1. 2. 4-OXADIAZOLE DERIVATIVES AND USES THEREOF

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 62/732,629, filed September 18, 2018, the contents of which is hereby incorporated in its entirety.

FIELD OF THE INVENTION

[0002] The present invention is in the field of medicine and pharmacology. More particularly, the invention relates to hemostasis and compounds useful for the treatment of cancer and disorders resulting from a disruption of hemostasis.

BACKGROUND OF THE INVENTION

[0003] Oxadiazole is a heterocyclic compound containing an oxygen atom and two nitrogen atoms in a five-membered ring. It is derived from furan by substitution of two methylene groups (=CH) with two pyridine type nitrogens (-N=). Oxadiazole has four isomers (l,2,4-oxadiazole, l,3,4-oxadizole and l,2,5-oxadiazole and l,2,3-oxadiazole, of which the 1,2,4- and l,3,4-oxadiazoles are stable.

[0004] l,2,4-oxadiazoles are flat, aromatic linkers able to bend a drug’s structure, and to exert an electron-withdrawing effect. They have been used as bioisosteric replacements of esters and amides, and have been used as a pharmacophore. Compounds containing 1,2,4- oxadizole cores have a broad biological activity spectrum. For example, certain 1,2,4- oxadiazole derivative are known to have biological activities useful in treating certain diseases and disorders, including antibacterial, antifungal, analgesic, anti-inflammatory, antiviral, anticancer, antihypertensive, anticonvulsant, and anti-diabetic properties. These compounds have also showed inhibitory activity for 5-lipoxygenase, fatty acid amide hydrolase, tyrosinase, transferases, monoamine oxidase B, acyl-Co A: Cholesterol O-acyltransferase, stearoyl-CoA desaturase 1, protein kinase CK2 , glycogen phosphorylase,

phosphodiesterase4b2, and cyclooxygenase. [0005] In living organisms, enzymes called proteases are produced to degrade proteins into peptides or amino acids to be used either as an energy source or as building blocks for resynthesize proteins. Proteases also modify cellular environments and facilitate cell migration in connection with wound repair and cancer, ovulation and implantation of the fertilized egg, embryonic morphogenesis, and involution of mammary glands after lactation.

In addition, proteases are regulators in processes such as inflammation, infection and blood clotting.

[0006] Proteases act on their natural substrates, proteins and peptides by hydrolyzing one or more peptide bond(s). This process is usually highly specific in the sense that only peptide bonds adjacent to certain amino acids are cleaved. Consequently, most proteolytic enzymes are highly specific for their substrates.

[0007] Mammalian serine proteases are one type of protease that may be divided into two families: the trypsins; and the subtilisins. The trypsin family contains, among others, trypsin and chymotrypsin, elastase, mast cell tryptase, and many of the factors regulating blood coagulation and fibrinolysis, including thrombin, Factor Xa, plasmin, tissue plasminogen activator (tA), urokinase plasminogen activator (uPA), and others.

[0008] It is well known that serine proteases play an important role in fibrinolysis.

Fibrinolysis is the degradation of the blood plasma protein, fibrin. Plasminogen is an inactive protein found in blood and is a precursor to plasmin. Plasmin is an enzyme that degrades blood plasma proteins such as fibrin, fibrinogen, Factors V, VIII, IX, XI, and XII, inter alia. Serine proteases are known to activate plasminogen to plasmin.

[0009] In addition, serine proteases are involved in the breakdown of the extracellular matrix, allowing for cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease plasmin and several matrix

metalloproteases. The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors. Thus, the generation of plasmin involves the pro-enzyme plasminogen, the urokinase type plasminogen activator, uPA, and its pro-enzyme, pro-uP A, the uPA inhibitor, PAI-l, the cell surface uPA receptor uPAR, and the plasmin inhibitor a2- anti plasmin.

[0010] The plasminogen system promotes tumor metastasis by several different mechanisms. One of these mechanisms is the uPA and uPAR (uPA receptor) system, which initiates the activation of MMPs as well as the conversion of plasminogen to plasmin followed by ECM degradation and reduced cellular interaction.

[0011] The uPA system is a conversion system from plasminogen into plasmin, which plays a key role in cancer invasion and metastasis dissemination by allowing malignant cells to invade the tumor site locally and spread to distant sites. This system includes the serine protease, uPA, membrane-linked receptor uPAR, and two serpin inhibitors, PAI-l and PAI-2. Thus, plasmin plays a role during multiple steps of cancer invasion and metastasis, by inducing the degradation of a number of ECM proteins and activating certain growth factors leading to aggressive cancers.

[0012] Plasminogen receptors also play a role in the proliferation and migration of tumor cells in many cancer types and may serve as prognostic and diagnostic markers. They are involved in mediating colocalization of plasminogen and its activators such as uPA and tPA on cell surfaces and markedly decrease the Km for plasminogen activation. Plasminogen receptors are expressed on the cell surface of most tumors and their expression frequently correlates with cancer diagnosis, survival and prognosis. Notably, they can trigger multiple specific immune responses in cancer patients, highlighting their role as tumor-associated antigens. Cell surface receptors loaded with plasmin, which is protected from inhibitors, play a key role in cancer progression.

[0013] What is needed are better methods of controlling serine proteases for stemming bleeding and for inhibiting the growth and metastasis of cancer. SUMMARY OF THE INVENTION

[0014] It has been discovered that certain 1,2,4 oxadiazole derivatives have serine protease inhibitory (SERPIN) activity that can inhibit plasminogen activation and the proteolytic activity of plasmin, tPA, and uPA, and thereby can stem hemorrhaging, and can inhibit the growth and metastasis of cancer in a mammals

[0015] These discoveries have been exploited to develop the present disclosure which, in part, is directed to 1,2,4- oxadiazole derivatives, therapeutic formulations containing such derivatives, and methods of using such derivatives to treat disorders resulting in uncontrolled bleeding, cancer, and metastasis.

[0016] In one aspect, the disclosure provides a compound of Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: X and Y are independently CH or N; X and Y are both not N, and X and Y both not CH; R is hydrogen, C _1-4 alkyl, arylamine, or piperadin-4-yl; R ¹ is hydrogen, -OH, C _1-4 alkyl, C _1-4 alkoxy, ketone, or -C(0)R ² ;

R ² is Ci- ₄ alkyl, C _1-4 alkoxy, NFE. N(H)C _I-4 alkyl, N(C _H alkyl) ₂ , hydrogen, or -OH; and - is an optional double bond, and wherein the compound is not 3-(l-(piperidin-4-yl)-lH-l,2,3- triazol-4-yl)-l,2,4-oxadiazol-5(4H)-one; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazol-5(4H)-one; or 3 -(l-ethyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)-one.

[0017] In one embodiment of the compound, X is N and Y is CH. In another embodiment of the compound, X is CH and Y is N. In still another embodiment of the compound, R ¹ is a ketone. [0018] In specific embodiments, the compound is selected from the group consisting of 3- (l-(piperidin-4-yl)-lH-l,2,3-triazol-3-yl)-l,2,4-oxadiazole; 3-(l -(piperidin-4-yl)-lH- 1,2,4- triazol-3-yl)-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazole; 3-(l-ethyl- lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazole;3-(l-propyl-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazo le;3-(l-ethyl-lH-l,2,4-triazol-3- yl)-l ,2,4-oxadiazol-5 (4H)-one; 3 -( 1 -propyl- 1 H- 1 ,2,3-triazol-4-yl)- 1 ,2,4-oxadiazol-5 (4H)-one;

3-(l-propyl-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)- one;3-(l-(piperidin-4-yl)-lH-l,2,3- triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,2,4- oxadiazol-5-ol; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3-(l-ethyl-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazol-5-ol; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3- (l-propyl-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5-ol; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-

4-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-4-yl)-5-methyl-l,2,4- oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,4- triazol-3-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,2,4- oxadiazole; 3-(l-propyl-lH-l,2,4-triazol-3-yl)-5-methyl-l,2,4-oxadiazole ; 3-(l-(piperidin-4- yl)-lH-l,2,3-triazol-4-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3- yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l- ethyl-lH-l,2,4-triazol-3-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-5- ethyl-l,2,4-oxadiazole; and 3-(l-propyl-lH-l,2,4-triazol-3-yl)-5-ethyl-l,2,4-oxadiazole.

[0019] In one particular embodiment, the compound is 3-(l-(piperidin-4-yl)-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazol-5(4H)-one.

[0020] Also provided by the disclosure is a pharmaceutical formulation comprising a therapeutically effective amount of at least one l,2,4-oxadiazole derivative and a

pharmaceutically acceptable carrier.

[0021] In some embodiments, the pharmaceutical formulation further comprises a therapeutically effective amount of at least one agent which treats a bleeding disorder. In other embodiments, the pharmaceutical formulation further comprises at least one therapeutic agent for treating a cancer. [0022] In another aspect, the disclosure provides a method of treating a patient afflicted with a bleeding disorder, comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising at least one compound of Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: X and Y are independently CH or N; X and Y are both not N, and X and Y both not CH; R is hydrogen, C _1-4 alkyl, arylamine, or piperadin-4-yl; R ¹ is hydrogen, -OH, C _1-4 alkyl, C _1-4 alkoxy, ketone, or - C(0)R ² ;R ² is Ci-4 alkyl, Ci-4 alkoxy, NH ₂ , N(H)CI-4 alkyl, N(CI-4 alkyl)2, hydrogen, or -OH; and - is an optional double-bond, and a pharmaceutically acceptable carrier.

[0023] In some embodiments, the method further comprises administering a therapeutically effective amount of a second therapeutic agent which treats a bleeding disorder.

[0024] In some embodiments, the bleeding disorders that are treated the pharmaceutical formulations and methods according to the disclosure include, but are not limited to, spontaneous bleeding, cardiac surgery (i.e., cardiopulmonary bypass), liver transplant, following therapeutic thrombolysis, congenital anti-plasmin deficiency, acquired anti-plasmin deficiency, hemophilia A and B, quantitative and qualitative platelet dysfunction, genitourinary bleeding, upper and lower urinary tract, dysfunctional uterine bleeding (essential menorrhagia and menorrhagia associated with intrauterine device), gastrointestinal bleeding (upper by varices, gastritis, ulcers, and lower by inflammatory bowel disease), mucous membrane bleedings for recurrent epistaxis or for excessive bleeding following tonsillectomy, traumatic hyphemia, trauma, general surgery, orthopedic surgery or cancer.

The methods are also useful to treat bleeding due to lack of coagulation factors, V, VII, VIII, or IX, or lack of von Willebrand’s factor. In addition, the methods according to the invention can be used to treat bleeding as the result of administration of an anticoagulant treatment. [0025] In yet another aspect, the disclosure provides a method of treating a patient afflicted with a cancer, comprising administering a therapeutically effective amount of a pharmaceutical formulation comprising a compound of Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: X and Y are independently CH or N; X and Y are both not N, and X and Y both not CH; R is hydrogen, Ci- ₄ alkyl, arylamine, or piperadin-4-yl; R ¹ is hydrogen, -OH, Ci- ₄ alkyl, Ci- ₄ alkoxy, ketone, or -C(0)R ² ;

R ² is Ci-4 alkyl, Ci-4 alkoxy, NFF. N(H)CI-4 alkyl, N(C H alkyl) ₂ , hydrogen, or -OH; and - is an optional double bond; and a pharmaceutically acceptable carrier.

[0026] In some embodiments, the method further comprises administering a second therapeutically effective amount of a second therapeutic agent which treats a cancer.

[0027] Also provided is a method of inhibiting the serine protease activity of an enzyme selected from the group consisting of tissue plasminogen activator, urokinase plasminogen activator, and plasmin, comprising contacting the enzyme with the compound of Formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein: X and Y are independently CH or N; X and Y are both not N, and X and Y both not CH; R is hydrogen, Ci-4 alkyl, arylamine, or piperadin-4-yl; R ¹ is hydrogen, -OH, Ci- ₄ alkyl, Ci- ₄ alkoxy, ketone, or -C(0)R ² ;

R ² is Ci- ₄ alkyl, Ci- ₄ alkoxy, NFF. N(H)C _I-4 alkyl, N(C _H alkyl) ₂ , hydrogen, or -OH; and - is an optional double bond. [0028] The disclosure also provides the use of any of the 1, 2, 4-oxadiazole compounds described above for the manufacture of a medicament for treating bleeding episodes. In some embodiments, the medicament is for reducing clotting time, in other embodiments, the medicament is for prolonging the clot lysis time. In yet other embodiments, the medicament is for increasing clot strength. In yet other embodiments, the medicament is formulated for topical, oral, or for intravenous or intramuscular injection administration.

[0029] In addition, the disclosure provides the use of any of the 1, 2, 4-oxadiazole compounds described above for the manufacture of a medicament for treating a cancer. In some embodiments, the medicament is for inhibiting the growth, reducing the size, or inhibiting the metastasis of, a cancer. In other embodiments, the medicament is formulated for topical, oral, or intravenous or intramuscular injection administration.

DESCRIPTION OF THE DRAWINGS

[0030] The foregoing and other objects of the present disclosure, the various features thereof, as well as the disclosure itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0031] FIG. 1 is a schematic representation of the structure of 3-[l-(l-ethanoyl-4- piperidy 1)- 1H- 1 ,2,3 -triazol-4-yl] -4H- 1 ,2,4-oxadiazol-5-one;

[0032] FIG. 1A are schematic representations of 1,2,4- oxadiazole derivatives of the disclosure where Rl is always -H and R2 could be different derivatives;

[0033] FIG. 1B are schematic representations of 1,2,4- oxadiazole derivatives of the disclosure where Rl is always -OH and R2 could be different derivatives;

[0034] FIG. 1C are schematic representations of 1,2,4- oxadiazole derivatives of the disclosure where Rl is always -CH ₃ and R2 could be difference derivatives; [0035] FIG. 1D are schematic representations of 1,2,4- oxadiazole derivatives of the disclosure where Rl is always -CFb - CFb and R2 could be different derivatives;

[0036] FIG. 1E are schematic representations of 1,2,4- oxadiazole derivatives of the disclosure where Rl is always -Carbonyl and R2 could be different derivatives;

[0037] FIG. 2A is a graphic representation showing the fibrinolytic activity of

representative 1, 2, 4- oxadiazole derivatives of the disclosure in which A is 3-(l-(piperidin-4- yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol; B is 3-(ethylamine-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazol; C is 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol -5(4H)-one; D is 3-(ethylamine-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)-o ne; marine blue represents the control (tissue factor, t-PA and calcium), and dark blue represents the blank (calcium and plasma);

[0038] FIG. 2B is a graphic representation showing the fibrinolytic activity of

representative 1, 2, 4- oxadiazole derivatives of the disclosure; wherein E is 3 -(propyl amine- lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)-one; F is 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol- 3-yl)-l,2,4-oxadiazol-5(4H)-hydroxyl; G is 3-(propylamine-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazol-5(4H)-hydroxyl; H is 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol - 5(4H) methyl; marine blue represents the control (tissue factor, t-PA and calcium), and dark blue represents the blank (calcium and plasma);

[0039] FIG. 2C is a graphic representation showing the fibrinolytic activity of

representative 1, 2, 4- oxadiazole derivatives of the disclosure; in which I is 3-(ethylamine- lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)-methyl; J is 3-(l-(piperidin-4-yl)-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazol-5(4H)-ethyl; K is 3-(ethylamine-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazol-5(4H)-ethyl; L is 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol - 5(4H)-carbonyl; M is 3-(propylamine-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)- carbonyl; marine blue represents the control (tissue factor, t-PA and calcium), sky blue is the and dark blue represents the blank (calcium and plasma); [0040] FIG. 3A is a graphic representation of the results of chromogenic assays showing the inhibitory effect of 1, 2, 4- oxadiazole derivatives of the disclosure as identified above in FIGS. 2A-2C;

[0041] FIG. 3B is a graphic representation of the results of chromogenic assays showing the inhibitory effect of 1, 2, 4- oxadiazole derivatives on the disclosure on t-pa activity, plasmin (S-2782) as identified above in FIGS. 2A-2C; and

[0042] FIG. 3C is a graphic representation of the results of chromogenic assays showing the inhibitory effect of 1, 2, 4- oxadiazole derivatives of the disclosure on uPA activity (S- 2444) as identified above in FIGS. 2A-2C.

DESCRIPTION

[0043] The disclosures of these patents, patent applications, and publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.

[0044] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0045] l,2,4-oxadiazoles have broad biological activity. However, to date, specific and direct serine protease inhibitor activity has not been reported for these compounds. The present disclosure provides certain l,2,4-oxadiazoles derivatives, and their salts and prodrugs, which are serine protease inhibitors (SERPINs) and which specifically and directly inhibit plasminogen activation and proteolytic activity of plasmin and plasminogen activation via direct inhibition of the serine proteases tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), and plasmin. As such, these 1,2,4- oxadiazole derivatives are useful in the treatment and/or prevention of hemorrhages in a mammalian subject and in the treatment of cancer and metastasis of cancer cells expressing high levels of plasmin and plasminogen activators activity. l.2.4-Oxadiazole Derivatives

[0046] The 1, 2, 4-oxadiazole derivatives according to the disclosure comprise a compound of formula I:

or a pharmaceutically acceptable salt or prodrug thereof, wherein:

X and Y are independently CH or N, wherein X and Y are both not N, and X and Y both not CH;

R is hydrogen, C _M alkyl, arylamine, or piperadin-4-yl;

R ¹ is hydrogen, -OH, C alkyl, CM alkoxy, ketone, or -C(0)R ² ;

R ² is CM alkyl, CM alkoxy, Nfl·. N(H)C _M alkyl, N(CM alkyl) ₂ , hydrogen, or -OH; and

- is an optional double bond; and salts and prodrugs thereof. Exemplary 1, 2, 4- oxadiazole derivatives according to the disclosure are substituted at position C5 and/or at position C3 (see FIGS. 2A-2B).

[0047] At position C5, the substitution is -H, =0, -OH, methanol, phenol, methyl, ethyl, phenyl, isopropyl, thienyl, ethanamine, ethylamine, methylamine, carboxyamide,

methanamine, methyletanamine, piperidine, methylmethanamine, pyrrolidine, pyridine, aniline, carboxylic acid, acetic acid, benzoic acid, propanoic acid, or and butanoic acid. At position C3, 1, 2, 4- oxadiazole can be substituted with -H, -Cl, methanol, phenol, ethyl, methyl, phenyl, fluorophenyl, bromophenyl, thienyl, isopropyl, pyridil, amine, aminomethyl, carboxiamidamide, methanamine, carbothiamide, triazol, carboxylic acid, or benzoid acid.

[0048] Representative compounds within the scope of the present disclosure include: 3-(l- (piperidin-4-yl)-lH-l,2,3-triazol-3-yl)-l,2,4-oxadiazole; 3-(l-(piperidin-4-yl)-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazole; 3-(l-ethyl- lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazole;

3-(l-propyl-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,4-triazol-3-yl)-l,2,4- oxadiazol-5(4H)-one; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)-one ; 3-(l- propyl- 1 H- 1 ,2,4-triazol-3 -yl)- 1 ,2,4-oxadiazol-5 (4H)-one; 3 -( 1 -(piperidin-4-yl)- 1H- 1,2,3- triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,2,4- oxadiazol-5-ol; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3-(l-ethyl-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazol-5-ol; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5-ol; 3- (l-propyl-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5-ol; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-

4-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-4-yl)-5-methyl-l,2,4- oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,4- triazol-3-yl)-5-methyl-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,2,4- oxadiazole; 3-(l-propyl-lH-l,2,4-triazol-3-yl)-5-methyl-l,2,4-oxadiazole ; 3-(l-(piperidin-4- yl)-lH-l,2,3-triazol-4-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3- yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-ethyl-lH-l,2,3-triazol-4-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l- ethyl-lH-l,2,4-triazol-3-yl)-5-ethyl-l,2,4-oxadiazole; 3-(l-propyl-lH-l,2,3-triazol-4-yl)-5- ethyl-l,2,4-oxadiazole; and 3-(l-propyl-lH-l,2,4-triazol-3-yl)-5-ethyl-l,2,4-oxadiazole, and salts and prodrugs thereof.

[0049] The salts of the compounds of formula (I) are be pharmaceutically acceptable salts such as other salts may, however, be useful in the preparation of the compounds according to the disclosure or of their pharmaceutically acceptable salts or prodrugs. Suitable

pharmaceutically salts of the compounds include acid addition salts which can be formed by mixing a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulfuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid. Furthermore, where the compounds of the disclosure carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.

[0050] Prodrugs of these 1 ,2, 4 oxadiazole derivatives are compounds that when cleared, are active as SERPINS, and are useful when activated to treat bleeding disorders and cancer. Such prodrugs include inactive intermediates in the synthetic scheme of the active derivatives, and which can be enzymatically or chemically modified in or on the body, when administered, to derive the active derivative. Some representative and non-limiting prodrugs include the l,2,4-oxadiazole derivatives as described herein further functionalized with acid and base labile functional groups that cleave in vivo. Some acid and base labile functional groups include, but are not limited to, carbonates, carbamates, ureas, and esters. Representative prodrugs include, but are not limited to: /er/-butyl 4-(4-(etho\ycarbonyl)- 1 H- 1 2.3-triazol- 1 - yl)piperidine-l-carboxylate; /e/ /-butyl (Z)-4-(4-0V-hydro\ycarbamimidoyl)- 1 H- 1 2.3-triazol- l-yl)piperidine-l-carboxylate; and /e/ /-butyl 4-(4-carbamoyl- 1 H- 1 2.3-triazol- 1 -yl)piperidine- l-carboxylate.

Synthesis of 1 2. 4- Oxadiazole Derivatives [0051] The 1, 2, 4 oxadiazole derivative according to the disclosure can be synthesized by any means known in the art (see, e.g., Sangshetti el al. (2009) Bioorg Med. Chem Lett. 19: 3564-3567). Usefule representative methods can be found in EXAMPLE 1.

The Role of Serine Proteases in Hemostasis

[0052] The 1, 2, 4 oxadiazole derivatives according to the disclosure are useful in part in controlling hemostasis, which maintains blood in a fluid state under physiologic conditions.

[0053] The oxadiazole derivative stem abnormal bleeding by affecting the two mechanism of hemostasis.

[0054] The first mechanism of hemostasis comprises two phases. The first phase is characterized by the occurrence of vasoconstriction at the vascular lesion site and platelet aggregation. In the second phase, the fibrin clot is formed due to the action of the different coagulation cascade proteolytic enzymes. This phase and it consists of several steps ending with fibrin polymer formation from fibrinogen hydrolysis due to the action of thrombin. Fibrin polymers are further stabilized by covalent isopeptide bonds formed by factor XIII activated (Factor XHIa) by thrombin. The mechanical strength of the fibrin gel is useful to impede blood loss when exposed to sheer forces in the circulation. There is a shift in the equilibrium between the formation of soluble fibrin polymers and the assembly of insoluble fibrin fibers. Factor XHIa lowers the fibrin concentration needed for an insoluble clot to form.

[0055] The second mechanism of hemostasis, the fibrinolytic system, is accomplished by localized activation of the plasminogen-plasmin enzyme, whereby it can heal a vascular lesion. Fibrinolysis counteracts the consequences of the coagulation process. The dissolution or solubilization of the fibrin clot at the correct time is needed for the orderly process of wound healing. Fibrinolysis is also required for angiogenesis as recanalization after clot formation. However, excessive local or systemic fibrinolytic activity can result in bleeding, as the weakened plug is dissolved. Conversely, an inadequate fibrinolytic response may retard lysis of a thrombus and contribute to its extension. By a balance of the simultaneous forces of coagulation and platelet aggregation, inhibition of coagulation, pro-fibrinolytic and anti- fibrinolytic reactions, and cellular mechanisms for both coagulation and lysis, the clot is gradually reduced.

[0056] Plasmin is a fibrinolytic serine protease that degrades fibrin, and is generated by activation of the zymogen, plasminogen (PLG). PLG is converted to plasmin by two serine protease enzyme plasminogen activators (PA): tissue-type plasminogen activator (tPA); and urokinase-type plasminogen activator (uPA). Secretion of t-PA by endothelial cells may be stimulated by fibrin, by thrombin bound to the thrombus, or by the effects of vessel occlusion, thereby increasing the local concentration of PA. tPA exerts high affinity for fibrin and increased PA activity, whereas, u-PA does not express any interaction with fibrin. tPA converts glu-PLG to the two-chain glu-plasmin. In response to fibrin, endothelial cells are capable of releasing t-PA thereby stimulating the activation of glu-PLG 500-fold, an effect that keeps PLG activation localized to the site of a clot. Once formed, glu-plasmin begins to digest the clot by catalyzing cleavages after selected arginine and lysine residues in the a, b and g-chains in regions connecting the D- and E- domains of the fibrin protomers.

[0057] Hemostasis also reacts to vascular injury to stem blood loss by normal

vasoconstriction (the vessel walls closing temporarily), by an abnormal obstruction (such as a plaque), or by coagulation or surgical means (such as ligation).

[0058] Abnormal bleeding occurs under certain disease conditions when normal clot formation fails to occur (e.g. hemophilia). Abnormal bleeding may also occur as the result of certain medications prescribed to treat another disorder. In addition, abnormal bleeding also occurs due to physical injuries sustained by otherwise healthy individuals. For example, surgery, dental procedures, accidents, and over-doses of anti-coagulant drugs can result in ruptured vessels and/or organs can result in abnormal bleeding.

[0059] In addition, abnormal bleeding may occur due to physically injured ruptured vessels or organs, and has treated by surgical ligation to repair the vessel or organ. However, when surgical ligation of bleeding fails, or is not possible, a number of hemostatic aids have been used. For example, abnormal bleeding can be treated with coagulant drugs such as thrombin, Tissue Factor, Factor VII, and Factor Vila. [0060] With tissue injury and bleeding, exposed collagen and released tissue factor cause activation of the intrinsic and extrinsic coagulation pathways. Both pathways lead to activation of Factor X which along with activated Factor V forms a complex that cleaves the

prothrombin protein into the active thrombin molecule. Thrombin production is the final coagulation step required to cleave fibrinogen into fibrin which provides a hemostatic lattice for platelet aggregation and thrombus formation at the site of injury. Thrombin is often used in conjunction with other hemostatic aids, including absorbable agents (e.g., gelfoam, collagen, and cellulose), and with fibrinogen in fibrin glue.

[0061] Factor VII initiates the process of coagulation in conjunction with Tissue Factor. Once bound to Tissue Factor, Factor VII is activated to Factor Vila by different proteases, including thrombin. Factor Vila has been used to treat uncontrolled bleeding in hemophilia patients, but there have been safety concerns. Other treatments include protamine sulfate, vitamin K, and plant substances such as leaf of nettle, and water pepper. Antagonists of anti coagulant drugs, such as protamine sulfate, vitamin K, and inhibitors of fibrinolysis such as aminocaproic acid, contrycal, and aprotinin, have also been used to stem abnormal bleeding.

Treatment of Bleeding Disorders

[0062] l,2,4-oxadiazole derivatives according to the disclosure affect hemostasis by directly inhibiting plasminogen activation and the proteolytic activity of plasmin, t-pa, and u- PA. As such these derivatives are useful in the treatment of bleeding disorders and abnormal bleeding.

[0063] The terms“bleeding disorder” and“abnormal bleeding” encompasses disorders and diseases affecting hemostasis and blood coagulation, spontaneous bleeding, cardiac surgery (i.e., cardiopulmonary bypass), liver transplant, following therapeutic thrombolysis, congenital anti-plasmin deficiency, acquired anti-plasmin deficiency, hemophilia A and B, quantitative and qualitative platelet dysfunction, genitourinary bleeding, upper and lower urinary tract, dysfunctional uterine bleeding (essential menorrhagia and menorrhagia associated with intrauterine device), gastrointestinal bleeding (upper by varices, gastritis, ulcers, and lower by inflammatory bowel disease), CCM, cerebral aneurysm, stroke, vasospasm after subarachnoid hemorrhage, spinal cord injury, mucous membrane bleedings for recurrent epistaxis or for excessive bleeding following tonsillectomy, traumatic hyphemia, trauma, general surgery, and orthopedic surgery.

[0064] The term“bleeding disorder” as used herein also encompasses physical trauma causing unwanted or uncontrolled bleeding in a subject such as, but not limited to, an accident causing an injury, surgery, dental procedure such as extractions, synovectomy, joint replacement, and in postoperative settings, drugs such as thrombolytic agents, -. The methods are also useful to treat bleeding due to lack of coagulation factors, V, VII, VIII, or IX, or lack of von Willebrand’s factor. In addition, the methods according to the invention can be used to treat bleeding as the result of administration of an anti-coagulant treatment.

[0065] The methods according to the disclosure are useful in the treatment of spontaneous bleeding, cardiac surgery (i.e., cardiopulmonary bypass), liver transplant, following therapeutic thrombolysis, congenital anti-plasmin deficiency, acquired anti-plasmin deficiency, hemophilia A and B, quantitative and qualitative platelet dysfunction,

genitourinary bleeding, upper and lower urinary tract, dysfunctional uterine bleeding

(essential menorrhagia and menorrhagia associated with intrauterine device), gastrointestinal bleeding (upper by varices, gastritis, ulcers, and lower by inflammatory bowel disease), CCM, cerebral aneurysm, stroke, vasospasm after subarachnoid hemorrhage, spinal cord injury, mucous membrane bleedings for recurrent epistaxis or for excessive bleeding following tonsillectomy, traumatic hyphemia, trauma, general surgery, orthopedic surgery or cancer.

The methods are also useful to treat bleeding due to lack of coagulation factors, V, VII, VIII, or IX, or lack of von Willebrand’s factor. In addition, the methods according to the invention can be used to treat bleeding as the result of administration of an anti-coagulant treatment.

The Role of Serine Proteases in Cancer

[0066] Breakdown of the extracellular matrix is involved in cancer invasion and metastasis. It is accomplished by the concerted action of several proteases, including the serine protease, plasmin, and several matrix metalloproteases. The activity of each of these proteases is regulated by an array of activators, inhibitors and cellular receptors. Thus, the generation of plasmin involves the pro-enzyme, plasminogen, the urokinase type plasminogen activator, uPA, and its pro-enzyme, pro-uP A, the uPA inhibitor PAI-l, the cell surface uPA receptor uPAR, and the plasmin inhibitor a2-anti plasmin.

[0067] This system promotes tumor metastasis by several different mechanisms. One of these mechanisms is the uPA and uPAR (urokinase plasminogen activator receptor) system, which initiates the activation of MMPs as well as the conversion of plasminogen to plasmin followed by ECM degradation and reduced cellular interaction.

[0068] Plasminogen receptors also play a role in the proliferation and migration of tumor cells in many cancer types and may serve as prognostic and diagnostic markers. They are involved in mediating colocalization of plasminogen and its activators such as uPA and tPA on cell surfaces and decrease the Km for plasminogen activation. Plasminogen receptors are expressed on the cell surface of most tumors and their expression frequently correlates with cancer diagnosis, survival and prognosis. They can trigger multiple specific immune responses in cancer patients, highlighting their role as tumor-associated antigens. Cell surface receptors loaded with plasmin, which are protected from inhibitors, play a key role in cancer progression.

[0069] The uPA system, which converts plasminogen into plasmin, plays a key role in cancer invasion and metastasis dissemination by allowing malignant cells to invade the tumor site locally and spread to distant sites. This system includes the serine protease, uPA, membrane-linked receptor uPAR, and two serpin inhibitors, PAI-l and PAI-2.

Treatment of Cancer

[0070] By inhibiting tPA, uPA, and plasmin, the l,2,4-oxadiazole derivatives according to the disclosure can be used to treat cancer and inhibit the metastasis of cancer cells. As such, with the l,2,4-oxadiazole derivatives according to the disclosure, are useful in treating cancers, such as, but are not limited to, carcinomas, sarcomas, lymphomas, leukemias, germ cell tumors, and blastomas. Pharmaceutical Formulations

[0071] The pharmaceutical formulations useful in the therapeutic methods according to the disclosure include a therapeutically effective amount of at least one 1, 2. 4- oxadiazole derivative, and/or a salt and/or a prodrug thereof.

[0072] A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic and/or prophylactic therapeutic effect for treating a bleeding disorder or trauma resulting in unwanted, uncontrolled bleeding.

[0073] In addition, the pharmaceutical formulations according to the disclosure may also comprise more than one l,2,4-oxadiazole derivative, and/or other known therapeutic agents for stemming bleeding. Such therapeutic include, but are not limited to, thrombin, Tissue factor, and/or Factor VII A. Different combinations of a therapeutically effective amount of at least one derivative according to the disclosure and a therapeutically effective amount of one or more therapeutic anti-bleeding agents can be applied together, e.g. topically.

[0074] A“therapeutically effective amount” of a 1, 2, 4-oxadiazole derivative, or salt or prodrug thereof, alternatively refers to that amount which treats, kills, and/or controls the growth and/or metastasis of a tumor or cancer cell affecting.

[0075] Likewise, the pharmaceutical formulations contain 1, 2, 4- oxadiazole derivatives according to the disclosure may comprise a therapeutically effective amount of at least one known anti-cancer genet or cancer therapeutic including, but not limited to, alkylating agents (including, but not limited to, cisplatin, chlorambucil, and procarbazine), antimetabolites (including, but not limited to, methotrexate, cytarabine, and gemcitabine), anti-microtubule agents (including, but not limited to, vinblastine and paclitaxel), topoisomerase inhibitors (including, but not limited to, etoposide and doxorubicin) and cytotoxic agents such as, but not limited to, bleomycin.

[0076] In the methods according to the disclosure, the pharmaceutical formulations including 1, 2, 4-oxadiazole derivatives of the disclosure can be administered alone or in combination with other known therapeutic agents. [0077] The pharmaceutical formulations according to the disclosure further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable carrier" is to be understood herein as referring to any substance that may, medically, be acceptably administered to a patient, together with a compound of this invention, and which does not undesirably affect the pharmacological activity thereof; a "pharmaceutically acceptable carrier" may thus be, for example, a pharmaceutically acceptable member(s) comprising of diluents, preservatives, solubilizers, emulsifiers, adjuvant, tonicity modifying agents, buffers as well as any other physiologically acceptable vehicle. These formulations are prepared with the pharmaceutically acceptable carrier in accordance with known techniques, for example, those described in Remington, The Science And Practice of Pharmacy (9th Ed. 1995).

[0078] For use in medicine, the salts of the compounds of formula (I) are pharmaceutically acceptable salts. Other salts may, however, be useful in the preparation of the compounds or of their pharmaceutically acceptable salts according to the disclosure. Suitable

pharmaceutically salts of the compounds of this invention include acid addition salts with may, for example, be formed by mixing a solution of a pharmaceutically acceptable acid such as hydrochloric acid, sulphuric acid, methanesulphonic acid, fumaric acid, maleic acid, succinic acid, acetic acid, benzoic acid, oxalic acid, citric acid, tartaric acid, carbonic acid or phosphoric acid.

[0079] Additionally, where the compounds of the disclosure carry an acidic moiety, suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g,. sodium or potassium salts; alkaline earth metal salts, e.g. calcium or magnesium salts; and salts formed with suitable organic ligands, e.g. quaternary ammonium salts.

[0080] The pharmaceutical formulations may also or instead comprise prodrugs of the derivatives according to the disclosure include inactive intermediates in the synthetic scheme of the active derivatives, and which can be enzymatically or chemically modified in or on the body, when administered, to derive the active derivative. Some representative and non- limiting prodrugs include the l,2,4-oxadiazole derivatives as described herein further functionalized with acid and base labile functional groups that cleave in vivo. Some acid and base labile functional groups include, but are not limited to, carbonates, carbamates, ureas, and esters.

[0081] The pharmaceutical formulation may be prepared for injectable use, topical use, oral use, intramuscular or intravenous injection, inhalation use, transdermal use,

transmembrane use, and the like.

[0082] These compositions are in unit dosage forms such as tablets, pills, capsules, powders, granules, sterile parenteral solutions or suspensions, metered aerosol or liquid sprays, drops, ampoules, auto-injector devices or suppositories; for oral parenteral, intranasal, sublingual topical or rectal administration, or for administration by inhalation or insufflation. Alternatively, the compositions may be presented in a form suitable for one-weekly or once- monthly administration; for example, an insoluble salt of the active compound, such as decanoate salt, may be adapted to provide a depot preparation for intramuscular injection. An erodible polymer containing the active ingredient may be envisaged.

[0083] For preparing solid compositions such as tablets, the principal active ingredient is mixed with a pharmaceutical carrier, e.g. conventional tableting ingredients such as com starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, di calcium phosphate or gums, and other pharmaceutical diluents, e.g. water, to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention, or a pharmaceutically acceptable salt thereof.

[0084] These formulations may be homogeneous, i.e., the l,2,4-oxadiazole derivative, or salt or prodrug thereof, is dispersed evenly throughout the composition so that the composition may be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules. This solid formulation composition is then subdivided into unit dosage forms of the type described above containing from 0.1 mg to about 500 mg of the active ingredient of the present invention. Some useful unit dosage forms contain froml to 100 mg, for example 1 mg, 2 mg, 5 mg, 10 mg, 25 mg, 50 mg, or 100 mg, of the derivative. The tablets or pills of the novel composition can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action. The liquid forms in which the novel compositions if the present invention may be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils as well as elixirs and similar pharmaceutical vehicles. In the treatment of bleeding episodes and cancer, a suitable dosage level is about 0.001 mg/kg to 250 mg/kg per day. The compounds may be administered on bolus and or a regimen of 1 to 4 times per day.

[0085] Injectable dosage forms may be sterilized in a pharmaceutically acceptable fashion, for example by steam sterilization of an aqueous solution sealed in a vial under an inert gas atmosphere at l20°C for about 15 minutes to 20 minutes, or by sterile filtration of a solution through a 0.2 mM or smaller pore-size filter, optionally followed by a lyophilization step, or by irradiation of a composition containing a compound of the present invention by means of emissions from a radionuclide source.

[0086] A therapeutically effective dosage of the 1, 2, 4- oxadiazole derivatives according to the disclosure or of another therapeutic which treats a bleeding disorder or cancer may vary from patient to patient, and may depend upon factors such as the age of the patient, the patient’s genetics, and the diagnosed condition of the patient, and the route of delivery of the dosage form to the patient. A therapeutically effective dose and frequency of administration of a dosage form may be determined in accordance with routine pharmacological procedures known to those skilled in the art. For example, dosage amounts and frequency of

administration may vary or change as a function of time and severity of the disorder. A dosage from about 0.1 mg/kg to 1000 mg/kg, or from about 1 mg/kg to about 100 mg/kg may be suitable.

Activity of Specific l.2.4-Oxadiazole Derivatives

[0087] The enzymatic and/or inhibitory activity of the l,2,4-oxadiazole derivatives according to the disclosure can be determined by assaying for their fibrinolytic potentials, their serine protease inhibitory activities, and their ability to inhibit cancer growth and migration of cancer cells. These activities can be determined by any assay known in the art, including the following assays. A. Fibrinolytic Activity Assays

[0088] Fibrinolytic activity in human plasma was determined by a one-step

spectrophotometric method (Gidron et al. (1978) J. Clin. Pathol. 3l(l):54-57) with minor modifications (see EXAMPLE 2). In this method, fibrin clot formation in anticoagulant citrate dextrose (ACD) plasma samples obtained from healthy volunteers is triggered by tissue factor (TF) and can be quantified by spectrometry as a significant increase in basal plasma absorbance at 340 nm.

[0089] As shown in FIGS. 2A - 2C, in the absence of tPA, plasma absorbance remains permanently elevated. In contrast, in the presence of tPA, plasma absorbance returns quickly to basal values, indicating that plasmin was able to lysis completely the formed fibrin clot.

B. Serine Protease Inhibitor Activity Assays

[0090] Spectrophotometric assays can be used to determine the inhibitory effect of 1,2,4 oxadiazole on serine proteases (see. e.g. Jespersen et al, eds. (1992) ECAT Assay

Procedures. A Manual of Laboratory Techniques. Springer Science). These

spectrophotometric assays use chromogenic substrates (peptides) that react with different proteases under the formation of color. The synthetically made substrates are designed to possess selectivity similar to that of the natural substrate for the enzyme. The ability of these chromogenic substrates to detect low enzyme concentrations makes them useful to investigate the specificity of SERPINs.

[0091] Measurements made using chromogenic substrates reflect specifically enzyme activity. Attached to the peptide portion of the chromogenic substrate is a chemical group which when released after the enzyme cleavage gives rise to color. The color change can be followed spectrophotometrically and is proportional to the proteolytic activity.

[0092] Selected synthetic chromogenic substrates for certain serine proteases were used to determine the specificity of inhibitory effect of the l,2,4-oxadizole derivatives according to the disclosure on tPA, uPA, plasmin, and other serine protease activities (EXAMPLE 3). [0093] The results show that the 1,2,4- oxadiazole derivatives according to the disclosure displayed specific inhibitory effects on tPA PA (S-2782) (FIG. 6), uPA (S-2444) (FIG. 7), and plasminogen activation by tPA Plasmin (S-2390) (FIG. 8). However, no effects on Thrombin (S-2238), Factor X activated (S-2787), Factor XI activated (S-2288), Kailikrem (S-2302), Granulocyte Elastase (S-2484), Trypsin (S-2222) and Chymotrypsin (S-2586) were detected.

B. Cancer Inhibition

The ability of 1,2,4- oxadiazole derivatives cording to the disclosure to inhibit cancer cell migration and proliferation in vitro can be measured, e.g., by a wound healing assay (Rodriguez et al, in Cell Migration: Developmental Methods and Protocols. Web el al, Guan ed., Humana Press 294:. 23-29). This assay is based on the observation that, upon the creation of an artificial gap on a confluent cancer cell monolayer, the cells on the edge of the created gap will start migrating and proliferating until new cell-cell contacts are established.

As described below in EXAMPLE 4, the l,2,4-oxadiazole derivatives according to the disclosure were able to significantly inhibit the proliferation and migration of fibrosarcoma cells in vitro.

Reference will now be made to specific examples illustrating the disclosure. It is to be understood that the examples are provided to illustrate exemplary embodiments and that no limitation to the scope of the disclosure is intended thereby.

EXAMPLES EXAMPLE 1

Synthetic of 1,2,4-Oxadiazole Derivatives

A. Synthesis of 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol -5(4H)-one

Step 1 : /e/V-butyl 4-((methylsulfonyl)oxy) piperidine- 1 -carboxylate

[0094] To a stirred solution of /e/V-butyl 4-hydroxypiperidine-l-carboxylate (Sigma- Aldrich, US) (1) (0.96 g, 4.8 mmol) in 10 mL dichloromethane was added triethylamine (1 mL) and stirred at room temperature for 5 minutes and then cooled to 0-5 °C. Methanesulfonyl chloride (0.67 g, 6.2 mmol) was added dropwise at 0-5 °C and after addition the reaction mixture was allowed to warm to room temperature and then stirred at room temperature for 2 h. After completion (Monitored by TLC), the reaction was quenched by addition of distilled water, additional dichloromethane was added and the dichloromethane layer after extraction was separated, dried (MgSCL) and concentrated in vacuo to yield 1.3 g of /er/-butyl 4- ((methylsulfonyl)oxy)piperidine-l-carboxylate (2) as a pale yellow solid. Yield 97 %.

!H-NMR (400 MHz, CDCb) d (ppm): 4.84 - 4.90 (m, 1H), 3.66 - 3.72 (m, 2H), 3.25 - 3.32 (m, 2H), 3.02 (s, 3H), 1.91 - 1.99 (m, 2H), 1.76 - 1.84 (m, 2H), 1.44 (s, 9H).

Step 2: fer/-butyl 4-azidopiperidine-l-carboxylate

[0095] To a stirred solution of /er/-butyl 4-((methylsulfonyl)oxy)piperidine-l-carboxylate (2) (2.48 g, 8.9 mmol) in 20 mL dimethyl formamide, sodium azide (1.74, 26.8 mmol) was added and the resulting suspension was stirred at 80 °C for 8 h. After completion (Monitored by TLC), the reaction was allowed to cool to room temperature and then poured into cold water with occasional stirring. The residue was extracted with of ethyl acetate (3 xlO mL). The combined organic layers were concentrated in vacuo and the residue was dissolved in hexane (50 mL) and an extraction with water (3 x 10 mL) was made. The combined organic layers were dried (MgSCL) and concentrated in-vacuo to yield 1.75 g of /e/V-butyl 4- azidopiperidine-l-carboxylate (3) as a yellow oil. Yield 87%.

^! H-NMR (400 MHz, CDCb) d (ppm): 3.81 - 3.76 (m, 2H), 3.51 - 3.56 (m, 1H), 3.02 - 3.09 (m, 2H), 1.79 - 1.86 (m, 2H), 1.49 - 1.56 (m, 2H), 1.44 (s, 9H). Step 3: tert-butyl 4-(4-(ethoxycarbonyl)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 -carboxyl ate

[0096] Tert-butyl 4-azidopiperidine-l-carboxylate (3) (0.66 g, 2.9 mmol) was taken in acetonitrile (15 mL) and to it ethyl propiolate (Sigma-Aldrich, US) (0.29 g, 2.9 mmol) was added, followed by Cul (0.11 g, 0.6 mmol) and the resulting solution was stirred at room temperature for 12 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water was added to the residue and then extracted with 3 portions (3 x 10 mL) of ethyl acetate. The combined organic layers were dried (MgSCb) and concentrated in vacuo to yield 0.79 g of the desired compound tert-butyl 4-(4-(ethoxycarbonyl)- 1 H- 1 2.3-triazol- 1 - yl)piperidine-l-carboxylate (4) as a pale yellow solid. Yield 84%.

^! H-NMR (400 MHz, CDCb) d (ppm): 8.08 (s, 1H), 4.62 - 4.70 (m, 1H), 4.40 (q, J=7. lHz, 2H), 4.23 - 4.31 (m, 2H), 2.89 - 2.93 (m, 2H), 2.18 - 2.23 (m, 2H), 1.88 - 1.98 (m, 2H), 1.46 (s, 9H), 1.39 (t, J=7. lHz, 3H).

Step 4: tert-butyl 4-(4-carbamoyl- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 -carboxyl ate

[0097] To a solution of tert-butyl 4-(4-(ethoxycarbonyl)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- l-carboxylate (4) (2.04 g, 6.3 mmol) in 20 mL of methanol, 40 mL of ammonia in methanolic solution was added and the resulting solution was stirred at room temperature for 12 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water was added to the residue. It was extracted with 3 portions (3 x 10 mL) of dichloromethane. The combined organic layers were dried (MgSCL) and concentrated in vacuo to yield 1.69 g the desired compound tert- butyl 4-(4-carbamoyl- \H- \ 2.3-triazol- 1 -yl (piperidine- 1 -carboxyl ate (5) as a white solid. Yield 91 %. ¹ H-NMR (400 MHz, CDCb) d (ppm): 8.10 (s, 1H), 7.00 (s, 1H), 5.70 (s, 1H), 4.62 - 4.70 (m, 1H), 4.24 - 4.31 (m, 2H), 2.89 - 3.01 (m, 2H), 2.19 - 2.23 (m, 2H), 1.88 - 1.99 (m, 2H), 1.44 (s, 9H).

Step 5: fer/-butyl 4-(4-cyano- 1 H- 1 2.3-tria/ol- 1 -yl (piperidine- 1 -carboxylate

[0098] A solution of tert-butyl 4-(4-carbamoyl- 1 H- 1 2.3-tria/ol- 1 -> 1 (piperidine- 1 - carboxylate (5) (0.21 g, 0.7 mmol) and 0.4 mL of triethylamine in 9 mL of dichloromethane was stirred at room temperature. The resulting solution was then cooled to 0-5 °C in an ice bath. To this cooled solution, trifluoroacetic anhydride (0.30 g, 1.5 mmol) was added. The resulting solution was stirred for 5 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water added to the residue and then extracted with 3 portions (3x1 OmL) of dichloromethane. The organic layer was washed with saturated NaHCCb solution, dried (MgSCri) and concentrated in vacuo to yield 0.19 g of the desired compound /e/V-butyl 4-(4-cyano- \ H- \ 2.3-tria/ol- 1 -yl)piperidine- 1 -carboxylate (6) as a brown oil. Yield 97%.

^! H-NMR (400 MHz, CDCb) d (ppm): 8.12 (s, 1H), 4.61 - 4.72 (m, 1H), 4.25 - 4.30 (m, 2H), 2.89 - 3.03 (m, 2H), 2.20 - 2.26 (m, 2H), 1.92 - 2.04 (m, 2H), 1.48 (s, 9H).

Step 6: tert- butyl (Z)-4-(4-(7V-hydroxycarbamimidoyl)-li/-l,2,3-triazol-l- yl)piperidine- 1 -carboxylate

[0099] To a stirred solution of te/7-butyl 4-(4-cyano- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 - carboxylate (6) (0.14 g, 0.5 mmol) in 10 mL methanol, hydroxylamine hydrochloride (0.07 g,

1 mmol) and sodium bicarbonate (0.07 g, 0.9 mmol) was added. The resulting mixture was heated under reflux for 14 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water added to the residue and then extracted with 3 portions (3 x lOmL) of dichloromethane. The organic layer dried (MgSCL) and concentrated in vacuo. A small portion of ethyl acetate was added and stirred at room temperature for 30 min. The resulting solid, tert- butyl (Z)-4-(4-(iV'-hydrox> carbamimido> l)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 - carboxylate (7), was filtered. 0.04 g of compound 7 were obtained. Yield 23%.

¾-NMR (400 MHz, de-DMSO) d (ppm): 9.50 (s, 1H), 8.35 (s, 1H), 5.71 (s, 2H), 4.69 - 4.77 (m, 1H), 4.03 - 4.07 (m, 2H), 2.95 (s, 2H), 2.05 - 2.09 (m, 2H), 1.82 - 1.93 (m, 2H), 1.42 (s, 9H).

Step 7 : fe/7-butyl 4-(4-(5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl)-li/-l,2,3-tria zol-l- yl)piperidine- 1 -carboxylate

[0100] To a suspension of / -butyl (Z)-4-(4-(/V-hydroxycarbamimidoyl)-li/-l,2,3-triazol- l-yl)piperidine-l -carboxylate (7) (0.16 g, 0.5 mmol) in tetrahydrofuran (15 mL) NN ^~ - carbonyldiimidazole (CDI) (0.08 g, 0.5 mmol) was added at room temperature and the mixture was stirred for 20 h under reflux. After completion (Monitored by TLC), solvent was removed by evaporation and then water was added to the residue. The precipitate was collected and washed with water. Recrystallization of the residue from ethyl acetate gave 0.01 g of tert- butyl 4-(4-(5 -oxo-4, 5-dihy dro- 1 2.4-oxadiazol-3-yl)- 1 H- 1 ,2,3 -triazol- 1 -yl)piperidine- 1 -carboxylate (8). Yield 6%.

^! H-NMR (400 MHz, de-DMSO) d (ppm): 8.57 (s, 1H), 6.92 (s, 1H), 4.71 - 4.77 (m, 1H), 4.00 - 4.03 (m, 2H), 2.82 - 3.01 (m, 2H), 2.03 - 2.08 (m, 2H), 1.81 - 1.89 (m, 2H), 1.38 (s, 9H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 159.6, 154.2, 151.9, 150.6, 139.8, 79.4, 57.5, 42.9, 31.6, 28.4. HRMS (E SI-FI A-T OF) : m/z calcd for Ci ₄ H2iN ₆ 0 ₄ 337.1624, found 337.1619.

Step 8: 3-(l-(piperidin-4-yl)-li/-l,2,3-triazol-4-yl)-l,2,4-oxadiazo l-5(4i7)-one

[0101] A mixture of /e/V-butyl 4-(4-(5-oxo-4.5-dihydro- 1.2.4-oxadia/ol-3-yl)- 1 H- 1.2.3- tria/ol-l-yl)piperidine-l-carboxylate (8) ( 10 mg, 0.03 mmol) and 4N HC1 in dioxane (2 mL) was stirred at 23°C for 2 h. The solvent was removed in vacuo and the resulting yellow solid was triturated with EtOAc to provide 7 mg of the hydrochloride of 3-( 1 -(piperidin-4-yl)- 1 H- l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(47 )-one (9) as a white solid. Yield 90%.

^! H-NMR (400 MHz, CDCb) d (ppm): 13.28 (s, 1H), 9.10 (s, 1H), 8.90 (s, 1H), 8.89 (s, 1H), 4.92 (m, 1H), 3.32 (m, 2H), 3.09 (m, 2H), 2.33 - 2.24 (m, 4H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 159.4, 151.5, 131.9, 124.4, 55.1, 41.7, 30.6, 28.3.

B. Synthesis of 3-(l-(piperidin-4-yl)-lH-l,2,4-triaz.ol-3-yl)-l,2,4-oxadiaz. ol-5(4H)-one

Step 1 : /e/V-butyl 4-(3-(methoxy carbonyl)- 1 H- 1 2.4-tria/ol- 1 -\ 1 /piperidine- 1 -carboxylate

[0102] NaH (1 g; 41.7 mmol) was added to a solution of methyl 1 //~l ,2,4-triazole-3- carboxylate (Sigma-Aldrich, US) (2.8 g; 22 mmol) in DMF (130 mi.). The reaction mixture was stirred at 25°C for 20 min followed by 1 h at 70 °C. fe/ -butyi 4-iodopiperidme-l- carboxylate (Alfa Aesar, US) (6 g, 19.3 mmol) was added and the reaction mixture was heated at 70 °C for 48 h. The solution was cooled to 0°C and the insoluble material was removed by filtration. The filtrate was diluted with DCM and washed with water, brine, dried over Na ₂ S0 ₄ , filtered and evaporated to dryness. The residue was purified by chromatography over silica gel (mobile phase: petroleum ether/ethyl acetate 1 /5) to render 1.5 g of intermediate 14.

Yield 25%.

¹ H-NMR (400 MHz, CDCb) d (ppm): 7.98 (d, J= 0.6 Hz, 1H), 5.32 (s, 1H), 4.24 (t, J= 16.7 Hz, 2H), 4.01 (s, 3H), 2.91 (s, 2H), 2.

Step 2: fer/-butyl 4-(3 -carbamoyl- 1 H- 1 2.4-triazol- 1 -yl)piperidine- 1 -carboxylate

[0103] To a solution of /er/-butyl 4-(3-(methoxYcarbonyl)- 1 H- 1 2.4-tria/ol- 1 -vl)piperidine- l-carboxylate (14) (0.85 g, 2.6 mmol) in 10 mL of methanol, 20 mL of ammonia in methanolic solution was added and the resulting solution was stirred at room temperature for 12 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water was added to the residue. It was extracted with 3 portions (3 x 10 mL) of dichloromethane.

The combined organic layers were dried (MgSCL) and concentrated in vacuo to render 0.75 g of the desired compound tert-butyl 4-(3-carbamoyl- 1 H- 1 2.4-tria/ol- 1 -yl)piperidine- 1 - carboxylate (15) as a white solid. Yield 96 %.

^! H-NMR (400 MHz, de-DMSO) d (ppm): 13.08 (s, 2H), 8.20 (s, 1H), 5.19 - 4.89 (m, 1H),

4.19 - 3.82 (m, 2H), 2.80 (m, 2H), 1.95 (s, 2H), 1.88 - 1.69 (m, 2H), 1.39 (s, 9H).

Step 3: /e/7- butyl 4-(3-cyano- 1 H- 1 2.4-triazol- 1 -yl)piperidine- 1 -carboxylate

[0104] A solution of ter/-butyl 4-(3 -carbamoyl- \H- 1,2, 4-triazol-l -yl)piperidine-l - carboxylate (15) (0.9 g, 3.0 mmol) and 1.7 mL of triethylamine in 80 mL of dichloromethane was stirred at room temperature. The resulting solution was then cooled to 0 - 5°C in an ice bath. To this cooled solution, trifluoroacetic anhydride (l.3g, 6.2 mmol) was added in 20 mL of dichloromethane. The resulting solution was stirred for 5 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water added to the residue and then extracted with 3 portions (3 x 10 mL) of dichloromethane. The organic layer was washed with saturated NaHCCb solution, dried (MgSCri) and concentrated in vacuo to render 0.7 g of the desired compound /er/-butyl 4-(3-cyano- \H- 1 2.4-tria/ol- 1 -yl)piperidine- 1 -carboxylate (16) as a brown oil. Yield 91%.

¹ H-NMR (400 MHz, CDCb) d (ppm): 8.04 (s, 1H), 4.62 (tt, J = 11.4, 4.3 Hz, 1H), 4.29 (s, 2H), 2.92 (t, J= 13.1 Hz, 2H), 2.13 (dddd, J= 13.1, 12.0, 11.3, 4.5 Hz, 2H), 2.00 (m, 2H), 1.47 (s, 9H).

Step 4: /e/V-butyl (Z)-4-(3 -(/V -hydroxy carbamimidoyl)- \H- 1,2, 4-triazol-l - yl)piperidine- 1 -carboxylate

[0105] To a stirred solution of /er/-butyl 4-(3-cyano-li/-l,2,4-triazol-l-yl)piperidine-l- carboxylate (16) (0.38 g, 1.3 mmol) in 10 mL methanol, hydroxylamine hydrochloride (0.47g, 6.8 mmol) and sodium bicarbonate (0.93 g, 6.8 mmol) was added. The resulting mixture was heated under reflux for 14 h. After completion (Monitored by TLC), solvent was removed in vacuo and distilled water added to the residue and then extracted with 3 portions (3 x lOmL) of dichloromethane. The organic layer dried (MgSCri) and concentrated in vacuo. A small portion of ethyl acetate was added and stirred at room temperature for 30 min. The solid precipitated out (0.33 g) was collected as /e/V-butyl (Z)-4-(3-GV-hydro\ycarbamimidoyl)- l //- l,2,4-triazol-l-yl) piperidine- l-carboxylate (17). Yield 82%.

*H-NMR (400 MHz, de-DMSO) d (ppm): 10.21 (s, 1H), 7.99 (s, 1H), 5.93 (s, 2H), 5.25 - 5.08 (m, 1H), 4.02 (s, 2H), 2.79 (s, 2H), 1.95 - 1.72 (m, 4H), 1.39 (s, 9H).

Step 5: /er/-butyl 4-(3-(5-oxo-4,5-dihydro-l,2,4-oxadiazol-3-yl)-li/-l,2,4-tria zol-l- yl)piperidine- 1 -carboxylate

[0106] To a suspension of / -butyl (Z)-4-(3-(/V-hydroxycarbamimidoyl)-li/-l,2,4-triazol- l-yl)piperidine- l-carboxylate (17) (0.33 g, 1.0 mmol) in tetrahydrofuran (15 mL) NJT- carbonyldiimidazole (CDI) (0.2 g, 1.2 mmol) was added at RT, and the mixture was stirred for 20 h under reflux. After completion (Monitored by TLC), solvent was removed by evaporation and then water was added to the residue. The precipitate was collected and washed with water. Recrystallization of the residue from ethyl acetate gave 0.3 g of /e/V-butyl 4-(3-(5-oxo-4,5- dihydro-l,2,4-oxadiazol-3-yl)-li/-l,2,4-triazol-l-yl)piperid ine-l-carboxylate (18). Yield 90%.

*H-NMR (400 MHz, de-DMSO) d (ppm): 9.47 (s, 1H), 7.95 (s, 1H), 5.54 - 5.30 (m, 1H), 4.02 (d, J= 13.5 Hz, 2H), 2.84 (s, 2H), 1.95 - 1.70 (m, 2H), 1.70 - 1.49 (m, 2H), 1.39 (s, 9H). ¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 159.8, 153.7, 151.9, 132.1, 123.7, 79.0, 57.8, 42.3, 31.6, 28.0.

HRMS (E SI-FI A-T OF) : m/z calcd for Ci ₄ H _2i N ₆ 0 ₄ 359.1624, found 337.1619.

Step 6: 3 -( 1 -(piperidin-4-yl)- 1 H- 1 ,2,4-triazol-3-y 1)- 1 ,2,4-oxadiazol-5 (4H)-one

[0107] A mixture of /e/V-butyl 4-(3-(5-o\o-4.5-dihydro- 1.2.4-oxadia/ol-3-yl)- 1 H- 1.2.4- triazol-l-yl) piperidine- l-carboxylate (21) (100 mg, 0.3 mmol) and 4N HC1 in dioxane (2 mL) was stirred at room temperature for 2 h. The solvent was removed in vacuo and the resulting yellow solid was triturated with EtOAc to provide 77 mg of 3-(l-(piperidin-4-yl)-lH-l,2,4- triazol-3-yl)-l,2,4-oxadiazol-5(4H)-one (19) as a yellow solid. Yield 95%.

^! H-NMR (400 MHz, de-DMSO) d (ppm): 9.09 (s, 2H), 8.90 (d, J = 4.5 Hz, 1H), 7.67 (s, 2H), 4.93 (s, 1H), 3.13 - 3.03 (m, 2H), 2.38 - 2.29 (m, 2H), 2.24 (s, 2H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 153.5, 151.3, 145.4, 139.9, 54.0, 41.8, 27.9.

EXAMPLE 2

Fibrinolytic Activity in Human Plasma

[0108] The effect of l,2,4-oxadiazole derivatives of the disclosure on fibrinolytic activity in human plasma was determined by a one-step spectrophotometric method as follows.

[0109] Blood from healthy volunteers (15 ml) was anticoagulated with ACD and plasma was separated by centrifugation 3,000 rpm for 10 min at RT.

[0110] Each vial of Thromborel S (Siemens, USA) containing a lyophilized extract of rabbit-brain thromboplastin (tissue factor) was reconstituted with 10 ml of a 3.2 mM solution of CaCh _. Each vial of human recombinant t-PA (Sigma-Aldrich, US) was reconstituted with chUO to a final concentration of 100 pg/ml.

[0111] Fibrin clot formation in human plasma was induced as follows: An aliquot of 150 pl of plasma was incubated in a 96- well plate at 37°C during 30 min. in the presence of 50 pl of reconstituted Thromborel S reagent (Fisher Scientific, USA), and 20 pl of 1 mM Tris-HCl buffer. Clot formation was quantified spectrophotometrically at 340 nm.

[0112] The following derivatives according to the disclosure showed IC50 values below 50Nm, showing their ability to cause the formation of fibrin clots (FIGS 2A - 2C):

3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol ; 3-(ethylamine-lH-l,2,3-triazol-

4-yl)-l,2,4-oxadiazol; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol -5(4H)- one; 3-(ethylamine-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)-o ne; 3 -(propylamine- 1E1-

1.2.4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)-one; 3-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-

1.2.4-oxadiazol-5(4H)-hydroxyl; 3-(propylamine-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol- 5(4H)-hydroxyl; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol -5(4H) methyl; 3- (ethylamine-lH-l,2,4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)-met hyl; 3-(l-(piperidin-4-yl)-lH-

1.2.4-triazol-3-yl)-l,2,4-oxadiazol-5(4H)-ethyl; 3-(ethylamine-lH-l,2,3-triazol-4-yl)-l,2,4- oxadiazol-5(4H)-ethyl; 3-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol -5(4H)- carbonyl; and 3-(propylamine-lH-l,2,3-triazol-4-yl)-l,2,4-oxadiazol-5(4H)- carbonyl.

EXAMPLE 3

Determination of Specificity of Inhibitory Effect on Serine Protease Activity

The effect of a l,2,4-oxadiazole derivative on the serine protease activities of plasmin, tPA, and uPA was determined by the following chromogenic assay. The serine proteases and chromogenic substrates used (Diapharma, USA) are described below in TABLE 1.

TABLE 1

[0113] Each vial of human recombinant t-PA (Sigma- Aldrich, US) was reconstituted with dEbO to a final concentration of 100 pg/ml. Each vial of human plasmin (Roche, Sigma- Aldrich, USA) in 0.5 ml of working solution (Roche, Sigma-Aldrich, USA) pH 8.6 (provided by the supplier) has the activity of 5 U Each vial of recombinant human uPA (Sigma- Aldrich, USA) containing 50 pg was reconstituted with 1 ml of PBS to a final concentration of 50 pg/ml. Each vial of recombinant human thrombin (Sigma-Aldrich, USA) containing 100 NTH units/mg of protein was reconstituted with cUrhO to a final concentration of 100 NIH units/mg of protein per ml. Each vial of trypsin from human pancreas (Sigma-Aldrich, USA) containing 1,000 BAEE units was reconstituted with cUrhO to a final concentration of 1,000 BAEE units/ml. Each vial of a-chymotrypsin from human pancreas (Sigma-Aldrich, USA) containing 10 BTEE units was reconstituted with cUrhO to a final concentration of 10 BTEE units/ml. Each vial of coagulation Factor Xa from human plasma (Sigma-Aldrich, USA) containing 170 units/mg of protein was reconstituted with cUrhO to a final concentration of 170 units/mg of protein per ml. Each vial of Kallikrein form human plasma (Sigma-Aldrich,

USA) containing 5 units/ mg of protein was reconstituted with cUrhO to a final concentration of 5 units (mg) of protein per ml. Each vial of chromogenic substrates (Diapharma, USA) and working solutions were prepared and reconstituted following the instructions of the manufacturer.

[0114] Chromogenic assays were performed as follows: An aliquot of 900 pi of working solution containing each reconstituted chromogenic substrate was incubated in a 96 well-plate at 37°C during 30 min in the presence of 90 pi of each reconstituted proteolytic enzyme and 10 pi of 1 mM Tris-HCl buffer, mix and read each 2 min the absorbance at 405 nm.

[0115] The effect of 1,2,4- oxadiazole derivatives on proteolytic activity by chromogenic assays was determined as follows: An aliquot of 900 pi of working solution containing reconstituted each chromogenic substrate was incubated in a 96 well-plate at 37°C during 30 min in the presence of 90 pi of each reconstituted proteolytic enzyme and 10 pi of each 1,2,4- oxadiazole derivate at different concentrations and inhibitory effect was quantified spectrophotometrically at 405 nm.

[0116] The results are shown in FIGs. 3A - 3D. EXAMPLE 4

Anti-Cancer Activity Assays

[0117] The following assays were performed to evaluate the inhibitory effect mediated by l,2,4-oxadiazol derivatives on cancer cell proliferation and migration (Rodriguez el al, “Wound-Healing Assay, pp. 23-29, in Cell Migration Jun-Lin Guam, ed., Humana Press).

[0118] HT-1080 human cells (about lOVwell) (fibrosarcoma cells ATCC-CCL-121)

(Manassas, VA) were cultured in Eagle's Minimum Essential Medium (ATCC-formulated medium, Catalog No. 30-2003) containing fetal bovine serum at 10% during several periods. Wound healing assays with supplemented medium containing l,2,4-oxadiazole derivatives were performed to evaluate the inhibitory effect on proliferation and migration of cancer cells as follows: In a confluent cell layer a gap formation was done manually with a scrapper and cell proliferation and migration was determined recording a time-lapse video for 20 hr with a time interval of 30 min. The microscopic pictures were manually analyzed for obtaining information about the proliferation and migration characteristics of the cultured cells and the image analysis detects the cell covered area. Plotting the cell covered area against the time showed the process of gap closure and the proliferation and migration of HT-1080 was determined.

[0119] Gap formation in the monolayer is created manually with a scrapper and cell proliferation and migration is determined recording a time-lapse video for 20 hr with a time interval of 30 min. The microscopic pictures are manually analyzed for obtaining information about the proliferation and migration characteri stics of the cultured cells and the image analysis detects the cell covered area.

[0120] Plotting the ceil covered area against the time showed the process of gap closure. Then the proliferation and migration of HT-1080 was determined.

[0121] All the compounds of the present invention were able to inhibit significantly the proliferation and migration at below 100 mM concentration. EXAMPLE 5

In Vivo Testing of Coagulation Activities of Derivatives

[0122] To test the anti-bleeding effect of l,2,4-oxadiazole derivatives, in vivo experiments were performed using mice with congenital coagulation anomalies.

[0123] The tails of 5 normal JAX mice (The Jackson Laboratory, Bar Harbor, ME) and 3 transgenic mice with genetic deficiencies for hemophilia deficient in FVIII (F8 ^r ) (The Jackson Laboratory, Bar Harbor, ME) are severed. Either buffer (phosphate buffered saline (PBS)) (control) or a 1 ,2,4-oxadiazol derivative according to the disclosure in this buffer is applied topically at a dose of 100 mg derivative on the wound together a bolus intravenously of derivative in buffer administered at a dose of 10 mg/kg or the same volume of buffer (control). The mortality rates in the group that received the derivative relative to control is then determined.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.