Design, Synthesis, and Characterization of 4-Undecylpiperidine-2-carboxamides as Positive Allosteric Modulators of the Serotonin (5-HT) 5-HT2C Receptor

Article

pubs.acs.org/jmc

Cite This: J. Med. Chem. XXXX, XXX, XXX −XXX

Design, Synthesis, and Characterization of 4‑Undecylpiperidine-2-

carboxamides as Positive Allosteric Modulators of the Serotonin (5-

HT) 5‑HT_2CReceptor

Christopher T. Wild,^†,‡,∥Joanna M. Miszkiel,^†,∥Eric A. Wold,^†,‡,∥Claudia A. Soto,^†Chunyong Ding,^†,‡

Rachel M. Hartley,^†Mark A. White,^§Noelle C. Anastasio,*^,†,‡Kathryn A. Cunningham,*^,†,‡

and Jia Zhou*^,†,‡

‡

§

^†Center for Addiction Research, Department of Pharmacology and Toxicology, and Sealy Center for Structural Biology and

Molecular Biophysics, University of Texas Medical Branch, Galveston, Texas 77555, United States

S

* Supporting Information

ABSTRACT: An impaired signaling capacity of the serotonin (5-HT) 5-HT_2Creceptor (5-HT_2CR) has been implicated in the

neurobehavioral processes that promote relapse vulnerability in cocaine use disorder (CUD). Restoration of the diminished 5-

HT_2CR signaling through positive allosteric modulation presents a novel therapeutic approach. Several new molecules with the 4-

alkylpiperidine-2-carboxamide scaﬀold were designed, synthesized, and pharmacologically evaluated, leading to the discovery of

selective 5-HT_2CR positive allosteric modulators (PAMs). Compound 16 (CYD-1-79) potentiated 5-HT-evoked intracellular

calcium release in cells stably expressing the human 5-HT_2CR but not the 5-HT_2AR cells. A topographically distinct allosteric site

was identiﬁed based on the newly solved 5-HT_2CR structure. Compound 16 modulated 5-HT_2CR-mediated spontaneous

ambulation, partially substituted for the training dose of the 5-HT_2CR agonist WAY163909, synergized with a low dose of

WAY163909 to substitute fully for the stimulus eﬀects of WAY163909, and attenuated relapse vulnerability as assessed in a

rodent self-administration model, indicating its therapeutic promise for CUD.

Dysfunctional serotonin (5-hydroxytryptamine; 5-HT) neu-

rotransmission contributes to the neurobiological states that

precipitate relapse events in CUD and is an important target in

the quest to develop eﬀective medications for CUD.^2,3

Serotonin is involved in essential brain functions as diverse as

appetite, cognition, reward, and motor control.⁴The actions of

5-HT are transduced by 14 receptor subtypes, which are

classiﬁed into seven receptor families based upon the

conjunction of genetic and molecular structure, intracellular

transduction mechanisms, and pharmacological criteria. The 5-

HT₂receptor (5-HT₂R) subtype family of 5-HT receptors is

composed of three G protein-coupled receptors (GPCRs), 5-

HT_2AR, 5-HT_2BR, and 5-HT_2CR, that share a high level of

INTRODUCTION

■

The United States is reeling from a public health crisis driven

by the misuse of psychoactive drugs, the pervasive trajectory to

substance use disorders (SUDs), and the limited access to

eﬃcacious treatment strategies. A 61% increase in adults

initiating cocaine use coupled with the highest cocaine

overdoses reported since 2006 reveal alarming trends¹and

signal the potential for a resurgence in the incidence of cocaine

use disorder (CUD), an acquired brain disorder marked by

habitual cocaine-seeking despite adverse consequences. Core

challenges in recovering from CUD include the risk for relapse

promoted in part by the attentional orientation toward

motivationally relevant, drug-associated stimuli (cue reactivity)

that support drug-seeking during abstinence.²New, eﬀective,

and accessible modalities for the treatment of CUD are needed

to combat relapse risk to alleviate the chronic psychological and

physical burdens of this disorder.

Special Issue: Allosteric Modulators

Received: March 12, 2018

Published: April 5, 2018

A

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Figure 1. Initial design strategy for novel 5-HT_2CR positive allosteric modulators based on 1.

amino acid sequence homology within transmembrane

regions.⁵In particular, hypofunctional 5-HT_2CR neurotrans-

mission is implicated as a core mediator of relapse vulnerability,

and selective 5-HT_2CR agonists are eﬃcacious to blunt cocaine

cue reactivity as well as the rewarding eﬀects of cocaine in

preclinical models^2,3,6−12but lack abuse liability on their

own.¹³⁻¹⁵However, achieving receptor subtype selectivity

versus the highly homologous 5-HT_2AR and 5-HT_2BR is an

essential requisite for the successful development of 5-HT_2CR

ligands because activation of the related 5-HT_2A/2BR systems

can result in signiﬁcant adverse CNS (5-HT_2AR) and

cardiovascular (5-HT_2BR) eﬀects.¹⁶Despite advances in the

past decade,^17,18attaining high 5-HT_2CR selectivity remains a

challenge yet to be solved. For instance, dexfenﬂuramine is a 5-

HT_2CR agonist that was previously approved for antiobesity

therapy. However, this drug was later withdrawn from the

market because of side eﬀects caused by its potent agonist

activity at the 5-HT_2BR.¹⁶Lorcaserin, a conformationally

restricted analogue of dexfenﬂuramine, is a 5-HT_2CR agonist

that has recently been approved by the FDA for treatment of

obesity. However, lorcaserin is classiﬁed as a Schedule IV drug

due to potential side eﬀects, and like most other 5-HT_2CR

agonists, its selectivity for 5-HT_2CR (K_i= 15 nM) is only

modest with a 7.5-fold selectivity over the 5-HT_2AR (112 nM)

and 11.6-fold selectivity versus the 5-HT_2BR (174 nM).¹⁹

Most therapeutic compounds that target GPCRs interact at

the orthosteric site, which accommodates the endogenous

ligand, to control downstream intracellular signaling. However,

with an increasing emphasis on cellular functional screens, a

growing number of allosteric modulators of GPCRs have been

discovered and are under investigation as potential medications.

These small molecules do not bind to the orthosteric ligand site

but instead act at a topographically distinct allosteric site on the

receptor and either potentiate or inhibit the binding or

signaling of an orthosteric ligand. There are theoretical reasons

that allosteric ligands may be preferred therapeutics, including

the prospects for increased subtype selectivity, avoidance of

receptor desensitization, better control of physiological systems,

and upper ceiling eﬀects, as well as separate control of aﬃnity

and eﬃcacy of orthosteric ligands.²⁰In recent years, multiple

allosteric modulators of GPCRs have been developed and

predicted to have robust eﬀects in a variety of CNS

disorders.²¹⁻²³The recent preclinical indications of eﬃcacy,

coupled with the launch of cinacalcet²⁴and maraviroc²⁵as the

ﬁrst marketed GPCR allosteric modulators, provide strong

validation of the clinical utility of both positive and negative

allosteric modulators, respectively. The validated proof of

concept is fueling the discovery of highly selective ligands for

other GPCRs that have been previously intractable. Therefore,

allosteric modulators of 5-HT_2CR present a novel pharmaco-

logical strategy to ﬁne-tune binding or signaling in response to

endogenous 5-HT or synthetic ligands in a site- and event-

speciﬁc manner. Such a strategy may address issues concerning

receptor subtype selectivity, toxicity, desensitization, and long-

term changes in receptor up or down regulation that orthosteric

ligands may promote.²⁶

An analogue of the antibiotic lincomycin, PNU-69176E (1,

Figure 1), was identiﬁed via a chemical library screen as a 5-

HT_2CR positive allosteric modulator (PAM).²⁷Nevertheless,

little eﬀort has been made on structural modiﬁcations of

chemical lead 1 to optimize its PAM activity, and thus the

relevant structure−activity relationship (SAR) studies remain

sparse. Structurally, 1 consists of two moieties directly attached

to the piperidine-2-carboxamide core scaﬀold, an undecyl

lipophilic tail (LT) and a polar head (PH) moiety including a

complex α-^D-galactopyranoside, neither of which are optimal

for displaying drug-like properties such as cLogP, tPSA, and

bioavailability [see Supporting Information (SI), Table S1]. We

have established the only reported method to readily access 1

and its diastereomer and demonstrated that 1 exhibits a proﬁle

of 5-HT_2CR PAM activity with no intrinsic agonist activity at

the 5-HT_2CR or 5-HT_2AR.²⁸While the lead 5-HT_2CR PAM

recently described was shown to suppress locomotor activity,

food intake, and body weight gain,²⁹the impact of 5-HT_2CR

PAMs remains to be explored with in vivo models to uncover

interaction with exogenous 5-HT_2CR stimulation and to

determine eﬃcacy to suppress relapse vulnerability in CUD.

These combined observations clearly justify our structural

optimization campaign to further explore the SAR of structural

modiﬁcations of 1 and develop new 5-HT_2CR putative PAMs

with an enhanced pharmacological proﬁle for the treatment of

CUD. Herein, we describe our eﬀorts for the rational design,

chemical synthesis, and pharmacological evaluation of novel,

simpliﬁed 5-HT_2CR PAMs based on 1 as the chemical lead. The

identiﬁcation of small molecule 5-HT_2CR PAMs aﬀords novel

chemical probes for elucidating the neurobiology of 5-HT_2CR

signaling and new opportunities for proof of concept studies to

develop pharmacotherapies for CUD arising from deﬁcient 5-

HT_2CR signaling capacity.

RESULTS AND DISCUSSION

■

Chemistry. Very little is known about allosteric modulation

of the 5-HT_2CR, and the binding site of 1 remains unclear. In

our drug design strategy, we speculate that 1 consists of three

possible moieties important for binding, including the

previously described PH and LT, and the N−H of the core

piperidine, as depicted in Figure 1. This hypothesis was

formulated initially on the basis of the available pharmaco-

phore-directed homology modeling of the human 5-HT_2CR^30,31

and the limited SAR studies on 1 that showed that shorter alkyl

chains (methyl to n-hexyl groups) or the undecyl derivatives

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a

Scheme 1. Synthesis of PNU-69176E Analogs with Simpliﬁed Polar Heads (PH)

a

Reagents and conditions: (a) see ref 28; (b) 1-undecyne, CuI, Pd(PPh₃)₂Cl₂, Et₃N, rt, 12 h, 94%; (c) H₂(60 psi), PtO₂, HCl/MeOH/H₂O, rt, 16 h,

95%; (d) (i) (Boc)₂O, Et₃N, MeOH, rt, 16 h, 90%; (ii) LiOH, THF/H₂O (v/v = 2:1), rt, 48 h, 85%; (e) (i) amino alcohols, HBTU, DIPEA, DMF,

rt, 16 h; (ii) TFA, CH₂Cl₂, rt, 63−88% (two steps).

with the headgroup of N-phenyl, N,N-diethyl, N-[2-

(dimethylamino)ethyl]-N-methyl, or N-(4-acetoamido-1-naph-

thylsulfonyl) in place of the speciﬁc polar sugar moiety failed to

exert positive allosteric modulation on 5-HT_2CR.²⁷This

suggests that the PH and LT are likely important

pharmacophore groups required for allosteric modulation.

Considering the poor drug-like properties of 1, our initial

eﬀort was directed to simplify and optimize the PH. The N−H

of the piperidine was kept intact so that the target compounds

could form HCl salts for improved aqueous solubility.

We ﬁrst designed a series of new analogues to optimize the

PH by keeping the n-undecyl LT intact while replacing the

sugar moiety of 1 with a variety of simple amino alcohols

(Scheme 1). These simpliﬁed PHs structurally resemble

fragments of the polar α-^D-galactopyranoside moiety of 1,

which presumably undergo selective binding via hydrogen

bonding or dipole−dipole interactions near the membrane

surface. These new analogues, with simpliﬁed and readily

accessible PHs, have enhanced drug-like properties (Table S1)

and development potential. According to our previously

established synthetic route,²⁸a four-step protocol aﬀords 3,

which undergoes Sonogashira coupling with 1-undecyne,

resulting in the alkynylated picolinic derivative 4. Metal-

catalyzed hydrogenation readily provided the (cis-2,4)-piperidyl

carboxylate 5. Boc-protection and hydrolysis of 5 provided 6 as

the key intermediate in high yield on the multigram scale (68%

yield from 3). Direct deprotection of 6 aﬀorded the parent

amino acid 7. With 6 in hand, amidation coupling with a variety

of amino alcohols using HBTU/DIPEA followed by Boc-

deprotection with TFA generated a series of novel derivatives

8−22 (Table 1) with diverse, simpliﬁed PHs in 63−88% yields

(2 steps, Scheme 1). It was noted that the diastereomers 12−15

could be separated through preparative TLC (PTLC) when the

phenyl amino alcohols were employed as the PHs. The other

compounds were inseparable enantiomeric or diastereomeric

mixtures of isomers by TLC.

To further explore the SAR of the LT attached to the core

piperidinyl scaﬀold, replacement of the long alkyl chain with

other saturated moieties (cyclohexyl, cyclohexylethyl or 4-

methylcyclohexylethyl) were investigated. The 2-amino-1-

phenyl-1,3-propanediol PHs consistently resulted in separable

isomers when coupled with the piperidine scaﬀold bearing an

undecyl tail; therefore, these polar heads were retained with the

cyclohexyl tails with the hope that the isomers would again be

separable by TLC. As shown in Scheme 2, Suzuki coupling of 3

with phenylboronic acid or Sonogashira coupling of 3 with

ethynylbenzene or 1-ethynyl-4-methylbenzene aﬀorded requi-

site substituted picolinic derivatives 23−25. Metal-catalyzed

hydrogenation of compounds on a Parr hydrogenator at 60 psi

in the presence of 0.4 equiv of PtO₂and 1 equiv of 37% aq HCl

readily provided compounds 26−28. Boc-protection and

hydrolysis of 26−28 provided 29−31 as the key intermediates

in high yield on the multigram scale. Amidation coupling with

(1S,2S)- or (1R,2R)-(−)-2-amino-1-phenyl-1,3-propanediol

followed by removal of Boc groups with TFA furnished a

series of novel derivatives 32−37 with compact LTs in 83−86%

yields (two steps). As previously observed, each amidation

coupling reaction also produced a pair of diastereoisomers,

which could be separated by PTLC after the Boc-deprotection.

It was our expectation that the compact LTs would result in

ﬁnal compounds that could form crystals suitable for X-ray

diﬀraction. To our delight, two of the six compounds were

crystallized and characterized. As shown in Figure 2, the

absolute stereochemistry of compounds 32 and 35 was

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Table 1. Eﬀects of 4-Alkylpiperdine-2-carboxamides (1 nM) on 5-HT-Induced Ca_i²⁺Release in h5-HT_2CR-CHO Cells

a

Maximum 5-HT-induced Ca_i²⁺release (E_max) in the presence of 1 nM of test compound; the screen utilized concentrations of 5-HT (vehicle, 10⁻¹¹

to 10⁻⁶M) to establish the E_maxof 5-HT in the presence of the test compound as described in Figure S2. * indicates p < 0.05. The E_maxfor the test

compound plus 5-HT was normalized to the E_maxfor 5-HT alone. Subsequent post hoc comparisons between means for E_maxwere made using an

unpaired t test with Welch’s correction (GraphPad Prism). All statistical analyses were conducted with an experiment-wise error rate of α = 0.05.

unambiguously determined by their X-ray crystallography

analyses (Table S2, Figure S1), from which the stereochemistry

of the other diastereoisomers could also be deduced as

described in our previous work.²⁸

signaling pathway. The Ca_i²⁺levels can be regarded as a primary

readout, as has been utilized in a multitude of 5-HT_2CR drug

discovery projects.³³Our assay was conducted in Chinese

hamster ovary (CHO) cells stably transfected with the human

5-HT_2CR (unedited INI isoform; h5-HT_2CR-CHO cells).^28,34,35

The maximum capacity of 5-HT to promote Ca_i²⁺release

(E_max) was established and set to 100% response. All

compounds are reported relative to the E_maxof 5-HT. Previous

titration studies with compound 1 revealed an upward shift of

5-HT-evoked Ca_i²⁺release at the concentration eﬀective to

evoke 20% response (EC₂₀), while 1 nM of 1 produced a

leftward shift of the 5-HT-evoked Ca_i²⁺response curve.²⁸

Therefore, all compounds were initially screened at 1 nM for

In Vitro Ca_i²⁺Release Assay in h5-HT_2CR-CHO Cells.

The best-characterized intracellular pathway of 5-HT_2CR

signaling is the activation of phospholipase Cβ (PLCβ) via

G_αq/11proteins followed by the production of inositol-1,4,5-

trisphosphate (IP₃) and diacylglycerol (DAG), leading to

increased Ca_i²⁺release from intracellular stores.³²Therefore, in

vitro functional characterization of these synthetic compounds

can be determined by utilizing a ﬂuorescence-based, live cell

Ca_i²⁺release assay to measure activation of the 5-HT_2CR

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a

Scheme 2. Synthesis of PNU-69176E Analogs with Compact Lipophilic Tails (LT)

a

Reagents and conditions: (a) Suzuki coupling, (i) phenylboronic acid, Pd(PPh₃)₄, Na₂CO₃, EtOH/H₂O/PhMe = 2:1:1, reﬂux; (ii) H₂SO₄,

methanol, reﬂux, 98% (two steps); or Sonogashira coupling, ethynylbenzene or 1-ethynyl-4-methylbenzene, CuI, Pd(PPh₃)₂Cl₂, Et₃N, rt, 12 h, 88%

to 97%; (b) (i) H₂(60 psi), PtO₂, HCl/MeOH/H₂O, rt, 16 h, 97−98%; (ii) (Boc)₂O, Et₃N, MeOH, rt, 16 h, 96−98%; (c) LiOH, THF/H₂O (v/v =

2:1), rt, 48 h, quantitative yield; (d) (i) amino alcohols, HBTU, DIPEA, DMF, rt, 16 h; (ii) TFA, CH₂Cl₂, rt, 83−86% (two steps).

Figure 2. Determination of absolute conﬁguration of chiral carbons 2 and 4 of the piperdinyl ring. X-ray crystallography structures of analogues

(2S,4R)-32 and (2R,4S)-35.

Figure 3. Eﬀects of select compounds on Ca_i²⁺release in live h5-HT_2CR-CHO cells in the absence (black circles) and in the presence of the test

compound (red triangles) against the concentration−response curve for 5-HT, vehicle (blue circle), or vehicle in the presence of test compound

(green triangle). (A) Compound 14 (1 nM), representative example of a potential NAM; (B) compound 16 (1 nM), representative example of a

potential PAM; (C) compound 16a (1 nM), representative example of an inactive compound. The maximum 5-HT-induced Ca_i²⁺release in the

absence of the test compounds was set as 100%. The E_maxof 5-HT in the presence of the test compounds are listed in Table 1. Concentration−

response curves for remaining compounds are in Figure S2.

their ability to enhance 5-HT-induced Ca_i²⁺release evoked by

increasing concentrations of 5-HT in h5-HT_2CR-CHO cells

(Figure S2). The cells were pretreated for 15 min with each test

compound to establish an absence of intrinsic agonist activity in

each assay. The analysis of novel compounds on maximum 5-

HT-induced Ca_i²⁺release in h5-HT_2CR-CHO cells is

summarized in Table 1.

None of the 24 new analogs screened exhibited intrinsic

eﬃcacy at 1 nM to induce Ca_i²⁺release in h5-HT_2CR-CHO

cells in the absence of 5-HT (Figure S2). Compound 7, the

deprotected amino acid of the key intermediate 6 (Scheme 1),

exhibited no signiﬁcant PAM activity (Table 1). Topographical

analysis of 1 revealed 2- or 3-carbon long chains to the nearest

heteroatom (O and Cl) in the sugar-like PH. Therefore, the

selection of PHs emphasized the incorporation of −OH(s) two

carbons away from the amide linker. The coupling of

ethanolamine with the key intermediate aﬀorded 8. Similar to

7, no signiﬁcant PAM activity with 8 was observed (Table 1).

Incorporation of two −OH groups (a 1,3-propandiol fragment,

9) provided a negligible increase in 5-HT-induced Ca_i²⁺release,

though statistically signiﬁcant. Various analogues that retained

the 1,3-propanediol fragment with the addition of a methyl or

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Figure 4. Eﬀects of 16 on Ca_i²⁺release in live h5-HT_2CR-CHO cells (A) and h5-HT_2AR-CHO cells (B) against the concentration−response curve

for 5-HT. (A) Compound 16 (1 pM, 1 nM, 1 μM) demonstrates an upward shift in h5-HT_2CR-CHO cells. (B) Compound 16 (1 pM, 1 nM, 1 μM)

does not alter 5-HT-induced signaling in h5-HT_2AR-CHO cells. The maximum 5-HT-induced Ca_i²⁺release in the absence of the test compounds

was set as 100%.

phenyl group (10−15) did not provide for the desired PAM

activity (Table 1). However, compound 14, which was a

separable isomer with the (2S,4R) conﬁguration at the

piperidine ring and the (1R,2R) conﬁguration for the PH

moiety, displayed signiﬁcant activity consistent as a negative

allosteric modulator (NAM, Figure 3A, Table 1). Next, the use

of an additional carbon spacer between the amide linker and

the most distal −OH was explored through the incorporation

of a 1,2-propanediol fragment (16). The glycol moiety proved

fruitful. Compound 16 displayed signiﬁcant PAM activity and

increased the 5-HT-evoked Ca_i²⁺release ∼23% above the 5-HT

E_maxvalue (Figure 3B). The enantiomerically pure versions of

the PH of 16 were incorporated into the scaﬀold and were

tested and showed a stereochemical preference for PAM

activity. Compound 16a, the R-variant at the PH, lacked

appreciable PAM activity (Figure 3C), while the S-conﬁg-

uration (16b), like 16, displayed signiﬁcant PAM activity

(Table 1). Incorporation of a terminal −Cl abolished

statistically signiﬁcant activity (17). Additional attempts to

incorporate various hydrogen bond donor/acceptor moieties as

PHs did not produce compounds with PAM activity [N-ethyl

morpholino (18), methyl esters of threonine (19), serine (20),

tyrosine (21), and tryptophan (22)]. The lactate dehydrogen-

ase assay was employed to predict the cytotoxicity of

compounds at 0.1 nM and 1 μM; none of the tested

compounds exhibited values in this assay indicative of cellular

toxicity (Table S3).

Compound 16 promoted an upward shift of 5-HT-evoked

Ca_i²⁺release at multiple concentrations (Figure 4A) without a

leftward shift, an indication that 16 promotes improved 5-HT

eﬃcacy but did not impact 5-HT potency at the 5-HT_2CR

(Figure 4A). To determine the selectivity of 16 for allosteric

modulation of the 5-HT_2CR over the 5-HT_2AR, multiple

concentrations of 16 were employed to assess 5-HT-evoked

Ca_i²⁺release in h5-HT_2AR-CHO cells (Figure 4B).^28,34

Compound 16 produced no change in the 5-HT E_maxor

EC₅₀at the 5-HT_2AR (Figure 4B). These data provide the

conceptual framework for the continued development of

selective PAMs of the 5-HT_2CR given the ability of 16 to

aﬀect 5-HT eﬃcacy at the 5-HT_2CR (Figure 4A), but not the 5-

HT_2AR (Figure 4B).

while changing the LT from the undecyl tail to an

ethylcyclohexyl moiety, aﬀorded a NAM that promoted a

slight but signiﬁcant decrease in 5-HT-induced Ca_i²⁺release

(32, 96.0% 1.9%). All other attempts to utilize various LTs

failed to produce any compounds with allosteric eﬀects (34−

37). Exploration of the remaining chemical space, in particular

with regard to the LT, will be reported in due course.

Molecular Docking to 5-HT_2CR. To explore a potential

binding mode and provide additional lines of evidence that the

described compounds are allosteric modulators, molecular

docking using the Schrodinger Drug Discovery Suite was

̈

employed to identify the allosteric binding site relative to the

orthosteric binding site (see SI for details on docking protocol).

Until recently, the only solved structures of serotonin receptors

were the 5-HT_1BR and 5-HT_2BR.³⁶⁻³⁸However, the very

recently disclosed 5-HT_2CR structure has aﬀorded the

opportunity to conduct the ﬁrst allosteric-5-HT_2CR docking

study.³⁹With compound 16b displaying positive allosteric

modulation, molecular docking using the active state of the 5-

HT_2CR was used to search the extracellular portion of the

receptor for a suitable allosteric site.

Figure 5A depicts a global view of the 5-HT_2CR and the

docked endogenous ligand 5-HT (pink space-ﬁll) in the

orthosteric site engaging residues consistent with previous

reports based on site-directed mutagenesis studies.⁴¹The red

cluster overlay of 16b (red wire-representation of top ﬁve poses

using Schrodinger Glide scoring function) highlighted a

̈

possible binding pocket for the proposed PAM as being

topologically distinct from the orthosteric site.

The allosteric site (Figure 5A) is near extracellular loop 2

(EL2) accommodating the PH of the proposed PAM, an

observation also made when describing similar binding modes

of other class A GPCRs.⁴²Figure 5B depicts the 1,2-diol PH

moiety with the S-conﬁguration engaging in a bidentate, H-

bonding interaction with the backbone carbonyl L209 of EL2, a

residue conserved in the 5-HT₂R subfamily. Additionally, the

ionizable N atom of the piperidine ring engages in an H-bond

with the −OH side chain of S334 of transmembrane helix VI

(TMH VI), which is not present in either the 5-HT_2AR or 5-

HT_2BR, thus suggesting a predicted molecular basis for the 5-

HT_2CR subtype functional selectivity over the 5-HT_2AR as

described in our cell-based studies. The predicted bridging

eﬀect of 16b between EL2 and TMH VI may result in a

drawing in of the extracellular portion of TMH VI and, thereby,

promote the outward shift of the intracellular portion of TMH

VI, consistent with reports that this helical movement is

The compounds synthesized according to Scheme 2 retained

the 1-phenyl-1,3-propandiol PH in an eﬀort to produce

separable isomers to further explore the impact of changes

made to the LT. Preserving the PH used in the potential NAM

14, which produced a decrease in 5-HT-mediated Ca_i²⁺release,

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Table 2. Displacement of Radioligand Binding by

Compound 16 (10 μM) in a Broad Panel of Receptors and

a

Transporters

receptor or

transporter

% inhib (10

K_i

(μM)

b

c

radioligand

μM)

5-HT_1A

5-HT_1B

5-HT_1D

5-HT_1E

5-HT_2A

5-HT_2B

5-HT_2C

5-HT₃

5-HT_5A

5-HT₆

5-HT₇

D₁

[³H]-8-OH-DPAT

[³H]-GR125743

[³H]-5-HT

[³H]-ketanserin

[³H]-LSD

25.7

−18.1

5.5

e

0.7

d

37.1

d

15.1

d

[³H]-mesulergine

[³H]-LY278584

[³H]-LSD

9.8

27.0

17.2

18.1

26.8

29.3

28.8

[³H]-LSD

Figure 5. Binding poses of (A) global view of 5-HT_2CR (PDB 6BQG)

with 5-HT (pink space-ﬁll representation) at the orthosteric site and

16b (red stick representation overlay of top ﬁve poses) at the

proposed allosteric site; (B) representative pose of 16b (red stick

representation) engaged with residues of proposed allosteric site

(superscript designation represents Ballesteros−Weinstein nota-

tion);⁴⁰(C) representative pose of 16a (red stick representation).

[³H]-LSD

[³H]-SCH23390

D₂

[³H]-N-

methylspiperone

D₃

D₄

[³H]-N-

71.4

4.7

methylspiperone

[³H]-N-

−14.2

e

methylspiperone

D₅

[³H]-SCH23390

[³H]-WIN35428

[³H]-citalopram

32.5

66.3

24.8

e

associated with the active state of the receptor.^39,43,44

Compound 16a, containing the 1,2-diol PH with the R-

conﬁguration, does not form the same EL2-to-TMH VI bridge

(Figure 5C), providing a potential explanation as to why 16a is

inactive in the described cell-based assay.

DAT

SERT

1.8

e

a

National Institute of Mental Health (NIMH) Psychoactive Drug

Screening Program (PDSP).⁴⁵Receptor binding proﬁles were

generously provided by NIMH PDSP, Contract no. HHSN-271-

2013-00017-C. The PDSP is directed by Bryan L. Roth, M.D., Ph.D.,

at the University of North Carolina at Chapel Hill and Project Oﬃcer

In Vitro Assessment of Oﬀ-target Eﬀects of Com-

pound 16. Preliminary data from a broad-panel assay

generously provided by the National Institute of Mental Health

(NIMH) Psychoactive Drug Screening Program (PDSP)⁴⁵

showed that the most promising PAM, compound 16 assessed

at 10 μM displayed no signiﬁcant ability to displace binding to a

variety of related receptors (Table 2). Speciﬁcally, at 10 μM, 16

did not displace binding of radioligands to 5-HT receptors (5-

HT_1AR, 5-HT_1BR, 5-HT_1DR, 5-HT_1ER, 5-HT_2AR, 5-HT_2BR, 5-

HT_2CR, 5-HT₃R, 5-HT_5AR, 5-HT₆R, 5-HT₇R), dopamine

receptors (D₁, D₂, D₄, D₅), or monoamine transporters [5-

HT transporter (SERT) and norepinephrine transporter

(NET)]. Compound 16 (10 μM) shows weak to no

displacement of binding to other evaluated receptors and

monoamine transporters (Table S4). Compound 16 (10 μM)

did display signiﬁcant inhibition (deﬁned as greater than 50%)

of radioligand binding at the dopamine transporter (DAT;

66.3%), dopamine D₃receptor (71.7%), or α_2Aand α_2B

receptors (85.1% and 80.7%, respectively). However, the

concentration (10 μM) tested in the radioligand displacement

assay is signiﬁcantly higher than the concentrations of 16 (1

pM, 1 nM, 1 μM) screened for activity in the functional assay.

Therefore, in total, compound 16 appears to be promising with

regard to displaying minimal oﬀ-target eﬀects, a commonly

postulated advantage of allosteric modulators of GPCRs.²⁰

Moreover, 16 showed little to no interaction with the

orthosteric sites of the tested 5-HT receptor subtypes.

b

Jamie Driscoll at NIMH, Bethesda MD, USA. Data represent mean %

inhibition (n = 4) for compound 16 assessed for displacement of

binding at targets. Inhibition of binding >50% at 10 μM of compound

16 resulted in determination of K_i. Data represent K_i(μM) values

obtained from nonlinear regression of radioligand competition

isotherms. K_ivalues are calculated from best ﬁt IC₅₀values using the

Cheng−Prusoﬀ equation. Data for 5-HT₂receptor subtypes

represent the average of the mean % inhibition (n = 8) for compound

c

d

e

16. Not tested.

concentration (T_max) after 10 mg/kg po was 3.3 0.58 h. The

plasma clearance (CL) of 16 was 5.37 0.61 L h⁻¹kg⁻¹after 5

mg/kg iv, while the volume of distribution plasma distribution

(V_ss) was 35.07

maximum serum concentration (C_max) of 68.1

4.66 L/kg. Compound 16 displayed a

6.8 ng/mL

after 10 mg/kg po, while 16 exhibited moderate plasma

exposure (AUC_0−inf) after administration of 5 mg/kg iv (939

108 ng·h·mL⁻¹) or 10 mg/kg po (737

56 ng·h·mL⁻¹).

Decent oral bioavailability (F) was observed (10 mg/kg po;

39.1%). We also calculated the central nervous system (CNS)

multiparameter optimization (MPO) value for compound 16 to

be 4.5; collective scores range from 0 to 6 with higher MPO

scores predicted to be more desirable for CNS medication

candidates (Table S5).⁴⁶Based upon these data, compound 16

was selected for subsequent evaluation of in vivo eﬃcacy.

Eﬀects of Compound 16 on Spontaneous Locomotor

Activity. Our candidate 5-HT_2CR PAM could be expected to

enhance the eﬀects of endogenous (5-HT) or exogenous 5-

HT_2CR agonists in vivo. Selective and nonselective agonists of

the 5-HT_2CR induce dose-dependent suppression of sponta-

neous locomotor activity in rodents, which is reversed by

selective 5-HT_2CR antagonists.^9,47,48In the present experiment,

we tested the hypothesis that 16 would evoke a modest

In Vivo Pharmacokinetic (PK) Proﬁle of Compound 16.

The PK proﬁle of 16 was assessed in a pilot study to investigate

whether this class of molecules exhibits favorable drug-like

properties. The PK parameters of 16 were evaluated in rats

after single iv (5 mg/kg) or po (10 mg/kg) administration. As

summarized in Table 3, Compound 16 displays a decent half-

life (T_1/2) after administration of 5 mg/kg iv (t_1/2= 6.59 0.26

h) or 10 mg/kg po (5.82 0.37 h). The time of maximum

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a

Table 3. In Vivo Pharmacokinetic Proﬁle of Compound 16

dose (mg/kg)

T_1/2(h)

T_max(h)

CL (L h⁻¹kg⁻¹

)

V_ss(L/kg)

C_max(ng/mL)

AUC_0−inf(ng·h·mL⁻¹

)

F (%)

5, iv

6.59 0.26

5.82 0.37

b

5.37 0.61

35.07 4.66

939 108

737 56

10, po

3.3 0.58

b

68.1 6.8

39.1

a

Values are the average of three runs. Vehicle, DMSO/Tween 80/saline (v/v 1:3:6). T_1/2, half-life; T_max, time of maximum concentration; CL,

plasma clearance; V_ss, volume of distribution; C_max, maximum concentration; AUC_0−inf, area under the plasma concentration−time curve; F, oral

b

bioavailability. Not determined.

Figure 6. Eﬀects of compound 16 on spontaneous locomotor activity. (A) Groups of rats (n = 7−8/group) naive to the activity monitors were

injected with a single dose of saline or compound 16 (0.5, 1, or 5 mg/kg; ip) immediately prior to the start of locomotor assessment. (B) Groups of

rats (n = 9−10/group) naive to the activity monitors were injected with a single dose of the 5-HT_2CR antagonist SB242084 (1 mg/kg; ip; 15 min

pretreatment) and the 5-HT_2CR agonist WAY163909 (1 mg/kg; ip) in combination with compound 16 (5 mg/kg; ip) immediately prior to the start

of locomotor assessment. The mean total ambulations ( SEM) in 30 min are plotted. *p < 0.05.

suppression of motor activity in adult male Sprague−Dawley

rats similar to the observations made for a recently evaluated

candidate 5-HT_2CR PAM.²⁹We assessed the eﬀects of

compound 16 (0.5, 1, or 5 mg/kg ip) on spontaneous

locomotor activity over a 90 min session. As the eﬀects of

compound 16 were conﬁned to the initial 30 min of the

session, Figure 6 presents ambulations totaled for that 30 min.

A main eﬀect of compound 16 was observed for mean total

ambulations ( SEM) [F_(3,25)= 3.44, p = 0.03]. A priori

comparisons revealed that 5 mg/kg of compound 16

signiﬁcantly reduced mean total ambulations ( SEM) vs saline

(p < 0.05; Figure 6A). A main eﬀect of compound 16 treatment

was also observed for mean total vertical activity ( SEM) in 30

min [F_(3,25)= 9.14, p = 0.0003]. A priori comparisons revealed

that 5 mg/kg of compound 16 signiﬁcantly reduced mean total

vertical activity ( SEM) vs saline (p < 0.05; data not shown).

Ambulation and vertical activity are often positively correlated;

however, these measures are actually independent of one

another, can be pharmacologically dissociated, and appear to be

mediated by diﬀerent neural circuitry.⁴⁹⁻⁵²

Figure 6B illustrates the allosteric eﬀects of compound 16 (5

mg/kg) in combination with the selective 5-HT_2CR orthosteric

agonist WAY163909 (1 mg/kg) on spontaneous locomotor

activity in the presence or absence of the 5-HT_2CR antagonist

SB242084 (1 mg/kg). Pretreatment with SB242084 inhibits

WAY163909-evoked suppression of ambulations, but not

vertical activity (data not shown). A main eﬀect of treatment

was observed for ambulations totaled across the 30 min session

[F_(3,34)= 8.25, p = 0.0003]. A priori comparisons revealed that

the combination of compound 16 plus WAY163909 reduced

total ambulations versus WAY163909 alone (p < 0.05; Figure

6B); this eﬀect was prevented by SB242084 (p < 0.05; Figure

6B). A main eﬀect of treatment was observed for vertical

activity totaled across the 30 min session [F_(3,34)= 19.19, p <

0.0001]; a priori comparisons revealed that the combination of

compound 16 plus WAY163909 reduced vertical activity versus

WAY163909 alone (p < 0.05; data not shown); this eﬀect was

only marginally prevented by SB242084 pretreatment (p <

0.05; data not shown).

These data demonstrate that the 5-HT_2CR is engaged in the

eﬀects of compound 16 to suppress motor activity in the

presence of exogenous 5-HT_2CR stimulation with WAY163909.

Furthermore, compound 16 may augment endogenous 5-

HT_2CR signaling in vivo to suppress spontaneous ambulations,

as might be expected of a PAM.

Eﬀects of Compound 16 in a WAY163909 vs Saline

Drug Discrimination Assay. We employed the drug

discrimination assay to explore the impact of compound 16

in the context of endogenous or exogenous 5-HT_2CR

stimulation. The drug discrimination assay is widely recognized

as one of the major methods in neuropharmacology and drug

discovery.⁵³⁻⁵⁵This assay has face validity for modeling the

subjective eﬀects of drugs that penetrate the blood−brain

barrier as well as to assess the inﬂuence of the novel

compounds on the interoceptive eﬀects of receptor-selective

agonists or other ligands, including allosteric modulators.⁵⁶⁻⁵⁹

Drug discrimination procedures have been extensively used to

characterize the neuropharmacological proﬁle of novel small

molecules as well as 5-HT₂R subtype agonists, including 5-

HT_2CR agonists (e.g., m-chlorophenylpiperazine, Ro 60-

0175).⁶⁰⁻⁶³Here, we trained rats to discriminate 0.75 mg/kg

of the selective 5-HT_2CR agonist WAY163909 from saline in a

two-lever drug discrimination protocol. The mean number of

sessions required to meet the criterion for acquisition of the

WAY163909 (0.75 mg/kg, ip; 15 min pretreatment) versus

saline discrimination was 44 training sessions (range 39−57).

During dose−response tests, WAY163909 (0.125−1.0 mg/kg,

ip) elicited a dose-dependent increase in WAY163909-

appropriate responding whereas saline resulted in <10%

WAY163909-appropriate responding (Figure 7A, closed

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Figure 7. Dose−response relationship of WAY163909, compound 16, and the combination in a WAY163909 vs saline drug discrimination assay.

The dose−response curves (n = 7−9/dose) for (A) WAY163909 (ip) and (B) compound 16 (ip) are presented. Closed circles (solid lines) denote

the mean percentage of WAY163909-appropriate responding ( SEM; left axis), and open triangles (dotted lines) denote the mean responses/min

( SEM; right axis). *p < 0.05 vs 0.75 mg/kg WAY163909. (C) The results of substitution tests in which WAY163909 alone or in combination with

compound 16 are presented. The dotted line represents the % WAY163909-lever response observed for the training dose of WAY163909 (0.75 mg/

kg). *p < 0.05 vs 0.5 mg/kg of compound 16; p < 0.05 vs 0.5 mg/kg of WAY163909.

̂

circles). Greater than 95% WAY163909-appropriate responding

was observed at the training dose (0.75 mg/kg, ip). Response

rates were stable across all test doses of WAY163909 (Figure

7A, open triangles). The time course of WAY163909-

appropriate responding at 0.75 mg/kg (Figure S3) indicated

that the stimulus eﬀects of the training dose of WAY163909

(0.75 mg/kg, ip) are maximal at a 15 min pretreatment time

and decline until 120 min at which time rats cannot

discriminate this dose of WAY163909 (Figure S3). The dose

predicted to elicit 50% of WAY163909-lever responding (ED₅₀)

in rats is 0.53 mg/kg.

eﬃcacy as a 5-HT_2CR agonist and are consistent with other

ﬁndings regarding the discriminative stimulus eﬀects of

allosteric modulators.⁶⁵

We also postulated that a 5-HT_2CR PAM would enhance the

eﬀects of the 5-HT_2CR agonist WAY163909 to evoke its

discriminative stimulus eﬀects. To further probe this premise,

rats were injected with low doses of WAY163909 (0.5 mg/kg)

in combination with compound 16 (0.5 mg/kg; Figure 7C);

each evokes ∼30% WAY163909-appropriate responding with

rates of ∼30 responses/min. Interestingly, the coadministration

of 0.5 mg/kg of WAY163909 plus 0.5 mg/kg of compound 16

synergized to evoke a full substitution for WAY163909 (∼95%

WAY163909-lever responding). The response rate seen with

the combination (15 2.0 responses/min) did not diﬀer from

0.5 mg/kg of WAY163909 (31.0 4.4 responses/min), but was

signiﬁcantly lower than 0.5 mg/kg of compound 16 (27.4 5.8

responses/min) administered alone (p < 0.05). Taken together,

these data suggest that compound 16 acts in vivo to augment 5-

HT_2CR agonist-mediated signaling in vivo.

Eﬀects of Compound 16 on Cocaine Cue Reactivity.

We employed a rat model of cocaine self-administration to

establish the eﬃcacy of compound 16 to suppress relapse

vulnerability assessed by responding for the discrete cue

complex paired with cocaine intake (cue reactivity).^8,9,12,66,67

Selective 5-HT_2CR agonists eﬀectively suppress cocaine cue

reactivity and cocaine intake in the self-administration

paradigm,^{9,12,47,68,69}with no evidence of abuse liability on

their own.¹³⁻¹⁵Relapse during abstinence from cocaine use is a

major hurdle for the successful treatment of CUD; an eﬀective

pharmacotherapy that improves inhibitory control in the

context of exposure to cocaine-associated cues would represent

a ﬁrst-in-class drug useful for those suﬀering from CUD.^2,3

The self-administration assay has the greatest face validity for

modeling human drug-taking and cue-reactivity and for

therapeutic drug discovery. Cocaine self-administration training

consisted of 14 daily 180 min sessions during which rats were

trained to lever press for cocaine infusions (0.75 mg/kg per 0.1

mL infusion, iv).^6,8,9,11Schedule completions on the active lever

resulted in delivery of a cocaine infusion paired simultaneously

with illumination of the house and stimulus lights and

activation of the infusion pump (discrete cue complex paired

with cocaine delivery); inactive lever presses produced no

scheduled consequences. Following stable acquisition, rats were

Compound 16 alone did not stimulate Ca_i²⁺release in live

h5-HT_2CR-CHO cells, indicating an inability to act as a 5-

HT_2CR agonist in vitro; therefore, we hypothesized that

compound 16 would not substitute fully for the full 5-HT_2CR

agonist WAY163909 in rats trained to discriminate

WAY163909 from saline. Compound 16 (0.125−0.5 mg/kg;

ip) evoked a maximum of 45% 20% (SEM) WAY163909-

appropriate responding at a dose of 1 mg/kg (Figure 7B, closed

circles). All doses of 16 elicited WAY163909-lever responding

that was signiﬁcantly diﬀerent than the training dose (0.75 mg/

kg, p < 0.05); this lack of substitution of 16 suggests a

dissociation between the stimulus eﬀects of the full 5-HT_2CR

agonist WAY163909 and compound 16. Response rates

(Figure 7B, open triangles) were stable across the lowest

doses of compound 16 (0.125−0.5 mg/kg). At the highest dose

of compound 16 tested (1 mg/kg), seven out of the nine rats

tested completed the lever press response criterion; the

response rate for these rats was signiﬁcantly decreased relative

to the training dose of WAY163909 (p < 0.05). While this

observation may reﬂect motor suppression evoked by 16, the

maximal dose of 16 (1 mg/kg) employed in drug

discrimination studies was not associated with suppressed

ambulations (Figure 6A). In general, responding in an operant

task is relatively more sensitive than is locomotor activity, and it

is important to note that modiﬁcations in response rates are

dissociable from response choice.^60,64

The observation that compound 16 partially substituted for

WAY163909 may not be unexpected given that the eﬀects of

endogenous 5-HT could be enhanced by a 5-HT_2CR PAM.

Coupled with data that compound 16 does not have intrinsic

activity in the h5-HT_2CR-CHO assay, these data support the

contention that compound 16 does not exhibit appreciable

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reintroduced to the self-administration chambers 24 h later and

assayed in a cue reactivity test session comprised of two

sequential components. The ﬁrst component (10 min)

evaluated whether rats would exhibit diﬀerential levels of

lever presses when placed in the context in the absence of the

discrete cue complex. The second component (60 min) was

signaled by a single, nonresponse contingent delivery of the

discrete cue complex presented immediately at the termination

of the ﬁrst component.⁶

We assessed the eﬀects of compound 16 (1 mg/kg; ip) on

cocaine cue reactivity across both components of the test

session. A main eﬀect of treatment [(F_(1,14)= 7.28, p = 0.0173],

time [(F_(13,182)= 82.07, p < 0.0001], and a treatment × time

interaction [(F_(13,182)= 4.58, p < 0.0001] for previously active

lever presses was observed. Previously active lever presses

reinforced by the discrete cue complex were signiﬁcantly lower

following pretreatment with 16 vs saline (Figure 8; p < 0.05);

site of the 5-HT_2CR; a topographically distinct site was

identiﬁed by our molecular docking analyses. Compound 16

exhibited a favorable overall pharmacokinetic proﬁle such as

decent oral bioavailability and half-life, and a respectable CNS

MPO value (4.5 out of 6). Compound 16 evoked a modulation

of 5-HT_2CR-mediated spontaneous ambulations, partially

substituted for the training dose of WAY163909, synergized

with a low dose of WAY163909 to substitute fully for the

stimulus eﬀects of this selective 5-HT_2CR agonist, and

attenuated cocaine cue reactivity in rats. Our current knowledge

of the selectivity and speciﬁcity of compound 16 is driving our

future studies to develop drug-like 5-HT_2CR PAMs with

constrained oﬀ-target eﬀects.

Our success in the chemical design, synthesis, and

pharmacological evaluation of these new molecules opens

new doors to a greater understanding of 5-HT_2CR allosteric

modulation and the development of novel pharmacotherapeu-

tics for CUD characterized by deﬁcient 5-HT_2CR signaling

capacity.^2,3To the best of our knowledge, this report includes

the only in vivo investigation of a 5-HT_2CR PAM in a proof-of-

concept study toward the treatment of CUD and is the only

study that proposes allosteric binding sites for candidate PAMs

on the 5-HT_2CR. As impaired signaling capacity of the 5-

HT_2CR may contribute to the neurobehavioral processes that

underlie chronic health issues such as depression, impulsivity

disorders, obesity, and schizophrenia, the full potential for

novel molecules with actions as selective 5-HT_2CR PAMs

remains to be elucidated. Continued medicinal chemistry

eﬀorts toward structural optimization, in particular at the LT,

and in vitro and in vivo cellular evaluations are underway.

Figure 8. Eﬀects of compound 16 on cocaine cue reactivity in rats (n =

7/treatment group). Mean ( SEM) previously active lever are

presented for the cue reactivity test session following administration

for saline or compound 16. The ﬁrst component of the cue reactivity

session (initial 10 min) evaluated lever presses in the absence of the

discrete cue complex. The second component was signaled by the

single delivery of the discrete cue complex (denoted by arrow). Presses

on the previously active lever in the 60 min second component were

reinforced by the discrete cue complex; inactive lever presses were

recorded but produced no scheduled consequences. Rats were injected

with saline (1 mL/kg, ip) or compound 16 (1 mg/kg; ip) 15 min prior

to a cocaine cue reactivity test session. Compound 16 suppressed cue

reactivity vs saline (*p < 0.05).

EXPERIMENTAL SECTION

■

Chemistry. General. All commercially available starting materials

and solvents were reagent grade and used without further puriﬁcation.

Reactions were performed under a nitrogen atmosphere in dry

glassware with magnetic stirring. Preparative column chromatography

was performed using silica gel 60, particle size 0.063−0.200 mm (70−

230 mesh, ﬂash). Analytical TLC was carried out employing silica gel

60 F254 plates (Merck, Darmstadt). Visualization of the developed

chromatograms was performed with detection by UV (254 nm). NMR

spectra were recorded on a Bruker-600 (¹H, 600 MHz; ¹³C, 150 MHz)

spectrometer or Bruker-300 (¹H, 300 MHz; ¹³C, 75 MHz). H and

1

¹³C NMR spectra were recorded with TMS as an internal reference.

Chemical shifts were expressed in ppm, and J values were given in Hz.

High-resolution mass spectra (HRMS) were obtained from Thermo

Fisher LTQ Orbitrap Elite mass spectrometer. Parameters include the

following: Nano ESI spray voltage was 1.8 kV; capillary temperature

was 275 °C, and the resolution was 60 000; ionization was achieved by

positive mode. Melting points were measured on a Thermo Scientiﬁc

electrothermal digital melting point apparatus and were uncorrected.

Purity of ﬁnal compounds was determined by analytical HPLC, which

was carried out on a Shimadzu HPLC system (model CBM-20A LC-

20AD SPD-20A UV/vis). HPLC analysis conditions were as follows:

Waters μBondapak C18 (300 mm × 3.9 mm); ﬂow rate 0.5 mL/min;

UV detection at 270 and 254 nm; linear gradient from 30% acetonitrile

in water (0.1% TFA) to 100% acetonitrile (0.1% TFA) in 20 min

followed by 30 min of the last-named solvent. All newly synthesized

compound 16 did not signiﬁcantly alter inactive lever presses vs

saline (data not shown). These in vivo data provide the ﬁrst

reported in vivo evidence that a putative 5-HT_2CR PAM

suppresses a key facet of relapse vulnerability in CUD.

CONCLUSIONS

■

A series of structurally simpliﬁed analogues have been rationally

designed and synthesized based on the 5-HT_2CR PAM 1. The

allosteric modulatory SAR of the 5-HT_2CR was explored using a

ﬂuorescence-based, 5-HT-evoked Ca_i²⁺release assay. The

stereochemistry of the synthesized compounds was secured

by X-ray crystallographic analysis of compounds 32 and 35.

Simpliﬁcation of the PH of 1 is tolerable or even favorable for

potentiation of 5-HT_2CR-evoked Ca_i²⁺release. None of the

compounds exhibited 5-HT_2CR agonist eﬃcacy when tested

alone, and 16 (CYD-1-79) potentiated 5-HT-evoked Ca_i²⁺

release in h5-HT_2CR-CHO cells with receptor subtype

selectivity versus the 5-HT_2AR. Radioligand binding results

suggest that these compounds do not bind to the orthosteric

compounds have been fully characterized with H NMR, ¹³C NMR,

HRMS, and HPLC analyses to ensure a purity >95% prior to the

biological evaluations.

(cis-2,4)-4-Undecylpiperidine-2-carboxylic acid (7). Compound 6

(54 mg, 0.14 mmol) was dissolved in CH₂Cl₂(1 mL). TFA (0.25 mL)

was added to the solution at rt. The reaction mixture was stirred for 2

h at rt, and the reaction was judged complete by TLC. The reaction

mixture was then concentrated under reduced pressure to aﬀord an

oily , which was partitioned between CH₂Cl₂(15 mL) and a saturated

1

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solution of NaHCO₃(5 mL). The organic layer was treated with

anhydrous Na₂SO₄(5 mL), ﬁltered, and concentrated under reduced

pressure. The crude mixture was loaded on a PTLC plate (hexanes/

EtOAc; 5:1) which aﬀorded 7 (22.5 mg, 40%) as a white solid; mp

1H), 1.43 (m, 1H), 1.25 (m, 19H), 1.17 (m, 3H), 1.08 (m, 2H), 0.88

(t, 3H, J = 7.2 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 174.8, 174.2,

67.8, 67.7, 63.7, 61.0, 60.4, 54.9, 45.7, 45.3, 37.0, 36.9, 36.0, 35.7, 32.2,

31.9, 29.9, 29.7, 29.4, 26.5, 26.4, 22.7, 20.5, 20.4, 14.1. HRMS Calcd

for C₂₁H₄₂N₂O₃: [M + H]⁺371.3274; found 371.3258.

1

185−187 °C. H NMR (300 MHz, CDCl₃) δ 9.55 (s, 1H), 8.77 (s,

1H), 3.44 (t, J = 12.4 Hz, 2H), 3.01−2.76 (m, 1H), 2.34 (d, J = 13.2

Hz, 1H), 1.81 (d, J = 11.5 Hz, 1H), 1.64−1.42 (m, 2H), 1.30 (s, 21H),

0.93 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.12, 59.64,

43.72, 36.17, 35.05, 33.50, 31.91, 29.68, 29.64, 29.62, 29.34, 28.70,

26.48, 22.68, 14.09. HRMS (ESI) calcd for C₁₇H₃₃NO₂[M + H]⁺

284.2584; found 284.2580.

(2R,4S)-N-((1S,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-unde-

cylpiperidine-2-carboxamide ((2R,4S)-12) and (2S,4R)-N-((1S,2S)-

1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carbox-

amide ((2S,4R)-13). Compounds (2R,4S)-12 (14 mg, 35%) and

(2S,4R)-13 (15 mg, 35%) were prepared (2 steps) by a procedure

similar to that used to prepare compound 8. These two isomers could

be separated by preparative TLC as colorless amorphous gels. (2R,4S)-

cis-N-(2-Hydroxyethyl)-4-undecylpiperidine-2-carboxamide (8).

To a solution of 6 (70 mg, 0.18 mmol) and 2-aminoethanol (11

mg, 0.18 mmol) in 4 mL of DMF was added HBTU (89 mg, 0.23

mmol) and DIPEA (58 mg, 0.45 mmol). The resulting mixture was

stirred at room temperature for 16 h. The DMF was removed under

vacuum to give a brown oily residue, which was partitioned between

CH₂Cl₂(50 mL) and 10% citric aqueous solution (10 mL). The

organic layer was separated and washed with saturated aqueous

NaHCO₃(10 mL). After drying over anhydrous Na₂SO₄, the solvent

was removed under vacuum to give an oily residue. This residue was

puriﬁed with a silica gel column (5% MeOH in CH₂Cl₂) and aﬀorded

the Boc-protected amide (58 mg, 75%). The amide (58 mg, 0.13

mmol) was dissolved in CH₂Cl₂(1 mL), followed by the addition of

TFA (250 μL). The resulting mixture was stirred at room temperature.

After 2 h, TLC showed that the starting material had disappeared. The

solvent was removed under vacuum to give an oily residue. The

residue was partitioned between CH₂Cl₂(30 mL) and saturated

NaHCO₃aqueous solution (10 mL). The organic layer was dried over

anhydrous Na₂SO₄, ﬁltered, and concentrated to give an oily residue.

This residue was puriﬁed with a silica gel column (eluting with 10%

MeOH in CH₂Cl₂), aﬀording 8 (34 mg, 73%) as a colorless

1

12. H NMR (600 MHz, CDCl₃+ CD₃OD) δ 7.37 (m, 2H), 7.32 (t,

2H, J = 7.2 Hz), 7.24 (t, 1H, J = 6.6 Hz), 4.98 (m, 1H), 4.03 (d, 1H, J

= 4.2 Hz), 3.69 (m, 1H), 3.62 (m, 1H), 3.10 (d, 2H, J = 11.4 Hz), 2.60

(t, 1H, J = 12.0 Hz), 1.78 (d, 1H, J = 12.6 Hz), 1.67 (d, 1H, J = 12.6

Hz), 1.33 (m, 1H), 1.26 (m, 20H), 1.18 (m, 1H), 1.01 (m, 1H), 0.88

(t, 3H, J = 6.6 Hz), 0.75 (t, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz,

CDCl₃+ CD₃OD) δ 174.7, 141.2, 128.0 (2C), 127.3, 125.8 (2C),

72.3, 62.1, 60.2, 56.2, 45.2, 36.7, 36.1, 35.5, 31.9, 31.7, 29.6, 29.5 (2C),

29.4 (2C), 29.1, 26.1, 22.5, 13.8. HRMS Calcd for C₂₆H₄₄N₂O₃: [M +

H]⁺433.3425; found 433.3415. (2S,4R)-13. ¹H NMR (600 MHz,

CDCl₃) δ 7.41 (m, 1H), 7.38 (m, 1H), 7.31 (t, 2H, J = 7.2 Hz), 7.25

(m, 1H), 5.06 (s, 1H), 4.08 (m, 1H), 3.80 (m, 4H), 3.22 (d, 1H, J =

10.8), 3.03 (d, 1H, J = 12.0 Hz), 2.54 (t, 1H, J = 12.0 Hz), 1.77 (d, 1H,

J = 12.0 Hz), 1.61 (d, 1H, J = 12.6 Hz), 1.27 (m, 20H), 1.13 (m, 2H),

0.95 (m, 1H), 0.88 (t, 3H, J = 6.6 Hz), 0.81 (m, 1H). ¹³C NMR (150

MHz, CDCl₃) δ 173.6, 141.4, 128.2 (2C), 127.4, 125.8 (2C), 73.3,

63.2, 60.0, 56.4, 44.9, 36.7, 36.2, 35.2, 31.8, 31.6, 29.7, 29.6 (3C), 29.3

(2C), 26.2, 22.6, 14.0. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺

433.3425; found 433.3427.

(2S,4R)-N-((1R,2R)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-unde-

cylpiperidine-2-carboxamide ((2S,4R)-14) and (2R,4S)-N-((1R,2R)-

1,3-Dihydroxy-1-phenylpropan-2-yl)-4-undecylpiperidine-2-carbox-

amide ((2R,4S)-15). Compounds (2S,4R)-14 (35 mg, 43%) and

(2R,4S)-15 (37 mg, 45%) were prepared (2 steps) by a procedure

similar to that used to prepare compound 8. These two isomers could

be separated by preparative TLC as colorless amorphous gels. (2S,4R)-

14. ¹H NMR (600 MHz, CDCl₃+ CD₃OD) δ 7.67 (br s, 1H), 7.40 (d,

2H, J = 7.8 Hz), 7.31 (t, 2H, J = 7.8 Hz), 1.24 (t, 1H, J = 7.8 Hz), 4.94

(d, 1H, J = 4.8 Hz), 4.10 (m, 1H), 3.67 (dd, 1H, J = 6.0 Hz, 11.4 Hz),

3.52 (dd, 1H, J = 6.0 Hz, 11.4 Hz), 3.17 (d, 1H, J = 12.6 Hz), 2.71 (m,

1H), 1.93 (d, 1H, J = 13.2 Hz), 1.75 (d, 1H, J = 13.8 Hz), 1.46 (br s,

1H), 1.28 (s, 20H), 1.14 (m, 1H), 0.97 (q, 1H, J = 12.6 Hz), 0.89 (t,

3H, J = 7.2 Hz). ¹³C NMR (150 MHz, CDCl₃): δ 172.9, 141.8, 128.0

(2C), 127.3, 126.0 (2C), 71.7, 61.4, 59.5, 56.7, 44.5, 36.5, 35.5, 35.1,

31.7, 30.6, 29.6 (4C), 29.4, 29.1, 26.1, 22.4, 13.5. HRMS Calcd for

1

amorphous gel. H NMR (600 MHz, CDCl₃) δ 7.54 (d, 1H, J = 4.8

Hz), 4.11 (br s, 2H), 3.71 (s, 2H), 3.46 (d, 2H, J = 10.2 Hz), 3.32 (m,

1H), 3.24 (d, 1H, J = 12.0 Hz), 2.78 (t, 1H, J = 11.4 Hz), 2.09 (d, 1H,

J = 12.6 Hz), 1.73 (d, 1H, J = 13.2 Hz), 1.48 (br s, 1H), 1.25 (m,

22H), 0.88 (t, 3H, J = 7.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 173.5,

61.2, 60.0, 45.1, 42.2, 36.8, 36.0, 35.3, 31.9, 31.4, 29.8, 29.7, 29.6, 29.4

(4C), 26.4, 22.7, 14.1. HRMS Calcd for C₁₉H₃₈N₂O₂: [M + H]⁺

327.3006; found 327.3010.

cis-N-(1,3-Dihydroxypropan-2-yl)-4-undecylpiperidine-2-carbox-

amide (9). Compound 9 (50 mg) was prepared in 76% yield (2 steps)

by a procedure similar to that used to prepare compound 8. The title

compound was obtained as a colorless amorphous gel. ¹H NMR (600

1

MHz, CD₃OD) δ H NMR (600 MHz, CDCl₃+ CD₃OD) δ 3.89 (t,

1H, J = 4.2 Hz), 3.66 (m, 4H), 3.15 (m, 1H), 2.62 (m, 1H), 1.99 (d,

1H, J = 12.0 Hz), 1.71 (d, 1H, J = 12.6 Hz), 1.43 (m, 1H), 1.26 (m,

20H), 1.02 (m, 2H), 0.88 (t, 3H, J = 6.6 Hz). ¹³C NMR (150 MHz,

CDCl₃+ CD₃OD) δ 174.5, 60.9, 60.1, 52.1, 45.1, 36.7, 36.4, 35.6, 32.1,

31.5 (2C), 29.4, 29.3 (3C), 29.0 (2C), 26.0, 22.3, 13.5. HRMS Calcd

for C₂₀H₄₀N₂O₃: [M + H]⁺357.3112; found 357.3119.

1

C₂₆H₄₄N₂O₃: [M + H]⁺433.3425; found 433.3427. (2R,4S)-15. H

NMR (600 MHz, CDCl₃) δ 7.40 (d, 1H, J = 8.4 Hz), 7.35 (d, 2H, J =

7.2 Hz), 7.28 (m, 2H), 7.21 (t, 1H, J = 7.8 Hz), 5.02 (d, 1H, J = 3.0

Hz), 4.52 (br s, 2H), 4.08 (m, 1H), 3.77 (m, 1H), 3.71 (m, 1H), 3.13

(dd, 1H, J = 1.8 Hz, 12.0 Hz), 2.94 (d, 1H, J = 11.4 Hz), 2.43 (m, 1H),

1.71 (d, 1H, J = 12.6 Hz), 1.56 (d, 1H, J = 12.0 Hz), 1.26 (m, 20H),

0.99 (m, 2H), 0.88 (t, 3H, J = 7.2 Hz), 0.73 (q, 1H, J = 12.0 Hz). ¹³C

NMR (150 MHz, CDCl₃): δ 173.8, 141.7, 128.2 (2C), 127.4, 125.9

(2C), 72.9, 63.0, 60.0, 56.3, 44.9, 36.8, 36.4, 35.4, 31.9, 31.8, 29.8, 29.7

(4C), 29.4, 26.3, 22.7, 14.1. HRMS Calcd for C₂₆H₄₄N₂O₃: [M + H]⁺

433.3425; found 433.3427.

cis-N-((2S,3S)-1,3-Dihydroxybutan-2-yl)-4-undecylpiperidine-2-

carboxamide (10). Compound 10 (48 mg) was prepared in 71% yield

(2 steps) by a procedure similar to that used to prepare compound 8.

1

The title compound was obtained as a colorless amorphous gel. H

NMR (600 MHz, CDCl₃) δ 7.59 (m, 1H), 4.78 (br s, 3H), 4.06 (m,

1H), 3.74 (m, 3H), 3.5 (m, 1H), 3.22 (m, 1H), 2.73 (q, 1H, J = 13.8

Hz), 2.06 (m, 1H), 1.74 (t, 1H, J = 13.8 Hz), 1.47 (m, 1H), 1.25 (m,

19H), 1.15 (m, 5H), 0.87 (t, 3H, J = 7.2 Hz). ¹³C NMR (150 MHz,

CDCl₃): δ 173.7, 172.9, 67.6, 67.4, 63.4, 60.4, 59.7, 55.5, 45.3, 44.8,

36.8, 36.2, 36.0, 35.6, 35.2, 31.9, 31.3, 30.9, 29.8, 29.6, 29.4, 26.5, 26.4,

22.7, 20.4, 14.1. HRMS Calcd for C₂₁H₄₂N₂O₃: [M + H]⁺371.3274;

found 371.3270.

cis-4-Undecyl-piperidine-2-carboxylic Acid (2,3-

Dihydroxypropyl)amide (16). Compound 16 (45 mg) was prepared

in 68% yield (2 steps) by a procedure similar to that used to prepare

compound 8. The title compound was obtained as a colorless

1

amorphous gel. H NMR (300 MHz, CDCl₃) δ 7.49 (m, 1H), 3.82

(m, 4H), 3.50 (m, 3H), 3.36 (m, 1H), 3.29 (d, 1H, J = 10.5 Hz), 3.15

(m, 1H), 2.66 (t, 1H, J = 11.1 Hz), 2.03 (m, 1H), 1.69 (d, 1H, J = 12.9

Hz), 1.42 (m, 1H), 1.25 (br s, 20 H), 1.02 (m, 2H), 0.88 (t, 3H, J =

6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.2, 70.9 (2C), 63.8, 63.7,

60.4, 45.6, 42.0, 37.0, 36.7, 35.8, 32.2, 31.9, 29.8, 29.7, 29.4, 26.5, 22.7,

14.1. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺357.3112; found

357.3116.

cis-N-((2R,3R)-1,3-Dihydroxybutan-2-yl)-4-undecylpiperidine-2-

carboxamide (11). Compound 11 (52 mg) was prepared in 77% yield

(2 steps) by a procedure similar to that used to prepare compound 8.

1

The title compound was obtained as a colorless amorphous gel. H

NMR (600 MHz, CDCl₃) δ 7.27 (m, 1H), 4.10 (m, 1H), 3.77 (m,

6H), 3.31 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 3.25 (dd, 1H, J = 2.4 Hz, 11.4

Hz), 3.15 (m, 1H), 2.65 (t, 1H, J = 12.6 Hz), 2.05 (m, 1H), 1.68 (m,

K

DOI: 10.1021/acs.jmedchem.8b00401

J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

Article

cis-N-((R)-2,3-Dihydroxypropyl)-4-undecylpiperidine-2-carboxa-

mide (17). Compound 17 (54 mg) was prepared in 80% yield (2

steps) by a procedure similar to that used to prepare compound 8. The

26.45, 26.40, 22.69, 14.12. HRMS (ESI) calcd for C₂₁H₄₅N₂O₃[M +

H]⁺385.3061; found 385.3058.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-^L-tyrosinate

(23). Compound 23 (32 mg) was prepared as a white solid in 86.0%

yield (2 steps) by a procedure similar to that used to prepare

1

title compound was obtained as a colorless amorphous gel. H NMR

(300 MHz, CDCl₃) δ 7.54 (m, 1H), 3.99 (br s, 3H), 3.77 (d, 1H, J =

2.1 Hz), 3.56 (m, 1H), 3.50 (m, 1H), 3.35 (s, 1H), 3.33 (m, 1H), 3.29

(d, 1H, J = 5.4 Hz), 3.15 (d, 1H, J = 5.4 Hz), 2.66 (t, 1H, J = 6.0 Hz),

2.02 (d, 1H, J = 5.7 Hz), 1.69 (d, 1H, J = 6.3 Hz), 1.43 (br s, 1H), 1.25

(br s, 20 H), 1.04 (m, 2H), 0.88 (t, 3H, J = 3.6 Hz). ¹³C NMR (75

MHz, CDCl₃): δ 175.0, 70.8, 63.8 (2C), 60.4, 45.5, 42.0, 37.0, 36.7,

35.7, 32.1, 31.9, 29.9, 29.7, 29.4, 26.5, 22.7, 14.1. HRMS Calcd for

C₂₀H₄₀N₂O₃: [M + H]⁺357.3112; found 357.3113.

1

compound 8; mp 129.0−129.6 °C. H NMR (300 MHz, CDCl₃) δ

7.07 (dd, J = 36.9, 8.4 Hz, 1H), 6.95 (dd, J = 8.4, 3.6 Hz, 2H), 6.67

(dd, J = 13.6, 8.5 Hz, 2H), 4.91−4.75 (m, 1H), 3.74 (d, J = 1.8 Hz,

3H), 3.24−3.03 (m, 3H), 2.95 (td, J = 14.2, 7.0 Hz, 1H), 2.61 (ddd, J

= 12.1, 9.8, 5.6 Hz, 1H), 1.99 (t, J = 14.3 Hz, 1H), 1.74−1.55 (m, 1H),

1.27 (s, 22H), 1.10−0.93 (m, 3H), 0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR

(75 MHz, CDCl₃) δ 174.38, 173.99, 172.24, 172.20, 155.80, 130.34,

130.17, 126.93, 126.70, 115.66, 115.61, 60.52, 60.39, 53.01, 52.94,

52.40, 45.79, 45.46, 37.22, 36.95, 36.91, 36.65, 35.90, 35.75, 32.37,

32.29, 31.92, 29.81, 29.68, 29.66, 29.36, 26.43, 26.37, 22.69, 14.13.

HRMS (ESI) calcd for C₂₇H₄₄N₂O₄[M + H]⁺461.3374; found

461.3362.

cis-N-((S)-2,3-Dihydroxypropyl)-4-undecylpiperidine-2-carboxa-

mide (18). Compound 18 (44 mg) was prepared in 67% yield (2

steps) by a procedure similar to that used to prepare compound 8. The

1

title compound was obtained as a colorless amorphous gel. H NMR

(300 MHz, CDCl₃) δ 8.34 (m, 1H), 5.77 (br s, 3H), 3.82 (s, 2H), 3.52

(m, 4H), 3.20 (dd, 1H, J = 3.0 Hz), 2.89 (m, 1H), 2.12 (d, 1H, J = 4.5

Hz), 1.79 (d, 1H, J = 5.1 Hz), 1.57 (br s, 1H), 1.36 (m, 1H), 1.25 (br

s, 22H), 0.88 (t, 3H, J = 3.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ

171.6, 70.8, 70.6, 63.7, 59.1, 44.6, 42.3, 36.5, 34.7, 32.0, 29.9, 29.7,

29.4, 26.5, 22.7, 14.1. HRMS Calcd for C₂₀H₄₀N₂O₃: [M + H]⁺

357.3112; found 357.3115.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-^L-tryptopha-

nate (24). Compound 21 (43 mg) was prepared as a white solid in

83% yield (2 steps) by a procedure similar to that used to prepare

1

compound 8; mp 112.6−113.8 °C. H NMR (300 MHz, CDCl₃) δ

8.56 (d, J = 13.5 Hz, 1H), 7.56 (dd, J = 7.6, 3.3 Hz, 1H), 7.35 (d, J =

7.9 Hz, 1H), 7.15 (ddd, J = 14.8, 10.9, 6.9 Hz, 3H), 6.97 (s, 1H), 4.96

(td, J = 10.1, 5.7 Hz, 1H), 3.71 (d, J = 5.6 Hz, 3H), 3.43−3.23 (m,

2H), 3.12 (dt, J = 11.5, 3.1 Hz, 1H), 3.05−2.92 (m, 1H), 2.62−2.46

(m, 1H), 2.08−1.93 (m, 1H), 1.59 (d, J = 11.0 Hz, 2H), 1.29 (s, 20H),

1.21−1.11 (m, 2H), 1.06−0.82 (m, 4H). ¹³C NMR (75 MHz, CDCl₃)

δ 174.22, 174.10, 172.49, 172.42, 136.15, 136.12, 127.73, 127.70,

122.81, 122.72, 122.11, 119.47, 119.39, 118.69, 118.47, 111.42, 111.32,

110.12, 110.03, 60.66, 60.61, 52.61, 52.52, 52.34, 45.77, 37.05, 36.97,

36.74, 36.71, 36.01, 35.94, 32.65, 32.46, 31.94, 29.85, 29.82, 29.66,

29.37, 27.71, 27.57, 26.44, 22.71, 14.14. HRMS (ESI) calcd for

C₂₉H₄₅N₃O₃[M + H]⁺484.3534; found 484.3517.

cis-N-((S)-3-Chloro-2-hydroxypropyl)-4-undecylpiperidine-2-car-

boxamide (19). Compound 19 (43 mg) was prepared in 63% yield (2

steps) by a procedure similar to that used to prepare compound 8. The

1

title compound was obtained as a colorless amorphous gel. H NMR

(300 MHz, CDCl₃) δ 7.38 (br s, 1H), 3.89 (br s, 3H), 3.53 (m, 3H),

3.36 (m, 2H), 3.16 (m, 1H), 2.69 (t, 1H, J = 11.4 Hz), 2.07 (m, 1H),

1.70 (m, 1H), 1.43 (m, 1H), 1.26 (br s, 20H), 1.06 (m, 2H), 0.88 (t,

3H, J = 6.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.2, 70.7, 70.5,

60.4, 46.4 (2C), 45.6, 45.5, 43.0, 42.9, 36.9, 36.6, 35.7, 35.6, 32.0, 31.9,

29.8, 29.7, 29.6, 29.4, 26.4, 22.7, 14.1. HRMS Calcd for

C₂₀H₃₉ClN₂O₂: [M + H]⁺375.2773; found 375.2776.

cis-N-(3-Morpholinopropyl)-4-undecylpiperidine-2-carboxamide

(20). Compound 20 (55 mg) was prepared in 74% yield (2 steps) by a

procedure similar to that used to prepare compound 8. The title

compound was obtained as a colorless amorphous gel. ¹H NMR (600

MHz, CD₃OD) δ 4.05 (d, 2H, J = 12.6 Hz), 3.79 (m, 3H), 3.48 (d,

2H, J = 10.8 Hz), 3.37 (m, 2H), 3.22 (m, 5H), 2.98 (m, 1H), 2.21 (d,

1H, J = 13.2 Hz), 1.94 (m, 3H), 1.66 (br s, 1H), 1.25 (m, 22H), 0.84

(t, 3H, J = 7.2 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 169.2, 63.8, 58.0,

54.8, 52.1, 51.9, 43.6, 36.2, 36.1, 35.8, 33.9, 33.4, 31.7, 29.5 (2C), 29.4

(2C), 29.2, 28.0, 26.2, 23.7, 22.4, 13.2. HRMS Calcd for C₂₄H₄₇N₃O₂:

[M + H]⁺396.3585; found 396.3588.

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-^L-allothreoni-

nate (21). Compound 21 (14 mg) was prepared as a whitish wax in

87.5% yield (2 steps) by a procedure similar to that used to prepare

compound 8. ¹H NMR (300 MHz, CDCl₃) δ 7.43 (t, J = 9.8 Hz, 1H),

4.58 (td, J = 9.1, 2.6 Hz, 1H), 4.44−4.30 (m, 1H), 3.78 (d, J = 1.1 Hz,

3H), 3.30 (dd, J = 11.6, 2.7 Hz, 1H), 3.22−3.11 (m, 1H), 2.68 (tt, J =

11.6, 2.6 Hz, 1H), 2.45 (s, 3H), 2.11 (d, J = 13.4 Hz, 1H), 1.70 (d, J =

12.9 Hz, 1H), 1.52−1.37 (m, 1H), 1.27 (s, 22H), 1.11−0.98 (m, 2H),

0.89 (t, J = 6.7 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.76,

171.48, 67.99, 67.80, 60.91, 60.71, 57.07, 56.89, 52.54, 45.86, 45.70,

36.98, 36.64, 35.95, 32.52, 32.34, 31.92, 29.81, 29.68, 29.64, 29.35,

26.45, 22.69, 20.05, 14.12. HRMS (ESI) calcd for C₂₂H₄₂N₂O₄[M +

H]⁺399.3217; found 399.3208.

(2R,4S)-4-(2-Cyclohexylethyl)-N-((1R,2R)-1,3-dihydroxy-1-phenyl-

propan-2-yl)piperidine-2-carboxamide ((2R,4S)-35) and (2S,4R)-4-

(2-Cyclohexylethyl)-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)-

piperidine-2-carboxamide ((2S,4R)-34). Compounds (2R,4S)-35 (53

mg, 44%) and (2S,4R)-34 (50 mg, 42%) were prepared from 33 in 2

steps by a procedure similar to that used to prepare compound 8.

These two isomers could be separated by preparative TLC as colorless

1

amorphous gels. Compound 35. H NMR (600 MHz, CDCl₃) δ 7.77

(d, 1H, J = 6.0 Hz), 7.32 (d, 2H, J = 7.2 Hz), 7.26 (m, 2H), 7.19 (t,

1H, J = 7.2 Hz), 5.50 (br s, 2H), 4.97 (d, 1H), 4.12 (d, 1H, J = 3.0

Hz), 3.74 (m, 2H), 3.39 (d, 1H, J = 11.4 Hz), 3.03 (d, 1H, J = 9.0 Hz),

2.50 (m, 1H), 1.69 (m, 6H), 1.58 (d, 1H, J = 9.6 Hz), 1.19 (m, 10H),

0.97 (m, 1H), 0.86 (m, 2H), 0.74 (q, 1H, J = 12.0 Hz). ¹³C NMR (150

MHz, CDCl₃): δ 172.3, 141.5, 128.1 (2C), 127.3, 125.8 (2C), 72.6,

62.7, 59.2, 56.5, 44.3, 37.8, 35.2, 34.9, 34.0, 33.7, 33.4 (2C), 30.3, 26.7,

26.4 (2C). HRMS Calcd for C₂₃H₃₆N₂O₃: [M + H]⁺389.2799; found

1

389.2802. Compound 34. H NMR (600 MHz, CDCl₃+ CD₃OD) δ

7.69 (br s, 1H), 7.39 (d, 2H, J = 7.8 Hz), 7.31 (t, 2H, J = 7.8 Hz), 7.23

(t, 1H, J = 7.8 Hz), 4.97 (d, 1H, J = 4.2 Hz), 4.09 (m, 1H), 3.70 (m,

1H), 3.5 (m, 1H), 3.15 (dd, 1H, J = 2.4 Hz, 12.0 Hz), 3.11 (d, 1H, J =

12.0 Hz), 2.63 (m, 1H), 1.80 (m, 1H), 1.69 (m, 7H), 1.20 (m, 10H),

0.89 (m, 1H), 0.82 (q, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz,

CDCl₃): δ 174.5, 141.9, 127.9 (2C), 127.2, 125.9 (2C), 71.5, 61.5,

60.1, 56.2, 45.0, 37.7, 36.3, 35.8, 33.9, 33.2 (2C), 31.7, 29.3, 26.5, 26.2

(2C). HRMS Calcd for C₂₃H₃₆N₂O₃: [M + H]⁺389.2799; found

389.2802.

(2S,4S)-N-((1S,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-(2-(4-

methylcyclohexyl)ethyl)piperidine-2-carboxamide ((2R,4S)-36) and

(2S,4R)-N-((1R,2S)-1,3-Dihydroxy-1-phenylpropan-2-yl)-4-(2-(4-

methylcyclohexyl)ethyl)piperidine-2-carboxamide ((2S,4R)-37).

Compounds (2R,4S)-36 (40 mg, 41%) and (2S,4R)-37 (42 mg,

43%) were prepared from 33 in 2 steps by a procedure similar to that

used to prepare compound 8. These two isomers could be separated

Methyl((cis-2,4)-4-undecylpiperidine-2-carbonyl)-^L-serinate (22).

Compound 22 (16 mg) was prepared as a whitish wax in 89.0%

yield (2 steps) by a procedure similar to that used to prepare

1

compound 8. H NMR (300 MHz, CDCl₃) δ 7.51 (dd, J = 20.8, 7.7

Hz, 1H), 4.70−4.58 (m, 1H), 4.07−3.83 (m, 2H), 3.79 (s, 3H), 3.25

(d, J = 11.5 Hz, 1H), 3.15 (d, J = 12.1 Hz, 1H), 2.83 (s, 2H), 2.66 (t, J

= 12.2 Hz, 1H), 2.07 (t, J = 10.4 Hz, 1H), 1.69 (d, J = 13.0 Hz, 1H),

1.41 (s, 1H), 1.27 (s, 20H), 1.14−0.94 (m, 2H), 0.89 (t, J = 6.7 Hz,

3H). ¹³C NMR (75 MHz, CDCl₃) δ 174.61, 174.21, 170.98, 170.92,

62.86, 60.64, 54.48, 54.45, 52.67, 45.88, 45.65, 37.03, 36.99, 36.77,

36.54, 35.99, 35.85, 32.57, 32.51, 31.92, 29.81, 29.68, 29.64, 29.35,

1

by preparative TLC as colorless amorphous gels. Compound 36. H

NMR (300 MHz, CDCl₃) δ 7.32 (m, 5H), 4.99 (d, 1H, J = 7.2 Hz),

4.08 (m, 1H), 3.70 (m, 1H), 3.62 (m, 1H), 3.34 (m, 1H), 3.09 (m,

2H), 2.61 (m, 1H), 1.46 (m, 4H), 1.27 (m, 13H), 0.89 (m, 3H), 0.76

(q, 1H, J = 12.6 Hz). ¹³C NMR (75 MHz, CDCl₃): δ 175.6, 142.2,

L

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Article

128.5, 127.8, 126.4, 72.4, 62.4, 60.9, 56.9, 45.8, 38.0, 37.0, 36.5, 35.7,

35.1, 34.7, 33.8, 33.7, 33.3, 32.7, 31.1 (2C), 30.7, 29.2, 29.1, 22.8, 20.4.

HRMS Calcd for C₂₄H₃₈N₂O₃: [M + H]⁺403.2955; found 403.2955.

Compound 37. ¹H NMR (600 MHz, CDCl₃) δ 7.31 (m, 5H), 7.22 (t,

1H, J = 7.2 Hz), 5.03 (d, 1H, J = 3.6 Hz), 4.08 (m, 1H), 3.93 (br s,

3H), 3.78 (m, 1H), 3.72 (m, 1H), 3.13 (dd, 1H, J = 2.4 Hz, 12.0 Hz),

2.99 (d, 1H, J = 10.8 Hz), 2.48 (m, 1H), 1.74 (m, 1H), 1.67 (m, 1H),

1.60 (m, 2H), 1.44 (m, 3H), 1.20 (m, 10H), 0.91 (d, 3H, J = 6.6 Hz),

0.87 (m, 2H), 0.78 (q, 1H, J = 12.6 Hz). ¹³C NMR (150 MHz,

CDCl₃): δ 174.0, 141.6, 128.2, 127.4, 125.9, 73.1, 63.1, 60.2, 56.4, 50.5,

45.1, 37.5, 36.4, 35.7, 35.3, 34.6, 34.2, 34.0, 33.3, 32.9, 32.0, 30.8, 30.1,

28.7, 22.6, 20.2. HRMS Calcd for C₂₄H₃₈N₂O₃: [M + H]⁺403.2955;

found 403.2953.

delivery of compound (20 μL/well) was followed 15 min later by 5-

HT (10 pM to 10 μM; 25 μL/well). A baseline was established for

each well before addition of the test compound and again before

addition of 5-HT. The ﬂuorescence read after compound addition was

used to establish potential intrinsic agonist activity of the compounds.

The ﬂuorescence read following the addition of 5-HT was used to

assess allosteric modulation of 5-HT-evoked Ca_i²⁺release. Fluo-

rescence was measured using a FlexStation 3 (Molecular Devices) or

FLIPR^TETRA(130 gain, 60% intensity, 0.3 s exposure). For the

FlexStation 3, a 17 s baseline was established before addition of

compounds following which ﬂuorescence was recorded every 1.7 s for

240 s. Maximum peak height was determined by the SoftMax software

(Pro 5.4.5) for each well. For the FLIPR^TETRA, a 10 s baseline was

established before addition of compounds following which ﬂuo-

rescence was recorded every 1 s for 120 s following compound or for

360 s following 5-HT. Maximum peak height was determined by

ScreenWorks 4.0 software for each well. After the ﬁnal readings, cells

were ﬁxed in 2% paraformaldehyde (Sigma) overnight. The maximum

5-HT-induced Ca_i²⁺release (E_max) in the presence of test compound

was determined using 4-parameter nonlinear regression analysis

(GraphPad Prism 7.04) and calculated from at least three biological

replicates, each conducted in technical triplicates. The E_maxfor the test

compound plus 5-HT was normalized to the E_maxfor 5-HT alone.

Subsequent post hoc comparisons between means for E_maxwere made

using an unpaired t test with Welch’s correction (GraphPad Prism). All

statistical analyses were conducted with an experiment-wise error rate

of α = 0.05.

In Vivo Pharmacokinetics and Behavior. Pharmacokinetic

Analysis. Male Sprague−Dawley rats were randomly assigned into

control and treatment groups (n = 3/group). Rats in the control group

were injected with vehicle [DMSO/Tween 80/saline (v/v 1:3:6)], and

rats in the treatment groups were administered compound 16 at 5 mg/

kg iv or 10 mg/kg po. Blood samples (0.3 mL) were collected from the

retro-orbital sinus vein before dosing and at 5 min and 0.25, 0.5, 1.0,

2.0, 3.0, 4.0, 5.0, 7.0, 9.0, and 24 h postdosing for iv administration and

0.25, 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0, 9.0, and 24 h postdosing for po

administration. Blood samples were placed in heparinized tubes and

immediately centrifuged at 12 000g for 5 min at 4 °C. All samples were

stored at −20 °C until analysis.

(2S,4R)-4-Cyclohexyl-N-((1R,2R)-1,3-dihydroxy-1-phenylpropan-

2-yl)piperidine-2-carboxamide (38) and (2R,4S)-4-Cyclohexyl-N-

((1R,2R)-1,3-dihydroxy-1-phenylpropan-2-yl)piperidine-2-carboxa-

mide (39). Compounds 38 (70 mg, 40%) and 39 (74 mg, 43%) were

prepared from 33 (2 steps) by a procedure similar to that used to

prepare compound 8. These two isomers could be separated by

preparative TLC as colorless amorphous gel. Compound 38. ¹H NMR

(600 MHz, CDCl₃) δ 7.48 (d, 1H, J = 8.4 Hz), 7.38 (d, 2H, J = 7.2

Hz), 7.28 (m, 2H), 7.22 (t, 1H, J = 7.8 Hz), 4.98 (d, 1H, J = 4.8 Hz),

4.30 (br s, 3H), 4.08 (m, 1H), 3.70 (m, 1H), 3.62 (m, 1H), 3.11 (dd,

1H, J = 2.4 Hz, 12.0 Hz), 2.97 (m, 1H), 2.46 (m, 1H), 1.72 (m, 2H),

1.59 (m, 4H), 1.13 (m, 5H), 0.99 (m, 2H), 0.84 (m, 3H). ¹³C NMR

(150 MHz, CDCl₃): δ 174.5, 141.7, 128.2 (2C), 127.4, 126.2 (2C),

72.9, 62.7, 61.0, 56.6, 45.5, 42.7, 41.0, 33.3, 29.8, 28.6 (2C), 26.6, 26.5

(2C). HRMS Calcd for C₂₁H₃₂N₂O₃: [M + H]⁺361.2486; found

1

361.2488. Compound 39. H NMR (600 MHz, CDCl₃+CD₃OD) δ

7.48 (s, 1H), 7.37 (d, 2H, J = 7.2 Hz), 7.30 (m, 2H), 7.23 (t, 1H, J =

7.2 Hz), 4.99 (d, 1H, J = 3.6 Hz), 4.10 (m, 1H), 3.72 (m, 1H), 3.62

(m, 1H), 3.13 (m, 2H), 2.58 (dt, 1H, J = 3.0 Hz, 12.6 Hz), 1.75 (m,

3H), 1.66 (m, 4H), 1.15 (m, 6H), 0.93 (m, 2H), 0.87 (q, 1H, J = 12.0

Hz). ¹³C NMR (150 MHz, CDCl₃): δ 173.9, 141.7, 128.0 (2C), 127.2,

125.8 (2C), 71.9, 62.0, 60.0, 56.3, 45.1, 42.7, 41.0, 33.5, 29.8, 29.7,

28.7, 26.5, 26.4 (2C). HRMS Calcd for C₂₁H₃₂N₂O₃: [M + H]⁺

361.2486; found 361.2487.

In Vitro Pharmacology. Intracellular Calcium (Ca_i²⁺) Release

Assay in Live h5-HT₂R-CHO Cells. Chinese hamster ovary (CHO)

cells stably transfected with human unedited (INI) h5-HT_2CR (h5-

HT_2CR-CHO cells) or the human 5-HT_2AR (h5-HT_2AR-CHO cells)

were a generous gift of Drs. Kelly A. Berg and William P. Clarke

(University of Texas Health Science Center, San Antonio). Cells were

grown at 37 °C, 5% CO₂, and 85% relative humidity environment in

GlutaMax-MEM medium (Invitrogen, Carlsbad CA) containing 5%

fetal bovine serum (Atlanta Biologicals, Atlanta GA) and 100 μg/mL

hygromycin (Mediatech, Manassas VA) and were passaged when they

reached 80% conﬂuence. The Ca_i²⁺release assay was performed

according to our recent publications.^9,28,70Brieﬂy, cells (150 μL;

passages 6−16) were plated in serum-replete medium at a density of

14 000−16 000 (FlexStation 3; Molecular Devices) or 30 000 cells/

well (FLIPR^TETRA; Molecular Devices) in black-wall 96-well culture

plates with optically clear ﬂat bottoms. To ensure even plating of cells,

the source reservoir was frequently agitated or triturated, and plates

were maintained on a rotary shaker at low speed for 20 min after

plating and returned to the incubator overnight. Approximately 24 h

following plating, the medium was replaced with serum-free (SF)

GlutaMax-MEM medium supplemented with 20 nM to 100 μM

putrescine (Sigma-Aldrich, St. Louis, MO), 20 nM to 100 μM

progesterone (Sigma-Aldrich), and 1:100 ITS (1000 mg/L human

recombinant insulin, 550 mg/L human recombinant transferrin, 0.67

mg/L selenious acid; Corning Inc., Corning, NY) (SF+ medium).

Following a 3 h incubation, SF+ medium was replaced with 40 μL of

Hank’s balanced saline solution (HBSS; without CaCl₂or MgCl₂, pH

7.4) plus 40 μL of Calcium 4 dye solution (FLIPR No-wash kit,

Molecular Devices, Sunnyvale, CA, part no. R8142) supplemented

with 2.5 mM of water-soluble probenicid (Sigma) to inhibit

extracellular transport of the dye. Plates were incubated for 60 min

at 37 °C followed by 15 min at room temperature in the dark. Drug

dilutions were prepared at 5× ﬁnal concentration in 1× HBSS;

The pharmacokinetic parameters of compound 16 were calculated

according to a noncompartmental model using WinNonlin (Pharsight

Corporation, ver 5.3, Mountain View, CA, USA). The peak

concentration (C_max) and time of peak concentration (T_max) were

directly obtained by visual inspection of the plasma concentration−

time proﬁle. The elimination rate constant (λ) was obtained by the

least-squares ﬁtted terminal log−linear portion of the slope of the

plasma concentration−time proﬁle. The elimination half-life (T_1/2

)

was evaluated according to 0.693/λ. The area under the plasma

concentration−time curve from 0 to time t (AUC_0−t) was evaluated

using the linear trapezoidal rule and further extrapolated to inﬁnity

(AUC_0−inf) according to the following equation: AUC_0−inf= AUC_0−t

+

C_last/λ. The pharmacokinetic parameters were presented as mean

SD.

Eﬀects of Compound 16 on Spontaneous Locomotor Activity.

Animals. A total of 67 male Sprague−Dawley rats (Harlan, Inc.,

Indianapolis, IN) weighing 225−325 g at the start of the experiments

were used. Rats were housed two per cage and allowed to acclimate for

5−7 days in a colony room at a constant temperature (21−23 °C) and

humidity (45−50%) on a 12 h light−dark cycle (lights on 0700−1900

h). Food and water were available ad libitum. Rats were handled for 1

week prior to the start of behavioral testing. All experiments were

carried out in accordance with the National Institutes of Health Guide

for the Care and Use of Laboratory Animals (2011) and with the

approval of the UTMB Institutional Animal Care and Use Committee.

All eﬀorts were made to minimize animal suﬀering, to reduce the

number of animals used, and to utilize alternatives to in vivo

techniques, when available.

Drugs. WAY163909 [(7b-R,10a-R)-1,2,3,4,8,9,10,10a-octahydro-

7bH-cyclopenta[b][1,4]diazepino[6,7,1hi]indole] was a gift from

Pﬁzer, Inc. (New York, NY) and dissolved in 0.9% NaCl (vehicle

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employed for comparison to WAY163909). SB242084 [6-chloro-5-

methyl-1-[[2-(2-methylpyrid-3-yloxy)pyrid-5-yl]carbamoyl]indoline

dihydrochloride; Sigma Chemical Co., St. Louis, MO, USA] was

dissolved in saline containing 10 mmol/L citric acid (Sigma Chemical

Co.) and 8% 2-hydroxypropyl-β-cyclodextrin (Trappsol Hydroxpropyl

Beta Cyclodextrin, pharmaceutical grade, Cyclodextrin Technologies

Development Inc., High Springs, FL, USA) with the ﬁnal pH of the

solution adjusted to 5.6. SB242084, WAY163909, and compound 16

were injected intraperitoneally (ip) at a volume of 1 mL/kg.

Locomotor Activity Assessments. Locomotor activity was moni-

tored and quantiﬁed under low light conditions using a modiﬁed open

ﬁeld activity system (San Diego Instruments, San Diego, CA)

according to previous publications with minor modiﬁcations.⁴⁷Clear

plexiglass chambers (40 × 40 × 40 cm³) were surrounded by a 4 × 4

photobeam matrix positioned 4 cm from the chamber ﬂoor. Total

ambulations were quantiﬁed as the sum of consecutive photobeam

breaks that occurred within the inner central 16 × 16 cm²perimeter

and in the surrounding outer peripheral 16 × 16 cm²perimeter of the

activity monitor. Vertical activity counts (rearing) were quantiﬁed as

the sum of breaks in the upper row of 16 photobeams positioned 16

cm from the monitor ﬂoor.

Rats were trained to discriminate an injection of WAY163909 (0.75

mg/kg; 1.0 mL/kg, ip) from saline (1.0 mL/kg, ip) administered 15

min before start of training sessions (see Supporting Information for

WAY163909 time course). Daily sessions lasted 15 min and were

conducted Monday through Friday. During the phase of errorless

training, only the stimulus-appropriate (drug or saline) lever was

present. Training began under a ﬁxed ratio 1 (FR1) schedule of water

reinforcement, and the FR requirement was incremented until all

animals were responding reliably under an FR20 schedule for each

experimental condition. For half of the rats, left lever responses were

reinforced after WAY163909 administration, whereas right lever

responses were reinforced after saline administration; conditions

were reversed for the remaining animals. During this phase of training,

WAY163909 and saline were administered irregularly with the

restriction that neither condition prevailed for more than three

consecutive sessions. After responding stabilized, both levers were

presented simultaneously during 15 min training sessions. The rats

were required to respond on the stimulus-appropriate (correct) lever

to obtain water reinforcement. There were no programmed

consequences for responding on the incorrect lever. This phase of

training continued until the performance of all rats attained criterion

(deﬁned as mean accuracies of at least 80% stimulus-appropriate

responding for ten consecutive sessions).

Test sessions were initiated and conducted once or twice per week

following attainment of criterion. Training sessions were run during

the intervening days to maintain discrimination accuracy. Rats were

required to maintain accuracies of at least 80% correct for saline and

WAY163909 maintenance sessions, which immediately preceded a

test. During test sessions, animals were placed in the chambers and,

upon completion of 20 responses on either lever, a single reinforcer

was delivered and the house lights were turned oﬀ. The rat was

removed from the chamber, returned to the colony, and allowed free

access to water for 15 min beginning 15−30 min after the end of each

test. The test sessions were terminated after 15 min if the rats did not

complete 20 responses on either lever; only data from rats that

accomplished the FR20 during test sessions within 15 min were

employed in data analysis.

Time course, substitution and combination tests were conducted.

To determine the time course of WAY163909, rats were pretreated

with WAY163909 (0.75 mg/kg, ip) 15, 30, 60, or 120 min prior to

placement into the chambers. In substitution tests, rats were

administered WAY163909 (0.125, 0.25, 0.5, 0.75, or 1.0 mg/kg, ip),

compound 16 (0.125, 0.25, 0.5, 1.0 mg/kg, ip), or saline 15 min prior

to test. In combination tests, rats were tested for lever selection

following simultaneous ip administration of compound 16 (0.5 mg/

kg) and WAY163909 (0.5 mg/kg) and placed in the operant chamber

15 min later. The doses of WAY163909 and compound 16 employed

were based upon the results of the substitution tests as producing

<50% drug-appropriate responding. Full substitution was deﬁned as

≥80% drug-appropriate responding and not statistically diﬀerent from

the training drug, and partial substitution as ≥40% and <80% drug-

appropriate responding.

Statistical Analyses. Accuracy was deﬁned as the percentage of

correct responses to total responses before the delivery of the ﬁrst

reinforcer. During test sessions, performance was expressed as the

percentage of drug-lever responses to total responses upon completion

of an FR20 on either lever. The response rate (responses per minute)

was calculated as the total number of responses emitted before

completion of the ﬁrst FR20 divided by the number of minutes taken

to complete the ﬁrst ratio. For compound 16 dose−response tests,

Student’s t-test for repeated measures was used to compare the

percentage of drug-lever responding and response rate during test

sessions with the corresponding values for the previous saline session

(substitution tests). A two-way ANOVA for repeated measures was

employed to analyze the eﬀects of pretreatment with compound 16

(factor 1) and WAY163909 dose (factor 2; SAS for Windows, version

9.4). A priori comparisons were calculated to compare the eﬀects of

each dose of WAY163909 in the presence versus absence of

compound 16 using a Bonferonni-corrected test. Log-probit analyses

were used to estimate the dose of WAY163909 predicted to elicit 50%

(i) A between-subjects design was employed to study the eﬃcacy

of compound 16 to impact spontaneous motor activity. Rats (n

= 29) were administered vehicle (saline, 1 mL/kg, ip) or

compound 16 (0.5, 1, or 5 mg/kg, ip) immediately prior to

placement in activity monitors on the test day; ambulations and

vertical activity counts were monitored in 5 min bins for 90

min.

(ii) A between-subjects design was used to investigate the

selectivity of compound 16 to allosterically modulate

WAY163909-mediated suppression of spontaneous motor

activity. Rats (n = 38) were pretreated with SB242084 (1

mg/kg, ip) or vehicle (8% 2-hydroxypropyl-β-cyclodextrin) 15

min prior to ip administration of WAY163909 (1 mg/kg) plus

compound 16 (5 mg/kg) and immediately placed into activity

monitors on the test day; ambulations and vertical activity

counts were monitored in 5 min bins for 90 min.

Statistical Analyses. Locomotor activity data are presented as mean

total ambulations ( SEM) summed in 30 min. The main eﬀect of

treatment on total ambulations or vertical activity was analyzed with a

one-way analysis of variance using the GLM procedure (SAS version

9.4 for Windows). Subsequent a priori comparisons to vehicle were

made with Dunnett’s procedure (experiment i) or a Bonferroni

multiple comparisons test (experiment ii). All statistical analyses were

conducted with an experiment-wise error rate of α = 0.05.

Eﬀects of Compound 16 in a WAY163909 versus Saline Drug

Discrimination Assay. Animals. Male Sprague−Dawley rats (n = 9;

Harlan, Inc.) weighing 300−325 g at the beginning of the experiment

were housed two per cage in a temperature- (21−23 °C) and

humidity- controlled (45−50%) environment; lighting was maintained

under a 12 h light−dark cycle (0700−1900 h). Rats were maintained

at 80−90% of their free-feeding weights by restricting access to water.

Rats received water during daily training sessions (5−6 mL/rat/

session), in the afternoon several hours after training (20 min), and

over the weekend (36 h). Experiments were conducted during the

light phase of the light−dark cycle (between 0900 and 1200 h) and

were carried out in accordance with the National Institutes of Health

Guide for the Care and Use of Laboratory Animals and with the approval

of the Institutional Animal Care and Use Committee at University of

Texas Medical Branch.

Drug Discrimination Procedures. Standard two-lever, water-

reinforced drug discrimination procedures were used (Med Associates,

St. Albans, USA). Each chamber was equipped with a water-ﬁlled

dispenser mounted equidistantly between two retractable response

levers on the wall and housed in a light- and sound-proof cubicle.

Illumination came from a 28-V house light; ventilation and masking

noise were provided by a ventilation fan. A computer with Med-PC IV

software was used to run programs and record all experimental events.

N

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WAY163909-appropriate responding (ED₅₀).⁴⁸Only data from

animals that completed the FR20 during the test sessions were used.

All statistical analyses were conducted with an experiment-wise error

rate of α = 0.05.

Eﬀects of Compound 16 on Cocaine Cue Reactivity. Animals.

Male, outbred Sprague−Dawley rats (n = 17; Harlan, Houston, TX)

weighing 250−275 g upon arrival were housed two per cage under a

12-h light−dark cycle with controlled temperature (21−23 °C) and

humidity (40−50%). Animals were acclimated for 7 days to the colony

room prior to the start of handling and experimental procedures. All

experiments were conducted in accordance with the NIH Guide for

the Care and Use of Laboratory Animals (2011) and with the

University of Texas Medical Branch Institutional Animal Care and Use

Committee approval.

compound 16 (1.0 mg/kg, ip) 15 min prior to the cue reactivity

session.

Statistical Analyses. The data from the self-administration and cue

reactivity phases were analyzed separately. For the cue reactivity tests,

a two-way ANOVA for repeated measures was employed to analyze

the eﬀects of treatment with compound 16 (factor 1) and time (factor

2; SAS for Windows, version 9.4) on previously active or inactive lever

presses. A priori comparisons were calculated to compare the eﬀects of

compound 16 at each time point using Fishers LSD test. All statistical

analyses were conducted with an experiment-wise error rate of a α =

0.05.

ASSOCIATED CONTENT

* Supporting Information

■

S

Drugs. (−)-Cocaine (National Institute on Drug Abuse, Research

Triangle Park, NC) was dissolved in 0.9% NaCl.

The Supporting Information is available free of charge on the

ACS Publications website at DOI: 10.1021/acs.jmed-

chem.8b00401.

Cocaine Self-Administration and Cue Reactivity Analyses.

Cocaine self-administration studies employed standard operant

conditioning chambers (Med-Associates, Inc., St. Albans, VT, USA)

housed in ventilated, sound-attenuating cubicles with fans (Med-

Associates, Inc.). Each chamber was outﬁtted with two retractable

response levers, a stimulus light above each response lever, a

houselight opposite the levers, and a magazine-type pellet dispenser.

The cocaine infusions were delivered via syringes attached to infusion

pumps (Med Associates, Inc.) located outside the cubicles. The

infusion pumps were connected to liquid swivels (Instech, Plymouth

Meeting, PA, USA) that were fastened to the catheters via

polyethylene 20 tubing encased inside a metal spring leash (Plastics

One, Roanoke, VA).

Rats were anesthetized (8.6 mg/kg of xylazine, 1.5 mg/kg of

acepromazine, 43 mg/kg of ketamine in bacteriostatic saline) and

implanted with intravenous catheters with back mounts and allowed to

recover for 5−7 days.^6,8,9,47Catheter patency was maintained by daily

ﬂushes with a solution of 0.1 mL of bacteriostatic saline containing

heparin sodium (10 U/mL; American Pharmaceutical Partners, East

Schaumburg, IL), streptokinase (0.67 mg/mL; Sigma Chemical), and

ticarcillin disodium (66.67 mg/mL; Research Products International,

Mt. Prospect, IL) immediately following daily cocaine self-admin-

istration sessions.

Cocaine self-administration training consisted of 14 daily 180 min

sessions during which rats were trained to lever press for cocaine

infusions (0.75 mg/kg per 0.1 mL infusion).^6,8,9,47Schedule

completions on the active lever resulted in delivery of a cocaine

infusion over a 6 s period paired simultaneously with illumination of

the house and stimulus lights and activation of the infusion pump

(discrete cue complex paired with cocaine delivery); inactive lever

presses produced no scheduled consequences. Following reinforcer

delivery, the stimulus light as well as the infusion pump were

inactivated; the house light remained on for an additional 20 s to

indicate a timeout period during which lever presses had no scheduled

consequences. Rats were trained on a FR1 schedule of reinforcement

and progressed to an FR5 schedule after achieving seven infusions per

hour with less than 10% variability for three consecutive days. Upon

achieving stability on the FR5 schedule (less than 10% variability for a

minimum of three consecutive days), rats were pseudorandomly

assigned to either vehicle or compound 16 and returned to their home

cages for the appropriate time period. Rats that exhibited diminished

catheter patency were excluded from analysis (n = 3).

Rats were reintroduced to the self-administration chambers 24 h

after the last self-administration session and assayed in a test session

comprised of two sequential components. In the ﬁrst component,

responses on either lever were recorded, but no discrete cues (e.g.,

stimulus light, pump) were present nor delivered during the 10 min

period. The second component was signaled by the single delivery of

the discrete cue complex presented at the termination of the ﬁrst 10

min component. To assess cocaine cue reactivity, presses on the

previously active lever in the 60 min second component were

reinforced by the discrete cue complex on an FR1; inactive lever

presses were recorded but produced no scheduled consequences.⁶Rats

received an injection of vehicle (0.9% NaCl, 1 mL/kg, ip) or

Calculated physicochemical properties, radioligand bind-

ing data, cytotoxicity data, additional cellular assay data,

CNS-MPO values, docking protocol, drug discrimination

1

time course study, H and ¹³C NMR spectra for the

compounds described in this paper, X-ray data for

compounds 32 and 35 (PDF)

Molecular formula strings (CSV)

AUTHOR INFORMATION

Corresponding Authors

*Tel: (409) 772-9748. Fax: (409) 772-9648. E-mail: jizhou@

utmb.edu (J. Zhou).

■

*Tel: (409)-772-9629. Fax: (409)-772-7050. E-mail:

kcunning@utmb.edu (K. A. Cunningham).

*Tel: (409)-772-9656. Fax: (409)-772-7050. E-mail:

ncanasta@utmb.edu (N. C. Anastasio).

ORCID

Eric A. Wold: 0000-0001-6434-7562

Claudia A. Soto: 0000-0002-5214-9136

Noelle C. Anastasio: 0000-0001-5579-4213

Kathryn A. Cunningham: 0000-0002-4257-1739

Jia Zhou: 0000-0002-2811-1090

Author Contributions

^∥C.T.W., J.M.M., and E.A.W. contributed equally to this work.

J.Z., K.A.C., and N.C.A. developed the concepts and

approaches as well as supervised the work. C.T.W., E.A.W.,

and C.D. synthesized, puriﬁed, and characterized the reported

compounds. J.M.M., C.A.S., and R.M.H. performed and

analyzed the in vitro cellular studies. N.C.A. and K.A.C.

analyzed the in vitro and in vivo studies. C.T.W., N.C.A., K.A.C.,

and J.Z., wrote the manuscript.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This work was supported by Grants R21 MH093844 (J.Z./

K.A.C.), R01 DA038446 (J.Z./K.A.C.), K05 DA020087

(K.A.C.), P30 DA28821 (K.A.C.), T32 DA07287 (C.T.W.

and E.A.W.), F31 DA038922 (C.T.W.), and F31 DA045511

(E.A.W.) from the National Institutes of Health, R.A. Welch

Foundation Chemistry and Biology Collaborative Grant from

Gulf Coast Consortia (GCC) for Chemical Genomics, a

training fellowship from the Keck Center for Interdisciplinary

Bioscience Training of the GCC (NIGMS Grant T32

GM089657-03, C.T.W.), Sealy and Smith Foundation grant

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(to the Sealy Center for Structural Biology and Molecular

Biophysics), John Sealy Memorial Endowment Fund, and the

Center for Addiction Research at UTMB. We thank Lawrence

C. Sowers, Jacob A. Theruvathu, and Tianzhi Wang for the

NMR spectroscopy assistance, Robert G. Fox, Gongliang

Zhang, and Sonja J. Stutz for performing and analyzing in

vivo studies, Claudia M. Crawford, Carrie E. McAllister, and

Konrad Pazdrak for conducting in vitro assays, and Marcy B.

Jordan for helpful discussions during the development of this

project. Receptor binding proﬁles and agonist functional data

was generously provided by the National Institute of Mental

Health’s Psychoactive Drug Screening Program, Contract no.

HHSN-271-2013-00017-C (NIMH PDSP). The NIMH PDSP

is directed by Bryan L. Roth at the University of North Carolina

at Chapel Hill and Project Oﬃcer Jamie Driscoll at NIMH,

Bethesda MD, USA.

ABBREVIATIONS USED

■

5-HT, serotonin; 5-HT_2AR, 5-HT_2Areceptor; 5-HT_2BR, 5-HT_2B

receptor; 5-HT_2CR, 5-HT_2Creceptor; CNS, central nervous

system; GPCR, G protein-coupled receptor; FDA, U.S. Food

and Drug Administration; PAM, positive allosteric modulator;

NAM, negative allosteric modulator; SAR, structure−activity

relationship; tPSA, total polar surface area; HBTU, N,N,N′,N′-

tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexaﬂuorophos-

phate; DIPEA, N,N-diisopropylethylamine; TFA, triﬂuoroacetic

acid; PTLC, preparative thin layer chromatographic; PLCβ,

phospholipase Cβ; IP3, inositol-1,4,5-trisphosphate; DAG,

diacylglycerol; CHO, Chinese hamster ovary; CCR5, C−C

chemokine receptor type 5; PLD, phospholipase D; PK,

pharmacokinetics; PD, pharmacodynamics; ADMET, absorp-

tion, distribution, metabolism, excretion, and toxicity; EL,

extracellular loop; TMH, transmembrane helix; MPO, multi-

parameter optimization; TMS, tetramethylsilane; DMF,

dimethylformamide

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