Discovery of a Novel Highly Selective Histamine H4 Receptor Antagonist for the Treatment of Atopic Dermatitis

Article abstract of DOI:10.1021/acs.jmedchem.7b01855

The histamine H4 receptor (H4R), a member of the G-protein coupled receptor family, has been considered as a potential therapeutic target for treating atopic dermatitis (AD). A large number of H4R antagonists have been disclosed, but no efficient agents controlling both pruritus and inflammation in AD have been developed yet. Here, we have discovered a novel class of orally available H4R antagonists showing strong anti-itching and anti-inflammation activity as well as excellent selectivity against off-targets. A pharmacophore-based virtual screening system constructed in-house successfully identified initial hit compound 9, and the subsequent homology model-guided optimization efficiently led us to discover pyrido[2,3-e]tetrazolo[1,5-a]pyrazine analogue 48 as a novel chemotype of a potent and highly selective H4R antagonist. Importantly, orally administered compound 48 exhibits remarkable efficacy on antipruritus and anti-inflammation with a favorable pharmacokinetic (PK) profile in several mouse models of AD. Thus, these data strongly suggest that our compound 48 is a promising clinical candidate for treatment of AD.

Full text of DOI:10.1021/acs.jmedchem.7b01855

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Discovery of a novel highly selective histamine H4
receptor antagonist for the treatment of atopic dermatitis
Kwangseok Ko, Hye-Jung Kim, Pil-Su Ho, Soon Ok Lee, Ji-Eun Lee, Cho-Rong Min, Yu Chul Kim, Ju-
Han Yoon, Eun-Jung Park, Young-Jin Kwon, Jee-Hun Yun, Dong-Oh Yoon, Jung-Sook Kim, Woul-Seong
Park, Seung-Su Oh, Yu-Mi Song, Woon-Ki Cho, Kazumi Morikawa, Kyoung-June Lee, and Chan-Hee Park
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01855 • Publication Date (Web): 26 Mar 2018
Downloaded from http://pubs.acs.org on March 27, 2018
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Discovery of a novel highly selective histamine H4
receptor antagonist for the treatment of atopic
dermatitis
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AUTHOR NAMES
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$
1$
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Kwangseok Ko, Hye-Jung Kim, Pil-Su Ho, Soon Ok Lee, Ji-Eun Lee, Cho-Rong Min, Yu
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Chul Kim, Ju-Han Yoon, Eun-Jung Park, Young-Jin Kwon, Jee-Hun Yun, Dong-Oh Yoon,
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Jung-Sook Kim, Woul-Seong Park, Seung-Su Oh, Yu-Mi Song, Woon-Ki Cho, Kazumi
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Morikawa, Kyoung-June Lee, Chan-Hee Park *
* Corresponding author
AUTHOR ADDRESS
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C&C Research Laboratories, DRC, Sungkyunkwan University, 2066, Seobuꢀro, Janganꢀgu,
Suwonꢀsi, Gyeonggiꢀdo, 16419, Korea
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JW Pharmaceutical Co., Ltd., 2477, Nambusunhwanꢀro, Seochoꢀgu, Seoul, 06725, Korea
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Chugai Pharmaceutical Co., Ltd., Fuji Gotemba Research Labs, 1ꢀ135 Komakado, Gotemba,
Shizuoka, 412ꢀ8513, Japan
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KEYWORDS
Atopic dermatitis; Histamine H4 receptor antagonist; Pharmacophore model; Virtual screening;
Homology modeling; Structureꢀactivity relationships; Offꢀtarget selectivity
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ABSTRACT
The histamine H4 receptor (H4R), a member of Gꢀprotein coupled receptor family, has been
considered as a potential therapeutic target for treating atopic dermatitis (AD). A large number of
H4R antagonists have been disclosed, but no efficient agents controlling both pruritus and
inflammation in AD have been developed yet. Here, we have discovered a novel class of orally
available H4R antagonists showing strong antiꢀitching and antiꢀinflammation activity as well as
excellent selectivity against offꢀtargets. Pharmacophoreꢀbased virtual screening system
constructed in house successfully identified initial hit compound 9, and the subsequent homology
modelꢀguided optimization efficiently led us to discover pyrido[2,3ꢀe]tetrazolo[1,5ꢀa]pyrazine
analogue 48 as a novel chemotype of potent and highly selective H4R antagonist. Importantly,
orally administered compound 48 has a remarkable efficacy on antiꢀpruritus and antiꢀ
inflammation with favorable pharmacokinetic (PK) profile in several mouse models of AD. Thus,
these data strongly suggest that our compound 48 is a promising clinical candidate for treatment
of AD.
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INTRODUCTION
Atopic dermatitis (AD) is a pruritic inflammatory skin disease, which is characterized by eczema,
epidermal thickness, itching and predominant expression of the inflammatory Th2 cytokines
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including ILꢀ4, ILꢀ5 and ILꢀ13.
Especially, mechanical skin injury caused by scratching
aggravates the defect in skin barrier function and leads to the release of proꢀinflammatory
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cytokines causing skin inflammation.
The current firstꢀline therapy for AD includes topical corticosteroids, topical calcineurin
inhibitors, and adjuvant treatment of antihistamines (H1R antagonists) for pruritus. However,
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, 5
these agents cause doseꢀdependent side effects or have little efficacy on itching. Recently, new
biologics such as antiꢀILꢀ4Rα antibody and antiꢀILꢀ31RA antibody are also being developed for
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AD as an injection.
However, orally available drug with sufficient efficacy for AD is still
desired to be developed as widelyꢀused one. Therefore, the large unmet medical need in the
treatment of AD is the development of an orally available systemic agent to mitigate pruritus and
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inflammation in skin, simultaneously.
5, 9
Histamine is closely associated with pruritus and inflammation in AD. Its receptors belong to
a class of Gꢀprotein coupled receptors (GPCRs), and four subtypes have been identified so far
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(H1R, H2R, H3R, and H4R).
In particular, H4R, the most recently discovered subtype, is
highly expressed in variety of organs and the several types of immune cells such as eosinophils,
dendritic cells and Th2 cells that play important roles in allergic immune responses and
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14ꢀ16
diseases.
H4R knockout mice showed the attenuated skin inflammation in AD models,
and its antagonists substantially reduced Th2 cytokine production, pruritus and skin
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15, 17, 18
inflammation in several murine models mimicking human AD.
These findings suggest
that H4R is a promising therapeutic target for the treatment of AD.
Up to now, a number of H4R antagonists have been discovered through ligandꢀbased or
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homology modelꢀbased virtual screening.
Among the representative H4R antagonists (Figure
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), compound 4, 5 and 6 have advanced into Phase II clinical study. Compound 4 (Toreforant,
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JNJꢀ38518168) has entered in clinical trials for the treatment of asthma, rheumatoid arthritis,
and psoriasis. It was known that compound 4 (toreforant) could not inhibit histamineꢀinduced
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scratching in mice associated to AD. Although compound 5 (JNJꢀ39758979) has showed a
profound antiꢀpruritic effect in phase II clinical trial for moderate AD patients, the further
development was discontinued due to drugꢀinduced agranulocytosis caused by offꢀtarget
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effects. Compound 6 (ZPLꢀ3893787) with very similar structure to 5 substantially reduced
eczema area and severity index (EASI) score in Phase IIa proofꢀofꢀconcept study, but its antiꢀ
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pruritic efficacy was not statistically significant. Meanwhile, another aminopyrimidineꢀtype
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H4R antagonists, compound 7 (Aꢀ940894) and 8 exhibited crossꢀactivity on serotonin 5ꢀHT3
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receptor (5ꢀHT3R) which shares very similar ligand recognition pattern with H4R. Since 5ꢀ
HT3R is widely expressed in the central and peripheral nervous system and controls various
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physiological functions, its inhibition may cause CNSꢀrelated side effects.
Thus, the
development of oral H4R antagonist with high selectivity having dual effect of antiꢀpruritus and
antiꢀinflammation in AD still remains a big challenge.
In this work, we developed a novel class of highly selective H4R antagonist. For a new scaffold
hopping, a stratified virtual library with structural diversity was established from large sized
commercially available database, and a pharmacophore modelꢀbased virtual screening was
carried out. And then, the homology modelꢀguided optimization of compounds followed to
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improve the inhibitory activity for H4R and selectivity over offꢀtargets, especially against 5ꢀ
HT3R. The medicinal chemistry campaign resulted in the lead compound 48 presenting profound
antiꢀpruritic and antiꢀinflammatory efficacies in pruritogenꢀinduced acute itching models and
oxazoloneꢀinduced mice AD model. This report describes the discovery and structureꢀactivity
relationship (SAR) of pyrido[2,3ꢀe]tetrazolo[1,5ꢀa]pyrazine derivatives as new H4R antagonists,
and a structural rationale to achieve the selectivity against 5ꢀHT3R based on computational
models.
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RESULTS AND DISCUSSION
Pharmacophore model-based virtual screening
In order to find a novel chemotype of H4R antagonists, we took advantage of a pharmacophore
modelꢀbased virtual screening. Figure 2 shows a schematic representation of the virtual
screening cascade. Initially, we constructed a structurally diverse representative library for a new
scaffold hopping by conducting iterative clustering with Tanimoto similarity (similarity
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coefficient ≥ 0.9) in the ZINC12 database, which is a free database of commerciallyꢀavailable
compounds for virtual screening. The total size of inꢀhouse set for virtual screening (~500k
molecules) is about 2% of the original database (~22 million molecules), but it still maintains
structural and chemical diversity analyzed by principal component analysis of molecular
fingerprints, HB donor, acceptors, rotatable bonds, MW, and clogP as compared to the original
database. It allowed us to efficiently screen a huge size database without reducing the chance to
find new chemotypes. In the next step, we generated a pool of pharmacophore models from
known H4R antagonists depicted in Figure 1. The lowest energy conformation of each molecule
was regarded as a bioactive conformation, and potential pharmacophore hypotheses were
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carefully assigned based on our prior knowledge acquired by scaffold alignment and SAR of the
published H4R antagonists. A total of 8 pharmacophore models with four features were created
and used for virtual screening. Virtual hit structures with fitꢀscore better than 2.7 were visually
inspected, and 291 compounds with novel scaffolds were subjected to competitive radioligand
binding assay for H4R. Among hit compounds with IC value less than 10 ꢁM (data not shown),
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we have identified [1,2,4]triazolo[4,3ꢀa]quinoxaline compound 9 (Table 1) which bears a novel
tricyclic scaffold distinct from known H4R antagonists.
H4R homology model-guided optimization
Since H3R has the highest sequence homology with H4R among histamine receptors and many
H3R antagonists also exhibit inhibitory activity for H4R, we included the competitive binding
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assay for H3R as well as H4R in our primary screening.
As an initial hit compound, 9
displayed good inhibitory activity for H4R and selectivity over H3R, 2.3 ꢁM and >100 µM of
IC , respectively (Table 1). To obtain the detailed insight for structureꢀguided rational design,
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we built a H4R homology model based on the recently published H1R crystal structure
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(
3RZE.pdb). The sequence identity and similarity between H4R and H1R is 24% and 47%,
respectively. The homology model of H4R was constructed as similar as possible to the template,
and was refined using MD simulation as detailed in experimental section.
The predicted docking pose of 9 in H4R homology model indicated a small space around 8ꢀ
position of the [1,2,4]triazolo[4,3ꢀa]quinoxaline scaffold (Figure 3a). Thus, 8ꢀposition of
compound 9 was a good starting point for structural modification to improve activity. Targeting
the small pocket at 8ꢀposition, we first introduced a subset of electronꢀwithdrawing group or
donating group (10-12). Surprisingly, 10 substituted with a chlorine atom displayed about 100ꢀ
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fold enhanced inhibitory activity for H4R (IC = 0.022 ꢁM) compared to 9. Compound 11 with
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nitro group and 12 with methyl group also showed the slightly improved activity compared to 9,
likely due to filling effect of small pocket. The dramatic improvement of 10 in inhibitory activity
compared to 11 and 12 strongly suggested the possibility of halogen bonding.
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The predicted binding mode of 9 also suggests another important interaction, hydrogen bond
between nitrogen in X position (Table 1) and amide of Gln347 (Figure 3a). To validate the
hypothesis from the predicted binding mode in homology model, we synthesized and evaluated
compound 13 lacking hydrogen bond acceptor capable of interacting with Gln347. As expected,
the inhibitory activity of 13 was decreased extremely, and it may be due to the absence of
hydrogen bonding source interacting with Gln347.
Although 10 showed potent inhibitory activity against H4R and acceptable selectivity over H3R,
it had moderate metabolic stability in human and mouse liver microsome and decreased FaSSIF
solubility compared to 9 (Table 1). To investigate the effect of nitrogen inserted in the A ring on
activity, metabolic stability and solubility, we synthesized 14 and 15 with nitrogen atom in the A
ring of 9. Intriguingly, compound 15 with nitrogen inserted at 6ꢀposition of the A ring had
markedly improved metabolic stability in human and mouse liver microsome while maintaining
inhibitory activity against H4R and selectivity over H3R. This effect of nitrogen insertion was
also confirmed in the experiment with 10 and 16. Nitrogen inserted compound 16 displayed a
strong inhibitory activity against H4R and the better selectivity against H3R, metabolic stability,
and FaSSIF solubility compared to 10. These improvements in metabolic stability and solubility
are presumably due to the increased hydrophilicity of 16 (experimental logD7.4 = 0.95) compared
to 10 (experimental logD = 2.12).
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The predicted binding mode of 16 was described in Figure 3b. The basic amine of Nꢀ
methylpiperazine is expected to form ionic interaction with Asp94, which is essential for binding
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to H4R and conserved in other aminergic GPCR ligands. In addition, the methyl group attached
to piperazine occupies a small hydrophobic pocket composed of aromatic residues, Tyr72,
Phe344, and Trp348 (Figure 3b). The nitrogen atom at Xꢀposition makes a strong hydrogen
bonding with Gln347 as described previously. The pyrido[2,3ꢀe][1,2,4]triazolo[4,3ꢀa]pyrazine
ring is shown to favorably form piꢀpi interaction with Tyr95, Trp316, Tyr319, and Phe168 in
extracellular loop 2. It also would form OHꢀpi interaction with Tyr319 and CHꢀpi interaction
with Leu175. As shown in Figure 3b, the chlorine atom at 8ꢀposition is predicted to be located
within the distance where chlorine atom could form the halogen bond with the carbonyl oxygen
of Ser179. The predicted OꢀCl distance is very close to the optimal distance 3.3Å for such
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interactions.
The inhibitory activities of 15-18 strongly support this interaction model. The
IC values of 15-18 were excellently correlated with the strength of the halogen bond by the
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magnitude of sigmaꢀhole: H << Cl < Br < I. The significant decrease in H4R inhibitory activities
of 19-21 may be attributed to loss of the halogen bond. Meanwhile, the introduction of small size
substituents at the 7ꢀ and 9ꢀpositions (22-26) decreased inhibitory activity compared to
compound 16, which allowed us to confirm that there is limited space around those positions as
expected in our binding model. As a result, the initial modification of the A ring part based on
the homology model led to 8ꢀchloropyrido[2,3ꢀe][1,2,4]triazolo[4,3ꢀa]pyrazine derivative 16
presenting good inhibitory activity against H4R, selectivity over H3R, and favorable metabolic
stability and solubility.
Further optimization of amine group
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A positively charged amine, an essential pharmacophoric feature of H4R ligands for the receptor
binding, makes ionic interaction with Asp94 conserved in aminergic GPCRs. To investigate the
effect of the basicity on the ionic interaction, we first replaced Nꢀmethylpiperazine (16, ACD
pKa = 9.9 ± 0.4) with piperazine (27, ACD pKa = 8.9 ± 0.8), which resulted in slightly decreased
inhibitory activity against H4R (Table 2). In addition to basicity, the methyl group itself is also
predicted to contribute to inhibitory activity by optimal fitting to a small hydrophobic pocket
composed of aromatic residues (Figure 3aꢀb). The size effect of methyl group was illustrated by
the loss in inhibitory activity of 28 substituted with cyclopropyl causing steric clash with Tyr72.
Additional methyl group in piperazine (29-30) and the homopiperazine derivatives (31-32) also
resulted in decrease of inhibitory activity against H4R. It indicates that the binding site around
positively charged amine would be spatially limited. The replacement of Nꢀmethylpiperazine
with (R)ꢀ3ꢀamino pyrrolidines as in compounds 33-35 resulted in a reduction in H4R inhibitory
activity likely due to the repulsion between Phe344 and the puckered conformation of
pyrrolidine ring. Interestingly, compound 37 with 3ꢀmethylaminoꢀazetidine, whose threeꢀ
dimensional coordinate of positively charged amine is very close to that of Nꢀmethylpiperazine,
showed comparable inhibitory activity to that of 16. However, addition or removal of a methyl
group in 3ꢀmethylaminoꢀazetidine caused a loss of inhibitory activity against H4R as shown in
compound 36 and 38. The lower activities of 39-40 against H4R relative to 16 are expected as a
result of the small steric hindrance preventing the Asp94 from optimally interacting with the
basic amine. Although 41 with the bicyclic piperazine group showed similar inhibition activity
against H4R compared to 16 with the methylpiperazine group, it demonstrated rather lower
selectivity against H3R. Considering both inhibitory activity for H4R and selectivity over H3R,
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we concluded that the Nꢀmethylpiperazine (16) and Nꢀmethylꢀ3ꢀaminoꢀazetidine (37) are optimal
amine substituents for pyrido[2,3ꢀe][1,2,4]triazolo[4,3ꢀa]pyrazine scaffold.
Improvement of selectivity based on 5-HT3R homology model
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In order to explore the possibility of sideꢀeffects coupled with offꢀtarget binding, we carried out
competitive binding assay of compound 16 against a broad panel of GPCR, ion channel, and
transporters. Intriguingly, 16 showed very strong inhibitory activity against both 5ꢀHT3R (IC =
50
0
.057 ꢁM) and H4R (IC = 0.077 ꢁM) while it did not exhibit any significant inhibitory activity
50
against other 55 membrane proteins (data not shown). This is consistent with the previous report
for fragment library screening that illustrated remarkable similarities in ligand recognition
27
between H4R and 5ꢀHT3R. To obtain structural clues for selectivity against 5ꢀHT3R, we
constructed a human 5ꢀHT3R homology model based on Xꢀray crystallized mouse structure.
Figure 4a (left panel) displays the predicted docking pose of 16 in 5ꢀHT3R homology model. In
the predicted binding mode, the pyrido[2,3ꢀe][1,2,4]triazolo[4,3ꢀa]pyrazine scaffold is expected
to form piꢀpi interaction with Trp85, CHꢀpi with Ile66 and strong hydrogen bond with Arg87.
Moreover, the halogen bonding between the chlorine atom and side chain oxygen of Asp199 is
also observed at a distance 3.5Å. The triazole moiety is embedded between Val202 and Met223
via CHꢀpi or hydrophobic interaction. Compound 16 showed very similar interaction patterns in
5
ꢀHT3R with those in H4R. However, the basic amine of Nꢀmethylpiperazine is oriented for
making cationꢀpi interactions with Trp178 and Tyr229 in 5ꢀHT3R, while it forms ionic
interaction with Asp94 in H4R. These interactions of basic amine in 5ꢀHT3R are consistent with
previous observation that a cationꢀpi interaction between the primary amine of serotonin and
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Trp178 (Trp183 in mouse 5ꢀHT3R) critically contributes to ligand recognition of 5ꢀHT3R.
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In aqueous media, nonꢀcovalent interactions between protein and ligand must compete with the
desolvation penalty of ligand. The negative contribution on binding affinity of desolvation
energy is further maximized when the ligand binds to the aromatic box such as Trp178, Tyr229
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and Tyr148 in 5ꢀHT3R. In the previous study, Salonen and coꢀworkers reported that the
stepwise Nꢀalkylation of ammonium ions making cationꢀpi interactions at active site of Factor
4
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Xa dramatically improved binding affinity, which was related to desolvation cost of ligands.
These reports prompted us to calculate desolvation energies of basic amines including
methylpiperazine and Nꢀalkylated azetidines. As shown in Figure 4a (right panel), the
desolvation energies, minus of solvation energies, are strongly correlated with % inhibition at 10
µM against 5ꢀHT3R. In addition, the negative contribution of hydrophilicity on the inhibitory
activity against 5ꢀHT3R is supported by calculated ACD logD7.4 values. Surprisingly, the Nꢀ
methylꢀ3ꢀaminoꢀazetidine of 37 displayed significantly reduced inhibitory activity against 5ꢀ
HT3R compared to 16 while maintaining potent inhibitory activity against H4R. The Nꢀmethylꢀ
3
ꢀaminoꢀazetidine of 37 has much higher desolvation energy than Nꢀmethylpiperazine of 16 and
the high desolvation cost of 37 could not be overcome by the energy gain in cationꢀpi
interactions with 5ꢀHT3R. On the contrary, Nꢀmethylꢀ3ꢀaminoꢀazetidine of 37 forms strong ionic
interaction with Asp94 in H4R, and it would be sufficient to compensate for the desolvation
penalty of the basic amine. All the molecular pairs in Table 3 indicate that the Nꢀmethylꢀ3ꢀ
aminoꢀazetidine group has better selectivity against 5ꢀHT3 than Nꢀmethylpiperazine without
exception.
From the predicted binding mode and interaction pattern of 16 in H4R and 5ꢀHT3R (Figure 4b),
we hypothesized another modification point to improve selectivity against 5ꢀHT3R. In the
interaction modes in H4R and 5ꢀHT3R, triazole of 16 (I part in Figure 4b) forms a piꢀpi
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interaction with Tyr95, Phe168 and Tyr319 in H4R while it makes hydrophobic interaction via
CHꢀpi interaction with Met223 and Val202 in 5ꢀHT3R. To utilize this discrepancy in interactions
with H4R and 5ꢀHT3R for selectivity improvement, we modified triazole ring of 16 and
summarized assay results in Table 3. Compound 43 and 46 showed decreased inhibitory
activities against H4R relative to those of the parent 16 and 37 without improving selectivity
over 5ꢀHT3R. It was thought that the relative positions of three nitrogen atoms on triazole ring
negatively affect to the displaced piꢀpi interaction with Phe168 (Figure 3). However, the
replacement of triazole with a tetrazole ring (44 and 47) clearly showed the improvement of
selectivity over 5ꢀHT3R without loss of inhibitory activity against H4R. The tetrazole ring is
expected to weaken hydrophobic or CHꢀpi interaction with Met223 and Val202 in 5ꢀHT3R
relative to triazole ring. Eventually, we derived compound 48 as a lead compound by introducing
bromine instead of chlorine atom to reinforce halogen bond, which exhibits strong inhibitory
activity against H4R and excellent selectivity over H3R as well as 5ꢀHT3R.
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Chemistry
Compounds 10ꢀ48 were synthesized through synthetic routes summarized in Schemes 1ꢀ6.
Intermediates, 2,3ꢀdichloropyrido[2,3ꢀb]pyrazine analogues were obtained from corresponding
pyridineꢀ2,3ꢀdiamines and diethyl oxalate, and followed by chlorination (Scheme 1). Triazole
ring formation progressed from condensation with corresponding hydrazinyl compounds and
trimethyl orthoformate (or triethyl orthoformate). And then, amination was performed from
2
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respective amines with or without base (General procedure A in Scheme 2). According to
chemical reactivity of intermediates shown in Scheme 2, triazole ring formation and amination
was conducted in reverse order (General procedure B). 13 was prepared as described in Scheme
2
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3. 13b, which was a mixture of regioisomer was used without further purification. Desired 13c
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was purified by column chromatography after the chlorination. The final step was performed
through copperꢀcatalyzed amination. 27ꢀ42 were prepared as shown in Scheme 4. 29, 33, 36, and
39 were obtained by additionally reductive alkylation from 30, 35, 37, and 40, respectively. 43-
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4
8 were prepared by Scheme 5 and 6. Intermediate, 43f was produced by bromination,
palladiumꢀcatalyzed cyanation, followed by ring closure, and chlorination. Each final step
progressed in the same way as Scheme 2. 44, 45, 47, and 48 were obtained from cyclization
using sodium azide in good yield (69ꢀ81%). It is recommended to be aware that intermediates
1
0b, 12b, 13d, 14d, and 43f should be handled carefully during synthesis because their
4
4
molecular structures are similar to known substances that induce chloracne.
Profile of compound 48
The lead compound 48 displayed a potent inhibitory activity against human H4R with an IC of
5
0
0
.027 ꢁM while it did not show any noticeable binding affinity to other subtypes of histamine
receptors, H1R, H2R, and H3R (Supporting Information, Table S1). In addition, the competitive
binding assay against a wider panel of GPCR, ion channel, and transporters at the concentration
of 10 ꢁM revealed that compound 48 is highly selective for H4R (Table S1). The inhibitory
activity of 48 against mouse H4R (IC = 0.29 ꢁM) was about 10 times weaker than that for
50
human H4R. The pharmacological differences in species have been known to result from
45
significant sequence variation between human and mouse H4R (68%). In order to evaluate the
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functional behavior of compound 48 for human and mouse H4R, [ S]GTPγS binding assay was
performed in parallel with H4R agonist histamine and H4R antagonist thioperamide (mouse) or
46
JNJꢀ10191584 (human). Tested in antagonist mode, compound 48 showed a potent antagonism
for both human and mouse H4R with IC value of 0.028 ꢁM and 0.69 ꢁM, respectively, while
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no activity for human and mouse H4R was observed in agonist mode (data not shown). The
degrees of protein binding in human and mouse plasma are 73.5% and 65.2%, respectively.
The pharmacokinetic profiles of compound 48 and its precursors were assessed in ICR mice after
oral administration (Table 4 and Figure 5). The plasma exposure of compound 16 at 50 mg/kg
was dramatically increased compared to compound 10, possibly due to improved mouse
metabolic stability by inserting nitrogen atom in 6ꢀposition of the A ring (Table 1). Its plasma
levels reached a Cmax of 4.63 ꢁg/mL at 0.5 h (Table 4). At the same dose, compound 48 showed
higher plasma exposure than 16 with a maximum plasma levels reaching a Cmax of 7.69 ꢁg/mL at
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.5 h. There was no difference in pharmacokinetic properties between ICR and BALB/c mice
(data not shown). The doseꢀdependent increase in systemic exposures (AUC) was also confirmed
at doses of 2, 10 and 50 mg/kg.
In vivo efficacy of compound 48
4
7ꢀ49
Because histamine is a major mediator in the pruritus of AD,
antiꢀpruritic effect of
compound 48 was evaluated using the histamineꢀinduced pruritus model. Intradermal injection of
histamine increased the incidence of scratching in mice, and the number of scratching was
significantly decreased by oral administration of compound 48 in a dose dependent manner
3
5
(
Figure 6a). In the [ S]GTPγS functional assay, compound 48 showed the inhibitory activity
against mouse H4R with an IC of 0.69 µM, and it exhibited about 50% reduction of number of
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scratching at 10 mg/kg in the histamineꢀinduced pruritus model where the free concentration of
compound 48 is 1.89 µM. Therefore, the plasma free concentration required for 50% reduction
of scratching is about 3ꢀfold higher than the in vitro IC value. Figure 6a clearly shows that the
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antiꢀpruritic efficacy of compound 48 is correlated well with its plasma free concentration and in
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vitro activity. Compound 48 completely suppressed the histamine induced scratching behavior at
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0 mg/kg. Antiꢀpruritic effect of compound 48 was also evaluated in the different acute pruritus
mouse models mediated by substance P and compound 48/80 (C48/80) known as histamineꢀ
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7ꢀ49
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dependent triggers.
Oral administration of compound 48 dramatically decreased scratching
behavior at 50 mg/kg (Figure 6bꢀc) in both models as observed in histamineꢀinduced model.
These results suggest that our lead compound 48 efficiently suppresses itching induced by
various pruritogens associated with AD.
We also evaluated antiꢀpruritic and antiꢀinflammatory efficacy of compound 48 in the
oxazoloneꢀinduced murine AD model displaying structural barrier alterations and clinical signs
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highly similar to human AD. First, chronic AD was induced by repeated challenge with
oxazolone in the ear skin of BALB/c mouse as described in experimental section. And then,
compound 48 was orally administered twice a day for 3 weeks, and its antiꢀpruritic and antiꢀ
inflammatory efficacy were evaluated by counting the number of scratching and by measuring
ear thickness, respectively. Challenge of oxazolone to the mice significantly increased scratching
behavior and ear thickness, whereas treatment of compound 48 at 50 mg/kg significantly reduced
scratching behavior and ear thickness (Figure 7aꢀb). In addition, the histological examination of
challenged ear skin also revealed that compound 48 profoundly reduced epidermal thickness and
hyperplasia shown in AD skin lesions (Figure 7c). In summary, our lead compound 48 presented
dual antiꢀpruritic and antiꢀinflammatory effects in the in vivo pruritus and dermatitis murine
models.
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CONCLUSION
Here, we have detailed the lead identification and optimization process of a novel class of potent
and highly selective H4R antagonist. Firstly, hit compound 9 with a novel tricyclic scaffold has
been found from virtual screening using a set of knowledgeꢀbased pharmacophore models. The
construction of representative subset library by iterative clustering allowed us to efficiently
screen the large ZINC database without loss of structural diversity. The molecular modification
was supported by homology modelꢀbased design, leading to the rapid identification of compound
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6 with dramatically improved inhibitory activity for H4R and metabolic stability. However,
compound 16 exhibited the selectivity issue for 5ꢀHT3R as shown in many developed H4R
antagonists due to the very similar ligand recognition pattern of 5ꢀHT3 receptor with H4R. A
structural clue to avoid this selectivity issue was obtained by comparison of interaction modes
predicted in homology model of H4R and 5ꢀHT3R, and the desolvation penalty of positively
charged amine forming cationꢀpi interaction as in 5ꢀHT3R could be a good target point for
selectivity improvement. To our best knowledge, this is the first report improving inhibitory
activity and selectivity based on structural rationale derived from homology modelling of H4R
and 5ꢀHT3R. The final lead compound 48 has a structurally unique scaffold and was illustrated
as a highly potent and selective H4R antagonist without noticeable activity on offꢀtargets
including other histamine receptors and 5ꢀHT3R. In the in vivo test, 48 exhibited a good
pharmacokinetic profile and remarkably reduced the number of scratching in acuteꢀitching
murine models induced by pruritogens such as histamine, substance P and C48/80. Furthermore,
compound 48 showed the significant antiꢀpruritic and antiꢀinflammatory efficacy in oxazoloneꢀ
induced murine model mimicking human AD. In conclusion, compound 48 is expected to be a
promising candidate for the treatment of AD.
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EXPERIMENTAL SECTION
Chemistry
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Commercially available reagents and solvents were used without further purification. Hit
compound 9 was purchased from Synnovator. Thin layer chromatography (TLC) analyses were
1
13
carried out on Merck Kieselgel 60F254 plates or NH plates of Fuji Silysia, Ltd.. H and C NMR
spectra were verified by a Bruker DRX 300 (300 MHz), a Bruker Avance III 400 (400 MHz)
using tetramethylsilane and residual solvents as internal standards. Chemical shifts (δ) are
expressed as parts per million (ppm) and coupling constants (J) as herz (Hz). Resonance patterns
are reported with the notation s (singlet), d (doublet), t (triplet), q (quartet), dd (doublet of
doublets), td (triplet of doublets), br (broad peak), brs (broad singlet) and m (multiplet). Some
carbon peaks of piperazine ring are frequently merged with DMSO peak or overlapped each
other and not observed. Analytical reverseꢀphase ultraꢀhighꢀperformance liquid chromatography
mass (UPLCꢀMS) was carried out on Ascentis Express C18 column (50 × 2.1 mm, 2.7 ꢁm) using
Waters Acquity UPLC and PDA instruments and highꢀperformance liquid chromatography mass
(HPLCꢀMS) was carried out on ACE 5C18 column (50 × 4.6 mm, 5.0 ꢁm) using Waters 2767
LC/MS system. Normal phase and reverseꢀphase flash column chromatography were performed
with flash cartridge with Yamazen medium pressure liquid chromatography (MPLC) system,
FRꢀ260 and Biotage SNAP cartridges equipped with Isora One, respectively. The yields reported
were not optimized. The purities of all compounds are >95% at two wavelengths (225 and 254
nm) and are determined on Xbridge column (150 × 4.5 mm, 5 ꢀm) equipped with HPLC,
Shimadzu LCꢀ2010A system. Analytical conditions are as follows (Eluent A: CH CN ꢀ 1% to
3
100%, 0.1% TFA containing, gradient; Eluent B: H O ꢀ 99% to 0%, 0.1% TFA containing,
2
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o
gradient; analytical time: 20 min; oven temperature, 25 C; flow rate, 1.0 mL/min). Highꢀ
resolution mass (HRMS) was measured with JEOL, JMSꢀ700 spectrometer. Synthetic procedures
and analytic data for compound 9ꢀ48 were described in Supporting Information.
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Pharmacophore Model
The structures of seven known H4R antagonists except 4 (Figure 1) were built using the
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Schrödinger suite implemented in Maestro molecular modeling software. After ionized at pH =
.0 ± 2.0 using Epik module, the structures were minimized using the PolakꢀRibiereꢀconjugateꢀ
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gradient method with default parameters in MMFFs force field. The conformational searches
were done using Systematic PseudoꢀMonte Carlo (SPMC) method by applying the generalized
Born/solvent accessible surface (GB/SA) continuum solvation model. The lowest energy
conformation for each H4R antagonist was validated by analyzing distribution for torsional
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angles of similar structures stored in the CSD (Cambridge Structure Database). Based on prior
knowledge of scaffold alignment and SAR analysis of analogues from original literature (data
not shown), a pool of potential common feature pharmacophore hypotheses for each lowest
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energy conformation was created using Discovery Studio, assuming that the biologically active
conformations are fairly close to the global minimum energy conformations. Five functional
features were considered in assigning pharmacophore variants: hydrogen bond acceptor,
hydrogen bond donor, hydrophobic group, aromatic ring, and positively charged group.
Library Preparation and Virtual Screening
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A representative subset of all purchasable ZINC database was created using pipeline pilot. All
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purchasable molecules of the ZINC12 database containing ~22 million were sorted by
ascending molecular weight. Tanimoto similarity index of the first ranked molecule and the rest
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of molecules was calculated, and the molecules with similarity higher than a specified cutoff, 0.9
were classified into the same cluster. It was iteratively conducted to all remaining compounds of
the database. Finally, the most diverse of 1,126 clusters were created with the least redundancy.
As an inꢀhouse virtual screening set, total of 493,834 molecules were selected in proportion to
the size of each cluster. Conformational database for virtual screening was generated in
Discovery studio with default parameters (maximum 250 conformers and fast search method).
Virtual screening for a pool of pharmacophore models was conducted with fast search method.
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Homology Modeling and Molecular Docking
The H1R Xꢀray structure (pdb code: 3RZE) was selected as a template structure for H4R
homology model and was obtained from the RSCB (Research Collaboratory for Structural
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Bioinformatics) Protein Data Bank (http://www.rcsb.org/). The T4ꢀlysozyme inserted to assist
crystallization was removed and was not modeled. The amino acid sequence of human H4R
(accession number: Q9H3N8) was retrieved in UniProtKB sequence database
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(
http://www.uniprot.org). The target and template sequences were aligned using ClustralW
method, followed by manual adjustment to avoid gaps in transmembrane. Threeꢀdimensional
structure of human H4R was generated with adjusted alignment result and default parameters in
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Prime of Schrödinger suite. The loops were refined using the refine loops tool with the
extended highꢀloop refinement procedure. Five models were generated and were scored by
probability density function (PDF) total energy. The model with the lowest energy value, which
indicates the highest similarity with the template, was selected as a final model. The lowest
energy conformation of compound 3 was aligned to the 3D structure of doxepin, a ligand bound
to H1R in Xꢀray structure. The initial structure of H4R–compound 3 complex was minimized
using CHARMm implemented in Discovery studio with all backbone atoms fixed. Through the
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subsequent molecular dynamics simulation performed with restraints on protein backbone atoms
in the NPT ensemble for 10ns, we derived the final homology model of human H4R complexed
with compound 3 which was used to generate pharmacophore finding hit compound 9. The
centroid coordinates of compound 3 within refined homology model were used for generating
the receptor grid box to define ligand binding site for molecular docking. The hit compound 9
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was docked into grid box using standard precision mode of Glide program in Schrödinger suite.
The OPLS_2005 force field and all default parameters were applied during docking calculation.
The docking pose with the lowest Glide score was minimized again with CHARMm program
and used for analogue docking. The human 5ꢀHT3R homology model (accession number:
P46098ꢀ1) of extracellular Nꢀterminal domain that comprises the orthosteric ligandꢀbinding site
was built based on the mouse 5ꢀHT3R Xꢀray structure (pdb code: 4PIR) in the same way as
described in H4R homology model.
PAINS
All of the biologically tested compounds (Table 1ꢀ3) were checked for the presence of PAINS
56
substructures using PAINSꢀRemover online web server. None of the compounds were
classified as a panꢀassay interference compound.
Calculation of Solvation Energy
The conformation search of the protonated amine structure was performed utilizing MMFFs
force field. Conformations within 2 kcal/mol of the observed lowest conformation were further
optimized with MP2/6ꢀ31G** basis set using the density functional theory (DFT) program
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Jaguar in Schrödinger package. For each amine structure with the lowest single point energy,
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electrostatic solvation energy in water was computed using the standard PoissonꢀBoltzmann
continuum solvation model in Jaguar program.
Histamine Receptor Radioligand Binding Assay
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Competitive radioligand binding assays for H3R and H4R were performed using the
commercially available membrane preparation of CHOꢀK1 cells that express human or mouse
H4R and human H3R (Perkin Elmer, ESꢀ393ꢀM400UA for human H4R, ESꢀ392ꢀM400UA for
3
human H3R, and multispan, Cm1030 for mouse H4R). [ H]Histamine (Perkin Elmer, NET732)
3
was used for H4R binding assay and [ H]Nαꢀmethylhistamine (Perkin Elmer, NET1027) was for
H3R binding assay. All binding assays were performed in 50 mM TrisꢀHCl buffer (pH 7.4 at
room temperature) in the presence or absence of competing ligands. The mixtures of cell
membrane lysate, radioligand and competing ligands were incubated in a total assay volume of
2
00 µL in 96ꢀwell plates for 30 min at 27 °C and then harvested on 96ꢀwell glass fiber plates
(Millipore, MSFBN6B50) that were pretreated with 0.5% PEI, followed by washing for three
times using iceꢀcold 50 mM TrisꢀHCl buffer (pH=7.4 at 4 °C). The radioactivity retained on the
filters was measured by liquid scintillation counting in a Microbeta counter (PerkinꢀElmer Life
and Analytical Sciences, Inc., USA).
Off-Target Selectivity Assay of Compound 48
Binding affinities to various proteins including receptors, ion channels and transporters were
assessed based on competitive radioligand binding technique. These assays were performed by
Eurofins Cerep, in study number 100040480 (Celle l'Evescault, France). The experiment was
accepted in accordance with Eurofins validation Standard Operating Procedure.
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GTPγ S Functional Assay of Compound 48
3
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The functional activity on recombinant mouse and human H4R was evaluated using GTPγ S
binding assays. The agonistic and antagonistic activities of compound 48 were assessed using
reference agonist (Rꢀαꢀmethyl histamine for both of human and mouse H4R) and reference
antagonists (JNJ10191584 for human and thioperamide for mouse H4R). The assays were
performed by Euroscreen FAST, Ogeda SA (Belgium).
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Intrinsic Clearance (CL ) Determination in Microsomes
int
The metabolic stability of each compound was evaluated in pooled liver microsomes of CDꢀ1
mice and humans. Test compounds (5 ꢁM) were incubated with mice (Corning, 452701) and
human (Corning, 452117) liver microsomes activated with 1 mM NADPH, and loss of
compound was followed at 0, 10, 20, and 30 min. The percentages of remaining compounds
were determined by UPLCꢀUV. Intrinsic clearance (CL ) based on microsomal protein
int
concentration was calculated from the elimination rate constant assuming first order kinetics.
Solubility Assay
FaSSIF (pH=6.5) and SGF (pH=1.2) solutions of test compounds were incubated at 37 °C for 0.5
and 2 h, respectively. Samples were collected using a 0.45 ꢁm syringe filter and the
concentration was measured by UPLCꢀUV.
Animals
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All experiments were conducted in accordance with the National Research Council’s Guidelines
(
IACUC, Korea) for the Care and Use of Laboratory Animals. The experimental protocol was
approved by the Animal Experiments Committee of Sungkyunkwan University.
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PK Study in ICR Mice
Pharmacokinetic studies were performed on ICR mice (n=3 for each group). After oral
administration of test compounds, blood was sampled at 0.5, 1, 2, 4, 7, and 24 h post dose.
Plasma concentrations were quantitated by LCꢀMS/MS. Pharmacokinetic parameters were
calculated from nonꢀcompartmental analysis using Phoenix WinNonlin (Pharsight, Palo Alto,
CA).
Pruritogen-induced Acute Pruritus in Mouse and Quantification of Scratching Behavior
Pruritic responses to histamine, substance P and compound 48/80 (C48/80) were examined in
female ICR mice (n=8ꢀ10 per group). For the induction of scratching, the hair on the back skin
was clipped 24 h before intradermal injection of pruritogens. Mice were received an oral dose of
compound 48, dissolved in a vehicle of 20% hydroxylpropylꢀβꢀcyclodextrin, 30 min before
intradermal injection of histamine (300 nmol), substance P (100 nmol) and C48/80 (100 ꢀg),
respectively. Histamine, substance P and C48/80 (Sigma Chemical Co.) were dissolved in saline.
Immediately after intradermal injection of histamine, the mice were returned to an acrylic cage
(approximately 30 × 30 × 30 cm, access production) and scratching responses were videoꢀ
recorded from above and was quantified by counting the number of bouts of scratching for 20
min immediately after the intradermal injection. A bout of scratching was defined as 3 or more
rapid back and forth motions of the hindpaw directed to the site of sensitization
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Oxazolone-induced AD Model
The hair on the abdominal skin of each female BALB/c mice (n=8 per group) was shaved with a
clipper 1 day before experiments. Mice were then sensitized on day 0 by the topical single
application of 50 ꢁL of 1% oxazolone (Sigma Chemical Co.) in acetone/corn oil (4:1) on the
shaved abdominal skin. On day 7, 25 ꢁL of 0.2% oxazolone and acetone/corn oil vehicle was
applied repeatedly to the right and left ear, respectively, 3 times per week. Compound 48 was
orally administered 30 min before the challenge, twice a day for 3 weeks. Scratching behavior
was quantified by counting the number of bouts of scratching for one hour after application of
oxazolone to the ear on day 25, as described above. On days 27, the ear thickness was measured
with the dial thickness gauge (IDC1012, Mitutoyo) and the ears were removed for analysis.
Change in ear thickness (ꢂ ear thickness) was expressed by thickness of the right (oxazoloneꢀ
challenged ear) − thickness of the left ear (acetone/corn oilꢀchallenged ear). For histological
analysis of challenged ear skin, ear tissue was fixed with 4% paraformaldehyde, embedded in
paraffin and sectioned at 3 ꢀm. Sections were stained with hematoxylinꢀeosin.
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Statistical Analysis
All data are represented as the mean ± S.E.M. Student’s tꢀtest was used to compare two groups.
Dunnett’s test or KruskalꢀWallis test in GraphPad Prism 7.0 (GraphPad Software, Inc., San
Diego, CA) was used for multiple comparisons. Values of p < 0.05 were considered statistically
significant.
ASSOCIATED CONTENT
Supporting Information
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Selectivity data of compound 48 against offꢀtargets; synthetic procedures and analytical data
for compound 9ꢀ48 (PDF)
Molecular formula strings and some data (CSV)
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Predicted binding mode of compound 16 in H4R homology model (PDB)
Predicted binding mode of compound 16 in 5ꢀHT3R homology model (PDB)
AUTHOR INFORMATION
Corresponding Author
* Eꢀmail: chpark@cnclab.co.kr
Author Contributions
$
These authors contributed equally to this work (coꢀfirst author).
The manuscript was written through contribution of all authors. All authors have approved to the
final version of manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
We would like to express our great gratitude to Drs. Tatsumi Yamazaki, JaeꢀKwang Chun, and
Hisafumi Okabe for insightful scientific discussion and the proofing of this manuscript. This
work was supported by JW Pharmaceutical Co., Ltd. and Chugai Pharmaceutical Co., Ltd.
ABBREVIATIONS
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AD, Atopic dermatitis; DIPEA, N,Nꢀdiisopropylethylamine; EtOH, ethanol; H4R, histamine H4
receptors; 5ꢀHT3R, Human ligandꢀgated ion channel serotonin 5ꢀHT3 receptors; MeOH,
methanol; SAR, Structureꢀactivity relationships; TEA, triethylamine.
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REFERENCES
(
1) Jin, H.; He, R.; Oyoshi, M.; Geha, R. S. Animal models of atopic dermatitis. J. Invest.
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Etoh, T.; Mihara, R.; Yoshida, H.; Stewart, J.; Kabashima, K. Antiꢀinterleukinꢀ31 receptor A
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(
10) Leurs, R.; Smit, M. J.; Timmerman, H. Molecular pharmacological aspects of histamine
receptors. Pharmacol. Ther. 1995, 66, 413ꢀ463.
11) Hough, L. B. Genomics meets histamine receptors: new subtypes, new receptors. Mol.
Pharmacol. 2001, 59, 415ꢀ419.
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antagonists: the new antihistamines? Curr. Opin. Investig. Drugs 2004, 5, 1174ꢀ1183.
13) de Esch, I. J.; Thurmond, R. L.; Jongejan, A.; Leurs, R. The histamine H4 receptor as a new
(
(
(
therapeutic target for inflammation. Trends Pharmacol. Sci. 2005, 26, 462ꢀ469.
(14) Rossbach, K.; Schaper, K.; Kloth, C.; Gutzmer, R.; Werfel, T.; Kietzmann, M.; Baumer, W.
Histamine H4 receptor knockout mice display reduced inflammation in a chronic model of atopic
dermatitis. Allergy 2016, 71, 189ꢀ197.
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