Discovery of quinazolines as histamine H4 receptor inverse agonists using a scaffold hopping approach

Article abstract of DOI:10.1021/jm800876b

From a series of small fragments that was designed to probe the histamine H₄ receptor (H₄R), we previously described quinoxaline-containing fragments that were grown into high affinity H ₄R ligands in a process that was guided by pharmacophore modeling. With a scaffold hopping exercise and using the same in silico models, we now report the identification and optimization of a series of quinazoline-containing H₄R compounds. This approach led to the discovery of 6-chloro-N-(furan-3-ylmethyl)2-(4-methylpiperazin-1-yl)quinazolin-4-amine (VUF10499, 54) and 6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmethyl) quinazolin-4-amine (VUF10497, 55) as potent human H₄R inverse agonists (pK_i ) 8.12 and 7.57, respectively). Interestingly, both compounds also possess considerable affinity for the human histamine H ₁ receptor (H₁R) and therefore represent a novel class of dual action H₁R/H₄R ligands, a profile that potentially leads to added therapeutic benefit. Compounds from this novel series of quinazolines are antagonists at the rat H₄R and were found to possess anti-inflammatory properties in vivo in the rat.

Full text of DOI:10.1021/jm800876b

J. Med. Chem. 2008, 51, 7855–7865
7855
Discovery of Quinazolines as Histamine H4 Receptor Inverse Agonists Using a Scaffold Hopping
Approach
Rogier A. Smits,† Iwan J. P. de Esch,† Obbe P. Zuiderveld,† Joachim Broeker,‡ Kamonchanok Sansuk,† Elena Guaita,§
Gabriella Coruzzi,§ Maristella Adami,§ Eric Haaksma,†,‡ and Rob Leurs*,†
Department of Pharmacochemistry, Faculty of Exact Sciences, Leiden/Amsterdam Center for Drug Research (LACDR), DiVision of Medicinal
Chemistry, Vrije UniVersiteit Amsterdam, De Boelelaan 1083, 1081 HV Amsterdam, The Netherlands, Department of Medicinal Chemistry,
Boehringer Ingelheim RCV GmbH & Co. KG, Dr. Boehringergasse 5-11, Vienna, Austria, and Department of Human Anatomy, Pharmacology
and Forensic Medicine, Section of Pharmacology, UniVersity of Parma, Via Volturno 39, 43100 Parma, Italy
ReceiVed July 16, 2008
From a series of small fragments that was designed to probe the histamine H4 receptor (H4R), we previously
described quinoxaline-containing fragments that were grown into high affinity H4R ligands in a process that
was guided by pharmacophore modeling. With a scaffold hopping exercise and using the same in silico
models, we now report the identification and optimization of a series of quinazoline-containing H4R
compounds. This approach led to the discovery of 6-chloro-N-(furan-3-ylmethyl)2-(4-methylpiperazin-1-
yl)quinazolin-4-amine (VUF10499, 54) and 6-chloro-2-(4-methylpiperazin-1-yl)-N-(thiophen-2-ylmeth-
yl)quinazolin-4-amine (VUF10497, 55) as potent human H4R inverse agonists (pKi ) 8.12 and 7.57,
respectively). Interestingly, both compounds also possess considerable affinity for the human histamine H1
receptor (H1R) and therefore represent a novel class of dual action H1R/H4R ligands, a profile that potentially
leads to added therapeutic benefit. Compounds from this novel series of quinazolines are antagonists at the
rat H4R and were found to possess anti-inflammatory properties in vivo in the rat.
Introduction
Histamine plays an important role in physiology and exerts
its effects through the modulation of four known G-protein-
coupled receptors (GPCRsa).1 The H1R receptor has long been
known to be involved in allergic conditions. Its activation causes
several responses such as vasoconstriction and increased
vascular permeability leading to the characteristic symptoms
in allergic rhinitis.2 Several histamine H1 receptor (H1R)
antagonists (e.g., cetirizine and loratidine) have reached block-
buster status because of their successful use in the treatment of
allergic rhinitis.3 The histamine H2 receptor (H2R) was discov-
ered in 1972 and is well-known for its role in the regulation of
the secretion of gastric acid.4,5 Like the H1R antagonists, H2R
antagonists (e.g., cimetidine and ranitidine) have also been
widely prescribed, in this case for the treatment of gastric
Figure 1. Several H4 ligands and their reported potencies at the human
H4R.
ulcers.5 The H3R was discovered in 1983, but major drug
discovery efforts were only initiated after the identification of
the H3R gene in 1999.6,7 The H3R is mainly but not exclusively
The H4R is mostly expressed on cells of the immune system
and blood forming organs. Several lines of evidence suggest
that the H4R is a potential target for the treatment of asthma,
pruritis, and rheumatoid arthritis.14-17 It has also been suggested
that in some cases (e.g., pruritis) additional clinically beneficial
effects might be expected when H1R and H4R receptors are
blocked simultaneously.18
Soon after the discovery of the H4R receptor and the notion
of its potential therapeutic value, combined high throughput
screening and medicinal chemistry efforts identified the first non-
imidazole antagonists, some with low nanomolar affinities
(Figure 1).19 The indole JNJ7777120 (1) is currently the
prototypic H4R antagonist used in most of the studies despite
its very short half-life.20 The more distinct benzimidazole (2)
shows an improved H4R affinity but has so far not been used
in vivo.21 Using a fragment-based approach, we recently
reported the discovery of a series of quinoxalines as potent H4R
ligands (Figure 1). The 6,7-dichloro-substituted VUF10214 (3)
is the most potent compound in this series.22 Moreover, using
expressed in the central nervous system (CNS). Currently, H3R
ligands are studied in clinical trials for a variety of potential
indications such as attention deficit hyperactivity disorder
(ADHD) and narcolepsy.8
The histamine H4 receptor (H4R) is the latest addition to the
family of G-protein-coupled histamine receptors. Although the
receptor protein shows moderate homology to the H3R (31%
overall, 54% in the transmembrane region), it has a distinct
pharmacological profile and a very different tissue distribution.9-13
* To whom correspondence should be addressed. Phone: +31(0)205987600.
Fax: +31(0)205987610. E-mail: leurs@few.vu.nl.
†
Vrije Universiteit Amsterdam.
Boehringer Ingelheim RCV GmbH & Co. KG.
University of Parma.
‡
§
a Abbreviations: GPCRs, G-protein-coupled receptors; H1R, histamine
H1 receptor; H2R, histamine H2 receptor; H3R, histamine H3 receptor; H4R,
histamine H4 receptor; SAR, structure-activity relationship; CRE, cyclic
AMP responsive element.
10.1021/jm800876b CCC: $40.75
2008 American Chemical Society
Published on Web 11/18/2008

7856 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smits et al.
Table 1. Quinazolines Substituted at the 4-Position with a Variety of Substituents
a Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R. pKi values are calculated
from at least three independent measurements as the mean ( SEM.
an in-house developed pharmacophore model based on the
flexible alignment of 1 and a rigid clozapine analogue,23 we
hypothesized the presence of a hydrophobic pocket in the H4R
active site that could be occupied by substituents on the
2-position of the quinoxaline heterocycle (Figure 2A). Indeed,
several aromatic rings and aliphatic groups are tolerated on the
quinoxaline scaffold, leading to, for example, VUF10148 (4),
which possesses low, nanomolar H4R affinity.22
In the previous fragment based approach we observed that
besides the quinoxaline scaffold, a quinazoline heterocycle
(Figure 1, compound 5) also binds the H4R with micromolar
affinity.22 Given the high structural similarity between the

Quinazolines as InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7857
Figure 2. Scaffold hopping from quinoxalines to quinazolines as potential new ligands for the histamine H4 receptor. Quinoxaline (A) (6, pKi )
6.57 ( 0.05) and quinazoline (B) (7) are proposed to have similar binding modes in this three pocket model. The flexible alignment of 6 and 7 (C)
indicates that a similar low energy conformation can be adopted.
quinazoline and quinoxaline heterocycles, we assumed that both
scaffolds might have overlapping binding modes in the H4R
binding site. The H4R affinity of the quinoxaline series is
determined by three important structural elements: a basic
aminergic cyclic amine, a heterocyclic core with a small
lipophilic substituent, and to a lesser extent the side chain on
the 2-position (e.g., compound 6, Figure 2A). On the basis of
these structural elements, a scaffold hopping approach was
attempted to obtain a new series of substituted quinazolines with
high H4R affinity.
Since in other series of H4R antagonists the N-methylpip-
erazine moiety was found to be crucial for H4R affinity, this
basic amine was left unchanged in our initial series of
quinazolines.19,22-24 A chlorine atom was introduced on the
6-position (Figure 2B), since such a substituent is known to
increase H4R affinity in the quinoxaline series but also in
chemically more distinct H4R ligands such as indoles and
benzimidazoles.19,24 Substituents on the 4-position of the
quinazoline heterocycle were expected to be tolerated to some
extent or even to increase the H4R affinity, as had been the
case in the quinoxaline series.22 Introducing a benzyl-
amine group at the 4-position would give a ligand (7, Figure
2B) with a relatively flexible aromatic group that would be able
to adopt a similar conformation as quinoxaline 6 (Figure 2C).
In order to explore the SAR of such substituted quinazolines,
we synthesized an initial series of compounds with diverse
substituents ranging from aliphatics and aromatics to groups
that are capable of forming hydrogen bonding interactions such
as alcohols and esters (Tables 1 and 2).
cording to a modified procedure reported in literature.26 2,4-
Dichloroquinazoline (23) was synthesized according to a
procedure described in literature, whereas substituted dichlo-
roquinazolines (24-28) were obtained by a modified procedure
to give the desired products in very high purity.27
Disubstituted quinazolines 29-64 were prepared from their
respective 2,4-dichlorosubstituted precursors (23-28, Scheme
2) and a set of commercially available primary amines using a
one-pot procedure with microwave assisted heating. Intermediate
2-chloro substituted quinazoline 65 was synthesized by reacting
24 with methylamine. Subsequently, compound 65 was used
for the preparation of compounds 66-81 by microwave assisted
heating in the presence of diisopropylethylamine (DIPEA) while
using N-methylpyrrolidone as a solvent.
The 2,4-dichloroquinazoline intermediates (23-28) could be
selectively substituted at position 4 because of the lower
reactivity of the carbon position 2. In a first step, we selectively
introduced amines or alcohols at the 4-position at room
temperature (rt). The reactivity of the alcohols was increased
by deprotonation with sodium hydride (NaH) in dimethylfor-
mamide (DMF) in order to successfully react at room temper-
ature. The selective introduction of the primary amines at the
4-position of 23-28 was effected at room temperature in the
presence of DIPEA. These conversions were typically very high,
and upon completion, excess N-methylpiperazine was subse-
quently introduced at the 2-position with microwave assisted
heating. With this one-pot procedure, no workup of the 2-sub-
stituted quinazoline intermediate was required. In some cases
the amines for substituting the 4-position needed to be used in
excess (for compounds 29, 30, 38, and 40-42) because of the
lack of reactivity of the 4-position at room temperature. In such
cases, an intermediate workup to remove excess amine was
required.
Chemistry
Starting from 2-amino-5-chlorobenzoic acid (8), thioxo-
quinazoline 9 (Scheme 1) was prepared according to a procedure
described in literature.25 Intermediate 9 was subsequently
alkylated with methyl iodide in aqueous sodium hydroxide to
give intermediate 10.25 Heating of thioether 10 in neat N-
methylpiperazine finally gave quinazolin-4-one 11.
Results and Discussion
In the present study we focussed on applying a scaffold
hopping approach and developing a new series of quinazolines
as H4R antagonists on the basis of the known SAR of related
Commercially available anthranilic acids 8 and 12-16 were
used to synthesize quinazolinediones 17-22 (Scheme 2) ac-

7858 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smits et al.
Table 2. Quinazolines Substituted at the 4-Position with a Variety of Substituents
a Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R. pKi values are calculated
from at least three independent measurements as the mean ( SEM.
Scheme 1a
Surprisingly, the related quinazoline 11 has low affinity,
indicating that the hydroxyl group of 11 is detrimental for H4R
affinity. In contrast, the introduction of a hydroxyl group in the
quinoxaline series is very beneficial for H4R affinity, leading
to compounds with pKi values in the range 8-9 (e.g., compound
3, Figure 1).22 Aromatic substitution of the hydroxyl group
resulted in some gain in H4R affinity, but the compounds
remained only moderately active (31 and 32, Table 1). Phenyl
substitution of the 4-amino group as in 33 was also not well
tolerated, resulting in a 0.6 log unit loss in affinity. However,
introducing an alkyl spacer between the amino group and the
aromatic ring increased the affinity almost 10-fold again (34,
Table 1). The introduction of a chlorine atom on the 6-position
of these quinazolines leads to a further increase in H4R affinity
(compare 34 with 7 and 35 with 36, Table 1). This beneficial
effect is seen throughout this series and is in line with our
hypothesis that a 6-chloro substituent on the heterocyclic
quinazoline core would increase H4R affinity. It is worth
mentioning that quinazoline 7 was found to have a pKi very
similar to that of quinoxaline 6 (Figure 2), which was used as
the original template for its design.
a Reagents and conditions: (a) SOCl2, reflux; (b) NH4SCN, acetone, rt;
(c) CH3I, aqueous NaOH, rt; (d) N-methylpiperazine, reflux.
quinoxalines.22 Initially, we introduced various substituents at
the 4-position of the quinazoline scaffold. Substitution of the
4-position (R1 in Table 1) of quinazoline 5 with an amino
function (29) leads to a 3-fold increase in affinity. Introducing
an additional 6-chloro substituent (30) further boosts the activity
up to a pKi of 6.98 (Table 1), making compound 30 an
interesting starting point for further optimization.
Aliphatic substitution of the amine function of 30 is generally
not well tolerated and mostly leads to a reduction in H4R affinity
(Table 1, 37-39). The only exception to this trend was the

Quinazolines as InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7859
Scheme 2a
a Reagents and conditions: (a) urea, 160 °C; (b) 0.2 M NaOH, NaCl; (c) N,N-diethylaniline or DIPEA, POCl3, reflux; (d) amine (R1H), tetrahydrofuran
or methanol or ethanol or ethyl acetate, DIPEA, rt; (e) ethyl acetate, N-methylpiperazine, 120 °C, 10 min; (f) amine (R3H), N-methylpyrrolidone, DIPEA,
microwave, 150 °C, 10 min.
methyl-substituted quinazoline 40, which has a pKi of 7.15.
Since the H4R affinity decreases when the size of the alkyl
substituent increases, we speculate that in these compounds
steric hindrance prevents the amino group from optimally
interacting with the H4R. We propose that the 4-amino group
on the quinazoline scaffold acts as a hydrogen bond donor in
binding to the H4R. This proposal is supported by the observa-
tion that the sterically hindered aminophenylquinazoline 33 also
shows moderate H4R affinity. Increasing the length of the spacer
between the amino group and a bulky amino substituent can
regain some of the H4R affinity that was lost as a result of steric
shielding of the amino group (compare 33 with 34 and 35).
Replacement of the hydrogen atom on the methylamino group
with an additional methyl group (38) results in the loss of the
putative hydrogen bonding interaction, and the H4R affinity
drops approximately 8-fold (Table 1, compare 38 and 40). Given
the structural similarity of these 4-aminoquinazolines with the
previously reported indolecarboxamide series (e.g., 1, Figure
1), we suggest that the 4-aminoquinazolines make use of a
similar hydrogen bonding interaction with the H4R.28 The basic
amine in the N-methylpiperazine moiety of the quinazolines
Following the discovery of 50, some quinazolines with five-
membered aromatic rings were synthesized to explore the SAR
of such small aromatic heterocycles (52-64, Table 2). Moving
the oxygen atom in the furan moiety of 50 from the 2- to the
3-position resulted in quinazoline 54 with 3-fold higher H4R
affinity. Replacement of the furan group of 50 by a thiophene
moiety increased H4R affinity 11-fold and resulted in the highly
potent quinazoline analogue 55 with 7.5 nM affinity. Moving
the sulfur atom of 55 from the 2- to the 3-position resulted in
a loss of H4R affinity (compare 55 and 56). All other structural
changes made to 50 and 55 such as extending the spacer length
(57), introducing methyl substituents on the aromatic ring
(58-61), introducing one or more additional heteroatoms
(61-63), and increasing the heterocycle size (64) did not further
improve the H4R ligands.
The SAR of N-methylpiperazine replacements for H4R
activity has been reported for the indole and benzimidazole
scaffolds.19,22-24 From this work, we initially concluded that
the N-methylpiperazine moiety is crucial for good H4R activity.
Nevertheless, some patent applications have described several
H4R ligands in which the N-methylpiperazine moiety has been
successfully replaced with other cyclic amines.29-32 We there-
fore investigated to what extent the quinazoline series would
allow replacement of the N-methylpiperazine group. For cou-
pling to the quinazoline scaffold we selected a set of cyclic
amines (Table 3) with the following three structural elements:
(1) at least two nitrogen atoms of which one could be directly
attached to the aromatic heterocycle (proximal nitrogen atom),
(2) a distal basic nitrogen atom potentially alkylated with a small
aliphatic substituent, (3) an aliphatic ring system that would
introduce a conformational restraint of the distal nitrogen atom.
The 16 selected amines were coupled to 2,6-dichloro-N-
methylquinazolin-4-amine (65) by a parallel synthesis method
and evaluated for H4R affinity in a [3H]histamine displacement
assay. Interestingly, compounds 73 and 74 displace [3H]hista-
mine binding to the H4R with potency comparable to that of
the parent compound 40 of this series. Further pharmacological
evaluation (n ) 3) resulted in a pKi of 6.81 ( 0.02 (n ) 3) for
would then interact with aspartic acid residue Asp94 (Asp3.32
)
in transmembrane helix 3 (TM3) through a salt bridge, and the
hydrogen bond donor would interact with glutamic acid residue
Glu182 (Glu5.42) in TM5 through formation of a hydrogen
bond.28
To further optimize 30, a series of substituted benzylamines
was synthesized (43-47, Table 1), but no major improvement
was obtained. From this series one might expect opposing effects
of electron-donating or -withdrawing substituents on H4R affinity
(Table 1). Compounds 48-51 were synthesized in an attempt
to allow an additional hydrogen bond (e.g., with Glu182
(Glu5.42)). Interestingly, the 2-furfurylaminoquinazoline 50 had
a comparable affinity to 30, indicating that a small aromatic
ring was tolerated at this position. Also for 50 the 6-chloro atom
is of substantial importance for the relatively high H4R affinity
(compare 50 with 51, Table 1). Other substitution patterns did
not lead to an increase in affinity (52 and 53, Table 2).

7860 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smits et al.
Table 3. Affinity Screen of Quinazolines with N-Methylpiperazine Bioisosteresa
a Measured by displacement of [3H]histamine binding using membranes of HEK cells transiently expressing the human H4R. pKi values are calculated
from at least three independent measurements as the mean ( SEM. Substitutions with more than a 10-fold loss (pKi < 6) in affinity compared to 40 are
considered unsuccessful. Compounds 73 and 74 were found to have H4R affinities very similar to that of parent compound 40.
compound 73 and a pKi of 6.85 ( 0.02 (n ) 3) for compound
74 compared to a pKi of 7.15 ( 0.06 (n ) 3) for the parent
compound 40. Because no difference in affinity was found
between the S-enantiomer 73 and its racemic mixture 74, we
conclude that there is no significant affinity difference between
the R- and S-enantiomers of diazabicyclo[4.3.0]nonane substi-
tuted quinazolines. Interestingly, several of the amines in Table
3 that have been reported to be tolerated on other H4R scaffolds
(e.g., 66, 69, 72, and 77) are very detrimental to H4R affinity
on the quinazoline scaffold.31-33 From the tested series of cyclic
amines, we conclude that the N-methylpiperazine moiety in our
4-aminoquinazoline scaffold can be replaced with its bioisostere
diazabicyclo[4.3.0]nonane without significant loss of affinity.
For the most potent examples from the optimized quinazoline
scaffold, we selected compounds 54 and 55 for further evalu-
ation. As explained in the Introduction, our studies were guided
by our three-pocket pharmacophore model (Figure 2) on the
basis of the flexible alignment of 1 and a close analogue
(VUF6884, 82, H4R pKi) 7.55 ( 0.10) of the antipsychotic
drug clozapine (Figure 3A).23 The van der Waals interaction
surface of this flexible alignment model was generated to map
the surface of the binding site of the H4R. The two pockets in
which the N-methylpiperazine and aromatic heterocycles bind
the H4R are clearly visible as well as the proposed third
hydrophobic pocket (Figure 3A). After flexible alignment of
quinazolines 54 and 55 with compounds 1 and 82 it was found
that the quinazolines can adopt a low energy conformation in
which the furan (54) and thiophene (55) can occupy the
proposed hydrophobic third pocket while occupying the other
two pockets with the N-methylpiperazine and quinazoline
moieties (Figure 3B).
On the basis of the same model, we previously developed
benzylquinoxaline 4, which appears to combine good H4R
affinity with a reasonable H1R affinity (H1R pKi ) 6.0).22
Clozapine analogue 82 also possessed high H1R affinity (pKi
) 8.11),23 prompting us to speculate that filling the third putative

Quinazolines as InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7861
agonist thioperamide (pKi ) 7.28 ( 0.02, Figure 4A). Quinazo-
line 54 (pKi ) 7.57) has an affinity close to that of thioperamide.
Next, we examined the functional activities of the new
compounds on the human H4R. In an H4R-driven CRE-ꢀ-
galactosidase reporter gene assay, histamine shows full agonistic
behavior (R ) 1) (Figure 4B), whereas thioperamide behaves
as an invere agonist (R ) -1). Compound 54 displays pro-
nounced inverse agonistic behavior (R ) -1.49), whereas 55
(R ) -1.06) shows inverse agonism comparable to thiopera-
mide. At the rat H4R, 54 and 55 have respective pKi values of
7.61 ( 0.21 and 7.80 ( 0.02 and behave as neutral antagonists
(data not shown). Compound 55 was thereafter selected to study
the anti-inflammatory effect on this new series of H4R ligands
in the carrageenan induced paw edema model for inflamma-
tion.34 After 2 h, compound 55 caused a significant inhibition
of carrageenan-induced edema at both 10 and 30 mg/kg when
compared to vehicle (Figure 5). This effect was still significant
4 h after administration.
In conclusion, this work describes the design and synthesis
of a novel series of quinazolines as inverse agonists for the
histamine H4 receptor. These quinazolines are closely related
to a series of quinoxalines reported by our group in a previous
disclosure.22 The quinoxalines provided the basis for the
molecular design and subsequent scaffold hopping that led to
this novel series of quinazoline compounds. After pharmaco-
logical evaluation, they were found to have high affinity up to
the single-digit nanomolar range while showing inverse agonistic
behavior at the human H4R. In addition, we were able to
successfully replace the most commonly encountered structural
element of H4R ligands, the N-methylpiperazine group, with a
suitable diazabicyclo[4.3.0]nonane bioisostere while retaining
H4R affinity. Since the most potent compounds possessed
considerable affinity for the histamine H1 receptor subtype, the
quinazoline scaffold may be a good starting point for the
development of a new class of dual action H1R/H4R antihista-
mines. The three-pocket pharmacophore model apparently
allows for the design of dual action ligands. Such compounds
might be used to treat the acute symptoms of inflammatory
conditions. For example, allergic rhinitis could be treated by
blockade of the H1R, similar to the mode of action of the H1R
antiallergic antihistamines (e.g., loratidine and cetirizine), while
simultaneously targeting the H4R to modulate the underlying
disease mechanism by blocking the influx of proinflammatory
cells.18 Such dual action H1R/H4R ligands may prove to be
superior to the selective H1R antihistamines or selective H4R
antihistamines that are exclusively targeted at one of either
receptor.18
Figure 3. Model of the H4R binding site. The hydrophobic (van der
Waals) surface is depicted in yellow, mild polar surface in blue, and
potential sites for hydrogen bonding interaction in red. (A) The model
based on the alignment of 1 and 82 reveals three pockets, with the
proposed third hydrophobic pocket on the right-hand side. (B) Quinazo-
lines 54 (green) and 55 (brown) fit the binding site and occupy the
third pocket with their furan and thiophene moieties, respectively.
Table 4. Affinity and Functional Profile of Compounds 54 and 55 at the
Humane Histamine H1 and H4 Receptors
54
55
receptor
pKi ( SEMa
receptor
pKi ( SEMa
human H4
human H1
7.57 ( 0.05
7.01 ( 0.10
human H4
human H1
8.12 ( 0.02
7.70 ( 0.10
a Measured by displacement of radioligand binding using previously
described methods from literature.35,22 pKi values are calculated from at
least three independent measurements as the mean ( SEM.
Experimental Section
General Remarks. Chemicals and reagents were obtained from
commercial suppliers and were used without further purification.
The amines for compounds 67, 68, 70, 71, and 74-81 were
generously donated by Boehringer Ingelheim RCV GmbH & Co.
KG, Vienna, Austria. Yields given are isolated yields unless
mentioned otherwise. Flash column chromatography was typically
carried out on an Argonaut Flashmaster II flash chromatography
system, using prepacked Silicycle Flash Si II columns with the UV
detector operating at 254 nm. All melting points are uncorrected
and were measured on an Optimelt automated melting point system
from Stanford research systems. All 1H NMR and 13C NMR spectra
were measured on a Bru¨ker 200 or Bru¨ker 250. Analytical
HPLC-MS analyses were conducted using a Shimadzu LC-8A
preparative liquid chromatograph pump system with a Shimadzu
SPD-10AV UV-vis detector with the MS detection performed with
a Shimadzu LCMS-2010 liquid chromatograph mass spectrometer.
The buffer mentioned under conditions I and II is a 0.4% (w/v)
pocket (Figure 3), e.g., with aromatic groups, greatly increases
H4R affinity of quinazoline 30 but might also lead to substantial
H1R affinity. This hypothesis is supported by the observation
that quinoxalinone 3 (Figure 1) has very high H4R affinity but
does not bind the H1R.22 Quinoxalinone 3 fits in two pockets
of the pharmacophore model but does not occupy the third
pocket with an aromatic substituent, thereby explaining its lack
of H1R affinity. The pharmacology of compounds 54 and 55
was therefore studied at the human histamine H1R subtype
(Table 4). Interestingly, both 54 and 55 were found to possess
considerable affinity for the H1R and to be almost equipotent
at both the H1R and H4R.
With a pKi of 8.12, quinazoline 55 is significantly more
effective (9-fold) in displacing [3H]histamine binding from the
H4R compared to the standard imidazole-containing inverse

7862 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smits et al.
Figure 4. (A) Compounds 54 and 55 bind to the hH4R with high affinity as determined by [3H]histamine displacement. (B) Quinazolines 54 (R
) -1.49) and 55 (R ) -1.06) show inverse agonistic behavior in a functional assay (CRE-galactosidase reporter gene) performed in parallel with
H4R agonist histamine and H4R inverse agonist thioperamide. The R values for histamine and thioperamide have been arbitrarily set at 1 and -1,
respectively. Corresponding pIC50 values for 54 and 55 are 6.84 ( 0.12 and 7.16 ( 0.04, respectively (n ) 3).
Figure 5. Anti-inflammatory effects of compound 55 on paw edema induced by subplantar injection of carrageenan (1% in CMC) in rats. Data are
expressed as the mean ( SEM, n ) 6 rats per group. Comparisons between multiple groups were made by using one-way analysis of variance
(ANOVA), followed by Dunnett’s test: (/) P < 0.05 and (//) P < 0.01 compared with vehicle-treated animals (Student t test for grouped data).
NH4CO3 solution in water, adjusted to pH 8.0 with NH4OH. The
analyses were performed using the following two conditions.
Condition I: an Xbridge (C18) 5 µm column (100 mm × 4.6 mm)
with solvent A (90% MeCN-10%) and solvent B (90% water-10%
buffer), flow rate of 2.0 mL/min, start 5% A, linear gradient to
90% A in 10 min, then 10 min at 90% A, then 10 min at 5% A,
total run time of 30 min. Condition II: Xbridge (C18) 5 µm column
(100 mm × 4.6 mm) with solvent A (MeCN with 0.1% formic
acid) and solvent B (water with 0.1% formic acid), flow rate of
2.0 mL/min, start 5% A, linear gradient to 90% A in 10 min, then
5 min at 90% A, then 5 min at 5% A, total run time of 20 min.
Compound purities under both conditions were calculated as the
percentage peak area of the analyzed compound by UV detection
at 254 nm. Analytical HPLC-MS analyses for condition III were
carried out on Agilent 1100 series HPLC-MS system including
an Agilent G1315B DAD. Condition III: XBridge (C18) 3.5 µm
column (2.1 mm × 50 mm) with solvent A (a 5 mM solution of
NH4HCO3 in water set to pH 9.0 using 19 mM NH3) and solvent
B (100% ACN), MS detection with an Agilent G1956B LC/MSD
SL using a multimode ion source, flow rate of 1.2 mL/min, start
95% A, linear gradient to 5% A in 1.25 min, then 0.75 min at 5%
A, followed by 1.0 min 95% A, 5% B, total run time of 3 min.
Preparative HPLC-MS separations were carried out on an Agilent
1100 series preparative HPLC-MS system including a G1968D
active splitter, G1315B DAD, and a G1946D LC/MSD using an
ESI ion source. Elution was done over a Waters X-Terra MS C18
5 µm column (19 mm × 100 mm) with the solvent A (a 10 mM
solution of NH4HCO3 in water set to pH 9.5 using 38 mM NH3)
and solvent B (100% ACN), flow rate of 30 mL/min, gradient of
30% B f 75% B, start 70% A, 30% B, 1.3 min isocratic elution,
6.0 min to 25% A, 75% B followed by 0.7 min to 95% A, 5% B,
then 1.0 min at 5% A and 95% B, followed by 1.8 min 75% A,
30% B, total run time of 10.8 min.
In Vitro Pharmacology. Radioligand Displacement Studies
at the Human H1 and H4 Receptors. The pKi values at the H1R
and H4R were determined as described before.35,22
H4R CRE-ꢀ-galactosidase Assay. Five million HEK 293T cells
(in a suspension of 500 000 cell/mL) were transfected with a
mixture containing 2.5 µg of pCRE-gal, 5 µg of receptor plasmid,
and 35 µg of a 25 kDa linear polyethyleneimine and transferred
into a transparent 96-well plate. Twenty-four hours after transfec-
tion, cells were stimulated with ligands in the presence of 1 µM
forskolin for 6 h. Thereafter, the medium was discarded, the cells
were lysed in 100 µL of assay buffer (100 mM sodium phosphate
buffer at pH 8.0, 4 mM ONPG, 0.5% Triton X-100, 2 mM MgSO4,
0.1 mM MnCl2, 40 mM ꢀ-mercaptoethanol) and incubated at room
temperature for up to 4 h. The ꢀ-galactosidase activity was
determined at 420 nm with a Powerwave X340 plate reader (Bio-
Tek Instruments, Inc.).36 The data were analyzed using Prism 4.0
(Graphpad Software Inc.).
Flexible Alignment Models. The flexible alignment models were
created with Molecular Operating Environment 2006.08 (MOE)
from Chemical Computing Group (Montreal, Canada). The com-
pounds in Figure 2 were aligned with the flexible alignment module
of MOE using default parameters and similarity term “partial
charge” added with a weight factor (WF) of 1. The alignment with
lowest alignment score S and average strain energy score U (S )
227.7783, U ) 98.9762, F (similarity score) ) 128.8201) was
selected and refined further using the “refine existing alignment”
option with an energy cutoff of 7.0 kcal/mol. Compounds 1 and
82 were aligned with the following similarity terms and associated
weight factors: H-bond donor (WF ) 3), aromaticity (WF ) 3),

Quinazolines as InVerse Agonists
Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24 7863
polar hydrogens (WF ) 1), and volume (WF ) 3). The lowest
alignment score S was then selected and refined further using the
“refine existing alignment” option with an energy cutoff of 7.0 kcal/
mol. The van der Waals surface map for Figure 3A was generated
with a distance of 4.5 Å. The final alignment of 1 and 82 was then
fixed, and compounds 54 and 55 were both separately aligned with
1 and 82 using the flexible alignment module with the similarity
terms described for the alignment of 1 and 82. The final alignments
were selected on the basis of the best fit: for 54, lowest objective
function score S (S ) 73.2264, U ) 17.9301, F ) 55.3363); for
55, third lowest objective function score S (S ) 71.3573, U )
19.0853, F ) 52.1195).
In Vivo Pharmacology. Carrageenan-Induced Edema Model.
Male Wistar rats (180-200 g; Harlan-Italy, Milan, Italy) were
housed under controlled standard conditions (23 °C, 12 h light/
dark cycle, and 65% humidity). Food and water were provided ad
libitum. The experiments received the approval of the local Animal
Ethics Committee of the University of Parma, Italy. Inflammation
was induced in fasted rats by subplantar injection of carrageenan
(0.1 mL of 1% suspension in carboxymethycellulose) into the left
hind paw. As previously described, carrageenan-induced edema was
measured with a plethysmometer (Basile, Comerio, Italy) im-
mediately prior to the injection of carrageenan and thereafter at 2,
4, and 6 h.34 Edema was expressed for each animal as percent
increase in paw volume after carrageenan injection relative to the
preinjection value, considered as 100. Equivalent volumes (0.1 mL
per 100 g) of compound 55 or vehicle were administered subcu-
taneously (sc) in separate groups of rats immediately prior to
carrageenan injection. Before use, a solution of the histamine H4
receptor ligand in dimethyl sulfoxide was freshly prepared. Data
are expressed as the mean ( SEM. P < 0.05 was considered
statistically significant. Prism GraphPad 3.0 (GraphPad Software
Inc., San Diego, CA) was used to process data.
the residue was purified over SiO2 (EtOAc 90%, Et3N 5%, MeOH
5%), yielding 20 mg (4%) of a light-yellow solid. Mp 102.2-103.1
°C; 1H NMR (CDCl3) δ (ppm) 8.09 (d, J ) 8.2 Hz, 1H), 7.67-7.38
(m, 4H), 7.27-7.16 (m, 4H), 3.72 (t, J ) 5.0 Hz, 4H), 2.38 (t, J )
5.0 Hz, 4H), 2.29 (s, 3H); 13C NMR (CDCl3) δ (ppm) 166.82,
157.81, 154.01, 152.48, 133.93, 129.08, 125.15, 124.94, 123.64,
122.01, 121.83, 111.01, 54.64, 45.80, 43.34; MS (ESI) m/z 321
(M + H)+.
General Method C. The following method is representative for
the synthesis of 2,4 diamino substituted quinazolines 66-81.
6-Chloro-4-methylamino(4-methyl-1,4-diazepan-1-yl)quinazo-
line (69). A microwave tube was charged with 2,6-dichloro-N-
methylquinazolin-4-amine (100 mg, 0.44 mmol), diisopropylethy-
lamine (113 µL, 0.66 mmol), N-methylpyrrolidinone (200 µL), and
N-methylhomopiperazine (75 µL, 0.60 mmol). The mixture was
then heated at 150 °C for 10 min, and the obtained solution was
transferred to a LC-MS vial using a small amount of N-
methylpyrrolidinone. The reaction mixture was directly purified with
preparative LC-MS and freeze-dried to yield the title compound
as a white solid. Compounds 66, 70-72, and 77 were synthesized
from tert-butyl octahydro-1H-pyrrolo[3,4-b]pyridine-1-carboxylate,
(R)-tert-butyl pyrrolidin-3-ylcarbamate, (S)-tert-butyl pyrrolidin-
3-ylcarbamate, tert-butyl methyl(pyrrolidin-3-yl)carbamate and
(3aR,6aS)-tert-butyl hexahydropyrrolo[3,4c]pyrrole-2(1H)-carboxy-
late, respectively. After purification by LC-MS the Boc-protected
intermediates were deprotected by stirring in 2 M dioxane/HCl until
LC-MS indicated complete conversion. Removal of the solvent
yielded the desired amines as hydrochloride salts.
General Method D. The following method is representative for
the synthesis of chloroquinazoline-2,4(1H, 3H)-diones 19-22.
6-Chloroquinazoline-2,4(1H,3H)-dione (18). A flask containing
urea (15.0 g, 0.25 mol) and 2-amino-5-chlorobenzoic acid (4.25 g,
24.8 mmol) was heated at 140 °C. After being stirred for 6 h, the
reaction mixture was cooled to 100 °C and an equal volume of
water was added. The obtained suspension was left to stir for 10
min, after which it was cooled to room temperature. The precipitate
was filtered off and was dissolved in an aqueous 0.2 M NaOH
solution (∼100 mL). The solution was then heated at 100 °C for 5
min, causing a white precipitate to form. After being stirred at room
temperature overnight, the solution was acidified to pH 7 with
concentrated HCl and a white solid was filtered off. The obtained
solid was washed with water, triturated with hot EtOH (100 mL),
and cooled to room temperature. After filtration of the suspension
and drying of the product in vacuo, the title compound was obtained
Synthetic Methods. General Method A. The following method
is representative for the synthesis of 2,4-disubstituted quinazolines
29, 33, 35-39, and 43-64.
N-Benzyl-2-(4-methylpiperazin-1-yl)quinazolin-4-amine (34).
2,4-Dichloroquinazoline (171 mg, 0.86 mmol) was added to a
microwave tube containing EtOAc (3.0 mL) and DIPEA (0.32 mL,
mmol). Benzylamine (94 µL, 0.86 mmol) was added, and the
resulting mixture was stirred at room temperature until TLC
indicated complete conversion of the starting material to the
monosubsituted quinazoline. N-Methylpiperazine (1.0 mL) was
added, and the reaction mixture was heated at 120 °C for 10 min
under microwave irradiation. The obtained suspension was then
diluted with EtOAc (50 mL) and washed with water and brine.
Drying of the organic phase with Na2SO4 and evaporation of the
solvent gave the crude product that was purified over SiO2 (90%
EtOAc, 5% Et3N, 5% MeOH) to yield 398 mg (79%) mg of the
1
as 2.81 g (14.3 mmol, 58%) of a white solid. H NMR (DMSO-
d6) (ppm) δ 11.37 (app bs, 2H), 7.80 (d, J ) 2.5 Hz, 1H), 7.68
(dd, J ) 8.7 Hz, J ) 2.5 Hz, 1H), 7.18 (d, J ) 8.7 Hz, 1H).
General Method E. The following method is representative for
the synthesis of 2,4-dichloroquinazolines 25-28.
1
title compound as a beige solid. Mp 132.4-134.6 °C; H NMR
2,4,6-Trichloroquinazoline (24). 6-Chloro-2,4-dihydroxyquinazo-
line (1.41 g, 7.17 mmol), N,N-diethylaniline (2.3 mL), and POCl3
(5.0 mL) were heated at reflux. After 3 h the mixture was cautiously
poured over crushed ice. The obtained suspension was stirred. The
solids were extracted with DCM, and the combined organic phases
were washed with brine. Drying over Na2SO4 and evaporation of
the solvent yielded 1.52 g (6.51, 91%) of a yellow solid. The solid
was dissolved in DCM and filtered over a pad of silica using DCM
as eluent to yield the title compound as a white solid. Mp
(CDCl3) δ 7.50-7.24 (m, 7H), 7.07-7.00 (m, 1H), 5.80 (m, 1H),
4.78 (d, J ) 5.4 Hz, 2H), 3.91 (t, J ) 5.0 Hz, 4H), 2.45 (t, J ) 5.0
Hz, 4H), 2.32 (s, 3H); 13C NMR (CDCl3) δ (ppm) 159.48, 158.62,
151.92, 138.63, 132.45, 128.53, 127.74, 127.33, 125.65, 120.88,
120.56, 110.19, 54.99, 46.10, 45.03, 43.71; MS (ESI) m/z 334 (M
+ H)+.
General Method B. The following method is representative for
the synthesis of 2,4-disubstituted quinazoline ether 32.
2-(4-Methylpiperazin-1-yl)-4-phenoxyquinazoline (31). 2,4-
Dichloroquinazoline (300 mg, 1.52), phenol (170 mg, 1.81 mmol),
and NaH (55 mg of a 60% dispersion in mineral oil, 2.28 mmol)
in DMF (5.0 mL) were stirred at room temperature. After 2 h the
reaction mixture was diluted with EtOAc and water. The aqueous
phase was extracted with ethyl acetate, and the combined organic
layers were washed with H2O and brine. After the mixture was
dried over Na2SO4 and after evaporation of the solvent, the obtained
product was transferred to a microwave tube containing N-
methylpiperazine (2.0 mL). The mixture was heated at 140 °C for
5 min under microwave irradiation. The product was diluted with
ethyl acetate and washed with H2O and brine. After the mixture
was dried over Na2SO4 and subsequent evaporation of the solvent,
1
129.1-130.8 °C; H NMR (CDCl3) (ppm) δ 8.23-8.22 (m, 1H),
7.97-7.87 (m, 2H).
6-Chloro-2-(4-methylpiperazin-1-yl)quinazolin-4(3H)-one (11).
6-Chloro-2-(methylthio)quinazolin-4(3H)-one (10) (1.0 g, 4.41
mmol) was added to N-methylpiperazine (15.2 mL), and the
resulting mixture was heated at reflux. After 4 h the mixture was
diluted with water and washed with EtOAc. The aqueous phase
was then acidified with concentrated HCl to pH 7, causing the
desired product to crystallize from the solution. The solid was
filtered off and recrystallized from EtOH to yield 567 mg (2.03
mmol, 46%) of white crystals. 1H NMR (DMSO-d6) δ (ppm) 7.82
(d, J ) 2.5 Hz, 1H), 7.60 (dd, J ) 2.5 Hz, J ) 8.8 Hz, 1H), 7.30

7864 Journal of Medicinal Chemistry, 2008, Vol. 51, No. 24
Smits et al.
(d, J ) 8.8 Hz, 1H), 3.62 (t, J ) 4.9 Hz, 4H), 2.37 (t, J ) 4.9 Hz,
4H), 2.20 (s, 3H); MS (ESI) m/z 279 (M + H)+.
purified over SiO2 (EtOAc 90%, Et3N 5%, MeOH 5%) to yield
186 mg (87%) of the title compound. H NMR (CDCl3) δ (ppm)
1
2,4,7-Trichloroquinazoline (25). 7-Chloroquinazolin-2,4(1H,3H)-
dione (1.03 g, 5.21 mmol), DIPEA (1.91 mL, 10.95 mmol), and
POCl3 (5.0 mL) were heated at reflux. After 4 h the hot mixture
was carefully poured onto crushed ice, causing a precipitate to form
after vigorous stirring. The formed suspension was extracted with
CH2Cl2, and the organic phases were combined. After the mixture
was washed with brine and dried over Na2SO4, the solvent was
removed and the title compound was obtained as a yellow solid
that was used in the next step without further purification. Yield:
1.124 g (96%). 1H NMR (DMSO-d6) δ (ppm) 8.17 (d, J ) 9.0 Hz,
1H), 7.96 (d, J ) 2.0 Hz), 7.66 (d, J ) 2.0 Hz, J ) 9.0 Hz, 1H).
6-Chloro-2-(4-methylpiperazin-1-yl)quinazolin-4-amine (30).
2,4,6-Trichloroquinazoline (300 mg, 1.28 mmol) was added to a
saturated solution of ammonia in MeOH (5.0 mL) and stirred at
room temperature. After 16 h the mixture was diluted with EtOAc
(50 mL) and washed with water and brine. After the mixture was
dried over Na2SO4, the organic phase was concentrated (about 3
mL) and transferred to a microwave tube containing N-methylpip-
erazine (1.0 mL). The mixture was heated at 140 °C for 5 min
using microwave irradiation. The formed suspension was then
diluted with EtOAc and washed with water and brine. Subsequent
drying over Na2SO4 and evaporation of the solvent yielded an
orange solid that was purified over SiO2 (EtOAc 90%, Et3N 5%,
MeOH 5%). The title compound was obtained as 276 mg (78%)
7.44-7.35 (m, 3H), 5.45 (d, J ) 4.2 Hz, 1H), 5.06-4.89 (m, 2H),
3.18-3.02 (m, 6H), 2.74-2.65 (m, 1H), 2.29-2.11 (m, 2 H),
2.02-1.74 (m, 4H), 1.56-1.50 (m, 1H); 13C NMR (CDCl3) δ (ppm)
159.46, 158.76, 150.50, 132.73, 127.14, 125.41, 120.07, 110.97,
62.53, 53.43, 52.00, 48.25, 43.10, 27.99, 27.14, 21.06; MS (ESI)
m/z 318 (M + H)+.
Acknowledgment. Thanks goes to Debora Granemann for
synthetic assistance. The authors also greatly appreciate the
technical assistance of Frans de Kanter.
Supporting Information Available: Purity data for compounds
5, 7, 11, and 29-64 as determined by LC-MS; experimental details
for compounds 7, 19-22, 26-29, 32, 33, 35-39, and 41-64. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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of a light-yellow solid. Mp 176.0-178.5 °C; H NMR (CDCl3) δ
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°C; H NMR (CDCl3): δ (ppm) 7.42-7.24 (m, 3H), 5.42 (s, 1H),
3.92 (t, J ) 4.8 Hz, 4H), 3.10 (d, J ) 4.8 Hz, 3H), 2.46 (t, J ) 4.8
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2,6-Dichloro-N-methylquinazolin-4-amine (65). To a suspen-
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mL) was added methylamine (1.91 mL of 40% w/w in water). The
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the solution was concentrated to a volume of about 30 mL. The
reaction mixture was then diluted with water (200 mL) and extracted
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