The use of 2-phenyl-3-koivistoinen to increase expression of thrombomodulin


(57) Abstract:

A new tool to increase expression of thrombomodulin, representing a compound of formula l

< / BR>
in which R1and R3independently represent hydrogen, -CH3,

< / BR>
where Ar represents optionally substituted phenyl; R2selected from the group consisting of pyrrolidino, hexamethyleneimino and piperidino; or its pharmaceutically acceptable salt or solvate. The invention extends the range of means for inhibiting thrombotic disorders by expression of thrombomodulin. 3 C.p. f-crystals, 8 PL.

The process of blood clotting and thrombus formation is called a complex proteolytic cascade leading to the formation of thrombin. Thrombin proteoliticeski removes the activation peptides of A - and B-chains of fibrinogen, which dissolves in the blood plasma, initiating the formation of insoluble fibrin.

The anticoagulation is usually achieved by the introduction of heparins and coumarins. Parenteral pharmacological regulation of coagulation and clot formation is based on the inhibition of thrombin by the use of heparins. The heparins indirectly vandenhaute thrombin/. Because the level of anti-thrombin III in the plasma is changed, and because of surface-bound thrombin is stable to such indirect mechanism of action, treatment with heparins may be ineffective. Because I believe some testing coagulation are efficient and reliable, the level of heparin is usually controlled using tests for coagulation /in particular, determining the time activated partial thromboplastin (the activated partial thromboplastin time /APTT/assay)/. Coumarins inhibit the generation of thrombin, blocking posttranslational gamma-carboxylation in the synthesis of prothrombin and other proteins of this type. Due to the mechanism of their effects, the effects of coumarins may occur only slowly, after 6 to 24 hours after injection. In addition, they are not selective anticoagulants. Coumarins also require control by testing for coagulation /in particular, the determination of prothrombin time/.

For a better understanding of the invention the following is a brief description of the system the enzymatic coagulation. The coagulation system, sometimes also called the "cascade", is best seen as a chain reaction involving sequential activation is on. Thrombin through limited proteolysis converts plasma fibrinogen into an insoluble gel fibrin. Two key moments in the coagulation cascade is the conversion of coagulation factor X and Xa by means of coagulation factor IXa and the conversion of prothrombin to thrombin by the action of coagulation factor Xa.

Both reactions occur on the surface of cells, most noticeable on the surface of the lamellar endothelial cells, and both reactions require cofactors. The main cofactors factor V and VIII - circulate as a relatively inactive precursors, but when the formation of the first few molecules of thrombin, thrombin by limited proteolysis activates cofactors. Activated cofactors Va and VIIIa - accelerate by about three orders of magnitude as the conversion of prothrombin to thrombin, and the conversion of factor X into factor Xa.

Activated protein C in most cases prefers two plasma protein-substrate, it hydrolyzes and irreversibly destroys. These plasma protein substrates are activated forms of coagulation cofactors V and VIII /cofactors Va and VIIIa, respectively/. Activated protein C only minimally disrupts inactive predshestvennikami main physiological fibrinolytic enzyme tissue plasma activator.

However, activation of protein C affects thrombin, the final serine protease in the coagulation cascade and is associated with the membranes of endothelial cell glycoprotein - thrombomodulin. Thrombomodulin forms a strong stoichiometry is 1:1 complex with thrombin. Thrombomodulin, when it forms a complex with thrombin, significantly modifies the properties of thrombin. Thrombin in the coagulation cascade usually roll fibrin, activates platelets and turns the coagulation cofactors V and VIII in their active forms Va and VIIIa. Thrombin, when one acts as an activator of protein C, but only very slowly and inefficiently. In contrast, thrombin in complex 1: 1 with thrombomodulin not able to move fibrin, activates platelets and does not make the clotting factors V and VIII in their active forms. The complex of thrombin with thrombomodulin promotiom activation of protein C, but the rate constant for the activation of protein C 20,000 times higher for the complex of thrombin C thrombomodulin than thrombin.

Therefore, activated protein C is an antithrombotic agent with a wider therapeutic index than the other and the protein C, neither activated protein C are not effective as long as no separate place degenererede thrombin. Activated protein C is actually ineffective without thrombin, since thrombin is needed in order to make the clotting factors V in Va, and factor VIII clotting - VIIIa. As noted, the activated form of the two cofactors are the preferred substrate for activated protein C. When administered to patients zymogen protein C will remain inactive until such time as it is generated thrombin. Without complex thrombomodulin with thrombin zymogen protein C is converted to activated protein C with a very low speed. The present invention is directed to the disclosure compounds, which are defined below, which increase the expression of thrombomodulin, and biologically suitable for oral administration.

The present invention provides methods for increasing the expression of thrombomodulin, including introduction to people in need of such treatment, an effective amount of the compounds of formula I

< / BR>
in which R1and R3independently represent hydrogen, -CH3,

< / BR>
where Ar represents an optional samisen pharmaceutically acceptable salt or solvate.

The invention also covers a method of inhibiting thrombotic complications or cases, which includes an introduction to the needy in this person an effective amount of the compounds of formula I.

The invention also covers a method of increasing the rate of activation of protein C, which includes the introduction of compounds of formula I.

The present invention relates to the discovery that 2-phenyl-3-aeroelasticity /benzothiophene/ selected group having the formula I, suitable for increasing the expression of thrombomodulin. The ways offered by the present invention, in practice, represent the introduction of a human or mammal in need this, doses of the compounds of formula I or its pharmaceutically acceptable salt, or MES, which is effective to increase expression of thrombomodulin. The present method includes both medical treatment and/or prophylactic treatment, depending on the purpose.

The term "inhibition" usually includes values that include delay, warning, braking, slowing down or reversal.

Raloxifene connection of the present invention represents a hydrochloride ASS="ptx2">

Usually the connection is formulated with conventional excipients, diluents or carriers, and compressed into tablets, or formulated as elixirs or solutions for conventional oral administration, or administered intramuscularly or intravenously. Connections can be introduced percutaneously and can be formulated in dosage forms with prolonged release, etc.

Compounds used in the methods of the present invention can be obtained in accordance with the developed techniques, such as are described in U.S. patent N 4133814, 4418068 and 4380635, which are all included in the present invention as references. Generally, the method begins with benzo[b] thiophene containing the 6-hydroxyl group and 2-(4-hydroxyphenyl) group. In the original connection introducing a protective group, alkylate or acelerou, and remove the protective group, to obtain compounds of formula I. Examples of the preparation of such compounds are given in the aforementioned U.S. patents. Optionally substituted phenyl includes phenyl and phenyl substituted by one or two groups from among (C1-C6)-alkyl, (C1-C4)-alkoxyl, hydroxy-group, nitro group, chlorine, fluorine or three(chloro or fluoro)methyl.

With the wide near organic acids and inorganic acids and bases, which are often used in pharmaceutical chemistry. Such salts are also part of the present invention. Typical inorganic acids used for the formation of such salts include hydrochloric acid, Hydrobromic, idiscovered, nitric, sulfuric, phosphoric, hypophosphorous acid, etc. can Also be used salts derived from organic acids such as aliphatic mono - and dicarboxylic acids, fatty one - and dibasic hydroxy acid, aromatic acids, aliphatic and aromatic sulfonic acids. Thus, pharmaceutically acceptable salts include acetates, phenylacetate, triptoreline, acrylates, ascorbate, benzoate, chlorobenzoate, dinitrobenzoate, hydroxybenzoate, methoxybenzoate, methylbenzoate, acetoxybenzoic, naphthalene-2-benzoate, bromides, isobutyrate, phenylbutyrate-hydroxybutyrate, butyl-1,4-dioate, hexyne-1,4-dioate, caprate, caprylate, chlorides, cinnamate, citrate, formate, fumarate, glycolate, heptanoate, hippurate, lactates, maleate, malate, hydroxylate, malonate, mandelate, mesylates, nicotinate, isonicotinate, nitrates, oxalates, phthalates, terephthalate, postface, monohydrogenphosphate, dihydrophosphate, metaphosphate, proposterosly, sulfites, bisulfite the sulfonates, bansilalpet, p-bromophenylacetate, chlorobenzenesulfonate, econsultancy, 2-hydroxyethanesulfonic, methansulfonate, naphthalene-2-sulfonates, naphthalene-2-sulfonates, p-toluensulfonate, cellsurface, tartratami, etc., the Preferred salt is the hydrochloric salt.

Pharmaceutically acceptable salt accession acids, as a rule, be obtained by interaction between the compounds of formula I with an equimolar or excess amount of acid. The reactants are typically combined in a joint solvent such as diethyl ether or benzene. Salt usually falls out of solution within one hour and up to 10 days, and can be separated by filtration or solvent may be removed by conventional means.

Grounds generally applicable to the formation of salts include ammonium hydroxide and the hydroxides of alkali and alkaline earth metals, carbonates and aliphatic primary, secondary and tertiary amines, aliphatic diamines. The Foundation is particularly suitable for the production of salts of joining bases include ammonium hydroxide, potassium carbonate, methylamine, diethylamine, Ethylenediamine and cyclohexylamine.

Pharmaceutically priemlemim way often more suitable for formulations in the form of liquids or emulsions.

The pharmaceutical preparations can be obtained by techniques known in the art. For example, the compounds can be introduced into the preparations with conventional excipients, diluents or carriers, and formed into tablets, capsules, suspensions, powders, etc., Examples of excipients, diluents, and carriers that are suitable for such drugs include the following materials: fillers and dry diluents, such as starch, sugars, mannitol and silica-containing derivatives; binding agents such as carboxymethyl cellulose and other cellulose derivatives, alginates, gelatin and polyvinylpyrrolidone; moisturizing agents such as glycerin; a means of preventing caking, such as calcium carbonate and sodium bicarbonate; agents to slow the dissolution such as paraffin; accelerators resorbtive, such as Quaternary ammonium compounds; surface active agents such as cetyl alcohol, glycerol monostearate; adsorption media, such as kaslin and bentonite; and lubricants, such as talc, calcium stearate and magnesium and solid polyethylene glycols. Connections may also be spewy for parenteral administration, for example intramuscular, subcutaneous or intravenous route. In addition, the compounds are well suited for drugs in dosage forms with delayed release of drugs, etc., Drugs can be developed so that they can release the active ingredient only or preferably in a particular part of the gastrointestinal tract, possibly over a certain period of time. Coatings, membranes and protective bases can be manufactured, for example, polymeric substances or waxes.

The specific dosage of the compounds of formula I required to increase expression of thrombomodulin, inhibition of thrombotic complications or event, or increasing the rate of activated protein C, in accordance with the present invention will depend on the severity and nature of the condition, the route of administration and other factors, which will be determined by staff physicians hospitals. Generally, accepted and effective daily dose will be from 0.1 to 1000 mg/day, and more typically from 50 to 200 mg/day. Such dosages will be administered to a subject in need of treatment from one to three times daily, or more frequently as required, to effectively increase the EC of the Sabbath.

It is generally preferable to introduce the compounds of formula I in salt form accession acid that is familiar with the introduction of pharmaceuticals, bearing this core group, as piperidino ring. Also it is advantageous to introduce such a connection through the mouth. For this purpose, a suitable dosage forms for oral administration, the following.


In drugs, referred to hereinafter, the "active ingredient" means a compound of formula I.

Preparation 1. Gelatin capsules

Prepare gelatin capsules, using the following ingredients (see tab.1).

The ingredients are mixed, passed through sieve No. 45 mesh /US/ and fill hard gelatin capsules.

Examples of specific capsule formulations with raloxifene, which is prepared include those listed below.

The drug 2. Capsule with raloxifene (see tab. 2).

Preparation 3. Capsules raloxifene (see tab. 3).

Preparation 4. Capsule with raloxifene (see tab. 4).

The drug 5. Capsule with raloxifene (see tab. 5).

Specific above, the wording can be changed within reasonable limits.

Fo the and ( see table. 6 )

The components are mixed and compressed to obtain tablets.

On the other hand, tablets, each containing 0.1 to 1000 mg of active ingredient, are prepared as follows.

Preparation 7. Tablets ( see tab. 7).

The active ingredient, starch and cellulose are passed through sieve No. 45 mesh /USA/ and thoroughly mix. The solution of polyvinylpyrrolidone is mixed with the resultant powders which are then passed through sieve No. 14 mesh /USA/. Thus obtained granules are dried at 50o- 60oC, and pass through a sieve No. 18 mesh /USA/. Then the beads add natrocarbonatite, magnesium stearate and talc, previously passed through sieve # 60 mesh /USA/ and after mixing, are compressed on a tablet press machine, and get pills.

Suspensions containing 0.1 - 1000 mg of the active ingredient in 5-ml dose is prepared as follows.

Preparation 8. Suspension ( see tab. 8).

The active ingredient is sifted through a sieve No. 45 mesh /USA/, and mixed with the sodium carboxymethyl cellulose and syrup, get a homogeneous paste. A solution of benzoic acid, perfume and dye diluted with some water and add pasta when Augie embodiments of the invention relate to the treatment, a person or animal, state when you want the inhibition of thrombotic disorders. Thrombotic disorders or cases, and thromboembolic diseases include a wide variety of acquired medical conditions, including intravascular coagulation, including deep thrombophlebitis, pulmonary embolism, cardiac ischemia, myocardial infarction, cerebral thrombosis, non-status, status with local hypercoagulation and widespread tissue injury related to inflammation, capillary, arterial thrombosis, peripheral arterial thrombosis, embolism, originating from the heart or peripheral arteries, acute myocardial infarction, thrombotic strokes and diffuse intravascular coagulation. Diffuse intravascular coagulation occurs as a complication, many painful conditions, including extensive trauma, extensive surgery, thermal shock, septicemia, acute and chronic filling of the liver, malignant diseases, including solid tumors, leukemias and lymphomas, with a wide variety of bacterial, fungal, parasitic and viral infectious diseases, obstetric complications, hemolytic about the mustache snakes, collagen vascular disorders, purple lightning, acute pancreatitis, allergic vasculitis, polycythemia vera, trombozitemia and ulcerative colitis, among others. Although it is not yet solved, congenital deficiency in the expression of thrombomodulin can be prodemonstrirovana in patients with thromboembolic problems. Exacerbation of these patients will be treated by the compounds of formula I.

I believe that the compounds of formula I are also suitable for the treatment of thrombotic strokes. The level of thrombomodulin in the vessels of the brain is low, thus increasing it will be important for thrombotic stroke. Today kicks, as a rule, conventional anticoagulants is not treated. Treatment of shock or heparin or oral anticoagulants although sometimes successful, however, carries a high risk of bleeding in the injured brain, and the blow is accompanied by deterioration in neurological status.

In addition, suppose that the compounds of the present invention is suitable for the treatment of acute myocardial infarction, because it is obvious that the main proposed mechanism of action of thrombomodulin and derivatives, i.e. anticoagulation through activation of protein C, with the tion, as well as reducing reperfusion injury. During the tests with thrombolytic agents in acute myocardial infarction and from experiments on animals, it will become clear that the heparin as an auxiliary therapeutic agent, is relatively ineffective as antithrombotic agents on arterial side of the circulation.

It is also assumed that the compounds of the present invention is suitable for the treatment of diffuse intravascular coagulation /"DIC"/. There are experimental data that the mediators in inflammation, as well as II - I, F and I P-endotoxin, sharply reduce the expression of thrombomodulin on endothelial cells, which in turn leads to insufficient activation of the protein C anticoagulant cascade. Heparin and other oral anticoagulants were given to patients with diffuse intravascular coagulation in advanced clinical trials, but the results of these tests disappoint. Characteristically, patients with diffuse intravascular coagulation clots are widely distributed, including capillary circulation, associated with often serious bleeding problems that are the result of "spending" important factory microvascular fibrin clots.

Since thrombomodulin falls through various intermediaries from inflammation, the preferred use of compounds consists in the regulation of inflammatory conditions associated with capillary thrombosis. In a broader sense, this would include any state, marked by dysfunction of vascular endothelium, as occurs in sepsis, damage, including extensive tissue damage, and injury, syndrome, systemic inflammatory response syndrome, sepsis, septic shock syndrome and dysfunction of many organs, including DIC.

There is some evidence that regular antikoaguliruyuschee drugs, particularly warfarin, suitable for the treatment of invasive malignant tumors. Many tumor cells produce substances that cause activation of the coagulation system, resulting in a local deposits of fibrin. These fibrine sediments function as "nests" in which cancer cells can divide, forming a metastatic lesion. In one clinical study found that patients receiving warfarin in addition to anti-cancer chemotherapy in the treatment of small cell lung cancer live longer and have less extensive metastatic the ri this study, was less intense than that which today is considered optimal in clinical Oncology. More intensive forms of cancer chemotherapy is almost always give a sharp deterioration in the platelet count, and thrombocytopenia, in combination with warfarin treatment gives the patient an unacceptably high risk of serious complications such as bleeding.

It is expected that the compounds are useful in other diseases, when the coagulation of blood can be a major contributing process or source of secondary pathology, such as cancer, including metastasis, inflammatory diseases, including arthritis, and diabetes. Anticoagulate compound is administered orally, parenterally, e.g. by intravenous infusion /iv/, intramuscular injection /im/ or adjustably /SC/.

The method of the present invention in practice is also used together with dissolving the blood clot by means of, for example, tissue plasma activator /tPA/ modified tPA, streptokinase or urokinase to reduce reperfusion injury. In cases where the formation of clots and blocked artery or vein /or partially or completely/ typically use dissolving thrombus, credco application, one or together with a solvent by means of, in addition, preferably it is administered together with aspirin to prevent a recurrence of blood clots.

The method of the present invention in practice is also used in combination with an antagonist trombotsitnoy glycoprotein IIb-IIIa receptor, which inhibits platelet aggregation. The compound of the invention can be introduced before or together with the antagonist IIb-IIIa receptor, or after application to prevent or renewal of blood clots.

The method of the present invention is also used in practice in combination with aspirin. The compound of the invention can be introduced before or together with aspirin, or after it is applied, to prevent the occurrence or the resumption of blood clots. As mentioned above, the compound of the present invention preferably in combination with dissolving thrombus agent and aspirin.

Test 1

To further understand the steps /actions/ compounds of the formula I on the inner membrane of the smooth muscle cells and their role in enhancing anticoagulative blood is needed to examine changes in the activity of thrombomodulin TM on the surface of Shaporenko, which lead to a decline in activity of TM in the TM surface of such cells.

Approximately 40000 - 80000 previously passaged endothelial /arterial, venous or capillary/ cells, or cells of intimal smooth muscle, sown and grown to confluence in 24-cell culture plates. The cell monolayer is then washed 2 to 3 times or buffer saline Hanks /HBSS/ or serum-free medium /SFM/. After 24 hours, cells are added, in three parallel cases, different concentrations of the compounds of formula I /in the range from micromolar to subpicomolar/. Cells in the control cells /negative control/ incubated in serum-free medium with the amount of media that is equivalent in all cells.

Measurement of activity of TM on the surface of cells performed using the two-stage amylolyticus test (two-phase amidolitic assay). During the first stage of the test, followed by rinsing cells in HBSS or SFM, the monolayer add 0.4 ml of SFM containing human protein C (final concentration of 11.2 ug/ml), alpha-thrombin (final concentration of 0.1 NIHU/ml), and incubated at 37oC and 5% CO2. After 15, 30 and 45 minutes of each cell selected 100 ul environment, and injected into the cell microtinae the action of thrombin.

As a negative control in the absence of cells using SFM with protein C and alpha-thrombin, as described above, and treated in a similar way.

In the second stage tests 50 ul of 3 mm 2366, chromogenic substances of protein C, add to the mixtures of concentrated environments with hirudin, and with the help of automatic reading device to measure the optical density at 405 nm /OD405/ to examine the kinetics of activity of TM for 4 minutes. After completing this kinetic tests are conducted by measuring total protein using the BCA method. The final activity of the TM is expressed as % increase. Compound a is a compound of formula I, in which R1and R3are hydrogens, R2is a 1-pyrrolidinone.

Compound a (the concentration (μm) - TM Activity (% increase)

110-4- 14

110-2- 15

110-1- 15

1,00 - 17

10,00 - 17

Test 2

To show the effect of compounds of the formula I as antithrombotics and their ability to correct endothelial dysfunction induced by inflammation, use Avianova model of sepsis induced by E. coli, as described in U.S. patent N 5009889 /vinuesa positive influence on the expression of thrombomodulin, thrombotic disorders or characteristics of the speed of activation of protein C, which is found in the above tests.

1. The use of compounds of General formula (I)

< / BR>
in which R1and R3independently are hydrogen, -CH3,

< / BR>
where Ar represents optionally substituted phenyl;

R2selected from the group consisting of pyrrolidino, hexamethyleneimino and piperidinium,

or its pharmaceutically acceptable salt or MES to increase expression of thrombomodulin.

2. Application under item 1, characterized in that the compound is a hydrochloride.

3. Application under item 1, characterized in that the introduction is preventive.

4. Application under item 1, characterized in that the connection specified is a

< / BR>
or its hydrochloride.


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