Development of Novel Quinoxaline-Based κ-Opioid Receptor Agonists for the Treatment of Neuroinflammation

Article abstract of DOI:10.1021/acs.jmedchem.8b01609

Neuroinflammatory disorders, such as multiple sclerosis or experimental autoimmune encephalomyelitis (EAE), an established mouse model mimicking part of the human pathology, are characterized by inflammatory infiltrates containing T helper 1 (T_H1) and T_H17 cells, which cause demyelination and neurodegeneration. Disease onset and perpetuation are mediated by peripherally generated autoreactive T cells infiltrating into the central nervous system, where they are restimulated by antigen-presenting cells. Here, we show that newly designed peripherally active, potent, and selective κ-opioid receptor (KOR) agonists comprising the ethylenediamine KOR pharmacophore in a perhydroquinoxaline scaffold exhibit potent anti-inflammatory capacities in primary antigen presenting cells as well as T cells. In the EAE model, the secondary amine 12 and the triazole 14 were able to ameliorate disease severity and to delay disease onset by blocking effector T cell activation. Importantly, the beneficial effects were mediated via signaling through KOR because off-target effects were excluded by using KOR-deficient mouse mutants.

Full text of DOI:10.1021/acs.jmedchem.8b01609

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Article
Development of novel quinoxaline-based kappa-opioid receptor
(KOR) agonists for the treatment of neuroinflammation
Giovanni Tangherlini, Dmitrii V. Kalinin, Dirk Schepmann, Tao Che,
nadine mykicki, Sonja Ständer, Karin Loser, and Bernhard Wünsch
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b01609 • Publication Date (Web): 13 Dec 2018
Downloaded from http://pubs.acs.org on December 13, 2018
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Development of novel quinoxaline-based -opioid receptor (KOR) agonists for the
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treatment of neuroinflammation
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Giovanni Tangherlini,a Dmitrii V. Kalinin,a Dirk Schepmann,a Tao Che,b Nadine Mykicki,c,d
Sonja Ständer,c Karin Loser,c,d,e,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
Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill,
NC 27599, USA.
c
Department of Dermatology, University of Münster, von-Esmarch-Str. 58, D-48149
Münster, Germany
d CRC1009 Breaking Barriers and CRC-TR 128 Multiple Sclerosis, University of
Münster, Germany
e
Cells-in-Motion Cluster of Excellence (EXC 1003 – CiM), Westfälische Wilhelms-
Universität Münster, D-48149 Münster, Germany.
Abstract
Neuroinflammatory disorders, such as multiple sclerosis (MS) or experimental
autoimmune encephalomyelitis (EAE), an established mouse model mimicking part of the
human pathology, are characterized by inflammatory infiltrates containing T helper 1
(TH1) and TH17 cells, which cause demyelination and neurodegeneration. Disease onset
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and perpetuation are mediated by peripherally generated autoreactiveT cells infiltrating
into the central nervous system, where they are re-stimulated by antigen-presenting cells.
Here, we show that newly designed peripherally active, potent and selective -opioid
receptor (KOR) agonists comprising the ethylenediamine KOR pharmacophore in a
perhydroquinoxaline scaffold exhibit potent anti-inflammatory capacities in primary
antigen presenting cells as well as T cells. In the EAE model the secondary amine 12 and
the triazole 14 were able to ameliorate disease severity and to delay disease onset by
blocking effector T cell activation. Importantly, the beneficial effects were mediated via
signaling through KOR since off-target effects were excluded by using KOR deficient
mouse mutants.
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Key words
KOR agonists; perhydroquinoxaline; selectivity; agonistic activity; structure-activity
relationships; docking; anti-inflammatory activity; TH1 or TH17 effector cells; experimental
autoimmune encephalomyelitis; KOR deficient mouse mitants
1. Introduction
Multiple sclerosis (MS) is one of the most common chronic, autoimmune, inflammatory
and demyelinating disorders of the central nervous system (CNS) affecting 2.5 million
people worldwide with a high incidence in young adults.1-3 MS is considered a T-cell-
mediated disease, where peripherally generated autoreactive T cells producing
interferone- (IFN-) or interleukin-17 (IL17) are supposed to infiltrate into the CNS and
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to be responsible for the initiation of the disease.2 After entering the CNS, these T cells
are restimulated by antigen-presenting cells resulting in local expansion of pathogenic T
helper 1 (TH1) and TH17 cells, tissue damage to oligodendrocytes, and a subsequent
infiltration of bystander immune cells, controlling disease perpetuation.3 In experimental
autoimmune encephalomyelitis (EAE), a mouse model mimicking part of the human MS
pathology, the lung has been identified as a niche, where pathogenic T cells are
stimulated, proliferate and acquire migratory properties to enter the CNS. Thus, peripheral
organs seem to contribute to the activation of CNS-reactive T cells and their transition
into a migratory state.4 In MS as well as in EAE regulatory T cells (Treg) have been shown
to be involved in limiting the proliferation and infiltration of pathogenic TH1 and TH17 cells
into the CNS.5 However, a modified phenotype and an impaired suppressive activity of
Treg has been associated with disease progression.6–8
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Since MS lesions can be detected in different parts of the brain, a big variety of symptoms
are observed in patients, such as sensory or motor disturbances, acute or chronic pain,
depression and optic neuritis.2,9
Since the launch of the first drug against MS in 1993, several immunomodulatory
treatments were developed.10 Interferon--1b (IFN-β),11 glatiramer acetate (GLAT),12
DNA intercalating drugs such as mitoxantrone,13 spingosine-1-phosphate (S1P) receptor
antagonists, such as fingolimod,14 and dimethyl fumarate15 finally induce anti-
inflammatory pathways via different mechanisms of action. Additionally, various
monoclonal antibodies,16,17 including natalizumab and ocrelizumab, are able to inhibit the
infiltration of immune cells into the CNS. All these drugs have been shown to reduce the
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relapse rate and to improve the disability in relapsing-remitting MS.18 However, they are
not able to stop disease perpetuation.2 In addition, all drugs currently used for MS
treatment can have adverse effects ranging from mild, such as flu-like symptoms or
irritations at the injection site, to serious, like progressive multifocal leukoencephalopathy
(PML), a viral disease of the CNS.19 For this reason, novel drugs addressing new
mechanisms of action are urgently needed.
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Recent studies suggested the endogenous opioid system to play a special role in the
pathogenesis of MS inflammatory neurodegeneration.20–22 The effects of this system are
predominantly mediated by three G-protein-coupled receptors, called µ- (MOR), - (DOR)
and -opioid receptor (KOR).23 They are present in the CNS but also in peripheral tissues
and cells, such as skin, intestine and immune cells, including T cells and antigen
presenting cells.23,24 The opioid system is commonly associated with pain transmission
and with different psychiatric disorders, such as depression, anxiety and addiction.23,25
During the last years, KOR acquired increasing attention in research and emerged as an
alternative target for the development of safer analgesics.26,27 In particular, KOR
activation results in strong analgesia, but does not lead to typical MOR-mediated side
effects, such as euphoria, respiratory depression and dependelnce.26,27 In 2009, the
selective KOR agonist nalfurafine was approved in Japan for the treatment of uremic
pruritus.28 Clinical studies showed that KOR partial agonists or antagonists, such as
buprenorphine and CERC-501, are promising drugs for the treatment of major depression
and abuse disorders.25,27,29 A few recent studies suggest a potential role of KOR in
inflammatory neurodegeneration. Screening of mRNA in Theiler’s murine
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encephalomyelitis, a virus model mimicking part of the MS pathology showed a
decreased level of KOR mRNA.21 Additionally, KOR has the ability to down-regulate the
proliferation, activation and secretion of cytokines in immune cells pointing to its
antiinflammatory activity.30 Furthermore, very recent data in EAE showed the ability of
activated KOR to prevent neuronal damage and to promote remyelination. Moreover,
ligation of KOR by a selective agonist seems to be involved in the differentiation of
oligodendrocyte progenitor cells (OPC) towards myelinated oligodendrocytes.20 Due to
their ability to regulate cytokine production and immune cell activation, KOR agonists may
represent a novel promising approach for the treatment of neuroinflammatory or
neurodegenerative disorders such as MS or EAE.20
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Cl
Cl
N
O
5
N
8a
N
1
( )n
H3CO2C
1a
1b
n = 0: (4aR,5S,8aS)-
n = 1: (4aR,5S,8aS)-
Figure 1: KOR agonists (4aR,5S,8aS)-1a,b with cis-configuration of the annulated ring
system and trans-orientation of the pyrrolidine ring.
Recently, we have reported that perhydroquinoxalines (4aR,5S,8aS)-1a,b (Figure 1)
represent potent and selective KOR agonists (Ki(1a) = 0.25 nM, EC50 = 2.0 nM, (Ki(1b) =
0.70 nM). The cis-configuration of the annulated rings and the relative trans-orientation
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of the pyrrolidine ring are crucial for high KOR affinity and agonistic activity.31,32 In the
arachidonic acid-induced ear inflammation model, enantiomerically pure quinoxaline
(4aR,5S,8aS)-1a and some analogous KOR agonists showed anti-inflammatory activity
comparable to those of indomethacin or hydrocortisone.33
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With the aim to contribute to potential further clinical development towards novel MS
therapeutics, a further improvement of quinoxaline-based KOR agonists of type 1 was
envisaged. In particular, the polarity of the ligands should be increased by introduction of
a second basic amino moiety (4-position) or a polar triazole ring within the substituent in
4-position. Moreover, the interaction with KOR, the anti-inflammatory effect on primary
mouse and human immune cells as well as the in vivo efficacy of the new KOR agonists
in EAE were investigated.
2. Result and discussion
2.1. Synthesis
In order to get fast access to perhydroquinoxaline derivatives with various substituents in
1-position (Figure 1) the strategy of late-stage diversification was pursued. For this
purpose the secondary amine 12 was selected as building block, which allows the
introduction of diverse substituents in 1-position. (Scheme 1)
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OTBDPS
HO
O
N
N
N
N
N
(d)
O
(c)
(a)
(b)
N
N
N
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O
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S
OH
OTBDPS
OTBDPS
O
H
N
H
N
H
N
4
N
(e)
(f)
(g)
N
N
N
N
H
1
Boc
Boc
Boc
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Cl
Cl
Cl
Cl
N
N
N
O
O
H
N
(j)
(h)
(i)
N
N
N
N
N
H
Boc
Boc
12
11
10
Scheme 1: Synthesis of central building block 12.
Reagents and conditions: (a) m-CPBA, CH2Cl2, rt, 22 h, 89 %; (b) 1: acetic anhydride,
120 °C, 5 h; 2: aq. NaOH 4 M, CH3OH, rt, 12 h, 71 %; (c) TBDPSCl, imidazole, 4-DMAP,
CH2Cl2, rt, 16 h, 98 %; (d) H2 (5 bar), PtO2, CH3OH, AcOH, rt, 5 h, 88 %; (e) Boc2O,
CH2Cl2, rt, 18 h, 87 %; (f) TBAF, THF, rt, 4 h, 73 %; (g) 1: SOCl2, imidazole, CH2Cl2, -78
°C, 17 h; 2: NaIO4, RuCl3.H2O, CH3CN/EtOAc 6:1, 0 °C, 12 h, 88 %; (h) pyrrolidine,
CH3CN, 80 °C, 16 h, 72 %; (i) 2-(3,4-dichlorophenyl)acetyl chloride, CH2Cl2, 0 °C, 2-5 h,
84 %; (j) CF3CO2H, CH2Cl2, rt, 16 h, 98 %.
Only one enantiomer of the racemic mixtures is shown.
In order to obtain higher yields and adapt the conditions to our research laboratory, the
originally reported industrial synthesis of compounds 132,33 was modified. In Scheme 1
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the modified synthesis starting with tetrahydroquinoxaline (2) is displayed. At first, the N-
oxide 3 was prepared by oxidation of quinoxaline 2 with m-CPBA. The subsequent
Boekelheide rearrangement34 of N-oxide 3 was performed with Ac2O instead of
trifluoroacetic anhydride which gave higher yields of benzylic alcohol 4 after hydrolysis
with NaOH and methanol. The hydroxy moiety of 4 was then protected by a silyl group.
Instead of the tert-butyldimethylsilyl protective group33 the tert-butyldiphenylsilyl
(TBDPS)35 protective group was chosen for four main reasons: (1) the very large
TBDPSO group should direct the hydrogenation from the opposite site leading to cis,cis-
configured perhydroquinoxaline 6 with high diastereoselectivity; (2) the large TBDPSO
group should shield the adjacent NH-group of product 6 in order to achieve high
regioselectivity during the Boc-protection; (3) the TBDPSO-group should be stable under
the harsh conditions during hydrogenation, which requires a pressure of 5 bar H2 and the
addition of HOAc to the methanolic solution; (4) The UV-absorption of the phenyl rings
allows an easy detection of educts and products by TLC and HPLC.
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As expected, hydrogenation (H2, 5 bar, PtO2, CH3OH, HOAc) of the TBDPS-protected
tetrahydroquinoxalinol 5 led exclusively to the cis,cis-configured perhydroquinoxaline 6 in
88 % yield. This transformation represents the key step of the overall diastereoselective
synthesis, since only one diastereomer (cis,cis) of four theoretically possible
diastereomers was formed by the backside attack of H2 at the aromatic pyrazine ring.
Next, perhydroquinoxaline 6 was reacted with (Boc)2O to afford the mono-Boc-protected
quinoxaline 7. The large TBDPSO group inhibits the acylation of the NH moiety in 4-
position resulting in exclusive formation of 7. After having fulfilled its task, the TBDPS-
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protective group of 7 was removed with tetrabutylamonium fluoride (TBAF) to provide the
β-amino alcohol 8.
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According to literature, -amino alcohol 8 was converted into the cyclic sulfuric acid ester
amide 9 using SO2Cl2. In our hands a two-step procedure36 involving the reaction of -
amino alcohol 8 with SOCl2 and subsequent oxidation with NaIO4 and RuCl3 gave higher
yields (88 %) of the desired 1,2,3-oxathiazolidine 9. It has to be noted that the NMR
spectra of the intermediate after reaction of 8 with SOCl2 is rather complex, since the S-
atom represents an additional center of chirality. After oxidation, clear signals in the NMR
spectra confirm the structure of the cyclic sulfuric acid derivative 9. The cyclic sulfuric acid
derivative fulfills two tasks. First of all, it protects the secondary amino moiety in 4-
position. Secondly, it transformed the OH moiety in 5-position into a good leaving group
suitable for SN2 substitution.
The nucleophilic substitution of 9 with pyrrolidine was the second key step in the synthetic
route. The transformation proceeded as pure SN2 reaction with inversion of configuration
at C-5 affording the desired cis,trans configuration in quinoxaline 10.33 After acylation of
the free secondary amine 10 with 2-(3,4-dichlorophenyl)acetyl chloride (11), the Boc
protective group was cleaved off using trifluoroacetic acid in order to isolate the secondary
amine 12 as promising building block in 98 % yield.
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Cl
Cl
Cl
Cl
6
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8
9
N
O
N
O
(a) or (b)
(e)
N
N
N
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N
H
4
(f)
CO2CH3
12
1
rac-
(c)
Cl
Cl
Cl
Cl
Cl
Cl
Cl
Cl
N
O
O
N
O
O
N
O
(g)
N
O
N
N
N
N
N
N
N
N
R
O
Cl
F
13a-g
13h
(d)
15
(h)
16
13
Cl
R
Cl
Cl
a
b
c
d
e
f
CH3
Cl
Cl
CH2CH3
Cl
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2F
CH2CH2CH2CH2F
4-F-benzyl
N
O
N
O
N
N
N
O
O
(i)
N
N
N
N
g
O
N
N
N
N
N
N3
N
F
F
14
17
18
Scheme 2: Late stage diversification of building block secondary amine 12.
Reagents and conditions: (a) RCH=O, NaBH3CN, CH3CO2H, CH3OH, rt, 16 h, 54 % (13a),
69 % (13b), 66 % (13c), 85 % (13d), 42 % (13g). (b) RCH=O, NaBH(OAc)3, CH2Cl2, 0
°C, 17 h, 65 % (13e), 51 % (13f); (c) propargyl bromide, Cs2CO3, DMF, 40 °C, 16 h, 92
%; (d) 2-fluoroethyl azide, CuSO4, sodium ascorbate, DMF/H2O, rt, 4 h, 35 %; (e) methyl
chloroformate, pyridine, CH2Cl2, 0 °C, 1 h, 98 %; (f) 2-chloroacetyl chloride, Et3N, CH2Cl2,
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0 °C, 5 h, 73 %; (g) 2-fluoroethanol, NaH, THF, 0 °C, 40 h, 39 %; (h) NaN3, DMF, rt, 20
h, 91 %; (i) 5-fluoropent-1-yne, CuSO4, sodium ascorbate, DMF/H2O, rt, 3 h, 17 %.
Only one enantiomer of the racemic mixtures is shown.
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Scheme 2 shows the formation of diverse test compounds using the common building
block 12. Quinoxaline derivatives 13a-g bearing various alkyl side chains in 4-position
were obtained by reductive alkylation of secondary amine 12 with various aldehydes and
NaBH3CN or NaBH(OAc)3. Since alkylation of 12 with fluoroalkyl halides failed to give
13e and 13f, fluoroacetaldehyde and 4-fluorobutyraldehyde were prepared in situ by
oxidation of the corresponding alcohols with Dess-Martin-Periodinane and the crude
aldehydes were directly used in the reductive alkylation reaction.31,37 The fluorobenzyl
derivative 13g was synthesized to investigate the effect of a large and more lipophilic
fluorobenzyl moiety on the interaction with KOR.
The propargyl derivative 13h was obtained by alkylation of secondary amine 12 with
propargyl bromide. Cu+-catalyzed 1,3-dipolar cycloaddition38,39 of alkyne 13h with 2-
fluoroethyl azide provided the triazole derivative 14. The triazole moiety led to increased
size and polarity of the side chain. The increased polarity is of particular importance, since
we are interested in KOR agonists which are not able to pass the blood-brain-barrier.
Acylated quinoxalines 1 and 15 were obtained by acylation of secondary amine 12 with
methyl chloroformate and 2-chloroacetyl chloride, respectively. The racemic methyl
carbamate 1 was prepared for comparison of its KOR affinity and activity with those of
the newly synthesized KOR ligands. Reaction of the chloroacetamide 15 with 2-
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fluoroethanol and NaH40 afforded the fluoroethyl ether 16 in 80 % yield. Nucleophilic
substitution of 15 with NaN3 led to the azide 17, which underwent a 1,3-dipolar
cycloaddition (click reaction)38,41 with 5-fluoropent-1-yne to produce fluoropropyltriazole
18.
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2.2. Pharmacological evaluation
KOR affinity
The KOR affinity of the synthesized quinoxaline derivatives and some reference
compounds was investigated in competitive radioligand binding assays using guinea pig
brain homogenates as source of KOR and [3H]U-69,593 as competitive radioligand. The
data in Table 1 summarize the KOR affinity of the test compounds.42–44
Table 1: Affinities of perhydroquinoxalines and reference compounds towards KOR and
related receptors.
Cl
Cl
N
O
1
4
N
N
R
Ki ± SEM [nM] a) b)
compd.
R
KOR
MOR
DOR
1
2
[3H]U-69,593 [3H]DAMGO [3H]DPDPE [3H]-(+)-pentazocine [3H]DTG
rac-1a
CO2CH3
1.3 ± 0.4 0 % 250 0 % 8 %
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CO2C(CH3)3
H
3.8 ± 1.2
260
28 %
0 %
438
205
12 %
0 %
16 %
5 %
0.54 ± 0.15
2.3 ± 1.3
4.2 ± 1.7
1.4 ± 0.2
6.6 ± 3.1
2.2. ± 0.2
8.0 ± 3.0
81 ± 21
13a
13b
13c
13d
13e
13f
13g
14
CH3
333
0 %
4 %
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13
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CH2CH3
3300
407
1100
992
11 %
1800
876
16 %
2400
1300
3 %
CH2CH2CH3
CH2CH2CH2CH3
CH2CH2F
2600
12 %
8 %
2500
5 %
12 %
0 %
CH2CH2CH2CH2
F
17 %
397
8 %
4-F-Bn
0 %
1600
2 %
0 %
CH2-triazole-
CH2CH2F
5.6 ± 0.6
15 ± 3
573
413
0 %
C(=O)CH2OCH2
CH2F
16
0 %
1100
13 %
0 %
C(=O)CH2-
triazole-
CH2CH2CH2F
18
3.3 ± 1.3
947
425
0 %
0 %
U-69,593
naloxone
0.97 ± 0.40
-
-
-
-
7.3 ± 0.40
2.3 ± 1.1
103
-
-
morphine
-
-
-
-
5.2 ± 1.6
-
-
-
SNC80
-
-
-
1.2 ± 0.5
-
-
(+)-pentazocine
haloperidol
-
-
5.4 ± 0.5
6.6 ± 0.9
-
78 ± 2.3
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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).
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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.
With exception of the 4-fluorobenzyl derivative 13g and the fluoroethoxy derivative 16 the
Ki values reflecting KOR affinity of all new test compounds are lower than 10 nM indicating
very high KOR affinity. The length of the side chain of the N-4 substituent seems not to
play a crucial role for the interaction with KOR, as the quinoxalines 13a-13d with
homologous alkyl substituents display almost the same KOR affinity. Even the
quinoxaline 11 with the large and bulky tert-butoxycarbonyl moiety shows high KOR
affinity (Ki = 3.8 nM). It is postulated that the N-4-substituent is oriented towards the
entrance channel and the surface of KOR. This hypothesis is supported by the triazole
derivatives 14 and 18 showing KOR affinity of 5.6 nM and 3.3 nM, respectively.
Hydrophilic interactions of surrounding water molecules with the polar triazole rings within
the large N-4-substituents may explain the high KOR affinity of 14 and 18.
Moreover, KOR accepts both alkyl and acyl moieties at N-4. Although the basicity and the
geometry of differently substituted derivatives are quite diverse, the KOR affinity of pairs
with analogous alkyl and acyl moieties are quite similar. As an example, the KOR affinity
of 13c with a propyl moiety at N-4 is 1.4 nM and the KOR affinity of the methoxycarbonyl
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derivative 1 is 1.3 nM. This observation supports the hypothesis that the binding pocket
of KOR accepts substituents with different size at N-4. Furthermore, a second basic
amino group (N-4) in the ligand is also well accepted by KOR.
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Since a fluorinated PET tracer for selective labeling of KORs represents a long term aim
in our group, fluorine atoms were introduced at different positions. In general, high KOR
affinity was found for quinoxalines with a F-atom at the N-4 alkyl side chain (e.g. 13e, 13f)
or at the triazole side chain (14, 118).
Interactions of 14 with KOR (docking)
To rationalize the observed KOR affinity of triazole 14, the compound was docked into
the crystal structure of KOR in its active state (PDB: 6B7366). The obtained docking pose
of 14 (Figure 2) is consistent with previously reported docking modes of its
conformationally unrestricted analogues U50,488 and U69,593.66 Thus, protonated
tertiary amino moiety of the pyrrolidine ring of 14 forms an ionic interaction with Asp138,
whereas the O-atom of the carbonyl moiety forms an H-bond with Gln115. The pyrrolidine
ring is additionally involved in the lipophilic contacts with a number of amino acid residues
e.g. Met142, Tyr320, Trp287, and Asp138. The perhydroquinoxaline scaffold of 14 from
one side of the pocket forms hydrophobic contacts with Ile290, Ile294, and Trp287, and
from the other side with Tyr139. The dichlorophenyl moiety of 14 occupies a hydrophobic
site of the pocket undergoing lipophilic interactions with Leu135 and Gln115. Finally, the
triazole ring of 14 forms nonpolar interactions with Lys227 and Tyr139 pointing fluoroethyl
moiety toward the extracellular region of the receptor.
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Figure 2: Binding mode (docking pose) of compound 14 (orange stick model) in the active
state of KOR (PDB: 6B7366). Amino acid residues are depicted as blue stick models.
Oxygen, nitrogen, sulfur, fluorine, and chlorine atoms are colored in red, blue, yellow, light
green, and green, respectively. Hydrogen bonds are depicted as dashed lines (black).
Selectivity over MOR, DOR and  receptors
In order to analyze the selectivity of the potent KOR agonists, competitive radioligand
binding studies were performed to determine MOR, DOR, 1 and 2 affinity of the ligands.
In the MOR and DOR assay, receptor preparations from rat brain and the radioligands
[3H]DAMGO and [3H]DPDPE were employed, respectively. 1 and 2 receptor affinity
were recorded using guinea pig brain and rat liver membrane preparations and [3H](+)-
pentazocine and [3H]di-o-tolylguanidine as radioligands, respectively.64,65 At first
quinoxalines were tested at a concentration of 1 µM. The complete competition curves
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were recorded only when the binding of the radioligand was reduced by more than 50 %.
Otherwise, the inhibition of radioligand binding (in %) at a test compound concentration
of 1 µM is given in Table 1.
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The quinoxaline derivatives show very low affinity towards MOR and DOR (Ki > 200 nM)
resulting in high KOR:MOR and KOR:DOR selectivity. (Table 1) Extraordinarily high
selectivity was recorded for quinoxalines with particular relevance for this project, i.e.
secondary amine 12 (KOR:MOR > 1800, KOR:DOR = 380), fluoroethyl derivative 13e
(KOR:MOR > 4500, KOR:DOR > 450), fluoroethyltriazole 14 (KOR:MOR = 100,
KOR:DOR = 74) and fluoropropyltriazole 18 (KOR:MOR > 285, KOR:DOR = 130).
It has been reported that small modifications of KOR agonists led to high σ receptor
affinity. For example, the change of the trans-configured prototypical KOR agonist U-
50,488, to its cis-configured analog generated a ligand with high σ receptor affinity.45
Moreover, reduction of the phenylacetamide moiety of monoacylated ethylenediamine
KOR agonists to a phenylethylamino group further increased the σ affinity and decreased
the KOR affinity.46 For this reason, σ1 and σ2 affinity of the novel KOR agonists were also
recorded. (Table 1) The data in Table 1 clearly demonstrate that the quinoxaline-based
KOR agonists do not substantially interact with σ1 and σ2 receptors. The highest σ1 and
σ2 affinities were found for the butyl derivative 13d (Ki(1) = 876 nM, Ki(2) = 1300 nM).
Nevertheless, the KOR:1 and KOR:2 selectivity of 13d is 135 and 230, respectively.
The extraordinarily high KOR:1 and KOR:2 selectivity of the important quinoxalines 12,
13e, 14 and 18 of greater than 180 should be emphasized here.
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In conclusion, the newly synthesized KOR agonists show high selectivity against MOR,
5
6
DOR, σ1 and σ2 receptors.
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Functional activity
In order to further explore the pharmacological potential of quinoxaline-based KOR
agonists, the KOR affinity of the most promising ligands was investigated in a second
binding assay. In this assay, the human KOR was transiently expressed in HEK 293T
cells and [3H]diprenorphine was used as radioligand.47 Table 2 correlates the affinity data
obtained with human and guinea pig KORs.
Table 2: Activity of selected KOR agonists correlated with KOR affinity.
Cl
Cl
N
O
N
N
R
rac.
Ki ± SEM [nM]
cAMPb)
β-arrestin-2c)
KORa)
KORb)
compd.
R
[3H]U-
69,593
[3H]diprenor EC50 ± SEM Emax± SEM EC50 ± SEM Emax± SEM
d)
d)
-phine
[nM]
%
[nM]
%
11
12
CO2C(CH3)3 3.8 ± 1.2
18 ± 1.2
8.1 ± 0.8
94 ± 2
103 ± 2
102 ± 2
103 ± 2
102 ± 1
42 ± 1.2
102 ± 4
93 ± 1
H
0.54 ± 0.15 4.8 ± 0.9
1.2 ± 0.4
3.4 ± 0.5
2.0 ± 0.4
1.6 ± 0.3
3.8 ± 0.8
17 ± 0.7
12 ± 0.6
7.9 ± 0.9
13a
13b
13c
CH3
2.3 ± 1.3
4.2 ± 1.7
11 ± 1.0
11 ± 0.5
9.2 ± 0.8
101 ± 2
99 ± 3
CH2CH3
CH2CH2CH3 1.4 ± 0.2
108 ± 4
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CH2CH2CH2
CH3
13d
13g
6.6 ± 3.1
81 ± 21
16 ± 1.3
42 ± 2.6
6.0 ± 0.8
32 ± 1.8
102 ± 3
97 ± 3
33 ± 1.3
106 ± 5
105 ± 5
4-F-Bn
247 ± 4.2
CH2-
triazole-
CH2CH2F
C(=O)CH2O
CH2CH2F
14
5.6 ± 0.6
46 ± 2.1
2.8 ± 0.2
103 ± 1
97 ± 2
43 ± 1.4
86 ± 3
16
15 ± 3
77 ± 4.7
-
10 ± 0.6
227 ± 3.8
103 ± 4
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U-69,593
0.97 ± 0.40
-
-
-
-
Diprenor
phine
salvinorin
A
0.69 ± 0.08
-
-
0.015
5.1
a)
Guinea pig brain membrane preparations.
Human HEK 293T cells.
b)
c)
d)
Human HTLA cells.
The Emax values refer to salvinorin A (100 %).
With exception of the fluorobenzyl derivative 13g, the Ki values obtained at human KORs
(transfected HEK-293T cells) are generally 3-9-fold higher than the Ki values recorded
with guinea pig brain preparations. This is due to the different properties of the
radioligands used in the binding assays. While [3H]U69,593 at 1 nM labels only the high-
affinity sites and thus Kobserved is the Khigh, [3H]diprenorphine at 1.5 nM could label both
high- and low-affinity sites, so Kobserved is the average of both. This is also supported by
the same tendencies observed in both assay systems, e.g. the tertiary amines 13a and
13b have Ki values of 11 and 11 nM at the human KOR and Ki values of 2.3 and 4.2 nM
at the guinea pig KOR.
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In order to detect potency and efficacy in KOR-mediated signaling pathways, the
functional activity of the quinoxaline-based KOR agonists was recorded in a cAMP
inhibition assay and a Tango β-arrestin-2 recruitment assay.67,68
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In Table 2 the EC50-values of the quinoxaline-based KOR agonists are displayed and
compared with the EC50-values of reference compound salvinorin A. The quinoxaline
analogs are all full agonists at KOR. The tested compounds show remarkably high
activation of the Gi-dependent pathway with EC50 values below 10 nM. As expected from
the low KOR affinity, the fluorobenzyl derivative 13g has rather low agonistic activity (EC50
= 32 nM). Regarding the β-arrestin-2 recruitment, most of the tested quinoxalines show
slightly lower β-arrestin-2 activation than salvinorin A.
The very high KOR activation of the secondary amine 12 with EC50-values of 1.2 nM and
4.8 nM in the cAMP and β-arrestin-2 recruitment assay, respectively, should be
emphasized. In addition to 12, the fluoroethyltriazole 14 shows also high KOR affinity and
KOR agonistic activity in the cAMP assay. The calculated clogP values for 12 (clogP =
3.70) and 14 (clogP = 3.80) are in the same ragne as the clogP value of 1b (clogP = 3.81).
As 1b did not show any central effects in vivo,33 low CNS penetration of 12 and 14 was
expected.
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Anti-inflammatory activity of the quinoxaline-based KOR agonists in vitro
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8
Several studies revealed that β-arrestins are functionally involved in inflammation and
autoimmunity. Accordingly, mice deficient for β-arrestin-2 showed exacerbated symptoms
of EAE, which. was mediated by decreased numbers of Foxp3+ regulatory T cells, a cell
subset, which usually controls the activation and expansion of pathogenic TH1 or TH17
effector cells .48 Also cAMP has been associated with EAE perpetuation. In addition to a
direct impact of the cAMP pathway on CD4+ T cells by inhibiting the proliferation and
IFN- secretion in encephalitogenic T cells, stimulation of the cAMP pathway polarized
macrophages towards an M2-like phenotype, leading to indirect inhibition of effector T
cell activation.49 Based on these observations and since we already identified
decahydroquinoxaline KOR agonists with a strong anti-inflammatory potential,33 we
intended to characterize the effect of the secondary amine 12 and the fluoroethyltriazole
derivative 14 on immune cells involved in the perpetuation of neuroinflammation and to
assess the therapeutic potential of both compounds in EAE.
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Therefore, we first investigated the anti-inflammatory properties of compounds 12 and 14
on T cells and dendritic cells (DC), which were purified from peripheral blood of healthy
human donors or secondary lymphatic organs of mice. The cells were stimulated with
phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin (PMA/Iono) to up-
regulate the expression of typical activation markers and the secretion of pro-
inflammatory cytokines. In MS as well as in EAE naive T cells differentiate into effector
cells in the peripheral immune system by direct contact to mature DC that express co-
stimulatory receptors such as CD40, CD80 or CD86 and secrete pro-inflammatory
cytokines including IFN-, IL-12, IL-23 or IL-6.50,51 These effector T cells are able to cross
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the blood-brain barrier (BBB), migrate into the CNS and promote demyelination, axonal
damage and finally, the formation of inflammatory foci.50 MS and EAE have long been
considered as T cell-mediated diseases and it has been shown that particularly TH1 and
TH17 cells secreting INF- and IL-17 are markedly increased in cerebrospinal fluid (CSF)
from MS patients and in CNS tissue from EAE mice.51–54
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Figure 3: Fluoroethyltriazole 14 significantly reduced the expression of pro-inflammatory
cytokines in human and mouse immune cells via binding to KOR. (A, B) DC (A) and T
cells (B) were sorted from human peripheral blood mononuclear cells (PBMC) by
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magnetic beads and activated with PMA + ionomycin (PMA/Iono) for 12 h as described.
Subsequently, cells were stimulated with compounds 12 or 14 (5 µg/mL each) for
additional 48 h. Control cells received an equal amount of PBS. Percentages of IFN-+
cells in total DC (A) or total T cells (B) from n=3 healthy donors are shown. Cells were
gated for HLA-DR+CD11c+ (A) or CD3+ (B) and IFN- staining was performed after cell
permeabilization. Data are presented as means ± SEM; *, p < 0.05 vs. PBS stimulation.
(C) Immunomodulatory effects of compound 14 as assessed by the down-regulation of
pro- and the up-regulation of anti-inflammatory cytokines. Cytokine quantification in cell
culture supernatants from PBMC of healthy human donors after activation with PMA/Iono
and stimulation with compounds 12 or 14. Data from n=3 donors are shown (*p < 0.05 vs.
PBS). (D) Percentages of IFN-+ cells in total bone-marrow-derived DC (bmDC) from n=5
wild-type (WT) or KOR deficient (Oprk–/–) mice. Cells were gated on MHC-II+CD11c+ and
IFN- staining was performed after cell permeabilization. Data are presented as means ±
SEM; *, p < 0.05 vs. PBS. (E, F) Flow cytometry of T cells from WT or Oprk–/– mice after
activation with PMA/Iono and stimulation with compounds 12 or 14. Representative FACS
plots (E) and statistics from n=5 mice per group (F) are shown. Cells are gated on CD3;
data are presented as means ± SEM; *, p < 0.05 vs. PBS (G) Cytokine secretion in
isolated T cells from WT or Oprk–/– mice after activation with PMA/Iono and stimulation
with compounds 12 or 14. Data from n=5 mice per group are shown; *p < 0.05 vs. PBS.
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To assess the anti-inflammatory capacity of the secondary amine 12 and the
fluoroethyltriazole derivative 14, pre-activated T cells and DC were stimulated with both
compounds. As shown in Figures 3A and 3B compound 14 significantly reduced the
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expression of IFN- in human T cells and DC as evidenced by intracellular flow cytometry
whereas compound 12 seemed to have only a minor but not significant anti-inflammatory
effect on human immune cells (Figures 3A and 3B).
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Interestingly, the fluoroethyltriazole 14 might not only have anti-inflammatory properties
but also immunomodulatory capacities since besides the down-regulated IFN  levels in
cell culture supernatants from human PBMC stimulated with compound 14, we detected
a significant up-regulation of the anti-inflammatory cytokine IL-10 (Figure 3C). This
observation could possibly point to the induction of tolerogenic DC or regulatory T cells
by the fluoroethyltriazole 14. It is well known that opioid receptor agonists are able to
modulate immune cell functions. In this context endogenous opioids have been shown to
inhibit NF-B activation in T cells, to induce IL-10 in DC or to prevent DC maturation and
thus, reduce their TH1 priming capacity.55–57
DC are central in the regulation of immune responses during autoimmune
neuroinflammation since they control the balance between the initiation of immune
activation via inducing effector T cells, and immune tolerance via expanding regulatory T
cells.58 In particular IL-10 producing tolerogenic DC, which can be generated by blocking
NF-B signaling, are critically involved in the reprogramming of immune responses during
autoimmunity.59,60 Hence, it might be conceivable that the fluoroethyltriazole 14, by
inducing IL-10 as well as by inhibiting NF-B activation and the secretion of pro-
inflammatory cytokines, could have modulated the immune phenotype. This hypothesis
is strengthened by the observation that immunomodulatory properties have already been
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described for other kappa opioids. Hu et al. for instance, summarized the
immunomodulatory and neuroprotective capacities of KOR ligands in the context of
human immunodeficiency virus (HIV) infection.61
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Next, we assessed whether the anti-inflammatory and immunomodulatory effects of
compound 14 were indeed mediated by binding to KOR or whether off-target effects might
be detectable. For this purpose, we purified DC and T cells from wild-type mice as well
as KOR deficient mouse mutants (Oprk–/–), activated the cells with PMA/Iono, stimulated
them with the secondary amine 12 or the fluoroethyltriazole derivative 14 and quantified
cell activation as well as cytokine secretion. As expected, compound 14 significantly
down-regualted the IFN- secretion in wild-type DC and suppressed the activation of wild-
type T cells (Fig. 3D-3F). However, the fluoroethyltriazole derivative 14 had no effect on
the cytokine secretion or expression of activation markers in PMA/Iono stimulated DC or
T cells from Oprk–/– mice (Figures 3D-3F) demonstrating the specificity of compound 14
for KOR. Worth mentioning, similar to human immune cells (Figure 3C) the
fluoroethyltriazole 14 showed immunomodulatory properties in wild-type cells since it
down-regulated the IFN- levels in primary mouse T cells and simultaneously up-
regulated the IL-10 expression (Figure 3G). The immunomodulatory capacity of
compound 14 was not an off-target effect but dependent on binding to KOR because we
did not observe any impact of compound 14 on the cytokine secretion in activated T cells
from Oprk–/– mice (Figure 3G).
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The fluoroethyltriazole derivative 14 and the secondary amine 12 ameliorated EAE in
mice
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To characterize the impact of the fluoroethyltriazole 14 and the secondary amine 12 on
the development and perpetuation of inflammatory neurodegeneration in vivo and to
further assess the therapeutic potential of both compounds, we immunized wild-type mice
with myelin oligodendrocyte glycoprotein (MOG) peptide and systemically treated them
with compound 12 or 14. The test compounds 12 and 14 were intraperitoneally injected.
In contrast to our in vitro observations made in primary mouse immune cells (Figures 3D-
3G), the secondary amine 12 delayed disease onset and moreover, reduced EAE severity
(Figure 4A). However, the anti-inflammatory effect of the fluoroethyltriazole derivative 14
was even more pronounced since contrary to mice treated with compound 12, animals
that received injections of compound 14 only developed partial tail paralysis (clinical score
1.5-2) and did not show any kind of hind limb paralysis (starting at clinical score 3-4). The
beneficial in vivo effects of both KOR agonists were mediated by signaling through KOR
because treatment of MOG-immunized Oprk–/– mice with either of the two compounds did
not modulate EAE perpetuation (Figure 4B).
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Figure 4: Compounds 12 and 14 reduced severity EAE by down-regulating effector T cell
activation. (A, B) Wild-type (WT) mice (A; n=9 per group) and KOR deficient mutants
(Oprk–/–; B; n=7 per group) were immunized with MOG peptide and treated daily with
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compounds 12, 14 (i.p. injection of 100 µl of a 20 µM solution once a day) or PBS starting
at day 1 after immunization. Mean clinical scores are shown and data are presented as
means ± SEM; *, p < 0.05 (day 9-16 after immunization) and ***, p < 0.01 vs. PBS-treated
controls (day 9-18 after immunization). (C) Representative hematoxylin and eosin (H&E)
staining in lumbar spinal cord at disease maximum (scale bar, 1 mm). Inflammatory foci
and the infiltration of mononuclear cells are indicated by black arrows. (D, E) Flow
cytometry analyses of total CD45+ leukocytes in the CNS at disease maximum.
Representative FACS plots (D) and statistics from n=5 mice per group (E) are shown.
Data are presented as means ± SEM; *, p < 0.05 vs. PBS-treated controls; FSC, forward
scatter. (F, G) Immunofluorescence staining (F) and flow cytometry (G) of CD4+IL-17+
effector T cells (TH17) in the CNS at disease maximum. Representative
immunofluorescence staining of lumbar spinal cord tissue using antibodies against CD4
(green) and IL-17 (red) are shown; scale bar, 20 µm; TH17 cells are indicaed by white
arrows (F). Statistical evaluation of TH17 cells in the CNS from n=5 mice per group; data
are presented as means ± SEM; *, p < 0.05 vs. PBS-treated controls; cells are gated on
CD45+CD4+ and IL-17 staining was performed after cell permeabilization (G). (H) Flow
cytometry of CD4+Foxp3+ regulatory T cells (Treg) in the CNS at disease maximum.
Statistics of Treg numbers from n=5 mice per group are shown, cells are gated on
CD45+CD4+ and Foxp3 staining was performed after cell permeabilization. Data are
presented as means ± SEM; *, p < 0.05 vs. PBS treatment.
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The anti-inflammatory properties of the secondary amine 12 and the fluoroethyltriazole
14 were associated with reduced inflammatory foci and immune cell infiltration into the
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