Design, Synthesis, and Biological Evaluation of Bivalent Ligands Targeting Dopamine D2-Like Receptors and the μ-Opioid Receptor

Accepted Article

Title: Design, synthesis and biological evaluation of novel bivalent

ligands targeting Dopamine D2-like receptors and µ opioid

receptor

Authors: Mingcheng Qian, Lakshmi Vasudevan, Jelle Huysentruyt,

Martijn D. P. Risseeuw, Christophe Stove, Patrick M. L.

Vanderheyden, Kathleen Van Craenenbroeck, and Serge Van

Calenbergh

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content of this Accepted Article.

To be cited as: ChemMedChem 10.1002/cmdc.201700787

Link to VoR: http://dx.doi.org/10.1002/cmdc.201700787

A Journal of

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10.1002/cmdc.201700787

ChemMedChem

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Design, synthesis and biological evaluation of novel bivalent

ligands targeting Dopamine D₂-like receptors and µ opioid

receptor

Mingcheng Qian,^[a,b]Lakshmi Vasudevan,^[b]Jelle Huysentruyt,^[b]Martijn D. P. Risseeuw,^[a]Christophe

Stove,^[b]Patrick M. L. Vanderheyden,^[c]Kathleen Van Craenenbroeck^[b]and Serge Van Calenbergh*^[a]

[a]

M. Qian; Dr. M. D. P. Risseeuw; Prof. S. Van Calenbergh

Laboratory for Medicinal Chemistry, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium

E-mail: Serge.VanCalenbergh@UGent.be

[b]

[c]

M. Qian; L. Vasudevan; J. Huysentruyt; Prof. C. Stove; Prof. K. Van Craenenbroeck

Laboratory of Toxicology, Ghent University, Ottergemsesteenweg 460, B-9000 Ghent, Belgium

Prof. P. M. L. Vanderheyden Department

Research Group of Molecular and Biochemical Pharmacology, Faculty of Sciences and Bioengineering Sciences, Vrije Universiteit Brussel, VUB–MBFA,

Pleinlaan 2, B-1050 Brussels, Belgium

Supporting information for this article is given via a link at the end of the document

Abstract: Nowadays, there is mounting evidence that intermolecular

receptor-receptor interactions may result in altered receptor

recognition, pharmacology and signaling. Heterobivalent ligands have

been proven useful molecular probes for confirming and targeting

heteromeric receptors. This paper describes the design and synthesis

of novel heterobivalent ligands for dopamine D₂-like receptors (D₂-

likeR) and the µ opioid receptor (µOR) and their evaluation using

radioactive ligand binding and functional assays. Interestingly, we

identified a potent bivalent ligand 12d containing a short 18-atom

linker that combined good potency with high efficacy both in β-

arrestin2 recruitment for µOR and MAPK-P for D₄R. Furthermore, this

compound was characterized by a biphasic competition binding curve

for the D₄R-µOR heterodimer, indicative of a bivalent binding mode.

As compound 12d possibly bridges the D₄R-µOR heterodimer, it could

be used as a pharmacological tool to further investigate the

interactions of D₄R and µOR.

mounting evidence that also intermolecular receptor-receptor

interactions may result in altered receptor recognition,

pharmacology and signaling. Heterobivalent ligands have been

proven useful molecular probes for confirming and targeting

heteromeric receptors,^6,7,8such as the κ opioid receptor (KOR)-

δOR heteromers.⁷

There are five dopamine receptor subtypes that can be divided

into two distinct subfamilies: D₁-like (D₁R and D₅R) and D₂-like

(D₂R, D₃R and D₄R). In this study we focus on the

heterodimerization of the dopamine D₂-like receptors (D₂-likeR)

and the μ opioid receptor (µOR). D₂-likeR and µOR are GPCRs,

expressed in the brain, which play a major role in schizophrenia,

Parkinson’s disease, addiction and pain. Heterobivalent ligands

are valuable tools to demonstrate the existence of receptor

heteromers even in native tissue and can be used to study a

specific GPCR dimer behavior without any receptor

modification.^9,10,11Such compounds may eventually also evolve to

useful pharmacological agents.¹⁰Heterobivalent ligands with a

spacer of optimal length are envisaged to exhibit a potency that is

greater than that derived from its two monovalent

pharmacophores and may allow the targeting of certain

heteromeric subtypes, increasing the selectivity of drug

action.^11,12,13

Introduction

G protein-coupled receptors (GPCRs) represent the largest family

of membrane proteins. About half of the clinically used drugs

today recognize GPCRs. It is now widely accepted that different

GPCRs can interact with each other and form complexes, so

called heteromers. The first hypothesis on GPCR heteromers was

already raised in the early eighties after observations of

neuropeptide-monoamine receptor-receptor interactions.^{1, 2}The

existence of heteromers was confirmed almost twenty years later

for two non-functional GPCR monomers, gamma amino butyric

acid (GABA) receptors, GABAB1 and GABAB2, which assemble

at the cell surface in a signaling heterodimer, the GABAB

receptor. ³Now, dimerization has been described for many

GPCRs in in vitro settings although evidence in native tissue is

still sparse.^4,5

The dopaminergic and opioid peptide system are

pharmacological targets for the treatment of addiction and chronic

pain. They show an impressive co-distribution in many nuclei of

the brain, which enables intermolecular receptor-receptor

interactions^14,15that may be relevant for the treatment of addiction

and chronic pain. In vivo studies indicate that there exists a cross-

regulation between the D₂-likeR and the µOR. Activation of

dopaminergic receptors causes a transient reduction of the µOR

immunoreactivity. Additionally, D₂-likeR/µOR interactions also

modulate morphine-induced upregulation of certain transcription

factors c-Fos, deltaFosB and P-CREB. ^16,17,18,19These results can

be explained by the existence of direct D₂-like-R-µOR interactions.

Furthermore, we have identified heterodimerization of D₂R-μOR

and D₄R-μOR by co-immunoprecipitation (co-IP) and

In classical pharmacology allosteric mechanisms were only

discussed in terms of intramolecular interactions within a receptor

between orthosteric and allosteric sites. Nowadays, there is

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bioluminescence resonance energy transfer (BRET) studies in

mammalian transfected cells (unpublished results). Therefore, all

results showed above indicate that D₂-likeR (especially D₂R and

D₄R) and µOR heterodimers could be potentially therapeutic

targets for the treatment of addiction and chronic pain.

units to the spacer relies on two criteria: the feasibility of the

chemical modification and the compatibility of the modification

with the biological activity (SAR data) of the pharmacophore.

Here we report the design and synthesis of a series of

heterobivalent

µOR

agonist/antagonist-D₂-likeR

agonist/antagonist ligands as pharmacological tools to further

study µOR-D₂-likeR (µOR-D₂R and µOR-D₄R) heteromers. In

these compounds, the D₂-likeR agonist/antagonist and the µOR

agonist/antagonist were linked through a spacer of variable length.

The spacers were based on PEG unit repeats, and their size was

varied (from 18 to 24 atoms) to obtain the best interaction with the

µOR-D₂-likeR heteromers. The binding properties of these

compounds were determined by radioligand binding studies in

membrane preparations and intact cells. Furthermore, MAPK

phosphorylation and β-arrestin2 recruitment assays were

performed to test the pharmacological response of the ligands for

D₂-likeR and µOR, respectively. Experiments to evaluate the

binding of the heterobivalent ligands to µOR and D₂-likeR were

performed in cells expressing both µOR and D₂-likeR.

Figure 1. Overview of used monovalent ligands and spacer molecules with

appropriate ligation handles.

As indicated above, the selected D₂-likeR ligands need to be

functionalized with an alkyne moiety to allow conjugation to the

azide group of the PEG linkers. The intrinsic activity of DAPs for

D₂-likeR is determined by an aromatic headgroup and an amine

moiety, which forms a strong hydrogen bond to the crucial

Design of heterobivalent ligands based on

structure of D₂-likeR ligands and µOR ligands

As depicted in Figure 1, the bivalent ligands were derived from

the µOR specific agonist hydromorphone (HM) and the

structurally related µOR antagonist naltrexone (NTX). As D₂-likeR

Asp3.32 residue in the transmembrane helix 3 (TM3) of D₂R.²⁰

A

lipophilic appendage is necessary for enhancing ligand affinity.

Because proper elongation of this appendage is expected to lead

to the “entrance region” of the receptor and from there to the

binding pocket of a neighboring protomer, the para-position of an

aromatic moiety terminating this appendage is considered an

appropriate point of attachment for a linker unit.²⁰Vanilline allows

for easy introduction of an alkyne group in para-position, while

reductive amination permits coupling to the phenylpiperazine

moiety.

ligands we used the antagonist 1,4-disubstituted aromatic

20

piperazines (DAPs, 3)

and the agonist 5-hydroxy-2-

(dipropylamino)tetralin (DPAT, 7)¹¹that were equipped with an

appropriate ligation handle.

DPAT is a prototypical D₂R/D₃R agonist suitable for constructing

bivalent ligands.²¹Site-directed mutagenesis has demonstrated

that the 2-amino tetralin moiety interacts with an agonist binding

domain involving TM3 and TM5 for activation of either D₂R or D₃R.

The basic nitrogen and the 5-OH group are critically important for

binding affinity. A known aniline derivative of DPAT¹¹was coupled

with 4-pentynoic acid to afford compound 7 with an alkyne group

for click reaction.

Figure 1. Designed heterobivalent ligands.

The dimeric ligands were constructed around PEG spacers of

variable length that were equipped with a carboxylic acid on one

and an azide on the other end (Figure 2). The use of PEG linkers

precludes cumulative incremental increases in hydrophobicity

that would occur upon homologation if an alkyl chain were

employed. Using existing procedures both opioid ligands 1a and

1b were converted to their corresponding 6’-R-amino derivatives

2a and 2b. The introduced amine groups allow coupling to the

PEG spacers via an amide bond, whereas the azido group on the

PEG linker allows facile connection to the alkyne derived

Results and Discussion

Chemistry

Individual components of the bivalent ligands

dopamine ligands via

a

copper-catalyzed azide–alkyne

cycloaddition (CuAAC) reaction. The choice of the position and

nature of the attachment points for linking the two pharmacophore

As µOR ligands the known R-amines 2a and 2b were prepared

from NTX (1a) and HM (1b) respectively according to published

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procedures.²²Briefly, compounds 1a and 1b were converted to

the corresponding oximes, which were reduced to 6R-amines 2a

and 2b in the presence of ZrCl₄and NaBH₄. Although previous

studies showed little stereoselectivity in opioid binding for 6R

23

versus 6S amine diastereomers,

the predominant R-

diastereomers were separated from their S-epimers by silica gel

chromatography, to facilitate further characterization.

For the synthesis of the first alkyne functionalized D₂-likeR ligand,

DPAT (7), commercially available 5-methoxyl-2-tetralone (4) was

converted to the 1,2,3,4-tetrahydronaphthalen-2-amine derivative

5 in 4 steps following existing procedures.¹¹Arylamine 5 was

coupled with 4-pentynoic acid to give amide 6. Deprotection of the

methyl ether with Me₂SBF₃in CH₂Cl₂gave the desired compound

7 (Scheme 1). The alkyne functionalized D₂-likeR ligand DAP (3)

was synthesized starting from vanillin as described earlier by

Kühhorn et al.²⁰The bifunctional PEG linkers were prepared as

described earlier. ^24,25,26

Scheme 3. Reagents and conditions: a) sodium ascorbate, CuSO₄,

triethylamine, tris[(1-benzyl-1,2,3-triazol-4-yl)methyl]amine, dimethylformamide.

Scheme 1. Reagents and conditions: a) 4-pentynoic acid, (3-

dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, triethylamine, CH₂Cl₂,

RT, overnight; b) Me₂SBF₃, CH₂Cl₂, RT, overnight.

Ligand binding assay

Bivalent ligands synthesis.

Ligand binding assay to evaluate the binding of the bivalent

ligands to the D₂-likeR

The bivalent ligands were generated by first condensing each of

the two µOR ligands with each of the three PEG linkers in the

presence of the coupling agent EDC and triethylamine to yield the

six intermediate azides 11a-f (Scheme 2). The series of bivalent

ligands was finalized by conjugating each of the six azides to both

DPAT and DAP through CuAAC (Scheme 3). This yielded a

concise series of 12 bivalent D₂-likeR-µOR ligands in which each

of the sets of ligands and the three linkers are systematically

represented.

A saturation binding assay for [³H]spiperone, a non-selective

antagonist of D₂-likeR, was described previously.^27,28Here we

performed a competition assay to evaluate the binding of the

bivalent ligands to the D₂-likeR (mainly focused on D₂R and D₄R).

In a first set of ligand binding experiments the binding affinity of

the bivalent ligands 12a-f and 13a-f along with their alkynylated

DAP and DPAT precursors

3 and 7 was measured by

displacement of the radioligand [³H]spiperone from the D₄R

expressed in HEK 293T cells, indicating that all the bivalent

ligands bind to the D₄R (Table 1). However, a clear reduction in

the D₄R binding affinities of the bivalent ligands was detected

when compared with the alkynes 3 and 7. When comparing the

binding affinities of 12d, 12e and 12f to D₄R, compound 12d with

a short spacer length (18-atom) showed a slightly higher affinity

for D₄R than 12e and 12f. Likewise, this trend also can be found

for the other bivalent ligands (12a, 13a and 13d) possessing the

same spacer as 12d. In a next step we wanted to compare the

affinity of the bivalent ligands for D₄R in the absence and

presence of overexpressed µOR. Unexpectedly, no major

differences in K_ivalues were found between both experimental

set-ups, except for 12d. Interestingly, a biphasic competition

curve was observed for 12d from which two individual affinity

constants could be derived (K_{i high}1.2 ± 0.3 nM and K_{i low}207 ± 51

nM, Figure 3a). Such biphasic competition-binding curves are

indicative of a bivalent binding mode.¹¹Specifically, the high-

affinity K_ivalue represents a bivalent receptor-bridging binding

mode of 12d to D₄R/µOR heterodimer, whereas the low-affinity K_i

value reveals a monovalent-binding mode to D₄R. Bivalent ligand

Scheme 2. Reagents and conditions: a) (3-dimethylaminopropyl)-N-

ethylcarbodiimide hydrochloride, 1-hydroxybenzotriazole, triethylamine, CH₂Cl₂.

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12d thus displays a 170-fold preference for the high-affinity

bivalent interaction with the D₄R/µOR heterodimer over

monovalent-binding mode to D₄R.

Table 2. Binding affinities (K_i) for D₂R and D₂R-µOR.^[a]

Compound

K_i(nM) for D₂R

22 ± 5

K_i(nM) for D₂R-µOR

3

32 ± 11

12d

62 ± 3

101 ± 10

By following the same approach and still using [³H]spiperone as

the radioligand, we also tested the affinity of the bivalent ligand

12d and its corresponding monovalent ligand 3 to the D₂R and

D₂R-µOR receptors. The affinities of compound 12d to D₂R and

D₂R-µOR decreased 3-fold compared to the ligand 3 for both

[a] Binding affinities (K_i) were obtained by competitive displacement of

radiolabeled [³H]spiperone binding to HEK293T D₂R and HEK293T D₂R-µOR

membranes. All values are expressed as the mean ± SEM of at least two

independent assays.

receptors (Table 2). Unfortunately, we only obtained

a

monophasic competition-binding curve for 12d to D₂R-µOR and

no major differences in binding affinity were observed between

cells expressing only D₂R and cells expressing both D₂R and µOR

(Figure 3b).

1 2 0

1 0 0

8 0

6 0

Next, following a similar protocol, we determined the affinity of

monomeric ligand 3 and bivalent ligand 12d on cells expressing

D₂R and D₂R-µOR, utilizing an alternative radioligand, i.e.

[³H]raclopride. Raclopride that acts as a selective D₂R antagonist

is more hydrophilic and has substantially less non-specific binding

in intact cells as compared with spiperone. Furthermore it is not

cell permeable, rendering the assay possibly more sensitive as

binding of the radioligand to be displaced is restricted to receptors

in the plasma membrane. In this experiment (Table 3) the affinities

of 12d to D₂R and D₂R-µOR were 32.4 ± 8.5 nM and 38.6 ± 3.2

nM, respectively, while the affinities of the monovalent alkyne 3

were 43.1 ± 9.0 nM and 49.9 ± 5.8 nM, respectively. Still, only

one-site binding curves for 12d were obtained from cells

monoexpressing D₂R and cells coexpressing D₂R and µOR

(supporting information). However, the affinities of 12d for both

receptors were comparable to that of compound 3. The saturation

binding data for [³H]raclopride can be found in supporting

information. Moreover, when comparing the affinity of the bivalent

ligand 12d for D₂-likeR using [³H]spiperone, we found that the

affinity of 12d to D₄R decreased 100-fold in comparison to the

monovalent ligand 3. Interestingly, the K_ivalue of 12d binding to

D₂R only decreased 3-fold compared to that of 3.

4 0

D

₄R

2 0

₄R - µ O

R

0

- 1 2

- 1 1

- 1 0

- 9

- 8

- 7

- 6

- 5

- 4

L o g [ 1 2

d

]

( M

)

b

1 2 0

1 0 0

8 0

6 0

4 0

2 0

0

D

₂R

₂R - µ O

R

- 1 2

- 1 1

- 1 0

- 9

- 8

- 7

- 6

- 5

- 4

L o g [ 1 2

d

] ( M )

Figure 3. Representative competition curves for bivalent ligand 12d. D₂-likeR

binding of the bivalent ligand 12d was measured by displacement of the

radiolabeled [³H]spiperone from membranes of HEK293T cells coexpressing

D₄R/µOR (or D₂R/µOR) or monoexpressing D₄R (or D₂R) only. (a) D₄R binding

of 12d in the presence (filled squares, K_{i high}1.2 ± 0.3 nM, K_{i low}207 ± 51 nM) or

absence (open squares, K_i184 ± 35 nM) of µOR. (b) D₂R binding of 12d in the

presence (filled circles, K_i101 ± 10 nM) or absence (open circles, K_i62 ± 3 nM)

of µOR. Data points represent the mean ± SEM of three independent assays,

each performed in duplicate.

Table 1. Binding affinities (K_i) for D₄R and D₄R-µOR.^[a]

Compd

R₁

R₂

n

D₄R

K_i(nM)

D₄R-µOR

K_i(nM)

2.1 ± 0.3

nd^[b]

219 ± 10

nd

3

-

1.6 ± 0.2

117 ± 12

214 ± 11

226 ± 18

184 ± 35

369 ± 42

339 ± 50

15 ± 6.1

118 ± 12

157 ± 30

138 ± 25

110 ± 9.3

191 ± 9.5

199 ± 16

12a

12b

12c

12d

12e

12f

7

13a

13b

13c

13d

13e

13f

OH

H

CH₂cPr

CH₃

3

4

5

3

4

5

-

3

4

5

3

4

5

-

368 ± 10

417 ± 8.8

16 ± 2.5

101 ± 1.3

nd

394 ± 12

493 ± 11

Table 3. Binding affinities (K_i) for D₂R and D₂R-µOR.^[a]

-

Compound

K_i(nM) for D₂R

43.1 ± 9.0

32.4 ± 8.5

K_i(nM) for D₂R-µOR

49.9 ± 5.8

OH

H

CH₂cPr

CH₃

3

12d

38.6 ± 3.2

[a] Binding affinities (K_i) were obtained by competitive displacement of

radiolabeled [³H]raclopride binding to HEK293T D₂R and HEK293T D₂R-µOR

intact cells. All values are expressed as the mean ± SEM of at least two

independent experiments.

[a] Binding affinities (K_i) were obtained by competitive displacement of

radiolabeled [³H]spiperone binding to HEK293T D₄R and HEK293T D₄R-µOR

membranes. All values are expressed as the mean ± SEM of three independent

assays using 11 different concentrations of ligands, each performed in duplicate.

[b] not determined.

Ligand binding assay to evaluate the binding of the bivalent

ligands to µOR

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We performed competitive receptor binding assays in cell

membranes expressing µOR to determine the binding affinity of

bivalent ligands, using the tracer [³H]diprenorphine, which is a

nonselective opioid antagonist. Saturation binding assays

showed that the Kd value and Bmax of [³H]diprenorphine were

0.21 ± 0.064 nM and 489 ± 28 fmol/mg for µOR, respectively. In

the competition assay the binding affinities for the monovalent

ligands HM and NTX were 4.4 ± 0.21 nM and 7.8 ± 0.16 nM,

respectively (Table 4). Compounds 12a-c and 13a-c, which were

derived from NTX, exhibited a binding affinity for µOR comparable

to NTX. Whereas, bivalent ligands 12b and 13b with a 21-atom

spacer displayed relatively lower K_ivalues than the other NTX-

based ligands. HM-based bivalent ligands 12d-f and 13d-f

showed a small reduction in the binding affinities for µOR

compared to the corresponding monovalent compound HM,

except for 12e (3.7 ± 0.02 nM) and 13e (7.2 ± 0.53 nM) with a

medium spacer length (21 atoms). Therefore, the affinities of

bivalent ligands with a medium length of spacer (21 atoms) to

µOR were relatively higher than the other ligands with shorter (18

atoms) or longer linkers (24 atoms).

In order to assess functional activation of the D₂R and D₄R by the

above described bivalent ligands, we used a HEK293 cell line

stably expressing the D₂R and D₄R, respectively.³⁰Unexpectedly,

the signal band of MAPK-P for D₂R was quite weak (see

supporting information). Nevertheless, we found

a strong

immunoreactive band (Figure 4) for MAPK-P upon activation of

D₄R with the agonist dopamine (DA), and with the alkynylated

DAP (3) and DPAT (7). Next, all bivalent ligands were tested in

this cell system (Figure 5 and Table 5). We can conclude that all

bivalent ligands activate the MAPK signaling pathway.

Interestingly, compound 12d with an 18-atom linker (shown in

Table 5) displayed modestly high potency (EC₅₀=0.12 ± 0.04 µM)

and relatively high efficacy (92 ± 1%) as compared with the

monovalent ligand 3 (EC₅₀= 0.21 ± 0.12 µM, E_max= 75 ± 2%),

while the potency of compounds 12a-c decreased 8-16 fold

compared to 3. In addition, all the DAP-bond ligands, except for

12d, showed a comparable efficacy (E_max= 72-85%) relative to

compound 3. On the other hand, DPAT-based bivalent ligands

had a potency (EC₅₀= 0.45-0.95 µM) and efficacy (E_max= 86-

108%) comparably to that of 7 (EC₅₀= 0.37 ± 0.17 µM, E_max= 100

± 3%). Overall, bivalent ligand 12d with a short linker length (18

atoms) was the most potent compound to activate the MAPK

phosphorylation of D₄R.

Table 4. Binding affinities (K_i) for µOR.^[a]

Compd

Hydromorphone

Naltrexone

12a

R₁

H

R₂

CH₃

n

-

K_i(nM)

4.4 ± 0.21

7.8 ± 0.16

6.3 ± 1.1

4.6 ± 0.25

9.2 ± 0.41

7.5 ± 0.95

3.8 ± 0.84

7.6 ± 0.39

16 ± 0.87

3.7 ± 0.02

11 ± 0.57

13 ± 1.2

OH

H

CH₂cPr

CH₃

3

4

5

3

4

5

3

4

5

3

4

5

12b

12c

13a

13b

13c

12d

12e

12f

13d

13e

7.2 ± 0.53

17 ± 3.4

13f

H

CH₃

[a] Binding affinities (K_i) were obtained by competitive displacement of

radiolabeled [³H]diprenorphine binding to HEK293T µOR. All values are

expressed as the mean ± SEM of three independent assays.

Figure 4. Stimulation of MAPK phosphorylation upon D₄R agonist treatment, as

determined by immunoblotting assays. Cells were treated with serum-free

media (SFM) for 12 h. Next, D₂-likeR agonists (10 µM), SFM or vehicle control

were added for 5 min. Cells were washed and lysed as explained in the

Experimental section. Phosphorylated MAPK was detected by immunoblotting

using rabbit anti-phospho-p44/42 MAPK antibody and mouse anti-p44/42

MAPK antibody was used for demonstrating equal protein loading. The results

shown are representative of three independent experiments. DA=dopamine.

Functional assay

MAPK phosphorylation to study D₂-likeR activation by the

newly developed bivalent ligands

The mitogen-activated protein kinase (MAPK) phosphorylation

assay was used to define the functional activity of the bivalent

ligands. The MAPK pathway, also known as the extracellular-

signal regulated kinase (ERK) pathway consists of an intracellular

chain of proteins that transfer the signal from the cell surface

receptor to the nuclear DNA or other subcellular targets causing

cellular responses. Signaling molecules in this pathway

communicate with each other by adding a phosphate group to the

neighboring protein. This phosphorylation event functions as an

“on/off” switch, leading to the activation or inhibition of the next

signaling molecule in the chain.²⁹The most commonly studied

element of the MAPK pathway, initiated by GPCR activation, is

the phosphorylation of p44/42 MAPK (ERK1/2).

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To assess the ligand induced interaction of β-arrestin2 with µOR,

H

E K 2 9 3

D

₄R

4

3

2

1

0

overexpression of GRK2 in cell culture was performed. Our

results show that DAMGO induces

a robust β-arrestin2

recruitment even in the absence of GRK2. On the other hand, HM

could promote β-arrestin2 recruitment only when GRK2 was

overexpressed in HEK 293 cells. Both results are in line with those

found in the literature (see supporting information Figure 2).

Upon stimulation with a known agonist of µOR, HM, µOR-SmBiT

showed a concentration dependent interaction with LgBiT-βarr2

in the presence of GRK2 (Figure 6). Also, for the bivalent ligands

containing the agonistic HM-based monomer, concentration-

dependence was obtained and EC₅₀values were determined as

a measure of relative potency (Table 6 and Figure 6). Ranging

from 12.73 nM to 57.06 nM, all the EC₅₀values of the bivalent

ligands were comparable to that of the parent HM (EC₅₀= 30.09

nM). Amongst the DAP-based ligands, compound 12d, having the

shortest linker (18-atom), showed excellent potency (EC₅₀= 12.73

nM) and high efficacy (85 ± 3%). Remarkably, all DPAT-based

ligands showed lower efficacies than DAP-bond ligands, with

compound 13d, having the same spacer as 12d, displaying the

lowest efficacy (24 ± 1%) amongst the DPAT-based ligands.

f

3

a

c

e

a

3

c

e

3

1

7

b

d

b

d

3

A

M

2

3

2

3

F

D

1

S

Figure 5. Efficacy of bivalent ligands to phosphorylate MAPK in HEK293 D₄R

cells. Cells were treated with serum-free media (SFM) for 12 h. Next, D₂-likeR

agonists (10 µM) and SFM were added for 5 min. Cells were washed and lysed

as explained in the Experimental section. Phosphorylated MAPK was detected

by immunoblotting using rabbit anti-phospho-p44/42 MAPK antibody and

mouse anti-p44/42 MAPK antibody was used for demonstrating equal protein

loading. The top panel shows a representative result from three independent

experiments; the bottom panel is given as mean ± SEM. DA=dopamine.

Table 5. Potency (EC₅₀) of D₂-likeR agonists induced MAPK-P in HEK293 D₄R

cells

[b]

Compd

Dopamine

3

12a

12b

12c

12d

12e

12f

7

13a

R₁

-

OH

H

-

OH

H

R₂

-

CH₂cPr

CH₃

-

CH₂cPr

CH₃

n

-

EC₅₀µM^[a]

0.037 ± 0.012

0.21 ± 0.12

3.4 ± 0.2

1.9 ± 0.3

1.6 ± 0.1

0.12 ± 0.04

0.31 ± 0.02

0.80 ± 0.06

0.37 ± 0.17

0.55 ± 0.24

0.48 ± 0.06

0.88 ± 0.01

0.95 ± 0.03

0.57 ± 0.14

0.45 ± 0.01

E_max%

D

o s e r e s p o n s e c u r v e f o r r e c r u i t m e n t o f B a r r 2

100 ± 4

75 ± 2

78 ± 3

77 ± 2

72 ± 1

92 ± 1

85 ± 5

76 ± 2

100 ± 3

89 ± 4

86 ± 4

90 ± 1

97 ± 1

101 ± 6

108 ± 2

1 4 0 0 0 0

H M

3

4

5

3

4

5

-

3

4

5

3

4

5

1 2 0 0 0 0

1 0 0 0 0 0

8 0 0 0 0

6 0 0 0 0

4 0 0 0 0

2 0 0 0 0

0

1 2 d

1 2 e

1 2 f

1 3 d

1 3 e

1 3 f

13b

13c

13d

13e

- 1 1

- 1 0

- 9

- 8

- 7

- 6

M

- 5

- 4

C

o m p o u n d ( lo g

)

13f

Figure 6. Concentration-dependent interaction of µOR with β-arrestin2 upon

stimulation with agonists of µOR. Data are given as mean ± SEM of at least two

independent experiments. Main Text Paragraph.

[a] Data shown represent the mean ± SEM of three independent experiments.

[b] E_maxrelative to the effect of the reference agonist dopamine at 10 μM.

β-arrestin2 recruitment to µOR

Table 6. Potency (EC₅₀) and maximal effect of µOR agonists on β-arrestin2

recruitment for µOR.

Compd

HM

12d

12e

12f

13d

13e

13f

R₁

H

R₂

n

-

EC₅₀(95% C.I.) nM^[a]

30.09 (24.51-36.94)

12.73 (7.612-21.28)

40.38 (30.41-53.61)

34.86 (25.76-47.18)

13.41 (8.286-23.15)

57.06 (38.49-84.58)

14.09 (10.14-21.08)

% HM^[b]

100

CH₃

To assess activation of the µOR, HEK 293 cell line was used to

stably express the µOR. Unfortunately, we did not obtain any

good results from MAPK-P for µOR (data not shown). Then we

performed a β-arrestin2 recruitment assay based on the NanoLuc

Binary Technology (NanoBiT). ³¹Our research group recently

reported on the application of this assay for the monitoring of

GPCR activation, via ligand induced interaction of βarr2 with CB1

and CB2 cannabinoid receptors.³²Here, we optimized this assay

for ligand induced interaction of βarr2 with µOR. Agonists of µOR,

such as DAMGO, have previously been shown to induce robust

receptor phosphorylation, β-arrestin2 recruitment and µOR

trafficking.^33,34On the other hand, morphine is a poor inducer of

3

4

5

3

4

5

85 ± 3

68 ± 1

64 ± 1

24 ± 1

28 ± 1

38 ± 1

[a] EC₅₀values are presented as a measure of potency for β-arrestin2

recruitment. Data are given as EC₅₀values (95% CI proﬁle likelihood). [b]

Percent maximal stimulation relative to the agonist HM at 10 μM.

Conclusions

receptor

phosphorylation,

β-arrestin2

recruitment

and

In summary, we have designed and synthesized a series of novel

heterobivalent ligands based on the chemical structure of two

distinct D₂-likeR ligands and a µOR agonist and antagonist.

Ligation of µOR ligands does not perturb the affinity for µOR. µOR

internalization of µOR. However, upon overexpression of G

protein-coupled receptor kinase (GRK2) in cell culture, morphine

gained the capacity to induce µOR phosphorylation, accompanied

by the rescue of β-arrestin2 recruitment.^34,35

6

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bivalent ligands derived from the HM agonist are still capable of

activating the µOR signaling pathway as demonstrated by β-

arrestin2 recruitment. Furthermore, bivalent ligand 12d containing

the shortest linker (18-atom) showed excellent potency and high

efficacy both in β-arrestin2 recruitment for µOR and MAPK-P for

D₄R. On the other hand, ligation of D₂-likeR ligands negatively

influences the affinity for D₂R and D₄R when using [³H]spiperone

as the radioligand. However, a biphasic competition-binding curve

was observed for 12d to D₄R-µOR, which indicates a bivalent

binding mode.^11,43Hence, compound 12d could bridge the D₄R-

µOR heterodimer.

standard. ¹H- and ¹³C-APT-NMR spectra were recorded with a

Varian Mercury-300BB (300/75 MHz) spectrometer. Chemical

shifts are given in ppm (δ) relative to tetramethylsilane as an

internal standard (¹H NMR) or the NMR solvent (¹³C NMR).

Coupling constants are given in Hertz (Hz). Weak signals in ¹³C

NMR are indicated as (w). LC-MS analyses were carried out on a

Waters AutoPurification System equipped with PDA and ESI-MS

detection and using a Waters CORTECS C18 Column (4.6×100

mm, 2.7µm) and a water/acetonitrile/formic acid linear gradient

system at a flow rate of 1.44 mLmin^-1.

General procedure 1: Carbodiimide-mediated amide formation.

Unexpectedly, we were unable to identify bivalent ligands that

show a biphasic binding mode or a significant increase in affinity

for cells expressing both the D₂R and the µOR, compared to cells

that only express D₂R. This indicates that none of the bivalent

ligands is capable of binding both receptors simultaneously,

possibly owing to a suboptimal length or nature of the selected

linkers, which, however, were based on the spacers of class A

GPCR bivalent ligands.^{20, 36}On the other hand it cannot be

excluded that subtle differences such as differences in the

dissociation rate of the ligands remain undetected by comparing

equilibrium dissociation constants. In a study investigating

adenosine A₃receptors, it was found that ligand binding kinetics

is found to be influenced in membrane microdomains as a

consequence of receptor dimerization.³⁷

To a solution of (3-Dimethylaminopropyl)-N-ethylcarbodiimide

hydrochloride (1.5 eq.) and 1-hydroxybenzotriazole (1.4 eq.) in

CH₂Cl₂was added a solution of the PEG spacer (1.2 eq.) in

CH₂Cl₂(0.2 M) and cooled to 0°C under an argon atmosphere.

After 15 min at 0°C, amines 2a-b (1.0 eq.) were added and

triethylamine (2 eq.) was added dropwise; the reaction was slowly

warmed to room temperature and stirred overnight. The reaction

was diluted with CH₂Cl₂, washed successively with water, HCl

(5% aq.), NaHCO₃(sat. aq.), NaCl (sat. aq.), dried over Na₂SO₄,

filtered and solvent was evaporated under reduced pressure. The

crude product was purified by silica gel chromatography

(NH₄OH/MeOH/CH₂Cl₂, 1:5:94 v/v/v) to yield the amides 11a-f.

General procedure 2: Copper mediated azide-alkyne

cycloaddition.

While future studies will try to address these shortcomings, the

current study already gives access to the monomeric ligands that

are equipped with appropriate ligation handles to construct

second-generation bivalent ligands with alternative spacers. In

conclusion, the present results provide useful insights into

development of new bivalent ligands as tools to investigate the

µOR-D₄R heterodimer.

To

a

solution of the intermediate azide (1.0 eq.) in

dimethylformamide (0.1 M) was added the alkyne (1.5 eq.),

sodium ascorbate (1.0 eq., 0.5 M), CuSO₄(0.2 eq., 0.05 M),

triethylamine (3.0 eq.) and a catalytic amount of tris[(1-benzyl-

1,2,3-triazol-4-yl)methyl]amine. The reaction mixture was stirred

overnight at room temperature in the dark under an argon

atmosphere. The solvent was evaporated under reduced

pressure and the residue was redissolved in water and extracted

with CH₂Cl₂. Then combined organic fractions were pooled,

washed with brine and dried over Na₂SO₄. The crude compound

was purified by silica gel chromatography (NH₄OH/MeOH/CH₂Cl₂,

1:5:94 v/v/v) to give the final compound as a white solid (yield, 40-

57%).

Experimental Section

Chemistry

All reactions described were performed under an N₂atmosphere

and at ambient temperature unless stated otherwise. All regents

and solvents were purchased from Sigma-Aldrich (Diegem,

Belgium), Fisher Scientific (Merelbeke, Belgium), TCI Europe

(Zwijndrecht, Belgium) or Apollo Scientific (Bredbury, Stockport,

United Kingdom) and used as received. NMR solvents were

acquired from Eurisotop (Saint-Aubin, France). Reactions were

monitored by TLC analysis using TLC aluminium sheets

(Macherey-Nagel, Alugram Sil G/UV254) with detection by

spraying with a solution of (NH₄)₆Mo₇O₂₄·4H₂O (25 gL^-1) and

(NH₄)₄Ce(SO₄)₄·2H₂O (10 gL^-1) in H₂SO₄(10 % aq.) followed by

charring or an aqueous solution of KMnO₇(20 gL^-1) and K₂CO₃

(10 gL^-1) or an ethanolic solution of ninhydrin (2 gL^-1) and acetic

acid (1% v/v) followed by charring. Solution pH values were

estimated using universal indicator paper (Merck). Silica gel

column chromatography was performed using a Grace Reveleris

X2 system and the corresponding silica gel cartridges. ESI-HRMS

spectra were measured with a Waters LCT Premier XE Mass

spectrometer calibrated using leucine enkephalin as an external

N-(4-(2-((5-methoxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)pent-4-ynamide (6)

To a solution of (3-Dimethylaminopropyl)-N-ethylcarbodiimide

hydrochloride (1.1 g, 4.92 mmol) in CH₂Cl₂(20 mL) was added a

solution of 4-pentynoic acid (0.39 g, 3.93 mmol) in CH₂Cl₂(5 mL)

followed by a solution of 5 (1.1 g, 3.28 mmol) in CH₂Cl₂(5 mL).

The mixture was cooled to 0 ºC and triethylamine (0.68 mL, 4.92

mmol) was added dropwise; the reaction was slowly warmed to

room temperature and stirred overnight. The reaction was diluted

with CH₂Cl₂, washed successively with water, HCl (5% aq.),

NaHCO₃(sat. aq.), NaCl (sat. aq.), dried over Na₂SO₄, filtered and

solvent was evaporated under reduced pressure. The crude

product was purified by silica gel chromatography (Hexane/EtOAc,

1

3:2 v/v) to give 6 as a pale yellow solid (1.32 g, 95%). H NMR

7

10.1002/cmdc.201700787

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FULL PAPER

(300 MHz, CDCl₃) δ ppm 7.42 (d, J = 8.4 Hz, 2H), 7.15 (d, J = 8.4

Hz, 2H), 7.07 (t, J = 7.9 Hz, 1H), 6.69 (d, J = 7.8 Hz, 1H), 6.62 (d,

J = 8.1, 1H), 3.78 (s, 3H), 3.04-2.91 (m, 2H), 2.91-2.80 (m, 1H),

2.79-2.63 (m, 6H), 2.63-2.53 (m, 7H), 2.05 (d, J = 3.2 Hz, 2H),

1.61-1.41 (m, 3H), 0.88 (t, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz,

CDCl₃) δ 169.4, 157.2, 137.9, 137.0, 135.7, 129.2, 126.1, 125.2,

121.6, 120.1, 106.9, 82.9, 69.5, 56.7, 55.2, 52.9, 52.6, 36.1, 35.4,

32.3, 25.7, 23.8, 22.2, 14.8, 11.9. HRMS (ESI) m/z: calculated for

C₂₇H₃₅N₂O₂[(M+H)⁺] 419.2699; found 419.2705.

3.01 (d, J = 18.4 Hz, 1H), 2.70 – 2.55 (m, 2H), 2.38 (d, J = 6.6 Hz,

2H), 2.26 – 2.10 (m, 2H), 1.87 (td, J = 12.5, 2.9 Hz, 1H), 1.72 –

1.54 (m, 2H), 1.46 (dt, J = 11.3, 2.8 Hz, 2H), 0.91 – 0.79 (m, 2H),

0.58 – 0.49 (m, 2H), 0.17 – 0.09 (m, 2H). ¹³C NMR (75 MHz,

CDCl₃) δ 170.0, 142.5, 140.1, 130.9, 124.1, 119.0, 117.8, 93.2,

70.9, 70.6, 70.6, 70.5, 70.5, 70.4, 70.30, 70.0, 70.0, 62.3, 59.1,

50.8, 50.6, 47.6, 44.1, 30.5, 30.1, 24.3, 22.6, 9.3, 3.9, 3.8. HRMS

(ESI) m/z: calculated for C₃₂H₄₈N₅O₉[(M+H)⁺] 646.3452; found

646.3458.

N-(4-(2-((5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)pent-4-ynamide (7)

20-azido-N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-3,6,9,12,15,18-

hexaoxaicosanamide (11c)

To a solution of 6 (1.05 g, 2.51 mmol) in CH₂Cl₂(20 mL) was

added boron trifluoride methyl sulfide complex (4.77 mL, 45.21

mmol). The reaction was stirred overnight under nitrogen at room

temperature. The mixture was treated with a saturated aqueous

NaHCO₃solution, extracted with CH₂Cl₂, dried over Na₂SO₄and

the solvent was evaporated under reduced pressure. The crude

Compound 11c was subjected to general procedure 1. Pale

yellow solid, 56%. H NMR (300 MHz, CDCl₃) δ 6.71 (d, J = 8.1

Hz, 1H), 6.56 (d, J = 8.1 Hz, 1H), 4.45 (d, J = 7.5 Hz, 1H), 4.00 (s,

2H), 3.75 – 3.60 (m, 22H), 3.38 (t, J = 5.1 Hz, 2H), 3.10 – 2.98 (m,

1

product was purified by silica gel chromatography (Hexane/EtOAc, 2H), 2.59 (dd, J = 19.1, 6.3 Hz, 2H), 2.36 (d, J = 6.5 Hz, 2H), 2.24

1:1 v/v) to yield 7 as a white foam (750 mg, 74%). ¹H NMR (300

MHz, CDCl₃) δ ppm 7.41 (d, J = 8.4 Hz, 2H), 7.14 (d, J = 8.4 Hz,

2H), 6.97 (t, J = 7.8 Hz, 1H), 6.66 (d, J = 7.5 Hz, 1H), 6.59 (d, J =

7.8 Hz, 1H), 3.02-2.94 (m, 2H), 2.90-2.82 (m, 1H), 2.79 -2.66 (m,

6H), 2.62-2.53 (m, 7H), 2.05 (d, J = 3.2 Hz, 2H), 1.66-1.56 (m,

1H), 1.53-1.46 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz,

CDCl₃) δ ppm 169.1, 153.5, 138.3, 137.1, 135.4, 129.2, 126.3,

– 2.08 (m, 2H), 1.90 (qd, J = 12.7, 3.2 Hz, 1H), 1.72 – 1.53 (m,

2H), 1.46 (dd, J = 9.0, 4.4 Hz, 2H), 0.83 (dp, J = 13.0, 6.3 Hz, 2H),

0.56 – 0.49 (m, 2H), 0.12 (d, J = 4.7 Hz, 2H). ¹³C NMR (75 MHz,

CDCl₃) δ 169.9, 142.4, 140.0, 131.0, 124.3, 119.0, 117.7, 93.3,

70.9, 70.6, 70.6, 70.6, 70.5, 70.5, 70.4, 70.4, 70.3, 70.0, 70.0,

62.3, 59.2, 50.8, 50.6, 47.7, 44.0, 30.6, 30.1, 24.4, 22.6, 9.4, 3.9,

3.7. HRMS (ESI) m/z: calculated for C₃₄H₅₂N₅O₁₀[(M+H)⁺]

123.0, 121.6, 120.0, 111.9, 82.8, 69.7, 56.6, 52.7, 52.6, 36.2, 35.2, 690.3714; found 690.3744.

32.2, 25.7, 23.5, 22.0, 14.8, 11.9. HRMS (ESI) m/z: calculated for

C₂₆H₃₃N₂O₂[(M+H)⁺] 405.2542; found 405.2545.

14-azido-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

14-azido-N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-3,6,9,12-

tetraoxatetradecanamide (11a)

e]isoquinolin-7-yl)-3,6,9,12-tetraoxatetradecanamide (11d)

Compound 11d was subjected to general procedure 1. Offwhite

solid, 62%. ¹H NMR (300 MHz, CDCl₃) δ 6.66 (d, J = 8.1 Hz, 1H),

6.56 (d, J = 8.1 Hz, 1H), 4.41 (d, J = 7.9 Hz, 1H), 3.96 (s, 2H),

3.72-3.57 (m, 14H), 3.37 (t, J = 5.6 Hz, 2H), 3.11 (br s, 1H), 2.98

(d, J = 18.4 Hz, 1H), 2.54 (dd, J = 11.9, 4.4 Hz, 1H), 2.39 (s, 3H),

2.34-2.14 (m, 2H), 1.92-1.75 (m, 2H), 1.71-1.48 (m, 3H), 1.42-

1.23 (m, 2H), 1.14-1.00 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ

169.9, 142.8, 140.3, 129.3, 125.2, 119.4, 117.5, 93.2, 70.8, 70.6,

70.5, 70.4, 70.4, 70.4, 70.2, 70.0, 59.4, 51.5, 50.6, 47.2, 44.9,

43.4, 42.6, 35.2, 28.8, 24.1, 20.1. HRMS (ESI) m/z: calculated for

C₂₇H₄₀N₅O₇[(M+H)⁺] 546.2928; found 546.2923.

Compound 11a was subjected to general procedure 1. Pale

1

yellow solid, 68%. H NMR (300 MHz, CDCl₃) δ 6.71 (d, J = 8.1

Hz, 1H), 6.56 (dd, J = 8.2 Hz, 1H), 4.45 (d, J = 7.5 Hz, 1H), 4.01

(s, 2H), 3.74-3.66 (m, 14H), 3.39 (t, J = 5.6 Hz, 2H), 3.10-3.04 (m,

2H), 2.99 (s, 1H), 2.67-2.54 (m, 2H), 2.40-2.34 (m, 4H), 2.24-2.13

(m, 2H), 1.89 (dd, J = 12.8, 3.1 Hz, 2H), 1.70-1.40 (m, 4H), 0.90-

0.77 (m, 1H), 0.57-0.48 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ

169.9, 142.4, 140.0, 131.0, 129.0, 128.2, 125.2, 119.1, 117.7,

93.5, 70.9, 70.6, 70.5, 70.5, 70.4, 70.3, 70.0, 62.3, 59.2, 50.7,

50.6, 47.7, 44.0, 30.6, 30.1, 24.4, 22.6, 9.4, 3.9, 3.7. HRMS (ESI)

m/z: calculated for C₃₀H₄₄N₅O₈[(M+H)⁺] 602.3190; found

602.3205.

17-azido-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-3,6,9,12,15-pentaoxaheptadecanamide (11e)

17-azido-N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-3,6,9,12,15-

pentaoxaheptadecanamide (11b)

Compound 11e was subjected to general procedure 1. Offwhite

solid, 53%. ¹H NMR (300 MHz, CDCl₃) δ 6.59 (d, J = 8.1 Hz, 1H),

6.51 (d, J = 8.1 Hz, 1H), 4.36 (d, J = 7.9 Hz, 1H), 3.92 (s, 2H),

3.73 – 3.54 (m, 18H), 3.34 (t, J = 5.6 Hz, 2H), 2.95 (d, J = 19.1

Hz, 1H), 2.49 (dd, J = 12.5, 4.2 Hz, 1H), 2.39 – 2.29 (m, 2H), 2.20

(s, 3H), 1.79 (dd, J = 12.4, 4.6 Hz, 2H), 1.68 – 1.55 (m, 2H), 1.33

– 1.20 (m, 2H), 1.07 (t, J = 7.2 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃)

δ 169.8, 143.0, 140.7, 129.3, 124.8, 119.2, 117.5, 92.8, 70.8, 70.6,

70.5, 70.4, 70.4, 70.3, 70.3, 70.2, 69.9, 59.3, 51.6, 47, 45.0, 43.4,

Compound 11b was subjected to general procedure 1. Pale

1

yellow solid, 62%. H NMR (300 MHz, CDCl₃) δ 6.70 (d, J = 8.1

Hz, 1H), 6.54 (d, J = 8.2 Hz, 1H), 4.45 (d, J = 7.5 Hz, 1H), 3.99 (s,

2H), 3.73 – 3.60 (m, 18H), 3.37 (t, J = 5.6 Hz, 2H), 3.11 (br s, 1H),

8

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N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-20-(4-((2-methoxy-4-

((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-

1H-1,2,3-triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosanamide (12c)

42.7(d, J = 5.2 Hz), 38.9, 35.3, 28.9, 24.1, 20.0, 15.49. HRMS

(ESI) m/z: calculated for C₂₉H₄₄N₅O₈[(M+H)⁺] 590.3190; found

590.3192.

20-azido-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-3,6,9,12,15,18-hexaoxaicosanamide (11f)

Compound 12c was subjected to general procedure 2. Offwhite

solid, 50%. LC-HRMS: t_R=5.63 min (10–100% MeCN, 15 min run),

95.23 %. HRMS (ESI) m/z: calculated for C₅₆H₇₉N₇O₁₃[(M+2H)²⁺]

528.7862; found 528.7854.

Compound 11f was subjected to general procedure 1. Offwhite

solid, 51%. ¹H NMR (300 MHz, CDCl₃) δ 6.64 (d, J = 8.1 Hz, 1H),

6.54 (d, J = 8.1 Hz, 1H), 4.42 (d, J = 8.0 Hz, 1H), 3.95 (s, 2H),

3.76-3.49 (m, 22H), 3.36 (t, J = 5.6 Hz, 2H), 3.10 (br s, 1H), 2.93

(s, 2H), 2.86 (d, J = 0.6 Hz, 1H), 2.58-2.46 (m, 1H), 2.37 (s, 3H),

2.26-2.10 (m, 2H), 1.85 (dt, J = 12.0, 6.0 Hz, 1H), 1.70-1.59 (m,

1H), 1.51 (dd, J = 13.4, 3.5 Hz, 1H), 1.45-1.20 (m, 2H), 1.06 (ddd,

J = 15.3, 10.5, 2.4 Hz, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 169.9,

142.8, 140.3, 129.4, 125.2, 119.3, 117.4, 93.1, 70.8, 70.6, 70.6,

70.5, 70.5, 70.4, 70.4, 70.4, 70.3, 70.2, 69.9, 59.3, 51.7, 50.6,

47.2, 43.4, 42.7, 35.2, 28.7, 24.1, 20.0. HRMS (ESI) m/z:

calculated for C₃₁H₄₈N₅O₉[(M+H)⁺] 634.3452; found 634.3444.

N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-14-(4-((2-methoxy-4-((4-(2-

methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-1H-1,2,3-

triazol-1-yl)-3,6,9,12-tetraoxatetradecanamide (12d)

Compound 12d was subjected to general procedure 2. Offwhite

solid, 42%. LC-HRMS: t_R=5.23 min (10–100% MeCN, 15 min run),

96.94 %. ¹H NMR (300 MHz, CDCl₃) δ 7.85 (s, 1H), 7.11 (d, J =

8.2 Hz, 1H), 7.01 – 6.94 (m, 2H), 6.94 – 6.88 (m, 2H), 6.83 (ddd,

J = 8.1, 5.5, 1.7 Hz, 2H), 6.68 (d, J = 8.1 Hz, 1H), 6.56 (d, J = 8.1

Hz, 1H), 5.28 (d, J = 6.7 Hz, 2H), 4.53 (t, J = 5.7 Hz, 2H), 4.42 (d,

J = 8.0 Hz, 1H), 3.91 (d, J = 1.4 Hz, 2H), 3.87 (t, J = 5.5 Hz, 2H),

3.84 (s, 3H), 3.82 (s, 3H), 3.66 – 3.51 (m, 14H), 3.09 (br s, 4H),

2.98 – 2.85 (m, 1H), 2.66 (br s, 3H), 2.52 (dd, J = 12.1, 4.6 Hz,

1H), 2.39 (s, 3H), 2.19 (dt, J = 9.8, 5.3 Hz, 2H), 1.82 (td, J = 12.2,

5.1 Hz, 1H), 1.58 (ddd, J = 33.7, 12.6, 3.5 Hz, 2H), 1.45 – 1.21

(m, 1H), 1.17 – 0.98 (m, 1H). ¹³C NMR (75 MHz, CDCl₃) δ 169.8,

152.2, 149.4, 146.7, 144.1, 142.7, 141.2, 140.1, 131.2, 129.3,

125.3, 124.25, 122.8, 121.7, 120.9, 119.5, 118.2, 117.7, 113.7,

112.9, 111.1, 93.0, 70.8, 70.5, 70.4, 70.3, 70.3, 70.2, 69.4, 63.1,

62.8, 59.4, 55.8, 55.3, 53.2, 51.3, 50.3, 50.2, 47.2, 43.4, 42.8,

42.7, 35.3, 28.7, 24.1, 20.0. HRMS (ESI) m/z: calculated for

C₄₉H₆₇N₇O₁₀[(M+2H)²⁺] 456.7469; found 456.7434.

N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-14-(4-((2-methoxy-4-

((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-

1H-1,2,3-triazol-1-yl)-3,6,9,12-tetraoxatetradecanamide (12a)

Compound 12a was subjected to general procedure 2. Offwhite

solid, 55%. LC-HRMS: t_R=5.43 min (10–100% MeCN, 15 min run),

96.49 %. ¹H NMR (300 MHz, CDCl₃) δ 7.85 (s, 1H), 7.20 (d, J =

8.7 Hz, 1H), 7.00 – 6.79 (m, 6H), 6.68 (d, J = 8.1 Hz, 1H), 6.53 (d,

J = 8.1 Hz, 1H), 5.28 (d, J = 7.1 Hz, 2H), 4.53 (t, J = 5.6 Hz, 2H),

4.42 (d, J = 7.5 Hz, 1H), 3.93 (s, 2H), 3.87 (t, J = 5.2 Hz, 2H), 3.84

(s, 3H), 3.82 (s, 3H), 3.68 – 3.56 (m, 14H), 3.53 (s, 2H), 3.09-3.03

(m, 5H), 2.95 (d, J = 6.4 Hz, 1H), 2.72 – 2.53 (m, 6H), 2.35 (d, J

= 6.5 Hz, 2H), 2.21 – 2.11 (m, 2H), 1.86 (qd, J = 12.6, 3.3 Hz, 1H),

1.74 – 1.62 (m, 1H), 1.61 – 1.37 (m, 3H), 0.90 – 0.75 (m, 1H),

0.55 – 0.47 (m, 2H), 0.15 – 0.07 (m, 2H). ¹³C NMR (75 MHz,

CDCl₃) δ 169.8, 152.2, 149.4, 146.8, 144.1, 142.5, 141.2, 140.2,

131.1, 130.9, 124.2, 124.08, 122.8, 121.7, 120.9, 119.0, 118.2,

N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-17-(4-((2-methoxy-4-((4-(2-

methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-1H-1,2,3-

118.0, 113.7, 112.9, 111.1, 92.8, 70.8, 70.5, 70.4, 70.4, 70.3, 70.3, triazol-1-yl)-3,6,9,12,15-pentaoxaheptadecanamide (12e)

70.0, 69.4, 63.1, 62.7, 62.3, 59.1, 55.8, 55.3, 53.1, 51.0, 50.3,

50.2, 47.6, 44.0, 30.7, 30.0, 24.4, 22.6, 9.4, 3.9, 3.7. HRMS (ESI)

Compound 12e was subjected to general procedure 2. Offwhite

m/z: calculated for C₅₂H₇₁N₇O₁₁[(M+2H)²⁺] 484.7600; found

solid, 46%. LC-HRMS: t_R=5.30 min (10–100% MeCN, 15 min run),

484.7572.

88.20 %. HRMS (ESI) m/z: calculated for C₅₁H₇₁N₇O₁₁[(M+2H)²⁺]

478.7600; found 478.7562.

N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-17-(4-((2-methoxy-4-

((4-(2-methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-

1H-1,2,3-triazol-1-yl)-3,6,9,12,15-pentaoxaheptadecanamide

N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-20-(4-((2-methoxy-4-((4-(2-

methoxyphenyl)piperazin-1-yl)methyl)phenoxy)methyl)-1H-1,2,3-

(12b)

triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosanamide (12f)

Compound 12b was subjected to general procedure 2. Offwhite

solid, 52%. LC-HRMS: t_R=5.44 min (10–100% MeCN, 15 min run),

92.62%. HRMS (ESI) m/z: calculated for C₅₄H₇₅N₇O₁₂[(M+2H)²⁺]

Compound 12f was subjected to general procedure 2. Offwhite

solid, 40%. LC-HRMS: t_R=5.37 min (10–100% MeCN, 15 min run),

92.86 %. ¹H NMR (300 MHz, CDCl₃) δ 7.84 (s, 1H), 7.22 (d, J =

506.7731; found 506.7713.

9.7 Hz, 1H), 7.02 – 6.88 (m, 4H), 6.82 (t, J = 8.0 Hz, 2H), 6.66 (d,

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FULL PAPER

J = 8.0 Hz, 1H), 6.55 (d, J = 8.1 Hz, 1H), 5.26 (s, 2H), 4.71 (s, 0H),

4.52 (t, J = 5.1 Hz, 2H), 4.43 (dd, J = 8.0, 3.6 Hz, 1H), 3.92 (s,

2H), 3.85 (d, J = 4.3 Hz, 5H), 3.81 (d, J = 3.0 Hz, 3H), 3.77 – 3.45

(m, 29H), 3.19 – 2.98 (m, 6H), 2.98 – 2.92 (m, 1H), 2.65 (t, J = 4.8

Hz, 4H), 2.51 (dd, J = 11.9, 4.4 Hz, 1H), 2.38 (s, 3H), 2.18 (dt, J

= 15.9, 5.0 Hz, 2H), 1.82 (dt, J = 11.6, 5.3 Hz, 2H), 1.71 – 1.47

(m, 2H), 1.44 – 1.23 (m, 1H), 1.15 – 0.99 (m, 1H). ¹³C NMR (75

MHz, CDCl₃) δ 169.9, 152.2, 149.4, 146.8, 144.1, 142.7, 141.3,

140.2, 131.3 (d, J = 4.5 Hz), 129.3, 125.3, 124.2, 122.8, 121.6,

120.9, 119.4, 118.2, 117.5, 113.7, 112.8, 111.1, 93.1, 70.8, 70.5,

70.4, 70.3, 70.2, 69.4, 63.1, 62.8, 59.4, 55.8, 55.3, 53.2, 51.8,

50.3 (d, J = 14.4 Hz), 47.2, 43.4, 42.8 (d, J = 8.3 Hz), 35.3, 28.7,

24.2, 20.1. HRMS (ESI) m/z: calculated for C₅₃H₇₅N₇O₁₂

[(M+2H)²⁺] 500.7731; found 500.7742.

146.2, 142.5, 140.2, 138.2, 136.5, 136.1, 130.9, 129.0, 126.2,

124.1, 123.1, 122.8, 121.4, 119.8, 119.0, 118.0, 112.0, 92.7, 77.2,

70.8, 70.5, 70.5, 70.4, 70.3, 70.3, 70.1, 69.4, 62.3, 59.1, 56.6,

52.8, 52.5, 51.0, 50.2, 47.7, 44.0, 36.5, 35.2, 32.1, 30.6, 30.0,

25.7, 24.4, 23.5, 22.6, 21.9, 21.3, 11.9, 9.4, 3.9, 3.7. HRMS (ESI)

m/z: calculated for C₅₈H₈₁N₇O₁₁[(M+2H)²⁺] 525.7991; found

525.8002.

N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-20-(4-(3-((4-(2-((5-

hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-3,6,9,12,15,18-hexaoxaicosanamide (13c)

N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-14-(4-(3-((4-(2-((5-

hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-3,6,9,12-tetraoxatetradecanamide (13a)

Compound 13c was subjected to general procedure 2. Offwhite

solid, 43%. LC-HRMS: t_R=5.53 min (10–100% MeCN, 15 min run),

98.35 %. HRMS (ESI) m/z: calculated for C₆₀H₈₅N₇O₁₂[(M+2H)²⁺]

547.8122; found 547.8098.

14-(4-(3-((4-(2-((5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-3,6,9,12-tetraoxatetradecanamide (13d)

Compound 13a was subjected to general procedure 2. Offwhite

solid, 60%. LC-HRMS: t_R=5.43 min (10–100% MeCN, 15 min run),

99.45%. H NMR (300 MHz, CDCl₃) δ 7.40 (d, J = 8.2 Hz, 2H),

1

7.22 (d, J = 8.9 Hz, 1H), 7.08 (d, J = 8.4 Hz, 2H), 6.94 (t, J = 7.8

Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.62 (d, J = 7.9 Hz, 2H), 6.53 (d,

J = 8.2 Hz, 1H), 4.47 (t, J = 5.1 Hz, 2H), 4.41 (d, J = 7.5 Hz, 1H),

3.92 (s, 2H), 3.83 (t, J = 5.2 Hz, 2H), 3.64-3.56 (m, 14H), 3.07 (dt,

J = 15.4, 6.3 Hz, 3H), 2.99 – 2.85 (m, 2H), 2.76 (dt, J = 14.2, 7.4

Hz, 6H), 2.56 (dt, J = 17.0, 7.8 Hz, 4H), 2.35 (d, J = 6.5 Hz, 2H),

2.20 – 1.99 (m, 3H), 1.85 (q, J = 12.8 Hz, 1H), 1.71 – 1.36 (m, 6H),

1.25 (s, 1H), 0.89 (t, J = 7.3 Hz, 3H), 0.56 – 0.48 (m, 2H), 0.11 (q,

J = 5.0 Hz, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 170.6, 170.1, 153.8,

146.2, 142.5, 140.3, 137.9, 136.2 (d, J = 9.1 Hz), 130.3, 129.1,

126.3, 124.0, 123.1, 122.9, 121.3, 119.9, 119.1, 118.0, 112.1,

92.6, 77.2, 74.6, 70.8, 70.5, 70.4, 70.4, 70.3, 70.3, 70.1, 69.4,

62.3, 59.1, 56.8, 53.4, 52.7 (d, J = 18.9 Hz), 51.0, 50.2, 47.6, 44.1,

36.5, 34.9, 32.0, 30.6, 30.0, 25.6, 24.5, 23.5, 22.6, 21.7, 21.3,

11.9, 9.4, 3.9, 3.7. HRMS (ESI) m/z: calculated for C₅₆H₇₇N₇O₁₀

[(M+2H)²⁺] 503.7860; found 503.7833.

Compound 13d was subjected to general procedure 2. Offwhite

solid, 52%. LC-HRMS: t_R=5.26 min (10–100% MeCN, 15 min run),

98.63 %. ¹H NMR (300 MHz, CDCl₃) δ 8.23 (s, 1H), 7.59 (s, 1H),

7.39 (d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.1 Hz, 2H), 6.96 (t, J = 7.7

Hz, 1H), 6.72 (d, J = 8.1 Hz, 1H), 6.64 (d, J = 7.6 Hz, 1H), 6.58

(dd, J = 8.0, 4.2 Hz, 2H), 4.47 (t, J = 5.0 Hz, 2H), 4.37 (d, J = 7.9

Hz, 1H), 3.92 (s, 2H), 3.82 (t, J = 5.0 Hz, 2H), 3.69 – 3.50 (m,

14H), 3.11 (d, J = 6.9 Hz, 3H), 2.98 – 2.85 (m, 3H), 2.83 – 2.66

(m, 6H), 2.55 (dd, J = 8.6, 6.5 Hz, 3H), 2.41 (s, 3H), 2.19 (d, J =

12.6 Hz, 2H), 2.02 (d, J = 12.6 Hz, 1H), 1.81 (td, J = 12.2, 11.3,

7.1 Hz, 2H), 1.69 – 1.57 (m, 1H), 1.50 (q, J = 7.1 Hz, 3H), 1.25 (s,

3H), 0.89 (t, J = 7.3 Hz, 3H). ¹³C NMR (75 MHz, CDCl₃) δ 170.6,

169.9, 146.2, 153.8, 142.8, 140.2, 136.6, 136.0, 129.1, 126.3,

123.2, 122.8, 121.3, 119.8, 119.5, 117.8, 112.0, 93.0, 70.7, 70.5,

70.4, 70.4, 70.2, 70.2, 69.4, 59.5, 56.7, 53.4, 51.6, 50.1, 47.2,

43.3, 42.5, 36.6, 35.2, 32.1, 29.6, 28.7, 25.7, 24.0, 23.5, 21.9,

21.4, 20.1, 11.9. HRMS (ESI) m/z: calculated for C₅₃H₇₃N₇O₉

[(M+2H)²⁺] 475.7729; found 475.7702.

N-((4R,4aS,7R,7aR,12bS)-3-(cyclopropylmethyl)-4a,9-

dihydroxy-2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-

methanobenzofuro[3,2-e]isoquinolin-7-yl)-17-(4-(3-((4-(2-((5-

hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-3,6,9,12,15-pentaoxaheptadecanamide (13b)

17-(4-(3-((4-(2-((5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-3,6,9,12,15-pentaoxaheptadecanamide (13e)

Compound 13b was subjected to general procedure 2. Offwhite

solid, 47%. LC-HRMS: t_R=5.48 min (10–100% MeCN, 15 min run),

1

95.65%. H NMR (300 MHz, CDCl₃) δ 7.59 (s, 1H), 7.40 (d, J =

8.4 Hz, 2H), 7.08 (d, J = 8.3 Hz, 2H), 6.95 (t, J = 7.8 Hz, 1H), 6.72

(d, J = 8.1 Hz, 1H), 6.62 (t, J = 7.8 Hz, 2H), 6.54 (d, J = 8.1 Hz,

1H), 3.92 (s, 2H), 3.81 (t, J = 5.0 Hz, 2H), 3.72 – 3.48 (m, 18H),

3.17 – 3.02 (m, 3H), 3.02 – 2.85 (m, 2H), 2.85 – 2.65 (m, 5H),

2.63 – 2.49 (m, 4H), 2.35 (d, J = 6.5 Hz, 2H), 2.15 (s, 1H), 1.61 –

1.38 (m, 3H), 0.89 (t, J = 7.3 Hz, 3H), 0.55 – 0.47 (m, 2H), 0.14 –

0.09 (m, 2H). ¹³C NMR (75 MHz, CDCl₃) δ 170.6, 170.0, 153.6,

Compound 13e was subjected to general procedure 2. Offwhite

solid, 48%. LC-HRMS: t_R=5.33 min (10–100% MeCN, 15 min run),

95.43 %. HRMS (ESI) m/z: calculated for C₅₅H₇₇N₇O₁₀[(M+2H)²⁺]

497.7860; found 497.7826.

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Human embryonic kidney (HEK) 293T cells were cultured in

Dulbecco's modified Eagle's medium (DMEM; Invitrogen),

supplemented with 10% fetal bovine serum, penicillin (100 U/ml),

and streptomycin (100 µg/ml) in a controlled environment (37 °C,

98% humidity, 5% CO₂). For ligand binding experiments and β-

arrestin2 recruitment assay, HEK293T cells were transiently

transfected using the Polyethylenimine (PEI) method as

described before.³⁰A total amount of 10 µg of DNA was used for

transfection of cells in one 10-cm dish. HEK293S cells stably

expressing HA D_4.2R³⁰were grown in DMEM/F12 (Gibco,

Invitrogen) supplemented with 10% fetal bovine serum, penicillin

(100 U/ml), streptomycin (100 µg/ml), and 0.5 mg/ml G418

(Geneticin, Gibco) in a controlled environment (37 °C, 98%

humidity, 5% CO₂).

20-(4-(3-((4-(2-((5-hydroxy-1,2,3,4-tetrahydronaphthalen-2-

yl)(propyl)amino)ethyl)phenyl)amino)-3-oxopropyl)-1H-1,2,3-

triazol-1-yl)-N-((4R,4aR,7R,7aR,12bS)-9-hydroxy-3-methyl-

2,3,4,4a,5,6,7,7a-octahydro-1H-4,12-methanobenzofuro[3,2-

e]isoquinolin-7-yl)-3,6,9,12,15,18-hexaoxaicosanamide (13f)

Compound 13f was subjected to general procedure 2. Offwhite

solid, 44%. LC-HRMS: t_R=5.37 min (10–100% MeCN, 15 min run),

1

97.02 %. H NMR (300 MHz, CDCl₃) δ 8.48 (d, J = 7.4 Hz, 1H),

7.59 (s, 1H), 7.40 (d, J = 7.9 Hz, 2H), 7.07 (d, J = 8.0 Hz, 2H),

6.93 (t, J = 7.7 Hz, 1H), 6.69 (d, J = 8.1 Hz, 1H), 6.58 (q, J = 8.6

Hz, 3H), 5.29 (s, 1H), 4.52 – 4.35 (m, 3H), 3.90 (s, 2H), 3.79 (t, J

= 5.0 Hz, 2H), 3.71 – 3.42 (m, 22H), 3.10 (t, J = 6.7 Hz, 3H), 2.94

(td, J = 13.9, 13.5, 7.0 Hz, 2H), 2.83 – 2.62 (m, 6H), 2.62 – 2.44

(m, 4H), 2.38 (s, 3H), 2.18 (dt, J = 12.5, 4.2 Hz, 2H), 2.02 (d, J =

11.2 Hz, 1H), 1.80 (t, J = 11.9 Hz, 1H), 1.69 – 1.40 (m, 4H), 1.27

(d, J = 12.9 Hz, 1H), 1.03 (t, J = 12.9 Hz, 1H), 0.88 (t, J = 7.3 Hz,

3H). ¹³C NMR (75 MHz, CDCl₃) δ 170.6, 170.1, 154.1, 146.3,

142.8, 140.3, 138.1, 136.4, 136.2, 129.2, 129.0, 126.2, 125.0,

123.4, 122.9, 121.0, 119.8, 119.4, 117.7, 111.9, 92.9, 70.7, 70.5,

70.4, 70.3, 70.3, 70.2, 69.3, 59.4, 56.7, 52.8, 52.6, 51.6, 50.2,

47.2, 43.3, 42.5 (d, J = 11.1 Hz), 36.4, 35.1 (d, J = 8.9 Hz), 32.2,

28.7, 25.8, 24.1, 23.7, 22.0, 21.3, 20.1. HRMS (ESI) m/z:

calculated for C₅₇H₈₁N₇O₁₁[(M+2H)²⁺] 519.7991; found 519.7953.

Membrane Preparation and Protein Determination.

Membrane suspensions from HEK293T cells transiently

transfected with the receptors of interest were prepared as

described previously.^27,28Cells were disrupted with a Polytron

homogenizer for two 10 s periods in 10 volumes of ice-cold Tris-

HCl buffer (50 mM, pH 7.4). Membranes were obtained by

centrifugation at 16500g for 20 min at 4 °C, and the pellet was

resuspended and centrifuged under the same conditions. The

resulting pellet was stored at –80 °C and was washed once more

as described above and resuspended in Tris-HCl buffer (50 mM,

pH 7.4) for immediate use. Protein was quantified by the BCA

method using bovine serum albumin dilutions as the standard.

Plasmids and receptor fusion constructs

The plasmids HA D_4.2and HA D₂were described previously.³⁸The

NanoBiT constructs (NB MCS1) and (NB MCS2) were procured

from Promega. These plasmids encode for the split fragments of

Nanoluciferase: LargeBiT (LgBiT) and SmallBiT (SmBiT). The

human µOR was PCR amplified and SmBiT was cloned at the C-

[³H]Spiperone Binding.

Saturation binding assay for [³H]spiperone was performed as

previously.^27,28For competition binding assays, membranes (20

μg) were incubated in binding buffer (50 mM Tris-HCl containing

5 mM KCl, 1 mM EDTA, 1.5 mM CaCl₂and 4 mM MgCl₂, pH 7.4)

with 0.2 nM [³H] spiperone (specific activity = 76 Ci/mmol,

PerkinElmer, USA) in the absence or presence of 11 different

concentrations (0.1 nM-10 µM) of unlabeled ligands for 1 h at

22 °C. Nonspecific binding was determined in the presence of 1

μM haloperidol. Incubation was terminated by rapid filtration

through GF/C glass-fiber filters (Whatman Schleicher and Schuell,

Keene, NH) embedded in 0.1% polyethylenimine and mounted on

a Brandel cell harvester. Filters were washed three times with cold

50 mM Tris-HCl, pH 7.4 buffer and transferred to vials containing

3 mL of scintillation cocktail to soak the filter overnight. The

radioactivity counts were measured with a Tri-Carb 2800TR liquid

scintillation analyzer (PerkinElmer).

terminus

of

the

receptor.

A

flexible

linker

(GSSGGGGSGGGGSSG) is present in between µOR and SmBiT.

In order to clone µOR-SmBiT, specific primers were generated

with a HindIII restriction site at the 5’ terminus and a XhoI site at

the 3’ terminus of the µOR coding sequence by removing the stop

codon. PCR was performed with 20 ng of plasmid with Pfu DNA

Polymerase (2.5 U/µL, Thermo Fisher Scientific) according to the

manufacturer’s protocol. The reactions were performed in a

Mastercycler™ Nexus Thermal Cycler (Eppendorf, Hamburg,

Germany) and then purified using QIAquick Gel Extraction Kit

(QIAGEN, Germany). The resultant amplicon and the vector (NB

MCS2) were digested with HindIII and XhoI for 3 h at 37°C. The

digested PCR product was cleaned using “High Pure PCR

Product Purification Kit” (Roche, Germany) and then ligated into

the corresponding digested NB MCS2. After transformation of the

ligated product into competent MC 1061 “E. coli” strain, the

ampicillin resistant clones were screened by performing PCR with

primers against µOR. Furthermore, a digestion was performed

with HindIII and XbaI, which are the restriction sites flanking µOR-

SmBiT. The constructs were verified by sequencing. LgBiT-Barr2

has been previously described.³²

[³H]Raclopride Binding.

[³H]raclopride saturation binding assay was performed as

39

described before.

Briefly, a suspension of whole cells

(corresponding a total protein amount of 20 µg) were incubated

with different concentrations (0–20 nM) of [³H]raclopride in 50 mM

Tris-HCl, pH 7.4 buffer (containing 5 mM KCl, 100 mM NaCl, 1.5

mM CaCl₂, 4 mM MgCl₂and 1 mM EDTA) at 37°C for 1 h. For

competitive binding experiments, 2.0 nM [³H]raclopride (specific

activity = 73.8 Ci/mmol, PerkinElmer, USA) was incubated with 20

Cell culture and transfection

11

10.1002/cmdc.201700787

ChemMedChem

FULL PAPER

µg of whole cells in the absence or presence of varying

concentrations (0.1 nM-10 µM) of unlabeled ligands at 37°C for 1

h. Nonspecific binding was determined in the presence of 1 μM

haloperidol. Free and cell-bound ligand were separated by rapid

filtration through GF/C glass-fiber filters (Whatman Schleicher

and Schuell, Keene, NH) embedded in 0.1% polyethylenimine

and mounted on a Brandel cell harvester. Filtration and measuring

were as described above.

Imaging system. In the analysis, the phospho-p44/42 MAPK

signal is normalized against the total p44/42 MAPK signal after

quantification by Image J.

β-arrestin2 recruitment

HEK293T cells were seeded in 10-cm dishes with 2.5 x 10⁶

cells/dish and transiently transfected with pµOR-SmBiT (4 µg),

pLgBiT-βarr2 (1 µg), pGRK2 (1 µg) and pcDNA3 (4 µg) per 10-

cm dish. Forty-eight hours after transfection, the cells were

washed with warm PBS, a cell suspension was prepared with

warm HBSS and proteins were estimated by the bicinchoninic

acid method (BCA, Thermo Scientific™) using bovine serum

albumin dilutions as the standard. The cell suspension was added

to a 96-well plate (100 µl cell suspension in each well

corresponding to 800 ng protein). The Nano-Glo Live Cell reagent,

a nonlytic detection reagent containing the cell permeable

furimazine substrate, was prepared by diluting the Nano-Glo Live

Cell substrate 20× using Nano-Glo LCS Dilution buffer, and 25 μl

was added to each well. Subsequently, the plate was placed in

[³H]Diprenorphine Binding.

[³H]diprenorphine saturation binding assay was adapted from a

published protocol.⁴⁰Membranes (10 µg) were incubated with

different concentrations (0–6 nM) of [³H]diprenorphine in 50 mM

Tris-HCl, pH 7.4 buffer at 25°C for 1 h. For competitive binding

experiments, 0.5 nM [³H]diprenorphine (specific activity = 25.8

Ci/mmol, PerkinElmer, USA) was incubated with 10 µg of

membranes in the absence or presence of varying concentrations

(0.1 nM-10 µM) of unlabeled ligands at 25°C for 1 h. Nonspecific

binding was determined in the presence of 10 μM naloxone.

Filtration and measuring were as described above.

the

luminodetecter

(CLARIOstar)

for

measurement.

Luminescence was monitored during the equilibration period until

the signal was stabilized before adding the agonists of µOR (0.1

nM-10µM, 14 µl). After adding the test compounds, luminescence

was continuously detected for 20-22 cycles. Solvent controls were

run in all experiments.

Data Analysis. Results are expressed as means ± SEM for at

least three experiments, each performed in duplicate. Statistical

analysis and curve fits of dose-response curves were performed

using GraphPad Prism 7. Ki values were calculated by using the

Cheng-Prusoff equation.⁴¹

Acknowledgements

MAPK phosphorylation

This project is financially supported by China Scholarship

Council and FWO (G021715N).

HEK293S cells stably expressing HA D_4.2R were seeded in 6-well

plates at 400,000 cells per well. When the cells were 70%

confluent, cells were starved overnight with serum free medium.

The ligands (1 nM-10µM) were added sequentially to the cells at

the concentrations indicated in the legend for 5 min. To stop the

reaction, medium was aspirated, the cells were placed on ice and

washed with ice-cold PBS. RIPA buffer [150 mM NaCl; 50 mM

Tris/HCl pH 7.5; 1 % NP-40; 0.1 % SDS; and 0.5 % deoxycholic

acid sodium salt (Acros Organics: 218591000). protease

inhibitors: aprotinin (2.5 μg/ml, Sigma-Aldrich: A6279), pefablock

(1 mM, Sigma-Aldrich: 76307), leupeptin (10 μg/ml, Acros

Organics: 328350050), and phosphatase inhibitor β-glycerol

phosphate disodium salt pentahydrate (10 mM, Fluka

BioChemika: 50020) was used to prepare the cell lysate for

western blotting according to the protocol described before.⁴²

After western blotting, the membrane was incubated with blocking

buffer (LI-COR Biosciences) for 1h at RT. Then, primary antibody

rabbit polyclonal phospho-p44/42 MAPK (1/2000) (Cell Signaling,

cat. no. 9101L) or mouse monoclonal p44/42 MAPK (1/2000)

(clone L34F12; Cell Signaling, cat. no. 4696S) was added to

develop the blot overnight at 4 °C. Next, the blot was washed with

washing buffer (PBS + 0.05% Tween 20 from Sigma-Aldrich) for

three times and then secondary antibodies goat anti-rabbit

IRDye680 LT (1/10000) (cat. no. 926–68021, LI-COR

Biosciences) or goat anti-mouse IRDye800 (1/10000) (cat.

no.926–32210, LI-COR Biosciences) were added for 1h at RT in

the dark. The blot was washed three times with washing buffer

and the fluorescent signal was detected with the Odyssey Infrared

Keywords: Dopamine D₂-like receptors • µ opioid receptor •

Bivalent ligands • Ligand binding • Signal transduction

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Entry for the Table of Contents

A series of heterobivalent µOR agonist/antagonist-D₂-likeR agonist/antagonist ligands were designed and synthesized as

pharmacological tools to study µOR-D₂-likeR heterodimers. The most promising bivalent ligand 12d displayed a biphasic competition-

binding curve for D₄R-µOR heterodimer and showed excellent potency both in MAPK-P for D₄R and β-arrestin2 recruitment for µOR.

14

Article Doi

Design, Synthesis, and Biological Evaluation of Bivalent Ligands Targeting Dopamine D2-Like Receptors and the μ-Opioid Receptor

DOI: 10.1002/cmdc.201700787

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Article abstract of DOI:10.1002/cmdc.201700787

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