Discovery of Potent and Orally Bioavailable Small Molecule Antagonists of Toll-like Receptors 7/8/9 (TLR7/8/9)

Article abstract of DOI:10.1021/acsmedchemlett.0c00264

The toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system, responsible for the early detection of foreign or endogenous threat signals. In the context of autoimmunity, the unintended recognition of self-motifs as foreign promotes initiation or propagation of disease. Overactivation of TLR7 and TLR9 have been implicated as factors contributing to autoimmune disorders such as psoriasis, arthritis, and lupus. In our search for small molecule antagonists of TLR7/9, 7f was identified as possessing excellent on-target potency for human TLR7/9 as well as for TLR8, with selectivity against other representative TLR family members. Good pharmacokinetic properties and a relatively balanced potency against TLR7 and TLR9 in mouse systems (systems which lack functional TLR8) made this an excellent in vivo tool compound, and efficacy from oral dosing in preclinical models of autoimmune disease was demonstrated.

Full text of DOI:10.1021/acsmedchemlett.0c00264

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Letter
Discovery of Potent and Orally Bioavailable Small Molecule
Antagonists of Toll-like Receptors 7/8/9 (TLR7/8/9)
Christopher P. Mussari, Dharmpal S. Dodd, Ratna Kumar Sreekantha, Laxman Pasunoori,
Honghe Wan, Shana L. Posy, David Critton, Stefan Ruepp, Murali Subramanian, Andrew Watson,
Paul Davies, Gary L. Schieven, Luisa M. Salter-Cid, Ratika Srivastava, Debarati Mazumder
Tagore, Shailesh Dudhgaonkar, Michael A Poss, Percy H. Carter, and Alaric J. Dyckman
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.0c00264 • Publication Date (Web): 29 Jul 2020
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ACS Medicinal Chemistry Letters
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Discovery of Potent and Orally Bioavailable Small Molecule
Antagonists of Toll-like Receptors 7/8/9 (TLR7/8/9)
Christopher P. Mussari1, Dharmpal S. Dodd1, Ratna Kumar Sreekantha2, Laxman Pasunoori2, Honghe Wan1, Shana L. Posy1,
David Critton1, Stefan Ruepp1, Murali Subramanian2, Andrew Watson1, Paul Davies1, Gary L. Schieven1, Luisa M. Salter-
Cid1, Ratika Srivastava2, Debarati Mazumder Tagore2, Shailesh Dudhgaonkar2, Michael A. Poss1, Percy H. Carter1 and
Alaric J. Dyckman*1
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1. Bristol Myers Squibb Research and Development, P.O. Box 4000, Princeton, NJ 08543
2. The Biocon Bristol Myers Squibb Research Center (BBRC), Bangalore, India.
ABSTRACT: The toll-like receptor (TLR) family is an evolutionarily conserved component of the innate immune system,
responsible for the early detection of foreign or endogenous threat signals. In the context of autoimmunity, the unintended recognition
of self-motifs as foreign promotes initiation or propagation of disease. Overactivation of TLR7 and TLR9 have been implicated as
factors contributing to autoimmune disorders such as psoriasis, arthritis and lupus. In our search for small molecule antagonists of
TLR7/9, 7f was identified as possessing excellent on-target potency for human TLR7/9 as well as for TLR8, with selectivity against
other representative TLR family members. Good pharmacokinetic properties and a relatively balanced potency against TLR7 and
TLR9 in mouse systems (systems which lack functional TLR8) made this an excellent in vivo tool compound, and efficacy from oral
dosing in pre-clinical models of autoimmune disease was demonstrated.
KEYWORDS: Toll-like receptor, TLR, autoimmunity, innate immunity, psoriasis, lupus
The innate immune system, a first line of host-defense, includes
several families of pattern recognition receptors (PRRs) such as
the toll-like receptor (TLR) family. PRRs recognize pathogen-
associated molecular patterns (PAMPs) from invading
microbes and danger-associated molecular patterns (DAMPs)
released from stressed or damaged host cells.1 TLRs are
transmembrane proteins composed of an extracellular (or intra-
TLR7 and 9 again showed enhanced protection, supporting the
potential benefit of dual inhibition of these targets for the
treatment of lupus.6 Among the other endosomal TLRs, TLR3
signaling has not been as strongly associated with autoimmune
disorders, while deciphering the role of TLR8 has been
complicated by species differences. Unlike TLR7 or TLR9, the
roles of TLR8 in human are not recapitulated in mouse—mouse
TLR8 does not respond to single-stranded RNA ligands which
do stimulate TLR8 in human.4, 7 As such, an understanding of
the function of TLR8 in autoimmune diseases has lagged behind
that of other TLRs.8
endosomal) ligand-binding domain connected via
a
transmembrane region to a cytosolic toll/interleukin (TIR)
signaling domain.2 Binding of ligands (PAMPs/DAMPs) to the
ectodomain (ECD) of hetero- or homodimeric TLR pairs
initiates signaling events via recruitment of adaptor proteins to
the TIR domain. This activates signal transduction cascades
resulting in cellular proliferation as well as production of pro-
inflammatory cytokines, type-I interferons (IFN) and
N
N
Ra
NH
NH
N
OMe
chemokines.
To date, 13 mammalian TLRs have been
Cl
N
Cl
3
identified including nine sub-types (TLR1–9) with defined
functions in human.2 These PRRs are localized either on the
plasma cell membrane (TLR1, 2, 4, 5, 6) or on endosomal
membranes (TLR3, 7, 8, 9), with the endosomal TLRs
responding to double-stranded RNA (TLR3), single-stranded
RNA (TLR7 and 8), or hypomethylated double-stranded DNA
(TLR9) as activating ligands. In autoimmune disorders,
sustained activation of TLR7/9 has been shown to play a key
role in immune dysfunction leading to enhanced disease states.3
In mouse models, overexpression of TLR7 led to autoimmune
disease progression, while TLR7 deficiency protected against
disease development in the lupus–prone MRL/lpr
background.4,5 Although specific knockout of TLR9 led to
worsening of disease in lupus–prone mice, the dual knockout of
1: Ra = H (Chloroquine)
Quinacrine
2: Ra = OH (Hydroxychloroquine)
H
N
R4
H3C
N
F
N
R3
3
5
(R2)n
2
N
N
H
R
R1
4a
R = OMe
4b
R = Cl
5, 6, 7
TLR9; 7 IC50 (M) 0.61; 23.3
TLR4; 3 (inhib. at 10 M) NT; <5%
0.69; 21.7
<10%; 23%
Figure 1. Indole-5-piperidine chemical series identified from
screening
Herein, we detail the identification of potent and selective small
molecule antagonists of TLR7/8/9 based on a 2-phenyl-indole
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chemical series. While TLR8 function is not captured in mouse (Figure 1; 4a,b). This scaffold afforded an opportunity to
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models, the in vivo studies presented here support at least the
potential utility of small molecule antagonists of TLR7/9 as
therapeutics for the treatment of autoimmune disorders.
Examples of potent small molecule inhibitors of endosomal
TLR signaling were quite limited at the outset of our efforts,
with none showing the desired TLR selectivity profile that we
were seeking.9 Antimalarials (e.g., chloroquine, quinacrine)
and structurally related compounds have been shown to inhibit
TLR9, but were reported to exert their effects through binding
to the nucleic acid ligands, resulting in limited selectivity
against TLR3 as well as inhibition of other cytosolic DNA
sensors.10,11 We initiated a screen of a proprietary compound
collection utilizing cellular reporter assays to monitor for
inhibition of TLR activity, focused on the identification of
antagonists of TLR7 and TLR9, given the body of data
connecting their dysregulation to autoimmune disorders. The
screen utilized the HEK293 BlueTM secreted embryonic alkaline
phosphatase (SEAP) assay12, in which the cells were engineered
to overexpress the TLRs of interest. The functional nature of
this screen necessitated deconvolution to filter out general
pathway inhibitors with alternative mechanisms of action.
Counter-screening against additional endosomal (TLR3) and
cell-surface (TLR4) family members helped to eliminate non-
specific pathway inhibitors or compounds with assay
interference.
readily investigate changes to several positions, including at N-
1, on the C-2 phenyl, at C-3, and the nitrogen of the C-5
piperidine.
Structure-activity relationship (SAR) studies began with
variation of the indole C-2 group as shown in Table 1. Mono-
and di-substituted phenyl patterns were explored using both
electron-donating and electron-withdrawing groups. The
tolerability for substitution of the C-2 phenyl was narrow for
TLR7, with unsubstituted and mono-substituted systems,
regardless of substituent position or electronics, showing poor
TLR7 activity. Of the single substitution patterns evaluated, a
methoxy group in the meta or para positions (5f; 5g) were the
most encouraging. Combination of these substitutions into the
3’,4’-dimethoxyphenyl compound 5n provided a dramatic
improvement in TLR7 potency. Constraining these substituents
as in dioxolane 5p reduced TLR7 activity, as did increasing the
size of the alkoxy groups, as in 5o. Specific positioning of the
dimethoxy groups was critical, with the 2’,4’- (5l) and 2’,3’-
dimethoxy (5m) patterns yielding substantially reduced TLR7
activity.
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Table 2. C-5 Piperidine SAR
R
N
CH3
OMe
OMe
N
H
Table 1. C-2 SAR of C-3-Methyl-Indoles
TLR7 IC50
(nM)b
TLR9 IC50
(nM)b
a
Cmpd
5n
pKa
R
H
N
H
N
H3C
N
pKa1 = 10.0
pKa2 = 6.6
CH
3 R1
R2
250 ± 160
430 ± 260
H3C
R3
H
N
6a pKa1 = 10.0
380 ± 250 4,845 ± 3,220
H
O
TLR7 IC50
(nM)a
>10,000
TLR9 IC50
(nM)a
1,800 ± 820
Cmpd R1
R2
R3
6b
6c
NA
1,310 ± 290
790 ± 230
>50,000
H3C
5a
5b
5c
5d
H
Me
H
H
H
H
H
H3C
pKa1 = 10.6
pKa2 = 8.0
N
H
550 ± 230
>10,000
>10,000
>10,000
>10,000
2,140 ± 1,150
2,620 ± 1,550
5,390 ± 3,770
1,100*
H3C
Me
H
H
N
pKa1 = 10.2
pKa2 = 7.1
6d
6e
730 ± 300
850 ± 480
216*
H
Me
H
5e OMe
H
CH3
N
pKa1 = 10.0
pKa2 = 6.0
5f
5g
5h
5i
H
H
OMe
H
H
6,170 ± 1,200 1,870 ± 1,360
740 ± 370
OMe
H
5,460 ± 960
>10,000*
>10,000
500 ± 420
600*
HN
CN
H
H
pKa1 = 9.5
pKa2 = 3.0
6f
1,720 ± 1,250 2,340 ± 2,520
920 ± 380 2,710 ± 1,440
CN
H
H
460 ± 130
520 ± 210
270 ± 60
2,330 ± 420
2,200 ± 690
430 ± 260
1,660*
H3C
N
5j
H
CN
CONH2
OMe
H
7,010 ± 220
16,600*
pKa1 = 8.1
pKa2 = 5.5
6g
5k
5l
H
H
N
OMe
H
>10,000
H
N
pKa1 = 8.0
pKa2 = 6.2
6h
3,490 ± 1,090
190*
H3C
5m OMe OMe
>10,000
N
5n
5o
H
H
OMe OMe
250 ± 160
5,550*
H3C
pKa1 = 7.4
pKa2 = 3.8
OEt OiPr
N
6i
6j
3,690 ± 2,690 850 ± 920
1,680 ± 530 3,360 ± 1,450
O
N
5p
650 ± 100
1,205 ± 470
O
pKa1 = 8.6
pKa2 = 3.5
N
aIC50 values are
determinations except those marked with *.
a mean of two or more individual
aACDpKa calculated by Instant JChem software (ChemAxon);
showing values within a range of 0-15. bIC50 values are a mean of
two or more individual determinations except those marked with *.
Given the human-to-mouse TLR8 functional disconnect
discussed above, activity on that receptor was not tracked for all
compounds, with only advanced examples subsequently
evaluated. From these screening efforts, a 2-phenyl-indole-5-
piperidine chemical series was identified with examples
demonstrating encouraging dual antagonism of TLR7/9 and an
indication that selectivity against TLR3/4 could be achieved
Activity against TLR9 was more accommodating to changes in
C-2 phenyl substitution, although the SAR was flat in
comparison to TLR7 activity. Across the range of changes
described in Table 1, at least modest TLR9 antagonism was
retained, spanning a range of approximately 300–5,000 nM.
Single substitutions at any position of the C-2 phenyl with a
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ACS Medicinal Chemistry Letters
methyl group led to a slight (3-fold or less) decrease in activity,
whereas introduction of methoxy group was most
nitrogen via a methylene linker. In each case, the basicity of
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5
6
7
8
a
the piperidine nitrogen is attenuated by the substituent, which
includes a second nitrogen of varying basicity. While 1-methyl-
5-yl-imidazole 6g showed modest activity for TLR7 and TLR9,
2-methylimidazole 6h was 4-fold reduced in TLR7 potency but
demonstrated very good TLR9 activity, despite only a slight
change in calculated basicity of these constitutional isomers.
Imidazole 6i, regioisomeric to 6g, also exhibited reduced TLR7
but increased TLR9 activity, despite a reduction in calculated
pKa values.
Compound 5n also served as the basis for optimization at the
N-1 and C-3 indole positions (Table 3). The N-1 position was
found to be intolerant to substitution in regards to TLR7 activity
as evidenced by methyl indole 7a (IC50 5,260 nM) whereas
TLR9 activity was only slightly reduced by this change (IC50
740 nM). Modification of the C-3 position proved much more
rewarding, with a range of variations being well tolerated for
TLR9 activity and select changes leading to dramatic
improvement in TLR7 potency. While chloro (7b) or cyano
(7c) replacements at C-3 reduced TLR7 activity, extension of
the alkyl group to ethyl (7d) or trifluoroethyl (7e) improved
TLR7 activity by 2- to 4-fold. This improvement was even
more pronounced with isopropyl substituted 7f which provided
low nanomolar TLR7 potency (IC50 10 nM). Homologation to
isobutyl substitution (7g) led to a precipitous decline in TLR7
potency (IC50 1,290 nM), although that extension had little
effect on TLR9 activity.
advantageous when placed at the para position (5g). Nitrile
substitution was also beneficial for TLR9 activity, regardless of
position (5h–j). An additional electron withdrawing group, the
primary carboxamide, was explored only at the para position,
providing further improved activity (5k). For the di-substituted
examples, the 3’4’-dimethoxy provided favorable results, as
was observed for TLR7 activity (5n). The constrained (5p) and
bulkier (5o) analogs were once again found to exhibit reduced
activity relative to the 3’4’-dimethoxy substitution pattern.
Table 3. SAR of Indole N-1 and C-3 Positions
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H
N
H3C
N
R2
OMe
OMe
N
R1
TLR7 IC50
(nM)a
TLR9 IC50
(nM)a
Cmpd
R1
R2
5n
7a
7b
7c
7d
7e
7f
H
Me
H
Me
Me
250 ± 160
430 ± 260
5,260 ± 1,540 740 ± 170
Cl
1,880 ± 710
>10,000
130 ± 100
70 ± 40
430 ± 170
260 ± 200
490 ± 370
750 ± 270
700 ± 390
830 ± 770
H
CN
H
CH2CH3
CH2CF3
isopropyl
H
Table 4. TLR Cellular and Whole Blood Activity Data for 7f
H
10 ± 7
Assay
Receptor
IC50 (nM)a
7g
H
isobutyl 1,290 ± 770
TLR9
TLR8
TLR7
TLR4
TLR3
700 ± 390
17 ± 37
aIC50 values are
determinations.
a mean of two or more individual
HEK293; NF-kB Reporter
(Antagonist mode)
10 ± 7
Compound 5n was profiled for selectivity and exhibited
reduced activity against TLR3 (IC50 2,160 nM) and TLR4 (IC50
14,970 nM) relative to TLR7/9. Based on the encouraging dual
7/9 antagonist profile of 5n, the 3’,4’-dimethoxyphenyl group
was retained at C-2 for subsequent studies. Efforts turned to the
evaluation of C-5 piperidine capping groups (Table 2). In
comparison to the relatively steep and unaccommodating TLR7
SAR noted for the substitution pattern of the C-2 phenyl, a high
level of chemical diversity was tolerated off of the C-5
piperidine. For TLR9, the antagonist activity was noted to be
influenced by the basicity of the region, specifically the number
of basic atoms and the varying level of basicity associated with
each. Removal of the piperidine capping group to give mono-
basic 6a was tolerated for TLR7 but not for TLR9 (>10-fold
reduction in activity). Acetylation of the piperidine to afford a
non-basic C-5 group (6b) was somewhat detrimental to TLR7
activity (5-fold reduced vs 5n) but highly detrimental to TLR9
activity (IC50 >50,000 nM). The spacing between the basic
atoms and nature of their arrangement was flexible, as
highlighted by examples 6c–6f. Homologated compound 6c
demonstrated activity against both TLRs that was close to that
of 5n, particularly for TLR9. Cyclic constraint of the terminal
amine chain such as piperidines 6d and 6e were comparable in
activity to homologated 6c. As compared to the piperidine (6d),
constraint as an azetidine (6f) led to slightly reduced TLR7
activity (~2-fold) but TLR9 potency was 10-fold reduced,
possibly corresponding to the reduction in pKa of both amines
introduced by this moiety.
15,100 ± 16,500
5,440 ± 5,000
HEK293; NF-kB Reporter
(Agonist mode)
TLR7,8,9
>50,000
TLR9
TLR7
510 ± 80
4.8 ± 3.6
PBMCs; IL-6
pDC (ODN2216); IFN
pDC (Flu Virus); IFN
TLR9
TLR7
210 ± 390
11 ± 9
TLR9
TLR8
TLR7
320 ± 97
5.1 ± 5.6
5.7 ± 6.3
Human Whole Blood; IL-6
TLR9
TLR7
510 ±150
43 ± 45
Mouse Whole Blood; IL-6
aIC50 values are
a mean of two or more individual
determinations. See supporting information for assay details
As described above, 7f was found to be a potent antagonist of
TLR7 with modest activity against TLR9. Further evaluation
against other TLRs in the HEK293 reporter system (Table 4)
showed 7f to also possess potent activity against TLR8 (IC50 17
nM) but much reduced activity against endosomal TLR3 (IC50
5,440 nM) and cell-surface TLR4 (IC50 15,100 nM). In agonist
mode, 7f was inactive up to 50,000 nM against TLR7, 8, or 9.
The antagonist activity of 7f was tested in additional cellular
and whole blood assays. In a peripheral blood mononuclear cell
(PBMC) assay measuring IL-6 production, inhibition of TLR7-
or 9-driven responses were very similar to that observed in the
The remaining compounds in Table 2 describe the effects of
introducing a pendant heteroaryl group off of the piperidine
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HEK293 reporter assays. Inhibition of the TLR9-stimulated
production of IFN in plasmacytoid dendritic cells (pDCs) was
found to be more pronounced (IC50 210 nM) than for other
cellular assays. As pDCs express both TLR9 and TLR7, this
cell type was also used for the evaluation of TLR7 inhibition.
The TLR7 agonist employed in the pDC assay was inactivated
flu virus, rather than a small molecule agonist as utilized in the
other assays. The excellent response of 7f in this assay (IC50 11
nM) with a more physiologically relevant TLR7 ligand
highlights that the potency appears to be independent of the
nature of the TLR7 agonist. This differentiates from the
antimalarials, for which poor inhibitory activity was observed
against non-nucleic acid based agonists of TLR7.10 In human
whole blood (hWB) assays, 7f demonstrated inhibition of IL-6
production consistent with the cellular responses for TLR7, 8,
9. In anticipation of in vivo studies in mouse, the activity of 7f
was evaluated in mouse whole blood (mWB) assays. As
described above, TLR8 is non-functional in mouse and for that
reason only TLR7 and TLR9 responses were examined in this
mWB assay. The TLR9 inhibitory response of 7f in mWB (IC50
510 nM) was similar to that found in hWB, however the TLR7
inhibition in mWB (IC50 43 nM) was ~7-fold reduced relative
to hWB, indicating a more balanced TLR7/9 dual antagonist
profile in mouse.
treatment with 7f was compared to dexamethasone (3 mg/kg qd)
or vehicle control (Figure 3).
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Table 5. Mouse Pharmacokinetic Profile of 7fa
dose (mg/kg)
3 (IV)
15 (PO)
0.5
Tmax (h)
Cmax (nM)
Cl (mL/min/kg)
Vss (L/kg)
T1/2 (h)
Figure 2. Efficacy of 7f in TLR7 (Panel A) and TLR9 (Panel B)
PD experiments measuring suppression of agonist-induced IL-6.
**p<0.01; ***p<0.001; One-way ANOVA with Dunnett’s test
compared to the vehicle. N = 8 mice per group.
1628
7.7
20
32
Significant and dose-dependent suppression of multiple
endpoints was observed, with maximal activity of 7f exceeding
the efficacy shown by dexamethasone. As shown for the
scaling endpoint, treatment with 7f led to a reduction of up to
~70% relative to vehicle at the top dose. Exposures from day 5
(Supporting Information) show that achieving partial coverage
above mWB TLR7 IC50 as observed for the 3 mg/kg dose
appears sufficient to elicit ~50% inhibition in scaling whereas
sustained coverage above the mWB IC50, as achieved for the 10
and 30 mg/kg doses, provided further improvement.
Scaling
F (%)
62
aIV vehicle: 70/30 PEG400/water. PO vehicle: 80/20
PEG400/200 mM citrate buffer (pH 3.0).
Assessment of pharmacokinetic (PK) properties for 7f in mouse
showed good oral bioavailability (62 %F) and a flat exposure
profile with a 24h peak-to-trough ratio of ~4 (Table 5). Based
on exposures in blood, the time to maximal concentration
(Tmax) for oral dosing was rapid (0.5 hours) with maximal
concentration (Cmax) of 1,628 nM (15 mg/kg). The low
clearance and high volume of distribution (20 L/kg) led to a
long terminal half-life of 32 hours. Compound 7f was found to
partition preferentially into the blood (blood-to-plasma ratio
4.6:1 at 2 hr and 6.9:1 at 7 hr) and also into the central nervous
system (7 hr brain to plasma ratio 1.4:1).
In mouse pharmacodynamic (PD) models, oral dosing with 7f
was able to effectively block cytokine production elicited by
treatment with agonists of TLR7 (gardiquimod) or TLR9
(ODN1585) (Figure 2). A dose-dependent decrease in agonist-
induced IL-6 production was observed, with plasma EC50 of 2.4
nM (TLR7) or 25 nM (TLR9). This was noted to be 4- to 5-
fold more potent than the mWB assay results, when factoring in
the blood-to-plasma ratio, possibly reflecting the preferential
distribution of the compound into tissues.
2.5
2.0
1.5
1.0
0.5
0.0
Veh.
1
3
7f (mg/kg)
10
30
**
***
Dex. 3 mg/kg
***
***
***
1
2
3
4
5
6
Days
Figure 3. Efficacy of 7f (1, 3, 10, 30 mg/kg) or dexamethasone (3
mg/kg) vs. vehicle in imiquimod-induced mouse model of psoriasis
showing changes to scaling **p<0.01; ***p<0.001; One-way
ANOVA with Dunnett’s test compared to the vehicle. N = 7–10
mice per group.
7f was then advanced to rodent models of autoimmune
disorders. Topical application of imiquimod (a TLR7 agonist)
is a well-precedented method of eliciting skin changes
characteristic of human psoriasis.13 In this model, imiquimod
cream (5%) was applied to the shaved back region of mice once
a day for 6 days and the effect of concurrent once-daily oral
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Figure 4. Efficacy of 7f (1, 3, 10, 30 mg/kg) or prednisolone (10 mg/kg) vs. vehicle in a MRL/lpr mouse model of lupus showing changes
to proteinuria levels (A), anti-dsDNA antibody titer (B) and plasma IL-10 (C). *p<0.05, **p<0.01; ***p<0.001; One-way ANOVA with
Dunnett’s test compared to the vehicle. N = 12 mice per group.
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Compound 7f was subsequently progressed to a disease model
with a more extensive duration of dosing. MRL/lpr mice
spontaneously develop disease with manifestations that are
reminiscent of human lupus, including proteinuria,
study (Supporting Information) indicated that sustained
coverage above the mWB EC50 for TLR7 was achieved with a
dose of 3 mg/kg, where notable impact on proteinuria (55%
reduction) was observed and modest reduction (30–35%) of the
other endpoints was seen. At the higher doses (10, 30 mg/kg),
coverage above the TLR9 mWB EC50 was achieved as well, and
more robust efficacy was demonstrated.
Subsequent to the identification of 7f as a potent antagonist of
TLR7/8/9, structural evidence was obtained informing on the
direct binding mode of inhibition for this class of
compounds. The availability of hTLR8 protein enabled the
generation of co-crystal structures with compound 17c, as
detailed in Figure 5.
lymphadenopathy,
splenomegaly,
elevated
cytokine
production, and increased antibody production.14 Figure 4
shows the effect of 8 weeks of treatment with 7f (1, 3, 10, 30
mg/kg, qd) as compared to treatment with prednisolone (10
mg/kg, qd) on the urinary marker proteinuria (Panel A), anti-
double stranded DNA (dsDNA) antibody titer (Panel B), and
the serum cytokine IL-10 (Panel C) in the MRL/lpr mice.
For each endpoint, a dose-dependent decrease in severity was
noted, with a maximal response comparable to that shown by
the steroid positive control. Exposures of 7f at the end of the
B.
A.
C.
Figure 5. X-ray crystal structure of 17c bound to TLR8. (A) Structure of 17c in complex with human TLR8 (RCSB PDB: 6V9U). The two
protein chains are shown as light green and light blue ribbons, respectively. The two ligand molecules are shown in space-filling
representation, with the carbon atoms of one molecule in green and the other in cyan. (B) Detailed view of one of the two 17c binding sites.
Colors are as in (A), with hydrogen bonds indicated as dashed orange lines. (C) The complex of TLR8 bound to 17c is shown aligned to
agonist-bound TLR8 (RCSB PDB code 3W3J, orange color); apo TLR8 (3W3G, pink); and TLR8 bound to antagonist CU-CPT9b (5WYZ,
purple).
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TLR8 crystallized as a dimer with two symmetric ligand (33% sequence identity overall and 25% in the binding site),
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3
4
5
6
7
8
binding sites located at the dimer interface, consistent with
previously reported TLR8 X-ray structures15,16 (Panels A and
C). Interestingly, 17c stabilizes an inactive conformation of
TLR8 in which the C-termini of the ECD are positioned far
apart; the structure aligns well with the apo conformation of the
protein (Panel C middle). The open, inactive conformation
observed in this study is differentiated from both the closed,
active agonist-bound conformation (Panel C left) and from a
which is reflected in the more disparate SAR for TLR7/8 vs
TLR9 inhibition.
Preparation of indole-based antagonists is depicted in Scheme
1. Variation of the C-3 position was accomplished through the
use of commercially available 5-bromoindoles. The C-3 methyl
indole (9a) was prepared via LiAlH4 reduction of 5-bromo-
indole-3-carboxaldehyde (8a), while the C-3 alkyl indoles 9b–
d were prepared by exposing 8b to the appropriate secondary
amine in the presence of Shvo’s catalyst. These indole cores
9a–d, along with the commercially available 3-cyano-5-
bromoindole (9e) were further elaborated to the general
structure 10 by Suzuki-Miyaura cross-coupling and subsequent
hydrogenation. Preparation of C-3 chloro intermediate 10f
began with cross-coupling and reduction of 5-bromoindole (8b)
in a manner similar to that described above, followed by NCS-
mediated chlorination. Bromination of 10a–f with NBS
occurred selectively at the C-2 position to afford intermediates
11a–f, which were transformed into the final compounds by one
of several routes. Functionalization of the indole C-2 position
required the removal of the piperidine Boc protecting group via
treatment of 11a–c with HBr/acetic acid in DCM. Addition of
TEA, followed by aldehyde 12 under reductive amination
conditions afforded scaffolds 13a–c, which were subjected to
cross-coupling with phenylboronic acids 14 to afford, after Boc
deprotection, analogs 5a–p and 7d,f. Alternatively, 11a,c–f
were reacted with 3, 4-dimethoxyphenylboronic acid under
cross-coupling conditions, followed by Boc removal via
treatment with HCl to give 6a or 17c–f.
recently reported
antagonist-bound structure which
9
demonstrated an inactive, moderately open conformation
(Panel C right).17 Panel B shows a detailed view of one of the
binding sites. The 3’,4’-dimethoxyphenyl moiety forms a
bidentate hydrogen bond to the Gly-351 backbone NH and
stacks between Tyr-348 of one chain and Phe-494/Phe-495 of
the second chain. These interactions account for the SAR
favoring the 3’,4’-dimethoxy substitution pattern on the C-2
phenyl. The importance of the indole NH for activity (e.g.,
indole N-methyl 7a; TLR8 IC50 958 nM) can be understood in
light of the hydrogen bond between the backbone carbonyl of
Phe-494 and the indole NH. The protonated piperidine is able
to interact with the Glu-427 sidechain and is oriented towards
the solvent-exposed interior of the ectodomain, consistent with
the flexible SAR observed for substitution of the C-5
position. TLR7 and TLR8 share 40% sequence identity in their
ectodomains, and 50% of the residues in the 17c binding site
are identical. TLR7 and TLR8 activity align well for the
compounds tested, and homology modeling of the TLR7
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12
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binding site supports
a similar set of protein-ligand
interactions. The TLR9 ECD is related more distantly to TLR8
Scheme 1. Synthesis of TLR Antagonistsa
8b
(c),(d),(e)
Boc
Boc
N
N
R1
R1
G
R1
Br
Br
(f)
(c)
(d)
8a
(a)
(b)
Br
or 8b
N
N
N
N
H
H
H
H
9a–e
10a–f
` R= Me, Et, iPr, iBu, CN, Cl
11a–f
R= Me, Et, iPr, iBu, CN, Cl
8a
8b
G = CHO
G = H
R= Me, Et, iPr, iBu, CN
Boc
(j), (k)
(g), (h)
N
N
H3C
O
12
Boc
N
H3C
CF3
HN
R1
OMe
R1
OMe
OMe
(l)
(m)
Br
(c),(d)
OMe
OMe
Br
OMe
(o)
N
N
N
H
H
H
15
16
6a; 17c–g
13a–c
R1= Me, Et, iPr
R1= Me; R1= iPr, iBu, CN, Cl, CH2CF3
O
O
18
(R2)n
Boc
(i)
(q)
(HO)2B
14
Me
R3
Cl
Cl
12
N
(n), (o)
or
(n), (p)
or
or
19
20
or (r)
or (s)
CH3
O
R4
R5
H
N
H
N
R6
H3C
N
H3C
N
N
R1
R1
OMe
OMe
Me
OMe
OMe
(R2)n
N
X
N
N
H
H
5a–p,
7d,
7f,
R1= Et; R1= iPr
R1= Me;
7b,c,e–g
6b–j
X= H; R1= Cl, CN, CH2CF3, iPr, iBu
7a X= Me; R1= Me
aReagents and conditions: (a) LiAlH4, THF, reflux, 45% (b) Shvo's catalyst, amine, K2CO3, 155 oC, 21%–72% (c) tert-butyl 4-(4,4,5,5-
tetramethyl-1,3,2-dioxaborolan-2-yl)-3,6-dihydropyridine-1(2H) carboxylate, 3.0 M K3PO4, Pd(dppf)Cl2, THF, 70–84% (d) 10% Pd/C, H2,
EtOAc, 85–97% (e) NCS, DCM, 84%. (f) NBS, DCM then 10% aqueous Na2SO3, 20–75% (g) HBr(AcOH), DCM (h) TEA, AcOH,
Na(OAc)3BH, DCM, 85% 2-steps (i) 3.0 M K3PO4, Pd(dppf)Cl2, THF, then TFA, DCM, 2–66% (j) 3.0 M K3PO4, 3,4-
dimethoxyphenylboronic acid, Pd(dppf)Cl2, THF, 1 h (k) 4.0 M HCl in dioxane, 44–100% (2 steps) (l) trifluoromethylbutanoic acid, PPA,
toluene, 43% (m) 4-bromophenylhydrazine, PPA, 155 oC, 38% (n) TEA, AcOH, Na(OAc)3BH, DCM (o) 4.0 M HCl in dioxane, 12–95%.
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(p) NaH, DMF, then MeI, then 50% TFA in DCM, 52% (q) TEA, DCM, 25–73% (r) DBU, DCM, 6% (s) TEA, AcOH, Na(OAc)3BH,
DCM, 11–88%
1
2
3
4
5
6
7
8
9
Synthesis of C-3-trifluoroethyl indole 17g began with the
reaction of 3,4-dimethoxybenzene with
(2) O'Neill, L.; Golenbock, D.; Bowie, A. The history of Toll-
like receptors — redefining innate immunity. Nat. Rev.
Immunol. 2013, 13, 453–460.
(3) Marshak-Rothstein, A.; Rifkin, I. Immunologically active
autoantigens: The role of Toll-like receptors in the development
of chronic inflammatory disease. Ann. Rev. Immunol. 2007, 25,
419–441.
(4) Heil, F.; Hemmi, H.; Hochrein, H. Species specific
recognition of single-stranded RNA via Toll-like receptor 7 and
8. Science, 2004, 303, 1526–1529.
trifluoromethylbutanoic acid and polyphosphoric acid (PPA).
The resulting intermediate was heated with 4-
bromophenylhydrazine and PPA to afford 16. A three-step
sequence (coupling/hydrogenation/deprotection) analogous to
that described for the conversion of 9 to 10 above was applied
to 16 to afford 17g. Reaction of intermediates 17 with acetyl
chloride (18), alkyl chlorides 19, or aldehyde or ketones 20
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afforded 6b–j.
Intermediates 6a/17c–g were separately
subjected to reductive with a subsequent Boc deprotection to
give final compounds 7b,c,e–j. N-Methyl indole 7a was
prepared by reductive amination of 6a and 12, followed by
exposure to sodium hydride and MeI prior to Boc removal.
In conclusion, we discovered 7f as a promising lead for a small
molecule TLR7/9 dual antagonist program aimed at the
treatment of lupus and other autoimmune diseases. Through an
HTS campaign we identified a 2-phenyl-indole-5-piperidine
series and conducted optimization studies, which led to the
incorporation of dimethoxyphenyl substitution at C-2 to drive
dual potency against TLR7 and TLR9. Potency was further
enhanced through modifications of alkyl substituents at C-3,
while changes to C-5 provide an area for tuning of potency as
well as PK and off-target liabilities through modification of
compound basicity and polarity. The excellent potency and oral
PK properties of 7f in mouse enabled its use in multiple in vivo
models for the evaluation of modulating TLR7/9 driven
pharmacodynamic responses (with TLR8 not able to be
evaluated in mouse systems), and also for efficacy in rodent
disease models for psoriasis and lupus. These results establish
small molecule inhibition of the TLR7/9 pathway as a
promising approach for treatment of autoimmune disorders.
(5) Subramanian, S.; Tus, K.; Li, Q. Z.; Wang, A.; Tian, X. H.;
Zhou, J.; Liang, C.; Bartov, G.; McDaniel, L. D.; Zhou, X. J.;
Schultz, R. A.; Wakeland, E. K. A TLR7 translocation
accelerates systemic autoimmunity in murine lupus. Proc. Natl.
Acad. Sci. 2006, 103, 9970–9975.
(6) Christensen, S.; Shupe, J.; Nickerson, K.; Kashgarian, M.;
Flavell, R.; Shlomchik, M. Toll-like receptor 7 and TLR9
dictate autoantibody specificity and have opposing
inflammatory and regulatory roles in a murine model of
lupus. Immunity 2006, 25, 417–428.
(7) Liu, J.; Xu, C.; Hsu, L.; Luo, Y.; Xiang, R.; Chuang, T. A
five-amino-acid motif in the undefined region of the TLR8
ectodomain is required for species-specific ligand
recognition. Mol. Immunol. 2010, 47, 1083–1090.
(8) Cervantes, J.; Weinerman, B.; Basole, C.; Salazar, J. TLR8:
The forgotten relative revindicated. Cell. Mol. Immunol. 2012,
9, 434–438.
(9) Patinote, C.; Karroum, N.; Moarbess, G.; Cirnat, N.;
Kassab, I.; Bonnet, P.; Deleuze-Masquéfa, C. Agonist And
Antagonist Ligands Of Toll-Like Receptors 7 And 8: Ingenious
Tools For Therapeutic Purposes. Eur. J. Med. Chem. 2020, 193,
112238.
(10) Kužnik, A.; Benčina, M.; Švajger, U.; Jeras, M.; Rozman,
B.; Jerala, R. Mechanism Of Endosomal TLR Inhibition By
Antimalarial Drugs And Imidazoquinolines. J. Immunol. 2011,
186, 4794–4804.
(11) An, J.; Woodward, J.; Sasaki, T.; Minie, M.; Elkon, K.
Cutting Edge: Antimalarial Drugs Inhibit IFN-Β Production
Through Blockade Of Cyclic GMP-AMP Synthase–DNA
Interaction. J. Immunol. 2015, 194, 4089–4093.
ASSOCIATED CONTENT
Supporting Information
Supporting Information is available free of charge on the ACS
Publications website.
Preparation and characterization of compounds 5–7. Biologic
assays. X-ray co-crystallographic data and refinement statistics
for compound 17c bound to TLR8.
Accession Codes
(12)
HEK-Blue™
TLR
Cells.
Atomic coordinates for the X-ray structure of compound 17c:
(PDB: 6V9U) in TLR8 is available from the RCSB Protein Data
Bank (www.rscb.org). Authors will release the atomic
coordinates upon article publication.
https://www.invivogen.com/hek-blue-tlr-cells. (accessed Mar.
14, 2019).
(13) van der Fits, L.; Mourits, S.; Voerman, J.; Kant, M.; Boon,
L.; Laman, J.; Cornelissen, F.; Mus, A.; Florencia, E.; Prens, E.
Imiquimod-induced psoriasis-like skin inflammation in mice is
mediated via the IL-23/IL-17 axis. J. Immunol. 2009, 182,
5836–5845.
(14) Perry, D.; Sang, A.; Yin, Y.; Zheng, Y.; Morel, L. Murine
models of systemic lupus erythematosus. J. Biomed.
Biotechnol. 2011, 1–19.
(15) Tanji, H.; Ohto, U.; Shibata, T.; Miyake, K.; Shimizu, T.
Structural reorganization of the Toll-like receptor 8 dimer
induced by agonistic ligands. Science 2013, 339, 1426–1429.
(16) Tanji, H.; Ohto, U.; Shibata, T.; Taoka, M.; Yamauchi, Y.;
Isobe, T.; Miyake, K.; Shimizu, T. Toll-like receptor 8 senses
degradation products of single-stranded RNA. Nat. Struct. Mol.
Biol. 2015, 22, 109–115.
AUTHOR INFORMATION
Corresponding Author: *E-mail: alaric.dyckman@bms.com.
Phone: 609-252-3593.
ABBREVIATIONS
TLR, toll-like receptor; PRR, pattern recognition receptor;
PAMP, pathogen-associated molecular patterns; DAMP,
danger-associated molecular patterns; ECD, extracellular
domain or ectodomain
REFERENCES
(1) Baccala, R.; Gonzalez-Quintial, R.; Lawson, B.; Stern, M.;
Kono, D.; Beutler, B.; Theofilopoulos, A. Sensors of the innate
immune system: Their mode of action. Nat. Rev.
Rheumatol. 2009, 5, 448–456.
(17) Zhang, S.; Hu, Z.; Tanji, H.; Jiang, S.; Das, N.; Li, J.;
Sakaniwa, K.; Jin, J.; Bian, Y.; Ohto, U. Small-molecule
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TOC Graphic
MRL/lpr mouse lupus model
H
N
H3C
CH3
H3C
N
200
100
0
OCH3
OCH3
36%
55%
76%
N
H
7f
82%
30
85%
TLR7 IC50
TLR9 IC50
Inhib. of IL-6 production
(mouse Whole Blood)
43 nM
510 nM
Veh
1
3
10
Pred
Compund 7f (mg/kg)
8
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