U.S. patent application number 15/156819 was filed with the patent office on 2016-09-08 for acylated 4-amidino- and 4-guanidinobenzylamines for the inhibition of plasma kallikrein.
The applicant listed for this patent is The Medicines Company (Leipzig) GmbH. Invention is credited to Andrea SCHWEINITZ, Torsten STEINMETZER, Joerg STUERZEBECHER.
Application Number | 20160257708 15/156819 |
Document ID | / |
Family ID | 32602564 |
Filed Date | 2016-09-08 |
United States Patent
Application |
20160257708 |
Kind Code |
A1 |
STUERZEBECHER; Joerg ; et
al. |
September 8, 2016 |
ACYLATED 4-AMIDINO- AND 4-GUANIDINOBENZYLAMINES FOR THE INHIBITION
OF PLASMA KALLIKREIN
Abstract
The invention relates to the use of acylated 4-amidino- or
4-guanidinobenzylamine in accordance with the general formula I
P4-P3-P2-P1 (I) where P4 is a monosubstituted or polysubstituted or
unsubstituted benzylsulfonyl group, P3 is a monosubstituted or
polysubstituted or unsubstituted, natural or unnatural
.alpha.-amino acid or .alpha.-imino acid in the D configuration, P2
is a monosubstituted or polysubstituted or unsubstituted, natural
or unnatural .alpha.-amino acid or .alpha.-imino acid in the L
configuration, and P1 is a monosubstituted or polysubstituted or
unsubstituted 4-amidino- or 4-guanidinobenzylamine group, for
inhibiting plasma kallikrein (PK), factor XIa and factor XIIa, in
particular for preventing the activation of coagulation at
synthetic surfaces and for systemic administration as
anticoagulants/-antithrombotic agents, in particular for preventing
the activation of coagulation at synthetic surfaces for the purpose
of averting thromboembolic events.
Inventors: |
STUERZEBECHER; Joerg;
(Erfurt, DE) ; STEINMETZER; Torsten; (Jena,
DE) ; SCHWEINITZ; Andrea; (Jena, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The Medicines Company (Leipzig) GmbH |
Leipzig |
|
DE |
|
|
Family ID: |
32602564 |
Appl. No.: |
15/156819 |
Filed: |
May 17, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10540958 |
Jan 3, 2006 |
9365613 |
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PCT/EP04/00247 |
Jan 15, 2004 |
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15156819 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61K 31/401 20130101;
A61K 31/435 20130101; A61P 9/02 20180101; A61P 37/08 20180101; A61P
7/02 20180101; A61P 9/00 20180101; A61P 9/10 20180101; A61P 37/00
20180101; A61K 47/60 20170801; C07K 5/06095 20130101; C07K 5/0606
20130101; C07K 5/06034 20130101; A61K 31/198 20130101; C07K 5/06078
20130101; A61P 43/00 20180101 |
International
Class: |
C07K 5/065 20060101
C07K005/065; C07K 5/062 20060101 C07K005/062 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 15, 2003 |
DE |
103 01 300.8 |
Claims
1. (canceled)
2. An acylated 4-amidinobenzylamine or 4-guanidinobenzylamine
according to formula I P4-P3-P2-P1 (I), or a salt thereof, wherein
P4 is a monosubstituted or polysubstituted or unsubstituted
benzylsulfonyl group; P3 is a monosubstituted or polysubstituted,
unnatural .alpha.-amino acid residue or .alpha.-imino acid residue
in the D configuration, or a monosubstituted, polysubstituted, or
unsubstituted, natural .alpha.-amino acid residue or .alpha.-imino
acid residue in the D configuration, wherein, when P3 has a
structure according to the following formula: ##STR00033## R.sub.2
is not an unsubstituted branched alkyl radical having 1-6 C atoms;
P2 is (a) a monosubstituted or polysubstituted natural or unnatural
.alpha.-amino acid residue or .alpha.-imino acid residue in the L
configuration, wherein the substituent at substituted P2 is a
substituted or unsubstituted, branched or linear aralkyl radical
having 1-10 C atoms, or P2 is (b) Asp, hGlu, Dap, Dap(Z), Lys,
Lys(Z), Arg, Thr, Thr(Bzl), Ser(Bzl), hSer(Bzl), Phe or hPhe; and
P1 is a monosubstituted or polysubstituted or unsubstituted
4-amidinobenzylamine or 4-guanidinobenzylamine group; wherein a
linker group is optionally and additionally coupled to P4 or P2;
wherein (a) the linker group, when present, together with the
substituent on P4 or P2, is of formula II U--Z--Y--X-- (II),
wherein U is H.sub.2N--, HOOC--(CH.sub.2).sub.n--CO--NH--, HOOC--,
H.sub.2N--(CH.sub.2).sub.n--NH--CO-- or HS--, and Z is
--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k--, or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.vO--(CH.sub.2).sub.m--
-(NH--CO--CH.sub.2--O--CH.sub.2).sub.k--, wherein n=1 to 10, d=1,
2, 3 or 4, v is an integer from 1 to 1000, m=0, 1, 2, 3 or 4, and
k=0 or 1; U is CH.sub.3--O--, and Z is
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--O--CH.sub.2).sub.k--, wherein d=1, 2, 3 or 4;
v is an integer from 1 to 1000; m=0, 1, 2, 3 or 4; and k=0 or 1; or
U and Z are absent; Y is --CO--NH--, --NH--CO--, --SO.sub.2--NH--,
--NH--SO.sub.2--, --S--S--, or --S--, or, if U and Z are absent, Y
is H.sub.2N--, HOOC--, HS--, HO--, or halogenated alkyl; and X is
absent, --(CH.sub.2).sub.n--, wherein n=0, 1, 2, 3, or 4, or
--(CH.sub.2).sub.n--O--, wherein n=1, 2, 3 or 4, and wherein, when
X is --(CH.sub.2).sub.n--O-- linked to P4, X is bonded to the
benzyl radical by way of the oxygen atom; or (b) the linker group,
when present, together with P2, is of formula III: ##STR00034##
wherein q is 0, 1, 2, 3, 4 or 5, and D is U--Z--Y--, wherein U is
H.sub.2N--, HOOC--(CH.sub.2).sub.n--CO--NH--, HOOC--,
H.sub.2N--(CH.sub.2).sub.n--NH--CO-- or HS--, and Z is
--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k--, or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--CH.sub.2).sub.k--, wherein n=1 to 10, d=1, 2,
3 or 4, v is an integer from 1 to 1000, m=0, 1, 2, 3 or 4, and k=0
or 1; U is CH.sub.3--O--, and Z is
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--O--CH.sub.2).sub.k--, wherein d=1, 2, 3 or 4;
v is an integer from 1 to 1000; m=0, 1, 2, 3 or 4; and k=0 or 1; or
U and Z are absent; Y is --CO--NH--, --NH--CO--, --SO.sub.2--NH--,
--NH--SO.sub.2--, --S--S--, or --S--, or, if U and Z are absent, Y
is H.sub.2N--, HOOC--, HS--, HO--, or halogenated alkyl; wherein
optionally two linker groups of two compounds of formula I join
together to form a single connector, each of the two linker groups
independently having the structure of formula II or formula III;
wherein each substituent at the substituted P4, P3, and P1 is: (a)
a halogen, (b) a substituted or unsubstituted, branched or linear
alkyl radical having 1-6 C atoms, or a substituted or
unsubstituted, branched or linear aralkyl radical having 1-10 C
atoms, or (c) hydroxyl, amino, cyano, amidino, guanidino,
methyloxycarbonyl, benzyl, benzyloxycarbonyl, aminomethyl,
glutarylamidomethyl, succinylamidomethyl, oxyalkylcarbonyl,
carboxyl, carboxymethyl, carboxyethyl group, where appropriate
esterified with a lower alkyl radical or oxyalkylcarbonyl,
carboxyl, carboxymethyl, or carboxyethyl which is present as
unsubstituted amide or amide which is substituted by an alkyl or
aryl group; wherein the sulfur atom of the benzylsulfonyl group of
P4 is linked to the .alpha.-amino or .alpha.-imino group of P3, the
carbon atom of the carbonyl group of P3 is bonded to the
.alpha.-amino or .alpha.-imino group of P2 or, when .alpha.-amino
or .alpha.-imino group is absent in P2, to the .beta.-amino or
.beta.-imino group of P2, and the nitrogen atom of benzylamine of
P1 is linked to the C-terminal carbonyl group of P2.
3. The compound as claimed in claim 2, wherein the linker group is
additionally coupled to P4 or P2, with the linker group being
coupled to P4 by way of said substituent or coupled directly to a
functional group of P2.
4. The compound as claimed in claim 2, wherein the compound
comprises the linker group coupled to P4.
5. The compound as claimed in claim 3, wherein, if the linker group
is coupled to P4, P2 is glycine, alanine, or serine.
6. The compound as claimed in claim 3, wherein the linker group is
coupled to P2, and P2 is of formula III.
7. The compound as claimed in claim 2, wherein said acylated
4-amidinobenzylamine or 4-guanidinobenzylamine is according to
formula V, ##STR00035## wherein q=0 or 1; R.sub.1 is: (a) hydrogen,
(b) a halogen, (c) a substituted or unsubstituted, branched or
linear alkyl radical having 1-6 C atoms, (d) a hydroxyl, amino,
cyano, amidino, guanidino, methyloxycarbonyl, benzyl,
benzyloxycarbonyl, aminomethyl, glutarylamidomethyl,
succinylamidomethyl, and an oxyalkylcarbonyl, carboxyl,
carboxymethyl or carboxyethyl group, where appropriate esterified
with a lower alkyl radical, oxyalkylcarbonyl, carboxyl,
carboxymethyl or carboxyethyl which is present as unsubstituted
amide or amide which is substituted by an alkyl or aryl group, or
(e) a group of formula (II): U--Z--Y--X-- (II), wherein U is
H.sub.2N--, HOOC--(CH.sub.2).sub.n--CO--NH--, HOOC--,
H.sub.2N--(CH.sub.2).sub.n--NH--CO-- or HS--, and Z is
--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k--, or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--O--CH.sub.2).sub.k--, wherein n=1 to 10, d=1,
2, 3 or 4, v is an integer from 1 to 1000, m=0, 1, 2, 3 or 4, and
k=0 or 1; U is CH.sub.3--O--, and Z is
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--OCH.sub.2).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CHO--CH.sub.2).sub.k--, wherein d=1, 2, 3 or 4; v is an
integer from 1 to 1000; m=0, 1, 2, 3 or 4; and k=0 or 1; or U and Z
are absent; Y is --CO--NH--, --NH--CO--, --SO.sub.2--NH--,
--NH--SO.sub.2--, --S--S--, or --S--, or, if U and Z are absent, Y
is H.sub.2N--, HOOC--, HS--, HO--, or halogenated alkyl; X is
absent, --(CH.sub.2).sub.n--, wherein n=0, 1, 2, 3, or 4, or
--(CH.sub.2).sub.n--O--, wherein n=1, 2, 3 or 4, and wherein, when
X is --(CH.sub.2).sub.n--O-- linked to P4, X is bonded to the
benzyl radical by way of the oxygen atom; R.sub.2 is: (a) hydrogen,
(b) a substituted branched, substituted linear, or unsubstituted
linear alkyl radical having 1-6 C atoms, or (c) a hydroxyl, amino,
cyano, amidino, guanidino, methyloxycarbonyl, benzyl,
benzyloxycarbonyl, aminomethyl, glutarylamidomethyl,
succinylamidomethyl, and an oxyalkylcarbonyl, carboxyl,
carboxymethyl or carboxyethyl group, where appropriate esterified
with a lower alkyl radical, oxyalkylcarbonyl, carboxyl,
carboxymethyl or carboxyethyl which is present as unsubstituted
amide or amide which is substituted by an alkyl or aryl group;
R.sub.3 together with P2 is (a) a monosubstituted or
polysubstituted natural or unnatural .alpha.-amino acid residue or
.alpha.-imino acid residue in the L configuration, wherein the
substituent at substituted P2 is a substituted or unsubstituted,
branched or linear aralkyl radical having 1-10 C atoms, (b) P2 is
Asp, hGlu, Dap, Dap(Z), Lys, Lys(Z), Arg, Thr, Thr(Bzl), Ser(Bzl),
hSer(Bzl), Phe, or hPhe, or (c) a group of formula (III):
##STR00036## wherein q is 0, 1, 2, 3, 4 or 5, and D is U--Z--Y--,
wherein U is H.sub.2N--, HOOC--(CH.sub.2).sub.n--CO--NH--, HOOC--,
H.sub.2N--(CH.sub.2).sub.n--NH--CO-- or HS--, and Z is
--(CH.sub.2).sub.n--,
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2]O--(CH.sub.2).sub.m--(NH--CO---
CH.sub.2--O--CH.sub.2).sub.k--, or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.vO--(CH.sub.2).sub.m--
-(NH--CO--CH.sub.2--CH.sub.2).sub.k--, wherein n=1 to 10, d=1, 2, 3
or 4, v is an integer from 1 to 1000, m=0, 1, 2, 3 or 4, and k=0 or
1; U is CH.sub.3--O--, and Z is
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.vO--(CH.sub.2).sub.m--
-(NH--CO--CH.sub.2--O--CH.sub.2).sub.k--, wherein d=1, 2, 3 or 4; v
is an integer from 1 to 1000; m=0, 1, 2, 3 or 4; and k=0 or 1; or U
and Z are absent; Y is --CO--NH--, --NH--CO--, --SO.sub.2--NH--,
--NH--SO.sub.2--, --S--S--, or --S--, or, if U and Z are absent, Y
is H.sub.2N--, HOOC--, HS--, HO--, or halogenated alkyl; and
R.sub.4 is: (a) hydrogen, (b) a substituted or unsubstituted,
branched or linear alkyl radical having 1-6 C atoms, or (c) a
hydroxyl, amino, cyano, amidino, guanidino, methyloxycarbonyl,
benzyl, benzyloxycarbonyl, aminomethyl, glutarylamidomethyl,
succinylamidomethyl, and an oxyalkylcarbonyl, carboxyl,
carboxymethyl or carboxyethyl group, where appropriate esterified
with a lower alkyl radical, oxyalkylcarbonyl, carboxyl,
carboxymethyl, or carboxyethyl which is present as unsubstituted
amide or amide which is substituted by an alkyl or aryl group; or
each of R.sub.1 and R.sub.3 is independently and optionally a
linker group, with the linker group being coupled to P4 by way of a
substituent as defined for R.sub.1 or coupled directly to a
functional group of P2.
8. The compound as claimed in claim 7, wherein the compound is:
##STR00037## wherein n=1 to 8, and q=0 or 1.
9. The compound as claimed in claim 7, wherein the compound is:
##STR00038## wherein n=1 to 1000, r=0 to 3 and q=0 or 1.
10. The compound as claimed in claim 7, wherein the compound is:
##STR00039## wherein p=0, 1, 2 or 3, q=0 or 1, n=1 to 1000, m=1 to
3.
11. The compound as claimed in claim 7, wherein the compound is:
##STR00040## wherein n=0 to 5; ##STR00041## wherein n=0 to 9;
##STR00042## wherein n=1 to 6; ##STR00043## wherein n=0 to 3;
##STR00044## wherein n=0 to 3 and m=0 to 1000; ##STR00045## wherein
n=1 to 1000; or ##STR00046## wherein n=1 to 3 and m=1 to 1000;
wherein q, in each case, is 0 or 1.
12. The compound as claimed in claim 7, wherein the compound is:
##STR00047## wherein n=0 to 4 and m=10 to 1000; ##STR00048##
wherein n=1 to 4, p=2 to 4 and m=1 to 1000; ##STR00049## wherein
n=1 to 3 and m=10 to 1000; ##STR00050## wherein m=10 to 1000; or
##STR00051## wherein m=10 to 1000; and wherein q is 0 or 1.
13. The compound as claimed in claim 2, wherein the compound is
##STR00052## wherein D-Ser(tBu) in position 3 is optionally
replaced with D-Cha or D-Phe, and succinyl at P2 is optionally
replaced with glutaryl; ##STR00053## wherein D-Cha in position P3
is optionally replaced with D-Phe or D-Ser(tBu); or ##STR00054##
wherein D-Cha in position P3 is optionally replaced with D-Phe or
D-Ser(tBu).
14. The compound as claimed in claim 2, wherein, in formula I, P4
carries a radical R at the aromatic radical, P3 is D-Ser,
D-Ser(tBu), D-Phe, or D-Cha, and P2 is a natural or unnatural amino
acid Aaa, wherein R is H--; 4-, 3-, or 2-COOH; 4-, 3-, or 2-COOMe;
4-, 3-, or 2-AMe; 4-, 3-, or 2-glutaryl-AMe; or 4-, 3-, or 2-CN;
and Aaa is Asp, hGlu, Dap, Dap(Z), Lys, Lys(Z), Arg, Thr(Bzl),
Ser(Bzl), hSer(Bzl), Phe, or hPhe.
15. The compound as claimed in claim 14, wherein, when P3 is D-Ser,
Aaa is Dap, Dap(Z), Lys, Lys(Z), Ser(Bzl), Phe, or hPhe, and R is
H; when P3 is D-Ser(tBu), Aaa is Gin, Dap, Dap(Z), Lys, Lys(Z),
Arg, Thr(Bzl), Ser(Bzl), hSer(Bzl), Phe, or hPhe, and R is H; when
P3 is D-Cha, Aaa is Lys or Glu, and R is H; or when Aaa is
--NH--CH--[CH.sub.2--CH.sub.2--CO--NH--(CH.sub.2).sub.3--[O--(CH.sub.2).s-
ub.2].sub.3--CH.sub.2--NH.sub.2]--CO--, R is H.
16. The compound as claimed in claim 2, wherein the substituent at
substituted P2 is a substituted or unsubstituted, branched or
linear aralkyl radical having 1-10 C atoms.
17. A compound of formula I: P4-P3-P2-P1 (I), wherein P1 is
unsubstituted 4-amidinobenzylamine; and (1) P4 is unsubstituted
benzylsulfonyl, P3 is D-Ser or D-Ser(iBu), and P2 is Asp, GIn,
hGlu, Dap, Dap(Z), Lys, Lys(Z), Arg, Thr, Thr(Bzl), Ser(Bzl), Phe,
or hPhe; (2) P4 is benzylsulfonyl substituted with 4-COOH or
4-COOMe, P3 is D-Ser, and P2 is Ser; (3) P4 is benzylsulfonyl
substituted with 4-CN, P3 is D-Ser, and P2 is Pro; (4) P4 is
unsubstituted benzylsulfonyl, P3 is D-Cha, and P2 is Glu, Lys, or
Lys(Z); or (5) P4 is benzylsulfonyl substituted with 3-CN,
3-aminomethyl, or 3-(Glut-NHCH.sub.2), P3 is D-Cha, and P2 is Pro;
wherein the sulfur atom of the benzylsulfonyl group of P4 is linked
to the .alpha.-amino group of P3, the carbon atom of the carbonyl
group of P3 is bonded to the .alpha.-amino group of P2 or, when P2
is hSer, to the n-amino group of P2, and the nitrogen atom of
benzylamine of P1 is linked to the C-terminal carbonyl group of
P2.
18. The compound as claimed in claim 17, wherein P4 is
unsubstituted benzylsulfonyl.
19. The compound as claimed in claim 18, wherein P3 is D-Ser.
20. The compound as claimed in claim 19, wherein P2 is hPhe.
21. A compound: ##STR00055## or a salt thereof.
Description
[0001] The invention relates to the use of acylated
4-amidinobenzylamine or 4-guanidinobenzylamine in accordance with
the general formula P4-P3-P2-P1 (I), with P4 being a
monosubstituted or polysubstituted or unsubstituted benzylsulfonyl
group, P3 being a monosubstituted or polysubstituted or
unsubstituted, natural or unnatural, .alpha.-amino acid or
.alpha.-imino acid in the D configuration, P2 being a
monosubstituted or polysubstituted or unsubstituted, natural or
unnatural, .alpha.-amino acid or .alpha.-imino acid in the L
configuration, and P1 being a monosubstituted or polysubstituted or
unsubstituted 4-amidino- or 4-guanidinobenzylamine group, for
inhibiting plasma kallikrein (PK). In this connection, the novel PK
inhibitors are employed for prevention of the activation of
coagulation at synthetic surfaces and for systemic administration
as anticoagulants/antithrombotic agents, especially for prevention
of the activation of coagulation at synthetic surfaces, in order to
prevent thromboembolic events.
[0002] The present invention furthermore relates to the novel
acylated 4-amidino- or 4-guanidinobenzylamines per se, with
preference being given, in particular, to those which possess a
linker group at P2 or P4, with these linker groups preferably
being, in particular, oligo- or polyalkylene glycols.
[0003] The present invention also relates to the use of the
abovementioned acylated 4-amidino- or 4-guanidinobenzylamines for
inhibiting factor XIa and/or factor XIIa. The use of the
abovementioned compound for inhibiting thrombin and prothrombin is
also described within the context of the present invention.
[0004] PK is a multifunctional, trypsin-like serine protease for
which several physiological substrates are known. Thus, PK can, by
means of proteolytic cleavage, liberate the vasoactive peptide
bradykinin from high molecular weight kininogen and activate the
proteases coagulation factor XII, prourokinase, plasminogen and
Pro-MMP 3. It is therefore assumed that the PK/kinin system plays
an important role in a variety of syndromes, for example in
thromboembolic situations, disseminated intravasal coagulation,
septic shock, allergies, the postgastrectomy syndrome, arthritis
and ARDS (adult respiratory distress syndrome) (Tada et al., Biol.
Pharm. Bull 24, 520-524, 2001).
[0005] As a result of activating coagulation factor XII, thus
transforming it into factor XIIa, PK plays an especial role in the
activation of the intrinsic coagulation cascade. The intrinsic
coagulation cascade can become activated if blood which is present
in extracorporeal blood circulations comes into contact with
synthetic surfaces, for example in connection with hemodialysis or
in connection with using oxygenators. As a result of factor XII
being bound to, in particular, negatively charged surfaces and/or
synthetic surfaces, the intrinsic coagulation cascade is triggered
by means of autoactivation or by traces of PK (Kaplan, Prog.
Hemostasis Thromb. 4, 127-175, 1978). The activated factor XII (F
XIIa) catalyzes the conversion of plasma prekallikrein to PK,
which, in the sense of a positive feedback, brings about further
formation of factor XIIa (Griffin, Proc. natl. Acad. Sci. USA 75,
1998-2002, 1978). In conformity with the significance of factor
XIIa and PK in the early phase of the intrinsic coagulation
cascade, inhibitors of these enzymes should also have a
coagulation-inhibiting effect. During this early phase in the
activation of intrinsic coagulation, factor XIIa activates factor
XI thereby converting the latter into factor XIa.
[0006] Anticoagulants of the heparin type, vitamin K antagonists or
hirudin are used as inhibitors of both the intrinsic and the
extrinsic coagulation cascades and thus for the prophylaxis and
therapy of the abovementioned syndromes, such as thromboembolic
situations, disseminated intravasal coagulation, septic shock,
allergies, the postgastrectomy syndrome, arthritis and ARDS. Since,
however, the current anticoagulants do not meet all the
requirements placed on an "ideal" antithrombotic agent, for example
because of their low specificity, because of bleeding complications
which arise, because of a low half-life or because of inadequate
oral availability, attempts are being made to use small-molecule
inhibitors of the coagulation proteases thrombin and factor Xa to
develop alternatives. Factor VIIa, which is the initial enzyme in
the extrinsic coagulation pathway, is another target enzyme which
is being investigated in a variety of ways for the purpose of
developing inhibitors (Robinson and Saiah, Ann. Rep. Med. Chem. 37,
85-94, 2002). However, an inhibitor of thrombin and F Xa, or an
inhibitor of F VIIa as a specific inhibitor of the extrinsic
coagulation cascade, does not have any inhibitory effect on the
activation of the intrinsic coagulation cascade which is induced,
for example, by contact of the blood with synthetic surfaces.
[0007] There are only a few approaches with regard to searching for
inhibitors for the two enzymes factor XIIa and PK, which institute
intrinsic coagulation following activation at a charged surface.
The guanidinoalkylcarboxylic acid derivative FOY (Isobe, Blood
& Vessel 12, 135-138, 1981), leupeptin, the thrombin inhibitor
Na-dansyl-L-arginine-4-ethylpiperidide (Ratnoff, Blood 57, 55-58,
1981) and a variety of tripeptides (esters and amides) (Fareed et
al. Ann. N. York Acad. Sci. 370, 765-784, 1981; Silverberg and
Kaplan, Blood 60, 64-70, 1982) have been reported to have some
degree of inhibitory effect on factor XIIa. Amides of
Na-substituted 4-amidinophenyl-.alpha.-aminobutyric acid have been
reported to be more active inhibitors (Sturzebecher et al.,
Zentralbl. Pharm. Pharmakother. Lab. Diagn. 122, 240-241,
1983).
[0008] A variety of bisbenzamidines such as pentamidine and related
compounds having K.sub.i values around 50 .mu.M have been found to
be active PK inhibitors (Ashgar et al., Biochim. Biophys. Acta 438,
250-264, 1976). Esters of .omega.-amino- and
.omega.-guanidinoalkylcarboxylic acids have also been reported to
be PK inhibitors having micromolar Ki values (Maramatu and Fuji,
Biochim. Biophys. Acta 242, 203-208, 1971; Muramatu and Fuji,
Biochim. Biophys. Acta 268, 221-224, 1972; Ohno et al. Thromb. Res.
19, 579-588, 1980; Muramatu et al. Hoppe-Seyler's Z. Physiol. Chem.
363, 203-211, 1982; Satoh et al. Chem. Pharm. Bull. 33, 647-654,
1985; Teno et al. Chem. Pharm. Bull. 39, 2930-2936, 1991). The
first highly selective competitive inhibitors, which are derived
from arginine or phenylalanine, were developed by Okamoto et al.
(Thromb. Res., Suppl. VIII, 131-141, 1988) and inhibit PK with
K.sub.i values around 1 .mu.M. Okada's group has published several
studies on the development of competitive PK inhibitors, with the
most active compounds, which are derived from
trans-4-aminomethylcyclohexanecarbonyl-Phe-4-carboxymethylanilide,
having inhibitory constants around 0.5 .mu.M (Okada et al.,
Biopolymers 51, 41-50, 1999; Okada et al., Bioorg. Med. Chem. Lett.
10, 2217-2221; 2000, Tsuda et al., Chem. Pharm. Bull. 49,
1457-1463, 2001). A feature possessed in common by the
abovementioned PK inhibitors is their relatively high K.sub.i
value. WO 00/41531 described potent PK inhibitors which have
inhibitory constants around 1 nM and which possess a
4-amidinoaniline as the P1 radical. However, these inhibitors
described in WO 00/41531 are not suitable for being coupled
covalently to synthetic surfaces. PK inhibitors have also been
described in WO 94/29336. The essential difference as compared with
the compounds in accordance with the present invention is that the
compounds described in WO 94/29336 do not contain the crucial
benzylsulfonyl radical (P4). Furthermore, WO 94/29336 did not
describe any coupling of the compounds to, for example, synthetic
surfaces.
[0009] By now, some transition state-analogous PK inhibitors, which
possess an arginal (e.g. adamantyloxycarbonyl-D-Phe-Phe-arginal,
K.sub.i 12 nM, Garrett et al., J. Pept. Res. 52, 60-71, 1998) or
arginyl trifluoromethyl ketone (e.g.
adamantyloxycarbonyl-D-tert-butylglycine-Phe-Arg-CF.sub.3, K.sub.i
2 nM, Garrett et al., Bioorg. Med. Chem. Lett. 9, 301-306, 1999) as
the P1 radical, have also been described. The boroarginine
derivative DuP 714 (Ac-D-Phe-Pro-boroarginine), which was
originally developed as a thrombin inhibitor, has also been found
to be a powerful inhibitor of PK (K.sub.i 1.6 nM) (Kettner et al.,
J. Biol. Chem. 265, 18289-18297). However, these transition
state-analogous protease inhibitors suffer from the disadvantage
that they can only be obtained by means of elaborate syntheses and
tend to racemize, and are very nonspecific inhibitors.
[0010] PK is also inhibited irreversibly by a variety of
chloromethyl ketones. H-Ala-Phe-ArgCH.sub.2Cl and
H-Pro-Phe-ArgCH.sub.2Cl have been reported to be the most reactive
compounds (Kettner and Shaw, Biochemistry 17, 4778-4784, 1978).
However, peptidyl chloromethyl ketones are only suitable for
research purposes since, in vivo, they are only stable for a few
minutes (Lawson et al., Folia Haematol. (Leipzig) 109, 52-60, 1982;
Collen et al., J. Lab. Clin. Med. 99, 76-83, 1982).
[0011] The invention is therefore based on the object of providing
active compounds which are suitable for therapeutic applications,
which inhibit plasma kallikrein with a high degree of activity and
specificity and which, following coupling to a synthetic surface or
following parenteral, enteral or topical administration, in
particular intravenous or subcutaneous administration, have a
coagulation-inhibiting effect.
[0012] It has been found, surprisingly, that acylated 4-amidino- or
4-guanidinobenzylamine in accordance with the general formula
P4-P3-P2-P1 (I), with P4 (following the definition in accordance
with Schechter and Berger, Biochem. Biophys. Res. Comm. 27,
157-162) being a monosubstituted or polysubstituted or
unsubstituted benzylsulfonyl group, P3 being monosubstituted or
polysubstituted or unsubstituted, natural or unnatural,
.alpha.-amino acid or .alpha.-imino acid in the D configuration, P2
being a monosubstituted or polysubstituted or unsubstituted,
natural or unnatural, .alpha.-amino acid or .alpha.-imino acid in
the L configuration, and P1 being a monosubstituted or
polysubstituted or unsubstituted 4-amidino- or
4-guanidinobenzylamine group, inactivates plasma kallikrein very
effectively, has a coagulation-inhibiting effect even after being
coupled to a synthetic surface and can be used either parenterally,
enterally or topically, in particular intravenously or
subcutaneously.
[0013] A particular advantage of the acylated 4-amidino- or
4-guanidinobenzylamine derivatives according to the invention is
consequently their ability to inactivate PK with high activity even
after binding to a synthetic surface. The compounds according to
the invention therefore constitute a novel group of highly active
and, in particular, couplable plasma kallikrein inhibitors.
[0014] Within the meaning of the present invention, a synthetic
surface is a surface which is composed, for example, of cellulose
diacetate, cellulose triacetate, poly(ether sulfone), poly(aryl
ether sulfone), regenerated cellulose, cuprophan, hemophan,
poly(sulfone), poly(acrylonitrile), poly(vinyl alcohol),
poly(carbonate), poly(amide), poly(methyl methacrylate),
poly(ethylene-co-vinyl alcohol) or another material which is used
in appliances such as dialyzers, oxygenators, catheters and
membranes, and/or the hose systems and air traps which belong to
the appliances, which come into contact with blood, particularly in
extracorporeal circulations, with the surface materials being
modified, where appropriate, with functional groups, e.g. amino
groups, aminoalkyl groups, carboxyl groups, carboxyalkyl groups,
mercapto groups, mercaptoalkyl groups, hydroxyl groups or
hydroxyalkyl groups in order to permit covalent coupling of the
inhibitors.
[0015] According to a preferred embodiment, the substituent at the
substituted P4, P3, P2 and/or P1 is hydrogen and/or a halogen,
preferably fluorine, chlorine and/or bromine, and/or a substituted
or unsubstituted, branched or linear alkyl radical having 1-6 C
atoms, preferably 1-3 C atoms, in particular methyl, or a
substituted or unsubstituted, branched or linear aralkyl radical
having 1-10 C atoms, with the substituent of the substituted,
branched or linear alkyl radical or aralkyl radical preferably
being a halogen, hydroxyl, amino, cyano, amidino, guanidino and/or
carboxyl group, where appropriate esterified with a lower alkyl
radical, in particular with methyl or ethyl, and/or being a
hydroxyl, amino, cyano, amidino, guanidino, methyloxycarbonyl,
benzyl, benzyloxycarbonyl, aminomethyl or glutaryl or
succinylamidomethyl group, and/or being an oxyalkylcarbonyl,
carboxyl, carboxymethyl or carboxyethyl group, where appropriate
esterified with a lower alkyl radical, in particular with methyl or
ethyl, or an oxyalkylcarbonyl, carboxyl, carboxymethyl or
carboxyethyl group which is present as unsubstituted amide or amide
which is substituted by an alkyl or aryl group.
[0016] Unless otherwise stated, an alkyl radical within the meaning
of the present invention is always to be understood as being an
alkyl radical having 1-12 C atoms, while an aryl radical is always
to be understood as being an aryl radical having 6-10 C atoms and
an aralkyl radical is always to be understood as being an aralkyl
radical having 6 to 12 C atoms.
[0017] Within the meaning of the present invention, a lower alkyl
radical is understood as being an alkyl radical having 1 to 6 C
atoms, preferably 1-3 C atoms.
[0018] A linker group can additionally be coupled to P4 or P2, with
the linker group being coupled to P4 by way of one of the
above-described substituents or coupled directly to a functional
group of P2, in particular by way of a --NH-- or --CO-- group.
[0019] A linker group within the meaning of the present invention
is defined as being a chemical structure which exhibits at least
one functional group for covalent coupling to an acylated
4-amidino- or 4-guanidinobenzylamine by way of P4 or P2 and, in
addition, exhibits either at least one second functional group for
simultaneous covalent coupling to a synthetic surface or for the
simultaneous coupling of a second molecule of the acylated
4-amidino- or 4-guanidinobenzylamine and/or exhibits an oligo- or
polyalkylene glycol group which is able to couple noncovalently to
the synthetic surface by interacting with it.
[0020] A linker group according to the present invention is
therefore preferably a dicarboxylic acid, an aminocarboxylic acid,
a diamine, a disulfonic acid or an aminosulfonic acid having an
alkyl, aryl or aralkyl skeletal structure, with the alkyl skeletal
structure exhibiting from 1 to 12 C atoms, in particular 2-6 C
atoms, the aryl skeletal structure exhibiting 6-10 C atoms, in
particular phenyl, and the aralkyl skeletal structure exhibiting
6-12 C atoms, in particular benzyl, or an aminoalkyl or
carboxyalkyl group having 2-12 C atoms, in particular 2-6 C atoms;
or with the linker group at P4 or P2 being an oligo- or
polyalkylene glycol chain, in particular being a poly- or
oligoethylene or poly- or oligopropylene glycol chain, with the
oligo- or polyalkylene glycol exhibiting a functional group, in
particular a substituted or unsubstituted amino, carboxyl and/or
mercapto group, at least at both ends, or with the oligo- or
polyalkylene glycol exhibiting a functional group, in particular a
substituted or unsubstituted amino, carboxyl and/or mercapto group,
at least at one end, and being modified with a CH.sub.3 group at
the other end.
[0021] When the linker group is coupled to P4, the linker group is
preferably coupled to P4 by way of a --NH-- group, --NH-alkyl group
having from 1 to 6 C atoms, in particular methyl, a --CO-- group, a
--CO-alkyl group having 2-6 C atoms, in particular --CO-methyl, a
--CO--O-alkyl group having 1-6 C atoms, in particular methyl, a
--S-- group, a --S-alkyl group having from 1 to 6 C atoms, in
particular methyl, a --O-alkyl group having 1-6 C atoms, in
particular methyl, a --SO.sub.2-- group or a --SO.sub.2-- alkyl
group having 1-6 C atoms, in particular methyl.
[0022] Instead of being coupled to P4, the linker group can also be
coupled to P2, with P2 preferably being lysine or its homologs
having 1-5 C atoms in the side chain, in particular ornithine,
homolysine, .alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, .alpha.-diaminoglycine or
glutamic acid or its homologs having 1-5 C atoms in the side chain,
in particular aspartic acid, glutamic acid or homoglutamic acid or
cysteine or homocysteine or serine or threonine.
[0023] According to a preferred embodiment of the present
invention, the linker group which is coupled to P4 exhibits,
together with the substituent for the coupling to P4, the general
formula U--Z--Y--X-- (II), where U is an H.sub.2N--,
HOOC--(CH.sub.2) n-CO--NH--, HOOC--,
H.sub.2N--(CH.sub.2).sub.n--NH--CO-- or HS-group, with Z being
--(CH.sub.2).sub.n--, in which n=1 to 10, in particular 1-5, or Z
being an oligo- or polyalkylene glycol of the general formula
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--O--CH.sub.2).sub.k-- in which d=1, 2, 3 or 4,
v=an integer of from 1 to 1000, preferably of from 1 to 50, in
particular of from 2 to 10, m=0, 1, 2, 3 or 4 and k=0 or 1 or U is
a CH.sub.3--O-group with Z being an oligo- or polyalkylene glycol
of the general formula
--(CH.sub.2).sub.d--[O--CH.sub.2--CH.sub.2].sub.vO--(CH.sub.2).sub.m--(NH-
--CO--CH.sub.2--O--CH.sub.2).sub.k-- or
--(CH.sub.2).sub.d--[O--CH(CH.sub.3)--CH.sub.2].sub.v--O--(CH.sub.2).sub.-
m--(NH--CO--CH.sub.2--O--CH.sub.2).sub.k-- in which d=1, 2, 3 or 4,
v=an integer of from 1 to 1000, preferably of from 1 to 50, in
particular of from 2 to 10, m=0, 1, 2, 3 or 4 and k=0 or 1; Y is a
--CO--NH-- group, a --NH--CO-- group, a --SO.sub.2--NH-- group, a
--NH--SO.sub.2-- group, a --S--S-- group or a --S-- group, or, if U
and Z are not present, is a H.sub.2N-- group, HOOC-- group, HS--
group, HO-- group or halogenoalkyl group; X is a
--(CH.sub.2).sub.n-- group in which n=0, 1, 2, 3 or 4, in
particular n=1, or is a --(CH.sub.2).sub.n--O-- group having a bond
to the benzyl radical by way of the oxygen and n=1, 2, 3 or 4. The
coupling of the linker group to the benzyl radical is from X, if
present, or from Y, if X is not present.
[0024] If the linker is coupled to P4, P2 is then glycine, alanine,
proline, homoproline or azetidinecarboxylic acid.
[0025] According to another preferred embodiment, the linker group
is coupled to P2, with P2 exhibiting the general formula III
##STR00001##
where q=0, 1, 2, 3, 4 or 5 and D is the formula
U--Z--Y-- (IV)
where U, Z and Y have the same meaning as in formula II.
[0026] According to a particularly preferred embodiment, the
acylated amidino- or guanidinobenzylamine exhibits the general
formula V or VI
##STR00002##
where m=1 to 3 and q is 0 or 1, in particular 0, and where R.sub.1,
R.sub.2, R.sub.3 and/or R.sub.4 is hydrogen and/or a halogen,
preferably fluorine, chlorine and/or bromine, and/or a substituted
or unsubstituted, branched or linear alkyl radical having 1-6 C
atoms, preferably 1-3 C atoms, in particular methyl, with the
substituent of the substituted, branched or linear alkyl radical
preferably being a halogen, hydroxyl, amino, cyano, amidino,
guanidino and/or carboxyl group, where appropriate esterified with
a lower alkyl radical, in particular with methyl or ethyl, and/or
being a hydroxyl, amino, cyano, amidino, guanidino,
methyloxycarbonyl, benzyl, benzyloxycarbonyl, aminomethyl or
glutaryl or succinylamidomethyl group and/or being an
oxyalkylcarbonyl, carboxyl, carboxymethyl or carboxyethyl group,
where appropriate esterified with a lower alkyl radical, in
particular with methyl or ethyl, or being present as unsubstituted
amide or amide which is substituted by an alkyl or aryl group.
[0027] Within the context of the present invention, the hydroxyl
radical, an amino radical and an alkoxycarbonyl radical, in
particular an alkoxycarbonyl radical having from 2 to 10 C atoms,
are particularly preferred as radicals R.sub.4.
[0028] R.sub.1 and/or R.sub.3 can additionally be a linker group,
where the linker group is coupled to P4 by way of one of the
above-described substituents or coupled directly to a functional
group of P2, in particular by way of a --NH-- or a --CO-- group,
with the linker group preferably being a dicarboxylic acid, an
aminocarboxylic acid, a diamine, a disulfonic acid or an
aminosulfonic acid having an alkyl, aryl or aralkyl skeletal
structure, with the alkyl skeletal structure exhibiting from 1 to
12 C atoms, in particular 2-6 C atoms, the aryl skeletal structure
exhibiting 6-10 C atoms, in particular phenyl, and the aralkyl
skeletal structure exhibiting 6-12 C atoms, in particular benzyl,
or an aminoalkyl or carboxyalkyl group having 2-12 C atoms, in
particular 2-6 C atoms; or with the linker group at P4 or P2 being
an oligo- or polyalkylene glycol chain, in particular a poly- or
oligoethylene or poly- or oligopropylene glycol chain, with the
oligo- or polyalkylene glycol exhibiting a functional group, in
particular a substituted or unsubstituted amino, carboxyl and/or
mercapto group, at least at both ends, or with the oligo- or
polyalkylene glycol exhibiting a functional group, in particular a
substituted or unsubstituted amino, carboxyl and/or mercapto group,
at least at one end and being modified with an alkyl group having
1-4 C atoms, in particular CH.sub.3 group, at the other end, and/or
R.sub.1 additionally exhibiting the formula (II) as defined above
and P2 together with R.sub.3 additionally exhibiting the formulae
(III) and (IV) as defined above.
[0029] Preferred exemplary embodiments of acylated amidino- and/or
guanidinobenzylamines in accordance with the general formula I
having a linker group at P4 in accordance with the general formula
II preferably exhibit one of the following structures:
##STR00003##
in which n=1 to 10, m=1 to 3 and q=0 or 1, in particular 0, where
R.sub.2 and R.sub.3 have the abovementioned meanings. By means of
the presence of a second functional group, such as H.sub.2N-- or
HOOC--, the above-listed substances can be coupled covalently to
synthetic surfaces concomitantly with the coupling to P4.
[0030] Other preferred exemplary embodiments of acylated amidino-
and/or guanidinobenzylamines in accordance with the general formula
I having a linker group at P4 in accordance with the general
formula II preferably exhibit the following structures:
##STR00004## ##STR00005##
in which p=0, 1, 2 or 3, q=0 or 1, in particular 0, n=1 to 1000 and
m=1 to 3, where R.sub.2 and R.sub.3 in each case have the
abovementioned meanings. Because of the absence of a second
functional group, the above-listed substances can, aside from the
covalent coupling to P4, only be coupled noncovalently to synthetic
surfaces. This takes place by the oligo- or polyalkylene group of
the linker group interacting with the synthetic surface.
[0031] Within the meaning of the present invention, interaction of
the linker group, in particular of a linker group which contains an
oligo- or polyalkylene group, with a synthetic surface is to be
understood as meaning a noncovalent interaction of this linker
group with the synthetic surface, for example by way of
water-mediated hydrogen bonds, hydrophobic interactions or van der
Waals' interactions.
[0032] Within the meaning of the present invention, the substances
in which two molecules of the formula I are coupled to an oligo- or
polyalkylene group are termed doubly inhibitor-functionalized
oligo- or polyalkylene glycols.
[0033] Another advantage of oligo- and/or polyalkylene derivatives
which are present as pure monomethyl ethers at one end, and are
thus not suitable for covalent coupling, consists in their extended
half-life in the circulation following systemic administration.
[0034] Preferred exemplary embodiments of acylated
amidinobenzylamines in accordance with the general formula I having
a linker group at P2 in accordance with the general formulae III
and IV preferably exhibit one of the following structures:
##STR00006##
in which n=0 to 5, preferably 1 or 2, or
##STR00007##
in which n=0 to 11, or
##STR00008##
in which n=1 to 6, or
##STR00009##
in which n=0 to 3 and m=0 to 1000 or
##STR00010##
in which n=1 to 1000, or
##STR00011##
in which n=1 to 3 and m=1 to 1000, where q is in each case 0 or 1,
in particular 0, and R.sub.2 has in each case the abovementioned
meanings. As a result of the presence of a second functional group,
the above-listed substances can be coupled covalently to synthetic
surfaces or to a second molecule of the general formula I
concomitantly with the coupling to P2.
[0035] Another preferred exemplary embodiment of an acylated
amidino- and/or guanidinobenzylamine in accordance with the general
formula I having a linker group at P2 in accordance with the
general formulae III and IV preferably exhibits one of the
following structures:
##STR00012##
in which n=0 to 4 and m=10 to 1000, or
##STR00013##
in which n=1 to 4, p=2 to 4 and m=1 to 1000, or
##STR00014##
in which n=1 to 3 and m=10 to 1000, where q is 0 or 1, in
particular 0, and R.sub.2 in each case has the abovementioned
meanings. As a result of the absence of a second functional group,
the above-listed substances can, aside from the covalent coupling
at P2, only be coupled noncovalently to synthetic surfaces. This
takes place by means of the oligo- or polyalkylene group of the
linker group interacting with the synthetic surface, for example on
the basis of hydrogen bonds, hydrophobic interactions or van der
Waals' interactions. Within the meaning of the present invention,
the substances in which two molecules of the formula I are coupled
to one oligo- or polyalkylene group are termed doubly
inhibitor-functionalized oligo- or polyalkylene glycols.
[0036] Another advantage of these oligo- and/or polyalkylene
derivatives which are present as pure monomethyl ethers at one end
and are thus not suitable for covalent coupling consists, as in the
case of the derivatives in which the linker group is coupled to P4,
in their extended half-life in the circulation following systemic
administration.
[0037] When the coupling to the synthetic surface takes place by
way of P2, the substituent at P4 is, in particular, H, a halogen,
an amino group, an hydroxyl group or a linear or branched alkyl
group having from 1 to 6 carbon atoms.
[0038] A particularly preferred embodiment of an acylated
amidinobenzylamine in accordance with the general formula I having
a linker group at P4 in accordance with the general formula II
preferably exhibits the following structure:
##STR00015##
where D-Cha in position P3 can, in particular, also be D-Phe or
D-Ser(tBu), and glutaryl at P4 can also be succinyl. This compound
is suitable for simultaneous covalent coupling to a synthetic
surface.
[0039] Another particularly preferred embodiment of an acylated
amidinobenzylamine in accordance with the general formula I having
a linker group at P2 in accordance with the general formulae III
and IV preferably exhibits the following structure:
##STR00016##
where D-Ser(tBu) in position P3 can, in particular, also be D-Cha
or D-Phe, and succinyl at P2 can also be glutaryl. This compound is
suitable for simultaneous covalent coupling to a synthetic
surface.
[0040] Another particularly preferred embodiment of an acylated
amidinobenzylamine in accordance with the general formula I having
a linker group at P2 in accordance with the general formulae III
and IV preferably exhibits one of the following structures:
##STR00017##
where D-Cha in position P3 can, in particular, also be D-Phe or
D-Ser(tBu). These compounds are suitable for simultaneous covalent
coupling to a synthetic surface or for covalent coupling to a
second molecule of the general formula I.
[0041] Further possible exemplary embodiments of acylated
aminobenzylamines which inhibit PK with high activity and
specificity are compounds in accordance with formula I where P4
carries a radical R, P3 is D-Ser, D-Ser(tBu), D-Phe or D-Cha and P2
is a natural or unnatural amino acid Aaa, where R is H--, 4-, 3- or
2-, preferably 4- or 3-COOH, 4-, 3- or 2-, preferably 4- or
3-COOMe, 4-, 3- or 2-, preferably 4- or 3-AMe, 4-, 3- or 2-,
preferably 4- or 3-glutaryl-AMe or 4-, 3- or 2-, preferably 4- or
3-CN, and Aaa is Gly, Ala, Pro, Asp, Glu, Gln, hGlu, Dap, Dap(Z),
Lys, Lys(Z), Arg, Thr, Thr(Bzl), Ser, Ser(Bzl), hSer, hSer(Bzl),
Phe or hPhe.
[0042] In this connection, particular preference is given to the
acylated aminobenzylamines where, when P3 is D-Ser, Aaa is
preferably Gln, Dap, Dap(Z), Lys, Lys(Z), Ser(Bzl), hSer, Phe or
hPhe, in particular Lys(Z), and R is H or, when Aaa is Ala or Ser,
R is HOOC--;
or, when P3 is D-Ser(tBu), Aaa is Pro, Gln, Dap, Dap(Z), Lys,
Lys(Z), Arg, Thr, Thr(Bzl), Ser(Bzl), hSer(Bzl), Phe or hPhe, in
particular Pro, Gln, Lys, Lys(Z), hSer(Bzl), Phe or hPhe, and R is
H or, when Aaa is Gly or Ala, R is HOOC-- or, when Aaa is Pro, R is
CN--; or, when P3 is D-Cha, Aaa is Lys or Glu and R is H or, when
Aaa is Pro, R is glutaryl-AMe, in particular, when Aaa is
--NH--CH--[CH.sub.2--CH.sub.2--CO--NH--(CH.sub.2).sub.3--[O--(CH.sub.2).s-
ub.2].sub.3--CH.sub.2--NH.sub.2]--CO--, R is H.
[0043] As a rule, the acylated 4-amidino- or 4-guanidinobenzylamine
derivatives according to the invention are present in the form of a
salt, in particular of a mineral acid, for example sulfuric acid or
hydrochloric acid, or of a suitable organic acid, for example
acetic acid, formic acid, methylsulfonic acid, succinic acid, malic
acid or trifluoroacetic acid, in particular as hydrochloride,
sulfate or acetate.
[0044] Another preferred embodiment of the present invention is the
reaction of an H.sub.2N group of a linker group coupled to the
acylated 4-amidino- or 4-guanidinobenzylamine with a dicarboxylic
anhydride, preferably the anhydride of succinic acid or of glutaric
acid, with the formation of an HOOC-- group, or the reaction of an
HOOC-- group of a linker group coupled to the acylated 4-amidino-
or 4-guanidinobenzylamine with a diamine with the formation of an
H.sub.2N group. These reactions are carried out using standard
methods which are known to the skilled person.
[0045] The conversion of an H.sub.2N group into an HOOC-- group,
and of an HOOC-- group into an H.sub.2N group, which these
reactions make possible extends the opportunities for coupling the
compounds of the general formula I to synthetic surfaces or to a
second molecule of the general formula I.
[0046] In a particularly preferred embodiment of the present
invention, the linker group which is coupled covalently to P4 or P2
can, in the presence of a second functional group, in particular a
substituted or unsubstituted amino, carboxyl and/or mercapto group,
be simultaneously coupled covalently to synthetic surfaces or,
provided the linker group is an oligo- or polyalkylene glycol,
coupled covalently to a second molecule of the general formula I
with the formation of a doubly inhibitor-functionalized oligo- or
polyalkylene glycol termed.
[0047] According to a preferred embodiment of the present
invention, the synthetic surface to which the acylated 4-amidino-
or 4-guanidinobenzylamine derivatives can be coupled is composed of
cellulose diacetate, cellulose triacetate, poly(ether sulfone),
poly(aryl ether sulfone), regenerated cellulose, cuprophan,
hemophan, poly(sulfone), poly(acrylonitrile), poly(vinyl alcohol),
poly(carbonate), poly(amide), poly(methyl methacrylate),
poly(ethylene-co-vinyl alcohol) or another material which is used
in appliances such as dialyzers, oxygenators, catheters or
membranes, and/or the hose systems and/or air traps which belong to
the appliances, for the surfaces which come into contact with
blood, with the surface material, for the covalent coupling of the
molecule of the general formula I by way of the linker group
coupled to P4 or P2, being modified, where appropriate, with
functional groups, e.g. amino groups, aminoalkyl groups, carboxyl
groups, carboxyalkyl groups, mercapto groups, mercaptoalkyl groups,
hydroxyl groups or hydroxyalkyl groups, with the alkyl radical
exhibiting 1-10, in particular 1-6, C atoms.
[0048] According to another preferred embodiment of the present
invention, the acylated 4-amidino- or 4-guanidinobenzylamine
derivatives are coupled to synthetic surfaces of, for example,
appliances such as dialyzers, oxygenators, catheters and/or
membranes for the purpose of preventing blood coagulation at the
surfaces of these appliances.
[0049] The coupling of the acylated 4-amidino- or
4-guanidinobenzylamine derivatives is preferably effected by
covalently or noncovalently coating the synthetic surface(s) by way
of one of the above-described linker groups which is bonded to a
substituent on P4 and/or where appropriate bonded directly to the
side chain of P2 of the general formula I.
[0050] Within the meaning of the present invention, an appliance is
any device which comes into contact with blood and its
constituents.
[0051] Another preferred embodiment of the present invention is the
use of one or more of the acylated 4-amidino- or
4-guanidinobenzylamine derivatives according to the invention for
producing a pharmaceutical for use as an anticoagulant and/or
antithrombotic agent for preventing and/or treating cardiac
infarction, cerebral stroke, embolisms, deep leg vein thromboses,
e.g. following hip joint operations and/or knee joint replacement,
unstable angina, and complications as a consequence of angioplasty,
in particular percutaneous transluminal coronary angioplasty
(PTCA).
[0052] Within the meaning of the present invention, anticoagulant
is to be understood as meaning any substance which inhibits blood
coagulation. Within the meaning of the present invention,
antithrombotic agents are to be understood as being substances
which are to be used in thrombosis prophylaxis. Within the meaning
of the present invention, angioplasty is to be understood as
meaning a dilatation of blood vessels, in particular using
catheters such as balloon catheters.
[0053] Another embodiment is the use of one or more of the
above-described acylated 4-amidino- or 4-guanidinobenzylamines for
producing a pharmaceutical for use as an anticoagulant and/or
antithrombotic agent for the purpose of preventing and treating
disseminated intravascular coagulation, septic shock, allergies,
the postgastrectomy syndrome, arthritis and ARDS (adult respiratory
distress syndrome).
[0054] According to a preferred embodiment of the present
invention, the acylated 4-amidino- or 4-guanidinobenzylamine
derivatives are used for producing a pharmaceutical for inhibiting
plasma kallikrein and/or factor XIa and/or factor XIIa in a
parenteral use form, in particular in an intraarterial,
intravenous, intramuscular or subcutaneous form, in an enteral use
form, in particular for oral or rectal use, or in topical use form,
in particular as a skin treatment agent. Preference is given to
intravenous or subcutaneous use forms in this connection. The
inhibition of plasma kallikrein is preferred, for example.
[0055] The acylated 4-amidino- or 4-guanidinobenzylamine
derivatives according to the invention can be used, in particular,
for producing a pharmaceutical for inhibiting plasma kallikrein,
which pharmaceutical is in the form of a tablet, a sugar-coated
tablet, a capsule, a pellet, a suppository, a solution, in
particular a solution for injection or infusion, of eye, nose and
ear drops, of a juice, of a capsule, of an emulsion or suspension,
of globuli, of styli, of an aerosol, of a powder, of a paste, of a
cream or of an ointment.
[0056] In addition to the inhibitor according to the invention, the
pharmaceutical can comprise further pharmaceutically suitable
auxiliary substances and/or additives. Suitable auxiliary
substances and/or additives which serve, for example, to stabilize
and/or preserve the pharmaceutical are well-known to the skilled
person (e.g. Sucker H. et al., (1991) Pharmazeutische Technologie
[Pharmaceutical technology], 2nd edition, Georg Thieme Verlag,
Stuttgart). They include, for example, physiological sodium
chloride solutions, Ringer glucose, Ringer lactate, demineralized
water, stabilizers, antioxidants, complexing agents, antimicrobial
compounds, proteinase inhibitors and/or inert gases.
[0057] Another embodiment of the present invention is the use of
acylated amidinobenzylamine of the general formula V or VI, in
which R.sub.4 is, in particular, HO-- and R.sub.1 and R.sub.3 are
not an oligo- or polyalkylene group, for producing a pharmaceutical
for use as an anticoagulant and/or antithrombotic agent in
connection with the abovementioned indications, with the active
compound being present in the form of a prodrug for oral
administration.
[0058] Within the meaning of the present invention, a prodrug is an
acylated amidino- or guanidinobenzylamine according to the general
formula I which is present as a pharmaceutically inactive
derivative of the corresponding pharmaceutically active substance
and, after having been administered orally, is biotransformed
spontaneously or enzymically with the pharmaceutically active
substance being released.
[0059] In addition to the preferred use of the described acylated
amidino- or guanidinobenzylamines for inhibiting plasma kallikrein,
they can also be used for inhibiting other trypsin-like serine
proteases such as thrombin, factor XIIa, factor factor XIa, Xa,
factor IXa, factor VIIa, urokinase, tryptase and plasmin as well as
trypsin-like serine proteases of the complement system.
[0060] The present invention also relates to acylated 4-amidino- or
4-guanidinobenzylamine in accordance with the general formula
P4-P3-P2-P1 (I), with the substance being bound, covalently or
noncovalently, to a synthetic surface by way of one of the
above-described linker groups at P4 and/or at P2. In this
connection, the substance is preferably bound covalently to a
synthetic surface by way of an amide or sulfonamide bond, a
disulfide bridge or the alkylation of a mercapto group, in
particular by way of an amide bond. The substance is bound
noncovalently to a synthetic surface preferably by way of an oligo-
or polyalkylene glycol group, in particular an oligo- or
polyethylene glycol group, interacting with a synthetic
surface.
[0061] The present invention also relates to a synthetic surface,
with the surface being coated covalently or noncovalently with an
acylated 4-amidino- or 4-guanidinobenzylamine according to the
invention. The present invention also relates to an appliance, for
example a dialyzer, oxygenator, catheter or a membrane, together
with the appurtenant hose systems and/or air traps, which con
LdinLs a synLheLic surfadC which is covalently or noncovalently
coated with an acylated 4-amidino- or 4-guanidinobenzylamine
according to the invention.
[0062] The acylated 4-amidino- or 4-guanidinobenzylamine
derivatives according to the invention are synthesized using
methods known to the skilled person. For example, methods known to
the skilled person are used to obtain Boc-protected
4-(acetyloxamidino)benzylamine from the commercially obtainable
4-cyanobenzylamine (Showa Denko, Japan). Another possibility is
that of directly coupling 4-cyanobenzylamine to the Boc- or
Z-protected P2 amino acid and converting the cyano group into the
acetyloxamidine at this stage. After the Boc protecting group has
been eliminated, standard coupling methods are used to couple on
the other amino acids using Boc as the N-terminal protecting group.
The P3 amino acid can also be coupled directly as an N-aryl- or
N-aralkylsulfonyl-protected amino acid. Most of the intermediates
crystallize well and can be readily purified in this way. The
inhibitors are finally purified at the last stage, preferably by
way of preparative, reversed-phase HPLC.
[0063] In that which follows, the invention will be explained in
more detail, without this restricting it, with the aid of the
appended exemplary embodiments and tables.
ABBREVIATIONS EMPLOYED
[0064] Aaa amino acid [0065] Ac acetyl [0066] AcOH acetic acid
[0067] CNA acetonitrile [0068] Amba amidinobenzylamine [0069] AMe
aminomethyl [0070] ARDS adult respiratory distress syndrome [0071]
Boc tert-butyloxycarbonyl [0072] Bzl benzyl [0073] Bzls
benzylsulfonyl [0074] Can canavanine [0075] Cha cyclohexylalanine
[0076] IBCC isobutyl chlorocarbonate [0077] CNBzls
cyanobenzylsulfonyl [0078] Dab .alpha.,.gamma.-diaminobutyric acid
[0079] Dap .alpha.,.beta.-diaminopropionic acid [0080] Dap(Z)
benzyloxycarbonyl-.alpha.,.gamma.-diaminobutyric acid [0081] DCM
dichloromethane [0082] DIEA diisopropylethylamine [0083] DMF
N,N-dimethylformamide [0084] D-Ser D-serine, other amino acids
correspondingly [0085] D-Ser(tBu) D-(tert-butylserine) [0086] F XIa
factor XIa [0087] F XIIa factor XIIa [0088] Glut glutaryl [0089]
GuMe guanidinomethylene [0090] hAla(4-Pyr)
homo-4-pyridylalanine
[0090] ##STR00018## [0091] hGlu beta-homoglutamic acid
[0091] ##STR00019## [0092] hPhe homophenylalanine
[0092] ##STR00020## [0093] hSer beta-homoserine
[0093] ##STR00021## [0094] hTyr homotyrosine
[0094] ##STR00022## [0095] n.d. not determined [0096] PEG
polyethylene glycol [0097] Phe phenylalanine [0098] PK plasma
kallikrein [0099] Pro-MMP 3 pro-matrix metalloprotease 3 [0100]
PyBop benzotriazol-1-yl-N-oxytris(pyrrolidino)phosphonium [0101]
hexafluorophosphate [0102] RT room temperature [0103] Ser(Blz)
serine(benzyl) [0104] Suc succinyl [0105] TFA trifluoroacetic acid
[0106] Tfa trifluoroacetyl [0107] Z benzyloxycarbonyl
Analytical Methods:
[0108] Analytical HPLC: Shimdazu LC-10A system, column; Phenomenex
Luna C.sub.18, 5 .mu.m (250.times.4 mm), solvent A: 0.1% TFA in
water; B: 0.1% B in ACN, gradient: 10% B to 70% B in 60 min, 1
ml/min flow rate, detection at 220 nm.
[0109] Preparative HPLC: Shimdazu LC-8A system, column: Phenomenex
Luna C.sub.18, 5 .mu.m (250.times.30 mm), solvent A: 0.1% TFA in
water; B: 0.1% B in ACN, gradient: 10% B to 55% B in 120 min, 10
ml/min flow rate, detection at 220 nm.
[0110] Mass spectroscopy: The mass spectra were either measured on
a Kompact probe from Kratos (Manchester, UK) using a time of flight
measurement detector and .alpha.-cyanohydroxycinnamic acid as
matrix or using an ESI-MS LCQ from Finnigan (Bremen, Germany).
EXEMPLARY EMBODIMENT 1
Synthesizing
3-(glutarylamidomethyl)benzylsulfonyl-D-Cha-Pro-4-amidinobenzylamide.time-
s.TFA
##STR00023##
[0111] 1a) 3-(Cyano)benzylsulfonic acid, sodium salt
[0112] 30 g (153 mmol) of 3-cyanobenzyl bromide (Aldrich) were
suspended in 150 ml of water and boiled under reflux for 8 h after
21.2 g (168.3 mmol) of Na.sub.2SO.sub.3 had been added. The mixture
was filtered in the hot state and the water was evaporated to some
degree in vacuo. The mixture was stored in a refrigerator overnight
for crystallization; after that, the crystals were filtered off
with suction and recrystallized once again from water. The crystals
were filtered off with suction and dried in vacuo.
[0113] Yield: 17.1 g (78 mmol), HPLC: 18.2% B
1b) 3-(Cyano)benzylsulfonyl chloride
[0114] 5 g (22.83 mmol) of 3-cyanobenzylsulfonic acid sodium salt
were moistened with approx. 20 ml of phosphoryl chloride, after
which 5.2 g (25.11 mmol) of PCI.sub.5 were added and the mixture
was stirred for 15 min while being cooled with ice. The mixture was
then heated at 80.degree. C. for 4 h. After that, the mixture was
poured onto ice and stirred vigorously for 30 min in connection
with which the product sedimented as a white solid on the ice.
After the ice had been partially thawed, the mixture was filtered
through a frit and the product/ice mixture which remained was
washed several times with water. The crystals which remained were
dried in vacuo and used directly for the next step in the
synthesis.
[0115] Yield: 3.4 g (15.8 mmol)
1c) 3-(Cyano)benzylsulfonyl-D-Cha-OH
[0116] 3.775 g (22 mmol) of H-D-Cha-OH were suspended in 100 ml of
dry DCM after which 6.316 ml (50 mmol) of trimethylsilyl chloride
and 8.7 ml (50 mmol) of DIEA were added. The mixture was boiled
under reflux for 1 h and cooled in an ice bath. 5 g (23.18 mmol) of
3-cyanobenzylsulfonyl chloride and 5 ml (28.75 mmol) of DIEA were
then added within the space of 30 min. The mixture was stirred for
a further 30 min while being cooled with ice and then stirred for a
further 3 h at room temperature. The solvent was removed in vacuo
after which the residue was dissolved in water (brought to pH 8.5-9
with 1 N NaOH) and this solution was extracted 2.times. with ethyl
acetate. The ethyl acetate phase was then extracted once again with
alkaline water (pH 9, NaOH). The combined alkaline water phases
were then acidified (pH approx. 3) with a concentrated solution of
HCl and extracted 3.times. with ethyl acetate. The combined ethyl
acetate phase was washed, in each case 3.times., with a 5% solution
of KHSO.sub.4 and a saturated solution of NaCl and then dried over
Na.sub.2SO.sub.4. The solvent was removed in vacuo.
[0117] Yield: 6.99 g of oil which crystallizes slowly in the
refrigerator, HPLC: 53.9% B
1d) H-Pro-4-(Acetyloxamidino)benzylamide.times.HBr
[0118] 75 ml of HBr solution (33% strength in acetic acid) were
added, at room temperature, to 5 g of
Z-Pro-4-(acetyloxamidino)benzylamide (synthesized as described in
WO 02/059065). The mixture was left to stand for one 20 hour while
being shaken occasionally. After that, ether was added to the
mixture and the precipitated product was filtered off with suction
and washed several times on the frit with ether. The product was
dried in vacuo.
[0119] Yield: 4.3 g (11.16 mmol), HPLC 18.3% B
1e)
3-(Cyano)benzylsulfonyl-D-Cha-Pro-4-(acetyloxamidino)benzylamide
[0120] 2.5 g (7.13 mmol) of 3-cyanobenzylsulfonyl-D-Cha-OH and 2.74
g (7.13 g) of H-Pro-4-(acetyloxamidino)benzylamide.times.HBr were
dissolved in 50 ml of DMF. 3.71 g (7.13 mmol) of PyBop and 3.7 ml
of DIEA were added while cooling with ice. The mixture was stirred
for 30 min while being cooled with ice and then stirred at RT for 3
h. The solvent was removed in vacuo after which the mixture was
taken up in ethyl acetate and this solution was washed, in each
case 3.times., with 5% KHSO.sub.4, NaCl-saturated water, a
saturated solution of NaHCO.sub.3 and, once again, with
NaCl-saturated water. The ethyl acetate phase was dried with
Na.sub.2SO.sub.4 and the solvent was then removed in vacuo. The
crude product was used without any further purification for the
next step in the synthesis.
[0121] Yield: 3.3 g of oil, HPLC at 53.77% B
[0122] MS: calculated 578.27 (monoisotopic). found 579.4
[M+H].sup.+.
1f)
3-(Aminomethyl)benzylsulfonyl-D-Cha-Pro-4-(amidino)benzylamide.times.2
HCl
[0123] 1 g of
3-cyanobenzylsulfonyl-D-Cha-Pro-4-(acetyloxami-dino)benzylamide
crude product was dissolved in 500 ml of acetic acid after which
150 ml of 1 N HCl were added. After that, 200 mg of catalyst (10%
palladium on active charcoal) were added and the mixture was
hydrogenated with hydrogen at 50.degree. C. for 15 h. The catalyst
was filtered off and the solvent was evaporated in vacuo. Toluene
was added to the residue and the solvent was removed in vacuo; the
procedure was repeated a further 2.times.. The residue was
dissolved in a little methanol and the product was precipitated by
adding ether and filtered off with suction. The product was washed
with ether and dried in vacuo. The crude product was used without
further purification for the next step in the synthesis.
[0124] Yield: 0.8 g, HPLC at 34.28% B
[0125] MS: calculated 582.30 (monoisotopic). found 583.5
[M+H].sup.+.
1 g)
3-(Glutarylamidomethyl)benzylsulfonyl-D-Cha-Pro-4-(amidino)benzylamid-
e.times.TFA
[0126] 38 mg (0.33 mmol) of glutaric anhydride and 115 .mu.l (0.66
mmol) of DIEA in 5 ml of DMF were added, while cooling with ice, to
200 mg (approx. 0.3 mmol) of
3-(aminomethyl)benzylsulfonyl-D-Cha-Pro-4-(amidino)benzylamide.times.2
HCl crude product. The mixture was stirred for 30 min while being
cooled with ice and then stirred for a further 3 h at RT. The
solvent was removed in vacuo and the crude product was purified by
means of preparative reversed-phase HPLC.
[0127] Yield: 125 mg, HPLC at 40.1% B
[0128] MS: calculated 696.33 (monoisotopic). found 697.8
[M+H].sup.+.
EXEMPLARY EMBODIMENT 2
Synthesizing benzylsulfonyl-D-Ser(tBu)-Lys
(succinyl)-4-Amba.times.TFA
##STR00024##
[0129] 2a) Boc-Lys(Tfa)-4-(acetyloxamidino)benzylamide
[0130] 5 g (14.61 mmol) of Boc-Lys(Tfa)-OH were dissolved in 100 ml
of THF after which 1.767 ml (16.10 mmol) of NMM and 1.899 ml (14.61
mmol) of IBCC were added at -15.degree. C. The mixture was stirred
at -15.degree. C. for 10 min after which 3.74 g (15.33 mmol) of
4-(acetyloxamidino)benzylamine.times.HCl (prepared as described in
WO 01/96282 A2) and, once again, 1.767 ml (16.10 mmol) of NMM were
added. The mixture was stirred for a further hour at -15.degree. C.
and then overnight at room temperature. The solvent was removed in
vacuo and the mixture was taken up in ethyl acetate; this solution
was then washed, in each case 3.times., with 5% KHSO.sub.4,
NaCl-saturated water, a saturated solution of NaHCO.sub.3 and, once
again, with NaCl-saturated water and then dried with
Na.sub.2SO.sub.4. The solvent was removed in vacuo and the product
was crystallized from ethyl acetate.
[0131] Yield: 6.82 g (12.83 mmol) of white crystals, HPLC: 43.87%
B
2b) H-Lys(Tfa)-4-(Acetyloxamidino)benzylamide.times.HCl
[0132] 5 g (9.41 mmol) of
Boc-Lys(Tfa)-4-(acetyloxamidino)benzylamide were solubilized in a
little glacial acetic acid after which 100 ml of 1 N HCl in glacial
acetic acid were added. After the mixture had been standing at room
temperature for 45 min, part of the solvent was evaporated off and
the product was precipitated by adding diethyl ether; it was then
filtered off with suction and washed again with diethyl ether. The
product was dried in vacuo.
[0133] Yield: 4.65 g (10.78 mmol) of white solid, HPLC: 25.52%
B
2c) Bzls-D-Ser(tBu)-Lys(Tfa)-4-(Acetyloxamidino)benzylamide
[0134] 1.93 g (6.107 mmol) of Bzls-D-Ser(tBu)-OH and 3 g (6.412
mmol) of H-Lys(Tfa)-4-(acetyloxamidino)-benzylamide.times.HCl were
dissolved in 30 ml of acetonitrile after which 3.337 g (6.412 mmol)
of PyBop and 3.187 ml (18.32 mmol) of DIEA were added at 0.degree.
C. The mixture was stirred for 30 min at 0.degree. C. and for a
further 4 h at room temperature. The solvent was removed in vacuo
and the residue was taken up in ethyl acetate; this solution was
then washed, in each case 3.times., with 5% KHSO.sub.4,
NaCl-saturated water, a saturated solution of NaHCO.sub.3 and, once
again, with NaCl-saturated water and then dried with
Na.sub.2SO.sub.4. The solvent was removed in vacuo. A slightly
yellow, amorphous crude product remained, with this product being
used directly, without further purification, for the next step in
the synthesis.
[0135] Yield: 5.88 g (crude product), HPLC: 52.93% B
2d)
Bzls-D-Ser(tBu)-Lys(Tfa)-4-(Amidino)benzylamide.times.acetate
[0136] 5.88 g of
Bzls-D-Ser(tBu)-Lys(Tfa)-4-(acetyloxamidino)benzylamide (crude
product) were dissolved in 150 ml of 90% acetic acid after which
500 mg of catalyst (10% Pd/C) were added. The mixture was
hydrogenated with hydrogen for 6 h, at room temperature and under
standard pressure. The catalyst was then filtered off and the
solvent was partially evaporated; the product was then
precipitated, by adding diethyl ether, filtered off with suction
and washed once again with diethyl ether. The white crystalline
precipitate was dried in vacuo.
[0137] Yield: 4.36 g (5.962 mmol), HPLC: 43.50% B
2e) Bzls-D-Ser(tBu)-Lys-4-(Amidino)benzylamide.times.2 TFA
[0138] 5 ml of a 1 M aqueous solution of piperidine were added,
while cooling with ice, to 0.2 g of
Bzls-D-Ser(tBu)-Lys(Tfa)-4-(amidino)benzylamide.times.acetate crude
product and the mixture was stirred for 3 h. After that, the
solvent was evaporated off in vacuo and the remaining residue was
purified by means of preparative reversed-phase HPLC.
[0139] Yield: 72 mg, HPLC: 30.9% B
[0140] MS: calculated 574.29 (monoisotopic). found 575.7
[M+H].sup.+.
2f)
Bzls-D-Ser(tBu)-Lys(Succinyl)-4-(amidino)benzylamide.times.TFA
[0141] 2 ml of DMF, 7.8 mg (0.078 mmol) of succinic anhydride and
27.1 .mu.l (0.156 mmol) of DIEA were added, while cooling with ice,
to 60 mg (0.075 mmol) of
Bzls-D-Ser(tBu)-Lys-4-(amidino)benzylamide.times.2 TFA. The mixture
was stirred for a further 30 min while being cooled with ice and
then for 3 h at room temperature. The solvent was removed in vacuo
and the product was purified by means of preparative reversed-phase
HPLC.
[0142] Yield: 41 mg, HPLC: 35.8% B
[0143] MS: calculated 674.31 (monoisotopic). found 675.9
[M+H].sup.+.
EXEMPLARY EMBODIMENT 3
Synthesizing
benzylsulfonyl-D-Cha-Lys(CO--CH.sub.2--O--CH.sub.2--CO--NH--CH.sub.2--CH.-
sub.2-hexaethylene
glycol-CH.sub.2--CH.sub.2--NH.sub.2)-4-Amba.times.2 TFA
##STR00025##
[0144] 3a) Benzylsulfonyl-D-Cha-OH
[0145] 6 g (35.1 mmol) of H-D-Cha-OH were suspended in 120 ml of
dry DCM after which 9.75 ml (77.2 mmol) of trimethylsilyl chloride
and 13.4 ml (77.2 mmol) of DIEA were added. The mixture was boiled
under reflux for 1 h and then cooled in an ice bath. 7.02 g (36.85
mmol) of benzylsulfonyl chloride and 7.83 ml (45 mmol) of DIEA were
then added within the space of 30 min. The mixture was stirred for
a further 30 min while being cooled with ice and, after that, for a
further 3 h at room temperature. The solvent was removed in vacuo
and the residue was dissolved in water (brought to pH 8.5-9 with 1
N NaOH); this solution was then extracted 2.times. with ethyl
acetate. The alkaline aqueous phase was then acidified (pH approx.
3) with a concentrated solution of HCl and extracted 3.times. with
ethyl acetate. The combined ethyl acetate phase was washed, in each
case 3.times., with a 5% solution of KHSO.sub.4 and an
NaCl-saturated solution and then dried with Na.sub.2SO.sub.4. The
solvent was removed in vacuo.
[0146] Yield: 9.2 g of oil (crystallizes slowly in the
refrigerator), HPLC: 55.8% B
3b) Boc-Lys(Z)-4-(Acetyloxamidino)benzylamide
[0147] 4.41 g (11.59 mmol) of Boc-Lys(Z)--OH were dissolved in 125
ml of DMF after which 1.275 ml (11.59 mmol) of NMM and 1.506 ml
(11.59 mmol) of IBCC were added at -15.degree. C. The mixture was
stirred at -15.degree. C. for 10 min after which 2.97 g (12.17
mmol) of 4-(acetyloxamidino)benzylamine.times.HCl (prepared as
described in WO 01/96286 A2) and, once again, 1.34 ml (12.17 mmol)
of NMM were added. The mixture was stirred for a further hour at
-15.degree. C. and overnight at room temperature. The solvent was
removed in vacuo and the mixture was taken up in ethyl acetate;
this solution was then washed, in each case 3.times., with 5%
KHSO.sub.4, NaCl-saturated water, a saturated solution of
NaHCO.sub.3 and, once again, with NaCl-saturated water and then
dried with Na.sub.2SO.sub.4. The solvent was removed in vacuo and
the amorphous substance which remained was dried in vacuo.
[0148] Yield: 5.2 g, HPLC: 51.12% B
3c) H-Lys(Z)-4-(Acetyloxamidino)benzylamide.times.HCl
[0149] 100 ml of 1 N HCl in glacial acetic acid were added to 5 g
of Boc-Lys(Z)-4-(acetyloxamidino)benzylamide. After the mixture had
been standing at room temperature for 45 min, the solvent was
partially evaporated and the product was precipitated by adding
diethyl ether; it was then filtered off with suction and washed
once again with diethyl ether. The product was dried in vacuo.
[0150] Yield: 4.2 g (8.3 mmol) of white solid, HPLC: 33.81% B
3d) Bzls-D-Cha-Lys(Z)-4-(acetyloxamidino)benzylamide
[0151] 2 g (6.146 mmol) of Bzls-D-Cha-OH and 3.13 g (6.146 mmol) of
H-Lys(Z)-4-(acetyloxamidino)benzylamide.times.HCl were dissolved in
50 ml of DMF after which 3.198 g (6.146 mmol) of PyBop and 3.2 ml
(18.43 mmol) of DIEA were added at 0.degree. C. The mixture was
stirred for 30 min at 0.degree. C. and for a further 5 h at room
temperature. The solvent was removed in vacuo and the residue was
taken up in ethyl acetate; it was then washed, in each case
3.times., with 5% KHSO.sub.4, NaCl-saturated water, a saturated
solution of NaHCO.sub.3 and, once again, with NaCl-saturated water,
and then dried with Na.sub.2SO.sub.4. The solvent was removed in
vacuo. The crude product was used directly, without further
purification, for the next step in the synthesis.
[0152] Yield: 3.7 g (crude product), HPLC: 61.84% B
3e) Bzls-D-Cha-Lys-4-(Amidino)benzylamide.times.2 HBr
[0153] 3.5 g of Bzls-D-Cha-Lys(Z)-4-(acetyloxamidino)-benzylamide
(crude product) were dissolved in 175 ml of 90% acetic acid after
which 400 mg of catalyst (10% Pd/C) were added. The mixture was
hydrogenated with hydrogen for 6 h at room temperature and under
standard pressure. The catalyst was then filtered off and the
solvent was evaporated off; toluene was added to the residue and
the solvent was evaporated off again in vacuo. 50 ml of hydrogen
bromide solution (33% strength in acetic acid) were added to the
residue; the mixture was shaken occasionally. After an hour, the
product was precipitated by adding diethyl ether, filtered off with
suction and washed several times with diethyl ether. The resulting
solid (faintly yellowish) was dried in vacuo. The crude product was
used for the next step in the synthesis.
[0154] Yield: 2.3 g of crude product, HPLC: 34.77% B.
[0155] Part of the crude product was purified by means of
preparative reversed-phase HPLC.
[0156] MS: calculated 584.31 (monoisotopic). found 585.4
[M+H].sup.+.
3f) Bzls-D-Cha-Lys
(CO--CH.sub.2--O--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2-Hexaethylene
glycol-CH.sub.2--CH.sub.2--NH-Boc)-4-(amidino)benzylamide.times.HBr
[0157] 0.318 g (approx. 0.427 mmol) of
Bzls-D-Cha-Lys-4-(amidino)benzylamide.times.2 HBr crude product and
250 mg (0.4275 mmol) of
O--(N-Boc-2-aminoethyl)-O'--(N-diglycolyl)-2-aminoethyl)hexaethylene
glycol (Novabiochem, order no.: 01-63-0102) were dissolved in 10 ml
of DMF. While cooling with ice, 0.222 g (0.4275 mmol) of PyBop and
149 .mu.l (0.855 mmol) of DIEA were added. The mixture was stirred
for 15 min while being cooled with ice and for a further 4 h at
room temperature. After that, the solvent was evaporated off in
vacuo and the residue was taken up in approx. 350 ml of ethyl
acetate and 75 ml of a saturated solution of NaHCO.sub.3. The ethyl
acetate phase was washed once again with a saturated solution of
NaHCO.sub.3 and then 2.times. with a saturated solution of NaCl; it
was then dried with Na.sub.2SO.sub.4. The solvent was removed in
vacuo, resulting in a yellow oil which was used without further
purification for the next step in the synthesis.
[0158] Yield: 446 mg, HPLC: 47.03% B
[0159] Part of the compound was purified by means of preparative
HPLC.
3g) Bzls-D-Cha-Lys
(CO--CH.sub.2--O--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2-hexaethylene
glycol-CH.sub.2--CH.sub.2--NH.sub.2)-4-(amidino)benzylamide.times.2
TFA
[0160] 10 ml of 1 N HCl in acetic acid were added to 400 mg of
compound 3f
(Bzls-D-Cha-Lys(CO--CH.sub.2--O--CH.sub.2--CO--NH--CH.sub.2--CH.sub.2-hex-
aethylene
glycol-CH.sub.2--CH.sub.2--NH-Boc)-4-(amidino)benzylamide.times.-
HBr crude product). After an hour at room temperature, the product
was precipitated by adding diethyl ether, filtered off with suction
and purified by means of preparative HPLC.
[0161] Yield: 210 mg, HPLC: 37.2% B
[0162] MS: calculated 1050.57 (monoisotopic). found 1051.6
[M+H].sup.+.
EXEMPLARY EMBODIMENT 4
Synthesizing
benzylsulfonyl-D-Cha-Glu(NH--[CH.sub.2].sub.3--[O--CH.sub.2--CH.sub.2].su-
b.2--O--[CH.sub.2].sub.3--NH.sub.2)-4-Amba.times.2 TFA
##STR00026##
[0163] 4a) Boc-Glu(OBzl)-4-(Acetyloxamidino)benzylamide
[0164] 3.37 g (10 mmol) of Boc-Glu(OBzl)-OH were dissolved in 100
ml of DMF after which 1.1 ml (10 mmol) of NMM and 1.3 ml (10 mmol)
of IBCC were added. The mixture was stirred at -15.degree. C. for 8
min after which 2.44 g (10 mmol) of
4-(acetyloxamidino)benzylamine.times.HCl (prepared as described in
WO 01/96286 A2) and, once again, 1.1 ml (10 mmol) of NMM were
added. The mixture was stirred for a further hour at -15.degree. C.
and overnight at room temperature. The solvent was removed in vacuo
and the mixture was taken up in ethyl acetate; this solution was
then washed, in each case 3.times., with 5% KHSO.sub.4,
NaCl-saturated water, a saturated solution of NaHCO.sub.3 and, once
again, with NaCl-saturated water and then dried with
Na.sub.2SO.sub.4. The solvent was removed in vacuo and the compound
was crystallized from ethyl acetate.
[0165] Yield: 3.8 g (7.2 mmol), HPLC: 52.34% B
4b) H-Glu(OBzl)-4-(Acetyloxamidino)benzylamide.times.HCl
[0166] 80 ml of 1 N HCl in glacial acetic acid were added to 3 g (6
mmol) of Boc-Glu(OBzl)-4-(acetyloxamidino)benzylamide. After the
mixture had been standing at room temperature for 45 min, the
solvent was partially evaporated off and the product was
precipitated by adding diethyl ether, filtered off with suction and
washed once again with diethyl ether. The product was dried in
vacuo.
[0167] Yield: 2.5 g (5.4 mmol) of white solid, HPLC: 31.07% B
4c) Bzls-D-Cha-Glu(OBzl)-4-(Acetyloxamidino)benzylamide
[0168] 0.84 g (2.59 mmol) of Bzls-D-Cha-OH and 1.2 g (2.59 mmol) of
H-Glu(OBzl)-4-(acetyloxamidino)benzylamide.times.HCl were dissolved
in 40 ml of DMF after which 1.35 g (2.59 mmol) of PyBop and 1.35 ml
(7.77 mmol) of DIEA were added at 0.degree. C. The mixture was
stirred for 30 min at 0.degree. C. and for a further 4 h at room
temperature. The solvent was removed in vacuo and the residue was
taken up in ethyl acetate; this solution was then washed, in each
case 3.times., with 5% KHSO.sub.4, NaCl-saturated water, a
saturated solution of NaHCO.sub.3 and, once again, with
NaCl-saturated water, after which it was dried with
Na.sub.2SO.sub.4. The solvent was removed in vacuo.
[0169] Yield: 1.35 g (oil), HPLC: 63.16% B
4d) Bzls-D-Cha-Glu-4-(Amidino)benzylamide.times.HCl
[0170] 1.2 g of
Bzls-D-Cha-Glu(OBzl)-4-(acetyloxamidino)-benzylamide were dissolved
in 200 ml of 90% acetic acid after which 200 mg of catalyst (10%
Pd/C) were added. The mixture was hydrogenated with hydrogen for 24
h at 45.degree. C. and under standard pressure. The catalyst was
then filtered off and the solvent was evaporated off; toluene was
added to the residue and the solvent was evaporated off once again
in vacuo. The residue was dissolved in 25 ml of a 1 N solution of
HCl in glacial acetic acid and the product was precipitated by
adding diethyl ether, filtered off with suction and washed several
times with diethyl ether. The resulting solid was dried in
vacuo.
[0171] Yield: 0.82 g, HPLC: 40.55% B.
[0172] A part of the crude product was purified by means of
preparative reversed-phase HPLC.
[0173] MS: calculated 585.26 (monoisotopic). found 586.5
[M+H].sup.+.
4e) Bzls-D-Cha-Glu
(NH--[CH.sub.2].sub.3--[O--CH.sub.2--CH.sub.2].sub.2--O--[CH.sub.2].sub.3-
--NH-Boc)-4-(Amidino)benzylamide.times.HCl
[0174] 0.4 g (0.643 mmol) of
Bzls-D-Cha-Glu-4-(amidino)benzylamide.times.HCl and 0.216 g (0.675
mmol) of
Boc-NH--(CH.sub.2).sub.3--(O--CH.sub.2--CH.sub.2).sub.2--O--
(CH.sub.2).sub.3--NH.sub.2 (obtained from Quanta Biodesign, Powell,
Ohio) were dissolved in 10 ml of DMF after which 0.335 g (0.643
mmol) of PyBop and 224 .mu.l (1.29 mmol) of DIEA were added at
0.degree. C. The mixture was stirred for 30 min at 0.degree. C. and
for a further 6 h at room temperature. The solvent was removed in
vacuo and the residue was taken up in a mixture of ethyl acetate
and a saturated solution of NaHCO.sub.3. The mixture was shaken in
a separating funnel and the alkaline phase was separated off. The
ethyl acetate phase was washed once again with a saturated solution
of NaHCO.sub.3. The ethyl acetate was removed in vacuo and the
remaining residue was used without purification for the next step
in the synthesis.
[0175] Yield: 0.35 g (oil), HPLC: 49.17% B
4f) Bzls-D-Cha-Glu
(NH--[CH.sub.2].sub.3--[O--CH.sub.2--CH.sub.2].sub.2--O--[CH.sub.2].sub.3-
--NH.sub.2)-4-(Amidino)benzylamide.times.2 TFA
[0176] 20 ml of 1 N HCl in glacial acetic acid were added to the
crude product of compound 4e
(Bzls-D-Cha-Glu(NH--[CH.sub.2].sub.3--[O--CH.sub.2--CH.sub.2].sub.2--O--[-
CH.sub.2].sub.3--NH-Boc)-4-(amidino)benzylamide.times.HCl). The
mixture was left to stand for 45 min and, after that, the product
was precipitated by adding diethyl ether and filtered off with
suction. The resulting solid was purified by means of preparative
reversed-phase HPLC and the product was lyophilized.
[0177] Yield: 0.21 g of lyophilisate, HPLC: 36.33% B
[0178] MS: calculated 787.43 (monoisotopic). found 788.5
[M+H].sup.+.
EXEMPLARY EMBODIMENT 5
Determining the Inhibitory Constants (K.sub.i Values in M)
[0179] The inhibitory effect for the individual enzymes was
determined in analogy with a method which has already been
described (Storzebecher et al., J. Med. Chem. 40, 3091-3099,
1997).
[0180] Specifically for determining the inhibition of PK, 200 .mu.l
of Tris buffer (0.05 M, 0.154 M NaCI, 5% ethanol, pH 8.0; contains
the inhibitor), 50 .mu.l of substrate (Bzl-Pro-Phe-Arg-pNA in
H.sub.2O) and 25 .mu.l of PK were incubated at 25.degree. C. After
3 min, the reaction was terminated by adding 25 .mu.l of acetic
acid (50%) and the absorption at 405 nm was determined using a
Microplate Reader (Labsystems iEMS Reader MF). The K.sub.i values
were determined, in accordance with Dixon (Biochem. J. 55, 170-171,
1953), by means of linear regression and using a computer program.
The K.sub.i values are the mean of at least three
determinations.
[0181] The inhibition of factor XIa and factor XIIa was determined
in an analogous manner. When determining the inhibitory constants
for human factor XIa (Haemochrom Diagnostica GmbH, Essen, Germany),
H-D-Lys(Z)-Pro-Arg-pNA (Chromozym PCa, Roche Diagnostics GmbH,
Mannheim, Germany) was used as the substrate.
[0182] H-D-HHT-Gly-Arg-pNA (Chromozym XII, Roche Diagnostics GmbH,
Mannheim, Germany) was used as the substrate for measuring the
inhibitory constants of human factor XIIa (Haemochrom Diagnostica
GmbH, Essen, Germany).
TABLE-US-00001 TABLE 1 Inhibition of PK, factor XIIa, factor XIa
and thrombin by compounds of the
(R)-benzylsulfonyl-D-Ser-Aaa-4-Amba type K.sub.i, .mu.M No. Aaa R
PK F XIIa F XIa Thrombin 1 Gly H 1.7 16 2.2 13 2 Ala H 0.070 9.2
0.11 0.11 3 Pro H 0.054 5.1 0.10 0.012 4 Asp H 3.7 >1000 n.d.
>1000 5 Glu H 1.1 >1000 n.d. 38 6 Gln H 0.047 25 0.13 0.49 7
hGlu H 20 >1000 11 >1000 8 Dap H 0.050 15 0.39 0.65 9 Dap(Z)
H 0.042 13 0.28 6.9 10 Lys H 0.016 21 0.89 4.3 11 Lys(Z) H 0.0035
15 0.3 0.18 12 Arg H 0.079 16 0.77 4.7 13 Thr H 0.24 51 0.25 4.0 14
Thr(Bzl) H 0.091 23 0.33 0.30 15 Ser H 0.16 80 0.30 14 16 Ser(Bzl)
H 0.025 9.8 0.30 0.48 17 hSer H 0.020 >1000 n.d. 8.5 18 Phe H
0.021 0.97 0.92 1.6 19 hPhe H 0.048 2.8 0.084 1.2 20 Gly 4-COOH
0.70 >1000 0.60 170 21 Gly 4-COOMe 4.2 42 8.1 9.4 22 Ala 4-COOH
0.016 17 0.015 2.3 23 Ser 4-COOH 0.029 >1000 0.17 120 24 Ser
4-COOMe 0.16 19 0.87 4.2 25 Gly 4-AMe 6.3 17 6.0 8.0
TABLE-US-00002 TABLE 2 Inhibition of PK, factor XIIa, factor XIa
and thrombin by compounds of the
(R)-benzylsulfonyl-D-Ser(tBu)-Aaa-4-Amba type K.sub.i, .mu.M No.
Aaa R PK F XIIa F XIa Thrombin 26 Gly H 0.34 2.6 1.4 0.22 27 Ala H
0.061 2.0 0.030 0.0021 28 Pro H 0.0065 0.49 0.036 0.0020 29 Asp H
0.91 >1000 0.39 6.0 30 Glu H 0.36 19 0.079 2.6 31 Gln H 0.0092
6.3 0.067 0.021 32 hGlu H 8.0 >1000 8.2 >1000 33 Dap H 0.022
4.0 0.19 0.0094 34 Dap(Z) H 0.025 0.93 0.31 0.37 35 Lys H 0.0036
4.4 0.51 0.055 36 Lys(Z) H 0.0094 5.4 0.48 0.024 37 Arg H 0.040 2.6
0.34 0.065 38 Thr H 0.032 14 n.d. 0.044 39 Thr(Bzl) H 0.044 17 0.40
0.019 40 Ser H 0.052 6.0 0.20 0.047 41 Ser(Bzl) H 0.012 1.4 0.20
0.012 42 hSer H 0.21 >1000 0.74 13 43 hSer(Bzl) H 0.0082 80 0.61
0.50 44 Phe H 0.0055 4.6 0.26 0.16 45 hPhe H 0.0045 1.3 0.083 0.048
46 Gly 4-COOH 0.029 7.5 n.d. 2.2 47 Gly 4-COOMe 1.1 4.8 1.6 0.36 48
Ala 4-COOH 0.0062 9.5 0.0069 0.044 49 Ala 4-COOMe 0.054 4.7 0.079
0.0043 50 Gly 4-AMe 4.0 1.8 2.9 0.12 51 Pro 4-CN 0.0094 1.6 0.0091
0.000064
TABLE-US-00003 TABLE 3 Inhibition of PK, factor XIIa, factor XIa
and thrombin by compounds of the
(R)-benzylsulfonyl-D-Cha-Aaa-4-Amba type K.sub.i, .mu.M No. R Aaa
PK F XIIa F XIa Thrombin 52 3-CN Pro 0.086 13 n.d. <0.0010 53 H
Lys 0.0023 0.83 0.15 0.010 54 H Lys(Z) 0.020 4.0 0.34 0.015 55
3-AMe Pro 0.090 0.47 0.17 0.0032 56 3-(Glut-NHCH.sub.2) Pro 0.044
5.6 0.052 <0.0010 57 H Glu 0.030 4.0 0.020 0.081
[0183] Inhibitory constants for PEG-coupled compounds in .mu.M:
[0184] Inhibitor No. 58:
##STR00027##
[0185] PK 0.059; F XIIa 2.0, F XIa 0.23, thrombin 0.0080
[0186] Inhibitor No. 59:
##STR00028##
[0187] PK 0.015; F XIIa 0.98, F XIa 0.040, thrombin 0.015
TABLE-US-00004 TABLE 4 Inhibition of PK, factor XIIa, factor XIa
and thrombin by compounds of the (4-R)-benzylsulfonyl-P3-Aaa-4-Amba
type ((4-R) denotes the 4 position of the radical R in Table 4 on
the phenyl ring of the benzylsulfonyl radical) K.sub.i, .mu.M No. R
P3 Aaa PK F XIIa F XIa Thrombin 60 H D-hAla(4-Pyr) Glu(OBzl) 0.0055
0.094 0.031 0.17 61 COOH D-Ser Pro 0.0091 29 0.014 0.24 62 H
D-Ser(tBu) Lys(Tfa) 0.011 6.1 n.d. 0.0029 63 H D-Cha Gly 0.011 0.70
25 0.0090 64 H D-Ser(tBu) His 0.014 61 n.d. 0.12 65 COOH D-Ser(tBu)
Ser 0.015 17 0.030 2.0 66 CH.sub.2COOH D-Ser(tBu) Pro 0.016 4 n.d.
0.0018 67 H D-hPhe Ser 0.019 0.63 3.0 0.55 68 H D-Ser(tBu) Can
0.019 6.8 n.d. 0.038 69 H D-hAla(4-Pyr) Ser 0.020 1.6 n.d. 0.91 70
COOH D-Ser(tBu) Pro 0.025 2.4 n.d. 0.0023 71 H D-Cha Lys(Suc) 0.029
11 n.d. 0.0021 72 H D-hTyr Glu 0.22 0.36 0.028 19 73 H D-hTyr Ser
0.13 0.28 0.078 1.4 74 NO.sub.2 D-hPhe Gly 0.051 0.39 0.093 0.71 75
H D-hTyr Gly 0.12 0.78 0.61 1.5 76 H D-hPhe Gly 0.39 0.15 0.27
0.047 77 H D-Phe(3-amidino) Gly 0.082 0.19 0.25 0.085 78 NH.sub.2
D-hPhe Gly 0.045 0.26 0.12 0.26 79 H D-Phe(3-GuMe) Gly 0.075 0.31
0.22 0.059 80 H D-norarginine Gly 0.068 0.34 0.49 2.1 81 H D-Arg
Gly 0.074 0.35 0.70 1.4 82 H D-Cha Gly 0.10 1.4 0.33 0.023 83 H
D-indanylglycine Gly 0.075 0.37 n.d. 0.14 84 COOCH.sub.3
D-Phe(3-amidino) Gly 0.14 0.38 0.70 0.53 85 H D/L-hAla(4-Pyr) Gly
0.13 0.40 1.1 2.0 86 H D-Ser Lys(Glut) 0.39 n.d. n.d. 2.8
[0188] Inhibitor 87:
##STR00029##
[0189] K.sub.i values in .mu.M: PK 0.42; F XIIa 0.16; F XIa 0.33,
thrombin 3.6
[0190] Inhibitor 88
##STR00030##
[0191] K.sub.i values in .mu.M: PK 0.22; F XIIa 21; F XIa 0.4,
thrombin 1.2
[0192] Inhibitor 89
##STR00031##
[0193] K.sub.i values in .mu.M: PK 0.19; F XIIa 79; thrombin 1.72.
PEG.sub.5000 denotes a polyethylene glycol having an average
molecular weight of 5000 daltons.
EXEMPLARY EMBODIMENT 6
Preventing the Activation of Prothrombin in Hirudin-Anticoagulated
Plasma
[0194] Venous blood from healthy voluntary donors was mixed,
immediately after removal, with hirudin solution (2000 ATU/ml, 0.9%
NaCI solution) in a ratio of 10:1 and this mixture was centrifuged
at 250.times.g for 10 min. 950 .mu.l of plasma were mixed with 20
.mu.l of inhibitor solution (5 or 0.5 mM) and incubated at
37.degree. C., for 5 h in polypropylene tubes. 30 .mu.l of kaolin
(PTT reagent, diluted 1:1000; Roche Diagnostics, Penzberg, Germany)
were added in order to augment the activation at the synthetic
surface.
[0195] An enzyme immunoassay (Enzygnost-F 1+2, DadeBehring GmbH,
Marburg, Germany) in accordance with the Sandwich principle was
used for determining the prothrombin fragment F 1+2. The
prothrombin fragment binds to fixed antibodies directed against F
1+2. Peroxidase-conjugated antibodies directed against prothrombin
bind in a second step and the bound enzyme activity is determined
chromogenically. The concentration of prothrombin fragment F 1+2
was ascertained from a calibration curve.
TABLE-US-00005 TABLE 5 Influence of different compounds on the
activation of prothrombin in hirudin- anticoagulated plasma in
polypropylene tubes in the added presence of kaolin. The quantity
of the prothrombin fragment F 1 + 2 (in nM) which was detected
after 5 h in the presence of kaolin was set at 100%. Prothrombin
fragment F 1 + 2 (%) Kaolin + Kaolin + Kaolin + Inhibitor inhibitor
inhibitor inhibitor no. +kaolin -kaolin 100 .mu.M 10 .mu.M 1 .mu.M
45 100 0.64 0.11 0.59 n.d. 11 100 0.49 0.15 110.9 n.d. 53 100 0.46
0.08 0.09 0.59 59 100 0.46 0.03 0.20 59.3 75 100 0.14 n.d. 0.01
0.07 73 100 0.14 n.d. 0.04 0.07
EXEMPLARY EMBODIMENT 7
Use of a PK Inhibitor for Affinity Chromatography as a Model for
Modifying a Synthetic Surface
[0196] The material for an affinity chromatography was prepared by
coupling the inhibitor benzylsulfonyl-D-Ser-Lys-4-Amba to
CH-Sepharose 4B (Pharmacia). For this, 16 g of swollen CH-Sepharose
4B were first of all suspended in 65 ml of MES buffer (0.1 M, pH
4.75) after which the inhibitor (50 mg in 2 ml of buffer) was
added. 2.837 g of N-cyclohexyl-N'-(2-morpholino-ethyl)carbodiimide
metho-p-toluenesulfonate (Acros Organics) were added to the mixture
(corresponds to 0.1 M in the mixture) and the whole was incubated
at room temperature for 24 h. The Sepharose was then washed with
MES buffer and water and equilibrated with Tris buffer (0.05 M,
contains 0.75 M NaCl, pH 7.5). After the column (1.4.times.19 cm)
had been packed and equilibrated, 100 .mu.g of PK (Haemochrom
Diagnostics, Essen, Germany) were loaded on in 1 .mu.l of buffer.
The column was then washed firstly with Tris buffer and then with a
3 M NaCl solution, with no PK being eluted in this connection. 41%
active PK was eluted by means of a subsequent benzamidine gradient
(0.1-2.5 M).
[0197] A comparable result can be obtained when using an affinity
chromatography column in which the inhibitor depicted below is
coupled on covalently.
##STR00032##
* * * * *