Preparation and biological evaluation of indole, benzimidazole, and thienopyrrole piperazine carboxamides: Potent human histamine H4 antagonists

Article abstract of DOI:10.1021/jm0502081

Three series of H₄ receptor ligands,- derived from indoly-2-yl-(4-methyl-piperazin-1-yl)-methanones, have been synthesized and their structure-activity relationships evaluated for activity at the H ₄ receptor in competitive binding and functional assays. In all cases, substitution of small lipophilic groups in the 4 and 5-positions led to increased activity in a [₃H]histamine radiolabeled ligand competitive binding assay. In vitro metabolism and initial pharmacokinetic studies were performed on selected compounds leading to the identification of indole 8 and benzimidazole 40 as potent H₄ antagonists with the potential for further development. In addition, both 8 and 40 demonstrated efficacy in in vitro mast cell and eosinophil chemotaxis assays.

Full text of DOI:10.1021/jm0502081

J. Med. Chem. 2005, 48, 8289-8298
8289
Preparation and Biological Evaluation of Indole, Benzimidazole, and
Thienopyrrole Piperazine Carboxamides: Potent Human Histamine H4
Antagonists
Jennifer D. Venable,* Hui Cai, Wenying Chai, Curt A. Dvorak, Cheryl A. Grice, Jill A. Jablonowski,
Chandra R. Shah, Annette K. Kwok, Kiev S. Ly, Barbara Pio, Jianmei Wei, Pragnya J. Desai, Wen Jiang,
Steven Nguyen, Ping Ling, Sandy J. Wilson, Paul J. Dunford, Robin L. Thurmond, Timothy W. Lovenberg,
Lars Karlsson, Nicholas I. Carruthers, and James P. Edwards
Johnson & Johnson Pharmaceutical Research and Development, L.L.C., 3210 Merryfield Row, San Diego, California 92121
Received March 7, 2005
Three series of H4 receptor ligands, derived from indoly-2-yl-(4-methyl-piperazin-1-yl)-
methanones, have been synthesized and their structure-activity relationships evaluated for
activity at the H4 receptor in competitive binding and functional assays. In all cases, substitution
of small lipophilic groups in the 4 and 5-positions led to increased activity in a [3H]histamine
radiolabeled ligand competitive binding assay. In vitro metabolism and initial pharmacokinetic
studies were performed on selected compounds leading to the identification of indole 8 and
benzimidazole 40 as potent H4 antagonists with the potential for further development. In
addition, both 8 and 40 demonstrated efficacy in in vitro mast cell and eosinophil chemotaxis
assays.
Introduction
Until recently, the pharmacological effects of the
endogenous chemical mediator histamine were attrib-
uted to its interaction with three known G-protein
coupled receptors H1, H2 and H3.1 The human histamine
H1 receptor is expressed widely throughout the body and
is involved in numerous functions including smooth
muscle and endothelial cell contraction causing in-
creases in vascular permeability.2,3 These effects account
for the role of the H1 receptor in allergic and inflam-
matory responses and consequently for the application
of H1 receptor antagonists for the treatment of allergies.
Although the H2 receptor also participates in vasodila-
tion, due to its expression in vascular smooth muscle,
the major physiological role of this receptor is in the
gastrointestinal tract where histamine stimulates acid
secretion.4 Antagonists of this receptor are widely used
for treatment of ulcerative conditions. The histamine
Figure 1. Dual H3/H4 Ligands Thioperamide and (R)-R-
Methylhistamine and H4 Selective Antagonist 3.
H3 receptor behaves as a presynaptic autoreceptor on
histamine neurons and as a heteroreceptor on nonhis-
tamine neurons controlling the release of histamine and
play a key role in inflammation and regulation of the
other neurotransmitters in the central nervous sys-
immune system.2,9,14,15 The H4 receptor is known to
tem.5,6 While the therapeutic importance has not been
mediate chemotaxis of eosinophils and mast cells14-16,35,36
clinically defined, it has been implicated in various
and is also implicated in histamine-induced release of
conditions involving deficits in arousal, vigilance, and
interleukin-16 from CD-8+ T cells.2 Additionally, a
cognition. The fourth human histamine receptor was
selective H4 receptor antagonist demonstrated antiin-
disclosed in 2000 as a 390 amino acid GPCR with a 37-
flammatory activity in a mouse peritonitis model.16
43% sequence homology to the human H3 receptor.7-12
A screen of our corporate compound collection in a
This high degree of similarity is reflected by the affinity
[3H]histamine radiolabeled ligand competitive binding
of well-known H3 ligands thioperamide (1) and (R)-R-
assay resulted in the identification of piperazine 3,
methylhistamine (2) (Figure 1) for the H4 receptor.13
which displayed a Ki ) 38 nM at the H4 receptor (Figure
While the H3 receptor is found in the peripheral and
1) with 240-fold selectivity over the H3 receptor. Ana-
central nervous systems, the H4 receptor is primarily
logues of indole 3 were synthesized and found to exhibit
expressed in eosinophils, mast cells, dendritic cells, and
other leukocytes, implying that the H4 receptor may
excellent affinity (4-150 nM) for the H4 receptor with
100-1500-fold selectivity over the H3 receptor. Further
profiling in vitro and in vivo studies revealed a promis-
* To whom correspondence should be addressed. Phone: +1-858-
320-3365. Fax: +1-858-784-3116. jvenable@prdus.jnj.com.
ing metabolic and pharmacokinetic profile for this series
10.1021/jm0502081 CCC: $30.25 © 2005 American Chemical Society
Published on Web 11/23/2005

8290 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Venable et al.
Table 1. Binding Affinity and Functional Data for
Indole-Substituted Analogues
of indolylpiperazines. Using these data, modifications
were made to the heterocyclic nucleus of the pharma-
cophore with the intent of maintaining activity at the
H4 receptor while exploring what effects this had on the
metabolic and pharmacokinetic parameters. Therefore,
each ring of the fused heterocycle was alternately
replaced with an appropriate bioisostere, resulting in
the development of the thienopyrroles and benzimida-
zole carboxamides as two additional series of potential
H4 receptor ligands.
Results and Discussion
Initial examination of the high-throughput screening
hit, indolylpiperazine 3, revealed the N-methylpipera-
zine moiety as the optimal amine terminus, with all
other modifications yielding decreased affinity for the
H4 receptor, as previously disclosed.13 Thus, attention
turned toward the indole core and substituent effects
on the arene ring. As a result, a variety of substituted
indolyl N-methylpiperazine-carboxamides (4-24, Table
1) were constructed, the synthesis of which was de-
scribed in the previous report.13 The indole carboxamide
analogues were first examined in a recombinant human
histamine H4 competitive binding assay using [3H]-
histamine as the radioligand (Table 1). For compounds
with Ki values of less than 75 nM, the functional
antagonism was measured by Schild analysis of a
forskolin-stimulated cAMP-mediated reporter gene as-
say in SK-N-MC cells and reported as a pA2 value. The
parent N-methylpiperazine carboxamide 4 was es-
sentially equipotent (Ki ) 17 nM) to the high-through-
put screening hit 3 (Ki ) 38 nM) and displayed encour-
aging results in the functional assay with a pA2 ) 7.4.
A considerable amount of information could be obtained
by examining substitution at various positions around
the indole nucleus. Placement of a bromine atom in the
4-position gave 5, which was equipotent to the unsub-
stituted indole 4 in the binding assay, whereas bromine
substitution at C(6) (6) resulted in decreased affinity
for the receptor. However, the 5-bromoindole 7 displayed
increased affinity for the receptor (Ki ) 8 nM) and
showed a marked improvement in the functional assay
(pA2 ) 7.8). With this information in hand, we surveyed
a variety of substituents at the indole 5-position. The
5-chloro and 5-fluoro compounds 8 and 9 were both
found to be potent ligands for the H4 receptor, with
indole 8 having excellent efficacy in the functional assay
with a pA2 ) 8.1. Nonhalo substituents at C(5) (10-
12) reduced affinity for the receptor 10-100 fold com-
pared to 4. Small electron-donating groups were also
tolerated at this position, with the 5-hydroxy compound
13 and 5-aminoindole 14 retaining potent H4 activity
(Ki ) 23 nM and 15 nM, respectively). Introduction of
similar substituents at the 7-position (15-18) also
showed improved activity in the binding and functional
assays when compared with the parent 4. In general,
the 5-position appears to have the greatest effect on in
vitro potency. Interestingly, once the requisite 5-position
is occupied, additional substituents in the 4 or 7-posi-
tions (19-20 and 21-24 respectively) are tolerated
without a loss in H4 binding affinity.
a Displacement of [3H]histamine from the recombinant hista-
mine H4 receptor. Ki values are the geometric mean ( SEM of
three or more independent determinations and calculated accord-
ing to Cheng and Prusoff.17 b Antagonism of histamine inhibition
of forskolin-stimulated cAMP-mediated reporter gene activity in
SK-N-MC cells expressing the human histamine H4 receptor.
analogous thienopyrrole carboxamides. The desired
thienopyrrole system has two possible regioisomers: one
isomer positions the sulfur and nitrogen on the same
ring fusion carbon atom (head-to-head), alternatively
sulfur and nitrogen may be placed on different carbons
(head-to-tail). The head-to-head thienopyrroles were
synthesized via Hemetsberger reaction of thiophene 27,
derived from the condensation of thiophene-3-carbox-
aldehyde and ethyl azidoacetate (Scheme 1).20 Ester 28
could be functionalized further by treatment with N-
chlorosuccinamide (NCS) to give the 5-chloro compound
29, yielding a similar substitution pattern to the indoles
already described. After cyclization, the esters were
hydrolyzed and coupled with N-methylpiperazine to give
the desired amide products 30 and 31. The head-to-tail
thienopyrroles were synthesized utilizing the same
With the structure-activity relationships around the
indole core evaluated, bioisosteric replacement18,19 of the
indole arene ring with thiophene afforded a series of

Human Histamine H4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8291
Table 2. Activity of Thienopyrroles
Scheme 1a
a Reagents and conditions: (i) EtONa, EtOH, 0 °C; (ii) xylenes,
145 °C; (iii) NCS (1 equiv), AcOH, CHCl3, rt; (iv) LiOH, THF/
MeOH/H2O, 65 °C; (v) (COCl)2, cat. DMF, CH2Cl2, rt; or EDCI,
CH2Cl2; (vi) N-methylpiperazine, CH2Cl2, rt.
Scheme 2a
a Displacement of [3H]histamine from the recombinant hista-
mine H4 receptor. Ki values are the geometric mean ( SEM of
three or more independent determinations and calculated accord-
ing to Cheng and Prusoff.17 b Antagonism of histamine inhibition
of forskolin-stimulated. cAMP-mediated reporter gene activity in
SK-N-MC cells expressing the human histamine H4 receptor.
has also been taken by Terzioglu et al.29 There are
relatively few literature methods for constructing ben-
zimidazole 2-carboxylic esters, acids and amides.23-29
One synthesis involves oxidation of 2-hydroxymethyl
benzimidazole to the carboxylic acid30 followed by
standard amide coupling. Unfortunately, many of the
benzimidazole-2-carboxylic acids were found to be un-
stable, readily decarboxylating upon standing and under
coupling conditions, giving only moderate to low yields
of the desired compounds. Following the work of Holan,31
an alternative two-step route was developed avoiding
this labile intermediate. Commercially available phe-
nylenediamines were treated with methyl 2,2,2-trichlo-
roacetimidate in glacial acetic acid to afford 2-trichlo-
romethylbenzimidazoles,29,32 followed by ammination/
hydrolysis directly to the desired amides with N-methyl-
piperazine and excess sodium hydrogencarbonate in wet
acetonitrile (Scheme 3).
The benzimidazoles synthesized by this method were
tested for affinity at the H4 receptor (Table 3). For
simplicity, the numbering system in Table 3 follows that
of the indole series. However, when comparing data for
the benzimidazoles to that of the indole series, one must
keep in mind that C(4) of the benzimidazole is equiva-
lent to C(7) due to tautomerization of the benzimidazole
moiety. Likewise, C(5) and C(6) are also interchange-
able. The parent benzimidazole 36 was found to be
comparable in activity to the parent N-methylpiperazi-
nylindole 4. Contrary to the indole and thienopyrrole
series, introduction of substituents in the 5-position
(39-42) did not greatly improve affinity for the receptor.
a Reagents and conditions: (i) EtONa, EtOH, 0 °C; (ii) xylenes,
145 °C; (iii) LiOH, THF/MeOH/H2O, 65 °C; (iv) (COCl)2, cat. DMF,
CH2Cl2, rt; (v) N-methylpiperazine, CH2Cl2, rt.
methodology starting with variously substituted thio-
phene-2-carboxaldehydes (Scheme 2).21,22
As postulated, replacement of the benzene portion of
indole with thiophene did indeed yield potent antago-
nists at the human histamine H4 receptor (Table 2),
although the unsubstituted compounds 30 and 32
displayed a 3-4-fold decrease in affinity in comparison
with the parent indole 3. In the indole series, the most
consistently active compounds were obtained from
5-substituted indoles (e.g. 8, Ki ) 4 nM, pA2 ) 8.1). A
similar trend was found in both regioisomers of the
thienopyrrole analogues; when either R4 or R5 of the
thiophene moiety was substituted, activity at the H4
receptor was increased as demonstrated by compound
31 in the head-to-head series (R5 ) Cl) and compound
33 (R4 ) Cl) in the head-to-tail series with Kis of 25 and
40 nM, respectively. Substitution of R5 in the head to
tail series also led to the H4 ligand 34 (Ki ) 21 nM).
These effects appeared to be additive in this series with
di-substituted thienopyrrole 35 having a 10-fold increase
in affinity for the H4 receptor (Ki ) 3 nM) over either
monosubstituted analogue 33 or 34. However this did
not translate to a marked difference in the functional
assay.
Replacement of the pyrrole ring of the indole carboxa-
mides for imidazole was also examined. This approach

8292 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Venable et al.
Table 4. In Vitro Metabolism and Phamacokinetic Data
Scheme 3a
a Reagents and conditions: (i) Methyl 2,2,2-trichloroacetimidate,
AcOH, rt; (ii) LiOH, EtOH; N-methylpiperazine, NaHCO3, AcOH/
H2O, rt.
Table 3. Activity for Benzimidazole-2-carboxamides
thienopyrrole and benzimidazole systems, thienopyrrole
31 and benzimidazole 4033 (JNJ 10191584), were se-
lected for further profiling. All three compounds were
found to be potent ligands for the H4 receptor (Ki ) 4
nM, 25 nM, and 26 nM, respectively) and showed
excellent functional antagonism in H4 receptor trans-
fected cells with pA2’s ranging from 7.7 to 8.1. In vitro
metabolism studies in both rat and human liver mi-
crosomes and S9 fraction were performed with each
heterocyclic nucleus for comparison with the observed
in vivo pharmacokinetic data (Table 4). The indolylpip-
erazine 8 showed a moderate rate of metabolism in both
human and rodent with a t1/2 ) 28 min in human liver
microsomes and t1/2 ) 64 min in rodent S9 fraction. This
compares with an in-vivo half-life of 2h when dosed
orally in rats at 10 mg/kg with a Cmax ) 1.8 µM. The
oral bioavailability of the compound was confirmed by
its activity in the zymosan induced mouse peritonitis
model.16 From the in vitro metabolism results, it ap-
peared that the chlorosubstituted head-to-head (31)
thienopyrrole was metabolized similarly to the indole
8. Unfortunately, in vivo pharmacokinetic studies of 31
in rodents unexpectedly showed the compound had no
oral bioavailability at 10 mg/kg and an extremely short
intravenous half-life (t1/2 ) 6 min at 2 mg/kg). Replace-
ment of the pyrrole ring of indole 8 with imidazole to
give the benzimidazole 40 did show increased stability
a Displacement of [3H]histamine from the recombinant hista-
mine H4 receptor. Ki values are the geometric mean ( SEM of
three or more independent determinations and calculated accord-
ing to Cheng and Prusoff.17 bAntagonism of histamine inhibition
of forskolin-stimulated cAMP-mediated reporter gene activity in
SK-N-MC cells expressing the human histamine H4 receptor.
This core was also less tolerant of substitution in
general, as demonstrated by the reduced affinity of the
5-methylbenzimidazole 41 (Ki ) 528 nM). Di-substituted
5-fluoro-4-methylbenzimidazole 43 maintained similar
activity to the corresponding indole analogue (20). The
4,6-disubstituted compound 45 maintained good affinity
for the receptor but showed diminished activity in the
functional assay. Interestingly, the 4,6-dichlorobenz-
imidazole 46 was 20-fold less active than the corre-
sponding 5,7-substituted indole 22.
In Vitro Metabolism and In Vivo Pharmacoki-
netics. On the basis of these results, the 5-chloroindole
8 (JNJ 7777120), and its direct analogues from the

Human Histamine H4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8293
imidazole carboxamides. Specifically, the activity of the
indoles was matched in the head-to-tail series of the
thienopyrroles by substitution with small lipophilic
groups (e.g. 35, Ki ) 3 nM), whereas analogues from
the benzimidazole series were generally found to be
5-10-fold less potent than the corresponding indole
carboxamide (e.g. 40, Ki ) 26 nM).
Comparison of the chloro-substituted indole 8, thieno-
pyrrole 31, and benzimidazole 40 in in vitro metabolism
studies demonstrated the effect of the heterocyclic core
on in vitro stability. The benzimidazole 40 showed an
increased stability over the indole 8 and thienopyrrole
31, with half-lives 2-4-fold longer in human liver
microsomes and 1.5-2-fold longer in rodent S9 fraction;
however, the same was not observed in vivo. The indole
and benzimidazole displayed similar bioavailability with
Cmax ) 1.8 µM in both cases, however the benzimidazole
had a 2-fold shorter half-life. The thienopyrrole 31 had
no oral bioavailability in-vivo in rat. The efficacy of the
two orally bioavailable compounds, indole 8 and benz-
imidazole 40, were examined in both the human eosi-
nophil and mouse bone-marrow mast cell chemotaxis
assays. These compounds were able to block chemotaxis
of these inflammatory cells in vitro with IC50s from 40
to 530 nM. Additionally, compounds 8 (JNJ 7777120)
and 40 (JNJ 10191584) have been shown to be selective
for the H4 receptor over the H3 receptor (Ki ) 5125 (
1087)16 and 14053 ( 4878 nM, respectively) and dis-
played no cross-reactivity in a CEREP panel of 50 other
receptors, enzymes, transporters, and ion-channels. We
hope to utilize the compounds developed from all three
series of potent human histamine H4 antagonists to
further discern the role of the newest histamine receptor
in inflammatory pathways.
Figure 2. Inhibition of mast cell (A) and eosinophil (B)
chemotaxis by 8 (b)and 40 (O). The results shown are the
average of at least two experiments and the error bars
represent the S. E. M.
in both human and rodent liver microsomes (3-4 fold)
and had added stability in rodent S9 fraction. However,
this improvement in vitro did not translate to a longer
half-life in vivo in rodent. Oral administration of ben-
zimidazole 40 at 10 mg/kg in rats gave a profile similar
to that of 8 with a Cmax ) 1.8 µM, but with a shorter
half-life (1 h).
In Vitro Mast Cell and Eosinophil Chemotaxis.
As previously mentioned, the H4 receptor is primarily
expressed on hematopoietic cells such as eosinophils and
mast cells.15,34,35 Both types of effector cells are impor-
tant during an inflammatory response and have been
implicated in the pathology of several diseases including
asthma and rheumatoid arthritis. In recent publica-
tions, we described the in vitro histamine-induced
chemotaxis of mouse bone-marrow mast cells and hu-
man eosinophils.14-16 Using specific histamine receptor
antagonists, as well as mast cells derived from H4
receptor deficient mice, this chemotaxis was determined
to be an H4-mediated effect. The induction of eosinophil
chemotaxis by histamine is also mediated by the H4
receptor.35,36 We have previously shown that 8 can
inhibit both eosinophil and mast cell chemotaxis with
IC50 values of 86 nM and 40 nM, respectively.16,35 In
the present study a second H4 antagonist, 40, was
shown to be efficacious in both assays, inhibiting
chemotaxis of eosinophils and mast cells with IC50
values of 530 nM and 138 nM, respectively (Figure 2).
The relative potency of the indole 8 and benzimidazole
40 in the chemotaxis assays is consistent with the
differences in their affinity for the H4 receptor.
Experimental Section
General Experimental Information. 1H NMR spectra
were recorded on Bruker DPX-400 or DPX-500 NMR spec-
trometers in the specified deuterated solvent. Column chro-
matography was performed employing an Isco SG100 auto-
mated flash chromatography system using prepacked silica gel
columns purchased from either Isco, Inc. or Biotage, Inc. Low
resolution mass spectra (MS) were determined on an Agilent
HP1100/MSD. High-resolution mass spectra (HRMS) were
determined on a Bruker MicroTOF with Eksigent capillary
HPLC express-100 using a Zorbax 300SB-C18 (3.5 microme-
ter, 50 × 0.3 mm), 100 nL injection, 100% ACN (with 0.1%
formic acid) for 3 min at 10 µL/min. Elemental analyses were
performed by Desert Analytics, Tucson, AZ, or Numega
Laboratories, San Diego, CA, and are within (0.4% of the
calculated values unless otherwise stated. IR spectra were
obtained on a Thermo-Nicolet Avatar 360. Reagents were
purchased commercially and used without purification unless
otherwise stated. Abbreviations used below include CDI (1,1′-
carbonyldiimidazole), DMF (N,N-dimethylformamide), EDCI
(1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochlo-
ride), HATU (O-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethy-
luronium hexafluorophosphate), HOAT (1-hydroxy-7-azaben-
zotriazole), HOBT (1-hydroxybenzotriazole hydrate), TFA
(trifluoroacetic acid), and THF (tetrahydrofuran). The synthe-
sis of compounds 3-9, 12-19, and 21-23 has been previously
described.13
Conclusion
After determining the indolyl piperazine 3 was a
potent human histamine H4 receptor antagonist, a
medicinal chemistry effort based on this series led to
the synthesis of the 5-chloroindole 8 (Ki ) 4 nM).
Alternately replacing each ring of the indole moiety with
an appropriate bioisostere yielded two additional series
of potent H4 antagonists, the thienopyrrole and benz-
(5-Methyl-1H-indol-2-yl)-(4-methyl-piperazin-1-yl)-meth-
anone (10). A mixture of 5-methylindole-2-carboxylic acid (1.0
g, 5.7 mmol) and EDCI (1.6 g, 8.3 mmol) in CH2Cl2 (10 mL)
was treated with 1-methylpiperazine (0.63 mL, 5.7 mmol) and
stirred at room temperature for 18 h. The reaction mixture
was quenched with saturated NaHCO3 solution and extracted
with CH2Cl2 and dried over MgSO4, filtered, and concentrated

8294 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Venable et al.
under reduced pressure to yield the crude product as a pale
yellow solid. The crude product was purified via silica gel
chromatography (0-100% acetone/CH2Cl2) to give the title
in CH2Cl2 (0.2 M) and allowed to stir at room temperature for
3 h. The reaction mixture was partitioned between CH2Cl2 and
sat. NaHCO3. The organic layer was separated, washed with
H2O, dried over Na2SO4, and concentrated. The crude product
was further purified with silica gel column chromatography.
1
compound (1.16 g, 79%). H NMR (400 MHz, CDCl3): δ 9.04
(br s, 1H), 7.39 (d, J ) 0.7 Hz, 1H), 7.29 (d, J ) 8.4 Hz, 1H),
7.09 (dd, J ) 8.4, 1.4 Hz, 1H), 6.67 (m, 1H), 3.92 (br s, 4H),
2.47 (t, J ) 5.1 Hz, 4H), 2.42 (s, 3H), 2.32 (s, 3H); Anal.
(C15H19N3O) C, H, N.
6H-Thieno[2,3-b]pyrrole-5-carboxylic Acid Ethyl Ester
(28). Thiophene-3-carbaldehyde (2.24 g, 20 mmol) was annu-
lated according to General Procedure A to provide 6H-Thieno-
[2,3-b]pyrrole-5-carboxylic acid ethyl ester (1.2 g, 31%) as a
(5-Trifluoromethyl-1H-indol-2-yl)-(4-methyl-piperazin-
1-yl)methanone (11). The title compound was prepared
according to the procedure described for compound 10. 1H
NMR (400 MHz, CDCl3): δ 10.94 (s, 1H), 7.93 (s, 1H), 7.48
(dd, J ) 13.1 Hz, 9.0 Hz, 2H), 6.84 (d, J ) 1.6 Hz, 1H), 4.00
(br s, 4H), 2.53 (t, J ) 4.9 Hz, 4H), 2.36 (s, 3H); MS
(electrospray): mass calculated for C15H16F3N3O, 311.12; m/z
found, 312.1 [M+ + H], Anal. (C15H16F3N3O); C, H, N.
(5-Fluoro-4-methyl-1H-indol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (20). The title compound was prepared
according to the procedure described for compound 10. 1H
NMR (400 MHz, CDCl3): δ 9.98 (br s, 1H), 7.18 (dd, J ) 4.89
Hz, 3.9 Hz, 1H), 6.99 (t, J ) 9.8 Hz, 1H), 6.74 (d, J ) 2.2 Hz,
1H), 3.99 (br s, 4H), 3.51 (t, J ) 5.3 Hz, 4H), 2.44 (s, 3H), 2.36
(s, 3H); MS (electrospray): exact mass calculated for C15H18-
FN3O, 275.14; m/z found, 276.2 [M+ + H], Anal. (C15H18FN3O);
C, H, N.
(7-Chloro-5-methyl-1H-indol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (24). The title compound was prepared
according to the procedure described for compound 10.1H NMR
(400 MHz, CDCl3): δ 9.71 (br s, 1H), 7.20 (s, 1H), 7.30 (s, 1H),
7.11 (s, 1H), 6.71 (d, J ) 2.3 Hz, 1H), 3.95 (br s, 4H), 2.50 (t,
J ) 4.80 Hz, 4H), 2.42 (s, 6H), 2.35 (s, 3H); MS (electrospray):
mass calculated for C15H18ClN3O, 291.11; m/z found, 292.2 [M+
+ H], Anal. (C15H18ClN3O) C, H, N.
General Procedures for the Preparation of Com-
pounds 30-35. General Procedure A: Annulation of
Aldehyde with Ethyl Azidoacetate. A solution of the
aldehyde (1 equiv) and ethyl azidoacetate (4 equiv) was added
dropwise to a solution of NaOEt (4 equiv) in ethanol (0.15 M)
at 0 °C. The reaction mixture was stirred at 0 °C for 1 h and
room temperature for an additional 1 h. The reaction mixture
was then poured into sat. NH4Cl and extracted with ether.
The combined organics were dried (Na2SO4) and concentrated
in vacuo. The residue was purified by silica gel column
chromatography to provide the desired acrylate. A solution of
the resultant acrylate in xylene (0.2 M) was heated at 145 °C
for 10-60 min and then allowed to cool to room temperature.
The xylene solution was either cooled further to induce product
crystallization or directly subjected to silica gel column chro-
matography to obtain the desired annulation product.
General Procedure B: Ester Hydrolysis. A solution (0.2
M) of the ethyl ester (1 equiv, from General Procedure A) and
LiOH (5 equiv) in THF/MeOH/H2O (3:1:1) was heated at 65
°C overnight, cooled to room temperature, acidified with 2 N
HCl, and extracted with EtOAc. The organic layer was
separated, dried over Na2SO4, and concentrated to give the
desired crude acid which was taken to the next step without
further purification.
1
white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.50. H
NMR (CDCl3, 400 MHz): δ 10.30 (br s, 1H), 7.10 (d, J ) 1.9
Hz, 1H), 6.96 (d, J ) 5.4 Hz, 1H), 6.87 (d, J ) 5.4 Hz, 1H),
4.39 (q, J ) 7.1 Hz, 2H), 1.38 (t, J ) 7.1 Hz, 3H).
(4-Methyl-piperazin-1-yl)-(6H-thieno[2,3-b]pyrrol-5-
yl)-methanone (30). 6H-Thieno[2,3-b]pyrrole-5-carboxylic
acid ethyl ester (835 mg, 4.3 mmol) was hydrolyzed according
to General Procedure B to provide the crude acid as a pale-
1
yellow solid. H NMR (CD3OD, 400 MHz): 7.02 (s, 1H), 6.96
(s, 1H), 6.95 (s, 1H). The carboxylic acid (60 mg, 0.35 mmol)
was coupled with N-methylpiperazine according to General
Procedure C to provide the title compound (44 mg, 50%) as a
light yellow solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4.
MS (electrospray): mass calculated for C12H15N3OS, 249.34;
m/z found, 250.1 [M+ + H]. 1H NMR (CD3OD, 400 MHz, TFA
salt): 6.97 (s, 1H), 6.96 (s, 1H), 6.85 (s, 1H), 4.20-3.10 (m,
8H), 2.96 (s, 3H). Anal. (C12H15N3OS) C, H, N, S.
2-Chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic Acid Eth-
yl Ester (29). A solution of compound 28 (580 mg, 3.0 mmol)
in acetic acid (6 mL) and CHCl3 (6 mL) was treated with three
portions of N-chlorosuccinimide (total 415 mg, 3.15 mmol) at
0 °C over 2 h. The reaction mixture was slowly warmed to room
temperature and stirred overnight. CHCl3 was then removed,
and the residue was basified with 4 N NaOH and extracted
with EtOAc. The combined organics were washed with sat.
NaHCO3, dried over Na2SO4, and concentrated. Column chro-
matography (silica, 5-10% EtOAc/hexanes) gave 600 mg (88%)
of 2-chloro-6H-thieno[2,3-b]pyrrole-5-carboxylic acid ethyl ester
as a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.5.
1H NMR (CDCl3, 400 MHz): δ 10.50 (br s, 1H), 6.97 (d, J )
2.0 Hz, 1H), 6.85 (s, 1H), 4.39 (q, J ) 7.2 Hz, 2H), 1.35 (t, J )
7.2 Hz, 3H).
(2-Chloro-6H-thieno[2,3-b]pyrrol-5-yl)-(4-methyl-pi-
perazin-1-yl)-methanone (31). Compound 29 (102 mg, 0.45
mmol) was hydrolyzed (General Procedure B) and coupled with
N-methylpiperazine (procedure D) to provide the title com-
pound (102 mg, 80% for two steps) as an off-white solid. TLC
(silica, 10% MeOH/CH2Cl2): Rf ) 0.4. HRMS (electrospray):
calculated for C12H14ClN3OS, 284.0619; m/z found, 284.0611
[M+ + H]. 1H NMR (CDCl3, 400 MHz): δ 10.50 (br s, 1H), 6.87
(s, 1H), 6.61 (d, J ) 1.8 Hz, 1H), 3.92 (t, J ) 5.1 Hz, 4H), 2.50
(t, J ) 5.1 Hz, 4H), 2.35 (s, 3H). Anal. (C12H14ClN3OS‚C4H4O4)
calcd C, 48.06; H, 4.54; N, 10.51, S, 8.02. Found C, 47.58. H,
4.07; N, 10.95; S, 8.50.
(4-Methyl-piperazin-1-yl)-(4H-thieno[3,2-b]pyrrol-5-
yl)-methanone (32). A solution of thiophene-2-carbaldehyde
(1.10 mL, 11.7 mmol) was annulated according to General
Procedure A to provide 4H-thieno[3,2-b]pyrrole-5-carboxylic
acid ethyl ester as a yellowish solid. 1H NMR (400 MHz, CDCl3)
δ 9.06 (br s, 1H), 7.33 (d, J ) 5.3 Hz, 1H), 7.14 (m, 1H), 6.96
General Procedure C: Amide Formation Using 1-(3-
Dimethylaminopropyl)-3-ethylcarbodimide Hydrochlo-
ride (EDCI). A mixture of acid (1 equiv, from General
Procedure B), amine (1.5 equiv), and EDCI (2.0 equiv) in CH2-
Cl2 (0.2 M) was stirred at room-temperature overnight and
then partitioned between CH2Cl2 and sat. NaHCO3. The
organic layer was separated, washed with H2O, dried over Na2-
SO4, and concentrated. The crude product was further purified
with silica gel column chromatography.
General Procedure D: Amide Formation via Acyl
Chloride Intermediate. A mixture of acid (1 equiv, from
General Procedure B) in CH2Cl2 (0.5 M) was treated at 0 °C
with oxalyl chloride (1.2 equiv) followed by 1-2 drops of DMF.
The reaction mixture was stirred at 0 °C for 30 min then slowly
warmed to room temperature and stirred for an additional 1
h. All volatiles were removed to provide the crude acyl chloride.
The resultant acyl chloride was treated with amine (5.0 equiv)
(m, 1H), 4.37 (q, J ) 7.3 Hz, 2H), 1.39 (t, J ) 7.3 Hz, 3H). 13
C
NMR (100 MHz, CDCl3) δ 161.3, 140.9, 129.2, 126.9, 124.6,
110.9, 107.3, 60.4, 14.2. The ethyl ester (98.5 mg, 0.50 mmol)
was then hydrolyzed according to General Procedure B and
coupled with N-methylpiperazine (General Procedure C) to
1
provide the title compound in 51% yield. H NMR (400 MHz,
CDCl3) δ 9.26 (br s, 1H), 7.26 (m, 1H), 6.97 (m, 1H), 6.74 (m,
1H), 3.99 (m, 4H), 2.59 (m, 4H), 2.43 (br, 3H). MS (electro-
spray): mass calculated for C12H15N3OS, 249.34; m/z found,
250.1 [M+ + H]. Anal. (C12H15N3OS) C, H, N, S.
(2-Chloro-4H-thieno[3,2-b]pyrrol-5-yl)-(4-methyl-pi-
perazin-1-yl)-methanone (33). 5-Chloro-thiophene-2-carbal-
dehyde (2.92 g, 20 mmol) was annulated according to General
Procedure A to provide the annulated product (2.8 g, 61%) as
a white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.48.

Human Histamine H4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8295
1H NMR (CDCl3, 400 MHz): δ 9.10 (br s, 1H), 7.04 (dd, J )
1.9, 0.7 Hz, 1H), 6.89 (d, J ) 0.7 Hz, 1H), 4.37 (q, J ) 7.2 Hz,
2H), 1.39 (t, J ) 7.2 Hz, 3H). The ethyl ester (230 mg, 1.0
mmol) was hydrolyzed (General Procedure B) and coupled with
N-methylpiperazine (General Procedure C) to provide com-
pound 33 (128 mg, 45% for two steps) as an off-white solid.
TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. HRMS (electro-
spray): mass calculated for C12H14ClN3OS, 284.0619; m/z
found, 284.0615 [M+ + H]. 1H NMR (CDCl3, 400 MHz): δ 10.1
(br s, 1H), 6.88 (s, 1H), 6.64 (d, J ) 1.4 Hz, 1H), 3.91 (t, J )
4.4 Hz, 4H), 2.49 (t, J ) 5.1 Hz, 4H), 2.35 (s, 3H). Anal. (C12H14-
ClN3OS‚C4H4O4) calcd C, 48.06; H, 4.54; N, 10.51, S, 8.02.
Found C, 47.53; H, 3.89; N, 11.25; S, 8.60.
was washed with water (2 × 30 mL) and dried under vacuum
to afford 1.90 g (88%) of 2-trichloromethyl-1H-benzoimidazole
which was used without further purification. MS (electro-
spray): mass calculated for C8H5Cl3N2, 234.0; m/z found 235.0,
[M + H]+. 1H NMR (400 MHz, CDCl3): δ 13.45 (br s, 1H), 7.68
(br m, 2H), 7.35 (m, 2H).
4-Methyl-2-trichloromethyl-1H-benzoimidazole. The re-
action was carried out as described in General Procedure E
employing 2,3-diaminotoluene. Purification via silica gel chro-
matography (40% EtOAc/hexanes) afforded 830 mg (34%) of
the title compound. MS (electrospray): mass calculated for
1
C9H7Cl3N2, 247.97; m/z found 249.0, [M + H]+. H NMR (400
MHz, CDCl3): δ 9.78 (s, 1H), 7.52 (br s, 1H), 7.27 (d, J ) 7.4,
8.1 Hz, 1H), 7.17 (d, J ) 7.4 Hz, 1H), 2.64 (s, 3H).
(4-Methyl-piperazin-1-yl)-(3-methyl-4H-thieno[3,2-b]-
pyrrol-5-yl)-methanone (34). A solution of 3-methylth-
iophene (6.76 mL, 70 mmol) in ether (70 mL) was treated with
n-butyllithium (2.5 M in hexanes, 28.6 mL, 71.4 mmol) at such
a rate that a slight reflux was maintained. The reaction
mixture was heated to reflux for 15 min and DMF (7.0 mL, 91
mmol) in ether (30 mL) was added. After stirring for 4 h, the
reaction was quenched with addition of sat. NH4Cl (200 mL).
The organic layer was separated and washed with brine, H2O,
dried over Na2SO4, and concentrated. Column chromatography
(silica, 5-10% EtOAc/hexanes) provided a mixture of 4-methyl-
thiophene-2-carbaldehyde and 3-methyl-thiophene-2-carbal-
dehyde (4.4:1, 8.1 g, 92%) as a light yellow oil. TLC (silica,
10% EtOAc/hexanes): Rf ) 0.55. For 4-methyl-thiophene-2-
carbaldehyde: 1H NMR (CDCl3, 400 MHz): δ 9.95 (s, 1H), 7.58
(d, J ) 1.2 Hz, 1H), 7.37-7.35 (m, 1H), 2.32 (s, 3H). For
3-methyl-thiophene-2-carbaldehyde: 1H NMR (CDCl3, 400
MHz): 10.02 (s, 1H), 7.64 (d, J ) 4.6 Hz, 1H), 6.97 (d, J ) 4.6
Hz, 1H), 2.58 (s, 3H). The mixture of 4-methyl-thiophene-2-
carbaldehyde and 3-methyl-thiophene-2-carbaldehyde (2.84 g,
22.5 mmol) was annulated according to General Procedure A
to provide the 3-methyl-4H-thieno[3,2-b]pyrrole-5-carboxylic
acid ethyl ester (2.5 g, 65%) as a white solid. TLC (silica, 20%
EtOAc/hexanes): Rf ) 0.45. 1H NMR (CDCl3, 400 MHz): δ
9.95 (br s, 1H), 7.12 (d, J ) 1.9 Hz, 1H), 6.90 (d, J ) 1.2 Hz,
1H), 4.39 (q, J ) 7.2 Hz, 2H), 2.35 (s, 3H), 1.39 (t, J ) 7.2 Hz,
3H). The ethyl ester (200 mg, 0.96 mmol) was then hydrolyzed
(Procedure B) and coupled with N-methylpiperazine (General
Procedure D) to provide compound 34 (197 mg, 78% for two
steps) as an off-white solid. TLC (silica, 10% MeOH/CH2Cl2):
Rf ) 0.4. HRMS (electrospray): mass calculated for C13H17N3-
OS, 264.1165; m/z found, 264.1155 [M+ + H]. 1H NMR (CDCl3,
400 MHz): δ 11.10 (br s, 1H), 6.76 (d, J ) 1.2 Hz, 1H), 6.69
(d, J ) 2.0 Hz, 1H), 3.94-3.90 (m, 4H), 2.47 (t, J ) 5.1 Hz,
4H), 2.33 (s, 3H), 2.25 (s, 3H). Anal. (C13H17N3OS‚C4H4O4) calcd
C, 53.81; H, 5.58; N, 11.07; S, 8.45. Found C, 49.81; H, 5.50;
N, 13.86; S, 7.58.
5-Fluoro-2-trichloromethyl-1H-benzoimidazole. The re-
action was carried out as described in General Procedure E
using 4-fluoro- 1,2-phenylenediamine (1.0 g, 8.12 mmol) Tritu-
ration of the resulting precipitate afforded 1.20 g (60%) of the
title compound. MS (electrospray): mass calculated for C8H4-
Cl3FN2, 251.9; m/z found 253.0, [M + H]+. 1H NMR (400 MHz,
CDCl3): δ 7.64 (br s, 1H), 7.31 (br s, 1H), 7.07 (dt, J ) 2.27,
9.1 Hz, 1H).
5-Methyl-2-trichloromethyl-1H-benzoimidazole. The re-
action was carried out as described in General Procedure E
using 3,4-diaminotoluene Purification via silica gel chroma-
tography (40% EtOAc/hexanes) afforded 980 mg (36%) of the
title compound. MS (electrospray): mass calculated for C9H7-
Cl3N2, 247.97; m/z found 249.0, [M + H]+. 1H NMR (400 MHz,
CDCl3): δ 9.77 (br s, 1H), 7.60 (br s, 1H), 7.43 (br s, 1H), 7.19
(dd, J ) 1.3, 8.6 Hz, 1H), 2.50 (s, 3H).
2-Trichloromethyl-5-trifluoromethyl-1H-benzoimida-
zole. The reaction was carried out as described in General
Procedure E using 4-(trifluoromethyl)-1,2-phenylenediamine
(1.0 g, 5.68 mmol). Purification via silica gel chromatography
(40% EtOAc/hexanes) afforded 930 mg (54%) of the title
compound. MS (electrospray): mass calculated for C9H4-
Cl3F3N2, 301.94; m/z found 303.0, [M + H]+. 1H NMR (400
MHz, CDCl3) rotamers: δ 10.16 (br s, 1H), 8.18 (br s, 0.55H),
7.98 (br d, J ) 8.1 Hz, 0.5H), 7.83 (br s, 0.45H), 7.64 (m, 1.5H).
General Procedure F. Preparation of (1H-Benzoimid-
azol-2-yl)-(4-methyl-piperazin-1-yl)-methanone (36). To a
suspension of 2-trichloromethyl-1H-benzoimidazole (100 mg,
0.42 mmol) in acetonitrile/water (3:1 ratio, 4.0 mL) was added
N-methylpiperazine (0.93 mL, 0.84 mmol) followed by 4 M K2-
CO3 (0.30 mL). The reaction was stirred for 24 h before diluting
with NaHCO3 (3 mL) and extracting with dichloromethane (3
× 5 mL). The organic extract was dried and evaporated to give
a crude product which was purified by chromatography over
silica gel (eluent: 4% MeOH/CH2Cl2) to afford 54 mg (52%) of
the title compound. MS (electrospray): mass calculated for
1
C13H16N4O, 244.1; m/z found 245.2, [M + H]+. H NMR (400
(2-Chloro-3-methyl-4H-thieno[3,2-b]pyrrol-5-yl)-(4-meth-
yl-piperazin-1-yl)-methanone (35). 5-Chloro-4-methyl-thio-
phene-2-carbaldehyde (5.36 g, 33 mmol) was annulated ac-
cording to Procedure A to provide 2-chloro-3-methyl-4H-thieno-
[3,2-b]pyrrole-5-carboxylic acid ethyl ester (4.4 g, 55%) as a
MHz, DMSO): δ 13.20 (s, 1H), 7.74 (d, J ) 7.3 Hz, 2H), 7.53
(d, J ) 8.3 Hz, 2H), 7.30 (m, 2H), 4.43 (m, 2H), 3.71 (t, J ) 5.2
Hz, 2H), 2.41 (m, 4H), 2.22 (s, 3H). 13C NMR (400 MHz,
DMSO): 158.1, 145.5, 142.3, 133.2, 124.1, 122.4, 120.1, 112.2,
55.0, 54.4, 46.0, 45.5, 42.3. Anal. (C13H16N4O), C, H, N.
(4-Methyl-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (37). The reaction was carried out as
described in General Procedure F using 4-methyl-2-trichlo-
romethyl-1H-benzoimidazole (100 mg, 0.40 mmol). Purification
afforded 51 mg (50%) of the title compound. MS (electro-
spray): mass calculated for C14H18N4O, 258.15; m/z found
259.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.53
(br s, 1H), 7.64 (d, J ) 8.3 Hz, 0.5H), 7.32 (d, J ) 8.3 Hz, 0.5H),
7.22 (m, 1H), 7.10 (t, J ) 7.3 Hz, 1H), 4.85 (br m, 1H), 4.78 (br
m, 1H), 3.94 (m, 2H), 2.66 (s, 1.5H), 2.57 (m, 4H), 2.50 (s, 1.5H),
2.36 (s, 3H). Anal. (C14H18N4O), C, H, N.
1
white solid. TLC (silica, 20% EtOAc/hexanes): Rf ) 0.45. H
NMR (CDCl3, 400 MHz): δ 12.30 (br s, 1H), 7.04 (s, J ) 1H),
4.37 (q, J ) 7.2 Hz, 2H), 2.28 (s, 3H), 1.32 (t, J ) 7.2 Hz, 3H).
The ethyl ester (243 mg, 1.0 mmol) was hydrolyzed (Proce-
dure B) and coupled with N-methylpiperazine (Procedure D)
to provide compound 35 (178 mg, 60% for two steps) as a white
solid. TLC (silica, 10% MeOH/CH2Cl2): Rf ) 0.4. MS (electro-
spray): mass calculated for C13H16ClN3OS, 297.80; m/z found,
1
298.1 [M+ + H]. H NMR (CDCl3, 400 MHz): 11.1 (br s, 1H),
6.62 (d, J ) 1.4 Hz, 1H), 3.92 (m, 4H), 2.48 (t, J ) 5.1 Hz,
4H), 2.33 (s, 3H), 2.18 (s, 3H). Anal. (C13H16ClN3OS‚C4H4O4)
C, H, N, S.
General Procedure E: Preparaton of 2-Trichloro-
methyl-1H-benzimidazoles. 2-Trichloromethyl-1H-ben-
zoimidazole. Methyl 2,2,2-trichloroacetimidate (1.63 mL, 9.22
mmol) was added to a solution of phenylenediamine (1.0 g,
9.2 mmol) in acetic acid (30 mL), which was then stirred at
room temperature for 1 h. Water was added (20 mL) to the
mixture, and the resultant precipitate was collected. The solid
(4-Methyl-piperazin-1-yl)-(4-nitro-1H-benzoimidazol-
2-yl)-methanone. The reaction was carried out as described
in General Procedure F employing 4-nitro-2-trichloromethyl-
1H-benzoimidazole (1.18 g). Filtration and purification by
recrystallization from ethyl acetate afforded 800 mg (66%) of
the title compound. MS (electrospray): mass calculated for
C13H15N5O3, 289.12; m/z found 290.4, [M + H]+. 1H NMR (400

8296 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26
Venable et al.
MHz, MeOD): δ 7.37 (d, J ) 8.2 Hz, 1H), 7.27 (t, J ) 7.6 Hz,
1H), 7.37 (d, J ) 7.6 Hz, 1H), 4.12 (m, 2H), 3.82 (m, 2H), 2.45-
(m, 4H), 2.36 (s, 3H).
lated for C13H14F2N4O, 280.11; m/z found 281.2, [M + H]+. 1H
NMR (400 MHz, CDCl3) rotamers: δ 11.90 (br s, 1H), 7.32 (br
m, 1H), 7.17 (m, 1H), 4.79 (m, 2H), 3.97 (m, 2H), 2.60 (m, 4H),
2.38 (s, 3H). Anal. (C13H14F2N4O) C, H, N, F.
(4-Amino-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (38). The reaction was carried out as
described for compound 14 employing 4-Methyl-piperazin-1-
yl)-(4-nitro-1H-benzoimidazol-2-yl)-methanone (640 mg, 2.21
mmol) to afford 519 mg (91% yield) of the title compound. MS
(electrospray): mass calculated for C13H17N5O, 259.14; m/z
found 260.4, [M + H]+. 1H NMR (400 MHz, CDCl3): δ 12.1
(br s, 1H), 7.12 (t, J ) 7.9 Hz, 1H), 7.89 (m, 1H), 7.53 (d, J )
7.9 Hz, 1H), 4.84 (m, 2H), 4.40 (m, 2H), 3.93 (m, 2H), 2.55(m,
4H), 2.35 (s, 3H). Anal. (C13H17N5O), C, H, N.
(5-Fluoro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (39). The reaction was carried out as
described in General Procedure F using 5-fluoro-2-trichlorom-
ethyl-1H-benzoimidazole (100 mg, 0.39 mmol). Purification
afforded 28 mg (27%) of the title compound. MS (electro-
spray): mass calculated for C13H15FN4O, 262.12; m/z found
236.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.55
(br s, 1H), 7.74 (br s, 0.5H), 7.46 (br s, 1H), 7.18 (br m, 0.5H),
7.08 (br m, 1H), 4.78 (m, 2H), 3.94 (m, 2H), 2.58 (m, 4H), 2.37
(s, 3H). Anal. (C13H15FN4O) C, H, N, F.
(5-Chloro-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (40). The reaction was carried out as
described in General Procedure F with commercially available
5-chloro-2-trichloromethyl-1H-benzoimidazole (100 mg, 0.37
mmol). Purification afforded 65 mg (63%) of the title com-
pound. MS (electrospray): mass calculated for C13H15ClN4O,
278.1; m/z found 279.2, [M + H]+. 1H NMR (400 MHz,
DMSO): 13.29 (s, 1H), 7.67 (br s, 2H), 7.33 (d, J ) 8.6 Hz,
2H), 4.49 (m, 2H), 3.71 (t, J ) 4.6 Hz, 2H), 2.40 (m, 4H), 2.22
(s, 3H). Anal. (C13H15ClN4O), C, H, N, Cl.
(4-Chloro-6-methyl-1H-benzoimidazol-2-yl)-(4-methyl-
piperazin-1-yl)-methanone (45). 4-Chloro-6-methyl-2-trichlo-
romethyl-1H-benzoimidazole was prepared as described in
General Procedure E using 4-chloro-6-methyl-1,2-diaminoben-
zene (1.0 g, 6.41 mmol). MS (electrospray): mass calculated
for C9H6Cl4N2, 281.93; m/z found 283.6, [M + H]+. The crude
product was utilized in Procedure F affording 703 mg of the
title compound (38% yield). MS (electrospray): mass calculated
for C14H17ClN4O, 292.11; m/z found 293.3, [M + H]+. 1H NMR
(400 MHz, CDCl3) rotamers: δ 11.20 (br s, 1H), 7.62 (s, 0.5
H), 7.32 (s, 0.5 H), 7.03 (s, 1H), 4.74 (m, 2H), 3.91(m, 2H),
2.62-2.55 (m, 7H), 2.37 (s, 3H). Anal. (C14H17ClN4O) C, H, N,
Cl.
(4,6-Dichloro-1H-benzoimidazol-2-yl)-(4-methyl-piper-
azin-1-yl)-methanone (46). 4-Chloro-6-methyl-2-trichlorom-
ethyl-1H-benzoimidazole was prepared as described in General
Procedure E using 4,6-dichloro-1,2-diaminobenzene (1.0 g, 6.64
mmol). The crude product (200 mg, 0.65 mmol) was utilized
in Procedure F affording 59 mg of the title compound (29%
yield). MS (electrospray): mass calculated for C14H17Cl2N4O,
313.18; m/z found 315.1, [M + H]+. 1H NMR (400 MHz,
CDCl3): 11.7 (br s, 1H), 7.65 (br s, 1 H), 7.36 (s, 1H), 4.74 (m,
2H), 3.91(m, 2H), 2.57 (m, 4H), 2.36 (s, 3H). Anal. (C13H14-
Cl2N4O) C, H, N, Cl.
Biological Materials. SK-N-MC cells were stably trans-
fected with human H4 receptor as previously described.5,9,34
All of the radiolabeled ligands were purchased from Perkin-
Elmer (Boston, MA).
Binding Assays. Radioligand binding assays were run as
previously described.9,16 Cell pellets from SK-N-MC cells
transfected with the human H4 receptor were used. Cell pellets
were homogenized in 50 mM TRIS pH 7.5 containing 5 mM
EDTA and supernatants from an 800g spin were collected and
recentrifuged at 30 000g for 30 min. Pellets were rehomog-
enized in 50 mM TRIS pH 7.5 containing 5 mM EDTA. For
the H4 competition binding studies, human cell membranes
were incubated with 10 nM, 40 nM, and 150 nM [3H]histamine
(specific activity 23 Ci/mmol), respectively, with or without test
compounds for 45 min at 25 °C. Nonspecific binding was
defined using 100 µM unlabeled histamine. The Kd values for
the human H4 receptor was determined to be 5 nM and the
Bmax value was determined to be 1.12 pmol/mg protein. For
all studies the Ki values were calculated based on an experi-
mentally determined appropriate Kd values according to Cheng
and Prusoff.17
Cell-Based cAMP Assays. Cell-based functional assays
were run as previously described.9,16 SK-N-MC cell lines were
created that express a reporter gene construct and the human
H4 receptor full-coding region. The reporter gene was â-galac-
tosidase under the control of cyclic AMP responsive elements.
Cells were plated in 96-well plates the night before the assay.
Histamine was used as the agonists for all assays. For the H4
receptor the inhibition of forskolin-stimulated cAMP produc-
tion was measured. For determination of antagonists activity
compounds were added 10 min prior to the addition of agonist,
which was added directly to the cell medium. In the case of
the H4 receptor assay, forskolin (5 µM final concentration) was
added 10 min after the addition of histamine. Cells were
returned to the incubator for 6 h at 37 °C. The medium was
then aspirated, and the cells were washed with 200 mL of
phosphate buffered saline (PBS). Cells were lysed with 25 µL
of 0.1× assay buffer (10 mM sodium phosphate, pH 8, 0.2 mM
MgSO4, 0.01 mM MnCl2) and incubated at room temperature
for 10 min. Cells were then incubated for 10 min with 100 µL
of 1× assay buffer containing 0.5% (v/v) Triton and 40 mM
â˜-mercaptoethanol. Color was developed using 25 µL of 1 mg/
mL substrate solution (chlorophenol red â-D-galactopyrano-
side; Roche Molecular Biochemicals, Indianapolis, IN). Color
was quantitated on a microplate reader by measuring the
absorbance at 570 nm. The data from each concentration
(5-Methyl-1H-benzoimidazol-2-yl)-(4-methyl-piperazin-
1-yl)-methanone (41). The reaction was carried out as
described in General Procedure F using 5-methyl-2-trichlo-
romethyl-1H-benzoimidazole (100 mg, 0.40 mmol). Purification
afforded 36 mg (35%) of the title compound. MS (electro-
spray): mass calculated for C14H18N4O, 258.15; m/z found
259.2, [M + H]+. 1H NMR (400 MHz, CDCl3) rotamers: δ 11.24
(br s, 1H), 7.69 (d, J ) 8.3 Hz, 0.6H), 7.60 (br s, 0.4H), 7.39 (d,
J ) 8.3 Hz, 0.4H), 7.29 (br s, 0.6H), 7.18 (d, J ) 8.3 Hz, 0.4H)
7.13 (d, J ) 8.3 Hz, 0.6H), 4.79 (br m, 2H), 3.94 (m, 2H), 2.57
(m, 4H), 2.49 (s, 3H), 2.36 (s, 3H). Anal. (C14H18N4O), C, H, N.
(4-Methyl-piperazin-1-yl)-(5-trifluoromethyl-1H-ben-
zoimidazol-2-yl)-methanone (42). The reaction was carried
out as described in General Procedure F using 2-trichloro-
methyl-5-trifluoromethyl-1H-benzoimidazole (100 mg, 0.33
mmol). Purification afforded 42 mg (41%) of the title com-
pound. MS (electrospray): mass calculated for C14H15F3N4O,
312.12; m/z found 313.2, [M + H]+. 1H NMR (400 MHz,
CDCl3): δ 8.01 (br s, 1H), 7.72 (br m, 1H), 7.58 (dd, J ) 1.3,
8.6 Hz, 1H), 4.78 (m, 2H), 3.95 (m, 2H), 2.59 (m, 4H), 2.37 (s,
3H). Anal. (C14H15F3N4O) C, H, N, F.
(5-Fluoro-4-methyl-1H-benzoimidazol-2-yl)-(4-methyl-
piperazin-1-yl)-methanone (43). (5-Fluoro-4-methyl-2-trichlo-
romethyl-1H-benzoimidazole was prepared as described in
General Procedure E using 5-fluoro-4-methyl-1,2-diaminoben-
zene (500 mg, 3.57 mmol) to afford 900 mg of a crude orange
powder. The crude reaction product (100 mg, 0.37 mmol) was
used without purification in General Procedure F to afford 43
mg (31% yield) of the title compound. MS (electrospray): mass
calculated for C14H17FN4O, 276.14; m/z found 277.8, [M + H]+.
1H NMR (400 MHz, CDCl3) rotamers: δ 11.70 (br s, 1H), 7.57
(m, 0.5H), 7.26 (m, 0.5 H), 7.03 (m, 1H), 4.75 (m, 2H), 3.93 (m,
2H), 2.56 (m, 4H), 2.37 (s, 3H). Anal. (C14H17FN4O) C, H, N,
F.
(4,5-Difluoro-1H-benzoimidazol-2-yl)-(4-methyl-piper-
azin-1-yl)-methanone (44). (4,5-Difluoro-2-trichloromethyl-
1H-benzoimidazole was prepared as described in General
Procedure E using 4,5-difluoro-1,2-diaminobenzene (200 mg,
1.38 mmol). The crude reaction product was used without
purification in General Procedure F to afford 168 mg (44%
yield) of the title compound. MS (electrospray): mass calcu-

Human Histamine H4 Antagonists
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 26 8297
response curve were fitted to a sigmoidal curve to obtain the
maximum response, Hill coefficient and EC50, using Prism
(GraphPad Software, San Diego, CA). Dose ratios were cal-
culated from individual concentration response curves of
agonists at three antagonist concentrations. An apparent pA2
values were calculated using a Schild plot.
analytical expertise. We also thank Drs. Daniel Buzard
and Ronald Wolin for the editing of this manuscript.
Supporting Information Available: Microanalysis data
of all final compounds are available free of charge via the
Internet at http://pubs.acs.org.
Bone-Marrow Mast Cell Culture. Mast cells were dif-
ferentiated from bone marrow derived from BALB/c mice as
previously described.15,16 Briefly bone marrow cells were
cultured in RPMI with 10% (v/v) fetal calf serum, 0.1 mM
nonessential amino acids, 50 µg/mL penicillin/streptomycin,
and 20% (v/v) conditioned medium from WEHI-3 cells (ATCC).
After 16 h culture, the nonadherent cells were transferred to
a new flask. Four weeks later, the cells were analyzed by flow
cytometry for IgE receptor and CD117 (c-kit) expression to
confirm the differentiation to mast cells. Greater than 99% of
the cells were IgE receptor and CD117 positive. Mast cells of
4-8 weeks of age were used for the experiments.
In Vitro Mast Cell Chemotaxis Assay. Mast cell chemo-
taxis assays were run as previously described.15,16 Transwells
(Costar, Cambridge, MA) of a pore size 8 µm were coated with
100 µL of 100 µg/mL bovine fibronectin (Sigma, St. Louis, MO)
for 2 h at room temperature. After removal of the fibronectin,
600 µL of RPMI with 0.5% (w/v) bovine serum albumin in the
presence of histamine was added to the bottom chamber, and
histamine receptor antagonists were added to both chambers.
Mast cells (2 × 105/well) were added to the top chamber. The
plates were incubated for 3 h at 37 °C. Transwells were
removed and the number of cells in the bottom chamber was
counted for 1 min using a flow cytometer.
Purification of Human Eosinophils. Eosinophils were
purified from blood samples collected from healthy volunteers
as previously described.35 Briefly, platelet-rich plasma was
removed by centrifugation of heparinized whole blood. Poly-
morphonuclear leukocytes (PMNL), which are enriched with
neutrophils and eosinophils, were separated from PBMC by
centrifugation at 2000 rpm for 20 min over a discontinuous
plasma-Percoll gradient (density1.082 g/mL). Red blood cells
in PMNL were removed by hypotonic shock lysis. PMNL were
stained with Hematoxylin and Eosin (H&E), and a differential
cell count was performed. Eosinophil counts ranged from 2 to
10% of the total PMNL number. Eosinophils were purified
from the PMNL by negative selection. PMNL were incubated
with a cocktail of anti-CD16, anti-CD3, anti-CD19, and anti-
CD14 conjugated micro-beads in PBS containing 0.5% BSA
and 2 mM EDTA, which selectively bind to neutrophils, T cells,
B cells, and monocytes, respectively, in the PMNL suspension.
Eosinophils were purified by removing cells bound to micro-
beads in the AutoMACS system, resulting in eosinophil
populations of >97.5% purity according to H&E stain. Purified
eosinophils were washed once in buffer (PBS containing 10
mM Ca2+ and Mg2+, 10 mM HEPES, 10 mM glucose, and 0.1%
BSA, pH 7.2-7.4) and used immediately for experiments.
In Vitro Eosinophil Chemotaxis Assays. Eosinophil
chemotaxis assays were carried out as previously described.35
Transwells (Costar, Cambridge, MA) with 5 µm pore size were
coated with 100 µL of 100 ng/mL human fibronectin (Sigma)
for 2 h at room temperature. After removal of excess fibronec-
tin, 600 µL of RPMI-1640 medium containing 0.5% BSA and
different concentrations of histamine (0.01-100 µM) were
added to the bottom chamber. Eosinophils (2 × 105/well) were
added to the top chamber. Compounds were added to both the
top and bottom chambers. The plates were incubated for 2 h
at 37 °C, and the number of cells migrated to the bottom
chamber was counted for 1 min using flow cytometer.
Statistics. Experimental data are presented as mean (
standard deviation (SD) from the number (n) of independent
samples. The IC50 or EC50 values were calculated from the
concentration-effect curves by nonlinear regression analysis
using GraphPad Prism (GraphPad Software Inc., Philadelphia,
PA).
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