AU4508601A - Inhibitors of collagen-induced platelet aggregation - Google Patents

Description

WO 01/41754 PCT/USOO/42370 Inhibitors of Collagen-Induced Platelet Aggregation 5 This application is being filed as a PCT International Patent application in the name of Parker Hughes Institute, a U.S. national corporation, (applicant for all countries except US), and Fatih M. Uckun, a U.S. citizen (applicant 10 for US only), on 29 November 2000, designating all countries. Field of the Invention 15 The present invention relates to a therapeutic method for treating or preventing a disease or condition of platelet aggregation in a subject wherein the method includes administering a pharmaceutically effective amount of a compound that inhibits platelet aggregation and specifically, collagen-induced platelet aggregation. 20 Background of the Invention Heart disease, a common cause of death in today's society, is often a result of ischemic syndromes that are produced by atherosclerosis and arteriosclerosis including myocardial infarction, chronic unstable angina, transient 25 ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and other cardiovascular devices. These syndromes represent a variety of stenotic and occlusive vascular disorders thought to be initiated by platelet aggregation on vessel walls or within the lumen by 30 blood-born mediators thereby forming collagen that restrict blood flow. The basic mechanism of platelet aggregation has been well-studied. The mechanism starts with a blood vessel injury such as narrowing of the lumen, plaque formation, and the presence of foreign bodies/medical instruments. This injury leads to platelet activation and binding of fibrinogen and ligands. 35 Bruton's tyrosine kinase (BTK), a member of the BTK/Tec family of protein tyrosine kinases (PTKs) is a cytoplasmic PTK involved in signal transduction pathways regulating growth and differentiation of B-lineage lymphoid WO 01/41754 PCT/USOO/42370 cells (Rawlings, D. J., and Witte, 0. N. (1994) Immunol. Rev. 138, 105-119; Kurosaki, T. (1997) Curr Opin. Immunol. 9, 309-318; and Uckun, F. M. (1998) Biochemical Pharmacology, et al., 56, 683-691). BTK participates in signal transduction pathways initiated by the binding of a variety of extracellular ligands to 5 their cell surface receptors. Following ligand binding of B cell antigen receptors (BCR), BTK activation by the concerted actions of the PTKs Lyn and Syk (Kurosaki, T. (1997) Curr Opin. Immunol. 9, 309-318) is required for induction of phospholipase C-y2 mediated calcium mobilization (Kurosaki, T. (1997) Curr Opin. Immunol. 9, 309-318). BTK participates in the collagen receptor glycoprotein VI 10 (GP VI)-Fc receptor gamma (FcRy) chain coupled signaling. Tyrosine phosphorylation of the immune-receptor tyrosine based activation motif (ITAM) of the FcRy chain leads to phosphorylation and activation of phosphoslipase C gamma 2 (PLCy 2). Activated PLCy 2 converts PI-4,5-bisphospate (PIP2) to inositol triphosphate (IP3), leading to intracellular calcium mobilization. 15 Thrombin induced platelet aggregation describes platelet aggregation in response to the enzyme thrombin, which is formed in blood from prothrombin. Collagen induced platelet aggregation describes platelet aggregation in response to the protein collagen. Gelotte, U.S. Pat. No. 5,972,967 and Scarborough, et al. U.S. Patent 20 No. 5,968,902 have described certain compounds and compositions that inhibit binding to a platelet by limiting the binding of fibrinogen. Nevertheless, there still is a need for finding compounds and improved methods to treat or prevent a condition of platelet aggregation. 25 Summary of the Invention In accordance with the purpose(s) of this invention, as embodied and broadly described herein, this invention, in one aspect, relates to a method for inhibiting platelet aggregation by administering an effective amount of a compound of the formula: 2 WO 01/41754 PCT/USOO/42370 CN H Br Me N OH 0 Br or a pharmaceutically acceptable acid addition salt thereof. In a second aspect, the invention relates to a method of preventing or treating a disease or condition of platelet aggregation in a subject comprising 5 administering to a subject an effective amount of a compound of the formula: CN H Br Me N OH 0 Br or a pharmaceutically acceptable acid addition salt thereof. Additional advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention. The advantages of the invention will be 10 realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. The accompanying drawings, which are incorporated in and 15 constitute a part of this specification, illustrate several experimental examples and together with the description, serve to explain the principles of the invention Brief Description of the Drawings Figures la- If are graphs showing experimental results for Example 20 1, including the platelet aggregation effects of collagen at 5 micrograms per milliliter for 20 minutes (Fig. la) and 24 hours (Fig. ld) and collagen at 2 3 WO 01/41754 PCT/USOO/42370 micrograms per milliliter for 20 minutes (Fig. 1b) and 24 hours (Fig. le) and thrombin for 20 minutes (Fig. 1 c) and 24 hours (Fig. 1 f). Figure 2 is a graph showing experimental results for Example 2, including the selective, dose-dependent inhibition of collagen-induced platelet 5 aggregation by LFM-A1 3. Description of the Preferred Embodiments The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and 10 the Examples included therein and to the Figures and their previous and following description. In this specification and in the claims that follow, reference will be made to a number of terms that shall be defined to have the following meanings: Reference in the specification and concluding claims to parts by 15 weight of a particular component in a composition, denotes the weight relationship between the component and any other components in the composition for which a part by weight is expressed. By "platelet aggregation" is meant the clumping together of platelets or red blood cells. As used herein, "inhibiting platelet aggregation" includes 20 slowing platelet aggregation, as well as completely eliminating and/or preventing platelet aggregation. Additionally, "inhibiting platelet function" includes decreasing platelet function, as well as completely eliminating and/or preventing the platelet function. Conditions of platelet aggregation include, but are not limited to, embolus formation, thrombolytic complications, disseminated intravascular comgelopathy, 25 thrombosis, coronary heart disease, thromboembolic complications, myocardial infarction, restenosis, and atrial thrombosis formation in atrial fibrillation, chronic unstable angina, transient ischemic attacks and strokes, peripheral vascular disease, arterial thrombosis, preeclampsia, embolism, restenosis and/or thrombosis following angioplasty, carotid endarterectomy, anastomosis of vascular grafts, and chronic 30 exposure to cardiovascular devices. Such conditions may also result from thromboembolism and reocclsion during and after thermbolytid therapy, after angioplasty, and after coronary artery bypass. 4 WO 01/41754 PCT/USOO/42370 "Thrombin induced platelet aggregation" includes platelet aggregation in response to the enzyme thrombin, which is formed in blood from prothrombin. "Collagen induced platelet aggregation" includes platelet aggregation 5 in response to the protein collagen. As used throughout, by "contacting" is meant an instance of exposure of at least one cell (e.g., a neural cell, a stem cell, a cardiac cell) to an agent (e.g., a compound that inhibits platelet aggregation and specifically, collagen induced platelet aggregation). 10 The term "subject" is meant an individual. Preferably, the subject is a mammal such as a primate, and more preferably, a human. Thus, the "subject" can include domesticated animals (e.g., cats, dogs, etc.), livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), and laboratory animals (e.g., mouse, rabbit, rat, guinea pig, etc.). 15 In general, "therapeutically effective amount", "therapeutically effective dose" and "effective amount" means the amount needed to achieve the desired result or results (treating or preventing platelet aggregation). One of ordinary skill in the art will recognize that the potency and, therefore, an "effective amount" can vary for the various compounds that inhibit platelet aggregation and 20 specifically, collagen induced platelet aggregation used in the invention. One skilled in the art can readily assess the potency of the compounds. By "pharmaceutically acceptable" is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected bicyclic compound without causing any 25 undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. A preferred compound for use in the present invention is ax-Cyano-p hydroxy-p-methyl-N-(2,5-dibromophenyl)-propenamide (LFM-A13), and is structurally shown below (formula I): 5 WO 01/41754 PCT/USOO/42370 CN H Br Me N OH 0 Br (I) or a pharmaceutically acceptable salt thereof. Characterization data of a.-Cyano-p-hydroxy-p-methyl-N-(2,5 5 dibromophenyl)-propenamide (LFM-A13) is shown below: mp: 148 - 150'C; IR (KBr): 3353, 2211, 1648 and 1590 cm- 1 ; 'H NMR (DMSO-d 6 ): 6 11.41 (s, 1H, NH), 8.57 (d, J= 2.4 Hz, 1H, ArH), 7.55 (d, J= 8.7 Hz, 1H, ArH), 7.14 (dd, J= 8.7, 2.4 Hz, 1H, ArH), 7.10 (s br, 1H, OH), 2.17 (s, 3H, CH 3 ); MS (El) 10 m/z 362 (M*+ 4), 360 (M*+ 2), 358 (M*), 253, 251, 249, 150. Pharmaceutically acceptable salts of x-Cyano-p-hydroxy-p-methyl N-(2,5-dibromophenyl)-propenamide (LFM-A13), or any other compound useful in the present invention, may be used in the present invention. Examples of acceptable 15 salts are organic acid addition salts formed with acids, which form a physiological acceptable anion, including, but not limited to, tosylate, methanesulfonate, acetate, citrate, malonate, tartarate, succinate, benzoate, ascorbate, a-ketoglutarate, and C glycerophosphate. Suitable inorganic salts may also be formed, including, but not limited to, hydrochloride, sulfate, nitrate, bicarbonate, and carbonate salts. 20 Acceptable salts may be obtained using standard procedures well known in the art, for example by reacting a sufficiently basic compound such as an amine with a suitable acid affording a physiologically acceptable anion. 25 Synthetic Methods: The compound of the present invention is readily synthesized using techniques generally known to synthetic organic chemists. Suitable experimental 6 WO 01/41754 PCTIUSOO/42370 methods for making and derivatizing the compound is described in PCT Published Application No. W099/54286 to Uckun et al., the disclosure of which is hereby incorporated by reference. 5 Utility and Administration: The therapeutic method included herewith is useful for treating or preventing a condition of platelet aggregation, in a subject comprising administering a pharmaceutically effective amount of a compound or composition that inhibits BTK and that inhibits platelet aggregation, specifically, collagen induced platelet 10 aggregation. The condition of platelet aggregation includes cardiovascular, hematopoietic and cerbrovascular diseases such as, but not limited to, embolus formation, thrombolytic complications, disseminated intravascular comgelopathy, thrombosis, coronary heart disease, thromboembolic complications, myocardial 15 infarction, restenosis, or atrial thrombosis formation in atrial fibrillation. Such platelet aggregation inhibition may selectively target the collagen pathway, over other pathways including thrombin induced platelet aggregation. The methods include contacting the cells with such compound or compositions, or administering to the subject a therapeutically effective amount of 20 these compound or compositions. In one embodiment, the cells are part of the blood and immune system including: red blood cell, megakaryocytes, macrophages (e.g. monocytes, connective tissue macrophages, Langerhans cells, osteoclasts, dendritic cells, microglial cells), neutrophils, eosinophils, basophils, mast cells, T lymphocytes (e.g. helper T cells, suppressor T cells, killer T cells), B lymphocytes 25 (e.g. IgM, IgG, IgA, IgE), killer cell, and stem cells and committed progenitors for the blood and immune system. In another embodiment, the cells are contractile cells such as skeletal muscle cells (e.g. red, white, intermediate, muscle spindle, satellite cells), heart muscle cells (e.g. ordinary, nodal, Purkinje fiber), smooth muscle cells, and myoepithelial cells. 30 It is well known in the art how to determine the inhibition of platelet aggregation using the standard tests described herein, or using other similar tests. Preferably, the method would result in at least a 10% reduction in collagen-induced platelet aggregation, including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount in between, more preferably by 90%. 7 WO 01/41754 PCT/USOO/42370 Similarly, the method would result in at least a 10% reduction in collagen-induced intracellular calcium mobilization including, for example, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. Similarly, the method would result in at least a 10% reduction in the level of phosphorylated PLCg 2 including, for example, 5 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%. The reduction can be measured, for example, by comparing the optical impedence in a chronology platelet aggregometer. Any other known measurement method may also be used. For example, (1) upon collagen stimulation, the level of collagen-induced intracellular calcium mobilization increases over time 10 and so the measurement may include measuring the level of collagen-induced intracellular calcium or (2) upon collagen stimulation, the level of phosphorylated PLCg 2 increases over time and so the measurement may include measuring the level of phosphorylated PLCg 2. The cells can be contacted in vitro, for example, by adding the 15 compound to the culture medium (by continuous infusion, by bolus delivery, or by changing the medium to a medium that contains the agent) or by adding the agent to the extracellular fluid in vivo (by local delivery, systemic delivery, inhalation, intravenous injection, bolus delivery, or continuous infusion). The duration of "contact" with a cell or population of cells is determined by the time the compound 20 is present at physiologically effective levels or at presumed physiologically effective levels in the medium or extracellular fluid bathing the cell or cells. Preferably, the duration of contact is 1-96 hours, and more preferably, for 24 hours, but such time would vary based on the half life of the compound and could be optimized by one skilled in the art using routine experimentation. 25 Pharmaceutical Formulations The compound useful in the present invention can be formulated as pharmaceutical compositions and administered to a mammalian host, such as a human patient or a domestic animal in a variety of forms adapted to the chosen route 30 of administration, i.e., orally or parenterally, by intranassalyy by inhalation, intravenous, intramuscular, topical or subcutaneous routes. The compound of the present invention can also be administered using gene therapy methods of delivery. See, e.g., U.S. Patent No. 5,399,346, which is incorporated by reference in its entirety. Using a gene therapy method of 8 WO 01/41754 PCT/USOO/42370 delivery, primary cells transfected with the gene for the compound of the present invention can additionally be transfected with tissue specific promoters to target specific organs, tissue, grafts, tumors, or cells. Thus, the present compound may be systemically administered, e.g., 5 orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They may be enclosed in hard or soft shell gelatin capsules, may be compressed into tablets, or may be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound may be combined with one or more excipients and used in the form of 10 ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations may, of course, be varied and may conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such 15 therapeutically useful compositions is such that an effective dosage level will be obtained. The tablets, troches, pills, capsules, and the like may also contain the following: binders such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato 20 starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such as sucrose, fructose, lactose or aspartame or a flavoring agent such as peppermint, oil of wintergreen, or cherry flavoring may be added. When the unit dosage form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier, such as a vegetable oil or a polyethylene glycol. Various other 25 materials may be present as coatings or to otherwise modify the physical form of the solid unit dosage form. For instance, tablets, pills, or capsules may be coated with gelatin, wax, shellac or sugar and the like. A syrup or elixir may contain the active compound, sucrose or fructose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any 30 material used in preparing any unit dosage form should be pharmaceutically acceptable and substantially non-toxic in the amounts employed. In addition, the active compound may be incorporated into sustained-release preparations and devices. 9 WO 01/41754 PCTIUSOO/42370 The active compound may also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, 5 and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile 10 injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form must be sterile, fluid and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), 15 vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, 20 phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin. 25 Sterile injectable solutions are prepared by incorporating the active compound in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying 30 techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. For topical administration, the present compound may be applied in pure form, i.e., when they are liquids. However, it will generally be desirable to 10 WO 01/41754 PCT/USOO/42370 administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which may be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina and the like. Useful liquid carriers include 5 water, hydroxyalkyls or glycols or water-alcohol/glycol blends, in which the present compound can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and 10 other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers. Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, 15 and the like, for application directly to the skin of the user. Examples of useful dermatological compositions which can be used to deliver the compound of formula I to the skin are known to the art; for example, see Jacquet et al. (U.S. Pat. No. 4,608,392), Geria (U.S. Pat. No. 4,992,478), Smith et al. (U.S. Pat. No. 4,559,157) and Wortzman (U.S. Pat. No. 4,820,508). 20 Useful dosages of the compound can be determined by comparing their in vitro activity, and in vivo activity in animal models. Methods for the extrapolation of effective dosages in mice, and other animals, to humans are known to the art; for example, see U.S. Pat. No. 4,938,949. Generally, the concentration of the compound(s) of formula I in a 25 liquid composition, such as a lotion, will be from about 0.1-25 wt-%, preferably from about 0.5-10 wt-%. The concentration in a semi-solid or solid composition such as a gel or a powder will be about 0.1-5 wt-%, preferably about 0.5-2.5 wt-%. The amount of the compound, or an active salt or derivative thereof, required for use in treatment will vary not only with the particular salt selected but 30 also with the route of administration, the nature of the condition being treated and the age and condition of the patient and will be ultimately at the discretion of the attendant physician or clinician. Also the dosage of the compound varies depending on the target cell, tumor, tissue, graft, or organ. 11 WO 01/41754 PCT/USOO/42370 In general, however, a suitable dose will be in the range of from about 0.5 to about 100 mg/kg, e.g., from about 10 to about 75 mg/kg of body weight per day, such as 3 to about 50 mg per kilogram body weight of the recipient per day, preferably in the range of 6 to 90 mg/kg/day, most preferably in the range of 15 to 5 60 mg/kg/day. The compound may conveniently be administered in unit dosage form; for example, containing 5 to 1000 mg, conveniently 10 to 750 mg, most conveniently, 50 to 500 mg of active ingredient per unit dosage form. Ideally, the active ingredient should be administered to achieve peak 10 plasma concentrations of the active compound of from about 0.0005 to about 300 pLM, preferably, about .001 to 100 ptM, more preferably, about 1 to about 100 [tM. This may be achieved, for example, by the intravenous injection of a concentration of the active ingredient, optionally in saline, or orally administered as a bolus. Desirable blood levels may be maintained by continuous infusion to provide about 15 0.0005-50.0 mg/kg/hr or by intermittent infusions containing about 0.004-150 mg/kg of the active ingredient(s). The desired dose may conveniently be presented in a single dose or as divided doses administered at appropriate intervals, for example, as two, three, four or more sub-doses per day. The sub-dose itself may be further divided, e.g., into 20 a number of discrete loosely spaced administrations; such as multiple inhalations from an insufflator or by application of a plurality of drops into the eye. An administration regimen could include long-term, daily treatment. By "long-term" is meant at least two weeks and preferably, several weeks, months, or years of duration. Necessary modifications in this dosage range may be 25 determined by one of ordinary skill in the art using only routine experimentation given the teachings herein. See Remington's Pharmaceutical Sciences (Martin, E.W., ed. 4), Mack Publishing Co., Easton, PA. The dosage can also be adjusted by the individual physician in the event of any complication. The invention will now be illustrated by the following non-limiting 30 Examples. Experimental The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how the 12 WO 01/41754 PCT/USOO/42370 compounds, compositions, articles, devices, and/or methods claimed herein are made and evaluated, and are intended to be purely exemplary of the invention and are not intended to limit the scope of what the inventors regard as their invention. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, 5 temperature, etc.) but some errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, temperature is in 'C or is at ambient temperature, and pressure is at or near atmospheric. Example 1 10 Platelets treated with 100pM LFM-A13 for 20 minutes or 24 hours and were stimulated with 0.1 U/ml thrombin or 10pg/ml collagen. Platelet aggregation with respect to control was monitored in a Chronolog Model 560 Dual Chamber Platelet Aggregometer. LFM-A13 significantly reduced the platelet response to high and low doses of collagen (as shown in Fig. la, 1d and Fig. Ib, le). 15 No significant effect with LFM-A13 was noted on the thrombin induced platelet aggregation (shown in Fig. 1 c, 1f). Platelets treated with LFM-A 13 for 24 hours responded in the same manner as those treated with the compound for 20 minutes. The results are shown on Fig. 1. 20 Example 2 Platelets treated with varying concentration s of LFM-A13 were stimulated with 2 and jtg/ml collagen and 0.1 U/ml thrombin. Platelet aggregation with respect to control was monitored in a Chronolog Model 560 Dual Chamber Platelet Aggregometer. LFM-A13 significantly reduced the platelet response to 2 25 ptg/ml collagen with an IC 50 value of 2.78tM and the platelet response to 5 Vtg/ml collagen with an IC 50 value of 66.1 tM. No significant effect was noted on the thrombin-induced platelet aggregatory response. The results are charted on Fig. 2. Example 3 30 Platelets were incubated with 100 ptM LFM-A13 or DMSO for 30 minutes at 37 degrees C and then stimulated with 2 ptg/ml collagen. The samples were subjected to immunoprecipiation utilizing antibodies raised against BTK. The BTK immune complexes were subjected to immune kinase assays. Additional BTK 13 WO 01/41754 PCTIUSOO/42370 immune complexes were collected, boiled in 2x SDS reducing sample buffer, fractionated on 8% polyacrylamide gels, transferred to PVDF membranes and examined for the presence of BTK by western blotting analysis. The enzymatic activity (activity index) of BTK was estimated by comparing the 5 autophosphorylation (PIU) to the relative density of the protein bands in densitometric scanning units (DSU). The results are shown in Table 1 and indicate that LFM-A13 significantly inhibited BTK kinase activity. Table 1 Measurement DMSO DMSO LFM-A13 LFM-A13 0 secs 90 secs 0 secs 90 secs PIU 1912 3012 78 68 DSU 2489 1590 1236 1432 Activity 0.77 1.89 0.17 0.05 10 Example 4 Platelets were incubated with 100 ptM LFM-A13 or DMSO for 30 minutes at 37 degrees C and then stimulated with 2 tg/ml collagen. The samples were subjected to immunoprecipiation utilizing antibodies raised against PLCg2 and 15 immunoblotted against antibodies against phosphotyrosine. The membrane was stripped and reprobed with antibodies raised against PLCg2. An increase in the level of phosphorylated PLCg2 was seen between 30 and 45 seconds following collagen stimulation. No phosphorylation of PLCg2 was noted with LFM-Al3 treatment. Thus, LFM-A13 inhibited collagen induced PLCg2 tyrosine 20 phosphorylation. Example 5 Platelets were loaded with 3 mM fura-3 (Molecular Probes, Eugene Oregon) for 15 minutes at 37 degrees C. Following a short wash (10 minutes, 3000 25 rpm), the platelets were resuspended in Hepes buffer (1x10 8 cells/ml) and stimulated with collagen (20 [tg/ml) in the presence or absence of 100 tM LFM-A13. The changes in fluorescence were monitered on a spectrofluorimeter. LFM-A13 significantly reduced (inhibited) collagen-induced calcium mobilization. 14 WO 01/41754 PCTUSOO/42370 Example 6 Platelets (1x10 9 ) were incubated with and without 100 tM LFM-A13 for 30 minutes at 37 degrees C. Platelets were stimulated at 37 degrees C with 2 5 tg/ml collagen for various times. Activation was stopped by the addition of ice cold 20% perchloric acid. The amount of inositol 1,4,5-triphosphate production was measured using a detection kit from Amersham. The results show that LFM-Al 3 inhibited collagen-stimulated PI-PLC activity. The results are shown in Table 2 below. Treatment % control inositol 1,4,5- Standard % inhibition triphosphate production error (peak response) saline 100 0 LFM-A13 75 23 collagen 321 23 collagen and 75 23 100 LFM-A13 10 Example 7 The clotting times of C57BL/6 mice both chronically (25mg/kg/36 days) and acutely (40 mg/kg/i hour) treated with LFM-A13 were compared with the clotting times of XID and control mice. The blood of XID mice clotted 15 approximately two minutes later than the CBA control mice. Following this pattern, blood from chronically treated mice also clotted two minutes later than control. The results show that LFM-A13 decreases blood clotting time. Results are shown below in Table 3. 15 WO 01/41754 PCT/USOO/42370 Table 3 Mouse Type Mean Clotting Standard Error % Control Time (min) n=3 CBA 1.3 0.11 XID 3.2 0.27 246 C57BL/6 DMSO 1.6 0.20 control C57BL/6 25mg/kg 3.7 0.27 231 chronic 36 days C57BL/6 40mg/kg 2.6 0.43 164 acute 1 hour Throughout this application, various publications are referenced. The 5 disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pertains. It will be apparent to those skilled in the art that various modifications and variations can be made in the present invention without departing 10 from the scope or spirit of the invention. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with a true scope and spirit of the invention being indicated by the following claims. 16

Claims (6)

1. A method for inhibiting platelet aggregation in a subject comprising 5 administering an effective amount of a compound of the formula: CN H Br Me N OH 0 Br or a pharmaceutically acceptable acid addition salt thereof.

2. The method of claim 1, wherein the compound selectively inhibits collagen induced platelet aggregation. 10

3. The method of claim 1, wherein the method specifically acts to inhibit collagen-induced platelet aggregation without significantly inhibiting thrombin-induced platelet aggregation. 15

4. The method of preventing or treating a disease or condition of platelet aggregation in a subject comprising administering to a subject an effective amount of a compound of the formula: 17 WO 01/41754 PCTIUSOO/42370 CN H Br Me N OH 0 Br or a pharmaceutically acceptable acid addition salt thereof

5. The method of claim 4, wherein the disease or condition comprises a cardiovascular, cerbrovascular, or hematologic disease at risk for 5 thromboembolic complications.

6. The method of claim 5, wherein the condition of platelet aggregation comprises embolus formation, thrombolytic complications, disseminated intravascular comgelopathy, thrombosis, coronary heart disease, 10 thromboembolic complications, myocardial infarction, restenosis, or atrial thrombosis formation in atrial fibrillation. 18