U.S. patent application number 11/854532 was filed with the patent office on 2008-01-03 for amidinophenylalanine derivatives as thrombin inhibitors.
This patent application is currently assigned to LEK PHARMACEUTICALS D.D.. Invention is credited to Gregor Mlinsek, Tomaz Solmajer, Mojca Stegnar, Bakija Alenka Trampus, Uros Urleb, Anamarija Zega.
Application Number | 20080004256 11/854532 |
Document ID | / |
Family ID | 20432799 |
Filed Date | 2008-01-03 |
United States Patent
Application |
20080004256 |
Kind Code |
A1 |
Urleb; Uros ; et
al. |
January 3, 2008 |
Amidinophenylalanine Derivatives as Thrombin Inhibitors
Abstract
The compounds of the formula I ##STR1## and pharmaceutically
acceptable salts thereof and a process for preparing the same and
pharmaceutical compositions containing the same are described
wherein the substituents have the meaning as specified in the
description. The compounds are used as thrombin inhibitors.
Inventors: |
Urleb; Uros; (Ljubljana,
SI) ; Zega; Anamarija; (Ljubljana, SI) ;
Stegnar; Mojca; (Ljubljana, SI) ; Trampus; Bakija
Alenka; (Ljubljana, SI) ; Solmajer; Tomaz;
(Ljubljana, SI) ; Mlinsek; Gregor; (Skofja Loka,
SI) |
Correspondence
Address: |
BROMBERG & SUNSTEIN LLP
125 SUMMER STREET
BOSTON
MA
02110-1618
US
|
Assignee: |
LEK PHARMACEUTICALS D.D.
Verovskova 57
Ljubljana
SI
1526
|
Family ID: |
20432799 |
Appl. No.: |
11/854532 |
Filed: |
September 12, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10433358 |
Jun 3, 2003 |
7285547 |
|
|
PCT/IB01/02600 |
Dec 20, 2001 |
|
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11854532 |
Sep 12, 2007 |
|
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Current U.S.
Class: |
514/217.11 ;
514/237.5; 514/255.01; 514/330; 514/423; 540/607; 544/159; 544/386;
546/245; 548/536 |
Current CPC
Class: |
C07D 295/215 20130101;
A61P 7/02 20180101; C07C 311/49 20130101; A61P 43/00 20180101 |
Class at
Publication: |
514/217.11 ;
514/237.5; 514/255.01; 514/330; 514/423; 540/607; 544/159; 544/386;
546/245; 548/536 |
International
Class: |
A61K 31/55 20060101
A61K031/55; A61K 31/40 20060101 A61K031/40; A61K 31/445 20060101
A61K031/445; A61P 7/02 20060101 A61P007/02; C07D 211/60 20060101
C07D211/60; C07D 295/00 20060101 C07D295/00; C07D 223/04 20060101
C07D223/04; C07D 207/16 20060101 C07D207/16; A61K 31/495 20060101
A61K031/495; A61K 31/5375 20060101 A61K031/5375 |
Foreign Application Data
Date |
Code |
Application Number |
Dec 22, 2000 |
SI |
P-200000326 |
Claims
1. The compound of the formula I ##STR52## wherein R.sup.1
represents a residue of the formula ##STR53## wherein R.sup.4 is
selected from the group consisting of H, alkyl (C.sub.1-C.sub.3),
OH, O-alkyl (C.sub.1-C.sub.3), and NH.sub.2; R.sup.2 is selected
from the group consisting of a residue of the formula ##STR54##
wherein R.sup.5 is selected from the group consisting of H, alkyl
(C.sub.1-C.sub.3), CF.sub.3, and COOR.sup.9; R.sup.6 is selected
from the group consisting of H, alkyl (C.sub.1-C.sub.3), CF.sub.3,
and COOR.sup.9; R.sup.7 is selected from the group consisting of
alkyl (C.sub.1-C.sub.3), cycloalkyl and (C.sub.3-C.sub.6); R.sup.8
is selected from the group consisting of alkyl (C.sub.1-C.sub.3),
and cycloalkyl (C.sub.3-C.sub.6); R.sup.9 is selected from the
group consisting of H, and alkyl (C.sub.1-C.sub.3); R.sup.10 is
selected from the group consisting of H, alkyl (C.sub.1-C.sub.3),
and benzyl; R.sup.11 is selected from the group consisting of H,
SO.sub.2Me, and CO.sub.2Me; R.sup.3 is selected from the group
consisting of a residue of the formula ##STR55## wherein R.sup.12
is selected from the group consisting of H, H.sub.3C--, and acetyl
and pharmaceutically acceptable salts thereof.
2. A compound as claimed in claim 1, of formula I wherein R.sup.2
is selected from the group consisting of: ##STR56##
3. A compound as claimed in preceding claim 1 or claim 2 wherein
R.sup.3 is selected from the group consisting of: ##STR57##
4. A compound as claimed in claim 1 wherein said compound is
selected from the group consisting of:
N'-hydroxy-3-{[1-[(2-methyl-I -piperidinyl)carbonyl]
-2-(2-naphthylsulfonyl) hydrazino]methyl}benzenecarboximidamide;
N'-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]
-1-[(2-methyl-1-piperidinyl)
carbonyl]hydrazino}methyl)benzenecarboximidamide; Imino
[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]
-1-[(2-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)phenyl]methanaminiu-
m chloride; N'-Hydroxy-3-{[1-[(4-methyl-1-piperidinyl)carbonyl]
-2-(2-naphthylsulfonyl) hydrazino]methyl}benzenecarboximidamide;
Imino(3-{[ 1-[(4-methyl-I -piperidinyl)carbonyl]
-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)methanaminium
chloride; N'-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]
-1-[(4-methyl-1-piperidinyl)
carbonyl]hydrazino}methyl)benzenecarboximidamide; Imino
[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]
-1-[(4-methyl-1-piperidinyl)carbonyl]hydrazino}methyl)phenyl]methanaminiu-
m chloride.
5. A method for inhibiting blood coagulation comprising contacting
a sample of blood or plasma with a compound of formula I according
to claim 1.
6. A process for preparing a compound of the formula I according to
claim 1, comprising: converting 3-cyanobenzaldehyde of formula (II)
##STR58## with BOC-carbazate of the formula (III) ##STR59## to a
compound of formula (IV), ##STR60## reducing the compound of
formula (IV) by catalytic hydrogenation or with NaCNBH.sub.3 to a
compound of formula (V), ##STR61## reacting the compound of formula
(V) with triphosgene and an amine of formula (VI), ##STR62##
wherein R.sup.5 and R.sup.6 have the same meanings as in the
formula (I), or with triphosgene and an amine of W+e formula (VII
or VIII), ##STR63## or with triphosgene and an amine of the formula
(IX), ##STR64## wherein R.sup.7 and R.sup.8 have the same meanings
as in the formula (I), or with triphosgene and an amine of We
formula (X), ##STR65## wherein R.sup.10 has the same meaning as in
the formula (I), to yield a compound of formula (XI), ##STR66##
wherein R.sup.2 has the same meaning as in formula (I); removing
the protecting BOC group with HCl (g) in HOAc at room temperature
to obtain a compound of formula (XII), ##STR67## wherein R.sup.2
has the same meaning as in the formula (I); reacting the compound
of formula (XII) with aromatic sulfonylchloride to yield a compound
of formula (XIII) ##STR68## wherein R.sup.2 and R.sup.3 have the
same meanings as in the formula (I); and converting the compound of
formula (XIII) to the a compound of formula (I) ##STR69##
7. A method for treating blood coagulation disorders in man and
other mammals comprising administering a therapeutically effective
amount of a compound of formula I as claimed in claim 1, to a
subject in need thereof.
8. A pharmaceutical composition comprising a therapeutically
effective amount of a compound of formula I as claimed in claim 1
and pharmaceutically acceptable auxiliary substances.
9. A pharmaceutical composition according to claim 8, for
inhibiting thrombin in man and other mammals, wherein the compound
of formula I has a trypsin K.sub.i to thrombin K.sub.i ratio of
about 5 or more.
10. A pharmaceutical composition according to claim 8, for
inhibiting thrombin in man and other mammals, wherein the compound
of formula I has a thrombin K.sub.i of about 0.6 .mu.M or less.
11. A method for inhibiting thrombin in the blood of man and other
mammals comprising administering a therapeutically effective amount
of a compound of formula I of as claimed in claim 49.
12. A method for inhibiting thrombin in man and other mammals
comprising administering a therapeutically effective amount of a
compound of formula I as claimed in claim 10.
Description
TECHNICAL FIELD
[0001] The invention belongs to the field of pharmaceutical
industry and relates to novel azaphenylalanine derivatives,
procedures for their preparation and pharmaceutical compositions
containing them. Novel azaphenylalanine derivatives have the
enzymatic activity (serine proteases) and anticoagulant effect.
Technical Problem
[0002] There exists a constant need for new medicinal products with
anticoagulant activity that may be administered either orally or
parenterally and that are highly selective and of low toxicity.
Recently, low molecular weight thrombin inhibitors appear to be of
growing importance in this area.
PRIOR ART
[0003] Thrombin as a senne protease is one of key enzymes in the
processes of blood coagulation and in the development of thrombosis
(Edit, J. F.; Allison, P.; Noble, S.; Ashton, J.; Golino, P.;
McNard, J.; Buja, L. M.; Willerson, J. T., J. Clin. Invest. 1989,
84,18.).
[0004] The crystalline structure of serine proteases of thrombin
and serine is known (Bode, W.; Turk, D.; Karshikov, A. J., Protein
Sci. 1992, 1, 426).
[0005] Currently used anticoagulant agents are toxic and of limited
effectiveness.
[0006] Low molecular weight thrombin inhibitors should have
selective efficacy and can be used in oral administration. There
are reversible and irreversible thrombin antagonists (Kimball, S.
D., Curr. Pharm. Design 1995, 1, 441, Das, J.; Kimball, S. D.,
Bioorg. Med. Chem. 1995, 3, 999, Kimball, S. D., Blood Coagulation
and Fibrinolysis 1995, 6, 511, Breznik, M.; Pe{hacek over (c)}ar,
S., Farm. Vestn. 1997, 48, 545, Sanderson, P. E. J.; Naylor-Olsen,
A. M.; Current Med. Chem. 1998, 5, 289, Rewinkel, J. B. M.; Adang,
A. E. P., Curr. Pharm. Design 1999,5,1043,Wiley, M. R.; Fisher, M.
J.; Exp. Opin. Ther. Patents 1997, 7, 1265, Menear, K.; Current
Med. Chem. 1998, 5, 457.
[0007] The majority of reversible thrombin antagonists derive from
peptidomimetically modified structure D-Phe-Pro-Arg. The aim of the
modification is to provide chemical stability, selectivity and
effectiveness.
[0008] The active substance used in Japan and the USA is argatroban
(Kikumoto, R.; Tamao, Y.; Tezuka, T.; Tonomura, S.; Hara, H.,
Ninomiya, K.; Hijikata, A.; Okamoto, S., Biochemistty 1984, 23,
85).
[0009] Amidinophenylalanine derivatives and aminopyridylalanine
derivatives have selective antithrombotic activity (Danilewicz, J.,
et al. WO 95/13274 (1995)).
[0010] By replacing amidino-pyperidine P1 group with a weakly basic
2-amino-6-metylpyridine structure, a selective and effective
thrombin inhibitor that can be used in oral administration has been
obtained (Sanderson, P. E. J.; et al., Bioorg. Med. Chem. 1998, 8,
817).
[0011] SI 20025 describes thrombin inhibitors which are the
derivatives of azaphenylalanine.
[0012] Accordingly, the object of the present invention is to
overcome the problems encountered in the prior art.
DESCRIPTION OF THE SOLUTION OF TECHNICAL PROBLEM INCLUDING
EXAMPLES
[0013] The above problems are solved e.g. by a compound as defined
in claim 1 and a process as defined in claim 6 as well as
pharmaceutical compositions as defined in claim 8.
[0014] The invention relates particularly to novel azaphenylalanine
derivatives and analogs thereof of the general formula (I) ##STR2##
wherein [0015] R.sup.1 represents a residue of the formula ##STR3##
[0016] wherein R.sup.4=H, alkyl (C.sub.1-C.sub.3), OH, O-alkyl
(C.sub.1-C.sub.3), NH.sub.2; [0017] R.sup.2 represents a residue of
the formula ##STR4## [0018] wherein R.sup.5=H, alkyl
(C.sub.1-C.sub.3), CF.sub.3, COOR.sup.9,
[0019] R.sup.6=H, alkyl (C.sub.1-C.sub.3), CF.sub.3,
COOR.sup.9,
[0020] R.sup.7=alkyl (C.sub.1-C.sub.3), cycloalkyl
(C.sub.3-C.sub.6),
[0021] R.sup.8=alkyl (C.sub.1-C.sub.3), cycloalkyl
(C.sub.3-C.sub.6),
[0022] R.sup.9=H, alkyl (C.sub.1-C.sub.3),
[0023] R.sup.10=H, alkyl (C.sub.1-C.sub.3), benzyl
[0024] R.sup.11=H, SO.sub.2Me, CO.sub.2Me; [0025] R.sup.3
represents a residue of the formula ##STR5##
[0026] wherein R.sup.12=H. alkyl (C.sub.1-C.sub.3), acetyl and
pharmaceutically acceptable salts thereof.
[0027] In comparison to the compounds, described in SI 20025, the
compounds of the present invention are of greater activity. Ki
thrombin (.mu.M) of the compound a, according to the patent SI
20025, is 0.032, Ki thrombin (.mu.M) of the compound b, according
to the present invention, is 0.005. ##STR6##
[0028] The invention also particularly relates to a process for the
preparation of azaphenylalanine derivatives and analogs of the
general formula (I).
[0029] They may be suitably prepared as follows:
[0030] a) 3-cyanobenzaldehyde of formula (II) ##STR7##
[0031] is converted with BOC-carbazate of the formula (III)
##STR8##
[0032] to the compound of the formula (IV), ##STR9##
[0033] which is subjected to reduction by catalytic hydrogenation
or with NaCNBH.sub.3 and is converted to the compound (V),
##STR10##
[0034] which reacts with triphosgene and amine of the formula (VI),
##STR11##
[0035] wherein R.sup.5 and R.sup.6 have the same meanings as in the
formula (I), or with triphosgene and amine of the formula (VII or
VIII), ##STR12##
[0036] or with triphosgene and amine of the formula (IX),
##STR13##
[0037] wherein R.sup.7 and R.sup.8 have the same meanings as in the
formula (I), or with triphosgene and amine of the formula (X),
##STR14##
[0038] wherein R.sup.10 has the same meaning as in the formula
(I),
[0039] in "one pot" reaction to the compound (XI) ##STR15## [0040]
wherein R.sup.2 has the same meaning as in the formula (I).
[0041] The protecting BOC group in the compound (XI) is removed
with HCl (g) in HOAc at room temperature to obtain the compound
(XII), ##STR16##
[0042] wherein R.sup.2 has the same meanings as in the formula (I)
which reacts with aromatic sulfonylchloride up to the compound
(XIII), ##STR17##
[0043] wherein R.sup.2 and R.sup.3 have the same meanings as in the
formula (I).
[0044] The compound (XIII) is converted to the compound of the
formula (I) ##STR18##
[0045] in a usual manner. This can be achieved e.g. by converting
compound (XIII) by addition of a hydroxylamine and an anhydrous
alcohol such as e.g. ethanol or a mineral acid such as e.g. HCl(g)
and e.g. ammonium acetate, thus obtaining compound (I).
[0046] When the compound (XIII) reacts with hydroxylamine in
anhydrous ethanol, R.sup.4 denotes OH group, after the reaction of
the compound (XIII) with HCl (g) and ammonium acetate R.sup.4
denotes hydrogen.
[0047] The starting compounds are prepared, unless otherwise
directed, according to the procedures described in the literature;
e.g., the compound of the formula IV as described by A. Fassler,
et. al., J. Med. Chem. 1996, 39, 3203-3215.
[0048] The invention further relates to the use of compounds of the
formula I as therapeutically active compounds. The novel compounds
are preferably thrombin inhibitors. They inhibit thrombin and
formation of fibrin. They are useful in the treatment or prevention
of a variety of thrombosis forms: (i) venous thromboembolism due to
formation of a thrombus within a vein (venous thrombosis)
associated with acquired (prolonged bedrest, surgery, injury,
malignancy, pregnancy and postpartum states or inherited
(deficiency of natural coagulation inhibitors) risk factors,
obstruction or occlusion of a lung artery by a detached thrombus
(pulmonary embolism), (ii) cardiogenic thromboembolism due to
formation of a thombus in the heart associated with cardiac
arrythmia, heart valve defect, prosthetic heart valves or heart
disease, embolism of peripheral arteries caused by a detached
thrombus, most commonly in the brain (ischemic stroke), (iii)
arterial thrombosis due to underlying atherosclerotic processes in
the arteries which obstructs or occludes an artery and causes
myocardial ischemia (angina pectoris, acute coronary syndrome) or
heart muscle death cell (myocardial infarction), obstructs or
occludes a peripheral artery (ischemic peripheral artery disease)
and obstructs or occludes the artery after the procedure on the
blood vessel (reocclusion or restenosis after transluminal coronary
angioplasty, reocclusion or restenosis after percutaneous
transluminal angioplasty of peripheral arteries) and (iv) in the
number of states (e.g., in complications in pregnancy, in
metastazing malignant diseases, after extensive injuries, in
bacterial sepsis) when thrombogenic activation causes widespread
formation of thrombi within the vascular system (disseminated
intravascular coagulation).
[0049] The compounds of the present invention may be also used as
an adjunct therapy in conjunction with thrombolytic therapy in
recent myocardial infarction, in combination with aspirin in
patients with unstable angina pectoris designed to undergo
percutaneous transluminal angioplasty and in the treatment of
patients with thrombosis and with heparin-induced
thrombocytopenia.
[0050] The compounds of the present invention may further be used
for the prevention of coagulation of blood which is in contact with
nonbiological surfaces (vascular prosthesis, vascular stents,
prosthetic heart valves, extracorporeal circulation systems,
hemodialysis) and in vitro to prevent coagulation in biological
samples for testing or storage.
[0051] The present invention also relates particularly to
pharmaceutical compositions comprising the compounds of the formula
I. They can be formulated as injectable or oral formulations. The
active drug component may be combined with suitable standard
additives with respect to the intended form of administration. The
pharmaceutical compositions may be prepared according to the
standard procedures. The preparation may be formulated in such a
manner as to permit controlled and sustained release of the active
ingredient. Dosage, frequency and mode of administration depend on
a variety of factors, they also depend on individual active
ingredient and its pharmacokinetic parameters and on the patient's
therapeutic needs.
In Vitro Assays for Determining Proteinase Inhibition
[0052] Coagulation assays
[0053] The Anticoagulant effect of thrombin inhibitors was measured
by thrombin time (TT), activated partial thromboplastin time
(aPTT), and prothrombin time (PT) assays. Inhibitors were added to
normal pooled human plasma over a range of concentrations and
clotting was recorded in an automated coagulometer (Fibrintimer,
Dade/Behring). The concentration of inhibitor that doubled the
clotting time was determined for each assay.
[0054] 1. Thrombin time (TT)
[0055] a) Principle of Method
[0056] In a plasma sample thrombin converts fibrinogen into fibrin
and the clot is formed. The time for clot formation was measured.
Thrombin time is prolonged due to disorders in fibrin
polymerisation or due to the presence of thrombin inhibitors.
[0057] b) Reagents
[0058] Thrombin (Test Thrombin Reagent, 1.5 IU/mL, Dade/Behring):
lyophilized bovine thrombin was dissolved in 5 mL of HEPES (25
mmol/L, pH 7.4). The reagent was warmed to 37.degree. C. prior to
the assay.
[0059] Normal pooled plasma: venous blood from 11 apparently
healthy volunteers was collected (1 part 0.11 mol/L sodium citrate
solution with 9 parts of blood) and centrifuged immediately at
2000.times.g for 30 minutes at 4.degree. C. Plasma was removed,
pooled and stored in aliquots at -70.degree. C. until use.
[0060] Inhibitors: were dissolved in DMSO (10 mM stock solution)
and diluted with distilled water to working solutions (the highest
concentration 100 .mu.M).
[0061] c) Method
[0062] Inhibitors (10 .mu.L working solution, concentrations from
2.5 .mu.M to 100 .mu.M) and pooled plasma (90 .mu.L) were incubated
at 37.degree. C. for 5 minutes and then thrombin (200 .mu.L) was
added. Clot formation was measured in a coagulometer in
duplicate.
[0063] 2. Activated partial thromboplastin time (aPTT)
[0064] a) Principle of method
[0065] Incubation of plasma with the optimal quantity of
phospholipids and a surface activator leads to activation of
factors of the intrinsic coagulation pathway. The addition of
calcium ions triggers the coagulation process. The time of fibrin
clot formation was measured. aPTT was used as a screening test for
coagulation disorders of the intrinsic coagulation pathway. It is
prolonged due to intrinsic coagulation factors deficit or due to
the presence of inhibitors.
[0066] Reagents
[0067] Pathromtin SL (Dade/Behring): silicon dioxide particles,
vegetable phospholipids, sodium chloride (2.4 .mu.L), Hepes (14.3
.mu.L, pH 7.6), sodium azide (<1 g/L). The reagent was used at
room temperature (15-25.degree. C.).
[0068] Calcium chloride solution: 0.025 mol/L, warmed to 37.degree.
C.
[0069] Normal pooled plasma: venous blood from 11 apparently
healthy volunteers was collected (1 part 0.11 mol/L sodium citrate
solution with 9 parts of blood) and centrifuged immediately at
2000.times.g for 30 minutes at 4.degree. C. Plasma was removed,
pooled and stored in aliquots at -70.degree. C. until use.
[0070] Inhibitors: were dissolved in DMSO (10 mM stock solution)
and diluted with distilled water to working solutions (the highest
concentration 100 .mu.M).
[0071] c) Method
[0072] Inhibitor (10 .mu.L working solution, concentrations from 5
.mu.M to 100 .mu.M) and pooled plasma (90 .mu.L) were incubated at
37.degree. C. for 5 minutes. Pathromtin (100 .mu.L) was added and
the sample was incubated for another 2 minutes at 37.degree. C. The
addition of calcium chloride (100 .mu.L) triggered the coagulation
process and clot formation was detected with a coagulometer in
duplicate.
[0073] 3. Prothrombin Time (PT)
[0074] a) Principle of Method
[0075] An optimal amount of thromboplastin and calcium are added to
plasma sample and the time of fibrin clot formation wass measured.
PT is a rapid, sensitive screening test for coagulation disorders
of the extrinsic pathway. It is well suited for the induction and
monitoring of oral anticoagulant therapy, for diagnosing genetic or
acquired deficiencies in coagulation factors and checking the
synthesis performance of the liver in hepatic diseases. PT is
prolonged due to extrinsic coagulation factors deficit or due to
the presence of inhibitors.
[0076] b) Reagents
[0077] Thromboplastin (Thromborel S, Dade/Behring): was dissolved
in 4 mL of distilled water. Reagent was at 37.degree. C. prior to
use at least 30 minutes.
[0078] Normal pooled plasma: venous blood from 11 apparently
healthy volunteers was collected (1 part 0.11 mol/L sodium citrate
solution with 9 parts of blood) and centrifuged immediately at
2000.times.g for 30 minutes at 4.degree. C. Plasma was removed,
pooled and stored in aliquots at -70.degree. C. until use.
[0079] Inhibitors: were dissolved in DMSO (10 mM stock solution)
and diluted with distilled water to working solutions (the highest
concentration 100 .mu.M).
[0080] c) Method
[0081] Inhibitor (10 .mu.L working solution, concentrations from 5
.mu.M to 100 .mu.M) and pooled plasma (90 .mu.L) were incubated at
37.degree. C. for 5 minutes and then Thromborel S (200 .mu.L) was
added. Clot formation was measured with a coagulometer in
duplicate.
[0082] Enzyme assay
[0083] The enzyme inhibitory effect of the compounds can be
identified by determination of inhibition constant Ki. It denotes
the degree of dissociation of the enzyme-inhibitor complex. Low
dissociation constant means high potency of inhibitor. Ki can be
determined during reaction of the enzyme with a specific
chromogenic substrate which under hydrolysis by the enzyme develops
color. Reaction in time is recorded by spectrophotometry and Ki is
calculated from kinetic parameters (Vmax, Km, reaction rate).
[0084] 1. Determination of Thrombin Ki
[0085] a) Principle of Method
[0086] The ability of a compound to act as an inhibitor of human
thrombin catalytic activity was assessed by determination of
Ki.
[0087] b) Reagents
[0088] Buffer: HBSA, pH 7.5 (10 mM Hepes, 150 mM NaCl, 0.1% w/v
bovine serum albumin)
[0089] Substrate (S-2238: H-D-Phe-Pip-Arg-pNA HCl, 25 mg;
Chromogenix): dissolved in distilled water to 1 mM concentration.
(Km is 2.6 .mu.M)
[0090] Human thrombin (308 NIH; Sigma): dissolved in saline to give
a stock solution of 20 NIH/mL.
[0091] Inhibitors: were dissolved in DMSO (10 mM stock solution)
and diluted with distilled water to working solutions (the highest
final concentration of DMSO was 3%).
[0092] c) Method
[0093] A mixture of 50 .mu.L of buffer, 50 .mu.L of inhibitor in
water (final concentrations from 10 to 100 .mu.M) and 50 .mu.L of
thrombin (0.5 NIH U/mL f.c.) was incubated for 15 minutes at room
temperature. The reaction was started with 50 .mu.L of S-2238 (20
.mu.M or 40 .mu.M f.c.) and the absorbance of each sample at 405 nm
(at 25.degree. C.) was measured in triplicate every 10 seconds for
a period of 15 minutes using a microtiter plate reader (Tecan
Sunrise).
[0094] Thrombin activity was determined from the change in
absorbance in the linear part of the velocity graph. Ki was
calculated according to Cheng and Prussof (Biochem Pharmacol, 1973)
where Ki is IC.sub.50/(1+S/Km). The Km for the substrate was
determined under the test conditions with at least 6 substrate
concentrations varying around Km and calculated with the non-linear
regression programme Curve expert.
[0095] 2. Determination of Trypsin Ki
[0096] a) Principle
[0097] The ability of a compound to act as an inhibitor of trypsin
catalytic activity was assessed by determination of Ki.
[0098] b) Reagents
[0099] Buffer: HBSA, pH 7.5 (10 mM Hepes, 150 mM NaCl, 0.1% w/v
bovine serum albumin)
[0100] Substrate (S-2222: N-benzoyl-Ile-Glu-Gly-Arg-pNA HCl, 25 mg;
Chromogenix): dissolved in distilled water to 2mM concentration.
(Km is 21 .mu.M)
[0101] Trypsin (6000 E/mg prot.; Sigma): dissolved in distilled
water to give a stock solution of 300 E/mL.
[0102] Inhibitors: were dissolved in DMSO (10 mM stock solution)
and diluted with distilled water to working solution (the highest
final concentration of DMSO was 10%).
[0103] c) Method
[0104] 50 .mu.L of buffer, 50 .mu.L of test compound in water
(final concentrations from 50 to 300 .mu.M) and 50 .mu.L of trypsin
(3 mE/mL f.c.) were incubated for 15 minutes at room temperature.
The reaction was started with 50 .mu.L of S-2222 (50 .mu.M or 100
.mu.M f.c.) and the absorbance of each sample at 405 nm (at
25.degree. C.) was measured in triplicates every 10 seconds for a
period of 15 minutes using a microtiter plate reader (Tecan
Sunrise).
[0105] Trypsin activity was determined from the change in
absorbance in the linear part of velocity graph. Ki was calculated
according to Cheng and Prussof (Biochem Pharmacol, 1973) where Ki
is IC.sub.50/(1+S/Km). The Km for the substrate was determined
under test conditions with at least 6 substrate concentrations
varying around Km and calculated with the non-linear regression
program Curve expert.
[0106] 3. Selectivity to Thrombin
[0107] By the use of other serine protease like trypsin with the
appropriate chromogenic substrate, selectivity of inhibitors with
respect to thrombin was determined. Selectivity of an inhibitor is
expressed as a ratio of Ki for trypsin to Ki for thrombin.
[0108] In vivo Assays
[0109] 1. Stasis-Induced Thrombosis
[0110] Principle of Method
[0111] Using a combination of thrombogenic challenge (0.045 ng/kg
of tissue factor) and stasis, compounds were tested for their
abillity to affect thrombus formation in a venous thrombosis model
in the rat.
[0112] Method
[0113] Thrombus formation by a combination of stasis and
hypercoagulability was induced as described by Vogel et al.
(Thromb. Res., 1989, 54, 399-410). Male sprague-Dawley rats
(250-300 g) were anaesthetized with sodium pentobarbitone (30
mg/kg, i.p.). The abdomen of the animals was surgically opened and
after careful dissection, the vena cava was exposed and dissected
free from surrounding tissue. Saline, or the various compounds were
administered i.v. 5 min before thrombosis induction. Two loose
sutures were prepared 0.7 cm apart on the inferior vena cava and
all collateral veins were ligated. Human tissue factor (0.045
ng/kg, i.v.) was injected into the dorsal vein of the penis. Ten
seconds after the end of the injection, stasis were established by
tightening the two, firstly the proximal and then the distal. The
abdominal cavity was provisionally closed and stasis was maintained
for 20 minutes. The cavity was then reopened, the ligated segment
was opened longitudinally and the thrombus formed was removed,
rinsed, blotted on filter paper, dried overnight at 60.degree. C.
and weighed. Under these experimental conditions, control thrombus
weight was 5.5.+-.0.1 mg (n=15).
[0114] 2. Bleeding Time
[0115] Principle of Method
[0116] The hemorrhagic risk associated to a treatment with selected
compounds were determined using an experimental model of bleeding:
transection of the tail of rats.
[0117] Method
[0118] Bleeding time was determined by transection of the tail, 2
mm from the tip, of phenobarbital-anaestetised rats (30 mg/kg
i.p.). The compounds were injected i.v. at the indicated doses 5
min before tail transection. Blood was carefully blotted every 15 s
on a filter paper. Haemostasis was considered to be achieved when
no more blood stain was observed over 1 min.
EXAMPLES
[0119] The invention is further described by means of the following
examples, but not in any limitative case:
Example 1
tert-Butyl
2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]-1-hydraz-
inecarboxylate
[0120] ##STR19##
[0121] Triphosgene (2.296 g) was dissolved in 15 ml of anhydrous
dichloromethane and cooled to -5.degree. C. While stirring under
argon atmosphere, a solution of
N-1-(tert-butoxycarbonyl)-N-2-[(3-cyanophenyl)methyl]hydrazine
(3.833 g) and N,N-diisopropylethylamine (4.040 ml) in 25 ml of
dichloromethane was added dropwise. After addition, the reaction
mixture was stirred for another 5 minutes, and a mixture of
4-methyl piperidine (1.836 ml) and N,N-diisopropylethylamine (4.040
ml ) in 25 ml dichloromethane was then added at once and stirred at
room temperature for another 20 minutes. The solvent was evaporated
in vacuo and the residue was dissolved in 50 ml of ethyl acetate,
washed with 25 ml of 5% citric acid, with 25 ml of 5% solution of
NaHCO.sub.3 and with 25 ml of brine. The organic phase was dried
over MgSO.sub.4, filtered and the filtrate evaporated under reduced
pressure.
[0122] The resulting mixture was suspended in 20 ml of diethylether
and 5 ml of hexane was added. The precipitate was filtered by
suction.
[0123] Yield: 1.756 g (30.4%)
[0124] Melting range: 147-150.degree. C.
[0125] IR (KBr, cm.sup.-1): 3275.0, 2922.7, 2229.3, 1722.3, 1632.3,
1497.3, 1445.5, 1367.6, 1252.9, 1157.0, 979.2, 805.1, 736.2,
573.7
[0126] NMR (DMSO-d.sub.6) ):.delta. (ppm): 0.86 (d, J=6,4 Hz, 3H,
CH.sub.3), 0.90-1.05 (m, 2H, Pip-H.sup.3,5), 1.35 (s, 9H, Boc),
1.45-1.60 (m, 4H, Pip-H.sup.3',5'), 2.60-2.75 (m, 2H,
Pip-H.sup.2,6), 3.70-3.85(m, 2H, Pip-H.sup.2',6'), 4.52 (br s, 2H,
CH.sub.2), 7.58-7.66 (m, 1H, Bn--H.sup.5), 7.79-7.85 (m, 1H,
Bn--H.sup.6), 7.93-8.05 (m, 2H, Bn--H.sup.24), 11.05 (s, 1H,
NH).
[0127] Molecular weight of C.sub.20 H.sub.27 N.sub.4 03:
calculated: 372; found: 373 (MH.sup.+)
Example 2
2-(3-Cyanobenzyl)-2-[(4-methyl-1-pipendinyl)carbonyl]hydrazinium
chloride
[0128] ##STR20##
[0129] terc-butyl
2-(3-cyanobenzyl)-2-[(4-methyl-1-pyperidinyl)carbonyl]hydrazine
carboxylate (1.173 mg) was dissolved in 30 ml of glacial acetic
acid and HCl gas was bubbled through the solution for 20 minutes.
The progress of the reaction was monitored by TLC. Glacial acetic
acid was evaporated on a rotavapor, and diethylether was poured
over the oily product. The liquid phase was removed, the resulting
precipitate is dried at 50.degree. C. and desiccated over NaOH for
24 hours.
[0130] Yield: 0. 875 g (%) of the mixture
[0131] Melting range: 170-173.degree. C.
[0132] IR (KBr, cm.sup.-1): 3417.8, 2934.6, 2664.1, 2227.3, 1711.6,
1641.3, 1433.7, 1402.3, 1249.6, 1132.0, 1017.8
[0133] NMR (DMSO-d.sub.6):.delta. (ppm): 0.92 (d, J=6,4 Hz, 3H,
CH.sub.3), 0.99-1.16 (m, 2H, Pip-H.sup.3,5), 1.52-1.71 (m, 4H,
Pip-H.sup.3',5'), 2.90-3.05 (m, 2H, Pip-H.sup.2,6), 3.80-3.95 (m,
2H, Pip-H.sup.2',6'), 4.62 (s, 2H, CH.sub.2), 7.59-7.69 (m, 1H,
Bn--H.sup.45), 7.79-7.88 (m, 1H, Bn--H.sup.2,6).
[0134] Molecular weight of C.sub.14H.sub.19ClN.sub.4O: calculated:
294.78; found: 295(MH.sup.+)
Example 3
N'-(3-Cyanobenzyl)-N'-[(4-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulf-
onohydrazide
[0135] ##STR21##
[0136]
2-(3-cyanobenzyl)-2-[(4-methyl-i-pyperidinyl)carbonyl]hydrazine
(0.870 g) was dissolved in 50 ml of dichloromethane, cooled to
-5.degree. C. and while stirring, 1.190 ml of triethylamine and
0.640 g of naphthalen-2-sulfonylchloride were added. The reaction
mixture was stirred at -5.degree. C. for 30 minutes and then at
room temperature for another 24 hours. The solvent was evaporated
under reduced pressure and 100 ml of ethyl acetate was added to the
residue. In the separatory funnel it was washed twice with 50 ml of
water, with 50 ml of 10% of citric acid and with 50 ml of brine.
The organic phase was dried over Na.sub.2SO.sub.4, filtered and the
filtrate was evaporated in vacuo. The resulting product was
suspended in diethylether and the precipitate was filtered by
suction. The precipitate was then recrystalized from ethanol and
the crystals were filtered by suction.
[0137] Yield: 0.464 g (35.6%)
[0138] Melting range: 92-95.degree. C.
[0139] IR (KBr, cm.sup.-1): 3191.7, 2931.6, 2221.0, 1680.5, 1433.5,
1333.1, 1168.8, 857.9, 749.4, 692.2
[0140] .sup.1H NMR (DMSO-d.sub.6) ):.delta. (ppm): 0.10-0.35 (m,
2H, Pip-H.sup.3,3',5,5'), 0.47 (d, 3H, J=6.0 Hz, CH.sub.3),
1.22-1.32 (m, 3H, Pip-H.sup.3,3',4,4',5,5'), 2.30-2.45 (m, 1H,
Pip-H.sup.2,2'), 2.65-2.80 (m, 1H, Pip-H.sup.6,6'), 3.45-3.65 (m,
2H, Pip-H.sup.2',2 6',6), 4,38 (br d, 2H, CH.sub.2), 7.47-7.55 (m,
2H, Bn--H.sup.5,6), 7.58-7.63 (m, 1H, Bn--H.sup.2), 7.63-7.80 (m,
4H, Bn--H.sup.4 in Nf-H.sub.3,6,7), 8.02-8.18 (m, 3H,
Nf-H.sup.4,58), 8.43 (s, 1H, Nf-H.sup.1), 9.60 (s, 1H, NH). Two
sets of signals
[0141] Molecular weight of C.sub.25H.sub.26N.sub.4O.sub.3S:
calculated: 462.6; found: 463 (MH.sup.+)
Example 4
N'-Hydroxy-3-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)-
hydrazino]methyl}benzenecarboximidamide
[0142] ##STR22##
[0143]
N'-(3-cyanobenzyl)-N'-[(4-metyl-1-pyperidinyl)carbonyl]-2-naphthal-
ene sulfonohydrazide (0.223 g ) was suspended in 20 ml of anhydrous
ethanol under argon atmosphere and 0.032 g of hydroxylamine was
added. The reaction mixture was heated at reflux temperature for 24
hours and evaporated under reduced pressure. The resulting product
was purified by column chromatography (eluent: chloroform/methanol
=9/1) to yield white crystals.
[0144] Yield: 0.215 g (38%)
[0145] Melting range: 169-173.degree. C.
[0146] IR (KBr, cm.sup.-1): 3365.8, 3194.6, 2943.8, 2864.7, 1652.6,
1447.5, 1332.2, 1169.3,1076.1, 756.2, 700.3, 553.2
[0147] .sup.1H-NMR (DMSO-d.sub.6):.delta. (ppm): 0.01-0.36 (m, 2H,
Pip-H.sup.3,5), 0.44 (d, 3H, J=6.4 Hz, CH.sub.3), 1.15-1.27 (m, 3H,
Pip-H.sup.3,4,5), 2.27-2.42 (m, 1H, Pip-H.sup.2), 2.56-2.75 (m, 1H,
Pip-H.sup.6), 3.45-3.63 (m, 2H, Pip-H.sup.2,6), 4.35 (br d, 2H,
CH.sub.2), 7.14 (d, 1H, J=7.9, Bn--H.sup.4), 7.25-7.40 (m, 1H,
Bn--H.sup.5), 7.48-7.59 (m, 2H, Bn--H.sup.2,6), 7.63-7.81 (m, 3H,
Nf-H.sup.3,6,7), 8.02-8.19 (m, 3H, Nf-H.sup.4,5,8), 8.46 (s, 1H,
Nf-H.sup.1), 9.40 (s, 1H, NH), 9.78 (s, 1H, OH).
[0148] Molecular weight of C.sub.25H.sub.30N.sub.5O.sub.4S:
calculated: 495.6; found: 496 (MH.sup.+)
Example 5
Imino(3-{[1-[(4-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)hydra-
zino]methyl}phenyl)methanaminium chloride
[0149] ##STR23##
[0150]
N'-(3-cyanobenzyl)-N'-[(4-methyl-1-pyperidinyl)carbonyl]-2-naphtha-
lene
[0151] sulfonohydrazide (0.200 g) was suspended in 10 ml of
ethanol, cooled in an ice bath and HCl was bubbled into the
suspension for 30 minutes. The mixture was stirred at room
temperature for 4 hours and then the solvent was evaporatedunder
reduced pressure. The solid residue was washed with isopropyl ether
and filtered by suction. The resulting precipitate was dissolved in
10 ml ethanol and ammonium acetate (0.092 g) was added. The
resulting mixture was stirred at room temperature for 20 hours and
then HCl was passed through the solution for 30 minutes. After 24
hours the white precipitate was formed which was filtered by
suction.
[0152] Yield: 50.4 mg (52%)
[0153] Melting range: 148-152.degree. C.
[0154] IR (KBr, cm.sup.-1): 3396.1, 3101.7, 2945.8, 1669.1, 1435.8,
1335.9, 1165.9, 1072.4, 749.4, 668.4
[0155] .sup.1H-NMR (DMSO-d.sub.6):.delta. (ppm): 0.01-0.40 (m, 2H,
Pip-H.sup.3,5), 0.47 (d, 3H, J=6.4 Hz, CH.sub.3), 1.15-1.29 (m, 3H,
Pip-H.sup.3,4,5), 2.27-2.42 (m, 1H, Pip-H.sup.2), 2.56-2.75 (m, 1H,
Pip-H.sup.6), 3.45-3.60 (m, 2H, Pip-H.sup.2,6), 4.41 (br d, 2H,
CH.sub.2), 7.14 (d, 1H, J=7.9, Bn--H.sup.4), 7.25-7.40 (m, 1H,
Bn--H.sup.5), 7.48-7.59 (m, 2H, Bn--H.sup.2,6), 7.63-7.81 (m, 3H,
Nf-H.sup.3,6,7), 8.02-8.19 (m, 3H, Nf-H.sup.4,5,8), 8.46 (s, 1H,
Nf-H.sup.1), 9.40 (s, 1H, NH).
[0156] Molecular weight of C.sub.24H.sub.27N.sub.5O.sub.3S (free
base): calculated: 465.57; found: 466 (MH.sup.+)
Example 6
tert-Butyl 2-(3-cyanobenzylidene)-1-hydrazinecarboxylate
[0157] ##STR24##
[0158] 3-Cyanobenzaldehyde (40.0 mmol) suspended in EtOH (100 mL)
was added to a stirred solution of tert-butylcarbazate (40.0 mmol)
in EtOH. The mixture was heated at reflux temperature and after 4 h
the EtOH was partially evaporated in vacuo. Water (100 mL) was
added and the precipitated product was collected by filtration and
washed with diethylether to give tert-butyl
2-(3-cyanobenzylidene)-1-hydrazinecarboxylate as a white solid.
[0159] Yield: 77%.
[0160] MP: 115-158.degree. C.
[0161] IR (KBr): 3297, 2977, 2232, 1703, 1530, 1477, 1376, 1248,
1160, 1061, 941, 865 cm.sup.-1.
[0162] MS (FAB): MH.sup.+=246.
[0163] .sup.1H NMR (300 MHz CDCl.sub.3): 1.55 (s, 9H, Boc-H), 7.55
(m, 2H, Bn--H), 7.93 (m, 3H, Bn--H in CH), 8.24 (s, 1H, NH).
[0164] Elemental analysis: calculated for
C.sub.13H.sub.15N.sub.3O.sub.2: C, 63.66; H, 6.16; N, 17.13, found
C, 63.38; H, 6.18; N, 17.09.
Example 7
tert-Butyl 2-(3-cyanobenzyl)-1-hydrazinecarboxylate
[0165] ##STR25##
[0166] tert-Butyl 2-(3-cyanobenzylidene)-1-hydrazinecarboxylate
(60.0 mmol) was dissolved in MeOH (250 mL) and 10% Pd/C (10 w/w %)
was added. The mixture was hydrogenated for 6 h. The catalyst was
filtered off and the filtrate was evaporated in vacuo to yield oily
tert-butyl 2-(3-cyanobenzyl)-1-hydrazinecarboxylate which
crystallized overnight.
[0167] Yield: 95%.
[0168] MP: 75-80.degree. C.
[0169] IR (KBr): 3368, 2980, 2226, 1679, 1482, 1366, 1286, 1166,
793 cm.sup.-1
[0170] MS (FAB): MH.sup.+=248.
[0171] .sup.1H NMR (300 MHz CDCl.sub.3): 1.47 (s, 9H, Boc-H), 4.04
(br s, 2H, CH.sub.2), 4.28 (br s, 1H, NH), 6.09 (brs, 1H, NH),
7.12-7.41(m, 4H, Bn--H).
[0172] Elemental analysis: calculated for
C.sub.13Hl.sub.7N.sub.3O.sub.2.1/4H.sub.2O: C, 62.01; H, 7.00; N,
16.69; found C, 61.90; H, 7.22; N, 16.69.
Example 8
tert-Butyl
2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]-1-hydraz-
inecarboxylate
[0173] ##STR26##
[0174] The product was prepared from tert-butyl
2-(3-cyanobenzyl)-1-hydrazinecarboxylate and 2-methylpiperidine
using the procedure described in EXAMPLE 1.
[0175] Yield: 42%.
[0176] MP: oil
[0177] IR (KBr): 3271, 2941, 2230, 1725, 1644, 1428 cm.sup.-1.
[0178] MS (FAB): MH.sup.+=373.
[0179] .sup.1H NMR (300 MHz, CDCb.sub.3): 1.22 (d, J=6,8 Hz, 3H,
CH.sub.3), 1.45 (s, 9H, Boc), 1.45-1.79 (m, 5H, Pip-H), 2.97-3.10
(m, 1H, Pip-H), 3.44-3.54 (m, 1H, Pip-H), 3.63-3.76 (m, 1H, Pip-H),
4.18-4.32 (m, 1H, Pip-H), 4.48 (s, 2H, CH.sub.2), 6.30 (s, 1H, NH),
7.46 (t, J=7.9 Hz, 1H, Bn--H), 7.56-7.67 (m, 3H, Bn--H).
Example 9
2-(3-Cyanobenzyl)-2-[(2-methyl-1-pipendinyl)carbonyl]hydrazinium
chloride
[0180] ##STR27##
[0181] The product was prepared from tert-butyl
2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]-1-hydrazinecarboxy-
late using the procedure described in EXAMPLE 2.
[0182] Yield: 91%.
[0183] MP: 135-137.degree. C.
[0184] IR (KBr): 3429, 2949, 2229, 2708, 2230, 1694, 1526, 1420
cm.sup.-1.
[0185] MS (FAB): MH.sup.+=273.
[0186] .sup.1H NMR (300 MHz, CDCl.sub.3): 1.23 (d, J=6,8 Hz, 3H,
CH.sub.3), 1.40-1.51 (m, 1H, Pip-H) 1.52-1.79 (m, 5H, Pip-H),
2.98-3.10 (m 1H, Pip-H), 3.60-3.79 (m, 1H, Pip-H), 4.25-4.38 8 (m,
1H, Pip-H), 4.64 (s, 2H, CH.sub.2), 7.53 (t, J=7.9 Hz, 1H, Bn--H),
7.61-7.76 (m, 3H, Bn--H), 9.60 (br s, 2H, NH.sub.2).
Example 10
tert-Butyl
2-(1-azepanylcarbonyl)-2-(3-cyanobenzyl)-1-hydrazinecarboxylate
[0187] ##STR28##
[0188] The product was prepared from tert-butyl
2-(3-cyanobenzyl)-1-hydrazinecarboxylate and azepane using the
procedure described in EXAMPLE 1.
[0189] Yield: 63%.
[0190] MP: oil.
[0191] IR (KBr): 3287, 2925, 2223, 1726, 1634 cm.sup.-1. MS (FAB):
MH.sup.+=373.
[0192] .sup.1H NMR (300 MHz, CDCl.sub.3): 1.45 (s, 9H, Boc),
1.53-1.62 (m, 4H, Pip-H.sup.45), 1.69-1.80 (m, 4H, Pip-H 3),
3.39-3.46 (m, 4H, Pip-H 2), 4.50 (s, 2H, CH.sub.2), 6.25 (br s, 1H,
NH), 7.45 (t, J=7.9 Hz, 1H, Bn--H), 7.56-7.69 (m, 3H, Bn--H).
Example 11
2-(1-Azepanylcarbonyl)-2-(3-cyanobenzyl)hydrazinium chloride
[0193] ##STR29##
[0194] The product was prepared from tert-butyl
2-(1-azepanylcarbonyl)-2-(3-cyanobenzyl)-1-hydrazinecarboxylate
using the procedure described in EXAMPLE 2.
[0195] Yield: 66%.
[0196] MP: oil.
[0197] IR (KBr): 2926, 2702, 2230 1703, 1521 cm.sup.-1.
[0198] MS (FAB): MH.sup.+=273.
[0199] .sup.1H NMR (300 MHz, CDCl.sub.3): 1.53-1.64 (m, 4H,
Pip-H.sup.4,5), 1.65-1.81 (m, 4H, Pip-H.sup.3,6), 3.38-3.46 (m, 4H,
Pip-H.sup.2,7), 4.70 (s, 2H, CH.sub.2), 7.48-7.64 (m, 2H, Bn--H),
7.69-7.80 (m, 2H, Bn--H), 9.60 (br s, 2H, NH.sub.2).
Example 12
N'-(3-cyanobenzyl)-N'-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesulf-
onohydrazide
[0200] ##STR30##
[0201] The product was prepared from
2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]hydrazinium
chloride and naphthalene-2-sulfonyl chloride using the procedure
described in EXAMPLE 3.
[0202] Yield: 14%.
[0203] MP: 116-119.degree. C.
[0204] IR (KBr): 3231, 2943, 2227, 1653 cm.sup.-1.
[0205] MS (FAB): MH.sup.+=273.
[0206] .sup.1H NMR (300 MHz CDCl.sub.3): 0.35-0.55 (m, 2H, Pip-H),
0.75-0.90 (m, 2H, Pip-H), 1.03-1.45 (m, 6H, Pip-H in CH.sub.3),
2.54-2.70 in 2.75-2.95 (m, 1H, Pip-H) 3.50-3.75 in 3.75-4.00 (m,
1H, Pip-H), 4.31 (br d, 2H, CH.sub.2), 7.35-7.70 (m, 7H, Bn--H in
Nph-H), 7.75-7.85 (m, 1H, Nph-H), 7.86-8.00 (m, 3H, Nph-H), 8.42
(s, 1H, Nph-H), (two sets of signals).
[0207] Elemental analysis: calculated for C.sub.25 H.sub.26 N.sub.4
O.sub.3 S: C, 64.91; H, 5.67; N, 12.11; found: C, 65.07; H, 5.58;
N, 11.93.
Example 13
N'-hydroxy-3-{[1-[(2-methyl-1-piperidinyl)carbonyl]-2-(2-naphthylsulfonyl)-
hydrazino]methyl}benzenecarboximidamide
[0208] ##STR31##
[0209] The product was prepared from
N'-(3-cyanobenzyl)-N'-[(2-methyl-1-piperidinyl)carbonyl]-2-naphthalenesul-
fonohydrazide using the procedure described in EXAMPLE 4.
[0210] Yield: 48%.
[0211] MP: 101-103 OC.
[0212] IR (KBr): =3370, 2932, 1651 cm.sup.-1.
[0213] MS (FAB): MH.sup.+=496.
Example 14
N'-(3-Cyanobenzyl)-6-methoxy-N'-[(2-methyl-1-piperidinyl)carbonyl]-2-napht-
halenesulfonohydrazide
[0214] ##STR32##
[0215] The product was prepared from
2-(3-cyanobenzyl)-2-[(2-methyl-1-piperidinyl)carbonyl]hydrazinium
chloride and 6-methoxy-2-naphthalenesulfonyl chloride using the
procedure described in EXAMPLE 3.
[0216] Yield: 26%.
[0217] MP: 169-173.degree. C.
[0218] IR (KBr): 3172, 2934, 2229, 1671, 1623 cm.sup.-1.
[0219] MS (FAB): MH.sup.+=493273.
[0220] .sup.1H NMR (300 MHz, CDCl.sub.3): 0.47-0.60 (m, 2H, Pip-H),
0.79-0.92 (m, 2H, Pip-H), 1.12-1.50 (m, 6H, Pip-H in CH.sub.3),
2.50-2.75 in 2.80-3.15 (m, 1H, Pip-H), 3.55-3.76 in 3.80-3.95 (m,
1H, Pip-H), 3.95 8s, 3H, OCH.sub.3); 2.55-2.74 (m, 1H, Pip-H),
3.45-3.65 (m, 2H, Pip-H), 3.84 (s, 3H, CH.sub.3), 4.32 (br d, 2H,
CH.sub.2), 7.28-7.33 (m, 1H, Nph-H), 7.44-7.60 (m, 5H, Bn--H and
Nph-H), 7.75-7.88 (m, 3H, Nph-H), 8.34 (d, 1H, J=1.88 Hz,
Nph-H).
Example 15
N'-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(2-methyl-1-piperidin-
yl)carbonyl]hydrazino}methyl)benzenecarboximidamide
[0221] ##STR33##
[0222] The product was prepared from
N'-(3-Cyanobenzyl)-6-methoxy-N'-[(2-methyl-1-piperidinyl)carbonyl]-2-naph-
thalenesulfonohydrazide using the procedure described in EXAMPLE
4.
[0223] Yield: 45%.
[0224] MP: 172-175.degree. C.
[0225] IR (KBr): 3368, 2937, 1647, 1160 cm.sup.-1.
[0226] MS (FAB): MH.sup.+=526.
[0227] .sup.1H NMR (300 MHz, CDCl.sub.3): 0.35-0.50 (m, 2H, Pip-H),
0.79-1.00 (m, 2H, Pip-H), 1.12-1.50 (m, 6H, Pip-H in CH.sub.3),
2.40-2.75 in 2.80-3.00 (m, 1H, Pip-H), 3.55-3.76 in 3.80-3.95 (m,
1H, Pip-H), 3.95 (s, 3H, OCH.sub.3); 4.35 (br d, 2H, CH.sub.2),
5.04 (s, 2H, NH.sub.2), 7.15-7.65 (m, 6H, Nph-H in Bn--H),
7.74-7.87 (m, 3H, Nph-H), 8.34 (s, 1H, Nph-H).
[0228] Elemental analysis: calculated for
C.sub.26H.sub.3lN.sub.5O.sub.5S EtOH : C, 58.83; H, 6.52; N, 12.25;
found: C, 59.41; H, 5.96; N, 12.09.
Example 16
N'-(3-Cyanobenzyl)-6-methoxy-N'-[(4-methyl-1-piperidinyl)carbonyl]-2-napht-
halenesulfonohydrazide
[0229] ##STR34##
[0230] The product was prepared from
2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]hydrazinium
chloride using the procedure described in EXAMPLE 3.
[0231] Yield: 50%.
[0232] MP: 169-173.degree. C.
[0233] IR (KBr): 3168, 2932, 2232, 1671 cm.sup.-1.
[0234] MS (FAB): MH.sup.+=493.
[0235] .sup.1H NMR (300 MHz, DMSO-d.sub.6): 0.15-0.40 (m, 2H,
Pip-H), 0.53 (d, 3H, J=6.0 Hz, CH.sub.3), 1.25-1.39 (m, 3H, Pip-H),
2.25-2.38 (m, 1H, Pip-H), 2.55-2.74 (m, 1H, Pip-H), 3.45-3.65 (m,
2H, Pip-H), 3.84 (s, 3H, CH.sub.3), 4.32 (br d, 2H, CH.sub.2),
7.28-7.33 (m, 1H, Nph-H), 7.44-7.54 (m, 3H, Bn--H and Nph-H), 7.60
(d, 1H, J=1.88 Hz, Bn--H), 7.60-7.76 (m, 2H, Bn--H and Nph-H), 7.94
(d, 1H, J=8.66 Hz, Nph-H), 8.05 (d, 1H, J=8.66 Hz, Nph-H ), 8.34
(d, 1H, J=1.88 Hz, Nph-H.sup.1), 9.46 (s, 1H, NH).
Example 17
N'-Hydroxy-3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidin-
yl)carbonyl]hydrazino}methyl)benzenecarboximidamide
[0236] ##STR35##
[0237] The product was prepared from
N'-(3-cyanobenzyl)-6-methoxy-N'-[(4-methyl-1-piperidinyl)carbonyl]-2-naph-
thalenesulfonohydrazide using the procedure described in EXAMPLE
4.
[0238] Yield: 36%.
[0239] MP: 154-156.degree. C.
[0240] IR (KBr): 3473, 3365, 2950, 1646 cm.sup.-1.
[0241] MS (FAB): MH.sup.+=526
[0242] .sup.1H NMR (300 MHz, CDCl.sub.3): 0.30 (br d, 3H, Pip-H),
0.58 (d, 3H, J=6.0 Hz, CH.sub.3), 1.26 (br d, 3H, Pip-H), 2.45 (br
s, 1H, Pip-H), 2.63 (br d, 1H, Pip-H), 3.74 (br d, 2H, Pip-H), 3.97
(s, 3H, OCH.sub.3), 4.34 (br d, 2H, CH.sub.2), 4.94 (s, 2H,
NH.sub.2) 7.17-7.35 (m, 4H, Nph-H and Bn--H), 7.49-7.55 (m, 2H,
Bn--H), 7.77-7.89 (m, 4H, Bn--H and Nph-H), 8.35 (m, 1H, Bn--H).
8.65 (br s, 1H, NH), 9.02 (s, 1H, OH).
Example18
Imino[3-({2-[(6-methoxy-2-naphthyl)sulfonyl]-1-[(4-methyl-1-piperidinyl)ca-
rbonyl]hydrazino}methyl)phenyl]methanaminium chloride
[0243] ##STR36## The product was prepared from
N'-(3-cyanobenzyl)-6-methoxy-N'-[(4-methyl-1-piperidinyl)carbonyl]-2-naph-
thalenesulfonohydrazide using the procedure described in EXAMPLE
5.
[0244] Yield: 26%.
[0245] MP: 151-154.degree. C.
[0246] IR (KBr): 3054, 1677, 1624 cm.sup.-1.
[0247] MS (FAB): MH.sup.+=510.
[0248] .sup.1H NMR (300 MHz, CDCb.sub.3): 0.28 (br d, 3H, Pip-H),
0.54 (d, 3H, J=6.0 Hz, CH.sub.3), 1.27 (br d, 3H, Pip-H), 2.45 (br
s, 1H, Pip-H), 2.64 (br s, 1H, Pip-H), 3.66 (br s, 2H, Pip-H), 3.95
(s, 3H, OCH.sub.3), 4.32 (m, 2H, CH.sub.2), 7.16-7.28 (m, 2H,
Nph-H), 7.36-7.40 (m, 2H, Bn--H.sup.26), 7.73-7.87 (m, 4H,
Bn--H.sup.5 and Nph-H), 7.95 (m, 1H, Bn--H.sup.4), 8.78 (br s, 2H,
NH.sub.2), 8.16 (br s, 2H, NH.sub.2).
Example 19
N'-(1-Azepanylcarbonyl)-N'-(3-cyanobenzyl)-2-naphthalenesulfonohydrazide
[0249] ##STR37##
[0250] The product was prepared from
N'-(1-azepanylcarbonyl)-N'-(3-cyanobenzyl)-2-naphthalenesulfonohydrazide
and naphthalene-2-sulfonyl chlonde using the procedure described in
EXAMPLE 3.
[0251] Yield: 43%.
[0252] MP: 128-130.degree. C.
[0253] IR (KBr): 3190, 2942, 2223, 1674 cm.sup.-1.
[0254] MS (FAB): MH.sup.+=463.
[0255] .sup.1H NMR (300 MHz, DMSO-d.sub.6): 0.63-1.40 (m, 8H,
Pip-H.sup.3,455.sup.6), 2.90-3.20 (m, 4H, Pip-H.sup.2J), 4.35 (br
d, 2H, CH.sub.2), 7.45-5.55 (m, 2H, Nph-H in Bn--H), 7.50-7-75 (m,
5H, Nph-H in Bn--H), 8.02-8.15 (m, 3H, Nph-H), 8.45 (s, 1H,
Nph-H),9.56 (s, 1H, NH).
[0256] Elemental analysis: calculated for za C.sub.25 H.sub.26
N.sub.4 03 S: C, 64.91; H, 5.67; N, 12.1 1; found: C, 64.65; H,
5.78; N, 12.10.
Example 20
3-{[1-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}-N'-hydr-
oxybenzenecarboximidamide
[0257] ##STR38##
[0258] The product was prepared from
N'-(1-azepanylcarbonyl)-N'-(3-cyanobenzyl)-2-naphthalenesulfonohydrazide
using the procedure described in EXAMPLE 4.
[0259] Yield: 79%.
[0260] MP: 152-155.degree. C.
[0261] IR (KBr): 3365, 2924, 1646 cm.sup.-1.
[0262] MS (FAB): MH.sup.+=496.
[0263] .sup.1H NMR (300 MHz, DMSO-d.sub.6): 0.65-1.40 (m, 8H,
Pip-H.sup.3,4,5,6), 2.90-3.20 (m, 4H, Pip-H.sup.2,7), 4.34 (br d,
2H, CH.sub.2), 5.87 (br s, 2H, NH.sub.2), 7.45-5.55 (m, 2H, Nph-H
in Bn--H), 7.50-7-75 (m, 5H, Nph-H in Bn--H), 8.02-8.15 (m, 3H,
Nph-H), 8.45 (s, 1H, Nph-H),9.35 (br d, 1H, NH), 9.67 (br s, 1H,
OH).
Example 21
(3-{[1-(1-Azepanylcarbonyl)-2-(2-naphthylsulfonyl)hydrazino]methyl}phenyl)-
(imino)methanaminium chloride
[0264] ##STR39##
[0265] The product was prepared from
N'-(1-azepanylcarbonyl)-N'-(3-cyanobenzyl)-2-naphthalenesulfonohydrazide
using the procedure described in EXAMPLE 5.
[0266] Yield: 36%.
[0267] MP: 151-154.degree. C.
[0268] IR (KBr): 3374, 3054, 2926, 1654, 1420, 1160 cm.sup.-1.
[0269] MS (FAB): MH.sup.+=480.
[0270] .sup.1H NMR (300 DMSO-d.sub.6): 0.60-1.45 (m, 8H,
Pip-H.sup.3,4,5,6), 2.90-3.20 (m, 4H, Pip-H.sup.2,7), 4.40 (br d,
2H, CH.sub.2), 7.44 (d, J=8,3 Hz, 2H, Bn--H.sup.2,6), 7.62-7.80 (m,
5H, Bn--H.sup.3,5 in Nph-H), 8.02-8.18 (m, 3H, Nph-H), 8.45 (s, 1H,
Nph-H), 9.09 (s, 2H, NH.sub.2), 9.35 (s, 1H, NH)
Example 22
N'-(1-Azepanylcarbonyl)-N'-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfonoh-
ydrazide
[0271] ##STR40##
[0272] The product was prepared from
2-(3-cyanobenzyl)-2-[(4-methyl-1-piperidinyl)carbonyl]hydrazinium
chloride using the procedure described in EXAMPLE 3.
[0273] Yield: 15%.
[0274] MP: 128-130.degree. C.
[0275] IR (KBr): 3169, 2939, 2229, 1670 cm.sup.-1.
[0276] MS (FAB): MH.sup.+=493.
[0277] .sup.1H NMR (300 MHz, DMSO-d.sub.6): 0.65-1.50 (m, 8H,
Pip-H.sup.3,4,5,6), 2.85-3.20 (m, 4H, Pip-H.sup.2,7), 3.91 (s, 3H,
OCH.sub.3) 4.35 (br d, 2H, CH.sub.2), 7.28-7.32 (m, 1H, Bn--H),
7.42-7.52 (m, 3H, Bn--H in Nph-H), 7.60 (br s, 1H, Nph-H),
7.66-7.71 (m, 2H, Nph-H), 7.92 (d, 1H, J=8.7, Nph-H), 8.04 (d, 1H,
J=8.7, Nph-H), 8.33 (d, 1H, J=1.5, Nph-H), 9.44 (s, 1H, NH).
[0278] Elemental analysis: calculated for C.sub.26 H.sub.28 N.sub.4
O.sub.4 S: C, 63.14; H, 6.1 1; N, 11.33; found: C, 63.26; H, 5.87;
N, 11.06.
Example 23
3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}met-
hyl)-N'-hydroxybenzenecarboximidamide
[0279] ##STR41##
[0280] The product was prepared from
N'-(1-azepanylcarbonyl)-N'-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfono-
hydrazide using the procedure described in EXAMPLE 4.
[0281] Yield: 24%.
[0282] MP: 173-175.degree. C.
[0283] IR (KBr): 3459, 3368, 2932, 1648, cm.sup.-1.
[0284] MS (FAB): MH.sup.+=526.
[0285] .sup.1H NMR (300 MHz, CDCl.sub.3): 0.65-1.50 (m, 8H,
Pip-H.sup.3,4,1.sup.6) 2.85-3.20 (m, 4H, Pip-H.sup.2,7), 3.97 (s,
3H, OCH.sub.3) 4.35 (s, 2H, CH.sub.2), 4.95, (s, 1H, NH) 7.28-7.32
(m, 1H, Bn--H), 7.42-7.52 (m, 3H, Bn--H in Nph-H), 7.60 (br s, 1H,
Nph-H), 7.66-7.71 (m, 2H, Nph-H), 7.92 (d, 1H, J=8.7 Hz, Nph-H),
8.04 (d, 1H, J=8.7 Hz, Nph-H), 8.33 (d, 1H, J=1.5 Hz, Nph-H), 8.10
(br s, 1H, NH), 8.35 (s, 1H, OH).
[0286] Elemental analysis: calculated for C.sub.26 H.sub.31 N.sub.5
O.sub.5 S: C, 59.18; H, 6.30; N, 13.27; found: C, 59.68; H, 6.16;
N, 12.88.
Example 24
[3-({1-(1-Azepanylcarbonyl)-2-[(6-methoxy-2-naphthyl)sulfonyl]hydrazino}me-
thyl)phenyl](imino)methanaminium chloride
[0287] ##STR42##
[0288] The product was prepared from
N'-(1-azepanylcarbonyl)-N'-(3-cyanobenzyl)-6-methoxy-2-naphthalenesulfono-
hydrazide using the procedure described in EXAMPLE 5.
[0289] Yield: 21%.
[0290] MP: 151-154.degree. C.
[0291] IR (KBr): 3057, 2929, 1677,1623, 1474, 1157 cm.sup.-1.
[0292] MS (FAB): MH.sup.+=510.
[0293] .sup.1H NMR (300 MHz CDCl.sub.3): 0.65-1.40 (m, 8H,
Pip-H.sup.345',6), 2.95-3.20 (m, 4H, Pip-H.sup.2,7), 3.97 (s, 3H,
OCH.sub.3) 4.35 (br s, 2H, CH.sub.2), 7.44 (d, J=8,3 Hz, 2H,
Bn--H.sup.2',6), 7.62-7.80 (m, 5H, Bn--H.sup.3,5 in Nph-H),
8.02-8.18 (m, 3H, Nph-H), 8.45 (s, 1H, Nph-H), 9.03 (s, 2H,
NH.sub.2), 9.35 (s, 2H, NH.sub.2).
[0294] Results of biological in vitro testing for selected
compounds TABLE-US-00001 Ki thrombin Ki trypsin Ki trypsin/ aPTT PT
TT (.mu.M) (.mu.M) Ki thrombin (.mu.M) (.mu.M) (.mu.M) ##STR43##
0.469 >68.3 146 -- -- -- ##STR44## 0.004 0.112 28 -- -- --
##STR45## 0.011 -- -- 2.1 2.7 2.0 ##STR46## 0.594 32.236 54 -- --
-- ##STR47## 0.005 0.143 29 -- -- -- ##STR48## 0.61 17.945 29 -- --
-- ##STR49## 0.009 0.053 5.84 -- -- --
Example 27
[0295] Results of biological in vivo testing for selected compounds
TABLE-US-00002 Stasis-induced Bleeding thrombosis (fold increase
(ED.sub.50 .+-. SD - mg/kg) at 1 mg/kg) ##STR50## 290 .+-. 11 3.3
##STR51## 70 .+-. 5 9.4
* * * * *