6416
S. Harusawa et al. / Bioorg. Med. Chem. Lett. 23 (2013) 6415–6420
kyl-N-alkylisothioureas.19 In the present paper, using this synthetic
S
NH
method, we first examined whether substitution of the imidazole
of clobenpropit with various cyclic amines affected the potency
and affinity of hH3R. We then evaluated the effect of varying the
length of the alkyl spacer between the isothioureylene and 4-chlo-
rophenyl group. Further, the effects of para-substituents on the
phenyl group, and conversion of the isothiourea central core into
a carbamoyl group were examined. Among the molecules evalu-
ated, novel isothiourea derivative 19 (OUP-186) and related struc-
tures were found to exhibit potent and selective H3R antagonistic
activities, whilst being inactive against hH4R. However, 19 and
its homologs did not have any effect on histamine release when
assessed using in vivo rat brain microdialysis. This discrepancy
between in vitro and in vivo studies prompted us to investigate
the structural differences in the ligand-binding cavities of hH3R
and rH3R using molecular modeling.
N
N
H
S
N
H
HN
N
N
Cl
HN
N
clobenpropit
thioperamide
CH3
OH
N
N
O
O
Cl
N
H
imoproxifan
BF2.649 (pitolisant)
H
Me
N
O
NC
N
N
Me
O
N
O
We designed the new nonimidazole H3R antagonists by modifi-
cation of clobenpropit, substituting the imidazole with typical
secondary cyclic amines: pyrrolidine, morpholine, piperidine,
methylpiperadine, and piperazine (Scheme 1). The synthesis of
an important intermediate 4-chlorophenylbutyl amine 5,
commenced from commercially available bromopropylphthali-
mide 1. Using microwave (MW) irradiation in the four synthetic
steps, 4-(4-chlorophenyl)butan-1-amine 5 was synthesized in
rapid and straightforward manner through formation of phospho-
nium salt 2 (85%), subsequent Wittig olefination (65%), catalytic
reduction of alkene 3 (98%), and deprotection of phthalimide 4
with hydrazine (82%). The H3R antagonist target, N-[(4-chloro-
phenyl)butyl]-S-[3-(piperidin-1-yl)propyl]isothiourea 19, was syn-
thesized from amine 5 by using the protocol that we previously
reported for the preparation of S-alkyl-N-alkylisothioureas.19 Reac-
tion of 5 with 3-phenylpropionyl isothiocyanate (PPI)19 as an inter-
calating agent of SCN atoms afforded thiourea 6 (46%). This
thiourea 6 was subsequently converted into isothiourea 7 (76%)
via Mitsunobu S-alkylation with 1-piperidinepropanol using
N,N,N0,N0-tetramethylazodicarboxamide (TMAD) and tributylphos-
phine. Selective cleavage of the N–CO bond of 7 with hydrazine
hydrate, with retention of the fissile S-alkyl moieties, produced
target compound 19 (52%),20a which was subsequently treated
with hydrochloric acid to give a dihydrochloride.
In addition, amide compound 24 was easily prepared via three
steps starting from piperidine, as outlined in Scheme 1. The other
S-alkyl-N-alkylisothioureas (10–18 and 20–23) and amide analogs
25 and 26 were successfully synthesized in good yields using the
reaction conditions detailed for 19 and 24, respectively. All final
compounds (10–26) were provided as di- or monohydrochlorides,
and their structures were confirmed using standard spectral tech-
niques (1H and 13C NMR, IR, and HRMS).20b
The seventeen synthesized compounds were tested consisting
of an in vitro functional assay using LANCE kit (PerkinElmer) with
ES-392-C and ES-393-C cells that stably express hH3R and hH4R,
respectively (PerkinElmer). All tested compounds showed antago-
nistic activities for hH3R. Potencies (pIC50) were determined by
analyzing concentration–response curves obtained for the test
CEP-26401 (irdabisant)
ABT-239
Figure 1. Structures of imidazole or nonimidazole H3R antagonists.
hH3R.15 Consequently, most imidazole-containing histamine H3R
ligands also have a high affinity with H4Rs.16
An extremely potent H3R inverse agonist, clobenpropit is
composed of an imidazole ring, a propyl spacer and an isothiourea
central core that is connected to a lipophilic 4-chlorobenzyl group
(Fig. 1).5,17 During our investigations into novel and potent H3/H4R
ligands,18 we have reported an efficient synthetic method for S-al-
O
O
a
b
N
(CH2)3 Br
N
(CH2)3 PPh3
Br
MW
MW
O
1
2 (85%)
O
O
O
Cl
c
d
N
(CH2)2
3 (65%)
N (CH2)4
Cl
MW
O
O
4 (98%)
O
O
S=C=N
HN
(PPI)
e
H2N (CH2)4
Cl
S
N
H
(CH2)4
Cl
5 (82%)
6 (46%)
O
N
OH
N
g
N
S
N
H
(CH2)4
Cl
f
7 (76%)
NH
S
N
N
(CH2)4
19
(52%)
Cl
H
compounds in the presence of agonist R-a-methylhistamine at
i
h
OEt
NH
OH
. HCl
N
N
NH
EC80 concentration (see Supplementary data, Section 2). As 3-pip-
eridino- or pyrrolidinopropyl groups were identified as being the
key pharmacophores in the nonimidazole antagonists that have
reached clinical trials (Fig. 1),10 the spacer between the cyclic
amines and central isothiourea was set as three methylene–car-
bons in the first step (Table 1). The respective pIC50 values for pyr-
rolidine and piperidine derivatives 10 and 12 were 7.7 and 7.1
(entries 1 and 3), but those of morpholine, methylpiperazine, and
piperazine derivatives 11, 13, and 14 gave lower values of
5.2–5.8 (entries 2, 4, and 5). Among these compounds, piperidine
analog 12 was previously reported as FUB661 (pA2 = 7.4) by Stark
O
(64%)
O
8
9
(96%)
j
Cl
(CH2)4
24 (57%)
N
O
Scheme 1. Synthesis of isothiourea 19 and amide 24. Reagents and conditions: (a)
Ph3P, CH3CN, MW, 200 °C, 15 min; (b) 4-chlorobenzaldehyde, NaOMe, DMF, MW,
200 °C, 15 min; (c) H2/Pd–C, THF–MeOH, 50 min; (d) NH2NH2ꢀH2O, EtOH, MW,
120 °C, 15 min; (e) PPI, toluene, 60 °C, 0.5 h; (f) 1-piperidinepropanol, TMAD, Bu3P,
THF, rt, 16 h; (g) NH2NH2ꢀH2O, EtOH, rt, 16 h; (h) ethyl 4-bromobutyrate, MeCN,
reflux, 2 h; (i) 6NHCl, reflux, 1.5 h; (j) 5, (EtO)2P(O)CN, Et3N, DMF, 14 h.