Triaryl Pyrazole Toll-Like Receptor Signaling Inhibitors: Structure–Activity Relationships Governing Pan- and Selective Signaling Inhibitors

Article abstract of DOI:10.1002/cmdc.201800417

The immune system uses members of the toll-like receptor (TLR) family to recognize a variety of pathogen- and host-derived molecules in order to initiate immune responses. Although TLR-mediated, pro-inflammatory immune responses are essential for host defense, prolonged and exaggerated activation can result in inflammation pathology that manifests in a variety of diseases. Therefore, small-molecule inhibitors of the TLR signaling pathway might have promise as anti-inflammatory drugs. We previously identified a class of triaryl pyrazole compounds that inhibit TLR signaling by modulation of the protein–protein interactions essential to the pathway. We have now systematically examined the structural features essential for inhibition of this pathway, revealing characteristics of compounds that inhibited all TLRs tested (pan-TLR signaling inhibitors) as well as compounds that selectively inhibited certain TLRs. These findings reveal interesting classes of compounds that could be optimized for particular inflammatory diseases governed by different TLRs.

Full text of DOI:10.1002/cmdc.201800417

Accepted Article
Title: Triaryl Pyrazole Toll-Like Receptor Signaling Inhibitors: Structure-
Activity Relationships Governing Pan- and Selective Signaling
Inhibitors
Authors: Julie A Pollock, Naina Sharma, Sirish K Ippagunta, Vanessa
Redecke, Hans Häcker, and John Katzenellenbogen
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201800417
Link to VoR: http://dx.doi.org/10.1002/cmdc.201800417
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Triaryl Pyrazole Toll-Like Receptor Signaling Inhibitors:
Structure-Activity Relationships Governing Pan- and Selective
Signaling Inhibitors
Julie A. Pollock[a,b]‡, Naina Sharma[a]‡, Sirish K. Ippagunta[c,d], Vanessa Redecke[c], Hans Hӓcker*[c], and
John A. Katzenellenbogen*[a]
[a]
Dr J. A. Pollock, Dr N. Sharma, Prof John A. Katzenellenbogen
Department of Chemistry
University of Illinois
505 S. Mathews Ave, Urbana, IL 61801, USA
E-mail: jkatzene@illinois.edu
[b]
[c]
Dr J. A. Pollock
Department of Chemistry
University of Richmond
28 Westhampton Way, Richmond, VA 23173, USA
Dr S. K. Ippagunta, Dr V. Redecke, Dr H. Hӓcker
Department of Infectious Diseases
St. Jude Research Hospital
262 Danny Thomas Place, Memphis, TN 38105, USA
E-mail: Hans.Haecker@stjude.org
Present address: Dr S. K. Ippagunta
Department of Biotechnology
[d]
All India Institue of Medical Sciences
Ansari Nagar, New Belhi, 110029, India
‡These authors contributed equally to the work.
Supporting information for this article is given via a link at the end of the document.
Abstract: The immune system uses members of the toll-like receptor
(TLR) family to recognize a variety of pathogen- and host-derived
molecules in order to initiate immune responses. Although TLR-
mediated, pro-inflammatory immune responses are essential for host
defense, prolonged and exaggerated activation can result in
inflammation pathology that manifests in a variety of diseases.
Therefore, small molecule inhibitors of the TLR signaling pathway
might have promise as anti-inflammatory drugs. We have previously
identified a class of triaryl pyrazole compounds that inhibit TLR
signaling by modulation of the protein-protein interactions essential to
the pathway. We have now systematically examined the structural
features essential for inhibition of this pathway, revealing
characteristics of compounds that inhibited all TLRs tested (pan-TLR
signaling inhibitors) as well as compounds that selectively inhibited
certain TLRs. These findings reveal interesting classes of compounds
that could be optimized for particular inflammatory diseases governed
by different TLRs.
are responsible for sensing bacterial lipoproteins and
lipopolysaccharide (LPS), respectively.[1] TLRs also sense
endogenous ligands, such as S100A8/S100A9 proteins (TLR4),
in order to detect sterile tissue injury or to amplify pathogen-
mediated immune responses.[2] TLRs are expressed by different
cell types and control production of various effector mechanisms,
including pro- and anti-inflammatory cytokines and interferons.[3]
While inflammation triggered by infections is essential for
immune defense and health, prolonged TLR-mediated
inflammation has been linked to a variety of diseases, including
bacterial sepsis, systemic lupus erythematosus, cerebral malaria,
and pancreatitis.[4] In the case of such pathologic inflammation, it
is important to have effective pharmacologic means to moderate
TLR activity. One potential therapeutic strategy is the
development of TLR-signaling inhibitors (TSIs); however, at least
in part due to the complexity of the signal transduction pathway,
there are no clinically approved TSIs available to date.
Signal transduction through TLRs is initiated by dimerization
of Toll/interleukin-1 receptor (TIR) domains contained in the
cytoplasmic part of TLRs and cytoplasmic adaptor proteins,
including MyD88 and TIRAP.[5] While some receptors, such as
TLR7 and TLR9, bind directly to MyD88, TLR2 and TLR4 use the
intermediate protein TIRAP to activate MyD88.[6] Dimerization of
MyD88 leads to recruitment of members of the IRAK family,
whose oligomerization in turn leads to recruitment and activation
of the ubiquitin ligase TRAF6 to transduce signaling to
downstream pathways.[6] As such, the up-stream mechanisms
involved in TLR signaling rely heavily on protein-protein
Introduction
Toll-like receptors (TLRs) comprise a family of receptors
that play key functions in the immune defense against pathogens.
In humans, there are 10 functional TLRs that detect different
pathogen- and host-derived molecules in order to mediate
inflammation and initiate an immune response. For example,
TLR9 detects viral and bacterial unmethylated CpG-DNA motifs,
while TLR7 detects viral single stranded RNA; TLR2 and TLR4
1
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interactions (PPI), which are inherently challenging targets for
drug development.
Recently, we have employed a drug screening method that
relies on the inducible dimerization of the hierarchically acting
proteins TIRAP, MyD88 and TRAF6 to identify TSIs.[7] Specific
activation of these proteins allows assignment of compound
activity to the level of signal transduction. Using this approach, we
identified a class of compounds, i.e. triarylpyrazoles appended
with a basic side chain, which interfere with TLR signaling at the
level of TIR interaction. Specifically, we elucidated that these TSIs
act mechanistically upstream of TRAF6 by interfering with TIR
dimerization. Interestingly, initial structure-activity-relationships
(SAR) showed that alterations to the structure could lead to
compounds with selective activity against certain TIR-domain
interactions, and thus selective TLR inhibition.[7] Herein, we
describe the expansion of the SAR for the development of TSI
with general TLR inhibitory activity (Pan-TLR), and TSI with more
selective TLR inhibitory profile.
Results and Discussion
1,3,5-Triaryl pyrazoles: inhibition of Toll-Like Receptor
signaling vs. binding to the estrogen receptor. From our initial
screen of 4364 unique compounds, we identified the lead
compound, methyl-piperidino-pyrazole (MPP), a basic side chain-
containing pyrazole (BSC-pyrazole) that had been previously
optimized as a high affinity estrogen receptor-alpha (ERα)
selective antagonist.[7-8] Therefore, our initial goal was to decouple
ER binding affinity from activity against TLR signaling. In total, we
investigated 90 pyrazoles. Initially, each compound was
evaluated for ERα and ERβ binding affinities by a competitive
radiometric ligand-binding assay (expressed as a relative binding
affinity (RBA) value, with estradiol being 100) and for TLR9
inhibitory activity based on CpG-DNA-mediated TNFα production
in RAW264.7 macrophage cells. Compounds that showed
promising inhibition of TLR9-mediated TNFα release were then
subjected to additional dose-response inhibition assays that used
ligands for TLR7, TLR2 and TLR4, i.e., R848, Pam3Cys, and LPS,
respectively; in parallel, the toxicity (Tox.) of each compound was
measured using an Alamar blue assay. IC50 values were
established in all of the cell assays. For each TLR signaling
pathway, we were able to identify compounds with low ER affinity
(RBA < 0.003) and moderate to good TLR signaling inhibitory
potency (IC50 < 5 μM). Looking more deeply, it is clear that there
is no significant correlation between binding to either ERα or ERβ,
and TLR signaling inhibition (Figure 1), evidence being the
abundance of inhibitors for all of the TLR signaling pathways with
low IC50 values that also have low RBA values. These results
established that ER binding and TLR signaling modulation can be
decoupled, and selective TSIs can be optimized.
Figure 1. Correlation of RBA for ERα (a) and ERβ (b) with TLR signaling
inhibition. Binding values for ERα and ERβ are relative to that of estradiol (100).
TSI inhibition values (IC50), assayed for the four TLRs studied, are color coded,
as specified.
In our initial published study, we probed the requirement for
and placement of the BSC around the pyrazole core, revealing
the necessity of the BSC.[7] Although there was some activity
when the BSC was appended to the N1 and C4 positions, we
focused most of our early efforts on the derivatives with a BSC on
the C5 ring, as found in our initial hit molecule MPP (compound
1), and eventually moving to derivatives with a BSC on the C3
ring, as they exhibited less affinity for the ERs (Figure 2).[7] All of
the pyrazoles we studied contained three aromatic rings at
positions N1, C3, and C5, because early in our studies we found
that when the phenyl group appended to the N1 position was
removed, potency decreased significantly (Supplemental Table
S1). Therefore, the presence of three aryl rings appears important
for TLR signaling inhibition.
R1
2
N
O
1
N
1
MPP
( )
N
3
R1 R3
R3
=
OH
,
R4
R5
4
=
=
5
CH3
Basic Side Chain
(BSC)
R4
BSC
R5
Figure 2. 1,3,5-Triaryl pyrazoles studied, and structure of MPP (1), the initial hit.
Pyrazoles with BSC at C5. We screened a variety of C5
close derivatives of MPP from a library previously developed as
ER ligands to examine different alkyl lengths on C4 and side
chains identities (Table 1).[8-9] As the alkyl group on C4 decreased
in length, the TLR9 signaling inhibition potency increased
(compounds 1-4), with similar activity against TLR2/4, where
2
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10.1002/cmdc.201800417
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tested. The removal of the alkyl group on C4 (1) decreased affinity
for ER somewhat. The identity of the C5 appended side chain did
not influence the TLR activity significantly, as long as it was still
basic. The length (2 vs. 15), ring linking atom (1 vs. 14), ring size
(3 vs. 6), or the presence of a ring (vs. a dialkyl amine; compounds
7-9) on the BSC did not result in large differences in TLR inhibition.
Increasing the length of the BSC by one methylene unit (1 vs. 15)
reduced affinity towards ER without influencing TLR activity. We
confirmed the necessity of a basic side chain by replacing with
morpholino (10), lactam (11, 13), diol (12), and cyclohexyl (16)
groups, resulting in reduced or even loss of activity against TLR9.
Table 1. TLR signaling inhibition by MPP and derivatives with C5 basic side chains.
HO
N2
1
N
OH
3
5
4
R
BSC
RBAa
ERβ
IC50 (μM)
TLR2
Cmpd
2
R
BSC
TLR9
TLR7
TLR4
Toxicity
ERα
1.47 ±
0.25
3.01 ±
0.06
11.2 ±
0.3
8.67 ±
0.12
11.7 ±
3.7
5.72 ±
0.36
0.015 ±
0.001
H
N
O
O
O
1
2.33 ±
0.64
3.67 ±
0.18
10.2 ±
0.2
8.8 ±
1.5
11.5 ±
2.4
12.0 ±
2.0
0.05 ±
0.01
Me
Et
N
N
N
(MPP)
2.85 ±
0.05
2.08 ±
0.10
3.89 ±
2.41
11.5 ±
5.8
0.49 ±
0.13
3
n.d.
n.d.
4.72 ±
0.15
4.84 ±
0.13
29.3 ±
6.6
6.8 ±
1.1
0.54 ±
0.13
4
5
6
Pr
Me
Et
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
O
O
O
9.97 ±
0.38
16.1 ±
0.3
47.9 ±
3.0
1.15 ±
0.04
0.047 ±
0.010
N
3.32 ±
0.11
2.34 ±
0.45
12.4 ±
4.2
16.0 ±
2.6
0.17 ±
0.02
N
3.06 ±
0.33
3.70 ±
0.28
21.1 ±
6.4
7.77 ±
0.25
0.051 ±
0.008
N
7
8
9
Me
Et
(Et)2
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
O
3.04 ±
0.30
3.64 ±
0.29
16.6 ±
4.0
16.6 ±
3.5
0.17 ±
0.04
N
(Et)2
O
4.21 ±
0.27
30.9 ±
0.6
39.0 ±
2.2
9.12 ±
0.30
0.64 ±
0.14
N
(Me)2
O
Et
O
9.46 ±
0.77
0.083 ±
0.020
10
11
12
Et
Et
Et
> 50
> 50
> 50
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
> 50
> 50
> 50
N
O
O
13.3 ±
3.6
0.43 ±
0.07
N
O
7.0 ±
1.9
0.246 ±
0.008
HO
O
OH
O
1.61 ±
0.05
0.015 ±
0.005
N
N
13
Me
>50
n.d.
n.d.
n.d.
> 50
1.07 ±
0.02
1.37 ±
0.16
9.70 ±
0.10
8.91 ±
0.13
8.76 ±
1.38
5.8 ±
1.7
0.026 ±
0.001
14
15
Me
H
1.44 ±
0.27
3.23 ±
0.33
7.12 ±
0.54
9.26 ±
0.36
12.4 ±
2.1
1.75 ±
0.43
0.011 ±
0.001
N
O
12.7 ±
0.3
10.8 ±
0.4
15.8 ±
0.2
20.9 ±
2.5
0.45 ±
0.12
0.037 ±
0.010
16
H
n.d.
O
aRBA = relative binding affinity values, where estradiol = 100.
n.d. = not determined
3
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In addition, we screened the methoxy precursors to the MPP
(SAR). First, as observed above, the smaller group at C4 provided
more potent activity against TLR (17 vs.18). Extending the BSC
by one methylene retained similar activity (19 vs. 17), with some
increased activity towards TLR7. Removal of all oxygen-
containing substituents decreased activity against TLR9/7 (22).
The presence of the electron-withdrawing chloro group on the N1
ring also decreased TLR activity across the board (23, 24).
derivatives above (Table 2). As expected, the relative binding
affinity for ERs decreased significantly when the phenols on the
MPP derivatives were masked as methoxy groups. However, it
may not be prudent to rely on this functional group, because
methoxy groups can readily be metabolized into phenols in vivo,
restoring high affinity for ERα. Nevertheless, these compounds
gave us additional insight into the structure-activity-relationships
Table 2. TLR signaling inhibition by other MPP derivatives with C5 basic side chains.
R1
N2
1
N
R3
3
5
4
R4
N
O
n
RBAa,b
IC50 (μM)
TLR2
Cmpd
17
R1
R3
R4
n
TLR9
TLR7
TLR4
Tox.
ERα
ERβ
2.46 ±
0.97
12.6 ±
0.4
9.85 ±
0.05
16.2 ±
3.4
OMe
OMe
H
1
> 50
n.d.
n.d.
n.d.
4.15 ±
0.56
29.3 ±
0.6
18
19
20
21
22
23
24
OMe
OMe
H
OMe
OMe
OMe
OH
Me
H
1
2
1
1
1
1
1
> 50
n.d.
n.d.
n.d.
n.d.
n.d.
3.58 ±
0.20
10.9 ±
0.5
10.1 ±
0.4
10.0 ±
0.3
0.002
0.004
0.002
n.d.
0.001
0.001
0.001
n.d.
3.26 ±
0.18
14.4 ±
1.0
31.3 ±
1.5
17.2 ±
3.0
H
3.31 ±
0.26
27.7 ±
0.8
8.94 ±
0.18
10.2 ±
0.1
26.2 ±
2.0
H
H
8.92 ±
0.33
8.03 ±
0.32
23.9 ±
2.4
H
H
H
n.d.
n.d.
n.d.
> 50
9.31 ±
0.36
31.4 ±
0.3
29.2 ±
0.1
26.4 ±
0.6
Cl
OMe
H
0.002
0.007
0.001
0.002
3.04 ±
0.30
Cl
OH
H
n.d.
> 50
> 50
aRBA = relative binding affinity values, where estradiol = 100. bsingle determinations for RBA < 0.01
n.d. = not determined
Due to their low binding affinity for ER, we focused most of
our synthetic efforts on diversifying the C3 BSC-containing
pyrazole derivatives. From our initial screen for C5 BSC
containing compounds, the size of the alkyl chain influenced
activity, with smaller groups being preferred. Therefore, we
O
O
O
a)
b)
R5
+
H3C
H
R5
OH
OH
25
26
27
focused on compounds without an alkyl chain at C4. These
compounds were assembled by a four-step modular synthesis
that allowed for easy derivatization on the C5 and N1 rings
(Scheme 1). In the first step, an aldol condensation was
R1
O
d)
c,
N
N
R5
N
N
O
O
performed between 4-hydroxyacetophenone (26) and the
R5
28
29-71
(42
pyrazoles)
appropriate benzaldehyde (25). Then, the basic side chain was
installed on the chalcone (27). Reaction of the BSC-chalcone (28)
Scheme 1. General synthetic scheme for C3 4-O(CH2)2-piperdinyl pyrazole
derivatives. Reagents and conditions: a) KOH, TBAB, MeOH, reflux, overnight.
b) 1-(2-chloroethyl)-piperidine hydrochloride, K2CO3, Cs2CO3, DMF, overnight.
c) phenylhydrazine hydrochloride, DMF, 85 °C, overnight. d) MnO2, benzene,
reflux, 5-10 h.
with the appropriate hydrazine resulted in the corresponding
pyrazoline that was oxidized to the pyrazole (29-71) by treatment
with manganese oxide. A total of 42 pyrazoles containing a 4-
O(CH2)2-piperdinyl BSC at C3 (Tables 3-6, Supplemental Table
S2) were synthesized and examined for their ability to inhibit TLR
signaling. In addition, each compound had low ER binding affinity
(RBA < 0.06, Supplemental Table S1).
The position and number of free phenols on the aromatic
rings had some influence on activity (Table 3). Removal of the p-
OH from the N1 ring was well tolerated (30 vs. 29); however, on
the C5 ring, removal of the p-OH (31) or movement of it to the
meta position (32) decreased activity. Replacement of the phenol
4
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on N1 with a methyl group (33 vs. 29) or electron-withdrawing
chloro group (34 vs. 29) retained activity against TLR9 but
reduced activity against TLR7/2/4. When additional electron-
donating groups were added to the C5 ring (35 vs. 29), the activity
against TLR4 increased but the general toxicity was also
increased. The replacement of the phenol on C5 with an amide
(29 vs. 36) improved activity against TLR9 but also resulted in
increased toxicity.
groups (52) seemed to decrease activity, with some selectivity for
TLR9/7.
Table 4. TLR signaling inhibition by C3 BSC pyrazoles with methoxy
substituents.
R1
N
N2
1
N
O
3
5
Table 3. TLR signaling inhibition by C3 BSC pyrazoles with free phenols.
4
R5
IC50 (μM)
TLR2
R1
Cmpd
37
R1
R5
N
TLR9
TLR7
TLR4
Tox.
N2
1
N
28.2
±
2.8
O
4.26 ±
0.04
28.4 ±
0.1
3
5
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
H
> 50
n.d.
4
R5
15.8
±
2.9
4.44 ±
0.36
4.96 ±
0.33
28.6 ±
0.1
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
H
n.d.
n.d.
IC50 (μM)
TLR2
Cmpd
29
R1
R5
TLR9
TLR7
TLR4
Tox.
14.0
±
2.8
m-
OMe
5.75 ±
0.23
12.3 ±
0.1
11.6 ±
0.3
2.65 ±
0.39
7.01 ±
1.41
9.13 ±
3.88
6.73 ±
0.66
35.0 ±
3.2
p-OH
p-OH
p-OH
H
13.3
±
2.5
3.95 ±
0.11
11.3 ±
0.1
24.3 ±
0.6
17.7 ±
0.7
p-Me
2.61 ±
1.37
2.99 ±
0.62
6.73 ±
1.97
5.38 ±
0.64
33.3 ±
2.6
30
31
32
33
34
35
36*
H
16.6
±
2.3
p-
OCF3
5.81 ±
0.55
12.1 ±
0.5
16.0 ±
0.1
19.1 ±
0.4
6.80 ±
0.65
15.5 ±
1.0
7.94 ±
2.19
15.5 ±
3.1
19.9 ±
5.2
p-OH
p-OH
p-Me
p-Cl
H
20.0
±
5.1
4.49 ±
0.69
34.7 ±
2.4
16.0 ±
0.1
p-Cl
n.d.
30.2 ±
0.1
m-OH
p-OH
p-OH
n.d.
> 50
n.d.
> 50
31.4
±
3.1
3.02 ±
0.11
22.3 ±
0.9
26.5 ±
1.6
26.6 ±
0.3
p-NO2
3.97 ±
0.27
18.4 ±
0.6
23.5 ±
0.6
22.8 ±
0.7
26.3 ±
3.7
4-
NHBoc
3.00 ±
0.24
> 50
> 50
> 50
> 50
3.79 ±
0.30
34.8 ±
0.1
11.3 ±
0.1
16.7 ±
0.5
23.6 ±
0.4
15.8
±
1.4
4.08 ±
0.38
19.6 ±
0.8
13.1 ±
1.0
19.3 ±
1.7
3,5-
(OMe)2-
4-OH
4-NH2
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
p-OMe
3.23 ±
0.08
10.7 ±
0.4
5.66 ±
0.75
2.92 ±
0.22
17.7 ±
4.4
22.6
±
3.5
4.61 ±
0.23
27.4 ±
0.6
> 50
n.d.
p-NH
C(O)Me
1.18 ±
0.18
7.67 ±
0.21
11.0 ±
0.5
6.17 ±
0.14
9.72 ±
0.15
p-OH
8.61
±
1.91
2,4-
(OMe)2
3.73 ±
0.11
3.51 ±
0.07
15.5 ±
0.8
13.5 ±
0.7
*compound 36 was a 1:1 mixture of pyrazole:pyrazoline
n.d. = not determined
10.7
±
0.8
We next expanded the SAR to include a variety of methoxy
derivatives at N1 (R1) and C5 (R5) positions, retaining the same
4-O(CH2)2-piperidinyl BSC on C3 and a H at C4 (Table 4). The
removal of the para-methoxy on the N1 phenyl group increased
activity against TLR7 (38). Placement of the methoxy on the meta
position slightly improved activity against TLR2 and TLR7 (39). All
substitutions at the para position regardless of electronics (38, 40-
45) retained comparable activity against TLR9; however, the
activity against the other TLRs was variable, albeit not very potent.
Of particular note was the N-Boc protected derivative (44) that
was completely selective for TLR9 and exhibited no toxicity in the
cells. Overall, changes to the C5 (R5) position were generally well
tolerated, although they resulted in some increased toxicity
(compounds 46-52). Of note, increased electron-donating
methoxy groups tended to increase activity towards TLR7, TLR2,
and TLR4 (47, 49, 50), with the tri-methoxy (49) being a pan-TLR
inhibitor. An amide group on C5 also produced a pan-TLR
inhibitor (48). Replacement of some methoxy groups with an
electron-withdrawing chloro (51) or slightly donating methyl
4-NH
C(O)Me
1.24 ±
0.07
3.19 ±
0.08
5.18 ±
0.48
5.60 ±
0.34
14.7
±
0.1
3,4,5-
(OMe)3
3.60 ±
0.49
3.14 ±
0.63
3.79 ±
1.10
1.26 ±
0.39
10.1
±
2.1
3,4-
(OMe)2
3.03 ±
0.38
17.0 ±
1.1
17.9 ±
0.7
13.5 ±
0.6
18.1
±
1.2
3-Cl-
4-OMe
6.00 ±
0.04
7.30 ±
0.33
> 50
> 50
> 50
> 50
3,5-
(CH3)2-
4-OMe
17.1
± 1.
4
7.53 ±
0.49
8.42 ±
0.55
n.d. = not determined
Next, we focused on carboxylic acid derivatives on the C5
position, keeping a phenyl ring at N1 (Table 5). The methyl ester
(53) lost some potency, while the carboxylic acid (54) was
completely inactive. The amide (55) and free amine (57) were
5
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10.1002/cmdc.201800417
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selective for TLR9/7, while the nitrile (56) afforded selectivity for
TLR9. Additional modifications of the amine as a carbamate (58)
or amide (59, 60) retained selectivity for TLR9/7.
Table 6. TLR signaling inhibition by C3 BSC pyrazoles with electron-donating
groups.
N
Table 5. TLR signaling inhibition by C3 BSC pyrazoles with C5 carboxylic acid
derivatives.
N2
1
N
O
3
5
4
R
N
N2
1
N
IC50 (μM)
TLR2
Cmpd
61
R
O
TLR9
TLR7
TLR4
Tox.
3
5
4
8.26 ±
0.34
4.68 ±
1.16
40.9 ±
6.4
3,4-(OMe)2
3,4,5-(OMe)3
2,4,5-(OMe)3
3,4,6-(OMe)3
2,3,4-(OMe)3
> 50
> 50
R5
IC50 (μM)
TLR2
Cmpd
53
R5
5.45 ±
0.80
2.25 ±
0.27
2.49 ±
0.06
1.13 ±
0.39
16.90
± 0.01
62
63
64
65
66
67
68
69
70
71
TLR9
TLR7
TLR4
Tox.
6.29 ±
0.19
16.9 ±
0.4
15.7 ±
0.5
30.8 ±
1.3
CO2Me
CO2H
> 50
7.62 ±
0.41
5.97 ±
1.33
27.3 ±
2.8
22.8 ±
7.0
> 50
> 50
> 50
> 50
> 50
> 50
> 50
54
55
56
57
58
59
60
> 50
n.d.
n.d.
n.d.
> 50
> 50
> 50
> 50
> 50
8.40 ±
0.01
26.5 ±
4.3
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
> 50
3.43 ±
0.15
5.96 ±
0.74
27.8 ±
2.5
17.7 ±
7.7
CONH2
CN
10.8 ±
0.1
> 50
> 50
7.89 ±
0.36
33.5 ±
3.5
> 50
> 50
> 50
> 50
> 50
> 50
3,5-(OMe)2-4-
OEt
9.68 ±
0.82
3.81 ±
0.30
5.93 ±
0.04
33.1 ±
3.5
NH2
11.0 ±
1.1
1.48 ±
0.02
20.0 ±
0.2
3,4,5-(OEt)3
3.46 ±
0.31
3.18 ±
0.21
25.7 ±
5.9
NHCO2Me
NHC(O)Me
NHC(O)Et
12.8 ±
0.3
3-OMe-4-OEt
> 50
> 50
> 50
3.38 ±
0.14
7.25 ±
0.21
17.4 ±
0.33
14.9 ±
2.4
5-OMe-3,4-
dioxymethylene
7.03 ±
0.29
0.79 ±
0.09
2.93 ±
0.84
7.92 ±
0.15
38.8 ±
4.5
> 50
3,5-(Me)2-4-
OMe
7.04 ±
0.24
2.25 ±
0.17
15.3 ±
0.3
17.3 ±
0.5
13.9 ±
0.9
n.d. = not determined
7.78 ±
0.34
19.9 ±
1.4
3-Cl-4-OMe
> 50
> 50
> 50
As the increased electron-donating capability on C5
seemed to have some effect on activity, we examined a wide
range of derivatives with electron-donating groups on various
positions on the C5 ring (Table 6). Depending on the position,
number, and identity of the ethers, we could obtain TLR9-
selective (compounds 64-66, 68, 71), TLR9/7-selective
(compounds 61, 63, 67, 69, 70), or pan-TLR (62) inhibition. Many
of the TLR9/7-selective inhibitors showed a preference for TLR7
inhibition (67, 69, 70). We also examined some different aromatic
rings, 6-methoxyl-2-naphthyl and 3-thienyl, appended to C5,
which showed moderate preference for TLR9 (Supplemental
Table S2).
From our investigations of the C5 site above, we found that
variation in the BSC, as long as it was basic, was well tolerated.
Therefore, we appended different basic groups in the C3 position
(Table 7). There were not significant changes to TLR signaling
inhibition with modifications of the length or identity of the linker
to the piperidinyl group, although there was a modest increase in
toxicity (72-74). Surprisingly, replacement of the piperidinyl group
with a pyrazole (75) resulted in a completely TLR7-specific
compound without any toxicity issues. As reported elsewhere, we
have examined this compound further in biological studies.[7]
6
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10.1002/cmdc.201800417
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screens
(http://www.cbligand.org/PAINS/
and
Table 7. TLR signaling inhibition by pyrazoles with different C3 basic side
http://zinc15.docking.org/patterns/home). In the third screen
(http://fafdrugs3.mti.univ-paris-diderot.fr/), the two aniline-
containing compounds (45, 57) were identified as high risk, and
the phenol-containing compounds and thiophene compound (S4)
were identified as low risk. As discussed above, future studies will
not focus on phenolic compounds due to their off-target effects on
ER. In addition, derivatives 45, 57, and S4 were not among the
most promising TSIs. These results, coupled with our toxicity and
ER RBA values, make us confident that the SAR concluded here
is appropriate.
chains.
R
N2
1
N
BSC
3
5
4
R
IC50 (μM)
TLR2
Cmpd
72
R
BSC
TLR9
TLR7
TLR4
Tox.
N
3.36 ±
0.09
4.62 ±
0.12
5.88 ±
0.31
6.11 ±
0.48
26.0 ±
0.5
OH
Conclusions
N
3.42 ±
0.34
3.52 ±
0.03
12.8 ±
0.3
8.69 ±
2.41
73
74
75
OMe
OMe
OMe
n.d.
n.d.
> 50
Previously, we identified triaryl-pyrazoles with an appended
basic side chain (BSC) as TLR signaling inhibitors through the
inhibition of MyD88 dimerization at the TIR domain.[7] Here, we
expanded the SAR of these pyrazole-based TLR signaling
inhibitors. We conclude that three aryl rings are necessary for
activity, the BSC is optimal on the C3 or C5 ring, smaller
substituents at the C4 position are best tolerated, and
adjustments to the electronics around the aromatic groups result
in variable TLR signaling inhibition (Figure 3). Binding to the
estrogen receptor is essentially eliminated in the more extensively
investigated series with the BSC on the C3 ring.
N
N
O
3.46 ±
0.18
5.55 ±
0.22
4.99 ±
1.70
n.d.
2.27 ±
0.95
N
O
> 50
> 50
> 50
n.d. = not determined
In order to avoid any promiscuous chemotypes that might
fail in future studies, we subjected all of our compounds to three
publicly available in silico filters to identify Pan Assay INterference
compoundS (PAINS).[10] All of the compounds passed two
Various electronics
tolerated at N1;
Must have
ring
aryl
R1
or
methylene longer
N2
with
chain
1
N
Replacement
retains activity but increases toxicity
O
3
5
4
R4
N
essential;
Basic side chain
Teriary amine is best
R5
Shorter alkyl
chains at
C4 better;
H is best
Para-substitution at C5 is best;
Nitrogen containing amine,
amide, carbamate) increase Pan-activity;
groups
(i.e.
Additional electron-donating
groups
result
ethers)
in TLR7-selectivity
(i.e.
Figure 3. Summary of Structure-TSI Activity-Relationships of triaryl-pyrazoles.
Interestingly, we identified some TSI with selectivity for one
signaling pathway over the others. For example, we identified
compounds with selectivity for nucleic acid-recognizing TLRs, i.e.,
TLR7 and TLR9. Most interesting are TLR7-selective inhibitors
that result from additional electron-donating groups on the C5
position or replacement of the piperidinyl BSC with a pyrazolyl
BSC (compound 76). Both TLR7 and TLR9, possibly acting in a
complementary fashion, have been linked to the autoimmune
disease systemic lupus erythematosus, and compounds targeting
their signaling pathways are expected to have therapeutic
potential.[11] On the other hand, other inflammatory diseases, such
as polymicrobial sepsis or cerebral malaria, are likely driven by
the combined activity of different TLRs, including TLR2, TLR4 and
TLR9, making Pan-TLR inhibitors therapeutically attractive
candidates.[12] While we speculate that TSI with selectivity for
specific TLRs, e.g. TLR7, target specific TIR-interactions, more
detailed molecular studies will be required to explore their
mechanism of action.
Experimental Section
General Synthetic Methods. All reagents were used as purchased. DMF
used in reactions was dried using a solvent delivery system (neutral
alumina column).[13] MeOH and benzene used in reactions were
anhydrous, purchased from Aldrich. Solvents used for extraction and flash
chromatography were reagent or ultima grade, purchased from either
7
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10.1002/cmdc.201800417
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Aldrich or Fisher Scientific. All reactions were run under dry N2 atmosphere
except where noted. Flash column chromatography was performed on
Silica P Flash Silica Gel (40-64 μM, 60 Å) from SiliCycle® or using a
Teledyne ISCO CombiFlash MPLC system equipped with Redisep Gold
silica gel columns. 1H NMR and 13C NMR spectra were obtained on 400 or
500 MHz Varian® FT-NMR spectrometers. The chemical shifts are
reported in ppm and are referenced to either tetramethylsilane or the
solvent. Except where noted, both low and high resolution mass spectra
were obtained using electrospray ionization on either a Micromass Q-Tof
Ultima or Waters Quattro instrument.
stimulation. Cell viability (Tox.) was analyzed by the Alamar blue assay
system (Invitrogen). Data are presented as means
independent experiments.
± SD from 3
Estrogen Receptor Binding Assays. Competitive radiometric ligand
binding assays were performed on 96-well microtiter filter plates (Millipore),
using full-length human estrogen receptor α and β, with titrated estradiol
as tracer, as previously described.[14] After incubation on ice for 18-24 h,
ER-bound tracer was absorbed onto hydroxyapatite (BioRad), washed
with buffer, and measured by scintillation counting. Most RBA values are
the average of 2-3 determinations ± SD. As indicated in Table footnotes,
single determinations were done with compounds having <1/2000 or
<1/10,000 the affinity of estradiol (RBA <0.05 or <0.01).
The basic synthetic transformations outlined in Scheme 1 are given below.
The detailed synthesis and spectroscopic characterization of specific
compounds are given in the Supporting Information.
Acknowledgements
Chalcone Formation (Scheme 1 step i) Typical Procedure: To a
solution of p-hydroxyacetophenone (7.34 mmol) and the appropriate
benzaldehyde (8.81 mmol) in methanol (70 mL), KOH (29.38 mmol) and
catalytic tetrabutylammonium bromide (TBAB) was added. The reaction
mixture was refluxed overnight. After the completion of reaction, the
mixture was cooled and poured into iced cold water to which dil. HCl was
added. The precipitate obtained was then filtered and washed with excess
of water, methanol, dried in air and finally recrystallized from methanol to
obtain pure chalcones in 65-90 % yield.
This work was supported by the National Institutes of Health (PHS
5R01 DK015556 to JAK and T32ES007326 to JAP), the St. Jude
Children’s Infection Defense Center (CIDC) and the American
Lebanese Syrian Associated Charities (ALSAC).
Keywords: toll-like receptor signaling • immune response •
heterocycles • structure-activity-relationships • drug design
BSC Installment (Scheme 1 step ii) Typical Procedure: To the solution
of the respective chalcone (2.36 mmol) in DMF (12 ml), K2CO3 (3.54 mmol)
and Cs2CO3 (2.36 mmol) were added, followed by 1-(2-
chloroethyl)piperidine-hydrochloride (2.60 mmol). Reaction mixture was
allowed to stir for overnight at room temperature. After completion of the
reaction, extraction was done using ethyl acetate. The organic layer was
washed with brine, dried over sodium sulfate, concentrated under vacuum,
and partially purified by flash column chromatography using
methanol/DCM to remove the nonpolar impurities or any traces of
unreacted chalcone. The synthesized chalcones with basic side chains
(85-95 % yield) were then directly taken to the next step.
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9
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Entry for the Table of Contents
Dysregulation and sustained toll-like receptor (TLR) signalling is linked to a variety of inflammatory diseases. Herein, we report the
structure-activity-relationships of triaryl pyrazole-core compounds with basic side chain appendages that inhibit TLR signalling.
Interestingly, some compounds influence downstream activity of all TLRs whereas others are selective, making them promising
candidates for further development as anti-inflammatory therapeutics.
10
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