Novel and selective TLR7 antagonists among the imidazo[1,2- a]pyrazines, Imidazo[1,5- a]quinoxalines, and Pyrazolo[1,5- a]quinoxalines Series

Article abstract of DOI:10.1021/acs.jmedchem.9b00411

The Toll-like receptors (TLRs) 7 and 8 play an important role in the immune system activation, and their agonists may therefore serve as promising candidate vaccine adjuvants. However, the chronic immune activation by excessive TLR stimulation is a hallmark of several clinically important infectious and autoimmune diseases, which warrants the search for TLR antagonists. In this study, we have synthesized and characterized a variety of compounds belonging to three heterocyclic chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline, and pyrazolo[1,5-a]quinoxaline. These compounds have been tested for their TLR7 or TLR8 agonistic and antagonistic activities. Several of them are shown to be selective TLR7 antagonists without any TLR7 or TLR8 agonistic activity. The selectivity was confirmed by a comparative ligand-docking study in TLR7 antagonist pocket. Two compounds of the pyrazolo[1,5-a]quinoxaline series (10a and 10b) are potent selective TLR7 antagonists and may be considered as promising starting points for the development of new therapeutic agents.

Full text of DOI:10.1021/acs.jmedchem.9b00411

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Article
Novel and selective TLR7 antagonists among the imidazo[1,2-a]pyrazines,
imidazo[1,5-a]quinoxalines and pyrazolo[1,5-a]quinoxalines series
Nour Bou Karroum, Georges Moarbess, Jean-françois Guichou, Pierre
Antoine Bonnet, Cindy Patinote, Hasnaa Bouharoun-Tayoun, Soulaima
Chamat, Pierre Cuq, Mona Diab-Assaf, Issam Kassab, and Carine Masquefa
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 08 Jul 2019
Downloaded from http://pubs.acs.org on July 8, 2019
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Novel and selective TLR7 antagonists among the
Imidazo[1,2-a]pyrazines, Imidazo[1,5-
a]quinoxalines and Pyrazolo[1,5-a]quinoxalines
series
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Nour Bou Karroum , Georges Moarbess , Jean-François Guichou , Pierre-Antoine
1*
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Bonnet , Cindy Patinote , Hasnaa Bouharoun-Tayoun , Soulaima Chamat , Pierre
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Cuq , Mona Diab-Assaf , Issam Kassab , Carine Deleuze-Masquefa .
1. Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 F16, CNRS,
Université de Montpellier, Faculté de Pharmacie, 15 avenue Charles Flahault, BP
14491, 34093 Montpellier Cedex 5, France.
2. Tumorigenèse et Pharmacologie Antitumorale, Lebanese University, EDST, BP
90656, 1202 Fanar Jdeideh, Lebanon.
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. CNRS, UMR5048, INSERM, U105, Université de Montpellier, Centre de Biochimie
Structurale, F-34090 Montpellier, France.
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. Laboratory of Immunology and Vector-Borne diseases, Faculty of Public Health,
Lebanese University, 1202 Fanar Jdeideh, Lebanon.
5. Faculty of Medical Sciences, Lebanese University, 1500 Hadath, Lebanon.
KEYWORDS: Toll-like receptor 7 (TLR7), imidazo[1,2-a]pyrazine, imidazo[1,5-
a]quinoxaline, pyrazolo[1,5-a]quinoxaline, TLR7 antagonist, autoimmune diseases,
cancer.
ABSTRACT: The Toll-like receptors (TLRs) 7 and 8 play an important role in the
immune system activation, and their agonists may therefore serve as promising
candidate vaccine adjuvants. However, the chronic immune activation by excessive
TLR stimulation is a hallmark of several clinically important infectious and autoimmune
diseases, which warrants the search for TLR antagonists. In this study, we have
synthesized and characterized a variety of compounds belonging to three heterocyclic
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chemical series : imidazo[1,2-a]pyrazine, imidazo[1,5-a]quinoxaline and pyrazolo[1,5-
a]quinoxaline. These compounds have been tested for their TLR7 or TLR8 agonistic
and antagonistic activities. Several of them are shown to be selective TLR7
antagonists without any TLR7 or TLR8 agonistic activity. The selectivity was confirmed
by a comparative ligand-docking study in TLR7 antagonist pocket. Two compounds of
the pyrazolo[1,5-a]quinoxaline series (10a and 10b) are potent selective TLR7
antagonists and may be considered as promising starting points for the development
of new therapeutic agents.
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INTRODUCTION
Innate immunity, also called natural or non-specific immunity, is the first line of
defense against pathogens, recognizing highly conserved pathogen-associated
molecular patterns (PAMPs) found in invading organisms. It also plays a major role in
elimination of infected, stressed or dying cells, by recognizing damage-associated
molecular patterns (DAMPs). PAMPs and DAMPs are recognized by pattern
recognition receptors (PRRs) mainly expressed by the innate immunity cells 1,2.
Several classes of PRRs can be distinguished in mammals including Toll-like
receptors (TLRs), RIG-I-like receptors (RLRs), Nod-like receptors (NLRs), C-type
lectin receptors (CLRs) and intracellular DNA sensors such as cyclic GMP-AMP
synthase (cGAS) and Absent In Melanoma 2-like (AIM2-like) receptors (ALRs) 3,4,5,6.
Toll-like receptors (TLRs) are a particularly important group of PRRs that exist in
humans and other mammals and are homologous to the Drosophila Toll gene product
7,8. Each TLR is composed of an extracellular domain with leucine-rich repeats (LRRs)
that is responsible for the recognition of PAMPs and/or DAMPs, a transmembrane
domain and a cytoplasmic Toll/IL-1 receptor (TIR) domain that initiates downstream
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signaling 9,10. Upon ligand binding, TLRs recruit adaptor proteins, such as Myeloid
Differenciation Primary Response 88 (MyD88) and TIR-domain-containing adaptor-
inducing interferon- (TRIF), which trigger proinflammatory signaling cascades,
resulting in the activation of nuclear factor (NF)-B and the upregulation of
inflammatory cytokines and chemokines or in the production of type I interferons
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(
IFNs) . Ten different TLRs (TLR1 through 10) have been identified in humans. They
are mainly expressed in innate immune cells such as dendritic cells (DCs) and
macrophages, as well as in non-immune cells such as fibroblasts and epithelial cells
11,12.
TLRs are divided into two groups based on their cellular localization. Cell surface
TLRs include TLR1, TLR2, TLR4, TLR5, TLR6, and TLR10 13,14, whereas intracellular
TLRs are localized in the endosome membrane and include TLR3, TLR7, TLR8 and
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TLR9 . Cell surface TLRs recognize microbial membrane components such as lipids,
lipoproteins, and proteins 3,16,17,18. Intracellular TLRs recognize viral and endogenous
double-stranded RNA (dsRNA; TLR3) 19,20, singled-stranded RNA (ssRNA; TLR7/8)
21,22,
or unmethylated CpG sequences in DNA (TLR9) 23.
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Among the endosomal TLRs, TLR7 and TLR8 have a high degree of sequence
homology and function similarity 24,25,26,27. Both receptors recognize viral ssRNA, as
well as synthetic tricyclic imidazoquinoline derivatives such as imiquimod (a TLR7
agonist) and resiquimod (a TLR7/8 agonist) 26 (Figure 1A). Many reports in literature
have shown that TLR7/8 agonists belonging to different chemical series 28,29,30,31,32,33,34
strongly induce the production of T helper 1-polarizing cytokines and may therefore
serve as promising candidate adjuvants for viral vaccines, especially for the very
young and the elderly 35. Nevertheless, chronic immune activation is involved in
several inflammatory and autoimmune diseases. For example, since TLR7 and TLR8
can detect endogenous ssRNA derived from apoptotic cells, they may trigger the
development of autoimmune diseases such as Systemic Lupus Erythematosus (SLE)
36,37,38. Several studies have shown that TLR7 has also protumorigenic effects and is
highly expressed in pancreas, liver and lung cancer cells, where its stimulation induces
tumor progression and carcinogenesis and reduces the expression of several
antitumor molecules implicated in apoptosis. In addition, high TLR7 expression in
primary tumors was shown to confer resistance to chemotherapy in cancer patients
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39,40.
Consequently, the development of TLR7 antagonists may play an important role
in the therapy of these diseases. In 2013, a series of synthetic nucleotides has been
designed by Agrawal and coworkers as TLR7, 8 and 9 antagonists 41,42. They inhibit
TLR 7-, 8- and 9-mediated NF-kB activation and the production of a broad range of
pro-inflammatory cytokines, including tumour necrosis factor alpha (TNF-α),
interleukin (IL)-12, IL-6, interferon alpha (IFN-α), and IL-1β 42. In addition, these
oligonucleotides effectively inhibited inflammatory pathways involved in the
development of Systemic Lupus Erythematosus (SLE) and were presented as a
potential therapeutic approach for the treatment of SLE, psoriasis and other
autoimmune diseases 41,43. More recently, IMO-8400, one of these oligonucleotides,
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demonstrated clinical activity in patients with moderate to severe plaque psoriasis .
Besides these oligonucleotides, only very few small-molecule inhibitors of TLR7 and
TLR8 have been reported in the literature. In 2009, two imidazoquinolines were first
reported as TLR7 antagonists but unfortunately without any indication on their activity
45
on TLR8 . Further to this preliminary report, the same team later described a very
limited series of imidazoquinolines or dimeric imidazoquinolines as dual TLR7/TLR8
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antagonists (Figure 1B) 46,47. In 2015, Švajger et al. have identified new TLR7
antagonists through 3D ligand-based virtual screening. The most active compound
belongs to the chromeno[3,4-d]imidazole-4-one series, it exhibited a low micromolar
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IC50 (IC50 = 23 µM) . More recently, Zhang et al. have developed selective TLR8
antagonists belonging to the pyrazolo[1,5-a]pyrimidine and quinolines series (Figure
1B). In their study, they showed the existence of a new antagonist binding site distinct
from the described agonist site. Upon binding to this site, the small-molecule
antagonists stabilize the preformed TLR8 dimer in its resting state, preventing its
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activation .
In the present study, we have synthesized a wide variety of compounds belonging
to three chemical series, imidazo[1,2-a]pyrazine, pyrazolo[1,5-a]quinoxaline and
imidazo[1,5-a]quinoxaline, that share structural similarity with already reported TLR7/8
antagonists (Figure 1). These compounds have been tested for their TLR7 and/or
TLR8 agonistic and antagonistic activities using HEK-Blue™-hTLR7 and HEK-Blue™-
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hTLR8 cells. These genetically-engineered cell lines stably express the human TLR7
or TLR8 respectively. Modeling and ligand docking studies were also performed.
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A
O
N
N
N
NH2
N
N
N
NH2
Imiquimod (TLR7 agonist)
Resiquimod (TLR7/8 agonist)
B
HN
HN
C H
OH
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OH
N
N
N
N
N
N
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.
2CF COOH
. 2CF COOH
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3
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NH2
N
N
N
TLR7 antagonist
Dual TLR7/8 antagonist
TLR7 antagonist
Dual TLR7/8 antagonist
Bn
N
N
N
H CO
3
N
N
N
Bn
N
NH2
N
N
N
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N
O
N
NH2
NH2
O
O
OH
Dual TLR7/8 antagonist
Dual TLR7/8 antagonist
TLR8 antagonist
TLR8 antagonist
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Figure 1: Structures of representative TLR7 and TLR8 agonists (A) and antagonists
(B)
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RESULTS AND DISCUSSION
We have designed and synthesized new molecules that share structural similarity
with known TLR7 and TLR8 ligands. In the literature, several TLR7/8 ligands have a
NH group and an alkyl chain, preferably a butyl or pentyl chain, which are essential
2
for agonistic or antagonistic activity 31,35,45,46. So, we have synthesized a variety of
compounds belonging to three chemical series: imidazo[1,2-a]pyrazine, imidazo[1,5-
a]quinoxaline and pyrazolo[1,5-a]quinoxaline, with an NH group in position 8 for the
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imidazo[1,2-a]pyrazine (I) and in position 4 for the pyrazolo[1,5-a]quinoxaline (II) and
1
imidazo[1,5-a]quinoxaline (III), and an alkyl chain R on the pyrazole or imidazole ring
(Figure 2).
R1
R1
R1
N
N
N
N
N
N
N
III
NH2
N
II
NH2
N
I
NH2
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Figure 2: Chemical structures of the synthesized compounds.
Synthesis of imidazo[1,2-a]pyrazine derivatives
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Several synthetic pathways have been described in the literature in order to obtain
imidazo[1,2-a]pyrazine derivatives 50,51,52,53. We used the synthetic route depicted in
Scheme 1. The imidazo[1,2-a]pyrazine scaffold was built up from 2-amino-3-
chloropyrazine and in situ generated bromoacetaldehyde affording compound 1,
according to reference 49. Compound 1 was iodinated with N-iodosuccinimide (NIS)
to give compound 2. After amination of compound 2 under microwave assistance to
obtain 3, the Sonogashira cross-coupling reaction was used to introduce a variety of
alkyl chains in position 3 of the imidazole ring in the presence of copper and palladium
catalysts. The reduction of the alkyne function gave the final compounds 5a-f.
I
Br
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N
N
N
N
NH2
N
N
+
O
O
N
a
b
Cl
N
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Cl
N
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R1
I
R
N
N
N
N
N

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2
3
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Scheme 1. Synthesis of imidazo[1,2-a]pyrazines: (a) 48% aq. HBr solution, reflux, 1.5
h, NaHCO /IPA, then 2-amino-3-chloropyrazine, reflux, 3 h, 74%; (b) NIS, DMF, rt, 12
3
o
h, 65%; (c) 30% aq. NH OH solution, ACN, MW (140 C, 2h), 62%; (d) Alkynes,
4
o
ACN/Et N (2/1), Pd(PPh ) , CuI, 70 C, 12h, 25-75%; (e) H , Pd/C, EtOH, 50 psi, 24h,
3
3 4
2
25-60%.
Synthesis of pyrazolo[1,5-a]quinoxaline derivatives
Pyrazolo[1,5-a]quinoxaline derivatives were prepared according to the procedure
presented in Scheme 2. Dimers 6a and 6b resulted from the bimolecular condensation
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of the commercially available 5-substituted 1H-pyrazole-3-carboxylic acids in
presence of thionyl chloride. Product 6a was coupled with fluoroaniline derivatives to
give the intermediates 7a, 7c and 7d, whereas product 6b was coupled with ortho-
fluoroaniline to give the intermediate 7b. The tricyclic compounds 8a-d were obtained
via an intramolecular cyclization of 7a-d, under strong basic conditions. Treatment of
compounds 8a-d with phosphorus oxychloride and N,N-diethylaniline gave
compounds 9a-d. Displacement of the chlorine of 9a-d with aqueous ammonia
afforded the final compounds 10a-d.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
R2
R3
F
R2
NH2
R3
F
2
3
R = R = H
O
O
2
3
HOOC
R = CH3, R = H
N
NH
N
N
2
3
H
R1
R
1 R = H, R = CH
3
NH
N
N
a
N
O
N
c
b
R1
R1
1
6
a: R = butyl
1
2
3
7
7
7
7
a: R = butyl, R = R = H
1
6
b: R = isobutyl
1
2
3
b: R = isobutyl, R = R = H
1
2
3
c: R = butyl, R = CH , R = H
3
1
2
3
d: R = butyl, R = H, R = CH
3
R1
1
R1
R
R2
R2
R2
N
N
N
R3
N
R3
R3
N
N
d
N
Cl
e
N
NH2
N
H
a-d
O
8
9a-d
1
0a-d
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Scheme 2. Synthesis of pyrazolo[1,5-a]quinoxaline derivatives: (a) SOCl , reflux, 18
2
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
h, 25%, 43%; (b) NaHMDS, THF, 5 h, 60-82%; (c) NaH, DMA, reflux, 48 h; (d) POCl3,
o
o
DEA, MW (130 C, 15 min); (e) 30% aq. NH OH solution, ACN, MW (140 C, 2h), 25-
4
100%.
Synthesis of imidazo[1,5-a]quinoxaline derivatives
The imidazo[1,5-a]quinoxaline derivatives were prepared using a new synthetic
pathway (Scheme 3), in order to introduce a variety of alkyl chains in position 1 of the
imidazole ring. After the mono protection of the amino group of the 2-iodo-1H-
imidazole reagent, Sonogashira cross couplings allowed us to obtain the alkylated
imidazoles 12a-c. After a deprotection step, imidazoles 13a-c were coupled with ortho-
fluorobenzene with an excess of potassium carbonate to give 14a-c respectively. After
reduction of the nitro group and the alkyne function, compounds 16a-c were obtained
by intramolecular cyclization of 15a-c in presence of carbonyldiimidazole. The
chlorination of 16a-c by phosphorus oxychloride followed by a substitution with
aqueous ammonia afforded compounds 18a-c. The compound 18d bearing a butyl
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chain on the imidazole ring was prepared according to the synthetic pathway
previously described by us 54,55 (Scheme S1).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
O
O
I
I
R
O
O
HN
N
N
N
N
N
a
b
c
12a : R = 1-pentynyl
12b : R = 1-isobutynyl
12c : R = 1-hexynyl
11
NO2
F
R
NO2
NH2
N
N
N
HN
N
N
R1
R
e
d
f
1
3a-c
1
4a-c
1
1
1
1
5a : R = pentyl
1
5b : R = isopentyl
1
5c : R = hexyl
R1
R1
R1
N
N
N
N
N
N
g
N
O
N
Cl
h
N
NH2
H
1
7a-c
18a-c
16a-c
Scheme 3. Synthesis of imidazo[1,5-a]quinoxaline derivatives : (a) Boc O, DMF/ACN,
2
o
Et N, rt, 24h, 80%; (b) Alkynes, DMF, Pd(PPh ) , CuI, 70 C, 12h, 33-67%; (c) TFA,
3
3 4
o
CH Cl , 0 C, 1h; (d) ACN, reflux, 24 h, 28-33%; (e) H , Pd/C, EtOH, 50 psi, 24h, 81-
2
2
2
o
8
7%; (f) CDI, PhCl , reflux, 24 h, 62-84%; (g) POCl , DEA, MW (130 C, 15 min); (h)
2
3
o
30% aq. NH OH solution, ACN, MW (140 C, 2h), 38-98%.
4
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Biological activity
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
All the derivatives were counter-screened for TLR7 or TLR8 agonistic and
antagonistic activities in vitro using HEK-Blue™-hTLR7 and HEK-Blue™-hTLR8 cells.
To test the agonistic activity, HEK-Blue™-hTLR7/8 cells were incubated either in the
absence or in the presence of different concentrations of each compound. Resiquimod
(
TLR7/8 agonist) and CL264 (TLR7 agonist) were used as positive controls. None of
our novel compounds displayed any TLR7 or TLR8 agonistic activity, in the range of
tested concentrations. All compounds up to 100 µM demonstrated very low to
negligible cytotoxicity at the tested concentrations, using an MTT assay (Figure S1).
TLR7 or TLR8 antagonistic activities were assessed by incubating HEK-Blue-hTLR7
or HEK-Blue-hTLR8 cells with graded concentrations of tested compounds in the
presence of 1 µg/mL of CL264 or resiquimod, respectively. All new compounds
showed a selective TLR7 antagonistic activity (Figure 3A, Figure S2), with no TLR8
antagonistic activity (Figure S3), in the range of tested concentrations. Results are
summarized in Table 1, showing concentrations which produced 50% TLR7 inhibition
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(
IC50 values) for the imidazo[1,2-a]pyrazine (I), pyrazolo[1,5-a]quinoxaline (II) and
imidazo[1,5-a]quinoxaline (III) series.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
A structure-activity relationship analysis for the IC values showed general trends
50
in TLR7 selective activity. Compounds 4a-c and 5a-c belonging to the imidazo[1,2-
a]pyrazine series showed a TLR7 antagonistic activity with IC values in the range of
50
2
3 to 40 M. The introduction of an alkyl chain with a heteroatom in position 3 of the
imidazole ring (compounds 4d-f and 5d-f) resulted in a weaker TLR7 antagonistic
effect.
In comparison to the imidazo[1,2-a]pyrazine series, a modest increase in the
antagonistic activity is noted for the imidazo[1,5-a]quinoxaline compounds where the
alkyl chain length reaches 4 to 5 carbon atoms (butyl, pentyl and isopentyl
substituents). Compounds 18a, 18b and 18d displayed TLR7 antagonistic activity with
IC values of 22.0, 17.7 and 17.3 µM respectively. However, the antagonistic activity
50
was reduced when the alkyl chain was lengthened beyond 5 carbon atoms to hexyl,
i.e. compound 18c (IC50 = 45 M). The greatest TLR7 antagonistic potencies are
shown with compounds from the pyrazolo[1,5-a]quinoxaline series. Compounds 10a
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8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
and 10b, bearing a butyl or isobutyl chain respectively, exhibited TLR7 antagonist
activity with low micromolar IC50 values of 8.2 and 10.0 µM, respectively (Table 1).
This activity was not significantly affected by the introduction of a methyl group in
position 8 in compound 10d (IC = 16.3 µM), while it was weakened by the introduction
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
50
of a methyl group in position 9 in compound 10c (IC = 30.0 µM). Although these three
50
chemical series share structural similarities, significant differences in the antagonistic
activity have been detected. In fact, the position of the nitrogen atom on the five-
membered ring as well as the position of the alkyl chain, led to differences in activity.
According to our results, the vicinal position of the two nitrogen atoms in the five-
membered ring, like pyrazole ring, is the best position leading to high antagonistic
activity. The series of pyrazolo[1,5-a]quinoxaline is little described and has been
reported in the literature to have weak biological activity. In 1993, Colotta et al. have
synthesized pyrazolo[1,5-a]quinoxaline derivatives and showed that one of the tested
compounds has some benzodiazepine receptor activity 56. Then in 2008, several
pyrazolo[1,5-a]quinoxaline derivatives were synthesized in our laboratory, and tested
for their potential as tumor-targeting agents; they showed direct anti-tumoral activity
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similar to that of imiquimod 54. Recently, we synthesized and tested pyrazolo[1,5-
a]quinoxaline compounds for their capacity to inhibit the IKK pathway; none of these
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
55
compounds exhibited any inhibitory activity on IKK1 or IKK2 . In the present work,
we have discovered an exquisite activity for pyrazolo[1,5-a]quinoxaline derivatives as
selective TLR7 antagonists.
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5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. TLR7-antagonistic activities of imidazo[1,2-a]pyrazines (I), pyrazolo[1,5-
a]quinoxalines (II) and imidazo[1,5-a]quinoxalines (III).
R1
R1
R1
R2
R2
N
N
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
N
R3
R3
N
N
N
N
NH2
N
NH2
N
NH2
Imidazo[1,2-a]pyrazine (I)
Pyrazolo[1,5-a]quinoxaline (II)
Imidazo[1,5-a]quinoxaline (III)
Compounds
R1
R2
R3
IC50 a (M)
Imidazo[1,2-a]
pyrazines (I)
2
3.6 ± 5.8
9.0 ± 2.8
-
-
-
-
4
a
b
3
4
2
3.5 ± 0.7
-
-
-
-
-
-
4
c
O
4d
42.5 ± 21.9
OH
49.5 ± 3.5
4e
OH
42.5 ± 6.3
40.0 ± 7.0
-
-
-
-
4
f
5a
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26.0 ± 9.5
-
-
-
-
5b
32.5 ± 3.5
5c
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
84.0 ± 15.5
O
-
-
-
-
5d
5
0.0 ± 21.2
8.5 ± 13.4
-
5e
OH
4
-
5f
OH
Pyrazolo[1,5-a]
quinoxalines (II)
10a
H
H
H
H
8.2 ± 1.6
10b
10.0 ± 1.7
30.6 ± 17.5
10c
CH
H
3
H
10d
CH
3
16.3 ± 6.8
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Imidazo[1,5-a]
quinoxalines (III)
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18a
H
H
H
H
H
H
H
H
22.0 ± 11.0
17.7 ± 6.0
45.0 ± 18.3
17.3 ± 5.0
18b
18c
18d
a
IC : concentration of the compound (µM) producing 50% TLR7 inhibition after 24
50
h of compound co-incubation with the cells. Each experiment was repeated at least
three times and the results are presented as average values ± standard deviation
In order to assess the selectivity of 10a and 10b towards TLR receptors, both
compounds were tested for their capacity to inhibit NF-kB translocation in HEK-Blue
cells overexpressing either TLR7 or TLR8. At 15 M, 10a and 10b induced
approximately 50% inhibition of NF-kB translocation in HEK-Blue cells overexpressing
TLR7, as shown by decreased expression of the alkaline phosphatase reporter gene,
while the TLR8 mediating signaling was not affected (Figure 3B). The physiologically
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normal antagonistic activity of 10a and 10b was further confirmed by assessing their
capacity to inhibit the expression of the proinflammatory cytokines IL-6 and TNF-.
For this, HEK-blue TLR7 cells were activated by the TLR7 selective agonist CL264, in
the presence or absence of 50 µM 10a or 10b and mRNA levels of the cytokines were
measured by quantitative real-time PCR (RT-PCR). Addition of 50 M 10a inhibited
the induced expression of both TNF- and IL-6 by 48% and 51% respectively.
Similarly, 10b inhibited 54% of TNF- and 56% of IL-6 expression (Figure 3C).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
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Figure 3: 10a and 10b significantly inhibit TLR7.
(A) Concentration-response curves of 10a and 10b in HEK-Blue TLR7 cell line. Data
was normalized to control (data are mean ± SD; n = 3 independent experiments). (B)
Specificity test for 10a (15µM) and 10b (15M) with TLR7 and TLR8 -specific agonists
used to selectively activate HEK-Blue hTLR7, and hTLR8 cells in the presence or
absence of 10a or 10b (data are mean ± SD; n = 3 independent experiments; **: P-
value <0.01). (C) TNF-, and IL-6 mRNA level in CL264 treated HEK-Blue TLR7 cells
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in the presence and absence of 50µM concentrations of 10a and 10b. Average values
of duplicates are presented.
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Comparative modeling and ligand docking in TLR7
49
Recently, Zhang et al. synthesized for the first time selective TLR8 antagonists . In
their research, they found the existence of a new antagonist site, distinct from the
agonist one. Based on these results, we did a comparative modeling and ligand
docking in TLR7 in order to highlight the TLR7 selectivity of our compounds. Docking
simulation was performed using PLANT 57 with a box of 20 Å x 20 Å x 20 Å to cover
all the antagonist sites of the protein with no constraint. This modeling study performed
on TLR7 showed the presence of a new binding site for the antagonists, as for TLR8.
This new binding site is located in the dimerization interface and binds antagonists
through several chemical interactions (Figure 4).
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Figure 4: Overall structure of TLR7 with compound 10a bound to the antagonist site.
Color coding: magenta and green for TLR7 monomers, nitrogen atoms blue, oxygen
atoms red carbon atoms yellow for compound 10a. PDB code is 5WYZ.
The overall binding mode of all compounds with an alkyl chain (5a-f, 10a-d, 18a-d)
on the five membered ring was found to be conserved in the TLR7 antagonist site.
Figure 5A shows the interactions between the compound 5b and the TLR7 antagonist
site. The pyrazine ring was sandwiched by the side chain of PHE-1302 (chain B),
forming a face-to-face π-π stacking interaction. Hydrogen bonding occurred between
the NH group and the VAL-333 (chain A) main chain. The alkyl chain is engaged in
2
Van der Waals interactions with the hydrophobic pocket formed by the side chains of
the residues PHE-386, VAL-359 and PHE-329 from the chain A and TYR-1374 from
the chain B.
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In the case of tricyclic compounds (10a-b, 18a-d), an additional Van der Waals
interaction is observed between the benzene ring and TYR-242 (Figure 5B).
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A
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Figure 5: Structures of human TLR7 ectodomain in complex with compound 5b (A),
compound 10a (B), compound 4a (C) and compound 10d (D), showing key
interactions in the antagonist binding site. Hydrogen bonds are showed by black dash
line. PDB code is 5WYZ.
On the other hand, the presence of an alkyne group on the five-membered ring
(
compounds 4a-f) changes the orientation of the molecule in the active site. Figure 5C
shows the interactions between compound 4a and the TLR7 antagonist site. The
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pyrazine ring was sandwiched by the side chains of PHE-1302 and TYR-242, forming
a face-to-face π-π stacking interaction with PHE-1302. The NH group was involved
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to form the hydrogen bond with the oxygen of TYR-242. The alkyne group is involved
in Van der Waals interactions with the hydrophobic pocket formed by the PHE-386,
VAL-359, PHE-329 and TYR-1374.
The presence of a methyl group on the benzene ring of compounds 10c and 10d
changes the orientation of the molecule in the TLR7 antagonist site (figure 5D). Van
der Waals interactions between the aromatic ring and the hydrophobic pocket formed
by PHE-1302, PHE-1301 and PHE-329 are noted. The same interactions were found
between the alkyl chain and the hydrophobic pocket formed by the TYR-242 and LEU-
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31. The NH group was involved to form the hydrogen bond with the oxygen of VAL-
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333. The methyl group shows Van der Waals contacts with TYR-1374 and LEU-1323
residues.
In order to propose a rational for the selective antagonist effect of 10a, a deep study
of the antagonist binding site of TLR7 and TLR8 was performed. These antagonist
pockets share a high degree of similarity, however one amino-acid can be point out:
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THR384 (chain A) for TLR7 and ILE403 (chain A) for TLR8 (Figure S4 panel A). This
difference could play a role in the selectivity of compound 10a since isoleucine
contains a bulky side chain. Docking simulation was performed for both TLR and
analysis using DSX-online confirmed the quality of the docking poses with no major
clashes and numerous favorable interactions for compound 10a with TLR7 (Figure S4
panel B). The same analysis for TLR8 showed an unfavorable interaction with ILE403
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(Figure S4 panel C). This result suggested that these residues in TLR7 and TLR8 were
involved in establishing a ligand preference of each receptor that should be confirmed
by X-ray crystallography.
The different biological results as well as the docking study allowed us to carry out
a study of the structure-activity relationship which is illustrated in Figure 6.
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