Design, Synthesis, and Pharmacological Characterization of Heterobivalent Ligands for the Putative 5-HT2A/mGlu2Receptor Complex

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Design, Synthesis, and Pharmacological Characterization of

Heterobivalent Ligands for the Putative 5‑HT_2A/mGlu₂Receptor

Complex

Christian B. M. Poulie, Na Liu, Anders A. Jensen,* and Lennart Bunch*

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ABSTRACT: We report the synthesis of the ﬁrst series of

heterobivalent ligands targeting the putative heteromeric 5-HT_2A/

mGlu₂receptor complex, based on the 5-HT_2Aantagonist MDL-

100,907 and the mGlu₂ago-PAM JNJ-42491293. The functional

properties of monovalent and heterobivalent ligands were

characterized in 5-HT_2A-, mGlu₂/Gqo5-, 5-HT_2A/mGlu₂-, and 5-

HT_2A/mGlu₂/Gqo5-expressing HEK293 cells using a Ca²⁺imaging

assay and a [³H]ketanserin binding assay. Pronounced functional

crosstalk was observed between the two receptors in 5-HT_2A/

mGlu₂and 5-HT_2A/mGlu₂/Gqo5 cells. While the synthesized monovalent ligands retained the 5-HT_2Aantagonist and mGlu₂ago-

PAM functionalities, the seven bivalent ligands inhibited 5-HT-induced responses in 5-HT_2A/mGlu₂cells and both 5-HT- and Glu-

induced responses in 5-HT_2A/mGlu₂/Gqo5 cells. However, no deﬁnitive correlation between the functional potency and spacer

length of the ligands was observed, an observation substantiated by the binding aﬃnities exhibited by the compounds in 5-HT_2A, 5-

HT_2A/mGlu₂, and 5-HT_2A/mGlu₂/Gqo5 cells. In conclusion, while functional crosstalk between 5-HT2A and mGlu2 was

demonstrated, it remains unclear how these heterobivalent ligands interact with the putative receptor complex.

nervous system, 5-HT_2Aand mGlu₂have both been pursued

intensively as drug targets in several cognitive and psychiatric

disorders over the years. Atypical antipsychotics act in part as

5-HT_2Aantagonists, and augmentation of 5-HT_2Asignaling has

in recent years gained renewed therapeutic interest in light of

remarkable eﬀects exhibited by classical hallucinogens in

clinical trials for depression and other psychiatric disor-

ders.^13,14The therapeutic potential in the augmentation of

signaling through the mGlu₂receptors within indications such

as schizophrenia and depression has been investigated for

mGlu_2/3agonists in clinical trials.¹⁵In addition to the

respective physiological functions and therapeutic potential

of the two receptors, several lines of evidence from

electrophysiological, biochemical, and behavioral studies have

indicated substantial negative functional crosstalk between 5-

HT_2Aand mGlu₂in the frontal cortex where the two receptors

exhibit an overlapping distribution. While this crosstalk could

arise from several diﬀerent origins, including synaptic

mechanisms and/or crosstalk between diﬀerent stages of the

signaling pathways activated through the two receptors,^16,17

INTRODUCTION

■

G-protein-coupled receptors (GPCRs) mediate signaling of a

diverse range of neurotransmitters and hormones and are

divided into ﬁve major classes (class A, class B, class C,

adhesion, and frizzled/taste receptors).¹GPCRs exert their

functions as monomers but may also assemble into functional

dimeric or oligomeric complexes of homomeric or heteromeric

compositions.²While the signaling properties of monomeric

and homodimeric GPCRs are often diﬃcult to dissect from

one another, GPCR heterodimers have, in several cases, been

reported to exhibit distinct functional properties compared to

those of their two parent monomeric receptors.²⁻⁷Expression

and unequivocal demonstration of the existence of GPCR

dimer/oligomers in vitro are challenging because of the cell

line-dependent receptor expression levels and other exper-

imental factors.⁸Moreover, studies of GPCR dimerization/

oligomerization in vivo are associated with additional

challenges,⁹and with the exception of a few examples of

truly well-established GPCR heterodimerization in vivo,^10,11

the actual extent and physiological importance of GPCR

dimerization/oligomerization in native tissues are thus still

largely unknown.

Received: June 19, 2020

The present study focuses on the putative heteromeric

complex of the serotonin 2A (5-HT_2A) receptor and the

metabotropic glutamate 2 (mGlu₂) receptor.¹²As key

mediators of neurotransmission mediated by serotonin (5-

hydroxytryptamine, 5-HT) and glutamate (Glu) in the central

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A

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Figure 1. (A) Chemical structures of 5-HT_2Ahead group MDL-100,907 and mGlu₂head group JNJ-42491293. (B) Cartoon drawing of a

heterobivalent ligand.

a

Scheme 1. Synthesis of 5-HT_2AHead Group

a

Reagents and conditions: (a) TBDPS-Cl, imidazole, DCM, rt, 20 h (96%); (b) O-methylhydroxylamine, EDCI, HOBt, Et₃N, DMF, rt, 18 h

(94%); (c) (i) nBuLi, THF 0 °C → rt, 2 h and (ii) 4, −42 °C → rt, 18 h (59%); (d) (i) TFA, DCM, 0 °C → rt, 2 h and (ii) NH₄OH (96%); (e)

NaI, NaHCO₃, DMF, 65 °C, 16 h (48%); (f) MeOTs, K₂CO₃, DMF, 60 °C, 17 h (47%); and (g) KI, K₂CO₃, bromo-2-methoxyethane, DMF, 65

°C, 17 h (56%).

the existence of a heteromeric 5-HT_2A/mGlu₂complex

An attractive strategy to enable selective targeting of a

heteromeric receptor complex over its two parent receptors is

the design of a heterobivalent ligand, which comprises two

receptor-selective head groups separated by a chemical spacer

of suitable length and ﬂexibility.³³⁻³⁵The bivalent strategy was

pioneered by Portoghese and colleagues in their development

of selective ligands for studies of opioid receptor dimeriza-

tion^28,36,37and has subsequently been pursued in the design of

ligands for other GPCR dimers.³In the present work, we

report the design, synthesis, and pharmacological character-

ization of a series of heterobivalent ligands aimed at the

putative 5-HT_2A/mGlu₂heteromer.

́

proposed by Gonzalez-Maeso and colleagues in 2008 oﬀered

an attractive molecular mechanism by which this crosstalk

could be explained.^12,18,19Furthermore, both of these receptors

are known to form homodimers²⁰as well as heteromeric

complexes with other receptors. For example, 5-HT_2Ahas been

reported to form functional heterodimers with other class A

GPCRs, speciﬁcally, the dopamine D₂receptor^21,22and the

cannabinoid CB₁receptor.²³Analogous to other class C

GPCRs, mGlu₂is expressed as a constitutive dimer²⁴and, in

addition to its homodimeric assembly, it has also been shown

to form heteromeric complexes with other mGlu recep-

tors.²⁵⁻²⁷Although the reported eﬃcient complex formation in

vitro between two structurally diﬀerent and distantly related

GPCRs is quite remarkable, other family A and C GPCRs have

also been proposed to assemble into heteromeric com-

plexes.²⁸⁻³⁰Interestingly, the reported extent and character-

istics of the functional crosstalk between the two receptors in

this putative 5-HT_2A/mGlu₂heteromer in heterologous

expression systems have varied considerably in previous

studies (this will be outlined in detail in Results and

Discussion).^{18,19,27,31,32}As a possible explanation for this

variation, the relative expression ratio of the two receptors in

the cells has been proposed to be important for the exact

functional properties exhibited by the putative heteromer

assembled from them.^16,18,19

RESULTS AND DISCUSSION

■

Design. The heterobivalent ligands were designed to

comprise the potent 5-HT_2A-selective antagonist MDL-

100,907 (volinanserin) as the 5-HT_2Ahead group (Figure

1A). This compound is attractive for this purpose, as it has

previously been incorporated successfully into homobivalent 5-

HT_2Aligands, using the meta-methoxy position as the anchor

point for the spacer.³⁸⁻⁴⁰Furthermore, it has been reported

that the secondary chiral alcohol in MDL-100,907 can

successfully be substituted for a carbonyl group.⁴⁰

For the mGlu₂head group, the low-nanomolar potent

mGlu₂-selective allosteric agonist and positive allosteric

modulator (ago-PAM) JNJ-42491293 (Figure 1A) was

B

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a

Scheme 2. Synthesis of Heterocycle 16

a

Reagents and conditions: (a) hydrazine·hydrate, dioxane, 70 °C, 16 h (64%); (b) oxalyl chloride, DCM, rt, 4 h (98%); (c) Et₃N, DCM, rt, 2 h

(63%); and (d) dimethylsulfone, sulfolane, 160 °C, 3 h (68%).

a

Scheme 3. Synthesis of Methoxy and 2-Methoxyethoxy Derivatives of the mGlu₂Head Group

a

Reagents and conditions: (a) BBr₃(33 wt% in AcOH), reﬂux, 2 h (91%); (b) (Boc)₂O, Et₃N, DCM, rt, 18 h (94−96%); (c) KI, K₂CO₃, bromo-2-

methoxyethane or MeI, DMF, 60 °C, 17 h (68−88%); (d) (i) TFA, DCM, rt, 2 h and (ii) NH₄OH (97−99%); and (e) 0.05 equiv Pd(OAc)₂, 0.1

equiv XantPhos, 2.4 equiv Cs₂CO₃, dioxane, reﬂux, 36 h (22−78%).

selected.⁴¹Extensive structure−activity relationship (SAR)

information is available for this compound class.⁴¹⁻⁵⁰Derived

from all these data, it is apparent that the heterocycle of this

compound class interacts deeply within the allosteric binding

pocket in the 7-transmembrane domain of mGlu₂, and that the

anisole moiety points toward the solvent. Nevertheless, we

decided to extend the SAR by synthesizing analogues 31−36,

in order to determine the optimal orientation of the anisole

moiety (described in detail below).

For the chemical spacer, a polyethylene glycol (PEG) chain

was selected as it oﬀers a balanced lipophilicity proﬁle because

of its alternating ethers and hydrocarbons and allows for

signiﬁcant ﬂexibility between the two head groups.^33,51Largely,

because of the unknown structure of this putative heteromeric

complex, the main focus of the present study was to address

the optimal spacer length and, as such, we have selected seven

spacer lengths ranging from 3 to 12 PEG units (9−36 atoms).

Chemistry. The synthesis of 5-HT_2Ahead group 8 followed

the previously reported method,^52,53and it commenced with

the protection of commercially available guaiacol (1) as its tert-

butyldiphenylsilyl (TBDPS) ether 2 (Scheme 1). Directed

ortho metalation of 2 followed by quenching with the Weinreb

amide 4⁵⁴(obtained from 3) gave rise to 5 in 59% yield.^52,53

The product was obtained as a single regioisomer, which is in

accordance with literature reports.⁵⁵Subsequently, 5 was

deprotected with triﬂuoroacetic acid (TFA) in dichloro-

methane (DCM) and converted to its corresponding free

base 7. Alkylation of 7 with bromide 6 was performed under

previously reported conditions.⁵³However, in our hands, the

TBDPS ether was also cleaved, and head group 8 could be

isolated directly without the need for an additional

deprotection step.^52,53

The methyl ether analogue 9 was now prepared by

methylation of 8 with MeOTs, and 2-methoxyethoxy ether

10 was obtained using bromo-2-methoxyethane as the

alkylating agent. Interestingly, no formation of the quaternary

ammonium salt was observed, despite the fact that 2 equiv of

the bromide alkylating agent was used.

The synthesis of mGlu₂head group 37 was based on a

previously reported work by Cid and colleagues,⁵⁰and it

commenced with the reaction of 2,3-dichloro-4-iodopyridine

(11) with hydrazine·hydrate to give 12 (Scheme 2). Acid

chloride 14 was prepared from 2-cyclopropylacetic acid (13)

and oxalyl chloride and isolated by direct distillation of the

reaction mixture. Acylation of 12 with 14 gave intermediate

15, which was then set up for a cyclocondensation reaction.

C

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a

Scheme 4. Synthesis of Bivalent Ligands

a

Reagents and conditions: (a) NaH, ditosyl-(PEG)_n, THF, rt, 17 h (42−51%) and (b) NaH, DMF, rt, 17 h (10−23%).

Following the reported conditions, this step proceeded with

low isolated yields (21%) of 16. However, addition of

dimethylsulfone and sulfolane greatly improved the isolated

yield (68%).

(PEG)₄and 5-HT_2Ahead group 8 could be readily isolated

using preparative thin-layer chromatography (TLC). Thus,

intermediates 38−44 were ﬁrst prepared in 42−49% yield.

Subsequently, phenolic mGlu₂head group 37 was deproto-

nated with 2.0 equiv of NaH in DMF, followed by addition of

appropriate intermediates 38−44. The target bivalent ligands

(45−51) were isolated by preparative high-performance liquid

chromatography (HPLC) in 10−23% isolated yield as the

corresponding TFA salts (Scheme 4).

Pharmacological Characterization. Construction of

Stable HEK293 Cell Lines Expressing 5-HT_2A, mGlu₂, and 5-

HT_2A/mGlu₂. In this study, we wanted to characterize the

functional properties of the synthesized monovalent and

bivalent ligands at cell lines expressing the putative 5-HT_2A/

mGlu₂heteromer and its two parent receptors using the same

functional assay. For this purpose, four stable HEK293 cell

lines expressing 5-HT_2Aand mGlu₂on their own or in

combination were established, and a Ca²⁺imaging assay using

the ﬂuorescent calcium dye Fluo-4 was applied. A previously

constructed stable 5-HT_2A-HEK293 cell line was used to assay

this receptor,⁵⁶and the other parent receptor was assayed

using a HEK293 cell line coexpressing mGlu₂and the chimeric

Gα-protein Gqo5 that directs the Gα_i/o-signaling of mGlu₂into

the Gα_q/11-pathway and intracellular Ca²⁺mobilization.⁵⁷To

facilitate testing of the ligands at the putative 5-HT_2A/mGlu₂

complex, the following two cell lines coexpressing the two

receptors were established: 5-HT_2A/mGlu₂- and 5-HT_2A/

mGlu₂/Gqo5-HEK293 cells. The expressed receptors and the

origin of intracellular Ca²⁺mobilization measured in the four

cell lines are illustrated in Figure 2A.

The series of methoxy and 2-methoxyethoxy derivatives

(31−37) (Scheme 3) were prepared directly by a Buchwald−

Hartwig coupling of 16 with the corresponding aniline. The

reported yield for this reaction is in the range of 20−50%,⁵⁰for

which reason, we sought to ﬁrst optimize the reaction

conditions using commercially available 4-(3-methoxyphenyl)-

piperidine (17). From this study, we concluded that the best

conditions were 0.05 equiv of Pd(OAc)₂, 0.1 equiv of

XantPhos, 2.4 equiv of Cs₂CO₃, and dry dioxane stirred

under reﬂux for 36 h under inert conditions, which gave an

isolated yield of 78% (Scheme 3).

Free ortho-phenol 18 was obtained by deprotection of

commercially available 17 by reﬂuxing in 33 wt % hydrogen

bromide/acetic acid (HBr/AcOH). The ortho- and (commer-

cially available) meta-hydroxy analogues were both Boc-

protected under standard conditions to give 19 and 20,

respectively. tert-Butyl-4-(4-hydroxyphenyl)piperidine-1-car-

boxylate was commercially available. With all the three Boc-

protected phenol piperidines in hand, the 2-methoxyethoxy

moieties could be installed, as well as the remaining para-

methoxy analogue (21−24) (Scheme 3). The deprotection of

the Boc groups was performed with TFA in DCM; the reaction

mixtures were quenched with NH₄OH (25% in H₂O) and

yielded the free amines in near quantitative yields (26−29).

These secondary amines were cross-coupled with 16, under the

optimized reaction conditions established beforehand. Regret-

tably, the yield for these reactions varied signiﬁcantly, with the

yields of the 2-methoxyethoxy analogues being somewhat

lower compared to their corresponding methoxy analogues

(Scheme 3).

Basic Functional Characterization of 5-HT2A, mGlu2, and

the Putative 5-HT2A/mGlu2 Complex. Prior to the testing of

monovalent and bivalent compounds at 5-HT_2A, mGlu₂, and

the putative 5-HT_2A/mGlu₂heteromer in the four cell lines, we

delineated the basic pharmacology of the receptors in the

Ca²⁺/Fluo-4 assay using reference ligands. 5-HT evoked robust

responses in 5-HT_2A-HEK293 cells in a concentration-

dependent manner (Figure 2B), and in a previous study, the

functional properties exhibited by numerous reference 5-HT_2A

agonists and antagonists on this cell line in the Ca²⁺/Fluo-4

assay have been found to be in good agreement with literature

With both the 5-HT_2Aand mGlu₂head groups in hand, the

construction of the heterobivalent ligands (45−51) was

commenced (Scheme 4). Initial experimentation showed that

the reaction between ditosyl-(PEG)₄and mGlu₂head group

37 proceeded with high conversion in dimethylformamide

(DMF), but the product isolation proved tedious. On the

other hand, the product of the reaction between ditosyl-

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Figure 2. 5-HT_2A-, mGlu₂/Gqo5-, 5-HT_2A/mGlu₂-, and 5-HT_2A/mGlu₂/Gqo5-HEK293 cell lines. (A) Receptors and the origin of intracellular

Ca²⁺mobilization in the four cell lines. (B) Concentration−response relationships for 5-HT and Glu in the four stable HEK293 cell lines. 5-HT

and Glu concentrations on the X-axis are given in nM and μM, respectively. Data are given in % of the R_maxof 5-HT (5-HT_2A-, 5-HT_2A/mGlu₂-,

and 5-HT_2A/mGlu₂/Gqo5-HEK293) or the R_maxof Glu (mGlu₂/Gqo5-HEK293) in the same plate. Data are given as mean SD values from

representative experiments out of a total of at least four independent experiments.

data.⁵⁶Analogously, Glu mediated robust increases in

intracellular Ca²⁺levels via the chimeric Gα-protein Gqo5 in

mGlu₂/Gqo5-HEK293 cells (Figure 2B), and a selection of

reference mGlu₂ligands [Glu, the mGlu_2/3-selective agonist

(1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylic

acid (LY 354740/eglumegad), the mGlu_2/3-selective compet-

itive antagonist (1S,2S)-2-((S)-1-amino-1-carboxy-2-(9H-

xanthen-9-yl)ethyl)cyclopropane-1-carboxylic acid (LY

341495), the mGlu₂-selective negative allosteric modulator

(NAM) (Z)-1-(2-(cycloheptyloxy)-2-(2,6-dichlorophenyl)-

vinyl)-1H-1,2,4-triazole (Ro 64-5229), and the ago-PAM N-

(4′-cyano-[1,1′-biphenyl]-3-yl)-N-(pyridin-3-ylmethyl)-

ethanesulfonamide (CBiPES)] also exhibited functional

properties at the cell line in concordance with previously

reported values (Figure S16 and Table S1).⁵⁸⁻⁶²Importantly,

the 5-HT_{2 A}agonists 5-HT and 1-(4-iodo-2,5-

dimethoxyphenyl)propan-2-amine (DOI) (Figure S16) did

not evoke signiﬁcant responses in mGlu₂/Gqo5-HEK293 cells

(shown for 5-HT in Figure 2B), and the 5-HT_2Aantagonist 2-

methyl-1,2,3,4,10,14b-hexahydrodibenzo[c,f ]pyrazino[1,2-a]-

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Figure 3. Functional crosstalk between 5-HT_2Aand mGlu₂in the 5-HT_2A/mGlu₂- and 5-HT_2A/mGlu₂/Gqo5-HEK293 cell lines. (A)

Concentration−response relationships for 5-HT on the 5-HT_2A/mGlu₂-HEK293 cells in the absence and presence of various concentrations of the

mGlu₂ligands Glu, LY 354740, LY 341495, and Ro 64-229. The agonists Glu and LY 354740 were coapplied together with 5-HT on the cells,

whereas the antagonists LY 341495 and Ro 64-229 were applied on the cells prior to the application of 5-HT. (B) Concentration−response

relationships for Glu on the 5-HT_2A/mGlu₂/Gqo5-HEK293 cells in the absence and presence of various concentrations of the 5-HT_2Aligands 5-

HT and mianserin. 5-HT was coapplied with Glu on the cells, whereas the antagonist mianserin was applied on the cells prior to the application of

Glu. (A,B) Data in the ﬁgure are given as mean SD values from representative experiments for each cell line. Responses are given normalized to

the R_maxvalue of 5-HT (A) or Glu (B) in the absence of another ligand on the same plate, and data are given as mean SD values from

representative experiments out of a total of 3−4 independent experiments.

azepine (mianserin) did not inhibit Glu-induced responses in

the cells (data not shown). Conversely, neither Glu, LY

354740 nor CBiPES mediated signiﬁcant responses in 5-

HT_2A-HEK293 cells (shown for Glu in Figure 2B), and LY

341495 and Ro 64-5229 did not inhibit 5-HT-induced

responses in these cells (data not shown).

The concentration−response relationships exhibited by 5-

HT as an agonist on 5-HT_2A/mGlu₂- and 5-HT_2A/mGlu₂/

Gqo5-HEK293 cells in the Ca²⁺/Fluo-4 assay did not diﬀer

substantially from those displayed by it on 5-HT_2A-HEK293

cells (Figure 2B). In contrast to its complete inactivity in 5-

HT_2Acells, Glu induced small but signiﬁcant responses in 5-

HT_2A/mGlu₂cells, although the response was very minute

compared to that evoked by 5-HT (Figure 2B). As expected,

Glu mediated robust responses in 5-HT_2A/mGlu₂/Gqo5 cells

because of the Gqo5 expression in these cells, exhibiting an

half maximal eﬀective concentration (EC₅₀) value for these

cells comparable to that at the mGlu₂/Gqo5 cell line (Figure

2B). Next, the crosstalk between 5-HT_2Aand mGlu₂in the two

cell lines in the assay was investigated by determining the

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agonist properties of 5-HT and Glu in the presence of various

ligands targeting mGlu₂and 5-HT_2A, respectively. Co-

application of 5-HT with Glu or LY 354740 resulted in a

substantial left shift in the 5-HT concentration−response

relationship at the 5-HT_2A/mGlu₂-HEK293 cells (Figure 3A).

In contrast, the 5-HT-evoked signaling in these cells was not

aﬀected signiﬁcantly by the presence of the competitive

antagonist LY 341495 or the NAM Ro 64-5229 (Figure 3A).

In analogous experiments on the 5-HT_2A/mGlu₂/Gqo5-

HEK293 cell line, the putative eﬀects of 5-HT and the 5-

HT_2Aantagonist mianserin on Glu-mediated signaling were

investigated. The concentration−response relationship ex-

hibited by Glu on the cells was substantially potentiated in a

concentration-dependent manner by co-application of 5-HT,

and Glu-induced signaling in the cells was inhibited in a

concentration-dependent manner by mianserin (Figure 3B).

While the substantial eﬀects mediated by Glu and LY 354740

on 5-HT-signaling in 5-HT_2A/mGlu₂cells and by mianserin on

Glu-signaling in 5-HT_2A/mGlu₂/Gqo5 cells were observed in

the eﬀective concentration ranges of the ligands at their

respective target receptors (Figure 3A,B), 5-HT robustly

enhanced both the Glu potency and eﬃcacy on 5-HT_2A/

mGlu₂/Gqo5 cells at concentrations below those displaying

intrinsic activity on 5-HT_2A-HEK293 cells (Figure 2B).

5-HT_2Aantagonist and the ineﬀectiveness of mGlu₂antago-

nists to inhibit 5-HT_2Asignaling observed here are in excellent

agreement with previous ﬁndings.¹⁹

The diverse types of functional cross-signaling reported for

coexpressed 5-HT_2Aand mGlu₂in HEK293 cells in these

studies are likely to arise from a complex background of

diﬀerences in relative receptor expression levels, in the speciﬁc

HEK293 strains used, and/or in the functional read-outs and

speciﬁc characteristics of the assays used. For the purpose of

this study, however, we argue that the robust functional

crosstalk between the two receptors in the Ca²⁺/Fluo-4 assay

provides a solid foundation to characterize the functional

properties of the bivalent 5-HT_2A/mGlu₂ligands at the

putative heteromeric complex.

Functional Characterization of Monovalent and Bivalent

Ligands at 5-HT_2A, mGlu₂, and 5-HT_2A/mGlu₂. Monovalent

Ligand SARs. First, we studied the SAR of the two head

groups, mGlu₂ago-PAM 31 and competitive 5-HT_2A

antagonist 9, on 5-HT_2A- and mGlu₂/Gqo5-HEK293 cell

lines. This served the following two purposes: to identify the

most optimal positions in the two head groups to introduce a

linker without severely aﬀecting their pharmacological activity

and to be able to compare the functional properties of

monomeric ligands at the respective receptors with those

exhibited by the bivalent ligands.

Comparison of Basic Functional Properties of the Four

Cell Lines with Findings from Previous Studies. As

mentioned in the Introduction, the functional crosstalk

observed between 5-HT_2Aand mGlu₂when coexpressed in

heterologous expression systems, including HEK293 cells, have

mGlu₂Head Group. With its EC₅₀values of 37 nM (when

tested as an agonist) and 5.2 nM (when tested as PAM) on the

mGlu₂/Gqo5-cell line, the functional properties of 31 were in

agreement with a previous report.⁴¹Analogous to what has

been reported for its close structural analogue JNJ-

40411813,⁶³31 displayed antagonist activity at 5-HT_2A, albeit

its inhibitory potency at this receptor was 35- and 250-fold

lower than its agonist and PAM potencies at mGlu₂,

respectively (Table 1). The optimal orientation of the anisole

moiety in the mGlu₂head group was investigated using meta-

and para-position analogues (32, 33); both the analogues

exhibited slightly reduced agonist potencies but similar PAM

potencies compared to 31 at mGlu₂. The meta-substituted

analogue 32 displayed a slightly reduced antagonist potency at

5-HT_2Acompared to 31, whereas 33 was completely devoid of

activity at 5-HT_2A(Table 1). All three anisole analogues

displayed comparable agonist potencies and slightly (3−5 fold)

weaker PAM potencies on mGlu₂/5-HT_2A/Gqo5 cells

compared to those on mGlu₂/Gqo5 cells. The corresponding

2-methoxyethoxy analogues (34−36) did not display signiﬁ-

cant decrease in the mGlu₂ago-PAM activity compared to

their parent methoxy analogues. 34 and 35 displayed slightly

reduced antagonist potencies at 5-HT_2A, whereas 36,

analogous to 33, was completely inactive at 5-HT_2A(Table

1). The introduction of a hydroxy substituent in the para

position (37) resulted in a 16-fold decreased PAM potency at

mGlu₂compared to 31. On mGlu₂/5-HT_2A/Gqo5 cells, this

compound did not display signiﬁcant agonism, but exhibited a

slightly increased PAM potency compared to its PAM potency

on mGlu₂/Gqo5 cells. None of the seven mGlu₂head group

analogues displayed signiﬁcant agonist or PAM activity on

mGlu₂/5-HT_2Acells but were weak antagonists of 5-HT-

induced signaling in the cells (at mid-micromolar concen-

trations).

́

varied considerably in previous studies. Gonzalez-Maeso and

collaborators have reported inverse cross-signaling between 5-

HT_2Aand mGlu₂in Xenopus oocytes with agonists for

suppressing one receptor and antagonists for potentiating the

signaling of the other receptor³¹and that mGlu_2/3antagonists

potentiate 5-HT_2Asignaling in HEK293 cells,¹⁸although the

latter observation was found to be highly dependent on

receptor expression levels and cellular colocalization of the

receptors. Moreno et al.¹⁹have found that both 5-HT- and

mGlu_2/3-selective agonists induced robust Gα_q/11-mediated

responses in 5-HT_2A/mGlu₂cells, and in contrast to the lack of

eﬀect of the mGlu_2/3-selective antagonist LY 341495 on the 5-

HT-evoked response, the 5-HT_2Aantagonist MDL-100,907

inhibited mGlu_2/3agonist-induced signaling in the cells. Delille

et al.²⁷observed no signiﬁcant eﬀects of coexpression of the

two receptors in HEK293 cells on the pharmacological

properties of mGlu₂ligands in a cyclic adenosine mono-

phosphate (cAMP) accumulation assay and of 5-HT_2Aligands

in the Ca²⁺imaging assay, but they did not investigate the

eﬀects of ligands of one receptor on the response evoked

through the other. Finally, Murat et al. has recently reported

that selective mGlu_2/3agonist-stimulated phosphorylation of a

speciﬁc serine residue in mGlu₂requires coexpression of 5-

HT_2Ain HEK293 cells.³²With this diversity of functional

manifestations of the crosstalk between the two receptors in

previous studies, the data presented in Figure 3 are bound to ﬁt

with some observations and not with others. We clearly do not

see the inverse cross-signaling reported by some studies,^18,31

and although we do see a minute Glu-induced response in 5-

HT_2A/mGlu₂cells (Figure 2B), it is much less pronounced

than that reported by Moreno et al.¹⁹Instead, we see a robust

potentiation of signaling through each of the receptors being

mediated by agonists of the other receptor (Figure 3). On the

other hand, the inhibition of mGlu₂signaling mediated by the

5-HT_2AHead Group. In concordance with the literature,⁶⁴

the carbonyl analogue of the 5-HT_2Aantagonist MDL-100,907

(9) displayed an half maximal inhibitory concentration (IC₅₀)

value of 0.81 nM on 5-HT_2Acells in the Ca²⁺/Fluo-4 assay

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(Table 1). Replacing the methoxy moiety in the 3-position

with a 2-methoxyethoxy functionality (10) or a phenol (8)

group resulted in ∼10-fold reduction in the inhibitory potency

at 5-HT_2Ain both cases (Table 1), which is also in agreement

with previously reported values.⁴⁰In concordance with these

properties, all three analogues were antagonists of 5-HT EC₈₀-

induced signaling in both the 5-HT_2A/mGlu₂and 5-HT_2A/

mGlu₂/Gqo5 cells, exhibiting comparable or slightly higher

IC₅₀values for these cell lines compared to the 5-HT_2Acells.

All three analogues were completely inactive on the mGlu₂/

Gqo5 cell line and, in agreement with this, they did not exhibit

activity as agonists or PAMs (co-applied with Glu EC₂₀) on the

5-HT_2A/mGlu₂/Gqo5 cell line. Interestingly, however, they

exhibited potent antagonism (IC₅₀values of 23−29 nM) for

Glu EC₈₀-induced signaling in 5-HT_2A/mGlu₂/Gqo5 cells

(Table 1). Thus, analogous to the observed inhibition of Glu-

induced signaling in the 5-HT_2A/mGlu₂/Gqo5 cells mediated

by the reference 5-HT_2Aantagonist mianserin (Figure 3B),

binding of 8−10 to the orthosteric site in 5-HT_2Ainhibited the

signaling through mGlu₂in these cells.

Based on these SAR data for the mGlu₂head group, we have

determined that the orientation of the anisole moiety only

marginally reduced their potency as ago-PAMs for mGlu₂.

Furthermore, the introduction of the 2-methoxyethoxy moiety,

in any position, did not alter their potency. However, because

of the weak 5-HT_2Aantagonism of both ortho- and meta-

anisole and the lack of any activity of para-anisole at the 5-

HT_2Areceptor, this orientation was selected as the appropriate

anchor point for the spacer.

Heterobivalent Ligands. In the outline below, the func-

tional properties exhibited by the bivalent 5-HT_2A-mGlu₂

ligands on the four cell lines will be compared with those of

the two monovalent ligands that best represent the head

groups in the bivalent ligands, 36 (mGlu₂) and 10 (5-HT_2A).

All seven bivalent ligands (45−51) retained both the 5-HT_2A

antagonist activity and the mGlu₂ago-PAM activity of their

two head groups on the 5-HT_2A- and mGlu₂/Gqo5-cell lines

(Figure 4A,B, Table 1). However, they were considerably less

potent as such, with the seven ligands exhibiting 30−120-fold

higher IC₅₀values as antagonists than 10 at 5-HT_2Aand 2−8-

and 40−60-fold higher EC₅₀values as agonists and PAMs than

36 at mGlu₂(Table 1). This is probably best explained by the

entropic penalty caused by the large unbound section of the

opposing head group in these ligands. Nevertheless, the

qualitative functionalities of the two head groups at both 5-

HT_2Aand mGlu₂were clearly retained in the bivalent ligands.

The functional properties exhibited by the seven bivalent

ligands on the 5-HT_2A/mGlu₂and 5-HT_2A/mGlu₂/Gqo5 cell

lines diﬀered substantially from their properties on the mGlu₂/

Gqo5-cell line. None of the ligands displayed signiﬁcant ago-

PAM activities on 5-HT_2A/mGlu₂or 5-HT_2A/mGlu₂/Gqo5

cells. Instead, they antagonized 5-HT-induced signaling in 5-

HT_2A/mGlu₂cells and both 5-HT- and Glu-induced signaling

in 5-HT_2A/mGlu₂/Gqo5 cells (Figure 4C,D, Table 1). While

45 was the least potent antagonist, displaying IC₅₀values of 5−

10 μM in the three antagonist assays (5-HT_2A/mGlu₂with 5-

HT as the agonist, 5-HT_2A/mGlu₂/Gqo5 with 5-HT or Glu as

agonists), the other six bivalent ligands (46−51) displayed

IC₅₀values in the high-nanomolar to low-micromolar range

(140−2400 nM). The rank orders of antagonist potencies

exhibited by these six bivalent ligands in the three antagonist

assays were fairly similar and essentially comparable to their

IC₅₀values at 5-HT_2A(Table 1).

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Figure 4. Functional properties displayed by monovalent and bivalent ligands in the 5-HT_2A-, mGlu₂/Gqo5-, 5-HT_2A/mGlu₂-, and 5-HT_2A/mGlu₂/

Gqo5-HEK293 cell lines in the Ca²⁺/Fluo-4 assay. Data for the monovalent 5-HT_2A(9, 10) and mGlu₂(33, 36) head groups are given in blue and

red, respectively, and data for the seven bivalent ligands (45−51) are in black. Data in the ﬁgure are from representative experiments performed in

duplicate (error bars are omitted for reasons of clarity) out of a total of 3−5 independent experiments. Averaged data are given in Table 1. (A)

Concentration−inhibition relationships for the ligands tested as antagonists at 5-HT_2A-HEK293 cells. The compounds were applied at the cells

prior to the application of 5-HT EC₈₀. (B) Concentration−response relationships for the ligands tested as PAMs on mGlu₂/Gqo5-HEK293 cells.

The compounds were coapplied with Glu EC₂₀on the cells, and responses are given normalized to the R_maxvalue of Glu in the same plate. (C)

Concentration−inhibition relationships for the ligands tested as antagonists on 5-HT_2A/mGlu₂-HEK293 cells. The compounds antagonists were

applied to the cells prior to the application of 5-HT EC₈₀. (D) Concentration−inhibition relationships for the ligands on 5-HT_2A/mGlu₂/Gqo5-

HEK293 cells. Left: Concentration−response relationships for the ligands tested as PAMs (coapplied with Glu EC₂₀on the cells), and responses

are given normalized to the R_maxvalue of Glu on the same plate. Middle-Right: Concentration−inhibition relationships for the ligands tested as

antagonists using Glu EC₈₀(middle) or 5-HT EC₈₀(right) as the agonist. The compounds were applied to the cells prior to the application of the

agonist.

Binding Properties of Selected Monovalent and Bivalent

Ligands at 5-HT_2Aand 5-HT_2A/mGlu₂. To estimate the overall

5-HT_2Areceptor expression levels in the 5-HT_2A-, 5-HT_2A/

mGlu₂and 5-HT_2A/mGlu₂/Gqo5-HEK293 cells and to

determine the binding aﬃnities of selected monovalent and

bivalent ligands, we next performed saturation and competition

binding experiments on membranes from the three cell lines

using the 5-HT_2Aantagonist radioligand [³H]ketanserin.

Because of the lack of the commercially available mGlu₂

radioligand, we were unable to perform analogous binding

experiments focused on this receptor.

reasonable to assume that 5-HT_2Areceptor levels on the cell

surface in the three cell lines are largely comparable.

The rank order of the binding aﬃnities displayed by the

monovalent 5-HT_2Ahead groups 9 and 10 and the seven

bivalent ligands 45−51 at 5-HT_2A-, 5-HT_2A/mGlu₂-, and 5-

HT_2A/mGlu₂/Gqo5-cell membranes in the [³H]ketanserin

competition binding assay was very much comparable, and the

absolute K_ivalues displayed that each of the ligands across the

three cell lines were also very similar (Table 2 and Figure 5B).

Moreover, the binding properties of both monovalent and

bivalent ligands aligned very well with their antagonist

properties on the 5-HT_2A-containing cell lines. 9 and 10

exhibited substantially higher aﬃnities (10−400-fold) for all

three cell lines than 45−51, and while 45 was a substantially

weaker binder than the six other bivalent ligands, 46−51

exhibited essentially similar K_ivalues on the 5-HT_2A, 5-HT_2A/

mGlu₂, and 5-HT_2A/mGlu₂/Gqo5 cells. Notably, none of the

seven bivalent ligands exhibited signiﬁcantly diﬀerent K_ivalues

on either of the two 5-HT_2A/mGlu₂-expressing cell lines

compared to those on 5-HT_2Acells (Table 2 and Figure 5B).

Thus, all in all, the binding aﬃnities exhibited by the analogues

corroborated their functional properties as antagonists on the

three cell lines (Table 1 and Figure 4).

In the saturation binding experiments, [³H]ketanserin

displayed comparable K_Dvalues (1.2−1.4 nM) for the 5-

HT_2A, 5-HT_2A/mGlu₂, and 5-HT_2A/mGlu₂/Gqo5 cell mem-

branes, and these values are in good agreement with previously

reported K_Dvalues for [³H]ketanserin at 5-HT_2A(Table 2 and

Figure 5A).^65,66The B_maxvalues exhibited by [³H]ketanserin

on the three cell lines were within a 2.5-fold range (1.37, 3.25,

and 2.52 pmol/mg protein for 5-HT_2A, 5-HT_2A/mGlu₂, and 5-

HT_2A/mGlu₂/Gqo5, respectively). Thus, while it is important

to stress that the determined 5-HT_2Areceptor levels represent

both cell surface-expressed and intracellular receptor pools, it is

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Table 2. Binding Properties of [³H]Ketanserin and Monovalent and Bivalent Ligands on 5-HT_2A-, 5-HT_2A/mGlu₂-, and 5-

a

HT_2A/mGlu₂/Gqo5-HEK293 Cell Membranes in the [³H]Ketanserin Binding Assay

5-HT_2A

5-HT_2A/mGlu₂

5-HT_2A/mGlu₂/Gqo5

[³H]Ketanserin

K_D(nM)

B_max(pmol/mg protein)

1.39 0.09

1.37 0.08

1.36 0.06

3.25 0.12

1.20 0.09

2.52 0.14

K_i(nM) [pK_iSEM]

5-HT_2A

5-HT_2A/mGlu₂

5-HT_2A/mGlu₂/Gqo5

9

10

0.52 [9.28 0.05]

3.0 [8.53 0.07]

360 [6.44 0.08]

33 [7.48 0.06]

130 [6.89 0.05]

100 [7.00 0.06]

110 [6.92 0.08]

79 [7.10 0.04]

200 [6.70 0.04]

3.0 [8.52 0.08]

6.5 [8.19 0.07]

840 [6.08 0.05]

100 [6.99 0.08]

180 [6.75 0.07]

120 [6.93 0.08]

120 [6.94 0.10]

70 [7.14 0.08]

170 [6.78 0.04]

8.7 [8.06 0.09]

13 [7.88 0.05]

1040 [5.98 0.04]

110 [6.94 0.07]

320 [6.50 0.03]

190 [6.71 0.03]

160 [6.88 0.08]

120 [6.93 0.04]

170 [6.76 0.01]

45 (PEG_n= 3)

46 (PEG_n= 4)

47 (PEG_n= 5)

48 (PEG_n= 6)

49 (PEG_n= 8)

50 (PEG_n= 10)

51 (PEG_n= 12)

a

Binding properties of [³H]ketanserin, 9−10, and 45−51 at 5-HT_2A-HEK293, 5-HT_2A/mGlu₂-HEK293, and 5-HT_2A/mGlu₂/Gqo5-HEK293 cell

membranes in the [³H]ketanserin binding assay. K_DSEM and B_maxSEM values for [³H]ketanserin are given in nM and pmol/mg protein,

respectively. K_ivalues for the monovalent and bivalent ligands are given in nM (with pK_iSEM values in brackets). The saturation binding data

are based on four independent experiments performed in duplicate, and the competition binding data are based on three independent experiments

performed in duplicate.

Figure 5. Binding properties displayed by [³H]ketanserin and by monovalent and bivalent ligands on 5-HT_2A-, 5-HT_2A/mGlu₂- and 5-HT_2A/

mGlu₂/Gqo5-HEK293 cell membranes in the [³H]ketanserin binding assay. Data in the ﬁgure are from representative experiments performed in

duplicate out of a total of 3−4 independent experiments. Averaged data are given in Table 2. (A) Saturation binding of [³H]ketanserin at 5-HT_2A,

5-HT_2A/mGlu₂, and 5-HT_2A/mGlu₂/Gqo5. Data are from an individual experiment and given as mean

SD values based on duplicate

determinations. (B) Concentration−inhibition relationships exhibited by the monovalent 5-HT_2Ahead groups (9, 10, in blue) and the seven

bivalent ligands (45−51, in black) on 5-HT_2A-, 5-HT_2A/mGlu₂-, and 5-HT_2A/mGlu₂/Gqo5-HEK293 cell membranes in the [³H]ketanserin

competition binding assay using a radioligand concentration of 1.52 nM on the day. Data are from an individual experiment and given as mean

SD values based on duplicate determinations.

Overall Discussion of the SAR. The loss of activity

displayed by the monovalent analogues 8, 10, 32−37 and

the bivalent ligands (45−51) at the parent 5-HT_2Aand mGlu₂

receptors compared to the respective head group leads (9 and

31) was not surprising (Tables 1 and 2). The addition of the

other head group in the bivalent ligands further reduced the 5-

HT_2Aantagonist activity and the mGlu₂ago-PAM activity at

the two parent receptors. These observations are in line with

previous works in which bivalent ligands have been developed

from head groups already optimized for activity at their

respective receptors, where introduction of a linker in the head

group molecule or the fusion of two head groups via a linker

most often have resulted in reduced receptor activity.^67,68

The functional properties displayed by the monovalent

analogues at the two cell lines expressing the putative 5-HT_2A/

mGlu₂heteromer were also very much in line with those at

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their respective parent receptors, with 8−10 being slightly less

potent as antagonists of the 5-HT-induced responses in 5-

HT_2A/mGlu₂and 5-HT_2A/mGlu₂/Gqo5 cells and with 31−37

being slightly less potent as direct agonists and PAMs of the

Glu-induced responses in the 5-HT_2A/mGlu₂/Gqo5 cells. As

for the inhibition of the Glu-induced responses in 5-HT_2A/

mGlu₂/Gqo5 cells mediated by the monovalent 5-HT_2A

analogues 8−10, on one hand, it is a strong reﬂection of the

functional crosstalk between the two receptors in these cells,

regardless of the basis, but on the other, it also presents a

challenge for interpretation of the functional properties

exhibited by the bivalent ligands.

thus, enabling functional proﬁling of ligands at the two parent

receptors and the putative 5-HT_2A/mGlu₂heteromer in assays

measuring Gα_q/11-mediated signaling, and basic functional

characterization of these cell lines revealed pronounced

functional crosstalk between 5-HT_2Aand mGlu₂in 5-HT_2A/

mGlu₂- and 5-HT_2A/mGlu₂/Gqo5-cell lines in a Ca²⁺/Fluo-4

assay. Analogous to previous studies developing bivalent

ligands for other GPCRs, the introduction of a spacer in the

head groups as well as the fusion of the two head groups in

bivalent ligands resulted in substantially reduced potencies

compared to the head groups in the parent mGlu₂and 5-HT_2A

receptors. Importantly, however, the bivalent ligands exhibited

the same 5-HT_2Aantagonist and mGlu₂ago-PAM function-

alities as the respective head groups. In contrast, the bivalent

ligands inhibited both the 5-HT- and the Glu-induced

responses through the putative 5-HT_2A/mGlu₂heteromer,

suggesting a predominant role of the 5-HT_2Ahead group in the

ligands. None of the bivalent ligands exhibited increased

potencies as antagonists compared to the 5-HT_2Ahead group,

and no deﬁnitive correlation between the functional potency

and spacer length of the ligands could be observed. Thus, all in

all, the observed functional crosstalk between 5-HT_2Aand

mGlu₂in the 5-HT_2A/mGlu₂-cell lines in this study supports

previous reports, even though the actual characteristics of it

represents yet another phenotype of this crosstalk. As for the

seven bivalent ligands designed for the putative 5-HT_2A/mGlu₂

complex, it remains a question whether they truly act by

targeting binding sites in both receptors in this heteromer, or

whether they have preference for the orthosteric 5-HT_2Asite

and thus mediates their inhibition of the 5-HT- and Glu-

evoked responses in the 5-HT_2A/mGlu₂-expressing cells

exclusively through binding to this receptor.

The mGlu₂PAM activity observed for the monovalent

analogues 31−37 on 5-HT_2A/mGlu₂/Gqo5 cells is clearly

eliminated in the bivalent ligands which all displayed

antagonism for both 5-HT- and Glu-induced responses in

these cells. However, in light of the antagonism of the Glu-

induced response in 5-HT_2A/mGlu₂/Gqo5 cells mediated by

monovalent 5-HT_2Aanalogues 8−10, this cannot be taken as

an unequivocal proof of the bivalent ligands acting through

both the orthosteric site in 5-HT_2Aand the allosteric site in

mGlu₂. Considering the potencies displayed by the bivalent

ligands compared to 8−10 as antagonists at 5-HT_2A, it is not

necessarily a surprise that a similar pattern is observed in the 5-

HT_2A/mGlu₂and 5-HT_2A/mGlu₂/Gqo5 cells. On the other

hand, fusion of the mGlu₂head group to 8 in the bivalent

ligands clearly does not increase the antagonist potencies at the

putative 5-HT_2A/mGlu₂complex nor does it increase the

binding aﬃnity to the 5-HT_2Areceptor in the cells (Tables 1

and 2). It is clear from the functional properties of the bivalent

ligand in the mGlu₂/Gqo5 cells that the mGlu₂head group in

this ligand is able to bind the allosteric binding site in mGlu₂

and that it actually does this in an ago-PAM binding mode.

Regardless of whether the functional crosstalk observed

between 5-HT_2Aand mGlu₂(Figure 3) arises from the

formation of this putative heteromer or from another

mechanism, concomitant binding of the bivalent ligand to

the two binding sites in such as heteromer would be expected

to result in substantially higher antagonist potency of it

compared to the monovalent 5-HT_2Ahead group. The most

obvious explanation for the lack of synergistic eﬀect of the

bivalent ligand compared to the monovalent ligand is that the

two head groups in the ligand are unable to target the 5-HT_2A

and mGlu₂binding sites in the heteromer simultaneously.

There could be several molecular explanations for this,

including steric clashes between mGlu₂and the mGlu₂head

group or the linker when the bivalent ligand is anchored

through binding to the 5-HT_2Abinding site. Alternatively,

concomitant binding of the 5-HT_2Ahead group in the bivalent

ligand to 5-HT_2Ain the putative 5-HT_2A/mGlu₂heteromer

could be envisioned to limit the conformational ﬂexibility of

the mGlu₂head group, thus disabling it from adopting the

optimal binding orientation. Another possible explanation

could be that binding of the bivalent ligands disrupts the

heterodimer complex formation by shifting the equilibrium to

monomeric complexes.

EXPERIMENTAL SECTION

■

Chemistry. All reactions involving dry solvents or sensitive agents

were performed under a nitrogen atmosphere, and glassware was

dried prior to use. Commercially available chemicals were used

without further puriﬁcation. Solvents were dried prior to use with an

SG water solvent puriﬁcation system or by standard procedures, and

reactions were monitored by analytical TLC (Merck silica gel 60 F₂₅₄

aluminum sheets). Dry-column vacuum chromatography (DCVC)

was carried out using Merck silica gel 60A (15−40 μm),⁶⁹and ﬂash

chromatography was carried out using Merck silica gel 60A (35−70

1

μm). H nuclear magnetic resonance (NMR) spectra were recorded

on a 400 MHz Bruker Avance III or 600 MHz Bruker Avance III HD

and ¹³C NMR spectra on a 101 MHz Bruker Avance III or 151 MHz

Bruker Avance III HD. Analytical HPLC was performed using an

UltiMate HPLC system consisting of an LPG-3400A pump (1 mL/

min), a WPS-3000SL autosampler, and a 3000 diode array detector

installed with a Gemini-NX C18 (250 × 4.60 mm, 3 μm) column.

Solvent A: H₂O + 0.1% TFA; solvent B: MeCN−H₂O 9:1 + 0.1%

TFA. For HPLC control, data collection, and data handling,

Chromeleon software v. 6.80 was used. Preparative HPLC was

carried out on an UltiMate Thermo Scientiﬁc HPLC system with an

LPG-3200BX pump, a Rheodyne 9721i injector, a 10 mL loop, an

MWD-3000SD detector (200, 210, 225, and 254 nm), and a Gemini-

NX C18 (250 × 21.2 mm, 5 μm) column for preparative puriﬁcations

or a Gemini-NX C18 (250 × 10.00 mm, 5 μm) column for

semipreparative puriﬁcations. Solvent A: H₂O + 0.1% TFA; solvent B:

MeCN−H₂O 9:1 + 0.1% TFA. For HPLC control, data collection,

and data handling, Chromeleon software v. 6.80 was used. Chiral

preparative HPLC was performed using the same instrumentation

mentioned above. Ultraperformance liquid chromatography−tandem

mass spectrometry (UPLC−MS) was performed using an Acquity

UPLC H-Class Waters series solvent delivery system equipped with

an autoinjector coupled to Acquity QDa and TUV detectors and

CONCLUSIONS

■

In summary, in the present work, we successfully synthesized

both the 5-HT_2Aand mGlu₂head groups for bivalent ligands

and conﬁrmed the most suitable attachment points in them for

the spacer. Moreover, four HEK293 cell lines expressing 5-

HT_2A, mGlu₂, and the two receptors together were developed,

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installed with an Acquity UPLC BEH C18 (50 × 2.1 mm, 1.7 μm)

column. Solvent A: 5% aq. MeCN + 0.1% HCO₂H; solvent B: MeCN

+ 0.1% HCO₂H. Usually, gradients of A/B from 1:0 to 1:1 (5 min) or

A/B from 1:0 to 0:50 (5 min) were performed depending on the

polarity of the compounds. For data collection and data handling,

MassLynx software was used. Optical rotations were determined in a

thermostated cuvette on an Anton Paar MCP 300 modular circular

polarimeter. Compounds were dried under high vacuum or freeze-

dried using a ScanVac CoolSafe freeze drier. The purity of

compounds submitted for pharmacological characterization was

21.81 mmol, 1.0 equiv) was dissolved in DMF. N,O-Dimethylhy-

droxylamine hydrochloride (3.2 g, 32.71 mmol, 1.5 equiv) was added,

followed by Et₃N (6.1 mL, 43.62 mmol, 2.0 equiv). The reaction was

stirred for 10 min, and subsequently hydroxybenzotriazole (HOBt;

3.5 g, 26.17 mmol, 1.2 equiv) and 1-ethyl-3-(3-

dimethylaminopropyl)carbodiimide (EDCI; 4.63 mL, 26.17 mmol,

1.2 equiv) were added. The reaction mixture was stirred for an

additional 18 h at rt. HCl (1 M in H₂O, 200 mL) was added and the

crude was extracted with EtOAc (3 × 200 mL). The combined

organic fractions were washed with sat. NaHCO₃(100 mL) and brine

(150 mL) and dried over MgSO₄. The crude was puriﬁed by DCVC

(heptane/EtOAc 1:1), which aﬀorded the title compound (5.6 g,

20.50 mmol, 94%). MS (ESI) m/z: 273.1 [M + H]⁺; R_f= 0.21

1

determined by H NMR and HPLC to be >95%, unless otherwise

speciﬁed.

General Experimental Procedures. General Procedure I. To a

solution of 37 (1.0 equiv) in DMF is added NaH (60%) (2.0 equiv)

portion wise. The reaction is cooled to 0 °C and stirred for 10 min,

followed by the addition of tosylate 38−44 (1.0 equiv). The reaction

is allowed to warm to ambient temperature and stirred for an

additional 15 h. The reaction is quenched with MeOH, diluted with

TFA (0.1% v/v in H₂O), ﬁltered, and subjected to preparative HPLC

puriﬁcation. The fractions containing the desired product were

lyophilized.

General Procedure II. Phenol (1.0 equiv) was dissolved in dry

DMF to which were added K₂CO₃(2.0 equiv) and KI (2.0 equiv),

followed by 1-bromo-2-methoxyethane (1.2 equiv). The reaction was

stirred for 15 h at 60 °C. The crude was extracted with EtOAc (3×).

The combined organic fractions were washed with H₂O (2×) and

brine and dried over MgSO₄. The crude was puriﬁed by DCVC (5 →

25% EtOAc in heptane).

General Procedure III. N-Boc-arylpiperidine (1.0 equiv) was

dissolved in dry DCM. An equal volume of TFA was added dropwise

and the reaction was stirred for 2 h at room temperature (rt). The

reaction was cooled to 0 °C and quenched with dropwise addition of

NH₄OH (25% in H₂O) until pH 8−9. The crude was extracted with

DCM (3×). The combined organic fractions were washed with brine

and dried over MgSO₄. No additional puriﬁcation was needed.

General Procedure IV. Pd(OAc)₂(0.05 equiv), XantPhos (0.1

equiv), 16 (1.0 equiv), arylpiperidine (1.0 equiv), and Cs₂CO₃(2.4

equiv) were placed in a closed vial and dry and degassed dioxane was

added. The reaction was heated to reﬂux and stirred for 72 h, under

an argon atmosphere. After cooling to rt, the reaction mixture was

diluted with EtOAc and ﬁltered over Celite. The Celite was washed

with EtOAc (3×). The ﬁltrate was concentrated, and the crude was

puriﬁed by preparative HPLC. The isolated TFA salt was dissolved in

HCl (1 M in H₂O, 3×) and concentrated. The concentrate was

lyophilized.

General Procedure V. A solution of 8 (1.0 equiv) in dry

tetrahydrofuran (THF) was cooled to 0 °C to which is added NaH

(2.0 equiv). The reaction mixture was stirred for 10 min. Ditosyl-

(PEG)_n(5.0 equiv) was added to dry THF. The reaction mixture was

allowed to warm to rt and stirred for 15 h. The reaction mixture was

directly applied to a preparative TLC plate (100% EtOAc with 0.1%

Et₃N).

1

(heptane/EtOAc 1:1); H NMR (600 MHz, CDCl₃): δ 4.27−4.01

(m, 2H), 3.71 (s, 3H), 3.18 (s, 3H), 2.86−2.66 (m, 3H), 1.76−1.62

(m, 4H), 1.46 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 175.9, 154.8,

79.6, 61.7, 43.2, 38.3, 32.4, 28.6, 28.2.

tert-Butyl 4-(3-((tert-Butyldiphenylsilyl)oxy)-2-methoxybenzoyl)-

piperidine-1-carboxylate (5). n-BuLi (1.71 M in hexanes) (15.78 mL,

26.98 mmol, 1.05 equiv) was added to a stirred solution of 2 (9.8 g,

26.98 mmol, 1.05 equiv) in THF (75 mL) at 0 °C. The reaction

mixture was allowed to warm to rt and stirred for 2 h. After cooling to

−42 °C, 4 (7.0 g, 25.70 mmol, 1.00 equiv) in THF (5 mL) was added

dropwise to the stirred reaction mixture. The reaction mixture was

allowed to warm slowly to rt and stirred for 2 h. The reaction was

quenched with sat. NH₄Cl (5 mL). The crude was extracted with

EtOAc (3 × 20 mL). The combined organic fractions were washed

with brine (250 mL) and dried over MgSO₄. The crude was puriﬁed

by DCVC (0 → 4% EtOAc in heptane), which aﬀorded the title

compound (7.7 g, 13.42 mmol, 59%). MS (ESI) m/z: 574.3 [M +

H]⁺; R_f= 0.40 (heptane/EtOAc 3:1); ¹H NMR (600 MHz, CDCl₃):

δ 7.74−7.71 (m, 4H), 7.46−7.42 (m, 2H), 7.39−7.35 (m, 4H), 6.86

(dd, J = 1.6, 7.6 Hz, 1H), 6.87 (t, J = 7.9 Hz, 1H), 6.65 (dd, J = 1.6,

8.1 Hz, 1H), 4.16−3.98 (m, 2H), 3.92 (s, 3H), 3.19 (tt, J = 3.7, 11.0

Hz, 1H), 2.91−2.79 (m, 2H), 1.80 (dd, J = 2.4, 12.9 Hz, 2H), 1.57

(dd, J = 4.9, 8.8 Hz, 2H), 1.46 (s, 9H), 1.14 (s, 9H); ¹³C NMR (151

MHz, CDCl₃): δ 206.2, 154.9, 149.1, 148.7, 135.6, 134.8, 132.4,

130.2, 128.0, 124.1, 123.7, 121.1, 79.6, 62.2, 48.0, 28.6, 28.0, 26.7,

19.7.

(3-((tert-Butyldiphenylsilyl)oxy)-2-methoxyphenyl)(piperidin-4-

yl)methanone (7). 5 (7.8 g, 13.58 mmol, 1.0 equiv) was dissolved in

DCM (100 mL) and cooled to 0 °C. TFA (50 mL) was added slowly.

After the addition, the reaction mixture was allowed to warm to rt and

stirred for 1.5 h. The reaction mixture was cooled to 0 °C and

quenched with NH₄OH (25% in H₂O) until pH 8−9. The crude was

extracted with DCM (3 × 100 mL). The combined organic fractions

were washed with brine (200 mL) and dried over MgSO₄, which

aﬀorded the title compound (6.18 g, 13.05 mmol, 96%). MS (ESI) m/

1

z: 474.5 [M + H]⁺; R_f= 0.29 (DCM/MeOH 5:1); H NMR (600

MHz, CDCl₃): δ 7.74−7.69 (m, 4H), 7.47−7.41 (m, 2H), 7.37 (t, J =

7.4 Hz, 4H), 6.87 (dd, J = 1.6, 7.7 Hz, 1H), 6.70 (t, J = 7.9 Hz, 1H),

6.64 (dd, J = 1.6, 8.1 Hz, 1H), 3.92 (s, 3H), 3.72 (br s, 1H), 3.26−

3.21 (m, 1H), 3.19 (dt, J = 3.9, 12.7 Hz, 2H), 2.78 (ddd, J = 3.0, 11.0,

12.4 Hz, 2H), 1.94−1.88 (m, 2H), 1.67 (dt, J = 3.7, 10.7 Hz, 2H),

1.13 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 205.9, 148.9, 148.6,

135.5, 134.5, 132.3, 130.1, 127.8, 124.0, 123.6, 121.0, 62.0, 47.2, 45.3,

27.9, 26.6, 19.5.

(1-(4-Fluorophenethyl)piperidin-4-yl)(3-hydroxy-2-

methoxyphenyl)methanone Hydrochloride (8). 7 (3.80 g, 8.02

mmol, 1.0 equiv) was dissolved in DMF (50 mL) to which was added

1-(2-bromoethyl)-4-ﬂuorobenzene (1.24 mL, 8.82 mmol, 1.1 equiv),

followed by KI (2.66 g, 16.04 mmol, 2.0 equiv) and K₂CO₃(2.22 g,

16.04 mmol, 2.0 equiv). The reaction mixture was heated to 85 °C

and stirred for 17 h. The reaction was quenched with sat. NaHCO₃

(50 mL) and the crude was extracted with EtOAc (3 × 100 mL). The

combined organic fractions were washed with H₂O (2 × 250 mL) and

brine (300 mL) and dried over MgSO₄. The crude was puriﬁed by

DCVC (50 → 100% EtOAc in heptane). The isolated free base was

dissolved in HCl (2 M in dioxane, 40 mL) and concentrated, which

aﬀorded the title compound (1.52 g, 3.86 mmol, 48%). MS (ESI) m/

Experimental Procedures and Characterization. tert-Butyl(2-

methoxyphenoxy)diphenylsilane (2). Guaiacol (10.0 g, 80.55 mmol,

1.0 equiv) was dissolved in 250 mL of DCM and imidazole (13.7 g,

201.39 mmol, 2.5 equiv) was added, followed by tert-butyldiphenyl-

chlorosilane (22.7 mL, 88.61 mmol, 1.1 equiv). The reaction mixture

was stirred for 20 h at rt. H₂O (200 mL) was added and extracted

with DCM (3 × 100 mL). The combined organic fractions were

washed with H₂O (2 × 100 mL) and brine (250 mL) and dried over

MgSO₄. The crude was puriﬁed by DCVC (heptane/EtOAc 9:1),

which aﬀorded the title compound (28.0 g, 77.23 mmol, 96%). MS

1

(ESI) m/z: 363.6 [M + H]⁺; R_f= 0.72 (heptane/EtOAc 4:1); H

NMR (400 MHz, CDCl₃): δ 7.75−7.70 (m, 4H), 7.43−7.32 (m, 6H),

6.83 (ddd, J = 1.7, 7.2, 8.0 Hz, 1H), 6.78−6.71 (m, 2H), 6.65 (ddd, J

= 1.7, 7.2, 7.9 Hz, 1H), 3.56 (s, 3H), 1.12 (s, 9H); ¹³C NMR (101

MHz, CDCl₃): δ 150.7, 145.3, 135.5, 133.8, 129.7, 127.6, 121.7,

120.8, 120.5, 112.6, 55.5, 26.8, 19.9.

tert-Butyl 4-(Methoxy(methyl)carbamoyl)piperidine-1-carboxy-

late (4). 1-(tert-Butoxycarbonyl)piperidine-4-carboxylic acid (5.0 g,

M

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1

z: 358.0 [M + H]⁺; H NMR (600 MHz, D₂O): δ 7.32 (dd, J = 5.5,

N’-(3-Chloro-4-iodopyridin-2-yl)-2-cyclopropylacetohydrazide

(15). 12 (5.34 g, 19.82 mmol, 1.0 equiv) was dissolved in DMF (60

mL) and cooled to 0 °C. Et₃N (6.19 mL, 49.54 mmol, 2.5 equiv) was

added, followed by 14 (2.58 g, 21.80 mmol, 1.1 equiv). The reaction

was allowed to warm to rt and was stirred for 16 h. Sat. NaHCO₃(25

mL) was added to quench the reaction and the crude was extracted

with DCM (3 × 50 mL). The combined organic fractions were

washed with H₂O (2 × 75 mL), followed by brine (100 mL), and

dried over MgSO₄. No additional puriﬁcation was needed and the title

compound was obtained (4.39 g, 12.48 mmol, 63%). MS (ESI) m/z:

8.5 Hz, 2H), 7.19−7.07 (m, 5H), 3.81 (s, 3H), 3.72 (d, J = 12.7 Hz,

2H), 3.55 (tt, J = 3.7, 12.2 Hz, 1H), 3.42−3.34 (m, 2H), 3.17−3.10

(m, 2H), 3.09−3.02 (m, 2H), 2.21 (d, J = 14.6 Hz, 2H), 1.92−1.81

(m, 2H); ¹³C NMR (151 MHz, MeOD): δ 207.4, 163.7, 162.0, 150.5,

146.7, 133.1, 133.0, 131.4, 131.3, 126.3, 122.0, 121.3, 116.7, 116.5,

62.9, 58.8, 53.1, 45.4, 30.0, 26.5.

(2,3-Dimethoxyphenyl)(1-(4-ﬂuorophenethyl)piperidin-4-yl)-

methanone Hydrochloride (9). 8 (30 mg, 0.08 mmol, 1.0 equiv) was

dissolved in DMF (2 mL). K₂CO₃(26 mg, 0.19 mmol, 2.5 equiv) was

added, followed by methyl 4-methylbenzenesulfonate (16 mg, 0.08

mmol, 1.1 equiv). The reaction was heated to 65 °C and stirred for 15

h. The reaction was quenched with sat. NaHCO₃(2 mL) and the

crude was extracted with EtOAc (3 × 5 mL). The combined organic

fractions were washed with H₂O (2 × 5 mL) and brine (10 mL) and

dried over MgSO₄. The crude was puriﬁed by preparative HPLC. The

isolated TFA salt was dissolved in HCl (1 M in H₂O, 3 × 15 mL) and

concentrated. The concentrate was lyophilized, which aﬀorded the

title compound (15 mg, 0.04 mmol, 47%). MS (ESI) m/z: 372.1 [M

1

351.7 [M + H]⁺; R_f= 0.26 (heptane/EtOAc 1:1); H NMR (600

MHz, CDCl₃): δ 8.36 (d, J = 5.4 Hz, 1H), 7.68 (d, J = 5.2 Hz, 1H),

7.48 (d, J = 5.2 Hz, 1H), 7.25 (d, J = 5.2 Hz, 1H), 2.29 (d, J = 7.1 Hz,

2H), 1.14−1.04 (m, 1H), 0.69−0.62 (m, 2H), 0.28 (dt, J = 4.6, 6.03

Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 170.7, 152.9, 145.5, 126.8,

120.4, 110.2, 39.6, 7.0, 4.9.

8-Chloro-3-(cyclopropylmethyl)-7-iodo-[1,2,4]triazolo[4,3-a]-

pyridine (16). Dimethylsulfone (5.5 g) and sulfolane (0.7 g) were

combined with 15 (4.1 g, 11.66 mmol, 1.0 equiv) and stirred for 3 h

in a preheated ﬂask at 160 °C. After cooling to rt, H₂O (100 mL) was

added and the crude was extracted with DCM (3 × 150 mL). The

combined organic fractions were washed with H₂O (2 × 100 mL) and

brine (150 mL) and dried over MgSO₄. The crude was puriﬁed by

DCVC (0 → 100% EtOAc in heptane), which aﬀorded the title

compound (2.7 g, 8.09 mmol, 68%). MS (ESI) m/z: 333.7 [M + H]⁺;

+ H]⁺; H NMR (600 MHz, MeOD): δ 7.35−7.29 (m, 2H), 7.23

1

(dd, J = 1.6, 8.2 Hz, 1H), 7.15 (t, J = 8.0 Hz, 1H), 7.08 (dd, J = 7.4,

10.0 Hz, 2H), 7.05 (dd, J = 1.6, 7.8 Hz, 1H), 3.90 (s, 6H), 3.77−3.70

(m, 2H), 3.54−3.45 (m, 1H), 3.37−3.32 (m, 2H), 3.19−3.09 (m,

2H), 3.09−3.01 (m, 2H), 2.24−2.17 (m, 2H), 1.93−1.81 (m, 2H);

¹³C NMR (151 MHz, MeOD): δ 204.9, 164.3, 162.7, 154.4, 148.8,

1

R_f= 0.45 (EtOAc); H NMR (600 MHz, CDCl₃): δ 7.67 (d, J = 7.1

133.9, 133.4, 131.7, 131.6, 125.6, 121.3, 117.4, 116.7, 116.6, 62.1,

59.1, 56.5, 53.5, 46.4, 30.5, 27.0.

Hz, 1H), 7.16 (d, J = 7.1 Hz, 1H), 3.07 (d, J = 6.7 Hz, 2H), 1.23−

1.12 (m, 1H), 0.68−0.59 (m, 2H), 0.33 (q, J = 5.3 Hz, 2H); ¹³C

NMR (151 MHz, CDCl₃): δ 148.9, 148.4, 128.2, 123.3, 120.5, 95.8,

29.6, 8.6, 5.3.

(1-(4-Fluorophenethyl)piperidin-4-yl)(2-methoxy-3-(2-

methoxyethoxy)phenyl)methanone Hydrochloride (10). 8 (50 mg,

0.13 mmol, 1.0 equiv) was dissolved in DMF (2 mL), NaH (60% in

mineral oil) (5 mg, 0.13 mmol, 1.0 equiv) was added, and the reaction

mixture was stirred for 10 min. 1-Bromo-2-methoxyethane (24 μL,

0.25 mmol, 2.0 equiv) was added and the reaction mixture was stirred

for 6 h at rt. The reaction mixture was quenched with sat. NaHCO₃

(2 mL) and the crude was extracted with EtOAc (3 × 5 mL). The

combined organic fractions were concentrated and the crude was

puriﬁed by preparative HPLC. The isolated TFA salt was dissolved in

HCl (1 M in H₂O, 3 × 15 mL) and concentrated. The concentrate

was lyophilized, which aﬀorded the title compound (32 mg, 0.07

2-(Piperidin-4-yl)phenol (18). 4-(2-Methoxyphenyl)piperidine

(400 mg, 2.09 mmol, 1.0 equiv) was dissolved in HBr (33 wt % in

AcOH) (1.3 mL, 7.32 mmol, 3.5 equiv) and stirred under reﬂux for 2

h. After cooling to rt, the reaction mixture was neutralized to pH 8

with sat. Na₂CO₃. The crude was extracted with DCM (3 × 50 mL).

The combined organic fractions were washed with brine (75 mL) and

dried over MgSO₄. No additional puriﬁcation was needed and the title

compound was obtained (337 mg, 1.90 mmol, 91%). MS (ESI) m/z:

1

178.1 [M + H]⁺; H NMR (600 MHz, CDCl₃): δ 7.14 (dd, J = 1.6,

mmol, 56%). MS (ESI) m/z: 416.1 [M + H]⁺; H NMR (600 MHz,

1

7.7 Hz, 1H), 7.04 (td, J = 1.5, 7.7 Hz, 1H), 6.84 (td, J = 1.4, 7.6 Hz,

1H), 6.70 (dd, J = 1.3, 7.8 Hz, 1H), 5.12−4.09 (br s, 2H), 3.26−3.06

(m, 2H), 3.13−2.99 (m, 1H), 2.89−2.71 (m, 2H), 1.84−1.72 (m,

4H); ¹³C NMR (151 MHz, CDCl₃): δ 155.2, 153.2, 132.0, 127.2,

127.1, 121.0, 115.5, 79.7, 31.9, 28.7.

MeOD): δ 7.33 (dd, J = 5.2, 8.4 Hz, 2H), 7.23 (dd, J = 1.6, 8.2 Hz,

1H), 7.13 (t, J = 7.9 Hz, 1H), 7.07 (q, J = 8.6 Hz, 3H), 4.24−4.17 (m,

2H), 3.95 (s, 3H), 3.83−3.78 (m, 2H), 3.74 (d, J = 12.4 Hz, 2H),

3.49 (td, J = 6.0, 11.8 Hz, 1H), 3.44 (s, 3H), 3.38−3.32 (m, 2H),

3.20−3.12 (m, 2H), 3.12−3.02 (m, 2H), 2.21 (d, J = 14.7 Hz, 2H),

1.97−1.83 (m, 2H); ¹³C NMR (151 MHz, MeOD): δ 204.9, 164.3,

162.7, 153.5, 149.1, 134.0, 133.5, 133.4, 131.7, 131.6, 125.4, 121.7,

118.8, 116.7, 116.6, 72.2, 69.6, 62.2, 59.2, 59.1, 53.5, 46.5, 30.5, 27.0.

3-Chloro-2-hydrazinyl-4-iodopyridine (12). 2,3-Dichloro-4-iodo-

pyridine (20.0 g, 73.02 mmol, 1.0 equiv) was dissolved in dioxane

(500 mL) to which was added hydrazine·hydrate (21.25 mL, 438.13

mmol, 5.0 equiv). The reaction was heated to 70 °C and stirred for 16

h. After cooling to rt, NH₄OH (25% in H₂O, 250 mL) was added and

the mixture was concentrated in vacuo. The crude was dissolved in

warm EtOH and the residue was collected by ﬁltration, which

aﬀorded the title compound (12.6 g, 46.74 mmol, 64%). MS (ESI) m/

tert-Butyl 4-(2-Hydroxyphenyl)piperidine-1-carboxylate (19). 18

(330 mg, 1.86 mmol, 1.0 equiv) was dissolved in DCM (15 mL).

Et₃N (0.55 mL, 3.91 mmol, 2.1 equiv) was added, followed by di-tert-

butyl dicarbonate (447 mg, 2.05 mol, 1.1 equiv). The reaction mixture

was stirred for 8 h at rt. H₂O (20 mL) was added and the crude was

extracted with DCM (3 × 25 mL). The combined organic fractions

were washed with HCl (1 M in H₂O, 50 mL) and brine (75 mL) and

dried over MgSO₄. No additional puriﬁcation was needed and the title

compound was obtained (495 mg, 1.78 mmol, 94%). MS (ESI) m/z:

1

178.2 [M − Boc + H]⁺; R_f= 0.38 (heptane/EtOAc 3:1); H NMR

(600 MHz, CDCl₃): δ 7.13 (dd, J = 1.6, 7.7 Hz, 1H), 7.07 (td, J = 1.7,

7.7 Hz, 1H), 6.90 (td, J = 1.2, 7.5 Hz, 1H), 6.76 (dd, J = 1.1, 8.0 Hz,

1H), 4.24 (s, 2H), 3.04 (tt, J = 3.5, 12.2 Hz, 1H), 2.91−2.73 (m, 2H),

1.83 (d, J = 13.1 Hz, 2H), 1.67−1.58 (m, 2H), 1.49 (s, 9H); ¹³C

NMR (151 MHz, CDCl₃): δ 155.2, 153.2, 132.0, 127.2, 127.1, 121.0,

115.4, 79.7, 35.8, 31.8, 28.7, 27.6.

z: 269.8 [M + H]⁺; R_f= 0.46 (heptane/EtOAc 1:3); H NMR (600

MHz, CDCl₃): δ 7.72 (d, J = 5.2 Hz, 1H), 7.14 (d, J = 5.2 Hz, 1H),

6.34 (br s, 1H), 3.98 (br s, 2H); ¹³C NMR (151 MHz, CDCl₃): δ

155.8, 145.6, 124.7, 119.4, 109.4, 67.2.

1

tert-Butyl 4-(3-Hydroxyphenyl)piperidine-1-carboxylate (20). 3-

(Piperidin-4-yl)phenol (4.05 g, 22.87 mmol, 1.0 equiv) was dissolved

in DCM (100 mL) and Et₃N (3.51 mL, 25.16 mmol, 1.1 equiv) was

added, followed by di-tert-butyl dicarbonate (5.49 g, 25.16 mol, 1.1

equiv). The reaction mixture was stirred for 8 h at rt. H₂O (100 mL)

was added and the crude was extracted with DCM (3 × 100 mL). The

combined organic fractions were washed with HCl (1 M in H₂O, 150

mL) and brine (200 mL) and dried over MgSO₄. No additional

puriﬁcation was needed and the title compound was obtained (6.09 g,

2-Cyclopropylacetyl Chloride (14). 2-Cyclopropylacetic acid (6.51

mL, 69.92 mmol, 1.0 equiv) was dissolved in DCM (30 mL). Oxalyl

chloride (11.83 mL, 139.83 mmol, 2.0 equiv) was added dropwise to

the solution and the reaction was stirred at rt for 4 h. The solvent and

reagent were removed by Kugelrohr distillation (20 mbar, 25 °C), and

the title compound was isolated by Kugelrohr distillation (20 mbar,

1

65 °C) (8.12 g, 68.49 mmol, 98%). H NMR (600 MHz, CDCl₃): δ

2.78 (d, J = 7.0 Hz, 2H), 1.16−1.09 (m, 1H), 0.67−0.63 (m, 2H),

0.25 (dt, J = 4.9, 6.1 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 173.5,

52.0, 7.2, 4.7.

1

96%). 178.2 [M − Boc + H]⁺; H NMR (600 MHz, CDCl₃): δ 7.16

N

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(td, J = 1.4, 7.5 Hz, 1H), 6.76 (dt, J = 1.2, 7.5 Hz, 1H), 6.71−6.67 (m,

2H), 4.23 (br s, 2H), 2.79 (br s, 2H), 2.60 (tt, J = 3.6, 12.2 Hz, 1H),

1.81 (ddd, J = 2.6, 4.8, 12.8 Hz, 2H), 1.65−1.53 (m, 2H), 1.49 (s,

9H); ¹³C NMR (151 MHz, CDCl₃): δ 156.3, 155.1, 147.7, 129.8,

119.2, 113.7, 113.5, 79.9, 44.4, 42.7, 31.4, 28.6.

s, 1H), 3.77 (s, 3H), 3.30 (dt, J = 2.7, 12.5 Hz, 2H), 2.81 (td, J = 2.7,

12.5 Hz, 2H), 2.61 (tt, J = 3.7, 12.1 Hz, 1H), 1.87 (d, J = 12.7 Hz,

2H), 1.76 (qd, J = 4.0, 12.9 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃):

δ 157.1, 136.9, 126.6, 112.9, 54.3, 45.2, 40.3, 32.2.

4-(2-(2-Methoxyethoxy)phenyl)piperidine (27). The title com-

pound was prepared according to general procedure III (149 mg, 0.63

tert-Butyl 4-(4-Methoxyphenyl)piperidine-1-carboxylate (21).

tert-Butyl 4-(4-hydroxyphenyl)piperidine-1-carboxylate (500 mg,

1.80 mmol, 1.0 equiv) was dissolved in DMF (3 mL), to which

were added K₂CO₃(498 mg, 3.61 mmol, 2.0 equiv) and methyl

iodide (0.12 mL, 1.98 mmol, 1.1 equiv). The reaction mixture was

stirred for 15 h at 60 °C. The crude was extracted with EtOAc (3 ×

25 mL). The combined organic fractions were washed with H₂O (2 ×

50 mL) and brine (50 mL) and dried over MgSO₄. The crude was

puriﬁed by DCVC (5 → 25% EtOAc in heptane), which aﬀorded the

title compound (414 mg, 79%). MS (ESI) m/z: 192.4 [M − Boc +

H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.12 (d, J = 8.59 Hz, 2H), 6.85

(d, J = 8.60 Hz, 2H), 4.23 (br s, 2H), 3.79 (s, 3H), 2.79 (br s, 2H),

2.59 (tt, J = 3.62, 12.17 Hz, 1H), 1.79 (d, J = 13.05 Hz, 2H), 1.63−

1.55 (m, 2H), 1.48 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 158.2,

155.0, 138.2, 127.8, 114.0, 79.5, 55.4, 44.6, 42.0, 33.6, 28.6.

tert-Butyl 4-(2-(2-Methoxyethoxy)phenyl)piperidine-1-carboxy-

late (22). The title compound was prepared according to general

procedure II (319 mg, 0.95 mmol, 88%). MS (ESI) m/z: 236.3 [M −

Boc + H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.19−7.12 (m, 2H), 6.93

(t, J = 7.5 Hz, 1H), 6.86 (d, J = 8.1 Hz, 1H), 4.23 (br s, 2H), 4.13 (t, J

= 4.8 Hz, 2H), 3.76 (t, J = 4.8 Hz, 2H), 3.45 (s, 3H), 3.11 (tt, J = 3.5,

12.2 Hz, 1H), 2.82 (br t, J = 13.0 Hz, 2H), 1.81 (d, J = 12.8 Hz, 2H),

1.59 (qd, J = 4.2, 12.8 Hz, 2H), 1.48 (s, 9H); ¹³C NMR (151 MHz,

CDCl₃): δ 156.2, 155.1, 134.4, 127.2, 126.7, 121.2, 112.0, 79.5, 71.4,

67.9, 59.4, 44.8, 35.7, 31.9, 28.6.

1

mmol, 99%). MS (ESI) m/z: 236.1 [M + H]⁺; H NMR (600 MHz,

CDCl₃): δ 7.18 (ddt, J = 2.0, 4.2, 6.0 Hz, 2H), 6.95 (td, J = 1.2, 7.5

Hz, 1H), 6.86 (dd, J = 1.2, 8.5 Hz, 1H), 6.37 (br s, 1H), 4.15−4.10

(m, 2H), 3.78−3.72 (m, 2H), 3.48−3.39 (m, 5H), 3.23−3.14 (m,

1H), 2.96 (td, J = 3.9, 12.3 Hz, 2H), 2.00−1.88 (m, 4H); ¹³C NMR

(151 MHz, CDCl₃): δ 155.0, 132.1, 126.6, 125.7, 120.3, 111.0, 70.3,

66.8, 58.3, 44.6, 33.5, 29.0.

4-(3-(2-Methoxyethoxy)phenyl)piperidine (28). The title com-

pound was prepared according to general procedure III (48 mg, 0.49

mmol, quantitative). MS (ESI) m/z: 236.1 [M + H]⁺; ¹H NMR (600

MHz, CDCl₃): δ 7.21 (t, J = 8.1 Hz, 1H), 6.84−6.80 (m, 2H), 6.78−

6.74 (m, 1H), 4.14−4.09 (m, 2H), 3.77−3.72 (m, 2H), 3.45 (s, 3H),

3.21 (dt, J = 2.9, 12.2 Hz, 2H), 2.75 (td, J = 2.5, 12.3 Hz, 2H), 2.59

(tt, J = 3.7, 12.1 Hz, 1H), 2.43 (br s, 1H), 1.84 (d, J = 13.1 Hz, 2H),

1.66 (qd, J = 4.0, 12.5 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ

159.0, 148.3, 129.5, 119.6, 113.7, 112.0, 71.2, 67.3, 59.4, 47.1, 43.1,

34.3.

4-(4-(2-Methoxyethoxy)phenyl)piperidine (29). The title com-

pound was prepared according to general procedure III (313 mg, 1.33

1

mmol, 97%). MS (ESI) m/z: 236.1 [M + H]⁺; H NMR (600 MHz,

CDCl₃): δ 7.12 (d, J = 8.6 Hz, 2H), 6.86 (d, J = 8.6 Hz, 2H), 4.10−

4.07 (m, 2H), 3.90 (br s, 1H), 3.74−3.71 (m, 2H), 3.43 (s, 3H), 3.24

(dt, J = 3.1, 12.3 Hz, 2H), 2.77 (td, J = 2.6, 12.4 Hz, 2H), 2.57 (tt, J =

3.7, 12.1 Hz, 1H), 1.83 (d, J = 13.8 Hz, 2H), 1.69 (dtd, J = 4.0, 12.1,

13.3 Hz, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 157.3, 138.7, 127.7,

114.7, 71.2, 67.4, 59.3, 46.8, 41.8, 34.0.

4-(4-((tert-Butyldiphenylsilyl)oxy)phenyl)piperidine (30). The

title compound was prepared according to general procedure III

(3.27 g, 7.87 mmol, 74%). MS (ESI) m/z: 416.2 [M + H]⁺; ¹H NMR

(600 MHz, CDCl₃): δ 7.75−7.69 (m, 4H), 7.44−7.38 (m, 2H),

7.39−7.33 (m, 4H), 6.93 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.5 Hz,

2H), 3.18 (dt, J = 3.0, 12.4 Hz, 2H), 2.71 (td, J = 2.5, 12.4 Hz, 2H),

2.49 (tt, J = 3.7, 12.1 Hz, 1H), 2.21 (br s, 1H), 1.77 (ddq, J = 2.3,

4.30, 12.8 Hz, 2H), 1.63−1.53 (m, 2H), 1.09 (s, 9H); ¹³C NMR (151

MHz, CDCl₃): δ 153.9, 139.0, 135.7, 135.0, 133.3, 129.9, 127.8,

119.6, 47.0, 42.0, 34.3, 26.7, 19.6.

tert-Butyl 4-(3-(2-Methoxyethoxy)phenyl)piperidine-1-carboxy-

late (23). The title compound was prepared according to general

procedure II (246 mg, 0.75 mmol, 68%). MS (ESI) m/z: 236.3 [M −

Boc + H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.21 (t, J = 8.1 Hz, 1H),

6.81−6.78 (m, 2H), 6.78−6.75 (m, 1H), 4.23 (br s, 2H), 4.13−4.09

(m, 2H), 3.78−3.71 (m, 2H), 3.45 (br s, 3H), 2.78 (s, 2H), 2.60 (tt, J

= 3.6, 12.2 Hz, 1H), 1.81 (d, J = 13.1 Hz, 2H), 1.64−1.57 (m, 2H),

1.48 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 159.1, 155.0, 147.6,

129.5, 119.6, 113.8, 112.1, 79.6, 71.2, 67.3, 59.4, 42.9, 28.6.

tert-Butyl 4-(4-(2-Methoxyethoxy)phenyl)piperidine-1-carboxy-

late (24). The title compound was prepared according to general

procedure II (507 mg, 1.51 mmol, 84%). MS (ESI) m/z: 236.3 [M −

Boc + H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.10 (d, J = 8.6 Hz, 2H),

6.87 (d, J = 8.6 Hz, 2H), 4.22 (br s, 2H), 4.12−4.07 (m, 2H), 3.76−

3.71 (m, 2H), 3.44 (s, 3H), 2.78 (br t, J = 10.7, 21.3 Hz, 2H), 2.58 (tt,

J = 3.6, 12.2 Hz, 1H), 1.82−1.75 (m, 2H), 1.57 (qd, J = 4.4, 12.8, 13.7

Hz, 2H), 1.48 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 157.4, 155.0,

138.4, 127.7, 114.7, 79.5, 71.2, 67.4, 59.3, 44.6, 42.0, 33.5, 28.6.

tert-Butyl 4-(4-((tert-Butyldiphenylsilyl)oxy)phenyl)piperidine-1-

carboxylate (25). tert-Butyl 4-(4-hydroxyphenyl)piperidine-1-carbox-

ylate (2.5 g, 9.01 mmol, 1.0 equiv) and imidazole (1.53 g, 22.53

mmol, 2.5 equiv) were dissolved in DCM (50 mL), followed by tert-

butyldiphenylchlorosilane (2.54 mL, 9.91 mmol, 1.1 equiv). The

reaction mixture was stirred for 16 h at rt. The crude was extracted

with DCM (3 × 50 mL). The combined organic fractions were

washed with brine (100 mL) and dried over MgSO₄. The crude was

puriﬁed by DCVC (0 → 20% EtOAc in heptane), which aﬀorded the

title compound (4.64 g, 8.99 mmol, quantitative). MS (ESI) m/z:

8-Chloro-3-(cyclopropylmethyl)-7-(4-(2-methoxyphenyl)-

piperidin-1-yl)-[1,2,4]triazolo-[4,3-a]pyridine Hydrochloride (31).

The title compound was prepared according to general procedure

1

IV (184 mg, 0.42 mmol, 47%). MS (ESI) m/z: 397.0 [M + H]⁺; H

NMR (600 MHz, MeOD): δ 8.48 (d, J = 7.5 Hz, 1H), 7.35 (d, J = 7.5

Hz, 1H), 7.23−7.17 (m, 2H), 6.96 (d, J = 7.7 Hz, 1H), 6.92 (td, J =

1.1, 7.5 Hz, 1H), 4.22 (dt, J = 2.2, 13.1 Hz, 2H), 3.85 (s, 3H), 3.37

(td, J = 2.5, 12.6 Hz, 2H), 3.07 (d, J = 6.9 Hz, 2H), 2.05−1.96 (m,

2H), 1.92 (qd, J = 3.7, 12.3 Hz, 3H), 1.35−1.22 (m, 1H), 0.73−0.66

(m, 2H), 0.39 (dt, J = 4.6, 6.0 Hz, 2H); ¹³C NMR (151 MHz,

MeOD): δ 158.3, 154.2, 149.4, 147.1, 134.1, 128.5, 127.4, 125.1,

121.8, 113.8, 111.7, 98.3, 55.8, 52.3, 36.4, 33.2, 29.7, 8.4, 5.5.

8-Chloro-3-(cyclopropylmethyl)-7-(4-(3-methoxyphenyl)-

piperidin-1-yl)-[1,2,4]triazolo-[4,3-a]pyridine Hydrochloride (32).

The title compound was prepared according to general procedure

1

IV (98 mg, 0.23 mmol, 78%). MS (ESI) m/z: 397.0 [M + H]⁺; H

1

416.1 [M − Boc + H]⁺; R_f= 0.49 (heptane/EtOAc 3:1); H NMR

NMR (600 MHz, MeOD): δ 8.50 (d, J = 7.6 Hz, 1H), 7.37 (d, J = 7.6

Hz, 1H), 7.23 (t, J = 7.9 Hz, 1H), 6.87 (d, J = 7.7 Hz, 1H), 6.84 (t, J =

2.1 Hz, 1H), 6.8 (dd, J = 2.17, 8.22 Hz, 1H), 4.24 (dp, J = 1.9, 13.3

Hz, 2H), 3.79 (s, 3H), 3.37 (td, J = 2.4, 12.7 Hz, 2H), 3.07 (d, J = 6.9

Hz, 2H), 2.89 (tt, J = 3.8, 12.1 Hz, 1H), 2.04 (ddd, J = 1.9, 4.0, 12.6

Hz, 2H), 1.92 (qd, J = 3.8, 12.8 Hz, 2H), 1.35−1.23 (m, 1H), 0.74−

0.65 (m, 2H), 0.40 (dt, J = 4.6, 6.1 Hz, 2H); ¹³C NMR (151 MHz,

MeOD): δ 161.4, 154.4, 149.4, 148.1, 146.9, 130.6, 125.2, 120.1,

113.9, 113.9, 112.6, 98.1, 55.6, 52.0, 43.3, 34.6, 29.7, 8.4, 5.5.

8-Chloro-3-(cyclopropylmethyl)-7-(4-(4-methoxyphenyl)-

piperidin-1-yl)-[1,2,4]triazolo-[4,3-a]pyridine Hydrochloride (33).

The title compound was prepared according to general procedure

(600 MHz, CDCl₃): δ 7.73−7.70 (m, 4H), 7.44−7.40 (m, 2H),

7.38−7.34 (m, 4H), 6.91 (d, J = 8.5 Hz, 2H), 6.69 (d, J = 8.5 Hz,

2H), 4.19 (br s, 2H), 2.74 (br s, 2H), 2.50 (tt, J = 3.6, 12.1 Hz, 1H),

1.74 (dd, J = 3.9, 13.4 Hz, 2H), 1.51 (td, J = 4.4, 12.7 Hz, 2H), 1.46

(s, 9H), 1.09 (s, 9H); ¹³C NMR (151 MHz, CDCl₃): δ 155.0, 154.1,

138.4, 135.7, 133.3, 123.0, 127.9, 127.5, 119.7, 79.5, 44.6, 42.0, 33.5,

28.7, 26.7, 26.7, 19.6.

4-(4-Methoxyphenyl)piperidine (26). The title compound was

prepared according to general procedure III (255 mg, 1.33 mmol,

97%). MS (ESI) m/z: 192.3 [M + H]⁺; ¹H NMR (600 MHz,

CDCl₃): δ 7.13 (d, J = 8.6 Hz, 2H), 6.84 (d, J = 8.5 Hz, 2H), 5.24 (br

O

https://dx.doi.org/10.1021/acs.jmedchem.0c01058

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

IV (35 mg, 0.08 mmol, 31%). MS (ESI) m/z: 397.0 [M + H]⁺; H

NMR (600 MHz, MeOD): δ 8.48 (d, J = 7.6 Hz, 1H), 7.35 (d, J = 7.7

Hz, 1H), 7.22−7.16 (m, 2H), 6.89−6.85 (m, 2H), 4.23 (dp, J = 2.1,

13.2 Hz, 2H), 3.77 (s, 3H), 3.35 (td, J = 2.4, 12.7 Hz, 2H), 3.06 (d, J

= 6.9 Hz, 2H), 2.85 (tt, J = 3.8, 12.1 Hz, 1H), 2.04−1.98 (m, 2H),

1.89 (qd, J = 3.8, 12.6 Hz, 2H), 1.33−1.23 (m, 1H), 0.72−0.66 (m,

2H), 0.39 (dt, J = 4.6, 6.1 Hz, 2H); ¹³C NMR (151 MHz, MeOD): δ

159.8, 154.4, 149.4, 146.9, 138.6, 128.7, 125.3, 115.0, 114.0, 98.0,

55.7, 52.1, 42.4, 34.9, 29.7, 8.4, 5.5.

8-Chloro-3-(cyclopropylmethyl)-7-(4-(2-(2-methoxyethoxy)-

phenyl)piperidin-1-yl)-[1,2,4]triazolo[4,3-a]pyridine Hydrochloride

(34). The title compound was prepared according to general

procedure IV (34 mg, 0.08 mmol, 26%). MS (ESI) m/z: 441.1 [M

+ H]⁺; ¹H NMR (600 MHz, MeOD): δ 8.33 (d, J = 7.5 Hz, 1H), 7.24

(dd, J = 1.6, 7.6 Hz, 1H), 7.20−7.16 (m, 1H), 7.15 (d, J = 7.5 Hz,

1H), 6.98−6.91 (m, 2H), 4.18−4.13 (m, 2H), 3.94 (dq, J = 2.5, 12.2

Hz, 2H), 3.80−3.77 (m, 2H), 3.44 (s, 3H), 3.26 (ddd, J = 7.3, 9.6,

15.9 Hz, 1H), 3.23−3.17 (m, 2H), 3.06 (d, J = 6.9 Hz, 2H), 2.00−

1.94 (m, 4H), 1.28−1.21 (m, 1H), 0.69−0.62 (m, 2H), 0.36 (dt, J =

4.6, 6.1 Hz, 2H); ¹³C NMR (151 MHz, MeOD): δ 157.6, 154.4,

149.4, 146.9, 134.5, 128.5, 127.7, 125.2, 122.1, 113.9, 113.1, 97.8,

72.4, 68.8, 59.3, 52.3, 36.9, 33.1, 29.7, 8.4, 5.5.

679.3 [M + H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.79 (d, J = 8.3 Hz,

4H), 7.33 (d, J = 8.2 Hz, 4H), 4.17−4.12 (m, 4H), 3.67−3.59 (m,

20H), 3.57 (s, 8H), 2.44 (s, 6H); ¹³C NMR (151 MHz, CDCl₃): δ

144.9, 133.2, 130.0, 128.1, 70.9, 70.74, 70.69, 70.6, 69.4, 68.8, 21.8.

3,6,9,12,15,18,21,24,27-Nonaoxanonacosane-1,29-diyl Bis(4-

1

methylbenzenesulfonate) (53). To

a solution of

3,6,9,12,15,18,21,24,27-nonaoxanonacosane-1,29-diol (250 mg, 0.55

mmol, 1.0 equiv) in THF, tosyl chloride (312 mg, 1.64 mmol, 3.0

equiv) was added at rt and then cooled to 0 °C. To this mixture,

potassium hydroxide (6.6 equiv) in H₂O (0.9 g/mL) was added

dropwise over 1 h. Then the reaction mixture was kept at 0 °C for 30

min and some white precipitation appeared. Afterward this mixture

was further stirred at rt until TLC analysis indicated complete

consumption of the diol. The reaction mixture was neutralized with a

saturated ammonium chloride solution and concentrated in vacuo.

The residue was absorbed on the silica gel and further puriﬁed by

column chromatography to obtain the pure ditosylate (241 mg, 0.31

1

mmol, 58%). MS (ESI) m/z: 767.3 [M + H]⁺; H NMR (400 MHz,

CDCl₃): δ 7.82−7.77 (m, 4H), 7.37−7.32 (m, 4H), 4.18−4.13 (m,

4H), 3.70−3.67 (m, 4H), 3.63 (d, J = 7.85 Hz, 24H), 3.58 (s, 8H),

2.45 (s, 6H). ¹³C NMR (101 MHz, CDCl₃): δ 144.8, 133.1, 129.8,

128.0, 70.8, 70.6, 70.6, 70.5, 69.2, 68.7, 21.6.

8-Chloro-3-(cyclopropylmethyl)-7-(4-(3-(2-methoxyethoxy)-

phenyl)piperidin-1-yl)-[1,2,4]triazolo[4,3-a]pyridine Hydrochloride

(35). The title compound was prepared according to general

procedure IV (108 mg, 0.22 mmol, 54%). MS (ESI) m/z: 441.1

[M + H]⁺; ¹H NMR (600 MHz, MeOD): δ 8.47 (d, J = 7.4 Hz, 1H),

7.34 (d, J = 7.4 Hz, 1H), 7.23 (t, J = 7.8 Hz, 1H), 6.90−6.85 (m, 2H),

6.80 (dd, J = 2.1, 8.2 Hz, 1H), 4.19 (d, J = 14.1 Hz, 2H), 4.13−4.09

(m, 2H), 3.76−3.72 (m, 2H), 3.42 (s, 3H), 3.39−3.33 (m, 2H), 3.07

(d, J = 6.9 Hz, 2H), 2.88 (tt, J = 3.7, 12.2 Hz, 1H), 2.07−1.99 (m,

2H), 1.92 (qd, J = 3.7, 12.5 Hz, 2H), 1.36−1.22 (m, 1H), 0.72−0.65

(m, 2H), 0.39 (dt, J = 4.6, 5.9 Hz, 2H); ¹³C NMR (151 MHz,

MeOD): δ 160.5, 153.9, 149.4, 148.2, 147.3, 130.6, 125.0, 120.4,

114.6, 113.7, 113.3, 99.0, 72.3, 68.3, 59.2, 52.1, 43.3, 34.6, 29.7, 8.5,

5.5.

3,6,9,12,15,18,21,24,27,30,33-Undecaoxapentatriacontane-1,35-

d i y l B i s ( 4 - m e t h y l b e n z e n e s u l f o n a t e ) ( 5 4 ) .

3,6,9,12,15,18,21,24,27,30,33-Undecaoxapentatriacontane-1,35-diol

(1000 mg, 1.83 mmol, 1.0 equiv) was dissolved in dry DCM (150

mL). Tosyl chloride (732 mg, 3.84 mmol, 2.1 equiv) was added,

followed by DMAP (469 mg, 3.84 mmol, 2.1 equiv), and the reaction

was stirred for 16 h at rt. The reaction mixture was quenched with sat.

NH₄Cl (100 mL), diluted with H₂O (100 mL), and extracted with

DCM (3 × 100 mL). The combined organic fractions were washed

with brine (100 mL) and dried over MgSO₄. The crude was puriﬁed

by DCVC, which aﬀorded the title compound (1192 mg, 1.40 mmol,

76%). MS (ESI) m/z: 855.2 [M + H]⁺; ¹H NMR (400 MHz,

CDCl₃): δ 7.79 (d, J = 8.3 Hz, 4H), 7.34 (d, J = 8.1 Hz, 4H), 4.19−

4.11 (m, 4H), 3.69−3.60 (m, 36H), 3.58 (s, 8H), 2.44 (s, 6H); ¹³C

NMR (151 MHz, CDCl₃): δ 144.9, 133.2, 130.0, 128.1, 70.9, 70.8,

70.73, 70.70, 70.66, 69.4, 68.8, 21.8.

2-(2-(2-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-

methoxyphenoxy)ethoxy)ethoxy)ethyl 4-Methylbenzenesulfonate

(38). The title compound was prepared according to general

procedure V (27 mg, 0.04 mmol, 42%); MS (ESI) m/z: 644.3 [M

+ H]⁺, ¹H NMR (600 MHz, CDCl₃): δ 7.78 (d, J = 8.2 Hz, 2H), 7.32

(d, J = 8.0 Hz, 2H), 7.16−7.12 (m, 2H), 7.07−7.00 (m, 2H), 6.96

(dddd, J = 2.0, 6.7, 8.7, 15.10 Hz, 3H), 4.18−4.12 (m, 4H), 3.88 (s,

3H), 3.86 (d, J = 4.7 Hz, 2H), 3.71−3.64 (m, 4H), 3.61 (dd, J = 3.3,

5.8 Hz, 2H), 3.08 (tt, J = 3.8, 11.1 Hz, 1H), 2.97 (dt, J = 3.8, 11.8 Hz,

2H), 2.78−2.73 (m, 2H), 2.57−2.52 (m, 2H), 2.43 (s, 3H), 2.10 (td,

J = 2.62, 11.8 Hz, 2H), 1.93−1.86 (m, 2H), 1.78−1.70 (m, 2H); ¹³C

NMR (151 MHz, CDCl₃): δ 206.6, 162.3, 160.7, 152.0, 147.5, 145.0,

134.6, 133.1, 130.2, 130.0, 128.01 124.3, 120.9, 116.7, 115.3, 71.0,

70.9, 69.9, 69.3, 68.9, 68.6, 61.8, 60.8, 53.4, 48.3, 33.0, 28.2, 21.8.

2-(2-(2-(2-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-

methoxyphenoxy)ethoxy)ethoxy)ethoxy)ethyl 4-Methylbenzene-

sulfonate (39). The title compound was prepared according to

general procedure V (32 mg, 0.05 mmol, 46%); MS (ESI) m/z: 688.3

[M + H]⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.79 (dd, J = 2.1, 8.5 Hz,

2H), 7.33 (dd, J = 3.6, 8.4 Hz, 2H), 7.17−7.12 (m, 2H), 7.07−7.00

(m, 2H), 7.00−6.91 (m, 3H), 4.16 (ddd, J = 4.0, 5.7, 18.1 Hz, 4H),

3.90−3.89 (m, 1H), 3.88 (s, 3H), 3.73−3.70 (m, 2H), 3.70−3.65 (m,

3H), 3.65−3.61 (m, 2H), 3.60−3.57 (m, 4H), 3.08 (tt, J = 3.9, 11.1

Hz, 1H), 2.97 (dt, J = 3.9, 11.3 Hz, 2H), 2.79−2.73 (m, 2H), 2.59−

2.46 (m, 2H), 2.43 (s, 3H), 2.11 (t, J = 10.8 Hz, 2H), 1.90 (d, J = 13.9

Hz, 2H), 1.79−1.70 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 206.6,

162.3, 160.7, 152.0, 147.5, 144.9, 134.6, 133.2, 130.2, 129.9, 128.01,

124.3, 120.8, 116.6, 115.3, 70.9, 70.9, 70.8, 70.7, 69.8, 69.3, 68.8, 68.6,

61.8, 60.8, 53.4, 48.2, 33.0, 28.2, 21.8.

8-Chloro-3-(cyclopropylmethyl)-7-(4-(4-(2-methoxyethoxy)-

phenyl)piperidin-1-yl)-[1,2,4]triazolo[4,3-a]pyridine Hydrochloride

(36). The title compound was prepared according to general

procedure IV (42 mg, 0.09 mmol, 22%). MS (ESI) m/z: 441.1 [M

+ H]⁺; ¹H NMR (600 MHz, MeOD): δ 8.50 (d, J = 7.3 Hz, 1H), 7.37

(d, J = 7.3 Hz, 1H), 7.23−7.17 (m, 2H), 6.93−6.87 (m, 2H), 4.24 (d,

J = 12.8 Hz, 2H), 4.12−4.07 (m, 2H), 3.76−3.71 (m, 2H), 3.42 (s,

3H), 3.41−3.33 (m, 2H), 3.07 (d, J = 6.8 Hz, 2H), 2.86 (tt, J = 3.7,

12.1 Hz, 1H), 2.07−1.95 (m, 2H), 1.97−1.84 (m, 2H), 1.38−1.22

(m, 1H), 0.75−0.63 (m, 2H), 0.45−0.35 (m, 2H); ¹³C NMR (151

MHz, MeOD): δ 158.9, 154.4, 149.4, 146.9, 138.9, 128.7, 125.2,

115.7, 114.0, 97.9, 72.3, 68.4, 59.2, 52.1, 42.4, 34.9, 29.7, 8.4, 5.5.

4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-a]-

pyridin-7-yl)piperidin-4-yl)phenol Hydrochloride (37). The title

compound was prepared according to general procedure IV (42 mg,

1

0.09 mmol, 27%). MS (ESI) m/z: 420.1 [M + H]⁺; H NMR (600

MHz, MeOD): δ 8.37 (d, J = 7.6 Hz, 1H), 7.33 (d, J = 8.0 Hz, 1H),

7.23−7.14 (m, 2H), 6.84 (dt, J = 2.8, 9.2 Hz, 2H), 4.21 (d, J = 12. Hz,

2H), 3.23−3.14 (m, 2H), 3.05 (d, J = 7.5 Hz, 2H), 2.89−2.80 (m,

1H), 1.99 (d, J = 13.2 Hz, 2H), 1.85 (dd, J = 11.0, 14.8 Hz, 2H),

1.26−1.18 (m, 1H), 0.71−0.61 (m, 2H), 0.36 (dt, J = 5.1, 7.6 Hz,

2H); ¹³C NMR (151 MHz, MeOD): δ 155.1, 154.0, 149.4, 146.5,

138.5, 128.9, 124.5, 116.3, 113.9, 97.5, 51.7, 49.4, 41.6, 34.3, 29.3, 7.9,

5.3.

3,6,9,12,15,18,21-Heptaoxatricosane-1,23-diyl Bis(4-methylben-

zenesulfonate) (52). 3,6,9,12,15,18,21-Heptaoxatricosane-1,23-diol

(1000 mg, 2.70 mmol, 1.0 equiv) was dissolved in dry DCM (150

mL). Tosyl chloride (1081 mg, 5.67 mmol, 2.1 equiv) was added

followed by 4-dimethylaminopyridine (DMAP) (693 mg, 5.67 mmol,

2.1 equiv) and the reaction was stirred for 16 h at rt. The reaction

mixture was quenched with sat. NH₄Cl (100 mL), diluted with H₂O

(100 mL) and was extracted with DCM (3 × 100 mL). The

combined organic fractions were washed with brine (100 mL) and

dried over MgSO₄. The crude was puriﬁed by DCVC, which aﬀorded

the title compound (1295 mg, 1.91 mmol, 71%). MS (ESI) m/z:

14-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-methoxy-

phenoxy)-3,6,9,12-tetra Oxatetradecyl 4-Methylbenzenesulfonate

(40). The title compound was prepared according to general

procedure V (29 mg, 0.04 mmol, 40%); MS (ESI) m/z: 732.4 [M

P

https://dx.doi.org/10.1021/acs.jmedchem.0c01058

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

+ H]⁺, ¹H NMR (600 MHz, CDCl₃): δ 7.78 (dt, J = 1.5, 2.0, 8.6 Hz,

2H), 7.33 (dt, J = 1.4, 7.8 Hz, 2H), 7.17−7.11 (m, 2H), 7.06−6.99

(m, 2H), 6.99−6.91 (m, 3H), 4.20−4.14 (m, 4H), 3.88 (s, 3H),

3.74−3.69 (m, 2H), 3.69−3.65 (m, 4H), 3.64−3.60 (m, 5H), 3.57 (s,

4H), 3.07 (tt, J = 3.9, 11.1 Hz, 1H), 2.96 (dt, J = 3.7, 11.7 Hz, 2H),

2.76 (dd, J = 6.2, 10.0 Hz, 2H), 2.59−2.50 (m, 2H), 2.45−2.42 (m,

4H), 2.16−2.05 (m, 2H), 1.95−1.83 (m, 2H), 1.78−1.69 (m, 2H);

¹³C NMR (101 MHz, CDCl₃): δ 206.6, 162.7, 160.3, 152.0, 147.45

1.77−1.67 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 206.4, 162.2,

160.6, 151.9, 147.4, 144.8, 136.1, 134.5, 133.1, 130.1, 130.0, 129.9,

128.0, 124.2, 120.7, 116.6, 115.2, 115.0, 72.6, 71.4, 70.83, 70.77,

70.68, 70.65, 70.64, 70.60, 70.5, 70.4, 69.7, 69.3, 68.7, 68.5, 61.73,

61.65, 61.6, 60.7, 53.3, 48.1, 42.8, 32.9, 28.0, 21.7.

(3-(2-(2-(2-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo-

[4,3-a]pyridin-7-yl)piperidin-4-yl)phenoxy)ethoxy)ethoxy)ethoxy)-

2-methoxyphenyl)(1-(4-ﬂuorophenethyl)piperidin-4-yl)methanone

Bis(2,2,2-triﬂuoroacetate) (45). The title compound was prepared

according to general procedure I (4 mg, 0.003 mmol, 10%); MS (ESI)

144.9, 134.6, 133.2, 130.2, 129.9, 128.1, 124.3, 120.8, 116.6, 115.3,

70.9, 70.9, 70.8, 70.7, 70.7, 70.7, 69.8, 69.4, 68.8, 68.6, 61.8, 60.9,

53.5, 48.3, 33.0, 28.2, 21.8.

1

m/z: 584.6 [M + H]⁺; H NMR (600 MHz, MeOD): δ 8.46 (d, J =

7.6 Hz, 1H), 7.39 (d, J = 7.4 Hz, 1H), 7.35−7.30 (m, 3H), 7.26 (dd, J

= 1.1, 7.9 Hz, 1H), 7.16−7.03 (m, 5H), 6.88−6.83 (m, 2H), 4.32 (t, J

= 4.2 Hz, 2H), 4.28−4.11 (m, 2H), 4.10−4.06 (m, 2H), 3.91−3.88

(m, 3H), 3.85 (s, 1H), 3.83−3.81 (m, 2H), 3.75−3.57 (m, 10H),

3.56−3.38 (m, 4H), 3.07 (d, J = 6.9 Hz, 2H), 2.80 (tt, J = 3.9, 12.2

Hz, 1H), 2.27 (t, J = 12.2 Hz, 2H), 2.07−1.91 (m, 4H), 1.85 (qd, J =

3.4, 12.7, 13.3 Hz, 2H), 1.36−1.23 (m, 3H), 0.72−0.65 (m, 2H), 0.39

(dt, J = 4.6, 6.0 Hz, 2H); ¹³C NMR (151 MHz, MeOD): δ 202.1,

164.3, 162.7, 162.5, 158.8, 153.9, 149.4, 147.6, 147.4, 139.1, 133.4,

131.70, 131.67, 128.8, 125.0, 124.7, 122.7, 122.0, 116.9, 116.8, 116.6,

115.9, 113.6, 99.2, 71.8, 71.8, 70.9, 70.9, 70.8, 70.7, 70.1, 69.6, 68.8,

68.7, 59.2, 52.1, 50.0, 42.4, 34.9, 30.5, 29.7, 8.5, 5.5.

(3-(2-(2-(2-(2-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]-

triazolo[4,3-a]pyridin-7-yl)piperidin-4-yl)phenoxy)ethoxy)ethoxy)-

ethoxy)ethoxy)-2-methoxyphenyl)(1-(4-ﬂuoro phenethyl)piperidin-

4-yl)methanone Bis(2,2,2-triﬂuoroacetate) (46). The title com-

pound was prepared according to general procedure I (10 mg, 0.008

mmol, 20%) (purity 90%); MS (ESI) m/z: 898.3 [M + H]⁺; ¹H NMR

(600 MHz, MeOD): δ 8.45 (d, J = 7.6 Hz, 1H), 7.30 (dt, J = 3.0, 8.4

Hz, 3H), 7.22 (dd, J = 1.6, 8.1 Hz, 1H), 7.21−7.16 (m, 2H), 7.15−

7.02 (m, 4H), 6.92−6.85 (m, 2H), 4.22−4.17 (m, 2H), 4.19−4.12

(m, 2H), 4.11−4.07 (m, 2H), 3.95 (s, 3H), 3.91−3.78 (m, 5H),

3.75−3.56 (m, 12H), 3.47 (dd, J = 10.5, 13.9 Hz, 1H), 3.36−3.31 (m,

1H), 3.16−3.00 (m, 6H), 2.82 (tt, J = 3.8, 12.0 Hz, 1H), 2.33−1.95

(m, 4H), 1.94−1.79 (m, 4H), 1.29 (tdd, J = 1.6, 3.2, 8.1 Hz, 1H),

0.68 (dt, J = 4.8, 6.1, 8.0 Hz, 2H), 0.39 (q, J = 4.7, 6.1 Hz, 2H); ¹³C

NMR (151 MHz, MeOD): δ 204.9, 164.3, 162.7, 158.9, 153.8, 153.5,

149.4, 147.5, 139.0, 133.9, 133.44, 133.41, 131.7, 131.6, 128.8, 125.4,

124.9, 121.7, 118.8, 116.8, 116.6, 115.8, 113.5, 99.4, 71.8, 71.7, 71.6,

70.9, 70.8, 69.8, 68.7, 62.3, 59.1, 53.5, 52.1, 46.8, 42.4, 34.9, 30.5,

29.7, 27.0, 8.5, 5.5.

17-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-methoxy-

phenoxy)-3,6,9,12,15-pentaoxaheptadecyl 4-Methylbenzenesulfo-

nate (41). The title compound was prepared according to general

1

procedure V (38 mg, 0.05 mmol, 49%); MS (ESI) m/z: 776.3; H

NMR (600 MHz, CDCl₃): δ 7.78 (d, J = 8.3 Hz, 2H), 7.33 (d, J = 7.9

Hz, 2H), 7.14 (dd, J = 5.6, 8.4 Hz, 2H), 7.07−7.00 (m, 2H), 6.99−

6.92 (m, 3H), 4.18−4.14 (m, 4H), 3.88 (s, 3H), 3.73−3.70 (m, 2H),

3.69−3.65 (m, 5H), 3.64−3.62 (m, 5H), 3.62−3.59 (m, 4H), 3.58−

3.56 (m, 4H), 3.07 (tt, J = 3.8, 11.1 Hz, 1H), 2.96 (dt, J = 3.5, 11.8

Hz, 2H), 2.78−2.72 (m, 2H), 2.56−2.51 (m, 2H), 2.43 (s, 3H), 2.10

(t, J = 10.8 Hz, 2H), 1.92−1.82 (m, 2H), 1.77−1.68 (m, 2H); ¹³C

NMR (151 MHz, CDCl₃): δ 206.6, 162.3, 160.7, 152.0, 147.5, 144.9,

134.6, 133.2, 130.2, 129.9, 128.1, 124.3, 120.8, 116.6, 115.3, 70.9,

70.9, 70.8, 70.8, 70.7, 70.7, 70.7, 70.6, 69.8, 69.4, 68.8, 68.6, 61.8,

60.8, 53.4, 48.3, 33.0, 28.2, 21.8.

23-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-methoxy-

phenoxy)-3,6,9,12,15,18,21-heptaoxatricosyl 4-Methylbenzenesul-

fonate (42). The title compound was prepared according to general

procedure V and puriﬁed by ﬂash chromatography (0 → 3% MeOH

in DCM) (109 mg, 0.13 mmol, 45%). MS (ESI) m/z: 864.3 [M +

1

H]⁺; H NMR (600 MHz, CDCl₃): δ 7.78 (d, J = 8.3 Hz, 2H), 7.33

(d, J = 7.8 Hz, 2H), 7.17−7.11 (m, 2H), 7.07−6.99 (m, 2H), 6.98−

6.90 (m, 3H), 4.17 (dd, J = 5.7, 4.1 Hz, 2H), 4.16−4.12 (m, 2H),

3.91−3.86 (m, 5H), 3.74−3.70 (m, 2H), 3.68−3.57 (m, 20H), 3.57

(s, 4H), 3.08 (t, J = 11.4 Hz, 1H), 2.97 (dt, J = 11.7, 3.9 Hz, 2H),

2.81−2.74 (m, 2H), 2.61−2.51 (m, 2H), 2.43 (s, 3H), 2.22−2.08 (m,

2H), 1.91 (d, J = 12.6 Hz, 2H), 1.81−1.71 (m, 2H); ¹³C NMR (151

MHz, CDCl₃): δ 206.5, 162.3, 160.7, 152.0, 147.5, 144.9, 136.1,

134.5, 133.2, 130.2, 130.1, 129.9, 128.1, 124.3, 120.8, 116.7, 115.3,

115.1, 72.7, 71.5, 70.92, 70.86, 70.8, 70.74, 70.73, 70.69, 70.6, 69.8,

69.4, 68.8, 68.6, 61.7, 60.7, 53.3, 48.1, 42.8, 32.9, 28.0, 21.7.

29-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-methoxy-

phenoxy)-3,6,9,12,15,18,21,24,27-nonaoxanonacosyl 4-Methyl-

benzenesulfonate (43). The title compound was prepared according

to general procedure V and puriﬁed by ﬂash chromatography (0 →

3% MeOH in DCM) (33 mg, 0.03 mmol, 43%). MS (ESI) m/z: 952.1

[M + H]⁺, 447.0 [M + 2H]²⁺; ¹H NMR (600 MHz, CDCl₃): δ 7.81−

7.77 (d, J = 12.0 Hz 2H), 7.33 (d, J = 8.0 Hz, 2H), 7.16−7.12 (m,

2H), 7.06−7.01 (m, 2H), 6.99−6.92 (m, 3H), 4.18 (t, J = 4.9 Hz,

2H), 4.16 (t, J = 4.9 Hz, 2H), 3.90−3.88 (m, 5H), 3.72 (dd, J = 3.7,

5.9 Hz, 2H), 3.69−3.60 (m, 28H), 3.57 (s, 4H), 3.09 (ddt, J = 4.2,

7.7, 11.1 Hz, 1H), 2.98 (dt, J = 3.9, 11.7 Hz, 2H), 2.77 (dd, J = 6.3,

10.0 Hz, 2H), 2.57 (dd, J = 6.0, 10.0 Hz, 2H), 2.44 (s, 3H), 2.19−

2.08 (m, 2H), 1.91 (d, J = 13.4 Hz, 2H), 1.82−1.70 (m, 2H). ¹³C

NMR (151 MHz, CDCl₃): δ 206.4, 162.2, 160.6, 151.9, 147.3, 144.8,

136.0, 134.4, 133.0, 130.1, 130.0, 129.8, 128.0, 124.2, 120.7, 116.5,

115.2, 115.0, 70.8, 70.7, 70.7, 70.6, 70.6, 70.6, 70.6, 70.5, 70.5, 70.5,

69.7, 69.2, 68.7, 68.4, 61.6, 60.6, 53.2, 48.0, 32.8, 29.7, 28.0, 21.6.

35-(3-(1-(4-Fluorophenethyl)piperidine-4-carbonyl)-2-methoxy-

phenoxy)-3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl

4-Methylbenzenesulfonate (44). The title compound was prepared

according to general procedure V and puriﬁed by ﬂash chromatog-

raphy (0 → 3% MeOH in DCM) (148 mg, 0.14 mmol, 51%). MS

(3-((14-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-

a ] p y r i d i n - 7 - y l ) p i p e r - i d i n - 4 - y l ) p h e n o x y ) - 3 , 6 , 9 , 1 2 -

tetraoxatetradecyl)oxy)-2-methoxyphenyl)(1-(4-ﬂuorophen-ethyl)-

piperidin-4-yl)methanone Bis(2,2,2-triﬂuoroacetate) (47). The title

compound was prepared according to general procedure I (5 mg,

1

0.004 mmol, 12%); MS (ESI) m/z: 942.3 [M + H]⁺; H NMR (600

MHz, CDCl₃): δ 8.46 (d, J = 7.6 Hz, 1H), 7.36−7.27 (m, 3H), 7.24−

7.15 (m, 3H), 7.11 (t, J = 8.3 Hz, 1H), 7.09−7.01 (m, 3H), 6.88 (d, J

= 8.6 Hz, 2H), 4.23−4.14 (m, 4H), 4.12−4.04 (m, 2H), 3.97−3.92

(m, 3H), 3.92−3.75 (m, 6H), 3.75−3.57 (m, 16H), 3.57−3.41 (m,

2H), 3.14−3.09 (m, 1H), 3.09−3.00 (m, 4H), 2.82 (tt, J = 3.9, 12.3

Hz, 1H), 2.19 (d, J = 14.3 Hz, 2H), 1.99 (dd, J = 3.7, 13.8 Hz, 2H),

1.93−1.80 (m, 3H), 1.32−1.24 (m, 2H), 0.71−0.65 (m, 2H), 0.42−

0.35 (m, 2H); ¹³C NMR (151 MHz, CDCl₃): δ 204.9, 164.3, 162.7,

158.9, 154.0, 153.5, 149.4, 147.2, 138.9, 134.0, 133.5, 133.4, 131.7,

131.6, 128.8, 125.4, 125.0, 121.7, 118.8, 116.7, 116.6, 115.8, 113.7,

98.7, 71.74, 71.68, 71.66, 71.61, 71.60, 71.58, 71.56, 71.5, 70.9, 70.7,

69.8, 69.8, 68.7, 62.3, 62.1, 59.1, 53.5, 52.1, 46.5, 42.4, 34.9, 30.5,

29.7, 27.0, 8.4, 5.5.

(3-((17-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-

a]pyridin-7-yl)piper-idin-4-yl)phenoxy)-3,6,9,12,15-

pentaoxaheptadecyl)oxy)-2-methoxyphenyl)(1-(4-ﬂuoro-

phenethyl)piperidin-4-yl)methanone Bis(2,2,2-triﬂuoroacetate)

(48). The title compound was prepared according to general

procedure I (11 mg, 0.009 mmol, 20%); MS (ESI) m/z: 986.3 [M

1

(ESI) m/z: 1040.4 [M + H]⁺, 520.7 [M + 2H]²⁺; H NMR (600

MHz, CDCl₃): δ 7.76 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.3 Hz, 2H),

7.14−7.09 (m, 2H), 7.04−6.98 (m, 2H), 6.96−6.89 (m, 3H), 4.15

(dd, J = 5.7, 4.1 Hz, 2H), 4.14−4.10 (m, 2H), 3.88−3.84 (m, 5H),

3.74−3.56 (m, 38H), 3.55 (s, 4H), 3.06 (tt, J = 10.9, 3.7 Hz, 1H),

2.94 (dt, J = 10.8, 3.8 Hz, 2H), 2.77−2.72 (m, 2H), 2.56−2.50 (m,

2H), 2.41 (s, 3H), 2.09 (t, J = 11.0 Hz, 2H), 1.91−1.84 (m, 2H),

1

+ H]⁺; H NMR (600 MHz, MeOD): δ 8.46 (d, J = 7.5 Hz, 1H),

7.34−7.27 (m, 3H), 7.22 (dd, J = 1.6, 8.2 Hz, 1H), 7.21−7.17 (m,

2H), 7.12 (t, J = 8.0 Hz, 1H), 7.10−7.02 (m, 3H), 6.92−6.87 (m,

2H), 4.21−4.15 (m, 4H), 4.09 (t, J = 4.9 Hz, 2H), 3.95 (s, 3H),

Q

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J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Article

3.90−3.84 (m, 3H), 3.82 (t, J = 4.7 Hz, 2H), 3.75−3.67 (m, 6H),

3.66−3.59 (m, 14H), 3.55−3.42 (m, 2H), 3.35−3.33 (m, 1H), 3.14−

3.03 (m, 5H), 2.83 (tt, J = 3.7, 12.1 Hz, 1H), 2.31−2.13 (m, 2H),

2.02−1.95 (m, 2H), 1.90−1.81 (m, 3H), 1.37−1.25 (m, 2H), 0.71−

0.66 (m, 2H), 0.39 (dt, J = 4.7, 6.2 Hz, 2H); ¹³C NMR (151 MHz,

MeOD): δ 204.9, 164.3, 162.7, 158.9, 154.0, 153.5, 149.4, 147.3,

138.9, 134.0, 133.4, 133.4, 131.6, 131.6, 128.8, 125.4, 125.0, 121.7,

118.8, 116.8, 116.6, 115.8, 113.6, 98.2, 71.75, 71.70, 71.62, 71.59,

71.58, 71.56, 71.5, 70.9, 70.8, 69.8, 68.7, 62.3, 59.1, 53.5, 52.1, 46.5,

42.4, 34.9, 30.5, 29.7, 27.0, 8.5, 5.5.

71.72, 7166, 71.59, 71.56, 71.55, 71.54, 70.9, 70.8, 69.8, 68.7, 62.3,

59.1, 53.5, 52.2, 46.5, 42.5, 34.9, 30.5, 29.7, 27.0, 8.6, 5.4.

Pharmacology. Materials. Culture media, serum, antibiotics, and

buﬀers for cell culture were obtained from Invitrogen (Paisley, UK).

The Fluo-4/AM dye and pluronic acid were obtained from Molecular

Probes (Eugene, OR). Glu, 5-HT, and probenecid were purchased

from Sigma-Aldrich (St. Louis, MO), whereas mianserin, clozapine,

LY 354740, LY 341495, CBiPES, and Ro 62-5229 were obtained

from Tocris Cookson (Bristol, UK). [³H]Ketanserin (speciﬁc activity:

41.9 Ci/mmol) was obtained from PerkinElmer (Waltham, MA). The

h5-HT_2A-pCDNA3.1neo and Gqo5-pCDNA3neo plasmids were kind

(3-((23-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-

a]pyridin-7-yl)piperidin-4-yl)phenoxy)-3,6,9,12,15,18,21-

heptaoxatricosyl)oxy)-2-methoxyphenyl)(1-(4-ﬂuorophenethyl)-

piperidin-4-yl)methanone Bis(2,2,2-triﬂuoroacetate) (49). The title

compound was prepared according to general procedure I (3 mg,

0.002 mmol, 9%). MS (ESI) m/z: 1074.7 [M + H]⁺, 538.0 [M +

̈

gifts from Drs. H. Brauner-Osborne and B. R. Conklin, respectively.

The construction of the stable 5-HT_2A-HEK293 cell line has been

described previously.⁵⁶

Molecular Biology. The complementary deoxyribonucleic acid

(cDNA) encoding for the human mGlu₂receptor was cloned from the

IMAGE clone 8322671 (Source Bioscience, Nottingham, UK) and

subcloned into the human cytomegalovirus (CMV) promoter-

associated multicloning site in the bistronic mammalian expression

vector pBudCE4.1 (Thermo Fisher Scientiﬁc, Waltham, MA) by use

of the restriction enzymes HindIII and XbaI, yielding the mGlu₂-

pBudCE4.1 construct. The cDNA for the human 5-HT_2Areceptor

was subcloned from 5-HT_2A-pCDNA3.1neo into the EF-1α

promoter-associated multicloning site in mGlu₂-pBudCE4.1 by use

of the restriction enzymes NotI and XhoI, yielding the 5-HT_2A/mGlu₂-

pBudCE4.1 construct. The integrity and the absence of unwanted

mutations in all cDNAs created by the polymerase chain reaction

were veriﬁed by DNA sequencing (Euroﬁns MWG Operon,

Martinsried, Germany).

1

2H]²⁺; H NMR (600 MHz, MeOD): δ 8.40 (d, J = 7.6 Hz, 1H),

7.34−7.28 (m, 2H), 7.25−7.22 (m, 2H), 7.21−7.18 (m, 2H), 7.12 (t,

J = 7.9 Hz, 1H), 7.10−7.02 (m, 3H), 6.91−6.87 (m, 2H), 4.25−4.17

(m, 4H), 4.13−4.09 (m, 2H), 3.95 (s, 3H), 3.91−3.85 (m, 2H),

3.84−3.79 (m, 2H), 3.77−3.67 (m, 6H), 3.67−3.57 (m, 20H), 3.52−

3.44 (m, 2H), 3.38−3.32 (m, 3H), 3.18−3.10 (m, 2H), 3.08−3.01

(m, 4H), 2.80 (tt, J = 12.0, 3.9 Hz, 1H), 2.25−2.15 (m, 2H), 2.01−

1.95 (m, 2H), 1.94−1.79 (m, 4H), 1.37−1.23 (m, 1H), 0.71−0.63

(m, 2H), 0.38 (dt, J = 6.0, 4.6 Hz, 2H). ¹³C NMR (151 MHz,

MeOD): δ 204.9, 164.3, 162.7, 158.9, 154.0, 153.5, 149.4, 147.4,

139.1, 134.0, 133.40, 133.38, 131.7, 131.6, 128.8, 125.5, 124.5, 121.7,

118.8, 116.8, 116.6, 115.8, 113.0, 98.4, 71.8, 71.73, 71.67, 71.60,

71.59, 71.57, 71.56, 70.9, 70.8, 69.8, 68.7, 62.3, 59.1, 53.5, 52.2, 46.5,

42.5, 35.0, 30.5, 29.7, 27.0, 8.7, 5.4.

Construction and Culture of Stable Cell Lines. Stable HEK293

cell lines coexpressing mGlu₂and Gqo5 (termed mGlu₂/Gqo5-

HEK293); 5-HT_2Aand mGlu₂(5-HT_2A/mGlu₂-HEK293); and 5-

HT_2A, mGlu₂, and Gqo5 (5-HT_2A/mGlu₂/Gqo5-HEK293) were

constructed using the mGlu₂-pBudCE4.1, 5-HT_2A/mGlu₂-

pBudCE4.1, and Gqo5-pCDNA3neo constructs. HEK293 cells were

transfected with various combinations of these plasmids using the

PolyFect transfection reagent (Qiagen, Hilden, Germany) and

maintained in a culture medium (Dulbecco’s modiﬁed Eagle’s

medium-Glutamax-I with penicillin [100 U/mL], streptomycin [100

μg/mL], and 5% dialyzed fetal bovine serum) supplemented with 100

μg/mL zeocin (5-HT_2A/mGlu₂-HEK293), or with 100 μg/mL zeocin

and 1 mg/mL G418 (mGlu₂/Gqo5-HEK293 and 5-HT_2A/mGlu₂/

Gqo5-HEK293). After approximately 3 weeks of culture, antibiotic-

resistant colonies were picked up and cultured further with selected

antibiotics. Various clones were screened for activity by determination

of concentration−response relationships for Glu (mGlu₂/Gqo5-

HEK293), 5-HT (5-HT_2A/mGlu₂-HEK293), and both Glu and 5-

HT (5-HT_2A/mGlu₂/Gqo5-HEK293). The three cell lines and the 5-

HT_2A-HEK293 cell line⁵⁶were cultured under a humidiﬁed

atmosphere at 37 °C and 5% CO₂in a culture medium supplemented

with 1 mg/mL G-418 (5-HT_2A-HEK293), with 100 μg/mL zeocin (5-

HT_2A/mGlu₂-HEK293) or with 100 μg/mL zeocin and 1 mg/mL

G418 (mGlu₂/Gqo5-HEK293 and 5-HT_2A/mGlu₂/Gqo5-HEK293).

Ca²⁺/Fluo-4 Assay. Reference ligands and test compounds were

characterized functionally with various cell lines in the ﬂuorescence-

based Ca²⁺/Fluo-4 assay essentially as previously described.⁵⁶Brieﬂy,

the cells were split into poly-^D-lysine-coated black 96-well plates with

a clear bottom (6 × 10⁴cells/well). The following day, the culture

medium was aspirated and the cells were incubated in 50 μL of assay

buﬀer (Hank’s Balanced salt solution containing 20 mM HEPES, 1

mM CaCl₂, 1 mM MgCl₂, and 2.5 mM probenecid; pH 7.4)

supplemented with 6 mM Fluo-4/AM at 37 °C for 1 h. Then, the

buﬀer was aspirated, the cells were washed once with 100 μL of assay

buﬀer, and then 100 μL of assay buﬀer was added to the cells (in the

antagonist experiments, the antagonist was added at this point). The

96-well plate was assayed in a FLEXStation³(Molecular Devices,

Crawley, UK) measuring emission [in ﬂuorescence units] at 525 nm

caused by excitation at 485 nm before and up to 90 s after the

addition of 33.3 μL of agonist solution in assay buﬀer. To characterize

the PAM activity of ago-PAMs, the compounds were coapplied with

(3-((29-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-

a]pyridin-7-yl)piperidin-4-yl)phenoxy)-3,6,9,12,15,18,21,24,27-

nonaoxanonacosyl)oxy)-2-methoxyphenyl)(1-(4-ﬂuorophenethyl)-

piperidin-4-yl)methanone Bis(2,2,2-triﬂuoroacetate) (50). The title

compound was prepared according to general procedure I (6 mg,

0.005 mmol, 12%). MS (ESI) m/z: 1162.9 [M + H]⁺, 582.4 [M +

1

2H]²⁺; H NMR (600 MHz, MeOD): δ 8.47 (d, J = 7.6 Hz, 1H),

7.35−7.28 (m, 3H), 7.24 (dd, J = 8.2, 1.6 Hz, 1H), 7.23−7.17 (m,

2H), 7.13 (t, J = 7.9 Hz, 1H), 7.11−7.03 (m, 3H), 6.93−6.87 (m,

2H), 4.23−4.16 (m, 4H), 4.13−4.08 (m, 2H), 3.95−3.94 (m, 3H),

3.92−3.88 (m, 2H), 3.85−3.80 (m, 2H), 3.74 (d, J = 12.9 Hz, 2H),

3.72−3.68 (m, 4H), 3.67−3.60 (m, 28H), 3.47 (tt, J = 12.0, 3.40 Hz,

1H), 3.37−3.32 (m, 4H), 3.16−3.08 (m, 2H), 3.07−3.04 (m, 4H),

2.75 (tt, J = 12.1, 3.8 Hz, 1H), 2.21 (dd, J = 14.4, 3.6 Hz, 2H), 2.05−

1.97 (m, 2H), 1.87 (pd, J = 3.8, 13.2, 14.2 Hz, 4H), 1.32−1.26 (m,

1H), 0.72−0.66 (m, 2H), 0.39 (dt, J = 6.1, 4.6 Hz, 2H); ¹³C NMR

(151 MHz, MeOD): δ 204.9, 164.3, 162.7, 158.9, 154.1, 153.5, 149.4,

149.2, 147.2, 138.9, 134.0, 133.45, 133.43, 131.7, 131.6, 128.8, 125.5,

125.0, 121.7, 118.8, 116.8, 116.6, 115.8, 113.7, 98.8, 71.8, 71.7, 71.65,

71.62, 71.59, 71.54, 71.53, 71.47, 70.9, 70.8, 69.8, 68.7, 62.3, 59.1,

53.5, 52.1, 46.4, 42.4, 34.9, 30.5, 29.7, 27.0, 24.6, 8.5, 5.5.

(3-((35-(4-(1-(8-Chloro-3-(cyclopropylmethyl)-[1,2,4]triazolo[4,3-

a ] p y r i d i n - 7 - y l ) p i p e r i d i n - 4 - y l ) p h e n o x y ) -

3,6,9,12,15,18,21,24,27,30,33-undecaoxapentatriacontyl)oxy)-2-

methoxyphenyl)(1-(4-ﬂuorophenethyl)piperidin-4-yl)methanone

Bis(2,2,2-triﬂuoroacetate) (51). The title compound was prepared

according to general procedure I (18 mg, 0.012 mmol, 23%). MS

1

(ESI) m/z: 625.9 [M + 2H]²⁺, 418.0 [M + 3H]³⁺; H NMR (600

MHz, MeOD): δ 8.44 (d, J = 7.6 Hz, 1H), 7.33−7.26 (m, 3H), 7.23

(dd, J = 8.2, 1.6 Hz, 1H), 7.22−7.18 (m, 2H), 7.12 (t, J = 7.9 Hz,

1H), 7.10−7.02 (m, 3H), 6.92−6.87 (m, 2H), 4.24−4.18 (m, 2H),

4.17−4.07 (m, 4H), 3.96−3.93 (m, 3H), 3.91−3.87 (m, 2H), 3.85−

3.81 (m, 2H), 3.78−3.68 (m, 6H), 3.68−3.55 (m, 34H), 3.56−3.43

(m, 2H), 3.37−3.32 (m, 2H), 3.30−3.27 (m, 2H), 3.20−3.09 (m,

2H), 3.09−3.02 (m, 4H), 2.82 (tt, J = 12.1, 3.8 Hz, 1H), 2.30−2.08

(m, 3H), 2.05−1.95 (m, 2H), 1.95−1.82 (m, 4H), 1.30−1.24 (m,

1H), 0.70−0.65 (m, 2H), 0.38 (dt, J = 6.2, 4.6 Hz, 2H); ¹³C NMR

(151 MHz, MeOD): δ 204.9, 164.3, 162.7, 158.9, 153.55, 153.49,

149.4, 149.1, 147.7, 139.0, 134.0, 133.47, 133.45, 131.7, 131.6, 128.8,

125.4, 124.8, 121.7, 118.8, 116.7, 116.6, 115.8, 113.4, 99.9, 71.8,

R

https://dx.doi.org/10.1021/acs.jmedchem.0c01058

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Y = Bottom + (Top − Bottom)/(1 + 10^{((log IC}^−X)×n^H⁾), where Y is the

speciﬁc radioligand binding, Top and Bottom are the plateau values of

the curves, X is the test compound concentration, IC₅₀is the

equilibrium aﬃnity of the test compound, and n_His the Hill slope. K_i

values were calculated from the determined IC₅₀values using the

Cheng−Prusoﬀ equation:⁷¹K_i= IC₅₀/(1 − ([RL]/K_D)), where [RL]

is the radioligand concentration used for the speciﬁc experiment and

K_Dis the dissociation constant determined in the saturation binding

experiments.

50

the agonist EC₁₀, and the antagonist activity was tested by adding the

compounds on the cells in the assay buﬀer prior to agonist

application. The compounds were characterized for various forms of

activities in duplicate at least 3 times at the receptors.

[³H]Ketanserin Binding Assay. The 5-HT_2Areceptor expression

levels in 5-HT_2A-, 5-HT_2A/mGlu₂,- and 5-HT_2A/mGlu₂/Gqo5-

HEK293 cells and the binding properties of selected monovalent

analogues and all seven bivalent analogues at these cell lines were

characterized by the [³H]ketanserin binding assay essentially as

previously described.⁷⁰The three cell lines were cultured until ∼90%

conﬂuent, scraped into ice-cold assay buﬀer (50 mM Tris−HCl, 10

mM MgCl₂, and 0.1 mM ethylenediaminetetraacetic acid; pH 7.4),

homogenized on ice using an IKA T18 Basic Ultra-Turrax for 10 s,

and centrifuged for 20 min at 50.000g at 4 °C. Cell pellets were

resuspended in a fresh assay buﬀer, homogenized on ice, and

centrifuged at 50.000g at 4 °C for another 20 min, after which the

membranes were stored at −80 °C until use.

ASSOCIATED CONTENT

* Supporting Information

■

sı

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01058.

Analytical HPLC traces of all tested compounds;

chemical structures of the reference ligands; basal

characterization of the mGlu2/Gqo5-HEK293 cell line;

and graphs showing the potency/aﬃnity−spacer length

correlations (PDF)

Molecular formula strings with pharmacological data

(CSV)

On the day of the assay, cell membranes were resuspended in the

assay buﬀer, and protein concentrations were determined using the

Bradford protein assay. The membranes were incubated with various

concentrations of [³H]ketanserin (determined by scintillation

counting) and test compounds in a total assay volume of 225 μL in

the wells in the 96-well plate, and the amount of membrane protein

added to wells was adjusted so that the bound/free ratio of

[³H]ketanserin was always <10%. In the saturation binding experi-

ments, 0.03−20 nM [³H]ketanserin were incubated with cell

membranes in the absence (total binding) or presence of 20 μM

clozapine (nonspeciﬁc binding). In the competition binding experi-

ments, a ﬁxed [³H]ketanserin concentration ∼1.5 nM (1.3−1.8 nM)

was incubated with the cell membranes in the presence of various

concentrations of test compounds. The reactions were incubated for 2

h at rt while shaking, and binding reactions were terminated by rapid

ﬁltration through UniFilter 96-well GF/C plates (PerkinElmer,

Waltham, MA) using a FilterMate Harvester (PerkinElmer), followed

by four washes of each with ∼300 μL of ice-cold wash buﬀer (0.9%

w/v NaCl and 50 mM Tris−HCl; pH 7.4). Then, the ﬁlters were

dried at least for 1 h at 50 °C and 25 μL of MicroScint-O

(PerkinElmer) was added to each well in the ﬁlters, which were then

shaken gently overnight. The following day, the amount of bound

radioactivity on the ﬁlters was determined using a TopCount NXT

scintillation counter (PerkinElmer).

AUTHOR INFORMATION

Corresponding Authors

■

Anders A. Jensen − Department of Drug Design and

Pharmacology, Faculty of Health and Medical Sciences,

University of Copenhagen, 2100 Copenhagen OE, Denmark;

Phone: +45 23 96 24 34; Email: aaj@sund.ku.dk

Lennart Bunch − Department of Drug Design and

Pharmacology, Faculty of Health and Medical Sciences,

University of Copenhagen, 2100 Copenhagen OE, Denmark;

orcid.org/0000-0002-0180-4639; Phone: +45 35 33 62

44; Email: lebu@sund.ku.dk

Authors

Christian B. M. Poulie − Department of Drug Design and

Pharmacology, Faculty of Health and Medical Sciences,

University of Copenhagen, 2100 Copenhagen OE, Denmark;

orcid.org/0000-0003-2662-9803

Na Liu − Department of Drug Design and Pharmacology,

Faculty of Health and Medical Sciences, University of

Copenhagen, 2100 Copenhagen OE, Denmark

Data Analysis. The data from the Ca²⁺/Fluo-4 assay was analyzed

using KaleidaGraph 3.6 (Synergy Software). The concentration−

response and concentration−inhibition relationships exhibited by

various ligands in the Ca²⁺/Fluo-4 assay were extracted as the

diﬀerence in relative ﬂuorescence units (ΔRFU) between the

maximum ﬂuorescence level measured after ligand application and

the basal level measured before ligand application. Concentration−

response curves for compounds tested as agonists and PAMs were

ﬁtted to a nonlinear regression curve ﬁt with a variable slope

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.0c01058

[Response = Bottom + (Top − Bottom)/(1 + 10^{((log EC}^−X)×n^H⁾)],

50

Author Contributions

where the Top and Bottom values are plateaus in units of the

response axis, X is the concentration of the ligand, EC₅₀is the

concentration of the ligand that gives a response half way between

Bottom and Top, and n_His the Hill slope. Concentration−inhibition

curves for compounds tested as antagonists were ﬁtted to a nonlinear

regression curve ﬁt with variable slope Response = Bottom + (Top −

C.B.M.P., medicinal chemistry and manuscript preparation;

N.L., medicinal chemistry; A.A.J., pharmacology and manu-

script preparation; L.B., medicinal chemistry and manuscript

preparation.

Notes

Bottom)/(1 + 10^{((log IC}^−X)×n^H⁾), where Top and Bottom values are

50

The authors declare no competing ﬁnancial interest.

plateaus in units of the response axis, X is the concentration of the

ligand, IC₅₀is the concentration of the ligand that gives a response

half way between Bottom and Top, and n_His the Hill slope.

ACKNOWLEDGMENTS

■

The data from the [³H]ketanserin binding assay were analyzed

using GraphPad Prism 7.0c (GraphPad Software, Inc. La Jolla, CA).

Speciﬁc binding from the saturation binding experiments was

analyzed with a nonlinear regression one site-speciﬁc binding model

given by the equation: Y = B_max× X/(K_D+ X), where Y is the speciﬁc

[³H]ketanserin binding, B_maxis the total amount of binding, X is the

[³H]ketanserin concentration, and K_Dis the dissociation constant.

The competition binding data were analyzed with a nonlinear

regression one site-ﬁt log IC₅₀model given by the following equation:

The authors thank the University of Copenhagen, the Aase og

Ejnar Danielsens Foundation, the Augustinus Foundation, and

the China Scholarship Council (CSC) for ﬁnancial support.

ABBREVIATIONS

■

5-HT_2AR, serotonin 2A receptor; CNS, central nervous system;

DCVC, dry-column vacuum chromatography; HEK, human

embryonic kidney; mGlu₂R, metabotropic glutamate 2

S

https://dx.doi.org/10.1021/acs.jmedchem.0c01058

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

́

Lopez-Gimenez, J. F.; Robakis, N. K.; Logothetis, D. E.; Meana, J. J.;

receptor; PEG, polyethylene glycol; TBDPS, tert-butyldiphe-

nylsilyl

́

Gonzalez-Maeso, J. Allosteric signaling through an mGlu2 and 5-

HT2A heteromeric receptor complex and its potential contribution to

schizophrenia. Sci. Signaling 2016, 9, ra5.

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Design, Synthesis, and Pharmacological Characterization of Heterobivalent Ligands for the Putative 5-HT2A/mGlu2Receptor Complex

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