U.S. patent application number 09/214143 was filed with the patent office on 2001-11-29 for compounds.
Invention is credited to KARLSSON, OLLE, LINSCHOTEN, MARCEL, NYSTROM, JAN-ERIK.
Application Number | 20010046981 09/214143 |
Document ID | / |
Family ID | 20409280 |
Filed Date | 2001-11-29 |
United States Patent
Application |
20010046981 |
Kind Code |
A1 |
KARLSSON, OLLE ; et
al. |
November 29, 2001 |
COMPOUNDS
Abstract
There is provided compounds of formula I, 1 wherein R.sup.1,
R.sup.2, R.sup.3, R.sub.x, Y, n and B have meanings given in the
description which are useful as competitive inhibitors of
trypsin-like proteases, such as thrombin, and in particular in the
treatment of conditions where inhibition of thrombin is required
(e.g. thrombosis) or as anticoagulants.
Inventors: |
KARLSSON, OLLE; (MOLNDAL,
SE) ; LINSCHOTEN, MARCEL; (VASTRA FROLUNDA, SE)
; NYSTROM, JAN-ERIK; (LINDOME, SE) |
Correspondence
Address: |
NIXON & VANDERHYE
1100 NORTH GLEBE ROAD
8TH FLOOR
ARLINGTON
VA
222014714
|
Family ID: |
20409280 |
Appl. No.: |
09/214143 |
Filed: |
December 29, 1998 |
PCT Filed: |
December 1, 1998 |
PCT NO: |
PCT/SE98/02187 |
Current U.S.
Class: |
514/183 |
Current CPC
Class: |
A61K 38/00 20130101;
A61P 7/02 20180101; C07D 207/16 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/183 |
International
Class: |
C12P 019/12; A61K
031/33; A01N 043/00 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 5, 1997 |
SE |
9704543-9 |
Claims
1. A compound of formula I, 21wherein R.sup.1 represents H,
C(O)R.sup.11, SiR.sup.12R.sup.13R.sup.14 or C.sub.1-6 alkyl (which
latter group is optionally substituted or terminated by one or more
substituents selected from OR.sup.15 or (CH.sub.2).sub.qR.sup.16);
R.sup.12, R.sup.13 and R.sup.14 independently represent H, phenyl
or C.sub.1-6 alkyl; R.sup.16 represents C.sub.1-4 alkyl, phenyl,
OH, C(O)OR.sup.17 or C(O)N(H)R.sup.18; R.sup.18 represents H,
C.sub.1-4 alkyl or CH.sub.2C(O)OR.sup.19; R.sup.15 and R.sup.17
independently represent H, C.sub.1-6 alkyl or C.sub.7-9
alkylphenyl; R.sup.11 and R.sup.19 independently represent H or
C.sub.1-4 alkyl; and q represents 0, 1 or 2; R.sup.2 and R.sup.3
independently represent H, C.sub.1-4 alkyl, cyclohexyl or phenyl;
R.sub.x represents a structural fragment of formula IIa, IIb or
IIc, 22wherein k, l and m independently represent 0, 1, 2, 3 or 4;
R.sup.4 and R.sup.5 independently represent H, Si(Me).sub.3, 1- or
2-naphthyl, a polycyclic hydrocarbyl group, CHR.sup.41R.sup.42 or
C.sub.1-4 alkyl (which latter group is optionally substituted by
one or more halo substituents), or C.sub.3-8 cycloalkyl, phenyl,
methylenedioxyphenyl, benzodioxanyl, benzofuranyl,
dihydrobenzofuranyl, benzothiazolyl, benzoxazolyl, benzimidazolyl,
coumaranonyl, coumarinyl or dihydrocoumarinyl (which latter twelve
groups are optionally substituted by one or more of C.sub.1-4 alkyl
(which latter group is optionally substituted by one or more halo
substituents), C.sub.1-4 alkoxy, halo, hydroxy, cyano, nitro,
SO.sub.2NH.sub.2, C(O)OH or N(H)R.sup.43); R.sup.41 and R.sup.42
independently represent cyclohexyl or phenyl; R.sup.6 and R.sup.7
independently represent H, C.sub.1-4 alkyl, C.sub.3-8 cycloalkyl,
phenyl (which latter group is optionally substituted by one or more
of C.sub.1-4 alkyl (which latter group is optionally substituted by
one or more halo substituents), C.sub.1-4 alkoxy, halo, hydroxy,
cyano, nitro, SO.sub.2NH.sub.2, C(O)OH or N(H)R.sup.44) or together
with the carbon atom to which they are attached form a C.sub.3-8
cycloalkyl ring; R.sup.43 and R.sup.44 independently represent H or
C(O)R.sup.45; and R.sup.45 represents H, C.sub.1-4 alkyl or
C.sub.1-4 alkoxy; Y represents CH.sub.2, (CH.sub.2).sub.2,
CH.dbd.CH, (CH.sub.2).sub.3, CH.sub.2CH.dbd.CH or
CH.dbd.CHCH.sub.2, which latter three groups are optionally
substituted by C.sub.1-4 alkyl, methylene, oxo or hydroxy; n
represents 0, 1, 2, 3 or 4; and B represents a structural fragment
of formula IVa, IVb, IVc or IVd, 23wherein X.sup.1, X.sup.2,
X.sup.3 and X.sup.4 independently represent CH, N or N--O; X.sup.5
and X.sup.6 independently represent a single bond or CH.sub.2; one
of X.sup.7, X.sup.8 and X.sup.9 represents S, O or NH, and the
other two independently represent --CH.dbd., .dbd.CH--, --N.dbd.,
.dbd.N--, --N(O).dbd. or .dbd.N(O)--; R.sup.31 represents, in all
cases, one or more optional substituents selected from halo,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy or
--O--(CH.sub.2).sub.p--C(O)N(R.sup.32)(R.sup.33); p represent 0, 1,
2, 3 or 4; and R.sup.32 and R.sup.33 independently represent H,
C.sub.1-6 alkyl or C.sub.3-7 cycloalkyl; or a pharmaceutically
acceptable salt thereof.
2. A compound of formula I, as defined in claim 1, wherein, when B
represents a structural fragment for formula IVa (in which X.sup.1,
X.sup.2, X.sup.3 and X.sup.4 all represent CH), a structural
fragment of formula or IVb or a structural fragment of formula or
IVc, R.sup.31 represents one or more substituents selected from
halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy or
--O--(CH.sub.2).sub.p--C(O)N(R.sup.32)(R.sup.33).
3. A compound of formula I, as defined in claim 1 or claim 2,
wherein B represents a structural fragment for formula IVa.
4. A compound of formula I, as defined in any one of the preceding
claims, wherein the fragment 24is in the S-configuration.
5. A compound of formula Ia, 25wherein B.sup.a represents a
structural fragment of formula IVe, IVf, IVg or IVh 26wherein
D.sup.1 and D.sup.2 independently represent, in each case, H, OH,
OR.sup.a, OC(O)R.sup.b, OC(O)OR.sup.c, C(O)OR.sup.d, C(O)R.sup.e,
and R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e independently
represent C.sub.1-12 alkyl (which latter group is optionally
interrupted by oxygen and/or substituted by halo), phenyl,
naphthyl, C.sub.1-3 alkylphenyl (which latter three groups are
optionally substituted by C.sub.1-4 alkyl, C.sub.1-4 alkoxy, nitro,
or halo) or --(C(R)(R.sup.f)).sub.2OC(O)C(R.sup.h), R.sup.f,
R.sup.g and R.sup.h independently represent H or C.sub.1-4 alkyl,
and R.sup.1, R.sup.2, R.sup.3, R.sub.x, Y, n, X.sup.1, X.sup.2,
X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8, X.sup.9 and
R.sup.31 are as defined in claim 1, or a pharmaceutically
acceptable salt thereof, provided that D.sup.1 and D.sup.2 do not
both represent H.
6. A pharmaceutical formulation including a compound as defined in
any one of the preceding claims, or a pharmaceutically acceptable
salt thereof, in admixture with a pharmaceutically acceptable
adjuvant, diluent or carrier.
7. A compound as defined in any one of claims 1 to 5, or a
pharmaceutically acceptable salt thereof, for use as a
pharmaceutical.
8. A compound as defined in any one of claims 1 to 5, or a
pharmaceutically acceptable salt thereof, for use in the treatment
of a condition where inhibition of thrombin is required or
desired.
9. A compound as defined in any one of claims 1 to 5, or a
pharmaceutically acceptable salt thereof, for use in the treatment
of thrombosis.
10. A compound as defined in any one of claims 1 to 5, or a
pharmaceutically acceptable salt thereof, for use as an
anticoagulant.
11. The use of a compound as defined in any one of claims 1 to 5,
or a pharmaceutically acceptable salt thereof, as active ingredient
in the manufacture of a medicament for the treatment of a condition
where inhibition of thrombin is required or desired.
12. The use as claimed in claim 11, wherein the condition is
thrombosis.
13. The use of a compound as defined in any one of claims 1 to 5,
or a pharmaceutically acceptable salt thereof, as active ingredient
in the manufacture of an anticoagulant.
14. A method of treatment of a condition where inhibition of
thrombin is required or desired which method comprises
administration of a therapeutically effective amount of a compound
as defined in any one of claims 1 to 5, or a pharmaceutically
acceptable salt thereof, to a person suffering from, or susceptible
to, such a condition.
15. A method as claimed in claim 14, wherein the condition is
thrombosis.
16. A method as claimed in claim 14, wherein the condition is
hypercoagulability in blood and tissues.
17. The use of a compound as defined in claim 5 as a prodrug.
18. A process for the preparation of a compound of formula I which
comprises: (a) the coupling of a compound of formula V, 27 wherein
R.sup.1, R.sup.2, R.sup.3 and R.sub.x are as defined in claim 1
with a compound of formula VI, 28 wherein Y, n and B are as defined
in claim 1; (b) the coupling of a compound of formula VII, 29
wherein R.sup.1, R.sup.2, R.sup.3, R.sub.x and Y are as defined in
claim 1 with a compound of formula
VIII,H.sub.2N--(CH.sub.2).sub.n--B VIII wherein n and B are as
defined in claim 1; (c) for compounds of formula I in which B
represents a structural fragment of formula IVa, IVb or IVc,
reaction of a compound of formula IX, 30 wherein B.sup.1 represents
a structural fragment of formula IVa.sup.1, Ivb.sup.1 or IVc.sup.1
31 wherein R.sup.y represents C.sub.1-4 alkyl and R.sup.1, R.sup.2,
R.sup.3, R.sub.xY, n, X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.5,
X.sup.6 and R.sup.31 are as defined in claim 1, with ammonia gas;
(d) for compounds of formula I in which B represents a structural
fragment of formula IVa, IVb or IVc, reduction of a compound of
formula X, 32 wherein B.sup.2 represents a structural fragment of
formula IVa.sup.2, IVb.sup.2 or IVc.sup.2 33 and R.sup.1, R.sup.2,
R.sup.3, R.sub.x, Y, n, X.sup.1, X.sup.2, X.sup.3, X.sup.4,
X.sup.5, X.sup.6 and R.sup.31 are as defined claim 1; or (e) for
compounds of formula I wherein X.sup.1, X.sup.2, X.sup.3, X.sup.4,
X.sup.7, X.sup.8 and/or X.sup.9 represent N-O, oxidation of a
corresponding compound of formula I in which X.sup.1, X.sup.2,
X.sup.3, X.sup.4, X.sup.7, X.sup.8 and/or X.sup.9 (as appropriate)
represent N.
Description
FIELD OF THE INVENTION
[0001] This invention relates to novel pharmaceutically useful
compounds, in particular competitive inhibitors of trypsin-like
serine proteases, especially thrombin, their use as medicaments,
pharmaceutical compositions containing them and synthetic routes to
their production.
BACKGROUND
[0002] Blood coagulation is the key process involved in both
haemostasis (i.e. the prevention of blood loss from a damaged
vessel) and thrombosis (i.e. the formation of a blood clot in a
blood vessel, sometimes leading to vessel obstruction).
[0003] Coagulation is the result of a complex series of enzymatic
reactions. One of the ultimate steps in this series of reactions is
the conversion of the proenzyme prothrombin to the active enzyme
thrombin.
[0004] Thrombin is known to play a central role in coagulation. It
activates platelets, leading to platelet aggregation, converts
fibrinogen into fibrin monomers, which polymerise spontaneously
into fibrin polymers, and activates factor XIII, which in turn
crosslinks the polymers to form insoluble fibrin. Furthermore,
thrombin activates factor V and factor VIII leading to a "positive
feedback" generation of thrombin from prothrombin.
[0005] By inhibiting the aggregation of platelets and the formation
and crosslinking of fibrin, effective inhibitors of thrombin would
therefore be expected to exhibit antithrombotic activity. In
addition, antithrombotic activity would be expected to be enhanced
by effective inhibition of the positive feedback mechanism.
PRIOR ART
[0006] The early development of low molecular weight inhibitors of
thrombin has been described by Claesson in Blood Coagul. Fibrinol.
(1994) 5, 411.
[0007] Blombck et al. (in J. Clin. Lab. Invest. 24, suppl. 107, 59,
(1969)) reported thrombin inhibitors based on the amino acid
sequence situated around the cleavage site for the fibrinogen
A.alpha. chain. Of the amino acid sequences discussed, these
authors suggested the tripeptide sequence Phe-Val-Arg (P9-P2-P1,
hereinafter referred to as the P3-P2-P1 sequence) would be the most
effective inhibitor.
[0008] Thrombin inhibitors based on dipeptidyl derivatives with an
.alpha.,.omega.-aminoalkyl guanidine in the P1-position are known
from U.S. Pat. No. 4,346,078 and International Patent Application
WO 93/11152. Similar, structurally related, dipeptidyl derivatives
have also been reported. For example International Patent
Application WO 94/29336 discloses compounds with, for example,
aminomethyl benzamidines, cyclic aminoalkyl amidines and cyclic
aminoalkyl guanidines in the P1-position; European Patent
Application 0 648 780, discloses compounds with, for example,
cyclic aminoalkyl guanidines in the P1-position.
[0009] Thrombin inhibitors based on peptidyl derivatives, also
having cyclic aminoalkyl guanidines (e.g. either 3- or 4-
aminomethyl-1-amidinopiperidine) in the P1-position are known from
European Patent Applications 0 468 231, 0 559 046 and 0 641
779.
[0010] Thrombin inhibitors based on tripeptidyl derivatives with
arginine aldehyde in the P1-position were first disclosed in
European Patent Application 0 185 390.
[0011] More recently, arginine aldehyde-based peptidyl derivatives,
modified in the P3-position, have been reported. For example,
International Patent Application WO 93/18060 discloses hydroxy
acids, European Patent Application 0 526 877 des-amino acids, and
European Patent Application 0 542 525 O-methyl mandelic acids in
the P3-position.
[0012] Inhibitors of serine proteases (e.g. thrombin) based on
electrophilic ketones in the P1-position are also known. For
example, European Patent Application 0 195 212 discloses peptidyl
.alpha.-keto esters and amides, European Patent Application 0 362
002 fluoroalkylamide ketones, European Patent Application 0 364 344
.alpha.,.beta.,.delta.-tri- ketocompounds, and European Patent
Application 0 530 167 .alpha.-alkoxy ketone derivatives of arginine
in the P1-position.
[0013] Other, structurally different, inhibitors of trypsin-like
serine proteases based on C-terminal boronic acid derivatives of
arginine and isothiouronium analogues thereof are known from
European Patent Application 0 293 881.
[0014] More recently, thrombin inhibitors based on peptidyl, and
amino acid, derivatives have been disclosed in European Patent
Application 0 669 317 and International Patent Applications WO
95/35309, WO 95/23609, WO 94/29336, WO 97/02284, WO 97/46577, WO
98/06740 and WO 98/06741.
[0015] However, there remains a need for a effective inhibitors of
trypsin-like serine proteases, such as thrombin. There is a
particular need for compounds which are both orally bioavailable
and selective in inhibiting thrombin over other serine proteases.
Compounds which exhibit competitive inhibitory activity towards
thrombin would be expected to be especially useful as
anticoagulants and therefore in the therapeutic treatment of
thrombosis and related disorders.
DISCLOSURE OF THE INVENTION
[0016] According to the invention there is provided a compound of
formula I, 2
[0017] wherein
[0018] R.sup.1 represents H, C(O)R.sup.11,
SiR.sup.12R.sup.13R.sup.14 or C.sub.1-6 alkyl (which latter group
is optionally substituted or terminated by one or more substituents
selected from OR.sup.15 or (CH.sub.2).sub.qR.sup.16);
[0019] R.sup.12, R.sup.13 and R.sup.14 independently represent H,
phenyl or C.sub.1-6 alkyl;
[0020] R.sup.16 represents C.sub.1-4 alkyl, phenyl, OH,
C(O)OR.sup.17 or C(O)N(H)R.sup.18;
[0021] R.sup.18 represents H, CH.sub.1-4 alkyl or
CH.sub.2C(O)OR.sup.19;
[0022] R.sup.15 and R.sup.17 independently represent H, C.sub.1-6
alkyl or C.sub.7-9 alkylphenyl;
[0023] R.sup.11 and R.sup.19 independently represent H or C.sub.1-4
alkyl; and
[0024] q represents 0, 1 or 2;
[0025] R.sup.2 and R.sup.3 independently represent H, C.sub.1-4
alkyl, cyclohexyl or phenyl;
[0026] R.sub.x represents a structural fragment of formula IIa, IIb
or IIc, 3
[0027] wherein
[0028] k, l and m independently represent 0, 1, 2, 3 or 4;
[0029] R.sup.4 and R.sup.5 independently represent H, Si(Me).sub.3,
1- or 2-naphthyl, a polycyclic hydrocarbyl group,
CHR.sup.41R.sup.42 or C.sub.1-4 alkyl (which latter group is
optionally substituted by one or more halo substituents), or
C.sub.3-8 cycloalkyl, phenyl, methylenedioxyphenyl, benzodioxanyl,
benzofuranyl, dihydrobenzofuranyl, benzothiazolyl, benzoxazolyl,
benzimidazolyl, coumaranonyl, coumarinyl or dihydrocoumarinyl
(which latter twelve groups are optionally substituted by one or
more of C.sub.1-4 alkyl (which latter group is optionally
substituted by one or more halo substituents), C.sub.1-4 alkoxy,
halo, hydroxy, cyano, nitro, SO.sub.2NH.sub.2, C(O)OH or
N(H)R.sup.43); R.sup.41 and R.sup.42 independently represent
cyclohexyl or phenyl;
[0030] R.sup.6 and R.sup.7 independently represent H, C.sub.1-4
alkyl, C.sub.3-8 cycloalkyl, phenyl (which latter group is
optionally substituted by one or more of C.sub.1-4 alkyl (which
latter group is optionally substituted by one or more halo
substituents), C.sub.1-4 alkoxy, halo, hydroxy, cyano, nitro,
SO.sub.2NH.sub.2, C(O)OH or N(H)R.sup.44) or together with the
carbon atom to which they are attached form a C.sub.3-8 cycloalkyl
ring;
[0031] R.sup.43 and R.sup.44 independently represent H or
C(O)R.sup.45; and
[0032] R.sup.45 represents H, C.sub.1-4 alkyl or C.sub.1-4
alkoxy;
[0033] Y represents CH.sub.2, (CH.sub.2).sub.2, CH.dbd.CH,
(CH.sub.2).sub.3, CH.sub.2CH.dbd.CH or CH.dbd.CHCH.sub.2, which
latter three groups are optionally substituted by C.sub.1-4 alkyl,
methylene, oxo or hydroxy;
[0034] n represents 0, 1, 2, 3 or 4; and
[0035] B represents a structural fragment of formula IVa, IVb, IVc
or IVd, 4
[0036] wherein
[0037] X.sup.1, X.sup.2, X.sup.3 and X.sup.4 independently
represent CH, N or N--O;
[0038] X.sup.5 and X.sup.6 independently represent a single bond or
CH.sub.2;
[0039] one of X.sup.7, X.sup.8 and X.sup.9 represents S, O or NH,
and the other two independently represent --CH.dbd., .dbd.CH--,
--N.dbd., N--, --N(O).dbd. or .dbd.N(O)--; R.sup.31 represents, in
all cases, one or more optional substituents selected from halo,
C.sub.1-4 alkyl, C.sub.1-4 alkoxy or
--O--(CH.sub.2).sub.p--C(O)N(R.sup.32)(R.sup.33); p represent 0, 1,
2, 3 or 4; and
[0040] R.sup.32 and R.sup.33 independently represent H, C.sub.1-6
alkyl or C.sub.3-7 cycloalkyl;
[0041] or a pharmaceutically acceptable salt thereof.
[0042] The compounds of formula I may exhibit tautomerism. All
tautomeric forms and mixtures thereof are included within the scope
of the invention.
[0043] The compounds of formula I may also contain one or more
asymmetric carbon atoms and may therefore exhibit optical and/or
diastereoisomerism. All diastereoisomers may be separated using
conventional techniques, e.g. chromatography or fractional
crystallisation. The various stereoisomers may be isolated by
separation of a racemic or other mixture of the compounds using
conventional, e.g. fractional crystallisation or HPLC, techniques.
Alternatively the desired optical isomers may be made by reaction
of the appropriate optically active starting materials under
conditions which will not cause racemisation or epimerisation, or
by derivatisation, for example with a homochiral acid followed by
separation of the diastereomeric derivatives by conventional means
(e.g. HPLC, chromatography over silica). All stereoisomers are
included within the scope of the invention.
[0044] Alkyl groups which R.sup.1, R.sup.2, R.sup.3, R.sup.4,
R.sup.5, R.sup.6, R.sup.7, R.sup.11, R.sup.12, R.sup.13, R.sup.14,
R.sup.15, R.sup.16, R.sup.17, R.sup.18, R.sup.19, R.sup.31,
R.sup.32, R.sup.33 and R.sup.45 may represent, and with which
R.sup.4, R.sup.5, R.sup.6, R.sup.7 and Y may be substituted; alkoxy
groups which R.sup.31 and R.sup.45 may represent and with which
R.sup.4, R.sup.5, R.sup.6 and R.sup.7 may be substituted;
cycloalkyl groups which R.sup.2, R.sup.3, R.sup.4, R.sup.5,
R.sup.6, R.sup.7, R.sup.32, R.sup.33, R.sup.41 and R.sup.42 may
represent; and alkylphenyl groups which R.sup.15 and R.sup.17 may
represent may be linear or branched, and may be saturated or
unsaturated. Alkylene groups represented by --(CH.sub.2).sub.k--,
--(CH.sub.2).sub.1--, --(CH.sub.2).sub.m--, --(CH.sub.2).sub.n--,
--(CH.sub.2).sub.p-- and --(CH.sub.2).sub.q-- in compounds of
formula I may be linear or branched, and may be saturated or
unsaturated.
[0045] Halo groups which R.sup.31 may represent, with which
R.sup.4, R.sup.5, R.sup.6 and R.sup.7 may be substituted, and with
which the substituents on R.sup.4, R.sup.5, R.sup.6 and R.sup.7 may
be substituted, include fluoro, chloro, bromo and iodo.
[0046] In the structural fragments of formulae IIa, IIb and IIc,
the dots indicate the carbon atom which is bonded to the --C(O)--
group and to the carbon atom bearing --OR.sup.1, R.sup.2 and
R.sup.3 in a compound of formula I (for the avoidance of doubt,
there is no further H atom bonded to the carbon atom so
indicated).
[0047] The wavy line on the bond in the fragments IVa, IVb, IVc and
IVd signifies the bond position of the fragments. For the avoidance
of doubt, when one or more substituent R.sup.31 is/are present,
it/they replace(s) one or more H atoms of CH, CH.sub.2 and/or NH
groups in the appropriate rings.
[0048] The skilled person will appreciate that, in the structural
fragment IVd, two double bonds must be present in the five-membered
ring, the position of which double bonds will depend upon which of
X.sup.7, X.sup.8 and X.sup.9 represents S, O or NH.
[0049] Abbreviations are listed at the end of this
specification.
[0050] Preferred compounds of the invention include those in which,
when B represents a structural fragment for formula IVa (in which
X.sup.1, X.sup.2, X.sup.3 and X.sup.4 all represent CH), a
structural fragment of formula or IVb or a structural fragment of
formula or IVc, R.sup.31 represents one or more substituents
selected from halo, C.sub.1-4 alkyl, C.sub.1-4 alkoxy or
--O--(CH.sub.2).sub.p-- C(O)N(R.sup.32)(R.sup.33) (i.e. the
substituent(s) is/are not optional).
[0051] Preferred compounds of the invention include those in which
B represents a structural fragment for formula IVa.
[0052] Compounds of formula I in which the fragment 5
[0053] is in the S-configuration are preferred. The wavy lines on
the nitrogen and carbon atom in the above fragment signify the bond
position of the fragment.
[0054] Preferred compounds of formula I include the compounds of
Examples 1 and 2.
Preparation
[0055] According to the invention there is also provided a process
for the preparation of compounds of formula I which comprises:
[0056] (a) the coupling of a compound of formula V, 6
[0057] wherein R.sup.1, R.sup.2, R.sup.3 and R.sub.x are as
hereinbefore defined with a compound of formula VI, 7
[0058] wherein Y, n and B are as hereinbefore defined, for example
in the presence of a coupling system (e.g. oxalyl chloride in DMF,
EDC, DCC, HBTU or TBTU), an appropriate base (e.g. pyridine, DMAP,
TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane,
acetonitrile or DMF);
[0059] (b) the coupling of a compound of formula VII, 8
[0060] wherein R.sup.1, R.sup.2, R.sup.3, R.sub.x and Y are as
hereinbefore defined with a compound of formula VIII,
H.sub.2N--(CH.sub.2).sub.n--B VIII
[0061] wherein n and B are as hereinbefore defined, for example in
the presence of a coupling system (e.g. oxalyl chloride in DMF,
EDC, DCC, HBTU or TBTU), an appropriate base (e.g. pyridine, DMAP,
TEA or DIPEA) and a suitable organic solvent (e.g. dichloromethane,
acetonitrile or DMF);
[0062] (c) for compounds of formula I in which B represents a
structural fragment of formula IVa, IVb or IVc, reaction of a
compound of formula IX, 9
[0063] wherein B.sup.1 represents a structural fragment of formula
IVa.sup.1, IVb.sup.1 or IVc.sup.1 10
[0064] wherein R.sup.y represents C.sub.1-4 alkyl and R.sup.1,
R.sup.2, R.sup.3, R.sub.x, Y, n, X.sup.1, X.sup.2, X.sup.3,
X.sup.4, X.sup.5, X.sup.6 and R.sup.31 are as hereinbefore defined,
with ammonia gas, for example at room temperature in the presence
of a suitable organic solvent (e.g. methanol or ethanol);
[0065] (d) for compounds of formula I in which B represents a
structural fragment of formula IVa, IVb or IVc, reduction of a
compound of formula X, 11
[0066] wherein B.sup.2 represents a structural fragment of formula
IVa.sup.2, IVb2 or IVc.sup.2 12
[0067] and R.sup.1, R.sup.2, R.sup.3, R.sub.x, Y, n, X.sup.1,
X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6 and R.sup.31 are as
hereinbefore defined, in the presence of a suitable reducing agent
(for example by catalytic hydrogenation in the presence of e.g.
Pd/C or TiCl.sub.3) and an appropriate organic solvent (e.g.
ethanol); or
[0068] (e) for compounds of formula I wherein X.sup.1, X.sup.2,
X.sup.3, X.sup.4, X.sup.7, X.sup.8 and/or X.sup.9 represent N--O,
oxidation of a corresponding compound of formula I in which
X.sup.1, X.sup.2, X.sup.3, X.sup.4, X.sup.7, X.sup.8 and/or X.sup.9
(as appropriate) represent N, for example under conditions which
are well known to those skilled in the art.
[0069] Compounds of formula I in which B represents a structural
fragment of formula IVd may be prepared analogously to methods
described herein or, alternatively, analogously to the methods
described in international patent applications WO 95/23609 and WO
98/06741.
[0070] Compounds of formula V are commercially available, are well
known in the literature, or are available using known techniques.
For example, compounds of formula V may be prepared by hydrolysis
of a compound of formula XI, 13
[0071] wherein R is C.sub.1-6 alkyl or C.sub.1-3 alkylphenyl and
R.sup.1, R.sup.2, R.sup.3 and R.sub.x are as hereinbefore defined,
for example at room temperature in the presence of a suitable base
(e.g. lithium hydroxide) and an appropriate solvent (e.g. THF
and/or water).
[0072] Compounds of formula VI may be prepared by reaction of a
compound of formula XII, 14
[0073] wherein Y is as hereinbefore defined with a compound of
formula VIII as hereinbefore defined, for example under conditions
such as those described hereinbefore for synthesis of compounds of
formula I (process steps (a) and (b)).
[0074] Compounds of formula VII are readily available using known
techniques.
[0075] For example, compounds of formula VII may be prepared by
reaction of a compound of formula V as hereinbefore defined with a
compound of formula XII as hereinbefore defined, for example under
conditions such as those described hereinbefore for synthesis of
compounds of formula I (process steps (a) and (b)).
[0076] Compounds of formula IX may be prepared by known techniques.
For example, compounds of formula IX in which B.sup.1 represents a
structural fragment of formula IVa.sup.1 or IVc.sup.1 may be
prepared by reaction of a compound of formula XIII, 15
[0077] wherein B.sup.3 represents a structural fragment of formula
IVa.sup.3 or IVc.sup.3, 16
[0078] and R.sup.1, R.sup.2, R.sup.3, R.sub.x, Y, n, X.sup.2,
X.sup.2, X.sup.3, X.sup.4 and R.sup.31 are as hereinbefore defined,
with HCl(g) and a C.sub.1-4 alkyl alcohol, for example at or below
room temperature.
[0079] Compounds of formula X may be prepared by known techniques.
For example, compounds of formula X in which B.sup.2 represents a
structural fragment of formula IVa.sup.2 or IVc.sup.2 may be
prepared by reaction of a compound of formula XIII as hereinbefore
defined with HCl(g) and methanol, for example at or below room
temperature, followed by reaction with hydroxylamine, or a
hydrohalide salt thereof, for example at or around room temperature
in the presence of an appropriate base (e.g. TEA) and a suitable
solvent (e.g. MeOH).
[0080] Compounds of formula XI in which R.sup.1 and R.sup.3 both
represent H may be prepared by reduction of a compound of formula
XIV, 17
[0081] wherein R, R.sub.x and R.sup.2 are as hereinbefore defined,
for example at below room temperature (e.g. between -70.degree. C.
and -5.degree. C.) in the presence of a suitable reducing agent
(e.g. sodium borohydride) and an appropriate organic solvent (e.g.
MeOH or EtOH).
[0082] Compounds of formula XI in which R.sup.1 represents H and
R.sup.3 represents C.sub.1-4 alkyl, cyclohexyl or phenyl may be
prepared by reaction of a compound of formula XIV as hereinbefore
defined with an organometallic reagent of formula XV
R.sup.3aM XV
[0083] wherein R.sup.3a represents C.sub.1-4 alkyl, cyclohexyl or
phenyl, M represents Li or MgHal and Hal is Cl, Br or I, under
conditions which are well known to those skilled in the art in the
presence of an appropriate organic solvent (e.g. THF).
[0084] Compounds of formula XI in which R.sup.1 represents H may
also be prepared by reaction of a compound of formula XVI,
RO--C(O)--R.sub.xH XVI
[0085] wherein R and R.sub.x are as hereinbefore defined with a
compound of formula XVII,
R.sup.2--C(O)--R.sup.3 XVII
[0086] wherein R.sup.2 and R.sup.3 are as hereinbefore defined
under conditions which are well known to those skilled in the
art.
[0087] Compounds of formula XI in which R.sup.1, R.sup.2 and
R.sup.3 all represent H, R.sub.x represents a structural fragment
of formula IIa, as hereinbefore defined, in which neither k nor 1
represent 0, may be prepared by reduction of a compound of formula
XVIII, 18
[0088] wherein R.sub.xa represents a structural fragment of formula
IIa, as hereinbefore defined, in which neither k nor 1 represent 0,
and R is as hereinbefore defined, in the presence of a suitable
reducing agent (e.g. borane) in the presence of an appropriate
organic solvent (e.g. THF).
[0089] Compounds of formula XIII may be prepared by coupling a
compound of formula VII as hereinbefore defined to a compound of
formula XIX,
H.sub.2N--(CH.sub.2).sub.n--B.sup.3 XIX
[0090] wherein n and B.sup.3 are as hereinbefore defined, for
example under conditions such as those described hereinbefore for
synthesis of compounds of formula I (process steps (a) and
(b)).
[0091] Compounds of formula XIV are either known from, or may be
prepared analogously to, the methods described in J. Org. Chem.,
54, 3831 (1989).
[0092] Compounds of formula XVIII are well known in the literature
or may be prepared using known techniques, for example by reaction
of a suitable malonic acid derivative with an alkylating agent of
formula XX,
R.sub.xaL XX
[0093] in which L is a leaving group (e.g. halo (Cl, Br, I) or
tosyl) and R.sub.xa is as hereinbefore defined, for example in the
presence of a suitable base (e.g. sodium hydride or sodium
ethoxide) and an appropriate organic solvent.
[0094] Compounds of formula VIII, XII, XV, XVI, XVII, XIX and XX
and derivatives thereof, are either commercially available, are
known in the literature, or may be obtained either by analogy with
the processes described herein, or by conventional synthetic
procedures, in accordance with standard techniques, from readily
available starting materials using appropriate reagents and
reaction conditions.
[0095] Substituents on inter alia phenyl groups contained in
compounds of formulae I, V, VI, VII, VIII, IX, X, XI, XIII, XIV,
XVI, XVIII, XIX and XX may be inter-converted using standard
techniques.
[0096] Compounds of formula I may be isolated from their reaction
mixtures using conventional techniques.
[0097] It will be appreciated by those skilled in the art that in
the process described above the functional groups of intermediate
compounds may need to be protected by protecting groups.
[0098] Functional groups which it is desirable to protect include
hydroxy, amino and carboxylic acid. Suitable protecting groups for
hydroxy include trialkylsilyl or diarylalkylsilyl groups (e.g.
t-butyldimethylsilyl, t-butyldiphenylsilyl or trimethylsilyl) and
tetrahydropyranyl. Suitable protecting groups for carboxylic acid
include C.sub.1-6 alkyl or benzyl esters. Suitable protecting
groups for amino, amidino and guanidino include t-butyloxycarbonyl
or benzyloxycarbonyl. Amidino and guanidino nitrogens may be either
mono- or diprotected.
[0099] The protection and deprotection of functional groups may
take place before or after coupling.
[0100] For example, the compounds of formula I may be prepared by
processes comprising the coupling of an N-acylated amino acid or a
N-protected amino acid. When a N-protected amino acid is used the
acyl group may be added after coupling and deprotection of the
nitrogen atom may then be effected using standard methods
thereafter.
[0101] Protecting groups may be removed in accordance with
techniques which are well known to those skilled in the art and as
described hereinafter.
[0102] The use of protecting groups is fully described in
`Protective Groups in Organic Chemistry`, edited by J W F McOmie,
Plenum Press (1973), and `Protective Groups in Organic Synthesis`,
2nd edition, T W Greene & P G M Wutz, Wiley-Interscience
(1991).
[0103] Certain protected derivatives of compounds of formula I,
which may be made prior to a final deprotection stage to form
compounds of formula I, are novel.
[0104] According to a further aspect of the invention there is
provided a compound of formula Ia, 19
[0105] wherein B.sup.a represents a structural fragment of formula
IVe, IVf, IVg or IVh 20
[0106] wherein D.sup.1 and D.sup.2 independently represent, in each
case, H, OH, OR.sup.a, OC(O)R.sup.b, OC(O)OR.sup.c, C(O)OR.sup.d,
C(O)R.sup.e, and R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e
independently represent C.sub.1-12 alkyl (which latter group is
optionally interrupted by oxygen and/or substituted by halo),
phenyl, naphthyl, C.sub.1-3 alkylphenyl (which latter three groups
are optionally substituted by C.sub.1-4 alkyl, C.sub.1-4 alkoxy,
nitro, or halo) or --(C(R.sup.f)(R.sup.g)).sub.2OC(O)C(R.sup.h),
R.sup.f, R.sup.g and R.sup.h independently represent H or C.sub.1-4
alkyl, and R.sup.1, R.sup.2, R.sup.3, R.sub.x, Y, n, X.sup.1,
X.sup.2, X.sup.3, X.sup.4, X.sup.5, X.sup.6, X.sup.7, X.sup.8,
X.sup.9 and R.sup.31 are as hereinbefore defined, or a
pharmaceutically acceptable salt thereof, provided that D.sup.1 and
D.sup.2 do not both represent H.
[0107] Alkyl groups which R.sup.a, R.sup.b, R.sup.c, R.sup.d,
R.sup.e, R.sup.f, R.sup.g and R.sup.h may represent, and with which
R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e may be substituted,
may be linear or branched, may be saturated or unsaturated, and may
be cyclic, acyclic or part cyclic/acyclic. Alkoxy groups with which
R.sup.a, R.sup.b, R.sup.c, R.sup.d and R.sup.e may be substituted,
may be linear or branched, may be saturated or unsaturated, and may
be cyclic, acyclic or part cyclic/acyclic. The alkyl part of
alkylphenyl groups which R.sup.a, R.sup.b, R.sup.c, R.sup.d and
R.sup.e may represent, may be linear or branched, and may be
saturated or unsaturated.
[0108] Halo groups with which R.sup.a, R.sup.b, R.sup.c, R.sup.d
and R.sup.e may be substituted include fluoro, chloro, bromo and
iodo.
[0109] The wavy line on the bond in the fragments IVe, IVf, IVg and
IVh signifies the bond position of the fragments.
[0110] Preferred compounds of formula Ia include those wherein
D.sup.2 represents H and D.sup.1 represents OH, OCH.sub.3,
OC(O)R.sup.b or C(O)OR.sup.d, wherein R.sup.b and R.sup.d are as
hereinbefore defined.
[0111] Structural preferences mentioned hereinbefore for compounds
of formula I also apply to compounds of formula Ia.
[0112] Compounds of formula Ia may also be prepared directly from
compounds of formula I in accordance with techniques well known to
those skilled in the art. For example compounds of formula Ia in
which B.sup.a represents a structural fragment of formula IVe, IVf
or IVg, and in which D.sup.1 or D.sup.2 represent OH, may be
prepared as described hereinbefore for compounds of formula X, or
by analogous methods.
[0113] Alternatively, compounds of formula Ia in which B.sup.a
represents a structural fragment of formula IVe, IVf or IVg, and in
which D.sup.1 or D.sup.2 represent OH or OR.sup.a, wherein R.sup.a
is as hereinbefore defined, may be prepared from compounds of
formula XIII as defined hereinbefore by reaction with a compound of
formula XXI,
H.sub.2NOR.sup.al XXI
[0114] wherein R.sup.al represents H or R.sup.a and R.sup.a is as
hereinbefore defined, for example at between 40 and 60.degree. C.,
in the presence of a suitable base (e.g. TEA) and an appropriate
organic solvent (e.g. THF, CH.sub.3CN, DMF or DMSO).
[0115] Compounds of formula Ia may alternatively be prepared via
other protected derivatives of formula Ia in accordance with
techniques well known to those skilled in the art. For example
compounds of formula Ia in which D.sup.1 or D.sup.2 represents
OC(O)OR.sup.c, and R.sup.c is as hereinbefore defined, may be
prepared by reaction of a corresponding compound of formula Ia in
which D.sup.1 or D.sup.2 (as appropriate) represents OH with a
compound of formula XXII,
R.sup.cIC(O)--O--C(O)R.sup.c XXII
[0116] wherein R.sup.c is as hereinbefore defined, for example at
room temperature in the presence of a suitable base (e.g. TEA,
pyridine or DMAP) and an appropriate organic solvent. Moreover,
compounds of formula Ia in which D.sup.1 or D.sup.2 represents OH
may be prepared by reaction of a corresponding compound of formula
Ia in which D.sup.1 or D.sup.2 (as appropriate) represents
COOR.sup.d and R.sup.d is as hereinbefore defined with
hydroxylamine (or a hydrohalide salt thereof), for example at
40.degree. C. in the presence of a suitable base (e.g. TEA) and an
appropriate organic solvent (e.g. THF). Compounds of formulae XXI
and XXII are commercially available, are well known in the
literature, or are available using known techniques.
[0117] It will also be appreciated by those skilled in the art
that, although such protected derivatives of compounds of formula I
(e.g. compounds of formula Ia) may not possess pharmacological
activity as such, they may be administered parenterally or orally
and thereafter metabolised in the body to form compounds of the
invention which are pharmacologically active. Such derivatives may
therefore be described as "prodrugs". All prodrugs of compounds of
formula I are included within the scope of the invention.
[0118] Protected derivatives of compounds of formula I which are
particularly useful as prodrugs include compounds of formula
Ia.
[0119] Moreover, certain compounds of formula I may act as prodrugs
of other compounds of formula I.
[0120] Compounds of formula I, pharmaceutically-acceptable salts,
tautomers and stereoisomers thereof, as well as prodrugs thereof
(including compounds of formula Ia which are prodrugs of compounds
of formula I), are hereinafter referred to together as "the
compounds of the invention".
[0121] Persons skilled in the art will appreciate that, in order to
obtain compounds of the invention in an alternative, and, on some
occasions, more convenient, manner, the individual process steps
mentioned herein may be performed in a different order, and/or the
individual reactions may be performed at a different stage in the
overall route (i.e. substituents may be added to and/or chemical
transformations performed upon, different intermediates to those
associated hereinbefore with a particular reaction). This will
depend inter alia on factors such as the nature of other functional
groups present in a particular substrate, the availability of key
intermediates and the protecting group strategy (if any) to be
adopted. Clearly, the type of chemistry involved will influence the
choice of reagent that is used in the said synthetic steps, the
need, and type, of protecting groups that are employed, and the
sequence for accomplishing the synthesis.
Medical and Pharmaceutical Use
[0122] The compounds of the invention are useful because they
possess pharmacological activity. They are therefore indicated as
pharmaceuticals.
[0123] According to a further aspect of the invention there is thus
provided the compounds of the invention for use as
pharmaceuticals.
[0124] In particular, the compounds of the invention are potent
inhibitors of trypsin-like proteases, especially thrombin, either
as such or, in the case of prodrugs, after administration to
mammals including man, for example as demonstrated in the tests
described below.
[0125] The compounds of the invention are thus expected to be
useful in those conditions where inhibition of thrombin is
required.
[0126] The compounds of the invention are thus indicated in the
treatment or prophylaxis of thrombosis and hypercoagulability in
blood and tissues of animals including man.
[0127] It is known that hypercoagulability may lead to
thrombo-embolic diseases. Conditions associated with
hypercoagulability and thrombo-embolic diseases which may be
mentioned include activated protein C resistance, such as the
factor V-mutation (factor V Leiden), and inherited or acquired
deficiencies in antithrombin III, protein C, protein S, heparin
cofactor II. Other conditions known to be associated with
hypercoagulability and thrombo-embolic disease include circulating
antiphospholipid antibodies (Lupus anticoagulant), homocysteinemi,
heparin induced thrombocytopenia and defects in fibrinolysis. The
compounds of the invention are thus indicated both in the
therapeutic and/or prophylactic treatment of these conditions.
[0128] The compounds of the invention are further indicated in the
treatment of conditions where there is an undesirable excess of
thrombin without signs of hypercoagulability, for example in
neurodegenerative diseases such as Alzheimer's disease.
[0129] Particular disease states which may be mentioned include the
therapeutic and/or prophylactic treatment of venous thrombosis and
pulmonary embolism, arterial thrombosis (e.g. in myocardial
infarction, unstable angina, thrombosis-based stroke and peripheral
arterial thrombosis) and systemic embolism usually originating from
the atrium during arterial fibrillation or from the left ventricle
after transmural myocardial infarction.
[0130] Moreover, the compounds of the invention are expected to
have utility in prophylaxis of re-occlusion (i.e. thrombosis) after
thrombolysis, percutaneous trans-luminal angioplasty (PTA) and
coronary bypass operations; the prevention of re-thrombosis after
microsurgery and vascular surgery in general.
[0131] Further indications include the therapeutic and/or
prophylactic treatment of disseminated intravascular coagulation
caused by bacteria, multiple trauma, intoxication or any other
mechanism; anticoagulant treatment when blood is in contact with
foreign surfaces in the body such as vascular grafts, vascular
stents, vascular catheters, mechanical and biological prosthetic
valves or any other medical device; and anticoagulant treatment
when blood is in contact with medical devices outside the body such
as during cardiovascular surgery using a heart-lung machine or in
haemodialysis.
[0132] In addition to its effects on the coagulation process,
thrombin is known to activate a large number of cells (such as
neutrophils, fibroblasts, endothelial cells and smooth muscle
cells). Therefore, the compounds of the invention may also be
useful for the therapeutic and/or prophylactic treatment of
idiopathic and adult respiratory distress syndrome, pulmonary
fibrosis following treatment with radiation or chemotherapy, septic
shock, septicemia, inflammatory responses, which include, but are
not limited to, edema, acute or chronic atherosclerosis such as
coronary arterial disease, cerebral arterial disease, peripheral
arterial disease, reperfusion damage, and restenosis after
percutaneous trans-luminal angioplasty (PTA).
[0133] Compounds of the invention that inhibit trypsin and/or
thrombin may also be useful in the treatment of pancreatitis.
[0134] According to a further aspect of the present invention,
there is provided a method of treatment of a condition where
inhibition of thrombin is required or desired, which method
comprises administration of a therapeutically effective amount of a
compound of the invention, or a pharmaceutically acceptable salt
thereof, to a person suffering from, or susceptible to such a
condition.
[0135] The compounds of the invention will normally be administered
orally, intravenously, subcutaneously, buccally, rectally,
dermally, nasally, tracheally, bronchially, by any other parenteral
route or via inhalation, in the form of pharmaceutical preparations
comprising active compound either as a free base, or a
pharmaceutical acceptable non-toxic organic or inorganic acid
addition salt, in a pharmaceutically acceptable dosage form.
Depending upon the disorder and patient to be treated and the route
of administration, the compositions may be administered at varying
doses.
[0136] The compounds of the invention may also be combined and/or
co-administered with any antithrombotic agent with a different
mechanism of action, such as the antiplatelet agents
acetylsalicylic acid, ticlopidine, clopidogrel, thromboxane
receptor and/or synthetase inhibitors, fibrinogen receptor
antagonists, prostacyclin mimetics and phosphodiesterase inhibitors
and ADP-receptor (P.sub.2T) antagonists.
[0137] The compounds of the invention may further be combined
and/or co-administered with thrombolytics such as tissue
plasminogen activator (natural, recombinant or modified),
streptokinase, urokinase, prourokinase, anisoylated
plasminogen-streptokinase activator complex (APSAC), animal
salivary gland plasminogen activators, and the like, in the
treatment of thrombotic diseases, in particular myocardial
infarction.
[0138] According to a further aspect of the invention there is thus
provided a pharmaceutical formulation including a compound of the
invention, in admixture with a pharmaceutically acceptable
adjuvant, diluent or carrier.
[0139] Suitable daily doses of the compounds of the invention in
therapeutic treatment of humans are about 0.001-100 mg/kg body
weight at peroral administration and 0.001-50 mg/kg body weight at
parenteral administration.
[0140] The compounds of the invention have the advantage that they
may be more efficacious, be less toxic, be longer acting, have a
broader range of activity, be more potent, produce fewer side
effects, be more easily absorbed than, or that they may have other
useful pharmacological properties over, compounds known in the
prior art.
Biological Tests
Test A
Determination of Thrombin Clotting Time (TT)
[0141] Human thrombin (T 6769, Sigma Chem. Co.) in buffer solution,
pH 7.4, 100 .mu.L, and inhibitor solution, 100 .mu.L, were
incubated for one min. Pooled normal citrated human plasma, 100
.mu.L, was then added and the clotting time measured in an
automatic device (KC 10, Amelumg).
[0142] The clotting time in seconds was plotted against the
inhibitor concentration, and the IC.sub.50TT was determined by
interpolation.
[0143] IC.sub.50TT is the concentration of inhibitor in the test
that doubles the thrombin clotting time for human plasma.
Test B
Determinaton of Thrombin Inhibition with a Chromogenic, Robotic
Assay
[0144] The thrombin inhibitor potency was measured with a
chromogenic substrate method, in a Plato 3300 robotic microplate
processor (Rosys AG, CH-8634 Hombrechtikon, Switzerland), using
96-well, half volume microtitre plates (Costar, Cambridge, Mass.,
USA; Cat. No. 3690). Stock solutions of test substance in DMSO (72
.mu.L; 1 mmol/L) were diluted serially 1:3 (24+48 .mu.L) with DMSO
to obtain ten different concentrations, which were analysed as
samples in the assay. 2 .mu.L of test sample was diluted with 124
.mu.L assay buffer, 12 .mu.L of chromogenic substrate solution
(S-2366, Chromogenix, Molndal, Sweden) in assay buffer and finally
12 .mu.L of .alpha.-thrombin solution, (Human .alpha.-thrombin,
Sigma Chem. Co.) both in assay buffer, were added, and the samples
mixed. The final assay concentrations were: test substance
0.00068-13.3 .mu.mol/L, S-2366 0.30 mmol/L, .alpha.-thrombin 0.020
NIHU/mL. The linear absorbance increment during 40 minutes
incubation at 37.degree. C. was used for calculation of percentage
inhibition for the test samples, as compared to blanks without
inhibitor. The IC.sub.50-robotic value, corresponding to the
inhibitor concentration which caused 50% inhibition of the thrombin
activity, was calculated from a log dose vs. % inhibition
curve.
Test C
Determinaton of the Inhibition Constant K.sub.i for Human
Thrombin
[0145] K.sub.i-determinations were made using a chromogenic
substrate method, performed at 37.degree. C. on a Cobas Bio
centrifugal analyser (Roche, Basel, Switzerland). Residual enzyme
activity after incubation of human .alpha.-thrombin with various
concentrations of test compound was determined at three different
substrate concentrations, and was measured as the change in optical
absorbance at 405 nm.
[0146] Test compound solutions (100 .mu.L; normally in buffer or
saline containing BSA 10 g/L) were mixed with 200 .mu.L of human
.alpha.-thrombin (Sigma Chem. Co.) in assay buffer (0.05 mol/L
Tris-HCl pH 7.4, ionic strength 0.15 adjusted with NaCl) containing
BSA (10 g/L), and analysed as samples in the Cobas Bio. A 60 .mu.L
sample, together with 20 .mu.L of water, was added to 320 .mu.L of
the substrate S-2238 (Chromogenix AB, Mondal, Sweden) in assay
buffer, and the absorbance change (.DELTA.A/min) was monitored. The
final concentrations of S-2238 were 16, 24 and 50 .mu.mol/L and of
thrombin 0.125 NIH U/mL.
[0147] The steady state reaction rate was used to construct Dixon
plots, i.e. diagrams of inhibitor concentration vs. 1/(A/min). For
reversible, competitive inhibitors, the data points for the
different substrate concentrations typically form straight lines
which intercept at x=-K.sub.i.
Test D
Determination of Activated Partial Thromboplastin Time (APTT)
[0148] APTT was determined in pooled normal human citrated plasma
with the reagent PTT Automated 5 manufactured by Stago. The
inhibitors were added to the plasma (10 .mu.L inhibitor solution to
90 .mu.L plasma) followed by the reagent and calcium chloride
solution and APTT was determined in the mixture by use of the
coagulation analyser KC10 (Amelumg) according to the instructions
of the reagent producer. The clotting time in seconds was plotted
against the inhibitor concentration in plasma and the IC.sub.50APTT
was determined by interpolation.
[0149] IC.sub.50APTT is defined as the concentration of inhibitor
in human plasma that doubles the Activated Partial Thromboplastin
Time.
Test E
Determination of Thrombin Time ex vivo
[0150] The inhibition of thrombin after oral or parenteral
administration of the compounds of the formula I and Ia, dissolved
in ethanol:Solutol.TM.:water (5:5:90), were examined in conscious
rats which, one or two days prior to the experiment, were equipped
with a catheter for blood sampling from the carotid artery. On the
experimental day blood samples were withdrawn at fixed times after
the administration of the compound into plastic tubes containing 1
part sodium citrate solution (0.13 mol per L.) and 9 parts blood.
The tubes were centrifuged to obtain platelet poor plasma. The
plasma was used for determination of thrombin time as described
below.
[0151] The citrated rat plasma, 25 .mu.L, was diluted with a saline
solution, 0.9%, 25 .mu.L, and plasma coagulation was started by the
addition of human thrombin (T 6769, Sigma Chem. Co., USA) in a
buffer solution, pH 7.4, 25 .mu.L. The clotting time was measured
in an automatic device (KC 10 A-micro, Amelumg, Germany).
[0152] Where a compound of formula Ia was administered,
concentrations of the appropriate active thrombin inhibitor of
formula I in the rat plasma were estimated by the use of standard
curves relating the thrombin time in the pooled citrated rat plasma
to known concentrations of the corresponding "active" thrombin
inhibitor dissolved in saline.
[0153] Based on the estimated plasma concentrations of the active
thrombin inhibitor of formula I (which assumes that thrombin time
prolongation is caused by the aforementioned compound) in the rat,
the area under the plasma concentration-time curve after oral
and/or parenteral administration of the corresponding compound of
formula Ia was calculated (AUCpd) using the trapezoidal rule and
extrapolation of data to infinity.
[0154] The bioavailability of the active thrombin inhibitor of
formula I after oral or parenteral administration of the compound
of formula Ia was calculated as below:
[(AUCpd/dose)/(AUCactive,parenteral/dose].times.100
[0155] where AUCactive,parenteral represents the AUC obtained after
parenteral administration of the corresponding active thrombin
inhibitor of formula I to conscious rats as described above.
Test F
Determination of Thrombin Time in Urine ex vivo
[0156] The amount of the active thrombin inhibitor of formula I
that was excreted in urine after oral or parenteral administration
of the compounds of the invention, dissolved in
ethanol:Solutol.TM.:water (5:5:90), was estimated by determination
of the thrombin time in urine ex vivo (assuming that thrombin time
prolongation is caused by the aforementioned compound).
[0157] Conscious rats were placed in metabolism cages, allowing
separate collection of urine and faeces, for 24 hours following
oral administration of compounds of the invention. The thrombin
time was determined on the collected urine as described below.
[0158] Pooled normal citrated human plasma (100 .mu.L) was
incubated with the concentrated rat urine, or saline dilutions
thereof, for one minute. Plasma coagulation was then initiated by
the administration of human thrombin (T 6769, Sigma Chem. Co.) in
buffer solution (pH 7.4; 100 .mu.L). The clotting time was measured
in an automatic device (KC 10; Amelumg).
[0159] The concentrations of the active thrombin inhibitor of
formula I in the rat urine were estimated by the use of standard
curves relating the thrombin time in the pooled normal citrated
human plasma to known concentrations of the aforementioned active
thrombin inhibitor dissolved in concentrated rat urine (or saline
dilutions thereof). By multiplying the total rat urine production
over the 24 hour period with the estimated mean concentration of
the aforementioned active inhibitor in the urine, the amount of the
active inhibitor excreted in the urine (AMOUNTpd) could be
calculated.
[0160] The bioavailability of the active thrombin inhibitor of
formula I after oral or parenteral administration of the prodrug
was calculated as below:
[(AMOUNTpd/dose)/(AMOUNTactive,parenteral/dose].times.100
[0161] where AMOUNTactive,parenteral represents the amount excreted
in the urine after parenteral administration of the corresponding
active thrombin inhibitor of formula I to conscious rats as
described above.
[0162] The invention is illustrated by way of the following
examples.
EXAMPLES
General Experimental Procedures
[0163] Mass spectra were recorded on a Finnigan MAT TSQ 700 triple
quadrupole mass spectrometer equipped with an electrospray
interface (FAB-MS) and VG Platform II mass spectrometer equipped
with an electrospray interface (LC-MS). .sup.1H NMR and .sup.13C
NMR measurements were performed on BRUKER ACP 300 and Varian UNITY
plus 400, 500 and 600 spectrometers, operating at .sup.1H
frequencies of 300.13, 399.96, 499.82 and 599.94 MHz respectively,
and at .sup.13C frequencies of 75.46, 100.58, 125.69 and 150.88 MHz
respectively. Preparative HPLC was performed on reverse phase
columns (250 mm, 20 or 50 mm; 5 to 7 .mu.M phase Chromasil C8) with
flow rates of 10 to 50 mL/min using a UV detector (240 to 290
nm).
Example 1
(2,5-diMeO)Ph-(S)- or (R)-CH(CH.sub.2OH).mu.13
CO--Pro-Pab(2-Cl).times.HOA- c
(i) 4-Azidomethyl-3-chlorobenzonitrile
[0164] A mixture 8.0 g (0.035 mol) of
4-bromomethyl-3-chlorobenzonitrile (J. Pharm. Sci., (1986) 75,
410), 2.7 g (0.042 mol) of sodium azide, 1.2 g (3.4 mmol) of
tetrabutylammonium hydrogen sulfate, 0.30 g (3.4 mmol) of sodium
hydrogen carbonate, 7 mL of water and 20 mL of toluene was stirred
vigorously for 3 days. The phases were separated and the aqueous
layer was extracted three times with ether. The combined organic
phase was washed with water, dried over sodium sulfate and
evaporated to give 6.7 g (100%) of the sub-title compound.
[0165] .sup.1H-NMR (300 MHz; CDCl.sub.3): .delta.4.60 (s, 2H), 7.57
(d, 1H), 7.61 (m, 1 H), 7.70 (d, 1H)
(ii) 4-Aminomethyl-3-chlorobenzonitrile
[0166] 4-Azidomethyl-3-chlorobenzonitrile (1.0 g; 5.2 mmol; from
step (i) above), was dissolved in 9 mL of water and 1 mL of
ethanol. Triphenylphosphine (1.5 g) was added, and the mixture was
stirred overnight. The ethanol was evaporated and the residue was
partitioned between 1M HCl and benzene. The aqueous layer was
extracted several times with benzene and then freeze dried. The
yield of the amine hydrochloride was 0.54 g (51%).
[0167] .sup.1H-NMR (400 MHz; D.sub.2O) HCl salt: .delta.4.42 (s,
2H), 7.69 (d, 1H), 7.81 (dd, 1 H), 7.98 (d, 1 H)
(iii) (R,S)-3-Hydroxy-2-(2,5-dimethoxyphenyl)propionic acid
ethylester
[0168] To a solution of 3-oxo-2-(2,5-dimethoxyphenyl)propionic acid
ethyl ester (7.6 g; 30 mmol; prepared according to the method
described in J. Org. Chem. 54, 3831 (1989)) in ethanol was added
NaBH.sub.4 (2 equivalents) at -15.degree. C. After stirring for 2 h
at -15.degree. C., and 4 h at -5.degree. C., water was added, the
reaction mixture was concentrated and the resultant extracted with
ethyl acetate. The organic phase was washed with brine, dried
(MgSO.sub.4) and concentrated to give the sub-title compound. Yield
7.7 g (100%).
(iv) (2,5-diMeO)Ph-(R,S)CH(CH.sub.2OH)--COOH
[0169] The ethylester from step (iii) above (7.4 g; 29 mmol) was
dissolved in THF:water (1:1). LiOH.times.H.sub.2O (2 eq.) was added
and the reaction mixture was stirred for 2 h at room temperature.
The reaction mixture was concentrated and extracted with
CH.sub.2Cl.sub.2. The aqueous phase was acidified (pH 2) with HCl
(2M) and extracted three times which CH.sub.2Cl.sub.2. The organic
phases were combined, dried (NaSO.sub.4) and concentrated to give
the sub-title compound. Yield 5.4 g (82%).
[0170] .sup.1H-NMR (400 MHz; CDCl.sub.3): .delta.6.89 (d, 1H); 6.85
(d, 1H); 6.78 (dd, 1H); 4.86 (broad, 2H); 4.14 (dd, 1H); 3.98 (dd,
1H); 3.78 (s, 3H); 3.72 (s, 3H); 3.67 (dd, 1H)
(v) (2,5-diMeO)Ph-(R,S)CH(CH.sub.2OH)--CO-Pro-OBn
[0171] 1.8 mL (9.6 mmol) of diisopropylethylamine was added to an
ice-cooled mixture of 0.50 g (2.2 mmol) of
(2,5-diMeO)Ph--(R,S)CH(CH.sub.- 2OH)--COOH (from step (iv) above),
0.58 g (2.4 mmol) of H-Pro-OBn and 0.77 g (2.4 mmol) of TBTU in 10
mL of DMF. The mixture was stirred at room temperature overnight,
and was then poured into 1M HCl and extracted twice with ethyl
acetate:toluene (1:1). The combined organic layer was washed with
NaHCO.sub.3 (aq.) and water, dried (Na.sub.2SO.sub.4) and
evaporated. The product was pure and was used directly in the next
step. Yield: 0.91 g (100%).
[0172] .sup.1H-NMR (500 MHz;CDCl.sub.3) diastereomeric mixture:
.delta.1.8-2.3 (m, 4H), 3.0-3.1 (m, 1H), 3.18 (m, 1H,
diastereomer), 3.34 (m, 1H, diastereomer), 3.46 (m, 1H,
diastereomer), 3.65-3.7 (m, 1H), 3.75-3.9 (m, 7H, thereof 4
singlets at 3.77, 3.80, 3.85 and 3.87 ppm), 3.95-4.05 (m, 1H), 4.39
(m, 1H, diastereomer), 4.46 (m, 1H, diastereomer), 4.58 (m, 1H,
diastereomer), 4.63 (m, 1H, diastereomer), 5.18 (d, 1H,
diastereomer), 5.26 (s, 1H, diastereomer), 5.32 (d, 1H,
diastereomer), 5.37 (s, 1H, diastereomer), 6.8-7.0 (m, 3H), 7.2-7.5
(m, 5H)
(vi) (2,5-diMeO)Ph-(R,S)CH(CH.sub.2OH)--CO-Pro-OH
[0173] (2,5-diMeO)Ph-(R,S)CH(CH.sub.2OH)--CO-Pro-OBn (0.91 g; 2.2
mmol; from step (v) above), was hydrogenated for 2 h at atmospheric
pressure over 50 mg of 10% Pd/C in 25 mL of ethanol. The mixture
was filtered through celite and evaporated to give 0.71 g (100%) of
the sub-title compound.
[0174] .sup.1H-NMR (500 MHz; CDCl.sub.3) diastereomeric mixture:
.delta.1.8-2.3 (m, 4H), 3.02 (m, 1H, diastereomer), 3.15 (m, 1H,
diastereomer), 3.47 (m, 1H, diastereomer), 3.7-3.8 (m, 5H, thereof
the other diastereomer corresponding to 3.47, and two singlets at
3.74 and 3.76 ppm), 3.83-3.85 (m, 3H), 4.0-4.1 (m, 1H), 4.38 (m,
1H, diastereomer), 4.50 (m, 1H, diastereomer), 4.56 (m, 1H,
diastereomer), 4.63 (m, 1H, diastereomer),6.8-6.9 (m, 3H)
(vii) (2,5-diMeO)Ph-(R and
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Cl,4-C- N)
[0175] 0.76 mL (4.4 mmol) of diisopropylethylamine was added to an
ice-cooled mixture of 0.35 g (1.1 mmol) of
(2,5-diMeO)Ph-(R,S)CH(CH.sub.2- OH)--CO-Pro-OH (from step (vi)
above), 0.22 g (1.1 mmol) of 4-aminomethyl-3-chlorobenzonitrile
(from step (ii) above) and 0.35 g (1.1 mmol) of TBTU in 10 mL of
DMF. The mixture was stirred at room temperature for two days,
poured into 1M HCl and extracted twice with ethyl acetate:toluene
(1:1). The combined organic layer was washed with NaHCO.sub.3 (aq.)
and water, dried (Na.sub.2SO.sub.4) and evaporated. The crude
product was flash chromatographed on silica gel with EtOAc:MeOH
(99:1) as eluant. The diastereomers separated. Total yield: 0.48 g
(94%). The initial diastereomer (A) was isolated in a yield of 0.22
g.
[0176] Diastereomer A
[0177] .sup.1H-NMR (400 MHz; CDCl.sub.3): .delta.1.7-2.1 (m, 3H),
2.34 (m, 1H), 2.90 (m, 1H), 3.12 (m, 1H), 3.61 (m, 1H), 3.73-3.8
(m, 4H, thereof one singlet at 3.75), 3.82 (s, 3H), 4.01 (m, 1H),
4.44 (m, 1H), 4.56 (m, 1H), 4.65 (m, 1H), 6.75-6.85 (m, 3H), 7.53
(d, 1H), 7.56 (dd, 1 H), 7.65 (d, 1H), 7.68 (m, 1H)
(viii) (2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Cl,4-C- (NH)OMe)
(2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Cl,4-CN)
[0178] (0.22 g; 0.47 mmol; diastereomer A from step (vii) above)
was dissolved in 25 mL of methanol saturated with hydrogen chloride
gas and put in the refrigerator for two days. Evaporation yielded a
crude material which was used in the next step without further
purification.
[0179] MS (M+1).sup.+504
(ix) (2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-Pab(2-Cl).times.HOAc
[0180] Half of the crude material from step (viii) above was
dissolved in 25 mL of methanol saturated with ammonia and allowed
to stand at room temperature for five days. The solvent was
evaporated and the residue was purified on preparative HPLC with
CH.sub.3CN:0.1 M NH.sub.4OAc (30:70). Freeze drying from water gave
9 mg (7%) of the desired product.
[0181] .sup.1H-NMR (400 MHz; D.sub.2O ) rotamers: .delta.1.8-2.4
(m, 7H, thereof one singlet at 1.93 ppm), 3.35 (m, 1H), 3.63 (m,
1H), 3.7-3.9 (m, 8H, thereof singlets at 3.80, 3.81 and 3.86 ppm
(rotamers)), 4.05 (m, 1H), 4.4-4.65 (m, 3H), 6.82 (d, 1H, major
rotamer), 6.86 (d, 1H, minor rotamer), 6.95-7.05 (m, 1H), 7.08 (d,
1H, major rotamer), 7.23 (d, 1H, minor rotamer), 7.59 (d, 1H), 7.63
(dd, 1H, minor rotamer), 7.73 (dd, 1H, major rotamer), 7.82 (d, 1H,
minor rotamer), 7.98 (d, 1H, major rotamer) .sup.13C-NMR (100 MHz;
D.sub.2O ) carbonyl and amidine carbons: .delta.176.2, 174.6,
166.7
Example 2
(2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-Pab(2-Me).times.HOAc
(i) 3-Methyl-4-vinylbenzonitrile
[0182] 0.75 g (0.65 mmol) of
tetrakis(triphenylphosphine)palladium(0) was added to a solution of
5.1 g (0.026 mol) of 4-bromo-3-methylbenzonitrile and 8.3 g (0.026
mol) of vinyltributyltin in 250 mL of toluene under argon, and the
reaction was heated at reflux overnight. The mixture was filtered
through a layer of celite and evaporated. The residue was flash
chromatographed on silica gel with heptane:EtOAc (1:1) as eluant.
Yield: 4.0 g (100%).
[0183] .sup.1H-NMR (300 MHz; CDCl.sub.3): .delta.2.36 (s, 3H), 5.46
(d, 1H), 5.73 (d, 1H), 6.90 (dd, 1H), 7.4-7.5 (m, 2H), 7.52 (d,
1H)
(ii) 4-Hydroxymethyl-3-methylbenzonitrile
[0184] 3-Methyl-4-vinylbenzonitrile (0.40 g; 2.8 mmol; from step
(i) above) was dissolved in 50 mL of methanol and cooled to
-70.degree. C. Ozone (2 eq.) was bubbled through, and then 0.20 g
(5.3 mmol) of sodium borohydride and 5 mL of water were added and
the cooling bath was removed. After 4 h the methanol was evaporated
and the residue was partitioned between 1M HCl and ether. The
aqueous layer was extracted twice with ether and the combined
organic phase was washed with water, dried (Na.sub.2SO.sub.4) and
evaporated to yield 0.37 g (90%) of the desired product, which was
used in the next step without further purification.
[0185] .sup.1H-NMR (400 MHz; CDCl.sub.3): .delta.2.29 (s, 3H), 3.07
(broad, 1H), 4.69 (s, 2H), 7.37 (s, 1H), 7.45 (d, 1H), 7.53 (d,
1H)
(iii) 4-Methanesulfonyloxy-3-methylbenzonitrile
[0186] 1.2 g (0.010 mol) of methylsulfonyl chloride was added
dropwise to a solution of 1.5 g (0.010 mol) of
4-hydroxymethyl-3-methylbenzonitrile (from step (ii) above) and 1.0
g (0.010 mol) of triethylamine in 50 mL of methylene chloride at
0.degree. C. The reaction mixture was stirred at room temperature
for 3 h, washed with 1M HCl, water, dried (Na.sub.2SO.sub.4) and
evaporated. Yield: 2.1 g (91%).
[0187] .sup.1H-NMR (300 MHz; CDCl.sub.3): .delta.2.41 (s, 3H), 3.03
(s, 3H), 5.28 (s, 2H), 7.4-7.6 (m, 3H)
(iv) 4-Azidomethyl-3-methylbenzonitrile
[0188] 1.0 g (0.015 mol) of sodium azide and 10 mL of water were
added to a solution of 2.1 g (9.3 mmol) of
4-methanesulfonyloxy-3-methylbenzonitri- le (from step (iii) above)
in 20 mL of DMF. The reaction mixture was stirred for 1.5 h at room
temperature, poured into 200 mL of water and extracted three times
with ether. The combined organic phase was washed several times
with water, dried (Na.sub.2SO.sub.4) and evaporated. Yield: 1.4 g
(87%).
[0189] .sup.1H-NMR (300 MHz; CDCl.sub.3): .delta.2.39 (s, 3H), 4.40
(s, 2H), 7.40 (d, 1H), 7.45-7.55 (m, 2H)
(v) 4-Aminomethyl-3-methylbenzonitrile
[0190] The sub-title compound was prepared according to the method
described in Example l(ii) above from 1.4 g (8.1 mmol) of
4-azidomethyl-3-methylbenzonitrile (from step (iv) above) and 2.3 g
(9.0 mmol) of triphenylphosphine in 18 mL of ethanol and 2 mL of
water. Yield: 0.60 g (contaminated by triphenylphosphine oxide;
estimated actual yield: 0.36 g (28%)).
[0191] .sup.1H-NMR (300 MHz; CDCl.sub.3): .delta.2.38 (s, 3H), 3.90
(bs, 2H), 7.3-7.4 (m, 3H) obscured by triphenylphosphine oxide
(vi) (2,5-diMeO)Ph-(R and
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Me,4-CH- )
[0192] The sub-title compound was prepared according to the method
described in Example l(vii) above from 0.37 g (1.2 mmol) of
(2,5-diMeO)Ph-(R,S)CH(CH.sub.2OH)--CO-Pro-OH (see Example 1(vi)
above), 0.42 g of 4-aminomethyl-3-methylbenzonitrile (from step (v)
above; estimated content of pure material: 0.25 g (1.7 mmol)), 0.38
g (1.2 mmol) of TBTU and 0.62 g (4.8 mmol) of diisopropylethylamine
in 10 mL of DMF. The crude product was flash chromatographed on
silica gel with EtOAc:acetone (9:1) as eluant. The two
diastereomers separated. Total yield: 0.58 g contaminated by
triphenylphosphine oxide; estimated actual yield: 0.45 g (87%). The
first diastereomer to be eluted was isolated as diastereomer A.
[0193] Diastereomer A (yield: 0.30 g; contaminated by
triphenylphosphine oxide; estimated actual yield: 0.20 g)
[0194] .sup.1H-NMR (600 MHz; CDCl.sub.3): .delta.1.7-2.1 (m, 3H),
2.3-2.35 (m, 4H, thereof one singlet at 2.32 ppm), 3.1-3.2 (m, 1H),
3.65 (m, 1H), 3.7-3.8 (m, 4H, thereof one singlet at 3.73 ppm),
3.80 (s, 3H), 3.98 (m, 1H), 4.4-4.5 (m, 3H), 4.65 (m, 1H), 6.7-6.9
(m, 3H), 7.37 (d, 1H), 7.41 (s, 1H), the remaining aromatic signal
obscured by triphenylphosphine oxide
(vii) (2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Me,4-C(- NH)OMe)
(2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-NHCH.sub.2-Ph(2-Me,4-CN)
[0195] (0.30 g; estimated content of pure material: 0.20 g (0.44
mmol); diastereomer A from step (vi) above) was dissolved in 25 mL
of methanol saturated with hydrogen chloride gas and allowed to
stand at room temperature overnight. Evaporation yielded a crude
material which was used without further purification. According to
TLC (EtOAc:acetone; 9:1) no starting material was left.
(viii) (2,5-diMeO)Ph-(R or
S)CH(CH.sub.2OH)--CO-Pro-Pab(2-Me)(OH)
[0196] The crude product from step (vii) above was dissolved 10 mL
of methanol and 0.14 g (2.0 mmol) of hydroxylamine hydrochloride
and 0.40 g (4.0 mmol) of triethylamine were added. The mixture was
allowed to stand at room temperature for two days. Evaporation and
flash chromatography on silica gel with CH.sub.2Cl.sub.2:MeOH (9:1)
gave 0.13 g (60%) of the desired compound.
[0197] .sup.1H-NMR (600 MHz; CDCl.sub.3): .delta.1.80 (m, 1H), 1.93
(m, 1H), 2.08 (m, 1H), 2.20 (s, 3H), 2.27 (m, 1 H), 3.22 (m, 1H),
3.65-3.8 (m, 5H, thereof one singlet at 3.74 ppm), 3.80 (s, 3H),
4.06 (m, 1H), 4.32 (dd, 1H), 4.38 (dd, 1H), 4.51 (m, 1H), 4.71 (m,
1H), 4.93 (broad, 2H), 6.78 (dd, 1H), 6.81 (d, 1H), 6.85 (d, 1H),
7.08 (d, 1H), 7.16 (d, 1H), 7.20 (s, 1H), 7.81 (bt, 1H)
[0198] .sup.13C-NMR (100 MHz; CDCl.sub.3) carbonyl and amidine
carbons: .delta.174.7, 172.8, 154.9
(ix) (2,5-diMeO)Ph-(R or S)CH(CH.sub.2OH)--CO-Pro-Pab(2-Me)
(2,5-diMeO)Ph-(R or S)CH(CH.sub.2OH)--CO-Pro-Pab(2-Me)(OH)
[0199] (65 mg; 0.13 mmol; from step (viii) above) was dissolved in
5 mL of ethanol. HOAc (8 drops from a Pasteur pipette) and 40 mg of
10% Pd/C were added. The reaction mixture was hydrogenated at
atmospheric pressure for two days. The reaction mixture was
filtered through celite and evaporated. The residue was dissolved
in water, washed with ethyl acetate and freeze dried. Yield: 46 mg
(65%).
[0200] .sup.1H-NMR (500 MHz; D.sub.2O ) rotamers: .delta.1.8-2.1
(m, 6H, thereof one singlet at 1.92 ppm), 2.2-2.4 (m, 4H, thereof
two singlets at 2.24 (minor rotamer) and 2.38 (major rotamer)),
3.35 (m, 1H, minor rotamer), 3.6-3.7 (m, 1H), 3.75-3.85 (m, 7H,
thereof three singlets at 3.77, 3.79 and 3.84 ppm(rotamers)),
4.0-4.1 (m, 1H), 4.4-4.5 (m, 3H), 4.8 (m, 1H partially obscured by
the HDO peak), 6.80 (d, 1H, major rotamer), 6.85 (d, 1H, minor
rotamer), 6.9-7.1 (m, 2H), 7.45 (d, 1H, major rotamer), 7.51 (d,
1H, minor rotamer), 7.55-7.65 (m, 2H)
[0201] .sup.13C-NMR (75 MHz; D.sub.2O ) carbonyl and amidine
carbons: .delta.175.4, 174.5 (minor), 174.2 (minor), 174.1,
167.1
Example 3
[0202] The title compounds of Examples 1 and 2 were tested in Test
A above and were both found to exhibit an IC.sub.50TT value of less
than 0.3 .mu.M.
[0203] Abbreviations
1 aq = aqueous Bn = benzyl DCC = dicyclohexylcarbodiimide DIPEA =
diisopropylethylamine DMAP = N,N-dimethyl amino pyridine DMF =
dimethylformamide DMSO = dimethylsulphoxide EDC =
1-(3-dimethylaminopropyl)-3-ethylcarbod- iimide hydrochloride Et =
ethyl EtOAc = ethyl acetate EtOH = ethanol h = hours HBTU =
[N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium
hexafluorophosphate] HOAc = acetic acid H-Pab =
4-amidinobenzylamine H-Pab(Z) = 4-(N-benzyloxycarbonylamidino)benz-
ylamine H-Pab(2-Cl) = 4-amidino-2-chloro-benzylamine H-Pab(2-Me) =
4-amidino-2-methyl-benzylamine HPLC = high performance liquid
chromatography Me = methyl MeOH = methanol Ph = phenyl Pr = propyl
i-PrOH = i-propanol TBTU =
[N,N,N',N'-tetramethyl-O-(benzotriazol-1-yl)uronium
tetrafluoroborate] TEA = triethylamine THF = tetrahydrofuran Z =
benzyloxy carbonyl
[0204] Prefixes n, s, i and t have their usual meanings: normal,
iso, sec and tertiary. The stereochemistry for the amino acids is
by default (S) if not otherwise stated.
* * * * *