Enantiomerically Pure Quinoline-Based κ-Opioid Receptor Agonists: Chemoenzymatic Synthesis and Pharmacological Evaluation

Article abstract of DOI:10.1002/cmdc.202000300

Racemic _K-opioid receptor (KOR) agonist 2-(3,4-dichlorophenyl)-1-[(4aRS,8SR,8aSR)-8-(pyrrolidin-1-yl)-3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-one ((±)-4) was prepared in a diastereoselective synthesis. The first key step of the synthesis was the diastereoselective hydrogenation of the silyl ether of 1,2,3,4-tetrahydroquinoin-8-ol ((±)-9) to afford cis,cis-configured perhydroquinoline derivative (±)-10. Removal of the TBDMS protecting group led to a β-aminoalcohol that reacted with SO₂Cl₂ to form an oxathiazolidine. Nucleophilic substitution with pyrrolidine resulted in the desired cis,trans-configured perhydroquinoline upon inversion of the configuration. In order to obtain enantiomerically pure KOR agonists 4 (99.8 % ee) and ent-4 (99.0 % ee), 1,2,3,4-tetrahydroquinolin-8-ols (R)-8 (99.1 % ee) and (S)-8 (98.4 % ee) were resolved by an enantioselective acetylation catalyzed by Amano lipase PS-IM. The absolute configuration was determined by CD spectroscopy. The 4aR,8S,8aS-configured enantiomer 4 showed sub-nanomolar KOR affinity (K_i=0.81 nM), which is more than 200 times higher than the KOR affinity of its enantiomer ent-4. In the cAMP assay and the Tango β-arrestin-2 recruitment assay, 4 behaved as a KOR agonist. Upon incubation of human macrophages, human dendritic cells, and mouse myeloid immune cells with 4, the number of cells expressing co-stimulatory receptor CD86 and proinflammatory cytokines interleukin 6 and tumor necrosis factor α was significantly reduced; this indicates the strong anti-inflammatory activity of 4. The anti-inflammatory effects correlated well with the KOR affinity: (4aR,8S,8aS)-4 was slightly more potent than the racemic mixture (±)-4, and the distomer ent-4 was almost inactive.

Full text of DOI:10.1002/cmdc.202000300

Accepted Article
Title: Chemoenzymatic synthesis and pharmacological evaluation of
enantiomerically pure quinoline-based κ-opioid receptor (KOR)
agonists
Authors: Bernhard Wünsch, Benedikt Martin, Dirk Schepmann, Freddy
A. Bernal, Thomas J. Schmidt, Tao Che, and Karin Loser
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
published online in Early View as soon as possible and may be different
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.202000300
Link to VoR: https://doi.org/10.1002/cmdc.202000300
01/2020

10.1002/cmdc.202000300
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FULL PAPER
Chemoenzymatic synthesis and pharmacological evaluation of
enantiomerically pure quinoline-based κ-opioid receptor (KOR)
agonists
Dr. Benedikt Martin,[a] Dr. Dirk Schepmann,[a] Dr. Freddy A. Bernal,[b] Prof. Thomas J. Schmidt,[b] Prof.
Tao Che,[c] Prof. Karin Loser,[d,e] Prof. Bernhard Wünsch[a,e]
*
[a]
Institut für Pharmazeutische und Medizinische Chemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
Tel.: +49-251-8333311; Fax: +49-251-8332144; E-mail: wuensch@uni-muenster.de
[b]
[c]
Institut für Pharmazeutische Biologie und Phytochemie der Universität Münster, Corrensstraße 48, D-48149 Münster, Germany.
Department of Anesthesiology, Washington University in St. Louis, St. Louis, Missouri; Center for Clinical Pharmacology, St. Louis College of Pharmacy and
Washington University School of Medicine, St. Louis, Missouri. Department of Pharmaceutical and Administrative Sciences, St. Louis College of Pharmacy,
St. Louis, Missouri; United States.
[d]
[e]
Department of Dermatology, University of Münster, von-Esmarch-Street 58, D-48149 Münster, Germany.
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelms-Universität Münster, D-48149 Münster, Germany.
δ-opioid receptor (DOR, vas deferens).[1] Initially, the σ receptor
Abstract: Racemic -ppioid receptor (KOR) agonist 2-(3,4-
dichlorophenyl)-1-[(4aRS,8SR,8aSR)-8-(pyrrolidin-1-yl)-
was considered as an additional opioid receptor subtype, but this
classification was later corrected and the σ receptor was
reclassified as non-opioid receptor type.[1] All opioid receptors
belong to the class A (rhodopsin-like) γ subfamily of G-protein-
coupled receptors (GPCRs).[2] They share a common seven-
transmembrane architecture and are coupled predominantly to
heterotrimeric Gi/Go proteins. Cloning of the four opioid receptors
revealed a homology of more than 60 % with respect to their
amino acid sequences.[3]
3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-one ((±)-4)
was prepared in a diastereoselective synthesis. The first key step
of the synthesis was the diastereoselective hydrogenation of the
silyl ether of 1,2,3,4-tetrahydroquinoin-8-ol ((±)-9) affording
cis,cis-configured perhydroquinoline deriviative (±)-10. Removal
of the TBDMS protective group led to an β-aminoalcohol which
reacted with SO2Cl2 to torm an oxathiazolidine. Nucleophilic
substitution with pyrrolidine resulted in the desired cis,trans-
configured perhydroquinoline upon inversion of the configuration.
In order to obtain enantiomerically pure KOR agonists 4
(99.8 %ee) and ent-4 (99.0 %ee), 1,2,3,4-tetrahydroquinolin-8-ols
(R)-8 99.1 % ee) and (S)-8 (98.4 % ee) were resolved by an
enantioselective acetylation catalyzed by Amano lipase PS-IM.
The absolute configuration was determined by CD spectroscopy.
Activation of MOR, KOR and DOR leads to strong analgesia, but
also to subtype specific side effects.[4] Most of the clinically used
analgesics are MOR agonists, such as morphine and methadone,
which cause euphoria, constipation, respiratory depression,
development of tolerance and addiction side effects.[5] In contrast,
KOR agonists do not elicit these typical MOR-mediated side
effects. Therefore, KOR represents an interesting target for the
development of safer analgesics.[5] However, KOR agonists are
not devoid of side effects, since they induce centrally mediated
dysphoria, sedation, and diuresis.[6] A high density of KOR is not
only found in the central nervous system, but also in peripheral
cells (e.g. immune cells, including T cells and antigen presenting
cells)[4] and peripheral tissues (e.g. skin). KOR agonists are also
of interest for the treatment of itching skin diseases, including
pruritus, atopic dermatitis and psoriasis. In Japan, the
morphinane derivative nalfurafine activating KOR in the sub-
nanomolar range (Ki = 0.17 nM)[7] is approved for the treatment of
uremic pruritus. Its antipruritic activity is linked to activation of
peripheral KOR.[8]
The (4aR,8S,8aS)-configured enantiomer
4 showed sub-
nanomolar KOR affinity (Ki = 0.81 nM), which is more than 200-
fold higher than the KOR affinity of its enantiomer ent-4. In the
cAMP assay and the Tango -arrestin-2 recruitment assay, 4
behaved as KOR agonist. Upon incubation of human
macrophages, human dendritic cells and mouse myeloid immune
cells with 4, the number of cells expressing co-stimulatory
receptor CD86 and proinflammatory cytokines interleukin 6 and
tmnor necrosis factor  was significantly reduced, which indicates
the strong anti-inflammatory activity of 4. The anti-inflammatory
effects correlated well with the KOR affinity: (4aR,8S,8aS)-
configured 4 was slightly more potent than the racemic mixture
(±)-4, and the distomer ent-4 was almost inactive.
In a very recent study, the pharmacological effects of the
prototypical KOR agonist U-50,488 (1, Ki = 0.34 nM, Figure 1)[9]
in experimental autoimmune encephalomyelitis (EAE) were
investigated. EAE is a commonly used animal model of multiple
sclerosis (MS). KOR activation by U-50,488 (1) alleviated the
symptoms of EAE by preventing neuronal damage and promoting
remyelination.[10] The anti-inflammatory properties of KOR
agonists are mainly based on the downregulation of cell
1. Introduction
The group of opioid receptors consists of four subtypes. They are
termed according to their prototypical agonists μ-opioid receptor
(MOR, morphine), κ-opioid receptor (KOR, ketocyclazocine) and
opioid receptor-like 1 (ORL1) receptor (NOR, nociceptine) or
according to the tissue, in which the receptor was detected first,
1
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10.1002/cmdc.202000300
ChemMedChem
FULL PAPER
proliferation and the inhibition of immune cell activation, which
leads to a reduced secretion of proinflammatory cytokines.[11]
Very recently, we have shown that the KOR agonist (±)-2 (Ki = 5.6
nM, EC50 = 2.8 nM, Figure 1), which we successfully synthesized
agonists with a perhydroquinoline framework. In this series of
compounds, the highest KOR affinity was found for racemic (±)-4
(IC50 = 2.7 nM) with a cis-configured bicyclic framework and
trans-oriented pyrrolidinyl moiety (IC50 = 2.7 nM).[20] (Figure 1)
Since the Ki value of the KOR agonist (±)-4 was not reported in
according to
a
diastereoselective pathway, significantly
decreased the expression of classical activation markers as well
as interferon-gamma (IFN- ), tumor-necrosis-factor-alpha (TNF-
), and interleukin 17A (IL-17A). In parallel, the compound
increased the release of anti-inflammatory cytokines like IL-10 in
different mouse and human immune cell subsets pointing to an
immunomodulatory effect of (±)-2. In support of this, (±)-2
significantly reduced disease perpetuation in EAE. Experiments
in KOR deficient mouse mutants confirmed that the observed
beneficial effects of (±)-2 were indeed mediated by binding of the
compound to KOR.[12]
the patent, a direct comparison of the KOR affinity of
enantiomerically pure 3 and racemic (±)-4 was not possible.
In the patent, the racemic KOR agonist (±)-4 was obtained by a
non-stereoselective synthesis and subsequent laborious and
time-consuming separation of racemic diastereomers. In this
manuscript we would like to report an efficient diastereoselective
synthesis of (±)-4. A chemoenzymatic route should provide
enantiomerically pure KOR agonists 4 and ent-4, which will be
thoroughly characterized in various biological assays.
F
N
CH3
O
N
O
N
N
N
N
N
2. Results and Discussion
Cl
Cl
Cl
Cl
CH3
Cl
Cl
Cl
Cl
N
N
N
N
O
N
O
O
N
O
2.1. Diastereoselective synthesis of racemic
(±)-4
(±)-2#
1
3
(±)-4#
U-50,488
Figure 1: Important ethylenediamine-based KOR agonists.
# Only one enantiomer of racemic mixtures (±)-2 and (±)-4 is shown.
The diastereoselective synthesis of cis,trans-configured racemic
KOR agonist (±)-4 started with 5,6,7,8-tetrahydroquinoline (5),
which was oxidized with m-chloroperoxybenzoic acid (mCPBA) to
afford the N-oxide 6. (Scheme 1) Boekelheide rearrangement[21]
of N-oxide 6 was performed with Ac2O at 120 °C providing the
acetate (±)-7. After hydrolysis of acetate (±)-7 with NaOH, the
alcohol (±)-8 was protected with a silyl group to give silyl ether (±)-
9. The bulky tert-butyldimethylsilyl protective group was chosen,
since it should direct the hydrogenation of the aromatic pyridine
ring from the opposite side. Thus, hydrogenation (5 bar) of (±)-9
in the presence of catalyst Rh/Al2O3 in the solvent HOAc afforded
diastereoselectively the cis,cis-configured perhydroquinoline (±)-
The known KOR agonists belong to four structurally diverse
classes of compounds: peptides derived from the endogenous
KOR agonist dynorphin A,[13] morphinoids,[14] the non-basic
natural product salvinorin A,[15] and ethylenediamines with U-
50,488 (1)[16] being the first synthetic KOR agonist. Whereas one
N-atom of the ethylenediamine-based KOR agonists should be
connected with a (3,4-dichlorophenyl)acetyl moiety, the second
N-atom should display basic properties. Usually, this N-atom is
part of a pyrrolidine ring, which after protonation forms a crucial
ionic interaction with Asp138 in the KOR binding site.[17] The (3,4-
dichlorophenyl)acetyl moiety at the other end of the
ethylenediamine pharmacophore interacts with a hydrophobic
pocket of KOR.
10 in 94
%
yield..[22] In addition to the control of the
stereoselectivity, the silyl protective group should be stable during
the hydrogenation conditions in pure HOAc used as solvent,
which activates the pyridine ring and accelerates its
hydrogenation[23] providing increased yields of (±)-10. The
hydrogenation represents the key step in the nine-step reaction
sequence, since it establishes the configuration of all three
centers of chirality with excellent diastereoselectivity.
Recently, we have reported
constrained KOR agonists, in which the cyclohexane ring of U-
50,488 (1) is embedded in a perhydroquinoxaline scaffold.[18,19]
a series of conformationally
A
very high KOR affinity (Ki = 0.25 nM, EC50 = 2.0 nM) was found
for the (4aR,5S,8aS)-configured perhydroquinoxaline 3 with a cis-
configured bicyclic ring system.[18] (Figure 1) This result is in good
accordance with the results of a patent describing racemic KOR
2
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(a)
(b)
(c)
(e)
(d)
N
N
O
N
N
OTBDMS
OR
9
(±)-
5
6
(±)-7: R = Ac
(±)-8: R =H
(i)
(g)
(h)
Cl
N
N
O
N
N
H
H
N
O S
O
N O
Cl
OR
(±)-10: R = TBDMS
(±)-11: R =H
12
(±)-
13
(±)-
4
(±)-
Scheme 1: Synthesis of racemic KOR agonist (±)-4. Reagents and reaction conditions: (a) mCPBA, CH2Cl2, rt, 19 h, 81 %; (b) (Ac)2O, 120 °C, 1 h, 88 %; (c) aqu
NaOH 1 M, CH3OH, rt, 1 h, 98 %; (d) TBDMSCl, imidazole, DMF, rt, 16 h, 63 %; (e) H2 (5 bar), Rh/Al2O3, AcOH, rt, 25 h, 94 %; (f) TBAF, THF, rt, 18 h, 88 %; (g)
SO2Cl2, triethylamine, CH2Cl2, 0 °C  rt, 20 h, 34 %; (h) pyrrolidine, CH3CN, 80 °C, 18 h, 82 %; (i) 2-(3,4-dichlorophenyl)acetyl chloride, ethyldi(isopropyl)amine,
CH2Cl2, rt, 1 h, 56 %. Only one enantiomer of the racemic mixtures is shown.
The relative configuration of (±)-10 was determined by nuclear
Overhauser effect. (Figure 2) Irradiation at the resonance
frequency of 8a-H ( = 2.85 ppm, t, J = 3.5 Hz) led to increased
signals of 2-Haxial ( = 2.59 ppm), 4a-H/7-Haxial ( = 1.64 ppm) and
8-Haxial ( = 3.27 ppm) in the recorded NOE difference spectrum.
Additional interactions between 6-Haxial ( = 1.31 ppm) and 8-H (
= 3.27 ppm) were observed in a two-dimensional NOESY
spectrum. Both spectra are shown in the supporting information.
These NOEs clearly prove the cis-orientation of the protons 4a-H,
8a-H and 8-Haxial, which unequivocally confirms the cis,cis-
configuration of the perhydroquinoline ring of (±)-10.
The TBDMS protective group of (±)-10 was removed with
tetrabutylammonium fluoride (TBAF) to yield β-aminoalcohol (±)-
11, which was reacted with SO2Cl2 to give oxathiazolidine (±)-12
in 34 % yield. In the cyclic sulfuric ester amide (±)-12 the original
OH moiety of the β-aminoalcohol (±)-11 was activated for a SN2
reaction and the secondary amino moiety was protected. The
nucleophilic substitution was carried out with pyrrolidine giving the
diamine (±)-13 upon removal of the N-protective group during
work-up under acidic conditions. This SN2 reaction of (±)-12
represents the second key step, as the inversion of the
configuration at C-8 during the nucleophilic attack led to the
desired cis,trans-configured perhydroquinoline system. Finally,
the secondary amine (±)-13 was acylated with 2-(3,4-
dichlorophenyl)acetyl chloride to afford the racemic KOR agonist
(±)-4.
H
H
H
2.2. Chemoenzymatic synthesis of
4a
6
5
enantiomerically pure KOR agonists 4 and ent-4
4
A lipase-catalyzed kinetic resolution of racemic alcohol (±)-8 was
planned as key step for the synthesis of the enantiomerically pure
KOR agonists 4 and ent-4. In a previous study, the lipase-
catalyzed kinetic resolution of alcohol (±)-8 had been carried out
using Candida antarctica lipase as biocatalyst and vinyl acetate
as acetylating reagent at 60 °C for 30 h.[24] Under these conditions
the acetate (R)-7 and the alcohol (S)-8 were obtained in 43 % and
44 % yield, respectively, with an enantiomeric purity >99 %ee in
both cases.
8
H
7
8a
3
O
HN
TBDMS
H
1
2
Figure 2: Neighborhood of protons of cis,cis-configured perhydroquinoline (±)-
10 determined by nuclear Overhauser effect (NOE).
3
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Figure 3: Lipase-catalyzed kinetic resolution of racemic alcohol (±)-8. 3A: Enantioselective acetylation of (±)-8 using isopropenyl acetate in the presence of Amano
lipase PS-IM. 3B: Amounts [%] of remaining alcohol 8 and formed acetate 7 during the reaction period. 3C: Time course of the ee-values of both, alcohol (S)-8 and
acetate (R)-7 during the transformation.
Herein, Amano lipase PS-IM was used for the kinetic resolution of
racemic alcohol (±)-8, which allowed milder reaction conditions,
i.e. reaction at room temperature, a longer reaction time, and
isopropenyl acetate as acetylating agent. (Figure 3A) Although
alcohol (S)-8 (97.9 %ee) and acetate (R)-7 (97.3 %ee) were
present in the ratio 50:50 after a reaction time of 70 h (Figures 3B
and 3C), excellent enantiomeric purity of remaining alcohol (S)-8
(98.4 % ee) was obtained after a very long reaction time of 140 h.
(Figure 3C) As expected, the Amano lipase PS-IM acetylated
almost exclusively the (R)-configured alcohol (R)-8. Thus, a long
reaction time led to conversion of even traces of alcohol (R)-8 into
acetate (R)-7 providing excellent enantiomeric excess of the
remaining alcohol (S)-8. On the other hand, small amounts of
alcohol (S)-8 were also acetylated by the lipase resulting in
decreased enantiomeric purity of the acetate (R)-7. After a
reaction period of 140 h, acetate (R)-7 (96.2 %ee) and alcohol
(S)-8 (98.4 %ee) were isolated in 45 % and 32 % yield,
respectively. Chiral HPLC of (S)-8 is shown in Figure S2.
In order to increase the enantiomeric purity of (R)-configured
alcohol (R)-8, acetate (R)-7 (96.2 %ee) was hydrolyzed with
NaOH and the resulting alcohol (R)-8 (94.0 %ee) was used in a
second lipase-catalyzed acetylation reaction. (Figure 4) Since the
lipase converted predominantly the (R)-configured alcohol (R)-8
into its acetate (R)-7, (Figure 4A) the amount of (R)-8 decreased
rapidly as the reaction proceeded. (Figure 4B) However, the
enantiomeric purity of the acetate (R)-7 was very high from the
beginning. After a reaction period of 52 h, the transformation was
stopped and the acetate (R)-7 was obtained in 88 % yield and
99.5 %ee. (Figure 4C) Finally, alcohol (R)-8 (99.1 %ee) was
obtained by ester cleavage with NaOH. (Figure S3)
4
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Figure 4. Saponification and second lipase-catalyzed acetylation of enantiomerically enriched alcohol (R)-8 (94.0 %ee). 4A: Transformations of (R)-7 (96.2 %ee).
4B: Amounts [%] of remaining alcohol 8 and formed acetate 7 during the reaction period. 4C: Development of the ee-values of alcohols (R)-8/(S)-8 and acetate (R)-
7 during the transformation. The decreased ee-value of acetate (R)-7 at the beginning of the transformation is due to very small amounts of acetate 7 (0.04 %) in
the starting material (R)-8.
Interestingly, the ee-value of the (R)-configured alcohol (R)-8
decreased continuously until it reached a minimum (< 5 %ee)
after 32 h. At this time point the amounts of desired (R)- and non-
desired (S)-configured alcohols (R)-8 and (S)-8 are almost
identical. After passing the minimum, ee-values for (S)-configured
alcohol (S)-8 increased, since (R)-8 was transformed selectively.
5
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Figure 5: Recorded CD spectra of enantiomeric TBDMS ethers (R)-9 and (S)-9 and TDDFT calculated CD spectrum of (S)-configured TMS-ether (S)-14 as model
compound.
In order to determine the absolute configuration of the
enantiomeric alcohols (R)-8 and (S)-8, CD spectra of the
corresponding enantiomeric silyl ethers (R)-9 and (S)-9 were
recorded in acetonitrile (Figure 5) Time-dependent density
functional theory (TDDFT) calculation at the B3LYP/6-31G(d,p)
level were used to correlate the observed Cotton effects with the
corresponding absolute configuration. The trimethylsilyl ether (S)-
14 was employed as model compound due its significantly higher
conformational restrictions compared to (R)-9 and (S)-9 (but
bearing the same chromophore), which translated in lower overall
computational costs. As a result, the absolute configuration of
both silyl ethers (R)-9 and (S)-9 was unequivocally assigned.
(Figure 5) This configurational assignment perfectly agrees with
the observation that Amano lipase PS-IM preferably acetylates
(R)-configured secondary alcohols (in case the larger substituent
has higher CIP priority than the smaller substituent).[24]
additional purification of the enantiomers 4 and ent-4 by chiral
preparative HPLC using a Chiralpak® IA column was performed
resulting in enantiomerically pure KOR agonists 4 (99.8 %ee) and
ent-4 (99.0 %ee).
2.3. KOR affinity, selectivity and agonistic
activity
The KOR affinity of the synthesized quinoline derivatives and
some reference compounds was investigated in competitive
radioligand receptor binding assays using guinea pig brain
homogenates as source of KOR and [3H]U-69,593 as competitive
radioligand.[25–27] Table
1 summarizes the affinity data of
perhydroquinolines, related perhydroquinoxalines and reference
compounds.
The KOR affinity of racemic (±)-4 (Ki = 1.2 nM) is approximately
5-fold higher than the KOR affinity of triazole-based KOR agonist
(±)-2, but 5-fold lower than those of enantiomerically pure methyl
carbamate 3. The KOR affinity of (±)-4 resides almost exclusively
in the (4aR,8S,8aS)-configured enantiomer 4 (Ki = 0.81 nM),
which is more than 200-fold more active than the distomer ent-4.
The Ki value of the eutomer 4 is in the same range as the Ki values
of the prototypical KOR agonists U-50,488 and the methyl
carbamate 3. (Table 1)
With both enantiomerically pure alcohols (R)-8 (99.1 %ee) and
(S)-8 (98.4 %ee) in hands the already described synthetic route
(Scheme 1) was performed in order to obtain the KOR agonists 4
[(4aR,8S,8aS)-configuration]
and
ent-4
[(4aS,8R,8aR)-
configuration]. The corresponding reaction Scheme is shown in
the Supporting Information. (Scheme S1) The final control of the
enantiomeric purity showed some racemization of both
enantiomers (4: 80.3 %ee, ent-4: 66.3 %ee). We assume that
partial racemization occurred during hydrogenation of the TBDMS
ethers (R)-9 and (S)-9 under acidic conditions. Therefore,
6
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Table 1: Affinities of racemic and enantiomerically pure perhydroquinolines and reference compounds towards KOR and related receptors.
Cl
Cl
N
N O
Ki ± SEM [nM] a) b)
compd.
configuration
racemate
KOR
[3H]U-69,593
5.6 ± 0.6
0.25 ± 0.08
1.2 ± 0.6
0.81 ± 0.32
195 ± 67
0.34 ± 0.07
7.3 ± 0.40
-
MOR
DOR
1
2
[3H]DAMGO
[3H]DPDPE
(+)-[3H](+)-pentazocine
[3H]DTG
(±)-2[12]
3[19]
573
413
2 %
0 %
43 ± 9.2
58 ± 8.4
0 %
8 %
(±)-4
racemate
4aR,8S,8aS
4aS,8R,8aR
1S,2S
1200
1400
0 %
0 %
4
0 %
0 %
676
3300
ent-4
0 %
0 %
0 %
12 %
U-50,488
naloxone
morphine
SNC80
-
-
-
-
2.3 ± 1.1
103
-
-
5.2 ± 1.6
-
-
-
-
-
-
-
1.2 ± 0.5
-
-
(+)-pentazocine
-
-
5.4 ± 0.5
-
haloperidol
-
-
6.6 ± 0.9
78 ± 2.3
a) A value in % reflects the inhibition of the radioligand binding at a test compound concentration of 1 μM. Ki values without SEM values
represent the mean of two experiments (n = 2) and Ki values with SEM values represent the mean of three experiments (n = 3).
b)
Guinea pig brain membrane preparations were used in the KOR, MOR and 1 assay. In the DOR assay rat brain and in the 2 assay
rat liver membrane preparations were used.
ligands were also recorded.[28] Very low 1 and 2 affinity was
The enantiomerically pure KOR agonist 4 as well as the racemic
mixture (±)-4 and the distomer ent-4 showed very low affinity
towards MOR and DOR, indicating high selectivity for KOR over
the other two opioid receptor subtypes MOR and DOR. Due to the
historical and ligand structure relationship of  and opioid
receptors, in particular KOR, 1 and 2 receptor affinity of the
found for the (4aR,8S,8aS)-configured enantiomer 4. However,
the Ki values at both receptors indicate a more than 800- (1)
and more than 4000-fold (2) selectivity for KOR.
7
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Table 2: Correlation of KOR affinity and KOR activity of 4 and U-50,488.
Cl
Cl
N
N O
KORa)
cAMPb)
β-arrestin-2c)
compd.
[3H]U-69,593
Ki ± SEM
[nM]
EC50 [nM]
0.029
Emax (%)d)
103
EC50 [nM]
1.09
Emax (%)d)
4
0.81 ± 0.32
0.34 ± 0.07
64
U-50,488
0.16
100
7.85
100
a)
b)
c)
d)
Guinea pig brain membrane preparations.
Human HEK 293T cells.
Human HTLA cells.
The Emax values refer to U-50,488 (100 %).
The KOR agonistic activity was investigated for the eutomer 4 with
(4aR,8S,8aS)-configuration. For this purpose, two assays
recording the reduction of cAMP production (human HEK293T
cells) and -arrestin-2 recruitment (Tango assay using human
HTLA cells) were performed. (Table 2)
CD28 (T cells) to upregulate the expression of typical activation
markers and induce the production of pro-inflammatory cytokines.
Upon activation, antigen-presenting cells, such as DC or
macrophages, upregulate co-stimulatory markers of the tumor-
necrosis factor (TNF) or B7 families including CD40 or CD86,
respectively and start to secrete INF-, IL-6, IL-12 or TNF-. In
systemic inflammation, these activated antigen-presenting cells
get into direct contact to naive T cells, resulting in the priming of
effector T cells (T helper or cytotoxic T cells). Effector T cells
including Th1 or Th17 cells migrate to the site of inflammation and
thus, can be found in target organs. Under infectious conditions
they are critically involved in the elimination of bacteria or virus-
containing cells. However, in chronic and complex inflammatory
diseases, Th1 and Th17 are also considered to mediate tissue
destruction as shown in psoriasis, atopic dermatitis, rheumatoid
arthritis or multiple sclerosis.[30]
In Table 2, the KOR affinity and agonistic activity of
enantiomerically pure 4 and the prototypical KOR agonist U-
50,488 are compared. Whereas the KOR affinity of 4 is slightly
lower than the KOR affinity of U-50,488, its effect on the cAMP
production is higher than that of U-50,488. With an EC50 value of
0.029 nM 4 shows very high KOR agonistic activity. In the -
arrestin-2 recruitment assay 4 is also approximately 7-fold more
active than U-50,488. In the cAMP assay 4 reaches the same total
effect as U-50,488 (100 % intrinsic activity), whereas in the -
arrestin-2 recruitment assay 4 attains only 64 % of the U-50,488
effect. These data indicate, that 4 does not prefer the activation
of one of the two pathways (no biased activation).
To assess the anti-inflammatory capacity of the KOR agonists (±)-
4, 4 and ent-4 pre-activated T cells, macrophages, neutrophils
and DC were stimulated with the KOR agonists in a concentration
of 1 M after having confirmed that this concentration is sufficient
to markedly downregulate the expression of the early and
transient activation marker CD69 (Figure S7, Supporting
Information). Interestingly, in activated human macrophages we
observed significantly reduced numbers of cells expressing the
co-stimulatory receptor CD86 as well as the cytokines IL-6 and
TNF- after stimulation with the compounds (±)-4 and 4 (Figure
6A and 6B). In contrast, compound ent-4 did not show anti-
inflammatory properties since comparable levels of cells
expressing co-stimulatory receptors or pro-inflammatory
cytokines were found in ent-4 stimulated cultures and controls
(Figure 6A and 6B). Worth mentioning that the potent anti-
inflammatory effect of (±)-4 and 4 was not limited to human
macrophages since we observed a similar reduction in the
expression of typical surface markers characteristic for cell
In analogous MOR and DOR cAMP and -arretin-2 recruitment
assays, 4 did not exhibit significant activity, indicating the
selective activation of these pathways via interaction with KOR.
2.4. Effects of KOR agonists (±)-4 and its
enantiomers 4 and ent-4 on immune cells
To
further
characterize
the
anti-inflammatory
and
immunomodulatory properties of the newly synthesized
compounds different immune cell subsets were isolated including
T cells, dendritic cells (DC) or macrophages, which are known to
be involved in the development and progression of various
(systemic) inflammatory disorders.[29] Immune cell populations
were purified from lymph nodes and spleen of wild-type (WT) mice
or from peripheral blood of healthy human donors, stimulated with
lipopolysaccharide (LPS; myeloid cells) or anti-CD3 plus anti-
8
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10.1002/cmdc.202000300
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activation and classical pro-inflammatory cytokines in human DC
and mouse myeloid immune cell subsets (Figure 6C – 6F).
Figure 6: Compounds (±)-4 and 4 significantly reduced the activation and cytokine production in myeloid human and mouse immune cells. (A,B) Human
macrophages were sorted from peripheral blood mononuclear cells (PBMC) by magnetic beads and activated with LPS for 12 h as described (activated
macrophages). Subsequently, cells were stimulated with compounds (±)-4, 4 and ent-4 (1 µM each) for additional 48 h. Control cells received an equal amount of
PBS. Representative dot-plots (A) and percentages of cells expressing the pro-inflammatory cytokines TNF- and IL-6 or the co-stimulatory surface marker CD86
from n = 4 healthy human donors (B) are shown. Cells are gated for CD11b+CD68+ macrophages and IL-6 as well as TNF- staining was performed after cell
permeabilization. Data are presented as mean ± SEM; *, p < 0.05. (C,D) Mouse macrophages were isolated from lymph node and spleen tissue and stimulated with
LPS for 12 h as described (activated macrophages). Subsequently, cells were cultured in the presence of the compounds (±)-4, 4 and ent-4 (1µM each) for additional
48 h. Control cells received an equal amount of PBS. Representative dot-plots (C) and percentages of cells expressing the pro-inflammatory cytokines TNF- and
IL-6 or the co-stimulatory surface marker CD86 from n = 4 individual mice (D) are shown. Cells are gated for CD11b+F4/80+ macrophages and IL-6 as well as TNF-
 staining was performed after cell permeabilization. Data are presented as mean ± SEM; *, p < 0.05. (E,F) Human (E) or mouse (F) dendritic cells were purified
from peripheral blood or spleen tissue, respectively and stimulated with LPS for 12 h. Subsequently, cells were incubated with the compounds (±)-4, 4 and ent-4
(1µM each) for additional 48 h or received an equal amount of PBS. Percentages of human (E) or mouse (F) dendritic cells expressing the pro-inflammatory cytokine
IL-12 or the co-stimulatory surface marker CD40 from n = 4 healthy human donors (E) or n = 4 individual mice (F) are shown. Cells are gated for HLA-DR+ (E) or
MHC-II+F4/80–CD19– (F) and IL-12 staining was performed after cell permeabilization. Data are presented as mean ± SEM; *, p < 0.05.
It is well known that opioid receptor agonists not only control the
phenotype and function of myeloid cells but also modulate
lymphoid immune cell subsets and thereby, impact on the innate
as well as the adaptive immune system. In line with this, it has
been shown that besides impairing DC maturation, KOR agonists
are able to prevent effector T cell priming.[12,31]
even more pronounced compared to (±)-4 and was not limited to
human CD4+ T cells since we observed a similar reduction of Th1
and Th17 cells in murine cultures (Figure 7D and 7E). However,
although compound 4 (and to a lesser extend also compound (±)-
4) markedly downregulated the cytokine secretion and
transcription factor expression in activated mouse and human
CD4+ T cells, both compounds did not exhibit immunomodulatory
capacities. Immunomodulation would for instance, require the
switch from activated effector to immunosuppressive regulatory
To assess the effects of the compounds (±)-4, 4 and ent-4 during
T
cell activation and to characterize their potential anti-
inflammatory capacity in more detail we purified CD4+ T cells from
peripheral blood of healthy human donors or spleen and lymph
node tissue of WT mice, activated the cells with anti-CD3 plus
anti-CD28 and stimulated them with the three KOR agonists.
Notably, the numbers of both, Th1 and Th17 cells were
significantly downregulated in cultures treated with (±)-4 or 4
whereas we did not observe reduced levels of CD4+ T cells
expressing IFN- and the transcription factor T-bet (Th1 cells) or
IL-17 and the transcription factor ROR-c (Th17 cells) in T cell
cultures that were stimulated with compound ent-4 (Figure 7A and
7B). Of note, the anti-inflammatory effect of compound 4 was
CD4+
T cells (Treg), which among other markers are
characterized by the expression of the transcription factor Foxp3.
Neither compound (±)-4 nor 4 was able to modulate Foxp3
expression in CD4+ T cells (Figure 7C and 7F). Two subsets of
Treg are known; thymus-derived Treg (tTreg) and peripherally
induced Treg (pTreg) that can be generated from conventional
CD4+ T cells and are converted in peripheral tissues to cells that
express Foxp3 and acquire suppressive ability. The transcription
factor Helios, a member of the Ikaros transcription factor family,
is believed to be a marker of tTreg.[32] Therefore, we analyzed the
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Helios+ and Helios- Treg subsets separately in T cell cultures
stimulated with the compounds (±)-4, 4 and ent-4 but did not
detect any significant difference in cell numbers compared to non-
stimulated controls (Figure 7C and 7F), confirming that (±)-4 and
4 show broad and potent anti-inflammatory effects but none of the
-agonists seems to have immunomodulatory properties.
Figure 7: Compounds (±)-4 and 4 significantly reduced the cytokine production and transcription factor expression in Th1 and Th17 cells but did not exhibit
immunomodulatory capacities. (A-C) Human CD4+ T cells were sorted from peripheral blood and activated with anti-CD3 and anti-CD28 for 48 h (activated CD4).
Subsequently, cells were stimulated with compounds (±)-4, 4 and ent-4 (1 µM each) for additional 48 h. Control cells received an equal amount of PBS.
Representative dot-plots (A) and percentages of cells expressing the Th1 markers IFN- and T-bet, the Th17 markers IL-17 and ROR-c from n = 4 healthy human
donors (B) or the Treg markers Foxp3 and Helios (C) are shown. Cytokine and transcription factor staining was performed after cell permeabilization. Data are
presented as mean ± SEM; *, p < 0.05. (D-F) Mouse CD4+ T cells were sorted from spleen and lymph node tissue of wild-type mice, activated with anti-CD3 and
anti-CD28 for 48 h (activated CD4), and stimulated with compounds (±)-4, 4 and ent-4 (1µM each) for additional 48 h. Representative dot-plots (D) and percentages
of cells expressing the Th1 markers IFN- and T-bet, the Th17 markers IL-17 and ROR-t from n = 4 individual mice (E) or the Treg markers Foxp3 and Helios (F)
are shown. Cytokine and transcription factor staining was performed after cell permeabilization. Data are presented as mean ± SEM; *, p < 0.05.
not induce immunomodulatory effects. The enantiomerically pure
KOR agonist 4 showed broad and potent anti-inflammatory
3. Conclusion
activity, but did not induce immunomodulatory effects.
A
diastereoselective synthesis of the cis,trans-configured
perhydroquinoline-based KOR agonist (±)-4 was developed.
Since the racemate (±)-4 exhibited high KOR affinity (Ki = 1.2 nM),
Very interestingly, all these different anti-inflammatory effects
correlate nicely with the KOR affinity of the KOR agonists 4, (±)-
4, and ent-4. In general, the racemate (±)-4 was slightly less
potent than the eutomer 4 and the distomer ent-4 did not exhibit
anti-inflammatory effects.
enantiomerically pure KOR agonists
4 and ent-4 were
synthesized by a lipase-catalyzed kinetic resolution of racemic
alcohol (±)-8. The (4aR,8S,8aS)-configured ligand 4 represents
the eutomer with a KOR affinity of 0.81 nM. Moreover, 4 shows
high selectivity for KOR over MOR, DOR, 1 and 2 receptors.
Whereas 4 has slightly lower KOR affinity than the prototypical
KOR agonist U-50,488, it shows higher KOR agonistic activity
than U-50,488 in the cAMP and -arrestin-2 assay. A bias for the
cAMP or -arrestin-2 pathway could not be observed.
4. Experimental
4.1. Chemistry, General Methods
Oxygen and moisture sensitive reactions were carried out under
nitrogen, dried with silica gel with moisture indicator (orange gel,
VWR, Darmstadt, Germany) and in dry glassware (Schlenk flask
or Schlenk tube). Thin layer chromatography (tlc): tlc silica gel 60
F254 on aluminum sheets (VWR). Flash chromatography (fc):
Silica gel 60, 40–63 µm (VWR); parentheses include: diameter of
the column (∅), length of the stationary phase (l), fraction size (v)
and eluent. Melting point: Melting point system MP50 (Mettler
Toledo, Gießen, Germany), open capillary, uncorrected. MS:
MicroTOFQII mass spectrometer (Bruker Daltonics, Bremen,
Germany); deviations of the found exact masses from the
The (4aR,8S,8aS)-configured KOR agonist 4 showed strong anti-
inflammatory activity. After activation of human macrophages,
human dendritic cells and mouse myeloid immune cells, the
number of cells expressing co-stimulatory receptor CD86 and
proinflammatory cytokines IL6 and TNF was significantly
reduced. The numbers of Th1 and Th17 cells was significantly
reduced by incubation of activated CD4+ T cells with KOR agonist
4. Moreover, 4 led to downregulation of the cytokine secretion and
transcription factor expression (anti-inflammatory activity), but did
10
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10.1002/cmdc.202000300
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calculated exact masses were 5 ppm or less; the data were
analyzed with DataAnalysis® (Bruker Daltonics). NMR: NMR
spectra were recorded in deuterated solvents on Agilent DD2 400
MHz and 600 MHz spectrometers (Agilent, Santa Clara CA, USA);
chemical shifts (δ) are reported in parts per million (ppm) against
the reference substance tetramethylsilane and calculated using
the solvent residual peak of the undeuterated solvent; coupling
constants are given with 0.5 Hz resolution; assignment of 1H and
13C NMR signals was supported by 2-D NMR techniques where
necessary.IR: FT/IR IR Affinity®-1 spectrometer (Shimadzu,
Düsseldorf, Germany) using ATR technique. Optical rotation:
Polarimeter 341 (Perkin Elmer); 1.0 dm tube; concentration c in
g/100 mL; T = 20 °C; wavelength 589 nm (D-line of Na light); the
HPLC method 5 to separate the KOR-agonists 4 and ent-4 on a
preparative scale: Equipment: UV/Vis detector: L-7400, pump: L-
7150A, autosampler: L-7200, data acquisition: HSM-software
(LaChrom, Merck-Hitachi); column: Daicel Chiralpack® IA
(250x20 mm, 5 μm particle size); flow rate: 15 mLmin-1; injection
volume: 400 μL; detection at λ = 275 nm; solvent: isohexane/
ethanol 90:10 + 0.1 % diethylamine, isocratic.
4.3. Synthetic procedures
(4aRS,8SR,8aSR)-8-(Pyrrolidin-1-yl)-decahydroquinoline[33]
((±)-13)
Pyrrolidine (0.34 mL, 4.14 mL, 6.0 eq) was added to a solution of
(±)-12 (150 mg, 0.69 mmol, 1.0 eq) in dry acetonitrile (1.5 mL).
After stirring for 18 h at 80 °C, additional pyrrolidine (0.17 mL, 2.07
mmol, 3.0 eq) was added and the mixture was stirred for another
2 h at 80 °C. The solvent was removed in vacuo, toluene was
added and evaporated in vacuo several times (azeotropic
evaporation of excess pyrrolidine). After addition of HCl (1 M, 1
mL), the mixture was washed with Et2O (2 x 2 mL). The pH value
of the aqueous layer was then set to pH 10 by addition of NaOH
(1 M, 1 mL) and extracted with Et2O (2 x 2 mL). After additional
extraction with EtOAc (5 x 2 mL) the combined organic layers
were dried (Na2SO4), filtered and evaporated in vacuo. The
residue was purified by flash column chromatography (d = 2 cm,
h = 20 cm, V = 10 mL, CH2Cl2/CH3OH = 98:2  95:5 and then
CH2Cl2/CH3OH/Et3N = 94:5:1, Rf = 0.11 (CH2Cl2/CH3OH = 95:5)).
Yellow viscous oil, yield 118 mg (82 %). Chemical formula:
C13H24N2 (208.35 g/mol).
T
unit of the specific rotation ([]D grad.mL.dm-1.g-1) is omitted for
clarity. CD-spectroscopy: Jasco (Pfungstadt, Germany) J-815
spectropolarimeter; concentration 1 mg / mL in acetonitrile.
4.2. HPLC methods
HPLC method 1 to determine the purity of compounds: Equipment
1: Pump: L-7100, degasser: L-7614, autosampler: L-7200, UV
detector: L-7400, interface: D-7000, data transfer: D-line, data
acquisition: HSM-Software (all from LaChrom, Merck Hitachi);
Equipment 2: Pump: LPG-3400SD, degasser: DG-1210,
autosampler: ACC-3000T, UV-detector: VWD-3400RS, interface:
DIONEX UltiMate 3000, data acquisition: Chromeleon
7
(Thermo Fisher Scientific); column: LiChropher® 60 RP-select B
(5 µm), LiChroCART® 250-4 mm cartridge; flow rate: 1.0 mL/min;
injection volume: 5.0 µL; detection at λ = 210 nm; solvents: A:
demineralized water with 0.05 % (V/V) trifluoroacetic acid, B:
acetonitrile with 0.05 % (V/V) trifluoroacetic acid; gradient elution
(% A): 0 - 4 min: 90 %; 4 - 29 min: gradient from 90 % to 0 %;
29 - 31 min: 0 %; 31 - 31.5min: gradient from 0 % to 90 %;
31.5 - 40 min: 90 %. The purity of all test compounds is greater
than 95 %.
(4aR,8S,8aS)-8-(Pyrrolidin-1-yl)-decahydroquinoline (13)
Oxathiazole 12 (188 mg, 0.97 mmol, 1.0 eq) was reacted with
pyrrolidine (0.64 mL, 7.79 mmol, 8.0 eq) in CH3CN (2 mL) as
described for (±)-13. Dark yellow oil, yield 107 mg (53 %). Specific
rotation: ꢀ ꢂꢃ = +26.6 (c = 3.2 mg/mL, CH3OH).
ꢁ
HPLC method 2 to determine the enantiomeric purity of alcohols
(R)-8 and (S)-8: Equipment: UV/Vis detector: L-7400, pump: L-
7150A, autosampler: L-7200, data acquisition: HSM-software
(LaChrom, Merck-Hitachi); column: Daicel Chiralpack® AD-H
(250x4.6 mm, 5 μm particle size); flow rate: 1.0 mLmin-1; injection
(4aS,8R,8aR)-8-(Pyrrolidin-1-yl)-decahydroquinoline (ent-13)
Oxathiazole ent-12 (205 mg, 0.94 mmol, 1.0 eq) was reacted with
pyrrolidine (0.47 mL, 5.72 mmol, 6.1 eq) in CH3CN (2 mL) as
described for (±)-13. Dark yellow oil, yield 90 mg (46 %). Specific
rotation: ꢀ ꢂꢃ = -16.9 (c = 3.4 mg/mL, CH3OH).
ꢁ
volume: 10 μL; detection at
λ
=
266 nm; solvent:
isohexane/CH3OH 95:5, isocratic.
2-(3,4-Dichlorophenyl)-1-[(4aRS,8SR,8aSR)-8-(pyrrolidin-1-
yl)-3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-
one[33] ((±)-4)
HPLC method 3 to determine the enantiomeric purity of acetates
(R)-7 and (S)-7: Equipment: UV/Vis detector: L-7400, pump: L-
7150A, autosampler: L-7200, data acquisition: HSM-software
(LaChrom, Merck-Hitachi); column: Daicel Chiralpack® IA
(250x4.6 mm, 5 μm particle size); flow rate: 1.0 mLmin-1; injection
Ethyldiisopropylamine (0.09 mL, 0.53 mmol, 2.0 eq) was added to
a solution of (±)-13 (57 mg, 0.27 mmol, 1.0 eq) in dry CH2Cl2 (2.5
mL). 2-(3,4-Dichlorophenyl)acetyl chloride (122 mg, 0.55 mmol,
2.0 eq) dissolved in dry CH2Cl2 (0.5 mL) was added dropwise at
0 °C. The mixture was stirred for 1 h at room temperature. After
addition of H2O (3.0 mL), the mixture was extracted with CH2Cl2
(3 x 5 mL). The combined organic layers were dried (Na2SO4),
filtered and concentrated in vacuo. The residue was purified by
flash column chromatography (d = 1 cm, h = 15 cm, V = 5 mL,
CH2Cl2/CH3OH = 95:5, Rf = 0.31 (CH2Cl2/CH3OH = 95:5)). Yellow
solid, mp 83 °C, yield 61 mg (56 %). Chemical formula:
C21H28Cl2N2O (395.4 g/mol).
volume: 10 μL; detection at
isohexane/CH3OH 95:5, isocratic.
λ
=
266 nm; solvent:
HPLC method 4 to determine the enantiomeric purity of KOR-
agonists 4 and ent-4: Equipment: DAD detector: L-7455, pump:
L-6200A, injector: Rheodyne 7725i, data acquisition: HSM-
software (LaChrom, Merck-Hitachi); column: Daicel Chiralpack®
IA (250x4.6 mm, 5 μm particle size); flow rate: 1.0 mLmin-1;
injection volume: 5 μL; detection at λ = 275 nm; solvent:
isohexane/isopropyl alcohol 90:10 + 0.1 % diethylamine, isocratic.
2-(3,4-Dichlorophenyl)-1-[(4aR,8S,8aS)-8-(pyrrolidin-1-yl)-
3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-one (4)
11
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10.1002/cmdc.202000300
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FULL PAPER
Secondary amine 4 (81 mg, 0.39 mmol, 1.0 eq) was reacted with
ethyldiisopropylamine (0.13 mL, 0.76 mmol, 1.9 eq) and 2-(3,4-
dichlorophenyl)acetyl chloride (174 mg, 0.78 mmol, 2.0 eq) in dry
CH2Cl2 (3 mL) as described for (±)-4. The product was obtained
as a yellow oil with a yield of 110 mg (71 %, 80.3 %ee (HPLC
method 4)). Therefore, a purification by chiral HPLC (HPLC
method 5) was performed. Yellow oil, yield 63 mg (41 %). Purity
(HPLC method 1): 96.8 % (tR = 18.96 min). Enantiomeric purity
(HPLC, method 4): 99.8 %ee (tR = 7.15 min). Specific rotation:
4.4.3. Preparation of membrane homogenates
from rat brain
5 rat brains (species: Sprague Dawley rats) were homogenized
with the potter (500-800 rpm, 10 up and down strokes) in 6
volumes of cold 0.32 M sucrose. The suspension was centrifuged
at 1,200 x g for 10 min at 4 °C. The supernatant was separated
and centrifuged at 23,500 x g for 20 min at 4 °C. The pellet was
resuspended in 5-6 volumes of buffer (50 mM TRIS, pH 7.4) and
centrifuged again at 23,500 x g (20 min, 4 °C). This procedure
was repeated twice. The final pellet was resuspended in 5-6
volumes of buffer and frozen (-80 °C) in 1.5 mL portions
containing about 1.5 mg protein/mL.
ꢀ
ꢂꢃ = -22.6 (c = 6.4 mg/mL, CH3OH).
ꢁ
2-(3,4-Dichlorophenyl)-1-[(4aS,8R,8aR)-8-(pyrrolidin-1-yl)-
3,4,4a,5,6,7,8,8a-octahydroquinolin-1(2H)-yl]ethan-1-one
(ent-4)
Secondary amine ent-13 (67 mg, 0.32 mmol, 1.0 eq) was reacted
with ethyldiisopropylamine (0.11 mL, 0.65 mmol, 2.0 eq) and 2-
(3,4-dichlorophenyl)acetyl chloride (144 mg, 0.64 mmol, 2.0 eq)
in dry CH2Cl2 (3 mL) as described for (±)-4. The product was
obtained as a yellow oil with a yield of 71 mg (56 %, 66.3 %ee
(HPLC method 4)). Therefore, a purification by chiral HPLC
(HPLC method 5) was performed. Yellow oil, yield 37 mg (29 %).
Purity (HPLC method 1): 97.2 % (tR = 19.02 min). Enantiomeric
purity (HPLC method 4): 99.0 %ee (tR = 8.75 min). Specific
4.4.4. Protein determination
The protein concentration was determined by the method of
Bradford,[34] modified by Stoscheck.[35] The Bradford solution was
prepared by dissolving 5 mg of Coomassie Brilliant Blue G 250 in
2.5 mL of EtOH (95 %, v/v). 10 mL deionized H2O and 5 mL
phosphoric acid (85 %, m/v) were added to this solution, the
mixture was stirred and filled to a total volume of 50 mL with
deionized water. The calibration was carried out using bovine
serum albumin as a standard in 9 concentrations (0.1, 0.2, 0.4,
0.6, 0.8, 1.0, 1.5, 2.0 and 4.0 mg /mL). In a 96 well standard
multiplate, 10 µL of the calibration solution or 10 µL of the
membrane receptor preparation were mixed with 190 µL of the
Bradford solution, respectively. After 5 min, the UV absorption of
the protein-dye complex at  = 595 nm was measured with a plate
reader (Tecan Genios®, Tecan, Crailsheim, Germany).
rotation: ꢀ ꢂꢃ = +23.3 (c = 3.5 mg/mL, CH3OH).
ꢁ
4.4. Receptor binding studies
4.4.1. Materials
Guinea pig brains and rat brains were commercially available
(Harlan-Winkelmann, Borchen, Germany). Homogenizers:
Elvehjem Potter (B. Braun Biotech International, Melsungen,
Germany) and Soniprep® 150 (MSE, London, UK). Centrifuges:
Cooling centrifuge model Eppendorf 5427R (Eppendorf, Hamburg,
Germany) and High-speed cooling centrifuge model Sorvall® RC-
5C plus (Thermo Fisher Scientific, Langenselbold, Germany).
Multiplates: standard 96 well multiplates (Diagonal, Muenster,
Germany). Shaker: self-made device with adjustable temperature
and tumbling speed (scientific workshop of the institute).
Harvester: MicroBeta® FilterMate 96 Harvester. Filter: Printed
Filtermat Typ A and B. Scintillator: Meltilex® (Typ A or B) solid
state scintillator. Scintillation analyzer: MicroBeta® Trilux (all
Perkin Elmer LAS, Rodgau-Jügesheim, Germany).
4.4.5. General procedures for the binding
assays
The test compound solutions were prepared by dissolving
approximately 10 µmol (usually 2-4 mg) of test compound in
DMSO so that a 10 mM stock solution was obtained. To obtain
the required test solutions for the assay, the DMSO stock solution
was diluted with the respective assay buffer. The filtermats were
presoaked in 0.5 % aqueous polyethylenimine solution for 2 h at
rt before use. All binding experiments were carried out in
duplicates in the 96 well multiplates. The concentrations given are
the final concentration in the assay. Generally, the assays were
performed by addition of 50 µL of the respective assay buffer,
50 µL of test compound solution in various concentrations (10-5,
10-6, 10-7, 10-8, 10-9 and 10-10 mol/L), 50 µL of the corresponding
radioligand solution and 50 µL of the respective receptor
preparation into each well of the multiplate (total volume 200 µL).
The receptor preparation was always added last. During the
4.4.2. Preparation of membrane homogenates
from guinea pig brain
5 guinea pig brains were homogenized with the potter (500-
800 rpm, 10 up and down strokes) in 6 volumes of cold 0.32 M
sucrose. The suspension was centrifuged at 1,200 x g for 10 min
at 4 °C. The supernatant was separated and centrifuged at
23,500 x g for 20 min at 4 °C. The pellet was resuspended in 5-6
volumes of buffer (50 mM TRIS, pH 7.4) and centrifuged again at
23,500 x g (20 min, 4 °C). This procedure was repeated twice.
The final pellet was resuspended in 5-6 volumes of buffer and
frozen (-80 °C) in 1.5 mL portions containing about 1.5 mg
protein/mL.
incubation, the multiplates were shaken at
a speed of
500-600 rpm at the specified temperature. Unless otherwise
noted, the assays were terminated after 120 min by rapid filtration
using the harvester. During the filtration, each well was washed
five times with 300 µL of water. Subsequently, the filtermats were
dried at 95 °C. The solid scintillator was melted on the dried
filtermats at a temperature of 95 °C for 5 min. After solidifying of
the scintillator at rt, the trapped radioactivity in the filtermats was
measured with the scintillation analyzer. Each position on the
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filtermat corresponding to one well of the multiplate was
measured for 5 min with the [3H]-counting protocol. The overall
counting efficiency was 20 %. The IC50 values were calculated
with the program GraphPad Prism® 3.0 (GraphPad Software, San
thawed membrane preparation of rat liver (about 100 μg of
protein) was incubated with various concentrations of the test
compound, 3 nM [3H]DTG and buffer containing (+)-pentazocine
(500 nM (+)-pentazocine in 50 mM TRIS, pH 8.0) at room
Diego, CA, USA) by non-linear regression analysis. Subsequently, temperature. The non-specific binding was determined with 10
the IC50 values were transformed into Ki values using the equation
of Cheng and Prusoff.[36] The Ki values are given as mean value
± SEM from three independent experiments.
μM non-labeled DTG. The Kd value of [3H]DTG is 17.9 nM.[38]
4.5. In vitro functional assays
4.5.1. cAMP inhibition assay
4.4.6. KOR assay
To measure KOR Gαi-mediated cAMP inhibition, HEK 293T
(ATCC CRL-11268) cells were co-transfected with human KOR
along with a luciferase-based cAMP biosensor (GloSensor;
Promega) and assays were performed similar to previously
described.[39] After 16 h, transfected cells were plated into Poly-
lysine coated 384-well white clear bottom cell culture plates with
DMEM + 1% dialyzed FBS at a density of 15,000-20,000 cells per
40 μL per well and incubated at 37 °C with 5% CO2 overnight. The
next day, drug solutions were prepared in fresh drug buffer (20
mM HEPES, 1X HBSS, 0.3% bovine serum albumin (BSA), pH
7.4) at 3X drug concentration. Plates were decanted and received
20 μL per well of drug buffer (20 mM HEPES, 1X HBSS) followed
by addition of 10 μL of drug solution (3 wells per condition) for 15
min in the dark at room temperature. To stimulate endogenous
cAMP via β adrenergic-Gs activation, 10 μL luciferin (4 mM final
concentration) supplemented with isoproterenol (400 nM final
concentration) were added per well. Cells were again incubated
in the dark at room temperature for 15 min, and luminescence
intensity was quantified using a Wallac TriLux microbeta (Perkin
Elmer) luminescence counter. Results (relative luminescence
units) were plotted as a function of drug concentration, normalized
to % SalA stimulation, and analyzed using “log(agonist) vs.
response” in GraphPad Prism 5.0.
The assay was performed with the radioligand [3H]U-69,593 (55
Ci/mmol, BIOTREND). The thawed guinea pig brain membrane
preparation (about 100 μg of the protein) was incubated with
various concentrations of test compounds, 1 nM [3H]U-69,593,
and TRIS-MgCl2-buffer (50 mM TRIS, 8 mM MgCl2, pH 7.4) at
37 °C. The non-specific binding was determined with 10 μM
unlabeled U-69,593. The Kd value of U-69,593 is 0.69 nM.[27]
4.4.7. MOR assay
The assay was performed with the radioligand [3H]DAMGO (51
Ci/mmol, Perkin Elmer). The thawed guinea pig brain membrane
preparation (about 100 μg of the protein) was incubated with
various concentrations of test compounds, 3 nM [3H]DAMGO, and
TRIS-MgCl2-buffer (50 mM TRIS, 8 mM MgCl2, pH 7.4) at 37 °C.
The non-specific binding was determined with 10 μM unlabeled
naloxone. The Kd value of DAMGO is 0.57 nM.[26]
4.4.8. DOR assay
The assay was performed with the radioligand [3H]DPDPE (69
Ci/mmol, BIOTREND). The thawed rat brain membrane
preparation (about 75 μg of the protein) was incubated with
various concentrations of test compounds, 3 nM [3H]DPDPE, and
4.5.2. Tango β-arrestin-2 recruitment assay
The KOR Tango constructs were designed and assays were
performed as previously described.[40] HTLA cells expressing TEV
fused-β-arrestin2 (kindly provided by Dr. Richard Axel, Columbia
Univ.) were transfected with the KOR Tango construct. The next
day, cells were plated in DMEM supplemented with 1% dialyzed
FBS in poly-L-lysine coated 384-well white clear bottom cell
culture plates at a density of 10,000-15,000 cells/well in a total of
40 µL. The cells were incubated for at least 6 h before receiving
drug stimulation. Drug solutions were prepared in drug buffer (20
mM HEPES, 1X HBSS, 0.3% BSA, pH 7.4) at 3X and added to
cells (20 µL per well) for overnight incubation. Drug solutions used
for the Tango assay were exactly the same as used for the cAMP
assay. The next day, media and drug solutions were removed and
20 µL per well of BrightGlo reagent (purchased from Promega,
after 1:20 dilution) was added. The plate was incubated for 20 min
at room temperature in the dark before being counted using a
luminescence counter. Results (relative luminescence units) were
plotted as a function of drug concentration, normalized to % SalA
stimulation, and analyzed using “log(agonist) vs. response” in
GraphPad Prism 5.0.
TRIS-MgCl2-buffer (50 mM TRIS,
8 mM MgCl2, pH 7.4)
supplemented with SIGMAFAST® protease inhibitor mix (Sigma
Aldrich Biochemicals, Hamburg, Germany; 1 tablet dissolved in
100 mL of buffer) at 37 °C. The non-specific binding was
determined with 10 μM unlabeled morphine. The Kd value of
DPDPE is 0.65 nM.[25]
4.4.9. σ1 receptor assay
The assay was performed with the radioligand [3H]-(+)-
Pentazocine (22.0 Ci/mmol; Perkin Elmer). The thawed
membrane preparation of guinea pig brain cortex (about 100 μg
of protein) was incubated with various concentrations of test
compounds, 2 nM [3H]-(+)-Pentazocine, and TRIS buffer (50 mM,
pH 7.4) at 37 °C. The non-specific binding was determined with
10 μM unlabeled (+)-Pentazocine. The Kd value of (+)-
Pentazocine is 2.9 nM.[37]
4.4.10. σ2 receptor assay
The assays were performed with the radioligand [3H]DTG
(specific activity 50 Ci/mmol; ARC, St. Louis, MO, USA). The
4.6. Immune cell isolation and multicolor flow
cytometry
13
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10.1002/cmdc.202000300
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GL1), MHC II (major histocompatibility complex II; clone M5/114),
CD69 (clone H1.2F3), F4/80 (clone BM8), CD40 (clone 3/23), and
CD19 (clone 6D5; all purchased from Biolegend). Intracellular
staining of Foxp3 (clone FJK-16s), Helios (clone 22F6), IFN-
(clone XMG1.2), IL-12 (clone C17.8), IL-17 (clone TC11-18H10.0),
ROR-t (clone REA278), T-bet (clone 4B10), IL-6 (clone MP5-
20F3), and TNF- (clone MP6-XT22; all purchased from
Biolegend or Miltenyi Biotech) was performed after cell
permeabilization using the Fix/Perm Buffer Set (Biolegend)
according to the manufacturer’s instructions.
4.6.1. Purification and stimulation of mouse
immune cell populations
C57BL/6 mice (wild-type, WT; purchased from Janvier Labs, Le
Genest-Saint-Isle, France) were used at the age of 8 to 12 weeks
and housed under specific pathogen–free (SPF) conditions in
microisolator cages. Mice were given chow and water ad libitum
and animal experiments were performed with the approval of the
State Review Board of North Rhine-Westphalia according to the
German law for animal welfare (reference number 81-
02.05.50.17.015). After sacrifice of mice spleen and peripheral
lymph nodes were removed to isolate T cells, macrophages or DC.
For this purpose, single cell suspensions of the tissues were
prepared according to standard methods. Subsequently, total T
cells were isolated from using the Pan T Cell Isolation Kit II, DC
were purified using the Pan Dendritic Cell Isolation Kit mouse,
macrophages were separated from single cell suspensions with
anti-F4/80 microbeads (all purchased from Miltenyi Biotech,
Bergisch Gladbach, Germany), activated, stimulated with the
compounds (±)-4, 4 and ent-4, and subjected to flow cytometry
analyses (see below). After isolation, mouse macrophages or DC
were activated for 12 h with LPS (10 µg/mL) and cultured for
additional 48 h in the presence of compounds (±)-4, 4 and ent-4
at indicated concentrations or PBS as a control. Mouse T cells
were activated with plate-bound anti-CD3 and soluble anti-CD28
(clones 145-2C11 and 37.51; 0.5 µg/mL each antibody, both
purchased from Biolegend, San Diego, CA). Finally, cells were
subjected to flow cytometry (see below).
Human cells were stained in PBS using antibodies against CD4
(clone OKT4), CD11b (clone M1/ 70), CD86 (clone BU63), CD40
(clone 5C3), and HLA-DR (clone L243; all purchased from
Biolegend). Intracellular staining of Foxp3 (clone 206D), Helios
(clone 22F6), IFN- (clone 4S.B3), IL-12 (clone C11.5), IL-17
(clone BL168), ROR-c (clone REA278), T-bet (clone 4B10), IL-6
(clone MQ2-13A5), and TNF- (clone Mab11; all purchased from
Biolegend or Miltenyi Biotech) was performed after cell
permeabilization using the Fix/Perm Buffer Set (Biolegend)
according to the manufacturer’s instructions. Isotype-matched
controls were included in each staining, and apoptotic cells were
identified using an annexin V apoptosis detection kit.
4.6.4. Statistics
All values are expressed as means ± SEM. Statistically significant
differences were assessed by one-way analysis of variance
(ANOVA) test, comparing more than two groups. The alpha-level
was set at a value of <0.05 in all cases. SigmaPlot 14 or
GraphPad Prism 8 was used to analyze, plot, and illustrate data.
4.6.2. Purification and stimulation of human
immune cells
4.7. Computational methods – Simulation of
electronic CD spectra
Human macrophages, DC and T cells were isolated from
peripheral blood mononuclear cells (PBMC). Therefore, PBMC
were purified from fully anonymized leukapheresis reduction
chambers, obtained from the blood bank with the informed
consent of healthy donors by Ficoll gradient centrifugation
according to standard methods (Ficoll reagent was purchased
from Merck, Darmstadt, Germany). Total macrophages, DC or T
cells were selected using CD14 microbeads (macrophages) or
negatively enriched using the Pan-DC Enrichment Kit or the Pan
T Cell Isolation Kit (Miltenyi Biotech). All experiments were carried
out according to the declaration of Helsinki and were approved by
the ethical committee of the University of Münster Medical School
(2008-180-f-S). After isolation, human macrophages or DC were
activated for 12 h with LPS (10 µg/mL) and cultured for additional
48 h in the presence of compounds (±)-4, 4 and ent-4 at indicated
concentrations or PBS. Human T cells were activated with plate-
bound anti-CD3 and soluble anti-CD28 (clones UCHT1 and
CD28.2; 1 µg/mL each antibody, both purchased from Biolegend,
San Diego, CA). Finally, cells were subjected to flow cytometry.
A three-dimensional (3D) molecular model of (S)-14 was built with
the Molecular Operating Environment (MOE) software (version
2018.0101).[41] The structure was then energy minimized using
the MMFF94x force field. Compound (S)-14 was then subjected
to a conformational search using the low mode molecular
dynamics (LowMD) method and the MMFF94x force field, as
implemented in MOE, with an energy window of 5 kcal/mol. The
structures of the generated conformers (six low-energy
conformers) were stepwise-optimized in Gaussian 03.[42] During
the first step, the AM1 semi-empirical method was employed.
Calculation at higher level of theory using the B3LYP density
functional and a 6-31G(d,p) basis set constituted the second
optimization step. The population of the resulting conformers
were calculated from their final total energies by a Boltzmann
distribution. TDDFT calculations were performed for the first two
conformers (representing 95% of the population) using the same
density functional and basis set as above, considering the 30
lowest-energy electronic transitions for each conformer. All
calculations were run in vacuo. The ECD curve, expressed as the
differential molar extinction, Δε (in M-1cm-1) as function of the
energy, E (in eV), was obtained by applying a Gaussian-type
function[43] in Microsoft Excel over the corresponding rotatory
strength vectors (length). A width of the band at 1/e height of 0.15
eV was chosen. The final spectrum was obtained as a weighted
4.6.3. Multicolor flow cytometry analyses
The expression of cell surface and intracellular markers was
analyzed by multicolor flow cytometry on a GalliosTM flow
cytometer (Beckman Coulter) using the Kaluza software. For flow
cytometry, mouse cells were stained in PBS using antibodies
against CD4 (clone RM4-5), CD11b (clone M1/ 70), CD86 (clone
14
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average of the spectra of two conformers according to the
calculated Boltzmann distribution, applying a blue-shift of 0.55 eV.
Supporting Information
The Supporting Information contains the Scheme for the
synthesis of enantiomerically pure KOR agonists 4 and ent-4,
procedures and analytical data of synthesized compounds,
HPLC chromatograms of enantiomerically pure acetates 7,
alcohols 8 and KOR agonists 4, effects of (±)-4 on CD4+ T cells
and 1H and 13C NMR spectra of all prepared compounds.
Author Information
Corresponding author
*Tel:
+49-251-8333311.
Fax:
+49-8332144;
Email:
wuensch@uni-muenster.de
ORCID: Bernhard Wünsch 0000-0002-9030-8417
Acknowledgement
We would like to thank Meike Steinert and Kerstin Vischedyk for
excellent technical assistance. Financial support of this project by
the Deutsche Forschungsgemeinschaft (DFG, SFB-TR 128
project A10 to K.L., SFB 1009 project B07 to K.L., project 817/5-
1 to K.L., Cluster of excellence Cells in Motion, project FF-2016-
11 to K.L. and B.W.) is gratefully acknowledged.
Conflict of interest
The authors declare no conflict of interests.
15
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Graphical Abstract
KOR
1. Amano lipase PS IM
isopropenyl acetate
2. 6 steps
affinity: Ki = 0.81 nM
Cl
Cl
N
activity (cAMP): EC50 = 0.029 nM
activity (-arrestin): EC50 = 1.09 nM
high selectivity (MOR, DOR, 1, 2)
anti-inflammatory activity
N
N O
OH
cis,trans-Configured perhydroquinoline-based enantiomerically pure -opioid receptor (KOR) agonists were prepared in a stereoselective
synthesis. Amano lipase PS-IM was used for the enantioselective acetylation of racemic tetrahydroquinolin-8-ol with isopropenyl
acetate. (4aR,8S,8aS)-Configured enantiomer (structure see graphic) showed sub-nanomolar KOR affinity (Ki = 0.81 nM), which is
more than 200-fold higher than the KOR affinity of its enantiomer. In two different functional assays, it behaved as KOR agonist. The
strong anti-inflammatory effect was shown upon incubation of various human immune cells.
17
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