Substituted oxazolidinones and their use in the field of blood coagulation

Description of the invention

The invention relates to the field of blood coagulation. The present invention relates to novel oxazolidinone derivatives, a method for their preparation, medicaments containing them, their use in the preparation of medicaments and their use for the prevention of ex vivo blood clotting.

Blood clotting is the body's defense mechanism by which defects in the vessel wall can be 'sealed' quickly and reliably. In this way, blood loss can be prevented or minimized. Stopping bleeding after vessel damage occurs essentially through the coagulation system, which triggers the enzymatic cascade of complex plasma protein reactions. A plurality of blood clotting factors are involved, each of which, after activation, each transforms the next inactive pre-stage into active form. At the end of the cascade, the conversion of soluble fibrinogen to insoluble fibrin occurs so that a blood clot is formed. Traditionally, a distinction is made in blood coagulation between the endogenous and extrinsic systems which meet in a final common reaction pathway. The key role here is played by factor Xa, which is formed from the proenzyme of factor X, because it binds both clotting pathways. The activated serine protease Xa cleaves prothrombin into thrombin. The resulting thrombin, in turn, cleaves fibrinogen to fibrin, which is a fibrous-jelly-like clotting substance. Moreover, thrombin is a potent trigger of thrombocyte aggregation and also contributes significantly to hemostasis.

Maintaining normal hemostasis - between bleeding and thrombosis - is subject to a complex regulatory mechanism. Uncontrolled activation of the coagulation system or defective inhibition of the activation processes can cause the formation of local thrombi or emboli in the vessels (arteries, veins, lymph vessels) or in the heart cavities. This can lead to serious conditions such as heart attack, angina pectoris (including unstable angina), re-closure and restenosis following angioplasty or aortic bypass surgery, stroke, transient ischemic attacks, peripheral arterial obstruction, pulmonary embolism or deep thrombophlebitis; these diseases are also collectively referred to as thromboembolic disorders. In addition, systemic hypercoagulability in coagulopathy from the consumption of coagulation factors may lead to disseminated intravascular thrombus.

These thromboembolic conditions are the most common cause of morbidity and mortality in most industrialized countries (Pschyrembel, Klinisches Worterbuch, 257th edition, 1994, Walter de Gruyter, pp. 199 ff., Entry "Blutgerinnung"; Rompp Lexikon Chemie, version 1.5, 1998, publishing house Georg Thieme Stuttgart, entry "Blutgerinnung"; Lubert Stryer, Biochemie, Spektrum der Wissenschaft Verlagsgesellschaft mbH Heidelberg, 1990, pp. 259 et seq.).

The anticoagulants known in the art, i.e. substances for inhibiting or preventing blood clotting, have various, sometimes aggravating, disadvantages. An effective method of treatment or prevention in the case of thromboembolic diseases is therefore very difficult and unsatisfactory in practice.

In the treatment and prophylaxis of thromboembolic diseases, heparin is used, which is administered parenterally or subcutaneously. Due to favorable pharmacokinetic properties, low molecular weight heparin is used more and more today; however, the following known disadvantages that occur during treatment with heparin cannot be omitted here as well. Thus, heparin is orally inactive and has, by comparison, only a small half-life. Since heparin simultaneously inhibits many factors of the blood clotting cascade, the effect is non-selective. In addition, there is a high risk of bleeding, in particular, there may be bleeding in the brain and bleeding in the gastrointestinal tract, and may result in thrombocytopenia, drug-induced alopecia or osteoporosis (Pschyrembel, Klinisches Worterbuch, 257th edition, 1994, Walter de Grayter, pp. 610, password "Heparyn"; Rompp Lexikon Chemie, version 1.5, 1998, publishing house Georg Thieme Stuttgart, entry "Heparin").

The second class of anticoagulants are vitamin K antagonists. These include, for example, 1,3-indanedione, but above all compounds such as Warfarin, Phenprocoumon, Dicumarol and other coumarin derivatives, which non-selectively inhibit the liver synthesis of various products of certain vitamin K dependent coagulation factors. However, the action due to this mechanism is very slow (latency to onset of action is 36-48 hours). Although the compounds can be administered orally, due to the high risk of bleeding and the narrow therapeutic index, individual adjustments and monitoring are necessary.

PL 200 413 B1 of the patient. In addition, further side effects such as gastrointestinal disturbances, hair loss and skin necrosis are described (Pschyrembel, Klinisches Worterbuch, 257th edition, 1994, Walter de Gruyter, p. 292 et seq., Entry "Cumarinderivate"; Ullmann's Encyclopedia of Industrial Chemistry , 5th edition, publishing house VCH Verlagsgesellschaft, Weinheim, 1985 - 1996, entry "Vitamin K").

Recently, a new type of therapy has been described for the treatment and prevention of thromboembolic diseases. The goal of a new type of therapy is to inhibit factor Xa (e.g. WO-A-99/37304; WO-A-99/06371; J. Hauptmann, J. Stiirzebecher, Thrombosis Research 1999, 93, 203; F. Al-Obeidi, JA Ostrem, Factor Xa inhibitors by classical and combinatorial chemistry, DDT 1998, 3, 223; F. Al-Obeidi, JA Ostrem, Factor Xa inhibitors, Exp. Opin. Ther. Patents 1999, 9, 931; B. Kaiser, Thrombin and factor Xa inhibitors, Drugs of the Future 1998, 23, 423; A. Uzan, Antithrombotic agents, Emerging Drugs 1998, 3, 189; B.-Y. Zhu, RM Scarborough, Curr. Opin. Card. Pulm. Rhenium. Inv. Drugs 1999,1 (1), 63). It has been shown that various compounds, both peptide and non-peptide, are active in animal tests as inhibitors of factor Xa.

The object of the invention is therefore to provide new substances for the control of diseases which exhibit a wide range of therapeutic effects.

Such compounds should in particular be suitable for the more effective prophylaxis and / or treatment of thromboembolic disorders and should - at least in part - not suffer from the above-described disadvantages of the prior art, the term "thromboembolic disorders" in the context of the present application being particularly understood. stressful diseases such as heart attack, angina pectoris (including unstable angina), re-closure and restenosis following angioplasty or aortic coronary bypass, stroke, transient cerebral ischemic attacks, peripheral arterial occlusion disorders, pulmonary embolism, or deep thrombophlebitis.

It is a further object of the invention to provide novel anticoagulants which inhibit blood coagulation factor Xa with increased selectivity and overcome - at least in part - the problems of prior art therapeutic methods for thromboembolic diseases.

The invention therefore relates to substituted oxazolidinones of the general formula I

<img file="PL200413B1_D0001.tif" />

wherein

R¹ represents a 2-thiophene group that is substituted at the 5-position by a substituent selected from the group consisting of chlorine, bromine, methyl or trifluoromethyl,

R² means the DA- group, where the symbol "A" means a phenylene group, the symbol "D" means a 2-oxopyrrolidin-1-yl, 2-oxo-1,3-oxazolidin-3-yl, 3-oxomorpholin-1-yl group , 2-oxo-1,3-oxazinan-3-yl or 2-oxopiperidin-1-yl, which is linked to "A" via the nitrogen atom, with the group "A" as defined above meta in relation to the bond to the oxazolidinone may be optionally one or two substituted with a substituent selected from the group consisting of fluoro, chloro, amino, trifluoromethyl, methyl or cyano,

R³, R⁴, R⁵, R⁶, R⁷ and r⁸ are hydrogen atoms.

PL 200 413 B1

A compound of the following formula is especially preferred:

<img file="PL200413B1_D0002.tif" />

So far, oxazolidinones have been described essentially only as antibiotics, in particular also as MAO inhibitors and fibrinogen antagons (reviewed: Riedl, B., Endermann, R., Exp. Opin. Ther. Patents 1999, 9 (5), 625), where for antibacterial activity, a small 5- [acylaminomethyl] group (preferably 5- [acetylaminomethyl]) appears to be significant.

Substituted aryl and heteroarylphenyl oxazolidinones, where the N atom of the oxazolidinone ring is bonded with a monosubstituted or multisubstituted phenyl group and which may have an unsubstituted N-methyl-2-thiophenecarboxamide group at the 5-position of the oxazolidinone ring, and their use as antibacterial substances are known from US patents US Am. No. US-A-5929248, US-A-5801246, US-A-5756732, US-A-5654435, US-A-5654428 and US-A-5565571.

Furthermore, benzamidine-containing oxazolidinones are known as synthetic intermediates for the preparation of factor Xa inhibitors or fibrinogen antagonists (WO-A-99/31092, EP-A-623615).

The compounds according to the invention of general formula I, depending on the substitution pattern, may exist in stereoisomeric forms which either behave as image and mirror image (enantiomers) or which do not behave as image and mirror image (diastereomers). The invention relates both to the enantiomers or diastereomers and to the mixtures in question. Racemic forms and diastereomers can be separated into stereoisomerically homogeneous components in a known manner.

Furthermore, certain compounds of the general formula I may exist in tautomeric forms. This is known to those skilled in the art and such compounds are also included in the invention.

The invention also relates to a process for the preparation of the compounds of the general formula I according to the invention.

<img file="PL200413B1_D0003.tif" />

wherein R², R³, R⁴, R⁵, R⁶, R⁷ and r⁸ are as defined above, are reacted with carboxylic acids of general formula III

HO R¹ (Ul),

Y ₁ in which R.¹ has the meaning given above,

With carboxylic acid halides, preferably carboxylic acid chlorides, or with symmetrical or mixed carboxylic acid anhydrides of the above-defined carboxylic acids of general formula III, in inert solvents, optionally in the presence of an activating or coupling agent and / or in the presence of a base to give compounds of general formula I

<img file="PL200413B1_D0004.tif" />

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and r⁸ are as defined above or according to another variant [B] the compounds of general formula IV

<img file="PL200413B1_D0005.tif" />

wherein R¹, R³, R⁴, R⁵, R⁶, R⁷ and r⁸ are as defined above, converting with a selective oxidizing agent in an inert solvent into the corresponding epoxy compound of the general formula V

<img file="PL200413B1_D0006.tif" />

and by reaction in an inert solvent, optionally in the presence of a catalyst, with an amine of general formula VI

R² - NH2 (VI) where R² is as defined above, firstly compounds of general formula VII are obtained

<img file="PL200413B1_D0007.tif" />

wherein R¹, R², R³, R⁴, R⁵, R⁶, R⁷ and r⁸ have the meaning given above,

And then in an inert solvent in the presence of phosgene or phosgene equivalents such as e.g. carbonyldiimidazole (CDI) is cyclized to compounds of general formula I

<img file="PL200413B1_D0008.tif" />

The methods according to the invention can be illustrated by the following schemes:

<img file="PL200413B1_D0009.tif" />

PL 200 413 B1

The above-described, possibly following oxidation step can be exemplified by the following scheme:

<img file="PL200413B1_D0010.tif" />

Suitable solvents for the processes described above are organic solvents which are inert under the reaction conditions. Hydrogen halides such as dichloromethane, trichloromethane, tetrachloromethane, 1,2-dichloroethane, trichlorethane, tetrachloroethane, 1,2-dichloroethylene or trichlorethylene, ethers such as diethyl ether, dioxane, tetrahydrofuran, dimethyl ether of ethylene glycol or glycol ethylene glycol are mentioned here. diethylene, alcohols such as methanol, ethanol, n-propanol, isopropanol, n-butanol or t-butanol, hydrocarbons such as benzene, xylene, toluene, hexane or cyclohexane, dimethylformamide, dimethyl sulfoxide, acetonitrile, pyridine, hexamethylphosphoric triamide, or water.

It is also possible to use mixtures of the solvents mentioned.

The usual reagents used as activating or coupling agents in the above-described processes are, e.g.₂About and the like.

Suitable bases are known inorganic or organic bases. Preference is given to alkali metal hydroxides, such as, for example, sodium or potassium hydroxide, or alkali metal carbonates, such as sodium or potassium carbonate, or sodium or potassium methoxide, or potassium t-butoxide, or amides, such as sodium amide, Lithium (trimethylsilyl) amide or lithium diisopropylamide or amines such as triethylamine, diisopropylethylamine, diisopropylamine, 4-N, N-dimethylaminopyridine or pyridine.

The base can be used in an amount of 1-5 moles, preferably 1-2 moles, based on 1 mole of the compounds of general formula II.

The reaction can generally be carried out at a temperature from -78 ° C to reflux, preferably from 0 ° C to reflux.

The reaction can be carried out at normal, elevated or reduced pressure (e.g. in the range 0.5-5 bar). In general, the process is carried out under normal pressure.

Suitable oxidizing agents for the preparation of epoxy compounds as well as for the optionally carried out oxidation to a sulfone, sulfoxide or N-oxide are, for example, m-chloroperbenzoic acid (MCPBA), sodium metaperiodate, N-methylmorpholine N-oxide (NMO), monoperoxy phthalic acid or osmium tetroxide.

Conditions conventionally employed in such reactions are used to prepare epoxy compounds.

With respect to more detailed data on the reaction conditions for the optionally performed oxidation to a sulfone, sulfoxide or N-oxide, reference can be made to the following literature: MR Barbachyn et al., J. Med. Chem. 1996, 39, 680 and WO-A-97/10223.

In addition, reference is made to examples 14 to 16 given in the experimental section.

The optionally used amidination is carried out under conventional conditions. For details, reference is made to Examples 31-35 and 140-147.

Compounds of general formulas II, III, IV and VI are known to the person skilled in the art and can be prepared in a known manner. For oxazolidinones, especially the 5- (aminomethyl) -2-oxooxazolidinones used see WO-A-98/01446, WO-A-93/23384, WO-A-97/03072, JA Tucker et al., J. Med. Chem. 1998, 41, 3727; SJ Brickner et al., J. Med. Chem. 1996, 39, 673; WA Gregory et al., J. Med. Chem. 1989, 32, 1673.

PL 200 413 B1

The compounds according to the invention of the general formula I exhibit an unpredictable, valuable pharmacological range and are therefore suitable for use in the prevention and / or treatment of diseases.

The compounds of the general formula I according to the invention act in particular as anticoagulants and can therefore be used in medicaments for the prevention and / or treatment of thromboembolic diseases. "Thromboembolic disorders" within the meaning of the invention include particularly severe conditions such as myocardial infarction, angina pectoris (including unstable angina), re-closure and restenosis following angioplasty or aortic bypass surgery, stroke, transient ischemic attacks, associated with peripheral arterial obstruction, pulmonary embolism, or deep thrombophlebitis.

Furthermore, the compounds of the general formula I according to the invention are suitable for the treatment of disseminated intravascular thrombus (DIC).

Furthermore, the compounds according to the invention of the general formula are suitable for the prevention and / or treatment of arteriosclerosis and arthritis as well as for the prevention and / or treatment of Alzheimer's disease and cancer.

The compounds according to the invention of the general formula I act in particular as selective inhibitors of the blood coagulation factor Xa and do not inhibit or inhibit other serine proteases, such as thrombin, plasmin or trypsin only at markedly higher concentrations.

"Selective" in the context of the invention are those inhibitors of blood coagulation factor Xa in which the IC50 values ​​for factor Xa inhibition, compared to the IC50 values ​​for the inhibition of other serine proteases, in particular thrombin, plasmin and trypsin, are 100 times, preferably 500 times. times, in particular 1000 times lower, with reference to the methods for testing selectivity, referring to the test methods described in Examples A-1) a.1) and a.2).

The compounds of the invention of general formula I can furthermore be used to prevent coagulation ex vivo, e.g. in the case of preserving blood or biological samples containing factor Xa.

The invention therefore relates to the oxazolidinones of the formula I, which in particular exhibit an unexpected, strong and selective inhibition of factor Xa, the same also being true of compounds excluded in the discussion of product protection.

The invention also further relates to medicaments and pharmaceutical preparations which contain at least one compound according to the invention of general formula I together with one or more pharmacologically acceptable auxiliaries or carriers and which can be used for the above-mentioned indications.

The agents according to the invention are used for the prevention and / or treatment of diseases of the human or animal body, in particular of the above-mentioned diseases.

The invention further relates to the use of compounds of general formula I for the preparation of medicaments or pharmaceutical compositions for the prevention and / or treatment of diseases positively influenced by the inhibition of factor Xa, in particular arteriosclerosis, arthritis, Alzheimer's disease or cancer or in case of thromboembolic diseases, especially such as a heart attack, angina (including unstable angina), re-closure and restenosis after angioplasty or aortic coronary bypass, stroke, transient cerebral ischemic attacks, peripheral arterial occlusive disease, pulmonary embolism, or deep thrombophlebitis.

The invention further relates to the use of compounds of general formula I for the preparation of medicaments or pharmaceutical compositions for the treatment of disseminated intravascular thrombi (DIC).

The invention further relates to the use of compounds of general formula I for the prevention of ex vivo blood clotting.

The invention further relates to the use of compounds of general formula I for the prevention of ex vivo blood clotting in biological samples containing factor Xa.

A method of preventing blood clotting in vitro, especially in the case of preserving blood or in the case of biological samples containing factor Xa, consists in adding compounds of general formula I.

For the administration of the compounds according to the invention, all known administration forms are contemplated. Oral, intra-lingual, sublingual, buccal, rectal or parenteral administration (i.e. bypassing the intestinal tract, i.e. intravenous, intraarterial, intracardiac, intradermal, subcutaneous, transdermal, intraperitoneal or intramuscular) is preferably used. Especially

Oral and intravenous administration is preferred. Oral administration is especially preferred, which is an additional advantage over the prior art therapy of thromboembolic diseases.

The new active substances of the general formula I can be converted into known preparations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable carriers or solvents.

The therapeutically active compound should be present in each case in a concentration of about 0.1-95% by weight, preferably 0.5-90% by weight, especially 1-85% by weight of the total mixture, i.e. in an amount sufficient to achieve the dosage range indicated.

Nevertheless, it may possibly be desirable to deviate from the above-mentioned amounts, namely depending on the body weight or the type of administration route, on individual drug behavior, on the type of preparation and on the point or time interval over which administration occurs. Thus, in some cases it may be sufficient to use less than the above-mentioned minimum amounts, while in other cases the upper limit mentioned should be exceeded. When larger amounts are to be administered, several single doses per day may be recommended.

The formulations are prepared, for example, by mixing the active ingredients with solvents and / or carriers, optionally with the use of emulsifiers and / or dispersants, whereby, for example, when water is used as the diluent, organic solvents can optionally be used as auxiliary solvents.

In general, when administered intravenously, it has proven advantageous to administer about 0.001-10 mg / kg, preferably about 0.01-10 mg / kg, especially about 0.1-8 mg / kg body weight, in order to obtain effective results.

In general, when administered orally, it has proven advantageous to administer about 0.01-50 mg / kg, preferably about 0.1-10 mg / kg, especially about 0.5-8 mg / kg body weight, in order to obtain effective results.

Nevertheless, it may possibly be desirable to deviate from the abovementioned amounts when administering intravenously or orally, namely depending on the body weight or the type of administration route, on the individual behavior towards the drug, on the type of preparation and on the point or time interval over which it takes place. administration. Thus, in some cases it may be sufficient to use less than the above-mentioned minimum amounts, while in other cases the upper limit mentioned should be exceeded. When larger amounts are administered, it may be advisable to split such a dose over the course of the day, namely by administering several single doses or as a long-term infusion.

The compounds according to the invention of the general formula I - including the compounds excluded in the discussion of product protection - are distinguished compared with known formulations for the treatment of thromboembolic diseases in particular in that a greater therapeutic range is achieved by selective inhibition of factor Xa. For the patient this means a lower risk of bleeding and for the treating physician a better patient positioning. In addition, there is a faster - dependent on the mechanism - action to start. Above all, however, the compounds according to the invention can be administered orally, which is a further advantage of the therapy with the compounds according to the invention.

The invention is explained in more detail in the following examples, which, however, in no way limit the invention.

A. Assessment of physiological action

1. General testing methods

The particularly advantageous biological properties of the compounds according to the invention can be ascertained by the following methods.

a) Overview of the test (in vitro)

a.1) Factor Xa inhibition measurement

The enzymatic activity of human factor Xa (FXa) is measured by the reaction of an FXa-specific chromogenic substrate, where factor Xa cleaves p-nitroaniline from the chromogenic substrate. The determinations are performed in the microtiter plates as follows.

The test substances are dissolved in DMSO at various concentrations and incubated with human FXa for 10 minutes (0.5 nmol / liter dissolved in 50 mmol / liter Tris buffer [C, C, C-tris- (hydroxymethyl) -aminomethane], 150 mmol / liter NaCl, 0.1% BSA (Bovine Serum Albumin, pH = 8.3) at 25 ° C. Pure DMSO was used as a control. The chromogenic substrate (150 µmol / liter Pefachrome® FXa from Pentapharm) is then added. After 20 minutes of incubation at 25 ° C

The extinction at 405 nm is determined. The extinction of the test samples with the test substance is compared with the control samples without test substance and the IC50 values ​​are calculated from this.

a.2) Determination of selectivity

To determine the selective inhibition of FXa, test substances are tested for inhibition of other human serine proteases such as thrombin, trypsin, plasmin. To determine the enzymatic activity of thrombin (76 mU / ml), trypsin (500 mU / ml) and plasmin (3.2 nmol / liter), these enzymes are dissolved in Tris buffer (100 mmol / liter, 20 mmol / liter CaCl2, pH = 8.0) and incubated with the test substance or solvent for 10 minutes. Then by adding an appropriate specific chromogenic substrate (Chromozym Thrombin^® from Boehringer Mannheim, Chromozym Trypsin^® by Boehringer Mannheim, Chromozym Plasmin^® from Boehringer Mannheim) the enzymatic reaction is started and the extinction is determined after 20 minutes at 405 nm. All determinations are carried out at 37 ° C. The extinction of the test samples with the test substance is compared with the control samples without test substance and the IC50 values ​​are calculated from this.

a.3) Determination of the anticoagulant activity

The anticoagulant activity of test substances is determined in vitro in human plasma. For this, human blood is collected using a 0.11 molar sodium citrate solution in a 1/9 sodium citrate / blood mixture. The blood is mixed well immediately after collection and centrifuged for 10 minutes at about 2000 g. The supernatant is removed by pipette. The prothrombin time (PT, synonym: thromboplastin time, Quick test) is determined in the presence of different concentrations of the test substance or an appropriate solvent using a commercially available test kit (Neoplastin^® from Boehringer Mannheim). Test compounds are incubated with plasma for 10 minutes at 37 ° C. Coagulation is then induced by the addition of thromboplastin and the time point for the onset of clotting is determined. The concentration of the test substance is determined which doubles the prothrombin time.

b) Determination of anticoagulant activity (in vivo) b.1) Arteriovenous fistula model (rat)

Male fasted rats (strain: HSD CPB: WU) weighing 200-250 g are anesthetized with the Rompun / Ketavet solution (12 mg / kg / 50 mg / kg). An arteriovenous fistula thrombus is induced based on the method described by Christopher N. Berry et al., Br. J. Pharmacol. (1994), 113, 1209-1214. For this purpose, the left jugular vein and the right carotid artery are dissected. An extracorporeal fistula is placed between the two vessels using a 10 cm long polyethylene (PE 60) hose. This polyethylene hose is connected in the center to a further 3 cm long polyethylene hose (PE 160) which contains a roughened and looped nylon thread to obtain a thrombogenic surface. The extracorporeal circulation lasts for 15 minutes. The fistula is then removed and the nylon thread with the thrombus is immediately weighed. The weight of the nylon thread itself is determined before starting the experiment. The test substances are administered to the sleepy animals, prior to the introduction of extracorporeal circulation, either intravenously via the tail vein or orally via an esophageal tube. The results are summarized in Table 1.

Table 1

Anticoagulant activity in a venous-arterial fistula model (rat) after oral or intravenous administration

<td>Example</td><td>ED50 [mg / kg] orally</td><td>ED50 [mg / kg] intravenously</td>

<td> 17</td><td></td><td> 6</td>

<td> 44</td><td> 3</td><td></td>

<td> 95</td><td></td><td> 3</td>

<td> 114</td><td></td><td> 3</td>

<td> 115</td><td></td><td> 3</td>

<td> 123</td><td> 3</td><td></td>

<td> 162</td><td></td><td> 3</td>

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b.2) Arterial thrombosis model (rat)

Male fasted rats (strain: HSD CPB: WU) are anesthetized as described above. The average weight of the rats is approximately 200 g. The left carotid artery (approximately 2 cm) is dissected. An arterial thrombus is induced by mechanical injury to the vessel according to the method described by K. Meng et al., Naunyn-Schmiedeberg's Arch. Pharmacol. (1977), 301, 115-119. Then, the dissected carotid artery is clamped, inhibiting the blood flow, cooled in a metal trough to -12 ° C for 2 minutes, and in order to standardize the size of the thrombus, it is simultaneously compressed with a weight of 200 g. Then, the blood flow is additionally reduced by means of a clamp placed on the carotid artery. distal to the injured section of the vessel. The proximal clamp is removed, the wound is closed and reopened after 4 hours to remove the injured section of vessel. This section of the vessel opens longitudinally and the thrombus is removed from the injured section of the vessel. The wet weight of the thrombi is determined immediately. At the beginning of the experiment, the test substances are administered intravenously via the tail vein or orally via an oesophageal gavage at the beginning of the experiment.

b.3) Venous thrombus model (rat)

Male fasted rats (strain: HSD CPB: WU) are anesthetized as described above. The average weight of the rats is approximately 200 g. The left jugular vein (approximately 2 cm) is dissected. Venous thrombus formation is induced by mechanical injury to the vessel according to the method described by K. Meng et al., Naunyn-Schmiedeberg's Arch. Pharmacol. (1977), 301, 115-119. Then, the prepared jugular vein is clamped to inhibit the blood flow, cooled in a metal trough to -12 ° C for 2 minutes and simultaneously compressed with a weight of 200 g to standardize the size of the thrombus. The blood flow is reopened and the wound is closed. After 4 hours, the wound is reopened to remove the thrombus from the injured section of the vessel. The wet weight of the thrombi is determined immediately. At the beginning of the experiment, the test substances are administered intravenously to the sleepy animals either via the tail vein or orally via an esophageal tube.

B. Manufacturing Example

Starting compounds

A method for producing 3-morpholinone is described in US 5349045.

The preparation of N- (2,3-epoxypropyl) -phthalimide is described in J.-W. Chern et al., Tetrahedron Lett. 1998, 39, 8483.

Substituted anilines can be prepared by reacting, for example, 4-fluoronitrobenzene, 2,4-difluoronitrobenzene or 4-chloronitrobenzene with the appropriate amines or amides in the presence of a base. This process can also be carried out using Pd catalysts such as Pd (OAc) 2 / DPPF / NaO-t-Bu (Tetrahedron Lett. 1999, 40, 2035) or copper (Renger, Synthesis 1985, 856; Aebischer et al., Heterocycles 1998, 48, 2225). Similarly, haloaromatics without a nitro group can first be converted into the corresponding amides and then nitrated at the 4-position (US 3,279,880).

I. 4- (4-Morpholin-3-onyl) -nitrobenzene

<img file="PL200413B1_D0011.tif" />

2 moles (202 g) of morpholin-3-one (E. Pfeil, U. Harder, Angew. Chem. 79, 1967, 188) are dissolved in 2 liters of N-methylpyrrolidone (NMP). 88 g (2.2 mol) of sodium hydride (60% in paraffin) are then added portionwise over the course of 2 hours. After the evolution of hydrogen had ceased, 282 g (2 mol) of 4-fluoronitrobenzene were added dropwise with cooling at room temperature in 1 hour and the reaction mixture was stirred overnight. Then 1.7 liters of liquid volume are distilled off at 12 mbar pressure and at 76 ° C., the residue is poured into 2 liters of water and this mixture is extracted twice with 1 liter of ethyl acetate each time. After washing the combined organic phases with water, it is dried over sodium sulphate and the solvent is distilled off under reduced pressure. Purification is carried out by chromatography 12

Preparation on silica gel with hexane / ethyl acetate (1: 1) and by subsequent recrystallization from ethyl acetate. 78 g of product are obtained as a colorless to brown solid with a yield of 17.6% of theory.

¹H-NMR (300 MHz, CDCl3): 3.86 (m, 2H, CH2CH2), 4.08 (m, 2H, CH2CH2), 4.49 (s, 2H, CH2CO), 7.61 (d, 2H , ³J = 8.95 Hz, CHCH), 8.28 (d, 2H, ³J = 8.95 Hz, CHCH)

MS (rI%) = 222 (74, M.⁺), 193 (100), 164 (28), 150 (21), 136 (61), 117 (22), 106 (24), 90 (37), 76 (38), 63 (32), 50 (25)

The following compounds were prepared in an analogous manner: 3-fluoro-4- (4-morpholin-3-onyl) -nitrobenzene, 4- (N-piperidonyl) -nitrobenzene, 3-fluoro-4- (N-piperidonyl) -nitrobenzene, 4- (N-pyrrolidonyl) -nitrobenzene, 3-fluoro-4- (N-pyrrolidonyl) -nitrobenzene.

II. 4- (4-Morpholin-3-onyl) -aniline

<img file="PL200413B1_D0012.tif" />

In an autoclave, 63 g (0.275 mol) of 4- (4-morpholin-3-onyl) -nitrobenzene are dissolved in 200 ml of tetrahydrofuran, 3.1 g of Pd / C (5%) are added and hydrogenated for 8 hours at temperature 70 ° C and 50 bar hydrogen pressure. After filtering off the catalyst, the solvent is distilled off in vacuo and the product is purified by crystallization from ethyl acetate. The product precipitates at 20 g as a colorless to bluish solid with a yield of 37.6% of theory.

Purification can also be carried out by chromatography on silica gel with hexane / ethyl acetate.

¹H-NMR (300 MHz, CDCl3): 3.67 (m, 2H, CH2CH2) 3.99 (m, 2H, CH2CH2), 4.27 (s, 2H, CH2CO), 6.68 (d, 2H, ³J = 8.71Hz, CHCH), 7.03 (d, 2H, ³J = 8.71Hz, CHCH)

MS (rI%) = 192 (100M⁺) 163 (48), 133 (26), 119 (76), 106 (49), 92 (38), 67 (27), 65 (45), 52 (22), 28 (22)

The following compounds were prepared in an analogous manner: 3-fluoro-4- (4-morpholin-3-onyl) -aniline, 4- (N-piperidonyl) -aniline, 3-fluoro-4- (N-piperidonyl) -aniline, 4- (N-pyrrolidonyl) -aniline, 3-fluoro-4- (N-pyrrolidonyl) -aniline.

General method for preparing 4-substituted anilines by reacting 1-fluoro-4-nitrobenzenes and 1-chloro-4-nitrobenzenes with primary or secondary amines and subsequent reduction

<img file="PL200413B1_D0013.tif" />

Equimolar amounts of the fluoronitrobenzene or chloronitrobenzene and the amine are dissolved in dimethyl sulfoxide or acetonitrile (0.1 M to 1 M solution) and stirred overnight at 100 ° C. After cooling to room temperature, the reaction mixture was diluted with ether and washed with water. The organic phase was dried over MgSO4, filtered and concentrated. If a precipitate forms in the reaction mixture, it is filtered off and washed with ether or acetonitrile. If the product is also found in the mother liquor, it is worked up as described above with ether and water. The crude products can be purified by chromatography on silica gel (mixtures of dichloromethane / cyclohexane and dichloromethane / ethanol).

PL 200 413 B1

For the subsequent reduction, the nitro compound is dissolved in methanol, ethanol or ethanol / dichloromethane mixtures (0.01 M to 0.5 M solution), palladium on carbon (10%) is added and stirred overnight under normal hydrogen pressure. Then it is filtered and concentrated. The crude product can be purified by chromatography on silica gel (dichloromethane / ethanol mixtures) or by preparative reverse phase HPLC (acetonitrile / water mixtures).

Iron powder can also be used as a reducing agent. For this, the nitro compound is dissolved in acetic acid (0.1 M to 0.5 M solution) and 6 equivalents of iron powder and water (0.3-0.5 times the volume of acetic acid) are added in portions at 90 ° C. within 10-15 minutes. After a further 30 minutes at 90 ° C, it is filtered and the filtrate is concentrated. The residue was extracted with ethyl acetate and 2N sodium hydroxide solution. The organic phase is dried over magnesium sulfate, filtered and concentrated. The crude product can be purified by chromatography on silica gel (dichloromethane / ethanol mixtures) or by preparative reverse phase HPLC (acetonitrile / water mixtures).

The following starting compounds are prepared in an analogous manner:

III-1. 1- (4-Aminophenyl) -L-proline t-butyl ester

MS (ESI): m / z (%) = 304 (M + H + MeCN, 100), 263 (M + H, 20);

HPLC (method 4): rt = 2.79 minutes

III-2. 1- (4-Aminophenyl) -3-piperidinecarboxamide

MS (ESI): m / z (%) = 220 (M + H, 100);

HPLC (method 4): rt = 0.59 minutes

III-3. 1- (4-Aminophenyl) -4-piperidinecarboxamide

MS (ESI): m / z (%) = 220 (M + H, 100);

HPLC (method 4): rt = 0.57 minutes

III-4. 1- (4-Aminophenyl) -4-piperidinone

MS (ESI): m / z (%) = 191 (M + H, 100);

HPLC (method 4): rt = 0.64 minutes

III-5. 1- (4-Aminophenyl) -L-prolinamide

MS (ESI): m / z (%) = 206 (M + H, 100);

HPLC (method 4): rt = 0.72 minutes

III-6. [1- (4-Aminophenyl) -3-piperidinyl] methanol

MS (ESI): m / z (%) = 207 (M + H, 100);

HPLC (method 4): rt = 0.60 minutes

III-7. [1- (4-Aminophenyl) -2-piperidinyl] methanol

MS (ESI): m / z (%) = 207 (M + H, 100);

HPLC (method 4): rt = 0.59 minutes

III-8. 1- (4-Aminophenyl) -2-piperidinecarboxylic acid ethyl ester

MS (ESI): m / z (%) = 249 (M + H, 35), 175 (100);

HPLC (method 4): rt = 2.43 minutes

III-9. [1- (4-Aminophenyl) -2-pyrrolidinyl] methanol

MS (ESI): m / z (%) = 193 (M + H, 45);

HPLC (method 4): rt = 0.79 minutes

III-10. 4- (2-Methylhexahydro-5H-pyrrolo [3,4-d] isoxazol-5-yl) -phenylamine

This compound is prepared starting from 2-methylhexahydro-2H-pyrrolo [3,4-d] isoxazole (Ziegler, Carl B. et al; J. Heterocycl. Chem., 25, 2, 1988, 719-723)

MS (ESI): m / z (%) = 220 (M + H, 50), 171 (100);

HPLC (method 4): rt = 0.54 minutes

III-11. 4- (1-Pyrrolidinyl) -3- (trifluoromethyl) -aniline

MS (ESI): m / z (%) = 231 (M + H, 100);

HPLC (method 7): rt = 3.40 minutes

III-12. 3-Chloro-4- (1-pyrrolidinyl) -aniline

MS (ESI): m / z (%) = 197 (M + H, 100);

HPLC (method 4): rt = 0.78 minutes

III-13. 5-Amino-2- (4-morpholinyl) -benzamide

MS (ESI): m / z (%) = 222 (M + H, 100);

HPLC (method 4): rt = 0.77 minutes

PL 200 413 B1

III-14. 3-Methoxy-4- (4-morpholinyl) -aniline

MS (ESI): m / z (%) = 209 (M + H, 100);

HPLC (method 4): rt = 0.67 minutes

III-15. 1- [5-Amino-2- (4-morpholinyl) -phenyl] -ethanone

MS (ESI): m / z (%) = 221 (M + H, 100);

HPLC (method 4): rt = 0.77 minutes

General method for the preparation of 4-substituted anilines by the reaction of 1-fluoro-4-nitrobenzenes with amides and subsequent reduction

<img file="PL200413B1_D0014.tif" />

The amide is dissolved in DMF and 1.5 equivalents of potassium t-butoxide are added. The mixture is stirred for 1 hour at room temperature, then 1.2 equivalents of 1-fluoro-4-nitrobenzene are added portionwise. The reaction mixture was stirred overnight at room temperature, diluted with ether and ethyl acetate, and washed with saturated aqueous sodium bicarbonate solution. The organic phase is dried over magnesium sulfate, filtered and concentrated. The crude product can be purified by chromatography on silica gel (dichloromethane / ethanol mixtures).

For the subsequent reduction, the nitro compound is dissolved in ethanol (0.01 M to 0.5 M solution), palladium on carbon (10%) is added and stirred overnight under normal hydrogen pressure. Then it is filtered and concentrated. The crude product can be purified by chromatography on silica gel (dichloromethane / ethanol mixtures) or by preparative reverse phase HPLC (acetonitrile / water mixtures).

Iron powder can also be used as a reducing agent. For this, the nitro compound is dissolved in acetic acid (0.1 M to 0.5 M solution) and 6 equivalents of iron powder and water (0.3-0.5 times the volume of acetic acid) are added in portions at 90 ° C. within 10-15 minutes. After a further 30 minutes at 90 ° C, it is filtered and the filtrate is concentrated. The residue was extracted with ethyl acetate and 2N sodium hydroxide solution. The organic phase is dried over magnesium sulfate, filtered and concentrated. The crude product can be purified by chromatography on silica gel (dichloromethane / ethanol mixtures) or by preparative reverse phase HPLC (acetonitrile / water mixtures).

The following starting compounds are prepared in an analogous manner:

IV-1. 1- [4-Amino-2- (trifluoromethyl) -phenyl] -2-pyrrolidinone

MS (ESI): m / z (%) = 245 (M + H, 100);

HPLC (method 4): rt = 2.98 minutes

IV-2. 4- [4-Amino-2- (trifluoromethyl) -phenyl] -3-morpholinone

MS (ESI): m / z (%) = 261 (M + H, 100);

HPLC (method 4): rt = 2.54 minutes

IV-3. 4- (4-Amino-2-chlorophenyl) -3-morpholinone

MS (ESI): m / z (%) = 227 (M + H, 100);

HPLC (method 4): rt = 1.96 minutes

IV-4. 4- (4-Amino-2-methylphenyl) -3-morpholinone

MS (ESI): m / z (%) = 207 (M + H, 100);

HPLC (method 4): rt = 0.71 minutes

IV-5. 5-Amino-2- (3-oxo-4-morpholinyl) -benzonitrile

MS (ESI): m / z (%) = 218 (M + H, 100);

HPLC (method 4): rt = 1.85 minutes

IV-6. 1- (4-Amino-2-chlorophenyl) -2-pyrrolidinone

MS (ESI): m / z (%) = 211 (M + H, 100);

HPLC (method 4): rt = 2.27 minutes

PL 200 413 B1

IV-7. 4- (4-Amino-2,6-dimethylphenyl) -3-morpholinone is prepared starting from 2-fluoro-1,3-dimethyl-5-nitrobenzene (Bartoli et al., J. Org. Chem. 1975, 40, 872 )

MS (ESI): m / z (%) = 221 (M + H, 100);

HPLC (method 4): rt = 0.77 minutes

IV-8. 4- (2,4-Diaminophenyl) -3-morpholinone is prepared starting from 1-fluoro-2,4-dinitrobenzene

MS (ESI): m / z (%) = 208 (M + H, 100);

HPLC (method 4): rt = 0.60 minutes

IV-9. 4- (4-Amino-2-chlorophenyl) -2-methyl-3-morpholinone is prepared starting from 2-methyl-3-morpholinone (Pfeil, E .; Harder, U .; Angew. Chem. 1967, 79, 188 )

MS (ESI): m / z (%) = 241 (M + H, 100);

HPLC (method 4): rt = 2.27 minutes

IV-10. 4- (4-Amino-2-chlorophenyl) -6-methyl-3-morpholinone is prepared starting from 6-methyl-3-morpholinone (EP 350002)

MS (ESI): m / z (%) = 241 (M + H, 100);

HPLC (method 4): rt = 2.43 minutes. Synthesis Example

The following examples 17, 42-46 and 55-57, 255 relate to variant [A]. (Example 47 does not pertain to the compounds of the invention and is only given to explain how the compounds of Examples 55-57 are prepared).

Example 17. 5-Chloro-N - ({(5S) -2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

<img file="PL200413B1_D0015.tif" />

From 1- (4-aminophenyl) -pyrrolidin-2-one (for preparation see Reppe et al., Justus Liebigs Ann. Chem .; 596; 1955; 209) analogously to a known synthesis scheme (see SJ Brickner et al., J. Med. Chem. 1996, 39, 673) after reaction with benzyloxycarbonyl chloride, subsequent reaction with R-glycidyl butyric acid ester, reaction with potassium phthalimide, hydrazinolysis in methanol and reaction with 5-chlorothiophene-2-carboxylic acid finally give 5-chloro N - ({(5S) -2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} methyl) -2-thiophenecarboxamide. Thus obtained 5-chloro-N - ({(5S) -2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl ) -2-thiophenecarboxamide has an IC50 value = 4 nM (Test method for IC50 values ​​according to Example A-1.a.1 described above) "Factor Xa inhibition measurement").

Melting point: 229 ° C;

Rf value (SiO2, toluene / ethyl acetate 1: 1) = 0.05 (educt = 0.0);

MS (ESI): 442.0 (21%, M + Na, pattern Cl), 420.0 (72%, M + H, pattern Cl), 302.3 (12%), 215 (52%), 145 (100%);

¹H-NMR (d 6 -DMSO, 300 MHz): 2.05 (m, 2H), 2.45 (m, 2H), 3.6 (t, 2H), 3.77-3.85 (m, 3H ), 4.15 (t, 1H), 4.75-4.85 (m, 1H), 7.2 (d, 1H), 7.5 (d, 2H), 7.65 (d, 2H) , 7.69 (d, 1H), 8.96 (t, 1H).

The individual steps of the above-described synthesis of Example 17 together with the respective preliminary steps are as follows:

To 4 g (22.7 mmol) of 1- (4-aminophenyl) -pyrrolidin-2-one and 3.6 ml (28.4 mmol) of N, N-dimethylaniline in 107 ml of tetrahydrofuran are slowly added at -20 ° C. C 4.27 g (25.03 mmol) chloroformic acid benzyl ester. The mixture was stirred for 30 minutes at -20 ° C and then allowed to reach room temperature. 0.5 liters of ethyl acetate are added and the organic phase is washed with 0.5 liters of saturated NaCl solution. The separated organic phase is dried over MgSO4 and the solvent is evaporated off in vacuo. The residue is triturated with diethyl ether and suction filtered

PL 200 413 B1. 5.2 g (73.8% of theory) of benzyl 4- (2-oxo-1-pyrrolidinyl) phenylcarbamate are obtained in the form of light beige crystals, m.p. 174 ° C.

To 1.47 g (16.66 mmol) of isoamyl alcohol in 200 ml of tetrahydrofuran is added dropwise under argon at -10 ° C to 7.27 ml of a 2.5 M solution of n-butyllithium (BuLi) in hexane, and the need for further 8 ml of BuLi solution until the color of the added N-benzylidenebenzylamine indicator changes color. It is stirred for 10 minutes at -10 ° C, cooled to -78 ° C and a solution of 4.7 g (15.14 mmol) of benzyl 4- (2-oxo-1-pyrrolidinyl) phenylcarbamate is slowly added. . Then 4 ml of n-BuLi solution is added again until the indicator turns pink. The mixture is stirred for 10 minutes at -78 ° C, and 2.62 g (18.17 mmol) of butyric acid R-glycidyl ester are added and the mixture is stirred for 30 minutes at -78 ° C.

The mixture is allowed to reach room temperature overnight, 200 ml of water are added to the mixture and the THF is evaporated off in vacuo. The aqueous residue is extracted with ethyl acetate, the organic phase is dried over MgSO4 and evaporated down i. Vac. The residue is triturated with 500 ml of diethyl ether and the precipitated crystals are suction filtered.

There are obtained 3.76 g (90% of theory) of (5R) -5- (hydroxymethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-2-one at the temperature mp 148 ° C and Rf value (SiO2, toluene / ethyl acetate 1: 1) = 0.04 (educt = 0.3).

3.6 g (13.03 mmol) of (5R) -5- (hydroxymethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-2-one and 2, 9 g (28.67 mmol) of triethylamine in 160 ml of dichloromethane are stirred at 0 ° C. 1.79 g (15.64 mmol) of methanesulfonic acid chloride are added with stirring and the mixture is stirred for 1.5 hours at 0 ° C and for 3 hours at room temperature.

The reaction mixture is washed with water and the aqueous phase is extracted again with methylene chloride. The combined organic extracts are dried over MgSO4 and evaporated. Then the residue (1.67 g) was dissolved in 70 ml of acetonitrile, 2.62 g (14.16 mmol) of potassium phthalimide were added and the mixture was stirred in a sealed vessel in a microwave oven for 45 minutes at 180 ° C.

Undissolved components are filtered off from the mixture, the filtrate is evaporated in vacuo, the residue (1.9 g) is dissolved in methanol and 0.47 g (9.37 mmol) of hydrazine hydrate is added. The mixture was boiled for 2 hours, a saturated sodium bicarbonate solution was added and the mixture was extracted six times with a total of 2 liters of methylene chloride. The combined organic extracts of the crude (5S) -5- (aminomethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-2-one are dried over MgSO4 and evaporated in vacuo .

Final compound, 5-chloro-N - ({(5S) -2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide, prepared by preparing 0.32 g (1.16 mmol) of the above-prepared (5S) -5- (aminomethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl ] -1,3-oxazolidin-2-one, 5-chlorothiophene-2-carboxylic acid (0.19 g, 1.16 mmol) and 1-hydroxy-1H-benzotriazole hydrate (HOBT) (0.23 g, 1 , 51 mmol) is dissolved in 7.6 mL of DMF. 0.29 g (1.51 mmol) of N '- (3-dimethylaminopropyl) -N-ethylcarbodiimide (EDCI) is added and 0.3 g (0.4 ml, 2.32 mmol, 2 eq.) Is added dropwise at room temperature. diisopropylethylamine (DIEA). The mixture was stirred overnight at room temperature.

The mixture is evaporated to dryness in vacuo, the residue is dissolved in 3 ml of DMSO and chromatographed on RP-MPLC with a gradient of acetonitrile / water / 0.5% TFA. Acetonitrile is evaporated off from the appropriate fractions and the precipitated compound is suction filtered. 0.19 g (39% of theory) of the target compound is obtained.

General method for the preparation of acyl derivatives starting from 5- (aminomethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-2-one and carboxylic acids

<img file="PL200413B1_D0016.tif" />

PL 200 413 B1

To 2.9 equivalents of resin bound carbodiimide (PS-Carbodiimide, Argonaut Technologies) was charged the appropriate carboxylic acid (about 2 equivalents) and an absolute dichloromethane / DMF mixture (about 9: 1). After approximately 15 minutes of gentle shaking at room temperature, 5- (aminomethyl) -3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-2-one (from example 45) is added. (1.0 eq.) And the mixture was shaken overnight, the resin was filtered off (washed with dichloromethane) and the filtrate was concentrated. The resulting product is optionally purified by preparative RP-HPLC.

The following is produced in an analogous way:

Example 42 5-Methyl-N - ({2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) - 2-thiophenecarboxamide

LC-MS: m / z (%) = 400 (M + H, 100).

LC-MS (method 6): rt (%) = 3.23 (100),

IC50: 0.16 μΜ

Example 43 5-Bromo-N - ({2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) - 2-thiophenecarboxamide

LC-MS: m / z (%) = 466 (M + H, 100).

LC-MS (method 5): rt (%) = 3.48 (78)

IC₅₀: 0.014 µM

Example 44 5-Chloro-N - ({(5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

<img file="PL200413B1_D0017.tif" />

PL 200 413 B1

a) 2 - ((2R) -2-hydroxy-3 - {[4- (3-oxo-4-morpholinyl) -phenyl] -amino} propyl) -1H-isoindole-1,3- (2H) - dione

Suspension of 2 - [(2S) -2-oxiranylmethyl] -1H-isoindole-1,3 (2H) -dione (A. Gutcait et al., Tetrahedron Asym. 1996, 7, 1641) (5.68 g, 27.9 mmol) and 4- (4-aminophenyl) -3-morpholinone (5.37 g, 27.9 mmol) in ethanol-water (9: 1, 140 ml) is heated to reflux for 14 hours (the precipitate goes into solution, after some time a precipitate re-forms). The precipitate (desired product) is filtered off, washed three times with diethyl ether and dried. The combined mother liquors are concentrated in vacuo and after the addition of a second portion of 2 - [(2S) -2-oxiranylmethyl] -1H-isoindole-1,3 (2H) -dione (2.84 g, 14.0 mmol) in ethanol- water (9: 1, 70 ml) is suspended and the mixture is refluxed for 13 hours (the precipitate goes into solution, after some time re-forms). The precipitate (desired product) is filtered off, washed three times with diethyl ether and dried. Total yield: 10.14 g, 92% theory.

MS (ESI): m / z (%) = 418 ([M + Na]⁺, 84), 396 ([M + H]⁺, 93),

HPLC (method 3): rt (%) = 3.34 (100).

b) 2 - ({(5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -1H-isoindole 1,3 (2H) -dione

N, N'-carbonyldiimidazole (2.94 g, 18.1 mmol) and dimethylaminopyridine (catalytic amount ). The reaction slurry is stirred at 60 ° C for 12 hours (the precipitate goes into solution, after some time re-forms), a second portion of N, N'-carbonyldiimidazole (2.94 g, 18.1 mmol) is added and the mixture is stirred for a further 12 hours at 60 ° C. The precipitate (desired product) is filtered off, washed with tetrahydrofuran and dried. The filtrate was concentrated in vacuo and the product was purified by flash chromatography (dichloromethane-methanol mixtures). Total yield: 3.32 g, 87% of theory).

MS (ESI): m / z (%) = 422 ([M + H]⁺, 100),

HPLC (method 4): rt (%) = 3.37 (100).

c) 5-chloro-N - ({(5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) - 2-thiophenecarboxamide

Methylamine (40% strength in water, 10.2 ml, 0.142 mol) is added dropwise to a suspension of oxazolidinone (4.45 g, 10.6 mmol) in ethanol (102 ml) at room temperature. The reaction mixture was refluxed for 1 hour and concentrated in vacuo. The crude product is used in the next step without further purification.

5-Chlorothiophene-2-carboxylic acid chloride (2.29 g, 12.7 mmol) is added dropwise to a solution of the amine in pyridine (90 ml) under argon at 0 ° C. Ice cooling is removed and the reaction mixture is stirred for 1 hour at room temperature and mixed with water. After adding dichloromethane and phase separation, the aqueous phase is extracted with dichloromethane. The combined organic phases are dried (sodium sulfate), filtered and concentrated in vacuo. The desired product is purified by flash chromatography (dichloromethane-methanol mixtures). Total yield: 3.92 g, 86% theory.

Melting point: 232-233 ° C, ¹H-NMR (DMSO-d⁶, 200 MHz): 9.05-8.90 (t, J = 5.8 Hz, 1H), 7.70 (d, J = 4.1 Hz, 1H), 7.56 (d, J = 9 , 0 Hz, 2H), 7.41 (d, J = 9.0 Hz, 2H), 7.20 (d, J = 4.1 Hz, 1H), 4.93-4.75 (m, 1H ), 4.27-4.12 (m, 3H), 4.02-3.91 (m, 2H), 3.91-3.79 (dd, J = 6.1 Hz, 9.2 Hz, 1H), 3.76-3.66 (m, 2H), 3.66-3.54 (m, 2H);

MS (ESI): m / z (%) = 436 ([M + H] +, 100, pattern Cl);

HPLC (method 2): rt (%) = 3.60 (100);

[and]²¹D = -38 ° (c = 0.2985, DMSO); ee: 99%;

IC₅₀: 0.7 nM

The following is produced in an analogous way:

Example 45 5-Methyl-N - ({(5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 831 ([2M + H]⁺0.1 100), 416 ([M + H]⁺, 66);

HPLC (method 3): rt (%) = 3.65 (100);

IC50: 4.2 nM

Example 46 5-Bromo-N - ({(5S) -2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 480 ([M + H]⁺, 100, Br pattern);

HPLC (method 3): rt (%) = 3.87 (100);

IC₅₀: 0.3 nM

PL 200 413 B1

Example 47 5-Chloro-N - {[(5S) -3- (3-isopropyl-2-oxo-2,3-dihydro-1,3-benzoxazol-6-yl) -2-oxo- 1,3-oxazolidin-5-yl] methyl} -2-thiophenecarboxamide

<img file="PL200413B1_D0018.tif" />

200 mg (0.61 mmol) 6 - [(5S) -5- (aminomethyl) -2-oxo-1,3-oxazolidin-3-yl] -3-isopropyl-1,3-benzoxazol-2 (3H) hydrochloride -one (EP 738726) is suspended in 5 ml of tetrahydrofuran and 0.26 ml (1.83 mmol) of triethylamine and 132 mg (0.73 mmol) of 5-chlorothiophene-2-carboxylic acid chloride are added. The reaction mixture is stirred overnight at room temperature and then concentrated. The product was isolated by column chromatography (silica gel, methylene chloride / ethanol = 50/1 to 20/1). 115 mg (43% of theory) of the desired compound are obtained.

MS (ESI): m / z = 436 (M + H, 100);

HPLC (method 4): rt = 3.78 minutes.

The following compounds are prepared in an analogous manner:

<td> 55</td><td>0 Q «Μ from 3- (4-amino-phenyl) -oxazolidin-2-one (Artico, M. et al; Farmaco Ed. Sci. 1969,24; 179)</td><td> 256</td><td> 0,04</td>

<td> 56</td><td>Q eM</td><td> 218</td><td> 0,004</td>

<td> 57</td><td>□ Oari</td><td> 226</td><td> 0,58</td>

<td> 255</td><td></td><td> 228-230</td><td></td>

The following examples 95, 97, 112-116, 118, 119, 123 and 127-130a relate to variant [B].

General method for the preparation of 3-substituted 5-chloro-N - [(2-oxo-1,3-oxazolidin-5-yl) -methyl] -2-thiophenecarboxamide derivatives starting from substituted N- (3-amino-2-hydroxypropyl derivatives) ) -5-chloro-2-thiophenecarboxamide

PL 200 413 B1

<img file="PL200413B1_D0019.tif" />

To a solution of the substituted N- (3-amino-2-hydroxypropyl) -5-chloro-2-thiophenecarboxamide derivative (1.0 eq.) In absolute THF (about 0.1 mol / l) carbodiimidazole (1 2-1.8 eq.) Or a comparable phosgene equivalent. The mixture is stirred at room temperature or, if appropriate, at elevated temperature (up to 70 ° C) for 2-18 hours and then concentrated under reduced pressure. The product can be purified by chromatography on silica gel (dichloromethane-methanol mixtures or cyclohexane-ethyl acetate mixtures).

The following is produced in an analogous way:

Example 95 5-Chloro-N - ({3- [3-fluoro-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

<img file="PL200413B1_D0020.tif" />

260 mg (0.608 mmol) 5-chloro-N- (3 - {[3-fluoro-4- (3-oxo-4-morpholinyl) -phenyl] -amino} -2-hydroxypropyl) -2-thiophenecarboxamide (from example 61 ), 197 mg (1.22 mmol) of carbonyimidazole and 7 mg of dimethylaminopyridine in 20 ml of dioxane are refluxed for 5 hours. Then 20 ml of acetonitrile are added and the mixture is stirred in a microwave oven in a closed vessel for 30 minutes at 180 ° C. The solution is centrifuged and chromatographed on an RP-HPLC column. 53 mg (19% of theory) of the target compound are obtained.

NMR (300 MHz, d6-DMSO): δ = 3.6-3.7 (m, 4H), 3.85 (dd, 1H), 3.95 (m, 2H), 4.2 (m, 1H) ), 4.21 (s, 2H), 4.85 (m, 1H), 4.18 (s, 2H), 7.19 (d, 1H, thiophene), 7.35 (dd, 1H), 7 , 45 (t, 1H), 7.55 (dd, 1H), 7.67 (d, 1H, thiophene), 8.95 (t, 1H, CONH).

Example 97 5-Chloro-N - ({2-oxo-3- [4- (3-oxo-4-morpholinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) - 2-thiophenecarboxamide is prepared starting from Example 63.

MS (ESI): m / z (%) = 458 ([M + Na]⁺, 66), 436 ([M + H]⁺, 100), standard Cl;

HPLC (method 3): rt (%) = 3.89 (100)

IC50: 1.4 nM.

Example 112 5-Chloro-N - ({2-oxo-3- [4- (2-oxo-1-pyrrolidinyl) -3- (trifluoromethyl) -phenyl] -1,3-oxazolidin-5- yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 488 (M + H, 100);

HPLC (method 4): rt = 3.64 minutes.

Example 113 5-Chloro-N - ({3- [3-chloro-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 470 (M + H, 100);

HPLC (method 4): rt = 3.41 minutes.

Example 114 5-Chloro-N - ({2-oxo-3- [4- (3-oxo-4-morpholinyl) -3- (trifluoromethyl) -phenyl] -1,3-oxazolidin-5- yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 504 (M + H, 100);

HPLC (method 4): rt = 3.55 minutes.

Example 115 5-Chloro-N - ({3- [3-methyl-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 450 (M + H, 100);

HPLC (method 4): rt = 3.23 minutes.

PL 200 413 B1

Example 116 5-Chloro-N - ({3- [3-cyano-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 461 (M + H, 100);

HPLC (method 4): rt = 3.27 minutes.

Example 118 5-Chloro-N - ({3- [3-chloro-4- (2-oxo-1-pyrrolidinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 454 (M + H, 100);

HPLC (method 4): rt = 3.49 minutes.

Example 119 5-Chloro-N - ({3- [3,5-dimethyl-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5- yl} -methyl) -2-thiophenecarboxamide

MS (ESI): m / z (%) = 464 (M + H, 100);

HPLC (method 4): rt = 3.39 minutes.

Example 123. N - ({3- [3-Amino-4- (3-oxo-4-morpholinyl) -phenyl] -2-oxo-1,3-oxazolidin-5-yl} -methyl) - 5-chloro-2-thiophenecarboxamide

MS (ESI): m / z (%) = 451 (M + H, 100);

HPLC (method 6): rt = 3.16 minutes.

The following compounds are also prepared by opening the epoxy bond with an amine and then cyclizing to the corresponding oxazolidinone:

<td>Example no</td><td>Building</td><td>Tt [° C]</td><td>IC50 [pM]</td>

<td> 127</td><td></td><td> 159</td><td> 0,0007</td>

<td> 128</td><td></td><td> 198</td><td> 0,002</td>

<td> 129</td><td>q-o4ut ^⁰ 0</td><td> 196</td><td> 0,001</td>

<td> 130</td><td></td><td> 206</td><td> 0,0033</td>

The following example 162 relates to the derivatization of an amine group of oxazolidinone.

Example 162.

5-Chloro-N - ({(5S) -2-oxo-3- [4- (2-oxo-1-piperidinyl) -phenyl] -1,3-oxazolidin-5-yl} -methyl) -2- thiophenecarboxamide

<img file="PL200413B1_D0021.tif" />

Under neutral conditions, 5 ml of DMSO are introduced with 30 mg of 60% NaH in paraffin oil and heated for 30 minutes at 75 ° C until gas evolution ceases. Then a solution of 290 mg (0.617 mmol) of 5-chloro-N - [((5S) -3- {4 - [(5-chloropentanoyl) -amino] -phenyl} -2-oxo-1,3-oxazolidin- is added dropwise. 5-yl) methyl] -2-thiophenecarboxamide

PL 200 413 B1

<img file="PL200413B1_D0022.tif" />

ml of methylene chloride and stirred overnight at room temperature. The reaction was stopped and the mixture was poured into 100 ml of water and extracted with ethyl acetate. The organic phase was evaporated and chromatographed on an RP-8 column, eluting with acetonitrile / water. 20 mg (7.5% of theory) of the target compound are obtained.

Melting point: 205 ° C;

NMR (300 MHz, d6-DMSO): δ = 1.85 (m, 4H), 2.35 (m, 2H), 3.58 (m, 4H), 3.85 (m, 1H), 4. 2 (t, 1H), 4.82 (m, 1H), 7.18 (d, 1H, thiophene), 7.26 (d, 2H), 7.5 (d, 2H), 2.68 (d , 1H, thiophene), 9.0 (t, 1H, CONH);

IC₅₀: 2.8 nM.

All products of the solid phase synthesis are characterized by LC-MS. The following separation system is typically used for this purpose: HP 1100 with UV detector (208-400 nm), oven temperature 40 ° C, Waters-Symmetry C18 column (50 mm x 2.1 mm, 3.5 μm), eluent A : 99.9% acetonitrile / 0.1% formic acid, eluent B: 99.9% water / 0.1% formic acid; gradient:

<td>Time</td><td>AND:%</td><td>B:%</td><td>Flow</td>

<td> 0,00</td><td> 10,0</td><td> 90,0</td><td> 0,50</td>

<td> 4,00</td><td> 90,0</td><td> 10,0</td><td> 0,50</td>

<td> 6,00</td><td> 90,0</td><td> 10,0</td><td> 0,50</td>

<td> 6,10</td><td> 10,0</td><td> 90,0</td><td> 1,00</td>

<td> 7,50</td><td> 10,0</td><td> 90,0</td><td> 0,50</td>

Detection of substances is carried out with a Micromass Quattro LCZ MS, ionization: ESI positive / negative.

In the above-mentioned structures containing one or more · or -O residues, a function is always understood

<img file="PL200413B1_D0023.tif" />

or -OH.