Synthesis and evaluation of a 2-benzothiazolylphenylmethyl ether class of histamine H4 receptor antagonists

Article abstract of DOI:10.1016/j.bmcl.2016.09.049

Synthesis and biological evaluation of a new class of histamine H₄receptor ligands, distinct from the previously reported chemotypes, are described. A virtual screening of our corporate compound collection identified a hit with an undesired dual H3R/H4R activity. Chemical exploration led to the discovery of a more potent and selective 2-benzothiazolylphenylmethyl ether lead compound.

Full text of DOI:10.1016/j.bmcl.2016.09.049

Accepted Manuscript
Synthesis and evaluation of a 2-benzothiazolylphenylmethyl ether class of his-
tamine H4 receptor antagonists
Olivier Labeeuw, Nicolas Levoin, Xavier Billot, Denis Danvy, Thierry Calmels,
Stéphane Krief, Xavier Ligneau, Isabelle Berrebi-Bertrand, Philippe Robert,
Jeanne-Marie Lecomte, Jean-Charles Schwartz, Marc Capet
PII:
S0960-894X(16)30991-X
http://dx.doi.org/10.1016/j.bmcl.2016.09.049
BMCL 24273
DOI:
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
7 July 2016
16 September 2016
17 September 2016
Please cite this article as: Labeeuw, O., Levoin, N., Billot, X., Danvy, D., Calmels, T., Krief, S., Ligneau, X., Berrebi-
Bertrand, I., Robert, P., Lecomte, J-M., Schwartz, J-C., Capet, M., Synthesis and evaluation of a 2-
benzothiazolylphenylmethyl ether class of histamine H4 receptor antagonists, Bioorganic & Medicinal Chemistry
Letters (2016), doi: http://dx.doi.org/10.1016/j.bmcl.2016.09.049
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Synthesis and evaluation of a 2-benzothiazolylphenylmethyl ether class of
histamine H4 receptor antagonists
Olivier Labeeuw,* Nicolas Levoin, Xavier Billot, Denis Danvy†, Thierry Calmels,
Stéphane Krief, Xavier Ligneau, Isabelle Berrebi-Bertrand, Philippe Robert, Jeanne-Marie
Lecomte, Jean-Charles Schwartz, Marc Capet
Bioprojet-Biotech, 4 rue du Chesnay Beauregard, BP 96205, 35762 Saint-Grégoire, France
† Present address: Interor, IRCOF, UMR 6014 CNRS LFAOC, Faculté des Sciences et techniques, 1 rue
Tesnière, 76821 Mont Saint-Aignan Cedex, France
Abstract:
Synthesis and biological evaluation of a new class of histamine H4 receptor ligands, distinct
from the previously reported chemotypes, are described. A virtual screening of our
corporate compound collection identified a hit with an undesired dual H3R/H4R activity.
Chemical exploration led to the discovery of a more potent and selective 2-
benzothiazolylphenylmethyl ether lead compound.
Keywords: Histamine, Histamine H4 receptor, asthma, atopic dermatitis
Since its characterization and cloning in 2000 by Nakamura et al.1 and Liu et al.2 the
histamine H4 (H4R) receptor has been widely studied and reviewed.3 This G-protein-
coupled receptor is mainly expressed on a variety of immune cells such as eosinophils,
dendritic cells, mast cells and leukocytes4 and represent an interesting pharmacological
target for autoimmune or inflammatory disorders. Figure 1 describes a selection of H4R
ligands that already entered clinical trials for the treatment of asthma, rheumatoid arthritis,
atopic dermatitis and psoriasis.5
NH2
NH2
N
N
N
N
N
N
N
N
H
N
N
N
N
NH2
N
H
H
N
H
Toreforant/JNJ-38518168
JNJ-39758979
Phase II (discontinuated)
PF-3893787/ZPL-3893787
Phase II
Phase II
Figure 1. A selection of histamine H4 ligands in clinical trials.
2

In this paper we describe the discovery of a new type of H4R ligands, distinct from the
previously reported chemotypes.6 We started our research program by a virtual screening of
our corporate compound collection based on a 3D model of the receptor.7 Representative
elements of the more promising series were then evaluated in an in vitro hH4R
[35S]GTPS binding assay and among several hits we identified compound 1 (Kb= 19 nM)
(Figure 2) as a very interesting starting point for a medicinal chemistry program. We first
disclosed this 3-(1H-imidazol-4-yl)propyldiphenylmethyl ether in 1996 as a selective (over
histamine H1 and H2 receptors) and potent histamine H3 receptor antagonist (hH3 binding
Ki= 17 nM).8 The fourth histamine receptor was unknown at this time. A dual H3R/H4R
activity is not surprising since these two paralogues are found to share the highest degree of
homology within the histamine receptor family.1 Several other research groups also
identified H3R ligands with H4R affinity.9
O
HN
N
Compound 1
hH4R Kb= 19 nM
hH3R Ki= 17 nM
selectivity: 0.9
Figure 2. Hit compound
Here we report our efforts to design potent histamine H4 ligands with a better selectivity
over the histamine H3 receptor. We first investigated the replacement of one phenyl group
by a small selection of aryl or hetaryl rings. Thus we prepared different benzydrol analogs
by condensation of various aryl carboxaldehydes with aryl or hetaryl lithium or Grignard
reagents according to standard methods. These intermediates were chlorinated with SOCl2
and the resulting chlorides treated with 3-(1H-imidazol-4-yl)propan-1-ol hydrochloride10 to
afford compounds 2-6 (Scheme 1).
3

Ar, Het
Ar, Het
c
a, b
ArCHO
or
HetCHO
Cl
O
HN
N
N
S
N
N
N
1
2
3
4
5
6
Scheme 1. Synthesis of compounds 1-6. Reagents and conditions: (a) PhMgCl, THF, rt or 40°C, 1h or
benzothiazole/BuLi, THF, -78°C, 2h (b) SOCl2, DCM, rt, 2h; (c) 3-(1H-imidazol-4-yl)propanol
hydrochloride, ACN, reflux, 24h.
Histamine H4 receptor binding affinities of compounds 1-6 were evaluated in a
[35S]GTPS binding assay (stimulation with the hH4R agonist Imetit) on membranes of
SH-SY5Y cells stably expressing the human H4 receptor. Results are reported in Table 1.
In this test, ligands 1-15 proved to be neutral antagonists (intrinsic activities of zero).
Table 1
hH4R binding data for the aryl or hetaryl selection (compounds 1-6).
Ar, Het
O
HN
N
hH4R
Kb (nM)
Compound
Ar, HetAr
19
17
1
2
3
100
4

S
N
1.1
29
32
4
5
6
N
N
N
Among the first synthesized ligands, the 2-benzothiazolyl group (compound 4) proved to be
the more promising hetaryl ring with a tenfold improved affinity for H4R in comparison
with the phenyl of the hit compound. Figure 3 describes the possible binding mode of 4 at
the H4R. The imidazole could form a salt bridge with Aspartate 94 of TM3, the oxygen of
the linker a H-bond with the phenol moiety of Tyrosine 95 (TM3) and the nitrogen atom of
benzothiazolyl group would establish another H-bond with Tyrosine 319 (TM6).
Figure 3. Detail of the predicted binding mode of compound 4. The binding site is represented by its
electrostatic potential on the Connoly surface. Several residues are hidden to improve the visibility.
5

We logically kept this benzothiazole to explore the western part of the scaffold. We
investigated a shorter linker (compounds 7 and 8), N- or C-methylated imidazoles
(compounds 9-12) and N-imidazole branched linkers (compounds 13-15).
The synthesis of compounds 7 and 8 are described in scheme 2. Etherification of
commercially available (1H-imidazol-4-yl)methanol and 2-(1H-imidazol-4-yl)ethan-1-ol
with benzothiazol-2-yl(phenyl)methanol was performed in the presence of p-
toluenesulfonic acid and Dean-Stark apparatus.
Compounds 9-12 were prepared analogously to 7 and 8. Starting N(1), N(3), C(2) or C(5)
methyl-imidazol-4-yl-propan-1-ols were synthesized in a 3-step procedure from the
corresponding imidazole-carboxaldehydes (Scheme 3).
Ligands 13-15 were obtained by alkylation with imidazole of chlorinated intermediates
(Scheme 4).
N
S
N
n
S
n
a
( )
OH
, HCl
( )
+
HO
O
HN
N
HN
N
7-8
Scheme 2. Synthesis of compounds 7-8. Reagents and conditions: (a) APTS, toluene, reflux with Dean-Stark
apparatus, 20h.
6

N
S
3
3
R1
R1
d
R1
CHO
a,b,c
( )
( )
OH
N R4
O
N R4
R3 N
R3 N
N R4
R3 N
R2
R2
R2
9 (R1=Me, R2=H)
10 (R1=H, R2=Me)
11 (R3=Me, R4=/)
12 (R3=/, R4=Me)
Scheme 3. Synthesis of compounds 9-12. Reagents and conditions: (a)
(Carbethoxymethylene)triphenylphosphorane, toluene, reflux, 6h (b) H2, Pd/C 10%, MeOH, rt, 15h (c)
LiAlH4, THF, reflux, 3h (d) benzothiazol-2-yl(phenyl)methanol, APTS, toluene, reflux with Dean-Stark
apparatus, 20h.
N
n
S
N
S
N
n
S
a
b
( )
( )
Cl
O
HO
N
O
N
13-15
Scheme 4. Synthesis of compounds 13-15. Reagents and conditions: (a) 2-Chloroethan-1-ol (n=2) or 3-
chloropropan-1-ol (n=3) or 4-chlorobutan-1-ol (n=4), APTS, toluene, reflux with Dean-Stark apparatus, 20h;
(b) imidazole, DMF, 3-15h
Table 2
hH4R binding data for compounds 7-16.
7

N
S
n
( )
Im
O
hH4R
Kb (nM)
Compound
Im
n
1
2
3
3
399
3.7
1.1
1.6
7
8
4
9
HN
HN
HN
HN
HN
N
N
N
N
N
3
> 10 000
10
3
3
2
> 10 000
> 10 000
> 10 000
11
12
13
N
N
N
N
N
N
8

3
4
> 10 000
> 10 000
14
15
N
N
N
N
Reduced affinities of compounds 7 and 8 versus ligand 4 illustrate the progressive loss of
the salt bridge interaction of imidazole with D94 when linker is shortened. C5-methylated
imidazole (compound 9) displayed equivalent potency whereas the substitution of C2 is
sterically unfavorable (compound 10).11 What is more surprising is the loss of affinity for
N-methylated compounds 11 and 12. It is reminiscent of the discrepancy between binding
interactions of histamine (HA) and its metabolite tele-methylhistamine (tMeHA) at the
Histamine H3 receptor (Ki = 12.8nM for HA and Ki = 20µM for tMeHA)12 or at the
Histamine H4 receptor (Ki = 1.6nM for HA and Ki = 11µM for tMeHA).13 Analogously N-
imidazole branched linkers (compounds 13-15) are totally inactive at the H4R whatever the
alkyl length.
The Histamine H3 receptor binding affinity of the more promising ligand (compound 4 –
BP1.2106) was evaluated by displacement of [125I]-iodoproxyfan (IPX) binding to
membranes of HEK-293 cells stably expressing the human H3 receptor14 (Figure 4). Ligand
4 retained nearly comparable H3R binding affinity (34.0 nM versus 18.7 nM for hit
compound) but displayed improved hH3R/hH4R selectivity (31 versus 0.9).
N
O
S
HN
N
Compound 4 (BP1.2106)
hH4R Kb= 1.1 nM
hH3R Ki= 34 nM
selectivity: 31
Figure 4. hH3R/hH4R selectivity for compound 4
9

In summary, we have discovered a new class of histamine H4 receptor antagonists, distinct
from the previously reported chemotypes. Starting from a hit suffering from a dual
H3R/H4R activity, introduction of 2-benzothiazolyl group allowed to reach a nanomolar
hH4R affinity and an improved selectivity over hH3R. Thus compound 4 (BP1.2106) was
selected as a very promising starting point for lead generation. Further improvements will
be reported in due course
Acknowledgements: The authors gratefully acknowledge Catherine Dartois, Isabelle
Delimoge, Marie Noelle Legave, Isabelle Léger, Marcel Morvan, Aurélien Boudeau and
Jean-Claude Camelin for technical assistance in the synthesis and the evaluation of
compounds.
References and notes
1 Nakamura, T.; Itadani, H.; Hidaka, Y.; Ohta, M.; Tanaka, K. Biochem. Biophys. Res. Commun 2000, 279,
615.
2
Liu, C.; Ma, X.-J.; Jiang, X.; Wilson, S.J.; Hofstra, C.L.; Blevitt, J.; Pyati, J.; Li, X.; Chai, W.; Carruthers,
N.; Lovenberg, T.W. Mol. Pharmacol. 2001, 59, 420.
3
(a) Histamine H4 receptor: A novel drug target in immunoregulatory and inflammatory diseases; Stark, H.,
Ed, Versita, London, 2013; (b) Salcedo, C.; Pontes, C.; Merlos, M. Front. Biosci. 2013, 5, 178. (c) Marson,
C.M. Chem. Rev. 2011, 111, 7121; (d) Engelhardt, H.; Smits, R.A.; Leurs, R.; Haaksma, E.; de Esch, I.J.
Curr. Opin. Drug Discov. Devel. 2009, 12, 628; (e) Thurmond, R.L.; Gelfand, E.W.; Dunford, P.J. Nat. Rev.
Drug Discov. 2008, 7, 41; (f) Liu, W.L. Drug Discov. Today. 2014, 19, 1222; (g) Thurmond, R.L. Front
Pharmacol. 2015, 6,65.
4 Konttinen, Y.; Husu, H.; Han, X.; Beatrice Passani, M.; Ballerini, C.; Stegaev, V.; Sillat, T.; Mackiewicz, Z.
In Histamine H4 receptor: A Novel Drug Target For Immunoregulation and Inflammation; Stark, H., Ed,
Versita, London, 2013, Chapter 7.
5 (a) A Study of the Safety and Effectiveness of JNJ-39758979 in the Treatment of Adults With Persistent
Asthma (NCT00946569) (www.clinicaltrials.gov); (b) A Study of JNJ-39758979 in Adult Japanese Patients
With Moderate Atopic Dermatitis (NCT01497119) (www.clinicaltrials.gov); (c) A Study to Evaluate Safety
and Efficacy of Toreforant (JNJ-38518168) in Participants With Moderate to Severe Plaque-type Psoriasis
(NCT02295865) (www.clinicaltrials.gov); (d) A Dose Range Finding Study of JNJ-38518168 in Patients
With Active Rheumatoid Arthritis in Spite of Treatment With Methotrexate (NCT01679951)
(www.clinicaltrials.gov); (e) A Study to Determine the Efficacy of ZPL-3893787 in Subjects With Atopic
10

Dermatitis (NCT02424253) (www.clinicaltrials.gov); (f) A Study to Determine the Efficacy of ZPL-3893787
in Subjects With Plaque Psoriasis (NCT02618616) (www.clinicaltrials.gov).
6 (a) Schreeb, A.; Łażewska, D.; Dove, S.; Buschauer, A.; Kieć-Kononiwcz, K.; Stark, H. In Histamine H4
receptor: a novel drug target for immunoregulation and inflammation; Stark, H., Ed.; Versita, London, 2013,
Chapter 2. (b) Kiss, R.; Keseru, G.M. Expert Opin. Ther. Pat. 2014, 24, 1185.
7 Levoin et al., manuscript submitted.
8 Hüls, A.; Purand, K.; Stark, H.; Ligneau, X.; Arrang, J.-M.; Schwartz, J.-C.; Schunack, W. Bioorg. Med.
Chem. Lett. 1996, 6, 2013.
9 (a) Kobayashi, T.; Watanabe, M.; Yoshida, A.; Yamada, S.; Ito, M.; Abe, H.; Ito, Y.; Arisawa, M.; Shuto, S.
Bioorg. Med. Chem. 2010, 18, 1076. (b) Lim, H.D.; Istyastono, E.P.; van de Stolpe, A.; Romeo, G.; Gobbi, S.;
Schepers, M.; Lahaye, R.; Menge, W.M.; Zuiderveld, O.P.; Jongejan, A.; Smits, R.A.; Bakker, R.A.;
Haaksma, E.E.; Leurs, R.; de Esch I.J. Bioorg. Med. Chem. 2009, 17, 3987. (c) Wijtmans, M.; de Graaf, C.;
de Kloe, G.; Istyastono, E.P.; Smit, J.; Lim, H.; Boonnak, R.; Nijmeijer, S.; Smits, R.A.; Jongejan, A.;
Zuiderveld, O.; de Esch, I.J.; Leurs, R. J. Med. Chem. 2011, 54, 1693. (d) Więcek, M.; Kottke, T.; Ligneau,
X.; Schunack, W.; Seifert, R.; Stark, H.; Handzlik, J.; Kieć-Kononowicz, K. Bioorg. Med. Chem. 2011, 19,
2850. (e) Kottke, T.; Sander, K.; Weizel, L.; Schneider, E.H.; Seifert, R.; Stark, H. Eur. J. Pharmacol. 2011,
654, 200. (f) Igel, P.; Schnell, D.; Bernhardt, G.; Seifert, R.; Buschauer, A. Chem. Med. Chem. 2009, 4, 225.
(g) Geyer, R.; Buschauer, A. Arch. Pharm. 2011, 344, 775.
10 Stark, H.; Purand, K.; Hüls, A.; Ligneau, X.; Garbarg, M.; Schwartz, J.-C.; Schunack, W. J. Med. Chem.
1996, 39, 1220.
11 See the supplementary material for details of the binding mode of imidazole moiety.
12 Baker, J. G. BMC Pharmacol. 2008, 8:9.
13 Unpublished data.
14 (a) Ligneau, X.; Garbarg, M.; Vizuete, M. L.; Diaz, J.; Purand, K.; Stark, H.; Schunack, W.; Schwartz, J. C.
J. Pharmacol. Exp. Ther. 1994, 271, 452; (b) Ligneau, X.; Morisset, S.; Tardivel-Lacombe, J.; Gbahou, F.;
Ganellin, C. R.; Stark, H.; Schunack, W.; Schwartz, J.-C.; Arrang, J.-M. Br. J. Pharmacol. 2000, 131,
1247; (c) Ligneau, X.; Perrin, D.; Landais, L.; Camelin, J.; Calmels, T. P.; Berrebi-Bertrand, I.; Lecomte, J.
M.; Parmentier, R.; Anaclet, C.; Lin, J. S.; Bertaina-Anglade, V.; La Rochelle, C. D.; d’Aniello, F.; Rouleau,
A.; Gbahou, F.; Arrang, J.M.; Ganellin, C. R.; Stark, H.; Schunack, W.; Schwartz, J.-C. J. Pharmacol. Exp.
Ther. 2007, 320, 365.
11

N
O
S
O
HN
N
HN
N
Hit Compound
Compound 4
hH4R Kb= 19 nM
hH3R Ki= 17 nM
selectivity: 0.9
hH4R Kb= 1.1 nM
hH3R Ki= 34 nM
selectivity: 31
1