6-peptidylamino-1-naphthalenesulfonamides useful as fluorogenic proteolytic enzyme substrates

• US 5,399,487 A
• Filed: 03/04/1993
• Issued: 03/21/1995
• Est. Priority Date: 03/04/1993
• Status: Expired due to Term

• Current Assignee: Haematologic Technologies Incorporated
• Original Assignee: Haematologic Technologies Incorporated
• Inventors: Butenas, Saulius, Lawson, Jeffrey H., Mann, Kenneth G.
• Primary Examiner(s): Wityshyn, Michael G.
• Assistant Examiner(s): Leary, Louise N.
• Application Number:
  US08/027,294
• Time in Patent Office: 747 Days
• Field of Search: 435/13, 435/4, 514/19, 514/822, 514/20, 514/319, 514/183, 514/145, 424/244, 424/177, 546/145, 544/67, 544/88, 544/179, 544/145
• US Class Current: 435/13
• CPC Class Codes: C07D 209/48 with oxygen atoms in positions 1 and 3, e.g. phthalimide
• CPC Class Codes: C07K 5/0808 the side chain containing 2 to 4 carbon atoms, e.g. Val, Ile, Leu
• CPC Class Codes: C12Q 1/37 involving peptidase or proteinase
• CPC Class Codes: C12Q 1/56 involving blood clotting factors, e.g. involving thrombin, thromboplastin, fibrinogen
• CPC Class Codes: C12Q 2337/30 Naphthyl amides, e.g. beta-NA, 2-NA, 4-methoxy-beta-naphthylamine, i.e. 4MNA

Abstract

The present invention encompasses compounds of formula I: ##STR1## and the pharmaceutically acceptable non-toxic salts thereof; wherein R₁ is hydrogen, lower alkyl, alkenyl, alkynyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, or phenylalkyl;

R₂ is hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, alkylcycloalkyl, cycloalkylalkyl, or phenylalkyl; or

NR₁ R₂ forms a nitrogen heterocycle; and

R₃ is hydrogen, an amino acid or a peptide residue.

These compounds may be employed as substrates in assays for determining proteolytic enzyme activity or as enzyme inhibitors. The invention also encompasses methods for determining proteolytic enzyme activity using the compounds of formula I.

Claims

1. A compound of the formula:

##STR28## and the pharmaceutically acceptable non-toxic salts thereof;

wherein R₁ is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, cycloalkylalkyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula ##STR29## wherein R₅ represents an amino acid side chain and R₄ is hydroxy, an amino acid or a peptide residue;

R₂ is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, cycloalkylalkyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula ##STR30## wherein R₅ represents an amino acid side chain and R₄ is hydroxy, an amino acid or a peptide residue;

or

NR₁ R₂ forms a nitrogen heterocycle; and

R₃ is an amino acid or a peptide residue.

2. A compound according to claim 1, wherein R₃ is lysine or arginine.

3. A compound according to claim 1, where R₃ is a peptide having arginine or lysine at the carboxy terminus.

4. A compound according to claim 3, which is 6-L-arginylamino-1-(N-ethyl)naphthalenesulfonamide.

5. A compound according to claim 3, which is 6-L-arginylamino-1-(N-n-propyl)naphthalenesulfonamide.

6. A compound according to claim 3, which is 6-L-arginylamino-1-(N-isopropyl)naphthalenesulfonamide.

7. A compound according to claim 3, which is 6-L-arginylamino-1-(N-n-butyl)naphthalenesulfonamide.

8. A compound according to claim 3, which is 6-L-arginylamino-1-(N-isobutyl)naphthalenesulfonamide.

9. A compound according to claim 3, which is 6-L-arginylamino-1-(N-tert-butyl)naphthalenesulfonamide.

10. A compound according to claim 3, which is 6-L-arginylamino-1-(N-n-pentyl)naphthalenesulfonamide.

11. A compound according to claim 3, which is 6-L-arginylamino-1-(N-cyclohexyl)naphthalenesulfonamide.

12. A compound according to claim 3, which is 6-L-arginylamino-1-(N-benzyl)naphthalenesulfonamide.

13. A compound according to claim 3, which is 6-L-arginylamino-1-(N,N-diethyl)naphthalenesulfonamide.

14. A compound according to claim 3, which is 1-[(6-L-arginylamino-1-naphthalenyl)sulfonyl]-piperidine.

15. A compound according to claim 3, which is 1-[(6-L-arginylamino-1-naphthalenyl)sulfonyl]-morpholine.

16. A compound according to claim 3, which is 1[(6-L-arginylamino-1-naphthalenyl)sulfonyl]-pyrrolidine.

17. A compound according to claim 3, which is 6-L-arginylamino-1-(N,N-dimethyl)naphthalenesulfonamide.

18. A compound according to claim 3, which is 6-L-arginylamino-1-(N-methyl)naphthalenesulfonamide.

19. A compound according to claim 3, which is 6-(t-butoxycarbonyl-prolyl-phenylalanyl-arginylamino)-1-(N-n-propyl)naphthalenesulfonamide.

20. A compound according to claim 3, which is 6-(prolyl-phenylalanyl-arginylamino)-1-(N-n-propyl)naphthalenesulfonamide.

21. A compound according to claim 3, which is 6-(t-butoxycarbonyl-phenylalanyl-phenylalanyl-arginylamino)-1-(N-n-pentyl)naphthalenesulfonamide.

22. A compound according to claim 3, which is 6-(phenylalanyl-phenylalanyl-arginylamino)-1-(N-n-pentyl)naphthalenesulfonamide.

23. A compound according to claim 3, which is 6-(carbobenzyloxy-glycyl-glycyl-arginyl-arginylamino)-1-(N-cyclohexyl)naphthalenes

24. A compound according to claim 3, which is 6-(glycyl-glycyl-arginyl-arginylamino)-1-(N-cyclohexyl)naphthalenesulfonamide.

25. A compound according to claim 3, which is 1-[(6-methanesulfonyl-D-leucinyl-phenylalanyl-arginylamino-1naphthalenyl)sulfonyl]-piperidine.

26. A compound according to claim 3, which is 1[(6-benzyloxycarbonyl-D-leucinyl-phenylalanyl-arginylamino-1-naphthalenyl)sulfonyl]-piperidine.

27. A compound according to claim 3, which is 1-[(6-D-leucinyl-phenylalanyl-arginylamino-1-naphthalenyl)sulfonyl]-piperidine.

28. A compound according to claim 3, which is 6-(N.sup.α

-t-butoxycarbonyl, N.sub.ω

-benzyloxycarbonyl-lysylarginylamino)-1-(N,N-dimethyl)naphthalenesulfonamide.

29. A compound according to claim 3, which is 6-(N¹⁰⁷ -benzyloxycarbonyl-lysyl-arginylamino)-1-(N,N-dimethyl)-1-naphthalenesulfonamide.

30. A compound according to claim 3, which is 6lysyl-arginylamino)-1-(N,N-dimethyl)napthalenesulfonamide.

31. A compound according to claim 3, which is 6(benzoyl-isoleucinyl-glycinyl-arginylamino)-1-(N-isopropyl)napthalenesulfonamide.

32. A compound according to claim 3, which is 6-(toluenesulfonyl-glycyl-phenylalanyl-arginylamino)-1-(N-isobutyl)naphthalenesulfonamide.

33. A compound according to claim 3, which is 6-(methanesulfonyl-D-leucinyl-glycinyl-arginylamino)-1-(N,N-diethyl)naphthalenesulfonamide.

34. A method for determining proteolytic enzyme activity comprising the steps of:

(a) contacting a proteolytic enzyme with a substrate compound of the formula;

##STR31## wherein R₁ is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, cycloalkylalkyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula ##STR32## wherein R₅ represents an amino acid side chain and R₂ is hydroxy, an amino acid or a peptide residue;

R₂ is hydrogen, straight or branched chain lower alkyl having 1-6 carbon atoms, straight or branched chain alkenyl having 2-8 carbon atoms, straight or branched chain alkynyl having 2-8 carbon atoms, cycloalkyl having 3-7 carbon atoms, alkylcycloalkyl where the alkyl portion has 1-6 carbon atoms, cycloalkylalkyl where the alkyl portion has 1-6 carbon atoms, or phenylalkyl where the alkyl portion is straight or branched chain alkyl having 1-6 carbon atoms, or a group of the formula ##STR33## wherein R₅ represents an amino acid side chain and R₄ is hydroxy, an amino acid or a peptide residue;

or

NR₁ R₂ forms a nitrogen heterocycle; and

R₃ is an amino acid or a peptide residue; and

(b) measuring fluorescence intensity changes as a result of substrate compound hydrolysis.

35. A method according to claim 34, where R₃ is lysine or arginine.

36. A method according to claim 34, where R₃ is a peptide having arginine or lysine at the carboxy terminus.

37. A method according to claim 36, wherein the proteolytic enzyme is a serine protease involved in blood coagulation.

38. A method according to claim 34, wherein the proteolytic enzyme is factor VIIa, tissue plasminogen activator, urokinase, factor IIa, factor IXa, factor Xa, plasmin, or activated protein C.