REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. provisional application 62/732,719, 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, cancer, and compounds useful for the treatment of disorders resulting from a disruption of hemostasis and for treating cancer.

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-oxadiazole and l,2,5-oxadiazole and 1,2,3 oxadiazole, of which the 1, 3, 4-oxadiazoles and 1,2, 4-oxadiazoles are stable.

[0004] The 1,3, 4-oxadiazoles have been used as bioisosteric replacements of esters and amides, and have been used as a pharmacophore. These compounds have a broad biological activity spectrum. For example, certain l,3,4-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-CoA: Cholesterol O-acyltransferase, stearoyl-CoA desaturase 1, protein kinase CK2 , glycogen phosphorylase, phosphodiesterase4b2, and cyclooxygenase. Two examples of compounds containing the l,3,4-oxadiazole unit currently used in clinical medicine are:

Raltegravir®, an antiretroviral drug and Zibotentan® an anticancer agent. [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 (tPA), 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 serine protease inhibitors

(“SERPINs”), PAI-l and PAI-2.

[0012] 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.

[0013] 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.

[0014] What is needed are better methods for stemming bleeding and for inhibiting the growth and metastasis of cancer. SUMMARY OF THE INVENTION

[0015] It has been discovered that certain l,3,4-oxadiazole derivatives have SERPIN activity. As such these derivatives can inhibit plasminogen activation and the proteolytic activity of plasmin, tPA and uPA activities thereby stemming uncontrolled bleeding or hemorrhaging and inhibiting tumor growth and metastasis in a mammalian subject.

[0016] These discoveries have been exploited to develop the present disclosure, which, in part, is directed to certain l,3,4-oxadiazole derivatives and their use thereof in treating disorders resulting in uncontrolled bleeding or hemorrhaging, and in treating cancer.

[0017] In a specific embodiment, the compound is hydrochloride 5-(l-(piperidin-4-yl)-lH-

1.2.4-triazol-3-yl)-l,3,4-oxadiazol-2(3H)-one. In certain embodiments, the compound is selected from a group consisting of: 2-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazole; 2-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol e; 2-(l-ethyl-lH-l,2,4- triazol-3-yl)-l,3,4-oxadiazole; 2-(l-ethyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazole; 2-(l-propyl- lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazole; 2-(l-propyl-lH-l,2,4-triazol-3-yl)-l,3,4- oxadiazole; 5 -( 1 -(piperidin-4-yl)- 1H- 1 ,2,4-triazol-3-yl)- 1 ,3 ,4-oxadiazol-2(3H)-one; 5 - (ethylamine- 1H- 1 ,2,3 -triazol-4-yl)- 1 ,3,4-oxadiazol-2(3H)-one; 5 -(ethylamine- 1H- 1,2,4- triazol-3-yl)-l,3,4-oxadiazol-2(3H)-one; 5-(propylamine-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazol-2(3H)-one; 5-(propylamine-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2(3H)- one; 5-(l- (piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2-ol ; 5-(l-ethyl-lH-l,2,3-triazol-4-yl)-

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

1.2.3-triazol-4-yl)-l,3,4-oxadiazol-2-ol; 5-(l-propyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2- ol; 2-methyl-5-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazole; 2-methyl -5 -(1- (piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazole; 2-(l-ethyl-lH-l,2,3-triazol-4-yl)-5- methyl-l,3,4-oxadiazole; 2-(l-ethyl-lH-l,2,4-triazol-3-yl)-5-methyl-l,3,4-oxadiazole; 2-(l- propyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,3,4-oxadiazole; 2-(l-propyl-lH-l,2,4-triazol-3-yl)-5- methyl-l,3,4-oxadiazole; 2-ethyl-5-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazole; 2-ethyl-5-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,3,4-o xadiazole; 2-ethyl-5- (l-ethyl-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazole; 2-ethyl-5-(l-ethyl-lH-l,2,4-triazol-3-yl)-

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

In another aspect, the disclosure provides a pharmaceutical formulation comprising a 1,3,4- oxadiazole derivative and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical formulation further comprises a therapeutic agent for treating a bleeding disorder in a patient. In other embodiments, the pharmaceutical formulation further comprises a therapeutic agent for treating a cancer in a patient.

[0018] In yet 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 a l,3,4-oxadioazole derivative to the patient. In some embodiments, the pharmaceutical formulation further comprises at least one therapeutically effective amount of an agent which treats a bleeding disorder. In still other embodiments, the method further comprises administering to the patient a therapeutically effective amount of at least one agent which treats a bleeding disorder.

[0019] 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.

[0020] In other embodiments, the bleeding episodes treated with the l,3,4-oxadiazole derivative according to the disclosure include, but are not limited to , spontaneous bleeding, cardiac surgery, including 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, lack of coagulation factors, V, VII, VIII, or IX, or lack of von Willebrand’s factor, and anticoagulant treatment.

[0021] 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 l,3,4-oxadiazole derivative to the patient. In some embodiments, the pharmaceutical formulation further comprises at least one therapeutically effective amount of an agent which treats a cancer. In still other embodiments, the method further comprises administering to the patient a therapeutically effective amount of at least one agent which treats a cancer.

[0022] In some embodiments, the cancer treated with the pharmaceutical formulations and methods of the invention include, but are not limited to, alkylating agents (including, but not limited to, cisplatin, chlorambucil, and procarbazine) , antimetabolites (including, but not limit 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 (including, but not limited to, bleomycin).

[0023] The disclosure also provides the use of any of the l,3,4-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 intravenous or intramuscular injection administration.

[0024] In addition, the disclosure provides the use of any of the l,3,4-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. [0025] The disclosure also provides 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 a compound of formula 1.

DESCRIPTION OF THE DRAWINGS

[0026] 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:

[0027] FIG. 1A displays schematic representations of l,3,4-oxadiazole derivatives including 1,2,3- and 1 ,2,4-triazole derivatives, where Rl is always -H, and R2 can be different derivatives;

[0028] FIG. 1B displays schematic representations of 1,3,4- oxadiazole derivatives including 1, 2, 3- and 1, 2, 4- triazole derivatives where Rl is always -OH, and R2 can be different derivatives;

[0029] FIG. 1C displays schematic representations of l,3,4-oxadiazole derivatives where Rl is -CH _3, and R2 can be different derivatives;

[0030] FIG. 1D displays schematic representations of l,3,4-oxadiazole derivatives including 1,2,3- and 1 ,2,4-triazole derivatives, where Rl is -CH ₂ CH ₃ and R2 can be different derivatives;

[0031] FIG. 1E displays schematic representations of l,3,4-oxadiazole derivatives, where Rl are is -CO (C=0, Carbonyl) and R2 can be different derivatives; and

[0032] FIG. 2 is a graphic representation of the results of fibrinolytic assays.

DESCRIPTION

[0033] 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.

[0034] 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.

[0035] The present invention relates to certain l,3,4-oxadiazoles derivatives, and their salts and prodrugs thereof, which are SERPINs, and which specifically and directly inhibit plasminogen activation and proteolytic activity of plasmin and plasminogen activation via direct inhibition of tPA and uPA)activities. As such, these 1,3,4- oxadiazole derivatives are useful in the treatment and/or prevention of hemorrhages in a mammalian subject and in the treatment of cancer and metastasis in cancer cells expressing high levels of plasmin and plasminogen activators activity.

1.3.4- Oxadiazole Derivatives

[0036] 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, Ci-4 alkyl, arylamine, or piperadin-4-yl;

RHs hydrogen, -OH, Ci-4 alkyl, CM alkoxy, ketone, or C(0)R ² ;

R ² is hydrogen, C14 alkyl, C1-4 alkoxy, NH ₂ , N(H)Ci4 alkyl, N(Ci4 alkyl) ₂ , or -OH; and - is an optional double bond, wherein the compound is not 5-(l-(piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazol - 2(3H)-one or 5 -( 1 -(piperidin-4-yl)- 1 H- 1 ,2,3-triazol-4-yl)- 1 ,3 ,4-oxadiazol-2-ol

[0037] In some embodiments, X is N and Y is CH, or X is CH and Y is N. In some embodiments, R ¹ is a ketone.

[0038] Specific compounds within the scope of the present disclosure include: 2-(l- (piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazole; 2-(l-(piperidin-4-yl)-lH-l,2,4- triazol-3-yl)-l,3,4-oxadiazole; 2-(l-ethyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazole; 2-(l-ethyl- lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazole; 2-(l-propyl-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazole;2-(l-propyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazo le; 5-(l-(piperidin-4-yl)-lH- l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2(3H)-one; 5-(ethylamine-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazol-2(3H)-one; 5-(ethylamine-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2(3H)-o ne; 5- (propylamine-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazol-2(3H)-on e; 5-(propylamine-lH-l,2,4- triazol-3-yl)-l,3,4-oxadiazol-2(3H)-one; 5-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-l,3,4- oxadiazol-2-ol; 5-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazol-2-ol; 5-(l-ethyl-lH-l,2,4- triazol-3-yl)-l,3,4-oxadiazol-2-ol; 5-(l-propyl-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazol-2-ol; 5- (l-propyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2-ol; 2-methyl-5-(l-(piperidin-4-yl)-lH-

1.2.3-triazol-4-yl)-l,3,4-oxadiazole; 2-methyl-5-(l-(piperidin-4-yl)-lH-l,2,4-triazol-3-yl)-

1.3.4-oxadiazole; 2-(l-ethyl-lH-l,2,3-triazol-4-yl)-5-methyl-l,3,4-oxadiazole; 2-(l-ethyl-lH-

1.2.4-triazol-3-yl)-5-methyl-l,3,4-oxadiazole; 2-(l-propyl-lH-l,2,3-triazol-4-yl)-5-methyl-

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

1.2.4-triazol-3-yl)-l,3,4-oxadiazole; 2-ethyl-5-(l-ethyl-lH-l,2,3-triazol-4-yl)-l,3,4- oxadiazole; 2-ethyl-5-(l-ethyl-lH-l,2,4-triazol-3-yl)-l,3,4-oxadiazole; 2-ethyl-5-(l-propyl- lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazole; 2-ethyl-5-(l-propyl-lH-l,2,4-triazol-3-yl)-l,3,4- oxadiazole; 5 -( 1 -(piperidin-4-yl)- 1H- 1 ,2,3 -triazol-4-yl)- 1 ,3 ,4-oxadiazol-2(3H)-one; 5-(l- (piperidin-4-yl)-lH-l,2,3-triazol-4-yl)-l,3,4-oxadiazol-2-ol ; and salts and prodrugs thereof.

Synthesis of Oxadiazole Derivatives

[0039] The l,3,4-oxadiazole derivatives according to the disclosure can be synthesized by any methods known to those with skill in the art and as exemplified in EXAMPLE 1.

The Role of Serine Proteases in Hemostasis

[0040] The l,3,4-oxadiazole derivatives according to the disclosure are useful in part in controlling hemostasis, which maintains blood in a fluid state under physiologic conditions. These oxadiazole derivatives stem abnormal bleeding by affecting the two mechanisms of hemostasis.

[0041] 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 Xllla lowers the fibrin concentration needed for an insoluble clot to form.

[0042] 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.

[0043] 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 tPA 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, uPA 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 tPA, and 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.

[0044] 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).

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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

[0049] The l,3,4-oxadiazole derivatives according to the disclosure affect hemostasis by directly inhibiting plasminogen activation and the proteolytic activity of plasmin, tPA, and uPA. As such, the 1,3,4- oxadiazole derivatives are useful in the treatment of spontaneous bleeding, cardiac surgery (e.g., 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 (e.g., essential menorrhagia and menorrhagia associated with intrauterine device), gastrointestinal bleeding (e.g., 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

[0050] Breakdown of the extracellular matrix is important 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.

[0051] Furthermore, the regulation of extracellular proteolysis in cancer involves a complex interplay between cancer cells and non-malignant stromal cells in the expression of the molecular components involved. For some types of cancer, this cellular interplay mimics that observed in the tissue of origin during non-neoplastic tissue remodeling processes.

[0052] 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.

[0053] 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.

[0054] The uPA system is a conversion system from plasminogen into plasmin, which plays important physiological roles. It also 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 SERPINs, PAI-l and PAI-2.

Treatment of Cancer

[0055] By inhibiting the serine proteases tPA, uPA, and plasmin, the 1,3,4 oxadiazole derivatives according to the disclosure can treat cancer and inhibit the metastasis of cancer cells. As such, with the l,3,4-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

[0056] The pharmaceutical formulations useful in methods of treating bleeding disorders and cancer according to the disclosure comprise a therapeutically effective amount of a 1,3,4- oxadiazole derivative in a pharmaceutically acceptable carrier.

[0057] 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.

[0058] In addition, the pharmaceutical formulations according to the disclosure may also comprise more than one l,3,4-oxadiazole derivative in addition to other known therapeutic agents for stemming bleeding. Such therapeutic include, but are not limited to, thrombin, Tissue factor, and/or Factor VIIA. 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.

[0059] In addition, the pharmaceutical formulations according to the disclosure may alternatively comprise a therapeutically effective amount of other known therapeutic agents for treating cancer, 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 pacbtaxel), topoisomerase inhibitors (including, but not limited to, etoposide and doxorubicin) and cytotoxic agents such as, but not limited to, bleomycin.

[0060] A "therapeutically effective amount" as used herein refers to that amount which provides a therapeutic and/or prophylactic therapeutic effect for treating a cancer, including inhibiting its growth, reducing its size, and/or inhibiting its metastasis.

[0061] The term "pharmaceutically acceptable carrier” refers to any substance that may, medically, be acceptably administered to a patient, together with a l.3.4-oxadiazole derivative and which does not undesirably affect the pharmacological activity thereof. A

"pharmaceutically acceptable carrier" may thus be, for example, a pharmaceutically acceptable member(s) selected from the group comprising or consisting of diluents, preservatives, solubilizers, emulsifiers, adjuvant, tonicity -modifying agents, buffers as well as any other physiologically acceptable vehicle.

[0062] Formulations according to the disclosure are prepared with a pharmaceutically acceptable carrier in accordance with known techniques, for example, those described in Remington. The Science And Practice of Pharmacy (9th Ed. 1995).

[0063] 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.

[0064] 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.

[0065] 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 butyl 4-(4-(etho\y carbonyl)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 - carboxylate, tert- butyl 4-(4-(5-o\o-4.5-dihydro- 1 3.4-oxadiazol-2-yl)- 1 H- 1 2.3-triazol- 1 - yl)piperidine-l-carboxylate, and / -butyl 4-(4-(etho\ycarbonyl)- 1 H- 1 2.3-triazol- 1 - yl)piperidine- 1 -carboxylate

[0066] The pharmaceutical formulations may take different 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 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.

[0067] 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.

[0068] 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.

[0069] Formulations suitable for oral administration may be presented in discrete units or dosage forms, such as capsules, cachets, lozenges, tablets, sublingual tablets, pills, powders, granules, chewing gum, suspensions, solutions, and the like. Each dosage form contains a predetermined amount of 1,3,4- oxadiazole derivative. If in the form of a solution, the pharmaceutically acceptable carrier may be an aqueous liquid, such as buffered with a pharmaceutically acceptable pH buffer, or in non-aqueous liquid such as DMSO, or be prepared as an oil-in-water or water-in-oil emulsion.

[0070] 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.

[0071] When referring to these preformulation compositions as“homogeneous”, it is meant that the active ingredient 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 preformulation 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. Favored unit dosage forms contain froml to 100 mg, e.g., 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.

[0072] A therapeutically effective dosage of the l,3,4-oxadiazole derivatives according to the disclosure and/or of another therapeutic agent which treats a bleeding disorder or which treats 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. In the treatment of bleeding episodes and cancer, a suitable dosage level is about O.OOlmg/kg per day to about 250 mg/kg per day. The compounds may be administered on bolus and or a regimen of 1 to 4 times per day.

Activity of Specific l.3.4-Oxadiazole Derivatives

[0073] The activity of the l,3,4-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.

A. Fibrinolytic Activity Assays

[0074] 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).

[0075] 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. As shown in FIG. 3, 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 is able to lysis completely the formed fibrin clot. B. Serine Protease Inhibitor Activity Assays

[0076] Spectrophotometric assays can be used to determine the inhibitory effect of 1,3,4 oxadiazole on serine proteases (see. e.g. Jespersen et ah, edr. (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.

[0077] 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.

[0078] Selected synthetic chromogenic substrates for specific serine proteases were shown to be specific for the inhibitory effect of the l,3,4-oxadizole derivatives according to the disclosure on tPA, mRA, plasmin, and other serine protease activities (see EXAMPLE 3).

C. Cancer Inhibition

[0079] The ability of 1,3,4- oxadiazole derivatives cording to the disclosure to inhibit cancer cell migration and proliferation in vitro can be measured by a wound healing assay (EXAMPLE 4) (Rodriguez et al, in Cell Migration: Developmental Methods and Protocols Web et al., Guan ed., Humana Press 294:. 23-29).

[0080] This assay showed that the 1,3,4- oxadiazole derivatives according to the disclosure displayed specific inhibitory effects on tPA, uPA, and plasminogen activation by tPA. [0081] 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 Methodologies for 1,3,4-Oxadiazole Derivatives

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

Step 1 : Tert-butyl 4-((methylsulfonyl)oxy) piperidine- l-carboxylate

[0082] To a stirred solution of tert- butyl 4-hydroxypiperidine- l-carboxylate [Sigma- Aldrich US1 (1) (0.96 g, 4.8 mmol) in 10 mL dichloromethane was added triethylamine (1 mL) and stirred at RT (RT) for 5 min 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 RT and then stirred at RT for 2 h. After completion (Monitored by TLC), the reaction was quenched with distilled water and diluted with dichloromethane. The organic extract was dried (MgSCri) and concentrated in vacuo to yield 1.3 g of tert- butyl 4- ((methylsulfonyl)oxy)piperi dine- l-carboxylate (2) as a pale yellow solid. Yield 97 %.

¹ 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: Tert-butyl 4-azidopiperi dine- l-carboxylate

[0083] To a stirred solution of tert-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 RT and then poured into cold water with occasional stirring. The residue was extracted with ethyl acetate (3x10 mL). The combined organic layers were concentrated in vacuo and the residue was dissolved in hexane (50 mL) and washed with water (3x10 mL). The combined organic layers were dried (MgSCL) and concentrated in vacuo to yield 1.75 g of tert-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: T ert-buty 1 4-(4-(ethoxy carbonyl)- \ H- \ ,2,3 -triazol- 1 -yl)piperidine- 1 -carboxylate

[0084] Tert-butyl 4-azidopiperidine-l -carboxylate (3) (0.66 g, 2.9 mmol) was dissolved in acetonitrile (15 mL). 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 RT for 12 h.

After completion (Monitored by TLC), solvent was removed in vacuo. Distilled water was added to the residue and then the crude material was extracted with ethyl acetate (3x10 mL). The combined organic layers were dried (MgSCL) and concentrated in vacuo to yield 0.79 g of the desired compound tert-butyl 4-(4-(etho\y carbonyl)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 - carboxylate (4) as a pale yellow solid. Yield 84%. ¹ 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-(hydrazinecarbonyl)- 1 H- 1 ,2,3-triazol- 1 -yl)piperidine-l -carboxylate

[0085] Tert-butyl 4-(4-(etho\ycarbonyl)- 1 //- 1 2.3-triazol- 1 -yl)piperidine- 1 -carboxylate (1 g, 3.1 mmol) and hydrazine hydrate (0.5 g) were combined in 20 mL of «-butanol and refluxed for 3 h. The solvent was removed in vacuo. The residue was dissolved in dichloromethane and washed with water. The organic phase was dried (MgSCb) and the solvent removed under reduced pressure. The resulting solid (0.93 g) was washed with cold ethanol. Yield 97%.

^! H-NMR (400 MHz, CDCb) d (ppm): 8.08 (s, 1H), 4.63 (tt , J = 11.6, 3.6 Hz, 1H), 4.27 (s, 2H), 4.03 (s, 1H), 2.94 (t, J = 13.0 Hz, 2H), 2.21 (d, J = 12.8 Hz, 2H), 1.94 (qd, J = 12.0, 4.4 Hz, 2H), 1.47 (d, J= 0.6 Hz, 9H).

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

[0086] Tert-butyl 4-(4-(hydrazinecarbonyl)- 1 H- 1 2.3-triazol- 1 -yl) piperidine- 1 -carboxylate (10) (0.5 g, 1.6 mmol) was added to a mixture of 20 mL of THF and 2 mL of DMF. NJT- carbonyldiimidazole (CDI) (0.4 g, 2.5 mmol ) and triethylamine (0.32 g, 3 mmol) were added. After refluxing for 15 hr, the solvent was removed in vacuo. The residue was treated with dichloromethane and washed with water. The organic phase was dried (MgSCri) and the solvent removed under reduced pressure. Chromatography yielded 0.5 g of tert-butyl 4-(4-(5- oxo-4.5-dihydro- 1 3.4-oxadiazol-2-yl)- 1 H- 1 2.3-triazol- 1 -yl)piperidine- 1 -carboxylate. Yield 94%.

¹ H-NMR (400 MHz, CDCb) d (ppm): 9.52 (s, 1H), 8.05 (s, 1H), 4.70 (tt , J = 11.7, 4.1 Hz, 1H), 4.29 (d, J= 11.9 Hz, 2H), 2.96 (t , J = 12.7 Hz, 2H), 2.38 - 2.17 (m, 2H), 2.17 - 1.88 (m, 2H), 1.47 (s, 9H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 154.5, 154.1, 148.7, 133.6, 124.1, 79.4, 58.2, 42.2, 32.0, 28.50.

HRMS (ESI-FIA-TOF): m/z calcd for Ci ₄ H ₂ oN ₆ Na0 ₄ 359.1444, found 359.1438.

Step 6: Hydrochloride 5-( 1 -(piperidin-4-yl)- 1 //- 1 2.3-triazol-4-yl)- 1 3.4-oxadiazol-2(3//)-one

A mixture of tert-butyl 4-(4-(5-oxo-4.5-dihydro- 1 3.4-oxadiazol-2-yl)- 1 H- 1 2.3-triazol- 1 - yl)piperidine-l-carboxylate (11) (100 mg, 0.3 mmol) and 4N HC1 in dioxane (2 mL) was stirred at RT for 2 h. The solvent was removed in vacuo and the resulting yellow solid was triturated with EtOAc to provide 74 mg of the hydrochloride of 5-( 1 -(piperidin-4-yl)- 1 H- l,2,3-triazol-4-yl)-l,3,4-oxadiazol-2(377)-one as a white solid. Yield 91%. ¹ H-NMR (400 MHz, de-DMSO) d (ppm): 13.24 (s, 1H), 9.26 (s, 1H), 9.10 (s, 1H), 8.24 (s, 1H), 5.28 - 5.02 (m, 1H), 3.38 (d, J= 12.7 Hz, 2H), 3.05 (q, J= 11.7 Hz, 2H), 2.40 - 1.99 (m, 4H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 154.0, 148.1, 133.1, 124.2, 54.9, 41.7, 28.2.

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

Step 1 : Tert-butyl 4-(3-(methoxycarbonyl)-li/-l, 2, 4-triazol-l-yl)piperidine-l -carboxylate

[0087] NaOH (1.0 g; 41.7 mmol) was added to a solution of methyl l//~i,2,4~triazole-3- carboxylate [Sigma- Aldrich, US] (2.8 g: 22 mmol) in DMF (130 mL). The reaction mixture was stirred at 25 °C for 20 minutes followed by 1 b at 70 °C, /erf-butyl 4-iodopiperidine-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 and brine. The organic extract was 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%.

¾-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: Tert-butyl 4-(3-(hydra/inecarbonyl)- 1 H- 1 2.4-tria/ol- 1 -yl)piperidine- 1 -carboxylate

[0088] Tert-butyl 4-(3-(methoxycarbonyl) lH !, 2, 4-iriazoI l-yl)piperidme-l -carboxylate

(14) (0.5 g, 1.6 mmol) and hydrazine hydrate (0.25 g) in 20 mL of «-butanol were refluxed for 3 h. The solvent was removed in vacuo. The residue was dissolved in DCM and washed with water. The organic phase was dried (MgSCri) and the solvent was removed under reduced pressure. The resulting solid (0.5 g) was washed with cold ethanol. Yield 95%.

¹ H-NMR (400 MHz, CDCb) d (ppm): d 8.53 (s, 1H), 7.86 (s, 1H), 5.51 (tt , J= 11.4, 4.2 Hz, 1H), 4.27 (s, 2H), 4.03 (s, 2H), 2.90 (s, 2H), 2.16 - 2.02 (m, 2H), 1.96 (d, J= 11.8 Hz, 2H), 1.47 (s, 9H).

Step 3: T ert-butyl 4-(3 -(5 -oxo-4,5 -dihy dro- 1 ,3,4-oxadiazol-2-yl)- 1 H- 1 ,2,4-triazol- 1 - yl)piperidine- 1 -carboxylate

[0089] Tert-Butyl 4-(3-(hydrazinecarbonyl)- 1 H- 1 2.4-triazol- 1 -yl)piperidine- 1 -carboxylate (20) (0.25 g, 0.8 mmol) was dissolved in a mixture of 10 mL THF and 1 mL of DMF. NJT- carbonyldiimidazole (CDI) (0.2 g, 1.2 mmol) and triethylamine (0.16 g, 1.6 mmol) were added. After refluxing for 15 h, the solvent was removed by evaporation under vacuum. The residue was treated with DCM and washed with water. The organic phase was dried (MgSCL) and the solvent removed under reduced pressure. Chromatography yielded 0.24 g of tert- butyl 4-(3-(5-o\o-4.5-dihydro- 1.3.4-oxadia/ol-2-yl)- 1 H- 1 2.4-tria/ol- 1 -yl)piperidine- 1 -carboxyl ate (21). Yield 90%.

³ H-NMR (400 MHz, CDCb) d (ppm): 8.04 (s, 1H), 5.00 (d, J = 11.3 Hz, 1H), 4.29 (s, 2H), 2.90 (s, 2H), 2.24 - 1.92 (m, 4H), 1.49 (s, 9H).

¹³ C-NMR (100.6 MHz, de-DMSO) d (ppm): 153.9, 153.7, 151.2, 145.6, 139.7, 78.9, 56.6, 54.9, 31.1, 28.04.

HRMS (ESI-FIA-TOF): m/z calcd for Cw^oNeNaCri 359.1444, found 359.1438.

Step 4: Hydrochloride of 5-( 1 -(piperidin-4-yl)- 1 H- 1 2.4-triazol-3-yl)- 1 3.4-oxadiazol-2(3//)- one

[0090] A mixture of /er/-butyl 4-(3-(5-oxo-4.5-dihydro- 1 3.4-oxadiazol-2-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 RT for 2 h. The solvent was removed in vacuo and the resulting yellow solid was triturated with EtOAc to provide 77 mg of the hydrochloride of 5-( 1 -( pi peridin-4-y l )- 1 H- l,2,4-triazol-3-yl)-l,3,4-oxadiazol-2(377)-one (22) as a white solid. Yield 95%.

^! H-NMR (400 MHz, de-DMSO) d (ppm): 13.24 (s, 1H), 9.25 (s, 1H), 9.10 (s, 1H), 8.24 (d, J = 0.6 Hz, 1H), 5.18 - 5.03 (m, 1H), 3.38 (d, J= 12.7 Hz, 2H), 3.05 (q, J= 11.7 Hz, 2H), 2.33 - 2.08 (m, 4H).

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

Derivative Effect on Fibrinolytic Activity in Human Plasma [0091] The effect of l,3,4-oxadiazole derivatives of the disclosure on fibrinolytic activity in human plasma was determined by a one-step spectrophotometric method as follows.

[0092] Blood from healthy volunteers (15 ml) was anticoagulated with ACD and plasma was separated by centrifugation 3,000 rpm for 10 min at RT.

[0093] 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 dEhO to a final concentration of 100 mg/ml.

[0094] Fibrin clot formation in human plasma was induced as follows: An aliquot of 150 ml of plasma was incubated in a 96-well plate at 37°C during 30 min in the presence of 50 ml of reconstituted Thromborel S reagent (Fisher Scientific, USA), and 20 ml of 1 mM Tris-HCl buffer and clot formation was quantified spectrophotometrically at 340 nm.

[0095] The effect of 1,3,4- oxadiazole derivatives on both fibrin clot formation and clot lysis was determined as follows: An aliquot of 150 ml of plasma was incubated in a 96 well- plate at 37°C for 30 min in the presence of 50 ml of reconstituted Thromborel S reagent (Siemens Healthineers), 10 ml of reconstituted human recombinant t-PA and 10 ml of each 1,3,4- oxadiazole derivate (below in Table 1 at different concentrations and clot formation followed by clot lysis were quantified spectrophotometrically at 340 nm.

TABLE 1

[0096] The results are shown in Table 2.

TABLE 2

All of the tested derivatives of the present disclosure had IC50 values below 100 mM.

EXAMPLE 3

Specificity of Inhibitory Effect on Serine Protease Activity

[0097] The effect of a 1,3,4-oxadiazole derivative on several serme protease activities was determined by chromogenic assays. The serine proteases and chromogenic substrates used (Diapharma, USA) are described below in TABLE 3.

TABLE 3

[0098] Each vial of human recombinant t-PA (Sigma- Aldrich, US) was reconstituted with dEhO 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) at 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 mi of PBS to a final concentration of 50 pg / l. Each vial of recombinant human thrombin (Sigma- Aldrich, USA) containing 100 NIH units/mg of protein was reconstituted with CIH2O to a final concentration of 100 NIH units/mg of protein per ml. Each vial of trypsin from human pancreas (Sigrna- Aldrich, USA) containing 1,000 BAEE units was reconstituted with CIH2O 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 dfhO to a final concentration of 10 BTEE units/ml. Each vial of coagul ation Factor Xa from human plasma (Sigma- Aldrich, USA) containing 170 units/mg of protein was reconstituted with dH ₂ 0 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 w as reconstituted with dH ₂ 0 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.

[0099] Chromogenic assays were performed as follows: An aliquot of 900 mΐ 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 mΐ of each reconstituted proteolytic enzyme and 10 mΐ of 1 mM Tris-HCl buffer, mix and read each 2 min the absorbance at 405 nm.

[0100] The effect of 1,3,4- oxadiazole derivatives on proteolytic activity by chromogenic assays was determined as follows: An aliquot of 900 mΐ 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 mΐ of each reconstituted proteolytic enzyme and 10 mΐ of each 1,3,4- oxadiazole derivate at different concentrations and inhibitory effect was quantified spectrophotometrically at 405 nm.

[0101] The results are shown in TABLE 4

TABLE 4

compounds at 00 mM able to inhibit chromogenic substrate hyc rolysis mediated specifically by each serine protease

compounds at 1000 mM unable to inhibit chromogenic substrate hydrolysis mediated specifically by each serine protease

[0102] As shown in Table 4, 1,3,4- oxadiazole derivatives of present disclosure displayed specific inhibitory effect on Plasmin, tPA and uPA activities and Plasminogen activation mediated by tPA at below' 100 mM concentration. How- ever, no effects at 1000 mM of concentration on Thrombin, Factor X activated, Kaliikrein, Trypsin and Chymotrypsin, were detected.

EXAMPLE 4

Anti-Cancer Activities of Derivatives

[0103] The following assays were performed to evaluate the inhibitory effect mediated by l,3,4-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).

[0104] 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,3,4-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 hours 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.

[0105] All the compounds of the present invention were able to inhibit significantly the proliferation and migration at below 100 mM concentration.

EXAMPLE 5

Coagulation and Fibrinolytic Activities of Derivaives

[0106] Human blood (15 ml) was anticoagulated with ACD and plasma was separated by centrifugation 3,000 rpm for 10 min at RT.

[0107] Both fibrin clot formation and clot lysis assays in human plasma were performed simultaneously as follows: 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), 10 mΐ of reconstituted human recombinant t-PA and 10 mΐ of 1 mM Tris-HCl buffer and clot formation followed by clot lysis were quantified spectrophotometrically at 340 nm.

[0108] The effect of 1,3,4- oxadiazole derivatives on both fibrin clot formation and clot lysis was determined as follows: An aliquot of 150 mΐ of plasma was incubated in a 96 well- plate at 37°C during 30 min in the presence of 50 mΐ of reconstituted Thromborel S reagent, 10 mΐ of reconstituted human recombinant t-PA and 10 mΐ of each 1,3,4- oxadiazole derivate at different concentrations and clot formation followed by clot lysis were quantified

spectrophotometrically at 340 nm. The results are shown in FIG 2.

EXAMPLE 6

Testing of Coagulation Activities of Derivative in Mouse Model

[0109] To test the anti-bleeding effect of l,3,4-oxadiazole derivatives, in vivo experiments were performed using mice with congenital coagulation anomalies.

[0110] 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 l,3,4-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

[0111] 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.