Synthesis, Radiosynthesis and Biological Evaluation of Buprenorphine-Derived Phenylazocarboxamides as Novel μ-Opioid Receptor Ligands

Article abstract of DOI:10.1002/cmdc.202000180

Targeted structural modifications have led to a novel type of buprenorphine-derived opioid receptor ligand displaying an improved selectivity profile for the μ-OR subtype. On this basis, it is shown that phenylazocarboxamides may serve as useful bioisosteric replacements for the widely occurring cinnamide units, without loss of OR binding affinity or subtype selectivity. This study further includes functional experiments pointing to weak partial agonist properties of the novel μ-OR ligands, as well as docking and metabolism experiments. Finally, the unique bifunctional character of phenylazocarboxylates, herein serving as precursors for the azocarboxamide subunit, was exploited to demonstrate the accessibility of an ¹⁸F-fluorinated analogue.

Full text of DOI:10.1002/cmdc.202000180

Accepted Article
Title: Synthesis, biological evaluation and radiosynthesis of
buprenorphine-derived phenylazocarboxamides as novel μ-opioid
receptor ligands
Authors: Jasmin Krüll, Stefanie K. Fehler, Laura Hofmann, Natascha
Nebel, Simone Maschauer, Olaf Prante, Peter Gmeiner,
Harald Lanig, Harald Hübner, and Markus Rolf Heinrich
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
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to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000180
Link to VoR: https://doi.org/10.1002/cmdc.202000180
01/2020

10.1002/cmdc.202000180
ChemMedChem
Synthesis, biological evaluation and radiosynthesis of buprenorphine-derived
phenylazocarboxamides as novel μ-opioid receptor ligands
Jasmin Krüll,a Stefanie K. Fehler,a Laura Hofmann,a Natascha Nebel,b Simone Maschauer,b
Olaf Prante,b Peter Gmeiner,a Harald Lanig,c Harald Hübner,a Markus R. Heinricha,*
aDepartment of Chemistry and Pharmacy, Pharmaceutical Chemistry, Friedrich-Alexander-
Universität Erlangen-Nürnberg, Nikolaus-Fiebiger-Str. 10, 91058 Erlangen, Germany
bDepartment of Nuclear Medicine, Molecular Imaging and Radiochemistry, Friedrich-
Alexander-Universität Erlangen-Nürnberg, Schwabachanlage 6, 91054 Erlangen, Germany
cCentral Institute for Scientific Computing (ZISC), Friedrich-Alexander-Universität Erlangen-
Nürnberg, Martensstr. 5a, 91058 Erlangen, Germany
markus.heinrich@fau.de
Abstract
Targeted structural modifications led to a novel type of buprenorphine-derived opioid receptor
ligands displaying an improved selectivity profile for the μ-OR subtype. On this basis, it is
shown that phenylazocarboxamides may serve as useful bioisosteric replacements for widely
occuring cinnamide units, without loss of OR binding affinity or subtype selectivity. The
present study further includes functional experiments, pointing to weak partial agonist
properties of the novel μ-OR ligands, as well as docking and metabolism experiments. Finally,
the unique bifunctional character of phenylazocarboxylates, herein serving as precursors for the
18
azocarboxamide subunit, was exploited to demonstrate the accessibility of an F-fluorinated
analogue.
Introduction
Opioid receptors (OR) play an important role in medicinal chemistry[1-5] and continuous efforts
are made to develop new drug candidates[1, 6-8] as well as ligands designed for investigations in
the chemical biology of these receptors. Among the major subtypes μ,[9] κ,[10] δ,[11] and the
nociceptin receptor[12] the selective addressing of the µ subtype is of interest for a number of
applications.[13-18] Exemplarily, the selective µ-OR agonist PZM21 has been proposed for pain
treatment with reduced side effects.[19] PZM21 is not only subtype selective, but also displays
biased signaling with minimal β-arrestin recruitment.[20,21] Further analogs of PZM21 were
recently reported by Shi and co-workers.[22] The partial µ-OR agonist NAP, which consists of
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10.1002/cmdc.202000180
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a morphinan scaffold, also shows biased signal transduction.[23] NAP was developed for the
treatment of opioid-induced constipations. A first-in-human clinical trial was already performed
applying the biased µ-OR agonist TRV130.[24,25] Although pain management with reduced side
effects such as respiratory depression was proven within this clinical trial, the value of TRV130
has proven to be controversial.[26] Upon optimization of a piperidine benzimidazolone scaffold,
promising µ-OR agonists with exceptionally high bias factors have recently been discovered by
Bohn and Bannister.[27] Regarding the recently developed OR ligands from a structural point of
view, non-morphinan-derived[19,22,24,27] as well as morphinan-derived[23,28-31] scaffolds have
been used. Among the latter ones, scaffolds based on morphine (1),[32] diprenorphine (2)[33] and
β-funaltrexamine (β-FNA) (3)[34,35] still represent valuable starting points in the development
of opioid receptor ligands (Figure 1).[36]
Figure 1. Selected examples of morphinan-derived opioid receptor ligands.
With regard to subtype selectivity, the particular importance of the side chain on the
diprenorphine scaffold can be derived from the binding data summarized in Table 1.[33,34, 37,38]
Table 1. Binding affinities of known ligands derived from diprenorphine (2).[33, 34, 37,38]
compound
R1 =
Ki (nM)


0.14

0.73
2[33]
4[37]
5[34]
6[38]
7[38]
8[38]
H
H
2.0
2.19±0.65
0.7±0.25
14.4
4.15±1.3
2.6±0.0
0.15
3.66±0.92
0.7±0.05
89.3
H
Me
Me
Me
1.91
25.8
1753
1.87
0.74
--
When aiming at μ subtype selectivity, the 3-hydroxy substituted ligands diprenorphine (2), 4
and 5 (R1 = H) do not appear as preferred lead structures since these compounds show limited
subtype discrimination. Methylation of the 3-hydroxy group (R1 = Me), however, changes the
influence of the substituent R2. As demonstrated by the diprenorphine derivatives 6-8,[38] and
in particular by the biphenyl derivative 7, a suitably chosen side chain R2 can now lead to
preferred binding to the μ subtype.
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For the present study, we thought to combine the high binding affinity of the cinnamide 5[34]
towards the μ-OR (Ki = 0.7 nM, see Table 1) with methylation at the 3-hydroxy group to shift
the selectivity towards the μ subtype (Scheme 1). Moreover, the cinnamide substructure should
be replaced by a phenylazocarboxamide so that ligands of the general structure 9 would be
obtained.
Scheme 1. Structural modifications of cinnamide ligand 5 and synthetic approach to
azocarboxamides 9 from amine 10 and tert-butyl phenylazocarboxylates 11.
Besides our general interest in the question whether phenylazocarboxamides can serve as
bioisoteric replacements for cinnamide substructures,[39,40] the successful preparation of the
target compounds 9 from amine 10 and azo esters 11 would further enable a straightforward
access to the corresponding 18F-fluorinated analogue (R = 4-18F) (Scheme 1). This is due to the
good availability of the 18F-labeled tert-butyl phenylazocarboxylate 11 (R = 4-18F),[41-43]
whereat the corresponding 18-fluorine labeled azocarboxamide 9 (R = 4-18F) could then serve
as a µ-OR radioligand for in vivo PET imaging studies.[42-46] For all OR ligands of the general
structure 9, non-radiolabeled as well as radiolabeled derivatives, we envisaged the coupling of
the amine 10 with the corresponding tert-butyl phenylazocarboxylate 11 as final synthetic
step.[41] In addition to chemical and radiochemical syntheses, computational docking studies
were carried out to give insights into the factors responsible for binding affinity and subtype
selectivity. Besides the potential cytotoxicity, we further investigated the metabolic stability of
the novel ligands using an in vitro rat microsomal stability assay.
Synthesis
The synthetic route to the azocarboxamide and cinnamide ligands 9a-d and 16a-b evaluated in
this work is depicted in Scheme 2. For some reactions, the conditions are based on those
previously established by Kok et al.,[47] and for other steps, the reaction conditions were derived
from the work by Derrick et al.[34]
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Scheme 2. Synthesis of azocarboxamide ligands 9a-d and reference compounds 16a,b.
Reagents and conditions: i) mCPBA, HCl, ferroceneacetic acid (12 mol%), CHCl3/i-PrOH
(3:1), -5 °C to 50 °C, 28 h, 80%; ii) (bromomethyl)cyclopropane, NaHCO3, DMF, 90 °C, 20 h,
65%; iii) ethyl acrylate, 100 °C, 15 h, 78%; iv) HCl (6 M), 100 °C, quant.; v) oxalyl chloride,
DMF (cat.), CHCl3, 0 °C to r.t., 23 h; vi) benzylamine, NEt3, CHCl3, 0 °C to r.t. 22 h, 79%
(over two steps); vii) LiAlH4, THF, 70 °C, 20 h, 51%; viii) Pd/C (10 %), ammonium formate,
EtOH, 90 °C, 1.5 h, 81%; ix) tert-butyl phenylazocarboxylate 11a-d, K2CO3 or NEt3, EtOH,
r.t., 3-120 h, 42-87%; x) cinnamic acid chloride 17a,b, NaHCO3, CH2Cl2, r.t., 28 h, 25-63%.
The ligand synthesis started with the demethylation of thebaine (12) using ferroceneacetic acid,
mCPBA and hydrochloric acid to yield a secondary amine, followed by an N-alkylation of this
amine with (bromomethyl)cyclopropane in the presence of NaHCO3 to obtain the alkylated
compound 13. Afterwards, ethyl acrylate was used as dienophile for a hetero Diels-Alder
reaction with diene 13 to give ester 14. The ester 14 was hydrolyzed under acidic conditions,
the resulting carboxylic acid was activated with oxalyl chloride, and the acid chloride was
trapped with benzylamine to furnish the amide 15. The primary amine 10 was prepared from
15 via reduction of the amide moiety by LiAlH4 and subsequent hydrogenation of the double
bond which was accompanied by cleavage of the benzyl protecting group. At this point, it is
worth to note that the carbon-carbon double bond was already partially reduced under the
LiAlH4 conditions of step vii). For step viii), the procedure by Derrick[34] recommended the use
of H2 and Pd/C at 45 °C under a H2 pressure of 30 psi (ca. 2.1 bar) to give a yield of 76%. We
initially conducted the hydrogenation under atmospheric pressure conditions (H2 balloon),
leaving all other reagents and conditions unchanged, and obtained the primary amine 10 in 68%
yield. Even after extended reaction times, only partial conversion of the starting material had
taken place, and after the addition of a larger excess of acidic acid, we detected the opening of
the cyclopropyl ring via LC/MS analysis. Thus, the conditions were changed to Pd/C and
ammonium formate at a reaction temperature of 90 °C. Under these conditions, the primary
amine 10 was obtained in 81% yield after 2.5 h, and the desired product could now be easily
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10.1002/cmdc.202000180
ChemMedChem
purified by column chromatography. The synthesis of the azocarboxamide ligands 9a-d was
accomplished by a coupling of the primary amine 10 to the corresponding tert-butyl
phenylazocarboxylates 11a-d in the presence of sodium bicarbonate or triethylamine. Among
the four different azocarboxylic esters used in step ix), slightly modified conditions were
required for the tert-butyl 4-fluorophenylazocarboxylate (11b), as the long reaction time of
three days led to a partial substitution of the 4-fluoro substituent by an ethoxy group originating
from the solvent ethanol. Since ethanol turned out to be the best choice compared to other
solvents, the synthesis of the fluorinated ligand 9b was conducted at increased concentrations
and with a larger excess of the azocarboxylate 11b. In this way, the reaction time could be
reduced from three days to 2.5 h and the side reaction to the aryl ethyl ether could be suppressed.
In step x), the cinnamide ligands 16a and 16b were prepared from amine 10 and the related
cinnamic acid chlorides 17a,b in the presence of sodium bicarbonate.
Ligand binding and functional assays
The binding affinities of the azocarboxamides 9a-d and the cinnamides 16a,b to the µ-, κ- and
δ-OR subtypes were determined by radioligand competition binding assays using
[3H]diprenorphine and membrane preparations derived from HEK 293T cells transiently
expressing the related receptor subtypes. Displacement curves resulted in Ki values for 9a-d,
16a,b and the reference -FNA (3) (Table 2).
Table 2. Binding affinities of compounds 3, 5, 9a-d and 16a,b towards the µ-, κ- and δ-OR
subtypes.
compound
Ki (nM±SEM)a
κ
selectivityb
μ
δ
μ/κ
2
μ/δ
20
17
1
β-FNA (3)c
0.4±0.05
1.5±0.21
0.7±0.25
2.3±0.26
1.5±0.45
1.3±0.30
4.0±0.39
4.9±1.5
3.3±0.8
0.9±0.05
10±2.3
2.6±0.0
19±1.9
10±2.2
10±2.6
11±1.4
18±5.7
25±5.1
7.7±2.4
26±7.4
0.7±0.05
30±2.8
36±6.5
28±3.0
63±9.9
54±9.1
58±11
3
5c
6.7
4
9a
8.3
6.7
7.7
2.8
3.7
7.6
13
24
22
16
11
18
9b
9c
9d
16a
16b
aBinding affinities are displayed as mean Ki values ±SEM derived from 3 to 11 individual experiments each done
in triplicate. bSubtype selectivity for -OR expressed as ratio of Ki values. cBinding affinities as reported by Derrick
et al.[34]
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In the previous biological evaluation of cinnamide 5,[34] which served as a starting point for the
design of our azocarboxamide ligands 9a-d, β-FNA (3) was used as a reference compound
(Table 2). Under our assay conditions, the Ki values determined for β-FNA (3) were all
increased by factors in the range of 3 to 11 (×4 for the μ-OR, ×11 for the κ-OR and ×3.5 for the
δ-OR) thereby suggesting that our assay is less sensitive for all three OR subtypes. At this point
it is important to remember that β-FNA (3) is known to be an irreversible ligand at the μ-OR,
so that the equilibrium binding affinity to this particular subtype can be highly sensitive to
deviations in the assay conditions. Since the Ki values for all three OR subtypes are however
altered by comparable factors, and binding of β-FNA (3) to the κ-OR and the δ-OR subtype is
reversible, a certain comparison of our data with that available for 5[34] appears possible.
Taking into account the above mentioned sensitivity factors (×4 for the μ-OR, ×11 for the κ-
OR and ×3.5 for the δ-OR), one can assume that cinnamide 5 and our ligands 9a-d and 16a,b
show comparable binding affinities at the μ-OR and κ-OR subtypes. At the δ-OR, in contrast,
the binding of 5 is significantly stronger. Methylation at the 3-hydroxy group, which constitutes
the general structural difference between the reference compound 5 and all of our ligands 9a-d
and 16a,b, thus leads to an improved selectivity profile for the μ towards the δ subtype, but not
for μ compared to κ. This analysis is also reflected by the selectivity ratios κ/μ and δ/μ reported
in Table 2.
Among our ligands 9a-d and 16a,b neither di-aza substitution nor ring substitution on the
azocarboxamide (c.f. Scheme 1) led to strong changes in binding affinity at any of the three OR
subtypes, thereby indicating that these structural modifications are tolerated and that no major
ligand-receptor interactions are changed. Upon comparison of the newly prepared
azocarboxamides 9a-d and cinnamides 16a,b, the 4-fluoro and the 4-bromo azocarboxamide
derivatives 9b and 9c showed the most favorable biological profiles with regard to the μ-OR.
The highest binding affinities were determined for 9b and 9c and the subtype selectivity ratios
κ/μ and δ/μ for these ligands were also among the best values.
Since a covalent binding mode to the µ-OR was originally considered for the reference
compound 5,[34] radioligand depletion assays were performed for the novel azocarboxamide 9b
and its corresponding cinnamide 16b. Through these assays it turned out that a covalent binding
to the µ-OR is neither likely for 9b, nor for 16b, whereas 16b is more closely related to reference
compound 5 due to the common cinnamide substructure.
Functional assays at µ-OR were performed with the selected test compounds 9b and 16b
applying an inositol phosphate (IP) accumulation assay for G-protein mediated signaling (IP-
One assay®) and an arrestin recruitment assay for receptor stimulated recruitment of -arrestin-
2 (PathHunter assay®). In both signaling pathways the cinnamide 16b revealed as a neutral
antagonist while the azocarboxamide 9b showed antagonist properties for arrestin recruitment
but a weak partial agonist effect with an efficacy of 19 % for G-protein mediated signaling
(Figure 2).
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10.1002/cmdc.202000180
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µ O R s tim u la te d G 
a c tiv a tio n
µ O R m e d ia te d  -a rre s tin 2 re c ru itm e n t
q i
A
B
1 0 0
1 0 0
7 5
5 0
2 5
0
7 5
5 0
2 5
0
DAM GO
DAM GO
9 b
9 b
9 b
+
30 nM DAM G O
9 b + 1 µM DAM GO
-1 2
-1 1
-1 0
-9
-8
-7
M
-6
-5
-1 1
-1 0
-9
-8
-7
-6
-5
-4
lo g [c o m p o u n d ],
lo g [c o m p o u n d ],
M
C
D
1 0 0
7 5
5 0
2 5
0
1 0 0
7 5
5 0
2 5
0
DAM GO
DAM GO
1 6 b
1 6 b
1 6 b
+
30 nM D AM G O
1 6 b + 1 µM DM AGO
-1 2
-1 1
-1 0
-9
-8
-7
M
-6
-5
-1 1
-1 0
-9
-8
-7
-6
-5
-4
lo g [c o m p o u n d ],
lo g [c o m p o u n d ],
M
Figure 2. Functional investigation of the selected test compounds applying an IP accumulation
assay (IP-One®) for testing G protein signaling and an arrestin-2 recruitment assay (Path
Hunter®). A, C) G-protein signaling was determined in HEK293T cells transiently co-
transfected with µ-OR and the hybrid G-protein Gqi (Gq protein with the last five amino acids
at the C-terminus replaced by the corresponding sequence of Gi). While 9b (red line) shows a
weak partial agonist effect (A), 16b (blue line) behaves as an antagonist (C). Both effects were
confirmed when 9b (red dots) and 16b (blue dots) completely inhibited the agonist activity of
DAMGO (EC80 concentration of 30 nM for IP and 1 µM for arrestin). B, D) Arrestin recruitment
was measured by a luminescence based enzyme complementation assay in HEK-293 cells
stably expressing the enzyme acceptor (EA) tagged -arrestin-2 fusion protein and the
transiently transfected ProLink tagged -OR-PK1. Both 9b (B) and 16b (D) show neutral
antagonist properties (lines) and a strong inhibitory effect on an EC80 concentration of
DAMGO (dotted line).
These antagonist/partial agonist results could be confirmed by inhibition experiments when the
activity of a fixed concentration of the reference agonist DAMGO was fully diminished dose-
dependently by both 9b and 16b (Figure 2). The resulting IC50 values (for 9b: 20±6.3 nM for
IP accumulation, 22±5.3 nM for arrestin recruitment; for 16b: 9.2±5.3 nM for IP, 23±3.2 nM
for arrestin) are in good agreement with the observed Ki values from the binding experiments.
Cytotoxicity assay
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Due to the potential reactivity of the unsaturated azocarboxamide substructure of 9b and the
cinnamide entity of 16b we investigated both compounds on cytotoxicity in comparison to the
µ-OR reference naloxone. For that we incubated HEK293T cells with 100 nM of 9b, 16b or
naloxone for 24 hrs and determined the number of cells indicating any influence on cell growth
resulting in cell densities of 92 ± 6 % (n = 6, mean ± SEM) for 9b, 106 ± 6 % (n = 6) for 16b
and 84 ± 5 % (n = 6) for naloxone relative to the effect of vehicle (DMSO). Complemented by
the verification of viability of the cells by optical controls these results reveal no cytotoxic effect
of the ligands.
Docking studies
First, the binding modes of the ligands 5, 9a-d, 16a and 16b within the µ-OR, the κ-OR the δ-
OR subtype were compared (Figures S5, S6, and S7, see Supporting Information) using
available X-ray structures (PDB database entries µ-OR: 4DKL; δ-OR: 4N6H; κ-OR: 4DJH) as
templates. All three structures were obtained by co-crystallization with antagonists, which
ensures that the receptor geometries represent the inactive state and are therefore directly
comparable by geometric superposition. Within the µ-OR subtype, the morphinan scaffold of
the ligands 5, 9a-9d, 16a and 16b adopts the same orientation, whereas the side chain flips
dependent on the attached substituents, in particular for 9c and 9d bearing larger substituents
on the azocarboxamide (Figure S3). The preferred binding modes found for 5, 9a-9d, 16a and
16b in the κ-OR do not show a significant dependence on the structural variations (Figure S4).
In comparison to the binding modes predicted for the µ-OR subtype (Figure S3), those at the κ-
OR (Figure S4) however suggest a completely different position of the morphinan scaffold,
which could be due to the fact the κ-OR binding pocket is narrower than those of the µ-OR and
δ-OR. All ligands 5, 9a-d and 16a,b are also likely to present very similar binding modes within
the δ-OR subtype (Figure S5), whereat the overall orientation of the ligands is comparable to
binding mode 2, which was only observed for the two bulkier ligands 9c and 9d in the µ-OR
subtype (Figure S3).
The key interactions of azocarboxamide 9b, which is one of the two ligands 9b and 9c with the
most favorable binding profile (Table 2), to the µ-OR, κ-OR and the δ-OR subtype were
analyzed and are shown in Figure 3.
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10.1002/cmdc.202000180
ChemMedChem
Figure 3. Surrounding of the best ranked pose of azocarboxamide 9b (purple) in a) the µ-OR,
b) the κ-OR and c) the δ-OR subtype. The structures were visualized using PyMOL 1.3. The
poses which are adopted by 9b subtype are shown as Lewis structures in d) the µ-OR, e) the κ-
OR and f) the δ-OR subtypes.
The interactions of the protonated amine of 9b to Asp1493.32 (µ-OR, Figure 3a) and
Asp1283.32 (δ-OR, Figure 3c) as well as between the bridging ether oxygen of 9b to Tyr1503.33
(µ-OR, Figure 3a) and Tyr1293.33 (δ-OR, Figure 3c) are comparable in both subtypes. Although
the distances between the ether oxygen of 9b and the hydroxy group on the adjacent Tyr1503.33
or Tyr1293.33 (3.7 Å for the µ-OR and 3.4 Å for the δ-OR) appear as too long for a hydrogen
bond, it is important to note that amino acid side chains are not flexible in our docking setup,
so that geometric relaxation of the docked complex by energy minimization could indeed enable
this interaction. Additionally, the hydrophobic environment is very similar in both subtypes,
leading to an almost identical placement of the morphinan scaffold with its cyclopropyl side
chain within the binding pocket. Interestingly, the 4-fluoro substituent on the
phenylazocarboxamide side chain is likely to form a σ-hole based halogen bond to the carbonyl
unit of Asn1292.62 (TM2) within the µ-OR subtype, which could induce subtype selectivity. The
δ-OR possesses a bulkier Lys1083.32 without halogen bond capability at position of Asn1292.62
in the µ-OR subtype. This results in a different binding mode of the flexible 4-fluorophenyl
side chain due to steric restrictions. Within the κ-OR subtype, the geometry of the binding
pocket does not allow an orientation of the morphinan scaffold as in the other subtypes (see
also Figure S4). Especially the residues Ile2906.51, Ile2946.55, and Tyr3127.35 are responsible for
these geometric restrictions. In the alternative binding mode, compound 9b is able to form a
hydrogen bond from the CONH group to Asp1383.32. With only minor reorientation of this Asp
side chain, an additional hydrogen bond to the protonated amine nitrogen is possible,
characterizing Asp1383.32 as a key residue for the ligand recognition. Despite the different
orientation of 9b within the other subtypes, the 4-fluorophenyl side chain finds itself in a
stabilizing hydrophobic environment formed by the residues Trp2876.48, Ile2906.51, Ile3167.39
,
and Tyr3207.43. Unfortunately, the bridging oxygen atom of the morphinan scaffold is not
directly involved in interaction to the protein. However, this deficiency is compensated by the
strong polar interactions of the ligand to Asp1383.32
.
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Metabolism
To get an impression about the metabolic stability of the newly synthesized azocarboxamide
and cinnamide ligands, we treated azocarboxamide 9b and its cinnamide analog 16b with rat
liver microsomes. To enable a largely independent evaluation of the phenylazocarboxamide
and the cinnamide motif, the simple piperidine-derived azocarboxamide 18 and its
corresponding cinnamide 19 were also prepared. In Table 3, the determined half-lives (t1/2) and
intrinsic clearances (CL’int) of the azocarboxamides and cinnamides are summarized together
with the values obtained for the reference compound imipramine (20).
Table 3. Metabolic stability of azocarboxamides 9b and 18 and cinnamides 16b and 19 in the
presence of rat liver microsomes.
half-lifea
[min]
intrinsic clearancea
[µL×min-1×mg-1]
compound
166 ± 16
252 ± 94
73 ± 12
85 ± 18
52 ± 6
42 ± 4
28 ± 5
9b
16b
18
97 ± 15
85 ± 20
132 ± 4
19
20
aThe metabolic stability was determined via the half-life and the intrinsic clearance, which were calculated
depending on the amount of rat microsomes that was used (see Supporting Information).
The positive control imipramine (20) showed the shortest half-life (52 ± 6 min) and
corresponding to that also the highest clearance with 132 ± 4 µL×min-1×mg-1 (Table 3). The
two piperidine-derived compounds 18 and 19 displayed slightly higher, but comparable
stabilities (18: t1/2 = 73 ± 12 min; 19: 85 ± 18 min), thus demonstrating that the bioisosteric
replacement of a cinnamide unit by a phenylazocarboxamide does only result in a weakly
reduced life-time under the chosen assay conditions. For each compound 18 and 19, LC/MS
analysis of the reaction mixture revealed the formation of two oxidized species, whereat the
mass difference of m/z + 16 points to hydroxylation or, is the case of the cinnamide, possibly
also to epoxidation. The trend that the cinnamide derivative shows a higher metabolic stability
than the corresponding phenylazocarboxamide also turned out to be true for the ligands 16b
and 9b, albeit with larger relative deviation than previously found for 18 and 19. For the
azocarboxamide 9b displaying the favorable binding profile (Table 2), a half-life of
166 ± 16 min and an intrinsic clearance of 42 ± 4 µL×min-1×mg-1 were determined, suggesting
sufficient metabolic stability of [18F]9b for in-vivo use as a PET imaging agent. The metabolite
which was primarily detected by LC/MS for 9b is likely to be the N-dealkylated species (m/z –
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10.1002/cmdc.202000180
ChemMedChem
54, cleavage of cyclopropylmethyl group) along with small amounts of a reduced derivative
(m/z + 2) that could result from reduction of the azo unit to the corresponding hydrazine. For
the cinnamide analog 16b showing the slightly higher metabolic stability (t1/2 = 252 ± 94 min,
CL’int = 28 ± 5 µL×min-1×mg-1), only one metabolite corresponding to N-dealkylation (m/z –
54) was found. Interestingly, oxidized species (m/z +16), as they were observed as main
metabolites for 18 and 19, were neither detected for 9b nor for 16b.
Radiosynthesis of [18F]9b
The radiosynthesis shown in Scheme 3 takes advantage of the excellent availability of the
18F-labeled tert-butyl phenylazocarboxylate [18F]11b, which can readily be prepared in a single
step from the quaternary ammonium triflate 21 in only 30 seconds and in high radiochemical
yield.[41] Based on our previously developed radiosyntheses,[40-43] we herein studied the
coupling of [18F]11b to the primary amine 10, aiming at an optimum radiochemical yield of
[18F]9b.
Scheme 3. Radiosynthesis of the 18F-labeled azocarboxamide [18F]9b.
Nucleophilic substitution of azo ester [18F]11b with the primary amine 10 in ethanol at room
temperature in the presence of cesium carbonate afforded [18F]9b within a short reaction time
of only 5 min, whereat the identity of [18F]9b was confirmed through co-injection with the
reference compound 9b by HPLC methods (see Supporting Information). A concentration of
amine 10 of 54 mM led to a radiochemical yield of [18F]9b of 11 ± 2 % (n = 3) after 5 minutes
reaction time, which did not increase any further after 10 minutes. The concentration of amine
10 was therefore increased to 108 mM, which led to an only slightly higher radiochemical yield
of 13 ± 2 % (n = 3) after 5 minutes. The relatively low radiochemical yield of the second
reaction step could be due to the formation of [18F]fluorobenzene as a major byproduct and has
kept us from further experiments with [18F]9b. Nevertheless, [18F]9b was proven to be stable
under the reaction conditions and could be successfully isolated by HPLC in radiochemically
pure form. Provided that the radiosynthesis could be automated using a synthesis module,
[18F]9b could be made available via the use of tert-butyl 4-[18F]fluorophenylazocarboxylate
[18F]11b in larger amounts for further characterization by in vitro rat brain autoradiography or
extended in vivo small animal PET imaging studies. Since the opioid receptor, especially the µ
subtype, is of high interest in the field of brain research concerned with addiction or pain
processing,[48] subtype selective μ-OR radioligands for imaging studies by positron emission
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10.1002/cmdc.202000180
ChemMedChem
tomography (PET) are valuable tools to study the regulation of the µ subtype in vivo. The
11
11
11
currently available C-labeled OR receptor ligands for PET, such as C-carfentanil or C-
PEO,[44,49] suffer from the short half-life (20.3 min) and their use is clearly restricted to
18
18
institutions with cyclotrons. The radiosyntheses of most F-labeled OR ligands, such as F-
FE-DPN or 18F-FE-PEO,[46,50] are rather laborious and give low yields. Therefore, an alternative
18F-labeled μ-OR ligand, available by a more straightforward radiosynthesis is still desirable.
In summary, a novel type of buprenorphine-derived opioid receptor ligands was obtained upon
targeted structural modification. Besides the introduction of a methyl group on the 3-hydroxy
functionality of the buprenorphine core, the cinnamide side chain of the lead structure 5 was
exchanged for various phenylazocarboxamide moieties, also with the aim to explore future
18
suitability for F-radiosynthesis. Ligand binding studies at the three OR subtypes μ, κ and δ
revealed an even increased subtype selectivity for ligands 9 compared to 5 at a basically
unchanged affinity to the μ subtype. Evaluation of the azocarboxamide 9b in functional assays
showed a weak bias for 9b, as this compound acts as an antagonist in the β-arrestin pathway,
but as a weak partial agonist in G protein activation. Its cinnamide analogue 16b, in contrast,
behaved as a neutral antagonist in both pathways. Docking studies gave an impression on the
possible binding modes thereby supporting the experimental observation that methylation at the
3-hydroxy group of the buprenorphine core is not decisive for ligands of type 9 or 16. Further
assays revealed a reasonable metabolic stability of azocarboxamide 9b compared to 16b and
related reference compounds. Finally, the course of the 18F-radiosynthesis of an opioid receptor
radioligand candidate for PET was demonstrated by the straightforward synthesis of [18F]9b.
Experimental Section
Chemistry
Reactions which are sensitive to air or water were evacuated under traditional Schlenk
conditions while heating and under an inert argon atmosphere. Solvents and reagents for
sensitive reactions classified as “extra pure”, “dry” or “extra dry” or with water contents lower
than 0.1% were applied. For reactions which were insensitive towards water, chemicals
classified as “pure” or “for synthesis” were used. The applied chemicals were used as purchased
by commercial sources (Alfa Aesar, Acros Organics, Merck, Sigma Aldrich). Thin-layer
chromatography (TLC) was performed on purchased plates from Merck (aluminum foil,
0.25 mm Kieselgel 60, F254). The substances were determined applying fluorescence detection
under ultraviolet (UV) light with wavelengths of λ = 254 and 360 nm [UV]. Liquid flash
column chromatography was performed on silica gel with a particle size between 40 and 63 µm
(230-400 mesh ASTM, Si 60) from Merck under a pressure between 1.0 and 1.5 bar. The
applied eluents are described in the respective procedure, whereby gradients are presented by
arrows (→). For mass spectrometry (MS) a Waters Alliance e2695 Separations Module system
was used with a Waters 2998 Photodiode Array Detector for detection at wavelength of λ =
220 nm and 254 nm. A binary solvent gradient of 0.1% formic acid in acetonitrile/0.1% formic
acid in water was applied on a Waters XBridge C18 (4.6 mm×50 mm, 3.5 μm). The runs for the
measuring of the metabolites for metabolism studies were performed on a longer Phenomenex
Gemini NX-C18 (110Å, 4.6×250 mm, 3.5 μm) column. Masses were detected by electro spray
ionization (ESI) in a Waters ACQUITY QDa detector. For ESI-TOF high mass accuracy and
resolution experiments an AB Sciex Triple TOF660 SCiex or a Bruker maXis MS was applied.
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10.1002/cmdc.202000180
ChemMedChem
High Performance Liquid Chromatography (HPLC) was used for purification by preparative
RP-HPLC was performed on AGILENT 1100 Preparative Series which is equipped with a
VWD detector with wavelengths of λ = 230 nm and 254 nm. A column from MACHEREY-
NAGEL Varioprep VP 250/32 NUCLEODUR C18 HTec with 5 μm particles [C18] with a flow
rate of 32 mL/min was applied. Nuclear magnetic resonance (NMR) spectra were recorded with
Avance 600 (1H NMR: 600 MHz, 13C NMR: 151 MHz) and Avance 360 (1H NMR: 400 MHz,
13C NMR: 101 MHz) instruments from Bruker at 300 K in deuterated solvents from Deutero
GmbH. The chemical shifts δ are presented in ppm and are calibrated either in accordance to
tetramethylsilane (TMS) or to the used deuterated solvents:[51] Signal multiplicities are
abbreviated with the following letters: s: singlet, bs: broad singlet, d: doublet, t: triplet, q:
quartet, dd: doublet of doublets, dt: doublet of triplets, dq: doublet of quartets, m: multiplet.
Coupling constants J are listed in Hz as experimentally determined differences of the
frequencies. Coupling between fluoro and carbon atoms in 13C spectra are presented as JCF. The
determination of the spectra was performed using Mestre-C and TopSpin.
N-Northebaine (12’): Thebaine (12, 550 mg, 1.77 mmol) was dissolved in chloroform/iso-
propanol (3:1, 40 mL) and subsequently meta-chloroperoxybenzoic acid (407 mg (75%
content), 1.77 mmol) was added at -5 °C. After 15 minutes of stirring, the mixture was treated
with hydrochloric acid (36 M, 156 μL) and ferrocene acetic acid (57 mg, 12.5 mol%) and stirred
at 50 °C for further 28 h. After cooling to r.t. aqueous sodium hydroxide solution (5 %, 15 mL)
was added and the crude product 12’ was extracted with chloroform (3×50 mL). The combined
organic layers were washed with saturated aqueous sodium chloride solution and dried over
anhydrous sodium sulfate. The solvent was removed under reduced pressure. Afterwards, the
crude product 12’ was purified by column chromatography (desactivated with
dichloromethane, then 40:1:0.430:1:0.4 dichloromethane/methanol/NH3 (25%)). The desired
product 12’ was obtained as grey solid (421 mg, 1.42 mmol, 80%). Rf = 0.3 (35:1:0.4
1
dichloromethane/methanol/NH3 (25%) [ninhydrin]. H NMR (600 MHz, CDCl3) δ = 6.67 (d,
J = 8.2 Hz, 1H), 6.61 (d, J = 8.1 Hz, 1H), 5.48 (d, J = 6.4 Hz, 1H), 5.26 (s, 1H), 5.03 (d,
J = 6.4 Hz, 1H), 3.89 (dd, J = 5.3, 2.5 Hz, 1H), 3.85 (s, 3H), 3.60 (s, 3H), 3.19 (td, J = 13.2,
3.5 Hz, 1H), 3.15 – 3.08 (m, 2H), 2.91 (dd, J = 13.8, 4.1 Hz, 1H), 2.06 (td, J = 12.6, 5.1 Hz,
1H), 1.82 (dt, J = 16.1, 8.1 Hz, 1H), 1.59 (bs, 3H) ppm. 13C NMR (91 MHz, CDCl3) δ = 152.6
(Cq), 144.9 (Cq), 142.9 (Cq), 133.5 (Cq), 127.9 (Cq), 119.3 (CH), 113.0 (CH), 110.2 (CH), 95.9
(CH), 89.3 (CH), 56.5 (CH3), 55.0 (CH3), 54.0 (CH3), 46.8 (CH2), 40.8 (Cq), 38.6 (CH2),
37.9 (CH2) ppm.
N-(Cyclopropylmethyl)northebaine (13): N-Northebaine (12’, 266 mg, 0.89 mmol), sodium
carbonate (300 mg, 3.58 mmol) and (bromomethyl)cyclopropane (133 mg, 96 µL, 0.98 mmol)
were dissolved in DMF (4.5 mL). The resulting mixture was stirred for 20 h at 90 °C. After
cooling to r.t., water was added and the crude product was extracted with chloroform
(3×75 mL). The combined organic layers were dried over sodium sulfate. After the solvent was
removed under reduced pressure, the crude product was purified by column chromatography
(49:130:1 dichloromethane/methanol). The desired product 13 was obtained as light brown
solid (281 mg, 0.80 mmol, 90%). Rf = 0.5 (20:1 dichloromethane/methanol) [ninhydrin].
1H NMR (600 MHz, CDCl3) δ = 6.66 (d, J = 8.2 Hz, 1H), 6.60 (d, J = 8.2 Hz, 1H), 5.58 (d,
J = 6.4 Hz, 1H), 5.29 (d, J = 4.8 Hz, 1H), 5.04 (d, J = 6.4 Hz, 1H), 3.96 (d, J = 6.5 Hz, 1H),
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3.85 (s, 3H), 3.60 (s, 3H), 3.30 (d, J = 17.8 Hz, 1H), 2.93 (dt, J = 17.2, 8.4 Hz, 1H), 2.87 (dd,
J = 20.8, 6.1 Hz, 1H), 2.75 (dd, J = 17.9, 7.0 Hz, 1H), 2.53 (d, J = 6.3 Hz, 2H), 2.21 (td,
J = 12.6, 5.2 Hz, 1H), 1.73 (dd, J = 12.7, 1.9 Hz, 1H), 1.00 – 0.91 (m, 1H), 0.62 – 0.53 (m, 2H),
0.21 – 0.14 (m, 2H) ppm. 13C NMR (151 MHz, CDCl3) δ (ppm) 152.8 (Cq), 144.8 (Cq), 143.0
(Cq), 133.6 (Cq), 127.7 (Cq), 119.4 (CH), 113.1 (CH), 96.0 (CH), 89.3 (CH), 59.0 (CH2),
58.8 (CH), 56.6 (CH3), 55.1 (CH3), 46.6 (Cq), 44.3 (CH2), 36.6 (CH2), 30.9 (CH2), 9.4 (CH),
4.1 (CH2), 4.0 (CH2) ppm.
Ethyl 17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-endoethenylene-morphin-
an-7α-carboxylate (14): N-(Cyclopropylmethyl)northebaine (13, 241 mg, 0.68 mmol) was
dissolved in ethyl acrylate (1.84 g, 2.00 mL, 18.4 mmol) and the mixture was stirred for 15 h at
100 °C. After completion of the reaction, the excess of ethyl acrylate was removed under reduced
pressure. The crude product 14 was purified by column chromatography (40:1
dichloromethane/methanol). The desired product 14 was obtained as brown solid (266 mg,
1
0.59 mmol, 87%). Rf = 0.5 (35:1 dichloromethane/methanol) [CAM]. H NMR (600 MHz,
CDCl3) δ = 0.10 – 0.16 (m, 2H), 0.47 – 0.54 (m, 2H), 0.79 – 0.86 (m, 1H), 1.25 (t, J = 7.1 Hz,
3H), 1.47 (dd, J = 6.4, 12.7 Hz, 1H), 1.82 – 1.87 (m, 1H), 1.97 (dt, J = 5.5, 12.6 Hz, 1H),
2.32 – 2.45 (m, 4H), 2.70 (dd, J = 4.9, 12.1 Hz, 1H), 2.82 – 2.86 (m, 1H), 3.08 – 3.12 (m, 2H),
3.54 (d, J = 6.5 Hz 1H), 3.62 (s, 3 H), 3.82 (s, 3H), 4.09 – 4.19 (m, 2H), 4.60 (d, J = 1.4 Hz,
1H), 5.56 (d, J = 8.8 Hz, 1H), 5.84 (d, J = 8.8 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H), 6.62 (d,
J = 8.1 Hz, 1H) ppm. 13C NMR (91 MHz, CDCl3) δ = 173.6 (Cq), 148.2 (Cq), 142.0 (Cq), 135.6
(CH), 134.5 (Cq), 128.4 (Cq), 126.6 (CH), 119.5 (CH), 113.6 (CH), 94.1 (CH), 81.2 (Cq), 60.7
(CH2), 59.9 (CH2), 57.2 (CH), 56.8 (CH3), 53.0 (CH3), 48.1(Cq), 44.2 (CH2), 43.4 (CH), 43.1
(Cq), 33.8 (CH2), 31.1 (CH2), 23.4 (CH2), 14.4 (CH3), 9.6 (CH), 4.2 (CH2), 3.6 (CH2) ppm.
17-(Cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-endoethenylenemorphinan-7α-
caboxylic acid hydrochloride (14’): A solution of the ester 14 (266 mg, 0.59 mmol) in
hydrochloric acid (6 M) was stirred for 6 h at 105 °C. After cooling to r.t. the hydrochloric acid was
removed under reduced pressure. The crude product 14’ (260 mg, 0.57 mmol, 96%) was used for
1
the next step without further purification. H NMR (600 MHz, (CD3)2SO) δ = 12.25 (s, 1H), 8.73
(s, 1H), 6.74 (d, J = 8.2 Hz, 1H), 6.61 (d, J = 8.2 Hz, 1H), 5.65 (d, J = 8.8 Hz, 1H), 5.57 (d,
J = 8.8 Hz, 1H), 4.93 (s, 1H), 4.42 (d, J = 6.7 Hz, 1H), 3.72 (s, 3H), 3.46 (d, J = 7.0 Hz, 3H),
3.45 – 3.39 (m, 3H), 3.14 – 2.91 (m, 5H), 2.26 (td, J = 14.5, 5.1 Hz, 1H), 3.45 – 3.39 (m, 1H) 1.48
(dt, J = 14.8, 7.4 Hz, 1H), 1.15 – 1.05 (m, 1H), 0.86 (dd, J = 8.4, 5.5 Hz, 1H), 0.77 – 0.64 (m, 2H),
0.61 – 0.41 (m, 2H) ppm.
N-Benzyl-17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethenylenemorphinan-
7α-carboxamide (15): A solution of carboxylic acid 14’ (216 mg, 0.47 mmol) in dry chloroform
(8.5 mL) was cooled to 0 °C and treated with oxalyl chloride (907 mg, 0.61 mL, 7.14 mmol). The
resulting mixture was stirred at r.t. for 23 h. Afterwards, the solvent was removed under reduced
pressure. The remaining crude acid chloride 14’’ was dissolved in dry chloroform (7 mL).
Benzylamine (474 mg, 0.48 mL, 4.42 mmol) and triethylamine (352 mg, 0.48 mL, 3.48 mmol)
were added at 0 °C and the reaction mixture was stirred at r.t. for 22 h. After completion of the
reaction, the mixture was filtered, and the filtrate was concentrated under reduced pressure. The
crude product 15 was purified by column chromatography (200:1:2150:1:2100:1:2
dichloromethane/methanol/NH3(25%)). The desired product 15 was obtained as brown solid
(191 mg, 0.37 mmol, 79%). Rf = 0.5 (20:1 dichloromethane/methanol) [UV]. 1H NMR
(400 MHz, CDCl3) δ = 7.37 – 7.27 (m, 5H), 6.62 (d, J = 8.1 Hz, 1H), 6.52 (d, J = 8.1 Hz, 1H),
6.39 (t, J = 5.5 Hz, 1H), 5.90 (d, J = 8.8 Hz, 1H), 5.59 (d, J = 8.8 Hz, 1H), 4.56 (d, J = 1.4 Hz,
1H), 4.52 – 4.37 (m, 2H), 3.81 (s, 3H), 3.61 (s, 3H), 3.59 – 3.55 (m, 1H), 3.17 (dd, J = 13.2,
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10.1002/cmdc.202000180
ChemMedChem
9.7 Hz, 1H), 3.14 – 3.07 (m, 1H), 2.76 – 2.65 (m, 2H), 2.47 – 2.29 (m, 4H), 2.00 (td, J = 12.6,
4.9 Hz, 1H), 1.84 (dd, J = 13.0, 2.3 Hz, 2H), 1.60 (dd, J = 13.3, 6.2 Hz, 1H), 0.89 – 0.79 (m,
1H), 0.54 – 0.47 (m, 2H), 0.13 (d, J = 4.9 Hz, 2H) ppm. 13C NMR (91 MHz, CDCl3) δ = 172.5
(Cq), 148.0 (Cq), 141.9 (Cq), 138.6 (Cq), 137.3 (CH), 134.4 (Cq), 128.6 (2×CH), 128.3 (Cq),
127.5 (2×CH), 127.3 (CH), 125.6 (CH), 119.5 (CH), 113.5 (CH), 94.9 (CH), 80.7 (Cq),
59.8 (CH2), 57.1 (CH), 56.6 (CH3), 53.1 (CH3), 47.9 (Cq), 44.9 (CH), 44.0 (CH2), 43.6 (CH2),
43.0 (Cq), 33.6 (CH2), 30.8 (CH2), 23.2 (CH2), 9.5 (CH), 4.2 (CH2), 3.4 (CH2) ppm.
N-Benzyl-17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethenylenemorphinan-
7α- methanoamine (15’): Amide 15 (212 mg, 0.413 mmol) was dissolved in dry THF (1.5 mL)
and subsequently added to a suspension of lithium aluminum hydride (2.4 M in THF, 1.10 mmol,
0.45 mL). The reaction mixture was stirred for 20 h under reflux. After completion of the reaction,
an aqueous sodium sulfate was slowly added, and the precipitating solid was removed by filtration.
The filtrate contained the crude product 15’ which was purified by column chromatography
(100:1:1 dichloromethane/methanol/NH3(25%)). The desired product 15’ was obtained as
yellow oil (127 mg, 0.26 mmol, 62%). Rf = 0.5 (100:3:1 dichloromethane/methanol/
1
NH3(25%)) [KMnO4]. H NMR (600 MHz, CDCl3) δ = 7.34 – 7.30 (m, 4H), 7.26 – 7.22 (m,
1H), 6.60 (d, J = 8.1 Hz, 1H), 6.49 (d, J = 8.1 Hz, 1H), 5.72 (d, J = 8.7 Hz, 1H), 5.45 (d,
J = 8.8 Hz, 1H), 4.59 (d, J = 1.3 Hz, 1H), 3.81 (s, 3H), 3.78 (s, 1H), 3.56 (s, 3H), 3.45 (d,
J = 6.6 Hz, 1H), 3.11 – 3.01 (m, 2H), 2.77 (dd, J = 11.8, 6.2 Hz, 1H), 2.70 (dd, J = 12.0, 4.9 Hz,
1H), 2.42 – 2.35 (m, 3H), 2.35 – 2.29 (m, 1H), 2.29 – 2.23 (m, 1H), 2.15 – 2.09 (m, 1H), 1.99
(td, J = 12.7, 5.5 Hz, 1H), 1.81 (dd, J = 13.0, 2.3 Hz, 1H), 0.93 – 0.76 (m, 4H), 0.55 – 0.47 (m,
2H), 0.17 – 0.08 (m, 2H) ppm.
7α-(Aminomethyl)-17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-
morphinan (10): Compound 15’ (160 mg, 0.32 mmol) and ammonium formate (121 mg,
1.92 mmol) were dissolved in absolute ethanol (5 mL) and the resulting mixture was treated
with palladium on carbon (10%, 34.0 mg, 319 µmol). The reaction mixture was stirred for 1.5 h
under reflux. Afterwards, the remaining palladium on carbon was removed by filtration over
Celite and the solvent was removed under reduced pressure. The crude product was purified by
column chromatography (20:1:0.2 dichloromethane/methanol/NH3(25%)) yielding amine 10 as
yellow oil (106 mg, 0.26 mmol, 81%). Rf = 0.5 (91:7.5:1 dichloromethane/methanol/
1
NH3(25%)) [KMnO4]. H NMR (360 MHz, CDCl3) δ = 6.70 (d, J = 8.1 Hz, 1H), 6.55 (d,
J = 8.1 Hz, 1H), 4.50 (d, J = 1.6 Hz, 1H), 3.87 (s, 3H), 3.42 (s, 3H), 3.03 – 2.95 (m, 3H), 2.65
(dd, J = 11.8, 5.2 Hz, 1H), 2.37 – 2.20 (m, 5H), 2.05 (td, J = 12.7, 5.6 Hz, 1H), 1.90 (s), 1.66
(dd, J = 12.9, 2.5 Hz, 1H), 1.58 – 1.36 (m, 3H), 1.24 – 1.19 (m, 1H), 1.11 – 0.97 (m, 2H),
0.79 – 0.68 (m, 2H), 0.52 – 0.45 (m, 2H), 0.13 – 0.06 (m, 2H) ppm. 13C NMR (151 MHz,
CDCl3) δ = 147.0, 141.8, 132.8, 128.7, 119.0, 113.8, 93.0, 60.0, 58.7, 56.7, 51.2, 45.8, 43.8,
43.7, 42.7, 38.3, 35.6, 35.5, 33.4, 29.3, 22.8, 18.8, 9.4, 4.0, 3.6 ppm. HRMS (ESI) calculated
for C25H35N2O3 [M+H]+: 411.2642, found: 411.2647.
N-(Phenyl)-17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethanemorphinan-
7α-yl-methyl)azocarboxamide (9a): Amine 10 (51.0 mg, 0.12 mmol), tert-butyl (E)-2-
phenyldiazene-1-carboxylate (11a, 51.3 mg, 0.24 mmol) and potassium carbonate (85.7 mg,
0.62 mmol) were dissolved in dry ethanol (3 mL) and stirred at r.t. for 3 d. After addition of
water, the reaction mixture was extracted with ethyl acetate (3×20 mL) and dried over sodium
sulfate. The crude product was purified by column chromatography (100:1:1
dichloromethane/methanol/NH3(25%)) yielding 9a as yellow powder (48.0 mg, 0.09 mmol,
1
71%). H NMR (400 MHz, CD3CN) δ = 7.92 – 7.87 (m, 2H), 7.67 – 7.57 (m, 3H), 7.18 (bs,
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10.1002/cmdc.202000180
ChemMedChem
1H), 6.75 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.1 Hz, 1H), 4.57 (d, J = 2.1 Hz, 1H), 3.81 (s, 3H),
3.60 (dt, J = 13.4, 5.1 Hz, 1H), 3.44 – 3.39 (m, 1H), 3.38 (s, 3H), 3.02 – 2.89 (m, 3H), 2.66 (dd,
J = 11.8, 5.2 Hz, 1H), 2.33 – 2.20 (m, 5H), 2.12 – 2.02 (m, 1H), 1.59 (dd, J = 13.1, 2.5 Hz, 1H),
1.51 – 1.41 (m, 1H), 1.35 – 1.25 (m, 2H), 1.19 (dd, J = 13.3, 5.8 Hz, 1H), 1.10 (td, J = 12.6,
6.3 Hz, 1H), 0.80 – 0.71 (m, 1H), 0.71 – 0.62 (m, 1H), 0.47 – 0.42 (m, 2H), 0.12 – 0.05 (m, 2H)
ppm. 13C NMR (151 MHz, CD3CN) δ = 163.2, 152.4, 148.0, 142.7, 134.3, 133.9, 130.5, 130.1,
124.2, 120.2, 114.8, 91.9, 77.6, 60.6, 59.8, 57.1, 50.8, 46.5, 44.4, 43.0, 36.4, 36.2, 35.2, 33.6,
29.8, 23.5, 20.3, 10.2, 4.3, 4.1 ppm. HRMS (ESI) calculated for C32H38N4O4 [M+H]+: 542.2893,
found: 543.2968. Purity: 99%.
N-(4-Fluorophenyl)-(17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-
morphinan-7α-yl-methyl)azocarboxamide (9b): Amine 10 (14.0 mg, 34.0 µmol), tert-butyl
(E)-2-(4-fluorophenyl)diazene-1-carboxylate (11b, 33.6 mg, 0.15 mmol) and potassium
carbonate (23.6 mg, 0.17 mmol) were dissolved in dry ethanol (0.15 mL). The reaction mixture
was stirred at r.t. for 2.5 h. After the addition of water, the crude product was extracted with
ethyl acetate (3×20 mL) and the combined organic layers were dried over anhydrous sodium
sulfate. Afterwards, the crude product was purified by preparative HPLC (5:95100:0
methanol/H2O+0.1% TFA over 30 min, flow rate: 20 mL/min) to give 9b as yellow powder
(16.5 mg, 29.4 μmol, 87%). 1H NMR (600 MHz, CDCl3) δ = 10.54 (bs, 1H), 8.04 (dd, J = 8.7,
5.2 Hz, 2H), 7.22 (t, J = 8.4 Hz, 2H), 6.84 (d, J = 8.2 Hz, 1H), 6.70 (d, J = 8.2 Hz, 1H), 4.66 (s,
1H), 3.93 (s, 3H), 3.90 (d, J = 5.9 Hz, 1H), 3.77 (d, J = 13.1 Hz, 1H), 3.67 – 3.53 (m, 2H), 3.50
(s, 3H), 3.35 – 3.22 (m, 2H), 3.11 (d, J = 19.2 Hz, 1H), 3.05 – 2.99 (m, 1H), 2.91 (dd, J = 19.0,
5.8 Hz, 1H), 2.86 – 2.79 (m, 1H), 2.38 (s, 1H), 1.95 (d, J = 12.0 Hz, 1H), 1.67 – 1.58 (m, 2H),
1.55 – 1.46 (m, 1H), 1.42 – 1.33 (m, 1H), 1.28 (s, 1H), 1.23 – 1.14 (m, 1H), 0.96 – 0.88 (m,
1H), 0.85 – 0.76 (m, 2H), 0.58 – 0.49 (m, 1H), 0.46 – 0.38 (m, 1H) ppm. 13C NMR (151 MHz,
CDCl3) δ = 168.7 (d, J = 256.0 Hz), 165.2, 147.4, 143.2, 130.0, 126.5 (d, J = 9.8 Hz, 2×C),
122.9, 119.8, 116.4 (d, J = 23.2 Hz, 2×C), 115.4, 90.5, 75.57, 59.7, 59.1, 56.7, 51.0, 45.4, 44.2,
42.2, 35.8, 34.3, 31.9, 31.4, 29.7, 29.2, 24.6, 18.6, 5.6, 5.4, 3.8 ppm. 19F NMR (339 MHz,
CDCl3) δ = -108.1 ppm. HRMS (ESI) calculated for C32H38FN4O4 [M+H]+: 561.2872, found:
561.2866. Purity: 99%.
N-(4-Bromophenyl)-(17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-
morphinan-7α-yl-methyl)azocarboxamide (9c): To a solution of amine 10 (19.5 mg, 0.048
mmol) in dry ethanol (0.4 mL), tert-butyl (E)-2-(4-bromophenyl)diazene-1-carboxylate (11c,
27.1 mg, 0.10 mmol) in dry ethanol (0.6 mL) and triethylamine (13.2 μL, 0.10 mmol) were
added. After the reaction mixture was stirred for 5 d at r.t., the solvent was removed under
reduced pressure. The crude product was purified by preparative HPLC (5:95100:0
methanol/H2O + 0.1% TFA over 30 min, flow rate: 20 mL/min) to give 9c (12.5 mg,
1
0.02 mmol, 42%) as yellow oil. H NMR (600 MHz, CD3CN) δ = 9.45 (bs, 1H), 7.82 (d,
J = 8.0 Hz, 2H), 7.78 (d, J = 7.4 Hz, 2H), 7.46 (bs, 1H), 6.88 (d, J = 8.2 Hz, 1H), 6.71 (d,
J = 8.2 Hz, 1H), 4.73 (d, J = 2.1 Hz, 1H), 3.89 (d, J = 8.7 Hz, 1H), 3.85 (s, 3H), 3.65 – 3.59 (m,
1H), 3.43 – 3.40 (m, 1H), 3.38 (s, 3H), 3.38 – 3.34 (m, 1H), 3.21 (d, J = 19.7 Hz, 1H),
3.16 – 3.02 (m, 3H), 2.91 – 2.82 (m, 2H), 1.93 – 1.89 (m, 1H), 1.58 – 1.52 (m, 1H), 1.49 (dd,
J = 13.2, 5.2 Hz, 1H), 1.40 – 1.26 (m, 3H), 1.15 – 1.06 (m, 1H), 0.79 – 0.70 (m, 2H),
0.67 – 0.61 (m, 1H), 0.50 – 0.45 (m, 1H), 0.42 – 0.35 (m, 1H) ppm. 13C NMR (151 MHz,
CD3CN) δ = 148.2, 143.5, 133.7, 131.4, 128.3, 125.9, 125.8, 120.9, 118.2, 115.8, 90.1, 76.6,
59.8, 59.6, 57.0, 50.8, 46.0, 45.1, 42.5, 36.6, 32.5, 32.1, 30.8, 29.60, 3.13, 19.9, 6.4, 5.5, 3.8
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10.1002/cmdc.202000180
ChemMedChem
ppm (one signal is missing due to overlap with solvent signals). HRMS (ESI) calculated for
C32H38BrN4O4 [M+H]+: 621.2071, found: 621.2068.
N-(4-Methoxyphenyl)-(17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-
morphinan-7α-yl-methyl)azocarboxamide (9d): tert-Butyl (E)-2-(4-methoxyphenyl) -
diazene-1-carboxylate (11d, 8.90 mg, 0.04 mmol) and triethylamine (5.2 μL, 0.04 mmol)
dissolved in dry ethanol (0.3 mL) were added to a solution of amine 10 (7.70 mg, 0.02 mmol)
in dry ethanol (0.2 mL). After the reaction mixture was stirred for 5 d at r.t., the solvent was
removed under reduced pressure. The crude product was purified by preparative HPLC
(5:95100:0 methanol/H2O + 0.1% TFA over 30 min, flow rate: 20 mL/min) to yield the
1
desired product 9d (4.6 mg, 8.0 μmol, 42%) as yellow oil. H NMR (600 MHz, CD3CN) δ =
9.23 (bs, 1H), 7.91 (d, J = 8.4 Hz, 2H), 7.32 (bs, 1H), 7.11 (d, J = 7.9 Hz, 2H), 6.88 (d,
J = 8.2 Hz, 1H), 6.71 (d, J = 8.2 Hz, 1H), 4.73 (d, J = 2.1 Hz, 1H), 3.90 (s, 3H), 3.88 (d,
J = 7.1 Hz, 1H), 3.85 (s, 3H), 3.63 – 3.57 (m, 1H), 3.45 – 3.36 (m, 5H), 3.23 – 3.18 (m, 1H),
3.15 (dd, J = 13.4, 7.0 Hz, 1H), 3.06 – 2.98 (m, 2H), 2.91 – 2.82 (m, 2H), 1.59 – 1.45 (m, 2H),
1.40 – 1.19 (m, 2H), 1.13 – 1.05 (m, 1H), 0.81 – 0.70 (m, 2H), 0.68 – 0.62 (m, 1H), 0.51 – 0.43
(m, 1H), 0.42 – 0.35 (m, 1H) ppm (four signals are missing due to overlap with water signals).
13C NMR (151 MHz, CD3CN) δ = 165.1, 148.2, 143.5, 131.4, 126.7 (2×C), 125.8, 120.9, 115.8,
115.6 (2×C), 90.1, 76.7, 59.9, 59.6, 57.0, 56.6, 50.8, 45.9, 45.1, 42.4, 36.6, 34.3, 32.4, 31.9,
30.8, 29.6, 25.1, 20.0, 6.4, 5.4, 3.9 ppm (two signals are missing due to overlap with solvent
signals). HRMS (ESI) calculated for C33H40N4O5 [M+H]+: 573.3072, found: 573.3085.
N-17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-morphinan-7α-yl-
methyl)cinnamide (16a): Sodium bicarbonate (32.0 mg, 0.38 mmol) and cinnamoyl chloride
(17a, 23.0 mg, 0.14 mmol) were added to amine 10 (10.5 mg, 25.6 µmol) dissolved in dry
dichloromethane (1.2 mL). After stirring for two days at r.t. water was added, and the crude
product was extracted with ethyl acetate (3×20 mL). After the combined organic layers were
dried over sodium sulfate and the solvent was removed under reduced pressure, the crude
product
was
firstly
purified
by
column
chromatography
(49:110:1
dichloromethane/methanol) and afterwards by preparative HPLC (5:95100:0 methanol/H2O
+ 0.1% TFA over 28.5 min, flow rate: 20 mL/min). Compound 16a was received as yellow oil
1
(3.55 mg, 6.57 μmol, 25%). H NMR (600 MHz, CDCl3) δ = 7.63 (d, J = 15.0 Hz, 1H), 7.53
(d, J = 6.9 Hz, 2H), 7.42 – 7.30 (m, 3H), 7.06 – 6.97 (m, 1H), 6.82 (d, J = 8.2 Hz, 1H), 6.67 (d,
J = 8.2 Hz, 1H), 6.64 – 6.53 (m, 1H), 4.65 (s, 1H), 3.91 (s, 3H), 3.90 – 3.87 (m, 1H), 3.84 – 3.80
(m, 1H), 3.68 (d, J = 12.6 Hz, 1H), 3.53 – 3.47 (m, 1H), 3.46 (s, 3H), 3.26 – 3.13 (m, 1H),
3.13 – 2.99 (m, 2H), 2.94 – 2.84 (m, 2H), 2.65 – 2.49 (m, 1H), 2.38 – 2.26 (m, 1H), 2.02 – 1.91
(m, 1H), 1.64 – 1.53 (m, 2H), 1.35 – 1.22 (m, 2H), 1.22 – 1.14 (m, 1H), 0.88 – 0.74 (m, 3H),
13
0.54 – 0.47 (m, 1H), 0.44 – 0.37 (m, 1H) ppm. C NMR (91 MHz, CDCl3) δ = 147.6, 143.2,
141.4, 134.9, 132.5, 130.1, 129.6, 128.7, 128.0, 122.9, 119.8, 115.4, 90.3, 75.3, 59.7, 59.2, 56.8,
50.8, 45.7, 44.3, 41.1, 35.8, 34.0, 32.0, 31.6, 29.2, 24.7, 24.6, 18.7, 5.6, 5.5, 3.7 ppm (one signal
is missing due to overlap with solvent signals). HRMS (ESI) calculated for C34H41N2O4
[M+H]+: 541.3061, found: 561.3061.
N-(4-Fluorophenyl)-(17-(cyclopropylmethyl)-4,5α-epoxy-3,6-dimethoxy-6,14-ethane-
morphinan-7α-yl-methyl)cinnamide (16b): Amine 10 (15.0 mg, 37 µmol), 4-
fluorocinnamoyl chloride (17b, 34.1 mg, 0.19 mmol) and sodium hydrogen carbonate
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10.1002/cmdc.202000180
ChemMedChem
(46.6 mg, 0.56 mmol) were dissolved in dry ethanol (1.5 mL) and the reaction mixture was
stirred at r.t. for 15 h. After the addition of water, the crude product was extracted with ethyl
acetate (3×20 mL) and the combined organic layers were dried over anhydrous sodium sulfate.
The solvent was removed under reduced pressure. The crude product was purified by column
chromatography (100:1:1 dichloromethane/methanol/NH3(25%)) and 16b (13.0 mg, 23.0 µmol,
63%) was obtained as grey solid. Rf = 0.4 (80:1:1 dichloromethane/methanol/NH3(25%)) [UV].
1H NMR (600 MHz, CDCl3) δ = 7.58 (d, J = 15.6 Hz, 1H), 7.49 (dd, J = 8.6, 5.4 Hz, 2H),
7.10 – 7.02 (m, 2H), 6.72 (d, J = 8.1 Hz, 1H), 6.57 (d, J = 8.0 Hz, 1H), 6.49 (d, J = 5.8 Hz, 1H),
6.29 (d, J = 15.6 Hz, 1H), 4.48 (s, 1H), 3.89 (s, 3H), 3.76 – 3.69 (m, 1H), 3.51 (s, 3H),
3.41 – 3.30 (m, 1H), 3.09 – 2.94 (m, 3H), 2.65 (d, J = 10.4 Hz, 1H), 2.39 – 2.20 (m, 4H),
2.12 – 1.97 (m, 2H), 1.71 – 1.46 (m, 6H), 1.10 – 1.02 (m, 1H), 0.84 – 0.72 (m, 2H), 0.55 – 0.41
(m, 2H) ppm. 13C NMR (151 MHz, CDCl3) δ = 165.6, 163.6 (d, JCF = 250.1 Hz), 147.0, 142.0,
141.9, 139.6, 132.9, 131.3 (d, J = 3.4 Hz), 129.7 (2×C, d, JCF = 8.3 Hz), 121.0 (d, J = 2.2 Hz),
119.3, 116.02 (2×C, d, J = 21.9 Hz), 114.0, 94.1, 60.1, 58.6, 56.9, 51.9, 46.03, 43.9, 41.9, 36.4,
19
35.7, 35.5, 33.4, 29.9, 29.3, 22.9, 18.1, 9.6, 4.2, 3.7 ppm. F NMR (565 MHz, CDCl3) δ = -
111.4 ppm. HRMS (ESI) calculated for C34H39FN2O4 [M+H]+: 559.2967, found: 559.2968.
Purity: 100%.
Reference compounds for microsomal stability assay
(E)-((4-Fluorophenyl)diazenyl)(piperidin-1-yl)methanone (18): Phenylazocarboxylic ester 11b
(112 mg, 0.50 mmol), potassium carbonate (346 mg, 2.50 mmol) and piperidine (0.15 mL, 1.50
mmol) were dissolved in dry ethanol (2 mL). The reaction mixture was stirred for 20 min at 40°C.
After completion of the reaction, diluted hydrochloric acid (3 M) was added, until a pH value of 3
was reached. The crude product 18 was extracted with ethyl acetate (3×50 mL). The combined
organic layers were washed with aqueous saturated sodium chloride solution and dried over
anhydrous sodium sulfate. After removal of the solvent under reduced pressure, the crude product
was purified by column chromatography (3:1 hexane/ethyl acetate) yielding 18 (112 mg,
0.48 mmol, 95%) as orange solid. Rf = 0.4 (hexane/ethyl acetate = 2:1) [UV]. 1H NMR (360 MHz,
CDCl3) δ = 1.60-1.66 (m, 2 H), 1.71-1.74 (m, 4 H), 3.57-3.60 (m, 2 H), 3.72-3.75 (m, 2 H), 7.20
(dd, JHF = 8.2 Hz, J = 9.0 Hz, 2 H), 7.95 (dd, JHF = 5.2 Hz, J = 9.1 Hz, 2 H) ppm. 13C NMR (91
MHz, CDCl3) δ = 24.3 (CH2), 25.5 (CH2), 26.1 (CH2), 44.6 (CH2), 46.0 (CH2), 116.3 (d, JCF = 23.1
Hz, 2 × CH), 125.8 (d, JCF = 9.4 Hz, 2 × CH), 148.7 (Cq), 161.4 (Cq), 165.6 (d, JCF = 255.2 Hz, Cq).
19F NMR (338 MHz, CDCl3) δ = -106.4 ppm. HRMS (ESI) calculated for C12H15FN3O [M+H]+:
236.1190, found: 236.1194.
(E)-3-(4-Fluorophenyl)-1-(piperidin-1-yl)prop-2-en-1-one
(19):
(E)-3-(4-Fluorophenyl)-
acryloyl chloride (17b, 50.0 mg, 0.27 mmol) was dissolved in dry dichloromethane (1 mL).
Afterwards, the mixture was treated with a solution of piperidine (19.6 mg, 22.0 µL, 0.23 mmol)
and triethylamine (31.0 µL, 0.23 mmol) dissolved in ice cold dichloromethane (2 mL). The reaction
mixture was stirred at room temperature for 1 hour. After completion of the reaction, saturated
aqueous sodium bicarbonate solution (10 mL) was added and the crude product was extracted with
dichloromethane (3×30 mL). Purification by flash column chromatography (0%→1%→5%→10%
methanol in dichloromethane) gave the desired cinnamide 19 (30.9 mg, 0.17 mmol, 62%) as a
yellow solid. Rf = 0.3 (10:1 dichloro-methane/methanol) [UV]. 1H NMR
(600 MHz, CDCl3) δ
= 7.63 (d, J = 15.4 Hz, 1H), 7.56 – 7.49 (m, 2H), 7.07 (t, J = 8.6 Hz, 2H), 6.84 (d, J = 15.4 Hz, 1H),
3.64 (bs, 4H), 1.74 – 1.67 (m, 2H), 1.67 – 1.61 (m, 4H) ppm.
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10.1002/cmdc.202000180
ChemMedChem
Radioligand binding studies
Radioligand binding studies with the human opioid receptors OR, OR and OR were
performed as described previously.[52,53] In brief, competition binding experiments were done
using membranes of HEK293T cells transiently transfected with the cDNA of the human OR
(gift from the Ernest Gallo Clinic and Research Center, UCSF, CA), OR and OR receptor
(cDNA Resource Center, Bloomsberg, PA), respectively. Radioligand displacement assays
were performed in binding buffer (50 mM Tris, pH 7.4) with [3H]diprenorphine (specific
activity 31 Ci/mmol, PerkinElmer, Rodgau, Germany) at final concentrations of 0.15-0.50 nM.
The assays were carried out at protein concentrations of 1-10 µg/assay tube, a Bmax value of
3700±730 fmol/µg, a KD value of 0.15±0.026 nM for OR, 1-10 µg protein/assay tube, Bmax
of 1800±440 fmol/µg, KD of 0.24±0.036 nM for OR, and of 1-6 µg protein/assay tube, Bmax
of 6800±2700 fmol/µg, KD of 0.12±0.019 nM for OR respectively. Unspecific binding was
determined in the presence of 10 µM naloxone, protein concentration was established by the
method of Lowry using bovine serum albumin as standard.[54] The resulting competition curves
of the receptor binding experiments were analyzed by nonlinear regression using the algorithms
in PRISM 6.0 (GraphPad Software, San Diego, CA). The data were initially fit using a sigmoid
model to provide an IC50 value, representing the concentration corresponding to 50% of
maximal inhibition. IC50 values were transformed to Ki values according to the equation of
Cheng and Prusoff.[55]
Radioligand depletion assay
Tests on covalent blocking of the receptor were carried out as described previously.[56]
Membranes from HEK 293T cells transiently expressing the human OR were preincubated
in binding buffer at 37°C at a protein concentration of 100 µg/mL and the test compounds 9b
and 16b at concentrations of 60 nM and 100 nM, respectively roughly representing the 40-fold
Ki value derived from the binding experiment. As a reference -FNA was used at 1 µM.
Preincubation was run for 60 min. Generally incubation was stopped by centrifugation and the
amount of reversibly bound ligand was washed out for four times (resuspension of the
memebranes in buffer for 30 min followed by centrifugation). Washed membranes were used
for radioligand binding experiments with [3H]diprenorphine to determine the remaining
specific binding at the receptor according to the standard protocols for radioligand binding.
Non-specific binding was determined in the presence of 10 µM naloxone. Data analysis was
performed by normalizing the receptor bound radioactivity derived from unspecific binding
equal to 0% and total binding equal to 100%.
Average values from three individual experiments determined in quatruplicates show no
blocking of the radioligand binding site of the receptor by 9b or 16b.
Accumulation of inositol mono phosphate (IP-One Assay).
Determination of the activation of -OR was measured applying the IP-One HTRF® assay
(Cisbio, Codolet, France) according to the manufacturer’s protocol and as described
previously.[53] In brief, HEK-293T cells were grown to confluence of approximately 70% and
transiently transfected with the cDNA of the human -OR receptor applying the TransIT-293
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10.1002/cmdc.202000180
ChemMedChem
Mirus transfection reagent (Peqlab, Erlangen, Germany). After one day cells were detached
from the culture dish with Versene (Life Technologies GmbH, Darmstadt, Germany), seeded
into black 384-well plates (10000 cells/well) (Greiner Bio-One, Frickenhausen, Germany) and
maintained for 24 h at 37 °C. After incubation with the test compounds dissolved in stimulation
buffer (final range of concentration from 1 pM up to 10 µM) for 180 min at 37 °C the detection
reagents were added (IP1-d2 conjugate and Anti-IP1cryptate TB conjugate each dissolved in
lysis buffer) and incubation was continued for further 1 hr at room temperature. Time resolved
fluorescence resonance energy transfer (HTRF) was determined using the Clariostar plate
reader (BMG, Ortenberg, Germany). In the agonist mode each compound was tested in
duplicates in 5-6 individual experiments in comparison to the reference compound DAMGO
(n=10). Antagonist properties were determined after preincubation of the test compound for 30
min, subsequent addition of a fixed concentration of the reference agonist carbachol at a final
concentration of 30 nM and continued incubation for 90 min at 37°C (5 experiments each). The
resulting dose response curves were analyzed by nonlinear regression using the algorithms in
PRISM 6.0 (GraphPad software, San Diego, CA), fitted with a sigmoid model and normalized
to basal activity (0 %) and the maximal effect caused by the reference full agonist DAMGO
(100 %) (agonist mode).
Recruitment of -arrestin-2 (PathHunter Assay).
Measurement of arrestin-2 recruitment was done applying the PathHunter® assay (DiscoverX,
Birmingham, U.K.) according to the manufacturer’s protocol and as described previously.[53,57]
In brief, HEK-293 cells stably expressing the enzyme acceptor (EA) tagged -arrestin-2 fusion
protein were transiently co-transfected with the ProLink tagged -OR-PK1 and GRK2 (1:1)
employing the Mirus TransIT-293 transfection reagent. After 24 h cells were transferred into
white clear bottom 384-well plates (5000 cells/well) (Greiner Bio-One) and maintained for
further 24 h at 37 °C, 5 % CO2. To start receptor stimulated arrestin recruitment test compounds
were added to the cells to get a final concentration in a range of 10 pM to 100 μM. Incubation
was continued for 90 min at 37°C. Stimulation was stopped by addition of the detection mix
and incubation was continued for 60 min at room temperature. Chemiluminescence was
determined using a Clariostar plate reader. Agonist mode was measured in duplicates (3
individual experiments for each compound in comparison to DAMGO (n=9). Antagonist
properties were determined after preincubation of the test compound for 30 min, subsequent
addition of 1 µM of DAMGO and continued incubation for 90 min at 37°C (4 experiments
each). Data analysis was done as described above.
Test on cytotoxicity.
Determination of cytotoxic effects was measured with untransfected HEK293T cells in
presence of 100 nM of the test ligands 9b, 16b and the reference naloxone (65-fold Ki, 30-fold
Ki and 25-fold Ki, respectively). For this, cells were seeded in a 12-well plate (300,000 per well)
and kept growing for 24 hours in full medium (DMEM/F12 supplemented with 10 % FBS,
penicillin-streptomycin and L-glutamine; Life Technologies, Darmstadt, Germany). Test
compounds were diluted in PBS with an equal amount of DMSO and added to the cells at a
final concentration of 100 nM followed by a continued incubation for further 24 hours. Finally,
cells were thoroughly observed in a microscope for any change in morphology and growing
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10.1002/cmdc.202000180
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behavior different to vehicle or reference. After that cells were detached (Versene, Life
Technologies) and the cell number of each well was determined in an automated cell counter
(Countess, Life Technologies). The number of cells from six individual experiments was
normalized relative to vehicle (100%) subsequently calculating an average mean value ± SEM.
Microsomal stability assay
The metabolism studies were performed with pooled microsomes from male rat liver (Sprague-
Dawley) which were used as purchased from Sigma Aldrich and were stored at -75 °C until
usage. Nicotinamide adenine dinucleotide phosphate (NADPH) from Sigma Aldrich was stored
at -8 °C and also used as purchased. The microsomal degradation experiments were performed
in polyethylene tubes from Eppendorf with 1.5 mL volume size. The incubation mixture with
a total volume of 1 mL contained the azocarboxamides, cinnamides or imipramine as test
substances (40 μM stock, pre-diluted from a 0.1 mM stock in DMSO), microsomes in a
concentration of 1 mg of microsomal protein/mL of incubation mixture and Tris-MgCl2 buffer
(48 mM Tris, 4.8 mM MgCl2×6H2O, pH 7.4). The microsomal reactions were initiated by the
addition of a NADPH solution (100 μL, final concentration of 1.1 µM) and performed at 37 °C.
After time intervals of 0, 20, 40 and 60 minutes, samples (100 µL) were drawn from which the
enzymatic reactions were terminated by addition of ice-cold acetonitrile (200 μL, containing
the internal standard 22), and precipitated protein was removed by centrifugation (15000 rcf for
3 min) and the solids removed by filtration. The supernatant was analyzed by LC/MS (flow rate
0.5 mL/min, binary solvent system of 0.1% formic acid in water and 0.1% formic acid in
acetonitrile, gradient: 20%→90% 0.1% formic acid over 30 minutes). Parallel control
incubations were conducted in the absence of enzyme cofactor NADPH as negative controls
and for the determination of unspecific binding to matrix. The concentrations of the remaining
substrates were calculated as a mean value ± SEM. of three independent experiments by
comparison of the area under the curve (AUC) of the remaining substrate in comparison to the
AUC of substrate at time 0. Estimating a similar ionization rate, internal fluctuations were
corrected by a factor calculated from the AUC of the internal standard at each time point.
Radiosynthesis
No-carrier-added [18F]fluoride in target water was provided by Universitätsklinikum Würzburg,
Klinik und Poliklinik für Nuklearmedizin, Germany. Thin layer chromatography (TLC) was
carried out on silica gel-coated plastic sheets (Polygram®, Sil G/UV254, Macherey Nagel).
Electronic autoradiography (Instant Imager TM, Canberra Packard) was used to analyze radio-
TLC data. The HPLC system (Series 1100, Agilent) was equipped with a VWD lamp (detection
at 254 nm) and additionally connected to a radio-detector (500 TR Series, Packard).
[18F]11b was prepared as described previously.[41] After completion of the reaction, the yellow
solution containing [18F]11b was diluted with HCl (0.2 M, 15 mL), passed through a cartridge
(SepPak tC18, Waters) and the cartridge was washed with CH3CN /0.2 M HCl (20:80, 5 mL)
and H2O (2 mL). [18F]11b was eluted with 1 mL of ethanol in a reaction vial which was prepared
with the required reactants (the primary amine 10 (54 μmol or 108 μmol) and Cs2CO3 (7.5 mg,
23 μmol)). The reaction was stirred at room temperature and the radiochemical yield was
determined from aliquots taken from the reaction mixture after 2, 5 and 10 min by radio-TLC
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10.1002/cmdc.202000180
ChemMedChem
(ethanol/dichloromethane = 9:1, Rf ([18F]9b) = 0.4). After 10 min [18F]9b was isolated by semi-
preparative radio-HPLC (Kromasil C8, 125 x 8 mm, 4 mL/min, solvent: A: water (0.1 % TFA),
solvent B: acetonitrile (0.1 % TFA), gradient A/B: 75:25 to 40:60 in 30 min, tR=11.4 min) and
coinjected together with reference standard 9b.
Supporting Information
Receptor model generation, additional docking studies, determinations of half-life and intrinsic
clearance.
Corresponding Author
*E-mail: markus.heinrich@fau.de. Tel: (+49) 9131-8565670.
Acknowledgements
The authors are grateful for the financial support of this research project by the Deutsche
Forschungsgemeinschaft (DFG) (HE5413/3-3 and GRK1910/B3). H. L. gratefully
acknowledges support from DFG (German Science Foundation) through the Cluster of
Excellence "Engineering of Advanced Materials (EAM)", project EXC 315, and the Central
Institute for Scientific Computing (ZISC).
Abbreviations
OR: opioid receptor; TM: transmembrane domain; PET: positron emission tomography; IP:
inositol phosphate; EA: enzyme acceptor.
References
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Graphical abstract
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