Identification and hit-to-lead exploration of a novel series of histamine H4 receptor inverse agonists

Article abstract of DOI:10.1016/j.bmcl.2010.02.097

The identification and hit-to-lead exploration of a novel, potent and selective series of histamine H₄ receptor inverse agonists is described. The initial hit, 3A (IC₅₀ 19 nM) was identified by means of a ligand-based virtual screening approach. Subsequent medicinal chemistry exploration yielded 18I which possessed increased potency (R-enantiomer IC₅₀ 1 nM) as well as enhanced microsomal stability.

Full text of DOI:10.1016/j.bmcl.2010.02.097

Bioorganic & Medicinal Chemistry Letters 20 (2010) 2516–2519
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Identification and hit-to-lead exploration of a novel series of histamine H4
receptor inverse agonists
a
a
a
a
a
Sue Cramp a, , Hazel J. Dyke , Christopher Higgs , David E. Clark , Matthew Gill , Pascal Savy ,
Neil Jennings a, Steve Price a, Peter M. Lockey a, Dennis Norman a, Soraya Porres a, Francis Wilson b,
Alison Jones b, Nigel Ramsden b, Raffaella Mangano b, Dan Leggate b, Marie Andersson b, Richard Hale b
*
a Argenta, 8/9 Spire Green Centre, Flex Meadow, Harlow, Essex, CM19 5TR, UK
b Cellzome Ltd, Chesterford Research Park, Little Chesterford, Cambridge, CB10 1XL, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
The identification and hit-to-lead exploration of a novel, potent and selective series of histamine H4
receptor inverse agonists is described. The initial hit, 3A (IC50 19 nM) was identified by means of a
ligand-based virtual screening approach. Subsequent medicinal chemistry exploration yielded 18I which
possessed increased potency (R-enantiomer IC50 1 nM) as well as enhanced microsomal stability.
Ó 2010 Elsevier Ltd. All rights reserved.
Received 22 December 2009
Revised 25 February 2010
Accepted 26 February 2010
Available online 3 March 2010
Keywords:
Hit-to-lead
Hiatamine H4
Since its relatively recent discovery, the histamine H4 receptor
(H4R) has been the focus of much attention.1 The H4R is primarily
expressed on cells that are involved in inflammation and immune
responses and there is mounting evidence that targeting this
receptor may provide a valuable approach in the treatment of var-
ious allergic diseases such as allergic airway inflammation and
pruritis.2 Additionally, it is believed that modulating the H4R may
have potential in the treatment of autoimmune disorders.3 Thus,
the identification of novel ligands for the H4R is a topic of consid-
erable research and clinical interest.
with good selectivity over the closely related H3R. In addition, the
compounds possess attractive lead-like qualities. On the basis of its
superior potency, 3A was selected as the starting point for hit-to-
lead exploration (Fig. 2).
Further profiling of compound 3A indicated that metabolic sta-
bility in rat microsomes was very low with only 1% parent com-
pound remaining after 10 min incubation. Although metabolite
S
We have previously reported the successful application of li-
gand-based virtual screening to the search for novel MCH-1 recep-
tor antagonists.4 In this work, a similar approach was adopted
based on the structures of two H4R antagonists that had been re-
ported at the time the work was commenced (Fig. 1).5
N
N
N
H
N
O
HN
N
H
N
A database of commercially available screening compounds was
compiled from the catalogues of various suppliers and then inter-
rogated using these two query compounds and a variety of virtual
screening techniques. A set of 1177 compounds was initially se-
lected from the results of these searches. Following removal of
duplicates and assessment by an experienced medicinal chemist,
405 compounds were chosen for purchase. Upon screening,6 two
series of particular interest were identified. Both compounds re-
sulted from a pharmacophore search based on 1. In terms of their
properties, the compounds show good potency at the H4R together
1: JNJ 7777120 (Ki = 38nM)
2: Thioperamide (Ki = 27 nM)
Figure 1. H4R antagonists used as queries for ligand-based virtual screening. Ki
values are taken from Ref. 5.
N
Cl
N
N
O
N
3A
IC50 19nM
* Corresponding author. Tel.: +44 0 1279 645-645; fax: +44 0 1279 645 646.
E-mail address: Sue.cramp@argentadiscovery.com (S. Cramp).
Figure 2. Selected hit compound from ligand-based virtual screening.
0960-894X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2010.02.097

S. Cramp et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2516–2519
2517
Table 2
identification was not carried out, we considered that N-demethyl-
Analogues with exocyclic basic centre
ation was most likely to be a major contributor to the poor stabil-
ity. Thus the initial focus of the optimisation programme was
aimed at modifications of the amine designed to increase the met-
abolic stability while retaining potency. The first approaches were
designed to increase the steric bulk around the piperazine nitrogen
in the hope that this might reduce the propensity for N-demethyl-
ation. The results obtained for some of the compounds identified in
the original library had suggested that groups larger than methyl
on the piperazine nitrogen would result in a loss of potency. This
was in agreement with the results reported by Johnson and John-
son around JNJ 7777120.5 Thus alternative approaches were inves-
tigated. These approaches included incorporation of a group
adjacent to the N-methyl (B and C), bridging across the piperazine
ring (D to F) and incorporation of the substituent on the nitrogen
into a ring (G). Of these compounds, the bridged analogues and
the bicyclic derivative showed moderate potency, while those ana-
logues with adjacent methyl groups were weakly active or inactive.
Compound 3D showed moderate potency and improved micro-
somal stability compared to 3A (Table 1) whereas, surprisingly,
the bicyclic derivative 3G was unstable in microsomes.
Amines:
*
*
*
*
*
*
*
N
N
N
N
N
N
N
H
N
H
N
H
N
N
H
J
I
I-R
L
I-S
M
*
N
H
N
NH2
N
Et
N
K
*
*
*
H
N
N
N
N
NH
NH
N
O
P
N
N
Cl
NR1
O
2
The second approach involved moving the basic centre exocy-
clic to the ring. Thus a series of 3-amino-pyrrolidines was prepared
(3H–3M, Table 2). The NH-methyl analogue 3I was the most potent
of the compounds prepared and also showed some improvement
in microsomal stability. It is interesting to note that either increas-
ing the size of the alkyl group on the nitrogen (3K) or incorporating
3
Compd
IC50 (nM) H4R
% Remaining at 10 min rat microsomes
3H
3I
3I-R
3I-S
3J
3K
3L
3M
3N
3O
3P
21,000
95
33
285
126
>10,000
1400
1400
>10,000
>10,000
87
18
80
92
103
107
—
—
—
—
—
an
a-methyl group (3L) resulted in a loss in potency, suggesting
that the binding pocket for the basic centre is quite narrow and
confirming the earlier observations from the library compounds.
The primary amine (3J) was also potent and had increased micro-
somal stability. Potency was retained if the ring size was reduced
to four atoms (3P) but completely lost when the ring size was in-
creased to six atoms (3N and 3O). The two individual enantiomers
of compound 3I were also prepared. It was found that the R-enan-
tiomer (3I-R) was more active than the S-enantiomer (3I-S). How-
ever, the S-enantiomer was found to be more stable in rat
89
microsomes with a half-life of 73 min compared to a half-life of
19 min for the R-enantiomer.
Table 1
With improvements identified in the stability by modification
to the amine, our attention switched to investigation of the substi-
tuent on the benzene ring (compounds 4–10, Table 3). It was found
that activity was retained with small lipophilic groups in the 8-po-
sition of the benzene ring (e.g., 5I) but no improvement was ob-
served in the microsomal stability. Larger (e.g., 6I) or more polar
groups (e.g., 7I to 9I) generally provided a further increase in sta-
bility but resulted in a loss in potency. Moving the chlorine atom
to the 7-position (10I-R) also resulted in a reduction of potency.
Analogues designed to block N-demethylation
Amines
*
*
*
*
*
N
N
N
N
N
N
N
N
N
N
N
A
B
C
D
*
*
N
N
N
Table 3
Modifications to the aromatic substitution
E
F
G
N
9
8
N
N
R2
7
N
Cl
NR1
O
2
NR1
6
O
2
3
Compd R2
IC50 (nM)
H4R
% Remaining at 10 min rat
microsomes
Compd
IC50 (nM) H4R
% Remaining at 10 min rat microsomes
4I-R
5I-R
6I
7I
8I
8-Me
8-CF3
8-Ph
8-CO2H
8-CN
8-
CONHMe
7-Cl
86
30
>10,000
>30,000
6200
74
59
97
80
95
—
3A
3B
3C
3D
3E
3F
3G
19
1900
10,000
111
169
440
1
1
nt
75
66
62
0
9I
>30,000
236
10I-R
171
—

2518
S. Cramp et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2516–2519
Table 4
2-methyl analogue with N-methylpiperazine as the amine (11A)
Modifications to the 2-position
was equipotent with the 2-unsubstituted analogue (3A). However,
when the amine was replaced by the N-methylaminopyrrolidine (I),
the 2-methyl derivative (11I) showed a significant loss of potency.
A number of other analogues were prepared with either N-methyl-
piperazine or N-methylaminopyrrolidine as the amine and a similar
pattern was observed. Small lipophilic groups in the 2-position
were tolerated with N-methylpiperazine as the amine (e.g., 13A
and 14A) but not with N-methylaminopyrrolidine (e.g., 13I). As
was observed for the substituent on the benzene ring, larger and
more polar groups were generally not tolerated at all in the 2-posi-
tion (e.g., 12A, 15A and 16A). However, an amino group in the 2-po-
sition was an exception. It was found that this substituent provided
extremely good potency with N-methylaminopyrrolidine as the
amine (18I-R and 18I-S). Indeed, this provided the most potent ana-
logue prepared. It is interesting to see the similarity in structure of
these compounds to those recently disclosed by Cowart et al.7 Both
the R and S isomers of this compound were prepared but in this case
little difference in microsomal stability was observed between the
enantiomers, with the R-enantiomer having a half-life of 13 min
compared to a half-life of 21 min for the S-enantiomer.
R3
N
N
Cl
NR1
O
2
Compd R3
IC50 (nM) H4R % Remaining at 10 min rat microsomes
11A
11I
2-Me
2-Me
2-Ph
37
0
95
40
7
1600
>30,000
83
12A
13A
13I-R
14A
15A
16A
17A
18I-R
18I-S
2-CF3
2-CF3
14200
—
2-CycloPr 310
8
106
83
13
62
2-CO2H
2-OH
>10,000
1000
90
2-OMe
2-NH2
2-NH2
1
13
68
The majority of the compounds under investigation were pre-
pared according to Scheme 1. Reaction of the appropriately substi-
tuted cyanophenol with ethyl bromoacetate gave the phenoxyester
An investigation was then carried out into the nature of the
substituent in the 2-position of the pyrimidine ring (compounds
11–18, Table 4). Early in the investigation it was found that the
NH2
CN
OH
CN
O
i
R2
ii
R2
R2
CO2Et
O
CO2Et
20
19
iii
N
NH
O
N
N
R2
iv
v
N
N
R2
R2
O
NR1R1'
O
Cl
O
21
22
Scheme 1. Reagents and conditions: (i) BrCH2CO2Et, NaH, DMF room temperature; (ii) KOBut, THF, room temperature; (iii) (a) HC(OEt)3, microwave 200 °C 10 min, (b) 2 M
0
ammonia in MeOH, microwave 140 °C 10 min; (iv) POCl3, microwave 180 °C 15 min; (v) R1R1 NH, Et2NCH2–polystyrene, IMS, microwave 120 °C 30 min.
Cl
Cl
Cl
Cl
N
NH2
i
ii
+
N
NH2
N
NH2
N
B(OH)2
OMe
Br
N
OMe
N
Cl
OMe
Cl
Cl
23
iii
Cl
NH2
N
NH2
N
N
N
iv
v
Cl
Cl
N
NH2
O
R1'
N
Cl
OH
N
O
N
Br
Cl
25
24
R1' = Boc
R1' = H 18I
vi
Scheme 2. Reagents and conditions: (i) Pd(PPh3)4, Cs2CO3, DME, microwave 120 °C 10 min; (ii) NBS, acetonitrile, reflux; (iii) BCl3, DCM; (iv) CuCl, Cs2CO3, ButC(O)CH2C(O)But,
toluene, reflux 24 h; (v) N-methyl-N-Boc-aminopyrrolidine, Et2NCH2–polystyrene, IMS, microwave 120 °C 30 min; (vi) TFA, DCM.

S. Cramp et al. / Bioorg. Med. Chem. Lett. 20 (2010) 2516–2519
2519
Table 5
clearance (117 ml/min/Kg), a high volume of distribution (7 l/Kg)
and an oral bioavailability of 5%. However, in higher species the
PK properties were significantly improved. In dog, the compound
had an oral bioavailability of 36% and the corresponding value in
cynomolgus monkey was 99%. Clearance values of 52 and 62 ml/
min/Kg and volumes of distribution of 26 and 25 l/Kg were ob-
tained for the two species, respectively.
In conclusion, we have identified a lead series of histamine H4R
antagonists and inverse agonists using virtual screening. Modifica-
tions of the various parts of the structure have resulted in improve-
ments in both potency and stability leading to the discovery of a
highly potent and selective histamine H4R inverse agonist with
an acceptable oral PK profile in dog and monkey.
Functional activities for selected compounds
Compd IC50 (nM) H4R
assay
IC50 (nM) GTP
assay
c
S
Max %
inhib.
Response
3D
3I-R
111
33
70
41
105
151
Antagonist
Inverse
agonist
Inverse
agonist
Inverse
agonist
Antagonist
Inverse
agonist
Inverse
agonist
3I-S
285
87
292
76
122
149
3P
13A
18I-R
83
1
39
3
100
138
18I-S
13
4
170
References and notes
1. (a) Leurs, R.; Chazot, P. L.; Shenton, F. C.; Lim, H. D.; de Esch, I. J. P. Br. J.
Pharmacol. 2009, 157, 14; (b) Huang, J.-F.; Thurmond, R. L. Curr. Allergy Asthma
Rep. 2008, 8, 21; (c) Smits, R. A.; Leurs, R.; de Esch, I. J. P. Drug Discovery Today
2009, 14, 745.
2. (a) Thurmond, R. L.; Gelfand, E. W.; Dunford, P. J. Nat. Rev. Drug Disc. 2008, 7, 41;
(b) Kiss, R.; Keseru, G. M. Expert Opin. Ther. Patents 2009, 19, 119.
3. Zampeli, E.; Tiligada, E. Br. J. Pharmacol. 2009, 157, 24.
4. (a) Clark, D. E.; Higgs, C.; Wren, S. P.; Dyke, H. J.; Wong, M.; Norman, D.; Lockey,
P. M.; Roach, A. G. J. Med. Chem. 2004, 47, 3962; (b) Arienzo, R.; Clark, D. E.;
Cramp, S.; Daly, S.; Dyke, H. J.; Lockey, P.; Norman, D.; Roach, A. G.; Stuttle, K.;
Tomlinson, M.; Wong, M.; Wren, S. P. Bioorg. Med. Chem. Lett. 2004, 14, 4099; (c)
Arienzo, R.; Cramp, S.; Dyke, H. J.; Lockey, P. M.; Norman, D.; Roach, A. G.; Smith,
P.; Wong, M.; Wren, S. P. Bioorg. Med. Chem. Lett. 2007, 17, 1403.
19 which was cyclised to the benzofuran 20 on treatment with
base. The pyrimidine ring was formed by the reaction of the amin-
obenzofuran ester 20 with triethyl orthoformate followed by treat-
ment with ammonia. The pyrimidinone 21 was converted to the
chloropyrimidine 22 with phosphorus oxychloride then reaction
with the appropriate amine gave the target compounds. In some
cases, the amine required boc-protection in order to obtain the de-
sired target. Deprotection was then achieved using trifluoroacetic
acid in dichloromethane.
Some of the compounds could not be prepared in this manner
and required an alternative synthesis. The preparation of the 2-
amino derivatives (18I) exemplifies the alternative approach as
shown in Scheme 2. Suzuki coupling of 5-chloro-2-methoxyphenyl
boronic acid with 2-amino-4,6-dichloropyrimidine gave the biaryl
derivative 23. Bromination with NBS followed by demethylation of
the methoxy group gave the bromophenol derivative 24 which was
cyclised to give the benzofuranopyrimidine core 25. Reaction with
boc-protected aminopyrrolidine followed by treatment with triflu-
oroacetic acid gave the desired molecule 18I.
5. Jablonowski, J. A.; Grice, C. A.; Chai, W.; Dvorak, C. A.; Venable, J. D.; Kwok, A. K.;
Ly, K. S.; Wei, J.; Baker, S. M.; Desai, P. J.; Jiang, W.; Wilson, S. J.; Thurmond, R. L.;
Karlsson, L.; Edwards, J. P.; Lovenberg, T. W.; Carruthers, N. I. J. Med. Chem. 2003,
46, 3957.
6. Compound potency was assessed using a [3H]-histamine radioligand binding
SPA. Briefly, IC50 was determined from a 6-point semi-log dose response curve
performed in duplicate wells. All reagents were prepared in 50 mM Tris pH 7.5,
5 mM EDTA, 0.5% BSA and the assay performed in the presence of 1% DMSO (v/
v). Compounds were incubated for 2.5 h at room temperature in a clear-bottom,
white-walled 96-well isoplate in the presence of 20 nM [3H]-histamine
dihydrochloride (GE Healthcare), 25
lg human H4 receptor expressing
membranes (Euroscreen) and 0.75 mg SPA beads (GE Healthcare) in a total
volume of 200
ll. Non-specific binding was determined in the presence of 4
lM
A number of compounds that showed good potency in the pri-
mary assay and improved microsomal stability were selected for
further studies. Compounds were tested for their functional activ-
histamine dihydrochloride (Sigma–Aldrich). Plates were counted using
Microbeta scintillation counter (Perkin–Elmer).
a
7. Cowart, M. D.; Altenbach, R. J.; Liu, H.; Hsieh, G. C.; Drizin, I.; Milicic, I.; Miller, T.
R.; Witte, D. G.; Wishart, N.; Fix-Stenzel, S. R.; McPherson, M. J.; Adair, R. M.;
Wetter, J. M.; Bettencourt, B. M.; Marsh, K. C.; Sullivan, J. P.; Honore, P.;
Esbenshade, T. A.; Brioni, J. D. J. Med. Chem. 2008, 51, 6547.
ity in a GTPc
S assay8 and most were shown to be either antagonists
or inverse agonists. Results for selected compounds are shown in
Table 5.
8. Functional activity was determined using a [35S]-GTP
c
S SPA. All reagents were
prepared in 20 mM Hepes pH 7.4, 100 mM NaCl, 10 mM MgCl2, 20 g/ml
saponin and the assay performed in the presence of 1% DMSO (v/v). Compounds
were pre-incubated with 15 of H4 receptor expressing membranes
(Euroscreen) for 15 min at 30 °C in the presence of 10 GDP (Sigma–
l
Selectivity against other histamine receptors was also deter-
mined as well as activity against five isoforms of human CYP450.
In addition, selected compounds were tested in a rat PK study.
Compound 18I-R was the most interesting compound carried for-
ward. The compound showed excellent selectivity with IC50 values
lg
lM
Aldrich) in a clear-bottom, white-walled 96-well Isoplate. Agonist (150 nM
histamine dihydrochloride (Sigma–Aldrich)) was added for 30 min, 30 °C, before
addition of 1 nM [35S]-GTP
c
S (GE Healthcare) and 0.75 mg SPA beads (GE
Healthcare) for an additional 30 min. Non-specific binding was determined in
the presence of 10 M GTP S (Sigma–Aldrich). SPA beads were sedimented by
in H1, H2 and H3 binding assays of >30
respectively. In the CYP450 assay, activity was only observed
against CYP1A2 with 45% inhibition at 1 M. In a rat PK study at
an oral dose of 5 mg/Kg, compound 18I-R was shown to have high
lM, >30 lM and 5.8 lM
l
c
centrifugation and counted using a Microbeta scintillation counter (Perkin–
Elmer).
l