Structure-Based Design and Development of Chemical Probes Targeting Putative MOR-CCR5 Heterodimers to Inhibit Opioid Exacerbated HIV-1 Infectivity

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Structure-Based Design and Development of Chemical Probes

Targeting Putative MOR-CCR5 Heterodimers to Inhibit Opioid

Exacerbated HIV‑1 Infectivity

Boshi Huang, Huiqun Wang, Yi Zheng, Mengchu Li, Guifeng Kang, Victor Barreto-de-Souza,

Nima Nassehi, Pamela E. Knapp, Dana E. Selley, Kurt F. Hauser, and Yan Zhang *

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ABSTRACT: Crystal structures of ligand-bound G-protein-coupled receptors

provide tangible templates for rationally designing molecular probes. Herein,

we report the structure-based design, chemical synthesis, and biological

investigations of bivalent ligands targeting putative mu opioid receptor C−C

motif chemokine ligand 5 (MOR-CCR5) heterodimers. The bivalent ligand

VZMC013 possessed nanomolar level binding aﬃnities for both the MOR and

CCR5, inhibited CCL5-stimulated calcium mobilization, and remarkably

improved anti-HIV-1_BaLactivity over previously reported bivalent ligands.

VZMC013 inhibited viral infection in TZM-bl cells coexpressing CCR5 and

MOR to a greater degree than cells expressing CCR5 alone. Furthermore,

VZMC013 blocked human immunodeﬁciency virus (HIV)-1 entry in

peripheral blood mononuclear cells (PBMC) cells in a concentration-

dependent manner and inhibited opioid-accelerated HIV-1 entry more eﬀectively in phytohemagglutinin-stimulated PBMC cells

than in the absence of opioids. A three-dimensional molecular model of VZMC013 binding to the MOR-CCR5 heterodimer

complex is constructed to elucidate its mechanism of action. VZMC013 is a potent chemical probe targeting MOR-CCR5

heterodimers and may serve as a pharmacological agent to inhibit opioid-exacerbated HIV-1 entry.

Chronic opioid exposure tends to severely suppress both

innate and adaptive immune functions, resulting in signiﬁcantly

increased vulnerability to infectious pathogens, such as

HIV.^9,10Opioids can increase HIV replication and exacerbate

the progression of AIDS.¹¹⁻¹³One possible pathway for

opioid-mediated increased HIV replication occurs through the

opioid-dependent upregulation of the major chemokine

coreceptor CCR5.¹⁴⁻¹⁷As is well-known, opioid addiction

liability is predominantly mediated via actions at the mu opioid

receptor (MOR). Mounting studies have shown that MOR

agonists, such as [^D-Ala²-MePhe⁴-Gly(ol)⁵]enkephalin

(DAMGO),¹⁴morphine,^15,18−21and methadone,²²can induce

elevated CCR5 receptor expression in diﬀerent immune and

nonimmune cells. An increase in CCR5 expression furnishes

additional viral entry sites and thus promotes HIV-1 infection/

replication.

INTRODUCTION

■

The opioid epidemic in the United States is a signiﬁcant public

health crisis. A recent report suggested that opioids were

involved in 69.5% of all drug overdose deaths in the United

States during 2018.¹Acquired immune deﬁciency syndrome

(AIDS), caused by the human immunodeﬁciency virus (HIV)

infection, remains another major public health issue with

approximately 37.9 million people infected globally in 2018²

and an estimated 38,000 new cases reported in the United

States each year.³Currently, the opioid abuse epidemic is

closely associated with AIDS/HIV infection. Opioid use

disorder (OUD) increases the risk of transmission of HIV

infection directly through sharing needles, syringes, or other

drug injection materials.⁴It was reported that injection drug

abuse accounted for 7% of all new HIV diagnoses in 2018 in

the United States.⁵Conversely, HIV infection can elevate the

susceptibility to drug addiction as well.⁶It was estimated that

HIV-infected individuals prescribed opioids were 21−53%

more likely to have OUD than uninfected individuals.⁷

Moreover, long-term opioid use increases the risk of death

among the HIV-infected population compared to uninfected

individuals.⁸Therefore, it is important to uncover the

relationship between opioid use and enhanced HIV infectivity

with the goal of limiting the spread and pathophysiological

consequences of HIV.

Received: March 7, 2021

Published: May 24, 2021

https://doi.org/10.1021/acs.jmedchem.1c00408

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Figure 1. Chemical structures of naltrexone (red) and maraviroc (blue) within the previously reported bivalent ligand VZMC001 (the structure of

the intervening spacer is shown in black).

Accumulating in vitro studies suggest there are functional

crosstalks between the mu opioid receptor (MOR) and C−C

motif chemokine ligand 5 (CCR5), both belonging to the G-

protein-coupled receptor (GPCR) superfamily. Previous

reports have revealed that MOR-CCR5 crosstalk is, in part,

mediated by the formation of putative MOR-CCR5 hetero-

dimers.²³⁻²⁷For example, using a coimmunoprecipitation

approach, Suzuki et al. demonstrated that the MOR and CCR5

could form oligomers at the cell membrane of human or

monkey lymphocytes and the oligomerization can modulate

the function of both receptors.²³In addition, Chen and co-

workers investigated possible mechanisms of cross-desensitiza-

tion between the MOR and CCR5 coexpressed in Chinese

hamster ovary (CHO) cells. They proposed that MOR-CCR5

heterodimerization may contribute to the observed cross-

desensitization.²⁶Thus, putative MOR-CCR5 heterodimeriza-

tion aﬀects immune cell function and appears to contribute to

the synergistic eﬀects of opioids and HIV coexposure in

neuroHIV progression.

Bivalent ligands containing two discrete pharmacophores

tethered by an appropriate spacer are powerful chemical

probes to characterize GPCR dimerization.²⁸⁻³¹Hence,

bivalent ligands that are capable of interacting simultaneously

with both receptors would help facilitate the study of the

putative MOR-CCR5 heterodimer and its role in opioid

accelerated HIV entry/replication. We hypothesized that

bivalent ligands containing both an MOR and a CCR5

antagonist pharmacophore would eﬃciently inhibit opioid-

dependent increases in HIV entry/replication.

primary human astrocytes with or without morphine. Never-

theless, VZMC001 possessed less favorable binding aﬃnity

proﬁles for both the MOR and CCR5 compared to the

corresponding monomeric ligands. Moreover, results from our

molecular dynamics (MD) simulation showed that the

maraviroc portion of VZMC001 was partially dislodged from

the CCR5 binding pocket. We speculated that this limitation

stemmed from our original molecular design that was based on

the homology model of the CCR5.³⁴On the other hand, as a

proof-of-concept, VZMC001 served as a promising lead to

further design and develop more potent bivalent ligand(s)

targeting the putative MOR-CCR5 heterodimer. Herein, we

report the structure-based molecular design, synthesis, and

comprehensive biological investigations of the second-gen-

eration bivalent ligand VZMC013.

RESULTS AND DISCUSSION

■

Structure-Based Molecular Design. When we ﬁrst

initiated this project utilizing naltrexone and maraviroc as

pharmacophores, neither of the ligand-bound crystal structures

of the MOR and CCR5 were available for structure-based

molecular design. Given these constraints, it was critical to

choose appropriate spacers as well as the attachment points on

each pharmacophore to establish a desirable bivalent ligand.

Based on several successful cases,³⁷⁻³⁹the C6-position of

naltrexone was selected as the attachment point that could be

readily transformed to the 6β-amino group. Additionally,

molecular docking of maraviroc into a previously reported

CCR5 homology model by our group⁴⁰revealed that the

tropane core and the diﬂuorocyclohexyl moiety established

interactions with Glu283 and Ile198, respectively, in the

binding pocket. The 4′-position of the terminal phenyl ring in

maraviroc was therefore selected as the attachment point to

avoid disruption of these interactions. Furthermore, several

studies indicated that a spacer with 21 atoms may be ideal to

yield the optimal pharmacological eﬀects.^37,38,41,42Therefore,

the bivalent ligand VZMC001 with a 21-atom spacer linked

through the corresponding attachment points on both

pharmacophores was designed and prepared.³²Two mono-

To test our hypothesis and to understand how MOR-CCR5

dimerization uniquely alters the function of each receptor, we

previously designed and synthesized a bivalent ligand,

VZMC001 (Figure 1), containing the MOR antagonist

pharmacophore naltrexone (1) and the CCR5 antagonist

pharmacophore maraviroc (2).³²⁻³⁴Naltrexone, a selective

MOR antagonist, has been used in the treatment of OUD,³⁵

and maraviroc is the only marketed anti-HIV drug that blocks

the CCR5 coreceptor function.³⁶VZMC001 exhibited a 7- or

3.3-fold higher inhibition of HIV entry than maraviroc in

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Figure 2. (a) The binding mode of β-FNA in the MOR (adapted from PDB ID: 4DKL).⁴⁶(b) The binding mode of maraviroc in the CCR5

(adapted from PDB ID: 4MBS).⁴⁴(c) Structure-based molecular design of the second generation bivalent ligands VZMC013, VZMC017, and

VZMC019.

a

Scheme 1. Synthesis of Intermediate 9

a

Reagents and conditions: (a) Malonic acid (1.5 equiv), ammonium formate (2.5 equiv), EtOH, reﬂux, 59%; (b) (i) conc. H₂SO₄, MeOH, 0 °C to

r.t.; (ii) 2 M NaOH, pH 10, 0 °C. Two steps 99%; (c) ^L-(+)-tartaric acid (1 equiv), MeOH, 45 °C to r.t., 25%; (d) (i) benzyl chloroformate (1.2

equiv), Na₂CO_3(aq)(3.3 equiv), dichloromethane, 0 °C to r.t.; (ii) 2 M NaOH, pH 13, MeOH, 0 °C to r.t.; (iii) 2 M HCl, pH 1, 0 °C. Three steps

80%; (e) BH₃·THF (4 equiv), anhydrous THF, N₂, 0 °C to r.t., 58%; (f) sulfur trioxide pyridine complex (5 equiv), Et₃N (10 equiv), DMSO/

dichloromethane = 1/1, 0 °C, 87%.

valent ligands VZMC002 (containing the MOR antagonist

pharmacophore only, Figure S1) and VZMC003 (containing

the CCR5 antagonist pharmacophore only, Figure S1) were

also constructed as controls to examine the potential inﬂuence

of the spacer on the pharmacological eﬀects on each

pharmacophore.

Currently, there are mainly two approaches to characterize

putative GPCR dimerization: chemistry-based bivalent ligand

targeting approach and structural biology-based GPCR crystal

structure determination approach.³¹For the former approach,

bivalent ligands targeting putative GPCR dimers are

commonly synthesized empirically, regardless of the choices

of the attachment points on each monomeric pharmacophore

and the length and chemical composition of spacers.³¹For the

latter one, GPCR homodimer constructs have been observed

in some crystal structures,^43,44which provide further evidence

to support GPCR dimerization. To our knowledge, few

bivalent ligands reported have been designed in a rational

way, that is, with the support of structural biology, since the

concept of bivalent ligands was ﬁrst introduced in 1982.⁴⁵

High-resolution GPCR-ligand cocrystal structures oﬀer new

opportunities for structure-based molecular design of bivalent

ligands. In 2012, the crystal structure of the MOR in complex

with an epoxymorphinan antagonist β-funaltrexamine (β-FNA,

Figure S1) (PDB ID: 4DKL) was determined.⁴⁶It was shown

that the C6-position of β-FNA in the cocrystal structure

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a

Scheme 2. Synthesis of Intermediate 13

a

Reagents and conditions: (a) Thionyl chloride (2.5 equiv), MeOH, 0 °C to r.t., 62%; (b) Fmoc-Cl (1.05 equiv), Et₃N (3.75 equiv),

dichloromethane, 0 °C to r.t., 81%; (c) hydrazine monohydrate (5 equiv), MeOH, r.t., 49%.

a

Scheme 3. Synthesis of 2′-Aminoethyl Maraviroc Precursor (24)

a

Reagents and conditions: (a) Tetrahydro-2,5-dimethoxyfuran (1 equiv), benzyl amine (1.2 equiv), NaOAc, HCl_(aq), 0 °C to r.t., 48%; (b)

hydroxylamine hydrochloride (1 equiv), pyridine (1.1 equiv), EtOH, reﬂux, 51%; (c) sodium (10 equiv), n-pentanol, 120 °C, 72%; (d) isobutyryl

chloride (1.2 equiv), Na₂CO_3(aq)(2.8 equiv), dichloromethane, 0 °C to r.t., 49%; (e) (i) PCl₅(1.5 equiv), dichloromethane, 0 °C; (ii) 13 (0.77

equiv), tert-amyl alcohol, 0 °C to r.t.; (iii) AcOH, tert-amyl alcohol, 85 °C. Three steps 29%; (f) p-TsOH monohydrate (1 equiv), 30% Pd/C,

MeOH, 60 psi H₂, r.t.; (g) 9 (1.1 equiv), NaBH(OAc)₃(1.1 equiv), AcOH, dichloromethane, r.t. Two steps 53%; (h) 30% Pd/C, MeOH, 60 psi

H₂, r.t., 67%; (i) 4,4-diﬂuorocyclohexanecarboxylic acid (2 equiv), EDCI (2 equiv), HOBt (2 equiv), Et₃N (4 equiv), 4 Å molecular sieves (MS),

dichloromethane, 0 °C to r.t., 54%; (j) 20% piperidine/DMF, r.t., 42%.

pointed toward the extracellular end of transmembrane helix 5

(TM5) and TM6 (Figure 2a) of the MOR. Considering the

high structural similarity between naltrexone and β-FNA, we

believed that the C6-position of naltrexone may still be the

optimal attachment point on the MOR pharmacophore. One

year later, the crystal structure of CCR5 complexing with

maraviroc (CCR5/maraviroc, PDB ID: 4MBS) was reported.⁴⁴

Maraviroc was determined to bind within the pocket such that

the 3′-methyl group on the 1,2,4-triazol moiety is directed

away from the extracellular domains of TM1, 2, and 3 (Figure

2b). This observation revealed that the 3′-methyl group on the

1,2,4-triazol moiety, rather than the 4′-position of the phenyl

ring in maraviroc, may be more accessible from the

extracellular side and was therefore selected as the attachment

point for the CCR5 pharmacophore for this round of design.

With both attachment points designated, we utilized the same

21-atom length spacer as in VZMC001 to construct the novel

bivalent ligand, VZMC013 (Figure 2c). A new CCR5

monovalent control VZMC014 (containing the CCR5

antagonist pharmacophore only, Figure S1) was proposed,

while the MOR monovalent control ligand VZMC002 was

adopted from the previous study.³⁴

Meanwhile, to further investigate how the spacer length

aﬀects biological activities, two additional bivalent ligands

VZMC017 and VZMC019 (with their corresponding CCR5

monovalent controls VZMC018 and VZMC020, Figure S1)

bearing longer (23-atom) or shorter (19-atom) spacers relative

to VZMC013 were also designed.

Chemical Synthesis. We report the chemical syntheses of

the newly designed bivalent ligands (VZMC013, VZMC017,

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a

Scheme 4. Synthesis of the Bivalent Ligands VZMC013, VZMC017, and VZMC019

a

Reagents and conditions: (a) Benzyl chloroformate (1 equiv), dichloromethane/MeOH = 1/1, 0 °C to r.t., 30%−49%; (b) diglycolic anhydride

(1.05 equiv), THF, r.t., 70%−86%; (c) 6β-naltrexamine hydrochloride (0.5 equiv), EDCI (1.25 equiv), HOBt (1.05 equiv), Et₃N (2 equiv), 4 Å

MS, anhydrous DMF, 0 °C to r.t., 36%−61%; (d) 30% Pd/C, MeOH, 60 psi H₂, r.t., 90%−94%; (e) diglycolic anhydride (1 equiv), DMF, r.t.,

72%−81%; (f) 24 (1.2 equiv), EDCI (4 equiv), HOBt (4 equiv), Et₃N (4 equiv), 4 Å MS, anhydrous DMF, 0 °C to r.t., 20%−55%.

and VZMC019) and corresponding monovalent control

ligands (VZMC014, VZMC018, and VZMC020) in the

present study. The synthetic routes for these compounds are

depicted in Schemes 1−5. Synthesis of the 2′-aminoethyl

maraviroc precursor 24 was ﬁrst carried out step-wise

(Schemes 1−3). First, to prepare compound 9, a similar

synthetic route as reported was employed (Scheme 1).⁴⁷The

benzaldehyde 3 was condensed with ammonium acetate and

malonic acid to yield the amino acid 4, which was esteriﬁed in

methanol to give β-amino ester 5. The racemic β-amino ester 5

was resolved by using ^L-(+)-tartaric acid to aﬀord 6 as the L-

(+)-tartaric acid salt.⁴⁸Protection of 6 using benzyl

chloroformate was carried out under modiﬁed Schotten-

Baumann conditions,⁴⁹followed by hydrolysis to furnish the

Cbz-protected acid 7. The β-aminoaldehyde 9 was obtained

from 7 through borane-mediated reduction and subsequent

oxidation of the resulting alcohol under Parikh−Doering

conditions.

Then, commercially available β-alanine 10 was converted to

corresponding methyl ester hydrochloride 11,⁵⁰which was

protected using ﬂuorenylmethyloxycarbonyl chloride (Fmoc-

Cl) to provide 12.⁵¹Hydrazinolysis of 12 led to the Fmoc-

protected hydrazide 13 (Scheme 2).⁵²

Next, N-benzylnortropinone 15 was obtained from

acetonedicarboxylic acid 14, tetrahydro-2,5-dimethoxyfuran,

and benzylamine following the literature procedures⁵³and was

then transformed to oxime 16 using hydroxylamine hydro-

chloride. The oxime 16 was reduced by sodium in n-pentanol

to give amine 17.⁵⁴Acylation of 17 with isobutyryl chloride

supplied the subunit 18. Activation of 18 to its corresponding

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a

Scheme 5. Synthesis of the Monovalent Ligands VZMC014, VZMC018, and VZMC020

a

Reagents and conditions: (a) 31 (1.2 equiv), EDCI (1.5 equiv), HOBt (1.2 equiv), Et₃N (2 equiv), 4 Å MS, anhydrous DMF, 0 °C to r.t., 27%−

47%; (b) 10% Pd/C, MeOH, 60 psi H₂, r.t., 69%−100%; (c) diglycolic anhydride (1 equiv), DMF, r.t., 92%−100%; (d) 24 (1.2 equiv), EDCI (4

equiv), HOBt (4 equiv), Et₃N (4 equiv), 4 Å MS, anhydrous DMF, 0 °C to r.t., 21%−46%.

a

imidoyl chloride followed by trapping with hydrazide 13 and

acetic acid-catalyzed thermal cyclization in three successive

steps provided the triazole 19.⁴⁷Subsequent Bn-deprotection

of compound 19 by hydrogenolysis with p-toluenesulfonic acid

(p-TsOH) and 30% Pd/C aﬀorded the key intermediate 20,

which was directly used as a tosylate salt for the next step.

Reductive amination of amine 20 with the β-aminoaldehyde 9

was facilitated using sodium triacetoxyborohydride as a

reducing reagent to give compound 21.⁵⁵Removal of the

Cbz group of 21 by hydrogenolysis with 30% Pd/C in

methanol oﬀered free amine 22, which was then coupled with

the 4,4-diﬂuorocyclohexanecarboxylic acid to yield amide 23.

Treatment of 23 in 20% piperidine/DMF ﬁnally provided the

precursor 2′-aminoethyl maraviroc 24 (Scheme 3).⁵⁶

Table 1. MOR Radioligand Binding Aﬃnity

compounds

K_i(nM)

b

VZMC013

VZMC017

VZMC019

VZMC001

Naltrexone

6.05 0.22

11.2 1.92

4.23 0.27

51.8 7.9

0.7 0.1

c

a

b

[³H]Naloxone was used as the radioligand in the binding assay. The

values are the mean SEM of at least three independent experiments.

Data have been reported in ref 33 and are presented here for

c

comparison.

pharmacophore naltrexone (K_i= 0.7 nM), which was not

unusual based on previous reports from other research

groups.⁵⁸⁻⁶⁰Brieﬂy, VZMC013 eﬀectively recognized the

MOR.

With intermediate 24 in hand, we sought to synthesize the

naltrexamine-containing acids 30a−c to prepare the bivalent

ligands as in our previous reports^32,34with only minor

modiﬁcations. The bivalent ligands VZMC013, VZMC017,

and VZMC019 were obtained by coupling amine 24 with

corresponding acids 30a−c via the HOBt/EDCI method

(Scheme 4).⁵⁷In addition, the monovalent ligands VZMC014,

VZMC018, and VZMC020 were furnished following similar

synthetic protocols (Scheme 5).

MOR [³⁵S]GTPγS Functional Studies. The [³⁵S]GTPγS

functional assay was carried out in mMOR-CHO cells to

deﬁne the relative eﬃcacy of these bivalent ligands to activate

the MOR, as previously illustrated.^57,61E_maxvalues of all

ligands were measured relative to that of the maximal

stimulated response produced by the full MOR agonist

DAMGO. VZMC013 produced minimal stimulation of the

In Vitro MOR Radioligand Binding Studies. All bivalent

ligands were ﬁrst tested for their binding aﬃnity to the MOR.

The competitive radioligand binding assay was conducted

using monoclonal mouse MOR expressed in CHO cell lines

(mMOR-CHO). VZMC013 showed high MOR binding

aﬃnity (K_ivalue of 6.05 nM; Table 1), which was 8.6-fold

higher than that of VZMC001 (K_i= 51.8 nM), suggesting that

the structure-based reselection of the attachment point on the

CCR5 pharmacophore exerted partial inﬂuence on MOR

binding as well. Moreover, VZMC013 possessed relatively

higher binding aﬃnity than that of VZMC017 (K_i= 11.2 nM),

while similar to that of VZMC019 (K_i= 4.23 nM), indicating

that the chosen spacer length range was suitable to preserve

ligand binding aﬃnity to the MOR. The monovalent ligand

VZMC002 displayed a slightly lower MOR aﬃnity compared

to VZMC013 (Table S1). VZMC013 possessed a relatively

lower binding aﬃnity for the MOR compared to the parent

MOR and displayed insigniﬁcant apparent eﬃcacy (%E_max

=

9.22%), similar to those of the previously designed bivalent

ligand VZMC001 and the parent compound naltrexone (Table

2). VZMC017 also showed minimal eﬃcacy (%E_max= 4.27%),

Table 2. [³⁵S]GTPγS Binding Results

compounds

% E_maxof DAMGO

a

VZMC013

VZMC017

VZMC019

VZMC001

naltrexone

9.22 0.94

a

4.27 0.66

a

19.4 1.94

b

11.7 1.2

c

7.75 0.20

a

The values are the mean

SEM of at least three independent

b

experiments. Data have been reported in ref 32 and are presented

here for comparison. See ref 57.

c

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whereas VZMC019 exhibited slightly higher eﬃcacy (%E_max

=

S3a), which was 12-fold greater than the monovalent

compound VZMC014 (K_i= 41 nM) (Table S2 and Figure

S3b). In addition, VZMC013 possessed a relatively higher

binding aﬃnity for the CCR5 than VZMC017 and VZMC019.

Most importantly, VZMC013 showed a dramatically improved

binding aﬃnity for the CCR5 than VZMC001,³³demonstrat-

ing the success of the attachment point relocation on the

CCR5 pharmacophore via the structure-based molecular

design approach.

Calcium Mobilization Assay Results in HOS-CCR5

Cells. We then conducted the calcium mobilization assay to

further test both the agonist and antagonist properties of the

newly synthesized compounds in the HOS-CCR5 (stably

transfected for the expression of CCR5) cells using our

reported protocol,^33,34as calcium mobilization is associated

with the activation of CCR5. Prior to the assay, the HOS-

CCR5 cells were transiently transfected with a Gqi5 to boost

calcium signaling levels. As expected, none of these

compounds appeared to activate CCR5 within the range of

concentrations tested (represented by VZMC013 and

VZMC014, Figure S4a) compared to the control C−C motif

chemokine ligand 5 (CCL5/RANTES) under the same

conditions (CCL5 exhibited an agonism proﬁle with an EC₅₀

value of 155 6.58 nM, Figure S4a). In the antagonism assay,

these compounds were assessed for their ability to inhibit

CCL5-stimulated Ca²⁺mobilization. As shown in Table 5 and

19.4%) but was still a low-eﬃcacy partial agonist relative to

DAMGO. Overall, the results indicated that VZMC013 may

act as an MOR antagonist as designed, comparable to the

MOR neutral antagonist naltrexone.

Calcium Mobilization Assay Results in mMOR-CHO

Cells. The pharmacological proﬁles of the three newly

prepared MOR bivalent ligands were further characterized

for their eﬀects on MOR-dependent intracellular Ca²⁺signaling

in an mMOR-CHO cell line that was transfected with chimeric

Gqi5 protein as previously described.⁶¹No agonism was

observed for VZMC013 at varying concentrations (Figure

S2a), compared with the known MOR agonist DAMGO (EC₅₀

= 36.3

1.85 nM). Meanwhile, VZMC013 inhibited

DAMGO-induced increases in intracellular Ca²⁺concentration

[Ca²⁺]_ieﬀectively with moderate potency (Figure S2b and

Table 3). Additionally, VZMC013 showed comparable

Table 3. Inhibition of DAMGO Induced Ca²⁺Mobilization

compounds

IC₅₀(nM)

a

VZMC013

VZMC017

VZMC019

VZMC001

naltrexone

50.0 2.45

74.4 3.20

a

1103 7.11

b

40.0 4.8

a

6.62 1.45

a

The values are the mean

SEM of at least three independent

b

Table 5. Inhibition of CCL5-Stimulated Intracellular Ca²⁺

experiments. Data have been reported in ref 33 and are presented

a

here for comparison purposes.

Mobilization

compounds

IC₅₀(nM)

potency to the previously developed bivalent ligand

VZMC001 (IC₅₀= 40.0 nM) in inhibiting DAMGO-

stimulated Ca²⁺mobilization which conformed to our

molecular design. Moreover, VZMC013 was more potent

VZMC013

VZMC017

VZMC019

VZMC001

maraviroc

57.5 4.87

965 30.5

1260 77.6

b

126 28

than VZMC017 (IC₅₀= 74.4 nM) and VZMC019 (IC₅₀

=

0.77 0.20

1103 nM), further suggesting the critical role of spacer length

in designing bivalent ligands. In summary, the results of the

[³⁵S]GTPγS functional assay and the ability to inhibit

DAMGO-stimulated calcium mobilization demonstrated that

VZMC013 is a potent MOR antagonist.

a

The values are the mean

SEM of at least three independent

b

experiments. Data have been reported in ref 33 and are presented

here for comparison purposes.

In Vitro CCR5 Radioligand Binding Studies. To verify

the binding aﬃnity of the bivalent ligands to the CCR5, the

competitive radioligand binding assay was conducted in

recombinant rhesus macaque CCR5-expressing Chem-1 cells,

and macrophage inﬂammatory protein 1 beta (MIP-1β) was

used as a control. VZMC013 exhibited a reasonably high

binding aﬃnity with a K_ivalue of 3.29 nM (Table 4 and Figure

Figure S4b, VZMC013 demonstrated two-digit nanomolar

inhibitory potency (IC₅₀= 57.5 nM) and was 2.2-fold more

potent than VZMC001 (IC₅₀= 126 nM).³³Thus, as predicted,

the positional switch in the spacer attachment on maraviroc in

VZMC013 improved its inhibition of CCR5 agonist-induced

calcium mobilization relative to VZMC001. This prediction

was further supported by the enhanced potency of VZMC014

(IC₅₀= 116 nM) compared to the monovalent ligand

VZMC003 (IC₅₀= 622 nM)³³(Table S3 and Figure S4b).

VZMC013 also demonstrated a much higher potency than

those of VZMC017 and VZMC019, indicating the importance

of spacer length. In brief, VZMC013 acted as a potent CCR5

antagonist.

Anti-HIV-1_BaLActivity and Cytotoxicity of VZMC013

in GHOST CCR5 Cells. To further characterize the capacity of

VZMC013 to block HIV-1 entry by occupying the HIV

binding site on the CCR5, we utilized a well-established HIV-1

entry assay, in which the ability for small molecules to inhibit

HIV-1 entry is measured as a decrease in HIV-1 reverse

transcriptase (RT) activity that is equal to a decrease in

radioactivity output after standard radioactive incorporation of

tritiated thymidine triphosphate (needed for the synthesis of

viral DNA).⁶²This assay was run in GHOST-CCR5 cells using

a

Table 4. CCR5 Radioligand Binding Aﬃnity

b

compounds

K_i(nM)

VZMC013

VZMC017

VZMC019

VZMC001

MIP-1β

3.29 0.29

5.70 0.23

c

ND

239 56

d

0.056 0.006

a

b

[

¹²⁵I]MIP-1α was used as the radioligand in the binding assay. K_i

values were calculated using the Cheng−Prusoﬀ equation. The values

c

are the mean SEM of at least three independent experiments. Its

IC₅₀value is higher than 10,000 nM, the highest concentration tested.

d

Data have been reported in ref 33 and are presented here for

comparisons. It should be noted that the assay methods employed in

both studies are similar to minor diﬀerences.

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the CCR5-tropic viral strain HIV-1_BaL. Compounds

VZMC001, VZMC002, VZMC013, and VZMC014 (maravir-

oc tested as the control) were subjected to this assay. The

treatment concentrations for each compound were 0 and from

0.001 to 100 μM; in which the RT activity at 0 μM of tested

compound was deﬁned as 100%, and the results were

expressed as EC₅₀values. As shown in Table 6, the previously

HIV-1_BaLentry and integration under both circumstances

(Figure 3a,b). More importantly, VZMC013 showed nearly 2-

Table 6. Inhibition of HIV-1_BaLand Cytotoxicity in GHOST

a

CCR5 Cells

compounds

EC₅₀(μM)

TC₅₀(μM)

TI

VZMC013

VZMC001

maraviroc

0.093 0.004

>100

0.018 0.002

>100

>0.5

>1075

−

>28

a

Measured in triplicate.

developed bivalent ligand VZMC001 did not show any

signiﬁcant inhibition of HIV-1 entry at concentrations up to

100 μM (RT activity: 107.6−226.1%), whereas the newly

designed bivalent ligand VZMC013 acted as a potent inhibitor

in preventing HIV-1 entry, with an EC₅₀value of 0.093 μM.

The results from this assay clearly indicated that our structure-

based design strategy has yielded a bivalent ligand with a

markedly improved ability to inhibit HIV-1 entry in cells

compared to the previously designed bivalent compound, and

the proper selection of an attachment site on the CCR5

pharmacophore was critical for inhibiting HIV-1 entry.

These compounds were concurrently assessed for cytotox-

icity in GHOST-CCR5-expressing cells using the XTT assay.

The treatment concentrations for each compound were the

same as in the anti-HIV assay in which the viability of

untreated cells was deﬁned as 100%, and the results were

interpreted as TC₅₀(a 50% reduction in cell viability). The

therapeutic index (TI, TC₅₀/EC₅₀) values were calculated if

applicable. As depicted in Table 6 and Table S4, neither

VZMC013 nor other compounds exhibited cytotoxicity at any

concentration ≤100 μM (cell viability ∼100%). The bivalent

ligand VZMC013 possessed a TI value >1075.

Inhibitory Eﬀect of VZMC013 on HIV-1_BaLEntry into

MOR-CCR5 Coexpressed TZM-bl Cells. CCR5-expressing

TZM-bl cells coexpress an HIV-1 long-terminal repeat (LTR)-

ﬁreﬂy luciferase reporter that can be activated by trans-

activator of transcription (Tat) in cells expressing HIV.

Therefore, increases or decreases in pro-viral gene expression

coincide with increases or decreases, respectively, in luciferase

activity. To initially determine whether the coexpression of

MOR aﬀected HIV-1 entry/expression, OPRM1- or control

plasmid-transfected TZM-bl cells were exposed to varying

concentrations of HIV-1_BaL, and HIV-1 entry was calculated

based on LTR-driven luciferase activity. As seen in Figure S5,

viral entry in TZM-bl cells expressing MOR was enhanced

compared to non-MOR-expressing TZM-bl cells upon

exposure to identical amounts of virus. Our ﬁndings indicate

that MOR coexpression by itself can be suﬃcient to enhance

the infectivity of R5-tropic HIV in CCR5-expressing cells,

which could possibly result from a putative MOR-CCR5

heterodimer in addition to the CCR5 mediating the virus entry

into host cells.

Figure 3. Comparison of % inhibition of infection by VZMC013 in

(a) control plasmid, (b) OPRM1, and (c) OPRM1 (with 100 μM of

morphine added) transfected TZM-bl cells. % Inhibition of infection

was related to the decrease of RLU. Data analysis was performed

using GraphPad Prism version 8.0.1 for Windows.

fold higher inhibitory potency of virus infection in TZM-bl

cells containing OPRM1 (EC₅₀= 368 nM) than that of those

cells containing only control plasmid (EC₅₀= 610 nM). The

results further suggested that the CCR5 may heterodimerize

with the MOR, and the putative MOR-CCR5 complex may

play an essential role in facilitating viral entry. In turn, our

bivalent ligand VZMC013 may eﬃciently bind to the putative

MOR-CCR5 heterodimers expressed in TZM-bl cells and

more eﬀectively block HIV entry.

Furthermore, when morphine (100 μM) was added (Figure

3c), the ability of VZMC013 to inhibit HIV infectivity was

enhanced in TZM-bl cells containing OPRM1 (EC₅₀= 247

nM) compared to OPRM1-expressing TZM-bl cells without

morphine (EC₅₀= 368 nM). Therefore, this ﬁnding indicates

that morphine can exacerbate HIV-1 entry in an MOR-

dependent manner and that our bivalent ligand VZMC013 is

Next, the inhibitory eﬀect of HIV-1_BaLinfection of the

bivalent compound VZMC013 in OPRM1 and control plasmid

transfected TZM-bl cells was measured and compared.

VZMC013 demonstrated submicromolar potency in inhibiting

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more potent at suppressing viral entry in the presence of

morphine. This further supported our hypothesis that

functional MOR-CCR5 dimers may be inhibited eﬀectively

by a properly designed bivalent ligand.

Inhibitory Eﬀect of VZMC013 on HIV-1 Entry to PBMC

Cells. Peripheral blood mononuclear cells (PBMCs) include

large numbers of CCR5-expressing leukocytes and are

susceptible to infection by R5-tropic HIV-1 strains. To

determine whether VZMC013 would alter HIV infectivity in

nonstimulated PBMCs, we used an HIV-1_BaLEnv-pseudotyped

virus encoding a recombinant ﬁreﬂy luciferase gene (HIV-1-

Luc). VZMC013 inhibited HIV-1-Luc infectivity in a

concentration-dependent manner in PBMCs with a nearly

complete blockade at a concentration of 1000 nM (Figure 4a).

These results were as expected since VZMC013 has shown

potent CCR5 antagonism proﬁles in the radioligand binding

and calcium mobilization assays.

Inhibitory Eﬀect of VZMC013 on HIV-1 Entry into

PHA-Stimulated PBMCs. Phytohemagglutinin (PHA) can

stimulate PBMCs into cell cycle, and PHA-stimulated PBMCs

have been widely utilized for investigating the replication of

HIV primary isolates in vitro.⁶³The ability of VZMC013 to

inhibit HIV-1 entry into PHA-stimulated PBMCs was further

tested. As seen in Figure 4b, exposure to VZMC013 (100 nM)

inhibited viral entry, with a 22% reduction in luminescence.

While exposure to morphine (10 nM) or DAMGO (10 nM)

led to remarkable increases in HIV expression in PHA-

stimulated PBMCs, VZMC013 (100 nM) was more eﬀective

in preventing viral entry, displaying 55% or 69% decreases in

HIV expression, respectively, in cells coexposed to morphine

or DAMGO and HIV. These results were in agreement with

our ﬁndings evaluating HIV-1_BaLentry into MOR-CCR5

coexpressing TZM-bl cells discussed above. That is, MOR

agonists may enhance HIV invasion through activation of the

MOR-CCR5 dimer, while a bivalent ligand speciﬁcally

inhibiting the heterodimer may eﬀectively block the viral

invasion.

Molecular Dynamics Simulation Studies on VZMC013

with the MOR-CCR5 Heterodimer. To delineate the

possible binding mode between the bivalent ligand

VZMC013 and the putative MOR-CCR5 heterodimer, we

implemented molecular modeling studies including molecular

docking and MD simulation. Previous studies of the crystal

packing interactions of GPCRs revealed that the postulated

and observed homodimer interfaces of GPCRs involved TM1/

TM2/TM7, TM4/TM5, and TM5/TM6.⁶⁴The crystal

structure study of the MOR indicated that two diﬀerent

interfaces, TM1/TM2/helix 8 and TM5/TM6, were involved

in the dimerization of the MOR and TM5/TM6 is a more

prominent interface observed in the ligand-bound MOR crystal

structure.⁴⁶Moreover, TM5/TM6 as the interface of the MOR

was also observed in the MD simulation studies performed by

Meral et al.⁶⁵In our case, the attachment point for the spacer

of the bivalent ligands designed, that is, C6-position of

naltrexone pointed toward the TM5/TM6 in the MOR

binding pocket. Based on such observations, TM5/TM6 was

selected as the interface of the MOR to dimerize with the

CCR5. For the CCR5, Jin et al. conducted a site-direct

mutagenesis study and found that mutations with lysine on

TM5 and TM6 did not completely prevent the dimerization of

CCR5. Thus, they speculated that other interfaces have a

higher possibility to be the interface of the CCR5.⁶⁶Moreover,

the computational study together with the site-direct muta-

Figure 4. (a) Varying concentrations of VZMC013 were added

before PBMC cells were infected by HIV-1_BaLEnv-pseudotyped Luc-

expressing virus (HIV-1-Luc, 1.5 ng p24/well). Luciferase content was

expressed as RLU. Null: PBMC cells were infected by HIV-1-Luc,

with no ligand added. (b) PHA-stimulated PBMCs were exposed to

opioids 3 days prior to infection with HIV-1_BaLEnv-pseudotyped Luc-

expressing virus. Morphine (Morph) and DAMGO were used at a

concentration of 10 nM. Bivalent compound VZMC013 (100 nM)

was added 1 h before the cultures were infected with HIV and

maintained in vitro. RLU values of each treatment group were

normalized based on the null group (RLU values of the null group

were deﬁned as “1”). Null: PHA-stimulated PBMC cells were infected

by HIV-1-Luc, with no ligand added. Statistical analysis was

performed using GraphPad Prism version 8.0.1 for Windows. Data

were analyzed according to one-way ANOVA followed by Newman−

Keuls posthoc test; *p < 0.05 and **p < 0.01 are considered as

statistically signiﬁcant and ns: not signiﬁcant.

genesis study performed by Zhang et al. revealed that the

CCR5 homodimerization involved TM1, TM2, TM3, and

TM4.⁶⁷In the present study, considering the fact that the

attachment point for the spacer of the bivalent ligands

designed, that is, the 3′-methyl group on the 1,2,4-triazol

moiety of maraviroc pointed toward TM1, TM2, and TM3,

and the possible steric hindrance of extracellular loop 1

(ECL1) between TM2 and TM3, TM1/TM2 was therefore

selected as the plausible dimer interface of the CCR5. An

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Figure 5. (a) RMSD and (b) RMSF of the backbone atoms of the proteins in the MOR-CCR5_VZMC013 complex. The CCR5 portion was

labeled in green text, and the MOR portion was labeled in blue text.

MOR-CCR5 heterodimer model in which TM5/6 of the MOR

and TM1/2 of the CCR5 were selected as dimer interfaces in

complexing with VZMC013 (Figure S6a) was inserted into a

membrane-aqueous sodium chloride solution system (Figure

S6b) and subjected to further MD simulations.

amino acid residues from the two proteins within a 5 Å

proximity of the bivalent ligand VZMC013 (Figure 6 and

Table S5). Among them, 19 residues were associated with the

CCR5 pharmacophore, and 10 of them were in juxtaposition

to the MOR pharmacophore and the remaining 10 to the

spacer. As shown in Figure 6, the CCR5 pharmacophore

portion of VZMC013 interacted with amino acid residues from

all seven TM helices of CCR5, while the MOR pharmacophore

portion of VZMC013 occupied the bottom of a hydrophobic

pocket formed mainly by residues from TM3, TM6, and TM7

of the MOR. Residues from TM1 and TM2 of the CCR5 and

TM5 and TM6 of the MOR contributed mainly hydrophobic

interactions with the spacer.

After 100 ns MD simulations, the root-mean-square

deviation (RMSD) of the backbone atoms of the proteins

was applied to evaluate the dynamic equilibrium of the ligand−

receptor complex. As shown in Figure 5a, for the MOR-

CCR5_VZMC013 complex, the average RMSD value for

backbone atoms in the protein during the 50−100 ns MD

simulation was 2.84 Å. An average RMSD value of <3.0 Å for

the backbone atoms of the protein is reported to indicate

stable binding.⁶⁸Therefore, the average RMSD value

conﬁrmed the thermodynamic equilibrium of the MOR-

CCR5_VZMC013 complex after 100 ns MD simulations.

Furthermore, the root-mean-square ﬂuctuation (RMSF)

values of the backbone atoms of the protein in the MOR-

CCR5_VZMC013 complex are plotted in Figure 5b.

Apparently, residues located in the seven TMs of the MOR-

CCR5 dimer displayed a more stable conformation than other

domains of the two proteins, for example, intracellular loops

(ICLs) and extracellular loops (ECLs). This observation

agreed with the fact that the transmembrane spanning domains

of GPCRs generally form more rigid and stable conformations

than those of ICLs or ECLs. RMSF values obtained from 100

ns MD simulations may further substantiate the stability of the

MOR-CCR5_VZMC013 complex. Therefore, the conforma-

tions of the MOR-CCR5_VZMC013 complex after 100 ns

MD simulations were selected for further analyses.

For comparison purposes, the docking mode of naltrexone,

the MOR pharmacophore of VZMC013, in the inactive MOR

was depicted ﬁrst (Figure S7a and Table S6). It seemed that

naltrexone may form hydrophobic interactions with residues

M151^3.36, W293^6.48, I296^6.51, H297^6.52, I322^7.39, and Y326^7.43

.

The protonated nitrogen atom at the 17-position of naltrexone

formed an ionic interaction with the oxygen atom at the side

chain of D147^3.32. A hydrogen-bonding interaction was formed

between the dihydrofuran oxygen atom of naltrexone and the

phenolic group of Y148^3.33. Similar interactions were also

observed in the crystal structure of β-FNA bound to the

MOR.⁴⁶In the binding of VZMC013 with the MOR-CCR5

heterodimer, the MOR pharmacophore moved closer to TM5

and TM6 of the MOR. This conformational change may

somehow weaken the ionic interaction with D147^3.32, the

hydrogen-bonding interaction with Y148^3.33, and the hydro-

phobic interactions with residues W293^6.48and Y326^7.43. The

distance analyses in Table S7 further supported these

After MD simulations, the stable binding mode of the MOR-

CCR5_VZMC013 complex showed that there were mainly 39

observations. On the other hand, residues L232^5.38, V236^5.42

,

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Therefore, the relatively lower binding aﬃnity of VZMC013

than maraviroc to the CCR5 seemed reasonable.

On the other hand, the putative movement of the CCR5

pharmacophore toward TM1 and TM2 of the CCR5 may

result in amino acid residues C178^ECL2, S179^ECL2, S180^ECL2

,

and F182^ECL2from ECL2 to form stronger interactions with

the CCR5 pharmacophore, whereas those interactions seemed

less obvious in the crystal structure of maraviroc binding to

CCR5 (Table S6). Among these, residues C178^ECL2and

F182^ECL2formed hydrophobic interactions, and residues

S179^ECL2and S180^ECL2formed polar interactions with the

CCR5 pharmacophore. These interactions were also supported

by the distance analyses listed in Table S7. In previous studies,

it has been demonstrated that the interaction between the

ECL2 of CCR5 and the V3 loop of HIV gp120 was critical to

the process of HIV accessing the host cell membrane.^44,69

Particularly, residues R11 and S13 of the V3 loop were

involved in polar interactions with residues S179^ECL2and

S180^ECL2from the ECL2 of CCR5 (Figure S8, which showed

the putative binding between V3 loop of HIV gp120 and

CCR5).^70,71Therefore, the CCR5 pharmacophore interacted

with the ECL2 of CCR5, which may eﬀectively inhibit the V3

loop of gp120 binding to the ECL2 of CCR5 and further block

the attachment of HIV to the host cell.

Figure 6. Binding mode of VZMC013 in the MOR-CCR5

heterodimer complex after MD simulations. The MOR-CCR5

heterodimer (top ﬁgure) is shown as a cylinder model. The MOR-

CCR5 heterodimer (bottom ﬁgure) is shown as a cartoon model. The

key residues within a 5 Å proximity of VZMC013 are shown as

surface models in the top ﬁgure and stick models in the bottom ﬁgure,

respectively. Carbon atom: The key residues directly interacted with

the MOR were colored in cyan; and the key residues directly

interacted with the CCR5 were colored in light-blue. Compound

VZMC013 is shown as a stick and ball model (magenta).

CONCLUSIONS

■

Utilizing the available ligand-bound crystal structures of the

MOR and CCR5, we successfully designed and developed a

new bivalent ligand VZMC013 targeting putative MOR-CCR5

heterodimers. VZMC013 demonstrated prominent binding

aﬃnities for both MOR and CCR5 at nanomolar levels, which

were much higher aﬃnities than those of our previously

reported bivalent ligand VZMC001. [³⁵S]GTPγS and calcium

mobilization assays of MOR function conﬁrmed VZMC013 to

be a potent MOR antagonist as designed. In addition,

VZMC013 acted as a CCR5 antagonist and inhibited CCL5-

stimulated Ca²⁺transients in HOS-CCR5 cells more potently

than VZMC001, and this ﬁnding was further supported by its

signiﬁcantly improved anti-HIV-1_BaLactivity at the CCR5 HIV

coreceptor. Moreover, VZMC013 showed more potent

inhibitory activity of viral infection in TZM-bl cells

coexpressing CCR5 and MOR than in TZM-bl cells expressing

CCR5 alone, implying that the presence of MOR and putative

MOR-CCR5 heterodimeric complexes enhances HIV entry

and suggesting that MOR complexation with CCR5

fundamentally alters the functional properties of CCR5 as an

HIV coreceptor. Most importantly, VZMC013 was able to

block opioid-accelerated HIV-1 invasion more eﬀectively in

TZM-bl cells and PHA-stimulated PBMC cells than in controls

(lacking opioids), further suggesting its promising role in

inhibiting opioid exacerbated HIV-1 infectivity. Utilizing

molecular docking and MD simulation approaches, a possible

binding mode of VZMC013 in the newly constructed MOR-

CCR5 heterodimer model was postulated and helped explain

the underlying mechanism of inhibition of viral infection by

VZMC013. In summary, VZMC013 is a potent chemical

probe that can be used to investigate the speciﬁc functional

role of putative MOR-CCR5 heterodimers in viral entry and

may also serve as a pharmacological agent to alleviate opioid-

dependent increases in HIV entry. We believe that the

knowledge retrieved from this practice may be applicable to

designing novel bivalent chemical probes targeting other

V300^6.55, and W318^7.35seemed to form additional hydrophobic

interactions with the MOR pharmacophore, which partially

compensated for those weakened interactions. This provided a

plausible explanation for the reasonably high binding aﬃnity of

VZMC013 to the MOR.

From the crystal structure of maraviroc bound to CCR5,

maraviroc binds to the pocket formed by residues from TM1−

TM3 and TM5−TM7 of CCR5 (Figure S7b and Table S6).⁴⁴

E283^7.39formed an ionic interaction with the protonated

nitrogen atom of the tropane moiety. Five hydrogen bonds

were formed between maraviroc and the CCR5: two hydrogen

bonds between 1′-N, 2′-N of the triazole moiety and residues

Y37^1.39, Y89^2.63respectively, one hydrogen bond between the

nitrogen atom of the carboxamide group and Y251^6.51, and dual

hydrogen bonds between one ﬂuorine atom on the cyclo-

hexane ring and residues T195^5.39, T259^6.59. Moreover, the

phenyl group of maraviroc formed hydrophobic interactions

with Y108^3.32, F109^3.33, F112^3.36, W248^6.48, and Y251^6.51. In

comparison, an examination of the VZMC013 and MOR-

CCR5 heterodimeric binding complex revealed that the spacer

induced a rotation of the triazole moiety (the dihedral angle

changed from 177.7° shown in Figure S7b to 102.0° shown in

Figure S7c). Due to this rotation, the two hydrogen-bonding

interactions between the triazole moiety of the CCR5

pharmacophore and residues Y37^1.39and Y89^2.63of CCR5

seemed no longer possible. Moreover, the CCR5 pharmaco-

phore moved closer to the TM1 and TM2 of the CCR5, which

may slightly move the CCR5 pharmacophore away from its

original binding position thereby decreasing the hydrophobic

interactions between the phenyl group of maraviroc and

residues Y108^3.32, F109^3.33, F112^3.36, W248^6.48, and Y251^6.51

.

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Na₂SO₄, ﬁltered, and concentrated to provide a colorless oil. The oil

was dissolved in MeOH (4 mL), and the pH was adjusted to 13 with

2 M NaOH at 0 °C. The resulting mixture was stirred at this

temperature for 4 h. Five mL of water was added, and then the

solution was acidiﬁed to pH 1 with 2 M HCl. The mixture was

ﬁltered, and the precipitate was washed with water and then

redissolved in dichloromethane. The organic layer was dried over

Na₂SO₄, ﬁltered, and concentrated to oﬀer 7 as a white solid (0.24 g,

GPCR dimerization and characterizing their function and

pharmaceutical applications.

EXPERIMENTAL SECTION

■

Chemistry. All reagents were purchased from commercial

suppliers and with no further puriﬁcation when used. TLC analyses

were performed on the Analtech Uniplate F254 plates. Spots were

visualized by irradiation with UV light (λ 254 nm) and iodine vapor.

Flash column chromatography was carried out on columns packed

1

80%). H NMR (400 MHz, DMSO-d₆): δ 7.90 (d, J = 8.60 Hz, 1H,

exchangeable), 7.38−7.28 (m, 10H, Ph-H), 5.00−4.96 (m, 3H),

1

2.74−2.64 (m, 2H). H NMR (400 MHz, CDCl₃): δ 7.33−7.25 (m,

with silica gel (230−400 mesh, Merck). H NMR (400 MHz) and

¹³C NMR (100 MHz) spectra were obtained at ambient temperature

10H, Ph-H), 5.71−5.70 (m, 1H, exchangeable), 5.17−5.03 (m, 3H),

2.97−2.84 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 175.9, 155.7,

140.4, 136.2, 128.8, 128.5, 128.2, 127.8, 126.2, 67.1, 51.4, 40.2.

HRMS (ESI) m/z calcd for C₁₇H₁₆NO₄[M − H]⁻: 298.1079, found:

298.1081.

with tetramethylsilane (TMS) as the internal standard on a Bruker

Ultrashield 400 Plus spectrometer (Bruker, Germany). ¹⁹F NMR (376

MHz) spectra were not externally calibrated, and chemical shifts are

given as received from the automatic data processing with

MestReNova. Chemical shifts were expressed in δ units (ppm), and

J values were reported in hertz (Hz). HRMS spectra were acquired

from a PerkinElmer Flexar UHPLC with AxION 2 time of ﬂight

(TOF) mass spectrometer (PerkinElmer, USA). Analysis of the

sample purity was performed on a Varian Prostar 210 high-

performance liquid chromatography (HPLC) system using a column

Agilent Microsorb-MV 100-5 C18 column (250 × 4.6 mm). HPLC

eluent conditions: acetonitrile/water (with 0.1% triﬂuoroacetic acid),

ﬁxed at 40%/60%, or acetonitrile increased from 40% to 100% in

gradient within 20 min of test. Flow rate, 0.5 mL/min; UV detection,

210 nm; temperature, ambient; injection volume, 5 μL. The purity of

all ﬁnal compounds was identiﬁed as ≥95%.

Preparation of Compound 9. 3-Amino-3-phenylpropanoic

acid (4). To a mixture of malonic acid (1.56 g, 15 mmol) and

ammonium formate (1.58 g, 25 mmol) was added a solution of

benzaldehyde 3 (1.06 g, 10 mmol) in 30 mL of ethanol. The resulting

mixture was reﬂuxed for 5 h and then cooled to ambient temperature.

The mixture was stirred overnight and ﬁltered. The precipitate was

washed with cool ethanol to give 4 as a white solid (0.97 g, 59%). ¹H

NMR (400 MHz, D₂O): δ 7.52−7.44 (m, 5H), 4.66−4.63 (m, 1H),

2.94−2.79 (m, 2H). C₉H₁₁NO₂(165.0790).

Methyl 3-Amino-3-phenylpropanoate (5). To a solution of 3-

amino-3-phenylpropanoic acid 4 (7.74 g, 46.8 mmol) in MeOH (45

mL) cooled in an ice−water bath was dropwise added 5 mL of

concentrated sulfuric acid. The resulting mixture was warmed up to

ambient temperature and stirred for 6.5 h. The excess solvent was

removed under reduced pressure, 100 mL of dichloromethane was

added to the residue, and then the mixture was cooled to 0 °C. The

pH of the solution was adjusted to 10 using 2 M NaOH. After

separation and further extraction with dichloromethane, the organic

layers were combined, washed with water and brine, dried over

Na₂SO₄, and ﬁltered. The ﬁltrate was concentrated to provide 5 as a

yellow oil (8.31 g, 99%). ¹H NMR (400 MHz, CDCl₃): δ 7.38−7.32

(m, 4H), 7.28−7.24 (m, 1H), 4.42 (t, J = 6.4 Hz, 1H), 3.68 (s, 3H),

2.67 (d, J = 7.6 Hz, 2H), 1.73 (s, 2H). C₁₀H₁₃NO₂(179.0946).

Methyl (S)-3-Amino-3-phenylpropanoate ^L-(+)-Tartaric Acid Salt

(5·^L-(+)-Tartaric Acid) (6). A solution of L-(+)-tartaric acid (5.43 g,

36.2 mmol) in MeOH (42 mL) was heated to 45 °C. Then, a solution

of methyl 3-amino-3-phenylpropanoate 5 (6.48 g, 36.2 mmol) in

MeOH (13 mL) was added. The resulting mixture was cooled to

ambient temperature and was stirred overnight. The mixture was

ﬁltered, and the precipitate was washed with cool MeOH and

recrystallized in MeOH for two times, aﬀording 6 as a white

crystalline solid (2.98 g, 25%). [α]²_D⁰= −20.84 (c 1.12, CHCl₃).

C₁₄H₁₉NO₈(329.1111).

Benzyl (S)-(3-Hydroxy-1-phenylpropyl)carbamate (8). To a

solution of 7 (1.87 g, 6.24 mmol) in anhydrous THF (10 mL) was

added borane tetrahydrofuran complex solution (1.0 M in THF, 24.9

mmol) under N₂protection at 0 °C. The resulting mixture was stirred

for an additional 1 h at room temperature. Two mL of acetone and 20

mL of water were added. The excess solvent was removed under

reduced pressure, and 50 mL of saturated sodium bicarbonate

solution was then added. The mixture was extracted with ethyl acetate

(30 mL × 3). The combined organic extracts were washed with

saturated sodium bicarbonate solution (50 mL), followed by 0.1 M

HCl (30 mL) and brine (50 mL). After being dried over Na₂SO₄and

ﬁltered, the ﬁltrate was concentrated to yield a crude product, which

was recrystallized with ethyl acetate and hexane to furnish 8 as a white

1

powder (1.03 g, 58%). H NMR (400 MHz, CDCl₃): δ 7.36−7.27

(m, 10H, Ph-H), 5.39−5.38 (m, 1H), 5.15−5.03 (m, 2H), 4.96−4.95

(m, 1H), 3.70−3.66 (m, 2H), 2.63 (brs, 1H, OH), 2.12−2.04 (m,

1H), 1.92−1.84 (m, 1H). ¹³C NMR (100 MHz, CDCl₃): δ 156.6,

141.7, 136.3, 128.8, 128.7, 128.6, 128.2, 127.6, 126.4, 67.0, 59.2, 52.6,

39.1. HRMS (ESI) m/z calcd for C₁₇H₁₉NNaO₃[M + Na]⁺:

308.1263, found: 308.1277.

Benzyl (S)-(3-Oxo-1-phenylpropyl)carbamate (9). Sulfur trioxide

pyridine complex (1.73 g, 10.85 mmol) was added to a solution of 8

(0.619 g, 2.17 mmol) and triethylamine (2.196 g, 21.7 mmol) in

DMSO/dichloromethane (10 mL/10 mL) at 0 °C. The mixture was

stirred for 0.5 h. Water (100 mL) was added, and the mixture was

extracted with ethyl acetate (30 mL × 3). The combined organic

layers were washed with 0.5 M HCl and brine, dried over Na₂SO₄,

ﬁltered, and concentrated. Further puriﬁcation of the residue by

column chromatography led to 9 as a yellowish solid (0.53 g, 87%).

¹H NMR (400 MHz, CDCl₃): δ 9.72 (s, 1H, CHO), 7.35−7.27 (m,

10H, Ph-H), 5.45−5.43 (m, 1H, NH), 5.26−5.24 (m, 1H), 5.12−5.00

(m, 2H), 3.03−2.88 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ 199.9,

155.6, 140.6, 136.2, 128.9, 128.5, 128.2, 128.1, 127.9, 126.3, 67.0,

50.6, 49.5. IR (diamond, cm⁻¹): 3317.00, 3031.58, 2922.38, 1693.15,

1585.06, 1525.06, 1496.85, 1454.47, 1405.38, 1338.29, 1242.68,

1049.96, 1027.24, 913.83, 738.70. HRMS (ESI) m/z calcd for

C₁₇H₁₇NNaO₃[M + Na]⁺: 306.1106, found: 306.1105; calcd for

C₁₈H₂₁NNaO₄[M + MeOH + Na]⁺: 338.1368, found: 338.1395.

Preparation of Compound 13. Methyl 3-Aminopropanoate

Hydrochloride (11). Thionyl chloride (10.12 g, 75 mmol) was slowly

added to methanol (25 mL) at 0 °C. The resulting mixture was stirred

for 20 min. After the addition of 3-aminopropanoate 10 (2.67 g, 30

mmol), the mixture was stirred at room temperature for 14 h. The

solvent was evaporated to give a crude salt, which was further

recrystallized using methanol/ether to aﬀord 11 as a white solid (2.60

g, 62%). C₄H₁₀ClNO₂(139.0400).

(S)-3-(((Benzyloxy)carbonyl)amino)-3-phenylpropanoic Acid (7).

To a solution of 6 (0.33 g, 1.0 mmol) in dichloromethane (1.6 mL)

was added sodium carbonate aqueous solution (0.35 g of sodium

carbonate dissolved in 1.5 mL of H₂O) at 0 °C. Benzyl chloroformate

(0.21 g, 1.2 mmol) was then dropwise added in 1 min, and upon

completion of the addition, the reaction mixture was warmed up to

ambient temperature and was stirred at room temperature for 5 h.

The water phase was extracted with dichloromethane (5 mL × 3), and

the combined organic layers were washed with brine, dried over

Methyl 3-((((9H-Fluoren-9-yl)methoxy)carbonyl)amino)-

propanoate (12). Methyl 3-aminopropanoate hydrochloride 11

(0.56 g, 4 mmol) was placed in 10 mL of dichloromethane, and the

mixture was cooled in an ice−water bath. Triethylamine (1.50 g, 15

mmol) and a solution of Fmoc-Cl (1.09 g, 4.2 mmol) in

dichloromethane (10 mL) were added in sequence. The resulting

mixture was stirred at ambient temperature for 1.5 h, and then the

mixture was treated with 0.5 N HCl. The organic layer was washed

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with saturated sodium bicarbonate solution (30 mL), brine (30 mL),

dried over Na₂SO₄, ﬁltered, and concentrated. The crude product was

puriﬁed by column chromatography to supply 12 as a white solid

(1.05 g, 81%). ¹H NMR (400 MHz, CDCl₃): δ 7.77 (d, J = 7.52 Hz,

2H, ﬂuorene-H), 7.59 (d, J = 7.40 Hz, 2H, ﬂuorene-H), 7.40 (t, J =

7.40 Hz, 2H, ﬂuorene-H), 7.32 (td, J₁= 7.44 Hz, J₂= 0.72 Hz, 2H,

ﬂuorene-H), 5.30 (brs, 1H, NH), 4.39 (d, J = 6.96 Hz, 2H, OCH₂),

4.21 (t, J = 6.92 Hz, 1H, CH), 3.71 (s, 3H, OCH₃), 3.48 (q, J = 6.00,

2H, CH₂), 2.57 (t, J = 5.84, 2H, COCH₂). ¹³C NMR (100 MHz,

CDCl₃): δ 172.9, 156.3, 143.9, 141.3, 127.7, 127.1, 125.1, 120.0, 66.8,

51.8, 47.3, 36.6, 34.2. C₁₉H₁₉NO₄(325.1314).

aqueous layers were basiﬁed with 5 M NaOH to pH 10. The resulting

mixture was extracted with ethyl acetate (20 mL × 3). The combined

organic layers were washed with brine, dried over Na₂SO₄, ﬁltered,

and concentrated to provide 17 as an oil (0.31 g, 72%), which was

1

used for the next step without further puriﬁcation. H NMR (400

MHz, DMSO-d₆): δ 8.07 (brs, 2H, NH₂), 7.36−7.30 (m, 4H), 7.26−

7.21 (m, 1H), 3.58 (s, 2H, NCH₂), 3.31−3.22 (m, 1H), 3.17 (s, 2H),

1.97−1.94 (m, 2H), 1.73−1.70 (m, 4H), 1.57−1.52 (m, 2H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 139.8, 128.3, 128.1, 126.7, 57.0, 54.0,

43.0, 34.1, 26.3. HRMS (ESI) m/z calcd for C₁₄H₂₁N₂[M + H]⁺:

217.1705, found: 217.1710.

N-(8-Benzyl-8-azabicyclo[3.2.1]octan-3-yl)isobutyramide (18).

To a stirring mixture of 8-benzyl-8-aza-bicyclo[3.2.1]octan-3-amine

17 (0.22 g, 1.0 mmol) in dichloromethane (6 mL) was added sodium

carbonate solution (0.30 g, 2.8 mmol, in 6 mL of H₂O). A solution of

isobutyryl chloride (0.13 g, 1.2 mmol) in dichloromethane (3 mL)

was then added dropwise at 0 °C. The resulting mixture was warmed

up to room temperature and stirred for 0.5 h. The mixture was

adjusted to pH = 9 with saturated sodium bicarbonate solution and

extracted with dichloromethane (5 mL × 3). The combined organic

layers were washed with 1 M NaOH (5 mL), brine, dried over

Na₂SO₄, ﬁltered, and concentrated. The residue was recrystallized

with ethyl acetate/hexane to furnish 18 as a white solid (0.14 g, 49%).

¹H NMR (400 MHz, CDCl₃): δ 7.37−7.36 (m, 2H, Ph-H), 7.33−

7.29 (m, 2H, Ph-H), 7.26−7.22 (m, 1H, Ph-H), 5.25 (d, J = 6.64 Hz,

1H, NH), 4.20−4.09 (m, 1H), 3.53 (s, 2H, CH₂), 3.22 (brs, 2H),

2.27 (hept, J = 6.88 Hz, 1H, CH), 2.05−2.02 (m, 2H), 1.83−1.78 (m,

2H), 1.75−1.70 (m, 2H), 1.53−1.47 (m, 2H), 1.12 (d, J = 6.88 Hz,

6H, CH₃× 2). ¹³C NMR (100 MHz, CDCl₃): δ 176.4, 140.0, 128.7,

128.3, 127.0, 59.0, 56.5, 41.2, 38.7, 35.9, 26.5, 19.7. HRMS (ESI) m/z

calcd for C₁₈H₂₇N₂O [M + H]⁺: 287.2123, found: 287.2132.

(9H-Fluoren-9-yl)methyl 2-(4-(8-benzyl-8-azabicyclo[3.2.1]-

octan-3-yl)-5-isopropyl-4H-1,2,4-triazol-3-yl)ethyl)carbamate (19).

To a solution of 18 (0.69 g, 2.4 mmol) in 15 mL of anhydrous

dichloromethane over 1 h. After being stirred at room temperature for

5 h, the mixture was treated with addition of a solution of (9H-

ﬂuoren-9-yl)methyl(3-hydrazinyl-3-oxopropyl)carbamate 13 (0.60 g,

1.85 mmol) in tert-amyl alcohol (15 mL) over 1 h at 0 °C. The

resulting mixture was stirred at room temperature for 16 h. The

mixture was then treated with saturated sodium bicarbonate solution

(15 mL) and extracted with dichloromethane (30 mL × 3). The

combined organic layers were washed with brine, dried over Na₂SO₄,

and concentrated to ca. 10 mL. After the addition of acetic acid (1.2

mL), the mixture was heated at 85 °C for 2 h. The reaction was

quenched by the addition of saturated sodium bicarbonate solution

(50 mL) and extracted with ethyl acetate (50 mL × 3). The combined

organic phases were washed with brine, dried over Na₂SO₄, and

concentrated. The residue was further puriﬁed by column

chromatography to give 19 as a white crystalline solid (0.31 g,

29%). ¹H NMR (400 MHz, DMSO-d₆): δ 7.88 (d, J = 7.52 Hz, 2H),

7.67 (d, J = 7.44 Hz, 2H), 7.55 (t, J = 5.68 Hz, 1H, exchangeable),

7.43−7.38 (m, 4H), 7.33−7.27 (m, 4H), 7.20 (t, J = 7.24 Hz, 1H),

4.33−4.30 (m, 2H), 4.29−4.20 (m, 2H), 3.55 (brs, 2H), 3.40−3.36

(m, 2H), 3.23−3.17 (m, 3H), 2.96 (t, J = 7.48 Hz, 2H), 2.12−2.03

(m, 4H), 1.73−1.68 (m, 4H), 1.28 (d, J = 6.76 Hz, 6H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 158.3, 156.1, 151.0, 143.8, 140.6, 139.8,

128.1, 128.0, 127.5, 126.9, 126.5, 125.0, 120.0, 65.4, 58.3, 55.4, 46.6,

36.5, 26.3, 25.8, 25.0, 21.9. IR (Diamond, cm⁻¹): 3320.83, 2933.60,

2875.13, 2160.60, 1979.45, 1712.86, 1507.73, 1449.25, 1349.55,

1312,41, 1247.11, 1138.87, 1099.09, 1071.14, 1029.43, 969.64,

920.53, 876.36, 844.60, 797.40, 758.76, 738.58, 696.58. C₃₆H₄₁N₅O₂

(575.3260).

(9H-Fluoren-9-yl)methyl (3-hydrazinyl-3-oxopropyl)carbamate

(13). The intermediate 12 (0.32 g, 1.0 mmol) was suspended in

methanol (2.5 mL). A solution of hydrazine monohydrate (0.25 g, 5

mmol) in methanol (2.5 mL) was added, and the mixture was stirred

at room temperature for 30 h. The solvent was removed, and the

residue was reslurried in ethyl acetate and ﬁltered to aﬀord 13 as a

white solid (0.16 g, 49%). ¹H NMR (400 MHz, DMSO-d₆): δ 9.01 (s,

1H), 7.89 (d, J = 7.48 Hz, 2H), 7.69 (d, J = 7.36 Hz, 2H), 7.42 (t, J =

7.36 Hz, 2H), 7.37−7.30 (m, 3H), 4.28 (d, J = 6.76 Hz, 2H), 4.23−

4.19 (m, 1H), 3.22−3.17 (m, 2H), 2.21 (t, J = 7.36 Hz, 2H). ¹³C

NMR (100 MHz, DMSO-d₆): δ 169.8, 157.1, 142.5, 139.4, 137.4,

128.9, 127.3, 121.3, 120.0, 109.7, 37.0, 34.2. C₁₈H₁₉N₃O₃(325.1426).

Preparation of the 2′-Aminoethyl Maraviroc Precursor 24.

8-Benzyl-8-azabicyclo[3.2.1]octan-3-one (15). Tetrahydro-2,5-di-

methoxyfuran (1.32 g, 10 mmol) was placed in 10 mL of 0.1 M

HCl and the mixture was stirred at room temperature for 6 h. Then

benzylamine (1.29 g, 12 mmol) was mixed with 10 mL of water and 1

mL of concentrated hydrochloric acid, which was added to the above

mixture at 0 °C. The resulting mixture was adjusted to pH 4 using 1

M sodium acetate solution. After the introduction of 1,3-

acetonedicarboxylic acid 14 (1.46 g, 10 mmol), the resulting mixture

was stirred at room temperature overnight before being ﬁltered. The

ﬁltrate was washed with ether, followed by being adjusted to pH 10

with 2 N NaOH. The mixture was extracted with ethyl acetate (50

mL × 3). The combined organic layers were washed with brine, dried

over Na₂SO₄, ﬁltered, and concentrated. The residue was further

puriﬁed by column chromatography to supply 15 as an oil (1.03 g,

1

48%). H NMR (400 MHz, CDCl₃): δ 7.43−7.41 (m, 2H), 7.36−

7.32 (m, 2H), 7.29−7.25 (m, 1H), 3.75 (s, 2H), 3.51−3.48 (m, 2H),

2.69 (dd, J₁= 16.08 Hz, J₂= 4.44 Hz, 2H), 2.23−2.18 (m, 2H), 2.14−

2.07 (m, 2H), 1.66−1.60 (m, 2H). ¹³C NMR (100 MHz, CDCl₃): δ

210.8, 139.9, 128.9, 127.6, 59.1, 55.7, 48.8, 28.3. C₁₄H₁₇NO

(215.1310).

8-Benzyl-8-azabicyclo[3.2.1]octan-3-one oxime (16). Hydroxyl-

amine hydrochloride (1.03 g, 14.9 mmol) and pyridine (1.29 g, 16.3

mmol) were added to a solution of 15 (3.20 g, 14.9 mmol) in 60 mL

of ethanol. The resulting mixture was heated to reﬂux overnight and

allowed to cool to room temperature. The mixture was then diluted

with saturated sodium carbonate solution. After ﬁltration, the ﬁltrate

was evaporated under reduced pressure to remove the excess solvent.

The residue was treated with water (100 mL) and extracted with

dichloromethane (60 mL × 3). The combined organic layers were

washed with brine, dried over Na₂SO₄, ﬁltered, and concentrated. The

residue was recrystallized with ethanol to aﬀord 8-benzyl-8-

azabicyclo[3.2.1]octan-3-one oxime 16 as a crystalline solid (1.74 g,

51%). ¹H NMR (400 MHz, DMSO-d₆): δ 10.31 (s, 1H, OH), 7.40−

7.38 (m, 2H), 7.34−7.30 (m, 2H), 7.26−7.22 (m, 1H), 3.61 (s, 2H,

NCH₂), 3.26−3.22 (m, 2H), 2.85 (d, J = 15.16 Hz, 1H), 2.44 (dd, J₁

= 14.40 Hz, J₂= 3.04 Hz, 1H), 2.06−2.00 (m, 2H), 1.96−1.90 (m,

2H),1.49−1.45 (m, 1H), 1.36−1.29 (m, 1H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 153.3, 139.8, 128.3, 128.1, 126.7, 58.0, 57.3, 54.6, 36.9,

31.0, 27.4, 26.5. HRMS (ESI) m/z calcd for C₁₄H₁₉N₂O [M + H]⁺:

231.1497, found: 231.1502.

(9H-Fluoren-9-yl)methyl 2-(4-(8-azabicyclo[3.2.1]octan-3-yl)-5-

isopropyl-4H-1,2,4-triazol-3-yl)ethyl)carbamate (20·Tosylate). A

solution of compound 19 (0.46 g, 0.80 mmol) in anhydrous methanol

(10 mL) was hydrogenated in the presence of 30% Pd−C (0.14 g)

and p-toluenesulfonic acid monohydrate (0.15 g, 0.80 mmol) under a

hydrogen atmosphere (60 psi) at room temperature for 24 h. The

mixture was ﬁltered through Celite, and the ﬁltrate was concentrated

to aﬀord 20 as a tosylate salt, which was directly used for the next step

8-Benzyl-8-azabicyclo[3.2.1]octan-3-amine (17). To a stirring

solution of 16 (0.46 g, 2 mmol) in 8 mL of 1-pentanol was added

sodium (0.46 g, 20 mmol) in portions at 120 °C. The resulting

mixture was stirred at this temperature for 5 h and then allowed to

cool to 0 °C in an ice−water bath. The mixture was acidiﬁed to pH =

2 with 6 M HCl and then extracted with 6 M HCl. The combined

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due to a stability issue of the free amine. HRMS (ESI) m/z calcd for

C₂₉H₃₆N₅O₂[M + H]⁺: 486.2869, found: 486.2881; calcd for

C₂₉H₃₅N₅NaO₂[M + Na]⁺: 508.2688, found: 508.2701.

26.9, 26.8, 22.3, 22.23, 22.20. ¹⁹F NMR (376 MHz, CD₃OD): δ

−103.31, −102.67, −93.67, −93.04. HRMS (ESI) m/z calcd for

C₄₅H₅₅F₂N₆O₃[M + H]⁺: 765.4304, found: 765.4280; calcd for

C₄₅H₅₄F₂N₆NaO₃[M + Na]⁺: 787.4123, found: 787.4097.

Benzyl((S)-3-(3-(3-(2-((((9H-Fluoren-9-yl)methoxy)carbonyl)-

amino)ethyl)-5-isopropyl-4H-1,2,4-triazol-4-yl)-8-azabicyclo[3.2.1]-

octan-8-yl)-1-phenylpropyl)carbamate (21). To a mixture of the 20·

tosylate salt from the last step and compound 9 (0.25 g, 0.88 mmol)

in dichloromethane (10 mL) were added acetic acid (0.28 mL) and

sodium triacetoxyborohydride (0.19 g, 0.88 mmol) in sequence. After

being stirred at room temperature overnight, the reaction was

quenched by addition of saturated sodium bicarbonate solution, and

the mixture was extracted with dichloromethane (30 mL × 3). The

combined organic layers were washed with brine, dried over Na₂SO₄,

and concentrated. The residue was puriﬁed by column chromatog-

N-((S)-3-(3-(3-(2-Aminoethyl)-5-isopropyl-4H-1,2,4-triazol-4-yl)-

8-azabicyclo[3.2.1]octan-8-yl)-1-phenylpropyl)-4,4-diﬂuorocyclo-

hexane-1-carboxamide (24). A mixture of compound 23 (0.10 g,

0.13 mmol) in 20% piperidine/DMF (4 mL) was stirred at room

temperature for 1 h. The mixture was then concentrated under

reduced pressure to get a crude residue. The residue was further

puriﬁed by column chromatography to aﬀord 24 as a white solid (30

mg, 42%). ¹H NMR (400 MHz, CDCl₃): δ 7.38−7.34 (m, 2H),

7.29−7.27 (m, 3H), 6.38 (d, J = 7.36 Hz, 1H), 5.16 (q, J = 7.32 Hz,

1H), 4.35−4.26 (m, 1H), 3.49 (s, 1H), 3.40−3.35 (m, 2H), 3.23 (t, J

= 6.24 Hz, 2H), 3.05−2.93 (m, 3H), 2.43 (t, J = 6.72 Hz, 2H), 2.29−

2.12 (m, 5H), 2.07−1.93 (m, 4H), 1.90−1.77 (m, 6H), 1.66−1.61

1

raphy to supply 21 as a white solid (0.32 g, two-step yield: 53%). H

NMR (400 MHz, DMSO-d₆): δ 7.88 (d, J = 7.48 Hz, 2H), 7.86−7.81

(m, 1H), 7.65 (d, J = 7.36 Hz, 2H), 7.46−7.39 (m, 3H), 7.32−7.19

(m, 12H), 5.06−4.94 (m, 2H), 4.80−4.74 (m, 1H), 4.30 (d, J = 6.96

Hz, 2H), 4.23−4.17 (m, 1H), 3.42−3.35 (m, 2H), 3.30−3.28 (m,

1H), 3.25−3.22 (m, 2H), 3.20−3.17 (m, 1H), 2.88 (t, J = 7.12 Hz,

2H), 2.34−2.89 (m, 2H), 2.05−1.99 (m, 2H), 1.92−1.75 (m, 4H),

1.67−1.65 (m, 4H), 1.27−1.25 (m, 6H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 158.5, 156.1, 155.5, 150.9, 143.84, 143.83, 140.7,

128.25, 128.20, 128.15, 127.7, 127.61, 127.55, 127.0, 126.6, 126.3,

125.1, 120.1, 79.3, 78.9, 78.6, 66.4, 65.4, 65.1, 58.7, 58.3, 53.0, 46.8,

46.7, 36.5, 25.2, 22.0. HRMS (ESI) m/z calcd for C₄₆H₅₃N₆O₄[M +

H]⁺: 753.4128, found: 753.4135; calcd for C₄₆H₅₂N₆NaO₄[M +

Na]⁺: 775.3948, found: 775.3945.

(m, 4H), 1.39 (dd, J₁= 6.80 Hz, J₂= 1.20 Hz, 6H), 1.26 (s, 1H). ¹³

C

NMR (100 MHz, CDCl₃): δ 173.2, 159.1, 141.9, 128.9, 127.6, 126.5,

77.2, 59.1, 58.3, 52.0, 50.9, 48.0, 47.2, 42.9, 39.9, 36.0, 35.8, 34.9,

33.08, 33.05, 32.9, 32.83, 32.81, 32.79, 32.60, 32.56, 30.5, 26.8, 26.7,

26.1, 26.0, 25.9, 21.8, 1.0. ¹⁹F NMR (376 MHz, CDCl₃): δ −92.83,

−93.46, −100.30, −100.93. HRMS (ESI) m/z calcd for C₃₀H₄₅F₂N₆O

[M + H]⁺: 543.3623, found: 543.3630; calcd for C₃₀H₄₄F₂N₆NaO [M

+ Na]⁺: 565.3442, found: 565.3438.

Preparation of the Bivalent Ligands VZMC013, VZMC017,

and VZMC019. Benzyl-(7-aminoheptyl)carbamate (26a). To a

stirring solution of 1,7-diaminoheptane 25a (0.90 g, 6.91 mmol) in

MeOH (80 mL) at 0 °C was dropwise added the solution of benzyl

chloroformate (1.18 g, 6.91 mmol) in dichloromethane (80 mL)

within 5 h while keeping the temperature at 0 °C. The reaction

mixture was stirred at room temperature overnight, and then the

excess solvent was removed under reduced pressure. Dichloro-

methane (100 mL) and water (100 mL) were added, and the mixture

was adjusted to pH 2 with 6 N HCl. The layers were separated. The

aqueous layer was washed with dichloromethane (50 mL × 2), then

adjusted to pH 12 using 10 N NaOH, and extracted with

dichloromethane (50 mL × 3). The combined organic layers were

dried over Na₂SO₄, concentrated, and recrystallized with methanol to

give 26a as a white solid (0.55 g, 30%). ¹H NMR (400 MHz, DMSO-

d₆): δ 7.38−7.28 (m, 5H), 7.20 (brs, 1H, exchangeable), 5.00 (s, 2H),

2.98 (q, J = 2.4 Hz, 2H), 2.53−2.51 (m, 2H), 2.04 (brs, 2H,

exchangeable), 1.41−1.38 (m, 2H), 1.34−1.31 (m, 2H), 1.24 (s, 6H).

¹³C NMR (100 MHz, DMSO-d₆): δ 156.0, 137.3, 128.2, 127.6, 127.6,

65.0, 41.4, 33.0, 29.3, 28.6, 26.3, 26.2. C₁₅H₂₄N₂O₂(264.1838).

Benzyl (9-Aminononyl)carbamate (26b). This compound was

prepared in a similar way as 26a, using 1,9-diaminononane as the

starting material. White solid. Yield: 49%. ¹H NMR (400 MHz,

CDCl₃): δ 7.36−7.29 (m, 5H, Ph-H), 5.09 (s, 2H, CH₂), 4.74 (brs,

1H, exchangeable), 3.18 (q, J = 6.76 Hz, 2H), 2.68 (t, J = 6.96 Hz,

2H), 1.50−1.40 (m, 5H), 1.28 (brs, 11H). ¹³C NMR (100 MHz,

CDCl₃): δ 157.7, 138.9, 130.0, 129.4, 129.3, 66.7, 42.5, 41.7, 33.7,

31.0, 30.6, 30.5, 30.3, 28.0, 27.8. HRMS (ESI) m/z calcd for

C₁₇H₂₉N₂O₂[M + H]⁺: 293.2229, found: 293.2228; calcd for

C₁₇H₂₈N₂NaO₂[M + Na]⁺: 315.2048, found: 315.2038.

(9H-Fluoren-9-yl)methyl(2-(4-(8-((S)-3-amino-3-phenylpropyl)-8-

azabicyclo[3.2.1]octan-3-yl)-5-isopropyl-4H-1,2,4-triazol-3-yl)-

ethyl)carbamate (22). To a solution of compound 21 (0.10 g, 0.13

mmol) in anhydrous methanol (8 mL) was added 30% Pd/C (0.03

g). The mixture was hydrogenated under a hydrogen atmosphere (60

psi) at room temperature for 20 h. The mixture was ﬁltered through

Celite and washed with methanol. The combined ﬁltrate was

concentrated under reduced pressure to provide 22 as a white solid

(55 mg, 67%). Compound 22 could be used for next-step reaction

1

without any further puriﬁcation. H NMR (400 MHz, DMSO-d₆): δ

7.90−7.84 (m, 3H), 7.59 (d, J = 7.36 Hz, 1H), 7.44−7.26 (m, 8H),

7.20 (t, J = 7.36 Hz, 1H), 6.29 (s, 1H), 4.31−4.13 (m, 1H), 3.95 (q, J

= 7.52 Hz, 1H), 3.14−3.06 (m, 2H), 2.97−2.81 (m, 3H), 2.47−2.46

(m, 1H), 2.38−2.29 (m, 2H), 2.13−2.00 (m, 2H), 1.94−1.89 (m,

2H), 1.83 (brs, 2H), 1.76−1.64 (m, 6H), 1.47 (d, J = 7.40 Hz, 1H),

1.26 (d, J = 6.72 Hz, 6H). HRMS (ESI) m/z calcd for C₃₈H₄₇N₆O₂

[M + H]⁺: 619.3760, found: 619.3725.

(9H-Fluoren-9-yl)methyl (2-(4-(8-((S)-3-(4,4-diﬂuorocyclohex-

ane-1-carboxamido)-3-phenylpropyl)-8-azabicyclo[3.2.1]octan-3-

yl)-5-isopropyl-4H-1,2,4-triazol-3-yl)ethyl)carbamate (23). A mix-

ture of 4,4-diﬂuorocyclohexanecarboxylic acid (27 mg, 0.16 mmol),

EDCI (31 mg, 0.16 mmol), HOBt (22 mg, 0.16 mmol), trimethyl-

amine (33 mg, 0.32 mmol), and 4 Å molecular sieves in anhydrous

dichloromethane (3 mL) was stirred at 0 °C for 0.5 h. Then,

compound 22 (50 mg, 0.08 mmol) in dichloromethane (1 mL) was

slowly added to the mixture. The reaction temperature was warmed to

room temperature and stirred overnight. The mixture was ﬁltered, and

the ﬁltrate was concentrated under reduced pressure to get crude

residue. The residue was further puriﬁed by column chromatography

Benzyl (5-Aminopentyl)carbamate (26c). This compound was

prepared in a similar way as 26a, using cadaverine as the starting

1

material. White solid. Yield: 31%. H NMR (400 MHz, CDCl₃): δ

1

to get compound 23 as a white foam solid (33 mg, 54%). H NMR

7.36−7.28 (m, 5H, Ph-H), 5.09 (s, 2H), 4.90 (brs, 1H, exchangeable),

3.19 (q, J = 6.44 Hz, 2H), 2.69 (t, J = 6.80 Hz, 2H), 2.21 (brs, 2H,

exchangeable), 1.55−1.43 (m, 4H), 1.38−1.31 (m, 2H). HRMS (ESI)

m/z calcd for C₁₃H₂₁N₂O₂[M + H]⁺: 237.1603, found: 237.1609.

3,13-Dioxo-1-phenyl-2,15-dioxa-4,12-diazaheptadecan-17-oic

Acid (27a). To a stirring solution of benzyl-(7-aminoheptyl)-

carbamate 26a (2.76 g, 10.46 mmol) in THF (20 mL) was added

diglycolic anhydride (1.27 g, 10.98 mmol) in three portions. The

resulting mixture was stirred at room temperature for 21 h. The excess

solvent was removed under reduced pressure, and the residue was

recrystallized by ethyl acetate/hexane to provide 27a as a white solid

(400 MHz, CD₃OD): δ 7.68 (d, J = 7.60 Hz, 2H), 7.49 (d, J = 7.48

Hz, 2H), 7.29−7.11 (m, 9H), 4.97 (t, J = 7.48 Hz, 1H), 4.79 (d, J =

3.08 Hz, 1H), 4.36−4.27 (m, 1H), 4.19 (d, J = 6.96 Hz, 1H), 4.05 (t,

J = 6.84 Hz, 1H), 3.54−3.53 (m, 1H), 3.41−3.37 (m, 1H), 3.26−3.23

(m, 2H), 2.95 (t, J = 7.04 Hz, 2H), 2.36−2.31 (m, 1H), 2.29−2.19

(m, 1H), 2.15−2.07 (m, 2H), 2.00−1.83 (m, 6H), 1.75−1.59 (m,

9H), 1.25 (d, J = 6.84 Hz, 6H), 1.19 (s, 2H), 0.82−0.75 (m, 1H). ¹³C

NMR (100 MHz, CD₃OD): δ 176.7, 145.3, 144.0, 142.6, 129.7,

128.8, 128.3, 128.2, 127.73, 127.66, 126.2, 121.0, 78.3, 71.6, 67.9,

61.1, 60.8, 60.4, 56.3, 52.7, 52.6, 44.3, 43.7, 43.6, 40.5, 40.4, 38.4,

37.7, 37.6, 37.54, 37.48, 36.4, 36.3, 36.2, 34.14, 34.09, 34.07, 33.90,

33.89, 33.86, 33.85, 33.81, 33.7, 33.61, 33.58, 27.3, 27.2, 27.04, 26.98,

1

(3.10 g, 78%). H NMR (400 MHz, DMSO-d₆): δ 12.80 (s, 1H,

exchangeable), 7.80 (s, 1H, exchangeable), 7.38−7.28 (m, 5H), 7.19

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(s, 1H, exchangeable), 5.00 (s, 2H), 4.10 (s, 2H), 3.94 (s, 2H), 3.08

(q, J = 6.80 Hz, 2H), 2.98 (q, J = 6.80 Hz, 2H), 1.42−1.37 (m, 4H),

1.24 (s, 6H). HRMS (ESI) m/z calcd for C₁₉H₂₈N₂NaO₆[M + Na]⁺:

403.1845, found: 403.2027.

C₃₇H₄₉N₄O₈[M + H]⁺: 677.3550, found: 677.3562; calcd for

C₃₇H₄₈N₄NaO₈[M + Na]⁺: 699.3370, found: 699.3369.

Compounds 29a−c and 30a−c were prepared as previously

reported.^32,34

Bivalent Ligand VZMC013. The target compound was prepared

following the general amide coupling procedure reported by our

group⁷²by reacting the 2′-aminoethyl maraviroc precursor 24 with

the acid 30a in DMF overnight. The crude product was further

puriﬁed by column chromatography to aﬀord VZMC013 as a white

solid. Yield: 20%. Compound VZMC013 was converted to its

hydrochloride salt for biological assays. ¹H NMR (400 MHz, CDCl₃):

δ 8.18−8.16 (m, 1H), 7.56−7.52 (m, 1H), 7.34−7.27 (m, 4H), 7.04

(brs, 1H), 6.74−6.71 (m, 1H), 6.54 (d, J = 8.16 Hz, 1H), 6.49−6.47

(m, 1H), 5.12−5.07 (m, 1H), 4.43 (d, J = 6.08 Hz, 1H), 4.37−4.28

(m, 1H), 4.05 (s, 1H), 4.02 (s, 2H), 3.98 (s, 3H), 3.74−3.64 (m, 3H),

3.40 (brs, 2H), 3.34−3.25 (m, 5H), 3.11−3.09 (m. 1H), 3.04−3.00

(m, 4H), 2.65−2.60 (m, 2H), 2.43−2.37 (m, 4H), 2.25−2.11 (m,

8H), 2.08−2.05 (m, 2H), 2.02−1.97 (m, 2H), 1.92−1.85 (m, 4H),

1.84−1.74 (m, 5H), 1.67−1.65 (m, 6H), 1.62−1.60 (m, 1H), 1.57−

1.52 (m, 5H), 1.48−1.44 (m, 2H), 1.38 (d, J = 8.84 Hz, 6H), 1.33

(brs, 4H), 1.26 (s, 2H), 0.90−0.79 (m, 2H), 0.54 (d, J = 8.12 Hz,

2H), 0.13 (d, J = 4.56 Hz, 2H). ¹³C NMR (100 MHz, CDCl₃): δ

173.6, 169.0, 168.9, 168.8, 168.6, 159.4, 152.8, 143.5, 142.1, 140.0,

131.0, 129.0, 127.7, 126.7, 119.3, 118.2, 92.1, 71.3, 71.2, 71.1, 70.9,

70.2, 62.6, 59.5, 58.9, 51.9, 50.3, 48.7, 47.7, 47.5, 44.21, 44.19, 44.17,

43.0, 39.2, 39.1, 36.5, 33.0, 29.9, 29.4, 29.3, 28.7, 26.7, 26.6, 26.2,

26.15, 26.07, 26.0, 23.7, 22.9, 21.85, 21.83, 9.6, 4.2, 4.1, 4.0. ¹⁹F NMR

(376 MHz, CDCl₃): δ −92.75, −92.38, −100.27, −100.89. IR

(diamond, cm⁻¹): 3246.08, 2536.28, 2159.05, 2027.61, 1976.79,

1648.71, 1545.36, 1450.68, 1373.09, 1325.41, 1232.43, 1126.20,

1033.83, 962.12, 936.21, 916.27, 877.82, 856.61, 747.87, 702.53.

HRMS (ESI) m/z calcd for C₆₅H₉₃F₂N₁₀O₁₀[M + H]⁺: 1211.7044,

found: 1211.7048; calcd for C₆₅H₉₄F₂N₁₀O₁₀[M + 2H]2+: 606.3561,

found: 606.3527. HPLC purity: 99.43%. Rt: 6.768 min.

3,15-Dioxo-1-phenyl-2,17-dioxa-4,14-diazanonadecan-19-oic

Acid (27b). This compound was prepared in a similar way as 27a.

White solid. Yield: 86%. ¹H NMR (400 MHz, DMSO-d₆): δ 7.85 (t, J

= 5.60 Hz, 1H, exchangeable), 7.39−7.29 (m, 5H), 7.22 (t, J = 5.52

Hz, 1H, exchangeable), 5.00 (s, 2H), 4.10 (s, 2H), 3.94 (s, 2H), 3.09

(q, J = 6.72 Hz, 2H), 2.98 (q, J = 6.72 Hz, 2H), 1.42−1.39 (m, 4H),

1.24 (brs, 10H). ¹³C NMR (100 MHz, DMSO-d₆): δ 171.5, 168.5,

156.0, 137.3, 128.3 (Ph−C × 2), 127.7, 127.7 (Ph−C × 2), 70.2,

67.9, 65.0, 40.2, 38.1, 29.4, 29.1, 28.9, 28.7 (CH₂× 2), 26.3, 26.2.

HRMS (ESI) m/z calcd for C₂₁H₃₁N₂O₆[M − H]⁻: 407.2182, found:

407.2179.

3,11-Dioxo-1-phenyl-2,13-dioxa-4,10-diazapentadecan-15-oic

Acid (27c). This compound was prepared in a similar way as 27a.

White solid. Yield: 70%. ¹H NMR (400 MHz, DMSO-d₆): δ 7.87 (t, J

= 5.28 Hz, 1H, exchangeable), 7.39−7.29 (m, 5H), 7.22 (t, J = 5.52

Hz, 1H, exchangeable), 5.01 (s, 2H), 4.10 (s, 2H), 3.94 (s, 2H), 3.08

(q, J = 6.72 Hz, 2H), 2.98 (q, J = 6.72 Hz, 2H), 1.45−1.37 (m, 4H),

1.27−1.20 (m, 2H). HRMS (ESI) m/z calcd for C₁₇H₂₃N₂O₆[M-

H]⁻: 351.1556, found: 351.1551.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(3′,13′-

dioxo-1′-phenyl-2′,15′-dioxa-4′,12′-diazaheptadecanamido)-

morphinan (28a). The title compound was prepared following the

general amide coupling procedure by reacting acid 27a with 6β-

naltrexamine hydrochloride (prepared according to the method

reported by our group)⁶¹in DMF overnight. The crude product

was further puriﬁed by column chromatography to furnish 28a as a

white solid. Yield: 36%. ¹H NMR (400 MHz, CDCl₃): δ 7.48 (s, 1H),

7.30−7.35 (m, 5H), 6.82 (s, 1H), 6.73 (d, J = 8.04 Hz, 1H), 6.56 (d, J

= 8.08 Hz, 1H), 5.30 (s, 1H), 5.09 (s, 2H), 4.84 (s, 1H), 4.43 (s, 1H),

4.06−4.05 (m, 5H), 3.35−3.27 (m, 2H), 3.21−3.16 (m, 2H), 3.11−

3.06 (m, 1H), 3.01 (s, 1H), 2.66 (m, 2H), 2.37 (m, 2H), 2.21 (m,

2H), 1.68−1.63 (m, 3H), 1.53−1.48 (m, 7H), 1.33 (s, 7H), 0.82 (m,

1H), 0.55−0.54 (m, 2H), 0.15−0.14 (m, 2H). ¹³C NMR (100 MHz,

CDCl₃): δ 168.4, 168.2, 156.0, 142.1, 140.4, 137.3, 128.3, 127.7,

127.6, 118.4, 117.1, 90.5, 70.4, 70.4, 69.6, 65.0, 61.8, 58.4, 50.6, 47.0,

38.2, 30.3, 30.0, 29.3, 29.2, 28.4, 26.4, 26.2, 24.5, 22.2, 9.2, 3.7, 3.5.

HRMS (ESI) m/z calcd for C₃₉H₅₃N₄O₈[M + H]⁺: 705.3863, found:

705.3982.

Bivalent Ligand VZMC017. This target compound was prepared in

a similar way as VZMC013 by coupling the 2′-aminoethyl maraviroc

precursor 24 with 30b. White solid. Yield: 45%. Compound

VZMC017 was converted to its hydrochloride salt for biological

1

assays. H NMR (400 MHz, CDCl₃): δ 8.21−8.15 (m, 1H), 7.57−

7.55 (m, 1H), 7.37−7.27 (m, 4H), 7.17−7.12 (m, 1H), 6.94 (brs,

1H), 6.75−6.71 (m, 1H), 6.56−6.53 (m, 1H), 6.44−6.40 (m, 1H),

5.24 (brs, 1H), 5.14−5.07 (m, 2H), 4.41 (dd, J₁= 5.52 Hz, J₂= 2.00

Hz, 1H), 4.36−4.27 (m, 1H), 4.07−4.00 (m, 4H), 3.98 (s, 3H),

3.80−3.70 (m, 2H), 3.40 (brs, 2H), 3.36−3.29 (m, 3H), 3.28−3.21

(m, 3H), 3.19−3.15 (m, 2H), 3.11−3.10 (m. 1H), 3.06−2.98 (m,

4H), 2.68−2.60 (m, 2H), 2.41−2.38 (m, 3H), 2.21−2.15 (m, 5H),

2.09−1.98 (m, 5H), 1.92−1.76 (m, 8H), 1.67−1.64 (m, 5H), 1.56−

1.47 (m, 8H), 1.38−1.36 (m, 6H), 1.29 (brs, 9H), 0.85−0.79 (m,

1H), 0.56−0.52 (m, 2H), 0.15−0.11 (m, 2H). ¹³C NMR (100 MHz,

CDCl₃): δ 173.5, 168.9, 168.63, 168.57, 168.4, 159.3, 159.1, 152.6,

143.5, 142.0, 139.94, 139.90, 130.7, 128.84, 128.82, 127.58, 127.55,

126.5, 124.32, 124.30, 119.2, 118.1, 118.0, 91.8, 71.0, 70.9, 70.7, 70.1,

62.4, 59.4, 59.3, 59.1, 58.7, 58.3, 57.3, 51.8, 49.80, 49.76, 48.6, 48.3,

47.5, 47.3, 45.2, 43.9, 42.9, 39.6, 39.2, 39.1, 36.3, 36.2, 35.4, 35.0,

33.13, 33.09, 32.9, 32.8, 32.64, 32.60, 31.9, 29.4, 29.1, 28.9, 27.3,

26.70, 26.65, 26.6, 26.5, 26.1, 26.00, 25.96, 25.9, 23.4, 22.6, 21.7, 15.5,

9.5, 4.0, 3.8. ¹⁹F NMR (376 MHz, CDCl₃): δ −92.60, −93.23,

−100.30, −100.93. HRMS (ESI) m/z calcd for C₆₇H₉₇F₂N₁₀O₁₀[M +

H]⁺: 1239.7357, found: 1239.7338; calcd for C₆₇H₉₆F₂N₁₀NaO₁₀[M

+ Na]⁺: 1261.7177, found: 1261.7164; calcd for C₆₇H₉₈F₂N₁₀O₁₀[M

+ 2H]²⁺: 620.3718, found: 620.3594; calcd for C₆₇H₉₇F₂N₁₀NaO₁₀

[M + H + Na]²⁺: 631.3627, found: 631.3517. HPLC purity: 99.96%.

Rt: 7.300 min.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(3′,15′-

dioxo-1′-phenyl-2′,17′-dioxa-4′,14′-diazanonadecanamido)-

morphinan (28b). This compound was prepared in a similar way as

28a by coupling acid 27b with 6β-naltrexamine hydrochloride. White

1

solid. Yield: 61%. H NMR (400 MHz, CD₃OD): δ 7.34−7.27 (m,

5H), 6.63 (d, J = 8.12 Hz, 1H), 6.57 (d, J = 8.16 Hz, 1H), 5.06 (s,

2H), 4.53 (d, J = 7.60 Hz, 1H), 4.06 (s, 2H), 4.05 (s, 2H), 3.80−3.74

(m, 1H), 3.26 (t, J = 7.20 Hz, 2H), 3.14−3.06 (m, 4H), 2.70−2.61

(m, 2H), 2.46−2.36 (m, 2H), 2.29−2.22 (m, 1H), 2.18−2.12 (m,

1H), 1.95−1.86 (m, 1H), 1.61−1.43 (m, 8H), 1.32 (brs, 10H), 0.93−

0.84 (m, 1H), 0.58−0.49 (m, 2H), 0.20−0.12 (m, 2H). ¹³C NMR

(100 MHz, CD₃OD): δ 171.5, 171.4, 158.9, 143.7, 141.9, 138.6,

132.5, 129.5, 128.9, 128.8, 125.4, 120.1, 118.6, 92.9, 71.7, 71.6, 71.5,

67.3, 63.7, 60.3, 52.5, 48.9, 45.3, 41.8, 40.1, 31.9, 31.2, 30.9, 30.6,

30.5, 30.4, 28.0, 27.8, 25.5, 23.5, 10.3, 4.5, 4.2. HRMS (ESI) m/z

calcd for C₄₁H₅₇N₄O₈[M + H]⁺: 733.4176, found: 733.4187; calcd

for C₄₁H₅₆N₄NaO₈[M + Na]⁺: 755.3996, found: 755.3992.

17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6β-(3′,11′-

dioxo-1′-phenyl-2′,13′-dioxa-4′,10′-diazapentadecanamido)-

morphinan (28c). This compound was prepared in a similar way as

28a by coupling acid 27c with 6β-naltrexamine hydrochloride. White

1

solid. Yield: 60%. H NMR (400 MHz, CD₃OD): δ 7.37−7.27 (m,

Bivalent Ligand VZMC019. This target compound was prepared in

a similar way as VZMC013 by coupling the 2′-aminoethyl maraviroc

precursor 24 with 30c.White solid. Yield: 55%. Compound

VZMC019 was converted to its hydrochloride salt for biological

5H), 6.65 (d, J = 8.12 Hz, 1H), 6.59 (d, J = 8.16 Hz, 1H), 5.09 (s,

2H), 4.57 (d, J = 7.56 Hz, 1H), 4.11−4.05 (m, 4H), 3.82−3.76 (m,

1H), 3.32−3.28 (m, 2H), 3.17−3.12 (m, 3H), 2.99 (q, J = 7.24 Hz,

1H), 2.76−2.70 (m, 2H), 2.53−2.45 (m, 2H), 2.36−2.23 (m, 2H),

2.00−1.90 (m, 1H), 1.65−1.32 (m, 10H), 0.98−0.89 (m, 1H), 0.61−

0.58 (m, 2H), 0.23−0.22 (m, 2H). HRMS (ESI) m/z calcd for

1

assays. Hydrochloride salt. H NMR (400 MHz, DMSO-d₆): δ 11.27

(s, 1 H), 10.10 (s, 1H), 9.34 (s, 1H), 8.84 (s, 1H), 8.54 (d, J = 8.36

Hz, 1H), 8.28 (d, J = 8.40 Hz, 1H), 8.14 (t, J = 5.64 Hz, 1H), 8.07 (t,

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J = 5.80 Hz, 1H), 7.62 (d, J = 8.56 Hz, 2H), 7.33 (d, J = 8.52 Hz, 2H),

6.72 (d, J = 8.12 Hz, 1H), 6.65 (d, J = 8.24 Hz, 1H), 6.18 (s, 1H),

4.88−4.82 (m, 1H), 4.76 (d, J = 7.72 Hz, 1H), 4.50−4.40 (m, 1H),

4.23−4.21 (m, 1H), 4.16 (s, 2H), 4.05 (s, 2H), 3.97 (d, J = 3.16 Hz,

3H), 3.85 (d, J = 5.60 Hz, 1H), 3.73 (brs, 1H), 3.57−3.48 (m, 4H),

3.18−3.08 (m, 6H), 3.05−3.03 (m, 2H), 2.97−2.92 (m, 3H), 2.89−

2.83 (m, 2H), 2.75−2.69 (m, 5H), 2.47−2.42 (m, 3H), 2.40−2.34

(m, 2H), 2.28−2.21 (m, 3H), 2.17−2.09 (m, 4H), 2.05−2.02 (m,

1H), 1.89−1.71 (m, 5H), 1.67−1.55 (m, 2H), 1.53−1.43 (m, 6H),

1.29−1.27 (m, 8H), 1.08−1.04 (m, 1H), 0.70−0.65 (m, 1H), 0.62−

0.57 (m, 1H), 0.53−0.48 (m, 1H), 0.44−0.38 (m, 1H). ¹³C NMR

(100 MHz, DMSO-d₆): δ 173.5, 168.8, 168.4, 168.3, 167.8, 159.7,

151.5, 142.1, 141.3, 138.0, 137.1, 129.6, 126.6, 120.6, 119.8, 119.2,

117.9, 89.7, 70.7, 70.6, 70.32, 70.28, 69.7, 61.5, 61.2, 60.3, 56.6, 50.3,

49.8, 48.6, 47.0, 46.7, 46.5, 45.6, 40.8, 40.1, 38.14, 38.09, 32.9, 32.2,

32.0, 30.6, 29.4, 28.9, 28.8, 25.59, 25.50, 25.47, 24.33, 24.31, 23.8,

23.7, 23.49, 23.47, 23.0, 21.5, 11.7, 11.5, 5.7, 5.1, 2.6. ¹⁹F NMR (376

MHz, DMSO-d₆): δ −89.77, −90.40, −98.68, −99.30. ¹⁹F NMR (376

MHz, CDCl₃): δ −92.66, −93.29, −100.32, −100.95. HRMS (ESI)

m/z calcd for C₆₃H₈₉F₂N₁₀O₁₀[M + H]⁺: 1183.6731, found:

1183.6692; calcd for C₆₃H₈₈F₂N₁₀NaO₁₀[M + Na]⁺: 1205.6551,

found: 1205.6507; calcd for C₆₃H₉₀F₂N₁₀O₁₀[M + 2H]²⁺: 592.3405,

found: 592.3318; calcd for C₆₃H₈₉F₂N₁₀NaO₁₀[M + H + Na]²⁺:

603.3314, found: 603.3225. HPLC purity: 99.96%. Rt: 6.867 min.

Preparation of the Monovalent Ligands VZMC014,

VZMC018, and VZMC020. 2-(2-(Methylamino)-2-oxoethoxy)-

acetic Acid (31). To a 2 M solution of methanamine in THF (27.5

mL, 55 mmol) was added diglycolic anhydride (5.80 g, 50 mmol) in

portions. After being stirred for 15 h at room temperature, the

reaction mixture was concentrated under reduced pressure. The

residue was further recrystallized with methanol to aﬀord 31 as a faint

yellow solid (6.11 g, 83%). ¹H NMR (400 MHz, DMSO-d₆): δ 12.80

(brs, 1H, exchangeable), 7.76 (s, 1H, exchangeable), 4.09 (s, 2H),

3.94 (s, 2H), 2.62 (d, J = 4.80 Hz, 3H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 171.3, 169.1, 70.1, 67.8, 25.1. C₅H₉NO₄(147.0532).

Benzyl (7-(2-(2-(Methylamino)-2-oxoethoxy)acetamido)heptyl)-

carbamate (32a). The title compound was prepared following the

general amide coupling procedure by reacting acid 31 with the Cbz-

3H), 3.90−3.88 (m, 2H), 3.57−3.48 (m, 2H), 3.38−3.29 (m, 2H),

3.16−3.08 (m, 7H), 3.03−2.98 (m, 2H), 2.89−2.84 (m, 2H), 2.76 (s,

1H), 2.69 (s, 3H), 2.41−2.32 (m, 3H), 2.26 (s, 4H), 2.19−2.02 (m,

2H), 1.96−1.87 (m, 4H), 1.76−1.67 (m, 4H), 1.64−1.61 (m, 2H),

1.43 (brs, 3H), 1.25 (s, 7H), 1.12−1.19 (m, 1H), 1.02 (t, J = 6.68 Hz,

4H). ¹³C NMR (100 MHz, CD₃OD): δ 176.6, 172.1, 171.9, 171.5,

171.4, 153.4, 144.2, 129.6, 128.3, 127.7, 112.6, 71.55, 71.52, 71.48,

70.1, 66.9, 60.9, 60.4, 59.8, 58.1, 52.7, 50.1, 45.0, 43.7, 42.2, 40.1,

39.7, 38.2, 37.72, 37.69, 36.4, 33.9, 33.8, 30.40, 30.38, 30.0, 28.5, 27.9,

27.3, 27.2, 27.15, 27.06, 27.0, 25.9, 22.3, 15.4, 14.0. ¹⁹F NMR (376

MHz, CD₃OD): δ −93.06, −93.68, −102.67, −103.26. HRMS (ESI)

m/z calcd for C₄₆H₇₂F₂N₉O₇[M + H]⁺: 900.5523, found: 900.2709.

HPLC purity: 99.72%. Rt: 6.583 min.

Monovalent Ligand VZMC018. This target compound was

prepared in a similar way as VZMC014 by coupling the 2′-aminoethyl

maraviroc precursor 24 with 34b. White solid. Yield: 35%.

Hydrochloride salt. ¹H NMR (400 MHz, CDCl₃): δ 8.19 (t, J =

5.76 Hz, 1H), 7.38−7.35 (m, 2H), 7.30−7.28 (m, 3H), 7.11 (t, J =

5.72 Hz, 1H), 6.68−6.50 (m, 2H), 6.31−6.29 (m, 1H), 5.17−5.11

(m, 1H), 4.36−4.26 (m, 1H), 4.05 (s, 2H), 4.03 (s, 2H), 3.99 (s, 3H),

3.77 (q, J = 5.88 Hz, 2H), 3.39−3.34 (m, 4H), 3.29 (q, J = 6.80 Hz,

3H), 3.03−2.98 (m, 3H), 2.89−2.86 (m, 3H), 2.42−2.38 (m, 2H),

2.27 (s, 1H), 2.23−2.11 (m, 6H), 2.07−1.93 (m, 6H), 1.93−1.83 (m,

4H), 1.38 (d, J = 6.80 Hz, 6H), 1.29−1.21 (m, 16H). ¹³C NMR (100

MHz, CDCl₃): δ 173.4, 173.1, 169.2, 168.7, 168.6, 168.5, 168.4,

128.9, 127.7, 126.5, 77.2, 71.3, 71.24, 71.22, 70.8, 70.7, 58.4, 51.8,

47.4, 42.9, 39.20, 39.18, 39.1, 36.3, 36.1, 32.8, 29.71, 29.68, 29.50,

29.45, 29.1, 28.94, 28.92, 26.7, 26.64, 26.62, 26.58, 26.02, 25.95, 25.9,

25.7, 21.7. ¹⁹F NMR (376 MHz, CDCl₃): δ −92.70, −93.33, −100.45,

−101.05. HRMS (ESI) m/z calcd for C₄₈H₇₆F₂N₉O₇[M + H]⁺:

928.5836, found: 928.5843. HPLC purity: 95.89%. Rt: 8.062 min.

Monovalent Ligand VZMC020. This target compound was

prepared in a similar way as VZMC014 by coupling the 2′-aminoethyl

maraviroc precursor 24 with 34c. White solid. Yield: 21%.

1

Hydrochloride salt. H NMR (400 MHz, DMSO-d₆): δ 11.45 (brs,

1H), 10.11 (s, 1H), 8.56−8.54 (m, 1H), 8.14 (s, 1H), 8.06−8.01 (m,

2H), 7.62 (d, J = 8.60 Hz, 2H), 7.35−7.32 (m, 3H), 7.22 (brs, 2H),

7.10 (brs, 2H), 4.88−4.82 (m, 1H), 4.49 (brs, 1H), 4.24−4.23 (m,

1H), 4.15 (s, 2H), 4.05 (s, 2H), 3.92 (s, 3H), 3.16−3.09 (m, 4H),

2.97−2.92 (m, 2H), 2.76 (brs, 3H), 2.66 (d, J = 4.68 Hz, 2H), 2.56−

2.55 (m, 1H), 2.46−2.33 (m, 3H), 2.27−2.13 (m, 6H), 2.08−2.00

(m, 2H), 1.90−1.75 (m, 3H), 1.70−1.56 (m, 2H), 1.51−1.42 (m,

3H), 1.30−1.25 (m, 7H), 1.05 (d, J = 6.12 Hz, 1H). ¹³C NMR (100

MHz, DMSO-d₆): δ 168.9, 168.4, 168.3, 142.1, 141.3, 129.6, 120.5,

89.7, 70.4, 70.3, 69.6, 64.9, 61.6, 56.7, 50.3, 46.5, 45.6, 38.1, 38.04,

38.01, 29.4, 28.90, 28.89, 28.8, 25.1, 23.79, 23.75, 23.7, 23.4, 23.0,

21.2, 15.1, 11.5, 5.7, 5.1, 3.2, 2.6. ¹⁹F NMR (376 MHz, DMSO-d₆): δ

−89.81−90.42, −98.82, −99.46. C₄₄H₆₇F₂N₉O₇(871.5132). HPLC

purity: 98.62%. Rt: 6.589 min.

1

protected amine 26a. White solid. Yield: 47%. H NMR (400 MHz,

DMSO-d₆): δ 7.99−7.96 (m, 2H, exchangeable), 7.38−7.28 (m, 5H),

7.19 (brs, 1H, exchangeable), 5.00 (s, 2H), 3.91 (s, 2H), 3.90 (s, 2H),

3.10 (q, J = 6.80 Hz, 2H), 2.98 (q, J = 6.80 Hz, 2H), 2.65 (d, J = 8.80

Hz, 3H), 1.46−1.38 (m, 4H), 1.25 (brs, 6H). ¹³C NMR (100 MHz,

DMSO-d₆): δ 168.8, 168.2, 156.0, 137.3, 128.2, 127.6, 70.2, 65.0,

38.1, 29.3, 29.1, 28.4, 26.3, 26.1, 25.0. C₂₀H₃₁N₃O₅(393.2264).

Benzyl (9-(2-(2-(Methylamino)-2-oxoethoxy)acetamido)nonyl)-

carbamate (32b). This title compound was prepared in a similar

way as 32a, by reacting acid 31 with amine 26b. White solid. Yield:

36%. ¹H NMR (400 MHz, DMSO-d₆): δ 8.02−7.97 (m, 2H,

exchangeable), 7.39−7.29 (m, 5H, Ph-H), 7.21 (t, J = 5.44 Hz, 1H,

exchangeable), 5.01 (s, 2H), 3.92 (s, 2H), 3.91 (s, 2H), 3.11 (q, J =

6.76 Hz, 2H), 2.98 (q, J = 6.68 Hz, 2H), 2.66 (d, J = 4.72 Hz, 3H),

1.45−1.38 (m, 4H), 1.25 (brs, 10H). HRMS (ESI) m/z calcd for

C₂₂H₃₅N₃NaO₅[M + Na]⁺: 444.2474, found: 444.2461.

Benzyl (5-(2-(2-(Methylamino)-2-oxoethoxy)acetamido)pentyl)-

carbamate (32c). This title compound was prepared in a similar way

as 32a, by reacting acid 31 with amine 26c. White solid. Yield: 27%.

¹H NMR (400 MHz, DMSO-d₆): δ 8.02−7.97 (m, 2H, exchange-

able), 7.39−7.29 (m, 5H, Ph-H), 7.23 (t, J = 5.44 Hz, 1H,

exchangeable), 5.01 (s, 2H), 3.92 (s, 2H), 3.91 (s, 2H), 3.11 (q, J =

6.64 Hz, 2H), 2.99 (q, J = 6.68 Hz, 2H), 2.66 (d, J = 4.68 Hz, 3H),

1.47−1.38 (m, 4H), 1.29−1.21 (m, 2H). C₁₈H₂₇N₃O₅(365.1951).

Compounds 33a−c and 34a−c were prepared as previously

reported.^32,34

In Vitro MOR Radioligand Binding Assay. The competition

binding assay was conducted using monoclonal mouse MOR

expressed in Chinese hamster ovary (CHO) cell lines. In this assay,

30 μg of membrane protein was incubated with the radioligand

[³H]naloxone in the presence of diﬀerent concentrations of tested

compounds in TME buﬀer (50 mM Tris, 3 mM MgCl₂, and 0.2 mM

EGTA, pH 7.4) for 1.5 h at 30 °C. The bound radioligand was

separated by ﬁltration using a Brandel harvester. Speciﬁc (i.e., opioid

receptor-related) binding to the MOR was determined as the

diﬀerence in binding obtained in the absence and presence of 5 μM

of DAMGO. The IC₅₀values were determined and converted to K_i

values using the Cheng−Prusoﬀ equation: K_i= IC₅₀/[1 + ([L*]/

K_D)], where [L*] is the concentration of the radioligand and K_Dis the

K_Dof the radioligand was determined.⁷³

[³⁵S]-GTPγS Functional Assay. [³⁵S]-GTPγS functional assays

were conducted in the mouse MOR cell membrane used for the

receptor binding assays. Membrane protein (10 μg) was incubated

with varying concentrations of test compounds, GDP (15 μM), and

80 pM [³⁵S]-GTPγS in assay buﬀer for 1 h at 30 °C. Nonspeciﬁc

binding was determined with 10 μM unlabeled GTPγS. DAMGO (5

μM) was included in the assay for a maximally eﬀective concentration

Monovalent Ligand VZMC014. The target compound was

prepared following the general amide coupling procedure by reacting

the 2′-aminoethyl maraviroc precursor 24 with 34a. White solid.

1

Yield: 46%. Hydrochloride salt. H NMR (400 MHz, CD₃OD): δ

7.88 (brs, 1H), 7.57 (brs, 1H), 7.25−7.21 (m, 2H), 7.15 (brs, 1H),

4.99−4.95 (m, 1H), 4.43−4.30 (m, 1H), 4.24−4.13 (m, 1H), 3.92 (s,

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of a full agonist for the MOR. After incubation, the bound radioactive

ligand was separated from the free radioligand by ﬁltration through a

GF/B glass ﬁber ﬁlter paper and rinsed three times with ice-cold wash

buﬀer (50 mM Tris-HCl, pH 7.2) using the Brandel harvester. The

results were determined by utilizing a scintillation counter. All assays

were determined in triplicate and repeated at least three times.

Percent DAMGO stimulated [³⁵S]-GTPγS binding was deﬁned as

(net-stimulated binding by ligand/net-stimulated binding by 3 μM

DAMGO) × 100%.

(GraphPad Software, San Diego, CA). All experiments were repeated

at least three times.

Anti-HIV-1_BaLActivity (Reverse Transcriptase Activity) and

Cytotoxicity Assays in GHOST CCR5 Cells. 100 μL of GHOST

CCR5 cells at a density of 1 × 10⁴cells/well in 10% complete DMEM

(10% FBS with 1% ^L-glutamine, and 1% penicillin/streptomycin)

media were plated in a 96-well ﬂat bottom plate and incubated for 24

h at 37 °C/5% CO₂. Following the incubation, 100 μL of each

compound at 6 concentrations were added in triplicate to both

eﬃcacy and toxicity plates followed by 100 μL of HIV-1_BaLat a

predetermined titer to the plates being evaluated for eﬃcacy and 50

μL of complete DMEM to the plates being evaluated for cytotoxicity.

The cultures were incubated for 6 days at 37 °C/5% CO₂. Following

the incubation, 50 μL of cell culture supernatant was removed from

the wells of the eﬃcacy plates for subsequent evaluation of virus

content by reverse transcriptase activity assay. The toxicity plates

being evaluated for cytotoxicity were stained with 2,3-bis(2-methoxy-

4-nitro-5-sulfophenyl)-5-[(phenylamino)carbonyl]-2H-tetrazolium

hydroxide (XTT).

RT activity (EC₅₀values) was measured using standard radio-

activity incorporation polymerization assay. One μL of titrated

thymidine triphosphate (TTP) at 1 Ci/mL was used per enzyme

reaction. Poly rA and oligo dT were prepared at concentrations of 0.5

mg/mL and 1.7 units/mL, respectively, from a stock solution which

was kept at −20 °C. The RT reaction buﬀer was prepared fresh and

consists of 125 μL of 1 M EGTA, 125 μL of dH₂O, 125 μL of 20%

Triton X-100, 50 μL of 1 M Tris (pH 7.4), 50 μL of 1 M DTT, and

40 μL of 1 M MgCl₂. For each reaction, 1 μL of TTP, 4 μL of dH₂O,

2.5 μL of rAdT ,and 2.5 μL of reaction buﬀer were mixed. Ten μL of

this reaction mixture was placed in a round-bottom microtiter plate

with 15 μL of virus containing supernatant. The plate was incubated

at 37 °C in a humidiﬁed incubator for 60−90 min. Following the

incubation, 10 μL of the reaction volume was spotted onto a DEAE

ﬁlter mat (PerkinElmer, catalog no. 1450-522) in the appropriate

plate format, washed 5 times for 5 min each in a sodium (150 mM)

citrate (15 mM) buﬀer (Invitrogen, catalog no. 15557-036), 2 times

for 1 min each in deionized water (ImQuest), 2 times for 1 min each

in 70% reagent alcohol (Fisher, catalog no. L-7168), and then air-

dried. The dried ﬁltermat was placed in a plastic sleeve, and 4 mL of

Opti-Fluor O (PerkinElmer, catalog no. 1205-440) scintillation ﬂuid

was added to each sleeve. Incorporated radioactivity was quantiﬁed

using a Wallac 1450 Microbeta Trilux liquid scintilation counter.

TC₅₀values for the tested compounds were derived by measuring

the reduction of the tetrazolium dye XTT. XTT in metabolically

active cells is metabolized by the mitochondrial enzyme NADPH

oxidase to a soluble formazan product. XTT solution was prepared as

a stock of 1 mg/mL in RPMI-1640 without additives. Phenazine

methosulfate (PMS) solution was prepared at 0.15 mg/mL in DPBS

and stored in the dark at −20 °C. XTT/PMS stock was prepared

immediately before use by adding 40 μL of PMS per mL of XTT

solution. 50 μL of XTT/PMS was added to each well of the plate, and

the plate was incubated for 4 h at 37 °C. The plates were sealed and

inverted several times to mix the soluble formazan product, and the

plate was read at 450 nm (650 nm reference wavelength) with a

Molecular Devices SpectraMax Plus 384 96-well format spectropho-

tometer.

Calcium Mobilization Assay in mMOR-CHO Cells. A CHO cell

line stably expressing the mouse mu opioid receptor (mMOR-CHO)

was used for this assay. The cells were transfected with Gαqi5 for 4 h

and then plated (10,000 cells/well) to black 96-well plates with clear

bottoms (Greiner Bio-One). After 24 h of incubation, the culture

medium was removed, and the cells were washed with assay buﬀer

(50 mL of HBSS, 1 mL of HEPES, 250 μL of probenecid, 50 μL of 1

mM CaCl₂, and 50 μL of 1 mM MgCl₂). The tested compounds were

dissolved in DMSO as a stock solution for the assay (1 M). For

agonist assays, cells were then incubated with 50 μL/well loading

buﬀer (6 mL of assay buﬀer, 24 μL of Fluo4-AM solution

(Invitrogen), and 12 μL of probenecid solution) for 60 min.

Following incubation, diﬀerent concentrations of the tested

compounds were added by a FlexStation3 microplate reader

(Molecular Devices) and read at ex494/em516. Each concentration

was run in triplicate. For antagonism studies, the cells were incubated

with the same loading buﬀer as the agonist assay for 60 min. Then,

diﬀerent concentrations of the tested compounds (20 μL/well) were

manually added to each well followed by another 15 min of

incubation. After that, the solution of DAMGO in assay buﬀer (500

nM) or just assay buﬀer (blank) was added by a FlexStation3

microplate reader (Molecular Devices) and read at ex494/em516.

Each concentration was run in triplicate. The corresponding IC₅₀

value of each compound was calculated by nonlinear regression using

GraphPad Prism 8.0.1 (GraphPad Software, San Diego, CA).

In Vitro CCR5 Radioligand Binding Assay. The CCR5

competitive radioligand binding assay was conducted following

previously reported studies.^74,75Rhesus macaque chemokine CCR5

receptors expressed in Chem-1 cells are assayed in modiﬁed HEPES

buﬀer (50 mM HEPES, 1 mM CaCl₂, 5 mM MgCl₂, 0.2% BSA, pH

7.4). Three μg of membrane protein was incubated with 0.10 nM

[

¹²⁵I]MIP-1α in the presence of six concentrations of tested

compounds (10 μM, 1 μM, 0.1 μM, 10 nM, 1 nM, and 0.1 nM)

for 90 min at 25 °C. Nonspeciﬁc binding is estimated in the presence

of 0.1 μM MIP-1β. Membranes are ﬁltered and washed, and the ﬁlters

are then counted to determine [¹²⁵I]MIP-1α speciﬁcally bound, which

speciﬁc binding was deﬁned as 85% as a historical value. The IC₅₀

values were determined and converted to K_ivalues using the Cheng−

Prusoﬀ equation: K_i= IC₅₀/[1 + ([L*]/K_D)], where [L*] is 0.10 nM

and K_Dis 0.31 nM (historical value).

Calcium Mobilization Assay in HOS-CCR5 Cells. HOS-CCR5

cells were transfected with Gqi5 pcDNA16 using Lipofectamine 2000

(Invitrogen) according to the manufacturer’s recommended proce-

dure and maintained in RPMI 1640 supplemented with 10% fetal

bovine serum, 100 μg/mL penicillin, 100 μg/mL streptomycin, and 1

mg/mL G418 at 37 °C and 5% CO₂. 48 h after transfection, a total of

2,500,000 cells were spun down and brought back up in 8 mL of 50:1

HBSS:HEPES assay buﬀer. Cells were then plated at 25,000 cells per

well into a clear bottom, black 96-well plate (Greiner Bio-one) and 50

μL of Fluo4 loading buﬀer [40 μL of 2 μM Fluo4-AM (Invitrogen),

100 μL of 2.5 mM probenecid, in 5 mL of assay buﬀer] was added to

bring the volume up to 130 μL. After incubating for 45 min, 50 μL of

varying concentrations of tested compounds was added, and the plate

was incubated for an additional 15 min. Plates were then read on a

FlexStation3 microplate reader (Molecular Devices) at 494/516 ex/

em for a total of 120 s. After 16 s of reading, 20 μL of 200 nM CCL5

(Biosource) in assay buﬀer, or assay buﬀer alone, was added to the

wells to bring the total volume up to 200 μL. The changes in calcium

mobilization were monitored, and peak height values were obtained

using SoftMaxPro software (Molecular Devices). Nonlinear regression

curves and IC₅₀values were generated using GraphPad Prism 8.0.1

HIV-1_BaLInfection Assay in MOR-CCR5 Coexpressed TZM-bl

Cells. HIV-1_BaL.01was kindly donated by the NIH AIDS Reagent

Program, NIAID, NIH (by Drs. Suzanne Gartner, Mikulas Popovic,

and Robert Gallo) and expanded in PHA-activated PBMCs. The

amount of viral particles was determined by commercial ELISA for

p24 capsid antigen (Zeptometrix), and aliquots were stored at −80

°C. The TZM-bl, a cell line contained integrated copies of luciferase

under control of the HIV-1 long-terminal repeat (LTR), were kindly

donated by the NIH AIDS Reagent Program, NIAID, NIH (from Drs.

John Kappes and Xiaoyun Wu). Cells were transfected with plasmid

containing GFP-tagged human opioid receptor mu 1 gene (OPRM1;

Origene) or with the original empty vector also containing GFP

(control plasmid; Origene) and selected for 2 weeks with 2 mg/mL

G418 (Invivogen) in DMEM (Hyclone) supplemented with 1×

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MEM nonessential amino acids (Gibco), 10% heat-inactivated FBS

(Hyclone), and 100 U penicillin-0.1 mg/mL streptomycin (Sigma).

Then, GFP+ cells were sorted out twice by ﬂow cytometry

(BDFACSAria model, Becton-Dickinson) and maintained in the

conditions mentioned above with 800 μg/mL G418. For the

infection, cells were detached from 25 cm²ﬂasks with 0.25% Trypsin

(Sigma), washed by centrifugation (400g, 10 min), and viable cells

determined by Trypan blue (Sigma) exclusion. Cells were added in

96-well black walled plate at 1.5 × 10⁴cells/well in 90 μL volume of

DMEM containing 10% heat-inactivated FBS and 100 U penicillin-0.1

mg/mL streptomycin. After 6−10 h incubation at 37 °C in 5%

CO₂:95% air, the bivalent compound VZMC013 was added 1 h

before infection with HIV-1_BaLat 460 pg p24/well, in a ﬁnal volume

of 100 μL per well. Luciferase activity was evaluated following the

addition of 100 μL/well of 2× luciferase substrate containing lysis

buﬀer (Bright-Glo Luciferase Substrate, Promega) after 2−3 days of

infection. Luminescence, reported as relative luminescence units

(RLU), was evaluated in PHERAstar FS plate reader, and results

expressed as percentage of inhibition relative to vehicle-treated,

infected cells (100% infection).

HIV-1-Luc Infection Assay in Peripheral Blood Mononuclear

Cells. Isolated human PBMCs were obtained commercially from the

New York Blood Bank and exempt from Human Subjects criteria

(NIH Exemption 4: https://grants.nih.gov/sites/default/ﬁ les/

exemption_infographic_v7_508c-3-21-19.pdf). The adult PBMCs

are publicly available, and no information or identiﬁers are provided

so subjects cannot be identiﬁed.

HIV-1_BaLenv-pseudotyped Luciferase viruses were generated by

the cotransfecting HEK293TT cell line (kindly donated by Dr. John

Schiller, NIAID, NIH) with plasmids containing expression vectors

for HIV-1_BaL.01env and for HIV-1_NL4−3delta-env with Luciferase

inserted into nef gene. After 3 days in culture, supernatants were

harvested, ﬂoating cells removed by centrifugation, HIV-1 p24 Ag

measured by commercial ELISA (Zeptometrix), and viral aliquots

stored at −80 °C. Both plasmids were donated by NIH AIDS Reagent

Program, NIAID, NIH (HIV-1_BaL.01env by Dr. John Mascola and

HIV-1_NL4−3Luc R⁻E⁻by Dr. Nathaniel Landau).

PBMCs were isolated from buﬀy coats of healthy blood donors by

centrifugation in Ficoll gradient (MP Biomedicals). Viable cells,

determined by Trypan blue exclusion, were stimulated, or not, for 2

days with 5 μg/mL PHA (Sigma). Non-PHA stimulated cells were

distributed in 96-well black walled plates at 2 × 10⁵per well in RPMI-

1640 medium supplemented with 1 mM sodium pyruvate (Gibco),

1× MEM nonessential amino acids, 10% heat-inactivated FBS, 100 U

penicillin-0.1 mg/mL streptomycin, and 20 U/mL recombinant

human Interleukin-2 (Peprotech). Cells were infected with HIV-

1_BaL.01env-pseudotyped Luc virus at 1.5 ng p24/well in a ﬁnal volume

of 100 μL, and uninfected cells served as a negative control. The

bivalent compound VZMC013 was added 1 h before infection and

kept in the culture. After 2 days of infection, 100 μL of 2× Luciferase

substrate containing lysing buﬀer (Bright-Glo Luciferase Assay

System, Promega) was added per well, the plate was agitated for 2

min (700 rpm), and Luminescence was evaluated using a PHERAstar

FS plate reader (BMG Labtech).

HIV-1 Infection Assay in PHA-Stimulated PBMC Cells. In a

24-well plate, PHA-stimulated PBMC cells from healthy donors were

infected by incubation with the HIV-1_BaL.01env-pseudotyped Luc

virus. A concentration of HIV-1 p24 50 pg/10⁶cells was used, and

uninfected cells served as a negative control. Cells were treated with

and without morphine or DAMGO (10 nM) along with bivalent

compound VZMC013 (100 nM) 3 days before HIV-1 infection. After

approximately 18−20 h, the supernatant was removed and stored at

−80 °C, and cells were rinsed twice with PBS and lysed. The lysate

was subsequently tested for the relative Tat protein expression by

using a luciferase assay system (Promega). Luciferase activity was

measured using a PHERAstar FS plate reader (BMG Labtech).

Molecular Docking and Molecular Dynamics Simulation. To

generate the MOR-CCR5 heterodimer, the monomeric crystal

structures of CCR5 (PDB ID: 4MBS)⁴⁴and MOR (PDB ID:

4DKL)⁴⁶were downloaded from the Protein Data Bank.⁷⁶Prior to

performing the molecular docking, other parts of the two crystal

structures were removed with the exception of the N-terminus, the

seven transmembrane helices, and the C-terminus. In addition, the

missing residues in intracellular loop 3 (ICL3) of 4MBS and ICL3 of

4DKL were modeled by Sybyl 8.0. Naltrexone was ﬁrst docked into

the MOR to obtain its monomeric complex with MOR, named as

MOR_naltrexone complex. The remaining CCR5 and original ligand

maraviroc obtained the CCR5 monomer complexing with maraviroc,

referred to CCR5_maraviroc complex. Afterward, Hex 8.0.0 was

applied to dock the two complexes together to build the heterodimer

model.⁷⁷The process included four steps: (1) CCR5_maraviroc and

MOR_naltrexone complexes were put together and moved close

enough to each other; (2) CCR5_maraviroc complex was ﬁxed, and

its TM1/2 was selected as the dimer interface; (3) MOR_naltrexone

complex was rotated to ﬁnd its dimer interface at TM5/6 and further

to interact with TM1/2 of the CCR5; and (4) the new coordinates of

CCR5_maraviroc and MOR_naltrexone complexes were obtained

and saved as the starting model of the MOR-CCR5 heterodimer

complexing with their respective ligands.

The above heterodimer model was inserted into a 1-palmitoyl-2-

oleoylphosphatidylcholine (POPC) lipid membrane and solvated by

TIP3 water molecules to conduct an energy minimization

determination using Amber14.0. The energy minimization process

included three steps: (1) restraining the ligands, the heterodimer

complex, and the lipid membrane to minimize the water molecules

and ions; (2) restraining the backbone atoms of the heterodimer, the

ligands, and the lipid membrane to minimize the protein side chains,

the water molecules, and ions; and (3) energy minimizing the whole

system without restraint. The energy minimized heterodimer model

was applied as the starting structure to build the bivalent ligand

VZMC013 into the binding site of the MOR-CCR5 heterodimer.

In the energy minimized heterodimer model, the CCR5

pharmacophore maraviroc and the MOR pharmacophore naltrexone

were both in their respective binding pockets. The molecular graphics

software Sybyl-X 8.0 (TRIPOS Inc., St. Louis, MO) was applied to

connect the two pharmacophores by the spacer displayed in Figure 2c.

Finally, the MOR-CCR5 heterodimer complexing with the bivalent

ligand VZMC013 was obtained and named as the MOR-

CCR5_VZMC013 complex (Figure S6a). With the MOR-

CCR5_VZMC013 complex in hand, the bivalent ligand VZMC013

was ﬁrst extracted from the complex by Sybyl-X 8.0 (TRIPOS Inc., St.

Louis, MO), and then VZMC013 was examined with the correct

atom type, bond type, and hydrogen atoms. Next, the structure of

VZMC013 was optimized to its minimum energy conformation by

Gaussian 03. After that, VZMC013 was submitted to the antechamber

program in AMBER 14 to form its restrained electrostatic potential

charges descriptors and other force ﬁeld parameters. In the following,

a system including the MOR-CCR5_VZMC013 complex, the POPC

lipid membrane, the TIP3 water box, and 0.15 M sodium and chloride

ions was built by the CHARMM-GUI Web site service.⁷⁷Meanwhile,

the input ﬁles of the MD simulations were also generated. MD

simulations were performed by GROMACS (ver. 2018.2) package

(www.gromacs.org). The Amber 03 force ﬁeld was selected for the

simulations.⁷⁸The 20,000 steps of the steepest-descent energy

minimization were initially applied to the system. After that, the

system was heated to 310 K via two steps. First, heating from 0 to 100

K under a constant volume ensemble for 0.5 ns. Second, heating from

100 to 310 K under a constant pressure ensemble (NPT) for another

1.0 ns. Subsequently, a 100 ns MD simulation was conducted using

the NPT (P = 10⁵Pa, T = 310 K) ensemble. The long-range

electrostatic interactions were computed using the particle mesh

Ewald method.⁷⁹A smooth cutoﬀ of 10 Å was used to calculate the

nonbonded van der Waals interactions. The periodic boundary

conditions were applied during the MD simulations. The snapshots

were collected at 2 fs intervals.

7719

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J. Med. Chem. 2021, 64, 7702−7723

Journal of Medicinal Chemistry

pubs.acs.org/jmc

Article

Notes

ASSOCIATED CONTENT

* Supporting Information

The Supporting Information is available free of charge at

https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00408.

■

The content is solely the responsibility of the authors and does

not necessarily represent the oﬃcial views of the National

Institute on Drug Abuse or the National Institutes of Health.

The authors declare no competing ﬁnancial interest.

sı

Molecular formula strings (CSV)

Supplementary ﬁgures and tables, spectra of key

intermediate 24 and target compounds, and HPLC

analysis for target compounds (PDF)

ACKNOWLEDGMENTS

■

The authors are grateful to NIDA Drug Supply Program for

providing the free base of naltrexone. This work was supported

by NIH/NIDA grant R01 DA044855 (Y.Z. and K.F.H.) and

R01 DA034231 (P.E.K. and K.F.H.).

Accession Codes

The binding mode of VZMC013 in the MOR-CCR5

heterodimer complex (PDB). The MOR-CCR5_VZMC013

complex in the membrane-aqueous sodium chloride solution

system (PDB). The binding mode of VZMC013 in the MOR-

CCR5 heterodimer complex after MD simulations (PDB). The

docking pose of naltrexone in the inactive MOR (PDB).

ABBREVIATIONS USED

■

AIDS, acquired immune deﬁciency syndrome; Cbz, carbox-

ybenzyl; CCR5, C−C chemokine receptor type 5; CCL5, C−

C motif chemokine ligand 5; CHO, Chinese hamster ovary;

¹³C NMR, carbon nuclear magnetic resonance; DAMGO, [D-

Ala²-MePhe⁴-Gly(ol)⁵]enkephalin; ECL, extracellular loop;

EDCI, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide;

Fmoc, ﬂuorenylmethyloxycarbonyl; β-FNA, β-funaltrexamine;

¹⁹F NMR, ﬂuorine nuclear magnetic resonance; GPCRs, G-

protein-coupled receptors; HIV, human immunodeﬁciency

AUTHOR INFORMATION

Corresponding Author

Yan Zhang − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States; orcid.org/0000-0001-8934-7016;

Phone: 804-828-0021; Email: yzhang2@vcu.edu;

Fax: 804-828-7625

■

1

virus; H NMR, proton nuclear magnetic resonance; HOBt,

hydroxybenzotriazole; HPLC, high-performance liquid chro-

matography; HRMS, high-resolution mass spectrum; ICL,

intracellular loops; LTR, long-terminal repeat; Luc, luciferase

gene; MD, molecular dynamics; MIP-1β, macrophage

inﬂammatory protein 1 beta; MOR, mu opioid receptor;

OUD, opioid use disorder; PBMC, peripheral blood

mononuclear cells; PDB, Protein Data Bank; PHA, phytohe-

magglutinin; RMSD, root-mean-square deviation; RMSF, root-

mean-square ﬂuctuation; RT, reverse transcriptase; SEM,

standard error of the mean; Tat, trans-activator of tran-

scription; TI, therapeutic index; TM, transmembrane; TMS,

tetramethylsilane; TOF, time-of-ﬂight; TsOH, toluenesulfonic

acid

Authors

Boshi Huang − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States

Huiqun Wang − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States

Yi Zheng − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States

Mengchu Li − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States

Guifeng Kang − Department of Medicinal Chemistry, Virginia

Commonwealth University, Richmond, Virginia 23298,

United States

Victor Barreto-de-Souza − Department of Pharmacology and

Toxicology, Virginia Commonwealth University, Richmond,

Virginia 23298, United States

Nima Nassehi − Department of Pharmacology and

Toxicology, Virginia Commonwealth University, Richmond,

Virginia 23298, United States

Pamela E. Knapp − Department of Pharmacology and

Toxicology, Virginia Commonwealth University, Richmond,

Virginia 23298, United States; Department of Anatomy and

Neurobiology, Virginia Commonwealth University,

Richmond, Virginia 23298, United States

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Structure-Based Design and Development of Chemical Probes Targeting Putative MOR-CCR5 Heterodimers to Inhibit Opioid Exacerbated HIV-1 Infectivity

DOI: 10.1021/acs.jmedchem.1c00408

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