A novel series of histamine H4 receptor antagonists based on the pyrido[3,2-d]pyrimidine scaffold: Comparison of hERG binding and target residence time with PF-3893787

Article abstract of DOI:10.1016/j.bmcl.2013.02.091

In this work we describe the optimization of a lead compound based on the quinazoline template to give a new series of potent pyrido[3,2-d]pyrimidines as histamine H₄ receptor antagonists. The pyrido[3,2-d]pyrimidine ligands have significantly reduced hERG binding compared to clinical stage compound PF-3893787 while showing good affinities at the human and rodent histamine receptors. The receptor residence time of several of these new compounds was determined for the human H₄R and compared with JNJ7777120 and PF-3893787. The pyrido[3,2-d]pyrimidines showed residence times lower than JNJ7777120 but comparable to the residence time of PF-3893787. Overall, the pyrido[3,2-d]pyrimidines show an excellent in vitro profile that warrants their further investigation in relevant models of human disease.

Full text of DOI:10.1016/j.bmcl.2013.02.091

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2663–2670
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
A novel series of histamine H4 receptor antagonists based
on the pyrido[3,2-d]pyrimidine scaffold: Comparison of
hERG binding and target residence time with PF-3893787
Mounir Andaloussi a, Herman D. Lim a, Tiffany van der Meer a, Maarten Sijm a, Chris B. M. Poulie a,
Iwan J. P. de Esch a,b, Rob Leurs a,b, Rogier A. Smits a,
⇑
a Griffin Discoveries BV, Department of Medicinal Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
b VU University Amsterdam, Department of Medicinal Chemistry, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
In this work we describe the optimization of a lead compound based on the quinazoline template to give
a new series of potent pyrido[3,2-d]pyrimidines as histamine H4 receptor antagonists. The pyrido[3,2-
d]pyrimidine ligands have significantly reduced hERG binding compared to clinical stage compound
PF-3893787 while showing good affinities at the human and rodent histamine receptors. The receptor
residence time of several of these new compounds was determined for the human H4R and compared
with JNJ7777120 and PF-3893787. The pyrido[3,2-d]pyrimidines showed residence times lower than
JNJ7777120 but comparable to the residence time of PF-3893787. Overall, the pyrido[3,2-d]pyrimidines
show an excellent in vitro profile that warrants their further investigation in relevant models of human
disease.
Received 5 January 2013
Revised 16 February 2013
Accepted 20 February 2013
Available online 1 March 2013
Keywords:
Histamine H4 receptor
GPCR
Pyrido[3,2-d]pyrimidine
hERG
Ó 2013 Elsevier Ltd. All rights reserved.
Residence time
PF-3893787
The human histamine H4 receptor (hH4R) is mainly expressed
in various cells of the immune system including eosinophils,
T-lymphocytes, dendritic cells, mast cells, and basophils.1 On the
cellular level, H4R activation induces for example chemotaxis of
mast cells and eosinophils and triggers calcium mobilization in
mast cells, monocytes and eosinophils.2 Furthermore, the H4R
modulates the release of various inflammatory mediators. In differ-
ent animal models, blocking the H4R with selective antagonists
such as JNJ7777120 (1, Fig. 1) proved to be beneficial, for example,
in OVA-induced allergic airway inflammation, and zymosan-
induced peritonitis in the mouse or in a TNDB-induced colitis mod-
el in the rat.3 H4R antagonists were also shown to be more effective
in the attenuation of experimental histamine-induced pruritis than
H1R antagonists.4,5 All these results indicate that the hH4R has a
distinct pharmacological profile and plays an important role in dif-
ferent inflammatory and allergic disorders. Antagonists are consid-
ered as potential drugs for the treatment of diseases like allergic
asthma, allergic rhinitis, pruritis, pain, vestibular disorders and
inflammatory bowel disease.3,6
sition 4 and screening different halogen, (hetero)aromatic and
aliphatic groups at position 7 of the quinazoline (see Fig. 2 for scaf-
fold numbering).7 We observed that the introduction of aliphatic
and heteroaromatic groups on the 7-position gave compounds
with excellent H4R affinity in the low nanomolar range. This series
included, among others, 7-(furan-2-yl)-4-(piperazin-1-yl)quinazo-
lin-2-amine (2, VUF11489) a potent antagonist of histamine at the
H4R with 255-fold selectivity over the H3R and good oral bioavail-
ability in the mouse (47%). Unfortunately compounds in this series
have considerable affinity for the hERG channel (hERG pKi of com-
pound 2 is 5.49 in an astemizole radioligand displacement assay)
and this remained an issue to address. In literature several strate-
gies to reduce hERG binding are discussed, for example change the
steric environment of the basic group (i.e., the piperazine moiety of
compound 2), reduce lipophilicity and introduce polarity.8 Because
we were also interested in finding substituents for the metaboli-
cally labile N-methylpiperazine and piperazine moieties without
losing H4R affinity, this could potentially be achieved in parallel
with a reduction of the hERG affinity. Polarity was to be introduced
by replacing the quinazoline ring with a pyrido[3,2-d]pyrimidine
ring, because we had seen from an in-house fragment-like com-
pound set designed as H4R ligands that the pyrido[3,2-d]pyrimi-
dine scaffold was well tolerated by the H4R (compare pKi of
compounds 2a and 2b, Fig. 2).The furan group is associated with
undesired metabolic activation and covalent binding to proteins
Recently we reported the fragment-based discovery of a new
quinazoline scaffold as a potent H4R ligand. In that work we syn-
thesized a library of compounds by varying the basic amine on po-
⇑
Corresponding author. Tel.: +31 6 41402952.
E-mail address: rogier.smits@griffindiscoveries.com (R.A. Smits).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.02.091

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M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
H
N
H
N
N
N
H
N
N
N
H
N
Cl
N
O
N
N
N
NH2
O
NH2
JNJ7777120 (1)
VUF11489 (2)
PF-3893787 (3)
Figure 1. Examples of histamine H4R receptor ligands.
Variation basic amines
Introduction polarity
R4
R4
N
N
N
5
8
N
6
3
2
N
N
N
N
R7
N
1
R7
N
NH2
N
N
N
NH2
N
NH2
Optimisation of drug-like properties and
H4 receptor affinity
Compound 2b
pKi hH4R= 7.51
Variation R7 group
Compound 2a
pKi hH4R= 7.35
Figure 2. Optimization strategy to improve compound quality of quinazoline-based H4R antagonists.
O
O
OH
i
ii
OEt
NH2
OEt
NH2
N
O
Br
R7
R7
N
N
H
5 isopropyl
10 isopropyl
4a
6 cyclopropyl
7 ethyl
11 cyclopropyl
12 ethyl
iii
8 tert-butyl
9 cyclopentyl
13 tert-butyl
14 cyclopentyl
R1
OTs
N
ii
N
O
O
R7
N
N
H
R7
N
N
H
compounds 20-29
15 isopropyl
16 cyclopropyl
17 ethyl
18 tert-butyl
19 cyclopentyl
Scheme 1. Reagents and conditions: (i) (a) isopropenylboronic acid pinacol ester, Pd(PPh3)4, K2CO3, PhCH3, EtOH, Mw, 130 °C, or (b) cyclopropylboronic acid, PCy3, Pd(OAc)2,
KF, THF, rt. Other building blocks were obtained from commercial suppliers, (c) H2, Pd/C 5%, EtOH, rt; (ii) (a) chloroformamidine hydrochloride, dimethyl sulfone, sulfolane,
160 °C, (b) acetic anhydride, reflux; (iii) TsCl, K2CO3, CH3CN,
D; (iv) (a) various (Boc-protected) amines, DIPEA, 1,4-dioxane, rt, (b) dioxane/HCl 2 M, rt.
and was therefore replaced with a saturated hydrocarbon group.9
This replacement would concomitantly decrease the number of
SP2 atoms and improve the drug-like properties.10 We were also
interested in developing new compounds for comparison with
PF-3893787 (3), the clinical stage compound from Pfizer that has
recently been described in the literature.11 We therefore started
this work by exploring the tolerance of the H4R to various non-aro-
matic substituents on the 7-position and different amines on the 4-
position of the quinazoline moiety (Schemes 1–5). The importance
of the 7-position for H4R binding has been described in our earlier
work as well as by researchers from UCB.7,12
palladium on activated carbon to get corresponding isopropyl ana-
logue 5 (Scheme 1). For preparation of cyclopropyl analogue 6, a
combination of PCy3, Pd(OAc)2 and KF was used. Ethyl, tert-butyl
and cyclopentyl analogues were obtained from commercial
sources. Analogues 5–9 were then condensed with freshly pre-
pared chloroformamidine hydrochloride and acetylated with acetic
anhydride to give the N-protected 4-hydroxyquinazolines 10–14.
The 4-position was functionalized by the introduction of a tosyl
group, using tosylchloride under reflux with acetonitrile as solvent
(compounds 15–19). The conversion to the final products was done
by stirring tosylate intermediates 15–19 at room temperature in
the presence of various amines. The introduction of the different
amines could be done either directly or by using their Boc-
protected precursors. Piperazine was introduced directly by using
10 equiv and when the reaction mixture was heated at reflux, this
The first quinazoline analogue was synthesized from bromo
anthranilic acid 4a by introducing an isopropylene group in a Suzu-
ki cross-coupling reaction with 2-methyl ethene boronic acid. The
double bond was subsequently reduced with hydrogen gas and

M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
2665
O
O
OTs
N
duced early on in the synthesis, we decided to perform a Suzuki reac-
tion at a later stage of our synthetic route. Therefore, we initiated a
synthesis by the condensation of the 3-amino-5-bromopicolina-
mide (4c) with chloroformamidine hydrochloride, dimethyl sulfone
and sulfolane, at 165 °C to give 2-amino-7-bromopyrido[3,2-d]pyr-
imidin-4-ol (33, Scheme 3). To perform a selective chlorination at
the C-4 carbon atom using POCl3 and DIPEA in toluene at reflux, pro-
tection of the 2-amino group with an acetyl group was needed (com-
pound 34). Then the corresponding chlorinated compound was
treated directly with N,N-methyl-Boc-pyrrolidine, and DIPEA in
1,4-dioxane at room temperature to yield pyrrolidine analogue
(35). Compound 35 was then reacted with isopropenylboronic acid
pinacol ester in the presence of tetrakis(triphenylphosphine)palla-
dium(0) and potassium carbonate to form the corresponding olefin.
This was then reduced to isopropyl analogue 36 by treatment with
palladium 5% on carbon under a hydrogen atmosphere at room tem-
perature using ethanol as the solvent. To get the target compound
(37), Boc-deprotection using a 4 M HCl solution in 1,4-dioxane
was performed. Although our synthetic plan described in Scheme 3
allowed us to prepare target compound (37), the yield for the syn-
thesis of compound (35) was very low (9%). To increase the yield
of reacting the basic amine with the pyrido[3,2-d]pyrimidine scaf-
fold, we used pyrido[3,2-d]pyrimidine dione 38 as starting material
(Scheme 4). This starting material was then converted to dichloro
analogue 39, which was stirred overnight in a mixture of N,N-
methyl-Boc-azetidine, DIPEA, and 1,4-dioxane. The direct amination
on position 2 was done by using a saturated solution of ammonia in
ethanol to give the corresponding aminopyridopyrimidine (40) in
only 13% yield (two steps).
While the introduction of the ethyl group in intermediate 41
was performed according to the procedures used for the isopro-
pyl group in intermediate 36, the nitrile analogue was synthe-
sized by reacting the bromine compound with Zn(CN)2 and
tetrakis(triphenylphosphine)palladium(0) at 150 °C. The Boc-
deprotection was performed using the same conditions reported
in Scheme 2 to give compound 44. To circumvent the problem of
a low yield of the amination reaction using intermediate 39, we
performed the synthesis of 2,4,7-trichloropyridopyrimidine (47)
in two steps from 3-aminopicolinic acid (45) by a urea conden-
sation, followed by the introduction of the three chlorine atoms
on positions 2, 4 and 7 of pyrido[3,2-d]pyrimidine dione (46)
according to a previously reported procedure.13 With compound
47 in hand, the first substitution was done at the 4-position with
basic amine R4 (pyrrolidine or azetidine analogue) and DIPEA in
1,4-dioxane at room temperature to give 48 and 49. The
Buchwald–Hartwig reaction was employed in the synthesis of
N
N
N
i
ii
OEt
OEt
90%
15%
Br
NH2
4b
NH2
N
NH2
30
31
51% iii
N
N
N
N
N
NH2
32
Scheme 2. Reagents and conditions: (i) (a) potassium vinyltrifluoroborate,
Pd(PPh3)4, K2CO3, PhCH3/EtOH, Mw, 130 °C, 30 min, (b) H2, Pd/C 5%, EtOH, rt; (ii)
(a) chloroformamidine hydrochloride, dimethyl sulfone, sulfolane, 160 °C, (b) TsCl,
K2CO3, CH3CN,
D; (iii) N-methylpiperazine, DIPEA, 1,4-dioxane, rt.
excess amine was also able to remove the acetyl group to directly
give the deprotected 2-aminoquinazoline. The 3-aminomethyl pyr-
rolidine and 3-aminomethyl azetidine groups were introduced by
using (R)-3-(N-Boc-N-methylamino)pyrrolidine and 3-Boc-3-
methylamino azetidine respectively and the subsequent removal
of the acetyl group by heating the reaction mixture with a satu-
rated aqueous Na2CO3 under microwave irradiation. Removal of
the Boc-protecting group was finally done by stirring the Boc-pro-
tected amines in a 2 M solution of HCl in dioxane at room temper-
ature. To build the pyrido[3,2-d]pyrimidin-2-amine scaffold we
started our synthetic pathway by reacting ethyl 3-amino-5-bro-
mopicolinate (compound 4b, Scheme 2) with potassium vinyl-
trifluoroborate under Suzuki conditions. This reaction gave the
vinyl compound which was immediately reduced under
a
hydrogen atmosphere in ethanol to yield the desired ethyl 3-amino-
5-ethylpicolinate (30). The treatment of (30) with chloroformami-
dine hydrochloride, dimethyl sulfone, and sulfolane at 160 °C,
allowed the formation of the corresponding 2-aminopyrimidine.
To perform the amination at position 4 of the 2-aminopyridopyr-
imidine, we introduced a tosyl group by treating the corresponding
reagent with tosyl chloride and potassium carbonate. Finally, the
treatment of intermediate 31 with N-methylpiperazine yielded
the corresponding methylpiperazine compound (32) (Scheme 2).
Because of the low overall yield described in Scheme 2 and the
complication that diversity on the 7-position needed to be intro-
O
OH
Cl
N
N
N
ii
i
NH2
NH2
N
N
78%
67%
NHAc
Br
Br
N
NH2
Br
N
4c
33
34
9%
iii
NH
N
BOC
NH2
N
BOC
N
N
N
N
N
N
N
N
N
N
v
iv
54%
N
N
49%
NH2
Br
NHAc
37
36
35
; (iii) (a) POCl3, DIPEA, PhCH3,
Scheme 3. Reagents and conditions: (i) chloroformamidine hydrochloride, dimethylsulfone, sulfolane, 165 °C; (ii) Ac2O,
D
D, (b) (R)-3-(N-Boc-
methylamino)pyrrolidine, DIPEA, 1,4-dioxane, rt; (iv) (a) isopropenylboronic acid pinacol ester, Pd(PPh3)4, K2CO3, PhCH3, EtOH, Mw, 130 °C, (b) H2, Pd/C 5%, EtOH, rt. The
acetyl group was removed in the Suzuki reaction through the presence of excess base; (v) HCl (4 M) in 1,4-dioxane, 1,4-dioxane, rt.

2666
M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
O
O
N
N
O
O
Cl
N
i
ii
iii
N
N
N
N
NH
OEt
NH2
N
N
90%
54%
13%
Br
N
H
O
Br
Br
Cl
Br
N
NH2
4b
38
39
40
v
67%
iv
O
O
O
N
N
O
N
N
N
N
N
N
N
NC
N
NH2
NH2
42
41
vi
49%
vi
HN
N
HN
N
N
N
N
N
NC
25% two steps 44
N
NH2
N
NH2
43
Scheme 4. Reagents and conditions: (i) urea, 160 °C, 0.2 M NaOH; (ii) POCl3, DIPEA, PhCH3,
D; (iii) (a) 3-Boc-3-methylaminoazetidine, DIPEA, 1,4-dioxane, rt, (b) NH3/EtOH,
Mw, 150 °C; (iv) (a) potassium vinyltetrafluoroborate, Pd(PPh3)4, PhCH3, EtOH, Mw, 130 °C, (b) H2, Pd/C 5%; (v) Zn(CN)2, Pd(PPh3)4, DMF, Mw, 150 °C; (vi) HCl (2 M) in 1,4-
dioxane, rt.
4
R
O
O
Cl
N
N
N
N
N
OH
NH
N
i
ii
N
iii
NH2
N
H
46
O
Cl
N
Cl
Cl
Cl
56%
90%
91-94%
45
47
48 R4 is 3-(N-Boc-N-methylamino)-azetidine
49 R4 is (R)-3-(N-Boc-N-methylamino)-pyrrolidine
40-74% iv
4
4
4
R
R
N
R
N
N
N
vi
27-93%
v
35-62%
N
N
N
N
7
7
R
NH2
R
NH2
Cl
N
NHAc
52-56
57 R4 is (R)-3-(methylamino)pyrrolidine, R7 is ethyl
50-51
58 R4 is N-methylazetidin-3-amine, R7 is cyclopentyl
59 R4 is N-methylazetidin-3-amine, R7 is cyclopropyl
60 R4 is N-methylazetidin-3-amine, R7 is isopropyl
61 R4 is (R)-3-(methylamino)pyrrolidine, R7 is cyclopropyl
Scheme 5. Reagents and conditions: urea, 160 °C, 0.2 M NaOH; (ii) PCl5, POCl3, Mw, 160 °C; (iii) (a) corresponding amine, DIPEA, 1,4-dioxane, rt; (iv) Pd(OAc)2, xantphos,
K2CO3, AcNH2, Mw, 130 °C; (v) (a) cyclopentene boronic acid or isopropylene boronic acid, potassium vinyl tetrafluoroborate, Pd(PPh3)4, PhCH3, EtOH, Mw, 130 °C or
potassium cyclpropyl trifluoroborate, Pd(OAc)2, n-BuPAd2, PhCH3, H2O, Mw, 130 °C for the 7-cyclopropyl substituted analogues, (b) H2, Pd/C 5%, EtOH, rt; (vi) HCl (2 M) in 1,4-
dioxane, rt.
compounds 50 and 51. The introduction of the alkyl group was
carried out by a Suzuki reaction followed by hydrogenation with
palladium 5% on carbon and hydrogen gas. Cyclopropyl ana-
logues 59 and 61 were synthesized directly with a different pal-
ladium source and ligand Pd(OAc)2/n-BuPAd2, using potassium
cyclopropyl trifluoroborate.14
Interestingly, having an excess of base in the Suzuki
reaction (3 equiv) and heating at 130 °C led to the concomitant

M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
2667
Table 1
H4R SAR of quinazoline analogues substituted with aliphatic substituents on the 7-position
R4
N
R7
N
N
Compound no.
R4
R7
hH4R
hH3R
Ki H3R/H4R
hERG
pKi SEM
pKi SEM
pKi SEM
H
N
O
VUF11489
2
7.82 0.05a
7.91 0.17a
8.47 0.04a
7.45 0.11b
7.82 0.03b
7.94 0.02a
8.15 0.02a
7.83 0.06a
7.95 0.09a
5.47 0.05a
6.48 0.05a
6.80 0.06b
6.07 0.06b
6.31 0.10b
6.81 0.07b
6.16 0.07b
6.66 0.04a
6.21 0.083a
223
5.49 0.11a
5.47 0.04a
6.08 0.03a
n.d.
N
H
N
20
21
22
23
24
25
26
27
27
47
24
32
13
98
15
89
N
HN
N
H
N
N
HN
n.d.
N
H
N
5.33 0.07a
5.44 0.09a
5.82 0.06a
5.61 0.08a
N
HN
N
H
N
N
HN
N
N
N
HN
HN
28
29
7.77 0.07a
7.66 0.04a
6.22 0.20b
6.75 0.03b
56
8
5.56 0.03b
n.d.
a
Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R or H3R. pKi’s are calculated from at least three
independent measurements as the mean SEM, n P3 unless mentioned otherwise.
b
n = 2. n.d. = not determined.
deprotection of the acetamide on position 2 to give compounds
52–56. Finally, to perform the Boc-deprotection, 4 M HCl solution
in 1,4-dioxane was used to give target compounds 57–61.
(compound 21). This finding shows that it was indeed possible to
replace both the furan group and the piperazine moiety while
maintaining and even increasing H4R affinity. Both t-butyl substi-
tuted analogues 22 and 23 however showed lower affinities, indi-
cating that the t-butyl group at the 7-position is less well tolerated
than the cyclopentyl group. The combination of a cyclopropyl
group with both amines (compounds 24 and 25) gave a slight
improvement of H4R affinity compared with the t-butyl analogues.
If the cyclopropyl group is replaced with an ethyl group a similar
affinity is found (compare compounds 24 and 25 with compounds
26 and 27). It was found that the combination of a (R)-N-methyl-
pyrrolidin-3-amine moiety, common to many known H4R ligands
As mentioned in the introduction, VUF11489 was one of the H4R
ligands previously made in the quinazoline series that showed a
favourable selectivity over the H3R and had a pKi of 7.82.6 Never-
theless it contained a furan group and considerable hERG affinity,
which we considered a significant liability. Replacing the furan
by a cyclopentyl group on position 7 of the quinazoline heterocycle
provided compound 20 with a comparable pKi of 7.91. The replace-
ment of the piperazine group of 20 with a 3-aminomethylazetidine
group gave an increase in H4R binding of about 0.5log units

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M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
Table 2
H4R SAR of pyrido[3,2-d]pyrimidine analogues with reduced hERG binding
R4
N
N
N
R7
NH2
Compound no.
PF-3893787
R4
R7
hH4R
pKi SEM
hH3R
pKi SEM
Ki H3R/H4R
hERG
pKi SEM
Structure in Figure 1
7.99 0.08a
6.73 0.07a
22
5.46 0.22b
N
32
8.03 0.05a
6.67 0.09b
23
4.57 0.08a
N
HN
43
57
44
7.97 0.10a
8.04 0.12a
7.27 0.04a
6.11 0.12a
5.82 0.02a
5.02 0.02b
72
166
186
4.46 0.20b
4.36 0.22b
<4a
N
HN
N
HN
N
N
HN
58
59
60
8.19 0.09a
8.28 0.09a
7.60 0.03a
6.82 0.09a
6.20 0.05a
6.08 0.05a
23
120
33
<4a
N
HN
’
<4a
N
HN
4.89 0.15b
N
HN
61
37
7.99 0.06a
7.91 0.11a
6.00 0.06b
6.84 0.01b
98
12
<4a
N
HN
n.d.a
N
Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R or H3R. pKis are calculated from at least three
a
independent measurements as the mean SEM, n P3 unless mentioned otherwise.
b
n = 2. n.d. = not determined.
also gave potent compounds when combined with an ethyl or
cyclopropyl group (compounds 28 and 29).15,16 The H3R affinity
of this series of H4R ligands was determined as well and it was
found that the compounds have pKi’s between 5 and 7 for this
receptor with cyclopentyl and cyclopropyl analogues 21 and 25
having the highest affinity of pKi 6.80 and 6.81, respectively.
The compounds were also evaluated for their ability to displace
[3H]astemizole from the hERG channel and some compounds were
found to bind this off target with considerable affinity. PF-3893787
was also found to have significant affinity for the hERG channel
(pKi 5.46 Tables 1 and 2). The hERG affinity of this compound series
is not significantly reduced in comparison with VUF11489 by
changing the substituents on the 7-position or the steric environ-
ment around the basic center and, as mentioned in the introduc-
affinity of pKi 7.27. This SAR indicates that for this scaffold various
combinations of aliphatic substituents with aminergic moieties are
well tolerated by the H4R. Like the quinazoline series, the pyr-
ido[3,2-d]pyrimidine analogues also have considerable affinity for
the H3R, yet good H4R selectivity >100-fold was found, in particular
for analogues 57, 59 and 61. Interestingly, almost all analogues
based on the pyrido[3,2-d]pyrimidine scaffold were found to have
a much lower affinity for the hERG channel than the compounds
from the quinazoline series, having a hERG Ki lower than 10 lM
which is a good improvement in comparison with VUF11489 and
PF-3893787. The introduction of an additional nitrogen atom in a
closely related tricyclic aminopyrimidine scaffold developed by
Savall et al. also had a strong detrimental effect on the ability of
those compounds to bind the hERG channel, thereby greatly
improving the quality of the physicochemical and pharmacological
properties.16
Considering the prominent species differences that are found
histamine H4R orthologues and the possible complications this
may pose in preclinical development, several analogues from the
tion, we prepared
a series of pyrido[3,2-d]pyrimidines with
(cyclo)aliphatic substituents on the 7-position and various amines
on the 4-position (Fig. 2, Table 2). These new pyrido[3,2-d]pyrim-
idine analogues all maintained good affinities, with the only excep-
tion being the 7-cyano analogue (compound 44) that has an

M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
2669
Table 3
Affinity of quinazoline and pyrido[3,2-d]pyrimidine analogues at the histamine receptor subtypesa
Compound no.
hH1R
hH2R
hH3R
hH4R
mH4R
rH4R
Ki SEM
pKi SEM
pKi SEM
pKi SEM
pKi SEM
pKi SEM
PF-3893787
n.d.
n.d.
6.73 0.07
6.11 0.12
5.82 0.02
6.82 0.09
6.20 0.05
7.99 0.08
7.97 0.10
8.04 0.12
8.19 0.09
8.28 0.09
7.26 0.07
7.30 0.12
7.07 0.08
7.71 0.17b
7.82 0.18
7.26 0.10
7.15 0.16
6.86 0.12
7.56 0.01b
7.53 0.16
43
57
58
59
4.47 0.12
4.95 0.03
4.78 0.16
4.68 0.08
4.85 0.05
5.50 0.07
5.55 0.04
n.d.
a
Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R, H3R or H1R. pKi values are calculated from at
least three independent measurements as the mean SEM (n P3), unless mentioned otherwise.
b
n = 2. n.d. = not determined.
Table 4
[
120
100
80
60
40
20
0
35S]GTP
cS assay
b
Compound no.
hH4R
pKb
pIC50 SEMa
PF-3893787
7.27 0.06
6.95 0.07
6.90 0.07
7.01 0.14
7.15 0.10
8.46 0.06
8.14 0.07
8.08 0.09
8.20 0.14
8.33 0.10
43
57
58
59
43
57
58
59
Antagonism against 100 nM histamine at the human H4R.
a
n P5.
PF3893787
b
pKb is determined with the Cheng and Prusoff equation, wherein the EC50 of
-20
histamine is 6.95 nM.
-11 -10 -9
-8
-7
-6
-5
-4
-3
Log[Ligand(M)]
pyrido[3,2-d]pyrimidine series were selected for further profiling
at the different histamine receptor subtypes and rodent H4 recep-
tors (Table 3). It was found that the compounds have varying selec-
tivities between 186- and 12-fold for the hH4R over the other
human histamine receptor subtypes. All compounds also retained
good affinity for the rat and mouse H4R with only compound 57
having an affinity that is significantly lower than that of histamine
itself for the species orthologues (histamine pKi is 7.1 0.1 for the
mH4R and 7.0 0.1 for the rH4R).17 The selected compounds were
Figure 3. Antagonism at the human H4R. The compounds dose dependently inhibit
100 nM histamine-induced activity of the human H4R in [35S]GTP
cS assay.
Table 5
Binding kinetics at the human H4R determined in competitive kinetic [3H]histamine
binding assaya
b
c
d
Compound no.
kon
koff
pKD
s (min)
PF-3893787
JNJ7777120
VUF11489
43
57
58
59
2.00 0.23
3.64 0.43
5.46 1.99
7.89 2.95
5.68 2.72
9.93 0.13
6.18 4.76
0.052 0.01
0.017 0.01
0.193 0.04
0.077 0.05
0.108 0.02
0.102 0.00
0.055 0.02
7.60 0.15
8.40 0.10
7.39 0.08
8.07 0.13
7.67 0.15
7.99 0.01
7.87 0.32
15.0 3.9
62.0 13.6
4.10 0.8
14.0 8.4
6.60 1.1
6.80 0.1
13.7 3.8
also evaluated in a [35S]GTP
cS assay for their functional activities
at the hH4R. In this assay, the compounds show their ability to
antagonize 100 nM histamine-induced activity at the hH4R (Ta-
ble 4, Fig. 3). The calculated pKb values, derived from pIC50 values
with the Cheng–Prusoff equation, approximate the pKi values of
the corresponding compounds as determined with the radioligand
displacement assay.18
a
Data were analyzed with Graphpad Prism 5.0, n = 3 unless mentioned
otherwise.
Recently we have described the use of compound residence
time as a parameter to take into consideration when optimizing li-
gands for the hH4R.2 The widely used H4R ligand JNJ7777120 is a
compound that shows efficacy in many animal models. Interest-
ingly, JNJ7777120 often has the best efficacy in those models in
comparison with compounds from other structural classes, even
despite its short in vivo pharmacokinetic elimination half-life.19
We have also speculated that the efficacy of JNJ7777120 may be
the result of its relatively long residence time at the various H4R
receptors. Residence time is considered to be an important param-
eter to consider in the lead optimization process and we therefore
studied the residence time of compounds 43, and 57–59 to com-
pare with the clinical stage compound PF-3893787 and
JNJ7777120 (Table 5). When comparing the dissociation constant
and associated pKD from Table 5 with the pKi determined in the
radioligand binding assay we find closely matching values. It
should be taken into consideration that compounds with long res-
idence times may not be detected in standardized radioligand
binding assays, because these assays may not have an incubation
time that gives and accurate estimation of the compounds’ affinity
at equilibrium. In the case of longer residence times, compound
affinities may therefore be underestimated. We found that none
b
Data expressed as106 MÀ1 minÀ1
.
c
Data expressed as MÀ1 minÀ1
.
d
pKD obtained from KD = koff/kon
.
of the compounds has a residence time longer than the 62 minutes
found for JNJ7777120. However, compounds 43, 59 and PF-
3893787 all have residence times about fourfold lower than the
reference antagonist JNJ7777120 but fourfold higher than lead
compound VUF11489. It has been reported that longer residence
times can lead to improved compound efficacy and target selectiv-
ity in vivo.20 However, no preclinical data that describes the effi-
cacy of PF-3893787 in various disease models has been disclosed
so far. Therefore we aim to use the reference compounds and novel
pyrido[3,2-d]pyrimidines described in this work to further study
the relevance of target residence time for H4R drug development
and select a candidate compound for further development.
We have developed a series of new H4R ligands based on the
pyrido[3,2-d]pyrimidine scaffold from a lead-series containing a
quinazoline moiety and managed to greatly reduce the hERG bind-
ing of those compounds. In parallel we succeeded to introduce var-
ious basic amines to create a series of highly potent compounds

2670
M. Andaloussi et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2663–2670
7. Smits, R. A.; Lim, H. D.; van der Meer, T.; Kuhne, S.; Bessembinder, K.;
Zuiderveld, O. P.; Wijtmans, M.; de Esch, I. J. P.; Leurs, R. Bioorg. Med. Chem. Lett.
2012, 22, 461.
8. Shagufta, D. G.; Kaasse, E.; de Vries, H.; Brussee, J.; Nalos, L.; Rook, M. B.; Vos, M.
A.; van der Heyden, M. A. G.; Ijzerman, A. P. ChemMedChem 2009, 10, 1722.
9. Neuwirth, C.; Mosesso, P.; Pepe, G.; Fiore, M.; Malfatti, M.; Turteltaub, K.;
Dekant, W.; Mally, A. Mol. Nutr. Food Res. 2012, 56, 1363.
10. Walters, P. W.; Green, J.; Weiss, J. R.; Murcko, M. A. J. Med. Chem. 2011, 54,
6405.
11. Mowbray, C. E.; Bell, A. S.; Clarke, N. P.; Collins, M.; Jones, R. M.; Lane, C. A.; Liu,
W. L.; Newman, S. D.; Paradowski, M.; Schenk, E. J.; Selby, M. D.; Swain, N. A.;
Williams, D. H. Bioorg. Med. Chem. Lett. 2011, 21, 6596.
with excellent in vitro profiles at the various histamine receptor
subtypes. Although JNJ777120 remains the compound with the
longest known residence time at the hH4R of 62 min, we were able
to find several compounds with extended residence times compa-
rable to that of clinical candidate PF-3893787. Further work on this
novel compound series will focus on its characterization in vivo
and the selection of an optimized lead for further development.
Acknowledgments
12. Raphy, G. U.S. Patent US20100298289, 2010.
13. Tikad, A.; Aksirra, M.; Massip, S.; Leger, J.-M.; Jarry, C.; Guillaumet, G.; Routier,
S. Eur. J. Org. Chem. 2012, 24, 4523.
The authors would like to thank Maikel Wijtmans, Andreas Eh-
lers, Hans Custers and Andrea van de Stolpe for their unrelenting
support and fruitful discussions. This work was supported by COST
action BM0806.
14. Molander, G. A.; Gormisky, P. E. J. Org. Chem. 2008, 73, 7481.
15. Examples of compounds with different basic amines including the pyrrolidine
group are manifold. Various reviews give an overview of preferred roups. (a)
Marson, C. M. Chem. Rev. 2001, 111, 7121; (b) Engelhardt, H.; Smits, R. A.; Leurs,
R.; Haaksma, E.; de Esch, I. J. P. Curr. Opin. Drug Discov. Devel. 2009, 9, 132.
16. Savall, B. M.; Gomez, L.; Chavez, F.; Curtis, M.; Meduna, S. P.; Kearney, A.;
Dunford, P.; Cowden, J.; Thurmond, R. L.; Grice, C.; Edwards, J. P. Bioorg. Med.
Chem. Lett. 2011, 21, 6577.
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