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WO1995023811A1 - Nouveaux composes carbocycliques inhibant l'agregation plaquettaire par interaction avec le complexe recepteur gpiib/iiia - Google Patents

Nouveaux composes carbocycliques inhibant l'agregation plaquettaire par interaction avec le complexe recepteur gpiib/iiia Download PDF

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Publication number
WO1995023811A1
WO1995023811A1 PCT/US1995/002572 US9502572W WO9523811A1 WO 1995023811 A1 WO1995023811 A1 WO 1995023811A1 US 9502572 W US9502572 W US 9502572W WO 9523811 A1 WO9523811 A1 WO 9523811A1
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WIPO (PCT)
Prior art keywords
alkyl
compound
asp
substituted
formula
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PCT/US1995/002572
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English (en)
Inventor
William Frank De Grado
Ronald Herman Hoess
Sharon Anne Jackson
Shaker Ahmed Mousa
Karyn Thompson O'neil
Arlene Lynnae Rockwell
Original Assignee
The Du Pont Merck Pharmaceutical Company
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
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Application filed by The Du Pont Merck Pharmaceutical Company filed Critical The Du Pont Merck Pharmaceutical Company
Priority to AU19756/95A priority Critical patent/AU1975695A/en
Publication of WO1995023811A1 publication Critical patent/WO1995023811A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/745—Blood coagulation or fibrinolysis factors
    • C07K14/75—Fibrinogen
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/50—Cyclic peptides containing at least one abnormal peptide link
    • C07K7/54—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring
    • C07K7/56—Cyclic peptides containing at least one abnormal peptide link with at least one abnormal peptide link in the ring the cyclisation not occurring through 2,4-diamino-butanoic acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This invention is in the field of novel cyclic compounds containing carbocyclic ring systems, and particularly to cyclic peptides, which are useful as antagonists of the platelet glycoprotein IIb/IIIa receptor complex. Consequently, the present invention is also in the field of pharmaceutical compositions containing, and therapeutic methods of using, these compounds for the inhibition of platelet aggregation, as thrombolytics, and/or for the treatment of other thromboembolic disorders.
  • Activation of platelets and the resulting platelet aggregation and secretion of factors by the platelets has been associated with different pathophysiological conditions including cardiovascular and cerebrovascular thromboembolic disorders, for example, the thromboembolic disorders associated with unstable angina, myocardial infarction, transient ischemic attack, stroke, atherosclerosis and diabetes.
  • cardiovascular and cerebrovascular thromboembolic disorders for example, the thromboembolic disorders associated with unstable angina, myocardial infarction, transient ischemic attack, stroke, atherosclerosis and diabetes.
  • the contribution of platelets to these disease processes stems from their ability to form aggregates, or platelet thrombi, especially in the arterial wall following injury or plaque rupture.
  • Platelets are known to play an essential role in the maintenance of hemostasis and in the pathogenesis of arterial thrombosis. Platelet activation has been shown to be enhanced during coronary thrombolysis which can lead to delayed reperfusion and reocclusion. Clinical studies with aspirin, ticlopidine and a monoclonal antibody for platelet glycoprotein IIb/IIIa provide biochemical evidence for platelet involvement in unstable angina, early stage of acute myocardial infarction, transient ischemic attack, cerebral ischemia, and stroke.
  • Platelets are activated by a wide variety of agonists resulting in platelet shape change, secretion of granular contents and aggregation. Aggregation of platelets serves to further focus clot formation by concentrating activated clotting factors in one site.
  • endogenous agonists including adenosine diphosphate (ADP), serotonin, arachidonic acid, thrombin, and collagen, have been identified. Because of the involvement of several endogenous agonists in activating platelet function and aggregation, an inhibitor which acts against all agonists would represent a more efficacious antiplatelet agent than currently available antiplatelet drugs, which are agonist-specific.
  • Current antiplatelet drugs are effective against only one type of agonist; these include aspirin, which acts against arachidonic acid; ticlopidine, which acts against ADP; thromboxane A2 synthetase inhibitors or receptor antagonists, which act against thromboxane A 2 ; and hirudin, which acts against thrombin.
  • GPIIb/IIIa platelet glycoprotein Ilb/IIIa receptor complex
  • GPIIb/IIIa platelet glycoprotein Ilb/IIIa receptor complex
  • GPIIb/IIIa specific antiplatelet agent which inhibits the activation and aggregation of platelets in response to any agonist.
  • Such an agent should represent a more efficacious antiplatelet therapy than the currently available agonist-specific platelet inhibitors.
  • GPIIb/IIIa does not bind soluble proteins on unstimulated platelets, but GPIIb/IIIa in activated platelets is known to bind four soluble adhesive proteins, namely fibrinogen, von Willebrand factor, fibronectin, and vitronectin.
  • fibrinogen The binding of fibrinogen and von Willegrand factor to GPIIb/IIIa causes platelets to aggregate.
  • the binding of fibrinogen is mediated in part by the Arg-Gly-Asp (RGD) recognition sequence which is common to the adhesive proteins that bind GPIIb/IIIa.
  • RGD-containing peptides and related compounds have been reported which block fibrinogen binding and prevent the formation of platelet thrombi. For example, see Cadroy, et al. (1989) J. Clin. Invest. 84:939-944; Klein, et al . , U.S. Patent No. 4,952,562, issued 8/28/90; European Patent Application EP 0319506A; European Patent Application EP 0422938 Al; European Patent Application EP 0422937 Al; European Patent Application EP 0341915 A2 ; PCT Patent Application WO 89/07609; PCT Patent Application WO 90/02751; PCT Patent Application WO 91/04247; and European Patent Application EP 0343085 Al . Additional consensus sequences related to RGD have also been implicated as involved in blocking the binding of fibrinogen and other factors and proteins.
  • novel consensus sequences related to RGD are linked by bridging groups that give cyclized peptides that have high affinity and selectivity for GPIIb/IIIa.
  • the present invention provides novel cyclic compounds containing bridging groups useful as antagonists of the platelet glycoprotein Ilb/IIIa receptor complex, pharmaceutical compositions containing such cyclic compounds, the methods of using these compounds for the inhibition of platelet aggregation, as thrombolytics, and/or for the treatment of thromboembolic disorders, as well as for the inhibition and/or treatment of osteoporosis.
  • the present invention also relates to combination products, that is, pharmaceutical compositions containing the novel cyclic compounds of the invention in combination with anti-coagulants such as warfarin or heparin, or anti-platelet agents such as aspirin, piroxicam or ticlopidine, or thrombin inhibitors such as boropeptides, hirudin or argatroban, or thrombolytic agents such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations thereof, to pharmaceutical kits containing these combination products, and to methods of using these combination products for the inhibition of platelet aggregation, as thrombolytics, and/or for the treatment of thromboembolic disorders.
  • anti-coagulants such as warfarin or heparin
  • anti-platelet agents such as aspirin, piroxicam or ticlopidine
  • thrombin inhibitors such as boropeptides, hirudin or argatroban
  • the present invention still further relates to combination products, that is, pharmaceutical compositions containing the novel cyclic compounds of the invention in combination with anti-osteoporosis agents such as estrogens, androgens, and sodium and other fluoride salts, or combinations thereof, to pharmaceutical kits containing these combination products, and to methods of using these combination products for the inhibition and/or treatment of osteoporosis.
  • combination products that is, pharmaceutical compositions containing the novel cyclic compounds of the invention in combination with anti-osteorosis agents such as estrogens, androgens, and sodium and other fluoride salts, or combinations thereof, to pharmaceutical kits containing these combination products, and to methods of using these combination products for the inhibition and/or treatment of osteoporosis.
  • the present invention also provides compounds, kits comprising said compounds, detectably labeled compounds, and methods for the diagnosis and detection of tumors in an individual.
  • the provided compounds are compounds of Formula (I) that are conjugated to a chelating moiety (hereinafter, "conjugated compounds").
  • conjugated compounds are compounds of Formula (I) that have been directly labeled and conjugated compounds that have been labeled.
  • the method for detection of tumors is a substantially non-invasive method using the detectably labeled compounds
  • A, B, E, G and H are each, independently, either absent, except
  • R 1 is H or C 1 -C 8 alkyl
  • R 2 is K or C 1 -C 3 alkyl
  • R 3 is selected from:
  • aryl substituted with 0-2 R 12 a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, or O, said heterocyclic ring being substituted with 0-2 R 12 ;
  • R 11 is one or more members selected from the group consisting essentially of:
  • C 1 -C 5 alkyl, C 2 -C 4 alkenyl, C 3 -C 6 cycloalkyl, C 3 - C 6 cycloalkylmethyl, C 2 -C 6 alkoxyalkyl, C 3 -C 6 cycloalkoxy, C 1 -C 4 alkyl (alkyl being substituted with 1-5 groups selected independently from: -NR 13 R 14 , -CF 3 , NO 2 , -SO 2 R 13a , or -S( O)R 13a ), aryl substituted with 0-2 R 12 ,
  • heterocyclic ring system containing 1-4 heteroatams independently selected from N, S, and 0, said heterocyclic ring being substituted with 0-2 R 12 ;
  • R 12 is one or more members selected from the group consisting essentially of:
  • R 13 is selected independently from: H, C 1 -C 10 alkyl, C 3 - C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C 1 - C 10 alkyDaryl, C 3 -C 10 alkoxyalkyl, or alternatively, when attached to a carboxylic acid residue, is a carboxy protecting group;
  • R 13 groups when two R 13 groups are bonded to a single N, said R 13 groups may alternatively be taken together to form
  • R 13a is selected independently fromC 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C 1 -C 10 alkyl)aryl, or C 3 -C 10 alkoxyalkyl;
  • R 14 is as above OH, H, C 1 -C 4 alkyl, or benzyl;
  • R 15 is selected from:
  • R34 and R 35 are independently selected from:
  • R 34 and R 35 can alternatively be taken together to form: a cyclic boron ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally 1-4 heteroatoms independently selected from N, S, or O;
  • a divalent cyclic boron amide where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-4 heteroatoms independently selected from N, S, or O;
  • a cyclic boron amide-ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-4 heteroatoms independently selected from N, S, or O;
  • C is a D-isomer or L-isomer amino acid of structure
  • R 6 is H or CH 3
  • R 7 is selected from:
  • cyclohexyl is at the 3 or 4 position
  • X is selected from:
  • R 6 and R 7 can alternatively be taken together to form
  • n 0 or 1 and X is -NH 2 or
  • F is a D-isomer or L-isomer amino acid of structure
  • R 2 is as defined above, i.e., H, or C 1 -C 3 alkyl
  • R 8 is selected from:
  • R 13b is selected from:
  • R 36 is selected independently from: H, C 1 -C 8 alkyl, C 3 -C 10 cycloalkyl, phenyl, or benzyl;
  • R 37 is selected from:
  • aryl substituted with 0-2 groups independently selected from: halogen, phenyl,
  • R 38 is the same as R 37 , except that it may be H;
  • R 39 is selected from:
  • R 40 is selected from: H, C 1 -C 5 alkyl, or benzyl;
  • L a bridging group
  • R 31 is a C 6 -C 14 saturated, partially saturated, or aromatic carbocyclic ring system substituted with 0-4 R 10 or R 10a ;
  • R 10 and R 10a are selected independently from one or more of the following:
  • R 32 is selected from:
  • R 20 and R 22 are independently selected from the following:
  • aryl substituted with 0-2 R 12 a 5-10 membered heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S, and 0, said heterocyclic ring being substituted with 0-2 R 12 ;
  • R 21 and R 23 are independently selected from:
  • R 1 is as defined above, i.e., H or C 1 -C 8 alkyl; when either n' or n" is 2, R 20 or R 21 , or R 22 or R 23 , respectively, can alternatively and independently, be taken together with R 20 or R 21 , or R 22 or R 23 , respectively, on an adjacent carbon atom to form a direct bond, thereby forming a double or triple bond between said adjacent carbon atoms;
  • R 2 ⁇ and R 21 , as well as R 22 and R 23 can alternatively, and independently, join to form a 3-7 membered carbocyclic ring substituted with 0-2 R 12 ;
  • R 31a is selected from:
  • R 32 , R 2 , R 20 , R 22 , and R 1 are as defined above, and each R 2 is selected independent of the other; r is each independently 1-4; s is 0-1;
  • R 3a is selected independently from H or CH 3 ;
  • R 4 is selected from:
  • R 25 is selected from:
  • heterocyclic ring system containing 1-4 heteroatoms independently selected from N, S , and 0, said heterocyclic ring being substituted with 0-4 R 11 ;
  • R 5 is selected from:
  • R 100 is a 6-500 amino acid chain terminating in H or N - formyl-Met
  • R 101 is a 6-500 amino acid chain terminating in a carboxylate or carboxamide;
  • the present invention includes those compounds wherein the bridging group L is as defined in (a) thereunder, as part of [1] above, which is as follows:
  • R 31 is bonded to (C(R 23 )R 22 ) n " and (C(R 21 )R 20 ) n ' at two different atoms on said carbocyclic ring.
  • n" is 0 and n' is 1
  • n" is 0 and n' is 2 n" is 1 and n' is 0;
  • n" is 1 and n' is 1;
  • n" is 1 and n' is 2;
  • n" is 2 and n' is 0;
  • n" is 2 and n' is 1;
  • n" is 2 and n' is 2.
  • R 31 (including R 10 and R 10a ); n' and n"; R 20 , R 21 , R 22 and R 23 , and combinations thereof; R 11 , R 12 , R 13 , R 13a , R 14 , R 15 , R 34 and R 35 ; and D and F are as defined above under [1]; and
  • R 32 is selected from:
  • R 1 is H or CH 3 ;
  • R 2 is H or C 1 -C 3 alkyl
  • R 6 is H; R 7 is selected from:
  • each q is independently 0-2 and substitution on the cyclohexyl is at the 3 or 4 position;
  • R 6 and R 7 can alternatively be taken together to form
  • R 31 is selected from the group consisting of :
  • R 31 is selected from the group consisting of:
  • any of the bonds forming the carbocyclic ring may be a single or double bond, and wherein said carbocyclic ring is substituted independently with 0-4 R 10 ; (b) a 10 membered saturated, partially saturated , or aromat i c bi cycli c carbocyclic ring of formula :
  • any of the bonds forming the carbocyclic ring may be a single or double bond, and wherein said carbocyclic ring is substituted independently with 0-4 R 10 or R 10a .
  • any of the bonds forming the carbocyclic ring may be a single or double bond, and wherein said carbocyclic ring is substituted independently with 0-4 R 10 or R 10a .
  • This invention includes compounds of formula (I) wherein: R 31 is selected from (the dashed bond may be a
  • R 31 may be substituted independently
  • n" is 0 or 1;
  • n' is 0-2.
  • R 20 and R 22 are independently selected from C 1 -C 4 alkyl substituted with one R 11 , phenyl, benzyl, or phenyl (C 1 -C 4 ) alkyl;
  • the present invention includes compounds of formula (I), or a pharmaceutically acceptable salt or prodrug form thereof wherein:
  • R 15 is as defined above under [1]; and R 31 is selected from: ; ; ;
  • R 31 may be substituted independently
  • n' is 0-2;
  • R 20 and R 22 are independently selected from H, C 1 -C 4 alkyl, phenyl, benzyl, phenyl -(C 2 -C 4 ) alkyl, C 1 -C 4 alkoxy;
  • R 21 and R 23 are independently H or C 1 -C 4 alkyl
  • R 1 is H or C 1 -C 4 alkyl
  • R 13 is selected independently from: H, C1-C10 alkyl, C 3 - C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C 1 - C 10 alkyl)aryl, or C 3 -C 10 alkoxyalkyl;
  • R 13a is C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C 1 -C 10 alkyDaryl, or C 3 - C 10 alkoxyalkyl; when two R 13 groups are bonded to a single N, said R 13 groups may alternatively be taken together to form -(CH 2 ) 2-5 - or -(CH 2 )O(CH 2 )-;
  • R 14 is OH, H, C 1 -C 4 alkyl, or benzyl;
  • R 10 and R 10a are selected independently from: H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy;
  • R 2 is H or C 1 -C 3 alkyl
  • R 1 and R 3 can alternatively be taken together to form
  • R 2 and R 3 can alternatively be taken together to form
  • R 6 and R 7 can alternatively be taken together to form where
  • Preferred compounds of the invention are 1,3-disubstituted phenyl compounds of the formula (III):
  • R 14 as defined above under [10], and R 15 is as defined above under [1]; and the phenyl ring in formula (III.) may be further substituted with 0-3 R 10 ;
  • R 10 is selected independently from: H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy;
  • R 20 is H, C 1 -C 4 alkyl, phenyl, benzyl, or phenyl-(C 1 -C 4 )alkyl;
  • R 1 is H or methyl
  • R 13 and R 13a are as defined above under [10] ;
  • A, B, E, G and H are as defined above under [10], except that R 1 and R 3 can alternatively be taken together to form -CH 2 CH 2 CH 2 -;
  • R 40 is selected from: H, C 1 -C 5 alkyl, or benzyl.
  • R 20 is H, C 1 -C 4 alkyl, phenyl, benzyl, or phenyl-(C 2 -C 4 ) alkyl; Under the definition of A, B, E, G and H:
  • R 2 is H; and R 1 and R 3 can alternatively be taken together to form -CH 2 CH 2 CH 2 -; Under the definition of C: is H;
  • Y is NH or O
  • R 8 is selected from:
  • R 13b is selected independently from:
  • R 36 is selected independently from: H, C 1 -C 8 alkyl, phenyl, or benzyl;
  • R 37 and R 38 are as defined above under [1]; and R 39 is selected from: C 1 -C 5 alkyl, benzyl or phenyl.
  • Preferred compounds of the present invention are compounds of formula (III.) above, wherein all of the definitions are the same as in [12] above, except that: the phenyl ring in formula (III.) may be further substituted with 0-2 R 10 or R 10a ;
  • R 20 is H
  • R 1 is H
  • R 1 is H and R 3 is CH 3 , CH 2 CH 3 , CH(CH 3 ) 2 , CH(CH 3 )CH 2 CH 3 , CH 2 CH 2 CH 3 , CH 2 CH 2 CH 2 CH 3 , CH 2 CH 2 SCH 3 , CH 2 CH(CH 3 ) 2 ; or
  • C is an L-isomer amino acid of formula
  • R 2 attached to carbon is H or CH 3 ;
  • R 2 attached to nitrogen is H; R 8 is
  • R 13b is selected independently from:
  • R 36 is C 1 -C 4 linear alkyl or H; and R 39 is C 1 -C 4 alkyl, benzyl, or phenyl.
  • Preferred compounds of the present invention are compounds of formula (III.), or a pharmaceutically acceptable salt thereof, wherein:
  • R 20 and R 1 are both H;
  • A, B, G and H are each absent, and E is selected from L-Abu, D- and L-Val, L-Ile, and L-Met;
  • C is selected from Arg
  • Specifically preferred compounds of the present invent ion are the f ol lowing compounds and pharmaceutically acceptable salts thereof : a) The compound of formula (III.) wherein R 20 and R 1 are both H; A, B, G and H are absent; C is Arg; D is Gly; E is L-Ile; and F is:
  • R 22 and R 1 are both H; A, B, G and H are absent; C is Arg; D is Gly; E is L-Ile; and F is Asp;
  • R 20 and R 1 are both H; A, B, G and H are absent; C is Arg; D is Gly; E is L-Ile; F is Asp; and when either of R 10 or R 10a is H, the other is:
  • H are absent; C is Arg; D is Gly; E is L-Ile; F is Asp;
  • R 20 and R 1 are both H; A, B, G and H are absent; C is Arg; D is Gly; E is L-Ile; F is Asp;
  • More specifically preferred compounds of the present invention are those of Formula (III.) wherein R 20 and R 1 are both H; B and G are absent; and A, C, D, E, F and H are as follows, respectively:
  • the present invention includes those compounds having the subgeneric definitions set out above in [4] through [16], but with respect to the bridging group L defined by (b), rather than (a), in [1] above which is as follows: R 32 -(R 22 (R 23 )C) n réelle-(R 32 ) u , (N(R 13 )) u '(R 32 ) u '-R 31a - (C(R 21 )R 20 ) n '-NR 1
  • n" is 0 and n is 1
  • n" is 0 and n is 2
  • n" is 0 and n is 3
  • n" is 1 and n is 0
  • n" is 1 and n is 1
  • n" is 1 and n is 2
  • n" is 1 and n is 3
  • n" is 2 and n is 0
  • n" is 2 and n is 1
  • n" is 2 and n is 2 or
  • R 32 is selected from:
  • A, B, E, G and H are each, independently, either absent, except
  • R 1 , R 2 and R 3 and combinations thereof, are as defined above under [1];
  • R 31a is selected from:
  • n" is 0-2;
  • R 31a is selected from: -CH 2 CH 2 - substituted with 0-2 R 10 ;
  • n" is 0 or 1;
  • n' is 0-2;
  • R 20 and R 22 are independently selected from H, C 1 -C 4 alkyl, phenyl, benzyl, phenyl-(C 2 -C 4 )alkyl, C 1 -C 4 alkoxy;
  • R 21 and R 23 are independently H or C 1 -C 4 alkyl;
  • R 2 is H or C 1 -C 4 alkyl;
  • R 10 is H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy.
  • R 31a is selected from:
  • n" is 0;
  • n' 2;
  • R 20 , R 21 , R 22 and R 23 ; R 2 and R 10 are as defined above in [5a];
  • the present invention includes compounds of formula (XI.):
  • R 31a is selected from:
  • n' is 0-2;
  • R 20 , R 21 , R 22 and R 23 ; R 2 and R 10 are as defined above in [5a];
  • A, B, E, G and H are as defined in [3a] above; C and D are as defined in [10] above; F is as defined above under [1];
  • Preferred compounds of the invention are compounds of formula (XI.) and pharmaceutically acceptable salt forms thereof wherein:
  • R 31a , R 10 , R 20 , R 21 , R 22 and R 23 are as defined in [5a] above; and R 14 and R 15 are as defined in [1] above; n" is 0 or 1;
  • n' is 1 or 2;
  • A, B, E, G, and H are defined as in [3a] above, except that R 1 and R 3 can alternatively be taken together to form -CH 2 CH 2 CH 2 -;
  • R 3 and R 5 can alternatively be taken together to form -CH 2 CH 2 CH 2 -;
  • D is as defined above under [7a];
  • F is as defined above under [7a], except that under the definitions for R 13b , the meanings (q), (t), and (u) are not included.
  • Y is NH or O; and Under the definition of F, q' is 1;
  • R 8 is selected from:
  • R i 3b is selected independently from:
  • R 36 is selected independently from: H, C 1 -C 8 alkyl, phenyl, or benzyl;
  • R 39 is selected from: C 1 -C 5 alkyl, benzyl or phenyl.
  • Preferred compounds of the present invention are compounds of formula (XI.) above, wherein all of the definitions are the same as in [9a] above, except that: R 31a is selected from:
  • R 20 , R 21 , R 22 , R 23 are H; R 1 is H;
  • Preferred compounds of the present invention are compounds of formula (X.), or a pharmaceutically acceptable salt thereof, wherein:
  • R 31a is selected from:
  • R 10 is independently selected from: H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy; n" is 0-2;
  • n' is 0-2;
  • R 21 , R 22 , and R 23 are H;
  • R 20 and R 1 are independently selected from H or methyl
  • A, B, C, D, E, F, G, and H are each as defined above under [14].
  • Preferred compounds of the present invention are compounds of formula (XI.), or a pharmaceutically acceptable salt thereof, wherein: R 31a is selected from:
  • R 10 is independently selected from: H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy; wherein n" and n' are 1 or wherein n" is 0 and n' is 2;
  • R 21 , R 22 , and R 23 are H;
  • R 20 and R 1 are independently selected from H, methyl
  • a through G are as defined under [14] above.
  • Specifically preferred compounds of the present invention are the following compounds and pharmaceutically acceptable salts thereof: a) The compound of Formula (XI.) wherein n" and n' are 1 or wherein n" is 0 and n' is 2; R 1 , R 20 , R 21 , R 22 and R 23 are all H; A, B, G and H are absent; C is Arg; D is Gly; E is L-Ile; R 31a is selected from:
  • F is as defined under [15] a) above.
  • the present invention includes those compounds wherein the bridging group L is as defined in (c) thereunder, as part of [1] above, which is as follows:
  • the present invention includes those compounds above wherein : each r is 1 , and s is 1.
  • the present invention includes those compounds above of the formula :
  • R 11 , R 12 , R 13 , R 13 a, R 14 , R 15 , R 34 and R 35 ; and D and F are as defined above under [1] ;
  • R 1 is H or CH 3 ;
  • R 3a are each independently H or CH 3 ;
  • R 4 is selected from H
  • R 25 is C 1 - C 4 alkyl or aryl; R 5 is selected from (R 25 ) 2 N- and R 25 -O-, wherein:
  • R 25 is independently H, C 1 -C 4 alkyl, or aryl
  • A, B, C, D, E, F, G and H are each, independently as defined above under [10].
  • the present invention includes compounds of Formula (XII.), or a pharmaceutically acceptable salt or prodrug form thereof wherein: R 11 , R 12 , R 15 , R 34 and R 35 ; and F are as defined above under [1]; and
  • R 1 is H;
  • R 3a have the same definition when attached to the same carbon; R 4 and R 5 are as defined above under [3b];
  • R 13 is selected independently from: H, C 1 -C 10 alkyl, C 3 - C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C 1 - C 10 alkyDaryl, or C 3 -C 10 alkoxyalkyl;
  • R 13a is C 1 -C 10 alkyl, C 3 -C 10 cycloalkyl, C 4 -C 12 alkylcycloalkyl, aryl, -(C ⁇ -C ⁇ o alkyDaryl, or C 3 - C 10 alkoxyalkyl; when two R 13 groups are bonded to a single N, said R 13 groups may alternatively be taken together to form -(CH 2 ) 2-5 - or -(CH 2 )O(CH 2 )-;
  • R 14 is OH, H, C 1 -C 4 alkyl, or benzyl
  • R 10 and R 10a are selected independently from: H, C 1 -C 8 alkyl, phenyl, halogen, or C 1 -C 4 alkoxy;
  • A, B, E, G and H are each, independently as defined in [10] above.
  • Preferred compounds of the present invention are compounds of the Formula (XII.):
  • A, B, C, D, E, F, G and H are as defined above under
  • Specifically preferred compounds of the present invention are the following compounds and pharmaceutically acceptable salts thereof:
  • Trp or naphtlyl -Ala More specifically preferred compounds of the present invention are those of Formula (XIII.) wherein the definitions of the substituents are the same as in [7b] a) above, except: F is Asp;
  • More specifically preferred compounds of the present invention are those of Formula (XIII.) wherein the definitions of the substituents are the same as in [7b] a) above, except: F is Asp; and
  • R 5 is OH, (R 25 ) 2 N-, or R 25 -O-, where R 25 is independently C 1 -C 4 alkyl or aryl;
  • More specifically preferred compounds of the present invention are those of Formula (XIII.) wherein the definitions of the substituents are the same as in [7b] a) above, except:
  • R 3a and R 3b for both occurrences of each, are CH 3 ; i) The compounds under h) immediately above, except : F is Asp; and
  • R 5 is OH, (R 25 )2N-, or R 25 -O-, where
  • R 25 is independently C 1 -C 4 alkyl or aryl; k) The compounds under h) immediately above, except:
  • R 3a for both occurrences are H
  • R 3b for both occurrences are CH 3 ; m) The compounds under 1) immediately above, except:
  • F is Asp
  • R 5 is OH, (R 25 ) 2 N-, or R 25 -O-, where R 25 is independently C 1 -C 4 alkyl or aryl; o) The compounds under 1) immediately above, except:
  • R 3a for both occurrences are CH 3 .
  • F is Asp
  • F is Asp
  • R 5 is OH, (R 25 ) 2 -N-, or R 25 -O-, where R 25 is independently C 1 -C 4 alkyl or aryl; s) The compounds under p) immediately above, except:
  • the present invention includes compounds wherein the bridging group L is as defines in (d) thereunder, as part of [1] above, which is as follows:
  • A is any amino acid
  • B & H are absent .
  • GPIIb/IIIa glycoprotein IIb/IIIa
  • the compounds of the present invention inhibit the activation and aggregation of platelets induced by all known endogenous platelet agonists .
  • the present invention also provides methods for the treatment (including prevention) of conditions involving platelet activation and aggregation, such as arterial or venous cardiovascular or cerebrovascular thromboembolic disorders, including, for example, thromboembolic disorders associated with unstable angina, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, deep vein thrombosis, pulmonary embolism, "or diabetes, by administering to a host in need of such treatment a pharmaceutically effective amount of the compounds described above.
  • thromboembolic disorders associated with unstable angina, first or recurrent myocardial infarction, ischemic sudden death, transient ischemic attack, stroke, atherosclerosis, deep vein thrombosis, pulmonary embolism, "or diabetes
  • the compounds of the present invention are useful for inhibiting the binding of fibrinogen to blood platelets, inhibiting aggregation of blood platelets, treating thrombus formation or embolus formation, or preventing thrombus or embolus formation in a mammal.
  • the compounds of the invention may be used as a medicament for blocking fibrinogen from acting at its receptor site in a mammal.
  • the compounds of the present invention can also be combined or co-administered with suitable anticoagulant or coagulation inhibitory agents, such as heparin or warfarin, or anti-platelet or platelet inhibitory agents, such as aspirin, piroxicam or ticlopidine. Further, the compounds of this invention may be combined or co-administered with thrombin inhibitors such as boropeptides, hirudin or argatroban. The compounds of the present invention may also be combined or co-administered with thrombolytic or fibrinolytic agents, such as plasminogen activators, anistreplase, urokinase, or streptokinase .
  • suitable anticoagulant or coagulation inhibitory agents such as heparin or warfarin
  • anti-platelet or platelet inhibitory agents such as aspirin, piroxicam or ticlopidine.
  • thrombin inhibitors such as boropeptides, hirudin or argatroban.
  • the compounds of the present invention may also be combined or co-administered with combinations of the foregoing ents and/or with other therapeutic agents.
  • Such combination products may be employed to achieve synergistic effects or effects additive to those provided by the compounds of the present invention, such as, for example, in such uses as described above, particularly in the treatment, including prevention, of thromboembolic disorders.
  • the GPIIb/IIIa antagonists of the present invention inhibit platelet aggregation at the final common pathway required for platelet aggregation induced by any of the known platelet activators or even their combinations.
  • platelet granular secretions of various important biomolecules from the a-granule (PAI-1) or the dense granule (serotonin) are not affected by the GPIIb/IIIa antagonist.
  • PAI-1 a-granule
  • Serotonin dense granule
  • the GPIIb/IIIa antagonists of the present invention with high affinity for the platelet GPIIb/IIIa receptor are expected to be very effective not only in preventing thrombosis formation, but also in accelerating lysis of platelet rich thrombi, thereby providing a greater utility of such antiplatelet agents in the acute and chronic thromboembolic disorders.
  • Such a strategy may be an effective adjunct therapy with thrombolytic therapy. Indeed, platelet activation after thrombolytic therapy may have a significant role in the delay of reperfusion and abrupt closure (reocclusion).
  • anti-coagulant agents denotes agents that inhibit blood coagulation.
  • agents include warfarin, heparin, or low molecular weight heparin (LMWH), including pharmaceutically acceptable salts or prodrugs thereof.
  • LMWH low molecular weight heparin
  • the preferable anti-coagulant agents are warfarin or heparin or LMWH.
  • the warfarin employed herein may be, for example, crystalline warfarin or amorphous sodium warfarin.
  • the heparin employed herein may be, for example, the sodium or sulfate salts thereof.
  • anti-platelet agents denotes agents that inhibit platelet function such as by inhibiting the aggregation, adhesion or granular secretion of platelets.
  • agents include the various known non-steroidal anti-inflammatory drugs (NSAIDS) such as aspirin, ibuprofen, naproxen, sulindac, indomethacin, mefenamate, droxicam, diclofenac, sulfinpyrazone, and piroxicam, including pharmaceutically acceptable salts or prodrugs thereof.
  • NSAIDS non-steroidal anti-inflammatory drugs
  • ASA acetylsalicyclic acid
  • piroxicam which exerts its anti-platelet effect when dosed once daily, are preferred compounds, especially aspirin.
  • Piroxicam is commercially available from Pfizer Inc. (New York, NY), as FELDANETM.
  • Other suitable anti-platelet agents include ticlopidine, including pharmaceutically acceptable salts or prodrugs thereof. Ticlopidine is also a preferred compound since it is known to be gentle on the gastro-intestinal tract in use.
  • Still other suitable platelet inhibitory agents include thromboxane-A2-receptor antagonists and thromboxane-A2-synthetase inhibitors, as well as pharmaceutically acceptable salts or prodrugs thereof.
  • thrombin inhibitors denotes inhibitors of the serine protease thrombin.
  • various thrombinmediated processes such as thrombin-mediated platelet activation (that is, for example, the aggregation of platelets, and/or the granular secretion of plasminogen activator inhibitor-1 and/or serotonin) and/or fibrin formation are disrupted.
  • Such inhibitors include boropeptides, hirudin and argatroban, including pharmaceutically acceptable salts and prodrugs thereof.
  • the thrombin inhibitors are boropeptides.
  • boropeptides N-acetyl and peptide derivatives of boronic acid, such as C-terminal a-aminoboronic acid derivatives of lysine, ornithine, arginine, homoarginine and corresponding isothiouronium analogs thereof.
  • hirudin includes suitable derivatives or analogs of hirudin, referred to herein as hirulogs, such as disulfatohirudin.
  • Preferable boropeptide thrombin inhibitors include compounds described in Kettner et al., U.S. Patent No. 5,187,157 and European Patent Application Publication Number 293 881 A2, the disclosures of which are hereby incorporated herein by reference.
  • Other suitable boropeptide thrombin inhibitors include those disclosed in PCT Application Publication Number 92/07869 and European Patent Application Publication Number 471 651 A2, the disclosures of which are hereby incorporated herein by reference, in their entirety.
  • thrombolytics or fibrinolytic agents (or thrombolytics or fibrinolytics), as used herein, denotes agents that lyse blood clots (thrombi).
  • agents include tissue plasminogen activator, anistreplase, urokinase or streptokinase, including pharmaceutically acceptable salts or prodrugs thereof.
  • Tissue plasminogen activator (tPA) is commercially available from Genentech Inc., South San Francisco, California.
  • anistreplase refers to anisoylated plasminogen streptokinase activator complex, as described, for example, in European Patent Application No.
  • urokinase is intended to denote both dual and single chain urokinase, the latter also being referred to herein as prourokinase.
  • Combination products where the cyclic compounds of the invention are combined or co-administered with suitable anti-coagulant agents, antiplatelet agents, thrombin inhibitors, and/or thrombolytic agents, may afford an efficacy advantage over the compounds and agents alone, and may do so while permitting the use of lower doses of each.
  • a lower dosage minimizes the potential of side effects, thereby providing an increased margin of safety.
  • ASA acetylsalicylic acid
  • ASA platelet function
  • ticlopidine alone has also been demonstrated to have efficacy in treating TIAs.
  • thrombin inhibitors such as boropeptides
  • studies have demonstrated that such compounds provide excellent candidates for the control of thrombinmediated processes.
  • Studies with hirudin, another thrombin inhibitor, have shown this agent to be an effective compound in the treatment of venous and arterial thrombosis.
  • thrombolytics such as plasminogen activators such as tPA, streptokinase, or urokinase. These standard thrombolytics, when employed alone, promote the generation of plasmin, which degrades platelet-rich fibrin clots.
  • Thromboembolic disorders are known, however, to have a diverse pathophysiological makeup. There is a need for a therapeutic approach to the treatment of these disorders which takes into account the diverse pathophysiological makeup of such diseases, and which includes components ameliorating each of the various pathophysiological aspects.
  • an anti-coagulant agent such as warfarin or heparin
  • an antiplatelet agent such as aspirin, piroxicam or ticlopidine
  • a thrombin inhibitor such as a boropeptide, hirudin or argatroban
  • a thrombolytic agent such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations thereof, in combination with a novel
  • an anticoagulant agent and a compound of this invention or an anti-platelet agent and a compound of this invention, or a thrombin inhibitor and a compound of this invention, or a thrombolytic agent and a compound of this invention, or combinations thereof, are directed to meeting these, as well as other, needs.
  • GPIIb/IIIa is known to be overexpressed in metastatic tumor cells.
  • the compounds or combination products of the present invention may also be useful for the treatment, including prevention, of metastatic cancer.
  • the compounds described above as inhibitors of glycoprotein Ilb/IIIa, also find utility in the prevention and/or treatment of osteoporosis.
  • the vitronectin receptor is known to recognize the RGD- sequence motif of bone matrix proteins and is likely to bind to one or more substrates in bone.
  • osteoporosis The majority of bone diseases, including osteoporosis, can be characterized as bone loss which results directly from an imbalance in the rates of bone resorption and bone formation, which together comprise the dynamic process of bone remodeling. This process is performed by specialized cells, and the resorption aspect of it is carried out by osteoclasts, which attach to the surface of mineralized bone matrix and resorb bone. Osteoclast attachment to bone is mediated through cell surface adhesion receptors called integrins, and one osteoclast integrin has been identified as the vitronectin receptor, or a v b 3 ; Davies et al . (1989) J. Cell Biol. 109:1817-1826.
  • RGD-containing proteins have facilitated investigation of the role of RGD-interactive adhesion molecules, and in particular a 49-amino acid protein, echistatin, has been found to bind to the av ⁇ like integrin expressed in osteoclasts, and it has been shown in vitro that echistatin inhibits excavation of bone by rat osteoclasts; Sato et al. (1990) J. Cell Biol. 111:1713-1723.
  • the present invention also provides compounds, kits comprising said compounds, detectably labeled compounds, and methods for the diagnosis and detection of tumors in an individual.
  • the provided compounds are compounds of Formula (I) that are conjugated to a chelating moiety
  • conjugated compounds are compounds of Formula (I) that have been directly labeled and conjugated compounds that have been labeled.
  • the method for detection of tumors is a substantially non-invasive method using the detectably labeled compounds
  • any variable for example, R 1 through R 8 , m, n, p, X, Y, etc.
  • its definition on each occurrence is independent of its definition at every other occurrence.
  • said group may optionally be substituted with up to two R 11 and R 11 at each occurrence is selected independently from the defined list of possible R 11 .
  • each of the two R 13 substituents on N is independently selected from the defined list of possible R 13 .
  • stable compound or “stable structure” is meant herein a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and formulation into an efficacious therapeutic agent.
  • substituted means that any one or more hydrogen on the designated atom is replaced with a selection from the indicated group, provided that the designated atom's normal valency is not exceeded, and that the substitution results in a stable compound.
  • 2 hydrogens on the atom are replaced.
  • alkyl is intended to include both branched and straight-chain saturated aliphatic hydrocarbon groups having the specified number of carbon atoms;
  • alkoxy represents an alkyl group of indicated number of carbon atoms attached through an oxygen bridge;
  • cycloalkyl is intended to include saturated ring groups, including mono-,bi- or poly-cyclic ring systems, such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl and adamantyl; and
  • biycloalkyl is intended to include saturated bicyclic ring groups such as [3.3.0]
  • Alkenyl is intended to include hydrocarbon chains of either a straight or branched configuration and one or more unsaturated carbon-carbon bonds which may occur in any stable point along the chain, such as ethenyl, propenyl and the like; and "alkynyl” is intended to include hydrocarbon chains of either a straight or branched configuration and one or more triple carbon-carbon bonds which may occur in any stable point along the chain, such as ethynyl, propynyl and the like.
  • boronic acid means a group of the formula -B(R 34 )(R 35 ), wherein R 34 and R 35 are independently selected from: -OH; -F; -NR 13 R 14 ; or C 1 -C 8 -alkoxy; or R 34 and R 35 can alternatively be taken together to form: a cyclic boron ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-4 heteroatoms independently selected from N, S, or O; a divalent cyclic boron amide where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-4 heteroatoms independently selected from N, S, or O; a cyclic boron amide-ester where said chain or ring contains from 2 to 20 carbon atoms and, optionally, 1-4 heteroatoms independently selected from N, S, or O.
  • Such cyclic boron esters, boron amides, or boron amide-esters may also be taken together to
  • Boron esters include boronic acid protecting groups, including moieties derived from diols, for example pinanediol and pinacol to form pinanediol boronic acid ester and the pinacol boronic acid, respectively.
  • diols useful for deriving boronic acid esters are perf luoropinacol, ethylene glycol, diethylene glycol, 1,2-ethanediol, 1,3-propanediol, 1,2-propanediol, 1,2-butanediol, 1,4-butanediol, 2,3-butanediol, 2,3-hexanediol, 1,2-hexanediol, catechol, 1,2-diisopropylethanediol, 5,6-decanediol, 1,2-dicyclohexylethanediol.
  • Halo or "halogen” as used herein refers to fluoro, chloro, bromo and iodo; and "counterion” is used to represent a small, negatively charged species such as chloride, bromide, hydroxide, acetate, sulfate and the like.
  • aryl or “aromatic residue” is intended to mean phenyl or naphthyl.
  • carbocycle or “carbocyclic residue” is intended to mean any stable 3- to 7- membered monocyclic or bicyclic or 7- to 14-membered bicyclic or tricyclic or an up to 26 -membered polycyclic carbon ring, any of which may be saturated, partially unsaturated, or aromatic.
  • carbocyles include, but are not limited to, cyclopropyl, cyclopentyl, cyclohexyl, phenyl, biphenyl, naphthyl, indanyl, adamantyl, or tetrahydronaphthyl (tetralin).
  • heterocycle or “heterocyclic ring system” is intended to mean a stable 5- to 7- membered monocyclic or bicyclic or 7- to 10-membered bicyclic heterocyclic ring which may be saturated, partially unsaturated, or aromatic, and which consists of carbon atoms and from 1 to 4 heteroatoms selected independently from the group consisting of N, O and S and wherein the nitrogen and sulfur heteroatoms may optionally be oxidized, and the nitrogen may optionally be quaternized, and including any bicyclic group in which any of the above-defined heterocyclic rings is fused to a benzene ring.
  • the heterocyclic ring may be attached to its pendant group at any heteroatom or carbon atom which results in a stable structure.
  • the heterocyclic rings described herein may be substituted on carbon or on a nitrogen atom if the resulting compound is stable.
  • Examples of such heterocycles include, but are not limited to, benzopyranyl, thiadiazine, tetrazolyl, benzofuranyl, benzothiophenyl, indolene, quinoline, isoquinolinyl or benzimidazolyl, piperidinyl, 4-piperidone, 2-pyrrolidone, tetrahydrofuran, tetrahydroquinoline, tetrahydroisoquinoline, decahydroquinoline, octahydroisoquinoline, azocine, triazine (including 1,2,3-, 1,2,4-, and 1,3,5-triazine), 6H-1,2,5-thiadiazine, 2H,6H-1,5,2-d
  • fused ring and spiro compounds containing, for example, the above heterocycles are fused ring and spiro compounds containing, for example, the above heterocycles.
  • any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino or sulfhydryl means any group bonded to an O, N, or S atom, respectively, which is cleaved from the O, N, or S atom when the compound is administered to a mammalian subject to provide a compound having a remaining free hydroxyl, amino, or sulfhydryl group, respectively.
  • Examples of groups that, when administered to a mammalian subject, are cleaved to form a free hydroxyl, amino or sulfhydryl include but are not limited to, C 1 -C 6 alkyl substituted with 0-3 R 11 , C 3 -C 6 alkoxyalkyl substituted with 0-3 R 11 , C 1 -C 6 alkylcarbonyl substituted with 0-3 R 11 , C 1 -C 6 alkoxycarbonyl substituted with 0-3 R 11 , C 1 -C 6 alkylaminocarbonyl substituted with 0-3 R 11 , benzoyl substituted with 0-3 R 12 , phenoxycarbonyl substituted with 0-3 R 12 , phenylaminocarbonyl substituted with 0-3 R 12 .
  • Examples of groups that, when administered to a mammalian subject, are cleaved to form a free hydroxyl, amino or sulfhydryl include hydroxy, amine or
  • carboxylate protecting group means any group known in the art of organic synthesis for the protection of carboxylate groups, examples of which include, but are not limited to, the following: 1) alkyl esters such as C 1 to C 8 alkyl, C 5 to C 8 cycloalkylalkyl and t-butyl; 2) aryl esters such as benzyl, substituted benzyl, triphenylmethyl, diphenylmethyl, pentamethylbenzyl, tetramethylbenzyl, and trimethylbenzyl; 3) esters which can be cleaved by acidolysis, mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters; 4) other protecting groups such as CH 2 CH 2 CN, trialkylsilyl, phthalimidomethyl, anthrylmethyl, phenylfluorenyl, 4-picolyl and phenacyl.
  • alkyl esters such as C 1 to C 8 alkyl, C 5 to
  • amine protecting group means any group known in the art of organic synthesis for the protection of amine groups. Such amine protecting groups include those listed in Greene, “Protective Groups in Organic Synthesis” John Wiley & Sons, New York (1981) and “The Peptides: Analysis, Sythesis, Biology, Vol. 3, Academic Press, New York (1981), the disclosure of which is hereby incorporated by reference. Any amine protecting group known in the art can be used. Examples of amine protecting groups include, but are not limited to, the following: 1) acyl types such as formyl.
  • Mbs-NR 2 4-methoxybenzene-sulfonamide
  • Mts-NR 2 2,4, 6-trimethylbenzenesulf onamide
  • iMds-NR 2 2,6-dimethoxy-4-methoxybenzenesulfonamide
  • Pmc-NR 2 2,2,5,7,8-pentamethylchroman-6-sulfonamide
  • acyl groups such as azidobenzoyl, p-benzoylbenzoyl, o-benzylbenzoyl, p-acetylbenzoyl, dansyl, glycyl-p- benzoylbenzoyl, phenylbenzoyl, m-benzoylbenzoyl, benzoylbenzoyl.
  • pharmaceutically acceptable salts refer to derivatives of the disclosed compounds wherein the parent compound of formula (I) is modified by making acid or base salts of the compound of formula (I).
  • pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and the like.
  • Prodrugs are considered to be any covalently bonded carriers which release the active parent drug according to formula (I) in vivo when such prodrug is administered to a mammalian subject.
  • Prodrugs of the compounds of formula (I) are prepared by modifying functional groups present in the compounds in such a way that the modifications are cleaved, either in routine manipulation or in vi vo , to the parent compounds.
  • Prodrugs include compounds of formula (I) wherein hydroxy, amine, or sulfhydryl groups are bonded to any group that, when administered to a mammalian subject, cleaves to form a free hydroxyl, amino, or sulfhydryl group, respectively.
  • Examples of prodrugs include, but are not limited to, acetate, formate and benzoate derivatives of alcohol and amine functional groups in the compounds of formula (I); and the like.
  • compositions of the compounds of the invention can be prepared by reacting the free acid or base forms of these compounds with a ⁇ toichiometric amount of the appropriate base or acid in water or in an organic solvent, or in a mixture of the two; generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol, or acetonitrile are preferred. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, PA, 1985, p. 1418, the disclosure of which is hereby incorporated by reference.
  • amino acid as used herein means an organic compound containing both a basic amino group and an acidic carboxyl group. Included within this term are modified and unusual amino acids, such as those disclosed in, for example, Roberts and Vellaccio (1983) The Peptides, 5: 342-429, the teaching of which is hereby incorporated by reference.
  • Modified or unusual amino acids which can be used to practice the invention include, but are not limited to, D-amino acids,hydroxylysine, 4-hydroxyproline, ornithine, 2,4-diaminobutyric acid, homoarginine, norleucine,N-methylaminobutyric acid, naphthylalanine, phenylglycine, ⁇ -phenylproline, tert-leucine, 4-aminoeyelohexylalanine, N-methyl-norleucine,
  • amino acid residue means that portion of an amino acid (as defined herein) that is present in a peptide.
  • peptide as used herein means a linear compound that consists of two or more amino acids
  • peptide also includes compounds containing both peptide and non-peptide components, such as pseudopeptide or peptide mimetic residues or other non-amino acid components. Such a compound containing both peptide and non-peptide components may also be referred to as a "peptide analog”.
  • a “pseudopeptide” or “peptide mimetic” is a compound which mimics the structure of an amino acid residue or a peptide, for example, by using linking groups other than amide linkages between the peptide mimetic and an amino acid residue (pseudopeptide bonds) and/or by using non-amino acid substituents and/or a modified amino acid residue.
  • a "pseudopeptide residue” means that portion of an pseudopeptide or peptide mimetic (as defined herein) that is present in a peptide.
  • peptide bond means a covalent amide linkage formed by loss of a molecule of water between the carboxyl group of one amino acid and the amino group of a second amino acid.
  • peptide bonds includes peptide bond isosteres which may be used in place of or as substitutes for the normal amide linkage. These substitute or amide "equivalent” linkages are formed from combinations of atoms not normally found in peptides or proteins which mimic the spatial requirements of the amide bond and which should stabilize the molecule to enzymatic degradation by numerous endogenous peptides of various types .
  • the compounds of the present invention can be prepared in a number of ways well known to one skilled in the art of organic synthesis. Preferred methods include but are not limited to those methods described below.
  • Phg phenylglycine
  • Trp tryptophan
  • the compounds of the present invention can be synthesized using standard synthetic methods known to those skilled in the art. Preferred methods include but are not limited to those methods described below.
  • peptides are elongated by deprotecting the a-amine of the C-terminal residue and coupling the next suitably protected amino acid through a peptide linkage using the methods described. This deprotection and coupling procedure is repeated until the desired sequence is obtained.
  • This coupling can be performed with the constituent amino acids in a stepwise fashion, or condensation of fragments (two to several amino acids), or combination of both processes, or by solid phase peptide synthesis according to the method originally described by Merrifield, J. Am. Chem. Soc, 85, 2149-2154 (1963), the disclosure of which is hereby incorporated by reference.
  • the compounds of the invention may also be synthesized using automated peptide synthesizing equipment.
  • procedures for peptide synthesis are described in Stewart and Young, "Solid Phase Peptide Synthesis", 2nd ed, Pierce Chemical Co., Rockford, IL (1984); Gross, Meienhofer, Udenfriend, Eds., "The Peptides: Analysis, Synthesis, Biology, Vol. 1, 2, 3, 5, and 9, Academic Press, New York, (1980-1987); Bodanszky, "Peptide Chemistry: A Practical Textbook", Springer-Verlag, New York (1988); and Bodanszky et al. "The Practice of Peptide Sythesis” Springer-Verlag, New York (1984), the disclosures of which are hereby incorporated by reference.
  • the coupling between two amino acid derivatives, an amino acid and a peptide, two peptide fragments, or the cyclization of a peptide can be carried out using standard coupling procedures such as the azide method, mixed carbonic acid anhydride (isobutyl chloroformate) method, carbodiimide (dicyclohexylcarbodiimide, diisopropylcarbodiimide, or water-soluble carbodiimides) method, active ester (p-nitrophenyl ester, N-hydroxysuccinic imido ester) method, Woodward reagent K method, carbonyldiimidazole method, phosphorus reagents such as BOP-Cl, or oxidation-reduction method. Some of these methods (especially the carbodiimide) can be enhanced by the addition of 1-hydroxybenzotriazole. These coupling reactions may be performed in either solution (liquid phase) or solid phase.
  • the functional groups of the constituent amino acids must be protected during the coupling reactions to avoid undesired bonds being formed.
  • the protecting groups that can be used are listed in Greene, "Protective Groups in Organic Synthesis” John Wiley & Sons, New York (1981) and "The Peptides: Analysis, Sythesis, Biology, Vol. 3, Academic Press, New York (1981), the disclosure of which is hereby incorporated by reference.
  • the a-carboxyl group of the C-terminal residue is usually protected by an ester that can be cleaved to give the carboxylic acid.
  • These protecting groups include: 1) alkyl esters such as methyl and t-butyl, 2) aryl esters such as benzyl and substituted benzyl, or 3) esters which can be cleaved by mild base treatment or mild reductive means such as trichloroethyl and phenacyl esters.
  • an insoluble carrier usually polystyrene
  • insoluble carriers contain a group which will react with the carboxyl group to form a bond which is stable to the elongation conditions but readily cleaved later.
  • examples of which are: oxime resin (DeGrado and Kaiser (1980) J. Org. Chem. 45, 1295-1300) chloro or bromomethyl resin, hydroxymethyl resin, and aminomethyl resin.
  • oxime resin DeGrado and Kaiser (1980) J. Org. Chem. 45, 1295-1300
  • chloro or bromomethyl resin hydroxymethyl resin
  • aminomethyl resin aminomethyl resin.
  • Many of these resins are commercially available with the desired C-terminal amino acid already incorporated.
  • acyl types such as formyl, trifluoroacetyl, phthalyl, and p-toluenesulfonyl
  • aromatic carbamate types such as benzyloxycarbonyl (Cbz) and substituted benzyloxycarbonyls, 1-(p-biphenyl)-1-methylethoxycarbonyl, and 9-fluorenylmethyloxycarbonyI
  • aliphatic carbamate types such as tert- butyl oxy carbony 1 (Boc), ethoxycarbonyl, diisopropylmethoxycarbonyl, and allyloxycarbonyl; 4) cyclic alkyl carbamate types such as cyclopentyloxycarbonyl and adamantyloxycarbonyl; 5) alkyl types such as triphenylmethyl and benzyl; 6) trialkylsilane such as trimethylsilane; and 7) thiol containing types such as phenylthiocarbonyl and dithiasuccinoyl.
  • the preferred a-amino protecting group is either Boc or Fmoc. Many amino acid derivatives suitably protected for peptide synthesis are commercially available.
  • the a-amino protecting group is cleaved prior to the coupling of the next amino acid.
  • the methods of choice are trifluoroacetic acid, neat or in dichloromethane, or HCl in dioxane.
  • the resulting ammonium salt is then neutralized either prior to the coupling or in situ with basic solutions such as aqueous buffers, or tertiary amines in dichloromethane or dimethylformamide.
  • the reagents of choice are piperidine or substituted piperidines in dimethylformamide, but any secondary amine or aqueous basic solutions can be used.
  • the deprotection is carried out at a temperature between 0 °C and room temperature.
  • any of the amino acids bearing side chain functionalities must be protected during the preparation of the peptide using any of the above-identified groups.
  • Those skilled in the art will appreciate that the selection and use of appropriate protecting groups for these side chain functionalities will depend upon the amino acid and presence of other protecting groups in the peptide. The selection of such a protecting group is important in that it must not be removed during the deprotection and coupling of the a-amino group.
  • Boc when Boc is chosen for the a-amine protection the following protecting groups are acceptable: p-toluenesulfonyl (tosyl) moieties and nitro for arginine; benzyloxycarbonyl, substituted benzyloxycarbonyls, or tosyl for lysine; benzyl or alkyl esters such as cyclopentyl for glutamic and aspartic acids; benzyl ethers for serine and threonine; benzyl ethers, substituted benzyl ethers or 2-bromobenzyloxycarbonyl for tyrosine; p-methylbenzyl, p-methoxybenzyl, acetamidomethyl, benzyl, or t-butylsulfonyl for cysteine; and the indole of tryptophan can either be left unprotected or protected with a formyl group.
  • tert-butyl based protecting groups are acceptable.
  • Boc can be used for lysine, tert-butyl ether for serine, threonine and tyrosine, and tert-butyl ester for glutamic and aspartic acids.
  • the peptide When a solid phase synthesis is used, the peptide should be removed from the resin without simultaneously removing protecting groups from functional groups that might interfere with the cyclization process. Thus, if the peptide is to be cyclized in solution, the cleavage conditions need to be chosen such that a free a-carboxylate and a free a-amino group are generated without simultaneously removing other protecting groups. Alternatively, the peptide may be removed from the resin by hydrazinolysis, and then coupled by the azide method.
  • Another very convenient method involves the synthesis of peptides on an oxime resin, followed by intramolecular nucleophilic displacement from the resin, which generates a cyclic peptide (Osapay, Profit, and Taylor (1990) Tetrahedron Let t ers 43, 6121-6124).
  • the Boc protection scheme is generally chosen.
  • the preferred method for removing side chain protecting groups generally involves treatment with anhydrous HF containing additives such as dimethyl sulfide, anisole, thioanisole, or p-cresol at 0 °C.
  • the cleavage of the peptide can also be accomplished by other acid reagents such as trifluoromethanesulfonic acid/trifluoroacetic acid mixtures.
  • Unusual amino acids used in this invention can be synthesized by standard methods familiar to those skilled in the art ("The Peptides: Analysis, Sythesis, Biology, Vol. 5, pp. 342-449, Academic Press, New York (1981)). N-Alkyl amino acids can be prepared using procedures described previously (Cheung et al., (1977) Can . J. Chem. 55, 906; Freidinger et al., (1982) J. Org. Chem. 48, 77 (1982)), which are incorporated here by reference.
  • the compounds of the present invention may be prepared using the procedures further detailed below.
  • HBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
  • TBTU 2-(1H-Benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate
  • NMM N-methylmorpholine
  • Abu D-2-aminobutyric acid
  • DIEA trimethylacetylchloride
  • DIEA diisopropylethylamine
  • 3-cyanobenzoic acid and [2-(tert-butyloxycarbonyloxylimino)-phenylacetonitrile] (Boc-ON) were purchased from Aldrich Chemical Company.
  • Dimethylformamide (DMF), ethyl acetate, chloroform (CHCI 3 ), methanol (MeOH), pyridine and hydrochloric acid (HCl) were obtained from Baker.
  • Acetonitrile, dichloromethane (DCM), acetic acid (HOAc), trifluoroacetic acid (TFA), ethyl ether, triethylamine, acetone, and magnesium sulfate were purchased from EM Science.
  • Palladium on carbon catalyst (10% Pd) was purchased from Fluka Chemical Company. Absolute ethanol was obtained from Quantum Chemical Corporation.
  • Thin layer chromatography (TLC) was performed on Silica Gel 60 F254 TLC plates (layer thickness 0.2 mm) which were purchased from EM Separations.
  • TLC visualization was accomplished using UV light, iodine, and/or ninhydrin spray. Melting points were determined using a Thomas Hoover or Electrothermal 9200 melting point apparatus and are uncorrected. HPLC analyses were performed on either a Hewlett Packard 1090, Waters Delta Prep 3000, Rainin, or DuPont 8800 system. NMR spectra were recorsent.c QE-300, Varian 300, or Varian 400 spectrometer. Fast atom bombardment mass spectrometry
  • Acid Derivatives 3 and 4-substituted Boc-aminomethylbenzoic acid derivatives useful as intermediates in the synthesis of the compounds of the invention are prepared using standard procedures, for example, as described in Tett. Lett., 4393 (1975); Modern Synthetic Reactions , H.O. House (1972); or Harting et al. J. Am. Chem. Soc , 50: 3370 (1928), and as shown schematically below.
  • 3-Cyanobenzoic acid (10.0 g, 68 mmol) was dissolved in 200 ml ethanol by heating in a 35-50 C water bath. Concentrated HCI (6.12 ml, 73 mmol) was added and the solution was transferred to a 500 ml nitrogen-flushed round bottom flask containing palladium on carbon catalyst (1.05 g, 10% Pd/C). The suspension was stirred under an atmosphere of hydrogen for 38 hours, filtered through a scintered glass funnel, and washed thoroughly with H 2 O. The ethanol was removed under reduced pressure and the remaining aqueous layer, which contained a white solid, was diluted to 250 ml with additional H 2 O.
  • the title compound can be prepared according to standard procedures, for examples, as disclosed in Olsen, J. Org. Chem. (1970) 35: 1912), and as shown schematically below.
  • 2-Aminomethylbenzoic acid ⁇ HCI and 2- aminomethylphenylacetic acid -HCI useful as intermediates in the synthesis of the compounds of the invention are prepared using standard procedures, for example, as described in Naito et al J. Antibiotics, 30: 698 (1977); or Young and Sweet J. Am. Chem. Soc, 80: 800 (1958), and as shown schematically below.
  • 4-phenylbutyric acid (1) was converted to the ethyl ester (2) which was acylated via aluminum chloride and acetylchloride to give 4-acetylphenylbutyric acid ethyl ester (3).
  • This ester was subjected to saponification to give 4-acetylphenylbutyric acid (4).
  • the acetyl group was oxidized to give 4-carboxyphenylbutyric acid (5) which was converted to the 1-tetralin-7-carboxylic acid (6) using aluminum chloride in a Friedel-Crafts cyclization with resonably high yield.
  • the tetralone was split into two portions and some was converted to the oxime (7) using sodium acetate and hydroxylamine hydrochloride.
  • the oxime was subjected to hydrogenolysis to give the racemic mixture of 8-amino- 5,6,7,8-tetrahydro-2-naphthoic acid as the hydrochloride
  • Part E A mixture of 4-carboxyphenylbutyric acid (10.40 g, 0.05 mol), aluminum chloride (33.34 g, 0.25 mol) and sodium chloride (2.90 g, 0.05 mol) was heated with continual stirring to 190°C over 30 minutes. As the mixture cooled to 60°C, cold hydrochloric acid (1N, 250 mL) was carefully added. The mixture was extracted with dichloromethane. The combined organic layers were backwashed with dilute hydrochloric acid and water, dried over anhydrous magnesium sulfate and evaporated to dryness under reduced pressure.
  • Part F A solution of 1-tetralon-7-carboxylic acid (1.0 g, 0.0053 mol) and sodium acetate (1.93 g, 0.024 mol) and hydroxylamine hydrochloride (1.11 g, 0.016 mol) in a mixture of methanol and water (1:1, 15 mL) was stirred at reflux over 4 hours. The mixture was cooled and then added was more water (50 mL).
  • Part G A mixture of 1-tetralonoxime-7-carboxylic acid (0.75 g, 0.0037 mol) in methanol (25 mL) with concentrated hydrochloric acid (0.54 mL, 0.20 g, 0.0056 mol) and palladium on carbon catalyst (0.10 g, 5% Pd/C) was shaken for 20 hours at ambient temperature under an atmosphere of hydrogen (60 psi). The reaction mixture was filtered over Celite @ and washed with methanol.
  • the remaining tetralone was then converted to the methyl ester (9).
  • the tetralone methyl ester (9) was converted, first, to the cyanohydrin by treatment with trimethylsilylcyanide and zinc iodide and then, via the in si tu dehydration with phosphorous oxychloride in pyridine, to the methyl 8-cyano-5, 6-dihydro-2-naphthoate (11).
  • This naphthoate was divided into two portions and some was subjected to hydrogenolysis, N-BOC-protection and saponification to give N-(BOC)-8-aminomethyl-5,6,7,8-tetrahydro-2-naphthoic acid (12) as an intermediate for incorporation into the cyclic peptide.
  • Part C A mixture of methyl 8-cyano-5,6-dihydro-2-naphthoate (0.80 g, 0.0038 mol) in methanol (25 mL) with concentrated hydrochloric acid (0.56 mL) and palladium on carbon catalyst (0.40 g, 5% Pd/C) was shaken for 20 hours at ambient temperature under an atmosphere of hydrogen (50 psi). The reaction mixture was filtered over Celite and washed with methanol.
  • Part D A solution of methyl 8-aminomethyl-5,6,7,8-tetrahydro-2-naphthoate (0.78 g, 0.0036 mol) and triethylamine (0.55 mL, 0.40 g, 0.004 mol) in aqueous tetrahydrofuran (50%, 75 mL) was added, portionwise as a so l i d , 2 - (tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (0.99 g, 0.004 mol). All was stirred at ambient temperature over 3 hours. The solution was concentrated to half volume and extracted with diethylether.
  • the aqueous layer was then acidified to a pH of 1.0 using hydrochloric acid (1N) and then extraced with ethyl acetate.
  • the combined organic layers were dried over anhydrous magnesium sulfate and evaporated to dryness under reduced pressure.
  • the remaining naphthoate (11) was treated with 2 , 3-dichloro-5, 6-dicyano-l, 4-benzoquinone (DDQ) in dioxane to aromatize the adjacent ring to give the methyl 8-cyano-2-naphthoate (13) .
  • DDQ 4-benzoquinone
  • the nitrile was reduced via hydrogentation and the methyl ester saponified to the carboxylic acid.
  • This acid was then N- BOC-protected to give N- (BOC) -8-aminomethyl-2-naphthoic acid (14) as an intermediate for incorporation into the cyclic peptide.
  • Part B A mixture of methyl 8-cyano-2-naphthoate (1.0 g, 0.0047 mol) in methanol (35 mL) with concentrated hydrochloric acid (0.69 mL) andpalladium on carbon catalyst (0.20 g, 5% Pd/C) was shaken for 6 hours at ambient temperature under anatmosphere of hydrogen (50 psi). The reaction mixture was filtered over Celite ® and washed with methanol. The filtrate was evaporated to dryness under reduced pressure and the residue was triturated with hexane to give methyl 8-aminomethyl-2-naphthoate- (0.76 g, 0.0035 mol, 75%) as an oil. 1 H NMR
  • Part D A solution of 8-aminomethyl-2-naphthoic acid (0.50 g, 0.00025 mol) and triethylamine (0.038 mL, 0.028 g, 0.000275 mol) in aqueous tetrahydrofuran (50%, 5 mL) was added, portionwise as a solid, 2-(tert-butoxycarbonyloxyimino)-2-phenylacetonitrile (0.068 g, 0.000275 mol). All was stirred at ambient temperature over 5 hours. The solution was concentrated to half volume and extracted with diethylether.
  • Coupling times range from 15 to 96 hours.
  • the substitution level is then determined using either the picric acid test (Sarin, Kent, Tarn, and Merrifield,
  • NMM based on the amount of amino acid used
  • the coupling times range from 1 hour to several days.
  • the completeness of coupling is monitored by qualitative ninhydrin assay, or picric acid assay in cases where the amino acid was coupled to a secondary amine. Amino acids are recoupled if necessary based on these results.
  • the N-terminal Boc group is removed by treatment with 25% TFA in DCM for 30 minutes.
  • the resin is then neutralized by treatment with 10% DIEA in DCM.
  • Cyclization with concomitant cleavage of the peptide is accomplished using the method of Osapay and Taylor ((1990) J. Am. Chem. So c. , 112, 6046) by suspending the resin in approximately 10 ml/g of DMF, adding one equivalent of HOAc (based on the loading of the first amino acid), and stirring at 50-60 C for 60 to 72 hours.
  • the DMF filtrate is evaporated, redissolved in HOAc or 1:1 acetonitrile: H 2 O, and lyophilized to obtain protected, cyclized material.
  • the material may be dissolved in methanol and precipitated with ether to obtain the protected, cyclized material. This is then treated using standard procedures with anhydrous hydrogen fluoride (Stewart and Young (1984) "Solid Phase Peptide Synthesis", 2nd. edition, Pierce Chemical Co., 85) containing 1 ml/g m-cresol or anisole as scavenger at 0 C for 20 to 60 minutes to remove side chain protecting groups.
  • the crude product may be purified by reversed-phase HPLC using a 2.5 cm preparative Vydac C18 column with a linear acetonitrile gradient containing 0.1% TFA to produce pure cyclized material.
  • the following N-a-Boc-protected amino acids may be used for the syntheses: Boc-Arg(Tos), Boc-N-a-MeArg(Tos), Boc-Gly, Boc-Asp(OcHex), Boc-3-aminomethyl-4-iodo-benzoic acid, Boc-D-Ile, Boc-NMeAsp (OcHex), Boc-NMe-Mamb, Boc-D-Phg, Boc-D-Asp(OBzl), Boc-L-Asp(OcHex), Boc-aMe-Asp(OcHex), Boc-bMe-Asp(OcHex), Boc-L-Ala, Boc-L-Pro, Boc-D-Nle, Boc-D-Leu,
  • trans-5-Boc-aminopent-3-enoic Acid Analogs The preparation of trans-5-Boc-aminopent-3-enoic acid analogs useful as intermediates in the synthesis of the compounds of this invention are accomplished using standard procedures, for example, as described in J. Chem. Soc , Chem. Commun . 1980, 799; J. Chem. Soc. , Perkin Trans . I 1982, 307; Aust . J. Chem. 1985, 38, 1651; Janssen Chim. Acta 1985, 3, 3; J. Org. Chem. 1989, 54 , 4004; Tetrahedron Let t . 1990, 50, 7301; Int . J. Peptide Protein Res . 1991, 38 , 237; J. Org. Chem. 1991, 56, 4370; Tetrahedron Lett . 1992, 33 , 4487, and as shown schematically in Scheme I below.
  • step a vinylmagnesium bromide
  • step b NaBH 4 . CeCl 3 -78 °C
  • step c MeOH, HCI, then diisobutylaluminium hydride, PhMe, -78 °C, then vinylmagnesium bromide, 0 °C
  • step d dihydropyran, PPTs, DCM
  • step e O3, DCM/MeOH, -78 °C, then Zn, AcOH
  • step f (MeO) 2 PCH 2 CO 2 Me, NaH, THF
  • step g PPTs, MeOH
  • step h MsCl, DIEA, DCM, step i, RMgX, cat.
  • CuCN THF.
  • N-Methylation and ester saponification may then be accomplished according to standard procedures (see J. Org. Chem. 1970, 35, 1912, Tetrahedron Lett. 1984, 25, 5831).
  • trans-5-Boc-aminopent-3-ynoic acid analogs useful as intermediates in the synthesis of the compounds of this invention may be accomplished as follows. Treatment of a N-protected a-amino aldehyde (see J. Chem . Soc , Perkin Trans . I 1982, 307; Tetrahedron Let t . 1990, 31 , 1385, Tetrahedron Lett . 1990, 31 , 7359, Int . J. Peptide Protein Res . 1991, 38, 237; J. Org. Chem . 1991, 56, 4370; Tetrahedron Lett .
  • step a (MeO) 2 PCHN 2 , t-BuOK, THF; step b, n-BuLi, -78 °C, MeO 2 CCHR 22 X; step c, LiOH, THF-H 2 O.
  • step a R 10 MgX, THF, 0°C
  • step b Ph 2 P(0)N 3 , DMF
  • step c H 2 , Pd/C, EtOH, CHCI 3 .
  • the title compounds may be prepared via Wittig olefination of amino acid derived amino aldehydes (see
  • the title compound was prepared using the general solid phase peptide synthesis procedure described in BP6543-A.
  • the peptide was prepared on a 0.560 mmol scale to give the protected cyclic peptide (279 mg, 61.3%).
  • the peptide (273 mg) and 273 mL of anisole were treated with anhydrous hydrogen fluoride at 0°C for 20 minutes.
  • the crude material was precipitated with ether, redissolved in 75% aqueous acetonitrile, and lyophilized to generate the title compound (214 mg, > quantitative yield calculated as the HF salt).
  • Protein ligands with specificity for a given receptor can be generated by replacement of a segment of the protein template scaffold with the sequence
  • the receptor bound ligand allows one to choose an appropriate molecular scaffold to display the target sequence.
  • IIb/IIIa is known to recognize the RGD sequence and binds with highest affinity when this sequence is constrained to adopt a turn conformation, Pierschbacher, M.D., Ruoslahti, E., J. Biol. Chem. 262, 17294-17298 (1987).
  • the protein display scaffold chosen should contain a surface accessible loop whose sequence can be replaced with the RGD sequence so that the new hybrid protein will bind to Ilb/IIIa.
  • We and others have used various protein scaffolds to display the RGD sequence including Strp G2, lysozyme Yamada, T. , Matsushima, M. , Inaka, K..,
  • oligonucleotides that have been appropriately designed O'Neil, K.T., DeGrado, W.F., Mou ⁇ a, S.A., Ramachandran, N. and Hoess, R.H., Methods of Enzymology, in press.
  • An appropriate loop for replacement with the RGID sequence was identified by visual inspection of the structure of the StrpG domain, Gronenborn, A. M., Filpula, D.R., Essig, N.Z., Achari, A., Whitlow, M., Wingfield, P.T., Clore, G.M. Science 253, 657-659 (1991).
  • the loop chosen is located between ß strands 3 and 4 as
  • Fig. 1 Residues 56 through 59 of StrpG were replaced with a 10 residue insertion coding for the RGID sequence between cysteine residues that were flanked by glycine residues to insure flexibility of the turn sequence (Gly Cys Phe Arg Gly Ile Asp Trp Cys Gly). Replacement of the loop was achieved using an
  • oligonucleotide cassette that could be inserted between Bgl II and BstE II sites of the wild type gene as described, O 'Neil , K. T. , DeGrado, W. F. , Mousa, S.A. , Ramachandran, N. and Hoess, R . H. , Methods of Enzymology, in press .
  • the Strp G/RGID gene was cloned into an expression vector where overexpression was controlled by the T7 promoter, Sfcudier, F. W. , Rosenberg, A.H. , Dunn, J. J. , and Dubendorff, J. W. , Methods of Enzymology 185, 60-80 (1990) .
  • Fig. 1 Ribbon representation of the backbone of the StrpG domain. The coordinates used to generate the structure were from the Protein Data Bank, accession number 1GB1. In this orientation, the amino terminus is at the bottom of the figure and the loop where RGID insertions were made is adjacent to it, between ⁇ strands 3 and 4. The figure was generated using
  • the peptide was assembled on a Milligen 9050 automated peptide synthesizer.
  • the support used was Fmoc-PAL resin (Millipore, 0.55 g of 0.35 mmol/g) mixed with 2.2 g glass beads.
  • the following amino acid derivatives were used: Fmoc-Cys (Trt) -OPfp, Fmoc-Lys(Boc)-OPfp, Fmoc-Arg(Pmc)-OH, Fmoc-Gly-OPfp, Fmoc-Ile-OPfp, Fmoc-Asp(OtBu)-OPfp, and Fmoc-Trp-OPfp.
  • the peptide N-terminus was acetylated by treatment of the resin with 0.5M acetic anhydride/0.5M pyridine in DMF for 30 minutes.
  • the resin was cleaved using lmL anisole per gram of resin and 10 mL/g of anhydrous hydrogen fluoride.
  • the crude product was obtained by ether precipitation, redissolved in 50% aqueous acetonitrile and lyophilized. Oxidation of the cysteine residues was accomplished by overnight stirring of a 1 mg/mL solution of the peptide in 10 mM ammonium acetate, pH 8.5. The addition of MeOH (30%) may be necessary to enhance solubility.
  • the resulting oxidized peptide may be purified by reversed-phase HPLC (20% recovery) on a preparative Vydac C18 column (2.5 cm) using a 0.23%/min gradient of 18 to 27% acetonitrile containing 0.1% TFA and then lyophilized to afford the di-TFA salt of the title compound.
  • Mass spectrum: M+H 1033.6.
  • the compounds of this invention possess antiplatelet efficacy, as evidenced by their activity in standard platelet aggregation assays or platelet fibrinogen binding assays, as described below.
  • a compound is considered to be active in these assays if it has an IC 50 value of less than about 1 mM.
  • Platelet aggregation and fibrinogen binding assays which may used to demonstrate the antiplatelet activity of the compounds of the invention are described below.
  • Platelet-Fibrinogen Binding Assay Binding of 125 I-fibrinogen to platelets was performed as described by Bennett et al. (1983) Proc. Natl. Acad. Sci. USA 80:2417-2422, with some modifications as described below. Human PRP (h-PRP) was applied to a Sepharose column for the purification of platelet fractions. Aliquots of platelets (5 X 10 8 cells) along with 1 mM calcium chloride were added to removable 96 well plates prior to the activation of the human gel purified platelets (h-GPP).
  • Activation of the human gel purified platelets was achieved using ADP, collagen, arachidonate, epinephrine, and/or thrombin in the presence of the ligand, 125 I-fibrinogen.
  • the 125 i-fibrinogen bound to the activated, platelets was separated from the free form by centrifugation and then counted on a gamma counter.
  • the test compounds were added at various concentrations prior to the activation of the platelets.
  • novel cyclic glycoprotein IIb/IIIa compounds of the invention also possess thrombolytic efficacy, that is, they are capable of lysing (breaking up) already formed platelet-rich fibrin blood clots, and thus are useful in treating a thrombus formation, as evidenced by their activity in the tests described below.
  • Preferred cyclic compounds of the present invention for use in thrombolysis include those compounds having an IC 50 value (that is, the molar concentration of the cyclic compound capable of achieving 50% clot lysis) of less than about 1 mM, more preferably an IC 50 value of less than about 0.1 mM, even more preferably an IC5 0 value of less than about 0.01 mM, still more preferably an IC 50 value of less than about 0.001 mM, and most preferably an IC 50 value of about 0.0005 mM.
  • an IC 50 value that is, the molar concentration of the cyclic compound capable of achieving 50% clot lysis
  • IC 50 determinations may be made using a standard thrombolysis assay, as described below.
  • Another class of preferred thrombolytic compounds of the invention include those compounds which have a Kd of ⁇ 100 nM, preferably ⁇ 10 nM, most preferably 0.1 to 1.0 nM.
  • Thrombolytic Assay Venous blood was obtained from the arm of a healthy human donor who was drug-free and aspirin free for at least two weeks prior to blood collection, and placed into 10 ml citrated Vacutainer tubes. The blood was centrifuged for 15 minutes at 1500
  • PRP platelet rich plasma
  • the suspension was then assayed on a Coulter Counter (Coulter Electronics, Inc., Hialeah, FL), to determine the platelet count at the zero time point.
  • test compounds were added at various concentrations. Test samples were taken at various time points and the platelets were counted using the Coulter Counter. To determine the percent of lysis, the platelet count at a time point subsequent to the addition of the test compound was subtracted from the platelet count at the zero time point, and the resulting number divided by the platelet count at the zero time point. Multiplying this result by 100 yielded the percentage of clot lysis achieved by the test compound. For the IC 50 evaluation, the test compounds were added at various concentrations, and the percentage of lysis caused by the test compounds was calculated.
  • Platelet Granular Secretion Studies The role of the claimed platelet GPIIb/IIIa receptor antagonists on the modulation of platelet granular secretion from the a-granules, dense granules or intracellular Ca +2 binding proteins was examined. This class of compounds did not have any significant effect on platelet granular secretion of plasminogen activator inhibitor type-1 (PAI-1) from a-granules, the mobilization of intracellular calcium stores or the secretion of the vasoconstrictor serotonin from the dense granules.
  • PAI-1 plasminogen activator inhibitor type-1
  • antiplatelet agents such as aspirin or the antithrombin hirudin has been shown to inhibit platelet granular secretion of the antifibrinolytic (PAI-1) or the vasoconstrictor (serotonin) .
  • PAI-1 antifibrinolytic
  • surotonin vasoconstrictor
  • novel cyclic compounds of the invention are also useful in combination products, that is, in pharmaceutical compositions containing the novel cyclic compounds of the invention in combination with anti-coagulant agents such as warfarin or heparin, or antiplatelet agents such as aspirin, piroxicam or ticlopidine, or thrombin inhibitors such as boropeptides, hirudin or argatroban, or thrombolytic agents such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations thereof.
  • anti-coagulant agents such as warfarin or heparin
  • antiplatelet agents such as aspirin, piroxicam or ticlopidine
  • thrombin inhibitors such as boropeptides, hirudin or argatroban
  • thrombolytic agents such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations thereof.
  • h-GPP human gel-purified platelet
  • IC 50 5- 100 nM
  • an enzyme-linked immunosorbent assay (ELISA) using purified GPIIb/IIIa receptors obtained from human platelets the representative compounds of the invention demonstrated direct inhibition of fibrinogen binding to RGD recognition site(s), with an IC 50 of 0.5-10 nM.
  • the inhibitory efficacy of the presently claimed compounds on fibrinogen binding to the platelet GPIIb/IIIa receptor was shown to be related to the number of binding sites, as is evident from the decrease in IC 50 when platelet number was decreased.
  • the Compound of the present invention can be shown to displace l25 I-fibrinogen bound to activated platelets .
  • fibrinogen bound to activated platelets was incubated for 20 minutes prior to the addition of Compound A. This suggests a high affinity for Compound A in displacing fibrinogen from an already formed platelet-rich clot. This effect may explain the lytic efficacy of the compounds of the present invention.
  • Compound A was shown not only to inhibit platelet aggregation induced by agonists, but also to deaggregate platelets after the initiation of aggregation.
  • the deaggregation efficacy of Compound A was dependent on its concentration and the time of addition post-initiation of platelet activation. The earlier the addition of Compound A after the induction of aggregation, the greater its deaggregatory efficacy (Fig. Ia).
  • the cyclic glycoprotein IIb/III antagonist compounds of this invention have also been shown to displace 125 I-fibrinogen bound to activated platelets in a platelet-fibrinogen binding assay similar to the platelet-fibrinogen binding assay previously described.
  • the compounds of this invention can be administered by any means that produces contact of the active agent with the agent's site of action, glycoprotein IIb/IIIa (GPIIb/IIIa), in the body of a mammal. They can be administered by any conventional means available for use in conjunction with pharmaceuticals, either as individual therapeutic agents or in a combination of therapeutic agents, such as a second antiplatelet agent such as aspirin, piroxicam, or ticlopidine which are agonist-specific, or an anti-coagulant such as warfarin or heparin, or a thrombin inhibitor such as a boropeptide, hirudin or argatroban, or a thrombolytic agent such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations thereof.
  • the compounds of the invention, or compounds of the invention in combination with other therapeutic agents can be administered alone, but generally administered with a pharmaceutical carrier selected on the basis of the chosen route of administration and standard pharmaceutical
  • the dosage of the novel cyclic compounds of this invention administered will, of course, vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent and its mode and route of administration; the age, health and weight of the recipient; the nature and extent of the symptoms; the kind of concurrent treatment; the frequency of treatment; and the effect desired.
  • a daily dosage of active ingredient can be expected to be about 0.01 to 10 milligrams per kilogram of body weight.
  • Dosage forms contain from about 1 milligram to about 100 milligrams of active ingredient per unit.
  • the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.
  • the active ingredient can be administered orally in solid dosage forms, such as capsules, tablets, and powders, or in liquid dosage forms, such as elixirs, syrups, and suspensions. It can also be administered parenterally, in sterile liquid dosage forms.
  • Gelatin capsules contain the active ingredient and powdered carriers, such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • powdered carriers such as lactose, starch, cellulose derivatives, magnesium stearate, stearic acid, and the like. Similar diluents can be used to make compressed tablets. Both tablets and capsules can be manufactured as sustained release products to provide for continuous release of medication over a period of hours. Compressed tablets can be sugar coated or film coated to mask any unpleasant taste and protect the tablet from the atmosphere, or enteric coated for selective disintegration in the gastrointestinal tract.
  • Liquid dosage forms for oral administration can contain coloring and flavoring to increase patient acceptance.
  • parenteral solutions In general, water, a suitable oil, saline, aqueous dextrose (glucose), and related sugar solutions and glycols such as propylene glycol or polyethylene glycols are suitable carriers for parenteral solutions.
  • Solutions for parenteral administration preferably contain a water soluble salt of the active ingredient, suitable stabilizing agents, and if necessary, buffer substances.
  • Antioxidizing agents such as sodium bisulfite, sodium sulfite, or ascorbic acid, either alone or combined, are suitable stabilizing agents.
  • citric acid and its salts and sodium EDTA are also used.
  • parenteral solutions can contain preservatives, such as benzalkonium chloride, methyl- or propyl-paraben, and chlorobutanol.
  • Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences. Mack Publishing Company, a standard reference text in this field.
  • a large number of unit capsules are prepared by filling standard two-piece hard gelatin capsules each with 100 milligrams of powdered active ingredient, 150 milligrams of lactose, 50 milligrams of cellulose, and 6 milligrams magnesium stearate.
  • a mixture of active ingredient in a digestable oil such as soybean oil, cottonseed oil or olive oil is prepared and injected by means of a positive displacement pump into gelatin to form soft gelatin capsules containing 100 milligrams of the active ingredient.
  • the capsules are washed and dried.
  • a large number of tablets are prepared by conventional procedures so that the dosage unit was 100 milligrams of active ingredient, 0.2 milligrams of colloidal silicon dioxide, 5 milligrams of magnesium stearate, 275 milligrams of microcrystalline cellulose, 11 milligrams of starch and 98.8 milligrams of lactose.
  • Appropriate coatings may be applied to increase palatability or delay absorption.
  • the combination products of this invention such as the novel cyclic IIb/IIIa antagonist compounds of this invention in combination with an anti-coagulant agent such as warfarin or heparin, or an anti-platelet agent such as aspirin, piroxicam or ticlopidine, or a thrombin inhibitor such as a boropeptide, hirudin or argatroban, or a thrombolytic agent such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations
  • an anti-coagulant agent such as warfarin or heparin
  • an anti-platelet agent such as aspirin, piroxicam or ticlopidine
  • a thrombin inhibitor such as a boropeptide, hirudin or argatroban
  • a thrombolytic agent such as tissue plasminogen activator, anistreplase, urokinase or streptokinase, or combinations
  • the combination products of the invention can be in any dosage form, such as those described above, and can also be administered in various ways, as described above.
  • the combination products of the invention are formulated together, in a single dosage form (that is, combined together in one capsule, tablet, powder, or liquid, etc.).
  • the combination products are not formulated together in a single dosage form, the cyclic glycoprotein IIb/IIIa compounds of this invention and the anti-coagulant agent, anti-platelet agent, thrombin inhibitor, and/or thrombolytic agent may be administered at the same time (that is, together), or in any order, for example the compounds of this invention are administered first, followed by administration of the anti-coagulant agent, anti-platelet agent, thrombin inhibitor, and/or thrombolytic agent.
  • the administration of the compound of this invention and any anti-coagulant agent, anti-platelet agent, thrombin inhibitor, and/or thrombolytic agent occurs less than about one hour apart, more preferably less than about 30 minutes apart, even more preferably less than about 15 minutes apart, and most preferably less than about 5 minutes apart.
  • administration of the combination products of the invention is oral.
  • oral agent, oral inhibitor, oral compound, or the like, as used herein, denote compounds which may be orally administered.

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Abstract

L'invention concerne des nouveaux composés cycliques renfermant des systèmes à noyau carbocyclique de la formule (I), utiles comme antagonistes du complexe récepteur glycoprotéique plaquettaire IIb/IIIa. Dans cette formule, A, B, C, D, E, F, G, H et L ont les notations données dans les revendications. L'invention se rapporte également à des compositions pharmaceutiqeus contenant de tels composés ainsi qu'aux procédés thérapeutiques utilisant ces composés et compositions pour l'inhibition de l'agrégation plaquettaire, en tant que thrombolytiques, et/ou pour le traitement d'autres troubles thrombo-emboliques.
PCT/US1995/002572 1994-03-04 1995-03-01 Nouveaux composes carbocycliques inhibant l'agregation plaquettaire par interaction avec le complexe recepteur gpiib/iiia WO1995023811A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU19756/95A AU1975695A (en) 1994-03-04 1995-03-01 Novel carbocyclic compounds which inhibit platelet aggregation by interaction with the gpiib/iiia receptor complex

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US20543394A 1994-03-04 1994-03-04
US08/205,433 1994-03-04

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WO1995023811A1 true WO1995023811A1 (fr) 1995-09-08

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014220A3 (fr) * 1996-10-02 1998-07-02 Du Pont Merck Pharma PEPTIDES CYCLIQUES CONTENANT DES AGENTS CHELATANTS ET MARQUES AU TECHNETIUM 99 m
WO1999001472A1 (fr) * 1997-07-04 1999-01-14 Merck Patent Gmbh Azapeptides cycliques a action angiogene

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001331A1 (fr) * 1989-07-17 1991-02-07 Genentech, Inc. Inhibiteurs d'aggregation de plaquettes a base de petits peptides cycliques
WO1993007170A1 (fr) * 1991-09-30 1993-04-15 The Du Pont Merck Pharmaceutical Company COMPOSES CYCLIQUES UTILES COMME INHIBITEURS DU COMPLEXE GLYCOPROTEIQUE PLAQUETTAIRE IIb/IIIa

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1991001331A1 (fr) * 1989-07-17 1991-02-07 Genentech, Inc. Inhibiteurs d'aggregation de plaquettes a base de petits peptides cycliques
WO1993007170A1 (fr) * 1991-09-30 1993-04-15 The Du Pont Merck Pharmaceutical Company COMPOSES CYCLIQUES UTILES COMME INHIBITEURS DU COMPLEXE GLYCOPROTEIQUE PLAQUETTAIRE IIb/IIIa

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998014220A3 (fr) * 1996-10-02 1998-07-02 Du Pont Merck Pharma PEPTIDES CYCLIQUES CONTENANT DES AGENTS CHELATANTS ET MARQUES AU TECHNETIUM 99 m
WO1999001472A1 (fr) * 1997-07-04 1999-01-14 Merck Patent Gmbh Azapeptides cycliques a action angiogene
US6534478B1 (en) 1997-07-04 2003-03-18 Merck Patent Gesellschaft Mit Cyclic azapeptides with angiogenic effect

Also Published As

Publication number Publication date
AU1975695A (en) 1995-09-18

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