Novel Dual-Target μ-Opioid Receptor and Dopamine D3Receptor Ligands as Potential Nonaddictive Pharmacotherapeutics for Pain Management

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Novel Dual-Target μ‑Opioid Receptor and Dopamine D₃Receptor

Ligands as Potential Nonaddictive Pharmacotherapeutics for Pain

Management

Alessandro Bonifazi,* Francisco O. Battiti, Julie Sanchez, Saheem A. Zaidi, Eric Bow, Mariia Makarova,

Jianjing Cao, Anver Basha Shaik, Agnieszka Sulima, Kenner C. Rice, Vsevolod Katritch, Meritxell Canals,

J. Robert Lane, and Amy Hauck Newman *

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ABSTRACT: The need for safer pain-management therapies with decreased abuse liability

inspired a novel drug design that retains μ-opioid receptor (MOR)-mediated analgesia, while

minimizing addictive liability. We recently demonstrated that targeting the dopamine D₃

receptor (D₃R) with highly selective antagonists/partial agonists can reduce opioid self-

administration and reinstatement to drug seeking in rodent models without diminishing

antinociceptive eﬀects. The identiﬁcation of the D₃R as a target for the treatment of opioid use

disorders prompted the idea of generating a class of ligands presenting bitopic or bivalent

structures, allowing the dual-target binding of the MOR and D₃R. Structure−activity

relationship studies using computationally aided drug design and in vitro binding assays led to

the identiﬁcation of potent dual-target leads (23, 28, and 40), based on diﬀerent structural

templates and scaﬀolds, with moderate (sub-micromolar) to high (low nanomolar/sub-

nanomolar) binding aﬃnities. Bioluminescence resonance energy transfer-based functional

studies revealed MOR agonist−D₃R antagonist/partial agonist eﬃcacies that suggest potential

for maintaining analgesia with reduced opioid-abuse liability.

In the past, D₃R-selective antagonists, such as GSK598809,

have been investigated as potential treatments for psychosti-

mulant use disorder (e.g., cocaine);¹⁰however, the potentiation

of hypertensive eﬀects observed in dogs produced by cocaine

in the presence of this selective D₃R antagonist prevented

further development¹¹and suggested that this may be a class

eﬀect. We recently demonstrated that our novel D₃R

antagonists and partial agonists look promising for the

treatment of OUD.¹²⁻¹⁴Highly selective antagonists, such as

VK4-116 (1) and VK4-40 (2) (Figure 1), attenuate

oxycodone self-administration and reinstatement to drug

seeking, without compromising oxycodone’s antinociceptive

eﬀects, in rodents. Importantly, these D₃R antagonists/partial

agonists do not potentiate the cardiovascular eﬀects induced

by cocaine or oxycodone in rats.¹⁵In combination, these

studies support the development of D₃R antagonists/partial

agonists to reduce the risk of opioid misuse and the

consequent development of OUD.

INTRODUCTION

■

Over the last decade, the United States has faced a devastating

opioid epidemic with an estimate of over 130 people dying

from opioid overdose every day.¹The misuse and often

consequent addiction to opioids (e.g., prescription pain

relievers and synthetic opioids, in general) are a serious

national health, social, and economic emergency. Coupled with

the SARS-CoV-2 pandemic, the mortality rate involving opioid

overdose is increasing, and the need for new therapeutic

strategies is more urgent than ever.^2,3According to recent

reports,⁴>20% of patients being treated for chronic pain will

misuse their opioid prescriptions⁵and 8−12% develop opioid

use disorders (OUD).⁵Ultimately, an estimated 5% of patients

are reported to transition from misuse of prescription opioids

to heroin⁶⁻⁸or other synthetic opioids.

The National Institutes of Health (NIH) launched the

Helping to End Addiction Long-Term (HEAL) initiative to

promote and support innovative research addressing this

national health emergency. Moreover, the National Institute

on Drug Abuse (NIDA) recently proposed a list of the “ten

most wanted” medication development strategies to tackle the

opioid epidemic/crisis.⁹Among them, the dopamine D₃

receptor (D₃R) antagonists and partial agonists are a “new”

proposed class of ligands as therapeutics to attenuate opioid

self-administration.

Received: April 2, 2021

Published: May 20, 2021

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Figure 1. Drug design based on structural modiﬁcation of canonical synthons inspired by agonists, antagonists, and partial agonists selectively

targeting the MOR and D₃R. Orange boxes denote bivalent templates, and green boxes denote bitopic templates. Variables A, B, and C correspond

to the aromatic substitutions seen in compounds 1, 2, 7, 10, or 11. The general Ar group corresponds to the aromatic substituents in compounds 7,

9, 10, and 11. General R¹and R²groups represent H, Me, or OH or (O). Variables Y and Z represent C or N, and variable X is H, OMe, or OH or

(O).

Until now, the main target for pain-management therapies

and drug development has been the opioid system, in

particular, the μ-opioid receptors (MORs), belonging to the

G-protein coupled receptors (GPCR) family. However, due to

the abuse liability and development of tolerance associated

with the most common MOR agonists used in pain therapy,

including chronic pain, for which opioid agonists are largely

ineﬀective in the long term,¹⁶⁻¹⁸research eﬀorts have been

directed toward identifying speciﬁc physiological responses

associated with MOR agonists’ cellular activation pathways.

Functionally biased agonists have been posited to reduce the

side eﬀect proﬁle of classic opioid analgesics and augment their

utility not only as analgesics but also in the treatment of

OUD.¹⁹⁻²²

Design, synthesis, and pharmacological characterization of

signaling-pathway-biased agonists targeting the MORs allowed

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the identiﬁcation of highly selective G-protein-biased agonists,

with limited activation of β-arrestin pathways, highlighting

diﬀerent physiological eﬀects mediated by each independent

pathway.^19,23It has been suggested that the MOR G-protein

pathway seems to be the predominant mediator of the

analgesic eﬀects of MOR agonists; meanwhile, it has been

posited that the simultaneous hindering of β-arrestin recruit-

ment might reduce the respiratory depression and other side

eﬀects, such as constipation, associated with opioid-like

drugs.²⁴⁻²⁸However, this has been a topic of intensive

investigation, and recently, it has been reported that classical

MOR agonists, such as morphine and fentanyl, induce dose-

dependent respiratory depression and constipation in β-

arrestin-2 knock-out mice, similar to what is observed in

wild-type mice.²⁹⁻³¹Subsequent pharmacological evaluation

has also shown that MOR G-protein-biased agonists still have

abuse potential.^29,32−34It is still unclear whether the optimal

outcome and pharmacotherapeutic potential of MOR agonists

can be gained through selective activation of a particular

downstream signaling pathway (functional selectivity) or

whether an optimal level partial agonism at multiple pathways

may instead provide a route for the development of safer

opioids.^31,35Independent of the signaling pathways and

cellular mechanisms associated with respiratory depression

and constipation, abuse liability remains as a serious concern

that must be addressed with diﬀerent drug design approaches.

The recognition of D₃R antagonism/partial agonism as an

alternative and nonopioid approach for treatment of OUD,

modulating the abuse potential of common prescription

opioids (e.g., oxycodone),^12,13,15combined with the well-

established antinociceptive properties of MOR agonists,

prompted the idea of generating a novel class of dual-target

ligands directed to both the MOR and D₃R (Figure 1). In

theory, compounds that are both MOR agonists/partial

agonists and D₃R antagonist/partial agonists would have

analgesic activity without concomitant abuse liability. Our

approach aimed at maintaining the analgesic eﬀects of the

classic MOR agonists, while reducing the rewarding properties

and subsequent abuse liability as a result of D₃R antagonism.

This drug design may lead to the development of safer dual-

target drugs, bridging the most promising pharmacological

eﬀects of two classes of molecules/targets previously developed

independently. Of note, our drug design was to develop new

small molecules endowed with diﬀering ranges of aﬃnities for

both targets, independently modulating their physiology/

pharmacology rather than toward simultaneous binding of

the MOR and D₃R when in close proximity or in a heteromeric

conformation. Nevertheless, if MOR−D₃R heteromers are

demonstrated to exist and are physiologically relevant, these

molecules may be interesting tools to probe their pharmacol-

ogy.

bivalent or bitopic drug design or binding to the D₃R was not

described or presumably intended.³⁷

Moreover, methadone (6) (Figure 1) also showed low

micromolar D₂R and D₃R aﬃnity in our binding assays (Table

1), supporting the hypothesis that some of its structural

fragments could be used to target not only the MOR

orthosteric binding site (OBS) but the D₂R and D₃R as well.

Unlike 6, the synthetic opioid fentanyl, with its completely

diﬀerent structural features, showed an interesting moderate

aﬃnity for the dopamine D₄R subtype but total lack of

recognition for either the D₂R or D₃R (Table 1). This template

was not considered in our drug design for this reason and also

because, recently, bivalent ligands using fentanyl have been

reported to lack antinociceptive activity.^38,39

Due to the limited availability of reported structure−activity

relationships (SARs) for dual-target MOR−D₃R li-

gands,^38,40−42we used a fragment-based drug design approach,

supported by molecular docking, computer-aided drug design

(CADD), and extensive in vitro pharmacology to guide SAR,

hit optimization, and lead identiﬁcation.

Herein, we refer to bivalent dual-target analogues when an

MOR agonist primary pharmacophore (PP) is tethered with a

D₃R antagonist PP; both can bind their respective OBS,

eliciting their corresponding opioid agonist and dopaminergic

antagonist eﬀects. Consistent with our deﬁnition of bitopic

ligands,⁴³we classify these new analogues as bitopic when they

incorporate an MOR agonist PP, targeting its corresponding

OBS that also has structural features suitable for D₃R OBS

recognition, tethered to a D₃R secondary pharmacophore

(SP), identifying the D₃R secondary binding pocket (SBP).⁴⁴

Of note, this D₃R SP may also elicit binding interactions within

the MOR SBP. All new compounds represent carefully

designed linkers, as tethering fragments, with speciﬁc SAR

focusing on the linkers’ regiochemistry, stereochemistry, and

substituents.⁴³

All newly synthesized analogues were tested for their on-

target and oﬀ-target aﬃnities at the MOR, D₂R, D₃R, and D₄R,

in a combination of agonist and antagonist radioligand

competition binding assays. Compounds selected as hits, for

their promising dual-target and sub-micromolar aﬃnities, were

further evaluated in functional bioluminescence resonance

energy transfer (BRET) studies to assess their agonist and/or

antagonist potencies for the target of interest and possible

functional selectivity (biased agonism) for speciﬁc signaling

pathways.

RESULTS AND DISCUSSION

■

Chemistry. This novel class of compounds can be

subdivided, as depicted in Figure 1A−C, in three general

templates: (A) the N,N-dimethyl-2,2-diphenylacetamide, 2,2-

diphenylacetonitrile, and 1,1-diphenylbutan-2-one MOR PPs,

derived from 3, 4, and 6, respectively (Figure 1), were tethered

with suitably substituted PPs, inspired by selective and

nonselective D₃R antagonists [e.g., 1, 2, PG648 (7),

eticlopride (8) and SB269,652 (9); Figure 1], via a short

ethyl linker chain; (B) the same MOR OBS-binding agonist

PPs were linked with D₃R OBS antagonist PPs via a longer and

more complex butyl linker, substituted with a 3-hydroxyl

group, or the piperazine or pyrrolidine basic function in several

regiochemical combinations; and (C) the MOR agonist PPs

were replaced with the more rigid, stereochemically complex,

and hindered ethyl-2-(-5-(3-hydroxyphenyl)-2-

azabicyclo[3.3.1]nonan-9-ylidene)acetate and 5-(3-hydroxy-

Furthermore, several well-known MOR agonists, such as

loperamide (3) (peripherally limited potent MOR agonist,

FDA-approved for antidiarrhea treatment) and diphenoxylate

(4), share similar structural motifs with the highly potent

nonselective D₂R/D₃R antagonist haloperidol (5) (Figure

1).³⁶Substituted phenyl-piperazine and/or phenyl-piperidine

synthons, common to both classes of ligands, can exploit the

structural similarities between MOR and D₃R proteins, thus

achieving dual-target binding. Of note, a similar approach was

taken previously toward more eﬀective peripherally limited

analgesics, based on loperamide, although implementing a

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a

Scheme 1

a

(a) Appropriate primary or secondary amine, K₂CO₃, and acetonitrile (ACN), 130 °C, overnight, 8−53%; (b) Me₂NH·HCl, K₂CO₃, and ACN,

130 °C, overnight, 79%; (c) HBr 48% in H₂O, 100 °C, overnight; (d) N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC), 1-

hydroxybenzotriazole hydrate (HOBt), 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine,⁴⁷N,N-diisopropylethylamine (Hu

̈

nig’s base,

DIPEA), and dichloromethane (DCM), 25 °C, overnight, 6%; and (e) ethyl magnesium bromide (EtMgBr) 3 M in diethyl ether (Et₂O),

toluene, 0 to 110 °C, 3 h, 59%.

a

Scheme 2

a

(a) 1-(2,3-Dichlorophenyl)piperazine, 2-(6-chloro-1-H-benzotriazole-1-yl)-1,1,3,3-tetramethylaminium hexaﬂuorophosphate (HCTU), and

DCM, 25 °C, 3 h, 59%; (b) triﬂuoroacetic acid (TFA) and DCM, 25 °C, 24 h; and (c) 4-bromo-2,2-diphenylbutanenitrile, K₂CO₃, and ACN,

82 °C, overnight, 3% (over two steps).

phenyl)-2-azabicyclo[3.3.1]nonan-9-ol PP (10 and 11; Figure

1), with structural features reminiscent of previously published

D₃R ligands^45,46that we have found to have low micromolar

aﬃnity for the MOR as well. This new MOR PP presented key

functional groups for extending linkers of multiple lengths,

substitutions, rigidity and chirality, and tethering D₃R PPs (for

bivalent dual-target compounds) or D₃R SPs (e.g., 2-

indoleamide for bitopic dual-target compounds). When

possible and appropriate, a complete resolution of the chiral

centers at PP, SP, or linkers was performed, and the

stereochemical properties of the new analogues have been

taken into consideration when generating detailed SAR.

The ﬁrst series of compounds with the 2,2-diphenylbutane-

nitrile as the MOR PP, inspired by 4, were prepared as

depicted in Scheme 1. Starting from the commercially available

4-bromo-2,2-diphenylbutanenitrile, simple N-alkylation under

basic conditions yielded the ﬁrst group of bivalent MOR−D₃R

hybrids, where the D₃R PP was represented by 2-phenylethan-

1-amine (12), 1-(2,3-dichlorophenyl)piperazine (13), 4-(4-

(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (14), and

4-amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol

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a

Scheme 3

a

(a) Appropriate secondary amine, K₂CO₃, DIPEA (for 23 and 24), ACN, and tert-butyl methyl ether (TBME), reﬂux, 24 h, 16−91%; (b) 2 N

NaOH in H₂O, 25 °C, 5 min, 100%; (c) Dess−Martin periodinane (DMP), DCM, 0 to 25 °C, 1 h, 60%; and (d) appropriate primary or secondary

amine, catalytic acetic acid (cat. AcOH), sodium triacetoxyborohydride (STAB), and 1,2-dichloroethane (DCE), 25 °C, 2.5 h, 12−100%.

a

Scheme 4

a

(a) N-(4-Bromobutyl)phthalimide, K₂CO₃, cat. KI, ACN, 82 °C, overnight; (b) hydrazine (NH₂NH₂) and ethanol (EtOH), 80 °C, 3 h, 94%

(over 2 steps); and (c) N,N-dimethyl-4-oxo-2,2-diphenylbutanamide (26), cat. AcOH, STAB, and DCE, 25 °C, 2.5 h, 46%.

(15). This provided initial SAR deduction, by increasing the

structural complexity of the D₃R PP and simultaneously

modifying the linker length and substitution. To investigate the

optimal regiochemistry for introducing the linker and D₃R PP,

compound 18 was prepared, where the butyl-4-(2,3-

dichlorophenyl)piperazine synthon was introduced to replace

the nitrile, while maintaining the MOR 4-(dimethylamino)-

2,2-diphenylbutanamide moiety, as seen in both 3 and 6

(Figure 1). In detail, 4-bromo-2,2-diphenylbutanenitrile was

ﬁrst N-alkylated with dimethylamine hydrochloride (Me₂NH·

HCl) under basic conditions, followed by hydrolysis of the

nitrile in the presence of 48% HBr (aq solution) to yield the

HBr salt of amino acid 17. Subsequent amidation mediated by

EDC, HOBt, and 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)-

butan-1-amine⁴⁷yielded the desired product 18. Despite the

importance of the α-methyl group of 6 being well reported in

the literature,⁴⁸favoring the optimal binding pose within the

MOR OBS, we decided to study the SAR of structurally

simpliﬁed analogues. Thus, via simple Grignard addition to the

nitrile 16, we prepared the desmethyl-methadone 19.

In Scheme 2, in order to investigate the structural

requirement of the D₃R pharmacophore, in particular, the

eﬀect of replacing the basic butyl-4-(2,3-dichlorophenyl)-

piperazine, with the corresponding amide analogue, HCTU

mediated amide coupling of 5-((tert-butoxycarbonyl)amino)-

pentanoic acid, followed by removal of the Boc-protecting

group, and consequent mono N-alkylation with 4-bromo-2,2-

diphenylbutanenitrile aﬀorded the desired product 21. The

longer 5 carbon atom linker, instead of the canonical butyl

chain, was chosen because of the increased rigidity of the cyclic

amide function and need for extending the tethered PP to an

optimal distance.

The switch from 2,2-diphenylbutanenitrile (4-like ana-

logues) to N,N-dimethyl-2,2-diphenylbutanamide (3-like ana-

logues) as the MOR PP was achieved, as described in Scheme

3. Starting from the commercially available N-(3,3-diphenyldi-

hydrofuran-2(3H)-ylidene)-N-methylmethanaminium bro-

mide, simple ring opening with the appropriate primary or

secondary amines aﬀorded compounds 22, presenting the 6-

like dimethylamino function, 23, where the 2,3-dichlorophenyl

piperazine D₃R scaﬀold was introduced, as well as 24, whose

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a

Scheme 5

a

(a) N-(4-Bromobutyl)phthalimide, K₂CO₃, and ACN, 82 °C, overnight; (b) NH₂NH₂and EtOH, 80 °C, 3 h, 36% (over two steps); and (c)

K₂CO₃and ACN, 130 °C, overnight, 10−23%.

a

Scheme 6

a

(a) N-(4-Bromobutyl)phthalimide, K₂CO₃, and ACN, 82 °C, overnight, 85%; (b) TFA and DCM, 25 °C, 3 h; (c) HCTU and DCM, 25 °C, 48 h,

26%; (d) NH₂NH₂and EtOH, 80 °C, 3 h; and (e) cat. AcOH, STAB, and DCE, 25 °C, 12 h, 21% (over two steps).

1,2,3,4-tetrahydroisoquinoline-7-carbonitrile is another com-

mon D₂R/D₃R antagonist PP fragment, present in well-

characterized ligands, such as 9.⁴⁹⁻⁵²

Compound 34, containing the 1,2,3,4-tetrahydroisoquino-

line-7-carbonitrile pharmacophore was synthesized according

to Scheme 4, via preparation of intermediate 33 and reductive

amination with 26.

To investigate the eﬀect of the linker length and substitution

on the bivalent hybrid analogues, N-(3,3-diphenyldihydrofur-

an-2(3H)-ylidene)-N-methylmethanaminium bromide was

simply washed with 2 N NaOH in H₂O and extracted with

DCM to obtain the alcohol intermediate 25, which was then

oxidized to the aldehyde using DMP and then mono-N-

alkylated via reductive amination conditions with the

appropriate primary and secondary amines. This small library

of compounds (27, 28, 29, 30, 31, and 32) covered a large

structural variety of canonical D₃R antagonist PPs, as well as

butyl and hydroxyl substituted linkers, known for increasing

D₃R subtype selectivity.⁵³

In order to expand the library toward diﬀerent D₃R PPs, the

canonical piperazine and 1,2,3,4-tetrahydroisoquinoline, in-

spired by selective D₃R antagonists and partial agonists (1, 2,

7, and 9; Figure 1) were replaced in Scheme 5 with the highly

decorated phenyl-N-(pyrrolidin-2-ylmethyl)benzamide derived

from the eticlopride pharmacophore (8, Figure 1). Alkylation

of (S)-nor-eticlopride (obtained from the NIDA Drug Supply

Program) with 4-bromo-2,2-diphenylbutanenitrile yielded

compound 35, introducing the 2,2-diphenyl-nitrile MOR PP,

tethered by an ethyl linker. Meanwhile, the formation of

intermediate 36 allowed the synthesis of analogue 37, tethered

via the longer butyl-amino linker.

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a

Scheme 7

a

(a) LAH and tetrahydrofuran (THF), 0 to 25 °C, 15 h, 40%; (b) 2-chloroacetyl chloride, DIPEA, and THF, 0 to 25 °C, 1 h; (c) cat. KI, K₂CO₃

and ACN, 82 °C, 3 h, 74% (over two steps); (d) TFA and DCM, 25 °C, 2 h; (e) HCTU, DIPEA, and DCM, 25 °C, 3 h, 15% (over two steps); (f)

methyl chloroformate, DIPEA, and DCM, 25 °C, 1 h; (g) LAH and THF, 0 to 25 °C, 56% (over two steps); and (h) propionyl chloride, DIPEA,

and DCM, 40 °C, overnight, 29%.

a

Scheme 8

a

(a) Cat. AcOH, STAB, and DCE, 25 °C, 4 h; (b) TFA and DCM, 25 °C, overnight, 8.5% (over two steps).

Analogous to the approach described above for the

phenylpiperazine series, we investigated the replacement of

the diphenyl nitrile MOR PP, with the 3-like N,N-

dimethylamide functional group for the 8-based bivalent

analogues. In Scheme 6, the substituted benzoic acid

intermediate was prepared following previous literature

procedures.^14,54The benzoic acid was coupled with 2-(4-(2-

(aminomethyl)pyrrolidin-1-yl)butyl)isoindoline-1,3-dione,

which was freshly prepared in situ via selective Boc-

deprotection of 38, prior to the HCTU-mediated amide

coupling. The phthalimide group in intermediate 39 was

removed, and the resulting primary amine was mono-N-

alkylated via reductive amination to yield 40 as the racemic

mixture.

In this case, we decided to synthesize the racemic mixture,

starting from the commercially available tert-butyl (pyrrolidin-

2-ylmethyl)carbamate, because although 8 favors the (S)

absolute conﬁguration at the pyrrolidine ring, we did not want

to exclude the possibility of diﬀerent stereochemical require-

ments for this bivalent analogue, as we have seen in another

series of bivalent/bitopic D₃R ligands.⁴⁶

SAR for an alternative MOR PP, with a 3,3-diphenyl

substituted pyrrolidine, was investigated through the synthesis

of analogues in Scheme 7. The common starting material 4-

bromo-2,2-diphenylbutanenitrile was reacted with lithium

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a

Scheme 9

a

(a) Titanium tetrachloride (TiCl₄), triethylamine (TEA), and DCM, 0 to 25 °C, overnight, 58%; (b) 4-(4-(2,3-dichlorophenyl)piperazin-1-

yl)butan-1-amine, cat. AcOH, STAB, and DCE, 25 °C, 4 h, 88%; (c) H₂(50 psi), Pd/C (20% wt), and EtOH, 25 °C, 12 h; (d) HBr 33% in AcOH;

118 °C, 48 h, 13−31%; (e) 1-(2-chloro-3-ethylphenyl)piperazine,¹⁴cat. AcOH, STAB, and DCE, 25 °C, 4 h, 88%; and (f) H₂(30 psi), Pd/C (20%

wt), and EtOAc/EtOH (2:1), 25 °C, 3 h, 75%.

aluminum hydride (LAH) to give the primary amine and

consequent ring-closure in a one-pot step. Intermediate 41 was

either methylated via treatment with methyl chloroformate

followed by in situ LAH reduction to aﬀord 42 or reacted with

propionyl chloride to yield the cyclic amide synthon 43. This

rather simple PP allowed us to evaluate the eﬀect of structural

rigidity in 6- and 3-like MOR PPs and the eﬀect of replacing

the protonatable cyclic amine to a cyclic amide. To prepare the

bivalent analogue 46, 41 was initially acylated with 2-

chloroacetyl chloride. Subsequent alkylation of racemic tert-

butyl (pyrrolidin-2-ylmethyl)carbamate yielded 45. Finally,

Boc-deprotection and amide coupling aﬀorded the desired

product.

observation that the meta-hydroxy groups will engage a water-

mediated hydrogen bond network observed in several known

opioid ligands while being well tolerated by the D₃R SP

binding site.

Homologation of the commercially available bis(3-

methoxyphenyl)methanone in the presence of TiCl₄and

TEA⁵⁵allowed the formation of the α,β-unsaturated aldehyde

49. Reductive amination in the presence of 2,3-dichlorophenyl

piperazine yielded 50, which was hydrogenated in the presence

of Pd/C. The crude mixture was then subsequently O-

demethylated with 33% HBr in AcOH (Scheme 9).

High-resolution mass spectroscopy (HRMS) and NMR

analyses revealed the loss of both chlorine atoms in ﬁnal

product 51. Retrospective analyses of the previous synthetic

steps and intermediates showed that the loss of both chlorine

atoms occurred during hydrogenation while reducing the

styrenyl oleﬁn. Unfortunately, dehalogenation and concom-

itant hydrogenation appear to occur faster than the reduction

of the desired oleﬁn. Despite replacing Pd/C with PtO₂, the

solvent (from EtOH to EtOAc), and H₂pressure (from 50 to

15−20 psi), the same reaction outcome was observed.

Nevertheless, we proceeded in testing 51, as a proof of

concept to validate the biological activity of the newly

proposed bis-phenol PP to target the MOR.

The 1-(2-chloro-3-ethylphenyl)piperazine scaﬀold proved to

be resistant to hydrogenation conditions¹⁴and was introduced

as the D₃R PP tethered to the MOR bis-phenol scaﬀold

(Scheme 9). Intermediate 52 was readily prepared by reductive

amination, followed by milder hydrogenation conditions (30

psi H₂, using a mixture of EtOAc/EtOH 3:1 as a solvent, for 3

h of total reaction time) to obtain the saturated analogue 53.

Partial and total O-demethylation was again achieved in the

As consistently observed in previous work,⁴³when

generating bitopic or bivalent ligand SAR, it is essential to

study structural requirements not only for the PP and/or SP

but also for regiochemistry and stereochemistry of the linkers,

which can play a crucial role in their biological activity.

In Scheme 8, we approached a modiﬁcation of the

regiochemistry for the pyrrolidine D₃R PP scaﬀold. The ﬁnal

compound 48 presents the MOR diphenyl-N,N-dimethyl

amide PP tethered to the D₃R PP, via a butyl ether linker

fused in position-4 of the pyrrolidine nucleus, in a rel-trans

stereochemistry conﬁguration with respect to the 8 amide PP

appended in position-2. This was easily introduced as shown in

Scheme 8, starting from 47 via reductive amination and Boc-

deprotection to ultimately yield 48.

Extensive in silico docking studies and optimization (see the

section below) were directed toward improving the dual-target

aﬃnity of these new MOR−D₃R analogues and guided us to

the synthesis of 51 (Scheme 9). In particular, the hydroxy

substitution of the MOR diphenyl PP was based on the in silico

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a

Scheme 10

a

(a) N-(4-Oxobutyl)-1H-indole-2-carboxamide,⁵⁷cat. AcOH, STAB, and DCE, 25 °C, 1.5 h, 26−88%; (b) trans-N-(2-(2-formylcyclopropyl)-

ethyl)-1H-indole-2-carboxamide,⁴⁵cat. AcOH, STAB, and DCE, 25 °C, 1.5 h, 65%; and (c) preparative enantioselective high-performance liquid

chromatography (HPLC), ChiralPak AD-H column.

presence of 33% HBr in AcOH, and respective products 54

(obtained and tested as the racemic mixture) and 55 were

isolated and tested in vitro.

the importance of the rigid cyclopropyl linker to achieve

unique pharmacological proﬁles, and its stereochemistry is

essential in modulating favorable poses for D₃R target

recognition, binding aﬃnity, selectivity, and functional eﬃcacy.

Thus, both trans enantiomers of 58 were resolved via

preparative chiral HPLC (58a and 58b) and pharmacologically

evaluated. Due to the lack of signiﬁcant diﬀerences between

their biological proﬁles we did not proceed any further in

assigning the absolute conﬁguration of each trans-cyclopropyl

enantiomers for these analogues.

In silico docking predicted that replacing the ethyl acrylate

on the phenylmorphan ring with a less sterically hindered

group (e.g., ketone or hydroxyl group) would be tolerated

within the MOR OBS and could increase the aﬃnity of the

new hybrids for the D₃R. Moreover, since the bitopic

analogues 57 and 58 showed high aﬃnity for the MOR, but

moderate to low binding at D₂-like receptors (Table 2), we

aimed to study and invert the stereochemistry of the

phenylmorphan ring, while simultaneously evaluating all the

possible stereochemical combinations with the hydroxyl group.

We synthesized both diastereoisomers 61 and 62, starting from

the fully resolved 59 and 60, to study not only the eﬀect of the

hydroxyl substitution but of its stereochemistry too, in the

dual-target binding proﬁle.

To further expand SAR on the MOR PP, in Scheme 10, we

synthesized a new library of bitopic analogues presenting

diﬀerent phenylmorphan nuclei as PPs to target the MOR and

the well-known 2-indoleamide SP to target D₃R SBP. We were

motivated to use this combination of pharmacophores because

phenylmorphans are a well-characterized scaﬀold for obtaining

highly potent and selective MOR ligands,⁵⁶and they also

structurally resemble phenyl/pyridine morpholino moieties

that we extensively studied as D₃R OBS ligands.⁴⁵Moreover,

we recently demonstrated how the pyridine morpholine

scaﬀold designed as a D₃R PP can also be structurally tweaked

via the bitopic approach and linker tethering to target other

families of GPCRs, including the MOR.⁴⁶This suggested that

simple structural modiﬁcation of the phenylmorphan scaﬀold

might also exploit aﬃnity for both targets and ultimately be

suitable for the generation of bitopic and bivalent hybrids for

both the MOR and D₃R.

In Scheme 10, 56 was N-alkylated via reductive amination

with N-(4-oxobutyl)-1H-indole-2-carboxamide⁵⁷and trans-N-

(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carboxamide,⁴⁵to

obtain 57 and 58, respectively. We have previously published⁴⁵

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a

Scheme 11

a

(a) 2-Bromoethan-1-ol, K₂CO₃, cat. KI, and ACN, 82 °C, overnight, 60%; (b) p-toluenesulfonyl chloride (p-TsCl), DIPEA, and DCM, 25 °C,

overnight, 36%; (c) NaHCO₃and ACN, 82 °C, 6 h, 43%.

Figure 2. Binding mode of 5 (orange sticks) inside the (A) D₃R (cyan; PDB: 3PBL) and (B) binding mode of 3 (green sticks) inside the MOR

(white; PDB: 5C1M).

Figure 3. Binding modes of 23 (green) and 5 (orange) inside the (A) D₃R (cyan; PDB: 3PBL) and (B) docking mode of 23 (green) and 13

(orange) inside the MOR (white; PDB: 5C1M).

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Figure 4. Binding mode of 14 (orange sticks) and 28 (green sticks) inside the (A) D₃R (cyan; PDB: 3PBL) and (B) MOR (white; PDB: 5C1M).

Figure 5. Binding mode of 40 (orange sticks) inside the (A) D₃R (cyan; PDB: 3PBL) and (B) MOR (white; PDB: 5C1M).

Finally, as shown in Scheme 11, we prepared the bivalent

analogue of this phenylmorphan series, 65, presenting both the

MOR PP and the canonical D₃R PP 2,3-dichlorophenyl

piperazine for D₃R OBS, instead of the 2-indoleamide for SBP

binding. The alcohol intermediate 63 was prepared starting

from 1-(2,3-dichlorophenyl)piperazine hydrochloride and 2-

bromoethan-1-ol, under basic conditions. Tosylation of the

hydroxy group and subsequent nucleophilic substitution with

56 yielded the desired product 65.

to canonical MOR ligands such as morphine and methadone,

interacting with residues Y150^3.33, M153^3.35, V238^5.42

,

W295^6.48, I298^6.51, H299^6.52, V302^6.55, W320^7.35, I324^7.39, and

Y328^7.43. Although the chlorophenyl and hydroxy substituents

on the piperidine rings are common for both 5 and 3, these

moieties occupy distinct SBPs and interact with diﬀerent

residues in their cognate receptors. Attempts at cross-docking

these ligands, that is, dock 5 to the MOR and 3 to the D₃R,

were unsuccessful, corroborating selectivity of the PPs to their

corresponding OBS pockets.

Molecular Docking and CADD. We employed structure-

based molecular modeling methods to guide the rational

design of our dual-target compounds. All atom docking studies

on the inactive-state D₃R (PDB: 3PBL)⁴⁴and active-state

MOR (PDB: 5C1M)⁵⁸were performed, followed by local

optimization of receptor−ligand interactions via energy-based

Monte Carlo minimization protocols⁵⁹using ICM-Pro

(Molsoft LLC). Figure 2 shows optimized binding modes of

5 and 3 inside the D₃R and MOR, respectively, indicating salt

bridge interactions between basic nitrogen and conserved D^3.32

of the receptors. The ﬂuorophenyl butanone moiety of 5

occupies the OBS between TM3, TM5, and TM6 in the D₃R,

Figure 3 shows an overlay of MOR and D₃R docking modes

of 23, a compound with the N,N-dimethyl-diphenylbutana-

mide PP of the MOR tethered to a 2,3-dichlorophenyl-

piperazine PP of canonical D₃R antagonists/partial agonists. In

docking to the MOR (Figure 3B), we observed a perfect ﬁt of

the MOR PP in the corresponding OBS, while the D₃R PP of

23 was accommodated in the secondary site of the MOR. In

the D₃R, the lack of ﬂexibility of the N-linked 2,3-

dichlorophenyl of 23 prevented this D₃R PP from reaching

its OBS site (Figure 3A). However, the 2,3-dichlorophenyl

moiety still comfortably ﬁts the hydrophobic pocket lined by

V111^3.33, W342^6.48, F345^6.51, F346^6.52, and H349^6.55. The MOR

PP moiety of 23, N,N-dimethyl-diphenylbutanamide, is placed

lined by hydrophobic residues such as V111^3.33, V189^5.39

,

F345^6.51, F346^6.52, and H349^6.55. In the MOR, the diphenyl

butanamide moiety of 3 also occupies the OBS pocket similar

in a hydrophobic region of the D₃R surrounded by V86^2.61

,

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Figure 6. Binding mode of 65 (orange sticks) and 10 (green sticks) inside the (A) D₃R (cyan; PDB: 3PBL) and (B) MOR (white; PDB: 5C1M).

L89^2.64, C103^3.25, F106^3.28, C181^ECL2, Y365^7.35, and T369^7.39

and makes additional interactions resulting in comparable

docking scores for the MOR and D₃R, supporting the synthesis

of this compound.

In contrast, despite D₃R SAR that supported introduction of

the butyl linker between the tethered N,N-dimethyl-diphe-

nylbutanamide or 2,2-diphenylbutanenitrile MOR PP and the

2,3-dichlorophenyl-piperazine, as seen in compounds such as

14 and 28, this design had no eﬀect on the placement of the

compounds in the OBS of the D₃R, while the corresponding

SP motif moves further toward the extracellular region, away

from the hydrophobic residues of TM2, TM3, and ECL2

(Figure 4).

The meta-hydroxy compounds were designed to avail the

water-mediated hydrogen bonding network close to H299^6.52

and Y150^3.33in the MOR, such as 51, 54, and 55 indeed

indicate the presence of those predicted hydrogen bonds, while

the 2-chloro-3-ethyl-substituted phenyl-piperazine D₃R PP

motif is tolerated in the voluminous and solvent-accessible

MOR pocket (Figure S1). Furthermore, the highly decorated

8-based D₃R PP of compounds 40 and 48 are placed

expectedly in the OBS of the D₃R, and like the substituted

phenyl analogues, the N,N-dimethyl-diphenylbutanamide

motif occupies hydrophobic subpockets between TM2, TM3,

ECL2, and TM7 (Figure 5).

The conformationally restricted ethyl-2-(-5-(3-hydroxyphen-

yl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate MOR PP motif

of 10 expectedly docks in the MOR hydrophobic pocket

formed between TM3−TM5−TM6−TM7, and 3-hydroxy-

phenyl forms water-mediated hydrogen bonding interactions

with H299^6.52and Y150^3.33. The ethyl phenyl tail of 10 shares

the same hydrophobic subpocket between TM2 and TM3

engaged by fentanyl-like compounds. The docking mode of 65

overlaps completely with the binding mode of 10, except the

fact that extended 2,3-dichlorophenyl piperazine of 65 moves

slightly toward the extracellular region and is placed in the

subpocket between TM2−TM3−ECL2. The 2,3-dichloro-

phenyl piperazine of 65 is docked in the OBS of the D₃R,

while the ethyl-2-(-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]-

nonan-9-ylidene)acetate moiety is placed in the hydrophobic

SBP region (Figure 6).

hMOR (in competition with [³H]-DAMGO), hD₂R, hD₃R,

and hD₄R [in competition with [³H]-N-methylspiperone

([³H]-NMSP) for all the hD₂-like subtypes]. Moreover, a

subset of selected hits were further studied at hD₂R and hD₃R

using the agonist [³H]-(R)-(+)-7-OH-DPAT as the competing

radioisotope. We have previously observed and reported that

diﬀerences in aﬃnity due to the radioligand being an agonist or

antagonist can predict functional eﬃcacy proﬁles for the tested

compounds.^45,60

In Table 1, the binding data are reported for the ﬁrst series

of MOR−D₃R hybrid analogues, based on, 3-, 4-, and 6-like

PPs. Reference compounds, including fentanyl, 3, 4, 5, and 6,

are reported in the table for useful comparisons. Among the

reference compounds, it was interesting to observe how 3, a

well-known potent MOR agonist, despite presenting an SP

identical to the D₂-like antagonist 5 (Figure 1), binds with low

micromolar aﬃnity to all the D₂-like subtypes, as predicted in

the CADD studies. Fentanyl, 4, and 6 all have low nanomolar

aﬃnities for the MOR, as expected and consistent with the

literature.^61,62Fentanyl, however, presents moderate aﬃnity

for D₄R (K_i= 554 nM), while being inactive at both the D₂R

and D₃R (K_is > 10,000 nM); meanwhile, 6 is endowed with a

preferential low micromolar aﬃnity for the D₃R, being

completely inactive at D₂R and >10-fold selective over D₄R.

These data highlight how subtle structural changes in well-

characterized MOR agonists can induce diﬀerent binding

proﬁles and subtype selectivity for the D₂-like dopamine

receptors and that binding aﬃnities can be directed toward

dual-target proﬁles with well-designed structural modiﬁcations.

The docking studies showed that the MOR PP motifs (N,N-

dimethyl-diphenylbutanamide and 2,2-diphenylbutanenitrile)

occupy primarily hydrophobic pockets in both the D₃R and

MOR. In the case of the MOR, this pocket formed between

TM3, TM5, TM6, and TM7 is an accumulation of three

subpockets lined by (a) V238^5.42, M153^3.33, H299^6.52, I298^6.51

,

and V302^6.55, (b) M153^3.33, W295^6.48, and Y328^7.43, and (c)

W320^7.35, I324^7.39, I298^6.51, and V302^6.55. In the D₃R, these

MOR PP motifs occupy a hydrophobic SBP between TM2,

TM3, and ECL2 (Figures 3 and 4). In general, replacing the

nitrile group with the 3-like N,N-dimethylamide synthon

signiﬁcantly increased the aﬃnity proﬁles of all the analogues.

In particular, 23 presents one of the highest MOR aﬃnities

among all the new analogues (MOR K_i= 0.832 nM), and

Binding Studies and SARs. All the newly synthesized

compounds were tested for their binding aﬃnities at the

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Table 1. Radioligand Competition Binding Aﬃnity Data for All the MOR Diphenyl PP Analogues Based on 3, 4, and 6 at

a

hMOR and hD₂-like Receptor Subtypes

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Table 1. continued

a

All the aﬃnity values are expressed as K_istandard error of the mean (SEM), derived from IC₅₀values using the Cheng−Prusoﬀ equation,⁶³and

calculated as the mean of at least three independent experiments (n = number of independent experiments), each performed in triplicate. ND = not

determined. No inhibition of speciﬁc radioligand binding was observed at the highest tested concentration in one to three independent

experiments, each performed in triplicate. K_ivalue obtained from ref 61.

b

c

despite the shorter linker, which is generally less favorable for

D₂-like receptors aﬃnity, we still obtained a potent D₂-like

ligand (D₂R K_i= 74.7 nM, D₃R K_i= 171 nM, and D₄R K_i=

102 nM). A similar nanomolar binding proﬁle across the D₂R

and D₃R was also conﬁrmed when 23 was tested in the

presence of [³H]-(R)-(+)-7-OH-DPAT. The analogous nitrile

compound, 13, showed reduced MOR binding (∼320-fold; K_i

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Table 2. Radioligand Competition Binding Aﬃnity Data, for All the MOR-Substituted Phenylmorphan PP Analogues, at the

a

hMOR and hD₂-like Receptor Subtypes

a

All the aﬃnity values are expressed as K_iSEM, derived from IC₅₀values using the Cheng−Prusoﬀ equation,⁶³and calculated as the mean of at

b

least three independent experiments (n = number of independent experiments), each performed in triplicate. ND = not determined. No inhibition

of speciﬁc radioligand binding was observed at the highest tested concentration in one to three independent experiments, each performed in

triplicate.

= 266 nM) and reduced D₃R binding (∼13-fold; K_i= 2240

nM).

an analogous compound with substituted N,N-dimethylamide

in the MOR PP motif, shows signiﬁcant improvement in both

D₃R aﬃnity (K_i= 39.2 nM) and MOR binding (K_i= 23.8

nM). This was the ﬁrst hit analogue in this series to show a low

nanomolar dual-target aﬃnity for both the MOR and the D₂-

like receptors. In contrast, compound 18 showed similarly high

aﬃnity for the D₂-like receptors, but MOR aﬃnity was

diminished (K_i= 1470 nM), whereas compounds 15, 19, 21,

24, and 27 showed the opposite proﬁle, having higher aﬃnities

for the MOR than the D₂R or D₃R. Compounds 16 and 22

were poorly active at all receptors tested, reﬂecting an inability

to bind the OBS of either MOR or the D₂-like receptors.

Introduction of the hydroxy substituent in the butylamine

linker (compounds 29 and 32), as well as replacement of the

2,3-dichlorophenyl piperazine, with the 2-chloro-3-ethyl-

phenylpiperazine (compounds 31 and 32) either maintained

or slightly decreased the overall aﬃnity for all the D₂-like

The docking studies of butyl-linked compounds show that in

the D₃R, the N,N-dimethyl-diphenylbutanamide and 2,2-

diphenylbutanenitrile MOR PP motifs move further toward

the extracellular region, away from the hydrophobic residues of

TM2, TM3, and ECL2 (Figure 4). Perhaps, because of this

conformational change, N,N-dimethylamide to cyano sub-

stitutions on the extended linker molecules such as 14 (D₂R K_i

= 149 nM and D₃R K_i= 132 nM) are better tolerated at the

D₃R than the shorter linker compounds such as 13 (D₂R K_i=

2630 nM and D₃R K_i= 2240 nM). As in the D₃R, the extended

linker compounds are reasonably well tolerated inside the

MOR. However, in contrast to its binding proﬁle at the D₃R,

even the extended linker molecule with nitrile substitutions 14

shows reduced MOR binding (K_i= 490 nM). This was also

observed with the nitrile analogues, 35 and 37. Compound 28,

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Figure 7. Functional proﬁles of selected MOR−D₃R hybrids. Each panel shows a diﬀerent signaling readout: (A) Nb33 recruitment at the MOR,

(B) MOR-mediated Gα_i2protein activation, (C) antagonism at the MOR using GPA in the presence of 100 nM DAMGO, (D) arrestin-3

recruitment at the MOR in the presence of overexpressed GRK2, (E) D₃R-mediated Gα_oAprotein activation, and (F) antagonism at the D₃R using

GPA in the presence of 3 nM quinpirole. In order to ensure that test ligands achieved maximal receptor occupancy possible before agonist addition,

all ligands were added to the cells 3 h prior to activating the D₃R with quinpirole and measuring BRET signals with the exception of 5. Dotted lines

represent ﬁts where the bottom asymptote was constrained to be 0%.

receptor subtypes, when compared to 28 and shown in Figure

S2. The introduction of 1,2,3,4-tetrahydroisoquinoline-7-

carbonitrile as the D₃R PP (34) decreased aﬃnity for the

D₂-like receptors into the micromolar range. None of the

diphenyl-pyrrolidine analogues (compounds 42, 43, and 46)

were active. However, the only bivalent compound 46 did have

moderate aﬃnity for the D₃R (K_i= 288 nM).

Removal of either nitrile or amide functions from the

diphenyl MOR PP and introduction of the meta-hydroxy

substituents to the bis-phenyl system, 51, were tolerated as

suggested from the docking predictions (Figure S1). Despite

the presence of a simple unsubstituted phenylpiperazine

pharmacophore, 51 still maintained moderate aﬃnities at

both the MOR (K_i= 213 nM) and D₃R (K_i= 249 nM).

When the bis-meta-phenol MOR PP was used in

combination with the 2-chloro,3-ethyl-phenylpiperazine D₃R

PP, tethered via the shorter (two methylene units) linker (55),

moderate aﬃnity for all the targets of interest was maintained,

but this analogue was not signiﬁcantly diﬀerent from the longer

linker analogue 51. The presence of the meta-hydroxy

substituents also retained D₂-like aﬃnity for 55, similar to

that observed for 23 and 30 (containing the 3-like MOR PP).

In a continuation of a trend noted previously, we observed a

signiﬁcant loss of aﬃnity at the MOR, with 55 presenting a

binding K_i>300-fold less than 30 and 23. These results suggest

that while the bis-phenol MOR PP is well tolerated as an

alternative to the more canonical 3-like di-phenyl-N,N-

dimethylamide, the binding proﬁle is still dependent on the

linker length, rigidity, and overall substitutions on the D₃R PP

as well.

In agreement with the CADD, the meta-substitutions in bis-

phenyl-containing compounds are important for MOR

recognition. The methoxy analogues result in partial loss of

MOR aﬃnity with a general binding proﬁle of 53 (di-methoxy)

< 54 (mono-methoxy) ∼ 55 (di-hydroxy). This, however, does

not apply for D₂-like binding, where all three analogues show

moderate aﬃnities for all the subtypes independent of meta-

methoxy or meta-hydroxy substitutions on the bis-phenyl rings,

interestingly with higher aﬃnities at D₄R.

Shifting from a phenylpiperazine-based D₃R PP to a highly

decorated 8-based D₃R PP, to develop SAR around the

pyrrolidine scaﬀold, 40, containing a racemic pyrrolidin-2-

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ylmethyl-amide linker, and 48, presenting a butyl ether linker

chain in position 4 of the trans-pyrrolidine nucleus, were

synthesized. Compound 48 showed the highest D₂R/D₃R

aﬃnity among all the new analogues (D₂R K_i= 9.41 nM; D₃R

K_i= 2.21 nM); however, the regio- and stereochemistry of the

substituted pyrrolidine ring was detrimental for MOR binding,

with a K_iof 559 nM. On the other hand, 40 emerged as our

third lead, alongside 23 and 28, with its almost identical

aﬃnities for both the MOR (K_i= 106 nM) and D₃R (K_i= 135

nM), ∼4- and ∼25-fold selectivity over D₂R and D₄R,

respectively. This proﬁle distinguished 40 as one of the most

promising dual-target MOR−D₃R compounds in the series.

The binding data for the phenylmorphan analogues are

reported in Table 2. Compounds 10, 11, and the nor-

analogues 56, 59, and 60 were tested as reference compounds,

since most of them were key MOR PP building blocks for our

bivalent and bitopic drug design. Compound 10 showed the

highest MOR aﬃnity as an OBS PP, with K_i= 0.633 nM,

similar to the aﬃnities of 3 and the hybrid bivalent analogue,

23. Interestingly, 10 also showed moderately low micromolar

and sub-micromolar aﬃnities for the D₂R and D₃R, supporting

the hypothesis that the phenylmorphan ring structurally

resembles more ﬂexible phenyl-morpholino moieties, canonical

scaﬀolds for D₃R ligands.⁴⁵

Docking studies suggested that the steric clash between the

ethyl acrylate group and the backbone of the D₃R receptor

would limit and potentially challenge the development of

bitopic ligands 57 and 58 targeting D₃R, despite being

tolerated in the MOR OBS. Indeed, bitopic analogues 57 and

58 and the resolved enantiomers 58a and 58b all showed high

aﬃnities at the MOR (K_is ranging from 13.5 to 57 nM) but

micromolar K_is for all the D₂-like subtypes, independent of

linker rigidity or stereochemistry. The shorter bivalent

analogue 65 with a simple ethyl linker chain (n = 2) was

predicted by docking studies to be the optimal spacer to

resolve steric clashes of the MOR PP motif in the D₃R; Figure

6. Indeed, tethering the MOR phenylmorphan PP and the D₃R

2,3-dichlorophenyl piperazine PP presented one of the most

interesting dual-target candidates (65) with equivalent

aﬃnities at the MOR (K_i= 92.7 nM) and D₃R (K_i= 139 nM).

Replacement of the sterically hindered ethyl acrylate group

with a smaller hydroxy substituent allowed validation of the

docking observation that a small substituent would signiﬁcantly

improve D₃R binding. Overall, 61 and 62 adopt the same

docking as 65 inside the MOR but lose hydrophobic

interactions of the ethyl acrylate moiety with M153^3.36and

Y328^7.43(Figure S3). The hydrogen-bonding interactions

between the 9-OH and Y328^7.43provide limited compensation

for 62. However, for 61, this hydrogen bond is accompanied

by negative interactions due to proximity of the carboxylate

oxygen of D149^3.32(3.2 Å compared to 4.5 Å of S-chiral 62).

Furthermore, 61 and 62 adopt an interesting conformation

inside the D₃R with the indole carboxamide ring situated in the

OBS. In the absence of positively charged nitrogen, the

nitrogen of the amide forms a hydrogen bond with the

D110^3.32residue. Interestingly, the 9-OH is placed very close to

the backbone of TM7 for 62 and TM2 for 61. Therefore, the

substitution of this position with ethyl acrylate would cause

steric clashes with the D₃R, as indicated by the loss of aﬃnity

of 57. Bitopic analogue 62, with all the resolved stereochemical

conﬁgurations around the phenylmorphan moiety inverted

with respect to 57, showed a D₃R K_iof 79.6 nM, ∼172-fold

improved aﬃnity with respect to the ethyl acrylate analogue

57, maintaining a moderate aﬃnity (K_i= 464 nM) for the

MOR.

Across all the tested compounds, no signiﬁcant diﬀerences

were observed in the D₂-like aﬃnities determined using [³H]-

NMSP, and [³H]-(R)-(+)-7-OH-DPAT binding assays were

observed, unlike our previous observations for eﬃcacious

agonist ligands,^43,45consistent with our hypothesis that all the

new analogues are likely antagonists or low-eﬃcacy partial

agonists at the D₃R.

Based on their binding proﬁles, a select group of hits were

tested in functional assays to determine their agonist and

antagonist potencies for the multiple GPCR-related signaling

pathways, as well as to validate and conﬁrm the MOR agonism

and D₃R antagonism/partial agonism proﬁle, we were seeking

with these new hybrid molecules.

BRET Functional Studies at the MOR and D₃R. With

the binding studies and SAR established, we then characterized

the action of selected ligands to signal through both the MOR

and D₃R; these results are shown in Figure 7 and Table 3. The

action of these ligands was assessed through arrestin-3 (or β-

arrestin-2) recruitment at MOR and G-protein activation

(GPA) at MOR (Gα_i2) and D₃R (Gα_oA) assays. In addition,

the ability of the ligands to induce the active state of the MOR

was determined by measuring recruitment of a conformation-

ally selective nanobody that recognizes and binds to the active

conformation of the MOR, nanobody 33 (Nb33).⁶⁴Seven of

the newly synthesized MOR−D₃R hybrids (14, 23, 28, 40, 57,

58a, and 58b) were tested together with 10. The eﬃcacious

agonists DAMGO (D-Ala2, N-MePhe4, and Gly5-ol-enkepha-

lin), quinpirole, and dopamine were used as reference

agonists to normalize data at the MOR and D₃R. We included

the MOR partial agonist morphine to illustrate the relative

coupling eﬃciency and ampliﬁcation of the diﬀerent assays

(Figure 7A,B,D). Both known antagonists, naloxone (MOR)

and 5 (D₃R), inhibited agonist-stimulated GPA in a

concentration-dependent manner (Figure 7C,F).

The three substituted phenylmorphan MOR PP analogues

(57, 58a, and 58b), despite showing improved aﬃnities for the

MOR compared to 56, did not activate the MOR in any of the

three pathways tested (Figure 7A,B,D). Thus, we tested the

ability of these bitopic compounds to inhibit the action of 100

nM of DAMGO in the GPA i2 assay. As shown in Figure 7C,

all three ligands were able to inhibit DAMGO-mediated GPA

to the same extent as naloxone albeit with lower potencies.

When tested at the D₃R, 57 failed to demonstrate any D₃R

activity (up to 10 μM, Figure 7E,F), which is likely due to the

low aﬃnity of this compound for this receptor (Table 2).

Compounds 58a and 58b showed agonist activity at the D₃R

but with low (micromolar) potencies that reﬂect their aﬃnity

for the D₃R (Figure 7E, Table 3). Thus, these two

phenylmorphan hybrids display MOR antagonism and D₃R

agonism, rather than the MOR agonist/D₃R antagonist or

partial agonist proﬁle we targeted.

In contrast, all four MOR diphenyl PP analogues tested (14,

23, 28, and 40) showed agonist activity at the MOR, with 23

being the most potent and eﬃcacious compound. Compound

14 showed low-potency MOR agonism that could only be

detected at the highest concentration used of 10 μM in the

most ampliﬁed and sensitive GPA i2 assay. 40 displayed higher

potency and eﬃcacy in assays of MOR activation than 28, with

28 displaying no detectable agonism in the less-ampliﬁed Nb33

and arrestin-3 recruitment assays but an E_maxof 50% that of

DAMGO in the GPA i2 assay. Compound 40 gave a robust

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response (E_max= 84.4% of DAMGO) in the GPA i2 assay but

much weaker responses in the arrestin and Nb33 assays,

indicating that it is a less-eﬃcacious partial agonist than

morphine. All four bivalent compounds share a similar MOR

diphenyl PP based on 3 and 4, the N,N-dimethyl-

diphenylbutanamide PP being more favorable than the

diphenylbutanenitrile PP for MOR agonism. The major

structural diﬀerences are present in the D₃R PP and the type

and length of the linker between the two pharmacophores: a

shorter linker being more favorable for MOR agonism. In

general, across the series of compounds and morphine, we

observe higher maximal eﬀects (E_max) and potencies in the

GPA i2 assay as compared to that in the arrestin-3 recruitment

and Nb33 assays. Such a behavior is consistent with the action

of partial agonists at signaling endpoints with diﬀerent levels of

ampliﬁcation and coupling eﬃciency of the pathway. In

agreement with this, when these data were analyzed using

the Black and Leﬀ operational model of agonism and assessed

for biased agonism using DAMGO as the reference ligand,

none of the compounds displayed signiﬁcant bias between

these two pathways relative to the action of DAMGO (Table

S2).

When these four MOR diphenyl PP ligands were tested for

their ability to activate the D₃R (Figure 7E,F), 14 and 28

showed similar eﬃcacies (64 and 55% of dopamine,

respectively), with 28 being the most potent compound in

agreement with their relative aﬃnities (Table 1). Although 14

and 40 display similar aﬃnities for D₃R, 40 acted as an

antagonist with micromolar potency (IC₅₀= 1.5 μM), whereas

14 acted as a robust partial agonist (E_max= 63.8% of

quinpirole). Finally, 23, which was the most potent MOR

agonist and shares the same D₃R PP structure as 28 but with a

shorter linker, displayed weak partial agonism (E_max= 20% of

quinpirole) at the D₃R and sub-micromolar potency consistent

with its binding aﬃnity.

penetrability as the current lead compounds have central

nervous system multiparameter optimization^65,66scores of ∼2

and are predicted to be peripherally limited. This indeed was

what Komoto and colleagues³⁷found with their loperamide

analogues, which is perhaps unsurprising. Nevertheless, with

the proof-of-concept in hand, we have laid the groundwork for

an alternative pharmacological approach, using bivalent drug

design to engage both the MOR and D₃R in the pursuit of a

novel class of opioid analgesics with lower abuse potential.

EXPERIMENTAL METHODS

■

Chemistry. All chemicals and solvents were purchased from

chemical suppliers unless otherwise stated and used without further

puriﬁcation. All melting points were determined (when obtainable)

on an OptiMelt automated melting point system and are uncorrected.

Reactions were not yield optimized. The H and ¹³C NMR spectra

1

were recorded on a Varian Mercury Plus 400 instrument. Proton

chemical shifts are reported as parts per million (δ ppm) relative to

tetramethylsilane (0.00 ppm) as an internal standard or to deuterated

solvents. Coupling constants are measured in Hertz. Chemical shifts

for ¹³C NMR spectra are reported as parts per million (δ ppm)

relative to deuterated CHCl₃or deuterated MeOH (CDCl₃77.5 ppm

and CD₃OD 49.3 ppm). Chemical shifts, multiplicities, and coupling

constants (J) have been reported and calculated using VnmrJ Agilent-

NMR 400MR or MNova 9.0 software. Gas chromatography−mass

spectrometry (GC/MS) data were acquired (where obtainable) using

an Agilent Technologies (Santa Clara, CA) 7890B GC equipped with

an HP-5MS column (cross-linked 5% PH ME siloxane, 30 m × 0.25

mm i.d. × 0.25 μm ﬁlm thickness) and a 5977B mass-selective ion

detector in the electron-impact mode. Ultrapure-grade helium was

used as the carrier gas at a ﬂow rate of 1.2 mL/min. The injection port

and transfer line temperatures were 250 and 280 °C, respectively, and

the oven temperature gradient used was as follows: the initial

temperature (70 °C) was held for 1 min and then increased to 300 °C

at 20 °C/min and maintained at 300 °C for 4 min, with a total run

time of 16.5 min. Column chromatography was performed using a

Teledyne Isco CombiFlash RF ﬂash chromatography system or a

Teledyne Isco EZ-Prep chromatography system. Preparative thin-

layer chromatography was performed on Analtech silica gel plates

(1000 μm). When % DMA is reported as the eluting system, it stands

for % of methanol in DCM, in the presence of 0.5−1% NH₄OH.

Preparative chiral HPLC was performed using a Teledyne Isco EZ-

Prep chromatography system with the diode array detector (DAD)

and ELS detectors. HPLC analysis was performed using an Agilent

Technologies 1260 Inﬁnity system coupled with the DAD. For each

analytical HPLC run, multiple DAD λ absorbance signals were

measured in the range of 210−280 nm. Separation of the analyte,

purity, and enantiomeric/diastereomeric excess determinations were

achieved at 40 °C using the methods reported in each detailed

reaction description. Preparative and analytical HPLC columns were

purchased from Daicel Corporation or Phenomenex. HPLC methods

and conditions are reported in the descriptions of the chemical

reactions where they were applied. Microanalyses were performed by

Atlantic Microlab, Inc. (Norcross, GA) and agree with 0.4% of

calculated values. HRMS (mass error within 5 ppm) and MS/MS

fragmentation analysis were performed on an LTQ-Orbitrap Velos

(Thermo Scientiﬁc, San Jose, CA) coupled with an ESI source in the

positive-ion mode to conﬁrm the assigned structures and

regiochemistry. Unless otherwise stated, all the test compounds

were evaluated to be >95% pure on the basis of combustion analysis,

NMR, GC/MS, and HPLC-DAD. The detailed analytical results are

reported in the characterization of each ﬁnal compound.

CONCLUSIONS

■

The data obtained highlight a series of hit to lead candidates as

MOR−D₃R dual-target ligands. We have synthesized multiple

combinations of bivalent or bitopic ligands based on carefully

designed structural modiﬁcations and in silico-guided SAR

around the MOR PP, D₃R PP, and SP, as well as linkers, with a

particular focus on regio- and stereochemistry. Importantly, we

have identiﬁed compounds with a range of sub-nanomolar to

sub-micromolar binding aﬃnities for each receptor of interest

and thus provide a new approach to modulate the

pharmacological proﬁles of highly selective MOR agonists

through concomitant dual-target D₃R antagonism.

The functional studies revealed three lead analogues, 23, 28

and 40, which are partial agonists at the MOR and partial

agonists or antagonists at the D₃R. We and others have

suggested that low intrinsic eﬃcacy could explain the improved

therapeutic window observed on the most recent MOR

agonists, such as PZM21 and TRV-130,³¹and our three lead

compounds ﬁt this desired functional proﬁle with the added

feature of D₃R low-eﬃcacy partial agonism or antagonism,

which may prove beneﬁcial in avoiding the addictive liability of

opioid receptor-targeted drugs. Furthermore, evaluation and

thus a better understanding of the desired kinetic proﬁles at

both targets for the optimal pharmacological eﬀect will be

crucial in the development of future generations of dual-target

MOR−D₃R ligands. Indeed, current drug design is focused on

improving drug-like characteristics and blood−brain barrier

4-(Phenethylamino)-2,2-diphenylbutanenitrile (12). A suspen-

sion of 4-bromo-2,2-diphenylbutanenitrile (500 mg, 1.67 mmol), 2-

phenylethan-1-amine (605 mg, 5 mmol), and K₂CO₃(230 mg, from

1.67 mmol up to 10 equiv) in ACN (50 mL) was heated at 130 °C in

a sealed vessel overnight. The mixture was ﬁltered, the solvent was

removed under vacuum, and the residue was puriﬁed by ﬂash

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chromatography eluting with 15% DMA. The desired product was

obtained as colorless oil (300 mg, 53% yield). H NMR (400 MHz,

range of 210−280 nm, Rt 9.072 and 10.862 min, purity >95%, er

43:57 (absorbance at 254 nm).

1

4-(Dimethylamino)-2,2-diphenylbutanenitrile (16). The reaction

was performed following the same procedure described for 12,

starting from dimethylamine hydrochloride (2.0 g, 25 mmol) and 4-

bromo-2,2-diphenylbutanenitrile (5.0 g, 16.7 mmol). The desired

product was puriﬁed by ﬂash chromatography eluting with 5% DMA

and obtained as colorless oil (3.5 g, 79% yield). ¹H NMR (400 MHz,

CDCl₃): δ 7.66−6.75 (m, 10H), 2.68−2.50 (m, 2H), 2.50−2.33 (m,

2H), 2.25 (s, 6H). GC/MS (EI), Rt 10.499 min; 264.1 (M⁺), purity

>95%. The free base was converted into the corresponding oxalate

salt. CHN (C₁₈H₂₀N₂+ H₂C₂O₄) Calcd: C, 67.78; H, 6.26; N, 7.90.

Found: C, 67.66; H, 6.43; N, 7.96. mp: 163−169 °C.

3-Carboxy-N,N-dimethyl-3,3-diphenylpropan-1-aminium Bro-

mide (17). Compound 16 (2 g, 7.6 mmol) was dissolved in 48%

HBr aq solution (50 mL) and stirred under reﬂux overnight. The

solution was concentrated to half-volume and decanted, and the

residue was washed multiple times with Et₂O. The dried crude

material was used in the next step without further puriﬁcation.

N-(4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)-4-(dimethyla-

mino)-2,2-diphenylbutanamide (18). A solution of 17 (460 mg, 1.26

mmol), EDC hydrochloride (240 mg, 1.26 mmol), HOBt (170 mg,

1.26 mmol), and DIPEA (2.2 mL; 12.6 mmol) in DCM (20 mL) was

stirred at room temperature (RT) for 1 h, followed by dropwise

addition of 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine

(380 mg, 1.26 mmol) in DCM (20 mL). The mixture was stirred

at RT overnight, the solvent was evaporated under vacuum, and the

residue was puriﬁed by ﬂash chromatography eluting with 10% DMA.

The desired product was obtained as yellow oil (40 mg, 6% yield). ¹H

NMR (400 MHz, CDCl₃): δ 7.36−7.20 (m, 10H), 7.18−7.08 (m,

2H), 6.94 (dd, J = 6.6, 3.0 Hz, 1H), 6.74 (br s, 1H), 3.31−3.14 (m,

2H), 3.03 (br s, 4H), 2.62−2.53 (m, 6H), 2.40−2.27 (m, 2H), 2.19

(m, 8H), 1.53−1.36 (m, 4H); ¹³C NMR (101 MHz, CDCl₃): δ

173.99, 151.26, 143.81, 133.99, 128.74, 128.25, 127.47, 127.40,

126.81, 124.51, 118.55, 60.22, 58.03, 55.98, 53.23, 51.28, 45.53, 45.42,

44.87, 39.71, 36.80, 29.40, 27.36, 24.09. The free base was converted

into the corresponding oxalate salt. HRMS (C₃₂H₄₀ON₄Cl₂+ H⁺):

calcd, 567.26519; found, 567.26524 (error 1.3 ppm). CHN

(C₃₂H₄₀ON₄Cl₂+ 2H₂C₂O₄+ 3H₂O) Calcd: C, 53.93; H, 6.29; N,

6.99. Found: C, 53.89; H, 5.90; N, 7.31. mp: salt too hygroscopic to

determine melting point.

CDCl₃): δ 7.36−7.18 (m, 15H), 3.74 (t, J = 7.1 Hz, 2H), 3.07 (t, J =

6.2 Hz, 2H), 2.95 (t, J = 7.2 Hz, 2H), 2.60 (t, J = 6.2 Hz, 2H); ¹³C

NMR (101 MHz, CDCl₃): δ 171.34, 142.74, 139.55, 128.86, 128.57,

128.46, 128.38, 128.36, 128.12, 126.98, 126.18, 61.47, 53.40, 46.66,

46.18, 37.35, 33.47. The free base was converted into the

corresponding oxalate salt. HRMS (C₂₄H₂₄N₂+ H⁺): calcd,

341.20123; found, 341.20048 (error −0.7 ppm). CHN (C₂₄H₂₄N₂

+ 1.5H₂C₂O₄+ H₂O) Calcd: C, 65.71; H, 5.92; N, 5.68. Found: C,

65.76; H, 5.77; N, 5.57. mp: salt too hygroscopic to determine the

melting point.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-2,2-diphenylbutaneni-

trile (13). The reaction was performed following the same procedure

described for 12, starting from 1-(2,3-dichlorophenyl)piperazine (250

mg, 0.9 mmol) and 4-bromo-2,2-diphenylbutanenitrile (350 mg, 1.17

mmol). The desired product was puriﬁed by ﬂash chromatography

eluting with hexanes/ethyl acetate (hex/EtOAc 5:5) and obtained as

1

colorless oil (30 mg, 7.5% yield). H NMR (400 MHz, CDCl₃): δ

7.45−7.23 (m, 10H), 7.19−7.08 (m, 2H), 6.94 (dd, J = 6.8, 2.8 Hz,

1H), 3.04 (br s, 4H), 2.65 (dd, J = 10.5, 4.3 Hz, 6H), 2.57−2.49 (m,

2H). The free base was converted into the corresponding oxalate salt.

CHN (C₂₆H₂₅N₃Cl₂+ H₂C₂O₄+ 0.25H₂O) Calcd: C, 61.71; H, 5.09;

N, 7.71. Found: C, 61.67; H, 5.09; N, 7.85. mp: 202−207 °C.

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)amino)-2,2-di-

phenylbutanenitrile (14). The reaction was performed following the

same procedure described for 12, starting from 4-(4-(2,3-

dichlorophenyl)piperazin-1-yl)butan-1-amine (200 mg, 0.7 mmol)

and 4-bromo-2,2-diphenylbutanenitrile (105 mg, 0.35 mmol). The

desired product was puriﬁed by ﬂash chromatography eluting with

15% DMA and obtained as colorless oil (58 mg, 31% yield). ¹H NMR

(400 MHz, CDCl₃): δ 7.37−7.29 (m, 10H), 7.29−7.18 (m, 2H), 6.95

(dd, J = 6.4, 3.2 Hz, 1H), 4.84 (br s, 1H), 3.52 (t, J = 7.2 Hz, 2H),

3.26 (t, J = 6.2 Hz, 2H), 3.05 (t, J = 4.8 Hz, 4H), 2.71 (t, J = 6.2 Hz,

2H), 2.59 (br s, 4H), 2.44 (dd, J = 8.2, 6.7 Hz, 2H), 1.70−1.53 (m,

4H); ¹³C NMR (101 MHz, CDCl₃): δ 171.46, 151.32, 142.37,

133.99, 128.51, 128.45, 127.48, 127.39, 127.15, 124.48, 118.56, 61.65,

58.24, 53.24, 51.31, 46.34, 44.72, 37.36, 25.14, 24.26. The free base

was converted into the corresponding oxalate salt. HRMS

(C₃₀H₃₄N₄Cl₂+ H⁺): calcd, 521.22333; found, 521.22363 (error

0.3 ppm). CHN (C₃₀H₃₄N₄Cl₂+ 3H₂C₂O₄+ 0.5H₂O) Calcd: C,

54.01; H, 5.16; N, 7.00. Found: C, 53.97; H, 5.33; N, 7.15. mp: salt

decomposes above 80 °C.

6-(Dimethylamino)-4,4-diphenylhexan-3-one (19). Ethyl magne-

sium bromide (3 M solution in diethyl ether, 10 mmol) was added

dropwise to a solution of 16 (2.0 g, 7.5 mmol) in toluene (30 mL) at

0 °C. The mixture was slowly warmed to RT and then stirred at reﬂux

for 3 h. The reaction was quenched with 10 mL of 2 N HCl (aq

solution) at 0 °C and stirred at reﬂux for 30 min. The suspension was

basiﬁed with 2 N NaOH at 0 °C, the toluene was removed under

vacuum, and the aqueous phase was extracted with DCM/2-PrOH

(3:1). The organic layers were combined, dried over Na₂SO₄, ﬁltered,

and dried under vacuum to aﬀord the crude material, which was

puriﬁed by ﬂash chromatography eluting with 10% DMA. The desired

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-

amino)-2,2-diphenylbutanenitrile (15). The reaction was performed

following the same procedure described for 12, starting from 4-amino-

1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-2-ol⁴⁷(223 mg, 0.7

mmol) and 4-bromo-2,2-diphenylbutanenitrile (105 mg, 0.35

mmol). The desired product was puriﬁed by ﬂash chromatography

eluting with 15% DMA and obtained as colorless oil (53 mg, 28%

1

yield). H NMR (400 MHz, CDCl₃): δ 7.48−7.32 (m, 6H), 7.24−

1

7.10 (m, 6H), 6.95 (dd, J = 7.2, 2.3 Hz, 1H), 4.22−4.06 (m, 3H),

3.75−3.67 (m, 2H), 3.12 (br s, 4H), 2.96 (tt, J = 8.1, 4.2 Hz, 4H),

2.76 (d, J = 10.7 Hz, 2H), 2.67 (dd, J = 12.5, 3.2 Hz, 1H), 2.61−2.50

(m, 1H), 2.01 (s, 1H), 1.82 (ddd, J = 16.4, 13.9, 7.1 Hz, 1H); ¹³C

NMR (101 MHz, CDCl₃): δ 169.96, 150.78, 138.44, 138.40, 134.10,

129.50, 129.48, 128.91, 128.89, 127.88, 127.78, 127.58, 127.43,

124.83, 118.64, 64.45, 63.72, 62.31, 53.36, 50.83, 50.62, 45.63, 37.82,

31.51. HRMS (C₃₀H₃₄ON₄Cl₂+ H⁺): calcd, 537.21824; found,

537.21935 (error 1 ppm). HPLC analysis method A: Chiralpak AD-H

analytical column (4.5 mm × 250 mm5 μm particle size); mobile

phase: isocratic 30% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

4.944 min, purity >94.3% (absorbance at 254 nm). HPLC analysis

method B: Chiralpak OZ-H analytical column (4.5 mm × 250 mm

5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes;

ﬂow rate: 1 mL/min; injection volume: 20 μL; sample concentration:

∼1 mg/mL; multiple DAD λ absorbance signals measured in the

product was obtained as colorless oil (1.3 g, 59%). H NMR (400

MHz, CDCl₃): δ 7.51−7.06 (m, 10H), 2.52 (m, 2H), 2.30 (m, 2H),

2.15 (d, J = 0.9 Hz, 6H), 1.95 (m, 2H), 0.88 (td, J = 7.3, 0.9 Hz, 3H);

¹³C NMR (101 MHz, CDCl₃): δ 211.02, 141.48, 129.14, 128.26,

126.94, 65.04, 55.67, 45.52, 35.41, 32.47, 9.05. The free base was

converted into the corresponding oxalate salt. HRMS (C₂₀H₂₅NO +

H⁺): calcd, 296.20089; found, 296.20150 (error 0.4 ppm). CHN

(C₂₀H₂₅NO + H₂C₂O₄) Calcd: C, 68.55; H, 7.06; N, 3.63. Found: C,

68.51; H, 7.15; N, 3.62. mp: 161−165 °C.

tert-Butyl (5-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-5-

oxopentyl)carbamate (20). A solution of 5-((tert-butoxycarbonyl)-

amino)pentanoic acid (470 mg, 2.16 mmol), 1-(2,3-dichlorophenyl)-

piperazine (500 mg, 2.16 mmol), and HCTU (895 mg, 2.16 mmol) in

DCM (25 mL) was stirred at RT for 3 h. The solvent was removed

under vacuum, and the residue was puriﬁed by ﬂash chromatography

eluting with hex/EtOAc (40/60). The desired product was obtained

1

as yellow oil (550 mg, 59% yield). H NMR (400 MHz, CDCl₃): δ

7.23−7.11 (m, 2H), 6.92 (dd, J = 7.7, 1.8 Hz, 1H), 4.63 (br s, 1H),

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3.79 (t, J = 4.9 Hz, 2H), 3.68−3.60 (m, 2H), 3.15 (q, J = 6.6 Hz, 2H),

3.00 (dq, J = 10.7, 5.2 Hz, 4H), 2.39 (t, J = 7.4 Hz, 2H), 1.76−1.62

(m, 2H), 1.60−1.51 (m, 2H), 1.43 (s, 9H).

Hz, 1H), 3.49 (br s, 2H), 2.98 (br s, 3H), 2.82 (t, J = 5.9 Hz, 2H),

2.63 (t, J = 5.9 Hz, 2H), 2.55−2.46 (m, 2H), 2.40−2.26 (br s, 3H),

2.26−2.17 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 173.44, 140.62,

140.46, 136.71, 130.29, 129.39, 129.33, 128.42, 128.39, 128.07,

126.80, 119.18, 109.08, 59.74, 55.45, 55.42, 50.18, 42.84, 29.55. The

free base was converted into the corresponding oxalate salt. HRMS

(C₂₈H₂₉ON₃+ H⁺): calcd, 424.23834; found, 424.23765 (error −1.6

ppm). CHN (C₂₈H₂₉ON₃+ 1.5H₂C₂O₄+ 0.5H₂O) Calcd: C, 65.60;

H, 5.86; N, 7.40. Found: C, 65.76; H, 5.87; N, 7.29. mp: 178−181 °C.

4-Hydroxy-N,N-dimethyl-2,2-diphenylbutanamide (25). N-(3,3-

Diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bro-

mide (700 mg, 2 mmol) was suspended in 2 N NaOH (15 mL of aq

solution) and then stirred at RT for 5 min. The mixture was extracted

with DCM, and the organic layers were combined, dried over Na₂SO₄,

ﬁltered, and evaporated under vacuum to aﬀord the pure desired

4-((5-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-5-oxopentyl)-

amino)-2,2-diphenylbutanenitrile (21). TFA (1 mL, 12.8 mmol) was

added to a solution of 20 (550 mg, 1.28 mmol) in DCM (10 mL).

The mixture was stirred at RT for 24 h, basiﬁed with 2 N NaOH, and

extracted with DCM. The organic layers were combined, dried over

Na₂SO₄, ﬁltered, and dried under vacuum to aﬀord the crude primary

amine intermediate, which was dissolved in ACN (20 mL), followed

by the addition of 4-bromo-2,2-diphenylbutanenitrile (384 mg, 1.28

mmol) and K₂CO₃(10 equiv). The reaction mixture was stirred at

reﬂux overnight and ﬁltered, and the solvent was removed under

vacuum. The residue was puriﬁed by ﬂash chromatography eluting

with 15% DMA, and the desired product was obtained as yellow oil

1

(20 mg, 3% yield). H NMR (400 MHz, CDCl₃): δ 7.37−7.10 (m,

product in quantitative yield, as colorless oil. H NMR (400 MHz,

12H), 6.90 (dd, J = 7.8, 1.8 Hz, 1H), 3.78 (d, J = 5.8 Hz, 2H), 3.63 (t,

J = 4.9 Hz, 2H), 3.59−3.51 (m, 2H), 3.28 (t, J = 6.2 Hz, 2H), 2.97

(dt, J = 9.3, 5.0 Hz, 4H), 2.72 (t, J = 6.2 Hz, 2H), 2.50−2.41 (m, 2H),

1.71 (m, 4H); ¹³C NMR (101 MHz, CDCl₃): δ 171.48, 171.42,

150.64, 142.20, 134.16, 128.51, 128.47, 128.43, 127.74, 127.50,

127.25, 125.13, 118.75, 61.72, 51.69, 51.20, 46.41, 45.80, 44.41, 41.73,

37.36, 32.69, 26.68, 22.47. HRMS (C₃₁H₃₄ON₄Cl₂+ H⁺): calcd,

549.21824; found, 549.21825 (error 0.0 ppm). HPLC analysis

method: Chiralpak AD-H analytical column (4.5 mm × 250 mm

5 μm particle size); mobile phase: isocratic 30% 2-PrOH in hexanes;

ﬂow rate: 1 mL/min; injection volume: 20 μL; sample concentration:

∼1 mg/mL; multiple DAD λ absorbance signals measured in the

range of 210−280 nm, Rt 27.693 min, purity >95% (absorbance at

254 nm).

CDCl₃): δ 7.48−7.25 (m, 10H), 3.17 (q, J = 5.0 Hz, 2H), 3.04−2.95

(m, 3H), 2.57−2.49 (m, 2H), 2.35−2.27 (m, 3H). GC/MS (EI), Rt

11.847 min; 283.1 (M⁺).

N,N-Dimethyl-4-oxo-2,2-diphenylbutanamide (26). DMP was

added portionwise to a solution of 25 (200 mg, 0.71 mmol) in

DCM (10 mL) at 0 °C. The reaction mixture was slowly warmed to

RT and stirred for 1 h. The suspension was washed with 10%

NaHCO₃(aq solution), and the organic layer was dried over Na₂SO₄,

ﬁltered, and evaporated under vacuum. The residue was puriﬁed by

ﬂash chromatography eluting with hex/EtOAc (50/50) to aﬀord the

desired product as a white solid (120 mg, 60% yield). ¹H NMR (400

MHz, CDCl₃): δ 9.17 (t, J = 2.2 Hz, 1H), 7.46−7.25 (m, 10H), 3.11−

2.99 (m, 6H), 2.33 (s, 2H). GC/MS (EI), Rt 11.744 min; 281.1

(M⁺).

4-(Dimethylamino)-N,N-dimethyl-2,2-diphenylbutanamide (22).

Dimethylamine hydrochloride (1.18 g, 14.5 mmol) was added to a

suspension of N-(3,3-diphenyldihydrofuran-2(3H)-ylidene)-N-meth-

ylmethanaminium bromide (500 mg, 1.45 mmol) and K₂CO₃(2.0 g,

14.5 mmol) in TBME/ACN (25 mL/10 mL). The reaction mixture

was heated in a sealed vessel for 24 h, the solvent was removed under

vacuum, and the residue was puriﬁed by ﬂash chromatography eluting

with EtOAc/MeOH (95/5). The desired product was obtained as

N,N-Dimethyl-4-(phenethylamino)-2,2-diphenylbutanamide

(27). A solution of 26 (90 mg, 0.32 mmol), 2-phenylethan-1-amine

(77 mg, 0.64 mmol), and cat. AcOH in DCE (5 mL) was stirred at

RT for 30 min. STAB (97 mg, 0.48 mmol) was added portionwise,

and the mixture was stirred for additional 2 h. The solvent was

removed under vacuum, and the residue was puriﬁed by ﬂash

chromatography eluting with 5% DMA. The desired product was

1

obtained as colorless oil (quantitative yield). H NMR (400 MHz,

1

colorless oil (70 mg, 16% yield). H NMR (400 MHz, CDCl₃): δ

CDCl₃): δ 7.39−7.10 (m, 15H), 2.96 (m, 3H), 2.75 (br s, 7H), 2.48−

2.28 (m + br s, 4H + 1H); ¹³C NMR (101 MHz, CDCl₃): δ 176.89,

173.88, 140.40, 139.18, 128.82, 128.73, 128.54, 128.45, 128.04,

126.94, 126.22, 126.17, 60.51, 50.11, 46.28, 44.40, 43.46, 40.00, 35.13,

23.25. The free base was converted into the corresponding oxalate

salt. HRMS (C₂₆H₃₀ON₂+ H⁺): calcd, 387.24309; found, 387.24226

(error −2.1 ppm). CHN (C₂₆H₃₀ON₂+ 1.5H₂C₂O₄+ 0.1NH₄OH)

Calcd: C, 66.33; H, 6.43; N, 5.60. Found: C, 66.10; H, 6.61; N, 5.91.

mp: 112−117 °C.

7.43−7.24 (m, 10H), 2.97 (br s, 3H), 2.57−2.46 (m, 2H), 2.30 (m +

br s, 2H + 9H). GC/MS (EI), Rt 11.256 min; 310.1 (M⁺), purity

>95%. The free base was converted into the corresponding oxalate

salt. HRMS (C₂₀H₂₆ON₂+ H⁺): found, 311.21098 (error −2.4 ppm).

CHN (C₂₀H₂₆ON₂+ 1.5H₂C₂O₄+ 0.75H₂O) Calcd: C, 60.19; H,

6.70; N, 6.10. Found: C, 60.09; H, 6.36; N, 6.13. mp: 174−178 °C.

4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-N,N-dimethyl-2,2-di-

phenylbutanamide (23). 1-(2,3-Dichlorophenyl)piperazine hydro-

chloride (400 mg, 1 mmol) was added to a suspension of N-(3,3-

diphenyldihydrofuran-2(3H)-ylidene)-N-methylmethanaminium bro-

mide (500 mg, 1 mmol), K₂CO₃(967 mg, 7 mmol), and DIPEA (1

mL, 7 mmol) in ACN (20 mL). The reaction mixture was stirred at

reﬂux overnight, the solvent was removed under vacuum, and the

residue was puriﬁed by ﬂash chromatography eluting with 5% DMA.

The desired product was obtained as colorless oil (640 mg, 91%

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)butyl)amino)-N,N-di-

methyl-2,2-diphenylbutanamide (28). The reaction was performed

following the same procedure described for 27, starting from 4-(4-

(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (272 mg, 0.9

mmol). The desired product was puriﬁed by ﬂash chromatography

eluting with 25% DMA and obtained as colorless oil (300 mg, 65%

1

yield). H NMR (400 MHz, CDCl₃): δ 7.41−7.33 (m, 7H), 7.28−

1

yield). H NMR (400 MHz, CDCl₃): δ 7.46−7.34 (m, 10H), 7.34−

7.26 (m, 3H), 7.15−7.13 (m, 2H), 7.00−6.90 (m, 1H), 3.05 (s, 4H),

2.98 (s, 3H), 2.62 (br s, J = 6.0 Hz, 3H), 2.51 (t, J = 6.8 Hz, 6H),

2.45−2.37 (m, 4H), 2.29 (br s, 3H), 1.62−1.49 (m, 4H); ¹³C NMR

(101 MHz, CDCl₃): δ 178.09, 173.97, 151.17, 139.95, 133.94, 128.69,

128.65, 127.97, 127.48, 127.38, 127.15, 124.55, 118.73, 60.48, 57.93,

53.14, 51.06, 50.67, 47.97, 45.70, 43.25, 25.94, 24.19, 23.98. The free

base was converted into the corresponding oxalate salt. HRMS

(C₃₂H₄₀ON₄Cl₂+ H⁺): calcd, 567.26519; found, 567.26458 (error

−1.1 ppm). CHN (C₃₂H₄₀ON₄Cl₂+ 2.5H₂C₂O₄+ H₂O) Calcd: C,

54.82; H, 5.84; N, 6.91. Found: C, 54.89; H, 5.68; N, 6.83. mp: initial

decomposition ∼119 °C, complete melting 150−160 °C.

7.20 (m, 2H), 7.15−7.03 (m, 1H), 2.97 (br s, 6H), 2.56−2.43 (m,

8H), 2.19−2.10 (m, 2H), 1.69 (s, 2H); ¹³C NMR (101 MHz,

CDCl₃): δ 173.41, 151.42, 140.76, 133.89, 128.35, 128.10, 127.44,

127.30, 126.70, 124.29, 118.54, 59.70, 55.71, 53.16, 51.33, 42.24. The

free base was converted into the corresponding oxalate salt. HRMS

(C₂₈H₃₁ON₃Cl₂+ H⁺): calcd, 496.19169; found, 496.19052 (error

−2.3 ppm). CHN (C₂₈H₃₁ON₃Cl₂+ 1.5H₂C₂O₄) Calcd: C, 58.96; H,

5.43; N, 6.65. Found: C, 58.69; H, 5.33; N, 6.65. mp: 199−205 °C.

4-(7-Cyano-3,4-dihydroisoquinolin-2(1H)-yl)-N,N-dimethyl-2,2-

diphenylbutanamide (24). The reaction was performed following the

same procedure described for 23, starting from 1,2,3,4-tetrahydroi-

soquinoline-7-carbonitrile (100 mg, 0.63 mmol). The crude material

was puriﬁed by ﬂash chromatography eluting with 5% DMA, and the

desired product was obtained as colorless oil (130 mg, 40% yield). ¹H

NMR (400 MHz, CDCl₃): δ 7.46−7.18 (m, 12H), 7.10 (d, J = 7.9

4-((4-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-3-hydroxybutyl)-

amino)-N,N-dimethyl-2,2-diphenylbutanamide (29). The reaction

was performed following the same procedure described for 27,

starting from 4-amino-1-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-

2-ol (130 mg, 0.41 mmol). The desired product was puriﬁed by ﬂash

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chromatography eluting with 25% DMA and obtained as colorless oil

(80 mg, 34% yield). H NMR (400 MHz, CDCl₃): δ 7.37 (m, 8H),

1-yl)butan-2-ol (166 mg, 0.53 mmol). The desired product was

1

puriﬁed by ﬂash chromatography eluting with 25% DMA and

1

7.31−7.22 (m, 2H), 7.16−7.07 (m, 2H), 6.93 (dd, J = 6.5, 3.1 Hz,

1H), 3.82 (m, 1H), 3.04 (br s, 6H), 2.97 (br s, 1H), 2.83−2.67 (m,

4H), 2.59 (dt, J = 11.8, 6.1 Hz, 4H), 2.50−2.30 (m, 4H), 2.30−2.17

(m, 4H), 1.56−1.32 (m, 2H); ¹³C NMR (101 MHz, CDCl₃): δ

173.53, 151.32, 140.75, 140.71, 133.98, 128.38, 128.33, 128.12,

128.09, 128.04, 127.48, 127.36, 126.71, 126.68, 124.45, 118.56, 71.43,

68.18, 64.64, 62.95, 59.92, 53.04, 52.17, 51.35, 47.60, 47.30, 45.64,

43.84, 34.82, 34.04. HPLC analysis method A: Chiralpak AD-H

analytical column (4.5 mm × 250 mm5 μm particle size); mobile

phase: isocratic 20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

11.906 and 12.953 min, purity >99%, er 38:62 (absorbance at 254

nm). HPLC analysis method B: Chiralcel OD-H analytical column

(4.5 mm × 250 mm5 μm particle size); mobile phase: isocratic

20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min; injection volume: 20

μL; sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 12.011 min, purity

>99% (absorbance at 254 nm). HPLC analysis method C: Chiralcel

OZ-H analytical column (4.5 mm × 250 mm5 μm particle size);

mobile phase: isocratic 20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

12.172 min, purity >95% (absorbance at 254 nm). The free base was

converted into the corresponding oxalate salt. HRMS

(C₃₂H₄₀O₂N₄Cl₂+ H⁺): calcd, 583.26011; found, 583.26204 (error

2.3 ppm). CHN (C₃₂H₄₀O₂N₄Cl₂+ 2H₂C₂O₄+ 1.5H₂O) Calcd: C,

54.69; H, 5.99; N, 7.09. Found: C, 54.75; H, 5.71; N, 7.16. mp: salt

decomposes above 116 °C.

obtained as colorless oil (110 mg, 36% yield). H NMR (400 MHz,

CDCl₃): δ 7.43−7.32 (m, 7H), 7.32−7.22 (m, 3H), 7.13 (t, J = 7.8

Hz, 1H), 6.92 (ddd, J = 11.9, 7.8, 1.6 Hz, 2H), 3.82 (m, 1H), 3.03 (s,

6H), 2.98 (s, 2H), 2.82−2.69 (m, 5H), 2.61 (ddd, J = 11.7, 8.1, 5.3

Hz, 3H), 2.47−2.19 (m, 8H), 1.58−1.34 (m, 2H), 1.22 (t, J = 7.5 Hz,

3H); ¹³C NMR (101 MHz, CDCl₃): δ 173.55, 149.65, 143.17,

140.71, 128.67, 128.39, 128.05, 126.82, 126.72, 123.88, 117.98, 67.91,

64.64, 59.97, 53.73, 51.60, 47.48, 47.22, 45.49, 34.03, 27.45, 14.07.

HPLC analysis method A: Chiralpak AD-H analytical column (4.5

mm × 250 mm5 μm particle size); mobile phase: isocratic 20% 2-

PrOH in hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL;

sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 8.242 and 9.055

min, purity >99%, er 37:63 (absorbance at 254 nm). HPLC analysis

method B: Chiralcel OD-H analytical column (4.5 mm × 250 mm5

μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes;

ﬂow rate: 1 mL/min; injection volume: 20 μL; sample concentration:

∼1 mg/mL; multiple DAD λ absorbance signals measured in the

range of 210−280 nm, Rt 8.516 and 9.788 min, purity >99%, er 57:43

(absorbance at 254 nm). HPLC analysis method C: Chiralcel OZ-H

analytical column (4.5 mm × 250 mm5 μm particle size); mobile

phase: isocratic 20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

8.886 and 9.524 min, purity >99%. er 50:50 (absorbance at 254 nm).

The free base was converted into the corresponding oxalate salt.

HRMS (C₃₄H₄₅O₂N₄Cl + H⁺): found, 577.33059 (error 0.4 ppm).

CHN (C₃₄H₄₅O₂N₄Cl + 2H₂C₂O₄+ H₂O) Calcd: C, 58.87; H, 6.63;

N, 7.23. Found: C, 59.06; H, 6.46; N, 7.14. mp: 126−130 °C.

2-(4-Aminobutyl)-1,2,3,4-tetrahydroisoquinoline-7-carbonitrile

(33). A suspension of 1,2,3,4-tetrahydroisoquinoline-7-carbonitrile

(300 mg, 1.9 mmol), N-(4-bromobutyl)phthalimide (535 mg, 1.9

mmol), cat. KI (3.15 mg, 19 μmol), and K₂CO₃(2.6 g, 19 mmol) in

ACN (20 mL) was stirred under reﬂux overnight. The reaction

mixture was cooled down to RT and ﬁltered, the solvent was removed

under vacuum, and the residue was dissolved in EtOH (10 mL),

followed by the addition of hydrazine (0.175 mL). The solution was

stirred under reﬂux for 3 h, EtOH was evaporated, and the residue

was diluted with 20% K₂CO₃aq solution and extracted with DCM.

The organic layers were combined, dried over Na₂SO₄, ﬁltered, and

evaporated under vacuum. The crude material was used in the next

step without further puriﬁcation (300 mg, 94% yield).

4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-N,N-dimethyl-2,2-

diphenylbutanamide (30). The reaction was performed following the

same procedure described for 27, starting from 1-(2-chloro-3-

ethylphenyl)piperazine¹⁴(120 mg, 0.53 mmol). The desired product

was puriﬁed by ﬂash chromatography eluting with 15% DMA and

1

obtained as colorless oil (130 mg, 50% yield). H NMR (400 MHz,

CDCl₃): δ 7.44−7.34 (m, 8H), 7.29 (m, 2H), 7.13 (t, J = 7.8 Hz,

1H), 6.91 (ddd, J = 19.7, 7.8, 1.5 Hz, 2H), 3.07 (br s, 6H), 2.99 (br s,

2H), 2.74 (br s + q, 4H + 2H), 2.61−2.52 (m, 2H), 2.38 (m, 4H),

1.20 (t, J = 7.5 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 175.46,

173.36, 148.98, 143.16, 140.12, 128.63, 128.54, 128.49, 128.06,

126.98, 126.87, 124.19, 118.14, 59.68, 55.36, 52.67, 50.17, 40.52,

27.39, 22.19, 14.03. The free base was converted into the

corresponding oxalate salt. HRMS (C₃₀H₃₆ON₃Cl + H⁺): found,

490.26239 (error 0.9 ppm). CHN (C₃₀H₃₆ON₃Cl + 1.5H₂C₂O₄)

Calcd: C, 63.40; H, 6.29; N, 6.72. Found: C, 63.35; H, 6.46; N, 6.67.

mp: 183−186 °C.

4-((4-(7-Cyano-3,4-dihydroisoquinolin-2(1H)-yl)butyl)amino)-

N,N-dimethyl-2,2-diphenylbutanamide (34). The reaction was

performed following the same procedure described for 27, starting

from 33 (300 mg, 1.31 mmol) and 26 (368 mg, 1.31 mmol). The

desired product was puriﬁed by ﬂash chromatography eluting with

4-((4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)butyl)amino)-

N,N-dimethyl-2,2-diphenylbutanamide (31). The reaction was

performed following the same procedure described for 27, starting

from 4-(4-(2-chloro-3-ethylphenyl)piperazin-1-yl)butan-1-amine

(390 mg, 1.33 mmol). The desired product was puriﬁed by ﬂash

chromatography eluting with 15% DMA and obtained as colorless oil

1

10% DMA and obtained as colorless oil (300 mg, 46% yield). H

NMR (400 MHz, CDCl₃): δ 7.42−7.21 (m, 12H), 7.16 (d, J = 7.9

Hz, 1H), 3.57 (s, 2H), 3.47 (d, J = 0.7 Hz, 6H), 2.99−2.87 (m, 4H),

2.68 (t, J = 5.9 Hz, 2H), 2.49−2.35 (m, 7H), 1.50 (q, J = 7.7 Hz, 2H),

1.41 (q, J = 7.3 Hz, 2H); ¹³C NMR (101 MHz, CDCl₃): δ 173.55,

140.74, 140.36, 136.37, 130.37, 129.50, 129.46, 128.35, 128.04,

126.69, 119.13, 109.29, 59.89, 58.00, 55.45, 50.77, 50.10, 49.57, 47.45,

45.64, 29.41, 27.93, 24.82. The free base was converted into the

corresponding oxalate salt. HRMS (C₃₂H₃₈ON₄+ H⁺): found,

1

(90 mg, 12% yield). H NMR (400 MHz, CDCl₃): δ 7.43−7.30 (m,

8H), 7.30−7.21 (m, 2H), 7.13 (t, J = 7.8 Hz, 1H), 6.92 (ddd, J = 8.1,

6.6, 1.6 Hz, 2H), 3.03 (br s, 6H), 2.97 (br s, 2H), 2.76 (q, J = 7.5 Hz,

2H), 2.48−2.38 (m, 4H), 2.38−2.30 (m, 6H), 2.30−2.22 (m, 4H),

1.51−1.40 (m, 2H), 1.40−1.31 (m, 2H), 1.21 (t, J = 7.5 Hz, 3H); ¹³C

NMR (101 MHz, CDCl₃): δ 173.52, 149.69, 143.14, 140.85, 128.66,

128.32, 128.07, 126.83, 126.64, 123.85, 117.99, 59.90, 58.56, 53.42,

51.55, 49.76, 47.50, 45.75, 28.20, 27.46, 24.72, 23.03, 14.09. The free

base was converted into the corresponding oxalate salt. HRMS

(C₃₄H₄₅ON₄Cl + H⁺): calcd, 561.33547; found, 561.33350 (error

−4.4 ppm). CHN (C₃₄H₄₅ON₄Cl + 2H₂C₂O₄+ 1.75H₂O) Calcd: C,

59.06; H, 6.85; N, 7.25. Found: C, 59.09; H, 6.65; N, 7.17.

495.31212 (error 0.5 ppm). CHN (C₃₄H₄₅O₂N₄Cl + 2H₂C₂O₄

+

1.5H₂O) Calcd: C, 61.61; H, 6.46; N, 7.98. Found: C, 61.59; H, 6.36;

N, 7.98. mp: salt decomposes above 134 °C.

(S)-3-Chloro-N-((1-(3-cyano-3,3-diphenylpropyl)pyrrolidin-2-yl)-

methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (35). The reac-

tion was performed following the same procedure described for 12,

starting from (S)-nor-eticlopride (250 mg, 0.8 mmol) and 4-bromo-

2,2-diphenylbutanenitrile (240 mg, 0.8 mmol). The desired product

was puriﬁed by ﬂash chromatography eluting with hex/EtOAc (60/

4-((4-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-3-

hydroxybutyl)amino)-N,N-dimethyl-2,2-diphenylbutanamide (32).

The reaction was performed following the same procedure described

for 27, starting from 4-amino-1-(4-(2-chloro-3-ethylphenyl)piperazin-

1

40) and obtained as colorless oil (98 mg, 23% yield). H NMR (400

MHz, CDCl₃): δ 13.78 (s, 1H), 8.74 (s, 1H), 7.44−7.19 (m, 11H),

3.86 (s, 3H), 3.62 (ddd, J = 14.0, 6.9, 2.6 Hz, 1H), 3.29−3.12 (m,

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2H), 2.89−2.78 (m, 1H), 2.72−2.55 (m, 5H), 2.49−2.37 (m, 1H),

2.28 (q, J = 8.8 Hz, 1H), 1.90 (dq, J = 12.3, 8.1 Hz, 1H), 1.75 (t, J =

7.8 Hz, 2H), 1.67−1.51 (m, 1H), 1.32−1.15 (m, 3H); ¹³C NMR (101

MHz, CDCl₃): δ 169.44, 160.15, 152.40, 140.02, 139.45, 132.90,

130.91, 128.95, 128.01, 127.97, 126.64, 122.00, 116.06, 108.18, 62.15,

61.44, 54.04, 50.33, 50.08, 40.43, 38.52, 29.68, 28.12, 22.71, 22.55,

13.43. CHN (C₃₁H₃₄N₃O₃Cl + 0.4 hexanes) Calcd: C, 70.81; H, 7.05;

N, 7.42. Found: C, 70.49; H, 7.15; N, 7.08. HPLC analysis method A:

Chiralpak AD-H analytical column (4.5 mm × 250 mm5 μm

particle size); mobile phase: gradient from 10 to 40% 2-PrOH in

hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 7.799 min, purity >99%, ee

>99% (absorbance at 254 nm). HPLC analysis method B: Chiralpak

AD-H analytical column (4.5 mm × 250 mm5 μm particle size);

mobile phase: isocratic 10% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

9.043 min, purity >99%, ee >99% (absorbance at 254 nm). HRMS

(C₃₁H₃₄N₃O₃Cl + H⁺): calcd, 532.23615; found, 532.23691 (error 0.4

ppm).

3-Chloro-N-((1-(4-(1,3-dioxoisoindolin-2-yl)butyl)pyrrolidin-2-

yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide (39). A solu-

tion of 38 (940 mg, 2.34 mmol) and TFA (2.5 mL) in DCM (15 mL)

was stirred at RT for 3 h. The reaction was quenched with NaHCO₃

(sat. aq solution) and extracted with DCM/2-PrOH (3:1). The

organic layers were combined, dried over Na₂SO₄, ﬁltered, and

evaporated under vacuum. The crude material was dissolved in DCM

(15 mL), followed by dropwise addition of a solution of 3-chloro-5-

ethyl-6-hydroxy-2-methoxybenzoic acid⁵⁴(647 mg, 2.81 mmol) and

HCTU (1.16 g, 2.81 mmol) in DCM (15 mL). The reaction mixture

was stirred at RT for 48 h, the solvent was removed under vacuum,

and the crude material was puriﬁed by ﬂash chromatography eluting

with 5% DMA. The desired product was obtained as brown oil (310

1

mg, 26% yield). H NMR (400 MHz, CDCl₃): δ 13.12 (s, 1H), 9.11

(s, 1H), 7.80 (dd, J = 5.4, 3.0 Hz, 2H), 7.69 (dd, J = 5.5, 3.0 Hz, 2H),

7.26−7.15 (m, 1H), 3.96−3.78 (m, 5H), 3.72 (m, J = 14.0 Hz, 4H),

3.57 (br s, 1H), 3.48 (br s, 1H), 3.26 (br s, 2H), 2.66−2.51 (m, 3H),

2.22 (dt, J = 15.0, 7.6 Hz, 1H), 1.99 (tt, J = 13.4, 6.9 Hz, 2H), 1.80−

1.60 (m, 3H), 1.18 (dt, J = 10.7, 7.5 Hz, 3H).

3-Chloro-N-((1-(4-((4-(dimethylamino)-4-oxo-3,3-

diphenylbutyl)amino)butyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hy-

droxy-2-methoxybenzamide (40). Hydrazine (0.2 mL, 50−60% wt

in H₂O) was added to a solution of 39 (310 mg, 0.6 mmol) in EtOH

(20 mL), and the solution was stirred at reﬂux for 3 h. The solvent

was removed under vacuum, and the residue was diluted with 20%

K₂CO₃aq solution and extracted with DCM. The organic layers were

combined, dried over Na₂SO₄, ﬁltered, and evaporated under vacuum.

The obtained crude material was dissolved in DCE (10 mL) and

added to a solution of 26 (169 mg, 0.6 mmol) and catalytic AcOH in

DCE (10 mL). The mixture was stirred for 10 min at RT, and STAB

(190 mg, 0.9 mmol) was added portionwise. The reaction mixture

was stirred for additional 12 h, the solvent was removed under

vacuum, and the residue was puriﬁed by ﬂash chromatography eluting

with 10% DMA. The desired product was obtained as colorless oil

(S)-N-((1-(4-Aminobutyl)pyrrolidin-2-yl)methyl)-3-chloro-5-

ethyl-6-hydroxy-2-methoxybenzamide (36). The reaction was

performed following the same procedure described for 33, starting

from (S)-3-chloro-5-ethyl-6-hydroxy-2-methoxy-N-(pyrrolidin-2-

ylmethyl)benzamide [(S)-nor-eticlopride 250 mg, 0.8 mmol]. The

crude material was used in the next step without further puriﬁcation

(110 mg, 36% yield).

(S)-3-Chloro-N-((1-(4-((3-cyano-3,3-diphenylpropyl)amino)-

butyl)pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenza-

mide (37). The reaction was performed following the same procedure

described for 12, starting from 36 (110 mg, 0.29 mmol) and 4-bromo-

2,2-diphenylbutanenitrile (78 mg, 0.26 mmol). The desired product

was puriﬁed by ﬂash chromatography eluting with 10% DMA and

1

(80.5 mg, 21% yield). H NMR (400 MHz, CDCl₃): δ 8.79 (br s,

obtained as colorless oil (15 mg, 10% yield). H NMR (400 MHz,

1H), 7.40−7.22 (m, 11H), 3.82 (s, 3H), 3.72 (dt, J = 12.1, 4.0 Hz,

2H), 3.28−3.19 (m, 1H), 3.14 (dt, J = 9.5, 4.5 Hz, 1H), 2.95 (br s,

3H), 2.74−2.52 (m, 3H), 2.47−2.35 (m, 4H), 2.25 (d, J = 14.1 Hz,

4H), 2.12 (q, J = 8.6 Hz, 3H), 1.93−1.80 (m, 1H), 1.72 (d, J = 7.8

Hz, 2H), 1.69−1.52 (m, 1H), 1.40 (dt, J = 16.4, 8.3 Hz, 4H), 1.20

(dt, J = 27.3, 7.3 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 173.62,

169.49, 160.11, 152.51, 140.71, 140.65, 132.82, 130.67, 128.37,

128.17, 128.16, 128.03, 126.72, 116.01, 108.17, 62.27, 61.41, 60.04,

54.01, 53.80, 49.38, 47.15, 45.23, 40.32, 28.10, 27.73, 26.57, 22.58,

22.51, 13.41. HPLC analysis method A: Chiralpak AD-H analytical

column (4.5 mm × 250 mm5 μm particle size); mobile phase:

isocratic 30% 2-PrOH in hexanes; ﬂow rate: 1 mL/min; injection

volume: 20 μL; sample concentration: ∼1 mg/mL; multiple DAD λ

absorbance signals measured in the range of 210−280 nm, Rt 9.538

and 10.664 min, purity >99%, er 46:54 (absorbance at 254 nm).

HPLC analysis method B: Chiralpak AD-H analytical column (4.5

mm × 250 mm5 μm particle size); mobile phase: isocratic 15% 2-

PrOH in hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL;

sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 22.250 and 25.814

min, purity >99%, er 50:50 (absorbance at 254 nm). HRMS

(C₃₇H₄₉N₄O₄Cl + 2H)²⁺: found, 325.18021; (C₃₇H₄₉N₄O₄Cl +

H⁺): calcd, 649.35151; found, 649.35221 (error 1.0 ppm).

CDCl₃+ CD₃OD): δ 7.39−7.23 (m, 6H), 7.18−7.06 (m, 5H), 3.83

(s, 3H), 3.82−3.50 (m, 6H), 3.36−3.26 (m, 6H), 2.82 (p, J = 6.9 Hz,

2H), 2.53 (q, J = 7.5 Hz, 2H), 1.74 (m, 5H), 1.11 (ddd, J = 8.2, 7.1,

0.8 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 174.69, 163.12, 156.56,

143.10, 142.99, 137.03, 134.45, 132.70, 132.67, 132.08, 132.06,

131.89, 131.85, 120.19, 112.47, 67.26, 66.39, 64.97, 57.87, 54.60,

49.82, 43.72, 42.16, 31.41, 27.77, 27.47, 26.07, 25.77, 16.57. HPLC

analysis method A: Chiralpak AD-H analytical column (4.5 mm ×

250 mm5 μm particle size); mobile phase: gradient from 10 to 40%

2-PrOH in hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL;

sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 22.362 min, purity

>95%, ee >99% (absorbance at 254 nm). HPLC analysis method B:

Chiralpak AD-H analytical column (4.5 mm × 250 mm5 μm

particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; ﬂow

rate: 1 mL/min; injection volume: 20 μL; sample concentration: ∼1

mg/mL; multiple DAD λ absorbance signals measured in the range of

210−280 nm, Rt 14.389 min, purity >99%, ee >99% (absorbance at

254 nm). HRMS (C₃₅H₄₃N₄O₃Cl + 2H)²⁺: found, 302.15901;

(C₃₅H₄₃N₄O₃Cl + H⁺): calcd, 603.30965; found, 603.31020 (error

0.4 ppm).

tert-Butyl ((1-(4-(1,3-Dioxoisoindolin-2-yl)butyl)pyrrolidin-2-yl)-

methyl)carbamate (38). A suspension of tert-butyl (pyrrolidin-2-

ylmethyl)carbamate (500 mg, 2.5 mmol), N-(4-bromobutyl)-

phthalimide (775 mg, 2.75 mmol), cat. KI (4.15 mg, 25 μmol), and

K₂CO₃(3.45 g, 25 mmol) in ACN (20 mL) was stirred under reﬂux

overnight. The reaction mixture was cooled down to RT and ﬁltered,

the solvent was removed under vacuum, and the desired product,

presenting both N-Boc and N-phthalimide protecting groups, was

puriﬁed by ﬂash chromatography eluting with 10% DMA and

3,3-Diphenylpyrrolidine (41). A suspension of LAH (0.56 g, 14.8

mmol) in THF (50 mL) was cooled to 0 °C, and a solution of 4-

bromo-2,2-diphenylbutanenitrile (1.5 g, 5 mmol) in THF (20 mL)

was added dropwise. The mixture was stirred at RT for 15 h,

quenched with MeOH (5 mL) and sat. aq NaHCO₃solution (5 mL),

ﬁltered over Celite, and concentrated under vacuum. The residue was

suspended in DCM and washed with sat. Na₂CO₃solution. The

organic layer was dried over Na₂SO₄, ﬁltered, and evaporated under

vacuum. The crude material was puriﬁed by ﬂash chromatography

eluting with 10% DMA. The desired product was obtained as yellow

1

obtained as yellow oil (940 mg, 85% yield). H NMR (400 MHz,

CDCl₃): δ 7.84 (dd, J = 5.5, 3.0 Hz, 2H), 7.71 (dd, J = 5.5, 3.1 Hz,

2H), 5.00−4.95 (m, 1H), 3.77−3.64 (m, 2H), 3.27 (d, J = 10.9 Hz,

1H), 3.15−2.99 (m, 2H), 2.73 (dt, J = 11.9, 8.1 Hz, 1H), 2.47 (br s,

1H), 2.23−2.02 (m, 2H), 1.89−1.43 (m, 8H), 1.43 (s, 9H).

1

oil (0.450 g, 40% yield). H NMR (400 MHz, CDCl₃): δ 7.34−7.22

(m, 8H), 7.22−7.13 (m, 2H), 3.52 (s, 2H), 3.13 (t, J = 7.2 Hz, 2H),

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2.80−2.70 (br s, 1H), 2.52 (t, J = 7.2 Hz, 2H). GC/MS (EI), Rt 9.987

min; 223.2 (M⁺).

2.68−2.52 (m, 4H), 2.14−1.97 (m, 1H), 1.92−1.66 (m, 4H), 1.28−

1.14 (m, 3H); ¹³C NMR (101 MHz, CDCl₃) rotamer conformations

observed: δ 169.75, 160.07, 160.04, 152.47, 152.46, 144.94, 144.88,

144.79, 133.07, 130.79, 130.77, 128.57, 128.53, 128.51, 126.67,

126.62, 126.59, 126.58, 126.52, 116.16, 116.11, 107.98, 61.57, 61.49,

56.60, 56.14, 56.12, 55.10, 54.87, 54.68, 54.45, 53.40, 52.22, 44.57,

44.39, 40.88, 40.46, 37.52, 35.57, 29.68, 28.38, 28.26, 23.05, 22.86,

22.51, 13.39. HPLC analysis method A: Chiralpak AD-H analytical

column (4.5 mm × 250 mm5 μm particle size); mobile phase:

isocratic 10% 2-PrOH in hexanes; ﬂow rate: 1 mL/min; injection

volume: 20 μL; sample concentration: ∼1 mg/mL; multiple DAD λ

absorbance signals measured in the range of 210−280 nm, Rt 20.539

and 23.859 min, purity >99%, er 51:49 (absorbance at 254 nm).

HRMS (C₃₃H₃₈N₃O₄Cl + H⁺): calcd, 576.26236; found, 576.26297

(error 1.0 ppm).

1-Methyl-3,3-diphenylpyrrolidine (42). Methyl chloroformate (85

mg, 69 μL, 0.9 mmol) was added dropwise to a solution of 41 (100

mg, 0.45 mmol) in THF (10 mL), followed by excess of DIPEA (5

equiv). The mixture was stirred at RT for 1 h, the solvent was

evaporated under vacuum, and the residue was dissolved in THF (10

mL). This solution was added dropwise to a suspension of LAH (17

mg, 0.45 mmol) in THF (10 mL), at 0 °C. The mixture was warmed

to RT, quenched with MeOH/2 N aq NaOH (1:1 ratio, 2 mL), and

ﬁltered over Celite, and the solvents were evaporated under vacuum.

The crude material was puriﬁed by ﬂash chromatography eluting with

10% DMA to aﬀord the desired product as colorless oil (60 mg, 56%

1

yield). H NMR (400 MHz, CDCl₃): δ 7.26 (d, J = 4.6 Hz, 8H),

7.20−7.11 (m, 2H), 3.22 (s, 2H), 2.82 (td, J = 7.0, 0.6 Hz, 2H), 2.60

(dd, J = 7.7, 6.7 Hz, 2H), 2.42 (s, 3H). GC/MS (EI), Rt 9.650 min;

237.2 (M⁺), purity >99%.

3-Chloro-N-(((2S,4R)-4-(4-((4-(dimethylamino)-4-oxo-3,3-

diphenylbutyl)amino)butoxy)pyrrolidin-2-yl)methyl)-5-ethyl-6-hy-

droxy-2-methoxybenzamide (48). A solution of 47 (400 mg, 0.8

mmol), 26 (225 mg, 0.8 mmol), and cat. AcOH (0.05 equiv) in DCE

(20 mL) was stirred at RT for 1 h, followed by portionwise addition

of STAB (339 mg, 1.6 mmol). The mixture was stirred at RT for 3 h

and basiﬁed with 10% NH₄OH in MeOH, the solvent was evaporated

under vacuum, and the residue was puriﬁed by ﬂash chromatography

eluting with 10% DMA. The obtained intermediate was dissolved in

DCM (20 mL) and TFA (10 mL), and the solution was stirred at RT

overnight. The excess of TFA was removed under vacuum, and the

residue resuspended in aq NH₄OH (pH 9) and extracted with DCM/

2-PrOH (3:1). The organic layers were combined, dried over Na₂SO₄,

ﬁltered, and evaporated to aﬀord the crude material, which was

puriﬁed by ﬂash chromatography eluting with 25% DMA. The desired

1-(3,3-Diphenylpyrrolidin-1-yl)propan-1-one (43). A solution of

41 (70 mg, 0.31 mmol), propionyl chloride (58 mg, 0.63 mmol), and

DIPEA (5 equiv) in DCM (10 mL) was stirred under reﬂux

overnight. The solvent was removed under vacuum, and the residue

was puriﬁed by ﬂash chromatography eluting with hex/EtOAc (70/

30). The desired product was obtained as colorless oil (25 mg, 29%

yield). ¹H NMR (400 MHz, CDCl₃) rotamer conformations observed

(60:40; rotamer A/rotamer B): δ 7.34−7.14 (m, 10H), 4.16 (s, 2H,

rotamer A), 4.04 (s, 2H, rotamer B), 3.53 (t, J = 6.9 Hz, 2H, rotamer

B), 3.42 (t, J = 6.7 Hz, 2H, rotamer A), 2.63 (t, J = 6.7 Hz, 2H,

rotamer A), 2.52 (t, J = 6.9 Hz, 2H, rotamer B), 2.41 (q, J = 7.5 Hz,

2H, rotamer B), 2.26 (q, J = 7.5 Hz, 2H, rotamer A), 1.21 (t, J = 7.5

Hz, 3H, rotamer B), 1.16 (t, J = 7.5 Hz, 3H, rotamer A). GC/MS

(EI), Rt 12.154 min; 279.1 (M⁺), purity >99%.

1

product was obtained as colorless oil (45 mg, 8.5% yield). H NMR

(400 MHz, CDCl₃): δ 8.92 (s, 1H), 7.43−7.21 (m, 11H), 3.97 (t, J =

4.9 Hz, 1H), 3.89 (s, 3H), 3.57 (dq, J = 9.6, 4.7 Hz, 3H), 3.36 (dt, J =

5.8, 2.8 Hz, 3H), 3.22 (tt, J = 8.9, 4.5 Hz, 2H), 3.02−2.90 (m, 5H),

2.69−2.55 (m, 4H), 2.45 (dd, J = 21.5, 5.7 Hz, 4H), 2.30 (br s, 2H),

2.00 (dd, J = 13.7, 6.9 Hz, 1H), 1.70−1.50 (m, 5H), 1.31−1.14 (m,

3H); ¹³C NMR (101 MHz, CDCl₃): δ 174.57, 169.21, 160.06,

152.48, 139.97, 132.89, 130.72, 128.78, 127.96, 127.93, 127.26,

116.16, 108.17, 80.67, 68.17, 61.57, 56.10, 51.78, 43.34, 36.07, 27.16,

22.50, 21.93, 13.41. HPLC analysis method: Chiralpak AD-H

analytical column (4.5 mm × 250 mm5 μm particle size); mobile

phase: isocratic 20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min;

injection volume: 20 μL; sample concentration: ∼1 mg/mL; multiple

DAD λ absorbance signals measured in the range of 210−280 nm, Rt

18.518 min, purity >95%, ee >95% (absorbance at 210 nm). The free

base was converted into the corresponding oxalate salt. HRMS

(C₃₇H₄₉N₄O₅Cl + 2H)²⁺: calcd, 333.17685; found, 333.17656 (error

0.9 ppm); (C₃₇H₄₉N₄O₅Cl + H)⁺: calcd, 665.34642; found,

2-Chloro-1-(3,3-diphenylpyrrolidin-1-yl)ethan-1-one (44). 2-

Chloroacetyl chloride (126 mg, 1.12 mmol) was added dropwise to

a solution of 41 (250 mg, 1.12 mmol) in THF (10 mL) at 0 °C,

followed by dropwise addition of DIPEA (0.3 mL, 1.68 mmol). The

mixture was allowed to warm to RT and stirred for 1 h. The solvent

was removed under vacuum, and the residue was used in the following

step without further puriﬁcation. GC/MS (EI), Rt 12.740 min; 299.1

(M⁺).

tert-Butyl ((1-(2-(3,3-Diphenylpyrrolidin-1-yl)-2-oxoethyl)-

pyrrolidin-2-yl)methyl)carbamate (45). A mixture of 44 (290 mg,

0.97 mmol), tert-butyl (pyrrolidin-2-ylmethyl)carbamate (194 mg,

0.97 mmol), KI (161 mg, 0.97 mmol), and K₂CO₃(1.34 g, 9.67

mmol) in ACN (25 mL) was stirred under reﬂux for 3 h. The mixture

was ﬁltered, the solvent was evaporated under vacuum, and the

residue was puriﬁed by ﬂash chromatography eluting with 10% DMA.

The desired product was obtained as yellow oil (330 mg, 74% yield).

¹H NMR (400 MHz, CDCl₃) rotamer conformations observed: δ

665.34552 (error 1.4 ppm). CHN (C₃₇H₄₉N₄O₅Cl + 2H₂C₂O₄

+

7.34−7.15 (m, 10H), 5.30 (m, 1H), 3.69−3.38 (m, 4H), 3.29−2.93

(m, 3H), 2.71−2.17 (m, 4H), 1.90 (tt, J = 19.2, 8.6 Hz, 2H), 1.73 (m,

J = 17.3, 8.5 Hz, 4H), 1.50−1.38 (m, 9H).

1.5H₂O) Calcd: C, 56.45; H, 6.47; N, 6.42. Found: C, 56.25; H, 6.24;

N, 6.40. mp: salt decomposes above 90 °C.

3,3-Bis(3-methoxyphenyl)acrylaldehyde (49). A solution of bis(3-

methoxyphenyl)methanone (1.0 g, 4.13 mmol) and TiCl₄(1.0 M

solution in DCM, 16.5 mL, 16.5 mmol) in DCM was cooled to 0 °C.

TEA (2.3 mL, 16.5 mmol) was added dropwise, and the mixture was

warmed to RT and stirred overnight.⁵⁵The reaction was quenched

with sat. NH₄Cl aq solution and stirred for 30 min. The mixture was

extracted with DCM, and the organic layers were combined, dried

over Na₂SO₄, ﬁltered, and concentrated under vacuum. The crude

material was puriﬁed by ﬂash chromatography eluting with hex/

EtOAc (95/5). The desired product was obtained as colorless oil

(645 mg, 58% yield). ¹H NMR (400 MHz, CDCl₃): δ 9.60−9.53 (m,

1H), 7.42−7.25 (m, 2H), 7.07−6.77 (m, 6H), 6.64−6.56 (m, 1H),

3.89−3.74 (m, 6H). GC/MS (EI), Rt 11.647 min; 268.1 (M⁺).

N-(3,3-Bis(3-methoxyphenyl)allyl)-4-(4-(2,3-dichlorophenyl)-

piperazin-1-yl)butan-1-amine (50). A solution of 49 (302 mg, 1.12

mmol), 4-(4-(2,3-dichlorophenyl)piperazin-1-yl)butan-1-amine (340

mg, 1.12 mmol), and cat. AcOH (0.1 equiv) in DCE (20 mL) was

stirred for 1 h at RT, followed by portionwise addition of STAB (715

mg, 3.37 mmol). The reaction mixture was stirred for 3 h, the solvent

3-Chloro-N-((1-(2-(3,3-diphenylpyrrolidin-1-yl)-2-oxoethyl)-

pyrrolidin-2-yl)methyl)-5-ethyl-6-hydroxy-2-methoxybenzamide

(46). TFA (0.3 mL) was added to a solution of 45 (330 mg, 0.71

mmol) in DCM (10 mL). The mixture was stirred at RT for 2 h,

basiﬁed with NH₄OH (28% aq solution), and extracted with DCM.

The organic layers were combined, dried over Na₂SO₄, ﬁltered, and

evaporated under vacuum to aﬀord the crude material, which was

ﬁltered over a silica pad, eluting and washing with 25% DMA to

isolate the desired primary amine intermediate. The amine was

dissolved in DCM (10 mL) and added dropwise to a solution of 3-

chloro-5-ethyl-6-hydroxy-2-methoxybenzoic acid (70 mg, 0.3 mmol),

HCTU (0.2 g, 0.33 mmol), and DIPEA (1.5 equiv) in DCM (10 mL).

The mixture was stirred at RT for 3 h, the solvent was removed under

vacuum, and the residue was puriﬁed by ﬂash chromatography eluting

with 5% DMA. The desired product was obtained as colorless oil (26

mg, 15% yield). ¹H NMR (400 MHz, CDCl₃) rotamer conformations

observed: δ 13.68 (s, 1H), 8.82 (s, 1H), 7.31−7.12 (m, 11H), 4.23 (t,

J = 11.0 Hz, 1H), 4.02 (dd, J = 26.1, 11.5 Hz, 1H), 3.85 (ds, J = 17.4

Hz, 3H), 3.79−3.68 (m, 1H), 3.62−3.21 (m, 6H), 2.96 (br s, 1H),

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was evaporated under vacuum, and the residue was puriﬁed by ﬂash

ution of 53 (200 mg, 0.42 mmol) in 33% HBr (solution in AcOH, 5

mL) and DCM (10 mL) was stirred under reﬂux for 24 h. The solvent

was removed under vacuum, and the residue was basiﬁed with 28−

30% v/v aq NH₄OH and extracted with DCM/2-PrOH (3:1). The

crude material was puriﬁed by ﬂash chromatography eluting with 10%

DMA. 54 eluted ﬁrst as yellow oil (60 mg, 31% yield): ¹H NMR (400

MHz, CDCl₃): δ 7.20−7.07 (m, 3H), 6.94 (dd, J = 7.6, 1.5 Hz, 1H),

6.91−6.66 (m, 4H), 6.64 (d, J = 1.0 Hz, 1H), 6.64−6.47 (m, 2H),

3.83 (t, J = 7.6 Hz, 1H), 3.73 (s, 3H), 3.01 (br s, 4H), 2.76 (br s + q, J

= 7.5 Hz, 6H), 2.49−2.36 (m, 2H), 2.35−2.15 (m, 2H), 1.36−1.17 (t,

J = 7.5 Hz, 3H). HRMS (C₂₈H₃₃ClN₂O₂+ H)⁺: calcd, 465.23033;

found, 465.22945 (error 1.9 ppm). 55 eluted second as a yellow oil

(25 mg, 13% yield): ¹H NMR (400 MHz, CDCl₃): δ 7.09 (td, J = 7.8,

2.4 Hz, 3H), 6.93 (dd, J = 7.6, 1.5 Hz, 1H), 6.85−6.69 (m, 3H), 6.63

(ddd, J = 8.1, 2.5, 0.9 Hz, 2H), 6.55 (t, J = 1.9 Hz, 2H), 3.75 (q, J =

7.9 Hz, 1H), 2.97 (br s, 4H), 2.75−2.60 (br s + q, J = 7.6 Hz, 6H),

2.43 (br s, 2H), 2.26−2.15 (m, 2H), 1.30−1.16 (t, J = 7.5 Hz, 3H).

HRMS (C₂₇H₃₁ClN₂O₂+ H)⁺: calcd, 451.21468; found, 451.21429

(error 0.9 ppm).

chromatography eluting with 5% DMA. The desired product was

1

obtained as colorless oil (550 mg, 88% yield). H NMR (400 MHz,

CDCl₃): δ 7.26 (m, 1H), 7.16 (q, J = 7.6 Hz, 2H), 6.90−6.79 (m,

3H), 6.79−6.67 (m, 4H), 6.67−6.60 (m, 1H), 6.20−6.11 (m, 1H),

3.74 (d, J = 5.4 Hz, 6H), 3.38−3.33 (m, 4H), 3.08 (br s, 4H), 2.74

(m, 2H), 2.58 (br s, 2H), 2.42 (m, 2H), 1.47 (m, 2H), 1.28 (m, 2H);

¹³C NMR (101 MHz, CDCl₃): δ 175.73, 159.49, 159.43, 150.75,

142.64, 140.20, 134.01, 129.39, 129.11, 127.48, 127.47, 124.83,

122.01, 119.88, 118.71, 115.22, 113.32, 112.98, 112.85, 57.48, 55.20,

55.16, 53.39, 52.57, 52.48, 51.42, 50.27, 30.90, 23.33, 23.10, 21.27.

HPLC analysis method: Chiralpak AD-H analytical column (4.5 mm

× 250 mm5 μm particle size); mobile phase: isocratic 20% 2-PrOH

in hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 6.538 min, purity >95%

(absorbance at 254 nm). HRMS (C₃₁H₃₇N₃O₂Cl₂+ H)⁺: calcd,

554.23356; found, 554.23390.

3,3′-(3-((4-(4-Phenylpiperazin-1-yl)butyl)amino)propane-1,1-

diyl)diphenol (51). A suspension of 50 (250 mg, 0.45 mmol) and Pd/

C (20% wt wet, 0.05 equiv) in EtOH (10 mL) was shaken in a Parr

apparatus under 50 psi pressure of hydrogen gas for 12 h. The mixture

was ﬁltered through a pad of Celite, the solvent was evaporated under

vacuum, and the residue was puriﬁed trough a pad of silica eluting

with 5% DMA. This intermediate was dissolved in 33% HBr (solution

in AcOH, 2 mL) and stirred under reﬂux for 48 h. The solvent was

removed under vacuum, and the residue was basiﬁed with 10% v/v

NH₄OH solution in methanol. The crude material was puriﬁed by

ﬂash chromatography eluting with 25% DMA. The desired product

was obtained as yellow oil (6.8 mg, 29% yield). ¹H NMR (400 MHz,

CDCl₃): δ 7.28−7.19 (m, 1H), 7.09 (t, J = 7.8 Hz, 2H), 6.90−6.80

(m, 2H), 6.71−6.60 (m, 4H), 6.55 (s, 2H), 5.44 (br s, 2H), 3.81 (t, J

= 7.9 Hz, 1H), 3.09 (t, J = 5.0 Hz, 4H), 2.61 (s, 2H), 2.49 (dt, J =

11.8, 5.4 Hz, 6H), 2.25 (p, J = 7.5 Hz, 4H), 1.30−1.16 (m, 4H); ¹³C

NMR (101 MHz, CDCl₃): δ 172.49, 157.37, 150.94, 145.43, 129.75,

129.07, 119.90, 119.43, 116.11, 114.54, 114.47, 65.83, 58.11, 52.97,

34.26, 26.57, 25.34, 24.12, 22.59, 15.25. HPLC analysis method:

Chiralpak AD-H analytical column (4.5 mm × 250 mm5 μm

particle size); mobile phase: isocratic 20% 2-PrOH in hexanes + 0.1%

DEA; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 38.741 min, purity >95%

(absorbance at 254 nm). HRMS (C₂₉H₃₇N₃O₂+ H)⁺: found,

460.29628.

trans-Ethyl (Z)-2-((1S,5S)-2-(4-(1H-Indole-2-carboxamido)butyl)-

5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)acetate

(57). A solution of 56 (20 mg, 66 μmol), N-(4-oxobutyl)-1H-indole-

2-carboxamide⁵⁷(18 mg, 80 μmol), and catalytic AcOH (0.01 equiv)

in DCE (10 mL) was stirred at RT for 1 h, followed by portionwise

addition of STAB (30 mg, 66 μmol). The reaction mixture was stirred

for additional 30 min and basiﬁed with 10% NH₄OH solution in

MeOH (10 mL), the solvent was evaporated under vacuum, and the

residue was puriﬁed by ﬂash chromatography eluting with 20% DMA.

The desired product was obtained as colorless oil (30 mg, 88% yield).

¹H NMR (400 MHz, CDCl₃): δ 9.30 (s, 1H), 7.61 (dd, J = 8.0, 1.0

Hz, 1H), 7.46−7.38 (m, 1H), 7.32−7.08 (m, 3H), 6.97−6.78 (m,

4H), 6.78−6.70 (m, 1H), 5.30 (s, 1H), 5.16 (d, J = 17.9 Hz, 2H),

4.13−3.92 (m, 2H), 3.63−3.44 (m, 2H), 3.20 (ddd, J = 13.2, 8.9, 4.8

Hz, 1H), 2.73 (ddt, J = 17.8, 12.3, 5.8 Hz, 3H), 2.46 (ddd, J = 14.4,

8.7, 6.0 Hz, 1H), 2.27−2.00 (m, 5H), 1.90−1.50 (m, 6H), 1.14 (t, J =

7.1 Hz, 3H); ¹³C NMR (101 MHz, CDCl₃): δ 167.91, 166.96, 161.79,

155.61, 148.65, 136.14, 131.04, 129.25, 127.68, 124.33, 121.83,

120.55, 119.46, 115.00, 114.38, 113.60, 111.89, 102.22, 59.92, 55.45,

53.31, 48.76, 45.69, 40.19, 39.34, 38.73, 30.66, 27.22, 24.50, 20.48,

14.13. HPLC analysis method A: Chiralpak AD-H analytical column

(4.5 mm × 250 mm5 μm particle size); mobile phase: isocratic

20% 2-PrOH in hexanes; ﬂow rate: 1 mL/min; injection volume: 20

μL; sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 30.677 min, purity

>99%, ee >99% (absorbance at 254 nm). HPLC analysis method B:

Chiralpak AD-H analytical column (4.5 mm × 250 mm5 μm

particle size); mobile phase: isocratic 30% 2-PrOH in hexanes; ﬂow

rate: 1 mL/min; injection volume: 20 μL; sample concentration: ∼1

mg/mL; multiple DAD λ absorbance signals measured in the range of

210−280 nm, Rt 13.495 min, purity >99%, ee >99% (absorbance at

254 nm). HRMS (C₃₁H₃₇O₄N₃+ H⁺): calcd, 516.28568; found,

516.28475 (error −1.8 ppm).

trans-Ethyl (Z)-2-((2S,6R)-6-(((2-(2-(1H-Indole-2-carboxamido)-

ethyl)cyclopropyl)methyl)(ethyl)amino)-2-(3-hydroxyphenyl)-2-

methylcyclohexylidene)acetate (58). The reaction was performed

following the same procure described for 57, starting from 56 (20 mg,

66 μmol) and N-(2-(2-formylcyclopropyl)ethyl)-1H-indole-2-carbox-

amide⁴⁵(17 mg, 66 μmol). The desired product was obtained as

colorless oil (23 mg, 65% yield). ¹H NMR (400 MHz, CDCl₃)

mixture of diastereomers observed: δ 9.33 (s, 1H, dr 60:40), 7.62 (dd,

J = 8.1, 3.8 Hz, 1H), 7.47−7.39 (m, 1H), 7.34−7.25 (m, 1H), 7.23−

7.08 (m, 2H), 6.93 (d, J = 10.1 Hz, 1H), 6.89−6.69 (m, 3H), 6.60 (s,

1H), 5.25−5.10 (m, 2H), 4.13−3.95 (m, 2H), 3.72−3.49 (m, 2H),

3.16 (d, J = 12.3 Hz, 1H), 2.96−2.74 (m, 2H), 2.46 (dd, J = 14.2, 6.9

Hz, 1H), 2.37−2.27 (m, 1H), 2.25−1.98 (m, 7H), 1.64−1.49 (m,

4H), 1.38−1.24 (m, 1H), 1.15 (dt, J = 8.2, 7.1 Hz, 3H), 0.89 (d, J =

62.8 Hz, 1H, dr 60:40), 0.69 (d, J = 52.6 Hz, 1H, dr 60:40), 0.32 (dt,

J = 17.8, 5.9 Hz, 1H); ¹³C NMR (101 MHz, CDCl₃): δ 166.85,

166.83, 161.95, 161.69, 156.18, 155.88, 136.29, 136.20, 130.95,

130.50, 129.23, 129.13, 127.69, 127.56, 124.62, 124.40, 121.90,

1-(3,3-Bis(3-methoxyphenyl)allyl)-4-(2-chloro-3-ethylphenyl)-

piperazine (52). The reaction was performed following the same

procure described for 50, starting from 1-(2-chloro-3-ethylphenyl)-

piperazine¹⁴(180 mg, 0.78 mmol). The desired product was obtained

as colorless oil (330 mg, 88% yield). ¹H NMR (400 MHz, CDCl₃): δ

7.24−7.11 (m, 3H), 7.00−6.66 (m, 8H), 6.31 (t, J = 7.0 Hz, 1H),

3.79 (d, J = 14.0 Hz, 6H), 3.34 (d, J = 6.9 Hz, 2H), 3.14 (br s, 4H),

3.00−2.76 (br s, 4H), 2.76 (q, J = 7.5 Hz, 2H), 1.21 (t, J = 7.5 Hz,

3H).

1-(3,3-Bis(3-methoxyphenyl)propyl)-4-(2-chloro-3-ethylphenyl)-

piperazine (53). A suspension of 52 (330 mg, 0.69 mmol) and Pd/C

(20% wt wet, 0.05 equiv) in EtOAc/EtOH (10:5 mL) was shaken in a

Parr apparatus under 30 psi pressure of H₂for 3 h. The mixture was

ﬁltered through a pad of Celite, the solvent was evaporated under

vacuum, and the desired product was obtained as colorless oil (250

1

mg, 75% yield). H NMR (400 MHz, CDCl₃): δ 7.18 (dt, J = 20.9,

7.8 Hz, 3H), 6.94 (ddd, J = 11.1, 7.8, 1.5 Hz, 2H), 6.90−6.67 (m,

6H), 3.96−3.86 (m, 1H), 3.82−3.67 (br s, 6H), 3.10 (br s, 4H), 2.76

(br s + q, J = 7.5 Hz, 6H), 2.50 (dd, J = 9.9, 5.8 Hz, 2H), 2.06 (m,

2H), 1.29−1.15 (t, J = 7.5 Hz, 3H). HRMS (C₂₉H₃₅ClN₂O₂+ H)⁺:

calcd, 479.24598; found, 479.24505 (error 1.9 ppm). The free base

(50 mg) was converted into the corresponding oxalate salt. mp: salt

decomposes above 100 °C.

3-(3-(4-(2-Chloro-3-ethylphenyl)piperazin-1-yl)-1-(3-

methoxyphenyl)propyl)phenol (54) and 3,3′-(3-(4-(2-Chloro-3-

ethylphenyl)piperazin-1-yl)propane-1,1-diyl)diphenol (55). A sol-

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121.85, 120.74, 120.58, 115.31, 115.13, 113.95, 113.82, 112.01,

111.90, 102.62, 60.90, 60.56, 59.87, 59.81, 55.32, 54.77, 50.85, 48.21,

48.08, 45.64, 45.60, 40.24, 40.17, 40.09, 39.74, 33.47, 33.42, 30.86,

20.19, 18.92, 17.04, 16.55, 15.72, 14.17, 14.15, 10.91, 9.23. HPLC

analysis method: Chiralpak AD-H analytical column (4.5 mm × 250

mm5 μm particle size); mobile phase: isocratic 20% 2-PrOH in

hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 22.572 and 27.023 min,

purity >99%, dr 45:55 (absorbance at 254 nm). HRMS (C₃₃H₃₉O₄N₃

+ H⁺): calcd, 542.30133; found, 542.29988 (error −3.6 ppm). The

two diastereoisomers 58a and 58b were resolved and separated by

preparative chiral HPLC (Chiralpak AD-H 21 mm × 250 mm × 5

μm): mobile phase: gradient from 10 to 40% 2-PrOH in hexanes;

temperature: 25 °C; ﬂow rate: 15−18 mL/min; injection volume: 3

mL (∼15−20 mg/mL sample concentration); detection at λ 230 and

254 nm with the support of ELS detector. 58a eluted ﬁrst; HPLC

analysis method: Chiralpak AD-H analytical column (4.5 mm × 250

mm5 μm particle size); mobile phase: isocratic 20% 2-PrOH in

hexanes; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 28.905 min, purity >99%,

de >99% (absorbance at 254 nm). 58b eluted second; HPLC analysis

method: Chiralpak AD-H analytical column (4.5 mm × 250 mm5

μm particle size); mobile phase: isocratic 20% 2-PrOH in hexanes;

ﬂow rate: 1 mL/min; injection volume: 20 μL; sample concentration:

∼1 mg/mL; multiple DAD λ absorbance signals measured in the

range of 210−280 nm, Rt 34.205 min, purity >99%, de >99%

(absorbance at 254 nm).

stirred for 25 min under gentle heating, followed by dropwise addition

of 2-bromoethan-1-ol (257 mg, 2.06 mmol) and cat. KI. The

suspension was stirred under reﬂux overnight and ﬁltered, and the

solvent was evaporated under vacuum to aﬀord the crude material.

The desired product was puriﬁed by ﬂash chromatography eluting

with 100% EtOAc and obtained as yellow oil (310 mg, 60% yield). ¹H

NMR (400 MHz, CDCl₃): δ 7.21−7.10 (m, 2H), 6.96 (dd, J = 6.7,

2.8 Hz, 1H), 3.72−3.62 (m, 2H), 3.08 (br s, 4H), 2.71 (br s, 4H),

2.67−2.60 (m, 2H).

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethyl 4-Methylbenzene-

sulfonate (64). p-Toluenesulfonyl chloride (p-TsCl, 200 mg, 1.05

mmol) was added portionwise to a solution of 63 (240 mg, 0.87

mmol) and DIPEA (0.310 mL, 225 mg, 1.74 mmol) in DCM (15

mL). The mixture was stirred at RT overnight, the solvent was

evaporated under vacuum, and the crude residue was puriﬁed by ﬂash

chromatography eluting with hex/EtOAc (1/1). The desired product

1

was obtained as a white solid (135 mg, 36% yield). H NMR (400

MHz, CDCl₃): δ 7.81 (d, J = 7.9 Hz, 2H), 7.34 (d, J = 7.9 Hz, 2H),

7.18−7.08 (m, 2H), 6.91 (dd, J = 6.8, 2.9 Hz, 1H), 4.16 (t, J = 5.7 Hz,

2H), 3.06−2.99 (br s, 4H), 2.72 (t, J = 5.8 Hz, 2H), 2.61 (br s, 4H),

2.44 (s, 3H).

Ethyl (Z)-2-((1S,5S)-2-(2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)-

ethyl)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]nonan-9-ylidene)-

acetate (65). A suspension of 56 (50 mg, 0.175 mmol), 64 (71.5 mg,

0.166 mmol), and NaHCO₃(100 mg, 1.2 mmol) in ACN (7 mL) was

heated and stirred in a sealed vessel for 6 h. The reaction mixture was

ﬁltered, the solvent was evaporated under vacuum, and the residue

was puriﬁed by ﬂash chromatography eluting with 10% DMA. The

1

desired product was obtained as yellow oil (40 mg, 43% yield). H

NMR (400 MHz, CD₃OD): δ 7.33−7.14 (m, 3H), 7.14−7.05 (m,

1H), 6.92 (m, 2H), 6.65 (m, 1H), 5.48 (s, 1H), 4.08 (dq, J = 21.4, 7.1

Hz, 2H), 3.34−3.21 (m, 2H), 3.08 (br s, 4H), 2.97−2.70 (m, 10H),

2.56−2.42 (m, 1H), 2.22−1.98 (m, 4H), 1.74 (m, 1H), 1.65 (s, 1H),

1.19 (t, J = 7.2 Hz, 3H); ¹³C NMR (101 MHz, CD₃OD): δ 168.38,

167.92, 158.33, 152.58, 149.48, 134.91, 130.18, 129.03, 128.40,

125.82, 120.17, 119.71, 116.02, 115.67, 114.48, 111.42, 59.50, 57.16,

56.04, 54.80, 54.16, 52.08, 46.63, 41.34, 39.27, 31.73, 21.35, 14.54.

The free base was converted into the corresponding oxalate salt.

HRMS (C₃₀H₃₇N₃O₃Cl₂+ H⁺): calcd, 558.22847; found, 558.22925

(error 1.4 ppm). CHN (C₃₀H₃₇N₃O₃Cl₂+ 2.5H₂C₂O₄+ 2H₂O)

Calcd: C, 51.29; H, 5.66; N, 5.13. Found: C, 51.07; H, 5.28; N, 5.21.

mp: salt decomposes above 110 °C.

Radioligand Binding Studies. hD₂R, hD₃R, and hD₄R. Radio-

ligand binding assays were conducted similar to those previously

described.^45,67HEK293 cells stably expressing human D₂LR or D₃R

or D_4.4were grown in a 50:50 mix of Dulbecco’s modiﬁed Eagle

medium (DMEM) and Ham’s F12 culture media, supplemented with

20 mM N-(2-hydroxyethyl)piperazine-N′-ethanesulfonic acid, 2 mM

L-glutamine, 0.1 mM nonessential amino acids, 1× antibiotic/

antimycotic, 10% heat-inactivated fetal bovine serum (FBS), and

200 μg/mL hygromycin (Life Technologies, Grand Island, NY) and

kept in an incubator at 37 °C and 5% CO₂. Upon reaching 80−90%

conﬂuence, cells were harvested using premixed Earle’s balanced salt

solution with 5 mM ethylenediaminetetraacetic acid (EDTA) (Life

Technologies) and centrifuged at 3000 rpm for 10 min at 21 °C. The

supernatant was removed, and the pellet was resuspended in 10 mL of

hypotonic lysis buﬀer (5 mM MgCl₂, 5 mM Tris, pH 7.4 at 4 °C) and

centrifuged at 14,500 rpm (∼25,000g) for 30 min at 4 °C. The pellet

was then resuspended in binding buﬀer. Bradford protein assay (Bio-

Rad, Hercules, CA) was used to determine the protein concentration.

For [³H]-N-methylspiperone binding studies, membranes were

diluted to 500 μg/mL, in fresh EBSS binding buﬀer made from 8.7

g/L Earle’s balanced salts without phenol red (US Biological, Salem,

MA), 2.2 g/L sodium bicarbonate, pH to 7.4, and stored in a −80 °C

freezer for later use. For [³H]-(R)-(+)-7-OH-DPAT binding studies,

membranes were harvested fresh; the binding buﬀer was made from

50 mM Tris, 10 mM MgCl₂, and 1 mM EDTA, pH 7.4. On the test

day, each test compound was diluted into half-log serial dilutions

using the 30% dimethyl sulfoxide (DMSO) vehicle. When it was

necessary to assist solubilization of the drugs at the highest tested

N-(4-((1S,5S,9R)-9-Hydroxy-5-(3-hydroxyphenyl)-2-azabicyclo-

[3.3.1]nonan-2-yl)butyl)-1H-indole-2-carboxamide (61). The reac-

tion was performed following the same procure described for 57,

starting from (1S,5S,9R)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]-

nonan-9-ol (59; 40 mg, 0.17 mmol). The desired product was

puriﬁed by ﬂash chromatography eluting with 20% DMA and

1

obtained as a white solid (20 mg, 26% yield). H NMR (400 MHz,

CD₃OD): δ 7.57 (ddt, J = 9.0, 8.0, 1.0 Hz, 2H), 7.41 (dt, J = 8.2, 0.8

Hz, 2H), 7.24−6.94 (m, 4H), 6.89−6.73 (m, 2H), 6.66−6.58 (m,

1H), 4.25 (m, 1H), 4.07 (s, 1H), 3.74 (d, J = 0.5 Hz, 2H), 3.46 (d, J =

6.1 Hz, 1H), 3.44−3.25 (m, 3H), 2.96 (d, J = 9.8 Hz, 1H), 2.70 (br s,

3H), 2.48 (td, J = 13.5, 7.8 Hz, 2H), 2.31 (d, J = 12.8 Hz, 1H), 2.23−

2.08 (m, 1H), 1.93−1.61 (m, J = 4.2, 3.2 Hz, 6H), 1.28 (br s, 1H).

HPLC analysis method: Chiralpak AD-H analytical column (4.5 mm

× 250 mm5 μm particle size); mobile phase: isocratic 50% 2-PrOH

in hexanes + 0.1% DEA; ﬂow rate: 1 mL/min; injection volume: 20

μL; sample concentration: ∼1 mg/mL; multiple DAD λ absorbance

signals measured in the range of 210−280 nm, Rt 29.790, purity

>99%, ee >99% (absorbance at 254 nm). HRMS (C₂₇H₃₄O₃N₃+

H⁺): calcd, 448.25947; found, 448.25979.

N-(4-((1S,5S,9S)-9-Hydroxy-5-(3-hydroxyphenyl)-2-azabicyclo-

[3.3.1]nonan-2-yl)butyl)-1H-indole-2-carboxamide (62). The reac-

tion was performed following the same procedure described for 57,

starting from (1S,5S,9S)-5-(3-hydroxyphenyl)-2-azabicyclo[3.3.1]-

nonan-9-ol (60; 40 mg, 0.17 mmol). The desired product was

puriﬁed by ﬂash chromatography eluting with 20% DMA and

1

obtained as a white solid (20 mg, 26% yield). H NMR (400 MHz,

CD₃OD): δ 7.62−7.53 (m, 2H), 7.42 (t, J = 8.3 Hz, 2H), 7.25−7.12

(m, 2H), 7.12−6.80 (m, 4H), 6.40 (m, 1H), 4.30 (m, 1H), 4.07 (s,

1H), 3.75 (s, 2H), 3.49−3.37 (m, 4H), 2.98 (br s, 1H), 2.20−1.93

(m, 7H), 1.73−1.66 (m, 5H), 1.28 (br s, 2H). HPLC analysis

method: Chiralpak AD-H analytical column (4.5 mm × 250 mm5

μm particle size); mobile phase: isocratic 50% 2-PrOH in hexanes +

0.1% DEA; ﬂow rate: 1 mL/min; injection volume: 20 μL; sample

concentration: ∼1 mg/mL; multiple DAD λ absorbance signals

measured in the range of 210−280 nm, Rt 32.837, purity >99%, ee

>99% (absorbance at 254 nm). HRMS (C₂₇H₃₄O₃N₃+ H⁺): calcd,

448.25947; found, 448.25982.

2-(4-(2,3-Dichlorophenyl)piperazin-1-yl)ethan-1-ol (63). A sol-

ution of 1-(2,3-dichlorophenyl)piperazine hydrochloride (500 mg,

1.87 mmol) and K₂CO₃(1.29 g, 9.34 mmol) in ACN (30 mL) was

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concentration, 0.1% AcOH (ﬁnal concentration v/v) was added

alongside the vehicle. Membranes were diluted in fresh binding buﬀer.

Radioligand competition experiments were conducted in 96-well

plates containing 300 μL of fresh binding buﬀer, 50 μL of the diluted

test compound, 100 μL of membranes (for [³H]-N-methylspiperone

assays: 10−20, 10−20, and 20−30 μg/well total protein for hD_2LR,

hD₃R, and hD_4.4R, respectively; for [³H]-(R)-(+)-7-OH-DPAT

assays: 40−80 and 20−40 μg/well total protein for hD_2LR and

hD₃R, respectively), and 50 μL of radioligand diluted in binding

buﬀer ([³H]-N-methylspiperone: 0.4 nM ﬁnal concentration for all

the hD₂-like receptor subtypes; [³H]-(R)-(+)-7-OH-DPAT: 1.5 nM

ﬁnal concentration for hD₂L, and 0.5 nM ﬁnal concentration for hD₃;

PerkinElmer). Aliquots of radioligand solution were also quantiﬁed

accurately in each experiment replicate to determine how much

radioactivity was added, taking into account the experimentally

determined counter eﬃciency. Nonspeciﬁc binding was determined

using 10 μM (+)-butaclamol (Sigma-Aldrich, St. Louis, MO), and

total binding was determined with the 30% DMSO vehicle. All

compound dilutions were tested in triplicate, and the reaction was

incubated for 60 min ([³H]-N-methylspiperone assays) or 90 min

([³H]-(R)-(+)-7-OH-DPAT assays) at RT. The reaction was

terminated by ﬁltration through PerkinElmer UniFilter-96 GF/B,

presoaked for the incubation time in 0.5% polyethylenimine, using a

Brandel 96-well plates harvester manifold (Brandel Instruments,

Gaithersburg, MD). The ﬁlters were washed thrice with 3 mL (3 Å

1 mL/well) of ice-cold binding buﬀer. PerkinElmer MicroScint 20

scintillation cocktail (65 μL) was added to each well, and ﬁlters were

counted using a PerkinElmer MicroBeta microplate counter. IC₅₀

values for each compound were determined from dose−response

curves, and K_ivalues were calculated using the Cheng−Prusoﬀ

equation.⁶³K_dvalues were determined via separate homologous

competitive binding experiments. When a complete inhibition could

not be achieved at the highest tested concentrations, K_ivalues have

been extrapolated by constraining the bottom of the dose−response

curves (=0% residual speciﬁc binding) in the nonlinear regression

analysis. These analyses were performed using GraphPad Prism

version 8 for Macintosh (GraphPad Software, San Diego, CA). All

results were rounded to the third signiﬁcant ﬁgure. K_ivalues were

determined from at least three independent experiments and are

reported as the mean SEM.

hMOR. Radioligand binding experiments were conducted, and the

results were analyzed, as described above and similar to those

previously reported.⁶²HEK293 cells stably expressing the hMOR

were grown in a DMEM, supplemented with 10% FBS, 2 mM ^L-

glutamine, 1% penicillin−streptomycin (or antibiotic/antimycotic),

and hygromycin B (50 μg/mL). Upon reaching conﬂuence, the cells

were harvested and the membranes were prepared as detailed before.

The binding buﬀer was made of 50 mM Tris and 5 mM MgCl₂at pH

7.4. The experiments were performed in the presence of [³H]-

DAMGO (ﬁnal concentration 3 nM; PerkinElmer) and 30 μg/well of

membranes (ﬁnal concentration). The reactions were incubated for

60 min at RT and terminated by rapid ﬁltration through PerkinElmer

UniFilter-96 GF/B, presoaked for 60 min in 0.5% polyethylenimine.

The nonspeciﬁc binding was determined using 10 μM C-TOP or cold

DAMGO. The radioligand K_dwas measured via radioligand saturation

experiments.

for GPA,⁶⁹and 4 μg of arrestin-3-Venus, 2 μg of WT-GRK2, and 1 μg

of mMOR-Rluc8 for arrestin-3 recruitment. Cells were then allowed

to grow overnight at 37 °C, 5% CO₂. The next day, cells were plated

in Greiner poly-^D-lysine-coated 96-well plates (SLS) in media and

allowed to grow overnight. On the day of the assay (48 h post-

transfection), cells were washed once with D-PBS (Lonza, SLS) and

incubated in D-PBS for 30 min at 37 °C. For the antagonist-mode

assays that require preincubation, the cells were washed once with D-

PBS and incubated with ligands in D-PBS (supplemented with 10

mM glucose) at 37 °C, 5% CO₂for 3 h prior to starting the assay. The

Rluc substrate coelenterazine h (NanoLight) was added to each well

(ﬁnal concentration of 5 μM), and cells were incubated for 5 min at

37 °C. After 5 min, ligands (ﬁnal concentration from 10 μM to 1 nM

in D-PBS) were added to the plate and cells were incubated for a

further 10 min at 37 °C before reading the plate in a PHERAstar FSX

microplate reader (Venus and Rluc emission signals at 535 and 475

nm, respectively, BMG Labtech). The ratio of Venus/Rluc counts was

used to quantify the BRET signal in each well. Data were normalized

to the wells containing 10 μM DAMGO/quinpirole or no drug for

maximal or minimal response, respectively, and as indicated in the

ﬁgure legends. All experiments were performed in duplicate and at

least three times independently. All data points represent the mean,

and error bars represent the SEM and were ﬁtted using the built-in

log(agonist) versus response (three parameters) model in Prism 8.0

(GraphPad Software Inc., San Diego, CA).For agonist-mode assays,

data were ﬁtted to a three-parameter concentration−response model

where EC₅₀is the concentration of the agonist needed to elicit half

the maximal response of the particular agonist, deﬁned as E_max. For

the antagonist-mode assays, data points were ﬁtted using a three-

parameter concentration−response model where IC₅₀is the

concentration required to inhibit half the maximum response of the

agonist used at a particular concentration. Values of pEC₅₀or pIC₅₀

error are given as the error has a Gaussian distribution, whereas the

error associated with the antilog value does not. For some ligands for

which the lower asymptote of the curve was not well deﬁned within

the concentration range (represented by a dotted line), the bottom

was constrained to be equal to 0%.

Molecular Docking and CADD. The receptor structures

corresponding to PDB accession code 6CM4, 3PBL, and 5C1M

were extracted from RCSB for the inactive-state D₂R, inactive-state

D₃R, and active-state MOR, respectively. All the objects except the

receptor protein subunit, the crystallized ligand, and in the case of

active-state MOR, three crystallographic waters were deleted, and this

was followed by the addition of hydrogens and optimization of the

side-chain residues. Ligands were sketched, assigned formal charges,

and energy-optimized prior to docking. The ligand docking box for

potential grid docking was deﬁned as the whole extracellular half of

the protein, and all-atom docking was performed using the energy

minimized structures for all ligands with a thoroughness value of 10.

The best-scored docking solutions were further optimized by several

rounds of minimization and Monte Carlo sampling of the ligand

conformation, including the surrounding side-chain residues (within 5

Å of the ligand) and the three crystallographic waters in the MOR

orthosteric sites. All the abovementioned molecular modeling

operations were performed in ICM-Pro v3.8-5 molecular modeling

package.

BRET Studies. All reagents were purchased from Sigma-Aldrich-

Merck unless otherwise stated. BRET experiments were performed in

transiently transfected human embryonic kidney 293 T (HEK 293T)

cells, as described previously.^31,68Brieﬂy, cells were grown and

maintained at 37 °C in 5% CO₂in DMEM supplemented with 10%

(v/v) FBS. Cells were seeded in 10 cm Petri dishes (2.5 × 10⁶cells

per dish) and allowed to grow overnight in media at 37 °C, 5% CO₂.

The following day, cells were transiently transfected in media

supplemented with antibiotics (100 U/mL penicillin and 100 μg/

mL streptomycin, Gibco) using a 1:6 total DNA to PEI (PolySciences

Inc.) ratio. BRET constructs were as follows: 4 μg of Nb33-Venus and

1 μg of mMOR-Rluc8 for Nb33 recruitment, 2 μg of WT-Gα (i2 or

oA), 1 μg of Gβ1-Venus(156−239), 1 μg of Gγ2-Venus(1−155), 1 μg

of masGRK3ct-Rluc8, and 1 μg of receptor (SNAP-mMOR or hD3R)

ASSOCIATED CONTENT

■

sı

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00611.

Molecular formula strings (CSV)

PDB ﬁles of all the ligand/target complexes studied

(ZIP)

Analytical data summary of the products (combustion

elemental analyses, HRMS−MS/MS, GC/MS, and

HPLC), supporting table reporting biased agonism

7804

https://doi.org/10.1021/acs.jmedchem.1c00611

J. Med. Chem. 2021, 64, 7778−7808

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Program, National Institutes of Health, Bethesda, Maryland

20892, United States

analysis at MOR signaling pathways, and supplemental

ﬁgures of all the ligand/target complexes studied (PDF)

Vsevolod Katritch − Bridge Institute, Michelson Center for

Convergent Bioscience, Department of Chemistry, Department

of Biological Sciences, University of Southern California, Los

Angeles, California 90089, United States; orcid.org/

0000-0003-3883-4505

Meritxell Canals − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queen’s Medical Centre,

University of Nottingham, Nottingham NG7 2UH, U.K.;

Centre of Membrane Protein and Receptors, Universities of

Birmingham and Nottingham, Midlands NG2 7AG, U.K.;

orcid.org/0000-0002-7942-5006

J. Robert Lane − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queen’s Medical Centre,

University of Nottingham, Nottingham NG7 2UH, U.K.;

Centre of Membrane Protein and Receptors, Universities of

Birmingham and Nottingham, Midlands NG2 7AG, U.K.;

orcid.org/0000-0002-7361-7875

AUTHOR INFORMATION

Corresponding Authors

■

Alessandro Bonifazi − Medicinal Chemistry Section,

Molecular Targets and Medications Discovery Branch,

National Institute on Drug AbuseIntramural Research

Program, National Institutes of Health, Baltimore, Maryland

21224, United States; orcid.org/0000-0002-7306-0114;

Phone: (443)-740-2897; Email: alessandro.bonifazi2@

nih.gov; Fax: (443)-740-2111

Amy Hauck Newman − Medicinal Chemistry Section,

Molecular Targets and Medications Discovery Branch,

National Institute on Drug AbuseIntramural Research

Program, National Institutes of Health, Baltimore, Maryland

21224, United States; orcid.org/0000-0001-9065-4072;

Phone: (443)-740-2887; Email: anewman@

intra.nida.nih.gov; Fax: (443)-740-2111

Complete contact information is available at:

https://pubs.acs.org/10.1021/acs.jmedchem.1c00611

Authors

Francisco O. Battiti − Medicinal Chemistry Section, Molecular

Targets and Medications Discovery Branch, National

Institute on Drug AbuseIntramural Research Program,

National Institutes of Health, Baltimore, Maryland 21224,

United States

Julie Sanchez − Division of Physiology, Pharmacology and

Neuroscience, School of Life Sciences, Queen’s Medical Centre,

University of Nottingham, Nottingham NG7 2UH, U.K.;

Centre of Membrane Protein and Receptors, Universities of

Birmingham and Nottingham, Midlands NG2 7AG, U.K.

Saheem A. Zaidi − Bridge Institute, Michelson Center for

Convergent Bioscience, Department of Chemistry, Department

of Biological Sciences, University of Southern California, Los

Angeles, California 90089, United States

Eric Bow − Drug Design and Synthesis Section, Molecular

Targets and Medications Discovery Branch, National

Institute on Drug AbuseIntramural Research Program,

National Institutes of Health, Bethesda, Maryland 20892,

United States

Mariia Makarova − Drug Design and Synthesis Section,

Molecular Targets and Medications Discovery Branch,

National Institute on Drug AbuseIntramural Research

Program, National Institutes of Health, Bethesda, Maryland

20892, United States

Jianjing Cao − Medicinal Chemistry Section, Molecular

Targets and Medications Discovery Branch, National

Institute on Drug AbuseIntramural Research Program,

National Institutes of Health, Baltimore, Maryland 21224,

United States

Anver Basha Shaik − Medicinal Chemistry Section, Molecular

Targets and Medications Discovery Branch, National

Institute on Drug AbuseIntramural Research Program,

National Institutes of Health, Baltimore, Maryland 21224,

United States; orcid.org/0000-0001-9894-5544

Agnieszka Sulima − Drug Design and Synthesis Section,

Molecular Targets and Medications Discovery Branch,

National Institute on Drug AbuseIntramural Research

Program, National Institutes of Health, Bethesda, Maryland

20892, United States

Author Contributions

A.B. and A.H.N. designed the project; A.B., F.O.B., and A.H.N.

wrote the manuscript with input of all the authors; A.B.,

F.O.B., J.S., S.A.Z., A.S., K.C.R., V.K., M.C., J.R.L., and A.H.N.

designed and supervised the experiments and data analysis;

A.B., F.O.B., J.S., S.A.Z., E.B., M.M., J.C., A.B.S., and A.S.

performed experiments.

Notes

The authors declare no competing ﬁnancial interest.

ACKNOWLEDGMENTS

■

This project was supported by the National Institute on Drug

AbuseIntramural Research Program (ZIADA000609;

ZIADA000424), as well as grant funding R33DA038858 for

V.K. The authors thank Drs. Ludovic Muller and Shelley

Jackson from the Structural Biology Core at NIDA-IRP for

high-resolution MS/MS analyses, Dr. Ning Sheng Cai from the

Integrative Neurobiology Section at NIDA-IRP for assistance

with cell lines and tissue culture protocols, and Dr. Arthur

Jacobson from the Drug Design and Synthesis Section at

NIDA-IRP for insightful suggestions to the manuscript.

ABBREVIATIONS

■

DA, dopamine; D_2‑likeR, dopamine D_2‑likereceptors; OUD,

opioid use disorders; CADD, computer-aided drug design;

BRET, bioluminescence resonance energy transfer; HEK 293

cells, human embryonic kidney 293 cells; HPLC, high-

performance liquid chromatography; HRMS, high-resolution

mass spectrometry; DMA, dichloromethane, methanol, and

a m m o n i u m h y d r o x i d e ; E D C , 1 - e t h y l - 3 - ( 3 -

dimethylaminopropyl)carbodiimide; HOBt, hydroxybenzotria-

zole; HCTU, O-(1H-6-chlorobenzotriazole-1-yl)-1,1,3,3-tetra-

methyluronium hexaﬂuorophosphate; DIPEA, N,N-diisopro-

pylethylamine; STAB, sodium triacetoxyborohydride; DMP,

Dess−Martin periodinane; ACN, acetonitrile; LAH, lithium

aluminum hydride; GPA, G-protein activation

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https://doi.org/10.1021/acs.jmedchem.1c00611

J. Med. Chem. 2021, 64, 7778−7808

Article Doi

Novel Dual-Target μ-Opioid Receptor and Dopamine D3Receptor Ligands as Potential Nonaddictive Pharmacotherapeutics for Pain Management

DOI: 10.1021/acs.jmedchem.1c00611

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Full text of DOI:10.1021/acs.jmedchem.1c00611

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