Note: Descriptions are shown in the official language in which they were submitted.
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WO 03/053434 PCT/EP02/13921
Description
Substituted imidazolidines, method for the production thereof, use thereof as
a
medicament or a diagnostic tool, and medicament containing substituted
imidazolidines
The invention relates to substituted imidazolidines of the formula I,
R3
R4 ~ N~ R1
HN
R5 ~ ~R7 R2
R6
in which
R1 and R2
independently of one another are CN, (C1-C5)-alkyl, (C2-C5)-alkenyl, (C2-C5)-
alkynyl, (C3-Cg)-cycioalkyl or (C4-Cg)-cycloalkenyl
where all carbon chains and carbon rings are unsubstituted or,
independently of one another, are substituted by 1 - 11 fluorine atoms
or by up to two radicals selected from the group consisting of OH,
NH2, NHCH3, N(CH3)2 and OCHg;
or
R1 and R2
together with the finro carbon atoms to which they are attached are a five- to
eight-membered-saturated or unsaturated carbon ring,
but without any double bond between the two carbon atoms to which
R1 and R2 are attached,
and
where the ring is unsubstituted or substituted by 1 - 12 fluorine atoms
or by up to two radicals selected from the group consisting of CHg and
OCHg;
CA 02470856 2004-06-17
2
R3 is F, CI, Br, I, (C1 -C4)-alkyl, (C1-C4)-alkenyl, (Cg-Cg)-cycloalkyl, OH,
(C1-C4)-alkoxy, Ophenyl, CN, N02 or NH2;
where phenyl is unsubstituted or substituted by up to two radicals
selected from the group consisting of CH3, F, CI, Br, i, OH and OCHg;
and
where the carbon chains or carbon rings are unsubstituted or
substituted by 1 - 11 fluorine atoms;
R4 to R6
independently of one another are H, F, CI, Br, I, (C1-C4)-alkyl, (C1-C4)-
alkenyl, (Cg-Cg)-cycloalkyl, OH, (C1-C4)-alkoxy, CN, N02, NH2, (C1-C4)-
alkylamino or (C1-C4)-dialkylamino;
where the carbon chains or carbon rings are unsubstituted or
substituted by 1 - 11 fluorine atoms;
R7 is H, F, CI, Br, I, (C1-C4)-alkyl, (C1-C4)-alkenyl, (C3-Cg)-cycloalkyl, OH,
(C~-C4)-alkoxy, CN, N02 or NH2;
where the carbon chains or carbon rings are unsubstituted or
substituted by 1 - 11 fluorine atoms;
and their pharmaceutically acceptable salts, and the trifluoroacetic acid
salts.
Preference is given to compounds of the formula I in which
R1 and R2
independently of one another are (C1-C5)-alkyl, (C2-C5)-alkenyl, (C2-C5)-
alkynyl, (C3-C6)-cycloalkyl or (C4-Cg)-cycloalkenyl,
where all carbon chains and carbon rings are unsubstituted or,
independently of one another, substituted by 1 - 11 fluorine atoms or
by up to two radicals selected from the group consisting of NHCH3,
N(CH3)2 and OCHg;
or
R1 and R2
together with the two carbon atoms to which they are attached are a five- to
eight-membered saturated or unsaturated carbon ring,
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3
but without any double bond between the two carbon atoms to which
R1 and R2 are attached,
and
where the ring is unsubstituted or substituted by 1 - 12 fluorine atoms
or by up to two radicals selected from the group consisting of CH3 and
OCH3;
R3 is F, CI, Br, I, (C1 -C4)-alkyl, (C1-C4)-alkenyl, (C3-C6)-cycloalkyl, OH,
(C1-C4)-alkoxy, Ophenyl, CN, N02 or NH2;
where phenyl is unsubstituted or substituted by up to two radicals
selected from the group consisting of CH3, F, CI, Br, OH and OCH3;
and
where the carbon chains or carbon rings are unsubstituted or
substituted by 1 - 11 fluorine atoms;
R4 to R6
independently of one another are H, F, CI, Br, CH3, OH, OrH3, CN, N02,
NH2, NHCH3, N(CH3)2;
where the methyl groups are unsubstituted or substituted by 1 - 3
fluorine atoms;
R7 is H, F, CI, Br, I, (C1-C4)-alkyl, (C1-C4)-alkenyl, (C3-C6)-cycloalkyl, OH,
(C1-C4)-alkoxy, CN, N02 or NH2;
where the carbon chains or rings are unsubstituted or substituted by
1 - 11 fluorine atoms;
and their pharmaceutically acceptable salts, and the trifluoroacetic acid
salts.
Very particular preference is given to the following compounds of the formula
I:
traps-(2-chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol-2-
ylidene)amine
hydrochloride,
(S,S)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt,
cis-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt,
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4
(R,R)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt, traps-(octahydrobenzimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride,
traps-(2,6-dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)amine
hydrochloride,
traps-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene)amir!e
trifl~~ora~etic
acid salt, cis-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-
ylidene)amine
hydrochloride,
traps-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-ylidene)amine
hydrochloride,
(2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-ylidene)amine nitric acid
salt,
traps-(2,6-dichlorophenyl)-(hexahydrocyclopentaimidazol-2-ylidene)amine
trifluoroacetic acid salt.
Extraordinary preference is given to the following compounds of the formula I:
(S,S)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt,
cis-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt,
(R,R)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid
salt,
traps-(2,6-dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)amine
hydrochloride,
traps-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene)amine
trifluoroacetic
acid salt, cis-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-
ylidene)amine
hydrochloride, traps-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-
ylidene)amine
hydrochloride, (2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-ylidene)amine
nitric
acid salt,traps-(2,6-dichlorophenyl)-(hexahydrocyclopentaimidazol-2-
ylidene)amine
trifluoroacetic acid salt.
Suitable acid addition salts are salts of all pharmacologically acceptable
acids, for
example halides, in particular hydrochlorides, lactates, sulfates, citrates,
tartrates,
acetates, phosphates, methylsulfonates, p-toluenesulfonates, adipinates,
fumarates,
gluconates, glutamates, glycerolphosphates, maleates and pamoates. This group
also
corresponds to the physiologically acceptable anions; but also
trifluoroacetates.
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If the compounds of the formula I contain one or more centers of asymmetry,
the
compounds can be both S- and R-configured. The compounds can be present as
optical isomers, as diastereomers, as racemates or as mixtures thereof.
5 The compounds of the formula I can furthermore be present as tautomers or as
a
mixture of tautomeric structures.
This refers in particular to the following tautomers:
R3 H R3 H NI R4 R3 N N
R4 ~ N~N R1 R4 ~ N~ R1 I ~ ~ R1
I IN' /
R2
R5 I / R7HN R5 / R7 R5 R7
R2 R2 R6
R6 R6
If R1 and R2 are different and if the nitrogen-carbon double bond has
sufficient
configurational stability, it is also possible for two double-bond isomers to
exist:
R3 H R3
N N
R4 ~ N~ R1 R4 ~ N ~ R2
H_IN ~ / HN
R5 ~ ~R7 RZ R5 ~ ~R7 R1
R6 R6
The carbon radicals mentioned, or the partially or fully fluorinated or
substituted carbon
radicals, can be straight-chain or branched.
Also described are methods for preparing the compounds used.
Thus, the substances described by formula I can be prepared in a manner known
to
the person skilled in the art from the isothiocyanate II parent compounds and
the
appropriate diamines III.
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6
R4
R5 ~ R3 II H2N R1
III
R6 ~ 'NCS
R7 H2N R2
The thiourea derivative which is formed as an intermediate is cyclized using
methyl
iodide (Synthesis, 1974, 41 - 42) or carbodiimide (Synthesis, 1977, 864 - 865)
to give
the corresponding imidazolidine I. If the isothiocyanates II employed here are
not
commercially available, they can be prepared in a manner known from the
literature
from the corresponding anilines, using methods known to the person skilled in
the art,
for example by treatment with thiophosgene (J. Med. Chem., 1975, 18, 90-99) or
thiocarbonyl diimidazole (Justus Liebigs Ann. Chem., 1962, 657, 104).
In addition to the isothiocyanates II described above, it is also possible to
successfully
react the isoc;ranatas IV with aminES of the type of formula III to give
compounds of the
formula I. Here, the urea derivative which is formed as an intermediate is
cyclized
using phosphorus oxychloride to give the corresponding imidazolidines of the
formula
I.
R4
R3
R6' ~ NCO
R7
w
In the present invention, it was surprisingly possible to demonstrate that the
compounds described are potent inhibitors of the sodium/hydrogen exchanger
(NHE),
in particular of NHE3.
The NHE3 inhibitors known to date are derived from compounds of the
acylguanidine
type (EP-A 825 178, HOE 96/F226), of the norbornylamine type (DE 199 60 204.2-
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7
HMR 99 / L 073), of the 2-guanidinoquinazoline type (WO 01 79 186 A1 or of the
benzamidine type (WO 01 21582 A1, WO 01 72 742 A1 ). Squalamine, which has
also
been described as NHE3 inhibitor (M. Donowitz et al. Am. J. Physiol. 276 (Cell
Physiol.
45): C136 - C144) is, unlike the compounds of the formula !, .not effective
immediately
but reaches its maximum potency only after one hour.
Clonidine, which is similar to the compounds described here, is known as a
weak NHE
inhibitor. However, its action on the NHE3 of the rat is, with an ICSp of 620
NM,
extremely moderate. In contrast, it shows a certain selectivity for the NHE2,
where it
has an IC50 of 42,uM (J. Orlowski et al J. Biol. Chem. 268, 25536). It would
therefore
be more accurate to refer to clonidine as an NHE2 inhibitor. In addition to
the weak
NHE action, clonidine has a high affinity for the adrenergic alpha2 receptor
and the
imidazoline 11 receptor, mediating a strong hypotensive action (Ernsberger et
al Eur. J.
Pharmacol. 134, 1, 1987).
Compounds of the formula I have increased NHE3 activity and reduced 11 and
alpha2
activity.
NHE3 is found in the body of various species, preferably in the gall bladder,
the
intestine and the kidney (Larry Fliegel et al, Biochem. Cell. Biol. 76: 735 -
741, 1998),
but can also be detected in the brain (E. Ma et al. Neuroscience 79: 591 -
603).
Owing to this unexpected property, the compounds of formula I are suitable for
treating
disorders caused by oxygen deficiency. As a result of their pharmacological
properties,
the compounds are highly suitable for use as antiarrhythmics having a
cardioprotective
component, for infarct prophylaxis and infarct treatment and for treatment of
angina
pectoris, and they also inhibit, or strongly reduce, in a preventative manner,
the
pathophysiological processes which contribute to ischemically induced damage,
in
particular those which trigger ischemically induced cardiac arrhythmias. Owing
to their
protective action against pathological hypoxic and ischemic situations, the
compounds
of the formula I according to the invention can, as inhibitors of the cellular
Na+/H+
exchange mechanisms, be used as medicaments for treating all acute or chronic
damage caused by ischemia, or diseases induced primarily or secondarily by
this
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damage. This relates to their use as medicaments for surgical interventions,
for
example organ transplantations, where the compounds can be used both for
protecting
the organs in the donor before and during removal, for protecting organs that
have
been removed, for example by treatment with or storage in physiological bath
fluiCs,
and also during transfer into the recipient organism. The compounds are also
useful
medicaments with protective action during angioplastic surgical interventions,
for
example at the heart, but also in peripheral vessels. Owing to their
protective action
against ischemically induced damage, the compounds are also suitable for use
as
medicaments for treating ischemias of the nervous system, in particular of the
CNS,
where they can be used, for example, for treating stroke or cerebral edema.
Moreover,
the compounds of the formula I to be used according to the invention are also
suitable
for treating forms of shock, such as, for example, of allergic, cardiogenic,
hypovolemic
and bacterial shock.
Furthermore, the compounds induce an improvement in the respiratory drive and
are
therefore ~.~sed for the treatment of respiratory conditions in the following
clinical
conditions and diseases: disturbed central respiratory drive (e.g. central
sleep apnea,
sudden infant death, post operative hypoxia), muscular-related respiratory
disorders,
respiratory disorders after long-term ventilation, respiratory disorders
during adaptation
in a high mountain area, obstructive and mixed forms of sleep apnea, acute and
chronic lung diseases with hypoxia and hypercapnia.
The compounds additionally increase the muscle tone of the upper airways, so
that
snoring is suppressed.
A combination of an NHE inhibitor with a carboanhydrase inhibitor (e. g.
acetazolamide), the latter producing metabolic acidosis and thereby even
increasing
the respiratory activity, proves to be advantageous as a result of increased
action and
decreased use of active compound.
It has been shown that the compounds to be used according to the invention
have a
mild laxative action and accordingly can be used advantageously as laxatives
or in the
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9
case of threatening intestinal blockage, the prevention of ischemic damage
accompanying blockages in the intestinal region being particularly
advantageous.
It is furthermore possible to prevent gallstone formation.
Moreover, the compounds of the formula I to be used according to the invention
can
exert a strong inhibitory action on the proliferation of cells, for example
fibroblast cell
proliferation and the proliferation of the smooth vascular muscle cells. The
compounds
of the formula I are therefore suitable as valuable therapeutics for diseases
in which
cell proliferation is a primary or secondary cause, and can therefore be used
as
antiatherosclerotics, agents against diabetic late complications, cancers,
fibrotic
disorders such as pulmonary fibrosis, liver fibrosis or kidney fibrosis, organ
hypertrophy and hyperplasia, in particular in prostate hyperplasia or prostate
hypertrophy.
The compounds according to the invention are effective inhibitors of the
cellular
sodium/proton antiporter (Na/H exchanger) which is raised in numerous
disorders
(essential hypertension, atherosclerosis, diabetes etc.) even in those cells
which are
easily accessible to measurement, such as, for example, in erythrocytes,
platelets or
leukocytes. The compounds to be used according to the invention are therefore
suitable as outstanding and simple scientific tools, for example in their use
as
diagnostics for the determination and differentiation of certain forms of
hypertension,
but also of atherosclerosis, of diabetes, of proliferative disorders etc.
Moreover, the
compounds of the formula I are suitable for preventive therapy to prevent the
development of high blood pressure, for example of essential hypertension.
It has moreover been found that NHE inhibitors exhibit a favorable influence
on the
serum lipoproteins. It is generally recognized that for the formation of
artereosclerotic
vascular changes, in particular of coronary heart disease, excessively high
blood lipid
values, 'hyperlipoproteinemias', are an essential risk factor. The lowering of
increased
serum lipoproteins is therefore of extreme importance for the prophylaxis and
the
regression of atherosclerotic changes. The compounds to be used according to
the
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invention can therefore be used for the prophylaxis and regression of
atherosclerotic
changes by excluding a causal risk factor. With this protection of the vessels
against
the syndrome of endothelial dysfunction, compounds of the formula I are
valuable
pharmaceuticals for the prevention and treatment of coronary vascular spasm,
of
5 atherogenesis and of atherosclerosis, of left-ventricular hypertrophy and of
dilated
cardiomyopathy, and thrombotic disorders.
The compounds mentioned are therefore advantageously used for the production
of a
medicament for the prevention and treatment of sleep apneas and muscular-
related
10 respiratory disorders; for the production of a medicament for the
prevention and
treatment of snoring, for the production of a medicament for lowering the
blood
pressure, for the production of a medicament having laxative action for the
prevention
and treatment of intestinal blockages; for the production of a medicament for
the
prevention and treatment of disorders which are induced by ischemia and
reperfusion
of central and peripheral organs, such as acute kidney failure, stroke,
endogenous
states of shock, intesinal disorders etc; for tir a production of a medicament
for the
treatment of hypercholesterolemia; for the production of a medicament for the
prevention of atherogenesis and of atherosclerosis; for the production of a
medicament
for the prevention and treatment of diseases which are induced by raised
cholesterol
levels; for the production of a medicament for the prevention and treatment of
diseases
which are induced by endothelial dysfunction; for the production of a
medicament for
the treatment of attack by ectoparasites; for the production of a medicament
for the
treatment of the diseases mentioned in combination with blood pressure-
lowering
substances, preferably with angiotensin-converting enzyme (ACE) inhibitors and
angiotensin receptor antagonists. A combination of an NHE inhibitor of the
formula I
with an active compound lowering the blood lipid level, preferably with an HMG-
CoA
reductase inhibitor (e.g. lovastatin or pravastatin), the latter producing a
hypolipidemic
effect and thereby increasing the hypolipidemic properties of the NHE
inhibitor of the
formula I, proves to be a favorable combination having intensified action and
decreased use of active substance.
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11
The administration of sodium/proton exchange inhibitors of the formula I as
novel
pharmaceuticals for lowering raised blood lipid levels, and the combination of
sodium/proton exchange inhibitors with pharmaceuticals having a blood pressure-
lowering and/or hypolipidemic action is claimed.
Pharmaceuticals which contain a compound I can in this case be administered
orally,
parenterally, intravenously, rectally or by inhalation, the preferred
administration being
dependent on the particular clinical picture of the disorder. The compounds I
can in this
case be used on their own or together with pharmaceutical excipients, namely
both in
veterinary and in human medicine.
The person skilled in the art is familiar on the basis of his/her expert
knowledge with
excipients which are suitable for the desired pharmaceutical formulation. In
addition to
solvents, gel formers, suppository bases, tablet excipients and other active
compound
carriers, it is possible to use, for example, antioxidants, dispersants,
emulsifiers,
antifoams, flavor corrigents, preservatives, solubilizers or colorants.
For a form for oral administration, the active compounds are mixed with the
additives
suitable therefor, such as excipients, stabilizers or inert diluents, and
brought by
means of the customary methods into the suitable administration forms, such as
tablets, coated tablets, hard gelatin capsules, aqueous, alcoholic or oily
solutions. Inert
carriers which can be used are, for example, gum arabic, magnesia, magnesium
carbonate, potassium phosphate, lactose, glucose or starch, in particular
cornstarch. In
this case, preparation can be carried out either as dry or as moist granules.
Possible
oily excipients or solvents are, for example, vegetable or animal oils, such
as sunflower
oil or cod-liver oils.
For subcutaneous or intravenous administration, the active compounds used are
brought into solution, suspension or emulsion, if desired with the substances
customary therefor such as solubilizers, emulsifiers or further excipients.
Suitable
solvents are, for example: water, physiological saline solution or alcohols,
e.g. ethanol,
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12
propanol, glycerol, and in addition also sugar solutions such as glucose or
mannitol
solutions, or alternatively a mixture of the different solvents mentioned.
Pharmaceutical formulations suitable for administration in the form of
aerosols or
sprays are, for example, solutions, suspensions or emulsions of the active
compound
of the formula I in a pharmaceutically innocuous solvent, such as, in
particular, ethanol
or water, or a mixture of such solvents.
If required, the formulation can also contain still other pharmaceutical
excipients such
as surfactants, emulsifiers and stabilizers, and a propellant. Such a
preparation
customarily contains the active compound in a concentration of approximately
0.1 to
10, in particular of approximately 0.3 to 3, % by weight.
The dose of the active compound of the formula I to be administered and the
frequency of administration depend on the potency and duration of action of
the
compounds used; moreover also on the nature and severity of the disease to be
treated and on the sex, age, weight and individual responsiveness of the
mammal to
be treated.
On average, the daily dose of a compound of the formula I in the case of a
patient
weighing approximately 75 kg is at least 0.001 mglkg, preferably 0.1 mg/kg, to
at most
mg/kg, preferably 1 mg/kg, of bodyweight. In acute episodes of the diseases,
for
instance, directly after a myocardial infarct, even higher and especially more
frequent
doses may also be necessary, e.g. up to 4 individual doses per day. In
particular in the
25 case of i.v. administration, for example in the case of an infarct patient
in the intensive
care unit, up to 200 mglkg per day may be necessary.
Descriptions of the experiments and examples:
30 List of abbreviations used:
Rt retention time
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13
TFA trifluoroacetic acid
LCMS liquid chromatography mass spectroscopy
MS mass spectroscopy
CI+ chemical ionization, positive mode
ES+ electrospray, positive mode
General:
The retention times (Rt) stated below are based on LCMS measurements with the
following parameters:
Method A:
Stationary phase: Merck Purospher 3N 2 x 55 mm
Mobile phase: 95% H20 (0.05% TFA)~ 95% acetonitrile; 4 min; 95%
acetonitrile; 1.5 min --> 5% acetonitrile; 1 min; 0.5 ml/min.
Method B:
Stationary phase: YMC J'sphereODS H80 2 x 33 mm
Mobile phase: 95% H20 (0.05% TFA)~ 95% acetonitrile; 2.3 min; 95%
acetonitrile; 1 min -~ 5% acetonitrile; 0.1 min; 1 ml/min.
Preparative HPLC was carried out under the following conditions:
Stationary phase: Merck Purospher RP18 (10NM) 250 x 25 mm
Mobile phase: 90% H20 (0.05% TFA)-~ 90% acetonitrile; 40 min;
ml/min
25 If the compounds are enantiomerically pure, the configuration and/or the
sign of the
optical rotation is given. If these data are missing, the compounds are
racemates or
not optically active.
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Example 1: (S,S)-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt
CIHN
'N ~ TFA
CI
2,6-Dichlorophenyl isothiocyanate (600 mg) and (1S,2S)-(+)-1,2-
diaminocyclohexane
(336 mg) were dissolved in toluene (30 ml) and stirred at 70°C for 3 h.
The mixture
was allowed to stand overnight and the solvent was then removed under reduced
pressure, and ether was added to the residue. The resulting thiourea was then
filtered
off with suction. 840 mg of the desired product were isolated.
A fraction of the thiourea obtained in this manner (420 mg) was then admixed
with
toluene (15 ml) and briefly heated at reflux. N,N'-Dicyclohexylcarbodiimide
(226 mg),
dissolved in toluene (5 ml), was then added dropwise, and the mixture was
stirred at
7U°C for 5 h. The mixture was allowed to stand overnight and the
resulting precipitate
was then filtered off and the filtrate was concentrated to dryness. The
residue was then
purified by preparative HPLC. The pure fractions were combined, the
acetonitrile was
removed using a rotary evaporator and the aqueous phase was freeze-dried. This
gave 70 mg of the desired compound.
LCMS-Rt: 3.69 min, (A)
MS (ES+, M+H+): 284.2
Example 2: cis-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt
CIHN
I
N H
CI TFA
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2,6-Dichlorophenyl isothioeyanate (600 mg) and cis-1,2-diaminocyclohexane (336
mg)
were reacted and worked up as described in Example 1. Frorn the 900 mg of
thiourea
obtained in the first step,.454 mg were reacted further in the next step. This
gave
112 mg of the desired compound.
5 LCMS-Rt: 3.65 min, (A)
MS (CI+, M+H+): 284.1
Example 3: (R,R)-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
10 trifluoroacetic acid salt
/ CIHN".,.
~N ~ TFA
CI
2,6-Dichlorophenyl isothiocyanate (50 mg) and (R,R)-(-)-1,2-diaminocyclohexane
(28 mg) were initially charged in toluene (1.5 ml) and heated at reflux for 15
min.
15 N,N'-Dicyclohexylcarbodiimide (76 mg) was then added, and the mixture was
kept
further at reflux. The mixture was allowed to stand overnight and the toluene
was then
removed and the residue was purified by preparative HPLC. Since the first
purification
gave only contaminated fractions, chromatography was repeated using a
different
column (MN Nucleosil 100-5-C18 250 x 25 mm; flow rate 20 ml/min), but with
otherwise identical conditions. The pure fractions were combined, the
acetonitrile was
removed using a rotary evaporator and the aqueous phase was freeze-dried. This
gave 10 mg of the desired compound.
LCMS-Rt: 3.70 min, (A)
MS (CI+, M+H+): 284.0
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16
Example 4: traps-(Octahydrobenzoimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride
/
CIH
O
. , ~-N v /
a) 2-Phenoxyphenyl isothiocyanate
1.96 g (0.011 mol) of thiocarbonyl diimidazole were added to a solution of
1.85 g
(0.01 mol) of 2-phenoxyaniline in 50 ml of THF and the mixture was stirred at
room
temperature for 4 hours, giving, after removal of the solvent by distillation,
the
compound as a brown amorphous product.
b) N-(traps-2-Aminocyclohexyl)-N'-(2-pr~enoxyphenyl)thiourea
A solution of 1.6 g of 2-phenoxyphenyl isothiocyanate in 10 ml of THF was
added to a
solution of 0.8 g of traps-1,2-diaminocyclohexane in 30 ml of THF, and the
mixture was
stirred at room temperature for about 4 hours. The solvent was evaporated and
the
residue was subsequently subjected to column chromatography on silica gel
using a
mixture of 10 parts of ethyl acetate, 5 parts of n-heptane, 5 parts of
methylene
chloride, 5 parts of methanol and 1 part of concentrated aqueous ammonia
solution,
giving the desired compound as an amorphous oily product.
c) traps-(Octahydrobenzimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride
3.4 g of methyl iodide were added to a solution of 1.03 g of N-(traps-2-
aminocyclohexyl)-N'-(2-phenoxyphenyl)thiourea in 30 ml of ethanol, and the
reaction
mixture was kept at reflux for 5 hours. The mixture was allowed to stand
overnight and
the solvent was then distilled off and the residue was treated with water and
subsequently made alkaline using saturated sodium bicarbonate solution. The
aqueous phase was extracted with ethyl acetate and the organic extraction
phase was
evaporated, and the oily residue was then chromatographed on silica gel using
a
CA 02470856 2004-06-17
17
mixture of 10 parts of ethyl acetate, 5 parts of n-heptane, 5 parts of
methylene
chloride, 5 parts of methanol and 1 part of concentrated aqueous ammonia
solution.
This gave an oily product which was dissolved in ethyl acetate and acidified
using a
saturated solution of HCI gas in diethyl ether. The solvent was distilled eff
and the
residue was then dissolved in water and subjected to freeze-drying. This gave
0.49 g
of a solid of m.p. 110°C.
MS (ES+, M+H+): 308.2
Example 5: trans-(2-Chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol-2-
ylidene)amine hydrochloride
F F
/-N ~F
CIH CI
a) N-(trans-2-Aminocyclohexyl)-N'-(2-chloro-6-trifluoromethylphenyl)urea
A solution of 0.46 g of trans-1,2-diaminocyclohexane in 10 ml of THF was added
to a
solution of 1.6 g of 2-chloro-6-trifluoromethylphenyl isocyanate in 30 ml of
THF, and
the mixture was stirred at room temperature for about 3 hours. The mixture was
allowed to stand overnight and the solvent was then distilled off, giving 0.57
g of the
desired compound as a semi-solid yellow product.
b) traps-(2-Chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol-2-
ylidene)amine
hydrochloride
0.57 g of N-(traps-2-aminocyclohexyl)-N'-(2-chloro-6-
trifluoromethylphenyl)urea in
20 ml of phosphorus oxychloride (POC13) was boiled at reflux for 4-5 hours.
The
POCI3 was distilled off, water was added to the residue and the pH was
adjusted to 7-
8 using 2N NaOH. The mixture was then extracted with ethyl acetate, the
organic
solvent was distilled off and the residue was chromatographed on silica gel
using a
CA 02470856 2004-06-17
18
mixture of 20 parts of ethyl acetate, 10 parts of n-heptane and 3 parts of
glacial acetic
acid. After removal of the eluent by distillation, the white solid residue was
dissolved in
a little ethyl acetate and acidified using a saturated solution of HCI gas in
diethyl ether.
Removal of the solvent by distillation and treatment of the residuo with
diisopropyl
ether gave 0.4 g of the desired product as a solid of m.p. 160 - 165°C.
MS (CI+, M+H+): 318.3
Example 6: trans-(4,5-Di-tert-butylimidazolidin-2-ylidene)-(2,6-
dichlorophenyl)amine
hydrochloride
CI
N~ '~--,
CI CIH
2,6-Dichlorophenyl isothiocyanate (150 mg) and traps-2,2,5,5-tetramethylhexane-
3,4-
diamine (127 mg) - analogously to Synthesis 1999, 2, 228; in racemic form -
were
initially charged in toluene (1.5 ml) and heated at reflux for 15 min.
N,N'-Dicyclohexylcarbodiimide (126 mg), dissolved in 2 ml of toluene, was then
added,
and the mixture was kept at reflux. After standing overnight, the toluene was
removed
under reduced pressure and the residue was purified by preparative HPLC. The
pure
fractions were combined, the acetonitrile was removed using a rotary
evaporator and
the aqueous phase was neutralized with saturated potassium carbonate solution
and
extracted three times with ethyl acetate. The combined ethyl acetate phases
were
washed with saturated sodium chloride solution and then dried over magnesium
sulfate. The drying agent was filtered off, the mixture was concentrated and
the
residue was then taken up in water, 2N hydrochloric acid was added and the
mixture
was freeze-dried. This gave 111 mg of the desired compound.
LCMS-Rt: 4.43 min, (A)
MS (CI+, M+H+): 342.2
CA 02470856 2004-06-17
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Example 7: trans-(2,6-Dichlorophenyl)-(4,5-diisopropylimidazolidin-2-
ylidene)amine
hydrochloride
CI
N
CI
CIH
traps-2,5-Dimethylhexane-3,4-diamine (226 mg) - analogously to Synthesis 1999;
2,
228; in racemic form - was initially charged in THF (2.5 ml), and 2,6-
dichlorophenyl
isothiocyanate was added a little at a time (portions of 150, 80 and 40 mg) at
room
temperature. N,N'-Dicyclohexylcarbodiimide (324 mg) was then added, and the
mixture was further stirred at room temperature. To complete the reaction,
some more
N,N'-dicyclohexylcarbodiimide was added. The mixture was allowed to stand
overnight, and the resulting precipitate was then filtered off with suction
and the filtrate
was concentrated. The residue was purified by preparative HPLC. The pure
fractions
were combined, the acetonitrile was removed using a rotary evaporator and the
aqueous phase was neutralized with saturated potassium carbonate solution and
extracted three times with ethyl acetate. The combined ethyl acetate phases
were
washed with saturated sodium chloride solution and then dried over magnesium
sulfate. The drying agent was filtered off and the mixture was concentrated,
and the
residue was then taken up in water, 2N hydrochloric acid was added and the
mixture
was freeze-dried. This gave 220 mg of the desired compound.
LCMS-Rt: 1.93 min, (B)
MS (ES+, M+H+): 314.1
The compounds described in the table below were synthesized according to the
examples stated in each case:
CA 02470856 2004-06-17
Analo-
Ex- gously MS LCMS-
Salt
ample to Ex- [M+H~] Rt [min]
ample
D
309.0
8 I \ HN TFA 7 1.84 (B)
i ~~ (ES+)
~N
CI
D
ci
HN~."1 HCI 7 309.1 (ES+) 1.87 (B)
s ~ ~p Q
CI HN 286.1
10 ~ , ~ w~~ HCI 7 + 1.75 (B)
'N p (ES )
ci
H
CI N ::
11 ~ I N~N TFA 7 270.1 (ES+) 1.56 (B)
w CI H
cl
12 ~ ~ / HN03
N
CI
Pharmacological data:
Test description:
CA 02470856 2004-06-17
21
In this test, the recovery of the intracellular pH (pHi) after an
acidification, which starts
when the NHE is capable of functioning, even under bicarbonate-free
conditions, was
determined. For this purpose, the pH; was determined using the pH-sensitive
fluorescent dye BCECF (calbiochem, the precursor BCECF-AM is employed). The
cells were initially loaded with BCECF. The BCECF fluorescence was determined
in a
"ratio fluorescence spectrometer" (Photon Technology International, South
Brunswick,
N.J., USA) with excitation wavelengths of 505 and 440 nm and an emission
wavelength of 535 nm, and was converted into the pHi using calibration plots.
The
cells were incubated in NH4C1 buffer (pH 7.4) (NH4CI buffer: 115 mM NaCI, 20
mM
NH4CI, 5 mM KC1, 1 mM CaCl2, 1 mM MgS04, 20 mM Hepes, 5 mM glucose, 1 mg/ml
BSA; a pH of 7.4 is established with 1 M NaOH) even during the BCECF loading.
The
intracellular acidification was induced by addition of 975 NI of an NH4C1-free
buffer
(see below) to 25,u1 aliquots of the cells incubated in NH4C1 buffer. The
subsequent
rate of pH recovery was recorded in the case of NHE1 for two minutes, in the
case of
NHE2 for five minutes and in the case of NHE3 for three minutes. To calculate
the
inhibitory power of the tested substances, the cells were initially
investigated in buffers
in which complete or absolutely no pH recovery took place. For complete pH
recovery
(100%), the cells were incubated in Na+-containing buffer (133.8 mM NaCI, 4.7
mM
KCI, 1.25 mM CaCl2, 1.25 mM MgCl2, 0.97 mM Na2HP04, 0.23 mM NaH2P04, 5 mM
Hepes, 5 mM glucose, a pH of 7.0 is established with 1 M NaOH). To determine
the
0% value, the cells were incubated in an Na~-free buffer (133.8 mM choline
chloride,
4.7 mM KCI, 1.25 mM CaCl2, 1.25 mM MgCl2, 0.97 mM K2HP04, 0.23 mM KH2P04,
5 mM Hepes, 5 mM glucose, a pH of 7.0 is established with 1 M KOH). The
substances to be tested were made up in the Na+-containing buffer. Recovery of
the
intracellular pH at the tested concentration of a substance was expressed as a
percentage of the maximum recovery. Using the Sigma-Plot program, the IC value
of
the substance in question was calculated for the individual NHE subtypes using
the
percentages for pH recovery.
CA 02470856 2004-06-17
22
Results:
IC50 [NM1~
Example
(rNHE3)
19
7 1.1
12 -3
