CA2208773A1 - Heterocyclic keto arginine peptides as thrombin inhibitors - Google Patents
Heterocyclic keto arginine peptides as thrombin inhibitorsInfo
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- CA2208773A1 CA2208773A1 CA002208773A CA2208773A CA2208773A1 CA 2208773 A1 CA2208773 A1 CA 2208773A1 CA 002208773 A CA002208773 A CA 002208773A CA 2208773 A CA2208773 A CA 2208773A CA 2208773 A1 CA2208773 A1 CA 2208773A1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/06—Dipeptides
- C07K5/06008—Dipeptides with the first amino acid being neutral
- C07K5/06078—Dipeptides with the first amino acid being neutral and aromatic or cycloaliphatic
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Peptides Or Proteins (AREA)
Abstract
This invention relates to new and useful inhibitors of the enzyme thrombin of the formula (I): AS - X, and more particularly compound (1) in the preparation, and pharmaceutical compositions. As well, this invention relates to the use of such compounds and compositions in vitro as anticoagulants and in vivo as agents for the treatment and prophylaxis of thrombotic disorders such as venous thrombosis, pulmonary embolism and arterial thrombosis resulting in acute ischemic events such as myocardial infarction or cerebral infarction. Moreover, these compounds and compositions have therapeutic utility for the prevention and treatment of coagulopathis associated with coronary bypass operations as well as restenotic events following transluminal angioplasty.
Description
W O96/19491 PCT/CAg5/00711 E{ETEROCYC~IC RETO AR~jl N 1~ r ~ S
AS ~U~M~IN IN~IBITORS
FIELD OF T~E lNv~ ON
This invention relates to compounds useful for the treatment of thrombotic disorders, and more particularly to novel heterocyclic inhibitors of the enzyme thrombin.
R~t'~r.ROI~
Inordinate thrombus formation on blood vessel walls precipitates acute cardiovascular disease states that are the leading cause of death in economically developed societies. Plasma proteins such as fibrinogen, proteases and cellular receptors participating in hemostasis have emerged as important factors that play a role in acute and chronic coronary disease as well as cerebral artery disease by contributing to the formation of thrombus or blood clots that effectively ~;m;n;.sh normal blood flow and supply. Vascular aberrations stemming from primary pathologic states such as hypertension, rupture of atherosclerotic plaques or denuded endothelium, activate biochemical cascades that serve to respond and repair the injury site. Thrombin is a key regulatory enzyme in the coagulation cascade. It serves a pluralistic role as both a positive and negative feedback regulator. However, in pathologic conditions the former is amplified through catalytic activation of cofactors required for thrombin generation as well as activation of factor XIII necessary for fibrin cross-linking and stabilization.
In addition to its direct effect on hemostasis, thrombin exerts direct effects on diverse cell types that support and amplify pathogenesis of arterial thrombus disease.
The enzyme is the strongest activator of platelets causing them to aggregate and release substances (e.g. ADP T ~ NE) that further propagate the thrombotic cycle. Platelets in a fibrin mesh comprise the principal framework of a white thrombus. Thrombin also exerts direct effects on endothelial cells causing release of vasoconstrictor substances and translocation of a&esion molecules that become sites for attachment of immune cells. In addition, the enzyme causes mitogenesis of smooth muscle cells and proliferation of fibroblasts. From this analysis, it is apparent that inhibition of thrombin activity constitutes a viable therapeutic approach towards the attenuation of proliferative events associated with thrombosis.
The principal endogenous neutralizing factor for thrombin activity in m~mm~l s is antithrombin III (ATIII), a circulating plasma macroglobulin having low affinity for the enzyme. Heparin exerts clinical efficacy in venous thrombosis by ~nh~ncing ATIII/thrombin b;n~;ng through catalysis. However, heparin also catalyzes inhibition of other proteases in the coagulation cascade and its efficacy in platelet-dependent thrombosis is largely reduced or abrogated due to inaccessibility of thrombus-bound enzyme. Adverse side effects such asthrombocytopenia, osteoporosis and triglyceridemia have been observed following prolonged treatment with Heparin.
Hirudin, derived from the gl~n~ r secretions of the leech Hirudo medicinal is is one of the high molecular - weight natural anticoagulant protein inhibitors of thrombin activity (Markwardt F. Cardiovascular Drug Reviews, 10, 211, 1992). It is a biopharmaceutical that has demonstrated efficacy in experimental and clinical thrombosis. A potential drawback to the use of hirudin as a therapeutic agent is its weak antigenicity and lack of an effective method of neutralization, especially in view of its extremely tight b;n~;ng characteristics toward thrombin. The exceedingly high affinity for thrombin is uni~ue and is attributed to a simultaneous interaction with the catalytic site as well as a distal ~anion b;n~; ng exosite" on the enzyme.
Thrombin activity can also be abrogated by hirudin-like molecules such as hirulog (Maraganore, J.M. et al., ~ Biochemistry, 29, 7095, 1990) or hirutonin peptides (DiMaio, J. et al., J. Med. Chem., 35, 3331, 1992).
Thrombin activity can also be inhibited by low molecular weight compounds that compete with fibrinogen for thrombin's catalytic site, thereby inhibiting proteolysis lO of that protein or other protein substrates such as the thrombin receptor. A common strategy for designing enzyme inhibitory compounds relies on mimicking the specificity inherent in the primary and secondary structure o~ the enzyme's natural substrate. Thus, Blomback et al. first l5 designed a thrombin inhibitor that was modeled upon the partial se~uence of the fibrinogen Aa chain comprising its proteolytically susceptible region (Blomback, et al., J
Clin. Lab. Invest., 24, 59, 1969). This region of fibrinogen m;n;m~l ly includes the residues commencing with 20 phenylalanine:
Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly -Gly-Gly-Val-Arg-Gly-Pro-Arg ~ scissile bond Systematic replacement of amino acids within this region has led to optimization of the tripeptidyl inhibitory sequence exemplified by the peptide (D)-Phe-Pro-Arg which corresponds to interactions within the P3-P2-Pl local 30 b; n~; ng sites on thrombin (Bajusz S. et al. in Peptides: Chemistry r Structure and Biology: Proceedings of the Fourth American Peptide Symposium, Walter R., Meienhofer J. Eds. Ann Arbor . Science Publishers Inc., Ann Arbor MI, 1975, pp 603).
Bajusz et al. have also reported related compounds such as W O96/19491 PCT/CAg5/00711 - (D)Phe-Pro-Arg-(CO)H (GYKI-14166) and (D)MePhe-Pro-Arg-: (CO)H (GYKI-14766) (Peptides-Synthesis, Structure and Function: Proceedings of the Seventh American Peptide Symposium, Rich, D.H. & Gross, E. eds., Pierce Chemical Company , 1981, pp. 417). These tripeptidyl aldehydes are ef~ective thrombin inhibitors both in vitro and in vivo.
In the case of both GYKI-14166 and GYKI-14766, the aldehyde group is presumed to contribute strongly to inhibitory activity in view of its chemical reactivity toward thrombin's catalytic Ser,95 resi~ue, generating a hemiacetal intermediate.
Related work in the area of thrombin inhibitory activity has exploited the basic recognition binding motif engendered by the tripeptide (D)Phe-Pro-Arg while incorporating various functional or reactive groups in the locus corresponding to the putative scissile bond (i.e.
Pl-Pl ) .
In U.S. Patent 4,318,904, Shaw reports chloromethyl-ketones (PPACK) that are reactive towards Ser195 and Hiss7.
These two residues comprise part of thrombin's catalytic triad (Bode, W. et al., EMBO Journal 8, 3467, 1989).
Other examples of thrombin inhibitors bearing the (D)Phe-Pro-Arg general motif are those incorporating COOH-terminal boroarginine variants such as boronic acids or ~ boronates (Kettner, C. et al., J. Biol. Chem., 268, 4734, 1993).
Still other congeners of this motif are those bearing phosphonates (Wang, C-L J., Tetrahedron Letters, 33, 7667, 1992) and a-Keto esters (Iwanowicz, E.J. et al.,Bioorganic and Medicinal Chemistry Letters, 12, 1607, 1992).
W O96119491 PCT/CAg5/00711 Neises, B. et al. have described a trichloromethyl ketone thrombin inhibitor (MDL-73756) and Attenburger, J.M. et al. have revealed a related difluoro alkyl amide ketone (Tetrahedron Letters, 32, 7255, 1991).
Maraganore et al. (European 0,333,356; WO 91/02750; U.S.
5,196,404) disclose a series of thrombin inhibitors that incorporate the D-Phe-Pro- moiety and hypothesize that this preferred structure fits well within the groove adjacent to the active site of thrombin. Variations on these inhibitors are essentially linear or cyclic peptides built upon the D-Phe-Pro moiety.
Another series of patents and patent applications have described attempts to develop effective inhibitors against thrombosis by using alpha-ketoamides and peptide aldehyde analogs (EP 0333356jWO 93/15756; WO 93/22344; WO 94/08941;
WO 94/17817, EP 0479489, U.S. 5,380,713).
Still others have focused their attention on peptides, peptide derivatives, peptidic alcohols, or cyclic peptides as anti-thrombotic agents (WO 93/22344, EP 0276014; EP
0341607; EP 0291982). Others have examined ~m;n~;ne sulfonic acid moieties to achieve this same end (U.S.
4,781,866), while yet others have examined para or meta substituted phenlyalanine derivatives (WO 92/08709; WO
92/6549).
Many of the examples cited above are convergent by maint~;n;ng at least a linear acyclic tripeptidyl motif consisting of an arginyl unit whose basic side chain interacts with a carboxylate group located at the base of the Pl specificity cleft in thrombin. Two adjacent hydrophobic groups provide additional binding through favorable Van der Waals interactions within a contiguous hydrophobic cleft on the enzyme surface designated the P3-P2 site.
. .
An object of the present invention is to provide compounds that display inhibitory activity towards throm.bin.
S ~ RY OF T~IE lNV~;N~ lON
An aspect of the present invention relates to peptide derivatives represented by formula (I), and pharmaceutically acceptable salts thereof :' 10 AS - X
(I) wherein X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl; and AS is an active site inhibitor of thrombin having an argininyl residue or an analogue thereof connected to X.
In another aspect of the present invention, there is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment of vascular diseases in a m~mm~ 1 including human.
= 25 In a further aspect, there is provided a method for the - treatment of vascular diseases in a m~mm~ 1 including ~ humans, comprising administering to said m~mm~l an amount : of a compound of formula ~I) effective to treat vascular : diseases.
DET~TT~Fn DESCRIPTION OF T~E lNv~ ION
Compounds o~ the present invention include those compounds where X is one or more heterocycle which may be unsubstituted or substituted with amino, oxygen, alkyl, . .
.
WO96/19491 PCT/CAg5/00711 ~ralkyl, or aryl. X includes aromatic or non-aromatic heterocycli~ rings. X also includes one or more heterocycle which is optionally fused to another carbocycle or heterocycle.
Preferably X is selected from the group consisting of:
s ~ Rs X~ ~x~o wherein 1O X5, X10, Xll, and Xl~ are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C~ 4 alkyl, or Cs 8 aryl.
X6, and Xl3 are each independently selected from the group consisting of C, O, N, S, N-X" or CH-X, where X, is as defined above.
Rs is hydrogen, Cl,6 alkyl optionally carboxyl substituted, carboxyl, -C0l6~alkyl-CO2-Cll6alkyl / C6 20 aralkyl, C3 cycloalkyl, aryl or an aromatic heterocycle.
More preferably X is selected from the group consisting of:
:
CA 02208773 1997-06-20' f~N~[~Rs ~N~s 1~S s~ ~ S
~3 N~N S ~S~
N~ s$0~ Rs~ Rs~
_ _ ~N\N ~ N~ ~Rs wherein Rs is as defined above - Further preferably X is selected from the group consisting . 5 of:
f ~N ~ j ~ ~ ~ ~ 6 R, wherein Rs is 2S defined above.
; Even further preferably X is selected from the group : 10 consisting o~:
.
. _ W O96/19491 PCTICAg5/00711 wherein R5 is as defined above.
Most preferably X is ~N~ls or -.~ ~
AS ~U~M~IN IN~IBITORS
FIELD OF T~E lNv~ ON
This invention relates to compounds useful for the treatment of thrombotic disorders, and more particularly to novel heterocyclic inhibitors of the enzyme thrombin.
R~t'~r.ROI~
Inordinate thrombus formation on blood vessel walls precipitates acute cardiovascular disease states that are the leading cause of death in economically developed societies. Plasma proteins such as fibrinogen, proteases and cellular receptors participating in hemostasis have emerged as important factors that play a role in acute and chronic coronary disease as well as cerebral artery disease by contributing to the formation of thrombus or blood clots that effectively ~;m;n;.sh normal blood flow and supply. Vascular aberrations stemming from primary pathologic states such as hypertension, rupture of atherosclerotic plaques or denuded endothelium, activate biochemical cascades that serve to respond and repair the injury site. Thrombin is a key regulatory enzyme in the coagulation cascade. It serves a pluralistic role as both a positive and negative feedback regulator. However, in pathologic conditions the former is amplified through catalytic activation of cofactors required for thrombin generation as well as activation of factor XIII necessary for fibrin cross-linking and stabilization.
In addition to its direct effect on hemostasis, thrombin exerts direct effects on diverse cell types that support and amplify pathogenesis of arterial thrombus disease.
The enzyme is the strongest activator of platelets causing them to aggregate and release substances (e.g. ADP T ~ NE) that further propagate the thrombotic cycle. Platelets in a fibrin mesh comprise the principal framework of a white thrombus. Thrombin also exerts direct effects on endothelial cells causing release of vasoconstrictor substances and translocation of a&esion molecules that become sites for attachment of immune cells. In addition, the enzyme causes mitogenesis of smooth muscle cells and proliferation of fibroblasts. From this analysis, it is apparent that inhibition of thrombin activity constitutes a viable therapeutic approach towards the attenuation of proliferative events associated with thrombosis.
The principal endogenous neutralizing factor for thrombin activity in m~mm~l s is antithrombin III (ATIII), a circulating plasma macroglobulin having low affinity for the enzyme. Heparin exerts clinical efficacy in venous thrombosis by ~nh~ncing ATIII/thrombin b;n~;ng through catalysis. However, heparin also catalyzes inhibition of other proteases in the coagulation cascade and its efficacy in platelet-dependent thrombosis is largely reduced or abrogated due to inaccessibility of thrombus-bound enzyme. Adverse side effects such asthrombocytopenia, osteoporosis and triglyceridemia have been observed following prolonged treatment with Heparin.
Hirudin, derived from the gl~n~ r secretions of the leech Hirudo medicinal is is one of the high molecular - weight natural anticoagulant protein inhibitors of thrombin activity (Markwardt F. Cardiovascular Drug Reviews, 10, 211, 1992). It is a biopharmaceutical that has demonstrated efficacy in experimental and clinical thrombosis. A potential drawback to the use of hirudin as a therapeutic agent is its weak antigenicity and lack of an effective method of neutralization, especially in view of its extremely tight b;n~;ng characteristics toward thrombin. The exceedingly high affinity for thrombin is uni~ue and is attributed to a simultaneous interaction with the catalytic site as well as a distal ~anion b;n~; ng exosite" on the enzyme.
Thrombin activity can also be abrogated by hirudin-like molecules such as hirulog (Maraganore, J.M. et al., ~ Biochemistry, 29, 7095, 1990) or hirutonin peptides (DiMaio, J. et al., J. Med. Chem., 35, 3331, 1992).
Thrombin activity can also be inhibited by low molecular weight compounds that compete with fibrinogen for thrombin's catalytic site, thereby inhibiting proteolysis lO of that protein or other protein substrates such as the thrombin receptor. A common strategy for designing enzyme inhibitory compounds relies on mimicking the specificity inherent in the primary and secondary structure o~ the enzyme's natural substrate. Thus, Blomback et al. first l5 designed a thrombin inhibitor that was modeled upon the partial se~uence of the fibrinogen Aa chain comprising its proteolytically susceptible region (Blomback, et al., J
Clin. Lab. Invest., 24, 59, 1969). This region of fibrinogen m;n;m~l ly includes the residues commencing with 20 phenylalanine:
Ala-Asp-Ser-Gly-Glu-Gly-Asp-Phe-Leu-Ala-Glu-Gly -Gly-Gly-Val-Arg-Gly-Pro-Arg ~ scissile bond Systematic replacement of amino acids within this region has led to optimization of the tripeptidyl inhibitory sequence exemplified by the peptide (D)-Phe-Pro-Arg which corresponds to interactions within the P3-P2-Pl local 30 b; n~; ng sites on thrombin (Bajusz S. et al. in Peptides: Chemistry r Structure and Biology: Proceedings of the Fourth American Peptide Symposium, Walter R., Meienhofer J. Eds. Ann Arbor . Science Publishers Inc., Ann Arbor MI, 1975, pp 603).
Bajusz et al. have also reported related compounds such as W O96/19491 PCT/CAg5/00711 - (D)Phe-Pro-Arg-(CO)H (GYKI-14166) and (D)MePhe-Pro-Arg-: (CO)H (GYKI-14766) (Peptides-Synthesis, Structure and Function: Proceedings of the Seventh American Peptide Symposium, Rich, D.H. & Gross, E. eds., Pierce Chemical Company , 1981, pp. 417). These tripeptidyl aldehydes are ef~ective thrombin inhibitors both in vitro and in vivo.
In the case of both GYKI-14166 and GYKI-14766, the aldehyde group is presumed to contribute strongly to inhibitory activity in view of its chemical reactivity toward thrombin's catalytic Ser,95 resi~ue, generating a hemiacetal intermediate.
Related work in the area of thrombin inhibitory activity has exploited the basic recognition binding motif engendered by the tripeptide (D)Phe-Pro-Arg while incorporating various functional or reactive groups in the locus corresponding to the putative scissile bond (i.e.
Pl-Pl ) .
In U.S. Patent 4,318,904, Shaw reports chloromethyl-ketones (PPACK) that are reactive towards Ser195 and Hiss7.
These two residues comprise part of thrombin's catalytic triad (Bode, W. et al., EMBO Journal 8, 3467, 1989).
Other examples of thrombin inhibitors bearing the (D)Phe-Pro-Arg general motif are those incorporating COOH-terminal boroarginine variants such as boronic acids or ~ boronates (Kettner, C. et al., J. Biol. Chem., 268, 4734, 1993).
Still other congeners of this motif are those bearing phosphonates (Wang, C-L J., Tetrahedron Letters, 33, 7667, 1992) and a-Keto esters (Iwanowicz, E.J. et al.,Bioorganic and Medicinal Chemistry Letters, 12, 1607, 1992).
W O96119491 PCT/CAg5/00711 Neises, B. et al. have described a trichloromethyl ketone thrombin inhibitor (MDL-73756) and Attenburger, J.M. et al. have revealed a related difluoro alkyl amide ketone (Tetrahedron Letters, 32, 7255, 1991).
Maraganore et al. (European 0,333,356; WO 91/02750; U.S.
5,196,404) disclose a series of thrombin inhibitors that incorporate the D-Phe-Pro- moiety and hypothesize that this preferred structure fits well within the groove adjacent to the active site of thrombin. Variations on these inhibitors are essentially linear or cyclic peptides built upon the D-Phe-Pro moiety.
Another series of patents and patent applications have described attempts to develop effective inhibitors against thrombosis by using alpha-ketoamides and peptide aldehyde analogs (EP 0333356jWO 93/15756; WO 93/22344; WO 94/08941;
WO 94/17817, EP 0479489, U.S. 5,380,713).
Still others have focused their attention on peptides, peptide derivatives, peptidic alcohols, or cyclic peptides as anti-thrombotic agents (WO 93/22344, EP 0276014; EP
0341607; EP 0291982). Others have examined ~m;n~;ne sulfonic acid moieties to achieve this same end (U.S.
4,781,866), while yet others have examined para or meta substituted phenlyalanine derivatives (WO 92/08709; WO
92/6549).
Many of the examples cited above are convergent by maint~;n;ng at least a linear acyclic tripeptidyl motif consisting of an arginyl unit whose basic side chain interacts with a carboxylate group located at the base of the Pl specificity cleft in thrombin. Two adjacent hydrophobic groups provide additional binding through favorable Van der Waals interactions within a contiguous hydrophobic cleft on the enzyme surface designated the P3-P2 site.
. .
An object of the present invention is to provide compounds that display inhibitory activity towards throm.bin.
S ~ RY OF T~IE lNV~;N~ lON
An aspect of the present invention relates to peptide derivatives represented by formula (I), and pharmaceutically acceptable salts thereof :' 10 AS - X
(I) wherein X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl; and AS is an active site inhibitor of thrombin having an argininyl residue or an analogue thereof connected to X.
In another aspect of the present invention, there is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment of vascular diseases in a m~mm~ 1 including human.
= 25 In a further aspect, there is provided a method for the - treatment of vascular diseases in a m~mm~ 1 including ~ humans, comprising administering to said m~mm~l an amount : of a compound of formula ~I) effective to treat vascular : diseases.
DET~TT~Fn DESCRIPTION OF T~E lNv~ ION
Compounds o~ the present invention include those compounds where X is one or more heterocycle which may be unsubstituted or substituted with amino, oxygen, alkyl, . .
.
WO96/19491 PCT/CAg5/00711 ~ralkyl, or aryl. X includes aromatic or non-aromatic heterocycli~ rings. X also includes one or more heterocycle which is optionally fused to another carbocycle or heterocycle.
Preferably X is selected from the group consisting of:
s ~ Rs X~ ~x~o wherein 1O X5, X10, Xll, and Xl~ are each independently selected from the group consisting of N, or C-X, where X, is hydrogen, C~ 4 alkyl, or Cs 8 aryl.
X6, and Xl3 are each independently selected from the group consisting of C, O, N, S, N-X" or CH-X, where X, is as defined above.
Rs is hydrogen, Cl,6 alkyl optionally carboxyl substituted, carboxyl, -C0l6~alkyl-CO2-Cll6alkyl / C6 20 aralkyl, C3 cycloalkyl, aryl or an aromatic heterocycle.
More preferably X is selected from the group consisting of:
:
CA 02208773 1997-06-20' f~N~[~Rs ~N~s 1~S s~ ~ S
~3 N~N S ~S~
N~ s$0~ Rs~ Rs~
_ _ ~N\N ~ N~ ~Rs wherein Rs is as defined above - Further preferably X is selected from the group consisting . 5 of:
f ~N ~ j ~ ~ ~ ~ 6 R, wherein Rs is 2S defined above.
; Even further preferably X is selected from the group : 10 consisting o~:
.
. _ W O96/19491 PCTICAg5/00711 wherein R5 is as defined above.
Most preferably X is ~N~ls or -.~ ~
2~Rs wherein Rs is as defined above. In another embodiment, X is a 1,2 thiazole optionally substituted with Rs and\or is attached to J at the 2, 3, 4 or 5 position of the ring.
Preferably, Rs is hydrogen, or C, 4 alkyl.
Further preferably, R5 is hydrogen or CH3.
Most preferably, Rs is hydrogen.
Preferred compounds o~ formula (I) include those wherein the AS portion has the formula (II):
G - G -(II) wherein G~ is one or more amino acid, alkyl, aryl, aralkyl, or cycloalkyl.
G2 is arginyl radical or an analogue thereof;
- 25 with the proviso that AS is an inhibitor of the active site of thrombin. In particular embodiments G2 is selected from the following amino acid derivatives prepared according to the procedures described in Bioorg Med.
Chem., 1995, 3:1145.
.
. CA 02208773 1997-06-20 o "'N~T --- ~T ,.N~
(~NH (~NH (~NH
HN~ N~/NMe N\~2 NH2 H2N ( ~nl .; . .
O O ~
N~JI~ "~JI~T ' T
N~ NH2 '~Or N~\
~-N~J~T ~-N~JI~ N~J~
(~NH ~ N
N NH ~
\ / ~NH2 NH2 ~=N H2N
N~ ~N~J~
N~N ( ~ H,~NlNH~
o O T~o N~JI~ HN~T "~NJ '~
~NH2 T~O T~50 ~J ~ NH
HN~NH H2N NH HN~
"N~T ~" ~T N~T
~1 ~NH2 H2N ~3 "N~T -' ~T - ~T
Preferably, Rs is hydrogen, or C, 4 alkyl.
Further preferably, R5 is hydrogen or CH3.
Most preferably, Rs is hydrogen.
Preferred compounds o~ formula (I) include those wherein the AS portion has the formula (II):
G - G -(II) wherein G~ is one or more amino acid, alkyl, aryl, aralkyl, or cycloalkyl.
G2 is arginyl radical or an analogue thereof;
- 25 with the proviso that AS is an inhibitor of the active site of thrombin. In particular embodiments G2 is selected from the following amino acid derivatives prepared according to the procedures described in Bioorg Med.
Chem., 1995, 3:1145.
.
. CA 02208773 1997-06-20 o "'N~T --- ~T ,.N~
(~NH (~NH (~NH
HN~ N~/NMe N\~2 NH2 H2N ( ~nl .; . .
O O ~
N~JI~ "~JI~T ' T
N~ NH2 '~Or N~\
~-N~J~T ~-N~JI~ N~J~
(~NH ~ N
N NH ~
\ / ~NH2 NH2 ~=N H2N
N~ ~N~J~
N~N ( ~ H,~NlNH~
o O T~o N~JI~ HN~T "~NJ '~
~NH2 T~O T~50 ~J ~ NH
HN~NH H2N NH HN~
"N~T ~" ~T N~T
~1 ~NH2 H2N ~3 "N~T -' ~T - ~T
3~NH2H2N ~3 "N~ N~JI~ HN~T
Nq H~
~'N~J~T NJ~ "N~
.~ t~ (~ (~
- I~NJ~NH2 N~NH2 I~NH
HN
N~JI~ H~ , ,~T
~1 ~N~NH2 HN
" N~ ""HN~ "" N~J~
H2N~ NH2 O O
N~T N~ HN~JI~T
H(~O ~N~NH
: 12 _.
o o T ~-tlN~
H2N~NH
Il NH
wherein n=1-6, nl=1-2, n2=0-7 and T is a bond or a divalent linking moiety with X.
Suitable AS portions include amino acids 45-47 of hirudin and analogues thereof, and inhibitors of thrombin based on the D-Phe-Pro-Arg sequence and its analogues such as D-Cha-Pro-Arg, D-Phe-Pip-Arg, and D-Cha-Pip-Arg. Other inhibitors o~ the active site of thrombin which include an argininyl or an analogue thereof at the C-terminus may also be incorporated into formula (I) as AS.
More preferrably, compounds of the present invention include those compounds where AS is -Phe-Pro-Arg- or an analogue thereof.
Most preferably compounds of the present invention include those compounds where AS is (D-Phe)-Pro-Arg- or an analogue thereof.
It will be appreciated that compounds of the invention encompass all isomers, enantiomers, and mixtures thereof.
In a preferred embodiment, compounds of the invention are represented by formula (III):
X, N~ CO~
O )0-7 Z
~- (III) - wherein R1 is selected from the group consisting of one or more aryl or cycloalkyl which is unsubstituted or substituted with hydroxy, Cl6 alkyl, C48 aralkyl, C3 3 aryl, or C38 cycloalkyl.
R2 is selected from the group consisting of hydrogen, hydroxy, C,~ alkyl, C48 aralkyl, and unsubstituted or substituted amino group.
R3 is selected from the group consisting of hydrogen, - hydroxy, SH, C, 6 alkyl, C38 aryl and C48 aralkyl.
n is an integer from 0 to 2.
~ 15 Q is a bond or -NH-;
Z is C~ 4 alkoxy; cyano; -NH2; -CH2-NH2; -C(NH)-NH2; -NH-C (NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 cycloalkyl or aryl substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C (NH) -NH2; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-; C(NH)-NH2 or -CH2-NH-C(NH) -NH2; and X is as defined above.
Preferred embodiments of the present invention include - compounds of formula (III) wherein R1 is selected from the group consisting o~ one or more S or 6 membered aromatic or non-aromatic ring which may be unsubstituted or substituted with hydroxy, C, 4 alkyl, or C38 cycloalkyl.
More preferably Rl is a 6 membered aromatic or non-aromatic ring unsubstituted or substituted with C14 alkyl.
Most preferably Rl is phenyl unsubstituted or substituted with Cl4 alkyl.
Most preferably R1 is phenyl.
Preferably R~ is hydrogen, hydroxy, C16 alkyl, or amino ~ unsubstituted or substituted with hydroxy, or Cl6 alkyl.
More preferably R2 is hydroxy or NH2.
Most preferably R2 is NH2.
0 Preferably R3 is hydrogen, hydroxy, SH, or C16 alkyl.
More preferably R3 is hydrogen, or C1q alkyl.
Most preferably R3 is hydrogen.
Preferably n is 1 or 2.
Most preferably n is 1.
Preferably Q is a bond.
Preferably Z is linked via a methylene chain or 2-5 carbon atoms and is selected from the group consisting of -NH2 i -C(MH)-NH2; -NH-C(NH)-NH2; a C6 cycloalkyl or aryl substituted with -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C(NH)-NH2 or -CH~-NH-C(NH) -NH2; and a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C (NH)-NH2 or -CH2-NH-C(NH) -NH2 ~
More preferably Z is -NH-C(NH)-NH2, -NH2, and -C(NH)-NH2 linked via a methylene chain of 3-5 carbon atoms. Most preferably, Z is -NH-C(NH)-NH2 linked via a trimethylene chain.
- Preferred compounds of the invention include:
CA 02208773 l997-06-20 .
.' ~
NH o .. H2N~
NH
.'~ , 3'~
NH O
H2N_~
NH
H2N~
H2N~
NH
N~
;~
: NH O
2 ~
NH
.
-w 096rl9491 PCTICA95100711 0~
- ~ N~
O HN~
NH / O
H2N_~ H
More preferred compounds of formula (I) include:
(D-Phe)-Pro-alpha-benzothiazolo keto arginine; and (D-Phe)-Pro-alpha-thiazolo keto arginine.
The following abbreviations are referred to herein. These abbreviations are common and well known to those skilled in the art of peptide chemistry.
BOC - butoxy-carbonyl BuLi - butyl lithium DCM - dichloromethane DMF - dimethylformamide iPr2NEt - diisopropylethylamine THF - tetrahydrofuran As used in this application, the term "alkyl" represents a saturated or unsaturated, substituted (for example, by a halogen, hydroxyl, amino, oxygen, sulfur, or C620 aryl) or unsubstituted, straight chain, branched chain hydrocarbon moiety having 1 to 10 carbon atoms and preferably from 1 to 6 carbon atoms. This chain may be interrupted by one or more heteroatom such as N, O, or S.
The term "amino protecting groups", "oxygen protecting groups", and "protecting groups" are well known in the field o~ peptide synthesis. Such protecting groups may be found in T. Greene, Protective Grou~s In Orqanic - 25 Svnthesis, (John Wiley & Sons, 1981). The appropriate protecting group for a particular synthetic scheme will depend on many factors, including the presence of other reactive functional groups and the reaction conditions ; CA 02208773 1997-06-20 - desired for removal as well known by persons skilled in the art of peptide chemistry.
The term "aryl" represents a carbocyclic moiety which may be substituted by one or more heteroatom (for example N, 0, or S) and cont~;n;n~ one benzenoid-type ring preferably - containing from 6 to 15 carbon atoms (for example phenyl and naphthyl). This carbocyclic moiety may be interrupted - by one or more heteroatom such as N, O, or S.
The term "aralkyl" represents an alkyl group being uninterrupted or interrupted , unsubstituted or substituted by an aryl substituent (for example benzyl), preferably contA;n;n~ from 6 to 30 carbon atoms.
Unless specified otherwise, the term "amino acid" used herein includes naturally-occurring amino acids as well as non natural analogs commonly used by those skilled in the art of chemical synthesis and peptide chemistry. A list of ; 20 non natural amino acids may be found in "The Peptides", vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F. Vellaccio. It is to be noted that unless indicated - otherwise, the amino acids used in the context of the present invention are those in the L-configuration.
The term "cycloalkyl" represents cyclic hydrocarbon groups -' cont~;n~ng 3 to 12 carbon, preferably 3 to 8 carbon, which includes for example cyclopropyl, cyclobutyl, cyclohexyl, and cyclodecyl, any o~ which may be substituted with substituents such as halogen, amino, alkyl, and/or hydroxy.
The term "heterocycle" and "heterocyclic rings~' represents one or more aromatic or non-aromatic ring which includes one or more heteroatom such as nitrogen, oxygen, and sulfur and which may be substituted with substituents such as halogen, amino, alkyl, and/or hydroxy. Preferably, the ring is 5, 6, or 7 membered.
While it may be possible that, for use in therapy, a compound of the invention may be A~m; n; .~tered as the raw chemical, it is preferable to present the active ingredient as a ~hArmAceutical formulation.
The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) and pharmaceutically acceptable acid addition salt thereof together with one or more p~rmAceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
In another aspect of the present invention is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment of vascular diseases in a mAmmA1 including hllmAn~
In another aspect, there is provided a method for the treatment of vascular diseases in a mAmmA1 including human, comprising the administration of an effective amount of a compound of formula (I).
It will be appreciated by people skilled in the art that treatment extends to prophylaxis as well to the treatment of established vascular disease.
The compounds of the present invention are useful in combinations, formulations and methods for the treatment and prophylaxis of vascular diseases. These diseases include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, ~ WO96/19491 PCT/CA95/00711 restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, cancer and metastasis.
The term "combination" as used herein, includes a single dosage form cont~;n;n~ at least one compound of this invention and at least one thrombolytic agent, a multiple dosage form, wherein the thrombin inhibitor and the - 10 thrombolytic agent are administered separately, but concurrently, or a multiple dosage form wherein the two components are administered separately, but sequentially.
In se~uential a~m; n; ~tration, the thrombin inhibitor may be given to the patient during the time period ranging from about 5 hours prior to about 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is a~m;n;stered to the patient during . the period ranging from 2 hours prior to 2 hours following administration of the thrombolytic agent.-- Thrombolytic agents which may be employed in the combinations of the present invention are those known in the art. Such agents include, but are not limited to, tissue plasminogen activator puri~ied from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, purokinase, anisolated streptokinase plasminogen activator complex (ASPAC), ~ animal salivary gland plasminogen activators and known, ~ biologically active derivatives of any of the above.
-- The dosage and dose rate of the compounds of this -; invention will depend on a variety of factors, such as the weight of the patient, the specific pharmaceutical - composition used, the object o~ the treatment, i.e., - 35 therapy or prophylaxis, the nature of the thrombotic disease to be treated, and the judgment of the treating ' physician.
' 20 :- --WO96/19491 PCT/CA9~/00711 According to the present invention, a preferred pharmaceutically effective daily dose of the compounds of this invention is between about l~g/kg body weight o~ the patient to be treated ("body weight") and about 5 mg/kg body weight.
Most preferably, the therapeutic and prophylactic compositions o~ the present invention comprise a dosage of between about lO ~g/kg body weight and about 500 ~g/kg body weight of the compounds of this invention It should also be understood that a daily pharmaceutically effective dose of either the compounds of this invention or the thrombolytic agent present in combinations o~ the invention, may be less than or greater than the specific ranges cited above.
According to an alternate embodiment of this invention, compounds may be used in compositions and methods for coating the sur~aces o~ invasive devices, resulting in a lower risk of clot formation or platelet activation in patients receiving such devices. Surfaces that may be coated with the compositions of this invention include, for example, prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating these devices are known to those of skill in the art. These include chemical cross-linking or physical adsorption of the compounds of this invention-cont~; n; ng compositions to the surfaces of the devices.
According to a further embodiment of the present ~ invention, compounds may be used for ex vivo thrombus imaging in a patient. In this embodiment, the compounds of this invention are labeled with a radioisotope. The choice of radioisotope is based upon a number of well-known factors, ~or example, toxicity, biological half-life -and detectability. Preferred radioisotopes include, but are not limited to I, I and I. Techniques for labeling the compounds of this invention are well known in the art.
Most preferably, the radioisotope is 123I and the labeling is achieved using 123I-Bolton-Hunter Reagent. The labeled ~ thrombin inhibitor is a~m; n; .stered to a patient and allowed to bind to the thrombin contained in a clot. The clot is then observed by utilizing w~ll-known detecting ; means, such as a camera capable of detecting radioactivity - 10 coupled to a computer imaging system. This techni~ue also - yields images of platelet-bound thrombin and meizothrombin.
- This invention also relates to compositions cont~i n; ng the r ~
compounds of this invention and methods for using such compositions in the treatment of tumor metastases. The efficacy of the compounds of this invention for the treatment of tumor metastases is manifested by the inhibition inhibitors to inhibit thrombin-induced - 20 endothelial cell activation. This inhibition includes the repression o~ platelet activation factor (PAF) synthesis by endothelial cells. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which is thought to be mediated be PAF. Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage. Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses.
This invention also relates to the use of the above-~- described compounds, or compositions comprising them, as -~ anticoagulants for extracorporeal blood. As used herein, the term '~extracorporeal blood" includes blood removed in line from a patient, subjected to extracorporeal treatment, and then returned to the patient in such processes as dialysis procedures, blood filtration, or ., 22 W O96/19491 PCT/CAg5/00711 blood bypass during surgery. The term also includes blood products which are stored extracorporeally for eventual administration to a patient and blood collected from a patient to be used for various assays. Such products include whole blood, plasma, or any blood ~raction in ~ which inhibition of coagulation is desired.
The amount or concentration o~ compounds o~ this invention in these types of compositions is based on the volume of blood to be treated or, more preferably, its thrombin content. Pre~erably, an ef~ective amount o~ a compounds o~
this invention of this invention for preventing coagulation in extracorporeal blood is ~rom about 1 ~g/60 ml of extracorporeal blood to about 5 mg/60 ml of extracorporeal blood.
The compounds of this invention may also be used to inhibit clot-bound thrombin, which is believed to contribute to clot accretion. This is particularly important because commonly used anti-thrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot-bound thrombin. Finally, the compounds of this invention may be employed in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process suggestive of the rounding in shape changes of brain cells and implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.
Compounds of the present invention may be synthesized by various methods well known in the art. Suitable methods of synthesis will vary dep~n~;ng upon the AS and X
portions used in the compound. Suitable methods for synthesis of Phe-Pro-Arg type analogues are described below. However, other well known methods may be employed.
-. CA 02208773 1997-06-20 SCHE~E
H-X
O NH 1 HN ~X
HN ~Z J~ NH-Pg z ~
NH~NH
2 HN~ ~ ~ P3~ ~~ ~Nr$
HN 2 ~ CO2H
X~\Q 4 !j Y
Step 1:
The heterocyle 1 in solution was metalated with an appropriate metalating base such as n-BuLi to generate the corresponding metalated heterocylic compound. The cyclic activated arginine group 2 was added to this mixture.
Compound 2 was prepared according to procedures known in = the literature and described in, for example, R.T. Shuman, et al ., "Highly Selective Tripeptide Thrombin Inhibitors", J.Med.Chem, 1993, 36, 314. The compound yielded was - heterocyclic ketoarginine 3.
. .
Step 2:
- 20 The heterocyclic ketoarginine 3 is deprotected and coupled to the dipeptide 4 in the presence of a suitable coupling WO96/19491 PCT/CA9Sl00711 agent, solvent, and base. The dipeptide 4 can be purchased or prepared by methods common in the art and the peptide literature. Suitable coupling agents include BOP
and isopropylchloro~ormate. Suitable solvents include DCM
5 and DMF. Suitable bases include iPr2NEt and n-methyl t morpholine.
The resulting compound is deprotected with appropriate déprotecting agents to yield the heterocyclic ketoargininyl 5. Suitable deprotecting agents include 10 BBr3, HBr in acetic acid, and TMSI. Methods to remove the protecting groups are well known to people skilled in the art.
Scheme I is used where Z is N. Scheme II is used when Z is 15 carbon, linear carbon chain, or ~orms a ring with Q.
Where Z forms a ring with Q, the activated amino group 2 would be amended accordingly to include this ring. The steps in the process remain the same as described ~or Scheme I.
SCHE~DE II
H--X
HN ~1 'X
OCH z pg-y ,. P9 HNXl~ ~=o ~ CO2H
X~
Q_z 4 Y
The compounds of this invention and their intermediates may be purified during their synthesis and/or after their preparation by standard techniques well known to the skilled artisan. One preferred purification technique is HPLC. However, other chromatographic methods such as column chromatography may be used for purification o~ the compounds. Crystallization may also be used to purify the products as may w~c~; n~ with appropriate organic solvents.
It is well known in the art that the amino protecting groups are not necessary for the reaction to occur. The process may be carried out without protecting groups.
However, they are used to increase the yield of the desired compounds.
The process described above may use suitable protecting groups for compounds 2, 3, and 4. ~uitable deprotection conditions and protocols are described in the synthesis literature and are well known to chemists skilled in the art.
+
Desired R" Rz~ and R3 groups may be substituted onto the dipeptide 4 before it is coupled to heterocyclic ketoarginine 3 using techniques well known in the art of peptide ch~m;stry. Also, preferred analogues of each of the peptides or the dipeptide may be purchased with the desired Rl, R2, or R3 groups substituents already present.
In order that the invention described herein may be more fully understood, the ~ollowing examples are set ~orth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
.
EXl~MPLE
5~ o~ I
O NH 2. 2/-78 -20 C NH
~N NH{~ NH~NH
To a THF (75 mL) solution of benzothiazole (compound 1) (4.0 mL, 36.7 mmol) at -78 ~C was slowly added n-BuLi (1.6 M, 25 mL), resultant orange suspension was stirred at -78 ~C for 1.5 h. Then added solid compound 2 (3.55 g, 8.7 mmol). Reaction stirred at -78 ~C for 30 min followed by at -20 for 30 min then quenched with saturated aqueous NH4Cl .
Extraction with ethyl acetate followed by column chromatography afforded yellow ~oam (1.28 g) in 28% yield as compound 3.
NMR(CDCl3) d 1.45 (s, 9H), 1.5-1.8 (m, 2H), 3.1-3.23 (m, lH), 3.45-3.60 (m, lH), 5.1(d, 2H), 5.53-5.64(m, 2H), 7.02-7.15(m, 4H), 7.21-7.28(m, 2H), 7.56-7.65(m, 2H), 8.0-8.05(m, lH), 8.18-8.23(m, lH).
MS: (M+l) 526.8 ;
.
WO96/19491 PCT/CAg5/00711 NH ~ ~,N
NH W~
Cbz ~ 1. 4M Ha~DicD~cne ~\
'NH~NH EtSMe/3 - N
2. BOP/~MF/iPr2NEt HN
3. BBr3/DCM/-78 C /~S
Nq H~
~'N~J~T NJ~ "N~
.~ t~ (~ (~
- I~NJ~NH2 N~NH2 I~NH
HN
N~JI~ H~ , ,~T
~1 ~N~NH2 HN
" N~ ""HN~ "" N~J~
H2N~ NH2 O O
N~T N~ HN~JI~T
H(~O ~N~NH
: 12 _.
o o T ~-tlN~
H2N~NH
Il NH
wherein n=1-6, nl=1-2, n2=0-7 and T is a bond or a divalent linking moiety with X.
Suitable AS portions include amino acids 45-47 of hirudin and analogues thereof, and inhibitors of thrombin based on the D-Phe-Pro-Arg sequence and its analogues such as D-Cha-Pro-Arg, D-Phe-Pip-Arg, and D-Cha-Pip-Arg. Other inhibitors o~ the active site of thrombin which include an argininyl or an analogue thereof at the C-terminus may also be incorporated into formula (I) as AS.
More preferrably, compounds of the present invention include those compounds where AS is -Phe-Pro-Arg- or an analogue thereof.
Most preferably compounds of the present invention include those compounds where AS is (D-Phe)-Pro-Arg- or an analogue thereof.
It will be appreciated that compounds of the invention encompass all isomers, enantiomers, and mixtures thereof.
In a preferred embodiment, compounds of the invention are represented by formula (III):
X, N~ CO~
O )0-7 Z
~- (III) - wherein R1 is selected from the group consisting of one or more aryl or cycloalkyl which is unsubstituted or substituted with hydroxy, Cl6 alkyl, C48 aralkyl, C3 3 aryl, or C38 cycloalkyl.
R2 is selected from the group consisting of hydrogen, hydroxy, C,~ alkyl, C48 aralkyl, and unsubstituted or substituted amino group.
R3 is selected from the group consisting of hydrogen, - hydroxy, SH, C, 6 alkyl, C38 aryl and C48 aralkyl.
n is an integer from 0 to 2.
~ 15 Q is a bond or -NH-;
Z is C~ 4 alkoxy; cyano; -NH2; -CH2-NH2; -C(NH)-NH2; -NH-C (NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 cycloalkyl or aryl substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C (NH) -NH2; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-; C(NH)-NH2 or -CH2-NH-C(NH) -NH2; and X is as defined above.
Preferred embodiments of the present invention include - compounds of formula (III) wherein R1 is selected from the group consisting o~ one or more S or 6 membered aromatic or non-aromatic ring which may be unsubstituted or substituted with hydroxy, C, 4 alkyl, or C38 cycloalkyl.
More preferably Rl is a 6 membered aromatic or non-aromatic ring unsubstituted or substituted with C14 alkyl.
Most preferably Rl is phenyl unsubstituted or substituted with Cl4 alkyl.
Most preferably R1 is phenyl.
Preferably R~ is hydrogen, hydroxy, C16 alkyl, or amino ~ unsubstituted or substituted with hydroxy, or Cl6 alkyl.
More preferably R2 is hydroxy or NH2.
Most preferably R2 is NH2.
0 Preferably R3 is hydrogen, hydroxy, SH, or C16 alkyl.
More preferably R3 is hydrogen, or C1q alkyl.
Most preferably R3 is hydrogen.
Preferably n is 1 or 2.
Most preferably n is 1.
Preferably Q is a bond.
Preferably Z is linked via a methylene chain or 2-5 carbon atoms and is selected from the group consisting of -NH2 i -C(MH)-NH2; -NH-C(NH)-NH2; a C6 cycloalkyl or aryl substituted with -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C(NH)-NH2 or -CH~-NH-C(NH) -NH2; and a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C (NH)-NH2 or -CH2-NH-C(NH) -NH2 ~
More preferably Z is -NH-C(NH)-NH2, -NH2, and -C(NH)-NH2 linked via a methylene chain of 3-5 carbon atoms. Most preferably, Z is -NH-C(NH)-NH2 linked via a trimethylene chain.
- Preferred compounds of the invention include:
CA 02208773 l997-06-20 .
.' ~
NH o .. H2N~
NH
.'~ , 3'~
NH O
H2N_~
NH
H2N~
H2N~
NH
N~
;~
: NH O
2 ~
NH
.
-w 096rl9491 PCTICA95100711 0~
- ~ N~
O HN~
NH / O
H2N_~ H
More preferred compounds of formula (I) include:
(D-Phe)-Pro-alpha-benzothiazolo keto arginine; and (D-Phe)-Pro-alpha-thiazolo keto arginine.
The following abbreviations are referred to herein. These abbreviations are common and well known to those skilled in the art of peptide chemistry.
BOC - butoxy-carbonyl BuLi - butyl lithium DCM - dichloromethane DMF - dimethylformamide iPr2NEt - diisopropylethylamine THF - tetrahydrofuran As used in this application, the term "alkyl" represents a saturated or unsaturated, substituted (for example, by a halogen, hydroxyl, amino, oxygen, sulfur, or C620 aryl) or unsubstituted, straight chain, branched chain hydrocarbon moiety having 1 to 10 carbon atoms and preferably from 1 to 6 carbon atoms. This chain may be interrupted by one or more heteroatom such as N, O, or S.
The term "amino protecting groups", "oxygen protecting groups", and "protecting groups" are well known in the field o~ peptide synthesis. Such protecting groups may be found in T. Greene, Protective Grou~s In Orqanic - 25 Svnthesis, (John Wiley & Sons, 1981). The appropriate protecting group for a particular synthetic scheme will depend on many factors, including the presence of other reactive functional groups and the reaction conditions ; CA 02208773 1997-06-20 - desired for removal as well known by persons skilled in the art of peptide chemistry.
The term "aryl" represents a carbocyclic moiety which may be substituted by one or more heteroatom (for example N, 0, or S) and cont~;n;n~ one benzenoid-type ring preferably - containing from 6 to 15 carbon atoms (for example phenyl and naphthyl). This carbocyclic moiety may be interrupted - by one or more heteroatom such as N, O, or S.
The term "aralkyl" represents an alkyl group being uninterrupted or interrupted , unsubstituted or substituted by an aryl substituent (for example benzyl), preferably contA;n;n~ from 6 to 30 carbon atoms.
Unless specified otherwise, the term "amino acid" used herein includes naturally-occurring amino acids as well as non natural analogs commonly used by those skilled in the art of chemical synthesis and peptide chemistry. A list of ; 20 non natural amino acids may be found in "The Peptides", vol. 5, 1983, Academic Press, Chapter 6 by D.C. Roberts and F. Vellaccio. It is to be noted that unless indicated - otherwise, the amino acids used in the context of the present invention are those in the L-configuration.
The term "cycloalkyl" represents cyclic hydrocarbon groups -' cont~;n~ng 3 to 12 carbon, preferably 3 to 8 carbon, which includes for example cyclopropyl, cyclobutyl, cyclohexyl, and cyclodecyl, any o~ which may be substituted with substituents such as halogen, amino, alkyl, and/or hydroxy.
The term "heterocycle" and "heterocyclic rings~' represents one or more aromatic or non-aromatic ring which includes one or more heteroatom such as nitrogen, oxygen, and sulfur and which may be substituted with substituents such as halogen, amino, alkyl, and/or hydroxy. Preferably, the ring is 5, 6, or 7 membered.
While it may be possible that, for use in therapy, a compound of the invention may be A~m; n; .~tered as the raw chemical, it is preferable to present the active ingredient as a ~hArmAceutical formulation.
The invention thus further provides a pharmaceutical formulation comprising a compound of formula (I) and pharmaceutically acceptable acid addition salt thereof together with one or more p~rmAceutically acceptable carriers therefor and, optionally, other therapeutic and/or prophylactic ingredients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the recipient thereof.
In another aspect of the present invention is provided the use of a compound of formula (I) in the manufacture of a medicament for the treatment of vascular diseases in a mAmmA1 including hllmAn~
In another aspect, there is provided a method for the treatment of vascular diseases in a mAmmA1 including human, comprising the administration of an effective amount of a compound of formula (I).
It will be appreciated by people skilled in the art that treatment extends to prophylaxis as well to the treatment of established vascular disease.
The compounds of the present invention are useful in combinations, formulations and methods for the treatment and prophylaxis of vascular diseases. These diseases include myocardial infarction, stroke, pulmonary embolism, deep vein thrombosis, peripheral arterial occlusion, ~ WO96/19491 PCT/CA95/00711 restenosis following arterial injury or invasive cardiological procedures, acute or chronic atherosclerosis, edema and inflammation, cancer and metastasis.
The term "combination" as used herein, includes a single dosage form cont~;n;n~ at least one compound of this invention and at least one thrombolytic agent, a multiple dosage form, wherein the thrombin inhibitor and the - 10 thrombolytic agent are administered separately, but concurrently, or a multiple dosage form wherein the two components are administered separately, but sequentially.
In se~uential a~m; n; ~tration, the thrombin inhibitor may be given to the patient during the time period ranging from about 5 hours prior to about 5 hours after administration of the thrombolytic agent. Preferably, the thrombin inhibitor is a~m;n;stered to the patient during . the period ranging from 2 hours prior to 2 hours following administration of the thrombolytic agent.-- Thrombolytic agents which may be employed in the combinations of the present invention are those known in the art. Such agents include, but are not limited to, tissue plasminogen activator puri~ied from natural sources, recombinant tissue plasminogen activator, streptokinase, urokinase, purokinase, anisolated streptokinase plasminogen activator complex (ASPAC), ~ animal salivary gland plasminogen activators and known, ~ biologically active derivatives of any of the above.
-- The dosage and dose rate of the compounds of this -; invention will depend on a variety of factors, such as the weight of the patient, the specific pharmaceutical - composition used, the object o~ the treatment, i.e., - 35 therapy or prophylaxis, the nature of the thrombotic disease to be treated, and the judgment of the treating ' physician.
' 20 :- --WO96/19491 PCT/CA9~/00711 According to the present invention, a preferred pharmaceutically effective daily dose of the compounds of this invention is between about l~g/kg body weight o~ the patient to be treated ("body weight") and about 5 mg/kg body weight.
Most preferably, the therapeutic and prophylactic compositions o~ the present invention comprise a dosage of between about lO ~g/kg body weight and about 500 ~g/kg body weight of the compounds of this invention It should also be understood that a daily pharmaceutically effective dose of either the compounds of this invention or the thrombolytic agent present in combinations o~ the invention, may be less than or greater than the specific ranges cited above.
According to an alternate embodiment of this invention, compounds may be used in compositions and methods for coating the sur~aces o~ invasive devices, resulting in a lower risk of clot formation or platelet activation in patients receiving such devices. Surfaces that may be coated with the compositions of this invention include, for example, prostheses, artificial valves, vascular grafts, stents and catheters. Methods and compositions for coating these devices are known to those of skill in the art. These include chemical cross-linking or physical adsorption of the compounds of this invention-cont~; n; ng compositions to the surfaces of the devices.
According to a further embodiment of the present ~ invention, compounds may be used for ex vivo thrombus imaging in a patient. In this embodiment, the compounds of this invention are labeled with a radioisotope. The choice of radioisotope is based upon a number of well-known factors, ~or example, toxicity, biological half-life -and detectability. Preferred radioisotopes include, but are not limited to I, I and I. Techniques for labeling the compounds of this invention are well known in the art.
Most preferably, the radioisotope is 123I and the labeling is achieved using 123I-Bolton-Hunter Reagent. The labeled ~ thrombin inhibitor is a~m; n; .stered to a patient and allowed to bind to the thrombin contained in a clot. The clot is then observed by utilizing w~ll-known detecting ; means, such as a camera capable of detecting radioactivity - 10 coupled to a computer imaging system. This techni~ue also - yields images of platelet-bound thrombin and meizothrombin.
- This invention also relates to compositions cont~i n; ng the r ~
compounds of this invention and methods for using such compositions in the treatment of tumor metastases. The efficacy of the compounds of this invention for the treatment of tumor metastases is manifested by the inhibition inhibitors to inhibit thrombin-induced - 20 endothelial cell activation. This inhibition includes the repression o~ platelet activation factor (PAF) synthesis by endothelial cells. These compositions and methods have important applications in the treatment of diseases characterized by thrombin-induced inflammation and edema, which is thought to be mediated be PAF. Such diseases include, but are not limited to, adult respiratory distress syndrome, septic shock, septicemia and reperfusion damage. Early stages of septic shock include discrete, acute inflammatory and coagulopathic responses.
This invention also relates to the use of the above-~- described compounds, or compositions comprising them, as -~ anticoagulants for extracorporeal blood. As used herein, the term '~extracorporeal blood" includes blood removed in line from a patient, subjected to extracorporeal treatment, and then returned to the patient in such processes as dialysis procedures, blood filtration, or ., 22 W O96/19491 PCT/CAg5/00711 blood bypass during surgery. The term also includes blood products which are stored extracorporeally for eventual administration to a patient and blood collected from a patient to be used for various assays. Such products include whole blood, plasma, or any blood ~raction in ~ which inhibition of coagulation is desired.
The amount or concentration o~ compounds o~ this invention in these types of compositions is based on the volume of blood to be treated or, more preferably, its thrombin content. Pre~erably, an ef~ective amount o~ a compounds o~
this invention of this invention for preventing coagulation in extracorporeal blood is ~rom about 1 ~g/60 ml of extracorporeal blood to about 5 mg/60 ml of extracorporeal blood.
The compounds of this invention may also be used to inhibit clot-bound thrombin, which is believed to contribute to clot accretion. This is particularly important because commonly used anti-thrombin agents, such as heparin and low molecular weight heparin, are ineffective against clot-bound thrombin. Finally, the compounds of this invention may be employed in compositions and methods for treating neurodegenerative diseases. Thrombin is known to cause neurite retraction, a process suggestive of the rounding in shape changes of brain cells and implicated in neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease.
Compounds of the present invention may be synthesized by various methods well known in the art. Suitable methods of synthesis will vary dep~n~;ng upon the AS and X
portions used in the compound. Suitable methods for synthesis of Phe-Pro-Arg type analogues are described below. However, other well known methods may be employed.
-. CA 02208773 1997-06-20 SCHE~E
H-X
O NH 1 HN ~X
HN ~Z J~ NH-Pg z ~
NH~NH
2 HN~ ~ ~ P3~ ~~ ~Nr$
HN 2 ~ CO2H
X~\Q 4 !j Y
Step 1:
The heterocyle 1 in solution was metalated with an appropriate metalating base such as n-BuLi to generate the corresponding metalated heterocylic compound. The cyclic activated arginine group 2 was added to this mixture.
Compound 2 was prepared according to procedures known in = the literature and described in, for example, R.T. Shuman, et al ., "Highly Selective Tripeptide Thrombin Inhibitors", J.Med.Chem, 1993, 36, 314. The compound yielded was - heterocyclic ketoarginine 3.
. .
Step 2:
- 20 The heterocyclic ketoarginine 3 is deprotected and coupled to the dipeptide 4 in the presence of a suitable coupling WO96/19491 PCT/CA9Sl00711 agent, solvent, and base. The dipeptide 4 can be purchased or prepared by methods common in the art and the peptide literature. Suitable coupling agents include BOP
and isopropylchloro~ormate. Suitable solvents include DCM
5 and DMF. Suitable bases include iPr2NEt and n-methyl t morpholine.
The resulting compound is deprotected with appropriate déprotecting agents to yield the heterocyclic ketoargininyl 5. Suitable deprotecting agents include 10 BBr3, HBr in acetic acid, and TMSI. Methods to remove the protecting groups are well known to people skilled in the art.
Scheme I is used where Z is N. Scheme II is used when Z is 15 carbon, linear carbon chain, or ~orms a ring with Q.
Where Z forms a ring with Q, the activated amino group 2 would be amended accordingly to include this ring. The steps in the process remain the same as described ~or Scheme I.
SCHE~DE II
H--X
HN ~1 'X
OCH z pg-y ,. P9 HNXl~ ~=o ~ CO2H
X~
Q_z 4 Y
The compounds of this invention and their intermediates may be purified during their synthesis and/or after their preparation by standard techniques well known to the skilled artisan. One preferred purification technique is HPLC. However, other chromatographic methods such as column chromatography may be used for purification o~ the compounds. Crystallization may also be used to purify the products as may w~c~; n~ with appropriate organic solvents.
It is well known in the art that the amino protecting groups are not necessary for the reaction to occur. The process may be carried out without protecting groups.
However, they are used to increase the yield of the desired compounds.
The process described above may use suitable protecting groups for compounds 2, 3, and 4. ~uitable deprotection conditions and protocols are described in the synthesis literature and are well known to chemists skilled in the art.
+
Desired R" Rz~ and R3 groups may be substituted onto the dipeptide 4 before it is coupled to heterocyclic ketoarginine 3 using techniques well known in the art of peptide ch~m;stry. Also, preferred analogues of each of the peptides or the dipeptide may be purchased with the desired Rl, R2, or R3 groups substituents already present.
In order that the invention described herein may be more fully understood, the ~ollowing examples are set ~orth. It should be understood that these examples are for illustrative purposes only and are not to be construed as limiting this invention in any manner.
.
EXl~MPLE
5~ o~ I
O NH 2. 2/-78 -20 C NH
~N NH{~ NH~NH
To a THF (75 mL) solution of benzothiazole (compound 1) (4.0 mL, 36.7 mmol) at -78 ~C was slowly added n-BuLi (1.6 M, 25 mL), resultant orange suspension was stirred at -78 ~C for 1.5 h. Then added solid compound 2 (3.55 g, 8.7 mmol). Reaction stirred at -78 ~C for 30 min followed by at -20 for 30 min then quenched with saturated aqueous NH4Cl .
Extraction with ethyl acetate followed by column chromatography afforded yellow ~oam (1.28 g) in 28% yield as compound 3.
NMR(CDCl3) d 1.45 (s, 9H), 1.5-1.8 (m, 2H), 3.1-3.23 (m, lH), 3.45-3.60 (m, lH), 5.1(d, 2H), 5.53-5.64(m, 2H), 7.02-7.15(m, 4H), 7.21-7.28(m, 2H), 7.56-7.65(m, 2H), 8.0-8.05(m, lH), 8.18-8.23(m, lH).
MS: (M+l) 526.8 ;
.
WO96/19491 PCT/CAg5/00711 NH ~ ~,N
NH W~
Cbz ~ 1. 4M Ha~DicD~cne ~\
'NH~NH EtSMe/3 - N
2. BOP/~MF/iPr2NEt HN
3. BBr3/DCM/-78 C /~S
4. RP HPLC ~N~ \
BOC ~N ~ H2N ~
To a mixture of compound 3 (0.223 g, 0.43 mmol) and EtSMe (0.25 mL), at ambient temperature, was added 4M HCl solution in dioxane (10 mL). The reaction was stirred for 1 h. All the solvents removed and the yellow gummy solid was dried. To this yellow solid was added compound 4 (0.17 g, 0.47 mmol) and BOP (0.21 g, 0.48 mmol) in DMF (~mL) at room temperature then to this mixture was added iPr2NEt until the pH of the mixture reaches 8-9. The reaction was allowed to stir overnight. The reaction was extracted with ethyl acetate and washed with brine, subsequent column chromatography gave 0.129 g of the desired precursor to compound 5 which was dissolved in DCM (10 mL) and added lM BBr3 solution in DCM (1.7 mL, 1.66 mmol) at -78 ~C.
Reaction was stirred at -78 ~C for 30 min followed by for 3 h at room temperature. Cooled back to -78 ~C and added anhd. MeOH (2mL) followed by stirring at RT for lh. All the solvents removed the mixture extracted with water and washed with ether. The water fraction lyophilized and was subjected to reverse phase HPLC purification to yield compound 5 . The two compounds were isolated as individual -diastereomers analogue 1 and analogue 2 with identical - Mass spectra [~M+l) 536.5]
:- Ea~MP~E 2 .
Determination of K~ Values This assay was performed with a Perkin Elmer fluorometer model ~LS 50B using a fluorogenix thrombin substrate (Tos-Gly-Pro-Arg-AMC.HCl) purchased from Calbiochem. Human thrombin was also obtained from Calbiochem. Measurements were determined at excitation and emission wavelengths of 383 and 455nm respectively.
The assay was carried out in rllnn;~ buf~er consisting of 50mM Tris, lOO~M NaCl, 0.1% and Peg pH 7.8 at 24~C. Buffer, substrate and inhibitor were mixed and the reaction was initiated by adding the enzyme solution. Initial velocities were recorded at several inhibitor and substrate concentrations. Kinetic parameters were determined by ~itting the data to a general equation describing enzyme inhibition (Segel, Enzyme Kinetics, Wiley Interscience Publications, 1993).
Dixon and Lineweaver-Burk plots were used to estimate the kinetic parameters (~, V~, K~) using the MicrosoftTM
Excell~ program.
Binding is the establishment of the equilibria between enzyme, inhibitor, and enzyme-inhibitor complexes. In slow binding inhibition, this equilibrium is established slowly.
Equilibrium dissociation constant for compound 5 is shown in Table 1. The result is compared with known reported tripeptidyl based thrombin inhibitors.
7M _ Registered Tr~de Mark Compound 5 exhibited slow binding kinetics, however the inhibition constant was determined assuming rapid steady state kinetics. There~ore, the reported values are a ~ reliable estimate of the equilibrium inhibitory constants.
dTT assay Procedure:
Fibrinogen, and buffer solution were transferred to disposable tubes and placed in a water bath for about 15 to 30 minutes before the assay to allow equilibration to 37~C
The cuvette-strips were incubated ~or 3 minutes at 37~C. A
ball was dispensed to each cuvette. To the prewarmed cuvettes was added 75~1 bu~er, 50 ~1 inhibitor solution, and 50 ~l fibrinogen solution. The timer was started corresponding to the incubation column ~or an incubation of 60 seconds. The cuvettes were transferred to the test column area. The multipette was primed once with the start reagent (thrombin solution). The multipette was activated and 25 ~1 of thrombin solution was dispensed.
When the clotting times were determined, they were displayed and printed.
A time versus inhibitor concentrations curve was constructed and ICso values were extrapolated from the inhibitor concentration curves. The ICso is de~ined as the dose required to double the coagulation time compared to control.
The result showing ICso value is shown in Table 1.
TABLE I
COMPOUND Kl (nM) ICs0(dTT)(nM) 0.05-0.180 1.8-7.2 PPACK 0.017 2.5 3l W O96/19491 PCTICAg5/00711 Boc-D-Phe-Pro-Arg-H 45 ; D-l-Tiq-Pro-Arg-H 19 ;
The results in Table I ~mo~trate that a heterocyclic function such as is embidied in a benzothiazolo-keto-arginyl unit sp~nn;n~ the Sl-SI' sites of thrombin enhances enzyme affinity up to 1000 fold compared to other reported inhibitors. Compound 5 is equipotent to PPACK which is regarded as an irreversible inhibitor of thrombin that forms a covalent bond with the enzyme whereas compound 5 is a reversible inhibitor of thrombin.
~- Having now fully described the invention, it will be . apparent to one of ordinary skill in the art that numerous modifications can be made thereto without departing from the spirit or the invention as set forth herein.
BOC ~N ~ H2N ~
To a mixture of compound 3 (0.223 g, 0.43 mmol) and EtSMe (0.25 mL), at ambient temperature, was added 4M HCl solution in dioxane (10 mL). The reaction was stirred for 1 h. All the solvents removed and the yellow gummy solid was dried. To this yellow solid was added compound 4 (0.17 g, 0.47 mmol) and BOP (0.21 g, 0.48 mmol) in DMF (~mL) at room temperature then to this mixture was added iPr2NEt until the pH of the mixture reaches 8-9. The reaction was allowed to stir overnight. The reaction was extracted with ethyl acetate and washed with brine, subsequent column chromatography gave 0.129 g of the desired precursor to compound 5 which was dissolved in DCM (10 mL) and added lM BBr3 solution in DCM (1.7 mL, 1.66 mmol) at -78 ~C.
Reaction was stirred at -78 ~C for 30 min followed by for 3 h at room temperature. Cooled back to -78 ~C and added anhd. MeOH (2mL) followed by stirring at RT for lh. All the solvents removed the mixture extracted with water and washed with ether. The water fraction lyophilized and was subjected to reverse phase HPLC purification to yield compound 5 . The two compounds were isolated as individual -diastereomers analogue 1 and analogue 2 with identical - Mass spectra [~M+l) 536.5]
:- Ea~MP~E 2 .
Determination of K~ Values This assay was performed with a Perkin Elmer fluorometer model ~LS 50B using a fluorogenix thrombin substrate (Tos-Gly-Pro-Arg-AMC.HCl) purchased from Calbiochem. Human thrombin was also obtained from Calbiochem. Measurements were determined at excitation and emission wavelengths of 383 and 455nm respectively.
The assay was carried out in rllnn;~ buf~er consisting of 50mM Tris, lOO~M NaCl, 0.1% and Peg pH 7.8 at 24~C. Buffer, substrate and inhibitor were mixed and the reaction was initiated by adding the enzyme solution. Initial velocities were recorded at several inhibitor and substrate concentrations. Kinetic parameters were determined by ~itting the data to a general equation describing enzyme inhibition (Segel, Enzyme Kinetics, Wiley Interscience Publications, 1993).
Dixon and Lineweaver-Burk plots were used to estimate the kinetic parameters (~, V~, K~) using the MicrosoftTM
Excell~ program.
Binding is the establishment of the equilibria between enzyme, inhibitor, and enzyme-inhibitor complexes. In slow binding inhibition, this equilibrium is established slowly.
Equilibrium dissociation constant for compound 5 is shown in Table 1. The result is compared with known reported tripeptidyl based thrombin inhibitors.
7M _ Registered Tr~de Mark Compound 5 exhibited slow binding kinetics, however the inhibition constant was determined assuming rapid steady state kinetics. There~ore, the reported values are a ~ reliable estimate of the equilibrium inhibitory constants.
dTT assay Procedure:
Fibrinogen, and buffer solution were transferred to disposable tubes and placed in a water bath for about 15 to 30 minutes before the assay to allow equilibration to 37~C
The cuvette-strips were incubated ~or 3 minutes at 37~C. A
ball was dispensed to each cuvette. To the prewarmed cuvettes was added 75~1 bu~er, 50 ~1 inhibitor solution, and 50 ~l fibrinogen solution. The timer was started corresponding to the incubation column ~or an incubation of 60 seconds. The cuvettes were transferred to the test column area. The multipette was primed once with the start reagent (thrombin solution). The multipette was activated and 25 ~1 of thrombin solution was dispensed.
When the clotting times were determined, they were displayed and printed.
A time versus inhibitor concentrations curve was constructed and ICso values were extrapolated from the inhibitor concentration curves. The ICso is de~ined as the dose required to double the coagulation time compared to control.
The result showing ICso value is shown in Table 1.
TABLE I
COMPOUND Kl (nM) ICs0(dTT)(nM) 0.05-0.180 1.8-7.2 PPACK 0.017 2.5 3l W O96/19491 PCTICAg5/00711 Boc-D-Phe-Pro-Arg-H 45 ; D-l-Tiq-Pro-Arg-H 19 ;
The results in Table I ~mo~trate that a heterocyclic function such as is embidied in a benzothiazolo-keto-arginyl unit sp~nn;n~ the Sl-SI' sites of thrombin enhances enzyme affinity up to 1000 fold compared to other reported inhibitors. Compound 5 is equipotent to PPACK which is regarded as an irreversible inhibitor of thrombin that forms a covalent bond with the enzyme whereas compound 5 is a reversible inhibitor of thrombin.
~- Having now fully described the invention, it will be . apparent to one of ordinary skill in the art that numerous modifications can be made thereto without departing from the spirit or the invention as set forth herein.
Claims (22)
1. A thrombin inhibiting compound according to formula (I), and pharmaceutically acceptable salts thereof AS - X
(I) wherein X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl; and AS is an active site inhibitor of thrombin having an argininyl residue or an analogue thereof connected to X.
(I) wherein X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl; and AS is an active site inhibitor of thrombin having an argininyl residue or an analogue thereof connected to X.
2. A compound according to claim 1, wherein X is selected from the group consisting of:
wherein X5, X10, X11, and X12 are each independently selected from the group consisting of N, or C-X7 where X7 is hydrogen, C1-4 alkyl, or C5-8 aryl;
X6, and X13 are each independently selected from the group consisting of C, O, N, S, N-X7, or CH-X7; and R5 is hydrogen, C0-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
wherein X5, X10, X11, and X12 are each independently selected from the group consisting of N, or C-X7 where X7 is hydrogen, C1-4 alkyl, or C5-8 aryl;
X6, and X13 are each independently selected from the group consisting of C, O, N, S, N-X7, or CH-X7; and R5 is hydrogen, C0-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
3. A compound according to claim 2, wherein X is selected from the group consisting of:
R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
4. A compound accoring to claim 2, wherein X is selected from the group consisting of:
wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
5. A compound according to claim 2 wherein X is selected from the group consisting of:
wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16 alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
6. A compound according to claim 2 wherein x is selected from the group consisting of:
or wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
or wherein R5 is hydrogen, C1-16 alkyl optionally carboxyl substituted, carboxyl, -C0-16 alkyl-CO2-C1-16alkyl, C6-20 aralkyl, C3-7 cycloalkyl, aryl or an aromatic heterocycle.
7. A compound according to claim 1, wherein AS is a group of formula (II):
(II) wherein G1 is one or more amino acid, alkyl, aryl, aralkyl, or cycloalkyl; and G2 is arginyl radical or an analogue thereof.
(II) wherein G1 is one or more amino acid, alkyl, aryl, aralkyl, or cycloalkyl; and G2 is arginyl radical or an analogue thereof.
8. A compound accoring to claim 7, wherein G2 is an arginyl radical selected from:
wherein n=1-6, n1=1-2, n2=0-7 and T is a bond or a divalent linking moiety with X.
wherein n=1-6, n1=1-2, n2=0-7 and T is a bond or a divalent linking moiety with X.
9. A compound according to claim 1, wherein AS is the peptide fragment of hirudin 45-47 and analogues thereof.
10. A compound according to claim 1, wherein AS is selected from D-Phe-Pro-Arg; D-Cha-Pro-Arg; D-Phe-Pip-Arg;
and D-Cha-Pip-Arg.
and D-Cha-Pip-Arg.
11. A thrombin inhibiting compound according to formula (III):
( III ) wherein R1 is selected from the group consisting of one or more aryl or cycloalkyl which is unsubstituted or substituted with hydroxy, C1-6 alkyl, C4-8 aralkyl, C3-8 aryl, or C3-8 cycloalkyl.
R2 is selected from the group consisting of hydrogen, hydroxy, C1-6 alkyl, C4-8 aralkyl, and unsubstituted or substituted amino group.
R3 is selected from the group consisting of hydrogen, hydroxy, SH, C1-6 alkyl, C3-8 aryl and C4-8 aralkyl.
n is an integer from 0 to 2.
Q is a bond or -NH-;
Z is C1-4 alkoxy; cyano; -NH2; -CH2-NH2; -C(NH)-NH2;
-NH-C(NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 cycloalkyl or aryl substituted with cyano, -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C(NH)-NH2; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C(NH)-NH2; and X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl.
( III ) wherein R1 is selected from the group consisting of one or more aryl or cycloalkyl which is unsubstituted or substituted with hydroxy, C1-6 alkyl, C4-8 aralkyl, C3-8 aryl, or C3-8 cycloalkyl.
R2 is selected from the group consisting of hydrogen, hydroxy, C1-6 alkyl, C4-8 aralkyl, and unsubstituted or substituted amino group.
R3 is selected from the group consisting of hydrogen, hydroxy, SH, C1-6 alkyl, C3-8 aryl and C4-8 aralkyl.
n is an integer from 0 to 2.
Q is a bond or -NH-;
Z is C1-4 alkoxy; cyano; -NH2; -CH2-NH2; -C(NH)-NH2;
-NH-C(NH) -NH2; -CH2-NH-C (NH) -NH2; a C6 cycloalkyl or aryl substituted with cyano, -NH2, -CH2-NH2, -C(NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C(NH)-NH2; or a 5 or 6 member, saturated or unsaturated heterocycle optionally substituted with cyano, -NH2, -CH2-NH2, -C (NH) -NH2, -NH-C (NH) -NH2 or -CH2-NH-C(NH)-NH2; and X is one or more aromatic or non-aromatic heterocycle unsubstituted or substituted with one or more amino, oxygen, alkyl, aralkyl, or aryl.
12. A compound according to claim 11, wherein R1 is selected from the group consisting of one or more 5 or 6 membered aromatic or non-aromatic ring optionally substituted with hydroxy, C1-4 alkyl, or C3-8 cycloalkyl.
13. A compound according to claim 12, wherein R1 is phenyl;
R2 is hydroxy or NH2;
R3 is hydrogen, or C1-4 alkyl;
n is 1 or 2;
Q is a bond; and Z is -NH-C (NH) -NH2 , -NH2, and -C(NH) -NH2 linked via a methylene chain of 3-5 carbon atoms.
R2 is hydroxy or NH2;
R3 is hydrogen, or C1-4 alkyl;
n is 1 or 2;
Q is a bond; and Z is -NH-C (NH) -NH2 , -NH2, and -C(NH) -NH2 linked via a methylene chain of 3-5 carbon atoms.
14. A compound according to claim 1 selected from (D-Phe)-Pro-alpha-benzothiazolo keto arginine and (D-Phe)-Pro-alpha-thiazolo keto arginine.
15. The use of a compound according to any one of claims 1 to 14 in the manufacture of a medicament for the treatment of vascular diseases in a mammal including humans.
16. The use according to claim 15, wherein said vascular disease is thrombosis.
17. A method for the treatment or prophylaxis of thrombotic disorders in a mammal, comprising administering to said mammal an effective amount of a compound according to any of one claims 1 to 14.
18. The method according to claim 17, wherein said disorder is venous thrombosis.
19. The method according to claim 17, wherein said disorder is pumonary thrombolism.
20. The method according to claim 17, wherein said disorder is arterial thrombosis.
21. The method according to claim 17, wherein said disorder is myocardial infarction.
22. The method according to claim 17, wherein said disorder is cerebral infarction.
Applications Claiming Priority (8)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| GB9426038.7 | 1994-12-22 | ||
| GBGB9426038.7A GB9426038D0 (en) | 1994-12-22 | 1994-12-22 | Low molecular weight bicyclic thrombin inhibitors |
| GBGB9503136.5A GB9503136D0 (en) | 1994-12-22 | 1995-02-17 | Low molecular weight bicyclic thrombin inhibitors |
| GB9503136.5 | 1995-02-17 | ||
| GB9504403.8 | 1995-03-06 | ||
| GBGB9504404.6A GB9504404D0 (en) | 1995-03-06 | 1995-03-06 | Heterocyclic keto arginine peptides as thrombin inhibitors |
| GB9504404.6 | 1995-03-06 | ||
| GBGB9504403.8A GB9504403D0 (en) | 1995-03-06 | 1995-03-06 | Heterocyclic keto arginine peptides as thrombin inhibitors |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2208773A1 true CA2208773A1 (en) | 1996-06-27 |
Family
ID=27451243
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002208773A Abandoned CA2208773A1 (en) | 1994-12-22 | 1995-12-21 | Heterocyclic keto arginine peptides as thrombin inhibitors |
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| Country | Link |
|---|---|
| EP (1) | EP0799240A1 (en) |
| JP (1) | JPH10513151A (en) |
| AU (2) | AU4250896A (en) |
| CA (1) | CA2208773A1 (en) |
| IL (1) | IL116502A0 (en) |
| WO (1) | WO1996019491A1 (en) |
Families Citing this family (28)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US6069130A (en) * | 1995-06-07 | 2000-05-30 | Cor Therapeutics, Inc. | Ketoheterocyclic inhibitors of factor Xa |
| IL121474A0 (en) * | 1996-08-23 | 1998-02-08 | Akzo Nobel Nv | Thrombin inhibitors |
| AU4172397A (en) * | 1996-09-06 | 1998-03-26 | Biochem Pharma Inc. | Lactam inhibitors of thrombin |
| AU743735B2 (en) * | 1996-10-11 | 2002-02-07 | Millennium Pharmaceuticals, Inc. | Selective factor Xa inhibitors |
| WO1998016524A1 (en) * | 1996-10-11 | 1998-04-23 | Cor Therapeutics, Inc. | HETEROCYCLIC DERIVATIVES AS FACTOR Xa INHIBITORS |
| CA2268264A1 (en) * | 1996-10-11 | 1998-04-23 | Cor Therapeutics, Inc. | Selective factor xa inhibitors |
| US6369080B2 (en) | 1996-10-11 | 2002-04-09 | Cor Therapeutics, Inc. | Selective factor Xa inhibitors |
| US6063794A (en) * | 1996-10-11 | 2000-05-16 | Cor Therapeutics Inc. | Selective factor Xa inhibitors |
| US6262047B1 (en) | 1996-10-11 | 2001-07-17 | Cor Therapeutics, Inc. | Selective factor Xa inhibitors |
| US6194435B1 (en) | 1996-10-11 | 2001-02-27 | Cor Therapeutics, Inc. | Lactams as selective factor Xa inhibitors |
| AU5526098A (en) * | 1996-12-23 | 1998-07-17 | Biochem Pharma Inc. | Bicyclic thrombin inhibitors |
| EP0977773A1 (en) | 1997-04-14 | 2000-02-09 | Cor Therapeutics, Inc. | SELECTIVE FACTOR Xa INHIBITORS |
| JP2001521524A (en) * | 1997-04-14 | 2001-11-06 | シーオーアール セラピューティクス インコーポレイテッド | Selective factor Xa inhibitor |
| AU6896298A (en) | 1997-04-14 | 1998-11-11 | Cor Therapeutics, Inc. | Selective factor xa inhibitors |
| EP0994894A1 (en) * | 1997-08-11 | 2000-04-26 | Cor Therapeutics, Inc. | SELECTIVE FACTOR Xa INHIBITORS |
| NZ502877A (en) * | 1997-08-11 | 2001-11-30 | Cor Therapeutics Inc | Bicyclic aryl azepinone selective factor Xa inhibitors for treating thrombosis related diseases |
| US6333321B1 (en) | 1997-08-11 | 2001-12-25 | Cor Therapeutics, Inc. | Selective factor Xa inhibitors |
| AU753842B2 (en) * | 1997-08-11 | 2002-10-31 | Millennium Pharmaceuticals, Inc. | Selective factor Xa inhibitors |
| US6228854B1 (en) | 1997-08-11 | 2001-05-08 | Cor Therapeutics, Inc. | Selective factor Xa inhibitors |
| US6218382B1 (en) | 1997-08-11 | 2001-04-17 | Cor Therapeutics, Inc | Selective factor Xa inhibitors |
| US6323219B1 (en) | 1998-04-02 | 2001-11-27 | Ortho-Mcneil Pharmaceutical, Inc. | Methods for treating immunomediated inflammatory disorders |
| US8106094B2 (en) | 1998-07-06 | 2012-01-31 | Johnson & Johnson Consumer Companies, Inc. | Compositions and methods for treating skin conditions |
| US8093293B2 (en) | 1998-07-06 | 2012-01-10 | Johnson & Johnson Consumer Companies, Inc. | Methods for treating skin conditions |
| SK10742001A3 (en) | 1999-01-27 | 2002-08-06 | Ortho-Mcneil Pharmaceutical, Inc. | Peptidyl heterocyclic ketones useful as tryptase inhibitors |
| US8431550B2 (en) | 2000-10-27 | 2013-04-30 | Johnson & Johnson Consumer Companies, Inc. | Topical anti-cancer compositions and methods of use thereof |
| US7192615B2 (en) | 2001-02-28 | 2007-03-20 | J&J Consumer Companies, Inc. | Compositions containing legume products |
| WO2009102761A1 (en) | 2008-02-12 | 2009-08-20 | Bristol-Myers Squibb Company | 1,2,3-triazoles as 11-beta hydroxysteroid dehydrogenase type i inhibitors |
| CN106986862A (en) | 2010-12-22 | 2017-07-28 | Abbvie 公司 | Hepatitis c inhibitor and application thereof |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4191753A (en) * | 1978-11-06 | 1980-03-04 | University Of Miami | Anti-hypertensive peptide analogs |
| FR2663336B1 (en) * | 1990-06-18 | 1992-09-04 | Adir | NOVEL PEPTIDE DERIVATIVES, THEIR PREPARATION PROCESS AND THE PHARMACEUTICAL COMPOSITIONS CONTAINING THE SAME. |
-
1995
- 1995-12-21 WO PCT/CA1995/000711 patent/WO1996019491A1/en not_active Application Discontinuation
- 1995-12-21 JP JP8519385A patent/JPH10513151A/en active Pending
- 1995-12-21 AU AU42508/96A patent/AU4250896A/en not_active Abandoned
- 1995-12-21 EP EP95940926A patent/EP0799240A1/en not_active Withdrawn
- 1995-12-21 CA CA002208773A patent/CA2208773A1/en not_active Abandoned
- 1995-12-22 IL IL11650295A patent/IL116502A0/en unknown
- 1995-12-22 AU AU40627/95A patent/AU699679B2/en not_active Ceased
Also Published As
| Publication number | Publication date |
|---|---|
| IL116502A0 (en) | 1996-03-31 |
| MX9704594A (en) | 1998-07-31 |
| EP0799240A1 (en) | 1997-10-08 |
| AU699679B2 (en) | 1998-12-10 |
| JPH10513151A (en) | 1998-12-15 |
| WO1996019491A1 (en) | 1996-06-27 |
| AU4062795A (en) | 1996-06-27 |
| AU4250896A (en) | 1996-07-10 |
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| Date | Code | Title | Description |
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| FZDE | Discontinued |