Novel Pyrimidine Toll-like Receptor 7 and 8 Dual Agonists to Treat Hepatitis B Virus

Article abstract of DOI:10.1021/acs.jmedchem.6b00747

Toll-like receptor (TLR) 7 and 8 agonists can potentially be used in the treatment of viral infections and are particularly promising for chronic hepatitis B virus (HBV) infection. An internal screening effort identified a pyrimidine Toll-like receptor 7 and 8 dual agonist. This provided a novel alternative over the previously reported adenine and pteridone type of agonists. Structure-activity relationship, lead optimization, in silico docking, pharmacokinetics, and demonstration of ex vivo and in vivo cytokine production of the lead compound are presented.

Full text of DOI:10.1021/acs.jmedchem.6b00747

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Article
Novel Pyrimidine Toll-like Receptor 7 and
Dual Agonists to Treat Hepatitis B Virus
8
David Craig McGowan, Florence Herschke, Frederik Pauwels, Bart Stoops, Stefaan Last, Serge Pieters,
Annick Scholliers, Tine Thoné, Bertrand Van Schoubroeck, Dorien De Pooter, Wendy Mostmans,
Mourad Daoubi Khamlichi, Werner Embrechts, Deborah Dhuyvetter, Ilham Smyej, Eric Arnoult, Samuël
Demin, Herman Borghys, Gregory Fanning, Jaromir Vlach, and Pierre Jean Marie Bernard Raboisson
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.6b00747 • Publication Date (Web): 11 Aug 2016
Downloaded from http://pubs.acs.org on August 12, 2016
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Figure 3. Predicted binding mode of 19, 21, and 31 in the R848 binding site of hTLR8.
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Figure 4. TLR8 and its dimerization partner are green and cyan, respectively. Left: Best docking
conformation of 50d in the R848 binding site of hTLR8. Right: Molecular model of the binding conformation
of 50d in the R848 binding site (hTLR7 model).
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Figure 5. a) Mean maximum concentrations of 50d after p.o. dosing in C57Bl/6 mice.
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Figure 5 b) Kinetics of IFNalpha secretion.
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Figure 6a) Serum sAg levels over time (mean +/- SD).
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Figure 6b) Percent HBsAg+ hepatocytes in the liver at end of dosing (mean +/- SD).
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Figure 6c) Serum anti-HBs antibody levels over time (mean +/- SD).
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Figure 6d) Serum viral load over time (mean +/- SD).
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Figure 6e) Liver HBV viral load at end of dosing (qPCR, mean +/- SD).
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Figure Table of Contents. Superposition of hLTR7 model and hTLR8 structure with 50d.
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Novel Pyrimidine Tollꢀlike Receptor 7 and 8 Dual
Agonists to Treat Hepatitis B Virus
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David McGowan,* Florence Herschke,* Frederik Pauwels, Bart Stoops, Stefaan Last, Serge
Pieters, Annick Scholliers, Tine Thoné, Bertrand Van Schoubroeck, Dorien De Pooter, Wendy
‡
Mostmans, Mourad Daoubi Khamlichi, Werner Embrechts, Deborah Dhuyvetter, Ilham Smyej,
§
†
Eric Arnoult, Samuël Demin, Herman Borghys, Gregory Fanning, Jaromir Vlach, Pierre
Raboisson
Janssen Infectious Diseases Diagnostics BVBA, Turnhoutseweg 30, 2340 Beerse, Belgium
‡ Villapharma Research S.L., Parque Tecnológico de Fuente Álamo. Ctra. El Estrechoꢀ
Lobosillo, Km. 2.5ꢀ Av. Azul 30320 Fuente Álamo de Murcia, Murcia, Spain
KEYWORDS
TLR, TollꢀLike Receptor, Interferon, pyrimidines, HBV
ABSTRACT
Tollꢀlike receptor (TLR) 7 and 8 agonists can potentially be used in the treatment of viral
infections, and are particularly promising for chronic Hepatitis B virus (HBV) infection. An
internal screening effort identified a pyrimidine Tollꢀlike receptor 7 and 8 dual agonist. This
provided a novel alternative over the previously reported adenine and pteridone type of agonists.
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Structure activity relationship, lead optimization, in silico docking, pharmacokinetics and
demonstration of ex vivo and in vivo cytokine production of the lead compound are presented.
INTRODUCTION.
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The innate immune system in vertebrates has several classes of patternꢀrecognition receptors
(PRRs) allowing the host to recognize the first signs of attack by a foreign pathogen. Tollꢀlike
receptors (TLRs) are among the four classes of PRRs known to date. Characteristic components
of each pathogen are detected by dedicated TLRs leading to a signaling event and activation of
1
2
innate host defense. There are 10 TLR paralogs identified in humans. TLR7 and 8, expressed in
the membranes of endosomes in the cells, recognize singleꢀstranded viral RNA and their agonists
2
,3,4
induce one of the strongest T 1ꢀtype immune response.
In humans, TLR7 is mainly
H
expressed in plasmacytoid dendritic cells (pDC), inducing their activation and production of
IFNꢀα, and to a lower extent on B cells, inducing their proliferation and secretion of antibodies.
Conversely, TLR8 is mainly expressed in monocytes and myeloid DCs (mDCs), inducing their
5
, 6, 7
activation towards a strong T 1 profile.
Therefore, TLR7/8 agonists have been pursued for
H
the treatment of viral infections, tumors, asthma and as vaccine adjuvants.
Chronic hepatitis B virus (HBV) infection remains a major public health problem affecting over
350 million people worldwide, as it eventually leads to cirrhosis, liver failure, or hepatocellular
8
carcinoma (HCC), responsible for 1 million deaths every year. Less than 10% of patients
achieve functional cure, defined as HBsAg seroconversion, with approved treatments (e.g.
9
, 10
nucleosides and interferonꢀα therapies).
Thus, there is an acute need for better treatments.
The low cure rates are due to high viral antigen loads, inability of the host immune system to
11, 12
control HBV and the immunosuppressive environment of the liver.
The induction of an
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+
HBVꢀspecific CD8 T cell response of high magnitude and breadth of epitope recognition,
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functional in the liver, is key in controlling HBV infection.
However, most HBVꢀspecific
+
CD8 T cells are exhausted in chronic patients, unable to proliferate and/or display their full
cytotoxic activity. Ex vivo, ILꢀ12, antiꢀCD40 mAb and antiꢀOX40L mAb are to date, the only
immunostimulatory agents able to reinvigorate exhausted HBVꢀspecific T cells. Expression of all
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three are strongly induced by TLR7/8 agonists.
TLR8 agonists were also the most efficient
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at inducing IFNγ from human liver nonꢀparenchymal cells. Therefore TLR 7 and 8 agonists
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have received attention toward the treatment of chronic hepatitis B virus.
Several immunomodulators that induce endogenous IFNα have been described in the literature
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(Figure 1). These include the systemic imidazoquinoline TLR7/8 agonist R848 (1), and two
2
1, 22
TLR7ꢀselective series including 8ꢀoxopurines (e.g. 2),
and a pteridinone series of agonists
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represented by GSꢀ9620 (3), which features a basic amine. Our objective was to identify a
novel series of TLR7/8 agonists of equal potency on both receptors, with limited plasma
exposure, and no offꢀtarget effect to minimize the risk of systemic cytokine induction and
toxicity.
Figure 1. Described TLR 7/8 agonists.
RESULTS AND DISCUSSION
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In order to discover novel agonists, we performed a high throughput screen of approximately
00,000 compounds from our internal collection. The ability of compounds to activate human
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TLR 7 was assessed in a cellular reporter assay using HEK293 cells transiently transfected with
a TLR 7 expression vector and NFκBꢀluciferase reporter construct. For the high throughput
screen, the TLR7 expression construct expressed a mutant sequence comprising a deletion in the
second leucineꢀrich repeat (dLRR2) of the TLR7 protein. The mutant TLR7 protein was
described to be more susceptible to agonist activation thereby allowing a more sensitive tool to
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identify novel agonists. Dose response curves were generated from measurements performed in
quadruplicates. Lowest effective concentrations (LEC) values, defined as the concentration that
induces an effect which is at least two fold above the standard deviation of the assay, were
determined for each compound.
Figure 2. Hit from HTS screening of our internal compound repository.
The screening effort resulted in a single hit, compound 4 (Figure 2, LEC [TLR7ꢀdLRR2] = 1.6
ꢀM). This novel hit had the advantages of low molecular weight, and the potential for facile lead
optimization. Each position of the pyrimidine can be explored using a variety of well described
chemistry. 2,4ꢀdiaminoꢀ5ꢀalkoxypyrimidines have been reported in the literature as antifungals,
purinergic P2X antagonists for the treatment of pain, and more recently as JAK3
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24,25,26
inhibitors.
We embarked on a structure activity study to explore each position of the
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pyrimidine ring system. The dLRR2 mutant assay was abandoned and we focused our attention
on a TLR expression construct that expressed the respective wild type sequences of hTLR7 or
hTLR8. 4 was retested in the wild type assay, and was observed to have dual agonist activity
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(
LEC [hTLR7] = 4.7 ꢀM, LEC [hTLR8] = 1.1 ꢀM).
a
Scheme 1. Resynthesis of the hit and close analogs.
a
Reagents and conditions: (a) ethyl formate, THF, NaH, 0ºC, in situ addition of guanidine carbonate, EtOH, NaOEt,
reflux 16h, 66% over 2 steps; (b) POCl , 100ºC, 51%; (c) CH X(CH ) CH NHR, Et N, EtOH, 80ºC, 40ꢀ85%. See
WO2012136834.
3
3
2 n
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Resynthesis of hit 4 was done in parallel with a limited set of structurally resemblant analogs
Scheme 1). The synthesis of 4 began with the condensation of ethyl 2ꢀethoxyacetate with ethyl
(
formate leading to an ethyl 2, 3ꢀdiethoxyacrylate intermediate. A one pot, in situ cyclization was
effected via addition of guanidine carbonate under basic conditions then reflux to afford 2ꢀ
aminoꢀ5ꢀethoxypyrimidinꢀ4ꢀol (5) in modest yield (66%). This was converted to the
corresponding 4ꢀchloro intermediate via phosphorous oxychloride providing a building block (6)
for parallel functionalization via the addition of an amine, and base in EtOH at 80ºC.
Resynthesis of 4 confirmed its activity, however the 2ꢀmethoxyethylamine derivative (9),
although the same size, was inactive. This suggests that the nꢀbutyl alkyl chain is preferred, or
that the methoxy ether is not tolerated in this position on the alkyl chain. The lower limit in size
of the alkyl chain, the nꢀpropylamine analog (7) was bereft of activity, as well as the upper limit,
the nꢀhexylamine analog (11). Only the pentylamine analog (10) showed activity (LEC [hTLR7]
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=
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HEK cell culture, indicating that this region has a highly specific interaction with the protein and
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restricted to a 4ꢀ5 carbon alkylamine. Finally, N ꢀbutylꢀ5ꢀethoxyꢀN ꢀmethylpyrimidineꢀ2,4ꢀ
diamine (8) showed no activity, indicating that this Nꢀhydrogen donor was essential for binding
to the TLR proteins.
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Scheme 2. Synthesis of ether derivatives.
R
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f, g
N
h, i
Bn
N
Boc
N
H
N
NH2
N
N
N
N
H
NH2
H
Boc
15
16-33
1
4a, R = H
14b, R = CH3
a
Reagents and conditions: (a) ethyl formate, anhydrous THF, NaH, 0ºC; (b) CH COCl, NMI, TiCl ꢀ45ºC, 1h; (c)
3
4,
guanidine carbonate, EtOH, NaOEt, reflux 16h, 66% over two steps for 13a, 72% over two steps for 13b (d) POCl3,
100ºC, 40%; (e) nꢀbutylamine, Et N, EtOH, 80ºC; 35% yield for 14a, 59% yield for 14b (f) (Boc) O, DMAP, 69%
yield; (g) H , Pd/C, CH OH, 96%; (h) RꢀOH, THF, PPh , DEAD, 0ºC to rt 3h, 70ꢀ90% yields, or RꢀX, Cs CO ,
3
2
2
3
3
2
3
DMF or ACN, 50ꢀ90% yields; (i) HCl, CH OH.
3
Further lead optimization held the more active 4ꢀnꢀbutylamine appendage constant, allowing
the exploration of a variety of ethers in the 5ꢀposition (Scheme 2). The 5ꢀhydroxypyrimidine
intermediate 15 was designed to allow rapid parallel synthesis using readily available alcohols or
alkyl halides employing either standard Mitsunobu or standard alkylation conditions
respectively. Meanwhile, in a closely related synthetic procedure, the influence of the 6ꢀmethyl
group on activity was compared. The synthesis, outlined in Scheme 2, began with the
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functionalization of ethyl 2ꢀ(benzyloxy)acetate, by the insertion of a formyl group as in 12a or an
acetyl group as in 12b, by aldol condensation or acylation respectively. A subsequent
condensation of the ethyl ester intermediates with guanidine carbonate in ethanol in the presence
of sodium ethoxide led to pyrimidines 13a and 13b in modest yield. Treatment of the 4ꢀ
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13
14
15
16
17
18
19
20
21
22
23
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35
36
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42
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45
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47
48
49
50
51
52
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54
55
56
57
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hydroxylpyrimidines with POCl at 100ºC followed by work up and treatment with nꢀbutylamine
3
under basic conditions afforded agonists 14a and 14b. Further conversion of 14a to a versatile
building block was effected in two steps. The 2ꢀamino position was first bocꢀprotected and the
benzyl ether removed under standard hydrogenolysis conditions to afford 15. Alkylation of this
oxygen proceeded smoothly under standard alkylation conditions with an alkyl halide in a polar
aprotic solvent, in the presence of cesium carbonate. Alternatively, standard Mitsunobu
conditions were employed with the variable alcohol in THF.
Investigation into the structure activity relationship of the ether position explored the effect of
substituent size, and heteroatomic substitution on activity and selectivity between TLR 7 and 8.
The benzyl ether 14a showed dual agonist activity and suggested that larger, aromatic groups
were viable, furthermore that the phenyl ring was favorably placed at one carbon spacer
compared to three (17) and showed higher potency compared to the simple alkyl chains (16 to
18), or perhaps that it interfered less with the preferred bound conformation of the nꢀbutylamino
group. A comparison of 14a to the 6ꢀmethylpyrimidine analog 14b showed a marked decrease in
activity (LEC [hTLR7] = 18 ꢀM, LEC [hTLR8] = 7.5 ꢀM) reducing the interest in further
exploration in this position of the pyrimidine scaffold. The relatively small yet more crowded
isopropyl ether (18) brought TLR 8 selectivity. It was observed that heterocyclic rings could
alter the potency and selectivity depending on the heteroatom, the position on the ring and the
size of the ring. The heterocycles lacking a nitrogen in the 2ꢀposition, relative to the carbon that
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5
5
5
5
5
6
connects the heterocycle to the ether, showed potency in the micromolar range (19, 20), while
heterocycles with this attribute showed subꢀmicromolar LECs (22-33). The exception to this
observation was the 2ꢀoxazole analog 21. A docking comparison of 3,5ꢀdimethylisoxazole (19),
0
1
2
3
4
5
6
7
8
9
0
1
2
3
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5
6
7
8
9
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2ꢀoxazole (21), and benzimidazole (31) ether analogs into the R848 region of the crystal
structure of TLR 8 showed that the latter clearly has the best potential for πꢀstacking with
Tyr353 (Figure 3), and thus provided increased potency on hTLR 8 (see supporting information).
These heterocyclic ethers showed impressive potency on both TLRs 7 and 8, especially the
bicyclic examples 29, 30, and 31, rivaling those of described agonists. As a compliment to the
activity data generated by the HEK reporter cellꢀbased assay, the potential of compounds to
induce interferon was evaluated indirectly by measuring the antiviral activity in the HCV
replicon system upon incubation with conditioned media from human peripheral blood
mononuclear cells (hPBMC). Activation of human TLR7 results in robust production of
27
interferon by plasmacytoid dendritic cells present in blood.
a
Table 1. SAR of the ether variations.
Entry
R
Human TLR7 Human TLR8 hPBMC
(
0.9
LEC ꢀM)
(LEC ꢀM)
2.4
(LEC ꢀM)
0.39
1
1
1
1
1
4a
benzyl
nꢀpropyl
6
6.2
4.5
7.9
0.7
1.0
4.8
7
nꢀpropylꢀ3ꢀphenyl 1.5
0.25
0.5
8
isopropyl
10
9
3.1
0.38
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2
0
7
13.7
3.6
n.d.
n.d.
n.d.
21
2.87
0.46
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
22
1.58
23
0.7
0.92
0.39
2
4
5
0.29
0.13
0.74
0.6
0.07
2
0.022
26
0.51
2.28
0.16
27
28
29
0.52
0.3
0.83
0.89
0.14
0.12
0.09
0.003
0.008
3
0
1
0.014
0.032
0.18
0.65
0.011
0.012
3
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5
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3
2
0.21
0.28
1.3
2.6
0.037
33
0.11
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8
9
0
1
2
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7
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9
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8
9
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5
6
7
8
9
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1
2
3
4
5
6
7
8
9
0
aAll compounds showed CC of > 24ꢀM in the HEK 293 cytotoxicity assay described in the supporting
information. LEC values were averaged when determined in two or more independent experiments. n.d.; not done
50
Figure 3. Predicted binding mode of 19, 21, and 31 in the R848 binding site of hTLR8.
Our attention then shifted to amine variation while holding the ether moiety constant. In
addition, the importance of the pyrimidine donor/acceptor pattern in the 2ꢀposition was
challenged. A methyl ether was chosen as the smallest example to reduce the conformational
influence with the amines. The methyl ether products could be accessed via a commercially
available building block, 2,4ꢀdichloroꢀ5ꢀmethoxypyrimidine in only two steps (Scheme 3). The
variable amine was installed in the first step, displacing the chlorine by heating with base in
acetonitrile or an alcoholic solvent. Excess Nꢀmethylamine in THF was heated in a pressure
4
2
vessel to afford N ꢀbutylꢀ5ꢀmethoxyꢀN ꢀmethylpyrimidineꢀ2,4ꢀdiamine (34), an Nꢀmethylated
congener of 4. In spite of the small methyl group, and the same electronic character of the
amine, the compound was devoid of activity, and thus identified the unsubstituted 2ꢀamino
moiety critical for activity. The final 2,4ꢀdiaminopyrimidine products were afforded after
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reaction with ammonia in the presence of copper oxide in an autoclave leading to a small library
of TLR 7/8 agonists (35ꢀ44).
a
Scheme 3. Synthesis of amine derivatives.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Reagents and conditions: (a) nꢀbutylamine, DIPEA, CH CN, sealed tube, 120ºC, 30 min, 76% (b) CH NH in THF,
3
3
2
autoclave, 130ºC, 15 h, 44% (c) RNH , DIPEA, CH CN (d) Cu O, aq. NH , NH CO , autoclave, 120ºC, 18h, 30ꢀ
65% yields over two steps.
2
3
2
3
4
3
A variety of amines from straight and branched chain alkylamines to those containing
heterocycles were explored. With the exception of allylamine analog 35, further exploration
continued on the highly specific, four to five carbon range alkyl amines. Close analogs to
butylamine showed decreased activity on TLR 7 perhaps due to increased π character at the end
of the chain (36), branching at the alpha position which may have brought a change in
conformation (rac-37) or increased lipophilic character as in 4,4,4ꢀtrifluorobutanꢀ1ꢀamine analog
38. The agonist containing (R)ꢀ2ꢀaminopentanol (40) was considerably less active than its
racemic deꢀoxyalkyl analog 37, but proved to have more agonist potential in the (S)ꢀ
configuration (41), and was over 30 fold selective for TLR 8. The same activity was seen for the
corresponding homolog 44. The addition of a methyl group to the distal end of the carbon chain
of 41, as in the (S)ꢀleucinol congener 42, decreased activity. Activity on both TLR 7 and 8
improved in the agonist containing isoleucinol (43), essentially an insertion of a (S)ꢀ
methylenehydroxy group into the inactive compound 39. The hydroxyl group clearly plays more
of a role than just conformation and is possibly involved in hydrogen bonding to the protein. In
the following part of the SAR exploration we sought to observe the effects of oneꢀcarbon
homologation from the stereocenter on TLR agonism potential (Scheme 4).
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4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 4. Synthesis of γ-aminoalcohols.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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8
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1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
b
Reagents and conditions: (a) tꢀbutyl 2ꢀ(triphenylphosphoranylidene)acetate, THF, 16h, rt, 60% ; (b) (S)ꢀNꢀbenzylꢀ
1ꢀphenylethanamine, nꢀbuLi, THF, ꢀ78ºC, 45% ; (c) LAH, THF, 87% ; (d) 10% Pd/C, CH OH, 50ºC, 24h; (e)
(boc) O, Et N, CH Cl 55% over two steps ; (f) HCl, EtOAc, 83% . Yields shown are for the route starting from
butyraldehyde and are representative for analogs starting from 45a, 45b, and 45d.
b
b
3
b
b
b
2
3
2
2,
The stereoselective fourꢀstep synthesis of γꢀaminoalcohols started with commercially available
aldehydes 45a to 45d in a Wittig reaction with tꢀbutyl 2ꢀ(triphenylphosphoranylidene)acetate to
form the unsaturated esters. Subsequent 1,4ꢀaddition of the chiral ammonia surrogate (S)ꢀNꢀ
benzylꢀ1ꢀphenylethanamine afforded aminoesters 47a-d. Reduction of the ester via LAH
afforded the (S)ꢀaminoalcohols 48a-d. Cleavage of the pendant benzyl groups by catalytic
hydrogenation under standard conditions, then subsequent boc group protection facilitated
purification via silica gel chromatography. The isolated intermediates were succeeded by
deprotection under acidic conditions that afforded the amines of interest 49a-d as HCl salts.
Application of aminoalcohols 49a-d onto the pyrimidine scaffold was done in the same manner
as described above (Scheme 2) to afford γꢀaminoalcohol TLR 7/8 agonists 50a-d. Branched γꢀ
aminoalcohols 50a and 50b lost considerable potency on TLR 7 as was observed for their
branched alkyl chain βꢀaminoalcohol analogs (e.g. 42). Agonist 50c, containing an (S)ꢀ3ꢀ
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aminohexanol moiety, a homolog of 41, shared its selectivity for TLR 8 but to a lesser degree.
Finally, the homolog 50d was equipotent in its agonist effect on TLR 7 vs. TLR 8.
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17
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19
20
21
22
23
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25
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33
34
35
36
37
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39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
a
Table 2. SAR of amine variations.
R
Human TLR 7 Human TLR 8 hPBMC
(LEC ꢀM)
Entry
Variable amine
(LEC ꢀM)
(ꢀM)
35
>
25
8.5
6.2
36
>
25
20.9
2.8
6.8
1.7
N
H
37
>
25
3
8
9
6.5
6.6
1.6
3
>
25
>25
>24
40
>
25
9
n.d.
0.4
11
41
42
43
8
.4
0.25
16
>
25
1
7.8
0.66
1.7
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1
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2
2
2
2
2
2
2
2
2
3
3
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3
3
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4
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5
5
5
6
4
4
8.01
0.39
2.3
0.4
7.5
5
0a
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9
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9
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7
8
9
0
1
1
0.8
0.4
OH
5
0b
2.6
2.2
(
S)
N
H
5
0c
0d
7.3
1.4
1.6
0.8
0.5
5
1.6
aAll compounds showed CC of >24 ꢀM in the HEK 293 cytotoxicity assay described in the supporting
information. LEC values were averaged when determined in two or more independent experiments. n.d.; not done.
50
We wanted a target compound profile different from classical antivirals. We sought after a dual
TLR 7/8 agonist with a high clearance profile and low oral bioavailability. We wanted the
compound lead to be preferentially distributed to the liver, then be metabolized and cleared to
limit the incidence of compound entering the systemic circulation and inducing further undesired
2
8,29
immune stimulation which could give rise to adverse events or the risk of autoimmunity.
Strategies employing a high first pass effect have been previously described in the TLR agonist
3
0
field, including GSK2245035, a TLR 7 selective agonist for intranasal treatment of asthma.
Limiting systemic side effects of TLR agonists has prompted research in antedrugs, where an
ester group, for example, can drive metabolism to the corresponding carboxylic acid, thus
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31
clearing the compound rapidly. Our strategy was to target a compound that was inherently less
metabolically stable. Indeed, many analogs were completely metabolized in human liver
microsomes within the time constraint of the in vitro assay.
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18
19
20
21
22
23
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35
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
While several compounds showed intriguing potency (e.g. 29), they were unfortunately wrought
with offꢀtarget activity. Compound 50d had the desired equivalent ratio of TLR 7/8 agonism in
the HEK reporter assay, and was active in the hPBMC assay (0.5 ꢀM LEC). 50d was highly
ꢀ6
permeable (Papp A→B, 25 x 10 cm/s, efflux ratio of 1.3) and had an excellent in vitro offꢀtarget
profile showing no activity on a multireceptor panel, nor on multiple kinases. 50d was chosen for
further profiling for its balance of activity in the low micromolar range, no offꢀtarget activity,
excellent solubility and permeability. Selectivity of 50d across other TLR receptors in the
HEK293 cell lines, each of which expressed the wildꢀtype human TLR receptors 1 through 9
with the same reporter construct showed no activity up to 25 ꢀM. In addition, no direct antiꢀviral
activity was seen after in vitro stimulation of several replicating viruses in various cell lines. A
high content cytotoxicity screening (HCS) in a human hepatocarcinoma cell line (HepG2)
3
2
revealed low cytotoxicity (EC > 86 ꢀM). There was no potential for mutagenicity revealed by
2
0
the AMES II test, and no tendency for the formation of micronuclei in vitro in the presence and
3
3
absence of rat S9. The cytokine profile of 50d was measured ex vivo in healthy donor PBMCs
and was shown to induce IFNα, IFNγ, ILꢀ12p40, ILꢀ12p70, and TNFꢀα amongst other cytokines.
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9
0
Figure 4. TLR8 and its dimerization partner are green and cyan, respectively. Left: Best docking
conformation of 50d in the R848 binding site of hTLR8. Right: Molecular model of the binding
conformation of 50d in the R848 binding site (hTLR7 model).
The docked conformation of 50d makes similar contacts to that of R848 with the TLR8 dimer
(Figure 4). The pyrimidine scaffold is involved in πꢀstacking interactions with Phe 405(Phe408
in TLR7) and the 2ꢀamino group is making direct hydrogen bonds with Asp543 (Asp555 in
TLR7), and the backbone of Thr574(Thr586). The butylamine substituent is pointing into the
hydrophobic pocket formed by the amino acid residues Phe346, Tyr348, and Val378 (Phe349,
Phe351 and Val381) while the pendant chiral alcohol of 50d forms contacts with Asp545 and
Thr574 via a water mediated Hꢀbond. The proposed binding mode for 50d is relevant and
matches well with the SAR of this series.
Table 3 shows the low plasma protein binding across species and compares the in vitro
metabolic stability of 50d in liver microsomes vs. wholeꢀcell hepatocytes. A relatively low
intrinsic clearance (CL ) is seen in liver microsomes in contrast to a higher clearance from
int
whole cell hepatocytes.
a
Table 3. In vitro Metabolic Stability and Plasma Protein Binding of 50d.
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Species
CLint
microsomes
CLint
Plasma
hepatocytes ProteinBinding
6
(
ꢀL/min/mg protein) (ꢀL/min/10 cells)
(% bound)
Mouse
Rat
Dog
5
60
7
8
<4
20
68
99
103
18
34
33
14
34
20
10
11
12
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14
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23
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36
37
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41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
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58
59
60
Cynomolgus monkey
Human
a
Values were rounded to the nearest whole number.
A pharmacokinetic study in male SpragueꢀDawley rats investigated the plasma exposure of 50d
in the portal vein as well as in the systemic circulation following p.o. administration of 10 mg/kg
as a solution. The fraction of the dose absorbed was 55%, Tmax in plasma occurred 1 hour after
dosing (0.5 h in the liver) and Cmax was equivalent to 65 ng/mL in the systemic circulation, and
1710 ng/g in the liver. Systemic exposure of 50d was 183 ng.h/mL, and the liver to plasma ratio
was found to be 14. The hepatic extraction ratio of 0.9 confirmed the extensive metabolism of
the compound.
A mouse in vivo model was used to demonstrate the initial proof of concept to induce
endogenous IFNα. Single oral administration of 1, 5, 10, and 50 mg/kg doses of 50d were given
to healthy C57Bl/6 mice. The compound was found to be rapidly cleared, both Cmax and AUC
increased more than dose proportionally between 1 and 50 mg/kg, and Tmax was between 0.5
and 1 h (Figure 5a). Levels of IFNα peaked between one and two hours post administration in
both the plasma and in the liver. Increase in IFNα was more than doseꢀproportional from 1 to 5
mg/kg, and less than doseꢀproportional from 5 to 50 mg/kg, suggesting a possible saturation of
endogenous IFNα production (Figure 5b). The production of other cytokines was also measured
following 1 and 10 mg/kg oral doses (Table 4). ILꢀ12p70 and IFNγ were detectable from 10
mg/kg, suggesting the induction of a T 1 response. Proꢀinflammatory cytokines TNFα, ILꢀ6 and
H
ILꢀ1β were measured from 10 mg/kg, while the antiꢀinflammatory cytokine ILꢀ10 was measured
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starting from 1 mg/kg. Overall, 50d was able to induce IFNα at low dose, as well as T 1 and
Η
myeloid responses at higher doses (Table 4 and Figure 5).
Figure 5. a) Mean maximum concentrations of 50d after p.o. dosing in C57Bl/6 mice. b)
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a
Kinetics of IFNα secretion.
a
Groups of 15 mice were dosed with 1, 5, 10, or 50 mg/kg of 50d. At time points 0.5, 1, 2, 4 or 7h, three mice of
each group were sacrificed, and blood was drawn to measure compound concentrations and plasma IFNα by ELISA
(
PBL Assay Sciences, VeriKine™ Mouse Interferon Alpha ELISA kit, ref #42120).
Table 4. Mean Maximum Levels of Plasma Cytokines (pg/mL) Induced by 50d after p.o.
a
Dosing in C57Bl/6 Mice.
Dose (mg/kg)
TNFα
IL-6
BQL
BQL
7511
IL-1β
CXCL-1
BQL
IL-12p70
IFNγ
IL-10
BQL
33
Vehicle
1
BQL
BQL
50
62
BQL
BQL
BQL
369
BQL
1
0
64225
6
6602
326
145
880
a
BQL, below quantifiable limit. Groups of 15 mice were dosed with 1, or 10 mg/kg of 50d. At 1, 2, 6, 24 or 48h,
three mice of each group were sacrificed and blood was drawn to measure plasma cytokines, by
electrochemiluminescence (MesoScaleDiscovery, Mouse ProInflammatory 7ꢀplex UltraꢀSensitive kit and MSD6000
reader). The mean maximum cytokine level was calculated, at the timepoint that it was reached. Values were
rounded to the nearest whole number.
The potential of the TLR7 component of 50d to inhibit HBV replication in vivo was tested in the
3
4,35
adenovirusꢀassociated virus (AAV)ꢀHBV mouse model.
This model allows for a chronic
HBV infection to be established within 3ꢀ6 weeks postꢀinfection, with longꢀlasting (>12 months)
7
3
4
viral load ~10 copies/mL, HBeAg ~10 U/mL and HBsAg ~10 U/mL levels. Six weeks after
infection 50d was administered to C57Bl/6 mice for eight weeks at 5 mg/kg once per week
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(
QW). The dose was chosen based on robust cytokine production in healthy C57Bl/6 mice,
where IFNα and IL12p70 were observed (Table 4). The QW dosing regimen was chosen to
avoid possible safety issues. Mean plasma compound concentrations were found in the expected
range (e.g. 245 ng/mL one hour after dosing on day 42, and no compound was detected 164
hours after dosing, prior to the subsequent dose). Agonist 50d decreased HBsAg levels by 2.4ꢀ
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log at the end of the dosing period, while this effect was not observed with entecavir (ETV)
+
(Figure 6a). This decline was also observed in the liver, where the percent of HBsAg
hepatocytes was decreased at the end of dosing (Figure 6b). In parallel, serum antiꢀHBsAg
antibody levels increased from the third to the fourth week of dosing (Figure 6c). Though no
followꢀup phase has been included in this study, one might hypothesize that this seroconversion
might sustain a continued HBsAg decrease. 50d induced a maximum 2.55 log drop of HBV
viral load in the serum, but in a nonꢀcontinuous manner: three days postꢀdose the viral load
significantly decreased, then reincreased by seven days postꢀdose (prior to the subsequent dose)
(Figure 6d). HBV viral load was reduced in the liver, as shown by qPCR (Figure 6e) and by
Southern blot (not shown) at the end of dosing. However, no decrease in liver HBV RNA levels
+
nor in the percent of HBcAg hepatocytes were observed at the end of dosing (not shown). These
results suggest that 50d inhibits HBsAg secretion and HBV viral load by 2 distinct nonꢀcytotoxic
mechanisms. 50d was well tolerated (no clinical observations, and no relevant change in body
weight, food consumption, or body temperature were noted). Therefore, a more frequent dosing
regimen or a higher dose may be beneficial to induce stronger HBV inhibition. In comparison,
murine IFNα administered at a humanꢀequivalent dose (3 MIU/70 kg HBV patient) induced no
reduction in HBV viral load nor HBsAg levels, clearly showing that TLR7/8 agonists have
actions beyond IFNα induction. These results warrant further exploration of 50d to define the
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