U.S. patent application number 10/323799 was filed with the patent office on 2003-10-02 for substituted imidazolidines, process for their preparation, and their use as a medicament or diagnostic.
Invention is credited to Heinelt, Uwe, Hofmeister, Armin, Lang, Hans-Jochen, Wirth, Klaus.
Application Number | 20030187045 10/323799 |
Document ID | / |
Family ID | 28457402 |
Filed Date | 2003-10-02 |
United States Patent
Application |
20030187045 |
Kind Code |
A1 |
Heinelt, Uwe ; et
al. |
October 2, 2003 |
Substituted imidazolidines, process for their preparation, and
their use as a medicament or diagnostic
Abstract
The invention relates to novel compounds of the type of the
imidazolidines of the formula I 1 in which R1 to R7 are as defined
herein. In one embodiment, these compounds are used for the
treatment of the central nervous system, of lipid metabolism, of
infection by ectoparasites, of disorders of gall function and for
improving the respiratory drive such as for treating respiratory
distress. In another embodiment, the compounds increase the muscle
tone of the upper respiratory tract, thus suppressing snoring.
Inventors: |
Heinelt, Uwe; (Wiesbaden,
DE) ; Lang, Hans-Jochen; (Hofheim, DE) ;
Hofmeister, Armin; (Oppenheim, DE) ; Wirth,
Klaus; (Kriftel, DE) |
Correspondence
Address: |
Finnegan, Henderson, Farabow,
Garrett & Dunner, L.L.P.
1300 I Street, N.W.
Washington
DC
20005-3315
US
|
Family ID: |
28457402 |
Appl. No.: |
10/323799 |
Filed: |
December 20, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60353518 |
Feb 1, 2002 |
|
|
|
Current U.S.
Class: |
514/386 ;
514/387; 548/307.4; 548/329.1 |
Current CPC
Class: |
C07D 235/02 20130101;
C07D 233/50 20130101; C07D 235/30 20130101 |
Class at
Publication: |
514/386 ;
514/387; 548/307.4; 548/329.1 |
International
Class: |
A61K 031/4184; A61K
031/4172; C07D 233/91; C07D 235/22; C07D 235/24 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 21, 2001 |
DE |
10163239.8 |
Claims
We claim:
1. An imidazolidine of the formula 19in which R1 and R2
independently of one another are --CN, --(C.sub.1-C.sub.5)-alkyl,
--(C.sub.2-C.sub.5)-alke- nyl, --(C.sub.2-C.sub.5)-alkynyl,
--(C.sub.3-C.sub.6)-cycloalkyl or --(C.sub.4-C.sub.6)-cycloalkenyl
where the carbon chains and carbon rings, independently of one
another, are unsubstituted or substituted by 1-11 fluorine atoms or
by up to two radicals selected from the group consisting of --OH,
--NH.sub.2, --NHCH.sub.3, --N(CH.sub.3).sub.2 and --OCH.sub.3; 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, where the ring is unsubstituted or substituted by 1-12
fluorine atoms or by up to two radicals selected from the group
consisting of --CH.sub.3 and --OCH.sub.3, with the proviso that
there is no double bond between the two carbon atoms to which R1
and R2 are attached; R3 is F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, -(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--(C.sub.1-C.sub.4)-alkoxy, --O-phenyl, --CN, --NO.sub.2 or
--NH.sub.2; where the phenyl is unsubstituted or substituted by up
to two radicals selected from the group consisting of --CH.sub.3,
F, Cl, Br, I, --OH and --OCH.sub.3; and where the carbon chains or
carbon rings, independently of one another, are unsubstituted or
substituted by 1-11 fluorine atoms; R4 to R6 independently of one
another are H, F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, --(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--(C.sub.1-C.sub.4)-alkoxy, --CN, --NO.sub.2, --NH.sub.2,
--(C.sub.1-C.sub.4)-alkylamino or --(C.sub.1-C.sub.4)-dialkylamino;
where the carbon chains or carbon rings, independently of one
another, are unsubstituted or substituted by 1-11 fluorine atoms;
R7 is H, F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, -(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--(C.sub.1-C.sub.4)-alkoxy, --CN, --NO.sub.2 or --NH.sub.2; where
the carbon chains or carbon rings, independently of one another,
are unsubstituted or substituted by 1-11 fluorine atoms; or a
pharmaceutically acceptable salt thereof, or a trifluoroacetic acid
salt thereof, in any stereoisomeric form, or a mixture of any such
compounds in any ratio.
2. The compound as claimed in claim 1, in which R1 and R2
independently of one another are --(C.sub.1-C.sub.5)-alkyl,
--(C.sub.2-C.sub.5)-alkenyl, --(C.sub.2-C.sub.5)-alkynyl,
--(C.sub.3-C.sub.6)-cycloalkyl or --(C.sub.4-C.sub.6)-cycloalkenyl,
where the carbon chains and carbon rings, independently of one
another, are unsubstituted or substituted by 1-11 fluorine atoms or
by up to two radicals selected from the group consisting of
--NHCH.sub.3, --N(CH.sub.3).sub.2 and --OCH.sub.3; 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, where
the ring is unsubstituted or substituted by 1-12 fluorine atoms or
by up to two radicals selected from the group consisting of
--CH.sub.3 and --OCH.sub.3, with the proviso that there is no
double bond between the two carbon atoms to which R1 and R2 are
attached; R3 is F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, --(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--C.sub.1-C.sub.4)-alkoxy, --O-phenyl, --CN, --NO.sub.2 or
--NH.sub.2; where the phenyl is unsubstituted or substituted by up
to two radicals selected from the group consisting of --CH.sub.3,
F, Cl, Br, --OH and --OCH.sub.3; and where the carbon chains or
carbon rings, independently of one another, are unsubstituted or
substituted by 1-11 fluorine atoms; R4 to R6 independently of one
another are H, F, Cl, Br, --CH.sub.3, --OH, --OCH.sub.3, --CN,
--NO.sub.2, --NH.sub.2, --NHCH.sub.3, or --N(CH.sub.3).sub.2; where
the methyl group, independently of one another, are unsubstituted
or substituted by 1-3 fluorine atoms; R7 is H, F, Cl, Br, I,
--(C.sub.1-C.sub.4)-alkyl, --(C.sub.1-C.sub.4)-alkenyl,
--(C.sub.3-C.sub.6)-cycloalkyl, --OH, --(C.sub.1-C.sub.4)-alkoxy,
--CN, --NO.sub.2 or --NH.sub.2; where the carbon chains or carbon
rings, independently of one another, are unsubstituted or
substituted by 1-11 fluorine atoms; or a pharmaceutically
acceptable salt thereof, or a trifluoroacetic acid salt thereof, in
any stereoisomeric form, or a mixture of any such compounds in any
ratio
3. The compound as claimed in claim 1, chosen from:
trans-(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)-(octahyd-
robenzimidazol-2-ylidene)amine trifluoroacetic acid salt,
(R,R)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
trans-(octahydrobenzimidazol-2-ylidene)-(2-phe- noxyphenyl)amine
hydrochloride, trans-(2,6-dichlorophenyl)-(4,5-diisopropy-
limidazolidin-2-ylidene)amine hydrochloride,
trans-(2,6-dichlorophenyl)-(4-
,5-dicyclopropylimidazolidin-2-ylidene)amine trifluoracetic acid
salt,
cis-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene)amine
hydrochloride,
trans-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-ylid-
ene)amine hydrochloride,
(2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-- ylidene)amine
nitric acid salt, and trans-(2,6-dichlorophenyl)-(hexahydroc-
yclopentaimidazol-2-ylidene)amine trifluoroacetic acid salt.
4. The compound as claimed in claim 1, chosen from:
(S,S)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
cis-(2,6-dichlorophenyl)-(octahydrobenzimidazo- l-2-ylidene)amine
trifluoroacetic acid salt, (R,R)-(2,6-dichlorophenyl)-(o-
ctahydrobenzimidazol-2-ylidene)amine trifluoroacetic acid salt,
trans-(2,6-dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)amine
hydrochloride,
trans-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin--
2-ylidene)amine trifluoroacetic acid salt,
cis-(2,6-dichlorophenyl)-(4,5-d-
icyclopropylimidazolidin-2-ylidene)amine hydrochloride,
trans-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-ylidene)amine
hydrochloride,
(2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-ylidene)a- mine
nitric acid salt, and
trans-(2,6-dichlorophenyl)-(hexahydrocyclopenta-
imidazol-2-ylidene)amine trifluoroacetic acid salt.
5. A method for the treatment of disorders of the respiratory drive
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
6. A method for the treatment of respiratory disorders associated
with sleeping comprising, administering to a patient in need
thereof at least one compound as claimed in claim 1, with the
proviso that said compound is not a trifluoroacetic acid salt.
7. The method of claim 6, wherein the respiratory disorders
associated with sleeping is sleep apnea.
8. A method for the treatment of snoring comprising, administering
to a patient in need thereof at least one compound as claimed in
claim 1, with the proviso that said compound is not a
trifluoroacetic acid salt
9. A method for the treatment of acute and chronic renal diseases
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
10. The method of claim 9, wherein the acute and chronic renal
diseases is chosen form acute kidney failure and chronic kidney
failure.
11. A method for the treatment of disorders of intestinal function
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
12. A method for the treatment of disorders of gall function
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
13. A method for the treatment of ischemic states of the peripheral
and central nervous system and of stroke comprising, administering
to a patient in need thereof at least one compound as claimed in
claim 1, with the proviso that said compound is not a
trifluoroacetic acid salt.
14. A method for the treatment of ischemic states of peripheral
organs and limbs comprising, administering to a patient in need
thereof at least one compound as claimed in claim 1, with the
proviso that said compound is not a trifluoroacetic acid salt.
15. A method for the treatment of states of shock comprising,
administering to a patient in need thereof at least one compound as
claimed in claim 1, with the proviso that said compound is not a
trifluoroacetic acid salt.
16. A method for preserving and storing transplants for surgical
interventions comprising, contacting the transplant with at least
one compound as claimed in claim 1, with the proviso that said
compound is not a trifluoroacetic acid salt.
17. A method for the treatment of diseases in which cell
proliferation is a primary or secondary cause comprising,
administering to a patient in need thereof at least one compound as
claimed in claim 1, with the proviso that said compound is not a
trifluoroacetic acid salt.
18. A method for the treatment of disorders of lipid metabolism
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
19. A method for the treatment of infection by ectoparasites
comprising, administering to a patient in need thereof at least one
compound as claimed in claim 1, with the proviso that said compound
is not a trifluoroacetic acid salt.
20. A pharmaceutical comprising at least one compound as claimed in
claim 1 and at least one pharmaceutical carrier, with the proviso
that said compound is not a trifluoroacetic acid salt.
21. A method of protecting a transplant organ in a host, comprising
administering to the host in need thereof at least one compound as
claimed in claim 1, wherein the host is a transplant donor or a
transplant recipient, with the proviso that said compound is not a
trifluoroacetic acid salt.
Description
[0001] This application claims the benefit of foreign priority
under 35 U.S.C. .sctn.119 of German patent application no.
10163239.8, filed on Dec. 21, 2001 and the contents of which are
incorporated by reference herein. This application also claims the
benefit of priority of U.S. Provisional Application No. 60/353,518,
filed Feb. 1, 2002, the contents of which are incorporated by
reference herein.
DESCRIPTION
[0002] Substituted imidazolidines, process for their preparation,
and their use as medicament or diagnostic
[0003] The invention relates to substituted imidazolidines of the
formula I, 2
[0004] in which
[0005] R1 and R2
[0006] independently of one another are --CN,
--(C.sub.1-C.sub.5)-alkyl, -(C.sub.2-C.sub.5)-alkenyl,
--(C.sub.2-C.sub.5)-alkynyl, --(C.sub.3-C.sub.6)-cycloalkyl or
--(C.sub.4-C.sub.6)-cycloalkenyl
[0007] where the carbon chains and carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms or by up to two radicals selected from the group consisting
of --OH, --NH.sub.2, --NHCH.sub.3, --N(CH.sub.3).sub.2 and
--OCH.sub.3;
[0008] or
[0009] R1 and R2
[0010] together with the two carbon atoms to which they are
attached are a five- to eight-membered saturated or unsaturated
carbon ring, where the ring is unsubstituted or substituted by 1-12
fluorine atoms or by up to two radicals selected from the group
consisting of --CH.sub.3 and --OCH.sub.3,
[0011] with the proviso that there is no double bond between the
two carbon atoms to which R1 and R2 are attached;
[0012] R3
[0013] is F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alk- enyl, --(C.sub.3-C.sub.6)-cycloalkyl,
--OH, --(C.sub.1-C.sub.4)-alkoxy, --O-phenyl, --CN, --NO.sub.2 or
--NH.sub.2;
[0014] where the phenyl is unsubstituted or substituted by up to
two radicals selected from the group consisting of --CH.sub.3, F,
Cl, Br, I, OH and --OCH.sub.3; and
[0015] where the carbon chains or carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms;
[0016] R4 to R6
[0017] independently of one another are H, F, Cl, Br, I,
--(C.sub.1-C.sub.4)-alkyl, --(C.sub.1-C.sub.4)-alkenyl,
--(C.sub.3-C.sub.6)-cycloalkyl, --OH, --(C.sub.1-C.sub.4)-alkoxy,
--CN, --NO.sub.2, --NH.sub.2, --(C.sub.1-C.sub.4)-alkylamino or
--(C.sub.1-C.sub.4)-dialkylamino;
[0018] where the carbon chains or carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms;
[0019] R7
[0020] is H, F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, --(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--(C.sub.1-C.sub.4)-alkoxy, --CN, --NO.sub.2 or --NH.sub.2;
[0021] where the carbon chains or carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms;
[0022] and their pharmaceutically acceptable salts, and the
trifluoroacetic acid salts.
[0023] In one embodiment, compounds of the formula I are chosen
from
[0024] R1 and R2
[0025] independently of one another are --(C.sub.1-C.sub.5)-alkyl,
--(C.sub.2-C.sub.5)-alkenyl, --(C.sub.2-C.sub.5)-alkynyl,
--(C.sub.3-C.sub.6)-cycloalkyl or
--(C.sub.4-C.sub.6)-cycloalkenyl,
[0026] where the carbon chains and carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms or by up to two radicals selected from the group consisting
of --NHCH.sub.3, --N(CH.sub.3).sub.2 and --OCH.sub.3;
[0027] or
[0028] R1 and R2
[0029] together with the two carbon atoms to which they are
attached are a five- to eight-membered saturated or unsaturated
carbon ring, where the ring is unsubstituted or substituted by 1-12
fluorine atoms or by up to two radicals selected from the group
consisting of --CH.sub.3 and --OCH.sub.3,
[0030] with the proviso that there is no double bond between the
two carbon atoms to which R1 and R2 are attached;
[0031] R3
[0032] is F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alk- enyl, --(C.sub.3-C.sub.6)-cycloalkyl,
--OH, --(C.sub.1-C.sub.4)-alkoxy, --O-phenyl, --CN, --NO.sub.2 or
--NH.sub.2;
[0033] where the phenyl is unsubstituted or substituted by up to
two radicals selected from the group consisting of --CH.sub.3, F,
Cl, Br, --OH and --OCH.sub.3; and
[0034] where the carbon chains or carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms;
[0035] R4 to R6
[0036] independently of one another are H, F, Cl, Br, --CH.sub.3,
--OH, --OCH.sub.3, --CN, --NO.sub.2, --NH.sub.2, --NHCH.sub.3, or
--N(CH.sub.3).sub.2;
[0037] where the methyl group, independently of one another, are
unsubstituted or substituted by 1-3 fluorine atoms;
[0038] R7
[0039] is H, F, Cl, Br, I, --(C.sub.1-C.sub.4)-alkyl,
--(C.sub.1-C.sub.4)-alkenyl, --(C.sub.3-C.sub.6)-cycloalkyl, --OH,
--(C.sub.1-C.sub.4)-alkoxy, --CN, --NO.sub.2 or --NH.sub.2;
[0040] where the carbon chains or carbon rings, independently of
one another, are unsubstituted or substituted by 1-11 fluorine
atoms;
[0041] and their pharmaceutically acceptable salts, and the
trifluoroacetic acid salts.
[0042] In another embodiment, compounds of the formula 1 are chosen
from:
[0043]
trans-(2-chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol-2-y-
lidene)amine hydrochloride,
[0044]
(S,S)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0045]
cis-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0046]
(R,R)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0047]
trans-(octahydrobenzimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride,
[0048]
trans-(2,6-dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)a-
mine hydrochloride,
[0049]
trans-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene-
)amine trifluoracetic acid salt,
[0050]
cis-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene)a-
mine hydrochloride,
[0051]
trans-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-ylidene)amine
hydrochloride,
[0052]
(2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-ylidene)amine
nitric acid salt, and
[0053]
trans-(2,6-dichlorophenyl)-(hexahydrocyclopentaimidazol-2-ylidene)a-
mine trifluoroacetic acid salt.
[0054] In another embodiment, compounds of the formula 1 are chosen
from:
[0055]
(S,S)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0056]
cis-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0057]
(R,R)-(2,6-dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt,
[0058]
trans-(2,6-dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)a-
mine hydrochloride,
[0059]
trans-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene-
)amine trifluoroacetic acid salt,
[0060]
cis-(2,6-dichlorophenyl)-(4,5-dicyclopropylimidazolidin-2-ylidene)a-
mine hydrochloride,
[0061]
trans-(2,6-dichlorophenyl)-(4,5-diethylimidazolidin-2-ylidene)amine
hydrochloride,
[0062]
(2,6-dichlorophenyl)-(4,5-dimethylimidazolidin-2-ylidene)amine
nitric acid salt, and
[0063]
trans-(2,6-dichlorophenyl)-(hexahydrocyclopentaimidazol-2-ylidene)a-
mine trifluoroacetic acid salt.
[0064] Suitable acid addition salts include 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.
[0065] If the compounds of the invention contain one or more
centers of asymmetry, the compounds may be both S- and
R-configured. The compounds may be present as optical isomers,
enantiomers, as diastereomers, as racemates or as mixtures
thereof.
[0066] The compounds of the invention may furthermore be present as
tautomers or as a mixture of tautomeric structures.
[0067] In one embodiment, this refers to the following tautomers:
3
[0068] 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: 4
[0069] The carbon radicals mentioned, including the partially or
fully fluorinated or substituted carbon radicals, may be
straight-chain or branched.
[0070] Also described are methods for preparing the compounds
used.
[0071] Thus, the substances described by formula I, in one
embodiment, may be prepared in a manner known to the person skilled
in the art from the isothiocyanate II parent compounds and the
appropriate diamines III. 5
[0072] The thiourea derivative which is formed as an intermediate
may be 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 may 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).
[0073] In addition to the isothiocyanates II described above, it is
also possible to successfully react the isocyanates 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. 6
[0074] In one embodiment, it was surprisingly possible to
demonstrate that the compounds described are potent inhibitors of
the sodium/hydrogen exchanger (NHE), in particular of NHE3.
[0075] 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-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 some of the
compounds of the formula I, not effective immediately but reaches
its maximum potency only after one hour.
[0076] Clonidine is also known as a weak NHE inhibitor. However,
its action on the NHE3 of the rat is, with an IC.sub.50 of 620
.mu.M, extremely moderate. In contrast, it shows a certain
selectivity for the NHE2, where it has an IC.sub.50 of 42 .mu.M (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 I1 receptor,
mediating a strong hypotensive action (Ernsberger et al Eur. J.
Pharmacol. 134, 1, 1987). Some of the compounds of the formula I
have increased NHE3 activity and reduced I1 and alpha2
activity.
[0077] 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).
[0078] Owing to this unexpected property, the compounds of the
invention may be suitable for treating disorders caused by oxygen
deficiency. As a result of their pharmacological properties, the
compounds may be highly suitable for use as antiarrhythmics having
a cardioprotective component, for infarct prophylaxis and infarct
treatment and for treatment of angina pectoris, and they may 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 invention may also, as inhibitors of the cellular
Na.sup.+/H.sup.+ exchange mechanisms, be used as medicaments for
treating all acute or chronic damage caused by ischemia, or
diseases induced primarily or secondarily by this damage. This
relates to their potential use as medicaments for surgical
interventions, for example organ transplantations, where the
compounds may 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 fluids, and also during transfer into the recipient organism.
The compounds may also be 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, some of the compounds
may also be 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,
some of the compounds of the invention may also be suitable for
treating forms of shock, such as, for example, of allergic,
cardiogenic, hypovolemic and bacterial shock.
[0079] As used herein, treating or treatment includes the treating
of, for example, a patient inflicted with a disease or condition,
as well as the prevention, prophylaxis, or protective treatment of
a patient. Treatment also includes treating a subject susceptible
to or predisposed to developing a disease or condition, which could
include patients in whom a disease or condition has not yet
presented as well as patients in whom the disease has been
successfully treated but could redevelop or reoccur.
[0080] Furthermore, some of the compounds may induce an improvement
in the respiratory drive and therefore may be used 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.
[0081] In another embodiment, some of the compounds additionally
increase the muscle tone of the upper airways, so that snoring is
suppressed.
[0082] in a further embodiment, the 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 the active compound.
[0083] It has been shown that some of the compounds to be used
according to the invention may also have a mild laxative action and
accordingly may be used advantageously as laxatives or in the case
of threatening intestinal blockage, the prevention of ischemic
damage accompanying blockages in the intestinal region.
[0084] In one embodiment, it may be possible to prevent gallstone
formation.
[0085] In another embodiment, the compounds of the invention may
exert a strong inhibitory action on the proliferation of cells, for
example fibroblast cell proliferation and the proliferation of the
smooth vascular muscle cells. Some of the compounds of the formula
I may therefore be suitable as valuable therapeutics for diseases
in which cell proliferation is a primary or secondary cause, and
may 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.
[0086] Some of the compounds according to the invention may also be
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 may also be 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
invention may also be suitable for preventive therapy to prevent
the development of high blood pressure, for example of essential
hypertension.
[0087] It has moreover been found that NHE inhibitors may exhibit a
favorable influence on the serum lipoproteins. It is generally
recognized that for the formation of artereoscierotic 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. In one embodiment, the compounds to be
used according to the invention may 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, some of the compounds of
the invention may be valuable pharmaceuticals for the prevention
and treatment of coronary vascular spasms, of atherogenesis and of
atherosclerosis, of left-ventricular hypertrophy and of dilated
cardiomyopathy, and thrombotic disorders.
[0088] In another embodiment, the compounds mentioned may be used
for the production of a medicament for the prevention and treatment
of sleep apneas and muscular-related respiratory disorders; for a
medicament for the prevention and treatment of snoring, for a
medicament for lowering the blood pressure, for a medicament having
laxative action for the prevention and treatment of intestinal
blockages; for 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, intestinal disorders, etc; for a
medicament for the treatment of hypercholesterolemia; for a
medicament for the prevention of atherogenesis and of
atherosclerosis; for a medicament for the prevention and treatment
of diseases which are induced by raised cholesterol levels; for a
medicament for the prevention and treatment of diseases which are
induced by endothelial dysfunction; for a medicament for the
treatment of attack by ectoparasites; for 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
invention 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 invention, proves to be a favorable combination
having intensified action and decreased use of active
substance.
[0089] In another embodiment, sodium/proton exchange inhibitors of
the invention as 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 may be administered to a patient in need
thereof.
[0090] Pharmaceuticals which contain a compound of the invention
may be administered orally, parenterally, intravenously, rectally
or by inhalation, the preferred administration being dependent on
the particular clinical picture of the disorder. The compounds of
the invention in this case may also be used on their own or
together with pharmaceutical excipients, namely both in veterinary
and in human medicine.
[0091] 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.
[0092] For example, for a form for oral administration, the active
compounds may be 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 may 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.
[0093] For subcutaneous or intravenous administration, the active
compounds used may, for example, be 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, propanol, glycerol, and in
addition also sugar solutions such as glucose or mannitol
solutions, or alternatively a mixture of the different solvents
mentioned.
[0094] 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.
[0095] If required, the formulation may 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.
[0096] The dose of the active compounds of the invention 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.
[0097] On average, the daily dose of a compound of the invention in
the case of a patient weighing approximately 75 kg is at least
0.001 mg/kg, preferably 0.1 mg/kg, to at most 30 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 case of i.v.
administration, for example in the case of an infarct patient in
the intensive care unit, up to 200 mg/kg per day may be
necessary.
DESCRIPTION OF THE EXPERIMENTS AND EXAMPLES
[0098] List of Abbreviations Used:
1 Rt retention time TFA trifluoroacetic acid LCMS liquid
chromatography mass spectroscopy MS mass spectroscopy CI.sup.+
chemical ionization, positive mode ES.sup.+ electrospray, positive
mode
[0099] General:
[0100] The retention times (Rt) stated below are based on LCMS
measurements with the following parameters:
[0101] Method A:
2 Method A: Stationary phase: Merck Purospher 3.mu. 2 .times. 55 mm
Mobile phase: 95% H.sub.2O (0.05% TFA).fwdarw.95% acetonitrile; 4
min; 95% acetonitrile; 1.5 min .fwdarw. 5% acetonitrile; 1 min; 0.5
ml/min. Method B: Stationary phase: YMG J'sphereODS H80 2 .times.
33 mm Mobile phase: 95% H.sub.2O (0.05% TFA).fwdarw. 95%
acetonitrile; 2.3 min; 95% acetonitrile; 1 min .fwdarw. 5%
acetonitrile; 0.1 min; 1 ml/min.
[0102] Preparative HPLC was carried out under the following
conditions:
3 Stationary phase: Merck Purospher RP18 (10 .mu.M) 250 .times. 25
mm Mobile phase: 90% H.sub.2O (0.05% TFA).fwdarw.90% acetonitrile;
40 min; 25 ml/min
[0103] 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.
EXAMPLE 1
[0104]
(S,S)-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt 7
[0105] 2,6-Dichlorophenyl isothiocyanate (600 mg) and
(1S,2S)-(+)-1,2-diaminocyclohexane (336 mg) were dissolved in
toluene (30 ml) and stirred at 70.degree. 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.
[0106] 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
70.degree. 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.
[0107] LCMS-Rt: 3.69 min, (A)
[0108] MS (ES.sup.+, M+H.sup.+): 284.2
EXAMPLE 2
[0109]
cis-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt 8
[0110] 2,6-Dichlorophenyl isothiocyanate (600 mg) and
cis-1,2-diaminocyclohexane (336 mg) were reacted and worked up as
described in Example 1. From 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.
[0111] LCMS-Rt: 3.65 min, (A)
[0112] MS (Cl.sup.+, M+H.sup.+): 284.1
EXAMPLE 3
[0113]
(R,R)-(2,6-Dichlorophenyl)-(octahydrobenzimidazol-2-ylidene)amine
trifluoroacetic acid salt 9
[0114] 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.
N,N'-Dicyclohexylcarbod- iimide (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.times.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.
[0115] LCMS-Rt: 3.70 min, (A)
[0116] MS (Cl.sup.+, M+H.sup.+): 284.0
EXAMPLE 4
[0117]
trans-(Octahydrobenzoimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride 10
[0118] a) 2-Phenoxyphenyl isothiocyanate
[0119] 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.
[0120] b)
N-(trans-2-Aminocyclohexyl)-N'-(2-phenoxyphenyl)thiourea
[0121] 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
trans-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.
[0122] c)
trans-(Octahydrobenzimidazol-2-ylidene)-(2-phenoxyphenyl)amine
hydrochloride
[0123] 3.4 g of methyl iodide were added to a solution of 1.03 g of
N-(trans-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 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 HCl gas
in diethyl ether. The solvent was distilled off 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.degree. C.
[0124] MS (ES.sup.+, M+H.sup.+): 308.2
EXAMPLE 5
[0125]
trans-(2-Chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol-2-y-
lidene)amine hydrochloride 11
[0126] a) N-(trans-2-Aminocyclohexyl
)-N'-(2-chloro-6-trifluoromethyl phenyl)urea
[0127] 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.
[0128] b)
trans-(2-Chloro-6-trifluoromethylphenyl)-(octahydrobenzimidazol--
2-ylidene)amine hydrochloride
[0129] 0.57 g of
N-(trans-2-aminocyclohexyl)-N'-(2-chloro-6-trifluoromethy-
lphenyl)urea in 20 ml of phosphorus oxychloride (POCl.sub.3) was
boiled at reflux for 4-5 hours. The POCl.sub.3 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 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 HCl gas in diethyl ether. Removal of the
solvent by distillation and treatment of the residue with
diisopropyl ether gave 0.4 g of the desired product as a solid of
m.p. 160-165.degree. C.
[0130] MS (Cl.sup.+, M+H.sup.+): 318.3
EXAMPLE 6
[0131]
trans-(4,5-Di-tert-butylimidazolidin-2-ylidene)-(2,6-dichlorophenyl-
)amine hydrochloride 12
[0132] 2,6-Dichlorophenyl isothiocyanate (150 mg) and
trans-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.
[0133] 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.
[0134] LCMS-Rt: 4.43 min, (A)
[0135] MS (Cl.sup.+, M+H.sup.+): 342.2
EXAMPLE 7
[0136]
trans-(2,6-Dichlorophenyl)-(4,5-diisopropylimidazolidin-2-ylidene)a-
mine hydrochloride 13
[0137] trans-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.
[0138] LCMS-Rt: 1.93 min, (B)
[0139] MS (ES.sup.+, M+H.sup.+): 314.1
[0140] The compounds described in the table below were synthesized
according to the examples stated in each case:
4 Analogously to MS LCMS- Example Salt to Example [M + H.sup.+] Rt
[min] 8 14 TFA 7 309.0 (ES.sup.+) 1.84(B) 9 15 HCl 7 309.1
(ES.sup.+) 1.87(B) 10 16 HCl 7 286.1 (ES.sup.+) 1.75(B) 11 17 TFA 7
270.1 (ES.sup.+) 1.56(B) 12 18 HNO.sub.3
[0141] Pharmacological Data:
[0142] Test Description:
[0143] In this test, the recovery of the intracellular pH
(pH.sub.i) after an acidification, which starts when the NHE is
capable of functioning, even under bicarbonate-free conditions, was
determined. For this purpose, the pH.sub.i 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 pH.sub.i
using calibration plots. The cells were incubated in NH.sub.4Cl
buffer (pH 7.4) (NH.sub.4Cl buffer: 115 mM NaCl, 20 mM NH.sub.4Cl,
5 mM KCl, 1 mM CaCl.sub.2, 1 mM MgSO.sub.4, 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 .mu.l of an NH.sub.4Cl-free buffer (see
below) to 25 .mu.l aliquots of the cells incubated in NH.sub.4Cl
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.sup.+-containing buffer (133.8 mM NaCl, 4.7 mM
KCl, 1.25 mM CaCl.sub.2, 1.25 mM MgCl.sub.2, 0.97 mM
Na.sub.2HPO.sub.4, 0.23 mM NaH.sub.2PO.sub.4, 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.sup.+-free buffer
(133.8 mM choline chloride, 4.7 mM KCl, 1.25 mM CaCl.sub.2, 1.25 mM
MgCl.sub.2, 0.97 mM K.sub.2HPO.sub.4, 0.23 mM KH.sub.2PO.sub.4, 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.sup.+-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.sub.50 value
of the substance in question was calculated for the individual NHE
subtypes using the percentages for pH recovery.
5 Results: IC.sub.50 [.mu.M], Example (rNHE3) 5 19 7 1.1 12
.about.3
* * * * *