U.S. patent application number 12/829185 was filed with the patent office on 2011-01-06 for n-sulphonylated amino acid derivatives, method for the production and use thereof.
This patent application is currently assigned to The Medicines Company (Leipzig) GmbH. Invention is credited to Andrea Schweinitz, Torsten Steinmetzer, Anne Sturzebecher, Jorg Sturzebecher, Kerstin Uhland.
Application Number | 20110002992 12/829185 |
Document ID | / |
Family ID | 33440914 |
Filed Date | 2011-01-06 |
United States Patent
Application |
20110002992 |
Kind Code |
A1 |
Sturzebecher; Jorg ; et
al. |
January 6, 2011 |
N-SULPHONYLATED AMINO ACID DERIVATIVES, METHOD FOR THE PRODUCTION
AND USE THEREOF
Abstract
The present invention relates to N-sulfonylated amino acid
derivatives, where an aryl radical is linked via the sulfonyl group
N-terminally to the amino acid and a radical which comprises at
least one imino group and at least one further basic group which
represents an optionally modified amino, amidino or guanidino group
is linked C-terminally via the carbonyl group. The invention
likewise relates to processes for preparing these compounds and to
their use, in particular as inhibitors of matriptase.
Inventors: |
Sturzebecher; Jorg; (Erfurt,
DE) ; Steinmetzer; Torsten; (Jena, DE) ;
Schweinitz; Andrea; (Jena, DE) ; Sturzebecher;
Anne; (Weimar, DE) ; Uhland; Kerstin;
(Grafelfing, DE) |
Correspondence
Address: |
CLARK & ELBING LLP
101 FEDERAL STREET
BOSTON
MA
02110
US
|
Assignee: |
The Medicines Company (Leipzig)
GmbH
Leipzig
DE
|
Family ID: |
33440914 |
Appl. No.: |
12/829185 |
Filed: |
July 1, 2010 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10555821 |
Aug 8, 2006 |
7772251 |
|
|
PCT/EP2004/005291 |
May 17, 2004 |
|
|
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12829185 |
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Current U.S.
Class: |
424/474 ;
514/255.01; 514/330; 544/387; 546/226 |
Current CPC
Class: |
A61P 35/04 20180101;
C07D 307/79 20130101; A61P 43/00 20180101; C07D 205/04 20130101;
C07D 211/62 20130101; A61P 35/00 20180101; C07D 211/26 20130101;
C07D 295/185 20130101 |
Class at
Publication: |
424/474 ;
544/387; 514/255.01; 546/226; 514/330 |
International
Class: |
A61K 31/495 20060101
A61K031/495; C07D 295/00 20060101 C07D295/00; A61K 9/28 20060101
A61K009/28; C07D 211/06 20060101 C07D211/06; A61K 31/445 20060101
A61K031/445; A61P 35/00 20060101 A61P035/00 |
Foreign Application Data
Date |
Code |
Application Number |
May 16, 2003 |
DE |
103 22 191.3 |
Claims
1. A compound of the formula (I''), ##STR00147## or a salt of this
compound, wherein optionally at least one of the methylene groups
which are indexed with m or n in (I'') is substituted at least once
by a hydroxyl, a halogen, a pseudohalogen, or a COOR.sub.2' group,
and R.sub.2' is a linear, branched or cyclic alkyl group having 1
to 10 C atoms, and/or optionally at least one of the C atoms of the
methylene groups which are indexed with m or n in (I'') is replaced
by S, N, or O, and/or with retention of the imino group
C-terminally linked to the sulfonylated amino acid, optionally at
least one of the bonds forming the ring ##STR00148## in (I'') is a
double bond, and wherein (i) R.sub.1 is an optionally partially
hydrogenated aryl or heteroaryl group comprising at least one of
the atoms O, N, or S having 5 to 20 C atoms, or a linear, branched,
or cyclic alkyl group having 1 to 10 C atoms, where R.sub.1 is
optionally substituted by at least one halogen and/or pseudohalogen
group, and/or at least one linear, branched, or cyclic alkyl or
alkyloxy or alkylthio group having 1 to 10 C atoms, which is
optionally substituted at least once by a halogen, pseudohalogen,
hydroxyl, amino, cyano, amidino, guanidine, or carboxyl group,
where the carboxyl group is optionally esterified with a linear,
branched, or cyclic alkyl group having 1 to 10 C atoms, and where
the linear, branched, or cyclic alkyl group having 1 to 10 C atoms
optionally comprises at least one heteroatom selected from the
group consisting of O, N, and S, and/or at least one aryl or
heteroaryl group having 5 to 20 C atoms, where this aryl or
heteroaryl group is optionally substituted by at least one linear,
branched or cyclic alkyl group having 1 to 10 C atoms and/or at
least one COR.sub.2' and/or COOR.sub.2' group, where R.sub.2' is a
linear, branched, or cyclic alkyl group having 1 to 10 C atoms,
and/or at least one halogen group, and/or at least one
pseudohalogen group, and/or at least one alkoxy group or one
alkylthio group, where the alkyl radical has in each case 1 to 10 C
atoms, and/or at least one nitro group, and/or at least one
haloalkyl group having 1 to 10 C atoms, and where the aryl or
heteroaryl group is linked via an alkylene group having 1 to 3 C
atoms or via an oxygen atom or a sulfur atom to the radical
R.sub.1; at least one hydroxyl, amino, cyano, amidino, guanidino,
carboxyl, or carboxyalkyl group, where the amino group is
optionally acylated and/or where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms and/or the carboxyl group is
optionally esterified with a linear, branched, or cyclic alkyl
group having 1 to 10 C atoms or is amidated, and wherein said
amidino is optionally substituted by a hydroxyl or a
C.sub.1-C.sub.6-alkyloxycarbonyl group; (ii) R.sub.2 is an at least
monosubstituted aryl group having 1 to 10 C atoms, where optionally
at least one of these C atoms is replaced by S, N or O, at least
one substituent is a group according to R.sub.4, R.sub.2 is
optionally additionally substituted by a hydroxyl, COR.sub.2' or
COOR.sub.2' group, and R.sub.2' is a linear, branched or cyclic
alkyl group having 1 to 10 C atoms; (iii) R.sub.3 is a radical of
the following formula (II): A.sub.1-T-A.sub.2-R.sub.4 (II) where
A.sub.1 is either absent or an alkylene group having 1 to 4 C atoms
which is optionally substituted by at least one halogen and/or
pseudohalogen group, and/or at least one linear, branched, or
cyclic alkyl group having 1 to 10 C atoms, and/or at least one aryl
or one aralkyl group having 5 to 10 C atoms, and/or at least one
cycloalkyl group having 3 to 10 C atoms, and/or at least one
hydroxyl, cyano, alkyloxy or alkylthio having 1 to 10 C atoms,
carboxyl or carboxyalkyl group, where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms, and/or the carboxyl group
is optionally esterified with a linear, branched or cyclic alkyl
radical having 1 to 10 C atoms, or is amidated; T is either absent
or one of the following groups: ##STR00149## where R.sub.5 is
hydrogen or an alkyl group having 1 to 10 C atoms or an alkylene
group having 1 to 6 C atoms, which forms with A.sub.2 a ring
optionally comprising at least one heteroatom; A.sub.2 is a linear,
branched, or cyclic alkylene group having 1 to 10 C atoms or an
aryl-, heteroaryl-, or aralkylene group having 1 to 10 C atoms,
optionally comprising at least one heteroatom selected from the
group consisting of N, S, and O, which is optionally substituted by
at least one halogen and/or pseudohalogen group, and/or at least
one linear, branched or cyclic alkyl group having 1 to 10 C atoms,
and/or at least one aryl or one aralkyl group having 5 to 10 C
atoms, and/or at least one cycloalkyl group having 3 to 10 C atoms,
and/or at least one hydroxyl, cyano, alkyloxy or alkylthio group
having 1 to 10 C atoms, carboxyl or carboxyalkyl group, where the
alkyl group of the carboxyalkyl group has 1 to 10 C atoms, and/or
the carboxyl group is optionally esterified with a linear,
branched, or cyclic alkyl radical having 1 to 10 C atoms, or is
amidated; (iv) R.sub.4 is one of the following, optionally modified
basic groups: ##STR00150## where t=0, 1; R.sub.6 and R.sub.7 are
independently of one another hydrogen or an alkyl group having 1 to
6 C atoms or an alkylene group having 1 to 5 C atoms which forms a
ring with A.sub.2, or are a hydroxyl, amino, alkylamino, acyl, or
alkyloxycarbonyl group, where the alkylamino, acyl, and
alkyloxycarbonyl groups have independently of one another 1 to 6 C
atoms, and where R.sub.8 is hydrogen, an alkyl group having 1 to 3
C atoms, a hydroxyl group, or a C.sub.1-C.sub.6-alkyloxycarbonyl
group, or is an alkylene group having 1 to 3 C atoms which forms a
ring with R.sub.6; (v) Q is N; (vi) j=0, 1, or 2; (vii) k=0, 1, 2,
or 3; and (viii) m and n are independently of one another=0, 1, 2,
3, 4, or 5, where m+n=3, 4, 5; and where the compound of formula
(I'') is neither ##STR00151## with s=0, 1, 2, nor ##STR00152## with
s=0, 1.
2. The compound of claim 1, wherein T is present and T is one of
the groups defined as in claim 1, it being possible for the amide
and ester groups to be incorporated in both orientations.
3. The compound of claim 1, wherein j=0 and R.sub.1 is an at most
disubstituted aryl radical.
4. The compound of claim 1, wherein R.sub.1 is selected from the
group consisting of tert-butylphenyl, cyclohexylphenyl,
5,6,7,8-tetrahydronaphthyl, naphthyl, anthracyl, anthraquinoyl,
anthrahydroquinoyl, pyridyloxyphenyl, phenyloxypyridyl, and
pyridylalkylphenyl having a C.sub.1-C.sub.3-alkyl.
5. The compound of claim 1, wherein R.sub.2 is an at least
monosubstituted phenyl radical, thienyl radical, or pyridyl
radical.
6. The compound of claim 1, wherein k=1 and R.sub.2 is a phenyl
radical meta-substituted by an amidino group, and wherein the
3-amidinophenylalanine produced thereby has the L
configuration.
7. The compound of claim 1, wherein m=n=2.
8. The compound of claim 1, wherein A.sub.1 is absent and T is
##STR00153##
9. The compound of claim 1, wherein A.sub.2 is a methylene,
ethylene, or propylene group, and R.sub.4 is selected from
##STR00154##
10. The compound of claim 1, wherein said compound has a structure
according to: ##STR00155## wherein s is 1, 2, or 3.
11. The compound of claim 2, wherein m=n=2, and R.sub.3 is a
guanidinooxyalkyl radical or an aryl or heteroaryl radical, wherein
said aryl or heteroaryl radical is unsubstituted or substituted by
at least one halogen, at least one methoxy radical, or at least one
trifluoromethyl radical.
12. The compound of claim 1, wherein said compound is selected from
the group consisting of: ##STR00156## ##STR00157## ##STR00158##
##STR00159## ##STR00160## or a salt thereof, wherein Phe(3-Am) is
3-amidinophenylalanine.
13. A pharmaceutical composition comprising the compound of claim 1
and a pharmaceutically suitable excipient.
14. The pharmaceutical composition of claim 13, wherein said
composition is in the form of a tablet, a coated tablet, a capsule,
a pellet, a suppository, a solution, eye drops, nose drops, ear
drops, a syrup, an emulsion or suspension, a pessary, a stick, an
aerosol, a dusting powder, a paste, a cream, or an ointment.
15. A compound of the formula (I''), ##STR00161## or a salt of this
compound, wherein optionally at least one of the methylene groups
which are indexed with m or n in (I'') is substituted at least once
by a hydroxyl, a halogen, a pseudohalogen, or a COOR.sub.2' group,
and R.sub.2' is a linear, branched or cyclic alkyl group having 1
to 10 C atoms, and/or optionally at least one of the C atoms of the
methylene groups which are indexed with m or n in (I'') is replaced
by S, N, or O, and/or with retention of the imino group
C-terminally linked to the sulfonylated amino acid, optionally at
least one of the bonds forming the ring ##STR00162## in (1'') is a
double bond, and wherein (i) R.sub.1 is an optionally partially
hydrogenated aryl or heteroaryl group comprising at least one of
the atoms O, N, or S having 5 to 20 C atoms, or a linear, branched,
or cyclic alkyl group having 1 to 10 C atoms, where R.sub.I is
optionally substituted by at least one halogen and/or pseudohalogen
group, and/or at least one linear, branched, or cyclic alkyl or
alkyloxy or alkylthio group having 1 to 10 C atoms, which is
optionally substituted at least once by a halogen, pseudohalogen,
hydroxyl, amino, cyano, amidino, guanidine, or carboxyl group,
where the carboxyl group is optionally esterified with a linear,
branched, or cyclic alkyl group having 1 to 10 C atoms, and where
the linear, branched, or cyclic alkyl group having 1 to 10 C atoms
optionally comprises at least one heteroatom selected from the
group consisting of O, N, and S, and/or at least one aryl or
heteroaryl group having 5 to 20 C atoms, where this aryl or
heteroaryl group is optionally substituted by at least one linear,
branched or cyclic alkyl group having 1 to 10 C atoms and/or at
least one COR.sub.2' and/or COOR.sub.2' group, where R.sub.2' is a
linear, branched, or cyclic alkyl group having 1 to 10 C atoms,
and/or at least one halogen group, and/or at least one
pseudohalogen group, and/or at least one alkoxy group or one
alkylthio group, where the alkyl radical has in each case 1 to 10 C
atoms, and/or at least one nitro group, and/or at least one
haloalkyl group having 1 to 10 C atoms, and where the aryl or
heteroaryl group is linked via an alkylene group having 1 to 3 C
atoms or via an oxygen atom or a sulfur atom to the radical
R.sub.1; at least one hydroxyl, amino, cyano, amidino, guanidino,
carboxyl, or carboxyalkyl group, where the amino group is
optionally acylated and/or where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms and/or the carboxyl group is
optionally esterified with a linear, branched, or cyclic alkyl
group having 1 to 10 C atoms or is amidated, and wherein said
amidino is optionally substituted by a hydroxyl or a
C.sub.1-C.sub.6-alkyloxycarbonyl group; (ii) R.sub.2 is an at least
monosubstituted aryl group having 1 to 10 C atoms, where optionally
at least one of these C atoms is replaced by S, N or O, at least
one substituent is a group according to R.sub.4, R.sub.2 is
optionally additionally substituted by a hydroxyl, COR.sub.2' or
COOR.sub.2' group, and R.sub.2' is a linear, branched or cyclic
alkyl group having 1 to 10 C atoms; (iii) R.sub.3 is a radical of
the following formula (II): A.sub.1-T-A.sub.2-R.sub.4 (II) where
A.sub.1 is either absent or an alkylene group having 1 to 4 C atoms
which is optionally substituted by at least one halogen and/or
pseudohalogen group, and/or at least one linear, branched, or
cyclic alkyl group having 1 to 10 C atoms, and/or at least one aryl
or one aralkyl group having 5 to 10 C atoms, and/or at least one
cycloalkyl group having 3 to 10 C atoms, and/or at least one
hydroxyl, cyano, alkyloxy or alkylthio having 1 to 10 C atoms,
carboxyl or carboxyalkyl group, where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms, and/or the carboxyl group
is optionally esterified with a linear, branched or cyclic alkyl
radical having 1 to 10 C atoms, or is amidated; T is either absent
or one of the following groups: ##STR00163## where R.sub.5 is
hydrogen or an alkyl group having 1 to 10 C atoms or an alkylene
group having 1 to 6 C atoms, which forms with A.sub.2 a ring
optionally comprising at least one heteroatom; A.sub.2 is a linear,
branched, or cyclic alkylene group having 1 to 10 C atoms or an
aryl-, heteroaryl-, or aralkylene group having 1 to 10 C atoms,
optionally comprising at least one heteroatom selected from the
group consisting of N, S, and O, which is optionally substituted by
at least one halogen and/or pseudohalogen group, and/or at least
one linear, branched or cyclic alkyl group having 1 to 10 C atoms,
and/or at least one aryl or one aralkyl group having 5 to 10 C
atoms, and/or at least one cycloalkyl group having 3 to 10 C atoms,
and/or at least one hydroxyl, cyano, alkyloxy or alkylthio group
having 1 to 10 C atoms, carboxyl or carboxyalkyl group, where the
alkyl group of the carboxyalkyl group has 1 to 10 C atoms, and/or
the carboxyl group is optionally esterified with a linear,
branched, or cyclic alkyl radical having 1 to 10 C atoms, or is
amidated; (iv) R.sub.4 is one of the following, optionally modified
basic groups: ##STR00164## where t=0, 1; R.sub.6 and R.sub.7 are
independently of one another hydrogen or an alkyl group having 1 to
6 C atoms or an alkylene group having 1 to 5 C atoms which forms a
ring with A.sub.2, or are a hydroxyl, amino, alkylamino, acyl, or
alkyloxycarbonyl group, where the alkylamino, acyl, and
alkyloxycarbonyl groups have independently of one another 1 to 6 C
atoms, and where R.sub.8 is hydrogen, an alkyl group having 1 to 3
C atoms, a hydroxyl group, or a C.sub.1-C.sub.6-alkyloxycarbonyl
group, or is an alkylene group having 1 to 3 C atoms which forms a
ring with R.sub.6; (v) Q is a CH group; (vi) j=0, 1, or 2; (vii)
k=0, 1, 2, or 3; and (viii) m=n=1.
16. The compound of claim 15, wherein R.sub.1 is naphthyl.
17. The compound of claim 15, wherein R.sub.2 is an at least
monosubstituted phenyl radical.
18. The compound of claim 15, wherein k=1 and R.sub.2 is a phenyl
radical meta-substituted by an amidino group, and wherein the
3-amidinophenylalanine produced thereby has the L
configuration.
19. The compound of claim 15, wherein said compound is ##STR00165##
or a salt thereof, wherein Phe(3-Am) is 3-amidinophenylalanine.
20. A pharmaceutical composition comprising the compound of claim
15 and a pharmaceutically suitable excipient.
21. The pharmaceutical composition of claim 20, wherein said
composition is in the form of a tablet, a coated tablet, a capsule,
a pellet, a suppository, a solution, eye drops, nose drops, ear
drops, a syrup, an emulsion or suspension, a pessary, a stick, an
aerosol, a dusting powder, a paste, a cream, or an ointment.
22. A compound having a structure according to the following
formula, ##STR00166## or a salt of this compound, wherein Phe(3-Am)
is 3-amidinophenylalanine and Gly is glycine, optionally at least
one of the methylene groups which are indexed with m or n is
substituted at least once by a hydroxyl, a halogen, a
pseudohalogen, or a COOR.sub.2' group, and R.sub.2' is a linear,
branched or cyclic alkyl group having 1 to 10 C atoms, and/or
optionally at least one of the C atoms of the methylene groups
which are indexed with m or n in (I'') is replaced by S, N, or O,
and/or with retention of the imino group C-terminally linked to the
sulfonylated amino acid, optionally at least one of the bonds
forming the ring ##STR00167## in (I'') is a double bond, and
wherein (i) R.sub.1 is an optionally partially hydrogenated aryl or
heteroaryl group comprising at least one of the atoms O, N, or S
having 5 to 20 C atoms, or a linear, branched, or cyclic alkyl
group having 1 to 10 C atoms, where R.sub.1 is optionally
substituted by at least one halogen and/or pseudohalogen group,
and/or at least one linear, branched, or cyclic alkyl or alkyloxy
or alkylthio group having 1 to 10 C atoms, which is optionally
substituted at least once by a halogen, pseudohalogen, hydroxyl,
amino, cyano, amidino, guanidine, or carboxyl group, where the
carboxyl group is optionally esterified with a linear, branched, or
cyclic alkyl group having 1 to 10 C atoms, and where the linear,
branched, or cyclic alkyl group having 1 to 10 C atoms optionally
comprises at least one heteroatom selected from the group
consisting of O, N, and S, and/or at least one aryl or heteroaryl
group having 5 to 20 C atoms, where this aryl or heteroaryl group
is optionally substituted by at least one linear, branched or
cyclic alkyl group having 1 to 10 C atoms and/or at least one
COR.sub.2' and/or COOR.sub.2' group, where R.sub.2' is a linear,
branched, or cyclic alkyl group having 1 to 10 C atoms, and/or at
least one halogen group, and/or at least one pseudohalogen group,
and/or at least one alkoxy group or one alkylthio group, where the
alkyl radical has in each case 1 to 10 C atoms, and/or at least one
nitro group, and/or at least one haloalkyl group having 1 to 10 C
atoms, and where the aryl or heteroaryl group is linked via an
alkylene group having 1 to 3 C atoms or via an oxygen atom or a
sulfur atom to the radical R.sub.1; at least one hydroxyl, amino,
cyano, amidino, guanidino, carboxyl, or carboxyalkyl group, where
the amino group is optionally acylated and/or where the alkyl group
of the carboxyalkyl group has 1 to 10 C atoms and/or the carboxyl
group is optionally esterified with a linear, branched, or cyclic
alkyl group having 1 to 10 C atoms or is amidated, and wherein said
amidino is optionally substituted by a hydroxyl or a
C.sub.1-C.sub.6-alkyloxycarbonyl group; (ii) R.sub.3 is a radical
of the following formula (II): A.sub.1-T-A.sub.2-R.sub.4 (II) where
A.sub.1 is either absent or an alkylene group having 1 to 4 C atoms
which is optionally substituted by at least one halogen and/or
pseudohalogen group, and/or at least one linear, branched, or
cyclic alkyl group having 1 to 10 C atoms, and/or at least one aryl
or one aralkyl group having 5 to 10 C atoms, and/or at least one
cycloalkyl group having 3 to 10 C atoms, and/or at least one
hydroxyl, cyano, alkyloxy or alkylthio having 1 to 10 C atoms,
carboxyl or carboxyalkyl group, where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms, and/or the carboxyl group
is optionally esterified with a linear, branched or cyclic alkyl
radical having 1 to 10 C atoms, or is amidated; T is either absent
or one of the following groups: ##STR00168## where R.sub.5 is
hydrogen or an alkyl group having 1 to 10 C atoms or an alkylene
group having 1 to 6 C atoms, which forms with A.sub.2 a ring
optionally comprising at least one heteroatom; A.sub.2 is a linear,
branched, or cyclic alkylene group having 1 to 10 C atoms or an
aryl-, heteroaryl-, or aralkylene group having 1 to 10 C atoms,
optionally comprising at least one heteroatom selected from the
group consisting of N, S, and O, which is optionally substituted by
at least one halogen and/or pseudohalogen group, and/or at least
one linear, branched or cyclic alkyl group having 1 to 10 C atoms,
and/or at least one aryl or one aralkyl group having 5 to 10 C
atoms, and/or at least one cycloalkyl group having 3 to 10 C atoms,
and/or at least one hydroxyl, cyano, alkyloxy or alkylthio group
having 1 to 10 C atoms, carboxyl or carboxyalkyl group, where the
alkyl group of the carboxyalkyl group has 1 to 10 C atoms, and/or
the carboxyl group is optionally esterified with a linear,
branched, or cyclic alkyl radical having 1 to 10 C atoms, or is
amidated; (iii) R.sub.4 is one of the following, optionally
modified basic groups: ##STR00169## where t=0, 1; R.sub.6 and
R.sub.7 are independently of one another hydrogen or an alkyl group
having 1 to 6 C atoms or an alkylene group having 1 to 5 C atoms
which forms a ring with A.sub.2, or are a hydroxyl, amino,
alkylamino, acyl, or alkyloxycarbonyl group, where the alkylamino,
acyl, and alkyloxycarbonyl groups have independently of one another
1 to 6 C atoms, and where R.sub.8 is hydrogen, an alkyl group
having 1 to 3 C atoms, a hydroxyl group, or a
C.sub.1-C.sub.6-alkyloxycarbonyl group, or is an alkylene group
having 1 to 3 C atoms which forms a ring with R.sub.6; (iv) Q is N
or CH; (v) j=0, 1, or 2; and (vi) m and n are independently of one
another=0, 1, 2, 3, 4, or 5, where m+n=3, 4, 5.
23. The compound of claim 22, wherein k=1 and R.sub.2 is a phenyl
radical meta-substituted by an amidino group, and wherein the
3-amidinophenylalanine produced thereby has the L
configuration.
24. The compound of claim 22, wherein R.sub.1 is naphthyl or
anthracyl.
25. The compound of claim 22, wherein m=n=2.
26. The compound of claim 22, wherein said compound is selected
from the group consisting of: ##STR00170## or a salt thereof.
27. A pharmaceutical composition comprising the compound of claim
22 and a pharmaceutically suitable excipient.
28. The pharmaceutical composition of claim 27, wherein said
composition is in the form of a tablet, a coated tablet, a capsule,
a pellet, a suppository, a solution, eye drops, nose drops, ear
drops, a syrup, an emulsion or suspension, a pessary, a stick, an
aerosol, a dusting powder, a paste, a cream, or an ointment.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation of U.S. application Ser.
No. 10/555,821, filed Nov. 7, 2005, which is the U.S. National
Stage of International Application No. PCT/EP2004/005291, filed May
17, 2004, which claims benefit of German Application No. 103 22
191.3, filed May 16, 2003, each of which is hereby incorporated by
reference.
[0002] The present invention relates to N-sulfonylated amino acid
derivatives, where an aryl radical is linked via the sulfonyl group
N-terminally to the amino acid and a radical which comprises at
least one imino group and at least one further basic group which
represents an optionally modified amino, amidino or guanidino group
is linked C-terminally via the carbonyl group. The invention
likewise relates to processes for preparing these compounds and to
their use, preferably as medicaments and in this connection in
particular as inhibitors of matriptase.
[0003] Proteases regulate numerous physiological processes which
enable or stimulate the growth and metastasis of tumor cells. This
relates in particular to the proteolytic degradation of the
extracellular matrix proteins which surround the tumor cells, the
degradation making it possible for the tumor cells which have
migrated from tumors to invade adjoining tissues and the lymphatic
and blood systems. Proteases are also involved in the activation of
growth factors which, for example, stimulate the proliferation of
tumor cells or angiogenesis and thus make tumor growth possible.
These proteolytic enzymes include various matrix metalloproteases,
membrane-bound metalloproteases, lysosomal cysteine proteases and a
large number of serine proteases such as, for example, urokinase,
plasmin, elastase, thrombin or cathepsin G, and also the type II
transmembrane serine protease matriptase or MT-SP1 (Hooper et al.,
J. Biol. Chem. 276, 857-860, 2001).
[0004] There have been numerous attempts to inhibit the growth and
metastasis of tumors through the use of protease inhibitors, but
experiments with inhibitors of matrix metalloproteases have shown
hardly any effect in clinical studies (Coussens et al., Science
295, 2387-2392, 2002). Initial clinical investigations with
inhibitors of urokinase have also now been initiated, but no
results on their efficacy are known as yet.
[0005] Matriptase is a trypsin-like serine protease which was
originally isolated from breast cancer cells and preferentially
cleaves C-terminally peptide linkages of the basic amino acid
arginine (Shi et al., Cancer Res. 53, 1409-1415, 1993; Lin et al.,
J. Biol. Chem. 272, 9147-9152, 1997).
[0006] In 1998, the matriptase gene was cloned as putative tumor
suppressor by a subtractive hybridization method in which healthy
and carcinogenic intestinal tissue were used (Zhang et al.
Cytogenet. Cell Genet. 83, 56-57, 1998).
[0007] Matriptase and MT-SP1 (abbreviation for "membrane-type
serine protease 1) (Takeuchi et al., Proc. Natl. Acad. Sci. USA 96,
11054-11061, 1999; Takeuchi et al., J. Biol. Chem. 275,
26333-26342, 2000) have the same cDNA. However, owing to
alternative splicings, the protein sequence of matriptase is
truncated at the N terminus by 172 amino acids compared with
MT-SP1. The gene for MT-SP1 was isolated from an epithelial cell
line of a prostate tumor.
[0008] In the context of the present invention, the term
"matriptase" refers to every trypsin-like protein having a
molecular weight of from 72 to 92 kDa which is derived from the
gene sequences having the entry number AF118224, AF133086,
BANKIt257050 and NM021978 (GenBank/EBI Data Bank) and has been
described previously (Takeuchi et al., Proc. Natl. Acad. Sci. USA
96, 11054-11061, 1999; Lin et al., J. Biol. Chem. 274, 18231-18236,
1999). The term "matriptase" refers in particular to both the
single-chain and two-chain forms of the protein. The zymogenic,
inactive form of matriptase is a single-chain protein. The
two-chain form of matriptase is its active form having catalytic
activity. In the context of the present invention therefore the
term "matriptase" refers in particular to the original matriptase
described hereinbefore, as well as MT-SP1.
[0009] The enzyme is tethered by means of a transmembrane domain in
the membrane of epithelial or cancer cells, with the serine
protease domain of matriptase being located on the cell surface and
thus in the extracellular space (Hooper et al., J. Biol. Chem. 276,
857-860, 2001). It has therefore been supposed that matriptase
might be involved in the proliferation and metastasis of breast
cancer cells through degradation and transformation of
extracellular matrix proteins, in the activation of latent growth
factors and other proteolytic cascades (Shi et al., Cancer Res. 53,
1409-1415, 1993; Lin et al., J. Biol. Chem. 272, 9147-9152,
1997).
[0010] It has also been possible to isolate matriptase from human
milk but, in this case, it was in the form almost entirely of a
proteolytically inactive complex with the endogenous inhibitor
HAI-1 (Lin et al., J. Biol. Chem. 274, 18237-18242, 1999). In
contrast thereto, matriptase from breast cancer cells is very
substantially in an uncomplexed and thus catalytically active form,
and only a small part is bound to HAI-1.
[0011] The first potential substrates of matriptase have now been
described. Matriptase is able to activate hepatocyte growth factor
(HGF) which is also referred to as scattering factor (Lee et al.,
J. Biol. Chem. 275, 36720-36725, 2000). Pro-HGF is secreted by
cancer cells or stromal cells in inactive form as single-chain
protein and is converted in the extracellular space by cleavage
C-terminally of the Arg495 into the active two-chain form (HGF).
Binding of HGF results in the cell surface receptor c-Met being
activated and phosphorylated on particular tyrosine residues. It
has recently been demonstrated that there is a close correlation
between a high expression of c-Met, matriptase and HAI-1 and a poor
prognosis for breast cancer patients (Kang et al., Cancer Res. 63,
1101-1105, 2003). It has also been possible to show in the
investigation of ovarian tumors that matriptase is expressed to an
increased extent. It was moreover found that matriptase is
expressed almost exclusively without HAI-1 especially in advanced
tumors of type III/IV, in contrast to tumors of type VII. This
indicates that in the advanced stage there is an imbalance between
matriptase and the inhibitor HAI-1, thus enhancing the proteolytic
activity of matriptase and thereby probably also the invasive
potential of the tumor cells (Oberst et al., Clin. Cancer Res. 8,
1101-1107, 2002).
[0012] Besides activation of Pro-HGF, matriptase is possibly also
involved in activation of the plasminogen activator cascade. Thus,
matriptase is able to activate pro-urokinase to urokinase (uPA)
(Lee et al., J. Biol. Chem. 275, 36720-36725, 2000; Takeuchi et
al., J. Biol. Chem. 275, 26333-26342, 2000) which converts
plasminogen into plasmin. Plasmin is the principal activator of
matrix metalloproteases which are involved in the degradation of
extracellular matrix proteins, which is also regarded as a
precondition for metastasis.
[0013] Ihara et al. (J. Biol. Chem. 277, 16960-16967, 2002) were
able to show that stomach cancer cells show enhanced expression of
.beta.1-6-N-acetylglucosaminyltransferase (GnT-V) which is able to
glycosylate matriptase. This modification makes matriptase more
stable to degradation and is present in proteolytically active form
in increased concentration.
[0014] It can be inferred from these findings that development of
an effective and selective inhibitor of matriptase will make it
possible to inhibit the proliferation of tumors and their
metastasis. Although it has now been possible also to elucidate the
X-ray structure of the catalytic domain of matriptase complexed
with benzamidine and with the bovine pancreatic trypsin inhibitor,
to date only a few inhibitors of matriptase are known (Friedrich et
al., J. Biol. Chem. 277, 2160-2168, 2002).
[0015] Enyedy et al. (J. Med. Chem. 44, 1349-1355, 2001) described
bis-benzamidines where the most effective inhibitor has a K.sub.i
of 0.19 .mu.M.
[0016] WO 01/97794 describes a method for inhibiting progression of
carcinoma in which matriptase plays a part. The compounds employed
in this case comprise two groups able to carry a positive charge at
a physiological pH. These groups are moreover connected together by
a chemical structural unit which has a length of from 5 to 30,
preferably 15 to 24 Angstrom. Positively charged groups which are
disclosed are the amino, amidino, guanidino groups and a cyclic
group derived from the amidino or guanidino group. Amino acid
derivatives are not mentioned in WO 01/97794 and accordingly in
particular no sulfonylated amino acid derivatives. On the contrary,
the compounds explicitly disclosed in WO 01/97794 differ
fundamentally from the compounds claimed in the context of the
present invention.
[0017] Tripeptide aldehydes having C-terminal arginal are published
in WO 02/20475. After preincubation of matriptase with these
inhibitors for a period of 30 minutes, IC.sub.50 values of less
than 100 nM were determined for the most effective compounds,
although exact inhibitory constants were not stated. It is believed
that these inhibitors bind covalently to matriptase to form a
hemiacetal. In the case of the development of inhibitors for other
trypsin-like serine proteases such as, for example, thrombin or
factor Xa, however, it has been shown that such transition
state-analogous peptide aldehydes are unsuitable for developing an
active substance which can be employed medicinally.
[0018] Long et al. (Bioorganic. Med. Chem. Lett. 11, 2515-2519,
2001) have described the synthesis of a bicyclic peptide of 14
amino acids which was originally isolated from sunflower seed. The
peptide inhibits matriptase with an inhibitory constant of 0.92 nM,
but it must be assumed that these structures are unsuitable for
developing an active substance.
[0019] One of the objects on which the present invention is based
was therefore to provide an active substance which is also suitable
for therapeutic applications and which inhibits matriptase with
high activity and specificity.
[0020] Accordingly, the present invention relates to a compound of
the formula (I)
##STR00001##
or a salt or a prodrug of this compound, where [0021] (a) X.sub.1
and X.sub.2 are independently of one another hydrogen or an alkyl
radical having 1, 2 or 3 C atoms, and at least one of the radicals
X.sub.1 and X.sub.2 is a radical of the structure (I')
##STR00002##
[0021] where [0022] optionally at least one of the methylene groups
which are indexed with m or n in (I') is substituted at least once
by a hydroxyl, a halogen, a pseudohalogen or a COOR.sub.2' group,
and R.sub.2' is a linear, branched or cyclic alkyl group having 1
to 10 C atoms, and/or [0023] optionally at least one of the C atoms
of the methylene groups which are indexed with m or n in (I') is
replaced by S, N or O, and/or [0024] optionally at least one of the
bonds forming the ring
[0024] ##STR00003## [0025] in (I') is a double bond, or where
[0026] (b) X.sub.1 and X.sub.2 are bridged to form a ring in such a
way that the compound (I) has the structure (I'')
##STR00004##
[0026] where [0027] optionally at least one of the methylene groups
which are indexed with m or n in (I'') is substituted at least once
by a hydroxyl, a halogen, a pseudohalogen or a COOR.sub.2' group,
and R.sub.2' is a linear, branched or cyclic alkyl group having 1
to 10 C atoms, and/or [0028] optionally at least one of the C atoms
of the methylene groups which are indexed with m or n in (I'') is
replaced by S, N or O, and/or [0029] with retention of the imino
group C-terminally linked to the sulfonylated amino acid,
optionally at least one of the bonds forming the ring
[0029] ##STR00005## [0030] in (I'') is a double bond, and where
[0031] (i) R.sub.I is an optionally partially hydrogenated aryl or
heteroaryl group comprising at least one of the atoms O, N or S,
having 5 to 20 C atoms, or a linear, branched or cyclic alkyl group
having 1 to 10 C atoms, where R.sub.1 is optionally substituted by
[0032] at least one halogen and/or pseudohalogen group, and/or
[0033] at least one linear, branched or cyclic alkyl or alkyloxy or
alkylthio group having 1 to 10 C atoms, which is optionally
substituted at least once by a halogen, pseudohalogen, hydroxyl,
amino, cyano, amidino, guanidino or carboxyl group, where the
carboxyl group is optionally esterified with a linear, branched or
cyclic alkyl group having 1 to 10 C atoms, and where the linear,
branched or cyclic alkyl group having 1 to 10 C atoms optionally
comprises at least one heteroatom selected from the group
consisting of O, N and S, and/or [0034] at least one aryl or
heteroaryl group having 5 to 20 C atoms, where this aryl or
heteroaryl group is optionally substituted by [0035] at least one
linear, branched or cyclic alkyl group having 1 to 10 C atoms
and/or [0036] at least one COR.sub.2' and/or COOR.sub.2' group,
where R.sub.2' is a linear, branched or cyclic alkyl group having 1
to 10 C atoms, and/or [0037] at least one halogen group and/or
[0038] at least one pseudohalogen group and/or at least one alkoxy
group or one alkylthio group, where the alkyl radical has in each
case 1 to 10 C atoms, and/or [0039] at least one nitro group and/or
[0040] at least one haloalkyl group having 1 to 10 C atoms, [0041]
and where the aryl or heteroaryl group is linked via an alkylene
group having 1 to 3 C atoms or via an oxygen atom or a sulfur atom
to the radical R.sub.1; [0042] at least one hydroxyl, amino, cyano,
amidino, guanidino, carboxyl or carboxyalkyl group, where the amino
group is optionally acylated and/or where the alkyl group of the
carboxyalkyl group has 1 to 10 C atoms and/or the carboxyl group is
optionally esterified with a linear, branched or cyclic alkyl group
having 1 to 10 C atoms or is amidated; [0043] (ii) R.sub.2 is an at
least monosubstituted aryl group having 1 to 10 C atoms, where
[0044] optionally at least one of these C atoms is replaced by S, N
or O, [0045] at least one substituent is a group according to
R.sub.4, [0046] R.sub.2 is optionally additionally substituted by a
hydroxyl, COR.sub.2' or COOR.sub.2' group, and R.sub.2' is a
linear, branched or cyclic alkyl group having 1 to 10 C atoms;
[0047] (iii) R.sub.3 is a radical of the following formula
(II):
[0047] A.sub.1-T-A.sub.2-R.sub.4 (II) [0048] where [0049] A.sub.1
is either absent or an alkylene group having 1 to 4 C atoms which
is optionally substituted by [0050] at least one halogen and/or
pseudohalogen group and/or [0051] at least one linear, branched or
cyclic alkyl group having 1 to 10 C atoms and/or [0052] at least
one aryl or one aralkyl group having 5 to 10 C atoms and/or [0053]
at least one cycloalkyl group having 3 to 10 C atoms and/or [0054]
at least one hydroxyl, cyano, alkyloxy or alkylthio having 1 to 10
C atoms, carboxyl or carboxyalkyl group, where the alkyl group of
the carboxyalkyl group has 1 to 10 C atoms, and/or the carboxyl
group is optionally esterified with a linear, branched or cyclic
alkyl radical having 1 to 10 C atoms, or is amidated; [0055] T is
either absent or one of the following groups:
[0055] ##STR00006## [0056] where R.sub.5 is hydrogen or an alkyl
group having 1 to 10 C atoms or an alkylene group having 1 to 6 C
atoms, which forms with A.sub.2 a ring optionally comprising at
least one heteroatom; [0057] where the amide or ester linkage can
be incorporated in both orientations, that is to say the following
orientations are also included:
[0057] ##STR00007## [0058] A.sub.2 is a linear, branched or cyclic
alkylene group having 1 to 10 C atoms or an aryl-, heteroaryl- or
aralkylene group having 1 to 10 C atoms, optionally comprising at
least one heteroatom selected from the group consisting of N, S and
O, which is optionally substituted by [0059] at least one halogen
and/or pseudohalogen group and/or [0060] at least one linear,
branched or cyclic alkyl group having 1 to 10 C atoms and/or [0061]
at least one aryl or one aralkyl group having 5 to 10 C atoms
and/or [0062] at least one cycloalkyl group having 3 to 10 C atoms
and/or [0063] at least one hydroxyl, cyano, alkyloxy or alkylthio
group having 1 to 10 C atoms, carboxyl or carboxyalkyl group, where
the alkyl group of the carboxyalkyl group has 1 to 10 C atoms,
and/or the carboxyl group is optionally esterified with a linear,
branched or cyclic alkyl radical having 1 to 10 C atoms, or is
amidated; [0064] (iv) R.sub.4 is one of the following, optionally
modified basic groups:
[0064] ##STR00008## [0065] where t=0, 1; R.sub.6 and R.sub.7 are
independently of one another hydrogen or an alkyl group having 1 to
6 C atoms or an alkylene group having 1 to 5 C atoms which forms a
ring with A.sub.2, or are a hydroxyl, amino, alkylamino, acyl or
alkyloxycarbonyl group, where the alkylamino, acyl and
alkyloxycarbonyl groups have independently of one another 1 to 6 C
atoms, and where R.sub.8 is hydrogen or an alkyl group having 1 to
3 C atoms, or is an alkylene group having 1 to 3 C atoms which
forms a ring with R.sub.6; [0066] (v) Q is either a CH group or N;
[0067] (vi) j=0, 1, 2; [0068] k=0, 1, 2, 3; [0069] m, n are
independently of one another=0, 1, 2, 3, 4, 5, where m+n=3, 4, 5;
and where the compound of formula (I) is neither
##STR00009##
[0069] with s=0, 1, 2 nor
##STR00010##
with s=0, 1.
[0070] If the abovementioned compounds of the invention are in the
form of a salt, then salts with mineral acids and/or salts with
suitable organic acids are preferred. It is preferred inter alia
for the compounds of the invention to be in the form of
hydrochlorides or else of sulfates. Examples of suitable organic
acids are formic acid, acetic acid, methylsulfonic acid, succinic
acid, malic acid and trifluoroacetic acid. It is preferred inter
alia for salts of the compounds of the invention with suitable
organic acids to be acetates.
[0071] In a preferred embodiment, the compound of the invention has
a structure in which at least one of the radicals X.sub.1 and
X.sub.2 has a structure (I'). Within the scope of this embodiment
of the non-cyclic imines which are linked to the carbonyl group of
the amino acid in the center of (I), preferred compounds are those
in which exactly one of the radicals X.sub.1 and X.sub.2 has a
structure (I'). The non-cyclic radical in this case is particularly
preferably hydrogen, methyl, ethyl or n-propyl, further preferably
methyl or ethyl and particularly preferably methyl. In relation to
the substituted cyclic radical, preferred embodiments are those in
which m+n is equal to 3 or 4. The radical R.sub.4 which is
obligatorily present in the radical R.sub.3 can generally be chosen
as desired within the scope of the definitions made above. Very
particularly preferred radicals R.sub.4 are those which are
unmodified and which are selected from the group consisting of
##STR00011##
[0072] The indices m and n of the ring in (I') can be chosen so
that the radical R.sub.3 can in principle be located in the 2, 3
or, depending on the ring size, also in the 4 position relative to
the nitrogen linked to the carbonyl group of the central amino
acid. For example, the 3 or 4 position is preferred, and when m=n=2
the 4 position is particularly preferred.
[0073] Within the scope of the embodiment of the non-cyclic imines
linked to the carbonyl group of the amino acid in the center of
(I), radicals R.sub.3 which are further preferred are those in
which A.sub.2 is absent. Radicals R.sub.3 in which the functional
group T is either absent or is selected from
##STR00012##
are further preferred. Very particularly preferred embodiments are
those in which T is absent. In relation to the group A.sub.2,
preferred within the scope of the non-cyclic imines linked to the
carbonyl group of the amino acid in the center of (I) are alkylene
groups having 1, 2, 3, 4 or 5 C atoms, in particular the methylene,
ethylene, n-propylene, isopropylene, butylene and pentylene
group.
[0074] In the case where A.sub.2 is an aryl-, heteroaryl- or
aralkylene group, mention should be made for example of groups of
the structures
##STR00013##
where v and w can independently of one another be 0, 1 or 2, and
the two alkylene groups may also be positioned in the 1,2 or 1,3
position relative to one another. The 1,4 position is preferred,
for example. Both the aryl radical and at least one of the two
alkylene groups may be suitably substituted as defined above. If
A.sub.2 includes a heteroaryl group, this preferably has 1 to 3
heteroatoms.
[0075] The ring of (I') can in principle comprise at least one
heteroatom, those to be mentioned in this connection as preferred
being oxygen, nitrogen or sulfur. If the heteroatom is, for
example, nitrogen, this nitrogen may have as further radical for
example hydrogen or a linear, branched or cyclic alkyl group having
1 to 10 C atoms, or form a double bond with an adjacent C atom of
the ring. In a particularly preferred embodiment, none of the
methylene groups in (I') is substituted by a heteroatom.
[0076] If the ring of (I') is additionally substituted, preferred
additional substituents are, inter alia, carboxyalkyl groups of the
general structure --COOR.sub.2', it being further preferred for
R.sub.2' to be an alkyl group having 1, 2 or 3 C atoms and
particularly preferably a methyl or ethyl group.
[0077] In a preferred embodiment, the compounds of the invention
according to (b) have a cyclic imine linked to the carbonyl group
of the amino acid in the center of (I), and thus have a structure
(I'').
[0078] Accordingly, the present invention also relates to a
compound as described above, where this compound has the structure
(I'')
##STR00014##
[0079] The ring in (I'') has in this connection preferably 5, 6 or
7 ring atoms. It is accordingly conceivable for the radical R.sub.3
to be located in the 2, 3 or 4 position relative to the imine
nitrogen linked to the carbonyl group of the central amino acid.
Embodiments which are preferred inter alia are those in which the
ring of the cyclic amine has 5 or 6 ring atoms. A six-membered ring
is particularly preferred. In this particularly preferred
six-membered ring, the indices m and n can be chosen as desired.
Examples of possible combinations are for instance m=0 and n=4, m=1
and n=3, m=2 and n=2, m=3 and n=1, m=4 and n=0. In a very
particularly preferred embodiment of the compounds of the
invention, m=n=2. The group Q in the structure (I) is thus very
particularly preferably located in the 4 position relative to the
imine nitrogen linked to the carbonyl group of the central amino
acid.
[0080] Accordingly, the present invention also relates to a
compound as described above, which is characterized in that
m=n=2.
[0081] In the context of the present invention, the ring of the
cyclic imine may be suitably substituted. Among the substituents
described above, the COOR.sub.2' group is, inter alia, particularly
preferred, where R.sub.2' is in turn preferably an alkyl group
having 1, 2, 3, 4, 5 or 6 C atoms and particularly preferably a
methyl group or an ethyl group. If the ring is substituted by a
halogen, then fluorine, chlorine and bromine are particularly
preferred. The hydroxyl group is likewise a suitable substituent.
The ring may further be substituted by two or more identical or
different substituents, especially those mentioned as
preferred.
[0082] At least one of the methylene groups which are indexed with
m or n in the ring of (I'') may be substituted by a heteroatom,
preferably oxygen, nitrogen or sulfur. If the heteroatom is, for
example, nitrogen, this nitrogen may have as further radical for
example hydrogen or a linear, branched or cyclic alkyl group having
1 to 10 C atoms, or form a double bond with an adjacent C atom of
the ring. The ring in (I'') may very generally comprise at least
one double bond, which can be formed either between two
heteroatoms, two C atoms or one C atom and one heteroatom. In the
context of the present invention, accordingly, rings having at
least one double bond and in which the imine nitrogen linked to the
carbonyl group of the central amino acid is retained, that is to
say is linked by three single bonds to the adjacent atoms, are
described.
[0083] In a particularly preferred embodiment, none of the
methylene groups in (I'') is substituted by a heteroatom. In a
further especially preferred embodiment, the methylene groups from
which the ring is constructed are not substituted.
[0084] Accordingly, the present invention also relates to a
compound as described above, where the ring formed by X.sub.1 and
X.sub.2 has the following structure
##STR00015##
and m, n are independently of one another=0, 1, 2, 3, 4, 5, where
m+n=3, 4, 5. Further preferred in this connection are compounds in
which m and n are chosen so that a six-membered ring is formed.
Accordingly, in this case Q can, as described above, be located in
the 2, 3 or 4 position relative to the imine nitrogen, with the 4
position being particularly preferred. Particular preference is
further given to m=n=2.
[0085] In the context of the present invention, Q is a CH group or
nitrogen. The present invention likewise also describes compounds
in which this CH group bears a suitable substituent instead of
hydrogen. Substituents to be mentioned inter alia as preferred are
those with which the methylene groups of the ring can also be
substituted.
[0086] Accordingly, the present invention also describes a compound
as described above, which is characterized in that Q is a nitrogen
atom.
[0087] Within the scope of this embodiment, the group A.sub.1
present where appropriate in the radical R.sub.3 can be chosen as
desired within the scope of the definitions described above. A
particularly preferred group A.sub.1 is a methylene or ethylene or
propylene group, especially a methylene or ethylene group and is
optionally substituted, where as substituents inter alia
particularly preferred are alkyl radicals having 1, 2 or 3 C atoms,
especially methyl and ethyl, particularly preferably methyl, and/or
halogen, especially fluorine, chlorine and bromine, and/or
cycloalkyl radicals having preferably 5, 6 or 7 C atoms and/or
carboxyalkyl radicals, where the alkyl radical is preferably methyl
or ethyl, and the carboxyl group is preferably esterified with a
methyl or ethyl group, or is amidated.
[0088] In a very particularly preferred embodiment of the compounds
of the invention in which Q is a nitrogen atom, the group A.sub.1
is absent.
[0089] Accordingly, the present invention also describes a compound
as described above, which is characterized in that Q is a nitrogen
atom and A.sub.1 is absent.
[0090] Concerning the functional group T which is optionally
present in the radical R.sub.3 in the context of the present
invention, it is possible to choose all of the groups described
above. Especially in the case where Q is a nitrogen atom, preferred
functional groups T are groups of the structures
##STR00016##
[0091] Structures particularly preferred for T groups are
##STR00017##
where it is further preferred for R.sub.5 to be hydrogen. In
further particularly preferred embodiments, T is a group of the
structures
##STR00018##
where T is very particularly preferably a functional group of the
structure
##STR00019##
[0092] In this case it is possible for the amide or ester groups to
be incorporated in both orientations.
[0093] Accordingly the present invention also describes a compound
as described above, which is characterized in that Q is a nitrogen
atom and T is a functional group of the structure
##STR00020##
[0094] In relation to the group A.sub.2 which is obligatorily
present in the radical R.sub.3, there are no restrictions within
the definitions described above.
[0095] Especially in the case where Q is a nitrogen atom and
further especially where T is a carbonyl group, alkylene groups
having 1 to 6 C atoms are preferred for the group A.sub.2, and the
methylene, ethylene, propylene, butylene groups are particularly
preferred, and the methylene, ethylene and propylene groups are
especially preferred.
[0096] In the case where A.sub.2 is an aryl-, heteroaryl- or
aralkylene group, groups of the structures
##STR00021##
should be mentioned by way of example, where v and w can
independently of one another be 0, 1 or 2, and the two alkylene
groups can also be positioned in the 1,2 or 1,3 position relative
to one another. The 1,4 position is preferred for example. Both the
aryl radical and at least one of the two alkylene groups may be
suitably substituted. If A.sub.2 includes a heteroaryl group, this
preferably has 1 to 3 heteroatoms.
[0097] The group A.sub.2 can also be suitably substituted as
defined above. Substituents in this connection are particularly
halogens, preferably fluorine, chlorine or bromine, and/or alkyl
radicals having preferably 1, 2 or 3 C atoms such as methyl, ethyl,
n-propyl or isopropyl, especially preferably methyl and ethyl,
and/or cycloalkyl radicals preferably having 5, 6 or 7 C atoms
and/or carboxyalkyl radicals, where the alkyl radical is preferably
methyl or ethyl, and the carboxyl group is preferably esterified
with a methyl or ethyl group, or is amidated.
[0098] In a very particularly preferred embodiment of the present
invention, the group A.sub.2 is unsubstituted. Accordingly, the
present invention describes a compound as described above, which is
characterized in that A.sub.2 is a methylene, ethylene or propylene
group. In particular, the present invention describes a compound as
described above, which is characterized in that it has the
following structure:
##STR00022##
where s=1, 2, 3.
[0099] In a likewise preferred embodiment, the present invention
describes a compound as described above, which is characterized in
that it has as functional group T a group of the structure
##STR00023##
and thus has the following structure:
##STR00024##
where s=1, 2, 3, preferably s=2.
[0100] All the abovementioned structures can be employed as radical
R.sub.4 obligatorily present in the radical R.sub.3. In this
connection, t=0 is preferred. Especially preferred in this
connection are radicals R.sub.4 of the following structures:
##STR00025##
where it is further preferred for R.sub.6 and R.sub.7 to be equal
to hydrogen.
[0101] Very particularly preferred radicals R.sub.4 are those of
the following structures:
##STR00026##
where it is further preferred for R.sub.6 and R.sub.7 to be equal
to hydrogen.
[0102] Accordingly, the present invention also relates to a
compound as described above, which is characterized in that R.sub.4
is selected from the group consisting of
##STR00027##
[0103] In a likewise preferred embodiment of the present invention,
Q is a CH group. The present invention therefore also describes
compounds as described above, which are characterized in that Q is
a CH group.
[0104] Within the scope of this embodiment, the group A.sub.1
present where appropriate in the radical R.sub.3 can be chosen as
desired within the scope of the definitions described above. A
particularly preferred group A.sub.1 is a methylene or ethylene or
propylene group, especially a methylene or ethylene group and is
optionally substituted, where as substituents inter alia
particularly preferred are alkyl radicals having 1, 2 or 3 C atoms,
especially methyl and ethyl, and/or halogen, especially fluorine,
chlorine and bromine, and/or cycloalkyl radicals having preferably
5, 6 or 7 C atoms and/or carboxyalkyl radicals, where the alkyl
radical is preferably methyl or ethyl, and the carboxyl group is
preferably esterified with a methyl or ethyl group, or is
amidated.
[0105] In a very particularly preferred embodiment of the compounds
of the invention in which Q is a CH group, the group A.sub.1 is
absent.
[0106] Accordingly, the present invention also describes a compound
as described above, which is characterized in that Q is a CH group
and A.sub.1 is absent.
[0107] Concerning the functional group T which is optionally
present in the radical R.sub.3 in the context of the present
invention, it is possible to choose all of the groups described
above. Especially in the case where Q is a CH group, preferred
functional groups T are groups of the structures
##STR00028##
[0108] Structures particularly preferred for T groups are
##STR00029##
where it is further preferred for R.sub.5 to be hydrogen. In
further particularly preferred embodiments, T is a group of the
structures
##STR00030##
where T is very particularly preferably a functional group of the
structure
##STR00031##
[0109] In this case it is possible for the amide or ester groups to
be incorporated in both orientations.
[0110] Accordingly, the present invention also describes a compound
as described above, which is characterized in that Q is a CH group
and T is a functional group of the structure
##STR00032##
[0111] In the case where Q is a CH group and T is one of the
functional groups mentioned as preferred, the group A.sub.1 is
absent in a particularly preferred embodiment of the present
invention.
[0112] The present invention therefore also describes a compound as
described above, which has the following structure:
##STR00033##
[0113] Besides the 4 position of the radical R.sub.3 which is
explicitly described in the above structural formula, the 2 or 3
position are likewise possible, with preference for the 4
position.
[0114] In a likewise preferred embodiment of the present invention,
both group A.sub.1 and the functional group T are absent.
[0115] The present invention therefore also describes a compound as
described above, which has the following structure:
##STR00034##
[0116] Besides the 4 position of the radical R.sub.3 which is
explicitly described in the above structural formula, the 2 or 3
position are likewise possible, with preference for the 4
position.
[0117] In relation to the group A.sub.2 which is obligatorily
present in the radical R.sub.3, there are, as already discussed
above, no restrictions within the definitions described above.
[0118] Especially in the case where Q is a CH group and further
especially when either T is a group of the structure
##STR00035##
or is absent, alkylene groups having 1 to 6 C atoms are preferred
for the group A.sub.2, especially the methylene, ethylene,
propylene, butylene groups, further especially the methylene,
ethylene and propylene groups and very especially the ethylene
group.
[0119] In the case where A.sub.2 is an aryl, heteroaryl or
aralkylene group, groups of the structures
##STR00036##
should be mentioned by way of example, where v and w can
independently of one another be 0, 1 or 2, and the two alkylene
groups can also be positioned in the 1,2 or 1,3 position relative
to one another. The 1,4 position is preferred for example. Both the
aryl radical and at least one of the two alkylene groups may be
suitably substituted. If A.sub.2 includes a heteroaryl group, this
preferably has 1 to 3 heteroatoms.
[0120] The group A.sub.2 can also be suitably substituted as
defined above. Substituents in this connection are particularly
halogens, preferably fluorine, chlorine or bromine, and/or alkyl
radicals having preferably 1, 2 or 3 C atoms such as methyl, ethyl,
n-propyl or isopropyl, especially preferably methyl and ethyl,
and/or cycloalkyl radicals preferably having 5, 6 or 7 C atoms
and/or carboxyalkyl radicals, where the alkyl radical is preferably
methyl or ethyl, and the carboxyl group is preferably esterified
with a methyl or ethyl group, or is amidated.
[0121] In a very particularly preferred embodiment of the present
invention, the group A.sub.2 is unsubstituted.
[0122] Accordingly, the present invention also describes compounds
as described above, which are characterized in that they have the
following structure:
##STR00037##
[0123] Besides the 4 position of the radical R.sub.3 which is
explicitly described in the above structural formulae, the 2 or the
3 position are likewise possible, with preference for the 4
position.
[0124] The present invention therefore likewise also describes
compounds as described above, which are characterized in that they
have the following structures:
##STR00038##
[0125] Besides the 4 position of the radical R.sub.3 explicitly
described in the above structural formulae, the 2 or the 3 position
are also possible, with preference for the 4 position.
[0126] All the abovementioned structures can be employed as radical
R.sub.4 obligatorily present in the radical R.sub.3. In this
connection, t=0 is preferred. Especially preferred in this
connection are radicals R.sub.4 of the following structures:
##STR00039##
where it is further preferred for R.sub.6 and R.sub.7 to be equal
to hydrogen.
[0127] Very particularly preferred radicals R.sub.4 are those of
the following structures:
##STR00040##
where it is further preferred for R.sub.6 and R.sub.7 to be equal
to hydrogen.
[0128] Accordingly, the present invention also relates to a
compound as described above, which is characterized in that R.sub.4
is selected from the group consisting of
##STR00041##
[0129] The radical R.sub.4 present in the radical R.sub.3 is in the
context of the present invention particularly preferably of the
structure
##STR00042##
[0130] In relation to the radical R.sub.2, generally all the
radicals falling under the definition (ii) are possible.
Accordingly, conceivable aryl groups are for example the phenyl
group or the naphthyl group, where at least one of the C atoms of
this aryl group can be replaced by a heteroatom selected from the
group consisting of S, N and O. A phenyl radical is particularly
preferred as radical R.sub.2. Thienyl and pyridyl are preferred as
an aryl radical comprising at least one heteroatom.
[0131] Accordingly, the present invention relates to a compound as
described above, which is characterized in that R.sub.2 is an at
least monosubstituted phenyl radical, thienyl radical or pyridyl
radical.
[0132] In a particularly preferred embodiment, R.sub.2 is an at
least monosubstituted phenyl radical.
[0133] The aryl radical, particularly preferably the phenyl
radical, R.sub.2 has at least one substituent R.sub.4 as defined
above, where one of the structures
##STR00043##
is preferred.
[0134] A particularly preferred substituent R.sub.4 in this
connection is one selected from the group consisting of
##STR00044##
[0135] A substituent of the structure
##STR00045##
is very particularly preferred as radical R.sub.4. The substituent
R.sub.4 can generally be positioned at all positions of the aryl
radical. In relation to the particularly preferred phenyl radical,
accordingly the 2, 3 or 4 position of the radical R.sub.4 is
possible, where the 3 position of the radical R.sub.4 on the phenyl
radical is particularly preferred.
[0136] Besides the radical R.sub.4, the aryl radical may further
have at least one further substituent. In a particularly preferred
embodiment, the aryl radical has a single substituent.
[0137] The alkylene group to which the radical R.sub.2 on the
alpha-C atom of the central amino acid in (1) is linked generally
has 0 to 3 C atoms. This alkylene group preferably has 1, 2 or 3 C
atoms, particularly preferably 1 or 2 C atoms and very particularly
preferably 1 C atom.
[0138] Accordingly, the present invention describes a compound as
described above, which is characterized in that it has the
following structure:
##STR00046##
[0139] The structure shown above may generally have both the D and
the L configuration at the .alpha.-C atom of the
3-amidinophenylalanine. In the context of the present invention,
the L configuration is particularly preferred. The compounds (I) of
the invention may very generally have either the L or the D
configuration or be the racemate in relation to the central
alpha-amino acid.
[0140] Accordingly, the present invention also relates to a
compound as described above, which is characterized in that k=1 and
R.sub.2 is a phenyl radical meta-substituted by an amidino group,
where the 3-amidinophenylalanine resulting thereby is in the L
configuration.
[0141] It is possible in principle for the compounds (I) to have,
when a further asymmetric C atom is present besides the
abovementioned asymmetric C atom, or another center of asymmetry is
present, both the L and the D configuration or S or R
configuration. It is likewise possible for the compound (I) to be
in the form of a racemate. Mixtures of L and D configurations or S
and R configurations are furthermore possible, where the proportion
of molecules with the D configuration or the proportion of
molecules with L configuration or, respectively, the proportion of
molecules with the S configuration or the proportion of molecules
with the R configuration, predominates.
[0142] It is possible in general for every radical R.sub.1
described in the above definition (i) to be present as radical
R.sub.1.
[0143] If the radical R.sub.1 is substituted for example by at
least one halogen, in this case fluorine, chlorine and/or bromine
are preferred.
[0144] If the radical R.sub.1 is substituted for example by at
least one esterified carboxyl group, methyl esters and/or ethyl
esters are preferred.
[0145] If the radical R.sub.1 is substituted for example by at
least one amino group, this amino group may be acylated, in which
case the acetyl group is particularly preferred.
[0146] Particular preference is given for example to mono-, bi- or
tricyclic aryl radicals and heteroaryl radicals in which optionally
at least one double bond is hydrogenated and/or which comprise at
least one heteroatom selected from O, S and N, it also being
possible for a heteroaryl radical R.sub.1 to comprise two or more
identical or different heteroatoms. Examples of preferred aryl
radicals are for instance phenyl, naphthyl, anthracyl or
phenanthryl. These aryl radicals may optionally be in reduced
or/and oxidized form. In relation to the naphthyl radical, for
example, a 1,2-dihydronaphthyl, a 1,4,-dihydronaphthyl or else a
1,2,3,4-tetrahydronaphthyl radical is possible. In oxidized form,
the naphthyl radical may for example be in the form of a
1,4-naphthoquinoyl radical. The anthraquinoyl radical may in
oxidized form be for example in the form of a 1,4- or
9,10-anthraquinoyl radical or 1,4- or 9,10-anthrahydroquinoyl
radical, and the phenanthryl radical for example in the form of a
phenanthrenequinoyl radical. Examples of heteroaryl radicals are
for instance pyrrolyl, furanyl, thiophenyl, pyridyl, pyrimidyl,
pyrazyl, triazyl, imidazolyl, thiazolyl, oxazolyl, indolyl,
purinyl, pyronyl, pyridonyl, quinolyl, isoquinolyl. Also included
likewise are radicals R.sub.1 such as indenyl or
tetrahydroindenyl.
[0147] It is further possible for the radical R.sub.1 to be
suitably substituted, preferred substituents being for example
linear, branched or cyclic alkyl radicals having 1 to 10 C atoms.
Linear or branched alkyl radicals having 1, 2, 3, or 4 C atoms are
particularly preferred. Examples of particularly preferred
substituents are isopropyl and tert-butyl. Cyclic alkyl radicals
are likewise preferred as substituents, with cyclic alkyl radicals
having 5, 6 or 7 C atoms and, in particular, 6 C atoms being
particularly preferred. Mention should likewise be made of aryl or
heteroaryl groups as substituents, it being possible for the
heteroaryl groups to comprise one heteroatom selected from N, S and
O, and to comprise two or more identical or different heteroatoms.
The substituents of the radical R.sub.1 may in turn themselves be
suitably substituted.
[0148] Both heteroaryl and aryl radicals and alkyl radicals may in
this connection be linked by a sulfur bridging atom or an oxygen
bridging atom or via an alkylene chain having 1-3 C atoms to the
radical R.sub.1. Accordingly, the radical R.sub.1 may be
substituted for example by an alkyloxy, alkylthio, aryloxy,
arylthio, heteroaryloxy or heteroarylthio group.
[0149] Examples of substituted radicals R.sub.1 are for
instance
##STR00047## ##STR00048##
[0150] An aryl radical which is preferably present may in principle
also have more than one substituent. Particularly preferred
embodiments are those in which the aryl radical has no, one, two or
three substituents. If the aryl radical has for example three
substituents, then alkyl radicals having 1, 2 or 3 C atoms are
preferred inter alia. Alkyl radicals having 2 or 3 C atoms are
particularly preferred, especially preferably alkyl radicals having
3 C atoms and especially particularly preferably isopropyl.
Especially preferred in this connection is, for example, the
2,4,6-triisopropylphenyl radical.
[0151] If the aryl radical has for example one substituent, then
the tert-butyl radical is preferred inter alia.
[0152] In a further suitable embodiment, the radical R.sub.1 is an
aryl radical, preferably a phenyl radical, which is substituted by
a further aryl radical or heteroaryl radical via an oxygen bridging
atom or via an alkylene chain having 1-3 C atoms in turn having an
aryl, heteroaryl or alkyl radical. It is possible in this case for
the aryl or heteroaryl radical, for example a pyridine, to be
unsubstituted or likewise substituted at a suitable position, in
ortho, meta or para position, for example by at least one alkyl
group such as, for example, a methyl group and/or by at least one
halogen atom, preferably by one chlorine atom or two chlorine atoms
or one or two fluorine atoms and/or by at least one trihalomethyl
group, preferably by one or two trifluoromethyl group(s) and/or by
at least one alkoxy group, preferably a methoxy group.
[0153] Examples of such radicals R.sub.1 are for instance
##STR00049## ##STR00050## ##STR00051##
where the oxygen connecting the two aryls may be replaced by an
alkylene chain having 1-3 C atoms.
[0154] In a particularly preferred embodiment, the radical R.sub.1
is selected from tert-butylphenyl, cyclohexylphenyl,
5,6,7,8-tetrahydronaphthyl, naphthyl, anthracyl, anthraquinoyl and
anthrahydroquinoyl, pyridyloxyphenyl, phenyloxypyridyl,
pyridylalkylphenyl having a C.sub.1-C.sub.3-alkyl.
[0155] The alkylene group which links the radical R.sub.1 and the
sulfonyl group generally has no, one or two C atoms. It preferably
has no or one C atom, and especially preferably has no C atom and
is thus absent.
[0156] An alternative embodiment of the invention are compounds of
the formula I''
##STR00052##
where R.sub.1, R.sub.2, Q, j and k are defined as described above,
m=n=2, and R.sub.3 is an aryl or heteroaryl radical, and where the
aryl radical is preferably a benzyl or a phenoxy radical, and the
heteroaryl radical is preferably selected from a
pyridinylmethylene, pyridinyloxo, pyrimidinyloxo, pyrazinyloxo,
pyridinylthio radical and where the aryl or heteroaryl radical is
unsubstituted or substituted by at least one halogen, preferably
fluorine or chlorine, at least one alkoxy radical, preferably
methoxy radical, and/or at least one trifluoromethyl radical.
[0157] In a further preferred embodiment, the radical R.sub.3 is a
guanidinooxyalkyl radical.
[0158] Examples of such compounds are compounds in which the
structural unit
##STR00053##
in formula I'' is replaced by
##STR00054## ##STR00055## ##STR00056##
which are commercially available from Array Biopharma, Bolder
Colo., U.S.A.
[0159] Likewise included are compounds in which an amino acid,
preferably glycine, is incorporated between the sulfonyl group and
the 3-amidinophenylalanine group of the formula I.
[0160] The compounds described above can generally be prepared by
all suitable processes. The compounds of the invention are
preferably prepared by processes in which a sulfonyl chloride is
reacted in a first step with an amino acid or an amino acid
derivative.
[0161] Accordingly, the present invention relates to a process for
preparing a compound as described above, which comprises the
following step (S1):
[0162] (S1) reaction of a compound of the general structure
(E1')
##STR00057## [0163] with a compound of the general structure
(E1'')
[0163] ##STR00058## [0164] to obtain a compound of the general
structure (ZP1)
[0164] ##STR00059## [0165] where R.sub.2'' is the aryl radical
R.sub.2 substituted either by R.sub.4 or by R.sub.4 protected with
a suitable protective group, or by a substituent which is a
precursor of R.sub.4.
[0166] If the aryl radical R.sub.2 is substituted for example
preferably by an amidino group R.sub.4, then the substituent which
represents a precursor of the amidino group is for example a cyano
group which can be converted in one, preferably two or more
suitable process stages by reaction with hydroxylamine and
subsequent hydrogenation into an amidino group. This amidino group
can subsequently in turn be protected with a suitable protective
group which can be removed again in a suitable process step.
[0167] If compounds of the invention of the general structure (I'')
in which the group Q is equal to nitrogen, and the alkylene group
A.sub.1 is absent, are prepared, then preferred preparation
processes are those wherein a compound of the structure (ZP1) is
initially reacted in a step (S2') with a cyclic compound of the
general structure (E2')
##STR00060##
where W is a suitable protective group. In this case, a compound of
the general structure (ZP2')
##STR00061##
is obtained, from which the protective group W is preferably
eliminated in a subsequent step (S3'). In a next step (S4'), the
compound obtained in (S3') is reacted with a compound of the
general structure (E2'')
HO-T-A.sub.2-R.sub.4' (E2'')
resulting in a compound of the general structure (P1)
##STR00062##
where R.sub.4.sup.' is either R.sub.4 or R.sub.4 protected with a
suitable protective group, or a precursor of R.sub.4. If R.sub.4'
is a radical R.sub.4 protected with a protective group, then the
protective group is preferably removed after step (S4').
[0168] It is then possible according to the invention for an amino
group to be present as radical R.sub.4. It is then possible to
build up from this group a guanidino group in a next step by a
process known to the skilled worker, for example by reaction with
pyrazolecarboxamidine.
[0169] A likewise preferred process for preparing compound (I'') in
which the group Q is equal to nitrogen, and the alkylene group
A.sub.1 is absent, comprises a step (S2') in which a compound of
the general structure (E2''')
##STR00063##
where W is a suitable protective group, is reacted with a compound
of the general structure (E2'')
HO-T-A.sub.2-R.sub.4' (E2'')
resulting in a compound of the general structure (ZP2'')
##STR00064##
in which R.sub.4' is either R.sub.4 or R.sub.4 protected with a
suitable protective group, or a precursor of R.sub.4. In this
process therefore firstly the C-terminal radical of the central
amino acid in (I) is built up. In a next step (S3'), preferably the
protective group W is eliminated, and in a next step (S4') the
compound obtained in (S3') is reacted with a compound of the
general structure (ZP1) to result in a compound of the general
structure (P1). If the radical R.sub.4 in this case is an amino
group, then a guanidino group can be built up therefrom in this
case too, as described above.
[0170] If compounds of the invention of the general structure (I'')
in which the group Q is equal to CH are prepared, then preferred
preparation processes are those which start from compounds (ZP1)
which are reacted in a step (S2'') with a compound of the general
structure (E3)
##STR00065##
where R.sub.4' is either R.sub.4 or R.sub.4 protected with a
suitable protective group, or a precursor of R.sub.4. This
preferred reaction results in a compound of the general structure
(P2)
##STR00066##
[0171] If R.sub.4' is a radical R.sub.4 protected with a protective
group, then the protective group is preferably removed after step
(S2''). It is then possible according to the invention for an amino
group to be present as radical R.sub.4. It is then possible to
build up from this group a guanidino group in a next step by a
process known to the skilled worker, for example by reaction with
pyrazolecarboxamidine.
[0172] If compounds of the invention of the general structure (I'')
in which T is equal to --(C.dbd.O)--NH--, Q is equal to CH, and
A.sub.1 is absent, are prepared, then preferred preparation
processes are those in which firstly, in a step (S2'''), compounds
of the general structure (E3')
##STR00067##
are reacted with a compound of the general structure (E3'')
H.sub.2N-A.sub.2-R.sub.4' (E3'')
resulting in a compound of the general structure (ZP3)
##STR00068##
[0173] The radical R.sub.4' has the meaning described in the
further preparation processes described above. W is a suitable
protective group in this case too. In a next step, the protective
group W is removed by a suitable process preferably in a step
(S3'''). This is preferably followed by a step (S4''') in which the
compound obtained in (S3''') is reacted with a compound (ZP1),
resulting in a compound of the general structure (P3)
##STR00069##
[0174] If R.sub.4' is a radical R.sub.4 protected with a protective
group, the protective group is preferably removed after step
(S2'''). It is then possible according to the invention for an
amino group to be present as radical R.sub.4. It is then possible
to build up from this group a guanidino group in a next step by a
process known to the skilled worker, for example by reaction with
pyrazolecarboxamidine.
[0175] In a preferred embodiment of this last-described process
variant, it is possible to employ as compound of the general
structure (E3'') for example a compound which has as radical
R.sub.4' a precursor of a radical R.sub.4 of the invention, this
precursor being a --CN group. In one or more suitable steps, this
--CN group is built up to an amidino group further preferably by a
process known to the skilled worker, for example by reaction with a
hydroxylamine, subsequent reaction with acetic anhydride and
following hydrogenation. In another embodiment of the process of
the invention, the hydroxyamidino derivative can be converted
directly by hydrogenation into an amidine derivative, in which case
the amidino group is if appropriate protected intermediately with a
suitable protective group. The reaction with hydroxylamine very
particularly preferably takes place after step (S2''') and before
step (S3'''). It is further particularly preferred for the reaction
with acetic anhydride likewise to take place before step (S3''').
Hydrogenation to give the amidino group preferably takes place
after step (S4''').
[0176] It is very particularly preferred for the final purification
of the compounds prepared in this way to take place by preparative,
reversed-phase HPLC or by crystallization from a suitable solvent
or solvent mixture or by countercurrent distribution.
[0177] Besides the compounds described as above per se and
processes for their preparation, the present invention also relates
to a medicament which comprises one of the compounds indicated
above.
[0178] Accordingly, the present invention also relates to a
medicament comprising at least one compound (I) or a salt of this
compound and, where appropriate, pharmaceutically suitable
excipients and/or additives.
[0179] The present invention likewise describes a compound (I) for
use as medicament, where the medicament comprises where appropriate
in addition pharmaceutically suitable excipients and/or
additives.
[0180] Suitable excipients and/or additives which serve, for
example, to stabilize and/or preserve the medicament are disclosed
for example in H. Sucker et al., Pharmazeutische Technologie, 2nd
edition, Georg Thieme Verlag, Stuttgart (1991), the disclosure in
this regard being included by reference in the context of the
present invention. The pharmaceutically suitable excipients and/or
additives include for example physiologically saline solutions,
Ringer's dextrose, Ringer's lactate, demineralized water,
stabilizers, antioxidants, complexing agents, antimicrobial
compounds, proteinase inhibitors and/or inert gases.
[0181] The compounds (I) of the invention can generally be employed
in any form as medicaments. The medicament is employed in possible
embodiments of the invention for example in the form of a tablet,
of a coated tablet, of a capsule, of a pellet, suppository, of a
solution, in particular of a solution for injection or infusion, of
eye drops, nose and ear drops, of a syrup, of a capsule, of an
emulsion or suspension, of a pessary, stick, aerosol, dusting
powder, of a paste, cream or ointment.
[0182] The present invention therefore also relates to a medicament
as described above, which is characterized in that it is employed
in the form of a tablet, of a coated tablet, of a capsule, of a
pellet, of a suppository, of a solution, in particular of a
solution for injection or infusion, of eye drops, nose drops or ear
drops, of a syrup, of an emulsion or suspension, of a pessary, of a
stick, of an aerosol, of a dusting powder, of a paste, of a cream
or of an ointment.
[0183] The present invention therefore likewise also describes a
compound (I) for use as medicament, where the medicament is
employed in the form of a tablet, of a coated tablet, of a capsule,
of a pellet, of a suppository, of a solution, in particular of a
solution for injection or infusion, of eye drops, nose drops or ear
drops, of a syrup, of an emulsion or suspension, of a pessary, of a
stick, of an aerosol, of a dusting powder, of a paste, of a cream
or of an ointment.
[0184] In a particularly preferred embodiment of the present
invention, the above-mentioned compounds (I) and/or their salts or
the abovementioned medicaments comprising these compounds (I)
and/or their salts and, where appropriate, at least one
pharmaceutically suitable excipient and/or additive are employed
for the diagnosis and/or therapy of a tumor. Prophylaxis of a tumor
is likewise also possible additionally or alternatively, it being
possible to use the compounds in particular for preventing and/or
reducing the metastasis of tumors.
[0185] A compound (I) of the invention or its salt or a medicament
as described above can very generally furthermore be employed for
example in parenteral use form, especially in intraarterial,
intravenous, intramuscular or subcutaneous form, in enteral use
form, especially for oral or rectal use, or in topical use form,
especially as dermatological agent. Intravenous or subcutaneous
uses are preferred.
[0186] These use forms are particularly suitable for the diagnosis
and/or therapy and/or prophylaxis of a tumor.
[0187] The present invention therefore also relates to the use of a
compound (I) or of a salt of this compound or of a medicament as
described above for the diagnosis, therapy or prophylaxis of a
tumor and for preventing and/or reducing the metastasis of a tumor,
especially in oral, subcutaneous, intravenous or transdermal
form.
[0188] The present invention likewise describes the use of a
compound (I) or of a salt of this compound for producing a
medicament for the diagnosis, therapy or prophylaxis of a tumor and
for preventing and/or reducing the metastasis of a tumor.
[0189] The present invention further describes this use for
producing a medicament for oral, subcutaneous, intravenous or
transdermal use.
[0190] In a particularly preferred embodiment of the present
invention, a compound (I) of the invention is employed for reducing
tumor metastases.
[0191] Accordingly, the present invention relates to the use
described above of the compound (I) or of a salt of this compound
or of a medicament as described above, where the formation of tumor
metastases is reduced.
[0192] The present invention likewise describes a process as
described above for producing a medicament for reducing the
formation of tumor metastases.
[0193] The present invention therefore likewise relates in
particular to the use of a compound (I) or of a salt of this
compound or of a medicament comprising the compound (I) or a salt
of this compound for inhibiting matriptase.
[0194] The present invention relates in particular also to the use
of a compound (I) or of a salt of this compound or of a medicament
comprising the compound (I) or a salt of this compound for
inhibiting matriptase, where the matriptase is MT-SP1.
[0195] The present invention accordingly also describes a process
for producing a medicament comprising a compound (I) or a salt of
this compound for inhibiting matriptase.
[0196] The present invention likewise also describes a process for
producing a medicament comprising a compound (I) or a salt of this
compound for inhibiting matriptase, where the matriptase is
MT-SP1.
[0197] The compounds described above may also be in the form of
prodrugs, e.g. through modification of the amidino group with a
hydroxyl or a C.sub.1-C.sub.6-alkyloxycarbonyl group, which are
converted into the species having inhibitory activity only after
uptake in the body, spontaneously and/or by one or more endogenous
enzymes, it thus being possible to improve the bioavailability and
pharmacokinetic properties of the compounds.
[0198] The invention is explained in more detail in the following
examples and figures.
DESCRIPTION OF FIGURES
[0199] FIG. 1 shows the inhibition of the invasive growth by the
matriptase inhibitors 37 and 54 from Example 9.
[0200] FIG. 2 shows the inhibition of the proHGF induced
scatterings of PC-3 cells by the matriptase inhibitors 37 and 54
from Example 10.
EXAMPLES
Methods
[0201] Analytical HPLC: Shimadzu LC-10A system, column: Phenomenex
Luna C.sub.18, 5 .mu.m, 100 .ANG. (250.times.4.6 mm) solvent A:
0.1% TFA in water, B: 0.1% TFA in ACN, gradient: 10% B to 70% B in
60 min, 1 ml/min flow rate, detection at 220 or 215 nm.
[0202] Preparative HPLC: Shimadzu LC-8A system, column: Phenomenex
Luna C.sub.18, 5 .mu.m, 100 .ANG. (250.times.30 mm) solvent A: 0.1%
TFA in water, B: 0.1% TFA in ACN, gradient: 10% B to 55% B in 120
min, 10 ml/min flow rate, detection at 220 nm.
[0203] Mass spectroscopy: The mass spectra were measured on a
Kompact probe from Kratos (Manchester, England) with a time of
flight measuring detector and .alpha.-cyano-hydroxycinnamic acid as
matrix, or on an ESI-MS LCQ from Finnigan (Bremen, Germany).
Abbreviations Used
[0204] Ac acetyl [0205] AcOH acetic acid [0206] ACN acetonitrile
[0207] .beta.-Ala .beta.-alanine [0208] Boc tert-butyloxycarbonyl
[0209] DAE 1,2-diaminoethane [0210] DCM dichloromethane [0211] DIEA
diisopropylethylamine [0212] DMF N,N-dimethylformamide [0213] IBCC
isobutyl chlorocarbonate [0214] iNip isonipecotic acid [0215]
K.sub.i inhibition constant [0216] NMM N-methylmorpholine [0217]
Phe(3-AcOxam) 3-(acetyloxyamidino)phenylalanine [0218] Phe(3-Am)
3-amidinophenylalanine [0219] Phe(3-CN) 3-(cyano)phenylalanine
[0220] Phe(3-Oxam) 3-(oxamidino)phenylalanine [0221] PyBop
benzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium
hexafluoro-phosphate [0222] Pzd piperazide [0223] RT room
temperature [0224] TFA trifluoroacetic acid [0225] THF
tetrahydrofuran [0226] Tips 2,4,6-(triisopropyl)phenylsulfonyl
[0227] Z benzyloxycarbonyl
Example 1
Synthesis of anthracenesulfonyl-Phe(3-Am)-Pzd-.beta.-Ala.times.2
TFA (Compound 10 from Table 1)
##STR00070##
[0228] 1a) Boc-Pzd-.beta.Ala-Z
[0229] 2 g (8.96 mmol) of Z-.beta.Ala-OH were dissolved in 20 ml of
THF and, at -15.degree. C., 0.99 ml (8.96 mmol) of NMM and 1.17 ml
(8.96 mmol) of IBCC were added. The mixture was stirred at
-15.degree. C. for 10 min and then 1.67 g (8.96 mmol) of Boc
piperazine (Fluka) and additionally 400 .mu.l (3.6 mmol) of NMM
were added. The mixture was stirred at -15.degree. C. for a further
hour and further stirred at room temperature overnight.
Subsequently, the solvent was removed in vacuo, and the residue was
taken up in ethyl acetate, washed 3.times. each with 5% KHSO.sub.4,
saturated NaHCO.sub.3 solution and NaCl-saturated water and then
dried over Na.sub.2SO.sub.4. The solvent was removed in vacuo. A
pale oil remained as residue and crystallized overnight in a
refrigerator.
[0230] Yield: 3.2 g (8.17 mmol), HPLC: 51.69% B
1b) H-Pzd-.beta.Ala-Z.times.HCl
[0231] 3.2 g (8.17 mmol) of Boc-Pzd-.beta.Ala-Z were partly
dissolved in glacial acetic acid, mixed with 50 ml of 1N HCl in
glacial acetic acid and left to stand at room temperature, shaking
occasionally, for 1 h. The solvent was partly removed in vacuo, and
the product was precipitated by adding diethyl ether, and was
filtered off with suction, washed again with diethyl ether and
dried in vacuo.
[0232] Yield: 2.13 g (6.5 mmol) of white solid, HPLC: 28.19% B
1c) Boc-Phe(3-CN)--OH
[0233] 2.5 g (13.1 mmol) of H-Phe(3-CN)--OH were dissolved in 100
ml of dioxane and, at 0.degree. C., 13 ml (13 mmol) of 1N NaOH and
3.16 g (14.5 mmol) of Boc pyrocarbonate were added. The mixture was
stirred at 0.degree. C. for 20 min and then at room temperature for
4 h, during which a total of 7 ml (7 mmol) of 1N NaOH was added in
portions, thus keeping the pH constant at 8-8.5. The solvent was
removed in vacuo, and the residue was taken up in ethyl acetate,
washed in each case 3.times. with 5% KHSO.sub.4 and 3.times. with
NaCl-saturated water and then dried over Na.sub.2SO.sub.4. The
solvent was removed in vacuo. A white solid was obtained as
residue.
[0234] Yield: 2.11 g (7.3 mmol) of white solid, HPLC: 45.93% B
1d) Boc-Phe(3-AcOxam)-OH
[0235] 2.11 g (7.3 mmol) of Boc-Phe(3-CN)--OH were dissolved in 100
ml of methanol, and 760 mg (10.95 mmol) of hydroxylamine.times.HCl
and 1.9 ml (10.95 mmol) of DIEA were added. The mixture was stirred
under reflux for 6 h. A further 266 mg (3.84 mmol) of
hydroxylamine.times.HCl and 665 .mu.l (3.84 mmol) of DIEA were
added and the mixture was stirred under reflux for a further 3 h
and then at room temperature overnight. The solvent was then
removed in vacuo. A pale oil remained as residue and was dissolved
in 50 ml of glacial acetic acid, and 2 ml (22 mmol) of acetic
anhydride were added. The mixture was stirred at room temperature
for 30 min. The solvent was removed in vacuo, and the residue was
taken up in ethyl acetate, washed 3.times. each with 5% KHSO.sub.4
and NaCl-saturated water and then dried over Na.sub.2SO.sub.4. The
solvent was removed in vacuo.
[0236] Yield: 3.31 g (colorless oil), HPLC: 26.59% B
1e) Boc-Phe(3-AcOxam)-Pzd-.beta.Ala-Z
[0237] 0.92 g (2.8 mmol) of H-Pzd-.beta.Ala-Z.times.HCl and 1.02 g
(2.8 mmol) of Boc-Phe(3-AcOxam)-OH were dissolved in 40 ml of DMF
and, at 0.degree. C., 1.46 g (2.8 mmol) of PyBop and 1.46 ml (8.4
mmol) of DIEA were added. The mixture was stirred at 0.degree. C.
for 20 min and at room temperature for a further 2 h. The solvent
was then removed in vacuo, and the residue was taken up in ethyl
acetate, washed 3.times. each with 5% KHSO.sub.4, saturated
NaHCO.sub.3 solution and NaCl-saturated water and then dried over
Na.sub.2SO.sub.4. The solvent was removed in vacuo.
[0238] Yield: 2.49 g (pale oil), HPLC: 48.13% B
1f) H-Phe(3-AcOxam)-Pzd-.beta.Ala-Z.times.HCl
[0239] 2.49 g of Boc-Phe(3-AcOxam)-Pzd-.beta.Ala-Z (crude product)
were partially dissolved in glacial acetic acid, mixed with 30 ml
of 1N HCl in glacial acetic acid and left to stand at room
temperature, with occasional shaking, for 1 h. The solvent was
partly removed in vacuo, and the product was precipitated by adding
diethyl ether, and was filtered off with suction, washed again with
diethyl ether and dried in vacuo.
[0240] Yield: 1.32 g (2.3 mmol) of white solid, HPLC: 32.89% B
1g) Anthracenesulfonyl-Phe(3-AcOxam)-Pzd-.beta.Ala-Z
[0241] At 0.degree. C. 207.7 mg (0.361 mmol) of
H-Phe(3-AcOxam)-Pzd-.beta.Ala-Z, 127 .mu.l (0.73 mmol) of DIEA and
100 mg (0.361 mmol) of anthracenesulfonyl chloride (Fluka) were
dissolved in 10 ml of DMF. The mixture was stirred at 0.degree. C.
for 20 min and then at room temperature overnight. The solvent was
removed in vacuo, and the residue was taken up in ethyl acetate,
washed 3.times. each with 5% KHSO.sub.4, saturated NaHCO.sub.3
solution and NaCl-saturated water and then dried over
Na.sub.2SO.sub.4. The solvent was removed in vacuo. A pale oil
remained as residue and was employed directly, without further
purification, for the next synthesis step
[0242] HPLC: 57.65% B
1h) Anthracenesulfonyl-Phe(3-Am)-Pzd-.beta.Ala
[0243] The crude product 1 g was dissolved in 50 ml of 90% acetic
acid and 5 ml of 1 N HCl, and 30 mg of catalyst (10% Pd/C) were
added. The mixture was hydrogenated with hydrogen under atmospheric
pressure at 40.degree. C. overnight. The catalyst was then filtered
off, and the solvent was concentrated in vacuo. Part of the crude
product was purified by preparative reversed phase HPLC.
[0244] HPLC: 34.11% B
[0245] MS: calculated 586.24 (monoisotopic). found 587.79
[M+H].sup.+.
Example 2
Synthesis of
anthracenesulfonyl-Phe(3-Am)-Pzd-CO--CH.sub.2--CH.sub.2-guanidino.times.2
TFA (Compound II from Table 1)
##STR00071##
[0246] 2a)
Anthracenesulfonyl-Phe(3-Am)-Pzd-CO--CH.sub.2--CH.sub.2-guanidi-
no
[0247] About 115 mg of anthracenesulfonyl-Phe(3-Am)-Pzd-.beta.Ala
crude product (1 h) were dissolved in 10 ml of DMF and 90.3 mg
(0.616 mmol) of pyrazolecarboxamidine.times.HCl and 107 .mu.l
(0.616 mmol) of DIEA were added. The mixture was stirred overnight
and then the solvent was removed in vacuo. The remaining residue
was dried in vacuo and purified by preparative reversed phase HPLC
without further purification.
[0248] to HPLC: 35.09% B
[0249] MS: calculated 628.26 (monoisotopic). found 629.4
[M+H].sup.+.
Example 3
Synthesis of
2,4,6-triisopropylphenylsulfonyl-Phe(3-Am)-iNip-DAE-H.times.2 TFA
(Compound 3 from Table 1)
##STR00072##
[0250] 3a) Tips-Phe(3-CN)--OH
[0251] A solution of 1 eq. of Tips-Cl (97% pure, 4.69 g, 15 mmol)
in dioxane was added dropwise to a solution of 1.05 eq. of
H-L-Phe(3-CN)--OH (3 g, 15.8 mmol) in dioxane and 2.1 eq. of 1M
NaOH (31.5 ml) while stirring at RT. The pH of the solution was
monitored during this and kept at pH 8-9 with 1M NaOH. After 4 h,
the solvent was removed in vacuo, and the residue was taken up in
ethyl acetate and subjected to 3 acidic (5% KHSO.sub.4) and 3
neutral (saturated NaCl solution) washes. The organic phase was
dried over Na.sub.2SO.sub.4, the solvent was removed in vacuo, and
the resulting product was recrystallized from ethyl acetate/hexane
(yellowish crystalline compound).
[0252] Yield: 6.6 g (96.3%)
[0253] HPLC: 71.1% B
3b) Tips-Phe(3-OAm)-OH
[0254] 1 eq. of Tips-Phe(3-CN)--OH (3 g, 6.6 mmol), 1.5 eq. of
hydroxylamine.times.HCl (685 mg, 9.9 mmol) and 3 eq. of DIEA (3.4
ml, 19.8 mmol) were dissolved in absolute ethanol and boiled under
reflux for 4 h, and then stirred at RT while metering in further
hydroxylamine.times.HCl and base (DIEA, pH 8-9) until starting
material was no longer found in the HPLC. After removal of the
solvent, the residue was taken up in ethyl acetate and subjected to
3 acidic (5% KHSO.sub.4) and 3 neutral (saturated NaCl solution)
washes. The organic phase was dried over Na.sub.2SO.sub.4, the
solvent was removed in vacuo, and the resulting product was
recrystallized from ethyl acetate/hexane.
[0255] Yield: 1.63 g (white crystals; 50.4%),
[0256] HPLC: 51.0% B
3c) Tips-Phe(3-AcOAm)-OH
[0257] 1 eq. of Tips-Phe(3-OAm)-OH (2.5 g, 5.1 mmol) was dissolved
in 100 ml of glacial acetic acid and then 1.5 eq. of acetic
anhydride (724 .mu.l, 7.6 mmol) were added and the mixture was
stirred for 15 min. After removal of the solvent in vacuo, the
product is obtained as a white powder.
[0258] Yield: 2.7 g (99.4%)
[0259] HPLC: 64.5% B
3d) H-iNip-DAE-Z.times.HCl
[0260] 497 mg (2.17 mmol) of Boc-isonipecotic acid were dissolved
with 250 .mu.l (2.27 mmol) of NMM in 10 ml of dry THF. 296 .mu.l
(2.27 mmol) of isobutyl chloroformate were added at -15.degree. C.,
and the mixture was stirred for a further 10 min. Then 500 mg (2.17
mmol) of N--Z-1,2-diaminoethane.times.HCl and 250 .mu.l (2.27 mmol)
of NMM were added, and the mixture was stirred at -15.degree. C.
for a further 1 h and then at RT for 4 h. The solvent was removed
in vacuo, and the residue was taken up in ethyl acetate and
subjected to 3 acidic (5% KHSO.sub.4), 1 neutral (saturated NaCl
solution), 3 basic (NaHCO.sub.3 saturated) and 3 neutral (saturated
NaCl solution) washes. The ethyl acetate phase was then dried over
Na.sub.2SO.sub.4, and the solvent was removed in vacuo, resulting
in the product as amorphous substance (HPLC: 50.3% B). The crude
product was dissolved in 20 ml 1 N of hydrogen chloride in glacial
acetic acid and left to stand at RT for 1 h. The solvent was then
removed in vacuo, and the product was lyophilized.
[0261] Yield: 722 mg
[0262] HPLC: 26.4% B
3e) Tips-Phe(3-Am)-iNip-DAE-H.times.2 TFA
[0263] 150 mg (0.28 mmol) of Tips-Phe(3-AcOAm)-OH were dissolved
with 97 mg (0.28 mmol) of H-iNip-DAE-Z.times.HCl in 5 ml of DMF and
cooled to 0.degree. C. in an ice bath while stirring. 122 .mu.l
(0.70 mmol) of DIEA and 154 mg (0.29 mmol) of PyBOP were added to
the cooled solution. After 15 min, the ice bath was removed and the
mixture was stirred at RT for a further 2 h. The solvent was then
removed in vacuo, and the residue was taken up in ethyl acetate and
subjected to 3 acidic (5% KHSO.sub.4), 1 neutral (saturated NaCl
solution), 3 basic (NaHCO.sub.3 saturated) and 3 neutral (saturated
NaCl solution) washes. The ethyl acetate phase was then dried over
Na.sub.2SO.sub.4, and the solvent was removed in vacuo. The residue
was dissolved in 90% strength glacial acetic acid, mixed with 10
percent by weight catalyst (10% Pd/C) and hydrogenated with
hydrogen at RT overnight. The catalyst was filtered off, and the
solvent was removed to dryness in vacuo, and the residue was
purified by preparative reversed phase HPLC.
[0264] Yield: 92 mg
[0265] HPLC: 43.3% B
[0266] MS: calculated 626.36 (monoisotopic). found 628.1
[M+H].sup.+.
Example 4
Synthesis of 2,4,6-triisopropylphenylsulfonyl-Phe
(3Am)-iNip-NH--CH.sub.2--CH.sub.2-guanidino.times.2 TFA (Compound
34 from Table 1)
##STR00073##
[0267] 4a)
Tips-L-Phe(3-Am)-iNip-NH--CH.sub.2--CH.sub.2-guanidino.times.2
TFA
[0268] 85 mg (0.1 mmol) of Tips-Phe(3-Am)-iNip-DAE-H.times.2 TFA
were dissolved in 5 ml of DMF and 30 mg (0.2 mmol) of
pyrazolecarboxamidine and 55 .mu.l (0.3 mmol) of DIEA were added.
The mixture was stirred at RT overnight. The solvent was then
removed in vacuo, and the residue was purified by preparative
reversed phase HPLC.
[0269] HPLC: 43.7% B
[0270] MS: calculated 668.38 (monoisotopic). found 669.8
[M+H].sup.+.
Example 5
Synthesis of 2-Nas-Phe(3-Am) 4(aminoethyl)piperidide.times.2 TFA
(Compound 36 from Table 1)
##STR00074##
[0271] 5a) 2-Nas-Phe(3-CN)--OH
[0272] 2.49 g (11 mmol) of 2-Nas chloride (dissolved in dioxane)
were added dropwise over a period of 30 min to 1.9 g (10 mmol) of
H-Phe(3CN)--OH, dissolved in 100 ml of dioxane/water mixture and 22
ml of 1 N NaOH solution, at 0.degree. C. The mixture was stirred at
0.degree. C. for 1 h and further at room temperature overnight. The
solvent was then removed in vacuo, and the residue was dissolved in
water (adjusted to pH 8-9 with NaOH). The aqueous phase was
extracted 2.times. with diethyl ether and then the pH was adjusted
to pH 3-4 with 1 N HCl. The product was extracted 3.times. with
ethyl acetate, and the ethyl acetate phase was washed 3.times. each
with 5% KHSO.sub.4 and NaCl-saturated water and then dried over
Na.sub.2SO.sub.4. The solvent was removed in vacuo. A pale oil
remained and crystallized in a refrigerator.
[0273] Yield: 3.51 g (9 mmol), HPLC: 51.02% B
5b) 2-Nas-Phe(3-AcOxam)-OH
[0274] 3.5 g (9 mmol) of 2-Nas-Phe(3CN)--OH were dissolved in 100
ml of methanol, and 1.04 g (15 mmol) of hydroxylamine.times.HCl and
2.61 ml (15 mmol) of DIEA were added. The mixture was stirred under
reflux for 6 h. Then a further 700 mg (10 mmol) of
hydroxylamine.times.HCl and 1.74 ml (10 mmol) of DIEA were added.
The mixture was stirred under reflux for a further 4 h and then at
room temperature overnight. The solvent was then removed in vacuo.
A pale oil remained as residue and was dissolved in 50 ml of
glacial acetic acid and mixed with 2.83 ml (30 mmol) of acetic
anhydride. The mixture was stirred at room temperature for 1 h. The
solvent was removed in vacuo, and the residue was taken up in ethyl
acetate, washed 1.times. with 5% KHSO.sub.4 solution and 3.times.
with NaCl-saturated water and then dried over Na.sub.2SO.sub.4. The
solvent was virtually removed in vacuo. The product began slowly to
crystallize and was filtered off with suction.
[0275] Yield: 3.02 g (6.64 mmol) of pale solid, HPLC: 44.04% B
5c) 2-Nas-Phe(3-AcOxam) 4(aminoethyl)piperidide.times.HCl
[0276] 100 mg (0.22 mmol) of 2-Nas-Phe(3-AcOxam)-OH and 50 mg (0.22
mmol) of 4-(2-Boc-aminoethyl)piperidine (Tyger Scientific Inc.,
Princeton, N.Y.) were dissolved in 10 ml of DMF and, at 0.degree.
C., 115 mg (0.22 mmol) of PyBop and 115 .mu.l (0.22 mmol) of DIEA
were added. The mixture was stirred at 0.degree. C. for 20 min and
at room temperature for a further 3 h. The solvent was then removed
in vacuo. The residue was taken up in ethyl acetate, washed
2.times. with 5% KHSO.sub.4 solution, 1.times. with NaCl-saturated
water, 2.times. with sat. NaHCO.sub.3 solution and 3.times. with
NaCl-saturated water and then dried over Na.sub.2SO.sub.4. The
solvent was removed in vacuo.
[0277] The crude product was partly dissolved in glacial acetic
acid, mixed with 5 ml of 1N HCl in glacial acetic acid and left to
stand at room temperature, shaking occasionally, for 1 h. The
solvent was removed in vacuo, a pale oil remaining.
[0278] Yield: 105 mg of oil, HPLC: 35.66% B
5d) 2-Nas-Phe(3-Am) 4(aminoethyl)piperidide.times.2 TFA
[0279] The crude product 5c was dissolved in 50 ml of 90% acetic
acid, and 15 mg of catalyst (10% Pd/C) were added. The mixture was
hydrogenated with hydrogen under atmospheric pressure at 40.degree.
C. overnight. The catalyst was then filtered off, and the solvent
was concentrated in vacuo. A third of the crude product was
purified by preparative reversed phase HPLC.
[0280] Yield: 17.6 mg HPLC: 29.03% B
[0281] MS: calculated 507.23 (monoisotopic). found 508.4
[M+H].sup.+.
Example 6
Synthesis of 2-Nas-Phe(3-Am) 4(guanidinoethyl)piperidide.times.2
TFA (Compound 37 from Table 1)
##STR00075##
[0282] 6a) 2-Nas-Phe(3-Am) 4(guanidinoethyl)piperidide.times.2
TFA
[0283] About 80 mg of 2-Nas-Phe(3-AcOxam)
4(aminoethyl)piperidide.times.HCl crude product (5d) were dissolved
in 5 ml of DMF, and 65 mg (0.45 mmol) of
pyrazolecarboxamidine.times.HCl and 105 .mu.l (0.6 mmol) of DIEA
were added. After 3 h, a further 21.5 mg (0.15 mmol) of
pyrazolecarboxamidine.times.HCl and 35 .mu.l (0.15 mmol) of DIEA
were added, and the mixture was stirred further overnight. The
solvent was removed in vacuo, and the remaining residue was
purified by preparative reversed phase HPLC.
[0284] Yield: 42 mg HPLC: 31.19% B
[0285] MS: calculated 549.25 (monoisotopic). found 550.4
[M+H].sup.+.
Example 7
Preparation of the Catalytic Domain of Matriptase
[0286] Cloning: The catalytic domain of matriptase was amplified by
PCR using the following primer pair:
Sense Primer:
TABLE-US-00001 [0287]
5'-GGCAATTCCATATGAAACATCACCATCATCACCATGTTGTTGGGG GCACGGATGCG-3'
Antisense Primer:
TABLE-US-00002 [0288]
5'-GCATGAATTCTTATACCCCAGTGTTCTCTTTGATCCA-3
[0289] Sense primer and antisense primer were chosen so that an
NdeI cleavage site (bold) followed by the peptide sequence Met Lys
(His).sub.6 was introduced at the 5' end in front of the protease
domain (italic), and an EcoR1 cleavage site (bold) was inserted at
the 3' end of matriptase (italic). The PCR product was cloned via
NdeI and EcoRI into pET24 (Novagen), a vector for expression in
Escherichia coli.
[0290] The catalytic domain of matriptase was expressed in inactive
and insoluble form in Escherichia coli, and was purified, refolded
and then activated. The steps were in detail:
[0291] Expression and purification: BL21 (DE3) cells (Novagen)
which contained the vector from the cloning described above were
incubated in LB, 30 .mu.g/ml kanamycin at 37.degree. C. and 250
rpm. Expression was induced at an OD.sub.600 of 0.6 by adding 1 mM
IPTG, and incubation was continued for one hour. The cells were
then pelleted and disrupted with 5 ml Bug Buster.TM. protein
extraction reagent (Novagen), and the DNA was digested with 25 U/ml
per 1g of cell pellet of Benzonase.RTM. nuclease (Novagen). The
protein aggregates were washed and denatured with 5 ml of
denaturation buffer (6 M guanidinium HCL, 10 mM Tris HCl, 100 mM Na
phosphate, pH 8.0) per 1 g of pellet. Insoluble constituents were
removed by centrifugation (16 000 g, 30 min, 20.degree. C.), and
the supernatant was filtered (0.2 .mu.m), mixed with 10 mM
.beta.-mercaptoethanol and then put onto a metal chelate
chromatography column (1 ml NiNTA (Qiagen) per 10 ml of
supernatant) to purify the catalytic domain of matriptase. The
column was washed (8 M urea, 10 mM Tris HCl, 100 mM Na phosphate,
pH 6.3), and the partially purified protein was eluted with 8 M
urea, 10 mM Tris HCl, 100 mM Na phosphate, pH 4.5.
[0292] Refolding: The matriptase-containing fractions were
combined, derivatized with glutathione and then diluted for the
refolding in a final concentration of 50 .mu.g/ml in refolding
buffer (50 mM Tris HCl, 0.5 M L-arginine, 20 mM CaCl.sub.2, 1 mM
EDTA, 0.1 M NaCl, pH 7.5). After incubation at room temperature for
3 days, the refolding mixture was filtered and concentrated to a
concentration of >300 .mu.g/ml (Centricon Plus-80, Amicon), and
the buffer was changed to activation buffer (20 mM Na phosphate,
150 mM NaCl, pH 7.0) by gel filtration (PD 10 columns,
Pharmacia).
[0293] Activation: Since a correctly processed N terminus is a
precondition for the activity of serine proteases, it was necessary
to delete the peptide MK(His).sub.6 to activate the refolded
matriptase. For this purpose, the refolding mixture was incubated
with 2.5 mU per 50 .mu.g of protein of activated DAPase.TM.
(Qiagen) at 30.degree. C. for 2 h, and the activated matriptase was
separated from the non-activated matriptase and the DAPase by metal
chelate chromatography.
[0294] The yield of active matriptase was about 0.9 mg/l of
bacterial culture. The proteolytic activity was detected by
cleavage of the chromogenic substrate Pefachrome tPA
(Pentapharm).
Example 8
Determination of the Inhibitory Effect of Matriptase with the
Inhibitors Listed in Table 1
[0295] To determine the inhibitory effect, 200 .mu.l of Tris buffer
(0.05 M, 0.154 M NaCl, 5% ethanol, pH 8.0; contains the inhibitor),
25 .mu.l of substrate (CH.sub.3SO.sub.2-D-HHT-Gly-Arg-pNA; 2 and 1
mM) and 50 .mu.l of matriptase (0.5 .mu.g/ml) were incubated at
25.degree. C. After 3 min, the reaction was stopped by adding 25
.mu.I of acetic acid (50%), and the absorption at 405 nm was
determined using a Microplate Reader (Dynatech MR 5000). The
K.sub.i values were found by the Dixon method (Biochem. J. 55,
170-171, 1953) by linear regression using a computer program. The
K.sub.i values (Table 1) are the average of at least three
determinations.
TABLE-US-00003 TABLE 1 Determination of the K.sub.i values for the
inhibition of matriptase No. Structure of inhibitor K.sub.i/.mu.M 1
##STR00076## 1.5 2 ##STR00077## 0.16 3 ##STR00078## 0.053 4
##STR00079## 0.14 5 ##STR00080## 0.057 6 ##STR00081## 0.14 7
##STR00082## 0.056 8 ##STR00083## 0.69 9 ##STR00084## 0.33 10
##STR00085## 0.27 11 ##STR00086## 0.044 12 ##STR00087## 0.83 13
##STR00088## 0.21 14 ##STR00089## 0.40 15 ##STR00090## 0.3 16
##STR00091## 0.99 17 ##STR00092## 0.14 18 ##STR00093## 0.089 19
##STR00094## 0.60 20 ##STR00095## 0.25 21 ##STR00096## 0.47 22
##STR00097## 0.2 23 ##STR00098## 3.3 24 ##STR00099## 0.73 25
##STR00100## 2.4 26 ##STR00101## 0.4 27 ##STR00102## 0.46 28
##STR00103## 0.11 29 ##STR00104## 0.014 30 ##STR00105## 0.021 31
##STR00106## 0.013 32 ##STR00107## 0.0098 33 ##STR00108## 0.38 34
##STR00109## 0.14 35 ##STR00110## 0.16 36 ##STR00111## 0.11 37
##STR00112## 0.046 38 ##STR00113## 0.013 39 ##STR00114## 0.17 40
##STR00115## 7.2* 41 ##STR00116## 0.074 42 ##STR00117## 0.36 43
##STR00118## 0.18* 44 ##STR00119## 21 45 ##STR00120## 0.14 46
##STR00121## 0.088 47 ##STR00122## 3.9 48 ##STR00123## 0.36 49
##STR00124## 0.16 50 ##STR00125## 3.5 51 ##STR00126## 0.89 52
##STR00127## 53 ##STR00128## 0.073 54 ##STR00129## 0.032 55
##STR00130## 0.98 56 ##STR00131## 0.24 57 ##STR00132## 0.086 58
##STR00133## 0.55 59 ##STR00134## 0.061 60 ##STR00135## 0.31 61
##STR00136## 0.38 62 ##STR00137## 1.1 63 ##STR00138## 0.1 64
##STR00139## 0.014 65 ##STR00140## 0.83 66 ##STR00141## 13 67
##STR00142## 3.4 68 ##STR00143## 0.038 69 ##STR00144## 0.037 70
##STR00145## 0.12 71 ##STR00146## 0.45
Example 9
Inhibition of Invasive Growth by Matriptase Inhibitors (Matrigel
Assay)
[0296] A common test system for invasive growth at the cellular
level is the Matrigel invasion assay. This entails cells being
applied to an artificial extracellular matrix and investigation of
how many cells migrate through the latter within a defined
period.
[0297] It is shown here by way of example for the matriptase
inhibitors 37 and 54 that invasive growth is influenced and the
migration of matriptase-expressing colon carcinoma cell line DLD-1
through Matrigel is inhibited:
[0298] The wells of a `Transwell` plate were each coated with 10
.mu.g of Matrigel and 160 000 DLD-1 cells (Dexter et al, Cancer
Research 39: 1020-1025 (1979)) in 100 .mu.l of medium (RPMI 1640,
with 2% Ultroser HY serum substituted) were applied to each,
invasive growth was stimulated by adding ProHGF in 400 .mu.l of
medium with and without inhibitor (30 .mu.M). After incubation at
37.degree. C. and 5% CO.sub.2 for 48 hours, the cells which had
migrated through the matrix were fixed, stained and photographed at
100.times. magnification.
[0299] As FIG. 1 shows, the invasion of the DLD-1 cells through the
extracellular matrix is stimulated by addition of proHGF. This
indicates that the zymogen on the cell surface is activated. This
effect is clearly inhibited by the matriptase inhibitors.
Example 10
Inhibition of Cell Scattering by Matriptase Inhibitors
[0300] Because of its ability to induce the detachment of cells
from an isolated assemblage and the dissemination of the cells, HGF
is also referred to as scatter factor. This function can be
detected at the cellular level with the aid of the so-called
scatter assay. This entails the cells being seeded and their
dissemination stimulated by the addition of HGF being recorded
after a defined time. HGF can be formed through activation of the
inactive proform of HGF (proHGF). It is shown below by way of
example for the matriptase inhibitors 37 and 54 that activation of
proHGF by cellular matriptase is prevented and the scattering of
proHGF-stimulated cells is reduced.
[0301] For this purpose, 500 matriptase-expressing prostate
carcinoma cells (PC-3) were seeded in each well of a 96-well plate
and incubated (37.degree. C., 5% CO.sub.2) in 100 .mu.l of medium
without fetal calf serum (Nut Mix. F-12, 2% Ultroser HY) overnight,
and then the scattering was stimulated by adding proHGF with and
without matriptase inhibitors (3 .mu.M). After 6 days, the cells
were fixed and stained, and representative sections were
photographed at 100.times. magnification.
[0302] As FIG. 2 shows, the matriptase inhibitors 37 and 54 inhibit
the proHGF-induced scattering of PC-3 cells.
[0303] In the concentrations used, neither proHGF nor the
inhibitors have an influence on the proliferation of PC-3 cells.
This indicates that the effect shown derives from an altered
scattering behavior and not from an altered cell-doubling time.
Sequence CWU 1
1
3156DNAArtificial SequenceDescription of Artificial Sequence
Synthetic primer 1ggcaattcca tatgaaacat caccatcatc accatgttgt
tgggggcacg gatgcg 56237DNAArtificial SequenceDescription of
Artificial Sequence Synthetic primer 2gcatgaattc ttatacccca
gtgttctctt tgatcca 3738PRTArtificial SequenceDescription of
Artificial Sequence Synthetic peptide 3Met Lys His His His His His
His1 5
* * * * *