SI21658A - Antithrombotic active ingredients with dual mechanism of action basedon 3,4-dihydro-2h-1,4-benzoxazine skeleton - Google Patents

Abstract

The invention relates to compounds with the general formula (I) and their pharmaceutically acceptable salts, where substituents have meanings given in the description. The compounds are used as antithrombotic active ingredients with inhibiting action on aggregation of thrombocites and simultaneously, on inhibition of thrombine and/or Xa factor.

Description

University of Ljubljana, Faculty of Pharmacy Lek Pharmaceutical Company d.d.

ANTITROMBOTIC SUBSTANCES WITH DUAL MECHANISM OF OPERATION BASED ON 3.4-DIHYDRO-2H-1.4-BENZOXASINE SKELET

FIELD OF THE INVENTION

The invention is within the scope of the pharmaceutical industry and relates to novel heterocyclic mimetics of the Arg-Gly-Asp and D-Phe-Pro-Arg tripeptides that prevent fibrinogen binding to platelets via the fibrinogen receptor while inhibiting the serine proteases involved in the coagulation cascade. The compounds of the invention prevent platelet aggregation and blood coagulation and have antithrombotic activity. The invention also relates to processes for their preparation, to pharmaceutical products containing them and to their use in therapy.

A technical problem

Cardiovascular disease is the most common cause of hospital treatment and death in the Western world. In the treatment of cardiovascular diseases, antifungal and fractionated heparins and antagonists of vitamin K are used as anticoagulants, as acetylsalicylic acid, ticlopidine, clopidogrel and dipyridamole as anti-aggregating agents. Heparins inhibit coagulation factors indirectly by enhancing the inhibitory activity of endogenous antithrombin III. They have a narrow therapeutic window and a low half-life, which is why they are administered several times a day, and they also exhibit a weak correlation of the dose-anticoagulant effect, which includes mandatory monitoring of treatment by laboratory verification of coagulation parameters. Patients are also uncomfortable with parenteral or subcutaneous administration and serious side effects. Heparins express a limited inhibition of thrombin bound in the blood clot, which in turn retains enzymatic activity and causes local platelet activation, which is probably an important factor in the recurrence of thrombosis after discontinuation of treatment. Indirect anticoagulants inhibit the synthesis of coagulation factors. They block the regeneration of vitamin K, which is required for posttranslational γ-carboxylation of glutamic acid residues in biosynthesis of factors II, VII, IX and X. This prevents the formation of thrombin by reducing the concentration of prothrombin and other coagulation factors. Even with indirect anticoagulants, because of the large variations in response, monitoring of the treatment by measuring blood clotting parameters is required, in addition to exhibiting many side effects and frequent interactions with other drugs. As an anti-aggregating agent, acetylsalicylic acid enables irreversible acetylation of the hydroxyl group of serine in cyclooxygenase, thus blocking the pathway of arachidonic acid conversion and TXA ₂ formation. However, it does not prevent platelet activation by agonists within other signaling pathways and therefore does not allow complete inhibition of platelet activation. New approaches to the development of antithrombotic agents consider different sites to prevent thrombin formation as a major prothrombotic factor in the coagulation cascade and to inhibit platelet activation and aggregation processes as effectively as possible. Serum protease inhibitors of the coagulation cascade and fibrinogen receptor antagonists are topical antithrombotic agents. Due to the disadvantages of the currently used antithrombotic agents, there is a need for new antithrombotic agents that are effective, safe and exhibit activity after oral administration. A possible solution to the described technical problem are antithrombotic agents with a dual mechanism of action that prevent the binding of fibrinogen to the fibrinogen receptor of platelets and simultaneously inhibit the serine proteases of the coagulation cascade.

The state of the art

The fibrinogen receptor (integrin α ^ βδ) is a transmembrane heterodimeric glycoprotein from the integrin receptor family located predominantly on the platelet surface. It consists of non-covalently linked α and β subunits, where the / V-terminal domains of both subunits represent the extracellular part of the receptor and form a site for ligand interaction, and the shorter intracellular C-terminal domains are the site of cell signaling protein interaction with the membrane and allow membrane signaling. . Under normal conditions, the fibrinogen receptor is dormant. Under the influence of various agonists released from endothelial cells and platelets upon tissue damage, it is activated. Activation is reflected in a conformational change in the receptor, which results in an increased affinity for fibrinogen binding. The binding of fibrin molecules enables the binding of adjacent platelets and the formation of a blood clot.

Simultaneously with platelet aggregation, the blood clotting process is carried out by serine proteases within the coagulation cascade. Coagulation factors enable cascade activation of other factors in the coagulation cascade, which ultimately leads to the formation of factor Xa, or thrombin. Thrombin allows the fibrinogen to be cleaved to fibrin by subsequently forming transverse connections between the fibrin monomers and forming a mechanically stable blood clot. Thrombin activates FXIII, which further stabilizes the fibrin polymer. In addition, thrombin activates prothrombin, FV, FVIII, FXI, which represents positive feedback loops that provide the amplification mechanism required for the extremely rapid response and effectiveness of the coagulation process and activates protein C as a hemostasis inhibitory mechanism. Thrombin enables platelet activation for further adhesion and aggregation. In addition to these, thrombin has a number of cellular effects in the context of tissue repair processes. Because of its central role in the process of hemostasis, thrombin is an interesting target for the design of active substances in the field of cardiovascular therapy. Other serine proteases of the coagulation cascade, in particular factor Xa, are also relevant in the design of novel antithrombotic agents. It expresses proteolytic activity in the initial part of the amplification mechanism of the coagulation cascade, which means that the plasma levels of this enzyme are significantly lower than the plasma levels of thrombin in the coagulation process and that lower inhibitor levels are required for successful inhibition.

The starting point for the design of fibrinogen receptor antagonists and thrombin inhibitors is the amino acid sequences of natural substrates, the RGD (Arg-Gly-Asp) sequence for fibrinogen receptor antagonists, and the D-Phe-Pro-Arg sequence for thrombin inhibitors. The peptide nature of the two lead compounds is the reason for the low bioavailability, metabolic instability and indiscrimination of the peptide ligands prepared on this basis for modulation of hemostasis processes. The development of the fibrinogen receptor antagonists therefore proceeded from simple linear and cyclic peptides towards peptidomimetic agents. Various SAR studies have identified the need for the presence of the guanidine group Arg and the carboxyl group Asp, which are suitably oriented in the space 1.2-1.8 A in space (Ojima, I .; Chakravarty, S.; Dong, Q. Bioorg. Med. Chem. 1995, 3, 337-360). Following the pharmacophore requirement with a combination of peptidomimetic approaches, compounds with a completely non-peptide heterocyclic framework (benzofuran, thiazole, isoxazoline, indole, benzodiazepine, benzimidazole, benzopyran, etc. ring) were attached to which key pharmacophore groups are attached. Benzamidine or various cyclic amines are the most commonly used mimetic of arginine. Aspartate mimetics use a wide variety of more or less complex carboxyl group fragments. These approaches have produced a number of highly effective selective antagonists, many of which are effective after oral administration. (Ojima, I.; Chakravarty, S.; Dong, Q. Bioorg. Med. Chem. 1995, 3, 337-360; Eldred, CD; Judkins, BD Prog. Med. Chem. 1999, 36, 29-90; Zablocki, JA; Rao, S, N; Baron, DA; Flynn, DL; Nicholson, NS; Feigen, LP Curr. Pharm. Des. 1995, 7, 533-558; Wang, W .; Borchardt, RT; Wang , B. Curr. Med. Chem. 2000, 7, 437453; Scarborough, RM; Gretler, DDJ Med. Chem. 2000, 43, 1-21). The advantage of using the fibrinogen receptor antagonists is that they are able to inhibit all pathways leading to platelet aggregation and platelet plug formation. Tirofiban is a representative of low molecular weight non-peptide fibrinogen receptor antagonists approved for use in conditions of acute coronary syndrome.

Thrombin inhibitors were designed based on the D-Phe-Pro-Arg sequence in combination with the known structure of the active site of thrombin (Bode W., Mayr I., Baumann U., Huber R., Stone SR, Hofsteenge J. EMBO J. 1989 , 8, 3467-3475). The basic P1 inhibitor residues are thought to bind into the S1 pocket, the smaller lipophilic residues into the S2 pocket, and the larger lipophilic fragments into the S3 pocket thrombin. The first effective thrombin inhibitors were covalent inhibitors, which form a covalent bond with Ser 195 in the thrombin active site and are inadequate due to low selectivity to other serine proteases and slow binding kinetics for therapeutic use. Tendencies in the development of thrombin inhibitors led to reversible low molecular weight thrombin inhibitors; initially various peptide analogues, and further towards peptidomimetic inhibitors. The introduction of nonpeptide structures reduces the possibility of degradation by various peptidases and allows for higher lipophilicity and consequently higher bioavailability (Revvinkel, JBM; Adang, AEP Curr. Pharm.Des. 1995, 5, 1043-1075; Tucker, T.; Isaacs, CA JAI Press Inc. 1999, 2, 53-87; Sanderson, PEJ; Med. Res. Rev. 1999, 79, 179-197; Coburn, CA Exp. Opin. Ther. Patents 2001, 7 7, 721-738.). The result of these studies is argatroban as the first effective therapeutically useful thrombin inhibitor and ximelagatran.

Many highly active low molecular weight factor Xa inhibitors have also been developed based on knowledge of the structure of the active site of factor Xa and the structure of many natural inhibitors.

New trends in the development of antithrombotic agents are taking into account the possibility of preventing the formation of blood clots by simultaneously inhibiting multiple levels of the hemostasis process. In order to develop more effective antithrombotic therapy, some research groups are investigating active substances with a dual mechanism of antithrombotic action. Of particular interest are compounds that simultaneously inhibit two coagulation factors, e.g. thrombin and factor Xa, thrombin and factor Vila and factor Xa and Vila (Nar, H.; Bauer, M.; Schmid, A.; Stassen, J.-M.; Wienen, W.; Priepke, H.; Kauffmann, IK; Ries, UJ; Hauel, NH Structure 2001, 9, 29-37; Kranjc, A.; Kikelj, D. Curr. Med. Chem., In press). A combination of dual antithrombotic activity with thrombin inhibition and antagonist activity at the fibrinogen receptor has also been described (Wienen, W .; Nar, H .; Ries, UJ; Priepke, HWM; Hauel, NH; Stassen, JM Thromb. Haemost. (18th Cong. Intl Soc Thromb Haemostas, July 6-12, Paris, 2001) 2001, Suppl., Abst P756.).

Description of solution to a technical problem with implementation examples

The invention relates to novel compounds of general formula (I):

A is O, S, NH or CH ₂ ;

B is C = O or C = S;

R1 is H, branched or unbranched C1-C4 alkyl, benzyl group or OR group in which

R is H, branched or unbranched C1-C4 alkyl or benzyl;

R2 is H, COOR, CONHR or a substituent K which is one of the groups

where they mean:

X is CONH, NHCO, CH ₂ NH, NHCH ₂ , OCH ₂ or CH ₂ O; Y is CO or CH _2;

R52 is a group

H '

N-R1

NH, group

N-R1 —N— ( ^{with H} NH ² or COOR group,

N-R1 - ( ^Z

R62 is R, COOR or an ^NHs group wherein R and R1 have the meanings defined above;

R3 is H, branched or unbranched C1-C4 alkyl, benzyl, OH, COR or a substituent L which is one of the groups

wherein Z is CO, CH ₂ , a CH ₂ CONH group attached to the N atom of the bicycle via a CH ₂ CO fragment or a CH ₂ CONHCH _{2 group} attached to an N atom of the bicycle via a CH ₂ CO fragment; (CH ₂ ) _n , (CH ₂ ) _{n is} CO or CO (CH ₂ ) _{n is a} group wherein n is 1, 2, 3 or 4;

R53 is a group

N-R 1 < ^z

NH "

N-R1

-NC ^{With the} group

N-R1 - ( ^with NH or -COOR group,

R63 is R, COOR or a group wherein R and R1 have the meanings defined above;

R4 is a group attached at the site of 6 or 7 bicycle ring and may be

H, a group Q-CH (R7) -COOR, Q-CH (R7) CH ₂ COOR, Q-CH (R7) -CONH- (R9) or a substituent M which is one of the groups

where they mean:

Q is a group CON (R8), N (R8) CO, CH ₂ N (R 8), N (R 8) CH _2;

Y is CO or CH _2;

R7 is H, branched or unbranched C1-C4 alkyl, C4-C6 cycloalkyl, phenyl or benzyl;

R 8 is H or a group - [CH ₂ ] _n COOR, where n is 0, 1, 2, 3 or 4, and the substituent R has the meaning defined above;

R 9 is a group CH (R 10) COOR or a group CH ₂ CH (NHSO ₂ C ₆ H ₄ -4-R) COOR, where R has the meaning defined above and where

R10 is a group R, a CH ₂ COOR group or a CH ₂ CH ₂ COOR group with the meaning defined above for the substituent R;

N-R1 N-R1 - <-N- <

NH ^H NH

R54 is group ² , group ² or group -COOR,

N-R1

R64 is R, COOR or group ² wherein R and R1 have the meanings defined above provided that

- if R52 is a group

N-R1 - ( ^With NH ₂

N-R1 (/ ^NHz or group

N-R1 —N- ( ^z

H nh ² or if R62 is R or the group is R4 is H, the groups R53 and R63 are COOR, wherein R63 cannot be COOH;

N-R1 N-R1

- if R52 is a group ^NH or a group ^NH or if R62 is R or a group

N-R1 - ( ^With NH ₂ , R54 and R64 are COOR, where R64 cannot be COOH and R3 cannot be a group L;

N-R1 N-R1 < ^{Z _} H ~ 1 |

- if R53 is a group ^ΝΗζ or group ² or if R63 is an R or group

N-R1 - ( ^With NH ₂

J is R4 H, R52 and R62 are COOR, wherein R62 cannot be COOH;

N-R1 N-R1 - ( ^z -N- ( ^z

NH H NH

- if R53 is group ² or group ² or if R63 is R or group

N-R1 - ( ^With NH ₂ , R54 and R64 are COOR, where R64 cannot be COOH and R2 cannot be a group K;

N-R1 N-R1 —ΝΗ

-if R54 is a group ^NH or a group of ² or if R64 is R or a group

N-R1 - < ^z nh ₂

J is R52 and R62 COOR, wherein R62 cannot be COOH and R3 cannot be a group L;

N-R1 N-R1 - < ^z -NA H

-if R54 is a group ^NH or a group of ² or if R64 is R or a group

N-R1 - ( ^with NH ₂

J is R53 and R63 COOR, where R63 cannot be COOH and R2 cannot be group K

The compounds described in the invention have one or more chiral centers at which the absolute configuration may be R or S and may occur in the form of racemates, racemic mixtures, pure enantiomers, pure diastereomers or mixtures of diastereomers. The invention also relates to pharmaceutically acceptable salts of compounds of formula I.

The invention also relates to processes for the preparation of compounds of general formula I. The starting heterocyclic compounds for the preparation of the compounds of the present invention are prepared by known methods. Compounds of formula (I) wherein A is oxygen and B is a C = O group (e.g., compounds 2 and 27) are prepared from the corresponding phenols in analogy to the procedures described in the literature (Shridhar, DR; Gandhi, SS; Rao, KS Synthesis 1982 , 986-987). Ethyl 4 - [(2,2-dichloroacetyl) amino] -3 hydroxybenzoate (39) was prepared from ethyl 4-amino-3-hydroxybenzoate (38) by acylation with dichloroacetyl chloride (March, J. Advanced organic chemistry. Reactions, mechanisms and structure John Wiley & Sons, Inc., New York, 4th Ed, 1992, 417-419) and subsequent cyclization in an alkaline medium. (Honkanen, E .; Virtanen, AI Acta Chem. Scand. 1960, 14, 504-507). Compounds of formula (I) wherein B is a group C = S are prepared from the corresponding amides (B is a C = O group) by reaction with phosphorus pentasulfide (P ₄ S ₁₀ ), as is well known in the art (Becker, HGO Organicum: Organic-chemisches Grundpraktikum. WileyVCH, Veinheim, 21 st Ed, 2001, 506-507). Preparation of Compounds of Formula (I) wherein A is a group

NH, is described in a monograph (Urleb, U. Annulated 1,3- and 1,4-diazines: Quinoxalines. Ed. In: Schaumann, E. Methods of Organic Chemistry. Methoden der Organischen Chemie: Houben-Weyl: Hetarenes IV: six-membered and larger hetero-rings with maximum unsaturation. Vol. E 9b / part 2. 4th Ed. Stuttgart; New York: G. Thieme, 1998, 193-265). Compounds of formula (I) wherein A is sulfur (S) are prepared as described e.g. in the article (Leskovsek-Cizej, V.; Urleb, U. The synthesis of 3, 4-dihydro-2-methyl-3-oxo-2H-benzo1,4-thiazine-2-carboxylic acids. J. Heterocycl. Chem. 1996, 33, 97-101).

4 - [(7-Nitro-3-oxo-2,3-dihydro-4 H -1,4-benzoxazin-4-yl) methyl] benzonitrile (3) and 4 - [(7-formyl 3-oxo- 2,3-Dihydro-4H-1,4-benzoxazin-4-yl) methyl] -benzonitrile (28) is prepared by alkylation of the 2 H-1,4-benzoxazin-3 (4 H) -one system with 4- (bromomethyl) benzonitrile in the presence of potassium carbonate and benzyltriethylammonium chloride in acetonitrile (Rutar, A.; Kikelj, D. Synth. Commun. 1998, 28, 2737-2749). Ethyl 2-hydroxy-4-methyl-3-oxo-3,4-dihydro-2H-1,4benzoxazine-7-carboxylate (41) is prepared by alkylation of ethyl 2-hydroxy-3-oxo-3,4-dihydro2 / - / -1,4-Benzoxazine-7-carboxylate (40) with dimethyl sulfate in the presence of potassium carbonate in acetone (Bourlot, A.-S.; Sanchez, I.; Dureng, G.; Guillaumet, G.; Massingham, R. Monteil, A.; Winslow, E.; Pujol, M.D.; Merour, J.-YJ. Med. Chem. 1998, 41, 3142-3158).

Ethyl esters are prepared by the method of classical acid-catalyzed esterification in absolute ethanol with the addition of concentrated H ₂ SO ₄ (March, J. Advanced Organic Chemistry. Reactions, mechanisms and structure. John Wiley & Sons, Inc., New York, 4th Ed, 1992; 393-396). Ether 42 was prepared from alcohols 40 and 4- (bromomethyl) bezonitrile in the presence of potassium fluoride in DMF.

Reductions of nitro groups to amino groups for the preparation of compounds 4 and 38 are carried out, as is commonly known in the art, by catalytic hydrogenation using palladium on carbon as a catalyst. In the synthesis of 3-oxo-3,4-dihydro-2F / -1,4-benzoxazine-7-carbaldehyde (27), the reduction of the nitro group is carried out with tin (II) chloride in ethanol. Hydrolysis of esters is carried out according to the well-known alkaline hydrolysis processes.

Compounds of methyl 2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2W-1,4-benzoxazin-7yl] amino} -3-oxopropanoate (8), ethyl 2 - [(2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro12

2H-1,4-benzoxazin-7-yl] amino} -3-oxopropanoyl) amino] acetate (14), diethyl (2S) -2 - [(2benzyl-3 - {[4- (4-cyanobenzyl) -3 -oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl] amino} -3oxopropanoyl) amino] pentanedioate (19), diethyl (2S) -2 - {[(2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo3,4-dihydro-2H-1,4-benzoxazin-7-yl] methyl} amino) -3-phenylpropanoyl] amino} pentanedioate (34) and ethyl ( 3S) -3 - [({2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazin-7yl} carbonyl) amino] -3-Phenylpropanoate (45) was prepared from the corresponding carboxylic acids by the amide bond formation method using EDC (N-(3-dirnethylaminopropyl) - N-ethyl-carbodiimide) and HOBT (N-hydroxylbenzotriazole) (Bodanszky, M Principles of peptide synthesis. Springer-Verlag 1984, 36-49).

2-Benzyl 3-methoxy-3-oxopropanoic acid (7) is prepared by alkylation of benzyl methyl malonate (5) by a modified literature procedure (Chorevv, M.; Goodman, M. Int. J. Peptide Proteins Res. 1983, 21, 258- 268.) and with the following catalytic hydrogenation of 2benzyl benzyl methyl malonate (6).

Compounds of diethyl (2S) -2 - [((2S) -2 - {[(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H1,4-benzoxazine- 7-yl) methyl] amino} -3-phenylpropanoyl) amino] pentanedioate in the form of acetate (31), diethyl (2S) -2 - [((2S) -2 - {[(4- {4- [amino (imino ) methyl] benzyl} -3-oxo-3,4-dihydro-2 H -1,4-benzoxazin-7-yl) methyl] amino} -3-phenylpropanoyl) amino] pentanedioate in the form of acetate (35) and ethyl ( 3S) -3 - ({[2 - ({4- [amino (imino) methyl] benzyl} oxy) -4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7 -yl] carbonyl} amino) -3-phenylpropanoate in the form of acetate (46) is prepared under the conditions of Pinner synthesis of amidines from the corresponding nitriles 30, 34 and 45 according to the procedure described in the literature. (Dow, A. W. Organic Synthesis, Coli. Vol. 1, John Wiley & Sons, Inc., editor Gilman H.; New York, 2nd Ed., 1951, 5-7).

Amidine methyl 3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl} -amino] -2-benzyl-3 -oxopropanoate in the form of acetate (11), ethyl 2 - ({3 - [(4- {4 [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4-benzoxazine- 7-yl) amino] -2-benzyl-3-oxopropanoyl} amino) acetate in the form of acetate (17), diethyl (2S) -2 - ({3 - [(4- {4 [amino (imino) methyl] benzyl} - 3-Oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl) amino] -2-benzyl-3oxopropanoyl} amino) pentanedioate in the form of acetate (22) and (2S) -2 - ({3 - [(4- {4 [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl) amino] -2-benzyl-313 oxopropanoyl} amino ) pentanedioic acid in the form of acetate (24) is prepared by converting the corresponding nitriles 8, 14 and 19 to amidoximes 9, 15 and 20 with hydroxylamine or hydroxylammonium chloride, following the formation of 1,2,4-oxadiazol-5 (4H) -ones 10, 16 and 21 with ethyl chloroformate in the presence of an appropriate base and their reduction under the conditions of catalytic hydrogenation in acetic acid (Bolton, RE; Coote, SJ; Finch, H.; Low don, A.; Pegg, N.; Vinader, M. V. Tetrahedron Lett. 1995, 36, 4471-4474; Chiou, S.; Shine, H. J. J. Heterocyclic Chem. 1989, 26, 125-128).

Pharmaceutically acceptable salts of the compounds of general formula (I) are prepared by converting the compounds of general formula I with acids or bases to the corresponding salts according to methods generally known in the art.

The invention also relates to the use of compounds of general formula (I) as therapeutically effective compounds for the preparation of medicaments. The invention also provides pharmaceutical compositions comprising compounds of general formula (I) and the use of compounds of general formula (I) in therapy. The compounds of the present invention may be administered in the form of injections or oral preparations, and may also be formulated to allow controlled and prolonged release of the active substance. In addition to the active substance, they may contain standard additives. The method of administration, frequency of administration and dose depend on the individual active substance and its pharmacokinetic properties and the patient's condition.

The novel compounds of this invention are fibrinogen receptor antagonists and thrombin inhibitors and factor Xa. They prevent platelet aggregation by inhibiting the binding of fibrinogen to platelets and prevent the formation of a mechanically stable blood clot under secondary hemostasis under the influence of factor Xa and thrombin. They can be used to prevent and treat arterial and venous thrombosis, ischemic stroke, peripheral arterial disease, acute coronary syndrome, pulmonary emboli, and to prevent systemic emboli in cardiac patients, to prevent ischemic complications in patients with surgery, such as prevention of constipation in the arterial reconstruction procedures, prevention of blood clotting in the extracellular circulation and hemodialysis.

Biological tests

Enzyme method for the determination of the strength of serine protease inhibitors

1. The basics of the method

The enzyme amidolytic method for the determination of inhibitory activity on serine proteases is based on spectrophotometric determination of the absorbance of a product formed upon bond cleavage under the influence of an enzyme. The serine protease cleaves the chromogenic substrate to form a yellow-colored p-nitroaniline. The concentration of released p-aniline is a measure of the activity of the enzyme and is proportional to the absorbance of the sample measured at 405 nm. The inhibitor that binds to the active site prevents the enzymatic cleavage of the substrate, which is reflected as a decrease in the amidolytic activity of the enzyme. IC ₅₀ and Ki, representing a quantitative measure of inhibitor potency, were determined by spectrophotometric monitoring of the enzyme kinetics without the inhibitor and after the inhibitor was added (Cheng, YC; Prusoff, WH Biochem. Pharmacol. 1973, 22, 3099-3108).

Determination of inhibitory activity on serine proteases is carried out according to the same principle, and the procedures differ in the type and method of preparation of individual serine proteases and their corresponding chromogenic substrates. Given the structural similarity of the thrombin coagulation cascade factors and the trypsin factor Xa, it is reasonable to determine the trypsin inhibitory constants to assess the selectivity of the inhibitors.

2. Reagents

REAGENT MANUFACTURER AND MANUFACTURER'S CODE Thrombin Factor Xa Trypsin Chromogenic substrate for thrombin Chromogenic substrate for factor Xa Chromogenic trypsin substrate HBSA buffer bovine serum albumin human thrombin, 308 NIH units, Sigma human factor Xa, Chromogenix bovine trypsin, 6000 BAEE units / mg protein, Sigma S-2238, Chromogenix, 25 mg S-2251, Chromogenix, 25 mg S-2222, Chromogenix, 25 mg HEPES, Sigma 98% bovine serum albumin, Sigma

3. Job description

Thrombin preparation: Dilute the contents of the bottle with distilled water to obtain a stock solution of 20 NIH units / mL. The contents were pipetted over 0.5 mL and stored at

-70 ° C. Immediately before use, prepare a working solution of thrombin with activity of 2 NIH units / mL with HBSA buffer. The final activity of thrombin in the microtiter plate is 0.5 NIH units / mL.

Preparation of factor Xa: Dilute the contents of the bottle with distilled water to give a stock solution of 10 nkat / mL. The contents were pipetted over 0.5 mL and stored at -20 ° C. Immediately before use, a working solution of factor Xa with activity of 2 nkat / mL was prepared with HBSA buffer. The final activity of factor Xa in the microtiter plate is 0.5 nkat / mL.

Trypsin preparation: Dilute the contents of the bottle with distilled water to give a stock solution of 300 E / mL. The contents were pipetted over 0.2 mL and stored at -70 ° C. Immediately before use, a trypsin working solution of 4 mE / mL with HBSA buffer is prepared. The final trypsin activity in the microtiter plate is 1 mE / mL.

Preparation of the chromogenic substrate for thrombin: Prepare a 1 mM substrate solution, pipette it with 0.5 mL and store at -20 ° C. Prepare substrate solutions with concentrations of 80 and 160 μΜ before use with distilled water. The final substrate concentrations in the microtiter plate are 20 and 40 μ 40.

Preparation of the chromogenic substrate for factor Xa: Prepare a 2 mM substrate solution, pipette it with 0.3 mL and store at -20 ° C. Prior to use with distilled water, substrate solutions with concentrations of 400 and 800 μΜ are prepared. The final substrate concentrations in the microtiter plate are 100 and 200 μ 200.

Preparation of the chromogenic substrate for trypsin: Prepare a 2 mM substrate solution, scatter at 0.3 mL and store at -20 ° C. Prior to use with distilled water, prepare substrate solutions with concentrations of 200 and 400 μΜ. The final substrate concentrations in the microtiter plate are 50 and 100 μ 100.

Preparation of HBSA Buffer (pH: 7.5): Dissolve 10 mM Hepes buffer, 150 mM NaCl and 0.1% (w / v) and bovine serum albumin in bidestilled water. The pH was adjusted with a 0.1 M NaOH solution. Preparation of inhibitor solutions: Inhibitors are dissolved in DMSO to give a 10 mM stock solution. Working solutions of concentrations from 10 to 100 μΜ are prepared with distilled water. The concentration of DMSO in the microtiter plate should not exceed 3%.

Performance of measurements: Measurements are performed with two concentrations of inhibitor and two concentrations of substrate. We measure reaction rates without inhibitor and in the presence of inhibitor. Pipette 50 μΜ HBSA buffer, 50 μΜ inhibitor solutions of various concentrations and 50 μΜ serine protease solution into the wells of the microtiter plate. Incubate for 15 minutes at 25 ° C and add 50 μΜ of chromogenic substrate after incubation. Insert the microtiter plate into a spectrophotometer (Tecan, Sunrise) and measure the absorbance increase at a wavelength of 405 nm and a temperature of 25 ° C every 10 seconds.

4. Calculation of inhibitory constants

The inhibition constant is calculated by determining the change in absorbance from the initial linear part of the curve by the Cheng and Prusoff method. Inhibition constant Which is determined by the equation:

K _{m +} S V - / / - inhibitor concentration v (τ Ί (In Ί iv Ί S - substrate concentration ⁰ K _m - - + 1 + S —- + 1 ^ -1 K _m - Michaelis constant k) K) J V _o - initial reaction rate without inhibitor V _o - initial reaction rate in the presence of an inhibitor For each combination of concentrations we calculate Ki, which is ultimately given as

the average of the values obtained. The result can also be given as an IC ₅₀ value representing an inhibitor concentration that reduces enzyme activity by 50%.

Determination of the binding affinity of antagonists to the fibrinogen receptor

1. Principle

The method is based on the measurement of chemoluminescence induced by an enzyme-catalyzed reaction. The test is performed on microtiter plates to which a vitronectin or fibrinogen receptor is bound. A solution of the natural agonist (biotinized fibrinogen) and the solution of the tested antagonist are then added to initiate a competitive process of binding of the agonist and antagonist to the receptors. In the next stage, antibodies against biotin with peroxidase bound biotin-bound fibrinogen are applied to the plates. The antibodies have a peroxidase-bound enzyme which, when added to a specific peroxidase substrate, undergoes chemical conversion. The reaction is accompanied by light emission (chemoluminescence), the strength of which is measured by a luminometer and proportional to the amount of bound agonist (biotinized fibrinogen). Ratio of binding between antagonist and agonist to receptors in proportion to the ratio of binding affinities of fibrinogen and antagonist.

2. Reagents

INORGANIC AND ORGANIC REAGENTS BIOCHEMICAL REAGENTS NaCI (Acros) Membrane platelet glycoproteins, llb lilac, Human CaCI ₂ (Acros) Platelets (Calbiochem) MgCI ₂ (Acros) Fibrinogen, Human plasma (Calbiochem) MnCI ₂ (Acros) Biotin-NHS-succinimidyl ester (Calbiochem) NaHCO ₃ (Merck) Antibiotin (Goat) Peroxidase Conjugated Tween® 20 (Acros) (Calbiochem) T ris (hydroxymethyl) - BSA, Albumin, Bovine (Sigma) aminomethane (Acros) BM Chemoluminescence ELISA Substrate (POD) DMF (Merck) DMSO (Merck) (Roche Diagnostics, Boehringer Mannheim)

3. Job description

Preparation of Fibrinogen Receptor Tiles: A receptor solution stored at -70 ° C is warmed to room temperature. Dilute 10 μΐ_ of the fibrinogen receptor solution with 10.2 mL of Tris buffer (20 mM Tris, 150 mM NaCI, 1 mM CaCI ₂ , 1 mM MgCI ₂ , 1 mM MnCI ₂ , pH = 4). Apply 100 μΙ_ of the prepared solution of the receptor to each well on a Lumitrac 600 microtiter plate and store overnight at 4 ° C.

Preparation of biotinized fibrinogen: 5 mL of 0.3 M NaCI solution warmed to 30 ° C was added 100 mg of fibrinogen preheated to 30 ° C and stirred until the fibrinogen dissolved. Mix 300 μΙ_ of fibrinogen solution in NaCI with a solution (2 mg / 2mL) of biotin in DMF, add 600 μΐ_ 1M NaHCO ₃ solution and 5.1 mL of bi-distilled water. The solution was incubated for 1.5 h at 30 ° C. Dialysis of biotinylated fibrinogen in Tris buffer (20 mM Tris, 150 mM NaCI, pH = 7.4) was performed and the remaining solution was centrifuged for 5 minutes at 5000 rpm. The supernatant was stabilized with Tween 20 (0.005%) and stored at 4 ° C.

Enzyme-Immunoassay: Pour out the integrin solution and block free binding sites with a solution of 1% BSA in Tris buffer (200 pL / well) for 1 hour at room temperature. Wash with Tris buffer with the addition of 0.01% Tweene 20. Pipette 100 μΙ_ of inhibitor solutions into each well of the microtiter plate (concentrations 200 μΜ, 60 μΜ, 20 μΜ, 6 μΜ, 2 μΜ, 0.6 μΜ, 0.2 μΜ, 0.06 μΜ and 0.02 μΜ was prepared by diluting the initial 10 mM solutions in DMF with Tris buffer) and 100 μΙ_ of the biotinylated fibrinogen solution (1:10 dilution in Tris buffer). Only a solution of biotinylated fibrinogen (50 μΙ_; 1:10 dilution in Tris buffer) in Tris buffer (50 μΐ_) was applied as a positive control and Tris buffer solution (100 μΙ_) was applied as a negative control. Incubate for 2 hours at room temperature and wash the microtiter plate twice with Tris buffer supplemented with 0.01% Tweene 20. Then add 100 μΙ_ of horseradish peroxidase-conjugated anti-biotin antibody solution (1: 1000 dilution in Tris buffer with 0.1% BSA) and incubate for 1 hour at room temperature. Wash 3 times with Tris buffer supplemented with 0.01% Tvveen 20. Add 50 μΙ_ of the chemiluminescent reagent solution and measure the released chemiluminescence with Tecan Genios and Magellan software (Version 3.0) within 15 minutes. The measurements are performed three times.

Binding affinity calculation: Data is processed using Excell and Microcal Origin. We adapt to the sigmoid receptor binding model and determine the value of IC ₅₀ . IC ₅₀ is the inhibitor concentration that causes a 50% inhibition of fibrinogen binding to the receptor.

Implementation examples

The invention is explained, but in no way limited by the following embodiments:

Example 1: Synthesis of methyl 3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 / 7-1,4benzoxazin-7-yl} amino] -2 -benzyl-3-oxopropanoate in the form of acetate (compound)

Reaction scheme for the preparation of compound 11.

NH ₂ AcOH

Synthesis of 7-nitro-2H-1,4-benzoxazin-3 (4 / - /) - one (2)

A suspension of potassium fluoride (8.715 g, 150.0 mmol) and ethyl bromoacetate (6.68 mL, 60.00 mmol) in DMF (40 mL) was stirred at room temperature for 15 minutes, then 2-amino-5-nitrophenol (1) (9.248 g) was added while stirring. , 60.00 mmol) and heated at 60 ° C until the end of the reaction is detected by thin layer chromatography. The reaction mixture was poured into a mixture of ice and water (200 mL), thus eliminating the precipitate which was filtered off and recrystallized from ethanol. 9.436 g (81%) of compound 2 are obtained.

layout: brown crystals T tal 218-226 ° C ¹ H NMR (300 MHz, DMSOd, γ. δ [ppm] = 4.70 (s, 2H, -OCHzCON-), 7.05 (d, 1H, J = 8.8 Hz, Ar-H ₅ ), 7.74 (d, 1H, J = 2.5 Hz, Ar-H ₈ ). 7.89 (dd, 1H, J, = 8.8 Hz, J ₂ = 2.5 Hz, Ar-H _e ). MS (Å): m / z (%) = 194 (M ⁺ , 100), 165 (38), 148 (19), 136 (25), 119 (15), 106 (24), 92 (24), 78 (25). 65 (50). IR (KBr): v [cm ' ¹ ] = 3080, 2924, 1698, 1598, 1509, 1391, 1341, 1222, 1133, 1043, 930, 864, 826, 742, 526. HRMS: Calculated for 0 ₈ Η ₆ Ν ₂ 0 ₄ : 194.032757 Obtained: 194.033250

Synthesis of 4 - [(7-nitro-3-oxo-2,3-dihydro-4H-1,4-benzoxazin-4-yl) methyl] benzonitrile (3)

Suspensions of 7-nitro-2H-1,4-benzoxazin-3 (4H) -one (2) (1.942 g, 10.0 mmol), benzyltriethylammonium chloride (2.278 g, 10.0 mmol) and potassium carbonate (3.455 g, 25 mmol) in Acetonitrile (60 mL) was added with stirring 4- (bromomethyl) benzonitrile (2.157 g, 10.0 mmol) and heated at 60 ° C until the end of the reaction was detected by thin layer chromatography. The suspension was filtered and the nut was evaporated under reduced pressure. The residue was dissolved in dichloromethane (100 mL) and washed with 10% citric acid (2 x 50 mL), saturated NaHCO ₃ solution (2 x 50 mL) and saturated NaCl solution (1 x 50 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 2.586 g (84%) of compound 3 are obtained.

layout: yellow crystals T tal 190-193 ° C ¹ H NMR (300 MHz, DMSO-d6): δ [ppm] = 4.99 (s, 2H, -OCH2CON-), 5.32 (s, 2H, -CH _? - (4-CN) -Ar-H). 7.15 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), 7.51 (d, 2H, J = 8.7 Hz, -CH _? - (4-CN) -Ar-H). 7.80-7.87 (m, 3H, Ar-H ₆ + -CH ₂ - (4-CN) -Ar-H), 7.87 (d, 1H, J = 2.6 Hz, Ar-H ₈ ). MS (Å): m / z (%) = 309 (M ⁺ , 44), 116 (100), 89 (23), 63 (7). IR (KBr): v [cm ' ¹ ] = 3454, 2233, 1686, 1602, 1517, 1392, 1344, 1242, 1043, 881, 808, 747, 522. HRMS: Calculated for C ₁₆ H ₁₁ N ₃ O ₄ : 309.074956 Obtained: 309.075820

Synthesis of 4 - [(7-amino-3-oxo-2,3-dihydro-4,7-1,4-benzoxazin-4-yl) methyl] benzonitrile (4)

The compound was prepared from 4 - [(7-nitro-3-oxo-2,3-dihydro-4 / 7-1,4-benzoxazin-4yl) methyl] benzonitrile (3) (5.10 g, 16.49 mmol) by reduction under conditions of catalytic hydrogenation at room temperature in DMF (80 mL) as solvent and palladium on carbon (10 wt%) as catalyst. 3.370 g (73%) of compound 4 are obtained.

layout: dark green-brown crystals T tal Mp 179-183 ° C ¹ H NMR (300 MHz, DMSO-cfe): δ [ppm] = 4.67 (s, 2H, -OCH2CON-), 5:01 (rs, 2H, Ar-NH _2), 5.14 (s, 2H, -CH2- (4-CN) -Ar), 6.13 (dd, 1H, 7 = 8.5 Hz, J ₂ = 2.5 Hz, Ar-H ₇ ) 6.25 (d, 1H, J = 2.5 Hz, Ar-H ₈ ), 6.62 (d, 1H, J = 8.5 Hz, Ar-H ₅₎ ), 7.44 (d, 2H, J = 8.1 Hz, -CH ₂ - (4-CN) -Ar), 7.80 (d, 2H, J = 8.1 Hz, -CH ₂ - (4-CN) -Ar). MS (Å): m / z (%) = 279 (M ⁺ , 95), 163 (100), 135 (73), 116 (32), 105 (27), 89 (18), 80 (16), 66 (9). IR (KBr): v [cm ' ¹ ] = 3433, 3342, 2228, 1676, 1516, 1402, 1325, 1221, 1148, 1041, 857, 804, 614, 537. HRMS: Calculated for Ci ₆ H ₁₃ N ₃ O ₂ : 279.100777 Obtained: 279.101550

Synthesis of 1-benzyl 3-methyl 2-benzylmalonate (6)

Benzyl methyl malonate (5) (12.48 g, 60.0 mmol) was dissolved in anhydrous dioxane (90 mL), cooled in an ice bath and NaH (1.44 g, 6.00 mmol) was added while stirring. Remove the bath and allow the reaction to warm to room temperature, then add benzyl bromide (10.572 g, 7.36 mL) and heat for another 4 hours at 50 ° C. The reaction mixture was cooled, the dioxane was evaporated and the ether residue (150 mL) was added to the oily residue. Wash with water (2 x 50 mL) and saturated NaCl solution (1 x 50 mL), dry with Na ₂ SO ₄ , filter and evaporate under reduced pressure. After purification by column chromatography (MF:

light petroleum: hexane = 1: 1) yield 3.979 g (22%) of compound 6.

layout: colorless oil T tal / ¹ H NMR (300 MHz, DMSO-cfe): δ [ppm] = 3.26 (d, 2H, J = 7.9 Hz, -CHCH _? Ph). 3.70 (s, 3H, -COOCH ₃ ), 3.75 (t, 1H, J = 7.9 Hz, -CHCH ₂ Ph), 5.15 (s, 2H, COOCH ₂ Ph), 7.18-7.38 (m, 10H, -CHCH ₂ Ph + -COOCH ₂ Ph). IR (NaCI): v [cm ' ¹ ] = 2953, 1735, 1497, 1455, 1227, 1148, 750. MS (Å): m / z (%) = 298 (M ⁺ , 1), 222 (6), 180 (57), 163 (37), 131 (50), 91 (100). HRMS: Calculated for C ₁₈ H ₁₈ O ₄ : 298.120509 Obtained: 298.121420

Synthesis of 2-benzyl 3-methoxy-3-oxopropanoic acid (7)

The compound was prepared from 1-benzyl 3-methyl 2-benzylmalonate (6) (3.969 g, 13.31 mmol) by reduction under catalytic hydrogenation at room temperature and THF (50 mL) as solvent. 2.770 g (100%) of compound 7 are obtained.

layout: yellow oil T tal / ¹ H NMR (300 MHz, DMSOd ₆ ): δ [ppm] = 3.05-3.11 (m, 2H, -CHCH ₂ Ph), 3.60 (s, 3H, -COOCH ₃ ), 3.72 (t, 1H, J = 7.9 Hz, -CHCH ₂ Ph), 7.18-7.30 (m, 5H, -CHCH _? Ph), 12.91 (rs, 1H, -COOH). IR (NaCI): in [cm ¹ ] = 2955, 1741, 1496, 1438, 1163, 701. MS (Å): m / z (%) = 208 (M ⁺ , 23), 162 (68), 159 (19), 148 (28), 131 (79), 104 (67), 91 (100). HRMS: Calculated for CnH ₁₂ O ₄ : 208.073559 Obtained: 208.074020

Synthesis of methyl 2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7yl] amino} -3-oxopropanoate (8)

Solutions of 4 - [(7-amino-3-oxo-2,3-dihydro-4,7-1,4-benzoxazin-4-yl) methyl] benzonitrile (4) (1.397 g, 5.00 mmol) and 2-benzyl HOBT (0.811 g, 6.00 mmol) was added while stirring 3-methoxy-3-oxopropanoic acid (7) (1.041 g, 5.00 mmol) in DMF (40 ml_). Nmethylmorpholine was adjusted to pH above 7 and EDC (1.296 g, 6.76 mmol) was added and stirred at room temperature for at least 4 hours. Evaporate the solvent under reduced pressure and dissolve the residue in dichloromethane (100 ml_) and wash with 10% citric acid (2 x 50 mL), saturated NaHCO ₃ solution (2 x 50 mL) and saturated NaCl solution (1 x 50 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 0.764 g (33%) of compound 8 are obtained.

appearance: yellow crystals T tal 60 - 63 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 3.10-3.13 (m, 2H, -CHCH ₂ Ph), 3.62 (s, 3H, -COOCH ₃ ), 3.78 (t, 1H, J = 7.5 Hz, -CHCH ₂ Ph), 4.79 (s , 2H, -OCH _? CON-), 5.20 (s, 2H, -CH _? - (4-CN) -Ar). 6.88 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), 6.99 (dd, 1H, J, = 8.7 Hz, J ₂ = 2.3 Hz, ArH ₆ ), 7.17-7.28 (m, 5H, -CHCH _? Ph), 7.32 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.45 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-CN) -Ar-H), 7.80 (d. 2H, J = 8.3 Hz, -CH ₂ - (4-CN) Ar-H), 10.14 (s, 1H, -CONH-). MS (FAB): m / z (%) = 470 (MH ⁺ , 100), 440 (11), 307 (15), 280 (15), 163 (15), 154 (84), 136 (72), 107 (29). 91 (73), 71 (37), 91 (73). IR (KBr): v [cm ' ¹ ] = 3318, 2229, 1662, 1606, 1510, 1406, 1222, 1051,794, 700, 545. Elemental analysis: Calculated for C ₂₇ H ₂₃ N ₃ O ₅ x 1 / 2H ₂ O: 67.77% C 5.06% H 8.78% N Obtained: 67.95% C 5.11% H 8.55% N

Synthesis of methyl 3 - [(4- {4- [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl) amino] -2-benzyl-3- oxopropanoate (9)

Hydroxylammonium chloride (0.238 g, 3.422 mmol) was dissolved in EtOH (10 mL), Et ₃ N (0.48 mL, 3.422 mmol) was added and methyl 2-benzyl-3 - {[4- (4-cyanobenzyl) -3 was added with stirring. -oxo3,4-dihydro-2 / 7-1,4-benzoxazin-7-yl] amino} -3-oxopropanoate (8) (0.403 g, 0.858 mmol) dissolved in 10 mL of ethanol and heated at 50 ° C until s thin layer chromatography does not detect the end of the reaction. The solvent was evaporated, the oily residue was dissolved in dichloromethane (30 mL) and washed with 10% citric acid (2 x 20 mL). It is dried with Na ₂ SO ₄ , filtered and the solvent is evaporated under reduced pressure. 0.336 g (78%) of the compound are obtained

9.

layout: yellow crystals T tal 89-92 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 3.09-3.12 (m, 2H, -CHCH _? Ph). 3.62 (s, 3H, -COOCH ₃ ), 3.78 (t, 1H, J = 7.7 Hz, -CHCH ₂ Ph), 4.78 (s, 2H, -OCH _? CON-). 5.12 (s, 2H, -CH, - (4CN) -Ar), 5.75 (s, 2H, -C (= NOH) NH _? ). 6.93 (d, 1H, J = 8.9 Hz, Ar-H ₅ ), 7.00 (dd, 1H, Ji = 8.9 Hz, J ₂ = 2.3 Hz, Ar-H ₆ ), 7.16-7.26 (m, 7H, CHCH ₂ Ph + -CH ₂ - (4-amidoxime) -Ar), 7.30 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.61 (d, 2H, J = 8.3 Hz, -CH ₂ - (4- amidoxime) -Ar), 9.59 (s, 1H, = N-OH), 10.13 (s, 1H, -CONH-). MS (FAB): m / z (%) = 503 (MH ⁺ , 100), 487 (10), 339 (6), 313 (6), 281 (7), 207 (7), 159 (13), 149 (32). 136 (36), 91 (62), 69 (75), 55 (83). IR (KBr): v [cm ' ¹ ] = 3745, 3676, 2366, 2327, 1745, 1651, 1509, 1459, 1403, 1261. Elemental analysis: Calculated for C ₂ 7H ₂₆ N ₄ O ₆ x 1 / 2H ₂ O: 63.40% C 5.32% H 10.95% N Obtained: 63.75% C 5.52% H 10.62% N

Synthesis of methyl 2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazol-3yl) benzyl] -3,4 -Dihydro-2H-1,4-benzoxazin-7-yl} amino) propanoate (10)

Methyl 3 - [(4- {4- [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl) amino] -2-benzyl-3-oxopropanoate (9) (0.295 g, 0.587 mmol) was dissolved in pyridine (10 mL), cooled to -10 ° C and ethyl chloroformate (0.067 g, 0.616 mmol) dissolved in 3 mL of pyridine was gradually added while stirring and allowed to stir for 30 minutes at - 10 ° C. Then remove the bath and allow the temperature to rise to room temperature and bring it to a boil until the end of the reaction is detected by thin layer chromatography. The reaction mixture was cooled and the solvent was evaporated under reduced pressure. 0.208 g (67%) of compound 10 is obtained.

appearance: white crystals T tal 170-173 ° C ¹ H NMR (300 MHz, DMSO d ₆ y. δ [ppm] = 3.10-3.13 (m, 2H, -CHCH _? Ph). 3.62 (s, 3H, -COOCH ₃ ), 3.78 (t, 1H, J = 7.7 Hz, -CHCH ₂ Ph), 4.80 (s, 2H, -OCH _? CON-). 5.20 (s, 2H, -CH _? - (4-CN) -Ar). 6.90 (d, 1 H, J = 8.8 Hz, Ar-H ₅ ), 6.99 (dd, 1 H, J, = 8.8 Hz, J ₂ = 2.0 Hz, ArH ₆ ), 7.14-7.28 (m, 5H, - CHCH? Ph), 7.31 (d, 1H, J = 2.0 Hz, Ar-H ₈ ), 7.45 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 7.76 ( d, 2H, J = 8.3 Hz, CH ₂ - (4-oxadiazolinone) -Ar-H), 10.14 (s, 1H, -CONH-), 12.92 (rs, 1H, oxadiazolinone NH). MS (FAB): m / z (%) = 529 (MH ⁺ , 52), 401 (11), 281 (10), 207 (14), 147 (35), 136 (28), 109 (25), 91 (48). 81 (53), 73 (89), 55 (100). IR (KBr): in [cm ' ¹ ] = 2230, 1719, 1674, 1602, 1445, 1297, 1202, 1088, 988, 825, 753, 569. Elemental Calculated for C ₂₈ H ₂₄ N ₄ O7 63.63% C 4.58% H 10.60% N analysis: Obtained: 63.51% C 4.59% H 10.74% N

Synthesis of methyl 3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl} amino] -2-benzyl Acetate -3-oxopropanoate (11)

The compound is prepared from methyl 2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl) benzyl] -3 , 4-dihydro-2 / 7-1,4-benzoxazin-7-yl} amino) propanoate (10) (0.261 g, 0.049 mmol) by reduction under catalytic hydrogenation at room temperature in acetic acid as solvent and palladium on carbon (10 wt%) as catalyst. 0.229 g (85%) of compound 11 are obtained.

appearance: brown crystals T tal 145-148 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ); δ [ppm] = 1.84 (s, 3H, AcOH), 3.09-3.12 (m, 2H, -CHCH _? Ph). 3.62 (s, 3H, -COOCH ₃ ), 3.79 (t, 1H, J = 7.5 Hz, -CHCH ₂ Ph), 4.79 (s, 2H, -OCH _? CON-), 5.22 (s, 2H, CH ₂ - (4-amidine) -Ar), 6.92 (d, 1H, J = 8.9 Hz, Ar-H ₅ ), 7.01 (dd, 1H, J = 8.9 Hz, J ₂ = 2.2 Hz, Ar-He), 7.16- 7.25 (m, 5H, -CHCH _? Ph). 7.28 (d, 1H, J = 2.2 Hz, ArH ₈ ), 7.45 (d, 2H, J = 8.1 Hz, -CH ₂ - (4-amidin) -Ar), 7.72 (d, 2H, J = 8.1 Hz. CH ₂ - (4-amidine) -Ar), 10.19 (s, 1H, -CONH-). MS (FAB): m / z (%) = 487 (MH ⁺ , 100), 323 (14), 296 (10), 154 (35), 134 (36), 107 (16), 91 (25), 71 (42). 55 (48). IR (NaCI): v [cm ' ¹ ] = 3057, 1666, 1612, 1552, 1512, 1407, 1236, 1051,814, 701.

Elemental analysis: Calculated for C27H26N4O5 x 3AcOH: 59.45% C 5.75% H 8.40% N Obtained: 59.31% C 6.07% H 8.54% N

Example 2

Synthesis of ethyl 2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl) amino] -2- acetyl acetate benzyl-3-oxopropanoyl} amino) acetate (17)

Reaction scheme for the preparation of compound 17.

Ο, Ν

BrCH ₂ COOEt KF, DMF, 60 * C

Oh

BTEAC, K ₂ CO ₃ , CHjCN, 60 ^e C

Synthesis of 2-benzyl- N - [4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2 / 7-1,4-benzoxazin-7-yl] -3-oxoβ-alanine (12)

Methyl 2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazol-3-yl) benzyl] -3, Dihydro-2H-1,4-benzoxazin-7-yl} amino) propanoate (8) (3.852 g, 8.13 mmol) was dissolved in ethanol (50 mL), 2M NaOH (8.2 mL, 16.4 mmol) was added and stirred at room temperature. until the end of the reaction is detected by thin layer chromatography. The solvent was evaporated and the oily residue was dissolved in distilled water (100 mL). Acidify the reaction mixture with 1M HCl to pH = 3 and extract with ethyl acetate or dichloromethane (4 x 30 mL). The combined organic fractions were dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 3.28 g (89%) of compound 12 are obtained.

appearance: yellow crystals T tal 93-96 ° C ¹ H NMR (300 MHz, DMSO-be): δ [ppm] = 3.07-3.09 (m, 2H, -CHCH ₂ Ph), 3.67 (dd, 1H, = 8.3 Hz, J ₂ = 6.8 Hz, - CHCH ₂ Ph), 4.79 (s, 2H, -OCH ₂ CON-), 5.20 (s, 2H, -CH ₂ - (4-CN) -Ar), 6.88 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), 7.01 (dd, 1H, J = 8.7 Hz). , J ₂ = 2.3 Hz, Ar-H _e ), 7.167.27 (m, 5H, -CHCH ₂ Ph), 7.33 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.45 (d, 2H. J = 8.3 Hz, -CH ₂ - (4-CN) -Ar), 7.80 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-CN) -Ar), 10.08 (s, 1H, CONH- ). MS (FAB): m / z (%) = 456 (MH ⁺ , 33), 281 (10), 221 (11), 207 (11), 154 (32), 147 (34), 136 (50), 91 (33). 73 (100), 55 (37). IR (KBr): v [cm ¹ ] = 3306, 3062, 2229, 1733, 1685, 1608, 1512, 1403, 1222, 1051, 307, 700, 546. Elemental analysis: Calculated for C ₂₆ H ₂₁ N ₃ O ₅ x 1 / 2H ₂ O: 67.23% C 4.77% H 9.05% N Obtained: 67.38% C 4.77% H 9.19% N

Synthesis of ethyl 2 - [(2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2A7-1,4-benzoxazin-7yl] amino} -3-oxopropanoyl) amino ] acetate (14)

To a solution of 2-benzyl-3-oxo-N- {3-oxo-4- [4- (5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl) benzyl] 3,4 -Dihydro-2A7-1,4-benzoxazin-7-yl} -p-alanine (13) (1.139 g, 2.50 mmol) and glycine ethyl ester hydrochloride (15) (0.349 g, 2.50 mmol) in DMF (40 mL) add while stirring

HOBT (0.405 g, 3.00 mmol). Z / V-methylmorpholine was adjusted to pH above 7 and EDC (0.648 g, 3.38 mmol) was added and stirred at room temperature for at least 4 hours. Evaporate the solvent under reduced pressure and dissolve the residue in dichloromethane (40 mL) and wash with 10% citric acid (2 x 30 mL), saturated NaHCO ₃ solution (2 x 30 mL) and saturated NaCl solution (1 x 30 mL) . The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 0.838 g (62%) of compound 14 are obtained.

appearance: yellow oil T tal / ¹ H NMR (300 MHz, DMSO-cfe): δ [ppm] = 1.14 (t, 3H, J = 7.2 Hz, Glv-OCH _? CH ₃ ). 3.06-3.17 (m, 2H, -CHCH _? Ph). 3.62 (dd, 1H, Jt = 8.1 Hz, J ₂ = 6.6 Hz, -CHCH ₂ Ph), 4.06 (q, 2H, J = 7.2 Hz, GIv-OCH ₂ CH ₃ ). 4.79 (s, 2H, -OCH? CON-). 5.21 (s, 2H, -CH _? - (4CN) -Ar), 6.89 (d, 1H, J = 9.0 Hz, Ar-H ₅ ), 7.02 (dd, 1H, = 9.0 Hz, J ₂ = 2.3 Hz. Ar-H ₆ ), 7.14-7.28 (m, 5H, -CHCH _? Ph). 7.35 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.45 (d, 2H, J = 8.5 Hz, -CH ₂ - (4-CN) -Ar-H), 7.80 (d, 2H, J = 8.5 Hz, -CH ₂ - (4-CN) -Ar-H), 8.26 (t, 1H, J = 5.8 Hz, Gly-NHCO-), 9.88 (s, 1H, -CONH-). MS (FAB): m / z (%) = 541 (MH ⁺ , 100), 438 (9), 410 (9), 279 (16), 174 (13), 159 (35), 136 (35), 104 (43). 91 (79), 71 (47), 55 (56). IR (NaCI): v [cm ¹ ] = 3312, 2932, 2229, 1747, 1674, 1513, 1403, 1200, 1095, 895, 810, 702. Elemental analysis: Calculated for Ο ₃₀ Η ₂₈ Ν ₄ Ο ₆ x H ₂ O: 64.51% C 5.41% H 10.03% N Obtained: 64.27% C 5.61% H 9.61% N

Synthesis of ethyl 2 - ({3 - [(4- {4- [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl) amino] -2- benzyl-3-oxopropanoyl} amino) acetate (15)

Hydroxylamine (0.287 g, 8.69 mmol) was dissolved in EtOH (10 mL) and ethyl 2 - [(2-benzyl3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2H-1) was added. 4-Benzoxazin-7-yl] amino} -3oxopropanoyl) amino] acetate (14) (1.179 g, 2.18 mmol) was dissolved in 10 mL EtOH and heated at 50 ° C until the end of the reaction was detected by thin layer chromatography. Evaporate the solvent under reduced pressure. 1.029 g (82%) of compound 15 are obtained.

appearance: yellow crystals T tal 82-86 ° C ¹ H NMR (300 MHz, DMSO-d6): δ [ppm] = 1.14 (t, 3H, J = 7.1 Hz, Glv-OCH, CH ₃ ). 3.03-3.19 (m, 2H, -CHCH _? Ph). 3.63 (dd, 1H, Ji = 8.3 Hz, J ₂ = 6.8 Hz, -CHCH ₂ Ph), 3.84 (d, 2H, J = 6.0 Hz, Gly-aH), 4.06 (q, 2H, J = 7.1 Hz. Glv-OCH _? CHA 4.79 (s, 2H, -OCH _? CON), 5.21 (s, 2H, -CH ₂ - (4-CN) -Ar), 5.75 (s, 2H, -C (= NOH) NH _? ) 6.89 (d, 1H, J = 9.0 Hz, Ar-Hg), 7.02 (dd, 1H, J, = 9.0 Hz, J ₂ = 2.3 Hz, Ar-H ₆ ), 7.14-7.28 (m, 5H. -CHCH? Ph + -CH ₂ - (4-amidoxime) -Ar-H), 7.35 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.61 (d, 2H, J = 8.3 Hz, -CH _2- (4-amidoxime) -Ar-H), 8.29 (t, 2H, J = 5.8 Hz, Gly-NHCO-), 9.59 (rs, 1H, = N-OH), 9.92 (s, 1H, -CONH -). MS (FAB); m / z (%) = 575 (MH ⁺ , 40), 339 (5), 281 (9), 207 (12), 154 (56), 136 (59), 123 (21), 107 (28). 91 (47), 73 (100), 55 (93). IR (KBr); in [cm ¹ ] = 3360, 1749, 1653, 1540, 1508, 1404, 1198, 1050, 928, 700. Elemental analysis; Calculated for C ₃₀ H ₃₁ N ₅ O ₇ x 2 / 3H ₂ O 61.53% C 5.45% H 11.96% N Obtained: 61.27% C 5.84% H 11.78% N

Synthesis of ethyl 2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazol-3yl) benzyl] -3,4-dihydro-2H-1,4-benzoxazin-7-yl} amino) propanoyl] amino} acetate (16)

Ethyl 2 - ({3 - [(4- {4 [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2 / 7-1,4-benzoxazin-7-yl) amino] -2- benzyl-3-oxopropanoyl} amino) acetate (15) (0.300 g, 0.52 mmol) was dissolved in anhydrous dichloromethane (10 ml_) and cooled to -10 ° C. Triethylamine (0.058 g, 0.58 mmol) and ethylchloroformate (0.060 g, 0.55 mmol) dissolved in dichloromethane (5 mL) were gradually added while stirring and allowed to stir at -10 ° C for 30 minutes. We then wean the bath and allow the temperature to rise to room temperature. The reaction mixture was washed with 10% citric acid (2 x 20 mL), dried with Na ₂ SO ₄ and the solvent was evaporated under reduced pressure. The intermediate was purified by column chromatography (MF; dichloromethane / methanol = 50/1) and then cyclized by heating on boiling in toluene until the end of the reaction was detected by thin layer chromatography. After completion of the reaction, the solvent was evaporated, the product was purified by column chromatography (MF: dichloromethane / methanol = 9/1) to give 0.175 g (56%) of compound 16.

layout: yellow crystals T tal 109-112 ° C ¹ H NMR (300 MHz, DMSO d ₆ y. δ [ppm] = 1.14 (t, 3H, J = 7.2 Hz, Glv-OCH ₂ CH ₃ ). 3.04-3.25 (m, 2H, -CHCH _? Ph). 3.61 (t, 1H, J = 7.5 Hz, -CHCH ₂ Ph), 3.84 (d, 2H, J = 6.0 Hz, Gly-aH), 4.05 (q, 2H, J = 7.0 Hz, Glv-OCH ₂ CHa) . 4.80 (s, 2H, -OCH ₂ CON-), 5.20 (s, 2H, CH ₂ - (4-oxadiazolinone) -Ar), 6.90 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), 7.02 ( dd, 1H, 7 = 8.7 Hz, J ₂ = 2.3 Hz, Ar-H ₆ ), 7.13-7.27 (m, 5H, -CHCH.Ph). 7.35 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), 7.45 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 7.76 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 8.25 (t, 1H, J = 5.8 Hz, Gly-NHCO-), 9.87 (rs, 1H, -NHCO-), 12.91 (s , 1H, -oxadiazolinoneNH-). MS (FAB): m / z (%) = 575 (MH ⁺ , 21), 569 (3), 460 (3), 391 (9), 307 (23), 289 (11), 154 (100), 136 (77). 107 (28), 71 (36). IR (KBr): v [cm ' ¹ ] = 3320, 1782, 1684, 1616, 1512, 1400, 1218, 1051, 944, 893, 807, 700. Elemental analysis: Calculated for C3iH ₂ gN50g: 62.10% C 4.88% H 11.68% N Obtained: 62.51% C 5.40% H 11.34% N

Synthesis of ethyl 2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 H -1,4-benzoxazin-7-yl) amino] Acetate acetate (2-benzyl-3-oxopropanoyl} amino) acetate (17)

The compound is prepared from ethyl 2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4oxadiazol-3-yl) benzyl] -3,4-dihydro-2H-1,4-benzoxazin-7-yl} amino) propanoyl] amino} acetate (16) (0.150 g, 0.250 mmol) by reduction under catalytic hydrogenation at room temperature in acetic acid as solvent and palladium on carbon (10 wt.%) as catalyst. 0.134 g (87%) of compound 17 are obtained.

layout: light brown crystals T tal 130-135 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ); δ [ppm] = 1.14 (t, 3H, J = 7.2 Hz, Glv-OCH _? CH ₃ ). 1.77 (s, 3H, AcOH), 3.01-3.44 (m, 2H, -CHCH ₂ Ph), 3.68 (t, 1H, J = 7.5 Hz, CHCH ₂ Ph), 3.82 (d, 2H, J = 5.3 Hz. Gly-aH), 4.05 (q, 2H, J = 7.0 Hz, Glv-OCH ₂ CH ₃ ). 4.79 (s, 2H, -OCH2CON-), 5.22 (s, 2H, _CH? - (4-amidine) -N). 6.92 (d, 1H, J = 8.9 Hz, Ar-H ₅ ), 7.05 (dd, 1H, J, = 8.9 Hz, J ₂ = 2.2 Hz, Ar-H ₆ ), 7.13-7.27 (m, 5H, CHCH ? Ph), 7.34 (d, 1H, J = 2.2 Hz, Ar-H ₈ ), 7.44 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-amidine) -Ar-H), 7.72 (d , 2H, J = 8.3 Hz, -CH ₂ - (4-

amidine) -Ar-H), 8.60 (rs, 1H, Gly-NHCO-), 10.22 (rs, 1H, NHCO-). MS (FAB): m / z (%) = 558 (MH ⁺ , 100), 323 (29), 296 (9), 154 (24), 134 (44), 91 (27), 55 (44). IR (KBr): v [cm ' ¹ ] = 3302, 2982, 1684, 1616, 1558, 1512, 1403, 1196, 1050, 700, 538. Elemental analysis: Calculated for θδζΗββΝδΟβ x H ₂ O: 60.46% C 5.87% H 11.02% N Obtained: 60.58% C 5.67% H 11.04% N

Example 3

Synthesis of diethyl (2S) -2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl) amino ] -2-Benzyl-3-oxopropanoyl} amino) pentanedioate in acetate form (Compound 22)

Reaction scheme for the preparation of compounds 22 and 24

Synthesis of diethyl (2S) -2 - [(2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl] amino} -3 -oxopropanoyl) amino] pentanedioate (19)

To a solution of 2-benzyl-3-oxo-N- {3-oxo-4- [4- (5-oxo-2,5-dihydro-1,2,4-oxadiazol-3-yl) benzyl] 3,4 -Dihydro-2H-1,4-benzoxazin-7-yl} -p-alanine (12) (0.910 g, 2.00 mmol) and tosylate (18) L-glutamic acid ethyl ester (18) (0.479 g, 2.00 mmol) in DMF (40 mL) while stirring, HOBT (0.324 g, 2.40 mmol) was added. Z / V-methylmorpholine was adjusted to pH above 7 and EDC (0.518 g, 2.70 mmol) was added and stirred at room temperature for at least 4 hours. Evaporate the solvent under reduced pressure and dissolve the residue in dichloromethane (40 mL) and wash with 10% citric acid (2 x 30 mL), saturated NaHCO ₃ solution (2 x 30 mL) and saturated NaCl solution (1 x 30 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. The product was purified by column chromatography (MF: dichloromethane / methanol = 50/1) to give 1.127 g (88%) of compound 19.

appearance: yellow crystals T tal 56-59 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.12 (t, 3H, J = 7.2 Hz, Glu-OCH ₂ CH ₃ ). 1.18 (t, 3H, J = 7.2 Hz, GluOCH? CH ₃ ). 1.75-1.82 (m, 1H, Glu-βΗ), 1.93-2.06 (m, 1H, Glu-βΗ), 2.182.31 (m, 2H, Glu-γΗ), 3.09-3.13 (m, 2H, -CHCH _? Ph). 3.62 (t, 1H, J = 6.8 Hz, -CHCH ₂ Ph), 3.95-4.09 (m, 4H, Glu-OCH ₂ CH ₃ ). 4.23-4.27 (m, 1H, GluaH), 4.79 (s, 2H, -OCH _? CON-j, 5.21 (s, 2H, -CH _? - (4-CN) -Ar). 6.88 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), * 6.89 (d, 1H, J = 8.7 Hz, Ar-H ₅ ), * 6.99-7.05 (m, 1H, ArH ₆ ), 7.14-7.27 (m, 5H, - CHCH.Ph) 7.34 (d, 1H, J = 2.2 Hz, Ar-H ₈ ), * 7.35 (d, 1 H, J = 2.2 Hz, Ar-H ₈ ), * 7.45 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-CN) -Ar-H), 7.80 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-CN) -Ar-H), 8.25 (d, 1H. J = 6.8 Hz, Glu-NHCO), * 8.27 (d, 1H, J = 6.8 Hz, Glu-NHCO -), * 9.75 (s, 1H, Ar-NHCO -), * 9.84 (s, 1H, Ar- NHCO -). * MS (FAB): m / z (%) = 641 (MH ⁺ , 64), 391 (10), 307 (24), 289 (12), 225 (16), 154 (100), 136 (74), 107 (29). 91 (34). IR (KBr): in [cm * ¹ ] = 3320, 2981, 2229, 1737, 1685, 1512, 1401, 1195, 1048, 893, 807, 700, 546. Elemental analysis: Calculated for C ₃₅ H ₃₆ N ₄ O ₈ x 1/5 H ₂ O: 65.25% C 5.69% H 8.70% N Obtained: 64.89% C 5.67% H 8.64% N

Synthesis of diethyl (2S) -2 - ({3 - [(4- {4- [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2 / 71,4-benzoxazin-7-yl) amino) -2-benzyl-3-oxopropanoyl} amino) pentanedioate (20)

Hydroxylammonium chloride (0.477 g, 6.87 mmol) was dissolved in EtOH (10 mL), Et ₃ N (0.96 mL, 6.87 mmol) was added with stirring, diethyl (2S) -2 - [(2-benzyl-3 - {[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2F / -1,4-benzoxazin-7-yl] amino} -3-oxopropanoyl) amino] pentanedioate (19) (1.100 g, 1.72 mmol) dissolved in 10 mL of ethanol and heated at 50 ° C until the end of the reaction is detected by thin layer chromatography. The solvent was evaporated, the oily residue was dissolved in dichloromethane (30 mL) and washed with 10% citric acid (2 x 20 mL). It is dried with Na ₂ SO ₄ , filtered and the solvent is evaporated under reduced pressure. 0.925 g (80%) of compound 20 are obtained.

layout: yellow amorphous substance T tal 85-99 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.12 (t, 3H, J = 7.2 Hz, Glu-OCH, CH ₃ ). 1.17 (t, 3H, J = 7.2 Hz, GluOCH ₂ CH ₃ ). 1.75-1.82 (m, 1H, Hz, Glu-βΗ), 1.93-2.06 (m, 1H, Hz, GluβΗ), 2.21-2.34 (m, 2H, Hz, Glu-γΗ), 3.03-3.10 (m, 2H , -CHCH? Ph). 3.77 (t, 1H, J = 7.5 (7.53) Hz, -CHCH ₂ Ph), 3.92-4.07 (m, 4H, Glu-OCH, CH _a ). 4.21-4.23 (m, 1H, Glu-aH), 4.77 (s, 2H, -OCH _? CON-). 5.13 (s, 2H, -CH _? (4-amidoxime) -Ar), 5.76 (s, 2H, Ar-C (NH ₂ ) = NOH), 6.93 (d, 1H, J _y = 9.0, Ar-H ₅ ), * 6.95 (d, 1H, = 9.0, Ar-H ₅ ), * 7.06-7.09 (m, 1H, Ar-H ₆ ), 7.13-7.22 (m, 5H, -CHCH? Ph), 7.26 (d , 2H, J = 8.3 Hz, -CH ₂ - (4-amidoxime) -Ar), 7.37 (d, 1H, = 2.3 Hz, Ar-H ₈ ), * 7.38 (d, 1H, J, = 2.3 Hz. Ar-H ₈ ), * 7.61 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-amidoxime) -Ar-H), 8.59 (d, 1H, J = 7.6 Hz, Glu-NHCO-). 9.60 (s, 1H, Ar-C (NH ₂ ) = NOH), 10.16 (s, 1H, Ar-NHCO -), * 10.27 (s, 1H, Ar-NHCO -). * MS (FAB): m / z (%) = 674 (MH ⁺ , 27), 307 (13), 289 (6), 225 (7), 154 (51), 137 (35), 102 (100), 91 (13). IR (KBr): v [cm ¹ ] = 3336, 2980, 1605, 2498, 1734, 1684, 1513, 1399, 1193, 1036, 808, 701.

Synthesis of diethyl (2S) -2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4oxadiazole-3- yl) benzyl] 3,4-dihydro-2 / 7-1,4-benzoxazin-7yl} amino) propanoyl] amino} pentanedioate (21)

Diethyl (2S) -2 - ({3 - [(4- {4- [amino (hydroxyimino) methyl] benzyl} -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl ) amino] -2-benzyl-3-oxopropanoyl} amino) pentanedioate (20) (0.900 g, 1.470 mmol) was dissolved in anhydrous dichloromethane (10 mL), cooled to -10 ° C. Triethylamine (0.20 mL, 0.56 mmol) and ethylchloroformate (0.152 g, 1.403 mmol) dissolved in dichloromethane (5 mL) were gradually added while stirring and allowed to stir at -10 ° C for 30 minutes. We then wean the bath and allow the temperature to rise to room temperature. The reaction mixture was washed with 10% citric acid (2 x 20 mL), dried with Na ₂ SO ₄ and the solvent was evaporated under reduced pressure. The product was cyclized by heating on boiling in toluene until the end of the reaction was detected by thin layer chromatography. After completion of the reaction, the solvent was evaporated. The product was purified by column chromatography (MF: dichloromethane / methanol = 20/1) to give 0.797 g (85%) of compound 21.

appearance: yellow crystals T tal 150-158 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.11 (t, 3H, J = 7.1 Hz, Glu-OCHzCHa), 1.16 (t, 3H, J = 7.1 Hz, GluOCH _? CH ₃ ). 1.72-1.88 (m, 1H, Glu-βΗ), 1.93-2.04 (m, 1H, Glu-βΗ), 2.182.31 (m, 2H, Hz, Glu-γΗ), 3.09-3.13 (m, 2H, - CHCH _? Ph). 3.62 (t, 1H, J = 7.5 Hz, -CHCH ₂ Ph), 3.95-4.08 (m, 4H, Glu-OCH ₂ CH ₃ ). 4.21-4.28 (m, 1H, Glu-aH), 4.80 (s, 2H, -OCH ₇ CON-). 5.20 (s, 2H, -CH _? - (4-oxadiazolinone) Ar), 6.90 (d, 1H, J, = 8.8 Hz, Ar-H ₅ ), * 6.91 (d, 1H, J, = 8.8 Hz, Ar -H ₅ ), * 6.99-7.05 (m, 1H, Ar-H ₆ ), 7.13-7.27 (m, 5H, -CHCH _? Ph), 7.33-7.37 (m, 1H, Ar-H ₈ ), 7.46 ( d, 2H, J = 8.3 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 7.76 (d, 2H, J = 8.3 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 8.26 (d, 1H, J = 6.8 Hz, Gly-NHCO -), * 8.28 (d, 1H, J = 6.8 Hz, Glu-NHCO -), * 9.75 (s, 1H, ArNHCO -), * 9.84 (s , 1H, Ar-NHCO-), * 12.92 (rs, 1H, oxadiazolinone NH). MS (FAB): m / z (%) = 700 (MH ⁺ , 8), 659 (2), 613 (1), 391 (13), 307 (28), 289 (13), 154 (100), 137 (73). 107 (24), 71 (38). IR (KBr): v [cm ' ¹ ] = 3443, 1786, 1735, 1652, 1540, 1513, 1403, 1204, 1022, 942, 805, 700.

Synthesis of diethyl (2S) -2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 / 7-1,4benzoxazin-7-yl) ) amino] -2-benzyl-3-oxopropanoyl} amino) pentanedioate in the form of acetate (22)

The compound is prepared from diethyl (2S) -2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazole- 3-yl) benzyl] 3,4-dihydro-2 / 7-1,4-benzoxazin-7yl} amino) propanoyl] amino} pentanedioate (21) (0.230 g, 0.33 mmol) by reduction under catalytic hydrogenation at room temperature in acetic acid as solvent and palladium on carbon (10 wt%) as catalyst. 0.222 g (94%) of compound 22 are obtained.

appearance: light brown crystals T tal 120-124 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.11 (t, 3H, J = 7.2 Hz, Glu-OCH _? CHA. 1.16 (t, 3H, J = 7.2 Hz, GluOCH? CH ₃ ). 1.75 (s, 3H, AcOH), 1.79- 1.89 (m, 1H, Glu-βΗ), 1.92-2.01 (m, 1H, Glu-βΗ), 2.19-2.24 (m, 1H, Hz, Glu-γΗ), 2.27-2.33 (m, 1H, Hz, GluγΗ ), 3.02-3.11 (m, 2H, -CHCH ₂ Ph), 3.71 (t, 1H, J = 7.4 Hz, -CHCH ₂ Ph), 3.95-4.08 (m, 4H, Glu-OCH ₂ CH ₃ ). -4.26 (m, 1H, Glu-aH), 4.79 (s, 2H, -OCHsCON-), 5.23 (s, 2H, -CH _? - (4-amidin) -Ar). 6.92 (d, 1H, J = 9.0 Hz, Ar-H ₅ ), * 6.93 (d, 1H, J = 9.0 Hz, Ar-H ₅ ), * 7.04-7.09 (m, 1H, Ar-H ₆ ), 7.14-7.26 (m, 5H. -CHCH _? Ph), 7.33 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), * 7.35 (d, 1H, J = 2.3 Hz, Ar-H ₈ ), * 7.45 (d, 2H, J - 8.5 Hz, (4-amidine) -Ar-H), 7.72 (d, 2H, J = 8.5 Hz, (4-amidine) -Ar-H), 8.65 (rs, 1H, Glu-NHCO-), 10.18 ( rs, 1H, Ar-NHCO-), * 10.26 (rs, 1H, Ar-NHCO-). * MS (FAB): m / z (%) = 658 (MH ⁺ , 100), 427 (5), 323 (30), 296 (8), 154 (9), 134 (41), 91 (25). IR (KBr): v [cm ' ¹ ] = 3318, 1734, 1684, 1559, 1508, 14 I05, 1197, 1019, 863, 700. Elemental analysis: Calculated for C37H43N5O10 x 1 / 3H ₂ O: 61.40% C 6.08% H 9.68% N Obtained: 61.18% C 6.26% H 9.39% N

Example 4

Synthesis of (2S) -2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl) ) amino] -2-benzyl-3-oxopropanoyl} amino) pentanedioic acid in the form of acetate (Compound 24)

Synthesis of (2S) -2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4-oxadiazol-3yl) ) benzyl] -3,4-dihydro-2H-1,4-benzoxazin-7-yl} amino) propanoyl] amino} pentanedioic acid (23)

Diethyl (2S) -2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl) ) amino] -2-benzyl-3-oxopropanoyl} amino) pentanedioate in acetate form (22) (0.396 g, 0.566 mmol) was dissolved in ethanol (50 mL), 2M NaOH (0.6 mL, 1.2 mmol) was added and stirred at room temperature. temperature until the end of the reaction is detected by thin layer chromatography. The solvent was evaporated and the oily residue was dissolved in distilled water (100 ml_). Acidify the reaction mixture with 1M HCl to pH = 3 and extract with ethyl acetate or dichloromethane (4 x 30 mL). The combined organic fractions were dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 0.310 g (78%) of compound 23 are obtained.

appearance: brown crystals T tal 125-129 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.70-1.82 (m, 1H, Hz, Glu-βΗ), 1.91-1.99 (m, 1H, Hz, Glu-βΗ), 2.10-2.24 (m, 2H, Hz, Glu-γΗ). 3.06-3.13 (m, 2H, -CHCH ₂ Ph), 3.60-3.65 (m, 1H, CHCH ₂ Ph), 4.21-4.27 (m, 1H, Glu-aH), 4.80 (s, 2H, -OCH ₂ CON) -). 5.20 (s, 2H, -CH ₂ - (4-oxadiazolinone) -Ar). 6.89 (d, 1H, 7 = 8.9 Hz, Ar-H ₅ ), * 6.90 (d, 1H, 7 = 8.9 Hz, Ar-H ₅ ), * 6.99-7.03 (m, 1H, Ar-H ₆ ). 7.15-7.24 (m, 5H, -CHCHzPh), 7.32 (d, 1H, J = 2.4 Hz, Ar-H ₈ ), * 7.33 (d, 1H, J = 2.4 Hz, Ar-H ₈ ), * 7.46 ( d, 2H, J = 8.1 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 7.76 (d, 2H, J = 8.1 Hz, -CH ₂ - (4-oxadiazolinone) -Ar-H), 8.16 (d, 1H, J = 8.9 Hz, Glu-CONH-), 8.19 (d, 1H, J = 8.9 Hz, Glu-CONH-), 9.67 (rs, 1H, ArNHCO -), * 9.83 (rs, 1H , Ar-NHCO-), * 12.91 (rs, 1H, oxadiazolinone NH). MS (FAB): m / z (%) = 644 (MH ⁺ , 29), 631 (3), 603 (5), 514 (5), 497 (3), 307 (16), 154 (100), 136 (83). 107 (33), 91 (57). IR (KBr): v [cm ' ¹ ] = 3325, 1778, 1662, 1512, 1405, 1329, 1220, 1051, 946, 894, 750, 701.

Synthesis of (2S) -2 - ({3 - [(4- {4- [amino (imino) methyl] benzyl} -3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl) amino] -2-Benzyl-3-oxopropanoyl} amino) pentanedioic acid in the form of acetate (24)

The compound is prepared from (2S) -2 - {[2-benzyl-3-oxo-3 - ({3-oxo-4- [4- (5-oxo-4,5-dihydro-1,2,4oxadiazole-3 -yl) benzyl] -3,4-dihydro-2 H -1,4-benzoxazin-7-yl} amino) propanoyl] amino} pentanedioic acid (23) (0.235 g, 0.365 mmol) by reduction under catalytic hydrogenation conditions at room temperature in acetic acid as solvent and palladium on carbon (10% by weight) as catalyst. 0.184 g (83%) of compound 24 is obtained.

layout: light yellow crystals T tal 177-181 ° C ¹ H NMR (300 MHz, DMSO-c / δ): δ [ppm] = 1.91 (s, 3H, AcOH), 1.69-1.84 (m, 2H, Hz, Glu-βΗ), 2.02-2.18 (m, 2H, Hz, Glu-γΗ), 3.03-3.16 (m. 2H, -CHCH _? Ph). 3.95-4.02 (m, 1H, Glu-aH), 4.79 (s, 2H, -OCHpCON-). 5.23 (s, 2H, -CH ₂ - (4-amidine) -Ar), 6.90 (d, 1H, J = 7.7 Hz, Ar-H ₅ ), * 6.96 (d, 1H, J = 7.7 Hz, Ar- H ₅ ), * 7.12-7.28 (m, 5H, CHCH ₇ Ph, Ar-H _e ), 7.46-7.51 (m, 3H, (4-amidine) -Ar-H, H ₈ ), 7.69-7.73 (m , 2H, (4-amidine) -Ar-H), 10.21 (rs, 1H, Ar-NHCO-). [signal for -CHCH ₂ Ph is under water signal] MS (FAB): m / z (%) = 602 (MH ⁺ , 16), 556 (2), 498 (3), 473 (3), 391 (17), 307 (22), 289 (12), 154 (100). 137 (75), 107 (27), 71 (56), 57 (60). IR (KBr): v [cm ' ¹ ] = 3330, 1678, 1615, 1512, 1403, 1221, 1050, 895, 700, 537. Elemental analysis: Calculated for C31H31N5O10 x AcOH x H ₂ O: 58.31% C 5.49% H 10.30% N Obtained: 58.76% C 5.68% H 9.98% N

Example 5

Synthesis of [amino (4 - {[7 - ({[(1S) -1-benzyl-2-ethoxy-2-oxoethyl] aminio} methyl) -3-oxo-2,3-dihydro [H] -1. 4-benzoxazin-4-yl] methyl} phenyl) methylene] ammonium dichloride (Compound 31)

Reaction scheme for the preparation of compounds 31 and 35

Synthesis of ethyl 2- (5-formyl-2-nitrophenoxy) acetate (26)

To a suspension of potassium fluoride (8.65 g, 148.92 mmol) in anhydrous DMF (40 mL) was added ethyl 2-bromoacetate (9.99 g, 56.84 mmol). After stirring at room temperature for 15 minutes, 3-hydroxy-4-nitrobenzaldehyde (10.00 g, 59.83 mmol) was added and stirred at 60 ° C for 24 hours. The reaction mixture was then cooled to room temperature, poured onto a mixture of water and ice (120 g) and extracted with ethyl acetate (1 x 300 mL - to dissolve all the precipitate, then 3 x 20 mL). The combined organic fractions were washed with 0.1 M NaOH (4 x 25 mL) and 10% aqueous citric acid solution (4 x 25 mL), dried with anhydrous MgSO ₄ , filtered and the solvent evaporated under reduced pressure. 13.93 g (92%) of compound 26 are obtained.

appearance: green-yellow crystals T tal 94 - 97 ° C ¹ H NMR (300 MHz, CDCI ₃ ): δ [ppm] = 1.31 (t, 3H, J = 7.06 Hz, -CH2CH3), 4.29 (q, 2H, J = 7.06 Hz, -CH _? CH ₃ ), 4.86 (s, 2H, -OCH? CO-) . 7.50 (d, 1H, J = 1.52 Hz, Ar-H _e ), 7.61 (dd, 1H, J = 8.10 Hz, J ₂ = 1.52 Hz, Ar-H ₄ ), 7.97 (d, 1 H, J = 8.10 Hz, Ar-H ₃ ), 10.04 (s, 1H, Ar-CHO). MS (Å): m / z (%) = 254 ((M + 1) ⁺ , 13%), 223 (12%), 207 (75%), 195 (24%), 179 (100%), 166 (22%). 150 (50%), 134 (54%), 120 (59%), 109 (10%), 103 (29%), 92 (48%), 78 (65%), 63 (53%), 53 ( 34%). IR (KBr): v [cm ' ¹ ] = 3481, 2991, 1750, 1699, 1604, 1528, 1458, 1364, 1208, 1161, 1101, 1020, 838, 719. Elemental analysis: Calculated for CnHuNOe: 52.18% C 4.38% H 5.53% N Obtained: 52.07% C 4.41% H 5.41% N

Synthesis of 3-oxo-3,4-dihydro-2H-1,4-benzoxazine-7-carbaldehyde (27)

Ethyl 2- (5-formyl-2-nitrophenoxy) acetate (26) (6.35 g, 25.12 mmol) was dissolved in anhydrous ethanol (60 mL), tin (11 l) chloride dihydrate (28.34 g, 125.0 mmol) was added and heated with stirring. on an oil bath at 70 ° C for 30 minutes. The reaction mixture was then cooled to room temperature and the solvent was evaporated under reduced pressure. The dry residue was then dissolved in 100 mL of water and basified with 1 M NaOH solution to pH = 7 - 8. The desired compound was partially eliminated as a yellow-orange precipitate. The precipitated precipitate was filtered off by suction and the mother liquor was extracted with ethyl acetate (3 x 20 mL). Evaporate the solvent under reduced pressure and combine the residue with a precipitate. Purification by column chromatography (MF: dichloromethane: methanol = 9: 1) gave 3.41 g (64%) of compound 27.

appearance: yellow crystals T tal 218-221 ° C ¹ H NMR (300 MHz, DMSO-d6): δ [ppm] = 4.67 (s, 2H, -OCH? CQ -). 7.Q7 (d, 1H, J = 7.91 Hz, Ar-H ₅ ), 7.42 (d, 1H, J = 1.50 Hz, Ar- H ₈ ), 7.54 (dd, 1 H, = 7.91 Hz, J ₂ = 1.50 Hz, Ar-H ₆ ), 9.83 (s, 1H, Ar-CHO), 11.13 (s, 1H, Ar-NHCO-). MS (EI): m / z (%) = 177 (M ⁺ , 100%), 148 (85%), 136 (7%), 120 (11%), 92 (17%), 78 (20%), 65 (28%) ), 52 (18%). IR (KBr): v [cm ' ¹ ] = 3062, 2970, 1708, 1683, 1603, 1516, 1428, 1322, 1271, 1119, 1046, 946, 816, 739, 536. Elemental analysis: Calculated for C ₉ H ₇ NO ₃ : 61.02% C 3.95% H 7.91% N Obtained: 61.07% C 4.13% H 7.94% N

Synthesis of 4 - [(7-Formyl-3-oxo-2,3-dihydro-4 H -1,4-benzoxazin-4-yl) methyl] -benzonitrile (28)

Suspensions of 3-oxo-3,4-dihydro-2 / 7-1,4-benzoxazine-7-carbaldehyde (27) (2.24 g, 12.66 mmol), benzyltriethylammonium chloride (2.88 g, 12.66 mmol) and potassium carbonate (4.37 g , 31.65 mmol) in acetonitrile (50 mL) 4- (bromomethyl) -benzonitrile (2.48 g, 12.66 mmol) was added while stirring. The reaction mixture was stirred under an oil bath at 60 ° C while stirring and the reaction was monitored by thin layer chromatography. After three days, the precipitate was filtered off and the solvent was evaporated under reduced pressure. The residue was dissolved in dichloromethane (100 mL) and the solution was washed sequentially with 10% citric acid solution (2 x 25 mL), saturated NaHCO ₃ solution (2 x 25 mL) and saturated NaCl solution (1 x 25 mL). The organic phase is then dried with anhydrous Na ₂ SO ₄ , filtered and the solvent evaporated under reduced pressure. Purification by column chromatography (MF: dichloromethane: methanol = 100: 1) gave 3.12 g (84%) of compound (28).

appearance: white crystals T tal 204 - 207 ° C ¹ H NMR (300 MHz, DMSO-d6): δ [ppm] = 4.94 (s, 2H, -OCH ₂ CON-), 5.31 (s, 2H, -N-CHg-Ar-CN), 7.16 (d, 1H, J = 8.29 Hz, Ar-Hs). 7.49 (d, 1H, J = 1.51 Hz, Ar-H ₈ ), 7.50 (d, 2H, J = 8.48 Hz, Ar-CN), 7.53 (dd, 1H, Jt = 8.29 Hz, J ₂ = 1.51 Hz , Ar-H ₆ ), 7.82 (d, 2H, J =

8.48 Hz, Ar-CN), 9.85 (s, 1H, Ar-CHO). MS (Å): m / z (%) = 292 ((M + 1) ⁺ , 64%), 148 (17%), 136 (6%), 116 (100%), 89 (21%), 63 (8%) · IR (KBr): v [cm ' ¹ ] = 2227, 1683, 1605, 1514, 1398, 1286, 1050, 930, 782, 556. Elemental Calculated for C ₉ H ₇ NO ₃ : 69.86% C 4.11% H 9.59% N analysis: Obtained: 69.56% C 4.05% H 9.24% N

Synthesis of ethyl (2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7yl] methyl} amino) -3-phenylpropanoate (30)

Suspensions of 4 - [(7-formyl-3-oxo-2,3-dihydro-4H-1,4-benzoxazin-4-yl) methyl] benzonitrile (28) (3.70 g, 12.82 mmol) and L-phenylalanine ethyl ester of hydrochloride (29) (2.95 g, 12.82 mmol) in 1,2-dichloroethane (120 mL) was added anhydrous triethylamine (1.29 g, 12.82 mmol) and stirred until a clear solution was obtained. This was then purged with argon and sodium triacetoxyborohydride (3.80 g, 17.94 mmol) was added. The reaction mixture was stirred under an inert argon atmosphere at room temperature for 24 hours. The reaction was quenched by the addition of saturated NaHCO ₃ solution (100 mL) and the first organic fraction separated in a separator funnel. The aqueous phase was extracted with ethyl acetate (3 x 25 mL). The combined organic fractions were dried with anhydrous Na ₂ SO ₄ , filtered and the solvent evaporated under reduced pressure. Purification by column chromatography (MF: dichloromethane: methanol = 50: 1) gave 4.96 g (82%) of compound 30.

appearance: green viscous oil T tal / ¹ H NMR (300 MHz, CDCI ₃ ): δ [ppm] = 1.18 (t, 3H, J = 7.16 Hz, -CH ₂ CH ₃ ), 2.95 (d, 2H, J = 7.15 Hz, -NH-CH 2 -ar). 3.48 (t, 1H, J = 6.78 Hz, -CH-CH ₂ Ph), 3.54 (d, 1H, J = 13.19 Hz, -CH _? -Ph) and 3.75 (d, 1H, J = 13.19 Hz, -CH , -Ph). 4.12 (q, 2H, J = 7.16 Hz, -CH ₂ -CH ₃ ). 4.73 (s, 2H, -OCHgCONH-L 5.20 (s, 2H, -CH _? -Ar-CN). 6.66 (d, 1H, J = 8.29 Hz, Ar-Hs), 6.79 (dd, 1H, J, = 8.29 Hz, J ₂ = 1/70 Hz, Ar-H ₆ ), 6.94 (d, 1H, J = 1.70 Hz, 1H, Ar-H ₈ ), 7.18 - 7.26 (m, 5H, -CH ₂ -Ph) , 7.37 (d, 2H, 7 = 8.67 Hz, Ar-CN), 7.64 (d, 2H, J = 8.67 Hz, Ar-CN). MS (FAB): m / z (%) = 470 ((M + 1) ⁺ , 51%), 378 (14%), 352 (8%), 277 (68%), 236 (35%), 194 (6%). 176 (5%), 162 (14%), 120 (29%), 91 (24%), 55 (16%). IR (NaCI): v [cm ' ¹ ] = 2979, 2229, 1729, 1688, 1609, 1512, 1398, 1328, 1189, 1054, 810, 701.

Elemental analysis: Calculated for C28H27N3O4. % H ₂ 0: 70.28% C 5.90% H 8.78% N Obtained: 70.09% C 6.07% H 8.76% N

Synthesis of [amino (4 - {[7 - ({[(1S) -1-benzyl-2-ethoxy-2-oxoethyl] aminio} methyl) -3-oxo-2,3-dihydro4H-1,4-benzoxazine -4-yl] methyl} phenyl) methylene] ammonium dichloride (31)

Ethyl (2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl] methyl} amino) -3 solution -phenylpropanoate (4) (1.29 g, 2.75 mmol) in anhydrous ethanol (35 mL) was cooled in an ice bath and gaseous HCl was introduced under stirring for 15 minutes. The flask is then stoppered and stirred at room temperature until the next day. The solvent was evaporated under reduced pressure and the residue was suspended in anhydrous ether (40 mL). The ether was poured to remove excess HCI. The rinsing process is repeated three more times. The light gray crystals of the iminoether were dried in air. They were then dissolved in anhydrous ethanol (40 mL) and dry crystals of ammonium acetate (0.57 g, 7.42 mmol) were added, which were washed with ether. The reaction mixture was stirred at room temperature for 4 days. Then, under reduced pressure, two-thirds of the solvent is evaporated to give a white precipitate. The suspension was cooled on an ice bath and filtered off with suction. Recrystallization from ethanol gave 0.31 g (18%) of compound 31.

appearance: white crystals T tal Mp 160-163 ° C ¹ H NMR (300 MHz, DMSOd ₆ ): δ [ppm] = 1.04 (t, 3H, J = 7.16 Hz, -CH _? CH ₃ ). 2.82-2.86 (m, 2H, -NHCH? Ar). 3.45 (t, 1H, J = 7.16 Hz, -CH-CH ₂ Ph), 3.50 (d, 1H, J = 13.93 Hz, -CH ₂ Ph), 3.69 (d, 1H, J = 13.93 Hz, -CH _? Ph). 3.96 (q, 2H, J = 7.15 Hz, -CH2CH3), 4.79 (s, 2H, -OCH _? CON-) · 5.25 (s, 2H, -CHpArCN), 6.78 (dd, 1H, = 8.29 Hz, J ₂ = 1.50 Hz, Ar-H ₆ ), 6.88 (d, 1 H, J = 8.29 "Hz, Ar-H ₅ ), 6.89 (d, 1 H, J = 1.50 Hz, Ar-H ₈ ), 7.12 - 7.27 (m, 5H, -Ph), 7.48 (d, 2H, J = 8.64 Hz, Ar-H), 7.82 (d, 2H, J = 8.64 Hz, Ar-H). MS (FAB): m / z (%) = FAB, m / z (%): 487 ((M + 1) ⁺ , 17%), 477 (1%), 460 (3%), 391 (13%), 324 (9 %), 307 (25%), 289 (11%), 171 (46%), 154 (100%), 137 (74%), 124 (20%), 107 (23%), 89 (19%) , 78 (9%), 71 (42%), 55 (30%). IR (KBr): v [cm ' ¹ ] = 3141, 1724, 1662, 1616, 1404, 1053, 702. ri ²⁰ -7.06 "(c = 1.00g / 100 mL, MeOH)

Example 6

Synthesis of (2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7yl] methyl} amino) -3-phenylpropanoic acid (compound 32)

Ethyl (2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl] methyl} amino) -3-phenylpropanoate (4) ( 1.80 g, 3.84 mmol) was dissolved in 1,4-dioxane and 1 M NaOH (5.7 mL, 5.7 mmol) was added and stirred at room temperature for 8 hours. The dioxane was evaporated under reduced pressure and the residue dissolved in water (100 mL). The impurities present were extracted with ethyl acetate (20 mL). The aqueous phase was then neutralized with 1 M HCl (5.7 mL); in this case the product falls out as an insoluble precipitate, which is filtered off by suction. Recrystallization from ethanol (400 mL) gave 1.55 g (91%) of compound 32.

appearance: white crystals T tal 202-204 ° C ¹ H NMR (300 MHz, DMSO-d _e ) · δ [ppm] = 2.82 (dd, 1H, Jt = 13.57 Hz, J ₂ = 7.54 Hz, -NH-CH? -ar). 2.92 (dd, 1H, L = 13.57 Hz, J ₂ = 6.03 Hz, -NH-CH? -Ar), 3.30 (t, 1H, J = 6.78 Hz, -CH-CH ₂ Ph), 3.53 (d, 1H , J = 13.57 Hz, -CH _? ~ -Ph). 3.70 (d, 1H, J = 13.94 Hz, -CH _? -Ph). 4.79 (s, 2H, -O-CH ₂ -CON-), 5.22 (s, 2H, -CH ₂ -Ar-CN), 6.79 (dd, 1H, Jt = 8.29 Hz, J ₂ = 1.50 Hz, Ar- H _e ), 6.85 (d, 1 H, J = 8.29 Hz, Ar-H ₅ ), 6.91 (d, 1H, J = 1.51 Hz, Ar-He), 7.18-7.27 (m, 5H, Ph), 7.46 (d, J = 8.29 Hz, 2H, Ar-CN), 7.80 (d, J = 8.29 Hz, 2H, Ar-CN). MS (FAB): m / z (%) = 442 ((M + 1) ⁺ , 10%), 277 (13%), 137 (71%), 120 (15%), 107 (20%), 89 (14%), 77 (13%), 55 (11%). IR (KBr): v [cm ' ¹ ] = 3370, 3057, 2229, 1693, 1570, 1515, 1398, 1294, 1055, 894, 808, 698, 539. ri ²⁰ -7.06 ° (c = 1.00 g / 100 mL, MeOH) HRMS Calculated for C ₂₆ H ₂ 3N ₃ O4: 441.168857 Obtained: 441.169950

Synthesis of diethyl (2S) -2 - {[(2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7-yl] methyl} amino ) -3-Phenylpropanoyl] amino} pentanedioate (34)

(2S) -2 - ({[4- (4-cyanobenzyl) -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7-yl] methyl} amino) -3- of phenylpropanoic acid (5) (1.43 g, 3.24 mmol) and diethyl ester of L-glutamic acid hydrochloride (33) (0.78 g, 3.24 mmol) in anhydrous DMF (30 mL) were added HOBT (0.49 g, 3.65 mmol) and z / V-methylmorpholine was adjusted to pH 8. 8. EDC (0.84 g, 4.38 mmol) was then added and stirred at room temperature for 1 day. The solvent was evaporated under reduced pressure and the residue was dissolved in dichloromethane (40 mL). The solution was then washed sequentially with 10% citric acid solution (3 x 30 mL), saturated NaHCO ₃ solution (2 x 25 mL) and saturated NaCl solution (1x10 mL). The organic phase was dried with anhydrous Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. Purification by column chromatography (MF: dichloromethane: methanol = 40: 1) gave 1.63 g (80%) of compound 34.

appearance: light green crystals T tal Mp 105-108 ° C ¹ H NMR (300 MHz, DMSO-d _e y δ [ppm] = 1.16 and 1.17 (2 xt, 3H, J = 7.15 Hz, -CH ₂ CH ₂ COOCH ₂ CH ₃ , -CHCOOCH ₂ CH ₃ ). 1.77 - 1.87 (m, 1H, -CH-CH ₂ CH ₂ COOCH ₂ CH ₃ ), 1.96-2.05 (m, 1H, -CH-CH ₇ CH ₉ COOCH ₉ CH ₃ ) · 2.28 (t, 2H, J = 7.71 Hz, -CHCH _? CH _? COOCH ₂ CH ₃ ), 2.27 (rs, 1H, -NH), 2.71 (dd, 1H, J = 13.57 Hz, J ₂ = 7.89 Hz, -NH-CHg-Ar). 2.86 (dd, 1H, Jt = 13.57 Hz, J ₂ = 5.46 Hz, -NH-CH ₂ Ar), 3.19 - 3.25 (m, 1H, -CH-CH ₂ Ph), 3.40 (d, 1H, J = 13.18 Hz, -C ^ Ph). 3.60 (d, 1H, J = 13.18 Hz, -CHgPh). 4.05 (q, 2H, J = 7.15 Hz, CH9CH9COOCH9CH9), 4.07 (q, 2H, J = 7.15 Hz, -CH-COOCH ₂ CH ₃ ), 4.25 4.32 (m, 1H, -CH-CH ₂ CH ₂ COOCH ₂ CH ₃ ), 4.78 (s, 2H, -O-CH 9 -CO-). 5.22 (s, 2H, -CH _? -Ar-CN). 6.73 (dd, 1H, Jt = 8.29 Hz, J ₂ = 1.50 Hz, Ar-H ⁶ ), 6.82 (d, 1H, J = 8.29 Hz, Ar-H ⁵ ), 6.86 (d, 1 H, J = 1.50 Hz, Ar-H ⁸ ), 7.14 - 7.25 (m, 5H, -Ph), 7.46 (d, 2H, J = 8.29 Hz, Ar-CN), 7.81 (d, 2H, J = 8.29 Hz, Ar-CN) ), 8.23 (d, 1H, J = 8.28 Hz, -CONH-). MS (Å): m / z (%) = 627 ((M + 1) ⁺ , 44%), 396 (10%), 277 (100%), 162 (6%), 136 (10%), 116 (29%). 105 (6%), 91 (12%), 73 (18%). IR (KBr): v [cm ¹ ] = 3321, 2981, 2228, 1745, 1684, 1641, 1517, 1400, 1200, 1126, 1027, 895, 744, 698. ri ²⁰ MD -27.05 ° (c = 0.10 g / 100 mL, MeOH) HRMS Calculated for C ₃ 5H ₃₈ N ₄ O7 '. 67.09% C 6.07% H 8.94% N Obtained: 66.87% C 6.07% H 8.78% N

Synthesis of diethyl (2S) -2 - [((2S) -2 - {[(4- {4- [amino (imino) methylbenzyl} -3-oxo-3,4-dihydro-2 / N, 1,4-benzoxazine- 7-yl) methyl] amino} -3-phenylpropanoyl) amino] pentanedioate in the form of acetate (35)

Diethyl (2S) -2 - ({(2S) -2 - [({4- [4-cyanomethyl) benzyl] -3-oxo-3,4-dihydro-2 H-1,4-benzoxazin-7yl (methyl) amino] -3-phenylpropanoyl} amino) propanoate (34) (0.758 g, 1.21 mmol) was dissolved in anhydrous EtOH (20 ml_) and gaseous HCl was introduced while stirring on an ice bath for 30 minutes. The reaction mixture was sealed well and allowed to stir at room temperature. After the end of the reaction is detected by thin layer chromatography, the solvent is evaporated. The residue was washed several times with ether and dried well. The resulting iminoether was dissolved in anhydrous alcohol (EtOH, 20 mL), ammonium acetate (0.255 g, 3.31 mmol) was added and allowed to stir at room temperature until the end of the reaction was detected by thin layer chromatography. The solvent is then evaporated to 1/3 volume and left at 4 ° C for a few days. The precipitated crystals were filtered off and washed with the mother liquor or a little cold EtOH. 0.164 g (20%) of compound 35 is obtained.

appearance: white crystals T tal Mp 164-168 ° C ¹ H NMR (300 MHz, DMSO-d6): δ [ppm] = 1.13-1.20 (m, 6H, Glu-OCH, CH ₃ ). 1.78-1.85 (m, 1H, Hz, Glu-βΗ), 1.85 (s, 3H, AcOH), 1.97-2.03 (m, 1H, Hz, Glu-βΗ), 2.25-2.31 (m, 2H, Glu-γΗ ), 2.67-2.74 (m, 1H, -CHCH? Ph). 2.85 (dd, 1H, J _y = 13.9 Hz, J ₂ = 5.4 Hz "CHCH? Ph). 3.24 (dd, 1H, J, = 8.1 Hz, J ₂ = 5.4 Hz-CHCH ₂ Ph), 3.38 (d, 1 H, J = 13.7 Hz, Ar-CH? NH-b 3.60 (d, 1 H, J = 13.7 Hz, Ar-CH? NH-) 4.024.11 (m, 4H, Glu-OCH ₂ CH ₃ ), 4.25-4.32 (m, 1H, Glu-aH), 4.77 (s, 2H, OCH ₂ CON -), 5.17 (s, 2H, -CH ₂ - (4-amidin) -Ar) 6.73 (dd, 1H, J, = 8.1 Hz, J ₂ = 1.7 Hz, Ar-H ₆ ), 6.83-6.87 ( m, 2H, Ar-H ₅₈ ), 7.15-7.23 (m, 5H, CHCH? Ph) 7.33 (d, 2H, J = 8.5 Hz, (4-amidine) -Ar-H), 7.82 (d, 2H , J = 8.5 Hz, (4-amidine) -Ar-H), 8.23 (d, 1H, J = 8.3 Hz, -CONH-). MS (FAB): m / z (%) = 644 (MH ⁺ , 100), 552 (6), 414 (13), 324 (19), 307 (14), 295 (88), 154 (65), 134 (64). 107 (20), 91 (24). IR (KBr): v [cm ' ¹ ] = 3341, 2979, 1735, 1683, 1635, 1539, 1402, 1205, 1028, 897, 697. ri ²⁰ M « +16.9 (c = 0.100 g / 100 mL, MeOH)

Example 7

Synthesis of compound ethyl (3S) -3 - ({[2 - ({4- [amino (imino) methyl] benzyl} oxy) -4-methyl-3-oxo-3,4-dihydro-2 / 7-1,4- benzoxazin-7-yl] carbonyl} amino) -3-phenylpropanoate in the form of acetate (46)

Reaction Schemes for the Preparation of Compound 46.

Synthesis of ethyl 3-hydroxy-4-nitrobenzoate (37)

3-Hydroxy-4-nitrobenzoic acid (36) (11.11 g, 60.0 mmol) was dissolved in 50 mL absolute ethanol, 96% sulfuric acid (5 mL) was added and heated at reflux for 2 hours. The reaction mixture was cooled and the solvent was evaporated. The oily residue was dissolved in dichloromethane (150 mL) and washed with saturated NaHCO3 solution (3 x 50 mL) and saturated NaCl solution (1 x 50 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. 12.169 g (96%) of compound 37 are obtained.

appearance: yellow crystals T tal 80-83 C ° and _the ²¹⁴ ° C = 84 1 H NMR (300 MHz, DMSO-d ₆ y. δ [ppm] = 1.33 (t, 3H, J = 7.1 Hz, -CH _? CH _a ), 4.34 (q, 2H, J = 7.1 Hz, -CH? CHA 7.48 (dd, 1H, 7 = 8.7 Hz, J ₂ = 1.7 Hz, Ar-H ₆ ), 7.69 (d, 1H, J = 1.7 Hz, Ar-H ₂ ), 7.95 (d, 1H, J = 8.7 Hz, Ar-Hs). MS (ΕΙ): m / z (%) = 211 (M ⁺ , 49), 183 (65), 166 (100), 149 (8), 136 (9), 120 (25), 81 (18), 69 (26). 63 (26). IR (KBr): v [cm ' ¹ ] = 3312, 2976, 1728, 1590, 1526, 1479, 1325, 1227, 1148, 1070, 1017, 894, 846, 751,650.

Synthesis of ethyl 4-amino-3-hydroxybenzoate (38)

The compound was prepared from ethyl 3-hydroxy-4-nitrobenzoate (37) (5.279 g, 25.0 mmol) by reduction under catalytic hydrogenation conditions with palladium on carbon as a catalyst at room temperature and EtOH (100 mL) as a solvent. 4.53 g (100%) of compound 38 are obtained.

appearance: yellow crista i 1 tal 90-94 ° CT, _a , ^ref = 98 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.26 (t, 3H, J = 7.1 Hz, -CH ^ CHs). 4.19 (q, 2H, J = 7.1 Hz, -CH ₂ CH _a ), 6.60 (d, 1H, J = 8.0 Hz, Ar-H ₅ ), 7.25 (dd, 1H, 7 = 8.0 Hz, J ₂ = 1.7 Hz, Ar-H ₆ ), 7.27 (s, 1H, Ar-H ₂ ). MS (Å): m / z (%) = 181 (M ⁺ , 64), 153 (33), 136 (100), 108 (18), 80 (25). IR (KBr): in [cm ¹ ] = 3375, 1707, 1676, 1624, 1449, 1298, 1239, 1104, 1025, 942, 760.

Synthesis of ethyl 4 - [(2,2-dichloroacetyl) amino] -3-hydroxybenzoate (39)

Ethyl 4-amino-3-hydroxybenzoate (38) (78) (9.03 g, 49.8 mmol) was dissolved in anhydrous diethyl ether (100 mL), triethylamine (7.28 mL, 52.3 mmol) was added and cooled to -10 ° C. Dichloroacetyl chloride (4.79 mL, 49.8 mmol) was added gradually while stirring, stirring for 1 h in an ice bath and 4 hours at room temperature. The reaction mixture was washed with 10% citric acid (2 x 50 mL), dried with sodium sulfate, filtered and the solvent evaporated under reduced pressure. 8.757 g (60%) of compound 39 are obtained.

layout: yellow crystals T tal Mp 153-158 ° C ¹ H NMR (300 MHz, DMSO d ₆ y. δ [ppm] = 1.31 (t, 3H, J = 7.2 Hz, -CH? CH ₃ ). 4.28 (q, 2H, J = 7.2 Hz, -CH ₂ CH ₃ ), 7.01 (s, 1H, -C (O) CHCl ₂ ), 7.46 (d, 1H, J = 8.4 Hz, Ar-H ₅ ), 7.52 (d, 1H, J = 2.3 Hz, Ar-H ₂ ), 8.13 (dd, 1H, = 8.4 Hz, J ₂ = 2.3 Hz, Ar-H ₆ ), 10.00 (rs, 1H, Ar-OH). 10.68 (rs, 1H, -NHCO-). MS (Å): m / z (%) = 264 (M ⁺ , 8), 228 (2), 181 (100), 153 (19), 135 (12), 107 (9), 79 (22). IR (KBr): v [cm ' ¹ ] = 3290, 1682, 1610, 1543, 1435, 1297, 1230, 1099, 1011, 859, 768. HRMS: Calculated for C ₁₁ H ₁₁ NO ₄ : 45.23% C 3.80% H 4.79% N Obtained: 45.39% C 3.81% H 4.60% N

Synthesis of ethyl 2-hydroxy-3-oxo-3,4-dihydro-2 / 7-1,4-benzoxazine-7-carboxylate (40)

To a solution of ethyl 4 - [(2,2-dichloroacetyl) amino] -3-hydroxybenzoate (39) (6.670 g, 22.8 mmol) in distilled water (180 mL), NaHCO ₃ (3.84 g, 45.6 mmol) was added and heated to boiling 1 hour. The reaction mixture was cooled and acidified with 1M HCl to pH = 3. Extracted with EtOAc (3 x 50 mL), the combined organic fractions were dried with Na ₂ SO ₄ and the solvent was evaporated under reduced pressure. 4.501 g (83%) of compound 40 are obtained.

layout: brown amorphous substance Tfp 187-200 ° C ¹ H NMR (300 MHz, DMSO- d ₆ y δ [ppm] = 1.30 (t, 3H, J = 7.1 Hz, -CH? CH ₃ ). 4.28 (q, 2H, J = 7.1 Hz, -CH ₂ CH ₃ ). 5.54 (s, 1H, 2-H), 7.04 (d, 1H, J = 8.2 Hz, Ar-H ₅ ), 7.50 (s, 1H, Ar-H ₈ ), 7.63 (dd, 1H, = 8.2 Hz). J ₂ = 1.3 Hz, Ar-H ₆ ), 8.11 (rs, 1H, 2-OH). MS (Å): m / z (%) = 237 (M ⁺ , 24), 208 (100), 192 (29), 180 (49), 153 (16), 136 (48),

108 (14), 80 (19). IR (KBr): v [cm ' ¹ ] = 3289, 1702, 1614, 1518, 1302, 1077, 1010, 932, 851,768, 512. HRMS: Calculated for CnHnNOs: 237.063723 Obtained: 237.064150

Synthesis of ethyl 2-hydroxy-4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-7-carboxylate (41)

Prepare a solution of ethyl 2-hydroxy-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7-carboxylate (40) (3.74 g, 15.8 mmol) and K ₂ CO ₃ (4.37 g, 31.6 mmol) and dimethyl sulfate (1.66 mL, 17.4 mmol) was added with stirring and heated at 40 ° C overnight. The reaction mixture was filtered and the mother liquor was evaporated. After separation by circular radial chromatography (MF:

dichloromethane: methanol = 50: 1) 0.684 (17%) of compound 41.

appearance: light brown crystals T tal Mp 132-136 ° C ¹ H NMR δ [ppm] = (300 MHz, 1.32 (t, 3H, J = 7.1 Hz, -CH ₂ CH ₃ ). 3.35 (s, 3H, N-CH ₃ ), 4.30 (q, 2H, J = 7.1) DMSO- Hz, -CH _? CH ₃ ). 5.66 (s, 1H, 2-H), 7.33 (d, 1H, J = 8.5 Hz, Ar-H ₅ ), 7.53 (d, d ₆ ): 1H, J = 1.9 Hz, Ar-H ₈ ), 7.71 (dd, 1H, = 8.5 Hz, J ₂ = 1.9 Hz, Ar-H ₆ ), 8.16 (rs, 1H, 2-OH). MS (Å): m / z (%) = 251 (M ⁺ , 25), 234 (14), 222 (100), 206 (22), 194 (49), 150 (14). IR (KBr): v [cm ' ¹ ] = 3136, 1719, 1667, 1614, 1480, 1394, 1293, 1213, 1095, 1029, 904, 760, 579. HRMS: Calculated for C ₁₂ H ₁₃ NO ₅ : 57.37% C 5.22% H 5.58% N Found: 57.57% C 5.15% H 5.50% N

Synthesis of ethyl 2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7carboxylate (42)

Suspension of potassium fluoride (0.149 g, 2.57 mmol) and 4- (bromomethyl) benzonitrile (0.504 g,

2.57 mmol) in DMF (20 mL) was stirred at room temperature for 15 minutes, while ethyl 2-hydroxy-4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7 was added while stirring -carboxylate (41) (0.645 g, 2.57 mmol) and heated at 60 ° C until the end of the reaction is detected by thin layer chromatography. The solvent was evaporated and the oily residue was dissolved in dichloromethane (50 mL) and washed with 10% citric acid (2 x 30 mL), saturated NaHCO ₃ solution (2 x 30 mL) and saturated NaCl solution (1 x 30 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. The product was purified by circular radial chromatography (dichloromethane: methanol = 100: 1) to give 0.364 (38%) of compound 42.

appearance: yellow amorphous substance T tal 106-120 ° C ¹ H NMR (300 MHz, DMSO-b ₆ ): δ [ppm] = 1.33 (t, 3H, J = 7.1 Hz, -CH ₇ CH ₃ ). 3.40 (s, 3H, N-CH ₃ ), 4.31 (q, 2H, J = 7.1 Hz, -CHpCHA 4.91 (s, 2H, -OCH, -Ar). 5.79 (s, 1H, 2-H), 7.34 -7.42, 7.507.59, 7.71-7.81 (m, 8H, Ar-H, (4-CN) -Ar-H), 8.30 (rs, 1H, 2-OH). MS (Å): m / z (%) = 366 (M ⁺ , 21), 295 (20), 235 (18), 207 (52), 194 (44), 132 (47), 116 (100), 104 (68). 77 (38). IR (NaCI): v [cm ' ¹ ] = 2227, 1696, 1613, 1508, 1397, 1285, 1248, 1159, 1084, 1032, 968, 818, 767. HRMS: Calculated for C ₂₀ H ₁₈ N ₂ O ₅ : 366.121572 Obtained: 366.122110

Synthesis of 2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7 carboxylic acid (43)

Ethyl 2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-7-carboxylate (42) (0.564 g, 1.54 mmol) was dissolved in ethanol (50 mL), 2M NaOH (1.54 mL, 3.08 mmol) was added and stirred at room temperature until the end of the reaction was detected by thin layer chromatography. The solvent was evaporated and the oily residue was dissolved in distilled water (50 mL). Acidify the reaction mixture with 1M HCl to pH = 3 and extract with dichloromethane (4 x 20 mL). The combined organic fractions were dried with Na ₂ SO ₄ then filtered and the solvent was evaporated under reduced pressure. 0.360 g (69%) of compound 43 are obtained.

appearance: brown amorphous substance Tfp 125-150 ° C ¹ H NMR (300 MHz, DMSOde) '· δ [ppm] = 3.36 (s, 3H, N-CH ₃ ), 4.58 (s, 2H, -OCHp-Ar). 5.78 (s, 1H, 2-H), 7.33 -7.44 (m, 2H, Ar-H), 7.51 (d, 2H, J = 8.5 Hz, - (4-CN) -Ar-H), 7.71-7.83 (m, 3H, ΑΓΗ, - (4-CN) -Ar-H). MS (Å): m / z (%) = 338 (M ⁺ , 21), 295 (20), 235 (18), 207 (52), 194 (44), 132 (47), 116 (100), 104 (68). 77 (38). IR (NaCI): v [cm ^-1 ] = 3424, 2931, 2229, 1684, 1615, 1518, 1396, 1294, 1090, 1032, 826.

HRMS: Calculated for C ₁₈ H ₁₄ N ₂ O ₅ : 338.090272 Obtained: 338.091020

Synthesis of ethyl (3S) -3 - [({2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2H-1,4benzoxazin-7-yl} carbonyl) amino] -3-Phenylpropanoate (45)

Solutions of 2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2 H -1,4-benzoxazine-7 carboxylic acid (44) (0.360 g, 1.06 mmol) and ethyl (3S) -3-amino-3-phenylpropanoic acid hydrochloride (104) (0.244 g, 1.06 mmol) in DMF (40 mL) was added with stirring, HOBT (0.172 g, 1.28 mmol). Z / V-methylmorpholine was adjusted to pH above 7 and EDC (0.275 g, 1.44 mmol) was added and stirred at room temperature for at least 4 hours. Evaporate the solvent under reduced pressure and dissolve the residue in dichloromethane (40 mL) and wash with 10% citric acid (2 x 20 mL), saturated NaHCO ₃ solution (2 x 20 mL) and saturated NaCl solution (1 x 20 mL). The organic phase was dried with Na ₂ SO ₄ , filtered and the solvent was evaporated under reduced pressure. Purification by circular radial chromatography (dichloromethane: methanol = 100: 1) afforded 0.485 (89%) of compound 45.

appearance: yellow oil T tal / ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.09 (t, 3H, J = 7.1 Hz, -CH? CH ₃ ), 3.37 (s, 3H, N-CH ₃ ), 3.98-4.07 (m, 2H, CH ₂ CH ₃ ). 5.75 (s, 1H, 2-H), 7.49 (dd, 1H, 7 = 7.7 Hz, J ₂ = 1.9 Hz, Ar-H ₆ ), 7.31-7.44 (m, 7H, (4-CN) -Ar- H, Ph-H), 7.51 (d, 1H, J = 7.7 Hz, Ar-H, _8), 7.767.80 (m, 2H, (4-CN) -Ar-H). MS (Å): m / z (%) = 513 (M ⁺ , 4), 468 (5), 439 (3), 426 (3), 407 (4), 366 (4), 321 (52), 192 (100), 116 (44). IR (NaCI): v [cm ' ¹ ] = 3748, 3364, 2229, 1698, 1507, 1393, 1293, 1029. HRMS: Calculated for C ₂₉ H ₂₇ N ₃ O ₆ : 513.189986 Obtained: 513.190550

Synthesis of ethyl (3S) -3 - ({[2 - ({4- [amino (imino) methyl] benzyl} oxy) -4-methyl-3-oxo-3,4-dihydro-2H1,4-benzoxazin-7 -yl] carbonyl} amino) -3-phenylpropanoate in the form of acetate (46)

Ethyl (3S) -3 - [({2 - [(4-cyanobenzyl) oxy] -4-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-7yl} carbonyl) amino] - 3-Phenylpropanoate (45) (0.230 g, 0.448 mmol) was dissolved in anhydrous

EtOH (20 mL) and gaseous HCl was introduced while stirring on an ice bath for 30 minutes. The reaction mixture was sealed well and allowed to stir at room temperature. After the end of the reaction is detected by thin layer chromatography, the solvent is evaporated. The residue was washed several times with ether and dried well. The resulting iminoether was dissolved in anhydrous alcohol (EtOH, 20 mL), ammonium acetate (0.173 g, 2.241 mmol) was added and stirred at room temperature until the end of the reaction was detected by thin layer chromatography. The solvent was then evaporated to 1/3 volume and left at 4 ° C for several days. The precipitated precipitate was filtered off and washed with ether. 0.070 g (29%) of compound 46 are obtained.

appearance: white crystals 1 tal 170-175 ° C ¹ H NMR (300 MHz, DMSO-d ₆ ): δ [ppm] = 1.09 (t, 3H, J = 7.1 Hz, -CH? CH ₃ V 1.10 (t, 3H, J = 7.1 Hz, -CH ₂ CH ₃ ), * 1.74 (s, 3H, AcOH). 3.35 (s, 3H, N-CH ₃ ), 3.99-4.07 (m, 2H, -CH _? CH ₃ ) 5.71 (s, 1H, 2-H), 7.25-7.43 (m, 9H, Ar-H. (4-CN) -Ar-H, Ph-H), 7.65-7.74 (m, 3H, Ar-H, (4-CN) -Ar-H), 8.91 (d, 1H, J = 8.3 Hz, CONH ). MS (FAB): m / z (%) = 531 (MH ⁺ , 100), 321 (19), 307 (12), 154 (71), 136 (62), 107 (27), 71 (60), 55 (70). IR (KBr): v [cm ¹ ] = 3282, 1735, 1684, 1616, 1507, 1404, 1312, 1085, 1033, 764. Elemental analysis: Calculated for C31H34N4O8 * ^1/2 H ₂ 0: 62.09% C 5.88% H 9.34% N Obtained: 62.01% C 5.97% H 8.94% N

Example 8

Test results of biological activities of compounds

Compound Ki thrombi n (μΜ) Ki trypsin (μΜ) Ki FXa (μΜ) Ki trip / Ki thrombus Binding affinity to the fibrinogen receptor IC ₅ o (μΜ) 11 38.6 5.30 22.3 0.14 0.973 17 12.1 8.4 20.6 0.69 6.84 22 21.6 10.5 19.2 0.5 3.66 24 49.4 17.7 25.7 0.4 0.206 31 16.3 5.5 8.8 0.3 5.19 35 23.3 46.2 54.1 2 3.94

Claims (13)

1. Compounds of general formula (I): (I) where they mean: A is O, S, NH or CH _2; B is C = O or C = S; R1 is H, branched or unbranched C1-C4 alkyl, a benzyl group, or an OR group in which R is H, branched or unbranched C1-C4 alkyl or benzyl; R2 is H, COOR, CONHR or a substituent K, which is one of the groups where: X Y R52 is CONH, NHCO, CH ₂ NH, NHCH ₂ , OCH ₂ or CH ₂ O; is CO or CH _2; is a group N-R1 NH ² group N-R1 -NC ^{7 ΝΗ} * ² or COOR Group, R62 is R, COOR or a group N-R1 // NH ₂ where R and R 1 have the meanings defined above; R3 is H, branched or unbranched C1-C4 alkyl, benzyl, OH, COR or a substituent L, which is one of the groups where: Z is CO, CH ₂ , a CH ₂ CONH group attached to the N atom of the bicycle via the CH ₂ CO fragment, or a CH ₂ CONHCH _{2 group} attached to the N atom of the bicycle via the CH ₂ CO fragment; W is (CH ₂ ) _n , (CH ₂ ) _n CO or CO (CH ₂ ) _n group wherein n is 1, 2, 3 or 4; N-R1 N-R1 - < ^z - N— < ^z NH hi kih R53 is group ² , group ² or group -COOR, N-R1 - The Nile — I R63 is R, COOR or group ² wherein R and R1 have the meanings defined above; R4 is a group attached at the site of 6 or 7 bicycle ring and may be H, a group Q-CH (R7) -C00R, Q-CH (R7) CH ₂ COOR, Q-CH (R7) -CONH- (R9), or a substituent M, which is one of the groups where: Q is a group CON (R8), N (R8) CO, CH ₂ N (R 8), N (R 8) CH _2; Y is CO or CH _2; R7 H is branched or unbranched C1-C4 alkyl, C4-C6 cycloalkyl, phenyl or benzyl; R8 is H or a group - [CH ₂ ] _n COOR, where n is 0, 1, 2, 3 or 4, and the substituent R has the above defined meaning; R9 is a CH (R10) COOR group or a CH ₂ CH group (NHSO ₂ C ₆ H ₄ -4-R) COOR, where R the meaning defined above and wherein R 10 represents a group R, a CH ₂ COOR group, or a CH ₂ CH ₂ COOR group as defined by the substituent R; from above N-R1 N-R1 - < ^{Ζ _} [j- < ^Ζ NH Η ΝΗ R54 is group ² , group ² or group -COOR, N-R1 - ( ^z R64 is R, COOR or an ^NH3 group, wherein R and R1 have the meanings defined above provided that N-R1 N-R1 - < ^z - NNH and NH - if R52 is group ² or group ² or if R62 is R or group N-R1 - ( ^with nh ₂ » R4 is H, R53 and R63 are COOR, wherein R63 cannot be COOH; N-R1 N-R1 - < ^z -N- ( ^z NH hi nh - if R52 is group ² or group ² or if R62 is R or group N-R1 - ( ^with nh ₂ are R54 and R64 COOR, where R64 cannot be COOH and R3 cannot be a group L; N-R1 N-R1 - ( ^Z ~ N ^- < ^Z -if R53 is a group ^NH or a group ^NH or if R63 is R or a group N-R1 - ( ^Z nh ₂ is a group R4 H, groups R52 and R62 are COOR, where R62 cannot be COOH; N - R1 N - R1 - ( ^{Z -} N ^- ( ^Z -if R53 is a group ^NH or a group of ^NHS or if R63 is R or a group N-R1 - ( ^Z nh ₂ is R54 and R64 COOR, where R64 cannot be COOH and R2 cannot be a group K; N-R1 N-R1 - ( ^z —N— ( ^z NH H μη - if R54 is group ² or group ² or if R64 is R or group N-R1 - ( ^with nh ₂ are R52 and R62 COOR, where R62 cannot be COOH and R3 cannot be a group L; - if R54 is a group N-R1 - ( ^With NH ₂ N-R1 N-R1 \ ^- N ^- ( ^{Z NHz} or group or if R64 is R or group are R53 and R63 are COOR, where R63 cannot be COOH and R2 cannot be group K; in the form of pure enantiomers, pure diastereomers, mixtures of enantiomers and mixtures of distareomers and pharmaceutically acceptable salts thereof. Compounds of formula (I) according to claim 1 for use as therapeutically effective substances. Use of compounds of formula (I) according to claim 1 for the preparation of medicaments that inhibit platelet aggregation induced by the binding of fibrinogen to the fibrinogen receptor on platelets. Use of the compounds of formula (I) according to claim 1 for the preparation of medicaments that inhibit thrombin and fibrin formation and clot formation. Use of the compounds of formula (I) according to claim 1 for the preparation of medicaments that inhibit factor Xa and fibrin production and clot formation. Use of the compounds of formula (I) according to claim 1 for the preparation of medicaments that inhibit the coagulation factors thrombin and factor Xa and the formation of fibrin and the formation of blood clots. Use of the compounds of formula (I) according to claim 1 for the preparation of medicaments that inhibit platelet aggregation caused by the binding of fibrinogen to the fibrinogen receptor on platelets and simultaneously inhibit thrombin or factor Xa and fibrin formation and clot formation. Use of the compounds of formula (I) according to claim 1 for the preparation of medicaments used for the prevention and treatment of arterial and venous thrombosis, ischemic stroke, peripheral arterial disease, acute coronary syndrome, pulmonary emboli and for the prevention of systemic emboli in cardiac patients , to prevent ischemic events in patients with surgery, such as prevention of constipation in the arterial reconstruction procedures, prevention of blood clotting in the extracellular circulation and hemodialysis. Pharmaceutical compositions comprising a therapeutically effective amount of a compound of formula (I) according to claim 1 and pharmaceutically acceptable excipients. Pharmaceutical preparations according to claim 10, characterized in that they are used as antithrombotic drugs for humans and other mammals. 11. Method for inhibiting aggregation, characterized in that I use « 12. A method for inhibiting tromt, characterized in that the use of blood / human / other mammalian jit according to claim 1. * ** “II in the blood of man and other mammals 5jin8 $ torm * $ (I) according to claim 1. A method for the inhibition of thrombin or factor Xa and the simultaneous inhibition of human and other mammalian platelet aggregation y4 <rvi in order to prevent thrombus formation in humans if <other mammals, characterized in that a compound of Formula 1 is used.