, 65, and

120 min at 37 °C or 37b and 37c for 0, 2, 6, 24, 48, and 72 h at 37

°C) with a mixing speed of 400 rpm in an Eppendorf (Enﬁeld, CT)

ThermoMixer prior to analysis. The samples were analyzed by HPLC

at each internal of time.

Hydrolytic Stabilities of the Prodrugs in Aqueous Medium.

Solutions (1.0 mM) of the prodrugs (37a− 37c) in HEPES buﬀer (50

mM, pH 7.3) with 10% DMSO and 10% Kolliphor EL were examined

immediately and after 24 h at 37 °C using identical HPLC conditions.

ADMET Measurements: In Vitro. The procedures for the

ADMET testing of compound 32 were reported in ref 2. hERG

inhibition was determined by a patch clamp method (Supporting

Information) and a ﬂuorescence polarization assay (Predictor hERG

ﬂuorescence polarization assay system by Thermo Fisher, performed

by Jai Research Foundation (JRF), Department of Toxicology,

Valvada - 396 105, Dist. Valsad, Gujarat, India) and with a patch

clamp electrophysiological assay (performed by Charles River

Laboratories, Cleveland, OH).^29,30

ADMET Measurements: In Vivo. In vivo pharmacokinetic study

is summarized in Table 5. The vehicle was prepared as follows.

Compound 32 was dissolved as a stock solution in pure DMSO.

For intravenous administration through the tail vein of each male

Wistar rat (body weight, 211−257 g), the stock solution was diluted

with an aqueous solution of 20% 2-hydroxypropyl-beta-cyclodextrin

(HPBCD) to make ﬁnal volume ratio of 10:90 of DMSO/HPBCD.

For intraperitoneal administration, the stock solution was added to an

equal volume of Kolliphor EL (polyoxyl castor oil) followed by PBS.

The ﬁnal volume ratio of DMSO/Kolliphor EL/PBS was 15:15:70. A

sample of dose formulation from each dose group was taken and

stored at −70 °C.

The full procedures were approved by the Institutional Animal

Ethics Committee (IAEC) of JRF, according to Association for

Assessment and Accreditation of Laboratory Animal Care (AAALAC)

standards. For the intravenous group, blood was collected through a

jugular vein catheter at 0.083, 0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-

dosing. For the intraperitoneal groups, blood was collected similarly at

0.25, 0.5, 1, 2, 4, 8, 12, and 24 h post-dosing. Blood samples of ∼200

μL were collected from the jugular vein of each rat and collected in

microcentrifuge tubes with heparin (20 IU mL⁻¹blood) added as an

anticoagulant. Following inversion four to ﬁve times, blood samples

were cooled on wet ice and centrifuged at 9000 rpm at 4 °C for 10

min. Plasma was collected and frozen immediately at −70 10 °C.

The frozen plasma samples were analyzed using the following

validated bioanalytical method. The plasma samples were thawed at

room temperature and centrifuged at 6000 rpm at 4 °C for 5 min.

Each plasma sample (25 μL) was combined in an Eppendorf tube

with 25 μL of 25 ng mL⁻¹ﬂufenamic acid as an internal standard and

50 μL of 5 mM ammonium acetate (pH 8). After vortexing, 1 mL of

tert-butyl methyl ether was added as a liquid−liquid extraction

solvent. Each sample was vortexed for 5 min and centrifuged at 10,000

rpm at 4 °C for 10 min. Aliquots of 0.9 mL were removed from the

supernatant portion, and each was dried using a Caliper TurboVap LV

evaporator (Biotage) under a N₂stream at 40 °C. The residue was

reconstituted with 500 μL of mobile phase, vortexed, and transferred

to the HPLC (ExionLC, SCIEX) autosampler for injection (10 μL),

with MS/MS (QTRAP 5500, SCIEX) analysis. The mobile phase

consisted of acetonitrile/5 mM ammonium acetate, pH 8.0 (85:15, v/

v), with a Waters XTerra MS C18 column (3.5 μm, 4.6 × 150 mm) at

a ﬂow rate of 0.400 mL min⁻¹. A 12-point calibration curve for the

analyte area as a function of concentration injected had an r = 0.9947.

Pharmacokinetic analysis of the plasma concentration−time data was

performed using the noncompartmental model of WinNonlin

software (version 8.2). No mortality or morbidity was observed in

any of the groups, and all animals had normal clinical signs

throughout the experimental period.

In Vivo Eﬃcacy Studies. All in vivo eﬃcacy studies received prior

institutional approval. Several potent antagonists (1a, 1b, 3a, and 32)

and three prodrug derivatives (37a−37c) were tested in the protease

mouse model of allergic asthma (sensitization with ovalbumin/

Aspergillus) at NIEHS as described.^2,6,33The timeline used for ASP/

OVA sensitization, P2Y₁₄R antagonist treatment, and OVA challenge

is shown in the study of Jung et al.²

Compound 32 was tested in a chronic constriction injury mouse

model of neuropathic pain, using a protocol that was approved by the

IACUC at St. Louis University.⁷The antagonist was administered 7

days post-chronic constriction injury of the sciatic nerve in adult male

mice, at the time of peak pain, as reported for various P2Y₁₄R

antagonists.^2,7

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00164.

Synthesis (Schemes S1 and S2), pain model data,

esterase cleavage curves, oﬀ-target binding inhibition

curves, pharmacological data, and molecular modeling

5119

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

John C. R. Randle − Random Walk Ventures, LLC, Boston,

Massachusetts 02111, United States

Zhoumou Chen − Department of Pharmacology and

Physiology and the Henry and Amelia Nasrallah Center for

Neuroscience, Saint Louis University School of Medicine, St.

Louis, Missouri 63104, United States

Daniela Salvemini − Department of Pharmacology and

Physiology and the Henry and Amelia Nasrallah Center for

Neuroscience, Saint Louis University School of Medicine, St.

Louis, Missouri 63104, United States

Tadeusz P. Karcz − Immunity, Inﬂammation and Disease

Laboratory, National Institute of Environmental Health

Sciences, National Institutes of Health, Durham, North

Carolina 27709, United States

data including tables reporting analysis of MD

simulations, plots of ligand RMSD, ligand−receptor

interaction energy and number of contacts during MD

simulations, last frame complex structure for compounds

29, 32, and 33, ADME tox procedures and results,

predicted LogD and LogS, patch clamp hERG assay, and

NMR and mass spectra of triazole derivatives (PDF)

3D coordinates of hP2Y₁₄R in complex with 29,

obtained through molecular docking (PDB)

3D coordinates of hP2Y₁₄R in complex with 32,

obtained through molecular docking (PDB)

Video S1, 30 ns MD simulations of hP2Y₁₄R in complex

with compound 29 (AVI)

Video S2, 30 ns MD simulations of hP2Y₁₄R in complex

with compound 32 (AVI)

Molecular formula strings of compounds 1a−1d, 3a, 3b,

4−36, and 37a−37c (CSV)

Donald N. Cook − Immunity, Inﬂammation and Disease

Laboratory, National Institute of Environmental Health

Sciences, National Institutes of Health, Durham, North

Carolina 27709, United States

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00164

AUTHOR INFORMATION

■

Corresponding Author

Notes

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; Phone: 301-

496-9024; Email: kennethj@niddk.nih.gov; Fax: 301-480-

8422

The authors declare no competing ﬁnancial interest.

^⊥Y.-H.J. and V.S. contributed equally to this work.

∥

́

Jagiellonian University Medical College, 30-688Krakow,

Poland 31-007, Poland (T.P.K.).

ACKNOWLEDGMENTS

■

We thank the Intramural Research Program of the NIH,

NIDDK (ZIA DK31116), and NIEHS (ZIAES102025) and

Kantum Diagnostics (Boston, MA) for support through

NIDDK CRADA 17-0824. We thank Bryan L. Roth, National

Institute of Mental Health’s Psychoactive Drug Screening

Program (Univ. North Carolina at Chapel Hill, contract

#HHSN-271-2008-00025-C), for screening data. This work

utilized the computational resources of the NIH HPC Biowulf

cluster (http://hpc.nih.gov).

Authors

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

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

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

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

ABBREVIATIONS

■

ADME, absorption, distribution, metabolism, excretion, and

toxicology; CHO, Chinese hamster ovary; DCM, dichloro-

methane; DIPEA, diisopropylethylamine; DMEM, Dulbecco’s

modiﬁed Eagle’s medium; DMF, N,N-dimethylformamide; EL,

extracellular loop; HATU, 1-[bis(dimethylamino)methylene]-

1H-1,2,3-triazolo[4,5-b]pyridinium 3-oxide hexaﬂuorophos-

phate; IE, interaction energy; LAH, lithium aluminum hydride;

MD, molecular dynamics; PDSP, Psychoactive Drug Screening

Program; RMSD, root-mean-square deviation; SAR, struc-

ture−activity relationship; UDPG, uridine-5′-diphosphoglu-

cose; TBAF, tetrabutylammonium ﬂuoride; TEA, triethyl-

amine; TFA, triﬂuoroacetic acid; THF, tetrahydrofuran; TM,

transmembrane helix; TMS, trimethylsilyl

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

David I. Lieberman − 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

Varun Gopinatth − 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

REFERENCES

■

(1) Yu, J.; Ciancetta, A.; Dudas, S.; Duca, S.; Lottermoser, J.;

Jacobson, K. A. Structure-guided modification of heterocyclic

antagonists of the P2Y₁₄receptor. J. Med. Chem. 2018, 61, 4860−

4882.

(2) Jung, Y.-H.; Yu, J.; Wen, Z.; Salmaso, V.; Karcz, T. P.; Phung, N.

B.; Chen, Z.; Duca, S.; Bennett, J. M.; Dudas, S.; Salvemini, D.; Gao,

Z.-G.; Cook, D. N.; Jacobson, K. A. Exploration of alternative

scaffolds for P2Y₁₄receptor antagonists containing a biaryl core. J.

Med. Chem. 2020, 63, 9563−9589.

5120

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(3) Battistone, M. A.; Mendelsohn, A. C.; Spallanzani, R. G.;

Allegretti, A. S.; Liberman, R. N.; Sesma, J.; Kalim, S.; Wall, S. M.;

Bonventre, J. V.; Lazarowski, E. R.; Brown, D.; Breton, S. Pro-

inflammatory P2Y₁₄receptor inhibition protects against ischemic

acute kidney injury in mice. J. Clin. Invest. 2020, 130, 3734−3749.

(4) Xu, J.; Morinaga, H.; Oh, D.; Li, P.; Chen, A.; Talukdar, S.;

Mamane, Y.; Macini, J. A.; Nawrocki, A. R.; Lazarowski, E.; Olefsky, J.

M.; Kim, J. J. GPR105 ablation prevents inflammation and improves

insulin sensitivity in mice with diet-induced obesity. J. Immunol. 2012,

189, 1992−1999.

(18) Robichaud, J.; Fournier, J.-F.; Gagné, S.; Gauthier, J. Y.; Hamel,

M.; Han, Y.; Hénault, M.; Kargman, S.; Levesque, J.-F.; Mamane, Y.;

Mancini, J.; Morin, N.; Mulrooney, E.; Wu, J.; Black, W. C. Applying

the pro-drug approach to afford highly bioavailable antagonists of

P2Y₁₄. Bioorg. Med. Chem. Lett. 2011, 21, 4366−4368.

(19) Mazák, K.; Noszál, B. Physicochemical properties of

zwitterionic drugs in therapy. ChemMedChem 2020, 15, 1102−1110.

(20) Sabbadin, D.; Ciancetta, A.; Moro, S. Bridging molecular

docking to membrane molecular dynamics to investigate GPCR-

ligand recognition: The human A₂adenosine receptor as a key study.

A

(5) Jain, S.; Jacobson, K. A. Adipocyte speciﬁc ablation of P2Y₁₄R

improves glucose metabolism in mice with diet-induced obesity.

American Diabetes Assoc., 80th Scientiﬁc Session, Abstract; 2020.

(6) Karcz, T. P.; Whitehead, G. S.; Nakano, K.; Nakano, H.; Grimm,

S. A.; Williams, J. G.; Deterding, L. J.; Jacobson, K. A.; Cook, D. N.

UDP-glucose and P2Y₁₄receptor amplify allergen-induced airway

eosinophilia. J. Clin. Invest. 2021, 131, e140709.

(7) Mufti, F.; Jung, Y. H.; Giancotti, L. A.; Yu, J.; Chen, Z.; Phung,

N. B.; Jacobson, K. A.; Salvemini, D. P2Y₁₄receptor antagonists

reverse chronic neuropathic pain in a mouse model. ACS Med. Chem.

Lett. 2020, 11, 1281−1286.

J. Chem. Inf. Model. 2014, 54, 169−183.

(21) Strupczewski, J. T.; Allen, R. C.; Gardner, B. A.; Schmid, B. L.;

Stache, U.; Glamkowski, E. J.; Jones, M. C.; Ellis, D. B.; Huger, F. P.;

Dunn, R. W. Synthesis and neuroleptic activity of 3-(1-substituted-4-

piperidinyl)-l,2-benzisoxazoles. J. Med. Chem. 1985, 28, 761−769.

(22) Ballesteros, J. A.; Weinstein, H. [19] Integrated methods for the

construction of three-dimensional models and computational probing

of structure-function relations in G protein-coupled receptors.

Methods Neurosci. 1995, 25, 366−428.

(23) Pennington, L. D.; Moustakas, D. T. The necessary nitrogen

atom: A versatile high-impact design element for multiparameter

optimization. J. Med. Chem. 2017, 60, 3552−3579.

(8) Lin, J.; Liu, F.; Zhang, Y.-y.; Song, N.; Liu, M.-k.; Fang, X.-y.;

Liao, D.-q.; Zhou, C.; Wang, H.; Shen, J.-f. P2Y₁₄receptor is

functionally expressed in satellite glial cells and mediates interleukin-

1 β and chemokine CCL2 secretion. J. Cell. Physiol. 2019, 234,

21199−21210.

(24) Gardell, S. J.; Hopf, M.; Khan, A.; Dispagna, M.; Hampton

Sessions, E.; Falter, R.; Kapoor, N.; Brooks, J.; Culver, J.; Petucci, C.;

Ma, C. T.; Cohen, S. E.; Tanaka, J.; Burgos, E. S.; Hirschi, J. S.; Smith,

S. R.; Sergienko, E.; Pinkerton, A. B.; Boosting, N. A. D. ⁺with a small

molecule that activates NAMPT. Nat. Commun. 2019, 10, 3241.

(25) Kiselyuk, A. S. HNF4 alpha bioactive ligands discovered by a high-

throughput screen for modulators of the human insulin promoter

(Doctoral dissertation, pp. 65). University of California: San Diego,

2011. (https://escholarship.org/content/qt64k4q3gr/qt64k4q3gr.

pdf)

(9) Jacobson, K. A.; Delicado, E. G.; Gachet, C.; Kennedy, C.; von

Ku

̈

gelgen, I.; Li, B.; Miras-Portugal, T.; Novak, I.; Schöneberg, T.;

ller, C. E. Update of

Perez-Sen, R.; Thor, D.; Wu, B.; Yang, Z.; Mu

̈

P2Y receptor pharmacology: IUPHAR review “27 ”. Br. J. Pharmacol.

2020, 177, 2413−2433.

(10) Kinoshita, M.; Nasu-Tada, K.; Fujishita, K.; Sato, K.; Koizumi,

S. Secretion of matrix metalloproteinase-9 from astrocytes by

inhibition of tonic P2Y₁₄-receptor-mediated signal(s). Cell. Mol.

Neurobiol. 2013, 33, 47−58.

(11) Zhang, Z.; Hao, K.; Li, H.; Lu, R.; Liu, C.; Zhou, M.; Li, B.;

Meng, Z.; Hu, Q.; Jiang, C. Design, synthesis and anti-inflammatory

evaluation of 3-amide benzoic acid derivatives as novel P2Y₁₄receptor

antagonists. Eur. J. Med. Chem. 2019, 181, 111564.

(12) Hamel, M.; Henault, M.; Hyjazie, H.; Morin, N.; Bayly, C.;

Skorey, K.; Therien, A. G.; Mancini, J.; Brideau, C.; Kargman, S.

Discovery of novel P2Y₁₄agonist and antagonist using conventional

and nonconventional methods. J. Biomol. Screening 2011, 16, 1098−

1105.

(13) Wang, W.; Liu, C.; Li, H.; Tian, S.; Liu, Y.; Wang, N.; Yan, D.;

Li, H.; Hu, Q. Discovery of novel and potent P2Y₁₄R antagonists via

structure-based virtual screening for the treatment of acute gouty

arthritis. J. Adv. Res. 2020, 23, 133−142.

(26) Yan, Z.; Caldwell, G. Optimization in Drug Discovery: In Vitro

Methods (pp. 58); Humana Press.

(27) Valkó, K. Application of high-performance liquid chromatog-

raphy based measurements of lipophilicity to model biological

distribution. J. Chromatogr. A 2004, 1037, 299−310.

(28) Besnard, J.; Ruda, G. F.; Setola, V.; Abecassis, K.; Rodriguiz, R.

M.; Huang, X.-P.; Norval, S.; Sassano, M. F.; Shin, A. I.; Webster, L.

A.; Simeons, F. R. C.; Stojanovski, L.; Prat, A.; Seidah, N. G.;

Constam, D. B.; Bickerton, G. R.; Read, K. D.; Wetsel, W. C.; Gilbert,

I. H.; Roth, B. L.; Hopkins, A. L. Automated design of ligands to

polypharmacological profiles. Nature 2012, 492, 215−220.

(29) Wible, B. A.; Hawryluk, P.; Ficker, E.; Kuryshev, Y. A.; Kirsch,

G.; Brown, A. M. HERG-Lite ®: a novel comprehensive high-

throughput screen for drug-induced hERG risk. J. Pharmacol. Toxicol.

Methods 2005, 52, 136−145.

(30) Piper, D. R.; Duff, S. R.; Eliason, H. C.; Frazee, W. J.; Frey, E.

A.; Fuerstenau-Sharp, M.; Jachec, C.; Marks, B. D.; Pollok, B. A.;

Shekhani, M. S.; Thompson, D. V.; Whitney, P.; Vogel, K. W.; Hess,

S. D. Development of the predictor HERG fluorescence polarization

assay using a membrane protein enrichment approach. Assay Drug

Dev. Technol. 2008, 6, 213−223.

(31) Perez, C.; Daniel, K. B.; Cohen, S. M. Evaluating prodrug

strategies for esterase-triggered release of alcohols. ChemMedChem

2013, 8, 1662−1667.

(32) Bennett, G. J.; Xie, Y. K. A peripheral mononeuropathy in rat

that produces disorders of pain sensation like those seen in man. Pain

1988, 33, 87−107.

(33) Whitehead, G. S.; Thomas, S. Y.; Shalaby, K. H.; Nakano, K.;

Moran, T. P.; Ward, J. M.; Flake, G. P.; Nakano, H.; Cook, D. N.

TNF is required for TLR ligand −mediated but not protease-mediated

allergic airway inflammation. J. Clin. Invest. 2017, 127, 3313−3326.

(34) Mignani, S.; Rodrigues, J.; Tomas, H.; Jalal, R.; Singh, P. P.;

Majoral, J.-P.; Vishwakarma, R. A. Present drug-likeness filters in

medicinal chemistry during the hit and lead optimization process:

how far can they be simplified? Drug Discovery Today 2018, 23, 605−

615.

(14) Junker, A.; Balasubramanian, R.; Ciancetta, A.; Uliassi, E.;

Kiselev, E.; Martiriggiano, C.; Trujillo, K.; Mtchedlidze, G.; Birdwell,

L.; Brown, K. A.; Harden, T. K.; Jacobson, K. A. Structure-based

design of 3-(4-aryl-1H-1,2,3-triazol-1-yl)-biphenyl derivatives as P2Y₁₄

receptor antagonists. J. Med. Chem. 2016, 59, 6149−6168.

(15) Zhang, J.; Zhang, K.; Gao, Z.-G.; Paoletta, S.; Zhang, D.; Han,

G. W.; Li, T.; Ma, L.; Zhang, W.; Muller, C. E.; Yang, H.; Jiang, H.;

̈

Cherezov, V.; Katritch, V.; Jacobson, K. A.; Stevens, R. C.; Wu, B.;

Zhao, Q. Agonist-bound structure of the human P2Y₁₂receptor.

Nature 2014, 509, 119−122.

(16) Kiselev, E.; Barrett, M. O.; Katritch, V.; Paoletta, S.; Weitzer, C.

D.; Hammes, E.; Yin, A. L.; Zhao, Q.; Stevens, R. C.; Harden, T. K.;

Jacobson, K. A. Exploring a 2-naphthoic acid template for the

structure-based design of P2Y₁₄receptor antagonist molecular probes.

ACS Chem. Biol. 2014, 9, 2833−2842.

(17) Barrett, M. O.; Sesma, J. I.; Ball, C. B.; Jayasekara, P. S.;

Jacobson, K. A.; Lazarowski, E. R.; Harden, T. K. A selective high

affinity antagonist of the P2Y₁₄receptor inhibits UDP-glucose-

stimulated chemotaxis of human neutrophils. Mol. Pharmacol. 2013,

84, 41−49.

5121

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

(35) Dalvie, D. K.; Kalgutkar, A. S.; Khojasteh-Bakht, S. C.; Obach,

R. S.; O’Donnell, J. P. Biotransformation reactions of five-membered

aromatic heterocyclic rings. Chem. Res. Toxicol. 2002, 15, 269−299.

(36) Zhu, J.; Mo, J.; Lin, H.-z.; Chen, Y.; Sun, H.-p. The recent

progress of isoxazole in medicinal chemistry. Bioorg. Med. Chem. 2018,

26, 3065−3075.

(37) Sun, H.; Shi, M.; Zhang, W.; Zheng, Y.-M.; Xu, Y.-Z.; Shi, J.-J.;

Liu, T.; Gunosewoyo, H.; Pang, T.; Gao, Z. B.; Yang, F.; Tang, J.; Yu,

L.-F. Development of novel alkoxyisoxazoles as sigma-1 receptor

antagonists with antinociceptive efficacy. J. Med. Chem. 2016, 59,

6329−6343.

(54) Essmann, U.; Perera, L.; Berkowitz, M. L.; Darden, T.; Lee, H.;

Pedersen, L. G. A smooth particle mesh Ewald method. J. Chem. Phys.

1995, 103, 8577.

(55) Phillips, J. C.; Braun, R.; Wang, W.; Gumbart, J.; Tajkhorshid,

E.; Villa, E.; Chipot, C.; Skeel, R. D.; Kalé, L.; Schulten, K. Scalable

molecular dynamics with NAMD. J. Comput. Chem. 2005, 26, 1781−

1802.

(56) Williams, T.; Kelley, C. Gnuplot 5.0.[Sl]; http://www.gnuplot.

info.

(57) Tetko, I. V.; Tanchuk, V. Y. Application of associative neural

networks for prediction of lipophilicity in ALOGPS 2.1 program. J.

Chem. Inf. Comput. Sci. 2002, 42, 1136−1145.

(58) Belley, M.; Deschenes, D.; Fortin, R.; Fournier, J.-F.; Gagne, S.;

Gareau, Y.; Gauthier, J. Y.; Li, L.; Robichaud, J.; Therien, M.;

Tranmer, G. K.; Wang, Z. et al.. WO2009070873 A1, June 11, 2009.

(38) Ratnatilaka Na Bhuket, P.; Jithavech, P.; Ongpipattanakul, B.;

Rojsitthisak, P. Interspecies differences in stability kinetics and plasma

esterases involved in hydrolytic activation of curcumin diethyl

disuccinate, a prodrug of curcumin. RSC Adv. 2019, 9, 4626−4634.

̈

(39) Schrodinger Release 2019−3: Maestro, Schrodinger, LLC: New

York, NY, 2019.

(40) Sastry, G. M.; Adzhigirey, M.; Day, T.; Annabhimoju, R.;

Sherman, W. Protein and ligand preparation: Parameters, protocols,

and influence on virtual screening enrichments. J. Comput.-Aided Mol.

Des. 2013, 27, 221−234.

(41) Harder, E.; Damm, W.; Maple, J.; Wu, C.; Reboul, M.; Xiang, J.

Y.; Wang, L.; Lupyan, D.; Dahlgren, M. K.; Knight, J. L.; Kaus, J. W.;

Cerutti, D. S.; Krilov, G.; Jorgensen, W. L.; Abel, R.; Friesner, R. A.

OPLS3: A force field providing broad coverage of drug-like small

molecules and proteins. J. Chem. Theory Comput. 2016, 12, 281−296.

(42) Friesner, R. A.; Banks, J. L.; Murphy, R. B.; Halgren, T. A.;

Klicic, J. J.; Mainz, D. T.; Repasky, M. P.; Knoll, E. H.; Shelley, M.;

Perry, J. K.; Shaw, D. E.; Francis, P.; Shenkin, P. S. Glide: A new

approach for rapid, accurate docking and scoring. 1. Method and

assessment of docking accuracy. J. Med. Chem. 2004, 47, 1739−1749.

(43) Doerr, S.; Harvey, M. J.; Noé, F.; De Fabritiis, G. HTMD:

High-throughput molecular dynamics for molecular discovery. J.

Chem. Theory Comput. 2016, 12, 1845−1852.

(44) Humphrey, W.; Dalke, A.; Schulten, K. VMD: Visual molecular

dynamics. J. Mol. Graph. 1996, 14, 33−38 27.

(45) Lomize, M. A.; Pogozheva, I. D.; Joo, H.; Mosberg, H. I.;

Lomize, A. L. OPM Database and PPM web server: Resources for

positioning of proteins in membranes. Nucleic Acids Res. 2012, 40,

D370−D376.

(46) Jorgensen, W. L.; Chandrasekhar, J.; Madura, J. D.; Impey, R.

W.; Klein, M. L. Comparison of simple potential functions for

simulating liquid water. J. Chem. Phys. 1983, 79, 926.

(47) Harvey, M. J.; Giupponi, G.; Fabritiis, G. D. ACEMD:

Accelerating biomolecular dynamics in the microsecond time scale. J.

Chem. Theory Comput. 2009, 5, 1632−1639.

(48) Best, R. B.; Zhu, X.; Shim, J.; Lopes, P. E. M.; Mittal, J.; Feig,

M.; Mackerell, A. D., Jr. Optimization of the additive CHARMM all-

atom protein force field targeting improved sampling of the backbone

φ , ψ and side-chain χ₁and χ₂dihedral angles. J. Chem. Theory Comput.

2012, 8, 3257−3273.

(49) Klauda, J. B.; Venable, R. M.; Freites, J. A.; O’Connor, J. W.;

Tobias, D. J.; Mondragon-Ramirez, C.; Vorobyov, I.; MacKerell, A.

D., Jr.; Pastor, R. W. Update of the CHARMM all-atom additive force

field for lipids: Validation on six lipid types. J. Phys. Chem. B 2010,

114, 7830−7843.

(50) Vanommeslaeghe, K.; MacKerell, A. D., Jr. Automation of the

CHARMM general force field (CGenFF) I: Bond perception and

atom typing. J. Chem. Inf. Model. 2012, 52, 3144−3154.

(51) Vanommeslaeghe, K.; Raman, E. P.; MacKerell, A. D., Jr.

Automation of the CHARMM general force field (CGenFF) II:

Assignment of bonded parameters and partial atomic charges. J. Chem.

Inf. Model. 2012, 52, 3155−3168.

(52) CHARMM General Force Field (CGenFF) program, https://

cgen ﬀ .umaryland.edu/

(53) Kräutler, V.; van Gunsteren, W. F.; Hunenberger, P. H. A fast

̈

SHAKE algorithm to solve distance constraint equations for small

molecules in molecular dynamics simulations. J. Comput. Chem. 2001,

22, 501−508.

5122

https://doi.org/10.1021/acs.jmedchem.1c00164

J. Med. Chem. 2021, 64, 5099−5122

Article Doi

Structure-Activity Relationship of Heterocyclic P2Y14Receptor Antagonists: Removal of the Zwitterionic Character with Piperidine Bioisosteres

DOI: 10.1021/acs.jmedchem.1c00164

Source and publish data:

Authors:

Article abstract of DOI:10.1021/acs.jmedchem.1c00164

Full text of DOI:10.1021/acs.jmedchem.1c00164

Products guided by the article

R&D Labs maybe for 1008505-60-9

Relevant to this article

Hot Product